text
stringlengths 10
379k
|
|---|
Ca activation of cardiac contraction is regulated by the human cardiac troponin complex (cTn), consisting of the Ca-binding cTnC subunit, the inhibitory cTnI subunit and the tropomyosin (Tm)-binding cTnT subunit. During the systole, cytosolic [Ca] rises to ≥1 M and binds to the low affinity Ca-binding site II of cTnC, the so-called regulatory binding site. This leads to a conformational change of the N-terminal domain of cTnC, which rearranges its interaction with the C-terminal regulatory region of cTnI (). This structural rearrangement involves the C-terminal part and the inhibitory region of cTnI and leads to the removal of Tm from its blocking position on actin, allowing myosin to bind to actin in a force-generating manner. During the diastole, cytosolic Ca falls to submicromolar concentrations. The systolic process is reversed: Ca dissociation from cTnC induces a conformational rearrangement of the complex involving cTnI and cTnT, which immobilizes Tm on the thin filament (). In this way, Tm prevents myosin from binding de novo to actin in a force-generating manner, and as the remaining cross-bridges detach, the heart relaxes ().
Simultaneous measurements of force and intracellular [Ca] in intact cardiac trabeculae have shown that the rate of myoplasmic Ca increase/removal does not rate-limit force development/relaxation (,). Thus, in principle, two mechanisms could determine the time course of the contraction/relaxation of myocardial force: 1), the cross-bridge turnover kinetics, and 2), the kinetics of thin-filament activation/inactivation after Ca binding/dissociation.
Cross-bridge kinetics during activation/relaxation have been obtained from force transients induced either by flash photolysis of caged Ca and caged-Ca chelators in skinned cardiac fibers (–) or by rapidly changing the [Ca] in the environment of subcellular cardiac myofibrils (,). The observed rates of force increase/decrease and their dependence on interventions affecting cross-bridge mechanics indicated that cross-bridge turnover kinetics determine the time course of cardiac myofibrillar force development and relaxation. This implies, but does not prove, that the switch-on and switch-off kinetics of cTn are rapid processes compared to the time course of force changes.
Kinetics of thin filament activation/inactivation have been investigated extensively by measuring the Ca-induced conformational changes of cTn using isolated regulatory proteins like cTnC (e.g., (–)), or the whole cTn complex (), as well as more complex systems, like reconstituted thin filaments (,). Comparison of the kinetics observed with isolated cTnC, cTnC·cTnI, cTn complex, and the cTn complex in reconstituted thin filaments showed that the kinetics depends on the complexity of the system (,). However, it had not yet been determined whether and how switch-on and switch-off kinetics of cTn are altered when the complex is incorporated into the functional environment of the contractile sarcomere. Therefore, direct measurements of the switch kinetics in the sarcomere are required to test for the possible role of cTn in rate-limiting force development/relaxation.
Recently, Bell et al. () measured simultaneously the kinetics of the switch-on of cTnC and force development in skinned fibers. Ca activation was induced by flash photolysis of caged Ca and switch-on kinetics was monitored with fluorescence polarization. Their fluorescence polarization signal was biphasic, which provided evidence for two conformational changes. The fast conformational change (∼120 s) was too fast to rate-limit the contraction of the cardiac muscle, whereas the slow conformational change (∼5 s) had the same rate constant as the kinetics of the force development. The fast change was interpreted to reflect the kinetics of Ca binding to cTnC, though it was considerably slower than that measured for cTnC in solution. The slow change was interpreted to reflect a further activation of the thin filament when cross-bridges bind to it. However, it remained unclear whether the slow phase is a consequence of cross-bridge binding or represents an intrinsic part of thin filament activation that rate-limits the force increase. Thus, there is a need for an alternative technique to probe the Ca-induced thin filament activation in the sarcomere.
Furthermore, to fully understand the mechanism of cTn switch in the sarcomere, not only the kinetics of the switch-on but also that of the switch-off and its relation to force relaxation has to be determined. Such experiments are difficult to perform with skinned fibers because the Ca affinity of available caged-Ca chelators is limited (). These problems can be overcome with myofibrils, which are functional contractile units of muscle. In comparison to skinned fibers, they are small enough to perform kinetic studies by rapid-mixing techniques such as stopped-flow (,) and rapid-solution-change techniques (,). These two techniques allow rapid and defined changes from initial to final [Ca] by the use of Ca chelators such as 1,2-Bis(2-aminophenoxy)ethane-,,′,′-tetraacetic acid (BAPTA).
The main goal of this study was to elucidate whether the Ca-dependent conformational change of cTn rate-limits the kinetics of muscle contraction and relaxation. To determine the kinetics of conformational change of cTn, we labeled cTnC on Cys-84 with N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (IANBD), a fluorescence detector of protein conformational changes, and reconstituted it with cTnI and cTnT to form a heterotrimeric cTn complex (cTn labeled with IANBD at cysteine 84 cTnC (NBD-cTn)). NBD-cTn was then exchanged against the endogenous cTn in cardiac myofibrils. The stopped-flow technique allowed us to determine the switch-on and switch-off kinetics from the changes in NBD-Tn fluorescence, which occur after rapid increases and decreases in [Ca]. Because this technique can be applied in the same way to myofibrils and isolated proteins, it allowed us to investigate how the myofibrillar environment changes switch-on and switch-off kinetics.
In parallel to the stopped-flow measurements, myofibrillar force kinetics after rapid increase and decrease in [Ca] were measured to test whether the kinetics of force development and/or relaxation could be rate-limited by the Ca-dependent conformational changes of cTn.
The three human cardiac troponin (cTn) subunits cTnC, cTnT, and cTnI were separately expressed in and isolated as described previously by Kruger et al. (), with the modification of adding protease inhibitors (1 mM PMSF, 1 g/ml pepstatin A, 0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 10 M leupeptin, 14.5 M antipain, 5 g/ml aprotinin) to the buffer for the lysis of cells. The plasmids of the three subunits were kindly provided by C. S. Redwood (University of Oxford, Oxford, UK). Site-directed mutagenesis of Cys-35-Ser on cTnC allowed us to label specifically the remaining Cys-84 in cTnC with the fluorescent dye IANBD. cTnC (2.5 mg/ml) was dissolved for 2 h at 4°C in urea buffer containing 25 mM MOPS (3-(N-morpholino)propanesulfonic acid, 4-morpholinepropanesulfonic acid), 0.2 M NaCl, 6 M urea, 0.5 mM CaCl, 2 mM dithiothreitol (DTT), adjusted to pH 7.0 with NaOH, and then dialyzed (1:100) against buffer A but with 0.1 mM DTT for ∼3 h. IANBD was dissolved in dimethylformamide ([DMF] < 2%) and then added in a 5:1 molar ratio (IANBD/cTnC) to the protein solution. The mixture was incubated for 1 h at 4°C in the dark. The labeling reaction was stopped by adding ∼10 mM DTT. The labeled cTnC was then dialyzed (1:100 at 4°C for 2 h) against the same urea buffer containing 1 mM DTT to remove the free fluorescence label. Thereafter the labeled cTnC was reconstituted with cTnI and cTnT to the cTn complex, as described previously by Kruger et al. (). Briefly, for reconstitution, the cTn subunits (weight ratio of cTnC/cTnT/cTnI = 2.5:1:1) were dialyzed in a series of buffers containing 25 mM MOPS, 0.5 mM CaCl, and 1 mM DTT, adjusted to pH 7.0 with NaOH and decreasing in [NaCl] (buffer I, 1.0 M NaCl; buffer II, 0.75 M NaCl; buffer III, 0.5 M NaCl), with a final buffer (IV) containing 0.3 M KCl and no NaCl. Dialysis was performed three times in each buffer for 2 h respectively. After reconstitution, 20 mM DTT was added and the cTn solution was filtered (pore size 0.45 m). The filtrate was then stored at −80°C. Before use, the cTn solution was thawed and then dialyzed (1:10 for 1 h at 4°C) against Ca-free rigor buffer containing 10 mM Tris, 132 mM NaCl, 5 mM KCl, 1 mM MgCl, 1 mM NaN, and 5 mM EGTA (pH 7.1 with NaOH).
Guinea pigs (females of 1–4.5 months in age) were anaesthetized with isofluran before the heart was excised. The following steps were all carried out at 4°C. The heart (1.7–2.2 g in weight) was perfused for ∼10 h with the Ca-free rigor buffer. The ventricular walls were dissected into small pieces of ∼10 mm and homogenized in exchange buffer (100 mg/ml) for 5 s with a blender (Ultra-Turrax T25, Janke & Kunkel, Staufen, Germany). The homogenate was filtrated with meshes (pore sizes 40 m and 20 m), and then centrifuged (380 × for 10 min at 4°C). The pellets were resuspended in an equal volume of a skinning buffer that contained 1% Triton X-100, 10 mM Tris, 10 mM NaCl, 15 0 mM KCl, 1 mM NaN, 1 mM MgCl, and 5 mM EGTA, adjusted with NaOH to pH 7.1. After incubation for 5 min, the myofibrils were centrifuged (380 × for 10 min at 4°C) and the supernatant was discarded.
NBD-cTn was incorporated into myofibrils by replacing the endogenous cTn as a whole in the myofibrils according to the technique developed by Brenner et al. (). To exchange the endogenous cTn in the myofibrils against NBD-cTn, the myofibril pellet was resuspended in Ca-free rigor buffer including protease inhibitors (0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 10 M leupeptin, 14.5 M antipain, and 5 g/ml aprotinin) and 1 mg/ml of NBD-cTn. The mixture was shaken gently for 1 h at room temperature. To prevent overcontraction of the myofibrils after transferring them from the Ca-free rigor buffer (0 mM ATP) to the ATP-containing relaxation buffer, 10 mM of the actomyosin ATPase inhibitor BDM (,-butandione-2-monoxime) was added before centrifugation of the myofibrils (380 × for 10 min at 4°C). The pellet was then resuspended in approximately three volumes of one of two relaxation buffers. For switch-on kinetics, the buffer (single-mixed (SX) relaxation buffer, pCa >8) contained 10 mM imidazole, 3 mM MgCl, 47.7 mM NaCrP, 1 mM ATP, 3 mM BAPTA, and 20 mM DTT, adjusted to pH 7.0 with HCl plus 10 mM BDM. For switch-off kinetics, the buffer (double-mixed (DX) relaxation buffer, pCa >8) had the same constitution, but with 0.6 mM BAPTA instead of 3 mM BAPTA. The myofibrils were then washed twice by centrifugation (380 × for 10 min at 4°C) and resuspension in the respective relaxation buffer containing no BDM to remove the excess cTn and the BDM.
To obtain myofibrillar suspensions with a reproducible density of the myofibrils, the concentration of myosin heads was determined at their absorption maximum at ∼280 nm as in Herrmann et al. (). For switch-on kinetics (single mixing) the concentration of myosin heads was 3 M, and for switch-off kinetics (double mixing) it was 6 M.
The incorporation of NBD-cTn into myofibrils was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Therefore, samples of the myofibrils (∼10 g total protein/slot) before and after the exchange were reduced with 30 mM DTT and alkylated with 75 mM iodoacetamide at pH 8.5, according to the procedure of Lane et al. (). The samples were then transferred to Laemmli buffer, and the proteins were separated on a 12.5% SDS-PAGE and were visualized by Commassie-R250-staining. The different mobility of guinea pig and human cTnI made it possible to monitor the loss of endogenous guinea pig cTn and its replacement by the exogenous NBD-cTn.
The Ca-dependent fluorescence change of cTn was measured using a stopped-flow apparatus of Bio-Logic (Claix, France) model SFM-400/S equipped with a TC 100/10F-cuvette (10-mm light path, dead time of 2.2 ms at 14 ml/s flow rate). The sample was excited by a 75-W Hg-Xe-Lamp (Hamamatsu, Hamamatsu, Japan) coupled to a monochromator (TgK Scientific, Bradford-on-Avon, UK) set to 482 nm and a band-with of ∼18 nm. Emission of fluorescence was monitored using an OG515 nm cut-off filter and a 530/40-nm band-pass filter in front of a custom-made photomultiplier system. All measurements were performed at 10°C. To determine the switch-on kinetics, myofibrils in SX relaxation buffer (pCa >8) were mixed 1:1 with SX activation buffer (like relaxation buffer but containing, in addition, different [CaCl] up to 6 mM) using the single-mixing mode of the apparatus. The switch-off kinetics were determined by DX: myofibrils in DX relaxation buffer were first mixed 1:1 with DX activation buffer (like DX relaxation buffer, but with 1.2 mM CaCl) to induce Ca activation of the myofibrils at pCa 5.0. After an incubation time of 91 ms, the myofibrils were then mixed 1:1 with DX inactivation buffer (like DX relaxation buffer, but with 12 mM NaBAPTA), which leads to an instantaneous drop in [Ca] to pCa >8.
To determine the kinetics of the fluorescence change after mixing rigor-myofibrils with 1 mM ATP, exchanged myofibrils were washed twice by centrifugation (380 × for 10 min at 4°C) and resuspension with normal rigor buffer (as detailed above, but adjusted to pH 7.0 with HCl). These myofibrils were mixed 1:1 with rigor buffer containing 2 mM ATP using the single-mixing mode of the apparatus.
To determine the switch kinetics on isolated cTn, the procedure was exactly the same as for cTn in myofibrils, except that instead of myofibrils we used cTn. Before the measurements of the switch-on and -off kinetics cTn was dialyzed 1:10 against the respective relaxation buffer three times (1 h at 4°C). For switch-on and -off kinetics, the concentration of cTn was 75 g/ml in the cuvette.
Thin cardiac myofibril bundles (2.0–3.5 m in diameter, 25–70 m in length) with the incorporated NBD-cTn were mounted in an apparatus based on the principle of atomic force microscopy, as described previously by Stehle et al. (). The myofibrils had slack sarcomere lengths of 1.96 ± 0.05 m (mean ± SD), which is similar to that of native cardiac myofibrils (1.98 ± 0.04 m). Bundles were prestretched by 15% of their slack length and then Ca-activated and thereafter relaxed by fast solution changes (10–30 ms), based on a technique used by Colomo et al. (). Experiments were performed at 10°C. Activating or relaxing buffers were the same as for stopped-flow measurements.
Kinetic parameters of myofibrillar contraction () and relaxation (, , and ) were obtained as described previously (). Transients of force relaxation were fitted to a function consisting of a linear (, ) and an exponential term (), starting at the time of Ca removal.
The NBD-cTn was incorporated to the myofibrils according to the method developed by Brenner et al. for skinned fibers (), i.e., by replacing the endogenous cTn as a whole complex with the exogenous labeled NBD-cTn. The binding of NBD-cTn in the sarcomere after performing the cTn exchange was visualized by fluorescence microscopy. To test whether NBD-cTn had been bound to the actin and not to the myosin filaments, a myofibril was mounted in the mechanical setup and stretched to sarcomere length of 3.15 ± 0.05 m. Thereby the actin filaments were pulled out partly from their overlap with the myosin filaments. Comparison of the fluorescence with the corresponding bright-field image () shows that the fluorescence is preferentially localized along the I-band and the distal parts of the A-band. This fluorescence pattern suggests that NBD-cTn is predominantly bound to the actin filaments.
The exchange was probed by SDS-PAGE. Since endogenous guinea pig cTnI and exogenous human cTnI have different electrophoretic mobilities, they can be fully separated by SDS-PAGE and their amounts quantified. The gel showed that ∼68 ± 14% (SD, = 6) of the endogenous cTnI was replaced by the exogenously added NBD-cTn (). The efficiency of our exchange correlates well with the exchange described for cardiac fibers (,,). That the exchange is incomplete can be explained by the fact that under rigor conditions, in the absence of Ca, mainly the overlapping region is exchanged but not the nonoverlapping thin filament ().
To test to what extent changes in NBD-Tn fluorescence induced by mixing myofibrils with the activator Ca reflect specifically rather than unspecifically bound NBD-Tn, the fluorescent NBD-cTn in exchanged myofibrils was “chased” by incubating the myofibrils for 1 h in Ca-free rigor buffer with an excess (10 mg/ml) of unlabeled cTn. The myofibrils were then mixed in the stopped-flow apparatus with (pCa 4.6) or without Ca (pCa >8), respectively. The recorded fluorescence traces were compared with those obtained with “unchased” myofibrils treated in the same way as the chased ones but without adding the unlabeled cTn to the Ca-free rigor buffer. shows that mixing of unchased myofibrils with Ca induces an exponential fluorescence increase with a signal/noise ratio of >10. In comparison to the unchased myofibrils, chased myofibrils also exhibit a significant background fluorescence at pCa >8, suggesting that some of the NBD-cTn might be not specifically bound to the sarcomere and cannot reversibly be replaced by competitive binding of the unlabeled cTn. However, in contrast to unchased myofibrils, there was almost no change in fluorescence when the chased myofibrils were mixed with Ca. Thus, it is possible that NBD-cTn partly binds unspecifically, but if so, this does not appear to bias the interpretation of the fluorescence signal, as it seems not to significantly contribute to the Ca-induced fluorescence changes.
We have investigated whether the exchange of cTn impairs myofibrillar structure by examining myofibrils before and after the exchange by phase contrast microscopy under 500× magnification. We could detect no effect of exchange on either sarcomere length or other characteristics of myofibrillar morphology ().
To test whether the exchange of the endogenous cTn in myofibrils by the human, recombinant NBC-cTn affects the contractile properties of myofibrils, we determined the kinetic parameters of force development and relaxation for NBD-cTn exchanged myofibrils and compared them with the parameters of native myofibrils which were treated as exchanged myofibrils but without NBD-cTn. In both exchanged and native myofibrils, Ca induces a monoexponential force development with a rate constant . The values of were not significantly different between the two preparations (). In both preparations, Ca removal induced a biphasic force decay described by an initial slow, seemingly linear decline with a rate constant, , lasting for a duration, , followed by a rapid exponential decay with a rate constant . There were no significant differences in , , or between native and exchanged myofibrils (). In summary, the cTn exchange and the IANBD label on cTnC appears not to alter significantly the kinetics of contraction or relaxation of the myofibrils.
In contrast to this, a decrease in the force per cross-sectional area was observed in exchanged myofibrils by up to ∼1/3. A similar reduction of force upon cTn-exchange has been reported for skinned fibers (,).
We also determined the kinetics of ATP-induced cross-bridge detachment of native and NBD-cTn exchanged myofibrils by measuring the changes in their intrinsic tryptophan fluorescence induced by mixing myofibrils incubated under rigor conditions with Mg·ATP (). The rate constant of the fluorescence increase induced by 1 mM Mg·ATP for NBD-cTn exchanged myofibrils (32 ± 7 s, = 4) was not significantly different ( = 0.30) from that for native myofibrils (46 ± 11 s, = 4). In conclusion, neither the exchange nor the labeling changes fundamental kinetics properties of the myofibrils, although maximal force can be decreased.
To test whether the high shear forces during the stopped-flow measurements could damage the myofibrils, we compared the sarcomere length and morphology of the myofibrils before and after being mixed in the stopped-flow apparatus: no change in sarcomere length or in the morphology before and after mixing of the myofibrils could be detected even if the maximum flow rate of 14 ml/s was used ().
In a first series of experiments, we investigated whether the structural environment of the myofibrils changes the switch-on kinetics of cTn. For this, we compared the Ca-induced fluorescence increase of isolated and incorporated NBD-cTn after changing the pCa from >8 to different pCa (). In both isolated and incorporated cTn, the jump to saturating [Ca] induced a biphasic fluorescence increase with an initial fast phase that cannot be resolved (for details, see below) followed by a resolvable slow phase. At Ca activation with pCa 4.6, the slow phase of the fluorescence increases monoexponentially with a rate constant of ( = 7) for isolated NBD-cTn and ( = 6) for incorporated NBD-cTn (). Thus, incorporation of the NBD-cTn increased the kinetics of the slow phase about fourfold at maximum Ca activation.
The kinetics of the fast phase of the fluorescence increase was too rapid to be resolved. The existence of the unresolvable fast phases with isolated and incorporated NBD-cTn is clarified in , respectively, where the monoexponential functions () fitted to the fluorescence transients () are extrapolated to = 0 s, i.e., the time of mixing with Ca. It is evident, that the extrapolations do not reach the baseline levels obtained by mixing isolated or incorporated NBD-cTn with the Ca-free buffer (pCa >8). After mixing isolated NBD-cTn with high [Ca] (pCa 4.6), already 50 ± 5% (SD, = 5) of the total fluorescence increase occurred within the dead time of 2.2 ms provided by the standard mixing cuvette of the apparatus (). For incorporated NBD-cTn, 63 ± 14% (SD, = 6) of the total fluorescence increase could not be resolved (). Even using a microcuvette with a shorter dead time of ∼250 s did not allow us to record the initial fluorescence rise (data not shown). Thus, the rate constant of the fast phase has to be >2000 s. This could be explained if the fast phase represents the fast Ca binding to cTnC. We have supported this hypothesis by modeling and simulation of the Ca-induced fluorescence changes (for details, see Discussion).
The fluorescence increase after activation with different [Ca] shows that the contribution of the fast phase to the overall fluorescence change is reduced with decreasing [Ca] (). At pCa <6.0, almost the whole signal is resolvable. This observation suggests that the fast phase and the slow phase report two sequentially coupled conformational changes (for details see Modeling, in Discussion) and thus supports the hypothesis that the label senses the fast Ca binding to the regulatory Ca-binding site II, which then induces a slower, regulatory conformational change.
For isolated and incorporated NBD-cTn the Ca dependencies of the steady-state fluorescence amplitudes (Δ) and of the rate constants of the slow phase () can be fitted by sigmoidal dose-response curves (). For incorporated NBD-cTn, the Ca dependence of Δ gives a pCa of 6.4 ± 0.1 ( = 6) () and a Hill slope of 1.1 ± 0.1 ( = 6), whereas the Ca dependence of leads to a pCa of 5.9 ± 0.1 ( = 6) and a Hill slope of 2.3 ± 0.4 ( = 6). In comparison to this, the Ca dependence of Δ of isolated NBD-cTn has a pCa of 6.7 ± 0.1 ( = 5) and thus is ∼0.3 pCa units higher than incorporated NBD-cTn. As expected, the Hill slope is 1.0 ± 0.1 ( = 5). The Ca dependence of the rate constant has a pCa of 6.2 ± 0.1 ( = 5) and thus is also ∼0.3 pCa units higher than incorporated NBD-cTn. The Hill slope is 1.4 ± 0.4 ( = 5).
The kinetics of the conformational changes after Ca removal from cTnC was determined also with isolated NBD-cTn and NBD-cTn incorporated into myofibrils. For this, the isolated NBD-cTn or the exchanged myofibrils were first Ca-activated for 91 ms at pCa 5.0 and then inactivated at pCa >8. The activation time was chosen to be sufficiently long to reach steady-state fluorescence during activation and, on the other hand, to be sufficiently short to prevent overcontraction of the myofibrils.
, shows that after the reduction in [Ca], the fluorescence decays biphasically for isolated and incorporated NBD-cTn. As for the switch-on kinetics, the first, fast phase cannot be resolved and is followed by a second slower phase. Compared to the switch-on kinetics, the amplitude of the missed signal for incorporated NBD-cTn comprises only 28 ± 15% ( = 10) of the total amplitude. For isolated NBD-cTn the missed signal is 40 ± 14% ( = 5). The decay of the slow phase was fitted by a monoexponential yielding a rate constant of ( = 10) for incorporated NBD-cTn and ( = 5) for isolated NBD-cTn.
shows the fluorescence change of NBD-cTn after mixing rigor myofibrils with 1 mM ATP. The fluorescence decays with a rate constant of 33 ± 7 s ( = 3) and is similar to the after Ca inactivation.
To find out whether the kinetics of the Ca-dependent switch-on/switch-off of cTn rate-limit the dynamics of myofibrillar force development and/or relaxation, force kinetics of myofibrils exchanged with NBD-cTn after rapid (within ∼10 ms) changes in [Ca] were determined in the mechanical setup. depicts the Ca-induced force development after a change in pCa from >8 to 4.6. Force rises in a monoexponential manner with a rate constant = 1.0 ± 0.1 s ( = 24). Thus, at saturating [Ca] the force develops ∼300-fold slower than the slow phase of the fluorescence increase, which is presumed to reflect the regulatory conformational change of cTnC.
shows that the force decay after the Caremoval (changing pCa from 4.6 to >8) is faster than force development and, additionally, biphasic. First, during the “quasi-isometric” slow phase, the sarcomere lengthening is negligible. The rate constant of this slow phase is = 0.4 ± 0.1 s ( = 24) and has a duration of = 0.3 ± 0.1 s ( = 24). The slow phase is followed by a rapid relaxation phase, which is induced by the rapid, sequential lengthening of sarcomeres (). It has a rate constant of = 3.1 s ± 0.1 ( = 24). Thus is ∼80 times, and ∼10 times, slower than the fluorescence decay presumed to report the regulatory conformational change of cTn.
xref
#text |
In the course of the life cycle of human immunodeficiency virus 1 (HIV-1), two copies of genomic RNA dimerize via loop-loop interactions. This process starts at the dimerization initiation site (DIS), located at the 5′ untranslated region of the viral RNA. The DIS stem-loop is nine-nucleotides-long (residues A-A) and contains a six-nucleotide (nt) self-complementary sequence in the loop that is flanked by one conserved adenine base at the 3′ side (residue A) and two conserved purines at the 5′ side (residues A and R) (). The 6-nt sequence promotes genome dimerization by formation of a kissing loop-loop complex () (–), which may be converted into a more stable extended duplex form at higher temperature (55°C) or in the presence of nucleocapsid protein (–). The conserved purines are key for formation and stability of kissing complexes (,). Kissing tertiary interaction has been also reported for TAR elements of HIV-1 () and H3 stem-loops of Moloney murine leukemia virus (). Kissing complex motifs were also identified during replication of the ColE1 plasmid () and in the crystal of two tRNAAsp (between their anticodon loops) (). In addition, large ribosomal subunit shows one kissing complex close to the ribosome exit site (regions 412–428 and 2438–2454 of ) (), in which flanking bases mediate tertiary contacts with neighboring part of the 23S rRNA.
Crystal structures of HIV-1 subtype A and B DIS kissing-loop complex and extended duplex forms provided comprehensive views of this key region (–). Despite the difference in topology, both forms have similar overall shape but they differ in position of purine R273. Recently, kissing-loop complex structures were refined at higher resolution (). In addition, two structures of HIV-1 subtype F DIS kissing-loop complex were obtained in distinct crystal environments, showing some variation in the conformation of bulged-out purines at the 5′ side () (see below). Additionally to x-ray structures, two NMR structures of HIV-1 subtype B DIS (,) and one NMR structure of HIV-1 subtype B/F DIS () were obtained. While Lancelot's () and Baba's NMR () structures are generally in agreement with the x-ray data, Mujeeb's NMR subtype B structure () shows substantial differences in the overall geometry compared to x-ray () and MD structures (). However, all three NMR structures exhibit apparent differences in the positions of flanking bases (A and R and the symmetrical ones) compared to the x-ray structures. The x-ray structures consistently show the flanking bases to be in bulged-out arrangement while the NMR experiments suggest their bulged-in orientation.
The RNA atomic-resolution experiments can be complemented by computational molecular dynamics (MD) studies (). Modeling is limited by the accuracy of the force field and simulation timescale but carefully executed MD simulations can be quite useful (–).
HIV-1 DIS kissing-loop complexes were studied using molecular dynamics methods (,–). We have carried out a set of explicit solvent MD (AMBER force field (), 33 ns of simulations for HIV-1 DIS kissing-loop complexes and 21 ns for other kissing systems) of HIV-1 DIS kissing-loop complexes assuming the earlier x-ray (PDB codes 1JJN and 1JJM) and NMR (1BAU) structures as start (). The simulations predicted a novel four-adenine stack of the bulged-out bases (in the subtype B). This MD-predicted arrangement was subsequently confirmed by new x-ray structures of kissing-loop complex of subtype F and extended duplex form of subtype B (,), and termed “closed conformation.” The central pocket of the kissing complexes is characterized by a deep electrostatic potential (ESP) site. The simulations revealed that the pocket, in absence of divalent cations, is continuously occupied by 2–3 monovalent ions, a feature that was missed by the other MD studies. The ions smoothly exchange with the bulk solvent on a timescale ∼1–3 ns per ion while being delocalized in the pocket. Such flexible ion-binding sites are not likely to be captured by the x-ray technique which explains the absence of ions in many refined x-ray kissing-loop structures. The simulations revealed distortions of the oldest HIV-1 NMR DIS kissing-loop complex () and deformation of intermolecular basepairs in NMR kissing-loop complex of H3 stem loops of Moloney murine leukemia virus ().
Beaurain and Laguerre () performed an MD (CHARMM force field (), ∼15 ns total) study of both NMR and x-ray kissing-loop complexes of subtype B. In contrast to our work, they suggested that the starting NMR structure results in more stable trajectory than the x-ray structure. Aci et al. reported MD simulations (AMBER force field, ∼44 ns total) () of both structural forms of DIS. Extended duplex simulations (both NMR and x-ray) appeared stable while NMR kissing complex simulations showed large rearrangements at the stem-loop junctions. Surprisingly, this study reported rapid and peculiar destabilization (melting) of the stems when starting from the x-ray kissing complexes, which is in striking disagreement with our preceding results with the same force field (). This is a quite unusual simulation behavior and to the best of our knowledge would be the only reported case where RNA x-ray structures are degraded in AMBER explicit solvent simulations. Another MD (AMBER) study of x-ray kissing complex of subtype A and B DIS was performed on a rather short timescale (400 ps) (). Finally, the x-ray subtype B kissing complex () was recently merged in silico with the NMR structure of the internal loop in an attempt to obtain a complete SL1 stem-loop structure in dimer form ().
In view of the discrepancies among the earlier MD studies, availability of new x-ray and NMR structures, and the continuing disagreement between positions of flanking bases seen in x-ray structures and predicted by NMR, we substantially extend the preceding theoretical studies on RNA kissing complexes. We report multiple extended MD simulations (AMBER code and force field, 30–50 ns trajectories, 583 ns in total) to study conformations of flanking bases in HIV-1 subtypes A, B, and F DIS crystal structures (), in two recent DIS NMR structures (,), and in the ribosomal kissing complex (). The simulations are carried out under variable ion conditions. The standard simulations are further supplemented by locally enhanced sampling (LES) MD technique (94 ns total) (,–) to enhance the sampling of the flanking bases. The AMBER simulations are complemented by preliminary CHARMM (–) simulations (98 ns in total), to get insights into the dependence of the results on the force field.
Even with this considerable computational effort, we were unable to obtain a quantitative and converged description of the flanking base behavior (and other related studied should be viewed in this context). Nevertheless, our simulations quite clearly reveal that free flanking bases tend to self-associate via stacking while we identify several distinct substates (close in energy) that can be adopted by the flanking nucleotides. The LES technique considerably contributed to our ability to describe the conformational flexibility of the flanking bases, so we assume that our simulations identify essentially all substates that are sampled by them, albeit we cannot guarantee that their mutual balance is not affected by the force field and sampling limitations. The bulged-out geometry with consecutive stack of four bases, predicted first by our earlier simulations () and seen subsequently in new x-ray structures (), is the most prominent substate. It is encouraging to see that AMBER and CHARMM force fields provide a qualitatively similar description of the flanking base substates, albeit CHARMM shows a visible tendency to a partial melting of the ARNA stem ends.
Overview of all simulations is given in . X-ray structures of the subtypes A, B, and F (PDB codes 1XPF, 1XPE, 1ZCI, and 1YXP) () and NMR structures of the subtypes B and B/F (PDB codes 2F4X/model 1 and 2D19/model 11) were simulated using the AMBER program () version 8 () with parm99 () (simulations MD_A_1-2; MD_B_1-2; MD_F_1-2; and MD_nmr_1-2, respectively). Our preceding simulations () of x-ray subtypes A and B (PDB codes 1JJN and 1JJM ()) using the AMBER-6.0 were extended up to 30 ns (simulations MD_A_3-4 and MD_B_3-4). Note that PDB files 1JJN and 1JJM have meantime been withdrawn from the PDB database and replaced as PDB 2B8R and 2B8S because of a reassignment of metal ions. Ribosomal kissing complex (regions 412–428 and 2438–2454) was extracted from the x-ray structure of the 50S subunit of (PDB code 1JJ2) ().
All systems were neutralized by Na or K ions using the Xleap module of AMBER. Ions that were placed initially into major groove or binding pockets were manually shifted 5 Å away from the solute to avoid any initial bias. Some simulations were carried out with x-ray Mg and K ions. Box of TIP3P water molecules was added to a distance of 12 Å on each side of the solute. The following parameters were used: Na radius 1.868 Å and well depth 0.00277 kcal/mol; Mg radius 0.7926 Å and well depth 0.8947 kcal/mol; and K radius 2.658 Å and well depth 0.000328 kcal/mol (). Note that the parm99 DNA force field was very recently replaced by reparameterization of the / backbone torsional profiles, presently known as parmbsc0 (). The force-field refinement was necessitated by substantial imbalances occurring in B-DNA simulations with parm99 and parm94, which are eliminated by parmbsc0. In contrast to DNA, however, the parm99 force field shows a proper backbone behavior in RNA simulations (,,,), and in tests we performed so far, both parm99 and parmbsc0 are equally suitable for RNA simulations.
The standard simulations were carried out using the particle mesh Ewald technique () with 9 Å nonbonded cutoff and 2-fs integration time step. Equilibration started by 5000 steps of minimization followed by 200 ps of MD, with the atomic positions of the solute molecule fixed. Then, two series of minimization (1000 steps) and MD simulation (20 ps) were carried out with restraints of 50 and 25 kcal/(mol Å), which were applied to all solute atoms. In the next stage, the system was minimized in five 1000-step rounds with restraints (20, 15, 10, 5, and 0 kcal/(mol Å)) applied only to solute atoms. During the subsequent 100-ps unrestrained MD, the system was heated from 50 to 300 K. The production MD runs were carried out with constant pressure boundary conditions (relaxation time of 1.0 ps). Constant temperature of 300 K was maintained using the Berendsen weak-coupling algorithm with a time constant of 1.0 ps. SHAKE () constraints with a tolerance of 10 Å were applied to all hydrogens to eliminate the fastest X-H vibrations and allow a longer simulation time step. Translational and rotational center-of-mass motion was removed every 5 ps. Trajectories were analyzed using the Ptraj module of AMBER and structures were visualized using the VMD molecular visualization program (). The figures were prepared using VMD. Molecular ESP was calculated using the DELPHI program (), which solves the nonlinear Poisson-Boltzmann equation. The present DELPHI calculations of ESP were carried out assuming the reference zero ionic strength, which simplifies comparison with minima of electrostatic potentials calculated for other RNA systems (,,). (Inclusion of salt effects into the ESP calculations would change neither shapes nor positions of the ESP basins but would scale down the absolute values of the ESP minima.) Visualization of the potential maps was carried out using the program VMD.
To enlarge sampling of flanking bases we employed the LES technique (,–) in AMBER-7.0 (). The ADDLES module of AMBER was used to split the region of flanking bases (residues 272, 273 and symmetrical 272*, 273*) into five independent copies. We tested also MD LES simulations with only three copies. Force-field parameters for the copies were scaled, which results in lowering of the energy barriers on the potential energy surface (). To provide an initial kick to the five copies, the structure was heated to 500 K. Moreover, a long relaxation phase appears vital to provide sufficient freedom for the copies to settle in different regions of the conformational space. Thus, the temperature was gradually decreased from 500 K to 300 K over 1.5 ns (during the first 750 ps, the pressure was set to 100 atm), and the flanking base region was maintained with flat-well restraints (1 = 0.0, 4 = 6.0, 2 = 10.0, and 3 = 20.0; 2 and 3 depending on the actual distance between the restrained atoms (2 = − 0.5 Å, 3 = + 0.5 Å)) applied to heavy atoms forming H-bonds in basepairs. Control LES simulations were carried out without the initial kick, i.e., the heating was carried only up to 300 K.
CHARMM simulations were performed using the CHARMM program (), with the CHARMM27 nucleic acid force fields (,) and using newly refined x-ray subtype A and B DIS structures (1XPF and 1XPE) with preliminary ion distribution (see Fig. S1 and Supplementary Material). Molecules were overlaid with a box of TIP3P water molecules (size of 95.2 × 52 × 52 Å for subtype A and 91.2 × 53.4 × 53.4 Å for subtype B) (). Na ions were added to neutralize the system. Ions were placed by replacing the water molecules with the highest electrostatic energy and at the distance 3.5–4.8 Å to any RNA atom. The equilibration protocol started with 100 steps of steepest descent (SD) minimization followed by 10 ps of MD applied only to the water molecules whereas the RNA and ions were constrained. During the next 150 ps, the constraints placed on the ions are released allowing equilibration of the solvent around the RNA. The resulting system was subjected to five rounds of 100 steps of SD minimization with gradually reduced harmonic constraints on RNA (100, 20, 5, 2, and 1 kcal/(mol Å)). Finally, the whole system was minimized without any restraints for 100 SD steps and heated from 50 to 300 K in 12 ps by 50 K increments. In contrast to AMBER simulations, CHARMM production runs were performed in constant volume ensemble. We do not expect this difference having any impact on the results. NVE conditions were used in our (Sarzynska and Kulinski) preceding studies with CHARMM. We kept all our standard protocols unchanged for the purpose of this article. Recent comparison of NVE versus NPT CHARMM simulations () did not reveal any differences. The particle mesh Ewald technique was used for treatment of electrostatic interactions (). MD simulations were run with a 2-fs time step and SHAKE constraints were applied to all hydrogens ().
We defined -parameter () to monitor movement of the flanking bases (see for definition). When flanking bases are bulged-in or at the gate of the kiss pocket the -angle falls into the bulged-in range, ±30°. Other -values correspond to the bulged-out states.
HIV-1 DIS kissing-loop complexes subtype A (PDB code 1XPF), B (1XPE), and F (1ZCI and 1YXP) () were studied using 30–50 ns standard explicit solvent simulations. Each hairpin contains three unpaired residues (A, A/G, and A) (). A and A/G are the flanking bases. Flanking bases in the subtype A and B x-ray structures are bulged-out and stacked in pairs, forming thus a base-grip with an empty space between the stacked pairs (), known as the “open conformation” (). In the crystal unit, the gap is filled by a stacking pair from the adjacent kissing complex ( in ()). The subtype F was solved in two different crystal forms. While the 1YXP structure shows the open conformation the 1ZCI structure has four continuously stacked bulged-out flanking bases (). This “closed conformation” is unaffected by the crystal packing () and has been predicted by simulations ahead of its observation in the x-ray structures ().
We extended earlier () simulations of the original x-ray subtype A and B structures () (1JJN and 1JJM) to 30 ns (see Materials and Methods). Geometries of the original x-ray subtype A and B and newly refined structures are almost identical (RMSD of ∼0.2–0.3 Å), including positions of the unpaired bases. However, the new structures include a substantially reduced number of refined Mg ions compared to the older structures where some divalent cations were misassigned. The new structures contain extra Na and ions (the subtype A structure shows also binding of spermine molecule and the subtype B structure suggests one Cl ion).
NMR subtype B DIS () and subtype B/F DIS () were simulated for 20 ns. In subtype B structure, flanking bases are at the entrance of the kiss ion-binding pocket (entering gate for ions) between bulged-in and bulged-out geometry and stack reversely than in the x-ray structure. We termed such an arrangement the “reverse stacking” conformation (). In subtype B/F structure, all flanking bases are entirely inside the pocket (bulged-in geometry) and do not stack ().
summarizes the main conformations of flanking bases observed in at least two simulations or sampled for at least 30% in one simulation. Occupancy of individual conformations is listed in . PDB files with main conformations are provided in Supplementary Material.
Four subtype B simulations () were run (, simulations MD_B_1-4). In the simulation MD_B_1, the bases stayed in the open conformation until 14 ns and then A fluctuated between bulged-in and bulged-out geometries (). The three remaining bulged-out bases A, A, and A formed, at 22 ns, the 3R-bulged-out conformation (). At 40 ns, A moved into the pocket and A modestly shifted toward the gate of the pocket (); however, the stacking between A and A was maintained. The initial x-ray values of glycosidic torsions of A, A, A, and A were −165°, −118°, −113°, and −165°, respectively. When A and A moved inside, its changed to (65° and 40°, respectively). In 3R-bulged-out conformation, of bulges ranged from −80° to −100°. Similarly to the subtype A DIS, residues of the subtype B stayed in C3′- conformation except for G, A, A, and the symmetrical residues that sampled both C3′- and C2′- conformations. X-ray sugar pucker of G, A, and the symmetrical residues is C2′-, while that of A and A is C3′-.
In the simulation MD_B_2 lacking Mg, the flanking bases sampled bulged-in conformations. After 1 ns, A and A moved into the pocket and mutually stacked inside. In addition, A formed a pair with opposite A stabilized by an A(N1)-A(N6) H-bond. Symmetrical A and A stayed bulged-out until 10 ns, and then they occupied the cavity as well but they did not stack inside and were rather flexible. At 15 ns, the base A flipped over its glycosidic bond and left the pocket. For the rest of the simulations it fluctuated between bulged-in and bulged-out conformations (see development of parameter ). Its fluctuated, spanning the full range of 360°. Bases A, A were still stacked and their -torsions fluctuated at ∼−100°. A changed to geometry (−52°) when this base moved inside. In the simulation MD_B_3, during the first two nanoseconds the flanking bases formed the complete four-adenine stack. Residue A was, however, very dynamic and oscillated between bulged-out and bulged-in geometries. When being inside the cavity, A forms a basepair with the opposite A (A(N6)-A(N1) and A(N1)-A(N6) H-bonds). The simulation MD_B_4 carried out with Na ions exhibited a similar outcome to the simulation MD_B_2 run with K ions. Flanking bases stayed for the first 10 nanoseconds in the open conformation, and after that, they moved into the cavity but did not stack. Particularly, A moved inside at 11 ns and created A(N6)-A(N1) and A(N1)-A(N6) H-bonds with unpaired adenine A. The bases A, A, and A moved inside at 15, 17, and 17.5 ns, respectively, but did not form any contacts inside the cavity.
We, in addition, initially carried out two simulations (S_MD_A and S_MD_B) based on preliminary refined x-ray subtype A and B structures. While solute geometries are the same as in the final deposited files 1XPF and 1XPE, these preliminary x-ray structures had few incorrectly assigned ions. Specifically, the preliminary x-ray subtype A structure contained two incorrectly placed Mg ions, and the subtype B structure, three such Mg ions. These electron densities were later reinterpreted as or Na ions in the deposited PDB files. More details are provided in Supplementary Material Figs. S1 and S2. Both simulations are quite consistent with simulations MD_A_1-4 and MD_B_1-4 ().
The subtype F DIS differs by one base mutation (GA) from the subtype A (). The x-ray structure 1ZCI that shows the closed conformation of flanking bases was run with two x-ray K ions and added 42 K ions in 35-ns simulation MD_F_1. Flanking bases stayed in the initial closed conformation (see ) for the majority of the simulation time (time periods 0–11, 14–16, 17–30, and 33–35 ns), except of base A, which occurred three times inside the pocket (). The -torsions of A, A, A, and A in the closed conformation ranged from −80° to −100° while the initial x-ray values were −139°, −97°, −99°, and −136°, respectively. A in bulged-in geometry adopted a geometry ( ∼40°).
The x-ray structure 1YXP that shows the open conformation of flanking bases was run in 10-ns simulation (MD_F_2) with 44 K ions. The initially open conformation changed after 1 ns to closed conformation. At 3.5 ns, A moved into the pocket, while other flanking bases stacked until the end of the simulation (-- conformation, see ). The initial x-ray values of A, A, A, and A were −168°, −116°, −123°, and −163°, respectively. Similarly to the previous simulation in the closed conformation -torsions of bulges ranged from −80° to −100°. Bulged-in A had -torsion in geometry (∼50°). X-ray subtype F DIS structure with open conformation has two residues with C2′- conformation, G and A of each strand. According to the experiment in the literature (), formation of the closed conformation is coupled with C2′- → C3′- flip of the sugar of G. During the first two nanoseconds, we observed such repuckering. However, the sugar rings oscillated between C3′-/C2′- for the rest of the simulation. Interestingly, the x-ray density maps show some evidence of a population of C2′- conformations for G271 (E. Ennifar, unpublished data, 2007), although it was not reported in the original article ().
The 48-nt NMR subtype B DIS structure (PDB code 2F4X/model 1) () was simulated for 20 ns (MD_nmr_1). This NMR structure is in general agreement with the x-ray structure except for the area of flanking bases. First, these bases are placed at the gate of the central pocket, between bulged-out and bulged-in geometries (). Second, stacking is different than in the crystal—namely, A stacks with A from the opposite loop instead of the A. Likewise, symmetrical A stacks with A (). We termed this arrangement “reverse stacked” conformation (). The cross stacking attracts and twiddles the loops, resulting in closed entrance of the pocket and deformed backbone (the shortest distance between opposite phosphorus atoms at the gate of the pocket is only 3.8 Å compared with 7.5 Å in the x-ray structures). Due to the backbone distortion, we could not calculate the -parameters for the flanking bases. Within the first ns, A and A moved into the pocket. While A stayed inside for the whole simulation and restored stacking with opposite A base, A flipped around its glycosidic bond and left the pocket. For the rest of the simulation, it fluctuated between bulged-in and bulged-out geometry. Bases A and A attempted to move inside as well, but the pocket entrance was obstructed by the reverse A/A stacking, preventing A and A from moving in. It appears that 20-ns timescale is not sufficient to relax the central part of the complex and find optimal conformation. The starting NMR glycosidic torsions of A, A, A, and A were −24°, −18°, −24°, and −18°, respectively. During the simulations, they established values of −130°, −90°, −40°, and −140°, respectively.
The second NMR structure () (PDB code 2D19/model 11) has 34-nt since it has both stems truncated by three basepairs. Sequence of the stem corresponds to subtype B while sequence of the loop corresponds to subtype F, so we call it “B/F structure” (). The overall geometry is in meaningful agreement with the x-ray structures. Area of bulges is not closed (the shortest distance between opposite phosphorus atoms at gate of the pocket is 11 Å) and not deformed. Baba's B/F structure predicts the flanking bases to be entirely bulged-in, i.e., inside the pocket. Initial NMR positions of flanking residues A and A slightly differ, so that only A creates H-bonds with the unpaired A (A(N1)-A(N6) and A(N6)-A(N1)) in the pocket, while the symmetrical base A does not form any H-bond. At the beginning of the simulation (MD_nmr_2) the flanking bases stacked resulting in closed-like bulged-in conformation positioned at the gate of the pocket. It can be considered as a partly bulged-in arrangement (). This arrangement was then stable, except of several disruptions when A moved entirely outside the pocket (). The H-bonds between A and A remained stable, while at 16 ns the A-A basepair formed. The new pair, however, exhibits different H-bonds (A(N6)-A(N1) and A(N7)-A(N6)). The initial values of glycosidic torsions A = −104°, A = −106°, A = −139°, and A = −71° changed to −25°, −145°, −88°, and 53°, respectively.
Two control 20-ns simulations (MD_nmr_2_1 and MD_nmr_2_2) were carried out using different random number seeds (). The first simulation sampled closed-like bulged-in conformation for ∼90% of the simulation time, similar to our primary simulation. In the second simulation, a three-adenine bulged-in stack of A, A, and A was seen for only ∼7% of the simulation time. In other periods we observed the A/A stack while A oscillated nearby. A was close to the stacked bases and involved in H-bonding with A. The flanking bases nevertheless remained in the bulged-in conformation, so all three simulations appear to be reasonably mutually consistent. Note that the NMR starting structures are more difficult to relax by MD due to their lower accuracy compared to x-ray structures.
The 412–428 and 2438–2454 regions of 23S rRNA of form a kissing-loop complex (). We extracted this complex from the x-ray structure of the large subunit () and carried out 50-ns-long MD simulation (MD_ribosome). Like the HIV-1 DIS kissing complexes, the ribosomal complex is formed by 6-nt complementary sequences (see and ). It has only two unpaired flanking bases (G and C). These bulged-out bases are in the large ribosomal subunit of () (as well as in (), (), and () ribosomal crystal structures) involved in tertiary contacts with bases of adjacent stem-loop of 23S rRNA (). During the first nanosecond of the simulation, the flanking bases formed a stack () and remained in this arrangement until the end.
The LES method splits the selected part of the molecule into (three-to-five) copies that move independently in the simulation. This allows us to overcome barriers that cannot be crossed during standard simulations. The LES method is optimally suited for loop regions but appears to be promising also for the flanking regions (,). LES can lead to wrong geometry when the force field itself is not sufficiently accurate and does not provide the correct global minimum (). In addition, the LES method itself can poorly converge when struggling between competing minima. The experience with this technique so far is rather limited, but it is capable of providing striking insights. Thus, LES simulations were carried out for subtypes A and B DIS structures. We split regions of the flanking bases into five independent copies (see Materials and Methods) and run two 30-ns simulations LES5_A and LES5_B. In addition, two 10-ns simulations (LES3_A and LES3_B) with three copies were executed.
In the LES5_A simulation within the extended 1.5 ns equilibration phase (when the system is heated up to 500 K and cooled back to 300 K; see Materials and Methods), we observed conversion from the open x-ray conformation to the closed conformation, which was stable until 13 ns. Then the closed conformation was disrupted and the entrance of the pocket opened. Bases A and G moved into the pocket, and closed-like bulged-in/out conformation, with two bases bulged-in and another two bulged-out, formed (). This arrangement has been seen in standard simulation MD_A_2 for 11 ns. For the rest of the simulation, closed-like bulged-in/out conformation repeatedly disrupted and then restored itself (note that enhanced mobility is expected when applying LES). During a control 5-ns LES simulation performed without the initial 500 K heating, the closed conformation formed after the first two nanoseconds. Thus the initial heating did not affect the simulation outcome. During 10-ns LES3_A simulation (three copies), the flanking bases stayed in the open conformation.
Conversion to the closed conformation was observed during the 30-ns-long simulation LES5_B run. The closed conformation was then seen for the rest of the simulation, except for a few disruptions when A or A moved inside the pocket. Five-nanosecond control LES simulation, performed without the initial 500 K heating, resulted in formation of the closed conformation after the first three nanoseconds. In the 10-ns simulation LES3_B, closed conformation initially formed after 500 ps and was followed by open conformation until ∼9 ns. After that, three bases moved into the pocket and stacked, while the fourth adenine stayed bulged-out.
Newly refined HIV-1 subtype A and B DIS structures (1XPF and 1XPE) with preliminary ion distribution (see Supplementary Material) were simulated using CHARMM27 force field (). In CH_A (subtype A) simulations, flanking bases were observed both bulged-out and bulged-in in agreement with AMBER simulations. At the beginning of simulation CH_A_1, all flanking bases moved toward the pocket, which has been seen for subtype B but not for subtype A in AMBER simulations. The 12-ns-long CH_A_2 and CH_A_3 simulations sampled only the open conformation. In CH_B (subtype B) simulations, flanking bases were seen both bulged-out and bulged-in. Simulations CH_B_1 and CH_B_2 revealed formation of the consecutive AAAA closed stack that was frequently identified in AMBER simulations (see and ). Moreover, in CH_B_1 simulation, 3R-bulged-out and closed-like bulged-in conformation variants of closed conformation were sampled. Further, simulation CH_B_2 sampled locked stacked conformation seen in MD_A_1 and MD_A_2 simulations. More detailed analysis of CHARMM data will be given elsewhere, but qualitatively we can conclude that the CHARMM and AMBER data are, regarding the flanking base dynamics, quite consistent. However, CHARMM simulations revealed melting of 1–3 terminal basepairs, which could suggest underestimation of the duplex stability. In several cases the melting was, however, reversible.
We analyze structural dynamics of flanking bases in RNA kissing-loop complexes. The work is a major extension of our preceding study () considering the amount of simulations (775 ns) and the number of studied structures. We focus on conformations of flanking bases in the new x-ray HIV-1 DIS kissing complexes (subtypes A, B, and F) (), in the NMR complexes of the subtype B and subtype B/F (,), and in the kissing complex from the large ribosomal subunit of (). We test different ion conditions and two force fields (AMBER parm99 () and CHARMM27 (,)). In addition, we applied several long (up to 30 ns) LES runs to improve sampling of the flanking bases ().
The crystal structures show bulged-out flanking bases, either four base stack ( conformation, see ) or two separate stacks ( conformation) depending on crystal packing (). NMR studies suggest bulged-in positions of bases which, however, are mutually inconsistent (,).
The simulations identified six typical positions of the flanking bases as summarized in and . The most prevalent arrangement predicted by the MD simulations is the closed conformation (closely agreeing with the respective x-ray arrangement) or related geometries where, e.g., three bases are stacked and the fourth one samples mostly bulged-in geometries. However, other substates are also nonnegligibly populated, including bulged-in geometries. Note that when assessing the , one needs to consider the starting and final structures separately (highlighted in ), and also take into account that some structures are mutually structurally related. Vast majority of simulations started with the open bulged-out conformation, which then obviously dominates the overall percent of population. However, there is a clear trend to move toward the closed structures and related geometries. The simulations routinely achieve transitions from bulged-out starting x-ray geometries to bulged-in arrangements and even subsequent returns to bulged-out geometries. We have also evidenced bulged-out base excursion from a parent bulged-in NMR structure. All these movements indicate a meaningful sampling of movements of flanking bases in both directions.
Self-association of bulged bases was observed for all three subtypes when starting from x-ray structures. The simulations of subtype A DIS kissing complex show that the initial open bulged-out conformation () tends to convert to the closed (bulged-out) conformation or related conformations such as closed-like bulged-in/out and 3R-bulged-out (, ). Outcomes of simulations of the subtype A are not affected by presence or absence of Mg ions. In contrast, for subtype B the conversion from the open conformation to the closed conformation occurred in presence of Mg while in absence of Mg ions we rather evidenced bulged-out→bulged-in conversion of all flanking bases. This could indicate that the subtype B bulged-out geometries are getting some stabilization by Mg ions. Notably, the NMR structures were solved in presence of monovalent ions. Nevertheless, simulations with divalent ions should be taken with specific care due to a number of limitations () (see Materials and Methods). Note also that the divalent ions were considered by including only those ions seen in the crystal structures (see and Supplementary Material).
Subtype F DIS kissing complex was investigated with the two different conformations of bulged-out residues observed in x-ray structures. The closed conformation showing weak crystal contacts was basically stable except of mobility of A. Open conformation in which flanking bases mediate crystal contacts in asymmetric unit changed after the first nanosecond into the closed conformation. This observation is in agreement with the x-ray experiment (), indicating that self-association of all four flanking bases is preferable, unless it is prevented by the crystal packing.
The two recent NMR DIS structures (subtype B () and subtype B/F ()) show bulged-in geometry. We simulated these two NMR structures on a scale of 20 ns and found that they basically remained in their bulged-in conformations. Open question, however, is whether some structural deformations in the starting NMR structures (especially the local deformation of backbone area of bulges) are not affecting our simulations. For example, the Lancelot's structure exhibits visible local deformations, which are not repaired on our simulation timescale. Overall, there are substantial mutual differences among the three available NMR structures of the DIS kissing complexes (–). It also is not clear whether the NMR experiment would capture the bulged-out conformation, if coexisting. Flanking bases of the ribosomal kissing complex were essentially stable in bulged-out conformation.
All kissing complexes are associated with a very deep ESP minimum in the central pocket (see ), which is continuously occupied by 2–3 monovalent ions (in absence of divalents), and these ions are delocalized (). The RNA kissing complexes thus create one of the most intriguing cation-binding pockets visualized in RNA MD simulations so far.
CHARMM simulations (total 98 ns) essentially agreed with the picture from AMBER simulations. These simulations identified five of the six AMBER flanking base conformations and in addition with similar populations. This is a good agreement, taking into account the considerably shorter timescale of CHARMM simulations. No new substate was located. CHARMM simulations, however, revealed partial melting of stems (disruption of terminal 1–3 basepairs). Though some of the disruptions were reversible, such stem perturbations are most likely excessive. A recent study reported a difficulty with the CHARMM27 force field providing stable trajectories of folded Hammerhead ribozyme ().
We also applied LES to enhance the sampling of the flanking bases. The MD and LES results were quite consistent, which gives us a confidence that no significant substate was missed. We also attempted to use the molecular mechanics, generalized Born, and surface area free energy method to characterize the free energies of various conformations seen in MD trajectories. This approximate method, however, was not capable of providing conclusive results (see Supplementary Material). Limitations of various methods that can supplement standard simulations are discussed elsewhere ().
Several other MD studies have been performed with the aim of describing the subtype B DIS kissing complex on the nanosecond timescale (–). These studies did not report formation of the closed bulged-out conformation, which is seen in x-ray structures and is a major substate according to our simulations. It is likely caused by the short simulation timescales. Beaurain et al. () (using CHARMM) and Aci et al. () observed bulged-in geometries of flanking bases in the presence of Na ions similar to our corresponding simulations. Aci et al. also reported a peculiar instability of the AMBER simulations using x-ray starting structures, which was not observed in any of our kiss simulations and, in fact, in no other simulations starting from RNA x-ray structures.
sub
#text
ext-link
#text |
Major histocompatibility complex (MHC) class I molecules were initially believed to present peptides derived solely from endogenous proteins, that is, proteins synthesized by the cell itself, including viral proteins produced upon infection. Subsequently, it was discovered that exogenous antigens could be presented by MHC class I molecules and stimulate CD8 T cells. This process is called cross-presentation and is critical for the initiation of T-cell immunity to viral infections (). Dendritic cells (DCs) are the major cell types that mediate cross-presentation, and they can generate functional MHC class I–peptide complexes from exogenous proteins internalized via various endocytic mechanisms such as phagocytosis, macropinocytosis, or receptor-mediated endocytosis. They can cross-present exogenous antigens in the form of soluble proteins, immune complexes, or apoptotic bodies derived from virally infected cells (; ).
We previously showed that efficient internalization of exogenous antigens by DCs is followed by their translocation into the cytosol via an endoplasmic reticulum (ER)-associated degradation (ERAD)-related mechanism (; ). In the ERAD pathway, misfolded or unassembled proteins in the ER are targeted for degradation through the cytoplasmic ubiquitin–proteasome system (). Misfolded substrates are first selected and recognized by lumenal chaperones (e.g., BiP, GRP94) and then targeted for dislocation into the cytosol (retrotranslocation; ; ). Retrotranslocation probably uses the Sec61 channel or a related channel involving Derlin-1, and it is coordinated by the Cdc48p–Npl4p–Ufd1p complex, which constitutes a transport cascade for substrate delivery from the site of ubiquitination to the proteasome (, ; ; ). Whether derived from ER proteins or from exogenous proteins internalized by DCs, peptides generated by the proteasome may then be translocated into the ER by the transporter associated with antigen processing (TAP) and loaded onto MHC class I molecules using the classical TAP-dependent MHC class I assembly pathway (). MHC class I–peptide complexes are then transported via the Golgi apparatus to the cell surface and surveyed by CD8 T cells.
Here we show that in DCs, in addition to dislocation followed by immunogenic peptide generation, exogenous proteins can use the same mechanism for cytosolic access and be refolded in the cytosol rather than being degraded. At least partial unfolding appears to precede retrotranslocation. It has previously been proposed that cytosolic chaperones such as Hsp90 cooperate with the ERAD pathway to determine the fate of misfolded proteins (), and here we show a similar requirement for the functional refolding of exogenous proteins in the cytosol of DCs.
To investigate the kinetics of internalization of exogenous soluble proteins and their entry into the cytosol, we designed a new set of assays using soluble recombinant Firefly and luciferases as substrates. From previous studies we know that soluble luciferases are efficiently internalized by fluid-phase uptake by the DC-like cell line KG-1 (). To measure exogenous protein delivery to the cytosol, KG-1 cells that had internalized soluble luciferases were permeabilized with streptolysin- (SLO). SLO permeabilization allowed the measurement of cytosolic luciferase activity in the supernatants of the cells following centrifugation. We established that in KG-1 cells luciferase activity was present in the cytosol after 5 min of internalization. No luciferase activity could be detected in the cytosol of CEM cells, a human T lymphoblastoid cell line with poor macropinocytotic capacity (). To ensure that the SLO permeabilization procedure was specifically releasing cytosolic components without ER or lysosomal leakage, supernatants from untreated or SLO-treated cells were subjected to SDS–PAGE and immunoblotted for p44/42 MAP kinase (MAPK) as a cytosolic marker, GRP94 as an ER marker, and gamma-interferon-inducible lysosomal thiolreductase (GILT; ) as a lysosomal marker. clearly shows that supernatants from SLO-permeabilized cells contained the cytosolic marker p42/44 MAPK, whereas GRP94 and GILT were undetectable. We also showed that endosomal integrity was maintained upon SLO permeabilization: a 10-kDa fluorescein-conjugated Dextran internalized by KG-1 cells was undetectable in supernatants from SLO-treated cells, but was readily detectable in the residual cell pellet following permeabilization ().
We wanted to ensure that the luciferase activity present in the cytosol of KG-1 cells was dependent on active internalization. Macropinocytosis, the predominant mechanism of fluid-phase uptake by DCs, is an actin-dependent process (; ). KG-1 cells were therefore pretreated with the actin polymerization inhibitor cytochalasin D for 30 min before addition of luciferase for an additional 30 min. Exposure to cytochalasin D strongly reduced the luciferase activity liberated into the supernatants of SLO-permeabilized cells (, black bar) compared with control cells treated with vehicle (DMSO) alone (, light gray bar). No significant luciferase activity was detected in the supernatants of non-permeabilized cells (, white and dark gray bars). Cytochalasin D treatment also decreased significantly the total amount of Firefly luciferase internalized by KG-1 cells, as assessed by enzymatic activity measured after detergent lysis (). These data indicate that internalization of exogenous luciferase is actin-dependent. We interpret this to suggest that luciferase is internalized by macropinocytosis followed by its translocation into the cytosol of KG-1 cells.
To investigate the mechanism by which exogenous luciferase enters the cytosol of KG-1 cells, we used Exotoxin A (ExoA), a bacterial toxin derived from . ExoA reversibly inhibits retrotranslocation from the ER into the cytosol, a process putatively mediated by the Sec61 channel (). We have recently shown that ExoA is efficiently internalized by KG-1 cells expressing the mouse MHC class I allele H2-K, and that it can block cross-presentation of the soluble antigen ovalbumin (OVA) to a K-restricted T-cell hybridoma (). This finding was used in part to argue that ERAD-like mechanisms govern cross-presentation. To determine if luciferase accesses the cytosol using the same ExoA-inhibitable pathway, we mixed soluble ExoA, or BSA as a control, at increasing concentrations with a fixed concentration of Firefly luciferase and added them to KG-1 cells. The addition of ExoA significantly reduced the luciferase activity detectable in supernatants of SLO-permeabilized cells (, solid lines) compared with the BSA control (, dashed lines). No significant luciferase activity was detected in the supernatant of non-permeabilized cells (, black triangle). The effect was clearly dose-dependent, with reduced luciferase activity present at higher concentrations of ExoA. ExoA did not affect luciferase uptake or the capacity of the cytosol to mediate refolding (). These data strongly suggest that luciferase is dislocated into the cytosol by the same mechanism used by exogenous antigens during cross-presentation by DCs.
The assay we have developed is based on the ability to detect luciferase activity in cytosolic fractions derived from KG-1 cells. This implies that both Firefly and luciferases can access the cytosol in a folded and functionally active form. To investigate whether the luciferase activity detected in supernatants of SLO-permeabilized cells might depend on cytosolic refolding, we performed preliminary refolding experiments. Firefly or luciferases were chemically unfolded in 6 M guanidine–HCl in intracellular transport (ICT) buffer for 30 min at room temperature. This procedure completely abrogated the activities of both enzymes (, white circles). To determine if cytosolic components could mediate refolding, unfolded luciferases were added to serial dilutions of cytosol isolated from KG-1 cells that was dialyzed into ICT buffer. Both Firefly and luciferases were efficiently refolded after 30 min incubation at room temperature in the presence of KG-1 cytosol (, black triangles). No significant refolding was observed in the presence of ICT buffer alone (, black squares). Cytosol similarly isolated from CEM cells was also capable of refolding luciferases , indicating that no specific property of KG-1 cytosol was responsible (data not shown). Extending refolding reactions up to 1 h and adding protease inhibitors or lactacystin to the refolding reactions did not increase refolding efficiency significantly (data not shown). Taken together, these data suggest that after being internalized and retrotranslocated into the cytosol, Firefly and luciferases could potentially undergo refolding in the DC-like cell line KG-1.
Hsp90 in the cytosol seemed a likely candidate for mediating the luciferase refolding. We therefore examined the effect of the potent Hsp90 inhibitor radicicol (; ) on the refolding reaction. After chemical unfolding, we observed a dose-dependent inhibition of Firefly luciferase refolding by radicicol in the presence of KG-1 cytosol (, gray bars). No inhibition occurred in cytosol containing DMSO as control (, white bars), and no significant refolding was observed in 6 M guanidine–HCl or in ICT buffer alone (, first three columns). These data suggest that luciferase refolding is mediated by the cytosolic chaperone Hsp90.
We wished to determine if the recovery of active luciferase from the cytosol of KG-1 cells depended upon refolding. For this purpose, luciferase was denatured in 6 M guanidine–HCl in ICT buffer and dialyzed overnight against PBS. After dialysis against PBS, chemically unfolded luciferase, but not Firefly luciferase, remained soluble but enzymatically inactive (). However, when the inactive enzyme was added to KG-1 cells, we could detect significant luciferase activity in the cytosol released by SLO permeabilization (, solid black lines). The activity recovered after addition of 20 μg of unfolded luciferase was comparable to that recovered after adding 0.2 μg of native enzyme to the same number of KG-1 cells (, solid gray lines). No significant luciferase activity was detected in the supernatants from non-permeabilized cells incubated with either folded enzyme (, gray square symbols with dashed lines) or unfolded enzyme (, black circles with dashed lines), and no spontaneous refolding was observed in the cell culture medium (data not shown). These data strongly favor the hypothesis that cytosolic refolding occurs after internalization by KG-1 cells.
The refolding experiments suggested that Hsp90 was the critical chaperone. To determine whether this was the case for refolding, we again used the Hsp90 inhibitor, radicicol. KG-1 cells were pretreated with radicicol for 1 h before 15-min incubation with native Firefly or unfolded luciferase, followed by washing and SLO permeabilization. Radicicol pretreatment strongly reduced the cytosolic activity recovered for both enzymes (, gray bars). No significant luciferase activity was detected in supernatants of non-permeabilized cells (, white bars). Similar results were obtained with overnight radicicol treatment at lower concentrations (), arguing against nonspecific effects, which could have resulted from the relatively high radicicol concentration used in , chosen to allow short incubation times. Radicicol treatment did not affect cell macropinocytotic activity, as assessed by fluid-phase Lucifer Yellow uptake (), and no toxic effects of radicicol on KG-1 cells were observed, assessed both by cell recovery and viability (data not shown). Taken together, these data implicate the Hsp90 chaperone in the cytosolic refolding of exogenous luciferases internalized by KG-1 cells.
The fragility of primary DCs prevented us from using SLO permeabilization to evaluate cytosolic access by luciferase in these cells; significant ER and lysosomal leakage was observed upon SLO treatment (data not shown). We therefore used hypotonic lysis to release the total cellular contents rather than SLO permeabilization to analyze luciferase refolding in primary DCs. After 15-min incubation with inactive, denatured luciferase, DCs were washed in ice-cold PBS, resuspended in ice-cold 10 mM Tris-chloride, pH 7.4, for 5 min, and centrifuged at 2000 for 10 min. Significant luciferase activity could be detected in the supernatants derived from hypotonically lysed cells compared with those from an equivalent number of non-lysed cells resuspended in PBS (). The data suggest that luciferase is highly likely to refold in the cytosol of primary human DCs.
To further confirm the role of Hsp90 in cytosolic refolding, we attempted to independently ‘knock down' the Hsp90 α and β isoforms using small-hairpin RNA (shRNA) constructs. We were unable to significantly reduce expression of the Hsp90 α isoform because KG-1 cells transduced with a specific shRNA were impaired in their ability to grow, and we could not recover enough cells to perform the experiment (data not shown). Decreased growth of cells upon Hsp90 α knockdown has been previously reported (; ). Hsp90 β, however, was more tractable. KG-1 cells were transduced with an shRNA construct specific for the Hsp90 β isoform. A GFP-silencing sequence was used as control. After selection in puromycin, we recovered two KG-1 cell clones (Hsp90 β.1 and Hsp90 β.2) with stable reduction of Hsp90 β. Hsp90 α and β expression was quantitated by SDS–PAGE followed by quantitative western blotting on serially diluted cell extracts. Approximately 65% reduction of Hsp90 β was obtained for both Hsp90 β.1 and Hsp90 β.2 clones compared with GFP–shRNA control cells (, top panels). No significant decrease in Hsp90 α protein was observed (, middle panels); MHC class I heavy-chain blots served as a loading control and normalizer (, bottom panels).
To assess the effect of Hsp90 β reduction on refolding, the shRNA-expressing cells were incubated for 15 min with native or unfolded luciferase, followed by washing and SLO permeabilization. Significantly decreased cytosolic luciferase activity was recovered from KG-1 cells with reduced levels of Hsp90 β compared with GFP–shRNA control cells (, black bars). No significant luciferase activity was detected in supernatants of non-permeabilized cells (, white bars). No differences were observed in the macropinocytotic activity of the Hsp90 β shRNA-expressing cells compared with the shRNA control cells, as assessed by fluid-phase Lucifer Yellow uptake (). Together with the data showing inhibition by radicicol, these experiments strongly support a key role for the Hsp90 chaperone in the cytosolic refolding of exogenous luciferases internalized by KG-1 cells.
We previously showed that for cross-presentation in DCs, exogenous antigens are translocated into the cytosol using mechanisms related to ERAD (). For part of this work we purified phagosomes from KG-1 cells containing internalized Firefly luciferase and showed that they could transport the enzyme into the topological equivalent of the cytosol. The translocation process was ATP-dependent and required the active form of the AAA-ATPase, p97 (; ). Perhaps the most surprising aspect of this finding was that the translocated luciferase was enzymatically active. In the present work we have extended our previous findings to luciferases taken up by fluid-phase mechanisms. We showed that the appearance of active enzyme in the cytosol could be inhibited by radicicol and by reducing the levels of Hsp90 β. This indicates that cytosolic refolding is mediated by Hsp90, and implies that an unfolding step occurs before translocation (see model in ).
The SLO permeabilization approach provides a useful strategy for determining the rate at which soluble proteins internalized by the DC-like KG-1 cell line enter the cytosol. Problems with cell fragility prevented us from using the same technique with monocyte-derived DCs. However, hypotonic lysis of DCs after internalization of inactive luciferase released active enzyme, suggesting that DCs were similarly capable of cytosolic refolding of internalized proteins (). Previous work investigating cytosolic access of exogenous proteins used imaging techniques such as immunofluorescence and electron microscopy, and focused on single-cell analysis (; ). The SLO-permeabilization/luciferase release assay allowed us to estimate retrotranslocation efficiency in whole-cell populations over time, avoiding potential bias in imaging selection. We were careful to exclude that lysosomal or ER lumenal components were released by SLO (), and we determined that SLO did not cause endosomal permeabilization in KG-1 cells by using internalized fluorescein-conjugated dextran as an endocytic marker (). Pores formed by SLO are 30-nm diameter (), large enough to accommodate a dextran of 10 kDa. Overall, these data confirm that the luciferase activity detected in supernatants from SLO-permeabilized cells is genuinely derived from the cytosol.
It is unclear from which structures retrotranslocation into the cytosol occurs. Initial entry into the cells is likely to involve macropinocytosis, an actin-dependent process allowing internalization of large volumes of extracellular fluid. The ability of cytochalasin D to inhibit cytosolic luciferase accumulation in KG-1 cells (; ), as well as the inability of the non-macropinocytotic CEM cell line to mediate the process (), are consistent with this idea. After macropinocytosis, translocation into the cytosol could occur from macropinosomes directly or it could occur after exogenous protein entry into the ER. We have provided some evidence based on immunofluorescence analysis for the appearance of ER proteins in macropinosomes (), and also have shown that internalized proteins can access the ER (), so both mechanisms seem possible. However, luciferase entry into the cytosol of KG-1 cells was rapid, with enzymatic activity detectable as early as 5 min after initiation of internalization (, , and ). The speed of the retrotranslocation and refolding suggests that recruitment of the ER retrotranslocation machinery to macropinosomes is perhaps more likely to be involved than transport of the enzyme to the ER. Whether other cell types capable of macropinocytosis, such as neutrophils or endothelial cells (; ), can perform this function remains to be investigated.
The identity of the retrotranslocation channel for ERAD is still subject to debate (; ; ), and the same debate applies to the work we describe here. There is evidence, particularly from work with yeast, that the Sec61 channel is responsible (; ), but evidence from both yeast and mammalian cells suggests that the multiple membrane-spanning protein Derlin-1 may be involved (; ; ). ExoA has been reported to block the Sec61 channel and inhibit the retrotranslocation of peptides from ER vesicles (), and here we have shown that it inhibits the cytosolic recovery of luciferase (), but it is conceivable that other channels could be implicated in this process. However, regardless of the channel involved, the luciferase we find in the cytosol is active and therefore presumably intact; thus, the channel must be capable of translocating a protein of at least 62 kDa. This is the M for Firefly luciferase, which is folded into two compact domains (). The data showing that both radicicol and Hsp90 β knockdown inhibit refolding, and the observation that denatured luciferase can be reactivated after internalization, argue that unfolding is a pre-requisite for translocation and that Hsp90 then mediates cytosolic refolding. Thus, the diameter of the retrotranslocation channel need not be unusually large. However, glycans remain attached during retrotranslocation for ERAD (, ), and there are data arguing that folded domains, including EGFP (25 kDa) () and a folded dihydrofolate reductase domain (42 kDa) (), can be retrotranslocated. There is no reason to suppose that this would not also be true for retrotranslocation for cross-presentation. However, denatured luciferase appears in the cytosol more rapidly than the native form (), indicating that prior unfolding may significantly speed up the retrotranslocation process. Perhaps certain checkpoints involved in substrate detection, recognition, and unfolding are bypassed, allowing faster retrotranslocation to occur. However, the validity of this conclusion is tempered by the requirement for higher concentrations of the unfolded luciferase to achieve a usable signal. Conceivably this could be responsible for the more rapid appearance of the non-native form in the cytosol.
At least two ERAD pathways exist: the ERAD-L and the ERAD-C pathway (; ). During protein synthesis, the ERAD-C pathway monitors the folding status of cytosolic domains of membrane proteins and rapidly clears misfolded variants from the ER. This occurs without regard to the state of the lumenal domain. If the conformation of the cytosolic domain passes the ERAD-C checkpoint, the ERAD-L pathway monitors the state of the lumenal domain. If a lesion is detected, the protein is processed for ERAD using a distinct set of factors not required for the ERAD-C pathway. In DCs, exogenous proteins can be directly delivered to the ER lumen for retrotranslocation or can be retrotranslocated from ER-containing phagosomes through the ERAD pathway. In both cases, we might speculate that extracellular proteins delivered to ER lumenal compartments would preferentially use ERAD-L. Previous work in yeast showed the importance of Hsp90 in maintaining the stability of the ERAD-L substrate CFTR (). This would be in agreement with the key role of Hsp90 indicated by our luciferase cytosolic refolding data.
Firefly luciferase readily refolds in the presence of purified Hsp90, Hsp70, and the PA28 ATPase proteasomal subunit (, ). The use of radicicol as an inhibitor provides evidence for Hsp90 as the key cytosolic chaperone that mediates refolding after retrotranslocation. Radicicol did not affect the efficiency of luciferase internalization, indicating that decreased enzymatic activity in the cytosol was specifically due to Hsp90 inhibition (). The effect of radicicol is also unlikely to be mediated by the ER Hsp90 homologue GRP94, because even lower concentrations than those used in the primary experiments gave similar results (). It has been reported that the radicicol affinity for Hsp90 is fivefold higher than its affinity for GRP94 (). This argues against the possibility that the decreased cytosolic luciferase activity seen upon radicicol treatment is due to decreased retrotranslocation caused by GRP94 inhibition.
Unfortunately, we were unable to ‘knock down' both the Hsp90 α and β isoforms using either siRNA oligonucleotides or shRNA approaches in the KG-1 cell line. Our attempts to obtain significant and stable silencing for Hsp90 α in KG-1 cells were unsuccessful. The reduced rate of cell division previously reported (; ) combined with the high number of cells required for our experiments rendered the Hsp90 α shRNA strategy unusable. We were able to establish ∼65% stable knock down for the Hsp90 β isoform in KG-1 cells (). We observed no differences in Lucifer Yellow uptake between shRNA control and shRNA Hsp90 β cells (), and no effect on cell growth rate or survival was observed (data not shown). Reduced levels of Hsp90 β caused a significant decrease in enzymatic activities recovered in the cytosolic fraction using native or unfolded luciferase as exogenous protein substrates (), confirming the role of Hsp90 suggested by the potent inhibitor radicicol ().
Workers using OVA as a probe for cytosolic access in DCs found only a 30-kDa fragment of the protein in the cytosol, indicating that proteolysis had occurred, but there is no evidence that proteolysis is required for cytosolic access (). The reduced yield of active luciferase when denatured enzyme was internalized, compared with the activity recovered when native enzyme was used, might indicate that proteolysis is more likely when the retrotranslocated substrate is totally denatured. No significant differences in the luciferase activity recovered were observed in cells pretreated with concanamycin B or leupeptin to inhibit lysosomal proteolysis (data not shown). However, proteolysis of acquired proteins could still occur in endocytic vesicles as well as in the cytosol after translocation. Lactacystin pretreatment of KG-1 cells followed by SLO treatment caused some ER lumenal contamination of the released cytosol, as assessed by western blotting for GRP94 (data not shown), so we were unable to determine if proteasome inhibition resulted in an increase in cytosolic luciferase activity. However, upon prolonged incubation, cytosolic luciferase activity did decrease (), which suggests that the refolded enzyme is ultimately degraded by cytosolic proteolysis.
An estimate of retrotranslocation efficiency can be extrapolated by comparing the luciferase activity recovered in SLO-permeabilized samples () to the total amount of internalized enzyme measured by cell lysis (). Thirty minutes after internalization was initiated, at least 60% of the internal luciferase could be recovered in the cytosol, suggesting that the level of proteolysis, either pre- or post-retrotranslocation, is relatively low for luciferase. For other internalized proteins, degradation may well be the predominant fate. This is the fate that gives rise to the peptides that are transported into the ER by TAP, and therefore the key pathway involved in cross-presentation. However, the unfolding/refolding mechanism we have identified that allows exogenous proteins to regain their activity in the cytosol could potentially be exploited by DCs for some aspects of their regulation or function.
There are a few examples in the literature where cytosolic delivery of growth factors and signaling molecules can mediate biological effects. Fibroblast growth factor (FGF)-1 and FGF-2 are internalized through the FGF receptor and then dislocated into the cytosol. Subsequently FGF traffics to the nucleus and binds to specific DNA sequences, causing distinct cellular effects; cytosolic FGF triggers a signaling cascade temporally and physiologically different from stimulation via FGF receptor tyrosine kinase (, ). A similar phenomenon has also been reported for epidermal growth factor (EGF). EGF binding to the cell-surface EGF receptor (EGFR) triggers EGF–EGFR trafficking to the ER. EGF is then retrotranslocated into the cytosol, apparently through Sec61, signaling to the nucleus and inducing cyclin D gene expression (; ). It is tempting to speculate that similar principles may apply to exogenous molecules internalized by DCs. For example, proteins secreted by neighboring cells or contained in apoptotic bodies could be internalized by DCs, penetrate the cytosol, and modulate their ability to migrate or function as APCs. To date, however, there are no data supporting this notion.
Rat anti-GRP94mAb and rabbit anti-MAPK pAb were from Stressgen Biotechnologies and Cell Signaling, respectively. QuantiLum recombinant Firefly luciferase (14.9 mg/ml) and luciferin were purchased from Promega. and native coloenterazine (nCTZ) were purchased from Prolume, AZ. luciferase was resuspended in PBS; nCTZ (1 mg/ml stock in ethanol) was diluted 1:1000 in PBS for luciferase assays. SLO was from Aalto, Ireland. Cytochalasin D and radicicol were from Sigma and AG Scientific Inc., CA, respectively. ExoA was from EMD.
The human DC-like KG1 and human T lymphoblastoid cell line CEM were previously described (; ). Primary human DCs were generated () from human mononuclear cells isolated using lymphocyte separation medium (LSM; Cellgro) according to the manufacturer's protocol.
A total of 6 × 10 KG-1 or CEM cells in 1% FBS IMDM were incubated with soluble recombinant Firefly or luciferases for the indicated time points at the final concentrations of 49 and 0.13 μg/ml, respectively. After extensive washes in serum-free medium, cells were permeabilized with SLO as previously described (). Briefly, cells were resuspended in 200 μl serum-free medium and incubated for 15 min on ice in the presence of 0.3 mM DTT with or without SLO at 22 μg/ml. Cells were then washed three times with Dulbecco's phosphate-buffered saline without calcium and magnesium (DPBS; Gibco) and resuspended in 100 μl DPBS. Cells were then incubated at 37°C for 5 min to allow permeabilization. After centrifuging at 14 000 for 5 min at 4°C, 20 μl of supernatants from SLO-permeabilized cells were analyzed for luciferase activity with 100 μl of either nCTZ or luciferin (Promega) using a luminometer (GE).
refolding assays were performed as previously described (). Briefly, chemical unfolding was performed in 6 M guanidine–HCl in ICT buffer (50 mM HEPES, 78 mM KCl, 4 mM MgCl, 8.37 mM CaCl, and 10 mM EGTA) for 30 min at room temperature. Refolding reactions were performed for 30 min at room temperature with serial dilutions of KG-1 cytosol, isolated as previously described (), in ICT buffer in the presence or absence of radicicol. Refolding efficiency was calculated as regained luciferase activity, measured by luminometer, and expressed as percentage of luciferase input in the refolding reaction.
luciferase was chemically unfolded in 6 M guanidine–HCl in ICT buffer for 2 h and then dialyzed overnight against PBS, retaining its unfolded form. For experiments with KG-1 cells, 0.3 μg of native or 40 μg of chemically unfolded luciferases were added to KG-1 cells at the final concentration of 0.13 or 20 μg/ml, respectively. Cells were then washed three times with DPBS and resuspended in 100 μl DPBS before permeabilization with SLO as described above. After centrifuging at 14 000 for 5 min at 4°C, 20 μl of supernatants from SLO-permeabilized cells were analyzed for luciferase activity. For experiments with human DCs, 30 μg of chemically unfolded luciferase in PBS were added to 2 × 10 primary human DCs seeded in Ultra Low Cluster Plates (Costar). After 15-min incubation at 37°C to allow internalization, DCs were washed with cold PBS and resuspended in 100 μl of PBS or subjected to hypotonic lysis with 100 μl of 10 mM Tris, pH 7.4, both in the presence of protease inhibitors (EDTA-free; Roche). After centrifuging at 2000 for 10 min at 4°C, 20 μl of supernatants were analyzed for luciferase activity.
Supernatants from SLO-permeabilized cells or total cell lysates were separated by reducing SDS–PAGE (10% (w/v) acrylamide) and electrophoretically transferred to Immobilon-P membrane (Millipore). The membrane was blocked in TBS with 0.05% Tween 20 with 5% dehydrated milk and probed with rabbit anti-human GILT serum (1/10 000), rat anti-GRP94mAb, or anti MAPK rabbit Ab. Membranes were then washed, incubated with HRP-conjugated goat anti-rabbit or anti-rat IgG (1/5000; Jackson ImmunoResearch Laboratories) and ECL substrate (SuperSignal West Pico; Pierce), and exposed to film.
shRNA-expressing cells were generated as previously described () using the pSUPER.retro. puro vector (OligoEngine). The siRNA sequence specific for the Hsp90 β isoform has been previously described (); the shRNA construct targeting GFP used as control was a kind gift from Dr Hidde Ploegh (). Clonal selection was performed in 20 μg/ml puromycin. Control and Hsp90 β shRNA KG-1 cells were maintained in 20 μg/ml puromycin to maintain selection pressure. Knockdown was assessed by reducing SDS–PAGE followed by western blotting with Hsp90 β isoform-specific rabbit Ab (1/1000; Lab Vision Corporation). To ensure specificity, membranes were stripped for 4 min in 0.2 M NaOH and reprobed with Hsp90 α isoform-specific rabbit Ab (1/2000; Stressgen). Membranes were also probed with anti-MHC-I rat mAb 3B10.7 as a loading control and normalizer. Alkaline phosphatase-conjugated goat anti-rabbit or anti-rat IgG (1/5000; Jackson ImmunoResearch Laboratories) and ECF substrate (GE) were used for signal detection with a FluorImager (GE). |
DNA double-strand breaks (DSBs) are extremely cytotoxic lesions that can be generated by ionising radiation, reactive oxygen species and exposure to toxic chemicals (; ). Left unrepaired or incorrectly repaired, this damage can cause cell death and genome rearrangements, and these can in turn lead to cancer. Notably, DSBs also arise as intermediates during programmed genome rearrangement processes, such as site-specific V(D)J recombination that generates the antigen-binding repertoire of the mammalian adaptive immune system. Two pathways are mainly used to repair DSBs: homologous recombination that uses as the DNA repair template a homologous, undamaged DNA molecule such as the sister chromatid; and non-homologous end joining (NHEJ), a mechanism that can be used throughout the cell cycle but which is of particular importance in G1 and G0 ().
To date, the best characterised NHEJ factors are the Ku heterodimer (consisting of Ku70 and Ku80), the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs; ), the Artemis endonuclease, XRCC4 and DNA Ligase IV (). While DNA Ligase IV, XRCC4, Ku70 and Ku80 are conserved throughout all eukaryotic species known, DNA-PKcs and Artemis are not present in simpler eukaryotes such as yeast (). Ku80/70 heterodimers bind to broken DNA ends to initiate the NHEJ process (), and DNA-PKcs serves to bridge the broken DNA ends and promote ligation by XRCC4–Ligase IV. DNA-PKcs also mediates phosphorylation of Artemis, and it is thought that this allows Artemis to cleave off the damaged bases at the broken DNA ends (; ; ; ). After the actions of other processing enzymes such as polynucleotide kinase and DNA polymerases, the resulting DNA ends are finally ligated by DNA Ligase IV, which is bound to XRCC4 homodimer as a cofactor (; ). In addition to causing radio-sensitivity, inherited defects in NHEJ proteins cause severe-combined immune deficiency as a result of impaired V(D)J recombination (; ; ).
Although the above proteins complete the main functions required for NHEJ, in 2003 it became apparent that there was at least one further NHEJ factor (). Indeed, in 2006, two groups identified a previously uncharacterised 299-amino-acid residue protein, XLF/Cernunnos (henceforth called XLF) as being essential for NHEJ in human cells (; ). This new human NHEJ protein was named ‘XRCC4-like factor (XLF)' by one of the two groups based on an analysis with the Fugue alignment method () that gave 95% confidence for structural similarity between XLF and XRCC4 ( score of 4.75), despite the low sequence identity (13.7%) between the two proteins (). The tertiary structure of XRCC4 is a homodimer with N-terminal globular head domains and long extended α-helical coiled-coil regions (; ). Notably, homotypic interactions between XLF polypeptides have been established by pull-down experiments with two differently tagged versions of the protein (; ). In line with there being a specific relationship between XLF and XRCC4, yeast two-hybrid results and pull-down experiments suggested the existence of a large complex containing XLF, XRCC4 and Ligase IV (). Further biochemical investigations (; ) subsequently supported this contention and, furthermore, indicated that residues 1–128 of XLF bind to the head domain (residues 1–119) of XRCC4 (). Moreover, in the presence of Ku, XLF has been shown to enhance DNA end-joining by XRCC4–Ligase IV, and was reported to regulate DNA repair activity under conditions where base mismatches exist (). Notably, XLF is evolutionary and functionally conserved in diverse eukaryotes, and belongs to a superfamily of proteins that also contains the NHEJ factors Lif1 and Nej1, which interact with one another (; ).
While the suggested structural relationship between XLF and XRCC4 has led to speculation on how XLF functions in DSB repair, so far, it has not been clear whether and to what extent XRCC4 and XLF are structurally analogous, and little is known about precisely how XLF promotes NHEJ. To address these issues, we cloned, expressed and crystallised XLF, and herein describe its tertiary structure at 2.3-Å resolution. The structure reveals both similarities to and differences from the known three-dimensional structure of XRCC4. It supports the identification of the interacting region between XLF and XRCC4 suggested by biochemical studies () and provides important clues as to how XLF functions in concert with the Ligase IV-XRCC4 complex to bring about NHEJ.
Homologues of XLF identified in human, mouse, rat, frog, fish and yeast display conserved sequence features, revealing phylogenetic relationships between the respective proteins (). Protease digestion of human full-length (299 residues) XLF revealed that it can be truncated at the C terminus to give a stable fragment of ∼27 kDa (data not shown). Results from secondary structure predictions using Jpred (), Coils (), DisPredict-EMBL () and Foldingdex () indicate that residues after 245 in XLF may not have a defined structure (data not shown). In view of these results, we cloned, expressed, purified and crystallised the human XLF fragment containing residues 1–233, a region that is highly conserved among all XLF orthologues ().
XLF wild-type crystals diffracted to 2.9-Å resolution, in space group C2, with two protomers in the asymmetric unit. Phase information was obtained with SeMet-substituted crystals by using single-wavelength anomalous diffraction (SAD). However, SeMet-substituted crystals belonged to P2 space group, with four XLF subunits in each asymmetric unit. As the SeMet-substituted crystals diffracted to a better resolution, 2.3 Å, than the wild-type crystals, the data from these crystals were used for structure determination. The -value of the refined structure is 18.2%, and the -free is 23.9%. The wild-type crystal structure was later solved by molecular replacement (MR) by using the model generated from SeMet-substituted structure as the template ().
In the SeMet-substituted crystal structure, four protomers are organised as two dimers. In subunit A, residues 1–230 are clearly defined, while in subunits B, C and D residues 1–227, 1–227 and 1–229, respectively can be seen; interpretable electron density for residues 231–233 of all four subunits is absent, presumably due to disorder. Subunits A and B form a homodimer with a pseudo two-fold axis along the length of the molecule; a similar dimer is formed by subunits C and D. Each subunit has a globular head domain and a cone-shaped C-terminal part, comprised of a long α-helix, a reverse turn and two helices that wind their way around the dimeric coiled-coil ( and ). Structural features plotted against the sequence alignment of XLF orthologues are shown in ).
A stable XLF fragment containing the coding sequence for the TEV cleavage site and amino-acid residues 1–233 of human XLF was generated by PCR cloning into Gateway™ Destination Vectors (EMBL). The resulting plasmids included N-terminal His-MBP tag and His tag, and were named as XLF441 and XLF410, respectively.
XLF441 was expressed in Rosetta2 cells (Novagen). Thus, an overnight culture of 20 ml was grown at 37°C and diluted into two 1-l cultures to grow at 37°C till OD reached 0.6. Each culture was induced with 1 mM isopropyl-β--thiogalactopyranoside (IPTG) at 20°C overnight. Cell pellets were resuspended in 20 mM Tris pH 8.0, 300 mM NaCl, protease inhibitor (EDTA-free, Complete™; Roche). Cells were lysed by running through Emulsiflex at 2000 p.s.i. After centrifuging at 15 000 r.p.m. for 45 min, the supernatant was loaded onto 5 ml Ni-NTA beads. Imidazole (10 mM) was applied to the beads to wash away nonspecifically bound materials, and XLF was eluted with 100 mM imidazole. Eluate was dialysed in an imidazole-free buffer and treated with 400 U of TEV protease per milligram fusion protein to cleave off the N-terminal tags. Both tags in solution were removed by 5 ml Ni-NTA beads, and cleaved protein was loaded onto a Superdex-200 (16/60) column equilibrated with 20 mM Tris pH 8.0, 200 mM NaCl, 5 mM dithiothreitol (DTT). Peak fractions were concentrated to 10 mg/ml for crystallisation.
SeMet-substituted XLF441 was expressed by using a modified protocol. Thus, 20 ml culture was used as seed, 1 ml of these cells was diluted into 250 ml of M9 broth (containing 4.2 g/l FeSO, 1 mM MgSO, 10 ml of 40% -glucose and 100 μl of 0.5% thiamine per litre culture as supplementary) to grow at 37°C until OD reached 0.3. Then -lysine, -threonine and -phenylalanine (100 mg/ml each) and -leucine, -isoleucine, -valine and -selenomethionine (50 mg/ml each) were added into the cultures. Methionine synthesis was inhibited after 20 min at 37°C, and the cultures were induced with 1 mM IPTG and left shaking at 220 r.p.m. in 20°C overnight. SeMet-substituted XLF441 was purified in the same way as the native protein. After the last step, SeMet-XLF441 was concentrated to 5 mg/ml for crystallisation.
A Superdex-200 (16/60) column was equilibrated with 50 mM Tris pH 7.5, 100 mM KCl; the column bed volume () was 122 ml. Gel filtration molecular weight markers (MW-GF-200; Sigma) included horse cytochrome (12.4 kDa), bovine carbonic anhydrase (29 kDa), bovine albumin (66 kDa), yeast alcohol dehydrogenase (150 kDa) and sweet potato β-amylase (200 kDa). Column void volume () was measured with 2 mg (1 ml solution) blue dextran (2000 kDa), and the volume resulted was 43 ml. Protein samples were prepared in three groups: albumin (10 mg/ml), mixture of cytochrome (2 mg/ml) and β-amylase (4 mg/ml), mixture of carbonic anhydrase (3 mg/ml) and alcohol dehydrogenase (5 mg/ml). Sample (1 ml) was loaded onto the column for each run. Elution volumes () of cytochrome , carbonic anhydrase, albumin, alcohol dehydrogenase and β-amylase were 108, 85.5, 75, 67 and 63 ml, respectively.
Purified XLF410 (1–233) was concentrated to 0.5 mg/ml and dialysed against 20 mM HEPES pH 8.0, 200 mM NaCl and 5 mM DTT. Crosslinking was performed by BS. Stock solution containing 3% (w/v) BS was diluted to 1/2, 1/5, 1/10, 1/20, 1/50, 1/100, 1/200, 1/500 and 1/1000. Protein solution (10 μl) and BS (1 μl) solution (at different concentrations) were mixed in separate Ependorf tubes and left at room temperature for 30 min. Crosslinking was stopped by adding 1 μl of 1 M Tris pH 8.0 to each reaction and incubating for 15 min at room temperature. Protein gel loading buffer (3 ×) (6 μl) was then added and samples were loaded on 12% SDS–PAGE for analysis.
DLS measurements were performed using NanoS ZEN-1600 Instrument (Malvern Instruments Ltd) with 20 μM cleaved XLF441 in 20 mM Tris, 200 mM NaCl, pH 8.0. Measurements were taken at 20°C. Data were collected and analysed using the Dispersion Technology software V.5.02 (Malvern Instruments Ltd) and showed that XLF consisted of a monodisperse population of protein molecules.
Analytical ultracentrifugation was performed on an Optima XL-I (Beckman Coulter) centrifuge with an An-60 Ti rotor, double-sector centrepieces and an interference optical system for data acquisition. Sedimentation velocity experiments were performed at a speed of 55 000 r.p.m. at 20°C. Three concentrations of isolated XLF410 were used (0.4, 0.7 and 1.8 mg/ml) and the sample volume was 400 μl. Data were analysed using SEDFIT software (). The estimations of the partial specific volumes and molecular weight were achieved by SEDINTERP software ().
High- and low-angle scattering data were collected at Station 2.1, Synchrotron Radiation Source, Daresbury Laboratory, UK, using a two-dimensional multiwire proportional counter at sample-to-detector distances of 1 and 4.25 m and an X-ray wavelength of 1.54 Å with beam currents between 120 and 200 mA. Each sample was exposed for 25 min in 30 s frames. Frames at the beginning and the end of each data collection were compared to exclude the possibility of protein aggregation and/or radiation damage. The data reduction involved radial integration, normalisation of the one-dimensional data to the intensity of the transmitted beam, correction for detector artefacts and subtraction of buffer scattering (OTOKO, SRS, Daresbury). The -range was calibrated with an oriented specimen of wet rat-tail collagen (diffraction spacing of 670 Å) and silver behenate (diffraction spacing of 58.38 Å). XLF solutions at concentration ranging between 1 and 7 mg/ml were prepared in 20 mM Tris–HCl, 200 mM NaCl, 5 mM DTT, pH 8.0 and analysed at 4°C. The profiles collected at both camera lengths were merged so as to cover the momentum transfer interval 0.03 Å<q<0.77 Å. The modulus of the momentum transfer is defined as =4 π sin Θ/λ, where 2Θ is the scattering angle and λ is the wavelength used. The maximum scattering angle corresponds to a nominal Bragg resolution of approximately 8 Å. The forward scattering intensity, radius of gyration , the maximum particle dimension and intraparticle distance distribution function (()) were calculated from the scattering data using the indirect Fourier transform method program GNOM (). The crystal structure of XLF was compared to its conformation in solution using the program CRYSOL (), which simulates the scattering profile from atomic coordinates and provides a goodness-of-fit relating to the experimental data by inclusion of a hydration shell.
Far-UV CD spectra were recorded on an AVIV 62-S spectropolarimeter (AVIV, NJ, USA) previously calibrated with camphorosulphonic acid and equipped with a temperature control unit. In all experiments, spectra were recorded at 20°C in a 0.1-cm quartz cell using an average time of 0.5 s, a step size of 0.5 nm, 1-nm bandwidth and averaged over 20 scans. The dependence of CD signal on protein concentration was calculated by triplicate using independent samples of concentrations ranging between 50 and 600 μg/ml. After subtraction of the buffer baseline, the CD data were normalised and reported as molar residue ellipticity. For thermal denaturation experiments, five unfolding curves were recorded upon heating from 20 to 90°C at a rate of 1°C/min, and 80 s accumulation time. The apparent melting temperature, , was determined from differential melting curves of the function [θ]()/d. The concentration of protein solutions was determined from amino-acid composition analysis at the PNAC facility (Department of Biochemistry, University of Cambridge). Far-UV CD analysis of all proteins was carried out immediately after gel filtration chromatography.
Biosensor surface preparation, formation and dissociation of the XLF–XRCC4 complex were monitored with a BIAcore 2000 apparatus (BIAcore AB) using HBS (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA and 0.005% surfactant P20, pH 7.4) as the running buffer. After the surface activation with a freshly prepared mixture of 50 mM -hydroxysuccinimide and 195 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide for 4 min at 10 ml/min, purified XLF441 (cleaved) was diluted with 10 mM sodium acetate, 50 mM NaCl, pH 4.0 to a final concentration of 5 mM, and 40 μl of this sample was covalently bound to CM5 biosensor chips at 10 μl/min for 10 min; 3000 resonance units (RUs) were immobilised. Remaining activated carboxylic groups were deactivated by injecting 40 μl of 1 M ethanolamine hydrochloride, pH 8.6 for 7 min at 10 μl/min. Binding experiments were performed at 20°C in HBS at 10 μl/min (1-min injection time). After each run, the biosensor chip was regenerated using 1 M NaCl, 50 mM NaOH under the same injection condition. Five different concentrations of XRCC4 (5, 10, 12.5, 25 and 50 mM) were tested. Analysis of experimental data was performed with the interactive software BIAevaluation v3.1 (BIAcore). The simple biomolecular reaction model was used to simultaneously fit the data sets, where the analyte forms a 1:1 complex with its ligand.
The structure was solved using SAD with SeMet-substituted crystals. Phase information was calculated by PHENIX, and 36 Se atoms were found. An initial structure was auto-built also with PHENIX, in which 60% of total amount of residues were built. The -value was 27%, and -free value was 31%. More residues were traced during refinements by CNS and Refmac. After six cycles of refinement and rebuilding, 903 residues and 235 water molecules were included. Because of the lack of electron density, sequence difference remains between the crystal structure and the protein sequence, as shown in .
Protein sequences used for alignments were obtained from the proteomics server ExPASy (). Sequences were initially aligned by ClustalW () and manually adjusted using BioEdit software (). Conserved and identical residues in the sequence alignments were highlighted using analysis of multiply aligned sequences (, ). Secondary structure prediction was carried out using JPRED () and FoldIndex (). Sequences adopting coiled-coil conformation was calculated by COILS (). Disordered regions were predicted by DisPredict-EMBL (). Data files of crystal structures were retrieved from the PDB (). Phylogenetic analysis of XLF orthologues and mapping the evolutionary trace to XLF structure were done by Evolutionary Trace Server (TraceSuite II) (). Protein surface accessibility was calculated by ODA (). Superposition of protein tertiary structures are generated using COOT v1.3 (), and cartoon images are drawn in PyMOL v0.99rc6 (). Effect prediction of disease mutations to XLF was performed by SDM () with substitution tables updated by Catherine L Worth (). |
Species of are native to Australia with isolated
pockets of native forests also occurring in Papua New
Guinea and the Philippines (). Many spp.
centres of origin to new environments where they are typically propagated in
plantations for the production of paper, pulp and other wood products
(,
,
).
are susceptible to many pests and diseases including those known in their
areas of origin and others that have undergone host shifts
(,
).
plantations resulting in decreased revenue for commercial
forestry companies.
ascomycetes, accommodating more than 2000 species. Approximately 60
spp.
spp., and these are collectively referred to as
Mycosphaerella Leaf Disease (MLD) (, , ).
leaves and shoots of trees, where infection results in
premature defoliation, twig cankers and stunting of tree growth
(,
,
).
However, several spp.
foliage, and this has been attributed to their ability to
produce a proto-appressorium that enables direct cuticle penetration
(,
).
different spp.
Identification of spp.
to be difficult.
fruiting structures with highly conserved morphology, and they are
host-specific pathogens that grow poorly in culture.
morphological characters of the teleomorph and anamorph have been used in
species delimitation ().
() introduced ascospore
germination patterns as an additional characteristic to identify
spp., and Crous
() subsequently identified
14 different ascospore germination patterns for the
spp. occurring on .
() and Crous . () also
introduced features of these fungi growing in culture and especially anamorph
morphology as important and useful characteristics on which to base species
delimitation.
have also been employed to distinguish between species
occurring on (, ).
technique to identify spp.
employing DNA sequence data for species identification have relied on sequence
data from the Internal Transcribed Spacer (ITS) region of the ribosomal RNA
operon (Crous .
,
,
,
, Hunter .
,
).
gene sequences for this region have been useful, the resolution provided by
this region is not uniformly adequate to discriminate between individuals of a
species complex or to effectively detect cryptic species
().
Thus, recent studies have shown the importance of employing Multi-Locus
Sequence Typing (MLST) to effectively identify cryptic fungal species and to
study species concepts ().
phylogenetic unit ().
conserved and the anamorph morphology provides additional characteristics to
discriminate between taxa ().
morphology is often not congruent with phylogenetic data.
phylogenetic studies have led to the recognition of several species complexes
within (Crous .
,
,
).
Most of these studies have been based on comparisons of sequences for the ITS
regions of the ribosomal DNA operon.
emerged from them, it is well recognised that greater phylogenetic resolution
will be required for future taxonomic studies on
species.
concepts in spp. occurring on .
This was achieved by sequencing four nuclear gene regions, namely part of the
Large Subunit (D1–D3 of LSU) and ITS region of the nuclear rRNA operon,
and a portion of the Actin (ACT) and Elongation Factor 1-alpha (EF-1α)
gene regions.
spp.
possible.
collections of the Forestry and Agricultural Biotechnology Institute (FABI),
University of Pretoria, South Africa and the Centraalbureau voor
Schimmelcultures (CBS), Utrecht, The Netherlands
().
grown on 2 % (w/v) malt extract agar (MEA) (Biolab, South Africa), at 25
°C for approximately 2–3 mo to obtain sufficient mycelial growth for
DNA extraction.
cultures, freeze-dried for 24 h and then ground to a fine powder using liquid
nitrogen.
protocol as described in Hunter .
(,
).
the addition of absolute ethanol (100 % EtOH).
washing with 70 % Ethanol (70 % EtOH) and dried under vacuum.
used to resuspend the isolated DNA.
resuspended DNA and incubated at 37 °C for approximately 2 h to digest any
residual RNA.
Diagnostics, Mannheim), stained with ethidium bromide and visualised under
ultra-violet light.
DNA (. 20 ng) isolated from the spp.
used in this study was used as a template for amplification using the
Polymerase Chain Reaction (PCR).
volume of 25 μL containing 10× PCR Buffer (5 mM Tris-HCl, 0.75 mM
MgCl, 25 mM KCl, pH 8.3) (Roche Diagnostics, South Africa), 2.5 mM
of each dNTP (dATP, dTTP, dCTP, dGTP) (Roche Diagnostics, South Africa), 0.2
μM of forward and reverse primers (Inqaba Biotech, South Africa) and 1.25 U
Taq DNA Polymerase (Roche Diagnostics, South Africa) and DNA (20 ng/μL).
Sterilised distilled water was added to obtain a final volume of 25 μL.
The ITS-1, ITS-2 and the 5.8 S gene regions of the ITS region of the rRNA
operon were amplified using primers ITS-1 (5′– TCC GTA GGT GAA CCT
GCG G –3′) and LR-1 (5′- GGT TGG TTT CTT TTC CT –
3′) (, ).
of Crous .
() and Hunter . (,
).
amplified using primers LR0R (5'-ACC CGC TGA ACT TAA GC-3')
() and LR7 (5'-TAC TAC CAC CAA GAT CT-3')
().
PCR cycling conditions were as follows: an initial denaturation step of 96
°C for 2 min, followed by 35 cycles of denaturation at 94 °C for 30 s,
primer annealing at 62 °C for 30 s, primer extension at 72°C for 1 min
and a final elongation step at 72 °C for 7 min.
A portion of the EF-1α was amplified using the primers EF1-728F
(5'-CAT CGA GAA GTT CGA GAA GG-3') and EF1-986R (5'-TAC TTG AAG GAA CCC TTA
CC-3') ().
96 °C for 2 min, followed by 35 cycles of denaturation at 94 °C for 30
s, primer annealing at 56 °C for 30 s and primer extension at 72 °C
for 30 s. The reaction was completed with a final extension at 72 °C for 7
min.
A portion of the ACT gene was amplified using the primers ACT-512F (5'-ATG
TGC AAG GCC GGT TTC GC-3') and ACT-783R (5'-TAC GAG TCC TTC TGG CCC AT-3')
().
followed by 10 cycles of denaturation at 94 °C for 30 s, primer annealing
at 61 °C for 45 s and extension at 72 °C for 45 s.
by 25 cycles of denaturation at 94 °C for 30 s, primer annealing at 61
°C and elongation at 72 °C for 45 s with an increase of 5 s per cycle.
The reaction was completed with a final elongation step at 72 °C for 7
min.
an ABI PRISM™ 3100 Automated DNA sequencer (Applied Biosystems, Foster
City, CA). The ABI Prism Big Dye Terminator Cycle sequencing reaction kit v.
3.1 (Applied Biosystems, Foster City, CA) was used for sequencing reactions
following the manufacturer's instructions.
performed with the same primers used for PCR reactions.
case of the ITS region where two internal primers ITS-2 (5'-GCT GCG TTC TTC
ATC GAT GC-3') and ITS-3 (5'-GCA TCG ATG AAG AAC GCA GC-3')
()
were included for the sequencing reactions.
internal primers LR3R (5'-GTC TTG AAA CAC GGA CC-3') and LR-16 (5'-TTC CAC CCA
AAC ACT CG-3') were used for the sequencing reactions.
All resulting sequences were analysed with Sequence Navigator v. 1.0.1
(Applied Biosystems, Foster City, CA).
MAFFT (Multiple alignment program for amino acid or nucleotide sequences) v.
5.667 () and manually adjusted where necessary.
and most parsimonious trees were generated in PAUP v. 4.0b10
() by heuristic
searches with starting trees obtained through stepwise addition with simple
addition sequence and with the MULPAR function enabled.
Reconnection (TBR) was employed as the swapping algorithm.
as missing data and characters were assigned equal weight.
nodes was obtained by performing 1000 bootstrap replicates of the aligned
sequences.
length (TL), retention index (RI), consistency index (CI), rescaled
consistency index (RC) and homoplasy index (HI).
Grossenb. & Duggar was used as the outgroup to root all trees.
1000 replicates on all informative characters was conducted in PAUP to
determine if the LSU, ITS and EF-1α data sets were combinable.
sequences of spp.
deposited in GenBank ().
ACT have been deposited in TreeBASE (accession numbers: LSU = SN2535, ITS =
SN2534, EF-1α = SN2536, ACT = SN2537).
conducted in PAUP.
identical to those described earlier. For distance analyses, Modeltest v. 3.04
()
was used to determine the best evolutionary model to fit the combined DNA
sequence alignment.
was conducted in PAUP.
time-reversible (GTR) and the proportion of sites assumed to be invariable (I)
was 0.4919, identical sites were removed proportionally to base frequencies
estimated from all sites, rates of variable sites assumed to follow a gamma
distribution (G) with shape parameter of 0.6198.
broken randomly.
p
Results of this study represent the first attempt to employ DNA sequence
data from a relatively large number of nuclear gene regions in order to
consider the phylogenetic relationships for spp.
occurring on leaves.
entirely on sequence data of the ITS region (Crous .
,
,
, and
– this volume,
).
Although the ITS region offers sufficient resolution to distinguish most taxa,
it has not been adequate to separate cryptic taxa in
().
Results of the present study showed that combined DNA sequence data from the
LSU, ITS, EF-1α gene regions offer increased genetic resolution to study
species concepts in .
variation within some clades that were well supported by sequences of other
loci and morphological characteristics.
ACT data from the final analyses.
other studies including greater numbers of species
(Crous & Groenewald, unpubl. data).
grouped together in a well-supported clade in the phylogeny emerging from the
combined alignment. This was also true for the ITS, EF-1α and ACT
phylogenies where these isolates grouped in a distinct clade with a 100 %
bootstrap support. and
both have anamorphs, however,
produces a anamorph
(,
).
Interestingly, the anamorph of
was differentiated from only by the fact that
conidia are produced in a pycnidium as opposed to an acervulus
().
anamorphs of has previously been viewed with
circumspection especially because anamorphs can
produce different conidiomatal forms under differing environmental conditions
(,
– this volume).
anamorph in is questioned and it should be
re-evaluated in terms of its morphological similarities to
.
are all similar, with germ tubes that grow parallel to the
long axis of the spore and ascospores with a slight constriction at the median
septum, typical of a type C ascospore germination pattern
(,
,
).
Furthermore, overlap is seen in ascospore dimensions of the three species
where those of are (11–)12–14(–17)
× (2.5–)3.5–4(–4.5) μm, those of are (12–)14–15(–22)
×(3.5–)4.5–5(–6) μm and those of
9.5–16.5 × 2.5–4 μm
(,
,
).
Leaf lesions of the three species are also similar, pale brown to dark
red-brown with lesions of and often
producing a red margin that was, however, not observed in (,
,
).
Morphological features of and are also very similar.
the present study where these three species appear to represent a single taxon
and therefore suggest that and should be synonomised under , which is the
oldest epithet. We therefore reduce and to synonymy with as follows:
(Thüm.) Lindau,
Natürliche Pfanzenfamilie, 1: 424. 1897.
: Crous & M.J.
Wingf., Canad. J. Bot. 75: 670. 1997.
().
this fungus included in the present study grouped together with isolates of
in the LSU, ITS, EF-1α and combined data set with
high bootstrap support.
spp.
().
is also not known to produce an anamorph
().
Although these two species are phylogenetically similar, they can be
distinguished from one another based on different ascus and ascospore
dimensions, ascospore germination patterns and cultural characteristics
(,
).
Although morphologically distinct, it is interesting that these two taxa are
phylogenetically so closely related and might suggest a recent speciation
event.
together in a separate clade in all of the DNA sequence data sets in this
study. This has also been shown by Crous .
(), where isolates of
these two species grouped together in a distinct clade based on ITS DNA
sequences. was originally described from
in Australia, and recognised as a distinct species of
due to its lesion characteristics, and ascospore
morphology ().
() examined the type of
and and found the two species to be
congeneric, and reduced them to synonymy under .
the present study support the synonymy.
have anamorphs, showed various phylogenetic placements in
this study.
sister to a sub-clade.
EF-1α phylogenies the clade was situated basal to
the larger clade.
Crous . (,
) where the
clade also resided outside the larger monophyletic
clade.
conserved and to show less nucleotide differences than the ITS and EF-1α
gene regions.
conclusion that could be different from
., this proved not to be the case when LSU data
were considered. is morphologically identical to
, and the separation of these two genera no longer seems
tenable.
have anamorphs ().
these anamorphs only upon initial isolation, and those that are currently
available are sterile.
anamorphs readily produce those in culture, and they usually sporulate
profusely.
resolved once fresh, sporulating collections of either
or can be obtained.
spp.
grouped into three clades in all of the phylogenies generated in this study.
Species in the clades have short branch lengths
arising from a common internode, suggesting that they have speciated
relatively recently from a common ancestor
() and, most likely have co-evolved with their
hosts as suggested by Crous .
(). Ávila . () suggested that
may represent a monophyletic lineage.
of this and other studies () have shown that is
paraphyletic in and has evolved more than once in the
genus.
likely to resolve cryptic species and species complexes within
as has already been shown for the and the species complexes (Crous . ,
).
members of the species complex
(,
).
Previous studies based on ITS DNA sequence data have demonstrated the
phylogenetic relatedness of these four species
(,
).
However, bootstrap support for their phylogenetic placement was low
().
The phylogeny of combined DNA sequence data in this study showed that the four
species in the complex reside in a well-supported clade
(bootstrap support 97 %).
species have also recently diverged from a common ancestor.
also included isolates of and
.
for have been incorporated into a phylogeny.
most closely related to .
single conidiophores arising exclusively from secondary mycelium, which is
different to in which conidiophores arise from
loose or dense fascicles of a stroma
(,
).
Furthermore, conidia of are more septate, longer, and
more uniformly cylindrical in shape than those of (, ).
that species which are morphologically distinct, can be very closely
related.
was the placement of in relation to
and .
Sequences for all but the ACT gene region showed that these three taxa
represent the same phylogenetic species.
suggested that should have a anamorph
because of its proximity to
(),
the current data suggest that this anamorph could be .
() described from spermatogonia on lesions colonised by , but failed to link the two states because single-ascospore
cultures did not form an anamorph in culture. is morphologically similar to , and
probably represents the same taxon.
to synonymy with as follows:
Carnegie & Keane, Mycol. Res.
98: 413–416. 1994.
: Crous &
M.J. Wingf., Mycologia 88: 456. 1996.
have no known anamorphs.
suggested that and grouped close to
spp. with anamorphs.
been assumed that and would have
anamorphs if they were to be found
().
However, the phylogenies emerging from LSU, ITS and EF-1α sequences and
the combined data for the three regions showed that and consistently group separately from the
anamorphs, close to a clade of isolates with
and anamorphs.
association of these three taxa to is thus doubted.
Furthermore, the clade containing and is also well-supported and seems to represent a single evolving
lineage.
represent a single phylogenetic species.
species consistently grouped together in all phylogenies with grouping as a sister. was
described from leaves of in south-western Australia and
is known only from this location ().
produces asci and ascospores that are similar in size and morphology to However, the ascospores of are not
constricted at the median septum whereas those of had
such constrictions, and ascospores of are longer
(9–)11–12(–15) μm than those of
(7–)8–10(–11) μm
(,
).
Furthermore, produces lateral hyaline germ tubes that
grow parallel to the long axis of the ascospore and become slightly verrucose
to produce lateral branches upon prolonged incubation
().
that germinate in an irregular fashion producing distinctly dark verrucose
germ tubes from different positions of the distorted ascospore
().
that these two species, which are morphologically quite distinct, would
represent a single phylogenetic species.
are required to determine whether they represent two distinct taxa or are
conspecific.
().
(,
).
, collected from in Australia,
consistently grouped in a clade with isolates of and
and are known to have and
anamorphs, respectively
().
previous studies employing ITS sequence data, isolates of grouped sister to isolates of and
().
However, based on sequence data from the four gene regions employed in this
study, isolates of grouped in a distinct
well-supported clade with .
has an anamorph
().
and
Crous & G. Hunter are the only spp.
that are known to have
anamorphs (,
– this volume).
considered in this study suggest that constitutes
heterogenous groups of which only a few are closely linked to certain anamorph
genera.
genera within has been polyphyletic, and not
monophyletic as previously suggested.
evolution of anamorph genera such as and within
().
It would thus not be advisable to predict anamorph relationships based on the
phylogenetic position within .
morphology evolved more than once in the group, but disjunct anamorph
morphologies also frequently cluster together (Crous .
,
,
).
interpretation difficult, and predictions based on position in clades
unreliable.
occurring on leaves should serve
as a framework for the more accurate taxonomic placement of these fungi in
future.
become more evident in this genus in recent years (Crous .
,
,
– this volume).
generation of greater numbers of data sets should allow for increased
resolution at the species level.
species complexes and cryptic speciation.
groups in can in future utilise sequence data for the
LSU region that have not previously been available.
more lucid indication and support for phenotypic characters that are
phylogenetically informative. |
Grey leaf spot of maize is a serious foliar disease of in
many countries where it is cultivated, especially in the eastern U.S.A.
Africa (, ).
move” by Latterell & Rossi
(), grey leaf spot has
become increasingly important and is currently seen as one of the most serious
yield-limiting diseases of maize (, ).
regarded as Tehon & E.Y.
Ellis & Everh.
().
() referred to a var. Ellis & Everh., which is morphologically
similar to , but suspected to represent a distinct species
due to its lack of pathogenicity to sorghum.
accepted that more than one species of is associated with
grey leaf spot of maize, namely Group I, which is
dominant in the U.S.A.
and occurs in the U.S.A., Africa and possibly elsewhere
(,
,
).
species associated with grey leaf spot symptoms occurring on maize in South
Africa.
of several loci, namely the internal transcribed spacers (ITS1 & ITS2),
the 5.8S rRNA gene, the elongation factor 1-α, histone 3, actin and
calmodulin gene regions.
morphologically compared to those isolates from the U.S.A., and the type
specimen of .
cultured as explained in Crous
().
characteristics and morphology of isolates
() were determined on
plates containing 2% malt extract agar (MEA) (20 g/L), 2% potato-dextrose agar
(PDA), oatmeal agar (OA), and carnation leaf agar (CLA) [1% water agar (10
g/L) with autoclaved carnation leaves placed onto the medium]
().
Plates were incubated at 25 °C under continuous near-UV light, to promote
sporulation.
unidentified sp. () were used for phylogenetic analysis.
isolate genomic DNA from fungal mycelium of monoconidial cultures grown on MEA
in Petri dishes.
of the 18S rRNA gene, the first internal transcribed spacer (ITS1), the 5.8S
rRNA gene, the second ITS region and the 5' end of the 28S rRNA gene. To
obtain additional sequence information, four other loci were also sequenced.
Part of the elongation factor 1-α gene (EF) was amplified with primers
EF1-728F and EF1-986R, part of the actin gene (ACT) with primers ACT-512F and
ACT-783R, and part of the calmodulin gene (CAL) with primers CAL-228F and
CAL-737R ().
primers CylH3F and CylH3R (). Sequencing was done with the same PCR primers.
The PCR conditions, sequence alignment and subsequent phylogenetic analysis
followed the methods of Crous
().
added to a subset of the alignment (TreeBASE matrix M2038) of Crous . () and additional
sequences were obtained from GenBank.
and alignments in TreeBASE (S1509, M2712).
species described in the present study.
the development of a species-specific diagnostic test.
CylH3R were used as external primers and their amplification product functions
as a positive control. Three species-specific primers were designed for sp. nov.
species, respectively: CzeaeHIST (5'-TCGACTCGTCTTTCACTTG-3'), CzeinaHIST
(5'-TCGAGTGGCCCTCACCGT-3') and CmaizeHIST (5'-TCGAGTCACTTCGACTTCC-3'); all of
them species-specific.
the external primers, were used in separate PCR reactions in a total volume of
12.5 μl, containing 1 μl of diluted genomic DNA, 1× PCR buffer, 2
mM MgCl, 48 μM of each of the dNTPs, 0.7 pmol CylH3F, 3 pmol of
CylH3R, 4 pmol of the specific internal primer and 0.7 units (Bioline)
polymerase.
PCR System 9600 (Perkin-Elmer, Norwalk, Connecticut).
step was done at 94 °C for 5 min, followed by 15 cycles of denaturation at
94 °C (20 s), annealing at 58 °C (30 s) and elongation at 72 °C
(40 s) as well as 25 cycles of denaturation at 94 °C (20 s), annealing at
55 °C (30 s) and elongation at 72 °C (40 s).
at 72 °C (5 min) was included to ensure that full length products are
obtained.
under UV-light after ethidium bromide staining.
well as on host material. Structures were mounted in lactic acid, and 30
measurements at × 1000 magnification were made of each structure.
given in parentheses.
().
are maintained in the culture collection of the Centraalbureau voor
Schimmelcultures (CBS), Utrecht, the Netherlands.
deposited at the National Collection of Fungi in Pretoria (PREM), South Africa
().
.
the last four loci were not available for other isolates, a separate tree that
included more isolates was generated using only ITS sequences
().
homogeneity test showed that all loci could be combined (p = 0.747) into a
single analysis ().
isolates) and 487 characters including alignment gaps.
446 are constant.
(uncorrected “p”, Jukes-Cantor and HKY85) on the sequence data
yielded trees with similar topology and bootstrap values.
of the alignment yielded six most parsimonious trees (TL = 44 steps; CI =
0.955; RI = 0.986; RC = 0.942), one of which is shown in
.
were obtained. The first clade (86 % bootstrap support) contained and together with two isolates of var. and an undescribed sp.
(CPC 12062) from in South Africa. The second clade (98 %
bootstrap support) contained three isolates of the new species (, formerly Group II). The isolates of var. and had ITS sequences
similar to those of Group II (= ),
but there was no bootstrap support for this branch.
Group I).
provided trees with similar topologies (data not shown).
taxa) and 1643 characters including alignment gaps.
1227 were constant.
parsimonious trees (TL = 519 steps; CI = 0.948; RI = 0.986; RC = 0.935), one
of which is shown in .
Three distinct clades were obtained, the first (100 % bootstrap support)
containing clades with (90 % bootstrap support) and
(100 % bootstrap support) with sp. CPC
12062 as a sister taxon (100 % bootstrap support).
the and isolates formed distinct and
well-supported clades (each with a bootstrap support value of 100 %).
Neighbour-joining analysis using the three substitution models on the sequence
data yielded trees with similar topology and bootstrap values to that obtained
using parsimony (data not shown).
new sp. is possible using three multiplex PCR
amplifications.
present for all three species, while the second 284 bp fragment is only
observed for the species recognised by the specific
internal primer ().
Primers CzeaeHIST, CzeinaHIST, and CmaizeHIST are therefore specific for
and the sp.,
respectively, and can be used for their identification and detection.
Tehon & E.Y.
17: 248. 1925. .
to brownish spots, shape and size variable, often with a narrow brown border
line or margin. amphigenous, mostly hypophyllous,
punctiform to subeffuse, brown. internal.
lacking or small, with a few swollen substomatal brown cells.
in small to moderately large fascicles (3–14),
emerging through the stomata, usually divergent, erect, straight,
subcylindrical to flexuous, distinctly geniculate–sinuous, unbranched,
40–180 × 4–8 μm, obscurely (0–)1–8-septate,
uniformly pale olivaceous to medium brown, thin-walled, smooth; conidiogenous
cells integrated, terminal, occasionally intercalary, 10–40 μm long,
conidiogenous loci conspicuously thickened and darkened, 2–3 μm wide.
solitary, broadly obclavate–subcylindrical,
30–100 × 4–9 μm, 1–10-septate, hyaline,
thin-walled, smooth, apex obtuse, base obconically truncate, hila somewhat
thickened and darkened, 2–3 μm wide (based on type specimen).
: , Illinois, Alexander Co.,
McClure, on , 29 Aug. 1924, P.A. Young (ILLS 4276)
, BPI 442569 ; Indiana, Princeton, 2003, B.
Fleener, YA-03 = A358 = ; Delaware, 1997, B. Fleener, DE-97 = A359 =
;
Wisconsin, Janesville, 2002, B. Fleener, ,
,
JV-WI-02 = A360 = , culture ex-type; Iowa, Johnston, 2004, B.
JH-IA-04 = A361 = ; Tennessee, Union City, 1999, B. Fleener, UC-TN-99 = A362 =
;
Pennsylvania, New Holland, 1999, B. Fleener, NH-PA-99 = A363 =
;
Indiana, Princeton, 1999, B. Fleener, PR-IN-99 = A364 =
;
Missouri, Dexter, 2000, B.
Reinbeck, 1999, B. Fleener, RENBECK-IA-99 = A367 =
.
: Colonies on PDA reaching 15–25 mm
diam after 3 wk, and forming ample spermatogonia; colonies on MEA erumpent,
with sparse aerial mycelium; margins smooth, but irregular; surface
olivaceous-grey with irregular patches of white or smoke-grey; reverse
iron-grey; colonies fertile.
mycelium; margins smooth but irregular; surface red with patches of white and
pale olivaceous-grey; fertile.
:
: Azerbaijan, Brazil, Cameroon, Canada, China,
Colombia, Congo, Costa Rica, Ecuador, Ethiopia, Georgia, Guatemala, Kenya,
Malawi, Mexico, Mozambique, Nigeria, Panama, Peru, South Africa, Swaziland,
Tanzania, Trinidad and Tobago, Uganda, USA (CO, DE, IA, IL, KS, KY, MD, MN,
NC, OH, PA, SC, TN, VA, WI, WV), Venezuela, Zambia, Zimbabwe
().
Crous & U.
MycoBank .
.
variable in length from 5–40 mm; lesions becoming confluent, pale grey
to pale brown; borders indistinct, chlorotic in younger leaf spots.
fasciculate, amphigenous, punctiform to subeffuse, grey to
brown on leaves, up to 120 μm high and wide. internal,
consisting of pale brown, septate, branched, smooth hyphae, 3–4 μm
wide. lacking or small, a few swollen substomatal cells,
brown, up to 30 μm diam. aggregated (3–20) in
loose to semi-dense fascicles arising from the upper cells of an inconspicuous
brown stroma, emerging through stomata, usually divergent, erect, straight,
subcylindrical to flexuous, distinctly geniculate–sinuous, unbranched or
branched above, 40–100 × 5–7 μm, 1–5-septate,
uniformly pale olivaceous to medium brown, thin-walled, smooth; conidiogenous
cells integrated, terminal, 40–60 × 5–6 μm, with several
conidiogenous loci that are conspicuously thickened, darkened and refractive,
2–3 μm wide. solitary, broadly fusiform,
(40–)60–75(–100) × (6–)7–8(–9)
μm, (1–)3–5(–10)-septate, hyaline, thin-walled, smooth,
apex subobtuse, base subtruncate, hila somewhat thickened, darkened and
refractive, 2–3 μm wide (based on type specimen).
: , KwaZulu-Natal,
Pietermaritzburg, on , 2005, P.
, = CPC 11995, culture ex-type.
: Colonies on PDA reaching 10–15 mm
diam after 3 wk, and forming spermatogonia; colonies on MEA erumpent, with
sparse aerial mycelium; margins smooth, but irregular; surface olivaceous-grey
with irregular patches of white or iron-grey; reverse iron-grey; colonies
fertile. On OA colonies are spreading with moderate whitish aerial mycelium;
margins smooth but irregular, olivaceous-grey; fertile.
:
: South Africa, Uganda, U.S.A. (NC, NY, OH, VA),
Zambia, Zimbabwe (, ).
: has conidia of similar
dimensions to those of .
distinguished by having shorter conidiophores (up to 100 μm) and more
broadly fusiform conidia, versus longer conidiophores (up to 180 μm) and
broadly obclavate–subcylindrical conidia of .
Colonies of grow more slowly in culture and lack the red
pigment associated with cercosporin production, typical of ().
In a recent review of grey leaf spot of maize, Ward .
() discussed the
complexities and importance of this disease in the U.S.A., as well as in
Africa.
or more species (, , ).
possible species complexes associated with grey leaf spot, namely the complex ( and var.
), and the complex (Groups I and
II).
taxonomic uncertainty, by demonstrating that Group II is, in fact, a distinct
species () and that Group I, to which the name applies, apparently does not occur in South Africa.
collections from other African countries, as well as other locations in South
Africa would be required, however, to determine if is
truly absent from the continent.
Grey leaf spot disease was first recorded from South Africa in 1988
().
The possible source of inoculum was later postulated by Ward .
() to have been from
infested maize residues imported from the U.S.A.
& Levy (), if this was
indeed the case, such inoculum would have more likely contained , which dominates over throughout most of
the maize-producing areas of the eastern and midwestern U.S.A.
distribution of throughout Africa and the fact that there is
more genetic diversity of the pathogen in Africa than in the U.S.A.
(), it
was thought to be more likely that was introduced to the
U.S.A. from Africa, than .
() also considered a third
possibility, namely that was introduced to Africa and the
U.S.A. on another host, as maize is not native to Africa.
likely hypothesis may be that is indeed native to Africa,
but that it has jumped from another indigenous host (such as sorghum) onto
maize. It is interesting to note that the ITS sequence of the isolates was more similar to that of an isolate of var. than to that of the presumably American
species .
further comparisons between and are
needed.
generally assumed to be host-specific
(,
), some
species have been observed to also have the ability to colonise hosts other
than those on which they are assumed to be primary pathogens.
recently observed for the greasy leaf-spot pathogen of Whiteside, which was isolated from other hosts such
as and ().
formulation of the pogo stick hypothesis
(),
where species of can jump to another host as a
secondary colonizer, where they sporulate on lesions of the primary
pathogen, producing enough inoculum to enable them to
continue the search for their real host.
isolate from grey leaf spot lesions caused by .
was originally suspected that this isolate may represent (fast growing and forming a red pigment in agar), this has
proven to not be the case.
more similar to isolates in the complex ( and ).
this complex are known from culture, CPC 12062 proved distinct based on DNA
sequence data when compared to the more than 100 sequences currently available
in our unpublished database.
from another host that has “jumped” onto maize
().
By using the PCR-based method described here as a diagnostic tool, it is
relatively easy to identify the three species on maize
that are treated in this study.
ample spermatogonia on host tissue as well as in culture.
been an earlier report of a possible teleomorph
(),
this has remained unconfirmed. Wang .
() were unable to find
evidence of the mating type idiomorph in isolates of
, and our current mating studies with isolates
of and have also given negative
results.
level of variation present in populations, and whether sexual reproduction
occurs within populations of these two fungi.
the existence of cryptic sex, however, as Wang .
() reported the variation
to be rather low in populations of both species. |
In recent years, two species of Wollenw.
associated with black foot disease of grapevines ( spp.).
(Zinnsm.) Scholten [anamorph of
(Gerlach & L. Nilsson) Mantiri &
Samuels] was first recorded on grapevine in France in 1961
().
Since then it has been isolated from diseased vines in Tasmania
(), Sicily
(), Portugal
(, Rego ., ,
), Pennsylvania, U.S.A.
(),
New Zealand and South Africa ().
have also been isolated from young vines and from
declining vines with basal rot or root necrosis in Australia
(),
Chile (), Greece (), Spain () and South Africa
(,
).
disease.
Tasmania, New Zealand and Canada ().
(Cooke & Harkn.) Wollenw.
(, ).
morphology, has also been associated with the disease in South Africa
().
segregated species into four groups based on the
presence or absence of microconidia and chlamydospores. (Sacc.) Wollenw. (+ chlamydospores; – microconidia),
which is the anamorph of the type species of Wollenw.,
Wollenw. (– chlamydospores; –
microconidia), which is the type species of the genus ,
and members of species predominantly connected with
teleomorphs of the W. Phillips & Plowr.
(– chlamydospores; + microconidia) were core members of three of these
anamorphic groups delineated by Booth
().
centred on (+ chlamydospores; + microconidia), which
generally is accepted as the anamorph of .
(),
Mantiri ()
and Brayford
() recently transferred
representatives of all “” groups with
anamorphs into .
() analysed
mitochondrial small subunit (SSU) ribosomal DNA (rDNA) sequence data of some
of the species and concluded that the
/ species grouped together by this
reclassification were monophyletic.
this overall / clade included
distinct subclades corresponding to at least three of the four groups
delineated by Booth ().
Significant molecular variation among taxa with -like
anamorphs was found by Seifert
(), in a study on fungi
causing root rot of ginseng () and other hosts,
encountered significant molecular variation particularly among
-like strains and suggested that / may present a complex of various
species.
() added an additional
phylogenetic clade mainly comprising of root and rootstock pathogens of
grapevines, that conformed well to the morphological concept of . Although Halleen .
() referred to the primary
causal organism of black foot disease of grapevine as ,
the ex-type strain of
did not
form part of the clade comprising of grapevine isolates, nor did isolates from
, which is the host from which Booth
() selected the neotype of
.
correct identity of like isolates occurring on
grapevines.
countries were subjected to DNA analyses of their ITS and β-tubulin genes
and to mating studies .
diseased grapevines in Portugal (, ), France, South Africa and New Zealand
(), were obtained from the collection of the Centraalbureau
voor Schimmelcultures in Utrecht, the Netherlands (CBS)
().
associated with these isolates include various forms of decline as well as
typical black foot symptoms.
() and
DNA was extracted using the FastDNA® Kit (Bio 101, Carlsbad, CA, U.S.A.).
PCR amplification and sequencing of the partial β-tubulin gene introns
and exons and the ITS rDNA, was performed as described by Halleen ().
generated sequences have been deposited in GenBank
().
Additional sequences were obtained from GenBank and added to the alignment.
Sequences were manually aligned using Sequence Alignment Editor v. 2.0a11
().
accession numbers; newly generated sequences are indicated by CBS strain
numbers.
()
was used as outgroup.
PAUP* 4.0b10 ()
consisted of distance (using the uncorrected “p”, Jukes-Cantor and
HKY85 substitution models) and parsimony analyses as described by Halleen
. ().
the parsimony analyses, heuristic searches were performed with 100 random
taxon additions.
outgroup sequence) were coded as a single character in the ITS alignment in
TreeBASE (S1511, M2716).
Strains were grown in darkness or under continuous near-ultraviolet (nuv)
light (400–315 nm) (Sylvania Blacklight-Blue, Osram Nederland B.V.,
Alphen aan den Rijn, the Netherlands) at 20 °C.
nutrient-poor agar (SNA) with and without the addition of a 1 × 3 cm
piece of filter-paper to the colony surface, potato-dextrose agar (Difco PDA,
Becton Dickinson, Sparks, MD, U.S.A.), oatmeal agar (OA), and carnation leaf
agar (CLA) (), and malt extract agar (MEA) (Sigma-Aldrich Chemie BV,
Zwijndrecht, the Netherlands) using 9 cm diam Petri dishes.
colony diameters of cultures incubated in darkness were measured on PDA.
Characters such as size and shape of conidia, phialides, and chlamydospores
were determined from strains grown on SNA, PDA, or CLA after 14–21 d.
Structures were mounted in lactic acid, and 30 measurements at × 1000
magnification were made of each structure.
calculated, and the extremes of spore measurements given in parentheses.
Images were taken from slides mounted in lactic acid.
of colonies were described after 14 d; colour names are from Rayner
().
for growth were assessed on PDA incubated for 7 d in the dark at 4, 10, 15,
20, 25, 30 and 35 °C.
medium at 25 °C, using autoclaved birch toothpicks as explained by Guerber
& Correll ().
replicates were done for each cross.
(Pechiney Plastic Packaging, Menasha, WI, USA), incubated in a single layer
under a mixture of cool-white fluorescent and nuv light, and observed at
weekly intervals for a total period of 8 wk.
sexually compatible if they produced perithecia with viable, exuding masses of
ascospores within this time.
including alignment gaps.
parsimony-informative, 60 were variable and parsimony-uninformative, and 365
were constant.
trees [tree length (TL) = 224 steps; consistency index (CI) = 0.795; retention
index (RI) = 0.924; rescaled consistency index (RC) = 0.734], one of which is
shown in .
of the trees generated with neighbour-joining analyses using the three
substitution models were identical to each other and were also similar to the
trees obtained using parsimony (data not shown).
(), isolates that cluster
with the ex-type strain of grouped with a bootstrap
support value of 85 %. Two isolates of (99 %
bootstrap support) formed the closest sister clade (75 % bootstrap support).
Isolates of “” form two poorly supported
clades (57 and 54 %, respectively) separated by .
final clade is in a basal position and contains isolates of (94 % bootstrap support) with as closest
sister (82 % bootstrap support).
The manually adjusted β-tubulin alignment contained 55 taxa and 327
characters including alignment gaps.
parsimony-informative, 39 were variable and parsimony-uninformative, and 255
were constant.
parsimonious trees (TL = 103 steps; CI = 0.864; RI = 0.959; RC = 0.829), one
of which is shown in .
The topology of the trees generated with neighbor-joining analysis using the
three substitution models and the trees obtained using parsimony only differed
in the order of the isolates in the “”
clade (data not shown).
supported clade (56 % bootstrap support).
basal positions with a small number of defined clades with high bootstap
support values. is also included in this
clade.
.
,
.
:J.D. MacDon.
& E.E. Butler, Plant Disease 65: 156. 1981.
verruculosis, et peritheciis levibus vel verruculosis distincta.
(7–)9–11(–14) ×
(2.5–)3–3.5(–4) μm.
(not known from nature) formed heterothallically , disposed solitarily or in groups of up to six, developing directly
on the agar surface or on sterile pieces of beach wood or pine needles, ovoid
to obpyriform, with a flattened apex, up to 70 μm wide, orange to red,
becoming purple-red in 3 % KOH (positive colour reaction), smooth to warted,
up to 300 μm diam and high; with minute stroma of dark red
pseudoparenchymatous cells; perithecial wall consisting of two regions; outer
region 15–30 μm thick, composed of 1–3 layers of angular to
subglobose cells, 10–25 × 8–17 μm; cell walls up to 1
μm thick; inner region 10–15 μm thick, composed of cells that are
flat in transverse optical section and angular to oval in subsurface optical
face view, 7–15 × 3–5 μm; perithecial warts consisting of
globose to subglobose cells, 15–30 × 15–20 μm in surface
view. narrowly clavate to cylindrical, 45–60 ×
5–6 μm, 8-spored; apex subtruncate, with a minutely visible ring.
medianly 1-septate, ellipsoidal to oblong ellipsoidal,
somewhat tapering towards both ends, smooth to finely warted, hyaline, become
pale brown with age, (7–)9–11(–14) ×
(2.5–)3–3.5(–4) μm.
simple or complex, sporodochial.
conidiophores arising laterally or terminally from the aerial mycelium or
erect, arising from the agar surface, solitary to loosely aggregated,
unbranched or sparsely branched, 1–6-septate, rarely consisting only of
the phialide, 40–160 μm long; phialides monophialidic, cylindrical,
20–40 × 3–4 μm, 2–2.5 μm near the aperture.
Sporodochial conidiophores aggregated in pionnote sporodochia, irregularly
branched; phialides cylindrical, mostly widest near the base, 15–30
× 2.5–3.5 μm, 2–2.5 μm wide near the aperture.
and present on both types of
conidiophores. predominating, formed by both types of
conidiophores, predominantly (1–)3-septate, straight or sometimes
slightly curved, cylindrical, mostly with a visible, basal or slightly
laterally displaced hilum; 3-septate macroconidia,
(24–)35–40(–55) × (4.5–)5.5–6(–6.5)
μm (n = 116). sparsely produced on all media,
0–1-septate, ellipsoidal to subcylindrical to ovoid, more or less
straight, with a minutely or clearly visible lateral hilum; aseptate
subcylindrical to ellipsoidal microconidia, 5–15 × 2.5–4
μm; aseptate ovoid microconidia, 3–5 × 3–4 μm, formed
predominently on dense, penicillately branched conidiophores on CLA and twigs,
and then also without subcylindrical to ellipsoidal microconidia; occurring on
other media as a mixture with ovoid microconidia. formed in
heads on simple conidiophores, as hyaline masses on simple as well as complex
conidiophores. common, medium brown, ovoid to
ellipsoid, mostly in short, intercalary chains, 10–20 ×
10–17 μm.
: , heterothallic mating of
×
,
of ;
,
mating of × ; , mating of
×
;
,
mating of × ; , mating of
×
;
,
mating of × .
: , ,
coll./isol. C. Rego, ,
.
, from , coll./isol. P. Larignon, CBS
112591, 112610. , from , coll./isol.
F. Halleen (CBS 112596, 112602).
, ex-type
strain of .
: Colonies on PDA (surface and reverse)
cinnamon to sepia, with sparse aerial mycelium.
(surface and reverse).
temperature 20–25 °C, at which PDA colonies reach 30–42 mm
diam after 7 d in the dark; maximum temperature between 30–35 °C.
Yellow pigmentation not observed.
: (France,
Portugal, New Zealand, South Africa), sp. (The Netherlands),
(U.S.A., California).
: Typically isolated from roots and rootstocks of
grapevines, causing black foot disease.
in California, where it caused root rot,
while another was associated with bulb rot of a sp.
Netherlands.
: .
Species of Wollenw.
and regarded to be saprobes or weak pathogens of a wide range of herbaceous
and woody plants ().
grapevine in France in 1961, it has been recognised as a pathogen of
grapevines cultivated in various countries of different continents
(,
,
,
,
).
recent taxonomic study revising spp.
grapevines, the primary organism causing the black foot disease was identified
as (). Halleen .
() also described a
species now known as .
is likely that strains pathogenic to grapevine roots but in older literature
identified as
(, ) belong to this species.
-like species were removed from
and classified as
().
on grapevines, proved to be the species most commonly
isolated from diseased vines, and could possibly be more important than the
other pathogens in this disease complex.
underway, however, to investigate this aspect.
from the “” clade. Oliveira .
() inoculated rooted
cuttings of the grapevine cultivar Seara Nova by dipping the roots in a spore
suspension of “” (Cy1 =
).
Typical black foot disease symptoms were observed within 60 d.
were obtained when rooted cuttings of `99Richter' rootstock were inoculated
with 12 “” isolates, two of which were Cy
68 () and
Cy 76 ().
Inoculation significantly reduced plant height and the number of roots, whilst
isolate
was considered to be one of the most virulent isolates evaluated
().
Inoculation of 6-mo-old potted grapevine rootstocks (cv.
resulted
in death of 27.5 % of the plants 60 d after inoculation, whilst the remaining
plants suffered a dramatic reduction in root and shoot mass
().
tulip poplar () in California.
appeared severely stunted and the root systems were covered with black, dry,
scabby lesions that completely girdled or rotted off distal portions of some
roots ().
() reported that does not form microconidia.
be part of the -complex.
contrasting those of MacDonald & Butler
() because the ex-type
strain ()
did form microconidia; also, sequences of
were
identical to other isolates from vines (formerly identified as ) that also formed microconidia in culture.
originally described Wollenw. was originally described from rotting bulbs of collected in Sweden, of which an ex-type culture was available
for study () ().
() based on a North
American neotype from Kentucky, collected on (CUP
11985), for which there is no culture available.
. () showed
that there was more than one -like species on
.
the ex-type strains of or is a name available for the grapevine
pathogen, which clusters in its own well supported clade, for which the name
is introduced to accommodate its teleomorph.
clades in this complex, and additional sequence data of other loci need to be
generated.
future studies. |
Rooibos () is a leguminous shrub that is
indigenous to the Western Cape Province of South Africa, and used for the
production of rooibos tea.
Clanwilliam area was first observed in 1977 and officially reported in the
scientific literature by Smit & Knox-Davies
(,
), who identified the causal
organism as (Desm.) Sacc. [teleomorph:
(Cooke & Ellis) Sacc.].
rooibos was originally reported, it has developed into a disease of
considerable economic importance, affecting up to 89 % of plants in 3-yr-old
plantations (Lamprecht ., unpubl. data).
The genus (Sacc.) Bubák contains a large number
of cosmopolitan plant pathogens, many of which incite blights, cankers,
die-backs, rots, spots, and wilts in a wide assortment of plants of economic
importance (,
).
diseases usually manifest themselves in the production of
characteristic symptoms, some of which can culminate in the death of the host
plant ().
symptoms manifest themselves as a die-back of harvested branches, with
pycnidia forming on dead tissue, and a characteristic internal discoloration
of infected branches.
which perithecia form just below the soil surface
().
,
), preliminary surveys and
pathogenicity studies revealed the isolates associated with
the disease to be highly variable with regards to morphology and virulence,
indicating the possible existence of more than one species.
sustainable die-back management programme for the rooibos industry, it was
necessary to determine which species were involved in this disease complex and
which of these were the most important pathogens.
was to characterise the spp.
die-back symptoms of rooibos bushes.
data of the ITS region and partial translation elongation factor-1 alpha (TEF1
or EF1-α) gene and analysing these data with morphological and cultural
observations.
various species identified and to determine which of these were the most
virulent pathogens involved with the die-back disease of
.
ranging from Citrusdal in the south to Nieuwoudtville in the north.
were made from surface-disinfected host tissue onto Petri dishes containing 2
% potato-dextrose agar (PDA; Difco, Becton Dickinson, Sparks, MD, U.S.A.).
morphological groups recognised on PDA were selected for further molecular
characterisation.
they were isolated, are listed in .
Schimmelcultures in Utrecht, the Netherlands (CBS).
& Taylor (). PCR
amplification and sequencing of the ITS rDNA, as well as partial EF1-α
gene introns and exons, were performed as described by Van Niekerk ().
generated sequences have been deposited in GenBank
() and the alignment in
TreeBASE (S1506, M2708).
Sequences were manually aligned using Sequence Alignment Editor v. 2.0a11
().
sequences were obtained from GenBank and added to the alignment.
phylogenetic trees, downloaded sequences are labelled with GenBank accession
numbers; newly generated sequences are indicated with strain numbers.
datasets were created and analysed using PAUP v. 4.0b10
() as described
by Van Niekerk .
().
Strains were grown under continuous near-ultraviolet light (400–315
nm) (Sylvania Blacklight-Blue, Osram Nederland B.V., Alphen aan den Rijn, The
Netherlands) at 25 °C.
pieces of autoclaved twigs using 9 cm diam Petri dishes.
Growth rates and colony diameters of cultures incubated in darkness were
measured on PDA.
at × 1000 magnification were made of each structure.
levels were determined, and the extremes of spore measurements given in
parentheses. Images were taken from slides mounted in lactic acid.
characters of colonies were described after 14 d using the colour charts of
Rayner ().
pathogenicity trials.
pots in a pasteurised sand: soil: perlite medium (1: 1: 1) (3 plants per pot).
Plants were maintained in a glasshouse at 25 °C (night) and 30 °C
(day) temperature, and watered three times a week. Nitrosol (Fleuron)
(Universal selected services, Braamfontein, S.A.) fertiliser was applied every
second week at 200 mL/pot.
respective isolates were used to inoculate plants (three pots per isolate,
with three plants per pot). Plant stems were trimmed to a uniform length of 20
cm.
and an agar plug inserted into the cut, and sealed with Parafilm.
after inoculation, plants were evaluated for disease symptoms, and lesion
length measured. Survival of plants was also recorded.
from plant material with disease symptoms onto PDA amended with 0.02 %
novostreptomycin.
these data using SAS statistical software v. 6.08 (SAS Institute, Cary, NC).
The Shapiro-Wilk test was performed to test for normality
().
There was no evidence against normality and the original data were analysed.
Student's t-least significant differences were calculated at the 5 % level to
compare ranked means.
Approximately 510 and 340 bases were sequenced for ITS and EF1-α,
respectively.
which ITS sequences could be downloaded from GenBank, a tree was generated for
all of them using only ITS sequences ().
incongruence between the two genes (P = 0.7630) and the two genes were
combined into a single alignment for isolates with both gene sequences
().
outgroups did not change the clades presented in Figs
,
; nor did analyses using 1000
random taxon additions change the number of trees found or the scores
calculated (data not shown).
The ITS data matrix contains 78 taxa (including the outgroup) and 353
positions including alignment gaps (the sequence of the 5.8S rDNA gene of
strain FAU458 was not available on GenBank and
this region was therefore excluded from the analysis).
the and species were variable in this
excluded region.
variable and parsimony-uninformative, and 113 are constant.
analysis using substitution models representative of three different
assumptions (uncorrected “p”, Kimura-2-parameter and HKY85) on the
sequence data yielded trees with similar topology and bootstrap values.
Parsimony analysis of the alignment yielded 176 equally parsimonious trees
(; TL = 698 steps; CI =
0.626; RI = 0.896; RC = 0.561), most of which differed only in the order of
taxa within terminal clades.
parsimony and neighbour-joining trees, although the order of branching was not
always congruent (data not shown).
are present in six clades ().
Curzi, containing sequences from the ex-type strain
.
second clade (98 % bootstrap support) is identified as
sp. nov.
from .
isolates are described as
sp. nov., and sp. 9, with bootstrap support values of 100 %
and 88 %, respectively.
isolates group together with the isolate in the sp. 9 clade. isolates in the final two clades belong to (containing GenBank sequence AF230767 of the ex-epitype; 100 %
bootstrap support) and a sp. (100 % bootstrap support).
The Desm.
sequences of (Nitschke) Sacc.
GenBank.
The combined data matrix contains 31 taxa (including the outgroup) with 755
positions including alignment gaps.
parsimony-informative, 142 are variable and parsimony-uninformative, and 175
are constant.
parsimonious trees (TL = 1196 steps; CI = 0.823; RI = 0.931; RC = 0.766), one
of which is shown in .
The same six species that were identified in the ITS tree
() were found in the
analysis of the combined dataset.
available, the bootstrap support for the species is 100 %.
analysis using three substitution models (uncorrected “p”, Kimura
2-parameter and HKY85) on the sequence data yielded trees with similar
topology and bootstrap values, except for the position of the
Desm. sp.
sp. nov.
support) and HKY85 (59 % bootstrap support) analyses (data not shown).
trees obtained using neighbour-joining differed from those obtained using
parsimony only with respect to the branching of the deeper nodes, which are
not supported by bootstrap analysis (data not shown).
Nitschke, in Nitschke, : 311. 1867. . : (Sacc.) Traverso,
Fl. Ital. Cryptog., Pars 1: Fungi. 2: 266. 1906.
globose, solitary to aggregated, up to 500 μm diam.
dark brown to black, subcylindrical, smooth,
tapering towards the apex, up to 1000 μm long, 250 μm wide at the base,
70 μm wide at the apex; ostiole red-brown, obtusely rounded.
unitunicate, cylindrical–clavate with a refractive apical ring,
8-spored, biseriate, 50–60(–65) × 7–8(–9) μm.
constricted at the septa, unbranched, tapering towards the
apex with a rounded tip, extending above the asci, up to 150 μm long, and
up to 7 μm wide at the base, and 3–4 μm wide at the apex.
hyaline, smooth, fusoid–ellipsoidal, widest just
above the septum, tapering towards both ends, medianly septate, constricted at
the septum at maturity, with 1–2 guttules per cell,
(12–)13–15 × (3–)3.5–4 μm.
formed on PDA and on twigs.
subcylindrical, branched below or unbranched, 0–1-septate, 15–45
× 2–3 μm. ellipsoidal, biguttulate, with
an obtuse apex, tapering to an obtuse or bluntly rounded base with a visible
scar, 6–7(–8) × 2(–3) μm, corresponding to the
dimensions reported for the anamorph
().
not seen.
= CPC
2657; cultures homothallic.
: Colonies on OA olivaceous-black,
spreading with patches of white, with sparse aerial mycelium and cream
conidial masses; colonies on PDA flat, spreading, with sparse, white, dense
aerial mycelium; surface with solid patches of olivaceous-black in the central
part; outer region dirty-white to cream; aerial mycelium sparse, consisting of
a dense layer of dirty white to cream mycelium; reverse with solid, iron-grey
patches in the central part, also with isolated patches in the outer region,
surrounded by cream areas.
: , Nordrhein-Westfalen, Landkreis
Unna, on , Th. Nitschke, Aug. 1866, in
B. , on , S.
,
culture ex-epitype
(=
AF230767).
: As shown in the present study, the host range of is wider than originally suspected by Nitschke, but not as
extreme as stated by Wehmeyer.
containing perithecia of a sp. and , and some remnants of .
were observed, 12–15 × 3.5–4 μm, that were constricted at
the median septum, and guttulate. was originally
described from cankers on pear in Germany (Nitschke 1867), and the name was
subsequently used for the organism causing cankers on apples, pears and plums
in South Africa ().
.
.
: Named after , on which it causes a
prominent die-back disease.
globose, solitary, scattered to aggregated, up to 500
μm wide. black, cylindrical, mostly smooth, but
tapering near the apex, up to 800–1000(–2000) μm long, 150
μm wide at the base, 90 μm wide at the apex; ostiole widening once
spores discharge, 90–130 μm wide. unitunicate,
cylindrical with a refractive apical ring, 8-spored, biseriate,
52–55(–60) × 7–8(–10) μm.
septate, unbranched, tapering towards the apex with a rounded tip, extending
above the asci, up to 110 μm long, and up to 8 μm wide.
hyaline, smooth, fusoid, widest at the septum, tapering
towards both ends, medianly septate, not constricted at the septum, with
1–2 guttules per cell, (12–)13–15(–16) ×
3(–3.5) μm. formed on PDA and on
twigs. biguttulate, fusoid with
obtuse ends, (6–)7–8(–9) × (2–)2.5(–3)
μm. and absent.
;
cultures homothallic.
: On OA flat, spreading with sparse to no
aerial mycelium; surface with irregular patches of pale white to cream and
olivaceous-grey, with sparse strands of pale white aerial mycelium; on PDA
flat, spreading, with sparse to no aerial mycelium; surface smoke-grey;
reverse smoke-grey to olivaceous-grey.
: , Western Cape Province,
Clanwilliam, Langebergpunt, on , J.
Rensburg, , culture
.
: Isolates (see ) readily produce perithecia on PDA and on
twigs. var.
described as causing soybean stem canker in the South-eastern U.S.A.
(Fernández & Hanlin 1996), is not closely related to as earlier expected.
species clusters apart from the reference strain of
(Figs ,
). var. is also the main causal organism
of canker and die-back of rooibos, and not as reported
earlier (Smit & Knox-Davies
,
). The name Sacc.
similar to Nitschke (Wehmeyer 1933), and hence a new name,
is proposed here for the species pathogenic to
and soybean.
.
.
: Named after the primary use of the host substrate,
which is to make “rooibos” tea.
conical, convulated to unilocular, singly ostiolate, up to 400 μm wide;
pycnidial wall consisting of brown, thick-walled cells of ; conidial mass globose, pale-luteous to cream.
cylindrical, noticeably flexuous and tall,
well-developed, branched above or below, 1–3-septate, 30–80
× 3–5 μm. straight to curved,
tapering slightly towards the apex, collarettes slightly flaring, up to 3
μm long, with minute periclinal thickening, 10–35 × 1.5–2
μm. fusoid–ellipsoidal, apex bluntly rounded,
base obtuse to subtruncate, bi- to multiguttulate,
(10–)12–13(–14) × (3–)4(–5) μm;
and not observed.
.
: Colonies on OA flat, spreading, with
sparse, dirty white aerial mycelium; surface with irregular patches of
olivaceous-black and pale olivaceous-grey; on PDA flat, spreading, with sparse
aerial mycelium; surface smoke-grey to pale olivaceous-grey; reverse
smoke-grey.
: , Western Cape Province,
Clanwilliam, Kossakse werf, on , J.
Rensburg, , culture
.
: Colonies on OA flat, spreading with
sparse dirty-white aerial mycelium; surface and reverse with diffuse patches
of fuscous-black and dirty-white; colonies on PDA flat, spreading, with
sparse, dirty-white aerial mycelium at the edge of the dish; surface and
reverse having a translucent to ochreous central part; outer region umber.
Description based on .
: When CPC 5417 was deposited in the CBS collection as
117165, it was sterile, and thus could not be named in the present study.
Connecting to the numbering system used by Van Niekerk
(), it is thus referred to
as sp. 9.
strains isolated from , and grouped together with
this isolate, proving that this species may have a wider host range than just
.
of , which proved to be the most virulent species.
Significantly shorter lesions were observed for isolates of the other taxa
tested (P = 0.05) ().
Three months after inoculation, 95.56 % of plants inoculated with isolates
belonging to were dead, followed by 25.71 % of plants
inoculated with , and 16.67 % of plants inoculated with
the sp. Only 14.14 % of plants inoculated with isolates of
died.
sp. 9, proved to be the least virulent, and in both cases
only 8.33 % of the inoculated plants died.
successfully re-isolated from inoculated plants, which in many cases ended up
with dead tissue being covered in pycnidia and perithecia.
controls died, or showed any disease symptoms.
alone (Smit & Knox-Davies
,
), the current study has
revealed that up to five spp.
rooibos bushes, while a sixth -like taxon proved to be
better accommodated in , which clustered among
teleomorphs.
Mostert .
() isolated a species of
from grapevines, which also proved to be present on
and in countries such as Australia, Portugal
and South Africa.
eventually referred to as Taxon 3.
the study on grapevines by Van Niekerk .
(), where 15 species were
distinguished, and taxon 3 was referred to as sp. 1.
current study we finally managed to identify this species, as its ITS DNA
sequence is identical to that of the ex-type strain of
Curzi (),
which was originally described from in Italy
().
obviously has a wide host range and distribution, which once again underlines
the difficulties mycologists encounter when trying to identify species of
.
proved to be identical to var.
based on morphology and sequence data.
identified as by Smit & Knox-Davies
(,
), the reference strain
available to us of (Figs
,
) (treated as authentic by
F.A.
pathogen. Furthermore, as var.
is clearly not a variety of , and
as the name is already preoccupied, a new name is
proposed for this pathogen as .
matched GenBank sequences for
was originally described from branches of from
Germany, and was later associated with a canker of apple,
pear and plum rootstocks in South Africa
().
To reduce any further confusion surrounding this name, we have thus chosen to
designate an epitype specimen and ex-epitype culture in the present study,
from which DNA sequence data are derived.
Two new spp.
study, namely , and sp. 9.
culture of the latter proved to be sterile, further collections are required
before its taxonomy can be resolved.
Several isolates of a sp.
during the present study.
an anamorph of and Tul. & C. Tul.
this taxon grouped with spp.
data, no teleomorph was ever observed on host material or induced in
culture.
inoculated plants.
twigs, hyphae of spp.
().
Movement through the stem cortex takes place through the intercellular spaces,
and intracellularly through the parenchyma of the outer cortex.
cortex has been completely colonised, does the fungus invade the vascular
tissue and pith (). infection of sunflower follows the same
pattern where, after penetration in the host, infection hyphae invade the
intercellular spaces in the cortex
().
the phloem and parenchyma tissues which disintegrate completely
().
() suggested that the
formation of vast numbers of tyloses inside the xylem in advance of infection
together with phloem plugging due to gums and hyphae, causes the lesions
associated with die-back.
().
various conditions of stress and during invasion by a pathogen
().
these structures is an attempt by the plant to close off invaded cells to
limit fungal movement in the plant
().
flow of water from the roots upward.
toxins often result in external symptoms
().
reduction in biomass and eventually plant death.
preliminary, and need to be confirmed in the field, they clearly show that
was the most virulent taxon, producing the longest stem
lesions and also causing the most plant death.
sp. hardly caused any tissue discoloration.
could be due to the fact that symptoms were rated after 3 mo, and that species
from the complex, as observed in grapevines,
generally take much longer for symptom expression.
necessary, however, to fully resolve the phylogenetic status of the various
other spp. associated with die-back of ,
which in this study appeared to be of less importance than . |
Conifer-infesting bark beetles (Coleoptera: Scolytinae) are economically
important forest insects.
attack healthy living trees and have caused significant economic losses to the
global forestry industry ().
species, , and
infest various spp.
().
generally considered as secondary pests, although may
undergo maturation feeding on healthy living seedlings causing significant
losses during plantation establishment
().
species (, , ).
morphologically similar fungi, adapted for insect dispersal.
ophiostomatoid fungi are important pathogens of conifers
(,
, ), while many others can cause sapstain on logs and freshly
cut wood ().
& Halst., G.J. Marais & M.J. Wingf.,
P. Karst. and C.D. Viljoen, M.J.
Wingf. & K.
(, ), and Syd. & P. Syd.,
Goid. and H.P. Upadhyay &
W.B. Kendr., with their J.L. Crane & Schokn.,
Lagerb. & Melin, Hektoen &
C.F. Perkins and H.P. Upadhyay & W.B. Kendr.
anamorphs in the
().
(), of which at least 12
are associated with the three exotic pine-infesting bark beetle species in the
country ().
galleries and identified based on their morphological characteristics
().
Eight of these species belong to the genus (
)
or its anamorphs.
observed remained to be identified to species level
().
The aim of this study was to use DNA sequence comparisons to confirm the
identities of the spp.
()
from South African pine bark beetles, previously identified based only on
morphology ().
fungi associated with the three pine-infesting bark beetle species in South
Africa ().
Pretoria, Pretoria, South Africa.
deposited with the Centraalbureau voor Schimmelcultures (CBS), Utrecht,
Netherlands.
Biolab malt extract, 20 g Biolab agar, and 1000 mL deionised water).
extracted using PrepMan Ultra Sample reagent (Applied Biosystems) as described
by Aghayeva .
().
transcribed spacer) region of the ribosomal RNA operon was amplified using
primers ITS1-F () and ITS4 (). PCR products were sequenced with the same primers.
Conditions for PCR amplification and sequencing reactions were as described by
Zhou . ().
For comparisons, ITS sequences of closely related taxa
() were obtained from
GenBank.
All sequences were aligned using MAFFT v. 5.667
().
Phylogenetic relationships among the isolates were determined using distance
analyses in MEGA3
().
Trees were constructed using the Neighbour-joining tree-building algorithm
() and
rooted using GenBank sequences of M. J.
Wingf. & K. Jacobs (AY649782 and AY649783).
().
clade separate from the other isolates, all of which grouped with known taxa.
For these three isolates, part of the β-tubulin gene was amplified using
primers Bt2a and Bt2b ().
analyses were done separately, followed by a distance analysis of the combined
data set. A partition homogeneity test was performed in PAUP v. 4.0b8
(Phylogenetic Analyses Using Parsimony)
() to determine
the congruence of the two data sets.
phylogenetic clade of unknown identity were grown on 2 % WA (20 g Biolab agar
and 1000 mL deionised water) with sterilised pine twigs, and on 1.5 % oatmeal
agar (15 g oats powder, 20 g Biolab agar and 1000 mL deionised water) to
induce production of perithecia.
isolates (CMW 19362 and CMW 19363) on oatmeal agar.
made for each structure, and the ranges and averages were computed.
structures were observed on 7-d-old slide cultures
(), mounted in
lactophenol.
in size.
identities of seven spp.
(). These included (Robak) Nannf., Marm. & Butin,
(Fr.) Syd. & P. Syd.,
(Hedgc.) Syd. & P. Syd., (Georgev.) Nannf., Math.-Käärik, and
(Math.-Käärik) G. Okada & Seifert. The identity of (Rumbold) Nannf. (also included in the study) had previously been
confirmed based on DNA sequence comparisons
().
β-tubulin gene were amplified for the three unidentified isolates (CMW
19362, CMW 19363, and CMW 19364). The β-tubulin region included intron 5,
but no intron 4 was present. This corresponds with species in the -complex ().
species in this complex were thus selected for further phylogenetic analyses,
with as outgroup. spp.
outside the -complex were not included in these
analyses because of the presence of intron 4, but no intron 5
().
The partition homogeneity test ( = 0.003) confirmed that the ITS and
β-tubulin data sets were congruent.
data set showed that the three unidentified isolates grouped together with a
bootstrap support of 100 % (), and that they either represented an undescribed species or a
species for which no sequence data are available.
morphologically similar to each other and different from any other described
species.
anamorph in culture with swollen clavate conidia.
ascomata with allantoid rounded ascospores.
β-tubulin gene, as well as morphology, we conclude the three isolates
from infesting pines in South Africa represent an
undescribed taxon. This is described as follows:
X.D. Zhou & M.J. Wingf., MycoBank
.
.
:
().
: The type locality of this species is in Mpumalanga
Province, South Africa.
place where the sun rises”.
dawn, so the specific epithet is an oblique reference to the type
locality.
Coloniae in agaro 1.5 % avenae in medio 45 mm diam aetate duarum hebdomadum
in 25 °C, laete hyalinae vel albae. Mycelium aerium adest.
superficialia vel subimmersa in agaro 1.5 % avenae.
globosae, obscurae, 130–220(–350) μm diam, hyphis laete griseis
65–150(–280) μm longis, 1.5–2.0 (–2.5) μm latis
ornatae.
12–15(–27) μm lata. Hyphae ostiolares adsunt.
hyalinae, non septatae, allantoideae, in sectione transversali rotundae,
2–3(–3.5) x 1–1.5(–2) μm.
12–60(–85) x 1.5–2(–2.5) μm, ad apicem incrassatum
denticulos acres perferentes; conidia hyalina, unicellularia, clavata vel
guttuliformia, 3–4.5(–8) x 1–1.5(–2.5) μm.
diam (), ornamented with
light grey hyphae, 65–150(–280) μm long,
1.5–2(–2.5) μm wide. brown to black,
smooth, 340–800 μm long, 35–42(–58) μm wide at
the base, 12–15(–27) μm at the apex
(). present ().
hyaline, aseptate, allantoid, round in side view,
2–3(–3.5) x 1–1.5(–2) μm
().
(), micronematous, mononematous, hyaline,
12–60(–85) x 1.5–2(–2.5) μm, sharp denticles
present in the swollen apical part.
() hyaline, single
1–celled, clavate to guttuliform, 3–4.5(–8) x
1–1.5(–2.5) μm.
: Colonies on 1.5 % oatmeal agar reaching
on average 45 mm diam in two weeks at 25 °C.
cotton–white. Aerial mycelium present.
on or partially immersed in 1.5 % oatmeal agar.
: and infested bark of
.
: Mpumalanga Province, South Africa.
: , Mpumalanga Province,
, Sep. 1999, X.D.
= CMW
19364.
spp.
beetles , and in South
Africa. These fungi are and .
South Africa.
an undescribed taxon, for which the name has been
provided.
associates of conifer timber. was first
described from attacked by a
sp. in Mexico (), and was considered as an intermediate between and (R.
(). is considered economically
important to the forestry industry, and a colourless mutant of this species
has been marketed as biocontrol agent against sapstaining fungi
(). was described from galleries of
infesting in Sweden
(), and the species has been reported from Australia,
California, Canada, and New Zealand
(, ).
similar to species in the -complex
(,
Aghayeva . ,
).
have typical orange-section-shaped ascospores and
anamorphs. can be distinguished from other species
in the complex by its very obviously rounded ascospores and swollen clavate
conidia.
applied in identification.
analyses of ITS and partial β-tubulin gene sequences confirmed that
resides in a phylogenetic clade, distinct from all
morphologically similar spp.
available.
spp.
conifer-infesting bark beetles accidentally introduced into South Africa.
also highlight the fact that the introduction of what might initially appear
to be a single organism (plant, insect, fungus) is often considerably more
complex.
specifically associated with the insects in their areas of origin and like
their insect vectors, they are also introduced exotics.
woody substrates in South Africa could have invaded the bark beetle niche.
relationships, as has recently been found with
(Linnaeus) and M.
States ().
complex and dynamic environment that deserves further study. |
Members of the genus De Not. ()
() and their
Morgan () anamorphs are commonly
associated with a wide range of plant disease symptoms
().
represents the second in a series assessing the taxonomy of species of
by integrating morphology with DNA sequence data and
sexual compatibility studies ().
pathogens from a wide range of hosts in most subtropical to tropical countries
(,
Crous . ,
,
,
,
,
).
study, we obtained numerous isolates of from baited
soils collected in tropical areas.
were obtained from a biotic complex including root rot fungi and
plant-parasitic nematodes associated with toppling disease of banana
().
(Bugn.) Boesew.
with stem lesions, root breakage and toppling disease (Risède &
Simoneau ,
).
study was to analyze all available strains with
clavate vesicles using morphology and DNA sequence analysis of their
β-tubulin and histone H3 gene regions in order to resolve the status of
species with clavate vesicles.
identify the sp.
banana.
in Crous ().
characteristics and morphology were determined on plates containing 2 % malt
extract agar (MEA) (20 g/L), and carnation leaf agar (CLA) [1 % water agar (10
g/L) with autoclaved carnation leaves placed onto the medium] in the other
().
Plates were incubated for 7 d at 25 °C under continuous near-UV light, to
promote sporulation.
using the isolation protocol of Lee & Taylor
().
amplified and sequenced as explained in Crous .
(), namely, part of the
β-tubulin gene, amplified with primers T1
() and CYLTUB1R (); and part of the histone H3 gene using
primers CYLH3F and CYLH3R ().
obtained from GenBank
()
and TreeBASE
()
and the alignment was assembled using Sequence Alignment Editor v. 2.0a11
() with manual
adjustments for improvement made visually where necessary.
(Bat., J.L. Bezerra & M.M.P. Herrera)
Boesew. and Crous, M.J. Wingf. &
Alfenas were added to the alignments as outgroups.
The phylogenetic analyses of sequence data were done using PAUP
(Phylogenetic Analysis Using Parsimony) v. 4.0b10
().
analysis of both datasets in PAUP consisted of distance (using the uncorrected
“p”, Jukes-Cantor and HKY85 substitution models) and parsimony
analysis as described in Crous .
().
deposited in GenBank ()
and the alignments in TreeBASE (S1508, M2711).
Morphological examinations were made from cultures sporulating on CLA.
Structures were mounted in lactic acid, and 30 measurements at × 1000
magnification were made of each structure.
determined, and the extremes of spore measurements given in parentheses.
Colony reverse colours were noted after 6 d on MEA at 25 °C in the dark,
using the colour charts of Rayner
() for comparison.
cultures studied are maintained in the culture collection of the
Centraalbureau voor Schimmelcultures (CBS), Utrecht, the Netherlands
().
isolates indicated in .
The manually adjusted alignment contained 123 isolates (including the two
outgroups) and 533 characters including alignment gaps.
220 were parsimony-informative, 60 were variable and parsimony-uninformative,
and 253 were constant.
models, as well as parsimony analysis, yielded trees in which the same clades
were supported.
“p” and HKY85 substitution models, the basal order of the clades
were different (data not shown).
657 most parsimonious trees (TL = 881 steps; CI = 0.529; RI = 0.853; RC =
0.451), one of which is shown in .
the Peerally and (Bugn.) C.
Booth clades.
clades, namely Crous & D.
support), (Linder & Whetzel) Crous (98 %), El-Gholl & Alfenas (100 %), Crous
& El-Gholl (100 %), Crous & M.R.A.
(100 %), (Petch.) Subram. (100 %), Crous (100 %), Crous & M.J.
Wingf. (88 %), Crous (100 %),
(Bugn.) Boesew. (91 %), F.A. Wolf (99 %), Crous (93 %), (Crous, M.J.
Wingf. & Alfenas) Crous (100 %), (96 %), Crous & M.J. Wingf. (100 %),
Gadgil & M. Dick (100 %) and (80 %).
represented by a single taxon were placed as unsupported sister taxa to the
other clades in the tree.
(Tubaki) Tubaki, which grouped with the clade with a
bootstrap support value of 89 %.
observed between some clades, for example the clades containing grouped with a bootstrap support value of
79 %.
isolates in .
manually adjusted alignment contained 115 isolates (including the two
outgroups), and for each taxon 425 characters including alignment gaps were
analysed.
and parsimony-uninformative, and 248 were constant.
using the three substitution models, as well as parsimony analysis, yielded
trees in which the same clades were supported.
Jukes-Cantor and HKY85 substitution models yielded trees with identical
topologies, but the tree obtained from the uncorrected “p” model
had rearrangements at the deep nodes when compared with the other two trees
(data not shown).
parsimonious trees (TL = 917 steps; CI = 0.382; RI = 0.868; RC = 0.331), one
of which is shown in .
All of these trees resulted from reordering of taxa within the clade.
species clustered together in well-supported clades
().
supported at the deeper nodes.
Alfieri, El-Gholl & E.L.
Mycotaxon 48: 206. 1993.
: Crous, Syst.
Appl. Microbiol.18: 248. 1995.
arrangement of fertile branches, a stipe extension, and a terminal vesicle;
stipe septate, pale brown at base, hyaline, smooth, septate, 60–260
× 5–7 μm; stipe extensions septate, straight to flexuous,
120–450 μm long, 3–4 μm wide at apical septum, terminating
in a narrowly clavate vesicle, 4–5 μm diam. 70–120 μm long, 25–60 μm wide; primary
branches aseptate or 1-septate, 30–65 × 4–6 μm; secondary
branches aseptate or 1-septate, 30–50 × 3–6 μm, tertiary
and quaternary branches aseptate, 15–30 × 3–5 μm, each
terminal branch producing 1–4 phialides; phialides elongate doliiform to
reniform, hyaline, aseptate, 10–20 × 4–5 μm, apex with
minute periclinal thickening and inconspicuous collarette.
cylindrical, rounded at both ends, straight, (55–)68–75(–95)
× (5–)6(–7) μm (av. = 70 × 6 μm), 1-septate (but
up to 5-septate at germination), lacking a visible abscission scar, held in
parallel cylindrical clusters by colourless slime (description based on
isolates obtained from ).
: Florida, Lake Placid, roots and
stems of , 5 Apr. 1978, C.P. Seymour & E.L.
Barnard, PREM 51721 of , P078-1543 =
ATCC 66389 = STE-U 2536 = culture ex-type, heterothallic mating with P078-1261 = STE-U
2537 = ,
Florida, Lee County, root debris in non-sterilized peat, 4 Mar. 1978, D.
Ferrin, Aug. 1989, N.E. El-Gholl, FLAS F55430, of . , sp., J.M. Risède &
Ph. Simoneau, Gua12 = CPC 11351, CPC 11352 =
, Gua9 =
.
, sp., J.M. Risède & Ph.
Mar11 = CPC 11349 = , Mar23 = CPC 11348 =
, Mar8 =
CPC 11347 = . , sp., SLU2 =
, SLU5 =
CPC 11350 = .
: See Crous
().
: spp., Guadeloupe,
Martinique, Saint Lucia; , and root debris in
peat U.S.A. (Florida) ().
: is known to have conidia
that are straight to curved, (44–)50–70(–80) ×
(4–)5–6 μm (av. = 65 × 5 μm) and 1(–3)-septate.
The isolates obtained from differ from the ex-type strains by
having conidia that are up to 7 μm wide.
the isolates to represent an undescribed taxon, they clustered
in the same clade as those of .
were able to mate, and since its original description, it has not proven
possible to reproduce perithecia of in
culture.
Crous & K.D.
.
,
.
: Named after the country from which it was
collected.
unknown. consisting of a stipe
bearing a penicillate arrangement of fertile branches, a stipe extension, and
a terminal vesicle; stipe septate, hyaline, smooth, 60–150 ×
6–7 μm; stipe extensions septate, straight to flexuous, 300–450
μm long, 2.5–3 μm wide at the apical septum, terminating in a
clavate vesicle, (3.5–)5(–6) μm diam. 40–80 μm long, and 40–60 μm wide; primary
branches aseptate or 1-septate, 15–30 × 5–7 μm; secondary
branches aseptate, 12–20 × 5–6 μm, tertiary and
additional branches (–6), aseptate, 10–15 × 5–6 μm,
each terminal branch producing 1–4 phialides; phialides cylindrical to
allantoid, hyaline, aseptate, 10–15 × 3.5–4.5 μm; apex
with minute periclinal thickening and inconspicuous collarette.
cylindrical, rounded at both ends, straight,
(48–)57–68(–75) × (6–)6.5(–7) μm (av. =
63 × 6.5 μm), (1–)3-septate, lacking a visible abscission scar,
held in parallel cylindrical clusters by colourless slime.
and unknown.
: , Queensland, Topaz, Atherton
Tablelands, , 2 Apr. 2001, C. Pearce & B.
Paulus, , culture ex-type
= CPC
4714.
: Colonies fast growing with abundant
white aerial mycelium; surface and reverse sienna (13i), with moderate numbers
of chlamydospores.
: .
: Australia.
: This species can be confused with taxa in the
Peerally species complex that form
3-septate conidia of similar dimensions, and yellow
perithecia.
(48–)57–68(–75) × (6–)6.5(–7) μm than
[(45–)60–70(–80) ×
(4–)5(–6) μm], and Crous
[(42–)52–58(–65) × (3.5–)4–5 μm].
Another species that needs to be compared to is
(Petch) Subram., which again has larger conidia
(65–)70–88(–96) × 5–6(–7) μm, and also
forms megaconidia and a teleomorph with red perithecia in
culture ().
Crous & M.J. Wingf., MycoBank
.
,
.
: Named after Ecuador, where it appears quite commonly in
soil.
unknown. consisting of a stipe
bearing a penicillate arrangement of fertile branches, a stipe extension, and
a terminal vesicle; stipe septate, hyaline, smooth, 60–100 ×
5–7 μm; stipe extensions septate, straight to flexuous, 200–300
μm long, 2–3 μm wide at the apical septum, terminating in a
clavate vesicle, (3–)4(–5) μm diam. 30–100 μm long and wide; primary branches aseptate or
1-septate, 20–30 × 3–5 μm; secondary branches aseptate,
15–25 × 3–5 μm, tertiary branches aseptate, 12–17
× 3–5 μm, additional branches (–7), aseptate, 10–15
× 3–5 μm, each terminal branch producing 2–6 phialides;
phialides doliiform to reniform, hyaline, aseptate, 7–15 ×
3–4 μm; apex with minute periclinal thickening and inconspicuous
collarette. cylindrical, rounded at both ends, straight,
(45–)48–55(–65) × (4–)4.5(–5) μm (av. =
51 × 4.5 μm), 1(–3)-septate, lacking a visible abscission scar,
held in parallel cylindrical clusters by colourless slime.
and unknown.
: , soil, 20 Jun. 1997, M.J.
Wingfield, , culture ex-type
= CPC
1635; =
CPC 1628; = CPC 1648;
= CPC
1627; =
CPC 1657. , Belém, Cpatu, soil, 1996, P.W.
= CPC
1587.
: Colonies sienna on the surface, and
umber in reverse; chlamydospores extensive, dense, occurring throughout the
medium, forming microsclerotia, with moderate to extensive sporulation on the
aerial mycelium.
: Soil.
: ?Brazil, Ecuador.
: When first isolated, isolates of
were observed to also form a few conidia that were 3-septate when studied on
CLA.
1-septate conidia.
obtained from Brazil ().
obtained from Ecuador, its conidia are somewhat shorter (av. 44 μm) than
those from Ecuador (av. 51 μm), and it might very well end up representing
a cryptic species closely related to .
the (Bugn.) Boesew. species complex.
(45–)48–55(–65) × (4–)4.5(–5) μm (av. =
51 × 4.5 μm), thus longer and wider than those of Crous & G.R.A. Mchau [(35–)40–48(–60)
× 4–5(–6) μm (av. = 45 × 4.5 μm)], narrower than
those of [(38–)40–55(–65) ×
(3.5–)4–5(–6) μm (av. = 53 × 4.5 μm)], and
shorter than those of Crous
[(44–)50–70(–80) × (4–)5–6 μm (av. = 65
× 5 μm)].
treated as representative of Crous, which has
conidia of similar dimensions of [(40–)53–58(–65) ×
(3.5–)4–5 μm (av. = 56 × 4.5 μm)]and are
1(–3)-septate. can be distinguished
from based on its lower average conidial length, and
the absence of a state in culture
().
spp. with clavate vesicles (Crous .
,
,
,
,
,
).
focusing on taxa with sphaeropedunculate vesicles, Crous .
() described nine new
species from the species complex.
expected, only two new species could be resolved from all
strains with clavate vesicles available to us.
of the reason for this could be that the this complex has been studied in more
detail than that with sphaeropedunculate vesicles, but also that
teleomorphs are more common among species with
sphaeropedunculate vesicles than those with clavate vesicles, causing more
variation. Some taxa, for instance and proved to be quite variable for the loci sequenced.
we believe that it is currently premature to split these species into more
taxa, and that additional isolates and more loci will have to be investigated
to fully resolve their status.
F.A.
disease of banana (Risède & Simoneau
,
).
expected these strains to represent an undescribed species, we were surprised
to find that both β-tubulin and histone H3 datasets placed them in
(teleomorph ).
unexpected, as conidia of are
[(44–)50–70(–80) × (4–)5–6 μm (av. 65
× 5 μm)] (),
while those of the banana isolates are [(55–)68–75(–95)
× (5–)6–7 μm (av. 70 × 6 μm)].
this discrepancy, these isolates clustered together in a clade (100 % support)
in both data sets, suggesting that the original ex-type strains, which have
narrower, slightly curved conidia, might be atypical for what is commonly seen
in this species.
redo the crosses between the two mating testers of , or
to mate them with the newly collected isolates from banana, none of the
matings proved successful.
be attributed to , and that the latter species is
morphologically more variable than originally expected
().
another species to the // complex.
however, that there are yet more Australian species awaiting description, as
,
isolated from in Queensland
(), also clustered apart
from any known taxon.
60–90 × 5–6 μm.
strain sporulated rather poorly, making it difficult to determine its range of
morphological variation on CLA.
treated under the name .
overlap in general conidial dimensions, this is not surprising, as these two
species are rather similar, and can only be distinguished once the mean
conidial dimensions have been determined.
, which
again has smaller conidia than both and , suggests that there may be yet more cryptic taxa within
this complex that need to be resolved. |
Considerable confusion has surrounded the taxonomy of the so-called
ophiostomatoid fungi ever since the first descriptions of
Syd. & P. Syd. and Ellis & Halst.
().
these fungi are specifically adapted for dispersal by insects, they resemble
each other morphologically and they typically share similar niches linked to
their biological characteristics.
phylogenetic studies based on DNA sequence comparisons have been applied to
these fungi and they confirmed suggestions
(,
,
,
, 1984) that
the two keystone genera, and , have
polyphyletic origins (Hausner
,
,,
).
Went anamorphs and endoconidia arising from ring
wall-building conidium development (), clearly reside in in
the order Luttrell Benny & Kimbr.
(, , ).
containing rhamnose and cellulose in their cell walls, and with anamorphs
residing in Hektoen & C.F.
H.P. Upadhyay & W.B. Kendr.,
Lagerb. & Melin, or J.L.
& Schokn. emend. G.
the Benny & Kimbr.
(, ).
results in more than 140 species, exhibiting a large
variety of distinct teleomorph and anamorph features.
include the shape and size of the ascomata and ascospores, and the presence or
absence of sheaths surrounding the ascospores.
spp.
of sticky ascospores adapted for dispersal by insects
(,
,
).
In 1957, Parker described the genus A.K.
species that exhibits all the characters of but with
ascocarps cleistothecial, lacking necks and ostioles
().
three additional species were described in
().
() because the formation or length of necks, and the presence
of an ostiole, might be affected by the environment and were considered `less
reliable' taxonomic characters ().
species are closely related to spp.
anamorphs (, ).
spp. have ascospores with unusual shapes.
studies have applied this characteristic to define groups within the genus,
which at the time of these studies was treated as a synonym of
(, , , ).
short perithecial necks, Upadhyay & Kendrick
() established
H.P. Upadhyay & W.B. Kendr.
() argued that ascospore
shape should not be the sole character to delineate genera, and that it was
illogical to maintain as a separate genus because
contained many species with a variety of other, distinct
ascospore forms.
treated as a synonym of and that ascospore morphology
should only be one of several characteristics on which to base further
subdivisions in the genus. Hausner .
() proceeded to formally
reduce to synonymy with , based
on partial SSU and LSU rDNA sequences. These authors included 10
spp., but only one ( (Rumbold) Nannf.) and one ( Ellis & Halst.) species in the phylogenetic analysis of the
data.
ascospore morphology should not be used as the only character for taxonomic
grouping as similar ascospore shapes often originated more than once in a
genus (, ).
established anamorph morphology as a preferred characteristic to group species
in the genus (,
,
,
,
).
significant number of spp.
combinations of up to three of the four possible anamorph states associated
with the genus (,
).
for example, has a continuum of synanamorph states,
which based on current definitions range from
like and -like to
().
classified in (), Preuss
(),
Corda
(),
(),
(),
H.P. Upadhyay & W.B. Kendr.
(),
Link: Fr. (), and
().
accommodate spp.
Goidánich ()
established Goid.
anamorphs.
invalidly, without a Latin description
().
Goidánich validated the genus and at the same time corrected the
spelling to
().
Siemaszko () reduced
to synonymy with on the basis of
teleomorph morphology. has been treated in all subsequent
studies as synonym of either
(,
,
,
,
,
)
or (, ,
,
,
,
).
studies have placed three of the original four species,
Goid., (Grosmann) Goid. and (Rumbold) Goid., in
(,
,
,
2005). The fourth species, (Münch) Goid., has been
treated as a synonym of (Hedgc.) Syd. & P. Syd.
(,
,
,
,
) which, based on
phylogeny, also resides in
().
Amongst the four anamorph-genera associated with spp.,
appears to be the most common form, with conidia produced
sympodially on denticles arising from undifferentiated hyphae
().
the form that occurs most often as a synanamorph of spp.
(,
,
,
).
mononematous conidiophores and conidiogenous cells with prominent denticles,
thus the -like component of the anamorph
().
In a study showing that is phylogenetically distinct from
the synnematal anamorphs of spp., and where
was redefined to encompass all synnematous anamorphs of
, only the synnematous form was described
().
The form was thus treated as a distinct synanamorph of
(). However, Harrington .
() accepted the original
description of which included the -like
forms, but restricted the genus to anamorphs with affinities to the complex.
() also stated that the
synnemata of spp.
are loose aggregates of conidiophores, without the
fused stipe cells that are characteristic of the complex.
These synnematous species outside the complex often lack a
anamorph, although some species produce a mononematous
form without prominent denticles, resembling was described for the mononematous anamorphs of
and
(), where conidia are produced through sympodial proliferation,
leaving flat, ring-like scars on the surface of conidiogenous cells, as
opposed to the denticles visible in spp.
(,
).
Although these anamorph-genera can be defined broadly, the delimitation of
species groups based on anamorph morphology remains problematic, especially
because of intermediate and overlapping forms.
species within almost all the morphological groups (based on ascospores and
anamorphs) of the genus
(, , , , , , ).
employing ribosomal together with protein-coding genetic data have become the
norm.
that some of the morphological traits must be represented by monophyletic
lineages.
ribosomal DNA data have failed to support the definition of monophyletic
lineages in (Hausner
,
,
,
).
logically subdivided based on monophyly.
from domains 1 and 2 of the 5' end of the nuclear LSU gene, together with
partial sequences for the β-tubulin gene region.
representing all the ascospore forms and anamorph shapes
associated with the genus are included in the study.
(CBS), Utrecht, The Netherlands, as well as in the culture collection (CMW) of
the Forestry and Agricultural Biotechnology Institute (FABI) at the University
of Pretoria, South Africa. Cultures were grown on malt extract agar (MEA, 2 %
malt extract [Biolab, Merck] and 2 % agar [Biolab, Merck]) at 21–24
°C for DNA extraction.
suitability in the phylogenetic analysis used in this study.
three species of were selected as being the most
appropriate and these include:
(; LSU =
AF408338; β-tubulin = DQ246580),
(; LSU =
AF408339; β-tubulin = AY063478), and
(; LSU =
AF408341; β-tubulin = DQ120768).
by Castlebury
() in GenBank although we
recognize that Gryzenhout have shown that () is incorrectly identified and also represents .
method described by Aghayeva .
().
amplified for sequencing and phylogenetic analysis.
large subunit rDNA was amplified using the primers LR0R (5' ACCCGCTGAACTTAAGC
3') and LR5 (5' TCCTGAGGGAAACTTCG 3')
().
Part of the β-tubulin gene was amplified with primers T10 (5'
ACGATAGGTTCACCTCCAGAGAC 3') () or Bt2a (5' GGTAACCAAATCGGTGC GCTTTC 3') in
combination with Bt2b (5' GGTAACCAAATCGGTGCTGCTTTC 3')
().
Reaction volumes for the PCR amplification were 50 μL and contained 5 μL
10 × PCR reaction buffer (Super-Therm, JMR Holdings, U.S.A.), 2.5 mM
MgCl, 10 mM dNTP, 10 μM of each primer, 2 μL DNA and 2.5 U
Super-Therm Taq polymerase (JMR Holdings, U.S.A.).
amplification of both the LSU and β-tubulin genes included denaturing for
3 min at 94 °C, annealing at 47–52 °C for 1 min, and elongation
at 72 °C for 1 min.
elongation step at 72 °C for 5 min.
confirmed on a 1 % (w/v) agarose gel stained with ethidium bromide.
visualized under UV light. The PCR fragments were purified with QIAquick®
PCR purification kit (Quiagen®) eluting the DNA in water.
noted above and the Big Dye™ Terminator v. 3.0 cycle sequencing premix
kit (Applied Biosystems, Foster City, CA, U.S.A.).
on an ABI PRISIM™ 377 or ABI PRISIM™ 3100 Genetic Analyzer
(Applied Biosystems).
(Applied Biosystems) and aligned in CLUSTAL-X
() and then in T-Coffee
() using multiple alignment algorithms.
combine the alignment results of Clustal X with the local and global pairwise
alignments obtained in T-Coffee, to produce a multiple sequence alignment with
the best agreement of these methods.
used for the analysis.
PAUP v. 4.0b8 (Phylogenetic Analysis Using Parsimony)
() as follows:
for the analysis of the partial LSU gene, sequences were trimmed at the 5' and
3' ends to align with DNA sequences from GenBank used for the outgroups.
the partial β-tubulin gene the sequences were trimmed on the 5' end to
correspond with the beginning of exon 4 of the β-tubulin gene.
were carried out using parsimony, neighbour-joining and maximum likelihood
() and Bayesian
inference (MrBayes 3.0b4) ().
: For parsimony analysis, ambiguous and missing
nucleotides were eliminated and the remaining characters were weighted
according to the consistency index (CI).
tree-bisection-reconnection (TBR) branch swapping.
used to obtain a majority rule consensus tree.
estimated using Bootstrap analysis (1000 replicates) with the full consensus
option.
: Data were analysed using a Bayesian approach
based on a Markov chain Monte Carlo (MCMC) analysis.
(GTR+I+G) model as determined by AIC criteria of Modeltest
()
was used for the analysis.
invariable, while the rate of the remaining sites was drawn from a gamma
distribution with six categories. All parameters were inferred from the data.
Four Markov chains were initiated at random and the program was allowed to run
for 2000000 generations with a sample frequency of 100.
repeated six times and consensus trees obtained from the six independent
analyses were examined for consistency.
calculate a consensus tree with mean branch lengths.
convergence was determined and these sampled trees were discarded as burn in.
The following trees with their branch lengths were used to generate a
consensus tree based on 50 % majority rule with mean branch lengths and
posterior probabilities for the nodes using PAUP
().
: A distance tree was calculated using
Neighbour-joining analysis based on the evolutionary model that was determined
as GTR+I+G based on AIC criteria using the Modeltest 3.06
().
Distance settings were adjusted according to the Akaike information criteria
(AIC) model: proportion invariable sites were assumed to be 0.4369 and the
rates for variable sites were assumed to follow a gamma distribution with
shape parameter of 0.5593.
replicates.
the branch swap algorithm set to TBR (tree bisection reconnection).
: Likelihood settings were set according to
GTR+I+G model as determined by AIC criteria in Modeltest 3.06
().
Assumed proportion invariable sites were set to 0.4369.
were assumed to have a gamma distribution with a 0.5593 shape parameter.
search was performed heuristically with random stepwise addition and TBR
branch swapping.
(1000 replicates) determined by heuristic search and TBR branch swapping.
697–702 nucleotides.
LSU gene.
obtained from the partial β-tubulin gene. This region included exon 4,
exon 5 and the 5' end of exon 6, as well as intron 4 situated between exons 4
and 5, and intron 5 between exons 5 and 6.
were of equal length for all taxa studied.
variable in both nucleotide length and DNA sequence.
intron 4 or intron 5 or both introns.
(), accounted for the
large difference in β-tubulin sequence lengths.
: For the cladistic analysis of the combined data
set, 39 missing and ambiguous characters were excluded from the analysis.
characters were parsimony-uninformative and 239 characters were
parsimony-informative.
consistency index. This resulted in 751 characters with a weight of 1, and 144
characters with a weight other than 1.
obtained using maximum parsimony analysis with the tree bisection reconnection
(TBR) branch swapping algorithm.
trees, with slight variations in the topology of the terminal nodes.
four trees a 50 % majority rule consensus tree was compiled with the TBR
algorithm. The tree length was 383 steps, CI = 0.656, and the retention index
(RI) = 0.860.
().
The cladogram ()
showed that the taxa are grouped in distinct, well-supported clades.
states.
β-tubulin gene ().
Clade B (82 % bootstrap) consisted of several distinct groups.
described species J. Reid, Eyjólfsd. &
Hausner and J.
previously residing in the genus .
have short perithecial necks and falcate ascospores, and are sensitive to
cycloheximide. Two anamorph states are associated with taxa in this group.
They are , the anamorph of (J.R.
Bridges & T.J. Perry) Hausner, J.
associated with (R.W.
Davidson) Hausner, J.
(Olchowecki & J. Reid) Hausner, J.
and
Clade D () had a
relatively low bootstrap support of 66 %.
subdivided in numerous smaller clades with various levels of confidence
support.
anamorphs producing secondary conidia.
(Hedgc.) Syd. & P. Syd., Livingston & R.W.
Davidson, and (Hedgc. & R.W. Davidson) Hendr.
have naked (no sheath) reniform ascospores, and J.
& Hausner has naked, narrowly clavate ascospores.
bootstrap support (60 %) and consists of two subclades (G and H).
G, X.D. Zhou & M.J. Wingf., and (Rumbold) Arx, formed one well-supported group (Clade J, 98 %
bootstrap support).
sheath and a continuum of anamorphs including and
.
defined and consisted of members of the complex with
anamorphs and (Fr.) Syd. & P. Syd.
Other species in this clade were H. Solheim and (Butin) de Hoog & R.J. Scheff.
anamorphs, and (Davidson) de Hoog & R.J. Scheff.,
H.
anamorphs.
lacked intron 5 in the β-tubulin gene
().
anamorphs and ascospores varying from orange section to allantoid in shape.
The species in this clade all lacked intron 4 and had intron 5 in the
β-tubulin gene ().
In this clade, (R.W.
separately from the other taxa that formed a clade with 98 % bootstrap
support.
& C.F. Perkins, the type species for the anamorph-genus (Robak) Nannf., de Hoog, D.N. Aghayeva & M.J. Wingf. and D.N.
Aghayeva & M.J. Wingf.
within infructescences of spp. in South Africa, G.J. Marias & M.J. Wingf., G.J.
Marias & M.J. Wingf., and G.J. Marias & M.J.
Wingf., constituted a well-defined, smaller clade with strong bootstrap
support within Clade H.
: Consistent results were obtained in the six
runs of the Bayesian phylogenetic analysis (Model GTR+I+G).
the obtained trees differed only slightly in the terminal nodes where low
confidence values were obtained.
nodes supported by high confidence values.
chains was observed after 33000 generations.
(representing 200000 generations) were thus discarded and 18000 trees were
included to calculate the 50 % rule consensus tree for each run.
phylogenetic trees obtained is presented in
.
confidence values (posterior probabilities) are indicated above the relevant
nodes where support exceeded 50 %.
: Phylogenetic distance was determined by
Neighbour-joining (NJ) analyses based on the general time reversal model.
Statistical support for the nodes was calculated using 1000 NJ bootstrap
repeats.
().
from NJ is similar to that obtained from Bayesian inference.
exception of group E, clustering basal to group J and I closest to group G and
not basal to groupings G and H or within group F as observed on MP and
Bayesian analysis respectively.
: For the phylogenetic relationship estimated
using maximum likelihood (ML), the GTR+I+G evolutionary model determined by
Model Test based on Akaike Information Criteria (AIC) was applied.
proportion invariable sites (I) was set to 0.4369 and the shape parameter for
gamma distribution (G) was set to 0.5595 and no molecular clock was enforced
on the data set. Bootstrap values for the groupings were determined by 1000
bootstrap repeats.
indicated in italics ()
on the phylogenetic tree obtained by Bayesian inference. Groups A–C, E,
and H–J were supported by ML.
these groups differs significantly from MP and Bayesian inference. Groupings D
and G were not supported and group B had poor ML statistical support.
support for the hypothesis that the genus includes at
least three monophyletic lineages.
with a combination of both anamorph and teleomorph characters.
characters have also previously been recognised as taxonomically informative
and have been employed to define the two genera, and
.
clearly defined morphological characters, we re-instate these genera with
emended descriptions and establish the necessary new combinations.
description of the genus is emended to reflect these
taxonomic changes.
Syd. & P. Syd., Ann. Mycol. 17: 43. 1919.
emend. Z.W. de Beer, Zipfel & M.J. Wingf.
straight or flexuous, cylindrical, brown to black; ostiole often surrounded by
ostiolar hyphae. 8-spored, evanescent, globose to broadly
clavate. hyaline, aseptate, cylindrical, lunate,
allantoid, reniform, orange section- or pillow-shaped, sometimes with a
hyaline, gelatinous sheath. most commonly
and/or occasionaly
-like, rarely -like.
Phylogenetically classified in the .
: Fr.: Fr. Syd.
& P. Syd., Ann. Mycol. 17: 43. 1919.
: Fr., Syst Mycol. 2: 472.
1822.
:
().
H.P. Upadhyay & W.B. Kendr., Mycologia
67: 799. 1975. emend. Z.W. de Beer, Zipfel & M.J. Wingf.
subglobose; necks relatively short, mostly tapered toward the apex, sometimes
surrounded by a collar-like structure; ostiolar hyphae convergent or lacking.
8-spored, evanescent, fusiform, clavate or ellipsoidal, hyaline.
hyaline, aseptate, elongate, falcate, or slender with
obtuse ends, sometimes with bulbous swelling, most often with a hyaline
sheath. Sensitive to cycloheximide. or
-like.
within a monopyletic lineage including
.
genus consists of at least three groups representing
separate genera.
characteristics, we have re-instated the teleomorph-genera
and The former genus now
incorporates 11 species including three new combinations, and the latter 27
species including 24 new combinations.
even though some monophyletic groups are evident in the
larger genus.
evidence to support these subgroups amongst the species retained in
we have chosen not to subdivide the genus further at the
present time.
taxa that have short ascomatal necks, produce falcate ascospores with sheaths
and have (occasionally -like)
anamorphs.
() established
to separate taxa having these distinct
characteristics from taxa residing in the aggregate genus
Our data revealed a strongly supported, monophyletic
lineage with central to it, and with morphological
characters consistent with the original description of
.
intron 4 and lack intron 5.
and described as the complex by Hausner
(), and the nine species
in the complex were characterised by sensitivity to cycloheximide.
Amalgamating the data from this study and other published phylogenetic data,
accommodates 11 species.
() retained their earlier
view () that the group treated as in this
study, could not constitute a genus because some species with falcate
ascospores did not form part of this lineage.
falcate ascospores evolved more than once in the .
Amongst the species not monophyletic with , two () are completely unrelated to the
, no phylogenetic data exist for three species
(), and one has a
anamorph and resides in ().
closely related to spp.
we treat these as species of .
have shown that there is substantial, consistent phylogenetic evidence to
support a distinct generic taxon for .
form a monophyletic group including both (type species
of ) and (type species of
).
presence of intron 4 and absence of intron 5 in the β-tubulin gene.
Goidánich ()
established for four species with (=
) anamorphs.
recognised and most teleomorph species with anamorphs
were treated as , and more
recently, ().
() indicated that
spp.
together.
spp. that are related to the type of the genus, , as artificial.
the partial ribosomal SSU and LSU regions.
from the 5' region of the nuclear LSU gene, including the variable D1 and D2
regions, and partial DNA sequence data for β-tubulin, a coding gene.
These regions are more variable than those used by Hausner
().
consistently strong support for the group of species that incorporates and as well as 13 other species with
anamorphs.
produce synnematous synanamorphs together with a state,
or a continuum of forms between the two states.
seven of the nine species () were assigned to the
genus by Okada
(), applying their
inclusive definition of .
().
The teleomorph for this species, , was discovered only
recently () and represents one of the nine species that we have assigned
to .
synnematous anamorph is This species was not included
in the study of Okada .
().
() recognized that the
synnematous anamorph of dominates in culture, but
accepted the suggestion of Harrington
() to retain
for anamorphs of the complex. Zhou ., therefore, recommended that the state
be treated as the primary anamorph of .
Harrington .
() suggested that
synnemata evolved more than once in (
Harrington, including ).
fused stipe cells and a synanamorph were only formed by
species in the complex.
spp.
best viewed as a `loose aggregation of conidiophores'
().
conidiophores such as those defining
().
() described
Upadhyay, with the anamorph of
as type species, for species with both mononematous
(-like) and synnematous anamorphs.
the appropriate genus in which to accommodate the anamorphs of
spp.
species producing synnematous anamorphs that phylogenetically reside in
.
synanamorph together with a state,
is ().
not mentioned in the descriptions of the species by Upadhyay
() and Jacobs &
Wingfield (), possibly
indicating that this form is produced only rarely.
spp. without known teleomorphs, M.J. Wingf., Crous &
Tzean, and J.-J. Kim & G.-H.
-like synanamorphs
(,
).
Illustrations of
()
and () shows that conidiophores bearing denticulate
conidiogenous cells, become pigmented towards the base.
with species of (with as
type), defined as having hyaline conidiophores
().
spp.
the fungi that we now treat in
(,
), but
they do not consistently group in a monophyletic clade with each other or with
().
spp.
conidiophores, it is our view that this character is rare and inconsistent
with the definition of
The spp.
monophyletic group (Group D in Figs
,
) that consisted of a number
of strongly supported subgroups.
bootstrap values and included and .
distinct sheaths that distinguish them from all other species in
(, , ).
structures described as and
-like ().
with anamorphs.
that the synnematous anamorph of should not be referred to as
, since does not have a
synanamorph, which is also true for the other two species.
Hoog () that only
spp.
spp.) have anamorphs,
is supported by our data.
Another group of spp.
support (Group I) included the type species for the genus, together with and
that have anamorphs
().
synanamorphs
(). The remaining taxa in Group I are members of the complex (
) that have anamorphs.
represents species spanning the entire spectrum of the anamorph continuum;
those that have only a anamorph, those with anamorphs in
Harrington .
() (synnematal structures
as well as states), and those that have synnemata lacking
the state.
ascospores without sheaths that vary from cylindrical to orange
section-shaped.
intron 5 in the β-tubulin gene (). Harrington .
() defined the complex as a well-resolved monophyletic group containing nine
species with anamorphs.
species without anamorphs group in between species of the
so-called complex.
the conserved nature of the genes in our analyses.
sequence data including the introns, will be necessary to resolve the
phylogeny of the species in this group.
consistently high statistical support includes , and .
three species have long ascomatal necks with annuli and reniform ascospores
without sheaths (, , ). has a relatively
short perithecial neck with no annuli and elongated clavate ascospores without
sheath ().
anamorphs producing secondary conidia
(,
,
).
only anamorphs. This group included , the type species of the genus.
intron 4 and have intron 5 of the β-tubulin gene
().
known, ascospores are more or less reniform and not protected by a sheath
().
group are found in a diverse range of ecological niches.
(,
),
occurs in soil (), and and are wood-inhabiting
(,
,
).
Three species, and
have been reported only from infructescences in South Africa
().
Our data suggest that the species from might form a
monophyletic lineage within .
not supported where greater numbers of species from proteas were included
().
and from
This separation has been implemented and it will hopefully simplify the
application of names for the large number of species occurring in
.
our data provide relatively strong support for the view that it contains a
number of groups, supported by morphological and possibly ecological
characters.
further subdivide in a meaningful way.
convinced that addition of taxa and consideration of DNA sequence data for
additional gene regions will result in the emergence of further genera in
. |
spp.
Hemisphere, providing important sources of structural timber and fibre.
diseases have, however, had a negative impact on their cultivation in many
parts of the world ().
first diseases to seriously damage plantations of outside
their native range, leading to the abandonment of some species for plantation
development ().
shoot die-back, and even tree death.
(Cooke) Hansf. and (Cooke) Hansf.
(,
,
, ,
).
In recent years, it has become apparent that there are many more species of
Johanson occurring on eucalypts than previously
realised.
cause minor leaf spots, rarely resulting in severe disease
(,
).
Little is known regarding some of these less important species but some could
become more important in genetically uniform plantations of susceptible clonal
hybrids or where trees are exposed to conditions of stress.
names (), and
several thousand anamorphs that lack known teleomorphs
().
() and several additional
species have been described more recently
(,
,
,
,
).
Species of are usually assumed to be host-specific,
and presently there are little data available that can be used to refute this
supposition.
hosts (, ), most seem to have narrow host ranges.
Interestingly, where species have been reported to have wider host ranges
within a plant family, e.g.
by Braun (), DNA-based
techniques have clearly shown that in most cases these morphologically similar
taxa are phylogenetically quite distinct (Crous & Groenewald, unpubl.
data).
tissue in an attempt to jump to an ideal host when this becomes available.
Crous & Groenewald ()
have referred to this unusual behavioural pattern as the “pogo stick
hypothesis”.
for teleomorph as well as anamorph states.
colonising atypical substrates are collected without proving their
pathogenicity, incorrect conclusions pertaining to host range could arise.
(primary, secondary or opportunistic), saprobes, endophytes (saprobic or
plant-pathogenic), or have mutualistic (in lichen) associations (Crous . ,
).
levels of virulence, and appear to be secondary colonists of lesions caused by
other pathogens including species of
().
also appear to be hyperparasites on pustules of various
rust species ().
Because several species can co-inhabit the same lesion, either as primary or
secondary pathogens, saprobes or endophytes
(,
),
species identification based on the host can be extremely difficult.
ascospore germination patterns, anamorph morphology and cultures greatly
facilitate species identification, co-inhabitancy
()
makes it difficult to link these cultures and anamorphs to their correct
teleomorphs ().
species occurring on eucalypts.
study was to use comparisons of DNA sequence data to clarify as many as
possible of the formerly published host and distribution records
().
while previous descriptions focused on species associated with leaf spots,
this study also includes species from eucalypt leaf litter.
with leaf spots were chosen for study.
were soaked in water for approximately 2 h, after which they were placed in
the bottom of Petri dish lids, with the top half of the dish containing 2 %
malt extract agar (MEA) (Biolab, Midrand, South Africa).
patterns were examined after 24 h, and single-ascospore and conidial cultures
established as described by Crous
().
sub-cultured onto carnation leaf agar (CLA) [1 % water agar (Biolab) with
autoclaved carnation leaves placed onto the surface of the solidified medium]
and incubated at 25 °C under continuous near-ultraviolet light to promote
sporulation.
() was used to isolate
genomic DNA from fungal mycelium, grown on MEA in Petri dishes.
ITS1 and ITS4 () were used to amplify part of the nuclear rRNA operon
spanning the 3' end of the 18S rRNA gene, the first internal transcribed
spacer (ITS1), the 5.8S rRNA gene, the second ITS region and the 5' end of the
28S rRNA gene.
used by Crous .
().
sequences obtained from GenBank
()
and the alignment was assembled using Sequence Alignment Editor v. 2.0a11
() with manual
adjustments for visual improvement where necessary.
complexity of the original alignment, the sequences were split over four
smaller alignments, each containing genetically similar sequences.
datasets were each treated identically.
were done using PAUP (Phylogenetic Analysis Using Parsimony) v. 4.0b10
().
analysis of the aligned ITS sequence data consisted of neighbour-joining
analysis with the uncorrected (“p”), the Kimura 2-parameter and
the HKY85 substitution model in PAUP.
data and all characters were unordered and of equal weight.
encountered, ties were broken randomly.
GenBank () and the
alignments in TreeBASE.
of structures mounted in lactic acid, with the extremes of spore measurements
given in parentheses.
1 mo on MEA, oatmeal agar (OA) and potato-dextrose agar (PDA)
() at
25 °C in the dark, using the colour charts of Rayner
().
in this study are maintained in the culture collection of the Centraalbureau
voor Schimmelcultures (CBS) in Utrecht, the Netherlands
().
novelties and descriptions were deposited in MycoBank
<>.
isolates ().
resulting from each of the four alignments are depicted in Figs
,
,
,
.
102 taxa (including the two outgroups) and 544 characters including alignment
gaps.
parsimony-uninformative, and 212 are constant.
using the three substitution models yielded trees with similar topologies and
bootstrap values. Parsimony analysis yielded 243 most parsimonious trees (TL =
1038 steps; CI = 0.620; RI = 0.893; RC = 0.554).
trees differed from the trees obtained using parsimony mainly at the deeper
nodes (data not shown).
,
,
,
.
derived tree () includes
(100 % bootstrap support), species of
Crous & M.J. Wingf., the
(Thüm.) Lindau complex (95 % bootstrap support), the
Crous & M.J. Wingf. complex (100 % bootstrap support) and the Crous, F.A. Ferreira, Alfenas & M.J. Wingf. complex (100 %
bootstrap support).
species of are indicated.
The second alignment () contains 90 taxa (including the two outgroups) and 535
characters including alignment gaps.
parsimony-informative, 51 are variable and parsimony-uninformative, and 238
are constant.
yielded trees with identical topologies and similar bootstrap values.
Parsimony analysis yielded 481 most parsimonious trees (TL = 862 steps; CI =
0.613; RI = 0.927; RC = 0.568).
from the trees obtained using parsimony only in the placement of the
sp. CPC 11171 clade (data not shown).
alignment and derived tree mainly includes the Carnegie
& Keane complex (100 % bootstrap support), the Crous
complex (60 % bootstrap support), the R.F. Park &
Keane (100 % bootstrap support) and the R.F. Park &
Keane complex (100 % bootstrap support).
are indicated in the tree.
alignment gaps.
variable and parsimony-uninformative, and 221 are constant.
analysis using the three substitution models yielded trees with identical
topologies and similar bootstrap values.
parsimonious trees (TL = 853 steps; CI = 0.626; RI = 0.856; RC = 0.536).
topology of the distance trees differed from the trees obtained using
parsimony mainly at the deeper nodes (data not shown).
derived tree mainly includes species of Speg.,
Fresen., Sacc.
Syd.
, two in , and two in
.
The fourth alignment () contains 50 taxa (including the two outgroups) and 570
characters including alignment gaps.
parsimony-informative, 25 are variable and parsimony-uninformative, and 252
are constant.
yielded trees with identical topologies and similar bootstrap values.
Parsimony analysis yielded eight most parsimonious trees (TL = 627 steps; CI =
0.864; RI = 0.973; RC = 0.841).
to that of the topology of the trees obtained using parsimony (data not
shown).
de Hoog, Oorschot & Hijwegen, Crous & T.
new species.
Several collections represented spp.
morphologically and phylogenetically distinct from ex-type strains of the
morphological species to which they had originally been assigned.
are described as new taxa as follows:
Crous,
MycoBank .
.
: Refers to Stellenbosch, where the fungus was
collected.
(6.5–)7–9(–10) × (33.5(–4) μm, distincta.
amphigenous, circular to subcircular, 0.5–3 mm
diam, pale brown, with a raised border and red-purple margin.
amphigenous, pycnidial, medium brown, globose,
80–120 μm diam; wall of 3–4 layers of brown discrete, ampulliform to subcylindrical,
pale to medium brown, finely verruculose, proliferating 1–3 times
percurrently near the apex, 3–6 × 3–4 μm.
holoblastic, solitary, aseptate, ellipsoidal, with subobtuse
apex and subtruncate base with minute marginal frill, medium brown, finely
verruculose, widest below the middle, (6.5–)7–9(–10) ×
(3–)3.5(–4) μm.
: , Western Cape Province, Stellenbosch
Mountain, on leaves of sp., 4 Dec. 2004, P.W.
,
, culture ex-type
= CPC
10886.
: Colonies after 3 wk on MEA 15–40 mm diam; on PDA
erumpent, spreading, producing copious amounts of slime, olivaceous-black at
the centre, aerial mycelium olivaceous-grey, with a vinaceous-grey outer zone
and wide olivaceous-black margin that is smooth but uneven; reverse
olivaceous-black; on OA surface smoke-grey with a wide, grey-olivaceous
border, forming a characteristic yellow pigment; on MEA grey-white on surface,
with sectors of smoke-grey; margin thin, submerged, smoke-grey; reverse
olivaceous-black; aerial mycelium sparse to moderate, grey-white; colonies
fertile.
: sp.
: South Africa.
: Numerous species of Corda and several
species of cause spots on eucalypt leaves.
is easily distinguished from the
taxa occurring on eucalypt leaves (), and from representatives of the “” species complex specifically, based on its conidial
morphology.
complex that cause stem cankers on eucalypt trees
(
– this volume)
Crous,
MycoBank .
.
: H.J.
Trans. Brit. Mycol. Soc. 90: 289. 1988.
diam, discrete to confluent, medium brown, surrounded by raised, red-purple
margin. hypophyllous, embedded in a raised, black,
subepidermal stroma, ostiolate, becoming erumpent up to 120 μm diam.
subcylindrical, subsessile, straight or slightly incurved,
8-spored, 50–70 × 9–12 μm. bi- to
triseriate, overlapping, hyaline, thin-walled, straight, obovoid with rounded
ends, widest near the apex, medianly 1-septate, not to slightly constricted at
the septum, tapering toward both ends, but more prominently toward the base,
10–14 × 3–4 μm. of
embedded in the same black subepidermal stroma that
contains ascomata, subepidermal, ostiolate, up to 450 μm diam; wall of
2–3 layers of brown
subcylindrical to ampulliform or doliiform, 5–15 × 3–4
μm, medium brown, verruculose, proliferating several times percurrently
near the apex. solitary, brown, aseptate, verruculose,
thick-walled, oval with an obtuse apex and a truncate to subtruncate base with
a prominent basal frill, which can extend up to 2 μm from the brown basal
rim of the conidium, (8–)10–12(–14) ×
4.5–)5–6(–6.5) μm (av. 11 × 5.5 μm).
: intermingled between that of and ascomata of
phialidic, hyaline, subcylindrical to ampulliform, with visible periclinal
thickening, 8–15 × 2.5–3.5 μm. hyaline,
curved, subcylindrical, widest in the middle, apex bluntly rounded, obtuse,
base truncate, 17–30 × 2–1.5 μm.
: No cultures available.
: , Darling Ranges W.A.,
Mundlimup Block, on leaves of , 24 Nov. 1981, F.
Tay, DAR 58999, of and .
: Swart
() reported that this
fungus is associated with abundant leaf spots on saplings and the foliage of
recently felled trees.
unilocular and subepidermal, occurring in a stroma which could result in some
of them appearing as multilocular.
() considered the fungus
to be the stromatic counterpart of is clearly related to species in the
complex that occurs on eucalypts, having characteristic aseptate, brown,
verruculose conidia that arise from percurrently proliferating conidiogenous
cells. is unique by virtue of its stroma, that gives
rise to the uni- or multilocular conidiomata.
exude in slimy masses.
curved, subcylindrical conidia of a synanamorph.
produced from unilocular conidiomata that formed in the same stromata that
gave rise to .
investigated also contained ascomata of a species,
which most likely also belong to the same fungus.
described here as .
Crous & M.J. Wingf., MycoBank
.
.
: Referring to its host, .
12–17 × 3.5–4.5 μm, modo B germinantibus,
distinguenda.
diam, medium brown, with raised, brown borders, and thin, red-purple margins.
pseudothecial, amphigenous but predominantly epiphyllous,
single, black, erumpent, globose, up to 120 μm diam; apical ostiole
10–15 μm diam, with prominent periphyses lining the ostiolar channel;
wall of 2–3 layers of medium brown
aparaphysate, fasciculate, bitunicate, subsessile, obovoid to ellipsoid,
straight or slightly incurved, 8-spored, 35–50 × 8–12 μm.
tri- to multiseriate, overlapping, hyaline, guttulate,
thin-walled, straight to slightly curved, fusoid–ellipsoidal with obtuse
ends, medianly 1-septate, widest in middle of apical cell, not constricted at
the septum, tapering towards both ends, but more prominently towards the lower
end, (12–)14–15(–17) × (3.5–)4(–4.5) μm
; some ascospores with slightly asymmetrical apical cells, as
commonly observed in .
: , on leaves of sp.,
Mar. 2004, M.J.
= CPC
11174.
: Type B.
darkening on MEA, and germinating from both ends, with germ tubes parallel to
the long axis of the spore, not distorting, becoming slightly constricted upon
germination, becoming up to 4 μm diam.
: Colonies on MEA after 3 wk 25–30 mm diam; on MEA
flat, spreading, folding, with sparse aerial mycelium, olivaceous-grey,
margins smooth, regular, reverse iron-grey; on PDA slightly erumpent, centre
olivaceous-grey; outer zone pale olivaceous-grey; reverse iron-grey; on OA
with sparse to moderate pale olivaceous-grey aerial mycelium and patches of
olivaceous-grey.
: sp.
: Indonesia.
: Conidia of a anamorph were found on some
lesions.
anamorph structures in culture. is
phylogenetically closely related to a sp.
Colombia that forms in culture.
(12–17 × 3.5–4.5 μm) germinate
with a Type B germination pattern as observed in
(10–20 × 2–3 μm) () and (11–22.5 × 2–3.5).
morphology and growth characteristics in culture
().
.
.
: Named after the collector, well-known mycologist and
friend, Prof. dr Walter Gams.
brown, with a raised, dark brown border. pseudothecial,
amphigenous, but predominantly hypophyllous, single, black, subepidermal,
becoming erumpent, globose, up to 90 μm diam; apical ostiole 5–10
μm diam; wall of 2–3 layers of medium brown aparaphysate, fasciculate, bitunicate, subsessile, obovoid to
narrowly ellipsoid, straight or slightly incurved, 8-spored, 25–35
× 7–9 μm. tri- to multiseriate,
overlapping, hyaline, guttulate, thin-walled, straight,
fusoid–ellipsoidal, medianly 1-septate, widest in the middle of the
apical cell, constricted at the septum, tapering towards both ends, but more
prominently towards the lower end, (8–)9–10 × (2–)3
μm .
: , Palampur, on leaves of
sp., Mar. 2004, W. Gams & M.
,
, culture ex-type
= CPC
11138–11140. 5/6-6
: Type C.
darkening on MEA, and germinating from both ends, with germ tubes parallel to
the long axis of the spore, but also variable in direction; becoming
constricted upon germination, up to 5 μm diam.
: Colonies on MEA 28–35 mm diam after 3 wk; on MEA
spreading, folding, flat, with moderate smoke-grey aerial mycelium in the
centre; outer region olivaceous-grey; margins smooth, regular; reverse
iron-grey; on PDA with moderate aerial mycelium, pale olivaceous-grey, outer
region olivaceous-grey with drops of slime; reverse iron-grey; on OA with
moderate aerial mycelium, pale olivaceous-grey, with patches of
olivaceous-grey.
: sp.
: India.
: is phylogenetically closely
related to , but is distinguishable in having a Type
C ascospore germination pattern, as is found in species such as and has ascospores that are 8–10 ×
2–3 μm, thus shorter than those of the species listed above, and it
also lacks an anamorph in culture.
Crous & M.J. Wingf.,
MycoBank
.
.
: Referring to ascospores that germinate with germ tubes
growing 90° to the long axis of the spore.
diam, medium brown, frequently with a orange-red discoloration in the central
part; border raised, dark brown. pseudothecial, epiphyllous,
single, black, subepidermal, globose, up to 90 μm diam; apical ostiole
10–15 μm diam; wall of 2–3 layers of medium brown aparaphysate, fasciculate, bitunicate, subsessile,
obovoid to broadly ellipsoid, slightly incurved, 8-spored, 25–35 ×
7–8 μm. multiseriate, overlapping, hyaline,
guttulate, thin-walled, straight, fusoid–ellipsoidal with obtuse ends,
medianly 1-septate, widest in the middle of the apical cell, constricted at
the septum, tapering towards both ends, but more prominently towards the lower
end, (8–)9–10(–12) × (2.5–)3 μm .
: , Suiza, on leaves of , Jan. 2004, M.J.
,
, culture ex-type
= CPC
10983–10985.
: Type M.
darkening on MEA, and germinating from both ends, with germ tubes 90° to
the long axis of the spore, and distorting upon germination, becoming up to 5
μm wide.
: Colonies on MEA reaching 28–37 mm diam after 3 wk;
colonies folding, spreading, flat, with sparse aerial mycelium, which is
olivaceous-grey on the agar surface, and with smoke-grey aerial mycelium;
margins are smooth, regular; reverse iron-grey at the centre, olivaceous-grey
in the outer zone; on OA with moderate aerial mycelium, olivaceous-grey at
centre, greenish black in outer zone; on PDA olivaceous-grey with some drops
of slime, iron-grey in reverse.
: .
: Colombia.
: Germinating ascospores of have
a characteristic Type M germination pattern, similar to that of can easily be distinguished
from , however, by virtue of the fact that the
ascospores distort at germination.
whereas those of are at right angles.
Crous & J.P.
MycoBank
.
,
.
: Refers to the ascospores that have multiple germ tubes
when they germinate.
diam, pale to medium brown, surrounded by a thin, raised, dark brown border.
pseudothecial, hypophyllous, single, black, immersed
becoming erumpent, globose, up to 100 μm diam; apical ostiole 10–15
μm diam; wall of 2–3 layers of medium brown aparaphysate, fasciculate, bitunicate, subsessile, obovoid to
subcylindrical, straight to slightly incurved, 8-spored, 30–45 ×
7–10 μm. multiseriate, overlapping, hyaline,
prominently guttulate, thin-walled, straight, obovoid with subobtuse ends,
medianly 1-septate, widest at the middle of the apical cell, constricted at
the septum, tapering towards both ends, but more prominently towards the lower
end, (8–)9–10(–11) × 3(–4) μm .
: , on leaves of , Nov.
2004, J.P.
= CPC
11697).
: Type F.
darkening on MEA, and germinating from both ends, with germ tubes parallel to
the long axis of the spore, and distorting prominently upon germination,
becoming up to 11 μm diam; frequently germinating with more than two germ
tubes.
: Colonies after 3 wk 17–22 mm diam on MEA; on PDA
colonies forming copious amounts of slime; surface olivaceous-black with
patches of olivaceous-grey and pale olivaceous-grey; aerial mycelium sparse;
margins feathery, uneven; reverse iron-grey; on OA surface smoke-grey with
patches of olivaceous-grey; on MEA with sparse aerial mycelium, colonies
erumpent, iron-grey, margins feathery, irregular; reverse olivaceous-black;
colonies sterile.
: .
: Spain.
: is characterised by
its distinct ascospore germination pattern (Type F), but where ascospores form
more than two germ tubes, thus distinguishing it from other species like
that have more typical type F germination patterns.
Crous & T.
MycoBank
.
.
: Referring to its morphological similarity to .
diam, medium brown, surrounded by a thin, raised, concolorous border.
pseudothecial, hypophyllous, single, black, immersed
becoming erumpent, globose, up to 120 μm diam; apical ostiole 10–15
μm diam; wall of 2–3 cell layers of medium brown aparaphysate, fasciculate, bitunicate, subsessile,
narrowly ellipsoid to subcylindrical, slightly incurved, 8-spored, 35–45
× 7–9 μm. tri- to multiseriate,
overlapping, hyaline to pale brown, guttulate, thin-walled, straight to
slightly curved, smooth to finely roughened, fusoid–ellipsoidal with
subobtuse ends, medianly 1-septate, widest in the middle of the apical cell,
constricted at the septum, tapering towards both ends, but more prominently
towards the lower end, (8–)9–10(–11) × (2.5–)3
μm similar to the ascomata in morphology.
hyaline, smooth, rod-shaped with bluntly rounded ends,
3–4 × 1–1.5 μm.
: , on leaves of , Aug.
1995, T.
= CPC
1230; 1229–1231.
: Type G.
darkening and becoming verruculose on MEA; germinating from both ends as
observed in , with germ tubes irregular to the long axis
of the spore, and distorting prominently upon germination, becoming up to 8
μm wide.
: Colonies reaching 12–17 mm diam after 3 wk on MEA;
colonies erumpent, irregular, surface iron-grey with olivaceous-grey, sparse
aerial mycelium in central part; margins catenate, smooth; reverse greenish
black; on PDA colonies erumpent, olivaceous-black with sparse olivaceous-grey
aerial mycelium in the central part, margins smooth, catenate; reverse
greenish black; on OA olivaceous-grey with smooth, catenate margins and
green-olivaceous central part.
: .
: Zambia.
: Ascospores of (8–11 ×
2.5–3 μm) germinate with a Type G pattern similar to that observed in
(7–11 × 2–3 μm).
are more verrucose than those of , but both taxa have very similar ascospore dimensions and
germination patterns.
associated.
they lack the red-purple margin found in .
means to distinguish these taxa from each other is to compare their growth in
culture: colonies of are black, produce a brown pigment
in MEA, and form clusters of chlamydospores, whereas cultures of also produce clusters of chlamydospores on MEA, but are
iron-grey, and lack the diffuse brown pigment observed in colonies of .
MycoBank .
Figs ,
.
: sp.
: Morphologically similar to .
(11–)12–14(–15) × (3–)3.5(–4) μm, saepe
utrinque germinantibus, distinguenda.
amphigenous, irregular to subcircular, 0.5–2 mm
diam, pale brown, with a raised, red-brown margin.
pseudothecial, hypophyllous, arranged in dense clusters in pale brown areas
next to the leaf spots associated with conidiomata of the anamorph, black,
immersed, globose, up to 70 μm diam; apical ostiole 10–15 μm diam;
wall of 2–3 layers of medium brown
aparaphysate, fasciculate, bitunicate, subsessile, narrowly ellipsoid to
subcylindrical, straight or slightly incurved, 8-spored, 35–45 ×
9–11 μm. multiseriate, overlapping, hyaline,
granular, thin-walled, straight, fusoid–ellipsoidal with obtuse ends,
medianly 1-septate, widest at the middle of the apical cell, constricted at
the septum, tapering towards both ends, but more prominently towards the lower
end, (11–)12–14(–15) × (3–)3.5(–4) μm,
; frequently encased in an irregular mucous sheath.
internal, consisting of branched, septate, medium brown,
smooth, 3–4 μm wide hyphae. intermixed among
ascomata or separate, predominantly on the lower leaf surface, pycnidial,
substromatal, up to 120 μm diam; wall of 3–4 layers of brown
0–1-septate, but mostly
reduced to conidiogenous cells. discrete,
ampulliform to subcylindrical, medium brown, smooth to finely verruculose,
proliferating 1–3 times percurrently near apex, but also intercalary and
sympodially, 5–15 × 3–5 μm. holoblastic,
solitary, aseptate, fusoid with obtuse to subobtuse apices and truncate bases,
medium brown, finely verruculose, (10–)12–14(–17) ×
(3.5–)4(–6) μm; inconspicuous basal marginal frill present.
: , Wellington Botanical Garden, on
leaves of sp., Mar. 2004, J.A.
,
, culture ex-type
= CPC
11267; 11267–11269 (teleomorph), CPC 11264–11266 (anamorph).
: Type A.
smooth, becoming olivaceous on MEA, germinating predominantly from both ends,
with germ tubes at some angle to the long axis of the spore, and with a
constriction at the ascospore septum; ascospores becoming up to 7 μm
wide.
: Colonies slow growing, 3–8 mm diam after 3 wk on
MEA; on MEA colonies erumpent, aerial mycelium sparse to absent, margins
smooth, surface white-grey to smoke-grey, or with a reddish tinge in patches;
reverse fuscous-black; on PDA erumpent, white to smoke-grey with patches of
vinaceous-grey; reverse vinaceous-grey, with a diffuse red pigment visible in
the agar, up to 2 cm from colony margins; on OA pale grey-olivaceous with a
pale vinaceous grey pigment diffusing into the agar.
: sp.
: New Zealand.
: Ascospores of germinate with a
Type A pattern (as observed in ), except that they tend to
germinate from both ends.
have in the past been confused with those of .
culture, which is similar to .
10–17 × 3.5–6 μm, while ascospores of are 9–17.5 × 2–5.5 μm, and conidia are
8.5–18 × 4–6 μm.
(), and distinct from
.
Crous & G.
MycoBank
.
,
. :
sp.
: Named after its morphological similarity to .
similis, sed ascosporis modo C
germinantibus distinguenda.
2–5 mm diam, brown, with a raised, dark brown margin.
pseudothecial, amphigenous, black, subepidermal, erumpent to superficial,
globose, up to 120 μm diam; apical ostiole 5–10 μm diam; wall of
2–3 layers of medium brown
aparaphysate, fasciculate, bitunicate, subsessile, obovoid to broadly
ellipsoid, straight or slightly incurved, 8-spored, 30–40 ×
8–10 μm. multiseriate, overlapping, hyaline,
sparsely guttulate, thin-walled, straight to slightly curved,
fusoid–ellipsoidal with obtuse ends, medianly 1-septate, widest in the
middle of the apical cell, not to slightly constricted at the septum, tapering
towards both ends, but more prominently towards the lower end,
(8–)9–10(–11) × (2–)2.5–3 μm, internal, consisting of branched, septate, pale to medium
brown, smooth, 3–4 μm wide hyphae.
sporodochial, hyaline. aggregated, unbranched or
branched, hyaline, smooth, tapering to flat-tipped apical and lateral loci,
proliferating sympodially, 8–15 × 2–3.5 μm.
holoblastic, solitary, but frequently undergoing microcyclic
conidiation, giving rise to one or several additional conidia, smooth,
hyaline, obclavate, apex subobtuse, base long obconically subtruncate to
truncate, irregularly curved, 0–3-septate, 12–40 ×
1.5–2 μm; hila inconspicuous.
: , KwaZulu-Natal, Enon, Richmond, on
leaves of , 3 May 2000, G.
,
, culture ex-type
= CMW
9098.
: Type C.
smooth, not darkening on MEA, germinating from both ends, with germ tubes
parallel to the long axis of the spore, and with a constriction at the
ascospore septum; ascospores becoming up to 3.5 μm wide.
: Similar to those of
().
: .
: South Africa.
: has been known to
us for some time, but its formal description required a molecular comparison
with ex-type strains of (which it resembles in
anamorph morphology), and (which it resembles in
ascospore germination pattern). As can be seen here, ()
is clearly a distinct species, sharing features of both of these taxa.
Crous & M.J. Wingf.,
MycoBank
.
. :
sp.
: Morphologically similar to .
petioles. pseudothecial, single to aggregated, black,
immersed becoming erumpent, globose, up to 120 μm diam; apical ostiole
10–20 μm diam; wall of 3–6 layers of brown aparaphysate, fasciculate, bitunicate, subsessile,
obovoid to broadly ellipsoid, straight or slightly incurved, 8-spored,
35–45 × 12–16 μm. tri- to
multiseriate, overlapping, hyaline, guttulate, thick-walled, straight to
slightly curved, fusoid-ellipsoidal with obtuse ends, medianly 1-septate,
widest at the middle of the apical cell, constricted at the septum, tapering
towards both ends, but more prominently towards the lower end,
(11–)12–14(–15) × (3–)3.5(–4) μm ; frequently surrounded by an irregular mucous sheath.
: , on leaves and petioles of
sp., Apr. 2005, M.J.
,
, culture ex-type
= CPC
12085.
: Type H.
darkening and becoming verruculose on MEA, germinating from both ends, with
germ tubes primarily parallel to the long axis of the spore, and distorting
prominently upon germination, becoming up to 11 μm wide.
: Colonies extremely slow growing, erumpent, uneven,
black; aerial mycelium absent; colonies powdery, producing a
anamorph.
: sp.
: Uruguay.
: is morphologically
similar, and phylogenetically closely related to .
be distinguished by its ascospores that are slightly narrower (3–4 μm
3–6 μm), having a mucous sheath, and germinating via two
germ tubes (predominantly) that originate from the ends of the spore.
Germinating spores exude mucus, and become pale brown and verruculose, which
differs from the numerous germ tubes and dark brown ascospores observed in
.
and resistant to being cut, while those of are
powdery, producing a anamorph in culture.
phylogenetic data available, it appears that there may be more species within
the complex awaiting description
().
Crous & T.
MycoBank
.
.
: Refers to the fact that this fungus is phylogenetically
closely related to species of .
diam, pale brown, surrounded by a thin, raised, dark brown border; spots
becoming confluent with age. pseudothecial, hypophyllous,
single, black, immersed becoming erumpent, globose, up to 100 μm diam; wall
of 2–3 cell layers of medium brown
aparaphysate, fasciculate, bitunicate, subsessile, obovoid to broadly
ellipsoid, straight to slightly incurved, 8-spored, 35–50 ×
10–12 μm.
thin-walled, straight, obovoid with subobtuse ends, unequally 1-septate,
widest close to the apex of the apical cell, not constricted at the septum,
tapering towards both ends, but more prominently towards the lower end,
(10–)12–13(–14) × (3–)3.5(–4) μm ; apical cell 4–6 μm long, basal cell 6–8 μm
long.
: , on leaves of , May
1995, T.
= CPC
1098.
: Type F. Similar to .
: Colonies after 3 wk on MEA reaching 6–15 mm diam;
on MEA erumpent with sparse aerial mycelium, pale olivaceous-grey; margins
smooth, regular; reverse ochraceous with patches of pale olivaceous-grey; on
PDA erumpent, centres white to pale olivaceous-grey, outer zone
olivaceous-grey, margins irregular, feathery; reverse smoke-grey in the
central part, olivaceous-grey in the outer region; colonies sterile.
: .
: Tanzania.
: Ascospores of (10–14
× 3–4 μm) germinate with a Type F germination pattern, similar
to that observed in (11–16 × 3–4.5
μm), but are somewhat shorter, and also cluster phylogenetically apart
().
interest is the fact that it aligns with sequences of , for which no teleomorph is known.
re-examination of the original specimen also failed to reveal the presence of
a state.
teleomorph clustering with
other than
()
().
Crous & M.J. Wingf., MycoBank
.
.
: sp.
: -like.
: Referring to the -like
synanamorph.
diam, grey to medium brown, with a raised, dark brown border.
pseudothecial, amphigenous, single, black, immersed becoming
erumpent, globose, up to 90 μm diam; apical ostiole 5–10 μm diam;
wall of 2–3 layers of medium brown
aparaphysate, fasciculate, bitunicate, subsessile, obovoid to ellipsoid,
straight or slightly incurved, 8-spored, 25–30 × 7–9 μm.
tri- to multiseriate, overlapping, hyaline, guttulate,
thin-walled, straight, fusoid–ellipsoidal with subobtuse ends, medianly
1-septate, widest in the middle of the apical cell, constricted at the septum,
tapering towards both ends, but more prominently towards the lower end,
8–10 × (2.5–)3 μm internal and
external, consisting of septate, branched, verruculose hyphae, 2–3 μm
wide. fasciculate, amphigenous on the leaves, brown, up to
50 μm wide and 60 μm high. aggregated in loose
fascicles arising from the upper cells of a brown stroma up to 50 μm wide
and 30 μm high, or situated on the top of the ascomata; conidiophores
medium brown, finely verruculose, 1–4-septate, subcylindrical, straight
to geniculate–sinuous, unbranched, 20–40 × 2–4 μm.
terminal, unbranched, medium brown, smooth to
verruculose, tapering to the flat-tipped apical loci, proliferating
sympodially, 7–15 × 2–3 μm, with thickened, darkened,
refractive scars. solitary, or in simple chains, medium
brown, verruculose, subcylindrical to ellipsoidal, apex obtuse, base
subtruncate, 1–2-septate, frequently constricted at the septa,
7–15 × 3–3.5 μm; hila thickened, darkened, refractive.
Aerial mycelium disarticulating into hyaline, smooth arthroconidia that are
-like, 12–35 × 3–5 μm.
: , Angela Maria, on leaves of
, Jan. 2004, M.J.
, culture ex-type
= CPC
10998.
: Type I.
darkening on MEA, and germinating from both ends, with germ tubes parallel to
the long axis of the spore, lateral branches present, and spore distorting
upon germination, becoming up to 5 μm wide.
: Colonies on MEA reaching 18–30 mm diam after 3 wk;
colonies erumpent, folding, margin smooth, irregular, aerial mycelium
moderate, pale olivaceous-grey; reverse iron-grey; on PDA with moderate aerial
mycelium, olivaceous-grey with patches of pale olivaceous-grey; reverse
olivaceous-black; on OA pale olivaceous-grey with patches of olivaceous-grey
and iron-grey.
: .
: Colombia.
: Several other as yet undescribed species occur on
leaves in Colombia, and some, such as Crous & M.J. Wingf.
()
(), is still not known
from culture.
are distinct from CPC 11004.
back to ascomata due to several species being present on the same leaf spots.
Thus, further collections will be required before these taxa can be named.
the sp. represented by CPC 11002 and CPC 10986
().
above, however, we presently cannot name the latter species.
is also noteworthy based on the fact that
it forms a anamorph, as well as a -like
synanamorph in culture.
clusters of chlamydospores on their hyphal tips in culture () (), leading to the impression that they could develop
into -like anamorphs.
to form anamorphs.
form aerial mycelium that remain hyaline, with wide,
disarticulating cells, suggesting that this anamorph morphology may be more
prevalent in species of than previously realised.
Ascospores of germinate with a Type I pattern, but none
of the species on with this germination pattern form a
anamorph in culture.
Crous & A.C.
.
.
: Referring to the ecology of this fungus as a secondary
coloniser on lesions of .
amphigenous, single, inconspicuous, sparsely distributed, black, subepidermal,
rarely erumpent, globose, up to 90 μm diam. aparaphysate,
fasciculate, bitunicate, subsessile, obovoid to narrowly ellipsoid, straight
or slightly incurved, 8-spored, 20–30 × 7–9 μm.
tri- to multiseriate, overlapping, hyaline, guttulate,
thin-walled, straight, ellipsoidal with subobtuse ends, medianly 1-septate,
widest close to the apex of the apical cell, constricted at the septum,
tapering towards both ends, but more prominently towards the lower end,
8–10 × 2.5–3 μm .
: , Bahia, Teixeira de Freitas, on leaves of
sp., 8 Jun. 2004, A.C.
,
, culture ex-type
= CPC
11551–11553.
: Type D. Similar to .
: Colonies on MEA after 3 wk reaching 25–35 mm diam;
on MEA olivaceous-grey, flat, spreading, folding, with sparse aerial mycelium
and smooth, even margins; reverse iron-grey; on PDA iron-grey with
olivaceous-grey aerial mycelium in central part, and drops of slime
throughout; reverse iron-grey; on OA flat, spreading, olivaceous-grey.
: spp.
: Brazil, Colombia.
: When this species was initially collected in 1992 (CPC
504), it was noted that it occurred in lesions ascribed to , presumably as a secondary pathogen.
recollect this fungus where it had colonised lesions caused by , as well as those of
Sankaran & B. Sutton on eucalypts in Brazil.
clade accommodating from Brazil, isolates collected in
Colombia were also found which were apparently associated with lesions caused
by (). has thus far only been
collected in association with other species of that we
believe are the primary pathogens.
(ascospores 8–10 × 2.5–3 μm) was originally treated as
(ascospores 8–15 × 2–3.5 μm)
().
: , Picadao, Conceicao da
Barra, on leaves of , 27 Apr. 1992, A.C.
= CPC
504.
Crous & A.C.
.
.
: Refers to the occurrence of this fungus on leaf
litter.
absent, ascomata associated with leaf litter.
pseudothecial, amphigenous, but predominantly hypophyllous,
single, black, immersed becoming erumpent, globose, up to 120 μm diam.
aparaphysate, fasciculate, bitunicate, subsessile, narrowly
ellipsoid to subcylindrical, straight or slightly incurved, 8-spored,
25–40 × 7–8 μm. tri- to multiseriate,
overlapping, hyaline, guttulate, thin-walled, straight to slightly curved,
fusoid–ellipsoidal with subobtuse ends, medianly 1-septate, widest in
middle of apical cell, constricted at the septum, tapering towards both ends,
but more prominently towards the lower end, (8–)10–12(–13)
× 3(–3.5) μm, .
: , Minas Gerais, Belo Oriente, on leaf
litter of sp., 24 Jan. 2004, A.C.
,
, culture ex-type
= CPC
11545–11547.
: Type I.
darkening on MEA, and germinating from both ends, with germ tubes parallel to
the long axis of the spore, and lateral branches also present; ascospore
constricting at the septum, becoming up to 5 μm wide.
: Colonies on MEA reaching 20–27 mm diam after 3 wk;
on MEA colonies erumpent, spreading, aerial mycelium sparse, surface folding,
pale olivaceous-grey, with central part having patches of smoke-grey; margin
feathery, irregular, reverse greenish black; on PDA surface olivaceous-black
with patches of smoke-grey aerial mycelium in central part; margins feathery,
irregular, reverse greenish black; on OA olivaceous-black with smoke-grey
aerial mycelium; margins irregular, feathery.
: sp.
: Brazil.
: Ascospores of germinate with a Type I
pattern.
(), from which can be distinguished by its ascospore dimensions and cultural
characteristics. Phylogenetically it is closely related to ().
Crous & A.C.
MycoBank
.
.
: Latin = leaf litter, the substrate
from which this fungus was collected.
absent, associated with leaf litter.
pseudothecial, amphigenous, single, black, immersed becoming erumpent,
globose, up to 90 μm diam; apical ostiole 5–10 μm diam; wall of
2–3 layers of medium brown
aparaphysate, fasciculate, bitunicate, subsessile, narrowly ellipsoid to
subcylindrical, straight or slightly incurved, 8-spored, 30–35 ×
7–9 μm. tri- to multiseriate, overlapping,
hyaline, guttulate, thin-walled, straight, fusoid-ellipsoidal with subobtuse
ends, medianly 1-septate, widest in the middle of the apical cell, constricted
at the septum, tapering towards both ends, but more prominently towards the
lower end, (8–)9–10(–11) × 3(–3.5) μm, .
: , Bahia, Eunapolis, on leaf litter of
sp., 23 May 2004, A.C.
, culture ex-type
= CPC
11438–11440.
: Type I.
darkening on MEA, and germinating from both ends, with germ tubes parallel to
the long axis of the spore, and distorting prominently upon germination,
becoming up to 6 μm wide; lateral branches also present.
: Colonies on MEA reaching 22–38 mm diam after 3 wk;
colonies flat, spreading; aerial mycelium sparse; margins smooth, regular,
surface olivaceous-grey with drops of slime; reverse iron-grey; on OA pale
olivaceous-grey in the centre due to moderate aerial mycelium; olivaceous-grey
in the outer region; on PDA olivaceous-grey with drops of slime, margin thin,
iron-grey on surface and reverse.
: sp.
: Brazil.
: is phylogenetically
closely related to isolates CPC 727–728
(), which represent an
undescribed taxon from Indonesia. has
ascospores that germinate with a Type I pattern, thus being similar to those
of (11–15 × 3–4 μm), (8–11 ×2–3 μm),
(8–11 × 2–3 μm), (7–16
× 2–3 μm), (7–10 ×
1.5–2.5 μm) and (10–13 ×
2.5–4 μm). Ascospores of are 8–11
× 3–3.5 μm, and thus being wider than those of , and .
Furthermore, cultures of are sterile, while all the
other species listed here produce anamorphs in culture.
Crous & M.J. Wingf., MycoBank
.
.
: Refers to Sumatra, where this fungus was collected.
diam, pale brown with a dark brown, raised border, and thin, red-purple
margin. pseudothecial, amphigenous but predominantly
epiphyllous, single, black, subepidermal to erumpent, globose, up to 80 μm
diam; apical ostiole 15–20 μm diam; wall of 2–3 layers of
medium brown aparaphysate, fasciculate,
bitunicate, subsessile, obovoid, straight or slightly incurved, 8-spored,
30–40 × 9–11 μm. multiseriate,
overlapping, hyaline, guttulate, thin-walled, straight,
fusoid–ellipsoidal with obtuse ends, medianly 1-septate, widest in
middle of apical cell, not constricted at the septum, tapering towards both
ends, but more prominently towards the lower end,
(12–)13–15(–16) × (3–)4 μm, .
: , Northern Sumatra, on leaves of
sp., Feb. 2004, M.J.
,
, culture ex-type
= CPC
11171, =
CPC 11175, = CPC 11178.
: Type J.
darkening on MEA, and germinating from both ends, with germ tubes parallel to
the long axis of the spore, but also with one or two lateral branches forming
at the spore ends; ascospores becoming slightly constricted and up to 4 μm
wide.
: Colonies 8–19 mm diam on MEA after 3 wk; erumpent,
with sparse aerial mycelium, smoke-grey; margin smooth, but irregular; reverse
olivaceous-black; on PDA erumpent, olivaceous-grey with a thin whitish border;
iron-grey in reverse; on OA smoke-grey, appearing olivaceous-black in the
centre due to collapse of the aerial in copious amounts of slime.
: sp.
: Indonesia.
: is phylogenetically
distinct from other species occurring on
().
(12–16 × 3–4 μm) germinate with Type J germination
patterns, as do (11–15 × 3–4 μm)
and (7–11 × 2.5–3 μm).
ascospores of are larger than those of , and it has no anamorph, while occurs
in close association with its anamorph
().
Crous & M.J. Wingf.,
MycoBank
.
.
: Refers to , to which it is
morphologically similar.
diam, pale brown to grey, surrounded by a raised, dark brown border, and a
thin, red-purple margin. pseudothecial, amphigenous but
chiefly hypophyllous, single, black, immersed becoming erumpent, globose, up
to 60 μm diam; apical ostiole 10–15 μm diam; wall of 2–3
layers of medium brown aparaphysate,
fasciculate, bitunicate, subsessile, obovoid to narrowly ellipsoid, straight
or slightly incurved, 8-spored, 18–27 × 7–8 μm.
tri- to multiseriate, overlapping, hyaline, guttulate,
thin-walled, straight, ellipsoid with obtuse ends, medianly 1-septate, widest
in the middle of the apical cell, constricted at the septum, tapering towards
both ends, but more prominently towards the lower end,
(7–)8–9(–10) × 3(–3.5) μm .
: , Northern Sumatra, on leaves of
sp., Feb. 2004, M.J.
, culture ex-type
= CPC
11167, =
CPC 11169, = CPC 11170).
: Type E.
becoming dark brown and verruculose on MEA, and germinating from both ends,
with germ tubes irregular to the long axis of the spore; frequently with more
than two germ tubes, and distorting prominently upon germination, becoming up
to 9 μm diam.
: Colonies on MEA 12–22 mm diam after 3 wk;
erumpent, spreading, with smooth, uneven margins; upper surface cracking open;
aerial mycelium sparse to absent; colonies sectoring, olivaceous-grey; margin
thin, iron-grey; reverse greenish-black; on PDA with moderate aerial mycelium,
and spots of slime appearing spread over the iron-grey surface; reverse
greenish black; on OA colonies submerged; aerial mycelium almost completely
absent, greenish black; forming chains of dark brown, thick-walled
chlamydospores that aggregate into small microsclerotia (on all media);
colonies sterile.
: sp.
: Indonesia.
: is distinguished
from other taxa currently known from in that it has a
characteristic ascospore germination pattern.
brown and verruculose, but germinate with more than two germ tubes, which grow
irregular to the long axis of the spore (Type G, becoming type E with age).
Young ascospores just beginning to germinate can be confused with those of
, as they initially also have only two germ tubes, though
the ascospores are more distinctly verruculose than those of .
appear, and the pattern is more similar to that of Type E, which is seen in
is distinguished from
in that the germ tubes remain hyaline, and ascospores and
leaf spots are quite distinct from those of .
Z.Q. Yuan, de Little &
Mohammed, Nova Hedwigia 71: 416. 2000.
.
= U. Braun & M.
Zealand J. For. Res. 32: 228. 2002.
: , North Island, KeriKeri, on
leaves of , 17 Oct. 2003, M.A.
= CPC
10849.
: Colonies reaching 25–35 mm diam after 3 wk on MEA;
pale olivaceous-grey, erumpent, with moderate to extensive aerial mycelium;
margin regular, smooth, reverse iron-grey; on PDA pale olivaceous-grey, margin
thin, olivaceous-grey, reverse iron-grey; on OA central part erumpent, pale
olivaceous-grey, outer zone olivaceous-grey, flat and spreading.
: .
: New Zealand.
: is morphologically
similar to , and hence they are listed here as
synonyms.
by both U. Braun & M. Dick and .
Although the culture was obtained from a single germinating conidium, it is
sterile, and we were unable to rule out the possibility that it may represent
and not .
cultures are required to undertake DNA sequence comparisons with the
Speg.
Braun & Dick ().
.
,
.
: Refers to its host, .
absent, conidiomata associated with leaf litter.
internal, consisting of smooth, branched, septate, pale
brown, 2–2.5 μm wide hyphae. pycnidial,
immersed, brown, globose on leaves, up to 160 μm diam; wall consisting of
3–6 cell layers of lining the
inner layer of the conidioma, dense aggregated, subcylindrical, straight to
curved, 0–1-septate, mostly reduced to conidiogenous cells.
terminal, unbranched, hyaline, smooth,
subcylindrical, proliferating sympodially near the apex, 5–10 ×
2–2.5 μm. solitary , but undergoing
microcyclic conidiation , finely guttulate, subcylindrical to
narrowly obclavate, with obtuse to subobtuse apex, and long subtruncate base,
straight to curved, 1(–3)-septate, (8–)12–16(–22)
× 2(–2.5) μm; hila inconspicuous, 0.5–1 μm diam.
: , Palampur, on leaf
litter, Feb. 2004, W. Gams & M.
,
, cultures ex-type
= CPC
11282, CPC 11283.
: Colonies after 3 wk on MEA 30–40 mm diam; on MEA
pale white to smoke-grey; aerial mycelium sparse; colonies spreading, margins
even, smooth; reverse fuscous-black with patches of vinaceous-grey; on PDA
producing large amounts of slime, with thread-like tufts of aerial mycelium;
surface pale purplish grey (centre) with a zone of vinaceous-grey, and a pale
vinaceous-grey, flat, spreading marginal region; reverse vinaceous-grey with
patches of pale vinaceous-grey; on OA pale vinaceous-grey (centre) with a zone
of purplish grey, a wide, flat margin concolorous with the medium; conidiomata
frequently formed along circardian growth lines.
: sp.
: India.
: Sankaran .
() listed several species
of on , most of which have been
redisposed to other genera. The exceptions are G.
& Roum. (conidia filiform–acicular, 1-septate, 14–18 ×
1.5 μm) and Penz. & Sacc. (conidia
0–2-septate, 50–55 × 3–3.5 μm).
() recently described
Gadgil & M.
filiform, sigmoid or falcate, 1-septate conidia, 65–70 × 2–3
μm. is distinct from this species in having
conidia that are subcylindrical to narrowly obclavate, 1(–3)-septate,
8–22 × 2–2.5 μm.
(), show that
is closely allied to (on
, conidia filiform, 1–3-septate, 17–40 ×
1.5–3 μm) and (on , conidia
subcylindrical to narrowly obclavate, (0–)1–3(–4)-septate,
6–30 × 1.5–2 μm).
however, other loci will need to be sequenced, as the ITS domain is
insufficient to distinguish species complexes in .
.
.
: Refers to the Provence in France where the fungus was
collected.
veins, 1–6 mm diam, becoming confluent with age.
internal, consisting of smooth, branched, septate, hyaline, 3–4 μm
wide hyphae. amphigenous on leaves, pycnidial, immersed,
brown, globose, up to 200 μm diam; wall consisting of 2–4 cell layers
of lining the inner surface of the
conidioma, densely aggregated, subcylindrical to ampulliform, straight to
slightly curved, 0–2-septate, 6–25 × 3–5 μm.
terminal, unbranched, hyaline, smooth,
subcylindrical to ampulliform, proliferating sympodially or several times
percurrently near the apex, 6–10 × 3–5 μm.
solitary , finely guttulate, subcylindrical
to narrowly obclavate, with subobtuse apex, and obconically subtruncate base,
variously curved to irregular, mostly widest in the middle of the basal cell,
tapering towards the apex, (1–)2(–3)-septate,
(12–)30–40(–45) × 2.5–3(–4) μm.
: , Provence, Cheval Blanc camping site, on
juvenile leaves, 29 Jul. 2005, P.W.
,
, cultures ex-type
= CPC
12226, CPC 12227–12228.
: Colonies 10–15 mm diam after 3 wk on MEA; colonies
erumpent, surface irregular, catenate, olivaceous-grey with cream to pale
rosy-buff spore masses; aerial mycelium absent; margins smooth, regular, with
a thin outer zone that is pale olivaceous-grey to slightly rosy-buff; colonies
olivaceous-black in reverse.
: sp.
: France.
: Conidia of (12–45 ×
2.5–4 μm) are most similar to (50–55
× 3–3.5 μm), although on average, they are much shorter.
Crous & M.J. Wingf., MycoBank
.
.
: sp.
: Morphologically similar to and its
anamorph.
diam, pale brown, with a raised border. arising singly
from superficial mycelium, brown, smooth to finely verruculose,
1–4-septate, subcylindrical, straight to variously curved, unbranched,
15–60 × 3–4 μm. terminal,
unbranched, medium brown, smooth, tapering to flat-tipped apical loci that are
darkened and refractive, proliferating sympodially, 15–25 ×
2–3 μm. solitary to catenulate in simple chains,
medium brown, verruculose, cylindrical or narrowly obclavate, with subobtuse
apex, and long obconically subtruncate base, straight to curved,
1–5-septate, 20–50 × 2.5–3 μm; hila thickened,
darkened and refractive.
: , on leaves of sp.,
1995, M.J.
= CPC
1087; 1088–1092.
: Type D.
smooth, not darkening on MEA, germinating from both ends, with germ tubes
parallel to the long axis of the spore, and some lateral branches; ascospores
distorting, becoming up to 5 μm wide.
: Colonies after 3 wk on MEA 23–30 mm diam, pale
olivaceous-grey, spreading, with moderate aerial mycelium, and smooth,
irregular margins; colonies folding, erumpent; reverse olivaceous-black; on
PDA pale olivaceous-grey with moderate aerial mycelium and copious amounts of
slime; margins submerged in the agar; reverse olivaceous-grey; on OA pale
olivaceous-grey, colonies folding with moderate aerial mycelium, and a thin
olivaceous-grey margin.
: sp.
: Colombia.
: Several species of were present on
the lesions from which was isolated, and it was not
possible to trace the ascospores back to the specific ascomata.
description of the teleomorph thus has to await
further collections.
Type D pattern, which together with its anamorph, resulted
in it being identified as
().
is distinct from , and most closely
related to , which has a Type I germination pattern.
has shorter conidia (20–50 ×
2.5–3 μm) than (25–200 ×
2–2.5 μm) (, ).
Crous & M.J. Wingf., MycoBank
.
.
: sp.
: refers to the morphological similarity with and its anamorph.
diam, pale brown, with a raised border and thin, red-purple margin.
arising singly from superficial mycelium, medium brown,
finely verruculose, 1–2-septate, subcylindrical, straight to variously
curved, unbranched, 30–60 × 3–4 μm. terminal, unbranched, medium brown, verruculose, tapering to
flat-tipped apical loci that are darkened and refractive, proliferating
sympodially, 10–25 × 3–4 μm. catenulate
in branched chains, medium brown, verruculose, cylindrical or narrowly
obclavate, with subobtuse apex, and subtruncate base, straight to curved,
0–2-septate, 12–50 × 3–5 μm; hila thickened,
darkened and refractive.
: , on leaves of , Mar.
1996, M.J.
,
cultures ex-type = CPC 1300; 1299–1301.
: Type D. Similar to .
: Colonies after 3 wk on MEA 25–35 mm diam; on MEA
spreading, slightly erumpent, margins smooth but irregular; aerial mycelium
sparse to moderate; surface olivaceous-black, but central part grey due to
aerial mycelium; reverse olivaceous-black; on PDA olivaceous-black with mucous
droplets and aerial mycelium that is olivaceous-grey in the central part, but
has a reddish tinge in the outer region; reverse greenish black; on OA
iron-grey with sparse to moderate olivaceous-grey aerial mycelium.
: .
: Indonesia.
: The specimen on which this species is based was originally
identified as representing .
based on its characteristic leaf spots, ascospore germination patterns and
dimensions, and the presence of a anamorph.
material was not retained, and hence only the anamorph, which forms in
culture, can be named.
(20–50 × 2.5–3 μm) and (25–200
× 2–2.5 μm) (, ).
or its anamorphs from leaves.
arise from a re-examination of specimens and cultures treated previously
().
this earlier study had been described primarily on the basis of morphology and
without the support of DNA sequence comparisons.
() showing that there are
several species of on eucalypts that have distinct
cultural characteristics and can be separated based on phylogenetic analyses,
but that share the same symptoms, morphological characterisitics and ascospore
germination patterns.
species on eucalypts in the absence of DNA sequence
analyses.
characteristics must consequently be viewed with some circumspection.
on phenotypic characters is found in the case of .
present study, we reconsidered several collections originally identified as
based on symptoms, ascospore dimensions, germination
patterns, and the presence of a anamorph in culture.
“”-like isolates were consequently shown to
represent several species.
only two anamorph species and ,
could be named.
.
fusoid–ellipsoidal ascospores that are constricted at the septum, that
darken upon germination, and that produce colonies that are relatively
slow-growing. These isolates are described here as and .
cryptic species were found in the case of , which
is morphologically similar to ,
which is similar to , and ,
which is similar to .
In this study we have applied only DNA sequences of the ITS region.
Although this locus has been very useful in delimiting species of
from , it is not always sufficient
to derive conclusions for all species complexes
(,
– this volume).
species in anamorph genera such as and .
In contrast, sequences of the ITS region appear to be useful for
distinguishing species with and most
other anamorph genera that we have considered (Crous
& Groenewald, unpubl. data).
has evolved at different rates in different anamorph genera associated with
, and that it is more conserved in
and , two genera that always cluster together.
species from leaf litter.
species that sporulate once leaves have died.
biology of species suggests that these fungi are
probably not saprobes but rather that they infect living leaf tissue and only
sporulate after leaf fall.
species, and it would be intriguing to follow the
infection patterns of species that are not primary pathogens.
arising from this study.
but its unique nature was not confirmed previously. is always found on leaf spots caused by .
This is an unusual habit for a species of , and its
ecological role deserves further study.
(teleomorph: ) was
originally described as a potential hyperparasite of powdery mildew (De Hoog
.
isolated from many different hosts (). Jackson .
is not a hyperparasite of and
, the two species with which
frequently co-occurs. Jackson . also showed that can infect leaves. and occur on leaves of numerous
spp., and they are frequently found on leaf spots caused
by other species, as well as unrelated fungi (Crous
unpubl. data).
to be determined.
similar coelomycete genera.
accommodated in , particularly those that are
-like and to which is definitely
closely related if not congeneric.
relationship between these taxa remains to be proven, and hence the anamorph
is best retained in .
spp.
leaves and stems.
spp. on eucalypts as there are species of that genus.
This would imply that only 14 % of the species of from
eucalypts have presently been described.
challenges face the taxonomists who wish to distinguish
spp. form eucalypts in future.
studies, DNA sequence comparisons based on multiple genes will be required to
accurately identify these fungi.
clearly need to be directed to those species that are primary pathogens.
However, the primary pathogens are so easily confused with other less
important species, that all material will ultimately have to be thoroughly
studied and understood. |
The taxonomy of (Sacc.) Sacc.
with cankers of spp.
these fungi have undergone numerous revisions and changes in recent years
(,
Gryzenhout .
,
,
).
that the important canker pathogen, (Bruner) Hodges (, ,
, , ), is different from other spp.
been placed in a new genus, Gryzenh. & M.J. Wingf.,
that includes at least two distinct species, (Bruner)
Gryzenh. & M.J. Wingf. and Gryzenh. & M.J.
Wingf. ().
pathogen, M. Venter & M.J. Wingf.,
formerly known as (Schwein.: Fr.) Fr.
(),
now resides in the new genus Gryzenh. & M.J. Wingf.
as (M. Venter & M.J. Wingf.) Gryzenh. & M.J.
Wingf. ().
(), and on exotic spp.
,
,
), all of which reside in the family , as
well as on native and
belonging to the family
().
In South East Asia and Australia the pathogen has been reported from
spp. (, , , ) and
(,
).
In Africa, has been reported from Cameroon, Republic of
Congo, Democratic Republic of Congo and Unguja Island, Zanzibar on
spp.
(,
,
,
,
,
,
).
from South Africa.
South African tree species and non-native ornamental and plantation forest
trees (, , ).
spp.
() and has recently also been reported from the non-native
ornamental tree ()
()
and native and
() () in South Africa.
spp.
bark cankers on trees (,
).
and spp.
(,
),
where it has been associated with cankers and tree death
(,
,
).
spp.
because species in both genera have orange stromatal tissue in their
teleomorph states (, , , ) and they share the same hosts and
geographical distributions (, , , ).
However, there are distinct morphological differences between the genera.
For example, the conidiomata of are superficial,
fuscous-black, pyriform to orange with attenuated necks
(, ), whereas those of are semi-immersed,
orange and globose without necks (, ,
).
septate, whereas those of are aseptate.
analyses have also shown that the two genera form distinct, well-supported
groups (, Gryzenhout
,
), separate from each
other and from the genus in which both had been placed
previously.
pathogen of commercially grown spp.
(, ).
substantial damage to clonal plantation forestry, which has been partially
mitigated through the selection and planting of disease-resistant clones
(, ).
native and in South Africa has led
to a change of view regarding its possible origin.
to be an introduced pathogen (, ,
), there is now
substantial evidence to suggest that it is a native pathogen that could have
moved from native South African spp.
as and
(,
,
,
).
in South Africa.
the country to establish the occurrence of spp.
().
These surveys yielded a fungus similar to that was
collected from three hosts in three geographic areas of the country.
of this study were to characterise the unknown fungus based on morphology and
DNA sequence comparisons and to assess its pathogenicity in greenhouse
inoculations on plants of and
.
from from Tzaneen, from
Lydenburg and from Durban
(;
).
all isolates have been deposited in the culture collection (CMW) of the
Forestry and Agricultural Biotechnology Institute (FABI), University of
Pretoria, Pretoria, South Africa and duplicates in the collection of the
Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands.
specimens have been deposited in the National Collection of Fungi, Pretoria,
South Africa (PREM).
one plate each containing MEA (20 g/L malt extract and 20 g/L agar, Biolab,
Midland, Johannesburg) and 100 mg/L streptomycin sulfate (Sigma-Aldrich,
Chemie, Gmbh, Steinheim, Germany) using a sterile scalpel, and transferred to
1.5 mL Eppendorf tubes. DNA was extracted as described by Myburg ().
ITS1 and ITS4 (), the rDNA (ITS 1, 5.8S and ITS 2) regions were amplified,
while primer pairs Bt1A/Bt1B and Bt2A/Bt2B
()
were used to amplify the β-tubulin 1 and 2 gene regions respectively.
reactions were performed in a volume of 25 μL comprising of 2 ng DNA
template, 800 μM dNTPs, 0.15 μM of each primer, 5 U/μL
polymerase (Roche Diagnostics, Mannheim, Germany) and sterile distilled water
(17.4 μL).
().
The purified PCR products were sequenced in a reaction volume of 10 μL
consisting of 5× dilution buffer, 4.5 μL HO, DNA (50 ng
PCR product), 10× reaction mix BD (ABI Prism Big Dye Terminator v. 3.1
Cycle Sequencing Ready Reaction Kit, Applied Biosystems, Foster City, CA), and
∼ 2 pmol/μL of one of either the reverse or forward primers that were
used in the PCR reactions.
0.06 g/mL Sephadex G-50 (Sigma-Aldrich, Amersham Biosciences Limited, Sweden)
according to the manufacturer's protocol.
directions using the Big Dye Cycle Sequencing kit (Applied Biosystems, Foster
City, CA) on an ABI Prism™ 3100 DNA sequencer (Applied Biosystems).
The gene sequences were analysed and edited using Sequence Navigator v.
1.0.1™ (Perkin-Elmer Applied BioSystems, Foster City, CA).
were compiled into a matrix using a modified data set (S1128, M1935) of Myburg
. () as
template.
(),
(,
),
(,
),
Fr. (, ), Gryzenh. & M.J. Wingf.
() and Gryzenh., Glen & M.J. Wingf.
(,
) species were added to the data matrix.
representing an undescribed genus identified by Myburg .
() and originating from
clove in Indonesia were also added.
interface
()
of the alignment program MAFFT v. 5.667
(),
and deposited with TreeBASE as S1488 and M2673.
Phylogenetic analysis was performed using the software package PAUP
(Phylogenetic Analysis Using Parsimony) v. 4.01b
().
homogeneity test () to determine the similarity and combinability of
the data for the ITS and the β-tubulin 1 and 2 regions, was run.
parsimonious trees were obtained with heuristic searches using simple stepwise
addition and tree bisection and reconstruction (TBR) as the branch swapping
algorithms.
to zero were collapsed. Gaps were treated as a fifth character.
replicates were done on consensus parsimonious trees
().
isolates of Nitschke (CMW 5288 and CMW 5587) were
used as outgroup to root the tree ().
dissection microscope, boiled for 1 min and sectioned (12 μm thick) using a
Leica CM1100 cryostat (Setpoint Technologies, Johannesburg, South Africa) as
described by Gryzenhout
().
were also crushed on microscope slides in 85 % lactic acid or 3 % KOH in order
to study the asci, ascospores, conidia, conidiophores and conidiogenous cells.
Measurements were then taken for the above-mentioned structures.
holotype specimen PREM 58896 50 measurements were made for each character.
Only 20 measurements per character were made for the remaining specimens (PREM
58897–58901). A HRc Axiocam digital camera with Axiovision 3.1 software
(Carl Zeiss Ltd., Germany) was used to capture digital images and to compute
measurements.
for and (Gryzenhout .
,
).
characteristics.
young cultures were placed onto the centres of 90 mm diam Petri dishes
containing MEA.
temperatures ranging from 15 to 35 °C in 5 ° intervals.
per isolate were inoculated and two measurements perpendicular to each other
were taken daily until the fastest growing culture covered the plate.
isolate, the colony diameter was calculated as an average of eight readings.
Colour notations of Rayner
() were used for the
descriptions of cultures and fruiting bodies.
tested on 25 trees each of an clone (ZG14) that is known
to be highly susceptible to fungal pathogens
(), and seedlings respectively, in a
greenhouse set at 25 °C.
2 m tall while the seedlings were approx. 1 m tall.
(4 mm diam).
colonies were inserted into the wounds with the mycelium facing the xylem.
(Pechiney plastic packing, Chicago, USA).
controls and were inoculated with sterile water agar (WA: 20 g agar Merck,
South Africa / 1 L water).
taking measurements of the lengths of lesions in the xylem.
repeated after four months.
small pieces of discoloured xylem onto MEA.
nurseries is seldom achieved.
species, , could be obtained for pathogenicity tests.
Two isolates (CMW 13645 and CMW 13646) of the unknown fungus from were inoculated into the stems of two
trees respectively.
serve as a negative control.
used when inoculating and plants,
except that each of the three trees had two inoculation points, with the same
isolate, on opposite sides of the stem at the same height.
measured 8 wks after inoculation and reisolations were made using the same
procedures as with the and
inoculations.
(ANOVA).
().
trees near Tzaneen in the Limpopo Province.
structures occurred between structures of that were
also fruiting profusely on these trees.
six native trees exhibiting severe cankers and die-back
growing in the private Buffelskloof Nature Reserve near Lydenburg in
Mpumalanga Province.
().
collections were made from the stems of two non-native
trees from the Durban Botanic Gardens in KwaZulu-Natal Province.
displayed symptoms of branch die-back ().
approximately 500 bp in size.
approximately 600 bp in size.
test showed that the data for each gene region were significantly congruent
(p-value = 0.02). The aligned sequences of the combined regions generated 1532
characters of equal weight, with 812 constant characters, 32
parsimony-uninformative characters and 688 parsimony-informative characters.
Five most parsimonious trees were generated with similar branch lengths and
topology and one was chosen for presentation. This tree had a length of 1725,
a consistency index (CI) of 0.737 and retention index (RI) of 0.922
().
distinct and well-supported clades reflecting the different genera.
isolates of the unidentified fungus from
and in South Africa grouped separately from these genera
(100 % bootstrap support), specifically separate from isolates of and , which also occur on
in South Africa.
formed a clade with the isolates of an undescribed fungus from from Indonesia ().
sub-clades linked to the collections from different hosts.
constant single base pair differences between isolates from the different
hosts.
support), and the sub-clade (96 % bootstrap support) from
South Africa.
isolates and those from Indonesia (100 % bootstrap support), strongly
suggesting that they represent different species.
in South Africa is characterised by fruiting structures
(; Figs
,
) that are morphologically
very similar to those of species and the
anamorph of
().
necks are covered in umber tissue as they extend beyond the bark surface
() and limited
orange to cinnamon stromatic tissue can be seen at the bases of the necks
().
are 1-septate, hyaline, and oblong to ellipsoidal
().
of the unknown fungus, conidiomata are pulvinate to conical, fuscous-black and
superficial (Figs ,
), similar
() to the conidiomata of
the same shape and colour in
().
in several morphological characters ().
,
), while
spp.
().
slightly attenuated apices (Figs
,
), differing from those of
spp.
().
(Figs ,
) are not as prominent as
those of members of .
to ovoid and occasionally allantoid (Figs
,
), differing from those of
spp.
().
pseudoparenchymatous (),
differing from that of , which consists of larger cells
of ().
white with grey patches, eventually becoming umber to hazel to chestnut.
is different from cultures of spp., which are white with
cinnamon to hazel patches ().
cryptic species.
for fruiting structures among specimens linked to the isolates used in the
phylogenetic analyses.
(PREM 58898 and PREM 58899), (PREM 58896 and PREM 58897)
and (PREM 58900 and PREM 58901).
clear differences in cultural morphology.
Myburg . ()
from clove in Indonesia is related to the unknown fungus from South Africa,
which formed the focus of the present study.
compare the South African and the Indonesian fungus based on morphology,
because the latter fungus is known only from culture without any connection to
morphological structures on the bark
().
Some poorly formed conidiomata obtained for the Indonesian fungus by
artificially inoculating it into twigs
(),
however, suggested that the fungus is similar to the South African collections
and probably represents the same genus.
unknown fungus collected from and in South Africa can be distinguished from and other closely related genera.
fungus most closely resembles but clearly represents an
undescribed genus.
previously collected from clove in Indonesia
().
Based on these differences, a new genus is thus established for the fungi from
South Africa and Indonesia.
genus.
different from the South African isolates, but could not be described because
there are insufficient structures on which to base a meaningful description.
The isolates from the different hosts in South Africa formed a closely related
group in the genus, although three possibly cryptic species, representing the
isolates from three areas (Mpumalanga, Limpopo and KwaZulu-Natal Provinces)
and hosts ( and ),
respectively, could be identified based on sequence differences.
morphological differences could be observed for these apparent cryptic
species, and at present there is insufficient material or ecological
information available regarding these groups to support the separation of
three species.
collections in a single species.
not belong to this species, but must remain undescribed until fresh host
material bearing fungal structures can be collected.
and teleomorph, while specimens from and
have only the anamorph present.
study, a single species is described in a new genus, and this is based on
specimens from as the holotype.
genus and species follow:
Nakab., Gryzenh., Jol. Roux & M.J. Wingf.,
MycoBank
.
: Latin, , to hide, referring to the fact
that the fungus is difficult to find deliberately, and ,
destroyer, referring to its pathogenic nature.
tectis, textura stromatica limitata cinnamomea vel aurantiaca praesens.
uniseptatae, oblongo-ellipsoideae.
superficialia, juventia aurantiaca, matura fusco-nigra, pulvinata vel conica,
collis brevibus vel absentibus.
pseudoparenchymatosa. cylindrica, ramosa.
non septata, oblonga, cylindrica vel ovoidea, interdum
allantoidea.
bark tissue, with the cylindrical perithecial necks covered with umber tissue
as they protrude through the bark surface.
prosenchymatous to pseudoparenchymatous stromatic tissue present around the
upper parts of the perithecial bases, usually beneath the bark or erumpent
through the bark surface. 8-spored, fusoid to ellipsoid.
hyaline, with one median septum, oblong-ellipsoidal.
fuscous-black when mature, pulvinate to conical with or without short
attenuated necks, unilocular with even inner surface. pseudoparenchymatous. hyaline, branched
irregularly at the base or above into cylindrical cells, separated by septa or
not. phialidic, apical or lateral on branches
beneath the septa. hyaline, non-septate, oblong to
cylindrical to ovoid, occasionally allantoid, exuded as bright luteous
tendrils or droplets.
: Nakab., Gryzenh., Jol.
Roux & M.J. Wingf., sp. nov.
Nakab., Gryzenh., Jol. Roux & M.J.
Wingf., .
.
,
.
: Latin, , scattered, referring to the
conidiomata scattered on the bark surface.
extensis textura umbrina tectis, textura stromatica limitata aurantiaca vel
umbrina composita. uniseptatae, oblongo-ellipsoideae.
superficialia, pulvinata vel conica collis brevibus vel
absentibus, fusco-nigra. pseudoparenchymatosa.
cylindrica, ramosa, cellulae conidiogenae apicibus
attenuatae. non septata, oblonga, cylindrica vel ovoidea,
interdum allantoidea.
extending, umber, cylindrical perithecial necks, occasionally erumpent,
limited, orange to umber ascostromatic tissue covering the tops of the
perithecial bases; ascostromata extending 100–400 μm high above the
bark, 320–505 μm diam (Figs
,
).
cinnamon and pseudoparenchymatous at the edges, prosenchymatous in the centre
().
valsoid, 1–6 per stroma, bases immersed in the bark, black, globose to
subglobose, 100–300 μm diam, perithecial wall 30–50 μm thick
(Figs ,
).
black, periphysate, 80–100 μm wide (Figs
,
), emerging through the
stromatal surface, covered in umber stromatic tissue of (),
extended necks up to 50 μm long, 100–150 μm wide.
8-spored, biseriate, unitunicate, free when mature, non-stipitate with a
non-amyloid refractive ring, fusoid to ellipsoidal,
(19.5–)23.5–29.5(–33.5) ×
(4.5–)5.5–7(–7.5) μm (Figs
,
).
hyaline, with one median septum, oblong-ellipsoidal, with rounded ends,
(4.5–)6–7(–8) × (2–)2.5–3(–3.5)
μm (Figs ,
).
pulvinate to conical without necks, occasionally with a neck that is slightly
attenuated (Figs ,
), orange to scarlet when
young, fuscous-black when mature, conidiomatal bases above the bark surface
300–500 μm high, 200–1000 μm diam. with even to convoluted inner surfaces, occasionally
multilocular, locules 100–550 μm diam (Figs
,
).
pseudoparenchymatous ().
hyaline, branched irregularly at the base or above into
cylindrical cells, with or without separating septa,
(9.5–)12–17(–19.5) × 1.5–2.5 μm (Figs
,
). phialidic, determinate, apical or lateral on branches beneath a
septum, cylindrical with or without attenuated apices, (1.5–)2–3
μm wide, collarette and periclinal thickening inconspicuous (Figs
,
). hyaline,
non-septate, oblong to cylindrical to ovoid, occasionally allantoid,
(2.5–)3–4(–5.5) × (1–)1.5(–2.5) μm
(Figs ,
), exuded as bright luteous
tendrils or droplets.
: On MEA, appears
white with grey patches, eventually becoming umber to hazel to chestnut,
fluffy with an uneven margin, fast-growing, covering a 90 mm diam plate in a
minimum of 5 d at the optimum temperature of 25 °C.
sporulate after sub-culturing and teleomorph structures are not produced in
culture.
: Bark of and .
: South Africa
: , Limpopo Province,
Tzaneen, , 2003, M. Gryzenhout, PREM
58896, culture ex-type CMW 9976 =
, PREM
58897, living culture CMW 9978 =
;
KwaZulu-Natal Province, Durban, Durban Botanic Gardens, , M.
, G. Nakabonge, J. Roux & M.
Oct. 2003, PREM 58899, living culture CMW 13645 =
, PREM
58898, living culture CMW 13646.
observed on the stems of the clone (ZG 14) and on those of
().
These lesions were light to dark brown, and stretched up and down the stems
from the inoculation points.
the inoculation study.
and 29 mm for in the first experiment and 104 mm and 25
mm, respectively, in the second experiment.
hosts were significant ( < 0.001) and were similar in both
trials. was re-isolated from the lesions.
inoculation were closed by callus tissue (Figs
,
).
showed no obvious lesion development after eight wks.
developed on the controls.
represents a new genus and species related to, but distinctly different from,
.
supported by both morphological characteristics and DNA sequence data.
have clearly shown that isolates of form a clade distinct
from and other taxa, which it resembles
morphologically.
and may appear indistinguishable from
spp.
of light microscopy.
shape.
to cinnamon stromatic tissue and perithecial necks covered in umber tissue as
they extend beyond the bark surface.
that are expelled as bright luteous spore tendrils.
and are 1-septate, hyaline and
oblong to ellipsoidal.
hosts as .
morphologically similar also occurs
(,
).
However, to the best of our knowledge this is the first fungus belonging to
the group that has been collected from a species of .
morphological differences separate these two fungi.
perithecial necks, pulvinate to conical conidiomata without necks, conidia
that are oblong to cylindrical to ovoid, and pseudoparenchymatous stromatic
tissue in the conidiomatal base, distinguish from
spp. spp.
perithecial necks, the conidiomata are pyriform to pulvinate with attenuated
necks, conidia are oblong and uniform in shape, and stromatic tissue of the
conidiomatal base is of and that of the neck of
(). produces
cultures that are white with grey to chestnut-coloured patches, in contrast to
spp.
hazel patches.
relatively easy to distinguish from
spp.
fungi occur on exotic and native in South Africa.
, which is a highly pathogenic fungus on
spp.
(, ) and which also occurs on
()
and native (). has been
described in this study and occurs on native and exotic in South Africa.
fungus, , has been recorded only from
spp.
().
can easily be distinguished from and based on differences in the
colour and shape of conidiomata as well as cultural morphology
(,
, , ).
groups are represented by the isolates now treated as the single species
Thus, is represented by isolates
from and spp.
Africa, and these isolates form three closely related sub-clades.
sub-clade represents isolates from clove in Indonesia and was previously
studied by Myburg
().
data, this fungus clearly represents a distinct species, which could not yet
be described because of insufficient material available to characterize it.
The fact that the unknown Indonesian fungus is now known to reside in
should facilitate the collection of additional samples
from clove in Indonesia.
host genera ( and ) and areas
of collection (Lydenburg, Tzaneen and Durban).
represented by a limited number of isolates and a larger collection of
isolates will be required to better understand the relationship among them.
these three subclades and the comparison was also hindered by the absence of
teleomorph structures on the specimens from and .
sub-clades contained in must await the acquisition of
additional material and isolates.
these fungi in South Africa is also largely unknown, and such information
would be useful in studying the taxonomic status of these three subclades of
is a rare and endangered tree species in
South Africa.
pers. comm., ).
from dying trees in the Buffelskloof Nature Reserve near Lydenburg and it was
thought that the fungus might be responsible for the death of the trees.
However, pathogenicity tests conducted using a limited number of trees of a
closely related species, , showed that is not pathogenic to that species.
is more susceptible to than is
, the fungus might not be the cause of tree death at
Buffelskloof.
study.
endangered and is extremely difficult to propagate artificially.
tree mortality in the Buffelskloof Nature Reserve thus remains unclear.
possibility that another organism is responsible for the death of the trees
must also be investigated.
hosts.
than is thus a newly discovered
pathogen of these trees and it could become important on commercially grown
trees in South Africa.
both native and non-native in South Africa.
important issues pertaining to the origin and distribution of these fungi.
Both fungi are currently known only from southern Africa, and they also occur
on native African trees.
(,
)
and the same is probably true for .
virulent pathogens of exotic trees and their accidental
introduction into Australia, where spp.
are native, could result in an ecological disaster.
view is based on the fact that similar canker pathogens, such as
(Murrill) M.E.
losses to trees after being introduced into new environments
(,
).
also potentially threaten plantation trees wherever they
are grown commercially.
of and spp.
South Africa and on other parts of the African continent.
fungi are almost indistinguishable in the field will complicate such surveys,
and laboratory studies will be required for reliable identifications.
collections and associated isolates of might also lead to
the subdivision of this species into additional taxa.
thus add knowledge to the relatively poorly studied fungal biodiversity on the
African continent and especially on native African tree species. |
When we realise that fungi have an immense impact on our lives, we also are
aware that it is important to know more about them.
require is gathered through scientific study and, eventually, all the
knowledge about a specific fungus is linked to its name.
the tools that make it possible to successfully store or retrieve data.
Consequently, it is essential to identify organisms accurately and to name
them correctly.
an actual sample of that organism.
which research is published, it is necessary to preserve reference material.
Type material and other voucher specimens must be carefully preserved in a
reputable reference collection, where they are available for study by the
international scientific community.
develop new technologies, and they are important in research and teaching.
Conditions for the maintenance of fungi must therefore prevent morphological,
physiological or genetic changes while preserving the ability of the fungus to
grow when again placed in suitable conditions.
reflect the biodiversity of the region that they represent, and contain a
wealth of information – such as the range of hosts or substrates on
which a particular fungus can be found, where it occurs in that region, and
the conditions under which it prospers.
a National Collection of Fungi as part of a country's natural history and
heritage.
#text
T
h
e
p
r
i
m
a
r
y
m
a
n
d
a
t
e
o
f
P
R
E
M
,
n
a
m
e
l
y
b
i
o
s
y
s
t
e
m
a
t
i
c
r
e
s
e
a
r
c
h
o
n
a
g
r
o
-
e
n
v
i
r
o
n
m
e
n
t
a
l
l
y
i
m
p
o
r
t
a
n
t
f
u
n
g
i
,
h
a
s
r
e
m
a
i
n
e
d
v
a
l
i
d
.
I
t
i
s
a
n
e
x
c
i
t
i
n
g
c
h
a
l
l
e
n
g
e
t
o
m
e
e
t
t
h
e
b
r
o
a
d
e
n
e
d
a
n
d
m
o
r
e
c
o
m
p
l
e
x
r
e
s
p
o
n
s
i
b
i
l
i
t
i
e
s
o
f
t
h
e
n
e
w
c
e
n
t
u
r
y
:
s
t
a
y
i
n
g
s
u
f
f
i
c
i
e
n
t
l
y
f
o
c
u
s
e
d
o
n
b
i
o
s
y
s
t
e
m
a
t
i
c
r
e
s
e
a
r
c
h
b
u
t
a
l
s
o
p
r
o
v
i
d
i
n
g
d
e
m
a
n
d
d
r
i
v
e
n
r
e
s
e
a
r
c
h
w
i
t
h
i
n
t
h
e
f
r
a
m
e
w
o
r
k
o
f
t
h
e
g
o
v
e
r
n
m
e
n
t
s
e
t
p
r
i
o
r
i
t
i
e
s
.
D
u
e
t
o
g
r
o
w
i
n
g
d
e
m
a
n
d
s
f
o
r
b
i
o
s
y
s
t
e
m
a
t
i
c
s
e
r
v
i
c
e
s
,
i
t
i
s
c
r
u
c
i
a
l
t
o
m
a
i
n
t
a
i
n
t
h
e
n
e
c
e
s
s
a
r
y
s
y
s
t
e
m
a
t
i
c
c
a
p
a
c
i
t
y
a
n
d
b
a
c
k
u
p
s
y
s
t
e
m
s
.
W
i
t
h
s
u
s
t
a
i
n
e
d
c
a
p
a
c
i
t
y
a
n
d
g
o
v
e
r
n
m
e
n
t
a
l
s
u
p
p
o
r
t
,
a
c
t
i
v
i
t
i
e
s
a
t
P
R
E
M
w
i
l
l
c
o
n
t
i
n
u
e
t
o
b
e
h
i
g
h
l
y
r
e
l
e
v
a
n
t
w
i
t
h
i
n
t
h
e
s
c
i
e
n
c
e
c
o
m
m
u
n
i
t
y
.
T
h
e
r
e
i
s
a
h
u
g
e
s
c
o
p
e
f
o
r
i
n
p
u
t
s
i
n
m
u
l
t
i
d
i
s
c
i
p
l
i
n
a
r
y
i
n
v
e
s
t
i
g
a
t
i
o
n
s
s
i
n
c
e
v
e
r
y
l
i
t
t
l
e
i
s
k
n
o
w
n
a
b
o
u
t
e
n
d
o
p
h
y
t
e
s
,
s
o
i
l
p
o
p
u
l
a
t
i
o
n
s
i
n
v
a
r
i
o
u
s
a
g
r
i
c
u
l
t
u
r
a
l
s
y
s
t
e
m
s
,
f
u
n
g
i
w
i
t
h
o
t
h
e
r
b
e
n
e
f
i
c
i
a
l
p
r
o
p
e
r
t
i
e
s
,
p
o
p
u
l
a
t
i
o
n
s
i
n
e
x
t
r
e
m
e
e
n
v
i
r
o
n
m
e
n
t
s
a
n
d
p
a
t
h
o
g
e
n
s
o
f
i
n
d
i
g
e
n
o
u
s
c
r
o
p
s
.
W
i
t
h
t
h
e
e
x
c
e
p
t
i
o
n
o
f
o
n
e
o
r
t
w
o
s
t
u
d
i
e
s
,
i
n
v
e
s
t
i
g
a
t
i
o
n
s
o
f
f
u
n
g
i
r
e
l
a
t
e
d
t
o
i
n
d
i
g
e
n
o
u
s
b
i
o
t
a
h
a
v
e
a
l
s
o
b
e
e
n
n
e
g
l
e
c
t
e
d
o
v
e
r
t
h
e
p
a
s
t
2
0
y
e
a
r
s
.
R
e
s
e
a
r
c
h
p
r
o
j
e
c
t
s
w
h
e
r
e
d
i
f
f
e
r
e
n
t
d
a
t
a
s
e
t
s
s
u
c
h
a
s
h
o
s
t
p
r
e
f
e
r
e
n
c
e
s
,
d
e
s
c
r
i
p
t
i
v
e
m
o
r
p
h
o
l
o
g
y
,
e
n
v
i
r
o
n
m
e
n
t
a
l
d
a
t
a
,
b
i
o
c
h
e
m
i
c
a
l
-
a
n
d
m
o
l
e
c
u
l
a
r
c
h
a
r
a
c
t
e
r
i
s
a
t
i
o
n
a
r
e
i
n
t
e
g
r
a
t
e
d
s
h
o
u
l
d
p
r
o
v
i
d
e
v
a
l
u
a
b
l
e
p
r
o
d
u
c
t
s
t
o
t
h
e
e
n
d
-
u
s
e
r
s
o
f
t
h
i
s
s
c
i
e
n
c
e
.
here, as well as associated noteworthy incidents and the appointment dates of
the present Staff members of the Mycology Unit.
Acronyms and abbreviations used are the following: |
The Kruger National Park (KNP) is one of the world's most famous wildlife
reserves covering an area of about 20000 km.
to 132 free-ranging mammals (), among them well-known mega-herbivores and large predators,
but also provides the habitat for some 500 species of birds and 2000 species
of higher plants; almost 400 of South Africa's approximately 1100 tree species
can be found in the park (, ).
native trees in various parts of the world during the course of the last 100
years, very little is known regarding the health of native trees in southern
Africa, including of the KNP.
at increasing the basic understanding of the role that diseases might play in
the life cycle of a native tree species in southern Africa.
Southern African Savanna and Nama Karoo biome (biome definition according to
Rutherford ().
savanna habitat distribution is also mirrored by its overall distribution in
Africa ().
areas, it is found on many different soil types, but it is especially abundant
on brackish flats, along rivers on alluvial soils, and it also shows a special
preference for termite mounds (, ).
food source for a great variety of animals both browsing (e.g.
giraffe, black rhino, kudu) and fruit-eating (e.g. warthog, monkeys, birds).
Furthermore, plays a central role in the nutrition of a
number of insects, being for example a crucial food source for the larval
caterpillars of the Atlas Moths and
().
comprises about 100, mostly very drought-tolerant species that are used by
humans for many different purposes.
India where they had been grown for some 400 years.
species, especially in Africa, in
China and India, and in South America, are important
sources of traditional African, Chinese, Indian and South American medicine
and various medicinally active compounds, e.g.
parasites, have been isolated, recently (e.g.
,
,
,
).
were observed mainly on the branches and fruits of trees
in the southern part of Greater KNP.
of the fungus causing the epidemic gall disease on in
the KNP.
determined based on DNA sequence comparisons.
discussed and illustrated comprehensively, for the first time providing
photographic records, and its relative importance is considered.
Africa and extends over about 400 km from the Crocodile River in the south to
the Limpopo River in the north (Figs
,
).
state-owned core KNP of about 20 000 km and bordering private game
reserves that are connected to KNP at its western border, mainly south of the
Olifants River, adding approximately 2000 km.
geology, climate and vegetation structure of KNP can be found in e.g.
(), and Gertenbach
(,
).
Observations for this study were made in the southern part of Greater KNP.
To understand the overall distribution of the disease, we monitored the fungal
galls that could be seen from the park tracks.
distribution of the fungus with the help of GPS-devices during their routine
control tours in June and July 2004.
disease in the national park could be extrapolated although large parts are
not easily accessible.
again for the disease.
sites in the park, a census was conducted in Manyeleti (compare
) in mid May 2004.
the census plots were approximately 2 ha in size, representing dense bushveld,
a dense form of savanna, and the third was situated in a depression next to a
dam.
conspicuous fungal galls.
monitored at these sites.
the surface of the galls and detached spores were mounted either in water,
clear lactophenol, cotton-blue lactic acid or Hoyer's fluid
() and examined
using a Zeiss Axiovision microscope with phase contrast and interference
optics.
symptoms on branches and fruits.
malt-yeast-peptone agar () in Petri dishes.
room temperature or in incubators at 25° C: Cultures of the fungus have
been deposited in the culture collections of the Forestry and Agricultural
Biotechnology Institute (FABI), University of Pretoria (CMW 23046, CMW
23047).
() under that had been deposited in the National Mycological Herbarium in
Pretoria (PREM) were examined. These include herbarium accession numbers PREM
92, 1006, 1214, 2537, 5648, 8789, 10090, 11240, 11812, 15019, 20611, 30667.
Department of Agriculture, Southern Rhodesia, which is now the National
Herbarium of Zimbabwe.
PREM (PREM 59000-WM3450, PREM 59001-WM3488).
morphological characteristics, DNA sequence comparisons were made and
phylogenetic trees were inferred.
infected branches and fruits as well as from cultures using Qiagen Plant Mini
Kit (Qiagen, Hilden, Germany) following the manufacturer's protocols.
mechanical cell disruption, spores were crushed between microscope slides, or
in the case of culture material, by using a micro pestle in an Eppendorf cup,
which was cooled with liquid nitrogen.
strands of the 5' end of the large subunit of the ribosomal gene cluster was
performed using the primer pair LR 0R
() and LR 6 ().
those described by Ritz .
().
done on an ABI PRISM 3100™ sequencer (Perkin-Elmer, Warrington, U.K.).
Contigs of the double-stranded nucleotide sequences were obtained and edited
with the help of Sequencher 4.5 (Gene Codes Corporation, Ann Arbor, Michigan).
All available sequences of were obtained from
GenBank and accompanied by sequences from and
.
used to root the phylogenetic trees.
species names on the phylogenetic tree.
From the above sequences an alignment was produced with MAFFT 5.66
()
using the iterative refinement method with the following settings: the
Needleman-Wunsch algorithm active, 2 tree rebuilding steps, 1000 iterations
and default values for gap opening and gap extension penalties (NW-NS-i:
–nofft –retree 2 –maxiterate 1000 [–bl 62] –op
1.530000 –ep 0.123000).
derived by Bio Neighbour Joining (BioNJ
() with the help
of PAUP 4.0b10 ()
and by Bayesian inference using Metropolis Coupled Monte Carlo Markov Chains
(MC) and MrBayes 3.1.1
(, ), respectively.
joining was determined by 5000 bootstrap replicates.
fitting model (TIMG) of DNA substitution was determined with the Akaike
Information Criterion () implemented in Modeltest 3.7
()
and then used to obtain both the phylogram and the bootstrap consensus tree.
In the case of MC the GTR+I+G model
(, Yang
,
), as the most complex
model, was chosen according to the simulation study results of Huelsenbeck
& Rannala () and
default values for the settings. Three runs of MC
with 1.000.000 generations were performed, and every 100
generation was sampled resulting in 10001 trees.
discarded and the remaining 9000 trees were used, well after the chains had
converged to stationarity, to estimate the probability
distribution. One MC analysis was run over 6.000.000 generations
to marginalize the chance that we might have missed a higher plateau of
stationarity.
from 50.000 trees and 10.001 trees were discarded as “burn-in”.
The sequences derived in this study have been deposited in GenBank with the
following accession numbers (DQ334805, DQ334806), the alignment is lodged in
TreeBase (study accession number=S1474, matrix accession number=M2652).
predominantly infects branches and fruits but could also be found on the veins
and peduncles of leaves.
commonly affected while in most cases the infections were randomly distributed
throughout the trees.
that produced an abundance of white flour-like spores.
few millimetres in diameter to over the size of a golf ball, seemingly
correlated to the size of the plant organ affected (compare
).
Har. & Pat.
that resides in the .
easily recognizable.
out of diverse organs of and the morphology of the
multi-celled spores (Figs
,,).
The spores are frequently composed of four longitudinally arranged sections,
each of which is made up of several cells.
completely odd-numbered spores with extraordinary shapes (compare
) and the general spore
shape might be best characterized by “diverse and uneven”.
were (15-)18-23(-28) μm in size.
found elsewhere (, ).
of the presumed basidiospores is covered more or less densely with small warts
() that cannot be seen
with the light microscope.
producing conidia and yeast cells (for detailed microscopical descriptions of
these compare Malençon
().
slowly (. 1 mm diam after 5 – 7 d).
salmon-coloured compact hyphal mass displaying a brain-like surface
structure.
collected in the following provinces of South Africa: Limpopo, Mpumalanga,
KwaZulu-Natal and Gauteng. They include the type collection of (PREM 92).
labelled as P. Magnus before being transferred to Har. & Pat. The remaining three specimens (PREM 92, 2537,
5648) had been labelled as P. Magn.
the name that was used in Doidge's compendium on the Southern African fungi
().
not a valid name as discussed below.
() also lists a collection
from Zimbabwe that was reported from .
March 2004, in the area of the camps Skukuza, Orpen, and Lower Sabie.
of the disease was reached in May/June when it was detected to have spread
over a distance of about 200 km on the north-south axis and the entire
east-west extension of the park (). Infections remained clearly visible on trees until August.
census taken at the two plots representing dense bushveld revealed that all 43
and 53 trees, respectively, in these plots were diseased.
next to the dam, all 38 trees counted were diseased.
disease prevalence of 100 % in the region.
plots were heavily infected, however medium-infected trees and trees with
hardly any infection could also be found.
in the park.
trees undertaken during April and June 2005.
reference plots were, therefore, closely investigated on foot.
trees in the two bushveld reference plots that had displayed 100 % disease
incidence in the previous year showed fresh infections.
that could be germinated on MYP agar were obtained from two galls from
previous year infections.
infected branches, easily detectable by the presence of old galls, had died.
There was also practically no fruit production in 2005, while the trees had
produced abundant fruit at the same time in the previous year.
trees close to the dam had recovered more effectively than the trees at the
two other census sites.
small numbers of weak infections could be found on the fruits of six
trees.
fruits) of different trees from different sample sites as well as from
cultures grown from spores.
the nuc LSU rDNA with a length of about 1000 bp.
were identical.
in GenBank that had been deposited for a study of the
().
from material collected by Johannes van der Walt in 1990 around Skukuza camp,
also in KNP.
the D1/D2 region, due to the length of the sequences deposited in Genbank, and
comprised 508 base pairs.
MC were identical. Tree topologies obtained by MC
versus BioNJ were almost identical.
specimens were resolved as a monophyletic group in
MC, whereas in BioNJ was the sister
group to the three samples of together with
the spp. as a whole.
probabilities and bootstrap values were similar (compare
).
support values for this study are those for (1.00
posterior probability / 99 % bootstrap) and those for the split between
(1.0/100) and the rest of the
(1.00/100).
monophyletic by MC, but with low support values, while in BioNJ
only was monophyletic, however, also just weakly
supported by bootstrap support.
within and . cf. ,
respectively. The monophyly of the outgroup genera (1.0/100)
and (0.96/100) was highly supported.
did not form a monophyletic group with
, which resides in the same family, the
, but with , which resides in the
.
is an unusual fungus with poorly known ecology and
infection biology.
pathogen in an ecologically important and sensitive part of South Africa,
where the disease raised concern amongst rangers and naturalists.
an opportunity to critically review what has been known about it, to clarify
some misconceptions of earlier studies, and it thus provides a foundation for
future studies. has had a complicated systematic
history.
resided in the Ascomycota for a period of time.
(), in his exceptionally
thorough study on the life cycle and systematic relationships of the fungus,
gave also a first account of the systematic history of .
However, he was not aware how frequently the fungus had been collected in
South Africa.
occurrence.
=
() where it had been collected on fruits in March and November
1903 by Chevalier.
() described based on material collected by Pole Evans in 1906 at Zoutpansberg
(Limpopo Province), South Africa, on sp., which was later
also identified as
().
() cited the fungus under
(Magnus) Höhn.
justification for this new combination.
von Höhnel (1910, )
himself argued strongly for the conspecificity of and
with having priority, and therefore
never made a new combination for this fungus.
explanation for this inconsistency is that an error was inadvertently made
with the epithet of Magnus' invalid description being mistakenly attached to
the valid older genus name.
() is, therefore, a
synonymous for .
believed to be with the ascomycetes, and it was relegated to either the
(), the
(,
) or the
().
() noted that some
features of the hypertrophic growth resembled that of .
When Malençon ()
received abundant fresh material of the fungus collected by Th.
Dakar, Senegal, he performed an extensive morphological study concluding that
the “conidiophores” producing the abundance of white spores were
in reality basidia, and the spores hence basidiospores.
connected systematically with gall-producing fungi
described from of Central and South America in the genera
and as Maublanc had proposed
before him, but then still under the ascomycetous
().
he had re-examined and
, Malençon named the family
(now
() in honour of Alfred
Lendner.
affinities of one of its members and had introduced the name
(), which was reduced to synonymy with . H.
Sydow, like Maublanc, originally retained in the
, but was convinced by Lendner's interpretations,
concluding “Ich glaube nun, daß und
Basidiomyceten sind” and therefore transferred them
accordingly ().
recently was transferred from the
to the
().
Therefore, the currently comprise five genera and
seven species (compare ).
monophyletic by Begerow .
(), but the statistical
support for the group in that study was low (obtaining a maximum of 59 %
bootstrap).
both by bootstrap and Bayesian posterior probabilities.
obviously arisen from the larger taxon sampling within the
, while using the same gene region.
especially possible, because additional sequences of and
had been deposited to GenBank by Nagao, Sato and
Kakishima in 2004.
occurrence in arid savanna biomes.
of the that inhabit moist sub-tropical and tropical
forests outside Africa attacking various genera in the laurel family while
so far has only been reported with certainty from
, a member of the .
biology, regarding its biogeography, ecology and host specificity, is
reflected by the phylogenetic position of , which is a
sister taxon separated from the other members of the family that parasitize
Lauraceae, by a long genetic distance and perfect support values
().
genus should be considered.
() of infecting ?) is interesting
in terms of the capacity of the pathogen to move to new hosts.
validity of the report could not be tested in this study and is regarded as
rather doubtful.
implications for countries like China and India where are extensively grown for fruit production.
convinced that is a rare fungus (“en
réalité est un champignon peu commun”).
knew of no additional collections subsequent to the first collections from
Chad and South Africa in 1903 and 1906, respectively, and the material that
was sent to him from Senegal almost 50 years later.
collections made in South Africa over a considerable geographic range (compare
above) and one in Zimbabwe between 1910 and 1938 documented in Doidge
() clearly escaped
Malençon's notice.
fungus is based on the incorrect assumption that the fungus had been collected
only twice before he received the material from Dakar.
was also collected in more recent years in KNP by Johannes van der Walt in
1974 and again in 1990 close to the camp-sites “Skukuza” and
“Lower Sabie”, respectively.
the fungus was almost absent from KNP in 2005, thus showing great fluctuations
in its prevalence in different years.
extensive spread of the fungus in 2004 was boosted by much higher rainfalls
between January and April 2004, compared to the same months in 2005 (data not
shown).
disprove this hypothesis.
previously believed, is provided by old galls found on branches of .
in recent years.
probably mainly determined by the number and activity of mycologists in areas
of Africa, where grows and we assume that it most likely
could be found in the whole range of its host's distribution if extensively
looked for.
in tropical America where specimens have been
recollected in Costa Rica in the late 1990s after a period of about 60 years
absence of reports of these fungi
().
production of large galls that we predicted large-scale death during the dry
winter months.
appeared to have recovered well.
from the severe infection by C. in 2004 is consistent with
observations of rapid recovery and vigorous resprouting of Buffalo Thorn after
fire damage.
after they had been heavily infected at the two bushveld plots.
that stress due to infection by C. reduced plant vigour
and consequently flower and fruit production in 2005.
close to the dam had recovered well and produced abundant fruit, despite their
being heavily infected in 2004, is probably due to favourable edaphic
conditions at this site, with higher water availability, which reduced the
impact of stress due to the disease.
This study represents the first report of an epidemic caused by , a fungus previously believed to be extremely rare.
to views regarding its rarity, we were able to show that has been collected regularly, especially between 1906 and
1938, in various parts of South Africa.
the trees in reference plots in KNP is being monitored and it is hoped that
during coming years new knowledge concerning the ecology of the pathogen and
the conditions favouring its spread will emerge.
useful in developing hypotheses regarding modes of distribution and ecological
factors that might have an effect on the survival and spread of the
fungus. |
(M.J. Wingf., Crous & T.A. Cout.)
M.-N. Cortinas, M.J. Wingf. & Crous
()
causes a serious stem canker disease on species.
disease was first reported in 1987 in South Africa, and the pathogen was
described as a species of , namely
M.J. Wingf., Crous & T.A. Cout.
().
initially occurring only on a single clone, but
ultimately occurring in all parts of South Africa with a sub-tropical climate,
and on a wide variety of species and hybrids.
research has thus been undertaken to better understand the disease and to
develop disease-resistant planting stock through breeding and selection
programmes (Van Zyl
,
).
of disease.
green stem tissue in the upper parts of trees.
elliptical, and the dead bark covering them typically cracks, giving a
“cat-eye” appearance ().
giving rise to the production of epicormic shoots and ultimately trees with
malformed or dead tops.
they penetrate the cambium to form black kino-filled pockets.
pockets with irregular borders of infected tissue can be seen within the
infected wood of trees coincident with the annual rings
().
can be seen on the surface of dead bark tissue
(), from where black
conidial tendrils exude under moist conditions.
and dematiaceous, appearing black in colour when seen in mass on the host or
agar media.
disease has been found in many other countries.
South Africa was in Thailand where it is associated with typical symptoms on
().
other countries in Africa (Alemu
,
), South and Central
America (, ), as well as South-East Asia
(,
Cortinas ,
)
().
disease remains unknown in the areas of origin of ,
although it might occur there at very low and undetectable levels
(,
).
morphological characteristics of the pathogen.
pigmented aseptate, ellipsoidal conidia arising from percurrently
proliferating conidiogenous cells were consistent with species placed in
Corda.
possible to recognise that the fungus has a clear phylogenetic position in
Johanson ().
which are anamorphs of
spp. This realisation has led to the transfer of to Crous & M.J. Wingf.
() is a well-recognised
anamorph and its circumscription was amended to
include species with pycnidioid conidiomata.
including A. Maxwell, (Cooke) Hansf., (Thüm.) Lindau,
(Cooke) Hansf.
Crous & M.J. Wingf., and (Cooke & Massee) Crous, F.A. Ferreira & B.
().
variable in morphology ()
and pathogenicity to different clones (Van Zyl 1997,
, Van Zyl 2002a).
based on the nuclear ribosomal small subunit (18S) and internal transcribed
spacer regions and the ribosomal 5.8 gene (ITS1, 5.8S, ITS2) had shown that
was monophyletic (Van Zyl 2002b,
).
understanding of the geographical range of continues to
expand.
DNA sequence comparisons and these have provided the opportunity to
re-consider the taxonomic status of , and the variation
observed in its morphology and pathogenicity.
can be retained when applying multi-gene analyses using
a large collection of isolates not previously available.
objective, individual and combined phylogenetic analyses using the ITS region,
β-tubulin gene (BT2), the elongation factor 1-α (EF1α) gene,
and the mitochondrial ATPase 6 (ATP6) gene, were carried out.
and other phenotypic characters were also considered.
distribution of .
known to be closely related to
were also included ().
All these isolates were obtained from the culture collection (CMW) of the
Forestry and Agricultural Biotechnology Institute (FABI), Pretoria, South
Africa.
isolated from lesions taken from the stems of trees in
South Africa and Uruguay.
sterile distilled water, and spread on the surface of Petri dishes containing
MEA (20 g/L Biolab malt extract, 15 g/L Biolab agar).
germinating conidia were transferred to fresh MEA plates and incubated for 30
d at 25 °C.
deposited at the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The
Netherlands ().
Nomenclature, descriptions and illustrations were deposited in MycoBank.
Petri dishes, freeze dried, frozen in liquid nitrogen and ground to a fine
powder.
() was simplified as
follows: DBE extraction buffer (200 mM Tris-HCl pH 8, 150 mM NaCl, 25 mM EDTA
pH 8, 0.5 % SDS) was added directly to the ground mycelium and incubated for 2
h at 80 °C (or until pigments changed colour from green to red).
extraction-DNA enrichment procedure, one volume of phenol was used first and
one volume of a 1:1 phenol-chloroform solution thereafter.
().
ribosomal DNA was targeted using the primers ITS1: 5' TCC GTA GGT GAA CCT GCG
G and ITS4: 5' GCT GCG TTC TTC ATC GAT GC
().
Exons 3 to 6 and the respective introns (BT2) of the β-tubulin gene
region were amplified using the primers BT2A: 5' GGT AAC CAA ATC GGT GCT GCT
TTC and BT2B: 5' AAC CTC AGT GTA GTG ACC CTT GGC
().
The intron sequence of the EF1-α gene was amplified using the primers
EF1-728F: 5' CAT CGA GAA GTT CGA GAA GG and EF1-986R: 5' TAC TTG AAG GAA CCC
TTA CC () and intron 2 and exon 3 of the ATP6 gene was amplified using
the set of primers 5'ATT AAT TSW CCW TTA GAW CAA TT and 5'TAA TTC TAN WGC ATC
TTT AAT RTA developed by Kretzer & Bruns
().
PCR reactions were prepared in a total volume of 25 μL including 1.5
μL of genomic 1/10 dilution DNA, 1 U of polymerase, 10 ×
buffer, 10 pmol of each primer, 0.8 mM of each dNTPs, and 2.0 mM
MgCl (ITS) or 4.0 mM MgCl (BT2, EF1-α, ATP6).
PCR amplicons were visualised under UV light on 1 % or 2 % agarose gels.
Different cycling conditions were used for the various gene regions.
ITS region, 96 °C, 3 min initial denaturation and cycles of 95 °C, 30
s, 54 °C, 30 s, 72 °C, 1 min were repeated 10 times followed by 25
cycles of 95 °C, 30 s, 56 °C, 30 s, 72 °C, 1 min with 5 s
extension after two cycles.
also included.
the annealing temperature to 50 °C.
initial denaturation and cycles of 95 °C, 30 s, 57 °C, 45 s, 72
°C, 45 s were repeated 40 times.
cycles of 95 °C, 30 s, 54 °C, 45 s, 72 °C, 45 s were repeated 40
times with 5 s extension after two cycles.
72 °C was included.
Alignments of sequence data were made using Clustal W under MEGA 3.0
()
and manually adjusted.
GenBank ().
were deposited in TreeBASE.
individual and combined partitions.
generated using PAUP v. 4.0b10 ().
the steepest descent option and the TBR swapping algorithm.
were equally weighted and treated as unordered.
nodes in the trees was tested with 1000 bootstrap replicates.
analyses were conducted using MEGA 3.0
().
Pairwise distances were estimated using the Kimura with two parameters model
().
distribution α = 0.5 was used to take into account the differences in
mutation rate among sites, due to the mix of coding and non-coding sequences
present in the analysed fragments.
performed with Minimum Evolution ().
missing data.
statistical support of the nodes in the phylogenetic trees.
to midpoint.
().
sensitive to under-specification than over-specification of the evolutionary
model () when calculating the posterior probabilities.
a time-reversible complex model with gamma-distributed rate variation (GTR + I
+ G) was selected to combine the data sets.
allows the consideration of different rates of substitutions among sites,
different nucleotide frequencies, and differences in the rate of substitutions
among nucleotides. Therefore, four sets of analyses were run in MrBayes 3.1.1
(, ) calculating marginal posterior probabilities
using the selected time reversal GTR + I + G model of nucleotide substitution
(, Yang
,
) and default values for
the prior settings.
generations. Trees and parameters were recorded every 100 generations.
Likelihood stability was reached at 30 000 generations.
generations was then established as the “burn-in” period
(represented by 3001 trees).
from the remaining sampled trees.
times.
on the tree and the sequences of Crous
& M.J. Wingf. and Crous & M.J. Wingf.
as outgroups.
Descriptions are based on sporulation .
30 measurements (× 1000 magnification) were made of structures mounted
in lactic acid, the 95 % deviation determined, and the extremes of spore
measurements given in parentheses.
assessed after 25 d on MEA at 25 °C in the dark, using the colour charts
of Rayner ().
four different gene regions were aligned to study fixed polymorphisms.
Alignments of 469 bp (ITS), 308 bp (BT2), 254 bp (EF1-α) and 656 bp
(ATP6) were generated.
β-tubulin gene was missing in all isolates studied.
the characters defined two groups among the isolates based on the fixed,
shared polymophisms.
China, Thailand, Vietnam and Malawi and a second group comprised isolates from
Uruguay, Argentina, Hawaii, Uganda and Ethiopia.
the different fixed characters in the alignments for the various isolates are
shown in .
characters were found at the ITS region, eleven were found in the BT2 dataset,
eight were found at the EF1-α intron where a 20-base-pair indel was also
found ().
position was found in the ATP6 region.
produced very similar topologies to those of the distance trees.
only distance trees are presented ().
established.
for the isolates considered.
represented isolates from South Africa, Malawi, Mexico, Thailand, Vietnam and
China (Group1) and those from Uruguay, Argentina, Hawaii, Ethiopia and Uganda
(Group 2).
the ITS tree.
100 % support, respectively.
distinguished although only one of these had strong support (100 %).
having reasonable support included isolates from Vietnam, Mexico, Malawi,
China and South Africa.
the Group 1 and Group 2 clusters in the ITS, BT2 and EF1-α trees.
sub-clusters had greater than 70 % bootstrap support only in the BT2 tree.
assortment of isolates within the sub-clusters was different in different
trees.
were carried out ().
reconstructed trees included the collection of
isolates together with spp.
1 and a 100 % bootstrap value separated the isolates
from the rest of spp.
half-compatible trees showed two major groups representing isolates from South
Africa, Malawi, Mexico, Thailand, Vietnam and China (Group1) and those from
Uruguay, Argentina, Hawaii, Ethiopia and Uganda (Group 2) supported by
posterior probabilities of 1 and 0.95 and 98 % and 100 % bootstrap values,
respectively. A rich internal topology was found within these two groups.
Numerous sub-clusters were supported with high probabilities and bootstrap
values.
locality.
sub-clusters were formed.
was different at some of the tested temperatures after 6 wk
().
was found at 5 °C, optimal growth occurred between 20 and 25 °C, and
the diameters of colonies decreased when they were incubated at temperatures
of 30 °C and above.
seen at 10 °C where the Uruguayan isolates grew more rapidly than isolates
from South Africa.
achieved their maximum diameter.
smaller for the Uruguayan isolates.
African and Uruguayan isolates was observed at 35 °C.
the Uruguayan isolates hardly displayed growth whereas South African isolates
reached between 10 and 20 mm diam.
and Ethiopia, were very similar to those comparing isolates from South Africa
and Uruguay.
origins were obvious at 35 °C ().
phylogenetically related to those from South Africa and those from Ethiopia
are related to those from Uruguay.
morphologically variable in culture.
isolates from South Africa and Uruguay, but it was possible to recognise some
characteristics apparently exclusive to the Uruguayan isolates.
distinctly different conidial and conidiogenous cell characteristics were
found when isolates from Uruguay were compared with those of from South Africa ().
between [conidia (4–)4.5–5(–6) ×
2–2.5(–3.5) μm] and the isolates from Uruguay [conidia
(4–)5–6(–7.5) × (2–)2.5(–3) μm].
Uruguayan conidia, however, had a larger maximum length, reaching 7.5 μm (6
μm for ).
slightly wider (3.5 μm) as opposed to those from Uruguay, which were an
average of 3 μm.
Uruguay is that it has sympodial polyphialidic conidiogenous cells, which is
different to , which has percurrently proliferating
monophialidic conidiogenous cells.
isolates, encompassed within the fungus currently treated as .
Thailand, Vietnam, China and Mexico.
Uruguay, Argentina, Hawaii-U.S.A., Ethiopia and Uganda.
be separated by characteristics of growth in culture, morphology and growth at
different temperatures.
.
cultures were deposited.
DNA sequence data are available, and that are tied to herbarium specimens to
serve as epitypes.
represents a distinct taxon that is described below.
M.-N. Cortinas, Crous &
M.J. Wingf., MycoBank
.
,
.
: Named after the gauchos people of South America that
live in the same area where this species is distributed and where it was first
collected.
KwaZulu-Natal Province and the “Zulu” people of South Africa.
(4–)5–6(–7.5) × (2–)2.5(–3) μm et
phialidibus nonnumquam sympodialiter proliferentibus distincta.
diam, with a raised, red-brown border. pycnidial to
somewhat acervular, subepidermal, single, rarely aggregated, occurring in
necrotic tissue, globose to slightly depressed, becoming erumpent, up to 120
μm diam, exuding conidia in a long cirrus; conidiomatal walls composed of
2–3 layers of medium brown ; opening by a
central ostiole or irregular rupture; ostiolar region lined with thick-walled,
brown, smooth, septate hyphae that are sometimes branched below, 3–4
μm wide, with obtuse ends that flare apart (upper 1–6 cells).
subcylindrical, subhyaline to medium brown, smooth to
finely verruculose, 0–3-septate, unbranched or branched below,
10–20 × 3–6 μm. subhyaline
to medium brown, dolilform to subcylindrical, smooth to finely verruculose,
mono- to polyphialidic, proliferating percurrently, with several percurrent
proliferations near the apex. medium brown, thick-walled,
finely verruculose, broadly ellipsoidal, apex obtuse to subobtuse, base
subtruncate to bluntly rounded, (4–)5–6(–7.5) ×
(2–)2.5(–3) μm; base frequently with a minute marginal
frill.
: , El Tarugo, bark of 1-yr-old
tree, Feb. 2005, M.J.
, cultures ex-holotype CMW 17331–17332; La Herradura,
,
cultures = CMW 17542–17543; ibid.,
,
cultures
= CMW 17545, CMW 17544; La Juanita,
,
cultures
= CMW 17558, CMW 17559; ibid.,
,
cultures = CMW 17560–17561.
: Colony characteristics on MEA at 25
°C are variable. Colony colours were similar to those of (Van Zyl .
, 2002).
range from greyish yellow-green, dull green, isabelline, greenish olivaceous
to grey-olivaceous; colonies in reverse range from dark grey, dark olive-grey
to dark green ();
margins are smooth, regular or irregular.
characteristic white outer zone of aerial mycelium
().
develop smoother surfaces with white aerial mycelium; some strains produce a
diffuse yellow pigment in MEA.
: [conidia
(4–)5–6(–7.5) × (2–)2.5(–3) μm] can
readily be distinguished from [conidia
(4–)4.5–5(–6) × 2–2.5(–3.5) μm] by its
slightly longer conidia, and the presence of sympodial polyphialidic
conidiogenous cells (Figs ,
).
readily at 10 °C, with hardly any to no growth at 35 °C.
grows more slowly at 10 °C, and faster at 35 °C
than , and strains of do not form
conidiomata in culture.
(M.J. Wingf., Crous & T.A.
Cout.) M.-N. Cortinas, M.J. Wingf. & Crous, Mycol. Res. 110: 235. 2006.
Figs ,
. [as
].
: M.J. Wingf., Crous &
T.A. Cout., Mycopathologia 136: 142. 1997.
: South Africa, KwaZulu-Natal, Kwambonambi, Teza
nursery, bark of 1-yr-old tree, Jan. 1996, M.J.
IMI 370886 ; KwaZulu-Natal, Kwambonambi, ,
Feb. 2005, M.J.
= CMW
17531, CMW 17530; , culture
= CMW
17528, CMW 17529; , culture
= CMW
17320, CMW 17319; , culture
= CMW
17526, CMW 17527.
from different parts of the world and based on multiple gene regions have
shown clearly that this material represents at least two discrete taxa.
species are described based on material from South Africa and Uruguay, but
both taxa include collections from many different countries.
China and Mexico.
occurs not only in Uruguay but also in Argentina, Hawaii-U.S.A., Ethiopia and
Uganda.
can clearly be distinguished from each other based on morphological
characteristics and growth characteristics in culture.
of isolates used in this study into two
distinctive groups.
.
is absent in The p-distance
among the isolates considered in this study
displayed a range of 0 to 1 % divergence in ITS sequences, 0–8 % for BT2
sequences, 0–24 % for EF1- α sequences and 0–4 % for ATP6
data-matrices respectively.
variation within to suspect that more than one taxon
was represented in the collection of isolates.
consistent with values used in previous studies
(, Barnes
. 2005) to separate taxa.
Uruguay.
polyphialidic, sympodially and percurrently proliferating conidiogenous cells
as opposed to the monophialidic, percurrently proliferating conidiogenous
cells in .
consistently longer than those of (Figs
,
).
the contrary, isolates of grow well at 35 °C,
whereas those of barely grow at this temperature.
.
considered sibling species only in terms of the fact that they are
ecologically and morphologically very similar.
sibling species occur in taxonomic groups varies depending on the group of
fungi studied.
implemented (see ).
collections of morphologically similar taxa that can only be discriminated
based on minute morphological details or characteristics in pure culture.
“” H.J. Swart (Crous . ,
,
– this volume),
which will be treated elsewhere (Cortinas . in prep.).
and to a lesser extent showed internal structure in the
individual and combined trees.
well-supported in the BT2, ATP6 and combined trees.
phylogenetic species concept, it would be possible to recognise additional
species especially in this complex.
provide additional names before robust population biology studies are
available.
worldwide ().
in a very limited location and spread rapidly, resulting in very substantial
losses to the local forestry industry.
damage to plantations in other countries such as Argentina and Uruguay.
thus intriguing that there are two distinct fungi associated with
indistinguishable symptoms.
known to occur in the native range of The evidence from
this study shows that the two fungi are closely related and have adapted
differently based on some ecological factor.
spp.
origin would be on or a host closely related to it. A
similar situation has emerged for species of Gryzenh.
& M.J. Wing. ().
but that appear to have originated on a wide variety of
woody plants in the order
(,
, ).
previously been recognised as belonging to the single taxon has important
consequences for disease control and quarantine.
suggested that the fungus originated in South Africa, and that it was
restricted to that country ().
other countries has often been linked to the movement of plant material and
particularly seed to other countries.
experimentally that is moved on seed, this appears to be
a likely mode of global distribution.
seed, which is variably controlled and monitored.
and have now wide geographic
distributions and this implies that they have been spread from one or a number
of sources.
movement to new countries and areas. |
The (restios) is a monocotyledonous family
distributed in the Southern Hemisphere, which includes more than 30 genera and
about 400 species ().
in the south-western tip of South Africa
().
This area, comprising 90 000 km and known as the Cape Floral
Kingdom, is home to more than 8 500 plant species, of which 5 800 are endemic
().
contributing 80 % of its species.
fynbos are indigenous.
thatching, matting or brooms ().
initiated in 2000 with an emphasis on two major plant groups: the
dicotyledonous and the .
coelomycetous anamorphs including the so-called pestalotioid fungi.
Pestalotioid fungi are defined as those having multi-septate, more or less
fusiform conidia with appendages at both or either ends, resembling those taxa
accommodated in De Not.
Sacc., Clem., and
M.E. Barr.
growing in fynbos. Four new and four known species are treated.
phylogenetic relationships between these and other related pestalotioid fungi,
DNA sequence data were generated for the partial 28S gene and ITS region
(ITS1, 5.8S, ITS2) and phylogenetic analyses were applied.
undisturbed areas of the fynbos during 2000–2002.
collected in paper bags.
assistance of curators of the Kirstenbosch Botanical Garden or by using Intkey
().
Fungal isolates were grown in 1 mL 2 % malt extract broth in three 2 mL
Eppendorf tubes for up to 7 d.
following a modification of the method of Möller .
().
ITS4 () were used to amplify part of the nuclear rDNA spanning the
3'end of the 18S rDNA, the internal transcribed spacers, the 5.8S rDNA and a
part of the 5' end of the 28S rDNA.
amplify part of the large subunit nuclear rDNA
().
Amplification reactions were started with 3 min denaturation in 94 °C,
followed by 30 cycles of 30 s denaturation at 94 °C, 1 min annealing at 55
°C and 1.5 min extension at 72 °C, and 10 min extension at 72 °C.
For the amplification of partial 28S rDNA, the annealing temperature was
adjusted to 50 °C. For specimens that could not be cultivated, direct PCR
was performed from conidia with increased cycles (40 cycles).
were separated by electrophoresis at 80–90 V for 15 min in 1 % (w/v)
agarose gel in 1× TAE running buffer (0.1 mM Tris, 0.01 mM EDTA, 2 %
SDS, pH 8.0) and visualised under UV light.
().
using the same primers used in the amplification reactions except for the
reverse primer of the partial 28S rDNA where LR5 was used
().
Sequencing reactions were performed using a PRISM™ Dye Terminator Cycle
Sequencing Ready Reaction Kit (Perkin-Elmer, Warrington, U.K.).
sequence data were generated with an ABI Prism 3100™ automated DNA
Sequencer (Perkin-Elmer, Norwalk, Connecticut).
processed using the Sequence Navigator v. 1.0.1 software package (Perkin-Elmer
Applied BioSystems, Foster City, California).
Sequences were assembled and aligned using ClustalW algorithm in MEGA v.
3.1 () and finally optimised by eye.
sequence data were done in PAUP (Phylogenetic Analysis Using Parsimony) v.
4.0b10 ().
parsimony analysis, alignment gaps were treated as fifth character and all
characters were unordered and of equal weight.
for all data sets using the heuristic search option with 100 random taxa
additions and tree bisection and reconnection (TBR) as the branch-swapping
algorithm.
() was
performed with adjusted settings: proportion of invariable sites (I) = 0.6169,
gamma distribution (G) = 0.5970, base frequency equal, rate matrix 1.00,
2.3919, 1.00, 1.00, 5.5792 for partial 28S rDNA; I = 0, G = 0.3769, base
frequency equal, substitution model (Ti/tv ratio) 1.6846 for ITS regions.
These models were chosen as suggested by MODELTEST v. 3.5
().
Branches of zero length were collapsed and all multiple, equally parsimonious
trees were saved. The robustness of the trees obtained was evaluated by 1000
bootstrap replications ().
(RC).
listed in .
S1442).
: Approximately 550 bases were determined for the isolates as
indicated in .
manually adjusted alignment consisted of 29 taxa (including the two outgroups)
and 612 characters including alignment gaps, of which 247 were
parsimony-informative, 111 were variable and parsimony-uninformative, and 254
were constant.
parsimonious trees, one of which is presented
().
four clades referred to as a Steyaert clade, a
-A clade, a -B clade and a
Cooke clade with 99 %, 100 %, 100 % and 100 % bootstrap
support, respectively.
The clade consisted of two sub-clades.
sub-clade included five species from our collections (100
% bootstrap support). And the other included (Pers.) S.
Hughes and species of Tassi with 96 % bootstrap support.
The -A clade included six
(.) species having conidia with concolorous median cells, and
(Richatt) S.
versicolorous median cells.
species having conidia with versicolorous median
cells, and formed a sister clade to (Sawada) Steyaert,
which had conidia with concolorous median cells and knobbed apical appendages
(R. Jeewon, pers. comm.). The (.) clade
included S.
(.) (Loos) Shoemaker which has a characteristic
of , centric apical and excentric basal appendages.
distance tree gave the same topology.
for both parsimony and distance analyses except for the branches supporting
two isolates and four species
within the clade.
distance analysis (95 % and 92 %, respectively) than in parsimony analysis (63
% and 58 %, respectively).
: Approximately 850 bases were determined for the isolates as
indicated in .
manually adjusted alignment contained 26 taxa (including the two outgroups),
and 856 characters including alignment gaps, of which 106 were
parsimony-informative, 55 were variable and parsimony-uninformative, and 695
were constant.
of which is presented ().
Ingroups consisted of three clades: a clade, a
clade, and a basal clade with 94 %, 100 % and
51 % bootstrap support, respectively.
Corda species and a species
(teleomorphic state of either or
).
sub-clades with and (Westend.)
Steyaert as basal taxa. The one sub-clade included sp.,
- (Tubeuf) Steyaert, and a group of (Tubeuf) Steyaert and S. Lee &
Crous with 100 % bootstrap support.
clade contained a species of
L. Cai, R. Jeewon & K.D.
) and two species (teleomorph
connection unknown).
the parsimony trees in grouping three clades, except for the rearrangement of
taxa within each clade.
except for the branch supporting two isolates which
received higher support in distance analysis (99 %) than in parsimony analysis
(54 %).
acervuloid–pycnidioid conidiomata were collected in this study.
were identified as belonging to three known genera representing eight species.
Of these, four are treated as new taxa, and they are described below.
characteristics of the respective species are summarised in
.
(Richatt) S.
MycoBank
.
.
joined, sub-epidermal, remaining immersed, visible at the surface by dark
exuding conidial masses; in section subglobose to ellipsoid, 193–366
× 178–215 μm. pseudoparenchymatous, in
section 13–16(–28) μm thick, consisting of 3–several
layers of pale brown, moderately thick-walled cells of arising from the entire periphery of the inside of the
conidiomata, reduced to conidiogenous cells or poorly developed, branched at
the base, ampulliform. annellidic, hyaline,
discrete or integrated, smooth, lageniform to cylindrical, 7–12 ×
1–2 μm. fusiform,
(22–)24–25(–29.5) × (6.5–)7(–8.5) μm
(av. 24.5 × 7.2 μm, ratio 3.4: 1), 4-septate; apical cell hyaline,
conical to trapezoid, 3–5 × 3–4 μm, smooth, thin-walled;
median cells brown, versicoloured, with third and fourth cells from the base
darker than the second cell (at times the third cell darker than the fourth
cell), doliiform, 15–17 × 6–7 μm, smooth but lumpy
(possibly due to desiccation), moderately thick-walled; basal cell hyaline to
subhyaline, obconical, 4–5 × 4–5 μm, smooth, thin-walled.
2–3, inserted along the upper half of the
apical cell, arising at different points, unbranched, flexuous, 13–19
× 1 μm, attenuated. single, centric,
unbranched, 2–6 × 1 μm, attenuated.
: , Western Cape Province, De
Hoop Nature Reserve, culm litter of , 28 Feb.
2002, A.
= CMW
18285; Kirstenbosch National Botanical Garden, culm litter of
, 3 Dec. 2001, S.
culture
= CMW18022.
: (),
().
: The two collections are morphologically most similar to the
following seven species as treated by Nag Raj
() and Guba
(): Nag Raj, (Ces.) Steyaert, Nag Raj, (Gucevič) Nag Raj,
(.) Servazzi [= (Servazzi) Steyaert], Ellis & Everh.,
and .
appendages that originate in three levels (tiers) on the apical cell, has larger conidia (25–10 × 9–11 μm),
has smaller conidia (25–25 × 5.5–7
μm) and distinct striations on second and fourth cells, has verruculose, pale brown second and fourth cells, and
has longer apical appendages (16–26 μm).
has third and fourth cells that are always darker
than the second cell, whereas our collections often had the third cell being
darker than the fourth cell.
collections best fit the characteristics of .
(), and the
recircumscription of and by Nag
Raj (), it is clear that
resides in , a decision that is
also supported by the DNA sequence data presented in this study.
(McAlpine) Nag Raj, Coelomycetous
anamorphs with appendage-bearing conidia: 798. 1993.
.
≡ McAlpine, Proc. Linn. Soc. N. S. W.
79: 140. 1954.
remaining immersed, visible at the surface by dark exuding conidial masses,
lifting up the epidermis; in section low conoid, 187–366 μm wide.
pseudoparenchymatous, consisting of a few layers of
brown, thick-walled, globose to angular cells, 9.5–21 μm thick;
lateral tissue absent. arising from the basal stroma,
cylindrical, 4–10 × 2–3 μm.
cylindrical to lageniform, 14–20 × 2–4 μm.
fusiform, straight or slightly curved,
(15–)19–20.5(–25) × (5–)6–7 μm (av.
19.8 × 6.7 μm, ratio 3: 1), 4-septate; apical cell hyaline, conical,
2–3 μm long, 2.5–3.5 μm wide at the base, smooth,
thin-walled; median cells brown, concoloured, doliiform, 12.5–16 ×
7–8 μm (second cell from the base (4–)5–6(–7) μm
long, av. 5.4 μm; fourth cell (3–)5(–7) μm long, av. 5.0
μm), echinulate, thick-walled, at times wall extended like bubbles; basal
cell hyaline, obconical with truncate end, 2–4 μm long, 3–3.5
μm wide at the top, smooth, thin-walled. single,
centric, unbranched, 30–38 × 1–1.5 μm, flexuous,
attenuated. single, excentric, unbranched,
30–36 × 1–1.5 μm, flexuous, attenuated.
: , Western Cape Province,
Jonkershoek Nature Reserve, culm litter of cf.
, 31 July 2001, S. Lee, PREM 58863.
: (),
cf. ()
: Our collections from the resulted in
three specimens representing two species.
had long, single appendages at both ends.
dimensions, one species (PREM 58863) matched the
descriptions of and (Lind) Nag
Raj ().
character separating these two species in Nag Raj
() is the length of second
and fourth conidial cells from the base. has
equal length of cells (4–6 μm, av. 5 μm), whereas has unequal length (second cell (3.5–)4–6 μm,
av. 5 μm; fourth cell 4–4.5(–5) μm, av. 4.3 μm).
this difference is not obvious from Nag Raj's line drawings of these species
(), as some of
these cells in the depicted conidia of are also unequal
in length.
cells.
and furthermore the range of length fits best that of .
S.
MycoBank .
.
: in reference to its host genus, .
remaining immersed, visible at the surface by dark exuding conidial masses,
lifting up the epidermis; in section low conoid, 132–270 μm wide.
pseudoparenchymatous, consisting of a few layers of
brown, thick-walled, angular cells, 9–14 μm thick; lateral tissue
absent or present, when present similar to the basal stroma, 8–9 μm
thick. arising from the base and lateral tissue when
present, often reduced to conidiogenous cells or poorly developed.
annellidic, hyaline, discrete, smooth,
cylindrical to lageniform, (5.5–)8–10(–13) × 2–3
μm. fusiform to ellipsoid, straight or slightly curved,
(15–)17–18(–20) × (6–)7–7.5(–9)
μm (av. 17.1 × 7.3 μm, ratio 2.3: 1), 4–-septate; apical
cell hyaline, conical, 2–3 × 3 μm, smooth, thin-walled; median
cells brown, doliiform, 10–16 × 7–8 μm, echinulate,
thick-walled; basal cell hyaline, obconical with truncate end, 2.5–3
× 3 μm, smooth, thin-walled. single,
centric, unbranched, 27–38 × 1–1.5 μm, flexuous,
attenuated. single, excentric, unbranched,
25–40 × 1–1.5 μm, flexuous, attenuated.
: , Western Cape Province,
Jonkershoek Nature Reserve, culm litter of , 15 June
2001, S.
= CMW
17971; culm litter of cf.
2001, S.
= CMW
17984.
: cf. ().
: Three known species are morphologically close to the two
collections of . They are (B.
Sutton) M. Morelet, [≡ (B. Sutton)
], (Loos) M.
.
Based on Nag Raj's ()
descriptions, has shorter appendages (basal 12–29
μm, apical 18–33 μm) and smaller conidia (13–16.5 ×
6–7.5 μm), and has appendages of similar length
(basal 14–40 μm, apical 13–40 μm), but larger conidia
(18–24 × 6–7 μm) than those of is the closest in terms of conidia and appendages,
but the variable shapes of conidia with visible septal pores clearly
differentiate it from our collections ().
species to accommodate these two specimens.
(Morochk.) S.
.
.
remaining immersed, visible at the surface by dark exuding conidial masses; in
section low conoid, 50–67 μm high, 170–413 μm wide.
pseudoparenchymatous, in section 4–9 μm thick
throughout the conidioma, consisting of a few layers of pale brown, moderately
thick-walled, compressed cells of
arising from the entire periphery of the inside of the conidiomata, branched
at the base, cylindrical, 10–12(–20) × 1–2 μm.
annellidic, hyaline, integrated, smooth,
cylindrical, 4–7 × 2–2.5 μm. fusiform,
(15–)16–17(–19.5) × (5–)6–7(–8)
μm (av. 16.5 × 6.5 μm, ratio 2.5: 1), 3-septate; apical cell
hyaline, conical to trapezoid, 2–3 × 3–3.5 μm, smooth,
thin-walled, at times deciduous; median cells brown, doliiform, 12–15
× 7–8 μm, echinulate, thick-walled; basal cell hyaline,
obconical, 2–3 × 3–4 μm, smooth, thin-walled, at times
deciduous. 3–4, inserted in the topmost part
of the apical cell, arising at the same point, occasionally branched,
flexuous, 8–16 × 1 μm. absent.
: , Western Cape Province,
Kirstenbosch National Botanical Garden, culm litter of , S. Lee, 3 Dec. 2001, SL1015; Kogelberg Nature Reserve,
culm litter of , 3 Nov. 2000, S.
litter of , 11 May 2000, S. Lee, PREM 58866.
: (),
().
: The three collections are morphologically similar to two
known species: (Henn.) Nag Raj and (,
).
has similar conidial dimensions (15–18
× 7–7.5 μm) as the fungi in these three collections, but it has
much shorter and unbranched apical appendages (3–8 μm).
description of the type specimen of provided by Guba
() (conidia 15–22
×5.5–8 μm, apical appendages 8–21 μm) closely matches
the dimensions of our collections.
() suggests that
should be allocated to this genus.
collected in the present study also clustered in the
clade () with a high
bootstrap support.
(Tubeuf) Steyaert, Bull. Jard. Bot.
État Bruxelles 19: 298. 1949. .
().
remaining immersed, visible at the surface by dark exuding conidial masses; in
section spherical or occasionally conical, at times laterally joined,
106–156 × (73–)124–177 μm.
pseudoparenchymatous, in section 9–12 μm thick throughout the
conidioma, consisting of 3–5 layers of pale brown, moderately
thick-walled, compressed cells of
arising from the entire periphery of the inside of the conidiomata, branched
at the base, cylindrical, 0–4-septate, 11–25 × 2–3
μm. annellidic, hyaline, integrated, smooth,
cylindrical, 6–19 × 2 μm. fusiform,
(16–)17–18(–20) × (6–)7(–8) μm (av.
17.8 × 7.1 μm, ratio 2.5: 1), 3-septate; apical cell hyaline, conical
to trapezoid, 2.5–3 × 2.5–4 μm, smooth, thin-walled, at
times deciduous; median cells brown, doliiform, 13–14 × 7 μm,
echinulate, thick-walled; basal cell hyaline, obconical, 2–3 ×
2–3 μm wide, at times deciduous.
2–4(–5), inserted in the topmost part of the apical cell, arising
at the same point, flexuous, 26–31 × 1 μm, attenuated,
1–2 appendages often dichotomously branched.
absent.
: , Western Cape Province,
Jonkershoek Nature Reserve, culm litter of , 15 June
2001, S.
= CMW
17958; Kirstenbosch National Botanical Garden, culm litter of
, 3 Dec. 2001, S.
culture
= CMW 18093.
: (), ().
: The two collections obtained are very similar to and .
difference between these taxa is in the branching patterns of their apical
appendages (,
). has 1–3 simple or staghorn-like branches.
and have more than one
apical appendage, often irregularly or dichotomously branched. However, often has two equal branches that branch dichotomously again.
Based on their conidial dimensions and the branching pattern of their apical
appendages, our collections are best accommodated in .
S.
MycoBank .
.
: in reference to its large conidia.
remaining immersed, visible at the surface by dark exuding conidial masses; in
section subglobose to ellipsoid, 141–245 × 85–136 μm.
pseudoparenchymatous, in section 8.5–18 μm thick
throughout the conidioma, occasionally becoming thinner towards the apex,
consisting of 3–5 layers of pale brown to brown, moderately
thick-walled, highly and moderately compressed cells of arising from the entire periphery of the inside of the
conidiomata, branched at the base, 8–10 × 2 μm.
annellidic, hyaline, integrated, smooth,
cylindrical, 0–3-septate, 7–26 × 2–3 μm.
fusiform, (25–)30–31(–36) ×
(9–)11–12(–13) μm (av. 30.5 × 11.8 μm, ratio
2.6: 1), 3-septate; apical cell hyaline, trapezoid, 3–4 ×
3–5 μm, smooth, thin-walled; median cells brown, doliiform,
19–24 × 9–13 μm, echinulate, thick-walled; basal cell
hyaline, obconical, 5–7 × 3–4.5 μm, smooth, thin-walled.
(2–)3(–4), inserted in the top part of
the apical cell, arising at different points, unbranched, flexuous, 9–23
× 1–2 μm. absent.
: , Western Cape Province,
Kogelberg Nature Reserve, culm litter of , 3 Nov.
2000, S. Lee, PREM 58870, .
: ()
: is unusual in having larger
conidia than any other species in this genus.
similar conidial dimensions are (J.V. Almeida &
Sousa da Câmara) Nag Raj and (Speg.) Nag Raj
(≡ Speg.).
is, however, different from in having smaller conidia
(23–32 × 7.5–10 μm) and more roughly ornamented median
conidial cells (verruculose to rugulose)
(,
). has similar conidial dimensions (25–33.5 ×
8–11.5 μm), but can be distinguished from by
having 4-septate conidia as opposed to the 3-septate
().
S.
.
.
: in reference to its host family,
.
remaining immersed, visible at the surface by dark exuding conidial masses; in
section conoid, convoluted, 200–270 × 520–573 μm.
pseudoparenchymatous, 9–12.5 μm thick throughout
the conidioma, consisting of 3–5 layers of pale brown, moderately
thick-walled cells of arising from
the entire periphery of the inside of the conidiomata, branched at the base,
cylindrical, 5–12.5 × 2–3 μm. annellidic, hyaline, integrated, smooth, cylindrical,
(5–)14–31 × 2–3 μm. fusiform,
(21–)24–25.5(–29) × (5–)7(–8) μm (av.
24.9 × 6.8 μm, ratio 3.6: 1), 3-septate; apical cell hyaline, oblong
to trapezoid, 3–4.5 × 2–4 μm, smooth, thin-walled; median
cells brown, doliiform, 14–20 × 6–8 μm, echinulate,
thick-walled; basal cell hyaline, obconical, 4–5 × 3–4 μm
wide at the base, smooth, thin-walled.
(2–)3(–4), inserted in the top part or along the upper half of the
apical cell, arising at different points, rarely branched, flexuous,
22.5–55 × 1 μm, attenuated.
absent.
: , Western Cape Province,
Jonkershoek Nature Reserve, culm litter of cf.
, 31 July 2001, S. Lee, PREM 58871, ,
living ex-type culture CMW 18755; culm litter or ,
15 June 2001, S.
= CMW
17968.
: cf. ().
: is distinct in having
3-septate conidia with relatively long apical appendages.
considered close to the species.
Raj, (Harkn.) Nag Raj,
(Dearn. & Fairm.) Nag Raj, (Peck) Nag Raj and
(). The conidia of (25–36 ×
8–10 μm), (25–32 ×
8–10 μm) and (23–32 × 7.5–10
μm) are larger than those of .
has smaller conidia (19–23 ×
5.5–7.5 μm), and could thus be excluded from the comparisons.
closely matches the description of , although there are some differences between these two species.
is reported to have up to five apical
appendages, and to also have a basal appendage.
appendages were never observed.
to be congeneric with other species of
.
S.
MycoBank .
.
: in reference to its pale brown conidia.
remaining immersed, visible at the surface by means of dark exuding conidial
masses; in section conoid or low applanate, some laterally joined,
(96–)200–238 × 105–136 μm.
pseudoparenchymatous, (4–)6–9 μm thick throughout the
conidioma, consisting of a few layers of hyaline or slightly pigmented,
moderately thick-walled, compressed cells. arising from
the entire periphery of the inside of the conidiomata, branched at the base,
cylindrical, 0–2-septate, 11–20 × 2–3 μm.
annellidic, hyaline, integrated, smooth,
cylindrical, (6–)14–31 × 2–3 μm.
fusiform, (20–)21–22(–23) × (7–)8(–8.5)
μm (av. 21.4 × 7.8 μm, ratio 2.7: 1), 3-septate; apical cell
hyaline, conical to trapezoid, 3.5–4 × 4–5 μm, smooth,
thin-walled; median cells pale brown, doliiform, 14–16 ×
7–8.5 μm, echinulate, moderately thick-walled; basal cell hyaline,
obconical, 3–4 × 4–5 μm, smooth, thin-walled. 3–4, inserted in the top part of the apical cell,
arising at different points, unbranched, 12–16(–25) ×
1–1.5 μm, attenuated. absent.
: , Western Cape Province,
Jonkershoek Nature Reserve, culm litter of , 5
Apr. 2001, S. Lee, PREM 58873, .
: ().
: is unique in having pale
brown median cells, and apical appendages originating at distant loci on the
apical cell. Four species, (McAlpine) Nag Raj, (Tassi) Nag Raj, (Henn.) Nag Raj and
, are morphologically similar to
().
has elongated, obconical basal cells and narrower
conidia (17–24 × 5–7.5 μm), has
larger conidia (21–25.5 × 9–10 μm) with constricted
septa, has brown median cells and narrower conidia
(19–23 × 5.5–7.5 μm), and has larger
conidia (19–26 × 7–9 μm) and a distinctly different
origin of the apical appendages distinguishing them from .
xref
#text |
The southern tip of Africa is recognised for its floral diversity,
accommodating the world's smallest floral kingdom that is commonly referred to
as the Fynbos.
Region (CFR) in which approximately 9000 vascular plant species (
44 % of the southern African flora) are found
(,
, ).
330 species of in 14 genera, 10 of which are endemic to
the region (,
).
, including the genus (proteas), commonly
dominate plant communities of the Fynbos Biome
()
().
significant, but provide the basis for the South African protea cut-flower
industry that generates an annual income of more than US $ 10 million
(,
).
Florets of spp. are arranged in inflorescences.
stage that can last for several months
(), the inflorescences
will open to reveal the often brightly coloured involucral bracts that attract
many insect and bird pollinators ().
forcing the florets together in compact infructescences
().
infructescence may persist on the plants for several years, and act as an
above-ground seed bank () that opens to release seeds after a fire
().
time, the infructescences are colonised by many different arthropods (Myburg
,
, Myburg & Rust
,
, Coetzee & Giliomee
,
,
,
,
,
,
)
and micro-fungi (, Lee .
,
), some of which are
specific to their hosts.
Three species of Syd. & P. Syd.
from infructescences in South Africa, showing varying degrees
of host specificity. G.J. Marais & M.J.
Wingf.
(),
while G.J. Marais & M.J. Wingf.
infructescences of
().
G.J. Marais & M.J. Wingf., in contrast, has
been reported from , and
(,
).
All three species are characterised by Hekt. & C.F.
Perkins anamorphs, tolerance to high levels of the antibiotic cycloheximide
and contain rhamnose in their cell walls
().
Wingfield .
spp.
.
between these species and (Fr.) Syd. & P. Syd., the
type species of .
()
has, however, confirmed that the -associated species reside in
the (Robak) Nannf. clade of .
three known -associated spp., using
ribosomal ITS and partial β-tubulin gene sequences.
the phylogenetic position of these species at the generic level using
ribosomal large subunit (LSU) data.
spp.
collected from spp.
considered previously.
Infructescences of various spp.
different sites in South Africa between Feb. 2003 and Jun. 2005, and examined
for the presence of spp.
apices of ascomatal necks with a small piece of agar attached to the tip of a
dissecting needle and transferred to 2 % malt extract agar (MEA; Biolab,
Midrand, South Africa) amended with 0.05 g/L cycloheximide
().
purified, all cultures were maintained on Petri dishes containing MEA at 4
°C.
() have been deposited
in the culture collection of the Centaalbureau voor Schimmelcultures (CBS),
Utrecht, The Netherlands, and the culture collection (CMW) of the Forestry and
Agricultural Biotechnology Institute (FABI), University of Pretoria, South
Africa.
new species have been deposited in the National Fungus Collection (PREM),
Pretoria, South Africa ().
Perithecia of spp.
infructescences, and conidiophores and conidia of the
anamorphs formed in culture, were mounted on microscope
slides in clear lactophenol.
differential interference contrast (DIC).
DXM1200 digital camera mounted on the microscopes.
taxonomically useful structure were made and means (± standard
deviation) calculated.
LSU rDNA
(),
ITS1–F () and ITS4 () for the ITS and 5.8S regions.
for the rDNA amplifications were 50 μL consisting of: 32.5 μL
ddHO, 1 μL DNA, 5 μL (10×) reaction buffer
(Super-Therm, JMR Holdings, U.S.A.), 5 μL MgCl, 5 μL dNTP
(10 mM of each nucleotide), 0.5 μL (10 mM) of each primer and 0.5 μL
Super-Therm Taq polymerase (JMR Holdings, U.S.A.).
amplified using a Gene Amp®, PCR System 2700 thermal cycler (Applied
Biosystems, Foster City, U.S.A.).
denaturation step of 2 min at 95 °C, followed by 35 cycles of: 30 s
denaturation at 95 °C, 30 s annealing at 55 °C, and 1 min elongation
at 72 °C.
at 72 °C.
the same as for ribosomal DNA, except that 1.5 μL DNA, 32 μL of
ddHO and primers T10
() and Bt2b () were used.
β-tubulin was as follows: initial denaturation for 4 min at 95 °C, 35
cycles of denaturation at 95 °C for 1 min, annealing at 50 °C for 1.5
min, elongation at 72 °C for 1 min, and a termination step of 7 min at 72
°C.
() were compared to
sequences of species of and related genera from the study
of Zipfel .
().
β-tubulin sequences from the present study
() were compared with
sequences of closely related spp.
(,
Aghayeva . ,
).
using Clustal X v. 1.81.
: One thousand random stepwise addition heuristic
searches were performed using the software package PAUP v. 4.0 beta 10
() with Tree
Bisection-Reconnection (TBR) on and 10 trees saved per replicate.
node support was assessed using the bootstrap algorithm
(), with 1000
replicates of simple taxon addition.
: Relationships between taxa were determined
using distance analysis in PAUP.
sets were determined based on AIC (Akaike Information Criteria) using the
Modeltest 3.06 ().
invariable sites 0.6899 and rates for variable sites following a gamma
distribution with shape parameter of 1.0185) for LSU, TrN+I+G (proportion
invariable sites 0.4213 and rates for variable sites following a gamma
distribution with shape parameter of 0.6253) for ITS, and HKY+G (rates for
variable sites following a gamma distribution with shape parameter of 0.1783)
for β-tubulin.
tree-building algorithm () and statistical support was determined by 1000 NJ bootstrap
replicates.
: Data were analysed using Bayesian inference
based on a Markov chain Monte Carlo (MCMC) approach in the software package
MrBayes v. 3.1.1 ().
MrBayes, GTR+I+G (shape parameter using 4 rate categories) was used for the
analysis. All parameters were inferred from the data.
chains were initiated from a random starting tree.
generations with a sample frequency of 50 were implemented.
(first 20000 generations) were discarded and the remaining trees from both
runs were pooled into a 50 % majority rule consensus tree.
with 12 isolates from five spp.
collections by Wingfield and Marais ().
spp. in surveys that formed part of this study.
morphology.
spp.
infructescences.
isolates of that came from the same host, . Some old isolates of from and were newly identified.
that did not resemble any of the three species described
from proteas, or any other species.
group were commonly collected on the styles of and
The fungus often occurred sympatrically with
(M.J. Wingf. & P.S. van Wyk) G.J.
& M.J. Wingf.
found only in the insect-damaged involucral receptacles of ().
growth at 30 °C.
was 26 mm (± 1), while the species from and had a colony diameter of 18 mm
(± 1) at this temperature after 8 d in the dark.
species were tolerant to cycloheximide and were able to
grow on all tested concentrations of this antibiotic.
the species collected from declined from 27 mm (± 1)
on 0.05 g/L to 17 mm on 2.5 g/L cycloheximide.
species from and declined from
20 mm (± 1) on 0.05 g/L to 12 mm (± 1) on 2.5 g/L
cycloheximide.
709 characters for LSU, 531 characters for ITS, and 307 characters for part of
the β-tubulin gene.
phylogenetic analyses for each gene region was similar.
regions, the trees presented (Figs
,
,
) were obtained from
neighbour-joining analyses.
For the LSU region there were 98 parsimony-informative characters, 611
parsimony-uninformative characters, and 581 constant characters. For the ITS
region there were 98 parsimony-informative characters, 433
parsimony-uninformative characters, and 389 constant characters.
β-tubulin region there were 112 parsimony-informative characters, 195
parsimony-uninformative characters, and 194 constant characters.
using the parsimony algorithm yielded 38, 9990 and 9530 equally most
parsimonious trees of 291, 234 and 268 steps long for the LSU, ITS and
β-tubulin data sets respectively. The Consistency Indices were 0.765,
0.533 and 0.705, while the Retention Indices were 0.957, 0.856 and 0.940 for
the ITS, LSU and β-tubulin regions, respectively. Apart from group C
[(), (PP 1.0)], PP values
obtained for LSU were not statistically significant for the groups of interest
and were omitted.
other, and only the neighbour-joining tree
() is presented.
taxa from proteas formed four distinct, well-supported groups (A–D).
These groups did not form a monophyletic lineage, but were distributed among
various species of the complex in the genus
. The LSU data did not distinguish between and which formed a single group [(A),
()].
analyses, two isolates of were selected as outgroup
for the more focused ITS and β-tubulin analyses.
Analyses of the ITS data () confirmed the topology of the LSU tree.
formed four well-supported groups (A–D), with isolates of and grouping together (group A) similar
to the outcome of the LSU sequence comparisons.
arising from analyses of part of the β-tubulin gene region
() differed from both the
LSU and ITS trees (Figs ,
).
well-resolved with strong bootstrap support, but group A was sub-divided into
two distinct, well-supported sub-groups, representing
(group A1) and (group A2), respectively.
isolates from each other as well as from the three
species previously described from the infructescences of
spp.
closely related spp.
morphologically and phylogenetically distinct groups are described as new
species as follows:
Roets, Z.W. de Beer & M.J. Wingf.,
MycoBank
.
. :
sp.
: The epithet ( = ghost)
refers to the small and inconspicuous perithecia growing within a cryptic
habitat.
Ascomata superficialia, basi depressa globosa, atra, nuda, 35–70
μm diam, collo atro, 20–60 x 15–25 μm, sursum ad 10–15
μm angustato, hyphae ostiolares absentes. Asci envanescentes.
allantoideae, unicellulares, hyalinae, vagina gelatinosa carentes, 4–6 x
2 μm, aggregatae electrinae. Anamorphe sp., conidiis
ellipsoideis vel clavatis, 5–8 x 2–3 μm.
depressed–globose, wider at base, black without hyphal ornamentation,
35–70 (51 ± 8) μm diam; necks black, 20–60 (42 ±
10) μm long, 15–25 (19 ± 3) μm wide at the base,
10–15 (11 ± 2) μm wide at the apex, ostiolar hyphae absent
().
evanescent. allantoid, aseptate, hyaline,
sheaths absent, 4–6 (5 ± 1) μm, 2 μm
(), accumulating in a
hyaline gelatinous droplet at the apex of the neck, becoming amber-coloured
when dry.
at 25 °C in the dark, white to cream-coloured, effuse, circular with an
entire edge, surface smooth becoming mucoid, with a distinctive soapy odour,
hyphae semi-immersed ().
Growth reduced at temperatures below and above the optimum of 30 °C.
Sporulation profuse on MEA. arising directly from
hyphae on the surface of the agar and from aerial conidiophores, proliferating
sympodially, hyaline (). holoblastic and hyaline and of two
forms, one ellipsoidal to clavate, smooth, thin-walled, 5–8 x 2–3
μm () and the other
globose to obovate, smooth, thin-walled, 3–5 x 2–3 μm
().
singly, but aggregating into slimy masses, often also produced directly on
hyphae (5H–I).
: Confined to the dead styles and petals of florets
within serotinous infructescences of spp.
: South Africa, Western Cape Province.
: , Western Cape Province,
Stellenbosch, Jan S. Marais Park, on , Jun. 2005, F.
Roets, PREM 58941, culture ex-type CMW 20676 =
;
Stellenbosch, Jonkershoek NR, on , May 2004, F.
PREM 58943, culture ex-paratype CMW 20681 =
;
Bainskloof Pass, on , Aug. 2004, F.
PREM 58946, culture ex-paratype CMW 20689 =
;
Stellenbosch, Jonkershoek NR, on , Jul. 2004, F.
PREM 58944, culture ex-paratype CMW 20682 =
;
Giftberg top, on , Jun. 2005, F. Roets, culture CMW
20698; Giftberg top, on , Jun. 2005, F.
CMW 20699; Bainskloof Pass, on , Aug. 2004, F.
PREM 58945, culture CMW 20683; Piekenierskloof Pass, Aug. 2004, on , F. Roets, culture CMW 20684; Jonkershoek NR, Aug. 2004, on
, F.
Pass, Sep. 2004, on , F. Roets, PREM 58947, culture CMW
20690.
Roets, Z.W. de Beer & M.J.
Wingf., MycoBank
.
. :
sp.
: The epithet ( =
palm; = peak) refers to the palm-like hyphal ornamentation of
the ostiolar tip.
nonnumquam paucis hyphis circumdata, collo atro, 360–760 x 20–35
μm, sursum ad 10–15 μm angustato, 8–12 hyphis ostiolaribus
rectis vel curvatis, hyalinis vel subhyalinis, 10–25 μm longis palmam
fingentibus ornato. Asci evanescentes.
hyalinae, vagina gelatinosa carentes, 3.5–5.5 x 2.0–2.5 μm,
aggregatae incoloratae. Anamorphe sp., conidiis clavatis
3–11 x 1.5–2.5 μm.
plates after 2 mo of growth at 25 °C in the dark.
80–195 (146 ± 33) μm diam, occasionally with sparse hyphal
ornamentation; necks black, 360–760 (569 ± 114) μm long,
20–35 (28 ± 5) μm wide at the base, 10–15 (12 ±
2.5) μm wide at the apex (). 8–12 ostiolar hyphae, straight or slightly
curved, hyaline to sub-hyaline, 10–25 (16 ± 5) μm long
().
evanescent. allantoid, aseptate, hyaline, sheaths absent,
3.5–5.5 x 2–2.5 μm (), collecting in a hyaline gelatinous droplet at the apex of the
neck (), remaining
uncoloured when dry.
dark, white to cream-coloured, circular, effuse, with an entire edge and
somewhat rough surface, not producing an odour
().
temperatures below and above the optimum of 30 °C.
MEA. cells arising directly from hyphae on the surface
of the agar and from aerial cinidiophores, proliferating sympodially, hyaline,
becoming denticulate (). Denticles 0.5–2 μm (1 ± 0.5) long
().
holoblastic, hyaline, aseptate, clavate, smooth, thin-walled, 3–11 x
1.5–2.5 μm ().
Conidia forming singly, but aggregating in slimy masses, also produced
directly on hyphae ().
: Confined to the insect-damaged involucral receptacles
of infructescences.
: South Africa, Western Cape Province.
: , Western Cape Province,
Stellenbosch, Jan S. Marais Park, on , Jun. 2005, F.
PREM 58942, culture ex-type CMW 20677 =
;
Stellenbosch, Jan S. Marais Park, on , Jun. 2005, F.
PREM 58949, culture ex-paratype CMW 20693 =
;
Stellenbosch, Jan S. Marais Park, on , Jun. 2005, F.
PREM 58950, culture ex-paratype CMW 20694 =
;
Stellenbosch, Jan S. Marais Park, on , Jun. 2005, F.
PREM 58951, culture ex-paratype CMW 20697 =
;
Stellenbosch, Jan S. Marais Park, on , Jun. 2005, F.
culture CMW 20695; Stellenbosch, Jan S. Marais Park, on ,
Jun. 2005, F. Roets, culture CMW 20696.
The infructescences of spp.
unique and unusual habitat for spp.
poorly understood and knowledge of their relatedness to other species of
is only just emerging.
these fungi amongst their close relatives.
and
previously described from infructescences, represent
well-defined species of Zipfel .
().
form a monophyletic lineage within the -complex.
The spp.
morphologically similar and in this respect, analyses of DNA sequence data
enhance our ability to recognise distinct taxa.
spp.
during the period when the first of these fungi were discovered and described.
The two new species, and can
easily be distinguished from each other and from the other three
spp.
DNA sequence comparisons.
other and from the other three species, although these differences would have
been difficult to define in the absence of DNA sequence comparisons.
of this study also represent the first report of from
and .
between and This shows that the
two species are very closely related.
β-tubulin gene regions, however, support the notion that the two species
represent distinct taxa as defined by Marais & Wingfield
() based on morphological
characters.
that they share a common ancestor.
fact that they occur in the infructescences of closely related
spp.
(). appears to be specific to (Marais &
Wingfield ,
) that is classified in
the section and occurs in the eastern and northern
provinces of South Africa (). was previously thought to be
specific to (), but sequence data from the present study
show that it also occurs in the infructescences of
and .
in the section , and the latter species is restricted to
the Drakensberg mountain range.
ranges of both and , although is classified in a different section of the genus
, the
().
in this study suggest that is closely related to and has been
recorded from and
in the Western Cape Province
().
However, morphological data arising from this study (results not shown) show
that all isolates from non- hosts from
the culture collection (CMW) of the Forestry and Agricultural Biotechnology
Institute (FABI), were misidentified and belong in .
only exception was one isolate (CMW 2753) collected from It is suspected that in most of these cases, was confused with due to superficial
similarities in the teleomorph structures of these species
(,
).
We did not isolate from any species
other than .
to , which resides in the section .
explanation for the close phylogenetic relationship between and its northern counterparts, and will probably only be revealed once a robust phylogeny for the
genus becomes available.
.
were also observed in the infructescences of and .
to isolate spp. from these spp.
the perithecia were old and the ascospores appeared not to be viable.
we were unable to identify the species definitively, we believe that the
perithecia in and
represent .
with a number of different spp.
sections.
restricted host range of mirrors the situation in
is exclusively associated with
, whereas is associated with numerous
spp. ().
to the styles and petals of florets of the host plant and they were never
observed in insect tunnels commonly found in the bases of infructescences.
Similar to the species
and preferably colonise the styles and petals of florets
of their host plants.
and that has been collected from
the tunnels of insects found within the involucral receptacles of .
larvae ().
contrasting with the substratum in the infructescences.
ability of to exclusively exploit this substrate
probably results in reduced competition between this species, and that can colonise the same
infructescence simultaneously (pers. observ.). Whether is pathogenic to its host remains to be determined.
spp.
bases of their ascomata.
in sticky masses on the apices of the necks.
represent adaptations for arthropod-vectored dispersal
().
conifers are the most common vectors of spp.
(, , ).
some cases, lead to the death of the host plant
(, ).
complexity of these associations (Six & Paine
,
, Klepzig .
,
, Six
,
,
,
).
on similarities in morphology, the spp.
to share this mode of vectored spore dispersal, and may thus also be involved
in mutualistic associations with arthropods.
multi-organism interactions is currently being investigated.
studies and it has been necessary to develop specialised DNA-based techniques
to study the vector relationships of spp.
().
involved, at least occasionally, in transporting spores of
spp., and we expect that the discovery of new species of
will enhance our understanding of these fungi and the invertebrates that
transport them from one infructescence to another. |
(Bruner) M.E.
from spp. () in Cuba
().
() found this fungus on
bark of dead, injured or healthy trees, but it did not
appear to cause disease. was also found on
dead branches of mango ()
and avocado () lying on the
ground in the vicinity of the trees
().
exotic hosts, fruiting structures of were also found on
the bark of jobo (), a
plant native to Cuba ().
() found on plantations in Florida.
however, reported as (Berk. & Broome) Sacc.,
a name previously used for the species
(,
).
identification of the fungus as was based on the
presence of orange stromata, as well as conidial and ascospore dimensions that
resembled those of the type specimen from Cuba.
(Bruner) Gryzenh. & M.J. Wingf., a fungus previously known as
(Bruner) Hodges
() and a serious pathogen of spp.
(), was also
found in the same plantations (). was
mainly associated with dead coppice shoots in stands of while was the causal agent of basal
cankers and death of coppice shoots
().
Cuba and Florida, the name has also been used for
collections of a fungus from in Japan
(, ).
is also known from other host genera besides
(), namely
species of (),
() and ().
()
showed that the fungus referred to as in Japan is the
same as (G. H. Otth) M.E. Barr.
Myburg ()
did not, however, consider whether is the same as the
fungus referred to as from Cuba, where was originally described
().
with orange stromatic tissue are known from islands in
the Caribbean Sea and Atlantic Ocean (). is well-known from several
countries in Central and South America
(), including Cuba () where was first discovered.
(Vizioli) Micales & Stipes occurs as a
saprobe on twigs, branches and seeds of (sea grape,
) from Bermuda
() and Florida
(,
).
In the Azores and Madeira, an unidentified species of
has been associated with cankers on () (, ).
Trinidad (,
).
saprobic and has recently been transferred to the new genus
(). also includes a second new
species, Gryzenh. & M.J. Wingf., which is a
pathogen of trees in Ecuador
().
relationship with species of and closely related genera
remained unresolved ().
that could, with reasonable certainty, be attributed to this species.
problem was true for
(), which has been suspected to be a synonym of (Hodges & Gardner 1990).
()
also remains to be resolved.
spp.
known previously.
and enabled us to reconsider questions relating to the identity and the
phylogenetic position of .
collected from cankers and dead trees on the stems of and
an unidentified sp.
U.S.A.).
stems of the same spp.
associated with cankers on living trees. was
also common on cankered trees on the island of Hawaii.
Specimens of this fungus previously examined from the Hawaiian Islands were
all from Kauai (, ), and collections made in this study represent the first
record of from the island of Hawaii.
Chiapas, Mexico. An additional isolate from Mexico was received from Dr. E.L.
Barnard (Florida Division of Forestry, FDACS, Gainesville, Florida).
(collected as ) from plantations in
Florida, linked to the study of Barnard
(), was acquired from the
American Type Culture Collection (ATCC).
,
) linked to the report of a
species from in the Azores
()
were also included in this study.
isolates () of from in the Azores
().
Unfortunately, no isolates of could be obtained from
Cuba despite surveys aimed at re-collecting the fungus in that country.
Florida, a fungus with distinctive orange fruiting structures was found in the
vicinity of Fort Lauderdale, Key Biscayne, Dania and Oakland Park (Tables
,
).
profusely on branches and twigs, but was not associated with disease symptoms.
It was included in this study to determine whether it represents .
conidia and ascospores collected from the apices of pycnidia and perithecia,
respectively.
collection (CMW) of the Forestry and Agricultural Biotechnology Institute
(FABI), University of Pretoria, Pretoria, South Africa and representative
isolates not originally obtained from internationally recognised collections
have been deposited with the Centraalbureau voor Schimmelcultures, Utrecht,
Netherlands ().
original bark specimens from which cultures were made have been deposited in
the National Collection of Fungi (PREM), Pretoria, South Africa
().
extract, Biolab, Midrand, South Africa) as described by Myburg
().
derived for the internal transcribed spacer (ITS) regions ITS1 and ITS2,
including the conserved 5.8S gene of the ribosomal RNA (rRNA) operon, using
primer pair ITS1/ITS4 (), and β-tubulin genes using the primer pairs
Bt1a/Bt1b and Bt2a/Bt2b respectively ().
(),
respectively, were followed.
sequence reactions was done using a QIAquick PCR Purification Kit (Qiagen
GmbH, Hilden, Germany).
primers used in the PCR reactions, using the ABI PRISM™ Dye Terminator
Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase
(Perkin-Elmer, Warrington, UK). The sequencing reactions were run on an ABI
PRISM 3100™ automated DNA sequencer.
using Sequence Navigator v. 1.0.1 (Perkin-Elmer Applied BioSystems, Inc.,
Foster City, California, U.S.A.) software.
().
sequences obtained in this study of additional M. Venter & M.J. Wingf.
().
pathogen of trees in South Africa
(, ) and Australia
(,
).
sequences were compiled into a matrix using a modified data set (TreeBASE
accession numbers S1128, M1935) of Myburg
() as a template.
Additional sequences from other studies were also added to the data matrix.
These included sequences of Gryzenh. &
M.J. Wingf. (, ), and those of (Murrill)
M.E. Barr, (Tak. Kobay. & Kaz.
Itô) M.E.
isolates referred to as
().
() and (Berk. & Broome)
Gryzenh. & M.J. Wingf., the new genus that now contains (), were also added.
TreeBASE (S1490, M2675).
()
of the alignment program MAFFT v. 5.667
().
Phylogenetic analyses were made using PAUP (Phylogenetic Analysis Using
Parsimony) v. 4.0b10 ().
on the rRNA and β-tubulin gene sequence data sets (after the exclusion of
uninformative sites) to determine whether they could be analysed collectively
().
Phylogenetic analyses included parsimony and distance methods.
parsimony (MP) was inferred using the heuristic search option with the
tree-bisection-reconnection (TBR) branch swapping and MULTREES options (saving
all optimal trees) effective and a 100 random additions.
manual sequence alignment were treated as fifth character (NEWSTATE) in the
heuristic searches, and missing in distance analyses.
were excluded and remaining characters were reweighted according to the
individual Consistency Indices (CI) to reduce the number of trees.
distance analyses, the correct model for the datasets was found with MODELTEST
v. 3.5 ().
the Gamma distribution shape parameter (G) set to 0.9717 and frequency of
invariable sites (I) 0.4643; base frequencies of 0.1903, 0.3411, 0.2301 and
0.2385; and rate matrix of 1, 3.1147, 1, 1, 4.1643, 1.
nodes in the various phylogenetic trees was tested with a 1000 replicate
bootstrap analysis and is presented as a 70 % majority rule tree.
areas were included in the morphological comparisons
().
type specimen of (BPI 614275).
ascostromata were cut from bark specimens, rehydrated (1 min) in boiling water
and sectioned with a Leica CM1100 cryostat at –20 °C, 12–14
μm thick.
South Africa) was used, which was dissolved in water after sectioning.
acid (85 %) was used to prepare semi-permanent slides.
with a razor blade to more closely study conidiophore morphology.
structures were also mounted in 3 % KOH when conidiophores and asci could not
easily be observed.
conidiophores suspended in lactic acid or KOH, were taken for the specimens
and these are presented as (min–)(average – std. dev.) –
(average + std. dev.)(–max) μm.
size range from the largest and smallest structures was obtained.
assigned to structures using the charts of Rayner
().
of Nitschke (), which reside
in a different family in the
(), as outgroup.
of 335 constant, 10 parsimony-uninformative and 226 parsimony-informative
characters (g1 = – 0.4143), and the β-tubulin DNA sequence set (966
bp) consisted of 516 constant, 32 parsimony-uninformative and 418
parsimony-informative characters (g1 = – 0.3582).
the PHT analyses (P = 0.004) indicated that trees obtained with the different
gene regions were incongruent.
clade with the other clades was different
in each gene tree.
always highly supported with bootstrap values between 90 and 100 %.
reason we combined the data. The resultant dataset contained 1537
characters.
The heuristic search resulted in six most parsimonious trees (tree length =
1101.9, CI = 0.736, Retention index/RI = 0.943), which differed only in the
lengths of the branches.
analyses showed identical clades grouping isolates.
isolates, with high bootstrap values, were obtained when the more variable
regions, and thus potentially ambiguously aligned sequences of the introns and
ITS1 region, were excluded.
the complete dataset is presented in .
() and formed a discrete
clade (bootstrap support 100 %) separate from the clades representing species
of (Sacc.) Sacc., Fr.,
Gryzenh. & M.J. Wingf.,
Gryzenh. & M.J. Wingf. and Gryzenh., Glen & M.J.
Wingf.
() also included the
isolates from in the Azores (CMW 11301) and Madeira (CMW
14551, CMW 11300). isolates from Kauai
grouped separately from isolates from Kauai (CMW 1856,
CMW 11006, CMW 11008) and Hawaii (CMW 10889).
(bootstrap support 100 %) in the South East Asian sub-clade
()
of ().
().
believed to represent .
() from the Azores (CMW 14547, CMW 14548) grouped with
other isolates (CMW 7048, CMW 13749) in the
clade (bootstrap support 100 %;
).
support 100 %) separate from the clade defining
().
separated from the clades representing other genera.
from in Florida also formed a clade distinct from those
representing the other genera (bootstrap support 100 %), and did not group
with the isolates representing .
Florida and Kauai () were
indistinguishable from those on the type specimen of
from Cuba. Ascospores [(5.5–)7–9(–10) ×
(2–)2.5–3(–4) μm], asci
[(26.5–)29.5–34.5(–37) ×
(5–)5.5–7(–8) μm] and conidia
[(2.5–)3–4(–5) × 1–1.5 μm] also fell within
the range of those reported for the type specimen
().
confident that the collections from Mexico and Hawaii represent , although the phylogenetic relationship between the fungus in
Cuba and the isolates from Mexico and Kauai could not be determined due to the
lack of isolates from Cuba.
from the Azores and Madeira, linked to isolates (CMW 11300,
CMW 11301, CMW 14551) that also grouped with those from Mexico and Kauai, were
similar to those from Cuba, Hawaii and Mexico
().
Puerto Rico (NY 511), annotated as but shown by
Gryzenhout
() not to represent this
species, was also morphologically similar to .
Madeira and the Azores), and those previously labeled as from Japan.
level of bark) than those on specimens from Japan (250–1630 μm diam
above the level of the bark).
tissue development (), while structures on specimens from Japan were distinctly
semi-immersed with strongly developed, erumpent tissue.
specimens () occasionally had long extending perithecial necks (up
to 370 μm long) while those from Japan were consistently short (up to 130
μm long).
also had characteristically long, cylindrical conidiophores up to 57 μm
long, with the longest of these being sterile, resembling paraphyses
().
structures differed from conidiophores of the Japanese specimens that were up
to 29 μm long.
to different hosts, there are also differences, e.g.
paraphyses, that cannot be attributed to hosts.
likely represent robust characteristics to support the distinct phylogenetic
grouping () of specimens
representing from those of and other closely related genera.
() on various specimens
were similar to those thought to represent .
[(2.5–)3–4.5(–5.5) × 1–1.5 μm] and ascospores
[(6.5–)7.5–9(–10.5) ×
(2.5–)3–3.5(–4) μm] were similar to those of , and similar long (up to 62 μm) and cylindrical
conidiophores, with the longest sterile, were observed
().
with conidia of (3–)3.5–4.5(–5) × 1–1.5 μm
and labeled from bark of
(CUP 35081) in Bermuda, also contained structures similar to those of the
other specimens.
differed from those on bark () in being superficial and not semi-immersed.
[(32.5–)34.5–39(–41) ×
(5–)7–9.5(–10.5) μm] were longer and wider than those
measured for the majority of specimens
[(26.5–)29.5–34.5(–37) ×
(5–)5.5–7(–8) μm].
character since specimen PREM 57518, linked to isolate CMW 11298 grouping with
the other isolates, had asci of similar size
[(31.5–)32–39(–44.5) ×
(5.5–)6–7.5(–8.5) μm] to those of the specimens and thus longer than the other specimens.
morphologically different from those representing .
Conidiomata were pyriform to rostrate, often having a globose base with a long
to tapered cylindrical neck or more than one neck (Figs
,
).
from conidiomata of which are pulvinate without long
necks ().
Furthermore, necks of the conidiomata were often covered with short hairs
().
the Florida specimens (Figs ,
) also did not contain the
long, sterile paraphyses commonly found in locules of
(). No
teleomorph was observed for the Florida specimens on the bark.
or
although that fungus was closely related to these genera
in the DNA sequence comparisons.
resembled the rostrate conidiomata of
() most closely, but could be distinguished from
based on conidiomata that are more pyriform in shape, and
with necks more cylindrical.
in Florida also lacked the distinct tissue at the junction between neck and base in the conidiomata
of ().
prosenchymatous (), and
not of as is found in
().
with orange stromatic tissue.
Waterston () to represent
, an illustration previously used by Seaver &
Waterston () in their
description of a fungus named Seaver &
Waterston. These structures occurred on petioles and twigs of from Bermuda ().
perithecial necks extending from the orange stromata are black and not orange
as is the case for .
1–2-septate, guttulate and 11.5–14.5(–16) ×
(2.5–)3–4(–5) μm.
not colour purple in KOH and yellow in lactic acid, similar to structures of
.
synonym () of , but these are clearly distinct fungi.
shown clearly that cultures and specimens believed to represent do not reside in but represent
a distinct taxonomic group.
can be distinguished from species in by its
smaller and more superficial stromata, and long paraphyses between the
conidiophores.
closely related to .
provided.
Gryzenh. & M.J. Wingf.,
MycoBank
.
: Greek, , small, and , a
heap, thus referring to the small and pulvinate stromata.
tissue predominantly prosenchymatous but pseudoparenchymatous at edges.
dark-walled, with globose to sub-globose bases and slender
periphysate necks that emerge at the stromatal surface as black ostioles in
papillae covered with orange stromatal tissue. fusiform,
floating freely in the perithecial cavity, unitunicate with non-amyloid,
refractive apical rings. fusoid to ellipsoid, hyaline,
1-septate, often with a slight constriction at the septum.
orange, uni- to multilocular and convoluted, locules often occurring in the
same stroma that contains perithecia. cylindrical,
slightly tapering, often septate with or without lateral branches beneath the
septum, hyaline, often long with longest cells sterile and representing
paraphyses, conidiogenous cells phialidic. hyaline,
cylindrical, aseptate, expelled through opening at stromatal surface as orange
droplets or tendrils.
Typus: (Bruner) Gryzenh. & M.J.
Wingf., comb. nov.
(Bruner) Gryzenh. & M.J. Wingf.,
MycoBank
.
.
: Bruner, Mycologia 8:
241–242. 1916.
: , Santiago de las Vegas,
sp., 15 Feb. 1916, S.C. Bruner, BPI
614275, BPI 614273; , 25 Mar. 1916, C.L.
BPI 614278; sp., 28 Mar. 1916, C.L. Shear, BPI 614282;
Earle' s Herradura, , 5 Apr. 1916, C.L.
BPI 614283, BPI 614284; Santiago de las Vegas, , 6
Apr. 1916, C.L. Shear, BPI 614279, BPI 614280, 26 Mar. 1916, C.L. Shear, BPI
614281. , Las Chiapas, , 26 Feb. 1998,
C.S. Hodges, PREM 57518, living culture CMW 11298. , 1923,
F.J. Seaver & C.E. Chardon, NY 511.
sp., Sept. 2002, M.J.
culture CMW 10879 = , PREM 57522, living culture CMW 10885 =
.
Florida, Near Palmdale, Glades Co., , 1984, E.L.
Barnard & K.M. Old, FLAS 54261, ATCC 60862; ,
1984, E.L. Barnard & K.M. Old, FLAS 54263.
, 8 May 2000, C.S. Hodges, PREM 57523, living culture CMW
14551 = .
, Island of São Miguel, Mosteiro, C.S.
Hodges & D.E. Gardner, PREM 57524, living culture from same locality CMW
11301; Island of Pico, , 30 Jul. 1992, C.S. Hodges & D.E.
Gardner, PREM 57525, living culture from same locality CMW 11301; Island of
Pico, , 31 May 1985, C.S. Hodges & D.E.
Miguel, , 2 Aug. 1992, C.S. Hodges & D.E. Gardner, PREM
58811, living culture from same locality CMW 11301; Island of Terceiro, , 31 May 1987, C.S. Hodges & D.E.
culture from same locality CMW 11301; Island of Faial, , 27
May 1985, C.S. Hodges, PREM 58813, living culture from same locality CMW
11301.
: and have
been considered as synonyms when the latter fungus was still known as (,
).
has also been known as Shear & N.E.
was considered synonymous to (Shear
. 1917, , , ).
is, however, a distinct fungus from , as shown clearly in this study.
Specimens of resemble those of closely and clearly reside in the same genus.
similar spore dimensions, it is also probable that is
conspecific with .
that can be used to confirm the phylogenetic relationship of , we propose that retain its independent
taxonomic status for the present.
species clearly does not reside in .
(Vizioli) Gryzenh. & M.J. Wingf., MycoBank
.
.
: Vizioli, Mycologia 15: 115.
1923 (as ).
: , Grape Bay, fruit of
, 11 Dec. 1921, H.H. Whetzel, CUP
128; Grape Bay, fruit of , 11 Dec. 1921, H.H.
BPI 613756, NY 147, other specimen CUP 30512; Elbow Beach,
Fruit of , 28 Jan. 1926, Whetzel, Seaver & Ogilvie,
CUP 34658; South Shore, bark of , 25 Nov. 1940, F.J.
Seaver & J.M. Waterston, CUP 57366; Devonshire, , 2 Feb. 1926, Seaver, Whetzel & Ogilvie, CUP 35078; Devonshire
Bay, , 5 Feb. 1926, Seaver, Whetzel & Ogilvie,
CUP 35081.
study clearly does not represent .
morphological comparisons showed that a new genus should be provided for it
and the appropriate description is presented below.
found on the material, but based on DNA sequence comparisons the fungus
clearly belongs to the and is closely related to
and allied genera.
anamorphic fungus following Art. 59.2 of the International Code of Botanical
Nomenclature ().
Gryzenh. & M.J. Wingf.,
MycoBank
.
: Latin, , a bear, and latin,
, neck.
reminds of that of a bear.
slightly immersed in bark, unilocular, internally strongly convoluted, orange,
with one to three attenuated or cylindrical necks, tissue pseudoparenchymatous
but prosenchymatous in the neck. hyaline, delimited by
septa or not, cylindrical, conidiogenous cells phialidic, apical or lateral on
branches beneath the septum. cylindrical, hyaline,
aseptate.
: Gryzenh. & M.J.
Wingf., sp. nov.
Gryzenh. & M.J. Wingf., MycoBank
.
,
.
: Latin, , false.
conidiomata that appear to be false ascostromata.
three attenuated or cylindrical necks (Figs
,
), base 120–400
μm high, 190–550 μm diam, neck up to 400 μm long, 90–180
μm wide, superficial to slightly immersed, unilocular, internally
convoluted (Figs ,
).
pseudoparenchymatous (),
neck tissue prosenchymatous (). hyaline, cylindrical with or without
attenuated apex, cells delimited by septa or not, total length of conidiophore
(4.5–)5.5–19(–39) μm (Figs
,
). phialidic, apical or lateral on branches beneath the septum,
cylindrical to flask-shaped with attenuated apices, 1.5–2(–2.5)
μm wide, collarette and periclinal thickening inconspicuous (Figs
,
).
(2.5–)3–4(–5.5) × (1–)1.5(–2) μm,
cylindrical, aseptate, hyaline, exuded as orange droplets (Figs
,
).
: on MEA white, fluffy, margins even,
optimum for growth 25–30 °C, isolates covering 90 mm diam plates
after 5–6 d at optimum growth temperatures.
: Bark of
: Florida (U.S.A.).
: , Florida, Fort Lauderdale,
, 8 Mar. 2005, C.S.
Biscayne, , 10 Mar. 2005, C.S. Hodges, PREM 58841,
PREM 58842, living cultures CMW 18115 =
, CMW
18124 = ;
Oakland Park, , 11 Mar. 2005, C.S.
, 11 Mar. 2005, C.S.
culture CMW 18110.
study and that of Gryzenhout .
(), showed that isolates
representing from Australia and South Africa form a
clade distinct from other species in .
phylogenetic grouping is supported by discrete morphological characteristics
such as aseptate ascospores and small stromata, which are different to those
found in .
justification to erect a new genus for , and a
description is provided as follows:
Gryzenh. & M.J. Wingf.,
MycoBank
.
: Greek, , undivided, -,
secret, referring to undivided ascospores and the semi-immersed nature of the
stromata.
pseudoparenchymatous tissue at the edge of stromata, prosenchymatous tissue in
the centre. dark-walled, with globose to sub-globose bases
and slender periphysate necks that emerge at the stromatal surface as black
ostioles in papillae covered with orange stromatal tissue.
fusiform, floating freely in the perithecial cavity, unitunicate with
non-amyloid, refractive apical rings. cylindrical,
occasionally allantoid, hyaline, aseptate.
uni- to multilocular and convoluted, locules often occurring in same stroma
that contains perithecia. cylindrical with or without
inflated bases, tapering, often septate with or without lateral branches
beneath a septum, hyaline, paraphyses occurring between conidiophores,
conidiogenous cells phialidic. hyaline, cylindrical,
aseptate, expelled through an opening at the stromatal surface as orange
droplets or tendrils.
: (M. Venter &
M.J. Wingf.) Gryzenh. & M.J. Wingf., comb. nov.
(M. Venter & M.J. Wingf.)
Gryzenh. & M.J. Wingf., MycoBank
.
: M. Venter & M. J.
Wingf., Sydowia 54: 113–115. 2002.
Mtubatuba, Nyalazi estate, bark of GC747 clone of , 25 Feb.
1998, M. Venter, , PREM 56211, ex-type culture CMW 7034;
Dukuduku estate, bark of , Oct. 1998, M.
PREM 56214, PREM 56216; KwaMbonambi, Amangwe estate, bark of , Oct. 1998, M. Venter, PREM 56215,
living culture CMW 7033 = ; Mpumalanga, Sabie, bark of , Aug. 1998,
J. Roux, PREM 56212; Limpopo, Tzaneen, bark of , 6
Feb. 1999, M. Venter, PREM 56305, living culture CMW 7035. ,
Western Australia, Perth, , 1997, M.J.
PREM 56217, living culture CMW 7038 =
.
includes the fungi previously known as
and , while
represents the pathogen previously known as is a new genus that was discovered on in Florida while attempting to locate fresh specimens of .
primarily on the phylogenetic grouping of the isolates, which are distinct
from and other closely related genera such as
and
and are defined by the
following morphological characteristics.
stromata of and are similar to those
of but are much smaller.
also tend to be more superficial on the substrate than those found in
.
and , is that the conidiomata of both
fungi contain exceptionally long cells between the conidiophores.
previously referred to as paraphyses
(),
do not produce conidia. and are thus
morphologically quite similar but can be distinguished from each other based
on ascospore morphology. has single-septate ascospores,
while those of are aseptate. is
morphologically distinct from the anamorphs of
and other related genera because of its unique orange, pyriform to globose
conidiomata with cylindrical to attenuated necks.
was previously known as (Schwein.: Fr.) Fr. ().
because phylogenetic analyses indicated that isolates
of this fungus grouped more closely with than with
, the only two genera that it resembled at that time (Venter
,
).
grouping was supported morphologically by the semi-immersed stromata similar
to those of Consequently, the new species was placed
in despite the fact that its single-celled ascospores
were different from the two-celled ascospores characteristic of all other
species.
()
including more genera and species than those considered by Venter () did not
provide convincing evidence to separate from other
species.
additional taxa presented in this study and that of Gryzenhout .
(), which are
morphologically similar to those of , to reveal the
distinction between and species in the clade.
different from all species in with two-celled
ascospores, could thus be resolved.
defined because numerous isolates of could be
subjected to DNA sequence comparisons in this study.
examination of the herbarium specimens of have led us
to suspect that this fungus is a synonym of , the
taxonomic position of the former fungus has yet to be defined precisely.
(),
constriction at the ascospore septa and stromatal size
(), the length of
the perithecial necks (, ), and the small number of perithecia in the stromata
() have been used
to distinguish from other species in
.
specimens.
and , and stromatal morphology
varied greatly.
also observed.
in other specimens of This was despite the fact that
isolate CMW 11298, linked to PREM 57518, grouped with isolates linked to the
other specimens of the same species based on DNA sequence data.
feature that may have convinced previous authors that
represents a distinct taxon is the superficial fruiting structures on seeds.
stromatal morphology on the seeds () was superficial, while on bark it is semi-immersed
(,
).
is very similar, the pathogenicity and ecology of these two species have been
reported to be different.
sp.
freshly-cut branch sections of as successfully as isolates
obtained from , which have been shown in this study to
represent Likewise, the fungus from
did not grow in freshly-cut branch sections of although
the isolate was able to colonise this substrate.
().
Reciprocal inoculations on various hosts such as
spp. and spp.
that the isolates alone were able to infect resulting in cankers ().
may indicate that the two species are distinct, despite
their similar morphology.
from other closely related fungi is its prolific
colonization of fruits of , often while they are still
green.
allied genera have been found only on bark.
have chosen not to synonymise these species before isolates of can be obtained for DNA sequence comparisons.
fungus, , was found on this host.
new genus and species, which is closely related to and
allied genera, although no teleomorph structures were found for the fungus.
Morphological comparisons with showed that is distinctly different from .
related and morphologically similar fungi thus occur on ,
although it could also be possible that previous reports of in Florida actually represent .
complicate continuing surveys searching for on this
host in order to obtain isolates for later phylogenetic comparisons.
().
determine whether was the same as
in Cuba ().
substantial collection of isolates linked to additional specimens that we feel
confident to have the fungus previously known as .
clearly that the type of represents a fungus different
from that of from Japan. The fungus now known as thus does not occur in Japan.
and other hosts.
() described the fungus on
dead branches and twigs.
() also reported it as a
saprotroph on in Florida, while was
the cause of canker disease in the same plantations.
fungus was found only on dead, suppressed trees of , and
was not associated with cankers.
was associated with cankers on trees in the Azores
(), it
also occurs on dead trees, and may only play a saprotrophic role on cankers
().
the same locality as trees infected with .
consistent with the fact that both and were first described from Cuba in the same locality (Bruner
,
) and both occurred in the
same plantations in Florida () and Kauai.
relationship between and ,
deserves further consideration.
morphologically similar fungi, all with orange stromatic tissue, occur on
trees worldwide.
single genus , but most have now been transferred to new
genera. and have been
newly described in this study. occurs on
spp.
sp.
spp. in Japan ().
as the single species , also occur on
spp.
as and
().
The various spp.
spp.
(). Thus and the undescribed sp.
on are known from the Far East,
occurs in Australia and South Africa, and is now known
from Mexico, Cuba, Puerto Rico, Florida, Hawaii, Azores and Madeira.
Furthermore, the different species of occur in different
tropical and sub-tropical countries of the world
().
specifically in South Africa and occurs in Hawaii,
Central and South America, Central Africa, South East Asia and Australia
().
differ significantly in their pathogenicity to spp., which
is an ecologically important tree that also forms the basis of large forestry
industries. spp.
considered the most important pathogens in this group.
and the different spp.
saprophytes.
(), it is possible that
these fungi could be introduced into new areas.
has already moved from Australia, where it is presumed
to be native due to the widespread occurrence of in
native forests in Australia
(,
),
into plantation areas of South Africa
().
pathogens, every effort must be made to identify collections accurately.
underpins efforts to monitor the spread of diseases and to manage their
impact. |
During the 1950's, a shoot disease was observed on (then ) seedlings in New South
Wales, Australia. The causal fungus was later described as J.
().
South Africa.
() described the South
African fungus as a new species, M.J. Wingf.,
Crous & Swart.
() transferred to Hektoen & C.F. Perkins.
volume, a third species, U.
& Crous, isolated from leaf spots on in
Thailand, was described.
() distinguished the three
species based on morphology and host specificity.
species in (),
and not (),
was based largely on conidial scar morphology
().
() treatment of the
pathogens as species of had shown that
this genus accommodates superficially similar species with diverse
phylogenetic relationships (, ). The type species for the genus Hekt. & C.F.
Syd. & P. Syd., based on 18S rDNA sequences
().
More recently, Simpson ()
showed that isolates of are not cycloheximide-tolerant,
as is almost always the case with isolates with affinities
to ().
species of and , the dense growth of
white conidiophores on agar media and the host, and the absence of distinct
denticles on the conidiogenous cells, Simpson
() concluded that the
affinities of and the two related species, and , are not with the
.
J.A. Simpson, to accommodate the three species.
(), like Braun
(), distinguished the
species based on conidial morphology and specificity to their respective
or hosts.
apparent absence of dolipore septa in their hyphae observed by light
microscopy, he suggested that these fungi probably reside in either one of the
basidiomycete orders Henn., emend. R. Bauer &
Oberw., or G. Winter, emend. R. Bauer & Oberw.
().
in Australia by V.F. Brown.
sent to CBS in 1973 and was identified as de
Hoog & G.A.
().
Smith & Batenburg-Van der Vegte
() confirmed that and also de Hoog, have dolipores in
their septa and are thus the anamorphs of basidiomycetes.
and the presence of the basidiomycetous coenzyme Q-10 system
(),
Moore () erected a new
genus, R.T. Moore, for the two spp.,
with (de Hoog) R.T. Moore as generic type species.
first phylogenetic study that included the two spp.
showed that groups within the
Henn. based on LSU sequences ().
& G.A. de Vries) R.T. Moore grouped in the R.
Bauer & Oberw., and it was suggested that it could not be accommodated in
.
() thus described a new
genus, Sigler, with (de Hoog &
G.A. de Vries) Sigler as type species.
The aim of this study was to determine whether spp.
are monophyletic and what their relationship was to
using ITS sequences.
characters were used to determine an appropriate order in which species of
should reside.
For phylogenetic studies, two South African isolates of (M.J. Wingf., Crous & W.J. Swart) J.A.
the ex-type culture (CMW 1101 =
), were
compared with two isolates representing (J. Walker &
Bertus) J.A.
().
representing including the ex-type culture
(), were
also included.
study, are listed in .
GenBank accession numbers of sequences obtained in previous studies, are
indicated in Figs ,
.
were used ().
specimens had been deposited in the National Collection of Fungal Specimens,
Pretoria, South Africa (PREM). The holotype of (PREM
51089) consists of a dried culture on 2 % MEA.
morphological and ultrastructural characters are only expressed on host
tissue.
(PREM 58939), consists of symptomatic leaf tissue, collected from the same
host in the same location as the holotype
().
designated here as epitype for .
with the epitype ( = CMW 11678), was also included in the phylogenetic
analyses.
used for ultrastructural work, are underlined in
.
The ex-type culture of (U. Braun & Crous) J.A.
Simpson (CMW 8279) was found to be contaminated with a
species and could not be purified.
specimen (HAL) were not successful.
the study.
extract agar.
of PCR products, as well as DNA sequencing, were done as described by Aghayeva
().
internal transcribed spacer region (ITS1, the 5.8S rRNA gene and ITS2), was
amplified using PCR with the primers ITS1 and ITS4
().
The 5' end of the ribosomal large subunit (LSU) was amplified using primers
NL1 and NL4 (O'Donnell 1993).
Both alignments were assembled with MAFFT 3.85
()
using the accurate and iterative refinement method (FFT-NS-i settings).
trimming of both ends, the LSU alignment consisted of 572 bp and the ITS
alignment of 726 bp. Phylogenetic analyses were carried out using PAUP v.
4.0b10 ().
Modeltest 3.0 () was applied to determine a model of DNA substitution that
fits the data set.
for the LSU alignment (base frequencies: π = 0.2563,
π = 0.1950, π = 0.2911, π =
0.2576; substitution rates: A/C = 0.7670, A/G = 2.6760, A/T = 0.7823, C/G =
0.3153, C/T = 5.9744, G/T = 1.0000; gamma shape parameter = 0.7950; percentage
of invariant sites = 0.3790).
criterion for the ITS alignment (base frequencies: π = 0.2535,
π = 0.2188, π = 0.2157, π =
0.3120; substitution rates: A/C = 0.14911, A/G C/T = 5.2884, A/T = 2.1848, C/G
= 0.8252, G/T = 1.0000; gamma shape parameter = 1.6440; percentage of
invariant sites = 0.3892).
genetic distances according to the specified substitution model.
chains were run over 1 000 000 generations using the general time-reversible
model (six rate classes) including a proportion of invariant sites and
gamma-distributed substitution rates of the remaining sites (GTR+I+G) (for
description of models see ). Trees were sampled every 100
generation, resulting in an overall sampling of 10 000 trees.
first 3000 trees were discarded (as burn-in). MrBayes 3.0b3
() was used to compute a 50 % majority rule consensus of the
remaining trees to obtain estimates for the posterior probabilities.
().
electron microscopy (TEM), samples were fixed overnight with 2 %
glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) at 20 °C.
Following six transfers in 0.1M sodium cacodylate buffer, samples were
postfixed in 1 % osmium tetroxide in the same buffer for 1 h in the dark,
washed in bidistilled water, and stained with 1 % aquaeous uranyl acetate for
1 h in the dark.
were dehydrated in acetone, using 10 min transfers at 10, 25, 50, 70, 95, and
three times in 100 % acetone.
plastic and sectioned with a diamond knife.
mounted on formvar-coated, single-slot copper grids, stained with lead citrate
at room temperature for 5 min, and finally washed with bidistilled water.
samples were studied using a Zeiss EM 109 transmission electron microscope
operating at 80 kV.
topologies resolving all known orders of
Jülich, emend. R. Bauer & Oberw.
().
H. Kreisel R. Bauer & Oberw.
supported as sistergroup to the other orders.
resolved in all parts, the specimens of and
considered in this study clustered within the
as a highly supported monophylum in both datasets.
Gokhale clustered together with members of
the and it was, therefore, used as outgroup for the
ITS dataset of the .
(). (Patil) Begerow, R.
Bauer & Oberw., the only known member of the
Begerow, R. Bauer & Oberw., was sister to the other members of the
Niessl appeared paraphyletic in the LSU
and ITS analyses, and the relationship between the two
clusters was weakly supported.
positions of Sugiy., Tokuoka &
Komag. and .
and appeared to form a monophylum.
monophyly of and was
supported only in the ITS neighbour-joining analysis and was rejected by
maximum parsimony and Bayesian inference and by the LSU data. and the isolates formed three
separate, well-supported clusters.
isolates (ex-type and ex-epitype cultures) were identical, and also those of
the two isolates. The ITS sequences of two isolates differed from each other by 4 bp.
Begerow, R. Bauer & Oberw.
or less rounded pore lips, which were enclosed on both sides by membrane caps
().
the pores were also enclosed by membrane caps, but the septal pore apparatus
consisted of dolipores with swollen pore lips
().
anamorphic yeast, we found no septal
pores.
pores, but there was no cytoplasmic continuum between adjacent cells
().
the genus resides in the .
However, the ultrastructure of the septal pores of spp.
differ substantially from those of species in the
Jülich and .
family, to accommodate the species with dolipores.
Thus, the now include not only taxa having septa
with simple pores, but also taxa with dolipores or septa without pores.
Ultrastructural characteristics, together with LSU and ITS data, show that
is clearly monophyletic with the two sampled
spp. is therefore synonymised here
with and the necessary new combination is
established.
Z.W. de Beer, Begerow & R.
MycoBank
.
Socii Microstromatalium doliporos cum labiis pororum tumidis facientes.
lips.
J.A. Simpson, Australas. Mycol. 19:
60–61. 2000.
In this study we have produced phylogenetic evidence showing that infecting spp.
Africa, indeed represent two distinct species.
sets revealed that the two spp.
the basidiomycete order .
is supported by ultrastructural features.
differs from other genera in the
because it has dolipores with swollen pore lips in
the septa, and not simple pores with more or less rounded pore lips, which are
characteristic of the and
.
, in the to accommodate
spp.
of the
().
the are restricted to angiosperms, and most are
parasites of monocots ().
(), members of only two, the and the
do not form teliospores and occur on woody bushes
or trees ().
by the formation of complex interaction apparatuses
including interaction rings ().
including at least nine genera in four families
().
over 100 species occurring world-wide on flowering plants such as the
.
Poit., which includes more than 12 species, occurring
exclusively on (palms), also with a global distribution
(
and
).
A third genus of this order is O. Kamat &
Rajendren (). O.
was recently reported from South Africa for the first time, causing a
prominent leaf spot on native
().
and interaction apparatus ().
().
( and
) which is monotypic.
This fungus has such a unique morphology that it was placed in a family of its
own ().
including about 35 species occurring world-wide,
primarily on and
(
and
).
Only two spp. have been reported from South Africa:
(Desm.) Sacc. from , both exotic, and Syd. & P. Syd. from three native spp.
().
exotic spp.
from and, additionally, (Berenger) Sacc.
(,
). () is known
only from and has been reported widely from the Northern
hemisphere.
() has been found on
different genera belonging to the , with a global
distribution. has only been reported from
spp.
() and India
().
these species was not available for study.
Begerow .
() showed with LSU sequence
analyses that two anamorphic yeasts, (Buhagiar)
Rodr. Mir. & Weijman and (R.G. Shivas & Rodr.
Mir.) Rodr. Mir. & Weijman are phylogenetically closely related to
and , respectively.
results and show that might be the same species as . We included a third species, (R.G.
& Rodr. Mir.) Rodr. Mir. & Weijman, and it differs from in only 2 bp. ().
() in Australia, and were
described then as new species
().
() between the two species are small and they might represent
individuals of the same species. The three spp.
not be accommodated in the genus , because the type
species for (Fresen.) F.C.
phylogenetically (based on sequence data) placed in the
R.T.
().
time, since they might be linked to teleomorphs (probably
spp.) and could be more appropriately treated at a time when additional
material is available for study.
ultrastructural similarities () between the three spp.
in this study, is supported by the ecology of these species.
three species, as well as (not included), occur on tree
species native to Australia, suggests that Australia is the centre of origin
of these species.
tissues on several occasions, the fungus has not been associated with specific
disease symptoms of humans (, ).
demonstrate virulence of the fungus on mammals
().
The fungus is, therefore, rather regarded as an opportunist, and potentially
can be implicated in disease in immunocompromised patients
().
The recognition of spp.
widely considered because the teleomorph has never been observed.
teleomorph morphology of the closely related fungus is
considered (), it might be found that the teleomorph of
is masquerading as an anamorph.
as the anamorph and teleomorph states would be difficult to distinguish from
each other.
remains uncertain ( and
), is the anamorphic
yeast .
the nectar of an orchid in Japan ().
() resemble those of , its phylogenetic
position () sets it apart
from all the other members of the .
yeast forms pseudomycelia, occasionally with retraction septa, it is not
surprising that we did not observe pores
(),
but septa with median swellings ().
() reported simple pores
in , but the respective micrograph is insufficient.
The pore structure of the hyphal phase of is thus
unknown.
lineages that correspond with host families, follows a trend that has been
observed in other orders in the
().
The four families in the , for example, can be
distinguished based on basidial morphology and host range, but these
characteristics also match phylogenetic lineages based on LSU rDNA sequences
().
R. Bauer & Oberw.
shown ().
, additional fungal isolates from a wider variety of
hosts would need to be included in phylogenetic studies together with their
host species.
infects only and is restricted to
hosts in the genus .
phylogenetically distinct (, ) and it appears that the pathogens have specifically
evolved to infect them.
likely because they have not been considered an economically important group
of fungi.
disease caused by members of the in commercial
plantations in South Africa
(), Brazil and Uruguay
(,
),
and in plantations in Australia
(,
).
That we have only touched the “tip of the iceberg” of the
() should be regarded as a challenge, since so many
questions surrounding the biology and distribution of this intriguing group of
fungi remain unanswered. |
Angular leaf spot (ALS) of beans () is caused by
(Sacc.) Ferraris.
importance in tropical and subtropical areas, causing yield losses of up to 80
% (, , ).
particularly destructive in warm, humid areas
().
consist of circular to elliptical red-brown lesions, while leaf lesions start
as small, brown or grey spots that become angular and necrotic, being confined
by leaf veins.
(, ).
marketability of seed across bean-producing areas of the world
().
estimated to be around 374 800 t ().
selection of resistant varieties.
complicated, as the pathogen is highly variable with regard to pathogenicity,
which means that durable resistance is difficult to achieve
().
reported in by various authors
(, , , , ).
domestication events for the common bean, which in turn gave rise to two main
gene pools, namely large-seeded beans of Andean origin, and small to
medium-sized beans of Middle-American origin
(,
Gepts & Bliss ,
,
,
,
,
,
Singh . ,
,
,
,
).
, causal organism of ALS, (Sacc. & Magnus) Briosi & Cavara, the causal
organism of anthracnose, and (Pers.: Pers.)
Unger var.
undergone parallel micro-evolution with the host.
considerable variation within gene pools, differences are particularly evident
when the reactions of isolates to differential lines of known Andean and
Middle-American origin are compared.
virulent only on large-seeded lines, whereas those originating from countries
such as Central America, Mexico, Bolivia and Brazil are generally virulent on
lines from both groups (, , , , , , ).
() could distinguish two
groups in 55 isolates from Africa, the U.S.A.
America.
American isolates clustered in both groups.
both groups was reported from Africa
(,
),
which was also supported by data derived from isozyme analysis
().
Guzmán
() used RAPD analysis to
divide 62 isolates from Brazil, Wisconsin (U.S.A.) and
Malawi into two broad groups.
predominantly from Andean bean host genotypes, were more pathogenic on Andean
genotypes, whereas those from the second group, originating predominantly from
Middle-American bean genotypes, were more pathogenic on Middle-American bean
genotypes.
the 42 Malawian isolates belonged to the Andean group.
the preference for small-seeded beans in Brazil, and large-seeded beans in
Malawi.
(,
) appears to be
a variation of the Andean group ().
differences in conidial size and amount of septation between isolates.
However, he concluded that, due to the extent of variation within groups,
these characteristics could not be used for grouping isolates.
have attempted to associate lesion size with pathogenicity differences.
& Sharma () observed
two types of lesions in the field that differed in size, but found no
significant differences in the number and size of lesions caused by the two
groups of isolates, or in their radial growth in culture.
considerably, but Correa-Victoria
() found no significant
correlation between disease severity and lesion size, and no correlation
between spore production and lesion size, but reported it to be highly
dependent on the host cultivar
().
size may be affected by the interaction between host gene pool and pathogen
origin ().
differences between the Andean and Middle-American groups.
genus Ferraris for four -like
species, including Sacc.
(), the type
species, characterised by having synnematous conidiophore fascicles and
pigmented conidiophores and conidia.
elements were included in the genus (Ellis
,
,
).
() described a bean
pathogen in his monograph under Ellis &
Everh., but cited the older name as synonym.
In his notes he stressed to favour the retention of .
Deighton () reassessed the
genus, and considered the synnematous arrangement of conidiophores to be
unsuitable as sole character for generic differentiation.
confined to a few species similar to having non-geniculate conidiogenous cells with flattened, but
conspicuous scars.
conidiogenous cells and thickened, darkened scars in Fr.,
whereas taxa with quite inconspicuous conidiogenous loci were reallocated to
Speg.
() and Braun
(,
,
) preferred to maintain
based on synnematous conidiomata, but confined it to
species with conspicuous (slightly thickened, not darkened) conidiogenous
loci.
within Johanson, for which a
subset of isolates were subjected to DNA sequence analysis of the SSU region.
A further aim was to compare isolates of the Andean and Middle-American groups
to address the question if they represent two groups or species.
purpose isolates were compared by means of morphology, cultural
characteristics, and DNA sequence analysis of their internal transcribed
spacer region (ITS-1, ITS-2 and 5.8S), calmodulin, and actin regions.
South America, were studied ().
agar (MEA) (Biolab, Midrand, South Africa) as outlined by Crous
().
subcultured onto 2 % potato-dextrose agar (PDA;
) and
incubated at 25 °C under continuous near-ultraviolet light to promote
sporulation.
and the ITS, actin (ACT) and calmodulin (CAL) regions were amplified and
sequenced using the protocols and primers as described by Crous . ().
the 18S rRNA gene (SSU) was amplified and sequenced as described by Braun
. ().
sequences obtained from GenBank
()
and the alignment was assembled using Sequence Alignment Editor v. 2.0a11
() with manual
improvement of the alignment where necessary.
explained in Braun .
() using PAUP
(Phylogenetic Analysis Using Parsimony) v. 4.0b10
() with both
neighbour-joining and parsimony algorithms.
conducted with the uncorrected (“p”), the Kimura 2-parameter and
the HKY85 substitution models in PAUP.
broken randomly.
character states and all characters were unordered and of equal weight.
Heuristic searches were performed with 10 random taxon additions.
homogeneity test () was conducted in PAUP to consider the feasibility of
combining the ITS, actin and calmodulin data sets Sequence data were deposited
in GenBank () and the
alignments in TreeBASE (S1507, M2709-10).
characterisation on ALS differential lines from both the large- and
small-seeded gene pools, as published previously
(,
).
made of structures mounted in lactic acid, and the extremes of spore
measurements given in parentheses.
assessed after 14 d on PDA at 25 °C in the dark, using the colour charts
of Rayner ().
temperatures for growth (from 9–33 °C, in 3° intervals) were
determined on PDA plates as explained in Crous
().
in this study are maintained in the culture collection of the Centraalbureau
voor Schimmelcultures (CBS) in Utrecht, the Netherlands
().
(including the two outgroups) and 1029 characters including alignment gaps; of
these characters 38 are parsimony-informative, 57 are variable and
parsimony-uninformative, and 934 are constant.
using the three substitution models on the sequence data yielded trees with
identical topologies (data not shown).
obtained with the parsimony analysis, which yielded 13 most parsimonious trees
(TL = 135 steps; CI = 0.807; RI = 0.809; RC = 0.653), one of which is shown in
.
of and form a well-defined clade
(bootstrap support value of 83 %) within .
the species within the clade.
ITS sequence alignment contains 45 isolates (including the two outgroups) and
499 characters including alignment gaps; of these characters 168 are
parsimony-informative, 25 are variable and parsimony-uninformative, and 306
are constant.
on the sequence data yielded trees with identical topologies (data not shown).
Only the order and grouping of the deeper nodes differed between the
neighbour-joining and parsimony analyses (data not shown).
yielded 13 most parsimonious trees (TL = 293 steps; CI = 0.816; RI = 0.918; RC
= 0.749), one of which is shown in .
together with a bootstrap support value of 100 %, with the Middle-American
isolates ( f. ) grouping together
with a bootstrap support value of 84 %.
species (89 % bootstrap support), two strains of
(type species of , 95 % bootstrap
support) and a basal well-defined clade (bootstrap support value of 100 %) of
two GenBank sequences of .
and Andean origin can be distinguished phylogenetically, the ACT (235
characters) and CAL (316 characters) sequences were combined with the ITS
sequences.
combinable into a single analysis (P = 0.6550).
alignment consists of 1050 bases (including alignment gaps) and 30 isolates
(including the two outgroups).
parsimony-informative, 42 were variable and parsimony-uninformative, and 720
were constant.
analyses were identical to each other and also to that obtained from the
parsimony analysis (data not shown).
yielded three most parsimonious trees (TL = 353 steps; CI = 0.994; RI = 0.994;
RC = 0.988), one of which is shown in . The tree shows two distinct clades, namely f. and what we call here the f. clade.
results in support values of 53 % and 71 % for each clade, respectively.
values increase to 62 % and 98 %, respectively, if neighbour-joining with the
HKY85 substitution model is used for bootstrapping.
f. clade is further split into two groups (62 / 95 % and 52
/ 71 % bootstrap support, respectively), which is the result of three
characters that changed in the CAL sequence of isolates CPC 12238 and CPC
12239 (99.04 % sequence similarity to the other f.
isolates).
(Sacc.) Crous & U.
MycoBank
.
.
Basionum: Sacc., Michelia 1: 273. 1878.
: on , Italy, Selva, Aug. 1877,
Saccardo, Mycotheca Veneta 1247 (e.g., B, HAL, PAD).
: (Sacc.)
Crous & U. Braun, f.
: , on , F.S. Ngulu & C.
,
,
= CPC
10468. culture ex-epitype.
based on European material, and from our analysis, it appears that European
material is representative of f.
(Sacc.) Crous & U.
Crous & U.
MycoBank
.
Morphologically similar to f.
distinct by having a broader range of virulence on different bean types, and
being able to grow at or above 30 °C, which is not the case for f.
.
: , on , M.M.
,
, culture ex-type
= CPC
10463.
(): Gonzáles Fragoso
(: 339), Chupp
(: 295, as ), Ellis (:
269), Shin & Kim (:
151–153).
(): Saccardo, Fungi italici, Pl.
838, Padova 1881; Briosi & Cavara, Funghi parassiti delle piante coltivate
od utili, Fasc. I, No. 17, figs 1–2, Pavia 1888; Gonzáles Fragoso
(: 340, fig. 79); Ellis
(: 269, fig. 183);
Deighton (: 1098, figs
2–3); Shin & Kim
(: 153, fig. 65).
: On leaves, petioles, stems and pods; amphigenous, angular–irregular, rarely
subcircular–elliptical, mostly vein-limited, 1–8 mm wide, finally
sometimes confluent, forming larger patches, brown, ranging from pale
olivaceous, olivaceous-brown, yellowish brown, greyish brown to dark brown, on
pods often reddish brown and more regular, subcircular–elliptical,
margin indefinite, only delimited by veins, or surrounded by a narrow, dark
brown border or marginal line. on petioles, pods, stems
and leaves, amphigenous, mostly hypophyllous, usually scattered, occasionally
aggregated, conspicuous, punctiform, dark brown to blackish grey.
internal. almost lacking to
well-developed, subglobose, depressed to lacrimoid, up to 70 μm diam,
brown. numerous, up to approx. 40, in dense fascicles,
often forming synnematous conidiomata, erumpent, 100–500 ×
20–70 μm, rarely longer, olivaceous-brown, composed of a more or less
firm stipe of closely appressed conidiophores and a terminal, loose capitulum,
i.e.
usually up to 100 μm long, individual conidiophores filiform, appressed
threads 2–5 μm wide, up to 7 μm wide towards the apex,
pluriseptate, subhyaline to olivaceous-brown, thin-walled, occasionally
becoming rough-walled with age. integrated,
terminal, 20–100 μm long, subcylindrical to subclavate, usually not
or only barely geniculate, but moderately geniculate in some collections;
conidiogenous loci terminal and lateral, quite inconspicuous to
subconspicuous, i.e.
darkened-refractive, in surface view visible as minute circles, 1.5–2.5
μm diam, usually flat, non-protruding. solitary,
obclavate-cylindrical, broadly subfusiform, short conidia sometimes
ellipsoid-ovoid to short cylindrical, straight to curved,
20–75(–85) × 4–9 μm,
(0–)1–5(–6)-septate, usually not constricted at the septa,
rarely with slight constrictions, subhyaline to pale olivaceous or
olivaceous-brown, thin-walled, smooth, sometimes rough-walled, with obtuse
apex, and obconically truncate to rounded base, 1.5–2.5(–3) μm
wide, hila unthickened or almost so, at most somewhat refractive.
: Forma ; on OA colonies
flat to slightly erumpent, spreading with moderate aerial mycelium; margins
smooth, regular, surface with patches of olivaceous-grey and smoke-grey to
dirty-white; on PDA erumpent with moderate aerial mycelium, surface pale
olivaceous-grey to olivaceous-grey in the central part; margin iron-grey, and
also iron-grey in reverse. Cardinal temperature requirements for growth:
minimum 6 > °C, optimum = 24 °C, maximum < 30 °C.
; on OA flat to slightly erumpent, spreading, with
moderate aerial mycelium; margins irregular, feathery to smooth, even; surface
with the central part dirty-white to pale or darker olivaceous-grey, outer
region iron-grey; on PDA spreading, erumpent, with moderate aerial mycelium;
surface olivaceous-grey in the central part; outer region and reverse
iron-grey, margins feathery, irregular.
growth: minimum 6 > °C, optimum 24 °C, maximum > 30 °C.
: On , Japan,
Tokyo, Toyoda, Itino-machi, Minamitama-gun, 9 Aug. 1962, S. Takamoto (IMI
96372). On , Italy, Selva, Aug. 1877, Sacc.,
Mycoth. Ven. 1247 (HAL), type of ; Italy, Pavia,
Casatisma e Albaredo Arnaboldi, 1888, Briosi & Cavara, Funghi parass. 17
(HAL); Russia, Czernigov, Borzova, Aug. 1914, G. Nevodovsky, Petr. Mycoth.
gen. 249 (B); South Korea, Chunchon, 7 Oct. 2003, H.D. Shin (HAL). On
sp., Brazil, São Paulo, Botanical Garden, 26 Dec.
1901, Puttemans, No. 413 (B), type of ;
Italy, Bugellae et Vercellis, Cesati, Rabenh., Herb. mycol., Ed. 2, No. 327
(HAL), type of ; USA, N.J., Newfield, 27 Sep.
1894, J.B. Ellis (NY), type of .
host ( sp.?), Paraguay, Caá-guazú, Jan. 1882,
B. Balansa, No. 3492 (LSP 918), type of .
: ? ? ? (), worldwide, including Angola,
Argentina, Armenia, Australia, Austria, Bhutan, Brazil, Bulgaria, Burundi,
Cameroon, Canada, China, Colombia, Congo, Costa Rica, Croatia, Cuba, Dominican
Republ., Ecuador, El Salvador, Ethiopia, Fiji, France, Georgia, Germany,
Ghana, Great Britain, Greece, Guatemala, Haiti, Hungary, Jamaica, Japan,
India, Indonesia, Iran, Ireland, Israel, Italy, Ivory Coast, Jamaica, Japan,
Kenya, Korea, Laos, Latvia, Malawi, Madagascar, Malaysia, Mauritius, Mexico,
Mozambique, Nepal, Netherlands, Netherlands Antilles, New Caledonia, New
Zealand, Nicaragua, Nigeria, Norfolk Island, Panama, Papua New Guinea,
Paraguay, Peru, Philippines, Poland, Portugal, Puerto Rico, Réunion,
Romania, Russia, Rwanda, Saint Helena, Senegal, Sierra Leone, Singapore,
Slovenia, Solomon Islands, Somalia, South Africa, Spain, Sudan, Suriname,
Swaziland, Switzerland, Taiwan, Tanzania, Thailand, Trinidad and Tobago,
Turkey, Uganda, Ukraine, U.S.A. (CT, DE, Eastern states, FL, HI, IN, MA, MD,
ME, MI, MS, NC, NH, NJ, NY, OK, PA, SC, TX, VA, WI), Vanuatu, Venezuela,
Virgin Islands, Yugoslavia, Zambia, Zimbabwe
().
: As a consequence of molecular sequence analyses (Figs
,
,
), and re-examination and
reassessments of the synnematous conidiomata and scar and hilum structures
(, see Discussion),
proved to be congeneric with
.
presupposes acceptance of a formal proposal to
conserve the latter genus against the older names and
().
have already been treated and reallocated elsewhere
().
Rabenh.
this species, which appeared first on the printed label of `Rabenh., Herb.
mycol. 327, 1856'.
Schlechtendal () and
Saccardo (), but in all
cases without any description (.).
(: 339) was the first
author who correctly cited this name as synonym of , which we confirm after having re-examined Rabenhorst's original
material.
Speg. to synonymy with ,
but without any comments and references.
() examined type material
of this species and confirmed Deighton's
() synonymy.
disease of bean on pathological or molecular grounds, we were unable to find
enough morphological, cultural or phylogenetic support to separate these as
two species.
other type based on their host reaction on differential cultivars, we have
chosen to designate them as of the same species.
the Andean and Middle-American groups of the angular leaf spot pathogen of
beans.
between the two groups (other than cardinal temperatures for growth), nor
clear phylogenetic support for the separation based on various gene loci, we
have chosen to recognise these two operational units as of the
same species, namely f. and f. .
and , namely the structure
of the conidiomata and the type of conidiogenous loci and conidial hila.
the genus level for anamorphs of .
illustrated by the examples of Sacc. (pycnidia) and
Wallr. (acervuli)
(), Crous & M.J. Wingf.
(acervuli) and -like species with aseptate conidia and
pycnidia (), Unger (normal fascicles) and
Bonord. (synnemata) (Crous , unpubl. data),
which are all irregularly scattered among the cladogrames.
genus (pycnidia) always clusters basal to
Fresen. (fasciculate hyphomycete) (Crous .
,
).
synnemata is thus insufficient to separate from
(, ).
includes some synnematous species [e.g.
(Lév.) Speg.].
, but with inconspicuous conidial scars, have already
been reallocated in
().
also some other genera of hyphomycetes with synnematous as well as
non-synnematous species, e.g., Cif.
().
important character used for the distinction of and
.
basic feature in the taxonomy of cercosporoid genera (Deighton
,
,
,
),
was mainly or even solely based on the synnematous
arrangement of the conidiophores, combined with pigmented conidia formed
singly.
() transferred
Lév., the type species of
, to and thus
reduced to synonymy with .
The heterogeneity of is also reflected by the
exclusion of all species, except for the type species, ,
originally placed in this genus by Ferraris
(): Ellis (≡ (Ellis) Deighton
& M.B. Ellis), Ellis & Everh. (≡
(Ellis & Everh.) M.B. Ellis) and Earle (= (Schwein.) Deighton)
(see ).
(), Deighton
() and Braun
(,
,,
1998) considered the conidiogenous loci and conidial hila in
to be conspicuous or at least subconspicuous, i.e.,
barely to slightly thickened and darkened.
() already
placed the ALS pathogen in (conidiogenous loci
inconspicuous), although the wrong combination [ (Ellis & Everh.) J.M.
basionym, , cited as synonym.
in
() thus reduces
to synonymy with .
re-examined the scars and hila in in detail, based on a
wide range of samples and , including type
material of , and .
the conidiogenous cells are terminal to lateral, non-protruding, quite
inconspicuous to subconspicuous, i.e.
darkened-refractive.
conidiogenous loci, e.g.
.
quite inconspicuous to subconspicuous.
Deighton () is an example
of subconspicuous loci.
examinations, taxa with subconspicuous loci and hila (unthicked or almost so,
but slightly darkened-refractive or only the ultimate rim slightly thickened
and darkened) clustered together with species, so
that further segregate-genera like Deighton and
U.
(Crous .
,
;
).
() clusters with the type of
(), and the type of
Sacc. [ (Fuckel) Sacc.].
affinity of these three genera underlines earlier suspicions of mycologists
that criteria such as 1) slightly thickened conidial hila and scars, 2)
synnematous to fasciculate to sporodochial conidiomata, 3) transverse to
muriformly septate conidia, 4) euseptate to distoseptate conidia, 5) smooth
percurrent proliferations and sympodial proliferation, versus irregular, rough
percurrent proliferations on conidiogenous cells, are an insufficient basis to
separate anamorph genera in .
, and that they phylogenetically reside in the same
clade, the next predicament arises as to what name should be applied:
(1910; 1171 names), (1909,
65 names), or (1880, 161 names).
is the oldest name, is the most commonly used, and
many species of in fact represent other fungi.
, which also is older than ,
has been reduced to its type species, with most other species being placed in
either or .
predates .
to be strictly applied, all species in this complex should be transferred to
.
genus, we choose to avoid this upheaval, and support conservation of the
commonly used and accepted generic name,
().
latter genus should be used for the whole complex of hyphomycetes formerly
placed in and some of .
conservation proposal to this extent has been prepared for Taxon
(). |
The genus Ces. & De Not.
(), and is based on the
type species (Moug.: Fr.) Ces. & De Not.
(,
). is a species-rich genus with a
cosmopolitan distribution ().
dicotyledonous and gymnosperm hosts, on woody branches, herbaceous leaves,
stems and haulms of grasses, on twigs and in the thalli of lichens
().
habit from being saprobic, parasitic and endophytic
(,
),
and can cause die-back and canker diseases of numerous woody hosts
().
forming uni- to multilocular ascomata with multi-layered walls, occurring
singly or in clusters, often intermixed with conidiomata, which are pycnidial.
Asci are bitunicate, with a thick endotunica, stalked or sessile, clavate,
with a well-developed apical chamber, forming in a basal hymenial layer,
intermixed among hyaline pseudoparaphyses that are frequently constricted at
the septa.
brown and become septate and even slightly verruculose upon germination
(, , , , , ).
of the , which was not assigned to any specific
order.
(), and in 1917 Theissen & Sydow were of the opinion that
the should be united with the
().
formation of asci in locules embedded in stromata, and contained the
, a family established to accommodate multiloculate forms
like .
() placed
in the sub-family which was
placed in the ().
in the because true perithecial
walls were absent.
(with perithecia and paraphyses), the
(ascostromatic forms without paraphyses), and the
(ascostromatic forms with interthecial threads) and
assigned to the .
eight types of centrum development, and highlighted the taxonomic value of
sterile, interthecial tissues in the taxonomy of the .
and assigned to this order.
Luttrell's views were supported by Eriksson
() and Barr
().
Luttrell and Barr were not accepted by von Arx & Müller
() and von Arx
(), as they comprised a
mixture of unrelated genera ().
() only delimited the
, with two sub-orders and 24 families.
this was a more appropriate means of dealing with the taxonomy of this very
large heterogeneous group, at least until a more natural method of
classification could be developed.
maintained in the , but retained in the
.
accommodates in the
, and the
().
Although the is treated in the present study, its
ordinal position in the will be treated elsewhere as
part of the AToL (Assembling the Tree of Life) project (Schoch ., in prep.).
genera, of which only two were recognised by Denman .
().
subdivision was supported by comparisons of ITS sequence data, which separated
the examined spp.
those species with -like anamorphs and those with
-like anamorphs (, ).
and a larger suite of DNA-based markers supported this view
(,
,
).
by Denman & Crous (as (Wakef.) Denman & Crous with and
synanamorphs), which is morphologically and phylogenetically
distinct from representatives of the - and
-like groups ().
Ellis & Everh.
Fr., because of its distinct phylogenetic (usually ITS or
EF-1α) and morphological (striate conidia and paraphyses)
characteristics (). Recently, the name Sacc.
re-introduced as a distinct anamorph (conidia brown,
septate while still attached to the conidiogenous cells)
() and Cooke has been linked to
species with anamorphs
().
Many of the other 18 coelomycete genera linked to
remain untested in terms of phylogenetic association to the above groups.
remains poor.
comparisons have included limited numbers of species, not representing the
full anamorph diversity associated with .
the intron-dominated sequences of the ITS, β-tubulin and EF 1-α
loci (on which most previous studies were based) to infer phylogenetic
relationships across the diversity of the genus, is also unclear.
conserved mtSSU data have, for example, suggested that
and (Demaree & Wilcox) Arx & E. Müll.
unrelated to
() even
though they are typically assigned to this genus.
evolutionary radiations in the group, as exemplified by the morphologically
and phylogenetically distinct anamorph genera linked to it.
approach would be natural unit classification, also referred to as the
“genus for genus concept”
().
to unique teleomorphs on a one for one basis, correlating with phylogenetic
DNA data.
De Not. (), (Sacc.) Sacc. & D. Sacc.
( – this volume), Syd. & P. Syd.
(
– this volume) and
().
The primary aim of the present study is to delineate the phylogenetic lineages
of the , and to discuss the morphological
differences and generic concepts that can be ascribed to them.
purpose, we have chosen comparisons of sequences for the 28S rRNA gene (LSU)
because of its favourable size (approx. 900–1000 bp.
relatively conserved (medium–high level) nature, suitable to consider
taxonomic sub-divisions at the generic level.
on dead or dying twigs of various hosts as explained in Slippers . ().
isolates of representative spp.
Centraalbureau voor Schimmelcultures (CBS), Utrecht, the Netherlands and the
Culture Collection of the Tree Protection Co-operative Programme (CMW), FABI,
University of Pretoria, South Africa (Table 1).
determined on plates containing 2 % malt extract agar (MEA), 2 %
potato-dextrose agar (PDA), and oatmeal agar (OA)
().
() was used to extract
genomic DNA from fungal mycelia grown on MEA.
the PCR conditions recommended by the authors and spanning the 3' end of the
18S rRNA gene, the internal spacers, the 5.8S rRNA gene and a part of the 5'
end of the 28S rRNA gene. PCR products were separated by electrophoresis at 80
V for 1 h in a 0.8 % (w/v) agarose gel in 0.5× TAE running buffer (0.4
Tris, 0.05 NaAc, and 0.01 EDTA, pH
7.85) and visualised under UV light using a GeneGenius Gel Documentation and
Analysis System (Syngene, Cambridge, U.K.) following ethidium bromide
staining.
Band Purification Kit (Amersham Pharmacia Biotech Europe GmbH, Germany).
purified products were sequenced in both directions using an ABI PRISM Big Dye
Terminator v. 3.1 Cycle Sequencing Ready Reaction Kit (PE Biosystems, Foster
City, CA) containing AmpliTaq DNA Polymerase as recommended by the
manufacturer.
entire length of the amplicon. The resulting fragments were analysed on an ABI
Prism 3100 DNA Sequencer (Perkin-Elmer, Norwalk, CN).
GenBank sequences using Sequence Alignment Editor v. 2.0a11
(), and manual
adjustments for improvement were made by eye where necessary.
analyses of sequence data were done in PAUP (Phylogenetic Analysis Using
Parsimony) version 4.0b10 () and consisted of neighbour-joining analysis with the
uncorrected (“p”), the Kimura 2-parameter and the HKY85
substitution model in PAUP.
all characters were unordered and of equal weight.
randomly when encountered.
as both missing and as a fifth character state and all characters were
unordered and of equal weight.
the heuristic search option with simple taxa additions and tree bisection and
reconstruction (TBR) as the branch-swapping algorithm.
were collapsed and all multiple, equally parsimonious trees were saved.
robustness of the trees obtained was evaluated by 1000 bootstrap replications
().
length (TL), consistency index (CI), retention index (RI) and rescaled
consistency index (RC) were calculated and the resulting trees were printed
with TreeView v. 1.6.6 ().
distance analysis. First MrModeltest v. 2.2
() was used to
determine the best nucleotide substitution model.
performed with MrBayes v. 3 () applying a general time-reversible (GTR)
substitution model with gamma (G) and proportion of invariable site (I)
parameters to accommodate variable rates across sites.
Carlo (MCMC) analysis of 4 chains started from random tree topology and lasted
10 000 000 generations.
in 1000 saved trees.
likelihood values were stationary, leaving 950 trees from which the consensus
trees and posterior probabilities were calculated. PAUP 4.0b10 was used to
reconstruct the consensus tree, and maximum posterior probabilities were
assigned to branches after a 50 % majority rule consensus tree was constructed
from the 950 sampled trees.
agar (WA) with sterilised pine needles as substratum, at 25 °C under
near-UV light, to induce sporulation.
and 30 measurements at × 1000 magnification were made of each structure
where possible.
of spore measurements given in parentheses.
are maintained in the CBS culture collection.
HKY85 substitution model (). These are discussed in the Taxonomy and Discussion sections.
Parsimony analysis with gaps treated as missing characters yielded 79604
equally parsimonious trees (TL = 582 steps; CI = 0.509; RI = 0.905; RC =
0.460).
trees (TL = 603 steps; CI = 0.524; RI = 0.910; RC = 0.477).
consensus trees calculated from the equally parsimonious trees were identical
to each other and are shown in TreeBASE.
parsimony analyses, the same clades were supported with two exceptions.
first exception is D.E.
(), which
resides in Clade 3 (),
but is basal to Clades 1 to 6 in the strict consensus trees.
with a bootstrap analysis (data not shown).
clades ().
observed were related to the position of , which clustered
close to Clade 3 in the distance analysis, but clustered in Clade 4 in the
Bayesian analysis.
Clades 8–9 clustered basal to the .
presently recognised in the were subjected to DNA
sequence analysis. These analyses revealed 11 clades in the family.
phylogenetic clades can also be correlated with distinct morphological
features.
Clade 1 includes species with Sacc.
anamorphs clustering together.
gene regions such as ITS, EF-1α and β-tubulin, support the synonymy
of under , in various cases they
separate from the clade
(,
).
() in which species
having anamorphs could not be separated from those in
.
polytomy with low bootstrap support.
which teleomorph name is best suited for this clade, as the form genus
is known to be polyphyletic
(,
).
clustering of three species of Höhn.
was also unexpected.
, namely , and its anamorph
Corda. The genus N.E.
Stevens & Baechler, represented by a strain identified as (Desm.) Petr. (type species)
(),
clusters in this clade, as does the genus Cooke,
represented by a strain identified as (Mont.) Cooke
(type species) ().
(Tassi) Goid.
“” (Taubenh.) E.J.
which is shown to be a member of the .
have apical mucous appendages early in their development, which has in the
past led to confusion, and the allocation of this species to the genus
().
brown and slightly roughened, appearing more -like in
morphology.
Clade 4 represents (Penz.) D.F. Farr.
species, which has a large number of synonyms
(),
is unusual in having a -like coelomycete anamorph (with
mucoid apical appendages).
hyphomycete synanamorph that is lacking in other species in the
, typified by
Pesante () clusters outside of the .
However, DNA sequence data derived from the species
present in the CBS collection lead us to conclude that this genus is also
polyphyletic (results not given).
clusters most closely with species in either Clade 3 or Clade 4 in the various
analyses.
species residing in Clade 2 ().
apical appendages or discoloration found in species residing in Clade 3, nor
does it have a -like synanamorph occurring in species
residing in Clade 4.
unresolved.
septate ascospores for which the genus Höhn. is
available (Barr ,
).
reside in Sacc.
().
-like anamorphs and -like synanamorphs,
for which the name gen. nov. is introduced.
-like synanamorphs in this clade are characterised by
globose to pyriform conidia. The older, brown conidia in Clade 2
(.) are obovoid, ellipsoid or fusiform, never
globose or subglobose (, ).
Clade 7 represents isolates of “” Mohali, Slippers & M.J. Wingf.
().
This taxon is distinguished from and other
-like genera by having conidia that are enclosed in a
persistent mucous sheath, for which the genus is
proposed.
Clade 8 is represented by a single species, (Schwein.) Sacc., for which the genus Nitschke
ex Fuckel is available. Denman & Crous (anamorph
-like; synanamorph -like) (Clade 9) is
morphologically distinguished from by having
unilocular ascomata that develop under a clypeus.
Pers. anamorphs ().
genera, namely Schulzer [type =
(Hazsl.) Sacc.], “” Aa,
and other morphologically distinct taxa. “”
Earle and “”
(Berk.) Sacc. (Clade 12) are shown to be distinct from the
Syd. & P. Syd.
for them, as they cluster apart from the .
Surprisingly, they cluster in the although no teleomorph
connections are currently known for species of .
italic
sup
#text |
The genus () contains approximately
700 species (), most of which are known to host a range of incredibly
diverse and interesting microfungi (, ).
papers listing and describing the plant-pathogenic fungi occurring on
eucalypts in the various countries where these trees are grown as ornamentals,
or planted in plantations for timber and paper fibre
(,
).
enabled plant pathologists to revise numerous important pathogen complexes
such as Mycosphaerella leaf blotch (, Crous .
,
,
,
),
Cylindrocladium leaf blight (Crous
, 2004b), Cryphonectria
canker (), Botryosphaeria canker (Slippers .
,,),
(),
(),
and leaf spots (), to name but a few.
the saprobic microfungi have largely been neglected, and in spite of
checklists and descriptions, very few are in fact known from culture, or are
represented in freely accessible culture collections.
diverse genera will never be represented in international initiatives like
Assembling the Tree of Life (AToL), or the Consortium for the Barcoding of
Life (CBoL), and biologists will remain ignorant as to their distribution,
host range, importance and various ecological roles.
appears to harbour numerous undescribed and relatively unstudied fungal
species, it was decided to focus on this host substrate to obtain cultures for
inclusion in larger projects and international initiatives such as those cited
above.
eucalypt microfungi from culture, and recollecting and culturing those already
known (), to help elucidate their taxonomy, and resolve their
phylogenetic relationships.
Leaf litter as well as living, symptomatic leaves were chosen for study.
Leaves were incubated in moist chambers (Petri dishes with moist filter paper
on the laboratory bench), and inspected daily for microfungi.
coelomycetes were cultured on 2 % malt extract agar (MEA) plates (Gams 1989) by obtaining single conidial colonies as explained in Crous
().
ascospores were obtained and cultured using the technique as explained in
Crous ().
sub-cultured onto fresh MEA, oatmeal agar (OA), cornmeal agar (CMA) and
carnation leaf agar (CLA) plates (Gams 1989) and incubated at
25 °C under continuous near-ultraviolet light, to promote sporulation.
plates following the protocol of Lee & Taylor
().
ITS4 () were used to amplify part (ITS) of the nuclear rRNA operon
spanning the 3' end of the 18S rRNA gene (SSU), the first internal transcribed
spacer (ITS1), the 5.8S rRNA gene, the second ITS region and the 5' end of the
28S rRNA gene (LSU).
as explained in Crous .
(). Part of the 18S rRNA
gene was amplified and sequenced as explained in Braun .
() and part of the 28S rRNA
gene as explained in Lee .
().
subjected to a nucleotide-nucleotide BLAST
()
of the NCBI sequence database (BLAST-N 2.2.11;
).
The LSU and / or SSU sequences were also used in cases where ITS sequences did
not provide adequate BLAST results.
Fungal structures were mounted in lactic acid or in water when stated.
extremes of spore measurements (30 observations) are given in parentheses.
Colony colours (surface and reverse) were rated after 7–14 d on MEA and
OA at 25 °C in the dark, using the colour charts of Rayner
().
in this study are maintained in the culture collection of the Centraalbureau
voor Schimmelcultures (CBS) in Utrecht, the Netherlands
(), and type specimens
in the mycology herbarium (PREM) at the Biosystematics Division of the Plant
Protection Research Institute, Agricultural Research Council of South
Africa.
GenBank (). BLAST
searches resulted in associations with known fungal species or orders.
results are discussed in the descriptive notes below each of the treated
species.
.
: Resembling species accommodated in
.
: Crous, sp. nov.
thick-walled, smooth to finely verruculose, branched, septate, with swollen
cells giving rise to conidiophores; hyphododium-like structures present,
simple, intercalary. separate, erect, medium to dark
brown, smooth to finely verruculose, thick-walled, subcylindrical, straight,
septate. terminal or intercalary, monotretic or
polytretic, sympodial, with 1–2 conspicuous loci, thickened, darkened,
refractive, with a minute central pore, not protruding as in the case of
frequently remaining attached in long
acropetal chains, simple or branched, narrowly ellipsoidal to cylindrical or
fusoid, 0–1-septate, medium brown, thick-walled, finely verruculose,
apical conidium with rounded apex, additional conidia with 1–2 truncate,
conspicuous hila; thickened, darkened, refractive, with a minute central pore.
on MEA producing abundant amounts of diffusing red pigment.
absent.
.
,
.
surface, medium to dark brown, thick-walled, smooth to finely verruculose,
branched, septate, 2.5–3.5 μm wide, frequently forming a swollen cell
which gives rise to a conidiophore; hyphododium-like structures present,
simple, intercalary, 2.5–3.5 μm diam.
separate, erect, medium to dark brown, smooth to finely verruculose,
thick-walled, subcylindrical, straight, 1–4-septate, 15–60 ×
5–7 μm. terminal or intercalary,
monotretic or polytretic, sympodial, usually with 1–2 conspicuous loci,
1.5–2 μm wide, thickened, darkened, refractive, with a minute central
pore, 0.5–1 μm wide, scar usually within the cell outline, and not
protruding as in the case of ., finely
verruculose, medium brown, 10–17 × 4–5 μm.
frequently remaining attached in long acropetal chains,
simple or branched, narrowly ellipsoidal to cylindrical or fusoid,
0–1-septate, (11–)13–15(–22) ×
(2.5–)3–3.5(–4) μm, medium brown, thick-walled, finely
verruculose, apical conidium with rounded apex, additional conidia with
1–2 truncate, conspicuous hila, 1.5–2 μm wide, thickened,
darkened, refractive, with a minute central pore, 0.5 μm wide.
: Colonies on MEA producing abundant
amounts of diffusing red pigment that changes the colour of the medium to red;
colonies irregular, erumpent, with smooth, irregular margins; surface
iron-grey; reverse greenish black.
: sp., South Africa
(Western Cape Province).
: , Western Cape Province,
Stellenbosch Mountain, on leaf litter, 13 Dec. 2003, P.W.
Crous, , , cultures ex-type CPC 10953–10955 =
.
: The genus Link contains 772 names
(),
many of which represent elements not congeneric with the type species, (Pers.: Fr.) Link, which is an anamorph of
Crous & U. Braun (). The recent description of Seifert &
N.L.
with slightly thickened conidial scars proves this point.
resembles in general morphology, but lacks chlamydospores,
forms a distinct red pigment in culture, and clusters apart from the
complex (), the
Borelli complex (), or
the U. Braun complex ().
BLAST results of the ITS sequence of this species had an E-value of 1e-90 with
ITS sequences of Tul., De Not.
() and Arx & D.L.
().
E-value of 0.0 were obtained from the LSU data: (Peck) Seaver (), (Hoffm.) Müll. Arg. (),
(Davies) Vězda (),
Grossenb. & Duggar (), and others.
0.0 were also obtained from the SSU data:
Wollenz. & de Hoog (), (Penz.) B.
(),
Ondřej () and others.
.
: Named after its characteristic conidiomata and spore
masses that appear as orange candle flames once plant material is incubated in
moist chambers.
: Crous, sp. nov.
septate hyphae, forming brown stromata that give rise to conidiomata.
sporodochial, appearing as erect, orange, fusoid
structures; basal region consisting of pale brown
to , giving rise to thick-walled, pale brown
cells of becoming thin-walled, hyaline, and
radiating outwards from the narrower, semi-cylindrical sporodochial base,
branching sympodially to give rise to hyaline, smooth, thin-walled setae with
bluntly rounded ends; inner conidiomatal layer consisting of a mixture of
setae and conidiogenous cells. hyaline, smooth,
subcylindrical, proliferating blastically and sympodially.
subcylindrical, straight or slightly curved with obtuse ends, septate,
hyaline, smooth, guttulate.
.
.
septate hyphae, 1–1.5 μm wide; aggregating in the epidermis to form a
pale to dark brown stroma, up to 50 μm wide, which gives rise to a
conidioma. sporodochial, appearing as erect, orange,
fusoid structures on the leaf surface (like the flame of a candle), up to 100
μm diam and 200 μm high; basal region consisting of pale brown cells of
to , 3–7 ×
2–3 μm, giving rise to thick-walled, pale brown cells of , 6–15 × 2–3 μm, becoming thin-walled,
hyaline, and radiating outwards from the narrower, semi-cylindrical
sporodochial base, branching sympodially to give rise to hyaline, smooth,
thin-walled setae that terminate in bluntly rounded, obtuse ends, and give the
conidiomatal margin a feathery appearance; the inner layer of the conidioma
gives rise to a mixture of setae and conidiogenous cells. hyaline, smooth, subcylindrical, 7–15 × 1.5–2.5
μm, proliferating sympodially, with inconspicuous scars, giving rise to
additional conidiogenous cells, or to conidia.
subcylindrical, straight or slightly curved, 3-septate, hyaline, smooth,
guttulate, widest in the middle, with obtusely rounded ends,
(35–)43–55(–60) × 1.5–2 μm.
: Colonies on MEA spreading, erumpent,
folded, with sparse aerial mycelium; surface pale luteous to buff, with
diffuse strips of red; reverse luteous.
mycelium, spreading, appearing to grow more in the agar than on the surface,
pale luteous; colonies sporulated when freshly isolated, but became sterile
upon first transfer.
: sp., Spain.
: , on leaf
litter, Apr. 2004, M.J.
,
, cultures ex-type CPC 11243 =
, CPC
11244–11245.
: is similar to other genera with
sporodochial conidiomatal such as B.
Petr., Hansf.
Nag Raj (, ).
conidiophores, mode of conidiogenesis, presence of marginal, thin-walled setae
and its cylindrical conidia.
had an E-value of 5e-130 with the ITS sequence of a foliar endophyte of
.
(Berk. & Broome) Dennis (7e-123;
), sp. (2e-120;
) and Mouch. (2e-117;
). A number of similarities with an E-value of 0.0
were obtained from the LSU data: Fr.
(), (G. Winter)
Malloch & Cain (), F.H.
Beyma (), Burt
(), (Alb. & Schwein.) Dennis
() and others.
0.0 were also obtained from the SSU data: (G.G.
Hahn) DiCosmo, Nag Raj & W.B. Kendr. (),
spp. (),
H.S. Jacks. () and others.
(Pers.: Fr.) Fr., Herb. mycol., ed. 2:
no. 532. 1857.
Ascomata indistinct on host, intermingled with those of Crous & M.J. Wingf.
obtained on CLA. dark brown to black, up to 400 μm high
and 300 μm wide, flask-shaped with an elongated red-brown neck up to 70
μm long. numerous, cylindrical, bitunicate, with a prominent
foot cell, 120–160 × 4–6 μm.
hyaline, septate, constricted at the septa, 2–3.5 μm wide, not
extending beyond the asci. somewhat spiralled or twisted
in the asci, pale brown, subcylindrical, with tapering to subobtuse ends,
multiseptate (septa at approx. 10 μm intervals), 130–165 ×
1–1.5 μm.
: Colonies spreading on MEA, slightly
erumpent with moderate aerial mycelium and feathery margins; surface on PDA
and OA pale mouse grey to mouse grey; reverse chestnut on MEA, iron-grey on
OA.
fertile perithecia was obtained on CLA.
:
sp., Colombia.
: , on leaf
spots, associated with lesions of Crous
& M.J. Wingf., 16 Feb. 2004, M.J.
,
culture CPC 11006 = .
: Shoemaker
() listed numerous hosts
for (as (Pers.: Fr.) Sacc.),
and stated that it is often recognized by the red-purple stain it induces on
the host substrate, and the red-brown colour of the apical part of the
ascomatal neck.
and is suspected to be the teleomorph of Desm. var.
Foister.
had an E-value of 0.0 with an ITS sequence of on
GenBank (AF383951; 99 % similarity).
spp. () ranged from 9e-175 to 4e-109.
similarities with an E-value of 0.0 were obtained from the LSU data:
(G.F. Weber) O.E. Erikss., M.
() and others.
of 0.0 were also obtained from the SSU data: spp., (Berk.) Berk.,
(Cooke & Peck) Sacc. ()
and others.
H.J. Swart, Trans. Br. Mycol. Soc. 90:
288. 1988. .
obtained, and thus the description is based on features were sparingly formed on MEA, medium brown, globose, up to
400 μm diam. pale brown, cylindrical,
proliferating percurrently near the apex, 10–15 × 3–5 μm.
medium to dark brown, ovoid, smooth, guttulate, developing a
single supramedian septum, thick-walled, frequently constricted at the septum,
apex obtuse, base truncate with a visible scar, 2–3 μm wide,
(15–)17–19(–20) × (8–)10–12(–13)
μm.
: Colonies flat on MEA, spreading, with
moderate aerial mycelium and submerged, smooth margins.
on MEA, cream to pale white on OA; reverse with patches of luteous to umber on
MEA, pale luteous on OA; fertile on MEA.
: sp., New Zealand;
also known from spp.
().
: , on sp.,
2004, J.A. Stalpers, CPC 10945 =
.
: As far as we could establish, has not
previously been known from culture ().
E-value of 9e-98 with an ITS sequence of Nitschke
().
spp. (1e-96), Munt.-Cvetk.,
Mihaljč. & M.
sp. (6e-96; ).
with an E-value of 0.0 were obtained from the LSU data:
spp. (), spp.
(), spp. () and
others. A number of similarities with an E-value of 0.0 were also obtained
from the SSU data: (Nitschke) Höhn.
(), spp., Crous & C.L. Lennox, (Schwein.) Fr.
() and others.
(Thüm.) Steyaert, Bulletin
Jard. Bot. l Etat Bruxelles 19: 319. 1949.
.
on the surface of the colony. broadly fusoid to
fusoid-clavate, straight or somewhat curved, 5-celled, upper cell conical to
cylindrical, hyaline, fairly thin-walled, apical setulae central,
(2–)3(–4), rather stout, up to 1.2 μm wide, 11–20 μm
long, with a blunt tip, three intermediate cells concolorous or the upper two
intermediate cells slightly darker, dull olivaceous-brown to vinaceous-brown,
contents guttulate, walls smooth, slightly constricted at the septa when
mounted in water, and thickened up to 1 μm especially in the upper two
intermediate cells and in the septa, basal cell hyaline, thin-walled, tapering
into a filiform pedicel (2–)2.5–4.5(–5) μm long; conidium
body (18–)20–24(–25) × 6.5–7(–8) μm
(OA).
: Colonies on OA reaching 52–54 mm
diam in 7 d with an even, glabrous, colourless margin; immersed mycelium
colourless, aerial mycelium pure white, fluffy, covering most of the colony
surface, and very dense and high in the centre and in concentric zones after 7
d; reverse in the centre buff.
after 7 d, as on OA, but aerial mycelium less well-developed, and reverse
colourless.
slightly undulating colourless margin; immersed mycelium colourless, but
surface of the colony completely covered by a high, dense mat of pure white,
in the centre yellowish, fluffy aerial mycelium, the margin also covered by a
diffuse layer of aerial hyphae; reverse with a faint cinnamon tinge.
: , New
Zealand (North Island).
: , North Island, Kerikeri,
living leaves of , 17 Oct. 2003, M.A. Dick, CPC
10950 = ,
CPC 10951.
: BLASTn results of the ITS sequence of this species had an
E-value of 0.0 with ITS sequences of spp.
after 3–5 d (OA, MEA & CMA). narrowly fusoid to
fusoid-clavate, straight or somewhat curved, 5-celled, upper cell conical to
cylindrical, hyaline, fairly thin-walled, without visible cellular contents,
bearing (2–)3(–4) rather stout central apical appendages,
10–19 μm long, up to 1.2 μm wide, with a blunt tip, three
intermediate cells concolorous or the upper two intermediate cells slightly
darker, dull olivaceous-brown to vinaceous-brown, contents guttulate, walls
smooth, thickened up to 1 μm especially in the upper two intermediate cells
and in the septa, basal cell hyaline, thin-walled, tapering into a filiform
pedicel (3–)4–5(–6) μm long; conidium body
(19–)20–24(–27) × (5.2–)5.5–6 μm
(OA).
: Colonies on OA reaching 50–53 mm
diam in 7 d with an even to undulating, glabrous, colourless margin; immersed
mycelium colourless, aerial mycelium pure white, woolly-cottony, covering most
of the colony surface without distinct concentrical zonations, almost absent
in the marginal zone after 7 d; reverse concolorous, in the centre buff (where
sporulation occurs).
well-developed.
irregularly undulating, colourless, glabrous margin; immersed mycelium
colourless, but surface of the colony completely covered by a moderately high,
densely woolly mat of pure white, locally faintly sulphur-yellow, aerial
mycelium; reverse ochreous to fulvous, brown where conidiomata develop.
: ?,
Colombia.
: , living leaves of
, 2004, M.J.
,
cultures CPC 10969 = , CPC 10970–10971.
: BLASTn results of the ITS sequence of this species had an
E-value of 0.0 with ITS sequences of spp., including
and
(Speg.) Bissett (both 99 % similar).
The primary reason for the inclusion of these spp.
in the present paper is the presence of a synanamorph, which has never before
been reported for species of in the literature
().
unpublished notes in the CBS database, this has once before been observed for
a culture of a sp. in the collection.
observed in host tissue to exude a mixture of black and hyaline spores in a
typical cirrhus associated with conidiomata.
cirrhus consisted of two conidial types, namely typical
conidia (alpha), and long, narrow, bent, needle-like
cylindrical conidia (beta) resembling the beta conidia observed in species of
, or the conidia typically associated with
anamorphs. Conidia were 25–30 ×1–1.5
μm, widest in the middle, tapering to a subobtuse apex, and a truncate
base.
conidiogenous cells that terminated in an apex with 1–2 loci which gave
rise to conidia in a sympodial arrangement.
cells were situated on 1–3-septate conidiophores that were 10–20
× 2–3 μm.
Colombia.
to germinate on MEA (observed over 2 wk), while all alpha conidia germinated
within 1–2 d.
types was obtained from New Zealand.
induced to germinate, and thus their ecological role as potential conidia, or
spermatia, still needs to be resolved.
conidia could be induced to form beta conidia on MEA, OA or CLA.
regard it is interesting to note that, contrary to common opinion, it has only
recently been proven that beta-conidia of spp.
germinate in culture ().
B.
Hedwigia 26: 3. 1975. .
that occupy the stomatal chamber; wall consisting of two regions, the lower
region having thick-walled dark-brown cells, up to 5 layers thick, the upper
region consisting of thin-walled, paler cells, up to 5 layers thick.
restricted to the lower part of the basal wall,
3–7 × 2–3 μm, doliiform to lageniform, phalidic with
periclinal thickening, hyaline, with an indistinct collarette.
hyaline, aseptate, guttulate, subcylindrical, predominantly
straight, with obtuse ends, 11–17 × 1–1.5 μm.
: Colonies spreading on MEA, flat with
sparse aerial mycelium and smooth margins; surface sienna to umber with
patches of white, and dark brown conidiomata; reverse umber (centre) to sienna
(margins); on OA umber with no aerial mycelium, and dark brown
conidiomata.
: spp., Colombia,
Indonesia.
: , on leaf
litter, Feb. 2004, M.J.
,
cultures CPC 10972–10974.
litter, Mar. 2004, M.J.
,
cultures CPC 11017 = , CPC 11018–11019.
: The collections from Indonesia and Colombia are
morphologically similar.
Indonesian collection (11–17 × 1–1.5 μm) are similar to
those of the Colombian collection (12–14 × 1–1.5 μm), and
fit within the range given for the species, namely 11.5–15.5 ×
1–1.5 μm ().
clear that there are some base pair differences between these isolates,
suggesting that these strains may in fact represent different species.
only obvious morphological difference observed was that conidiomata of the
Colombian collection were pale brown, with cells at the margin of the wall
being up to 5 μm wide.
collection were darker brown, with cells at the margins being narrower, namely
3–4 μm wide.
to the differences observed in the DNA sequences, can only be resolved once
further collections have been obtained.
this species has E-values of 5e-167 to 1e-115 with ITS sequences of
unidentified leaf litter and mycorrhizal ascomycetes.
species include (Pers.) Fuckel (2e-110),
(Pers.) Seaver (9e-107; ), and
spp. (4e-106; ).
similarities with an E-value of 0.0 were obtained from the LSU data:
Fr. (), (G. Winter) Malloch & Cain (),
G.W. Beaton () and others. A
number of similarities with an E-value of 0.0 were also obtained from the SSU
data: (G.G. Hahn) DiCosmo, Nag Raj & W.B.
Kendr., spp., H.S. Jacks.
(all ) and others.
(Berk.) Spooner, Bibl. Mycol. 116:
322. 1987. Figs ,
.
erumpent, stipitate, arising from a subepidermal stroma visible around the
stipe as a dark discoloration. plane to convex, greyish brown to
olivaceous, smooth, 0.4–1.5 mm diam. cupulate,
concolorous but usually darker than the hymenium, bearing dark brown to
reddish brown setae. central, smooth and dark brown,
0.4–1.8 mm high. mostly 20–50 per apothecium,
(150–)200–250 μm long, smooth, with dark brown walls thickened
up to 1.5 μm, septate, paler at the blunt top, attenuated and bent at the
base. cylindrical-clavate, apex conical-rounded, the apical
apparatus blueing in Melzer's reagent, croziers present, 8-spored,
75–100 × 7–9 μm; fusoid, 0-septate,
narrowly rounded at both ends, contents guttulate, hyaline, each end provided
with a central, everted (umbrella-shaped) mucelaginous appendage, 17–25
× 3–4 μm; sometimes producing ellipsoid microspores 3–5.5
× 1.5–2 μm directly from apertures at one or both ends.
simple or branched near the base, obtuse, hyaline,
somewhat inflated and up to 3.5 μm wide at the top.
: on OA reaching a diam
of 15–20(–30) mm in 14 d, with an even to slightly ruffled,
glabrous and colourless margin; immersed mycelium at first colourless, then
very faintly yellowish (primrose) or reddish (apricot), after 10–20 d
gradually developing a mixture of several tinges, pale hazel, ochreous and
amber, in the centre sometimes also greyish to olivaceous buff, most of the
surface almost glabrous and without aerial mycelium, locally with patches of
woolly, pure-white aerial mycelium.
diam in 14 d, with a ruffled, glabrous, colourless margin; most of the colony
surface covered by a fairly dense, woolly but low mat of pure white aerial
mycelium; reverse in centre ochreous to umber, fading to the colourless
margin.
surface, most very similar in shape and size to those formed , but with less setae; however, large abnormally shaped apothecia
are also formed: hymenium convex, protruding from the agar surface as a
greyish-black, globular mass with a smooth surface, 1–2.5 mm diam,
receptacle reduced, hairs present or absent, lacking a stipe.
: developing on the
surface of globular ascomatal initials after 2–3 wk, smooth-walled,
variable, simple, but mostly branched near the base, 15–30 ×
2–4(–5) μm thick, hyaline or somewhat yellowish brown,
conidiogenesis blastic, sympodial, sometimes seemingly retrogressive,
apertures mostly terminal but also immediately below septa (acropleurogenous),
scars visible but not thickened or protruding; hyaline,
ellipsoid, broadly rounded at the top, slightly attenuated into a blunt base,
with one or two small guttules, 4–5.2(–6) ×
(1.5–)1.8–2 μm.
: sp.,
Indonesia.
: , on leaf
litter, in association with Sherwood, M.J.
Wingfield, Mar. 2004, , single-ascospore isolates, CPC 11049 =
, CPC
11050–11051.
: The material used in this study generally agrees well with
the description given by Spooner
().
some additional observations that were not reported by this author,
particularly, the presence of apical appendages on the ascospores, and the
production of microspores from liberated ascospores.
only two or three guttules per spore.
material often merged into larger bodies, and this could explain the
difference between our obervations and those of Spooner, which were based on
herbarium specimens.
ascospores were barely visible.
on observtions in pure culture.
showed plasticity in conidiogenesis making it very difficult to assign it to a
particular anamorph genus.
-like, although it lacks the denticles characteristic of
that anamorph, and it also differs by branched and septate conidiophores.
Hektoen & C.F.
Hektoen & C.F.
wood, but is linked to Syd. & P.
Syd. BLASTn results of the ITS sequence of this species had an E-value of 0.0
with ITS sequences of and (G.W. Beaton & Weste) Spooner (both 94 % similar).
Similarities with known species include (Bull.)
De Not. (2e-135), (Bull.) Fr. (8e-135) and
(Batsch) Dennis (6e-96; all Helotiales).
similarities with an E-value of 0.0 were obtained from the LSU data:
(Pers.) W.
(Alb. & Schwein.) Dennis (both ) and others. |
Defining the number of fungi on earth has always been a point of discussion
(,
),
but has gained prominence in scientific literature towards the latter part of
the twentieth century.
of peripheral importance, it is fundamental to understanding and protecting
the world's biodiversity.
focused on enumerating the world's fungal biodiversity
(,
,
,
,
,
,
,
,
,
Hawksworth ,
).
foundation for studies aimed at a better understanding of fungal biodiversity
worldwide, and results have been used to motivate for bioconservation and
fungal biodiversity studies.
and studied from most countries, regions and habitats.
comparison to plants and larger animals that are considerably easier to
collect and identify than fungi.
predicted numbers of fungi on earth would have been considerably greater than
the 1.5 M suggested by Hawksworth
().
authors that have considered the likely total number of fungi have had
differing views of an appropriate answer, but the discrepancy between the
results of most of these studies is not particularly great.
accepted that the 1.5 M estimate is highly conservative.
thus far been described, therefore represents no more than 7 % of the
estimated total.
is not a particularly long history for mycology as science.
widely recognised as one of the world's biodiversity “hotspots”,
including areas such as the Cape Floral Kingdom, which is the smallest and
most diverse biome presently known.
mycological developments that have happened in South Africa, as well as to
provide an estimate of the number of fungi in this country.
this it is never possible to comment on all activities and groups that have
been active over the past 100 years, and for many of these there will only be
a brief mention, chiefly because much of the information was not available to
us at the time this paper was written.
summary and estimate will not only be interesting, but also provide a
foundation to promote and guide future mycological activity in South Africa
and elsewhere.
() list just over 24 500
taxa (including those at infraspecific level) as occurring in the flora of the
southern African region, which includes South Africa, Botswana, Lesotho,
Swaziland and Namibia.
world's flora occurs in less than 2.5 % of the total land area of the world.
This represents an increase of about 500 taxa on the previous checklist of
this flora ().
“cryptic” taxa appearing as a result of new revisions, or new
invader plants appearing in our area.
unknown taxa in ever more inaccessible areas, sometimes embarrassingly close
to major centres of population.
recent papers by Edwards .
() detailing a new
from within the Durban municipal boundaries, and any one of
Van Jaarsveld's numerous recent discoveries (e.g.
; this issue of Aloe contains two other similar new
discoveries) from further afield.
point to , which became noticeable a few
years ago in Pretoria, and has already spread to Durban (mapped and described
without historical data by ).
(SANBI), much systematic research is directed to cataloguing plant diversity
by means of regional floras; some
(, ) already published and others still in various stages of
preparation.
departments.
some groups, notably some genera of legumes, have not been critically examined
since the pioneering work of W.H.
).
and there have been various studies of the vegetation of southern Africa from
the historically classic studies of Drège
() and Bolus
(), through Acocks
( and still the most
often-cited work in South African botany) to the recent studies of Low &
Rebelo () and Rutherford
& Mucina (in prep.).
of biomes and centres of diversity given by Van Wyk & Smith
() will be used (Figs
,
).
I.B. Pole Evans in 1905.
country were those of MacOwan and Medley Wood, consisting of some 765
specimens ().
Pole Evans established a national collection of fungi in Pretoria.
of publication of Doidge's book (), this collection included more than 35 000 fungal specimens.
Other fungal collections were housed at Stellenbosch (collections of P.A.
der Bijl and L. Verwoerd), Cape Town (P.
herbarium), and at several European herbaria, the most important of which are
Kew and the International Mycological Institute (CABI Bioscience).
also sent numerous collections to Europe, many to P. Hennings, P.
H. and P. Sydow. Several collections of larger fungi were also sent to C.G.
Lloyd in the U.S.A., and many duplicates can be found in Vienna.
() summarised the content
of her book in tabular form, listing (species) 93 , 77
, 835 , 1159 lichens, 1704
, 880 fungi imperfecti, making a grand total of 4748
species.
circumstances and personal danger, which is reported on in detail by Doidge
().
fungi causing plant diseases, with some attention to saprobic fungi, and those
found to be mycotoxigenic.
(,
,
,
), Van der Westhuizen
& Eicker (), and
Eicker & Baxter ()
provide lists of various groups of fungi compiled after Doidge
().
subsequent to 1999 can be obtained via the Internet-based electronic system of
CAB abstracts (CAB)
().
Several lists of plant diseases caused by fungi, bacteria and viruses in South
Afirica have been published.
() followed by Doidge
& Bottomley () and
Doidge . (),
which was chiefly based on Doidge
().
updated in a series of bulletins published by Gorter
(,
,
,
). Later, Crous . () published the
compilation “Phytopathogenic Fungi of South Africa”, which was
made available online by the Systematic Botany & Mycology Laboratory in
the U.S.A., and is searchable via
<>.
studied in South Africa.
the Doidge era ().
Subsequently, the emphasis changed () from broader-based data collection or taxonomic work and the
description of new species to the study of fungi that are important as plant
pathogens.
National Collection of Fungi was placed with the Plant Protection Research
Institute of the National Department of Agriculture.
on the (), Ces. & De Not. (Denman . ,
, Slippers .
,,,) Johanson (, Crous
,
,
), powdery mildews
() (Gorter
,
,
),
()
and Fuckel (,
,
), causing diseases on various crop plants.
miscellaneous pathogenic species were newly described, such as Marasas & I.H. Schum.
(), a powdery mildew from the indigenous shrub and D.B.
() from roots of
wheat. The teleomorph of (Berk. & M.A.
Curtis) M.B.
aetiology of “geeldikkop” and facial eczema – both of which
are photosensitization syndromes – was found on in the Karoo, and described as Cec. Roux (). The genus Berl.
W. Gams, Crous & M.J. Wingf., which was shown to
be one of the causal organisms of Petri disease of grapevines
().
().
(). Johannes P.
Centre of Scientific and Industrial Research (CSIR) made an enormous
international contribution to the knowledge of yeast taxonomy with numerous
publications on the distribution and diversity of South African fungi.
retired, J.P.
ascomycetous teleomorphs and anamorphs (14 genera).
still known only from South African isolates.
ascomycetous fungi following Doidge
() became broken by a
steady flow of contributions by plant- and forest pathologists
(), with
the genera Syd. & P. Syd.
& Halst. (, ) and
(,
, Crous
.
attention.
also received considerable attention, with the
description of numerous new species and genera
(, Lee
& Crous ,
,
, Lee .
,
).
occurring on litter, however, were less intensively
studied (Lee & Crous
,
,
).
Contributions from other South African scientists included L. Korsten, F.
Wehner, N. McClaren, B. Flett, N. Labuschagne and G.
valuable information pertaining to distribution records, host preferences and
new disease reports.
others. One such genus is .
() listed 21 species of
, and more than 100 cercosporoid anamorph species.
description of numerous new taxa from indigenous and exotic hosts
(,
,
).
cercosporoids occurring in South Africa were treated by Crous & Braun
(), who listed 159
species from diverse hosts.
by Taylor and co-workers in a series of papers focusing on
, and anamorph-genera such as
Rangel, and Marasas, P.S. van Wyk & Knox-Dav.
(,
).
Other than , the has also received some
attention.
were treated in several papers by Crous and co-workers
(,
,
,
Hunter .
in South Africa.
is indigenous to South Africa, was also investigated,
revealing four species of on this host
(,
,
).
exceptional species richness on hosts in the
and , it can be assumed that numerous
species await description once leaves of other hosts are studied in more
detail.
received considerable attention in recent years
().
Specific papers have addressed species occurring on hosts such as
(, Slippers .
,,,),
(),
(),
(), Southern Hemisphere conifers (such as
) (), various fruit trees
() and
(), to name but a few.
there are numerous unknown species of the in the
Southern Hemisphere, and specifically in South Africa, awaiting
description.
been treated in the genus (Sacc.) Sacc. & D. Sacc.
(Myburg
,
, Gryzenhout
,
,
,
– this volume).
These investigations arose from the first discovery of the serious
stem pathogen (Bruner)
Hodges in South Africa ().
restricted to the Northern Hemisphere (Myburg .
,
).
in Southern Hemisphere countries including South Africa, reside in various
genera such as Gryzenh. & M.J. Wingf.
(), Gryzenh. & M.J. Wingf.,
Gryzenh. & M.J. Wingf.
(), Gryzenh. & M.J. Wingf.
() and Gryzenh., Glen & M.J. Wingf.
().
closely related to , such as Nakab., Gryzenh., J. Roux and M. J. Wingf.
( – this volume).
: A cursory look through Doidge
() reveals that none of
the earlier collectors in South Africa took particular note of hyphomycetes.
Of the total of 4748 species that she listed, a mere 18.5 % represented
asexual forms.
genera Link (), Link
(),
Link (), entomophagous fungi
(),
dematiaceous fungi (, Sinclair .
,
,
,
,
), and nematode-trapping
fungi (). A
review of Fresen.
(),
revision of the genus Morgan and related genera
(Crous & Wingfield ,
,
), and
differentiation of species of Shoemaker K.J.
() was compiled
mostly from specimens in PREM.
crops and animal feeds such as lucerne
(), pastures (), natural Karoo pastures
(), as well as
toxigenic representatives of
(, ) and Berk. & Broome
(,
), and various synnematous and other hyphomycetes
(,
,
).
Other genera that received attention include Corda
(), Lagerb. & Melin (Wingfield
& Marasas ,
,
,
),
Link (, ), W.B. Kendr.
(), and cercosporoid fungi (Crous & Braun
,
,
).
and in the fynbos
(, , ).
the toxigenicity and phytopathologically important genus
including soil surveys (), and the description of numerous new species such as
Marasas, Rheeder, Lampr., K.A. Zeller & J.F.
Leslie (), and Klittich, J.F. Leslie, P.E.
Nelson & Marasas ().
fungi by Crous and co-workers () have been very extensive.
organism originally being ascribed to Sacc., then
Deighton, and Miura
(). Crous .
() erected the genus
Crous & W.
anamorphs, while their discomycete teleomorphs were placed in the genus
Crous & W. Gams. Schroers .
() recently characterised
the hyphomycetes associated with guava wilt, and identified the causal
organism as a species of Subram.
Hyphomycetes from have been intensively studied.
Halleen .
() treated the
Wollenw.
and introduced a new genus, Halleen, Schroers &
Crous. Crous .
() erected the genus
for species associated with grapevine decline disease
of grapevines and human infections, while Crous & Gams
() described the genus
Crous & W.
Petri disease.
Species of Morgan (teleomorph:
De Not.) () are common in tropical
and subtropical regions of the world, and cause disease problems on a wide
range of hosts. Schoch .
() placed teleomorphs of
Boesew. in Crous &
C.L. Schoch.
() reported
Schoult., El-Gholl & Alfieri as
causing Cylindrocladium root and petiole rot of , while
Schoch . ()
resolved the Viégas species
complex, describing the common soil-inhabiting species in South Africa as
C.L. Schoch & Crous.
and allied genera, Crous
() reported six
species and five species
from South Africa.
: In the Doidge era, investigators often
recorded coelomycetes only incidentally, usually alongside their sexual state.
Because they were mainly found on plant-pathological specimens, those that
were most often reported were commonly occurring members of the genera
Corda Pers.
De Not. ().
they were found on new substrates.
judged with robust systematic techniques currently applied.
poisonings or other maladies in farm animals i.e. (J.G.
() on lupins, Höhn.
(),
(Baxter
,
) Speg. (), and Tassi
().
(W.A. Campb.) Verkley was investigated
as an antagonist of the devastating pathogen, (Lib.) de Bary
().
most notorious coelomycetes in South Africa are (McAlpine) Aa, the cause of black spot of oranges
(),
and (Berk.) B. Sutton (syn. van der Bijl) causing black rot of maize and intoxication of sheep
().
Höhn. and Petr. & Syd.
(),
(),
Cooke (, ), Hulbary
(),
the anamorphs of
(,
Slippers .
,,,),
and (Sacc.) Bubák (Smit 1989a, b,
,
,
,
).
some attention, namely those occurring on
()
and ().
(,
Talbot ,
,
,
,
) and
() received some attention.
listed by Doidge () are
basidiomycetous.
() present a good overview
of work done on basidiomycetes from 1977 to 1999.
references to studies on the genera (Pat.) Pat. Alb. & Schwein. R. Heim Pers. Massee P.
Micheli ex L. Heinem. (Pers.)
Gray Singer Locq. ex
Singer Pat., Fr.
Lév.
yeasts were also newly described through the years by J.P.
colleagues.
resupinate and stereoid and a series of papers between
1951 to 1958, dealing with Pers. Kalchbr.
& MacOwan Jungh.
().
investigations also included a series of papers on tree pathogens and
wood-destroying
().
Argentina, namely species of Lév.
() ().
(, ) but a revision of the South African
species, and the (earth stars), which will to a large
part rely on material lodged in PREM, is in progress (J. Coetzee, pers.
comm.).
Significant contributions following Doidge were made by D.A.
Royal Botanic Gardens Herbarium, Kew.
specimens of species of described from South Africa in
PREM (), the van der Bijl Herbarium (now housed at PREM), and
elsewhere ().
has been the foundation for his documentation of South African mushrooms in
collaboration with Albert Eicker from the University of Pretoria.
productive partnership has provided us with a scientific guide to our edible
and poisonous mushrooms and other large fungi. In 1994, G.C.A.
Westhuizen's lifetime of mycological research and photography culminated in a
field guide (), with excellent colour photographs of some 160
species of local macrofungi.
data, like that published on species of R.
& Eicker ,
,
).
Studies during the early part of the last Century reported (Vahl: Fr.) P. Kumm.
(,
,
).
largely associated with an expanding plantation forestry industry and the fact
that this fungus resulted in tree death.
that the fungus killing trees in this country is Petch
()
and that there are probably at least two other species occurring in
neighbouring countries such as Zimbabwe
().
Hemisphere species, was introduced into South Africa,
probably by the early Dutch settlers
(). Likewise, the Northern Hemisphere species Marxm. & Romagn.
plants in the Kirstenbosch botanical gardens
().
G.L.I. Zundel of the U.S.A., who collaborated with Pole Evans and Doidge in
describing and re-investigating material found by South African mycologists,
extensively studied local smut fungi. K.
changed the taxonomic study of this group of fungi by advocating the use of
morphological characters obtained by germinating spores (Vánky
,
,
,
–,
).
we apply these methods to old herbarium specimens, however, because their
spores have lost the ability to germinate.
most striking smuts is maize boil smut, (DC.) Corda
[= (Link) Unger].
():Most of the rust fungi known from
southern Africa were treated and described by Doidge
(,
,
,
,
,
).
remain the basis for identification of these fungi in southern Africa, and are
relevant to the whole of the African continent.
in Doidge ().
474 species are listed from southern Africa, including 145 anamorphs.
species have been transferred to other genera, and a further 30 species names
have been reduced to synonymy.
Pers.
(), and three have been demonstrated to be endocyclic (Wood
,
,
),
whilst 11 anamorph species of Pers.
their teleomorphs.
() and various
publications summarised in Cummins
().
that the status of many more names will change as further taxonomic studies
progress ().
number of new species have been described by G. B. Cummins and H.
in various papers, none dealing exclusively with species from the region
(, Gjærum
& Reid ,
, Gjærum
,
,
).
been described recently (, ,
, , Mennicken
,
,
,
).
Taking these above-mentioned changes into account, there are currently 537
species of rust fungi, representing 40 genera and 10 families presently
recorded from southern Africa (A. Wood, unpubl. data).
increase, as several more new species and records await publication.
countries included, southern Angola, Botswana, Mozambique, Namibia and
Swaziland have few species recorded, and Lesotho has none.
and Zimbabwe are relatively well explored mycologically, though little or no
collecting has been done in large areas of even these two countries.
of 20 new species have been described from the Western and Northern Cape
Provinces of South Africa, and Namibia, in just the last 5 years
(,
, , Mennicken
,
,
,
),
demonstrating that many new species await discovery in the subregion where
there has been little or no collecting in the past.
species known to occur is probably not a gross underestimation of the actual
total present, as with the study of fresh specimens it is probable that a
number of species will be reduced to synonyms, and many anamorphic species
will be linked to their teleomorphs.
the number of vascular plants.
only 1.7 and 3.2 %, respectively ().
likely to rise considerably.
recorded from the subcontinent in comparison to other countries
()? There are a number
of possible explanations for this, the most likely including: Much of
South Africa, as well as Botswana and Namibia is semi-arid to desert.
rainfall associated with these areas would restrict opportunities available to
rust fungi to infect their hosts, which could result in the lower diversity of
rust fungi.
succulent Karoo biomes, despite the high plant diversity for which these
biomes are well known.
few but speciose genera and families with high turnover of species over short
distances (, ).
related plant species with a large distribution, or on numerous related plant
species, many of which have limited distributions. (Doidge) A.R. Wood, (Henn.) A.R.
& Crous () and Doidge are examples of
the former, whilst Massee, (Lév.)
G. Winter and Dippen. are examples of the latter (A.
Wood, unpubl. data).
in the eastern parts, associated with a greater amount of summer rain.
the ratio of rust fungi to plant species is probably approximately the same as
for Zimbabwe, which is ecologically similar.
(), one of which,
Thaxt., has not since been found.
important saprotrophic species, identified early on, such as the ubiquitous
(Ehrenb.) Vuill. – listed as Ehrenb. – which causes post-harvest decay, particularly
in sub-tropical fruit.
Went & Prins. Geerl., has been associated with mycoses in humans.
the most commonly found member of this group, particularly in fodder samples.
During the course of several years, PREM has supplied numerous cultures of
and participated in research focused on metabolite
studies.
()
culminating in patenting of a fermentation process.
risen to 20 ().
tract of arthropods, yielded several new records and new species
().
particular are relatively well-studied, though much needs to be done on the
microlichens and lichenicolous species.
Stizenberger
(), but the
major synthesis to date is that of Doidge
().
were made by Elise Esterhuysen (1912–) in the late 1940s–1950s
(material in the Bolus Herbarium, University of Cape Town), but the principal
contributions have been by Ove Almborn (1914–1992) from Lund (Sweden)
and Franklin A.
the National Herbarium in Pretoria.
Norman () but travelled extensively in the country, for example
spending 5 mo there in 1953 (), analyzing distribution patterns (Almborn
,
), and also issued an
important exsciccate, “Lichenes Africani”; this comprised six
fascicles from 1956–1991 including 150 numbers.
interest in the and described many species new to
science, especially effigurate-crustose species on rock later placed in the
endemic genus Hale (the species known from Australia appear
to belong elsewhere).
collected, amongst the most notable being Gunnar Degelius (1903–1993)
who specialized in
(), Ingvar
Kärnefelt (University of Lund) with a special interest in
Leif Tibell who specializes in caliciaceous groups
(who visited with colleagues from the University of Uppsala in 1997), and
Mason E.
new genera in the (e.g.
,
), although not all his
new genera have yet been evaluated by molecular methods.
also carried out during the International Association for Lichenology's field
meeting in Namibia and South Africa in 1986, but the results from that
excursion have not been synthesized.
carried out based on the collections of these and other lichenologists, for
example the monograph of Norman in South Africa by
Guderley & Lumbsch
().
government, an extensive survey of lichen biodiversity from the Cape Province
to northern Namibia is underway, the leading researcher involved being Tassilo
Feuerer (University of Hamburg).
by Ana Crespo (Universidad Complutense de Madrid) and focussing on the
made about 750 collections in May–June 2005 from
the Western Cape north to the Namibian border.
Africa today.
preliminary list compiled from the literature
()
contained 1716 species as of 1 March 2005.
future work but is very preliminary and includes both many early names copied
from the literature whose position and status needs to be reassessed, and
others that have been revised but have yet to be updated from other
literature.
species to be expected, by comparison with checklists from other regions of
the world with similarly diverse climatic regions and rock types
() would be about 2000 (excluding lichenicolous fungi).
be endemic, notably De Not.
with others requiring reassessment, for example
Esslinger Elix & Hale Hale, and
Hale.
especially in the and the The
lichen assemblages are very distinctive in different parts of the country, as
discussed by Almborn (),
comprising at least eight phytogeographic categories: ubiquitous, steppe and
desert (e.g. Namaqualand, Karoo), montane (over 1000 m alt.), oceanic (e.g.
Table Mountain, Drakensberg Mountains), tropical-oceanic (e.g.
KwaZulu-Natal, Mpumalanga), maritime (on coastal rocks), and endemic.
or saprobes, have hardly been reported from South Africa.
the 2005 preliminary checklist, not even representatives of such ubiquitous
genera as De Not. Petr. &
Syd. Saut. ex Körb., and Trevis.
There are, however, some scattered records, such as that of Hafellner & Calat.
() which is very common on (Vain.) Hale
species in the country (D.L. Hawksworth, pers. obs.), and an undescribed
on (Nyl.) Hale
().
Ana Crespo's group in 2005, but have yet to be fully identified.
with what is known of the species richness of lichenicolous fungi compared
with the number of potential host lichen species, for example 403 .
1677 lichen species in Great Britain and Ireland
(), around 475
species would seem to be a reasonable estimate of the number to be expected in
South Africa.
have been recorded from eight southern African countries during the period
1941 to 2001.
stem canker, crown rot, tip blight, leaf rot and fruit rot.
Pringsheim species date from 1931 to 2004.
have been reported from 65 different hosts, and
representative disease symptoms were damping off, seedling blight, root rot,
stem rot (foot rot), crown rot (heart rot), fruit rot, tuber rot and soft
rot.
: The first detailed study of
species with associated hosts and distribution patterns in South Africa, was
done by Wager (,
) describing (Lebert & Cohn) J.
Rands (R.E. Smith & E.H.
Bary Breda de Haan and (Klebahn)
Klebahn.
Wijers () reported on various hosts and included descriptions and disease symptoms.
Other studies confirmed species identification
(,
).
different vegetable and ornamental crops were recorded by Thompson
(), Thompson &
Phillips (), Ferreira
. (),
Thompson & Naudé
() and Thompson . ().
Morphological and molecular methods (RFLPs of total DNA) were used by Botha
() to confirm species
identity of on tree lucerne. Linde .
() applied isozymes to
distinguish two separate populations of determining
genotypic variation within A1 and A2 isolates, and established that sexual
reproduction was rare or absent in South African isolates. Linde . () used both
RAPDs and RFLPs to assess genotypic diversity and reveal DNA polymorphisms in
South African and Australian isolates of .
& von Broembsen ()
reported Foister from onions, followed by Sawada causing shoot tip blight of lemons in propagation
tunnels ().
performed by Marais () in
the Western Cape Province, surveying grapevine rootstock diseases.
Broembsen () did an
extensive survey of root rot in indigenous fynbos, and
from the major catchment rivers in the Western Cape.
(,
) surveyed alfalfa in
various provinces for E.M. Hansen & D.P.
and Tucker. Linde .
(,
) surveyed the major
commercial forestry areas in South Africa for root rot caused by oomycetes,
while Thompson .
() surveyed citrus roots
for and in the Mpumalanga and Limpopo
Provinces.
() reported Leonian causing wilt of pumpkin, and stem and root rot of tomato
().
() conducted an extensive
survey of Late Blight () in the major potato production
areas in South Africa, determining mating type and using various molecular
markers to characterise isolates.
: The first major contribution by Wager
() consisted of several
species descriptions and disease symptoms on hosts.
descriptions, temperature-growth measurements and hosts, was also reported by
Wager ().
() and Doidge . () compiled the
host range of numerous species, and this compilation was
updated by Gorter (,
) and Crous . ().
. () and
Darvas () added further
records of species with host records. Scott .
() and Scott
() made a major
contribution with regard to the occurrence and disease symptoms of
species from various small grain crops.
of all recorded species in South Africa from 1926 to 1989 was
compiled by Denman & Knox-Davies
() and included
distribution, host range and species diversity.
description of various species associated with vegetables was
reported by Botha & Coetzer
() and W.A.
sp. (). species have also been associated
with grapevine (Marais ,
), medics
(), lucerne seedlings
(), pines and eucalypts (Linde .
,
) and lettuce
().
viz.
homothallic species, e.g. Vaartaja, Buisman Drechsler Kouyeas & Theohari Drechsler and
Sawada, have been reported from closed hydroponic systems
in South Africa (Labuschagne
,
,
,
).
() to fungi recorded in
particular ecological niches.
from aquatic habitats such as fast-flowing rivers, submerged woods and twigs
in stagnant freshwater habitats, wood invading fungi in marine waters,
estuaries and from leaves in a mangrove.
phylloplane fungi, foliicolous fungi, airspora over pastures, coprophilous
fungi from domestic, captivated and wild animals, fungi associated with
cultivated mushrooms, undisturbed soil populations, fungi for the control of
invasive plants and from plant litter.
publications addressed reports of fungi causing human and animal diseases.
These include a study on mycoses, dermatophytes and fungi causing subcutaneous
infections.
attention in South Africa.
survey of arbuscular mycorrhiza made by Wehner in 1976
().
Subsequent studies include recordings of Gerd.
& Trappe, and investigations on fynbos soils by Mitchell, Reid, Straker,
van Greuning, Sinclair and later during a survey of the fungi associated with
by Van der Westhuizen and Eicker
().
Several papers were published on fungal endophytes. Crous .
() listed 55 endophytes
from while Serdani .
() treated the endophytic
Nees species associated with core rot of apples.
. ()
provided an overview of foliar endophytes in , and
Mostert .
() treated the endophytic
spp. occurring in grapevines. Smith .
() treated endophytic
species in and .
Swart . ()
reported on the fungal endophytes in cultivated
and , and Taylor .
() treated the endophytes
occurring in spp. grown in the wild.
(,
Sinclair .
,
,
, Sinclair ,
), or on specific hosts
such as (),
(),
and (, , ).
Fungi on commercially important exotic hosts such as and have been studied in detail. Crous . () listed the
shoot and needle diseases occurring on spp., while the leaf
pathogens occurring on were treated Crous .
().
summary of the fungal pathogens on and
seedlings in nurseries in South Africa.
() treated the species of
occurring on , and listed 14
species from South Africa, while Slippers .
() treated the
spp. occurring on this host.
() treated the fungi
causing diseases of , and found various species of fungi,
including Schwabe and M.J. Wingf., De Beer & M.J.
wilt disease of this host (, Roux .
,
).
(restios) are two of the most prominent plant families
in the Southern Hemisphere.
confined to the south-western corner of the country in 90 000 km,
the so-called “Cape Floristic Region” or “Cape Floral
Kingdom”.
endemic to the region (mostly members of the genus ) and
restios by 330 species, of which 94 % are endemic to the region
().
these two families, nature reserves and botanical gardens were visited over a
2-year period (2000–2001).
34 restio species (representing 15 genera), which consists of approx. 150
fungal genera and 180 species.
43 protea species (representing five genera), which consists of approx. 120
fungal genera and 185 species.
isolated, of which 190 were confined to either the one or the other host
family, while 40 fungal genera occurred on both families.
identified to species level, of which 355 were restricted to one or the other
family, while 25 species occurred on both families
().
strong host specificity, but rather have a broad range of host recurrence (or
host preference) such as mono/dicotyledonous plants in the tropics or
gymno/angiosperms in temperate forests
(,
).
A well-studied group in our collections, hyphomycetous fungi, showed a high
degree of host-recurrence ().
compositions of tissues between the two host groups, which reflect their
taxonomic distance and different lineage, might be a cause for a higher degree
of host-recurrence.
groups. This again complies with the view of Polishook .
() that much of the
host-recurrence in litter fungi is probably related to physical and chemical
characteristics of leaves rather than host-specificity.
() argued that over 50 %
of the fungi inhabiting microhabitats have possibly evolved in a very close
relationship with their host.
considered as miniature ecosystems, which accommodate different food chains
and trophic levels ().
during this study, which revealed a unique composition of fungi
().
on host plant habitat.
(), which has a riverine
habitat and thick twigs and branches.
isolated from (), which grows in
dry areas, and has culms of approx. 1 mm diam.
of saprobic microfungi for each species of or
thus far investigated
().
this as a minimal estimate because of the limitations of the damp-chamber
isolation technique, and the undersampling of microhabitats such as
infructescences.
associations, have been characterised in virtually all insect groups, and
especially in families such as and others ().
ubiquity and novelty of the fungi involved in these associations, they are
left out of most estimates of fungal diversity due to insufficient data.
lack of information is due to the difficulty of characterising these fungi
based on traditional criteria and, in some cases, ignorance of their existence
and importance.
() reported over 200
undescribed species of yeasts from the guts of 27 families of beetles; a novel
niche.
even half of the unique species in their samples.
tempting example of the potential fungal biodiversity that could exist in
South Africa.
species in 104 families, but this species number could well be 2–3 times
higher (). Extrapolating from the data of Suh .
(), this niche alone most
likely harbour hundreds, if not thousands, of undescribed yeast species in
South Africa.
equally poorly known from South Africa (see
, as
example of the potential diversity).
significant portion of the fungal biodiversity of the region.
will have to be ignored in the current paper, highlights the need for more
specific research focus on this area in South Africa and elsewhere.
than most, especially in the Northern Hemisphere, is that of the
ophiostomatoid fungi with bark and ambrosia beetles
(,
).
Africa, three Arx & Hennebert species have been
described from two native ambrosia beetles
(),
while at least 11 ophiostomatoid species have been reported from introduced
pine bark beetles (Zhou
,
).
number of ambrosia and bark beetle species in South Africa has not been
determined to date.
described from merely nine of the more than 300 spp.
Africa (, , ).
present in protea infructescences suggest that the species from proteas are
vectored by arthropods ().
Hektoen & C.F. Perkins and spp.
discovered () growing on fungal combs in 13 termite mounds, representing
only three of the at least 42 fungus-growing termite species
() in Southern Africa
().
ophiostomatoid fungi associated with beetles, spp. (and their
insects), as well as termites, suggest that the species richness of the
ophiostomatoid fungi, and the interaction between arthropods and fungi in
these niches, are under-explored.
spp.
arguably one of the best known fungi among non-specialists in South Africa, as
they are rather obvious, numerous and a well-loved delicacy.
species have been described from South Africa
().
However, not all species of R.
42 South African fungus growing termite species have been characterised.
these fabulous fungi might be more diverse than current numbers suggest.
Neither have the Hill ex Schrank species associated with
termite nests been characterised.
one species to the list of South African fungi, namely its mutualistic
symbiont, (Fr.) Boidin
().
damage to pine plantations throughout the country.
the role of exotic insects introducing more fungi to South Africa, potentially
with disastrous consequences.
that the ratio between the number of vascular plants and fungi from all
substrata in the British Isles, which is an intensively studied region on
which he based his estimates, was around 1: 6 using several different data
sets.
species resulted in an estimate of 1620 000 species of fungi.
that the 1.5 M estimated number of fungal species exist, we currently only
know around 7 % of these ().
a plant to fungus ratio of 1: 4, while the U.S.A. was estimated at around 1: 1
().
likely numbers of fungi occurring on insects were not taken into account due
to insufficient data, estimates were as high as 13.5 M fungal species
().
that there could be at least ten times as many fungi as vascular plants in
Australia, with 2.7 M species of fungi occurring world-wide.
there could be 1 M on tropical plants alone.
() considered endophytic
fungi, a group easily forgotten, and estimated that 1.3 M taxa might exist in
this niche alone.
() concluded that on this
group of plants there was most likely a ration of 1: 33 fungi per plant
species.
ratio of 1: 3.5 species of macromycetes.
() considered that there
were approx.
of palm, suggesting that the fungus to plant ration could be as high as 1: 26.
Hyde . ()
also reported that 75 % of all fungi collected on palms were new to
science.
fungi.
().
() reported 92 species
from of which 17 appeared unique, and 55 on , of which 28 were host-specific.
from (558 unique fungi if potential synonymies were
taken into account), and 282 species on , of which
150 were not known from other eucalypts.
have been describing more unique fungi from , which
suggests that the 282 figure reported by Hawksworth
() was an underestimate.
So far only very few ecological niches and hosts have been thoroughly studied
in South Africa.
and no host has been thoroughly treated (e.g.
stems, leaves, and litter, endophytes, epiphytes, and specific isolation
techniques for specific fungal groups).
very difficult to estimate the number of unique fungi per host.
using moist-chamber incubation to culture saprobic fungi, Crous . () described
four unique hyphomycetes from .
groups were seen, but not treated, thus from this one host, and one fraction
of a niche, the ratio is 1: 4.
hyphomycetes on leaf litter of , Crous . () described
five unique saprobic fungi, while in later studies a further five unique plant
pathogenic fungi were described from this host
(,
),
which increases the ratio to 1: 10, with several more host-specific fungi on
this host awaiting description.
occurring on , Crous .
() reported six unique
foliicolous species from , suggesting a ratio of 1:
6 as being an underestimate.
studied, they provide a ratio of 4–10 unique fungi per host, suggesting
that there could be an estimated seven unique species of fungi per indigenous
host.
seven unique species of fungi, many hosts might have some more, as this figure
is an estimate based on a very incomplete examination of these hosts.
that there are 24 500 species of plants in South Africa
(), this estimate would mean that there could be 171 500
species of fungi, before taking numbers of insects into account.
approach, however, the number of endemic fungi would be determined by the
percentage endemic plants (and insects once taken into consideration).
ratio compares quite favourably to the ratio proposed by Hawksworth
(), which was 1: 6,
Pirozynski () which was
1: 3–5, Pascoe (),
which was 1: 10, and Rossman
() which was around 1:
4.
we have attempted for the first time to use its botanical diversity to
estimate its fungal biodiversity.
estimate that there could be as many as 171 500 species of fungi.
definitely an underestimate, as no insect-associated fungi were taken into
account, which alone makes a case that this estimate is inordinately
conservative.
southern African flora, one would expect an equally high number of unique
fungi.
thus far been described from South Africa (Figs
,
).
that the study of South Africa's unique, indigenous fungal biodiversity has
never been regarded as a research priority.
support has been allocated to it from the various southern African funding
bodies.
plant-pathogenic fungi.
many diverse fungi exist that have unique ecological niches and roles yet to
be studied.
anthropophilic fungi (infectious to man), aquatic fungi (aquatic habitats),
bryophilous fungi (on bryophytes), coprophilous fungi (on dung), dermatophytes
(on skin, hair nails), endolithic fungi (on rocks), entomogenous fungi (on
insects), halotolerant fungi (tolerant to salt), hypogeous fungi (growing
below ground), keratinophilic fungi (on feathers, horns), lichens (some
studied, see ), marine
fungi (in marine and estuarine habitats), mesophilic fungi (growing between
10–40 °C), mycorrhizal fungi (symbiotic with plant roots),
mycoparasites (on other fungi), nematophagous fungi (parasitic on nematodes),
osmotolerant fungi (growing at high osmotic pressure), psychrophilic fungi (at
< 10 °C), pyroxylophilous fungi (on burnt areas and substrates),
resinicolous fungi (on resin), rumen fungi (in anaerobic rumen environment),
sewage fungi (polluted water), thermophilic fungi (at or above 45 °C),
water moulds (in water), and xerotolerant fungi (at < 0.85 aw) (see
).
unique southern African flora and fauna, it is clearly timely that some
thought, financial resources and research be focused on preserving the basal
links of the ecosystem, which are the fungi.
undescribed fungi represent a vast biological resource which has yet to be
collected, cultured and studied.
contain numerous beneficial biological properties and other attributes that
could be used to greatly improve the quality of life for all future
generations of humanity. |
Among the most common and conspicuous members of the genus
() are species that form
extensively effused, lightly or brightly coloured stromata on leaf litter,
soil, wood, and bracket fungi.
include (Pers.: Fr.) Fr., (Fr.: Fr.)
Fr., Fuckel, P. Karst., and Niessl.
(Pers.: Fr.) Fr., and phylogenetically related species
have not been determined.
effuse, typically yellow and asci contain eight 2-celled uniseriate
ascospores.
into subgenera, sections, subsections, and series based on
stroma characters. He recognized , and as distinct species and placed them in subgen.
sect. Yoshim. Doi. He included and in subsect. Yoshim.
while was placed in subsect Yoshim.
Doi.
conidiophores with hyaline conidia.
(,
) assigned acremonium- or
verticillium-like condiophores to sect Bissett. Fourteen species of Fr., with
stromata similar to that of , or considered as its synonym,
and anamorphs assignable to sect.
are newly described or redescribed, type specimens examined, and, where
possible, phylogenetic relationships to established.
() were used in making
identifications except for and .
lists the isolates used
in this study. Frequently cited collectors are abbreviated, B.E.
(B.E.O.) and G. J. Samuels (G.J.S.). All isolates with G.J.S.
were obtained by isolating single ascospores on CMD with the aid of a
micromanipulator. All isolates with B.E.O.
plating the entire contents of individual perithecia.
annotated, host and substrate information were obtained from herbarium labels,
and data were entered in as it appears on the
labels.
sequences: ITS1-5.8S-ITS2 (ITS), a partial sequence of translation elongation
factor (), delimited by the primer pair ef1/2, and a partial
sequence of RNA polymerase II subunit () delimited by the primer
pair frpb2-5f/-7cr. Mycelia were lyophilized prior to DNA extraction.
lyophilization protocol is described in Stewart
().
DNA was carried out using the phenol and chloroform extraction outlined in
Stewart
().
chain reaction (PCR) for ITS and was performed following the
conditions outlined by Chaverri
() using the following
primer pairs: for ITS, the primers utilized were ITS1
(5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3')
();
for , the primers were ef-1 (5'-ATGGGTAAGGA(A/G)GACAAGAC-3') and
ef-2 (5'-GGA(G/A)GTACCAGT(G/C)ATCATGTT-3')
().
conditions outlined by Chaverri
() was utilized for
generating a partial product.
primers were employed: frpb2-5f
(5'-GA(T/C)GA(T/C)(A/C)G(A/T)GATCA(T/C)TT(T/C)GG -3') and
frpb2-7cr(5'-CCCAT(A/G)GCTTG(T/C)TT(A/G)CCCAT-3')
().
Two percent dimethyl sulfoxide (DMSO) from AMRESCO® was added to each 50
μL PCR reaction. A PCR purification was carried out using a QIAquick®
PCR purification kit.
Reaction Kit with Amplitaq DNA Polymerase FS (PE Applied Biosystems) was
utilized for sequencing cleaned PCR products for each primer direction.
Performa® DTR gel filtration cartridges from Edge BioSystems were used for
cleaning cycle sequencing products.
University or sent to the Nucleic Acid Facility (Life Science Consortium, The
Pennsylvania State University). Sequence data were trimmed at the 5' and 3'
ends.
replicate isolates.
if there were no obvious morphological or locality differences noted from the
herbarium specimens.
aligned using Clustal W in DNA Star (DNA Star Inc., Madison, Wisconsin), and a
phylogenetic analysis was performed using PAUP* v. 4.0b8 (Sinauer Associates,
Sunderland, MA). Alignments were manually adjusted in PAUP*.
to be a phylogenetically appropriate outgroup taxon for
and allies and this species was used as outgroup in all
data sets.
search option with starting trees obtained via random addition sequence (10
replicates) and TBR branch swapping.
as missing data.
random sequence addition (10 replicates).
deposited in GenBank ().
established in the species tree () was carried out using 20 mL of three media: potato-dextrose
agar (PDA, Difco), cornmeal-dextrose agar (CMD, Difco), and SNA with filter
paper ().
Inoculum was prepared on CMD.
inoculum colony was placed near the edge of the plate.
medium for each culture was incubated at 20, 25, 30, and 35 °C in total
darkness.
at 24-h intervals for 4 d.
subsequent weeks.
descriptions.
specimens representative of each phylogenetic species distinguished in
.
were grown on PDA, CMD and SNA at 20 °C, with 12 h
fluorescent light and 12 h darkness to observe and measure microscopic
characters of the anamorph. Observations were made at ∼7–10 d.
Observations were discontinued for cultures that did not produce conidiophores
after 10 d on each respective medium.
phialide length, width of the phialides at the middle, and conidium length and
width.
characteristics were recorded. Herbarium specimens were rehydrated in 3 % KOH
for sectioning. Rehydrated stromata were supported by Tissue-Tek O.C.T.
Compound 4583 (Miles Inc., Elkhart, Indiana) and sectioned at a thickness of
approximately 15 μm with a freezing microtome.
measured were ascus length, ascus width, ascospore proximal length and width,
ascospore distal length and width, perithecial length and width, and stroma
length and width.
in response to 3 % KOH and lactic acid, were also recorded.
characters of the stroma were described and photographed.
continuous characters were made using the image-capturing software Scion Image
beta 4.0.2 (Scion Corporation, Frederick, Maryland).
micromorphological characters were made based on 30 measurements per specimen,
except where noted, with confidence intervals (a = 0.05), and minimum and
maximum values reported. Colour terminology was obtained from Kornerup &
Wanscher ().
teleomorph characters are included in the species descriptions.
morphological characters used in species recognition are discussed in the
comments section immediately following each species description.
from and (Figs
,
,
).
proposed neotype of from Europe
()
differed from North American isolates of in a single
nucleotide. The isolate G.J.S. 95-183 is identical to
other isolates from North America in the ITS tree.
from the gene tree in which the European
was
identical with the North American isolate B.E.O. 99-29
() and from the
and gene trees, in which isolate G.J.S. 95-183 had
a longer branch length than other isolates of (Figs
,
).
ITS sequences showed a single nucleotide polymorphism separating isolates of
from supported by a bootstrap
score of 63 % ().
and gene trees were more pronounced in placing
in a highly supported monophyletic group with , and with bootstrap
scores of 99 % for both trees.
All three data sets are concordant with similar homoplasy indices.
heuristic search of the most parsimonious tree for the ITS dataset yielded two
trees with 48 steps. The minimal possible tree length is 41
().
parsimony-uninformative and 28 characters are parsimony-informative.
heuristic search of the most parsimonious trees for the dataset
yielded six trees with 476 steps with the minimum possible tree length of 393
().
characters, 392 characters are constant, 61 variable characters are
parsimony-uninformative, and 242 characters are parsimony-informative.
heuristic search of the most parsimonious trees for the dataset
yielded two trees with 172 steps with the minimum possible tree length of 150
().
characters, 833 characters are constant, 56 variable characters are
parsimony-uninformative, and 80 characters are parsimony-informative.
and distinguished from
with a bootstrap score of 100 %
().
American isolates of formed two distinct subclades, each
supported by a bootstrap score of 100 %. and
formed a monophyletic group supported by a bootstrap
score of 87 % (I, II).
a bootstrap score of 100 % (Clade A).
(including the ex-neotype (IV)) formed a strongly supported monophyletic group
with a bootstrap score of 100 % (Clade B). (VI)
and (V) are situated in an unresolved sister clade
to supported by a bootstrap score of 100 % (Clade C).
Isolate B.E.O. 99-36 is divergent from other isolates of ; nevertheless, all isolates of formed a strongly supported monophyletic group (V) with a
bootstrap score of 100 %.
strongly supported clade with a bootstrap score of 100 %.
of the most parsimonious trees yielded one tree with 678 steps with the
minimum possible tree length of 575 ().
parsimony-informative.
sequences.
distinguished between North American and European isolates of , whereas ITS did not.
resolved North American and European isolates of isolate G.J.S. 95-183 grouped with North American and European
isolates of in the ITS tree, but had a significantly
longer branch in the and trees.
point of discordance that establishes a phylogenetic species boundary for
. The highest bootstrap scores for isolates of were obtained in all three datasets at the base of Clade B.
gene trees, it can be concluded that is phylogenetically
distinct from .
().
subclades are separated by long branches
().
the large branch length differences, that long-branch attraction may be
artificially inflating the bootstrap score uniting these taxa.
phylogenetic studies that should include , and Doi may help to more clearly
elucidate the relationship of and to .
highly concordant gene trees, consistently generating the same subgroupings.
ITS data from 3 out of 4 isolates of from Europe differed
from North American isolates in a single nucleotide.
() was identical with B.E.O. 99-29 in the gene
tree, but they differed in the tree. Furthermore, isolate G.J.S. 95-183, which is invariant in the ITS tree from
other North American isolates, had significant branch length differences in
the and gene trees from all other isolates.
can best be explained by sexual recombination.
for was determined to be at the point of discordance
separating clade B in the gene trees ().
nucleotide changes between North American and European isolates in the
and data sets.
American and European isolates in the and may
represent cryptic species or simply variations in population structure.
American and European isolates of are morphologically
indistinguishable, and the ITS sequences are identical.
analysis and morphological examination of European isolates will be required
to determine whether European isolates represent a distinct species.
ITS gene tree for , a single nucleotide change separates
isolates of from , while there are
larger differences in the and gene trees between
these species.
() have yet to be tested by
DNA sequence analysis.
and as defined by Doi are not
monophyletic assemblages of species. and () are not situated in the clade.
It is more difficult to determine the phylogenetic boundaries of Subsection
because Doi broke it up into several series from which
isolates could not be sequenced.
protrusions and near the stroma surface.
difficult to distinguish between hyphal protuberances and .
surface of the stromata and the found in and are homologous characters,
but both of these taxa have similar irregular verticillium-like anamorphs.
() classified in subsect.
, and in subsect.
.
proposed by Doi () are
artificial ().
available for sequencing.
of suggest that these taxa are more similar to
and has been sequenced and is not situated in the same clade as
(). Molecular data generated by Chaverri () place near a cold-loving species described by Müller () as . Doi ()
described a species from Japan, Yoshim.
has characters similar to those of , and .
ascospores, stroma anatomy, and yellow pulvinate stromata of are similar to the characteristics described by
Müller
() for which has a white gliocladium-like anamorph.
ascospores of , longitudinal elongate ostiolar
canals, and regularly verticillate conidiophores are similar to characters
found in , and .
The warted, thick-walled ascospores and long ostiolar canals of and are the morphological
characters uniting these taxa.
in the group of subsection
, series which has been shown to be
paraphyletic ().
to all other taxa included by Doi
() in Series
cannot be determined at this time.
() have
shown that Berk. & Broome, also series
, is situated in the clade.
also showed that and are
phylogenetically related.
mentioned above, it is possible that , and are
phylogenetically related, but more taxa have to be sequenced to establish the
limits of the “” clade.
species can be firmly attached to a litter-inhabiting fungus.
the molecular and morphological data that (= var. Canham) and , as
neotypified here, are not synonyms and that Fries
() and later Canham
() made a taxonomic error
in expanding the species concept of to include the
polyporicolous element. var.
is elevated to species status, , based
on morphological characters and a well-delimited phylogenetic species
boundary.
stroma colour and substrate.
narrowly defined for . Specimens BPI 1107145, G.J.S. 89-145
and
(), which are white to
light yellow and occur on soil, were found to be phylogenetically the same as
the neotype of , which is bright yellow and occurs on leaf
litter ().
specimen, B.E.O. 99-29, on the moss (Hedw.)
Warnst., was also found to be the same as the neotype based on molecular
results.
characters, a finding not suggested by the original protologue of was never observed growing on polypores in this
study.
are not consistent with the protologue in that they are not
white, as emphasized in the original protologue of .
Recommendations 9A.2 and 9A.3 of the ICBN clearly indicate that historical
specimens should not automatically be taken as type material if they are
discordant with the protologue.
() to include a whitish
gold to greyish orange form that Fries illustrated
() with a colour
lithograph two years after the original description and sanctioned
().
specimen has almost exactly the same shape and colour as the specimen of
considered here as the specimen
().
specimens of in Fries's herbarium can be identified as the
yellow form of based on morphological characters.
Molecular data have shown that could in fact be white and
could occur on soil just as Fries described in the protologue for .
characteristics defined by Fries for and is consistent with
Rifai & Webster's ()
conclusion that and are synonymous.
is not consistent with the present Articles 11.4 and 15.4
of the ICBN, which govern the priority when two sanctioned names compete,
giving priority to the older name.
lodged in herbaria after 1966 were generally labeled .
Specimens previously identified under the name should
therefore be reidentified as , the older name, which has
priority over .
the name .
() described , he did not cite a specific specimen, but indicated
as the substratum.
() indicated that the
substratum for Fuckel 2467, preserved at K was
, which recently was identified as
.
remain.
G are also overmature.
also is identified as being (= )
.
designated here as the for .
Measurements obtained from all historical specimens included in this study
were consistent with measurements from more recent collections of used in the molecular analysis.
here for the polyporicolous form and the application of the name is consistent with Weese
(), Rifai & Webster
(), and Doi
().
list |
Oudem.
species of Pers., the anamorph genus of
Fr. ().
report the involvement of this species in the biological control of plant
diseases caused by other fungi.
to produce 6-pentyl alpha pyrone, a spore germination inhibitor
().
() characterized
trichokonins, which are peptaibols that have antimicrobial activity, in .
fungus-induced plant disease.
Sri Lanka (). also benefits plant
health and nutrient uptake when it was determined to be highly active in
biomineralizing calcium oxalate crystals in soil
(), the first reference to indicate this species as a
biomineral-producing agent.
Pers.
prior to 1984 only 35 species were included in the genus, and before 1969 very
few of these were reported in the literature subsequent to their original
description. , described in 1902
(),
was included by Rifai ()
as one of the nine “aggregate” or “morphological”
species that he recognized.
() included it in
sect.
the genus, Pers., on the basis of the morphology of the
conidiophore.
() confirmed membership of
in sect. using ITS1 and 2 sequences
of the rDNA gene cluster, and PCR fingerprinting, a result that has been
affirmed in additional publications with other genes (e.g.
).
() showed that
infra-species variation was greater than inter-species in ITS in the aggregate species.
not helpful in separating closely related species of sect.
, but the authors found that UP-PCR fingerprinting could
distinguish from and other members of
sect. The first version of an
oligonucleotide barcode based on ITS1 and 2 implemented in KEY
program () is able to identify the species triplet and attribute it to the
“ A” clade after Kullnig-Gradinger ().
species among several sections. Among them was sect.
, which included .
towards the classification based on phylogenetic clades rather than dividing
the genus into sections.
for the type species of the genus, which included
members of sections and species from the
“Pachybasium A” Clade. The latter group includes (Bonord.) Bainier, the type species of
Sacc., and other species.
().
we refer to the combined “Rufa Clade” and the “Pachybasium
A” Clade as the “Viride Clade.” and the species discussed in the current paper belong to that
clade.
() narrowly defined the
morphology of and linked it to a teleomorph, Lieckfeldt . Lieckfeldt .
() and Lübeck () demonstrated
genetic diversity within the aggregate species.
Lieckfeldt .
() noted four additional,
morphologically similar and phylogenetically closely related species that they
identified as cf. or sp.
of the strains identified by Lieckfeldt
() as cf.
has since been described as B.S.
Lu & Samuels ().
() found
nine ITS haplotypes among isolates that conformed to the broadly defined
morphospecies , of which one was true
in the narrow sense of Lieckfeldt
().
() distinguished Samuels & Schroers from .
other members of the morphological aggregate on the basis
of sequences of the protein-encoding gene translation-elongation factor
1-α ( and conidium morphology. In addition to these -like species, Holmes .
() designated four clades
of collections that have the
morphology as “Tkon 20,” “Tkon 21,” “Tkon
22,” and “Tkon 3.”
Since the study of Lieckfeldt .
() we have received many
additional collections from geographically and biologically diverse sources
that can be assigned generally to sect. and specifically
to the aggregate species.
the phenotypic and phylogenetic diversity found within the aggregate species, and develop a taxonomy for those fungi by
combining results of morphological, cultural, and molecular-phylogenetic
analyses.
ascospores of specimens, direct isolations by a variety of
means from soil or dead herbaceous tissue, and as isolations as endophytes
from living stems of and related tree species as reported
by Evans
().
isolates was obtained from the American Type Culture Collection (ATCC),
Centraalbureau voor Schimmelcultures (CBS) and colleagues.
from single part-ascospores that were germinated on cornmeal agar with 2 %
dextrose (CMD, Difco cornmeal agar + 2 % dextrose w/v) and isolated using a
micromanipulator; usually two or more single-spore cultures were combined in a
single stock culture and polyspore cultures were used in all subsequent
analyses. Representative cultures are deposited in ATCC and CBS.
& Wanscher () was used
as the colour standard.
.
commonly cited collectors are abbreviated as G.J.S. (G.J. Samuels) and C.T.R.
(C.T. Rogerson).
less frequently SNA (without filter paper,
), at 20 or 25
°C for 7–10 d under alternating 12 h cool white fluorescent light
and 12 h darkness; in the descriptions that follow, these alternating light
conditions are referred to when the word “light” is used.
Approximately 20 mL of agar was poured into Petri dishes.
SNA but there was a tendency for more reliable conidial production on SNA than
on CMD.
media appeared to be more similar to how they appear in nature than conidia
formed on other commonly-used media, including potato-dextrose agar, malt agar
and oatmeal agar ().
material that was first hydrated in the case of herbarium material, or wetted
in the case of living cultures, in 3 % KOH.
by distilled water.
observe any differences between the two reagents.
each parameter were measured for each collection.
confidence intervals of the means (CI) are provided; this figure represents
the interval within which 95 % of the individuals of the parameter will be
found.
.
measured by inverting a 7–10 d old CMD culture on the stage of a
compound microscope and observing with a 40 × objective.
gathered using a Nikon DXM1200 digital camera and Nikon ACT 1 software and
measured using Scion Image (release Beta 4.0.2; Scioncorp, Frederick, MD).
blocks of substratum supporting stromata in 3 % KOH.
by Tissue Tek O.C.T. embedding medium 4583 (Miles, Inc., Elkhart, IN) and
sectioned at about 15 μm on a Microtome-Cryostat (International Equipment
Co., Needham Heights, MA).
placed on slides to make semi-permanent preparations following
Volkmann-Kohlmeyer & Kohlmeyer
().
with the specimens.
addition that cultures were also grown at 25 °C under 12 h darkness/12 h
cool white fluorescent light for 96–120 h.
repeated three times and the results of the three were averaged.
determined by linear regression.
in which the colony radius changes (x's) with the time (y's) when measurements
of colony radius are made.
as the various x measures change, the regression line is understood to
describe the regression of y (colony radius) over x (time of measurement).
This regression line is the slope of the growth curve; it is the predicted
value of each colony radius for each time of measurement and essentially
reports growth per hour (see
).
Statistical Package, version 1.131; Kovach Computing Services, U.K.), was
utilized to determine patterns of variation of phenotype within
phylogenetically defined groups.
and graphical output is
shown in .
standardized data used in PCA, and other data analyses, were obtained using
Systat version 10 (SPSS Inc., Chicago, IL, U.S.A.).
or part of a culture growing in 9-cm-diam Petri dish in a cardboard two-slide
micro-slide holder (e.g. VWR Scientific, West Chester, PA, U.S.A.) and drying
them for 2 h over low heat of a fruit dryer.
prepared so as to preserve essential characters of conidiophore branching and
phialides.
().
() was amplified using the primers EF1-728F
() and
TEF1 rev (), which resulted in a PCR product of approximately 600 bp,
and was sequenced in both directions.
calmodulin-encoding gene () were CAL-228F and CAL-737R
().
Initially a fragment of actin gene () was amplified using the
primers Fung.ACT.F1 and Fung.ACT.R1 and the conditions described by Wirsel
().
on the sequences obtained, two -specific act primers were
designed T1 (5'-TGGCACCACACCTTCTACAATGA) and T2
(5'-TCTCCTTCTGCATACGGTCGGA).
for all the isolates in this study.
primers for were designed called T500F
(5'-ATTCCGTGCTCCTGAG) and T511R (5'-CTCAGGAGCACGGAAT) and were
used for sequencing reactions.
amplified and sequenced as described by Chaverri & Samuels
() using fRPB2-5F and
fRPB2-7cR () as forward and reverse primers, respectively.
Sequences were edited and assembled using Sequencher 4.1 (Gene Codes, WI).
Clustal X () was used to align the sequences; the alignment of each locus
was manually edited using MacClade and GeneDoc 2.6
().
() and alignments were
deposited in TreeBase
(),
submission number SN 1008).
locus is also available at
The interleaved NEXUS file was formatted using PAUP* v. 4.0b10 (Sinauer
Associates, Sunderland, MA) and manually formatted for the MrBayes v3.0B4
program.
() was
implemented using MrBayes 3.0B4
().
()
was used to compare the likelihood of different nested models of DNA
substitution and select the best-fit model for the investigated data set.
modelblock3. nex which is compatible with the current version of PAUP* v.
4.0b10 was downloaded from
.
Both hierarchical LRT and AIC output strategies were considered, although the
preference was given to the last one. The unconstrained GTR + I + G
substitution model was selected for all tree loci.
with four incrementally heated chains with the default heating coefficient
λ = 0.2, heats for cold chains 1 and heated chains 2, 3 and 4 are 1,
0.83, 0.71 and 0.63, respectively) that were simultaneously run for 5 million
generations for the alignment, which comprised more than 200
sequences.
generations.
repeated at least three times.
examining the value of the marginal likelihood through generations.
Convergence of substitution rate and rate heterogeneity model parameters were
also checked.
removing the first 2000 trees for and the first 500 for
and using the “burn” command.
to the protocol of Leache & Reeder
(), PP values lower then
0.95 were not considered significant, while values below 0.9 are not shown on
phylograms and radial trees.
burning first samples were collected.
values were estimated as G↔T =1, C↔T = 3.33, C↔G = 1.14,
A↔T = 1.32, A↔G = 5.98, A↔C = 1.43; nucleotide frequencies were
estimated as 0.19(A), 0.28(C), 0.17(G), 0.36(T); alpha parameter of gamma
distribution shape was 0.23.
estimated as G↔T =1, C↔T = 4.43, C↔G = 0.83, A↔T = 1.15,
A↔G = 3.55, A↔C = 1; nucleotide frequencies were estimated as
0.26(A), 0.26(C), 0.24(G), 0.24(T); alpha parameter of gamma distribution
shape was 0.1.
high affinity to pyrimidine transitions (C↔T = 81.9); other transitions
were G↔T =1, C↔G = 0.3, A↔T = 0.85, A↔G = 0.83, A↔C =
0.61; nucleotide frequencies were estimated as 0.2(A), 0.3(C), 0.24(G),
0.26(T); alpha parameter of gamma distribution shape was 0.09.
distance was computed in PAUP* v. 4.0b10 under the GTR + I model.
r
w
o
r
k
l
e
a
d
s
u
s
t
o
r
e
c
o
g
n
i
z
e
s
e
v
e
r
a
l
s
p
e
c
i
e
s
,
m
a
n
y
u
n
d
e
s
c
r
i
b
e
d
.
I
n
t
h
e
f
o
l
l
o
w
i
n
g
w
e
h
a
v
e
a
n
t
i
c
i
p
a
t
e
d
t
h
e
f
o
r
m
a
l
t
a
x
o
n
o
m
y
b
y
a
d
o
p
t
i
n
g
t
h
o
s
e
n
a
m
e
s
i
n
o
r
d
e
r
t
o
f
a
c
i
l
i
t
a
t
e
t
h
e
p
r
e
s
e
n
t
a
t
i
o
n
o
f
t
h
e
r
e
s
u
l
t
s
.
We studied 86 strains of , any of which could have been
identified as following the schemes of Rifai
() or Gams & Bissett
().
reproductive isolation and clear morphological differentiation for detecting
species boundaries, the GCPSR concept
()
remains the only currently applicable choice.
phylogenetic position of a taxon among closely related other taxa based on at
least three unlinked loci.
not be contradicted by analyses of other loci.
phylogenetic markers that could reliably resolve groups of closely related,
apparently recently evolved species.
of phylogenetic markers for a particular group of fungi is a delicate task.
Druzhinina & Kubicek
() have listed eleven
phylogenetic markers attributed to eight DNA loci used in phylogenetic
analyses of / species.
transcribed spacers 1 and 2 (ITS1 and 2), which provide considerable
diagnostic properties in /
(), are insufficient for phylogenetic modelling even at the
intercladal level.
species, we have selected intron-rich fragments of the protein-encoding genes
, and that deliver higher levels of variation.
However, sufficiently high resolution was obtained only from both
introns, while the phylogenetic signal of and
was moderate.
loci are concordant with the selected tree based on
, however, overall statistical support of species nodes was
relatively low.
species was developed that adopts multivariate analyses
of phenotypic characters and patterns of geographical distributions as well as
phylogenetic inferences and oligonucleotide barcoding (see also
,
).
revealed several weakly or well supported clades representing taxa that could
be characterized and therefore recognized at least partly by phenotypic
characters and patterns of geographic distributions.
integrated approach, a taxonomy was developed that currently accepts twelve
species and one variety.
morphological characters, which have been the basis of species descriptions
since the genus was described more than 200 years ago.
characters derived from DNA, all of the isolates that we studied could have
been identified as in the sense of Rifai
().
geographic distribution and rate of growth in agar culture were among the most
significant characters separating species.
available characters of the anamorph (i.e.
conidia and phialides) did not result in a clustering of strains that was
consistent with the results of phylogenetic analysis.
the anamorph are variable within a phylogenetic species; others suggest
homoplasy in different phylogenetic species.
conidiophores; instead, conidiophores are aggregated within more or less
well-developed pustules and individual elements cannot be measured and are
difficult to characterize.
aggregation of “conidiophores”, in most species is highly variable
and the ability to form pustules may decline with length of time of strain
preservation or after successive transfers.
longer or shorter, wider or narrower, depending upon whether they are formed
near the surface of the pustule, where they are less crowded, or at the
interior of the pustule where they are more crowded.
crowded phialides can hardly be analyzed separately from each other.
present the most consistent morphological character because they can be
measured and because their morphology remains constant over successive
transfers.
the general lack of pigments in cultures, a character that has been used in
taxonomy of sect.
()
or other species-rich genera such as .
use in recognizing phylogenetically distinct clades.
species within a clade cannot be distinguished on the basis of the
morphological characters of the teleomorph.
have a distinctive teleomorph morphology.
() found that among species
with green ascospores, the same anatomy of the stroma was
found in phylogenetically distant groups, however, all of those teleomorphs
were very different from the teleomorphs reported here and from those formed
in sect.
().
collections studied here cannot be distinguished from (Pers.:
Fr.) Fr., the teleomorph of the closely related , and the
type species of Doi
() subdivided
of Japan on the basis of ascospore colour and stroma
anatomy, but phylogenetic analysis has not upheld those subdivisions.
species delineated by morphological characters is likely to comprise multiple
phylogenetic species.
those of the conidia, conidiophores and ascospores, we might have concluded
(as did ) that
there were potentially more than one “cryptic species” within the
morphological species .
however, to identify most of them unambiguously.
the ascomycetes and led Hawksworth
() to revise upward the
estimated number of species of fungi.
have, in an earlier time, been disregarded as insignificant, are now accepted
and sought for characterization of clades.
was distinguished from the cause of green mould of
mushrooms, Samuels & W.
the latter species to grow at 35 °C
().
but rather is likely to become increasingly found in the
ascomycetes in general as genera are subjected to phylogenetic analyses.
() found that salt
tolerance was the only phenotypic character to distinguish from the closely related
has received much attention recently.
distinguished between the phylogenetically closely related and
().
challenge to taxonomic revision ().
or are included in
phylogenetic analyses, increasing numbers of morphologically defined species
will be found to be paraphyletic or comprise numerous cryptic species.
as we would wish for a taxonomy that would permit accurate species
identification using only the microscope, we must face the reality that there
may not be enough characters in morphology and growth to reflect the
differences revealed in diverging DNA sequences.
characters may well be the result if genetically distinct lineages occupy the
same niche for a long time.
for the overall carbon utilization profiles (Biolog) did not correspond to the
phylogenetic analysis, while the analysis of the utilization of certain single
carbohydrates did ().
unrelated genus collected from different marine sites
(Dela Cruz & Druzhinina, unpublished).
narrow, too strongly influenced by phylogeny, but an example from the present
study argues against that. and are well separated in the phylogenetic analysis (Figs
,
).
sympatric in the Eastern U.S.A.
incompletely separated by a suite of phenotype characters.
separating the two species () are the L/W and length of conidia and the length of the
proximal part-ascospores.
characteristic of the respective species.
statistically significant, there will be many cases that cannot be identified
on the basis of their phenotype.
provide practical identification of the apparent large number of species.
Carbon utilization estimated using a Phenotype MicroArray technique may
provide additional characters (, ).
the ability to perform mating experiments, which would help
immensely in determining whether strains belong to the same or different
biological species.
of the diversity of the species-rich “Viride Clade.” Work is
continuing on taxonomy of this group.
in this study; the species is distinguished by its longer and narrower conidia
and slow rate of growth on SNA.
most commonly cited species in the genus, our results suggest that it is an
uncommon species of Europe and North America.
isolated directly from natural substrata and the species often used in
biological control applications is the closely related .
from South America and Africa (Ethiopia), but its teleomorph
has been found as far north as New York State. is the most commonly encountered species having a -like morphology.
“ sp.” in part
(), “ II”
() and
“ Tkon 21”
().
sp. 1” from the United Kingdom.
() strongly resembles
in the morphology of conidia, conidiophores and in the
formation of concentric rings in agar culture.
cited by Prof. Webster from CBS.
these cultures and sequences of , we can see that
sp. 1 was based on a mixture of two species.
();
the other cultures (Webster 2545 =
, 2617 =
and 2644
= ) are
all Bissett, the anamorph of
().
Neither of these species is closely related to members of the “Viride
Clade” ().
-like anamorph for Japanese collections of Hino & Katumoto.
– including those with surface ornamentation – described by him
lead us to suspect that more than one species was involved.
the collections cultured by Doi was taken from bamboo, which is the substratum
of the type collection of .
an unusual substratum supporting fungi that are not usually found on other
substrata, at least not on woody substrata, which was the source of specimens
reported by Doi.
(YAM) and conclude on the basis of its morphology that it is a member of the
“Viride Clade,” but there is no material with a living culture
available.
() for Two cultures isolated from rhizomorphs of, respectively,
(IFO 31288) and (IFO
31293) and identified by Y.
Several isolates of , represented by G.J.S. 01-10
and G.J.S. 01-11 but not included in the phylogenetic analysis, were isolated
in Ecuador from pods of that were infected by the
destructive parasite .
currently in field trials to protect cacao from the
(C. Suarez, pers. comm.). isolates G.J.S.
04-10 and 04-11 are effective in protecting cotton plants from infection by
in Texas (C.R. Howell, pers. comm.), and
isolate G.J.S. 05-462 (received too late to be included in the present study)
is showing potential for control of in maize
(I. Yates, pers. comm.). A single isolate of this species, G.J.S. 97-273 (=
BBA 65450), was isolated from soil in Germany.
for their ability to parasitize the cacao pathogen
(results not shown) following the
“preinoculated plate test” described by Evans
() and found that several
were able to parasitize the mycelium of , with the German
isolate being especially effective.
common in tropical regions.
,
the species can be seen to
comprise several well-supported internal branches; however, we were not able
to detect any geographic or phenotypic bias to any of the clades.
(designated as “DIS” in ) of were isolated as endophytes from
freshly exposed, living sap-wood of trunks of species of in
Brazil, Ecuador and Peru.
and were reisolated from woody tissue but not from the apical meristem.
isolate DIS 339c (from ) could be reisolated from all stem
sections of seedlings, including the apical meristems, and
it could be reisolated from inoculated pods of after 12
weeks, indicating a potential for protecting pods against infection by (K. Holmes, pers. comm.). Ecuadorian strains (G.J.S.
01-07–G.J.S. 01-12, ) were isolated from pods of that were
naturally infected with the parasite , the cause of frosty
pod rot, and have been included in a field trial in Ecuador against that
pathogen (C. Suarez, pers. comm.).
Large Koningii Branch (LKB) in the tree
(), although the
statistical support of this species on both and
trees is particularly low.
distribution of the species in tropical countries may indicate a relatively
intensive recombination process due to sexual reproduction.
majority of strains were isolated as anamorphs from
natural substrata.
region and from the U.S.A. Alternately, the paraphyly of could be explained if the species were relatively old.
Partially sympatric old, clonal lineages could occur sympatrically and, over
evolutionary time, accumulated mutations in the introns and other parts of the
DNA could explain the variation in the species.
sympatric in eastern North America. was
reported earlier as “ sp.” in part
(), “ Tkon 3” and “Tkon
22” in part (). was reported as
“ sp. and”
(). The similarity between these two species was noted above.
The most obvious difference between these very similar but phylogenetically
relatively distantly related species is that grows more
slowly on SNA than does ; moreover, conidia of are slightly shorter and broader than those of (95 % CI of L/W respectively 1.35–1.39,
1.40–1.46).
each other and, at least in gross morphology, they are indistinguishable from
(anamorph: ).
an uncommon species, albeit sympatric with the other two, despite the many
reports of its occurrence.
approx. 4.5–5 × 4–4.5 μm; proximal part-ascospores
approx. 5–6 × 3–4 μm). is
readily distinguished from and
by its subglobose, warted conidia.
and .
despite their phylogenetic distance from each other, it is important that they
may be reliably distinguished by the ITS1 and 2 oligonucleotide barcodes.
ovoidal conidia ().
species and was also isolated from a woody stem of the liana that was infected by ()
the cause of Witches' Broom disease of cacao in tropical
America ().
cacao is grown; it was not included in the phylogenetic analysis.
isolate (DIS 70a) reinfected and was reisolated from meristematic tissue of
, and inhibited radial growth of the frosty pod rot pathogen
() .
surface, and within tissues, of cocoa pods in the field for at least 10 weeks.
Initial field trials in Costa Rica, where conidia were applied as a spray,
indicated an ability to protect pods against infection by
().
() were isolated as
endophytes from woody stems of South American
and other species.
isolates were reported previously by Evans .
() and Holmes . () to be
endophytes of .
additional endophytes as members of the “Viride Clade” of
sect. was
especially well represented in the endophyte isolations but they did not fall
into an endophyte-specific lineage in this species. In contrast to and var.
are known only as endophytes of cacao and cacao relatives but which are not
common even in that niche, it is not surprising that a species that is as
common as should be found as an endophyte of a common
tropical tree.
not to find it as an endophyte of stems of other tropical trees.
would not be surprising to find additional isolates that have a biological
control potential for fungus-induced plant diseases.
Arnold & Herre () did
not report species as leaf endophytes in Panama.
of the aggregate species, (DIS 7,
DIS 8 in ) was extended
to Peru.
Cambodia, Malaysia). was originally
described as a parasite of the xylariaceous fungus in Puerto Rico in 1996.
expand the biological and geographic distribution of this species by discovery
of its teleomorph in Costa Rica and Ghana on bark and perithecia of
, and endophyte isolations from woody tissue of
species in Ecuador and Brazil and from in the United Kingdom.
seen in colony reverse on PDA, and fast growth rate characterize this
species.
var. is represented by
two collections (G.J.S. 97-3, G.J.S. 98-43); both are derived from ascospores
of specimens collected in, respectively, Guadeloupe and
Puerto Rico.
closely similar in phenotype and also genotype
().
var. forms a highly supported
clade with the other two isolates but is phenotypically and apparently
biologically distinct. It was isolated as an endophyte from stems of in Ecuador.
biogeography and habit, despite its phylogenetic proximity to the ascospore
isolates, lead us to recognize the endophyte as a variety, var.
.
varieties is possibly an artifact of sampling; additional sampling could
support their separation at the species rank.
At least three species occur in New Zealand and Australia, viz. and is the slowest-growing species in the present study; its
temperature optimum is 20–25 °C and the colony radius is < 5 mm
after 72 h at 30 °C.
forests whereas the third, was
found in the tropical Queensland coast and in forests of
New Zealand. Cooke was described from New Zealand
() and is reported
often in the literature, or on the World Wide Web, from diverse geographic
regions (e.g. Brazil, ; Japan, ; New Guinea, ). Ascospores in the type specimen of (K!)
are unusually large (distal part-ascospores 5.1–6.7 ×
5.0–5.5 μm; proximal part-ascospores 5.7–7.2 ×
4.6–5.3 μm), suggesting that most or all of the reports of this
species outside of New Zealand are based on misidentifications.
, is derived from a specimen received
from New Zealand (J.M. Dingley No. 3, Auckland, Te Aroha).
locate that specimen in CBS or PDD to confirm its identity.
specimens in New Zealand that conform to the type collection of and redescribe the species in another publication
().
geographically diverse, including lineages (Figs
,
) from tropical Australia
(Queensland, ), temperate New Zealand
(), Russia
(), and a
single lineage that includes one collection from the United States (Florida)
and one from Taiwan ().
clade (e.g. growth rates, , and ascospore measurements).
diversity of the isolates in this “ clade,
which occupies the terminal position of the SKB clade
(), suggests that more
than one taxon could be involved and that additional sampling would resolve
this clade.
Some of the species are represented by one or two strains.
normally describe a species based on such a small amount of material because
there is no way to estimate intraspecific variability. Nonetheless, , based on a single collection from Taiwan (G.J.S. 95-93), and
, based on two collections (G.J.S. 97-88 from Thailand
and G.J.S. 96-13 from Puerto Rico), are phylogentically distinct from all
other species that we have included.
are phenotypically distinct as shown by PCA
(). was formerly reported as “ cf.
/ sp.” in Lieckfeldt ().
(continuous characters used in the PCA are presented in
) |
Nine species of Fr. () with effused stromata from Japan, Australia, New Zealand,
North America, Europe, and Central America, are newly described or
redescribed.
acremonium- or verticillium-like conidiophores with hyaline conidia, and are
assignable to sect Bissett.
(Schw.) Sacc.
fungicolous species recorded from North America and Europe that occurs on
spp.
() considered Kalchbr. & Cooke, recorded from Africa, as a synonym of
the older , but this synonymy has never been critically
examined.
fungus with yellow effused stromata. The relationship between and has not been established.
() described f. Yoshim. Doi.
type material (NY) of this forma with collections of
from North America and Europe. A specimen identified as Syd. in De Wild.
Japan and redescribed. Speg.
appearance to , but occurs on
decorticated wood and has a tropical distribution. Speg. is a later homonym of Berk.
& Broome. The type material of Berk. & Broome
is indistinguishable from and therefore synonymous with (Berk. & M.A. Curtis) Höhn., a member of the
(). A new name is proposed for Speg.
studied. Their relationship to spp.
pseudoparenchymatous tissue was unclear.
these hyphal species to (Berk. & Broome)
Petch, which also has a hyphal stroma, had to be examined.
() showed that some
species with anamorphs in sect.
form a highly supported subclade of sect. and suggested that sections and
are phylogenetically indistinguishable.
included a limited number of taxa with acremonium- or verticillium-like
anamorphs.
() used partial sequences
of the RNA polymerase II subunit () and the large exon of
tef-1α (LE) and found that anamorphs referable to sect.
do not form a monophyletic group, as Berk. & Broome and S.T. Carey &
Rogerson were situated in the clade.
() showed that (Pers.: Fr.) Fr.
supported clade, the limits of which were not established.
the ITS1-5.8S-ITS2 rDNA (ITS) region, that and form two distinct subclades of a strongly supported but
phylogenetically unresolved clade. These authors did not conclude that sect.
and sect. were phylogenetically
indistinguishable.
() and Chaverri () support the
conclusion of Kullnig-Gradinger
() that section
is paraphyletic.
to selected species treated by Overton
() to establish the
phylogenetic limits of sect. .
species; to verify the phylogenetic relationship between and ; to verify the relationship
between and ; to determine
the relationships of two new hyphal species to ;
to investigate the phylogenetic boundaries of with
anamorphs in sect. ; and to
describe the phylogenetic species delineated in this study according to
criteria developed by Taylor
().
(,
,
) were used in making
initial species determinations. lists the accession numbers used in this study.
collectors are abbreviated: B.E. Overton (B.E.O.), G. J. Samuels (G.J.S.), and
K. Põldmaa (K.P.). All isolates with G.J.S.
by isolating single ascospores on CMD with the aid of a micromanipulator.
isolates with B.E.O.
contents of individual perithecia.
data are taken from herbarium labels.
same as in Overton
().
DNA sequence analysis was conducted using three gene sequences: ITS
1-5.8S-ITS2 (ITS), a partial sequence of the large exon of translation
elongation factor (LE), and a partial sequence of the RNA
polymerase II subunit ().
generated following the protocol and primers described in Overton . ().
primers were employed for amplifying the LE regions which differs
from the region amplified in Overton .
(): for LE,
EF1-983F (5'-GC(C/T)CC(C/T)GG(A/C/T)CA(C/T)GGTGA(C/T)TT(C/T)AT-3') (Carbone
& Kohn 1999), EF1-2218R (5'-ATGAC(A/G)TG(A/G)GC(A/G)AC(A/G)GT(C/T)TG-3')
(S.A. Rehner, pers. comm.).
AMRESCO® was added to each 50 μL PCR reaction.
purified and sequenced following the protocol in Overton
().
assembled using SeqMan® II option and aligned using Clustal W in DNA Star
(DNA Star Inc., Madison, Wisconsin), and a phylogenetic analysis was performed
using PAUP* v. 4.0 b4 (). Alignments were manually adjusted in PAUP*.
varied depending on the phylogenetic analysis to meet two different objectives
in this study.
LE were evaluated in single and combined analyses to establish
phylogenetic species limits.
with isolates of cf.
, and cf.
used as outgroup taxa.
isolates with sect.
anamorphs in phylogenetic context with other
/ species.
cf.
Yoshim. Doi., and Rogerson & Samuels were
used as outgroup taxa for the combined LE and
analysis with representative isolates from the different sections of
included in the analysis.
were done using the heuristic search option under the following conditions:
TBR branch swapping, 10 random addition sequences, and gaps
(insertions/deletions) treated as missing.
500 replicates with random sequence addition (10 replicates).
LE and analysis, sequences were trimmed to the same
starting position because some GenBank sequences not generated in this study
were significantly shorter.
GenBank ().
relationships were compared by the Kishino-Hasegawa (K-H) test
() in PAUP* for the
combined LE and data set.
trees recovered with and without constraints were compared by likelihood
scores ().
likelihood model implemented in the K-H test assumed equal rates of
substitution and empirical base frequencies.
tested and model parameters obtained for the LE, and
combined alignments using MODELTEST 3.06
()
as implemented in PAUP*.
test (LRT) implemented in MODELTEST, selected the TIM+I+G model with unequal
base frequencies; nucleotide frequencies were set to A: 0.2133, C: 0.3337, G:
0.2211, T: 0.2320; a gamma-shape parameter of 0.5234; and substitution rates
set to 1.0000 (A–C), 3.1252 (A–G), 1.6847 (A–T), 1.6847
(C–G), 10.5209 (C–T), and 1.0000 (G–T).
data, the LRT implemented in MODELTEST, selected the TrN+I+G
model with unequal base frequencies; nucleotide frequencies were set to A:
0.2413, C: 0.2787, G: 0.2551, T: 0.2248; a gamma-shape parameter of 1.1736;
and substitution rates set to 1.0000 (A–C), 6.5499 (A–G), 1.0000
(A–T), 1.0000 (C–G); 9.0762 (C–T), and 1.0000 (G–T).
For the combined LE and data set, the LRT
implemented in MODELTEST, selected GTR G+I model with unequal base
frequencies; nucleotide frequencies were set to A: 0.22590, C: 0.30330, G:
0.24090, T: 0.22990; a gamma-shape parameter of 0.87796; and substitution
rates set to 1.0000 (A–C), 5.2773 (A–G), 1.0000 (A–T),
1.0000 (C–G), 8.4309 (C–T), and 1.0000 (G–T).
likelihood (ML) tree was then obtained in PAUP* using 10 random sequence
addition replicates and the substitution model suggested by MODELTEST.
Bootstrap analysis was performed with 500 replicates and fast stepwise
addition.
specimens representative of each phylogenetic species.
were grown on PDA, CMD and SNA at 20°C, with 12 h
fluorescent light and 12 h darkness.
7–10 d post inoculation.
were measured following Overton .
() with the exception that
optimal growth temperatures were not determined.
obtained from Kornerup & Wanscher
().
characters used in species recognition are discussed in the comments section
immediately following each species description.
p
The combined phylogenetic analyses using ITS and partial sequences of
LE and show that , and represent phylogenetically distinct species that are
members of a strongly supported clade C (Figs
,
).
() suggested that
morphology could not be used to distinguish between and
and considered these species synonymous.
studies, we found the ascospores of to be consistently
shorter and narrower than those of In contrast,
is considered a facultative synonym of the older
Dingley deposited a culture of
() from
New Zealand in CBS.
ITS sequence as and represent Dingley's concept of .
Molecular phylogenetic results indicate that the Australian specimens and the
New Zealand culture () represent a new phylogenetic species, described here as
, that differs morphologically from and .
between Australian specimens of and North American
specimens of are striking, but the part-ascospores of
the Australian species are more strongly spinulose than the part-ascospores
found in .
occurred on spp., which is a common substrate in North
America.
informative species characters for members of the
subclade (clade C, ).
sect. are situated in different
clades. resides in clade A2
() with has a hyphal stroma and a
verticillium-like anamorph with conidia that are uniform in size and shape.
Anamorphs in sect. typically produce
conidia that are variable in size and shape.
resides in the clade () with species having pseudoparenchymatous stromata.
the clade generally produce conidia that are typically
more uniform in size and shape than those found in sect.
and have
hyphal stromata and verticillium-like anamorphs and are basal to other major
clades of .
extensive stromata, with pseudoparenchymatous tissue, except one, which is hyphal.
variable in size and shape, typical of sect.
, known to have a
pseudoparenchymatous stroma and a verticillium-like anamorph also resides in
the clade (C2, ), a finding consistent with Chaverri
().
hyaline after repeated transfers.
anamorphs that produce hyaline conidia variable in size and shape are located
in clade B2 ().
with hyphal stromata and anamorphs that produce uniform conidia (of similar
size and shape) are polyphyletic.
the genus Petch for species that have simple ascomata
immersed or seated upon a byssoid stroma with ascospores that disarticulate
into part-ascospores.
() described the anamorph
of as acremonium- or verticillium-like. resides in clade B2 () with other species with acremonium- and verticillium-like
anamorphs.
below the perithecia in specimens of .
teleomorphs of specimens examined varied in the degree of pseudoparenchymatous
tissue present, the part-ascospore measurements obtained are identical to
those published for by Rossman .
() and the anamorph
characteristics are identical to those described by Doi
() for .
The phylogeny of the major clades in /
is essentially unresolved based on the genes used in this study.
spp.
and acremonium- or verticillium-like conidiophores (hypocreanum-like), that
produce hyaline conidia variable in size and shape, can be accommodated in a
large monophyletic assemblage of species B2
().
()
suggested that sect. and sect.
should be merged as they are phylogenetically
indistinguishable.
belonging to sect.
relationship of sect. to sect.
could not be resolved in the combined LE and
dataset.
to anamorphs typical of sect. ;
nevertheless, these fungi do not belong to the major
clade B2 (), nor are they
phylogenetically related to members of sect.
.
() should serve as an
example for future phylogenetic analyses to determine sectional relationships,
but future studies should include a larger number of taxa and exclude ITS rDNA
sequences.
related species () and has been used for the revision of sections
and (Kuhls
,
; Kinderman 1998; Samuels
,
).
() demonstrated that ITS
rDNA, , and the region could establish phylogenetic
species limits, but the introns found in the region, delimited
by the primers ef-1 and ef-2, were highly divergent among morphologically
similar species.
(LE) were generated for the seven species, including several of
those treated by Overton
().
gene region and distinguished between North American and
European isolates of ; therefore LE is
better suited for phylogenetic studies than the region
previously sequenced by Overton
().
have been sequenced to date, with published accounts placing
an over-reliance on ITS rDNA sequence data.
sequences generated in this study, work by Chaverri (), and data from
other gene regions published by Kullnig-Gradinger
(), have helped to clarify
our understanding of the sectional relationships of
.
Lloyd, with anamorphs, need to be
sequenced before a complete phylogeny of can be
established.
anamorphs in sect. Samuels
() hypothesized that the
anamorphs referable to sect. may be
synanamorphs or spermatial states, suggesting that with
acremonium- or verticillium-like anamorphs with hyaline conidia have lost the
ability to produce a primary trichoderma-like anamorph, with pyramidally
branched conidiophores and green conidia.
() presented molecular
data suggesting that the more typical trichoderma-like anamorph with green
conidia may have evolved from genera having verticillium-like anamorphs, in
particular Barrasa and Z.
Moravec.
evolution of the anamorph, including those referable to
sect. .
considering the hypotheses promulgated by Kullnig-Gradinger
() and Samuels
(). has light green conidia that become completely hyaline in
subsequent transfers, suggesting an incomplete reversal to the primitive
verticillium-like form with hyaline conidia.
clade C2 () based on combined and LE gene
sequences and based on ITS sequence data, a finding consistent with
Kullnig-Gradinger .
(). produces a primary synnematous anamorph and a
verticillium-like synanamorph.
when considering the hypothesis of Samuels
() that the
verticillium-like anamorphs found in sect.
represent spermatial states, in which the primary
trichoderma-like anamorph was lost. is located
in an unresolved basal clade of
() and, based on the
molecular results of this study, it could not be excluded from the genus
.
other species sequenced in this analysis could suggest that
the ability to produce a synnematous primary anamorph has subsequently been
lost.
of Samuels () and
Kullnig-Gradinger .
(), leaving room for
speculation.
anamorphs can be more accurately determined.
list |
Pers. () is
one of the most commonly reported species of fungi.
abstracted by CAB.
few examples of activities include organochlorine degradation as a soil fungus
(), biological
control in fungus-induced plant disease
(;
),
and as the cause of disease in button mushrooms in India
().
(), and
enhance phosphorus uptake by plants
().
(),
degrades cellulosic agricultural waste to alcohol
(),
colonises leaf litter () and is a normal inhabitant of soils
(,
).
. ()
detected a shockingly high level of misidentification of strains that were
reported in the literature as If this experience is
representative of the genus, as it is likely, then not all of these reports
actually refer to .
the degree of inaccuracy in identification is that of a biocontrol fungus
reported in the literature as (Bastos
,
,
) that was ultimately
described as the new species Samuels &
Pardo-Schultheiss (); these two species are distantly related and morphologically
and biologically highly dissimilar.
identity of , otherwise the literature is meaningless.
.
view held sway until 1969 when Rifai
() monographed the genus
and characterised as the only species having globose,
warted conidia.
activity by species prior to 1969.
description of and both having
warted conidia and both being members of sect.
Bissett (), stood out because its
conidia were globose as compared to ellipsoidal in the other species.
electron microscopy () revealed the existence of two distinct patterns
of conidial ornamentation within strains identified as ,
viz. more and less strongly warted.
conidia were segregated as Samuels .
(; ).
, in addition to recognising and
., Lieckfeldt
() noted the existence of
two additional ITS-defined groups that had warted conidia, which they referred
to as Vd and Ve. The group Vd was very closely related to Vb in its ITS1 and 2
sequences and its morphology.
phenotypically diverse, some of the few included strains having smooth conidia
and others having warted conidia.
()
determined that the group Vb was “true” by
comparison with the over two-hundred-year-old type specimen of the species
that is preserved in Leiden.
() could
not see consistent phenotypic differences between Vb and Vd that would support
recognition of Vd as a separate taxon.
include / and its relatives in
sect. , including also Oudem. and P. Karst.
this group either as sect. (e.g.
) or more recently simply as “the viride clade”
(), has been
affirmed by DNA sequence analysis.
obtained many additional specimens and cultures referable to the viride clade
and are able to propose a revised taxonomy for this clade.
we re-evaluate groups Vb and Vd and recognise group Vd as a
distinct species.
(),
has been recognised as the anamorph of (Pers.: Fr.) Fr., the type species of Fr.
is possibly the most common name used in the
identification of specimens.
herbaria throughout the world are labelled “”.
plethora of species has been lumped under this name.
as B.S. Lu /
Bissett and Yoshim. Doi/
(Link: Fr.) Rifai have both been incorrectly identified as the only distantly
related .
first modern description of .
stroma that starts out semieffused and whitish to tan to reddish brown and
pruinose and with age becomes darker and cushion-shaped; the ascospores are
hyaline.
especially the young stroma of most members of the clade is distinctive of a
number of often sympatric species that are best distinguished by their
anamorphs ().
teleomorphs for both groups, Vb and Vd.
redefinition and redescription of .
refine the description of and provide an epitype for the
species, we describe as new a teleomorph for group Vd,
redescribe with its new anamorph ,
and describe the new species and .
investigated in this study are listed in
.
European countries are indicated with coordinates and map sheets (MTB =
Messtischblatt).
share the same or very similar alleles of internal
transcribed spacers 1 and 2 (ITS1 and 2), rendering this locus inappropriate
for recognition of some species within the section.
genetic diversity of the group we used intron
sequences of the translation elongation factor 1-alpha (), the
most powerful phylogenetic marker as yet established in the genus.
resulting Bayesian phylogram (), which was obtained from 238 sequences, corresponds well to the
previous analysis of related species with -like morphology
().
there are two diverged groups named “Large Viride” and
“Large Viridescens” clades, both of them with significant
statistical support.
composed of mainly European but also North American, Asian and Pacific strains
showing its cosmopolitan nature.
additional unresolved lineages that apparently represent unnamed species.
description of these taxa requires further sampling and therefore will be
discussed in subsequent publications.
single endophytic strain from the Galapagos Islands, sp.
nov., which belongs to the “Large Viride” clade but at the same
time occupies the most distant position from .
group on the tree, the “Large Viridescens” clade, splits into two
independent evolutionary lineages.
represents a compact and well defined subclade with significant statistical
support that contains isolates of the former Vd group
(), described as below.
this species has mainly European origin, also nearly all
primary European nodes include North American, Central American, Asian and
Pacific isolates, suggesting the absence of recent allopatric speciation in
this group of isolates. Another well-supported clade in the vicinity of is composed of isolates of .
“Large Viride” clade this branch contains representatives of
several well-supported speciation nodes composed of strains that are closely
related to and and undoubtedly
represent yet undescribed species of .
diversity will be discussed in subsequent publications following further
investigations and sampling.
sufficient to prove the existence of another phylogenetic species with
eidamia-like morphology that occupies the second independent lineage within
the “Large Viridescens” clade.
forms a homogeneous clade mainly represented by isolates from undisturbed
soils in Sardinia and Central Russia.
any geographic isolation since we also sampled isolates from North America and
Australia.
Viridescens” Clade, once again a single strain, as The detailed analysis of the highly variable intron
sequences of the gene has clearly shown that, despite their
close relationship, and a large
group of isolates that we describe here as represent
distinct sympatric phylogenetic species.
within one of these two large clades.
distantly related sect.
()
and clade Ve ().
will be discussed in a future publication.
Viridescens” clade are characterised by the formation of peculiar,
percurrently proliferating phialides that are diagnostic of , the ex-type of which
() falls
in
DNA sequences referred eighty-seven strains to the “Large
Viridescens” clade and thirty-four to the “Large Viride”
clade.
copious amounts of green conidia in pustules or in extensive
“lawns” on CMD, PDA and SNA.
form more quickly on SNA than on CMD or PDA and often conidia did not form on
either of the latter media while they did form on SNA.
reliable production of conidia.
produced conidia on SNA to which a 1 cm piece of sterile filter
paper had been added; the conidia only formed at the interface of the paper
and the agar and on the paper itself.
Conidia tended to form in pulvinate to hemispherical pustules <
1–3 mm diam.
on CMD.
less than 1 mm and often no pustules were formed, the conidiophores arising in
more or less continuous cottony lawns.
the larger pustules in the aerial hyphae and in minute tufts.
groups tended to be cottony, and individual fertile branches could be seen;
often conidiophores protruded beyond the surface of a pustule, producing a
single phialide or a few fertile branches near the tip, the rest of the
conidiophore remaining sterile or nearly so.
transparent under a 10 × objective.
produced on CMD, conidia often appeared to form at the surface of the pustule.
In all cases, after one week at 20–25 °C, conidia were deep green to
dark green 27–28D–F6–8, although lighter green conidia were
observed in younger cultures.
grown at 25 °C under alternating light on CMD and SNA, conidial masses
were yellow.
first.
became greenish yellow when mounted in 3 % KOH.
recognizable as typical of in producing green conidia in
abundance on most media.
produced conidia sparingly on SNA to which a 1 cm piece of sterile
filter paper had been added.
branched, similar to what was described for Samuels
() and for synanamorphs of pustulate species of
().
appeared yellow to pale green because of the conidia, at the tips of the
phialides.
work.
. Conidiophores are mostly formed in pustules.
noted above, pustules tend to be composed of intertwined hyphae that terminate
in fertile branching systems.
conidiophore is referred to as the terminal branching system of intertwined
hyphae that form the pustule.
in this study, and these were largely related to the medium and to the clade.
In (), a well-developed main axis was not readily visible, or it was
short and sometimes sinuous.
paired and phialides tended to arise singly from the main axis.
often hooked or sinuous (), cylindrical or somewhat swollen at or below the middle.
type of conidiophore was only found in the “Large Viride” clade,
especially in . The conidiophore (e.g.
,
,
,
) was formed by all clades.
In the Type 2 conidiophore there was a more or less readily discernable,
well-developed main axis, from which lateral branches arose at or near
90°; the lateral branches were longer with distance from the tip and
secondary branches were shorter with distance from the point of departure of
the branch from the main axis.
secondary branches in pairs.
cruciate whorls of 3–4.
somewhat swollen at or below the middle.
the “Large Viridescens” clade, including and , the most distinctive characteristic
is the production of percurrently proliferating phialides (Figs
;
;
;
), the branching
system itself is highly variable in extent and form.
phialide percurrently produces a second phialide
().
be continuing percurrent proliferation of phialides results in a submoniliform
chain of five or more cells (Figs
,
,
), each cell of the
chain being often abruptly swollen in the middle and separated by the cell
above and below by a conspicuous septum.
and was often reduced to a few, short, verticillately disposed branches or a
reticulum of branches (e.g.
;
,
,
).
form of the third branching type was observed in old pustules on CMD and PDA,
where chains of percurrently proliferating phialides having subglobose bases
and extremely long, cylindrical beaks arose from swollen, subglobose cells
(Figs ,
,
).
proliferating phialides having this morphology were also seen occasionally on
more typically branched conidiophores (Figs
;
), on conidiophores that
produced typical, non-proliferating, phialides.
rarely seen on SNA.
well-developed pustules; they formed a reticulum with short fertile branches.
The branches tended to be sinuous or curved and to be broader than is found in
other clades that are studied here.
unicellular lateral branches, each of which terminated in 2–4 phialides.
The phialides in DIS 328g are shorter than in any strain included in this
study and have a smaller L/W ratio; they were often hooked or sinuous.
, which
is the ex-type culture of A.S. Horne & H.S.
Williamson.
“Large Viridescens” clade, especially the extreme form described
above as Type 3 and illustrated in
() and ().
Conidiophores produced by this culture on SNA
() were typical of
, with a more or less uniformly branched conidiophore and
typical phialides.
odour and a diffusing yellow pigment.
bearing green conidia in appressed phialides developed, but no pustules and no
proliferating phialides were seen.
synanamorph conidiophores described by Chaverri .
() for species of
/ having conidiophore elongations.
nor restricted to any particular clade (e.g.
,
,
,
).
Various conidial types were observed in this study.
conidiophore types, were largely typical of clades.
produced warted conidia.
(), ()
and () are smooth. (Figs
;
), DIS 328g (Vb 1),
G.J.S. 04-40 (Vb 2), and
() have nearly
globose conidia that have a length/width ratio 1.0–1.2.
G.J.S. 03-151/G.J.S. 02-87 (Vd 1) are ellipsoidal, length/width of
1.2–1.4.
vary from subglobose to ellipsoidal (Figs
,
); although the mean
L/W of all collections in this clade varies from 1.1–1.3, there is
considerable overlap between this clade and DIS 328g. Conidia of and are unusual in being ellipsoidal.
of these species produce -like conidiophores and conidia.
Conidia of are much more coarsely warted than any of the
other clades considered here.
clade Ve. Conidia in this clade are subglobose to ellipsoidal.
conidia were observed for most members of this clade.
often large, are widely spaced and thus are not as conspicuous as in members
of the “Large Viride” and “Large Viridescens” clades
that are the focus of the present work.
Chlamydospores were inconsistently produced in most clades.
formed in abundance in
() and ().
Chamydospores were especially abundant in
().
were typical of in being globose to subglobose and
terminal at the ends of hyphae or intercalary within hyphal cells.
Optimal temperature for growth on PDA for all clades except and Vd 1 was 25 °C.
was 30 °C and the two isolates in Vd 1 exhibited considerable variation at
30 °C (35–70 mm radius after 72 h). was
unusual in having a temperature optimum of 20–25 °C and in reaching
no more than 5 mm colony radius after 4 days at 30 °C.
less, after 72 h at 25–30 °C. On SNA only DIS 328g (Vb 1), and demonstrated a clear optimum at 25
°C.
to show a temperature optimum on SNA. G.J.S. 04-40 (Vb 2) was the fastest
growing strain on SNA, reaching 65 mm after 72 h at 30 °C.
temperature differential was not observed on PDA.
(Vb 1), G.J.S. 04-40 (Vb 2), , Vd 1 and Vd 2 reached or
exceeded a radius of 45 mm after 72 h.
both and showed very little
variation in growth rate among their many isolates, both reaching a radius of
30–40 mm after 72 h at 25 °C.
both of these clades, as well as in and DIS 328g (Vb 1),
was more than 20 mm slower at 30 °C than at 20 °C. was the slowest growing, reaching only 10 mm on SNA after 72 h
at 25–30 °C and 18 mm on PDA at 30 °C.
isolate at 30 °C was G.J.S. 04-40 (Vb 2) reaching 45 mm, although G.J.S.
03-151 (Vd 1) reached a radius of 70 mm after 72 h at 30 °C.
distinct groups of isolates. The North American isolates (G.J.S. 00-67, G.J.S.
97-243) cannot be distinguished from in any of their
morphs and aspects. The Taiwanese isolates (G.J.S. 94-9 – G.J.S. 94-11)
grow significantly more slowly than .
anatomically so similar that they often cannot be distinguished.
stage, when it could be observed, was semieffused, velutinous to conspicuously
hairy and light tan in colour (Figs ; ).
or turbinate, and assumed a brown to rufous colour.
“albino” stromata, off-white to pale yellow, were observed in
() and
in (), in the latter only in an immature state.
scurf was also present on mature stromata, the result of short hyphal hairs
protruding from the stroma surface (Figs
;
;
).
usually not visible macroscopically, or were barely visible as lighter areas
on the stroma surface, sometimes with darker margins.
observed in the compound microscope, was composed of small
pseudoparenchymatous cells.
the walls of these cells giving a mottled appearance to the rehydrated stroma
(Figs ,
).
have a pigmented cortical layer underlain by a region of loosely arranged
hyphae.
contained 8 uniseriate ascospores.
disarticulated early to form two halves, or part-ascospores.
part-ascospores were dimorphic, the distal part was subglobose to broadly
conical and the proximal part was ellipsoidal or oblong to narrowly
wedge-shaped. Ascospore sizes were clade-specific. G.J.S. 02-87 (Vd 1), a
teleomorphic member of the “Large Viridescens” clade from Sri
Lanka, had the smallest ascospores.
longer in the distal part than in all other species and the width of its
proximal and distal parts was greater than in all others. Ascospores of and are nearly identical in size.
North Carolina.
phylogenetically distinct from , we did not observe any aspect
of their teleomorph, anamorph or cultural phenotypes that would serve to
distinguish them from that species.
biogeographic bias. was originally described from New
Zealand and, in this work, it is restricted to New Zealand and Australia.
“Large Viride” and “Viridescens” clades are widely
distributed but are more common in North America and Europe.
tropical fungi. has been found in Peru at
high elevation.
tropical region, i.e. G.J.S. 92-15, from Brazil.
“Large Viride” clade, DIS 328g (Ecuador) and G.J.S. 04-40
(Brazil), originated in South America.
apparently represent two distinct species.
was isolated in a tropical region in Peru.
is far more common and possibly more widespread than
and are common
in Europe as anamorphs, but uncommon as teleomorphs if compared to common
species like .
originating in a geographic area (e.g.
but there was an equally strong tendency for clades to comprise strains of
mixed origin (e.g. Japan, United Kingdom and U.S.A.). includes strains from widely separated locations, viz.
Tyrrhenian island of Sardinia (Italy), U.S.A. (Texas), Russia and Australia.
The clade Vd 3 comprises two biogeographically distinct sister clades.
Isolates G.J.S. 00-67 and G.J.S. 97-243 are from eastern U.S.A.
G.J.S. 94-9 – G.J.S. 94-11 were collected in Taiwan.
The isolates G.J.S. 04-40 and DIS 328g were isolated as endophytes from
trunks of and respectively, and
was isolated as an endophyte from woody, above-ground
tissue of .
described taxonomic diversity in a large part of sect.
.
merit taxonomic recognizion, in the current work we emphasize the “Large
Viride” and “Large Viridescens” clades.
Viridescens” clade.
have smooth, ellipsoidal conidia. is a common
species in Europe and North America. is only
known from a single culture that was collected in Peru as a hyperparasite on a
destructive pathogen of .
have smooth, ellipsoidal conidia.
and Ghana that have ellipsoidal, warted conidia. One of these, G.J.S. 02-87
(Sri Lanka), produces a -like stroma but it has smaller
ascospores than either or .
observe an eidamia-like morphology in Vd 1 or Vd 2.
primarily on the basis of its faster rate of growth and on its larger
ascospores. It has a conspicuous eidamia-morphology when grown on CMD.
Clade Vd 3 is phenotypically and biogeographically diverse.
originally included all of these isolates within . As
was noted above, the North American isolates (G.J.S. 00-67, G.J.S. 97-243)
cannot be distinguished from , whereas the remaining
isolates, all from Taiwan, have a noticeably slower rate of growth than Their relationship to is indicated
by the dotted line in .
that is common in Europe.
geographically diverse, but the phenotypic diversity overlapped to such an
extent that we were not able to subdivide the species.
/ is characterised by north- and
south-temperate distribution, relatively slow growth, conidiophores that tend
to produce paired branches on SNA, subglobose to nearly ellipsoidal, warted
conidia, a coconut odour on PDA and CMD, and the conspicuous
eidamia-morphology found on PDA and CMD.
The most distant point of the “Large Viride” clade is .
trunk of an endemic daisy tree in the Galapagos Islands.
conidia on conidiophores that are atypical in .
the absence of conidial development, it is recognizable as a
by its strong odour of coconut and also by the production
of abundant chlamydospores that are typical of .
A single clade that is sister to /
includes Vb 1, Vb 2 and Vb 3.
Atlantic states of the U.S.A. and they cannot be distinguished from (with which they are sympatric) morphologically.
phylogenetic difference indicated by sequences of we cannot
observe any way to taxonomically separate them from /.
endophytes from trunks of, respectively, and in Ecuador and Brazil.
/, a difference that is especially marked on
SNA, and Vb 2 grows faster than any of the clades included in the present
study.
.
typical of .
relatives, Vb 2, Vb 3 or .
endophytic strains represent distinct species; their taxonomy will be
discussed in a future publication.
As was the case with .// is phylogenetically and phenotypically diverse but we did not find
any hiatus in the characters that would enable us to recognise more than a
single species.
consistent, rather slow rate of growth, strongly warted, globose to subglobose
conidia and this is consistent with the type specimen of
( and
).
Moreover, the conidiophores found in , with often solitary,
hooked phialides, are consistent with what Tulasne & Tulasne
() illustrated for their
concept of and .
selected as being typical of , given the overlap in
phenotype characters of the anamorph, but conidia in this group are not so
strongly warted and the tendency is for ellipsoidal conidia rather than
globose.
() was
included in our analysis. Thus we name this clade ,
with sp. nov. as its teleomorph.
.
phylogenetically and taxonomically complex sect.
(Dodd .
,
,
, , , , Samuels .
,
,
).
In the introduction to the current work we suggested that reports of refer to more than one species.
classical morphological concept of , i.e.
species with globose, warted conidia, is paraphyletic.
distinguish the two most common species of that have
warted conidia and describe a new species that has warted conidia, .
to include the peculiar morphology.
During the course of the study, additional species having warted conidia or
that were closely related to or
were revealed to us.
cultures on SNA, would be reported as a sterile unknown fungus.
present results from sequencing of only a single gene, the
resulting clades conform to phenotypic apomorphies in defining species.
Clearly, from it can be
seen that additional species remain to be characterized.
discussed in forthcoming publications.
concerns (), the question that we have had to answer
was how to delimit taxonomic species.
and submit them to DNA sequencing and phylogenetic analysis, we find within
clades considerable homoplasy in the few morphological features that are
available for analysis.
species, this morph is so highly conserved within the viride clade as to be
virtually useless in species-level taxonomy while at the same time being
diagnostic of the clade.
the viride clade, defined here as “Type 2”, signals membership in
the clade, it varies only little among the species.
cannot fault earlier mycologists for having recognized few species of
, or even later mycologists for having failed to recognize
the differences between and .
recognition of taxonomic species was to closely integrate phenetic and
phylogenetic characters.
two laboratories, and therefore were unbiased.
the concordance between both approaches were found.
understand that the phylogeny of one single locus, in this case 1,
is useful to formulate a hypothesis concerning the phylogeny of the fungus, we
also understand that use of this single gene region is not powerful enough to
falsify a species hypothesis.
phenotype and genotype.
high observed phylogenetic diversity enabled us to recognize a species (e.g.
), while in other cases, when the diversity was low and both
sets of characters were unclear, we refrained from proposing a new taxonomy
(Vd 1–3).
this character is derived within and that smooth conidia
are the primitive state.
evolution of most phenotypic characters in , and conidium
ornamentation is not an exception.
completely unrelated species of , including and (both sect.
Samuels 1994), and Bissett
() illustrated warted
conidia in (sect. ) using scanning
electron microscopy.
conidiophores produced on rich media is apomorphic for the large viridescens
clade defined here, and the coconut-like odour produced by several members of
the entire viride clade.
than for any other genus of fungi.
identifying species on the basis of their phenotype,
despite the fact that morphology-based keys to species, and illustrations of
at least the most common species is available at
.
However, every species is represented in GenBank by sequences of two or more
genes and by a multiplicity of correctly identified strains
().
known species of may be identified using molecular
markers at
.
The majority of species can be safely identified by the DNA barcoding based on
ITS1 and 2 loci ().
section share the same or very similar alleles of ITS1
and 2, they should be identified by an integration of the barcode method
(KEY) and similarity search TrichoBLAST
() as described in Druzhinina .
().
access to a sequencing facility, there is no need to misidentify a species of
.
identity of strains that are of interest and we urge editors of journals
reporting properties of species to require that the
identity of the strains be verified by members of the International
Subcommittee on Taxonomy of (ISTH) which can be accessed at
. |
Fr.
in terms of number of species and host range
(Kobayasi ,
, Mains
,
).
be more than 400 species (, , ) although this is expected to be an underestimation of the
extant global diversity ().
arthropods to the truffle-like genus , although most
species are restricted to a single host species or a set of closely related
host species (Kobayasi ,
, Mains
,
).
cosmopolitan, including all terrestrial regions except Antarctica, with the
height of known species diversity occurring in subtropical and tropical
regions, especially East and Southeast Asia (Kobayasi
,
,
).
The genus is generally included in the family , based
on its cylindrical asci, thickened ascus apices, and filiform ascospores that
often disarticulate into part-spores
(,
,
,
).
is characterized and distinguished from other genera of the
family by its production of superficial to completely immersed perithecia on
stipitate and often clavate to capitate stromata and its ecology as a pathogen
of arthropods and the fungal genus
(, Mains
,
,
,
).
primarily on the taxonomic studies of Kobayasi
(,
) and Mains
() (but see
).
(,
) recognized three
subgenera ( subg. . subg.
, and . subg. ),
emphasizing arrangement of perithecia and morphology of asci, ascospores and
part-spores. Species of . subg. (type ) were characterized by the production of either immersed or
superficial perithecia produced at approximately right angles (ordinal) to the
surface of the stroma and ascospores that disarticulate into part-spores at
maturity. subg. (Petch) Kobayasi
(type Petch) was distinguished by the production of whole
ascospores that do not disarticulate into part-spores and, in some species,
asci lacking pronounced apical hemispheric caps. subg.
Kobayasi (type (Klotzsch ex
Berk.) Berk. & M.A.
oblique angles in the clava region of the stroma and ascospores that
disarticulate into part-spores upon maturity.
infrageneric classification with a different emphasis on diagnostic characters
and recognized two additional subgenera, . subg.
(Ces.) Sacc. and . subg. Mains.
subg. (type
(Ces.) Sacc.) included species that produce superficial perithecia and asci
with hemispheric to short cylindrical caps. subg.
(type Berk. & M.A.
perithecia in a palisade-like layer at more or less right angles to the
surface of the stroma.
. subg. and . subg.
.
(), who essentially
adopted the diagnosis of Petch
() but at the rank of
subgenus, Mains () placed
only and Wakef. in . subg.
based on their lack of a thickened ascus apex, thus
deemphasizing the importance of ascospore disarticulation at the subgeneric
level.
() did not recognize
. subg.
oblique perithecia in subg. sect.
subsect. .
subgenera subg. . subg.
, and . subg.
Kobayasi () have been
arguably the most widely used infrageneric taxa of
(,
, , , ) with the relatively recent addition of . subg.
O.E. Erikss., which is characterized by the production
of bola-ascospores (Eriksson 198).
likened to the South American bola or the East Asian ninchuk (martial arts
weapon), the overall form is best likened to that of a skipping rope.
handles of the skipping rope are two terminal sets of four cells.
relic quantities.
affiliation has played an important role in the classification of
(, ). species that parasitize the truffle
genus have been recognized as a unique taxon.
Fr.
species () although it is a homonym of Pers. 1807.
Kobayasi (,
) also recognized the
mycogenous species as taxonomic units (e.g., .
subg. sect. subsect.
ser. ) and emphasized the utility of
host affiliations in delimiting closely related species of arthropod
pathogens.
questioned whether morphologically similar species on different insect hosts
(e.g., Moureau and attacking
ants and wasps, respectively) are conspecific.
taxonomic character is complicated, however, due to the difficulty in
identifying immature hosts (e.g., larvae and pupae) and insufficient host
identification for many herbarium collections.
and refine the classification of .
restricted by both limited taxon sampling and the inadequate resolution power
of ribosomal DNA, resulting in limited conclusions regarding systematics of
the genus.
(, ) based on multiple independent loci provided a greater level
of resolution and support, and revealed that neither nor
the family is monophyletic.
of the clavicipitaceous fungi were recognized, all of which include species of
.
classification () and
indicate that the phylogenetic diversity of is
representative of the entire family
(, ).
the is necessary to reflect the current hypotheses of
phylogenetic relationships and to be predictive in nature.
provide a basis for the phylogenetic classification of and
the clavicipitaceous fungi.
in the context of phylogeny, 2) investigate the taxonomic
utility of the anamorphic forms in classification of and
better understand the teleomorph–anamorph connections, and 3) revise the
classification of and to be
consistent with phylogenetic relationships.
families of with (Stoneman)
Spauld. & H. Schrenk () and Kleb. () included as outgroups
().
cultures or herbarium specimens were conducted using a FastDNA kit (Qbiogene)
following the manufacturer's instruction, with minor modifications.
chain and sequencing reactions were performed as previously described
().
DNA sequence data unique to this study were determined from five genes,
including the nuclear ribosomal small and large subunits ( and
), the elongation factor 1α (), and the
largest and second largest subunits of RNA polymerase II ( and
).
were obtained from Sung
().
to voucher numbers concerning the sequences is provided in
.
Sequences were edited using SeqEd 1.0.3 (Applied Biosystems Inc.) and
contigs were assembled using CodonCode Aligner 1.4 (CodonCode Inc.).
of each gene partition were initially aligned with Clustal W 1.64
() and appended to an existing alignment
().
This initial alignment was manually edited as necessary in MacClade 4.0
().
concatenated into a single, combined data set (162-taxon -gene data set) with
ambiguously aligned regions excluded from phylogenetic analyses.
from two additional gene regions, β-tubulin () and
mitochondrial ATP6 (), from Sung
() were also combined with
the 162-taxon 7-gene data set to generate a supermatrix of 162-taxon 7-gene
data set.
sampled in this study, bootstrap proportions were used for each individual
data set with the 107 taxa that was complete for all five genes
().
proportions (BP) were determined in a maximum-parsimony framework using the
program PAUP* 4.0b10 ().
following search options: 100 replicates of random sequence addition, TBR
branch swapping, and MulTrees OFF.
significant if two different relationships for the same set of taxa were both
supported with greater than 70 % bootstrap proportions by different genes
(, ).
in some clavicipitaceous species
(), and Sung
() also showed that while
possessed conflicting data for a limited number of taxa, the
conflict was localized and the locus simultaneously provided increased level
of support for other nodes.
analyses of the five aforementioned loci, we also conducted phylogenetic
supermatrix analyses with and (162-taxon 7-gene) to
detect any increased nodal support provided by those two loci.
the 162-taxon 7-gene data set (, ).
characters were equally weighted and unordered.
using only parsimony-informative characters with the following settings: 100
replicates of random sequence addition, TBR branch swapping, and MulTrees ON.
Phylogenetic confidence was assessed by nonparametric bootstrapping
(Felsenstein 198).
bootstrap proportions; bootstrapping used the same search options with five
replicates of random sequence addition per bootstrap replicate.
Maximum likelihood (ML) analyses were performed with RAxML-VI-HPC v2.2.
using a GTRCAT model of evolution with 25 rate categories
().
partitions, which consisted of and the nine codon
positions of three protein-coding genes (, and
).
using 200 replicates.
(B-MCMCMC) analyses were performed on combined datasets using MrBayes 3.0b4
().
general time-reversible model, with invariant sites and gamma distribution
(GTR+I+Γ) and employed the model separately for each partition.
initial analysis, a B-MCMCMC analysis with five million generations and four
chains was conducted in order to test the convergence of log-likelihood.
were sampled every 100 generations, for a total of 50,000 trees.
analysis, five independent Bayesian runs with two million generations and
random starting trees were conducted to reconfirm log-likelihood convergence
and mixing of chains.
analyses were conducted in MP, ML, and Bayesian frameworks with different
taxon samplings (107- and 152-taxon 5-gene data sets) to address the potential
topological effects of missing data.
studies demonstrated that the phylogenetic analyses are often not negatively
affected if less than 50 % characters are missing for each taxon in the
phylogenetic analyses (, Phylippe 2004).
that the phylogenetic analysis is not confounded if the taxa were complete for
at least three out of five gene partitions.
() in the 162-taxon
5-gene data set that were complete for only two gene partitions were excluded
to generate the 162-taxon 5-gene data set.
does not contain any missing data in gene partitions was also prepared to
compare the phylogenetic relationships between 107-taxon and 152-taxon 5-gene
analyses.
(Figs ,
) showed that the clade is characterized by long-branch lengths relative to
the rest of the clavicipitaceous fungi.
members of the clade from the 152-taxon 5-gene data
set and constructed a 147-taxon 5-gene data set.
sequence data ( 1102 bp, 954 bp,
1020 bp, 803 bp, 1048 bp).
excluding ambiguously aligned regions, the final alignment comprised 4600 base
pairs ( 1088 bp, 767 bp, 1020 bp,
677 bp, 1048 bp), 1882 of which were
parsimony-informative ( 233 bp, 220 bp,
466 bp, 382 bp, 581 bp).
107 taxa were complete for all five genes and the number of complete taxa for
each gene was as follows: 158 taxa, 157 taxa,
149 taxa, 143 taxa, 122 taxa
().
parsimonious trees.
(CI) of 0.1598 and a retention index (RI) of 0.6131.
parsimonious trees is shown in .
with asterisks.
tree with a log-likelihood (–ln) of 92019.95.
the five-million generation analysis converged on the log-likelihood (harmonic
mean = –ln 9951.22) at approximately around 250,000 generations.
results from five of two-million generation analyses also showed a convergence
on the log-likelihood at approximately 2 0,000 generations and the topologies
were identical.
a 50 % majority-rule consensus tree.
A 50 % majority consensus tree () was generated from the million generation analysis.
topology of ML analyses (tree not shown) was nearly identical to that of the
Bayesian consensus tree of , the bootstrap proportions of ML analyses are provided above the
corresponding nodes in .
Previous studies have shown that in interpreting the supports of the
phylogenetic estimates of relationships, the posterior probability tends to
overestimate the phylogenetic confidence
(,
,
).
to bootstrap proportions.
supported when supported by both bootstrap proportions (BP ≥ 70 %) and
posterior probabilities (PP ≥ 0.95)
().
set recognized three well-supported clades of clavicipitaceous fungi (Figs
,
), designated here as
clades A, B, and C (Figs
,
), following the convention of
the previous phylogenetic studies
(, ).
bootstrap proportions of the MP (MP-BP) and ML (ML-BP) analyses and posterior
probabilities (PP) of the Bayesian analyses (clade A: MP-BP = 98 %, ML-BP = 99
%, PP = 1.00; clade B: MP-BP = 93 %, ML-BP = 98 %, PP = 1.00; clade C: MP-BP =
100 %, ML-BP = 100 %, PP = 1.00). A sister-group relationship between clades A
and B was also strongly supported (MP-BP = 72 %, ML-BP = 90 %, PP = 1.00).
monophyletic group of clade C and was moderately to
strongly supported (MP-BP = 63 %, ML-BP = 92 %, PP = 1.00).
well-supported subclades (Figs
,
,
).
,
, and
as the clade
(MP-BP = 73 %, ML-BP = 78 %, PP = 1.00), the clade (MP-BP =
95 %, ML-BP = 98 %, PP = 1.00), the clade (MP-BP = 99 %,
ML-BP = 99 %, PP = 1.00), the clade (MP-BP = 100 %,
ML-BP = 100 %, PP = 1.00), and the clade
(MP-BP = 100 %, ML-BP = 100 %, PP = 1.00).
. (),
internal relationships among these five subclades were not strongly supported
in MP and ML analyses (Figs ,
,
).
designated as the , and clades (Figs
,
,
).
in clade B were strongly supported by bootstrap proportions and posterior
probabilities ( clade: MP-BP = 97 %, ML-BP = 100 %, PP =
1.00; clade: MP-BP = 71 %, ML-BP = 88 %, PP =
1.00; clade: MP-BP = 100 %, ML-BP = 100 %, PP =
1.00, clade: MP-BP = 64 %, ML-BP = 76 %, PP = 1.00).
resolved in the MP analyses ().
clade, which is characterized by long-branch lengths
relative to the rest of the clavicipitaceous fungi.
subclade, ranging from a basal lineage of the
clade B to a terminal clade nested within the subclade, were present among the most parsimonious trees
(data not shown).
() indicate that the
subclade is either a sister-group of the subclade (107-taxon 5-gene data set) or in the terminal
group of the subclade (152-taxon 5-gene data set).
(), the subclade was placed as a terminal group of the subclade with strong support (MP-BP = 89 %, ML-BP = 94 %, PP
= 1.00) as seen in the previous analyses
().
In the light of long-branch attraction problems associated with the MP
analyses (), we use the
Bayesian tree () to
further discuss the relationships in clade B and we conclude that the subclade was best included as a member of the subclade (Figs
,
).
bootstrap proportions and posterior probabilities (MP-BP = 88 %, ML-BP = 88 %,
PP = 1.00) based on the results of 147-taxon 5-gene data set
().
subclades (Figs ,
,
).
subclade (MP-BP = 100 %, ML-BP = 100 %, PP = 1.00) consisted of isolates of
the anamorph genus , most of which were isolated as
parasites of other fungi. The subclade (MP-BP = 98 %, ML-BP
= 100 %, PP = 1.00) included numerous species of and
species of that produce pallid to brightly coloured
stromata with ascospore morphologies ranging from whole ascospores to
part-spores to bola-ascospores according to species.
clade C included and represents
the core clade. The remaining species, H.C.
with strong support (ML-BP = 91 %, PP = 1.00) in ML and Bayesian analyses
(Figs ,
), but could not be
confidently assigned to either subclade in MP analyses
().
(, ) have revealed that species in the
form three strongly supported monophyletic groups based on combined data sets
of six or seven genes (the genes analyzed herein with and without
).
consistent with the previous studies, recognizing three monophyletic groups
designated as clades A–C (Figs
,
).
also support the paraphyly of the as defined by the
monophyly of clade C and (Figs
,
).
the (clade C + ) was moderately
supported (MP-BP = 63 %) in the 162-taxon 5-gene MP analyses
(), it was strongly
supported (ML-BP = 92 %, PP = 1.00) in the ML and Bayesian analyses
() and more robustly
addressed in the previous MP analyses, which investigated localized conflicts
among gene partitions and compared bootstrap proportions among alternative
sampling strategies ().
infrafamilial classification of the .
() proposed three
subfamilies, , and
, based on the development of stromata, anamorphic
characters and host affiliations.
coincide with the three clades of the inferred in the
present analyses (Figs ,
). Clavicipitaceae clade A
includes members of all three subfamilies (e.g., of
of , and
of ), whereas the remaining clades
only comprise members of (e.g.,
and ).
of indicating that , like
, is not monophyletic (Figs
,
).
recognized well-supported clades (clades A–C) of the clavicipitaceous
fungi represent a robust phylogenetic framework for the taxonomic revision of
and the .
comprises four subgenera ( subg. subg.
. subg. and . subg.
) based on ascospore morphology and arrangement of the
perithecia in the stromata (Kobayasi
,
,
).
of these characters are not consistent with the new phylogenetic hypothesis
and are not diagnostic of monophyletic taxa (e.g., subgenera and genera) (Figs
,
).
(,
) emphasized ascospore
morphology and the lack of ascospore disarticulation into part-spores to
delimit subg. from the other subgenera.
Species with non-disarticulating ascospores, however, are included in all
three major clades ( Ravenel of clade B, G.H.
sp.
non-disarticulating ascospores are not phylogenetically informative at this
level (Figs ,
).
of diagnostic characters, in the previous and current classifications of
is necessary for the three major clades to provide a basis
for taxonomic revisions of and the
.
().
in the clade are included in the clade.
Species of in the clade possess partially or completely
immersed perithecia on clavate to cylindrical fertile parts of the stromata
(,
,
).
They produce ascospores that either disarticulate or remain intact at maturity
and include species that possess ordinal and obliquely embedded perithecia.
that were formerly classified in three subgenera of M. Zang, D. Liu & R.
possess disarticulating ascospores, consistent with . subg.
(, ). H.C.
nondisarticulating ascospores, consistent with . subg.
() Z.Q. Liang & A.Y.
a known teleomorph species linked to the anamorph genus
Sorokin, produces disarticulating ascospores and obliquely embedded perithecia
in the stromata, a trait used to recognize . subg.
(). Importantly, sp.
2135 (described below as ) produce
non-disarticulating ascospores and obliquely embedded perithecia in the
stromata, characters inconsistent with any of the subgenera in the current
classification.
perithecia are not phylogenetically informative in recognizing either the
clade, or higher clades of clavicipitaceous fungi.
they are more useful at species level classification.
phylogenetic analyses revealed that is closely related to
Z. Y. Liu, Z.Q. Liang, Whalley, Y.J. Yao &
A.Y.
().
Although these species are similar to each other in macromorphology (e.g.,
greenish clavate stromata), they differ in the arrangement of the perithecia.
possesses perithecia that are ordinally placed
in the stromata, whereas has obliquely embedded perithecia.
These results therefore suggest that arrangement of the perithecia in the
stromata is useful in delimiting these closely related species in the clade ().
disarticulating or non-disarticulating ascospores and produce superficial to
completely immersed perithecia that are ordinally or obliquely inserted in the
stromata.
includes members of the former . subg. (e.g.,
(Ehrh.) Link and Petch),
. subg. (e.g., and
(Tul. & C. Tul.) Sacc.), and . subg.
(e.g., Pat. and ).
produce wiry to pliant or fibrous stromata that typically are completely or
partially darkly pigmented and parasitize subterranean or wood-inhabiting
hosts, which are buried in soil or embedded in decaying wood.
this morphology and ecology do exist, however; for example, (Tul. & C. Tul.) Sacc.
stains darkly upon handling, and members of the
clade parasitize adult insects.
Clade B consists of five subclades.
or anamorphs with potential links to
(e.g., (Yasuda) Samson linked to Petch) (,
).
well-resolved tree in the present study
() provides the basis to
characterize three of the five subclades of clade B.
sampling, it is not possible to characterize the members of the
species in the and subclades.
three subclades that include sufficient numbers of
species.
species that parasitize species of the genus
(e.g., and (Holmsk.) Link) and the nymphs of cicadas (e.g., Kobayasi and Kobayasi) buried in
soil (,
, Kobayasi &
Shimizu ,
).
subclade produce partially or completely immersed perithecia, in clavate to
capitate fertile parts of stromata that are darkly pigmented with olivaceous
tints (Kobayasi & Shimizu
,
).
disarticulating ascospores and ordinal perithecia, all known species of this
clade are classified in . subg. .
Petch is unique to the subclade ().
arising from a rhizomorph-like structure from scarabaeid beetle larvae
().
It is the only member of the subclade that parasitizes beetles embedded in
decaying wood ().
ecology and morphology of its stromata from most other taxa in the clade, but it possesses several characters shared by its
close relative,
(,
).
Both species grow axenically on simple media, produce verticilliate anamorphs
( has a anamorph, whereas has verticillium-like conidiophores), possess nearly
identical part-spore morphologies, and produce stromata that are connected to
their hosts via rhizomorph-like structures.
culture, they are attached directly to the host, and an anamorph is
unknown.
() also includes
parasites of subterranean cicada nymphs (e.g., and
), which are grouped with their close relatives (e.g.,
S.
parasitize subterranean truffles of .
of inter-species relationships within the subclade
due to short branch lengths, and are morphologically more similar to and than to any other members
of the clade.
than capitate stromata like other members of the clade (e.g., and Mains). Many of these species (e.g.,
and ) are also known to connect
to their hosts via rhizomorph-like structures (Kobayasi & Shimizu
,
), supporting a close
phylogenetic relationship.
diverse assemblages of species
().
in the clade parasitize larval, pupal or nymph stages of arthropods
(,
).
clade produce superficial to completely immersed perithecia on the stromata
with morphologies ranging from capitate to clavate to filiform
(,
).
possess tough, pliant, or fibrous stromata that are entirely or partially
darkly pigmented, although some exceptions (e.g., and
) do exist, which produce brightly pigmented stromata
().
the clade (e.g., Hywel-Jones, Berk. & Broome, and ) are also
differentiated by aperithecial stromatal apices while the production of
perithecia occurs in subterminal regions of the stroma.
non-disarticulating (intact) ascospores.
subclade (e.g., (Berk.) Sacc.
and ) were formerly classified in subg.
.
species (e.g., A. Kawam.
(Hook.) Berk.) that are classified in . subg.
This indicates that, while ascospore morphology is useful in delimiting
closely related species and uniting others in species
complexes, it is not diagnostic of the subclade
itself ().
Most members of . subg.
treated by Kobayasi (,
) and others (e.g.,
, ), form a monophyletic group labelled as the subclade within the group
().
species in the subclade produce long, thin, pliant,
brightly coloured (or dark marasmioid in a few species) stromata, which
terminate in clavate to elongated fertile parts, and possess ascospores that
disarticulate into sixty-four part-spores (Kobayasi
,
,
).
this clade produce perithecia, which are partially or completely immersed in
the stromata at strongly oblique angles (Kobayasi
,
,
,
).
is one of the best characterized by its morphology (obliquely embedded
perithecia in a well-defined clava) and its ecology of parasitizing adult
stages of insects.
species of the genus
().
species in this clade are currently classified in
. subg. (Kobayasi
,
).
contains the members of the former . subg.
and . subg. , resulting in . subg.
being paraphyletic within clade C
(,
,
).
Species of in this clade produce three ascospore types,
including disarticulating ascospores (e.g., ), intact
ascospores (e.g., and
Hywel-Jones & Sivichai), and bola-ascospores (e.g., O.E. Erikss.).
C.H. Su & H.-H.
unispecific genus C.H. Su & H.-H. Wang.
was originally described based on bola-ascospores and
its host affiliation as a pathogen of
Hayata () plant seeds (Su & Wang 198).
this species is most similar to in that it produces
bola-ascospores typical of . subg. .
the phylogenetic analyses in this study reveal that species producing
bola-ascospores (e.g., and )
do not form a monophyletic group ().
species possessing disarticulating ascospores, most notably .
immersed perithecia on fleshy stromata that are pallid to brightly pigmented.
This is in contrast to species in clade B, which produce
darkly pigmented, wiry to pliant or fibrous stromata.
pigmentation and texture of stromata may be phylogenetically informative at a
higher level of classification.
species in clade C are morphologically similar to distantly
related species (e.g., and ) in stromatal pigmentation.
useful in recognizing species of clade C, the utility of
these characters for any future infrageneric classification is probably
limited ().
is macroscopically similar to and
All three species produce orangish to
red-coloured and fleshy stromata; however, these species differ in ascospore
and anamorph morphology ().
exhibiting considerable variability in stroma morphology
().
Potentially conspecific species, such as Kobayasi
& Shimizu and A. Kawam., differ in stroma
morphology, but are closely related to and possess
identical ascospore and ascus morphologies
(,
,
).
species identification for many taxa, as is the case for much of
.
between the anamorphic species Z.Q.
China, cf.
from Taiwan
().
is Petch, which was originally
described as producing disarticulating ascospores and reddish orange stromata,
parasitizing lepidopteran cocoons (, , ).
Although the isolate of was obtained from ascospores
(), the morphology
of the ascospores was not well characterized.
primarily based on its host affiliation and macroscopic characters.
study, . cf. EFCC 5197 and N.H.J. 10627 were
collected from the same host family (Lepidoptera, Limacodidae) in Korea and
Thailand.
() and bola-ascospores
and not the typical subg. part-spores.
ascospores and it is possible that the terminal cells of bola-ascospores could
easily be interpreted as part-spores.
for the Chinese, Korean and Thai collections and, if
further attempts fail to locate type material for , one of
these may have to be designated a neotype.
collections are closely related to and morphologically indistinguishable from
with the exception of host affiliation, suggesting
the possibility of host misidentification in the original description of
.
phylogenetic species diversity, i.e., the Korean, Thai, and Taiwanese material
may represent unique phylogenetic species
(), we retain the use of
both names until more detailed sampling and analyses have been conducted.
but also species of the genus , which
generally parasitize spiders and scale insects
().
The genus is morphologically characterized by the
production of superficial perithecia on a mycelial subiculum that partially or
completely surrounds the host (, ).
disarticulating (e.g., Humber & Rombach) and
intact (e.g., ) ascospores.
(Lebert) Maire, a pathogen of adult
, has been considered an intermediate species between
and
(,
,
).
analyses in this study indicate that the members of do
not form a monophyletic group within clade C and are interspersed among
species of .
have been gained or lost several times during the evolution
of these fungi.
been described and the members of genus are clearly
undersampled in this study ().
perithecia used in the current classification of the genus
are not congruent with the three higher clades inferred in these analyses.
These characters are likely to prove useful, however, in lower level
classifications, such as the delimitation of closely related species and
species complexes.
of clavicipitaceous fungi are texture, pigmentation and morphology of the
stromata, but with exceptions.
species into three major clades, it is difficult to characterize
species within the clade A due to
the relatively few teleomorph species that are part of this clade (see key on
p. 54).
but additional sampling is needed to more definitively characterize the
teleomorphs of these species.
,
).
darkly pigmented, wiry, pliant or fibrous stromata.
parasitism exhibited by these species is on subterranean or wood-inhabiting
hosts, buried in soil or embedded in decaying wood, such as larval and pupal
stages of arthropods.
brightly pigmented and fleshy stromata and parasitize their hosts in
relatively more accessible environments, such as leaf litter, moss, or the
uppermost soil layer.
are found in some species in clade B.
it bruises dark upon handling and its hosts are the larvae of cockchafers or
June beetles buried in soil (). parasitizes adult ants, but
is darkly pigmented with a wiry stroma and subterminal production of
perithecia, and members of the clade are at least
partially brightly pigmented and are restricted to adult stages of insects.
These findings suggest that the traits described above are not universally
informative, but collectively useful in characterizing
species within clade B.
diversifications of most if not all traits during the evolution of these
fungi, but general trends in character state evolution are evident.
more than 25 anamorph genera (e.g., Vuill.,
Pat., Petch, Fr.,
W.
W. Gams) (, , , ).
The anamorph genera of are hyphomycetes with conidiogenous
cells that are hyaline to brightly coloured and produce conidia in dry chains
or slimy drops (). Some anamorph genera (e.g., ) are
known as a useful diagnostic character in recognizing monophyletic groups of
species
(, Kobayasi
,
), while other anamorph
morphologies and genera are placed in more than one clade of the
.
discussed to evaluate their phylogenetic utility in characterizing the three
clades of and and to better
understand teleomorph–anamorph connections.
xref
#text
This key is designed to emphasize the most conspicuous field-, host-, and
macroscopic characters available to the user for
Kobayasi and Mains.
and is not a key to the species.
and adult stages of Arthropoda, the key begins with these
characters so as to expeditiously highlight or remove these taxa from
consideration.
descriptions of arthropod-pathogenic fungi.
the fungal specimen whenever possible, but this often proves problematic.
vast majority of arthropod-pathogenic fungi occur on immature stages (e.g.,
larvae, pupae) of arthropods.
particular specimen or collection, we suggest the user to begin with couplet
.
stromata are particularly phylogenetically informative, thus we place special
emphasis on these characters where possible but emphasize that, as with most
fungal taxa, exceptions are to be expected.
and morphology that may not be
intuitive:
consists almost entirely of pallid to
brightly coloured species that produce soft fleshy stromata (e.g., ).
Lepidoptera and Coleoptera in leaf litter, moss or upper soil layers.
species that produce highly reduced stromata, loosely organized hyphae, or a
subiculum on the host also occur in this genus (e.g. ),
some of which were previously classified in (e.g., ).
and closely related species that attack nymphs of
cicadas.
pathogens are remarkably similar and attest to the recent history of
inter-kingdom host-jumps in a common subterranean environment
().
The exception to this genus is which macroscopically
and ecologically is distinct from the rest of the species, but is well
supported as being a member of the genus based on molecular data and
micromorphology.
species, of which all but one are only known from East Asia.
colour of fresh specimens ranges from white to lilac, purple or green, and the
darker pigments are almost black in dried specimens.
stromata is fibrous and not fleshy like , and the
hosts are almost always buried in soil.
fungi.
buried in soil or in decaying wood.
traits among species that attack adult stages of hosts, however.
is common on adult ants and occurs on the under sides
of leaves, and is common on adult wasps and is found
in leaf litter.
wiry to clavate and fibrous, according to species, and many species produce
their perithecia in nonterminal regions of the stroma, either distinctly
superficial, or in broad irregular patches, or in lateral pads. |
In 2006 we successfully continued with the policy of publishing three
issues per year, but in accordance to the policy of the
chose to make its
papers freely available, though hard copies will still be sold via its online
.
high-impact journal focusing on monographs and revisions and, under special
circumstances, introducing specific topical issues.
strives to publish monographs and books formerly published in the
series (CABI), or the
series of the .
founded taxonomic novelties is criterion for acceptance.
in-house editorial treatment, each issue is reviewed by two external
referees.
seamlessly linked to regular online mycological journals such as
and , as well as , the , and online herbaria, to
name but a few.
.
, which would further assist us in our goal to freely
distribute published mycological literature, and to help promote mycology
internationally.
Since the genus W. Gams, Crous & M.J.
Wingfield was described in 1996, the genus has been conclusively linked to
phaeohyphomycosis of humans, as well as Petri disease and brown wood streaking
of grapevines, a disease complex that is the topic of biennial meetings by the
was shown to comprise anamorphs of the genus
(); it was monographed by Mostert . (), who treated
10 and 22 species.
several new species of and were
introduced, along with a .
mating strategy of several species was investigated, showing
several taxa to be homothallic, while others had a biallelic heterothallic
mating system.
, while the and the
clustered in the .
incentive for a special celebratory volume of , focusing on some
current fungal research activities underway in southern Africa.
it also led to the digitalization of “Doidge 1950” ,
which made all these old fungal records available online.
for papers treating the history of the National Collection of Fungi
(), and
another one speculating about the number of fungal species that exist at the
tip of Africa ().
trees were treated
(,
Crous . ,
,
,
,
, ).
(),
also received attention, along with soil-inhabiting genera such as
(), and
(). Furthermore, Zhou .
() and Zipfel () treated the
genus , and reinstated as distinct from
.
were treated by Crous .
(,
), showing that one species of
with two was associated
with angular leaf spot of bean, but several species of
were associated with grey leaf spot of maize.
A special issue of was dedicated to Joan M.
occasion of her 90 birthday.
status as mycologist for her excellent monographic work dealing with the
of New Zealand.
papers focusing on and their
teleomorphs (, Overton .
,
,
).
Overton .
(,
) dealt with some
conspicuous, mainly fungicolous species which have
inconspicuous anamorphs. Samuels .
() (
clade) and ( clade) dealt with some of the commonest,
but very complex species, in which the anamorph outweighs
the teleomorph in ecological success and differentiation.
some important biocontrol agents. |
Fresenius () described
Fresen.
sp.
() examined this species
and and stated that it only occurred on
dead stems of spp.
this species in detail, and reallocated it to the genus
Link.
A second, cladosporioid hyphomycete on spp.,
Pass., was collected by Passerini on living
leaves of (as ) in Italy, and
distributed in Thümen, Herbarium mycologicum oeconomicum, Fasc. IX, No.
416, together with the first valid description, which was repeated by
Passerini ().
Passerini collected this fungus on at Parma in
Italy and distributed it in Thümen, Mycotheca universalis, No. 670
.
a collection of this species on from Russia, Siberia,
which he later described as var.
Sacc. ().
accomplished by Meuli (),
followed by a treatment by de Vries
().
() described and
illustrated in their treatment of
macroconidiophores, agreeing with those of the original diagnosis and
illustration of , as well as
semi-macronematous conidiophores concurring with those of , although no mention was made of the latter name.
out comprehensive studies on on spp.
and , including detailed discussions of the
history of the taxa concerned, taxonomic implications and comprehensive
descriptions and illustrations.
a semi-macronematous form (synanamorph) of the latter species, and reduced
to synonymy with the latter species.
characters, conidiogenesis, and DNA sequence data of the ITS and 28S nrDNA
were used to confirm the identity of (the
periconioid morph) and (the cladosporioid morph), and
clarify their relation to
() (
,
).
stems, and cultured as detailed in Crous
().
characteristics and morphology of isolates
() were recorded from
plates containing either 2 % potato-dextrose agar (PDA) or synthetic
nutrient-poor agar (SNA) ().
continuous near-UV light to promote sporulation.
isolated following the protocol of Lee & Taylor
().
transcribed spacer (ITS1), the 5.8S rRNA gene, the second ITS region and the
5' end of the 28S rRNA gene (LSU).
quality sequences over the entire length of the amplicon.
sequence alignment and subsequent phylogenetic analysis followed the methods
of Crous .
().
gaps were treated as new character states.
GenBank () and
alignments in TreeBASE
().
symptomatic leaves and stems, as well as cultures sporulating on SNA.
Structures were mounted in water or Shear's solution
(),
and 30 measurements at × 1 000 magnification were made of each structure
under an Olympus BX 50 microscope (Hamburg, Germany).
levels were determined and the extremes of spore measurements given in
parentheses.
Institute of Zoology, Martin-Luther-University, Halle (Saale), Germany, using
a Hitachi S-2400.
sputter coater SCD 004 (200 s in an argon atmosphere of 20 mA, 30 mm distant
from the electrode).
().
were deposited in the culture collection of the Centraalbureau voor
Schimmelcultures (CBS), Utrecht, the Netherlands
().
were lodged with MycoBank
().
isolates listed in .
ITS region of the sequences was used to obtain additional sequences from
GenBank which were added to the alignment.
contained 26 sequences (including the two outgroup sequences) and 518
characters including alignment gaps.
phylogenetic analysis, 226 were parsimony-informative, 33 were variable and
parsimony-uninformative, and 259 were constant.
using three substitution models on the sequence data yielded trees supporting
the same clades but with a different arrangement at the deeper nodes.
nodes were supported poorly in the bootstrap analyses (the highest value
observed for one of these nodes was 64 %; data not shown).
Two equally most parsimonious trees (TL = 585 steps; CI = 0.761; RI =
0.902; RC = 0.686), one of which is shown in
, was obtained from the
parsimony analysis of the ITS region. The K.
Schub., U.
Crous & U.
Syd. & P. Syd.
by analyses of the first part of the 28S rRNA gene (see
-
this volume).
chains, and
coincided with previous concepts of
(,
), belonging to a
wide assemblage of genera classified by Kiffer & Morelet
() as
“”.
a result confirmed here by DNA sequence analyses.
conidiogenesis, structure of the conidiogenous loci and conidial hila, and a
comparison with , typified by (Pers. : Fr.) Link, revealed obvious differences: The
conidiogenous loci and conidial hila of are quite
distinct from those of by being denticulate or
subdenticulate, apically broadly truncate, unthickened or slightly thickened,
but somewhat darkened-refractive.
are, however, characteristically coronate, i.e., with a central convex dome
surrounded by a raised periclinal rim
(,
,
).
peony fungus has to be excluded from A
comparison with phaeoblastic hyphomycetous genera revealed a close similarity
of this fungus with the genus Crous, Schroers,
J.Z. Groenew., U. Braun & K. Schub.
Cladosporium speckle disease of banana
().
Both fungi have dimorphic fruiting, pigmented macronematous conidiophores
often with distinct basal swellings and densely branched terminal heads
composed of short branchlets and ramoconidia, denticulate or subdenticulate
unthickened, but somewhat darkened-refractive conidiogenous loci, as well as
phaeoblastic conidia, formed in simple or branched acropetal chains.
semi-macronematous leaf-blotching morph is close to and barely distinguishable
from Bonord.
, the peony fungus does not form rhizoid hyphae
at the base of conidiophore swellings and the conidia are amero- to
phragmosporous [0-5-septate 0(-1)-septate in
].
within the (with ) nor
within the (with ), but clusters
basal to the (see also
-
this volume).
genus .
K. Schub., U.
.
,
,
and
.
: in Greek = twofold.
basalibus saepe inflatis, sed sine hyphis rhizoidibus, conidiis amero- ad
phragmosporis (0-5-septatis).
(Fresen.) K.
Schub., U. Braun & Crous, comb. nov.
(Fresen.) K. Schub., U.
Braun & Crous, MycoBank
.
,
,
and
.
: Fresen., Beiträge
zur Mykologie 1: 21. 1850.
: Mason & Ellis
(: 123-126), McKemy &
Morgan-Jones (: 140-144),
Schubert (: 216).
: Fresenius
(: Pl.
(: 137,
; 141,
; 139, pl. 1; 143, pl.
2), Schubert (: 217, fig.
113; 275, pl. 34).
: on living leaves amphigenous, variable in shape and size,
subcircularoval to irregular, broad, oblong to expanded, up to 30 mm long and
20 mm wide, at times covering the entire leaf surface, forming
olivaceous-brown to blackish brown patches, rarely violet-brown, margin
usually indefinite, attacked areas turning dry with age, also occurring on
young, green stems. amphigenous, punctiform to effuse, loose
to dense, caespitose, brown, villose. immersed, subcuticular
to intraepidermal; hyphae sparingly branched, 4-7(-10) μm wide, septate,
sometimes with swellings and constrictions, swollen cells up to 13 μm diam,
subhyaline to pale brown, smooth, walls thickened, hyphae sometimes
aggregated; mycelium at first mainly immersed, later also
superficial, branched, 1-5(-7) μm wide, pluriseptate, often constricted at
septa and with swellings and constrictions, therefore irregular in outline,
smooth to verruculose or irregularly rough-walled, loosely verruculose with
distinct large warts. formed on
leaf-blotches solitary or in small, loose groups, arising from internal hyphae
or swollen hyphal cells, erumpent through the cuticle, occasionally emerging
through stomata, erect, straight to somewhat flexuous, oblong-cylindrical,
usually unbranched or occasionally branched, 13-80(-120) × (4-)5-8(-10)
μm, slightly attenuated towards the apex, septate, septa often dense,
unconstricted, pale to medium brown, sometimes paler towards the apex, smooth,
thick-walled, wall often with two distinct layers, often somewhat inflated at
the very base, up to 14 μm diam, occasionally proliferating
enteroblastically; conidiophores arising laterally from
plagiotropous hyphae or terminally from ascending hyphae, the latter usually
appearing more filiform than those arising laterally from plagiotropous
hyphae, erect, straight to slightly flexuous, cylindrical-oblong, not
geniculate, usually unbranched, rarely with a short lateral prolongation near
the apex, 18-60(-100) × 3-6 μm, slightly attenuated towards the apex,
septate, pale to medium brown or olivaceous-brown, smooth to asperulate, walls
somewhat thickened. integrated, terminal or
intercalary, subcylindrical, 7-45 μm long, proliferation sympodial, with
one to several conidiogenous loci, subdenticulate or denticulate, protuberant,
terminally broadly truncate, 1.5-3 μm wide, unthickened or almost so,
somewhat darkened-refractive. catenate, in simple or branched
chains, polymorphous, small conidia globose, subglobose, broadly obovoid, 3-9
× 3-5 μm, aseptate, pale to medium brown, smooth, intercalary conidia
limoniform, ellipsoid-fusiform, oblong, 5-23 × 3.5-6.5 μm,
0-2-septate, medium brown, smooth to minutely verruculose or irregularly
rough-walled, large conidia ellipsoid, oblong-cylindrical, ampulliform,
22-45(-52) × (4.5-)5-8 μm, 0-5-septate, medium brown, smooth to
minutely verruculose or irregularly rough-walled, walls somewhat thickened,
hila truncate, 1-3 μm wide, unthickened or almost so, somewhat
darkened-refractive; occasionally with microcyclic conidiogenesis; numerous, polymorphous, catenate, in loosely branched chains, small
conidia globose, subglobose, or obovoid, 3-8 × 3-4 μm, aseptate,
intercalary conidia limoniform to ellipsoid-fusiform, 9-18 × 3.5-4.5
μm, 0-1-septate, large conidia ellipsoid to cylindrical-oblong, 14-30(-38)
× 3-6(-7) μm, 0-3-septate, pale to medium brown, asperulate, minutely
verruculose to irregularly rough-walled, walls thickened, hila usually short
denticle-like, protuberant, truncate, in smaller conidia 0.5-1.8 μm wide,
in larger conidia (1.5-)2-3 μm wide, unthickened or almost so but usually
darkened-refractive; with occasional microcyclic conidiogenesis.
: formed on faded or dead stems in late autumn, winter or
after overwintering; colonies at first visible as reddish brown streaks, later
turning olivaceous-brown to black, sometimes linear, sometimes encircling the
stems, often occupying large stem segments, effuse, densely caespitose,
velvety. immersed, subcuticular to intraepidermal; hyphae at
first sparsely branched, 3-7 μm wide, septate, not constricted at the
septa, becoming swollen and wider, up to 11 μm wide, often branched, pale
to medium olivaceous-brown, walls thickened, forming loose to dense hyphal
aggregations; mycelium immersed to superficial, loosely
branched, 2-6(-7) μm wide, pluriseptate, usually without swellings and
constrictions, subhyaline to medium brown or olivaceous-brown, almost smooth
to asperulate or irregularly rough-walled, in older colonies on PDA up to 10
μm wide, sometimes single hyphal cells distinctly swollen, up to 16(-20)
μm wide, mainly at the base of conidiophores, sometimes covered by a slime
coat or enveloped in a polysaccharide-like layer.
well-developed, large and expanded, up to about 50-320 μm in length, 15-30
μm deep, composed of a single to several layers of swollen pale to medium
brown stromatic cells, 5-18 μm diam, thick-walled.
solitary or in loose groups, arising from swollen hyphal cells or stromata,
erumpent through the cuticle, erect, straight, rigid to slightly flexuous,
150-680 μm long, composed of a subcylindrical stipe, 13-24 μm wide at
the base, slightly attenuated towards the apex, 5-15 μm just below the
branched head, pluriseptate, not constricted at the septa, young conidiophores
pale medium olivaceous-brown, later medium to usually dark brown, sometimes
slightly paler at the distal end, smooth or almost so, often appearing
somewhat granular, roughened, walls distinctly thickened, 1.5-3(-4) μm
wide; apex with a roughly subglobose to ovoid head, about 35-70 μm diam,
composed of dense branchlets and ramoconidia, primary branchlets close to the
apex and below the first and sometimes second and third septa, solitary, in
pairs or small verticils, appressed against the stipe or somewhat divergent,
subcylindrical to ellipsoid-oval, aseptate, rarely 1-septate, pale olivaceous
to dark brown, 10-20 × 5-8.5 μm; conidiophores
initially micro- and semimacronematous, then progressively macronematous as
colonies age, arising laterally from plagiotropous hyphae or terminally from
ascending hyphae, sometimes also from swollen hyphal cells; micronematous
conidiophores filiform, narrowly cylindrical-oblong, unbranched, up to 150
μm long, 2-3.5 μm wide, septate, septa often appear to be darkened, pale
to pale medium olivaceous-brown, asperulate, walls slightly thickened;
semimacronematous conidiophores often resembling those formed by the
leaf-blotching (cladosporioid) morph on the natural host, subcylindrical to
cylindrical-oblong, straight to slightly flexuous, unbranched, rarely
branched, (10-)15-120 × 3-5(-6) μm, slightly attenuated towards the
apex, septate, medium brown, minutely verruculose to irregularly rough-walled,
walls more or less thickened; macronematous conidiophores formed in older
cultures on SNA, PDA and also MEA (according to
), but more prominent on PDA and MEA, resembling those formed
by the stem-rotting morph (i.e., the periconioid morph, in planta), consisting
of a long unbranched stipe and a subglobose head, but in culture the heads are
often more loosely branched than on the natural substratum, not always forming
a compact head, up to 580 μm long, 5-13 μm wide, attenuated towards the
apex, 4-8 μm just below the branched upper part, somewhat swollen at the
base, septate, medium to very dark brown, minutely verruculose, walls
distinctly thickened, two distinct wall layers visible, 1-2 μm thick.
holoblastic, integrated, terminal, intercalary or
even discrete, ellipsoid to cylindrical or doliiform, subdenticulate,
proliferation sympodial, multilocal, conidiogenous loci truncate, flat,
unthickened, 1-3 μm wide, somewhat darkened-refractive; in culture
conidiogenous loci appearing to be somewhat thickened and distinctly
darkened-refractive, 1-2.5(-3) μm wide. catenate, in long,
branched chains, straight, subglobose, aseptate, 3.5-7 μm diam, or
ellipsoid-ovoid, 6-15 × 4-9 μm, 0(-1)-septate, pale olivaceous to
olivaceous-brown, smooth to verruculose (under the light microscope), hila
flat, truncate, unthickened, (0.5-)1-2(-2.5) μm wide, not darkened, but
somewhat refractive; conidia numerous, catenate, formed in
long, branched chains, small conidia globose to subglobose, (2-)3-7 ×
(2-)3-4 μm, aseptate, intercalary ones ellipsoid-ovoid, 6-16 × 3.5-5
μm, 0(-1)-septate, ellipsoid to
cylindrical-oblong, (13-)15-34(-47) × (3-)4-6(-7) μm, 0-2-septate,
sometimes slightly constricted at the septa, medium olivaceous-brown,
verruculose or irregularly rough-walled, walls slightly to distinctly
thickened, hila more or less protuberant, subdenticulate to denticulate, in
small and intercalary conidia 0.5-1(-1.5) μm, in secondary ramoconidia
1-2.5(-3) μm, unthickened or somewhat thickened, darkened-refractive;
occasional microcyclic conidiogenesis.
: Colonies on PDA at first whitish or
smoke grey, reverse smoke-grey to olivaceous-grey, with age smoke-grey to
olivaceous or olivaceous-grey, sometimes even dark mouse-grey, reverse
iron-grey to dark mouse-grey or black, felty; margin white to smoke-grey,
narrow to more or less broad, regular to slightly undulate, glabrous to
somewhat feathery; aerial mycelium at first mainly in the colony centre, with
age abundantly formed, covering almost the whole colony, whitish, smoke-grey
to olivaceous, felty; growth low convex to raised; numerous small exudates
formed, sometimes becoming prominent; fertile.
: , Bohemia, Turnau, on
leaves of , 15 Sep. 1905, J.E.
Kabát & Bubák, Fungi Imperf. Exs. 396, B 70-6669.
, on dead stems of sp., 1901, ex Herbario Musei
Parisiensis, ex herb. Magnus, exs. Desmazières, Pl. Crypt. N.
Ed. 2, Ser. 1, 1621, HBG, as “”;
Chailly-en-Biere, Seine-et-Marne, Feuilleaubois, on stems of , 27 Mar. 1881, Roumeguère, Fungi Sel. Gall. Exs. 1803,
HBG, as “”. ,
Baden-Würtemberg, Kreis Tübingen, Drusslingen, on leaves of , Jun. 1935, Raabe, B 70-6670; Bayern, Freising, on leaves of
, Sep. 1918, Prof. Dr. J.E.
pathologicum, B 70-6663; Brandenburg, Schloßpark zu Tamsel, on leaves of
, 15 Aug. 1924, P. Vogel, Sydow, Mycoth. Germ. 2447,
M-57751, PH; Triglitz, on leaves of , 3 Oct. 1909,
Jaap, B 70-6668; Hessen, Frankfurt am Main, botanical garden, on leaves of
, 7 Oct. 2004, R.
Kassel, Hofgeismar, Garten von Prof. Grupe, on leaves of , 3 Sep. 1947, Schulz, B 70-6658; Mecklenburg-Vorpommern,
Rostock, neuer botanischer Garten, on leaves of (=
), 27 Aug. 1950, Becker, B 70-6662; Nordrhein-Westfalen,
Duisburg, Dinslake, private garden, on leaves of , 9 Aug.
2005, N. Ale-Agha, HAL 2014 F; Hamborn, botanical garden, on leaves of , 10 Aug. 2005, N.
of the university of Essen, on leaves of , 10 Aug.
2005, N. Ale-Agha, HAL 2013 F; on leaves of and , 11 Aug. 2005, N.
Königstein, in Gärten, verbreitet, on leaves of , Aug. 1896, W. Krieger, Krieger, Fungi Saxon. Exs. 1545,
M-57749; Aug., Sep. 1896, 1915, W.
of , 22 Jun. 2004, K.
deposited at the CBS, = CPC 11383; on leaves of , 22 Jun.
2004, K. Schubert, HAL 2012 F; on stems of , 16 Mar.
2005, K.
, culture
ex-neotype = CPC 11969; on dead stems of sp., Jan.
1873, G. Winter, Rabenhorst, Fungi Eur. Exs. 1661, HBG, as
“”; Thüringen,
Fürstlicher Park zu Sondershausen, on leaves of , 20
Aug. 1903, G. Oertel, Sydow, Mycoth. Germ. 196, PH.
Herb. Mycol. Oecon. 416, on living leaves of [=
] (M-57753), ; : Thümen,
Herb. Mycol. Oecon. 416; Padova, on leaves of , Aug.
1902, P.A. Saccardo, Saccardo, Mycoth. Ital. 1186, B 70-6660, SIENA; Parma, on
leaves of , Jul. 1876, Prof.
Mycoth. Univ. 670, B 70-6654, 70-6655, M-57752; Pavia, botanical garden, on
leaves of , summer 1889, Briosi & Cavara, Fung.
Paras. Piante Colt. Utili Ess. 78, M-57748; F.
Fungi Sel. Gall. Exs. 5193, mixed infection with , B 70-6656; Siena, Hort. Bot., on leaves of
sp., Nov. 1899, SIENA. , prov.
garden, on leaves of [= ], 28 Aug.
1936, J. Smarods, Fungi Lat. Exs. 799, M-57747.
from red leaf and stem lesions on sp., M.
.
, Râmnicu-Vâlcea, distr.
leaves of , 17 Aug. 1930, Tr. Săvulescu & C.
Sandu, Săvulescu, Herb. Mycol. Roman. 298, M-57742.
sp., isol. F.
= IMI
048108a. , on leaves of sp., Sep. 1878, Ellis,
N. Amer.
sp., 8 Aug. 1882, F.S. Earle, No. 91, B 70-6657; Kansas,
Topeka, on leaves of , 7 Jul. 1922, C.F. Menninger, US
Dept. Agric., Pathol. Mycol. Coll. 60085, B 70-6661, F; Montana, Columbia, on
leaves of , Aug. 1886, B.T. Galloway, Ellis &
Everh., N. Amer. Fungi Ser. II, 1991, PH; on leaves of sp.,
18 Oct. 1931, W.E. Maneval, F.
: On var. spp. (), Asia
(Armenia, China, Georgia, Kazakhstan, Russia), Europe (Belgium,
Czechoslovakia, Denmark, France, Germany, Italy, Latvia, Moldova, Poland,
Romania, Switzerland, U.K., Ukraine), North America (Canada, U.S.A.), New
Zealand.
: Type material of is not
preserved in the herbarium of G.
Senkenberg, Frankfurt a. M., Germany).
Botanical Garden of the Martin-Luther-University Halle (Saale), Germany, is
proposed to serve as neotype.
deposited at the CBS, Utrecht, the Netherlands as ex-neotype culture.
of the synanamorph, .
used to generate DNA sequence data.
(), and molecular sequence
analyses documented herein clearly demonstrate that , occurring on necrotic stems, and ,
causing leaf-blotch symptoms on living leaves of spp., are
two synanamorphs of a single species, which has to be excluded from
since the conidiogenous loci are quite distinct
from the characteristically coronate scars in the latter genus and because ITS
sequences indicate clear separation from .
stage of this fungus (, the periconioid morph)
closely resembles , recently introduced for the
Cladosporium speckle disease of banana. There are, however, some differences.
In (E.W.
type species, micronematous conidiophores occur and , and macronematous conidiophores occur on leaf-spots, whereas in
the semi-macronematous conidiophores usually
accompany leaf-blotch symptoms on living leaves and the macronematous
conidiophores occur in saprobic growth on old necrotic stems.
arising from the swollen basal cells of the macronematous conidiophores are
characteristic for , but lacking in , and the conidia in the latter species are 0-5-septate,
but only 0(-1)-septate in .
leaf-blotching stage (the cladosporioid morph) is barely distinguishable from
the present concept of , which includes species with
catenate conidia ().
.
, the clade to which
belongs, the differences observed here seem to be sufficient to place this
fungus in a new genus (also see - this volume).
() discussed differences
between and allied dematiaceous hyphomycete
genera and provided a key to the latter genus and morphologically similar
genera.
lead to .
Differences between morphologically similar genera have been discussed in the
paper by Crous
() and are also valid for
the new genus U.
& K. Schub., a fungicolous genus recently introduced to accommodate
Deighton, is also morphologically similar in
having apically, densely branched conidiophores and truncate, unthickened
conidiogenous loci and hila, but is quite distinct in not having micronematous
conidiophores (). |
Species of Borelli are relatively simple
hyphomycetes with brown hyphae that give rise to branched chains of pale brown
conidia.
(, ), an order containing numerous opportunists
(); teleomorph relationships are with Sacc.
in the .
synanamorphs are found accompanying black yeasts of the genus
J.W. Carmich. ().
() placed several saprobic
hyphomycetes in , and described U. Braun & Feiler as teleomorph of U. Braun & Feiler.
(), who
described an additional teleomorph, Dugan, R.G.
Roberts & Hanlin for (Matsush.) U.
Braun & Feiler.
() reduced to synonymy with , and placed them in
Sacc. ().
, which is based on Matsush. () is confused, however, and phylogenetic studies have revealed
that isolates attributed to this name in recent studies, were in fact
representatives of three different species in phylogenetically distinct genera
().
The separation of with teleomorphs
(; commonly isolated as human
pathogens), from predominantly saprobic or phytopathogenic isolates in the
was recognised by Braun
().
endophyte de Hoog was reported to be the
nearest neighbour of (Trejos) de Hoog ., a
major agent of human chromoblastomycosis
(), so that the main distinction between the two anamorph
genera remains in their phylogenetic positions.
and were again recognised as distinct species, and
placed in a new genus, U.
(), while their anamorphs were accommodated in
U. Braun. was primarily
distinguished from based on its distinct
anamorphs. Recently, Crous .
() introduced a third
genus, namely Crous & Seifert, which produces a
sympodiella-like anamorph in culture.
. ()
concluded, based on an ITS DNA phylogeny, that the morphology attributed to
the form genera Fr., E. Bald. &
Cif., and Bonord.
, and that a single anamorph genus should be used for
, namely (see
for additional generic synonyms).
In their treatment of anamorphs, Schubert .
() excluded
, and stated that its status needs to be confirmed
along with that of other genera such as B.
and Riess.
. () an
isolate of (
sp.) was included to confirm the link to the , though
this was not well resolved, nor was the status of the older generic names
mentioned above addressed.
DNA sequence comparisons in conjunction with morphology in an attempt to
clarify these generic issues, as well as to determine which morphological
characters could be used to distinguish from
.
moist chambers to promote sporulation.
extract plates (MEA; ), by obtaining single conidial colonies as explained in Crous
().
subcultured onto fresh MEA, oatmeal agar (OA), potato-dextrose agar (PDA) and
synthetic nutrient-poor agar (SNA) (), and incubated at 25 °C under continuous
near-ultraviolet light to promote sporulation.
isolated following the CTAB-based protocol described in Gams .
().
transcribed spacer (ITS1), the 5.8S rRNA gene, the second ITS region and the
5' end of the 28S rRNA gene (LSU).
sequences were obtained.
phylogenetic analysis followed the methods of Crous .
().
5.8S rRNA gene were only sequenced for isolates of which these data were not
available.
GenBank where applicable.
events for the phylogenetic analyses; the remaining gaps were treated as
missing data.
() and alignments in
TreeBASE
().
measurements given in parentheses.
assessed after 2-4 wk on OA and PDA at 25 °C in the dark, using the colour
charts of Rayner ().
cultures obtained in this study are maintained in the CBS collection
().
novelties and descriptions were deposited in MycoBank
().
listed in .
sequences were used to obtain additional sequences from GenBank which were
added to the alignment.
including alignment gaps (available in TreeBASE).
in the phylogenetic analysis, 326 were parsimony-informative, 79 were variable
and parsimony-uninformative, and 425 were constant.
using three substitution models on the sequence data yielded trees with
identical topologies to one another.
same clades as obtained from the parsimony analysis, but with a different
arrangement at the deep nodes, for example, the clade containing
(Sacc.) M.E.
() is
placed as sister to the using parsimony but basal to the
using neighbour-joining.
number of different strain associations in the clade (see
the small number of strict consensus branches for this clade in
), only the first 5 000
equally most parsimonious trees (TL = 1 752 steps; CI = 0.392; RI = 0.849; RC
= 0.333) were saved, one of which is shown in
.
general time-reversible (GTR) substitution model with inverse gamma rates and
dirichlet base frequencies. The Markov Chain Monte Carlo (MCMC) analysis of 4
chains started from a random tree topology and lasted 2 000 000 generations.
Trees were saved each 1 000 generations, resulting in 2 000 saved trees.
Burn-in was set at 500 000 generations after which the likelihood values were
stationary, leaving 1 500 trees from which the consensus tree
() and posterior
probabilities (PP's) were calculated.
frequencies was 0.06683 at the end of the run.
that observed using parsimony was obtained, with the exception of the position
of R.F. Castañeda & W.B. Kendr.,
which is placed between the and the
based on the Bayesian analysis.
results obtained using neighbour-joining, the clade containing
() is
placed as sister to the and not to the
.
are discussed below.
and , and these are
described below.
but that clustered elsewhere, are treated under excluded species.
Crous & A.D.
MycoBank
.
.
: Named after its country of origin, Australia.
: consisting of branched, septate,
smooth, pale brown, guttulate, 2-3 μm wide hyphae; hyphal coils not seen.
dimorphic; macroconidiophores mononematous,
subcylindrical, multi-septate, straight to curved, up to 150 μm long
(including conidiogenous cells), and 4 μm wide, pale to medium brown,
smooth, guttulate; microconidiophores integrated with hyphae, which terminate
in subcylindrical conidiogenous cells that give rise to branched chains of
conidia; conidiophores (including conidiogenous cells) up to 5-septate, 50
μm long, with terminal and lateral conidiogenous cells. pale to medium brown, smooth, guttulate, terminal and lateral,
subcylindrical, 20-35 × 2-3.5 μm, or reduced to indistinct
subtruncate to truncate loci, scars up to 2 μm wide, mono- to polyblastic,
proliferating sympodially, scars neither darkened, thickened, nor refractive.
pale to medium brown, guttulate, smooth; ramoconidia
subcylindrical, 0-1-septate, 20-35 × 2-3 μm, hila subtruncate,
inconspicuous, up to 2 μm wide, giving rise to branched chains of conidia;
conidia ellipsoid, pale brown, but becoming dark brown and thick-walled in
older cultures, guttulate, tapering towards subtruncate terminal loci,
0-1-septate, occurring in chains of up to 20 conidia, (7-)8-12(-15) ×
3-4 μm (older, dark brown conidia are ellipsoid, up to 5 μm wide).
: Colonies erumpent, somewhat spreading,
margins crenate, feathery, aerial mycelium sparse; colonies on PDA
olivaceous-grey to iron-grey (surface); reverse iron-grey; on OA and SNA
olivaceous-grey.
dark; colonies fertile. Not able to grow at 37 °C.
: , isolated from apple juice,
Dec. 1986, A.D.
,
culture ex-type = CPC 1377.
: is one of two novel
species of originally isolated from sports drinks in
Australia. spp.
disorders (, , ), and thus their occurrence in sports drinks is cause for
concern.
grow at 37 °C, and therefore it is not expected that they could pose a
danger to humans. Comparing ITS diversity, the species shows more than 12 %
difference to established pathogens such as and (Sacc.) de Hoog
(Grove) Crous & Arzanlou,
MycoBank
.
.
: Grove, J. Bot.
Lond. 24: 199. 1886.
: consisting of branched, septate,
smooth, medium brown hyphae, 2-3.5 μm wide. reduced
to conidiogenous cells, or a single supporting cell, 20-40 × 3-4 μm.
subcylindrical, erect, straight to irregularly
curved, medium brown, smooth, 15-30 × 3-4 μm. in
branched, acropetal chains with up to 30 conidia; subcylindrical to fusiform,
medium brown, smooth, tapering slightly at subtruncate ends, 1(-3)-septate,
thin-walled, becoming slightly constricted at septa of older conidia,
(20-)25-30(-45) × 3-4(-5) μm; conidia remaining attached in long
chains; hila neither thickened, nor darkened-refractive.
: Colonies erumpent, convex, spreading,
with sparse to dense aerial mycelium; margins smooth, undulate; on PDA
iron-grey (surface), margins olivaceous-black; reverse olivaceous-black; on OA
olivaceous-grey in the middle due to fluffy aerial mycelium, iron-grey in wide
outer margin; on SNA olivaceous-grey.
on PDA at 25 °C in the dark. Not able to grow at 37 °C.
: , Yunnan, Yiliang, isolated from
), decaying bamboo, freshwater, 6
Jul. 2003, L.
wood, 15 Jun. 2003, C. Lei, . , isolated from roots of ), isol. by D.S.
.
, Schleswig-Holstein, Kiel-Kitzeberg, isolated from wheat field
soil, isol. by W. Gams, = ATCC 16274 = MUCL 8310.
: Two cultures of were
originally deposited as L. Cai, McKenzie &
K.D.
().
Petr.
spotting fungi with chains of brown, disarticulating conidia
(),
which have phylogenetic affinities to several orders, obviously being
polyphyletic. The type species of Petr.,
is a plant pathogen on
() with hitherto
unknown phylogenetic position.
to the , was rather unexpected.
be similar to others placed in by having short,
lateral conidiogenous cells, and long chains of branched subcylindrical
conidia that largely remain attached.
other members of in having medium brown conidia, and
in lacking the ellipsoid conidia observed in several species.
Crous, U. Braun & H.D.
MycoBank
.
-.
: Epithet derived from the host genus,
.
: amphigenous, subcircular to somewhat
angular-irregular, 1-5 mm wide, scattered to aggregated, sometimes confluent,
pale to medium brown or with a reddish brown tinge, later greyish brown,
margin indefinite or on the upper leaf surface with a narrow slightly raised
marginal line or very narrow lighter halo, yellowish, ochraceous to brownish.
epiphyllous, punctiform to confluent, dingy greyish brown.
immersed, forming fusicladium-like hyphal strands or plates;
hyphae septate, sometimes with constrictions at the septa, thin-walled, pale
olivaceous, 1.5-7 μm wide. immersed, small, 10-40 μm
diam, composed of swollen hyphal cells, subcircular to somewhat
angular-irregular in outline, 2-8 μm diam, wall somewhat thickened, brown.
in small to moderately large fascicles, loose,
divergent to moderately dense, rarely solitary, arising from stromatic hyphal
aggregations, erumpent, erect, usually unbranched, rarely branched, straight,
subcylindrical to distinctly geniculate-sinuous, 5-40 × 2-5 μm,
0-6-septate, pale to medium olivaceous to olivaceous-brown, thin-walled, up to
0.5 μm, smooth. integrated, terminal,
5-15(-20) μm long, sympodial, conidiogenous loci rather inconspicuous to
subdenticulate, flat-tipped, 1-1.5 μm diam, unthickened or almost so, not
to slightly darkened-refractive. in simple or branched
chains, narrowly ellipsoid-subcylindrical, 10-15 × 1.5-3.5 μm,
0-1-septate, subhyaline to pale olivaceous, thin-walled, smooth, ends truncate
or with two denticle-like hila in ramoconidia, (0.75-)1-1.5(-2) μm diam,
unthickened or almost so, at most slightly darkened-refractive.
: composed of branched, smooth, pale
olivaceous to medium brown hyphae, frequently forming hyphal coils, guttulate,
septa inconspicuous, not constricted, hyphae somewhat irregular in width, 1-2
μm wide. reduced to conidiogenous cells, integrated
in hyphae, terminal, subcylindrical, pale olivaceous to pale brown, smooth,
0-1-septate, proliferating sympodially at apex via 1-2(-3) flat-tipped,
minute, denticle-like loci, 1-1.5 μm wide, 10-15 × 1.5-2 μm; scars
minutely darkened and thickened, but not refractive. in
extremely long chains (-60), simple or branched, subcylindrical, or narrowly
ellipsoid, smooth, pale olivaceous, 0-1-septate, (7-)10-15(-20) ×
(1.5-)2(-2.5) μm, hila truncate, 1-1.5 μm wide, minutely thickened and
darkened-refractive.
: Colonies on PDA erumpent, spreading,
with smooth, undulate margins and dense aerial mycelium; surface hazel
(middle), outer zone isabelline; reverse fuscous-black in middle, isabelline
in outer zone. Colonies reaching 25 mm diam on SNA, and 40 mm diam on PDA
after 1 mo at 25 °C in the dark; colonies fertile.
: , Pyongchang, (), 20 Sep. 2003, H.D. Shin, HAL 2030 F,
, culture ex-type SMK 19664, CPC 10737 =
, CPC
10738-10739.
: Although this species is morphologically similar to
(Deighton) Crous, U. Braun & K. Schub.
described below in this paper, is treated as a separate
taxon due to the differences in the length and width of its conidiophores and
conidia , as well as 17 bp differences in the ITS DNA
sequence data and a distinct ecology causing leaf-spots on a different,
unrelated host.
is a very unusual, unexpected member of the genus
, the mycelium forms obvious hyphal strands
and plates which are characteristic for species.
conidiophores and conidia are also fusicladium-like.
species clusters within the , i.e., it has to be
placed in the genus .
differentiation between and
(incl. ) almost impossible without sequence data.
Furthermore, the morphology of and shows remarkable differences in conidiophore morphology, i.e., the
growth is characteristically fusicladium-like (conidiophores
macronematous, long, septate), whereas habit is rather
pseudocladosporium-like (conidiophores less developed, usually reduced to
conidiogenous cells, short).
have also been observed to exibit a growth habit
in culture, suggesting this growth plasticity to be rather common, and
strongly influenced by growth conditions.
Crous & U.
.
-.
: Named after its ecology, namely occurring in soil.
: composed of branched, smooth, pale
olivaceous to pale brown hyphae, frequently forming hyphal coils, prominently
guttulate, not to slightly constricted at the septa, 1-2 μm wide, cells
somewhat uneven in width. solitary, mostly
inconspicuous and integrated in hyphae, varying from inconspicuously truncate
lateral loci on hyphal cells, 1-1.5 μm wide, to occasionally terminal
conidiophores, 0-3-septate, subcylindrical, proliferating sympodially, 10-30
× 1.5-3 μm, pale brown, smooth.
loci 1-1.5 μm wide, or conidiogenous cells subcylindrical with 1-3
sympodial loci (which appear as minute lateral denticles), 7-17 × 1.5-2
μm; scars inconspicuous, neither darkened, refractive nor thickened.
in short chains of up to 10, simple or branched,
subcylindrical to narrowly ellipsoid, 0-1-septate, (8-)11-14(-17) ×
(1.5-)2(-2.5) μm, pale olivaceous to olivaceous-brown or pale brown,
smooth, hila truncate, 1-1.5 μm wide, unthickened, neither darkened, nor
refractive.
: Colonies erumpent, spreading, with
uneven, feathery margins and dense aerial mycelium on PDA; pale
olivaceous-grey in the middle, becoming olivaceous-grey in the outer zone
(surface); reverse olivaceous-black, with grey-olivaceous margins.
reaching 7 mm diam after 2 wk at 25 °C in the dark; colonies fertile.
: , Brandenburg, Müncheberg,
from soil, Zaspel, Zalf & H.
,
culture ex-type BBA 65570 = .
: Phylogenetically is
closely related to Crous & de Hoog (see below).
Morphologically the two species can be distinguished in that lacks ramoconidia, and has 1-septate conidia, while those of
are 0-3-septate.
Crous & A.D.
MycoBank
.
-.
: Refers to its presence in fruit juices and sports
drinks.
: consisting of branched, septate,
smooth, pale brown, guttulate, 1.5-2.5 μm wide hyphae.
solitary, macronematous, well distinguishable under the
dissecting microscope from aerial mycelium, pale to medium brown,
subcylindrical, straight to somewhat curved, erect, with apical apparatus
appearing as a tuft due to extremely long conidial chains; conidiophores up to
5-septate, and 100 μm tall (excluding conidiogenous cells).
pale brown, smooth, terminal and lateral,
subcylindrical, tapering towards subtruncate to truncate loci, 1 μm wide,
somewhat darkened, thickened, but not refractive, loci appearing
subdenticulate on lateral conidiogenous cells, mono- to polyblastic,
proliferating sympodially, 10-35 × 1.5-2 μm. pale
brown, smooth, guttulate, occurring in branched chains of up to 60; hila
somewhat darkened and thickened, but not refractive, 0.5 μm wide;
ramoconidia subcylindrical, 0-1-septate, 15-17(-20) × 2.5-3 μm;
conidia ellipsoid, (6-)8-10(-13) × 2-3 μm.
: Colonies erumpent, spreading, with
smooth margins and dense aerial mycelium on PDA, olivaceous-grey (surface),
with a thin, olivaceous-black margin; reverse olivaceous-black; on OA
olivaceous-grey (surface) with a wide olivaceous-black margin.
reaching 25-30 mm diam after 1 mo at 25 °C in the dark; colonies fertile,
also sporulating in the agar. Not able to grow at 37 °C.
: , isolated from apple juice
drink, Dec. 1986, A.D.
,
culture ex-type = CPC 11048; , isolated from sports drink,
Feb. 1996, A.D.
sports drink, Feb. 1996, A.D.
= FRR
4946.
: Originally this taxon, isolated from fruit and sports
drinks, was thought to be an undescribed species of
(= , see below).
closer examination, this proved not to be the case.
distinct tufts under the dissecting microscope, and are readily
distinguishable from the superficial mycelium, as is normally observed in
species of , but the conidial chains are extremely long,
and the conidia tend to be more ellipsoid than the predominantly fusiform or
subcylindrical conidia observed in species of Hyphal
coils were also not observed in cultures of , but are
rather common in species of .
this taxon within the clade also supports
inclusion in the genus .
Viljoen & Crous, S. African J.
Bot. 64: 137. 1998. .
: consisting of branched, septate
hyphae, often forming strands, anastomosing, smooth to finely verruculose,
frequently constricted at septa, olivaceous, 3-4 μm wide; hyphal cells in
older cultures becoming swollen, up to 6 μm wide.
reduced to conidiogenous cells. holoblastic,
integrated, forming short, truncate protuberances, 2-3 × 1.5-2 μm,
concolorous with mycelium, subcylindrical. arranged
in long acropetal chains (up to 20), simple or branched, subcylindrical to
oblong-doliiform, (9-)13-17(-22) × 2.5-3(-4) μm on
MEA, (9-)16-22(-25) × (2.5-)3-4(-6) μm on SNA; 0-1(-4)-septate, pale
brown to pale olivaceous, smooth, hila subtruncate to truncate, not thickened,
but somewhat refractive.
: Colonies erumpent, with sparse aerial
mycelium on PDA; margins irregular, feathery; greyish rose, with patches of
pale olivaceous-grey (surface); reverse olivaceous-grey. Colonies reaching 10
mm diam after 2 wk at 25 °C in the dark; colonies fertile.
: , Western Cape Province,
Stellenbosch, J.S.
(), 26 Aug. 1996, L. Viljoen, PREM 55345,
culture ex-type .
: differs from species of
(= ) based on its colony
colour, the slimy nature of colonies, as well as its conidia that have
inconspicuous, unthickened hila () (), unlike those observed in species of .
Sequence data show that this species is not allied to the
, but to the .
(Deighton) Crous, U. Braun &
K. Schub., MycoBank
.
.
: Deighton, N. Zealand J.
Bot. 8: 55. 1970.
: see Schubert & Braun
() and Schubert . ().
: consisting of branched, septate,
smooth, green-brown to medium brown, guttulate hyphae, variable in width,
1.5-3 μm diam. lateral or terminal on hyphae, erect,
straight to slightly flexuous, solitary, in some cases aggregated,
subcylindrical, curved to geniculate-sinuous, unbranched, up to 55 μm long,
2-3 μm wide, 0-7-septate, septa in short succession, pale to medium brown,
somewhat paler towards apices, smooth.
integrated, terminal or lateral as individual loci on hyphal cells, straight
to curved, subcylindrical, up to 14(-18) μm long and 2 μm wide, pale to
medium brown, smooth, with a single or few subdenticulate to denticulate loci
at the apex due to sympodial proliferation, or reduced to individual loci,
0.8-1.5(-2) μm wide; scars minutely thickened and darkened, but not
refractive. occurring in long, unbranched or loosely branched
chains (-30), straight to slightly curved, ellipsoid to mostly narrowly
subcylindrical, obclavate in some larger, septate conidia, (5-)10-20(-35)
× 1.5-3 μm, 0-1(-3)-septate, sometimes slightly constricted at the
septa, subhyaline to pale brown, smooth, guttulate, tapering at ends to
subtruncate hila, 0.8-1.5 μm wide, minutely thickened and darkened, but not
refractive; microcyclic conidiogenesis occurring.
: Colonies erumpent, spreading, with
smooth, even margins and dense, abundant aerial mycelium on PDA;
grey-olivaceous (surface); reverse dark olivaceous.
olivaceous-grey to iron-grey, velvety, aerial mycelium sparse, diffuse.
Colonies reaching 20 mm diam on SNA, and 40 mm on PDA after 1 mo at 25 °C
in the dark; colonies fertile.
: , Levin, on (), 21 Dec. 1965, G.F.
of , 25 Apr. 2004, C.F.
.
: In culture forms a
pseudocladosporium-like state, though the scars are somewhat darkened and
thickened, but not refractive.
cells that are integrated in the mycelium, terminal or lateral, frequently
also as an inconspicuous lateral denticle, with a flat-tipped scar.
occur in long, branched chains, which are subcylindrical to narrowly
ellipsoid, and are up to 35 μm long, 1.5-3 μm wide, thus longer and
thinner than reported on the host, which were 0-3-septate, subcylindrical to
ellipsoid-ovoid, 7-22 × 2.5-4 μm.
this species , Schubert & Braun
() reallocated it to
.
characteristics, was almost identical to an
isolate obtained from leaf spots of in Korea.
isolates appeared to resemble species of , but
phylogenetically they clustered in the .
Therefore, “” was placed in
the genus .
are first reports of phytopathogenic species within the
genus .
.
.
: Refers to its host, .
brown hyphae, frequently forming hyphal coils, not to slightly constricted at
the septa, 1-2 μm wide. medium brown,
subcylindrical, flexuous, mononematous, multiseptate, up to 50 μm long, and
2-3 μm wide. apical, sympodial, pale brown,
5-12 × 2-3 μm; scars somewhat darkened and thickened, not refractive.
occurring in branched chains; ramoconidia up to 2 μm wide,
giving rise apically to disarticulating chains of conidia; smooth,
0-3-septate, pale olivaceous, subcylindrical, (7-)10-16(-20) × 1.5-2
μm, with truncate ends; hila somewhat darkened and thickened, not
refractive.
: Colonies erumpent on PDA, with smooth,
catenulate margins; iron-grey (surface); reverse greenish black.
reaching 15 mm diam after 1 mo at 25 °C in the dark; colonies fertile.
: , Kootwijk, needle litter of
(), 8 Nov. 1982, G.S.
, culture ex-type
.
: Morphologically
was
originally identified as Sacc., but the latter
is reported to have conidia that are 5-5.5 × 1 μm
(), which is much
smaller than that observed for the present isolate.
species of Riess
(), does
not cluster within the , thus suggesting that
is best
treated as a new species of
Crous, de Hoog & H.D.
MycoBank
.
.
: Named after its host genus, .
branched, smooth, 3-5 μm wide hyphae, constricted at septa.
phialidic, intercalary, appearing denticulate, 1
μm tall, 1.5-2 μm wide, with minute collarettes (at times proliferating
percurrently). sickle-shaped, smooth, medium brown,
guttulate, (1-)3(-5)-septate, constricted at septa, widest in middle, or lower
third of the conidium; apex subacutely rounded, base subtruncate, or having a
slight constriction, giving rise to a foot cell, 1 μm long, 0.5-1 μm
wide, subacutely rounded, (15-)25-35(-55) × (2.5-)3(-4) μm; a
marginal frill is visible above the foot cell, suggesting this foot cell may
be the onset of basal germination; conidia also anastomose and undergo
microcyclic conidiation in culture.
: Colonies slow-growing, slimy, aerial
mycelium absent, margins smooth, catenate; surface crumpled, olivaceous-black
to iron-grey.
on PDA, 12 mm on SNA; colonies fertile.
: , Yangpyeong, on leaves of
(), 4 Jun. 2003, H.D.
, SMK 19550, culture ex-type
.
: is related to the type
species of the genus, G.A.
also resides in the The genus
G.A.
B.C.
B.C.
melanized hyaline thalli.
due to unavailability of sequence data. Decock .
() synonymised the hyaline
genus M. Jacob & D.J.
() with
, but as no cultures of this fungus are available this
decision seems premature.
by Decock
() have been found to be
involved in cutaneous infections in humans.
species originally described as being environmental,
Walz & de Hoog, which is closely related to (Ajello
.) Decock and (C.K. Campbell & B.C.
Sutton) Decock known from proven human and animal infections. Decock . () added the
melanized species Decock & Delgado, isolated as a
saprobe from tropical leaf litter. is the
first species of the genus infecting a living plant host.
remote from those of the remaining species, the nearest
neighbour being G.A.
19.1 % distance (data not shown). can be
distinguished based on its conidial dimensions and septation.
larger than those of (11-20 × 2-2.5 μm,
1-2-septate), and those of (11-25 × 2-5 μm,
1-3-septate) (for a key to the species see
).
genus to accommodate species with brown, mononematous
conidiophores bearing apically aggregated, flat-tipped, subdenticulate
conidiogenous loci that give rise to chains of pale brown subcylindrical
conidia with thickened, darkened hila. He compared the type species, B.
, but did not compare it to ,
to which it is remarkably similar.
() introduced the genus
based on R.F.
& W.B. Kendr.
formation of subdenticulate conidiogenous loci distributed along the apical
region of the conidiophore, and by the relatively poorly defined appearance of
these loci.
, but we studied strains of
R.F.
(,
ex-type), and Crous & W.B. Kendr.
(,
ex-epitype), and found them to cluster adjacent to
().
distantly from all other species, confirming that the genus name
is not available for any of the taxa treated here.
with indistinct marginal frills, and these are obviously different from those
of anungitea- and fusicladium-like anamorphs, including and
.
belong to a sister clade of the
(, incl. ) clade.
(), which produces a sympodiella-like anamorph in
culture, is the only teleomorph of this clade hitherto known.
venturia-like habit of , connected with fusicladium- /
pseudocladosporium-like anamorphs distributed in both clades, indicates a
close relation between these clades, suggesting a placement in the
. Schubert .
() referred to the
difficulty to distinguish between and is undoubtedly heterogeneous.
P.M.
instance, intermediate between (conidiophores with a
terminal denticulate conidiogenous cell, but conidia disarticulating in an
arthroconidium-like manner) and B. Kendr. (conidiophores
distinctly sympodial, forming arthroconidia).
possess a distinctly swollen, lobed conidiophore base, e.g.
P.M.
genera, e.g., P.M.
(),
Crous & U.
-
this volume), and Varghese & V.G.
(,
).
depends, however, on the affinity of ,
the type species, of which sequence data are not yet available.
solution for this problem is the widened application of
(incl. ) to both sister clades, i.e., to the whole
.
anamorphs of both clades is impossible.
“fusicladium-like” growth is mainly characteristic for the
fruiting , above all in biotrophic taxa, whereas the more
“pseudocladosporium-like” habit is typical for the growth and in saprobic taxa, a phenomenon which is also evident in species
of the morphologically similar genus (see and ).
would render the genus a synonym of ,
but in the case of a quite distinct phylogenetic position a new
circumscription of this genus, excluding the anamorphs,
would be necessary.
be postponed, awaiting cultures and sequence analyses of its type species.
leaf litter of sp.
(), is
somewhat problematic.
within itself, and it does not fit into the current
morphological concept of (incl.
).
conidia and pale brown conidiogenous structures, it resembles species
accommodated in W.B. Kendr. & R.F.
Castañeda (,
).
Crous & R.F.
MycoBank
.
.
: Named after the host genus it was collected from,
.
1.5-2.5 μm wide. macronematous, mononematous,
solitary, erect, subcylindrical, straight to geniculate-sinuous, medium brown,
smooth, 35-70 × 2.5-4 μm, 1-5-septate.
terminal, integrated, pale to medium brown, smooth, 10-35 × 2-3 μm,
proliferating sympodially, with one to several flat-tipped loci, 1.5-2 μm
wide; scars somewhat darkened, minutely thickened, but not refractive.
solitary, subacicular to narrowly subcylindrical, apex
subobtuse, base truncate, or somewhat swollen, straight or curved, smooth,
subhyaline to very pale olivaceous, guttulate, (45-)60-70(-80) ×
2.5-3(-3.5) μm, (4-)6-8-septate; scars are somewhat darkened, minutely
thickened, but not refractive, 2.5-3 μm wide.
: Colonies erumpent, convex, with smooth,
lobed margins, and moderate, dense aerial mycelium on PDA; mouse-grey in the
central part, and dark mouse-grey in the outer zone (surface); reverse dark
mouse-grey. Colonies reaching 5 mm diam after 2 wk at 25 °C in the dark;
colonies fertile.
: , Canary Islands, leaf litter of
sp. (), 4 Jan. 1995, R.F.
, culture ex-type
.
: The present fungus differs from (de Hoog) W.B. Kendr. & R.F.
() in that the
conidiophores are much longer, the conidia are subhyaline to very pale
olivaceous, and the scars and hila are thin, slightly darkened, but not
refractive.
Sacc., Syll. fung. (Abellini) 1: 586. 1882.
For additional synonyms see Sivanesan, : 604. 1984.
: Bonord., : 80. 1851.
For additional synonyms, see Schubert .
().
: The genus , based on (U. Braun & Feiler) U.
saprobic, soil-borne venturia-like ascomycetes with numerous ascomatal setae,
and an anamorph quite distinct from
().
is based on (Berk. & Broome)
Petr., which clusters in the , adjacent to
, which has anamorphs.
was retained by Barr
() as separate from
based on its superficial ascomata with a thin, stromatic
layer beneath the ascomata.
separation of and from
is debatable, and the names (Berk.
& Broome) Ces. & de Not. and (U.
& Feiler) Unter. are available for these organisms.
, which is based on
(Schwein.) Höhn., clusters in the , as was to be
expected based on its anamorph
().
having ascospores strictly septate near the lower end
().
. ().
Morphological as well as molecular studies
()
demonstrated that the genus with its
anamorphs is monophyletic.
uniform subclades based on the previous anamorph genera and was not evident and could be rejected.
As in cercosporoid anamorphs of , features such as the
arrangement of the conidiophores (solitary, fasciculate, sporodochial), the
proliferation of conidiogenous cells (sympodial, percurrent) and shape, size
as well as formation of conidia (solitary, catenate) proved to be of little
taxonomic value at generic level. Hence, Schubert .
() proposed to maintain
as sole anamorph genus for .
genus Partridge & Morgan-Jones (type species:
Thüm.)
(), recently erected to accommodate fusicladium-like species
with catenate conidia, represents a further synonym of
.
are usually micronematous, conidia often appear to be
directly formed on the mycelium, unilocal, determinate, mostly reduced to
conidiogenous cells, sometimes forming a few percurrent proliferations,
whereas the conidiophores of species of are
mostly macronematous, but sometimes also micronematous.
initiated as short lateral, peg-like outgrowths of hyphae which proliferate
sympodially, becoming slightly geniculate, forming a single, several or
numerous subdenticulate to denticulate, truncate, unthickened or only slightly
thickened, somewhat darkened-refractive conidiogenous loci.
from by being saprobic and connected with a different
teleomorph, viz.
().
the type species of , with its anamorph
(U. Braun & Feiler) U.
clusters together with numerous species, the genus
should be reduced to synonymy with
.
and .
pseudocladosporium-like habit, characterised by forming solitary
conidiophores, often reduced to conidiogenous cells or even micronematous, and
conidia formed in long chains, is mainly found in culture, above all in
saprobic taxa.
conidiophores is usually more evident , above all in
biotrophic taxa.
two genera.
.
.
: Named after the continent from which it was collected,
Africa.
1.5-2 μm wide hyphae, frequently forming hyphal coils.
reduced to conidiogenous cells, solitary, pale to
medium brown, smooth, inconspicuous, integrated in hyphae, varying from small,
truncate lateral loci on hyphal cells, 1-1.5 μm wide, to micronematous
conidiogenous cells, 5-10 × 2-3 μm; mono- to polyblastic, sympodial,
scars inconspicuous, 1 μm wide. in long, branched chains
of up to 40, subcylindrical, 0(-1)-septate, pale brown, smooth; hila truncate,
1 μm wide, unthickened, neither darkened nor refractive; ramoconidia
(11-)15-17(-20) × 2-3(-3.5) μm; conidia (8-)11-17 × 2-2.5
μm.
: Colonies somewhat erumpent, with
moderate aerial mycelium and smooth, lobate margins on PDA, ochreous to umber
(surface); reverse dark umber; on OA umber; on SNA ochreous.
9 mm diam on PDA after 2 wk at 25 °C in the dark; colonies fertile.
: , Western Cape Province,
Malmesbury, leaf litter, Jan. 2006, P.W.
, cultures ex-type CPC 12828 =
, CPC
12829 = .
: is a somewhat atypical
member of the genus, as its conidial hila are quite unthickened and
inconspicuous.
wider morphological variation was found pertaining to the structure of the
conidiogenous loci and conidial hila, ranging from being indistinct,
unthickened and not darkened-refractive to unthickened or almost so, but
somewhat darkened-refractive (). was found
occurring with Crous & Seifert on
leaf litter in South Africa
().
(R.F. Castañeda & Dugan)
Crous, K. Schub. & U.
.
.
: R.F. Castañeda &
Dugan, Mycotaxon 72: 118. 1999.
: , Santiago de Cuba, La Gran Piedra,
fallen leaves of sp. (), 2 Nov. 1994,
R.F.
-)
, culture ex-type
= ATCC
200947 = IMI 367525 = INIFAT C94/155 = MUCL 39143.
: In culture has a typical
pseudocladosporium-like morphology, though the scars are neither prominently
thickened, nor refractive.
Sacc., Ann. Mycol. 11: 20.
1913.
Česká Mycol. 25: 171. 1971.
.
: Schubert
(: 37).
: unbranched or only sparingly
branched, 2-3 μm wide, septate, not constricted at septa, subhyaline to
pale brown, smooth, walls unthickened or almost so.
laterally arising from hyphae, erect, straight to somewhat flexuous, sometimes
geniculate, unbranched, (6-)12-75 × (2.5-)3-4.5 μm, aseptate or
septate, pale brown or pale medium brown, smooth, walls somewhat thickened,
sometimes only as short lateral conical prolongations of hyphae, occasionally
irregular in shape. integrated, terminal or
conidiophores reduced to conidiogenous cells, sometimes geniculate, 6-29 μm
long, proliferation sympodial, with several denticle-like loci, broadly
truncate, 1.5-2(-2.5) μm wide, unthickened, somewhat refractive or
darkened. occurring, 20-28 × 5 μm, 0-1-septate,
somewhat darker, pale medium brown, with a broadly truncate base, 3-4 μm
wide, usually with several denticle-like apical loci.
catenate, formed in unbranched or loosely branched chains, straight to
sometimes curved, cells sometimes irregularly swollen, fusiform,
subcylindrical, sometimes obpyriform, 13-35 × 3.5-5.5(-6) μm,
0-3-septate, occasionally slightly constricted at the median septum, few very
large conidia with up to five septa, up to 75 μm long, 4.5-6 μm wide,
subhyaline to pale brown, smooth, walls slightly thickened, slightly
attenuated towards apex and base, hila broadly truncate, 1-2 μm wide,
unthickened or only slightly thickened, somewhat darkened-refractive;
microcyclic conidiogenesis occurring, conidia often germinating.
: Colonies on PDA spreading, somewhat
erumpent, with moderate aerial mycelium and regular, but feathery margins;
surface fuscous black, and reverse dark fuscous black.
diam after 1 mo on PDA at 25 °C in the dark.
: , Libina, okraj pole pod
nadrazim (okr.
(), 7 Sep. 1970, Ondřej, BRA.
, on leaves of , 7 Nov. 2000,
C.F.
,
culture ex-epitype = CPC 3884 = IMI 383037.
: Conidiophores are somewhat longer and narrower than , and ramoconidia occur
(,
).
MycoBank .
.
: Named after its host, .
verruculose, branched, 2-3 μm wide hyphae.
integrated, terminal on hyphae, 0-1-septate, mostly reduced to conidiogenous
cells, also lateral, visible as small, protruding, denticle-like loci, 10-15
× 2-3.5 μm. subcylindrical, 5-15 ×
2-3.5 μm, pale to medium brown, smooth to finely verruculose, tapering to
1-3 apical loci, 1-1.5 μm wide; scars inconspicuous. pale
brown, smooth, guttulate, subcylindrical to narrowly ellipsoid, occurring in
simple or branched chains, 0-1(-2)-septate, tapering towards subtruncate ends,
1.5-2.5 μm wide, aseptate conidia (8-)11-17(-20) × 3-3.5 μm,
septate conidia up to 40 μm long and 4 μm wide; hila inconspicuous, i.e.
neither thickened nor darkened-refractive; microcyclic conidiation common in
older cultures.
: Colonies erumpent, spreading, with
abundant aerial mycelium on PDA, and feathery to smooth margins; isabelline to
patches of fuscous-black due to the absence of aerial mycelium, which
collapses with age (surface); reverse fuscous-black.
diam after 1 mo at 25 °C in the dark; colonies fertile.
: , Baarn, Maarschalksbosch,
decaying leaves of (), 1 Oct. 1984,
G.S.
,
culture ex-type = ATCC 200937.
: Isolate was until recently preserved at the CBS as representative
of Ellis & Everh., a species known from
bark of sp. in the U.S.A.
quite distinct in having somewhat larger, and more subcylindrical to ellipsoid
conidia.
0-3-septate, 5-15 × 4-7 μm (), possessing the typical cladosporioid scars with a central
convex dome and a periclinal rim which characterise it as a true member of the
genus Link, which has been confirmed by a re-examination
of type material of (on inner bark of railroad ties,
U.S.A., West Virginia, Fayette Co., Nuttallburg, 20 Oct. 1893, L.A.
Flora of Fayette County No. 172, NY; also Ellis & Everh., N. Amer.
3086 and Fungi Columb. 382, BPI, NY, PH).
(Crous & W.B. Kendr.) Crous,
Mycobank
.
.
: Crous & W.B. Kendr.
S. Afr. J. Bot. 63: 286. 1997.
: , Mpumalanga, from leaf
litter of sp. (), Oct. 1992, M.J.
Wingfield, PREM 51438 .
leaf litter, Apr. 1994, P.W.
,
, culture ex-epitype CPC 778 = IMI 362702 =
.
: Conidiophores are dimorphic in culture, being macronematous,
anungitopsis-like, and micronematous, more pseudocladosporium-like.
(U. Braun & C.F.
U. Braun & K. Schub., Mycobank
.
: U. Braun &
C.F. Hill, Australas. Pl. Pathol. 33: 492. 2004.
(M. Morelet) Ritschel & U.
Braun, Schlechtendalia 9: 62. 2003.
: M. Morelet, Ann. Soc.
Sci. Nat. Archéol. Toulon Var 45: 218. 1993.
: M. Morelet, Ann.
Soc. Sci. Nat. Archéol. Toulon Var 45: 219. 1993.
: Schubert
(: 62).
narrowly cylindrical-oblong, 1-4 μm wide, later somewhat wider, up to 7
μm, septate, sometimes slightly constricted at the septa, sometimes
irregular in outline due to small swellings, subhyaline to pale brown, smooth,
walls unthickened, sometimes aggregating, forming compact conglomerations of
slightly swollen hyphal cells. usually reduced to
conidiogenous cells, arising terminally or laterally from hyphae,
subcylindrical to cylindrical, unbranched, 9-20 × (2.5-)4-5(-6) μm,
aseptate, very rarely 1-septate, very pale brown, smooth, walls unthickened,
monoblastic, unilocal, determinate, later occasionally becoming percurrent,
enteroblastically proliferating, forming a few (up to five) annellations, loci
broadly truncate, (2-)3-5 μm wide, unthickened, not darkened.
solitary, straight to curved, fusiform to obclavate,
distinctly apiculate, 24-45(-57) × (6-)7-9(-10.5) μm,
(1-)2-4(-5)-septate, more or less constricted at septa, sometimes up to 85
μm long with up to 7 septa, septa often somewhat darkened, second cell
often bulging, pale medium to medium olivaceous-brown or brown, smooth, walls
somewhat thickened, somewhat attenuated towards the base, hilum broadly
truncate, (2-)3-5 μm wide, unthickened, not darkened; microcyclic
conidiogenesis not observed.
: Colonies on OA iron-grey to
olivaceous-grey due to aerial mycelium and sporulation (surface); reverse
iron-grey to black, somewhat velvety; margin glabrous, olivaceous; aerial
mycelium sparsely formed, loose, diffuse; sporulating.
: , Liaoning, on × , 17 Jun. 1992, M.
PC (PFN 1466); , 20 Apr. 1993, , culture ex-epitype
= CPC
3639 = MPFN 307.
: Conidiophores are densely fasciculate ,
forming sporodochial conidiomata, cylindrical to ampulliform, 5-7 ×
6-7.5 μm ().
MycoBank .
.
: Named after its host, .
consisting of smooth, medium brown, branched, 1.5-2 μm
wide hyphae, giving rise to solitary, micronematous conidiophores.
reduced to conidiogenous cells, medium to dark brown,
erect, thick-walled, smooth, subcylindrical, widest at the base, tapering to a
subtruncate apex, 5-15 × 2-3 μm; scars flat-tipped, somewhat darkened
and thickened, one to several in the apical region, somewhat protruding, 0.5-1
μm wide. in branched or unbranched chains of up to 15,
medium brown, smooth, subcylindrical, 0-1-septate, widest in the middle,
tapering to subtruncate ends, straight to slightly curved, (6-)10-12(-17)
× 1.5-2(-2.5) μm; hila somewhat darkened and thickened, not
refractive, 0.5-1 μm wide.
: Colonies erumpent, with sparse aerial
mycelium and smooth margins on PDA, greyish sepia (surface); reverse
fuscous-black; on OA patches of greyish sepia and fuscous-black (surface); on
SNA umber (surface). Colonies reaching 15 mm diam on PDA after 1 mo at 25
°C in the dark; colonies fertile.
: , Baarn, De Vuursche, needle
of (), 12 Apr. 1982, G.S.
, culture ex-type
.
: This fungus was originally maintained in the CBS collection
as Matsush.
pseudocladosporium-like state was observed.
conidiogenous cells, and have several apical loci as in ,
but are not subdenticulate; scars are somewhat darkened and thickened, not
refractive. Conidia of are (8-)11-17(-20) ×
2-3(-3.5) μm, thus similar, but somewhat larger than the mean conidial size
range (10-12 × 1.5-2 μm) observed in .
conidiogenous loci and conidial hila of are also
somewhat larger.
that of , the ITS sequence similarity is 97 % (572/585
nucleotides).
.
-.
: Named after the presence of its characteristic
ramoconidia.
consisting of branched, septate, 1.5-2 μm wide hyphae,
pale brown, smooth, frequently with hyphal coils.
integrated into hyphae, and reduced to small, lateral protruding conidiogenous
cells, concolorous with hyphae, or macronematous, dark brown, erect,
thick-walled, 10-40 × 3-4 μm, 0-3-septate. terminal, integrated, subcylindrical, tapering to a rounded apex,
concolorous with hyphae (as hyphal pegs), or dark brown on mononematous
conidiophores, smooth, 3-15 × 2-3(-4) μm; proliferating sympodially,
loci slightly thickened, darkened and refractive, 0.5-1 μm wide.
occurring in branched chains, narrowly ellipsoid to
subcylindrical, pale olivaceous, guttulate; ramoconidia (0-)1(-3)-septate,
(12-)15-17(-20) × 2(-3) μm; conidia occurring in short chains (-15),
0-1-septate, (8-)10-12(-16) × 2(-3) μm; hila slightly thickened and
darkened, not refractive, 0.5-1 μm wide.
: Colonies erumpent, with sparse aerial
mycelium and smooth margins on PDA, hazel to fawn (surface), with a thin,
submerged margin; reverse brown-vinaceous; on OA hazel to fawn (surface) with
a wide, fawn, submerged margin.
at 25 °C in the dark; colonies fertile.
: , Baarn, De Vuursche, needle
of sp. (), 12 Apr. 1982, G.S.
, culture ex-type
.
: This strain has been deposited in the CBS collection as
(Matsush.) U. Braun.
ramoconidia and conidia are smaller than those cited by Matsushima
() (ramoconidia up to 30
μm long, conidia 10-21 × 2-4 μm). Although it clusters with in the LSU phylogeny, there are 13 bp differences in their ITS
sequence data.
absent in , and has a faster growth rate, and hazel to fawn
colonies, compared to the greyish sepia colonies of .
well-developed, septate conidiophores and ramoconidia are reminiscent of
, which differs, however, by its longer and wider
ramoconidia, up to 30 × 6(-7) μm, as well as larger conidiogenous
loci and conidial hila, 1.5-3 μm diam.
Crous & M.J. Wingf., MycoBank
.
.
: Named after the Greek Island, Rhodos, where it was
collected.
branched, septate, 1.5-3 μm wide hyphae, frequently forming hyphal coils,
giving rise to solitary, micronematous conidiophores.
reduced to conidiogenous cells that are terminal or lateral on hyphae, medium
brown, smooth, subcylindrical, subdenticulate, erect, or more distinct, up to
15 μm tall, 1.5-2 μm wide, mono- to polyblastic; scars flat-tipped,
somewhat darkened and thickened, but not refractive. in
branched or unbranched chains of up to 15, pale brown in younger conidia,
becoming medium brown, smooth, subcylindrical, 0-3-septate, tapering slightly
towards the subtruncate ends, straight, but at times slightly curved,
(8-)12-16(-20) × (2-)2.5-3(-4) μm; ramoconidia (0-)1(-3)-septate,
12-20 × 3-4 μm; conidia (0-)1-septate, 8-17 × 2-3 μm; hila
somewhat darkened and thickened, not refractive, 1-1.5 μm wide.
: Colonies spreading, somewhat erumpent,
with moderate aerial mycelium and crenate margins on PDA, uneven, greyish
sepia (surface), margins fuscous-black; reverse fuscous-black; on OA smooth,
spreading, with sparse aerial mycelium and even, regular margins, greyish
sepia; on SNA spreading, smooth, even margins, sparse aerial mycelium, greyish
sepia (surface).
the dark; colonies fertile.
: , Rhodos, on branches of
), 1 Jun. 2006, P.W. Crous & M.J.
Wingfield, , culture ex-type
= CPC
13156.
: has a typical
pseudocladosporium-like morphology in culture, with conidial scars that are
somewhat darkened and thickened.
(U. Braun & Feiler) Unter.,
Mycologia 89: 129. 1997.
: U.
Microbiol. Res. 150: 90. 1995.
: (U.
& Feiler) Crous, U. Braun & K. Schub., MycoBank
.
: U. Braun &
Feiler, Microbiol. Res. 150: 84. 1995.
(Dugan, R.G. Roberts & Hanlin)
Crous & U.
.
.
: Dugan, R.G. Roberts &
Hanlin, Mycologia 87: 713. 1995.
: sp.
1.5-2.5 μm wide hyphae, pale brown, forming hyphal strands.
mostly reduced to conidiogenous cells, or if present,
micronematous, consisting of a supporting cell, and single conidiogenous cell.
integrated in hyphae as lateral loci, or
terminal, frequently disarticulating, subcylindrical, pale to medium brown,
smooth, mono- to polyblastic, loci 1-1.5 μm wide, 2.5 μm tall;
conidiogenous cells subcylindrical, up to 40 μm tall, and 2-2.5 μm wide.
in long chains of up to 60, branched or not, subcylindrical
to narrowly ellipsoid, pale olivaceous to pale brown, smooth; ramoconidia
0-1(-3)-septate, 15-20(-30) × 2-3(-3.5) μm; conidia 0(-1)-septate,
6-8(-12) × 2-3(-3.5) μm; hila 1-1.5 μm wide, inconspicuous to
somewhat darkened, subtruncate.
: Colonies erumpent, with sparse aerial
mycelium on PDA, and smooth, even margins; olivaceous-grey to iron-grey
(surface); reverse greenish black; on OA dark mouse-grey (surface), with even,
smooth margins.
dark; colonies fertile.
: , Washington, Wenatchee, on bing
cherry fruit, cv. (),
R.G. Roberts, culture ex-type, ATCC 96019 =
.
: Dugan
() commented that although
similar to , the conidia of
this species were predominantly aseptate and somewhat shorter than those
described by Matsushima
().
Riess, Bot. Zeitung (Berlin) 11:
138. 1853. .
: Colonies erumpent, spreading, aerial
mycelium sparse, margins smooth; colonies sienna to umber on PDA, with patches
of greyish sepia; reverse chestnut-brown; on OA whitish due to moderate aerial
mycelium, with diffuse umber pigment in the agar; whitish on SNA.
reaching 15 mm diam on PDA after 3 wk at 25 °C in the dark.
: , Schovenhorst, leaf litter
of (), 8 Nov. 1997, W.
, culture
.
: is the type species of
the genus .
investigated here to determine if would be available for
taxa that have a pseudocladosporium-like morphology.
within
the was surprising.
sterile, and therefore its identity could not be confirmed.
Isolate sporulated profusely.
olivaceous margins on PDA; conidiophores pale, and not dark brown as depicted
for in Ellis
(); conidial chains were
greenish yellow in mass, and pale olivaceous-green under the dissecting
microscope, somewhat roughened, polyblastic; on ITS sequence this isolate is
identical to U57492, (Alb. & Schwein.)
Svrček (), but the latter species should have a
phialidic anamorph, so it is possible that this GenBank sequence is incorrect.
The identity of therefore remains unresolved.
when it was collected show this isolate to be authentic for the species and
the genus .
Crous, Summerb. & Summerell
(;
), and is therefore unrelated to the
.
R.F. Castañeda, Mycotaxon 60: 285.
1996,
Hyphomycetes. mostly superficial, hyphae septate, brown
to olivaceous. absent.
differentiated, mononematous, erect, aseptate or septate, brown to olivaceous.
integrated, terminal, proliferation sympodial,
polyblastic, with subdenticulate, somewhat thickened and darkened scars.
solitary, fusiform to obclavate or cylindrical, septate,
asperulate to verrucose, olivaceous to brown, tips always hyaline,
thinner-walled and smooth, forming mucoid appandages, often only visible as a
thickened frill. present, micronematous.
short cylindrical, antenna or hyphopodium-like,
phialidic, colarette sometimes present, aseptate, subhyaline.
solitary, obovoid, ellipsoid, aseptate, brown to olivaceous, verruculose.
R.F.
Mycotaxon 60: 285. 1996. .
: internal to superficial,
unbranched to sparingly branched, 1.5-3 μm wide, loosely septate, septa
almost invisible, pale brown, smooth to asperulate, minutely verruculose,
walls unthickened, sometimes inflated at the base of conidiophores.
macronematous, arising usually laterally from
plagiotropous hyphae, erect, straight, subcylindrical or conical, not
geniculate, usually unbranched, rarely branched, 13-45 × 3-4(-5) μm,
slightly to distinctly attenuated towards the apex, tapered, aseptate, rarely
with a single septum, pale brown to pale medium brown, smooth or minutely
verruculose, walls unthickened, often somewhat constricted near the base.
integrated or conidiophores usually reduced to
conidiogenous cells, subcylindrical to conical, proliferation sympodial, with
a single or several subdenticulate to denticulate conidiogenous loci mostly
crowded at or towards the apex, protuberant, truncate, 0.8-1.2 μm wide,
thickened and darkened-refractive. solitary, straight to
curved, ellipsoid, fusiform to obclavate, distinctly tapered towards the apex,
apiculate, (12-)15-32 × 3.5-5.5 μm, (0-)1-2(-3)-septate, mainly
1-septate, usually constricted at the septa, pale brown to pale medium brown,
asperulate to verruculose, walls unthickened or almost so, tips always
hyaline, thinner-walled and smooth, forming mucoid appendages, often only
visible as a thickened frill, base somewhat rounded or slightly bulbous, hila
often situated on short peg-like prolongations, truncate, 0.8-1(-1.2) μm
wide, thickened, darkened-refractive; microcyclic conidiogenesis occurring,
conidia forming secondary conidiophores.
micronematous. reduced to
conidiogenous cells, numerous, occurring as short lateral prolongations of
hyphae, antenna or telescope-like, cylindrical, unbranched, conidiogenesis
unclear, at times appearing phialidic, or having one to two apical scars; up
to 5 μm long, 1-1.5 μm wide, aseptate, subhyaline, smooth.
of the micronematous anamorph quite different from the
conidia formed by the macronematous conidiophores, solitary, obovoid,
ellipsoid to somewhat fusiform, 5-9 × 2.5-3 μm, aseptate, pale to
pale medium brown, verruculose, somewhat attenuated towards the base, hila
flat, unthickened to somewhat thickened, appearing to have the ability to form
a slime appendage at the apex.
: Colonies on OA iron-grey to olivaceous
due to abundant sporulation (surface); reverse black, velvety; margin regular
to undulate, feathery; aerial mycelium absent or sparse, sporulation
profuse.
: , isolated from air, 2 Oct. 1994,
R.F.
= INIFAT
C94/114 = MUCL 39155 = IMI 367520.
: Within the course of the recent phylogenetic studies in
and the type culture of
has been included since it was
deposited at the CBS as “”.
When the culture was re-examined, the described short appressorium-like,
inflated hyphopodia with slightly warted to lobed apices
() could be recognised as conidiogenous cells of a synanamorph
forming a second conidial type.
unthickened and smooth, and have the ability to form mucoid appendages that
are often only visible as a thickened frill. These two features, viz., the
synanamorph and the conidia with mucoid appendages, easily distinguish this
genus from morphologically similar genera such as Maubl., and Fr.
clusters basal to the .
and spp., which appear
morphologically similar.
are identified based solely on microscopic and cultural characteristics.
results clarify that is allied to the
and (=
) to the ().
The plant-pathogenic species compose a separate
clade within the order ().
extremotolerant, rock-inhabiting species around the genus
Link (Cluster 5 of
).
Both clades are significantly distinct from the prevalently hyperparasitic or
oligotrophic, frequently opportunistic species of the remainder of the order
().
includes all teleomorphs sequenced to date, and is thus
likely to represent the family .
trends in each of the main clades of are thus quite
different ().
clade of , including two new species associated with
leaf spots. is distinguished from
, which clusters outside the
, and appears allied to
Crous, Summerb. & Summerell, a recently introduced genus for species
occurring on leaves
().
the , and is placed in
as a distinctively pigmented member of the genus.
and are newly described from a
range of substrates such as fruit juices, drinking water and leaf litter,
revealing the potential of these materials as ecological sources of inoculum
for taxa associated with opportunistic human and animal infections.
, and is best treated as a synonym of
, along with other genera as proposed by Schubert () and Beck
().
Although numerous isolates of the were included for
study, it was surprising to find relatively little variation within the
family, suggesting that previously proposed teleomorph genera such as
and should be best
treated as synonyms of .
extended with the inclusion of a novel sister clade of hyphomycetes with a
pseudocladosporium-like morphology, which are also referred to as
, thus widening the generic concept of the latter to
encompass all pseudocladosporium-like anamorphs within the family.
species assigned to proved to cluster within the
, but the type species of the latter genus, , clustered elsewhere and possesses distinct conidiogenous loci,
i.e., cannot be reduced to synonymy with
.
clade are morphologically rather close to taxa assigned to
.
are morphologically barely distinguishable
(), but the true affinity of depends on its
type species of which cultures and sequence data are not yet available.
together, suggesting that these are either different synanamorphs of the same
teleomorph genus, or that they may represent cryptic clades that will diverge
further once additional species are added in future studies.
appeared to represent quite a diverse assembledge
of morphotypes, the were again surprisingly uniform. |
The halophilic and halotolerant mycobiota from hypersaline aqueous habitats
worldwide frequently contain Link isolates
(, ).
but surprisingly, many of these isolates were identified
as Penz.
unpublished).
common air-borne, cosmopolitan species, was frequently
isolated from indoor and outdoor air (), dwellings
(),
and occasionally from humans () and plants
().
(a 0.816), while other cladosporia clearly preferred a higher,
less extreme water activity ().
predilection for osmotically stressed environments although is reported from a wide range of habitats including
osmotically non-stressed niches.
complex of species having either narrow or wide ecological amplitudes.
molecular diversity of strains identified as has
not yet been determined and isolates from humans have not yet been critically
compared with those from environmental samples.
was initiated with the aim to define phylogenetically and morphologically
distinct entities and to describe their osmotolerance and
their natural ecological preferences.
different sites of the Mediterranean basin (Slovenia, Bosnia and Herzegovina,
Spain), different coastal areas along the Atlantic Ocean (Monte Cristy,
Dominican Republic; Swakopmund, Namibia), the Red Sea (Eilat, Israel), the
Dead Sea (Ein Gedi, Israel), and the salt Lake Enriquillio (Dominican
Republic).
once per month in 1999.
river saltern (Spain) were taken twice (July and November) in 2000.
in Namibia and one in the Dominican Republic were sampled twice (August and
October) in 2002.
encountered in these ponds.
through membrane filters (pore diam 0.45 μm), followed by incubation of the
membrane filters on different culture media with lowered water activity
().
selective medium per sample were analysed further.
selected from different evaporation ponds, collected at different times, in
order to avoid sampling of identical clones.
the Culture Collection of Extremophilic Fungi (EXF, Biotechnical Faculty,
Ljubljana, Slovenia), while a selection was deposited at the Centraalbureau
voor Schimmelcultures (CBS, Utrecht, The Netherlands) and the Culture
Collection of the National Institute of Chemistry (MZKI, Ljubljana, Slovenia).
Reference strains were obtained from CBS, and were selected either on the
basis of the strain history, name, or on the basis of their ITS rDNA sequence.
Strains were maintained on oatmeal agar (OA; diluted OA, Difco: 15 g of Difco
255210 OA medium, 12 g of agar, dissolved in 1 L of distilled water) with or
without 5 % additional NaCl.
lyophilisation.
.
were point inoculated on potato-dextrose agar (PDA, Difco), OA and Blakeslee
malt extract agar (MEA, ) and incubated at 25 °C for 14 d in darkness.
Surface colours were rated using the colour charts of Kornerup & Wanscher
().
microscopic morphology, strains were grown on synthetic nutrient agar (SNA,
) in
slide cultures.
aseptically, placed upon sterile microscope slides, and inoculated at the
upper four edges by means of a conidial suspension
().
blocks were covered with sterile cover slips and incubated in moist chambers
for 7 d at 25 °C in darkness.
conidiophores were observed at magnifications × 100, × 200 and
× 400 in intact slide cultures under the microscope without removing the
cover slips from the agar blocks.
with aniline blue.
(), Kirk .
() and Schubert . ( - this volume).
Conidiophores in are usually ascending and sometimes
poorly differentiated.
could sometimes be determined only approximately, their lengths were in some
cases useful for distinguishing morphologically similar species when observed
in slide cultures.
unilateral.
electron microscopy (SEM).
determined by the numbers of conidia in unbranched parts, the nature of
ramoconidia as well as their distribution in conidial chains.
given as (i) n-n or (ii)
(n-)n-n(-n), with
n = minimum value observed; n = maximum value
observed; n/n = first/third quartile.
ramoconidia also average values and standard deviations are listed.
provided are based on at least 25 measurements for the conidiophores of each
strain, and at least 50 measurements for conidia.
MEA without and with additional NaCl at concentrations of 5, 10, 17 and 20 %
NaCl (w/v) and incubated at 25 °C for 14 d.
temperature requirements for growth, plates were incubated at 4, 10, 25, 30
and 37 °C, and colony diameters measured after 14 d of incubation.
For DNA isolation strains were grown on MEA for 7 d.
according to Gerrits van den Ende & de Hoog
() by mechanical lysis of
approx. 1 cm of mycelium.
Internal Transcribed Spacer region 1, 5.8S rDNA and the ITS 2 (ITS) was
amplified using the primers V9G () and LS266
(). Sequence reactions were done using primers ITS1 and ITS4
().
For amplification and sequencing of the partial actin gene, primers ACT-512F
and ACT-783R were applied according to Carbone & Kohn
().
sequencing of the β-tubulin gene primers T1 and T22 were used according
to O'Donnell & Cigelnik
().
cycle sequencing kit (Applied Biosystems, Foster City, CA, U.S.A.) was used in
sequence reactions. Sequences were obtained with an ABI Prism 3700 DNA
Analyzer (Applied Biosystems). They were assembled and edited using SeqMan v.
3.61 (DNAStar, Inc., Madison, U.S.A.).
indicated in the trees by their GenBank accession numbers; newly generated
sequences are indicated by strain numbers (see also
).
automatically aligned using ClustalX v. 1.81 (Jeanmougin .
1998). The alignments were adjusted manually using MEGA3
().
Phylogenetic relationships of the taxa were estimated from aligned sequences
by the maximum parsimony criterion as implemented in PAUP v. 4.0b10
().
the SSU rDNA, ITS rDNA and the β-tubulin and actin genes are analysed
separately. Species of .
various taxa of the using SSU rDNA sequences and
G. Winter () as outgroup.
The other data sets focus on ., using
Zalar, de Hoog & Gunde-Cimerman as an
outgroup, because this species was most deviant within
in the SSU rDNA analysis (see below).
characters, which were unordered and equally weighted.
missing characters. Starting tree(s) were obtained via stepwise, random, 100
times repeated sequence addition.
“MaxTrees” setting to 9 000, the tree-bisection-reconnection as
branch-swapping algorithm, and the “MulTrees” option set to
active.
addition bootstrap analysis.
their respective branches.
();
their accession numbers are listed in . Alignments and trees were deposited in TreeBASE
().
tree scores for each analysed sequence locus are summarised in
.
material such as ex-type or ex-neotype strains was analysed on the level of
SSU rDNA sequences.
members of were compared with related taxa of
the and .
somewhat more distantly related
() (: ) was selected as outgroup. R.F.
Castañeda & Dugan (now placed in Bonord., see
),
also a member of the , was included in the analyses.
taxa included in the SSU rDNA analysis belong to the
(),
within which the ingroup is represented by the orders
() and
() (see also
).
The genus , of which some species are linked to
Crous & U.
(),
forms a statistically strongly supported monophyletic group
().
this paper, namely, Zalar, de Hoog &
Gunde-Cimerman, Zalar, de Hoog & Gunde-Cimerman,
Zalar, de Hoog & Gunde-Cimerman, Zalar, de Hoog & Gunde-Cimerman,
Zalar, de Hoog & Gunde-Cimerman and Zalar, de Hoog
& Gunde-Cimerman ().
A sister group relationship of with a clade of
taxa characterised, among others, by Johanson
teleomorphs, containing various anamorphic genera such as
Sacc., Unger, Fresen.,
Speg., “” Corda
(now Crous & U. Braun) (see Crous .
,
- this volume) and the
somewhat cladosporium-like genus Seifert & N.L. Nick.
(), was statistically only moderately supported
(), whereas in an
analogous analysis by Braun .
(:
) it was highly
supported. These data also support the conclusion by Braun .
() and Crous . () that
is not a member of the distantly related
(), which is also
rich in cladosporium-like taxa ().
environments belonged to the . The SSU rDNA
sequences do not resolve a phylogenetic structure within .
somewhat distinguished from a statistically unsupported clade with (Pers. : Fr.) Link, (Fresen.) G.A.
de Vries, Berk. & Broome, ,
and , etc.
most distinct within the genus in analyses of the SSU
rDNA (), it was used as
outgroup in analyses of the ITS rDNA and the β-tubulin and actin
genes.
gene introns and exons supported the species clades of and (Figs
,
and
), of which
was distinguished in the β-tubulin tree by a particular long terminal
branch of the only sequenced strain (). also clustered as a
well-supported species clade in preliminary analyses using various
species as outgroup (not shown). All strains of (Fonseca, Leão & Nogueira) Vuill.
well distinguishable from other species by strikingly
slow-growing colonies at all tested temperatures and relatively large, oblong
conidia.
indicate that presents two cryptic species (Figs
-).
The species clade of is moderately supported in
analyses of the actin gene but highly by means of the β-tubulin gene.
,
and
) that
and are closely related species.
of , which is morphologically clearly
distinguished from all other species by its conspicuous ornamentation
consisting of digitate projections (), is supported by β-tubulin
() and actin
() sequence data but not
by those of the ITS rDNA ().
the complex.
introns and exons (Figs ,
and
) do not allow the full
elucidation of phylogenetic relationships among these
species.
analyses of the β-tubulin and actin genes is low (bootstrap values mostly
< 50 %).
and is highly supported in the analysis based on the
β-tubulin gene, analysis of the ITS rDNA indicate that these two species
are unrelated, and that is closely related to
.
morphologically resembling are not phylogenetically
closely related and that the data we present here do not allow their
classification in natural subgroups of the genus .
was placed in all analyses among species of the complex and all analyses supported close relatedness of and .
ranging from minutely verruculose () to verrucose () ().
The verrucose conidia of can be recognised also under
the light microscope and used as a distinguishing character.
minutely verruculose conidia are encountered in and
().
, a member of the species
complex, has conidia with a digitate ornamentation that can appear spinulose
under the light microscope; however, when using the SEM it became clear that
its projections have parallel sides and a blunt end
().
() who originally included
four species, of which is the type species of the genus
().
In 1950, von Arx reported a teleomorph connection for this species with
(De Not.) Johanson.
the majority of species, including the type species of
the genus, (Pers.) Starbäck, clustered within
the , a family separated from
().
Therefore, was reclassified as (De Not.) Crous & U.
All anamorphs with a cladosporium- and heterosporium-like
appearance and with a supposed relationship were
maintained under the anamorph name , morphologically
characterised by scars with a protuberant hilum consisting of a central dome
surrounded by a raised rim ().
been adopted from Schubert .
().
described here, ramoconidia have been observed often in , sometimes in
and , and only sporadically in all other species.
Therefore, ramoconidia can be seen as important for distinguishing species
although sometimes, they can be observed only with difficulty.
ramoconidia as a diagnostic criterion, colonies only from SNA and not older
than 7 d should be taken into account.
() from decaying
leaves and branches in Italy.
conidiophores having a length of 150-300 μm and a width of the main
conidiophore stipe of 3.5-4 μm, (ii) spherical to ellipsoid, acrogenously
formed conidia of 3.4-4 μm diam, and (iii) ramoconidia of 6-14 ×
3.5-4 μm. Penzig's original material is not known to be preserved.
culture derived from , originating from a human nail, was accepted as typical of
However, de Vries
(), incorrectly cited it
as “lectotype”, and thus the same specimen is designated as
neotype in this study (see below), with the derived culture
() used
as ex-neotype strain.
including plants or walls of bathrooms.
in bathrooms and of plants, colonised by , can have
a similar low water activity as salterns.
this species, however, grew under conditions at a water
activity of up to 0.860, while Hocking .
() and Aihara . () reported that
it can grow even at 0.815.
as halo- or osmotolerant.
proving that is a human pathogen.
possible that was not involved in any disease process but rather occurred
as a contaminant on dry nail material. is
a phylogenetically well-delineated species (Figs
,
and
).
substrata such as peanut cell suspension, tissue culture, bathroom walls and
as culture contaminants.
that is distributed by air and that it can colonise
whatever substrata available, although it may have its natural niche
elsewhere.
and other saline environments and it was also detected with molecular methods
(but not isolated) from skin of a salt water dolphin.
reports of this species from plants ().
species closely linked to salty or hypersaline environments although
additional sampling is necessary to prove that. is morphologically recognisable by relatively oblong to
spherical, coarsely rough-walled conidia.
the skin of a bottlenose dolphin, suffering from lobomycosis, is identical to
the sequences of .
Taborda, V.A. Taborda & McGinnis (GenBank AF035674)
by Haubold .
(), who apparently
concluded wrongly that a fungus with a cladosporium-like ITS rDNA sequence
similar to that of can be the agent of lobomycosis.
Later, Herr .
() showed that phylogenetically belongs to the on the basis of
amplified SSU rDNA and chitin synthase-2 gene sequences generated from tissue
lesions.
() who reclassified the
organism as O.M.
().
was not the main etiologic agent for the lobomycosis and it was colonising the
affected dolphin skin secondarily while inhabiting other seawater
habitats.
closely related but is particularly well
distinguishable from all other species by its slow
growing colonies (1-7 mm diam/14 d) and relatively large conidia (4-5.5
× 3-4 μm). has smaller conidia
(3-4 × 2.5-3 μm) but a similar length : width ratio and faster
expanding colonies (8-18 mm diam/14 d). is
most likely a complex of at least two species.
Arctic and the Antarctic may need to be distinguished from on species level.
analyses of the β-tubulin and actin genes (Figs
-).
, represented by an authentic strain
of Fonseca, Leão & Nogueira,
(), has been
isolated from a variety of substrata but is tolerating only up to 10 % NaCl.
It was originally described by da Fonseca .
(1927,
) and subsequently
reclassified as by Vuillemin
().
derived from an ulcerating nodular lesion on the arm of a human patient.
Because other strains of this species are ubiquitous saprobes originating from
various substrata, we suspect that is not an important
human pathogen. has been isolated from
hypersaline environments only, and tolerates up to 20 % NaCl in culture
media.
In general, the human- or animal-pathogenic role of the -like species described here seems to be limited.
possible that pathogenic species of Sacc.
misidentified as or as other species of
().
isolated as clinical strains could have been secondary colonisers since they
are able to dwell on surfaces poor in nutrients, possibly in an inconspicuous
dormant phase and may then be practically invisible.
be air-borne contaminations of lesions, affected nails etc.
() or are perhaps disseminated by insufficiently sterilised
medical devices, as melanised fungi can be quite resistant to disinfectants
().
at isolation and thus difficult to exclude as etiologic agents of a disease.
For example, in 2002, a case report on an intrabronchial lesion by in a healthy, non-asthmatic woman was described
(),
but we judge the identification of the causal agent to remain uncertain, as it
was based on morphology alone and no culture is available.
have the opinion that all clinical cases ascribed to
species need careful re-examination.
PDA and MEA 14 d at 25 °C, if not stated otherwise; microscopical
characters are from SNA slide cultures grown for 7 d at 25 °C. |
The ascomycete Parbery
() grows in
hydrocarbon-rich substrates such as jet fuel, cosmetics and wood preserved
with creosote or coal tar.
(Lindau) Arx & G.A.
synonym (Lindau) G.A. de Vries.
lightly pigmented, warty conidiophores, and branched, acropetally developing
chains of lightly pigmented ameroconidia lacking conspicuous scars
().
known colloquially as the “creosote fungus”, the “kerosene
fungus” or the “jet fuel fungus”; to avoid confusion caused
by the many heterotypic names with the epithet “”,
in this paper we generally will use the oldest of these informal names,
“creosote fungus”, when referring to or its
anamorph.
water, and the mycelium clogs fuel lines and corrodes metal parts.
Consequently, fuel tanks in airports are monitored for this fungus by private
companies using various physiological or biochemical tests.
(Fr.) Fr.
resin, comprising dark synnemata and an effuse mononematous synanamorph, both
with cladosporium-like conidiogenous cells and conidia.
the creosote fungus, the conidia of are dark brown
and the lateral walls are conspicuously thicker than the poles
().
the mononematous anamorph sometimes occur, and the mononematous anamorph can
be sparse on colonies bearing synnemata.
mononematous anamorph have identical pigmentation and lateral wall thickening
to that of the synnematous anamorph.
referred to by its own binomial name although, as we will show, there is a
species epithet available.
Parbery, generally we will refer to herein as “the resin fungus”.
disagreement about whether the creosote fungus is conspecific with the
mononematous synanamorph of the resin fungus (Parberry 1969).
anamorph of the creosote fungus is based on
Lindau ().
. ()
presented a study of a cladosporium-like fungus commonly isolated from wood
impregnated with creosote and coal tar and applied Lindau's name without
examining its type.
() employed the same name
for the same fungus.
Vries (, using the name
G.A.
for the creosote fungus (differing in the colours of their conidia, the
production of setae, or the total absence of conidia), each based on single
conidium isolates made from one parent culture.
() and Parberry
examined the holotype of and concluded that it
represented the creosote fungus.
(), prior to the
description of or Arx & G.A.
synanamorph of the resin fungus.
() is correct, then
neither the species , nor the genus that it
typifies, , can represent the creosote fungus, as intended
by Parberry or von Arx and de Vries (in
).
evidence that the resin fungus is a different species from the creosote
fungus.
nomenclature for these two species.
, is also considered in our discussion of generic
concepts.
Full details of herbarium material examined are listed below.
dried herbarium specimens were studied in 90 % lactic acid without stains;
preparations of some exsiccate and types were mounted in glycerin jelly.
Cultures were grown on potato-dextrose agar (PDA, Difco), oatmeal agar (OA,
),
Blakeslee's malt extract agar (MEA, ) and dichloran-18 % glycerol agar (DG-18,
).
Colony characters were taken from cultures grown at 25 °C in darkness.
Cultures are maintained in the Canadian Collection of Fungal Cultures (DAOM),
Agriculture & Agri-Food Canada, Ottawa.
[scr. Link]. E. Hbr. Link =
III. 341 [scr.?] (herb. Link, B).
Lindau, n. sp. Fl. v.
Harz an , Sachsenwald, leg. O. Jaap, 29-4-1906. [scr.
Lindau]. (DAOM 41888, slide prepared from the preserved in
B.)
Lindau nov. gen. et nov. spec.,
Kabát et Bubák: Fungi imperfecti exsiccati no. 99.
erhärteten Fichtenharz an Brockenweg, am Dreieckigen Pfahl in Harz,
Deutschland, leg. G. Lindau, 13.VIII. 1903 (, B).
Fries. E. Hbr. Link, Fries legi, Smol.
[scr. Fries]. (DAOM 41890, slide prepared from herb. Link, B).
presumed of , the basionym for the resin
fungus, .
synnemata, brown conidia with laterally thickened walls, and acropetal
conidial chains, allowing it to be recognised as the fungus we now know as
.
be differentiated from Link.
fungus was collected by Fries, presumably in Småland (a province of
Sweden), match the details in the protologue of this species.
. “Fungi Rhenani Fasc. II, 1863, L.
Fuckel, no. 129, ad Abietis resinam, raro Hieme, in sylva Hostrichiensi”
(as Fr., DAOM 55543 ex FH). “Flora Suecica,
2956, Ad resinam piceae, Småland: Femsjö, Prostgaidsshogen, 6 Aug.
1929, leg. J.A. Nannfeldt, s.n.” (as (Fr.)
Sacc., DAOM 41891 ex UPS). “Flora Suecica, 4709, Ad resinam abietinum,
Uppland: Bondkysko sin Valsätra, 9 May 1932, leg. J.A. Nannfeldt”
(as Lindau, DAOM 41889 ex UPS). “[on wood
scr. Berkeley] J.E.
Fr., DAOM 113425 ex K). “Sydow, Mycotheca germanica, 350.
Fichtenharz... am Brockenweg 30.9.1904, leg. P. Sydow” (DAOM 41893).
.
Central Park, on resin of , leg. S. & L.
Hughes, 17 Aug. 2000 (DAOM 228572a, 228573a); Cameron Lake, Cathedral Grove,
on , leg. isol. S.J. Hughes, 21 Aug. 1957 (DAOM
56088a). Ladysmith, Ivy Green Park, on resinous exudates, leg. R.J.
no. BC-978, 18 Apr. 1960, det. S.J. Hughes (DAOM 70462).
Valley Conservation Area, leg. det. S.J. Hughes, 1 Jul. 1975 (DAOM 139385);
North Vancouver, Lynn Valley Conservation Area, on bark of living conifer
(probably ), leg. isol. K.A. Seifert no. 1574,
26 May 2002 (single conidium isolate, culture and specimen DAOM 239134; ITS
GenBank EU030275, LSU GenBank EU030277); Terrace, near Kalum, on , leg. W.G. Ziller no. V-6549, 10 July 1950, det. S.J.
(DAOM 59657); Queen Charlotte Islands, east coast of Moresby Island, north
side of Gray Bay, 53°08' N, 131°47' W, on
leg. I. Brodo, M.J. Schepanek, W.B. Schofield, 28 Sep. 1973, det. S.J.
(DAOM 144757); Queen Charlotte Islands, Graham Island, Tow Hill area, on resin
of leg. S.A. Redhead no. 4440, 20 Sep. 1982, det.
G.P.
leg. W. Ziller V-6567 det. S.J. Hughes, 6 Jun. 1950 (DAOM 59710); Vancouver
Island, Cathedral Grove, Cameron Lake, on , leg.
det. S.J. Hughes, 21 Aug. 1957 (DAOM 56088a); Vancouver Island, Caycuse, on
resin of leg. det. S.J. Hughes, 17 Jul. 1972
(DAOM 139355); Vancouver Island, Lake Cowichan, Honeymoon Bay, on resin of
, leg. J Ginns, det. S.J. Hughes, 29 Oct. 1971
(DAOM 134968); Vancouver Island, Lake Cowichan, Mesachie Lake Forest
Experimental Station, leg. det. S.J. Hughes, 5 Jul. 1972 (DAOM 139277a, DAOM
139278) and 6 Jul. 1072 (DAOM 139281). ,
Ještěd near Liberec, leg. det. S.J.
, 10 May 1955 (DAOM 51723). ,
Oregon: Andrews' Experimental Forest, Forest Service Rd.
leg. det. S.J. Hughes, 10 May 1969 (DAOM 134565);
Andrews' Experimental Forest, Blue River, on resin of conifer, cut wood, leg.
det. K.A. Seifert no. 69, 10 Jul. 1981 (DAOM 228203); Oregon, del Norte Co.,
J. Smith's State Park, on , leg. det. S.J.
11 May 1069 (DAOM 134614); Devil's Elbow State Park, Cape Perpetus, on
leg. det. S.J. Hughes, 6 May 1969 (DAOM 134615);
Linn Co., near Cascadia, on leg. R.
det. S.J. Hughes, 14 May 1969 (DAOM 127885); U.S. Forest Service Rd. no. 126,
North fork Cape Creek, on resin of , leg. det. S.J.
Hughes, 7 May 1969 (DAOM 134852,134563); Willamette National Forest, McKenzie
Bridge Camp Grounds, leg. det. S.J. Hughes, 10 May 1969 (DAOM 134564).
Washington: Kittitas Co., Wanatchee National Forest, Rocky Run, on , leg. Field Mycology Class 1955, 22 Jul. 1955, det. S.J.
(mononematous synanamorph only, DAOM 118934 ex WSP 45210, as
sp.); Jefferson Co., Olympic National Forest, 10 mi
Camp, Sec. 17, T26N, R3W, on , leg.
Mycology Class, 22 Jul. 1955 (DAOM 113801 ex WSP 45212, as
); Grays Harbor Co., Twin Harbors Beach State Park,
resin of , leg. W.B. & V.G. Cooke, 24 Jul. 1951,
det. S.J. Hughes (DAOM 118970 ex WSP 28432).
. Isolated from jet fuel by P.
(culture, DAOM 170427 = ATCC 22711, ITS GenBank EU030278, LSU GenBank
EU030280). Canada, British Columbia, source unknown, isol. “Mrs.
Volkoff”, Jul. 1969 (culture, DAOM 194228, ITS GenBank EU030279).
spp.
Park, leg. S.J. Hughes, 17 Aug. 2000 (DAOM 228571); Vancouver, Stanley Park,
leg. K.A. Seifert no. 1571, 11 May 2002 (culture and specimen, DAOM 239136,
LSU GenBank EU030276). Munter, Kerry, near Glenbeigh
(. N 52° 03' W 9° 54'), leg. K.A. Seifert no. 3197, 26 Sep.
2006 (culture and specimen, DAOM 239135, ITS GenBank EU030273).
, Gelderland, Kröller-Müller Museum, leg. K.A.
Seifert no. 1235, 12 May 2000 (DAOM 227136). Wales,
Hafod Estate (ca. N 52° 22' W 3° 51'), leg. K.A. Seifert no. 3198, 1
Oct. 2006 (culture and specimen, DAOM 239137, ITS GenBank EU030274).
DNA was isolated using a FastDNA™ Kit and the FastPrep™ FP120
(BIO 101 Inc.) or an UltraClean™ Microbial DNA Isolation Kit (Mo Bio
Laboratories, Inc., Solana Beach, CA, U.S.A.) using mycelium removed from agar
cultures.
Genius™ thermocycler (Techne Cambridge Ltd.).
performed using Ready-To-Go™ Beads (Amersham Canada Ltd.) in 25 μL
volumes, each containing 20-100 ng of genomic DNA, 2.5 units pure
DNA Polymerase, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl,
200 μM of each dNTP, 0.2 μL of each primer (50 μM), and stabilizers
including bovine serum albumin.
denaturation for 4 min at 94 °C, followed by 30 cycles of 1.5 min
denaturation at 95 °C, 1 min annealing at 56 °C, 2 min extension at 72
°C, with a final extension of 10 min at 72 °C.
by ethanol/sodium acetate precipitation and resuspended as recommended for
processing on an ABI PRISM 3100 DNA Analyzer or an ABI 373 Stretch DNA
Sequencer (Applied Biosystems, Foster, CA).
sequenced using the BigDye v. 2.0™ Terminator Cycle Sequencing Ready
Reaction Kit (ABI Prism/Applied Biosystems) following the manufacturer's
directions.
cycle-sequenced using primers LR0R, LR3R, LR16 and LR6
(,
;
).
The complete ITS and 5.8S rRNA genes were amplified and sequenced using the
primers ITS5 and ITS4, with ITS2 and ITS3 primers used for cycle sequencing
when necessary ().
of the ITS5-LR6 region.
in PAUP v. 4.0b10 () with simple stepwise addition of taxa, and tree
bisection-reconnection (TBR) branch swapping.
removed for all analyses.
robustness of the phylogenies was tested using full bootstrap analyses (1 000
replications).
tree figures, and the sequences generated in this study are indicated in
bold.
our sequences for the three fungi of interest,
and ; was added as an
out-group to root the tree.
matrix, there is no implication that this data set represents the diversity of
the The alignment was calculated using MAFFT
()
and adjusted using S-A (Sequence Alignment Program v.
1.d1;
)
to maximise homology.
anamorphs used by Davey & Currah
(), originally produced
using MAFFT.
S-A to maximise homology, but still included several
areas where the homology of aligned sequences was difficult to evaluate.
related sequences using a BLAST search of GenBank, and these relevant
sequences were added to an alignment of Robak sequences
from the study of Hambleton
(), and then adjusted
using S-A.
mononematous synanamorph of (DAOM 228772a, 228573a),
to allow comparison of sequences obtained from cultures of the synnematous
synanamorph.
unsuccessful.
(, de Vries
,
,
) and
the rhododendron fungus
(,
, ) are well-described in the literature and will not be
repeated here.
overall cultural phenotypes.
coal-black, wrinkled, and restricted in growth, no matter what agar medium is
employed; even after 3 mo, the colonies are rarely more than 2 cm diam
().
form in our cultures; , the synnemata produce branched,
acropetal chains of conidia with laterally thickened walls
().
refractive or darkened secession scars were evident on individual conidia or
ramoconidia.
synnematous parts of a freshly collected specimen (DAOM 56088a) and grown on
PDA and sterilised conifer wood.
colonies derived from the two types of conidiophores, in all cases yielding
restricted black colonies, or in their microscopic characters.
conclude that these two types of conidiophores represent synanamorphs of one
fungus.
().
occurrence of this fungus in California, Oregon, and Washington State, U.S.A.
and British Columbia, Canada, on resinous exudates on and
Microscopic features from the holotype specimen of Lindau are shown in .
acropetally developing conidial chains.
pigmented, and the lateral walls are more conspicuously thickened and darkened
than the polar walls.
secession scars on any of the cells.
the characters of the conidia and conidium ontogeny are identical in Lindau's
specimen and the synnematous specimens of
examined.
spreading rather than restricted agar colonies.
are sandy brown (), planar and powdery, growing 4-4.5 cm diam in 10 d on PDA
().
rhododendron fungus are slower, growing 2.5-3.5 cm diam after 21 d on MEA (not
shown). They are planar and greyish brown, with an orange-brown reverse. No
synnemata were observed in our cultures of the rhododendron fungus on MEA, OA
or PDA, but cladosporium-like conidiation occurred in the aerial mycelium.
phylogenetic relationships of the resin fungus (DAOM
239134), the creosote fungus (DAOM 170427,
194228) and the rhododendron fungus (DAOM 239136),
and subsequent analyses of the internal transcribed spacers were used to
estimate more precise affinities. shows the LSU analysis and demonstrates that appears to be a member of the is related to the inoperculate discomycetes
() and is most closely
related to a sequence labelled A. Funk &
Dorworth, which is unrelated to .
parsimony analyses were conducted, one with informative characters from the
full alignment, the second with a subset with 179 characters excluded from
seven ambiguously aligned regions. The consistency indices (full 0.301,
partial 0.324), tree topologies, and bootstrap supports for the two analyses
were relatively similar.
tree presented here ().
The data matrix included 57 taxa, with 352 of 752 characters phylogenetically
informative. clearly is related to
and allied anamorph genera, as suggested by the LSU analysis.
well-supported clade with Samuels, that is a
well-supported sister group to species now in three different anamorph genera,
(M.A. Rich & A.M.
, and an undescribed species of
Petr.
The ITS matrix for included 42 taxa, with 171
phylogenetically informative characters in the 530 base alignment.
phylogenetic analysis confirmed the relationship of this species with the
, and provided a more precise hypothesis of its
family-level relationships (). DAOM 170427 and 194228 had
identical ITS sequences to another strain of the same species reported in
GenBank (AY251067, from ), and one bp substitution from a second strain
(AF393726 based on the isotype ATCC 200942 =
).
four sequences formed a sister group to two sequences of
“ Morgan-Jones (AF393683,
AF393684).
, was previously noted by Braun
().
clade within two well-supported clades of spp.
associated anamorph genus which comprise the family
has not been documented previously.
and differed by one bp.
significant homologies only with unidentified fungi, and lower probability
matches with various members of the .
taxonomically meaningful phylogenetic analysis can be presented with these ITS
sequences.
, but the putative relationship with
, suggested by the LSU analysis, could not be confirmed
with the ITS analysis.
phylogenetic analysis all support the conclusion that the mononematous
synanamorph of , the resin fungus, is different from
the anamorph of , the creosote fungus.
ribosomal DNA sequences, the creosote fungus is related to the family
, the genus and its
anamorphs ().
are paraphyletic, with and the
nested within them. appears to be
an additional anamorph genus phylogenetically associated with
(,
).
between the synnematous and mononematous morphs of was
verified by morphological comparison of polyspore isolates derived from the
two synanamorphs.
connection was not confirmed with single conidium isolations.
specimen of
() is the basis for the
application of the most frequently used anamorph epithet for the creosote
fungus. This specimen represents the mononematous synanamorph of not the anamorph of .
micromorphologies are so different.
and shape, but in both morphs of (Figs
,
), the lateral walls are
conspicuously thickened, a condition not present in the creosote fungus
(), and the conidia are
much darker.
() noted that single
conidium isolates of gave rise to four different colony
types.
darker resin fungus was the same as one of his mutant forms of the creosote
fungus, despite never having isolated such a dark spored form from any of his
cultures.
diversity of hydrocarbon-rich substrates favoured the thought that it would be
able to grow on conifer resin.
persisted for so long.
fungus are so different (Figs
,
) that it would difficult to
defend the idea that they were mutants of the same fungus.
in the cultures are reflected by the disparate phylogenetic affinities of what
now are clearly demonstrated to be two different species.
to the creosote fungus, a species of economic importance.
this species is the type of , a generic name that the
community concerned with this fungus has been slow to adapt to in the 30 years
since its introduction. There are several possible solutions to this problem.
The conventional solution would be to apply names based strictly on the type
specimens and accept as a synonym of or
to use it as a generic name for the mononematous synanamorph of the resin
fungus.
making G.A.
type.
with the creosote fungus, and the earlier literature citing would be misleading.
A more parsimonious solution is possible. Article 14.9 of the International
Code of Botanical Nomenclature (McNeil . 2006) allows for
conservation of a name with a different type from that designated by the
authors.
because the mononematous synanamorph of the resin fungus is rarely referred to
by a Latin binomial, and because is based on a
different type.
for Lindau, preferably the holotype of (MELU 7130).
unequivocal, maintain current species epithets and taxonomic authorities, and
ensure that most of the historical literature can be interpreted easily
without the need to consult complicated nomenclators
().
perpetuating the use of the epithet “, this
change would also perpetuate the misunderstanding that resin is a possible
substrate for the creosote fungus.
the teleomorph of the creosote fungus, , is
legitimate and valid, and unlikely ever to be changed.
A third option would be an intermediate one.
G.A.
would be possible to conserve this species as the type of .
This has the advantage of maintaining the familiar generic name
, in combination with a species epithet that has been
consistently applied.
about the application and correct author citation around the epithet
“” for the anamorph of creosote fungus to
recede.
published in Taxon.
more length in that venue.
taxonomic problems.
, which has been considered
since its description by Parbery
().
analysis suggests that this family sits within the is the older name, but
is well-entrenched in the mycological literature.
are paraphyletic with respect to the
.
species presently placed in this family, lacks the thick-walled appendages
that characterise most species of the .
the acropetal-blastic features of the anamorph of differ
from the thallic-arthric conidiogenesis of the other anamorphs associated with
the , principally .
morphological differences explain why the affinity of with
the was not noted before.
conserve as the name for this family might be prudent
eventually, but this should await analysis of additional genes to confirm the
phylogenetic relationship.
discoloured wallpaper, is actually a distinct species from
requires further study.
would be a member of rather than .
Apart from the study of additional specimens, it might be fruitful to attempt
to induce an -like teleomorph in the two available
cultures of , and to compare the morphology with that
of .
(),
includes both homothallic and heterothallic strains.
were not established with certainty in this study.
LSU analysis is a sequence identified as
Funk & Dorworth (U43480, based on the apparent type culture ATCC 64711),
but this sequence does not cluster with others representing the family
(data not shown).
the rhododendron fungus did not cluster with the many ITS sequences of
available.
precise affinities are uncertain.
classified in (a taxonomic synonym of
), and continued recognition of the monotypic genus
seems justified. |
(Trejos) de Hoog, Kwon-Chung &
McGinnis is one of the most frequent etiologic agents of human
chromoblastomycosis, a chronic cutaneous disease characterised by verrucose
skin lesions eventually leading to emerging, cauliflower-like eruptions.
species is particularly observed in arid and semi-arid climates of e.g.
and Central America () and Australia (, ).
chromoblastomycosis are rural workers who acquire the infection after being
pricked by cactus thorns or splinters
(,
).
() concerns traumatic
inoculation with thorns of “guazábara” (), a common xerophilic plant in semi-arid Venezuela.
hypothesised traumatic route of infection was later supported by
Richard-Yegres & Yegres
(; strain SR3 =
) and
Fernández-Zeppenfeldt .
(), who isolated strains
from litter. Borelli has
also been detected in association with spines of the common xerophyte and of the : and
(,
).
Thorny American cacti are important components of the xerophyte flora of the
arid climate of our study area in Falcon State, Venezuela
().
supposed pathogenic invasive form of fungi causing chromoblastomycosis
().
species of cold-blooded animals have shown the abundant production of the
characteristic muriform cells
().
related agent of chromoblastomycosis, (Brumpt)
Negroni. Marques .
() isolated this species
from the shells of Babassu coconuts ().
of local people to sit on these shells might explain the frequent occurrence
of lesions on the buttocks (). Salgado .
() found the species on
the thorns of a plant which a patient could identify as
the source of traumatic onset of his chromoblastomycosis.
identification, doubt has arisen about the correctness of this supposed route
of infection.
represent exactly the same species needs to be re-determined.
establish this for -associated chromoblastomycosis,
reference strains from the CBS culture collection, supplemented with a large
set of strains from semi-arid Venezuela, have been verified using molecular
tools that are currently routinely employed to answer taxonomic questions in
black yeasts and their filamentous relatives
(), particularly the internal transcribed spacer (ITS) region
of rDNA, the partial β-tubulin gene (BT2), and an intron in the
translation elongation factor 1-alpha (EF1).
experiments has been conducted concerning inoculation into and superficial
application onto germlings of obtained by
cultivation , mature plants of from the
wild, and in spines of collected in the semi-arid area of
study.
life cycle of its associated spp., and to determine
whether a link could be made to for obtaining a better
understanding of human chromoblastomycosis.
Strains studied are listed in .
identified as .
collection, as well as fresh isolates from patients and the environment have
been included.
after deposit at CBS.
grown on slants of 2 % malt extract agar (MEA) and oatmeal agar (OA) at 24
°C.
grown on potato-dextrose agar (PDA) () and mounted in lactophenol cotton
blue.
transferred to a 2 mL Eppendorf tube containing 300 μL TES-buffer (Tris 1.2
% w/v, Na-EDTA 0.38% w/v, SDS 2 % w/v, pH 8.0) and about 80 mg of a silica
mixture (Silica gel H, Merck 7736, Darmstadt, Germany/Kieselguhr Celite 545,
Machery, Düren, Germany, 2 : 1, w/w).
in a tight-fitting sterile pestle for approximately 1 min. Subsequently 200
μL TES-buffer was added, the mixture was vortexed, 10 μL proteinase K
was added and incubated for 10 min at 65 °C.
material was incubated for 30 min at 65 °C.
30 min on ice water and centrifuged for 10 min at 14 000 rpm.
was transferred to a new tube with 225 μL 5 M NH-acetate,
incubated on ice water and centrifuged again for 10 min at 14 000 rpm.
supernatant was transferred to another Eppendorf tube with 0.55 vol
isopropanol and spun for 5 min at 14 000 rpm.
washed with ice cold 70 % ethanol.
re-suspended in 48.5 μL TE buffer (Tris 0.12 % w/v, Na-EDTA 0.04 % w/v)
plus 1.5 μL RNAse 20 U/mL and incubated for 15-30 min at 37 °C.
(BT2) and translation elongation factor 1-α (EF1), were sequenced.
ITS sequencing, amplification was performed with V9G
(5'-TTACGTCCCTGCCCTTTGTA-3') and LS266 (5'-GCATTCCCAAACAACTCGACTC-3').
Sequencing reactions were conducted with ITS1 and ITS4 primers
().
For BT2 amplification and sequencing, primers Bt2a
(5'-GGTAACCAAATCGGTGCTGCTTTC-3') and Bt2b (5'-ACCCTCAGTGTAGTGACCCTTGGC-3')
were used () and for EF1 amplification and sequencing, primers EF1-728F
(5'CATCGAGAAGTTCGAGAAGG-3') and EF1-986R (5'-TACTTGAAGGAACCCTTACC-3')
().
Sequences were aligned in BN v. 4.5 (Applied
Maths, Kortrijk, Belgium), exported and converted into P
interleaved format ().
Calculation of ILD (incongruence length difference) was performed in PAUP
v. 4.0b10 (). A
combined data set of ITS, EF1 and BT2 sequences was created.
criterion was set to parsimony.
equal weight, while 677 characters were constant, and 396
parsimony-informative.
tree-bisection-reconnection (TBR) was used as branch-swapping algorithm.
Maximum number of trees was set to 100 and left unchanged.
select a substitution model.
calculating the Akaike Information Criterion (AIC), corrected Akaike
Information Criterion (AICc), Bayesian Information Criterion (BIC), and Akaike
weights for nucleotide substitution models and model uncertainty.
P
().
All 56 models implemented in M
()
were evaluated.
sites (I) and/or gamma distribution shape parameter (G).
M and MA is that the latter does
not evaluate all models on the same, approximate topology as in PAUP
().
P was used to try to find the maximum of the likelihood
function under all models.
the models.
to characters (Nchar/Nparameters < 40;
)
for all loci.
trees.
with P was the obtained accuracy of tree topology and the
greater calculation speed ().
number of populations in the complex was inferred with
S v. 2.2 () using genotype data of the ITS regions of
rRNA gene and of the partial EF1 and BT2 genes.
of 43 isolates were sorted on the basis of sequence similarity.
S is a model-based clustering method for using multilocus
genotype data to infer population structure and assign individuals to
populations.
set to 10, number of MCMC repeats after burn-in 30 000; the
ancestry model: admixture (individuals have mixed ancestry and is recommended
as starting point for most analyses). Uniform prior for ALPHA was set to 1.0
(default) and all allele frequencies were taken as independent among
populations with λ set to 1.0 (default).
estimating K) was also computed ().
MCMC repetitions after burn-in were set as 10 000 and 100 000, and admixture
model and allele frequencies correlated model were chosen for analysis.
number of populations (K) was assumed from two to four.
in BN.
population, index of association (I, a measure of multilocus
linkage disequilibrium) was calculated with M v. 1.2.2
().
The null hypothesis for this analysis was complete panmixia.
I were compared between observed and randomised data sets.
hypothesis would be rejected when p < 0.05.
(index: theta, θ) was also detected using the same software and a null
hypothesis for this analysis is no population differentiation.
θ is statistically significantly different from those of random datasets
(p < 0.05), population differentiation should be considered.
A reticulogram was reconstructed using T-
(,
)
()
on sp.
classical additive tree using one of the five available tree reconstruction
algorithms.
edge) was chosen that minimised the least-squares or the weighted
least-squares loss function; it was added to the growing reticulogram.
statistical criteria (Q1 and Q2) were proposed to measure the gain in fit when
reticulations were added.
stopping rule for addition of reticulations.
transfer) reticulogram reconstruction option
() the program
mapped the gene tree into the species tree using the least-squares method.
Horizontal transfers of the considered gene were then shown in the species
tree.
species tree and compared with a gene tree, EF1.
horizontal gene transfer were also visualised using
ST v. 4.8 software
().
Split decomposition () was applied on three loci of the entire complex.
characters transformation using uncorrected P-values, equal angles and
optimise box iterations set to 1.
development, while reticulation indicates genetic exchange.
the house of a patient with chromoblastomycosis due to strain UNEFM 9902 =
() in Sabaneta (Miranda, Falcón State, Venezuela), were
analysed. Four fragments of approx. 2 × 3 × 1 cm were excised from
each plant at brownish superficial lesions in upper branches.
fragments were soaked in mineral oil for 15 min at 23 °C under agitation
at 150 rpm ().
per sample on agar slants.
morphology similar to
()
were selected.
by determining the ability of strains to grow at 35, 37, 38 and 40 °C, and
whether they could break down 20 % gelatin
(,
). Environmental strain UNEFM-SgSR3 =
( sp.) and clinical strain UNEFM 9902 =
() were selected for the inoculation experiments.
(SGA) was transferred to 50 mL YPG medium (yeast extract 0.5 %, peptone 0.5 %,
glucose 2 %) (), shaken at 150 rpm and incubated for 3 d at 23 °C
().
Five mL aliquots of the starter culture were transferred serially every 4 d to
500 mL flasks containing 100 mL synthetic medium (-glucose 2 %,
KHPO 0.2 %, NHSO 0.1 %, urea
0.03 %, MgSO 0.03 %, CaCl 0.003 %; pH 6.2) shaken at
150 rpm at 23 °C.
conidia, were filtered through sterile gauze, ground in 50 mL 0.85 % saline,
centrifuged at 2 000 rpm, and repeatedly washed with saline until a clear
supernatant was obtained. The suspensions were adjusted to 5 ×
10 cells/mL (, ).
lactritmel medium ().
laboratory ()
by cultivation from seeds of a single cardon fruit collected near the house of
the patient infected with in the endemic area for chromoblastomycosis in Falcon
State, Venezuela.
contents washed by agitation for 10 min at 120 rpm in 250 mL sodium
hypochlorite 4 % (v/v), and subsequently with sterile distilled water at 120
rpm for 5 min.
seeds were incubated for 3 h, decanted and washed repeatedly with sterile
distilled water. Seeds were then dried for 24 h on filter paper at 37 °C.
Onset of germination was obtained by incubation of the seeds in a moist
chamber on filter paper for 15 d under alternately 8 h of continuous white
light (26 W) and 16 h of darkness; bud emergence was observed daily.
of 1 cm in length, with green colour and having two leaves were transplanted
to 128-container germinators until roots developed.
contained 5 parts Sogemix® and 1 part river soil from the region where the
fruit was collected.
every 10 d with 5 mL sterile tap water for 1 yr.
Fungal suspensions (0.1 mL) were either injected using a syringe (13
× 0.4 mm) at a depth of approximately 5 mm into cortical tissue
(), or superficially
applied onto () 96
randomly selected 1-yr-old plants: 50 % using clinical strain
() and 50 % using environmental strain
( sp.).
treated similarly, but using sterile saline (0.85 %).
inoculated plants stayed in the laboratory under the conditions specified
above.
plants of each treatment were sectioned longitudinally from the apex and
transversely by means of a hand-held microtome, examined directly in glycerin
water (25 %), and cultured in lactritmel medium
().
macroscopically visible lesions, were dug from an area within a 50 m radius of
the house of the patient with chromoblastomycosis as specified above.
were transported to the laboratory and transplanted individually into
polyethylene bags with a capacity of 1 kg, using as substrate river soil from
the same area.
laboratory to adjust at average temperatures of 32 °C and with natural
daylight.
period of 6 mo.
For inoculation purposes, 150 sharp, wooden toothpicks 4 × 0.3
× 0.2 cm were washed and boiled for 3 min in tap water to eliminate
resins (). This procedure was repeated three times.
50 toothpicks each were kept separate in Petri dishes.
for 15 d at 23 °C after inoculating each batch with 1 mL fungal suspension
(5 × 10 cells/mL) of either strain
or
, with
sterile water as control.
inoculated () halfway up
the shaft with a toothpick colonised with
(), ( sp.) or the control
().
15 d, and tissue samples taken at the point of inoculation, and from the
thorns directly adjacent to this area.
hypochlorite for 3 min, and subsequently washed in sterile distilled water for
re-isolations, and for histological study by means of light microscopy
().
Ninety cactus spines of 2.5 cm av.
plant located near the home of the patient infected with
, at
approx. 2.5 m height, superficially sterilised, and divided into three groups,
of which 30 spines were inoculated with
(), 30 with ( sp.) and 30 to be used as
control, inoculated with a saline solution
().
composed of 90 spines of 1.5 cm average length was collected at approx. 1 m
height.
(Whatman #1) with 2 mL saline solution; subsequently 0.1 mL fungal suspension
was applied.
sectioning with a hand-held microtome, cultured as above and observed
microscopically until day 75 post incubation.
were evaluated using the X-test (P = 0.05 was considered
significant).
Student's T-test (P = 0.01 was considered significant).
The rDNA ITS region was sequenced for 43 strains identified as based on morphology.
downloaded from GenBank.
cladophialophora-like species were added, with
as
outgroup. In , 203 positions were compared in ITS1, 158
in the 5.8S rRNA gene and 182 in ITS2
().
be aligned with confidence over their entire lengths.
(), the two remaining
being variable T-repeats near the ends of ITS1 and 2.
For ITS sequences the AICc selected the TrN+G model (TrNG;
).
base frequency of ITS: T = 0.2467, C = 0.2897, A = 0.2247, G = 0.2390, TC =
0.5364, AG = 0.4636.
base frequency of EF1: T = 0.2990, C = 0.2665, A = 0.2123, G = 0.2221, TC =
0.5655, AG = 0.4345. The best model for BT2 sequences was the SYM+I+G
(symmetrical model). The base frequency for BT2: T = 0.2255, C = 0.2953, A =
0.2463, G = 0.2328, TC = 0.5208, AG = 0.4792.
were calculated with PAUP using parsimony and with maxtrees set to 500 and 500
replicates (data not shown). Total number of characters was 191 of which 101
were parsimony-informative. Tree length was 365 and had the following indices:
Consistency Index = 0.685, Retention Index = 0.542 and Homoplasy Index =
0.315.
combined data, L was 1 062.
difference L = (L-L) was 7 (P = 0.24).
ILD was not significantly greater than expected by chance and it was concluded
that the sequences were congruent and could be used together in a combined
analyses.
recombination.
exception of separation of and with EF1 ().
, was analysed in more
detail. With ITS, four groups were recognised (A-D;
). (A) was the main
group with 36 strains/sequences; FMC 248 differed only by a small T-repeat and
was regarded as a member of (A).
from group (A) maximally by two consistent positions
().
comprised sequences from GenBank and all originated from Abliz .
().
group (C), IFM 4808, concerned a subculture of
, which
is an original isolate of Trejos
() representing . Re-sequencing indicated that it was a member of group (B).
Analysis of our electropherograms of this isolate was not suggestive of
heterothallism.
found to differ in group (D), which deviated in 16 mutations in ITS1 and 8 in
ITS2; 17 of the mutations were transitions, 7 were transversions and 7 indels.
Group (D) was clearly distinct from the complex of (A)-(C), with a total of 27
mutations.
compared. Sequences of the 205 bp long element of EF1 contained 32
phylogenetically informative mutations.
(I-III; ). With BT2,
three groups with the same composition were recognised.
(C) were not available for study.
On the basis of multilocus screening in BN,
concordant groups (A)-(D) were tested with the S programme.
When K was set at 4 or 5, consistent groupings were noted, indicated as I, II
and III (), corresponding
with ITS groups (A), (B) and (D), respectively in
.
morphologically similar but phylogenetically unrelated group of fungi was
excluded by SSU sequencing.
, such as Link,
Seifert & N.L. Nick.,
Munt.-Cvetk., U. Braun and Syd.
proved to be remote (data not shown).
rather than between groups (B) and (A), despite the high sequence similarity
of (A) and (B) ().
(A)-(C) generally had conidiophores that arise at right angles from creeping
hyphae (), while those of
(D) tend to be ascending, hyphae gradually becoming conidiophore-like.
slight correspondence was found in independent markers and phenetic criteria,
we considered group (D) to represent a separate species, which is described as
follows.
.
,
.
: Named after Francisco Yegres, Venezuelean
mycologist.
pulverulentae vel velutinae, margine integra; reversum olivaceo-atrum.
fertiles dilute olivaceo-virides, ascendentes, paulatim in catenas conidiorum
concolorium vertentes.
olivaceo-viridia, levia et tenuitunicata, 4.5-6 × 2.5 μm, faciliter
liberata, cicatricibus modice pigmentatis.
absentes. Synanamorphe phialidica non visa. Teleomorphe ignota.
Holotypus cultura sicca in herbarium CBS praeservatur.
velvety, with entire margin; reverse olivaceous black.
pale olivaceous green, ascending, gradually changing over into concolorous
chains of conidia. profusely branched.
pale olivaceous green, smooth- and thin-walled, 4.5-6 ×
2.5 μm, detached rather easily, with slightly pigmented scars.
and yeast cells absent.
observed. Teleomorph unknown.
: , Falcon state, from
asymptomatic cactus, G.
Fernández-Zeppenfeldt,
, culture ex-type
= UNEFM
SgSR3.
: Of the 48 dematiaceous isolates obtained from 36 fragments
of the cactus , four strains originating from four
different plants of presented morphological and
physiological key characteristics of or
(de Hoog .
,
).
was negative in all strains and the maximum growth temperature was 37 °C.
After identification to species level using sequence data
(), both and
appeared to be among the strains isolated.
spines, and an average height of 15 cm.
fungal suspensions of the test strains
(, clinical) and
(, environmental) remained without visible external lesions during
the year of experimentation.
consistently revealed internal growth of the fungi in their filamentous form.
Muriform cells were not observed, neither on the epidermis, nor in the
internal tissue, spines or roots.
viability of the fungi during the entire experimental process:
() was grown from 26 (54.16 %) of the plants and
() in 23 (47.90 %) of the plants.
reveal significant differences between the isolates (Xc = 0.0729
< Xt = 3.84).
lesions, and in the histological sections no internal or external fungal
elements were observed.
proved to be a species of
With 96 plants with superficial application of spore suspension (48 plants
for each isolate, either clinical or environmental) neither internal nor
external lesions were observed.
elements in or on plant tissue.
were occasionally seen around and inside the outer layers of the spines.
re-isolated strains proved that the fungi survived during the entire
experimental procedure: () was isolated from 32 (66.67 %)
plants and () from 33 (68.75 %).
isolates (Xc = 0.4375 < Xt = 3.84).
of 1.88 cm diam with and 1.33 cm diam with , around the point of inoculation.
100 plants, dark, septate hyphae with inflated elements were observed at the
points of inoculation. Muriform cells were not observed.
were evidence of isolate viability:
() was grown from 36 (72 %) plants and
() from 30 (60 %). The fungi could not be isolated from spines.
The 50 plants used as controls showed scarring of 1.06 cm diam on average
around the point of inoculation.
examinations and histological sections of these plants.
were negative.
environmental strain and control proved to be highly significant:
Clinical .
: þ
= 0.000832, P = 0.01;
Clinical . control: þ = 0.00003128, P = 0.01;
Environmental . control: þ = 0.005343, P = 0.01.
Spines 2.5 cm av.
() and (), developed toruloid hyphal elements
with some dark, swollen cells similar to muriform cells known in human tissue.
The re-isolated strains proved the species to survive during the experimental
procedure (< 75 d). Similar results were obtained with the spines 1.5 cm
av. in length.
controls.
that are consistently associated with pathology to humans belong to the
in the order
().
Within this order, the genus is polyphyletic.
Conidia of all species are produced in branched chains on poorly
differentiated hyphae.
confusion with morphologically similar but unrelated fungi that are
encountered as contaminants in the hospital environment.
comprises ubiquitous airborne fungi which mostly have
erect, more or less differentiated conidiophores, and dark conidial scars.
They are associated with Crous & U.
and belong to the , family
(,
).
was introduced by Braun
() with three species
differing from mainly by intercalary hyphal cells
with lateral extensions that bear conidial chains, having
U.
-
this volume).
in the
().
The anamorph genus comprises thermophilic saprobes with a
cladophialophora-like appearance and producing dark, multi-celled
chlamydospores alongside the hyphae.
the , in the
().
() on the basis of 46
strains from Venezuela, Australia and South Africa.
holotype.
first strain mentioned by Trejos
(), is selected here as
representative for .
been deposited as in the Herbarium of the Centraalbureau voor
Schimmelcultures as .
, proved
to be indistinguishable from , which was also known to be
able to produce phialides in addition to catenate conidia
().
Remarkably, a strain identified as from Samoa
(;
)
proved to be related to but consistently different from all strains of the
complex.
health carrying this fungus had a 5 × 3 cm erythematous, scaling lesion
on his arm.
corneum.
chromoblastomycosis.
taxon, as this is a synonym of .
formally described in a forthcoming paper.
correctly assigned to .
melanised acropetal chains of conidia, near absence of conidiophores, and
phylogenetic affinity to the order .
Negroni and Medlar.
From a point of view of human disease, the species of the clade were known as
agents of brain infection [ (Sacc.) de Hoog (M. Moore & F.P. Almeida) de Hoog ],
disseminated disease [ (A.A.
], cutaneous disease [ (Borelli) de Hoog
] and particularly chromoblastomycosis ().
groups (A)-(C) were separated on the basis of five mutations in the ITS
region, which were supported by mutations in EF1 and BT2, as confirmed by
analysis in S, where the same separation (K = 5) of
entities was observed. Furthermore, K = 4 unites groups (B/II) and (D/III),
despite the fact that the sequence of (B) is more close to those of (A).
T- software a similar relationship between [(B), ] and [(D), ] was noted, suggesting
horizontal gene flow between these entities.
while (D) is found in equally remote localities in Venezuela.
reticulation was observed in all genes with ST.
With ITS and BT2, and cluster closely together, while in the more variable EF1
data these are all widely apart, suggesting that in
other mechanisms than recombination may occur.
from a single study ().
of data from Abliz .
(), was the same isolate as
, which
was found repeatedly in group (B) in our data set
().
was observed with strain IFM 41444 =
, of
which GenBank deposition AB109169 consistently deviated from our data in a
frequently observed mutation.
sequence conflicts is heterozygosity.
supposed to be haploid (; ), some strains have a double DNA content in yeast cells
().
Teleomorphs are not known in and related black
yeasts, but many species are known to form profuse hyphal anastomoses
(), allowing parasexual processes and mitotic recombination.
However, all electropherograms including those from the study of Abliz . (), which were
kindly sent by K.
peaks.
frequent anastomoses in J.W. Carmich.
().
An alternative explanation might be the occurrence of paralogous ITS repeats,
as reported earlier in Link
().
S shows some geographical structuring of populations, in
that group (A) does not occur in Asia, group (B) is limited to Australia and
Africa, and group (C) has thus far only been reported from Asia.
distribution of most genotypes suggests, however, that worldwide occurrence is
likely to become apparent when more strains have been analysed.
zones where was isolated were semi-arid to arid,
desert-like.
thus a relatively rapid vector of dispersal has to be hypothesised enabling
the fungus to cross climate zones where the saprobic phase is unable to
survive. Kawasaki .
() analysed three further
loci in mtDNA using RFLP.
sequencing. These had all identical mtDNA profiles, with the exception of IFM
4808 = ,
that differed in two markers ().
(A) and (C) (see above), the conclusion is warranted that mtDNA allows
distinction of polymorphism at the same level of diversity as detected in this
study with ITS, EF1 and BT2.
South America harbours group (D) which represents a second species, .
to be restricted to living cactus plants.
. ()
published a strain from chromoblastomycosis in China which matched the
morphology of strains now classified as , but as far as we
are aware this strain has not been sequenced.
human infection and only occasionally on dead plant debris, mainly seceded
cactus needles.
isolated from living, asymptomatic plants surrounding the
cabin of a symptomatic patient from whom ,
was
isolated.
infections originate from puncture by plant material (e.g.,
),
it now becomes clear that the environmental look-alikes of clinical strains do
not necessarily belong to the same species
(,
),
but may be members of other, related taxa with slightly different ecology; an
unambiguous connection between a clinical and an environmental strain still
has to be proven.
, has a semi-arid climate, with average yearly temperatures
of 24 °C, scarce rainfall (up to 600 annual mL) and is located at moderate
altitude (up to 500 m) (, ).
xerophytes. is a columnar American cactus with a
very strong, protective external epidermis that allows the accumulation of
water in the shaft and enables tolerance of extreme drought.
produces ovoidal, thorny fruits of about 5 cm diam, which are commonly eaten
by the local population.
chromoblastomycosis acquire their infection by traumatic inoculation with
cactus spines, similar to the supposed infection process of (Laveran) Brumpt in the arid climate of Africa
().
The frequent occurrence of 16/1 000 for chromoblastomycosis in areas endemic
for in Venezuela
(;
) indicates a marked invasive potential for .
().
Nevertheless, virulence of is low when inoculated into
the footpads of mice (); also an environmental strain of failed to produce lesions in mice and in a volunteer
().
We performed inoculation experiments with and using freshly grown, healthy cacti in the greenhouse.
were followed over a 1-yr period; during all this time the control plants
remained without lesions.
produce infection when syringe-inoculated deep into young cactus tissue.
Histopathology showed septate hyphae between host cells, and the shaft was
maintained over prolonged periods without causing visible damage.
of appreciable destruction would categorise them as endophytes.
is rich in carbohydrates, vitamins and minerals
() which may promote endophyte growth.
inoculation into mature plants: the clinical strain was
consistently more virulent than that originated from the
same host plant.
cultures.
the epidermis is broken, as happens e.g.
sap-sucking birds or piercing insects.
to meristematic morphology
() when entering hard spine tissue.
conversion is the dominance of lignin in the spines.
is enhanced by their capturing of atmospheric water formed after nightly
condensation.
around and inside the spines suggests that the spines play a role in mechanic
dispersion of the fungi.
inoculation into living tissue of humans or animals, where the same muriform
cells are formed, defining the skin disease chromoblastomycosis.
questioned whether animal/human inoculation plays a role in the evolution of
the fungus. ITS differences between the two species are observed in 23
positions, with a ratio of transitions : transversions of 2 : 1
().
of mutations has taken place and the diversification can be regarded as an
example of recent sympatric speciation. is
widely distributed, and shows a higher degree of diversity than .
existence and then should be regarded as ancestral to
, with the latter showing a founder effect due to the
absence of polymorphisms.
humans to cactus) is difficult to imagine.
its muriform cells.
of with African and Australian rather than Venezuelean
strains of .
variation in is not a founder effect, but rather a
sampling effect, as living cacti have thus far not been studied outside the
framework of our study on the patient with
chromoblastomycosis.
which lives as a saprobe on dead cactus debris for part
of its life cycle, and is less adapted to an endophytic life style.
materials, such as wood chips of and wooden remains
of and
(,
,
).
pathogenicity to humans, as also pathogens like (Sanfelice) Vuill.
their life cycle in hollows of trees. produces diphenol oxidase to degrade lignin, an aromatic
polymer in the cell wall of plants and a component of wood
().
degradation pathways are present in
().
association with xerophytes has been proven, but their environmental route of
dispersal is still unknown.
place when the hyphae reach the spines and on dead spines, the muriform cell
apparently is the extremotolerant phase of the species.
can be found sporulating on rotten spines directly after rainfall
(), but as the fungus has thus far never been isolated from
outside air, it is still unclear how a new host plant is reached.
characteristic disease, chromoblastomycosis, of which the agents are limited
to the ascomycete family
()
is puzzling.
rather than hyphae, and thus humans do not seem a natural reservoir of the
fungus. Nevertheless some acquired cellular immunity seems to be involved.
Albornoz . ()
demonstrated that a significant share of the local population of goat keepers
()
is asymptomatically infected with ; Iwatsu () detected
cutaneous delayed hypersensitivity in rats experimentally-infected with agents
of chromoblastomycosis.
fungus , Ahrens .
() found enlargement and
metastasis of lesions in athymic but not in normal mice, or in mice with
defective macrophage function.
(,
,
) observed a
significant role of acquired cellular immunity in , while
Cardona-Castro & Agudelo-Flórez
() obtained chronic
infection in immunocompetent mice when inoculated intraperitoneally.
Pires . ()
noted an unbalance between protective Th1 and less efficient Th2 responses.
The possible host response leads to different clinical types, referred to as
tuberculoid and suppurative granuloma, respectively.
constitutional factors in susceptibility is underlined by a marked frequency
of family relationships among symptomatic individuals
().
possibly due to their high body temperature (≈ 39 °C).
hyphal fragments artificially inoculated into goats led to transformation into
muriform cells, but the lesions disappeared within 60 d
().
according to new taxonomy will be necessary to answer questions on the role of
the fungus on warm-blooded animals. |
Cladosporioid hyphomycetes are common, widespread fungi.
Link is based on the type species, (Pers.: Fr.) Link, which in turn has been linked to
Crous & U.
(,
- this volume). is one of the largest, most
heterogeneous genera of hyphomycetes, comprising more than 772 names
(),
and including endophytic, fungicolous, human pathogenic, phytopathogenic and
saprobic species.
ways.
of senescing and dead leaves and stems of herbaceous and woody plants, as
secondary invaders on necrotic leaf lesions caused by other fungi, are
frequently isolated from air, soil, food stuffs, paint, textiles and other
organic matters, are also known to be common endophytes
(,
,
) as well as
phylloplane fungi (, , , , ).
to be potential agents of medical relevance.
is, for instance, a common contaminant in clinical laboratories and causes
allergic lung mycoses (, - this volume).
modern revision of , but some attempts to revise and
monograph parts of it have been initiated during the last decade
(,
, , , , , , Schubert & Braun
,
2005,
,
,
,
Schubert 2005,
,
).
()
have shown spp.
monophyletic Johanson cluster, suggesting a position
apart from the latter genus.
() carried out more
comprehensive sequence analyses, based on ITS (ITS-1, 5.8S and ITS-2) and 18S
rDNA data, providing further evidence that
represents a sister clade of .
(, ,
, ),
reaching different conclusions.
, leading to a more natural concept of this genus, was
published by David (), who
carried out comprehensive scanning electron microscopic examinations of the
scar and hilum structure in and
Klotzsch ex Cook.
these structures, published by Roquebert
(), indicated that the
conidiogenous loci and conidial hila in are
characterised by having a unique structure.
() confirmed these
observations, based on a wide range of and
species, and demonstrated that the structures of the
conidiogenous loci and hila in the latter genus fully agree with those of
, proving that was indeed a
synonym of .
“coronate” for the scar type, which is
characterised by having a central convex part (dome), surrounded by a raised
periclinal rim (),
and showed that this type is confined to anamorphs, as far as experimentally
proven, connected with teleomorphs belonging in
“” .
confirmed in a later phylogenetic study by Braun .
(). was shown to be a sister clade to ,
for which the new teleomorph genus was proposed.
no clear morphological differences were reported between
and , a further study by Aptroot
() found ascospores of
to have characteristic irregular cellular inclusions
(lumina), which are absent in species of , along with
periphysoids and pseudoparaphyses
( - this volume).
by Schoch .
(), which employed DNA
sequence data of four loci (SSU nrDNA, LSU nrDNA, EF-1α, RPB2), revealed
species of to cluster in a separate family
() from species of ), with both families residing in the ), and not as always presumed.
The current circumscription of emend.
summarised as follows: Davidiella
The new concept of , supported by molecular
data and typical coronate conidiogenous loci and conidial hila, rendered it
possible to initiate a comprehensive revision of .
The preparation of a general, annotated check-list of was the first step in this direction
().
The aim of the present study, therefore, was to delineate .
.
standardised conditions on a set of predescribed media
( - this volume), and subjected to DNA sequence analysis of
the LSU nrRNA gene.
(CBS), or freshly isolated from various substrates
().
cultured on 2 % malt extract plates (MEA;
), by
obtaining single conidial colonies as explained in Crous
().
subcultured onto fresh MEA, oatmeal agar (OA), potato-dextrose agar (PDA) and
synthetic nutrient-poor agar (SNA) (), and incubated under near-ultraviolet light
to study their morphology.
on OA and PDA at 25 °C in the dark, using the colour charts of Rayner
().
novelties and descriptions were deposited in MycoBank
().
isolated following the CTAB-based protocol described in Gams .
().
spanning the 3' end of the 18S rRNA gene (SSU), the first internal transcribed
spacer (ITS1), the 5.8S rRNA gene, the second ITS region and the 5' end of the
28S rRNA gene (LSU).
overlapping sequences are obtained.
subsequent phylogenetic analysis followed the methods of Crous .
().
5.8S rRNA gene (ITS) were only sequenced for isolates of which these data were
not available.
GenBank where applicable.
events for the phylogenetic analyses; the remaining gaps were treated as
missing data.
() and alignments in
TreeBASE
().
of structures mounted in lactic acid or Shear's solution
(),
with the extremes of spore measurements given in parentheses.
observations were made from colonies cultivated for 7 d under continuous
near-ultraviolet light at 25 °C on SNA as explained in Schubert . ( - this
volume). Three classes of conidia are distinguished. are
defined as short apical branches (often conidiogenous cells) of a conidiophore
which secede and function as conidia.
truncate, undifferentiated base, i.e., they differ from true conidia by
lacking characteristic basal hila caused by conidiogenesis.
rise to branched or unbranched conidia. are
branched conidia with a narrowed base, bearing a true hilum, that can occur in
chains, giving rise to , which differ from secondary
ramoconidia with regards to shape, size and septation.
on and allied genera, the true ramoconidia have often
been classified as “ramoconidia .” whereas the
secondary ramoconidia have been named “ramoconidia .”
Amplicons of approximately 1 700 bases were obtained for the isolates
listed in .
generated sequences were used to obtain additional sequences from GenBank,
which were added to the alignment.
contained 73 sequences (including the two outgroup sequences) and 996
characters including alignment gaps.
phylogenetic analysis, 336 were parsimony-informative, 77 were variable and
parsimony-uninformative, and 436 were constant.
using three substitution models on the sequence data yielded trees with
identical topologies to one another.
same clades as obtained from the parsimony analysis, but with a different
arrangement at the deeper nodes, which were poorly supported in the bootstrap
analyses or not at all (for example, the and
are swapped around).
with gaps treated as new characters increases the number of equally
parsimonious trees to 94; the same topology is observed but with less
resolution for the taxa in the (data not shown).
Forty-four equally most parsimonious trees (TL = 1 572 steps; CI = 0.436; RI =
0.789; RC = 0.344), one of which is shown in
, were obtained from the
parsimony analysis of the LSU sequence data.
found to belong to the and as
sister taxa to the in the .
with sequences for (Matsush.)
P.M. Kirk and (Matsush.) P.M.
as well as related sequences from GenBank.
Bayesian analysis using a general time-reversible (GTR) substitution model
with inverse gamma rates and dirichlet base frequencies and the temp value set
to 0.5.
a random tree topology and lasted 1 000 000 generations.
1 000 generations, resulting in 1 000 trees. Burn-in was set at 200 000
generations after which the likelihood values were stationary, leaving 800
trees from which the consensus tree () and posterior probabilities (PP's) were calculated.
standard deviation of split frequencies was 0.018459 at the end of the run.
The same overall topology as that observed using parsimony was obtained, with
the main exception that the and
swapped around, as observed with the distance analysis.
phylogenetically unrelated to, and morphologically distinct from
).
below:
Phylogenetic studies conducted on species of
proved the genus to be highly heterogeneous
().
It could be demonstrated that various anamorphs, previously referred to as
, e.g. Cooke [≡
(Cooke) U.
since they clustered in the clade
().
human pathogenic species, including morphology,
biology/ecology, physiology and molecular data
(, , , , ; ), could also be confirmed.
phylogenetic analyses, it could be shown that the human pathogenic fungi
concerned formed a clade belonging to the Sacc./ Borelli).
anamorphs with catenate conidia, previously often assigned to , clustered together with other anamorphs of the
and formed a monophyletic clade
(,
,
).
has now also been shown to accommodate less well-known
anamorph genera such as , which represent an
additional synonym of Bonord.
( -
this volume).
() examined morphological,
ecological and molecular characters of
(W.B. Kendr.) M.B.
them in the new genus , which formed a monophyletic group
apart from the clade. Crous .
() erected the genus
Crous for a saprobic species ()
characterised by having narrowly ellipsoidal to cylindrical or fusoid,
0-1-septate, medium brown, thick-walled, finely verruculose conidia arranged
in simple or branched chains, with thickened, darkened, refractive hila, with
a minute central pore. E.W.
agent of banana speckle disease, has recently been shown to be allied to the
(), and was placed in a new genus,
with as type species.
U. Braun, Heuchert & K. Schub. (type species:
Steyaert) and U.
Braun, Heuchert & K. Schub. (type species:
Deighton) represent two new genera of hyperparasitic hyphomycetes, introduced
due to unique morphological features and striking differences to
(), but have as yet been excluded from
DNA-based studies due to the absence of cultures.
( - this volume)
introduced a new genus, K. Schub., U.
fungus with dimorphic fruiting that is pathogenic to spp.
present study introduced yet several additional cladosporium-like genera,
which could be distinguished based on their morphology and distinct DNA
phylogeny, namely ),
), and ).
been confused with ., the unique coronate scar
type of allows a critical revision of
cladosporioid hyphomycetes, based on reliable, distinctive morphological
characters. In all cases where cladosporium-like (.) hyphomycetes clearly clustered apart from in the phylogenetic analyses, it could be demonstrated that the
fungal groups concerned were also morphologically unambiguously distinguished,
above all with regard to the structure of the conidiogenous hila.
excluded groups of species, belonging in other genera, sometimes even in new
genera, are genetically as well as morphologically clearly distinct from |
EGFP-transgenic donor mice were used for BM transplantation (BMT) into irradiated recipient animals before these were subjected to infarction and G-CSF/SCF treatment. As expected, ligation of the left anterior descending coronary artery (LAD) caused anterior myocardial infarction as evident by replacement of myocardium with fibrotic scar tissue 5 wk after the infarction (). Morphometric analysis of the hearts of 15 BMT animals (4 G-CSF/SCF treated and 11 untreated) killed 5 wk after induction of myocardial infarction identified EGFP (BM-derived) cells predominantly in the area of myocardial infarction and its border zone (). Furthermore, G-CSF/SCF led to a twofold increase of homing of EGFP cells to the infarcted area and the border zone of myocardial infarction (P < 0.05 for G-CSF/SCF vs. controls). In agreement with this finding, G-CSF/SCF led to a threefold rise in white blood cell concentration in the peripheral blood, accompanied by a doubling of the neutrophil proportion in splenectomized mice (not depicted). In 10 BMT animals not subjected to infarction (five G-CSF/SCF treated and five untreated), the accumulation of EGFP cells 5 wk after G-CSF/SCF treatment was 4–6-fold lower than in the non-infarcted myocardial tissue of animals with myocardial infarction ().
98–99% of EGFP cells in the myocardium were white blood cells as evident by coimmunolocalization of CD45 (), regardless of location (infarcted vs. non-infarcted area), and protocol type (G-CSF/SCF vs. control). In >100 sections from the hearts of G-CSF/SCF and control animals, only one troponin T/EGFP cell with central nucleus, myofilaments, and cross-striation () was identified in an area of infarction. In the non-infarcted areas, troponin T/EGFP cells were casually found (up to five cells per cross section).
Because G-CSF and SCF were administered systemically, expression of the corresponding receptors was analyzed in the heart to find evidence for potential direct effects on myocardial tissue. G-CSFR expression was evaluated by immunofluorescence microscopy 1 d and 5 wk after induction of myocardial infarction as well as by quantitative real-time RT-PCR at six time points after infarction. 1 d after myocardial infarction, prominent G-CSFR expression in the non-infarcted area was predominantly detected in small interstitial and vascular cells (, ), whereas in the infarcted area, cardiomyocytes also exhibited weak G-CSFR immunofluorescence (, ). In contrast, 5 wk after induction of myocardial infarction, cardiomyocytes exhibited marked G-CSFR expression both in the non-infarcted area and in the border zone (, and ). Cardiomyocytes, immunopositive for G-CSFR, display a bright red corona, indicating membrane localization of the receptor. Animals investigated 1 d after myocardial infarction were chimeras with EGFP-expressing BM (see Materials and methods). Because the small interstitial cells, immunopositive for G-CSFR, were not EGFP, these cells were not of hematopoietic origin but were resident myocardial cells. In contrast to G-CSFR, the receptor for SCF (c-kit) was not detectable by immunofluorescence 5 wk after myocardial infarction (not depicted).
Real-time RT-PCR analysis of myocardial tissue of untreated animals revealed an up-regulation of G-CSF and G-CSFR mRNA levels within the first 24 h after induction of myocardial infarction, which was more pronounced in the affected tissue than in the non-infarcted area ().
Myocardial infarction induced by LAD ligation led to regional wall motion abnormalities of the anterior wall, reduced fractional shortening, and typical electrocardiogram (ECG) changes seen after anterior myocardial infarction in the mouse (). Infarction size was relatively homogeneous throughout the experiments and did not differ between G-CSF/SCF hearts and controls, neither in pathological measurements () nor in vivo, assessed by the akinetic area of the left ventricle on echocardiography (G-CSF/SCF treated 5.4 ± 0.8 mm vs. controls 6.7 ± 1.1 mm; P = 0.39). Also, left ventricular contractility did not differ between G-CSF/SCF-treated mice and controls. It is worthy to note that fractional shortening showed a trend toward better function, and septal wall width bordering the area of myocardial infarction showed a trend toward greater thickness in G-CSF/SCF mice. Despite the lack in infarct size reduction, cardiac output was significantly increased in G-CSF/SCF mice compared with controls ().
Based on reports of proarrhythmic effects provoked by the transplantation of skeletal myoblasts (), the proarrhythmic risk of the increased homing of BM-derived cells was assessed by observing spontaneous ventricular rhythm after atrioventricular nodal block (–). This protocol did not provoke more spontaneous arrhythmias in G-CSF/SCF-treated hearts compared with controls (7 out of 25 G-CSF/SCF-treated hearts with spontaneous arrhythmias vs. 7 out of 26 controls; P = NS). Furthermore, ventricular action potential durations were shorter in G-CSF/SCF mouse hearts when pacing rates were high, whereas action potential durations were not different between groups during long pacing cycle lengths ().
Ventricular arrhythmias after myocardial infarction are often a result of conduction slowing in the border zone of the infarcted myocardium and associated with decreased intercellular connections in this region (). Programmed stimulation, the best assay to provoke such reentrant arrhythmias, provoked less ventricular tachycardias in G-CSF/SCF-treated mice when either a single premature extra stimulus (, top) or two consecutive premature stimuli (, bottom) were applied (). As expected, a reduction of connexin43 expression (90% reduction) was found in the border zone of the infarction ( and Videos S1–S4, available at ), consistent with conduction slowing, the hallmark of inducible ventricular arrhythmias after myocardial infarction. G-CSF/SCF-treated hearts, in contrast, had markedly higher connexin43 levels in the border zone of the infarction when compared with untreated controls (25% of normal connexin43 levels; and Videos S1–S4).
G-CSF/SCF enhanced arterialization in the border zone of myocardial infarction as indicated by an increased number of vessels per field of view (). However, concordant with increased arteriogenesis, the mean vessel diameter was not affected. Cardiomyocyte hypertrophy was more pronounced in the border zone of the infarction after G-CSF/SCF treatment ().
G-CSF/SCF treatment increased the homing of BM-derived cells (mainly white blood cells) into the infarcted myocardium twofold. In addition, up-regulation of G-CSFR expression in the infarcted myocardium suggests a sensitization for direct effects of the cytokine. G-CSF/SCF increased cardiac output regardless of infarction size and integration of BM-derived cells into the myocardium. G-CSF/SCF reduced the inducibility of ventricular arrhythmias. This antiarrhythmic effect can be attributed to an increased expression of connexin43 in cardiomyocytes in the border zone of the infarction. Although provocation of afterdepolarizations was a specific aim of this study, there was no tendency for spontaneous arrhythmias or afterdepolarizations after G-CSF/SCF treatment. The observed physiological effects of cytokine treatment were associated with cardiomyocyte hypertrophy and an enhanced arterialization in the border zone of the infarction.
G-CSF/SCF treatment resulted in an increased homing of BM-derived cells (mainly white blood cells) into the infarcted myocardium and its border zone. In agreement with Murry et al. (), Balsam et al. (), and Nygren et al. (), because a significant degree of transdifferentiation was not observed, this allows us to speculate that the observed therapeutic effects may be due to the secretion of stimulatory factors by white blood cells. However, the debate as to how G-CSF/SCF-based cardiac regenerative therapies improve cardiac function (, –), and whether transdifferentiation of BM-derived cells is the underlying mechanism of myocardial regeneration (, ), has recently been extended by Harada et al. (). Their results indicate a significant survival-promoting effect of G-CSF treatment on cardiomyocytes and endothelial cells that prevented left ventricular remodeling after myocardial infarction, suggesting that G-CSF may directly act on cardiomyocytes and endothelial cells. However, in contrast to Orlic et al. () and our work, they did not use splenectomized mice for their experiments. Differences in neutrophil accumulation in the blood between splenectomized and nonsplenectomized mice might explain why they were unable to identify differences between the homing of BM-derived cells in G-CSF–treated and control hearts. Although the threefold rise in white blood cell count in peripheral blood measured in our G-CSF/SCF–treated animals is in the range observed in nonsplenectomized animals (), the boost in the proportion of neutrophils is less pronounced in nonsplenectomized animals (not depicted). Nevertheless, Adachi et al. (), who also did not use splenectomized animals for their experiments, reported an increased infiltration of BM-derived side population cells into the G-SCF–treated infarcted heart. Together with recent data () about the secretion by BM-derived mesenchymal stem cells of so far unknown paracrine factor(s), capable of reducing the infarct size in a rat model of permanent coronary artery occlusion, their results demonstrate that the question of the relative contribution of direct and paracrine effects to the therapeutic action of G-CSF is not definitely answered.
Our results regarding G-CSFR expression are in agreement with Harada et al. () as well as Schneider et al. () and suggest that the infarcted myocardium is sensitized for direct effects of G-CSF by an increased expression of the G-CSFR. Together with the parallel increase of local G-CSF expression after myocardial infarction (), these data suggest the existence of an endogenous mechanism of G-CSF signaling activation potentially mediating cardioprotective effects. Similar results were obtained in a model of brain infarction (). However, in view of the twofold-increased homing of BM-derived cells, they also do not allow to favor one of the explanations (paracrine vs. direct effects) but leave the alternative open that both mechanisms act in parallel. Nevertheless, the recent observation of a rise of G-CSF concentration in the blood accompanied by an increase of peripheral CD34 cells (endothelial progenitor cells) after acute myocardial infarction in humans () allows us to speculate that myocardial damage by itself leads to the activation of an endogenous BM mobilization mechanism, possibly enhancing the recruitment of BM-derived cells from the blood.
Concordant with results from some (), but not all (), groups, we found no change in infarct size after G-CSF/SCF treatment. In addition, we did not find evidence for the generation of new myocardium after G-CSF/SCF treatment. Cardiomyocyte hypertrophy in the border zone of the infarction was, however, increased in G-CSF/SCF-treated hearts. Increased binding of G-CSF to its receptor on cardiomyocytes activates the Akt (, ) as well as the Jak/STAT pathway (, ), which are both known to impart hypertrophic signaling in cardiomyocytes. For the Akt pathway, it has also been demonstrated that its transgene-mediated, constitutive activation in mouse cardiomyocytes improves their contractile function in vitro as well as in vivo ().
The increased vascularization in the border zone of the infarction () might have also contributed to the improved cardiac output by enabling increased supply of oxygen and nutrients. Angiogenesis might be at least in part induced by direct effects of G-CSF, indicated by distinct immunolocalization of G-CSFR in vessel wall cells 1 d after induction of myocardial infarction. This finding is in accordance with Harada et al. (), who detected G-CSFR expression in endothelial cells, and with Chen et al. (), who demonstrated that G-CSF induces proliferation of vascular smooth muscle cells. Activation of the Akt as well as the Jak/STAT pathway by the binding of G-CSF to its receptor also might have been responsible for the enhanced induction of arteriogenesis in the G-CSF/SCF-treated hearts as suggested by work of Iwanaga et al. () and Harada et al. ().
Despite the homing of a large number of BM-derived cells into the border zone of myocardial infarction (up to 30% of all nucleated cells), a dedicated protocol to provoke afterdepolarizations and ventricular arrhythmias did not provoke afterdepolarizations or spontaneous ventricular tachycardias in G-CSF/SCF-treated hearts. The lack of differentiation into myocytes of the majority of BM-derived cells, observed by us and others (–, ), and consideration of the electrotonic attenuation of abnormal electrical behavior of a single cell when it is integrated into the myocardial syncytium, can explain that we did not find afterdepolarizations in the intact heart.
G-CSF/SCF-treated hearts had less inducible ventricular arrhythmias during programmed ventricular stimulation than controls (). Slow conduction due to decreased intercellular coupling in the border zone of a healed myocardial infarction is the pivotal factor for reentrant ventricular tachycardias in this setting (, , ). Reentry due to slow zigzag conduction in the border zone of an infarction is mainly caused by a dramatic decrease in expression of gap junctional proteins, specifically in connexin43 (, ). Indeed, a reduction of connexin43 expression by ∼90% is sufficient to enhance inducibility of ventricular tachycardias in transgenic mice (). A comparable (90%) reduction of connexin43 expression was found in cardiomyocytes in the border zone of the infarction in the absence of G-CSF/SCF treatment (). G-CSF/SCF partially reversed the down-regulation of connexin43 expression () by increasing connexin43 levels in the border zone of the infarction to ∼25% of normal expression. This level of connexin expression is sufficient to maintain almost normal conduction velocities () and can explain why G-CSF/SCF-treated hearts were protected against induction of ventricular tachyarrhythmias in this study.
This study examined the midterm electrophysiological effects of G-CSF/SCF into the infarcted myocardium. Arrhythmic events, including sudden arrhythmic death, may occur late after myocardial infarction, and current revascularization therapies during acute myocardial infarction usually limit infarct size in the clinical setting. The G-CSF/SCF treatment protocol was chosen here in agreement with the pioneering work by Orlic et al. () to allow for comparison of our data with those published by this group. Further studies are needed to establish a protocol for BM mobilization that is clinically applicable, i.e., where treatment begins during the acute phase of a myocardial infarction.
114 mice, which were 58 CD1 outbred mice (Charles River Deutschland GmbH; body weight 23–30 g at the day of myocardial infarction) and 56 female C57BL/6 inbred mice (Charles River Deutschland GmbH; body weight 20–26 g at the day of BMT), were studied. C57BL/6 mice served as hosts for BM transplantation from EGFP-expressing transgenic donor mice (also C57BL/6 background; reference 45). These mice were used to analyze myocardial homing of BM-derived cells. All experiments were performed in accordance with the German Law on the Care and Use of Laboratory Animals and were approved by the local Institutional Review Board.
To investigate the effect of G-CSF/SCF on mobilization and myocardial homing of BM-derived cells, 34 recipient mice underwent total body irradiation at a dose of 11 Gy (0.98 Gy/min), and intravenous infusion of BM (2 × 10 mononuclear cells) was harvested from EGFP-expressing mice the next day. The procedural details have been described previously (). Blood cell chimerism determined 8 wk after BMT by FACS analysis showed >95% donor cells (EGFP) in all transplanted mice. Myocardial homing of BM-derived cells was studied 10 wk after BMT.
With the exception of eight C57BL/6 animals only used for the analysis of BM mobilization in nonsplenectomized mice, all experimental animals were splenectomized according to a published protocol () at least 2 wk before BMT or G-CSF/SCF treatment in non-BMT animals.
Recombinant mouse SCF (50 μg/kg/day; R&D Systems) and recombinant human G-CSF (200 μg/kg/day; Chugai Pharma Marketing Ltd.) were injected subcutaneously for 6 d. Control mice received daily injections of 0.9% saline. On the third day of G-CSF/SCF treatment, myocardial infarction was induced by ligation of the proximal LAD. To investigate basal accumulation of EGFP-expressing BM-derived cells in the myocardium, in 10 BMT animals (5 G-CSF/SCF-treated and 5 controls) no myocardial infarction was induced. For additional information see the supplemental Materials and methods, available at .
After 4 d of G-CSF/SCF treatment, 200 μl blood was obtained from the tail vein of 31 animals (15 controls and 16 treated with G-CSF/SCF). Automatic full blood count measurements using the KK-21 hematology analyzer (Sysmex GmbH) were performed. After Pappenheim staining of blood smears, the proportion of lymphocytes, neutrophils, and monocytes in at least 100 white blood cell counts was calculated.
Sedated mice (ketamine/xylazine) underwent Doppler echocardiographic studies 5 wk after infarction immediately before the electrophysiological study according to published procedures (). For additional details, see the supplemental Materials and methods.
The heart was excised under general anesthesia, and the aorta was cannulated and retrogradely perfused using 37°C Krebs-Henseleit buffer (in mmol/liters; NaCl 118, NaHCO3 24.88, KH2PO 41.18, glucose 5.55, Na-pyruvate 2, MgSO4 0.83, CaCl2 1.8, KCl 4.7) equilibrated with a 95% oxygen/5% carbon dioxide gas mixture. The heart was mounted on a vertical Langendorff apparatus (Hugo Sachs Electronic-Harvard Apparatus GmbH) and constantly perfused at 100 ± 5 mmHg perfusion pressure, corresponding to coronary flow rates of 4 ± 1 ml/min. An octapolar mouse electrophysiologic catheter (CIBER MOUSE; NuMED) was placed in the right atrium and ventricle for atrial and ventricular pacing. A tissue bath ECG was recorded from Ag-AgCl electrodes immersed in superfused sponges flanking the isolated heart. Three murine monophasic action potentials (MAPs) were continuously and simultaneously recorded from the right and left ventricular epicardium (–). One MAP was positioned within the border zone of the infarct to record afterdepolarizations. All recordings were obtained simultaneously throughout the experimental protocols. ECG signals were amplified and filtered by an ECG amplifier at a bandwidth of 0.1 to 300 Hz (Hugo Sachs Electronic-Harvard Apparatus GmbH). MAP signals were amplified using dedicated MAP amplifiers (Boston Scientific Inc.). Additional details of the experimental setup have been described previously (–).
After placing all catheters in a stable position, atrial pacing was performed at different pacing cycle lengths to measure steady-state action potential durations. To provoke ventricular arrhythmias initiated by afterdepolarizations, the atrioventricular node was ablated and the spontaneous ventricular rhythm was observed for 10 min (, ). Thereafter, to test the propensity to reentry, programmed ventricular stimulation was performed using up to two premature stimuli (S2 and S3) at different pacing cycle lengths (80–140 ms; reference 48). All signals were digitized at 1 or 2 KHz and stored on digital media for offline analysis.
After completion of the electrophysiological procedure, hearts were either fixed in Bouin's solution and stained using Goldner's Trichrom staining, or fixed in 3.7% formaldehyde, followed by dehydration in 10% sucrose solution, O.C.T. embedding, and cryoconservation for fluorescent detection of EGFP-expressing cells or immunofluorescent detection of various antigens according to an established protocol (). The following primary antibodies were used: rat anti–mouse CD45 as a marker for blood cells (AB3088; Abcam Limited), monoclonal anti–troponin T as a marker of differentiated cardiomyocytes (cardiac isoform AB-1, clone 13-11; Lab Vision), rabbit anti–rat connexin43 polyclonal antibodies for the detection of gap junctions (71-0700; Zytomed GmbH), monoclonal rat anti-reticular fibroblast antibodies (clone ER-TR7; Novus Biologicals Inc.), rabbit smooth muscle anti-actin antibody for the detection of arteries and arterioles (RB-9010; Lab Vision Corporation), rabbit c-kit (SCF receptor) antibody (sc-168; Santa Cruz Biotechnology, Inc.), and rabbit anti–G-CSFR antibody (sc-694; Santa Cruz Biotechnology, Inc.). For additional information see the supplemental Materials and methods.
Sections were examined using a confocal fluorescence microscope (Axioplan 2 and LSM 510 Meta; Carl Zeiss MicroImaging, Inc.). Images were digitized and transferred to a personal computer. For quantification of different cell populations, the number of EGFP cells and of EGFP cells coexpressing the immunohistological marker (anti-CD45 or anti–troponin T antibody) were counted in three randomly selected fields of view at the same magnification in the infarcted area, the border zone, and in the non-infarcted area of the left ventricle. For quantitative analysis of connexin expression, the number of connexin43 spots and the proportion of the connexin43 area in relation to the nonfibrotic myocardial area of the respective field of view were determined on digitized images taken at high magnification from at least two nonconsecutive slides per mouse heart (four control and five G-CSF/SCF-treated animals) using Adobe Photoshop CS (Adobe Systems GmbH) and ImageJ 1.32 imaging software (National Institute of Mental Health). A minimum of eight images from the border zone and the non-infarcted area was analyzed in each heart.
Quantitative analysis of the arterial vessel area (smooth muscle anti-actin area of vessels and enclosed lumina) was analyzed as described for the measurement of the connexin43 area (see above). In addition, the number of arterial vessels per field of view as well as the diameter of each vessel per field of view were determined.
Histological sections immunostained for fibroblasts/connective tissue were also used for short axis diameter measurement of cardiomyocytes at the nuclear level (100 cells per region and animal) using published methods () adapted for immunofluorescence; i.e., DAPI staining was used for the detection of nuclei and cardiomyocytes were identified based on their autofluorescence.
Infarcted hearts of male CD1 mice at different time points after surgery (30 min, = 2; 1 h, = 3; 4 h, = 3; 24 h, = 2; 1wk, = 2; 6 wk, = 3) were explanted and the area of infarction, including its border zone, was separated from the non-infarcted myocardial tissue. Ventricular tissues of seven explanted hearts of untreated male CD1 mice served as controls.
All procedures and analyses (functional and histological) were performed by a blinded experimenter with respect to treatment group (G-CSF/SCF vs. control). Depending on the existence of equal variances and normal distribution of data, treatment groups were compared using an unpaired test and one-way ANOVA, respectively, or the corresponding nonparametric test procedures (Mann-Whitney U test and Kruskal-Wallis test). A chi-square test and log rank test were used to compare nominal parameters (e.g., inducibility of arrhythmias or mortality).
QuickTime videos (Videos S1–S4) presenting 3D-animated sequences of confocal scans of myocardial sections immunolabeled for connexin43/connective tissue (same scale as in ) and supplemental Materials and methods are available at . |
We used a 70mer oligonucleotide array interrogating 6,800 genes to establish expression profiles for 10 classes of mouse B cell lineage lymphomas (, ). A test was used to identify genes that distinguished each subset from all the others. was the gene that best distinguished the histologically defined CB subset of diffuse large B cell lymphomas (unpublished data). Analyses of the relative expression of transcripts among the lymphoma classes showed that the highest levels were seen in CB and the other GC-derived lymphomas, with transcript levels in CB being around sixfold higher than in the lowest-expressing class, plasmacytomas (Fig. S1, available at ). These results paralleled earlier studies of normal human primary B cells that showed that the levels of transcripts were ∼10-fold higher in total tonsillar CD19 B cells than in BM plasma cells (). This suggested that the pattern of expression seen with mouse lymphoma subsets was likely to reflect differences related to the differentiative state of the cell of origin rather than a mechanistic contribution to transformation.
To evaluate a possible relationship between levels of expression and stages in normal B cell differentiation, we turned to studies of human primary tonsillar B cells. Previous studies demonstrated that eight subsets of CD19 B cells can be distinguished by flow cytometry using antibodies to IgD and CD38 () (, ): IgD preswitch populations (CD38IgD, CD38IgD, and CD38IgD), CD38IgD (GC dark-zone centroblasts), CD38IgD (GC light-zone centrocytes), CD38IgD, CD38IgD (plasmablasts), and CD38IgD (plasma cells).
Gene arrays were used to quantitate transcripts in primary human tonsillar and peripheral blood B cell (PBB) subsets (). We monitored levels of transcripts for genes known to change in expression in association with GC passage and subsequent maturation to memory and plasma cells: , , , , and () (). Transcripts for and were highest in CB and decreased in memory and plasma cells. In addition, transcripts for , , , and were highest in plasma cells. These results are completely in keeping with earlier studies of tonsillar B cell subsets using microarrays or serial analysis of gene expression (, ), validating the methods used to subset the tonsillar B cell populations. Notably, transcripts for were highest in CD38IgD centroblasts.
RNA prepared from the sorted tonsillar subsets and purified IgD and IgD PBB was then examined by quantitative RT-PCR (qPCR) for expression of transcripts, with results normalized against levels in sorted IgD PBB (). Of note, transcripts were higher in nonsecreting B cells in activated secondary lymphoid tissue than in circulating B cells. The results also showed that transcripts were highest in centroblasts (6-fold over PBB), lower in pre- and postswitch populations (2–4-fold), and lowest in Ig-secreting cells (∼0.5-fold).
We next evaluated expression of some of these genes at the protein level using immunohistochemistry to study frozen sections of human tonsil. As expected, IRF8 was expressed in cells of the myeloid lineage with high levels in extrafollicular elements including macrophages, monocytes, granulocytes, and DCs (). Cells of the follicular mantle zone were mostly negative except for a minor population of small lymphoid cells. In contrast, all GC were positive, with large centroblast-like cells staining most intensely. We used two-color studies to examine expression of CD23, a marker of follicular mantle cells, BCL6, which is expressed most highly in centroblasts, and IRF8 (). Within GC, antibodies to IRF8 showed intense nuclear staining of cells in the dark zone. This reactivity colocalized with nuclear staining for BCL6. In addition, IRF8 cells were almost all positive for Ki-67, a marker for the highly proliferative population of centroblasts (not depicted).
We applied three approaches to identifying genes regulated by IRF8 in B cell lineage cells. First, we transfected primary CD19 tonsillar B cells with a construct expressing an enhanced GFP (EGFP)–tagged WT mouse IRF8. Because IRF8 positively regulates its own expression (), this allowed us to monitor human IRF8 expression from the endogenous locus using species-specific primers. We also used a second construct that expressed only EGFP. The cells expressing either EGFP alone or EGFP-tagged IRF8 were sorted into EGFP and EGFP fractions (Fig. S2, , and the transcript levels for various genes were assessed by qPCR. Of note, FACS analyses of EGFP levels at 18 h after transfection showed that IRF8 was equally well expressed in the eight subsets of tonsillar B cells (unpublished data).
These studies showed that human transcripts in the EGFP population expressing mouse IRF8 were increased an average of 6.5-fold over the levels in cells expressing EGFP alone (), confirming the predicted autoregulation of the gene and validating the experimental system. Expression of two known IRF8 targets, () and (not depicted) (), was increased 5.6- and 2.4-fold, respectively. Further analyses showed that cells with enhanced expression of had increased levels of transcripts for mature B cell/GC-related genes and . These experiments suggested that and are transcriptionally regulated by IRF8, either as direct or indirect targets.
In a second approach to discovering genes regulated by IRF8, we used small interfering RNA (siRNA) to knock down expression in the mouse follicular lymphoma–derived B cell line NFS-202. The cells were infected with a retrovirus that expressed puromycin and either of two siRNA for IRF8 or a nonreactive siRNA. After selection for puromycin resistance, cloned cells were assayed for levels of transcripts by qPCR. We selected two independent clones, one expressing siRNA #2 and one expressing siRNA #5, in which transcripts were reduced to levels 10% or less of those expressed by control cells when normalized to levels of β-actin or (unpublished data). By immunoblotting, the siRNA construct reduced IRF8 protein to 10% or less of control levels with α-tubulin serving as a loading control ().
RNA prepared from these clones was examined by qPCR. Transcripts for and were strikingly reduced (), reinforcing the suggestion that IRF8 functions as a transcriptional activator for both genes. To investigate the possibility that IRF8 directly regulates and gene expression, we cloned putative promoter fragments flanking the start sites of both genes and placed them upstream of a luciferase reporter gene. Transient expression of the reporters in HeLa cells with increasing amounts of an IRF8 expression plasmid showed a greater than fivefold increase in activity of the reporter and a greater than threefold increase for the reporter ().
As a third approach to understanding genes regulated by IRF8 in B cells, we examined mice homozygous for a null mutation of the gene (−/−) or WT animals (+/+) (). Histologic studies of spleens and LN from young −/− mice showed the previously described accumulations of granulocytes, macrophages, and pseudo-Gaucher cells in association with lymphadenopathy and splenomegaly. GC were readily identified in lymph nodes and spleens of both +/+ and −/− mice. Those of +/+ mice were compact and well defined, with centrocytes and centroblasts exhibiting characteristic nuclear staining for BCL6 and surface staining for PNA (). GC of −/− mice exhibited comparable staining of the B cell population but were usually less well organized. Some, as shown in , had populations of BCL6PNA cells trailing away from a more dense collection of cells like those seen in +/+ GC. Others had substantial numbers of small BCL6 lymphocytes localized in small clusters and dispersed throughout a loosened structure of BCL6PNA cells along with scattered apoptotic bodies not seen in +/+ GC (unpublished data).
To determine how these histologic features related to expression of GC-associated genes, RNA prepared from purified splenic B cells of +/+ and −/− mice was analyzed by qPCR for levels of transcripts of and normalized to transcript levels for . The results () showed that, compared with levels in +/+ B cells, transcripts were reduced approximately two- and threefold, respectively. These findings support the conclusion that IRF8 is involved in the transcriptional regulation of and and showed that reduced expression of these genes was associated with poorly organized GC in mutant mice.
We performed ChIP analyses to determine whether IRF8 protein was bound in vivo to promoters of genes that it appeared to regulate. As likely positive targets, we selected regions in the promoters of the mouse and human and genes. Protein from the mouse NFS-202 cell line or human tonsillar B cells was cross-linked to DNA using formaldehyde, and immunoprecipitations were performed with anti-IRF8 or control antibodies. After cross-linking was reversed, the DNA was amplified with species- and gene-specific primers for the promoter regions of and . Primer pairs for and amplified DNA products of the expected sizes from the total input DNA at specific locations in the 5′ sequences but not at 10-kb sites 5′ of these targets (). Notably, bands were observed only with anti-IRF8–precipitated DNA. These data indicated that IRF8 bound the endogenous promoters of and in a mouse GC B cell–derived cell line and in human tonsillar B cells.
The GC reaction is the hallmark feature of the B cell response to T cell–dependent antigen. Studies using DNA microarray and SAGE analyses, primarily of human cells, have identified large numbers of genes that are differentially expressed as cells move into and through the GC, maturing from the naive state to CB, to centrocytes, and then to memory or plasma cells as they exit (, , , , ). A substantial number of these genes are transcription factors. Among them are a series of “master regulators”—PAX5, BCL6, PRDM1, and XBP1—situated at critical developmental decision points and forming a transcriptional regulatory cascade in which they function in a hierarchic as well as a combinatorial fashion to govern the differentiation of B cells involved in the GC reaction (–, ). The transcriptional functions of these master regulators are complex, involving both gene activation and repression as well as mutual antagonism.
The data from this study identify the transcription factor, IRF8, as another important regulator of the GC program of molecular differentiation by demonstrating two striking and previously unappreciated roles in B cell biology, the direct transcriptional activation of both and , critical determinants of GC development, and function and effects on GC organization. By activating , a known repressor of , IRF8 may also function to prevent GC B cells from initiating a plasma cell differentiation program. Studies of mice homozygous for a null allele of IRF8 showing that transcripts for both and were reduced approximately two- to threefold in isolated B cells strongly support a direct role for IRF8 in regulating their expression.
Neither BCL6 nor AICDA was previously identified as a transcriptional target of IRF8. BCL6 is absolutely required for GC formation, and control of expression is known to be regulated at multiple levels (–). Although transcripts can be detected at low levels in several tissues, protein expression is limited to lymphocytes (). Within the B cell lineage, BCL6 is expressed only in mature B cells of the GC. In B cells, protein levels are modulated by phosphorylation and ubiquitination, resulting in proteasomal degradation, whereas acetylation negatively regulates BCL6 function (, ). Transcriptional down-regulation of appears to be required for B cells to exit the GC, and our experiments indicate that this may be caused by synchronous reductions in the expression of IRF8 and activation of BLIMP1, a transcriptional repressor of ().
AICDA is a critical determinant of GC formation and function. Deficiencies in AICDA in humans and mice result in markedly impaired CSR and SHM and, in contrast to mice deficient in BCL6, very large GC (, ). transcripts are detected only in peripheral lymphoid tissues, where they are almost exclusively associated with GC (). AICDA can be induced in resting B cells by several exogenous stimuli including IL-4, CD40, TGF-β, or LPS (, ), BLys or APRIL (), or IL-21 () alone or in different combinations. Our data strongly suggest that IRF8 is downstream of the signaling cascades initiated by these agents in B cells and plays a role in regulating expression of in the GC. Earlier studies showed that the levels of switched Ig classes in activated cells from AICDA and AICDA mice were comparable, indicating that a 50% reduction in AICDA expression had no major effect on CSR (). The fact that AICDA transcripts were ∼37% of normal in B cells from the KO may explain why levels of switched Ig classes were close to normal in IRF8 mice ().
Immunohistochemical studies demonstrated that both IRF8 and BCL6 are expressed in GC B cells localized to the dark zone. In contrast, expression of the other lymphoid-restricted member of the IRF family, IRF4, is limited to a small population of centrocytic B cells in the light zone, as well as a few plasmablasts and plasma cells (). Expression of IRF4 in B cells is regulated primarily by pathways known to drive lymphocyte activation rather than by IFN, which is the case for most other IRF family members. Stimuli provided by IL-4, CD40, and Ig cross-linking received by cells during selection in the GC not only activate IRF4 but also repress BCL6, providing a likely explanation for the contrasting expression patterns of these two genes (). Whether down-regulation of IRF8 or altered expression of its binding partners plays a role in down-regulating the expression of BCL6 is currently not known but is worth considering. A mechanistic basis for the contrasting patterns of IRF8 and IRF4 expression has not been defined, but the promoter regions of the two genes are apparently regulated by distinct members of the STAT family (STAT1 in the case of IRF8 and STAT6 for IRF4) (). The nature of the signals that induce the expression and activity of IRF8 in the tonsil is currently unknown but of great interest.
The approaches used to define transcriptional targets of IRF8 all have limitations. First, the knockdown studies were based on the use of a mouse cell line derived from a lymphoma with associated uncertainties about changes in gene expression patterns that might have occurred during the process of transformation or on adaptation to tissue culture. In addition, the use of stable siRNA knockdowns makes it difficult to determine which effects of IRF8 on gene expression are primary or secondary.
Second, splenic B cells from the IRF8 KO mice developed in an abnormal cellular environment caused by extensive myeloid proliferation and a T helper type 2 cell–based cytokine milieu deficient in IL-12 and IFN-γ (). Although IRF8 has not been shown to play a role in the development or function of follicular DCs, it has profound effects on subsets of DCs and Langerhans cells (–). Irregularities in the organization of follicles and GC in secondary lymphoid tissue of IRF8-null mice may well be caused by IRF8-dependent changes in the development and function of follicular DCs.
Third, the studies of total tonsillar B cells for effects of IRF8 overexpression were performed 18 h after transfection. This again leaves open the issue of primary versus secondary targets of IRF8 regulation. Finally, there are several differences between the immunology of mice and humans (, ), and conclusions drawn from cross-species comparisons must be made with caution. Nonetheless, the studies of IRF8-deficient mice, the ChIP experiments, and the transcriptional reporter assays all argue that and are primary targets of IRF8 in both species.
In conclusion, our experiments identify IRF8 as a novel component of the regulatory cascade that moves B cells into and through the GC reaction. Increased levels of IRF8 in GC B cells appear to play a role in fostering the GC response by up-regulating BCL6 and AICDA, and down-regulation of expression may contribute to GC B cell entry into the plasma cell differentiation program. Because GC can form in IRF8-deficient mice, IRF8 appears to function as a rheostat that modulates B cell differentiation and function during the GC reaction rather than as a master regulator in the mode of BCL6 or PRDM1.
C57BL/6-Irf8 mice described previously () were studied at 6–8 wk of age under a protocol (Lip-4) approved by the National Institute of Allergy and Infectious Diseases (NIAID) Animal Care and Use Committee. Splenic B cells were purified by negative selection using standard techniques and were (95% pure. The NFS-202 cell line was cultured from the second generation passage of a spontaneous follicular B cell lymphoma from an NFS.V mouse. Plasmacytoma cell lines were provided by M. Potter (National Institutes of Health [NIH], Bethesda, MD). The origins and characteristics of primary B cell–lineage lymphomas from NFS.V congenic, B6.λ-MYC, SJL-β2m, and BALB/c- mice, and the techniques used for transcriptional profiling of the lymphomas using oligonucleotide arrays, were detailed previously (, ). Microarray data is available under accession no. GSE1908.
For negative selection of B cells, tonsillar mononuclear cells were prepared (, ) and were stained on ice with a StemSep human B cell–negative selection cocktail containing a mixture of dextran cross-linked to mAb specific for glycophorin A, CD2, CD3, CD14, CD16, and CD56, followed by exposure to a magnetic coil covalently linked to antidextran mAb (StemCell Technologies, Inc.). Polystyrene tubes (12 × 75 mm) containing the suspension were placed into purple EasySep magnets (Stem Cell Technologies). After 5 min at room temperature, the suspension of purified B cells was decanted into collection tubes. The cells were pelleted, resuspended in PBS 1% BSA, and counted.
For transfections, negatively selected tonsillar B cells were diluted to 5 × 10 ml, separated into 1-ml aliquots, and pelleted. The supernatant was removed, and the cells were resuspended in 100 μl of nucleofector solution (Amaxa Biosystems). 5 μg of plasmid expressing EGFP or IRF8 tagged with EGFP (), both gifts from K. Ozato (NIH, Bethesda, MD) was added to each tube and run on the U-15 program of the Amaxa nucleofector machine. 2 ml of room-temperature RPMI medium (Life Technologies) supplemented with 200 U/ml penicillin G, 10 μg/ml gentamycin, and 10% FCS was added, and cells were placed in a 12-well plate to incubate at 37(C overnight.
For sorting of EGFP and EGFP populations, cells were harvested and resuspended in RPMI medium (Life Technologies) supplemented with 200 U/ml penicillin G, 10 μg/ml gentamycin, and 10% FCS before using a cell sorter (MoFlo; DakoCytomation). For analysis, cells were stained with PE–Cy7 anti–human CD38 (HD37 mIgG1; Becton Dickinson) and A647 anti–human IgD (IA6-1 mIgG2a; BD Biosciences); the A647 conjugation kit was purchased from Invitrogen. PE–Cy7 and A647-conjugated isotype-matched mAbs were used as controls.
Affymetrix arrays were used to profile gene expression in human tonsillar B cell subsets. RNA was prepared from tonsillar B cell subsets purified as described previously (). We studied RNA from three experiments for centroblasts, plasma cells, and CD38 IgD cells and from four experiments for CD38IgD cells. The purified RNA was reverse transcribed into cDNA (Invitrogen). The template cDNA was purified for amplification and in vitro transcription reaction to cRNA (BioArray High Yield RNA transcript labeling kit [T7]; Enzo Life Sciences). cRNA was biotin labeled, purified, and hybridized to HG-U133A Affymetrix Genechips according to the Affymetrix protocol (available at ). Genechips were scanned on a high-resolution Affymetrix scanner using GCOS software (version 1.2). Microarray data were log transformed. Relative gene expression of tonsillar B cell subsets was determined by comparison with levels in IgD PBBs.
The mammalian expression vector pSUPER.retro.puromycin (OligoEngine) was used for expression of siRNA in NFS-202 cells. To generate the pSR.puro-IRF8 #2 and pSR.puro-IRF8 #5, the pSUPER.retro.puromycin vector was digested with II and d III and the annealed oligos (IRF8 #2, 5′-gatccccACCACCACCTGCCTTGAAGttcaagagaCTTCAAGGCAGGTGGTGGTtttt tggaaa-3′ and 5′-agcttttccaaaaaACCACCACCTGCCTTGAAGtctcttgaaCTTCAAGGCAGGTGGTGGTggg-3′; IRF8 #5, 5′-gatccccACTCATTCTGGTGCAGGTAttcaagagaTACCTGCACCAGAATGAGTtttttggaaa-3′ and 5′-agcttttccaaaaaACTCATTCTGGTGCAGGTAtctcttgaaTACCTGCACCAGAATGAGTggg-3′) were ligated into the vector. The 19-nucleotide IRF8 target sequences are indicated in capitals in the oligonucleotide sequences. A control vector (pSR.puro-negative) was constructed using 64-nucleotide sequences (5′-gatccccTGTAGATGGGTACGCGCTCttcaagagaGAGCGCGTACCCATCTACAtttttggaaa-3′ and 5′-agcttttccaaaaaTGTAGATGGGTACGCGCTCtctcttgaaGAGCGCGTACCCATCTACAggg-3′) with no substantial homology to any mammalian gene sequence and thus serves as a nonsilencing control. The constructs were transformed into DH5α-competent cells (Invitrogen) according to the manufacturer's instructions. Positives were confirmed by sequencing.
Ecotropic retroviral supernatants were produced by transfection of phoenix packaging cells by a reagent (LipofectAMINE 2000; Invitrogen) according to the manufacturer's instructions. After transfection (48 h), the tissue culture medium was filtered through a 0.45-μm filter, and the viral supernatant was used for infection of NFS-202 cells after addition of 4 μg/ml polybrene. Cells were infected for 24 h and allowed to recover for 24 h with fresh medium. Infected cells were selected with 4 μg/ml puromycin for 72 h and cloned for 2 mo. Cloned cells were assayed for levels of IRF8 transcripts by qPCR.
The 5′(flanking regions of (−1772/+245) and (−840/+79) were PCR amplified from genomic DNA extracted from NFS-202 cells and cloned into the pGL3–Basic Firefly luciferase reporter vector (Promega). (1 corresponds to the first nucleotide of exon 1 of the gene. A murine IRF8 expression vector (pCDNA3.1-IRF8) was provided by K. Ozato. The 800-ng reporter plasmid was used for transfection of HeLa cells grown in 12-well plates. The 50-ng pRL-SV40 Renilla luciferase vector (Promega) was used as an internal control. To test dosage effects, 100, 200, and 300 ng of pCDNA3.1-IRF8 expression vector were adjusted to a total 300 ng of plasmid with pCDNA3.1 empty vector. Luciferase activities were measured 22 h after transfection using the dual-luciferase reporter assay kit (Promega) according to the manufacturer's protocol. All samples were tested in triplicate.
Total cell lysates were prepared in lysis buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 0.2 mM EDTA, 0.1% Nonidet P-40, 1 mM DTT, 10% glycerol) supplemented with protease inhibitor cocktail solution (Pierce Chemical Co.). Lysates were cleared by centrifugation at 14,000 for 20 min at 4(C, and the protein contents were then determined by the BCA protein assay kit (Pierce Chemical Co.). For Western blotting, 20 μg of protein per lane was separated on a NuPage 10% Bis-Tris gel (Invitrogen) and transferred to a polyvinylidene difluoride membrane. After blocking with a 5% skim milk solution, the blot was incubated with anti-IRF8 antibody (Santa Cruz Biotechnology, Inc.) or anti–α-tubulin antibody (Sigma-Aldrich). These antibodies were detected with horseradish peroxidase–conjugated donkey anti–rabbit or anti–mouse secondary antibody (GE Healthcare) and developed by Super Signal detection kit (Pierce Chemical Co.) according to the manufacturer's instructions.
Total RNA was isolated using a modified version of the RNA extraction protocol detailed elsewhere (available at ). In brief, total RNA was collected using the initial steps of the TRIzol protocol (Invitrogen). After collecting the aqueous supernatant, the RNeasy column coupled with DNase set–based (QIAGEN) protocol was followed. Reverse transcription was performed using 2 μg total RNA, 25 μg/ml oligo (dT) primers, and 200 units of SuperScript II reverse transcriptase (Invitrogen). For qPCR, 2 ng cDNA was amplified using SYBR Green PCR Master Mix (Applied Biosystems) and 0.33 μM each of forward and reverse primers on the ABI PRISM 7900HT sequence detector system (Applied Biosystems) with published techniques (). Primers for qPCR were designed using the Primer Express software (Applied Biosystems) and synthesized at MWG-Biotec (Tables S1 and S2, available at ). All samples were tested in triplicate, and mean values were used for quantification. Analysis was performed using software (SDS version 2.1; Applied Biosystems) according to the manufacturer's instructions. Samples were normalized using the housekeeping gene GAPDH for human studies and β-actin for mouse. Results for human cells are expressed as the relative fold increase compared with normal IgD PBBs. Assays of peripheral blood and tonsillar B cells were run in separate plates, compared with β-actin as a control, and normalized to IgD PBBs.
ChIP assays were performed by using the ChIP assay kit (Upstate Biotechnology) according to the manufacturer's protocol. 4 × 10 cells were cross-linked with 1% formaldehyde for 10 min at room temperature before ChIP assays. The chromatin was immunoprecipitated with goat anti-IRF8 Ab or preimmune goat IgG (Santa Cruz Biotechnology, Inc.) was used as a control Ab. A 3-μl aliquot out of the 60-μl solution of DNA recovered from each immunoprecipitate was used for PCR, and the products were analyzed on a 2% agarose gel after 36 cycles of amplification. Input DNA (1:400) was used as a control. The following primers were used for PCR: mouse promoter (5′-GTGCCTAATACTCTAGCTGGAAGGAG-3′ and 5′-GCTCGGCCTCTGGAATTCT-3′), mouse 10-kb 5′ region (5′-CCAAAGTTCTGGAATGCCCA-3′ and 5′-CAGCATATCCGTGCATGTGC-3′), human promoter (5′-AAGTGCAGGAGAGACACACTTCAG-3′ and 5′-CATATGTAACAATCCCAGCCCC-3′), human 10-kb 5′ region (5′-TATTGTAATTTACTTAATCATTCTTCATCCAA-3′ and 5′-GCAGACCCTTTGACCCAGAG-3′), mouse promoter (5′-ACATGGTGGCTTTCAACCG-3′ and 5′-GCATCCAGAGAGTGAACTTTAGCC-3′), mouse 10-kb 5′ region (5′-TTCCTATGGCATGTGTACGGC-3′ and 5′-AACACTCTTCGGGCCAATGA-3′), human promoter (5′-TAGCATTGCATCCCTAGCACC-3′ and 5′-TGGTCTATTAAAGATTTTATTTCTCTCTCCT-3′), human 10-kb 5′ region (5′-GTCTCTACTGAAAATACAAAAAAATTGGCT-3′ and 5′-ACTGCAACCTCTGCCTCCC-3′), mouse promoter (5′-CACCCTGGCATTTTCTTCCA-3′ and 5′-GACCCAGAGACCTGAATGCTG-3′), and human promoter (5′-GCCTGAGCAGTCCGGTGT-3′ and 5′-GATCGGTGCTGGTTCCCA-3′).
Fig. S1 shows the relative expression of IRF8 in mouse lymphomas and plasmacytomas. Fig. S2 depicts flow cytometric analyses of CD19 tonsillar B cells transfected with vectors expressing EGFP only or WT IRF8 tagged with EGFP at 18 h after infection. Tables S1 and S2 show mouse and human primers, respectively, for qPCR. Online supplemental material is available at . |
MΦ phagocytosis of promastigotes and amastigotes is rapid and efficient (). In contrast, skin DCs preferentially ingest amastigotes, and this occurs slowly and inefficiently (). We generated bone marrow–derived DCs (BMDCs) using GM-CSF/IL-4 and confirmed our previous findings obtained with skin DCs. Day 6 immature DCs expressed CD11c, intermediate levels of MHC class II, and low levels of CD86 (). BMDCs, like skin DCs, internalized freshly isolated amastigotes in a time- and dose-dependent manner. Normal mouse serum (NMS)-opsonized promastigotes, in contrast, were not readily ingested (27 ± 6 vs. 8 ± 1% infected DCs with a DC/parasite ratio of 1:3 at 18 h; P ≤ 0.05, ). As expected, DC infection was associated with up-regulation of MHC class I/II and costimulatory markers (reference 7 and unpublished data).
Phagocytosis of by MΦ is CR3 dependent (). To investigate the role of CR3 and CR4 in uptake by DCs, we used CD18 mice. As expected, DCs generated from CD18 mice did not express CD11b or CD11c (unpublished data). No differences in the percentages of infected wild type or CD18 DCs () or the number of parasites/cell was observed after DCs and amastigotes were cocultured for 18 h.
We also assessed the involvement of other candidate receptors. Antibodies reactive with CD11b (clone M1/70) (), CD205 (clone NLDC145) (), or preincubation with mannan () were used at optimal concentrations. This concentration of mannan was able to completely inhibit the uptake of by MΦ (unpublished data) (). None of the inhibitors tested affected the uptake of by DCs (). Thus, CR3/CR4 and C-type lectins appear to be dispensable for phagocytosis of by DCs.
amastigotes are isolated from infected tissues, whereas metacyclic promastigotes are enriched from stationary phase in vitro cultures. Among the most prominent differences between surface characteristics is the large amount of Ig bound to the surfaces of amastigotes, but not promastigotes. To determine if Ig was involved in parasite uptake, we quantified the ability of amastigotes isolated from B cell–replete, wild-type BALB/c mice, μMT (B cell–deficient) mice and SCID (B cell– and T cell–deficient) mice to parasitize DCs. DCs readily phagocytosed amastigotes from BALB/c mice, but not amastigotes from μMT or SCID mice (). Opsonization with NMS did not affect uptake. Parasites from B cell–deficient mice efficiently entered DCs only after they had been preincubated with Ig-containing immune serum (IS) from –infected BALB/c mice (or C57BL/6 mice; unpublished data). Phagocytosis of amastigotes by MΦ was not affected by the presence or absence of Ig. Opsonization of amastigotes from B cell–deficient mice with IS () also induced enhanced release of IL-12p40 from DCs, whereas infection of MΦ did not promote IL-12 production. Ig-mediated uptake of amastigotes did induce IL-10 release from MΦ (), whereas little, if any, IL-10 was produced by infected DCs.
To determine if Ig-coated promastigotes could be ingested by DCs, metacyclic promastigotes were left untreated or were opsonized with NMS or IS for 10 min at 37°C. After washing, parasites were cocultured with DCs for 18 h. IS-treated promastigotes were efficiently taken up by DCs and induced IL-12 release, whereas untreated or NMS-treated parasites were not ingested (). Interestingly, complete transformation of promastigotes into amastigotes was not observed within all DCs, even after 18 h (), suggesting that there are differences in the phagosomal compartments of DCs and MΦ that influence this transition (e.g., differences in pH, content of proteolytic enzymes) (). Stimulation of antigen-specific, carboxyl fluorescein succinimidyl ester (CFSE)-labeled T cells with parasite-treated DCs revealed that DCs infected with NMS amastigotes or IS promastigotes induced similar expansion of both CD4 and CD8 T cells, whereas DCs treated with NMS promastigotes did not promote T cell proliferation ().
Amastigotes from infected tissue efficiently parasitize DCs. By flow cytometry, we detected both IgG1 and IgG2a/b on the surfaces of amastigotes when analyzed directly after isolation from C57BL/6 mice or after opsonization with NMS (). Additional opsonization with NMS or IS led to enhanced binding of IgM to amastigotes. Cell surface Ig was not detected on promastigotes. NMS-opsonized promastigotes exhibited surface-associated IgM, attributable to natural Ig found in the sera of naive mice (). Because both NMS- and IS-opsonized amastigotes were taken up to similar extents and NMS-opsonized promastigotes were not phagocytosed by DCs (compare with ), we conclude that IgM is not required for parasite uptake. Interestingly, promastigotes bound similar amounts of IgG1 and IgG2a/b when incubated with sera harvested from infected resistant C57BL/6 mice () or susceptible BALB/c mice (unpublished data).
To conclusively implicate immune IgG in DC-parasite uptake, we isolated total IgG from IS using protein G affinity columns and tested the capacity of IgG to trigger phagocytosis. Similar to IS, the IgG fraction mediated uptake of promastigotes, whereas parasites incubated with the IgG-depleted fraction were not phagocytosed (). In addition, parasite uptake was associated with IL-12p40 release (772 ± 324 pg/ml for DCs incubated with IgG promastigotes vs. 137 ± 27 pg/ml for DCs cocultured with promastigotes incubated with the IgG fraction; ≥ 3, P = 0.03).
IgG1-containing immune complexes bind preferentially to FcγRIII (and FcγRII) and IgG2a-containing complexes bind with higher affinity to FcγRI than to FcγRIII. FcγRII typically mediates endocytosis of soluble immune complexes (). DCs from knockout mice deficient for single FcγR family members ingested as efficiently as DCs from wild-type mice (). In addition, blocking antibodies directed against FcγRII/III (clone 2.4G2) did not have a dramatic effect on uptake by wild-type DCs (, left). However, significant inhibition of phagocytosis by DCs (up to 70%) was observed if DCs from FcγRI/III- or Fcγ-deficient mice were compared with wild-type cells (). Uptake of amastigotes and Ig-opsonized promastigotes was impaired to similar extents. Thus, FcγRI and FcγRIII each facilitate phagocytosis of by DCs, and these receptors can compensate for one another.
In the setting of physiologic low dose infections, we have shown that increased accumulation of both T cells and DCs at inoculation sites coincides with the onset of lesion involution (). In addition, infiltration with DCs was delayed as compared with MΦ recruitment and infection. DCs were identified in lesions beginning 5 wk after inoculation, and their number increased substantially during the healing phase.
To determine if immune IgG, which dramatically enhances infection of DCs in vitro, is present at the time that DCs are recruited to lesions, we infected C57BL/6 mice with 10 promastigotes and quantified the number of inflammatory cells in lesional skin as well as the appearance of -reactive IgG in sera at weekly intervals. shows that by weeks 5–6 after infection, the numbers of DCs as well as serum parasite-specific IgG levels were increased. This indicates that -specific IgG is available to opsonize parasites and enhance phagocytosis by DCs at the time that DCs are infected in vivo. Significant accumulation of CD19 B cells in lesional skin (>10 cells) was not detected within 8 wk after infection.
Previously, we and others have demonstrated that –infected DCs release IL-12 and effectively vaccinate against progressive disease (–). Therefore, infection of DCs in vivo earlier in the course of infection should accelerate development of Th1 immunity. To test this hypothesis, promastigotes were opsonized either with NMS or IS. After washing, low dose infections using 10 opsonized parasites were initiated in the ear skin of C57BL/6 mice (). Inflammatory dermal cells from lesional ear skin were studied weekly (). Interestingly, the numbers of CD11c DCs in IS promastigote–infected skin were significantly higher (7.9 ± 0.9 × 10/lesion) than those in NMS promastigote–treated ears (2.5 ± 0.5 × 10/lesion in week 1, = 3, P ≤ 0.005), especially at early time points. At later time points (week 3 and after), this difference was not evident. DCs were enriched by preparative flow sorting and the number of infected DCs was determined (). At early time points, the percentage of DCs containing intracellular amastigotes was low. However, by week 2, significantly more infected cells were found in ears of mice infected with IS- versus NMS-opsonized parasites (9.5 ± 1.3 vs. 3.4 ± 0.7%, = 3, P ≤ 0.002). By week 3, this difference also disappeared.
Lesion development in infected mice was monitored for >3 mo. Interestingly, cutaneous lesions of mice infected with IS-opsonized parasites were significantly smaller and resolved more quickly than those in mice that were infected with parasites opsonized with NMS (). In addition, lesional parasite loads were decreased in weeks 4 and 6 after infection in mice inoculated with IS-opsonized parasites compared with NMS-treated parasites (). Smaller lesion volumes were associated with increased Th1 immunity as measured by antigen-specific restimulation of LN cells at weeks 4 and 6 (). The IFN-γ/IL-4 ratio in IgG-parasite infected mice was Th1-predominant (week 6: 1,068 ± 250) as compared with mice infected with NMS-opsonized parasites (week 6: 382 ± 86, = 6, P ≤ 0.05). Collectively, these data suggest that enhanced IgG-mediated recruitment and infection of DCs in vivo leads to enhanced Th1 immunity and more rapid resolution of cutaneous lesions.
Because our data suggested that IgG mediates parasite uptake by DCs, we characterized infections in B cell–deficient μMT mice (). Herein, wild-type C57BL/6 or μMT mice were infected with physiologically relevant doses of (10 promastigotes). Compared with wild-type mice, μMT mice showed significantly enhanced lesion progression from week 6 after infection (). Lesion involution was delayed by ∼4 wk in μMT compared with control mice. Furthermore, the skin of μMT mice contained greater numbers of parasites reaching a peak load of 4 ± 2 × 10 parasites/ear at week 6 as compared with 3 ± 2 × 10 parasites/ear in wild types (P ≤ 0.05) (). The IFN-γ/IL-4 ratios of μMT LN cell cultures stimulated with soluble antigen (SLA) were also skewed toward a Th2 profile as compared with C57BL/6 cells. In weeks 6 and 8 after infection, μMT LN cells released significantly less IFN-γ and more IL-4 compared with C57BL/6 mice (e.g., 40.1 ± 12.6 in μMT compared with 100.7 ± 19.2 ng IFN-γ/ml in C57BL/6 mice in week 6, ≥ 9, P ≤ 0.05; ).
We also isolated inflammatory cells from infected ears of μMT and wild-type mice. No significant difference in the number of CD11c DCs that accumulated in the lesions of μMT mice as compared with C57BL/6 ears was found (unpublished data). However, lesions of μMT mice contained significantly fewer –infected DCs at several time points (). Finally, we sought to determine if there was a correlation between numbers of infected DCs and the ability to prime CD4 and CD8 T cells in situ. LN cells of infected C57BL/6 or μMT mice were isolated 6 wk after infection and labeled with CFSE. Antigen-specific expansion of CD4 and CD8 T cells was assessed 5 d after restimulation of LN cells with SLA. μMT LN cells exhibited decreased SLA-specific CD4 expansion as compared with C57BL/6 cells (3.7 ± 1% vs. 15.7 ± 3%; ). Interestingly, the number of -reactive CD8 T cells was also greatly reduced in the absence of B cells (SLA: 2.9 ± 0.5% compared with 14.1 ± 3.9% in C57BL/6 mice, = 5, P ≤ 0.05). In summary, enhanced lesion progression in the μMT mice was associated with decreased numbers of infected DCs and defective T cell priming.
To investigate whether the deficiency in B cells or the lack of antibody contributed to the phenotype of μMT mice, we infected μMT mice with 10 NMS- or IS-opsonized promastigotes. In this setting, μMT mice infected with developed lesions in the presence of immune IgG that were significantly smaller than those caused by NMS-opsonized parasites (). In parallel, decreased lesion volumes in IgG-opsonized parasite-infected μMT mice correlated with significantly smaller parasite burdens in week 6 (). In IS parasite–infected μMT mice, the IFNγ/IL-4 ratio was shifted from a Th2-predominant (828 ± 94) to a Th1 immune response (3,680 ± 1,515, = 4, week 6). Thus, the lack of host IgG is responsible for disease outcome in μMT mice. The skin of μMT mice infected with NMS-opsonized or IS-opsonized promastigotes was analyzed for the presence of infected CD11c DC (). As shown before, infection of maximally 5% of DCs was found in μMT mice infected with NMS-treated parasites. Interestingly, inoculation of IgG-containing parasites led to dramatically increased numbers of infected DCs in the early course of infection (), even higher than those found in wild types (compare with ).
The μMT mice were previously shown to contain Ig in the sera, at least when mice were of BALB/c genetic background (, ). The presence of soluble Ig is due to low-level leakiness of the locus (). To confirm critical experiments in a truly B cell–deficient mouse strain, we infected C57BL/6 JT mice characterized by deletion of the Ig heavy chain (). As shown in , increased lesion development was observed in JT mice over the course of 4 wk identical to the course of infections in μMT mice. This is in contrast with the findings of Miles et al., who reported that J mice on a BALB/c background were less susceptible to infection than their controls ().
Our data suggested that FcγR-mediated uptake of parasites by DCs mediates protection. Thus, we infected Fcγ chain–deficient mice lacking all three known activating FcR with physiologically low dose inocula of (). Lesions were monitored for >3 mo. Fcγ C57BL/6 mice developed more progressive lesions between weeks 4 and 9 as compared with wild-type controls. Maximum lesion sizes in Fcγ mice were detected in week 9, reaching 21 ± 2 mm (C57BL/6: 13 ± 1 mm, = 14, P ≤ 0.008). Increased lesion volumes were paralleled by significantly higher parasite burdens as determined in week 4 after infection (). Similar to the course of disease in B cell–deficient mice, lesion involution in Fcγ mice was normal and all mice ultimately healed their infection. This data suggests that FcR-mediated antibody effects are not an absolute requirement for healing.
Finally, we assessed the number of parasite-containing CD11c DC in lesions of Fcγ mice infected for 4 wk with low doses of (). Ear skin of FcR-deficient mice harbored fewer parasite-infected DC (10.5 ± 2.3%) as compared with wild-type DCs (20.2 ± 3.8%, = 4, P = 0.09). This finding confirmed our in vitro data obtained with BMDCs generated from Fcγ mice that demonstrated inhibited parasite uptake in cocultures with ().
Microbe-binding receptors orchestrate events that occur subsequent to phagocytosis by transducing specific cellular signals (). The main receptor for uptake of promastigotes by MΦ is CR3 (, ). In the initial stages of cutaneous leishmaniasis, most parasites are taken up by MΦ. CR3-mediated phagocytosis of by MΦ leads to selective inhibition of IL-12 release (, –). Production of IL-12 in leishmaniasis is delayed (), and we and others have suggested that DCs, rather than MΦ, are the primary source of this Th1-promoting cytokine. It has also been demonstrated that infected DCs are activated and effectively present antigen to both naive CD4 and CD8 T cells in vitro and vaccinate against leishmaniasis in vivo (, , , ).
Although MΦ and DCs are ontogenically related, their roles in initiation and propagation of immune responses against are distinctly different. Uptake of by DCs differed significantly from that by MΦ with regard to kinetics as well as efficiency. Therefore, we speculated that phagocytosis of parasites by DCs might be promoted by receptors other than CR3. However, a previous report suggested that uptake of amastigotes by Langerhans cells/DCs was mediated via CR3 (). In the present study, using both blocking antibodies as well as cells deficient for CR3 and CR4 (from CD18 mice), we were not able to detect CR3-mediated uptake of by DCs. Recently, C-type lectins (DC-SIGN, DEC-205, and Dectin-1) have also been implicated in the uptake of various pathogens by DCs (, –). We were unable to implicate mannan-binding C-type lectins in phagocytosis of by murine DCs.
In this study, we demonstrate that parasites are predominantly phagocytosed by DCs via FcγRI and FcγRIII. In line with several studies, FcγR ligation was associated with DC activation and IL-12 release (–). We have previously shown that DCs can cross-present antigen to CD8 T cells (), whereas CR3-mediated phagocytosis by MΦ leads exclusively to MHC class II–restricted antigen presentation. These results bear some similarity to experiments evaluating the role of FcγR in antitumor immunity. In Fcγ mice, effective cross-presentation of tumor antigens by DCs was also dependent on FcγR-dependent activation (). In addition, signaling through FcγRI/III facilitated efficient restimulation of tumor-reactive T cells (). Thus, cross-presentation of both tumor-derived and –associated antigens by DCs requires FcγR, and is presumably dependent on production of specific antibody as well.
In MΦ, ingestion of amastigotes, in contrast with CR3-phagocytosed promastigotes, appears to occur through both the FcγR and CR3 (, ). In our work and consistent with prior findings, IgG did not play an important role in the uptake of amastigotes from SCID versus BALB/c mice by inflammatory skin MΦ (). Our results also confirm the finding that IgG-mediated phagocytosis of by MΦ leads to strong release of IL-10, and no IL-12 synthesis (), which might promote parasite survival (). Thus, FcγR-mediated uptake by MΦ and DCs has opposing roles in initiating immune responses in cutaneous leishmaniasis.
The role of B cell–derived IgG in cutaneous leishmaniasis in vivo is not fully understood yet. Polyclonal activation of human B cells leads to the production of large amounts of parasite-specific and nonspecific Ab, particularly IgM and IgG (). Also, amastigotes released into lesional tissue from infected and lysed MΦ appear to be coated with antiparasite antibodies (). In this study, we show that -specific IgG was present in sera at the time of DC accumulation in lesions. Consistent with prior findings, intradermal infection with IgG-opsonized parasites led to enhanced early recruitment of CD11c DCs into the lesions (), most likely by IgG-triggered chemokine release from MΦ (, ). Administration of IgG-opsonized parasites also led to enhanced infection of DC, augmented T cell priming, and limited disease as compared with inoculation of IgG-free parasites.
Prior data and our experiments suggest that IgG-mediated effects differ significantly, dependent on the genetic background of the mice. B cell–deficient J BALB/c mice showed improved disease outcome after infection with supraphysiologic doses of and coinjection of anti- IgG reversed their phenotype (). Administration of IgG at or near the time of parasite inoculation worsened disease outcome in BALB/c mice (, , ). This is consistent with studies demonstrating that FcγR ligation on infected MΦ induced IL-10 release, which in turn prevented parasite elimination and promoted disease progression (, ). Final proof was provided by the demonstration that anti- IgG reconstitution of J BALB/c mice correlated with increased IL-10 production and blocking of IL-10R prevented antibody-mediated disease exacerbation ().
Mice on a -resistant background lacking functional B cells (e.g., μMT C57BL/6 mice) did not exhibit a phenotype with regard to lesion development after high dose infection with (, , , ). However, DeKrey et al. reported that C57BL/6 μMT mice infected with high-dose inocula of showed reduced IFN-γ production after pathogen challenge (). In our experiment, using physiologically relevant low dose inocula, μMT as well as JT C57BL/6 mice consistently exhibited enhanced lesion progression and delayed lesion involution, higher parasite loads, and cytokine profiles consistent with a Th2-predominant immune response as compared with C57BL/6 mice. In accordance with our in vitro data, significantly fewer infected DCs were found in lesions of μMT mice. In addition, we determined that in the absence of IgG-mediated infection of DCs, decreased numbers of -reactive CD4 and CD8 T cells developed. The defects observed in μMT mice were reversed by using IgG-opsonized parasites for infection indicating that the deficiency in Ig is responsible for worsened disease outcome in B cell–deficient mice.
As expected from the in vitro results obtained with BMDCs generated from Fcγ-deficient mice, we also found decreased numbers of infected DC in Fcγ mice paralleled by increased lesion volumes over the course of several weeks and higher parasite burdens. In contrast, in prior studies, improved disease outcome of Fcγ mice was observed using infections with or (, ). However, the mice used were on a BALB/c background and, thus, are not comparable to those used for this study. Data generated with -resistant mice might be more physiologically relevant in a clinical setting because the course of disease in, for example, C57BL/6 mice more closely mimics infections of humans.
In summary, we propose that the two predominant APCs in skin, MΦ and DCs, are sequentially engaged via different pathogen recognition receptors as cutaneous leishmaniasis evolves. Although in the initial “silent” phase, promastigotes are primarily phagocytosed by resident MΦ via CR3, FcγR and DCs become critically important in established infections. IgG-mediated uptake of by DCs leads to IL-12 production and priming of Th1/Tc1 cells, both of which are required for efficient parasite killing by lesional MΦ. In contrast, FcγR-mediated uptake of amastigotes by MΦ induces counterregulatory IL-10 production. This may facilitate activation of regulatory T cells, which, in turn, promotes parasite persistence and maintenance of T cell memory (, ). The balance between CR3 and FcγR-triggered anti- and proinflammatory mechanisms involving MΦ and DCs is critical for disease outcome. The unexpected identification of immune IgG production as a prerequisite for efficient cross-priming of -specific Th1/Tc1 cells is intriguing. In future experiments it will be important to assess the T cell dependence of -reactive antibody production, and to identify the APCs that are involved in B cell and, if relevant, Th priming.
6–8-wk-old BALB/c and C57BL/6 mice were purchased from the Central Animal Facility of the University of Mainz. CD18 mice () on a mixed C57BL/6J and 129/SV background were provided by K. Scharffetter-Kochaneck (Department of Dermatology, University of Ulm, Ulm, Germany). FcγRII mice () were obtained from H. Mossmann (Max Planck-Institut für Immunbiologie, Freiburg, Germany). Mice deficient for FcγRIII () and FcγRI () as well as FcγRI/III double deficient mice (all C57BL/6 background) were provided by S. Verbeek. C57BL/6 Fcγ were obtained from T. Saito (RIKEN Research Center for Allergy and Immunology, Yokohama, Japan) () or from Taconic. B cell–deficient mice (C57BL/6 SCID, μMT, JT) were gifts from M. Neurath, K. Steinbrink, and A. Waisman (all from University of Mainz, Mainz, Germany). All animals were housed in accordance with institutional and federal guidelines. All experiments were undertaken with approved license from the Animal Care and Use Committee of the Region Rheinland-Pfalz.
Inflammatory skin-derived MΦ (MΦ) were elicited by subcutaneous injection of polyacrylamide beads and enriched to homogeneity (). BMDCs were generated in GM-CSF– and IL-4–containing media () and harvested on day 6 of cell culture. The characteristics of the cell populations were assessed by flow cytometry using relevant surface markers. The following antibodies were used: anti–I-A/I-E (2G9), anti-CD11b (M1/70), anti-CD11c (HL3), anti-CD40 (3/23), anti-CD54 (3E2), anti-CD80 (1G10), anti-CD86 (GL1) (all from BD Biosciences/Becton Dickinson), anti-F4/80 (Serotec), and respective isotype control mAb.
Metacyclic promastigotes or amastigotes of clone VI (MHOM/IL/80/Friedlin) were prepared as described previously (, ). Amastigotes were prepared from infected footpads of BALB/c or C57BL/6 mice, or mice genetically deficient in B cells (μMT, SCID) to obtain parasites devoid of Ig. Isolated parasites were opsonized with 5% NMS or serum from 6 wk–infected BALB/c or C57BL/6 mice (immune serum, IS) for 10 min (37°C) and washed before in vitro or in vivo infections. Parasites were stained for surface-associated Ig using isotype-specific secondary antibodies reactive with mouse Ig: anti-IgM (Serotec), anti-IgG1 (A85-1), and anti-IgG2a/b (R2-40, all from BD Biosciences). After staining, parasites were washed with PBS/2% BSA, fixed, and analyzed by flow cytometry. Anti- IgG was prepared from pooled sera of ≥5–6-wk –infected BALB/c mice using protein G columns (Pierce Chemical Co.) following the manufacturer's protocol. Sera were stored at −20°C before IgG purification. Purified IgG was stored at 4°C (0.8 mg/ml) in PBS before use.
Isolated cells were subcultured in medium (RPMI 1640/5% FCS) at 2 × 10/ml and parasites were added at the parasite/cell ratios indicated. In some experiments, cells were preincubated for 60 min with mannan (Sigma-Aldrich, 1 and 5 mg/ml), anti-CD11b, anti-CD16/32, anti-CD205, or control rat IgG (all at 50 μg/ml, all from BD Biosciences). Cells were harvested after several hours and cytospins were prepared. DiffQuick-stained cells were analyzed for the presence of intra- and extracellular parasites. At least 200 cells were counted per sample. Supernatants from parasite/cell cocultures were collected and assayed for the presence of IL-12p40 or IL-10 by ELISA (BD Biosciences).
Groups of ≥5 C57BL/6 mice were infected intradermally in ear skin with 1,000 promastigotes. At several time points, ears were harvested and the number of B cells and DCs that had accumulated at the site of infection was determined (). In brief, ears were incubated with 2 mg/ml Liberase (Boehringer Ingelheim). After 2 h, cells were dissociated mechanically and counted and the frequency of CD19 and CD11c cells was assessed using flow cytometry. In addition, serum from infected mice was obtained at several time points and stored at −20°C.
-specific IgG in serum was quantified by ELISA. Flat-bottom 96-well plates (Nunc) were coated overnight with 0.5 mg/100 μl of soluble freeze-thaw lysate (SLA), blocked for 1 h with PBS/2% BSA/0.05% Tween 20, and incubated for 2 h with dilutions of sera or reference standard anti- IgG (prepared from pooled sera of immune infected mice). Subsequently, biotinylated goat anti–mouse IgG (Caltag) was added (125 ng/ml) for 2 h at 20°C. ELISA plates were developed using commercially available ELISA kit components (BD Biosciences) and reaction products were quantified spectrophotometrically.
C57BL/6, μMT, JT, or Fcγ mice were infected intradermally with 10 metacyclic promastigotes. In some experiments, parasites were opsonized for 10 min with either NMS or IS and washed. Lesion development was assessed weekly in three dimensions using a caliper, and lesional volumes are reported (in mm) as ellipsoids [(a/2 × b/2 × c/2) × 4/3π]. Organisms present in lesional tissue were enumerated using limiting dilution assays (). For measurement of cytokine production, 10 retroauricular LN cells/200 μl were added to 96-well plates in the presence of SLA (25 μg/ml). Antigen-specific IFN-γ and IL-4 production was determined after 48 h using ELISA (R&D Systems).
At several time points, ears were harvested and inflammatory cells isolated using Liberase and mechanical disruption (). The cells were counted and the frequencies of CD11c DC were determined using flow cytometry. CD11c cells were enriched to >98% purity using a high speed cell sorter (FACS Vantage SE System, Becton Dickinson) and cytospins were analyzed by light microscopy to estimate the number of infected DCs/ear.
The frequency of daughter cells of proliferating antigen-reactive compared with nonproliferating LN T cells was estimated using flow cytometry (–). 6 wk after infection, LN cells were harvested and 5 × 10 cells/ml were labeled with 1 μM CFSE (Invitrogen). LN cells were subsequently plated at 10/200 μl media in a 96-well U-bottom plate and left untreated or stimulated with SEB (10 μg/ml; Sigma-Aldrich), or SLA (). After 5 d, proliferation was determined using flow cytometry. T cells were selected for analysis using mAbs against CD4 (L3T4, RM4-5), CD8 (Ly2, 53–6.7), or isotype control mAb (all from BD Biosciences). For each mouse, the percentage of -reactive cells compared with nonproliferating cells was calculated.
Statistical analysis was performed using the unpaired Student's test. |
We first performed flow cytometry analysis and found that the majority of isolated bone marrow mononuclear cells (BMMNCs) expressed α4 integrin (). To investigate whether BM α4 integrin–positive populations contain primitive EPCs in the steady-state, we used FACS to obtain equal numbers of CD45α4 and CD45α4 cells from the BMMNCs, then conducted a two-step EPC colony culture assay. The EPC colonies were identified by double staining for DiI-acLDL uptake and isolectin B4-FITC binding (, left). Primitive EPCs with colony-forming potential were exclusively α4 integrin–positive (, right). Moreover, flow cytometric analysis of BMMNCs using triple staining for α4 (or CD45) with two surrogate EPC markers, Sca-1 and Flk-1, demonstrated that only α4 or CD45 populations, not α4 or CD45 cells, contain Sca-1 and Flk-1 double-positive cells (). These results are in agreement with the EPC colony assay () and further suggest a possible role of α4 integrin in EPC homeostasis in the BM.
To investigate whether blockade of α4 integrin mobilizes BM EPCs to the peripheral blood (PB), we injected PS/2, a monoclonal α4 integrin–blocking antibody (Ab) i.v. into wild-type mice. After 24 h, peripheral blood mononuclear cells (PBMNCs) were isolated, and flow cytometry analysis was performed using the EPC markers Sca-1 and Flk-1. We found a substantial increase in circulating Sca-1Flk-1 double-positive cells in the Ab-treated mice compared with control IgG-treated mice (). The circulating EPCs (circEPCs) were also evaluated by EPC culture assay using isolated PBMNCs. The Ab treatment significantly increased the number of circEPCs, indicated by the increase in adherent cells double positive for DiI-acLDL uptake and isolectin B4 binding after culture (). In fact, we detected a higher level of circEPCs for up to 3 d after a single injection of the anti–α4 Ab in the time course study (). We also performed the EPC culture assay using PBMNCs from conditional α4 integrin knockout mice, those in which ∼97% of the BMMNCs had lost α4 integrin expression after induction of cre expression (). The knockout mice demonstrated a significantly greater number of circEPCs compared with their WT littermates (). These data indicate that specific disruption of the α4 integrin molecule increases the number of circEPCs.
Because VCAM-1 has been shown to be a major ligand of stem cell α4 integrin in the BM, we investigated whether α4 integrin–blocking Ab interferes with the adhesion interaction between α4 integrin and VCAM-1, postulating that such interference could contribute to anti–α4 integrin Ab–induced EPC mobilization. We applied freshly isolated BMMNCs to immobilized recombinant VCAM-1 in cell culture plates. The anti-α4 integrin Ab not only blocked BMMNC adhesion to VCAM-1 when added before the cells, but also competed with this adhesion in a dose-dependent manner when added after the cells (, top). Cells suspended after the addition of Ab were harvested and seeded for an EPC colony–forming assay. The number of EPC colonies grown from the suspended cells was proportionate to the total number of suspended cells (, bottom), suggesting that VCAM-1 supports α4 integrin–mediated EPC attachment and that α4 integrin–blocking Ab fosters EPC release. We also performed adhesion assays using another α4 integrin ligand, fibronectin (FN), and another extracellular matrix molecule, intercellular cell adhesion molecule (ICAM)-1, in parallel with VCAM-1. FN and ICAM-1 conferred lower levels of BMMNC adhesion compared with VCAM-1 (). The anti–α4 Ab, however, did not significantly compete or block adhesion of BMMNCs to FN or ICAM-1 ().
Because other α4 integrin ligands in the BM in addition to VCAM-1 and fibronectin may be involved in α4 integrin–dependent adhesion between EPCs and the stroma, we performed an additional adhesion assay using single-layer stromal cells grown from total mouse BM. Again, anti–α4 integrin Ab significantly blocked and competed the adhesion of EPCs to the BM stroma in a dose-dependent manner (), suggesting that the Ab may release BM EPCs from α4 integrin–mediated attachment in the BM.
Because tissue ischemia has been shown to induce EPC mobilization (), we examined the effect of α4 integrin blockade on the level of circEPCs after ischemia. We surgically induced hind limb ischemia (HLI) by excision of the left femoral artery in mice and randomized them to receive immediate anti–α4 integrin Ab or control IgG twice per week for 3 wk. As shown in , anti–α4 integrin Ab significantly increased the degree and the duration of HLI-induced EPC elevation in the peripheral circulation.
We used two mouse ischemia models, Tie2/LacZ BM transplantation plus hindlimb ischemia (Tie2/LacZ-BMT+HLI) and Tie2/GFP BM transplantation plus myocardial infarction (MI) (Tie2/GFP-BMT+MI), to investigate whether BM EPC mobilization induced by α4 integrin blockade affects angiogenesis. In the Tie2/LacZ-BMT+HLI model, we reconstituted the BM of WT mice with BMMNCs genetically marked with Tie2/LacZ, which allows for easy detection of BM-derived cells with immunofluorescent staining for β-galactosidase (β-gal). After surgical induction of HLI, the mice received periodic injections with either anti–α4 integrin Ab or control IgG. As shown in , the Ab-treated mice exhibited accelerated blood flow recovery compared with the IgG-treated animals, when assessed on days 7, 11, and 14. In addition, there was a significantly greater number of endothelial cells (ECs) of BM origin in the ischemic limb in the Ab-treated mice compared with the IgG-treated mice, when examined 14 d after induction of HLI (). The overall capillary density was also significantly higher in the Ab-treated mice (). Ab treatment conferred better long-term preservation of muscle tissue, as assessed by the ratio of muscle weight in ischemic limbs to normal limbs at 60 d after induction of HLI (). The endothelial identity of BM-derived cells incorporated in the neovasculature of the ischemic tissues was further confirmed by immunofluorescent staining for another independent endothelial marker, CD31, along with β-gal ().
We further evaluated the EPC-mediated proangiogenic effect of α4 integrin blockade in the Tie2/GFP-BMT+MI model. MI was induced by permanent ligation at the middle of the left anterior descending (LAD) coronary artery. Again, Ab-treated mice exhibited a significantly greater number of BM-derived ECs in the infarcted heart, indicated by a greater number of cells staining positive for both Tie2-driven GFP and (BS) lectin I–Rhodamine (). Interestingly, we also detected significantly more preexisting capillaries that survived (BS lectin I–RhodamineGFP) in the infarcted area () and a significantly higher capillary density in the periinfarct area in the Ab-treated mice compared to controls (). α4 integrin blockade significantly reduced both the left ventricular fibrosis area () and left ventricular dilation () when examined 2 wk after infarction, suggesting a favorable effect of α4 integrin blockade on the remodeling of the infarcted murine heart.
α4 integrin may play an important role in leukocyte recruitment during tissue inflammation (). The recruitment of EPCs to ischemic tissue is a key feature in EPC-mediated vasculogenesis, but the role of α4 integrin has not been clear. Therefore, the increased number of BM EPCs in the ischemic neovasculature after systemic blockade of α4 integrin was intriguing. To investigate whether α4 integrin plays a role specifically in EPC recruitment to ischemic tissue, we designed an in vivo EPC tissue homing assay. Isolated BMMNCs were pretreated ex vivo with either α4 integrin–blocking Ab or control IgG, labeled with DiI, and directly injected into the peripheral circulation of mice that had undergone surgical HLI and splenectomy without irradiation. This experimental design was used to minimize sequestering of EPCs (in the spleen) thereby providing the best opportunity to examine the impact of α4 blockade on tissue homing. Interestingly, BMMNCs pretreated with α4 integrin–blocking Ab were as well represented as BMMNCs pretreated with control IgG in the neocapillaries formed in the ischemic limb. We found similar numbers of DiI-labeled and control EPCs incorporated in the neocapillaries () and equal numbers of labeled cells from the two groups circulating in the PB. Because an equal number of BMMNCs were injected into the two groups of mice, these data indicate that the ex vivo α4 integrin blockade did not affect EPCs homing or incorporation into neocapillaries, and further support the importance of the mobilization effect of α4 integrin blockade on changes in ischemic tissue repair. Similar results were also obtained in mice without splenectomy.
To further confirm this observation and to overcome certain limitations of Ab blockade, such as unanticipated effects in nontarget tissues, BMMNCs were isolated from α4 integrin conditional knockout mice. WT litter mates served as controls. We injected these BMMNCs into background-matched WT mice that had undergone surgical MI and splenectomy. Again, loss of α4 integrin did not impair incorporation of the injected BM EPCs into the neovasculature of infarcted cardiac tissue (), consistent with the results obtained from the in vivo homing assay using α4 integrin–blocking Ab. These results confirm that loss of α4 integrin does not impair EPC homing to ischemic tissue.
In this study, we demonstrated that blockade of α4 integrin promotes mobilization of BM EPCs to the peripheral circulation and promotes functional neovascularization after ischemia. Several lines of evidence support this conclusion. First, primitive, colony-forming EPCs in isolated BMMNCs are exclusively α4 integrin–expressing cells. Second, anti–α4 integrin Ab blocks and competes with the adhesive interaction between BM EPCs and immobilized VCAM-1 or BM stroma ex vivo. Third, systemic administration of anti–α4 integrin Ab or conditional knockout of α4 integrin in the BM significantly increases circEPCs. Fourth, after ischemic injury, anti–α4 integrin Ab fosters homing of BM-derived EPCs to the neovasculature at ischemic tissue and augments recovery of blood flow and tissue preservation. Our study establishes for the first time that α4 integrin plays an important role in EPC mobilization and that functional disruption of α4 integrin–mediated EPC lodgment in the BM causes a shift toward a distribution of EPCs that is more favorable for neovascularization.
Anti–α4 integrin blocking Ab has previously been shown to mobilize BM HPCs (). These cells, in turn, have been shown to contribute to neovascularization at ischemic sites by secreting a spectrum of growth factors and supporting the establishment of EPCs (, ). Because Tie2 expression has also been found in a subset of HPCs (, ), it is possible that the potent proangiogenic effect of anti–α4 integrin Ab treatment observed in our study may have resulted from the combined mobilization of circEPCs and HPCs.
Granulocytes, macrophages, and lymphocytes have been shown to secrete various pro- and antiangiogenic factors in ischemic tissue and play complex roles in recovery after ischemia (, ). It has also been shown that α4 integrin plays a role in cytokine-induced leukocyte–endothelium interactions (–) and that blockade of α4 integrin inhibits inflammatory cell recruitment (, –). Consistent with these prior observations, we noted a decrease in F4/80-positive cells in the ischemic limbs of the Ab-treated mice (Fig. S1, available at ). Although the mechanism of EPC migration to ischemic tissue has been under intensive investigation, the role α4 integrin plays in this process is not yet elucidated. Some evidence suggests that α4 integrin may be unimportant for stem/progenitor cell tissue homing and migration, because α4 integrin levels are substantially down-regulated on mobilized stem cells in PB (, , ), whereas activated, migrating inflammatory cells express abundant α4 integrin (). It has recently been shown that circEPCs isolated from human PB express low levels of α4 integrin () and that neutralizing Ab to VLA-4 significantly inhibits adherence of BM CD34, but not mobilized PB CD34 stem cells, to stromal cells, suggesting the existence of alternative cell adhesion molecules that mediate circulating stem cell binding (). In addition, it has been reported that α4 integrin is necessary for the homing and lodgment of stem/progenitor cells to BM, but not to spleen (, , ). However, α4 integrin may play a role in soluble VCAM1-induced migration and angiogenesis in HUVECs (), as recently reported.
In the current study, we found that neither ex vivo blockade nor genetic knockout of α4 integrin prevents EPC homing to ischemic tissue, suggesting that adhesion activity of α4 integrin is not essential for homing of circEPCs to ischemic tissue in the setting of acute ischemia. Coincidently, it has recently been shown that β2 integrin may play a more prominent role in the homing of EPCs to ischemic skeletal muscle ().
Despite the fact that several other β1 integrins are known to be essential to various angiogenesis processes, the direct role of α4β1 integrin in angiogenesis and vasculogenesis remains largely obscure (–). Limited studies in experimental models suggest that α4 integrin may play a role in angiogenesis induced by TNF-α and soluble VCAM-1 but not by basic fibroblast growth factor (, ). Studies currently underway in our lab suggest that TNF-α signaling is indeed required for ischemic angiogenesis (). Unfortunately, with the exception of TNF-α, the stimuli for angiogenesis during tissue ischemia are not well understood at this time. Nevertheless, the effect of anti–α4 integrin treatment on local angiogenesis warrants further investigation.
A VLA-4–dependent mechanism has previously been shown to play an important role in mononuclear leukocyte emigration during early atherosclerosis (), neointimal formation after vessel injury (), and neutrophil-mediated cardiac myocyte dysfunction (). Blockade of α4 integrin has been shown to attenuate atherosclerosis () and reduce postinjury intimal hyperplasia (, ) and neoadventitial formation () in animals. Our study indicates that α4 integrin blockade enhances the mobilization of EPCs and EPC-mediated neovascularization. These data suggest a novel therapeutic strategy for stimulating therapeutic neovascularization in acute and chronic ischemia. Moreover, the rapidity and durability of EPC mobilization induced by a single dose of anti–α4 Ab compare favorably with currently available agents such as G-CSF () and may make this approach a practical addition to the therapeutic armamentarium.
The antimurine α4 integrin mAb PS/2 was purified from cultured hybridoma cells (American Type Culture Collection) using Montage Antibody Purification kits. The antimurine ICAM-1 blocking Ab was purchased from R & D Systems. All other Abs and isotype controls were purchased from BD Biosciences. A second mAb against a different recognition site of α4 integrin was used to confirm the specificity of the purified PS/2 Ab with flow cytometry analysis. The isotype control IgG of PS/2, rat IgG2b, was dialyzed to remove sodium azide when used in vivo.
Male FVB/NJ and background-matched Tie2/LacZ or Tie2/GFP transgenic mice were purchased from the Jackson Laboratories. The conditional α4 integrin knockout mice (Mx.
α4) and control littermates (α4) were generated as described previously (). The mice were maintained and operated following protocols proved by the Caritas St. Elizabeth's Institutional Animal Care and Use Committee.
Mouse BMMNCs or PBMNCs were isolated with density-gradient centrifugation (). Flow cytometry analysis and FACS sorting of the isolated BMMNCs or PBMNCs were performed as previously described ().
Taking advantage of the late growth and high proliferative properties of primitive EPCs, we developed a two-step EPC colony assay. The isolated mouse BMMNCs were cultured in 0.1% vitronectin/gelatin-coated plates in EBM-2 complete medium (EBM-2 basal medium supplemented with the cytokine cocktail in endothelial growth medium-2–microvascular; SingleQuots, Clonetics, Inc.) (). To evaluate BM colony–forming EPCs, 5 × 10 FACS-sorted CD45α4 or CD45α4 cells were cultured in a 6-well plate for 4 d and then resplit into a 10-cm plate, cultured for another 7 d. DiI-acLDL (1:500) was added, and the cells were incubated for 4 h. The cells were then washed with PBS, fixed in 1% PFA, and stained with isolectin B4-FITC (1:200). Cell colonies double positive for DiI-acLDL uptake and isolectin B4-FITC binding were counted. The numbers of EPC colonies reflected the number of primitive EPCs in the initial sorted cell fractions.
To count circEPCs, an EPC culture assay was performed as previously described (). In brief, the PBMNCs isolated from a 500-μl sample of PB were cultured in vitronectin-coated 4-well chamber slides in EBM-2 complete media. On day 4 of the culture, DiI-labeled acLDL was added to the media. After incubating 4 h, the cells were fixed in 1% paraformaldehyde and counterstained with isolectin B4-FITC. Double-positive cells were counted as EPCs, the number of which reflected the number of initial circEPCs in the 500-μl sample.
90 6-well tissue culture plates were coated with 10 μg/ml recombinant murine VCAM-1 or ICAM-1 (R & D Systems), or 50 μg/ml rat plasma fibronectin (Sigma-Aldrich). Freshly isolated BMMNCs (5 × 10) were added to each well. Antibodies were added either just before addition of the BMMNCs to block adhesion, or 15 min after the addition of cells to compete with adhesion. The cells and antibodies were coincubated in a 5% CO incubator at 37°C for 30 min. After incubation, nonadherent and loosely attached cells were removed by tapping each plate and gently washing the wells three times with Dulbecco's phosphate-buffered saline. Cells in the group with 100% attachment were not washed. Attached cells were fixed in 5% glutaraldehyde, stained with 0.1% crystal violet, and solubilized in 10% acetic acid. A microplate reader was used to measure the absorbance at 564 nm. The background crystal violet staining level was subtracted from readings, and the values were expressed as the percentage of attachment. To examine the effect of α4 integrin–blocking Ab on BMMNC adhesion to BM stromal cells, a single layer of BM stroma was prepared as previously described (). Adherent cells were counted during microscopic examination, and the result expressed as the ratio of the number of adherent cells in each experimental group to the number in the 100% attachment group.
This procedure was preformed as previously described (–). See supplemental Materials and methods, available at , for details.
BM transplantation and quantification of engraftment were performed (see supplemental Materials and methods) using Tie2/GFP mice as donors. Myocardial infarction was induced in recipient mice under artificial ventilation by permanent ligation of the middle of the left anterior descending (LAD) coronary artery. Mice were randomized to receive i.v. injection of either 200 μg of anti–α4 integrin Ab or control IgG, twice weekly starting on day 1. On day 14, the mice were injected with 50 μl of BS lectin I–Rhodamine (Vector Laboratories) at the apex of the left ventricle (LV), and after 5 min the cardiac vasculature was perfused with 4% PFA through the right carotid artery with distal aortic arch clamped. Cardiac tissue was fixed for 1 h in 4% PFA, incubated in 30% sucrose solution overnight, snap frozen in liquid nitrogen, and preserved at −80°C. Serial cryosectioning was performed starting at 1 mm below the suture (used to ligate the LAD) moving toward the apex, with three consecutive sections per 1 mm to allow for quantitative pathohistological analysis at each level (see next paragraph). Three sections per ischemic heart and 9 fields per section (6 fields in the infarct border zone, 3 fields in the infarct area) were examined with BS lectin I–Rhodamine to quantify total capillary density or with RhodamineGFP to determine BM EPC-derived capillary density. Masson's Trichrome staining was performed as previously described (). The fibrosis area was calculated as the ratio of the length of fibrotic area to the length of LV inner circumference (, d/c), and the LV dimension was quantified histologically (, (a+b)/2). All surgical procedures and patho/histological analysis was performed by investigators blinded to treatment assignment.
BMMNCs were isolated from donor WT mice. Equal numbers of the cells were either blocked with anti–α4 integrin Ab or treated with control IgG. Cells were then labeled with DiI cell tracer and washed thoroughly. Recipient mice underwent excision of the left femoral artery to induce HLI and underwent splenectomy without irradiation. Mice were then randomized to immediately receive by tail vein injection either 15 × 10 BMMNCs that had been treated with anti–α4 integrin Ab or 15 × 10 cells that had been treated with control IgG. The circulating DiI-labeled cells in the PB were monitored on days 1, 3, and 7 by flow cytometry or fluorescent analysis of PBMNCs. No difference was found between the two groups of recipient mice (unpublished data). On day 7, the mice were injected with BS lectin I–FITC and killed. The capillaries derived from injected BMMNCs, which were double positive for BS lectin I–FITC and DiI, were examined microscopically and quantified. In another similar independent experiment, BMMNCs isolated from α4 integrin conditional knockout mice (∼97% of cells deficient for α4 integrin) or control WT littermates were used. 10 × 10 cells were injected i.v. into background-matched recipient mice that had undergone surgical MI and splenectomy. On day 7, the mice were injected with BS lectin I–FITC and killed. The ischemic cardiac tissue was processed as described before, and the neocapillaries derived from the injected BM EPCs in the ischemic area were quantified.
Data are presented as average ± SEM. Comparison between two means was performed with an unpaired Student's test. Comparisons of more than two means were performed using ANOVA with Fisher PLSD and Bonferroni Dunn Post Hoc analysis. Statistical significance was assigned if P < 0.05.
Supplemental materials and methods describe mouse Tie 2/LacZ-BMT and the hindlimb ischemia model. Fig. S1 depicts hindlimb mouse tissue injected with control IgG and anti-α4 Ab. Online supplemental material is available at . |
HSCs in 4,000 ckit Thy1.1 lineage Sca-1 Flk2 CD34 cells from CD45.1 mice were transplanted intravenously into five unnirradiated CD45.1 × CD45.2 (F1) and five unirradiated congenic CD45.2 mice. Every 4 wk after transplantation, peripheral blood was analyzed for granulocyte, B cell, and T cell chimerism. Donor granulocyte chimerism, which accurately reflects HSC chimerism (), was observed only in the genetically unreactive F1 recipients at all time points analyzed with a median chimerism at 16 wk of ∼0.1% (). These data demonstrate that the subtle antigenic differences that exist between these CD45 congenic strains of mice are sufficient to prevent productive HSC cross-engraftment.
To gauge the potential clinical importance of these rare available niches, we repetitively transplanted HSCs from GFP-transgenic mice into RAG2 and IL-2 receptor common γ chain–deficient (RAG2γc) mice, which lack B, T, and NK cells (). Enormous numbers of donor-derived B and T cells were found in the blood at all time points, leading to an overall donor chimerism of ∼50% until at least 30 wk after the final transplantation (). Donor NK cells were also detected in all transplanted animals (not depicted). Moreover, all RAG2γc mice displayed persistent donor-derived myeloid chimerism (ranging from 0.5 to 2.0% donor-derived granulocytes).
To confirm that the donor granulocyte frequencies accurately reflected bone marrow HSC chimerism, we killed animals at 30 wk after transplant and analyzed bone marrow. Donor cells comprised ∼0.8% of the total long-term (LT)-HSC pool (). This chimerism was essen-tially the same in the short-term reconstituting stem cells (ckit lineage Sca-1 CD34 Flk2) and the multipotent progenitors (ckit lineage Sca-1 CD34 Flk2; not depicted). We were unable to detect any cells with these surface phenotypes in the spleen, a major organ associated with extramedullary hematopoiesis (not depicted). These data confirmed that in these animals, peripheral donor granulocyte frequencies much more accurately reflect HSC chimerism than the overall donor contribution in the blood. The data also demonstrate that small numbers of bone marrow–engrafted HSCs can correct severe lymphoid deficiencies without prior cytoreductive conditioning. The HSC chimerism in the RAG2 γc mice was comparable to the chimerism seen in the genetically unreactive F1 wild-type mice (), demonstrating that there are not obviously greater numbers of available HSC niches in these animals.
To confirm that functional HSCs had engrafted, we isolated bone marrow at 31 wk after transplant from unirradiated RAG2γc mice that had received GFP HSC transplants and performed secondary transplants using either unfractionated or c-kit–enriched marrow, which increases the frequency of HSCs by ∼10-fold, into lethally irradiated wild-type mice, such that ∼6–20 GFP HSCs along with 1,000 RAG2γc HSCs were transferred into each secondary recipient. Donor-derived GFP cells were observed in all secondary recipients until at least 25 wk after transplantation, and 6 out of 13 secondary recipients maintained detectable levels of granulocyte chimerism (). These data confirmed that rare GFP HSCs within the bone marrow of the primary RAG2γc recipients had productively engrafted. In contrast, transplantation of large numbers of splenocytes into secondary recipients did not lead to sustained multilineage stem cell reconstitution (not depicted).
To determine the developmental stage at which donor B cells overtake host B cells in RAG2γc recipients, we analyzed donor frequencies in myeloid and lymphoid progenitor cells in the bone marrow. Common myeloid progenitors (CMPs; reference 20) and common lymphoid progenitors (CLPs; references –) showed donor chimerism that was comparable to HSC chimerism (), indicating that donor-derived cells do not have a competitive proliferative advantage at these early developmental steps. Donor chimerism at the granulocyte macrophage progenitor (GMP) and megakaryocyte erythrocyte progenitor (MEP) developmental steps were also similar to HSC chimerism (not depicted). Consistent with these results, the frequencies of endogenous HSCs, CLPs, CMPs, GMPs, and MEPs within the bone marrow are similar between untransplanted wild-type and RAG2 γc mice (). Analysis of the pro–B-A and pro–B-B cell fractions (, ), however, showed donor chimerism that was dramatically higher than the preceding CLP (). At the pro–B-B cell stage and all subsequent B cell stages, cells were exclusively donor-derived. Although IL-7 receptor, which uses γc for proper signaling, is expressed at the CLP stage, these results suggest that IL-7 signaling is not a requisite pathway for CLP development or expansion, consistent with previous observations ().
To verify that the immune system of the HSC-reconstituted RAG2γc mice had been restored and was capable of mounting appropriate immune responses, we immunized reconstituted recipients with alum-precipitated 4-hydroxy-3-nitrophenylacetyl (NP) conjugated to chicken γ globulin, which elicits a Th2-dependent humoral response (, ). Serum levels of NP-specific antibody were similar at 1 wk after immunization between HSC-transplanted RAG2γc and wild-type mice, demonstrating the immunocompetence of the transplanted RAG2γc recipients ().
To investigate whether the absence of γc was critical for HSC engraftment in unconditioned hosts, perhaps by imparting a competitive disadvantage on HSCs in RAG2 γc animals, we transplanted GFP HSCs into RAG2 mice, which have normal γc expression, as well as into RAG2γc mice. As shown in , similar levels of donor granulocyte contribution were seen in RAG2 and RAG2γc mice, likely excluding a direct role for γc in maintaining host HSCs within their niches. However, γc expression has been observed in normal HSCs (), suggesting that there may be a slight competitive advantage for HSCs with proper γc expression. The data also suggest that NK cells, which are present in normal numbers in RAG2 mice but absent in RAG2γc mice, are not mediating HSC rejection in this H2-identical system. Elimination of host NK cells is required for engraftment of HSCs that carry one or more unshared H2 haplotype (). Interestingly, donor-derived lymphocyte frequencies were significantly reduced in RAG2 recipients at early time points (), perhaps as a result of the occupation of the lymphoid stage–specific stromal environments by RAG2 lymphoid progenitors (). The number of donor-derived B cells in RAG2 recipients was reduced more than 10-fold relative to RAG2γc recipients at 4 wk after transplant, and peripheral T cells were not seen at all until 8 wk after transplant (not depicted). However, by 16 wk after transplantation, B and T cell numbers in RAG2 recipients reached the levels seen in their RAG2γc counterparts ( and ).
Because RAG2 mice lack both mature B and T cells, we sought to determine which of these cell types was primarily responsible for mediating the rejection of transplanted donor HSC grafts. Therefore, we transplanted purified HSCs from GFP donor mice into unnirradiated TCRαβ and Cμ mice. In these experiments, known antigenic differences between donor HSCs and recipient mice exist at the GFP, CD45, and Thy1 loci. Multilineage engraftment was observed in T cell–deficient mice, but not in B cell–deficient mice (). These experiments show that host αβ T cells are required for the rejection of HSC grafts with these minor histocompatibility mismatches. To determine which class of T cells is essential for this immunosurveillance, we transplanted HSCs into unconditioned I-A mice, which lack MHC II–restricted CD4 T cells (), and β-microglobulin (βm) mice, which are deficient in MHC I–restricted CD8 T cells (). The HSC-transplanted I-A mice showed sustained chimerism until at least 16 wk, whereas the βm recipient mice did not show chimerism at any time point (), thereby demonstrating that CD4 T cells are essential for the rejection of minor histocompatibility–mismatched HSC grafts in our system. LT-HSCs express MHC II () and the costimulatory molecule CD86 (), suggesting that host CD4 T cells may directly recognize HSCs with slight antigenic mismatches. Although it appears that CD8 T cells are not required for this rejection, we cannot exclude the possibility that residual hyperreactive MHC I–restricted T cells might also contribute to HSC graft rejection in the βm mice (–). Consistent with the role for CD8 T cells in mediating bone marrow graft rejection, Xu et al. () have shown that host CD8 deficiency enhances engraftment.
Interestingly, the granulocyte chimerism in the RAG2 recipients was indistinguishable from that seen in previous experiments in which 3,000 HSCs rather than 1,000 were transplanted (). In contrast, although transplantation of 20 HSCs led to detectable B and T cell production in these immunodeficient mice, granulocyte chimerism was barely detectable (not depicted). Thus, our experiments suggest that HSC engraftment and chimerism asymptotically approaches a maximum of ∼0.5% in a cell dose–dependent manner. The data show that in contrast to previous speculations, endogenous HSCs cannot be displaced from the niches they occupy by increasing transplanted HSC numbers above a threshold level. In repetitively transplanted mice, however, we have observed small increases in granulocyte chimerism relative to mice that were HSC-transplanted only once with doses above this threshold ( vs. ). This provides evidence that transplantation of an excess of HSCs does not preclude additional niches from being vacated in the future.
To determine if transient CD4 T cell removal would allow access of transplanted HSCs to appropriate niches, we treated Cμ mice with a depleting CD4 antibody that led to ∼95% depletion of peripheral blood CD4 T cells (). CD4-depleted mice were then transplanted with 800 HSCs and analyzed at various time points for donor chimerism. All mice that received anti-CD4 treatment showed donor granulocyte chimerism, whereas none of the untreated mice displayed any detectable donor cells at 8 wk after transplantation (). Significant numbers of donor B cells were observed in the treated mice at 6–8 wk after transplantation, again demonstrating the ability of small numbers of productively engrafted HSCs to restore lymphocyte numbers in immunodeficient mice. Because host CD4 T cells levels were noted to recover subsequent to immunodepletion (not depicted), these experiments demonstrate that transient depletion of these cells before transplantation is sufficient to allow for productive short-term stem cell engraftment without the need for the standard toxic cytoreductive drugs commonly used for B(-) non-SCID patients before bone marrow transplant (). However, by 12 wk after HSC transplantation, donor B cell, T cell, and myeloid chimerism was lost in all recipient mice (not depicted). These data suggest that either the α-CD4–mediated depletion of mature T cells was incomplete, or that sufficient numbers of donor-derived dendritic cells were not generated to mediate lasting tolerance through negative selection in the thymus ().
Aged individuals show marked reductions in thymus size and T cell function (). To determine if the reduced lymphoid function in aged mice would allow acceptance of transplants without conditioning, we repetitively transplanted HSCs from GFP-transgenic mice into old (22 mo) and young (2 mo) recipients. As seen in , short-term low-level myeloid chimerism was observed in all old recipients in contrast to the young recipients that showed no engraftment. However, donor-derived cells declined to undetectable levels in all but one of the old recipients with time, suggesting that rejection of the transplants did occur, but with significantly reduced kinetics relative to the younger animals (not depicted). The level of granulocyte chimerism in this experiment was similar to the low levels seen in transplants of younger, genetically unreactive or immunodeficient mice, suggesting that aged HSCs cannot be displaced from their endogenous niches. This is in contrast to previous studies performed with unfractionated bone marrow transplants () and suggests that reduced immune capacity is responsible for short-term engraftment of donor HSCs.
The remarkable ability of HSCs to sustain multilineage hematopoiesis for the lifetime of an individual constitutes the foundation for their routine use in a range of clinical applications, including the treatment of primary immunodeficiencies (, ), malignancies (–), as conditioners for transplantation tolerance of tissue or organ grafts from the donors (), and as a method to reverse some types of autoimmunity (). The success of such therapies relies on the ability of HSCs to home to unique niches leading to sustained multilineage hematopoiesis. The studies presented here have quantified the number of these HSC niches that are available for engraftment at any given point in unconditioned animals as ∼0.1–1.0% of all HSC niches. Assuming a total adult murine bone marrow cellularity of 5 × 10 cells () and an endogenous HSC frequency of 0.01% (), the number of open HSC niches can be estimated to be 50–500. This is strikingly similar to the number of HSCs estimated to be in circulation at any given point (). The data suggest that HSCs that circulate normally have exited and left vacant their previous HSC niche. Thus, a constant exchange may be occurring between endogenous HSCs under normal circumstances, perhaps to maintain hematopoietic balance between and within each bone marrow compartment. In support of this, we have found little difference in the granulocyte chimerism rates between experiments when a single transplant of HSCs is provided in doses ranging from 800 to 4,000 cells. Although previous reports have suggested that the cell doses of transplanted bone marrow correlate with total chimerism linearly, such data at most show replacement of bone marrow and mature cells in bulk and do not reflect replacement of HSCs, which represent only 0.01% of unfractionated marrow (). When HSCs are repetitively transplanted, however, we have observed increases in granulocyte chimerism ( vs. ). Thus, occupation of available HSC niches after transplantation of an excess of exogenous HSCs, which remain in circulation for ∼1–5 min after transplantation (), does not preclude additional niches from becoming available subsequently. Conceivably, continuous transfusion of low numbers of HSCs would be superior to singly administered boluses, as the rate of niche emptying and filling is high. Because there does not appear to be an obvious increase in granulocyte chimerism with time or cell dose above a threshold level, the data also suggest that transplanted HSCs must find their way rapidly to an appropriate niche and cannot recirculate indefinitely in search of empty niches without the loss of hematopoietic potential.
The ability of transplanted HSCs to self-renew for the lifetime of the organism ensures a constant production of normal lymphoid cells through each developmental stage. In the genetic mutants used in our work, host lymphocyte development is blocked or perturbed at defined developmental stages. At each developmental stage or thereafter, wild-type donor cells have a competitive advantage and can opportunistically expand or accumulate to ultimately give rise to large numbers of normal mature lymphocytes. Several factors have been implicated in the expansion of the early B cell and thymocyte lineages, including IL-7 (, ), stem cell factor (), Flt3 ligand (, ), and recently, various Wnt/Frizzled pathways (). In the case of γc animals, the pro–B-B population appears to have defects in IL-7–dependent expansion, providing a proliferative advantage to wild-type donor cells at these stages (, –). RAG2 mice likely reconstitute more slowly because their lymphocytes can develop normally through the pro–B cell as well as DN3 thymocyte stages and occupy the appropriate stromal microenvironments (). However, because RAG2 lymphocytes cannot advance past these stages (), small numbers of developing donor-derived cells can expand and accumulate without competition at the pre–B as well as DN4 thymocyte cell stages and all subsequent developmental steps. Although it is possible that the donor LT-HSCs will only persist for finite periods of time because of the considerable demand imposed by the ∼250,000-fold expansion to the lymphocyte stage, we have observed no meaningful decline in granulocyte or lymphocyte chimerism at any time point up to 30 wk after transplantation of primary recipients. Nonetheless, because we have observed some declines in HSC potency after secondary transplantation ( vs. ), in clinical settings it would be advisable to keep donor LT-HSCs stored in the event that grafts do not persist indefinitely. Additionally, the low levels of granulocyte chimerism achieved from a single transplant are unlikely to be clinically useful for patients suffering from myeloid deficiencies.
We also show conclusively that stable engraftment within these rare niches by minor histocompatibility–mismatched HSCs is tightly regulated by host CD4 T cells. HSCs from CD45.1 mice cannot productively engraft unirradiated congenic CD45.2 mice, yet they routinely engraft the genetically unreactive F1 strain (CD45.1 × CD45.2). To our knowledge, the only antigenic difference between these strains is the CD45 allele, which is normally considered to be a relatively innocuous congenic marker. Similarly, HSCs isolated from GFP-transgenic mice backcrossed to the C57BL/Ka genetic background cannot productively engraft wild-type C57BL/Ka mice. The only antigenic difference between these strains to our knowledge is the GFP gene product. These experiments prove that very small antigenic differences lead to a complete rejection of donor HSC grafts in the absence of cytoreductive conditioning.
Encouragingly, however, the elimination of CD4 T cell function allows for the functional and sustained engraftment of HSCs with minor histocompatibility mismatches in our system. Xu et al. () have shown that transient antibody-mediated depletion of host αβ T cells in mice enhances engraftment of donor bone marrow with minor histocompatibility mismatches. Spitzer et al. () have shown that conditioning haploidentical patients with a depleting α-CD2 antibody along with low-dose cytoreductive treatments before bone marrow transplantation allows, at the minimum, transient multilineage engraftment. Consistent with this, we demonstrate that transient antibody-mediated CD4 T cell depletion alone is sufficient to allow short-term engraftment of wild-type donor HSCs and restoration of B cells in a mouse model of non-SCID. More complete CD4 T cell depletions and/or better methods to increase donor-derived thymic dendritic cell contribution might allow for lasting donor hematopoiesis.
Even in the absence of inherited genetic mutations, both mice and humans develop diminished immune capacity with age. This progressive loss of immune function has recently been attributed to HSC-intrinsic defects in differentiation to lymphoid-primed progenitors (). Because we have demonstrated that a very small number of properly functioning HSCs can mask the defects in a much larger pool of HSCs, it is tempting to speculate that age-related immune defects don't become readily apparent until nearly all fully “young” HSCs are exhausted. The reintroduction of fully multipotent HSCs, perhaps obtained as an autologous sample earlier in life, might significantly delay age-related immune decline.
Numerous studies have shown how common conditioning treatments used before bone marrow transplantation, such as irradiation, cyclophosphamide, and busulfan, can cause serious side effects, including lowered platelet counts, infertility, and secondary malignancies (). When these cytotoxic therapies are used to treat hematologic malignancies, the side effects must unfortunately be tolerated as a byproduct of necessary chemotherapy. The necessity of such conditioning treatments for hematopoietic deficiencies before HSC transplantation, however, should be reconsidered. Although it is true that available niche space is low under normal conditions, we show that transplantation of modest numbers of highly purified HSCs can engraft the few niches that are available and correct lymphoid deficiencies. Thus, niche space is not an absolute limiting factor to HSC-mediated correction of B, T, or NK cell deficiencies.
Unlike the myeloablative regimens almost always performed on non-SCID immunodeficient patients, SCID patients who receive MHC-matched CD34-enriched or T cell–depleted bone marrow grafts generally do not receive cytoreductive conditioning before transplantation (, ). However, it has been suggested that HSC engraftment does not occur in these patients (). Because many of these patients show poor B cell lymphopoiesis and lose T cell counts with time, it has been proposed that the lymphoid correction occurs as a result of engraftment of short-lived progenitor cells, which along with mature cells constitute the vast majority of transplanted cells, rather than HSCs with full hematopoietic and self-renewal potential (, ). A careful examination of the data, however, shows that ∼0.8% of CD34 cells in an unconditioned SCID patient who received a bone marrow transplant are not of host origin (). Although the authors, understandably, did not consider this level of engraftment meaningful, our results suggest that small numbers of HSCs have engrafted in these patients and that eventual T cell loss may be a reflection of HSC exhaustion rather than an initial failure to engraft. This hypothesis is reinforced by suggestions that the process of physiological HSC circulation seems to be conserved between mice and humans (, , ). Alternatively, the lack of B and T lymphopoiesis has correlated well with graft-versus-host disease (GVHD) in previous studies (, ). In MHC-matched settings, bone marrow grafts are often transplanted without manipulation, whereas in HLA-mismatched settings, T cell depletions or CD34 enrichments of donor marrow can still leave up to 10 T cells/kg (). Our results show that even in MHC-matched congenic mouse model systems, immune responses can recognize and reject very slightly mismatched cells, suggesting that it is very likely that GVH responses occur in all patients that receive any mature T cells as part of their nonautologous graft. Although GVHD may not be classified as clinically significant or obviously symptomatic in all cases, subtle GVH effects on B and T lymphopoiesis might still occur. Thus, the use of purified HSC transplants, which do not cause GVHD (), may potentially avoid poor B lymphopoiesis. In our mouse model system, we have observed sustained B and T lymphopoiesis for the duration of our experiments after purified HSC transplantation.
The mechanism by which transplanted HSCs correct hematopoietic deficiencies in our unconditioned recipients is applicable to the correction of many types of both SCID and non-SCID immunodeficiencies, but these studies at the same time clearly demonstrate that very subtle minor histocompatibility differences can mediate the rejection of HSC grafts when host T lymphocytes are present. Our data suggest that transplantation of purified HSCs, in combination with highly specific lymphoablative treatments when necessary, can correct lymphoid deficiencies in immunodeficient patients without the undesired side effects, such as toxic conditioning and GVHD, often associated with current conditioning and transplantation regimens. Future experiments will determine if the same strategy can be applied to the correction of myeloid deficiencies.
All animal procedures were approved by the International Animal Care and Use Committee. C57BL/Ka-Thy1.1 CD45.2 (HZ) and C57BL/Ka-Thy1.1 CD45.1 (BA) strains were derived and maintained in our laboratory. eGFP transgenic mice used in these studies were backcrossed at least 20 generations to either the BA or HZ strain. C57Bl6/Harland mice used for the aging studies were obtained from the National Institute of Aging. The RAG2, RAG2γc, I-A, and βm mice have been described previously (, , , ) and were bred at least 20 generations onto the C57BL/Ka-Thy1.2 CD45.1, C57BL/Ka-Thy1.2 CD45.2, and C57BL/Ka-Thy1.1 CD45.2 backgrounds. TCRαβ mice were provided by J. Campbell and M. Davis (Stanford University, Stanford, CA). Cμ mice were provided by J. Tung and L. Herzenberg (Stanford University). Peripheral blood was sampled from the tail vein, and all HSC transplants were performed by injection into the retroorbital sinus of isoflurane-anesthetized mice. For repetitive transplants, between 1,750 and 4,000 HSCs were transplanted weekly for 6 wk. Donor mice were 4–6-wk old, and recipient mice ranged from 4–12 wk of age unless otherwise noted.
The following monoclonal antibodies were purified and conjugated using hybridomas maintained in our laboratory: 19XE5 (anti-Thy1.1), 2C11 (anti-CD3), GK1.5 (anti-CD4), 53-7.3 (anti-CD5), 53-6.7 (anti-CD8), 6B2 (anti-B220), 8C5 (anti–Gr-1), M1/70 (anti–Mac-1), TER119 (anti-Ter119), A20.1.7 (anti-CD45.1), AL1-4A2 (anti-CD45.2), 2B8 (anti–c-kit), E13-161-7 (anti–Sca-1), anti-CD16/CD32 (2.4G2), and A7R34 (anti–IL-7 receptor α). Antibodies were conjugated to biotin, PE, allophycocyanin (APC), Alexa 405, Alexa 430, or Alexa 488 (Invitrogen), according to the manufacturer's instructions. The following were purchased from eBiosciences: antibodies against CD3, CD4, CD8, B220, Mac-1, Ter119, and Gr-1 conjugated to PE-Cy5; anti–c-kit and anti–Mac-1 conjugated to PE-Cy7; anti–Sca-1 conjugated to PE-Cy5.5; anti-CD45.1 and anti-CD45.2 conjugated to APC-Cy5.5; anti-B220 conjugated to APC-Cy7; anti-Flk2 (A2F10) conjugated to PE or biotin; and streptavidin conjugated to APC. Anti-CD34 (RAM34) conjugated to FITC or biotin, anti-TCRβ (H57-597) conjugated to APC, anti-CD43 (1B11) conjugated to PE, anti–I-A/I-E (M5.114.15.2) conjugated to PE, and anti-Ly51 (6C3) conjugated to biotin were purchased from BD Biosciences. Streptavidin conjugated to APC-Cy7 was purchased from Caltag.
All cells were sorted on a FACSVantage or a FACS Aria (Becton Dickinson). All peripheral blood analysis was performed on an LSR-Scan (Becton Dickinson). Peripheral blood was obtained from the tail vein, red blood cells were sedimented with 2% dextran, and the remaining red blood cells were lysed with an ammonium chloride solution. The remaining white blood cells were stained with anti-CD45.2–Alexa 488, anti–CD45.1-PE, anti–Ter119-PE-Cy5, anti–Mac-1–PE-Cy7, anti–TCRβ-APC, and anti–B220-APC–Cy7. When peripheral blood containing eGFP cells was analyzed, anti-CD45.1–Alexa-488 was omitted and anti–Gr-1–PE was used instead of anti–CD45.2-PE if warranted by the experiment. For HSC isolation, bone marrow was first enriched using anti–c-kit beads and immunomagnetic selection was performed on an AutoMACS machine (Miltenyi Biotec). Enriched cells were stained with anti–CD34-FITC, anti–Flk2-PE, anti-lineage (CD3, CD4, CD8, B220, Ter119, Mac-1, Gr-1)–PE-Cy5, anti–Sca-1–PE-Cy5.5, and anti–c-kit–PE-Cy7, and cells were double sorted before transplantation. For analysis of HSC chimerism, CD45.1–APC-Cy5.5 was included, and CD34 staining was achieved with anti-CD34 biotin followed by streptavidin APC. For isolation of HSCs from eGFP-transgenic mice, cells were stained with anti-lineage–Cy5-PE, anti–Sca-1–PE-Cy5.5, anti–c-kit–PE-Cy7, anti–Flk2-PE, and anti-CD34 biotin followed by streptavidin APC. For analysis of eGFP HSC chimerism, anti–CD45.1-APC–Cy5.5 was included.
Mice were immunized intraperitoneally with 100 μg NP conjugated to chicken γ globulin (Biosearch Technologies) precipitated in 10% aluminum potassium sulfate (Sigma-Aldrich). NP-specific enzyme-linked immunosorbent assays were performed with serum obtained 1 wk after immunization on high-protein binding 96-well plates coated with 5 μg NP-BSA (Biosearch Technologies). Wells were developed with anti–mouse IgG-horseradish peroxidase (Southern Biotechnology Associates, Inc.) followed by 1 mg/ml ABTS reagent (Sigma-Aldrich), and the reactions were stopped by the addition of 0.1% sodium azide (Sigma-Aldrich). Absorbance was read at a wavelength of 405 nm.
Mice were treated consecutively for 3 d with 500 μg of intravenously injected purified anti-CD4 antibody (GK1.5). Peripheral blood was analyzed for TCRβ and CD8 expression to quantitatively assess CD4 T cell depletion relative to untreated animals. These mice were then transplanted with 800 GFP HSCs 1 d after the third injection. The mice were then given weekly injections of anti-CD4 for the first 3 wk after HSC transplantation and left untreated afterward. |
xref
#text
xref
sup
#text
Heidelberger later took on his first graduate student, Elvin Kabat. Together, they helped settle a long-standing debate concerning whether serum “precipitins” and “agglutinins” (antibodies that agglutinate bacteria) were the same or different. At the time, many people thought that these distinct functional properties of antibacterial antiserum could be ascribed to separate entities. Returning to the pneumococcal bacteria, the duo showed that precipitins and agglutinins were present in identical amounts in antipneumococcal serum. Reduction of one activity—by adsorbing the serum with a bacterial or polysaccharide solution—resulted in an equivalent reduction in the other activity, suggesting that the two functions were properties of the same antibody molecules (). Kabat went on to show that antibodies in serum came in two sizes—large and small (now known as IgM and IgG)—based on their mass and sedimentation rate ().
“Heidelberger's work,” said former colleague Herman Eisner (Massachusetts Institute of Technology) in a 2001 article, “changed the concept of the antibody from an essentially ill-defined set of serum activities to a protein molecule, measurable in conventional chemical units…whose recognition of antigens could be analyzed in molecular terms” (). Heidelberger, who was also a talented musician and linguist, worked in the lab until his death at age 103. At his 100th birthday party, he was reportedly asked how many papers he had published in his lifetime. “Three hundred and four,” he answered, adding slyly, “So far” (). |
The interaction of the TCR and self-peptides presented by MHC class II plays a critical role in development of CD4CD25 T reg cells in the thymus and function in the periphery (, ), suggesting that TCR-mediated signaling might also be important in the development of T reg cells. We asked whether the severe autoimmune phenotype of the LAT mice might be caused by a defect in development or function of T reg cells. We first examined whether CD4CD25 T reg cells were present in LAT mice. Cells from thymuses and spleens of LAT and WT mice were analyzed for expression of CD4, CD8, and CD25 by FACS. As previously reported (, ), LAT mice had a partial block in thymocyte development with accumulation of CD4CD8 cells (). Splenomegaly and enlarged lymph nodes became obvious when they were 4–6 wk old. In the periphery, the CD4 to CD8 ratio was skewed toward CD4 cells. Although discreet populations of CD4CD25 and CD4CD25 SP thymocytes could be observed in WT mice, most of CD4 SP thymocytes in LAT mice expressed low levels of CD25 (). The aberrant expression of CD25 in LAT thymocytes is likely caused by the partial block of thymocyte development in the DN3 (double negative) stage (, ), in which thymocytes are CD25CD44. Interestingly, double positive thymocytes in LAT mice also expressed CD25 (not depicted). Thymocyte development in LAT mice might proceed without down-regulation of CD25 expression.
Because thymocytes from LAT mice expressed CD25, we next examined whether CD4CD25 T cells are present in the periphery. CD4CD25 T cells were clearly missing in the periphery of these mice (), although CD4 T cells from LAT mice expressed high levels of GITR (not depicted). We further determined expression of Foxp3, an important transcription factor that is specifically expressed in CD4CD25 T reg cells, in LAT T cells. CD4 SP thymocytes and CD4 splenocytes cells were sorted from both WT and LAT mice. expression was determined by RT-PCR. The level of was decreased dramatically in CD4 SP LAT thymocytes compared with that in CD4 SP WT thymocytes. Decrease in the level of mRNA was also observed in CD4 T cells from the spleen of LAT mice (not depicted). Further quantitation of mRNA by real-time PCR showed that mRNA level in CD4 SP thymocytes from LAT mice was ∼10-fold less than in WT thymocytes, and RNA level in CD4 splenic T cells from LAT mice was ∼30-fold less (). To exclude the possibility that decreased Foxp3 expression in CD4 T cells from the mutant mice reflected dilution of Foxp3 cells because of the expansion of CD4 T cells, Foxp3 expression in thymocytes and splenocytes from WT and LAT mice was examined by intracellular staining with an anti-Foxp3 antibody followed by flow cytometry. As shown in , only CD4 SP thymocytes and CD4 splenocytes derived from WT, not those from LAT mice, expressed Foxp3. Thus, LAT mice lack CD4CD25 T reg cells.
To exclude the possibility that the severe autoimmune conditions cause disappearance of T reg cells, we also examined development of T reg cells in younger mutant mice in which the autoimmune disease was not severe. As a result of a partial block in thymocyte development, very few CD4 or CD8 T cells were found in the peripheral lymphoid organs of 17-d-old mice (not depicted); however, they appeared in the spleens from 24-d-old mice (). Interestingly, a high percentage of CD4 SP thymocytes expressed CD25 (23.6%). There was also a higher percentage (1.23%; ) of CD4CD25 splenocytes compared with 6-wk-old mice (0.1%; ). However, these CD4 cells from the mutant mice did not express Foxp3 as indicated by intracellular staining (). We also performed a mixed bone marrow chimeras experiment. Lethally irradiated LAT mice were transferred with mixed bone marrow cells from WT Thy1.1 and LAT Thy1.2 mice. 6 wk after transfer, these mice showed no apparent signs of lymphoproliferative disease, whereas LAT mice received bone marrow cells from LAT mice developed the autoimmune disease like LAT mice (not depicted). Because of the partial block in thymocyte development in LAT mice, fewer Thy1.2 T cells than Thy1.1 T cells were detected even though more bone marrow cells from LAT Thy1.2 mice were transferred. As expected, WT Thy1.1 bone marrow cells gave normal population of CD4CD25 T cells. A small population of Thy1.1 T cells expressed Foxp3, and these Foxp3 cells also expressed CD25 (). In contrast, very few Thy1.2 cells in thymuses and spleens, if any, expressed Foxp3. Interestingly, different from the 6-wk-old LAT mice with the autoimmune disease, a large percentage of Thy1.2 CD4 splenocytes expressed CD25. It is possible that CD25 expression is down-regulated with progression of the disease. Because these Thy1.2LAT T cells did not express Foxp3, they likely represented activated T cells. Collectively, these results indicate that LAT mice, which express a LAT mutant that does not bind PLC-γ1, have a defect in Foxp3 expression and development of CD4CD25 T reg cells.
To investigate whether the absence of CD4CD25 T reg cells is indeed one of the underlying mechanisms responsible for the lymphoproliferative syndrome in LAT mice, we purified CD4CD25 or CD4CD25 T cells from congenic Thy1.1 mice by FACS sorting. 2–3 × 10 CD4CD25 or CD4CD25 Thy1.1 T cells were transferred into 3-d-old Thy1.2 LAT neonatal mice by i.p. injection. These mice were analyzed at 7 wk after injection. Untreated LAT mice clearly developed a pathological lymphoproliferative syndrome at 7 wk of age as shown before (, ) (). Compared with untreated LATmice, LAT mice that received Thy1.1CD4CD25 T cells had a normal size spleen and lymph nodes. In contrast, LAT mice injected with CD4CD25 T cells developed a lymphoproliferative syndrome similar to untreated LAT mice ().
We further examined donor cell engraftment and expansion of adoptively transferred cells in LAT recipient mice. In normal mice, CD4CD25 T cells comprise ∼5–10% of the peripheral CD4 T cells or ∼1–2% of total cells in lymph node and spleen. FACS analysis 7–9 wk after adoptive transfer revealed that 1–2% of the total cells in the lymph node and spleen of LAT mice adoptively transferred with CD4CD25 T cells were of donor origin, whereas very few donor cells were detected in the thymus (not depicted). This level of engraftment corresponded to ∼4–9-fold expansion of the initial donor cell inoculum of 2 × 10 cells. A similar degree of donor CD4CD25 T cell expansion was observed after injection of CD4CD25 T cells into neonates (). Likewise, CD4CD25 injected into LAT neonates also underwent a considerable degree of expansion as the number of Thy1.1CD4CD25 T cells increased at least 20-fold (not depicted). In contrast, only very few donor-derived CD4CD25 and CD4CD25 cells were detectable when they were transferred into age-matched WT recipients, which had normal T cell compartment. This observation reconciles with the possibility proposed by Fontenot et al. that donor CD4CD25 T cells can expand to fill the available homeostatic niche despite a skewed T cell compartment in these mice (). In addition, adoptive transfer of WT CD4CD25 and CD4CD25 T cells into 3-wk-old LAT mice that have not fully developed lymphoproliferative disease also resulted in expansion of both populations of donor-derived T cells (not depicted). However, only the transfer of CD4CD25 T cells was able to partially suppress lymphoproliferative disorder in 3-wk-old LAT recipients (not depicted). Neonatal adoptive transfer might provide donor-derived CD4CD25 T cell population with sufficient time to proliferate and suppress LAT T cells to fully suppress lymphoproliferative disorder. FACS analysis showed that in both groups of treated LAT mice >99% of the engrafted donor cells were still CD4 T lymphocytes as expected. At least 75% of the donor CD4 T cells expressed high levels of CD25 in the LAT mice transferred with CD4CD25 T cells, whereas the absolute majority of the donor CD4 T cells were still CD25 in mice that received CD4CD25 T cells. Adoptive transfer of either population of donor T cells had no effect on expression of CD25 on the host T cells ().
To evaluate whether adoptive transfer of normal CD4CD25 T cells could change activated phenotypes or other intrinsic properties in the recipient T cells leading to suppression of the lymphoproliferative disorder, thymocytes and splenocytes from LAT mice that received the adoptive transfer were analyzed and compared with those of untreated WT and LAT mice. Thymocyte development in LAT mice that received CD4CD25 T cells was similar to that in untreated LAT mice (). In fact, no substantial number of donor T cells was detected in the thymus of these mice (not depicted). However, the LAT mice transferred with CD4CD25 T cells had a significantly lower percentage of CD4 T cells in the spleen (∼3%) compared with untreated WT (27%) and LAT (56%) mice. Despite a substantial reduction in the number of CD4 T cells in LAT mice, donor CD4CD25 T cells did not alter the activated phenotypes of LAT T cells as T cells from both treated and untreated LAT mice were identical (CD25TCRβCD62LCD44; ). In addition, T cells from treated LAT mice failed to mobilize Ca-like T cells from untreated LAT mice (not depicted). These results indicate that donor CD4CD25 T cells did not change the activated phenotypes of LAT T cells based on these parameters we tested. Instead, CD4CD25 T reg cells transferred into LAT mice likely suppress expansion of CD4 LAT T cells.
To determine if transfer of CD4CD25 T cells into LAT mice could correct the autoimmune disease, we performed a histological analysis of spleen, liver, and kidney from those mice. Histological sections from untreated LAT mice showed disorganized B and T cell zones in the spleen, lymphocyte infiltration in the majority of portal veins and liver sinusoids, and immune complex deposition in the glomeruli of the kidneys (). In contrast, tissue sections from LAT mice treated with CD4CD25 T cells appeared relatively normal, although many Thy1.2 T cells did appear in the B cell zone of the spleen (). Histological analysis of the spleen, liver, and kidney of LAT mice that received CD4CD25 T cells exhibited lymphoproliferative diseases similar to untreated LAT mice (not depicted).
Examination of serum antinuclear (not depicted) and anti–double stranded DNA antibodies revealed that LAT mice treated with CD4CD25 T reg cells had comparable levels to WT controls (). These data were in agreement with a reduction in the number of hyperactivated B cells (MHC class II or IgMB220) in LAT mice treated with CD4CD25 T cells (). In addition, the concentration of IgG1 () or IgE (not depicted), two antibody isotypes dramatically elevated in LAT mice as a result of increased B cell maturation (), was significantly reduced in mice treated with CD4CD25 T cells. Collectively, our data indicate that adoptive transfer of CD4CD25 T cells into LAT neonates can protect LAT mice from further developing lymphoproliferative syndrome.
CD4CD25 T reg cells are capable of suppressing autoimmune disease; however, how these cells exert their suppressive function is still not clear. Inhibitory cytokines such as IL-10 and TGF-β are considered to be the key molecules involved in T reg cell–mediated immunosuppression (). Recently, it was reported that CD4CD25 T cells up-regulate granzyme B in vitro upon stimulation with anti-CD3 and IL-2 (). Whether this happens in vivo has not been demonstrated. We asked whether CD4CD25 T reg cells up-regulate granzymes (A and B) or inhibitory cytokines when they are transferred into LAT mice. At 7 wk after adoptive transfer of CD4CD25 or CD4CD25 Thy1.1 T cells into LAT mice, donor cells were reisolated from these mice by FACS sorting. Because these Thy1.1CD4CD25 T cells were placed in the autoimmune environment and capable of correcting the disease, they must be activated to exert their suppressive function. They were labeled as “activated” in . We also isolated Thy1.2CD4CD25 and CD4CD25 T cells from WT mice as “resting” T reg cells and Thy1.2CD4CD25 T cells as a negative control. Total RNAs were prepared from these cells and used in RT-PCR.
Donor-derived Thy1.1CD4CD25 T cells expressed a high level of Foxp3 similar to Thy1.2CD4CD25 cells directly isolated from WT mice. Thy1.1 or Thy1.2 CD4CD25 T cells did not have a substantial level of Foxp3 expression (). Interestingly, Thy1.1CD4CD25 T cells from neonatally injected LAT mice expressed high levels of granzyme A, granzyme B, and TGF-β RNAs compared with Thy1.2CD4CD25 T cells (), whereas their IL-10 expression was comparable to that of resting T reg cells (not depicted). This result is consistent with recent findings in which a granzyme B–dependent mechanism was identified as contact-mediated suppression by CD4CD25 T cells in vitro (). Increased TGF-β expression could also function to suppress the expansion of CD4 T cells from LAT mice. It is possible that transferred Thy1.1CD4CD25 T cells use both cytokine-mediated and contact-mediated mechanisms to suppress hyperproliferative host CD4 T cells in vivo. Although we have not demonstrated that increased granzymes or TGF-β indeed mediates suppression in LAT mice, our results show that T reg cells indeed up-regulate these proteins in the autoimmune environment.
Ectopic expression of Foxp3 was previously shown to be sufficient to activate a program of immunosuppression in CD4CD25 T cells (). Because Foxp3 expression was significantly decreased in LAT T cells (), we asked whether reexpression of Foxp3 in LAT T cells could confer the regulatory function that normal CD4CD25 T cells have. Because LAT T cells express low levels of TCRs on their surface, they were difficult to be activated via the TCR. Instead, LAT T cells were stimulated with PMA and ionomycin and cultured in the presence of IL-2 for 48 h before transduction with retroviruses expressing Foxp3 and green fluorescence protein (GFP) (pHSpG-Foxp3) or GFP alone (pHSpG-Empty) (). After culture for an additional 48 h postretroviral transduction, GFPCD4 T cells were FACS sorted, and 2−3 × 10 purified GFPCD4 LAT T cells were i.p. injected into 3-d-old LAT neonates. Expression levels of TCR-β and the GITR on transduced LAT CD4 T cells with or without Foxp3 were similar, whereas those of CD25 were slightly higher on pHSpG-Foxp3–transduced cells (). Expression of high levels of CD25 in these cells is likely a consequence of stimulation by PMA and ionomycin. 6 wk posttransfer, spleens and lymph nodes were harvested and examined. Mice receiving purified LAT T cells expressing only GFP developed a severe lymphoproliferative syndrome like untreated LAT mice (). In contrast, mice that received LAT T cells expressing Foxp3 and GFP exhibited no sign of lymphoproliferative disease as judged by the gross appearance of secondary lymphoid organs (). Analysis of GFP T cells from mice received CD4 LAT T cells expressing Foxp3 and GFP revealed a dramatic decrease in CD4 T cells similar to that observed in LAT mice that received normal CD4CD25 T cells. On the other hand, GFP host T cells from LAT mice treated with CD4 LAT T cells expressing GFP alone were predominantly CD4 similar to those in LAT mice (). These data indicate that reconstitution of Foxp3 expression in LAT T cells is sufficient to induce suppressive function and protect LAT mice from lymphoproliferative disease.
In this study, we demonstrated an essential role of the adaptor protein LAT in Foxp3 expression and CD4CD25 T reg cell development. In LAT mice with a severe lymphoproliferative disease, CD4CD25 cells were nearly absent in peripheral lymphoid organs. Foxp3 expression was also dramatically decreased in the LAT T cells. Interestingly, in young LAT mice CD4CD25 cells could be found; however, they did not express Foxp3. Similar results were also seen in LAT mice that received mixed WT and LAT bone marrow chimeras. These data indicate that the LAT–PLC-γ1 interaction is required for Foxp3 expression and T reg cell development. Severe lymphoproliferative disease in LAT knock-in mice could be prevented by transfer of normal CD4CD25 T reg cells but not CD4CD25 T cells. Our results indicate that the lymphoproliferative disease associated with the LAT mutation is not only caused by abrogation of central tolerance (), but also by a breakdown in peripheral tolerance caused by a severe block of CD4CD25 T reg cell development.
Analysis of donor cell engraftment indicates that CD4CD25 and CD4CD25 T cells are able to proliferate and expand in LAT mice. Two studies using IL-2Rβ– and Foxp3-deficient mice have previously demonstrated a similar expansion after the transfer of CD4CD25 T reg cells (, ). A rich Th2 cytokine environment, a consequence of hyperactivated LAT CD4 T cells, may provide an ideal environment to support the expansion of transferred CD4CD25 and CD4CD25 T cells. In LAT mice, at 2–3 wk after birth both CD4 and CD8 T cells begin to fill in peripheral lymphoid organs as a result of a partial block in thymocyte development. Nonselective expansion of transferred T cells might be caused by the lymphopenic environment in neonatal LAT mice. Adoptive transfer of CD4CD25 and CD4CD25 T cells into 3-wk-old LAT recipient mice also allowed engraftment and expansion of both T cell populations. Thus, it is also possible that this expansion of transferred T reg cells may result from a proliferative response to fill a homeostatic niche for CD4CD25 T cells (, ). Although donor-derived CD4CD25 T cells expanded upon transfer into LAT neonates, only transfer of CD4CD25 T cells into LAT mice rescued the lymphoproliferative disease. Even though CD4CD25 T cells could convert into CD4CD25 T cells upon homeostatic proliferation (), CD4CD25 T cells injected into neonatal or 3-wk-old LAT mice neither converted into a considerable number of CD4CD25 T cells nor rescued the lymphoproliferative disease.
The appearance of CD4CD25 LAT T cells in peripheral lymphoid organs of mixed bone marrow chimeras or young mutant mice was unexpected because CD25 expression was almost absent on CD4 T cells from LAT mice older than 6 wk. However, in mice that received treatment of CD4CD25 at the neonatal stage CD25 expression on LAT CD4 T cells was still missing. Regardless of CD25 expression in LAT T cells from different mice, Foxp3 expression was not detected in these cells, suggesting that these cells are likely activated T cells, not real T reg cells. It is possible that IL-2 produced by WT T cells might induce or maintain a high expression level of CD25 on LAT T cells or the autoimmune environment in LAT mice may cause T cells to gradually lose CD25 expression. Neonatally injected T reg cells, which do not secret IL-2 (, , ), failed to restore CD25 expression on LAT T cells in these mice and provided only protective function against lymphoproliferative disease. Previous studies show that Foxp3 is up-regulated upon activation of CD4CD25 T reg cells, and that ectopic Foxp3 expression confers suppressor function upon peripheral CD4CD25 T cells (). Likewise, ectopic expression of Foxp3 conferred LAT CD4 T cells a regulatory function that prevents autoimmunity in these knock-in mice. Although ectopic expression of Foxp3 had no effect on the levels of both TCR-β and GITR, CD25 was slightly up-regulated on transduced CD4 T cells of LAT mice. It is difficult to conclude the effect of Foxp3 on CD25 in our experiments because we had to activate LAT T cells with PMA and ionomycin, which up-regulate CD25 in vitro, before transducing these cells with retroviruses. Nevertheless, our results suggest that Foxp3-mediated suppression does not require normal function of LAT, perhaps independent of the TCR. Our data strongly support the notion that Foxp3 is a master regulator gene that controls suppressor function in CD4CD25 T cells.
The LAT–PLC-γ1 interaction is important in TCR-mediated Ca flux and MAPK activation (, ). LAT T cells have abrogated Ca flux although TCR-mediated MAPK activation is normal (). As the influx of extracellular Ca after TCR engagement has been implicated in influencing the outcome of both positive and negative selection (–), it is also possible that TCR-mediated Ca mobilization and further NFAT activation might be required for induction of Foxp3 expression. Interestingly, the phenotype of LAT mice resembles that of mice lacking NFATc2 and NFATc3 (), which suggests that the autoimmune disease in these mice may be attributed, at least in part, to a decrease in NFAT activation. However, it has been demonstrated that combined NFATc2/c3 deficiency has no effect on development and function of CD4CD25 T cells but renders CD4CD25 T cells unresponsive to suppression (). Based on these findings, we speculate that the LAT–PLC-γ1 interaction provides signals other than NFAT activation to induce Foxp3 expression. Because LAT CD4 T cells can be suppressed in vivo, NFATc2/c3, which are required for CD4CD25 T cells to be suppressed, might be activated independent of the TCR. Although our results indicated that the LATY136F mutation affected Foxp3 expression and T reg cell development, we cannot rule out the possibility that the LAT T reg cells can develop, but they might not be able to survive in these mice. In addition, because T cell development is partially blocked in the LAT mice, it is possible that the defect in Foxp3 expression and T reg cell development might be indirect consequences of the block in thymocyte development. The signaling pathways that link the LAT–PLC-γ1 association and Foxp3 expression or T reg cell development remain to be explored in the future.
A dramatic decrease in peripheral CD4 T cells in treated LAT mice strongly indicates that these autoreactive CD4 T cells are the targets of CD4CD25 T cell–mediated suppression. Our data show that donor-derived CD4CD25 T cells from treated LAT mice expressed high levels of granzyme A, granzyme B, and TGF-β compared with CD4CD25 T cells isolated directly from WT mice (). In normal mice, maintenance of central tolerance mechanisms by negative selection leaves only a scanty number of these potentially harmful T cells in the periphery. The majority of T reg cells may never encounter these self-reactive T cells and thus remain at the resting status. Once placed or exposed to the autoimmune conditions, CD4CD25 T reg cells may trigger both contact-dependent and cytokine-mediated mechanisms by secretion of granzymes and TGF-β. Whether these proteins indeed function to kill or suppress CD4 LAT T cells in vivo remains to be determined in the future. In conclusion, our study indicates that the proximal signaling pathways downstream of the TCR mediated by the LAT–PLC-γ1 interaction play an important role in CD4CD25 T cell development and opens up the question of what might be the missing link between TCR and Foxp3 induction.
All mice were used in accordance with the National Institutes of Health guidelines. The experiments described in this study were reviewed and approved by the Duke University Institutional Animal Care and Use Committee. Mice were housed in specific pathogen-free conditions at the Duke University Animal Care facility.
Streptavidin-conjugated Texas red and PE-Cy7 and biotinylated FITC, PE, and PE-Cy5, APC-conjugated antibodies to TCR-β, CD4, CD8α, CD25, B220, CD62L, CD44, GITR, Thy1.1, Thy1.2, IgM, IA, and mouse Ig were purchased from BD Biosciences. The anti-Foxp3 antibody was from eBioscience.
Cells were maintained in complete RPMI 1640 medium with 10% FCS. To isolate CD4CD25 and CD4CD25 T cells, splenocytes and lymph node cells were isolated. CD8 T and B cells were depleted by magnetic beads using biotin-conjugated anti-CD8 and anti-B220. The enriched CD4 lymphocytes were stained with FITC–anti-CD4, PE–anti-CD25, and 7AAD. CD4CD25 and CD4CD25 T cells were purified by cell sorting using a FACSVantage SE flow cytometer (BD Biosciences).
2–3 × 10 WT Thy1.1-marked CD4CD25 or CD4CD25 T cells were injected i.p. into 3-d-old (Thy1.2) LAT pups or 3-wk-old LAT mice. These LAT pups were derived from breeding LAT females with LAT males. Injected pups were analyzed at 7−9 wk after the adoptive transfer. Donor cell recovery was calculated based on the total number of lymphocytes multiplied by the percentage of Thy1.1 cells as determined by FACS analysis.
T cell–depleted bone marrow cells from Thy1.2 LAT mice (3.0 × 10 cells) were mixed with Thy1.1 congenic mice (1.5 × 10 cells) and were then injected i.v. into irradiated LAT mice (900 rads). 6 wk after bone marrow reconstitution, thymuses and spleens were harvested and analyzed by FACS.
pHSpG and pHSpG/Foxp3 retroviral vectors were used to transfect the Phoenix-ecotropic virus packaging cell line using the calcium phosphate method to produce recombinant retroviruses. To transduce T cells from LAT mice, splenocytes from LAT mice were first activated using 40 ng ml PMA and 0.5 μg ml ionomycin and recombinant mouse IL-2 (100 ng ml) for 36 h. Activated lymphocytes were then transduced by mixing with the retroviral supernatant in the presence of 8 μg ml polybrene and recombinant mouse IL-2 (100 ng ml). Cells were then centrifuged at 1,300 g for 2 h at 22°C. After culturing those cells at 37°C for 24 h, the transduction procedure was repeated. At 48 h after viral transduction, GFPCD4 T cells were sorted by FACS and 2−3 × 10 GFPCD4 T cells were injected i.p. into LAT neonates. At 6 wk after injection, lymph node and spleen cells were isolated and analyzed.
Total RNAs were extracted with the Trizol reagent (Invitrogen) and reverse transcribed using Superscript II reverse transcriptase (Invitrogen). cDNAs were then used as templates in PCR amplification with Taq polymerase. The mRNA level was quantified using the LightCycler system (Roche). The primer pairs used in real-time PCR were the following: β-actin, 5′-ACTCCTATGTGGGTGACGAG-3′, 5′-CAGGTC-CAGACGCAGGATGGC-3′; Foxp3, 5′-CCCAGGAAAGACAGCAACCTT-3′, 5′-TTCTCA-CAACCAGGCCACTTG-3′. The primer pairs used in RT-PCR were the following: Foxp3, 5′-CAGCTGCCTACAGTGCCCCTAG-3′, 5′-CATTTGCCAGCAGTGGGTAG-3′; granzyme A, 5′-CTCAAGACCGTATATGGCTCT-3′, 5′-CCTGCACAAATCATGTTTAGT-3′; granzyme B, 5′-ACTTTCGATCAAGGATCAGCA-3′, 5′-ACTGTCAGCTCAACCTCTTGT-3′; TGF-β1, 5′-TGCTGCTTTCTCCCTCAACCT-3′, 5′-CACTGCTTCCCGAATGTCTGA-3′.
Whole spleens, livers, and kidneys were embedded in Tissue-Tek (Sankura Torrance) and sliced into 5-μm-thick section. Sections were applied to poly lysine–coated slides and fixed in acetone. Spleen and liver sections were then stained with FITC-conjugated anti-B220 or biotin-conjugated anti-Thy1.2 followed by alkaline phosphatase-conjugated anti-FITC and horseradish peroxidase–conjugated streptavidin (Sigma-Aldrich). Fast Blue BB and 3-aminoethylcarbazole (Sigma-Aldrich) solution were added for color development. Kidney sections were stained with FITC–anti–mouse IgG (BD Biosciences).
Anti–double-stranded DNA antibodies were detected using ELISA. 96-well plates were coated with 2.5 μg ml calf thymus DNA in Reacti-bind DNA coating solution (Pierce Chemical Co.). Anti-nuclear antibodies were detected using slides of Hep-2 cells adhered to slides from Antibodies Inc. |
There are three possible models for the stoichiometry of the murine γδTCR. Two of these () are based on the present models proposed for the stoichiometry of the αβTCR. The configuration in is based on the monovalent αβTCR model (–) and depicts the surface γδTCR complex with one TCRγδ heterodimer, two CD3γ dimers, and one TCRζ homodimer, for a total of eight subunits. The configuration in is based on the alternative bivalent αβTCR model (–) and depicts the surface γδTCR complex with 2 TCRγδ heterodimers, 2 CD3γ dimers, and 1 TCRζ homodimer, for a total of 10 subunits. It is also conceivable that the rules of γδTCR assembly and surface expression differ from those of the αβTCR, such that a γδTCR complex containing only one CD3γ dimer is transported to and stably expressed on the cell surface. This surface complex would contain one TCRγδ heterodimer, one CD3γ dimer, and one TCRζ homodimer, for a total of six subunits (). As the CD3/TCRγδ ratio varies in the configurations shown in , quantifying this ratio is the first step in solving the stoichiometry of the murine γδTCR. To this end, we developed a flow cytometric approach similar to those used by others to quantify the CD3/TCR ratio on primary mouse and human T cells (, ). This method takes advantage of the fact that the mAbs against CD3γ/δ dimers (2C11) and TCRγδ heterodimers (GL3, GL4, UC7-13D5, and UC3-10A6) are all hamster IgG antibodies containing κ light chains. As each primary antibody can be detected with the same anti–hamster Igκ secondary antibody, the relative expression levels of CD3 dimers and TCRγδ heterodimers on the surface of γδ T cells can be measured if saturating amounts of mAb are used. Our approach differs from those of previous studies in that we used a monoclonal anti–hamster antibody instead of polyclonal anti–hamster IgG antibodies, thereby restricting recognition to a single epitope on each primary antibody. A representative staining profile for anti-CD3γ/δ (2C11) and two anti-TCRγδ (GL3 and UC7-13D5) mAbs on gated CD4CD8CD19 LN cells from TCRβ mice is shown in . Note that the relative fluorescence of 2C11 mAb staining is approximately twice that of anti-TCRγδ mAb staining, regardless of which anti-TCRγδ mAb was used (GL3, GL4, UC7-13D5, or UC3-10A6; ). These results indicate that there are two CD3 dimers for every TCRγδ heterodimer on the surface of γδ T cells. Importantly, loss of CD3δ expression does not affect this ratio, because we also observed two CD3 dimers for every TCRγδ heterodimer on the surface of CD4CD8 TCRβCD19 LN cells from CD3δ mice (). This finding is consistent with previous results demonstrating that neither TCRδ nor TCRγ pairs efficiently to a CD3δ dimer (). The observed 2:1 ratio of CD3 dimers to TCRγδ heterodimers favors the monovalent TCR model shown in , in which there is one TCRγδ heterodimer and two CD3 dimers in each γδTCR complex. Thus, our findings indicate that the γδTCR has a signal transducing complex that is similar to that of the αβTCR, in that it contains two CD3 dimers.
Biochemical analysis suggests that a small percentage of surface γδTCR complexes contain CD3δ dimers (, ). The γδTCRs that contain CD3δ dimers could be restricted to a distinct subpopulation of γδ T cells or may represent a minor subset of TCRs on each γδ T cell. To discern between these two possibilities, we developed a second flow cytometric assay that uses an anti-CD3γ mAb (7D6), which has been reported to block the binding of the 2C11 mAb to CD3γ dimers but not to CD3δ dimers (). To confirm the specificity of the 7D6 mAb and its ability to block 2C11 mAb staining of CD3γ dimers, we assayed double-negative (DN) thymocytes from CD3δ, CD3γ, and CD3
mice (). The 7D6 mAb detected the intracellular CD3γ dimers present in CD3δ DN thymocytes but not the intracellular CD3δ dimers present in CD3γ DN thymocytes. In addition, pretreatment with the 7D6 mAb completely blocked 2C11 mAb staining of intracellular CD3γ dimers in CD3δ DN thymocytes but had no effect on 2C11 mAb staining of intracellular CD3δ dimers in CD3γ DN thymocytes. Next, we tested the efficacy of this flow cytometric approach. We first assayed γδ T cells from CD3δ mice, which express only CD3γ dimers on their cell surface (), and found that the 7D6 mAb was indeed able to completely block 2C11 mAb surface staining (). We then assayed CD4 αβ T cells from B6 mice, which express both CD3γ and CD3δ dimers on their cell surface (for review see reference ) and found, as expected, that the 7D6 mAb only partially blocked 2C11 mAb surface staining (). If expression of γδTCRs containing CD3δ dimers were limited to a subpopulation of γδ T cells, then pretreatment with purified 7D6 mAb should partially block 2C11 staining on this CD3δ subset and completely block 2C11 staining on the CD3δ subset. However, if CD3δ containing γδTCRs were a minor subset of TCRs expressed on each γδ T cell, then pretreatment with purified 7D6 mAb should almost completely block 2C11 staining on all γδ T cells. We found that when γδ T cells from TCRβ mice were assayed, the 7D6 mAb almost completely blocked the staining of the 2C11 mAb, indicating that the TCRs containing CD3δ dimers represent a minor subset of TCRs expressed on each γδ T cell (). In fact, the relative percentage of CD3γ dimers was calculated to be 99.2 ± 0.1% of all CD3 dimers.
If two CD3γ dimers are found in each surface γδTCR complex, then the loss of CD3γ should have profound effects on γδTCR assembly and surface expression. Indeed, Haks et al. have reported that γδ T cell development is severely affected in CD3γ mice (). We sought to expand these earlier experiments by performing a more detailed analysis of γδTCR surface expression on thymocytes and splenocytes from CD3γ mice and from CD3γ mice carrying a γδTCR transgene. Virtually no γδTCR cells were detected in the thymus and spleen of CD3γ mice (). Moreover, introduction of a γδTCR transgene into CD3γ mice did not increase the number of γδTCR thymocytes and splenocytes as it does when introduced into CD3γ mice, indicating that the absence of γδTCR cells in CD3γ mice cannot solely be caused by a failure to express productively rearranged TCRγ and -δ genes (). These findings demonstrate that γδTCR assembly and surface expression are absolutely dependent on the presence of CD3γ. Remarkably, unlike the γδTCR, the αβTCR can still be expressed on the surface of CD3γ thymocytes and splenocytes, albeit at reduced levels compared with CD3γ cells () (). Therefore, CD3γ mice reveal a difference in the requirement for CD3γ in αβ- and γδTCR assembly and surface expression. Importantly, this difference is consistent with the supposition that TCRγ and -δ chains each pair with a CD3γ dimer but not a CD3δ dimer.
Using quantitative immunofluorescence techniques, we have addressed the issue of murine γδTCR stoichiometry. We observed a 2:1 ratio of CD3 dimers to TCRγδ heterodimers on the surface of peripheral γδ T cells, a ratio that supports the monovalent TCR model (). We also present new evidence, in accordance with previously reported biochemical data (, ), demonstrating that the two CD3 dimers contained within the γδTCR are almost exclusively CD3γ dimers. Lastly, an analysis of γδTCR surface expression on CD3γ thymocytes and splenocytes revealed an absolute requirement for CD3γ dimers in γδTCR assembly. Together, these data strongly support the idea that, during γδTCR assembly, both TCRγ and TCRδ pair with a CD3γ dimer. In this study, the ratio of TCRζ homodimers to CD3 dimers or TCRγδ heterodimers was not measured and, therefore, the number of TCRζ homodimers contained within a surface γδTCR complex cannot be determined. However, based on the conservation of positively charged residues in the transmembrane regions of all four TCR chains that are required for association with the invariant TCR chains (for review see reference ), we propose that, like the αβTCR, the γδTCR contains one TCRζ homodimer.
The vast majority of murine γδTCRs, whether expressed on naive or activated γδ T cells, contain only CD3γ dimers ( and not depicted) (, ). It is not clear, however, whether human γδTCRs share the same bias for CD3γ dimers. Biochemical analysis of surface γδTCR complexes on primary human γδ T cells detected some CD3δ dimers but considerably less than the amount observed in surface αβTCR complexes on primary human αβ T cells (). Interestingly, biochemical analysis of surface γδTCR complexes on activated and expanded human γδ T cell clones detected CD3δ dimers in amounts comparable to those seen in surface αβTCRs (unpublished data) (). Unfortunately, CD3δ deficiency in humans does not resolve the issue of whether CD3δ is required for human γδTCR surface expression, because it is not known whether the absence of peripheral γδ T cells (, ) is caused by the loss of CD3δ or by the markedly reduced levels of the other invariant subunits that accompany CD3δ deficiency in the patients analyzed (). Nevertheless, these findings suggest that there may be important differences in the subunit requirements for murine and human γδTCR assembly. It is believed, based on sequence homology, that TCRδ is the counterpart to TCRα and, consequently, that TCRδ should pair preferentially with CD3δ dimers (for review see reference ). Accordingly, the inconsistency in murine and human γδTCR assembly can be explained by a difference in either the binding affinities of the respective TCRδ chains for CD3δ dimers or the binding affinities of the respective CD3δ dimers for TCRδ chains. Murine TCRδ pairs to a CD3γ dimer but not to a CD3δ dimer ( and ) (, ). However, this is not the case for human TCRδ, as a metabolic labeling study using TCRαβ Jurkat cells transfected with a human TCRδ gene shows that human TCRδ can associate with either human CD3γ or CD3δ (). Remarkably, in the same study, when a murine TCRδ gene was transfected into the TCRαβ Jurkat variant, the murine TCRδ was also shown to pair with either human CD3γ or CD3δ. Collectively, these data indicate that murine and human CD3δ dimers differ in their ability to bind to TCRδ chains.
Of the current models of TCR stoichiometry, the observed 2:1 ratio of CD3 dimers to TCRγδ heterodimers favors the monovalent TCR model for γδTCR stoichiometry (). However, we cannot rule out the possibility that monovalent γδTCR complexes cluster or aggregate on the cell surface to form higher order complexes. If these higher order complexes exist, they may provide an explanation for how signal transduction by the γδTCR is superior to that of the αβTCR in the absence of coreceptor involvement. The difference in the subunit composition of the αβ- and γδTCR signal transducing complexes may also explain the increased signaling proficiency of the γδTCR. As the amino acid sequence of the immunoreceptor tyrosine-based activation motif in each CD3 chain is unique (for review see reference ), it is conceivable that αβ- and γδTCR complexes recruit distinct signaling molecules. In addition, or alternatively, intrinsic differences in the signaling pathways coupled to αβ- and γδTCRs may provide a mechanism by which the γδTCR is capable of signaling better than the αβTCR.
B6.129P2-TCRβ (TCRβ) mice () were purchased from the Jackson Laboratory. C57BL/6-CD3δ (CD3δ) mice () were provided by D. Kappes (Fox Chase Cancer Center, Philadelphia, PA), and 129-CD3γ (CD3γ) mice () were provided by D. Wiest (Fox Chase Cancer Center). C57BL/6-Vγ6/Vδ1 γδTCR transgenic (Tg) mice (line 134) () were provided by B.J. Fowlkes (National Institutes of Health [NIH], Bethesda, MD). B6.129-CD3
(CD3
) () and C57BL/6 (B6) mice were generated in our colony. Mice were bred and maintained in an NIH Research Animal Facility in accordance with the specifications of the Association for Assessment and Accreditation of Laboratory Animal Care. Mouse protocols were approved by the NIH Animal Care and Use Committee. All mice were killed at 8–12 wk of age.
mAbs used for flow cytometric analysis included anti-CD4 (RM4-5), anti-CD8α (53-6.7), anti-TCRγδ (GL3, GL4, and UC7-13D5), anti-Vγ4 (UC3-10A6), anti-TCRβ (H57-597), anti-CD3 (145-2C11), anti-CD19 (1D3), and a hamster IgG isotype control, all of which were purchased from BD Biosciences. The secondary reagent, biotin-conjugated anti–hamster Igκ (HIG-29), was also purchased from BD Biosciences. The anti-CD3γ (7D6) hybridoma () was obtained from A. Singer (NIH, Bethesda, MD) and D. Wiest and was used to produce ascites. Protein A/G–purified 7D6 mAb was conjugated to AlexaFluor 488 according to the manufacturer's instructions (Invitrogen). AlexaFluor 488 conjugated to streptavidin was also purchased from Invitrogen.
Flow cytometric analysis for surface antigens was performed as previously described (). Intracellular staining for CD3 dimers was performed (Cytofix/Cytoperm kit; BD Biosciences) according to the manufacturer's instructions. The ratio of CD3 dimers to TCRγδ heterodimers was determined using an assay previously described for determining the ratio of CD3 dimers to TCRαβ heterodimers (, ). In brief, 1.5 × 10 lymph node cells were incubated with saturating amounts of purified anti-TCRγδ (GL3, GL4, UC7-13D5, or UC3-10A6) and anti-CD3 (145-2C11) mAbs for 30 min on ice. Saturating amounts of antibody are defined as the concentration of purified antibody required to completely block the binding of the same antibody conjugated to a fluorochrome. All five mAbs are hamster IgG that use the Igκ light chain. Accordingly, their relative binding intensities can be assayed using saturating amounts of the same anti–hamster secondary mAb, biotin-conjugated anti–hamster Igκ (HIG-29). The CD3/TCRγδ ratio was calculated using the following equation, where MFI stands for mean fluorescence intensity:
The relative percentage of CD3γ dimers in CD3 dimers on the surface of γδ T cells was determined using saturating amounts of anti-CD3γ (7D6; 300 μg/ml for intracellular staining and 200 μg/ml for surface staining) mAb to block the binding of CD3γ dimers by anti-CD3 (145-2C11) mAb (). The percentage of CD3γ dimers was calculated using the following equation:
For all experiments, 2–4 × 10 cells were collected (FACSCalibur; Becton Dickinson) using CellQuest software or LSR II using FACSDiva software (BD Immunocytometry Systems) and analyzed using FlowJo software (Tree Star, Inc.). Dead cells were excluded from analysis based on forward and side scatter profiles. |
Both mRNA and protein for C5aR were measured in cardiomyocytes from sham rats and CLP rats as a function of time after surgery. As shown in , mRNA for C5aR in extracts of cardiomyocytes from CLP rats showed progressive increases 12, 24, and 48 h after CLP, whereas any changes in C5aR mRNA in sham cardiomyocytes were much less evident. When homogenates from cardiomyocytes were evaluated by Western blot analysis, there were progressive increases 6–48 h after CLP (). Cardiomyocytes from sham surgery rats showed no increases in C5aR expression (unpublished data).
The studies were extended by the rise of immunostaining of cardiomyocytes for C5aR protein (). Confocal immunostaining revealed intense cortical staining in cardiomyocytes 12 h after CLP (), whereas cells obtained from sham animals show evidence of constitutive expression () that was much less intense. Surface immunostaining of individual cardiomyocytes for C5aR proteins revealed intense staining of cardiomyocytes obtained 12 h after CLP () and much less staining of sham cardiomyocytes (), consistent with the confocal staining data. When irrelevant rabbit IgG was used, there was very little staining of either CLP or sham () cardiomyocytes, respectively.
LVP was recorded in CLP and sham-operated rats treated with either anti-C5a or an equivalent amount of nonspecific IgG. The results are shown in . Rats subjected to CLP developed significantly lower mean LVPs than their sham-treated counterparts (P < 0.001, analysis of variance [ANOVA]) (). Antibody-induced in vivo blockade of C5a, which was given intravenously immediately after CLP, resulted in greatly improved mean, maximum, and minimum LVPs (LVP, LVP, LVP) when compared with septic rats treated with nonspecific IgG (P < 0.001, ANOVA). Injection of either anti-C5a or preimmune IgG into sham rats had no effect on LVP values. No differences were found in measured heart rates between the various groups (unpublished data). These data indicate that in vivo myocardial dysfunction is abolished by blockade of C5a in septic animals when compared with sham animals. Whether the mean LVP, the maximum LVP, or the minimum LVP values were used, the results were the same in all cases.
We next investigated whether polymicrobial sepsis (CLP) influences cardiomyocyte sarcomere contractility. Baseline sarcomere length values did not differ significantly among all cardiomyocytes analyzed. Cardiomyocytes from CLP-injured animals demonstrated an ∼50% decrease in relative peak sarcomere shortening compared with sham animals (peak height CLP + nonspecific IgG 0.099 ± 0.005 μm vs. sham 0.201 ± 0.008 μm, P < 0.001, ANOVA, ).
Analysis of single cell cardiomyocyte sarcomere shortening revealed a substantial improvement in peak shortening of cells isolated from CLP animals treated with anti-C5a compared with CLP treated with nonspecific IgG (peak height CLP + anti-C5a IgG, 0.168 ± 0.007 μm vs. CLP + nonspecific IgG, 0.086 ± 0.005 μm, P < 0.001, ANOVA, ). To assess whether the effects of anti-C5a were injury dependent, we also included a group of rats that underwent sham operation and received the blocking antibody intravenously. Relative peak sarcomere shortening values were not different after administration of IgG or anti-C5a in sham animals (). These results demonstrate that the presence of C5a, which is generated during sepsis, is linked to cardiomyocyte dysfunction of CLP.
In addition to relative peak sarcomere shortening, normalized maximum contraction and relaxation velocities were determined. Interestingly, infusion of anti-C5a greatly improved the maximum velocities of contraction (−dL/dt) and relaxation (+dL/dt) comparable to those measured in sham animals (). Cardiomyocytes from CLP + anti-C5a animals contracted significantly faster than animals treated with nonspecific IgG (CLP + anti-C5a 27.98 ± 0.92 s vs. CLP + nonspecific IgG 22.68 ± 0.41 s, P < 0.001, ANOVA) (). Similar results were obtained for values of relaxation velocities +dL/dt (). Cardiomyocytes derived from CLP + anti-C5a animals exhibited significantly faster relaxation velocity than cells isolated from CLP + IgG animals (CLP + anti-C5a 24.35 ± 0.91 s vs. CLP + nonspecific IgG 18.58 ± 0.42 s, P < 0.001, ANOVA). Furthermore, maximum contraction and relaxation velocities occurred significantly earlier in CLP animals treated with anti-C5a than in rats infused with nonspecific IgG (). These data indicate that CLP induces C5a-dependent dysfunction in cardiomyocytes and that blockade of C5a reverses these changes.
Cardiomyocytes were obtained as a function of time after sham surgery or after CLP, as described in . As shown in , contractility values in the sham cardiomyocytes not otherwise treated were essentially stable as a function of time (0–48 h after sham surgery). In striking contrast, as shown in , there was a progressive decrease in sarcomere shortening 6, 12, and 24 h after CLP, with a slight improvement at 48 h. When sham cardiomyocytes were exposed to either 5.0 or 10.0 nM recombinant rat (rr)C5a, there was a progressive lost of contractility, with an ∼30% reduction in contractility after incubation with 5.0 nM C5a and a 53% reduction after exposure to 10.0 nM C5a. In the case of CLP cardiomyocytes, as shown in , there was a very substantial loss of contractility which was progressive with time up to 24 h after CLP. The addition of 5.0 nM of C5a caused further reductions and greater reductions occurred with exposure to 10.0 nM C5a. Thus, it appears that both sham cardiomyocytes as well as CLP cardiomyocytes show adverse effects after exposure to C5a, but with greater decrements in function in CLP cardiomyocytes.
For these studies, cardiomyocytes were isolated from sham rats as well as CLP rats 24 h after sham surgery or CLP. Thereafter, they were exposed in vitro to 10.0 nM C5a for 0–60 min and the changes on contractility were measured immediately after the indicated exposure time. As shown in , with no exposure to C5a, the expected significant differences in contractility between sham and CLP cardiomyocytes were verified. In general, contractility measurements diminished as a function of time of exposure to C5a, especially up to 30 min, seeming to approach a plateau thereafter. Thus, the effects of C5a on induction of contractility defects in cardiomyocytes seem to require no more than an exposure time of 30 min.
In companion studies, sham or CLP cardiomyocytes were exposed to C5a for either 15 min or 3 h, followed by washing, and contractility measurements were performed. Similar to the data in , there were decrements in cardiomyocyte contractility after exposure to C5a. There was no difference in the reductions in contractility in cardiomyocytes exposed for only 15 min versus 3 h (unpublished data). These data suggest that the interaction of C5a with cardiomyocytes occurs rapidly and that removal of the C5a soon thereafter will not prevent the progressive loss in contractility.
During sepsis and septic shock, cardiac function is often impaired, placing the patient at risk for multi-organ dysfunction and eventual death. The complement system is activated during sepsis, resulting in generation of potent proinflammatory factors, which may contribute to impaired function of the immune system and vital organs. To what extent complement activation products can be linked to cardiac dysfunction during sepsis is not known. In an endotoxin shock model, C3 deficiency was associated with a decreased left ventricular ejection fraction (). The present study investigated possible protective effects of blocking the potent complement activation product, C5a, on cardiac performance during experimental sepsis. Our results demonstrate for the first time that the C5aR is expressed on the surface of single cardiomyocytes obtained from septic and sham animals. C5aR content was higher on CLP cardiomyocytes. Furthermore, we provide evidence that in vivo interception of C5a (with anti-C5a) given at the time of onset of CLP significantly improved cardiac performance both in vivo (), as reflected by restored levels of LVP (LVP, LVP, LVP), and in vitro as reflected by significantly restored peak sarcomere shortening levels and improved contractile velocities (±dL/dt, ).
The current literature provides evidence for complement activation in humans and animals with septic disorders based on elevated plasma levels of C3a, C4a, and C5a (–). Excessive generation of complement anaphylatoxin, C5a, and its interaction with C5aR induces a plethora of cellular stress response mechanisms such as the modulation of cytokine expression, apoptosis, and activation of the coagulation pathway (, –). Previously, our laboratory showed that C5a generation during sepsis plays a key role in the regulation of various pathophysiologic changes that can be reversed by blockade of C5a (, , ). Anti-C5a treatment and complement depletion with cobra venom factor led to an increased oxygen extraction ratio and oxygen consumption in a porcine model of sepsis (). Septic cardiomyopathy is a key pathophysiologic event during the course of sepsis and it frequently determines whether the patient survives or dies. Given the immense socioeconomic impact of sepsis in terms of morbidity, mortality, and cost to the health system (estimated $16.7 billion per year), the development of novel therapeutic strategies for septic cardiomyopathy is clearly warranted ().
Western blotting (for C5aR protein) and real-time PCR (C5aR mRNA content) of cardiomyocyte lysates revealed characteristic bands indicating expression of C5aR on rat cardiomyocytes. Constitutive expression of C5aR on cardiomyocytes was found in both CLP and sham animals, but higher levels were seen in cardiomyocytes obtained from CLP rats. This is in contrast with our findings in neutrophils where surface C5aR expression on PMNs was down-regulated in septic animals compared with shams (). These contrasting findings underline the polyfunctional properties of the C5a–C5aR system. The differences in the C5aR status in blood neutrophils and cardiomyocytes after CLP may simply reflect a higher concentration of C5a in blood than in the extravascular compartment. The central role of the neutrophil for innate immune function necessitates the presence and activation of neutrophils in the face of invading microorganisms. Interference in this system because of excessive C5a production has been shown to compromise host defenses (). In contrast, constitutive expression of C5aR on the cardiomyocyte surface ensures the rapid initiation of cellular stress response mechanisms (alteration of intracellular calcium, etc.). When excessive C5a generation occurs as in sepsis, these potentially protective mechanisms may turn into detrimental derangements both at the functional and cellular level. Additional support for this hypothesis was obtained by in vitro incubation of cardiomyocytes with C5a. The dose of 10.0 nM dramatically impaired contractility parameters in cardiomyocytes after sham surgery (). These data correlate with the serum levels of C5a found in septic humans, which have been shown to range between 1.0 and 15.0 nM (, ). Similarly, 10.0 nM C5a abolished the already impaired contraction of cardiomyocytes from septic animals (unpublished data).
CLP reproduces many of the characteristics of polymicrobial sepsis found in humans (). We investigated alterations in cardiac function during the hypodynamic phase of sepsis, which usually occurs 20–24 h after CLP (), when hemodynamic parameters are decreased (). In animal models, sepsis-induced deficits in cardiac contractility have been controversial. Decreased parameters of contractility independent of preload and changes in the myocardial structure have been reported in ex vivo studies on Langendorff-perfused hearts (). In contrast, no changes of cardiac contractility parameters or myocardial ultrastructure were reported during the hypodynamic phase of sepsis (). Using a rat CLP model, a reduction in cardiac contractility parameters such as LVP and ±dP/dt has been described previously (). Our results demonstrate significantly lower LVP values in CLP animals compared with shams, suggesting severely impaired cardiac contractility in sepsis. We provide evidence that infusion of anti-C5a prevented impairment of LVP in vivo in CLP animals. Our cellular studies show that the interaction of C5a–C5aR on cardiomyocytes is a major contributor to the reduced levels of LVP observed in vivo during sepsis. C5a and C3a have previously been shown to exert profound vasodilatative effects on the systemic and coronary vasculature, which might exacerbate the myocardial contractile deficits in sepsis and partially explain the low systemic blood pressure values seen in septic humans and animal models (, ).
A major finding in the present study is that cardiomyocytes isolated from CLP animals exhibited significantly decreased sarcomere contractility parameters (peak sarcomere shortening, ±dL/dt) compared with sham animals. At the sarcomere level, our study showed myocellular contractile deficits during CLP-induced sepsis. In our analysis, we used a standardized area that included at least 15–20 sarcomeres and acquired real-time sarcomere-shortening values. This technique is sarcomere specific and independent of cell size compared with the cellular edge detection shortening measurement used in a prior study ().
We sought to determine the role of C5a toward inhibition of sarcomere shortening in sepsis. Confirming our in vivo data, anti-C5a treatment of septic rats completely reversed the sepsis-induced reduction in sarcomere contraction and resulted in cardiomyocyte function comparable to that found in sham animals. Coupled with our finding of constitutive expression of C5aR on cardiomyocytes and the detrimental effect of C5a incubation in vitro on contractility parameters, these data strongly suggest that specific C5a–C5aR interaction occurs in septic rat cardiomyocytes, leading to impaired cardiac performance. In agreement with our already demonstrated beneficial effects of anti-C5a on survival after CLP (), improved cardiomyocyte contractility resulting in improved end-organ perfusion and oxygen supply may be the mechanistic cornerstone leading to improved survival rates.
Further studies are needed to evaluate intracellular signaling pathways involved after C5a–C5aR interaction in impaired myocardial function. Various mediators such as reactive oxygen intermediates and proinflammatory cytokines (TNF-α, IL-1β, IL-6) have been proposed to be myocardial depressant substances underlining that upstream modulation, such as interception of the C5a–C5aR interaction, might be useful to improve cardiac dysfunction during sepsis (, , , ). Because intracellular calcium is one of the most important factors for coordinated and efficient contractility, various studies have focused on alterations in calcium fluxes in cardiomyocytes. It has been suggested that septic cardiomyocyte dysfunction may be linked to altered calcium transient properties (). Recently, an abnormal Ca leakage from the sarcoplasmic reticulum has been found and may contribute significantly to the depressed cardiomyocyte shortening in sepsis (). The interaction between phospholamban and sarcoplasmic reticulum Ca-ATPase2a (SERCA2a), two major modulators of calcium-dependent contractility, has been shown to play a central role in the pathogenesis of congestive heart failure and septic myocardial dysfunction (, ). Therefore, it is intriguing to hypothesize that C5a–C5aR interaction may affect intracellular calcium handling, possibly via modulation of SERCA2a activity leading to functional deficits. Our in vitro results support this theory. The maximum contraction and relaxation velocities are significantly higher in cardiomyocytes from CLP rats treated with anti-C5a when compared with infusion of nonspecific IgG. The improved contraction and relaxation velocities and peak shortening levels may indicate improved intracellular calcium handling.
There are suggestions that TNFα may contribute to cardiac dysfunction after sepsis or endotoxemic shock (). After injection of LPS, circulating levels of TNFα reached 10 ng/ml, resulting in evidence of cardiac dysfunction (). Our own studies of CLP in rats suggest extremely low levels of TNFα in the serum (<5 pg/ml) (). Accordingly, on the basis of the CLP model used in the current studies it seems unlikely that TNFα is responsible for the cardiomyocyte defects.
Unless otherwise indicated, all reagents were purchased from Sigma Aldrich.
Adult male Sprague-Dawley rats (Harlan Inc.) weighing 300–350 g were used in all experiments. Animals were housed in a specific pathogen-free environment and allowed to acclimate to their surroundings for 1 wk. Standard rat chow and water were available to the animals during the course of the experiment ad libitum. All experiments were performed in accordance with the guidelines set forth by the National Institutes of Health for Care and Use of Animals. Approval for the experimental protocol was obtained from the University Committee on Use and Care of Animals at the University of Michigan.
Sepsis was induced by the CLP procedure as described previously (). In brief, rats were anesthetized with isoflurane (2–3%, oxygen flow 3L O/min). After a midline incision, the cecum was exposed and ligated ∼2/3 of the distance from the distal pole. The ligated cecum was punctured through and through with an 18-gauge needle and a small portion of feces wad expressed. The abdomen was closed in layers using 4–0 sutures (Ethicon Inc.) and metallic clips. Sham animals underwent the same procedure except for ligation and puncture of the cecum. Where indicated, animals received 500 μg anti-C5a antibody in 500 μl sterile DPBS by intravenous injection immediately after CLP. Control animals received a similar dose of nonspecific IgG antibody. Rats were killed at 6, 12, 24, or 48 h after CLP for in vivo and in vitro experiments.
The COOH-terminal end (amino residues 58–77) of the rat C5a molecule was coupled to keyhole limpet hemocyanin and was used to immunize goats for the production of anti-sera (). The polyclonal anti-C5a specific antibody was affinity purified, and cross-reactivity with rrC5a was confirmed in Western blots.
Under isoflurane anesthesia, the left carotid artery was exposed, the left vagus nerve was dissected and freed from the carotid artery, and microclips were positioned to gain proximal and distal vascular control. A 2.5 French microtip catheter (Millar Instruments) was inserted into the left carotid artery and advanced into the left ventricle. Correct positioning in the left ventricle was verified by fluoroscopy and the appearance of the characteristic pressure curve. Mean (LVP), maximum (LVP), and minimum (LVP) LVP as well as heart rate (beats per minute) were recorded for 5 min using a signal transduction and amplification system connected to a standard Microsoft Windows operating system PC equipped with the appropriate recording and analysis software (PowerLab 8SP Base, Bridge Amp, Chart 5 Software, ADInstruments).
CLP or sham-operated rats were anticoagulated using heparin sodium (1,000 U i.p.) (Abbott Laboratories) and deeply anesthetized using isoflurane (2–3%) at 12 or 24 h after CLP. After measurement of LVP, the heart was rapidly excised and rinsed in ice-cold, sterile Krebs-Henseleit buffer (containing [in mM] 118 NaCl, 4.7 KCl, 21 NaHCO, 1.8 CaCl, 1.2 MgSO, 1.2 KHPO, and 11 glucose, pH 7.40) supplemented with 5 mM 2-aminoethanesulfonic acid (taurine). The aortic stump was cannulated and the heart was mounted on a Langendorff perfusion system (ADInstruments Inc.). Cannulation and initiation of heart perfusion took place within 60 s of removal of the organ from the animal. Retrograde perfusion was initiated with calcium-containing (1.2 mM) Krebs-Henseleit buffer for 5 min to stabilize the heart on pump. Flow rate was set to 7.5 ml/min, temperature was kept at 38°C (±0.5°C), and perfusion pressure was continuously monitored. Perfusion with calcium-free Krebs-Henseleit buffer was performed for 3 min, and the flow rate increased to 9.0 ml/min. Collagenase type II (Worthington Biochemical Corp.) and hyaluronidase type II were added and the hearts were perfused as described previously (). Cardiomyocytes were counted using a hemocytometer, cell viability was assessed by trypan blue dye exclusion, and cell morphology. Myocytes with a rod-like shape, clearly defined edges and sharp striations were counted as viable cells, whereas cells with membrane blebbing, loss of striation pattern and rounded cells were classified as nonviable. Cell suspensions with a viability of >75% were used for all subsequent experiments.
Cells were plated onto laminin-coated (Invitrogen), sterile 22 × 22 mm glass coverslips at a density of 5 × 10 cells/coverslip/well. Subsequently, coverslips were cultured in six-well tissue culture plates (37°C, 5% CO) in DMEM containing 50 U/ml penicillin, 50 μg/ml streptomycin (pen/strep), and 5% fetal calf serum to allow for cell attachment and adhesion to the laminin matrix. Media was then replaced with serum-free M199 media supplemented with 10 mM glutathione, 0.2 mg/ml BSA (GIBCO BRL), 15 mM Hepes (Sigma-Aldrich), and 26 mM NaHCO and placed in an incubator (37°C, 5% CO) for 3 h. All media and other reagents used for the cardiomyocyte isolation were certified endotoxin-free by the manufacturers.
Plated cardiomyocytes that had been incubated for 3 h underwent single cell sarcomere contraction and relaxation analysis using a variable rate CCD video camera system (MyoCam, IonOptix Corp.) equipped with sarcomere length detection software (IonWizard, IonOptix Corp.). A coverslip with the plated cardiomyocytes was placed in a prefabricated chamber, which was filled with warm (37°C) M199 and mounted on the microscope system (Eclipse; Nikon Corp.) connected to the CCD video camera system. The chamber system was connected to a stimulator system (Grass Inc.) and electrical pacing stimulation was initiated (100 mV, 4 ms, 1 Hz). A total number of 1,247 cardiomyocytes were analyzed. Measurement of cardiomyocyte function, by groups, was performed in a randomized fashion if different experimental groups were present. A rectangle-shaped region of interest within each randomly chosen cardiomyocyte was then defined and sarcomeres within the focused region were selected for analysis. Typically, this region included ∼15–20 sarcomeres. Cardiomyocyte contractile parameters (relative peak sarcomere shortening, normalized maximum contraction velocity −dL/dt, normalized maximum relaxation velocity +dL/dt, time of −dL/dt and +dL/dt) were recorded for 75 s.
Total protein from cardiomyocyte lysates (50 μg, harvested 0, 6, 12, 24, or 48 h after CLP or sham operation) was separated by electrophoresis in a denaturing 10% polyacrylamide gel and then transferred to a polyvinylidene fluoride membrane. Equal loading was facilitated by protein estimation (BCA Protein Assay; Pierce Chemical Co.) and confirmed by detection of β-actin as housekeeping protein. Non-specific binding sites were blocked with TBST (40 mM Tris, pH 7.6; 300 mM NaCl, 0.1% Tween 20) containing 5% nonfat dry milk for 1 h at 4°C. The membrane was incubated with rabbit anti–rat C5aR antibody at a 1:1,000 dilution overnight. After five washes in TBST, the membrane was incubated in a 1:5,000 dilution of horseradish peroxidase–conjugated donkey anti–rabbit IgG as the secondary antibody (GE Healthcare). The membrane was developed by enhanced chemiluminescence technique according to the manufacturer's protocol (). The results were quantified using digital pixel density and image analysis software Kodak ID (Scientific Imaging Systems). The results were normalized to β-actin and expressed as a ratio of C5aR/β-actin pixel densities ().
Rat hearts were obtained 0, 6, 12, 24, or 48 h after the CLP or sham procedure and cardiomyocytes were isolated as described in previous paragraphs. Total RNA was isolated using the TRIzol method (Life Technologies Inc.) according to the manufacturer's instructions. Digestion of any contaminating DNA was achieved by treatment of samples with RNase-Free DNase (Promega Corp.). Reverse transcription was performed with 5 μg RNA using the Superscript II RNase H Reverse Transcriptase (GIBCO BRL; Life Technologies Inc.) according to the manufacturer's protocol. Real-time PCR was then performed with primers for C5aR: 5′ primer, 5′-TATAGTCCTGCCCTCGCTCAT-3′; and 3′ primer, 5′-TCACCACTTTGAGCGTCTTGG-3′. The primers were designed for 409-bp cDNA amplification in the middle region of the rat C5aR cDNA (positions 373–781). The primers for the “housekeeping” gene GAPDH were: 5′ primer, 5′-GCCTCGTCTCATAGACAAGATG-3′; and 3′ primer, 5′-CAGTAGACTCCACGACATAC-3′. Reactions were prepared in duplicates as 50 μl reactions using the iQ SYBR green Supermix reagent (Bio Rad Laboratories). After a “hot-start” for 3 min at 94°C, 45 cycles were used for amplification with a melting temperature of 94°C (15 s), an annealing temperature of 60°C (15 s), and an extending temperature of 72°C (30 s), followed by a melting curve generation starting at 60°C with gradual increase in the temperature up to 95°C in 0.2°C steps. An amplification plot was generated using twofold dilutions of the cDNA generated from a known amount (1 μg) of mRNA. The cycle threshold (C) was set above the baseline fluorescence. Plotting the log of the dilutions versus the C values then generated a standard curve. Quantitation of C5aR and GAPDH in the samples was determined using the standard curves. Purity of the products was assessed by generating melting curves. Furthermore, the PCR products were run out on a 1.2% gel to confirm that the amplicons generated were single bands at the expected size (unpublished data). The C5aR to GAPDH ratios were then plotted for the various time points in CLP and sham rats (). Real-time PCR was performed using a Smart cycler (Cepheid). C values, standard curves, and melting curves were generated using the software provided by the manufacturer (Cepheid).
Cardiomyocyte suspensions (5 × 10) isolated 0, 6, 12, 24, or 48 h after CLP or sham procedure were incubated with recombinant, endotoxin-free (<5 pg/ml) rat C5a at various doses for various times. Dose-dependent effects of C5a on cardiomyocyte contractile parameters were then recorded measured and analyzed using the MyoCam system.
Cardiomyocytes isolated 12 h after CLP or sham injury were plated on coverslips and cells were fixed with 4% paraformaldehyde. For confocal microscopy, nonspecific binding sites were blocked with 0.1% gelatin for 20 min and with 4% normal goat serum for 30 min. Cells were incubated with either rabbit anti-rat C5aR antibody (1:50 dilution) or preimmune serum (1:50) for 2 h, washed, and incubated with goat anti–rabbit Alexa-568 Fab (1:200) (Invitrogen). After adding SlowFade solution (Invitrogen), cells were visualized using a confocal fluorescence microscope. Both projection view and optical sections were developed electronically and processed digitally. Optical scanning and digital processing of the images were performed to determine the topographic distribution of the Alexa Fluor–conjugated IgG on the surface of cardiomyocytes.
A chromogenic Limulus amebocyte assay (QCL-1000, Cambrex Corp.) was performed to confirm endotoxin-free cell culture conditions. In brief, samples of the cell culture supernatants were mixed with the Limulus polyphemus reagent and chromogenic substrate reagent over a short incubation period (16 min) and read on a spectrophotometer at a wavelength of 405 nm. The assay has a sensitivity range of 0.1–1.0 EU/ml.
All statistical analysis was performed using STATA Statistics/Data Analysis 8.0 software (Stata Corporation). Results are expressed as the mean value ± SEM unless otherwise noted. ANOVA followed by Tukey's post-hoc tests was used to test for differences among the experimental groups for each of the grouping variables. Statistical significance was defined as P ≤(0.05. |
We used a bicistronic retroviral transduction system to transduce human into progenitor cells. The constructs carrying either or hu are shown in Fig. S1 A, available at . LinIL-7RαThy1.1c-Kit bone marrow cells that contain a heterogeneous fraction of progenitors, including multipotent progenitors and myeloid-committed cells, and are devoid of Thy1.1 HSCs, IL-7Rα lymphoid–committed cells, and mature lineage cells, were sorted into Flt3 (Flt3 progenitors also containing Flt3 MEPs) and Flt3 (Flt3 progenitors containing multipotent progenitors and Flt3 CMPs) cell fractions (; references , –) and consecutively retrovirally transfected as described in Materials and methods. Fig. S1 B shows typical 18-h coculture transduction efficiencies (18–26%) in Flt3 and Flt3 progenitors as determined by GFP expression.
To study the effects of enforced hu expression on IPC and DC development, Flt3 and Flt3 progenitors were transduced with control- or hu and cultured in human Flt3L-Ig fusion protein (huFlt3L-Ig)– and stem cell factor (SCF)-supplemented media. Cultures were analyzed for cell numbers and the presence of IPCs and DCs at days 4, 8, and 12. Freshly isolated Flt3 progenitors did not express CD11c or MHC class II (not depicted). As expected, unmanipulated Flt3 progenitors (not depicted) as well as -transduced Flt3 progenitors gave rise to no or very few CD11c MHC class II (up to 1.1%) and CD11cB220 (up to 0.5%) cells (, top). In contrast, hu–transduced Flt3 progenitors differentiated into CD11c MHC class II and CD11cB220 cells that increased in relative numbers from day 4 to 8 of culture (, bottom). Similarly, -transduced as well as hu-transduced Flt3 progenitor cells gave rise to CD11c MHC class II and CD11cB220 cells, with hu-transdced Flt3 progenitor cells producing slightly higher relative numbers of both cell types (). To quantify absolute CD11c cell production, cell numbers were determined at days 4, 8, and 12 of culture. Numbers peaked at day 8 of culture, with hu-transduced Flt3 progenitors producing the highest cell numbers (∼14-fold expansion of input cells), followed by intermediate expansion (6–9-fold) of both -transduced Flt3 and hu-transduced Flt3 progenitors, and low expansion (3–4-fold) of -transduced Flt3 progenitors (). The total cellular expansion was paralleled by a peak expansion of CD11c MHC class II and CD11cB220 cells at day 8 of culture (). Interestingly, hu-transduced Flt3 progenitors produced significantly higher total numbers of both CD11c MHC class II and CD11cB220 cells compared with -transduced Flt3 progenitors (), with somewhat higher relative CD11cB220 cell numbers ().
Next, we evaluated IPC- and DC-associated surface antigen expression and function of CD11cB220 and CD11cB220 cells derived from different progenitor cell populations at day 8 of culture. Consistent with typical mouse IPC and DC phenotypes, CD11cB220 cells expressed Gr-1, Ly6C, and CD45RA, whereas CD11cB220 cells expressed CD11b and intermediate levels of CD80 and CD86, respectively (). Furthermore, CD11cB220 cells produced substantial amounts of IFN-α upon stimulation with either influenza virus or CpG, whereas CD11cB220 cells did not (). CD11cB220 cells, but not CD11cB220 cells, displayed typical DC morphology () and were efficient stimulators in allogeneic MLR cultures, as evaluated by thymidine incorporation (). Therefore, CD11cB220 cells phenotypically and functionally were typical IPCs, whereas CD11cB220 cells were typical conventional DCs. Collectively, these results indicate that huFlt3 signaling rescues and enhances the development of functional IPCs and DCs from Flt3 and Flt3 hematopoietic progenitor cell populations, respectively. In addition, it suggests that strong Flt3 signaling slightly skews development toward an IPC phenotype.
In normal mouse and human hematopoiesis, IPC and DC developmental potentials are maintained from Flt3 CMPs to downstream Flt3 GMPs, but are lost in Flt3 MEPs (–, , ). Thus, we tested whether enforced hu expression in MEPs would be sufficient to rescue IPC and DC development. As comparator cell population, we used hu-transduced GMPs and -transduced CMPs (
-CMPs). As reported previously for unmanipulated MEPs, -transduced MEPs (
-MEPs) gave rise to no or very few CD11c cells (, top).
-MEPs gave rise to CD11cB220 cells and CD11cB220 cells at day 8 in huFlt3L-Ig– and SCF-supplemented cultures, at least as efficient as
-GMPs (). Similarly as from hu-transduced Flt3 progenitor cells, CD11cB220 and CD11cB220 cell development was significantly enhanced from hu-transduced GMPs ().
-MEP– and hu
-GMP–derived CD11cB220 cells produced IFN-α (), suggesting that these cells are functional.
As reported previously for untransduced CMPs (–, ),
-CMPs generated both IPCs and DCs.
-CMPs to produce these offspring cells was about threefold higher than that observed from hu
-GMPs ().
-GMPs. Furthermore, expression enhances IPC and DC development from GMPs, but not to levels observed in the upstream CMP population.
Because huFlt3 signaling in MEPs activated IPC and DC development, a differentiation option normally confined to Flt3 progenitors as CMPs and GMPs, we were interested to test whether huFlt3 signaling in MEPs would also reestablish myeloid CFU activity.
-transduced CMPs, GMPs, and MEPs gave rise to their respective colony types, but with somewhat lower plating efficacy as compared with freshly isolated CMPs, GMPs, and MEPs (; reference ). hu
-MEPs gave rise to not only erythroid-affiliated colonies but also granulocyte/macrophage (GM)-affiliated colonies, including CFU-G, CFU-M, and CFU-GM ().
-MEPs was lower; however, the diversity of colony formation resembled that of CMPs with the exception that no CFU-Mix colonies developed.
-GMPs compared with
-GMPs (). Thus, huFlt3 signaling in MEPs reestablishes myelomonocytic CFU activity, whereas huFlt3 signaling in GMPs does not affect their overall CFU activity and particularly does not reestablish megakaryocyte/erythrocyte read out.
-MEPs, hu
-MEPs, and
-CMPs. 2 × 10 cells of each progenitor population combined with 2 × 10 cells of host bone marrow cells were transplanted into lethally irradiated mice, and spleen progeny cells were analyzed on day 7.
-MEPs produced ∼0.7% of nucleated GFP spleen cells that consisted mostly of Ter119 erythroid cells, but no DCs, IPCs, or Gr-1 myeloid cells (, top; references , , and ).
-MEPs gave rise to ∼2.7% of nucleated GFP spleen cells containing CD11c MHC class II conventional DCs, low numbers of CD11cB220 IPCs, as well as Ter119 erythroid and Gr-1 myeloid cells (, middle).
-CMPs gave rise to ∼6.0% of nucleated GFP spleen cells that contained DCs, IPCs, as well as erythroid and myeloid cells (, bottom; references , , and ). This formally demonstrates that enforced expression of hu is sufficient to rescue in vivo IPC, DC, and myelomonocytic cell development from MEPs.
-MEPs.
-MEPs but was clearly up-regulated in hu
-MEPs.
-MEPs but not
-MEPs expressed myelomonocytic development–associated genes, such as the cytokine receptors for , , and , as well as the transcription factors and , similar to that found in
-GMPs, hu
-GMPs, and normal CMPs (; references and ). However, transcription factors , , and could not be detected in any of the retrovirus-transduced MEPs and GMPs, whereas they were detectable in unmanipulated CMPs ().
-MEPs and similar high levels of expression in hu
-MEPs,
-GMPs, hu
-GMPs, and CMPs ().
-MEPs increased to levels found in CMPs, whereas expression levels of this gene were somewhat higher in GM-committed
-GMPs and hu
-GMPs ().
-MEPs, although the expression of decreased ().
-GMP showed some transcription of Meg/E development–associated genes as β and .
-GMPs were not able to give rise to Meg/E lineage colonies ( and A). Collectively, these results demonstrate on a molecular level that MEPs have a latent IPC, DC, and myelomonocytic lineage potential that is inducible by enforced Flt3 signaling.
-MEPs, we performed intracellular phospho-STAT3 staining in retrovirus-transduced MEPs that were Flt3L deprived and then stimulated with huFlt3L-Ig.
-MEPs but not in
-MEPs, indicating that as expected () STAT3 is a downstream activated transcription factor of enforced huFlt3 signaling ().
-MEPs, we tested whether STAT3 and PU.1 could directly activate IPC, DC, and myelomonocytic development from MEPs. Mouse and were transduced into MEPs using retrovirus expression vectors, respectively (Fig. S2, available at ). However, survival of cells was low when cultured in SCF alone or SCF and huFlt3L-Ig (not depicted). To possibly support the survival of MEPs, we first added thrombopoietin (TPO), followed by TPO and huFlt3L-Ig to SCF in consecutive cultures.
-MEPs and
-MEPs differentiated into CD11cB220 and CD11cB220 cells at day 8 in both SCF as well as TPO (not depicted) and, with even higher efficacy in SCF, TPO, and huFlt3L-Ig, supplemented cultures (). Interestingly, enforced expression of or in MEPs in turn led to the up-regulation of mouse mRNA levels (Fig. S3, available at ).
-MEPs and
-MEPs were plated in methylcellulose assays.
-MEPs and
-MEPs gave rise to CFU-G, CFU-M, and CFU-GM colonies, but not to Meg/E-affiliated colonies ().
Finally, we evaluated the expression of , a nonredundant transcription factor, for megakaryocyte/erythrocyte development by real-time RT-PCR. expression was down-regulated in
-MEPs and
-MEPs compared with that in
-MEPs,
-MEPs, and CMPs (). Thus, enforced expression of and in MEPs reprogrammed them to differentiate into IPCs, DCs, and myelomonocytic cell lineages and inhibited Meg/E lineage potentials, indicating that strong Flt3 downstream signals were capable of inducing complete lineage conversion.
A standing question in early hematopoiesis is whether cytokine signaling is sufficient to induce cell fate decisions. Here, we showed that enforced expression of hu in Flt3 progenitors rescued their potential to differentiate into functional IPCs and DCs with comparable in vitro differentiation efficiency as Flt3 progenitors ( and ).
-GMPs ().
-MEPs differentiated into IPCs and DCs upon in vivo transfer, the most informative assay available to prove the robustness of in vitro observations (). Thus, these data demonstrate that enforced expression and signaling of hu in Flt3 progenitors deliver an instructive signal to activate latent IPC and DC differentiation programs.
Overexpression of hu in total Flt3 progenitors, in Flt3-expressing GMPs, and in CLPs (of which ∼70% are Flt3) led to a gain of higher relative and absolute (two- to threefold) numbers of IPCs and DCs in vitro (, and , and not depicted). Thus, beyond activation, increased Flt3 signaling also enhanced IPC and DC development. The gain in offspring cells was consistently higher for IPCs than for DCs ( and ), in line with our previous findings that after 10 d of in vivo Flt3L injection, spleen IPCs and DCs were expanded on average 28- and 21-fold, respectively (). This suggests that a continuous strong Flt3 signal might induce a shift toward relatively higher IPC levels.
We previously found that Flt3 is expressed in lymphoid- and myeloid-committed progenitor cells, and in vivo Flt3L application mediates the expansion of both cell types without changing their biology (). The enforced expression of hu in MEPs not only led to a gain of IPC and DC developmental capacity, but, with the exception of mixed colony formation, also to a gain of CFU activity of upstream myeloid progenitors as well as to differentiation of erythroid and myelomonocytic cells in vivo (). In contrast, huFlt3 signaling in GMPs did not activate megakaryocyte/erythrocyte potential (). This implies that beyond activation and enhancement of IPC and DC development, Flt3 signaling is not immediately deterministic but primarily opens access to an IPC, DC, and myelomonocytic differentiation program. Thus, we propose that final IPC and DC lineage outcome might be a gradual process, depending on continuous strong Flt3 signaling.
What are the Flt3 signaling–initiated downstream molecular events? It was shown that hematopoietic system–confined deletion of transcription factor leads to the inhibition of Flt3-driven IPC and DC development (). Furthermore, human transfection and stimulation with Flt3L in mouse myeloid 32Dcl3 cells leads to the induction of PU.1 and C/EBPα expression (). These transcription factors are indispensable for granulocyte and monocyte development (), and it was shown that PU.1 cooperatively with C/EBPα activates myeloid development–associated cytokine receptor genes, including , , and (). Interestingly, -deficient mice, in addition to other hematopoietic defects, lack either CD8α or both CD8α and CD8α DCs, depending on the type of PU.1 deletion (, ). Here, we showed that enforced huFlt3 signaling in MEPs results in enhanced expression of IPC, DC, and GM lineage development related transcription factors , , and , as well as expression of , , and ().
-MEPs but not
-MEPs resembled the gene expression profiles of CMPs (, ).
Importantly, enforced expression of or in Flt3 MEPs was again sufficient to permit the development of both IPCs and DCs (). This, however, was only possible once TPO was added to SCF or SCF and Flt3L in cultures. Thus, TPO possibly substitutes for a survival signal otherwise delivered by Flt3. In addition, or alternatively, TPO might be involved in the phosphorylation of overexpressed (). Interestingly, enforced expression of or in MEPs led to the up-regulation of mouse mRNA levels (Fig. S3). This in turn likely allowed culture-supplemented human Flt3L to cross-reactively stimulate - or transduced cells via mouse Flt3. These results suggest a self-sustaining effect of Flt3 signaling–induced transcription via downstream STAT3 and PU.1.
As enforced expression of hu in MEPs did not terminate megakaryocyte/erythrocyte differentiation potential, whereas hu expression in GMPs did not lead to a gain of these differentiation potentials (), how can Flt3 signaling be integrated in megakaryocyte/erythrocyte versus IPC, DC, and GM lineage commitment? By using reporter mice, PU.1 expression was recently mapped in early hematopoietic progenitor cells (). It was shown that PU.1Flt3 CMPs contain high myelomonocytic developmental potential, whereas PU.1Flt3 CMPs and PU.1 MEPs have high megakaryocyte/erythrocyte potential (). The data presented here suggests that Flt3 might be critical in PU.1 regulation, although this likely will not be an exclusive event. GATA-1 is a nonredundant transcription factor for megakaryocyte and erythrocyte development (). DNA binding activity of GATA-1 can be suppressed by enforced expression, resulting in a differentiation block and apoptotic cell death of an erythroid cell line (). Conversely, GATA-1 inhibits the binding of PU.1 to c-Jun, a critical coactivator of myeloid gene transactivation by PU.1 (). Furthermore, GATA-1 interferes with DNA binding activity of STAT3 and inhibits TPO-dependent growth of the Ba/F3 cell line (). Thus, as suggested previously for PU.1 and GATA-1 (, ), relative dosage of gene transcription and protein levels will likely determine lineage outcomes.
-MEPs were increased to levels of normal CMPs and were somewhat lower than those observed in
-GMPs or hu
-GMPs (). Thus, it is possible that MEPs with relatively lower hu and consecutive STAT3 and PU.1 expression do not fully inhibit GATA-1, whereas high Flt3 expressing and signaling cells develop to IPC, DC, or GM lineages. Of note, enforced expression of and in MEPs suppressed expression and inhibited megakaryocyte/erythrocyte development (). Hu overexpression in GMPs in turn induced some , β, and mRNA expression; however, this was not sufficient to reactivate megakaryocyte/erythrocyte development as demonstrated for enforced high-level expression in GMPs ( and ; reference ).
Because overexpression of hu in Flt3 progenitors does not occur under physiologic conditions, what do these findings imply for normal hematopoiesis? Flt3 is expressed on short-term HSCs, multipotent progenitors, CLPs, CMPs, and GMPs, and in vivo injection of Flt3L resulted in increased numbers of these cells as well as IPCs and DCs, whereas MEPs and their progeny remained unchanged (, ). The data presented here demonstrate that enforced Flt3 cytokine receptor signaling is sufficient to activate as well as enhance IPC and DC differentiation programs, suggesting that instructive cytokine signaling might indeed occur in hematopoiesis. Thus, we speculate that once Flt3 short-term HSCs and their offspring Flt3 cells are exposed to Flt3L-rich environments, these cells will be instructed to differentiate into IPCs and DCs. This might be enhanced by a self-sustaining process in which Flt3 downstream transcription factors STAT3 and PU.1 in turn maintain receptor expression. However, Flt3 signaling does not immediately silence other developmental options. It is likely that most Flt3-expressing progenitors will not continuously be stimulated via Flt3L but will receive and activate alternative signals, and thus consecutively acquire myeloid or lymphoid, but not IPC or DC, cell fates. Beyond previous studies, our data further emphasize that IPC and DC development does not fit into a deterministic “lymphoid” nor “myeloid” lineage, but rather a “Flt3-permissive” developmental model, where Flt3-expressing progenitors maintain IPC and DC differentiation options in response to Flt3L as long as no competing signal shuts these down. It will be of interest to test whether downstream dividing Flt3 common IPC and DC progenitors with silenced alternative developmental programs exist and which critical factors are involved in final IPC or DC lineage termination.
C57BL/6 (CD45.2), C57BL/Ka-Thy1.1 (CD45.1), and BALB/c mice (Charles River Laboratories) were maintained at the Institute for Research in Biomedicine animal facility in accordance with the Swiss Federal Veterinary Office guidelines.
Hematopoietic progenitors were isolated as described previously with minor modifications (, ). Bone marrow cells were immunomagnetically preenriched for c-Kit cells using APC-conjugated c-Kit antibodies (2B8; eBioscience) and APC microbeads (Miltenyi Biotec). Cells were then stained with monoclonal antibodies as indicated below. Flt3 and Flt3 hematopoietic progenitors were sorted as lineage (CD3ε, 145-2C11; CD4, GK1.5; CD8, 53-6.7; B220, RA3-6B2; CD19, MB19-1; CD11b, M1/70; Gr-1, RB6-8C5; and TER119, TER119), IL-7Rα (A7R34), Thy1.1 (19XE5), c-Kit, and Flt3 (A2F10.1) cells. Thus, Flt3 and Flt3 hematopoietic progenitors did not contain Thy1.1 HSCs or IL-7Rα lymphoid progenitors. Myeloid progenitors were sorted as LinSca-1 (E13-161-7) c-KitCD34 (RAM34) FcγR (2.5G2; CMPs), LinSca-1c-KitCD34FcγR (GMPs), and LinSca-1c-KitCD34FcγR (MEPs) cells. For IPC and DC analysis and sorting, additional monoclonal antibodies against the following antigens were used: CD11c (N418), MHC class II (I-A/I-E; M15/114.15.2), Ly6C (AL-21), CD45RA (A20.1.7), CD80 (16-10A1), and CD86 (GL-1). Cells were sorted and analyzed using a FACSCalibur and FACSAria (Becton Dickinson).
The full length of human , mouse , and cDNA was inserted into a retroviral expression vector, pMYs-IRES-, respectively (). These constructs were transiently transfected into Phoenix-Ampho cells by LipofectAMINE (Invitrogen). The amphotropic retrovirus supernatants were used to transduce GP+E-86 cells. After 2 d, the brightest GFP-expressing GP+E-86 cells were FACS sorted and expanded. For transduction of hematopoietic progenitor cells, GP+E-86 cells were 20-Gy irradiated and plated in 24-well plates at 1.5 × 10 cells per well for 24 h. Progenitor cells were transduced by coculture with GP+E-86 for 18 h in IMDM (Invitrogen) containing 2% FCS, 4 μg/ml polybrene (Sigma-Aldrich), 100 ng/ml human Flt3L-Ig fusion protein (huFlt3L-Ig), 10 ng/ml mSCF (R&D Systems), and 10 ng/ml mIL-11 (R&D Systems). Transduced cells were removed by gentle pipetting and then subjected to further assays.
Retrovirus-transduced Flt3 and Flt3 progenitors as well as CMPs, GMPs, and MEPs were cultured in IMDM, supplemented with 10% FCS, 10 M 2-ME, sodium pyruvate, and antibiotics, 100 ng/ml huFlt3L-Ig, and 10 ng/ml mSCF as indicated. Half of the media was replaced every 3 d and new cytokines were added. Human Flt3L-Ig fusion protein was produced in cells as described previously ().
-CMPs,
-GMPs, and
-MEPs were sorted after viral transduction and were cultured in IMDM-based methylcellulose media (Methocult H4100; StemCell Technologies Inc.), containing 30% FCS, 1% bovine serum albumin, 2 mM -glutamine, and 50 μM 2-ME, 10 ng/ml mSCF, 10 ng/ml mIL-3 (R&D Systems), 10 ng/ml mIL-11, 10 ng/ml mGM-CSF (R&D Systems), 10 ng/ml mTpo (R&D Systems), 1 U/ml hEpo (Roche), and 100 ng/ml huFlt3L-Ig. Colonies were determined and enumerated under an inverted microscope consecutively from day 3 to 8. In some cases, to confirm colony types, colonies were picked using fine-drawn pasteur pipettes, spun on slides, Giemsa stained, and evaluated by light microscopy.
2 × 10 CD45.2
-MEPs,
-MEPs, or
-CMPs each were injected intravenously into lethally irradiated (2 × 6 Gy in a 4-h interval from a Cesium 137 source; Biobeam 8000; STS GmbH) congenic mice (CD45.1) with 2 × 10 recipient-type CD45.1 whole bone marrow cells. Mice were killed on day 7. The progeny of donor-derived cells were isolated as described previously () and evaluated by FACS analysis.
Graded numbers of sorted, irradiated (25 Gy) IPCs or DCs were plated in U-bottom 96-well plates with 2 × 10 immunomagnetically selected (CD4 microbeads; Miltenyi Biotec) BALB/c spleen CD4 T cells in a final volume of 200 μl RPMI 1640 supplemented with 10% FCS. Cells were cultured for 5 d and pulsed with 1 μCi [H]thymidine (Amersham Biosciences) per well during the last 16 h of culture. [H]thymidine incorporation was measured on a β-plate counter (MicroBeta TriLux; EG&G WALLAC).
To evaluate IFN-α production, sorted CD11cB220 IPCs or conventional CD11cB220 DCs derived from retrovirus-transduced progenitors were cultured for 24 h at 10 cells/200 μl in U-bottom 96-well plates in RPMI 1640 supplemented with 10% FCS, 2-ME, penicillin G, and streptomycin. Either 40 HAU/ml influenza virus (strain A/Beijing/353/89; provided by I. Julkunen, National Public Health Institute, Helsinki, Finland) or 1 μM CpG-A-ODN (ggTGCATCGATGCAgggggG; lowercase letters indicate base with phosphorothioate-modified backbones) was added at start of culture and again at 12 h. Culture supernatants were assayed using an IFN-α ELISA kit (Performance Biomedical Laboratories).
Total RNA was extracted from sorted progenitors as indicated using TRIzol reagent (Invitrogen) followed by DNase I (Invitrogen) treatment. The cDNA was synthesized using random hexamers as well as SuperScript II reverse transcriptase (Invitrogen) and amplified using specific primers as described previously (). For real-time PCR, cDNA products equivalent to RNAs from 200 progenitors were amplified using an Applied Biosystems 7900HT Fast Real-Time PCR System. The data were normalized by the level of expression in each sample. Taqman probes for mouse , , , , and were purchased from Applied Biosystems.
Retroviral-transduced MEPs were cytokine starved for 24 h in 1% FCS-IMDM. Cells were then incubated with or without 100 ng/ml huFlt3L-Ig and analyzed at indicated times for phosho-STAT3 by FACS according to the manufacturer's instructions (Cell Signaling).
Results of experiments are reported as mean ± SD. Differences were analyzed using Student's test.
Fig. S1 shows the diagrams of pMY-IRES- and pMY-hu-IRES-GFP bicistronic retroviral expression vector constructs and virus transduction efficacy in progenitor cells. Fig. S2 shows the diagrams of pMY-m-IRES-GFP and pMY-m-IRES-GFP bicistronic retroviral expression vector constructs. Fig. S3 shows the analysis of mouse mRNA expression in -, hu-, -, and -transduced MEPs, as well as CMPs. Figs. S1–S3 are available at . |
Stimulation of host cells by iE-DAP alone induces very low levels of IL-6 and TNFα secretion (). Furthermore, high doses of iE-DAP, compared with TLR ligands, are required to induce chemokines in intestinal epithelial SW620 and oral epithelial HSC-2 cells (, ). The high dose of Nod1 ligand required for Nod1 stimulation has hampered the analysis of Nod1-mediated immune responses in vitro and in vivo. To circumvent this problem, we sought to develop synthetic compounds that posses an enhanced ability to stimulate Nod1. Previous reports suggest that the peripheral structures of iE-DAP–containing molecules () affect their ability to stimulate Nod1 (, ). Therefore, we initially characterized the structural requirement for Nod1 stimulatory activity using a published bioassay with human embryonic kidney (HEK) 293T cells transiently expressing Nod1 (). The monomer disaccharide tetrapeptide -acetyl-glucosamyl (GlcNAc)--acetyl-muramyl (MurNAc)--Ala-γ--Glu-DAP--Ala was Nod1 stimulatory, whereas the dimeric disaccharide tetrapeptide resulting from one linkage between the ɛ-amino residue in DAP and the α-carboxyl residue in -Ala was not (). We also found that the stimulatory activity of the tripeptide -Ala-γ--glutaminyl--DAP (iQ-DAP) was less than the dipeptide iQ-DAP (). These results indicate that NH-terminal peptide expansion of the core dipeptide recognized by Nod1 does not result in higher stimulatory ability.
Based on the results given in , we used the dipeptide iE-DAP as a seed structure to develop compounds with an enhanced ability to stimulate Nod1. Consistent with cytosolic localization of Nod1 (), microinjection or calcium phosphate–mediated incorporation of Nod1 ligands into cells enhances the ability of the ligands to stimulate Nod1 (, , ). Furthermore, is reported to inject Nod1 ligand through a type IV secretion system into host cells (). These observations suggest that hydrophobic acylation of Nod1 ligands may improve their membrane permeability and ability to stimulate Nod1. To test this hypothesis, we developed synthetic compounds containing various acyl residues at the NH terminus of iE-DAP (X in ). -myristoyl (C-14) iE-DAP, designated here as KF1B, as well as other acyl iE-DAP compounds (not depicted) were found to specifically stimulate Nod1 (), but not Nod2 or TLR4/MD-2, signaling using the HEK293T bioassay (). These results indicate that acylation of the iE-DAP dipeptide does not affect ligand recognition or receptor specificity. Further analysis showed that KF1B exhibited a several hundredfold higher ability to induce Nod1-dependent NF-κB activation than the original dipeptide iE-DAP, as determined by the amount of compound required for achieving 50% of maximum activity (). Testing of additional compounds revealed that pentadecanoyl (referred as KFC15; C-15) and palmitoyl (referred as KFC16; C-16) iE-DAP also possessed a similar ability to stimulate Nod1 as KF1B (unpublished data). Although efficient stimulation of Nod1 by natural ligands requires the coexistence of calcium phosphate particles under our culture conditions (, ), we found that KF1B no longer requires calcium phosphate particles to stimulate Nod1 signaling, which was consistent with our hypothesis that the enhanced ability of KF1B to stimulate Nod1 is caused by increased accessibility to the cytosol ().
To determine if KF1B enhances LPS-induced cytokine production through endogenous Nod1, human monocytic MonoMac6 cells that express Nod1 were stimulated with the iE-DAP–derived compounds and controls. Stimulation of MonoMac6 cells with KF1B alone at concentrations as high as 2,500 ng/ml induced neither the secretion of IL-6 nor IL-1β (). However, KF1B possessed a higher ability to enhance LPS-induced production of these cytokines than the original iE-DAP (). KF1B was effective in stimulating LPS-induced responses at concentrations as low as 25 ng/ml and was comparable to MurNAc--Ala-γ--Glu--Lys(stearoyl)-OH (AcMTP), a modified ligand of Nod2 (–). As observed with MonoMac6 cells, stimulation of BM-derived mouse macrophages and DCs with KF1B alone did not induce the secretion of CCL2/MCP-1 (), IL-1β, and IL-12 (not depicted). However, KF1B enhanced LPS-induced CCL2 secretion from DCs () and IL-6 secretion from macrophages (). Notably, the response of macrophages with KF1B was abolished in Nod1-deficient macrophages (), demonstrating that KF1B requires Nod1 for enhancement of CCL2 secretion.
Given that Nod1 is expressed in the intestinal epithelium, we tested whether KF1B alone can induce cellular responses in epithelial cells. We found that KF1B alone, but not DAP, Induced production of IL-8 in two human intestinal epithelial cell lines, LoVo and SW620 (). In contrast, LoVo cells did not respond after stimulation with LPS, CpG, and polyIC (), which was consistent with the lack of TLR signaling in most intestinal epithelial cells (). In addition, KF1B induced NF-κB in LoVo cells as determined with an NF-κB–dependent reporter assay (). ELISA, real-time PCR, and immunoblotting analyses showed that KF1B also induced CXCL1/Groα and CD83, two molecules that are important for the recruitment and interaction of immune cells ().
To further characterize events induced by Nod1 stimulation in LoVo intestinal cells, we determined global gene expression changes using a cDNA microarray containing ∼20,000 genes (). At 60 min after stimulation, KF1B induced 11 genes at >2-fold and 41 genes at >1.6-fold as compared with unstimulated cells (). Further analysis at 3 and 24 h after stimulation revealed induction of 6 and 17 genes at >1.6-fold levels, respectively. The intestinal epithelium is the first line of defense against invasive bacteria and mediates innate immune responses, at least in part through TNFα. Therefore, we next examined the gene expression profile induced by KF1B and TNFα in LoVo cells. TNFα induced 37 genes at >1.6-fold levels at 1 h after stimulation (). Notably, 11 of these genes induced by TNFα stimulation were also induced by KF1B (). The great majority of genes induced by both KF-1B and TNFα are known to be up-regulated through NF-κB and proinflammatory molecules (), which is consistent with the observation that KF1B and TNFα induce gene expression through NF-κB signaling. The genes induced by KF1B and TNFα included molecules involved in innate immune responses (CXCL1, MBL2, and CD83) and negative feedback regulators of inflammatory and apoptotic pathways (NFKBIA/IκBα, ATF3, TNFAIP3/A20, CFLAR/CLARP, and BIRC3/c-IAP2). Transient induction of CXCL1 mRNA in the early stage of Nod1 stimulation is consistent with the ability of KF1B to induce CXCL1 secretion (). Nod1 stimulation did not induce proinflammatory cytokines, including TNFα and IL-1s, suggesting that Nod1 mediates a restricted pattern of immune responses. The microarray results also revealed a group of genes that are induced specifically by Nod1 or TNFα stimulation. 31 genes were induced by Nod1 but not TNFα stimulation. These genes included signaling molecules of TLR and IL1R pathways (MAP3K7/TAK1 and MAIL/Iκ-Bζ), components of ubiquitin/proteasome pathway (UBE2M, UBE2D3, RYBP, and WSB1) and 6 novel genes, whereas TNFα stimulation induced 37 unique genes including signaling molecules of Ephrin pathways (EFNA1 and EPHA2), transcription factors (EGR1, FOSL2/Fra2, and ELF3), and innate immune factors (PLAU/plasminogen activator and TNFAIP2a; ). These results suggest that Nod1 and TNFα mediate unique and overlapping innate immune responses in epithelial cells.
To test whether KF1B mediates Nod1-dependent immune responses in vivo, we stimulated WT and Nod1-deficient mice with KF1B i.p. and measured production of chemokines and cytokines in the animals. Administration of KF1B induced rapid production of CCL2 () and CXCL2/Groβ in the serum (). KF1B was as effective as LPS or MDP in inducing secretion of CCL2 (). Notably, serum secretion of CCL2 in response to KF1B was abolished in Nod1-deficient mice (), indicating that production of CCL2 is totally dependent on Nod1 in vivo. The lack of response of Nod1-deficient mice was specific in that they produced CCL2 after administration of LPS or MDP (). Because Nod1 is highly expressed in airways and the intestine (), we also tested whether intranasal and oral administration of KF1B could induce immune responses. Consistent with the expression of Nod1 in these organs, production of CCL2 was induced in WT mice but not in Nod1-deficient mice after intranasal and oral administration of mice with KF1B (). In contrast to the production of chemokines, administration of KF1B did not induce secretion of IFN-γ, IL-3, CSF2 (M-CSF), IL-12 (), and IL-4, -10, -18, and -1β (not depicted) in the serum or tissues, including spleen, liver, and kidney, when compared with the levels found in control mice (). Further analysis did not reveal any production of these cytokines at 24 h after Nod1 stimulation (unpublished data), suggesting that the lack of response is not caused by delayed production of these cytokines. These findings are consistent with our results in vitro that Nod1 stimulation induces a restricted number of immune response genes. Because Nod1 stimulation induced chemokine secretion in vitro and in vivo, we next tested whether Nod1 stimulation can induce recruitment of specific types of immune cells into the peritoneal cavity after i.p. administration of the synthetic Nod1 ligand. 16 h after administration, Nod1 stimulation was found to induce recruitment of Gr1 neutrophils but not lymphocytes, macrophages, or other cell types as determined by morphological () and flow cytometric analyses (). The effect was Nod1 dependent in that the recruitment of neutrophils induced by KF1B was abolished in Nod1-deficient mice (). FK1B did not increase the number of macrophages or lymphocytes in the peritoneal cavity (). The recruitment of neutrophils mediated through Nod1 stimulation is consistent with the induction of CXCLs by KF1B. To determine whether resident cells found in the peritoneal cavity respond to KF1B, we prepared intraperitoneal cells and stimulated them with the Nod1 ligand. We found no detectable secretion of CXCL1 after stimulation of KF1B even though the cells responded to LPS (). Notably, analysis of peritoneal cavity tissues that are rich in mesothelial cells revealed induction of CXCL1 expression after KF1B stimulation in vivo (). These results suggest that mesothelial cells and/or other cells present in the tissue from the peritoneal cavity are the main sources of CXCL1.
We previously demonstrated that Nod1 is essential for host response against iE-DAP–containing small molecules (). These iE-DAP–containing molecules include intermediates and degradation products of PGN (, , ). In this paper, we show that the monomeric disaccharide tetrapeptide was Nod1 stimulatory, whereas the dimeric disaccharide tetrapeptide resulting from one linkage between the ɛ-amino residue in DAP and the α-carboxyl residue in -Ala was not. This linkage bond is important for the cross-linking among PGN chains (). The percentage of linked/free PGN chains varies among bacterial species and growth conditions in certain bacteria (). For example, ∼80% of the ɛ-amino acid residue of DAP remains free in (). Thus, our finding that cross-linking between DAP and -Ala eliminates Nod1-mediated host recognition suggests that the ability of bacterial PGN fragments to stimulate Nod1 varies depending on species and growth conditions of bacteria. Both hosts and bacteria have amidases that cleave the bond between the polysaccharide and oligopeptide chains of PGN, and bacteria also have enzymes that generate various oligopeptide species during the synthesis and remodeling of PGN (). A tripeptide -Ala-iE-DAP was reported to possess a higher ability to stimulate Nod1 than dipeptide iE-DAP using purified fractions (). We confirmed the latter result by comparing the Nod1 stimulatory activity of -Ala-iE-DAP and iE-DAP using synthetic equivalents (). However, we found that the stimulatory activity of the tripeptide -Ala-iQ-DAP was less than that elicited by the dipeptide iQ-DAP (). Therefore, the association between NH-terminal extension of the core structure and increased ability to stimulate Nod1 is not a general feature of Nod1 ligands.
Bacterial- and synthetic iE-DAP–containing molecules are known to induce innate and acquired immune responses (). However, because of the relatively poor immunostimulatory ability of iE-DAP–containing molecules, compared with that of LPS and MDP-related molecules (see ) (, , ), it has been difficult to investigate the role of Nod1 stimulation in immune responses. In this paper we developed acyl compounds that possess an enhanced ability to stimulate Nod1. These molecules, including myristoyl (C-14), pentadecanoyl (C-15), and palmitoyl (C-16) iE-DAP compounds (referred to as KF1B, KFC15, and KFC16, respectively), were found to exhibit a several hundredfold stronger ability to stimulate Nod1 than the original iE-DAP. Because these fatty acyl residues naturally exist in host membrane lipids, their potent stimulatory ability could be explained by increased translocation across the plasma membrane and delivery into the cytosol through cellular machinery that may include lipid flippases or related factors (). Using these compounds, we investigated the immune response induced upon Nod1 stimulation. We found that intestinal epithelial cell lines were highly responsive, whereas macrophages, DCs, and splenocytes did not respond to Nod1 ligands alone under our experimental conditions. These results are consistent with the tissue expression profile of Nod1 that showed high levels of Nod1 expression in epithelial tissues such as the intestine (). Several genes induced by Nod1 stimulation, including CXCL1, CD83, and MBL2, are known to be involved in the recruitment of immune cells and complement factors after bacterial infection. Similarly, our analysis in mice revealed that secretion of CCL2 was induced in serum and multiple tissues upon Nod1 stimulation in vivo. These results suggest that Nod1 functions as a pathogen recognition receptor to induce expression of proteins that play a critical role in the early stages of the innate immune response.
Nod1 is an intracellular protein, and, thus, recognition of bacteria through Nod1 would signal potentially harmful invasion of epithelial surfaces such as that lining the gut. In the case of the intestine, the mucosa is exposed to a large concentration of commensal bacteria that express a wide array of TLR ligands. However, inappropriate innate recognition and inflammatory responses are avoided, at least in part, by the lack of TLR signaling in the surface intestinal epithelium (). A model suggested by these results is that after invasion of the epithelium by pathogenic bacteria, TLR-positive innate immune cells are recruited, though Nod1-induced chemokines and stimulation of these cells results in the production of cytokines such as TNFα that amplify the host response. Thus, although both TLR and Nod proteins recognize bacterial products, they may play distinct roles in bacterial clearance. Intestinal epithelial cells are also likely to play an important role in this amplification loop of the host defense through the induction of genes that are unique to TNFα signaling. Comprehensive microarray analysis revealed that Nod1 and TNFα induced a different profile of gene expression that include both common and specific genes in epithelial cells. These observations suggest that primary and secondary responses against bacteria in intestinal epithelial cells are different. The genes induced by Nod1, but not TNFα, include TAK1 and Iκ-Bζ, which are critical factors in TLR and IL-1R pathways (–). Co-stimulation with Nod1 and Nod2 enhances TLR signaling (, ). The enhancement of TLR signaling by Nod1 might be due, at least in part, to the induction of TAK1 and Iκ-Bζ. Nod1 stimulation also induced several novel genes that have unknown functions. Further studies are required to reveal the physiological role of these Nod1-responsible genes. The molecular basis that accounts for the induction of unique genes by Nod1 and TNFα signaling is unclear. A possible explanation is that differences in gene expression reflect differential activation of intracellular signaling molecules. For example, RICK, a downstream factor of Nod1, has been shown to activate NF-kB through TRAF6, whereas TRAF2 is critical for TNFα signaling (–).
Nod1 gene variants have been recently associated with the development of asthma (). Moreover, genetic variations in Nod1 and Nod2 are associated with increased susceptibility to Crohn's disease (–). The Crohn's disease–associated Nod2 mutations are defective in their response to muramyl dipeptide (, ). Because both Nod1 and Nod2 share the same downstream components for signaling, the defective response observed in cells harboring Nod2 mutations could be rescued by Nod1 stimulation. Thus, ligands such as KF1B could be useful for the development of novel approaches to treat Nod1- and Nod2-associated inflammatory diseases.
Ligand-dependent NF-κB activation was determined using HEK293T cells transiently expressing Nod1, Nod2, or TLR4 and MD2 in the presence of pBxIV-luc and pEF1BOS-β-gal as described previously (). In brief, HEK293T cells were transfected with expression plasmids by the calcium phosphate method, and cells were treated with medium containing various ligands 8 h after transfection. 24 h after transfection, ligand-dependent NF-κB activation was determined with a reporter assay. LoVo cells were transfected with pBxIV-luc and pEF1-BOS-β-gal () by Lipofectamine 2000 (Invitrogen), and cells were treated with medium containing 2 μg/ml KF1B, 0.2 μg/ml AcMTP, or 10 ng/ml TNFα 3 h after transfection. 24 h after transfection, ligand-dependent NF-κB activation was determined with a reporter assay.
Highly purified fractions of whole soluble Cellosyl-treated PGN fragments (GlcNAc-MurNAc--Ala-γ--Glu-DAP--Ala, shown as fraction 3 in reference , or dimeric GlcNAc-MurNAc--Ala-γ--Glu--DAP--Ala cross-bridged between DAP and -Ala, shown as fraction 21 in reference ) from were prepared as described previously (). In brief, dibenzyl-2,6-diaminopimelate (dibenzyl-DAP) was first connected to 2-chlorotrityl resin, and the remaining amino group of DAP was reacted with Fmoc--Glu-OBn. After deprotection of Fmoc group, the acyl group (C-14, C-15, or C-16) was introduced to the liberated amino group to give the corresponding acylated iE-DAP on the resin. After cleavage from the resin with 10% trifluoroacetic acid, cleavage of the remaining protecting groups gave KF1B, KFC15, or KFC16, respectively. Synthetic compounds iE-DAP, iQ-DAP, CpG, and polyIC were described previously (). Synthetic Ac-(6-O-stearoyl)-MurNAc--Ala-γ--Glu (AcMDP), AcMTP, and -Ala-Gln-octadecyl ester (AcAiQ) were obtained from Bachem. O55:B5 LPS was purchased from Sigma-Aldrich. The LPS preparation that lacked contamination of substantial Nod1 and Nod2 stimulatory activity in the HEK293T bioassay was used in this study.
Human LoVo, SW620, and MonoMac6 cells were cultured and maintained in RPMI 1640 medium (Invitrogen). Macrophages and DCs were derived from BM using CSF1 and CSF2, respectively, as previously described (). Cells were treated with the indicated amounts per milliliter of various ligands for the times shown in the figure legends. The cytokine and chemokine levels were determined by sandwich ELISA kits using specific antibodies (R&D Systems).
8-wk-old C57BL/6 (B6) WT and Nod1 mice in B6 background were maintained at the University of Michigan Animal Facility. B6 background Nod1 mice used in this study were generated by backcrossing at least six times with the parental B6 strain. Mice were administrated with the indicated amounts of various ligands by routes described in the figure legends. At the times indicated in the figures, sera and organs were collected from killed mice, and homogenates of organs were prepared by sonication with PBS (Invitrogen) containing 1% Triton X-100 and a mixture of protease inhibitors (Sigma-Aldrich), followed centrifugation at 100,000 for 10 min. The cytokine and chemokine levels in sera and tissue homogenates from mice were determined by ELISA. For recruitment of immune cells at 24 h after i.p. administration of KF1B, peritoneal cells were collected from killed mice by PBS lavage. After centrifugation (Cytospin 2; Shandon Ltd.), cells were fixed and stained with a staining kit (Diff; Dade Behring Inc.) according to the manufacturer's recommendations. Cell types were determined by standard morphological features of stained cells. The number of GR1 cells in 10 cells was determined by flow cytometry (FACScan II; BD Biosciences) as previously described (). The mouse studies were approved by the University of Michigan Committee on the Use and Care of Animals. For estimation of CXCL1 mRNA, total RNA was isolated from peritoneal tissue of WT mice using Trizol (Invitrogen). In brief, several 1-cm fragments of the peritoneal membrane were isolated and immediately homogenized with Trizol solution with and without KF1B stimulation. To assess mRNA expression, a semiquantitative RT-PCR method was used, as previously described (). CXCL1 and control GAPDH gene fragments (355 and 191 bp, respectively) were amplified by 35 cycles of the PCR with 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min using gene-specific primers (CXCL1, 5′-CACCTCAAGAACATCCAGAGCT-3′ and 5′-CAAGCAGAACTGAACTACCATCG-3′; GAPDH, AACGACCCCTTCATTGAC-3′ and 5′-CCACGACATACTCAGCAC-3′) and subjected to 2% agarose electrophoresis.
LoVo cells were treated with 2 μg/ml KF1B, AcMTP, 10 ng/ml TNFα, or left alone for the times indicated in the figures. Total RNA was prepared, and Cy3- and Cy5-labeled cDNA was synthesized from total RNA as described previously (). Differential expression of 20,000 human genes (Invitrogen) was determined by hybridization of the probes with their cDNAs, followed by an analysis using a microarray scanner (GenePix 4000A; Molecular Devices) as described previously ().
Quantitative real-time PCR was performed using gene-specific primers (CD83, 5′-TTTAATGGCCGGCTGGAAATG-3′ and 5′-TTTAGCCCATGCAACAGCCTTGTG-3′; NFKBIA, 5′-CGCGACGGGCTGAAGAAGG-3′ and 5′-ATGGCCAAGTGCAGGAACGAG-3′; IL8, 5′-CAAGGGCCAAGAGAATATCCGAACT-3′ and 5′-TATCACATTCTAGCAAACCCATTCAA-3′; and CXCL1, 5′-CTCAATCCTGCATCCCCCATAGTTA-3′ and 5′-GTGGCCTCTGCAGCTGTGTCTCT-3′) and SYBR green dye with a real-time PCR system (model 7300; Applied Biosystems) as previously described (, ). The mean calculated quantity of the target gene for each sample was then divided by the mean calculated quantity of the housekeeping genes and corresponding to each sample to give a relative expression of the target gene for the sample (). All reactions were performed in duplicate. Immunoblotting analysis of CD83 in 50 μg total protein lysate from cells treated with 2 μg/ml KF1B, AcMTP or left alone was performed using anti-CD83 monoclonal antibody (BD Biosciences). Equal loading of proteins were confirmed by reversible staining with Ponceau S (Sigma-Aldrich) before immunodetection. |
Human primary monocyte–derived macrophages express low levels of APOBEC3G (), consistent with a cell nonpermissive to HIV in the absence of Vif (). To determine whether IFN-α directly influences APOBEC3G expression, macrophages were treated with IFN-α and by RT-PCR; a dose-dependent increase in APOBEC3G gene expression was detected (). A kinetic analysis revealed that a single treatment with 10 ng/ml IFN-α enhanced APOBEC3G gene expression within 1 h, which increased further at 4 h (). PKR gene expression and activation are prominent consequences of IFN activity (), and a similar time-dependent pattern of gene expression was observed in macrophages treated with IFN-α for both APOBEC3G and PKR by RT-PCR (), and APOBEC3G expression was confirmed by real-time PCR (). A corresponding IFN-α–induced increase in APOBEC3G protein was evident in macrophage lysates by Western blot using a monoclonal anti-APOBEC3G antibody (). Thus, by both RNA and protein analyses, IFN-α was able to induce elevated APOBEC3G in primary macrophages. This IFN-α response was consistent, although variable baseline levels of APOBEC3G were donor dependent. Furthermore, 2-aminopurine (2-AP), a PKR inhibitor in macrophages (), suppressed IFN-α–induced APOBEC3G mRNA to basal levels () with a subsequent reduction in protein expression (), suggesting that APOBEC3G expression may be downstream of PKR activation. Collectively, these data indicate that IFN-α achieves APOBEC3G augmentation by promoting gene expression rather than inhibiting proteasome-dependent degradation and that there is a connecting link between PKR and APOBEC3G.
Because IFN-α impedes HIV infection in macrophages (), induced APOBEC3G may represent a previously unrecognized mechanism underlying this antiviral activity. Associated with the dose-dependent enhancement of APOBEC3G (), IFN-α blocked HIV infection (). Although HIV typically suppressed APOBEC3G protein levels (, inset, day 3), consistent with the fact that Vif promotes APOBEC3G degradation, the addition of IFN-α reversed the loss of APOBEC3G protein in the infected cultures, coincident with reduced viral replication (). Whether the emergence of low APOBEC3G expression around 7 d after infection reflects diminished proteasomal degradation, endogenous IFN-α release (), and/or other sequelae, increased APOBEC3G in IFN-α–treated infected macrophages remained consistent with restricted HIV expansion (). In virus-free cells, APOBEC3G levels characteristically diminish with time in culture. The influence of IFN-α on HIV infection in macrophages appears to occur early in the viral life cycle. For example, a single treatment may prevent productive HIV infection for up to 7 d, compatible with targeting of an event(s) in the virus life cycle before massive replication. An early increase of APOBEC3G by IFN-α likely promotes enhanced APOBEC3G packaging into the initial viral particles, which in turn renders these virions defective in establishing new rounds of infection.
To establish whether IFN-α was a unique regulator of this antiviral molecule in macrophages, we analyzed a panel of cytokines for their ability to alter the expression profile of APOBEC3G protein. As shown in , of the cytokines evaluated, only IFN-α and IFN-γ were found to detectably enhance this retroviral restrictor, whereas the remaining inflammatory mediators (TNF-α, IL-1β, IL-8, MCP-1, RANTES, and TGF-β) tested did not promote APOBEC3G production nor augment PKR. Although only a limited number of proteins were analyzed and other potential inducers cannot be excluded, since its recent discovery, there has yet to be any report of APOBEC3G induction by other endogenous factors. Moreover, because IFN-α was reportedly ineffective in enhancing APOBEC3G expression in a T cell line () and did not increase frequency of G to A hypermutations in hepatitis B virus stably infected HepG2 cell lines (), this may represent a key macrophage anti-viral pathway.
Because both IFN-α and IFN-γ increase APOBEC3G and inhibit HIV infection in macrophages, and macrophages remain a crucial target and reservoir of HIV in the blood even after HAART (, ) as well as in the tissues (), we sought to determine the relative efficiency of the IFNs on their innate cellular defenses. Although IFN-α is clinically effective against HIV and multiple additional viruses, its toxicity represents a clinical concern (). By comparison, IFN-γ, functioning as an immune stimulant, has both anti–HIV-1 and PKR activation potential, but to a lesser extent than IFN-α (). To test the possibility that IFN-γ together with IFN-α might enable use of reduced levels of IFN-α, while achieving a comparable antiviral effect, we compared IFN-α and IFN-γ concentrations required for inhibition of HIV. IFN-α was typically severalfold more potent than IFN-γ in the in vitro infection assay. Costimulation with IFN-α and IFN-γ at suboptimal concentrations in a single application after viral inoculation resulted in complete or near complete antiviral activity, as compared with treatment with IFN-α or IFN-γ alone (, day 7 p24 shown). This dual treatment further bolstered APOBEC3G (, inset) expression that correlated with enhanced viral resistance. Nonetheless, as shown in , a single treatment with IFN-α, IFN-γ, or with IFN-α plus IFN-γ did not inhibit viral replication indefinitely, and reexposure to the IFNs was required to sustain APOBEC3G and maximum viral suppression beyond 1 wk. Whether administered individually or coordinately, these data provide the first indication that APOBEC3G-mediated interference with the production of a functional provirus can be regulated by a host cytokine as a new point of attack in the viral life cycle.
APOBEC3 represents a family of deaminase proteins, some of which reportedly restrict HIV infection (APOBEC3G and APOBEC3F) or are influenced by IFN (APOBEC3A; references –), and we examined whether IFN-α could trigger the expression of additional superfamily genes. In macro-phages from multiple donors, RT-PCR showed that IFN-α not only up-regulated APOBEC3G, but also APOBEC3A and APOBEC3F with a similar profile. Family members APOBEC3B and APOBEC3C were not consistently augmented by IFN-α (, two representative donors shown). Consequently, IFN-α appears to preferentially regulate members of the APOBEC3 cluster that reportedly have antiviral activity. In this regard, our analysis of the 5′ region of APOBEC3 family genes (National Center for Biotechnology Information, National Institutes of Health [NIH]) revealed differential expression of IFN-stimulated response elements (ISRE). We identified the existence of an ISRE sequence −47 to −59 from the APOBEC3G translation start codon (GAAAGTGAAAC), and an identical ISRE sequence is also found in APOBEC3F, reflecting their coordinate regulation. A potential promoter region in APOBEC3A, containing an ISRE (−1,787 to −1,800), is located further from the start codon relative to APOBEC3G and is consistent with IFN-α inducibility. Thus, our data support several APOBEC3 nucleic acid–editing enzymes (APOBEC3A, APOBEC3F, and APOBEC3G) as ISRE-competent IFN-α–inducible targets. Among them, both APOBEC3F and APOBEC3G can reportedly restrain Vif-dependent infection mechanisms (, , ). Although we observed that APOBEC3A is also coordinately expressed after macrophage exposure to IFN-α, to date, no antiviral activity has been ascribed to this family member.
IFN-α executes its antiviral activity through multiple mechanisms, including PKR and RNase L and/or RNA deaminases (). As our evidence implicates APOBEC3 family induction as one of the potential contributing mechanisms, we considered that removal of APOBEC3G would result in at least a partial block of IFN-α antiviral activity. Using siRNA synthesized based on the sequence of APOBEC3G, we show that transfection of siRNA into macrophages decreased APOBEC3G RNA and protein induction by IFN-α, in contrast to control siRNA (), without any effect on IFN-α–induced PKR. When the cells were depleted of APOBEC3G by siRNA and then treated with a low dose of IFN-α (1 ng/ml), IFN-α could no longer completely inhibit HIV in the absence of APOBEC3G (). By comparison, the same dose of IFN-α effectively inhibited HIV infection in both untransfected and negative control siRNA-transfected macrophages (), confirming that APOBEC3G is a key downstream anti-HIV mechanism induced by IFN-α. Perhaps not surprisingly, at a higher dose of 10 ng/ml, IFN-α inhibition of HIV infection in macrophages was not substantially altered by siRNA transfection of APOBEC3G (not depicted), likely a result of the multiple antiviral mechanisms triggered by IFN-α. Furthermore, by nested PCR, which detects early viral de novo DNA synthesis (, inset), it was apparent that depletion of APOBEC3G by siRNA resulted in substantially augmented reverse transcription with elevated levels of HIV proviral DNA (day 2 after exposure to HIV shown) once this obstacle was removed.
In what appears to be a unique and powerful pathway, IFN-α increases APOBEC3G expression as a component of its complex repertoire of antiviral mechanisms. This selective induction of APOBEC3G by IFN-α provides a potential mechanism to overcome HIV Vif sequestration of APOBEC3G, thus allowing enhanced APOBEC3G packaging into the budding virions to dampen further infection potential. The net effect is that IFN-α tilts the balance between APOBEC3G and Vif in favor of APOBEC3G as opposed to unimpaired Vif-driven APOBEC3G degradation, which enables HIV replication in subsequent host cells. Demonstration of IFN-α–inducible intracellular APOBEC3G suggests an important strategic approach to regulation of this newly recognized intracellular defense against the deadly virus. Beyond the role of APOBEC3G in retroviral infections, recent evidence documents that APOBEC3G also blocks hepatitis B virus replication, pointing to a broader antiviral profile (). Moreover, the evidence that multiple APOBEC3 family proteins are inducible by IFN-α indicates the potential for additional elements in the regulatory control of the viral life cycle. With the recent recognition of several new host cell–associated proteins controlled by and/or required for HIV infection, such as p21 and annexin II (, ), as well as those that represent potent opposition to HIV infection (TRIM5α, APOBEC3G, and lentivirus susceptibility-1; references and ), new classes of inhibitors may emerge as future antiviral candidates.
Human peripheral blood mononuclear cells, obtained by leukapheresis of normal volunteers at the Department of Transfusion Medicine (NIH), were diluted in endotoxin-free PBS without Ca and Mg (BioWhittaker) and separated by density centrifugation on lymphocyte sedimentation medium (Organon Teknika Corp.) at 400 for 30 min. The monocytes in the mononuclear cell layer were enriched by elutriation (, ) and plated at 1.5 × 10/ml per well in 48-well plates or 6 × 10/2 ml per well in 6-well plates (Corning) in DMEM (BioWhittaker) with antibiotics and glutamine for 3 h, followed by the addition of 10% FCS. Adherent monocytes were cultured for 7–10 d to enable differentiation into monocyte-derived macrophages.
Unless indicated otherwise, adherent macrophages were incubated with HIV-1(HIV) at 1–2 × 10/TCID/ml (Advanced Biotechnologies, Inc.) in DMEM containing 10% FCS for 2 h at 37°C. After infection, cells were washed with PBS three times and cultured in DMEM containing 10% FCS for up to 14 d (, ). IFN-α and IFN-γ (National Cancer Institute-Frederick Cancer Research and Development Center [NCI-FCRDC]) were added once at the indicated concentrations or every 2–3 d. To measure viral infectivity, supernatants (200 μl) were collected at the indicated intervals and replaced with fresh medium. Infection was measured by supernatant p24 using HIV-1 p24 ELISA kits (PerkinElmer) with data presented as mean ± SD and by nested PCR of extracted DNA as described previously (, ). PCR product from the second amplification was visualized by ethidium bromide staining after agarose gel electrophoresis.
6 × 10 macrophages cultured in six-well plates were treated with 1–100 ng/ml IFN-α and/or 1mM 2-AP (Biomol) for the times indicated. Total RNA was extracted using RNeasy Total RNA kit (QIAGEN). For RT-PCR, GeneAmp RNA PCR kit (Perkin Elmer, Branchburg, NJ) was used according to protocol. A total of 0.15–0.3 μgRNA from each sample was used for first-strand cDNA synthesis. The cDNA was then divided and used for PCR amplification of APOBEC3G and other APOBEC3 family genes. PKR and GAPDH were used as controls. All PCRs were performed by 30-cycle amplification, except that 25 cycles were used for GAPDH (94°C for 4 min, followed by 30 cycles at 94°C for 30 s, 55°C for 3 s, 72°C for 30 s, and finally extension at 72°C for 10 min). Primers were as follows: APOBEC3A: 5′-TTCTTTGCAGTTGGACCCGG-3′ (forward), 5′-CTCATCTAGTCCATCCCAGG-3′ (reverse); APOBEC3B: 5′-TGGTCGGAGCTACACTTGGC-3′ (forward), 5′-CAGACAGGAATTCGGCCAGC-3′ (reverse); APOBEC3C: 5′-CTTCCTCTCTTGGTTCTGCG-3′ (forward), 5′-CCATGATCTCCACAGCGACC-3′ (reverse); APOBEC3F: 5′-TACGCAAAGCCTATGGTCGG-3′ (forward), 5′-GCTCCAAGATGTGTACCAGG-3′ (reverse); APOBEC3G: 5′-TTACCTGCTTCACCTCCTGG-3′ (forward), 5′-TCATCTAGTCCATCCCAGGG-3′ (reverse); PKR: 5′-GCCTTTTCATCCAAATGGAATTC-3′ (forward), 5′-GAAATCTGTTCTGGGCTCATG-3′ (reverse); and GAPDH: 5′-CCTTGGAGAAGGCTGGGG-3′ (forward), 5′-CAAAGTTGTCATGGATGACC-3′ (reverse).
For real-time PCR, cDNA was prepared using the GeneAmp RNA PCR Core kit (Applied Biosystems), and cDNA corresponding to 7.5 ng RNA was used for each reaction in triplicate. The primers for APOBEC3G and GAPDH and the FAM dye-labeled probes were from Applied Biosystems. PCR reactions (15 s at 95°C for melting and 1 min at 60°C for annealing/extending; a total of 40 cycles) were conducted with the Real Time PCR System 7500 (Applied Biosystems). Gene expression was determined using the relative quantification: ΔΔC = (C − C) − (C − C). C is the fractional cycle number that reaches a fixed threshold, C is the test of interest, and C is the reference control (RNA from 2-h untreated cells). ΔC is the difference between gene expression in treated cells and reference control cells. The fold increase was calculated using 2 ().
Macrophages were treated with or without IFN-α, IFN-γ, and/or 2-AP for the times indicated, and cells were lysed with ice-cold buffer containing 10 mM Hepes, pH 7.9, 10 mM KCl, and 0.1 mM EDTA, and 0.1 mM EGTA, 0.5 mM PMSF, 1 mM DTT, and 0.5% NP-40. 15–75 μg protein was used for electrophoresis (10% polyacrylamide gel) and blotted onto nitrocellulose membranes. After blocking with 5% milk in Tris-buffered saline with 0.05% Triton X-100 (TBS-T), membranes were probed with monoclonal anti-APOBEC3G at 1:1,000 (Immunodiagnostics) or polyclonal anti-PKR at 1:1,000 (Cell Signaling) at 4°C overnight. Membranes were washed with TBS-T three times for 10 min, followed by secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Inc.), and detected by chemiluminescence (Pierce Chemical Co.). A similar protocol was followed for detection of APOBEC3G in cells treated with 100 ng/ml IFN-γ, TNF-α, IL-1β, IL-8, MCP-1, or RANTES (NCI-FCRDC) or 10 ng/ml TGF-β (R&D Systems) for 20 h.
Monocytes (30 × 10 cells) were treated with 36 μl siRNA (20 nM) or siRNA (control; QIAGEN) in 300 μl Dendritic Nucleofector Solution (Amaxa Biosystems) or siRNA buffer only at room temperature. APOBEC3G siRNA was synthesized based on the sequence r(CGGUCAAGAUGGACCAGCA)dTdT/r(UGCUGGUCCAUCUUGACCG)dAdG. 100-μl aliquots were added to a cuvette for electropora-tion using the U-02 program (, ). After electroporation, the cells from three replicate cuvettes were pooled and resuspended in 3 ml RPMI before 0.2 ml (2 × 10) was added into a 24-well plate containing 1 ml RPMI preincubated in a humidified 37°C/5% CO incubator for 30 min and incubated for 3 h. The medium was changed to complete DMEM (1.5 ml/well), and the cells were cultured for 6 d before infection with HIV and/or treatment with 0.1–10 ng/ml IFN-α. |
To examine the physiological function of MKP-1 in the regulation of the innate immune responses during bacterial infection, thioglycollate-elicited peritoneal macrophages from both
and
mice were stimulated with LPS. In
macrophages, LPS stimulation resulted in an increase in MKP-1 protein levels within 30 min, with levels peaking at ∼60 min. The increase of the MKP-1 level temporally coincided with the inactivation of p38, JNK, and ERK ().
macrophages, even with LPS stimulation (, top).
cells exhibited substantially prolonged p38 and JNK activation compared with
macrophages (). However, the activation kinetics of ERK were similar in both cell groups, indicating that MKP-1 does not play a significant role in ERK inactivation in this system. MK2, often referred to as MAP kinase–activated protein kinase 2, is a physiological target of p38 (). MK2 plays a critical role in the innate immune response to bacterial infection and is required for the production of many inflammatory cytokines in vivo after LPS challenge ().
macrophages, LPS stimulation also resulted in a prolonged MK2 activation in
macrophages (, third panel). The findings that –deficient cells had sustained p38 and JNK activities after LPS stimulation were reinforced by detailed analysis of the activation kinetics of p38, JNK, and ERK (). The activity of MK2 was also examined by immune complex kinase assays using Hsp25 as a substrate. MK2 activity in knockout macrophages was sustained after LPS stimulation relative to wild-type cells (). These results clearly indicate that MKP-1 plays a predominant role in the termination of the JNK and p38 pathways in vivo.
IFN-γ has been shown to activate macrophages, resulting in more robust production of inflammatory cytokines (). We found that the pretreatment of resident peritoneal macrophages with IFN-γ inhibited MKP-1 induction by LPS and resulted in sustained p38 and JNK activation in LPS-stimulated resident peritoneal macrophages (). This observation suggests that IFN-γ may increase the activity of macrophages in part through attenuation of MKP-1 induction. Collectively, our results indicate that MKP-1 is a primary regulator of MAP kinases, especially p38 and JNK, in peritoneal macrophages activated by the Gram-negative bacterial cell wall component, LPS.
Because MAP kinases are pivotal in modulating innate immune responses (), we investigated the effect of deficiency on cytokine production by several different types of innate immune cells.
and
mice, primed with IFN-γ, and stimulated with LPS. The concentrations of cytokines secreted into the media were determined by ELISA. In the absence of LPS stimulation, TNF-α and IL-6 production was undetectable (not depicted).
mice produced significantly more TNF-α and IL-6 than did their wild-type counterparts ().
mice with thioglycollate and isolated peritoneal macrophages from these mice.
mice also mounted a more robust TNF-α production than did wild-type cells upon LPS stimulation (, top left).
peritoneal macrophages was also significantly elevated compared with wild-type cells at 6 h, but not at 4 h, likely reflecting the delayed expression of IL-6 relative to TNF-α (, top right). IL-12 is a classic Th1 cytokine produced by macrophages that can promote the differentiation of naive Th cells into Th1 cells ().
peritoneal macrophages in response to LPS was significantly decreased relative to
peritoneal macrophages (, bottom).
resident peritoneal macrophages compared with their
counterparts ().
and
mice, primed with IFN-γ, and stimulated with LPS for 4 h. The expression levels of TNF-α, IL-6, IL-12p35, and IL-12p40 mRNAs were assessed by quantitative real-time RT-PCR (qRT-PCR; ).
macrophages were 3.5-fold higher than those in the LPS-stimulated
macrophages.
macrophages than those in their wild-type counterparts.
macrophages relative to their wild-type counterparts.
macrophages were decreased by 65 and 70%, respectively.
cells is likely due to the modulation of cytokine gene expression.
and
mice and stimulated with LPS to assess the effect of deletion on cytokine production.
mice produced considerable amount of TNF-α (, left).
splenocytes (, left). However, compared with the more robust TNF-α production by the –deficient splenocytes, the production of two classic Th1 cytokines, IFN-γ and IL-12, was actually attenuated in these cells relative to that in the wild-type splenocytes (, middle and right).
Dendritic cells are professional antigen-presenting cells that play a vital role in the development of adaptive immunity in response to pathogens by facilitating T lymphocyte activation and differentiation (). The effect of knockout on cytokine production in dendritic cells was examined.
and
mice by culturing in the presence of GM-CSF. LPS stimulation of dendritic cells resulted in significant production of TNF-α ().
cells produced ∼10-fold more TNF-α and 75% more IL-6 when compared with wild-type dendritic cells (, first and second panels).
cells relative to the wild-type cells (, third panel). In contrast, IL-12 production in dendritic cells was significantly attenuated in –deficient cells relative to wild-type cells (, fourth panel). Collectively, our studies indicate that loss of results in profound alteration in the cytokine expression profiles in a variety of immune cells.
and
mice were injected i.p. either with vehicle (PBS) or with different doses of LPS.
nor
mice injected with PBS produced a detectable amount of TNF-α or IL-6 in their plasma ().
mice were more sensitive to LPS and produced significantly higher levels of both TNF-α (, left) and IL-6 (, right) over a broad range of LPS doses.
and
mice. In the absence of LPS challenge, serum levels of TNF-α, IL-6, and IL-10 were undetectable ().
mice were substantially elevated within 1 h followed by a rapid decline thereafter (, left). In
mice challenged with LPS, serum TNF-α levels were 3.5-fold higher than those of
mice at 1 h, and the levels continued to increase at 2 h.
mice were ∼29-fold higher than those in
mice.
mice declined substantially by 3 h, the levels of TNF-α remained significantly higher than those in wild-type mice.
mice at 3 h were not significantly different from the maximal TNF-α levels observed in
mice at 1 h (, left). In
mice, serum IL-6 was elevated within 1 h and plateaued by 2 h (, middle).
mice,
mice produced substantially more IL-6.
mice continued to increase through the observed 3-h period.
mice (, right).
mice remained elevated throughout the 3-h experimental period (, right). Collectively, our results indicate that knockout has profound effects on the production of TNF-α, IL-6, and IL-10 in vivo.
TNF-α is a pivotal inflammatory cytokine that plays an important role in the pathogenesis of septic shock (, ).
mice exhibited a marked elevation in TNF-α upon LPS challenge, we examined the effect of deficiency on survival.
and
mice were injected i.p. with LPS, and survival of these animals was monitored over 4–5 d.
and
mice. For knockout mice, mortality was observed within 22 h after LPS challenge ().
mice had died, and no further death was noted through 120 h.
mice, the first mortality occurred at 34 h.
mice had died (). An LPS dose of 1.5 mg/kg body weight caused few signs of distress in the
mice, and all
mice survived through the 96-h period ().
mice given 1.5 mg/kg LPS were severely distressed, and 75% of them died over the 96-h experimental period ().
To examine the effects of Mkp-1 deficiency on the function of key organs, wild-type and Mkp-1/ mice were challenged with either vehicle (PBS) or LPS at a dose of 1.5 mg/kg body weight and killed 24 h after LPS injection. Blood samples were collected, and blood urea nitrogen (BUN) levels, a measurement of renal function, were measured (, left). BUN levels in vehicle-treated mice were similar in both Mkp-1/ and Mkp-1/ mice (16.3 ± 1.3 vs. 17.0 ± 3.4 mg/dl, respectively). LPS challenge did not result in a significant change in BUN for Mkp-1/ mice. However, a substantial increase in BUN levels was observed in Mkp-1/ mice after LPS challenge (89.1 ± 12.6 mg/dl). Blood alanine aminotransferase (ALT) activity, an indication of liver damage, was measured (, right). There was no significant difference in ALT levels between vehicle-treated Mkp-1/ and Mkp-1/ mice (25.8 ± 2.4 vs. 17.9 ± 2.4 U/liters, respectively). Although LPS challenge did not significantly change blood ALT levels in Mkp-1/ mice, challenge of Mkp-1/ mice with LPS resulted in a significant increase in blood ALT levels (, right). Although the ALT levels in LPS-challenged wild-type mice were 22.7 ± 2.8 U/liters, ALT levels in LPS-challenged Mkp-1/ mice were increased to 149.2 ± 3.6 U/liters.
Because respiratory failure is often associated with septic shock syndrome, we examined the effect of deficiency on lung histology. Lung tissues were fixed with formalin at a constant distending pressure of 25 cm HO. The lung sections were stained with hematoxylin and eosin.
mice appeared normal, and there were no differences observed in lung histology between vehicle-treated
and
mice (not depicted).
mice, but not in lungs from
mice ().
but not
mice are highly susceptible to the development of multiple organ failure syndrome after LPS administration.
Hypotension is a clinical characteristic of severe sepsis and plays an important role in the pathophysiology of septic shock and multiple organ failure syndrome.
and
mice after LPS challenge ().
mice, LPS challenge at a dose of 1.5 mg/kg resulted in no significant change in systolic blood pressure.
mice.
mice decreased from 94.2 ± 3.0 mmHg to 41.5 ± 12.5 mmHg (P < 0.005).
mice persisted at 24 h (). Nitric oxide plays a critical role in the regulation of vasculature function.
mice 24 h after LPS challenge (1.5 mg/kg body weight), and plasma nitrate levels were measured ().
mice than in
mice after LPS challenge. Collectively, these observations indicate a critical regulatory role of MKP-1 in modulating the inflammatory responses to LPS and demonstrate that lack of MKP-1 markedly sensitizes mice to septic shock.
In this report, we have demonstrated that MKP-1 is a critical negative regulator in the innate immune response to LPS. We have found that MKP-1 is induced by LPS and plays a critical role in the attenuation of both JNK and p38 in peritoneal macrophages (). We showed that deletion of the gene resulted in a prolonged activation of JNK and p38 after LPS stimulation, leading to augmented production of the proinflammatory cytokines TNF-α and IL-6 in macrophages (). Although the prolonged JNK and p38 activation was only demonstrated experimentally in thioglycollate-elicited peritoneal macrophages, we found that LPS stimulation also resulted in a substantial increase in the production of TNF-α and IL-6 in –deficient splenocytes and dendritic cells (). This observation strongly suggests that MKP-1 plays a similar regulatory role in these cell types. The fact that –deficient mice produced dramatically more TNF-α and IL-6 after LPS challenge further validates the critical role of MKP-1 in the control of these two proinflammatory cytokines in vivo (). The substantial increase in mortality after LPS challenge in –deficient mice illustrates the critical importance of the MKP-1–dependent regulatory mechanism in host defense (). The severe hypotension () and dysfunction of vital organs () in LPS-challenged –deficient mice are consistent with the clinical symptoms of septic shock. These results clearly indicate that MKP-1 acts as a vital suppressor of the inflammatory responses and thereby protects the host from shock, multiple organ failure, and mortality upon exposure to LPS. Our results also raise the possibility that variations in the gene may represent a susceptibility factor for septic shock.
What is the mechanism through which MKP-1 controls the inflammatory cascade and prevents shock and multiple organ failure in the host? First, by inactivating JNK and p38, MKP-1 determines the window of synthesis of proinflammatory cytokines, including TNF-α. In this sense, MKP-1 serves as a servocontrol mechanism for TNF-α production. In the absence of the MKP-1–mediated servocontrol mechanism, the signal directing TNF-α synthesis is not appropriately down-regulated, thus resulting in the overproduction of proinflammatory cytokines. Previously, it has been shown that both JNK and p38 positively regulate TNF-α biosynthesis by stabilizing TNF-α mRNA and enhancing its translation (, ).
cells, explaining the prolonged TNF-α biosynthesis in
mice after LPS challenge (). A potential explanation for the shock and multiple organ dysfunction is that the excessive TNF-α triggers a considerable elevation in nitric oxide synthase activity (, ), resulting in severe hypotension that leads to hypoperfusion and multiple organ failure ().
A balance between activation and subsequent deactivation of the immune system is of critical importance in the host immunological defenses. Although activation of the signal transduction cascades is critical for mounting an aggressive immune response to eliminate invading pathogens, deactivation of the signaling pathways restrains the potentially devastating actions of the immunological system on the host, thus preventing self-destruction. A variety of negative regulators operate at various steps in the critical signal transduction pathways downstream of TLRs. These negative regulators modulate the strength and duration of the transduced signals and control the production of inflammatory cytokines (). It has been shown that TLR4 is transiently suppressed in response to LPS (). In addition to the modulation at the receptor level, several antiinflammatory proteins are also induced, which include IL-1 receptor–associated kinase (IRAK)-M, suppressor of cytokine signaling (SOCS)-1, inhibitor-κB, MKP-1, and antiinflammatory cytokines such as IL-10 (). Through these inhibitory proteins, cells not only terminate the signaling cascade at the cell surface, but also switch off downstream mediators, thus silencing the signaling pathways leading to the production of proinflammatory cytokines. Therefore, a timely “switch-off” of the signaling events is crucial, as it not only prevents the overproduction of the potentially harmful cytokines, but also prepares the cells for responding to subsequent pathogenic infection. The discovery of MKP-1 as a crucial negative regulator of the innate immune responses both in vivo and in vitro places it in the center of the complex negative regulatory mechanism dictating endotoxin tolerance. Although similarities exist between phenotypes of knockout mice and mice lacking other negative regulators, there are also important differences. It appears that knockout of or leads to generalized hyperresponses of innate cells to LPS challenge (, ), whereas deletion in the gene changes the pattern of innate immune responses. For example, knockout of either or resulted in an increase in IL-12 production after LPS stimulation (, ). In contrast, knockout of leads to decreased LPS-induced IL-12 production ( and ). The phenotypical differences between knockout mice and or knockout mice likely reflect the different mechanisms on which these regulators operate.
There are at least 11 MKPs in mammalian cells (). Although this group of phosphatases exhibit differential substrate specificities toward different MAP kinases, many of the phosphatases also share substrates. For example, MKP-1 has been shown to prefer p38 and JNK as substrates (), although it was originally characterized as an ERK phosphatase (). In contrast, phosphatase of activated cells 1, an MKP predominantly expressed in hematopoietic cells, preferentially inactivates ERK and p38 (). In macrophages, at least four MKPs are expressed: MKP-1, MKP-2, phosphatase of activated cells 1, and MKP-5/MKP-M (, ). It is possible that multiple MKPs act cooperatively to control the MAP kinase cascades. In the absence of MKP-1 protein, JNK and p38 will be eventually inactivated by other MKPs, albeit at a much slower rate.
macrophages was delayed. The finding that inactivation of ERK was not affected by the knockout of the gene () further supports the notion that MKP-1 does not play a significant role in the inactivation of ERK in our system. Recently, Zhang et al. () demonstrated that MKP-5 acts as a JNK phosphatase and plays an important role in the regulation of both innate and adaptive immune responses. Whether knockout of has an appreciable impact on the adaptive immunity remains to be addressed. Although it has not been reported whether knockout of also sensitizes mice to endotoxin shock, it is tempting to speculate that the endotoxin-induced phenotype of knockout may be less severe than that of knockout. MKP-5 has been found to only regulate the JNK pathway, whereas MKP-1 regulates both the JNK and p38 pathways. Considering that the JNK and the p38 pathways regulate both related and distinct cellular functions (, ), knockout of may perturb more cellular processes and have a more severe consequence than the deletion. Perhaps reflecting different severity, the differences in blood TNF-α levels between knockout mice and their wild-type littermates were relatively modest (less than twofold; reference 35). In contrast, the difference between the blood TNF-α levels in –deficient mice and their wild-type littermates after LPS challenge was dramatic (almost 30-fold; ).
Another intriguing finding from this study is that deficiency also leads to elevated production of IL-10 both in isolated cells and in vivo. These results indicate that IL-10 is regulated in a fashion similar to TNF-α, suggesting that the IL-10–mediated antiinflammatory mechanism is hardwired in the cells to counterbalance the actions of proinflammatory cytokines. Interestingly, it has been shown that p38 is crucial for the production of IL-10 during LPS stimulation (, ).
cells may be a reflection of the increase in p38 activity. The down-regulation of IL-12 and IFN-γ observed in –deficient cells was also an interesting and unexpected finding ( and ). Although the mechanisms mediating their down-regulation are unclear, two potential mechanisms may be involved. First, IL-10 is a potent antiinflammatory cytokine (, ). The profound increase in IL-10 secretion after LPS stimulation may down-regulate the expression of the classic Th1 cytokines IL-12 ( D) and IFN-γ through an autocrine system. Indeed, it has been reported that IL-10 inhibits IL-12 expression in dendritic cells (). Second, although less likely, JNK and/or p38 may negatively regulate the expression of IL-12 and IFN-γ. Thus, loss of MKP-1–mediated attenuation of these pathways may lead to the down-regulation of these cytokines. It is worth noting that IFN-γ can also influence expression (). Such a regulation likely has important biological significance. It has long been known that IFN-γ can boost the antimicrobial activity of macrophages and substantially enhance the secretion of TNF-α by macrophages (). We found that IFN-γ can attenuate LPS-induced expression and prolong the activation of both JNK and p38. It is plausible that the prolongation of these MAP kinase pathways contributes to the biological activities of IFN-γ.
In summary, we found that knockout prolonged the activation of p38 and JNK in LPS-stimulated macrophages. This prolonged activation of p38 and JNK was associated with substantially elevated production of TNF-α and IL-6 in macrophages. Furthermore, knockout mice demonstrated a substantial increase in mortality after LPS treatment compared with wild-type mice. LPS treatment of knockout mice resulted in renal, hepatic, and pulmonary dysfunction. Collectively, our findings demonstrate the essential role of in containing the host immune response to bacterial products. The unexpected findings that deficiency resulted in decreased IL-12 and IFN-γ but increased IL-10 production suggest that regulation of the immune response is more complex than simply down-regulation of proinflammatory cytokine production. We speculate that may represent a pharmacological target for treatment of patients with endotoxic shock, and that polymorphisms in the gene may result in greater susceptibility to endotoxic shock.
) were provided by Bristol-Myers Squibb Pharmaceutical Research Institute and regenerated into mice at The Jackson Laboratory.
and
mice. All animals received humane care and all animal-related work was performed in accordance with National Institutes of Health guidelines. The experimental protocols were approved by the Institutional Animal Care and Use Committee of the Columbus Children's Research Institute.
or
mice by peritoneal lavage. The resident peritoneal macrophages were cultured overnight in RPMI 1640 medium (Mediatech) supplemented with 5% FBS (Hyclone Laboratories) and 50 U/ml IFN-γ (Calbiochem) before being stimulated with 100 ng/ml LPS ( c O127:B8) purchased from Sigma-Aldrich. To isolate elicited peritoneal macrophages, mice were injected i.p. (2 ml/mouse) with 3% Brewer Thioglycollate Medium (BD Diagnostic) and cells were harvested 4 d later. Splenocytes were isolated, cultured in RPMI 1640 medium containing 10% FBS, and stimulated with 100 ng/ml LPS. Bone marrow–derived dendritic cells were prepared as described previously (). In brief, bone marrow cells depleted of T and B cells were cultured for 5 d in RPMI 1640 medium supplemented with 5% FBS and 10 ng/ml of recombinant murine GM-CSF (BD Biosciences). Cells were replated and matured in 1 μg/ml LPS ( O55:B55; Sigma-Aldrich) for 24 h.
Western blot analysis was performed using antibodies against MKP-1 (Santa Cruz Biotechnology, Inc.), phosphorylated JNK, p38, and ERK, as well as phosphorylated MK2 (Cell Signaling Technology). MK-2 activity was measured by immune complex kinase assays using [γ-P]ATP and recombinant mouse Hsp25 (StressGen Biotechnologies) as a substrate as described previously (). TNF-α, IL-6, IL-10, IL-12, and IFN-γ in the culture medium were determined using ELISA as described previously ().
or
mice were primed overnight with 50 U/ml IFN-γ and then stimulated with 100 ng/ml LPS ( c O127:B8) for 4 h. Total RNA was isolated using Trizol (Invitrogen), digested with RNase-free DNase I, and purified with RNeasy MinElute Cleanup kit (QIAGEN). First-strand cDNA was synthesized with 1.5 μg total RNA using Moloney murine leukemia virus reverse transcriptase (Invitrogen). qRT-PCR was performed by the comparative threshold cycle (ΔC) method and normalized to GAPDH. The primers used for GAPDH, IL-6, IL-12p35, and IL-12p40 were as described previously (). The following primers were used for TNF-α: 5′-CCCCAAAGGGATGAGAAGTT-3′ (forward) and 5′-CACTTGGTGGTTTGCTACGA-3′ (reverse).
or
mice were injected i.p. with PBS or the designated doses of LPS dissolved in LPS. Plasma or serum was assayed for cytokine levels using ELISA. Plasma BUN and ALT activity was measured using Infinity Urea and ALT kits (Thermo Electron), respectively. Lungs were first perfused with 10% formalin at constant distending pressure of 25 cm HO for 10 min, excised from the animals, and then placed in 10% formalin overnight at 4°C. 4-μm sections were prepared and stained with hematoxylin and eosin. Systolic blood pressures were determined noninvasively by tail cuff monitor (). Plasma nitrate levels were measured by chemiluminescence ().
and
cells using one-way analysis of variance (ANOVA). Plasma ALT levels were log-transformed before analysis using one-way ANOVA.
and
mice.
and
mice with or without in vivo LPS challenge were analyzed using two-way ANOVA. When ANOVA demonstrated differences, a modified test was used to identify differences.
and
mice after LPS challenge were determined by Kaplan-Meier analysis. All tests were performed using SPSS 13.01 software (SPSS Inc.). A p-value <0.05 was considered significant. |
Using primary bone marrow–derived macrophages from WT and DUSP1 mice, we observed significantly higher production of IL-6 in response to titrated amounts of LPS (). The secretion of TNF-α by LPS-stimulated macrophages was less affected by DUSP1 deficiency but higher than in WT (not depicted). These data confirm and extend the recently reported increase in TNF-α production in DUSP1 alveolar macrophages () and demonstrate differential control of various cytokines by DUSP1. In a kinetic analysis of MAPK phosphorylation, we found primarily an effect of DUSP1 deficiency on the down-regulation of p38 activation that led to markedly increased phospho-p38 levels at later time points (). In contrast, the kinetics of ERK1/2 activation were similar in WT and DUSP1 macrophages, whereas JNK showed a transiently increased phosphorylation status in the absence of DUSP1. Thus, DUSP1 is essential for the control of p38 activation in LPS-activated macrophages, which is consistent with earlier data showing selectivity of this MAPK phosphatase for p38 over ERK1/2 () and corroborates data by Zhao et al. () that demonstrate prolonged p38 activation in DUSP1 alveolar macrophages. In addition to DUSP1, LPS induces DUSP2, DUSP10, and DUSP16 in macrophages (, , ); apparently, however, these cannot fully compensate for a lack of DUSP1 in terms of p38 regulation, but may be more effective in regulating JNK and ERK1/2 activity.
Based on these in vitro data, we asked whether DUSP1 determines the response to LPS in vivo. First, we observed that after i.p. injection of LPS, DUSP1 mRNA expression was induced rapidly in the spleen (), lung, and liver (not depicted). To test the hypothesis that induction of DUSP1 expression is required to limit the inflammatory response in a negative feedback loop, we used DUSP1 mice in a high-dose LPS shock model (). At an LPS dose of 25 mg/kg body weight, all the injected DUSP1 mice died between 20 and 48 h thereafter. In contrast, >90% of WT mice survived.
Serum levels of cytokines with a known role in the pathogenesis of endotoxin shock were analyzed to investigate the mechanism underlying the high susceptibility to LPS in the absence of DUSP1 (). Already 1.5 h after injection of LPS, the levels of TNF-α and IL-6 were significantly elevated in DUSP1. At the later time points, differences were even more pronounced for IL-6, whereas TNF-α was down-regulated in the DUSP1, although still higher than in WT (). Because DUSP1 and WT mice were on a mixed background, we confirmed the differences in IL-6 and TNF-α levels 6 h after LPS injection in mice backcrossed on pure backgrounds to exclude confounding by modifier genes (, right). We also analyzed IFN-γ and IL-12p40, both known to contribute to lethal outcome of endotoxin shock (–), and found no significant effect of DUSP1 deficiency 6 h after injection (; not depicted for IFN-γ). In contrast, IL-10 mice that are also highly susceptible to LPS shock () exhibited uncontrolled release of IFN-γ, IL-12, IL-6, and TNF-α ( and reference ).
To obtain a global view of the impact DUSP1 has on LPS-induced gene expression, transcriptional profiling was performed using spleen RNA prepared 6 h after LPS challenge. In both groups of mice, LPS induced substantial changes in gene expression, with a considerable overlap in the genes induced in WT or DUSP1 mice (, Venn diagram; see Table S1, available at , for a complete list of 608 up-regulated genes). However, in DUSP1 mice, nearly threefold more genes were uniquely up-regulated compared with WT, which is also evident from the hierarchical clustering analysis, with cluster C containing 229 genes induced more strongly in the absence of DUSP1 (; see also box plots for the various clusters in Fig. S1, available at ). The microarray data corroborate most of the cytokine data obtained by ELISA from serum samples. IL-6 was up-regulated in DUSP1 mice, whereas IL-12p40 and IFN-γ showed no difference in expression. A notable exception is TNF-α, whose expression was not increased in DUSP1 mice at the mRNA level, a result that was confirmed by Northern analysis (Fig. S2) and likely due to posttranscriptional effects (e.g., on mRNA stability or translation).
We mined the microarray data with regard to the question of how DUSP1 deficiency causes excess lethality in LPS challenge. Although some downstream effectors of LPS-induced lethality may not yet be expressed at the relatively early time point analyzed in this experiment, DUSP1 mice already appeared sicker clinically. Therefore, it seems reasonable to assume a contribution to the severe inflammatory response for at least some of the genes overexpressed in the absence of DUSP1. Among the cytokines that have been implicated as contributors to the pathogenesis of lethal endotoxin shock, IFNs type I and II as well as IL-12, TNF- α, IL-1α, and IL-18 were expressed at similar levels in WT and DUSP1 spleen (Table S2). On the other hand, the chemokines CCL3, CCL4, and CXCL2 were among the LPS targets overexpressed most strongly in DUSP1 spleens, a finding that was validated at the protein level by corresponding serum levels (), and may contribute to the development of shock. For example, CCL3 (MIP-1α) promotes leukocyte recruitment to the lungs and increases lethality (), an effect that may be enhanced by the closely related CCL4 (MIP-1β) and CXCL2 (MIP-2). In contrast, CXCL10 (IP-10) was not affected by the absence of DUSP1 at the mRNA or protein level ().
The microarray analysis identified the immunoregulatory cytokine IL-10 to be induced 3.3-fold stronger in the DUSP1 spleen, which was confirmed by Northern blotting (). This difference was strikingly more pronounced in the liver, which may account for the 10-fold higher serum IL-10 levels in DUSP1 compared with WT mice 6 and 20 h after LPS challenge (). Higher production of IL-10 in response to LPS was also observed in DUSP1 macrophages stimulated in vitro (not depicted). Among the genes overexpressed in DUSP1 spleens, we observed a considerable number of transcripts previously identified as IL-10–induced genes (), including SOCS3, NFIL3, Ndr1, and Gadd45γ (Table S1), probably reflecting secondary effects of the overshooting production of IL-10 and IL-6 that both activate transcription via Stat3 signaling. However, high IL-10 levels in DUSP1 mice were not effective in down-regulating synthesis of IL-6 or the chemokines CCL3, CCL4, or CXCL2 (). Thus, DUSP1 may be required for inhibition of at least some IL-10–regulated genes.
The finding of a nonredundant role for DUSP1 in balancing the inflammatory response suggests that the various members of the DUSP family of MAPK phosphatases fulfill specific regulatory roles in innate immune cells. The recent report of increased JNK activation in innate and adaptive immune cells in the absence of DUSP10 is another example of this apparent division of labor between different MAPK phosphatases (). In the case of DUSP1, the p38 MAPK pathway likely is the major target, and DUSP1 deficiency unleashes the expression of a rather selective set of TLR-induced genes that can be hypothesized to be direct or indirect targets of p38 MAPK. However, it is also possible that in addition to the various MAPKs, DUSP1 dephosphorylates other substrates as well. Surprisingly, the lethal outcome of LPS challenge in DUSP1 is correlated with excessive production of the antiinflammatory cytokine IL-10. This counterintuitive finding may indicate that important inhibitory effects of IL-10 on inflammation depend on DUSP1 function. Similarly, in the myeloid-specific Stat3-deficient mouse, abrogated IL-10 signaling leads to overshooting, lethal cytokine production that includes IL-10 (). Given the phenotype of DUSP1 mice in the high-dose LPS model reported here, it will be interesting to investigate the contribution of this phosphatase to the host response in models of infection and polymicrobial sepsis. Finally, the DUSP family emerges as a potential target for immunomodulation; identification of small molecule inhibitors has recently been reported (, ) and holds promise for selective therapeutic intervention.
DUSP1 blastocysts generated by the R. Bravo laboratory at Bristol-Myers Squibb Pharmaceutical Research Institute () were supplied by The Jackson Laboratory and subsequently bred on a mixed 129Sv × C57BL/6 background at the Forschungszentrum Karlsruhe. All experiments were performed according to European and German statutory regulations and approved by the Regierung von Oberbayern. The genotype of the mice was established by tissue biopsies and subsequent DNA analysis by PCR using two separate reactions with the allele-specific primers 5′-CAGGTACTGTGTGTCGGTGGTGCTAATG-3′ (WT) and 5′-AAATGTGTCAGTTTCATAGCCTGAAGAACG-3′ (mutant), which were used with the common reverse primer 5′-CTATATCCTCCTGGCACAATCCTCCTAG-3′, respectively.
For control experiments, mice backcrossed for seven generations onto C3H and 129Sv were used. Mice were used for LPS challenge at an age of 5–10 wk. Experimental groups were matched for age and sex. O55:B5 LPS (no. L2880; Sigma-Aldrich) was diluted in sterile PBS and injected i.p. Bone marrow–derived macrophages were differentiated for 5–7 d in M-CSF containing media as described previously ().
Serum and cell culture supernatants were analyzed for cytokine content using Duoset antibody pairs (R&D Systems) for detection of IL-6, TNF-α, IFN-γ, CXCL10, and IL-10. IL-12p40 production was measured with an OptiEIA kit from BD Biosciences, and CXCL2, CCL3, and CCL4 were measured with Quantikine kits from R&D Systems.
Bone marrow–derived macrophages were treated as indicated and processed for analysis by Western blot as described previously (). Antibodies for phosphorylated and total p38, JNK, and ERK1/2 were from Cell Signaling, and anti–γ-tubulin antibody was from Sigma-Aldrich and used at a 1:1,000 dilution.
Total spleen RNA (5 μg) was labeled and hybridized to Affymetrix MOE430A 2.0 GeneChips according to the manufacturer's instructions. Three biological replicates per condition were analyzed. CEL files were processed for global normalization and generation of expression values using the rma algorithm in the R affy package (). Normalized CEL expression values deposited in the Gene Expression Omnibus () as series GSE3565.
The list of significantly regulated genes was achieved by applying the SAM multiclass algorithm () of the samr package for R (FDR < 1%: 1,465 probe sets). Further filtering included a minimum fold-change criterion between all four experimental conditions of ±2 (1,372 probe sets) and a max (all mean expression values) − min (all mean expression values) filter of >50 (1,215 probe sets). Of these, 608 were up-regulated. Further data preparation was performed with the Spotfire DecisionSite software (Spotfire), and hierarchical clustering was performed using the program Genesis (release 1.1.3; reference ).
Northern blot analysis was performed as described previously () using cDNA probes obtained from Deutsches Ressourcenzentrum für Genomforschung.
A list of all 608 genes that were found up-regulated by microarray analysis in WT or DUSP1 spleen 6 h after LPS injection can be found in Table S1. The expression levels of cytokines, chemokines, and ILs, as well as their receptors, in this microarray dataset are depicted in Table S2. Marked in bold are the probe sets showing significant differences in expression between WT and DUSP1 mice after LPS challenge with a p-value of <0.01. Fig. S1 shows box plots complementing the microarray data from Fig. S2 contains Northern blot validations of microarray results. Supplemental material is available at . |
To investigate whether plaque-infiltrating CD4 T cells can directly damage VSMCs, T cell lines and VSMC lines were established from inflamed plaque tissue that was collected by carotid endarterectomy. CD4 T cell lines maintained by polyclonal stimulation were incubated on autologous and heterologous VSMC monolayers. As shown in , plaque-derived CD4 T cells effectively induced VSMC apoptosis. At an effector–target ratio of 20:1, 60–70% of autologous VSMCs were killed within 4 h of coculture. Even at a low effector–target ratio of 2.5:1, one third of the VSMCs underwent apoptosis. VSMC apoptosis was absolutely dependent on T cells; VSMC death in control cultures without T cells was minimal (). Apoptosis rates at low effector–target ratios increased with prolonged incubation for 24–48 h (not depicted). Cytolytic activity was observed with heterologous VSMCs that were not matched for MHC polymorphisms with the responding T cell population; cytolytic activity on autologous VSMCs derived from the T cell donor was only slightly higher (). All VSMC lines expressed MHC class I and HLA-DR molecules, as determined by confocal microscopy (unpublished data). Anti–HLA-DR antibodies blocked CD4 T cell–induced VSMC apoptosis in a dose-dependent manner; antibodies specific for MHC class I molecules had no effect (). These findings suggested that the VSMC killing involved interaction between MHC class II molecules and the T cell antigen receptor.
To examine whether the ability to induce VSMC apoptosis was restricted to plaque-infiltrating T cells, CD4 T cells were isolated from the peripheral blood of patients with ACS and added to VSMC monolayers. CD4 T cells induced apoptosis in 40–50% of VSMCs (). Peripheral blood CD4 T cells were also able to kill both autologous and allogeneic VSMCs.
To assess whether CD4 T cells from patients with ACS had a higher ability to kill VSMCs, a cohort of 50 patients with either myocardial infarction (72% male, 60.9 ± 12.1 yr old) or unstable angina (72% male, 66.9 ± 12.4 yr old) was compared with 33 age-matched controls (58% male, 57.3 ± 16.8 yr old). The VSMCs used for these experiments were derived from the coronary artery of a single donor (coronary artery smooth muscle cell [COR-SMC]). 20–25% of the VSMCs underwent apoptosis when exposed to PBMC-derived CD4 T cells isolated from controls at an effector–target ratio of 10:1. The rate of VSMC apoptosis was substantially increased with T cells collected from peripheral blood of ACS patients (P = 0.0002) (). A similar increase in VSMC apoptotic activity was not observed with CD4 T cells from patients with stable coronary artery disease (unpublished data). These data demonstrated that VSMCs were susceptible to T cell–mediated death, and that CD4 T cells from patients with ACS had higher VSMC cytotoxicity.
T cells can kill target cells by different pathways; e.g., by releasing lytic enzymes or by triggering of the death pathway (, , ). Clustering of cell surface death receptors initiates death pathway–induced apoptosis via FADD recruitment and eventual caspase-8 activation. As shown in , caspase-8/granzyme B blocking in VSMCs resulted in a marked inhibition of VSMC apoptosis (P = 0.006 in ACS patients). Inhibition of apoptosis induced by CD4 T cells from normal donors was more variable. To lend further support to the assertion that VSMCs were killed via death pathway triggering, CD4 T cells were incubated on VSMCs transfected with dominant-negative (DN)–FADD–GFP or control GFP plasmid (). Blocking the function of FADD led to a marked decrease in the apoptosis rate (P = 0.02). These experiments established that CD4 T cells induced VSMC apoptosis by triggering the death pathway.
The expression of death receptors on VSMCs was examined by RT-PCR and FACS analysis. RT-PCR yielded positive results for three death receptors, DR4, DR5, and Fas. The transcripts for DR5 and Fas were abundant, whereas the signal for DR4 was only faint (). To assess surface expression of these receptors, VSMCs were stained with antibody and analyzed by FACS. DR5 and Fas were present at high membrane density, whereas DR4 was not detected ().
To examine whether VSMCs are sensitive to death receptor–mediated killing, VSMCs were incubated with rh-TRAIL. Apoptotic cells were identified according to morphology and DAPI nuclear staining. Cells that were spread out on the surface and displaying normal nuclei were considered viable, whereas cells that had acquired a round shape and fragmented nuclei were considered apoptotic. Rh-TRAIL was highly effective in inducing apoptosis. After 12 h of exposure, >50% of cells had typical nuclear disintegration and lost adherence to the surface ().
To examine whether DR5 expression in VSMC lines appropriately reflected tissue resident cell phenotypes in the atherosclerotic plaque, serial frozen sections from carotid arteries were stained with anti-CD68 mAb, to identify tissue-infiltrating macrophages; with anti–α-smooth muscle cell actin, to identify VSMCs; and with anti-DR5 antibody (). Atherosclerotic plaques were identified by oil-red staining. The plaque-free carotid artery wall served as the control. DR5 was expressed on VSMCs in the plaque cap region, but not on VSMCs in normal carotid artery walls.
To determine whether TRAIL or FasL were involved in CD4 T cell–mediated VSMC injury, anti-TRAIL and anti-FasL antibodies were added to the T cell–VSMC cultures. As shown in , TRAIL-reactive antibody inhibited VSMC apoptosis. In the presence of control antibody, ∼45% of VSMCs were killed at an effector–target ratio of 5:1. To reach similar levels of apoptosis in the presence of anti-TRAIL mAb, four times as many T cells (effector–target ratio of 20:1) were necessary, suggesting that TRAIL was responsible for >75% of the cytotoxic activity. Antibodies blocking Fas–FasL interaction had no effect on the level of VSMC apoptosis. Inhibition by anti-TRAIL antibody was only seen for the CD4 T cells deriving from ACS patients. Apoptosis levels in control T cell cultures were unaffected by TRAIL blockade. Similar results were obtained in experiments using anti-DR5 antibodies. The rate of VSMC apoptosis induced by CD4 T cells from ACS patients was reduced by ∼50% when the TRAIL–DR5 interaction was inhibited by anti-DR5 Ab ().
To examine whether TRAIL-expressing CD4 T cells were increased in frequency in patients with ACS, PBMC from patients and age-matched controls were analyzed by FACS for the membrane expression of TRAIL. TRAIL expression on CD4 T cells required cell activation; spontaneous expression was absent in ACS patients and controls. This result was consistent with the observation that cytotoxic activity was dependent on the recognition of HLA-DR molecules (). After TCR triggering, TRAIL expression was significantly increased (P = 0.002) in ACS patients compared with controls ().
To test the relevance of CD4 T cell–mediated killing of VSMCs in vivo, we implanted carotid plaque tissue into immunodeficient mice and adoptively transferred CD4 T cells into the chimeras. Mice engrafted with carotid artery specimens lacking atherosclerotic plaques served as controls. T cell lines were established from a plaque of the same artery that was implanted into the mice. 7 d after implantation, full engraftment was achieved, and 6 × 10 CD4 T cells were injected intravenously into the chimeras; 24 h later, the human tissue grafts were explanted. In addition to the adoptive transfer experiments in which the T cells and atheroma tissue derived from the same donor, CD4 T cell lines were also injected into chimeras engrafted with heterologous tissue. Infiltration of adoptively transferred T cells into the graft tissue was assessed by quantifying TCR-specific transcripts. TRAIL expression in the tissue, before and after T cell transfer, was measured by real-time PCR. As shown in , T cell–derived TCR mRNA was low in control carotid wall grafts that lacked plaque formation. In contrast, TCR sequences in the tissue increased more than fourfold, from a median of 1,800 copies to a median of 7,508 copies when CD4 T cells were injected into chimeras implanted with actively inflamed carotid artery plaque. In parallel, the tissue expression of TRAIL in the plaque tissue increased 2.5-fold, whereas levels remained unchanged in control tissue. Results were similar, irrespective of whether adoptively transferred CD4 T cells and plaque tissues derived from the same or different patients. These data suggested that the inflamed plaque tissue facilitated CD4 T cell recruitment and retention and that the infiltration of CD4 T cells into the tissue site was associated with enhanced expression of TRAIL.
To determine whether adoptive transfer of CD4 T cells induced VSMC apoptosis, tissue sections of the explanted grafts were analyzed for the frequency of TUNEL cells. As expected, the frequency of TUNEL cells was higher in the plaques than in noninflamed control wall sections (). Areas with apoptotic cells were patchy and cosegregated with areas containing inflammatory infiltrates. After CD4 T cell transfer, distribution patterns of apoptotic cells continued to be discontinuous. Immunohistochemical stains showed that VSMCs accounted for the majority of apoptotic cells in the lesion (). The percentage of TUNEL cells in these patchy areas with inflammatory infiltrates increased from 20% to >60% in the adoptively transferred mice (), demonstrating that infusion of CD4 T cells resulted in massive VSMC injury.
To examine whether the TRAIL pathway was involved in CD4 T cell–mediated apoptosis in vivo, TRAIL-blocking antibodies were included in the adoptive transfer experiments. 6 × 10 CD4 T cells were coadministered with anti-TRAIL mAb into chimeras implanted with autologous carotid tissue. As controls, mice implanted with the same tissue were either treated with 6 × 10 CD4 T cells combined with isotype-matched control antibody or injected with control antibody only. Results shown in demonstrated that 10–20% of the cells in the inflamed plaque tissues recovered from mice treated with only control IgG antibody were apoptotic. Adoptive transfer of CD4 T cells in combination with control IgG antibody resulted in massive apoptosis in the lesion. After injecting CD4 T cells, >70% of VSMCs in the lesion stained TUNEL (). Apoptosis rates returned to baseline when CD4 T cells were coadministered with TRAIL-blocking antibody. In contrast, TCR mRNA in the tissues was equal, irrespective of whether anti-TRAIL or control Ab was administered, indicating that anti-TRAIL Ab did not influence T cell infiltration and survival.
To use a different measure of VSMC death, we analyzed the leakage of lactate dehydrogenase (LDH) from the arterial tissue. As shown in , the activity of LDH in the chimeras' blood remained stable for >24 h in mice treated with control antibody. Infusion of 6 × 10 CD4 T cells resulted in a twofold increase in LDH activity, supporting the interpretation that the T cells had a direct role in damaging VSMCs. This T cell–mediated enhancement of LDH activity could be blocked by coadministration of anti-TRAIL antibody. These data demonstrated that CD4 T cells have a role in inducing VSMC apoptosis in vivo and that they use TRAIL to trigger VSMC death.
The loss of VSMCs in the fibrous cap and accelerated degradation of the collagen matrix have been postulated as critical mechanisms in transforming stable plaque into rupture-prone lesions (, ). We have demonstrated that CD4 T cells kill cultured VSMCs by expressing the TNF-like ligand TRAIL and by triggering death receptors on the surface of VSMCs. Patients with ACS have higher frequencies of apoptosis-inducing CD4 T cells in circulation, suggesting an abnormality in T cell differentiation that predisposes these patients to T cell–mediated VSMC damage, plaque rupture, and ischemic manifestations of atherosclerosis.
Recent evidence suggests that the thin-cap fibroatheroma is the major precursor lesion to ACS (). Plaque-infiltrating macrophages have been implicated in plaque destabilization through the production of metalloproteinases and, possibly, other tissue-injurious mediators (–, ). Macrophages cultured with VSMCs for 7 d kill VSMCs in a Fas–Fas ligand and TNF-dependent manner (, ). Also, unstable lesions contain a high frequency of activated T cells, which could directly promote the death of plaque-residing cells (, , ). Apoptotic cells can be identified in the unstable plaque in vivo and include cells of the inflammatory infiltrate but also VSMCs (, , ); the absolute number of apoptotic VSMCs in these studies is likely an underestimate given that apoptotic cells are rapidly cleared by macrophages in vivo. In the present study, CD4 T cells equipped to induce apoptosis of cultured VSMCs not only accumulated in the plaque, but also circulated in the blood of patients with ACS. In vivo studies with human carotid plaque–SCID chimeras confirmed the ability of CD4 T cells to infiltrate into the lesion and mediate VSMC killing. The human–mouse chimera model likely is a more sensitive system to assess apoptosis than the natural host because the chimeric mice do not have circulating human monocytes, and murine monocytes hardly enter the human tissue. The inhibitory effect of anti-TRAIL, both in vitro and in vivo, established that this TNF-like molecule was the major inducer of VSMC apoptosis.
T cells have been implicated in the pathogenesis of atherosclerosis and probably contribute to multiple aspects of the disease process (). They consistently accumulate in complex lesions; and T cell deficiency reduces lesion size and cellular infiltration (, ). Also, multiple cytokines intimately involved in T cell function (such as IL-15, IL-12 and IL-18), are expressed in the lesions and directly regulate plaque inflammation and lesion size (–). We focused on T cells as effector cells and not on their role as key regulators of in situ immunity. CD4 T cells, the dominant lymphocyte population in human atheromas, are usually not considered to lyse neighboring cells. However, CD4 T cells isolated from lesions that cause fatal myocardial infarction have a unique phenotype and functional profile (, ). They lack expression of CD28 and, instead, have acquired a new set of immunoregulatory receptors. Stimulation of killer immunoglobulin-like receptors, selectively expressed on CD4 T cells in ACS, facilitates cytolysis and has been shown to lyse endothelial cells (, ).
TCR triggering induces TRAIL expression on T cells, but cytokines, such as IFNs, can modulate this response (). TRAIL is stored in cytoplasmic vesicles and can be rapidly brought to the cell surface. Spontaneous expression of surface TRAIL on CD4 T cells from patients with ACS is not increased, suggesting that cross-linking of the TCR is necessary to initiate the cascade leading to T cell–mediated cytotoxicity. This is further supported by the finding that the activity of CD4 T cells is dependent on the recognition of MHC class II molecules. Different antigens have been implicated in driving the response of T cells in the atherosclerotic lesion, including oxidized low density lipoprotein (, ) and chlamydial heat shock proteins (). CD4CD28 T cells proliferate when activated with HSP60 (). The possibility remains that a spectrum of antigens, not just a single antigen, is responsible for the stimulation of tissue-infiltrating T cells. T cells from patients with ACS are prematurely aged and have characteristics of senescent cells (). Senescent T cells are less stringent in antigen-specific responses to enter activation. Cumulative gene expression of regulatory molecules can partially or sometimes even completely substitute for TCR-mediated signals, rendering T cells responsive to antigen-nonspecific cues in their microenvironment (, ). This may also explain why CD4 T cell–induced apoptosis, although dependent on the recognition of MHC class II molecules, was not restricted by MHC polymorphisms.
TRAIL is best known for its tumoricidal activity. Recombinant soluble forms of TRAIL have been found to induce cell death predominantly in transformed cells while not affecting normal tissues. However, TRAIL receptors are widely expressed on many cell types, and the physiologic functions of this TNF family member appear to be rather broad (, ). Besides playing a suspected role in T cell and NK cell tumor immunosurveillance, TRAIL has been implicated in the thymic clonal deletion of autoreactive T cells and may be involved in peripheral homeostatic T cell compartment control (, ). Data presented here reinforce previous papers asserting that TRAIL causes tissue injury in untransformed cells. In cholestatic hepatocyte injury, bile acids facilitate TRAIL receptor oligomerization and activation (). Human endothelial cells were found to express DR4 and DR5, and ∼30% were susceptible to TRAIL-induced apoptosis. When TRAIL was injected into human skin xenografts, it caused focal injury to tissue-residing endothelial cells (). Other cell types susceptible to TRAIL-mediated apoptosis include developing erythrocytes (), prostate cells, and T lymphocytes. Controversial results have been reported regarding TRAIL's effect on normal T lymphocytes, ranging from apoptosis resistance () and induced cell cycle arrest without cell death () to TRAIL-stimulated apoptosis (). Also, studies on TRAIL's critical role in establishing and maintaining immunologic tolerance by regulating negative selection of thymocytes have not been confirmed by other investigators (). Insensitivity to TRAIL's apoptotic effects has been recently associated with the high expression of FLICE inhibitory protein, which provides a mechanism through which TRAIL-producing T cells and NK cells protect themselves while using TRAIL to kill DR5-expressing target cells ().
VSMCs, both in the atherosclerotic plaque as well as in vitro, strongly expressed DR5, whereas DR4 was essentially absent. Within hours, recombinant TRAIL induced apoptosis in the majority of cultured VSMCs. Such cultured DR5-expressing VSMCs were highly sensitive to TRAIL-expressing CD4 T cells. In vivo transfer of CD4 T cells resulted in an increase of apoptotic VSMCs in the plaque, which could be blocked by anti-TRAIL Ab. CD4 T cells able to be activated by VSMC and to express TRAIL on the cell surface are abundant in ACS patients. A previous study has shown that these T cell abnormalities are not short-lived, likely precede the acute event, and certainly persist over time (). Also, the presence of such T cells is not unique for ACS but is also found in other chronic diseases (). The sole presence of such T cells is therefore not sufficient to precipitate disease manifestations. Rather, rate-limiting steps may be the access of these T cells to the vascular wall compartment and expression of DR5 on VSMCs. The mechanisms of DR5 expression are unknown, but the susceptibility of VSMCs to TRAIL-mediated apoptosis was such that it should raise concerns when considering using TRAIL as an anti-tumor agent in humans. T cells may gain access to the atherosclerotic plaque in patients with ACS via one of two routes: they may enter from the macrolumen; or, more likely, they may enter from the network of microcapillaries that develops in the unstable plaque. The mouse chimera model only partially resembles this situation, because T cells must enter exclusively from the microvasculature. Regardless, recruitment will be driven by chemokines produced in the tissue, and these chemokines are likely responsible for the preferential recruitment of T cells into the plaque compared with normal artery tissue as shown in . Once in the tissue, CD4 T cells are being activated and exert effector functions through TRAIL ().
Implicating CD4 T cells and their ability to up-regulate TRAIL surface expression in plaque destabilization could have direct consequences for the therapeutic approach to patients with vulnerable lesions. Inhibiting TRAIL or TRAIL-producing CD4 T cells and restoring resistance to DR5 triggering in VSMCs could have therapeutic potential for the acute management of patients with unstable angina and myocardial infarction.
25 consecutive patients admitted with ST elevation myocardial infarction and 25 patients treated for unstable angina were enrolled in this study. Blood samples were drawn at the time of admission and processed immediately. 33 age- and sex-matched control subjects had no history of autoimmune disease and no risk factors for cardiovascular disease. Control patients with stable coronary artery disease were matched for common risk factors such as hyperlipidemia, smoking, diabetes mellitus, and hypertension. Carotid artery specimens were collected from patients undergoing endarterectomy procedures. All specimens were examined for the presence and absence of inflammatory infiltrates and plaque. End fragments of the specimens lacking plaque formation and mononuclear cell infiltrate were used as controls. The protocols were approved by the Mayo Clinic Institutional Review Board and the Emory University Institutional Review Board, and appropriate consent was obtained.
CD4 T cells were isolated from PBMC by negative selection (RosetteSep; StemCell Technologies, Inc.). Purity was 95–98%. Importantly, purified cells did not include any NK cells. Human COR-SMCs were grown on collagen-coated tissue culture plates in SmGM-2 smooth muscle medium (Cambrex). Human carotid artery smooth muscle cell lines (CA-SMC) were established from carotid artery plaque using previously described methods (). T cell lines were isolated from carotid endarterectomy samples by culturing tissue fragments with 50 U/ml recombinant human (rh) IL-2 (CHIRON). After 7 d, tissue-derived T cells were stimulated with 10/ml irradiated PBMCs, 1.5 × 10/ml irradiated EBV-transformed B cells, 30 ng/ml anti-CD3 mAb (Ortho-Clinical Diagnostics), and 50 U/ml rh-IL-2. CD4 T cells were purified by fluorescence-activated cell sorting. Phenotypically, tissue-derived CD4 T cells were different from peripheral CD4 T cells in that they all expressed memory markers and the frequency of CD28 loss was increased (unpublished data).
Apoptosis assays were performed using nuclear fragmentation and condensation of VSMC stained with 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich) as read-out. Pilot studies showed an excellent correlation of the apoptosis rates by DAPI staining and by Annexin V staining (Fig. S1, available at ). Also, costaining of Annexin-positive VSMCs with PI clearly indicated that cell damage was irreversible. To control for subjective interpretation, the reader was blinded as to whether the T cells derived from a patient or control individual. COR-SMCs and CA-SMCs that reached confluence were seeded into collagen-coated 96-well plates at a cell density of 10 SMCs/well and incubated with 1 μg/ml DAPI in RPMI supplemented with 1% FBS for 1 h at 37°C. Cultures were washed and purified. CD4 T cells or resting CD4 T cell lines were added to the VSMC monolayer for 4 h at 37°C. VSMCs were examined with fluorescence microscopy, and apoptosis rates were determined. Parallel assays were performed by staining for 4 h T cell–VSMC cocultures with Annexin V. In selected experiments, COR-SMCs were preincubated for 1 h with 1.25 μg/ml to 10 μg/ml anti-MHC class-ABC mAb (W6/32) or anti–HLA-DR mAb (L243) before coculture with T cells. In other experiments, COR-SMCs were transfected with a DN-FADD-GFP–containing plasmid (0.05 μg/well; pcDNA3-GFP-ΔFADD, a gift from G.J. Gores, Mayo Clinic, Rochester, MN) or a GFP-expressing control plasmid pE-GFP-N1 using Opti-MEM I (100 μl/well; Invitrogen-GIBCO) containing LipofectAMINE (0.4 μg/well; Invitrogen-GIBCO) and LipofectAMINE Plus reagent (0.6 μl/well; Invitrogen) (). Cells were incubated for 4 h at 37°C in a 5% CO, 95% air incubator. 24 h later, transfection efficiency was determined by analyzing GFP expression using fluorescence microscopy. In other experiments, COR-SMCs were treated with a caspase-8/granzyme B inhibitor (25 μg/ml; caspase-8 inhibitor I; Calbiochem) for 1 h before apoptosis analysis.
Tissue expression of TCR and TRAIL mRNA was analyzed by real-time PCR. The following primers were used in these experiments: TCR α-chain, 5′-CCTTCAACAACAGCATTATTCCAG -3′ and 5′-CGAGGGAGCACAGGCTGTCTTA -3′; and TRAIL, 5′-ACCAACGAGCTGAAGCAGAT -3′ and 5′-CAAGTGCAAGTTGCTCAGGA -3′. Transcripts for each gene were quantified using PCR instruments (Mx 3000 and Mx 4000; Stratagene). 1 μl cDNA was mixed with a total volume of 50 μl SYBR green master mix (5 μl 10 × PCR buffer, 2.5 mM MgCl, 0.2 mM each dNTPs, 0.025% BSA, 0.2 μl 5 U/μl Plt. Taq, 1:20,000 SYBR green, and 0.1 μM each primer). PCR amplification protocol involved 40 cycles of denaturation at 95°C for 30 s, primer annealing at 55°C, and primer extension at 72°C for 60 s. cDNA concentrations were adjusted to 2 × 10 copies of β-actin. All PCR reactions were performed in triplicate.
TRAIL expression on CD4 T cells was determined by two-color staining with anti-CD4-PerCP mAb and anti-TRAIL-PE mAb (both Becton Dickinson). PBMC were stimulated with 250 ng/ml anti-CD3 mAb (Ortho Biotech) or soluble IgG mAb for 2h in the presence of FcRII P815 cells. Again, liver tumor cells were used as positive controls.
The TUNEL technique was used to quantify apoptotic cells in the tissue. The plaque tissues were cut into 5-μm sections and analyzed by the In Situ Cell Death Detection Kit, AP (Roche). The percentage of TUNEL cells was calculated by counting TUNEL cells per total nuclei. Serial sections were stained with anti-CD3 (1:200; DAKO) and anti–α-SMC actin mAb (1A4, 1:200; DAKO) followed by secondary biotinylated goat anti–mouse Ig antibody (1:400), ABC-peroxidase, and DAB.
6–8-wk-old NOD.CB17-Prkdc/J mice (NOD-SCID) or NOD-129S7 (B6)-Rag1 () J mice (Jackson ImmunoResearch Laboratories) were engrafted with human carotid artery plaque tissue placed into a subcutaneous pocket on the midback. Engraftment of small tissue fragments (50–100 mm) is rapid, and the graft is vascularized by day 7 with or without minimal murine inflammatory infiltrate within the implanted human tissue. A previous study has shown that the microvasculature within the graft is of human origin connecting to murine granulation tissue encapsulating the graft (). All animal procedures were approved by the Animal Care and Use Committee. Noninflamed sections of carotid artery walls lacking plaque formation were used as controls. On day 7 after implantation, the mice were injected intravenously with 6 × 10 CD4 T cells or PBS. 24 h after the cell transfer, the carotid artery grafts were explanted and shock frozen in liquid nitrogen for mRNA analysis or embedded in OCT compound (Sakura Fine-Tek) for immunohistochemical analysis.
In selected adoptive transfer experiments, the carotid artery chimera mice were injected intravenously with control IgG mAb (300 μg/mouse), CD4 T cells (6 × 10 cells/mouse) combined with control IgG mAb (300 μg/mouse), or CD4 T cells combined with anti-human TRAIL mAb (300 μg/mouse; Becton Dickinson) on day 7 after tissue implantation. After 24 h, the carotid artery grafts were explanted, and tissue apoptosis rates were analyzed by TUNEL staining.
Data were analyzed by Student's test or Mann-Whitney U test, when appropriate. Results are shown as mean ± SD, when parametric, and as box plots with medians and percentiles, when nonparametric testing was used.
Fig. S1 shows apoptosis rates assessed by nuclear fragmentation of DAPI-stained VSMCs and by Annexin V staining that were highly correlated (r = 0.95). CD4 T cells were cocultured with VSMCs, and induction of apoptosis was assessed by fluorescence microscopy. Intermediate stages of apoptosis were determined by staining with Alexa Fluor-labeled Annexin V. Nuclear fragmentation was detected by DAPI staining. Membrane damage was measured by permeability to propidium iodide. The online supplemental material is available at . |
The number of Sμ-Sα fragments was determined from 10 PCR reactions run in parallel, using DNA (30 ng per reaction) from the same individual. This method can be used to estimate the number of clones that have switched to IgA (). Because of the polyclonal nature of the rearrangements at the Sμ-Sα region, differently sized switch fragments are amplified and visualized. The number of clones that have switched from IgM to IgA in ATRD patients appears to be similar to controls. A typical run is shown in , where at least 11 distinct Sμ-Sα fragments were amplified from the control and at least 10 fragments were amplified from the ATRD1 patient. The experiments were repeated three times for each patient. The average numbers of Sμ-Sα fragments generated from ATRD1 and ATRD2 were 11.3 and 8.7, respectively, with an average of 10.0, which is similar to controls (–, average:11.8). Thus, the proportion of cells that have switched to IgA in the peripheral blood in ATRD patients, at the time of sampling, appears to be normal.
We subsequently cloned and sequenced 40 S fragments (39 Sμ-Sα and 1 Sμ-Sγ-Sα) from ATRD1 and ATRD2, generated in the aforementioned PCR reactions. All the S fragment sequences were unique and therefore represent independent CSR events (Fig. S1, available at ). The S junctions from controls ( = 154), used for comparison, have been published previously (, ).
We subsequently analyzed the microhomology usage at the Sμ-Sα junctions in the ATRD patients. There was a significant increase in the extent of donor–acceptor homology at the Sμ-Sα junctions from ATRD patients; the average length of overlap being 3.0 ± 3.8 bp in ATRD and 1.8 ± 3.2 bp in control B cells (P < 0.05, Student's test). The junctions from ATRD patients showed a significantly increased usage of microhomology (≥4 bp) (, 26 + 10 + 3 = 39 vs. 20% in controls, χ test, P < 0.05) as compared with controls. Interestingly, this was mainly because of homologies encompassing 4–6 bp (separated in , 26 vs. 10% in controls, χ test, P < 0.01) and, to a much lesser degree, 7–9 bp. However, there was no increased usage of longer microhomologies (i.e., ≥10 bp) at the junctions from the patients as compared with controls ( and ). When the data was plotted as an accumulative curve, it is evident that the proportion of S junctions with longer homologies was almost the same as in controls ().
In ATRD patients, the number of S junctions with mutations was significantly smaller than in controls (18 vs. 39%, χ test, P < 0.05), whereas insertions were observed at a similar rate (23 vs. 25%) (). The frequencies of mutations or insertions around the junctions were 13.7 and 22.9/1,000 bp for ATRD and controls, respectively. The number of mutations and insertions available for analysis was too low in the patient group ( = 13) to allow a formal analysis of the pattern of nucleotide alterations.
The mutation pattern in the Sμ region (upstream of the breakpoints, referred to as SHM-like mutations) resembles that in the V regions and is clearly different from that at, or close to, the Sμ-Sα breakpoints (, ), suggesting that the former mutations occur at an early step during CSR (). In ATRD patients, SHM-like mutations in the Sμ region were observed at a significantly lower frequency than in controls (3.1/1,000 bp vs. 6.5/1,000 bp; χ test, P < 0.001). In the few mutations observed ( = 25), the general pattern of base substitution seems to be slightly different from controls, with a stronger, but statistically nonsignificant, preference for G/C sites (72 vs. 62% in controls), a reduced number of mutations at A nucleotides (12 vs. 21% in controls) and an increased frequency of mutations at G nucleotides (40 vs. 28%).
The mutations in the Sμ region from ATRD patients were less often associated with the previously described consensus hotspot for SHM, the RGYW/WRCY (R = A or G, Y = C or T, W = A or T) motif (), as compared with controls (56 and 70% for ATRD and controls, respectively), but not to a significant degree.
We have previously designed a series of PCRs that specifically amplify Sμ-Sγ1, Sμ-Sγ2, and Sμ-Sγ3 fragments from DNA from peripheral blood cells. The number of clones that have switched to the different IgG subclasses in ATRD patients appears to be similar to controls (not depicted). We subsequently cloned and sequenced 49 Sμ-Sγ fragments from the ATRD patients. 48 of these fragments were unique in their junctional sequences and most were a result of direct switching to IgG1 ( = 17), IgG2 ( = 8), or IgG3 ( = 23). One S fragment was a result of sequential switching to IgG2 through Sα1.
As in the Sμ-Sα junctions, there was a significant increase in donor–acceptor homology at the Sμ-Sγ junctions from ATRD patients (the average length of overlap being 1.8 ± 2.0 bp vs. 1.2 ± 1.2 bp in controls; P < 0.05, Student's test). There was a trend toward an increased usage of microhomologies (≥4 bp) in the ATRD patients, but not to a statistically significant degree ( and , 11 + 4 + 0 = 15 vs. 5% in controls). Furthermore, the number of Sμ-Sγ junctions that showed mutations (±15 bp) was lower as compared with in controls (28 vs. 41%), whereas insertions were observed at a similar rate (13 vs. 12%).
RNA was prepared from PBLs of all three patients. In the first set of experiments, a previously described PCR strategy () was used to amplify the VH3-Cγ transcripts from ATRD3. In total, 14 distinct VH-Cγ clones were generated and the majority of these clones carried at least 10 mutations. The average frequency of mutations in the VH genes derived from this patient was 9.2%, which is higher than that found previously in normal controls ( = 7, 1.5–6.6%, average 4.2%) (). One notable finding was that the A and T residues on the coding strand were targeted at a similar frequency in the patient (21.9 and 20.8%, respectively; 365 mutations) and thus did not display the DNA strand polarity observed in controls (29.6 and 13.6%, respectively; 619 mutations).
To verify these findings, we performed a second set of experiments, where all three patients were included. VH3-23-Cγ transcripts were selectively amplified to exclude the possibility that the difference in A/T base targeting observed were because of a differential usage of VH3 genes between patients and controls. In total, 33 and 40 distinct VH3-23-Cγ clones were generated from B cells of ATRD patients and controls, respectively (). Most of the VH3-23 clones from the patients were mutated (2–49 bp substitutions/clone) and only 6% of the VH3-23 genes exhibited unmutated sequences (0–1 bp substitutions/clone) (). The frequency of mutations in the VH3-23 genes derived from ATRD patients varied from 6.8 to 11.9%, which is similar to, or slightly higher than, the age-matched control donors (). The ratio of replacement vs. silent mutations (R/S) in CDRs and framework regions was similar in the patient and control groups, arguing against any major abnormalities in the antibody selection process in ATRD patients.
The distribution of mutations observed in ATRD patients (754 mutations in total, 22.8/VH) was largely similar to that found in normal controls (749 mutations in total, 18.7/VH), with major hotspots of mutation (AGC and GCT at codons Ser, Ala, and Ser), conforming to the previously described hotspot consensus RGYW/WRYC motif () (). The mutations showed a preference for transitions in both patients and controls (53 and 56%, respectively). However, there were significantly more transitions occurring at T residues in ATRD patients as compared with those in controls (11.1 vs. 7.7%, χ test, P < 0.05). Furthermore, as observed in the VH3 sequences from the first set of experiments, the A and T residues on the coding strand were targeted at a similar frequency (after correcting for base composition, 25.6 and 23.0%, respectively) and thus, the DNA strand polarity observed in controls was lost (29.9 and 16.8%, respectively) ().
To search for a potential difference in the targeting of specific sequences between the patients and controls, we first analyzed the frequency of mutations in the RGYW/WRYC motifs. A significantly higher than expected number of mutations (18%) was observed within these motifs, both in patients and controls (36 and 38%, respectively, χ test, P < 0.0001). However, there seems to be no major difference in targeting of the individual bases in these motifs between the patients and controls. We subsequently analyzed the targeting of the WA/TW motifs and the frequency of mutations within these motifs was almost identical between patients and controls (262/754 vs. 261/749, 34%). TA was the most targeted motif in both patients and controls. However, there was a significant difference in the targeting of the A or T nucleotide in this motif. In patients, the T and A are equally targeted (70 vs. 69 mutations, respectively) whereas in controls, A is clearly more targeted (51 T vs. 99 A mutations) (χ test, P < 0.01, ). This difference was not observed in the reverse complementary motif, AT, nor the other two WA/TW motifs, AA and TT (unpublished data). When the flanking sequences of each TA motif are taken into account, the difference in targeting of the A/T nucleotides within the motif was most striking in four short DNA sequences (, footnote a). In patients, the T is more targeted than A (34 vs. 18, respectively) whereas in controls, A is the preferred target (19 T vs. 38 A, ). Interestingly, the common feature of these four sequences is that the TA motif (bold) partially overlaps with the RGYW motif (underlined) (i.e., two are
and the remaining two are
T). This suggests that the loss of the A preference on the coding strand in ATRD patients might be a result of differences in the mechanism generating A/T mutation within these sequences.
We subsequently analyzed the frequency of mutations in each of the 64 possible trinucleotide combinations (Table S1, available at ). The most targeted trinucleotides, in both controls and patients, were AGC, GTA, GCT, TAG, and GGT. These are all related to the known SHM hotspot motifs (RGYW/WRYC and WA/TW) (, ).
and
T sequences, supporting the aforementioned observations on TA motif targeting. In addition, significant differences were found in targeting of the trinucleotide TTT ( in ATRD) and the T bases in two additional trinucleotides, AC ( in ATRD) and CC ( in ATRD) (χ test, P < 0.05 in all cases). These increases in mutations of T nucleotides may also contribute to the observed reduction of the A/T strand bias, although in this instance the triplet sequences are not associated with the previously recognized SHM hotspots.
Diversity in the CDR3 region in clones derived from the ATRD patients was also analyzed. The CDR3 contains contributions from the VH, D, and JH gene segments and nucleotides added by TDT. All clones were in-frame rearrangements. The average length of the CDR3 was 15.7 ± 2.9 aa in the ATRD clones, which is similar to those from controls (14.9 ± 4.2 aa). The average length of the N1 regions (VH-D junctions) was significantly longer in ATRD patients as compared with controls (Student's test, P < 0.05; Table S2, available at ). There was however no significant difference in the length of the N2 regions (D-JH junctions) and the frequency of P nucleotides observed between patients and controls (Table S2). Thus, the V(D)J coding joints seem to be largely normal in ATRD patients.
We have previously analyzed the in vivo patterns of CSR junctions and mutations in the VH regions in A-T patients (ATM defective) using a similar experimental design, (, ) and these results were compared with those derived from the ATRD patients ().
The number of Sμ-Sα fragments and serum levels of immunoglobulins are highly variable in A-T patients (). About half of the patients have no detectable serum IgA, whereas the remainder of patients show subnormal to normal levels of IgA. However, they all show aberrant S junctions, with increased usage of microhomology and reduced number of mutations/insertions at the Sμ-Sα junctions (). Thus, the defect in CSR in A-T patients is probably not only quantitative, but also qualitative. In ATRD patients, the number of Sμ-Sα fragments and serum levels of immunoglobulins are normal. However, ATRD2 showed low levels of antipneumococcal antibodies after immunization, which might be due to a less efficient switching to IgG2 (unpublished data). Furthermore, the Sμ-Sα junctions showed a similar trend in microhomology usage to those from A-T patients, although not as dramatic ( and and ). However, the almost complete absence of 0 bp homologies (4.5% in A-T vs. 41.0% in ATRD, χ test, P < 0.001) and the large proportion of Sμ-Sα fragments exhibiting ≥10 bp homology (30% in A-T vs. 3% in ATRD, χ test, P < 0.01) were not found in ATRD patients. Another notable difference is that although the frequency of junctional mutations (±15 bp) was reduced in both patient groups, the proportion of junctions with insertions was reduced in patients with A-T (2 vs. 25% in controls, χ test, P < 0.01), but not in those with ATRD (23%). Mutations close to the S junctions and 1–3 bp insertions thus seem to represent features of different repair mechanisms. These data indicate that both ATM and ATR are involved in end joining during CSR, but appear to play both similar and disparate roles in the process.
The SHM-like mutations in the Sμ regions are significantly reduced in both patient groups. The general pattern of base substitutions is altered in A-T patients, with more mutations occurring at A/T sites (56 vs. 37% in controls) and shows a strong preference for transitions (86 vs. 59%) (). In ATRD patients, there are more mutations occurring at G/C (72%) sites, although the rate of transitions is still normal (64%). It is also worth noting that the preferred targets for SHM-like mutations within RGYW/WRCY motifs (commonly AGCT motifs in these data) are also different between the two patient groups. Almost all the mutations in ATRD patients target the second and third nucleotides (93%), whereas close to half of the mutations from A-T patients target the last T in these motifs (42%). In controls, 83% of the mutations occur at the second and third nucleotides and 5% at the last T in the RGYW/WRCY motifs. Therefore, ATR, like ATM, seems to be involved in the generation of mutations in the Sμ region, although probably via a different mechanism.
A-T patients show a normal frequency and a largely normal pattern of SHM in the VH regions (), whereas in ATRD patients, the mutation pattern was clearly altered. As for the targeting of individual trinucleotides, A-T patients showed a similar pattern as controls (unpublished data), whereas in ATRD patients the targeting of A or T bases in certain trinucleotides was significantly altered.
In this study, we have, for the first time, shown a role for ATR in CSR and SHM. The study also revealed interesting functional differences between ATM and ATR-mediated signaling; the ATM-dependent pathway is mainly used in the end joining process in CSR, whereas the ATR-dependent pathway appears to be involved in both CSR and SHM.
The functional roles of ATM and ATR have been extensively studied in the general DNA damage response. Both are structurally and functionally related with overlapping substrates and have therefore been suggested to operate within the same pathway (, , ). However, while ATM exclusively responds to DSBs induced by ionizing radiation and some radiomimetic agents, ATR is also activated by single-stranded DNA (ssDNA) generated during DNA replication or by agents such as UV irradiation that produce bulky lesions (, ). There also seem to be kinetic differences between the two kinases when responding to different forms of DNA damage. Both ATM and ATR phosphorylate p53 on serine-15 in response to ionizing radiation, but ATM initiates the process whereas ATR dominates the phosphorylation process at later stages (). However, in response to a methylating agent, temozolomide, ATM is activated later than ATR (). Furthermore, the function of ATR is dependent on an accessory protein, ATRIP (ATR-interacting protein), which is phosphorylated by ATR ().
The different pattern of CSR junctions and SHM in the VH regions, when ATM or ATR is defective, could potentially be explained by the known differences between the two kinases. In CSR, a DSB intermediate appears to be part of the reaction (), and ATM may thus play a major role in the initial stage of the repair process. However, by responding to the ssDNA resulting from processed DSBs, ATR will reinforce the ATM response, resulting in a potentiation of the reaction. In SHM, DSBs seem not to be prominent intermediates, instead, SSBs or single strand nicks appear to be essential (–). ATM, which only responds to DSBs, is thus dispensable for the SHM process. Alternately, ATR, when complexed with ATRIP, is recruited to ssDNA by RPA (), a factor that targets AID to SHM motifs (). When ATR activity is absent, or impaired (as in our patients, who carry hypomorphic mutations), the SHM pattern is perturbed.
The different patterns of Sμ-Sα junctions in A-T and ATRD patients may help to further dissect the four end joining pathways we have previously proposed (, ). The first pathway entails a “blunt end” direct joining mechanism, where no sequence homology is needed and the two DNA ends are either joined precisely or where 1–3 bp (mostly 1 bp) insertions are introduced. The second pathway involves joining of partially complementary “staggered” DNA ends by a mechanism involving alignment of residual complementarities (1–3 bp) in 3′ or 5′ overhangs, filling in of any remaining gaps and ligation. This process is imprecise, and mutations are frequently introduced at, or close to, the switch junctions. The third pathway has precise joining of complementary DNA ends with a short sequence homology (1–3 bp), probably through simple religation, with no mutations or insertions being introduced at, or close to, the junctions. The fourth pathway is an alternative joining of noncomplementary ends, by resecting the DNA ends until homology suitable for end joining is revealed where at least 4 bp of microhomology is observed at the S junction. In B cells from healthy individuals, the first pathway is dominant (44% of Sμ-Sα events) and the other three are used with similar frequencies (∼20%). The first pathway, “blunt end” direct joining is markedly impaired in A-T patients, but normal in ATRD patients, suggesting that this process is strictly dependent on a rapid, efficient response by ATM. The second (“staggered end” joining involving misalignment and filling in) pathway is less often used in both A-T and ATRD patients (9 and 3%, respectively, as compared with 16% in controls), suggesting that both ATM and ATR are required for processing of partially complementary ends. The third pathway, involving simple religation, is not affected. However, usage of the fourth pathway, involving ≥4 bp microhomology, is increased in both patient groups (61 and 38%, respectively, as compared with 20% in controls). Interestingly, in contrast with A-T patients, the CSR junctions from ATRD patients only show an increased usage of microhomology up to 9 bp, suggesting that there may be yet another pathway or subpathway, requiring longer microhomologies (≥10 bp), that may be dependent on ATR.
Although the aforementioned end joining pathways in CSR are still speculative, it could provide a platform when comparing data from patients with genetic defects that affect specific DNA damage response pathways. In patients lacking DNA ligase IV, a critical component of the NHEJ machinery, the pattern of Sμ-Sα junctions is similar to that of A-T patients (). The first and second pathways are almost not used at all, the third pathway is not affected (20%), and the fourth pathway is dominant (73%), suggesting that the “blunt end” and “staggered end” joining with short, noncomplementary ends are strictly dependent on the classical NHEJ machinery. The third pathway, which is not affected by ATM, ATR, or DNA ligase IV, is impaired in NBS (Nbs1 defective, 4%) and, to a lesser degree, in ATLD patients (Mre11 defective, 11%) (), suggesting that simple religation involving short homologous ends in CSR is probably, as described in the yeast system (), dependent on the Mre11–Rad50–Nbs1 complex.
However, the balance between these putative different pathways is dependent not only on the factors available but also on the degree of homology between the S regions. The microhomology based end joining is much more prominent at Sμ-Sα as compared with Sμ-Sγ junctions in all patient groups, probably because of the higher degree of homology between Sμ and Sα as compared with Sμ and Sγ regions (). The likelihood of obtaining a 7-, 10-, or 15-bp microhomology between the Sμ-Sα regions is 8, 270, and >1,000-fold higher than in the Sμ-Sγ regions. Furthermore, the mutation pattern at, or close to, the Sμ-Sγ junctions is also different from the Sμ-Sα junctions in normal controls (, ). Moreover, in A-T and ATRD patients, the Sμ-Sγ junctions tend to use more microhomologies, whereas in Lig4D patients, the Sμ-Sγ junctions mainly show an increased frequency of 1-bp insertions (). Thus, the Sμ-Sα and Sμ-Sγ junctions are resolved differently in controls and patients with various defects in their DNA repair systems. In mice, the Sα regions also show a much higher degree of homology with Sμ, than does Sγ, and the microhomology-based pathway would be a more attractive alternative for Sμ/Sα recombination when the normal repair pathways is impaired. However, in the various mouse knockout models described to date, Sμ-Sα junctions have not been analyzed in as much as detail as Sμ-Sγ1 and Sμ-Sγ3 junctions (, , –, ). An analysis of Sμ-Sα junctions from mouse B cells deficient for H2AX and 53BP1, where no equivalent human disease model is available, would thus facilitate our understanding of the regulation of ATM–ATR-dependent pathways in CSR.
The loss of strand polarity in targeting A/T pairs in the VH regions from ATRD patients is an interesting finding. The mechanism underlying the strand polarity at A/T pairs (i.e., with mutations at A on the coding strand being nearly twice as frequent as mutations at T) is not well understood. One possibility is that the A/T bias is a consequence of an initial preferential targeting of dCs by AID in the nontranscribed strand (). Alternatively, it may reflect the strand preference of transcription-linked repair, where an abasic site created by excising a U residue from the transcribed DNA strand of U-A pairs is treated differently (repaired or mutated) from an abasic site located on the nontranscribed strand (). Because ATR and AID can both associate with RPA, this raises several possible explanations; ATR could be acting as early as at the initial AID targeting stage by phosphorylating RPA () or by regulating its intranuclear translocation (). Alternately, RPA recruits ATR–ATRIP complex to sites of DNA damage () so ATR involvement could be postinitiation. Two other PIKKs kinases, ATM and DNA-PKcs, have also been implicated in the DNA damage-induced phosphorylation of RPA (for review see reference 52). However, as the SHM patterns are largely normal in cells deficient in ATM or DNA-PKcs (, ), their kinase activities are probably not required in this process. An alternative role for ATR could be at a later stage, by interacting with or recruiting members of the mismatch repair pathway, such as MSH2 (, ) and MSH6 or polymerase (pol) η, all of which have been implicated in generating mutations at A/T sites (, –). Pol η in particular is thought to contribute to strand bias of A versus T nucleotides by generating more mutations from A than T on the nontranscribed strand (). However, cells deficient in various mismatch repair proteins and pol η show reduced rates of mutation at both A and T nucleotides (, ), whereas in ATRD cells, the mutation load on A and T nucleotides is normal but an increased ratio of T/A targeting is observed. Loss of strand bias has also previously been noted in a mutating B cell line, Ramos (). Interestingly, the gene is not expressed in this cell line, whereas , (unpublished data) and η () transcripts are readily detected. However, the pattern of mutations observed in Ramos cells is strongly biased to G/C (82%), similar to those in MSH2, MSH6, or pol η–deficient cells, suggesting that in addition to ATR, deficiency of other DNA repair factors may account for the mutation pattern observed in this cells line. Together, ATR is unlikely to be an “A/T mutator,” but rather, recruits “A/T mutators,” such as pol η, preferentially to the transcribed strand at specific sequences.
or
T sequences.
Another possibility that needs to be excluded is that the altered mutation pattern in ATRD cells is the result of a bias introduced by studying only the expressed VH genes. To address this question, an analysis of the noncoding regions of the heavy chain locus such as the JH4 intron sequence () was attempted. We were able to obtain a small number of purified CD27 B cells from two patients, ATRD1 and ATRD2, and amplified the JH4 intron sequence downstream of the rearranged VH3-23 gene as described previously (). In controls ( = 5), an average frequency of 16/1,000 bp of mutations was observed, which is similar to that published previously (). However, very few mutations were identified in the patients even after screening a large number of clones ( = 66), precluding the possibility to compare the mutation pattern. Nevertheless, although WA motifs are common in the 320 bp of the JH4 intron sequence, there are only three TA motifs and none of these is associated with the RGYW motif. Thus, one would not expect to reproduce the specific pattern observed in the VH regions in ATRD patients.
The Sμ region is another noncoding region that has been used to analyze nonselected, SHM-like mutations (, ). In the limited number of mutations we observed in the Sμ region, away from the breakpoints, we did observe a reduced number of mutations at A nucleotides (T/A ratio 1:1). However, the SHM-like mutations observed in the Sμ region are probably introduced at an early stage during CSR, in a process that may share some of the factors with SHM but which is still mechanistically different (). Thus, an altered mutation pattern in the Sμ region may not necessarily be the result of changes in the SHM machinery.
The Seckel syndrome is a clinically and genetically heterogeneous disorder where at least four susceptibility loci have been suggested (–). To date however, mutation in is the only genetic defect identified. Of the 70 patients described worldwide, only 5 patients carry mutations in the gene. However, a recent study has shown that there are remarkably overlapping phenotypes between a panel of Seckel syndrome cell lines (with no defect in ) and an ATRD-Seckel cell line (), suggesting that the disease is indeed caused by defects in the ATR-signaling pathway. It would therefore be of considerable interest to study the CSR and SHM patterns in additional patients with the Seckel syndrome, where the gene mutated may be one of the other factors involved in the ATR-dependent pathway, including , , , , and .
In conclusion, V(D)J recombination, SHM and CSR may share some of the molecular mechanisms involved but are differentially regulated. ATR is probably dispensable for V(D)J recombination, but may play some role in the end joining machinery in CSR and in SHM.
The study included three patients from one family with Seckel syndrome with mutations in the gene. The clinical details of the patients (ATRD1, ATRD2, and ATRD3 referred to as V3, V6 and V4 in reference 66) have been described previously (, ). The mutation in the gene in these patients is a homozygous replacement within exon 9 (2101 A>G) that alters splicing, resulting in markedly reduced levels of normal transcripts and protein (). The serum levels of immunoglobulin classes are normal in the studied patients (). Lymphocyte count was available from one patient, ATRD2, and the number of B cells was slightly above the upper normal range, whereas the number of T cells was at the lower normal range. The genetic characterization and immunoglobulin levels in the A-T patients discussed in this study have all been described previously (, ). The institution review board at the Karolinska Institute approved the study.
Genomic DNA was purified from peripheral blood cells from the ATRD patients. The amplification of Sμ-Sα fragments from in vivo switched cells was performed as described previously (, ). In brief, two pairs of Sμ- and Sα-specific primers were used in a nested PCR assay. The number of Sμ-Sα fragments was determined from 10 reactions run in parallel using DNA (30 ng per reaction) from each individual and represents a random amplification of in vivo switched clones. The number of IgA positive cells in 30 ng of genomic DNA prepared from normal peripheral blood cells was estimated to be 30–60 (assuming that 40% peripheral blood cells are lymphocytes, 10–20% of the lymphocytes are B cells and 5% of the B cells are IgA). One or two Sμ-Sα fragments were normally amplified in one lane, when 30 ng of DNA was added in one reaction. This sensitivity, 15–30 copies, is in the same order of sensitivity as we have previously estimated using a human IgA1–producing cell line (). The same cell line was also used to assess the fidelity of the nested PCR reaction and the PCR error rate was 0.9/1,000 nucleotides ().
The Sμ-Sγ fragments were amplified as described previously () with addition of Sγ1-specific and Sγ2-specific primers ().
The PCR-amplified S fragments were gel purified, cloned, and sequenced as described previously (). The breakpoints were determined by aligning the switch fragment sequences with the Sμ (X54713)/Sα1 (L19121)/Sα2 (AF030305) or Sμ/Sγ1 (U39737)/Sγ2 (U39934)/Sγ3 (U39935)/Sγ4 (Y12547-52) sequences. Analysis of microhomology usage at the junctions and mutations ±15 bp around the junction and upstream Sμ region were performed as described previously (, ).
Total RNA was extracted from PBL using RNeasy RNA purification kits (QIAGEN) and first-strand cDNA synthesis was performed with a CγA primer (5′-GTCCTTGACCAGGCAGCCCAG-3′) using a cDNA synthesis kit (GE Healthcare). The primers used for amplification of VH3-Cγ and VH3-23-Cγ transcripts were VH3-consensus (5′-aaGGTGCAGCTGGTGGAGTC-3′) or VH3-23 (5′-GGCTGAGCTGGCTTTTTCTTGTGG-3′) and CγB (5′-caAAGACCGATGGGCCCTTGGTGG-3′). The oligonucleotides contained a restriction site, (underlined, a XbaI site in the VH3-consensus or VH3-23 primer and a SalI site in the CγB primer) for directional cloning of the PCR products. Amplification was performed in 35 cycles, each cycle consisting of 94°C for 50 s, 62°C for 1 min, and 72°C for 1 min. A high fidelity Vent DNA polymerase (New England BioLabs) was used in all PCR amplifications of the VH region transcripts.
The PCR products were purified and cloned into the Bluescript II KS (+) vector (Stratagene) and transformed into JM 109 competent cells. The resulting clones were screened by PCR amplification (VH3-consensus/VH3-23 and CγB) and positive clones were sequenced by an automated fluorescent sequencer in the MWG Co. using a Bigdye terminator cycle sequencing kit (Perkin Elmer). Sequence analysis was performed using the IMGT/V-QUEST () () to align the VH-CγB sequences to their closest germline VH, D, and JH segment counterparts. The immunoglobulin V(D)J junctional sequences were analyzed by the IMGT/JunctionAnalysis tool, available at .
When analyzing the trinucleotides targeting, mutated VH3-23 sequences were aligned beneath the germline VH3-23 region gene and a raw test file of the alignment created. This file was imported into a Microsoft Excel spreadsheet and computations of the number of each type of nucleotide substitution and the composition of the flanking sequences around these substitutions were performed using macros in Excel (Visual Basic). Computations of percentage differences and χ analysis were also performed using Excel.
Table S1 shows the number of mutations at each base of all trinucleotides. Table S2 shows the characteristics of the CDR3 regions in cells from ATRD patients and controls. Fig. S1 shows the sμ-sα junctions from ATRD patients. Online supplemental material is available at . |
We initially assessed whether loss of specific HDAC enzymes induced glucocorticoid insensitivity on IL-1β–induced GM-CSF production, which is NF-κB mediated, as previously reported (, ) and from our data showing concentration-dependent inhibition by IκB kinase 2 inhibitor (AS602868; IC = 3.6 × 10 M). Class I HDAC enzymes (HDAC1, -2, -3, and -8) () were selectively knocked down by RNA interference in A549 cells (). When GM-CSF production was normalized to viable cell numbers, HDAC2 (1,057.1 ± 209.3 ng/10 cells), HDAC3 (910.5 ± 230.5 ng/10 cells), and HDAC8 (919.5 ± 64.8 ng/10 cells) knockdown (KD), but not HDAC1 KD, significantly enhanced IL-1β–induced GM-CSF production (vs. 335.0 ± 55.5 ng/10 cells in control; ). These initial results show that HDAC2, -3, and -8 regulate NF-κB–mediated GM-CSF induction. We previously showed that trichostatin A (TSA), a nonselective HDAC inhibitor, enhanced GM-CSF production in A549 cells ().
Furthermore, HDAC2 KD shifted the concentration-dependent inhibition of IL-1β–stimulated GM-CSF release by Dex to the right, indicating a reduction in glucocorticoid action (). A similar result was seen after treatment with TSA (). A similar action was also seen with suppression of IL-1β–induced IL-8 production (). In contrast, HDAC1, -3, and -8 KD had no effect on Dex action on either GM-CSF or IL-8 production (). Using individual short interference RNA (siRNA), or combinations of up to four duplexes against HDAC2, we were able to obtain different levels of HDAC2 reduction in cells (). This HDAC2 study showed that there was a significant negative correlation between HDAC2 expression after RNAi and Dex EC with respect to IL-1β–induced GM-CSF production (r = −0.771; P = 0.0020; ). This graded reduction in glucocorticoid responsiveness with reducing HDAC2 expression suggests that HDAC2 plays an important role in the antiinflammatory actions of glucocorticoids. Intriguingly, KD of HDAC2 does not affect the maximal response to Dex seen at very high concentrations (1 μM; ) and suggests that, at these supraphysiological concentrations, alternative mechanisms of action may be occurring ().
HDAC2 KD did not affect GR expression or nuclear translocation (). Loss of HDAC2 did not reduce GR-GRE binding (), GRE-dependent luciferase activity (), or GRE-mediated SLPI gene expression (). In addition, GR-GRE binding after Dex treatment was sustained longer in HDAC2 KD cells than in nontreated cells or scrambled oligonucleotide (Sc)–transfected cells ().
In addition, HDAC2 KD did not affect NF-κB expression, nuclear translocation (), or NF-κB–DNA binding (). We confirmed previous data showing that Dex increased GR and HDAC2 association with the p65–NF-κB complex () (). However, GR was not recruited to the p65–NF-κB complex after HDAC2 KD (). Chromatin immunoprecipitation (ChIP) analysis showed that Dex failed to inhibit histone 4 acetylation at the p65–NF-κB binding site in the GM-CSF promoter region after HDAC2 KD (). Thus, HDAC2 KD did not reduce the ability of GR to induce GR-sensitive gene expression but had a preferential effect on the suppression of NF-κB–mediated inflammatory gene expression.
Previous studies have shown that both estrogen receptor α and androgen receptor are acetylated within their hinge/ligand binding domains and that this can modulate hormone-induced gene induction (, ). This nuclear receptor acetylation site is conserved among members of related nuclear receptors and, based on these findings (), there is a potential acetylation site at aa 492–495 (KTKK) within the DNA binding domain/hinge region of GR. Indeed, we show that GR is acetylated after Dex binding (), and the acetylation levels were decreased when K494 and K495 on GR were mutated to alanine (A), asparagine (N), or glutamine (Q) (). These mutants did not further induce SLPI expression by Dex even though overexpression of native GR enhanced SLPI expression by Dex (). There was no difference in ability of native or mutant GR to bind to p65–NF-κB (). However, GR-mediated suppression of IL-1β–stimulated GM-CSF release was not affected by TSA with the K494 and K495 mutants () even though the repression was attenuated by TSA in native GR overexpression cells. This suggests that GR acetylation negatively regulates Dex-induced repression of NF-κB–dependent gene expression. In addition, we found that p65–NF-κB–associated GR was deacetylated (). Because GR is unable to associate with the p65–NF-κB complex in HDAC2 KD cells, this deacetylation must be important for GR-mediated transrepression (). Importantly, HDAC2 KD does not inhibit GR-GRE binding or SLPI transactivation (), indicating that the acetylated GR is still able to activate glucocorticoid-responsive genes, which may be involved in some of the deleterious side effects that limit the clinical use of these powerful drugs, as well as some minor antiinflammatory effects via induction of antiinflammatory molecules such as SLPI and MKP-1.
To determine whether acetylated GR was a substrate for HDAC2, acetylated GR was immunoprecipitated in the presence of Dex and incubated with immunopurified HDAC1, -2, -3, and -8 (adjusted to show the same degree of HDAC activity) in a nonisotopic in vitro assay. Acetylated GR was clearly a substrate for HDAC2, although it is also partially deacetylated by HDAC3 (). Thus, acetylated GR is a substrate of HDAC2, and deacetylation of GR by HDAC2 may be prerequisite for GR association with the p65–NF-κB–activated complex and subsequent suppression of inflammatory gene expression. This mechanism provides a molecular explanation for the ability of GR to distinguish between recruitment of coactivator and corepressor proteins, as previously demonstrated for GRIP (), and the subsequent ability to transactivate or repress gene transcription.
We previously reported that HDAC2 expression and activity is decreased in smokers () and patients with COPD (), who are known to be insensitive to the antiinflammatory effects of glucocorticoids (). In addition, there is a negative correlation between the repressive effect of Dex on cytokine production and total HDAC activity in alveolar macrophages from smokers and nonsmokers (). To determine whether HDAC2 is important for Dex actions in primary cells from patients with glucocorticoid-insensitive disease, we obtained alveolar macrophages from healthy nonsmokers, healthy smokers, and patients with COPD. There was no substantial difference in macrophage GR expression between normal and COPD samples (unpublished data). Total HDAC activity, immunoprecipitated HDAC2 activity (), and HDAC2 protein expression (, NT) were markedly decreased in cells from patients with COPD. The acetylation level in immunoprecipitated nuclear GR after Dex treatment (10 M) was increased in alveolar macrophages obtained from patients with COPD (). In these cells obtained from COPD patients, Dex did not inhibit NF-κB–dependent () LPS-induced GM-CSF production in vitro (, top). Overexpression of HDAC2 protein by 6.5 ± 1.1-fold in primary macrophages from COPD patients () restored Dex efficacy toward suppressing LPS-induced GM-CSF release to levels seen in cells from healthy control subjects (, middle). In contrast, HDAC2 overexpression by 3.3 ± 0.86-fold and 5.2 ± 1.1-fold in nonsmokers and smokers, respectively, did not further increase Dex efficacy toward LPS-induced GM-CSF production in cells from nonsmokers/smokers, presumably as these cells are already sensitive to Dex actions (). HDAC1 overexpression did not affect glucocorticoid sensitivity (, bottom). Furthermore, 40% KD of HDAC 2 in sputum macrophages from healthy nonsmokers by RNAi caused a reduction in the inhibitory effect of Dex (10 M) from 62 to 36% ().
The human lung adenocarcinoma type II cell line (A549 cells) was purchased from American Type Culture Collection. Healthy nonsmokers, current healthy smokers, and patients with stage 2–3 COPD were recruited. The study was approved by the Brompton Harefield and National Heart and Lung Institute Ethics Committees, and all subjects gave signed informed consent. Bronchoscopy, bronchoalveolar lavage, isolation of bronchoalveolar lavage, and sputum macrophages were performed as previously described (, ).
100-nM siRNA sequences were transfected using Gene Silencer (GTS Inc.) for A549 cells and jetSI (Polyplus-transfection SA) for sputum macrophages. siRNAs were prepared with a GeneSilencer kit (Ambion) or purchased from QIAGEN or Dharmacon. Duplexes used were HDAC1 (SMART pool [Dharmacon] and nt 89), HDAC2 (nt 96, 177, 642, and 813), HDAC3 (SMART pool [Dharmacon] and nt 411), and HDAC8 (nt 155 and 373). Nonspecific control duplex (Sc, 47% guanine-cytosine content) was also purchased from Dharmacon.
Plasmids (pcDNA3.1) containing the HDAC1 and HDAC2 genes were donated by S. Georas (Johns Hopkins University, Baltimore, MD). Alveolar macrophages (3 × 10 cells/well) were transfected using jetPEI-Man (Polyplus-transfection SA).
Total RNA extraction and reverse transcription were performed using an RNeasy kit (QIAGEN) and an Omniscript RT kit (QIAGEN). Gene transcript level of SLPI and GAPDH were quantified by real-time PCR using a QuantiTect SYBR Green PCR kit (QIAGEN) on a Rotor-Gene 3000 (Corbett Research).
IL-8 and GM-CSF concentrations were determined by sandwich ELISA (R&D Systems) and normalized to cell number as determined by MTT assay.
Biotinated oligonucleotide duplexes containing two GREs () were incubated in streptavidin-conjugated 96-well plates (Thermo Labsystems). GR binding to oligonucleotides were colormetrically detected in nuclear extracts () by an enzyme-immunosorbent method using an anti-GR antibody (Santa Cruz Biotechnology, Inc.).
Nuclear extracts and immunoprecipitated samples were prepared and evaluated by conventional SDS-PAGE/Western blotting (). Determination of HDAC protein expression after RNAi was performed by dot blot analysis.
GR was immunoprecipitated in whole cell extracts with anti-GR antibody–conjugated A/G agarose beads. GR was also precipitated with avidin-agarose beads conjugated with biotinylated oligonucleotides containing 2 × GRE (5′-aagattcaggtcatg-acctgaggaga-3′) or coimmunoprecipitated with p65–NF-κB antibody–conjugated A/G agarose beads in nuclear extracts in the presence of 10 M Dex and 1 ng/ml IL-1β.
ChIP with panacetylated histone 4 antibody (ChIP assay kit; Upstate Biotechnology) was performed at the GM-CSF promoter region as previously described () using the aforementioned real-time PCR system.
Site-directed mutagenesis was performed using Gene Tailor Site-Directed Mutagenesis system (Invitrogen).
HDAC activity was measured with HDAC Fluorescent Activity Assay kit (BIOMOL Research Laboratories, Inc.).
NF-κB activation was determined with NF-κB TransAM kit (Active Motif).
Plasmids containing two GRE-luciferases were donated by J. Bloom (University of Arizona, Tucson, AZ). Plasmids were transfected to A549 cells with pSV–β-galactosidase (Promega) using Lipofectamine 2000 (Invitrogen) as described previously ().
Results are expressed as means ± SEM. Analysis of variance was performed by Kruskal-Wallis analysis and, when significant comparisons were made, by Mann-Whitney U test using the analysis package SPSS 10.0 (SPSS Inc.). P < 0.05 was considered statistically significant. |
Unlike other thymocytes that actively express genes encoding receptors for IL-7 and other prosurvival cytokines, preselection DP thymocytes extinguish expression of genes encoding receptors for IL-7 and presumably for other prosurvival cytokines as well. Indeed, Northern blot analysis revealed that preselection DP thymocytes are devoid of IL-7Rα mRNA, although they do express mRNA encoding the common cytokine receptor γ, which is the other component of the IL-7R complex (). Consequently, to assess cytokine signal transduction in DP thymocytes, we used mice expressing an IL-7Rα transgene whose DP thymocytes express transgenically encoded IL-7Rα proteins on their surface () (). In IL-7Rα transgenic (IL-7RαTg) mice, preselection DP thymocytes express the same high-surface IL-7Rα level as other thymocytes () and peripheral T cells (not depicted). Because DP thymocytes constitutively express endogenously encoded γ (), DP thymocytes from IL-7RαTg mice expressed the two IL-7R components (IL-7Rα and γ) that are required to initiate IL-7 signal transduction. Nevertheless, analysis of IL-7RαTg thymocytes provided no indication that preselection IL-7R DP thymocytes had been signaled by IL-7 in vivo, as their differentiation into single-positive (SP) T cells was identical to that in WT B6 mice () (). Indeed, the only observable impact of transgenic IL-7Rα expression was a reduction in overall thymocyte numbers () that has been ascribed to arrested development of double-negative thymocytes before the DP stage of development (). One explanation for absent IL-7 signaling in IL-7RαTg DP thymocytes is the relative deficiency of IL-7–producing cells in the thymic cortex where DP thymocytes reside (). However, we also considered that cytokine signal transduction might be impaired in IL-7RαTg DP thymocytes despite their high expression of surface IL-7Rs.
Because IL-7 is a prosurvival cytokine, we first assessed the ability of exogenous IL-7 to promote survival of preselection DP thymocytes. We placed DP thymocytes from IL-7RαTg and B6 mice in single cell suspension cultures in the presence of exogenous IL-7 and assessed survival over time (). Exogenous IL-7 failed to promote the in vitro survival of either DP population, including IL-7R DP thymocytes from IL-7RαTg mice (, left), although exogenous IL-7 did dramatically enhance the in vitro survival of lymph node T cells (LNT cells) from IL-7RαTg mice (, right). To understand IL-7's failure to enhance the survival of IL-7R DP thymocytes, we examined IL-7's ability to up-regulate expression of the anti-apoptotic protein Bcl-2. Thymocytes and T cells from IL-7RαTg and B6 mice were cultured for 18 h with and without IL-7 and then stained for intracellular Bcl-2 protein (, right). IL-7 did not up-regulate Bcl-2 protein expression in normal B6 DP thymocytes, as these cells expressed few if any IL-7Rs (, right). However, IL-7 did up-regulate Bcl-2 protein expression in IL-7R DP thymocytes from IL-7RαTg mice, but the levels of Bcl-2 protein expression in DP thymocytes were only ∼10% of those induced in either CD8SP thymocytes or CD8 LNT cells (, right). Thus, IL-7R DP thymocytes could be signaled by IL-7 to up-regulate Bcl-2 protein expression, but their response to IL-7 was considerably blunted relative to CD8SP thymocytes and CD8 LNT cells and was insufficient to promote cell survival.
To examine proximal IL-7R signaling events, we assessed IL-7–induced Stat5 phosphorylation by briefly exposing cells to exogenous IL-7 for 20 min and then staining for intracellular phospho-Stat5 (p-Stat5; , left). Because they are deficient in surface IL-7Rs, IL-7 did not induce significant p-Stat5 in B6 DP thymocytes (, left). Importantly, however, IL-7 did induce p-Stat5 in IL-7R DP thymocytes from IL-7RαTg mice, but the amount of p-Stat5 induced in IL-7R DP thymocytes was only 10–25% of that induced by IL-7 in CD8SP thymocytes and CD8 LNT cells (, left). Because the diminished amount of p-Stat5 induced in DP thymocytes might have been caused by less Stat5 protein in DP thymocytes than in mature T cells, we examined the amount of Stat5 protein present in DP and LNT cells purified from IL-7RαTg mice by protein immunoblotting (). In fact, protein immunoblotting revealed that the amounts of Stat5 protein in DP and LNT cells were essentially identical even though the p-Stat5 induced by IL-7 in DP thymocytes was <25% of that induced in LNT cells (). Thus, IL-7 is substantially less efficient in inducing Stat5 phosphorylation in IL-7R DP thymocytes than in CD8SP thymocytes and LNT cells.
Having determined that IL-7 induced less Stat5 phosphorylation in IL-7R DP thymocytes than other cell types, we considered the possibility that p-Stat5 in DP thymocytes might be further impaired in translocating from cytosol to nucleus or that p-Stat5 in DP thymocytes might be inhibited in some other way from binding to Stat5 target DNA sequences. Consequently, we assessed nuclear extracts from IL-7–stimulated DP and LNT cells for nuclear proteins able to bind Stat5 target DNA sequences. We did this by performing electrophoretic mobility shift assays (EMSAs) with a labeled Stat5/6 consensus oligonucleotide (). Nuclear extracts from unstimulated DP and LNT cells failed to bind to the labeled oligonucleotide (, lanes 1 and 2). However, nuclear extracts from IL-7–stimulated DP and LNT cells both bound to the labeled oligonucleotide, indicating that IL-7 had induced Stat5 nuclear translocation in both cell types (, lanes 3 and 7). The protein–DNA complexes formed were specifically dependent on Stat5 target DNA sequences, as protein binding to the labeled oligonucleotide could be inhibited by competitor oligonucleotides with intact, but not mutated, Stat5 target binding sites (, lanes 3–5 and 7–9). Moreover, the protein–DNA complexes contained Stat5 proteins as they were supershifted by anti-Stat5 antibody (, lanes 6 and 10). And, finally, it was evident from their relative band intensities that nuclear extracts from IL-7–stimulated DP thymocytes bound quantitatively less labeled oligonucleotide than nuclear extracts from IL-7–stimulated LNT cells (, compare lane 3 with 7), commensurate with quantitatively less p-Stat5 induced by IL-7 stimulation of DP versus LNT cells. These data reveal that p-Stat5 induced in IL-7–signaled DP thymocytes does translocate to the nucleus and bind to Stat5 target DNA sequences. Collectively, these data demonstrate that proximal IL-7R signal transduction is impaired in IL-7R DP thymocytes as revealed by inefficient Stat5 phosphorylation.
Because SOCS-1 is known to inhibit phosphorylation of Stat proteins in cytokine-stimulated T cells (–) and SOCS-1 is constitutively expressed in preselection DP thymocytes (), inefficient Stat-5 phosphorylation in IL-7R DP thymocytes was likely caused by SOCS-1. Consistent with previous observations (), quantitative real-time RT-PCR revealed that DP thymocytes contained 10× the number of SOCS-1 transcripts as unstimulated LNT cells (). To determine whether constitutive expression of SOCS-1 was responsible for impaired IL-7 signaling in DP thymocytes, we bred the IL-7RαTg into SOCS-1 mice (which were necessarily also IFN-γ to survive SOCS-1 deficiency) (, ), generating three strains of mice that differed in their number of SOCS-1 alleles: IL-7RαTgSOCS-1, IL-7RαTgSOCS-1, and IL-7RαTgSOCS-1 (all of which were selected to be IFN-γ, as well). DP thymocytes from these mice contained graded levels of SOCS-1 transcripts, with DP thymocytes from SOCS-1 mice containing twice the number of SOCS-1 transcripts as DP thymocytes from SOCS-1 mice (). Thymocyte profiles of these mice are shown (). We stimulated thymocytes from these three mouse strains with either medium or IL-7 for 1 h and stained the cells for intracellular p-Stat5 (, left). Indeed, the amount of p-Stat5 induced by IL-7 in DP thymocytes increased in a dose-dependent fashion as SOCS-1 expression decreased, with the highest p-Stat5 levels in DP thymocytes from SOCS-1 mice (, left). Thus, SOCS-1 expression clearly interferes with p-Stat5 induction in IL-7R DP thymocytes. Even so, complete removal of SOCS-1 did not completely restore IL-7 signaling in IL-7R DP thymocytes, as p-Stat5 levels in cells from SOCS-1 mice were still quantitatively lower in DP than CD8SP thymocytes (note different scales for DP and CD8SP thymocytes; , left). It might be noted that, unlike SOCS-1's inhibitory effect in DP thymocytes, p-Stat5 and Bcl-2 levels in CD8SP thymocytes were unaffected by the presence or absence of SOCS-1 (, left), which was consistent with reduced constitutive SOCS-1 expression in postselection T cells relative to preselection DP thymocytes (, ).
We also assessed the effect of SOCS-1 on up-regulation of a downstream target of IL-7 signaling, Bcl-2. We cultured thymocytes from IL-7RαTg SOCS-1, IL-7RαTg SOCS-1, and IL-7RαTg SOCS-1 mice for 18 h with either medium or IL-7 and stained the cells for intracellular Bcl-2 protein (, right). In parallel with p-Stat5, Bcl-2 was up-regulated in DP thymocytes in a dose-dependent fashion as SOCS-1 expression decreased, with maximal Bcl-2 expression in DP thymocytes from SOCS-1 mice (, right). As with p-Stat5, Bcl-2 up-regulation in DP thymocytes from SOCS-1 mice that were devoid of SOCS-1 was still lower in DP than CD8SP thymocytes (, right).
IL-7 signaling not only activates Stat5 but also activates PI3 kinase, which is responsible for IL-7–induced increases in cell size (). Because SOCS-1 directly binds to JAKs and inhibits all further downstream signaling events, we reasoned that constitutive SOCS-1 expression would also prevent IL-7 from signaling an increase in the cell size of IL-7RαTg DP thymocytes. Indeed, overnight stimulation with IL-7 failed to signal IL-7R DP thymocytes from IL-7RαTg SOCS-1 mice to increase cell size (). In contrast, IL-7R DP thymocytes with decreased SOCS-1 expression responded to IL-7 by increasing their cell size, as IL-7 induced the forward light scatter of DP thymocytes from IL-7RαTg SOCS-1 mice to increase by ∼50% (). Thus, constitutive SOCS-1 expression contributed to suppression of IL-7–mediated PI3 kinase activation in DP thymocytes. Interestingly, these data also reveal that the uniquely small cell volume of DP thymocytes is due, at least in part, to absent IL-7 signaling. We conclude that high SOCS-1 expression impairs IL-7 signal transduction in preselection DP thymocytes, although other factors also contribute.
During differentiation of normal DP thymocytes into mature T cells, IL-7Rα expression is up-regulated and IL-7 responsiveness is restored (, ). Consequently, we considered that TCR-mediated positive selection signals in DP thymocytes might down-regulate SOCS-1 gene expression and improve IL-7 signaling. To examine the effect of TCR signaling on SOCS-1 expression, we placed purified DP thymocytes from B6 and IL-7RαTg mice in culture overnight with immobilized anti-TCR and anti-CD2 mAbs and determined their SOCS-1 mRNA levels by quantitative RT-PCR. Indeed, TCR signaling reduced SOCS-1 gene expression in DP thymocytes from both B6 and IL-7RαTg mice (). Importantly, this experiment necessarily underestimates the amount that SOCS-1 gene expression is actually reduced by TCR signaling of DP thymocytes, as 30% of DP thymocytes do not express surface TCR complexes and so would not have received any TCR signals at all (). Consequently, the actual level of SOCS-1 mRNA in DP thymocytes that could have received TCR signals in this experiment would only be 10% of starting levels, a 90% reduction.
To determine whether TCR signaling improved IL-7 signaling in DP thymocytes, we initially prestimulated IL-7RαTg ZAP70 DP thymocytes for 3 h with the pharmacologic mimic of TCR signaling, PMA, and ionomycin (P+I) and then assessed their subsequent ability to respond to IL-7 by up-regulating expression of Bcl-2 mRNA and Bcl-2 protein. Because in vitro P+I stimulation for <12 h is insufficient to signal positive selection (, ), prestimulation of DP thymocytes with P+I for 3 h in our present experiment did not itself up-regulate either Bcl-2 mRNA or Bcl-2 protein (). However, prestimulation with P+I substantially augmented the ability of DP thymocytes to subsequently respond to IL-7, as indicated by a 3–4-fold increase in both Bcl-2 mRNA and Bcl-2 protein expression (). Thus, TCR signaling down-regulates SOCS-1 expression and improves IL-7 signaling in preselection DP thymocytes.
DP thymocytes that have received TCR-mediated positive selection appear in vivo as CD48 cells, and it has previously been shown that SOCS-1 gene expression is down-regulated in CD48 thymocytes relative to DP thymocytes (). Consequently, we examined IL-7 induction of p-Stat5 in preselection DP and postselection CD48 thymocytes by culturing IL-7RαTg thymocytes with either medium or IL-7 for 20 min (). Neither DP nor CD48 thymocytes contained substantial amounts of p-Stat5 in the absence of IL-7, and exposure to exogenous IL-7 induced p-Stat5 in both cell populations. More importantly, IL-7 stimulation induced ∼5× more p-Stat5 in postselection CD48 than in preselection DP thymocytes (), demonstrating that IL-7 signaling was considerably improved in CD48 thymocytes. We also examined Bcl-2 protein levels after 18 h of stimulation with either medium or exogenous IL-7 (). In parallel with the p-Stat5, IL-7 stimulation induced considerably more Bcl-2 protein in CD48 than DP thymocytes (). However, it is interesting to note that even without exogenous IL-7, unstimulated CD48 thymocytes expressed both elevated p-Stat5 and Bcl-2 protein levels relative to DP thymocytes (), suggesting that postselection CD48 thymocytes had been stimulated in vivo by intrathymic IL-7. Thus, IL-7 signal transduction is suppressed in preselection DP thymocytes by SOCS-1 and other factors but is substantially improved by TCR-mediated positive selection signaling.
Active suppression of cytokine signal transduction in preselection DP thymocytes by constitutive expression of SOCS-1 seemed unnecessary if preselection DP thymocytes were in fact relatively deficient in surface receptors for prosurvival cytokines, expressing mainly isolated γ as is currently thought. Consequently, we reconsidered the possibility that normal preselection DP thymocytes might actually express substantial quantities of surface receptors specific for some prosurvival cytokine. In fact, we found that DP thymocytes from normal B6 mice do express high levels of endogenous IL-4Rα proteins on their surface that, together with surface γ proteins, should form intact IL-4Rs (). Although IL-4R expression has not previously been observed on DP thymocytes, we found that expression of endogenously encoded IL-4Rα proteins on the surface of DP thymocytes was higher than on any other thymocyte population, with levels of IL-4Rα expression slightly exceeding those on CD4SP and CD8SP thymocytes ().
To confirm that DP thymocytes produced the IL-4Rα protein they expressed, we performed Northern blot analyses of purified DP thymocytes and of LNT cells from normal B6 mice (). Northern blots revealed that DP thymocytes contained mRNA for both IL-4R components, IL-4Rα and γ but did not detectably contain mRNA for IL-7Rα or SOCS-3 ( and ). In addition, these Northern blots confirmed that DP thymocytes constitutively expressed SOCS-1 mRNA, whereas unstimulated LNT cells did not ().
Finally, we assessed surface IL-4Rs for their ability to transduce signals in DP thymocytes and whether constitutive SOCS-1 expression interfered with that signaling. We used thymocytes from SOCS-1, SOCS-1, and SOCS-1 (containing 2, 1, or 0 SOCS-1 alleles), stimulated them with IL-4 for 1 h, and then stained the cells for intracellular p-Stat5 and -6, as IL-4 can induce phosphorylation of both Stat molecules (, left and center). IL-4 stimulation was unable to induce either p-Stat5 or -6 in SOCS-1 DP thymocytes despite high surface levels of IL-4Rs, whereas IL-4 induced both p-Stat molecules in CD8SP thymocytes present in the same culture wells (, left and center). Importantly, as SOCS-1 expression decreased, IL-4's ability to induce p-Stat5 and -6 in DP thymocytes increased, with SOCS-1 DP thymocytes achieving the highest levels of both p-Stat5 and -6 (, left and center). In fact, p-Stat5 and -6 levels induced by IL-4 stimulation of SOCS-1 DP thymocytes approached levels induced in CD8SP thymocytes (note different scales for DP and CD8SP thymocytes; , left and center). Continuing IL-4 stimulation for 18 h permitted us to also examine IL-4 induction of Bcl-2 protein expression (, right). In parallel with p-Stat5 and -6 induction, IL-4 failed to induce Bcl-2 protein expression in SOCS-1 DP thymocytes but induced increasing amounts of Bcl-2 protein as SOCS-1 expression decreased in DP thymocytes, with SOCS-1 DP thymocytes achieving the highest Bcl-2 protein levels (, right). These results demonstrate that DP thymocytes express endogenously encoded IL-4Rs on their surface, but their ability to transduce IL-4 signals is suppressed in DP thymocytes largely by constitutively expressed SOCS-1.
This paper has assessed cytokine signaling in DP thymocytes by both transgenic and endogenous cytokine receptors. By using an IL-7Rα transgene to circumvent the down-regulation of IL-7Rα gene expression that normally occurs in preselection DP thymocytes, this study demonstrates that IL-7R signal transduction is actively suppressed in DP thymocytes and is unable to promote DP thymocyte survival even when IL-7Rs are highly expressed on the cell surface, in large part as a result of SOCS-1, which is constitutively expressed in DP thymocytes. Interestingly, poor IL-7R signal transduction in DP thymocytes is responsible for the uniquely small cell volume of these cortical thymocytes. IL-7R signal transduction in DP thymocytes is substantially restored by TCR-mediated positive selection signals that down-regulate SOCS-1 expression and enhance IL-7 induction of prosurvival molecules such as Bcl-2. Finally, this study demonstrates that normal DP thymocytes express high levels of endogenous IL-4Rs on their cell surface but that IL-4R signal transduction is similarly suppressed by SOCS-1 in DP thymocytes. Thus, DP thymocytes do express high levels of surface receptors for the prosurvival cytokine IL-4, but signal transduction by γ cytokine receptors is actively suppressed in preselection DP thymocytes and restored by TCR-signaled positive selection signaling.
The vast majority of DP thymocytes in the thymus fail to receive TCR-mediated positive selection signals and, thus, “die by neglect.” The ability of unselected DP thymocytes to die by neglect permits thymocytes with appropriate TCR specificities to be the only ones to survive and to differentiate into functionally mature T cells. However, the ability of DP thymocytes to die by neglect requires the absence of prosurvival factors such as Bcl-2 and glucose transporter–1, which can be induced by signals from prosurvival cytokines (IL-2, -4, -6, -7, and -15) (). As a result, death by neglect requires that DP thymocytes fail to be signaled by prosurvival cytokines.
Several mechanisms for avoiding cytokine induction of prosurvival factors in DP thymocytes have been suggested. One obvious mechanism is the down-regulation of IL-7Rα gene expression in DP thymocytes so that DP thymocytes are relatively deficient in IL-7Rα surface expression (). However, low levels of residual IL-7Rα protein may remain on the surface of DP thymocytes, albeit in barely detectable quantities, with the possibility that some DP thymocytes might still be able to bind IL-7. A second mechanism that would reduce the possibility of IL-7 signaling in DP thymocytes is for DP thymocytes to arise from precursor cells that are themselves unable to respond to IL-7. Indeed, IL-7 signals have recently been shown to inhibit the expression of transcription factors required for differentiation of immature SPs into DP thymocytes, so that DP thymocytes only arise from immature SPs that have not been signaled by intrathymic IL-7 (). A third mechanism for minimizing IL-7 signaling in DP thymocytes is the relative deficiency of IL-7–producing cells in the thymic cortex where DP thymocytes reside (). Importantly, all three mechanisms only minimize and do not eliminate the possibility of IL-7 signaling, and none prevents potential signaling of DP thymocytes by other prosurvival cytokines. Indeed, because normal DP thymocytes constitutively express γ, it was possible that they might express receptors for prosurvival cytokines other than IL-7. In fact, we found that DP thymocytes do express endogenously encoded receptors for IL-4 and they express IL-4Rs at higher levels than any other cell population in the thymus. We do not yet know why DP thymocytes highly express IL-4Rs, especially because their ability to transduce IL-4 signals is suppressed by constitutively expressed SOCS-1. Speculatively, it may simply be that no molecular mechanism exists to dynamically regulate IL-4R gene expression during thymocyte development the way that IL-7Rα gene expression is dynamically regulated during thymocyte development, with the result that once IL-4R gene expression is turned on, IL-4R gene expression remains on in preselection DP thymocytes. By whatever mechanism, high endogenous IL-4R expression would require that DP thymocytes contain a suppressive mechanism to prevent IL-4Rs from transducing prosurvival signals that would prevent them from undergoing death by neglect.
Consequently, the inability of surface cytokine receptors to transduce signals in DP thymocytes represents a fail-safe mechanism to prevent signaling of DP thymocytes by any prosurvival cytokine, and an important component of this suppression is SOCS-1. In mature T cells, SOCS-1 expression is induced by cytokine signals as a feedback mechanism to limit further cytokine signaling (–, ). In contrast, SOCS-1 expression in DP thymocytes is constitutive (, ). The concept of constitutive SOCS-1 expression as a fail-safe mechanism to prevent any possibility of cytokine signaling in preselection DP thymocytes is made all the more plausible by our finding that DP thymocytes do in fact highly express endogenous IL-4Rs on their surface. This concept is further supported by the fact that constitutive SOCS-1 expression in preselection DP thymocytes is down-regulated by TCR signals in vitro and by positive selection signals in vivo, so that positive selection restores cytokine signal transduction. Thus, TCR-mediated positive selection signals restore IL-7 responsiveness to DP thymocytes in two ways: by down-regulating constitutive SOCS-1 expression and by up-regulating IL-7Rα gene expression (, ).
This study also provides an explanation for the uniquely small cell volume of DP thymocytes. We found that IL-7R DP thymocytes from SOCS-1 mice responded to IL-7 by increasing their cell size, suggesting that the uniquely small size of most DP thymocytes results, at least in part, from absent IL-7 signaling. From this perspective, it is interesting that DP thymocytes were identically small in IL-7RSOCS-1, IL-7RSOCS-1, and IL-7RSOCS-1 mice (see ), even though the latter were able to considerably increase their cell size in response to exogenous IL-7, inferring that endogenous IL-7 was not present in sufficient amounts in the thymic cortex to increase DP thymocyte size.
SOCS-1 deficiency leads to perinatal lethality resulting from both IFN-γ and T cell–dependent inflammation and necrosis in multiple organs (, ), so that survival of SOCS-1 mice requires that they be additionally IFN-γ or Rag. However, SOCS-1 conditional knockout mice have been described in which conditional deletion of SOCS-1 in T lineage cells results in improved IL-7 signaling, increased positive selection of CD8 T cells (), and decreased positive selection of CD4 T cells in an IFN-γ–dependent manner (). Thus, SOCS-1 regulates cytokine responsiveness and importantly affects the lineage direction of positively selected thymocytes. This study now demonstrates that SOCS-1 prevents unselected DP thymocytes from transducing cytokine-mediated survival signals.
In this paper, complete SOCS-1 deficiency did not completely restore either IL-7 signaling in transgenic IL-7R DP thymocytes or IL-4 signaling in normal DP thymocytes, although SOCS-1 deficiency appeared to improve IL-4 signaling more than IL-7 signaling. Consequently, we think that other factors in DP thymocytes in addition to SOCS-1 also contribute to impaired cytokine signaling. It is possible that other SOCS proteins in DP thymocytes contribute to decreased cytokine responsiveness, although we found little SOCS-3 mRNA in DP thymocytes; it is possible that the somewhat lower surface expression of γ on DP than mature T cells may also contribute. Future studies will assess the contribution of components other than SOCS-1 to impaired cytokine signal transduction in preselection DP thymocytes.
This study documents that signal transduction by highly expressed cytokine receptors is actively suppressed in preselection DP thymocytes and that SOCS-1 is an important component of the suppressive mechanism. Our current observations with highly expressed cytokine receptors for IL-7 and IL-4 are consistent with, and substantially extend, the study by Chong et al. () that found that weak IL-7 signaling by the few endogenously encoded IL-7Rs present on DP thymocytes was enhanced by removal of SOCS-1. In contrast, our current observations conflict with those of Munitic et al. (), who observed that IL-7 signals in DP thymocytes from IL-7RαTg mice up-regulated Bcl-2 expression. Finally, we would note that the finding by van De Wiele et al. () that normal nontransgenic DP thymocytes were unresponsive to in vivo IL-7 as assessed by Stat-5 phosphorylation did not necessarily implicate a SOCS-1–mediated suppressive mechanism as it was simply consistent with deficient IL-7R expression on DP thymocytes.
In conclusion, DP thymocytes fail to transduce signals from highly expressed cytokine receptors, and their impairment is partly the result of SOCS-1–mediated suppression. We think that suppression of cytokine signaling in preselection DP thymocytes is necessary for death by neglect and reinforces TCR-specific repertoire selection in the thymus.
C57BL/6 (B6) mice were obtained from the Jackson Laboratory. ZAP70 mice were bred in our own colony. The IL-7Rα–transgenic construct was made by ligating an IL-7Rα cDNA into a human CD2 (hCD2) enhancer-promoter–based vector and was injected into fertilized B6 oocytes to generate IL-7Rα transgenic mice. SOCS-1IFN-γ mice () were provided by J. Ihle (St. Jude Children's Research Hospital, Memphis, TN) and were bred with IL-7Rα transgenic mice in our own colony. Animal studies were approved by the Animal Care and Use Committee of the Laboratory of Animal Sciences Program at the NCI.
Cells were harvested, stained, and analyzed on a FACSVantage SE (Becton Dickinson). Dead cells were excluded by forward light scatter gating and propidium iodide staining. Data were analyzed using software designed by the Division of Computer Research and Technology at the National Institutes of Health (NIH). Total fluorescence units (TFU) were calculated with this software using the empirically derived formula for the FACSVantage: TFU = A × 10 × 10,000, where A is the percent positive population and B is the median channel for each corresponding FITC fluorescence using 4-log amplification. Antibodies (all obtained from BD Biosciences) with the following specificities were used for staining: CD4 (GK1.5 and RM4.5); CD8α (53-6.7), TCRβ (H57-597), γ (4G3), p-Stat5 (clone 47), and Bcl-2 (3F11). Anti–IL-7Rα (A7R34) was obtained from eBioscience, and PE-conjugated anti–IL-4Rα (M1) was obtained from Research Diagnostics, Inc.
DP thymocytes were purified by panning with anti-CD8 mAb. Where indicated in the figures, DP thymocytes were also purified by electronic sorting. Lymph node T cells were purified by incubating lymph node cells with magnetic beads to which anti–mouse Ig had been adsorbed and selecting the nonadherent cells.
Purified DP thymocytes or LNT cells were placed in suspension cultures in medium alone or with 6 ng/ml IL-7 (R&D Systems) or with 20 ng/ml IL-4 (R&D Systems) for the times indicated in the figures. Where indicated in the figures, DP thymocytes were stimulated with 0.3 ng/ml PMA (Calbiochem) and 0.3 μg/ml ionomycin (Calbiochem) or with 10 μg/ml plate-bound anti-TCR (BD Biosciences) and 10 μg/ml anti-CD2 (BD Biosciences).
Cells were fixed and permeabilized first with 4% PFA and then with a 1:1 methanol/acetone mixture (vol/vol). Cells were stained with anti–Bcl-2 antibody followed by FITC-labeled secondary antibody. After intracellular staining, cells were further stained for surface proteins. For intracellular p-Stat5 staining, cells were incubated in medium alone or with IL-7 for either 20 min or 1 h. The cells were fixed and permeabilized and stained with FITC-labeled anti–p-Stat5 mAb, and then stained for surface proteins.
Total RNA was reversed transcribed using oligo dT and Superscript II reverse transcriptase (Invitrogen). The cDNA was subjected to real-time PCR amplification for 40 cycles, with each cycle consisting of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C.
Cells were lysed in SDS sample buffer, and cell lysates were resolved on 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane. Blots were incubated with anti–p-Stat5 antibody (Zymed Laboratories) or a mixture of anti-Stat5a and anti-Stat5b antibodies (Santa Cruz Biotechnology, Inc.), followed by horseradish peroxidase–conjugated protein A. Reactivity was revealed by enhanced chemiluminescence.
Equal amounts of nuclear extracts were incubated with P-labeled ATP Stat5/6 consensus oligonucleotides (Santa Cruz) either without or with 50× cold oligonucleotide competitors. For supershift assays, anti-Stat5 gel supershift antibody (Santa Cruz Biotechnology, Inc.) was added to the binding reactions. Protein–DNA complexes were electrophoresed through a 5% nondenaturing polyacrylamide gel, which was subsequently dried and exposed to film for autoradiography.
Total RNA was isolated from purified DP thymocytes and LNT cells using TriZol (Invitrogen). Equal amounts of RNA were resolved in a 3-(-morpholino) propanesulfonic acid–buffered agarose gel under denaturing conditions and subsequently blotted onto Hybond-N+ membranes (GE Healthcare) by capillary transfer. Radioactive probes were generated from cloned cDNA fragments of the corresponding genes by using the EZ-strip DNA kit (Ambion) and hybridized for 16 h with RNA-blotted membranes in UltraHyb solution (Ambion) at 42°C. Unbound probes were washed off two times with 2× SSC/0.1% SDS for 30 min and two times with 0.1× SSC/0.1% SDS at 55°C. Membranes were then exposed to a PhosphoImager screen (GE Healthcare) and analyzed. |
Three cohorts of patients, the Colorado WNV-seropositive cohort, the Arizona WNV-seropositive cohort, and the Arizona WNV-seronegative cohort, were identified that had symptomatic illness in which WNV was considered in the differential diagnosis. Inclusion criteria for patients in these cohorts are defined in the Materials and methods section and the clinical characteristics are given in . Genotypes were defined for 247 of the 261 WNV-seropositive Arizona cases (94.6%); for 148 of the 155 WNV-seropositive Colorado cases (95.5%); for 143 of 152 and 72 of 74 of the self-reporting Caucasian subsets of the Arizona and Colorado WNV-seropositive cohorts, respectively; for all WNV-seronegative random blood donors ( = 1,318); and for 145 of 154 WNV-seronegative subjects from Arizona (94.2%).
The genotype distribution observed for the combined Arizona and Colorado WNV-seropositive cohorts deviated strongly from Hardy-Weinberg equilibrium (P = 0.013), mainly because of increased frequency of homozygotes. 17 (4.3%) of the 395 informative cases in the combined Arizona and Colorado seropositive cohorts unstratified by race were homozygotes (). This value greatly exceeds the percentage of homozygotes reported for the general US Caucasian population (0.8–1.1%; reference ). To quantitate the significance of this genotype–disease association, we have used as a reference the group of 1,318 healthy Caucasian random blood donors from the US, in which 1.0% were homozygotes. Compared with this value, the increased frequency of homozygotes in both the Arizona and Colorado WNV-seropositive cohorts was highly statistically significant (). When all genotyped WNV-seropositive cases unstratified by race were compared with the US random blood donors, the odds ratios (OR) for this genetic factor were 4.7 for the Arizona cohort (95% confidence interval [CI], 2.1–10.6, P < 0.0001) and 4.2 for the Colorado cohort (95% CI, 1.6–11.3, P = 0.0018). It is important to note that this is a conservative analysis because the frequency of homozygotes is certain to be lower in non-Caucasian racial groups from these cohorts ().
Consistent with this, when the data were stratified to include only self-reporting Caucasians in both the WNV-seropositive cohorts ( = 215), the strength of the association increased by 30% (12 homozygotes, 5.6%). When the self-reporting Caucasian portions of the two WNV-seropositive cohorts were compared separately to the random blood donor benchmark, the OR was 4.4 in the Arizona cohort (95% CI, 1.6–11.8, P = 0.0013), and 9.1 in the Colorado cohort (95% CI, 3.4–24.8, P < 0.0001).
Although unlikely, it is possible that the large increase in frequency of homozygotes in these cohorts may be caused by an atypical geographic distribution of this allele in the general population of Arizona and Colorado. To directly address this possibility, we analyzed the distribution of in a symptomatic Arizona WNV-seronegative cohort ( = 145). Because the allele frequency in this cohort (7.2%) is only slightly lower than in the Arizona WNV-seropositive cohort (10.7%) and the US Caucasian random blood donors (8.7%), it is likely that this cohort contains only a slightly smaller percentage of Caucasians than the other two. Thus, given the size of the Arizona WNV-seronegative group, there is sufficient power at this allele frequency to meaningfully analyze the distribution of genotypes. Only one homozygote was identified in the cohort (0.7%). Moreover, unlike the WNV-seropositive cohort, the observed genotypic frequencies in the WNV-seronegative cohort did not deviate from Hardy-Weinberg equilibrium (P > 0.99). This strongly suggests that the general population in Arizona does not have an anomalous genetic structure with regard to the locus, with a skewed increase in homozygotes that could account for the high frequencies we observed in the WNV-seropositive cohorts. When the homozygote genotype frequency was compared between the race-unstratified Arizona WNV-seronegative and -seropositive cohorts, an OR of 6.7 was determined (95% CI, 0.86–52.6, P = 0.07).
With regard to the distribution of genotypes in symptomatic WNV-seropositive cases as a function of clinical outcome, no significant differences were observed compared with the distribution in the overall combined group () or in the Arizona or Colorado WNV-seropositive cohorts considered separately (not depicted), with the exception of death (). A total of 19 of 395 individuals in the two cohorts died from WNV neuroinvasive disease (4.8%), which is similar to the national average of mortality reported for WNV cases from the past three years: 2002 (4,156 cases, 284 deaths, 6.8%); 2003 (9,862 cases, 264 deaths, 2.7%); and 2004 (2,539 cases, 100 deaths, 3.9%). The combined total for this time interval in the US is 16,577 reported cases with 648 deaths (3.9%; references –). The Arizona WNV-seropositive cohort accounted for 8 of the 19 total deaths, 7 in Caucasians and 1 in a Hispanic (). The Colorado WNV-seropositive cohort accounted for 11 deaths, 1 in a Caucasian. Two of the 19 fatalities (10.5%) in the combined cohorts were homozygotes, both self-reporting Caucasians from the Arizona WNV-seropositive cohort. The ages of the two homozygotes who died were 70 and 74, similar to the average age of the other 17 fatal cases (74 yr). The two homozygote fatalities represent 25 and 29% of the race-unstratified ( = 8) and Caucasian ( = 7) Arizona WNV-seropositive cases who died, respectively. These values exceed the expected values based on the frequency of homozygotes among the race unstratified and Caucasian groups in the Arizona cohort (4.5 and 4.2%, respectively), and the differences are statistically significant: OR = 8.5 (95% CI, 1.5–48.2), P = 0.04, and OR = 13.2 (95% CI, 1.9–89.9), P = 0.03, respectively (). This association was not observed in the Colorado cohort. When Caucasians from both cohorts were analyzed together, the association test gave an OR of 6.6 (95% CI, 1.2–37), P = 0.07.
The present study provides the first evidence that the defective allele is a risk factor for symptomatic WNV infection, the first genetic risk factor identified for this disease. The association was restricted to homozygotes and was very strong, similar in magnitude to the association we and others have previously reported for homozygosity with the exposed, uninfected HIV resistance phenotype (e.g., OR = 6.04 [95% CI, 1.42–25.7], P = 0.02, in reference ). Although our results are based on retrospective analysis, they are unlikely to be because of chance for six reasons. First, the results are statistically strong (an OR greater than four). Second, the results were obtained in two independent as well as geographically and temporally distinct cohorts. Third, is a complete loss-of-function mutation and therefore homozygous individuals completely lack functional CCR5. Fourth, WNV infection is associated with infiltration of T cells and macrophages (cell types known to express CCR5) into brains of patients infected with WNV, suggesting biologically plausible involvement of CCR5 in regulating leukocyte migration to the brains of infected patients (, ). Fifth, WNV infection of mice induces expression of the CCR5 ligand CCL5 and accumulation of CCR5 leukocytes in the brain (, ). Sixth, WNV infection in CCR5 mice is uniformly fatal ().
Together these results imply that wild-type CCR5 functions as a host defense factor in WNV infection in man. CCR5 could potentially restrict WNV infection at the level of initial infection. This is not addressed by our retrospective study design and may not be feasible to resolve prospectively by measuring the association of homozygosity with asymptomatic WNV infection because this genotype and WNV infection are both uncommon in the general population. More likely, CCR5 restricts disease progression after initial infection so that its absence results in an increased likelihood of an infected patient coming to medical attention as a symptomatic case. Our data are consistent with this interpretation, and even suggest that a fatal outcome is more likely in the absence of CCR5 (). Additional work will be needed to more critically address the latter point because the positive association we observed between CCR5 deficiency and death in the Arizona cohort, although statistically significant, was based on only two cases, and could not be detected in the Colorado cohort, possibly because of its smaller size.
Another limitation of our study was the incomplete information about the racial background of cases (), which is needed to more precisely quantitate risk because is primarily found in Caucasians. We have addressed this by consistently applying a conservative analysis of the data that has enabled us to test boundary conditions for quantitating the strength of the genotype–disease association. Larger prospective studies will be needed to more precisely quantitate risk as well as to further define mechanisms in man.
Our data indicate that immunocompromised patients could be particularly susceptible to symptomatic WNV infection if CCR5 function is missing or blocked. Additional studies will be needed to address this issue, which should include close monitoring of AIDS patients treated with antagonists now in advanced stages of development targeting the HIV coreceptor activity of CCR5. Such agents are intended to imitate the homozygous genetic defect and could render AIDS patients particularly vulnerable to severe and possibly fatal WNV infection.
In summary, we have established that homozygous is a strong host genetic risk factor for symptomatic laboratory-confirmed WNV infection, the first one identified for this disease. In contrast, the homozygous genotype has previously been strongly associated with resistance to HIV. These genetic data imply that CCR5 plays opposite roles in HIV and WNV infection, facilitating the former and restricting the latter. Our results have important implications regarding the potential safety of CCR5-blocking agents now under development for the treatment of HIV/AIDS. Clinical care of individuals taking these medicines while residing in WNV-endemic areas may mandate strict measures to limit mosquito exposure and a high index of suspicion for symptoms consistent with WNV.
The study was approved by the Office of Human Subjects Research of the US National Institutes of Health. Four patient groups were defined: (a) healthy North American Caucasian random blood donors from the National Institutes of Health Department of Transfusion Medicine ( = 1,318) previously collected under an IRB approved protocol; (b) the Arizona WNV-seropositive cohort ( = 261), defined as patients presenting with acute illness in Arizona who tested positive for WNV but negative for St. Louis encephalitis virus (SLE) by specific IgM ELISA of serum or cerebrospinal fluid (CSF) at the Arizona Department of Health Services between 5/26/04 and 10/2/04, the dates for receipt by the Department of the first and last patient samples during mosquito season in the state of Arizona in 2004; (c) the Colorado WNV-seropositive cohort ( = 155), defined as patients presenting with acute illness to a physician in Colorado who tested positive for WNV but negative for SLE by specific IgM ELISA of serum or CSF at the Colorado Department of Public Health and Environment between 7/20/03 and 9/30/03, the dates for receipt by the Department of the first and last patient samples for 2003; (d) the Arizona WNV-seronegative cohort ( = 154), defined as patients presenting with acute illness to a physician in Arizona in whom WNV and SLE had been considered in the differential diagnosis but ruled out by serological testing at the Arizona Department of Health Services between 5/26/04 and 10/2/04. In the Arizona WNV-seropositive cohort, 89% of the samples collected were available and all of these were analyzed. In the Colorado WNV-seropositive cohort, only 164 samples remained that had a sufficient volume for purification of DNA and all of these samples were analyzed. This represented 14% of all Colorado samples originally collected. The following information was requested from the medical provider during case investigation by local health department communicable disease staff: age, sex, self-reported racial group, and clinical presentation based on the Centers for Disease Control–defined clinical parameters of disease; i.e., fever, meningitis, encephalitis, and death (). At the time of data collection each symptomatic patient was classified into one of the three disease categories: fever, meningitis, or encephalitis. Follow-up data on disease course and the maximal severity of symptomatic disease in each patient were not available. All fatal cases were caused by complications of WNV neuroinvasive disease. Study investigators were blinded to unique patient identifiers.
100 μl of serum or CSF was thawed for genomic DNA purification using a QiaAmp 96 DNA Blood kit according to the manufacturer's instructions (QIAGEN). Purified DNA was eluted into 100 μl of the recommended buffer and stored at 4°C until further use. DNA from random blood donors was isolated as previously described ().
Genotyping was performed by standard methods described previously (). In brief, 2 μl of patient DNA was amplified by PCR using primers that flank the site of the 32-bp deletion: 5′-GTCTTCATTACACCTGCAGCTCTC-3′ and 5′-GTCCAACCTGTTAGAGCTACTGC-3′. PCR products were analyzed by electrophoresis on a 3.0% agarose/TBE gel with known wild-type and amplicon controls (233 and 201 bp, respectively) and visualized with Gelstar (Cambrex) DNA staining. Each sample was tested in two independent PCR reactions, and results were concordant, as determined by two independent investigators.
OR and 95% confidence limits were calculated using a recessive genetic model (i.e., homozygotes compared with the combination of wild-type and heterozygotes) by cross-tabulation. With the exception of the death data, significance was determined by χ tests using a two-sided P value and Yates' continuity correction, and 95% CIs were estimated using the approximation of Woolf as implemented in the Prism statistics program version 4 (GraphPad Software). Association of homozygosity with death, due to small numbers, was analyzed by Fisher's exact test. Tests of Hardy-Weinberg equilibrium were performed using a χ test and two degrees of freedom after using the Hardy-Weinberg equation ( + + = 1, where and represent the frequency of the two alleles) to calculate expected frequencies of each of the three genotypes. |
is an invasive enteric pathogen that preferentially uses the M cells of the PP as portals of entry, after which the bacteria spread to the mesenteric LN and in mice, disseminate to cause a systemic disease (). We investigated whether innate SAbs block invasion of the gut epithelium by comparing the number of bacteria detected in the PP and small intestinal mucosa of B6 and pIgR mice 6 or 12 h after oral infection with 10 CFUs . The combined results of three independent experiments showed that invasion is significantly increased (P = 0.011) in pIgR mice (137 ± 129) compared with B6 mice (59 ± 88) ().
In pIgR mice, pIgA destined for secretion into the mucosal lumen accumulates in the blood, resulting in 100-fold greater serum levels than in B6 mice, whereas IgG and IgM concentrations are similar (, ). The use of pIgR serum allowed us to examine the interaction between “accumulated” innate SIgA in its dimeric form () and . We used an in vitro invasion assay to demonstrate that serum from pIgR mice blocked Madine-Darby canine kidney (MDCK) epithelial cell invasion by (). After preincubation of the MDCK cells with serum obtained from naive pIgR mice, a significantly reduced number of invaded was detectable (7.6 × 10 ± 1.5 × 10) compared with the effect of serum from naive B6 mice (2 × 10 ± 9 × 10; P = 0.04) or with no serum control (3 × 10 ± 3 × 10; P < 0.001). Similar results were obtained with heat-inactivated serum (unpublished data), suggesting that IgG antibodies inducing complement activation did not contribute to the obtained results. The inhibitory effect of pIgR serum was abolished by the addition of goat anti–mouse IgA antibodies but not by goat anti–mouse IgG antibodies, suggesting that IgA in pIgR serum was inhibiting invasion of MDCK cells by the bacteria ().
The production of IgA is induced in an antigen-unspecific manner by commensal flora (–). Although some reports have suggested that SIgA is polyreactive in nature, other findings point to a restricted specificity that may be cross-reactive (, –, , ). In further experiments, we examined the reactivity of dIgA in pIgR serum with . Serum samples were preabsorbed against by overnight incubation at 4°C, which resulted in a nonsignificant reduction in the amount of total IgA in the samples (6.13 ± 1.6 mg/ml versus 4.31 ± 0.85 mg/ml in preabsorbed serum). D shows that compared with untreated pIgR serum, preabsorbed pIgR serum was no longer able to effectively inhibit invasion of MDCK cells, suggesting that a small proportion of total pIgA was able to bind and block host cell invasion.
In previous studies, we characterized the composition of the ileal flora of pIgR mice, which was similar to that of B6 mice (). Subsequent studies demonstrated that pIgR have increased levels of serum IgA specific for members of the gut flora (unpublished data). To further investigate the polyreactive nature of pIgA in serum from pIgR mice, isolated species of normal gut flora were cultured (25; unpublished data) and used as antigens in an ELISA to measure binding of normal pIgR serum and pIgR serum preabsorbed against . E shows that after preabsorption binding of pIgR serum to flora isolates was either increased or not altered.
In contrast to findings by Bouvet et al. (, ), preabsorbtion of pIgR serum with only removed a small fraction of the total IgA and did not reduce the binding to other commensals. SIgA forms large multivalent complexes with mucins and/or pFv after secretion (), which may affect the quantitative analysis of binding of SIgA to antigens. Whereas we used serum samples containing dIgA, Bouvet et al. used samples of saliva and gut washings containing large aggregates of SIgA, and preabsorbing to a cross-reactive antigen may therefore have resulted in removal of a large proportion of IgA. Collectively, our results suggest that pIgA in serum of pIgR mice is not polyreactive but restricted in nature, and some of the innate pIgA antibodies are able to bind in a cross-reactive manner. Further investigations which are beyond the scope of this paper, e.g., analysis of IgV gene usage and the use of Ig allotype chimeric mice (), may provide more insight into the exact nature and origin of the dIgA that binds .
We further investigated whether the increased microbial invasion in vivo as a result of lack of innate SAbs augmented susceptibility of pIgR mice to infection with virulent . By orally infecting naive mice with decreasing doses of and monitoring their survival, we first determined whether the lack of SAbs in pIgR mice renders these animals more sensitive to infection with virulent bacteria than wild-type B6 mice (). At an oral dose of 10 or 10 CFUs, pIgR and B6 mice were equally susceptible to infection. However, infection of pIgR mice with 10 CFUs resulted in 100% mortality, whereas this dose was lethal in only 20% of B6 mice. Although this difference in mortality of pIgR mice compared with B6 mice was demonstrated using a low inoculation dose (), the in vivo invasion experiments ( A) were performed with a higher infectious dose to enable recovery of intracellular bacteria within 6 h after infection from the gut tissues (). Collectively though, these results strongly suggest that innate SAbs protect mice against pathogenic bacterial invasion.
We subsequently wished to mimic the natural fecal-oral route of transmission of infection by cohousing orally infected mice with naive animals. These experiments were performed with three cages in which one orally infected B6 mouse was cohoused with two naive B6 and two naive pIgR mice, and with three cages in which one orally infected pIgR mouse was cohoused with two naive B6 and two naive pIgR mice. B6 mice infected with shed 4 × 10 (±5 × 10) bacteria 1 d after infection; this increased to 2 × 10 (±700) bacteria at day 8 (). One out of three orally infected B6 mice was shedding significantly higher numbers of bacteria on day 5 after infection before it became moribund and was killed, resulting in an increased average bacterial count (10 ± 1.8 × 10) in the feces on this day. All infected B6 mice had succumbed to the infection by day 9 after oral inoculation ().
Only one out of the six naive B6 mice cohoused with the orally inoculated B6 mice acquired the infection and had detectable bacteria in its feces on day 9 but none of them died (). Conversely, three out of six naive pIgR mice cohoused with the orally infected B6 mice started shedding the at day 5 after infection, and the number of bacteria shed in the feces rapidly increased to ∼8 × 10 bacteria around day 9 after infection (); these mice all became moribund by day 15 ().
The number of bacteria detected in feces of orally inoculated pIgR mice () was at any time point significantly (P < 0.01) higher than that in feces of B6 mice (), starting at 2 × 10 (±2 × 10) bacteria one day after infection, and increasing to −6.5 × 10 (±2 × 10) bacteria on day 7 and 8 after infection (), at which point the animals had all been killed (). This suggested that in the absence of SAbs, the number of replicating was dramatically increased.
Naive pIgR mice cohoused with the –infected pIgR mice rapidly acquired the infection and were shedding bacteria in their feces as early as 1 d after being cohoused with the orally inoculated pIgR mice. After 5 d of cohousing, the naive pIgR mice were shedding >10 bacteria (), and by day 11 all had succumbed to the infection (). Interestingly, naive B6 mice cohoused with orally infected pIgR mice were also more sensitive to the infection compared with cohousing with orally infected B6 mice, suggesting that the lower number of bacteria shed from B6 mice less readily infected other animals compared with bacteria shed from pIgR mice. was detectable in feces of three out of six B6 mice cohoused with infected pIgR mice (), and the infection was lethal for these mice ().
It has been demonstrated that circulating natural IgM antibodies can protect against pathogenic microorganisms (). Natural (or innate) SIgA production is induced by colonization with commensal flora (–), and SAbs are thought to act by “immune exclusion” or “immune elimination” (, , , ). Because of its ability to bind multiple antigens, whether in a restricted or polyreactive manner, binding of innate dIgA to commensal flora or autoantigens and subsequent removal from the lamina propria via transport by the pIgR may prevent systemic recognition and undesirable pathological immune responses. In support of this hypothesis is the finding that SIgA in breast milk is reactive with members of the mother's gut microbiota that were present in the third trimester and at birth, providing the neonate protection against commensal organisms while the GALT is undeveloped ().
In line with this concept, our studies show that the large amounts of innate SAbs normally actively exported into the gut lumen by the pIgR provide a primitive type of front line defense against invading bacterial pathogens in the naive host. Our data demonstrate for the first time that, in the absence of SAbs, increased numbers of invade the gastrointestinal epithelium, suggesting that innate SAbs normally present in the gut block bacterial adhesion and penetration. Indeed, our in vitro experiments verified that serum obtained from naive pIgR mice, which contains pIgA that is normally destined for secretion to the mucosae (, ), binds to in a cross-reactive manner and inhibits the invasion of MDCK cells by .
More importantly, however, our study shows that the innate SAbs may also reduce the shedding of pathogens and the spread of infections to other individuals which may not only be because of increased numbers of bacteria, but could also be the result of the absence of a SAb “coat” on the excreted bacteria, which may normally prevent the bacteria from invading a new host (, , ). Therefore, we propose that although innate SAbs may protect locally at mucosal surfaces their major role may be to prevent the spread of pathogens throughout the population. This may explain why the immune system has evolved to produce much larger amounts of IgA than any other Ig class, and why such large amounts of SAbs are continuously transported across the mucosae into external secretions and breast milk.
In conclusion, our data demonstrates that the innate SAb system, which in evolutionary terms is considered a primitive front line defense against induction of autoimmunity and invasion by microbial pathogens (), is crucial for both the individual host and its herd. This finding has important implications for the development of novel immunotherapeutics designed to protect against pathogens that invade via the mucosae, which are often devised to activate the production of antigen-specific SIgA while ignoring the protective roles of innate SAbs.
pIgR mice were generated on a pure C57BL/6 (B6) genetic background as previously described (). All mice were bred and reared under conventional conditions at the animal facility of The University of Melbourne, Department of Microbiology and Immunology. Mice were age and sex matched for each experiment, and used at 6–8 wk of age. All animal experiments were approved by The University of Melbourne Animal Ethics and Experimentation Committee, and complied with the Prevention of Cruelty to Animals Act and the National Health and Medical Research Council (NHMRC) Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.
In this study, mice were orally infected with the virulent strain SL1344 as described before ().
To mimic the natural fecal-oral route of transmission, a single mouse was infected with 10 CFU SL1344 by oral gavage and placed in a cage with naive animals. The acquisition and development of infection in the naive animals was monitored by detection of in feces. Absence of fecal was confirmed before the start of each experiment in all animals.
The number of live in PP or entire small intestines was determined by plating serial dilutions of the homogenates on LB agar plates containing streptomycin, as described previously ().
Shedding of was determined by counting the number of bacteria in fresh fecal extracts. Individual mice were placed in clean boxes, and the collected fecal pellets were weighed, dissolved in sterile PBS (100 mg/ml), and vortexed vigorously. Serial dilutions were plated on selective indicator media (DCA and MacConkey agar plates containing streptomycin) to enumerate the number of in the samples.
Invasion of MDCK cells by SL1344 was determined as described before (). In brief, monolayers of MDCK cells were infected with mid-log growth phase at multiplicity of infection of 10. Serial dilutions of serum, pooled from three naive B6 or pIgR mice, were added to the MDCK cells 5 min before addition of the bacteria. After 1 h, MDCK cells were washed and incubated for 1.5 h with culture media containing 100 μg/ml gentamicin to kill extracellular bacteria. After this period, cells were lysed with 0.1% (wt/vol) Triton X-100 in PBS, and the number of bacteria present in the lysates was determined by viability count. Preabsorbed serum was prepared by incubating the serum with SL1344 for 12 h at 4°C while rotating, after which the bacteria were removed by centrifugation. In some experiments, 1 mg of goat anti–mouse IgG or goat anti–mouse IgA (Sigma-Aldrich), diluted in culture media, was mixed with 200 μl serum before use in the assay.
The nonparametric two-tailed Mann-Whitney U test was used for statistical analysis of the results. Differences were considered significant when P < 0.05. |
In initial experiments, COS-7 cells were transiently transfected with a plasmid containing the G1 scFv gene fused to an adenoviral secretory signal peptide sequence (pS.scFvG1; ). Protein expression was analyzed by the ability of permeabilized cells to bind the Seam 3 mAb (e.g., the antigen against which the G1 scFv was raised) using immunofluorescence flow cytometry. After treatment with Seam 3 followed by FITC-conjugated anti–mouse IgG, pS.scFvG1-transfected cells showed increased fluorescence relative to cells transfected with the empty plasmid (). Increased fluorescence was not detected when cells were treated with an irrelevant, isotype-matched mAb in place of Seam 3 (not depicted). These data indicated that pS.scFvG1 transfection resulted in the expression of the G1 scFv in a functional form, as defined by its ability to bind to the Seam 3 idiotope.
Next, mice were injected i.m. with different doses of pS.scFvG1 or empty plasmid, and serum bactericidal antibodies were measured at various times after immunization. Results were compared with those observed after immunization with the G1 scFv protein conjugated with KLH using Freund's adjuvant. Bactericidal titers were always <9 (i.e., below the detection limit of the assay) in serum samples obtained before or after immunization with the empty plasmid (). Only two out of eight animals developed a bactericidal response (with titers of 36 and 72) after immunization with G1 scFv–KLH, which was in agreement with previous experiments (). The effects of pS.scFvG1 immunization on serum bactericidal activity were markedly dose dependent (). Using 150 μg, five out of eight animals produced bactericidal responses with titers ranging from 36 to 144. These data indicated that immunization with the scFv gene but not with the corresponding protein frequently resulted in serum bactericidal activity even though only moderately elevated titers were observed.
One of the advantages of DNA immunization is the possibility to easily manipulate the immunogen to link it, for example, to additional epitopes or immunostimulatory peptides. Therefore, in further experiments, we fused the G1 scFv gene with a universal T helper cell sequence to generate plasmid pST.scFvG1 (). Constructs containing the scFv gene without the adenoviral leader peptide sequence were also tested (pT.scFvG1 and pscFvG1; ). After three immunizations with these plasmids, bactericidal titers were assessed in sera obtained at 56 d after the first administration. For comparison, we also tested sera from animals immunized with -propionylated MenB CP (N-Pr MenB) conjugated with tetanus toxoid (TT). We used this conjugate because it contains the epitope mimicked by the G1 scFv and can elicit high levels of anti–MenB CP antibodies when used in Freund's adjuvant (, ). Vaccination with plasmids devoid of a signal peptide sequence resulted in bactericidal titers that were markedly higher than those induced by the corresponding plasmids bearing such a sequence and that were similar to those of animals vaccinated with the N-Pr MenB–TT in Freund's adjuvant (). The presence of a T cell epitope sequence in the immunizing plasmid was associated with a slight increase in antibody titers, although this effect did not reach statistical significance. These data indicated that immunization with the G1 scFv gene devoid of a secretory signal sequence was effective in inducing high-level serum bactericidal activity.
Next, we verified that the antibodies induced by scFv gene immunization were directed against their intended target, that is the MenB CP. In these experiments, we focused on sera obtained from pT.scFvG1-immunized animals because these sera showed the highest titers (). The bactericidal activity of such sera was inhibited, in a dose-dependent fashion, by purified N-Pr MenB CP or the G1 scFv protein but not by a control, irrelevant scFv (designated H6; ). Moreover, bactericidal activity was observed only with encapsulated MenB strains, but not with serogroups MenA or MenC (). These data indicated that vaccination with the G1 scFv gene induced anti-scFv antibodies that specifically cross reacted against the MenB capsule, thereby producing bacterial killing. Although we have previously shown that the antibodies induced by our antiidiotypic protein did not cross react with human PSA (), it was of interest to verify that this also occurred after DNA immunization, especially in consideration that the latter can broaden the repertoire of recognized epitopes (). Therefore, sera from pT.scFvG1-immunized animals were tested against neuraminidase-treated or untreated cells from the CHP 212 human cell line expressing high levels of PSA. The Seam 26 mAb, which is known to react with both human and MenB PSA, was used as a positive control. As expected, this mAb showed strong reactivity against untreated, but not neuraminidase-treated, cells (). Immune sera were totally devoid of reactivity against either untreated or neuraminidase-treated CHP 212 cells, indicating that human PSA cross-reactive antibodies were not induced by scFv gene immunization.
Because the antibodies cross reacting with the MenB surface should be a fraction of anti–G1 scFv antibodies, in further experiments we determined whether there was a correlation between serum bactericidal activity and total anti-scFv titers. To this end, serum samples from animals immunized with either the scFv-KLH protein (from the experiments reported in ) or the pT.scFvG1 plasmid (from the experiments reported in ) were tested for antibody binding to plates sensitized with the G1 scFv. These groups of sera were chosen because they differed widely in bactericidal activity. Surprisingly, however, they did not differ in reactivity against the scFv (), suggesting that DNA immunization selectively increased the fraction of anti–G1 scFv antibodies that cross reacted with MenB and caused bactericidal activity.
To determine the isotype distribution of anti-MenB antibodies, we used an ELISA assay in which plates were sensitized with whole meningococcal cells. Preimmune sera showed some background reactivity that was mostly accounted for by IgM (). Antibody binding was significantly higher in immune sera, with a predominance of IgG2a (). These data suggested that immunization with the G1-containing plasmid induced a Th1-type response and that IgG2a, which can mediate complement-dependent bacterial killing (), likely accounted for the observed serum bactericidal responses.
To completely assess the functional properties of the antibody response induced by scFv gene immunization, we ascertained the ability of immune sera to passively protect infant rats against meningococcal bacteremia. In these experiments we measured the number of blood CFU in pups inoculated with pools of sera obtained before or after pT.scfvG1 immunization and challenged i.p. with MenB strain 2996. As positive controls, groups of pups were treated with a pool of sera from N-Pr MenB–TT–immunized animals or with the Seam 3 mAb. Pretreatment with the pT.scfvG1 immune serum pool (diluted up to 1:8) (P < 0.05) significantly protected pups from bacteremia (). These effects were similar to those observed with the serum pool from N-Pr MenB–TT–immunized animals. In contrast, animals inoculated with a preimmune serum pool or with a pool obtained after immunization with the empty plasmid were not protected (). These data indicated that immunization with pT.scFvG1 induced serum antibodies capable of affording passive protection against systemic spreading of MenB.
Capsule-based vaccines are not available for the prevention of MenB infection because the MenB CP is cross-reactive with human tissue and is not immunogenic, even after protein conjugation. This is a serious problem, because MenB strains can account for up to 80% of devastating meningococcal disease in developed countries (, ). Because distinct human cross-reactive and non–cross-reactive epitopes exist on the MenB surface (, ), it was possible to induce anticapsular antibodies and at the same time avoid the risk of autoimmunity by mimicking a single non–cross-reactive epitope of the MenB CP (). However, the antibodies induced by our surrogate antigen were of insufficient avidity and/or concentration to consistently induce bacterial killing, which is an accepted marker of vaccine-induced protection in humans. This is not surprising, because the induction of weak antibody responses has been a problem with many antigenic mimics, including peptide mimotopes or antiidiotypic antibodies ().
After exploring different strategies in this paper, it was possible to consistently induce bactericidal and protective activity in the sera of animals immunized with the gene encoding for our scFv mimic. Serum bactericidal activity was totally accounted for by scFv-specific antibodies that cross reacted with the MenB capsule, as shown by complete abrogation of killing by competing G1 scFv or MenB CP. Moreover, immune sera could not kill meningococci with MenA or MenC capsules. The antibodies induced by scFv gene immunization were predominantly of the IgG2a isotype, which is typical of a Th1 response. Finally, the induced antibodies were non–cross-reactive with human PSA.
The importance, in terms of protection in humans, of the bactericidal responses observed in our experiments after scFv gene vaccination remains, of course, to be established. It should be noted, in this respect, that bactericidal activity was tested using rabbit complement, which produces considerably higher bactericidal titers than human complement (). Nevertheless, it is encouraging that the antibodies induced by scFv gene immunization were protective in a well characterized in vivo model, such as the infant rat model.
This paper illustrates the potential of DNA vaccination–based strategies to augment antibody responses against protein mimetics. The versatility and ease of manipulation of gene vaccination allowed us to quickly and efficiently screen several approaches, including the addition of T helper epitopes (, ). Unexpectedly, the most successful strategy that resulted in markedly augmented bactericidal titers involved deletion of the secretory signal peptide sequence initially placed before the scFv (). In DNA vaccination, homologous or heterologous signal sequences are generally used to direct the antigen into the endoplasmic reticulum of the transfected cell, thus leading to secretion, with the rationale of augmenting availability of the immunogen for uptake by antigen-presenting cells. However, there are few data on the relationships between the cellular localization of the antigen and the type and extent of the immune response. In a comparative study, two plasmids either containing or lacking a signal sequence produced similar antibody levels despite differential intracellular targeting of the encoded antigen (). In another study, cytoplasmic ovalbumin induced lower IgG1 but higher IgG2a than secreted ovalbumin (). Interestingly, mouse immunization with a plasmid encoding a modified version of carcinoembryonic antigen (CEA), devoid of its signal peptide and fused to a T helper epitope, resulted in higher anti-CEA antibody levels relative to those observed using the wild-type CEA plasmid (). Further studies will be necessary to clarify whether the mechanisms underlying the effects reported in these papers, as well as in the present one, involve differential antigen uptake and/or processing by antigen-presenting cells. Irrespective of the mechanisms involved, our data strongly indicate that manipulation of secretory signals deserves further exploration in the challenging task of optimizing antiinfectious DNA vaccines. In our hands, removal of secretory signals recently proved highly effective in increasing the immunogenicity of two additional unrelated mimics (unpublished data). Thus, this strategy may be widely applicable in the field of peptide mimotopes or recombinant antiidiotypes.
Interestingly, in this study immunization with the G1 scFv gene induced total anti–G1 scFv antibody levels that were similar, by ELISA, to those induced by the corresponding protein (). Yet, as discussed above, bactericidal activity was markedly higher after gene vaccination. This suggests that (a) a heterogeneous response is induced by scFv immunization, in which only a portion of the induced antibodies is cross-reactive with the MenB CP, and (b) MenB cross-reactive antibodies are preferentially induced by gene, not protein, scFv immunization. One of the interesting features of genetic immunization, which may perhaps explain these findings, is its ability to change the hierarchy of immunodominance of the recognized epitopes relative to that induced by conventional immunization. For example, DNA encoding for the mycobacterial antigen Ag85 increased the number of recognized Ag85 T cell epitopes over those recognized after immunization with live bacteria and changed the hierarchy of immunodominance in favor of the newly recognized epitopes ().
Studies are underway to determine the epitope specificity and relative frequency of B cell and T cell subpopulations activated by scFv gene, compared with protein, immunization. We are also testing the hypothesis that the increased functional activity of the antibodies induced by gene vaccination is related to the adjuvant-like properties of the immunizing plasmids (). Interestingly, major adjuvant-dependent differences have been documented in the ability of N-Pr MenB, the antigen mimicked by our scFv, to induce bactericidal activity. For example, N-Pr MenB–TT induced bactericidal activity when given in Freund's adjuvant (, ) but not in alum (). In contrast, a conjugate of N-Pr MenB with porin B, a protein with adjuvant-like properties (), could induce bactericidal activity using either adjuvant ().
Our data are in general agreement with previous reports dealing with immunization with minigenes encoding for peptide mimotopes. DNA vaccination was recently used to redirect the immune system from a Th2 to a more effective Th1 response (). Moreover, a similar approach was successful in inducing serum bactericidal activity against MenC CP () and antibodies directed against the type 4 pneumococcal CP ().
Collectively, these data indicate that DNA immunization offers new ways of stimulating the immune system and suggest that these features can be exploited in the prevention of diseases caused by encapsulated bacteria. This is especially relevant for infections, such as those caused by MenB, for which no vaccine is available because of the failure of conventional approaches. Moreover, despite considerable success, existing conjugate vaccines are not without problems, including complexities in polysaccharide production and conjugation. These difficulties can become particularly challenging with vaccines consisting of multiple conjugates. In contrast, it seems relatively easy to clone different mimics in a single vector for vaccinating against pathogens with multiple serotypes. Thus, the global use of vaccines directed against encapsulated bacteria would be facilitated by the development of effective DNA vaccines, especially in consideration of some additional advantages such as their low cost and independence from the cold chain.
Meningococcal strains 2996, 8047, and MC58 (MenB), F8238 and A1 (MenA), and C11 (MenC) were provided by M.M. Giuliani (Chiron Corp., Siena, Italy). Purified N-Pr MenB CP and the Seam 3 and Seam 26 mAbs were provided, respectively, by A. Bartoloni and M. Mariani (Chiron Corp., Siena, Italy). The G1 and the irrelevant H6 scFvs were expressed recombinantly in and purified as previously described (, , ). For immunization experiments, the G1 scFv–KLH and the N-Pr MenB–TT conjugates were prepared as previously described ().
To generate plasmids for DNA vaccination (), we used pCI-neo, a mammalian expression vector (Promega). The G1 scFv–encoding sequence was PCR-amplified as previously described (–) and ligated into the multiple cloning site of pCI-neo, generating pscFvG1. In the design of pS.scFvG1, the leader sequence was inserted at the beginning (i.e., at the 5′ flanking site) of the G1 scFv gene. To produce the pT.scFvG1, a tetanus toxin universal T helper cell epitope () was inserted at the beginning of the scFv. Finally, a plasmid (pST.scFvG1) was generated containing both the secretory sequence and the T helper cell epitope before the scFv gene.
Plasmids for in vitro transfection or mouse immunization were grown in DH5α and purified using EndoFree Plasmid Maxi or Giga kits (QIAGEN). Each lot of plasmid DNA had a A/A ratio ≥1.8 (as determined by UV spectrophotometry), endotoxin content ≤0.1 EU/μg DNA (as determined by Amebocyte Lysate assay kit; Associates of Cape Cod Inc.), and a predominantly supercoiled form.
The ability of engineered DNA constructs to express functional G1 scFv was analyzed by flow cytometry. A subconfluent monolayer of COS-7 cells (CCL-70, a monkey kidney fibroblast cell line; American Type Culture Collection) was transiently transfected with 2.5 μg of vector DNA per 10 cells in a synthetic cationic lipid solution (TransFast; Promega). The pCI-neo mammalian vector (empty vector) was used as a control. After 48 h, the transfected cells were washed in PBS (0.01 M phosphate, 0.15 M NaCl, pH 7.2), trypsin treated, and fixed overnight with 1 ml paraformaldehyde (2.5 mg/ml). The cells were then permeabilized with 1 ml PBS containing 0.2% (vol/vol) Tween 20 (PBS-Tween) and incubated with the Seam 3 mAb (4 μg/ml in PBS) for 2 h at 37°C. 10 μg/ml FITC-labeled rabbit anti–mouse IgG (Abcam Ltd.) was used to detect bound Seam 3.
For DNA immunization, 6–8-wk-old BALB/c mice (Charles River Laboratories) were injected in the quadriceps muscle with purified DNA at the doses indicated in the figures in 50 μl of PBS. Mice were immunized on days 0, 21, and 42 with equal plasmid doses, and tail veins were bled on days 0, 36, and 56 to obtain sera. Moreover, groups of mice were immunized with scFv G1–KLH or with N-Pr MenB–TT conjugates in Freund's adjuvant, as previously described ().
The bactericidal assay was performed as previously described () with minor modifications. In brief, bacteria were grown to the early stationary phase and mixed in equal volumes with serially diluted (ranging from 1:3 to 1:768) heat-inactivated serum and undiluted baby rabbit complement (Cederlane). The reciprocal of the highest final serum dilution causing (50% killing of the inoculum was recorded as the bactericidal titer. Because the lowest final serum dilution tested was 1:9, the lower limit of detection of the assay was a titer of 9. To assess inhibition of bactericidal activity, mixtures of twofold serial dilutions of inhibitor and diluted serum were incubated for 20 min at 37°C. After adding complement and bacteria, the test was completed as described above.
To determine the isotype distribution of anti–MenB antibodies, we used a whole bacteria ELISA (). Anti–human PSA antibodies were detected by ELISA, using untreated or neuroaminidase-treated neuroblastoma CHP 212 cells, which express high levels of PSA. Both of these assays were performed exactly as previously described (). Binding of serum antibodies to the scFv G1 was measured by an identical ELISA test, with the exception that plates were sensitized with 2 μg/ml of purified scFv.
To study the protective effects of sera obtained from immunized animals, we used an infant rat model, exactly as described previously (). In brief, 5-d-old Wistar rats (Charles River Laboratories) were inoculated i.p. with serially diluted mouse sera and, 2 h later, challenged i.p. with 8 × 10 CFU MenB (strain 2996). Blood samples were obtained at 18 h after challenge, serially diluted, and plated onto chocolate agar (100 μl/plate). Because the lowest plated dilution was 1:10, the lower detection limit of the assay was 100 CFU/ml of blood. Pups were considered protected from bacteremia in the presence of sterile blood cultures. All animal experiments reported in this paper were approved by the Department of Pathology and Experimental Microbiology Committee for Animal Studies and Istituto Superiore di Sanità .
Bactericidal titers were converted to log titer values to calculate means and SDs and to assess statistical significance using one-way analysis of variance (ANOVA) and Student-Keuls-Newman test. For the purpose of calculating means and SDs, sera with bactericidal activities below the detection threshold (i.e., with a titer <9) were given an arbitrary titer of 4.5 (i.e., half the lower detection limit). Differences in the frequency of protected animals were analyzed using Fisher's exact test. |
Skin biopsies were obtained pretransplant and on day 0 to assess the effect of RIT and FIT regimens on LC density. Intact epidermal sheets were examined by confocal microscopy as shown in . The natural arrangement of the LC network in the epidermal plane is readily appreciated from a collapsed Z-stack image. Enumeration was precise (10.3% median variation between two 40× fields) and sensitive (minimum 5 LC per mm). RIT had very little effect on LC density as illustrated by ; overall, there was a 9% downward trend from 644 to 588 LC/mm (P = 0.061; ). FIT induced a 55% fall in LC from a median of 654 to 296 LC/mm (P = 0.001; ), a significant drop but far from complete ablation of the population. Patients undergoing FIT were younger: median age was 34 (range: 20–54) compared with RIT, for which the median age was 43 (range: 21–57; P = 0.020). Both had slightly reduced pretransplant counts compared with normal controls (754 LC/mm), but this difference was not significant. There was no difference in day 0 counts with respect to alemtuzumab in conditioning regimens (not depicted), consistent with the observation that LC do not express CD52 (, ).
The hypothesis that recipient LC are essential in the afferent arm of acute GVHD is difficult to prove in humans without an interventional study (). The retention of significant recipient LC on the day of transplantation is a necessary condition, but other APCs such as dermal DCs may also play a role in sensitizing donor lymphocytes in the draining lymph nodes. Although LC are depleted in GVHD (–), this reflects the efferent phase of GVHD (see next paragraph) and cannot be used as evidence of their role in GVHD induction.
Most LC survive RIT conditioning and, although cytokine activation may be diminished in RIT compared with FIT (), the greater survival of recipient APCs may offset this benefit. Indeed, some non T cell–depleted RIT regimens have a delayed yet high cumulative incidence of acute GVHD, approaching that of conventional transplants (, ).
The prospect of attenuating acute GVHD by depleting recipient APCs was first suggested >20 yr ago (). Our data imply that there is scope to accelerate or enhance LC depletion by novel conditioning therapies, such as UV light or monoclonal antibodies to DCs (, ). Others have argued that complete ablation of recipient APCs might abolish graft versus leukemia (GVL) effects (), but a selective benefit might be gained by regional therapy of a GVHD target organ such as the skin.
During recovery, LC density declined to a similar nadir during 14–21 d in both types of transplant (). Although precise resolution of this phase is hampered by access to specimens, it suggests that the earlier difference between RIT and FIT seen at day 0 is partly kinetic (the response to RIT may be delayed with respect to FIT owing to the late scheduling of melphalan in RIT). Conditioning has a protracted effect throughout 14–21 d, suggesting that the tissue response to injury and dynamics of resident APC populations is relatively slow or that the absence of myeloid precursors during the hypoplastic phase further promotes LC depletion ().
After 28 d, there was no statistical difference between RIT and FIT patients; so, the data were reanalyzed according to cutaneous GVHD at the time of biopsy (). Patients without GVHD recovered pretransplant median LC density by 40 d (537 LC/mm compared with 649 LC/mm pretransplant; P = 0.107), whereas those with GVHD remained significantly depressed at both 40 d (156 LC/mm; P = 0.018) and 100 d (404 LC/mm; P = 0.006). This suggests that the effect of conditioning, in which migration of one or more DC types primes the afferent arm of GVHD, is a distinct process to the depletion of LC that occurs later as a result of GVHD effector mechanisms or corticosteroid therapy. In earlier studies, the high rate of early acute GVHD or “engraftment syndrome” at days 14–21 may have obscured this distinction (–).
Congenic markers are not universally applicable to human transplants; therefore, genotype analysis must be used. In situ methods were avoided, following the observation that extensive activation of LC membrane occurs in the posttransplant period (). An alternate method of single cell genotyping was preferred in which cells migrating from epidermal sheets in vitro were subjected to two-step Giemsa/fluorescent in situ hybridization (FISH). The principal constituents of migratory cell preparations obtained in this way are keratinocytes and CD1a-positive LC with <1% T cells or macrophages (). LC can be accurately identified by Giemsa staining alone (); this was confirmed in preparatory experiments by comparison with fluorescent CD1a or DR staining (not depicted). Approximately 20% of LC were recovered by migration. Although in vitro culture can exert selective effects, there is no a priori reason to suspect that either donor or recipient cells will be favored.
Many patients at 100 d after transplant had full donor LC chimerism as illustrated in . At 40 d after transplant, it was possible to detect mixtures of male and female cells as shown in . At 1 yr, a minority of patients had occasional recipient cells. shows an example of 1 of 12 recipient cells detected in 1,746 interphase nuclei examined, a sensitivity of <1% for recipient LC.
A cohort of 32 patients (14 FIT and 18 RIT) was examined at 40 d, 100 d, and 1 yr after transplant (). At 40 d, the recovery after FIT was predominantly donor (median: 97% donor; range: 89–100%) compared with significant persistence of recipient cells after RIT (median: 36.5% donor; range: 0–85%; P = 0.004). However, by 100 d, the majority of LC in all transplants were donor in origin with a median score of 100% (range: 93–100%) after FIT and 97.5% (range: 90–100%) after RIT (P = 0.133). At 1 yr, both groups achieved median scores of 100% (overall range: 97–100%). Assuming recipient APCs are required to prime donor T cells for acute GVHD (, ) and cellular GVL responses (), these events must occur within the first 100 d of fludarabine-mephalan RIT and probably within the first 40 d of conventional FIT.
The delay in donor engraftment of LC in RIT is in contrast with rapid blood myeloid engraftment in all transplants by day 30 (). As expected, there was a trend for higher LC engraftment at day 100 in patients with complete myeloid chimerism (P = 0.080; ). However, three patients with a delayed lapse of blood myeloid chimerism at 60–100 d (associated with declining T cell engraftment) did not suffer a similar reversal of LC engraftment, although none achieved 100% chimerism. This suggests there is a posttransplant window for LC engraftment (which may be regimen-dependent) beyond which LC are able to maintain relative independence from the blood myeloid compartment.
Donor T cells promote LC engraftment in murine transplantation, under conditions of strict T cell depletion and MHC class II incompatibility (). This effect was not evident in the human setting of in vivo T cell depletion and geno-identical transplant either in the whole cohort (P = 0.759; ) or the RIT ( = 16) or sibling RIT subgroups ( = 12; not depicted). However, a significant correlation between prior cutaneous GVHD and LC engraftment was observed (P = 0.002; ). The induction of cutaneous GVHD, which was associated with donor T cell inoculum in the mouse, is a potential unifying mechanism for many variables that increase alloreactivity and enhance LC engraftment (). Although we could not resolve a direct effect of donor T cells, the correlation of GVHD with donor chimerism confirms that alloreactivity is relevant to LC engraftment in humans. Two implications of this argument are that the priming phase of GVHD will be self-limiting and that the strategy of delayed T cell add-back () will be compromised unless adjunctive treatment, such as UV light, is given to ensure complete LC chimerism ().
Our results show that transplant regimens in clinical use promote high levels of LC engraftment by day 100. There was no difference in event-free survival between patients who achieved 100% donor chimerism and those who did not, supporting the concept that GVHD and GVL are distinct but overlapping processes (Fig. S1, available at ). RIT patients have lower LC donor chimerism at day 40, but often first experience acute GVHD between 60–100 d during immunosuppression withdrawal. We have argued that GVHD or alloreactivity is important in promoting LC engraftment and predict that this is increasingly important as the intensity of conditioning is reduced. To test this, it would be informative to examine LC chimerism in minimal conditioning RIT regimens () in which acute GVHD manifestations are even further retarded ().
The observation of high donor chimerism at 100 d is consistent with the hypothesis that the transition of acute to chronic GVHD at 100 d is related to indirect antigen presentation by donor APCs (, ). However, it now seems unlikely that the increased toxicity of early DLI is related to persistence of recipient APC (, ), unless cells that have migrated have extended longevity in lymphoid tissue. An alternative explanation worthy of consideration is that active tolerance mechanisms evolve to render DLI increasingly safe with time.
Consecutive patients who gave consent for study between April 2003 and June 2005 were recruited from four centers in the United Kingdom: Newcastle; Leeds; Christie Hospital, Manchester; and The Royal Free Hospital, London. 4-5-mm skin biopsies were performed at routine day 100 bone marrow aspiration from the posterior iliac crest. Additional 2- or 4-mm biopsies from the posterior iliac crest were obtained from patients transplanted at Newcastle at varying intervals. 184 skin biopsies were obtained from 76 patients. 48 patients received RIT, the majority with fludarabine, melphalan, and alemtuzumab or fludarabine and melphalan (). 28 patients received FIT, the majority with a total body irradiation regimen () or busulfan and cyclophosphamide () with or without alemtuzumab. All unrelated transplants, either RIT or FIT, received a regimen containing alemtuzumab. There were 44 HLA-identical siblings and 32 HLA-matched unrelated donors. GVHD prophylaxis consisted of pulsed methotrexate and cyclosporine or cyclosporine alone if a patient had received alemtuzumab. All patients were in remission or first chronic phase at transplantation. The median interval between prior therapy and transplant was 3 mo (range: 1–10 mo); three patients treated within 3 mo of transplant had received systemic corticosteroids. Full details of the patients used for LC chimerism analysis are included in Table S1 (available at ). Normal control skin was obtained from patients undergoing plastic surgery. All procedures were performed with informed consent and ethical approval obtained from Newcastle and North Tyneside Local Research Ethics Committee, Leeds Health Authority Local Research Ethics Committee, South Manchester Local Research Ethics Committee, and Royal Free Hospital and Medical School Ethics Committee. Clinical chimerism analysis on bone marrow, whole blood, myeloid (CD15), and T cell (CD3) fractions was performed according to local protocols using magnetic bead fractionation and X/Y FISH or PCR of short tandem repeats. GVHD was assessed clinically at the time of biopsy or from case notes. Cutaneous GVHD stages 1–2 was treated with topical betamethasone valerate 0.122%; cutaneous GVHD stages 3–4 or any acute GVHD grades II–IV was treated initially with 2 mg/kg intravenous methylprednisone and then oral prednisone 1 mg/kg tapered according to response.
Skin was trimmed of excess dermis and incubated at 37°C for 60–90 min in RPMI 1640 (Invitrogen) with 1 mg/ml dispase (Invitrogen). Epidermal sheets were separated, fixed in acetone for 15 min, and rehydrated in PBS (Cambrex) for 15 min. Anti-CD1a monoclonal NA 1/34 (DakoCytomation) was used at 1/10 dilution for 2 h at room temperature or overnight at 4°C. Specimens were mounted in VectaStain containing DAPI (Vector Laboratories) and analyzed with a Leica TCS SP2 UV confocal microscope and LCS V 2.51 imaging software (Leica). Typically, 12–16 images were acquired over 20–30-μm depth at pixel resolution 1025 × 1025, 40× power, pinhole 1.5–1.8, and voltage offset 350–500 V. In most samples, counts were the mean of two 40× fields selected at random in the interfollicular epidermis (total area: 750 μm).
LC were allowed to migrate from 4-mm punch epidermal sheets by floatation on 400 μl of X-Vivo 10 medium (Biowhittaker) supplemented with 500 IU/ml GM-CSF (Peprotech) in a 48-well culture plate (Nunc) incubated at 37°C for 60 h. In sex-mismatched transplants, migrant cells were harvested onto cytospin slides at 800 revolutions/min for 4 min (Thermo Shandon) for FISH. Migratory cells from sex-matched transplants were analyzed by flow cytometry as described previously (). Approximately 1,000 cells were obtained from most biopsies; examination of the epidermal sheet after migration revealed none remaining. Cytospin slides were air-dried and either stained immediately with an automated stainer (brighter autofluorescence but weaker FISH) before storage at −20°C or stored first and stained manually after thawing with Leishman and Giemsa stains 10:1 (BDH) for 5 min at room temperature (less autofluoresence but better FISH). About 20 × 20 Giemsa-stained fields (100–300 LC) were located captured and annotated using Applied Imaging Cytovision software. Slides fixed for FISH in methanol/acetic acid 3:1 for 5 min, probed with CEP X/Y DNA probes (Vysis) according to manufacturer's instructions and mounted with VectaStain containing DAPI (Vector Laboratories). Giemsa-stained fields were reexamined and LC genotype scored. FISH images were recorded using Applied Imaging Smartcapture software.
Mann-Whitney tests were run on SPSS 12. Images were processed with Adobe Photoshop 7.0. Cytogenetic images were captured at high power and assembled as montages of several fields for clarity of comparison with Giemsa images. Square cropping and normalizing the background were the only image adjustments made.
Table S1contains patient characteristics. Fig. S1 shows event-free survival according to LC engraftment. Online supplemental material is available at . |
The identification of a B-1 cell hybridoma clone (IgM ) that binds ischemic tissue in the murine intestinal and skeletal muscle models of RI provided support for the hypothesis that ischemic tissue was altered relative to normal tissue and that neoepitopes expressed or exposed during ischemia were targets for an innate response to self (, ). Moreover, the availability of a specific IgM provided a means to identify the neoepitope. The rationale was that circulating IgM would bind self-antigens exposed during ischemia and that these complexes could be isolated and the antigens identified by proteomic techniques. RAG-1 mice were reconstituted with an optimal amount of IgM , treated for intestinal ischemia, and reperfused for 0 or 15 min before harvesting of tissues. Immune complexes of IgM antigen were isolated from lysates of jejuneum at the two time points and fractionated by SDS-PAGE under reducing conditions. Analysis of the stained gels indicated common bands at varying molecular masses for all time points, including the sham control (). However, at 15 min an apparent unique band at high molecular mass (∼250 kD) was identified (). Protein bands were excised from stained gels and enzymatically digested, and peptides were analyzed by tandem mass spectrometry as described previously (). Analysis of eluted peptides indicated that the common bands at ∼25, 50, and 75 kD represented immunoglobulin light chain and IgG and IgM Hc, respectively. The IgG Hc band at 50 kD was identified as goat and most likely represents Ig eluting from the beads, whereas the 75-kD band represents murine IgM Hc. Analysis of the high molecular mass band yielded peptide sequences homologous to NMHC-II isoforms A and C (). In similar experiments using lysates prepared from WT mice treated for 3 h in intestinal RI, a similar size band at 250 kD was also observed and sequence analysis identified NMHC-II A and C peptides (unpublished data).
Three forms (A, B, and C) of NMHC-II have been identified in the mouse and human genomes (, ). All eukaryotic cells express NMHC-II, but the distribution of the three isoforms varies. NMHC-II A and B are ∼85% homologous, whereas NMHC-II C is ∼65% similar to A and B (). The three isotypes are highly conserved among mice and humans. As a further test for binding of IgM to NMHC-II, an ELISA approach was followed. Plates were coated with antibody specific for each of the three forms of NMHC-II or with a pan-myosin antibody to capture the relevant antigen from lysates prepared from jejuneum of RAG-1 mice. Subsequently, IgM (or IgM ) was added and developed with a labeled anti–mouse IgM antibody. Above background binding of IgM but not IgM to all three of the isoforms of NMHC-II was observed (). The combined sequence analysis and ELISA results suggest that IgM recognizes a conserved region of the type II NMHC. As a further test of whether myosin is exposed to circulating antibody after ischemia, RAG-1 mice were reconstituted with a purified IgG fraction of rabbit anti–pan-myosin Hc. No evidence of rabbit IgG deposition was observed in tissues of reconstituted but sham-treated mice (unpublished data). In contrast, ischemic RAG-1 mice reconstituted with the myosin-specific IgG before reperfusion developed significant RI compared with saline controls (33 ± 11 vs. 11 ± 8; P < 0.05; ). These results provide further support that myosin is exposed to antibody in circulation after ischemia. However, they did not identify the epitope within NMHC-II.
As an alternative approach to identify the IgM target self-antigen, an M13 phage display library of random 12-mer amino acid sequences was screened with the specific IgM. After four rounds of positive selection with IgM and two rounds with negative selection with a control IgM (clone CM-75), 10 phage clones were isolated and the nucleotide sequence of the relevant M13 gene was analyzed. Notably, all 10 clones bore a codon sequence rich in asparagine. Four best binders to IgM were selected and one of these clones, P8 (which bound with the highest efficiency), was further characterized ( and ). A 12–amino acid peptide (P8) was synthesized based on the phage sequence and assayed for inhibition of phage P8 binding to IgM (). Titration of increasing amounts of P8 peptide yielded 50% inhibition at an estimated concentration of 10–100 μM. This assay indicates a reasonable overall avidity of binding based on multiple binding sites expressed on the phage surface. This result also suggests that IgM binding to phage P8 is specific for the peptide region and that the synthetic peptide could be used as a mimetope for the actual antigen. To further characterize binding of P8 peptide to IgM , ELISA plates were coated with the peptide and tested with varying concentrations of IgM or control IgM for binding (). At the lower concentration of 1 μg/ml, neither IgM bound above background. However, at 10 μg/ml significantly more IgM bound than IgM (P < 0.05).
To test whether synthetic peptide P8 blocked IgM in vivo, antibody-deficient (RAG-1) mice were reconstituted with pathogenic IgM with or without the peptide. Previous studies had demonstrated that intestinal RI in RAG-1 mice was IgM-dependent and that IgM alone was sufficient to restore injury (, ). As expected, reconstitution of RAG-1 mice with IgM but not saline before reperfusion resulted in RI (, A [i] and B). In contrast, mixing of IgM with P8 peptide before injection in ischemic mice significantly blocked apparent injury (mean pathology score = 6 ± 3 vs. 31 ± 13; P < 0.001; , A [ii] and B). Previous titration of peptide with IgM suggested that an optimal concentration of ∼10 μM of peptide was sufficient to block 200 μg IgM (0.1–0.2 μM; unpublished data). Immunohistological analyses of serial sections of reperfused intestinal tissue (jejuneum) after RI identified colocalization of IgM and complement C4 and C3 within the microvilli in RAG-1 mice reconstituted with IgM but no deposition in sections prepared from mice receiving P8 peptide (unpublished data). No binding of IgM or complement was observed in IgM –reconstituted sham controls, nor in RAG-1 mice reconstituted with control IgM or RAG-1 mice reconstituted with saline only (unpublished data) (). Thus, peptide P8 appears to block binding of IgM and induction of injury in vivo.
To test whether peptide P8 represented a mimetope for a major self-antigen, WT mice were pretreated with peptide P8 (serum concentration of ∼10 μM) 5 min before reperfusion in the intestinal model. Analysis of jejuneum tissues of mice treated with a similar concentration of control peptide or saline before reperfusion identified significant injury to the microvilli, as expected (P < 0.05; , iii). In contrast, pretreatment of WT mice with peptide P8 5 min before reperfusion blocked apparent injury relative to saline or control peptide (mean pathology score = 5 ± 3 vs. 24 ± 16 and 23 ± 19, respectively; P < 0.005 and P < 0.027, respectively; , A [iv] and B). As expected, IgM, C4, and C3 colocalized within microvilli of RI-treated WT mice but not in mice administered P8 peptide (unpublished data). These results suggest that the number of key epitopes required to initiate RI is limited as a single peptide blocks injury and deposition of IgM and complement.
Comparison of the sequences of the three NMHC-II isoforms with the P8 peptide sequence identified one region of apparent homology (). All three isoforms include a motif of NxxxxNxNx that suggested similarity with the P8 sequence. A 12–amino acid synthetic peptide (referred to as N2) sequence (represents the NMHC-II C isoform) was prepared for further study. To test whether this region bound to IgM , surface plasmon resonance (SPR) analysis was used (). N2 peptide was injected over a surface coupled with IgM () and generated a robust response, which corresponded to a KD of 123 ± 61 μM (mean ± SD; = 2) as calculated from the steady-state response levels (). In contrast, no binding was observed when a control peptide was injected over the specific IgM-coupled surface () or when the N2 peptide was injected over a surface coupled with the IgM control IgM ().
To test whether NMHC N2 peptide represents the major self-epitope in intestinal RI, WT mice were treated with saline or increasing concentrations of the synthetic peptide (final serum concentrations = 8, 16, 32, or 40 μM, respectively) 5 min before reperfusion in the intestinal model. Histological analysis of tissue sections of saline-treated WT mice identified injury that correlated with deposition of IgM and complement as expected (, A [i], B, and D [i–iv]). In contrast, treatment of WT mice with an increasing concentration of N2 peptide demonstrated a dose-dependent reduction in injury (mean pathology scores: saline, 22 ± 17% [ = 9]; 8 μM, 21 ± 7% [ = 3; P < 0.40]; 16 μM, 17 ± 5% [ = 3; P < 0.20]; 32 μM, 8 ± 6% [ = 3; P < 0.03]; and 40 μM, 7 ± 4% [ = 7; P < 0.01]) (, A [ii] and B). Thus, significant protection from injury was achieved with 32- and 40-μM concentrations of N2 peptide. Interestingly, P8 peptide was protective at a lower concentration (10 μM), correlating with its higher CM-22 binding affinity relative to N2.
To further evaluate the protection by N2 peptide, mice were characterized for intestinal permeability. In this assay mice are administered horseradish peroxidase (HRP) enterally 10 min before ischemia, and uptake into circulation is measured after RI treatment. Hart et al. recently reported using a similar assay to measure intestinal permeability in RI-treated mice but administered FITC–dextran (). Our results indicate significant uptake of HRP into circulation in WT mice treated with saline versus sham controls (195.4 ± 51.7 vs. 31 ± 17.3 [ = 8 and 5, respectively]; P < 0.05; ). In contrast, mice pretreated with an optimal amount of N2 peptide before reperfusion develop significantly less permeability than saline-treated mice (79.8 ± 30 vs. 195.4 ± 51.7 [ = 10 and 8, respectively]; P < 0.05; ). Thus, pretreatment of mice with N2 peptide protects from both histological injury and intestinal permeability.
Protection from injury correlated with a reduction in deposition of IgM and complement in mice treated with 40 μM N2 peptide (, v–viii). Notably, among the control mice not all animals developed full injury even though treatment with N2 (or P8) peptide gave substantial protection. We speculate that the range in injury observed among WT mice is caused by a variation in serum levels of specific natural IgM because this pattern is not observed in RAG- 1 mice treated with IgM (). Moreover, IgM and complement deposition correlate with the level of injury; i.e., WT mice with a low injury score have low level of IgM (unpublished data).
Previous reports identified vascular permeability of labeled albumin as a measure of severity of intestinal RI (, , ). To evaluate further the effects of treatment with N2 peptide in the intestinal RI model, we used an intravital approach combined with a novel miniaturized laser scanning confocal microscope to image leakage of microvessels within the villi of the jejunum. In this approach anesthetized mice are administered a fluorescent-labeled vascular probe (AngioSense 680) that circulates within normal blood vessels for up to 2 h. Besides its stability in circulation, an advantage of this novel probe is that it emits in the near infrared and, thus, imaging is in the low energy range. To image circulation within the microvilli, a scanning confocal stick lens (approximately the diameter of a biopsy needle) is inserted within a small incision in the wall of the jejuneum and placed in direct contact with the mucosal surface. Contrast is provided by pretreatment of mice i.v. with Rhodamine 6G, which is taken up by cells, especially the enterocytes lining the microvilli. Mice are injected with the vascular probe 5 min before reperfusion, and imaging is initiated 15 min after reperfusion. For example, among the sham controls, the blood vessels within the lamina propria of microvilli are identified by the circulating vascular probe (red), which is contrasted by the green stain of the enterocytes (Rhodamine 6G; , i). In experimental mice, significant leakage of the vascular probe is observed as early as 20 min after reperfusion, as indicated by the presence of red dye within the lamina propria and surrounding target tissues. The mean ratio of vessel (V) to background (B) is 149 ± 14 versus 16 ± 4 in arbitrary units for sham controls and saline-treated mice, respectively (P < 0.001; , A [ii] and B). In contrast, limited leakage of the vascular probe was observed in experimental mice pretreated with N2 peptide (153 ± 4 vs. 149 ± 14 units for N2- and saline-treated mice, respectively; P < 0.001; , A [iii] and B). Thus, administration of an optimal amount of N2 peptide 5 min before reperfusion effectively limited leakage of the vascular probe.
The identification of a single self-peptide that blocks intestinal RI in WT mice led to the general question of whether the N2 region of NMHC-II was also the target for pathogenic IgM among other ischemic tissues. It might be predicted that the number of antibodies specific for ischemic tissue is limited based on the current understanding that the repertoire of natural IgM is relatively small (–) and putatively selected through evolution (, ). Moreover, ligands of natural IgM are considered highly conserved structures and are probably limited in number.
To test whether NMHC-II represents the self-ligand target in the murine hindlimb model, mice were treated for 2 h of ischemia followed by 3 h of reperfusion as described previously (). As expected, WT mice administered saline or 40-μM control peptide () developed considerable injury. In contrast, injury was significantly reduced in mice administered 40 μM (final serum concentration) of N2 peptide before reperfusion (mean number of injured muscle fibers per 50 counted = 25 ± 4 and 22 ± 3 for saline and control peptide, respectively, vs. 12 ± 1 for N2 treated; P < 0.01 and P < 0.05, as determined by Student's test between saline and control peptide and N2 and control peptide, respectively; P < 0.05 for all groups, as determined by analysis of variance [ANOVA]) (). Thus, these results suggest that, as in the intestinal model, NMHC-II is the major self-ligand in ischemic skeletal muscle.
The recent identification of a natural antibody (IgM ) that initiates intestinal and skeletal muscle RI in antibody-deficient mice has provided an important reagent to isolate and characterize the self-antigen involved in initiation of acute inflammation (, ). In this paper, we provide five lines of evidence that support the identification of the self-antigen as a highly conserved region (represented by N2 peptide) within NMHC-II. Notably, pretreatment of WT mice with N2 peptide (or the mimetope P8) after ischemia but before reperfusion dramatically reduced RI within both intestinal and skeletal muscle models.
15 types of myosin Hc have been identified to date. Like other myosins, the NMHC-II form hexamers of two Hc (∼250 kD each) and two pair of regulatory light chains (20 and 17 kD each). Functionally, they all bear ATPase activity, form molecular motors within the cell, and are thought to be important in regulating cytokinesis, cell motility, and cell polarity (). Of the three isoforms, little is known of the functional importance of the recently identified NMHC-II C.
Studies in cell culture and in isolated hearts made ischemic and reperfused in the absence of blood cells or sera reveal evidence of cell injury to cardiomyocytes, suggesting that factors independent of inflammation can lead to cell injury (, ). One well-characterized pathway is poly(ADP ribose) synthetase (PARS), which is a nuclear enzyme involved in DNA strand break repair (). Damage to chromatin by free radicals resulting from ROS, which form during hypoxia, leads to activation of PARS that has multiple effects, including depletion of mitochondrial ATP and eventual cell injury. Although the effects of hypoxia on NMHC-II have not been reported, one possible link with generation of ROS and activation of the PARS pathway is that nonmuscle myosins, which are involved in maintaining cell morphology, migrate to the outer membrane after cell injury (). Thus, a general explanation for the combined pathways is that NMHC-II is mobilized to the cell surface by hypoxia-related events resulting in transient exposure of the N2 region on the outer cell surface of endothelium and muscle cells. This exposure would provide a target for circulating pathogenic IgM resulting in enhancement of cell injury beyond that caused by endogenous intracellular pathways such as PARS. We propose that the NMHC-II epitope is exposed on hypoxic cells and not released as a result of cell death. If the latter were the case, multiple other self-antigens would be released and also serve as targets for circulating natural IgM, which binds many intracellular antigens. Based on the direct visualization of vascular leakage, it seems most probable that injury is initiated at the endothelium surface.
Expression of novel cell surface molecules in response to stress is not unprecedented, although examples are generally based on in vitro study. For example, a recent report identified mouse and human NKG2D ligands up-regulated in nontumor cell lines by genotoxic stress and stalled DNA replication (). In their in vitro model, expression of the novel NK receptor ligands was dependent on the ataxia telangiectasia, mutated pathway. Thus, they propose “a novel link between the immune response and processes that regulate genome integrity” (). Alternatively, in our system, NMHC-II could be released into circulation or the extracellular space and bound by antibody. Attempts to measure specific release of NMHC were unsuccessful to date. As more specific reagents are developed, it will be important to identify the kinetics of exposure/release by hypoxic cells. Thus, in the absence of IgM as in RAG-1 mice or peptide blocking of IgM with N2 or P8, we speculate that cell injury is reversible at least within the conditions of ischemia used in the current experiments. Of course, prolonged hypoxia would lead to irreversible cell injury and cell death independent of the innate immune system.
Although our experiments suggest that NMHC-II is the major antigen exposed during reversible RI, other epitopes are likely exposed on the surface of injured endothelium but in the absence of specific natural IgM are insufficient to mediate inflammation. Support for additional antigens comes from recent reports by Fleming et al.(, ). Using a similar model of mesenteric RI, they reported that murine and human antibodies specific for phospholipids (PLs) and/or β-2 glycoprotein could restore injury in Cr2 or RAG-1 animals. Because the murine anti-PL and β-2 antibodies are of the IgG subclass and were isolated from autoimmune mice (NZW × BxSB F1), they likely represent high-affinity antibodies that arose as a result of autoimmune disease. Similarly, IgG antibodies from 5-mo-old but not 2-mo-old B6.MRL/lpr autoimmune-prone mice are pathogenic in RI-treated RAG-1 mice. Whether natural IgM of similar specificity occur in nonautoimmune animals is unclear. However, given that pretreatment of WT mice with N2 or P8 peptides blocks injury in our two models, antibodies specific for self-antigens, such as PL or β-2 glycoprotein I, are not likely involved in the initiating event. However, after induction of RI it is clear that many additional self-antigens are released and become targets for self-specific natural IgMs. This could explain the observation by Fleming et al. () that mesenteric ischemic in the B6.MRL/lpr strain is more severe than WT controls as they express a higher level of self-reactive antibodies of both IgM and IgG subclasses ().
The pharmokinetics of N2 peptide are yet to be completely elucidated, but an initial serum concentration of ∼32–40 μM is sufficient to block substantial injury in ischemic RAG-1 mice administered IgM or WT mice over the 3 h of reperfusion. Because the peptide likely has a very short half-life in serum, it either binds with sufficient high affinity to the IgM to inhibit recognition of ligand over the complete period of reperfusion, or the ligand is only exposed transiently on reperfusion and peptide is only required after initial reperfusion. The kinetics of peptide binding determined by Biacore assay suggest that the former is unlikely and, therefore, we favor the latter possibility. Earlier studies in a rat myocardial RI model identified increased myocardial injury 7 d after ischemia (). Injury was complement dependent, as pretreatment with a soluble inhibitor of C3 considerably reduced late injury. A limitation of the intestinal model is that injured microvilli are replaced by newly migrating enterocytes that emanate from stem cells in the crypts (). Although it seems likely that pretreatment with peptide will block late injury as it does acute, these studies will be important to follow up in the skeletal or cardiac muscle models.
Given the highly conserved nature of the N2 region, it is possible that exposure of other forms of myosin Hc, such as smooth muscle, could also serve as a target for natural antibody. However, it is notable that only NMHC-II A and C isoforms were identified in the immune precipitation analyses, suggesting that these are the major antigens exposed/expressed during ischemia, at least in the intestine. It will be important to characterize the IgM–self-antigen complexes in the hindlimb model. Natural IgM, which is a product of innate lymphocytes (i.e., B-1 cells), is considered a component of innate immunity because of its germ line–encoded repertoire, absence of somatic mutation, and specificity for structures highly conserved among both prokaryotes and eukaryotes (–). Therefore, it is not surprising that a highly conserved region of myosin could also serve as a target for natural IgM.
It is proposed that natural antibody recognition of the N2 epitope is conserved among vertebrates, as identified for other B-1 cells such as for T-15 specificity. Natural IgM of the T-15 specificity binds oxidized lipids and phosphoryl choline (). Moreover, like anti–phosphoryl choline natural IgM, the serum levels of N2-specific antibody probably also vary among mice. If so, it will be important to test whether N2-specific antibody levels correlate with susceptibility to RI. Furthermore, we propose that this innate response to a stress-induced self-antigen represents a general mechanism referred to as “innate autoimmunity” (, ). This novel concept was recently proposed to explain a role for Toll-like receptors in mediating sterile inflammation in response to self-ligands (). Our observation that a natural antibody recognizes a highly conserved self-antigen, resulting in sterile inflammation and tissue destruction, extends this notion to other pathways of innate recognition. Although a response to NMHC-II by Toll-like receptors was not examined, it is possible that they could participate in a secondary role as cells dying as a result of inflammation release other potential self-ligands. Similarly, other components of innate recognition such as mannan-binding lectin (MBL) (), surfactants (, ), and C1q (), which are known to bind and clear apoptotic cells, could play a secondary role after cell injury. Recently, Hart et al. reported the novel observation that injury in a mesenteric model of RI is dependent on MBL and complement C2 but not C1q (). Based on the importance of IgM and C4, it was assumed that injury was mediated via the classical pathway. We propose that IgM initiates the lectin pathway based on preliminary experiments indicating IgM deposition but an absence of C4 or C3 binding in MBL-a/c double-deficient mice (unpublished data). Thus, recognition of NMHC-II and deposition of IgM either facilitates binding of MBL to IgM or exposes an MBL binding site on ischemic tissue leading to its activation.
RI represents a major health problem because it affects most tissues, including the intestine, myocardium, and central nervous system, and at present there are no effective therapies or treatment (). Intestinal ischemia, particularly the acute form, has a high mortality rate (70–90%) (). The identification of a common epitope that is conserved among vertebrates and represents the target for natural IgM in a murine model of RI could provide a basis for development of a new category of therapeutics. Indeed, our finding that N2 peptide inhibits RI in two distinct tissues supports the feasibility of the approach.
In summary, we have identified a conserved region of myosin Hc that serves as the target for natural IgM in two murine models of RI. It is proposed that this pathway represents a novel response by the innate immune system to self-antigens induced by hypoxia or other forms of cell stress.
A 12-mer M13 phage display library (New England BioLabs, Inc.) was screened for four rounds with MBL beads coated with IgM and counterselected by one round of rat IgM followed by one round of IgM , according to the manufacturer's recommendation. Phage clones were selected from the enriched pool, and the nucleotide sequence of the relevant phage gene was determined for <10 clones. Selected peptides were synthesized with a purity >95% in the Harvard Proteomic Core or New England Peptide, Inc.
ELISA was performed as described previously (). In brief, IgM binding to phage or phage-specific peptides was determined by coating a 96-well plate with saturating amounts of antigen. Subsequent to blocking, 1 or 10 μg/ml IgM was added for 2 h at 37°C. Plates were washed and developed with alkaline phosphatase–labeled goat anti–mouse IgM (Sigma-Aldrich). To detect binding of IgM to NMHC-II, plates were coated with specific rabbit IgG, and fresh intestinal lysate was added as a source of antigen. Subsequently, plates were washed, a source of natural IgM (i.e., IgM or IgM ) was added, and binding was detected as described in the previous sentence. The following rabbit antibodies were used: NMHC-II A and B (Covance Inc.), NMHC-II C (a gift from R. Adelstein, National Institutes of Health [NIH], Bethesda, MD), and pan–myosin Hc (Sigma-Aldrich). Lysates were prepared as described in Immune precipitation.
RAG-1 and C57BL/6 mice used in this study were purchased from Jackson ImmunoResearch Laboratories and maintained within the mouse colony under specific pathogen-free conditions at Harvard Medical School according to NIH guidelines on animal welfare. Procedures involving animals were approved by the Institutional Review Boards at the CBR Institute, Harvard Medical School, Massachusetts General Hospital, and Brigham and Women's Hospital. Surgical protocol for RI was performed as previously described (). In brief, a laparotomy is performed, a microclip (125 pressure; Roboz) is applied to the superior mesenteric artery, and bilateral circulation is limited with silk sutures flanking a 20-cm segment of the jejuneum. After 40 min of ischemia, the microclip was removed, and reperfusion of the mesenteric vasculature was confirmed by the return of pulsation to the vascular arcade and a change to pink color. The incision was closed, and all animals were kept warm for 3 h. Reconstituted RAG-1 animals received either IgM mixed with peptide or saline in 0.2 ml volume i.v. 30 min before the initial laparotomy. WT animals were treated with saline or peptide i.v. 5 min before reperfusion. At the end of reperfusion, the ischemic segment of the jejunum was harvested, and the central 4 cm was cut for pathological analysis.
8–12-wk-old WT mice underwent 2 h of hindlimb ischemia and 3 h of reperfusion as previously described (). In brief, bilateral rubber bands (Latex O-Rings; Miltex Instruments) were applied above the greater trochanter. Rubber bands were removed, and limb reperfusion was confirmed by return of pink color. Tissues were fixed for 8 h in 4% paraformaldehyde and sectioned for Masson's Trichrome staining. 50 individual muscle fibers were counted per section, and the injury was reported as the number of damaged fibers per 50 fibers counted.
Cryostat sections of intestinal tissues were stained by hematoxylin and eosin and examined by light microscopy for mucosal damage. Pathology score was assessed based on a procedure modified from Chiu et al. () that included direct inspection of all microvilli over a 4-cm stretch of jejuneum, as described previously (). In brief, the integrated injury score = S/(V + [P × 25]) × 50% + D/(V + [P × 25]) × 100% + (P × 25)/(V + [P × 25]) × 100%, where V = total number of villi; S = villi appearing with subepithelial space, defined as an acellular space under a continuous epithelial layer and a milder form of damage; D = villi appearing with epithelial disruption, defined as discontinuation of an epithelial layer of villus and a more severe form of damage; and P = villi lost over the length of intestine measured using a 100× field (each field has an average of 25 jejunal villi; thus, by aligning the intestinal wall through the center of each power field, an estimate of the number lost can be made). For immunofluorescence, cryosections fixed with 4% (wt/vol) paraformaldehyde were incubated for varying periods with biotin-labeled anti–mouse IgM (Becton Dickinson), followed by 1 h with streptavidin–Alexa 568 (Invitrogen). C4 deposition was detected by staining with FITC-labeled rabbit anti-huC4c (DakoCytomation), followed by anti–rabbit–Alexa 488 (Invitrogen). The specificity of anti-C4c staining was confirmed by staining serial sections with biotin-labeled anti–mouse C4 for 1 h followed by streptavidin–FITC (Becton Dickinson). C3 deposition was detected by treating with FITC-labeled anti-C3 (DakoCytomation). Sections were mounted in antifade mounting medium with DAPI (Invitrogen).
IgM (IgM or ) antibody was immobilized by amine coupling in a chip (SPR CM5; Biacore) flowcell at a density of 33,400 response units per ∼33 ng/mm as described previously (). A reference flow cell was prepared by coupling of ethanolamine-HCl. Peptides, diluted in PBS running buffer, were flowed separately over the IgM-coupled surface and the reference at a rate of 10 μl/min at 25°C, with the data collection rate at 10 Hz. The injection phase had a duration of 240 s (the ends of injection phases are marked by arrow heads). Binding isotherms were derived by subtracting the response in the reference cell from the response of the IgM-coupled surface. After each run, the surface was regenerated by injecting 40 μl 0.05% (vol/vol) polyoxyethylenesorbitan monolaureate/PBS.
Frozen tissues were homogenized in a lysis buffer containing detergent and a cocktail of enzyme inhibitors. A sample of lysate is analyzed for total protein content (protein assay dye; Bio-Rad Laboratories) to ensure similar levels of protein for analysis. Lysates are mixed with sepharose beads coated with rat anti–mouse IgM for 1 h at 4°C. Subsequently, beads were pelleted gently, washed in lysis buffer, and boiled in SDS sample buffer under reducing conditions to elute bound complexes. Samples were fractionated on 8% (wt/vol) polyacrylamide SDS gels and subsequently fixed and stained with either Coomassie blue or silver stain to identify protein bands.
Individual Coomassie-stained bands were excised from SDS gels, destained, and subjected to enzyme digestion as described previously (). The peptides were separated using a nanoflow liquid-coupled chromatography system (CapLC; Waters), and amino acid sequences were determined by tandem mass spectrometer (Q-Tof micro; Waters). Mass spectrum (MS)/MS data were processed and subjected to database searches using Mascot (Matrix Science) against the UniProtKB/Swiss-Prot, TrEMBL-NEW, or the NCBI nonredundant databases.
Intestinal permeability was determined by measuring the appearance of HRP in blood (molecular mass = 40 kD; Sigma-Aldrich) administered by gavage as described previously (). The model used for investigating intestinal permeability was basically identical to the surgical protocol described in Intestinal RI model, except bilateral circulation limitation with silk sutures was not performed to maintain an open lumen during the study. In brief, 10 min before intestinal RI was induced, mice were administered 10 μl/g body weight of phosphate-buffered saline (pH 7.4) containing 2 mg/ml HRP by gavage. To test the blocking effect of N2 peptide in intestinal RI, WT C57BL/6 mice were treated with saline or peptide (final serum concentration = 40 μM) in a volume of 100 μl i.v. 5 min before reperfusion. Blood samples were obtained in a capillary tube 3 h after reperfusion by retroorbital puncture. Blood samples were allowed to clot overnight at 4°C and centrifuged (3,000 rpm) for 10 min. Serum samples were diluted and added to a 96-well microplate (Nalge Nunc) precoated with goat anti-HRP antibody (1:2,500 dilution; Sigma-Aldrich). The plate was then incubated for 2 h at 37°C, after which the wells were washed three times with phosphate-buffered saline (pH 7.4) and 0.1% Tween 20. HRP activity was determined by adding 100 μl of reagent (TMB One-Step Substrate System; DakoCytomation) for 15 min, and the reaction was stopped with 100 μl 2N HSO. The concentration of HRP was determined by spectrometry at 450 nm.
Leakage was determined in real time using an intravital imaging approach combined with a novel scanning laser confocal microscope (prototype scanner from Olympus Corp.) and a vascular probe (AngioSense 680; VisEn Medical) as described previously (). Mice were anesthetized with a 0.3-ml i.p. injection of 15 mg/ml ketamine Hcl and 3 mg/ml xylazine and injected i.v. with AngioSense 680 (10 nmol/mouse) and Rhodamine 6G (Invitrogen) 5 min before reperfusion. Mice treated for intestinal RI (see Intestinal permeability) and sham controls were prepared for imaging before reperfusion by making a small incision within the wall of the exposed jejuneum and inserting the “stick-lens” (1.3 mm diameter) of the prototype confocal setup. Imaging of the microvilli was initiated 15 min after reperfusion and continued for up to 45 min. Images were prepared at varying time points after reperfusion. Results from representative images of the three groups of mice (i.e., sham, saline, and N2 peptide) were analyzed by imaging software (Image J version 1.34; Wayne Rasband, NIH, Bethesda, MD) by taking 10 measurements of representative fields of view, and the intensity of red dye within the target vessels (V) versus surrounding background (B) tissue was determined. Results were calculated as ratio of V/B intensity ± SD.
Data in all figures (except , in which SD is used) are expressed as means ± SEM. Statistical comparisons between groups were made by Student's test, and ANOVA was used for comparisons of multiple groups. P < 0.05 was considered significant. |
xref
#text
A global analysis of histone Ac status in S regions of B cells indicates that activation of CSR is correlated with changes in total H3 and H4 Ac, in agreement with previous studies (, ). Our studies newly demonstrate that transcription, B cell activation, and AID expression differentially impact on chromatin modification of S DNA and these processes are hierarchically organized with respect to their influence on histone H3 and H4 Ac. Several observations support this conclusion. Our data demonstrate that in unstimulated splenic B cells in which μ and γ3 GLTs are constitutively expressed, the Sμ and Sγ3 loci contain acetylated H3 but not H4 histones, indicating that H3 Ac is preferentially targeted by germline transcription. B cell activation after LPS treatment stimulates both H3 and H4 Ac at Sγ1 in the absence of GLTs, demonstrating that histone Ac of these loci can occur by a transcription-independent process. Examination of S regions across the IgH locus in AID-deficient mice indicates that histone Ac is significantly reduced compared with that found in WT B cells, showing that AID expression also influences chromatin remodeling. We conclude that, in S regions, H3 and H4 Ac are most strongly correlated with germline transcription and AID expression, respectively. It should be stressed that these correlations are not absolute and some overlap of Ac potential for both H3 and H4 histones after B cell activation is also observed. It is also important to note that several acetylated lysines are detected with the αH3 and αH4 antisera used in our studies. When these modifications are further dissected, it is conceivable that some will be strongly correlated with transcription, whereas others will be clearly associated with AID expression.
Detailed analysis of the histone Ac status for the Iγ3-Sγ3-Cγ3 locus before and during CSR yielded several unanticipated findings. Histone Ac is localized to the Iγ3 exon, but not to the GLT promoter. It is now well established that, in many genes, histone Ac levels are highest at transcription start sites with a gradual decline over the length of the gene (–). Histone Ac is also focused to the Sγ3 region and not to intergenic areas or the Cγ3 exons, demonstrating that these chromatin modifications are targeted and not spread across the entire IgH locus. In 1.B4.B6 cells, the Iγ3 box and the Sγ3 region were differentially regulated with respect to histone Ac. We observed that, in response to IL-4, conditions that suppressed μ→γ3 CSR, H4 Ac was repressed in the Sγ3 region, but not the Iγ3 exon. Although these findings are limited to the 1.B4.B6 cell line, they nonetheless demonstrate that the I box and S region loci can be differentially targeted by histone acetyltransferases and deacetylases and imply that chromatin remodeling may specifically identify the S region for AID attack.
Our finding that transcriptionally active Sμ and Sγ3 are acetylated on H3 histones in unstimulated splenic B cells demonstrates that GLT expression is correlated with histone H3 Ac, but not H4 Ac. It is now well established that transcription disrupts chromatin through partial disassembly followed by rapid reassembly of the underlying nucleosomes (, ). Histone turnover and deposition of variant histone H3.3 occurs in transcriptionally active genes (–). H3.3 is highly enriched for modifications that are associated with active chromatin, indicating that transcription plays a role in establishing and maintaining an active epigenetic state (). It is, therefore, likely that increased H3 Ac at S regions is causally linked with GLT expression.
Chromatin is remodeled before and during transcription initiation by ATP-dependent remodelers and during RNA polymerase II transcription elongation (for review see reference ). There are two theories, which are not mutually exclusive, as to how histone Ac might facilitate transcription and accessibility. Ac could affect transcription by neutralizing histone charge, thus weakening histone–DNA interactions and reducing chromatin compaction (for review see reference ). Second, the resulting pattern or “code” of histone modifications may function as an interface for additional factors that regulate gene expression and DNA accessibility, including ATP-dependent remodeling complexes that alter nucleosome phasing, and heterochromatin-associated proteins that are thought to silence gene expression (–). Thus, S region accessibility, germline transcription, and histone Ac are per force coupled.
Chromatin modifications at S regions have been interpreted to represent histone marks that serve to target them for AID attack (, ). However, chromatin remodeling also occurs secondary to DNA lesions. For example, DNA DSBs induce phosphorylation of the variant histone, H2AX (γH2AX) (), and γH2AX repair foci have been found associated with both CSR and VDJ recombination, processes that involve a DSB intermediate (, ). Phosphorylated H2AX has been detected at Sμ regions in B cells undergoing CSR by ChIP assays () and CSR is defective in H2AX-deficient mice (). Strikingly, histone Ac and deAc have also been linked to alteration of chromatin topology in response to DNA damage (). The generation of DSBs has been correlated with histone H4 hyperAc in yeast and mammalian cells (, –). These observations compel us to consider the possibility that transcription-independent histone hyperAc at S regions undergoing CSR is linked to a DNA repair process and unrelated to the initial generation of accessibility. DSBs in S regions are detected after mitogen activation of B cells () and are AID and UNG dependent (, , ). Our studies clearly indicate that histone H4 Ac in S regions is linked to AID expression because AID deficiency leads to a reduction of H4 Ac in B cells stimulated to undergo CSR, whereas H3 Ac is more modestly affected. In yeast, Ac of histone H4 by EsaI is essential for repair of DSBs through both homologous recombination and NHEJ repair pathways (). The human homologue of EsaI, Tip60, is a component of the large multisubunit complex NuA4 that contains HAT activity and is also essential for DSB repair (, ). Further investigation is necessary to fully elucidate a causal relationship of H4 Ac in S DNA with DSB repair during CSR.
A major unresolved question is how AID is targeted specifically to its substrates: V regions in the case of SHM and S regions during CSR when both V and S regions are transcriptionally active. LPS-activated B cells transcribe both the VH and Sμ regions, but undergo only CSR and not SHM (). Our analysis of histone Ac patterns in 1.B4.B6 cells demonstrates higher levels of acetylated H3 histones in VH as compared with Sμ and this difference is maintained after activation of the CSR program. Furthermore, H4 Ac is not inducible for the VH or Sμ regions as it is for all S regions tested. Therefore, in 1.B4.B6 cells, which undergo CSR but not SHM, there is a skewed ratio of chromatin modifications in the VH and Sμ elements that serve to differentiate between these regions. A study using the GC-derived cell line BL2, capable of induced SHM, concluded that histone Ac is partially responsible for targeting SHM to the IgH V region because this region is hyperacetylated as compared with Cμ (). However, in that analysis, the Sμ region was not examined for histone Ac. More recently, H3 and H4 Ac levels were found to be similar in the VH and Sμ of naive and purified GC B cells. After induction of SHM, both VH and Sμ regions showed evidence of DNA damage by virtue of heightened levels of H2B, suggesting that in these cells both regions are targeted for AID attack (). LPS-activated mature splenic B cells are restricted to CSR. In contrast, GC B cells and GC B cell lines appear to be capable of both SHM and CSR, depending on the activation conditions (, ). Therefore, in GC B cells, the default chromatin topology may provide for accessibility to both VH and Sμ by AID. Additional work is required to determine whether the different levels of histone Ac in VH and Sμ regions are functionally meaningful.
The Animal Care and Institutional Biosafety Committee at the University of Illinois gave approval for the animal protocols used here. BALB/c (nu/nu) mice and C57B6×129 (AID WT) mice were purchased from Jackson ImmunoResearch Laboratories. AID KO mice, which were a gift from T. Honjo (Kyoto University, Kyoto, Japan), were bred under specific pathogen-free conditions in a fully accredited animal facility at the University of Illinois College of Medicine. All mice were used at 8–10 wk of age. Purified splenic B cells were obtained and stimulated as described previously (). Enrichment of B cells from mouse spleens was accomplished using Cellect Immunocolumns (Cedarlane Laboratories) according to the manufacturer's instructions. The concentrations of supplements used in this study were 50 μg/ml LPS (Sigma-Aldrich), 10 ng/ml rIL-4 (R&D Systems). 1B4.B6 cells were induced to switch μ→γ3 with LPS and CD40L (50% vol/vol) as described previously (). 1B4.B6 cells were activated to undergo μ→γ1 switching by stimulating cells with LPS and CD40L (10% vol/vol) and rIL-4 (10 ng/ml). Total RNA was extracted from cultured splenic B cells or 1B4.B6 cells with TRIzol (Invitrogen) according to the manufacturer's instructions, and cDNA was prepared by standard methods. Semi-quantitative RT-PCR was performed as described previously (). Primers for GLTs (μ, γ γ ɛ), and postswitch transcripts (for μ→γ3 μ→γ1 μ→ɛ switching) were described previously (, ).
Chromatin immunoprecipitation (ChIP) assays were performed according to the Upstate Biology protocol () with modifications. In brief, 10 splenic B cells were collected and washed twice with Hank's solution. The cells were cross-linked in 10 ml 1% formaldehyde in Hank's solution at 37°C for 10 min. The reaction was stopped by adding glycine to a final concentration 0.125M and incubated for 5 min at room temperature. The cells were rinsed with ice-cold Hank's solution and incubated on ice for 10 min in 4 ml of buffer (1% Triton X-100, 50 mM MgCl, 100 mM Tris-HCl, pH 7.1, 11% sucrose). Nuclei were pelleted by centrifugation at 780 for 15 min at 4°C, resuspended in 1% SDS, 50 mM Tris-HCl, 10 mM EDTA with fresh protease inhibitor cocktail (Roche), and incubated on ice for 10 min. Chromatin was sonicated (SonicDismembrator 550, Fisher Scientific Microtip) to an average length of 500 bp. To preclear the chromatin, the sonicated cell suspension was diluted 10-fold with a buffer containing 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl and incubated with 80 μl salmon sperm DNA/protein A Agarose 50% Slurry (Upstate Biotechnology Inc.) for 3 h with rotation at 4°C. The one third of the precleared chromatin was incubated with 2 μg of one of the following polyclonal antibodies overnight: anti-H3Ac, anti-H4Ac (Upstate Biotechnology Inc.), and control antibodies (Santa Cruz Biotechnology, Inc.). One tenth of the precleared chromatin was saved as input. Each immunoprecipitation was recovered, washed, eluted from the beads, and processed as described by the Upstate protocol. ChIP DNA pellets were resuspended in 60 μl of TE.
The samples were analyzed by semi-quantitative PCR by serially diluting the samples then amplifying using Taq polymerase (MBI) at 95°C for 30 s, 60°C for 45 s, and 72°C for 45 s for 29–33 cycles and analyzed by agarose gel analysis. ChIP DNA was also analyzed by qPCR analysis using an iCycler iQ Real-Time PCR Detection System (Optical Module, Bio-Rad Laboratories) with SYBR green Master Mix (Applied Biosystems) or iQ SYBR green Supermix (Bio Rad Laboratories) in the 96-well plate format. To ensure that a single PCR product was amplified from each primer pair, the dissociation curves were examined and the PCR products were run on agarose gels. Primers were designed using Primer Express software (ABI) and all the primers have approximately equal efficiency of amplification. Primers used in the semi-quantitative PCR or qPCR are listed in . All samples were analyzed in duplicate and averaged. The amount of PCR product amplified was calculated relative to a standard curve. The value of control IgG immunoprecipitation was subtracted from the specific IP before the calculations were made. All the calculations were based on ABI Prism 7700 sequence Detection System user bulletin no. 2 (Applied Biosystems). Histone Ac index = bound/ input. SDs were calculated using data from independent experiments.
In Fig. S1, histone H3 becomes hyperacetylated when 1.B4.B6 cells are induced to undergo CSR. Fig. S2 shows analysis of GLT expression in splenic B cells activated with LPS and LPS+IL-4 for 24 h. Online supplemental material is available at . |
We used a series of lymphoma and leukemia cells to study the mechanisms of GC-induced apoptosis. Susceptibility to GC-induced apoptosis was determined by appearance of subdiploid cells and caspase 3 activation. PD1.6 and 2B4 cells readily apoptose in response to GC, whereas B10, S49, NB4, and Jurkat cells are resistant to GC-induced apoptosis (). It should be noted that the S49 variant used in this study () differs from the S49 variant described by Gametchu et al. () in being resistant to GC-induced apoptosis. Because it has been proposed that mGR is a mediator of GC-induced apoptosis (), we compared mGR expression on GC-sensitive and resistant cells. It appears that S49, but not PD1.6, B10, 2B4, or Jurkat cells, express mGR (, subpanels A vs. C, E, G, and I, respectively). Also, the macrophage cell line RAW264.7 expresses a high level of mGR and is resistant to GC-induced apoptosis (unpublished data). Treatment with 100 nM dexamethasone (Dex) for 2 h did not induce expression of mGR on otherwise mGR-negative cells (, subpanels D, F, H, and J). The fact that GC-resistant S49 and RAW264.7 cells express mGR indicates that expression of mGR per se does not impose susceptibility to GC. The observation that GC-sensitive PD1.6 and 2B4 cells do not express mGR suggests that another mechanism is involved in this apoptotic process.
It has recently been shown that GR may be localized to the mitochondria (). As GC-evoked apoptosis is mediated via the mitochondrial pathway (), we asked whether GR translocation to the mitochondria may be a trigger of apoptosis. To answer this question, we analyzed the intracellular trafficking of GR in GC-sensitive and resistant cells after treatment with Dex. Dex-induced GR translocation to the mitochondria was demonstrated in GC-sensitive PD1.6 cells using two different methods (). First, immunofluorescence studies using M20 antibodies to GR and red mitotracker to visualize the mitochondria show that a certain fraction of GR localizes to the mitochondria in Dex-treated PD1.6 cells (). Second, PD1.6 cells treated with 100 nM Dex for 2 h were fractionated using the Oncogene cytosol/mitochondria fractionation kit. The quantities of GR in the cytosolic and mitochondrial fractions were analyzed by Western blotting (). The PA1-511A antibody to GR (epitope aa 346–367) resolves two major bands on Western blots. The top one is GRα, whereas the bottom band is of unknown character and considered in as a “background band.” GR expression in the mitochondrial fraction of PD1.6 cells is increased after Dex treatment (, subpanels C and D, lane 2 vs. lane 1), indicating mitochondrial translocation of GR. Contamination of the mitochondrial fraction with mGR is excluded, because PD1.6 cells do not express mGR even after Dex treatment (, subpanels C and D). As expected, the amount of GR in the cytosolic fraction is reduced by Dex (, subpanel A, lane 2 vs. lane 1). We also determined the GR expression in cytosolic and mitochondrial extracts of PD1.6TEC cells that display reduced sensitivity to Dex-induced apoptosis (). In contrast with the GC-sensitive parental PD1.6 cells, the GR level was not increased in the mitochondrial fraction of PD1.6TEC cells treated with Dex (, subpanels C and D, lane 4 vs. lane 3). These data indicate that selection of PD1.6 cells toward apoptotic resistance in response to Dex is accompanied with reduced mitochondrial translocation of GR.
Because the Oncogene kit does not yield nuclear extracts, we applied a cellular fractionation protocol that enabled comparison between mitochondrial and nuclear translocations of GR. Cross-contamination between the cytosolic, nuclear, and mitochondrial fractions was ruled out by using antibodies against α-tubulin, histone H2B, and VDAC, respectively (). As with the Oncogene kit, this method detected mitochondrial translocation of GR in Dex-treated PD1.6 cells (, subpanel E, lanes 2–3 vs. lane 1). A longitudinal follow-up () revealed mitochondrial translocation of GR as early as 5 min after addition of Dex, long before the onset of apoptosis. Thereafter, the mitochondrial GR level did not increase further with time ( [subpanel E, compare lanes 2 and 3] and F [subpanel E, compare lanes 2–5]). Also, the amount of nuclear GR already peaked at 5 min (, E and F, subpanel C). Similar results were obtained with PD1.6 cells treated with the naturally occurring GC corticosterone (unpublished data). The mitochondrial GR could also be detected on Western blot using the NH-terminal (aa 5–20)-reacting M20 and the COOH-terminal (aa 750–769)-reacting P20 antibodies (), indicating that this GR is in full-length. To exclude the possibility of nonspecific GR binding to the mitochondria, we studied whether this translocation is temperature sensitive. To this end, PD1.6 cells were exposed to Dex at 4°C for 5–15 min before subcellular fractionation. GR did not translocate to either the mitochondria or the nucleus at 4°C (, compare lanes 5 and 6 with lanes 2 and 3), indicating that the GR mitochondrial localization is specific and temperature dependent.
Next, we compared GR translocation to the mitochondria in GC-sensitive PD1.6 cells and GC-insensitive B10 cells (). PD1.6 and B10 cells are both derived from a thymic lymphoma, and express similar GR levels. The cells were exposed to 5 and 100 nM Dex for 4 h before subcellular fractionation. Dex induced GR translocation to the nucleus in both PD1.6 and B10 cells (, subpanel C, lanes 2, 3, 5, and 6). A dose-dependent mitochondrial translocation of GR was observed in PD1.6 cells (, subpanel E, lanes 2 and 3), but not in B10 cells (, subpanel E, lanes 5 and 6). In some experiments, a slight increase in GR expression was seen in mitochondrial extracts of B10 cells treated with 100 nM Dex (unpublished data), which reflects residual sensitivity (10–12%) of these cells to Dex (). Moreover, Dex induced GR translocation to both the nucleus and the mitochondria in GC-sensitive 2B4 cells () and thymocytes (). In 2B4 cells, nuclear translocation of GR was induced by as low as 1 nM Dex, a nontoxic concentration (, subpanel C, lane 2). This Dex concentration was insufficient to cause mitochondrial GR translocation in the same cells (, subpanel E, lane 2). However, at toxic Dex concentrations (10 and 100 nM), GR translocated to the mitochondria as well (, subpanel E, lanes 3 and 4 vs. lane 1).
Contrary to GC-sensitive cells, GC-resistant NB4 (, lanes 3–6) and S49 (, lanes 1–3) cells responded to Dex with GR translocation to the nucleus, but not to the mitochondria. We also treated NB4 cells with AsO, which stabilizes PML and degrades the PML–RAR fusion protein (). AsO stabilized GR in these cells (, subpanel A, lane 5 vs. lane 3) to a level comparable with that of PD1.6. Even after AsO treatment, Dex did not cause mitochondrial translocation of GR in NB4 cells (, subpanel E, lane 6). Although S49 cells express a high basal level of GR (, subpanel A, lane 1), no GR translocation to the mitochondria is seen (, subpanel E, lanes 2 and 3). Thus, mitochondrial GR translocation does not correlate with GR expression but rather with the apoptotic sensitivity to GC.
Jurkat cells express a very low level of GR (, subpanel A, lane 4), which may explain their unresponsiveness to the apoptotic effects of GC. It is well documented that GC-induced apoptosis requires a threshold level of GR (, ). Although GR translocates to the nucleus in Dex-treated Jurkat cells (, subpanel C, lanes 5 and 6), GR is barely seen in the mitochondria (, subpanel E, lanes 5 and 6). Moreover, Jurkat cells express one WT and one mutated (R477H) GR allele, as do the GC-sensitive CCRF–CEM cells ().
It should be noted that in most of the GR-expressing cell lines tested, a certain basal level of GR is detected in the mitochondria (e.g., [subpanel E, lanes 1 and 4], B [subpanel E, lane 1], and E [subpanel E, lane 1]). Altogether, the data demonstrate correlation between GR translocation to the mitochondria and sensitivity to GC-induced apoptosis.
The GR antagonist RU486 induces a different conformational change of GR than GC agonists (). It causes nuclear translocation of GR, but prevents its binding to GREs, thereby avoiding transactivation. We sought to find out whether RU486 affects mitochondrial translocation of GR. Initially, we analyzed the effect of RU486 on Dex-induced apoptosis of PD1.6 cells. As expected, RU486 prevented this apoptotic response (). When looking on the intracellular trafficking of GR, we found that RU486 induced both nuclear and mitochondrial translocation of GR in PD1.6 cells (, subpanel C and E, respectively, lane 3). A combination of RU486 and Dex had an additive effect on GR translocation to the mitochondria (, subpanel E, lane 4). These data indicate that a mere localization of GR to the mitochondria is insufficient for inducing apoptosis, which apparently requires a particular GR conformation.
We have recently shown that thymic epithelial cells (TECs) induce apoptosis of PD1.6 cells in a GR-dependent manner (). It was therefore of interest to study the intracellular trafficking of GR in PD1.6 cells cocultured with TECs. For this purpose, PD1.6 cells were incubated alone or on a TEC monolayer for 4 h. In PD1.6 cells grown alone, most of the GR is localized in the cytosol (, lane 1). After cocultivation with TECs, GR is found in the mitochondrial fraction, but, surprisingly, not in the nuclear fraction (, compare lane 2 in subpanel E vs. subpanel C). PD1.6 cells treated with 100 nM Dex for 4 h were used as a positive control for nuclear translocation. As expected, this treatment caused both nuclear and mitochondrial translocation of GR (, lane 7 in subpanels C and E, respectively). Cocultivation on TEC did not cause a reduction in the cytosolic GR level as seen with Dex (, compare lanes 2 and 7 with lane 1). It should be noted that a similar quantity of GR was observed in the mitochondrial fractions of PD1.6 cells cocultivated with TECs and of PD1.6 cells treated with 100 nM Dex (, compare lanes 2 and 7). This outcome is consistent with the similar extent of apoptosis induced by these two stimuli (). To exclude the possibility that GR detected in PD1.6 cells cocultured with TECs is due to TEC contamination, we included in the experiment Dex-resistant PD1.6Dex cells that express minute amount of GR (). No GR could be seen in the mitochondrial or nuclear fractions of these cells after cocultivation with TECs (, lane 4 in subpanels E and C, respectively). As a negative control, we used TEC-resistant PD1.6TEC cells (), which show only a slight reduction in GR expression level in comparison to the parental PD1.6 cells. Interestingly, TECs induced neither mitochondrial nor nuclear translocation of GR in PD1.6TEC cells (, lane 6 in subpanels E and C, respectively). Altogether, our data demonstrate that TEC induces GR translocation to the mitochondria in TEC-sensitive PD1.6 cells, but not in TEC-resistant PD1.6TEC cells. Because TEC causes apoptosis of PD1.6 cells in a GR-dependent manner () and induces mitochondrial (but not nuclear) GR translocation, we propose that GR translocated to the mitochondria is a trigger of apoptosis. Our findings also show that the nuclear and mitochondrial translocations of GR are differentially regulated.
Our data showing that, depending on circumstances, GR translocates either to the nucleus or to the mitochondria, suggest that nuclear and mitochondrial GR translocations are differentially regulated. We therefore searched for signals directing the intracellular trafficking of GR. To this end, we transfected GR-negative 293 cells with WT or various deletion mutants of human GR (hGR). In the absence of ligand, the transfected GR is expressed in cytosol, nucleus, and mitochondria (, lane 1). Addition of Dex did not alter the intracellular distribution of GR in these cells (unpublished data). This pattern of exogenous GR expression enabled the search for NLS and MLS. Using 293 cells transfected with GR deletion mutants, we found that NH-terminal GR fragments (1–488, 1–515, and 1–550) translocated to the nucleus, but not to the mitochondria (, lanes 2–4), indicating that these mutants contain NLS but not MLS. GRΔ428-490 (ΔDBD) and GRΔ490-515 translocated to the mitochondria, but barely to the nucleus (, lanes 5 and 6), indicating that NLS resides within aa 428–515 of hGR, which is in accordance with the three NLS described previously (). GRΔ550-600 translocated to the nucleus, but barely to the mitochondria (, lane 7), indicating that MLS resides, at least in part, within aa 550–600. A residual mitochondrial translocation observed with GRΔ550-600 suggests that the 550–600 domain may act in concert with another site within the COOH-terminal region of GR. Similar to WT GR, the Δ727-777 mutant distributed to both the nucleus and the mitochondria (, lane 8). Also, the GRΔ77-262 deletion mutant translocated to both the mitochondria and nucleus (unpublished data). Hence, NLS and MLS are located within different domains of the GR protein.
The 550–600 domain of GR comprises the α-Helix 3 (aa 558–580) of the LBD. This α-helix is characterized by a series of positively charged aa (arginine/lysine), hydrophilic aa (threonine), and hydrophobic aa (valine, isoleucine, and leucine), but lacks negatively charged aa. The positively charged and hydrophilic aa locate on the one side of the α-helix, whereas the hydrophobic aa locate on its other side (). This arrangement is compatible with the requirements for an MLS (). It shows some sequence similarity to the MLS of cytochrome C oxidase (COX) (). To verify that the MLS resides within aa 558–580 stretch, Arg564 and Arg575 of mouse GFP-GR (which correspond to human GRArg558 and Arg569, respectively) were each mutated to glycine. Arg575 was also mutated to aspartate. The ability of these mutants to translocate to the mitochondria was compared with that of GFP-GR WT. R564G and R575G showed reduced ability to enter the mitochondria, whereas R575D behaved as WT in this respect (). These data indicate that the secondary structure created by R564 and R575, rather than their charge, is important for the MLS integrity. When this MLS is placed in the crystal structure of dimerized ligand-bound LBD, the two MLS appear adjacent (). The differential location of MLS and NLS may explain the dissociated nuclear and mitochondrial translocations of GR observed in various cell types in response to diverse stimuli.
Our data suggest a role for mitochondrial GR in mediating apoptosis. To verify this hypothesis, we sought to distinguish between the effects exerted by GR in the mitochondria and in other intracellular compartments. To this end, we constructed a GR variant (MLS-GFP-GR) that exclusively localizes to the mitochondria. This variant was attained by adding the MLS of COX upstream to GFP-GR. We initially analyzed the intracellular localization of MLS-GFP-GR in the absence or presence of Dex. HeLa, H1299, and PC3 cells transfected with GFP-GR or MLS-GFP-GR were either untreated or treated with 100 nM Dex for 2 h. Thereafter, the cells were stained with red mitotracker to visualize the mitochondria and analyzed by confocal microscopy. The MLS-GFP-GR exclusively localized to the mitochondria both in the absence and in the presence of Dex (). Hence, this construct was indeed useful for studying the effect of mitochondrial GR on apoptosis. For this purpose, we transfected HeLa cervical carcinoma, PC-3 prostate adenocarcinoma, and L929 E8.2 A3 fibroblast-like cells with plasmids encoding either GFP-GR or MLS-GFP-GR. After 48 h, the percentage of apoptotic transfectants was compared with that of nontransfectants from the same sample. GFP-GR induced apoptosis of HeLa and L929 E8.2 A3 cells, but not of PC-3 cells (). MLS-GFP-GR induced apoptosis of HeLa and L929 E8.2 A3 cells more efficiently than GFP-GR (). Interestingly, PC-3 cells, which do not undergo apoptosis by GFP-GR, were sensitive to MLS-GFP-GR (). These results demonstrate that when GR is directed to the mitochondria, it is capable of inducing apoptosis.
The aforementioned data indicate that mitochondrial GR triggers apoptosis induced by GC. We cannot, however, exclude the possibility that GR may act at additional intracellular sites. To further study in which intracellular compartments GR may induce apoptosis, we analyzed the apoptotic ability of an NLS-defective GR mutant (pEGFP-ratGRK513-515A) and a nucleus-only directed GR variant (NLS-GFP-GR). The latter was constructed by adding the NLS of SV40 T antigen in triplet [(DPKKRKV)] upstream to GFP-GR of mouse origin.
The apoptotic ability of pEGFP-GRK513-515A was compared with that of the parental pEGFP-GR WT in HeLa cells. It should be noted that pEGFP-GRK513-515A is unable to translocate to the nucleus (). We observed that both GR WT and the NLS-defective GR variant induced apoptosis of HeLa cells (). pEGFP-F, used as a negative control, did not cause apoptosis of HeLa cells (). The ability of pEGFP-GRK513-515A to trigger apoptosis demonstrates that GR may mediate apoptosis by a nucleus-independent manner.
Last, we compared the apoptotic ability of the nucleus-directed NLS-GFP-GR to those of MLS-GFP-GR and parental GFP-GR. For this purpose, HeLa and H1299 cells were transfected with the respective plasmids. Both NLS-GFP-GR and MLS-GFP-GR were more efficient than GFP-GR in inducing apoptosis of these cells (). pEGFP-F did not induce apoptosis under the same circumstances (). These results suggest that an overexpressed GR may induce apoptosis when present either in the mitochondria or in the nucleus. Under physiological conditions, we anticipate that the mitochondrial GR cooperates with nuclear GR in inducing apoptosis.
Numerous studies have been performed to elucidate the mechanisms by which GC induces apoptosis (, , , ). Several biochemical changes occurring immediately after exposure to GC have been characterized. These include Ca mobilization, activation of Src and Cdk2 kinases, and activation of phosphatidylinositol-specific phospholipase C and acidic sphingomyelinase with subsequent ceramide generation (, –). Downstream effector mechanisms of GC-induced apoptosis have also been defined. These involve the mitochondria apoptotic pathway mediated by Bax, Bak, Bim, and tBid, and antagonized by Bcl-2 and Bcl-X (, ). Dissipation of the mitochondrial membrane potential (ΔΨ) is followed by release of cytochrome C and Smac/Diablo to the cytosol (, ), which in turn leads to the activation of caspase-9, caspase-3, and endonucleases (, ). With the good knowledge of downstream effectors in GC-induced apoptosis, little is known about the role and fate of GR in this response, as well as the reasons why some GR-expressing cells are sensitive, whereas others are not.
GR expression above a threshold level is necessary, but is not sufficient to activate GC-mediated apoptosis (, , ). Several studies have indicated that GC-resistant cells may express similar GR levels as GC-sensitive cells (, –). Likewise, in the present study, we show that GC-sensitive (PD1.6, 2B4, thymocytes) and some GC-resistant (B10, S49) cells express high GR levels. These observations suggest that the apoptotic response is regulated by additional factors acting downstream to ligand–receptor interaction. One possibility is that only a certain isotype of GR can initiate apoptosis. Indeed, various mRNA transcripts of GR have been observed in both mouse and human cells (, , ); among them, the 1A variant is most abundantly expressed in hematopoietic cells (, ). The 1A transcript is mainly translated to a ∼90-kD GR and a small proportion of a higher molecular mass GR (∼150 kD) (). The various cells analyzed in our study mainly express the ∼90-kD form corresponding to GRα. We also detected a small amount of ∼150-kD GR in the cytosolic fraction of PD1.6, 2B4, B10, and S49 cells (unpublished data). Hence, the ∼150-kD GR form is not expressed exclusively in cells sensitive to GC, but in GC-resistant cells as well.
Another factor that could affect GC-mediated apoptosis is the intracellular trafficking of GR. GC causes nuclear translocation of GR in both GC-sensitive and GC-resistant cells. Both transactivation-deficient and transrepression-deficient GR variants have been shown to restore GC sensitivity in human T-ALL cells (, , , ). These data together with our present finding that an NLS-deficient GR mutant is proapoptotic suggest that nuclear-independent mechanisms are likely involved in this death pathway.
It has been suggested that mGR is involved in GC-induced apoptosis based on the finding that a cDNA derived from full-length 1A transcript, imparted both mGR expression and GC sensitivity to some GC-resistant cells (). However, this cDNA also caused GR overexpression in the cytosol (), which may have contributed to the GC sensitivity of the cells. In the present paper, we have further studied the role of mGR in GC-induced apoptosis by analyzing mGR expression in GC-sensitive and resistant lymphoid cells. mGR was expressed on some cell types that were GC resistant, but not on the GC-sensitive cells studied. Thus, mGR is not required for GC-induced apoptosis, and its mere presence does not impose susceptibility to GC. Our findings are compatible with those of Gametchu et al. (), showing that mGR-negative lymphoma cells may be GC sensitive.
Another possibility could be that mitochondrial GR is the trigger of apoptosis. GR has been shown to be located within the mitochondria in some cell types (–). In these studies, GR was detected in the mitochondrial membranes and in the matrix space. Likewise, we also observed a basal mitochondrial GR expression in most of the lymphoma cells analyzed. However, Dex induces GR translocation to the mitochondria in GC-sensitive, but not in GC-resistant, cells. This is in contrast with nuclear translocation of GR that takes place in all cell types. This is the first qualitative difference in GR behavior described that distinguishes between GC-sensitive and resistant cells, suggesting a role for mitochondrial GR in apoptosis.
In GC-sensitive cells, mitochondrial and nuclear translocations of GR occur simultaneously within the first minutes after exposure to Dex. After its initial translocation, the amount of GR in the mitochondria was maintained at a steady state. Thus, the elevated mitochondrial GR level is sustained in GC-sensitive cells, which is in contrast with the transient residence of GR in the mitochondria (5–30 min) in HeLa cells and with the reduced amount of mitochondrial GR in a glioma cell line after Dex treatment (, ). The sustained expression of GR in the mitochondria of GC-sensitive cells may account for the apoptotic effects of GC. Another support for a role of mitochondrial GR in mediating apoptosis comes from the observation that TECs, which trigger apoptosis of PD1.6 in a GR-dependent manner (), induces GR translocation to the mitochondria, but not to the nucleus. Thus, GR translocation to the nucleus is not necessary for the apoptotic response. However, we cannot exclude the possibility that mitochondrial GR cooperates with nuclear GR in inducing apoptosis. The fact that a mitochondria-directed GR induces apoptosis suggests that exclusive expression of GR in the mitochondria is sufficient for triggering apoptosis. Conversely, a nucleus-directed GR is also proapoptotic. It should be noted that the latter data were obtained by a transient transfection assay where the proteins were overexpressed. Thus, the GR effects are more pronounced than at physiological GR levels. Nevertheless, it is a valuable approach that provides information on GR function. Altogether, our data show that mitochondrial translocation of GR correlates with apoptotic sensitivity.
This behavior of GR resembles that of the proapoptotic p53 and Nur77 proteins. These proteins have recently been shown to have a direct apoptogenic role at the mitochondria, besides their nuclear effects (, ). Our results add GR to the growing list of proteins that mediate apoptosis when localized to the mitochondria. Also, the thyroid, estrogen β and retinoid X receptors have been shown to be located in the mitochondria (, , ). Thus far, however, the mitochondrial localization of these receptors has not been implicated in apoptosis.
The mechanism by which mitochondrial GR mediates apoptosis is a matter for further study. GC has been shown to regulate mitochondrial transcription and energy production (), and GRE elements have been found in the mitochondrial genome (). It should be mentioned in this regard that some of the apoptotic effects of p53 and Nur77 occur independently of their transcriptional activities (, ). p53 and Nur77 interact with the protective Bcl-X and Bcl-2 proteins in the mitochondria (, ). It would, therefore, be interesting to find out whether mitochondrial GR has similar effects.
Another important finding of our study is that nuclear and mitochondrial GR translocations are differentially regulated. For instance, Dex induces both mitochondrial and nuclear GR translocations in GC-sensitive lymphoid cells, but only nuclear translocation in GC-resistant cells. In contrast, TEC induces mitochondrial, but not nuclear, GR translocation. Furthermore, we have partially characterized a putative MLS comprising the α-Helix 3 (aa 558–580) of LBD. This domain lies COOH terminally to the three NLSs (aa 428–515) (), and possesses several traits of an MLS (). The hydrophobic and positively charged residues are partitioned on opposite sides of the helix. Most of the proteins destined to the mitochondria contain an NH-terminal mitochondrial transfer peptide that is cleaved off after entering the mitochondrial matrix (). Some mitochondrial proteins, however, have a noncleavable internal MLS (). The GR detected by us in the mitochondria is of a similar size as cytosolic GR, indicating that it does not undergo cleavage upon mitochondrial translocation. Hence, GR contains a noncleavable internal mitochondrial targeting sequence possessing an amphipathic presequence-type helix. This target sequence resembles the internal MLS of BCS1 ().
It is conceivable that GR is transported to the mitochondria by a heat-shock protein, as its 558–580 domain overlaps with one of several characterized Hsp90-binding sites (), and both Hsp90 and Hsp70 function as chaperones that interact with the mitochondrial protein import receptor Tom 70 (). Further studies are required to verify the role of heat shock proteins in GR trafficking.
In summary, we conclude that mitochondrial GR acts independently of nuclear GR in inducing apoptosis, and that mitochondrial and nuclear GR translocations are differentially regulated. Given the multiple nuclear adverse effects of GC therapy, our findings propose further research focusing on the development of therapeutic modalities that preferentially direct GR to the mitochondria.
PD1.6 thymic lymphoma (), B10 thymic lymphoma (), S49 T lymphoma (provided by J. Hochman, The Hebrew University of Jerusalem, Jerusalem, Israel), TEC (provided by A. Kruisbeek, The Netherlands Cancer Institute, Amsterdam, Netherlands), 293 kidney epithelial cells, HeLa cervical carcinoma, and L929 E8.2 A3 fibroblast-like cells (provided by W.V. Vedeckis, Louisiana State University, New Orleans, LA) were grown in DMEM supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 10 mM Hepes, 1 mM sodium pyruvate, nonessential aa, antibiotics, and 50 μM β-mercaptoethanol. 2B4 T cell hybridoma, 4B2 PML, Jurkat ALL, thymocytes, H1299 lung adenocarcinoma, and PC-3 prostate adenocarcinoma were cultured in RPMI 1640 with the same supplements as for DMEM. PD1.6Dex cells were derived from PD1.6 cells by repeated exposure to increasing concentrations of Dex and PD1.6TEC cells were derived from PD1.6 cells repeatedly cocultured with TECs ().
The following plasmids were used: GFP-mouse GR (provided by L.J. Muglia, Washington University in St. Louis, St. Louis, MO); hGRΔDBD and hGRΔ77-262 (provided by W. Doppler, Universität Innsbruck, Innsbruck, Austria); hGR WT, hGR1-488, hGR1-515, hGR1-550, hGR418-777, hGRΔ490-515, hGRΔ550-600, hGR550-777, and hGRΔ727-777 (provided by T.D. Gelehrter, University of Michigan, Ann Arbor, MI); pEGFP-rat GR and pEGFP-ratGR K513-515A (provided by K.R. Yamamoto, University of California, San Francisco, CA); pCMV/myc/mito (Invitrogen) (provided by S. Ostrand-Rosenberg, University of Maryland, Baltimore, MD); pCMV/myc/nuc (Invitrogen) (provided by D. Bensi, Mayo Clinic, Rochester, MN); farnesylated GFP (pEGFP-F; CLONTECH) and pEGFP-N1 (CLONTECH) (provided by Y. Haupt, The Hebrew University of Jerusalem, Jerusalem, Israel). A mitochondria-directed GFP-GR variant (MLS-GFP-GR) was prepared by inserting the MLS of COX (MSVLTPLLLRGLTGSARRLPVPRAKIHSL) NH-terminal to GFP-GR. A PCR product containing this MLS was obtained from pCMV/myc/mito using 5′-primer CTAGCTAGCTGACGCAAATGGGCGGTAGGCGTG-3′ harboring a NheI site and 3′-primer CCCACCGGTTTGGCCCCATTCAGATCCTCTTC-5′ harboring a AgeI site. After double digestion with NheI and AgeI, the PCR product was inserted within the NheI/AgeI sites of GFP-GR. A nucleus-directed GFP-GR variant (NLS-GFP-GR) was prepared by inserting the NLS of SV40 T antigen in triplet ([DPKKRKV)]) NH-terminal to GFP-GR. pCMV/myc/nuc was used as template for the aforementioned primers. Mutations in GR were introduced by site-directed mutagenesis (QuickChange kit; Stratagene). Sequencing of plasmids was done at the DNA Sequencing Facility of The Hebrew University of Jerusalem, Israel.
Cell surface staining of GR was performed by incubating cells with M20 antibody to GR (Santa Cruz Biotechnology) followed by FITC-conjugated AffiniPure F(ab)-fragment of goat anti–rabbit IgG (Jackson ImmunoResearch Laboratories). Intracellular GR staining was performed on rehydrated methanol-fixed cells.
Apoptosis was assessed by cell cycle analysis and caspase 3 activation. For cell cycle distribution, cells were fixed with ice-cold methanol, rehydrated in PBS, and treated with 50 μg/ml RNase. After adding 5 μg/ml propidium iodide, the DNA content was analyzed by flow cytometry (FACSCalibur; Becton Dickinson). Subdiploid cells are regarded as apoptotic cells, and presented as percentage of the whole cell population. Caspase 3 activation was analyzed by incubating rehydrated methanol-fixed cells with antibody to cleaved caspase 3 (Asp 175; Cell Signaling Technologies) followed by FITC-conjugated AffiniPure F(ab)-fragment of goat anti–rabbit IgG (Jackson ImmunoResearch Laboratories).
Cell pellets were gently resuspended in cytoplasmic buffer (10 mM Hepes, pH 7.4; 1.5 mM MgCl, 10 mM KCl, 0.5 mM DTT, 10 mM NaMoO, 2 mM PMSF, 20 μg/ml aprotinin, 0.1% NP-40; 25 mM NaF, and 0.2 mM NaVO) and kept on ice for 10 min before centrifugation at 900 for 10 min. The nuclear pellets were processed as described below. The cytoplasmic supernatant was recentrifuged at 900 to ensure complete removal of nuclear material. The resulting supernatant was centrifuged at 10,000 for 30 min. The cytosolic supernatant was processed for Western blot by adding protein sample buffer (PSB) × 4.5. The mitochondrial pellet was washed with cytoplasmic buffer, recentrifuged at 10,000 , and dissolved in PSB × 1.5. The nuclear pellet was washed with cytoplasmic buffer and recentrifuged at 900 before their extraction in nuclear buffer (20 mM Hepes, pH 7.4, 1.5 mM MgCl, 420 mM NaCl, 25% glycerol (vol/vol), 0.2 mM EDTA, 0.5 mM DTT, 10 mM NaMoO, 2 mM PMSF, 20 μg/ml aprotinin, 25 mM NaF, and 0.2 mM NaVO). The nuclear extracts were cleared at 20,000 for 10 min, and processed for Western blot by adding PSB × 4.5. GR was detected on Western blot by using the PA1-511A (Affinity BioReagent), M20, or P20 antibodies to GR (Santa Cruz Biotechnology). The blots were reprobed with antibodies to α-tubulin (Sigma-Aldrich), cytochrome C (CytoC; Santa Cruz Biotechnology), VDAC (Oncogene Research Products), or histone H2B (LG2-2; provided by M. Monestier, Temple University, Philadelphia, PA) (). The Oncogene cytosol/mitochondria fractionation kit (QIA88; Oncogene Research Products) was used according to the manufacturer's instructions.
5 × 10 TECs were seeded in 75 cm tissue culture bottles (Nunc). On the following day, 10 PD1.6 cells were added to the TEC monolayer in a 40-ml medium. After 4 h, nonadherent PD1.6 cells were harvested and processed for cell fractionation as described before.
Cells were transfected with the given plasmids by using the calcium phosphate precipitation method. For apoptosis assay, the adherent cells were trypsinized and collected in culture supernatants. Flow cytometry was performed while gating on GFP positive or negative cells. Percentage of apoptotic transfectants (GFP-positive) was compared with that of nontransfectants (GFP-negative) from the same culture. For confocal microscopy, the cells were incubated with red mitotracker (50 nM; Invitrogen) during the last 30 min of incubation. For cell fractionation studies, adherent transfected cells were harvested with rubber policeman and centrifuged at 720 for 5 min. |
PD-1 has two known ligands, PD-L1 and PD-L2 (). We first investigated the expression of PD-1 and its ligands on antigen-presenting cells: monocytes, myeloid dendritic cells (mDCs) (), and plasmacytoid dendritic cells (pDCs) (–). We purified human CD11c mDCs and CD123 pDCs from elutriated monocytes (, ). In vitro activation of purified monocytes with LPS resulted in up-regulation of PD-L1, whereas PD-1 and PD-L2 were less affected (). Cross-linking of toll-like receptor (TLR)7 or 8 (with the TLR7/8 ligand Resiquimod) on mDCs substantially stimulated the expression of both PD-L1 and PD-L2 and caused a slight induction in PD-1 expression (). In marked contrast, TLR7/8-mediated activation of pDCs had no effect on either PD-1 or its ligands, suggesting that pDCs are not involved in regulating CD8 T cell function via PD-1–PD-L interactions (). Our data indicate that mDCs and monocytes, in addition to presenting antigen and costimulatory signals to virus-specific CD8 T cells, may also serve to regulate CD8 T cell function through the expression of PD-1–specific ligands.
We next assessed the expression of PD-1 on naive, memory, and virus-specific CD8 T cells using polychromatic flow cytometry. The gating scheme for identification of the various CD8 T cell subsets is shown in . Our results show that PD-1 expression on naive (CD27CD45RO) CD8 T cells is infrequent in both HIV and HIV donors, consistent with previous studies (, ) (). However, in HIV subjects, PD-1 expression was more frequent on CD27CD45RO memory CD8 T cells than on the other memory CD8 subsets (P = 0.02 and 0.0001 versus CD27CD45RO and CD27CD45RO memory CD8 T cells, respectively). This distinction was not present within memory CD8 T cells from HIV subjects. We next assessed PD-1 expression on virus-specific CD8 T cells after staining with HIV-, CMV-, EBV-, and vaccinia virus (VV)–specific tetramers (example shown in ). We found different levels of PD-1 expression on memory CD8 T cells according to their specificity. In general, HIV-specific and EBV-specific CD8 T cells were found to express PD-1 more frequently than CMV-specific CD8 T cells, and VV-specific CD8 T cells rarely expressed PD-1 (). Similar patterns of PD-1 expression with respect to virus antigen specificity were obtained when PD-1 expression was analyzed by mean fluorescence intensity, rather than the frequency of cells that express a given level of PD-1 (unpublished data). We did not see a correlation between plasma viral load and PD-1 expression on HIV-specific CD8 T cells in this small group of subjects (unpublished data), although in larger cohort studies such an association has been clearly demonstrated (, ).
We asked whether PD-1 expression on antigen-specific cells is related to their altered maturational status (, ). The vast majority of HIV-specific CD8 T cells was found to express a CD27 phenotype, in agreement with previously published data (). There was no difference in the maturational phenotype of the PD-1 and PD-1 fractions of HIV-specific CD8 T cells (). CMV-specific CD8 T cells were evenly distributed among the three memory phenotypes defined by CD27 and CD45RO; furthermore, PD-1 and PD-1 CMV-specific CD8 T cells exhibited the same distribution of these three memory phenotypes (). We also found that there was no difference in the expression of other homing and maturational markers (CCR7 and CD57) between PD-1 and PD-1 antigen-specific CD8 T cells (unpublished data). Our data therefore indicate that PD-1 expression is independent of the maturational state of antigen-specific memory CD8 T cells.
Knowing that sustained high levels of PD-1 are associated with a diminished ability of LCMV-specific CD8 T cells to produce cytokine in chronically infected mice (), we hypothesize that PD-1 expression on human HIV-specific CD8 T cells would similarly result in impaired cytokine production. We first confirmed that the ability of CD8 T cells to produce cytokines in response to antigen stimulation was impaired in those T cells known to express high levels of PD-1. We measured IFN-γ, TNF-α, and IL-2 production by CD8 T cells after HIV Gag, CMV pp65, and VV stimulation, and assessed the proportion of the cells that were able to produce any one of the three cytokines. As shown in , there was no difference in the proportion of the HIV Gag–, CMV pp65–, or VV-specific CD8 T cells that were able to produce IFN-γ, but there was lower production of TNF-α and IL-2 by CD8 T cells known to express higher levels of PD-1 (HIV > CMV > VV). This confirmed that PD-1 expression is associated with impaired cytokine (TNF-α and IL-2) production by antigen-specific CD8 T cells.
We next asked if the impaired cytokine function resulted directly from the expression of PD-1, or whether the two phenomena were only indirectly linked. The expression of PD-1 in relation to production of cytokines under short (6 h) stimulation was assessed in HIV- and CMV-specific CD8 T cells. Cells were stimulated with peptides corresponding to HLA-A2–defined optimal HIV and CMV epitopes, and the production of IFN-γ, TNF-α, and IL-2 was measured within tetramer cells according to PD-1 expression. The data demonstrate that IFN-γ and TNF-α are readily produced from PD-1 cells, whereas IL-2 production is rarely produced from either PD-1 or PD-1 CMV- or HIV-specific CD8 T cells (). We next gated on the PD-1 and PD-1 subsets of antigen-specific cells (defined by cytokine production and/or tetramer staining) and calculated the proportion of the cells that were nonfunctional for cytokine production after stimulation with a range of peptide concentrations (). For both HIV- and CMV-specific CD8 T cells at all levels of antigen stimulation, there was no difference in the production (or lack thereof) of IFN-γ, TNF-α, or IL-2 between PD-1 and PD-1 cells. Therefore, PD-1 expression does not appear to be directly responsible for the inability of some CMV- and HIV-specific CD8 T cells to produce these cytokines.
We postulated, however, that PD-1 may not be adequately engaged by its ligands during the ex vivo stimulations. We therefore performed 6-h peptide stimulations in the presence and absence of anti–PD-1 antibody to directly engage the PD-1 pathway during the time of antigen stimulation. Raw data for one subject and compiled data for three subjects are shown in the left and right panels, respectively, of , and show that engagement of PD-1 during antigen stimulation had no effect on the ability of the antigen-specific T cells to produce IFN-γ, TNF-α, or IL-2. Collectively, these data demonstrate that PD-1 does not directly influence the capacity of HIV- or CMV-specific CD8 T cells to produce cytokines upon stimulation.
The major effect of PD-1 blockade in mice was on antigen-specific CD8 T cell proliferation (). HIV-specific CD8 T cells are characterized by limited proliferative capacity in chronic HIV infection (, ), a defect that is linked to the depletion of IL-2–secreting HIV-specific CD4 and/or CD8 T cells (, , ). Therefore, we investigated whether stimulation through, or interference with, the PD-1–PD-L1 pathway could affect the proliferation of HIV- and CMV-specific CD8 T cells. CFSE-labeled cells were stimulated with HIV- or CMV-specific peptide pools in the presence or absence of antibodies against either PD-1 or its ligand PD-L1. After 6 d, the frequency of CFSE cells, in either all CD8 or tetramer cells, was compared between cultures that had received no blocking antibody, an anti–PD-1 antibody (that acts as a PD-1 agonist), or an anti–PD-L1 antibody (clone MIH1; reference ). Representative flow cytometry plots are shown in the left panel of and composite data from multiple experiments are shown in the right panel. The data show that stimulation of PD-1, in both HIV- and CMV-specific CD8 T cells, resulted in a decrease in proliferative capacity. Alternatively, blocking of the PD-1–PD-L1 interaction with an anti–PD-L1 antibody resulted in increased proliferation of both HIV- and CMV- specific CD8 T cells. There was variation in the amount of inhibition or augmentation of proliferation that was not directly associated with the simple level of PD-1 expression, suggesting that PD-1 is not the only factor regulating the ability of antigen-specific T cells to proliferate.
We postulated that the effect of PD-1 on proliferation of antigen-specific T cells would affect the number of cytokine- producing cells in a multiday assay. Therefore, the ability of cells to produce cytokines after antigen stimulation and 6 d of culture was examined. PD-1 cross-linking in the presence of HIV Gag peptide reduced the frequency of CD8 T cells producing IFN-γ and had minimal effect on the frequency of cells producing TNF-α (). On the other hand, anti–PD-L1 treatment resulted in an increased frequency of cells capable of producing both cytokines (). Collectively, these data indicate that any effect of PD-1 engagement on cytokine expression in a multiday assay is secondary to enhanced proliferation of cytokine-producing cells.
We next sought to determine if the previously described effects of PD-1 on apoptosis were responsible for the observed effects on proliferation. PD-1 expression was consistently associated with higher sensitivity of total CD8 T cells to both spontaneous and CD95/Fas-induced apoptosis, estimated by annexin V surface exposure (, representative data; , compiled data). Apoptosis sensitivity was found to be significantly higher in HIV-specific compared with CMV-specific CD8 T cells, in agreement with previously published data (, ), and PD-1 CD8 T cells were always more sensitive to apoptosis than PD-1 CD8 T cells irrespective of antigen specificity (). Of interest, PD-1 HIV-specific CD8 T cells often were more sensitive to apoptosis than PD-1 CMV-specific CD8 T cells (). We therefore asked whether this difference could be secondary to the different maturational phenotypes of HIV- and CMV-specific CD8 T cells. In fact we found that PD-1CD8 T cells were more susceptible to both spontaneous and CD95/Fas-induced apoptosis compared with PD-1CD8 T cells in all memory subsets tested, but that sensitivity varied between maturational phenotypes (CD27CD45RO > CD27CD45RO > CD27CD45RO; ), suggesting that PD-1 expression augments apoptosis sensitivity upon the background defined by maturation and activation status.
Another possible explanation for the greater apoptosis sensitivity of HIV-specific CD8 T cells than CMV-specific CD8 T cells is the absolute level of PD-1 expression on the cells. Although we had defined CD8 T cells as being either PD-1 positive or PD-1 negative, it was apparent that PD-1 expression was higher (as measured by mean fluorescence intensity) on HIV- than CMV-specific CD8 T cells. We therefore asked if the absolute level of PD-1 expression on CD8 T cells was the primary determinant of apoptosis sensitivity while further determining whether PD-1 ligation directly affected survival of CD8 T cells. To do this, we assessed apoptosis in the presence and absence of plate-bound anti–PD-1 in samples from six HIV donors. The results were consistent for all of the donors, and the results from one are shown in . We found that HIV-specific CD8 T cells had higher expression of PD-1 than total or CMV-specific CD8 T cells, but that irrespective of antigen specificity, the CD8 T cells with the highest expression of PD-1 were the most sensitive to apoptosis and were the cells that augmented their apoptosis to the greatest extent upon PD-1 ligation. In fact, at medium/low and low levels of PD-1 expression, there was low spontaneous apoptosis that did not increase with PD-1 ligation, whereas at high levels of PD-1 expression, PD-1 ligation augmented the apoptosis to 80% or greater irrespective of antigen specificity. Therefore, the absolute level of PD-1 expression on antigen-specific CD8 T cells is the primary indicator of apoptosis sensitivity and the major determinant of sensitivity to apoptosis upon PD-1 ligation. Collectively, our data strongly support that PD-1 is a critical regulator of CD8 T cell survival in HIV infection.
The balance between positive and negative signals delivered by costimulatory molecules to T cells appears to be critical for the ultimate fate of cellular immune responses (, ). Recent data (, , ) suggest that manipulation of T cell costimulatory pathways may present a novel approach for enhancing and restoring virus-specific CD8 T cell responses, especially in the context of a chronic infection like HIV. We report here on the role of PD-1, a negative costimulatory receptor of T cells, as a regulator of virus-specific CD8 T cells in HIV infection. To understand how PD-1 can affect the function of CD8 T cells, it is critical to identify which cells provide the ligand(s) for PD-1. Our data indicate that mDCs and monocytes may use the PD-1–PD-L system to regulate adaptive antiviral immunity. Our findings in this context are very preliminary and much more needs to be investigated. For instance, the relative kinetics of costimulatory molecule expression on mDCs and monocytes after activation and the impact of the “positive” and “negative” signals delivered by these molecules to responding T cells remains to be elucidated. In addition, it will be important to determine whether there is redundancy between the various costimulatory signals affecting CD8 T cells or if they act independently by stimulating separate intracellular pathways after initiation of virus-specific CD8 T cell responses.
We found remarkably high expression of PD-1 on HIV-specific CD8 T cells. The frequency of PD-1 expression on the different virus-specific CD8 T cells (HIV = EBV > CMV > VV) is consistent with PD-1 regulation according to antigen stimulation. Although it is unlikely that the level of antigen in chronic EBV infection reaches that which occurs in HIV infection, it has been shown that EBV is continuously shed into saliva by induction of the lytic cycle as B cells differentiate into plasma cells, thereby chronically stimulating lytic cycle antigen-specific CD8 T cells (). The mechanism by which CD8 T cells control HIV and EBV may differ. Therefore, it may not follow that expression of PD-1 on EBV- and HIV-specific CD8 T cells will similarly impact CD8-mediated control of these two different virus infections. Further experiments are needed to clarify the relative role of PD-1 in regulation of EBV-specific CD8 T cell responses and compare it to the regulation of HIV-specific responses. In summary, although we cannot conclude that chronic antigen stimulation is the sole factor determining PD-1 expression, our data reveal that HIV-specific CD8 T cells, because of their high expression of PD-1, may be vulnerable to negative signals delivered by PD-1, potentially leading to functional consequences in vivo.
Despite their differential expression of PD-1, no difference in IFN-γ production was found between HIV-, CMV-, and EBV-specific CD8 T cells. On the other hand, PD-1 expression is associated with significantly lower ability of HIV-specific CD8 T cells to produce TNF-α and even lower production of IL-2. This is in agreement with the finding that PD-1–PD-L1 blockage has a substantial impact on LCMV-specific CD8 T cells producing both IFN-γ and TNF-α, whereas it has only a slight effect on single IFN-γ producers. However, we found that PD-1 and PD-1 antigen-specific CD8 T cells were equally able to produce cytokines upon antigen stimulation, indicating that PD-1 expression has no direct effect on cytokine production. This was further supported by our finding that ligation of PD-1 during antigen stimulation had no effect on cytokine production by virus-specific CD8 T cells. Collectively these data clearly demonstrate that PD-1 has no direct effect upon the immediate ability of antigen-specific CD8 T cells to produce IFN-γ, TNF-α, or IL-2.
Manipulation of the PD-1–PD-L system was found to alter the proliferation of virus-specific CD8 T cells. This was accompanied by altered percentages of CD8 T cells producing cytokines. The change in proliferation could result from an altered ability of these cells to either survive or divide. Importantly, we found no relationship between PD-1 expression and the degree of change in proliferative capacity after manipulation of the PD-1–PD-L1 axis. In fact, in some instances where the expression of PD-1 was very high on HIV-specific CD8 T cells, only minor effects of PD-1 ligation on proliferation were observed. Therefore, although PD-1 has a demonstrable effect on the ability of virus-specific (CMV and HIV) CD8 T cells to proliferate, it is not the sole factor regulating this function. This is not surprising as other factors (i.e., TCR activation threshold, relative expression of other costimulatory molecules, or levels of adaptor proteins mediating the intracellular signaling delivered by PD-1) could also contribute to the ability of PD-1 ligation to affect proliferative capacity.
At least two interventions have now proven successful in vitro in restoring the proliferation of HIV-specific CD8 T cells: the addition of IL-2 (or CD4 T cells producing IL-2) and the manipulation of costimulatory pathways such as PD-1. This raises the question of whether these two different manipulations affect proliferation through overlapping intracellular mechanisms. In addition, whether they can act in a synergistic mode remains to be elucidated. Since IL-2 cannot overcome the proliferative defect in CD57CD8 T cells (), it is of particular interest to examine whether manipulation of PD-1–induced pathways could specifically restore their proliferative capacity.
Our data indicate that the primary mechanism by which PD-1 affects CD8 T cell function is by regulating the ability of these cells to survive. This is in agreement with the originally described role of PD-1 as an apoptotic factor () and the reduced survival that characterizes virus-specific CD8 T cells under conditions of chronic antigen stimulation (, ). We can speculate on how PD-1 expression could affect HIV-specific CD8 T cell survival. It is possible that stimulation through PD-1 can direct cells into a cell cycle resting state, as has been described for the PD-1–PD-L2 interaction (). We found that lack of PD-1 expression is associated with similar levels of spontaneous and CD95/Fas-induced apoptosis, whereas CD95/Fas-induced apoptosis is greatly augmented in CD8 T cells that express PD-1, indicating that there may be cross-talk between the signals induced by these two receptors. Previously published data have shown that the Fas–FasL interaction impacts PD-L1–induced apoptosis of activated T cells (). Although no direct link between PD-1 and CD95/Fas was described in that work (), the possibility that PD-1 could prime (especially under conditions of chronic stimulation) CD8 T cells to undergo CD95/Fas-induced apoptosis cannot be excluded. Therefore, clarification of the intracellular mechanism(s) governing the proapoptotic function of PD-1 is of particular interest. Furthermore, the role of such a function on CD4 T cell survival in HIV infection would significantly add to our understanding of HIV pathogenesis.
A clear conclusion from our results is that the absolute level of PD-1 expression is a major determinant of spontaneous apoptosis and sensitivity to PD-1 ligation. We conclude this despite our observation that PD-1 HIV-specific CD8 T cells are often more susceptible to apoptosis than PD-1 CMV-specific CD8 T cells (). Although it is known that sensitivity to apoptosis is also affected by other factors—specifically the level of T cell activation (defined by CD38 expression; unpublished data) and maturational state, which we have shown is independent of PD-1 expression (, A and B and )—our data indicate that PD-1 is a primary determinant of apoptosis sensitivity over and above these other factors. We conclude this because, within any population of CD8 T cells (defined by activation, maturation, or antigen specificity), the PD-1 population is more sensitive to apoptosis than the PD-1 population. In addition, although we have described PD-1 expression as either positive or negative in most of our data, expression really represents a continuum, with high expression being associated with greater impact upon PD-1–regulated functions (i.e., apoptosis; ). When CD8 T cells of different antigen specificities, but similar levels of PD-1 expression, are analyzed, they exhibit similar levels of spontaneous apoptosis and sensitivity to PD-1 ligation. In addition, cells with moderate to low expression of PD-1 have low levels of spontaneous apoptosis and are not affected by PD-1 ligation. What this means is that within any population of antigen-specific CD8 T cells, it is the absolute level of PD-1 that primarily dictates the rate of spontaneous apoptosis and sensitivity to PD-1 ligation. Therefore, what is unique about HIV-specific CD8 T cells is their high level of PD-1 expression leading to a profound (but potentially reversible) survival defect. Although the proliferative capacity of a CD8 T cell is determined by more than just an ability to resist apoptosis, it is not difficult to visualize how the level of PD-1 and associated sensitivity to apoptosis would impact on the ability of a cell to proliferate.
Overall, our data demonstrate that PD-1 is preferentially expressed on CD8 T cells specific for chronic viruses, and that PD-1 interaction with its ligands can regulate the ability of these virus-specific CD8 T cells to survive and proliferate. Therefore, manipulation of this axis may lead to at least partial restoration of antigen-specific cell numbers and function in chronic viral infections such as HIV. It is important to remember that our data do not support the ability of PD-1 manipulation to restore all of the T cell functions that define functional “exhaustion.” For instance, we have no evidence that PD-1 blockade will restore absent cytokine functions, and may only affect CD8 T cell proliferation to the degree possible in the context of other as yet undetermined defects in HIV-specific CD8 T cells. Therefore, although our data identify PD-1 as a potential therapeutic target for restoring functional capacity of HIV-specific CD8 T cell responses, it may not be capable of fully restoring function. In addition, it should be appreciated that the PD-1–PD-L1 axis likely evolved to attenuate potentially harmful CD8 T cell responses to both self-antigens and chronic pathogens. Given that many non–HIV-specific CD8 T cells express PD-1 (), it is likely that interventions to release all CD8 T cells from PD-1–mediated suppression will have untoward effects.
PBMCs were obtained from HIV and HIV volunteers and cryopreserved until use. None of the HIV-infected subjects in this study were on antiretroviral therapy, and they had a range of viral loads from <50 to 439,000 per ml. Signed informed consent approved by the relevant Institutional Review Board was obtained. Samples from VV-naive individuals, preimmunized with modified vaccinia virus Ankara followed by scarification with Dryvax, were also used. Samples were taken within 3 mo of Dryvax administration. Purification of DC subsets has been previously described ().
The following directly conjugated antibodies were used: CD3-Cy7APC, IFN-γ–FITC, TNF-α–Cy7PE, CD14-FITC, CD11c-PE, CD11c-APC, PD-1–PE, PD-L1–Cy7PE, PD-L2–APC (all from BD Biosciences) and CD45RO-TexasRedPE (Beckman Coulter). Annexin V-Cy5PE and the following antibodies were conjugated in our laboratory: IL-2–FITC, IL-2–APC, CD14–Cascade blue, CD19–Cascade blue, CD8-Qdot 705, and CD27-Cy5PE. The unconjugated mAbs were obtained from BD Biosciences. Cascade blue was obtained from Molecular Probes. Cy5 was obtained from BD Biosciences. Quantum Dots were obtained from the Quantum Dot Corporation. The following APC-labeled tetramers were prepared as previously described (): A2-Gag (SLYNTVATYL), A2-CMV (NLVPVMTV), A2-Vaccinia (KVDDTFYYV), B8-Nef (FLKEKGGL), and A2-EBV (GLCTLVAML).
PBMCs were thawed and rested overnight at 37°C. Viability by Trypan blue exclusion was typically ≥90%. 2 × 10 PBMCs were diluted to 1 ml with medium containing costimulatory antibodies (αCD28 and αCD49d) (1 μg/ml) (Becton Dickinson), monensin (0.7 μg/ml; BD Biosciences), brefeldin A (10 μg/ml; Sigma-Aldrich), in the absence or presence of indicated amounts (μg/μl) of A2-Gag (SLYNTVATL) or A2-CMV (NLVPVMTV) epitope peptides and incubated for 6 h. Cells were washed and incubated with pretitrated amounts of APC-labeled A2-gag or A2-Cmv tetramer for 15 min at 37°C. After washing, cells were surface stained with PD-1–PE, CD8–Qdot 705, CD14– and CD19–cascade blue, and 156 ng/ml violet amine reactive viability dye (vivid; InVitrogen). Following permeabilization (Cytofix/Cytoperm kit; BD Biosciences), cells were stained with IFN-γ–FITC or IL-2–FITC, TNF-α–PECy7, and CD3-Cy7APC. Alternatively, cells were left untreated or preincubated for 30 min at 37°C with an anti–human PD-1 antibody (AF 1086; R&D Systems) (20 μg/ml) and subsequently stimulated with peptides (15mers overlapping by 11) corresponding to full-length HIV-1 Gag (2 μg/ml each peptide, 5 μl/ml; National Institutes of Health AIDS Research and Reference Reagent Program) or cytomegalovirus antigen (60 μl/ml; Microbix Biosystems Inc.) for 6 h. After a washing step, cells were stained with vivid, CD14– and CD19–cascade blue, CD8-Qd705, permeabilized, and stained intracellularly with CD3-Cy7APC, IFN-γ–FITC, IL-2–APC, and TNF-α–Cy7PE. For CFSE studies, PBMCs were washed thoroughly and labeled with 0.25 μM CFSE (Molecular Probes). Cells were adjusted to 1.5 × 10 cells/ml and cultured in the presence of peptides (15mers overlapping by 11) corresponding to full-length HIV-1 Gag (2 μg/ml each peptide, 5 μl/ml; National Institutes of Health AIDS Research and Reference Reagent Program) or cytomegalovirus CF antigen (60 μl/ml; Microbix Biosystems Inc.). aCD28/aCD49d (1 μg/ml) was used for costimulation. An unstimulated and a positive control (culture with 1 μg/ml immobilized anti-CD3, clone UCHT1; R&D Systems) was included in each assay. Antibodies against human PD-1 (AF 1086; R&D Systems) (20 μg/ml) and human PD-L1 (16–5983; eBioscience) (25 μg/ml) were used for cross-linking of PD-1 and PD-L1, respectively. Cells were cultured for 6 d, harvested, and stained first with A2-Gag or A2-CMV tetramer–APC (15 min, 37°C) and subsequently with annexin V–Cy5PE, CD3-Cy7APC, and CD8-PE. 2.5 mM CaCl was included in all staining steps. Alternatively, cells were cultured under the same conditions, and on day 6 cells were stained with vivid and CD8-Qd705 and intracellularly with CD3-Cy7APC, IFN-γ–FITC, IL-2–APC, and TNF-α–Cy7PE.
1–1.5 × 10 PBMCs were cultured in 24-well plates (BD Biosciences) in the absence or presence of plate-bound anti–human CD95/Fas (IgM, CH11; Upstate Biotechnology) (5 μg/ml) or anti–human PD-1 (AF 1086, R&D Systems) (20 μg/ml) for 12 h at 37°C. Cells were harvested, washed, and surface stained with APC-labeled A2-Gag, A2-CMV, or B8-Nef tetramers, and subsequently with annexin V–Cascade blue, CD3-Cy7APC, CD8-Qd705, CD27-CyPE, CD45RO-TRPE, and PD-1–PE and green amine reactive viability dye (grivid; InVitrogen). 2.5 mM CaCl was included in all staining steps.
Cells were analyzed with a modified LSRII flow cytometer (BD Immunocytometry Systems). Between 200,000 and 1 million events were collected. Electronic compensation was conducted with antibody capture beads (BD Biosciences) stained separately with individual mAbs used in the test samples. Data analysis was performed using FlowJo version 6.0 (TreeStar). Forward scatter area (FSC-A) versus forward scatter height (FSC-H) was used to gate out cell aggregates. CD14 cells, CD19 cells, and dead cells were removed from the analysis to reduce background staining. The cells were then gated through a FSC-A versus side scatter height (SSC-H) plot to isolate small lymphocytes. Next, CD3 cells were selected and PD-1 expression was measured in gated total CD8 T cells and tetramer cells, and in relation to their differentiation level by using the CD27-Cy5PE and CD45RO-TexasRedPE memory markers. Tetramer cells were selected and the percentage of PD1 and PD1, IFN-γ, and TNF-α–producing cells was determined using gating criteria determined using the total CD3CD8 population. For CFSE analysis, after initial gating (FSC-A versus FSC-H), apoptotic (annexin V) cells were removed and small lymphocytes were identified. CD3 cells were selected, and the percentage of CFSE low cells was determined in gated total CD8 T cells and tetramer cells. For analysis of monocytes and DCs, the following combinations of titrated antibodies were used: CD11c-APC/CD14-FITC/PD-1–PE, CD11c-PE/CD14-FITC/PD-L1–Cy7PE/PD-L2–APC, and CD123-Cy5PE/PD-1–PE/PD-L1–Cy7PE/PD-L2–APC. PD-1, PD-L1, and PD-L2 levels were determined in CD14CD11c (monocytes), CD14CD11c (mDCs), and high CD123 (pDCs) cells.
Statistical analysis was performed using Student's test and Wilcoxon's paired test. P values < 0.05 were considered significant. The GraphPad Prism statistical analysis program was used. |
To study early B cell tolerance in SLE patients during clinical remission, we cloned, expressed, and tested antibodies from mature naive B cells from six adolescent patients (SLE100CR, SLE101CR, SLE122CR, SLE14CR, SLE21CR, and SLE33CR; ) and compared them to recombinant antibodies cloned from three previously published healthy controls (, ). Three of the remission patients described here had been studied at the time of diagnosis before any therapeutic intervention (SLE100, SLE101, and SLE122; reference ). All patients met the Revised Criteria of the American College of Rheumatology, and their treatment is summarized (; reference ). Remission was defined by resolution of clinical symptoms, normalization of laboratory findings, and minimal maintenance therapy, but we did not assay for remission of organ damage (). Samples were obtained at least 3 mo after initial remission, and 278 antibodies were cloned from cDNA libraries created from single mature naive B cells purified on the basis of surface markers (CD19CD10IgMCD27; Tables S1–S6 and Fig. S1, which are available at ; reference ). Sequence analysis confirmed that all clones were unrelated and derived from naive B cells because they lacked somatic hypermutation ().
Several different abnormalities in Ig gene usage have been reported for patients with SLE, including bias toward V3, V4-34, Vκ1, and Vκ4 gene family usage (, ). We found some of these abnormalities, but no consistent abnormalities and no consistent differences between active disease and remission (). For example, SLE122 initially showed increased V3 gene family representation and unusually short IgH CDR3 regions, and this pattern remained unchanged after treatment (P = 0.948 for V repertoire, P = 0.454 for IgH CDR3 length), but these abnormalities were not found in other patients (; reference ). In contrast, overrepresentation of Vκ4-1 was found in active disease in SLE100 and SLE122, but not in remission (; reference ). We conclude that there are no consistent abnormalities in the IgH or IgL repertoires in SLE patients with active disease or in remission, and that although some specific features such as long CDR3s are associated with increased self-reactivity, they are not predictive.
Antibodies reactive with HEp-2 cell lysates as measured in ELISA assays (used to detect ANAs, ANA ELISA) are significantly enriched in mature naive B cells from untreated SLE patients compared with healthy controls (). To measure the frequency of these antibodies in clinical remission, we tested the cloned antibodies in HEp-2 cell ELISA assays and confirmed positives by indirect immunofluorescence assay on HEp-2 cell–coated slides (, Tables S1–S6, and Fig. S2, which is available at ; references and ). Although there was individual variation, antibodies cloned from mature naive B cells from SLE patients in remission remained more self-reactive in the HEp-2 ELISA than healthy controls (34.25 vs. 19.7%, respectively, P = 0.025; ; references and ). In all three cases where paired samples were available, the frequency of HEp-2 reactive antibodies was lower in remission than in crisis, but the difference only reached statistical significance in SLE101, who showed near normal levels of HEp-2–reactive antibodies in clinical remission (22.4% in SLE101CR vs. 19.7% in control, P = 0.710, and vs. 50.0% in SLE101, P = 0.009). We conclude that there is variability in the HEp-2 reactivity of the antibodies produced by mature naive B cells among SLE patients in crisis and remission, but most SLE patients in clinical remission show persistent abnormalities in this respect.
Polyreactivity, i.e., antibody binding to diverse nonrelated antigens including self-antigens such as DNA and insulin, is another measure of self-reactivity. In normal humans, polyreactive antibodies comprise ∼6% of antibodies in the circulating mature naive B cell compartment, whereas in untreated SLE patients, these antibodies account for ∼31% of the repertoire (, , ). To determine whether this abnormality persists during remission, we tested our collection of remission-derived antibodies for binding with single-stranded DNA, double-stranded DNA, insulin, and LPS (). Although the number of polyreactive antibodies varied between individuals, all but one (SLE101CR) of the clinical remission patients showed significantly elevated levels (16.7% in SLE100CR and SLE122CR, P = 0.083 and 0.041, respectively; 17.8% in SLE21CR, P = 0.033; 19.0% in SLE33CR, P = 0.028; and 25.0% in SLE14CR, P < 0.001 vs. 6.2% in healthy controls; reference ). The one patient that differed from the rest, SLE101, had higher than normal levels of polyreactivity before treatment (25.8%, P = 0.004; reference ) but reverted to normal levels of polyreactivity in clinical remission (3.0% in SLE101CR vs. 6.2% in control, P = 0.499). The other paired samples showed decreased polyreactivity in remission, but the difference did not reach statistical significance (30.8% in SLE100 and 16.7% in SLE100CR, P = 0.444; 24.4% in SLE122 and 16.7% in SLE122CR, P = 0.433). Finally, when all remission samples, including SLE101CR, were combined, the overall level of polyreactivity in the mature naive B cell compartment was significantly greater than in healthy controls (15.5 vs. 6.2%, respectively, P = 0.009), but the frequency of polyreactive antibodies in clinical remission was significantly lower than at the time of diagnosis (15.5 vs. 31.5%, respectively, P < 0.001; ).
The abnormalities in early B cell self-tolerance in active SLE might be secondary to disease activity per se because lymphopenia and mediators of acute inflammation such as type I IFNs and TNF-α can directly alter B cell development in vivo (–). Indeed, we found a decrease in the number of HEp-2–reactive and polyreactive antibodies expressed by circulating mature naive B cells during remission. However, despite normal numbers of circulating lymphoid and myeloid cells (), the majority of patients in clinical remission continued to show persistent abnormalities in antibody tolerance in the mature naive B cell compartment, suggesting that defects in early tolerance defects are in part independent of active disease.
Primary Ig gene rearrangements frequently involve 3′ Vκ genes and 5′ Jκ genes (–). This allows for replacement of self-reactive antibodies by secondary recombination between 5′ Vκ genes and 3′ Jκ genes during receptor editing (–). Abnormalities in IgL chain receptor editing based on sequence analyses have been reported in patients with autoimmune diseases including SLE (, , ), but these reports did not discriminate between patients with active disease and patients in clinical remission. To determine whether receptor editing associated genomic alterations occurs in the Igκ locus in SLE patients, we analyzed the Vκ and Jκ usage of self-reactive versus nonself-reactive antibodies (). Vκ genes were divided into four groups according to their 5′ to 3′ orientation in the Igκ locus (VκA–VκD). Jκ genes were grouped into 5′ (Jκ1/2) and 3′ (Jκ3/4/5) genes. In healthy controls, few self-reactive or polyreactive antibodies were present in the mature naive B cell compartment (, ), and there was no apparent difference in Jκ usage between self-reactive and nonself-reactive antibodies (0.2 and 0.4, respectively; ). In contrast, untreated SLE patients with active disease showed high levels of self-reactive antibodies that used 5′ Jκ genes (ratio of self-reactive/nonself-reactive: 1.5 for SLE100, P = 0.006; 1.4 for SLE101, P = 0.01; and 1.3 for SLE122, P = 0.013 when compared with healthy controls; references and ) and a variable level of self-reactive antibodies that used 3′ Jκ genes (0.3 for SLE100, P = 1.0; 0.4 for SLE101, P = 1.0; and 1.5 for SLE122, P = 0.047). However, SLE patients in remission resembled healthy controls in terms of frequency of self-reactive antibodies that used 5′ Jκ genes and were significantly different from patients with active disease (0.4 in total SLECR vs. 1.4 in total SLE, P = 0.001; 0.4 in total SLECR vs. 0.2 in controls, P = 0.229; references , , and ). We found a significant bias toward 3′ Vκ gene usage in self-reactive antibodies in active patients, but not in remission (; P = 0.003 and 0.052, respectively; references and ). In summary, mature naive B cells from SLE patients with active disease express self-reactive antibodies that are biased to contain 3′ Vκ's in association with 5′ Jκ's, whereas those from patients in remission do not. Such antibodies are more likely to originate from primary rearrangements (–), suggesting that active SLE is associated with increased expression of unedited self-reactive antibodies by mature naive B cells.
B cell tolerance is regulated by distinct checkpoints in the bone marrow and in the periphery. Either of these might be altered in SLE (for review see reference ). Elegant studies with transgenic mice have demonstrated that nascent self-reactive B cells can be regulated by receptor editing, deletion, or anergy, and that receptor editing appears to be the preferred mechanism to establish early B cell self-tolerance (, , ). Consistent with the primary importance of editing in shaping the B cell repertoire, this mechanism produces 25–50% of all antibodies (, ). In patients with active SLE or rheumatoid arthritis, and in mice with chronic autoimmune disease, the increase in self-reactive antibodies is consistent with abnormalities in editing and/or selection (, , ). In SLE patients in clinical remission, normalization of Igκ gene usage is consistent with an overall decrease in the number of self-reactive antibodies in these patients.
Less is known about how B cell tolerance is established in the periphery. Both positive and negative selection mechanisms have been proposed (), and several genetic loci have been implicated in disease etiology (). Under normal conditions, one important determinant appears to be decreased expression of BAFF receptor on autoreactive B cells rendering them less able to compete for limiting amounts of BAFF, which is a key B cell survival factor (). Consistent with this idea, excess BAFF expression leads to autoimmunity in mice (–), and patients with SLE show high levels of BAFF that could enable self-reactive B cells to survive in the mature naive B cell repertoire ().
The study was performed in accordance with institutional review board–reviewed protocols of the UT Southwestern Medical Center (IRB no. 0199-017) and the Rockefeller University (SYU-0571-1005), and all samples were obtained after signed informed consent at the Division of Pediatric Rheumatology of UT Southwestern Medical Center. Control data and data from SLE100, SLE101, and SLE122 before treatment were previously published and are shown for direct comparison. Single B cell isolation was performed as described previously (, , ).
Single cell cDNA synthesis and RT-PCR reactions were performed as described previously (, , ). All sequences matched published germline sequences of human Ig genes or could be attributed to polymorphisms (not depicted).
Antibodies were expressed in vitro as described previously (, , ). Antibody concentrations were determined by ELISA (, , ). ELISAs were performed as described previously (, , ), and dilutions are indicated in the figures. Samples from SLE patients were considered negative if the OD did not exceed a threshold value as indicated in each graph at any of the four dilutions in at least three independent experiments (Tables S1–S6). Threshold values for reactivity with specific antigens were set in all assays using antibodies from healthy donors and included our previously published control antibodies, mGO53 (negative), eiJB40 (low positive), and ED38 (strong positive; references and ). HEp-2 ELISAs were performed as described previously (, , ). The threshold OD levels below which samples were considered negative are indicated in the graphs. Positive and negative controls included sera from patients and healthy individuals (INOVA Diagnostics) and were included in every experiment.
Indirect immunofluorescence assays were performed as described previously (). Controls included ED38 () and positive and negative sera (Bion Enterprises, Ltd.).
p-values for Ig gene repertoire analyses, analysis of positive charges in IgH CDR3, and antibody reactivity were calculated by 2 × 2 or 2 × 5 Fisher's Exact test or Chi-square test. p-values for IgH CDR3 length were calculated by Student's test.
Fig. S1 shows representative FACS profiles of B cells from SLE patients in clinical remission. Fig. S2 shows antibody staining patterns in HEp-2 cell immunofluorescence assay. Tables S1–S6 show IgH and IgL chain characteristics and antibody reactivity for mature naive B cells of SLE patients in clinical remission. The online supplemental material is available at . |
Because HO-1 is a well-known cytoprotective protein induced by inflammation or oxidative stress (), we evaluated the physiological properties of HO-1 in vivo by using HO-1 null (HO-1) mice. We stained kidneys and livers for CI:A3-1 as an indicator of mouse monocytes or macrophages, and for TNF-α by immunohistochemistry under basal untreated conditions. We observed significant infiltration of macrophages and TNF-α expression around the convoluted tubules in the cortex of the kidney from HO-1 mice (). In contrast, immunohistochemical staining for macrophages and TNF-α was negligible in the kidney of wild-type (HO-1) mice (). In the livers from HO-1 mice (), marked accumulation of macrophages was observed in the portal vein accompanied with cellular infiltration in the interlobular septum as compared with in the liver from HO-1 mice (). Kupffer cells, which also can be stained by the CI:A3-1 marker, are likely to represent the increased staining observed in the sinusoid of the liver from HO-1 mice relative to that from HO-1 mice (). TNF-α expression was also markedly induced, consistent with localization of the macrophages in the liver from HO-1 mice (), as compared with liver from HO-1 mice (). In addition, increased staining for macrophages and expression of TNF-α was observed in the intestines from HO-1 mice compared with control mice (unpublished data). These results suggest that systemic inflammation is chronically increased in the HO-1 mice (–), consistent with the known antiinflammatory properties of HO-1 and the involvement of HO-1 in regulating the functions of monocyte/macrophages (, ).
HO-1 is significantly induced by LPS (a ligand for TLR4) stimulation and has been shown to dampen the inflammatory effects of LPS-treated models (, , ). Therefore, we examined whether HO-1 could modulate the immune response in various TLR signaling pathways. HO-1–overexpressing RAW 264.7 macrophages produced significantly less TNF-α than the control cells in response to LPS, PamCSK4 (a ligand for TLR2), and CpG (a ligand for TLR9) (; references and ). However, when the HO-1–overexpressing cells were treated with LPS and hemoglobin, a scavenger of CO (), the cells failed to inhibit LPS-induced TNF-α production (). These data suggest that the antiinflammatory effect of HO-1 on TLR signaling pathways in macrophages is mediated primarily by CO ().
Because HO-1 inhibited TLR2, 4, and 9 ligand–induced TNF-α production (), and CO as a byproduct of heme catabolism by HO-1 was critically involved with the antiinflammatory effect of HO-1 (), we next investigated the effect of CO on various TLR ligand–induced cytokine production in RAW 264.7 cells (TLR ligands; TLR2, peptidoglycan or PamCSK4 [Pam]; TLR3, double-stranded RNA poly(I:C); TLR4, LPS; TLR5, flagellin [Fla]; and TLR9, CpG). CO exposure significantly suppressed TLR2, 4, 5, and 9 ligand–induced TNF-α production (), which is similar to the effect of HO-1 observed in . However, CO did not affect poly(I:C)-induced TNF-α production (). As previously described, the effect of CO on LPS-induced cytokine production was dose dependent over a range of 50–500 ppm (, ); however, cytokine production by poly(I:C) was not affected by CO exposure at any of the doses (). To further confirm the effect of CO on macrophages, mouse peritoneal macrophages were stimulated with the TLR ligands in the presence or absence of CO. The effect of CO on TNF-α production in the mouse peritoneal macrophages was consistent with that observed in RAW 264.7 macrophages (). Because poly (I:C) is recognized not only by TLR3 but also by the helicase domain of MDA5 or RIG-I (), we examined the relative role of TLR3 in poly(I:C)-induced cytokine production by using peritoneal macrophages from TLR3-deficient mice (
).
macrophages was reduced to nearly background levels, relative to the poly (I:C)-induced TNF-α production observed in wild-type macrophages ().
macrophages (). Activation of both TLR3 and TLR4 signaling cascades induces IFN-β through the activation of IFN regulatory factor 3 (IRF-3), leading to the production of IFN-inducible gene products, such as IFN-γ–inducible protein 10 (IP-10) and the regulated upon activation, normal T expressed, and presumably secreted protein (RANTES; references and ). Although CO significantly inhibited LPS-induced IFN-β gene expression and the production of IP-10 and RANTES, the induction of these cytokines by poly(I:C) was not inhibited by CO ().
Both NF-κB and IRF-3 are key transcription factors activated in TLR3 and TLR4 signaling pathways (, ). Because CO inhibited the TLR ligand–induced (except TLR3) cytokine production, we next examined the effect of CO on the ligand-induced activation of NF-κB and IRF-3. CO inhibited LPS-induced NF-κB activation as described previously (), and Pam-, Fla-, and CpG-induced NF-κB activation was also significantly suppressed by CO (). Translocation of IRF-3 to the nuclear fraction increased after LPS treatment (), and CO significantly suppressed its translocation (). However, CO had no effect on poly(I:C)-induced NF-κB activation and translocation of IRF-3 (). Consistent with the effect of CO on TLR ligand–induced cytokine production ( and ), CO inhibited the activation of transcriptional factors by LPS, but not by poly(I:C) (). These results indicate that CO differentially regulates TLR signaling pathways.
With the exception of TLR3, all the TLRs interact with an adaptor protein, MyD88 (). TLR3 uses only the adaptor molecule TIR domain–containing adaptor-inducing IFN-β (TRIF) to activate IRF-3, and TLR4 also activates IRF-3 through TRIF. To investigate the effect of CO on upstream events in TLR signaling pathways, we assessed the effect of CO on interactions of TLRs and adaptor molecules that are subsequently induced after ligand binding to TLR (, ). We observed increased interaction not only between TLR4 and MyD88 but also between TLR4 and TRIF at 5 min after LPS treatment (); however, CO markedly attenuated both interactions (). Although poly(I:C) treatment also increased the interaction of TLR3 and TRIF, CO did not suppress the TLR3 and TRIF interaction (). These experiments support the notion that the negative regulation of CO on TLR signaling pathways is likely to be dependent not on adaptor proteins, but on the specificity of TLRs.
Membrane rafts are involved with TLR signaling, including the activation of NF-κB and cytokine production (, ). To further address the involvement of lipid rafts on TLR signaling pathways and the potential role of CO on the membrane rafts, cells were stimulated with LPS or poly(I:C) in the presence or absence of CO and incubated with FITC–cholera toxin (CTx). CTx specifically binds to the glycosphingolipid 1 (GM1), which is enriched in lipid rafts (, ). In the resting RAW 264.7 cells, the distribution of GM1 on the plasma membrane was quite homogeneous and TLR4 localized, in a diffuse manner, both in the membrane and intracellular compartment (; reference ). After LPS treatment, a large fraction of TLR4 translocated to the plasma membrane, and colocalization of TLR4 and GM1 was also observed (). In CO-treated cells, the LPS-induced translocation of TLR4 to the membrane rafts was significantly inhibited (). In contrast, TLR3 localized in the intracellular compartment in the resting cells (), which is consistent with previous results (, ), and remained unchanged after poly(I:C) stimulation, even when CO was added (). The localization of TLR4 and TLR3 did not change in the cells treated with CO alone (unpublished data). Next, cells were incubated with FITC-CTx and cross-linked with anti-CTx. CTx specifically binds to the GM, and GM1-CTx can be crossed-linked to membrane patches with anti-CTx (, , ). These membrane patches have properties that are similar to those of biochemically isolated rafts (, , ).Without anti-CTx cross-linking, distribution of GM1 on the plasma membrane was homogeneous and TLR4 was expressed in a diffuse manner (Fig. S1 A, available at ). After the anti-CTx cross-linking at 37°C, a large part of GM1 translocated to a crescent-shaped patched area in the plasma membrane and a large fraction of TLR4 also translocated to the same area. Colocalization of TLR4 and GM1 was observed (Fig. S1 A). In contrast, TLR3 remained unchanged in the cytosolic fraction, not in the crescent-shaped patched area, after the cross-linking at 37°C (Fig. S1 B). To further investigate whether CO modulates the translocation of TLR4 and its adaptor molecules to lipid rafts, we isolated raft fractions and examined the translocation of the proteins involved in TLR signaling by immunoblotting. Flotillin-1, constitutively expressed in lipid rafts (), localized in fractions 3 and 4. LPS or CO treatment had no effect on its abundance among the sucrose density fractions (). Most of GM1 localized in the same fractions () and no alteration of the distribution was observed by LPS or CO treatment (unpublished data; references and ). TLR4 and its adaptor molecules (MyD88 and TRIF), as well as MD-2, IL-1 receptor–associated kinase, TNF receptor–associated factor (TRAF)-6, p65, and IκB-α, rapidly translocated to lipid rafts after LPS stimulation (, ), whereas TRAF-2 was not translocated (). In contrast, CO significantly inhibited the LPS-induced recruitment of TLR4 and other signaling molecules to lipid rafts (). The distribution of TLR4 was not affected by CO treatment alone (). Consistent with other data (), CD-14 was constitutively expressed mainly in lipid rafts of resting cells and its expression was unaffected by LPS or CO treatment (). To confirm the differential localization and translocation patterns of TLRs, TLR3, 4, and 9 were immunoprecipitated with each antibody and immunoblotted with the same antibody using Triton X-100 soluble and insoluble raft fractions. After the ligand stimulation, TLR4 and TLR9 translocated to the rafts fraction, whereas TLR3 did not (). In addition, the adaptor protein TRIF interacted with TLR4 in the rafts fraction by LPS stimulation, but not with TLR3 by poly(I:C) (). These results confirm the differential translocation of TLRs to lipid rafts by ligand stimulation and agree with our immunofluorescence data ( and Fig. S1). To examine the role of lipid rafts as a platform of ligand-induced TLR activation, we pretreated cells with MβCD and stimulated them with LPS, Pam, or poly(I:C). Although LPS- or Pam-induced TNF-α production was significantly reduced by MβCD treatment, poly(I:C)-induced cytokine production was unaffected (; references and ). In contrast, pretreatment of monodansylcadaverine (MDC), an inhibitor of the clathrin-dependent internalization pathway (), markedly suppressed TNF-α production by poly(I:C) treatment, but not by LPS or Pam in macrophages (). These data suggest that the involvement of lipid rafts in TLR signaling is dependent on the specificity of the TLRs.
ROS are known to be critical in intracellular signaling, including the TLR signaling pathway, and the scavenging of ROS or the inhibition of NADPH oxidase suppresses LPS-induced cytokine production (, ). To examine if ROS are involved with the TLR signaling pathway and trafficking of TLR to lipid rafts, RAW 264.7 cells were pretreated with the antioxidant -acetyl--cysteine (NAC) or a NADPH oxidase inhibitor, diphenylene iodonium (DPI), for 30 min, followed by incubation with TLR ligands. NAC and DPI suppressed LPS-induced TNF-α production in a dose-dependent manner (), and DPI significantly suppressed TNF-α production induced by LPS, Pam, and CpG, but not by poly(I:C) treatment (). DPI also inhibited LPS-induced interaction of TRIF and TLR4 (). In addition, we observed that the LPS-induced translocation of TLR4 to lipid rafts was inhibited by DPI treatment (). These results indicate that inhibition of NAPDH oxidase suppresses the TLR signaling pathway by modulating events upstream of the pathway. Furthermore, cellular stimulation with HO and PMA also recruit TLR4 to lipid rafts (), which is consistent with previously reported results (). To investigate the involvement of CO on TLR ligand–induced ROS generation, we detected TLR ligand–induced ROS production by using a fluorescence probe. CO significantly suppressed not only LPS- but also Pam-induced ROS production (). However, CO failed to inhibit poly(I:C)-induced ROS production in macrophages (). Upon stimulation, ROS are generated by NADPH oxidase, which forms a membrane-bound complex, including p22 and gp91 (cytochrome 558) and cytosolic proteins such as p47 (, ). The complex of gp91 and p47 started to increase 5 min after LPS treatment, whereas complex formation was inhibited by CO as well as DPI treatment (). Furthermore, superoxide anion production, detected in isolated membrane fractions from LPS-treated macrophages, was inhibited when the cells were also exposed to CO (). Because CO is known to bind to heme proteins such as hemoglobin (), we asked whether CO may interact with gp91, a heme protein (, ). To address this question, we partially purified cytochrome 558 from bovine neutrophils and performed a spectral analysis. An oxidized spectrum was observed with a major peak at 410 nm. Heme proteins show major peak of absorption spectra typically at 400–450 nm and minor peaks in the range of 500–650 nm (, ). After incubation of the extract with sodium dithionite, the major peak shifted to 421 nm, generating distinct difference spectra (Fig. S2, available at ). Exposure of CO to the dithionite-reduced form of cytochrome 558 extract decreased the absorption peak and shifted the major peak from 421 to 418 nm, generating distinct difference spectra (). To elucidate the functional role of NADPH oxidase in TLR signaling, peritoneal macrophages from gp91-deficient (gp91) mice were exposed with LPS in the presence or absence of CO. Cells from gp91 mice significantly produced less TNF-α in response to LPS than the cells from the wild-type control (). The effect of CO on LPS-induced TNF-α production was impaired in gp91-deficient cells (). In addition, we examined the interaction of TLR4 and gp91 by LPS. TLR4 interacted with gp91 in response to LPS treatment, whereas DPI treatment inhibited the interaction (). CO inhibited the LPS-induced complex formation of TLR4 and gp91 (). Finally, to confirm if NADPH oxidase is involved in the LPS-induced translocation of TLR4 to lipid rafts, we isolated lipid rafts from peritoneal macrophages from gp91 mice. TLR4 translocated to lipid rafts 5 min after LPS treatment in the wild-type cells; however, the translocation of TLR4 by LPS was suppressed in gp91-deficient cells (). These results suggest that gp91 is involved with LPS-induced translocation of TLR4 to lipid rafts and that the effect of CO on trafficking to lipid rafts is potentially mediated by the modulation of gp91 and the suppression of NADPH oxidase activity, leading to the inhibition of trafficking of TLR4 to lipid rafts.
Although several negative regulators of TLR signaling have been reported (), there are few reports that the modulation of TLR trafficking to lipid rafts correlates with the regulation of TLR signaling, and that elucidate the mechanism of trafficking (). Here we demonstrate that TLRs show differential patterns of trafficking after TLR ligand stimulation, and that NADPH oxidase is critically involved with translocation of TLR4 to lipid rafts and downstream TLR signaling. Our results showed that TLR4 translocated to lipid rafts by LPS, whereas TLR3 remained in the intracellular compartment after poly(I:C) treatment. In addition, CO significantly suppressed LPS-induced ROS generation, resulting in the inhibition of the recruitment of TLR4 to the rafts and of downstream pathways, including activation of transcription factors and cytokine production. In contrast, CO failed to regulate the TLR3 signaling pathway. Thus, CO differentially regulated TLR signaling pathways.
CO negatively regulated TLR2, 4, 5, and 9 signaling pathways as revealed by the inhibition of cytokine production and the inactivation of transcription factors (–
). In addition, the effect of CO was exerted at the initial signaling events, TLR4–MyD88 or TLR4–TRIF complex formation (). In contrast, CO had no effect on poly(I:C)-induced TLR3 signaling pathways from complex formation of TRIF and TLR3 to cytokine production (–
), which is consistent with the observation that HO-1–overexpressing cells failed to suppress poly(I:C)-induced cytokine production (unpublished data). Although recent studies show that the cytoplasmic proteins RIG-I and MDA5 have also been identified as double-stranded RNA detectors (, ), our results demonstrate that TLR3 plays the dominant role in poly(I:C)-induced cytokine production at early time points in macrophages (; reference ). It has been shown that several molecules act as negative regulators in multiple TLR signaling pathways and some of them exert their effects on the level of receptor or adaptor proteins (). ST2 negatively regulates TLR2, 4, and 9, but not TLR3 signaling (), which is similar with the differential effect of CO. Although ST2 suppresses TLR4 signaling by sequestration of the adaptor protein MyD88 and Mal, but not TRIF (), CO inhibited the LPS-induced interaction of TLR4 and TRIF as well as of TLR4 and MyD88. Although Triad3A also negatively regulates TLR4- and TLR9-mediated signal activation, it has no effect on the TLR2 signaling pathway (). Thus, the mechanism of negative regulation by CO on TLR signaling pathways is likely to be independent of the type of adaptor molecules as well as transcription factors, but dependent on the specificity of TLRs.
Our studies have shown that translocation of TLRs to lipid rafts is also dependent on their diversity (, ). Although IκB kinase α/β and IκB-α are recruited to the lipid rafts in B cell signaling (), our data showed that LPS stimulation increased the trafficking of not only TLR4 but also other important signaling molecules in TLR signaling pathways to the raft membrane in macrophages (). This conclusion is supported by previous findings that MyD88 is translocated from cytosolic fraction to membrane fraction by LPS in RAW 264.7 cells (). We showed that TRAF-6, a well-known important molecule in TLR signaling, translocated to the rafts 5 min after LPS stimulation, whereas TRAF-2 did not (). The differential trafficking patterns of TRAF-2 and TRAF-6 may be dependent on the specificity of ligand stimulation. In a similar fashion, CD40 engagement resulted in differential translocation patterns, such that TRAF-2 translocated to lipid rafts but TRAF-6 did not (). We showed that CpG stimulation translocated TLR9 to membrane rafts (), similar to the previous observation that a small portion of TLR9 becomes cell surface accessible after CpG treatment (). Together with previous results on the localization of TLR9 (, ), the recognition of CpG by TLR9 is likely to occur not only in the intracellular compartment but also in lipid rafts where TLR9 presumably clusters from cytosol after CpG stimulation in macrophages. Lipid rafts cross-linked with anti-CTx antibody immediately increase intracellular Ca, which is up-regulated by ROS stimulation and which is required for the trafficking of molecules from the cytosolic compartment to the plasma membrane (, , ). Although cross-linking with anti-CTx causes signaling events analogous to activation of T cell receptor signaling (, , ), the results suggest that cross-linking by anti-CTx at 37°C is likely to provoke trafficking of TLR4 to the crescent-shaped membrane patch similar to biochemically isolated rafts. Although the cross-linking at 37°C showed translocation of TLR4 to the crescent-shaped patched area, TLR3 remained in the intracellular compartment (Fig. S1, A and B). These diverse patterns of TLRs trafficking by the cross-linking are very similar to those by TLR ligand stimulation (). Moreover, the effect of MβCD and MDC on poly(I:C)-induced TNF-α production was opposite to the responses to LPS or Pam (). The signaling events of TLR3 are unlikely to occur in the raft membrane (, ), whereas the rafts are involved with the other TLR signaling pathways (, ).
Despite several results about the involvement of ROS in TLR signaling (, ), the role of ROS on trafficking to lipid rafts and on TLR signaling is still unclear. ROS such as superoxide or hydrogen peroxide are known to regulate activation of NF-κB or cytokine production (, ). We showed that LPS-induced NADPH oxidase–dependent ROS generation is critically involved in the translocation of TLR4 to lipid rafts and the initiation of the TLR signaling pathway. Furthermore, not only stimulation with hydrogen peroxide but also PMA, a potent NADPH oxidase activator (), can recruit TLR4 to lipid rafts (). Powers et al. () have reported that oxidative stress induced by hemorrhagic shock recruits TLR4 to the plasma membrane in macrophages. The present study shows that NADPH oxidase–dependent ROS generation induced by TLR ligand stimulation plays a critical role in the trafficking of TLR to lipid rafts and the initiation of downstream signaling pathways. In addition, the results suggest that the inhibitory effect of CO on the translocation of TLR4 to membrane rafts and on TLR signaling is mediated through the inhibition of NADPH oxidase activity. We observed similar differential effects of DPI and CO on TLR ligand–induced TNF-α production ( and ). In addition, we showed that CO significantly inhibited ROS production induced by LPS () and Pam, but not by poly(I:C), which is similar to the above data ( and ). Two integral membrane proteins, gp91 and p22, form a stabilizing complex, termed cytochrome 558, and heme incorporation into gp91 is essential for the heterodimer formation (). Recent studies showed that CO modulates the activity of heme proteins such as a potassium channel and soluble guanylate cyclase (, , ). Our spectral results also suggest that CO may form a complex with cytochrome 558 (). One of the critical roles of NADPH oxidase in professional immune cells is to generate ROS as a defense against invading microorganisms. It is known that gp91-deficient humans, such as patients with chronic granulomatous disease, lack the ability to produce ROS, including superoxide (). Our data showed that LPS-induced TNF-α production was significantly suppressed in macrophages from gp91-deficient mice. In addition to this physiological importance of gp91 on LPS-induced proinflammatory cytokine production, we demonstrated LPS-induced interaction between gp91 and TLR4 by using immunoprecipitation analysis (). DPI, an NADPH oxidase inhibitor, suppressed this interaction as well as inhibited LPS-induced TLR4 signaling and cytokine production (). These results confirm a crucial link between gp91 and TLR4 signaling pathways. Consistent with previous results (, ), we showed that CO inhibits LPS-induced superoxide generation, associated with NADPH oxidase activation, in the membrane fraction of macrophages (). Furthermore, our results indicate that CO inhibited LPS-induced complex formation of gp91 and p47, which is essential for assembling the active complex of NADPH oxidase (). The possible interaction of CO with cytochrome 558 may inhibit complex formation with cytosolic NADPH oxidase components. We showed that not only inhibition of NADPH oxidase activity but also lack of gp91 abolished the trafficking of TLR4 to the rafts by LPS ( and ). These results suggest that CO inhibits LPS-induced translocation of TLR4 to lipid rafts by inhibiting NADPH oxidase activity. Moreover, our results indicate that CO is likely to modulate gp91, which is supported by the following observations: CO failed to suppress LPS-induced TNF-α production in gp91-deficient macrophages; CO inhibited LPS-induced complex formation of gp91 and TLR4; and CO can form a complex with 558 in vitro ().
Negative regulation of TLR signaling pathways by CO is likely to be mediated by the modulation of trafficking of TLRs to membrane rafts, and CO inhibits the trafficking by inhibition of NADPH oxidase activity, potentially through gp91. First, CO suppressed TLR4 signaling, but not TLR3 (–
). Second, LPS stimulation rapidly translocated TLR4 to lipid rafts, but TLR3 did not translocate to the rafts in response to poly(I:C) as revealed by immunoblotting and immunofluorescence studies (). Third, CO blocked LPS-induced recruitment of TLR4 and other signaling molecules to lipid rafts (). Finally, CO suppressed LPS-induced ROS production including superoxide production (), and this effect of CO was impaired in gp91-deficient cells (). Thus, ROS generated through NADPH oxidase activation is critically involved in signal transduction of TLRs in lipid rafts.
Recent studies in HO-1–deficient humans and mice provide the strongest evidence that HO-1 is a crucially important molecule in host defense against oxidative stress induced by various stimuli including LPS (–). Against invading PAMPs from microorganisms, macrophages dominantly contribute to the production of proinflammatory mediators (). The high expression of TNF-α in the macrophages of HO-1 mice () and negative regulatory effects of HO-1 on TLR ligand–induced TNF-α production in the macrophages () suggest that HO-1 is likely to be an important molecule in regulating the activation of immune cells. Although biliverdin, the other byproduct of HO-1, also has cytoprotective effects (), our results showed that scavenging CO by hemoglobin abolished the effect of HO-1 in TLR signaling (). Thus, HO-1 has a potent antiinflammatory role in TLR-mediated immune responses, and that role is mainly mediated through HO-1–derived CO (, ).
In summary, we have demonstrated that TLR trafficking evoked by ligand stimulation differentially occurred and that HO-1–derived CO negatively regulated TLR signaling pathways by the inhibition of TLRs trafficking to membrane rafts, resulting in suppression of downstream signaling and cytokine production. In addition, CO inhibited trafficking of TLR by suppressing NADPH oxidase–dependent ROS generation. Because excessive inflammatory mediator production during sepsis causes severe tissue damage or organ dysfunction beyond essential host defense function (), it is critically important to control ROS production as a key cell signaling activator in the immune system. The present study suggests that CO, a gas molecule produced physiologically in cells, might have a potent role to regulate PAMP-induced proinflammatory cascades by modulating ROS generation, resulting in the suppression of inappropriate hyperreactive immune responses. We have previously reported that the effect of CO on LPS-induced cytokine production is mediated by p38 MAPK (), and recent results have suggested another mechanism of CO on the LPS-induced inflammatory response, which is mediated by PPAR-γ (). However, these results do not address the involvement of MAPK and PPAR-γ in the trafficking of TLR to lipid rafts. Although further studies are needed to investigate the precise mechanisms of CO action, our results show that CO is a novel candidate as a negative regulator in TLR signaling pathways.
RAW 264.7 cells and mouse peritoneal macrophages were maintained in DMEM containing 10% fetal bovine serum and antibiotics (). For CO treatment, CO at a concentration of 1% corresponding to 10,000 ppm in compressed air was mixed with compressed air containing 5% CO before delivery into the exposure chamber as described previously (). After 2 h of pretreatment with either CO (0–500 ppm) or air, LPS (Sigma-Aldrich), peptidoglycan (Fluka), PamCSK4 (InvivoGen), poly(I:C) (GE Healthcare), flagellin (InvivoGen), or CpG oligonucleotide was added to the culture media and the culture plates were returned to the chambers.
RAW 264.7 cells were stably transfected with a pSFFV/HO-1 plasmid construct as described previously ().
C57BL/6 wild-type mice and gp91-deficient mice (C57BL/6 background) were purchased from The Jackson Laboratory. HO-1–deficient mice were provided by S.-F. Yet (Brigham and Women's Hospital, Boston, MA; reference ). TLR3-deficient mice were derived as described previously (). Animals were housed according to guidelines from the American Association for Laboratory Animal Care and Research Protocols and were approved by the Animal Care and Use Committee (University of Pittsburgh School of Medicine).
Sucrose-gradient raft fractions were separated as described previously (). In brief, cells were lysed in ice-cold MBS buffer (25 mM MES, pH 6.5, 150 mM NaCl, 1% Triton X-100, 1 mM NaVO, and protease inhibitors). Lysates were adjusted to 4 ml of 40% sucrose by mixing with 2 ml of 80% sucrose and overlaid with 4 ml of 35% sucrose and 4 ml of 5% sucrose in MBS buffer. Samples were ultracentrifuged at 39,000 rpm for 18 h and fractionated into 12 subfractions.
Media were analyzed with ELISA kits purchased from R&D Systems.
Real-time PCR analysis was performed as described previously (). mRNA expression was quantified by SYBR Green two-step, real-time RT-PCR for IFN-β. The sequence of the primer for IFN-β is 5′AGCTCCAAGAAAGGACGAACAT, 3′GCCCTGTAGGTGAGGTTGATCT (). The expression of each gene was normalized to GAPDH mRNA content and calculated relative to control.
Nuclei extraction and electrophoretic mobility shift assay was performed as described previously (). A double-stranded oligonucleotide containing the consensus transcription factor–binding site for NF-κB was purchased from Promega. p65 NF-κB transcriptional activity was also analyzed with ELISA-based kits purchased from Active Motif.
The rabbit anti-TLR4, TRAF-6, TRAF-2, IL-1 receptor–associated kinase 1, CD-14, and MD-2 were purchased from Santa Cruz Biotechnology, Inc. The rabbit anti-p65, total Iκ-B, and IRF-3 were purchased from Cell Signaling Technology. The rabbit anti-TRIF and TLR9 were purchased from Abcam. The rabbit anti-MyD88 was purchased from Chemicon. The rabbit anti-TLR3 and HO-1 were purchased from StressGen Biotechnologies. The mouse anti–flotillin-1, p47, and gp91 were purchased from BD Biosciences. CTx B Subunit peroxidase conjugate was purchased from Sigma-Aldrich. Immunoprecipitation and SDS-PAGE was performed as described previously (, ).
After incubation with TLR ligands, the cells were washed with serum-free medium and incubated with 8 μg/ml FITC-conjugated CTx B (FITC-CTx; Sigma-Aldrich) on ice for 10 min. After fixation and permeabilization, cells were stained with the rabbit anti-TLR3 or anti-TLR4 (Santa Cruz Biotechnology, Inc.). After washing, samples were incubated with Alexa 594–coupled secondary antibody for 1 h (). For raft-aggregation experiments, cells were incubated with 8 μg/ml FITC-CTx on ice for 10 min, followed by incubation with 4 μl/ml goat anti-CTx (Calbiochem) for 15 min at 4 or 37°C (). Cells were placed on ice and washed with cold PBS and then fixed, permeabilized, and stained with anti-TLR3 or anti-TLR4, as described above. For analyzing ROS, the cells were preincubated with 10 μM CM-HDCFDA (Invitrogen) for 30 min, followed by incubation with TLR ligands (). Samples were viewed with an Olympus Fluoview 300 Confocal Laser Scanning head with an Olympus IX70 inverted microscope, as described previously (). Fluorescence intensity of cells exposed with CM-HDCFDA was analyzed by Olympus Fluoview (V. 3.1.16) software from Olympus Optical Co.
Livers and kidneys were fixed, embedded, and serially sectioned (5 μm) in toto. Paraffin-embedded tissues were hydrated, retrieved, and immunostained with anti–TNF-α (BD Biosciences) and monoclonal anti–CI:A3-1(RDI) for macrophage staining. Bound primary antibodies were visualized with diaminobenzidine staining by using ABC kits (Vector Laboratories).
Membrane fraction was isolated as described previously (). The protein (1 mg/ml protein) was incubated with 1.2 mg/ml acetylated cytochrome C, 1 mg/ml NADPH in the presence or absence of 1 mg/ml superoxide dismutase for 20 min at 37°C. The reactions were read at 550 nm, subtracting the A550 of the reaction containing superoxide dismutase. The production of superoxide was calculated assuming an extinction coefficient of 21 mM cm for reduced cytochrome C and normalized for mg protein and reaction time. Values were expressed as fold increase over control value.
Fresh bovine peripheral blood was harvested and the neutrophils were isolated by using the erythrocyte-granulocyte fraction. Bovine cytochrome 558 was prepared as described previously (). Isolated cytochrome 558 was analyzed on a double beam recording spectrophotometer (Shimadzu UV2501-PC). After determination of oxidized spectra, the sample cuvette was degassed and replaced with an atmosphere of argon. To obtain reduced spectra, the cuvette was injected through the septum with sodium dithionite solution (5 mM). To obtain CO spectra, 100% CO was bubbled into the cuvette through a septum for 30 s. Corresponding difference spectra were digitally generated.
We performed statistical analysis by using an unpaired Student's test. All Data are mean ± SD from three different experiments. We considered values of P < 0.05 to be statistically significant.
Fig. S1 shows the immunofluorescence image of TLRs after cross-linking with anti-CTx antibody in macrophages. Fig.S2 shows spectral analysis of cytochrome 558 isolated from bovine neutrophils. The online supplemental material is available at . |
One of the major questions in the NKT cell field is the identity of the glycolipid ligands responsible for the positive selection of NKT cells. CD1d molecules most likely bind their ligands as they recirculate through the endosomal/lysosomal pathway of the DP cells before returning to the cell membrane to present the ligands to developing NKT cells (–). The prototypic NKT cell antigen is α-galacytosylceramide (α-GalCer) (, ), which is recognized by most, if not all, NKT cells in mice and humans. α-GalCer, a glycosphingolipid derived from marine sponges, is a potent agonist ligand that can initiate NKT cell–dependent immune responses, leading to enhanced immunity to tumors and infectious organisms and suppression of certain autoimmune diseases (). Several other nonmammalian agonist glycolipid ligands for NKT cells have recently been identified (, ). Although these ligands provide important insight into targets for NKT cell–dependent immune responses, they cannot serve as endogenous ligands for NKT cell selection.
Two years ago, a report from Zhou et al. () provided a breakthrough in the field, offering strong evidence that iGb3, a mammalian glycosphingolipid, is a CD1d-dependent agonist for NKT cells from mice and humans. This report also demonstrated that mutant mice, a model for human Sandhoff disease, had markedly impaired NKT cell development (). The gene product is a key subunit of the enzymes (β-hexosaminidase A and B; see text box) necessary for the lysosomal degradation of iGb4 to iGb3 (), as well as for the production of other glycolipid products. Among these products, however, only iGb3 is an NKT cell agonist. Assuming NKT cells are selected by self-agonists, this result implicated iGb3 as the prime candidate NKT cell–selecting ligand () (, ).
The case for iGb3 as a mammalian NKT cell agonist ligand is strong and has been verified by several other studies (–), including a report that demonstrated a role for this ligand in the activation of NKT cells in the periphery (). However, whether this ligand is unique in its ability to mediate intrathymic NKT cell selection has yet to be definitively demonstrated. It is also unclear whether these results can be translated to humans, as the synthesis of iGb3 in humans has not been formally demonstrated. A recent paper stated (as unpublished data) that iGb3 synthase mRNA was not detectable in a range of human tissues, including thymus (). In contrast, however, Zhou et al. () demonstrated, using an inhibitory lectin, that NKT cells respond to human dendritic cells via an NKT cell antigen comprising a Galα1-3Gal linkage. Given that the only two enzymes that can produce this linkage are α-galactosyltransferase and iGb3 synthase and that humans lack the former (), this result strongly suggested the presence of iGb3 in human cells.
Gadola et al. provide intriguing new results that suggest an alternative explanation for the NKT cell deficiency observed in mutant Sandhoff mice (). Sandhoff disease is one of several diseases of lysosomal glycolipid processing, broadly classed as LSDs, in which impaired trafficking or degradation results in an accumulation of lysosomal glycolipids and impaired cellular function (). In their study, () Gadola et al. examined several mouse models of LSD, each carrying a mutation that affects a different aspect of lysosomal glycolipid processing. These included mice with deficiencies in the lysosomal enzymes β-hexaminidase A and B (
, a model of Sandhoff disease), β-hexaminidase A and S (
, a model of Tay-Sachs disease and late-onset Tay-Sachs disease), β-galactosidase (a model of GM1 gangliosidosis), and α-galactosidase (a model of Fabry disease). The group also studied a mouse model of Niemann-Pick disease type C1 in which the mutation causes impaired cholesterol and glycolipid trafficking from the late endosome, a very different cause of LSD. Whereas the mutants (Sandhoff) have impaired hydrolysis of iGb4 to iGb3, this step should be intact in mice lacking functional (Tay-Sachs), β-galactosidase (GM1 gangliosidosis), or α-galactosidase (Fabry). Indeed, α-galactosidase mutant cells accumulate globotriaosyl ceramides, such as iGb3, as the breakdown of these glycolipids to lactosylceramide is inhibited ().
Despite the diversity of these mutations, the pathways affected, and the glycolipids that are stored, NKT cell development was impaired in each model, albeit to varying extents.
(Sandhoff), β-galactosidase–deficient (GM1 gangliosidosis), and Niemann-Pick disease type C1 mice had impaired ability to process and present an exogenous disaccharide analogue of α-GalCer, Galα1-2GalCer galactosylceramide (which can be processed and presented as an NKT cell antigen by normal cells), even though this processing event is independent of and β-galactosidase.
mice is specifically responsible for impaired NKT cell development. They instead suggest that any disruption of lysosomal glycolipid processing that results in LSD will potentially affect CD1d loading and, consequently, impair NKT cell development ().
#text
The Gadola study highlights the potential importance of proper lysosome function and glycolipid processing for appropriate CD1d loading. Although it does not eliminate iGb3 as a candidate selecting ligand, it appears to weaken the evidence that iGb3 is the exclusive selecting ligand, a hypothesis originally based on the use of mutant mice (). It must be pointed out that some results in the study by Gadola et al. are in apparent conflict with other reports (, ). In particular, the original paper describing iGb3 as an NKT cell ligand () included experiments to test whether the development of LSD in mutants nonspecifically disrupted glycolipid processing. In contrast to the findings reported in the Gadola study (), Zhou et al. () showed that
antigen-presenting cells processed and presented disaccharide Galα1-2GalCer galactosylceramide normally, providing convincing evidence that this LSD did not cause a general disruption of glycolipid processing. Furthermore, whereas Gadola et al. showed that α-galactosidase (Fabry) mutant mice had impaired NKT cell development (consistent with an earlier report that claimed reduced NKT cell numbers in the spleen []), no such defect was observed in a 2004 paper from Zhou et al. ().
Reasons for these discrepancies are unclear but may relate to different disease states caused by mouse age, sex, or other variables. The Fabry disease mice are particularly interesting in the context of iGb3, because this disease should lead to an accumulation of this ligand in the lysosome. If iGb3 is a major selecting ligand for NKT cells, it might be predicted that NKT cell development would be enhanced in these mice. However, it is also possible that an excess of agonist ligand could cause negative selection of NKT cells (), which would represent a distinct cause of impaired NKT cell development.
(Tay-Sachs) mice only showed impaired NKT cell numbers in the liver, which appeared to correlate with a more mild LSD that affected the liver but spared the thymus and spleen. Although this seems reasonable, it is inconsistent with reports that peripheral homeostasis of NKT cells is largely independent of peripheral CD1d-mediated signals (, ).
Clearly, there is considerable controversy surrounding the use of these LSD mouse models, and it will be important to independently assess these variables, as they have a major bearing on the interpretation of studies of NKT cell development and function using such models.
iGb3 is currently the only mammalian glycolipid with clear agonist activity for a majority of NKT cells and remains the strongest candidate ligand for NKT cell selection. It is important to add that even if LSD by itself impairs NKT cell development, this does not automatically exclude iGb3 as the candidate ligand (). However, more definitive studies are clearly necessary to test the hypothesis that iGb3 is required for normal NKT cell development in mice and humans. The production of iGb3 synthase–deficient mice is the most obvious approach, as this defect would affect iGb3 biosynthesis in the early secretory pathway rather than its degradation in the lysosome, and the mice would thus be unlikely to develop LSD.
Regardless of whether iGb3 is the key selecting ligand in mice, it remains unclear whether humans express a functional iGb3 synthase gene and, more specifically, whether iGb3 is produced in human thymus. Given that human NKT cells are highly variable in frequency but are typically 10–100 times less frequent than mouse NKT cells (), the identification and measurement of the human NKT cell selecting ligands are challenging but important objectives.
An analysis of the NKT cell compartment of humans with various LSDs will also be very valuable. The prediction from the Gadola study would be that these individuals would have lower NKT cell numbers compared with healthy individuals. A recent report demonstrated that patients with Gaucher disease (an LSD caused by glucocerebrosidase deficiency) undergoing enzyme replacement therapy had a modest increase in the percentage of Vα24 cells within the CD4 T cell pool compared with healthy controls (), which appears to support this hypothesis. However, some caveats to this study are that Gaucher disease itself was not associated with reduced Vα24 cells compared with healthy controls and, furthermore, that Vα24 alone is a not a reliable marker of NKT cells. Further studies of patients with Gaucher disease and other LSDs are clearly necessary.
Finally, it will now be interesting to study NKT cell development and glycolipid presentation by CD1d in mice or cell lines that have other defects in lysosomal function to further test the extent to which LSDs generally disrupt NKT cell development. For example, analysis of mice with defects in the formation of the intraluminal vesicles () will provide a distinct model of LSD, and it may also provide some insights as to whether glycolipids are loaded onto CD1 proteins from the limiting membrane of the lysosome or from the intraluminal vesicles. |
Homology searches of the database identified two identical genes on chromosome 31 in tandem, LmjF31.3060 and LmjF31.3070 (designated and ; available from GenBank/EMBL/DDBJ under accession no. ; ), which share 30% identity and 53.8% similarity with from (available from GenBank/EMBL/DDBJ under accession no. ). PCR amplification and cloning of the corresponding 1.3-kb gene from genomic DNA of revealed extensive identity with the sequence ().
is an Fe transporter from the ZIP family, whose members range from 309 to 476 amino acids and are predicted to have a similar membrane topology, with eight transmembrane domains and the amino- and carboxy-terminal ends located on the extracellular side of the plasma membrane (). Completely consistent with these features, encodes a 432–amino acid protein of 50 kD, which is also predicted to contain eight transmembrane domains. The most conserved portion of proteins from the ZIP family is in the putative transmembrane domain IV. This region is predicted to form an amphipathic helix with a conserved histidine and an adjacent semipolar residue, which are thought to be essential components of the heavy metal binding site (, ). Extensive identity is observed throughout this region in two homologous genes, and (available from GenBank/EMBL/DDBJ under accession no. ), and the genes from both and (). Furthermore, there is complete conservation of all five residues shown to be essential for divalent metal transport by IRT1 (, residues boxed in red) (), reinforcing the possibility that the genes encode an iron transporter. When expressed in promastigotes, a GFP-tagged form of LIT1 was expressed in a pattern consistent with localization on the plasma membrane (). The GFP-LIT1 chimera was also detected in an intracellular compartment, which may correspond to the parasite's megasome, a lysosome-like compartment (, ).
Antibodies generated against the 15 amino-terminal amino acids of LIT1, a region of the protein that is predicted to be exposed extracellularly (), failed to detect endogenous LIT1 by immunofluorescence or Western blot in promastigotes and axenically grown amastigotes (not depicted). However, when immunofluorescence was performed on permeabilized bone marrow–derived macrophages (BMmø) from C57BL/6 mice infected with axenic amastigotes, a strong reaction with the antibodies was observed on parasites residing intracellularly for 24 h. This is a stage when the markedly enlarged parasitophorous vacuoles typical of infections are clearly visible (, bottom). LIT1 was detected around the periphery of intracellular amastigotes, which was consistent with its predicted plasma membrane localization (, bottom right, inset).
Consistent with what was observed in axenically grown promastigotes and amastigotes, LIT1 was not detected by immunofluorescence on amastigotes recently internalized in BMmø (, top). This finding strongly suggested that LIT1 expression is up-regulated in the intracellular environment. Expression of IRT1, the close homologue of LIT1 in , is induced under iron-deficient conditions (). Because Nramp1/Slc11a1 has been postulated to modulate the iron concentration of phagolysosomes (), we performed time-course infection experiments in BMmø from C57BL/10ScSn (Nramp1) or B10.L-Lsh (Nramp1) congenic mice and analyzed LIT1 expression by immunofluorescence. In the Nramp1 BMmø, LIT1 expression was low at 6 h after infection but was clearly detected in a punctate pattern after 12 h (, Nramp). In Nramp1 BMmø, LIT1 expression was detected earlier; punctate immunofluorescence associated with amastigotes was clearly visible at 6 h after infection (, Nramp1). Confirming what was seen in C57BL/6 BMmø (Nramp1), no LIT1-specific immunofluorescence was detected in BMmø derived from both mouse strains immediately after the 1-h infection period (, top). These results (see also Fig. S1, available at ) suggest that the intracellular expression of LIT1 may be accelerated by the Nramp1-mediated extrusion of iron from the -containing phagolysosome.
To confirm that LIT1 functions as an iron transporter, we performed assays of functional complementation in yeast. The Δ mutant strain is extremely sensitive to iron deprivation, because it lacks the FET3 multicopper oxidase required for high affinity Fe transport and the FET4 low affinity Fe transporter (). It grows in iron-rich medium (YPD) but not in medium containing the iron chelator bathophenanthroline disulfonic acid (BPS; ). The gene was originally identified in a functional complementation screen using this strain and was subsequently shown to encode an iron transporter with preference for Fe as a substrate (). Overexpression of using the yeast multicopy vector p426 () suppressed the Δ growth defect in iron-limited conditions (YPD + 20 μM BPS; ), directly demonstrating that Fe transport ability was restored. To examine the divalent metal substrate preference of LIT1, a wild-type strain was transformed with and grown in minimum medium (SCD) containing cadmium, which is toxic for yeast at high concentrations (). overexpression increased the sensitivity of to low concentrations of cadmium, but the growth defect was suppressed when cadmium and iron were provided simultaneously (). This result demonstrates, similar to what was previously shown for the IRT1 transporter (), that though LIT1 is capable of translocating cadmium, it has a preference for iron as a substrate.
The two identical genes are present in tandem within a 5,425-bp region, allowing us to generate a targeted deletion construct to simultaneously inactivate both copies (). Because species are diploid protozoan parasites, two rounds of gene disruption are required to generate null clones. After two rounds of transfection and selection, clonal lines were identified in which both alleles of the two copies had been replaced by insertion of the - and -selectable markers (). Independent null clones isolated in this manner behaved similarly, so only results with a single doubly disrupted clonal line, designated Δ, are shown. Null Δ promastigotes had no growth defect in culture as promastigotes () and generated comparable numbers of infective metacyclic promastigotes when the cultures reached stationary phase (slender, free-swimming metacyclics were counted after agglutination of procyclic promastigotes with the mAb 3A1; unpublished data) (). Similarly, no defects in differentiation and growth as axenic amastigotes were observed after the cultures were shifted to pH 5.5 and incubated at 32°C (unpublished data). Northern blots identified a transcript of ∼2.4–2.5 kb in wild-type parasites but not in Δ axenic amastigotes and promastigotes (). The LIT1 protein was only detected with antibodies in intracellular amastigotes (as described in the previous section; ) or in extracellular promastigotes and amastigotes overexpressing LIT1 (see the next section). These observations reinforce the possibility that LIT1 expression is regulated posttranslationally in response to iron deprivation, as previously shown for several transitional metal transporters (, , ).
The normal generation of infective axenic amastigotes from Δ promastigotes allowed us to perform macrophage infections in parallel with wild-type parasites. The numbers of intracellular parasites detected after 1 h of infection were similar in BMmø infected with the wild-type or Δ lines, indicating no defect in invasion. After 24 h, LIT1 could be visualized by immunofluorescence around the periphery of wild-type, but not of Δ, intracellular amastigotes (). Reinforcing the conclusion that reactivity with the antibodies reflects an increase in LIT1 expression, a band of the predicted molecular mass (∼50 kD) was detected in Δ promastigotes transfected with the pXG-SAT episomal expression vector () carrying (). When induced to differentiate into axenic amastigotes, Fe uptake activity was observed in these -overexpressing parasites (). These results directly demonstrate that LIT1 expression confers Fe uptake activity to amastigotes.
In addition to a normal capacity for entering BMmø and triggering initial expansion of the parasitophorous vacuole (), Δ intracellular parasites also expressed P4, a specific marker of intracellular amastigotes () (). P4 was expressed by all Δ intracellular amastigotes, regardless of whether the infections were initiated with axenic amastigotes () or with purified, infective metacyclic promastigotes (). These results show that the LIT1 transporter is not required for the intracellular transition of recently internalized into replicative amastigote forms.
Next, we examined the capacity of Δ mutants for intracellular replication. In BMmø infected with wild-type axenic amastigotes, the number of intracellular parasites increased progressively between 24 and 72 h of incubation, as expected (, open columns). In contrast, there was no evidence for intracellular replication of Δ amastigotes during the same period (, black-shaded columns). Importantly, the capacity for intracellular growth was completely restored in Δ parasites expressing an episomally encoded wild-type copy of (Δ + ) (, gray-shaded columns). Similar results were obtained when BMmø infections were performed using purified metacyclic promastigotes: wild-type parasites increased in number between 48 and 72 h after infection (, open columns), whereas the numbers of intracellular Δ parasites remained constant throughout this period (, black-shaded columns). Again, complementation with episomally encoded LIT1 completely restored the growth phenotype (, gray-shaded columns). In these experiments, the slower onset of replication reflects the time period required for the intracellular differentiation of metacyclic promastigotes into replicative amastigotes. The results of both sets of experiments indicate the LIT1 transporter does not appear to be required for intracellular survival within the initial 72 h, but it is essential for intracellular replication.
Next, we examined the replicative compartments containing wild-type, Δ, and Δ + parasites within macrophages. A remarkable feature of the intracellular lifestyle of is that it thrives within acidified, hydrolase-rich compartments that share numerous properties with degradative lysosomes (, ). As expected, the lysosomal glycoprotein Lamp1 was present on the membrane of vacuoles surrounding wild-type amastigotes at early time points after invasion, as well as on the dramatically expanded compartments containing replicating parasites 48 and 72 h after infection (). Lamp1 was also detected at all time points on the membrane of vacuoles surrounding Δ amastigotes, suggesting that deletion does not affect biogenesis of the phagolysosomal compartment. However, marked differences were noted on the morphology of the vacuoles. By 48 h after infection, although some vacuole expansion was apparent, the overall size of the compartments containing Δ parasites was much smaller and, generally, contained only one amastigote per vacuole. By 72 h after infection these compartments appeared shrunken, and some Δ parasites showed signs of degeneration (granulated appearance on phase-contrast and diffuse nuclear and kinetoplast staining; ). In contrast, when Δ parasites were complemented with a wild-type copy of , by 48 h after infection the expansion of the Lamp1-positive vacuoles was completely restored, and each vacuole contained numerous amastigotes (). Interestingly, overexpression of LIT1 by transfection with the episomal plasmid seemed to interfere with the sustained expansion of the replicative compartments. By 72 h after infection, the large parasitophorous vacuoles containing Δ + parasites appeared somewhat collapsed, as indicated by the irregular staining pattern with anti-Lamp1 mAbs (). However, the complemented Δ + intracellular amastigotes showed no signs of degeneration, which was consistent with the quantification assays that demonstrated a complete restoration in their capacity for intracellular replication ().
In vivo infections with lead to the development of cutaneous lesions, which are considered to be the result of parasite replication in tissue macrophages. To determine if the intracellular replication defect of -null amastigotes resulted in a loss of virulence in vivo, we injected high numbers of wild-type or Δ metacyclic promastigotes into the footpads of BALB/c mice and followed lesion development for >200 d. Inoculation of up to 10 Δ parasites yielded no pathology in 100% of the mice infected, with no footpad lesions detected for up to 6 mo in several independent experiments. In contrast, all mice inoculated with wild-type parasites developed progressive lesions within 2–3 wk and were killed by 42 d after infection. Confirming that LIT1 is required for lesion formation, complementation of the Δ mutant with either episomally encoded or a chromosome-integrated wild-type copy of the gene restored lesion formation (). A several week delay was observed in the development of lesions in mice infected with the complemented strains when compared with rapid lesion development seen after inoculation of wild-type parasites. It is a common finding that -complemented strains do not completely recover virulence, most likely because of the unregulated expression of the “add-back” proteins (–). In the experiment shown, one of five mice injected with Δ metacyclic promastigotes displayed measurable footpad swelling beginning at 13 wk that progressed slightly at 15 wk, when the mice were killed for quantitation of parasites in the footpad and in local draining lymph node (DLN). Interestingly, all of the mice inoculated with Δ parasites harbored substantial numbers of viable amastigotes in the inoculation site () and DLN (), including those without overt pathology. Nonetheless, there was at least a log-fold reduction in parasite loads in these tissues compared with mice infected with either of the complemented lines. These results are consistent with previous descriptions of in vivo persistence without pathology in infections (, ). Further analysis of the Δ promastigotes recovered from mice indicated that they retained the same properties of the original Δ inoculum, including hygromycin/neomycin resistance and no capacity for intracellular replication in macrophages (unpublished data).
Protozoan parasites belonging to the genus are responsible for a spectrum of serious infections in humans, ranging from cutaneous lesions to a very severe visceralizing disease (). Major gaps still exist in our understanding of the biology of different species and how their different properties correlate with the various clinical forms of the disease. One common aspect of most species, however, is their remarkable capacity for surviving and replicating within acidified, hydrolase-rich phagolysosomes of macrophages (). In this study, we make an important step toward understanding the molecular mechanisms involved in adaptation to this harsh intracellular environment by identifying the first membrane protein that functions intracellularly as a ferrous iron transporter. Our results show that the LIT1 transporter is essential for intracellular replication in macrophages and for the development of pathogenic lesions in mice.
LIT1 was identified in the genome database through its close homology to IRT1, a demonstrated high affinity Fe transporter from . IRT1 was the first member to be identified in the ZIP family of metal transporters, which is now known to be present not only in plants but also in yeast, , , and humans (). Null mutants of in have a severe growth defect in normal soil, which is rescued by the exogenous application of iron (). Additional reports demonstrated that iron is the preferred metal substrate for IRT1, although cadmium, cobalt, manganese, and zinc can also be transported (, ). As indicated by the preferential uptake of iron over cadmium when expressed in yeast (), LIT1 also functions as a divalent metal transporter with preference for iron.
The endocytic pathway in mammalian cells is thought to be iron poor, because acidification-induced release of Fe from transferrin is followed by reduction to Fe and translocation to the cytosol. It is well established that this function is performed by Nramp2 (also known as DCT1 or DMT1), the pH-dependent divalent cation transporter localized on the membrane of endosomes (, , ). Nramp1 (Slc11a1) is a close homologue of Nramp2, found on macrophage late endosomes/lysosomes. The similarity between Nramp1 and Nramp2 has led to the suggestion that both function as pH-dependent symporters, promoting the efflux of metal ions from endocytic compartments (). However, different studies of this issue have reached contradictory conclusions, with some of the evidence indicating that Nramp1 may be an antiporter (–). Moreover, although Nramp1 was shown to be a pH-dependent transporter capable of extruding Mn from macrophage phagolysosomes (), its postulated role in iron transport has not yet been directly demonstrated. Our findings provide new evidence in support of the notion that Nramp1 contributes to iron depletion from late endosomal compartments. Immunolocalization assays with antibodies specific for LIT1 failed to detect the protein in all extracellularly grown life cycle stages and in parasites recently internalized by macrophages. However, a strong immunofluorescence signal was detected on intracellular amastigotes 12–24 h after infection of Nramp1 macrophages. Importantly, the intracellular expression of LIT1 appeared to be accelerated in macrophages derived from congenic Nramp1 mice. Thus, similar to what was previously shown for IRT1 () and other eukaryotic divalent cation transporters (, ), our results suggest that expression of the LIT1 transporter is up-regulated by iron deprivation.
Although post-transcriptional regulation may also be involved, four lines of evidence suggest that LIT1 expression may be regulated posttranslationally by iron. First, LIT1 mRNA is present in axenic promastigotes and amastigotes, but no protein is detected with specific antibodies. Second, when overexpressed in promastigotes, GFP-LIT1 accumulates in intracellular compartments that may correspond to degradative lysosomes, in addition to the plasma membrane. Third, long-term residence in phagolysosomes, a compartment predicted to be iron poor, allows the detection of LIT1 with antibodies on the surface of amastigotes. Fourth, residence within phagolysosomes of Nramp1 macrophages, postulated to be further depleted in iron, accelerates LIT1 expression. Collectively, these observations suggest that exposure to iron may induce LIT1 internalization and degradation, effectively removing the protein from the plasma membrane. This is a mechanism that was previously linked to the regulation of several eukaryotic divalent metal transporters (, ). Further characterization of this process in will require the development of iron-free axenic culture conditions.
The exclusively intracellular expression pattern of LIT1 and the fact that it is dispensable for growth and differentiation in axenic culture suggests that alternative mechanisms for iron acquisition exist in the extracellular stages of . This function may be fulfilled by the products of LmjF28.1330 and/or LmjF33.3200, genes present in the genome (chromosomes 28 and 33, respectively) that contain predicted ZIP metal permease domains and share reasonable similarity with putative metal transporters. The intracellular expression of LIT1 also highlights the fact that it has to compete with host late endosomal transporters for the same substrate, ferrous iron. The fact that episomal expression of LIT1 completely rescues the intracellular growth of -null parasites indicates that the transporter is active in the acidic pH of the phagolysosome. This scenario is in agreement with several earlier studies, which revealed acidic pH optima for the transport of glucose, amino acids, and polyamines by amastigotes (–).
A unique property of , when compared with other species, is its capacity to induce the formation of dramatically expanded intracellular vacuoles, where the replicating amastigotes accumulate. It has been proposed that this enlargement of the parasitophorous vacuole may be linked to the unique ability of to survive in IFN-γ–activated macrophages (). Little is known about how these enlarged compartments are maintained and how expansion affects the concentration of microbicidal products and nutrients within the vacuoles. Interestingly, although complementation of -null with wild-type restored their ability to replicate within macrophages (, Δ + column), the swollen parasitophorous vacuoles appeared partially collapsed at later times after infection (, 72 h). This may be related to the overexpression of when introduced in an episomal vector, suggesting that vacuole expansion and maintenance may be affected by the rate by which Fe is translocated into the parasite versus Fe efflux from the vacuole.
Our results clearly show that the block in intracellular replication of lacking the LIT1 transporter is not caused by defects in invasion, early survival within macrophages, or in differentiation into replicative amastigotes. These findings reinforce previous suggestions that the ability to replicate as amastigotes is the most important requirement for virulence in (). Indeed, mutations in genes involved in macrophage infection and early survival (but not in growth as amastigotes) lead only to attenuation in virulence and delayed lesion formation (, ).
lacking phosphoglycans () and the Δ described in this paper, are avirulent.
and Δ can still persist in vivo without causing pathology ().
persisting population can give rise to compensatory mutants with restored virulence despite their consistent lack of phosphoglycan expression (). No evidence for such reversal of the avirulent phenotype was seen so far in our studies, suggesting that secondary mutations may not be able to compensate for the absence of LIT1. In future studies, it will be of great interest to take advantage of the defined iron transport defect of Δ to pursue the cellular basis of the compartment where avirulent parasites persist in host tissues.
D.L. Sacks provided the IFLA/BR/67/PH8 strain. Promastigotes were maintained in vitro at 26°C in M199 (pH 7.4; Invitrogen) supplemented with 20% heat-inactivated FBS, 5% penicillin-streptomycin, 0.1% hemin (25 mg/ml in 50% triethanolamine), 10 mM adenine (pH 7.5), and 5 mM -glutamine (M199/S). Axenic amastigotes were cultured at 32°C in the same medium supplemented with 0.25% glucose, 0.5% trypticase, and 40 mM Na succinate (pH 4.5). Metacyclic promastigotes were purified from 7-d-old promastigote stationary phase cultures using the mAb 3A1, which specifically agglutinates procyclic promastigotes but not metacyclics (, ). Parasites were washed twice with PBS, resuspended at 2.5 × 10 parasites/ml in 0.5 ml PBS containing a 1:500 dilution of 3A1 ascites for 30 min, and centrifuged at 250 for 5 min. Nonagglutinated parasites in the supernatant were washed twice, counted, and used for infection of macrophages.
BLAST homology searches of the database identified two identical genes in tandem, LmjF31.3060 and LmjF31.3070, that shared 30% identity and 53% similarity to the iron transporter IRT1. The following primers were used to amplify the corresponding 1.3-kb gene from genomic DNA of : forward, 5′-ATGGAGACGGCGAAACTG-3′; and reverse, 5′-CTACAGCCAGTTGCCCAC-3′ (underlined sequences indicate added HI sites). The PCR product was cloned into the pCR2.1-TOPO vector (Invitrogen) to generate a pCR-LIT1 plasmid, and the correct coding sequence was confirmed by sequencing.
The gene deletion constructs (LaKOLITHyg and LaKOLITNeo) required for sequential deletion of both alleles of the genes were based on the expression vectors pXG-hyg and pXG-neo (courtesy of S. Beverley, Washington University, St. Louis, MO) (). A 2-kb flanking sequence upstream of the open reading frame (ORF) was PCR amplified using the following primers: forward, 5′-GGAGYGCTTGTACGACCTCC-3′; and reverse, 5′-AGCAAGAGGGAGATAGAG-3′. A 1.2-kb flanking sequence downstream of the ORF was amplified using the following primers: forward, 5′-AGAGCGCATTGACTTGGT-3′; and reverse, 5′-GCTCTGCATATCTGCCATAC-3′. HI and I restriction sites were created in both fragments and used for cloning into the pCR2.1 vector (Invitrogen). The upstream fragment was excised from the vector and cloned into the HI-linearized construct containing the downstream sequence. To generate the deletion constructs, the spanning regions containing DHFR- (or )-TS were PCR amplified from the pXG-based vectors and ligated to the described construct, linearized by I digestion. Plasmid DNA from each gene-targeting construct was digested with HI and I to release the integrating fragments, and the linearized gene deletion constructs were gel purified.
Mid-log promastigotes were collected by centrifugation, washed once with PBS and once with ice-cold electroporation buffer (21 mM Hepes, pH 7.5, 0.7 mM NaHPO, 137 mM NaCl, 5 mM KCl, and 6 mM glucose), and resuspended at 10 cells/ml. A volume of 0.4 ml was mixed with 10 μg DNA and placed into a 0.2 ml gap cuvette (Bio-Rad Laboratories). The cuvette was chilled on ice for 10 min, electroporated using a Cellpulser (Bio-Rad Laboratories) set at 450 V, 500 μF, and returned to ice for 10 min, followed by transfer of the promastigotes to 10 ml of growth medium and incubation at 26°C. After 2 d, promastigotes were plated on agar dishes (2% agar in complete promastigote growth medium) containing the appropriate drug for selection and incubated at 27°C. Colonies visible after 15 d were picked and tested for integration. For generation of the Δ knockout, the region containing the two genes was replaced sequentially on both alleles by the hygromycin B phosphotransferase () and neomycin phosphotransferase () genes, which confer resistance to the antibiotics hygromycin B and G418, respectively. 100 μg/ml hygromycin B and/or 50 μg/ml G418 were added to the medium used to expand the isolated colonies after each round of homologous gene targeting. Southern blots were performed to determine integration of the selectable markers at the locus. The homozygous Δ strain was cultured in complete promastigote growth medium containing 100 μg/ml hygromycin B and 50 μg/ml G418. Complementation of Δ parasites with wild-type was achieved by transfection of promastigotes with the episomal vector pXGSAT- (generated by cloning a HI fragment containing the ORF into the pXGSAT vector []) or linearized pIR1SAT- (generated by inserting the ORF into the I site of the pIR1SAT vector that promotes integration into the ribosomal SSU locus [provided by S. Beverley]). Agar-grown clones resistant to 50 μg/ml nourseothricin were selected for further characterization.
For Northern blots, 10 μg of total RNA extracted from amastigotes and promastigotes (RNeasy Mini Kit; QIAGEN) were loaded onto 1.2% MOPS/formaldehyde agarose gels, transferred to a nylon membrane, and prehybridized for 2 h at 42°C in 10 ml of 5× Denhardt's solution/5× standard sodium phosphate with EDTA/1% SDS with 200 μg/ml salmon sperm DNA. Hybridization was performed overnight at 42°C in 10 ml of the same buffer containing 50% formamide and the P-labeled LIT1 probe. Filters were washed and exposed at −70°C for autoradiography.
Axenic amastigotes were washed twice with PBS, resuspended in uptake buffer (140 mM NaCl, 5 mM KCl, 1mM CaCl, 0.09% glucose, 10 mM MES, pH 5.5) at 5 × 10 parasites/ml, and placed in 100-μl aliquots in microcentrifuge tubes. Ascorbic acid (to reduce Fe to Fe) was added to the uptake buffer to a final concentration of 50 μM from a 1-mM freshly made solution. To maintain Fe (82.69 mCi/mg, 39 mCi/ml; PerkinElmer,) in solution for uptake, a FeCl–nitrilotriacetic acid (Fe-NTA) solution was prepared at a 1:50 molar ratio. To initiate iron uptake, 100 μl Fe-NTA (2 μM Fe) was added to the cell suspension, and the samples were incubated at 35°C or 4°C for various time intervals. At the end of the incubation period, parasites were washed three times with PBS, the pellets were resuspended in 100 μl PBS, and the cell-associated radioactivity was counted in a liquid scintillation counter (Wallac 1409; PerkinElmer). The protein concentration of the samples was determined by a bicinchoninic acid assay (Pierce Chemical Co.).
The ORF was placed under the control of the ADH promoter for overexpression in by cloning into the pAD4M (LEU2) or p426 (URA2) vectors () at the I or HI sites, respectively. The Δ yeast strain (provided by M.L. Guerinot, Dartmouth College, Hanover, NH) (, ) was transformed with p426- and selected on minimal essential plates without uracil before streaking on rich media (YPD) with or without the iron chelator BPS (final concentrations of 20 or 40 μM). A wild-type strain (W303) was transformed with pAD4M- and selected on minimal essential plates without leucine. The transformed yeast colonies were tested for the ability to grow at 30°C for 5 d on plates containing 100 μM CdCl, or 100 μM CdCl and 100 μM FeCl.
BMmø isolated from C57/BL6 mice (Charles River Laboratories) were prepared as described previously (), seeded onto 24-well plates containing coverslips at a density of 7 × 10 cells per well, and incubated overnight in RPMI 1640 with 10% FBS containing 5% L cell supernatant (as a source of M-CSF) at 37°C and 5% CO. Attached BMmø were washed with fresh RPMI 1640 and infected with 1.4 × 10 axenic amastigotes or 7 × 10 metacyclic promastigotes per well in 0.5 ml RPMI 1640 with 2% FBS (multiplicity of infection = 2 and 10 for amastigotes and metacyclic promastigotes, respectively). BMmø isolated from C57BL/10ScSn (Nramp1) or B10.L-Lsh (Nramp1) congenic mice (provided by J. Blackwell, Cambridge University, Cambridge, UK) () were cultured in DMEM with 10% FBS containing 15% L cell supernatant for 6 d, followed by replacement of half the medium with DMEM with 10% FBS at day 7 and infection on day 8 in DMEM with 10% FBS (). After 1 h at 34°C and 5% CO, free parasites were removed by three washes with PBS, and the cultures were further incubated in for various periods of time, followed by fixation with 2% paraformaldehyde (PFA) and staining with DAPI. The number of intracellular parasites (identified through the characteristic kinetoplast DNA stained with DAPI and localization within enlarged vacuoles by phase contrast) was determined at 100× with a microscope (Axiovert 200; Carl Zeiss MicroImaging, Inc.) in a minimum of 400 macrophages per coverslip in triplicate, and the data were expressed as the total number of intracellular parasites per 100 macrophages. The data were analyzed for statistical significance using an unpaired Student's test (P < 0.05 was considered significant).
To construct a GFP- gene fusion, the ORF was cloned into the pXG-GFP2 vector (courtesy of S. Beverley) (), which drives the expression in of proteins fused to GFP at the amino terminus. Transfected promastigote clones expressing GFP- were selected by growth in 100 μg/ml G418. Polyclonal antibodies against LIT1 were generated by immunizing a rabbit with the first 15 amino acids of LIT1 (METAKLSVEASTRHL) coupled to keyhole limpet hemocyanin (MBS cross-linking reagent; Pierce Chemical Co.), and affinity purified using the cognate peptide coupled to Affigel 15 (Bio-Rad Laboratories). For immunofluorescence, BMmø infected with the various strains (wild-type, Δ, and Δ + ) were fixed with 2% PFA, blocked with 50 mM NHCl and 2% goat serum in PBS, permeabilized in 0.1 mg ml saponin, and incubated for 1 h at room temperature with rabbit polyclonal antibodies against LIT1, P4 (courtesy of D. McMahon-Pratt, Yale University, New Haven, CT) (), or mAbs against mouse lysosomal-associated membrane protein 1 Lamp1 (1D4B; Developmental Studies Hybridoma Bank). After incubation with Alexa-conjugated goat anti–rabbit or –mouse IgG (Invitrogen), coverslips were mounted in antifade reagent (ProLong; Invitrogen) and examined by a fluorescence microscope (Axiovert 200) equipped with a CCD camera (CoolSNAP HQ; Photometrics) controlled by software (MetaMorph; Molecular Devices Corporation).
Female BALB/c mice were injected in the left hind footpad with 10 ficoll gradient–purified metacyclic promastigotes of (), and lesion progression was followed by blinded weekly measurements with a caliper. The total number of parasites in the injected footpad and local DLN 15 wk after infection was estimated by a limiting dilution assay, as previously described (). Infected footpad tissue was prepared after removal of the toes and bones by incubation for 2 h at 37°C in DME containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μg/ml Liberase CI enzyme blend (Boehringer). Footpad tissues were ground in a Medimachine (Beckton Dickinson). Popliteal lymph nodes were removed and mechanically dissociated using a pellet pestle in 100 μl DME containing 100 U/ml penicillin and 100 μg/ml streptomycin medium. Tissue homogenates of both infected tissues and DLNs were filtered using a 70-μm pore size cell strainer (Falcon Products, Inc.). Recovered cells were serially diluted in a 96-well flatbottom microtiter plate containing biphasic medium prepared using 50 μl NNN medium containing 20% defibrinated rabbit blood and overlaid with 100 μl M199/S medium. The number of viable parasites was determined from the highest dilution at which promastigotes could be grown out after 7–10 d of incubation at 26°C. Statistical significance between means of various groups was determined using a two-tailed test for independent samples.
The expression of LIT1 by amastigotes in BMmø from C57BL/10ScSn (Nramp1) or B10.L-Lsh (Nramp1) mice was examined by immunofluorescence with specific antibodies. LIT1 was detected earlier (6 h after infection) in B10.L-Lsh BMmø, suggesting that iron depletion from the parasitophorous vacuole mediated by the Nramp1 transporter accelerates LIT1 expression.
Fig. S1 shows Nramp1-dependent expression of by intracellular . Randomly acquired, independent microscopic fields showing immunofluorescence of C57BL/10ScSn (Nramp1) or B10.L-Lsh (Nramp1) BMmø after 1 (A) or 6 (B) h of infection with axenic amastigotes. is detected after 6 h of infection in Nramp1 BMmø, whereas expression levels remain low in Nramp1 BMmø at 1 and 6 h after infection. Antibodies to LIT1 are labeled in green, and the host cell and parasite's DNA are stained in blue (DAPI). Arrows point to infected macrophages. The images were acquired and enhanced for contrast under identical conditions. Online supplemental material is available at . |
To clarify the contribution of derepressed and to the self-renewal defect of
HSCs, we evaluated the competitive repopulation capacity of
HSCs.
,
, and
mice (C57BL/6-Ly5.2) were infused into lethally irradiated recipients (C57BL/6-Ly5.1) along with the same number of competitor BM cells from C57BL/6-Ly5.1 mice.
BM cells exhibited a mostly normal long-term repopulating activity of the recipient BM in both primary and secondary transplantations, whereas
BM cells did not contribute to long-term repopulation at all ().
BM cells fully repopulated recipients' BM in cellularity () and also manifested a full differentiation capacity along myeloid and lymphoid lineages (). As evident in Fig.
and
CD34c-KitSca-1lineage marker (KSL) cells, which are highly enriched for long-term repopulating HSCs (), were comparable with that of the wild type.
mice displayed a HSC frequency no less than that of the wild type, and
mice exhibited almost the same HSC frequency as that of the wild type. Thus, the number of HSCs infused was comparable among recipients in the competitive repopulation assay.
BM cells did not contribute to the repopulation at all, highlighting a severe defect of
HSC function (Fig. S2).
HSCs could be substantially rescued by the deletion of and , thus defining the / locus as a critical Bmi1 target for the maintenance of HSC self-renewal.
HSCs, and the chimerism of
hematopoietic cells in peripheral blood gradually decreased with time (unpublished data).
phenotypes in neural stem cell self-renewal and cerebellar granule neuron progenitor proliferation ().
HSCs, we purified the CD34KSL HSC fraction, and an in vitro single-cell culture was performed for 14 d in the presence of stem cell factor (SCF), IL-3, thrombopoietin (TPO), and erythropoietin (EPO).
HSCs contained 3.3-fold fewer high proliferative potential (HPP) colony-forming cells (CFCs) than the wild type,
HSCs contained a comparable number of HPP-CFCs with the wild type (). We have previously demonstrated that CFU-neutrophil/macrophage/erythroblast/megakaryocyte (nmEM), which retains multilineage differentiation capacity, is a major subpopulation among CD34KSL HSCs but not among CD34KSL multipotential progenitor cells and that its frequency is well correlated with that of functional HSCs ().
CD34KSL cells present a drastic reduction in their frequency of CFU-nmEM, whereas
HSCs show a substantial recovery in their frequency of CFU-nmEM compared with the wild type ().
HSCs exhibited a considerable but only partial recovery of proliferation (). In vitro culture systems are a kind of stringent condition in which numerous signaling entities are missing that are supportive for HSCs and are present in the in vivo microenvironment.
HSCs.
mice in detail.
mice recovered to the same level as that of the wild type ().
mice, and, unexpectedly, their numbers progressively decreased over time ().
mice as previously described ().
mice ().
HSCs is mostly normal (), these data indicate defects of the BM microenvironment in the absence of Bmi1.
mice.
recipients recovered to the wild-type level after transplantation, the histological analysis of recipients' femurs and their BM cell counts demonstrated that the
BM microenvironment is defective in supporting the BM repopulation by wild-type HSCs ().
BM microenvironment to support hematopoiesis by wild-type HSCs ().
The regulation of self-renewal and differentiation of HSCs requires a specific BM microenvironment. In BM, a subpopulation of osteoblasts has been implicated as an important component of the HSC niche, indicating that the bone surface is the major HSC niche (–). The size of the osteoblastic niche is largely dependent on the amount of trabecular bone (, ). In
BM, development of the trabecular bone was severely impaired, particularly in the metaphyseal area (). This indicates a profound reduction in the osteoblastic niche and raises the possibility of an insufficient production of osteoblasts.
osteoblasts.
osteoblasts showed a normal level of alkaline phosphatase activity, which is one of the representative osteoblastic differentiation markers (Fig. S3 A, available at ).
osteoblasts, although and were also derepressed in
osteoblasts (Fig. S3 B). We then took advantage of the knockdown technique. Osteoblasts were infected with a lentivirus expressing short hairpin RNA (shRNA) against , which efficiently inhibited the transcription of (). Consistent with in vivo trabecular bone formation, knockdown led to a reduced osteoblast proliferation (). Nonetheless, knockdown osteoblasts similarly supported the survival and multilineage differentiation capacity of CD34KSL HSCs during a 5-d ex vivo culture (Fig. S3 C). Collectively, these findings suggest that Bmi1 controls the BM microenvironment, at least in part, by regulating osteoblast niche size. In contrast with the case of HSCs, however, the deletion of both and again did not substantially restore the impaired development of the trabecular bone () or the impaired proliferation of knockdown osteoblasts (), confirming that the and genes are not the major targets for Bmi1 in the maintenance of the BM microenvironment, as demonstrated in (A and C). The BM microenvironment consists of not only osteoblasts but also stromal cells, endothelial cells (), and so on. It would be intriguing to ask whether Bmi1 also functions in the other components of the BM microenvironment.
Our findings in this study clearly demonstrate that the derepression of and genes is responsible for defective HSC self-renewal.
HSCs undergo the first cell division in a fashion similar to that of the wild type and showed no apoptosis in a single HSC culture.
mice ().
mice do not grossly affect the cell cycle or survival of HSCs.
It has been well recognized that the activated p16–Rb and p19–p53 pathways are profoundly associated with cellular senescence (). Cellular senescence is a program activated by normal cells in response to various types of stress. These include telomere attrition, DNA damage, oxidative stress, oncogenic stress, and others. Senescence of HSCs is supposed to be induced by telomere-dependent and -independent pathways (, ).
lineage marker immature cells and lineage marker differentiated cells by fluorescence in situ hybridization. The loss of did not alter the telomere length at all (). In the absence of Bmi1, the derepression of and genes causes the premature senescence of mouse embryonic fibroblasts (). knockdown osteoblasts indeed exhibited a higher senescence-associated (SA) β-galactosidase activity, which was canceled in the absence of and genes (Fig. S4, available at ), suggesting that Bmi1 controls the cellular senescence of osteoblasts by regulating the expression of and genes.
CD34KSL HSCs in terms of the SA–β-galactosidase activity and SA gene expression profiles, but all appeared negative (unpublished data). It is possible that the senescent HSCs do not express specific combinations of marker antigens for HSC identification any more. Thus, the possibility that derepressed and genes facilitate the premature senescence of HSCs remains to be determined.
In contrast to the strong impact of derepressed p16 and p19 on HSC self-renewal, the loss of p16 and p19 has been reported to have a limited role in this process ().
HSCs did not show any advantages in competitive BM repopulation assays either.
HSCs retained their self-renewal capacity better than the wild type during long-term ex vivo culture (unpublished data). These findings suggest that a tight repression of and genes by Bmi1 accounts for a positive effect of forced expression on HSC self-renewal and multipotential progenitor expansion ().
HSCs with a retrovirus, cultured for 10 d in the presence of SCF and TPO, and subjected the cells to colony assays.
HSCs again induced a similar mode of multipotential progenitor expansion to that in wild-type HSCs ().
HSCs in vitro, indicate that additional targets for Bmi1 exist other than and genes, which are implicated in the regulation of HSC self-renewal and multipotential progenitor expansion, although they are largely dispensable in vivo.
Together, all of these observations implicate Bmi1 in both the cell-autonomous and nonautonomous regulation of the HSC system.
mice could be ascribed to certain defects in the
microenvironment of the spleen and thymus (, ). Our findings further unveiled the differential impact of derepressed and on HSCs and their BM microenvironment in -deficient mice, thus defining and as the major targets for Bmi1 in the maintenance of HSC self-renewal but not of the BM microenvironment.
Finally, Bmi1 has been demonstrated to be essential for the maintenance of leukemic stem cells in a mouse model of acute myelogenous leukemia induced by the fusion gene (). It has also been demonstrated that the Rb and p53-dependent cellular senescence plays a critical role to oppose neoplastic transformation triggered by the activation of oncogenic pathways (). It will be important to investigate whether the up-regulation of Bmi1 contributes to repression of the oncogene-induced senescence pathway in the leukemic transformation and maintenance of the self-renewal capacity of leukemic stem cells.
mice (provided by R.A. DePinho, Harvard Medical School, Boston, MA) that had been backcrossed at least eight times onto a C57BL/6 (B6-Ly5.2) background were used. Mice congenic for the Ly5 locus (B6 Ly5.1) were bred and maintained at the Animal Research Center of the Institute of Medical Science (University of Tokyo). All experiments using mice received approval from the Tokyo University Administrative Panel for Animal Care.
Hematopoietic cells from B6-Ly5.2 mice were mixed with BM competitor cells (B6-Ly5.1) and were transplanted into B6-Ly5.1 mice irradiated at a dose of 9.5 Gy. Donor cell chimerism in the recipient peripheral blood cells was evaluated as previously described ().
microenvironment to support hematopoiesis was evaluated by transplanting 2 × 10 wild-type BM cells (B6-Ly5.1) into 4-wk-old mutant mice (B6-Ly5.2) sublethally irradiated (
and
mice, 4.5 Gy; others, 6.5 Gy).
Mouse HSCs (CD34KSL cells) were purified from BM cells of 8-wk-old mice on a flow cytometry system (FACSVantage; Becton Dickinson) as previously described (). Single CD34KSL cells were sorted clonally into 96-well plates containing 200 μl SF-O3 (Sanko Junyaku) supplemented with 5 × 10 M 2-β-mercaptoethanol, 2 mM -glutamine, 10% FBS, 20 ng/ml of mouse SCF, 20 ng/ml of mouse IL-3, 50 ng/ml of human TPO, and 1 unit/ml of human EPO (PeproTech).
Femurs and tibiae were cut into small pieces after BM cells were fully flushed out. Then, bone fragments were cultured in α-MEM supplemented with 2 mM -glutamine, 10% FCS, and 5 × 10 M 2-β-mercaptoethanol. Suspension cells were removed by replacing the medium. Osteoblastic phenotypes were evaluated by the expression of alkaline phosphatase. A lentivirus vector (CS-H1-shRNA-EF-1α-EGFP) expressing shRNA against mouse (target sequence TAAAGGATTACTACACGCTAATG) and was prepared, and the viruses were produced as previously described ().
Semiquantitative RT-PCR was performed using normalized cDNA with quantitative PCR using TaqMan rodent GAPDH control reagent (PerkinElmer) as previously described ().
Telomere length was quantified on a flow cytometer (FACSCalibur; BD Biosciences) using flow fluorescence in situ hybridization with a Telomere PNA Kit/FITC for flow cytometry (DakoCytomation).
The retrovirus vector pGCDNsam-ires- (provided by M. Onodera, University of Tsukuba, Ibaraki, Japan), the production and concentration of recombinant retrovirus, and the transduction of CD34KSL cells have been described previously (). After transduction, the cells were further incubated for 9 d in S-Clone SF-O3 supplemented with 5 × 10 M 2-β-mercaptoethanol, 2 mM -glutamine, 1% FBS, 50 ng/ml SCF, and 50 ng/ml TPO and subjected to in vitro colony assay using a methylcellulose medium (StemCell Technologies Inc.) supplemented with 20 ng/ml of mouse SCF, 20 ng/ml of mouse IL-3, 50 ng/ml of human TPO, and 1 unit/ml of human EPO. GFP colony numbers were counted on day 10. Colonies derived from HPP-CFCs (colony diameter of >1 mm) were recovered and morphologically examined. The transduction efficiency was >80% as judged from the GFP expression.
Fig. S1 provides data for flow cytometric profiles and frequencies of HSCs in mutant mice. Fig. S2 provides data for the competitive BM repopulating assay using 10 times more test cells than the competitor cells. Fig. S3 provides data for differentiation and the HSC-supporting capacity of osteoblasts in the absence of Bmi1. Fig. S4 provides data for the senescence of knockdown osteoblasts. Online supplemental material is available at . |
xref
#text
xref
#text
Effective antiviral CD8 T cells possess several functional properties including cytokine production (e.g., IFN-γ, TNF-α, IL-2), cytotoxic potential (e.g., perforin/granzyme granule exocytosis), high proliferative potential, low apoptosis, and, for memory T cells, the ability to self-renew via homeostatic turnover (). One of the key features of a memory CD8 T cell is the ability to rapidly reactivate multiple effector functions and undergo vigorous proliferation after reexposure to antigen. In contrast to the high functional capacity of effector and memory CD8 T cells generated after acute infection or vaccination, CD8 T cell function is often impaired or exhausted during chronic infections. Exhaustion was originally described during chronic LCMV infection as the persistence of virus-specific CD8 T cells that lacked effector functions (). CD8 T cell exhaustion appears to be a prominent feature not only of experimental chronic infections in mice but also during chronic infections in primates and humans (for review see references , ). For example, nonfunctional antigen-specific CD8 T cells have been observed during SIV infection of primates and human infection with HIV, hepatitis B, hepatitis C, and human T lymphotropic virus-1.
Studies both in mouse models and human chronic infections have demonstrated that exhaustion comprises a range of dysfunctions from relatively mild to extreme () (). Impaired proliferative potential is a key feature of exhaustion that often occurs when other functions, such as cytokine production, are largely intact. Indeed, high proliferative potential correlates with nonprogression during HIV infection () and may be a critical property of those T cells that can respond to therapeutic intervention (). There appears to be a distinct hierarchy of exhaustion with certain functional properties, such as IL-2 production and proliferative potential, which are lost first, and other functions, such as IFN-γ production, which are more resistant to inactivation (). The duration of infection, level of antigen exposure, and the availability of CD4 T cell help are all critical factors that affect the level of exhaustion during any given chronic infection.
APC type and quality may also change dramatically during chronic infections as, for example, professional APCs are killed and inflammatory signals change (–). It is likely that this altered APC repertoire and the associated changes in costimulation will influence T cell exhaustion during chronic infections. During HIV infection, surface expression of PD-L1 on APCs is increased and that of CD86 is decreased, tipping the balance between inhibitory and stimulatory signals delivered to T cells toward inhibition (). In addition, the inhibitory signals delivered by PD-L1 on epithelial and endothelial cells may take on added significance when professional APCs are reduced during chronic infection.
Recent studies have shown that PD-1 is highly expressed by CD8 T cells during chronic LCMV infection and that the PD-1–PD-L pathway plays a major role in regulating T cell exhaustion during this infection (). When antibodies were used to block the PD-1–PD-L pathway in vivo during chronic LCMV infection, virus-specific CD8 T cell responses were potently enhanced. Not only was the number of LCMV-specific CD8 T cells increased dramatically, but their function was also improved. After in vivo PD-1–PD-L blockade, virus-specific CD8 T cells produced more IFN-γ and TNF-α on a per cell basis. The consequence of this reversal of virus-specific CD8 T cell exhaustion was a considerable reduction in viral load. This study set the stage for investigations into the expression of PD-1, and the possible control of T cell exhaustion by the PD-1– PD-L pathway, during human chronic viral infections.
Several new studies suggest a role for the PD-1–PD-L pathway in exhaustion of virus-specific CD8 T cells during HIV infection. The study by Petrovas, et al. (on p. ; []), in this issue, and studies by Day et al. () and Trautmann et al. (), published recently in and , respectively, show that PD-1 expression is elevated on HIV-specific CD8 T cells and that blocking the PD-1–PD-L pathway leads to increased T cell proliferation and effector cytokine production (illustrated in ). Previous work has shown that PD-L1 is up-regulated in HIV infection (). Collectively, these observations suggest that the PD-1– PD-L pathway may indeed be operating during chronic HIV infection.
A large percentage of HIV-specific CD8 T cells expressed PD-1, and the expression of this receptor was elevated on a per cell basis. A large proportion of HIV-specific CD8 T cells also expressed CD27 and CD45RO, indicating previous activation. These CD8 T cells had lost expression of the costimulatory receptor CD28 and perforin and expressed only low levels of CCR7 and CD127 (IL-7 receptor α), which are important molecules for the maintenance of memory T cells. This phenotype suggests that the T cells are poorly functional, are not transiting into memory cells, and are particularly receptive to inhibitory signals.
CD8 T cells in individuals infected with HIV have previously been shown to be dysfunctional with reduced proliferative capacity and effector function (, , , ). Day et al. show that disease severity, as judged by viral load and declining CD4 counts, correlated with both the level of PD-1 expression on HIV-specific CD8 T cells and the percentage of cells expressing PD-1, providing a marker on CD8 T cells that correlates with disease severity (). The level of PD-1 expression was also associated with decreased CD8 T cell proliferation in response to in vitro stimulation with HIV antigen. Collectively, these findings show that the level of PD-1 correlates with the extent of T cell exhaustion. Day et al. () and Trautmann et al. () further demonstrated that PD-1 expression on HIV-specific CD8 T cells was reduced in patients undergoing effective highly active antiretroviral therapy, consistent with the notion that high antigen levels drive PD-1 expression and functional exhaustion. According to Trautmann et al., the CD8 dysfunction did not result from changes in TCR expression or stimulation with altered peptide ligands ().
There were striking differences in the levels of PD-1 expression on virus-specific T cells in chronic versus resolved infection. T cells specific for vaccinia virus, which causes a self-limiting acute infection, expressed little PD-1 compared with high levels of PD-1 expressed by HIV-specific T cells. In contrast, T cells specific for the chronic viruses, CMV and Epstein Barr virus, expressed moderate to high levels of PD-1, respectively. This suggests that sustained viremia and antigen presentation maintain the high levels of PD-1 expression. It will be important to determine if up-regulation of PD-1 and PD-L is a consequence of the antiviral interferon response, an indirect effect of T cell activation and inflammatory cytokine production, or whether HIV proteins directly up-regulate their expression.
All three papers demonstrate functional significance of PD-1 expression on HIV-specific T cells. Blocking PD-L1 with a monoclonal antibody led to increased T cell proliferation and production of TNF-α, IFN-γ, and granzyme B, indicating an overall increase in effector function. Although short-term blockade of the PD-1–PD-L pathway in a 6-h cytokine assay had little effect on HIV-specific CD8 T cell function, blockade of this pathway during a 6 d proliferation assay enhanced the proliferation of both HIV-specific CD8 and CD4 T cells and resulted in more functional T cells at the end of the culture. Whether proliferation is a prerequisite for the recovery of other T cell functions remains to be determined. Cytolytic activity, for example, was not examined and will be of great interest. It is also unclear whether the recovery of proliferative potential observed in the LCMV and HIV systems reflects greater cell division, less death, or both.
Petrovas et al. used a plate-bound, PD-1-specific polyclonal antibody to show that engagement of PD-1 reduces HIV-antigen specific T cell proliferation (). They examined apoptosis in the PD-1 HIV-specific CD8 T cells and found increases in both spontaneous and Fas-mediated apoptosis, suggesting that cross-talk may occur between PD-1 and Fas receptors. The interpretation of these findings, however, is complicated by the finding that PD-1 HIV-specific CD8 T cells also have increased susceptibility to apoptosis. It is possible that other factors such as the level of T cell activation are involved. Cells expressing very high levels of PD-1 were much more susceptible to death signals, suggesting that PD-1 expression leads to a survival defect in vivo.
Because of the link between CD4 help and CD8 function, Day et al. also examined the effects of PD-1 ligand blockade on CD4 T cell expansion (). Remarkably, in five of six patients with undetectable CD4 proliferative responses to HIV p24 antigen, blocking PD-L1 restored vigorous CD4 T cell expansion, suggesting that HIV-specific CD4 T cells may be present but so functionally impaired that they are undetectable in standard assays.
xref
sup
#text
The PD-1–PD-L pathway has a critical role in regulating the balance between T cell activation and tolerance (, ). Blocking or eliminating PD-1 or its ligands can accelerate and exacerbate autoimmune disease. Single nucleotide polymorphisms in PD-1 have been linked with several autoimmune diseases, although how PD-1 affects susceptibility to human autoimmune diseases is not yet clear. It will be important to learn how to modulate this pathway to reactivate antiviral T cells while minimizing the risk of autoimmunity and immunopathology.
How does PD-1 exert its inhibitory effects? Many reports have shown that PD-1 signaling inhibits T cell activation. Early reports found an effect on cell cycle arrest rather than cell death (), but recent studies, including the study by Petrovas et al., emphasize the role of PD-1 in promoting T cell death (, , ). PD-1 might directly engage a death pathway or more likely indirectly influence cell death by down-regulating survival signals and growth factors or synergizing with death pathways. For example, PD-1 ligation inhibits expression of the cell survival gene and several growth factors (–). The effects of PD-1 ligation on expression of death receptors and death pathways need further study.
The outcome of PD-1–PD-L interactions on T cell expansion and survival might also depend on the microenvironment and the type of APC that interacts with the T cell. When macrophages are used as APCs, anti–PD-L1 or anti–PD-1 monoclonal antibody increases IFN-γ and IL-2 production by T cells but, paradoxically, inhibits their proliferation. This inhibition was caused by the IFN-γ–dependent induction of nitric oxide production by macrophages, which inhibits T cell proliferation (). Increased IFN-γ production after PD-L1 blockade is seen in HIV-specific T cells and is a common result reported in many experimental systems. Thus, combining inducible nitric oxide synthase blockade with PD-1–PD-L blockade may have therapeutic potential as a means to augment immune responses and protect against the potential proinflammatory effects of nitric oxide.
To optimally manipulate this pathway during chronic viral infection, further work is needed to understand the functions of PD-L1 versus PD-L2, and the role of PD-1 on B cells and macrophages and on T cells. Since PD-1 is expressed on B cells and macrophages and PD-L1 is expressed on T cells, there might also be bidirectional signaling. Some reports describe reverse signaling of PD-L1 and PD-L2 into cells that express them (). There are also data that support stimulatory functions of PD-L1 or PD-L2, perhaps mediated by an as yet unidentified second receptor. The contributions of each of these interactions to the therapeutic efficacy of pathway manipulations need to be evaluated.
#text |
To inhibit signaling from all Notch receptors (Notch1–4), we generated DNMAML mice () (). These mice harbor a cassette encoding the GFP-tagged Notch inhibitor DNMAML downstream of a floxed sequence that prevents transcription of DNMAML. Cre recombinase results in DNMAML expression from the ubiquitously active promoter. Notch-deprived cells can be tracked through GFP detection. DNMAML mice were crossed to LC tg mice to generate LCD mice and compared with control LC littermates. GFP expression was detected in a small percentage of CD44CD25 DN2 cells (). In subsequent stages, DNMAML was induced in >50% of CD44CD25 DN3 cells and >95% of CD44CD25 DN4, TCRβCD8 immature single-positive and CD4CD8 DP cells.
LCD thymi had impaired αβ T cell development, as shown by a decreased percentage of DP and an increased percentage of DN cells (), translating into a four to fivefold decrease in thymic cellularity (). The absolute number of DN cells was preserved, whereas numbers of immature single-positive, DP, CD4 single positive (SP) and mature CD8 SP cells were decreased, which was consistent with a block at the DN-DP transition. Among CD3 thymocytes, the percentage of TCRγ cells was increased in LCD mice (), an effect secondary to decreased TCRβ cells because the absolute number of TCRγ cells was maintained (). This was observed despite expression of DNMAML in ∼80% of LCD γδ thymocytes (). Regarding αβ T cell development, our observations in LCD mice were consistent with findings reported after LC-mediated inactivation of Notch1 or CSL/RBP-J (, ). In contrast, the preservation of γδ thymocytes in LCD mice was similar to LC x Notch1 mice () but differed from the increase in absolute γδ cell numbers in LC x CSL/RBP-J mice (). This cannot be explained by Notch2–4 activity after deletion because signaling from all four Notch receptors is inhibited both by DNMAML () and the absence of CSL/RBP-J. A potential explanation is that Notch-independent CSL/RBP-J–mediated transcriptional repression plays a role during γδ development. This repressor function would be lost in the absence of CSL/RBP-J but is unaffected by DNMAML. Alternatively, subtle differences in genetic background or timing of Notch inactivation may account for the discrepant results.
We studied the phenotype of Lin DN thymocytes in LC and LCD mice (), using GFP to better define the effects of Notch deprivation. The proportions of DN2, DN3, and DN4 cells were similar in LC and LCD thymi (). LCD DN3 cells expressed higher levels of CD25 than LC DN3 cells (). Although the difference was modest, it was statistically significant (Fig. S1, available at ). In addition to DN3 cells, abnormal CD25 DN2 cells were also present in LCD mice. These findings were reminiscent of the CD25 DN2-DN3 population observed after LC-mediated inactivation of Notch1 or CSL/RBP-J (, ). This population was hypothesized to represent Notch-deprived DN2-DN3 cells that failed to undergo β-selection, as in mice lacking pre-TCR components (). However, all CD25 DN2-DN3 cells in LCD mice were GFP, whereas GFP DN3 cells expressed lower levels of CD25 than the GFP DN3 cohort (). These findings indicated that CD25 DN2-DN3 cells emerged in LCD mice not as a direct consequence of Notch deprivation but because of a non–cell autonomous effect; e.g., an abnormality in intrathymic niches occupied by DNMAML DN cells.
We next assessed LC/LCD DN3 and DN4 cells for i.c. TCRβ and TCRγ ( and Fig. S2, available at ). The percentage of i.c. TCRβ DN3 and DN4 cells showed a modest, though statistically significant, decrease in LCD as compared with LC mice. The percentage of i.c. TCRγ thymocytes was not significantly different.
We then examined LC/LCD DN3 cells for phenotypic changes associated with β-selection (). DN3 cells exposed to pre-TCR signals exhibit active cell cycling, increased cell size, and CD27 up-regulation (DN3b population) (). FSCCD27 or CD44CD27 DN3b cells were reduced in LCD as compared with LC thymi (). When assessed for DNA content, a smaller proportion of DN3 cells were in the S-G2M phases of the cell cycle () and fewer cells incorporated BrdU in LCD as compared with LC DN3 cells (). In contrast, no significant proliferation defect was detected in DN4 cells. The proportion of i.c. TCRβ cells was reduced among BrdU LCD as compared with LC DN3 cells (LC, 68 ± 3% vs. LCD, 43 ± 4%; mean ± SEM; P < 0.01), a finding that was consistent with reports of impaired proliferation, predominantly in αβ lineage cells in vitro (, ). Collectively, these findings indicate that Notch-deprived DN3 cells, although detected phenotypically as CD25 cells at the DN3-DN4 transition, failed to undergo the typical changes associated with β-selection. We recently reported that Notch directly up-regulates transcription in primary DN3 cells and T cell leukemia cell lines, suggesting that abrogation of the Notch–c-myc axis contributes to the β-selection defects in LCD mice ().
In vitro studies using Rag-deficient DN3 cells on OP9-DL1 stroma found that Notch signaling had important roles in cell metabolism and survival. In contrast, we did not detect consistent abnormalities in Annexin V staining and cellular bioenergetics, as measured by tetramethylrhodamine ethyl ester labeling in LCD thymi (unpublished data). This does not rule out Notch-related changes in these parameters because compromised/dying thymocytes are rapidly eliminated and have been notoriously difficult to detect in vivo ().
The in vivo DNMAML-related defects were reminiscent of in vitro findings using OP9 cells in which the main consequences of Notch deprivation were growth arrest, decreased cell size, and eventual cell death (, ). To better understand changes induced by the loss of Notch signaling, we assessed cell size and CD25 expression in cultures of LC DN3 or LCD GFP DN3 thymocytes with OP9-DL1 cells (). After 24 h, LCD GFP DN3 cells exhibited decreased cell size and CD25 expression when compared with LC DN3 cells. Therefore, these changes resulted from a transcriptional effect of Notch signaling mediated by the ICN-CSL-RBP-J–MAML complex. The rapid modulation of CD25 expression suggested that () is a direct transcriptional Notch target in thymocytes (). A chromatin precipitation (ChIP) assay showed that Notch1 associated with two conserved CSL/RBP-J binding sites in the locus; one is located immediately upstream of the transcription start site and the other in intron 3 () These findings confirm that is a Notch transcriptional target in developing thymocytes.
Collectively, these observations suggest a scenario in which Notch-deprived DN3 cells down-regulate CD25 and fail to proliferate. Thus, some CD44CD25 DN3-DN4 thymocytes in LCD mice might represent Notch-deprived DN3 cells that have aberrantly down-regulated CD25. However, these cells did not accumulate to a large extent in LCD mice, as shown by the nearly normal frequency of i.c. TCRβ cells among LCD DN4 thymocytes (). The minimal accumulation of Notch-deprived DN cells in LCD mice might be related to their growth arrest () and/or rapid elimination by apoptosis, which was consistent with in vitro work showing poor survival of cells lacking both Notch and pre-TCR signaling (). In comparison to LCD mice, the accumulation of aberrant CD25 DN3-DN4 cells appeared more prominent in LC x Notch1 and, to a lesser extent, in LC x CSL/RBP-J mice, even if overall impairment of T cell development was similar (, ). We hypothesize that residual Notch signaling through Notch2–4 receptors in LC x Notch1 mice, or preformed CSL/RBP-J protein in LC x CSL/RBP-J mice, contributed to enhanced accumulation of aberrant DN3-DN4 cells without allowing normal progression through β-selection.
As in the absence of Notch1 and CSL/RBP-J (, ), i.c. TCRβ expression was decreased in LCD DN3 cells (). To assess if this was functionally relevant in vivo, we crossed LCD mice to tg mice (). DNMAML did not affect transgene expression (Fig. S3, available at ). The transgene failed to restore cellularity of LCD thymi back to normal numbers (), despite a twofold increase in LCD/ tg mice. The percentage of LCD DP cells was decreased with and without the transgene (), although the decrease was slightly less pronounced with . However, we consistently observed a higher percentage of GFP cells among DP thymocytes of LCD/ tg than in non-tg LCD mice. This suggested that the transgene accelerated the transition through DN2-DN4 stages of development, allowing more DP cells to arise without being exposed to DNMAML. Together, these results indicated that a transgene did not rescue Notch inactivation at the β-selection checkpoint in vivo.
In addition to its effect on TCRβ, Notch might regulate pTα expression (). To assess if this played a limiting role in vivo, we crossed LCD mice to TcrAND tg mice (). These mice express transgenes that can substitute for pre-TCR function (), though with reduced efficiency (). Expression of the transgenes did not restore thymic cellularity (). Accordingly, the percentage of DP cells was reduced in LCD/TcrAND as compared with TcrAND mice (). Similar results were observed with DO11.10 tg mice (unpublished data).
These results show that Notch deprivation in vivo cannot be rescued by restoring pre-TCR function. Instead, they suggest that Notch and the pre-TCR act in parallel pathways. An important future task will be to characterize the interactions between Notch, pre-TCR signals, and other partners that are active during β-selection, such as E proteins. Of note, recent work indicates that Notch and E proteins cooperate during T lineage commitment ().
In vitro experiments suggested an absolute requirement for Notch at the DN-DP transition (, ). However, the generation of LCD DP cells was reduced but not abolished in vivo. This could be explained by a less stringent requirement for Notch signaling in vivo or by the precise kinetics of LC-mediated excision. To differentiate between these possibilities, we purified GFP and GFP DN3 cells from LCD mice and performed intrathymic injections (). Control LC DN3 cells gave rise to donor-derived DP/SP T cells 10 d after injection (, top). In contrast, Notch-deprived GFP LCD DN3 cells gave rise to no or barely detectable progeny (, middle). There was at least a 3-log reduction in donor-derived cells in the absence of Notch signaling (). When GFP LCD DN3 cells were injected, significant numbers of donor-derived DP/SP cells were observed at day 10 (, bottom), and >50% of these cells were GFP as a result of DNMAML induction in vivo between injection and analysis. These results indicate that the requirement for Notch during β-selection is as stringent in vivo as in vitro. Furthermore, the apparently partial differentiation block observed in vivo results from late Notch inactivation in a fraction of DN3-DN4 cells.
DNMAML mice were generated as previously described (). The DNMAML-GFP construct encodes amino acids 13–74 of MAML1 fused to GFP (, ). DNMAML mice were crossed to LC tg mice (Taconic). LCD mice were compared with LC littermates. B6.CD45.1 and tg mice expressing a TCRβ chain from the DO11.10 hybridoma were obtained from Taconic. Tg(TcrAND) and Tg(DO11.10) / tg mice were purchased from the Jackson Laboratory. Experimental protocols were approved by the University of Pennsylvania Office of Regulatory Affairs.
The following antibodies were from obtained from BD Biosciences or eBioscience: PE anti-CD25 (PC61), CD27 (LG.7F9), TCRβ (H57-597), TCRγ (GL3), CD4 (RM4-5), and CD3 (145-2C11); APC anti-CD4, TCRβ, CD44 (IM7), and BrdU; biotinylated anti-CD45.2, CD8 (53-6.7), TCRβ, TCRγ, CD4 (GK1.5), CD3, NK1.1 (PK136), B220 (RA3-6B2), CD19 (1D3), CD11b (M1/70), Gr1 (RB6-8C5), and CD11c (HL3); APC-Cy7 anti-CD25; and PE-Cy5.5 anti-CD44 and PE-Cy7 anti-CD45.1 (A20). Biotinylated antibodies were revealed with streptavidin-PerCP (BD Biosciences), Pacific Blue (Invitrogen), or PE–Texas red (Caltag). Lineage cells were defined with anti-CD8, TCRβ, TCRγ, NK1.1, CD3, B220, CD19, CD11b, Gr1, and CD11c.
Cells were stained in PBS/2% FCS. i.c. staining was performed with fixation/permeabilization or BrdU labeling kits (Becton Dickinson). 0.5 mg BrdU was administered i.p. 3 and 1 h before death. Cells were sorted on a FACS DiVa (Beckton Dickinson) or a MoFlo (DakoCytomation). Analysis was performed on a FACS Calibur or LSR II (Becton Dickinson). DAPI was used to exclude dead cells or assess DNA content in fixed cells. Files were analyzed with software (FlowJo; Tree Star, Inc.).
1–2 × 10 sorted DN3 cells were injected intrathymically in anesthetized B6.CD45.1 recipients given 500 rad 2–6 h before injection.
OP9-DL1 cells were provided by J.C. Zuniga-Pflucker (University of Toronto, Toronto, Canada) and used as previously described (). Progenitors were seeded into 24-well plates containing a stromal monolayer with 1 ng/ml mIL-7 (PeproTech).
ChIP was performed from Rag-2 DN3 cells using Notch1 TAD domain–specific antiserum (), anti-acetylated histone 4 (Upstate Biotechnology), or rabbit IgG (Santa Cruz Biotechnology, Inc.), as previously described (). Quantitative PCR was performed with SYBR green (Applied Biosystems) and the following -specific primers: 25K, 5′ CAGTCATTGGTTGGCCACTCT 3′ and 5′ GGACCTCCATGCAGACATCA 3′; promoter, 5′ TGTTGAGTCTTCTGGGGGAGAA 3′ and 5′ CTAGGAGGTGTGGGCAGTGTTT 3′; and intron 3, 5′ TGCAGCATGGGTCAAATGAA 3′ and 5′ AGGTCTCCCCAGGAAAAGTCAC 5′.
Fig. S1 depicts CD25 median fluorescence intensity in Lin CD44 DN3/DN4 LCD as compared with LC thymocytes. Fig. S2 shows expression of i.c. TCRβ and i.c. TCRγ in Lin DN3-DN4 LC and LCD thymocytes (a representative example is presented). Fig. S3 shows expression of i.c. TCRβ in the presence or absence of a transgene in LC and LCD DN thymocyte subsets. |
is suspected to be a causative bacteria for human sarcoidosis, and its cell wall components show strong immunoadjuvant activities, which induce monocyte migration into the liver and granuloma formation (–).
To examine the role of IL-15 in the granuloma formation in vivo, control WT and IL-15 mice were injected with heat-killed . Consistent with previous reports (–), granuloma formation was evident in the liver of WT mice on day 6 after injection, whereas it was hardly seen in that of IL-15 mice (, top left and top middle). Importantly, the granuloma formation was substantially restored by IL-15 injection into IL-15 mice (, top right). It has been reported that on infection circulating DC precursors migrate into the liver and, in cooperation with Kupffer cells, monocytes, and T cells, participate in the granulomatous reaction (). Indeed, –induced granulomas contained CD11c DCs (). The granuloma formation was observed in the liver of RAG-2 mice and NK cell–depleted RAG-2 mice but was absent in that of IL-15RAG-2 mice (, bottom; and Fig. S1, available at ), indicating that both T, B, and NK cells are dispensable in the process of granuloma formation. Because chemokine production is critical for monocyte migration into the liver, we next examined the chemokine production, in particular MCP-1 (CCL2) and MIP-1α/β (CCL3/4), which are critical chemoattractants for monocytes. On day 3 after injection, considerable levels of these chemokines were detected in the sera of WT, RAG-2, and NK cell–depleted RAG-2 mice ( and Fig. S2, available at ). In contrast, these chemokines were produced only marginally in IL-15 and IL-15RAG-2 mice (). Again, the chemokine production was restored by IL-15 injection into IL-15 mice. To further examine whether IL-15 directly controls the chemokine production, we analyzed IFN-γ mice. As reported previously (), –induced granuloma formation was not seen in the liver of IFN-γ mice (). In addition, we found that –induced IL-12p70 and IFN-γ production in IL-15 and CCL2 production in IFN-γ mice was impaired (), indicating that IL-15 indirectly induces chemokine production by regulating the IL-12–IFN-γ axis in vivo.
Zymosan is a yeast cell wall particle containing β-glucan and mannan as major components. As does, zymosan can activate and recruit monocytes, macrophages, and leukocytes (–), resulting in the secretion of inflammatory cytokines, hydrogen peroxide, and arachidonic acid (–). We also used zymosan to examine the role for IL-15 in the granuloma formation. Consistent with previous experiments (), zymosan recruited monocytes and DCs and induced granuloma formation in the liver of WT mice. Again, the granulomas were not seen in the liver of IL-15 mice (), likely because of the lack of chemokine production, such as CCL2 () (). Our results collectively indicate that IL-15 controls – and zymosan-induced granuloma formation, likely through the regulation of chemokine production in vivo.
LPS injection into –primed mice stimulates DCs and macrophages to produce large amounts of proinflammatory cytokines such as IL-12, IFN-γ, and TNF-α, which cause lethal endotoxin shock in vivo (–). Likewise, LPS injection into zymosan-primed mice induces shock and tissue injury (). We next examined the role of IL-15 in LPS-induced endotoxin shock. On day 6 after a 0.5-mg heat-killed injection, 1 μg LPS was injected into WT and IL-15 mice to induce lethal endotoxin shock. As reported (–), –primed WT mice were sensitive to LPS-induced endotoxin shock, and all mice died within a day. In contrast, IL-15 mice were strongly resistant and survived (). In addition, NK cell–depleted WT mice, RAG-2, mice and NK cell–depleted RAG-2 mice died just as control WT mice did, indicating that proinflammatory cytokines produced by T, B, and NK cells are dispensable for the endotoxin shock induction ().
IL-12, IFN-γ, and TNF-α are known to play important roles in induction of liver injury and/or endotoxin shock (, , –). We thus examined the production of proinflammatory cytokines, in particular IL-12p70, IFN-γ, and TNF-α, in control WT and IL-15 mice (). Shortly after LPS injection (2 h), these cytokines were detected in the sera of control WT mice, whereas only small amounts of these cytokines were produced in the sera of IL-15 mice (). Because these cytokines cause liver injury, the level of serum glutamic-pyruvic transaminase (GPT) and glutamic-oxaloacetic transaminase (GOT), an index for hepatocyte damage, was also measured. As expected from the minimal proinflammatory cytokine production, considerable reduction of GPT and GOT release was observed in the sera of IL-15 mice, as compared with those in WT mice (). Similar to these observations, zymosan-primed IL-15 mice exhibited strong resistance to LPS-induced lethality, which was consistent with the reduced production of IL-12, IFN-γ, and TNF-α ().
As recently reported that IL-15 regulates survival of DCs (), impaired inflammatory responses observed in IL-15 mice might be caused by the impaired DC survival. To examine this possibility, we examined the number of splenic DCs between WT and IL-15 mice before and after injection. The numbers of DCs in untreated WT and IL-15 mice were 0.83 × 10 ± 0.15 ( = 5) and 0.82 × 10 ± 0.13 ( = 5), respectively, and those in –injected WT and IL-15 mice (6 d after injection) were 1.39 × 10 ± 0.28 ( = 4) and 1.54 × 10 ± 0.16 ( = 4), respectively. Using annexin V and propidium iodide staining, we further analyzed the number of apoptotic DCs before and after injection and found no important difference between WT and IL-15 DCs (). These results suggested that impaired inflammatory responses observed in IL-15 mice are unlikely caused by the impaired DC survival in vivo.
As IL-15 was found to be a critical mediator for granuloma formation and endotoxin shock induction in vivo, it is important to quantitate the amount of IL-15 produced as a soluble protein in mice during the induction phase of granuloma formation and the eliciting phase of endotoxin shock. For this purpose, we generated rat mAbs specific for mouse IL-15 (for details see Materials and methods). Among 108 clones, 2 clones named AIO2 and AIO3 produced mAbs against mouse IL-15, which were suitable as coating antibodies for an ELISA system. Importantly, the newly developed ELISA system is specific for mouse IL-15 and does not cross react with other cytokines tested, which include IL-2, IL-4, IL-12, TNF-α, IFN-α, IFN-β, IFN-γ, and GM-CSF (). Using the ELISA system, we found that substantial amounts of IL-15 were produced in the sera of –injected WT mice, and the IL-15 levels were dramatically enhanced immediately after LPS injection into –primed WT mice (), which was consistent with the observation that the lack of IL-15 resulted in the impaired endotoxin shock.
The results shown in (A and D) and collectively raised a possibility of treatment of inflammatory diseases by blocking IL-15 activity in vivo. To this end, we examined whether anti–IL-15 mAbs were capable of blocking IL-15 activity (). It is well known that both IL-2 and IL-15 efficiently induce proliferation of a T cell line, CTLL-2. As shown in , IL-15–dependent proliferation of CTLL-2 was markedly reduced (>95% reduction) in the presence of AIO2 or AIO3, whereas IL-2–dependent proliferation of CTLL-2 was not affected at all by these mAbs, demonstrating that the mAbs efficiently and selectively block IL-15 activity in vitro. We further examined the effect of AIO2 in vivo (). WT mice that had been injected with AIO2 on days 0, 3, and 6 after injection (x3 AIO2) and only on day 6 (1 h before LPS injection, x1 AIO2) were strongly resistant to LPS-induced lethality and survived. The results clearly show that antibody capable of neutralizing IL-15 activity is effective in blocking endotoxin shock in vivo.
As DCs are present in granuloma regions (), we further examined the role of DCs in the granuloma formation using CD11c–diphtheria toxin receptor (DTR)–GFP transgenic (DTR tg) mice (). Because the mice carry a transgene encoding DTR-GFP fusion protein under the control of a mouse CD11c promoter, DT injection induces selective depletion of DCs in vivo () (). – and zymosan-induced (, B and C; and , respectively) granulomas were observed in the livers of untreated WT, DT-injected WT, and untreated DTR tg mice, as expected. In contrast, neither CD11c DCs nor granulomas themselves were observed in the DT-injected DTR tg mice (). Consistent with this observation, production of IL-12p70, IFN-γ, CCL2, and CCL3/4 was impaired in the DT-injected DTR tg mice, as observed in IL-15 mice (). Importantly, immunohistochemical analysis revealed that many IL-15–producing cells in granuloma regions were CD11c DCs (). Contrary to the case of IL-15 mice, however, IL-15 administration restored neither the –induced granulomatous liver disease nor CCL2 production in DC-depleted DTR tg mice (), implying that direct action of DC-derived IL-15 on DCs is necessary for the chemokine-mediated granuloma formation in vivo. To formally demonstrate the importance of DC-derived IL-15 in the granuloma formation, BM-derived DCs (BMDCs) of WT and IL-15 mice were adoptively transferred into IL-15 mice. Importantly, the granuloma formation was substantially restored by the injection of WT but not IL-15 BMDCs into IL-15 mice (), demonstrating that DC-derived IL-15 is essential for the granuloma formation.
Intracellular redox status affects the pattern of cytokine production by DCs and macrophages (–). For example, reductive DCs (and macrophages) with elevated intracellular glutathione (GSH) preferentially produce IL-12 and are involved in Th1 responses, whereas oxidative DCs with reduced GSH produce IL-10 and PGE, which lead to Th2 cell induction. As priming efficiently induces reductive status in DCs (), we next examined the role for IL-15 in determining redox status in DCs (Fig. S3, available at ). As previously shown (), injection clearly induced a reductive condition with elevated intracellular GSH levels in WT DCs. In contrast, such a reductive condition was not induced, if any, in IL-15 DCs on stimulation. These data indicate that IL-15 is one of the critical cytokines in reductive DC differentiation.
We also examined the role of DCs in endotoxin shock. 1 d after DT injection, control WT and DTR tg mice were primed with 0.5 mg . On day 3 after injection, these mice were injected with 1 μg LPS, and survival rate was monitored. On LPS injection, all –primed WT and DT-untreated DTR tg mice died of endotoxin shock within 24 h (). However, –primed and DT-injected DTR tg mice survived much longer than control mice, indicating that DCs play a pivotal role in endotoxin shock as well. Of note, DT-injected DTR tg mice survived relatively shorter than IL-15 mice (), which was likely because of the gradual recovery of DCs in DT-injected DTR tg mice as reported previously (). –primed DT-injected DTR tg mice showed impaired production of IL-15 and IL-15–regulated proinflammatory cytokines, IL-12p70, IFN-γ, and TNF-α on LPS stimulation (). In addition, WT BMDC-transferred IL-15 mice became sensitive to the endotoxin shock, and all mice died within a day, whereas IL-15 BMDC–injected IL-15 mice remained resistant (). Collectively, these results indicate that DC-derived IL-15 is critical for endotoxin shock induction in vivo.
Prolonged or aberrant activation of immune responses cause a variety of immunopathological disorders that are often mediated by effector cells and cytokines. We previously found that DC-derived IL-15 is required for the functional maturation of DCs, such as IL-12 production in response to LPS and agonistic anti-CD40 mAb combined with IL-4 stimulation in vitro (), and have recently shown that DC-derived IL-15 is essential for CpG-induced protective immune activation against pathogen infections in vivo (). In contrast to the beneficial effect of DC-derived IL-15, we maintain in this study that DC-derived IL-15 has harmful aspects and causes inflammatory diseases, such as granuloma formation and endotoxin shock in vivo.
IL-15 is involved in a variety of inflammatory and autoimmune diseases (, , -). To address whether development of these diseases attributes to IL-15, analyses using IL-15 mice, anti–IL-15 neutralizing antibody, soluble IL-15Rα, and IL-15 mutant/Fcγ2a fusion protein are in progress in both human and mouse models (, ). As IL-15 is produced by multiple cell types, which include DCs, macrophages, monocytes, and endothelial cells (), an advanced and unresolved question has been whether DC-derived IL-15 is exclusively required for the development of certain diseases in vivo. Numerous previous in vitro studies indicated that DC-derived IL-15 is capable of inducing activation of Th1 cells, CTL, NK cells, monocyte differentiation into DCs, and antigen-processing machinery in DCs (, ), but these studies did not directly prove the irreplaceable role of DC-derived IL-15 in disease development in vivo, where multiple IL-15 producers are present. To address this issue, it is important to show that the development of certain diseases is impaired in IL-15 or WT mice treated with reagents to block IL-15 activity, and to further restore the disease development by adoptively transferring WT but not IL-15 DCs into these mice. In this respect, only one paper has shown that IL-15 or WT mice treated with soluble IL-15Rα were impaired in CD8 T cell–dependent delayed-type hypersensitivity response, and the delayed-type hypersensitivity response was restored by injecting antigen-labeled WT DCs in vivo (). It is unclear, however, whether IL-15 is important for cytokine production or antigen presentation in this study (). Other groups have shown that IL-15Rα and IL-15 expression by hematopoietic cells is critical for the maintenance of antigen-specific memory CD8 T cells and bystander CD8 T cell proliferation through a “transpresentation” pathway (–), and implied DCs as the major source of IL-15 but never proved it. Accordingly, it remains unknown whether DC-derived IL-15 is essential in the maintenance of innate and acquired immune responses, and whether it causes inflammatory disease development in vivo. It is thus important to prove the in vivo role of DC-derived IL-15 for the development of antiinflammatory drugs that selectively block the DC-derived IL-15 activity.
Mechanisms of how IL-15 controls cytokine production are unknown. As shown in this paper, IL-15 increased the intracellular GSH levels in DCs. Intracellular GSH levels in DCs and macrophages play an important role in determining the profiles of proinflammatory cytokines (–). We have previously demonstrated that reductive DCs with high intracellular GSH levels preferentially produce IFN-γ, which in turn augment GSH levels in the cells (). As shown in this paper, IL-15 is also one of the positive regulators of intracellular GSH status in DCs, augmenting the production of proinflammatory cytokines. In future studies, it should be determined how IL-15 controls the amounts of intracellular GSH in DCs.
The number of –induced granulomas and LPS-induced hepatic necrosis after priming with are substantially reduced in IFN-γ mice () (). In addition, does not induce granuloma formation in TNF-RI mice and mice treated with soluble TNF-RI (). These studies clearly demonstrate the importance of IFN-γ and TNF-α in –induced liver diseases in vivo. In contrast, it has been shown that CCL3 attracts DC precursors in the blood into the sinusoidal granuloma and lets them participate in inflammatory responses in –primed mice (). In addition, CCL2 has also been reported as an important monocyte chemoattractant for granuloma models induced by zymosan, , and (–). These studies show that the chemokines play a critical role in granuloma models, though the importance of IFN-γ and TNF-α in chemokine production remains unclear. Notably, we determined that an IL-15–IL-12–IFN-γ–chemokine (CCL2/3/4) axis in innate immune system is essential, whereas T and B cells are dispensable for the development of granulomatous disease and/or liver injury. Immunohistochemical analysis revealed that DCs preferentially expressed IL-15 in the granuloma regions, and the granuloma formation was impaired in mice lacking DC and DC-derived IL-15 production, clearly demonstrating that DC-derived IL-15 is an initiator for the development of liver diseases.
Of note, IL-15 injection restored the granuloma formation in the liver of IL-15 mice but not DC-depleted DTR tg mice. These results suggest that a critical first step for the granuloma formation is to stimulate DCs with DC-derived IL-15 in an autocrine manner. Although Kupffer cells are critically involved as initial antigen-presenting ( and zymosan) cells (), Kupffer cells isolated from –primed mice were unable to produce IL-15 on LPS stimulation in vitro (unpublished data).
LPS-induced liver injury is closely coupled to endotoxin shock. Indeed, mice deficient in IL-12, IFN-γ, TNF-α, or the receptors for these cytokines displayed resistance to LPS-induced endotoxin shock (, , , ). We showed in this paper that DCs are essential for endotoxin shock, and DC-derived IL-15 controls endotoxin shock induction by controlling the production of IL-12, IFN-γ, and TNF-α, as DC-depleted mice produced these cytokines at much reduced levels, and WT DC-injected IL-15 mice became sensitive to endotoxin shock. Contrary to IL-15 mice, –primed IL-18 mice showed much higher TNF-α production and susceptibility to LPS-induced endotoxin shock (). Together with our results, IL-15 and IL-18 function apparently through distinct pathways in terms of TNF-α production, and IL-15 induces, whereas IL-18 suppresses, TNF-α production.
Collectively, we propose here that DC-derived IL-15 is a master regulator of inflammatory responses in granulomatous liver diseases and related endotoxin shock. Indeed, DC-derived IL-15 regulates the production of IL-12p70, IFN-γ, TNF-α, and downstream CCL2/3/4. Given that elevated IL-15 production and IL-15–expressing cells are evident in RA (, ), inflammatory bowel disease (), type C chronic liver disease (), sarcoidosis (), multiple sclerosis (), and celiac disease (), it is important to further investigate the roles of IL-15 in these inflammatory diseases and search for the propriety of IL-15 as a target for the development of antiinflammatory drugs.
B6–IL-15 (IL-15) mice () were purchased from Taconic, and B6-RAG2 (RAG2) mice were provided by Taconic and Central Laboratories for Experimental Animals. B6–IFN-γ mice were purchased from The Jackson Laboratory. To obtain B6–IL-15RAG2 mice (IL-15 × RAG2), F mice were backcrossed with RAG2 mice, and the obtained IL-15RAG2 mice were intercrossed. The offspring were genotyped for IL-15, and IL-15RAG2 mice were used for experiments. B6-CD11c-DTR-GFP mice () were provided by Steffen Jung, Dan R. Littman, and Richard A. Lang (New York University School of Medicine, New York, NY). All mice were maintained in our specific pathogen-free animal facility, and experiments were performed between 6–12 wk of age in accordance with the guidelines of the Institutional Animal Care Committee of Akita University and Keio University School of Medicine.
Frozen livers embedded in OCT compound (Sakura Finetek) were sliced into 5-μm-thick sections and fixed with 1% paraformaldehyde and stained with Mayer's hematoxylin and eosin (H&E). Sections of livers were observed with a microscope (DM4500 B; Leica). For CD11c immunostaining, acetone-fixed 5-μm fresh-frozen tissue sections were incubated with biotinylated anti-CD11c mAb (clone, N418; eBioscience) overnight at 4°C and with streptavidin-conjugated horseradish peroxidase (HRP; PerkinElmer). Sections were immunostained using 3,3′-diaminobenzidine (DAB) substrate liquid (DakoCytomation). Slides were counterstained with Mayer's hematoxylin. For CD11c and IL-15 double immunofluorescence staining, acetone-fixed 5-μm fresh-frozen tissue sections were incubated with FITC-conjugated anti-CD11c (clone, N418; eBioscience) and biotinylated goat anti–mouse IL-15 polyclonal antibody (R&D Systems) for 1 h at room temperature. Sections were further stained with streptavidin-conjugated PE (eBioscience) for 30 min at room temperature and observed by fluorescence microscopy.
Levels of IL-12p70, IFN-γ, TNF-α, and CCL2/3/4 in the sera were measured by ELISA kits (IL-12p70, IFN-γ, and TNF-α were obtained from BD Biosciences; CCL2/3/4 was obtained from R&D Systems), according to the manufacturer's instructions. The concentrations of cytokines were determined using a data analysis program (Softmax PRO; Molecular Devices). Serum GPT and GOT levels were determined with Fuji Dri-Chem 5500V (Fuji Medical System), according to the manufacturer's instructions.
To detect mouse IL-15 protein, mAbs specific for mouse IL-15 were generated by immunizing mouse IL-15 into Lewis rat. Using conventional methods, spleen cells isolated from the immunized rat were fused with X63-Ag8.653 myeloma cells, and limiting dilution for hybridoma cells was performed. Positive clones producing anti–IL-15 mAb were screened based on the binding capacity to coated mouse IL-15. Among the mAbs, AIO2 and AIO3 clones were further selected as neutralizing mAbs based on the inhibition of IL-15–dependent CTLL-2 cell proliferation. In brief, 5 × 10 CTLL-2 cells were cultured with 10 ng/ml IL-15 or IL-2 in the presence or absence of 10 μg/ml AIO2 and AIO3 for 24 h and pulsed with [H]thymidine for an additional 8 h. For mouse IL-15 sandwich ELISA, microwells were coated with AIO3 overnight at 4°C and incubated with Block Ace (Dainippon Pharmaceutical) for 90 min. The diluted serum samples were incubated for 2 h, then for 60 min with biotinylated goat anti–mouse IL-15 antibody (R&D Systems) and for 60 min with avidin-HRP (Sigma-Aldrich). The absorbance of substance released from the substrate was measured at 450 nm. The IL-15 concentrations in samples were determined using Softmax PRO, based on a standard curve of recombinant mouse IL-15.
Mice were injected with either 0.5 mg of heat-killed (American Type Culture Collection) or 1 mg of zymosan (Sigma-Aldrich). 6 d later, the amounts of LPS indicated in the figures ( O55:B5; Sigma-Aldrich) were further injected into – and zymosan-primed mice, respectively, to induce endotoxin shock. To deplete NK cells, 300 μg/200 μl of polyclonal anti-asialo GM1 (Wako) was injected. To neutralize IL-15 in vivo, 0.5 mg AIO2 was injected. For systemic DC depletion in vivo, CD11c-DTR-GFP mice were injected intraperitoneally with 100 ng/body of diphtheria toxin (Sigma-Aldrich).
DCs were prepared from spleens as previously described (). In brief, collagenase-digested spleen cells were suspended in a 28% BSA solution in 1.08 g/ml PBS, overlaid with 1 ml FCS-free RPMI 1640 medium (Sigma-Aldrich), and centrifuged at 9,500 for 20 min at 4°C. The cells at the interface were collected, washed, and resuspended. DCs were further purified using anti-CD11c (clone N418) microbeads with an autoMACS separation system (Miltenyi Biotec). To detect apoptotic DCs, 5 × 10 DCs were cultured in vitro for 6 h and incubated for 15 min at room temperature in 500 μl annexin V binding buffer with 150 ng/ml annexin V (R&D Systems) or for 10 min at 4°C in 500 μl PBS with 2 μg/ml propidium idodide (Sigma-Aldrich), respectively. For generation of BMDCs, WT and IL-15 BM cells were cultured at 1.5 × 10 cells/ml in 10% FCS RPMI 1640 medium in the presence of 10 ng/ml GM-CSF (RDI Division of Fitzgerald Industries). After 3 d of culture, half of the medium was exchanged with a fresh one. After 6 d of culture, BMDCs were purified using anti-CD11c microbeads with an autoMACS separation system. For adoptive transfer experiments, 1 × 10 BMDCs were intravenously injected into IL-15 mice.
The procedures have previously been described (). In brief, 300 μl of a suspension of splenic DCs, adjusted to a density of 3 × 10 cells/ml in an RPMI 1640 (phenol red free) medium, were applied into a chamber slide (Lab-Tek; Nunc) and incubated for 3 h. After washing, 300 μl of 10 μM monochlorobiman was added, and the reaction was conducted for 30 min. The fluorescence intensity was monitored by argon-ion laser cytometry with a workstation (ACAS 570; Meridian Instruments). Intracellular GSH levels were detected with an excitation wavelength of 350 nm and an emission wavelength of 460 nm.
Fig. S1 shows granuloma formation in the liver of NK cell–depleted RAG-2 mice. Fig. S2 shows the level of serum CCL2 in NK cell–depleted RAG-2 mice. Fig. S3 shows reductive status in –stimulated IL-15 DCs. |
Blimp-1 is encoded by the gene. mice and littermate controls were used to assess the role of Blimp-1 in B-1 cells. CD19-dependent gene deletion is very efficient in splenic B cells (), and deletion of the allele in splenic B cells is nearly complete ().
animals (). Primers were designed specifically to amplify floxed but not deleted alleles, and samples were normalized using a control single copy gene. Less than 15% of the floxed allele was detected in cells. Because flow cytometry showed that the cells we analyzed were >70% Mac1IgM B-1 cells (), we conclude that is efficiently deleted in B-1 cells of mice, hereafter referred to as conditional knockout (CKO) mice.
Flow cytometry was used to study B-1 cells in 6–10-wk-old CKO and control mice. No significant differences were observed in the frequency of PerC B-1a and B-1b subsets determined by staining for B220 and CD5 (). However, CKO mice had an increase in total cellularity in the PerC resulting in a 2.5-fold increase in total numbers of B-1 cells (). When total splenocytes were examined, no differences were seen in the frequency of CD5B220CD43 splenic B-1a cells () or in the overall cellularity of total splenocytes (not depicted). From these data, we conclude that Blimp-1 is not required for the formation or maintenance of B-1 cells in either the PerC or spleen. Moreover, we surmise that the absence of serum Ig observed in our initial study of naive CKO mice () was not due to the absence of B-1 cells.
To determine directly the antibody-secreting ability of Blimp-1–deficient B-1 cells, ELISA assays were performed on supernatants from purified PerC B-1 cells cultured ex vivo. Although control B-1 cells secreted IgM in this setting, IgM secreted by B-1 cells derived from CKO mice was nearly undetectable (; WT, 756 ± 73 ng/ml; CKO, 53 ± 13 ng/ml). Similar results were obtained from sort-purified, cultured PerC B-1a and B-1b cells (not depicted).
To estimate the portion of cells secreting Ig in these B-1 cultures, we stained permeabilized cells for cytoplasmic Ig and calculated the fraction of cytoplasmic Ig B-1 cells. This analysis showed that in control B-1 cell cultures, 31.7% (average from two experiments) were secreting, as indicated by the presence of cytoplasmic Ig. In the CKO cultures, this fraction was 3.2-fold lower or 9.7% (average from two experiments), consistent with the conclusion that Blimp-1 is required for Ig secretion by B-1 cells ( and Fig. S1, A and B, which is available at ). For comparison, ∼38.5% (average from two experiments) of LPS-activated B-2 splenocytes were found to be cytoplasmic Ig after 4 d in culture when similarly analyzed ( and Fig. S1 C).
Treatment of PerC B-1 cells in vitro with LPS causes their proliferation (), and in mice treated with LPS, PerC B-1 cells increase IgM secretion (). When purified PerC B-1 cells were cultured for 3 d in LPS, the cells proliferated (on average, cell numbers doubled during the 3-d treatment) and greater than fivefold more IgM measured by ELISA was secreted into the cultures compared with untreated cultures. Similarly, when CKO B-1 cells were treated with LPS they also doubled during the 3-d LPS treatment. However, the CKO B-1 cells secreted 38-fold less IgM than LPS-treated control B-1 cells, although there was a small increase in IgM secretion comparing untreated and treated CKO cultures ().
To study Ig secretion by B-1 cells in a more physiological context, we determined the relative serum levels of antibodies bearing the T15 idiotype in CKO and WT mice. T15 idiotype antibodies recognize phosphorylcholine-containing self-antigens derived from oxidized lipids on apoptotic cells and atherosclerotic lesions (, ), provide protection against the pathogen (), and are regarded as typical natural antibodies that are exclusively derived from B-1 cells (). ELISA assays were performed for T15 antibodies in serum from WT and CKO animals using a mixture of two rat anti-T15 antibodies, T139 and Tc54 (). Relative T15 antibody levels in the sera of unimmunized CKO mice were roughly equivalent to the level of detection of this assay in all but one of eight mice analyzed, whereas the T15 serum levels in all six unimmunized control animals were on average >2.8-fold above background (; WT, OD = 0.404 ± 0.11; CKO, OD = 0.144 ± 0.07). Collectively, these data demonstrate that Blimp-1 is required for normal antibody secretion by B-1 cells both ex vivo and in vivo.
We next explored the molecular mechanisms underlying the requirement for Blimp-1 in Ig secretion by B-1 cells. In B-2 cells, direct repression of leads to the derepression of the activator , which then functions as the critical proximal regulator of a complex secretory program (, ). Furthermore, Blimp-1 is required for the processing of primary μ transcripts to the μS form of mRNA ().
Quantitative RT-PCR was used to determine the steady-state levels of μS, Pax5, and XBP-1 mRNAs in CKO and control B-1 cells. Purified PerC B-1 cells were analyzed with and without treatment with LPS for 3 d. Pax5 mRNA was higher in CKO cells compared with WT cells, both without LPS and after LPS treatment. Furthermore, Pax5 mRNA decreased after LPS treatment in the WT but not in CKO cells (; 2.9-fold difference between unstimulated CKO/WT; 20.2-fold difference between LPS-stimulated CKO/WT). These data provide evidence that Blimp-1 is required to repress Pax5 mRNA in B-1 cells. CKO B-1 cells also had lower levels of XBP-1 mRNA without LPS treatment and failed to induce XBP-1 after LPS treatment compared with control B-1 cells (; 2.6-fold unstimulated WT/CKO; 5.6-fold LPS-stimulated WT/CKO). Finally, μS was not expressed normally in untreated cells nor was it induced normally in LPS-treated CKO B-1 cell (; 3.0-fold unstimulated WT/CKO; 8.6-fold LPS-stimulated WT/CKO) transcripts. Thus, we conclude that Blimp-1 is required in B-1 cells for Pax5 repression and XBP-1 induction, as well as for formation of μS mRNA.
A unique feature of PerC B-1 cells, in contrast to B-2 cells, is their ability to regenerate the entire B-1 cell compartment. Adoptive transfer of peritoneal B-1 cells by i.p. injection into immunodeficient mice leads to the stable, long-term reconstitution of the PerC and IgA lamina propria B-1 cell pools, as well as restoration of natural IgM titers (, ). Peripheral B-2 cells, on the other hand, lack this ability and can only be generated from BM progenitors. To investigate a possible role for Blimp-1 in the self-renewal capacity of B-1 cells, total PerC cells from WT and CKO mice were harvested and adoptively transferred i.p.
mice. Recipient mice were killed 6–8 wk after transfer and the frequency of PerC B-1 cells was measured by flow cytometry. A small number of mice, receiving either WT or CKO B-1 cells, failed to reconstitute. Those in which reconstitution was <10% were excluded from the study. The half-life of B-1 cells has been reported to be between 38 and 56 d (); therefore, recovery of >50% of donor B-1 cells after 6–8 wk indicates proliferation of the transferred B-1 cells.
mice () indicate that CKO cells can reconstitute
mice. CKO B-1 cells, as well as WT B-1 cells (; WT, 66.2 ± 17.1% recovered; CKO, 71.4 ± 13.9% recovered), proliferated and self-renewed in this experimental setting, and more total PerC cells were recovered from CKO-reconstituted
mice than WT-transferred
mice (; WT, 3.1 × 10 ± 0.55 total cells; CKO, 5.0 × 10 ± 0.81 total cells). In addition, T15 antibodies were detected in the sera of mice reconstituted with WT PerC cells at the time recipient mice were killed, demonstrating that transferred B-1 cells were functional (not depicted). Thus, we conclude that Blimp-1 is not required for the self-renewal/homeostatic proliferation of B-1 cells.
Baumgarth et al. () have elegantly demonstrated that both B-1 and B-2 cells are required for effective early immunity against influenza infection in mice. Specifically, B-1 cell– derived natural antibodies, present before infection, promote subsequent B-2 cell IgG2b responses and reduce mortality. This is probably because natural antibodies trap viruses and fix complement (–). Because Blimp-1 is required for natural antibody secretion by B-1 cells (), we hypothesized that B-1 cells lacking Blimp-1 would be defective in their ability to provide protection to influenza infection.
mice were reconstituted i.v. with B-2 cells from BM from WT mice. Mice were also given PerC B-1 cells i.p. from either WT or CKO mice. B-2 cell reconstitution was confirmed by flow cytometric analysis performed for peripheral blood B220 cells. 3 wk after reconstitution, mice were infected intranasally with an <LD dosage of A/WSN/33 influenza virus, and then monitored for 2 wk.
Weight loss was used as a criterion for susceptibility to influenza infection. In more than three independent experiments in which 27 mice were intranasally infected with influenza virus ranging from 4,500 to 7,000 PFU/g, we found only 1/13 WT-reconstituted mice (, top), but 9/14 CKO-reconstituted mice (, bottom) lost at least 30% of total body weight. Mice were killed when they lost 30% of their body weight to prevent excessive suffering, or on day 14 for analysis of the efficiency of B-1 cell reconstitution. Every mouse successfully reconstituted donor PerC B-1 cells as determined by flow cytometry analysis for surface IgM and Mac1 expression (not depicted). The increased susceptibility to influenza infection of mice receiving CKO B-1 cells demonstrates the physiological relevance of the requirement for Blimp-1 in antibody secretion by B-1 cells.
Our data reveal an essential role for Blimp-1 in antibody secretion by B-1 cells, both ex vivo and in vivo (). A requirement for Blimp-1 in antibody secretion by B-2 cells has been established previously (). Earlier studies have shown that Blimp-1 is necessary for full induction of IgH, J chain, and XBP-1 mRNAs in B-2 cells, presumably due to direct repression of by Blimp-1 () and subsequent derepression of these genes that are repressed by (–), although recent papers disagree on whether or not Pax5 represses XBP-1 (, ). XBP-1 then functions as the proximal regulator of the Ig secretion program, inducing genes encoding proteins responsible for targeting proteins to the ER, cleavage of signal peptides, proper protein folding, degradation of misfolded proteins, and protein glycosylation, as well as proteins needed for ER and other organelle biogenesis and increased cell size (, ). In addition, Blimp-1 is required for the formation of μS mRNA, although the mechanistic basis for this requirement is not currently understood ().
We therefore compared Pax5 mRNA repression, XBP-1 mRNA induction, and formation of μS mRNA in control versus CKO B-1 cells to determine if similar mechanisms were involved in Ig secretion by B-1 cells and B-2 plasma cells. We found that B-1 cells lacking Blimp-1 failed to repress Pax5 mRNA, failed to induce XBP-1 mRNA, and failed to form μS mRNA when compared with control B-1 cells (). These results provide strong evidence that the Blimp-1–dependent mechanisms we studied are important for Ig secretion in both B-1 and B-2 cells. This conclusion is also consistent with a previous study showing that mice lacking XBP-1 in their lymphocytes formed B-1 cells but failed to secrete IgM ().
Why do spontaneously secreting B-1 cells have significantly lower levels of mRNA encoding Blimp-1 and XBP-1 compared with Ig-secreting B-2 cells, as reported by Tumang et al. ()? The amount of IgM measured by ELISA in LPS-treated splenic B-2 cell supernatants is ∼18-fold higher than that in cultures of purified B-1 cells (unpublished data). Yet our data for purified B-1 cells in short-term culture (), and that of Tumang et al. for ex vivo–purified B-1 cells, show that a significant fraction (∼32 and ∼21%, respectively) of purified B-1 cells spontaneously secrete IgM. In addition, our data ( and Fig. S1 C) indicate that a comparable fraction of LPS-stimulated B-2 cells are secreting, as measured by cytoplasmic Ig (∼39%). These data suggest that B-1 cells secrete less Ig per cell than B-2 cells. This conclusion is consistent with the ∼55% smaller spot sizes seen in B-1 cell ELISPOT assays, further demonstrating that B-1 cells secrete less IgM than do LPS-treated B splenocytes (). Moreover, the morphology of B-1 cells is distinct from that of plasma cells. Although they have ample ER, B-1 cells lack the distinct arrays of rough ER characteristic of plasma cells (). Thus, we suggest that although B-1 cells use the same regulatory mechanisms for Ig secretion, because they have less ER and secrete less Ig per cell, they may require lower amounts of Blimp-1 and XBP-1 mRNA and protein compared with B-2 plasma cells.
Our data clearly show that although Blimp-1 mRNA in B-1 cells is relatively low, it is nevertheless functionally important because it is required for normal Ig secretion. This conclusion is strengthened by the demonstration that Blimp-1–deficient B-1 cells do not secrete normal amounts of T15 natural antibodies () and do not provide normal protection against influenza virus infection ().
Mice that cannot secrete IgM due to mutation in the μ-secreted exon and polyA sites have 1.5–2-fold increases in the frequency and total numbers of PerC B-1 cells (, ). The mice we studied have significantly reduced serum levels of all Ig isotypes including IgM (). In spite of this, we did not observe an increase in the frequency of B-1a or B-1b cells in the PerC or in B220CD5CD43 B-1 cells in the spleen of these mice. There were, however, more total cells in the PerC of the CKO mice, resulting in a 2.5-fold increase in the total number of B-1 cells. Hence, our data support the idea that a lack of serum IgM feeds back to cause an increase in total B-1 cells in the PerC. The mechanism responsible for this effect remains obscure.
hosts. We found no difference in the rate of recovery between CKO- and WT-transferred PerC B-1 cells after 6–8 wk after intraperitoneal transfer (), consistent with our previous observation that splenic B-2 cells from CKO mice proliferate well in response to LPS (). Although we cannot formally rule out the possibility, we do not believe that CKO B-1 cell reconstitution was the result of preferential proliferation of B-1 cells that failed to delete .
were found to have approximately ninefold greater T15 antibody levels than CKO-reconstituted mice (not depicted) and CKO-reconstituted
mice were functionally inferior to WT-reconstituted mice upon challenge with influenza virus ().
The B-1 cell compartment is heterogeneous and no single anatomical location or surface marker can define the entire population. B-1 cells are particularly uncharacterized in terms of two defining features: proliferation associated with self-renewal and Ig secretion. Further complexity is added by the fact that many B-1 cells are resting and perform neither function. Although in earlier studies Ig secretion was not detected in PerC B-1 cells (, ), Tumang et al. () showed by ELISPOT assay that ∼21% of naive, freshly sorted PerC CD5B220 B-1 cells secreted IgM over 3 h. Our results on primary B-1 cells in short-term culture confirm this (, A and B, and Fig. S1 A). Many fewer PerC B-1 cells, however, are cycling than were found to be secreting: 2.5% of CD5 PerC B cells depleted of T cells and macrophages were found to be in cycle in vitro, and when the proliferative capacity of PerC CD5 B cells was determined in vivo, only 0.5–1.0% were in S phase (, ). Thus, it is not clear whether B-1 cells that secrete Ig have lost their proliferative capability, retain it, or, after a period of secretion, can revert to cells with proliferative potential.
In B-2 cells, plasmablasts are highly proliferative and also capable of secreting Ig. However, terminally differentiated plasma cells do not divide. Blimp-1 has been shown to repress multiple genes required for cell cycle entry, DNA replication, and cell division, and it is thought to be important for establishing/maintaining the postmitotic state of plasma cells (, , –). Nonetheless, dividing plasmablasts also express Blimp-1, demonstrating that Blimp-1 expression is not incompatible with cell division if the cells receive strong mitogenic signals (, ). Although additional studies will be necessary to learn if B-1 cells are fundamentally different from B-2 cells with respect to terminal differentiation to an Ig-secreting, nonproliferating state, we suspect that the low level of Blimp-1 mRNA in B-1 cells, compared with B-2 plasma cells, does not preclude Ig-secreting B-1 cells in the PerC from dividing when they receive appropriate signals. PerC B-1 cells may simultaneously retain both secretory and proliferative abilities, or they may alternate between secretory and proliferative states, but more data will be required to test this hypothesis. It will also be interesting to learn how overexpression of Blimp-1 might affect Ig secretion and proliferation of B-1 cells.
Overall, this study has shown that B-1 cells, like B-2 cells, require Blimp-1 and Blimp-1–dependent derepression of XBP-1 to secrete Ig. Antibodies derived from B-1 cells are important for immunity to mucosal and air-borne pathogens and are also implicated in autoimmune diseases, including systemic lupus erythematosus (), Sjorgen's syndrome (), and rheumatoid arthritis (). Antibodies derived from B-2 cells are similarly critical for humoral immunity and involved in autoimmunity. Understanding that both B-1 and B-2 cells use common mechanisms to secrete antibodies suggests that compounds designed to modulate the expression or activity of Blimp-1 or XBP-1 could affect both B-1 and B-2 cells and would be effective for either vaccine design or treatment of autoimmunity.
Prdm1 mice were crossed with CD19 mice to generate experimental (CD19 prdm1) and control (CD19prdm1) mice. Rag1 (B6.129S7-Rag1/J) and muMT (B6.129S2-Igh-6/J) were from The Jackson Laboratory. All mouse procedures were approved by Columbia University's Institutional Animal Care and Use Committee. PerC cells were harvested in 4% FBS, 1% BSA in PBS. 3–5 × 10 PerC cells were resuspended in 1 ml PBS and transferred i.p. to Rag1 or muMT mice. The remaining PerC cells were stained with IgM and Mac-1 antibodies and analyzed by flow cytometry. For BM reconstitution, muMT mice were lethally irradiated with 2× 700 rads separated by 4 h. Mice were rested overnight and then reconstituted via tail vein injection with 10 total BM cells in 200 uL PBS harvested from CD19prdm1 control mice. Mice were fed water containing Baytril (enrofloxacin) for the remainder of the experiment. Influenza virus A/WSN/33 was provided by P. Palese (Mount Sinai School of Medicine, New York, NY). Influenza virus was cultured on Mardin-Darby Bovine Kidney cells in modified Eagle's medium supplemented with 0.2% BSA and tittered on Mardin-Darby Canine Kidney cells as described () but without trypsin. For influenza infections, mice were anesthetized with 5% isoflurane and maintained in 2% isoflurane with oxygen. 4,500–7,000 PFU/g body weight was administered to each mouse intranasally in 20 ul PBS. Mice were caged separately and weighed on days 0 and 4–14.
PerC cells were harvested in RPMI (10% FBS) and gentamycin sulfate and plated for 2 h to remove adherent cells. Thy1.2 T cells were removed using magnetic beads and B-2 cells did not survive in culture. On day 4, cells were replated at a density of 10 cells/ml. B-1 cultures were treated with 1 ug/ml LPS (Sigma-Aldrich) or left untreated for 3 d when supernatants were harvested for anti-IgM ELISA assays and cells were processed for either immunohistochemistry, cDNA, or genomic DNA preparations (see below). Splenocyte cultures were prepared as described previously () and treated with 1 ug/ml LPS for 4 d at which time live cells were harvested for immunohistochemical analysis.
The following unlabeled, biotinylated, fluorochrome-conjugated, and secondary detection antibodies were used: Mac-1-PE (M1/70), IgM-biotin (II/41), CD16/32, and B220-APC (Ra3-6B2; all from eBioscience), and CD5-biotin (Ly-1), CD43-PE (S7), and strepavidin-APC (all from BD Biosciences). All flow cytometry stains were performed by incubating 10 cells in 10 uL of 4% FBS plus 1% BSA in PBS with Fc block for 10 min followed by primary antibodies for 45 min, and then, after a brief wash, secondary antibodies for 30 min at 4°C in darkness. Analysis was performed on an LSRII (BD Biosciences) using WinMDI software (Joseph Trotter, Scripps Research Institute).
Anti-IgM ELISA assays were performed on supernatants from B-1 cell cultures as described previously (). Anti-T15–expressing hybridoma lines T139.2 and Tc54.8 were provided by M. Scharff (Albert Einstein College of Medicine, New York, NY). Hybridomas were grown and antibodies were purified using standard ammonium acetate precipitation techniques. For detection of serum T15, 96-well plates were coated with 50 uL of a mixture of 25 ug each of T139.2 and TC54.8 antibodies in PBS for 60 min, and then blocked with a solution of 2% BSA in PBS overnight at 4°C. Wells were washed once with 0.05% Tween-20 in PBS and exposed to mouse serum (1:2 dilutions) for 60 min at 37°C. Wells were washed four times and incubated with a 1:500 dilution of goat anti–mouse Ig(H+L)-AP secondary antibody (SouthernBiotech) in 1% BSA in PBS for 60 min at 37°C. Four additional washes followed by development with 0.8 mg/ml of Sigma 104 phosphatase substrate (Sigma-Aldrich) in -nitrophenyl phosphate buffer, and spectrophotometric measurements at OD were performed.
Purified B-1 cells or splenocytes were seeded on slides by cytospin at 800 rpm for 5 min, air dried, fixed, and permeablized with 1% paraformaldehyde plus 0.2% Tween-20 in PBS for 20 min. Egg white in PBS was incubated for 1 h to block, followed by incubation with 3% human serum (Sigma-Aldrich) plus 3% FBS plus 1% BSA in PBS for 20 min. Primary goat anti–mouse Ig(H+L) antibody (SouthernBiotech) was diluted at 1:5,000 in serum block and applied to slides overnight. The slides were washed for 45 min with TBST (50 mM Tris, pH 7.5, plus 0.2% Tween-20) and incubated with 1:100 diluted rabbit anti–goat IgG(H+L) alkaline phosphatase–conjugated secondary antibody (SouthernBiotech) for 1 h in serum block. Slides were washed as described above and developed by fast blue (Sigma-Aldrich) and napthol AsBi-phosphate substrate (Sigma-Aldrich) supplemented with levamisole (Sigma-Aldrich) in 100 mM Tris-HCl, pH 9.2. A Nikon Eclipse TE300 microscope and Openlab (Improvision) software were used for photographic analysis. Cells with darkly staining rings of cytoplasm or with darkly staining cytoplasmic caps were scored positive.
Quantitative real-time PCR was performed with a cycle of 50°C, 2 min; 95°, 10 min; 95°C, 15 s; 60°C, 1 min; and 81°C, 20 s for 40 cycles, recording data at 81°C and using primers for the unprocessed form of XBP-1 (5′-AGCACTCAGACTATGTGCACCTCT-3′, 5′-TCCAGAATGCCCAAAAGGATATC-3′), μS (5′-TCTGCCTTCACCACAGAAG-3′, 5′-TAGCATGGTCAATAGCAGG-3′), Pax5 (5′-CAACAAACGCAAGAGGG-3′, 5′-GGGCTCGTCAAGTTGG-3′), β-2 microglobulin (5′-AGACTGATACATACGCCTGCA-3′, 5′-GCAGGTTCAAATGAATCTTCAG-3′), Blimp-1 (5′-AGTAGTTGAATGGGAGC-3′, 5′-CAATGCTTGTCTAGTGTC-3′) and peptidyl prolyl isomerase A (5′-CTGAGCACTGGAGAGAAAGG-3′, 5′-CTTGCTGGTCTTGCCATTCC-3′). Quantitative real-time PCR was performed on an ABI7000 machine. Total RNA and cDNA were prepared from at least 0.15 × 10 purified B-1 cells by TRIzol and Superscript III reverse transcriptase according to the manufacture's instructions (Invitrogen). Genomic DNA was made by lysis of purified B-1 cells in 50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, and 12.5% SDS, followed by phenol/chloroform extraction and ethanol precipitation.
Data were expressed as the mean ± SEM. Statistical significance was determined by a two-tail, unpaired Student's test.
Fig. S1 shows representative photographs of cytoplasmic Ig staining of purified, cultured WT B-1 (A), CKO B-1 (B), and LPS-treated splenic B-2 (C) cells. It is available at . |
Ciofani et al. used the OP9/DL-1 culture system to define the developmental stage at which the αβ and γδ T cell lineages first diverge by assessing the clonogenic frequency of αβ-committed, γδ-committed, and αβ/γδ bipotential progenitors (). In these in vitro analyses, all DN1 thymocytes were bipotent, in contrast to ∼35% of the DN2 subset. About 60% of fetal DN2 progenitors were αβ committed, whereas <5% were γδ committed. The vast majority of DN3 cells were unipotent, with αβ progenitors out numbering γδ progenitors by about four to one. Thus, αβ/γδ T cell commitment is first evident in vitro among DN2 progenitors and is largely complete by the DN3 stage (). These clonal in vitro studies nicely complement the recent demonstration that γδ-expressing progenitors can first be visualized at the DN2 stage in vivo ().
To examine the influence of Notch signaling on αβ/γδ lineage divergence, the authors cultured DN2 and DN3 thymocytes on OP9 or OP9/DL-1 stromal cells and quantified production of γδ-expressing DN versus αβ-committed DP thymocytes. Interestingly, γδ development from DN3 cells was largely Notch/DL-1 independent, and even the DN2 subset generated reasonable numbers of mature γδ T cells in the absence of DL-1. Together with the finding that γδ T cell development is not impaired by conditional deletion of all CSL-dependent Notch signaling in DN3 thymocytes (), these findings reveal that generation of γδ-committed progenitors and their subsequent survival and maturation can occur in the absence of Notch signaling.
In contrast, survival and maturation of αβ-committed cells from both DN2 and DN3 progenitors was highly Notch dependent. Small numbers of TCRβ-expressing cells were generated in bulk cultures of DN2 or DN3 thymocytes on OP9 cells, but their progenitors were not clonogenic, demonstrating that survival and/or proliferation of αβ-committed progenitors is highly Notch dependent. Also using the OP9/DL-1 system, Taghon et al. demonstrated a distinction in the degree of Notch dependence of wild-type DN3s before and after expression of TCRβ (designated DN3a and DN3b, respectively) (). DN3a cells could not make DP thymocytes in the absence of Notch/DL-1 signals, whereas DN3b cells could make small DP populations. In contrast, both subsets made γδ T cells in the absence of DL-1.
Collectively, these in vitro findings reveal that αβ progenitors are highly Notch dependent both before and after αβ/γδ lineage divergence, whereas progenitors committing to the γδ lineage become Notch independent ().
DN3s, which cannot express a pre-TCR. (). It will thus be important to determine how this Notch1-induced survival pathway overlaps with or is distinct from the γc-cytokine–mediated prosurvival pathways that also operate during the DN1-DN3 stages ().
What could account for the exquisite and lineage-specific Notch1 dependence of αβ-committed T cell progenitors? One possibility is that CSL-dependent Notch1 signaling directly induces pre-TCR expression, which is needed for the DN3 to DP transition. Consistent with this idea, conditional deletion of Notch1 () or CSL () at the DN3 stage causes a partial block in the generation of DP thymocytes. DN3 thymocytes from these mice had abnormally low frequencies of TCRβ protein and to rearrangements. Since expression of pre-Tα was normal, it was concluded that Notch1 and CSL regulate recombination. However, since the defect was not absolute, to rearrangement may only partially depend on Notch1 activity. Alternatively, some DN3 thymocytes may have produced pre-TCRs and undergone selection for in-frame rearrangements (β-selection) before deleting Notch1. Therefore, studies to date have not clearly defined whether DN3 thymocytes require Notch1 activation upstream and/or downstream of pre-TCR signaling in vivo.
In this issue, Maillard et al. resolve the problem by targeting expression of DN Mastermind–like (DN-MAML) to DN3 thymocytes using a conditional strategy involving Lck-Cre (). MAML transcriptional coactivators are required for CSL-dependent signaling from all Notch receptors, so DN-MAML expression inhibits transcription induced by all four mammalian Notch receptors (). Importantly, the authors strategy also ensured that all thymocytes expressing DN-MAML were marked by coexpression of green fluorescent protein, allowing them to separately track the fate of DN-MAML versus DN-MAML DN3 thymocytes. Using this system, these investigators report only a partial inhibition of the DN3 to DP transition when DN-MAML is conditionally induced in DN3 thymocytes, similar to the effects of deleting CSL or Notch1 at this stage. However, purified DN-MAML DN3 thymocytes were absolutely defective in generating DP thymocytes 10 days after intrathymic injection, whereas DNMAML DN3 thymocytes generated substantial numbers of DP thymocytes using this in vivo assay. These findings definitively demonstrate that heterogeneity in the timing of Lck-Cre expression accounts for the incomplete block in the DN3 to DP transition when Notch1 or CSL are inactivated in DN3 thymocytes.
Notch signals influence expression of pre-TCR components earlier during T lineage specification (), but transgenic TCRβ did not restore the DP thymocyte pool in Lck-Cre/DN-MAML mice. Although Notch1/CSL signaling could regulate TCRβ recombination before the DN3 stage, there is an absolute in vivo requirement for CSL-dependent Notch activity downstream of pre-TCR expression in DN3 thymocytes. Nonetheless, ectopic Notch activation doesn't relieve the developmental arrest of DN3s in mice lacking RAG-2 (), although DP leukemias can eventually develop (). These data and other findings () demonstrate that cooperation between Notch activation and pre-TCR signaling is absolutely necessary to promote the DN3 to DP transition ().
In pre-TCR–deficient mice, γδ TCRs and αβ TCRs can function as alternative pre-TCRs, but they generate a much smaller DP thymocyte pool than bona fide pre-TCRs. To investigate the basis for this difference, Garbe et al. () cocultured OP9/DL-1 cells with DN3 or DN4 thymocytes that were engineered to express predominantly conventional pre-TCRs versus αβ or γδ TCRs. pre-TCRs promoted DP thymocyte differentiation and proliferation more effectively in this culture system than either αβ or γδ TCRs, similar to what has been described in vivo. Ciofani et al. also found that several γδ TCRs could induce the DN3 to DP transition in thymocytes cultured on OP9/DL-1 cells (). As mentioned already, production of DP cells, but not γδ T cells was highly dependent on Notch/DL-1 signaling. Moreover, there was a negative correlation between the strength of γδ TCR signaling and development of DP cells. These observations are consistent with previous findings showing that strong γδ TCR signals can prevent T cell progenitors from developing into αβ-committed DP cells (, ). However, the new studies additionally show that these alternative pre-TCRs, like conventional pre-TCRs, promote DP thymocyte development in a Notch-dependent fashion.
Garbe et al. then titrated various amounts of γ-secretase inhibitor (GSI) into the cultures to inhibit the generation of active N. Surprisingly, they found that DP thymocyte production from αβ- or γδ-expressing DN3 cells was highly sensitive to a given dose of GSI, much more so than DP production from pre-TCR–expressing DN3 cells (). Similarly, αβ-expressing DN4 cells were more sensitive to GSI than pre-TCR–expressing DN4 cells. The authors interpret these data to suggest that DN3s and DN4s expressing conventional pre-TCRs require less Notch signaling to proliferate and mature to the DP stage than progenitors expressing alternative pre-TCRs. However, an alternative interpretation is that DN3 thymocytes expressing conventional pre-TCRs are more effective at capturing Notch1 signals to promote proliferation and differentiation during the DN3 to DP thymocyte transition. Indeed, previous work from this group has shown that pre-TCR–expressing precursors profoundly out-compete αβ TCR-expressing precursors to contribute to the DP thymocyte pool in vivo (). The new data shows that this competitive advantage can be recapitulated on OP9/DL-1 cells in vitro, and is diminished when high doses of GSI were added to the cultures. Thus, it seems likely that T cell progenitors expressing conventional pre-TCRs exhibit stronger Notch1/DL-1 interactions than progenitors expressing αβ or γδ TCRs, endowing the former cells with greater resistance to the γ-secretase–dependent generation of N .
The molecular basis for the synergy between Notch and pre-TCR signaling remains to be determined. At least two nonmutually exclusive scenarios can be envisioned. One possibility is that pre-TCR signaling could more effectively down-modulate the expression or activity of molecules that specifically antagonize Notch activation, or molecules that generally inhibit proliferation. Candidates in the former category include Numb, a negative regulator of Notch activation that physically interacts with the TCR in mature T cells (). Candidates in the latter category include the E47 and Gfi-1 transcription factors, which both restrain proliferation of DN3 thymocytes (, ).
Alternatively or in addition, pre-TCR signaling could enhance the efficiency or avidity of Notch1 interactions with DLs, perhaps by modulating expression or activity of Fringe proteins (). Lunatic Fringe is highly expressed in DN3 and DN4 thymocytes, where it enhances competition for limiting intrathymic niches in vivo to homeostatically regulate the size of the DP thymocyte pool (). Moreover, Lunatic Fringe enhances the ability of DN3 and DN4 thymocytes to bind DL-1 without affecting Jagged-1 binding (). T cell progenitors lacking Lunatic Fringe can respond to OP9/DL-1, but they are more sensitive to GSI than wild-type progenitors (unpublished data). Collectively these findings reveal that Lunatic Fringe–Notch1 interactions regulate T cell progenitor competition for limiting DLs in vivo. Thus, both the pre-TCR and Lunatic Fringe enhance Notch1 interactions with DLs, increasing their resistance to GSI and their competitive fitness. It will therefore be important to determine whether pre-TCR signals directly regulate Lunatic Fringe expression in DN3 and DN4 thymocytes.
In summary, these findings complement previous work showing that DN1, DN2, and DN3 thymocytes must continuously compete for limiting Notch1 signals in vivo () and further suggest that Notch1 activation has different functions during these early stages of intrathymic T cell development. Notch activation is needed to maintain survival of αβ/γδ bipotent and αβ-committed progenitors before TCRβ expression. Commitment to the αβ or γδ T cell lineages, which likely occurs stochastically, is first evident at the DN2 stage and can occur in the absence of Notch1/DL interactions. However, αβ-committed cells remain highly dependent on Notch signals, which act cooperatively with pre-TCR signals to induce vigorous proliferation and maturation to the DP stage (). In contrast, γδ-committed cells become Notch independent. Since weak γδ TCR signals can promote maturation to the DP stage in a Notch-dependent fashion (), strong γδ TCR signals may be needed to terminate Notch1 dependency and promote full γδ T cell maturation. Alternative TCRs are inefficient at promoting the Notch-dependent generation of DP thymocytes because they do not synergize effectively with Notch signals, but the molecular basis of this effect is currently unknown. Importantly, the highly effective synergy between pre-TCR signals and Notch1 activation provides a selective mechanism to prevent αβ-committed progenitors that express αβ or γδ TCRs from effectively competing with pre-TCR–expressing progenitors for access to limiting DL niches in vivo.
The importance of Notch-induced survival and proliferation throughout the early stages of αβ T cell development likely explains why activating Notch1 mutations are found in >50% of T cell acute lymphoplastic leukemias (). In future work, it will be important to identify the targets of Notch1 at each developmental stage and to determine how Notch signaling interacts with other pathways regulating early T cell development, as this may provide new candidates for targeted therapy of T cell leukemia. |
We have characterized the gene expression profile of PPARγ-activated DCs using Affymetrix GeneChips looking for PPARγ- regulated metabolic and signaling pathways. Our global analyses revealed that PPARγ ligand modulates the expression of genes, which participate in lipid metabolism ( [], , ) and lipid antigen presentation (, ) in monocyte-derived DCs (). Unexpectedly, we observed that several genes involved in all-trans retinoic acid (ATRA) biosynthesis () were also up-regulated by treatment of DCs with rosiglitazone (RSG), a synthetic PPARγ activator (). All of the identified retinol dehydrogenases, regulated by RSG treatment, are members of the SDR protein family. Several of them catalyze only the reduction of retinal, i.e., DHRS3 (), whereas DHRS9 and retinol dehydrogenase (RDH)10 are able to catalyze retinol oxidation (, ) and thus may participate in the conversion of retinol to retinal (). The conversion of retinal to ATRA can be catalyzed by retinaldehyde dehydrogenase type 2 (RALDH2), which also appeared to be regulated by PPARγ. These findings prompted us to investigate the relationship of PPARγ, retinoid metabolism, and retinoid signaling in monocyte-derived DCs. First, we validated the enhanced gene expression of retinol dehydrogenases by real-time quantitative PCR (RT-Q-PCR). Elevated levels of mRNA expression of DHRS9 and RDH10 were detected in RSG-treated DCs after 5 d of differentiation (). To further characterize the onset of enzyme expression, we measured mRNA levels at earlier time-points. We reasoned that for a certain enzymatic reaction to contribute to DC differentiation and/or subtype specification it must be induced early during differentiation. Interestingly, only RDH10 was induced as early as 24 h (), suggesting that the PPARγ-regulated component of retinol oxidation might be . We also looked at the expression of the protein product of this gene and found an elevated level of RDH10 enzyme detected by immunohistochemistry (IHC) (). These results suggested that PPARγ-activated cells are likely to acquire an increased retinol to retinal conversion capacity. Our RT-Q-PCR data also revealed that RALDH2 (also known as ALDH1A2), the enzyme responsible for converting retinal to retinoic acid, was up-regulated upon PPARγ activation (). This effect was confirmed by IHC, which showed elevated protein expression of RALDH () using an anti-RALDH antibody, which can detect RALDH1 or RALDH2. It should be noted that the mRNA expression of RALDH1 was very low in monocyte-derived DCs (unpublished data), suggesting that the detected signal is from RALDH2. Collectively, our results indicated that some key proteins, responsible for bioactive retinoid synthesis, are coordinately up-regulated in PPARγ-instructed DCs.
To obtain direct evidence that PPARγ ligand–treated DCs could generate retinoids we determined the intracellular ATRA concentrations by using a sensitive and specific liquid chromatography–mass spectrometry (LC-MS) method (). Untreated DCs produced very low levels of ATRA, but an elevated amount of ATRA was detected in RSG-treated DCs (). This accumulation appeared to be PPARγ dependent, because a PPARγ antagonist (GW9662) blocked it. The estimated ATRA concentration (0.8–1.2 ng/g cell pellet, equivalent to 2–5 nM) was well within the range to activate RARs (). The concentration of all-trans retinol, the precursor of ATRA, was also determined in the cells, and no difference was detected between treated and control samples (20–30 ng/g cell pellet). In addition, a similar retinol concentration was detected in the culture medium (unpublished data). These data strongly suggested that activation of PPARγ leads to ATRA production and potentially to retinoid-regulated gene expression. In other words, it appeared likely that part of the PPARγ-induced changes in gene expression are indeed retinoid mediated. Next, we embarked on evaluating this scenario. The effects of retinoids on gene expression are mediated by nuclear receptors (RARs α, β, and γ and RXRs α, β, and γ) (). We assessed the transcript levels of these receptors in differentiated DCs. We observed that RARα and RXRα were highly expressed, tested both at mRNA () and protein levels (), whereas RXRβ and RARγ were barely detectable. We failed to detect RARβ or RXRγ (unpublished data). These results indicated that the RARα–RXRα heterodimer is likely to be the dominant retinoid receptor in developing DCs. To assess the function of these receptors we monitored the mRNA level of (), a well-established retinoid receptor target gene (). We examined the effects of retinoids in developing DCs by using synthetic and natural RAR agonists. ATRA, a natural ligand of RARs, and AM580, an RARα selective synthetic ligand, strongly induced the expression of TGM2, but the RARγ-specific agonist (CD437) did not (). These findings implied that PPARγ-treated DCs produce ATRA, and endogenous accumulation of this compound might trigger the retinoid response by the activation of the RARα nuclear hormone receptor.
Our data provided several hints that activation of PPARγ induces a retinoid response in human DCs. (a) Key proteins responsible for bioactive retinoid synthesis are up-regulated in PPARγ-instructed DCs. (b) PPARγ ligand–treated cells produce a detectable amount of ATRA. (c) Retinoid target genes, i.e., and (), were induced by PPARγ ligand ( and unpublished data). To assess the contribution of retinoid signaling to the PPARγ response we decided to use a combination of pharmacological activators and inhibitors of these pathways along with an unbiased approach—global gene expression profiling. Cells were treated with the synthetic PPARγ ligand RSG, or with RSG along with the RARα antagonist (AGN193109), to block RARα-mediated gene expression, or the RARα-specific agonists (AM580) alone. This design allows one to determine if retinoid signaling is a downstream event of PPARγ activation and what portion of PPARγ-regulated genes is regulated via induced retinoid signaling. We found that 553 probe sets were significantly changed in a PPARγ-dependent manner (microarray analyses are described in the supplemental Materials and methods, available at ). Datasets are available in the public Gene Expression Omnibus database (accession no. ). We performed K-means clustering to find characteristic gene expression patterns. Six clusters were generated by this analysis (); cluster 1 contains probe sets, which were exclusively PPARγ ligand regulated. The RARα antagonist did not decrease and the RARα agonist did not increase the expression of these genes. In this cluster several established PPARγ-responsive genes such as and were found; in addition, the newly identified target gene was also in this group. Our quantitative PCR analysis confirmed that FABP4 and RDH10 were regulated only by PPARγ agonist (). In cluster 3 (see gene list in Table S1, available at ) gene expression was regulated by both ligands, and the RARα antagonist abolished the effect of the PPARγ ligand. As expected, was in this cluster, but surprisingly the previously characterized PPARγ-regulated gene, (), also fell into this category. These genes appeared to be regulated indirectly by PPARγ via the activation of retinoid signaling. RT-Q-PCR validation of these findings indicated that the gene expression pattern of CD1d was very similar to that of TGM2 (), suggesting that CD1d is indeed regulated in an RARα-dependent manner. We also identified several genes, which were induced independently by either of the two types of ligands (cluster 2). Interestingly, a p450 enzyme, , was also in this group. We have previously analyzed the promoter region of this gene () and have now confirmed that it is regulated by either PPARγ or RAR. We obtained a similar set of expression patterns when we analyzed the down-regulated genes. Most prominently, cluster 6 contains probe sets of genes down-regulated by PPARγ ligand. This effect is abolished in the presence of the RARα antagonist (see gene list in Table S2, available at ). belong to this group, and the expression of was confirmed using RT-Q-PCR. If taken together, our global gene expression profiling data implies that ∼30% of all PPARγ ligand–responsive genes are regulated via the induction of retinoid signaling. Thus, PPARγ-regulated retinoid production significantly contributes to both induced and inhibited gene expression in DCs.
The finding that RARα antagonist abolished PPARγ ligand–induced CD1d expression suggested that the link between PPARγ and retinoid signaling is likely to be at the level of induced retinoid production. If this argument is correct, increased intracellular ATRA levels should correlate with the expression of CD1d in PPARγ ligand–treated DCs. We have performed time course experiments to address this. As shown in , the time course of intracellular ATRA production matches the induction of CD1d, providing further evidence that there is a causative relationship between retinoic acid production and CD1d induction. To gain further mechanistic insights, we used pharmacological means to probe the contribution of enzymatic steps in the regulation of gene expression. We used inhibitors of the enzymatic steps involved in retinoic acid synthesis. Retinal is converted to ATRA by aldehyde dehydrogenases (). This step can be blocked by 4-diethyl amino-benzaldehyde (DEAB) (), an inhibitor of RALDHs. ATRA is inactivated by CYP26, a p450 enzyme. This step can be inhibited by R115866, a specific CYP26 inhibitor (). We used these inhibitors in combination with activators of PPARγ or RARα. As a readout of activation, we measured the expression of the PPARγ target gene and the RARα-regulated genes and . We found that PPARγ ligand–mediated induction of CD1d and TGM2 was completely abolished by RALDH inhibitor (DEAB) treatment. Conversely, administration of R115866 (abbreviated as R115) led to an enhanced expression of CD1d (). At the same time DEAB did not abolish the induction of FABP4, indicating that this compound is not a general inhibitor of PPARγ-induced gene expression. We also examined the effect of RALDH inhibitor on CD1d and TGM2 expression upon retinoid treatment. When cells were cotreated with the synthetic RARα agonist AM580, DEAB had no effect on the induction (). These data provided further support for the argument that endogenously generated ATRA is likely to be responsible for TGM2 and CD1d induction by PPARγ ligands.
One of the unanticipated findings of our global gene expression analyses was that both CD1d and CD1a were regulated by PPARγ in an RAR-dependent manner. Next, we wanted to gain more insight into how retinoic acid and its receptor (RARα) regulated gene expression. First, we compared the time courses of CD1d induction by PPARγ activators and retinoids. We reasoned that if retinoid-induced CD1d expression is downstream of PPARγ, then there must be a shift between the two time courses. It appears to be the case, because activation of RARα induced expression of CD1d already after 1 h, suggesting the involvement of a direct transcriptional event (), whereas PPARγ ligand administration did not up-regulate the expression of CD1d within this time interval as we have previously reported (). This expression pattern is consistent with an indirect mechanism triggered by PPARγ activation. The analysis of CD1d expression, however, is complicated by the fact that freshly isolated monocytes also express large amounts of CD1d; thus, it is difficult to determine whether retinoids elicit a net induction or block the down-regulation of CD1d. To circumvent this issue we added the activators after 12 h of DC differentiation (when CD1d expression is already low). Significantly, we detected an early and robust induction of CD1d by retinoids (). In contrast, to this prompt effect, PPARγ ligand failed to activate this gene after 2 h. To see if retinoids have similar effects in cell types other than monocyte-derived DCs we extended our studies into other DC models. We characterized the expression of CD1d in blood DCs. We isolated CD1c myeloid DCs from monocyte-depleted PBMCs (). Cultured blood DCs express low amount of CD1d, but CD1d was highly up-regulated upon retinoid (AM580) treatment (). PPARγ activator was also effective albeit much less potent. Based on the evidence presented here, we propose that the effect of retinoids on CD1d gene expression is likely to be a “proximal” direct transcriptional event and the PPARγ-dependent regulation of CD1d is indirectly mediated through the activation of retinoid signaling.
We and others have recently shown that the expression of the CD1 gene family is coordinately regulated by PPARγ activators. PPARγ ligand–treated DCs express a reduced level of CD1a, whereas the level of CD1d is increased upon ligand treatment (, ). Our results presented here argued strongly for the possibility that some of the regulation on CD1 expression is mediated by retinoid signaling. Therefore, we decided to compare the effects of retinoids and PPARγ activators on the expression of cell surface molecules of DCs. We sought to characterize the retinoid-regulated events and compare those to PPARγ-regulated processes. First, we looked at the cell surface expression of CD1a and CD1d. Consistent with the mRNA expression pattern membrane, CD1d was up-regulated, whereas CD1a was down-modulated by both RARα and PPARγ ligands (). Importantly and also consistent with the mRNA expression pattern, we observed that RSG-elicited induction of CD1d was abolished by a RALDH inhibitor (DEAB) (). Next, we looked at the functional consequences of retinoid-regulated gene expression. CD1d can present lipid antigens to a specific T cell subtype, iNKT cells, which have the potential to regulate both inflammatory and antiinflammatory responses through the rapid secretion of cytokines (). We assessed the ability of DCs to induce iNKT cell proliferation in autologous mixed leukocyte reaction (MLR) cultures. DCs were loaded with a synthetic CD1d ligand (α-GalCer) () for 24 h followed by a co-culture with autologous PBMCs for 5 d. We found that either RARα or PPARγ activation leads to a DC subtype, which has an increased capacity to promote the expansion of iNKT cells (). In addition, we also loaded the cells with galactosyl(a1-2) galactosyl-ceramide (GGC), a lipid precursor of the α-GalCer antigen, which requires delivery to the lysosomes where it is hydrolyzed and converted to the active glycolipid (α-GalCer) (). PPARγ or RARα receptor agonist–treated DCs elicited an enhanced iNKT expansion when the cells were loaded with this precursor of α-GalCer (), indicating that activation of these pathways enhance the lipid antigen-presenting capacity independent of processing and endosomal loading. To provide further evidence that ligand-instructed DCs could activate iNKT cells we measured INFγ secretion of purified iNKT cells, activated by α-GalCer–loaded DCs, by ELISPOT analysis. We found that both RARα and PPARγ ligand–treated DCs trigger an enhanced INFγ response of iNKT cells (). Our results demonstrated that both PPARγ- and RARα-specific ligand–treated DCs express CD1d that is able to present α-GalCer for iNKT cells to elicit their activation. It should be noted that ligand-treated DCs without exogenous glycolipid loading failed to elicit any iNKT cell expansion ().
Besides presentation of lipid antigens, DCs are highly active in the presentation of processed peptides by MHC class I and/or class II proteins. The question remained whether activation of either the PPARγ or the RARα pathways had an effect on MHC-mediated T cell activation. PPARγ ligand–activated immature DCs exhibit an elevated expression of MHC class II molecules (). We confirmed these results and extended them by phenotyping retinoid (ATRA or AM580)-treated cells in combination with measuring MHC class I (HLA-ABC) membrane expression. Retinoid-treated cells showed a similar HLA-DR expression pattern to PPARγ-activated cells. Both ligands stimulated the cell surface expression of HLA-DR, suggesting that these cells might have a generally enhanced capacity to present peptide antigens (Fig. S1 A, available at ). In contrast to CD1s, the cell surface expression of MHC II was highly up-regulated upon maturation/activation of DCs (), and it was also suggested that retinoid-treated DCs had a mature/activated phenotype (, ). Consistent with this finding we also found that in immature DCs the cell surface expression of HLA-DR was increased upon retinoid treatment, but the bona fide maturation marker (CD83) was not detected (unpublished data). It is possible that enhanced HLA-DR expression is just a marker of the activation state of these cells, and its expression can be further elevated by proinflammatory cytokines. Indeed, we found that mature DCs (MDCs) exhibited elevated expression of HLA-DR, which was not modified by retinoids (Fig. S1 B). We also investigated the cell surface expression of HLA-ABC and found that both immature and mature DCs express high levels of MHC class I molecules not modified by ligand treatment. To see if activation of RARα had any effect on DC-mediated T cell activation we compared the intensity of MLR reactions induced by mature DCs generated with or without RARα or PPARγ agonist. We failed to observe any change in the level of MHC-dependent T cell activation using control or ligand-treated MDCs in an allogeneic MLR test (Fig. S1 C). We concluded that activation of these nuclear receptors did not confer enhanced antigen-presenting capacity per se, but the activated cells acquired a selectively enhanced NKT cell activating capacity caused by elevated levels of membrane CD1d accompanied by decreased CD1a expression.
Our studies uncovered an interrelated network of lipid signaling processes regulating DC gene expression and function including lipid antigen presentation. An intriguing question remained, however, whether natural sources of PPARγ activators such as oxidized low density lipoprotein (oxLDL) or other lipids () were able to elicit retinoid signaling in DCs, providing a biological context and significance for these mechanisms. To test this hypothesis differentiating DCs were treated with oxLDL alone or in combination with RAR antagonist or RALDH inhibitor, and the expression pattern of retinoid and PPARγ-regulated genes was measured. oxLDL treatment induced the expression of CD1d, TGM2, and FABP4 (). Treatment with the RAR antagonist and RALDH inhibitor blocked the up-regulation of TGM2 and CD1d, suggesting that retinoid signaling was involved, but it did not block the up-regulation of FABP4. It is important to note, however, that lipoproteins besides PPARγ activators might also contain retinyl-esters or other retinoid precursors (, ), and these compounds might participate in the activation of retinoid signaling independent of the activation of the PPARγ receptor. To test this we also analyzed the retinoid content of oxLDL by LC-MS. Although we could detect various retinyl-esters, only trace amounts of retinol was found and no ATRA was detected in 100 mg of oxLDL. Our lipid analyses also indicated that several oxidized retinoid species were present (unpublished data). These data imply that oxLDL is likely to contain retinoic acid precursors that can be converted to ATRA inside cells and elicit retinoid signaling. The conversion of this (these) precursor(s) are also, at least in part, under the control of PPARγ. In addition, we have previously defined a serum condition (human AB serum), which either contains and/or induces endogenous PPARγ ligands/activators (). Next we tested if this source of PPARγ activators induced retinoid signaling. In DCs cultured in the presence of human AB serum an elevated level of FABP4, TGM2, and CD1d was detected (). The PPARγ-specific antagonist (GW9662) efficiently decreased the transcript level of all of these genes. Importantly, administration of RAR antagonist or RALDH inhibitor (DEAB) diminished the expression of TGM2 and CD1d but not that of FABP4 (). These experiments collectively demonstrated that activation of PPARγ by biologically relevant normal or pathological serum lipids can also elicit a retinoid response, and it appears likely that in vivo these lipids might be relevant sources of CD1d regulation.
italic
#text
Cells were treated with the following ligands: AM580 (Biomol), rosiglitazone and GW9662, (Alexis Biochemicals), and AGN193109 (a gift from R.A.S. Chandraratna, Allergan Inc., Irvine CA). oxLDL was obtained from Intracel, R115866 was obtained from Janssen, DEAB was obtained from Fluka, and α-GalCer was obtained from Kirin Brewery Ltd. GGC was described previously ().
Monocytes (98% CD14) were obtained from Buffy coats by Ficoll gradient centrifugation and magnetic cell separation using anti-CD14–conjugated microbeads (VarioMACS; Miltenyi Biotec). DCs were prepared as described previously () with minor modifications. In brief, monocytes were resuspended into six-well culture dishes at a density of 1.5 × 10 cells/ml and cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 10% FBS (Invitrogen) containing 800 U/ml GM-CSF (Peprotech) and 500 U/ml IL-4 (Peprotech). In some experiments, FBS was replaced with human AB serum (Sigma-Aldrich). Cells were cultured for 5 or 6 d, and the IL-4 and GM-CSF addition was repeated at day 3. Ligands or vehicle control (50% DMSO/ethanol) were added to the cell culture starting from the first day or otherwise indicated. AGN193109 administration was always repeated at day 3. To obtain MDCs, DCs were treated with the following mix of cytokines for 24 h: 10 ng/ml TNF-α, 10 ng/ml IL-1β, 1,000 U/ml IL-6 (Peprotech), and 1 μg/ml PGE (Sigma-Aldrich).
Peripheral blood myeloid DCs were magnetically isolated with the CD1c (BDCA-1) Dendritic Cell Isolation kit (Miltenyi Biotec) from monocyte-depleted PBMCs. Blood myeloid DCs (10 cells/ml) were cultured for 2 d in RPMI 1640 supplemented with 10% FBS.
Monocytes, DCs, or RSG-treated DCs (6 × 10 cells/group) were pelleted and fixed in 4% paraformaldehyde (pH 7.3) for 24 h at 4°C. Cell blocks were then embedded in paraffin followed by serial sectionings (4 μm thick). After deparaffinization and dehydration, serial sections from each cell group, mounted on the same glass slides, were used for peroxidase-based indirect IHC. In brief, sections were treated with 3% HO in methanol for 15 min at room temperature to block the endogenous peroxidase. For antigen unmasking, sections were heated in antigen-retrieving citrate buffer (pH 6.0; Dako) for 2 min at 120°C using a pressure cooker. IHC stainings were with the standard ABC technique using the primary antibody-specific biotinylated secondary antibodies (Vectastain kits; Vector Laboratories). After blocking the nonspecific binding sites, sections were incubated with the primary antibodies for 1 h at room temperature before using the biotinylated secondary antibodies. The peroxidase-mediated color development was set up for 5 min using the VIP substrate (Vector Laboratories). Finally, the sections were counterstained with methylgreen. The following antibodies were applied: rabbit antibody to RDH10 () in a dilution of ×1/75 and goat antibody to ALDH1A1 (RALDH1, ab9883; Abcam) in a dilution of ×1/75.
20 μg protein whole cell extract was separated by electrophoresis in 12.5% polyacrylamide gel and then transferred to PVDF membrane (Bio-Rad Laboratories). Membranes were probed with anti-RARα (2ZH1920L; Perseus Proteomics) or anti-RXRα (2ZK8508H; Perseus Proteomics) antibodies, and then the membranes were stripped and reprobed with anti-GAPDH (ab8245-100; Abcam) according to the manufacturer's recommendations.
MDCs were collected, extensively washed, and used as stimulator cells for allogeneic PBMCs (2 × 10 cells/well). Stimulator cells were added in graded doses to the T cells in 96-well flat-bottom tissue culture plates. Cell proliferation was measured on day 5 by a 16-h pulse with [H]-thymidine (1 μCi/well; BD Biosciences).
Cell staining was performed using FITC- or PE-conjugated mAbs. Labeled antibodies for flow cytometry included anti–CD1a-PE, anti–CD1d-PE, anti–HLA-DR-PE, and isotype-matched controls (BD Biosciences), anti–Vβ11-PE and anti–Vα24-FITC (Immunotech), and anti–HLA-ABC-FITC (W6/32; Abcam). The cells were assessed for fluorescence intensity using EPICS Elite flow cytometer (Beckman Coulter) or with FACS Calibur cytometer (Becton Dickinson).
DCs were treated with 100 ng/ml α-GalCer (or with 100 ng/ml GGC) for 24 h to obtain α-GalCer–loaded DCs. α-GalCer–pulsed DCs (10 cells) were cocultured with 10 monocyte-depleted autologous PBMCs for 5 d in 24-well plates. The expansion of iNKT cells was monitored by quantifying Vα24 Vβ11 cells by FACS analysis.
Monocyte-depleted human PBMCs were incubated with anti–Vα24-FITC (Immunotech) for 20 min at 4°C, cells were washed, anti–mouse immunoglobulin microbeads were added, and immunomagnetic separation was conducted according to the manufacturer's recommendations (VarioMACS; Miltenyi Biotec).
α-GalCer–pulsed DCs (10 cells) were cocultured with 10 freshly isolated iNKT cells for 16 h in 96-well human INFγ ELISpot plates (R&D Systems). ELISpot plate development was performed according to the manufacturer's recommendations.
Concentrations of ATRA and retinol were measured in cell pellets by our LC-MS method described in detail previously (). In brief, 50–100 mg of cell pellet was diluted with a threefold volume of isopropanol, and the extracts were dried in a concentrator (Eppendorf 5301) at 45°C. The dried extracts were resuspended with 60 μl of methanol, diluted with 40 μl of 60 mM aqueous ammonium acetate solution, transferred into the autosampler, and subsequently analyzed with an LC-MS system. The LC-MS system consisted of a Waters 2695XE separation module, MS-MS detector (Micromass Quattro micro QAA0029; Waters), including an APCI ionising option (Ion sabre APCI; Waters).
Total RNA was isolated using Trizol Reagent (Invitrogen) and further purified by using the RNeasy kit (Qiagen). cRNA was generated from 5 μg of total RNA by using the SuperScript Choice kit (Invitrogen) and the High Yield RNA transcription labeling kit (Enzo Diagnostics). Fragmented cRNA was hybridized to Affymetrix (Santa Clara) arrays (HU133 Plus 2.0) according to Affymetrix standard protocols. Preliminary data analysis was performed with GCOS software (Affymetrix). Further analysis was performed using GeneSpring 7.2 (Agilent). Microarray data analyses are described in detail in the supplemental Materials and methods.
Total RNA was isolated using TRIZOL reagent (Invitrogen). Reverse transcription was performed at 25°C for 10 min, 42°C for 2 h, and 72°C for 5 min from 100 ng of total RNA using Superscript II reverse transcriptase (Invitrogen) and random primers (3 μg/μl; Invitrogen). Quantitative PCR was performed using real-time PCR (ABI PRISM 7900; Applied Biosystems): 40 cycles at 95°C for 12 s and at 60°C for 30 s using Taqman assays. All PCR reactions were done in triplicates with one control reaction containing no RT enzyme. The comparative Ct method was used to quantify transcripts and normalize to 36B4 or cyclophilin A. Values are expressed as mean ± SD of the mean. Where indicated significant differences between mean values were evaluated using two-tailed, unpaired Student's t test. In , Taqman qPCR low density arrays (TLDA; Applied Biosystems) were used to quantify the expression of RXR and RAR genes in DCs according to the manufacturer's instructions. The sequences of the primers and probes are described in the Table S3 (available at ).
Microarray data analyses are described in detail in the supplemental Materials and methods. Tables S1 and S2 show gene expression data obtained by Affymetrix GeneChip analyses on genes clustered into cluster 3 and 6, respectively. Table S3 contains the sequences of the primers and probes for RT-Q-PCR. Fig. S1 is a comparison of the expression of MHC I and II molecules and T cell activation capacity of retinoid and PPARγ ligand–activated DCs. Online supplemental material is available at . |
Selection of NKT cells occurs through interaction with CD1d molecules presenting endogenous GSLs (). The aberrant accumulation of GSLs in the lysosome in storage diseases therefore has the potential to negatively affect this process. We have investigated several GSL storage disease models that differ in terms of their primary etiology and store GSLs from different branches of the GSL catabolic pathway () ().
We determined the percentage of NKT cells in the thymus, spleen, and liver of different GSL storage mice and controls using CD1d tetramers loaded with the synthetic NKT cell ligand α-GalCer. The organ-specific flow cytometry data are summarized for all models tested in and Fig. S1 (available at ). In the mouse models of Sandhoff, GM1 gangliosidosis, Fabry, late-onset Tay-Sachs disease (LOTS), and NPC1, there was a significant reduction in the percentage of NKT cells across all organs tested (ranging from a 50 to 90% reduction, depending on the model and organ; ). Tay-Sachs mice showed a reduction in liver but not in the spleen or thymus. This mouse model has low levels of GM2 and GA2 storage relative to the other models and a normal life expectancy (). We confirmed these results by examining NKT cells as a percentage of total lymphocytes and the total cell number from the spleen and thymus from Sandhoff, GM1 gangliosidosis, NPC1, and Fabry mice. These data show a reduction in the NKT cells as both a percentage of total lymphocytes and as absolute numbers in each model tested (Fig. S1). In contrast, the NPC1 thymic NKT cell population was larger than that of other control animals. The percentage of NKT cells detected in the liver of NPC1 control mice (NPC1), which are on a BALB/c background, was reduced relative to the controls of the other models that are on a C57BL/6 background. However, there was no difference in terms of absolute NKT cell numbers relative to control strains (Fig. S1). Additionally, a bias toward a CD4 phenotype was observed in intrasplenic NKT cells of the genetically identical Tay-Sachs and LOTS mice but not in the genetic background control mice (unpublished data).
To determine whether the in vivo function of NKT cells is diminished in GSL storage mice, we examined the NKT-dependent response to injection of α-GalCer. Injection of α-GalCer in mice rapidly induces secretion of several different cytokines, including IL-4 and IFN-γ, into the serum (). Although both IL-4 and IFN-γ showed normal profiles in control mice treated with α-GalCer, Sandhoff and NPC1 mice (as well as Fabry mice; unpublished data) failed to produce detectable levels of either cytokine (). Furthermore, in vivo cytotoxicity assays () demonstrated that the elimination of α-GalCer–pulsed and C20:2 analogue–pulsed wild-type targets by NKT cells were severely reduced in the Sandhoff homozygote compared with control recipients (). These data are consistent with the low frequency of NKT cells detected in these mouse models and indicate that residual NKT cells are capable of antigen-specific lysis in vivo. Owing to surface loading of C20:2 onto CD1d (), this ligand is efficiently presented by splenocytes, which results in an increase in killing efficiency observed in both wild-type and Sandhoff mice. Consistent with these observations, we demonstrated that residual NKT cells from Sandhoff and GM1 gangliosidosis mice were capable of releasing IFN-γ in ELISPOT assays when stimulated by α-GalCer–pulsed bone marrow–derived DCs (BMDCs) from wild-type mice (Fig. S2, available at ). These data were confirmed by ELISA (unpublished data). Given these data, it is unlikely that the residual NKT cells have an inherent functional defect, but additional experiments need to be done to address this question more completely.
To further investigate the possible reasons for the reduced NKT cell frequencies in GSL storage mice, we measured the cell surface CD1d expression on thymocytes and splenic B cells in GSL storage models and age-matched controls. Cell surface expression of CD1d molecules, particularly in the thymus, was not substantially altered in storage disease mice (; and not depicted). The only exceptions were a 40% reduction in the thymus and a 20% reduction in the spleen of NPC1 mice, and a small increase in the thymus in Fabry mice ().
To determine whether storage disease mice had a generalized defect in APC numbers that could contribute to the NKT cell deficiency, we analyzed B cell (unpublished data), macrophage, and DC frequencies in these animals (). There was a small increase in the splenic macrophage population in Sandhoff, GM1 gangliosidosis, and Fabry mice, whereas the DCs were slightly reduced in Sandhoff and GM1 gangliosidosis mice. In the thymus, there were only subtle differences in APC frequencies between GSL storage models and controls (). The percentage of cortical thymocytes was generally unaffected in younger Sandhoff, GM1 gangliosidosis, and NPC1 mice, whereas a loss in NKT cells in both Sandhoff and NPC1 mice was already observed at this age (unpublished data). Therefore, we conclude that the deficiency of NKT cells in lysosomal GSL storage diseases is not caused by defective CD1d expression in either the thymus or periphery or a lack of CD1d APCs.
Processing and presentation of peptides onto MHC class II molecules is dependent on functional late endosomes/lysosomes (). Therefore, lipid storage has the potential to disrupt these processes. We tested the capacity of Sandhoff mice to generate functional CD4 T cell responses to a model antigen, OVA. The CD4 T cell responses against two different I-A–binding OVA peptides, OVA () and OVA (not depicted), were measured in an IFN-γ ELISPOT assay in Sandhoff and control mice. Mice were either 6 wk (before onset of neurological signs) or 10 wk of age (when the animals have a neurodegenerative phenotype). Strong OVA-specific responses of similar magnitude in Sandhoff mice (6 and 10 wk of age) and age-matched controls were detected after prime-boost immunization with OVA. These data rule out any impairment of OVA processing and presentation via MHC class II molecules. Although both Fabry () () and GM1 gangliosidosis mice made class II–restricted responses, NPC1 mice had a reduced ability to generate anti-OVA responses (not depicted). Furthermore, the proportion of thymic CD4 T cells in Sandhoff and control mice were similar, which was consistent with unimpaired H-2–dependent thymic selection (unpublished data).
Different maturation stages of NKT cells have recently been defined in mice (–). After positive selection (), α-GalCer/CD1d–specific thymocytes bearing the invariant TCR α chain (TCR Vα14-Jα18) sequentially express CD44 and NK1.1 molecules, respectively. To investigate whether GSL storage affects thymic development of NKT cells, we analyzed the populations of NKT cells at different stages of thymic maturation in Sandhoff and GM1 gangliosidosis mice and controls (). The different NKT cell populations observed in age-matched control mice were similar to previous reported data (). The majority of thymic NKT cells were of the “mature” CD44/NK1.1 phenotype, with subpopulations of the “semimature” CD44/NK1.1 and “immature” CD44/NK1.1 cells. All three maturation stages could be clearly identified in both strains of mice (percent of gated tetramer/TCR cells; ). However, both Sandhoff and GM1 gangliosidosis mice exhibited a striking reduction in absolute numbers of α-GalCer/CD1d–specific thymocytes at all three stages of development studied (), culminating in the most significant reduction at the mature CD44/NK1.1 stage. These results are consistent with impaired thymic-positive selection of NKT cells caused by GSL storage.
To further analyze whether thymocytes, necessary for the positive selection of NKT cells (), display inappropriate GSL accumulation in Sandhoff mice, we performed quantitative analysis of the levels of GSL storage in total thymus. As shown in , the levels of GM2 and GA2 were significantly elevated in 10–12-d-old Sandhoff thymus compared with age-matched controls, whereas the level of GM3 was reduced. As expected, the levels of GM1a, GM1b, and GA1, not substrates for β-hexosaminidase A/B, were unchanged.
It is known that macrophages accumulate considerable levels of GSLs within late endosomes/lysosomes in Sandhoff disease (unpublished data). Therefore, it was possible that the storage of GSLs observed in the thymus () was caused by resident macrophages and not thymocytes. To determine whether GSLs are also stored in thymocytes, we examined the size/number of lysosomes in thymocytes using LysoTracker staining. (). We found that the relative fluorescence intensity of staining was significantly elevated in Sandhoff thymocytes compared with wild-type controls. An increase in the number and/or size of lysosomes within these cells suggests that thymocytes are storing GSLs. Collectively, these data suggest that accumulation of GSLs within late endosomes/lysosomes in thymocytes may impair selection of NKT cells in Sandhoff mice.
To further examine the mechanism for the loss of NKT cells in the different GSL storage disease mice, we performed experiments using α-GalCer and an analogue that requires endosomal processing to stimulate NKT cells (, ). This was done to determine whether APCs from GSL storage disease mice had a defect in the capacity to load CD1d with ligands in the lysosome.
Sandhoff, GM1 gangliosidosis, Fabry, or NPC1 splenocytes or BMDCs were pulsed with α-GalCer (; and not depicted) and used to stimulate an NKT cell hybridoma (DN32-D3), or left unpulsed and used to stimulate a control, CD1d-restricted, noninvariant NKT cell hybridoma (TCB11; ). Splenocytes from both Sandhoff and GM1 gangliosidosis mice appeared to be defective in presenting α-GalCer to the DN32 hybridoma compared with wild-type controls (). Interestingly, the defect in ability to present α-GalCer observed with Sandhoff and GM1 gangliosidosis splenocytes is not apparent when the APCs used were cultured BMDCs ().
We also examined the capacity of APCs from GSL storage disease mice to process and present an analogue of α-GalCer that is strictly dependent on lysosomal processing, galactosyl(α1→2) galactosylceramide (Gal(1→2)GalCer) (, ). This analogue has a second galactose group linked to the primary galactose of α-GalCer and requires processing by α-galactosidase (deficient in Fabry disease) within the lysosome, before recognition in the context of CD1d can occur. It has previously been shown that Fabry mice are unable to process and present Gal(1→2)GalCer ().
These experiments were performed using splenocytes () or BMDCs () from Sandhoff and GM1 gangliosidosis mice, as well as NPC1 mice (unpublished data). The response of the DN32 hybridoma to splenocytes or BMDCs from Sandhoff and GM1 gangliosidosis mice pulsed with Gal(1→2)GalCer was reduced compared with wild-type controls but was greater than that seen with CD1d APCs. Similarly, NPC1 mice had a dramatically reduced capacity to present Gal(1→2)GalCer (unpublished data).
#text
The following mouse models of GSL storage (C57BL/6 background) were maintained and genotyped according to published methods: Tay-Sachs
(); LOTS
(); Sandhoff
(); Fabry
(); and GM1 gangliosidosis
(). Each mouse strain had been backcrossed at least eight times before use. LOTS mice are female Tay-Sachs mice that have been repeatedly bred before 6 mo of age (). Tay-Sachs (nonbred) mice are asymptomatic because of the presence of a bypass pathway (the combined effects of sialidase and hexosaminidase B). Pregnancy induces down-regulation of components of the bypass pathway, causing higher levels of storage relative to the Tay-Sachs mouse and clinical presentation in 100% of LOTS mice. NPC1 mice () are on a BALB/c background. Also used were mice lacking the Jα18 TCR gene segment (), which were devoid of Vα14 NKT cells while having other lymphoid cell lineages intact (NKT mice), and CD1d knockout mice (
) (), which were also devoid of Vα14 NKT cells. Heterozygote littermates and age-matched C57BL/6 or NPC1 mice, as appropriate, were used as controls. All mice were maintained in the Biological Services Unit, Department of Biochemistry, University of Oxford and used according to established University of Oxford institutional guidelines under the authority of a UK Home Office project license.
Intrahepatic mononuclear cells were separated from mouse livers according to the following protocol: livers were cut into small pieces using a scalpel, passed through a 100-mm metal mesh filter, washed twice with PBS, layered over Ficoll-Hypaque gradients, and centrifuged at 2,000 rpm at room temperature for 20 min. An analogous procedure was used to separate splenic and thymic mononuclear cells. Before staining for FACS, both intrahepatic and splenic mononuclear cells were incubated for 10 min at room temperature with 20 μg of unconjugated anti-FcR antibody (BD Biosciences). For all FACS staining experiments, three to five animals per group were used.
Cell suspensions were stained according to published methods (). In brief, 10 cells in 50 μl FACS buffer (PBS containing 1% bovine serum albumin and 0.02 M sodium azide) were incubated on ice for 30 min with monoclonal antibodies (all from BD Biosciences) or α-GalCer/CD1d tetramer (), followed by two washes in FACS buffer. The antibodies used were R-phycoerythrin (RPE)–conjugated rat anti–mouse CD1d (CD1.1, Ly-38), FITC-conjugated rat anti–mouse CD19 (1D3), hamster anti–mouse CD11c (HL3), and CD68. Allophycocyanin-conjugated streptavidin (Phykolink) and RPE-conjugated Extraavidin (Sigma-Aldrich) were used for the generation of CD1d tetramers. CD1d tetramers were generated as previously described (). Propidium iodide was used to gate out dead cells. Quantitation of binding sites was performed using fluorescent microbead standards according to published methods (). To analyze the maturation phenotype of NKT thymocytes, cells were stained with CD1d/α-GalCer tetramer–PE, NK1.1-PerCP, pan Vβ–FITC, and CD44-allophycocyanin (BD Biosciences). To analyze lysosomes, 10 cells from 10–12-d-old Sandhoff and control thymi were stained in 200 μl 200 nM LysoTracker-Green (Invitrogen) for 10 min at room temperature. After washing, the cells were analyzed by flow cytometry gating on thymocytes by forward and side scatter. Percentages of APCs were analyzed by staining cells from different tissues with CD11c and CD68 antibodies.
Animals were injected i.v. with 1 μg α-GalCer (dissolved in a vehicle solution of 0.5% Tween 20/PBS) or vehicle diluted in PBS. Blood samples were collected at the time points indicated in the figures, and serum levels of IL-4 and IFN-γ were determined using cytokine-specific capture ELISAs. Antibodies used for ELISAs were obtained from eBioscience and Pierce Chemical Co.
Target splenocytes were pulsed with 5, 0.5, and 0.05 μg/ml α-GalCer or C20:2 analogue for 2 h at 37°C and labeled with different concentrations (1.65, 0.3, and 0.07 nM) of CFSE (Invitrogen), as previously described (). A control vehicle-pulsed population was labeled with 10 μM chloromethyl-benzoyl-aminotetramethyl-rhodamine (CMTMR; Invitrogen). An equal mixture of the four populations of splenocytes was injected i.v. at 10 cells/mouse into Sandhoff homozygote mice, age-matched littermate controls, and NKT mice ( = 3–5 mice/group). 48 h after injection, blood samples were examined for the presence of fluorescent cells by flow cytometry (FACSCalibur; BD Biosciences). Data are expressed as percent survival of α-GalCer– or analogue-pulsed splenocytes compared with unpulsed controls. Mean percent specific lysis (±SEM) of α-GalCer–pulsed splenocytes compared with unpulsed controls was calculated by the following formula: 100 − ([pulsed/unpulsed] × 100).
Sandhoff homozygotes (6 and 10 wk of age) or other GSL storage mice and age-matched controls were immunized s.c. with 100 μg chicken OVA protein (Sigma-Aldrich) in CFA (Sigma-Aldrich). 11 d later, the mice were injected i.v. with 2 × 10 PFU/mouse UV-inactivated recombinant vaccinia virus encoding full-length chicken OVA. After 7 d, immune responses were assessed by performing ELISPOT using a mouse IFN-γ ELISPOT kit (Mabtech) and 10 μM of two different I-A–restricted OVA peptides (synthesized in house), OVA (TEWTSSNVMEERKIKV) and OVA (ISQAVHAAHAEINEAGR), according to the manufacturer's protocol.
Thymi from 10–12-d-old Sandhoff mice and controls were homogenized in 0.5 ml distilled water, and GSLs were extracted using the Svernnerholm method (). The equivalent of 200 μg protein was treated with ceramide glycanase (Calbiochem-Novabiochem). Released glycans were labeled with anthranillic acid (Sigma-Aldrich) and analyzed by HPLC as described previously ().
APCs used were either BMDCs, cultured for 7 d in the presence of 20 ng/ml GM-CSF/IL-4 (PeproTech) and matured from day 6 of culture with 10 ng/ml TNF-α (PeproTech) or splenocytes. APCs were pulsed with α-GalCer, Gal(1→2)GalCer, or vehicle for 6 h, washed, and 5 × 10 splenocytes or 5 × 10 BMDCs were added to 96-well flatbottom plates. The DN32-D3 NKT cell hybridoma or the control, CD1d-restricted, non–NKT cell hybridoma (TCB11) from A. Bendelac (University of Chicago, Chicago, IL) and S. Porcelli (Albert Einstein School of Medicine, New York, NY) were added (5 × 10) to each well. Cells were cultured for 18–24 h at 37°C, and the supernatant was analyzed for the presence of IL-2 by ELISA (antibodies used were anti–mouse IL-2 JES6-1A12 and biotinylated JES6-5H4; eBioscience).
In Fig. S2, the ability of iNKT cells from storage disorder mice to mount a cytokine response was assayed by performing an overnight IFN-γ ELISPOT using T cell–enriched splenocytes incubated with BMDCs loaded with α-GalCer. Splenocyte cell preparations were enriched for T cells by incubating for 1 h at 37°C, adhering out the macrophages. BMDCs derived from wild-type mice were pulsed for 3 h with 5 μg/ml α-GalCer. Online supplemental material is available at . |
Previous studies have demonstrated that the exposure of iDCs generated in vitro from healthy donors to various strains of HIV-1 or to HIV-1–Gp120 envelope before exposure to maturation stimuli results in the impairment of several mDC functions despite the normal expression of surface markers associated with a mature phenotype (–). On the basis of these studies, we sought to determine whether iDCs obtained from HIV-1–infected patients expressed normal levels of those surface markers that usually define an mDC when exposed to strong maturation stimuli. The evaluation of CD40, CD80, CD83, CD86, MHC-I, and MHC-II surface expression on in vitro–generated iDCs after 24 h in culture with LPS (the time frame within which maturation occurs) did not reveal substantial differences among viremic and aviremic HIV-1–infected patients or uninfected individuals (Fig. S1, available at ). Similar experimental results were obtained when triggering the maturation of iDCs with sCD40 ligand (unpublished data).
Despite the expression of cell surface markers associated with a mature phenotype, we found that mDCs derived from viremic patients exhibited several functional defects that could potentially impair the mDC-mediated activation of NK cells. In vitro matured DCs from viremic HIV-1–infected subjects secreted a markedly lower amount of IL-10 as well as IL-12, which is an important cytokine for the activation of NK cells (–, ), compared with mDCs of HIV-1–infected aviremic patients who were receiving ART (for IL-10) or with healthy donors (for IL-10 and -12; ). However, the production of IL-15 and -18 by mDCs did not substantially differ among the three study groups (unpublished data).
To determine the capacity of mDCs to activate immunoregulatory functions of NK cells, we measured the amount of IFN-γ secreted by NK cells in the presence of autologous mDCs. As shown in , resting NK cells from HIV-1–infected aviremic patients released IFN-γ in amounts comparable with those from uninfected donors. In contrast, freshly purified NK cells cocultured with autologous mDCs derived from viremic individuals showed a statistically significant lower secretion of IFN-γ (P < 0.001). Moreover, within the same group of viremic individuals, the ability to secrete IFN-γ was markedly lower (P < 0.001) in freshly purified CD56 NK cells compared with CD56/CD16 (CD56) NK cells (unpublished data). We could not detect substantial differences in the levels of TNF-α and GM-CSF secreted by NK cells or NK cell subsets primed with autologous mDCs among the three study groups (unpublished data).
As previously described, an important function of mDCs is to trigger the proliferation/activation of autologous resting NK cells (–, ). In coculture experiments, the ability of mDCs generated from viremic patients to induce the proliferation of autologous NK cells was significantly diminished compared with that of either HIV-1–infected aviremic individuals (P < 0.001) or uninfected donors (P < 0.001; ). This result was not likely caused by an inherent defect in the proliferative capacity of NK cells isolated from viremic patients because these cells displayed a normal proliferation in response to mDCs derived from aviremic HIV-1–infected or healthy subjects in heterologous cocultures. Furthermore, NK cells isolated from all HIV-1–infected or uninfected subjects exhibited significantly lower rates of proliferation when cultured with heterologous mDCs generated from viremic subjects compared with proliferation rates triggered by exposure to heterologous mDCs generated from healthy donors (P < 0.001) or HIV-1–infected aviremic individuals (P < 0.001; ). Therefore, these coculture experiments confirm that the defective NK cell proliferation observed in HIV-1–infected viremic patients was not caused by a primary impairment of NK cell responsiveness but by an impairment of the triggering of NK cells by mDCs.
To address whether mDCs had any role in the expansion of the CD56 NK cell subset, we analyzed the distribution of CD56 and CD16 on the surface of NK cells cocultured with heterologous mDCs. Under all combinations of DC and NK cell cocultures, mDCs were not able to modify the relative distribution of NK cell subsets. In particular, heterologous cocultures of DCs generated from HIV-1–infected viremic patients and NK cells purified from healthy donors did not result in the expansion of the CD56 NK cell subset (unpublished data).
A recent study showed that iMDDCs from HIV-1–infected patients are relatively resistant to killing by autologous NK cells (). To ascertain whether this phenomenon pertains to all HIV-1–infected individuals or is restricted to those with high levels of chronic HIV-1 viremia, we analyzed the baseline lysis of autologous iDCs by rIL-2–activated, unfractionated NK cells isolated from HIV-1 viremic and aviremic patients. The degree of NK cell–mediated lysis of autologous iDCs was calculated using the traditional Cr release assay and was also visualized by fluorescence microscopy (Fig. S2, available at ). Spontaneous killing of autologous iDCs by activated NK cells isolated from aviremic patients did not substantially differ from that observed with NK cells purified from healthy donors. In contrast, the elimination of autologous iDCs by NK cells isolated from HIV-1–infected viremic individuals was markedly reduced compared with that from either aviremic HIV-1–infected subjects or healthy controls. This result was not likely caused by an increased resistance of iDCs to the elimination by NK cells from HIV-1–infected viremic patients. As shown in , the susceptibility to NK cell–mediated killing of heterologous iDCs isolated from all HIV-1–infected or uninfected subjects did not differ if cocultured with NK cells purified either from aviremic or healthy individuals. Only NK cells purified from viremic patients displayed a markedly defective killing of heterologous iDCs isolated from all HIV-1–infected or uninfected donors.
Using NK cells isolated from viremic individuals, we further characterized the defect in NK cell–mediated lysis of iDCs by comparing rIL-2–activated CD56- and CD56-derived NK cell subsets for their ability to eliminate autologous iDCs. The iDC killing appeared to be relatively intact for the CD56 NK cell subset but was highly impaired for the CD56 subpopulation (Fig. S2).
For an NK cell to exert lytic activity, an inhibitory signal must be weak or lacking, and an activating signal must be present. The interactions between MHC-I molecules and iNKRs trigger an inhibitory signal and result in the abrogation of NK cell lytic activity even if activating NK cell receptors are triggered. Two major classes of MHC-I–specific surface inhibitory receptors have been identified on NK cells: (a) the killer cell Ig-like receptors (KIRs), which bind HLA/-B/-C alleles, and (b) CD94/NKG2A, which binds to nonclassic HLA-E alleles (–). In this regard, the lysis of autologous iDCs is confined to the subpopulation of NK cells that express CD94/NKG2A and lack KIRs (). Moreover, NKp30, one of the three natural cytotoxicity receptors, has been shown to be primarily responsible for the NK cell–mediated killing of autologous iDCs ().
Under physiological conditions, the maturation of DCs leads to the elevated surface expression of classic and nonclassic MHC-I molecules that render them resistant to NK cell–mediated cytolysis (, , ). As expected, the normal levels of HLA-A/ -B/-C and HLA-E (Fig. S1) expressed by mDCs generated from donors in all three study groups made them similarly resistant to lysis by activated NK cells. Given the importance of interactions between MHC-I and iNKRs in inhibiting the NK cell–mediated killing of autologous mDCs (, , ), the masking of HLA molecules using specific mAbs would theoretically enhance the susceptibility of mDCs to lysis by NK cells in vitro. Despite the complete blocking of all HLA-I alleles with specific mAbs, activated NK cells from viremic patients were still highly impaired in their cytolytic activity against autologous mDCs compared with NK cells from aviremic patients and uninfected individuals. Moreover, the cytotoxicity of the rIL-2–activated CD56-derived NK cell subset against autologous HLA-I–masked mDCs was virtually undetectable compared with that of activated CD56-derived NK cells (). These masking experiments performed on mDCs clearly indicate that NK cells from viremic patients are defective in their lysis of mDCs when the inhibitory effects of MHC-I interactions with KIRs are abolished by masking the MHC-I molecules. Therefore, these data strongly suggest that the defect in iDC lysis of NK cells from viremic patients is at the level of the activating receptors on the NK cell itself.
The surface expression of NKp30 is decreased on fresh and rIL-2–activated NK cells from viremic HIV-1–infected individuals compared with cells from aviremic and uninfected individuals (, ). Therefore, we analyzed the contribution of NKp30 to the cytolysis of autologous iDCs in the three study groups. The masking of NKp30 with a specific mAb induced a statistically significant reduction in the killing of iDCs by activated NK cells derived from both uninfected donors (P < 0.001) and HIV-1–infected patients with undetectable viral load (P = 0.006), confirming the leading role of NKp30 in this process (). As expected, blocking NKp30 had almost no effect in further reducing the already impaired rIL-2–activated NK cell–mediated lysis of iDCs in the cohort of HIV-1–infected viremic individuals because the expression of NKp30 on NK cell surfaces was already very low (, ). Moreover, the masking experiments highlighted that the impairment in NKp30 activity was much greater in the rIL-2–activated CD56-derived subset compared with the CD56-derived NK cell subset (). The reduced ability of the CD56 NK cell subset to lyse autologous iDCs correlated well with its weak NKp30 surface expression ().
It has been reported that the secretion of IL-15 and -12 by mDCs normally plays an important role in triggering the activation of autologous NK cells (). Unlike IL-12, the production of IL-15 by mDCs did not substantially differ among the three study groups, prompting us to test whether NK cells stimulated in vitro with rIL-15 had an increased NKp30 surface expression and NKp30-dependent killing of autologous iDCs. As shown in Fig. S3 (available at ), NK cells cultured in the presence of rIL-15 did not show any considerable increase in NKp30 surface expression on either CD56 or CD56 NK cell subsets compared with freshly purified and rIL-2–activated cells from HIV-1–infected viremic individuals. In contrast, both rIL-2– and -15–activated NK cells expressed markedly higher levels of NKp30 compared with freshly purified NK cells from either HIV-1–infected aviremic patients or normal donors. Consistent with the similar expression of NKp30 on both rIL2– and -15–activated NK cells from each donor within the three study groups, we did not observe differences in the NKp30-dependent killing of autologous iDCs between rIL-2– and -15–activated NK cells from HIV-1–infected patients or healthy donors (Fig. S4).
TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF ligand family that signals apoptosis via the death domain–containing receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). It is primarily expressed as a type II membrane protein and is also secreted in a soluble form (sTRAIL) only by activated T and NK cells. Moreover, in the mouse system, TRAIL contributes substantially to NK cell–mediated killing of iDCs in vivo and plays an important role in antiviral responses (, ). Therefore, we analyzed the surface expression and secretion of TRAIL from NK cells stimulated with rIL-2 for 6 d. The percentage of TRAIL NK cells and the amount of sTRAIL released in the culture supernatant did not substantially differ between HIV-1–infected aviremic patients and healthy donors. In contrast, both the levels of TRAIL surface expression and sTRAIL secretion were highly reduced in NK cells from HIV-1–infected viremic patients compared with healthy controls. Moreover, within the cohort of viremic HIV-1–infected individuals, the frequency of rIL-2–activated NK cells expressing TRAIL was markedly lower in the CD56 subpopulation compared with the CD56 subset. Furthermore, the concentration of sTRAIL in the culture supernatant of rIL-2–stimulated CD56 NK cells was considerably lower compared with that from activated CD56 cells ().
To understand whether the decreased expression/secretion of TRAIL by activated NK cells from viremic subjects affects NK cell function in the context of the elimination of autologous iDCs, we repeated the masking experiments using a specific anti-TRAIL mAb. Blocking TRAIL induced a statistically substantial reduction of the NK cell–mediated iDC cytolysis in both HIV-1–infected patients with undetectable viral load and healthy donors, which is in line with the high percentage of NK cells expressing and secreting TRAIL. In contrast, among viremic individuals, blocking TRAIL had almost no effect in further reducing the already low cytolysis of iDCs by NK cells. Again, this correlated with the low levels of TRAIL expression and secretion by NK cells isolated from this study group. Even the simultaneous masking of TRAIL and NKp30 did not result in any substantial reduction of iDC cytolysis by NK cells isolated from viremic patients, whereas the concomitant blocking of NKp30 and TRAIL virtually eliminated the NK cell–mediated lysis of autologous iDCs in the cohorts of HIV-1–infected aviremic individuals and healthy donors. Moreover, the impairment of TRAIL activity was much greater among the activated CD56-derived NK cells compared with the CD56-derived NK cell subset either in the absence or presence of NKp30 masking (). This reflected the negative or very low surface expression and secretion of TRAIL from the rIL-2–stimulated CD56 NK cell subset. Finally, we detected similar DR4 and DR5 expression on the surface of iDCs generated from viremic and aviremic infected patients compared with iDCs of healthy donors (unpublished data).
The aforementioned masking experiments clearly confirmed the crucial role of NKp30 in the NK cell–mediated lysis of autologous iDCs in healthy donors and in aviremic HIV-1–infected subjects. Moreover, they also delineated the substantial contribution of TRAIL to autologous iDC elimination by human activated NK cells purified from the same study groups. In contrast, the function of both of these molecules appeared to be highly impaired in NK cells isolated from viremic HIV-1–infected subjects. Nevertheless, NK cells from this latter cohort still exerted a considerable level of residual iDC cytolysis (). Therefore, we investigated the role of another activating NK cell receptor, DNAM-1, which contributes to the NK cell–mediated killing of iDCs (). The addition of a blocking mAb specific for DNAM-1 together with TRAIL and NKp30 reduced the lysis of autologous iDCs by unfractionated NK cells or NK cell subsets purified from viremic patients to almost undetectable levels (). Moreover, only in this latter cohort of patients, the masking of DNAM-1 alone similarly reduced the NK cell–mediated lysis of autologous iDCs to levels comparable with those achieved by simultaneously blocking NKp30, TRAIL, and DNAM-1. This phenomenon demonstrated the compensatory role of DNAM-1 in iDC lysis among HIV-1–infected viremic patients. In this regard, unlike NKp30 and TRAIL, DNAM-1 was expressed at normal levels on all NK cell subpopulations isolated from viremic subjects (unpublished data), which is in line with its conserved functional activity. Moreover, similar levels of expression of poliovirus receptor (CD155) and Nectin-2 (CD112), the two natural ligands for DNAM-1 (), were detected on the surface of iDCs generated from all three study groups (unpublished data).
This study demonstrates that numerous components of the bidirectional NK–DC cross talk between a CD56 subset of NK cells and autologous DCs is highly impaired in cells from viremic HIV-1–infected individuals compared with those from healthy donors and aviremic HIV-1–infected individuals who have been receiving ART for 2 yr or longer. As illustrated in the in vitro model proposed in Fig. S5 (available at ), actively replicating HIV-1 in peripheral tissue is associated with a markedly dysfunctional maturation of DCs, although these DCs appear to be phenotypically mature on the basis of the expression of several costimulatory markers and MHC class I and II molecules. These abnormally matured DCs are substantially impaired in their ability to secrete IL-10 and -12 and to prime the proliferation of neighboring autologous NK cells, which, in turn, fail to secrete adequate amounts of IFN-γ. It has been previously reported that NK cells from HIV-1–infected individuals are defective in eliminating autologous iDCs (). In this study, we show that NK cells purified from HIV-1–infected individuals with high levels of ongoing viral replication but not from aviremic patients are also markedly impaired in their ability to eliminate autologous iDCs. This phenomenon is largely caused by the high frequency of a dysfunctional CD56 NK cell subset, whose surface expression/secretion and function of NKp30 and TRAIL molecules are either extremely low or absent.
Under physiological conditions, the maturation of DCs is induced directly by microbial signals as well as by activated NK cells performing a regulatory role (). Consistent with our results, several studies have demonstrated that in vitro exposure of iDCs to various isolates of HIV-1 or HIV-1 envelope interferes with the complete functional maturation of DCs, although the surface expression of markers associated with the DC mature phenotype is not affected (–). We show here that the secretion of IFN-γ, a potent inducer of DC differentiation (), is highly impaired when NK cells are exposed to autologous mDCs generated from viremic but not aviremic HIV-1–infected individuals. It was recently demonstrated that the ability to promote DC maturation was essentially confined to NK cells expressing a KIR/NKG2A/NKp30 phenotype (). We previously reported that freshly purified NK cells from HIV-1 viremic individuals expressed increased levels of KIRs, whereas they expressed extremely low or negative levels of NKG2A and NKp30 (), particularly among the CD56 NK cell subset (). Given the association of these defects with the viremic state, together, these data suggest that HIV-1 directly or indirectly negatively interferes with the maturation process of DCs. In this regard, it remains to be determined whether the reduced NK cell activation, as observed in vitro in this study, plays a role in generating functionally impaired myeloid or plasmacytoid DCs freshly isolated from HIV-1–infected viremic patients as previously reported ().
The priming of NK and T cells by autologous mDCs represents two fundamental steps in the natural process that links the innate to the adaptive immune response (, , –). In this study, autologous mDCs generated from viremic HIV-1–infected subjects clearly exhibited a markedly impaired capacity to induce both NK cell expansion and the secretion of IFN-γ. IL-12 is an important cytokine for the activation and proliferation of NK cells (–, ). Thus, the reduced IL-12 production by mDCs generated from viremic HIV-1–infected individuals, as demonstrated in this study, may partially account for their defective priming of NK cells. In this regard, others have previously reported that MDDCs that had been infected in vitro with HIV-1 fail to produce IL-12 in response to CD40 ligand stimulation ().
Here, we demonstrate that mDCs generated from HIV-1–infected viremic individuals secreted a markedly lower amount of IL-10 compared with mDCs from aviremic patients. Although not addressed directly in this study, the lower secretion of IL-10, an HIV-1 inhibitory cytokine in most systems (, ), by mDCs as well as the reduced cytolytic activity of NK cells (, ) from HIV-1–infected viremic patients may impair the inhibition of HIV-1 replication in peripheral tissues. Furthermore, it has been shown that IL-10 inhibits the expression of DC costimulatory molecules (), and, therefore, the weak production of IL-10 by mDCs generated from viremic HIV-1–infected individuals may partly contribute to the mature phenotype of the markedly dysfunctional mDCs.
Whether DCs from HIV-1–infected individuals are also impaired in their ability to prime an effective T cell response remains controversial (). Nevertheless, several studies have reported a highly impaired capacity to activate autologous and heterologous T cells either by in vitro HIV-1–exposed mDCs generated from healthy individuals or by myeloid or plasmacytoid DCs freshly isolated from HIV-1–infected viremic individuals (, , ). Moreover, the in vitro observation that iDCs exposed to HIV-1 achieve an mDC phenotype, express high amounts of CCR7, and are able to chemotax in response to MIP-3β (CCL19) indicates that they are able to migrate to secondary lymphoid tissues, where they can participate in the priming of T cells (). It has also been shown in vitro and ex vivo that these phenotypically mature but functionally immature DCs are infected by HIV-1 (, ) and are also able to transmit the virus to autologous T cells (, , ). Collectively these data suggest that these phenotypically mature but dysfunctional DCs would generate a suboptimal adaptive T cell response and might also be able to spread the infection.
A potentially important mechanism through which NK cells are thought to impact the quality of DCs undergoing maturation in response to antigen uptake is by killing iDCs (, , –). It is likely that this phenomenon occurs primarily at sites of tissue inflammation and appears to be dependent on the NK cell/DC ratio (, , , ). Low ratios of NK cells/DCs lead to DC survival and maturation, whereas high ratios of NK cells/DCs result in the elimination of DCs and the inhibition of DC maturation (). A recent study showed that iMDDCs generated from HIV-1–infected patients can escape lysis by autologous NK cells (), but the underlying causes of this escape have not been identified. We address some of the cellular mechanisms that may account for impaired NK cell–mediated lysis of autologous iDCs and show that this phenomenon appears to be restricted to those HIV-1–infected individuals with persistent high viremia. Our data demonstrate that the markedly impaired expression/secretion and function of NKp30 and TRAIL largely account for the highly defective NK cell–mediated lysis of autologous iDCs generated from viremic HIV-1–infected subjects. Moreover, we show that within this study group, the overrepresentation of CD56 NK cells expressing the unusual KIR/NKG2A/NKp30/TRAIL phenotype (), a subset present at very low frequencies in aviremic HIV-1–infected patients as well as in healthy donors (), likely explains why this defective elimination of autologous iDCs by NK cells is confined to HIV-1–positive viremic patients.
DCs are distributed within all lymphoid and nonlymphoid tissues and in humans are classified into at least two major types, myeloid and plasmacytoid, which differ in terms of function and phenotype (). Plasmacytoid DCs, regardless of their state of differentiation/activation, are poorly susceptible to NK cell–mediated lysis () and do not efficiently present antigens to T cells, whereas myeloid DCs are potent inducers of adaptive immunity (, ). It has been reported recently that freshly isolated myeloid DCs from healthy individuals were susceptible to NK cell–mediated lysis, although to a lesser degree than were iMDDCs (). However, the NK cell/DC ratios found in that study were possibly too low, and it is conceivable that the observed differences between freshly isolated myeloid DCs and iMDDCs with regard to the susceptibility to lysis may have been reduced at higher NK cell/DCs ratios. In addition, the heterogeneity of freshly isolated myeloid DCs in their surface expression of MHC-I molecules and costimulatory markers reflects differences in the maturation or activation state (, ) and, therefore, may impact NK cell–mediated killing. Although MDDCs differ from freshly isolated myeloid DCs in certain respects, previous studies have found that MDDCs are very similar both phenotypically and functionally to myeloid DCs present in the blood (, ). Therefore, we feel that MDDCs are appropriate surrogates for freshly isolated myeloid DCs and represent a practical tool in vitro for investigating the role of NK–DC cross talk in the pathogenesis of human diseases.
The conserved numbers and phenotype of circulating monocytes among viremic patients compared with aviremic and healthy subjects (unpublished data) are consistent with previous studies showing that a very small proportion of blood monocytes harbor HIV-1 throughout the course of infection (, ). Nonetheless, it has been demonstrated that the infection of monocytes with HIV-1 can affect important functions of monocytes, such as chemotaxis and phagocytic capacity, suggesting that viral replication in monocytes/macrophages may contribute to the establishment and persistence of HIV-1 infection and may also activate and infect surrounding T cells (, ).
In summary, certain bidirectional NK–DC interactions that normally occur after an inflammatory insult are affected during HIV-1 infection, resulting in the abnormal maturation of DCs, impaired activation of NK cells, and deficient killing of unnecessary iDCs by NK cells. As demonstrated in this study, these defects are associated with an overrepresented CD56 subset of NK cells observed only in viremic and not in aviremic HIV-1–infected individuals or normal controls. Additional studies will be needed to verify the in vivo significance of this markedly defective NK–DC cross talk among cells from HIV-1–infected viremic individuals to fully appreciate the potential negative impact on the immune-mediated control of HIV-1.
Two cohorts of 15 viremic and 15 aviremic HIV-1–infected patients were studied (Table S1, available at ). The viremic group was composed of HIV-1–infected individuals who were either naive to therapy or had formerly been receiving ART but whose treatment regimen had been discontinued at the time of our study. The aviremic group had been receiving ART for at least 24 mo, and viremia remained undetectable over this time period. PBMCs were obtained by leukapheresis performed after obtaining signed consent forms and in accordance with clinical protocols approved by the Institutional Review Board (IRB) of the University of Toronto (13 patients) and of the National Institute of Allergy and Infectious Diseases (NIAID; 17 patients). As negative controls, PBMCs from 15 healthy donors seronegative for HIV-1 were obtained by apheresis provided by the Transfusion Medicine Department of the Mark O. Hatfied Clinical Research Center National Institutes of Health as a part of IRB-approved clinical studies (Table S1). All experiments performed in this study using cells derived from HIV-1–infected individuals and from healthy donors have been approved by the IRBs of the University of Toronto and the NIAID.
PBMCs were isolated over Ficoll-Hypaque gradients (lymphocyte separation medium; MP Biomedicals). NK cells were freshly isolated by negative selection (StemCell Technologies Inc.; reference ). Purified NK cells contained ≤3% contamination with other PBMC subsets as determined by the expression of CD3, TCR-α/β, TCR-γ/δ, CD19, or CD14. CD56 or CD56 NK cell subsets were separated by a magnetic cell-sorting technique (Miltenyi Biotec; reference ). The purities of CD56 or CD16 NK cell fractions were consistently >95 and 90%, respectively. Polyclonal NK cells and NK cell subsets were activated in vitro with recombinant IL-2 (rIL-2; Roche) at 200 UI/ml or with recombinant IL-15 (rIL-15; R&D Systems) at 10 ng/ml for 6 d ().
To generate iDCs, monocytes were isolated from PBMCs by immunomagnetic selection (Miltenyi Biotec). The purity of CD14 and CD1a monocytes was ≥98%. iMDDCs were then obtained by culturing the highly purified CD14 cells at 10 cells/ml in complete media plus IL-4 at 200 U/ml (PeproTech) and GM-CSF at 200 ng/ml (Leukine Liquid Sargramostin). After 6 d of stimulation in culture, CD14 and CD1a iMDDCs were induced to undergo maturation by incubation with LPS at 1 μg/ml (Sigma-Aldrich) or sCD40 liter at 2 μg/ml (Biosource International) for 24 h.
The following mAbs were used in this study: mAbs 289 (IgG2a anti-CD3), C218 (IgG1 anti-CD56), KD1 (IgG2a anti-CD16), Gl183 (IgG1 anti-p58.2/KIR2DL2), 11pb6 (IgG1 anti-p58.1/KIR2DL1), Z27 (IgG1 anti-p70/KIR3DL1), F278 (IgG1 anti-LIR1/ILT2), Z270 (IgG1 anti-NKG2A), Xa185 (IgG1 anti-CD94), Bab281 and KL247 (IgG1 and IgM anti-NKp46, respectively), Az20 and F252 (IgG1 and IgM anti-NKp30, respectively), Z231 and KS38 (IgG1 and IgM anti-NKp44, respectively), Kra236 and F5 (IgG1 and IgM anti-DNAM, respectively), L95 (IgG1 anti–poliovirus receptor), L14 (IgG2a anti–Nectin-2), and A6-136 (IgM anti–HLA-A/-B/-C). FITC- or PE-labeled anti-CD4 (IgG1), anti–TCR-α/β (IgG1), anti–TCR-γ/δ (IgG1), anti-CD19 (IgG1), anti-CD56 (IgG2b), anti-CD14 (IgG1), anti-CD1a (IgG1), anti-CD40 (IgG1), anti-CD80 (IgM), anti-CD83 (IgG1), anti-CD86 (IgG1), anti–HLA-A/-B/-C (IgG1), anti–HLA-DP/-DQ/-DR (IgG2a), and anti-TRAIL (IgG1) were purchased from BD Biosciences. The anti–Trail-R1, -R2, -R3, and -R4 mAbs were purchased from R&D Systems, and the anti–HLA-E mAbs (IgG1) were purchased from Novus Biologicals.
For one- or two-color cytofluorimetric analysis (FACSCalibur; BD Biosciences), rIL-2–activated NK cells, iDCs, and mDCs were stained with the appropriate mAbs followed by PE- or FITC-conjugated isotype-specific goat anti–mouse second reagent (Southern Biotechnology Associates, Inc.). Second appropriate antiisotypic mAbs stained with PE and/or FITC were used as negative controls. The data were analyzed using CellQuest software (BD Biosciences). After 6 d of activation with rIL-2, NK cells were tested for cytolytic activity in a 4-h Cr release assay as described previously (). Saturating concentrations (10 μg/ml) of specific mAbs blocking NK cell receptors were added for the masking experiments performed with autologous iDCs. The NK cell/iDC ratio was 10:1.
rIL-2–activated NK cells were stained with cell tracker CM-DiI (Invitrogen) and autologous iDCs with Hoechst 33342 (Invitrogen). Fluorescent cells were coincubated for 2 h in 1640 RPMI medium supplemented with 10% FCS in the presence of FLICA reagent (Invitrogen) on fibronectin-coated chamber slides (LAB-TEK) at 37°C and 5% CO atmosphere. The NK cell/iDC ratio was 10:1. NK cells and iDCs were then washed with PBS, suspended in medium, and sealed on the slides with coverslips. Snap-shot images were captured on an inverted epifluorescence microscope (Axiovert 135; Carl Zeiss MicroImaging, Inc.) equipped with a filter for red, green, and blue dyes using a 20× plan-Apochromat objective. Imaris 4.04 software (Bitplane AG) was used for image processing.
Freshly purified NK cells were cryopreserved until required as responders. Experiments were performed in triplicate in 96-well round plates with complete medium. NK cells were cocultured at a constant concentration of 2 × 10 NK cells/well with autologous and heterologous mMDDCs (stimulators) in serial dilutions (10–1.56 × 10 cells/well). After 4 d, NK cell proliferation was measured by [H]thymidine uptake (16 h).
The levels of IL-12p70 (IL-12), IL-10, -15, and -18 secreted by mDCs and the level of sTRAIL secreted by r-IL2–activated NK cells were measured from cell culture supernatant by ELISA (R&D Systems and Biosource International). To detect the production of IFN-γ, freshly purified NK cells were cryopreserved until required and cocultured with autologous LPS or sCD40L matured DCs in 96-well round-bottom plates with complete medium (). The mDC/NK cell ratio was 1:10. The supernatant of the cultures was collected after 24 h and assayed by ELISA (BD Biosciences).
The distributions of each immune response variable were compared between uninfected and HIV-1–infected viremic and aviremic individuals using the Mann-Whitney test. For each individual, the differences between the CD56 and CD56 NK cell subsets were evaluated using the Wilcoxon signed ranks test. All p-values are two sided.
Fig. S1 shows the surface expression of maturation markers on iDCs and mDCs generated in vitro from representative donors within the three study groups. Fig. S2 shows the degree of NK cell–mediated lysis and CD56 and CD56 NK subset–mediated lysis of autologous iDCs from representative donors within the three study groups using the traditional Cr release assay and fluorescence microscopy. Fig. S3 shows the NKp30 surface expression on freshly purified rIL-2– and -15–activated NK cells from representative donors within the three study groups. Fig. S4 shows the NKp30-dependent killing of autologous iDCs by rIL-2– and -15–activated NK cells from representative donors within the three study groups. Fig. S5 shows our proposed model of the impaired interactions between NK cells and DCs in HIV-1–infected viremic patients. Table S1 provides data on the profiles of viremic and aviremic HIV-1–infected patients and healthy donors. Online supplemental material is available at . |
We have used cryoinjury () or left coronary artery (LCA) ligation () to induce large left ventricular infarctions. For hemodynamic studies the first injury model is preferable, as highly reproducible lesions are generated even in the mouse (). Enhanced GFP (EGFP) BM cells (3 × 10) were injected into the center and the border zone of the infarction as previously described (, ), and hearts were analyzed 3–4 wk later. Catheterization revealed no improvement of left ventricular ejection fraction (LVEF) and other functional parameters (not depicted) compared with sham-injected mice ( O and Table S1, available at ), whereas morphological analysis of cryosections revealed considerable engraftment of EGFP cells in the scar region (). To test whether a higher percentage of BM-derived stem cells is required to enhance left ventricular function (LVF), recipient mice were lethally irradiated, reconstituted with transgenic BM cells (), and, after assessment of good engraftment (>80%), heart infarction (cryoinfarction or LCA ligation) and cytokine mobilization were performed. The integration of EGFP cells in and around the injury site was massive 3–4 wk after operation in both lesion models () and clearly higher than after direct injection (). However, measurements of LVEF also yielded no improvement in these mice compared with the sham-injected controls after cryoinjury ( and Table S1) and after LCA ligation (49.6 ± 6.1% [ = 4] in the mobilized vs. 54.6 ± 3.3% [ = 3] in the nonmobilized control group; Table S1). Note that lesion sizes in LCA-infarcted animals can vary.
To determine in more detail the effects of directly injecting or mobilizing BM cells, hearts were harvested and analyzed by immunohistochemistry. In accordance with function, no obvious morphological differences were noted in cryoinjured and LCA-ligated versus control hearts. Cytokine-induced mobilization led also to infiltration of EGFP cells in intact myocardium (). Two different shapes of EGFP cells, round and more elongated, were identified; the latter was more frequent in the border zone of the lesion and in the intact myocardium (). The EGFP cells in the scar region () and in the border zone () were positive for the panhematopoietic marker CD45 and negative for the muscle marker cardiac α-actinin. Consistent with our earlier findings (), a few morphologically intact EGFP cardiomyocytes originating through cell fusion were found. Large () and small () vessels in and around the scar region were stained with the endothelial markers platelet/endothelial cell adhesion molecule (PECAM) or von Willebrand factor (unpublished data) and with α–smooth muscle actin (ASMAC), and all theses vessels were found to be EGFP. Some of the small vessels lacked the smooth muscle layer (), which is a typical finding for newly formed vessels in the lesion. Occasionally, EGFP/PECAM/ASMAC cells were detected in the lumen or even below the endothelial cell layer of small vessels (), indicating transmigrating hematopoietic cells.
To further exclude potential initiation or ongoing cardiac transdifferentiation of EGFP cells with a different technique than immunohistochemistry, we characterized the functional expression of ion channels using patch clamp; this technique provides a highly sensitive readout of the functional and developmental status of cells (). Isolated cells were obtained from mobilized hearts after collagenase treatment, and both EGFP and EGFP cells were investigated. We found that all EGFP cells had a small membrane capacitance (7.1 ± 0.1 pA/pF; = 7) and functionally did not express voltage-dependent inward currents ( = 15; , right), a hallmark of cardiomyocytes starting from early embryonic development (, inset) (, ). In contrast, native cardiomyocytes harvested from the same hearts were EGFP, had distinct cross-striation (, inset), a large membrane capacitance (127.2 ± 22.4 pA/pF; = 7), and expressed I and I (). Accordingly, action potentials could be evoked by depolarizing voltage ramps in EGFP ( = 3; ) but not EGFP cells ( = 11; , left), where only outwardly rectifying K currents were found ( = 15; , right).
Because of contrasting earlier reports (–), we first determined the tumorigenicity of undifferentiated ES cells by injecting 10 cells ( = 2) into the tail vein or 10 cells ( = 14) directly into infarcted hearts. Large tumor masses were detected in all syngeneic ( = 13) and 2 out of 3 allogeneic animals within 4 wk (see ). The onset of tumor growth occurred as early as 10 d after ES cell transplantation. Next, we excised beating EGFP areas from transgenic α–myosin heavy chain (α-MHC)–EGFP embryoid bodies (EBs) () to enrich for cardiomyocytes and reduce the number of undifferentiated ES cells. After injection (10 cells), tumor growth was still detected in the majority of mice (five out of six; ; and see ) but with delayed onset. Histological analysis of the tumors revealed glandular (endoderm), squamous epithelium (ectoderm), and cartilage (mesoderm) differentiation (), hence typical histological features of teratomas. These results indicated that highly purified ES cell–derived tissue preparations are required to minimize the risk for tumor generation.
To purify and identify ES cell–derived cardiomyocytes, we generated a bicistronic vector in which the cardiac-specific promoter α-MHC drives the expression of both the puromycin resistance gene and the EGFP cassette (, top). Two clones (αPIG10 and αPIG44) were chosen for further study and differentiated in vitro using the hanging drop or mass culture protocol. The first clusters of EGFP cells were detected in the EBs on days 7–8 of development, and spontaneous beating began ∼12–24 h later. 10 μg/ml puromycin was added to the culture dishes on days 9–10 (, left), and EGFP fluorescence and contractile activity of the EGFP clusters intensified during the first 24–72 h, indicating cardiomyocyte enrichment (, middle). After 6 d of puromycin treatment, most of the EBs consisted of strongly beating EGFP clusters of cardiac cells (, right). Similar results were obtained with both hanging drops and mass culture in vitro differentiation protocols. Because the mass culture protocol typically yielded an order of magnitude more cardiomyocytes, it was used in all subsequent experiments.
As the cardiac cell mass appeared to increase during later puromycin selection (see and Video 1, available at ), we determined whether proliferation of ES cell–derived cardiomyocytes underlies this phenomenon. We found that the number of EGFP cells increased dramatically during the first 3 d of puromycin selection and thereafter reached a plateau level yielding 6–10 times higher numbers of cardiomyocytes than in the untreated counterparts ( = 4; ). We confirmed this observation by labeling cardiomyocytes in the S phase using BrdU for 24 h. We found that 32% of cardiomyocytes were BrdU by day 3 of puromycin treatment compared with only 2% before the initiation of selection (). Thereafter, their number decreased to ∼13%. In the untreated control cultures, no BrdU-labeled cardiomyocytes were detected at these stages of development ( = 2; ). To estimate the size of the proliferative pool of cardiomyocytes, the BrdU labeling time was increased from 24 to 72 h beginning on the third day of puromycin treatment ( = 2). This protocol revealed that 37% of αPIG44- and 61% of αPIG10-derived cardiomyocytes had entered the cell cycle during the labeling period. From these results, the cell cycle length was calculated to be 18 ± 4 h and that ∼50% of the cardiomyocytes cycled four to five times during the first 3 d of drug selection. This is in complete agreement with numbers obtained by cell counting (see ), which indicated a 6–10-fold increase in the total number of cardiomyocytes induced by purification.
RT-PCR analysis using Oct-4 as a “bona fide” marker of undifferentiated mouse ES cells () showed that after 10 d of puromycin treatment no Oct-4 transcripts were detected by PCR (). This was further corroborated with Trypan blue staining by which untreated EBs contained 75% viable (Trypan blue) noncardiac (EGFP) cells ( = 1462), whereas this fraction decreased to 0.7% ( = 884) after 10 d of puromycin selection (not depicted). Finally, to definitively assess the purity of drug-selected EBs, we counted the number of EGFP and α-actinin cardiomyocytes versus noncardiomyocytes (). Consistent with previous results (), only 1.8% of cells in the untreated EBs were cardiomyocytes (EGFP and α-actinin; = 1334; , left). After puromycin treatment for 10 d, 99.4% of the cells were cardiomyocytes, whereas only 0.6% of all nucleated cells were noncardiomyocytes ( = 168; , right). The identity of these puromycin-resistant noncardiomyocytes was not determined.
The potential damage of cardiomyocytes by our novel lineage selection protocol was excluded with immunohistochemistry where structural integrity and the typical crossstriation were found (). Furthermore, puromycin-selected cardiomyocytes displayed normal action potentials at early and late stages of development (). As reported earlier for ES cell–derived and mouse embryonic cardiomyocytes (), a significant shortening of the action potential duration at 90% repolarization (from 65.0 ± 8.1 ms in briefly puromycin exposed [ = 9] to 25.3 ± 4.9 ms in long puromycin exposed [ = 7]) and a significantly more negative maximal diastolic potential (−49.5 ± 2.1 mV in briefly exposed to −58.1 ± 1.7 mV after long puromycin exposure) were clear indications that these cells were physiologically intact cardiomyocytes and differentiated during cultivation (). Our data demonstrate that puromycin selection induces formation of high purity (>99%) of morphologically and physiologically intact cardiomyocytes.
We next investigated the potential of purified ES cell–derived cardiomyocytes for engraftment and repair of the injured myocardium. ES cell–derived cardiomyocytes (3 × 10–10) were transplanted 9–12 d after puromycin selection into infarcted hearts of syngeneic mice. Surprisingly, poor engraftment of the transplanted cells was observed, as only 22% of mice ( = 9) contained any detectable EGFP cells 3 wk after operation. We therefore reevaluated our earlier data in which embryonic heart–derived cells were found to stably engraft within the injured myocardium (); analysis revealed that approximately half of the cells in embryonic hearts are cardiomyocytes, whereas the remaining noncardiomyocytes are primarily fibroblasts (). We therefore determined whether fibroblasts might play a facilitating role for cardiomyocytes by co-cultivating purified ES cell–derived cardiomyocytes with fibroblasts. We found that purified cardiomyocytes did not adhere well to gelatin coated culture dishes and did not maintain their typical elongated shape of cardiomyocytes (, bottom). However, cardiomyocytes plated onto embryonic feeder cells maintained an elongated cell shape for weeks and even aligned with the fibroblasts (, top). Microelectrode array (MEA) recordings of field potentials in these co-cultures revealed synchronous signals from electrodes in contact with EGFP cardiomyocyte clusters over several weeks ( = 2; ). These results suggest that formation of a functional syncytium occurs between cardiomyocytes and fibroblasts.
Based on these findings, we analyzed the effect of injecting equal numbers of puromycin-selected ES cell–derived cardiomyocytes and syngeneic fibroblasts into injured myocardium. This approach resulted in a substantial increase of cell engraftment. Prominent EGFP fluorescence, a reliable parameter for engraftment, was detected in 68.3% of operated hearts ( = 60) up to 147 d (mean = 63 d) after cellular cardiomyoplasty (). Interestingly, the ratio of engraftment did not vary between middle (<100 d, 68.2%; = 44) and long-term (>100 d, 68.7%; = 16) transplantations, implying that graft rejection is not a prominent problem. Transplanted cardiomyocytes were localized within the border zones of damaged areas of the myocardium. In cryosections, an almost transmural distribution of the implanted cells could be observed (). The injected cells colocalized in clusters but could be clearly distinguished from the native cardiomyocytes because of their reduced size, distinct cell shape, and incomplete myofibrils shortly after transplantation, strongly supporting the conclusion that the EGFP cells did not result from fusion with native cardiomyocytes. In the long-term transplants, the ES cell–derived cardiomyocytes displayed elongated cell shapes and distinct cross-striation, proving that the engrafted EGFP cells further differentiate after the transplantation (). We next examined the basis for intercellular coupling by analyzing gap junction formation based on connexin 43 staining. As depicted in , gap junctions were detected between the transplanted cardiomyocytes.
Although no tumors developed in 95% ( = 60) of transplanted mouse hearts, three hearts harvested 45, 90, and 91 d after transplantation did reveal tumors, the latter two evolved from the same preparation of cells (). Histological analysis of two of these hearts revealed mesenchymal tumors closely resembling the pleomorphic variant of malignant fibrous histiocytoma and not teratomas (Fig. S1, available at ). Thus, our transplantation data indicate that undifferentiated ES cells are completely eliminated by our lineage selection method, leading to an abolishment of teratoma formation after transplantation.
To assess whether transplantation of ES cell–derived cardiomyocytes and fibroblasts resulted in enhancement of function in infarcted hearts, hemodynamics was determined 3–4 wk after surgery. Pressure-volume loops () revealed a significant enhancement of LVEF (P < 0.01; 51.6 ± 6.2% vs. 36.3 ± 11.1%) and reduction of the enddiastolic volume (P < 0.05; 46.5 ± 6.6 μl vs. 58.5 ± 18.4 μl) in mice with cardiomyoplasty ( = 12) versus sham injection ( = 11; and Table S1). As a further control, we transplanted 10 EGFP fibroblasts and analyzed LVF after 3–4 wk. In contrast to ES cell–derived cardiomyocytes, no difference in LVEF (53.6 ± 7.1% vs. 50.3 ± 6.1%; and Table S1) and enddiastolic volume (60.5 ± 12.5 μl vs. 57.0 ± 12.2 μl) between the fibroblast ( = 6) and sham-injected ( = 6) mice was observed, although histological analysis proved good engraftment of the EGFP fibroblasts (). We also tested the efficacy of transplanting EGFP skeletal myoblasts (10, = 5). These cells were found to engraft well into the lesion (Fig. S2, A and B, available at ), and LVEF was significantly enhanced (P < 0.01; 60.6 ± 2.8% [ = 5] vs. 48.0 ± 5.7% [ = 5]; Fig. S2 C and Table S1). Because skeletal myoblasts are proven to not integrate functionally into the myocardium, we presume that the improvement of heart function was mainly caused by the significant reduction of left enddiastolic volume (P < 0.05; 33.2 ± 9.2 μl vs. 51 ± 8.7 μl).
Recent work () suggests that transplanted cells may act mainly via paracrine mechanisms. To determine whether the enhancement of LVF of ES cell–derived cardiomyocytes in vivo may be caused by active contribution rather than exclusively paracrine mechanisms, we assessed whether purified ES cell–derived cardiomyocytes generate force and transfer this to the surrounding tissue. For this purpose we established an “in vitro transplantation” model in which the purified ES cell–derived cardiomyocytes were transferred to ischemic ventricular slices (see scheme in ) and measured isometric force generation 1–2 wk after co-culture. Although ventricular slices without ES cells neither produced spontaneously nor after electrical stimulation any force ( = 4; ,D), ES cell–derived cardiomyocytes generated a force of 6.1 ± 0.8 μN ( = 5) at a spontaneous rate of 2.6 ± 0.3 Hz and an even higher force of 8.6 ± 1.7 μN at a stimulation frequency of 3 ± 0.4 Hz ().
The aim of this paper was to identify the most promising cell type for treatment of the infarcted heart. The investigation was largely motivated by the controversy surrounding the efficacy of adult BM cells and ES cells for cardiomyoplasty (for review see references , ). Although BM cells are easily accessible and allow autologous transplantations, it is still unclear and controversial whether these cells can improve defective heart function and whether this treatment is entirely safe. In fact, studies in animals suggest that cytokine treatment may carry the risk of accelerated restenosis of coronary arteries (), and direct injection of BM cells may carry the risk of extended calcifications ().
ES cells represent an ideal stem cell source for ex vivo generation of cardiomyocytes; however, studies are still missing in which their long-term, teratoma/tumor-free engraftment and enhancement of LVF are carefully evaluated. To address this critical topic, we used a highly reproducible infarction model and assessed the fate and functional relevance of transplanting ES cell–derived cardiomyocytes and compared this with the effect of transplanted BM cells, as well as fibroblasts and noncardiomyocytes. To avoid immunological interference and to be able to determine the tumorigenic risk of highly purified ES cell–derived cardiomyocytes, a syngeneic transplantation model was chosen. Thus, in case our purification approach of ES cell–derived cardiomyocytes turned out to be safe, it would be even safer in the clinically more relevant allogeneic setting (). Nevertheless, the recent identification of pluripotent cells in testicles of adult mice revealed a therapeutically relevant new autologous ES cell–like source (). Moreover, ongoing experiments on nuclear cloning and nuclear cloning–derived ES cells will soon also bring this approach closer to clinical application ().
Our experimental results demonstrate that neither direct injection nor cytokine-induced mobilization of BM cells reduced scar formation and/or enhanced LVF 3–4 wk after injury. This time point was chosen to allow the engraftment and further differentiation of BM progenitors. Consistent with our earlier findings (), we could not detect transdifferentiation of BM cells into cardiomyocytes using immunohistochemistry. To date the fate of transplanted BM cells has only been investigated with this approach, and we therefore have also performed single cell analysis using the patch-clamp technique whereby even the functional differentiation of BM cells into early embryonic cardiomyocytes (>E8.5) was excluded. We also could not detect, in contrast to earlier studies in mice () and human transplanted hearts (), the contribution of BM cells to endothelial or smooth muscle cells of vessels in infarcted hearts. Our findings are corroborated by measurements of hemodynamics, where neither direct injection nor mobilization of BM cells had a positive impact in contrast to earlier reports (, ). Thus, in a large number of animals, our studies exclude prominent neogenesis of cardiomyocytes and vessels from BM cells transplanted into infarcted hearts. Importantly, our results concur with recent clinical trials in which neither application of BM cells into affected coronary arteries () nor BM mobilization () had beneficial effects on LVF.
In clear contrast to the BM cells, highly purified ES cell–derived cardiomyocytes were found to long-term engraft (4–5 mo) and to clearly improve LVF. Because paracrine effects have been claimed to be potentially responsible for the beneficial effects observed after cellular cardiomyoplasty (), we addressed this using an in vitro transplantation model. The experiments clearly proved that the ES cell–derived cardiomyocytes generate force and transfer this to the surrounding tissue. Thus, if massive engraftment of ES cell–derived cardiomyocytes into the infarcted myocardium is achieved, a strong functional improvement can be obtained.
In accordance with earlier work (), we found that skeletal myoblasts engraft well into the infarcted myocardium and augment LVF. Because these cells are known to not couple electrically with the native myocardium () because of a lack of connexin 43 expression (), their beneficial effect is most likely caused by stabilization of the scar region, resulting in a considerable reduction of the enddiastolic volume. However, their therapeutic use does not appear promising, as experimental data in vitro () and in vivo () indicate that skeletal myoblasts can induce ventricular arrhythmias via reentry; similar events have been also observed in the clinical trials ().
The major challenge for clinical development of ES cell–derived cardiomyocytes has been to develop safe and efficient methods for their enrichment in vitro, measures to ensure their long-term engraftment, and, most importantly, avoidance of tumorigenicity caused by contaminating ES cells. This prompted us to develop a cardiac lineage–specific selection to purify ES cell–derived cardiomyocytes. Unexpectedly, we discovered that elimination of noncardiomyocytes was accompanied by a burst of proliferation, which produced 6–10 times more cardiomyocytes. At this time we can only speculate about the underlying mechanisms and assume that noncardiomyocytes and/or undifferentiated ES cells normally produce factors that inhibit the proliferation of differentiating cardiomyocytes within the EBs.
Tumors are currently considered the major hurdle for ES cell–derived transplantation, as very few undifferentiated ES cells suffice for teratoma growth in the heart (this study), the brain (), and the kidney (). We have succeeded in establishing a highly purified ES cell–derived cardiomyocyte preparation (>99%) and show, to our knowledge for the first time, in a large number of animals that teratoma formation after ES cell–based transplantation can be completely avoided, even long-term (4–5 mo), in syngeneic recipients. In fact, only 3 out of 60 hearts developed tumors, of which 2 were not teratomas but rather poorly differentiated mesenchymal tumors, and these most likely arose from the co-transplanted embryonic fibroblasts (the malignant fibrous histiocytoma looks predominantly fibroblast-like) (). Our data also demonstrate that studies with much larger cohorts and longer follow-up periods than reported earlier (, ) are needed to assess the risk of tumor formation after transplantation of ES cell–derived cells.
Cellular replacement approaches in different tissues have proven to be of limited efficacy, as the transplanted cells are subject to a high rate of death within hours after transplantation (). To date, the mechanisms responsible for this have been unclear but do not appear to be exclusively immune mediated, as similar rates of cell death are observed in the presence of immunosuppressants (). Our experimental data imply that the extracellular matrix plays an important permissive role in cell survival and viability (). This likely reflects the fact that fibroblasts provide extracellular matrix support for developing cardiomyocytes but also possibly critical growth factors and cytokines, as recently reported for the in vitro differentiation of cardiac progenitor cells into cardiomyocytes ().
In conclusion, we show that ES cell–derived cardiomyocytes can be highly purified, enriched, and do long-term engraft in the infarcted heart without teratoma formation. Most importantly, our comparative study demonstrates that ES cell–derived cardiomyocytes are the most suitable candidates for cellular cardiomyoplasty, as these cells enhance, in contrast to BM cells, the contractile function of the lesioned myocardium.
l
a
n
i
m
a
l
e
x
p
e
r
i
m
e
n
t
s
w
e
r
e
a
p
p
r
o
v
e
d
b
y
t
h
e
l
o
c
a
l
a
n
i
m
a
l
c
a
r
e
c
o
m
m
i
t
t
e
e
s
o
f
t
h
e
U
n
i
v
e
r
s
i
t
i
e
s
o
f
B
o
n
n
,
C
o
l
o
g
n
e
,
a
n
d
L
u
n
d
. |
Low estrogen—during menopause in women or induced by ovary removal in rodents—causes osteoporosis. Roberto Pacifici (Emory University School of Medicine, Atlanta, GA) has put the blame for this bone loss squarely on T cells and, at the meeting, he provided corroborating evidence. But new data from Reinhold Erben (University of Veterinary Medicine, Vienna, Austria) seems to exonerate these cells.
Perhaps the strongest evidence that T cells cause bone loss when estrogen is in short supply comes from T cell–deficient (“nude”) mice. In past studies, Pacifici and colleagues showed that nude mice—unlike wild-type mice—do not develop osteoporosis when their ovaries are removed. Bone loss kicks in when T cells are transferred into the mice. This effect depends on T cell production of the cytokine TNF, which causes stromal cells to produce growth factors that stimulate bone-resorbing osteoclasts.
Bone loss is also a symptom of T cell–driven autoimmune diseases, such as rheumatoid arthritis (RA). And the symptoms of RA worsen after childbirth and menopause, when estrogen levels are low, also hinting at a possible connection between estrogen, T cells and bone loss. Pacifici's group has now found that ovariectomized mice treated with a T cell–inhibiting drug (Abatacept) have lower levels of circulating TNF and are spared the bone loss normally induced by ovary loss. When the drug was discontinued, bone loss resumed.
Erben does not dispute the mouse data, but rather questions their relevance to other species. “This may be true for mice,” says Erben. “But whether this model is predictive of human physiology is unclear.” Erben's data showed that thymectomized rats depleted of circulating T cells still developed osteoporosis when their ovaries were removed. And the rat, suggests Erben, may be a better model of postmenopausal osteoporosis than the mouse. In the rat model, as in humans, bone loss is sustained, whereas in mice, bone loss is transient.
Reference:
Bone marrow cavities fill up with fat in mice lacking the transcription factor EBF-1 (early B cell factor-1), according to data from Mark Horowitz (Yale University School of Medicine, New Haven, CT).
The evolutionarily conserved EBF transcription factors (EBF-1–4) are required for the development of olfactory neurons and B cells. EBF-1 also promotes fat cell (adipocyte) differentiation when overexpressed in preadipocyte cell lines, but the mechanism—and in vivo significance—of this finding was unclear.
To investigate the role of EBF-1 in fat formation, Horowitz and colleagues examined mice lacking the transcription factor. In addition to having no B cells, the mice had fat in all the wrong places. Subcutaneous fat was almost entirely missing, possibly explaining their previously described wasted appearance. The mice instead had fat where their bone marrow should be.
Excess fat wasn't their only problem. The EBF-1–deficient mice also had increased bone mass due to an overabundance of osteoblasts. The combined fat–bone phenotype prompted Horowitz to suggest that EBF-1 might regulate gene expression in a common adipocyte–osteoblast precursor cell, as these cells are known to arise from a common progenitor. But this hunch is difficult to test. Unlike in B cell development, few markers have been identified to distinguish osteoblast precursor cells at various stages of differentiation. In this respect, says Horowitz, “bone biologists are 15 years behind immunologists.”
The only targets of EBF-1 identified thus far are those involved in B cell development. One of those targets, the B cell–specific transcription factor encoded by Pax5, is also required for bone maintenance. But in bone, the relationship between EBF-1 and Pax5 is more complicated, as the absence of Pax5 causes severe bone loss—the opposite of the defect caused by EBF-1 deficiency.
Reference:
The formation of bone-resorbing osteoclasts requires cell–cell fusion between monocyte lineage precursor cells. But little is known about how this fusion is regulated and what proteins it requires. According to Yongwon Choi (University of Pennsylvania, Philiadelphia, PA), fusion among preosteoclasts requires a subunit of the vacuolar ATPase (v-ATPase) proton pump—but not the proton pumping action of the v-ATPase itself.
Choi and colleagues had previously identified a novel isoform of the v-ATPase d2 subunit (d2)—one of the five subunits that form the membrane-spanning proton channel—in a search for genes preferentially expressed in osteoclasts. Finding d2 was not surprising, as osteoclasts use plasma membrane v-ATPases to acidify the extracellular space between the cell and the bone surface—the first step in bone resorption.
The surprise came, as presented by Choi, when the group eliminated d2 from mice. The d2-deficient mice had increased bone mass, not because their osteoclasts were defective in resorption, but simply because there were too few of them—at least of the multinucleated sort that digest bone the best. The paucity of multinucleated osteoclasts did not reflect a block in differentiation, as precursor cells from the d2-less mice still developed into mononuclear osteoclasts, which retained the normal low-level resorption activity. But without d2, the mononuclear cells couldn't fuse.
The normal mechanism of d2 action remains a mystery. In other cell types, v-ATPases are found on intracellular membranes where they promote pH-dependent membrane fusion. But, so far, a role for v-ATPase subunits in cell–cell fusion has been documented only in . There, a v-ATPase subunit prevents the inappropriate fusion of embryonic cells by somehow blocking the expression of a fusion-promoting membrane protein.
Reference:
According to data from Steven Teitelbaum (Washington University, St. Louis, MO), glucocorticoids (GCs)—the most widely used class of antiinflammatory drug—prevent osteoclasts from rearranging their cytoskeletons and thus from digesting bone. And without proper osteoclast function, bone-building osteoblasts also shut down, which might help explain the crippling osteoporosis that often accompanies long-term steroid therapy.
Prolonged GC therapy impairs normal bone remodeling, an ongoing process in which osteoclasts dig small pits in bone and osteoblasts fill them up with new bone. This continual turnover is essential to maintain the structural integrity of the bone. In patients with GC-induced osteoporosis, these pits do not get fully filled. The mechanism behind this defect was controversial, particularly as treating osteoblasts with GCs in vitro actually enhances their activity.
GCs might also have an indirect effect on osteoblasts, reasoned Teitelbaum, as the activity of osteoblasts and osteoclasts are tightly linked—an increase or decrease in the activity of one cell type prompts a parallel change in the other. Teitelbaum found that mouse osteoclasts treated with the GC dexamethasone failed to spread out on bone surfaces and had decreased bone-resorbing capacity. This dampened osteoclast activity triggered a subsequent decrease in bone formation, as bone loss was less severe in mice whose osteoclasts (but not osteoblasts) lack the glucocorticoid receptor. Why the decrease in osteoblast activity ultimately outweighed the decrease in osteoclast activity is not yet clear.
The osteoclast defect was traced to a block in the activation of the GTPases RhoA and Rac, which are needed for osteoclasts to rearrange their actin cytoskeletons and thus form a tight seal with bone surfaces. GCs blocked the activation of GTPases by suppressing the phosphorylation of the osteoblast-specific guanine nucleotide exchange factor Vav3.
In the meantime, these data raise potential concerns about the use of antiresorptive agents (such as Fosamax) to treat osteoporosis. Although these agents curb bone loss, they have also been reported to decrease bone remodeling, which causes brittle, fracture-prone bones. “It is not only the amount of bone present [that is important],” says Teitelbaum, “but also the quality of the bone.”
Reference:
A give-and-take between the action of bone-building osteoblasts and bone-resorbing osteoclasts keeps bone mass constant. Both cell types are controlled by an array of regulatory proteins, only some of which have been identified. One of these regulators, described by Laurie Glimcher (Harvard Medical School, Boston, MA), suppresses bone-building activity by marking an osteoblast-specific transcription factor for destruction.
Glimcher's group had previously identified the regulator in question—the zinc finger protein schnurri-3 (Shn3)—as an adaptor protein expressed in CD4 T helper (Th)-1 cells that drives IL-2 expression. But when the group eliminated Shn3 from mice, they ran into an unexpected problem. “My postdoc, Dallas [Jones], was having trouble getting bone marrow from the deficient mice,” says Glimcher, “so we took an x-ray [to find out why].” The x-ray revealed an age-related increase in bone mass so severe that, by seven months of age, the mice had virtually no marrow left.
The Shn3-deficient mice had bulked-up bones due to overly active osteoblasts. Osteoblasts from these mice had increased expression of Runx2, the preeminent transcription factor that drives osteoblast differentiation and function. In normal mice, Glimcher showed, Shn3 binds to the ubiquitin ligase WWP1. Shn3 and WWP1 then team up to ubiquitinate Runx2, tagging it for proteasomal degradation.
Normally, osteoclasts get revved up to compensate for increased osteoblast activity. Why this fails to occur in the absence of Shn3 is unclear, as Shn3-deficient osteoclasts appeared to function normally. The team is now investigating this question.
Reference:
Data presented by Marco Colonna (Washington University, St. Louis, MO) and Mary Nakamura (UCSF, San Francisco, CA) raised more questions about the bone defects in a rare genetic disorder—Nasu-Hakola disease—than they answered.
Previous work by Colonna's group showed that monocytes from patients with Nasu-Hakola disease fail to form bone-resorbing osteoclasts in vitro, suggesting that TREM2 is required for osteoclast differentiation. Nakamura's new mouse data extended this finding, showing that blocking TREM2 with antibodies prevented both the differentiation of osteoclast precursors and the resorptive function of mature osteoclasts.
But Colonna's mouse studies suggest the opposite—precursor cells from mice lacking TREM2 differentiated into osteoclasts more efficiently than wild-type cells. “The problem [with the antibody data],” says Colonna, “is that we don't know whether the antibody is activating or inhibiting [TREM2 signaling].”
Indeed both inhibitory and activating functions for TREM2 have been reported. Ligation of TREM2 on brain cells, for example, increases phagocytosis. On macrophages, its ligation dampens cytokine production.
The avidity of a ligand for TREM2 might determine whether a positive or negative signal is transmitted. The hunt is on for these TREM2 ligands.
Reference:
Inflammatory disease and infection often go hand-in-hand with bone loss. Mucosal infection with the parasite , for example, can destroy bones in the face and palate. Bone destruction in a mouse model of this disease, according to Marlon Quinones (University of Texas, San Antonio, TX), is triggered by activated CD4 T cells, which spur on bone-digesting osteoclasts.
CD4 T cell differentiation is a known determinant of disease outcome in mouse models of leishmaniasis. A Th1 response—dominated by the CD4 T cell production of interferon (IFN)-γ—protects against the parasite, whereas a Th2 response—dominated by the production of interleukin (IL)-4, is associated with susceptibility.
This Th1–Th2 balance, according to Quinones, also contributes to infection-induced bone loss. Mice lacking IFN-γ developed local and systemic bone loss when infected intradermally with due to a massive accumulation of osteoclasts. The overabundance of osteoclasts was initiated by T cells, as transferring IFN-γ–deficient CD4 T cells into lymphocyte-deficient mice triggered bone loss.
Bone destruction did not depend on T cell production of the osteoclast growth factor RANKL, which contributes to bone loss in other diseases, such as rheumatoid arthritis. The real culprit, suspects Quinones, is IL-17, which was produced in excess by the IFN-γ-deficient T cells and is required in other T cell–driven diseases. The group, co-led by Seema Ahuja, is now testing their suspicion by blocking IL-17 in the IFN-γ–deficient mice. |
To identify potential Th17 cytokines, we differentiated naive (CD62LCD4) T cells purified from DO11.10 (DO11) mice to the Th1 (IL-12 and anti–IL-4), Th2 (IL-4 and anti–IFN-γ), and Th17 (TGF-β, IL-6, IL-1β, TNF-α, IL-23, anti–IFN-γ, and anti–IL-4) lineages for 7 d. We then examined the expression of cytokine transcripts by quantitative PCR after restimulation of T cells with PMA and ionomycin. Th1 cells expressed the highest amounts of IFN-γ transcript, Th2 cells had the highest abundance of IL-4, and Th17 cells produced the greatest abundance of IL-17A and IL-17F, demonstrating that these cells were successfully differentiated (). Of 22 additional ILs examined, IL-22 transcript expression was higher in Th17 cells relative to Th1 by 120-fold or Th2 by 700-fold (). In contrast, expression of IL-2, IL-3, IL-5, IL-6, IL-9, IL-10, IL-13, IL- 21, IL-24, IL-25, and IL-31 transcript was equivalent or more abundant in Th1 or Th2 cells relative to Th17 (). Non–T cell–derived cytokines, including IL-1, IL-7, IL-11, IL-15, IL-16, IL-18, IL-19, IL-20, IL-27, and IL-28, were not expressed highly in any of the T cell lineages (Fig. S1, available at ). In summary, IL-22 was expressed at higher amounts by Th17 cells than by Th1 or Th2.
IL-22 is a member of the IL-10 family, along with IL-10, IL-19, IL-20, IL-24, and IL-26 (). Activation of human CD4 T cells with IL-12 and anti–IL-4 enhances IL-22 transcript expression, suggesting that IL-22 is a Th1 cytokine (). However, the expression of IL-22 protein from T cells has not been reported. To study this, we generated monoclonal antibodies to murine IL-22. Naive DO11 T cells activated with irradiated splenocytes and OVA (OVAp) (Th0) produced minimal amounts of IL-22 (<100 pg/ml) as determined by ELISA (). Although IL-22 expression was enhanced during Th1 (110-fold) and Th2 (40-fold) differentiation as compared with Th0, activation with IL-17–inducing conditions resulted in an even greater increase in IL-22 production. TGF-β, IL-6, IL-1β, and TNF-α enhanced IL-22 expression by 360-fold, whereas activation with IL-23, anti–IFN-γ, and anti–IL-4 increased IL-22 production by 460-fold. A combination of these conditions (Th17) yielded the greatest expression of IL-22, 2,400-fold over Th0 and 22-fold higher than Th1. These data demonstrate that IL-22 protein is expressed most abundantly during Th17 differentiation.
Because some IL-22 was induced under Th1 and Th2 conditions during primary T cell activation, we examined whether a secondary stimulation of these cells could enhance IL-22 production. Naive DO11 T cells were activated for 7 d with Th1, Th2, and Th17 conditions, or with TGF-β, IL-6, IL-1β, and TNF-α. Upon restimulation with just OVAp, IL-2, and irradiated splenocytes, cells originally differentiated with TGF-β, IL-6, IL-1β, and TNF-α or with Th17 conditions produced at least fivefold more IL-22 than Th1 or Th2 cells (). The continued differentiation of Th17 cells along the Th17 lineage by restimulating in the presence of IL-23, anti–IFN-γ, and anti–IL-4 enhanced IL-22 production by at least 12-fold over restimulation of cells with OVAp alone or with IL-12, anti–IL-4. In contrast, IL-22 production was not enhanced by restimulation of Th1 cells with IL-12, anti–IL-4 or of Th2 cells with IL-4, anti–IFN-γ. These results show that further differentiation toward Th1 or Th2 does not enhance IL-22 production. In addition, restimulation of Th1 and Th2 cells with IL-23, anti–IFN-γ, and anti–IL-4 did not enhance IL-22 production to that observed with Th17 cells activated under the same condition. These data demonstrate that IL-23 is more potent than IL-12 in stimulating IL-22 expression and that Th17 cells are the major producers of IL-22.
We also examined whether IL-22 could modulate proliferation or IFN-γ, IL-4, and IL-17A production from naive, Th1, Th2, and Th17 cells. We did not observe any changes when T cells were treated with exogenous IL-22 (Fig. S2, available at ). The transcript for the high affinity receptor subunit, IL-22R1, was not detected in any T cell population. IL-17A or IL-17F also did not induce IL-22 expression from naive, Th1, Th2, or Th17 cells (not depicted). These results demonstrate that IL-22 and IL-17A/IL-17F do not modulate each other's expression by CD4 T cells.
We next performed intracellular cytokine staining to determine if IL-22 can be coexpressed with IL-17A. Cells activated under Th0, Th1, or Th2 conditions had minimal expansion of IL-22 cells (≤0.2%; ). Activation under Th17 conditions generated a substantial population of IL-22–expressing cells (8.7%), with 81% of IL-22 cells expressing IL-17A and only 1% expressing IFN-γ. We further examined the roles of individual cytokines under Th17 differentiation conditions. Only 0.2% of cells activated with exogenous TGF-β expressed IL-22 (). Activation with IL-6, IL-1β, and TNF-α enhanced IL-22 cells (1.9%). The use of a neutralizing antibody to TGF-β indicated that endogenous TGF-β is important for optimal expression of IL-22 induced by IL-6, IL-1β, and TNF-α (Fig. S3, available at ). The addition of exogenous TGF-β to IL-6, IL-1β, and TNF-α further increased IL-22 cells (2.8%), with 62% of IL-22 cells expressing IL-17A or IL-17F (). Activation with IL-23, along with TGF-β, IL-6, IL-1β, and TNF-α, led to a threefold increase in IL-22 cells (9.5%). 80% of IL-22 cells produced either IL-17A or IL-17F, with 44% expressing both IL-17A and IL-17F. In summary, these data demonstrate that IL-22 protein is produced at greater amounts by Th17 cells and that IL-22 is coexpressed with both IL-17A and IL-17F during Th17 differentiation.
To further examine how IL-23 enhances IL-22 expression, we differentiated CFSE-labeled naive DO11 T cells to Th17 with various cytokines and analyzed the expression of IL-22 from days 1 to 5 of culture. Cells activated with only TGF-β and IL-6 peaked in IL-22 (14%) expression on day 2 and decreased substantially by day 3 (). Neither TNF-α, IL-1β, nor IL-12 addition prevented the decrease in expression of IL-22 observed after day 2. In contrast, cells activated with IL-23 as well as TGF-β and IL-6 expressed at least fivefold more IL-22 on day 4. To examine if IL-23 was inducing the expansion of IL-22–producing cells, we analyzed the CFSE dilution profiles of cells expressing IL-22 and/or IL-17A on day 4 (). We did not observe any differences in CFSE between IL-22IL-17A and IL-22IL-17A cells activated with TGF-β and IL-6 alone or when supplemented with TNF-α, IL-1β, or IL-23. This suggests that there is no correlation between IL-17A expression and proliferation. When we examined the CFSE profiles of IL-22IL-17A and IL-22IL-17A cells activated with TGF-β and IL-6, we observed that these cells had proliferated less than IL-22 IL-17A and IL-22IL-17A cells. Similar findings were observed in cultures supplemented with IL-1β, TNF-α, or IL-12 ( and not depicted). In contrast, IL-23 in the context of TGF-β and IL-6 enhanced the proliferation and expansion of IL-22IL-17A and IL-22IL-17A cells (). These findings demonstrate that IL-23 drives the expansion of IL-22–producing cells in the Th17 lineage.
To examine if endogenous IL-23 is necessary for optimal IL-22 expression, we activated naive DO11 T cells with LPS-treated DCs in the presence of anti–IL-23R to block IL-23 signaling or anti–IL-12p40 to neutralize both IL-12 and IL-23. Neutralization of IL-23R reduced IL-22 production by 62% (at 1 μg/ml OVAp) as compared with isotype control (). A similar reduction of IL-22 expression was observed with anti–IL-12p40 (64%), suggesting that IL-23, and not IL-12, is responsible for the majority of IL-22 production. Collectively, these data demonstrate that IL-23 is required for optimal expansion of IL-22–producing cells.
Our in vitro data demonstrate that IL-22 is coexpressed with IL-17A and IL-17F. To examine if this population exists in vivo, we performed intracellular cytokine staining on draining LN cells harvested from C57BL/6 mice that had been immunized with OVA/CFA 7 d earlier. Immunization with OVA/CFA increased the expansion of IL-22 (0.34%), IL-17A (0.35%), and IL-17F (0.43%) cells as compared with unimmunized mice (). IL-22 was coexpressed with IL-17A (44% of IL-17A cells were IL-22) and IL-17F (45% of IL-17F cells were IL-22), but not with IFN-γ, IL-4, or IL-10 (). We also did not observe any coexpression of IL-17A or IL-17F with IFN-γ, IL-4, or IL-10 (not depicted). We did detect considerable, but not complete, coexpression between IL-17A and IL-17F (). These results demonstrate a heterogeneity of IL-17A and IL-17F expression within Th17 cells. We further examined IL-22 expression in different populations of IL-17A– and IL-17F–producing cells and observed the highest IL-22 expression in IL-17AIL-17F cells (53%; ). We also analyzed the expression of IL-17A and IL-17F in IL-22 cells. The majority (70%) of IL-22 cells expressed either IL-17A or IL-17F, with 45% of IL-22 cells expressing both (). These in vivo expression profiles among IL-17A, IL-17F, and IL-22 are similar to the expression profiles generated in vitro with TGF-β, IL-6, IL-1β, TNF-α, and IL-23 (), suggesting that this in vitro condition is sufficient to replicate in vivo Th17 differentiation. Similar results were also observed on days 4 and 10 after immunization (not depicted). These data demonstrate that IL-22 is not coexpressed with IFN-γ, IL-4, and IL-10 in vivo, but rather with IL-17A and IL-17F.
To examine if IL-12 or IL-23 stimulates IL-22 production from in vivo–primed T cells, LN cells were restimulated in the presence of exogenous IL-12 or IL-23. The addition of IL-23 enhanced the production of IL-22 by sevenfold compared with OVA alone, whereas IL-12 had no effect (). These data further demonstrate that IL-23, rather than IL-12, is the stimulus for enhancing IL-22 production.
One function of IL-22 is to enhance the expression of antimicrobial peptides associated with host defense, including β-defensin 2 (hBD-2), S100A7, S100A8, and S100A9 (, ). To examine whether IL-17A, IL-17F, and IL-22 can act cooperatively to regulate these genes, we treated primary keratinocytes with individual or paired combinations of these cytokines. IL-17A enhanced transcript expression of all four antimicrobial peptides (5–70-fold induction at 200 ng/ml; ). IL-22 also induced all four transcripts (two- to fivefold at 200 ng/ml), whereas IL-17F (200 ng/ml) induced hBD-2 by eightfold, S100A8 by 1.5-fold, and S100A9 by twofold, but did not up-regulate S100A7. We then cultured keratinocytes with paired cytokine combinations. Treatment with IL-22 (200 ng/ml) and IL-17A (20 ng/ml) led to a synergistic increase of hBD-2 (IL-22, fivefold; IL-17A, 70-fold; IL-22IL-17A, 180-fold) and S100A9 (IL-22, twofold; IL-17A, fivefold; IL-22IL-17A, 13-fold) and an additive increase of S100A7 and S100A8 expression (). Treatment with IL-22 (200 ng/ml) and IL-17F (20 ng/ml) also synergistically enhanced hBD-2 (IL-22, fivefold; IL-17F, twofold; IL-22IL-17F, 20-fold). Even though S100A7, S100A8, and S100A9 were not up-regulated by IL-17F (20 ng/ml) alone, IL-17F plus IL-22 enhanced the expression of these three peptides by twofold over IL-22 alone. These data demonstrate that IL-22 can act cooperatively, either synergistically or additively, with IL-17A or IL-17F. Keratinocytes treated with a combination of IL-17A and IL-17F enhanced S100A8, but did not further enhance expression of hBD-2, S100A7, or S100A9. The combination of IL-17A and IL-17F resulted in less induction of these genes than the combination of IL-22 with IL-17A or IL-17F. Expression of receptors for IL-22 (IL-22R1) or IL-17 (IL-17RA) were not altered by IL-22, IL-17A, or IL-17F (not depicted), suggesting that these effects are not related to changes in receptor expression. These data demonstrate that IL-22 in combination with IL-17A or IL-17F cooperatively enhances the expression of antimicrobial peptides.
IL-22 was initially characterized as a Th1 cytokine because IL-12 treatment of T cells up-regulates IL-22 mRNA (). Here, we show that IL-22 protein is expressed in the Th17 lineage at substantially higher amounts than Th1 or Th2, revealing a new effector cytokine secreted by Th17 cells. Despite IL-22 being a Th17 cytokine, the IL-22 locus is located ∼90 kb in proximity to IFN-γ. The distinct expression pattern of IL-22 and IFN-γ suggests cis-regulatory elements exist near these loci that regulate the differentiation of Th1 versus Th17 cells. Further analysis of these loci may improve our understanding of the elements that control T cell differentiation.
Our data also define a new function for IL-23: the induction of IL-22 from T cells. Although IL-17A is a critical cytokine induced by IL-23, certain data suggests that IL-17A may not account for all the downstream effects of IL-23. For example, IL-23p19–deficient mice are completely resistant to disease in collagen-induced arthritis, whereas IL-17A–deficient mice remain susceptible, albeit with a significantly reduced incidence and severity (, ). Also, IL-23p19–deficient mice are very susceptible to infection despite maintaining wild-type expression of IL-17A (). IL-22 may play a role in these in vivo model systems.
The coexpression of IL-22 with IL-17A and IL-17F from T cells and the presence of their receptors on fibroblast and epithelial cells suggest that these cytokines may act together to regulate local tissue inflammation. One mechanism is by the induction of cytokine and chemokine expression. Similar to IL-17A and IL-17F, IL-22 induces IL-6, CXCL8, and MCP-1 expression by fibroblast cells (, , ). On colonic myofibroblasts, IL-22 and IL-17A additively enhance expression of CXCL8, IL-6, and LIF, as well as activation of transcription factors, further demonstrating a cooperative signaling relationship between these cytokines (). IL-22, in contrast to IL-17A, did not enhance mRNA expression for a variety of chemokines from primary keratinocytes (not depicted), suggesting that the functions of IL-22 and IL-17A are cell type dependent. We have demonstrated that IL-22 can function in synergy with IL-17A or IL-17F to enhance keratinocyte expression of antimicrobial peptides. These peptides are known to have potent activity against bacteria such as , supporting the idea that Th17 cells may have evolved to combat extracellular pathogens (, ). Although these data point to a function for IL-22 in inflammation, the essential role of IL-22 and its interplay with IL-17A and IL-17F in vivo are not well understood. The coexpression of IL-22 with IL-17A and IL-17F adds a new layer of complexity and prompts new directions for future investigation on the development and functions of the Th17 lineage.
Murine IL-4, IL-6, IL-12, IL-23, TNF-α, and GM-CSF and human IL-17A were purchased from R&D Systems. Murine IL-2 and human TGF-β were purchased from Sigma-Aldrich. Murine IL-1β was obtained from Bender MedSystems. IL-22 and IL-17F cytokines were prepared by previously described methods (). Antibodies to IFN-γ (XMG1.2), IL-4 (BVD4-1D11), IL-17A (TC11-18H10), IL-10 (JES5-16E3), and CD4 (RM4-5) were purchased from BD Biosciences. Anti–IL-12p40 (C17.8) and anti–IL-23R were obtained from R&D Systems. Anti-DO11 antibody (KJ126) was purchased from Caltag Laboratories. Antibodies to murine IL-22 (Ab-01, Ab-02, and Ab-03) and murine IL-17F (RK015-1) were generated by methods as described previously.
BALB/cByJ, C57BL/6, and C.Cg-Tg(DO11.10)10Dlo TCR transgenic mice were purchased from The Jackson Laboratory. All mice were used between 6 to 10 wk of age. Mice were immunized subcutaneously in the flanks with 100 μg OVA (Sigma-Aldrich) emulsified in CFA (Sigma-Aldrich). 7 d later, inguinal LNs were harvested. All mice were maintained in strict accordance with Wyeth Research Institutional Animal Care and Use Committee regulations.
Naive (CD62LCD4) T cells were purified from the spleens of DO11 mice by CD4 negative selection followed by CD62L positive selection according to the manufacturer's directions (Miltenyi Biotec). In a 24-well plate, 2 × 10 DO11 T cells were cultured with 4 × 10 irradiated BALB/cByJ splenocytes (3,300 rad) and 1 μg/ml OVA peptide (OVAp; New England Peptide). Recombinant cytokines and neutralizing antibodies were used at the following concentrations: 10 ng/ml IL-12, 10 ng/ml IL-4, 1 ng/ml TGF-β, 20 ng/ml IL-6, 10 ng/ml IL-1β, 10 ng/ml TNF-α, 10 ng/ml IL-23, 10 μg/ml anti–IFN-γ, and 10 μg/ml anti–IL-4. In some cases, cells were labeled with CFSE (Invitrogen) according to the manufacturer's directions. For restimulation cultures, cells were harvested on day 7 of primary stimulation, washed extensively, and rested overnight. 2 × 10 DO11 T cells were restimulated with 4 × 10 irradiated splenocytes, 5 ng/ml IL-2, and various conditions as indicated. Conditioned media was collected on day 5 for optimal cytokine accumulation. For generation of DCs, BM cells were cultured with 10 ng/ml GM-CSF and 1 ng/ml IL-4 for 7 d. After purification by CD11c positive selection (Miltenyi Biotec), DCs were matured for 24 h with 1 μg/ml LPS ( serotype 0111-B4; Sigma-Aldrich). DCs were then washed, and 10 DCs were cultured with 2 × 10 purified naive DO11 T cells, OVAp, and 10 μg/ml anti–IL-12p40, anti–IL-23R, or relevant isotype controls in a 96-well plate. For ex vivo restimulations, 4 × 10 inguinal LN cells were restimulated with 200 μg/ml OVA alone or with 10 ng/ml exogenous IL-12 or IL-23. All lymphocyte cultures were grown in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 5 mM Hepes, 100 U/ml Pen-Strep, and 2.5 μM β-mercaptoethanol.
Naive DO11 T cells were differentiated to Th1, Th2, and Th17 as described above. On day 7, CD4 T cells were purified by positive selection (Miltenyi Biotec) and rested overnight. Cells were then restimulated with 50 ng/ml PMA (Sigma-Aldrich), 1 μg/ml ionomycin (Sigma-Aldrich), and with the following conditions: Th1 cells (IL-12, anti–IL-4), Th2 cells (IL-4, anti–IFN-γ), or Th17 (IL-23, anti–IFN-γ, anti–IL-4) for 6 h before RNA was isolated. Quantitative PCR for cytokine transcripts was performed using SYBR Green Platinum Taq (Invitrogen) and prequalified primers (QIAGEN). The ΔΔCt method was used to normalize transcript to HPRT and to calculate fold induction relative to purified, unactivated naive DO11 T cells.
Cells were restimulated with 50 ng/ml PMA, 1 μg/ml ionomycin, and GolgiPlug (BD Biosciences) for 6 h. Cells were first stained for surface antigens and then treated with Cytofix/Cytoperm (BD Biosciences) according to the manufacturer's directions. Intracellular cytokine staining was performed using PE-conjugated antibodies to IFN-γ, IL-4, IL-10, and IL-17A as described above. Anti–IL-22 (Ab-02) was labeled with Alexa 647 (Invitrogen) and anti–IL-17F (RK015-1) was labeled with FITC (Pierce Biotechnologies) according to the manufacturer's directions.
Antibody pairs (coating, detection) were used to detect IFN-γ (AN-18, R4-6A2; eBioscience), IL-17A (MAB721, BAF421; R&D Systems), and IL-22 (Ab-01, biotinylated Ab-03) by standard sandwich ELISA.
Primary human keratinocytes (ScienCell) were cultured in keratinocyte medium (ScienCell) on human fibronectin-coated plates (BD Biosciences). Cells were passaged at 80% confluency and all experiments were done between passages 2 and 4. For evaluation of cytokine effects, 15,000 cells were seeded into a 24-well plate and allowed to adhere for 48 h. Cells were then treated with human IL-22, IL-17A, and IL-17F for 44 h. RNA was purified and quantitative PCR was performed using Taqman Real-Time PCR and prequalified primer probes (Applied Biosystems). Fold induction was calculated as described above.
Fig. S1 shows the mRNA expression of non–T cell–derived cytokines by Th1, Th2, and Th17 differentiated cells. Fig. S2 shows the effects of IL-22 on naive and differentiated T cells. Fig. S3 shows the role of endogenous TGF-β on IL-22 expression induced by IL-6, IL-1β, and TNF-α. Figs. S1–S3 are available at . |
Polyoma virus (PyV) establishes a systemic persistent infection in mice () (). CD8 T cell responses to PyV infection are detected via binding of D tetramers complexed to an immunodominant epitope encoded by aa 359–368 of the viral large T protein (LT359) (). After the acute phase of infection, LT359-specific CD8 T cells are detected as late as 560 d after infection (). At 246 d after infection, this population of antigen-specific cells is heterogeneous for expression of CD27, CD62L, CD127, and bcl-2 (). In addition, viral DNA is detectable in the spleen and other organs for at least that length of time ( and not depicted) (). We asked whether this phenotypic heterogeneity represented differentiation of cells primed during the acute phase of infection or resulted from an amalgam with cells newly generated during persistent infection.
We assessed the capacity of antiviral CD8 T cells to survive during persistent infection. T cells from mice infected by PyV 150 d earlier were transferred into infection-matched congenic hosts (). LT359-specific CD8 T cells of both host and donor origin were monitored over time. Whereas host CD8 T cells were maintained at a constant level, donor cells decreased over time (); the most rapid decline occurred over the first 3 wk, followed by a slower rate of attrition. This finding was unexpected because of the slower kinetics of virus-specific CD8 T cell loss during persistent infection in unmanipulated mice (). The transferred cells did not divide over 34 d (), although PyV-infected mice support homeostatic division of conventional memory CD8 T cells (), indicating that this cell loss is a cell-intrinsic phenomenon. Similarly, CD8 T cells from mice persistently infected by lymphocytic choriomeningitis virus (LCMV) fail to undergo homeostatic proliferation when transferred to naive mice (). Because the PyV-specific host and donor CD8 T cells were elicited and maintained under identical conditions in this transfer experiment and coexist in the same environment, this data is inconsistent with the concept that antigen maintains the survival of antiviral CD8 T cells during chronic infection. To reconcile this discrepancy, we reasoned that host cells, but not donor cells, can be resupplied through thymic output, and perhaps new, naive PyV-specific CD8 T cells were being generated and subsequently primed during persistent infection.
To test this hypothesis, we induced partial hematopoietic chimerism in persistently infected mice using congenic bone marrow (). Such animals now have stem cells that give rise to a population of naive CD8 T cells distinguished either by host or donor allelic differences (). This protocol permits stem cell engraftment without irradiation, thereby limiting the perturbance of viral load or established T cell populations, as preexisting PyV-specific T cells are not affected by busulfan treatment alone (unpublished data) (). Moreover, the emergence of donor naive T cells beginning at ∼1 mo after busulfan conditioning is thymus dependent (). Additionally, this protocol does not replace all bone marrow stem cells, and the population of newly developing T cells will be a mix of donor and host cells. 50 d after infusion of congenic bone marrow in busulfan-conditioned mice infected by PyV 115 d previously, animals were analyzed for PyV-specific CD8 T cells. As shown in , host-derived tetramer cells were detected in chimeric animals. Interestingly, a population of PyV-specific CD8 T cells originating from the donor population was also detected. This was not caused by contaminating naive or memory CD8 T cells in the donor cell inoculum, because transfer of the same number of bone marrow cells or splenocytes without busulfan did not generate a detectable donor cell population (). It has been estimated that thymic education occurs over the course of 3–4 wk (). This is consistent with the inability to detect donor antigen-specific T cells earlier than 30 d after busulfan conditioning (unpublished data). Recruitment of recent thymic emigrants during the persistent stage of infection was not limited to PyV infection. We performed the same chimeric induction protocol in mice infected 120 d earlier by LCMV clone 13; at this time point, LCMV is only detected in the kidneys and brain (). It is important to mention that this LCMV–mouse model is different from other models of persistent LCMV infection in which virus is detectable in the thymus (e.g., congenitally infected carrier mice, CD4-depleted LCMV clone 13–infected adult mice, and LCMV clone 13–infected adult mice approximately day 30 after infection) (, ), but it more closely approximates the low-level viral replication seen in persistent PyV infection and many persistent viral infections in humans. depicts the presence of donor and host LCMV-specific CD8 T cells in chimeric animals.
To assess the contribution of newly primed T cells to the long-term pool of antiviral CD8 T cells, we thymectomized naive mice and determined the number of PyV-specific CD8 T cells after infection. Thymectomy significantly reduced the number of tetramer CD8 T cells (). This is consistent with our interpretation that continuous priming of new thymic emigrants is required for the maintenance of the population of virus-specific CD8 T cells. Our data also support the conclusions of a recent study exploring the contribution of thymic emigrants to the CD4 T cell response, where prolonged antigen presentation occurs, despite the lack of infectious virus ().
Because the T cell priming environment may vary between acute and persistent phases of infection and differentially affect T cell programming, we also analyzed PyV-specific CD8 T cells for the expression of markers of differentiation. As shown in , there are variations in expression of CD27, CD62L, CD127, and bcl-2 between the cell populations primed at different times. When the expression of either CD27 or CD62L is monitored over time on the bulk population of PyV-specific T cells, an interesting pattern emerges (). At the peak of CD8 T cell expansion, the majority of cells are CD27. These cells quickly down-regulate surface expression of this molecule, but the proportion of CD27 cells progressively increases over time. By 560 d after infection, the majority of PyV-specific CD8 T cells are CD27. A similar pattern is observed with CD62L. This is consistent with the concept that newly primed T cells, being CD27 and CD62L (), are productively contributing to the pool of antigen-specific CD8 T cells and modify the phenotype of the entire virus-specific CD8 T cell population. Current evidence suggests that CD27 T cells do not regain expression of CD27 and preferentially die (). The data in (A and B) are in line with the interpretation that the CD27 PyV-specific CD8 T cell population is undergoing progressive attrition. Because PyV antigen is present up to 300 d after infection (), it is likely that antigen modifies the population of tetramer cells in at least two ways: antigen is required to prime recent thymic emigrants, which have a CD27 CD62L phenotype, but antigen can also induce down-regulation of CD27 and CD62L. Antigen-independent events may modulate the expression of these molecules as well.
Newly recruited antiviral CD8 T cells may differ qualitatively from their older counterparts. We have found that the number of newly primed cells making IFN-γ closely corresponds to the number of D-LT359 tetramer cells (unpublished data). A comprehensive comparison of the functional attributes of new and old antiviral CD8 T cells should prove particularly informative in elucidating the contribution of each cell population to viral control.
Analysis of chimeric animals at late time points () reveals a narrowing in the phenotype of virus-specific CD8 T cells primed over the course of persistent infection (compare with ). Although some differences are still apparent, such as bcl-2 levels, by 140 d after induction of chimerism, splenic CD27 levels by host and donor PyV-specific T cells are similar. Because chimerism induction does not displace the original host stem cells (), new naive T cells can originate from either donor or recipient precursors. The priming environment is also dynamic over time in terms of antigen load and virus-associated inflammation, with these changes affecting the strength and frequency of T cell activation and, therefore, longevity. Jelley-Gibbs et al. recently demonstrated that influenza virus–specific CD4 T cells primed late in the response survive better than those recruited earlier, suggesting that early and protracted antigen encounter is detrimental to T cell survival (). Collectively, these findings imply that many of the virus-specific CD8 T cells late in persistent infection may be derived from more recently primed cells.
Recent studies indicate a dependence on antigen for maintenance of antiviral CD8 T cells in chronic infections, although there are some exceptions (–, ). Our data demonstrate that new, naive virus-specific CD8 T cells are primed to viral antigens long after initial infection. Priming of newly emerging CD8 T cells to persisting antigens has been suggested before, and a recent report shows de novo CD4 T cell responses to chronic infections in baboons after autologous bone marrow transplant (–). In a chronic LCMV infection model, continuous thymic output does not appear to be required to maintain CD8 T cell responses, although a T cell deficit was observed at day 30 after infection (). In contrast, thymectomy is associated with loss of CTL activity in cats persistently infected by a feline lentivirus (). Our data are consistent with reports describing antigen dependence for chronic pathogen-specific T cell maintenance, especially as newly primed cells may be important for preserving the population of virus-specific T cells () (–). Moreover, the new, naive antigen-specific CD8 T cells are able to survive thymic education, despite persisting antigen, and exit to the periphery.
It has been suggested that memory-like CD8 T cells in chronic infections go through an abortive differentiation pathway, as dissected through phenotypic analysis of CD8 T cells in infected individuals (, , ). The lineage relationship between these stages is comprehensible only if the end cell stems from the beginning cell or its progeny. Our data indicate that new naive cells are continuously being recruited during the chronic phase of infection and that the time point of priming influences the phenotype of CD8 T cells. These observations affect our understanding of T cell differentiation pathways during chronic infections, as the T cell population analyzed will potentially contain cells primed at any time after infection.
In summary, we propose that ongoing selection and priming of naive CD8 T cells during persistent infection contributes to the maintenance of chronic memory-like cells. Phenotypic heterogeneity indicates programming differences in cells primed more recently, and their continual entry into the virus-specific T cell reservoir complicates interpretation of current models of lineage differentiation. In other situations in which antigen persists, such as cancer, autoimmunity, and organ transplantation, it will be important to address whether naive T cells continue to be recruited. Our findings may offer new avenues for therapeutic vaccination, as newly recruited cells may be more amenable to boosting strategies.
C57BL/6 (B6) female mice were purchased from the National Cancer Institute. B6/CD45.1 female mice were purchased from Taconic or bred in our facility. P14 transgenic mice bearing the TCR specific for the gp33-41 epitope of LCMV were bred on a B6/CD90.1 background in our facility. All mice were used between 4 and 10 wk of age. Thoracic thymi were removed from anesthetized mice, which were rested for at least 2 wk before infection. All protocols for animal studies were approved by the Institutional Animal Care and Use Committee of Emory University.
DNA was extracted from frozen tissue using a DNA mini kit (QIAamp; QIAGEN). PyV genomic copy numbers were determined by quantitative PCR (TaqMan; Glen Research Corporation), as described previously (). PyV DNA quantity is expressed in genome copies per milligram of tissue and is calculated based on a standard curve of known PyV genome copy numbers versus threshold cycle of detection. The detection limit with this assay is 10 copies of genomic viral DNA.
Mice were infected with 2 × 10 PFU of PyV (strain A2) s.c.; 2 × 10 PFU LCMV clone 13 was administered i.v. Spleens from PyV-infected mice were harvested at the indicated time points and depleted of IgG cells by panning on plates coated with anti-IgG antibodies (The Jackson Laboratory). Cells were labeled with 5 μM CFSE (Invitrogen) for 10 min at 37°C. 50 × 10 CFSE-labeled cells were transferred to infection-matched congenic hosts. Naive B6/CD90.2 mice received 5 × 10 naive P14 cells i.v. 1 d before i.p. infection by 2 × 10 PFU of LCMV-Armstrong. 120 d after infection, memory P14 cells were isolated from spleens and labeled with CFSE, and 20 × 10 cells were transferred to naive B6/CD90.2 mice or B6/CD90.2 mice infected by PyV 200 d earlier.
Femurs and tibias were crushed to isolate bone marrow cells. Single cell suspensions were made of RBC-lysed spleens. Animals were perfused before lung and liver isolation. Lung pieces were treated with EDTA and digested with collagenase with subsequent density centrifugation to isolate lymphocytes, as previously described (). Single-cell suspensions were made from liver by passing tissue through nylon mesh, followed by density centrifugation to isolate lymphocytes (). Antibodies to CD8, CD90.1, and bcl-2 were purchased from Becton Dickinson and used according to the manufacturer's specifications. Antibodies to CD27, CD45.1, CD45.2, CD62L, and CD127 were purchased from eBioscience. Antibodies to KLRG1 were purchased from Southern Biotechnology Associates, Inc. Tetramers were constructed as previously described (). All samples were collected on a FACSCalibur (BD Biosciences) and analyzed with FlowJo software (Tree Star, Inc.).
Mice persistently infected either by PyV or LCMV clone 13 at least 35 d earlier received 600 μg busulfan i.p. (Busulfex; Orphan Medical). The next day, 25 × 10 cells isolated from the bone marrow of congenic mice were injected i.v.
Statistical significance for individual experiments was determined using the unpaired Student's test. The Mack-Skillings test was used to determine statistical significance of multiple experiments. A p-value ≤0.05 was considered statistically significant. |
Using a T cell transfer model of colitis triggered by adoptive transfer of CD4 T cells into Rag recipients, we previously described the appearance of disease-protective CD4 IL-10–secreting T regulatory cells after infection of WT animals (). Here we demonstrate that the CD4-mediated T regulatory cell mechanism revealed in the Rag transfer model also functions in intact WT hosts, as treatment of these animals with anti–IL-10R, but not a control mAb, led to the development of typhlocolitis similar to that seen in IL-10 mice (, and ). Uninfected WT mice given anti–IL-10R did not develop intestinal inflammation (not depicted), demonstrating that the endogenous flora by itself was not sufficient to induce colitis, but that was required for disease initiation.
In agreement with our earlier findings of a critical role for p40 in colitis induction (, ), p40 mice treated with anti–IL-10R failed to develop typhlocolitis (, and ). Importantly however, anti–IL-10R–treated p35 mice, which lack the ability to produce IL-12, developed intestinal inflammation comparable to that seen in anti–IL-10R–treated WT mice (, and ). Collectively, these findings demonstrate that although the IL-12p40 subunit is essential for colitis development, IL-12 in itself is dispensable for disease pathogenesis.
To investigate the role of IFN-γ, mice deficient in this cytokine were infected with and treated with anti–IL-10R. Although the degree of cecal inflammation was comparable to that observed in colitic WT and p35 animals ( and ), IL-10R blockade led to only a mild inflammatory response in the colon of IFN-γ animals ( and ), suggesting a role for IFN-γ in exacerbating disease at this site. This conclusion was supported by similar findings in an adoptive transfer model (see below), emphasizing a role for IFN-γ in promoting colonic inflammation.
To examine –specific T cell cytokine responses, mesenteric lymph node (MLN) cells were stimulated in vitro with soluble antigen (SHelAg) and levels of IFN-γ and IL-17 were measured. After bacterial antigen stimulation, MLN cells from IL-10 mice mounted a strong IFN-γ response and secreted substantial amounts of IL-17, whereas MLN cells from WT mice given control mAb failed to produce these cytokines (). Similarly, no IFN-γ or IL-17 was detected in SHelAg-stimulated MLN cultures from control mAb–treated p40, p35, or IFN-γ mice. In contrast, MLN cells from anti–IL-10R–treated WT mice secreted elevated amounts of both IFN-γ and IL-17 in response to SHelAg (). Interestingly, a similar picture was seen for anti–IL-10R–treated p35 mice, with a significant IFN-γ response and markedly enhanced IL-17 levels after SHelAg stimulation (). Importantly, although displaying attenuated colonic inflammation, MLN cells from anti–IL-10R–treated IFN-γ mice exhibited a strong SHelAg-induced IL-17 response (). Intracellular cytokine staining of MLN cells from anti–IL-10R–treated WT and IL-10 mice demonstrated that SHelAg-induced IL-17 and IFN-γ were mainly produced by distinct populations of CD4 cells, with a smaller fraction of cells staining positive for both cytokines (). In preliminary experiments, anti–IL-10R–treated IFN-γ mice demonstrated an increased frequency of IL-17 CD4 cells after SHelAg stimulation (1.8–4.7-fold increase compared with similarly treated WT mice; data from two independent experiments). Collectively, these results indicate that although Th17 responses may be sufficient to induce severe cecal inflammation, maximal inflammation in the colon also depends on IFN-γ.
To examine cytokine responses at the site of inflammation, mRNA levels for p19, p35, p40, IL-17A, IL-17F, and IFN-γ were measured in colon samples from WT mice given control or anti–IL-10R mAb. As shown in , both groups showed an approximately twofold increase in p19 levels compared with uninfected controls. In contrast, p35 levels were slightly reduced in both groups, whereas p40 levels were unchanged in control mAb–treated WT mice but increased >20-fold in anti–IL-10R–treated WT mice. Interestingly, mRNA levels for IL-17A, IL-17F, and IFN-γ were approximately 40-fold, ninefold, and 150-fold higher in anti–IL-10R–treated compared with control mAb–treated WT mice ().
To examine if the IL-12p40 subunit is also required for disease development in the absence of IL-12, p35 mice were infected with and treated with anti–IL-10R alone or anti–IL-10R plus anti-p40 mAb. As shown in , anti-p40 treatment completely abrogated both cecal and colonic inflammation in anti–IL-10R–treated WT or p35 mice. In addition, anti-p40 treatment blocked SHelAg-induced IFN-γ and IL-17 secretion observed in MLN cultures from the same groups of mice (). Importantly, anti-p40 treatment also prevented the cecal inflammation observed in IFN-γ mice given anti–IL-10R (), and this correlated with a complete loss of SHelAg-induced IL-17 production (). Collectively, these findings confirmed a crucial role for p40 in the development of –induced intestinal inflammation even in the absence of IL-12, and again indicated a correlation between IL-17, IFN-γ, and pathology.
In light of the shared p40 subunit between IL-12 and IL-23, we reasoned that the p40-dependent, but IL-12–independent induction of –triggered colitis was likely to be mediated by IL-23. As various cytokine-deficient Rag animals were available to us, we next turned to an adoptive transfer model of colitis using Rag mice deficient in IL-12 (p35Rag), IL-23 (p19Rag), or both IL-12 and IL-23 (p40Rag) to further elucidate the role of IL-23 in disease development. In this model, colitis is induced in an IL-10–sufficient setting in B6 or B10 Rag recipients by transfer of CD4 T cells (). We first confirmed a critical role for p40 in the adoptive transfer model, as p40Rag mice failed to develop typhlocolitis after transfer of WT CD4 CD45RB cells () or IL-10 CD4 cells (not depicted). In contrast, Rag and p35Rag mice developed severe typhlocolitis after reconstitution with the same T cell populations (, and not depicted). Importantly, anti-p40 treatment inhibited intestinal inflammation in T cell–reconstituted Rag and p35Rag animals (), demonstrating a requirement for p40 but not IL-12 in disease induction also in this model. In contrast, anti–IFN-γ treatment had no effect on cecal pathology () but significantly reduced the inflammation in the colon of the Rag and p35Rag recipients (). Similarly, although WT and IFN-γ CD4 CD45RB cells induced a similar degree of cecal inflammation in Rag mice (), recipients of IFN-γ CD4 cells showed reduced inflammation in the colon (). These findings are consistent with the previously observed difference in disease severity in the cecum versus colon of IFN-γ mice treated with anti–IL-10R (, and ), and further support an important role for T cell–derived IFN-γ in disease pathogenesis in the colon.
To directly assess the contribution of IL-23 in the development of colitis in the /Rag transfer model, an additional series of experiments was performed that included p19Rag recipients, which lack the ability to produce IL-23. As shown in , CD4 T cell–reconstituted p19Rag mice displayed attenuated intestinal inflammation compared with Rag recipients, demonstrating a crucial role for IL-23 in disease pathogenesis. However, compared with disease-free p40Rag recipients, some residual intestinal inflammation was observed in T cell–reconstituted p19Rag mice (). These data suggest that, although not absolutely required for intestinal inflammation, IL-12 contributes to the mild intestinal inflammation observed in the absence of IL-23. Nevertheless, of the two cytokines, IL-23 appears to be more important in the disease process, as the inflammation present in p19Rag recipients was significantly reduced compared with that observed in p35Rag hosts (). Importantly, flow cytometric analysis revealed no significant difference in CD4 cell numbers in the spleens of p19Rag and p35Rag recipients (not depicted), demonstrating that the attenuated pathology in p19Rag mice was not due to lower T cell reconstitution. Moreover, there was no correlation between degree of inflammation and colonization levels, as all groups harbored similar concentrations of in their cecal contents ().
We next quantitated cytokines in colon homogenates from the different groups of T cell–reconstituted Rag mice. As shown in , the absence of both IL-12 and IL-23 led to abolished TNF-α, IFN-γ, MCP-1, and IL-6 levels in colon homogenates of T cell–reconstituted p40Rag animals. IL-12–deficient p35Rag recipients showed similar or marginally decreased levels of proinflammatory cytokines compared with Rag recipients. Finally, the absence of IL-23 in p19Rag mice led to a further reduction in proinflammatory cytokine production (), emphasizing the crucial role for IL-23 in mediating this response.
Before the discovery of IL-23 and its recently documented role in autoimmune disorders (–), the common view in the field of Th1-mediated intestinal inflammation has been that IL-12, by initiating and maintaining Th1 responses, is crucial for disease pathogenesis. We here demonstrate that IL-23 and not IL-12 is essential for the induction of maximal bacterial-induced T cell–dependent colitis. This conclusion is based on results from two complementary models of intestinal inflammation triggered by . Using an established CD4 T cell adoptive transfer model of colitis (), we observed that although severe intestinal inflammation was still induced in IL-12–deficient (p35Rag) recipients, selective ablation of IL-23 (p19Rag) resulted in attenuation of disease. In addition, using a novel model of colitis involving anti–IL-10R treatment of WT animals that circumvents the lymphopenic environment of Rag mice, we confirm an essential role for p40 and not IL-12 in intestinal inflammation also in immune competent hosts.
IL-23, which is produced by DCs and macrophages in response to Toll-like receptor stimuli (), may contribute to intestinal pathology in multiple ways. By inducing the production of IL-1β, TNF, IL-12, and IFN-γ by cells of the innate immune system (, ), IL-23 can directly participate in triggering a proinflammatory cytokine cascade. A key role for IL-23 in driving innate intestinal pathology was indeed observed in a complementary study by Hue et al. (), in which depletion of IL-23p19 attenuated the T cell–independent typhlocolitis that develops in 129SvEvRag mice. IL-23 is also known to amplify and/or stabilize Th17 cells (), which through their secretion of IL-17 induce myeloid and endothelial cell expression of proinflammatory cytokines (IL-1, IL-6, IL-8, TNF-α, and GM-CSF) and chemokines (, , ). In addition to our findings of increased IL-17 responses in colitic mice, several of the effects we observed in our T cell–dependent model of colitis may be interpreted in terms of Th17 function and subsequent recruitment of inflammatory cells to the intestine. Thus, both cecal inflammation and SHelAg-specific IL-17 responses observed in anti–IL-10R–treated IFN-γ mice were inhibited by co-administration of anti-p40 mAb. Moreover, IFN-γ CD4 CD45RB cells were equivalent to their WT counterparts in terms of inducing cecal inflammation (), and these cells were also pathogenic in an IL-12–deficient setting in p35Rag hosts (not depicted). Collectively, these findings argue for an IL-23–driven IFN-γ–independent component in disease pathogenesis, a conclusion in agreement with the pathogenic role of Th17 cells in EAE and CIA (–, ).
Th17 cells represent a unique linage distinct from the Th1 and Th2 subsets, as their development is inhibited by IFN-γ and IL-4 and is independent of STAT4, STAT6, STAT1, and T-bet (, ). Three recent reports have now established that TGF-β, in the presence of IL-6, is critical for the development of Th17 cells, whereas IL-23 appears dispensable for Th17 commitment and initial IL-17 production (–). IL-23 is, however, clearly essential for a fully effective Th17 response in vivo, as exemplified in EAE and CIA as well as in various infections with extracellular bacteria (e.g., and ; references –, , , and ). Our findings of up-regulated IL-17A (and to a lesser extent IL-17F) mRNA levels in the absence of significant induction of IL-23p19 or p40 in colonic tissues of control mAb–treated WT mice that show no colitis () support these conclusions. The mechanism behind the IL-23 dependency for effective Th17 responses is still unknown. As IL-23R expression is up-regulated by TGF-β, thus downstream of signals that initiate Th17 differentiation (, ), one possibility is that IL-23 stabilizes and/or maintains Th17 cells, analogous to the role of IL-12 in stabilizing Th1 cells after their expression of IL-12Rβ2 (). So far, the role of IL-10 in Th17 development has not been carefully examined. To the best of our knowledge, the results presented here are the first to demonstrate an inhibitory role for IL-10 in the development of IL-17–producing CD4 T cells in vivo, as anti–IL-10R treatment of WT mice led to markedly increased antigen-specific IL-17 responses. The mechanism by which IL-10 exerts its down-regulatory effect on IL-17 production is currently unknown but was shown to be independent of IL-12 and IFN-γ, as anti–IL-10R–treated p35 and IFN-γ mice also displayed increased IL-17 responses. One possibility is that IL-10 inhibits IL-23 secretion by cells of the innate immune system, thereby limiting the amount of IL-23 available to maintain Th17 cells. Alternatively, IL-10 might have a down-regulatory effect on IL-23R expression on CD4 cells, as has been described for bone marrow–derived macrophages (), thereby rendering these cells less responsive to IL-23. In the study by Veldhoen et al. (), IL-17–secreting T cells were generated in vitro in cocultures of naive CD4 cells with naturally occurring CD25 CD4 T regulatory cells in the presence of Toll-like receptor stimuli. In contrast to our findings, blocking IL-10 in this model did not result in an increased frequency of IL-17–producing T cells (). However, as the percentage of IFN-γ–secreting CD4 cells increased in the same cultures (), a possible inhibitory effect of IFN-γ on the development of IL-17–producing cells cannot be excluded.
Although, as shown in this study, IL-23 and not IL-12 is essential for the development of maximal pathology in T cell–dependent bacterial-induced intestinal inflammation, there are some differences between our model and the EAE and CIA models in regard to the contribution of IL-12 and IFN-γ to disease pathogenesis. Thus, in the sole absence of IL-12 (p35 mice), disease severity was enhanced in EAE and CIA (, ), whereas for colitis induced by anti–IL-10R treatment of p35 mice or by T cell transfer into p35Rag recipients, this was not the case. One explanation for this difference could be that IL-17–secreting CD4 T cells are the major pathogenic population in the former two models, with IL-12–driven IFN-γ suppressing the generation of IL-17 Th cells (, , ), whereas in the colitis setting, both IL-17 and IFN-γ contribute to disease pathogenesis. Indeed, loss of IFN-γ or the IFN-γ receptor was associated with an increased disease severity in both EAE and CIA (–), whereas as shown here, IFN-γ deficiency led to largely unaffected cecal pathology but reduced colonic inflammation. These findings indicate that IFN-γ enhances rather than suppresses inflammation in the intestinal tract, a conclusion supported by previous studies demonstrating that anti–IFN-γ treatment prevents the development of colitis (, , ). Yen et al. () have recently reported that IL-23 is required for the development of spontaneous enterocolitis in IL-10 animals, a finding attributed to IL-17 and IL-6 based on mAb neutralization studies in an adoptive transfer model. Importantly, neutralization of IL-17 or IL-6 alone did not significantly reduce pathology, and although combined treatment attenuated the colitis, it did not completely inhibit the inflammation (). Based on our findings, we hypothesize that additional pathogenic effector mechanisms synergize with IL-17/IL-6 to induce severe intestinal inflammation. In particular, our results highlight that although not absolutely required for cecal disease, IFN-γ plays a synergistic role in the induction of colonic inflammation. The reason for the difference in cytokine requirements to induce maximal pathology in the cecum versus the colon is currently unclear, but it may reflect distinct levels of bacterial colonization () leading to different induction of cytokines and/or chemokines.
Interestingly, this study revealed a role for –induced IL-12–independent, but p40-dependent IFN-γ in intestinal pathology. Whether the molecular mechanism behind this finding involves p40 acting alone, as a homodimer, or in association with other chains remains unknown. However, it is possible that these observations reflect the involvement of IL-23 in triggering –induced CD4 IFN-γ production in IL-12–deficient mice. This conclusion is supported by recent findings from the model in which antigen-specific IFN-γ CD4 cells were shown to develop in infected p35 mice via a p19-dependent mechanism, contributing to the prolonged survival of these hosts compared with infected p40 animals (, ). The co-appearance of antigen-reactive IFN-γ CD4 and IL-17 CD4 cells in p35 mice in both the () and models suggests that the IL-23–induced IFN-γ detected in these animals may not be sufficient to block the generation of IL-17–secreting CD4 cells. Based on the data presented here, we instead propose a modified model in which IFN-γ may have either inhibitory or synergistic effects on Th17 development and/or effector function. Thus, in settings with large amounts of the cytokine, IL-12–induced IFN-γ exerts negative effects on Th17 development, as described by Harrington et al. (), Park et al. (), and Mangan et al. (), and as exemplified by IL-10 mice that mount markedly enhanced IFN-γ but reduced IL-17 levels compared with anti–IL-10R–treated p35 mice (). However, in settings with no IL-12, lower levels of IL-23–dependent IFN-γ (or IFN-γ induced by another p40-dependent mechanism) act together with IL-17 to enhance intestinal inflammation (, compare p35 with IFN-γ mice). It is important to point out that although IL-12 is dispensable for colitis induction, this cytokine may still contribute to intestinal pathology, as shown in the T cell transfer experiments in which p19Rag recipients displayed a significantly higher level of intestinal inflammation compared with p40Rag recipients (). Besides the role of IL-12 in Th1 development, the IL-12p40 subunit by itself (possibly as a homodimer) may also contribute to disease pathogenesis through its effects on myeloid cells (–) and its recently described role in DC migration and subsequent T cell priming ().
Collectively, the data presented in this study demonstrate that IL-23 and not IL-12 is essential for the pathogenesis of T cell–dependent bacterial-induced intestinal inflammation. Although IL-23 may constitute a potential therapeutic target for IBD, our findings further support a model in which multiple factors downstream of IL-23 (e.g., IL-17, IL-6, and IFN-γ) may contribute to intestinal inflammation and together synergize to induce maximal pathology. Further identification and characterization of these downstream components and mechanisms should help dissect the immunological pathways involved in IBD, and should aid in the development of new therapeutic treatments for patients with these chronic inflammatory diseases of the intestinal tract.
Female or male specific pathogen-free C57BL/6 (B6) WT, B6 p40, B6 IFN-γ, C57BL/10 (B10) IL-10, B10 Rag-2 (Taconic Farms), and B6 p35 mice (The Jackson Laboratory) were used at 6–10 wk of age. p40Rag and p35Rag animals were generated by crossing the above mentioned single cytokine-deficient mice, and the progeny were intercrossed to generate double-deficient offspring. For , Rag, p40Rag, p35Rag, and p19Rag animals on the B6 background were used (). The animals used tested negative for antibodies to specific murine viruses and were free of species as assessed by PCR. Animals were housed in sterile microisolator cages at the National Institute of Allergy and Infectious Disease (NIAID) animal facility under a study proposal approved by the NIAID Animal Care and Use Committee or in accredited animal facilities at the University of Oxford according to the UK Scientific Procedures Act 1986.
Mice were inoculated intragastrically with 0.5 ml of an suspension (NCI-Frederick isolate 1A [reference ] isolated from the same mouse colony as isolate Hh-1; American Type Culture Collection 51449 [reference ]) prepared to a McFarland turbidity standard of 1.0 in PBS representing 2.45 × 10 CFU/ml. For the experiments shown in , three doses of 5 × 10–2 × 10 CFU were given to mice on days 0, 2, and 4. Age- and sex-matched uninfected animals were included as controls.
To analyze the effect of in vivo neutralization of IL-10R, IL-12/IL-23 p40, or IFN-γ, mice were treated i.p. with 1 mg per injection of mAb 1B1.3a (anti–IL-10R), C17.8 (anti-p40), XMG-6 (anti–IFN-γ), or a control mAb, GL113 (anti–β-galactosidase), in 0.5 ml PBS on days 0, 7, 14, and 21 of infection or cell transfer (for T cell–sufficient mice and Rag recipients, respectively). 1 wk after the last mAb injection, mice were killed, MLNs were collected for in vitro culture, and intestinal tissues were collected in Bouin's fixative for histology and in RNA-later (Ambion) for real-time PCR analysis.
Single cell suspensions were prepared from MLNs, and cells were resuspended in complete tissue culture medium (). For purification of CD4 CD45RB cells, MLN cells from naive WT or IFN-γ mice were stained with anti–CD4-PE (clone RM4-5) or anti–CD4-CyChrome (RM4-5) and anti–CD45RB-FITC (16A; all from BD Biosciences) and sorted on a FACSVantage or a FACStarPlus (Becton Dickinson), gating on ∼50% of the brightest CD4 cells. For the experiment shown in , splenic CD4 CD45RB cells from WT mice were used. The sorted CD4 CD45RB cells were ≥99% pure.
MLN cells (5 × 10/ml) pooled from three to five mice/group were cultured in medium alone or with 5 μg/ml of SHelAg in 96-well flat-bottomed plates (0.2 ml/well) at 37°C and 5% CO, and supernatants were collected after 72 h. SHelAg was prepared as described previously (), boiled for 5 min (), and stored at −40°C until use. IFN-γ and IL-17 were measured by ELISA using mAb from BD Biosciences and R&D Systems, respectively.
Analyses of intracellular cytokine expression were performed on cells from the same SHelAg-stimulated MLN cultures used for cytokine secretion assays as described previously (, ). In brief, cells were incubated for an additional 18 h in fresh medium added to replace the culture supernatant collected at 72 h. Thereafter, cells were stimulated with 10 ng/ml PMA and 1 μg/ml ionomycin (both from Sigma-Aldrich) for 4.5 h with the addition of 10 μg/ml brefeldin A (Sigma-Aldrich) during the last 2 h. Intracellular staining was then performed as described previously (, ) using anti–IL-17–PE (TC11-18H10.1) and anti–IFN-γ–FITC (XMG1.2; both from BD Biosciences). Cell fluorescence was measured using a FACSCalibur (Becton Dickinson), and data were analyzed using CELLQuest software (Becton Dickinson).
Unless otherwise indicated, CD4 CD45RB cells (3 × 10/mouse) were transferred i.v. to naive or 2–4-d-infected Rag mice. Mice were killed 4 wk after cell transfer, and intestinal tissues were collected for histologic analysis and measurements of cytokines.
Intestinal tissues were processed and inflammation in the cecum (typhlitis) and ascending colon was scored in a blinded fashion by the same pathologist (A.W. Cheever) on a scale from 0 to 20 as described previously (). In brief, a longitudinal section of the entire cecum was made together with a cross section of the ascending colon ∼1 cm from the cecum, and a total score for the whole section was assigned based on a 0–4+ scoring system for different features with emphasis on the number of infiltrating cells for the lamina propria, the submucosa, and the serosa. In addition, crypt abscesses and ulcers were each given a score from 0 to 4. A total score was calculated by adding the individual scores.
Because, in general, the degree of inflammation in the colon proportionally reflects that observed in the cecum, the colon was chosen for real-time RT-PCR analyses (and cytokine protein determinations; see below) to avoid the lymphocyte patches present irregularly throughout the cecum while preserving the intact cecum for histological examination. Total RNA was isolated from polytron-homogenized colonic tissue (∼3 mm of ascending colon distal of the piece used for histology) using the RNeasy mini kit, including a DNase treatment step as recommended by the manufacturer (QIAGEN). 6–9 μg RNA was reverse transcribed using Superscript III reverse transcriptase and oligo-dT primers (both from Invitrogen). cDNA was amplified using TaqMan reagents and a Chromo4 detection system (MJ Research). Cytokine expression levels for each individual sample (run in triplicates) were normalized to HPRT using ΔCt calculations. Mean relative cytokine expression levels between control and experimental groups ( = 2–4 mice/group) were determined using the 2 formula. Specific primer pairs and FAM/TAMARA- or VIC/TAMARA-labeled probes were as follows: HPRT: primers 5′-GACCGGTCCCGTCATGC-3′, 5′-TCATAACCTGGTTCATCATCGC-3′, probe 5′-ACCCGCAGTCCCAGCGTCGTC-3′; IL-23p19: primers 5′-AGCGGGACATATGAATCTACTAAGAGA-3′, 5′-GTCCTAGTAGGGAGGTGTGAAGTTG-3′, probe 5′-CCAGTTCT- GCTTGCAAAGGATCCGC-3′; IL-12p35: primers 5′-TACTAGAGA- GACTTCTTCCACAACAAGAG-3′, 5′-TCTGGTACATCTTCAAGT- CCTCATAGA-3′, probe 5′-AGACGTCTTTGATGATGACCCTGTG- CCT-3′; IL-12p40: primers 5′-GACCATCACTGTCAAAGAGTTTCTAGAT-3′, 5′-AGGAAAGTCTTGTTTTTGAAATTTTTTAA-3′, probe 5′-CCACTCACATCTGCTGCTCCACAAGAAG-3′; IL-17A: primers 5′-GCTCCAGAAGGCCCTCAG-3′, 5′-CTTTCCCTCCGCATTGA- CA-3′, probe 5′-ACCTCAACCGTTCCACGTCACCCTG-3′; IL-17F: primers 5′-AGGGCATTTCTGTCCCACGTGAAT-3′, 5′-GCATTGA- TGCAGCCTGAGTGTCT-3′, probe 5′-CATGGGATTACAACATCACTCGAGACCC-3′; and IFN-γ: primers 5′-GGATGCATTCATGAGTATTGC-3′, 5′-GCTTCCTGAGGCTGGATTC-3′; probe 5′-TTT- GAGGTCAACAACCCACAGGTCCA-3′.
To measure cytokines at the protein level in the intestine, colonic tissues were snap frozen and then homogenized in PBS containing a cocktail of protease inhibitors (Protease Inhibitor Cocktail Tablets; Roche) using a tissue polytron. After centrifugation at 10,000 to pellet debris, levels of TNF-α, IFN-γ, MCP-1, and IL-6 were measured in supernatants using the cytometric bead assay (BD Biosciences). Cytokine amounts were normalized to the total amount of protein in each sample, as measured by Bradford assay (Bio-Rad Laboratories).
DNA was purified from cecal contents of mice using the DNA Stool kit (QIAGEN). DNA was quantified as described previously (, ) using a real-time quantitative PCR method based on the gene, performed with a Chromo4 detection system. Standard curves were constructed using DNA that was purified from bacterial cultures using the DNeasy kit (QIAGEN).
Colitis scores were compared using the nonparametric Mann-Whitney U test. Cytokine data and colonization levels were analyzed by Student's unpaired two-tailed test. Differences were considered statistically significant with P < 0.05. |
Previous studies reporting IL-10 production during chronic viral infections prompted us to examine the secretion of IL-10 during chronic and acute LCMV infections (, –). Splenocytes from mice infected with LCMV clone 13 or LCMV Armstrong were isolated at various time points after infection and cultured for 48 h in the absence of exogenous antigen (to allow for direct ex vivo presentation of viral antigens by splenic APCs). At the end of this culture period, we measured the concentration of IL-10 in the supernatants by ELISA. IL-10 production by splenocytes from mice infected with LCMV (both clone 13 and Armstrong strains) peaked at day 20 after infection (). However, splenocytes from mice infected with LCMV Armstrong secreted significantly less IL-10. This difference was apparent regardless of the initial dose of virus, as increasing the inoculum of LCMV Armstrong from 10 PFU to 2 × 10 PFU resulted in comparable kinetics of IL-10 secretion (unpublished data). By day 40, although IL-10 production was undetectable in LCMV Armstrong–infected mice, mice infected with LCMV clone 13 still produced significant amounts of this cytokine (). After in vitro LCMV clone 13 infection, we observed that IL-10 was produced by different immune cell types, including CD4 and CD8 T cells and CD11c DCs, whereas LCMV Armstrong infection induced less IL-10 production by CD4 T cells and CD11c DCs and none by CD8 T cells (). Interestingly, when splenic cell cultures were treated with a neutralizing anti–IL-10R monoclonal antibody (which specifically targets the IL-10R α chain) at the time of LCMV clone 13 infection in vitro, the levels of IL-10 produced by CD4 T cells were reduced to those found in LCMV Armstrong–infected mice ().
We then analyzed in vivo IFN-γ secretion by CD8 T cells at different time points after LCMV Armstrong and LCMV clone 13 infection (). Interestingly, CD8 T cells from LCMV clone 13–infected mice secreted IFN-γ only during the early phase of infection (day 5), at a time when no IL-10 was yet detected (). However, IFN-γ secretion was almost completely lost at later time points, and high levels of IL-10 were produced (). In contrast, IFN-γ production by CD8 T cells from LCMV Armstrong–infected mice peaked later (day 7) and was sustained in the form of a memory response () (). Secretion of TNF-α followed a similar profile (unpublished data). Increasing the dose of LCMV Armstrong did not induce earlier cytokine responses (unpublished data). These results indicate that LCMV clone 13 induces a faster antiviral response, possibly linked to a higher binding affinity of the virus to its α-dystroglycan receptor ().
It has recently been shown that high expression of PD-1 is a characteristic of exhausted CD8 T cells in LCMV clone 13–infected mice and that treatment with anti–PD-1 antibodies leads to the proliferation of antiviral T cells and the enhancement of viral clearance (–). We therefore analyzed PD-1 expression on T cells. We found that a small percentage of CD4 and CD8 T cells expressed PD-1 in naive mice (). Upon LCMV infection, significantly higher levels of PD-1 were expressed on CD8 T cells isolated from LCMV clone 13–infected mice compared with LCMV Armstrong–infected mice (). In addition, the mean fluorescence intensity of PD-1 was increased in T cells from LCMV clone 13–versus LCMV Armstrong–infected mice (unpublished data).
Based on these findings, we hypothesized that the immune suppression mediated by LCMV clone 13 infection was caused by a shift from antiviral IFN-γ production to the secretion of the immunosuppressive cytokine IL-10. To investigate whether IL-10 was directly involved in the suppression of the antiviral immune response, we infected IL-10–deficient (IL-10) mice with LCMV clone 13 and monitored viral clearance. 3 wk after LCMV clone 13 infection, we found that viral titers were significantly lower in liver and kidney from IL-10 mice compared with wild-type control mice when measured by a highly sensitive RT-PCR method (). Resolution of LCMV clone 13 infection in IL-10 mice was associated with a lower systemic IL-10 to IFN-γ ratio, as one would expect (). Sustenance of antiviral immune responses to clone 13 through genetic elimination of IL-10 has been described in detail in another study by Brooks et al. ().
IL-10 can inhibit immune responses by skewing the development of helper T cells and suppressing their function (, ). Based on our initial findings that cells from LCMV clone 13–infected mice secreted IL-10 and that mice deficient in IL-10 cleared this strain of LCMV more efficiently, we investigated whether blocking the IL-10–IL-10R signaling pathway could resolve LCMV clone 13–induced immune suppression and reestablish an antiviral Tc1/Th1 response. We therefore infected BALB/c mice with LCMV clone 13 and injected age-matched groups with either a neutralizing anti–IL-10R monoclonal antibody or an IgG isotype control antibody on days 0, 7, and 14, or with a therapeutic regimen on days 7 and 14 after infection. Disease severity was monitored by assessing bodyweight, spleen cell numbers, and viral titer over time. LCMV clone 13–infected mice treated with isotype control antibody lost body mass and weighed 30–40% less than age-matched naive mice (). These mice also exhibited a nonshiny, scruffy coat (unpublished data). In comparison, LCMV clone 13–infected mice treated with anti–IL-10R antibody lost weight less rapidly () and exhibited a healthy shiny coat (not depicted). In addition, we monitored viral loads in lymphoid and nonlymphoid organs isolated from persistently infected mice treated with anti–IL-10R or IgG. Viral titers were measured by conventional plaque assay (not depicted) and by a more recently developed, highly sensitive RT-PCR method (). At 6 and 26 wk after infection, numbers of LCMV genomic copies were low or nondetectable when measured by the sensitive RT-PCR method in organs from anti–IL-10R–treated mice; 36 wk after infection, viral titers remained undetectable in liver and kidney (). In contrast, viral genome copies were still detectable in these organs when mice were treated with IgG isotype antibody, indicating that resolution of chronic infection resulted from anti–IL-10R treatment rather than progressive viral elimination over time. Viral titers in organs from control IgG-treated mice remained high, and virus predominated in the kidney, characterizing the chronic infection (). Furthermore, extremely low viral titers were detected by LCMV plaque assay in kidney, liver, and lung 6 wk after LCMV clone 13 infection in anti–IL-10R–treated mice, and no virus was detected 26 wk after infection (unpublished data).
Importantly, we also administered anti–IL-10R antibody in a therapeutic setting at days 7 and 14 after infection during the onset of lymphopenia and weight loss (by day 7 after infection, the total number of splenocytes was reduced by 41% in LCMV clone 13–infected mice compared with naive mice; unpublished data). Anti–IL-10R treatment was therapeutically effective during established infection as treated mice lost weight less rapidly (), did not exhibit any overt signs of disease, and showed a substantial reduction in viral titers in the liver, lung, and kidney 11 wk after treatment (not depicted). Additionally, a two to three log reduction in viral titer was observed when anti–IL-10R was given on days 5 and 12 after LCMV clone 13 infection, as well as on days 10 and 17 after infection (unpublished data).
As reported previously (, ), we found that chronic infection was associated with deletion of virus-specific CD8 T cells, leading to a drastic decrease in the number of spleen cells (unpublished data). We discovered that this state of lymphopenia was reversed after anti–IL-10R therapy both when treatment was initiated at day 0 or therapeutically at day 7 after infection (). In addition, endogenous IL-10 production by splenocytes isolated 3 wk after LCMV clone 13 infection was significantly reduced in anti–IL-10R–treated mice, indicating that early blockade of this cytokine prevented the long-term immunosuppressive effects mediated by LCMV clone 13 infection ().
We therefore assessed the effect of anti–IL-10R treatment on the LCMV-specific T cell response in detail. Splenocytes from anti–IL-10R– and control IgG–treated BALB/c mice were isolated at various time points after LCMV clone 13 infection and stimulated with the MHC class I–restricted immunodominant LCMV peptide NP. The number of IFN-γ–secreting CD8 cells was measured by intracellular cytokine staining. Anti–IL-10R–treated mice had significantly higher numbers of IFN-γ–secreting CD8 T cells compared with IgG-treated mice, particularly at later time points (). Importantly, therapeutic treatment with anti–IL-10R on days 7 and 14 after LCMV clone 13 infection caused a similar enhancement of the LCMV-specific memory T cell response (). Thus, anti–IL-10R treatment resulted in reemergence of a potent antiviral IFN-γ CD8 T cell response, indicating that events occurring early during T cell responses can profoundly affect the quality of T cell memory. It will be difficult to determine whether the enhancement of viral-specific CD8 T cell responses was the cause or consequence of reduced viral loads; however, we believe that both alternatives are possible.
Finally, we investigated the effect of anti–IL-10R treatment on the PD-1 inhibitory pathway (–). To determine whether suppression of the PD-1 pathway could be involved in the enhancement of viral clearance after therapeutic anti–IL-10R treatment, we monitored expression of PD-1 by T cells in anti–IL-10R– and IgG-treated mice 90 d after LCMV clone 13 infection. Low levels of PD-1 expression were detected on T cells from naive mice (, top). In comparison, T cells from LCMV clone 13–infected mice treated with IgG isotype antibody exhibited as much as a 25-fold induction of PD-1 expression compared with naive mice (, bottom). Importantly, a threefold reduction of PD-1 expression was observed on T cells from anti–IL-10R–treated mice compared with IgG-treated mice (, middle). These results indicate that anti–IL-10R treatment decreased PD-1 expression on T cells, which could potentially contribute to abrogation of T cell exhaustion because treatment with anti–PD-1 antibodies in chronically infected mice has been shown to down-regulate PD-1 and circumvent virally induced CD8 T cell exhaustion (–).
To further investigate the effect of anti–IL-10R treatment on the LCMV-specific immune response, we examined absolute numbers of different splenic cell subsets over time in mice infected with LCMV clone 13 and treated with anti–IL-10R or control IgG antibody. We discovered that even though LCMV clone 13 infection caused a reduction in the numbers of cells present in the spleen at day 21 after infection regardless of treatment, by day 150 after infection the numbers of CD4, CD8, B220, and CD11c cells in anti–IL-10R–treated mice were similar to those in naive mice, whereas in IgG-treated mice these cells remained scarce (). Notably, the numbers of CD11b cells in the spleen at day 150 after infection were comparable between groups, suggesting that the decrease in viral titers after anti–IL-10R treatment was not solely caused by enhanced clearance of virally infected macrophages.
We hypothesized that the loss of antiviral T cells after LCMV clone 13 infection would be mediated by APCs. DCs are the most potent APCs and have been shown to play a crucial role in the priming of LCMV-specific cytotoxic T cells (). We therefore examined absolute numbers of different DC subsets (CD11cCD8α and CD11cCD8α) and their ratio after infection with LCMV Armstrong or LCMV clone 13. Over the course of infection, the number of CD8α and CD8α DCs increased in LCMV Armstrong–infected mice but returned to that found in naive mice within 2 wk after infection (). In contrast, although the number of CD8α DCs from LCMV clone 13–infected mice 6 wk after infection was also comparable to that of naive mice, CD8α DCs gradually disappeared over time (). It has been previously shown that >50% of CD11c DCs carry viral particles at this time point in clone 13–infected mice (), making these cells an excellent target for destruction and elimination by the immune system. In contrast, very few CD11c cells are infected at the same time point after LCMV Armstrong infection (). Importantly, the numbers of CD8α DCs were significantly lower in LCMV clone 13–infected mice 6 wk after infection compared with mice infected with LCMV Armstrong (). Because the CD8α DC subset declined in LCMV clone 13–infected mice and the CD8α DC subset remained at a stable level, the CD8α to CD8α DC ratio was skewed toward CD8α DCs 6 wk after infection (). To investigate whether this phenomenon was affected by therapeutic anti–IL-10R treatment, absolute numbers of CD8α and CD8α DCs were monitored 6 wk after LCMV clone 13 infection in mice treated with anti–IL-10R on days 7 and 14 after infection. Anti–IL-10R treatment decreased CD8α DC numbers more than twofold, as well as CD8α DC numbers to a lesser extent (). Hence, the resulting CD8α to CD8α DC ratio after anti–IL-10R treatment resembled the ratio in LCMV Armstrong–infected mice 6 wk after infection (). In summary, these findings show that anti–IL-10R treatment in LCMV clone 13–infected mice resulted in a skewing of the CD8α to CD8α DC ratio to levels seen in mice infected with the nonpersistent LCMV Armstrong strain.
Previous reports suggest that different DC subsets vary in their ability to prime effector T cells (, , ). In particular, evidence suggests that different DC subsets can induce T cells to produce different cytokines depending on the cytokine milieu in which they encounter antigen (, , ). We therefore investigated whether T cell priming by different DC subsets modulated the nature of the antiviral T cell response. CD11cCD3 splenic DCs were isolated from mice infected 7 d earlier with LCMV Armstrong or LCMV clone 13. In addition, one group of mice was treated with anti–IL-10R antibody at the time of infection. CD11cCD3 DCs from all groups were sorted into CD8α and CD8α DC subsets on day 7 after infection (). This early time point was chosen to allow for capture and processing of viral antigens by DCs directly in vivo during the early phase of LCMV Armstrong and LCMV clone 13 infection, which is associated with viral dissemination and replication. At this time, viral antigen could be detected by RT-PCR (unpublished data). Because we had previously observed that CD4 T cells from LCMV clone 13–infected mice produced elevated levels of IL-10, the isolated DCs were cultured for 5 d with naive GP-specific CD4 responder T cells isolated from TCR transgenic SMARTA mice (). No exogenous antigen was added to the cultures to ensure that only viral antigen processed in vivo was presented by the DCs. The ability of the different DC subsets to stimulate LCMV-specific CD4 T cells was determined by measuring the concentration of cytokines in the supernatants by ELISA at the end of the culture period. To rule out any contamination caused by cytokine release by APCs, DCs were irradiated, and levels of both IL-10 and IFN-γ were measured in the supernatants of DC cultures devoid of T cells. These background levels were <1 pg/ml (unpublished data).
IL-10 production by antiviral CD4 T cells was induced exclusively by DCs isolated from LCMV clone 13–infected mice and was preferentially mediated by the CD8α DC subset (), an observation that was confirmed when the different DC subsets were loaded with LCMV GP peptide (not depicted). Importantly, this corresponded to the time point at which CD8α DC numbers in LCMV clone 13–infected mice started to decline (). These results suggest that when CD8α DCs are infected and killed, the remaining CD8α DCs that are left to prime T cells induce IL-10 production instead of a potent antiviral response. In contrast, CD8α or CD8α DCs isolated from mice infected with LCMV Armstrong induced little to no IL-10 production by LCMV-specific CD4 T cells (), indicating that differences between the two LCMV infections could merely reside in the type of cytokine responses induced. In vivo anti–IL-10R treatment completely abrogated the capacity of CD8α DCs to induce IL-10 secretion in CD4 T cells (), indicating that signaling through the IL-10R was crucial for CD8α DCs to induce IL-10.
Additionally, both CD8α and CD8α DCs from LCMV clone 13– and LCMV Armstrong–infected mice were able to stimulate IFN-γ production by CD4 T cells, whereas CD8α DCs induced higher amounts of IFN-γ than CD8α DCs. Anti–IL-10R treatment did not have an effect on IFN-γ production induced by the different DC subsets ().
Importantly, although the amount of IL-10 induced by CD8α and CD8α DC subsets was dependent on the LCMV strain used (), the amount of IFN-γ was similar for both LCMV strains (). Therefore, the IL-10 to IFN-γ ratio was always significantly higher when DCs from LCMV clone 13–infected mice were used as APCs ().
Collectively, these data show that anti–IL-10R treatment abolished CD8α DC-mediated IL-10 production in responder T cells early after infection and decreased absolute CD8α DC numbers over time, thereby inducing a shift toward IFN-γ and Tc1/Th1 responses, enabling viral clearance. Our observations that anti–IL-10R antibody led to resolution of protracted infection, enhanced antiviral T cell responses, and abrogated the ability of CD8α DCs to induce IL-10 in CD4 T cells, in conjunction with observed lower viral titers in mice deficient in IL-10, underscores the important role IL-10 plays in maintaining a chronic viral infection.
This study is the first to report that inhibiting IL-10 signaling in vivo leads to enhancement of viral clearance in a mouse model of persistent viral infection. Our data demonstrate that upon challenge with LCMV clone 13, the strong antiviral response elicited early after infection rapidly subsides and is followed by a state of chronic infection associated with generalized lymphopenia. In this paper, we show that chronic infection and deterioration in health is associated with systemic increase in the production of IL-10. Although the implication for the role of IL-10 in the maintenance of chronic viral infections has hitherto been unclear, our results show that lymphopenia is reversed, bodyweight recovers, and viral load is significantly reduced upon blockade of IL-10–IL-10R signaling (P < 0.0001). Furthermore, we show that anti–IL-10R treatment results in a reduction of CD8α DC numbers and a subsequent abolishment of IL-10 production, thereby favoring a Tc1/Th1 cell–like environment inducing LCMV-specific IFN-γ T cell responses. Indeed, after LCMV clone 13 infection, CD8α DC numbers substantially were found to decrease because of viral infection and clearance, and we now show that CD8α DCs will preferentially induce IL-10 production by T cells. Thus, it is possible that the generalized state of lymphopenia of LCMV clone 13–infected mice is mediated by APCs inducing IL-10–producing T cells, which results in a negative feedback loop. Our results suggest that distinct DC subsets may differentially regulate the cytokine balance of the immune response in vivo. The precise mechanism by which these DC subsets mediate their effects might be related to levels of IL-10 present in the microenvironment. We show that CD8α DCs induce IL-10 production more efficiently than their CD8α counterparts. The ability to potentiate IL-10 secretion is drastically reduced upon blockade of signaling through the IL-10R. We believe that anti–IL-10R treatment modulates CD8α DCs early upon infection and, as a result, dictates the nature of the cytokine response induced early during antiviral immunity.
The mechanisms by which IL-10 enables LCMV clone 13 to persist are unknown. Several variables associated with T cell priming have been shown to be important and may program qualitative differences into the effector and memory populations (). IL-10 could either down-regulate proinflammatory responses in a general manner or, more specifically, inhibit the induction or expansion of antiviral CD8 effector T cells. Importantly, we report for the first time that anti–IL-10R antibody treatment reduces PD-1 expression on CD8 T cells, which has previously been shown to contribute to virally induced CD8 T cell exhaustion (–). Interestingly, stimulation through TLR2, which has been shown to bind to LCMV, leads to the production of IL-10 and inhibits IFN-inducible protein 10 and IL-12 secretion by APCs. Thus, stronger binding of LCMV clone 13 to TLR2, as opposed to weaker TLR2 binding by LCMV Armstrong (), could not only induce preferential production of IL-10 by DCs but also inhibit the secretion of cytokines that promote DC differentiation, maturation, and proliferation rather than survival (). In addition, it is possible that the DCs that remain after LCMV clone 13 infection express higher levels of TLR2, thus perpetuating the cycle.
Subclasses of DCs have been shown to have the potential to differentially skew T cell cytokine production toward Th1 or Th2 cell profiles (, , ). Notably, it has been suggested that CD8α DCs induce Th2 cell profiles, whereas CD8α DCs preferentially stimulate IFN-γ production and, therefore, induce Th1 cell profiles (, ). We now show that this may be true in the LCMV system, as we have analyzed the ability of CD8α and CD8α DCs to polarize LCMV-reactive CD4 T cells ex vivo. It appears that priming of cytotoxic T lymphocytes by DCs is achieved in a similar fashion in LCMV clone 13– and LCMV Armstrong–infected mice, but that stronger binding of LCMV clone 13 to its receptor on APCs, particularly CD8α DCs, leads to more efficient processing and presentation of viral antigen. Our finding that LCMV clone 13 infection leads to early induction of IFN-γ–secreting LCMV-specific CD8 T cells strengthens this hypothesis. The strong cytotoxic function of such activated Tc1 or Th1 cells upon infection with LCMV clone 13 may result in killing of the APC subset responsible for their induction; i.e., DCs belonging to the CD8α subset. This hypothesis correlates with our finding that the number of CD8α DCs is reduced as early as day 7 after infection with LCMV clone 13 but not LCMV Armstrong.
Our results suggest that as the number of CD8α DCs decreases after chronic LCMV clone 13 infection, the CD8α DC subset—which is more likely to prime T cells to produce IL-10 than Tc1/Th1 cytokines—will by default become the modulator of the T cell response. LCMV-specific CD4 T cells activated in this context may provide inappropriate or insufficient antiviral help to other cell types, particularly CD8 T cells, thus leading to persistent infection. Additionally, it is possible that the CD8α DCs, which appear ill-equipped to propagate antiviral effectors, will continue to support IL-10 production. The resulting high concentration of IL-10 in the milieu may thus lead to further modulation of DC function. In fact, IL-10 may directly decrease the viability of CD8α DCs, as has been previously suggested (, ). As a result, only disruption of IL-10 signaling will have the ability to break the vicious circle and enable the recovery of appropriate antiviral immunity by the infected host.
Mechanistically, our findings show that anti–IL-10R antibody treatment lowers the endogenous levels of IL-10, restores the ability to mount an antiviral CD8 T cell response, and results in enhanced viral clearance, which all highlight the important role IL-10 plays in the maintenance of chronic infection. Upon treatment with anti–IL-10R, we observed a profound decrease in CD8α and, to a lesser degree, in CD8α DC numbers, as well as a complete inability of CD8α DCs to induce IL-10 production by CD4 responder T cells, thereby favoring a systemic cytokine milieu that would better support IFN-γ and Tc1/Th1 cell responses.
Importantly, the inability of CD8α DCs to induce IL-10 production by CD4 T cells after anti–IL-10R treatment underscores that IL-10 and signaling through the IL-10R is indeed involved in shaping the cytokine milieu with a potential impact on immune responses. Our results imply that IL-10R blockade is of great interest for the therapeutic treatment of chronic viral infections in humans.
6-wk-old BALB/c, C57BL/6 (B6), and IL-10 (B6 background) mice were purchased from The Jackson Laboratory. LCMV-GP–specific CD4 TCR transgenic SMARTA mice () were obtained from S. Crotty (La Jolla Institute for Allergy and Immunology, La Jolla, CA) and housed under specific pathogen-free conditions at the La Jolla Institute for Allergy and Immunology. LCMV plaques were purified three times on Vero cells, and viral stocks were prepared by a single passage on BHK-1 cells. Age-matched mice were infected i.v. with a single dose of either 2 × 10 PFU LCMV clone 13, 2 × 10 LCMV Armstrong, or an i.p. dose of 10 PFU LCMV Armstrong. For IL-10 blocking experiments, mice were injected i.p. with 250 μg anti–mouse IL-10R (1B1.3a; Becton Dickinson) monoclonal antibody or isotype control antibody (rat IgG1; BD Biosciences). All animal experiments were approved by the La Jolla Institute for Allergy and Immunology Animal Care Committee.
Organs (kidney, liver, spleen, and lung) were snap-frozen, weighed, and homogenized. In brief, three different dilutions of homogenized organ were prepared, and triplicates were used. Dilutions were incubated at 37°C, 5% CO for 1 h with Vero cell monolayers grown in 6-well plates (Costar). The plates were then overlaid with 1% agarose in minimal essential medium 199 (Invitrogen) containing 10% FCS (HyClone) and incubated at 37°C, 5% CO for 5 d. The wells were treated with 25% formaldehyde and stained with 0.1% crystal violet for 2 min. The agarose overlay was removed, infectious centers were counted, and the counts were averaged. Additionally, viral LCMV stock was used as a positive control.
A detailed version of this assay will be published elsewhere (unpublished data). In brief, RNA was isolated from 50 μl of serum or 10 mg of tissue samples using RNAqueous (Ambion). All samples were frozen at −80°C until RNA extraction. RNA was eluted in a volume of 20 μl, and purified RNA was frozen at −80°C until further use. 10 μl RNA was used in a 20-μl cDNA reaction with SuperScript III reverse transcriptase (Invitrogen) and a gene-specific primer (GP-R, GCAACTGCTGTGTTCCCGAAAC). 5 μl cDNA was used as template for a 25-μl quantitative real-time PCR reaction on a GeneAmp 5700 (ABI), using primers GP-R and GP-F (CATTCACCTGGACTTTGTCAGACTC). A standard curve was generated using pSG5-GP plasmid (), a gift from J.C. de la Torre (Scripps Research Institute, San Diego, CA). Data were analyzed using linear regression analysis software (Prism; GraphPad).
Cytokine quantification was performed by sandwich ELISA of supernatants. 2–5 × 10 splenocytes were incubated for 48 h in complete RPMI 1640 (Invitrogen) supplemented with 10% FCS (Sigma-Aldrich), 2 mM -glutamine (Sigma-Aldrich), 50 μM 2-β-mercaptoethanol (Sigma-Aldrich), and 5 mM Hepes (Sigma-Aldrich) in 24-well plates (Invitrogen). For the detection of IL-10–secreting cell subsets from in vitro infected cultures, bulk splenocytes were infected in vitro with LCMV Armstrong or LCMV clone 13 (multiplicity of infection = 3) without or with 10 μg/ml anti–IL-10R antibody and incubated at 37°C in complete RPMI 1640. 48 h after infection, splenocytes were sorted into CD4, CD8, and CD11c populations by MACS, and equal numbers of each cell subset were incubated for 5 d at 37°C. Supernatant was removed at the time points indicated in the figures, and ELISA was performed in accordance with the manufacturer's recommendations and standardized with mouse recombinant cytokine (BD Biosciences). Plates were read at 405 nm (Spectra Max 250; Molecular Devices). The sensitivity of the IL-10 ELISA was 10 pg/ml. Data shown correspond to the concentration of IL-10 (pg/ml) per 10 cells.
To detect cytokine-producing cell subsets, splenocytes from LCMV-infected BALB/c or B6 mice were stimulated with 1 μg/ml NP, GP, or GP peptide, respectively, in complete RPMI 1640 containing Brefeldin A (BFA; Sigma-Aldrich) at 37°C. 6 h later, cells were resuspended in staining buffer containing 1% FCS and 0.2% NaN, labeled with anti-CD4 and anti-CD8, fixed in 1% paraformaldehyde, and permeabilized with 0.1% saponin buffer. Intracellular staining was performed with fluorescent antibodies to IFN-γ, TNF-α, or isotype controls. All antibodies were obtained from BD Biosciences. Additionally, PD-1 expression was detected on splenocytes by labeling with an anti–PE-conjugated PD-1 antibody (eBioscience). Events were acquired using a flow cytometer (FACSCalibur; Becton Dickinson) and analyzed using software (CellQuest; Becton Dickinson). 1–2 × 10 events were acquired, and live cells were gated based on forward/side scatter properties. When analyzing CD11c subsets, CD3-expressing cells were gated out to avoid contamination by T cells expressing myeloid markers (, ), and expression of the indicated cell surface molecules was detected on CD11c and CD8α cells. The number of cells was calculated by relating the frequency of cells from each subset to the overall number of cells per spleen.
Splenocytes from mice infected 7 d earlier with LCMV Armstrong or LCMV clone 13 with or without anti–IL-10R antibody treatment were incubated with HBSS medium containing 0.5 mg/ml collagenase D (Sigma-Aldrich) at 37°C, 5% CO for 30 min. 0.01 M EDTA was added to disrupt T cell–DC complexes. Next, cells were depleted of CD3-expressing cells (Dynal CD3-beads; Dynal), incubated with CD11c microbeads (Miltenyi Biotec), and positively selected using MACS columns. The enriched CD11c cells were labeled with APC-conjugated CD11c and PE-conjugated CD8α antibody (BD Biosciences) and sorted using a cell sorter (FACS Aria; Becton Dickinson) into CD8α and CD8α CD11c subsets. 1.5 × 10 sorted CD11c cells were placed in 96-well plates in complete RPMI 1640 medium and irradiated with 2,900 rad. Assessment of the polarization of antigen-specific CD4 T cells by DCs ex vivo was adapted from a previously described method (). LCMV GP-specific CD4 T cells isolated from TCR transgenic SMARTA mice () were purified by negatively depleting CD8-, B220-, and CD11b-expressing cells with Dynal beads and, after enrichment of CD4 T cells, with CD4-MACS beads (purity > 98%). 6 × 10 CD4 cells were added to DCs in the presence or absence of 1 μg/ml GP peptide. Supernatants were isolated 5 d later and analyzed for the presence of IL-10 and IFN-γ by ELISA, as reported earlier in this paper.
Statistical analyses were performed using the Student's test: , P < 0.01; , P < 0.001; and , P < 0.0001. |
Incubation of mature B cells with BAFF drastically changes the state of B cell metabolism. Treatment with BAFF increases B cell size, cellular protein content, and mitochondrial membrane potential (). BAFF-treated cells show changes in gene transcription that direct these cells toward production of proteins required for glycolytic metabolism and cell cycle progression. Analysis of polyribosome-associated and, hence, actively translated mRNAs revealed two large mRNA clusters that were particularly prominent in BAFF-treated B cells (; and Table S1, available at ). The first cluster contains mRNAs collectively related to glycolysis. The second cluster overrepresented in BAFF-stimulated B cells contains mRNAs for proteins controlling cell cycle, chromosome condensation, and mitosis. The BAFF-induced up-regulation of cell cycle progression proteins such as cyclin D and cyclin E, Cdk4, Mcm2 and 3, the proliferation marker Ki67, and Survivin was independently confirmed by protein expression analysis (). Analysis of total RNA from BAFF-treated cells showed that the abundance of these transcripts reflects overall changes in the pattern of gene expression rather then BAFF-mediated recruitment of the selected mRNAs into polyribosomes (Table S2).
Stimulation with BAFF resulted not only in the transcription and translation of genes required for cell cycle progression but also in phosphorylation of the key cell cycle controlling Rb protein (, bottom), which is prerequisite for the release of E2F and cell cycle entry into S phase (). Despite the substantial accumulation and modification of cell cycle–controlling proteins, BAFF does not induce B cell proliferation in vitro (). However, preincubation of B cells with BAFF accelerates proliferation in response to BCR stimulation compared with BCR-only triggered cells (). This result points to a role of BAFF in maintaining B cells in a state of immediate responsiveness to antigenic stimulation.
Increased protein synthesis in response to BAFF suggests that BAFF controls activation of proteins required for translation. Indeed, treatment with BAFF caused phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) and its inhibitor, eIF4E-binding protein 1 (4E-BP1; ). Both are required for active protein synthesis: phosphorylation of eIF4E increases its binding to capped mRNAs, whereas 4E-BP phosphorylation disrupts its binding to eIF4E. Treatment with BAFF also leads to phosphorylation of S6 ribosomal protein, a hallmark of active protein synthesis (–).
Phosphorylation of 4E-BP, which is known to be controlled by Akt and Pim-2 (), suggested an involvement of these kinases in BAFF signaling. Activation of Akt requires its recruitment to the plasma membrane, where it binds to the lipid second messenger phosphatidyl inositol-3,4,5-triphosphate via its pleckstrin homology domain. This induces a conformational change, which allows for Akt phosphorylation at threonine 308 by PDK1 and at serine 473 by a second kinase, whose identity is still controversial (, ).
We found that treatment with BAFF led to a rapid phosphorylation of Akt at both S473 and T308, which, after a transitory decline after 1 h of stimulation, regained a stably high level by 24 h and beyond ( and not depicted). Activation of Akt by BAFF was associated with the phosphorylation of several Akt targets known to control cell survival and metabolism (). First, treatment with BAFF lead to phosphorylation of the GTPase-activating protein tuberous sclerosis complex 2 (TSC2), which promotes protein synthesis through activation of mammalian target of rapamycin (, ). Second, BAFF induced the phosphorylation and, hence, inactivation of glycogen synthase kinase–3 (GSK-3). GSK-3 has been found to cause apoptosis by inducing MCL1 degradation and compromising mitochondrial membrane integrity (). Finally, stimulation of B cells with BAFF led to phosphorylation of FoxO1 and its subsequent degradation. Members of the FoxO family of transcription factors are known to play a key role in the regulation of genes required for cell cycle progression, glycolytic metabolism, and survival (, ).
Growth factor stimulation of hematopoietic cells induces Pim-2 expression, which confers resistance to atrophy and cell death (). Treatment with BAFF strongly induced all three isoforms of Pim-2 within 24 h of stimulation ().
Akt-activation requires its binding to specific phosphoinositides, which are generated by PI3K at the plasma membrane (). The predominant form of PI3K in B cells consists of the regulatory subunit p85α and the catalytic subunit p110δ (). We found that treatment with BAFF led to tyrosine phosphorylation of a p85-associated protein with a molecular weight of approximately p110 (). Sequence analysis of a protein that co-migrated with phospho-p110 confirmed that this protein represents the catalytic subunit p110δ (unpublished data). It is therefore possible that BAFF activates PI3K by inducing the tyrosine phosphorylation of its catalytic subunit. A similar phenomenon has previously been reported in B cell lines stimulated through the BCR (, ).
The activation of PI3K by BAFF is important for the regulation of Akt activation and B cell survival. Treatment of B cells with the PI3K-specific inhibitor LY294002 diminished BAFF-induced activation of Akt and phosphorylation of Akt target proteins ( and Fig. S1, available at ). Consistently, BAFF-mediated B cell survival was severely impaired by concentrations of LY294002, which abolish Akt activity (). The half-life of the phosphoinositide products of PI3K is limited by the lipid phosphatase and tensin homologue deleted on chromosome 10 (PTEN), but BAFF did not appear to affect PTEN activity (Fig. S2), as judged by phosphorylation of the inhibitory PTEN phosphorylation site serine 380 ().
Akt activation requires two sequential events. Initially, Akt binding to the phosphoinositide products of PI3K induces a conformational change in the protein. This allows for its phosphorylation at two essential sites: T308 in the activation loop is phosphorylated by PDK1, whereas the identity of the kinase that phosphorylates the C-terminal S473 residue remains to be determined (, ). In search of the kinase, which is responsible for Akt S473 phosphorylation and B cell survival in response to BAFF, we tested the role of PKCβ in BAFF-mediated signaling. This choice was based on two major reasons. First, coincubation with recombinant PKCβ leads to Akt phosphorylation at S473 but not T308 (). Second, PKCβ deficiency in mice causes a decline in B cell survival in vitro and in vivo (, ).
We initially assessed the ability of BAFF to activate PKCβ by analysis of BAFF-induced PKCβ translocation to the plasma membrane. This translocation is an activation hallmark for phospholipid-dependent classic PKCs, including PKCβ (–), and triggering of B cells through the BCR leads to translocation of PKCβ into specialized membrane microdomains called lipid rafts (). We found that treatment of B cells with BAFF increased the amount of membrane-associated PKCβ (). A similar level of BAFF-induced membrane translocation was observed for Akt (). Notably, membrane translocation of PKCβ in response to BAFF occurred outside lipid rafts (Fig. S3, available at ). Because lipid rafts of BAFF-triggered B cells did also not contain Akt or PDK1, we conclude that BAFF-induced Akt activation is a raft-independent event.
In addition to PKCβ translocation to the plasma membrane, BAFF promotes the association of PKCβ with Akt (). This association does not depend on PI3K activity (Fig. S4, available at ), but it is functionally important, as BAFF-induced Akt phosphorylation at S473 was greatly reduced in PKCβ-deficient B cells (). In contrast, Akt phosphorylation at the PDK1 target site T308 was similar between wild-type and mutant cells. The functional link between PKCβ and Akt is specific to BAFF signaling, as stimulation through the BCR induced Akt phosphorylation in PKCβ-deficient B cells at wild-type levels ().
PKCβ-deficient B cells exhibit poor survival in vitro in the absence of stimuli () (). Treatment with BAFF promotes the viability of mutant B cells, although they fail to reach wild-type survival levels regardless of the BAFF dose and the duration of treatment. Although PKCβ-deficient cells were partially responsive to the survival action of BAFF, its ability to support B cell growth in vitro was severed by lack of PKCβ (). These results suggest that the PKCβ-mediated Akt signaling element contributes mostly to BAFF-mediated B cell fitness rather than mere survival. Altered cellular responses of PKCβ-deficient B cells were not caused by defective surface expression of BAFF-R (), arguing for a cell-intrinsic BAFF signaling defect in the absence of PKCβ.
The involvement of PKCβ in BAFF-mediated signaling is indirectly supported by the pattern of changes in the PKCβ-deficient, peripheral B cell compartment. In the absence of BAFF signaling, B cell maturation arrests between the T1 (CD21) and the T2 (CD21) stages (, –). PKCβ deficiency impairs B cell maturation in a fashion similar to failed BAFF signaling, albeit at a lower magnitude. In addition, the size of the peripheral PKCβ-deficient B cell compartment falls to ∼70% of wild-type levels (), and immature B cells prevail over mature B cells in the spleen of mutant mice, as judged by surface IgM and IgD expression (). Collectively, these observations suggest a partial defect in BAFF-mediated signaling and peripheral B cell maturation in the absence of PKCβ.
BAFF's prosurvival function has been linked to its ability to activate the processing of the NF-κB2 protein p100 into the transcriptionally active form p52 (, ). We find that BAFF-induced p100-p52 processing was not affected by the absence of PKCβ (). We have previously shown that BAFF controls B cell survival through cytoplasmic retention of the proapoptotic kinase PKCδ (). However, the ratio of cytoplasmic versus nuclear PKCδ was not altered by PKCβ deficiency (). We conclude that the BAFF-mediated regulation of NF-κB and PKCδ occurs independent of PKCβ.
Our findings show that like cytokines such as IL-3 and IL- 7, which promote survival of hematopoietic cells, BAFF supports B cell survival by increasing metabolic fitness. Treatment of B cells with BAFF induces transcription of mRNAs that encode components of carbohydrate metabolism. The BAFF-induced metabolic bias toward glycolysis might be especially important for B cell survival in lymphoid organs and at inflammatory sites where oxygen tension is low compared with arterial blood (, ). In this environment, activation of glycolysis will provide energy to sustain the active protein synthesis and cell growth caused by BAFF. Indeed, prolonged BAFF treatment led to expression of the hypoxia-inducible factor α (unpublished data).
Normally, accumulation of proteins, particular those controlling cell cycle, and increase in cell volume precedes cell division. However, the lack of BrdU incorporation in BAFF-treated cells shows that BAFF does not induce DNA-replication, thus precluding cell division. Previous findings show persistent expression of Cdk inhibitor proteins p18 and p27 in BAFF-treated cells (). Therefore, it is likely that high expression of these and possibly other cell cycle inhibitors prevents the proliferation of BAFF-treated B cells in the absence of an antigenic signal.
BAFF-stimulated cells enter BCR-induced proliferation more readily than untreated cells. This result suggests that the accumulation of cell cycle–controlling proteins in response to BAFF prepares resting B cells for an immediate immune response upon antigenic challenge. It is also attractive to speculate that, in response to BAFF, B cells might establish a storage pool of certain cell cycle–controlling mRNAs or proteins, which could be used for several rounds of cell division. Such a mechanism would help explain the astonishingly short replication time (∼7 h) of B cells at the height of the germinal center response (, ).
Akt activation requires two potentially independent pathways. The PI3K-dependent pathway is shared both by BAFF-R and BCR. This feature of the pathway makes it particularly important in the regulation of B cell immunity and explains the poor survival of B cells in the absence of the p85 regulatory subunit of PI3K (, ). The pathway that involves PKCβ appears to be BAFF specific. Thus, PKCβ deficiency impairs phosphorylation of Akt on activating serine 473 in response to BAFF but not BCR stimulation. The residual BAFF-induced Akt S473 phosphorylation, which we observe in the absence of PKCβ, indicates an ability of other kinases to partially adopt this role. This would be expected, as a plethora of kinases has previously been implicated in mediating Akt S473 phosphorylation ().
Increased translocation of PKCβ to the plasma membrane and PKCβ association with Akt in response to BAFF suggest a direct involvement of PKCβ in BAFF signaling. Furthermore, PKCβ dependency of Akt phosphorylation at S473 but not T308 agrees with in vitro data on Akt phosphorylation by PKCβ () and suggests a direct action of PKCβ on S473 of Akt upon BAFF stimulation.
Involvement of PKCβ in BAFF and BCR signaling provides a mechanistic explanation for the poor survival and altered peripheral maturation of PKCβ-deficient B cells in vitro and in vivo. However, the relatively mild reduction of B cell numbers in the absence of PKCβ argues in favor of additional PKCβ-independent signaling pathways initiated by BAFF-R and/or BCR. Signaling from both receptors induces activation of NF-κB, albeit through distinct mechanisms. Although PKCβ has an important function in canonical NF-κB activation upon BCR-triggering, BAFF-induced NF-κB2 processing is independent of PKCβ. Another PKC family member, PKCδ, also plays an important role in the regulation of B cell survival, but it promotes cell death rather than survival. The proapoptotic potential of PKCδ is contained by BAFF, which prevents its accumulation in the nucleus. This BAFF-dependent survival mechanism can also function in the absence of PKCβ.
BAFF-mediated B cell survival likely represents the collective outcome of several BAFF-induced signaling features, including NF-κB activation, cytoplasmic retention of PKCδ, and Akt activation. It is thus conceivable that the loss of a single signaling branch can to some degree be compensated for. In this case, the loss of PKCβ would be expected to cause some damage to B cell survival, but it would be less severe than the complete abrogation of BAFF signaling. This could explain the more dramatic changes to the B cell compartment in BAFF- or BAFF-R–deficient mice than in PKCβ knockouts. Although PKCβ-deficient B cells were partially responsive to the survival action of BAFF, they appeared to be largely refractive to BAFF-mediated cell growth. This cellular process is closely associated with Akt- and mammalian target of rapamycin–dependent signaling pathways and represents an important distinction between growth factor–mediated cellular fitness on the one hand and mere cell survival on the other (, ). The latter can also be achieved through an altered ratio in the expression of pro- and antiapoptotic proteins, but, in this context, cells become progressively smaller (). It appears that, perhaps in contrast to BAFF-induced NF-κB activation and cytoplasmic retention of PKCδ, which mediate B cell survival (), PKCβ-dependent Akt activation preferentially regulates the fitness facet of BAFF-mediated cellular responses.
Our findings may have practical implications. There is increasing evidence that BAFF plays an important role in the control of autoreactive B cells (, , , , , ). Therefore, identification of Akt and PKCβ as components of BAFF signaling may suggest novel ways of pharmacological intervention in B cell–mediated autoimmune disorders.
Mice were housed in the Laboratory Animal Research Center at the Rockefeller University under specific pathogen-free conditions. C57BL/6 mice were used as a source of wild-type B cells. PKCβ-deficient mice on a C57BL/6 genetic background were used for analysis (, ). Mice were used at ages of 8–12 wk. Protocols were approved by the Institutional Animal Care and Use Committee at the Rockefeller University.
Resting mature CD43, CD62L B cells were isolated from spleen and lymph nodes by MACS (Miltenyi Biotec) according to the manufacturer's instructions. The purity of mature B cells was routinely >95%. Cells were cultured for 12–16 h in RPMI 1640 medium supplemented with 10% FBS, 2 mM -glutamine, 50 μM 2-mercapto-ethanol, and 100 U/ml penicillin/streptomycin at 37°C in a CO incubator to reduce the background BAFF signaling caused by endogenous BAFF in vivo. This preincubation did not alter B cell size. After preincubation, live cells were separated over a Ficoll gradient (Cedar Lanes), and their purity was assessed by trypan blue exclusion. Cells were stimulated in culture medium using 25 ng/ml BAFF (R&D Systems) or 1.3 μg/ml F(ab′) fragment goat anti–mouse IgM (Jackson ImmunoResearch Laboratories). LY294002 (Calbiochem) was used at a 10-μM concentration.
Cells were analyzed on a flow cytometer (FACSCalibur; Becton Dickinson) using CellQuest software (BD Biosciences). For measurement of mitochondrial membrane potential, cells were used after 12–16 h of preincubation without BAFF (time point 0) or after 72 h of BAFF stimulation. Cells were incubated in culture medium with or without BAFF and containing the potentiometric dye tetramethylrhodamine ethyl ester (TMRE; Invitrogen) at a 100-nM concentration. Carbonyl cyanide m-chlorophenyl hydrazone (Sigma-Aldrich) was added to control samples at a concentration of 50 μM as an uncoupler of the mitochondrial respiratory chain. After 30 min of incubation at 37°C in a CO incubator, cells were immediately analyzed by FACS. For BrdU incorporation, B cells were cultured in the presence of 10 μM BrdU, and fixed and stained using the FITC BrdU Flow Kit (BD Biosciences). For cell death analysis, an aliquot of ∼10 cells was removed from the B cell culture at various time points ranging from 0 to 96 h. TO-PRO-3 (Invitrogen) was added at a concentration of 10 nM to distinguish live (TO-PRO-3) and dead (TO-PRO-3) cells in the culture samples. Upon TO-PRO-3 addition, cells were immediately analyzed by FACS. B cell surface marker expression was analyzed using antibodies against IgD, CD21/35, B220 (BD Biosciences), BAFF-R (R&D Systems), or a F(ab′) fragment of goat anti–mouse IgM (Jackson ImmunoResearch Laboratories), as previously described ().
Cells were lysed in buffer containing 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10% glycerol, 1% NP-40, 10 μM NaF, 2 mM NaVO, and protease inhibitor cocktail (Sigma-Aldrich). Lysates were resolved by SDS-PAGE and transferred onto Immobilon-P membrane (Millipore) using standard procedures. Membranes were developed with the following antibodies against: pS6 (S235/236), S6, peIF4E (S209), eIF4E, p4E-BP1 (S65), 4E-BP1, pAkt (S473), pAkt (T308), pGSK-3β (S9), GSK-3β, pFoxO1 (S256), FoxO1, pTSC2 (T1462), pPTEN (S380), PTEN, and Erk (all obtained from Cell Signaling Technology); Pim-2, Akt, TSC2, and PKCβ (all obtained from Santa Cruz Biotechnology, Inc.); p85, p110δ, and pY (4G10; all obtained from Upstate Biotechnology); and tubulin (Sigma-Aldrich). Where applicable, membranes were first developed with phosphospecific antibodies, and stripped and reprobed with the respective control antibody. For Akt immunoprecipitation, lysates were precleared with goat-serum and protein G–Sepharose (GE Healthcare), followed by incubation with anti-Akt antibody overnight. Anti–ferritin heavy chain antibody (Santa Cruz Biotechnology, Inc.) was used as a goat immunoglobulin control. Immune complexes were precipitated with protein G–Sepharose, washed five times in lysis buffer, and eluted in SDS sample buffer for SDS-PAGE. For p85 immunoprecipitation, lysates were precleared with rabbit immunoglobulin and protein A–Sepharose (GE Healthcare), followed by incubation with p85 antibody–agarose conjugate overnight. Immune complexes were washed and eluted as described earlier in this section. For analysis of NF-κB2 p100 processing and for the expression of cell cycle proteins, cells were lysed in a buffer containing 350 mM NaCl, 20 mM Hepes/NaOH, pH 7.9, 1 mM MgCl, 0.2 mM EDTA, 0.1 mM EGTA, 1% NP-40, 20% glycerol, 10 μM NaF, 2 mM NaVO, and protease inhibitor cocktail (Sigma-Aldrich). Lysates were analyzed by SDS-PAGE, and membranes were incubated with antibodies against NF-κB2 p52 (Upstate Biotechnology) or cyclin D2, cyclin E, Cdk4, and Mcm3 (all obtained from Santa Cruz Biotechnology, Inc.); Mcm2 and Rb (obtained from BD Biosciences); Ki67 (DakoCytomation); and Survivin (Chemicon). Signal quantification was done using NIH Image 1.63 software.
Lipid rafts were prepared as described previously (). The purity of the preparation was assessed by the presence of Lyn and the lipid raft marker ganglioside GM1, which was detected using horseradish peroxidase–coupled cholera toxin B subunit (Sigma-Aldrich). Proteins were detected using antibodies against PKCβ, Akt, and PDK1 (all obtained from Santa Cruz Biotechnology, Inc.) and Lyn (a gift from C. Lowell, University of California, San Francisco, San Francisco, CA).
Fractionation of primary mouse B cells into cytoplasmic and nuclear extracts was performed as described previously (). Lysates were analyzed for expression of PKCδ using an antibody from Santa Cruz Biotechnology, Inc. Fraction purity was assessed by Western blot analysis using tubulin as cytoplasmic and lamin B (Santa Cruz Biotechnology, Inc.) as nuclear markers, respectively. For preparation of cytoplasmic and membrane fractions, cells were disrupted by hypotonic lysis for 15 min on ice in a buffer containing 10 mM Hepes, pH 7.4, 10 mM KCl, 1.5 mM MgCl, 0.1 mM EDTA, 10 μM NaF, 2 mM NaVO, and protease inhibitor cocktail (Sigma-Aldrich). Nuclei were removed by centrifugation at 400 for 10 min. The supernatant was then centrifuged at 95,000 for 1 h in a SW55Ti rotor (Beckman Coulter). The supernatant was removed and saved as the cytoplasmic fraction. The pellet containing the membrane fraction was resolved in SDS sample buffer. Fraction purity was assessed by Western blot analysis using tubulin as cytoplasmic and Lyn as membrane markers, respectively.
Polyribosomes were purified essentially as described previously () from unstimulated cells and those that had been cultured in the presence of BAFF for 36 h. In brief, cells were incubated in culture medium containing 100 μg/ml cycloheximide (CHX; Sigma-Aldrich) for 15 min, washed three times in PBS containing CHX, and lysed in 10 mM Hepes-KOH, pH 7.4, 150 mM KCl, 5 mM MgCl, 1% NP-40, 0.5 mM DTT, 100 μg/ml CHX, 200 U/ml RNAsin (Promega), 100 U/ml SUPERase (Ambion), and EDTA-free protease inhibitor cocktail (Roche). All chemicals were of molecular biology–grade purity and nuclease free. Nuclei were removed from the extracts by centrifugation at 2,000 for 10 min. An aliquot of the lysate was saved for preparation of total RNA. The remaining lysate was loaded onto a 20–50% wt/wt linear density sucrose gradient in 10 mM HEPES-KOH, pH 7.4, 150 mM KCl, and 5 mM MgCl. Gradients were centrifuged for 2 h at 40,000 at 4°C in a SW41 rotor (Beckman Coulter). Fractions of 0.7-ml volume were collected with continuous monitoring at 254 nm using an ISCO UA-6 UV detector. To identify polyribosome-containing fractions, the content of ribosomal S6 protein in the fractions after trichloroacetic acid precipitation was determined by Western blot. For RNA isolation, polysome-containing fractions were pooled, and RNA was isolated from total and polysomal samples using TRI LS (Invitrogen) according to the manufacturer's protocol.
Quality of RNA was confirmed before labeling by analyzing 20–50 ng of each sample using the RNA 6000 NanoAssay and a Bioanalyzer 2100 (Agilent Technologies). All samples had a 28S/18S ribosomal peak ratio of 1.8–2 and were considered suitable for labeling. 2 μg of total RNA was used for cDNA synthesis using an oligo(dT)-T7 primer and the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen). Synthesis, linear amplification, and labeling of cRNA were accomplished by transcription in vitro using the MessageAmp aRNA Kit (Ambion) and biotinylated nucleotides (Enzo Diagnostics). 10 μg of labeled and fragmented cRNA were hybridized to the mouse genome MOE430 2.0 array (Affymetrix), which interrogates ∼39,000 transcripts at 45°C for 16 h. Automated washing and staining were performed using the Fluidics Station 400 (Affymetrix), according to the manufacturer's protocols. Finally, chips were scanned with a high numerical aperture and flying objective lens in the scanner (GS3000; Affymetrix). Raw expression data were analyzed using Microarray Analysis software (version 5.1; Affymetrix). The complete datasets can be accessed at under accession no. . Data were normalized to a target intensity of 500 to account for differences in global chip intensity. Genes, which were differentially expressed between unstimulated and BAFF-treated cells, were initially identified using GCOS software. 377 transcripts showed a change of twofold or more (Table S2) and were selected for further analysis. Approximately three quarters of these genes were up-regulated upon BAFF stimulation, whereas one quarter was down-regulated. For a biological interpretation of the differentially regulated genes, the GoMiner gene ontology (GO) tool was used. GO is a genome project that describes gene products in terms of their associated biological processes, cellular components, and molecular functions (). GO categories are organized in directed acyclic graphs, a kind of hierarchy in which one category can have more than one “parent.” GoMiner identifies GO categories that are over- or underrepresented in lists of genes of interest, such as differentially expressed genes from a microarray experiment, and calculates statistical significance as the one-sided nominal unadjusted p-value from Fisher's exact test (). We report only those BAFF-induced biological processes in which the p-value was <0.001.
Gel-resolved proteins were digested with trypsin, batch purified on a reversed-phase microtip, and resulting peptide pools were individually analyzed by matrix-assisted laser desorption/ionization reflectron time-of-flight (MALDI-reTOF) mass spectrometry (MS; ultraflex TOF/TOF; Bruker Daltronics Inc.) for peptide mass fingerprinting (PMF), as previously described (). Selected peptide ions (m/z) were taken to search a nonredundant (NR) protein database (3,245,378 entries on 28 January 2006; National Center for Biotechnology Information) using the PeptideSearch algorithm (developed by Matthias Mann, Max-Planck-Institute for Biochemistry, Martinsried, Germany; an updated version of this program is currently available as PepSea from Applied Biosystems/MDS SCIEX). A molecular mass range up to twice the apparent molecular weight (as estimated from electrophoretic relative mobility) was covered, with a mass accuracy restriction of <35 ppm and a maximum of one missed cleavage site allowed per peptide. To confirm PMF results with scores ≤40, mass spectrometric sequencing of selected peptides was done by MALDI-TOF/TOF (MS/MS) analysis on the same prepared samples using the ultraflex instrument in “LIFT” mode. Fragment ion spectra were taken to search the NR protein database using the Mascot MS/MS ion search program (version 2.0.04 for Microsoft Windows; Matrix Science Ltd.) (). Any tentative confirmation (Mascot score ≥30) of a PMF result thus obtained was verified by comparing the computer-generated fragment ion series of the predicted tryptic peptide with the experimental MS/MS data.
Fig. S1 shows BAFF-induced Akt phosphorylation in the absence or presence of LY294002. Fig. S2 shows PTEN phosphorylation at serine 380, as well as total PTEN content in cell extracts prepared at various time points of BAFF stimulation. In Fig. S3, the distribution of PKCβ, Akt, and PDK1 into lipid rafts was analyzed before and after BAFF stimulation. Lipid raft–containing fractions are marked by the presence of Lyn and GM1. Fig. S4 shows BAFF-induced PKCβ-Akt association, which is not affected by the presence of LY294002. Table S1 lists the gene IDs for the GO categories presented in . Table S2 contains the complete list of Affymetrix probe sets that showed a fold change of two or more in BAFF-stimulated versus unstimulated cells. Results are given both for RNA samples isolated from polysomes and analyzed in (sheet 1 = polysome), as well as RNA samples derived from total cell lysates (sheet 2 = total). Online supplemental material is available at . |
CD40 is expressed by all mature B cells, as well as by dendritic cells, macrophages, fibroblasts, epithelial cells, and endothelial cells (, ). CD40 is crucial for the induction of effective adaptive immune and inflammatory responses (). The interaction between CD40 and CD40 ligand (CD40L; also known as CD154), which is expressed by activated T cells, activated B cells, and activated platelets, promotes both humoral and cell-mediated immune responses. Indeed, studies using CD40-deficient mice show that CD40 has an essential role in T cell–dependent immunoglobulin class switching, memory B cell development, and germinal center formation (, ). Engagement of CD40 by CD40L on the surface of human endothelial cells induces the activation of Ras and phosphatidylinositol 3–kinase (PI3; ). Activation of this signaling pathway leads to the expression of several angiogenic factors—such as vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2)—and promotes angiogenesis in xenografts in vivo (, , ).
These two functions of CD40, in promoting both immune responses and angiogenesis, have contradictory effects on the growth of a tumor. Tumor-specific immune responses limit tumor growth, whereas increased angiogenesis supports tumor growth by providing nutrients to the tumor cells and providing them with a route to distant organs. However, although CD40 is expressed by blood vessels of renal cell carcinomas () and Kaposi's sarcoma (), so far a requirement for CD40 in neovascularization of tumors has not been established experimentally. Determining whether CD40 has more of an effect on antitumor immune responses or tumor vascularization may have important implications for the design and implementation of therapeutics that target this molecule.
To test the role of CD40 in the development of mammary carcinomas, Chiodoni et al. crossed transgenic mice expressing the oncogene under the control of the mouse mammary tumor virus promoter with CD40-deficient mice, and compared tumor onset and progression in these mice with that in CD40-sufficient -transgenic mice (). Remarkably, the analysis of tumors in the CD40-deficient transgenic mice showed increased latency, reduced numbers of tumors, and decreased total tumor size in comparison to the CD40-sufficient mice. These findings were surprising. Because CD40 is crucial in mounting an immune response, one would predict that mice lacking this protein would be more prone to tumors than their CD40-sufficient counterparts.
The reduced tumorigenicity was not a result of a functional defect in CD40-deficient bone marrow–derived cells in the tumor microenvironment. Rather, a lack of CD40 on endothelial cells impaired tumor angiogenesis, leading to a block in tumor growth. Indeed, in Matrigel implantation assays, the authors found that the interaction between CD40L (or an antibody specific for CD40) and CD40 on mouse endothelial cells triggered vessel formation in vivo. When the formation of tumor-associated blood vessels was analyzed in the mice, striking defects in the size, number, and organization of these vessels were observed in the absence of CD40 ().
These data suggest, at least in this model, that CD40 is required to establish a blood supply for the tumor. The authors next investigated CD40L expression in the tumor tissue. It is well known that CD40L is expressed by activated CD4 T cells (, ). More recently, CD40L has also been detected on human platelets, which release the soluble form of CD40L upon activation (, ). Because CD4 T cells are absent from the tumors in -transgenic mice, the authors focused on platelets. Treatment of CD40-sufficient -transgenic mice with Clopidogrel, a drug that blocks platelet aggregation and the release of soluble CD40L (, ), decreased both the tumor size and number. These data led the authors to suggest that the effect of Clopidogrel on tumor growth is probably caused by the inhibition of platelet activation and, therefore, the inhibition of CD40L release ().
The tumor microenvironment has a major impact on tumor growth, invasion into the bloodstream, and metastasis. Different cell populations present in the microenvironment affect these processes in distinct ways. For example, recent studies (, ) have shown that myeloid cells within tumors are not passive structural elements. Rather, these cells can take up tumor antigens and, upon maturation and activation, migrate to draining lymph nodes, where they can elicit a tumor-specific immune response. In contrast, activation of bone marrow–derived cells in the tumor can induce an inflammatory reaction, which either promotes or inhibits tumorigenesis. Furthermore, bone marrow–derived progenitors in the tumor can generate endothelial cells and hematopoietic cells that support tumor growth and metastasis to different organs through angiogenesis ().
Soluble factors in the tumor micro-environment also influence tumor growth and metastasis. Neovascularization, for example, is regulated by factors that either promote or inhibit angiogenesis. VEGF, FGF1, and FGF2 are known to trigger angiogenesis by binding to tyrosine kinase receptors on endothelial cells, inducing proliferation of the vasculature (, ). A prototypical angiogenesis inhibitor is thrombospondin-1, which binds CD36, a transmembrane receptor expressed by endothelial cells that is coupled to intracellular SRC-like tyrosine kinases (). This signaling pathway leads to the expression of Fas ligand (also known as CD95L) by endothelial cells and their subsequent apoptosis ().
In general, the relative amounts of the different activators and inhibitors dictate whether an endothelial cell will be in a quiescent or proangiogenic state. During tumor development, neovascularization is initiated by an “angiogenic switch,” which alters the appropriate balance between the activating and inhibitory signals received by an endothelial cell and causes the endothelial cells to proliferate ().
Until recently, the predominant view on the vascularization of a tumor was “tumor centric,” whereby the tumor induces angiogenesis by either secretion of proangiogenic factors and/or by transcriptional down-regulation of the expression of angiogenic inhibitors. Now, the observations of Chiodoni et al. (), along with other recent publications (, ), show that the induction of proangiogenic factors by the tumor alone is not sufficient. Rather, the tumor and its environment have a dynamic relationship that results in neovascularization and the promotion of tumor growth.
The report by Chiodoni et al. () also demonstrates that the same protein can have different functions in distinct physiological compartments in the tumor environment and, thus, can either promote or inhibit tumorigenesis. In the breast cancer model used by Chiodoni et al., the proangiogenic role of CD40 is dominant over its role in generating an antitumor immune response, thus promoting tumor progression (). The opposite was true, however, when the authors transplanted fast-growing tumor cell lines derived from -transgenic mice into CD40-deficient or CD40-sufficient mice. Under these conditions, more aggressive tumors developed in CD40-deficient mice than in CD40-sufficient mice (). In this transplant model of breast cancer, the transplanted tumor cells have already been selected for tumorigenic and malignant potential, and the immunological role of CD40 prevailed over the proangiogenic effect. Hence, it is not possible to focus only on the proangiogenic aspect of CD40 function and to neglect its role in immunosurveillance. This study seems to indicate that the involvement of CD40 in immune responses is more important during later stages of tumorigenesis.
The absence of CD40 might therefore have different outcomes on tumor growth depending on the stage of tumor development. In this breast cancer model, a lack of CD40 before tumors arise inhibited tumor growth, whereas a lack of CD40 at later stages of tumor development enhanced tumorigenicity and caused a more severe outcome. However, this conclusion is somewhat surprising, as it is generally assumed that advanced tumors are more dependent on neovascularization than early stage tumors. It is therefore essential to clearly define the tumor stage at which the switch from CD40-dependent vascularization to CD40-independent vascularization takes place and to understand what causes CD40 independence of late-stage tumors.
Based on the observations of Chiodoni et al. (), it is conceivable that the activation of endothelial cells by CD40 is an early event in tumorigenesis, whereas multiple angiogenic stimuli become available later in tumor progression and render CD40 expression by endothelial cells dispensable. It is now necessary to identify these other angiogenic factors and to understand how they stimulate vascularization independently of CD40.
Experiments with antiangiogenesis factors in a mouse model of pancreatic islet carcinogenesis have shown that the efficacy of angiogenesis inhibitors depends on the stage of tumor development: some inhibitors of angiogenesis are successful for the treatment of early stage disease but do not cause regression of end-stage tumors, whereas others are effective at reducing the mass of end-stage tumors but have no effect on preventing progression during early stage disease (). Therefore, the actual mechanism of tumor neovascularization seems to depend on the stage of tumor development, and specific pro- and antiangiogenic factors seem to be relevant at different stages of the disease. It is interesting to speculate that the responsiveness of tumor-associated endothelial cells to stimulation through CD40 and/or their level of expression of CD40 have a role in mediating the outcome of different antiangiogenesis therapies.
Another important observation of the paper by Chiodoni et al. () is the possible identification of the cells that are responsible for CD40-triggered angiogenesis. Because treatment of CD40-sufficient -transgenic mice with the antiplatelet drug Clopidogrel reduced tumor growth, it seems probable that platelets are responsible for CD40-triggered angiogenesis in this model. However, cell types other than platelets also express CD40L, and these cells could activate CD40 if located in a tumor. The ability of such cells to reside in the tumor or be recruited to a tumor could differ depending on the type of tumor or the stage of tumor development. Indeed, although T cells were absent from the tumors analyzed by Chiodoni et al., T cells are often found in leukocyte infiltrates in human breast carcinoma (). Therefore, in different breast cancer models T cells could have a more prominent role as CD40L-expressing cells in the tumor. Furthermore, as endothelial cells express CD40L (), one could envision an “autocrine” CD40–CD40L stimulating circuit within the endothelium.
Tumor angiogenesis offers an attractive target for the treatment of individuals with cancer, and several antiangiogenic drugs are already in use, albeit with variable therapeutic success (). A combinatorial approach to treatment that targets both cancer cells and the surrounding stroma might prove more promising than using single antiangiogenic agents.
Chiodoni et al. suggest a novel and intriguing approach to the treatment of cancer: the use of anticoagulants to prevent the interaction between platelets and endothelial cell progenitors in the tumor, thereby blocking the CD40–CD40L interaction that causes platelets to promote tumor angiogenesis. So far, therapies targeting CD40 have been designed to trigger CD40 signaling and thus boost the immune response against the tumor (). But this approach might be a double-edged sword, because promoting CD40 expression might also promote angiogenesis.
Importantly, as in the previous paragraph, the effect of CD40 loss on tumorigenesis differs depending on the stage of tumor development, which therefore has implications for therapy. Although down-regulation of CD40 expression might be desirable during early tumor development, it could have catastrophic consequences at later stages, when a tumor is more aggressive, by inhibiting the tumor-specific immune response. It is also important to point out that this study focuses exclusively on mammary tumors. Although the authors show that CD40 is required for neovascularization and growth of carcinomas in a model of breast cancer, other tumor types, especially more aggressive and faster growing tumors, might not be dependent on CD40. For this reason, the use of a CD40-based therapy for cancer is premature at this stage. Nevertheless, this study clearly highlights the fact that a targeted therapy has to be tailored not only to the genetic makeup of the tumor but also to the particular stage of tumor progression and the intricate relationship between the tumor and its stromal environment. |
Previous studies using cell lines and platelets demonstrated that phosphorylation of β integrin cytoplasmic tyrosines is involved in outside-in integrin signaling. Therefore, we assessed whether EC adhesion to extracellular matrix proteins could induce tyrosine phosphorylation of β integrin. We isolated ECs from the lungs of WT and DiYF mice, plated them on various substrates, and assessed the phosphorylation of β integrin on Tyr747 and Tyr459 (). Very low levels of phosphorylation of WT β integrin were observed when cells were kept in suspension or plated on poly--lysine. Extracellular matrix proteins that serve as ligands for αβ such as vitronectin, fibronectin, fibrinogen, and gelatin strongly stimulated Tyr759 and Tyr747 phosphorylation of WT ECs. Laminin and collagen, which are recognized primarily by integrins other than αβ, induced lower levels of tyrosine phosphorylation. Sodium pervanadate, known to block phosphatase activity, was used as a positive control in this experiment. As anticipated, no tyrosine phosphorylation of β integrin was observed in DiYF EC under any conditions.
The role of the β subunit phosphorylation in extracellular matrix recognition was previously assessed in CHO cells and K562 cells. These systems do not provide insight into the roles of β integrin in angiogenesis and in the regulation of VEGF or FGF-induced EC responses. These processes can be only assessed in ECs expressing appropriate receptors and signaling intermediates. We sought to determine whether VEGF treatment affected the phosphorylation status of β integrin. Treatment of ECs with VEGF-A165 induced both Tyr747 and Tyr759 phosphorylation in a time-dependent manner (). Both time curves of β tyrosine phosphorylation followed a bell-shaped pattern, which is typical for growth factor–induced responses. Thus, not only integrin engagement, but also VEGF treatment stimulated tyrosine phosphorylation of β subunit in ECs, suggesting a possible regulatory role for this process in VEGF-induced angiogenesis.
We next evaluated whether β integrin cytoplasmic tyrosine motifs are crucial for a complete angiogenic response to VEGF in vivo. We implanted VEGF-A–containing Matrigel subcutaneously into WT and DiYF mice and assessed the angiogenic response based on the amount of hemoglobin extracted from Matrigel. As shown in , the hemoglobin concentration was at least fivefold lower in Matrigel plugs isolated from DiYF mice compared with WT counterparts. The vascular density in Matrigel implants was assessed by von Willebrand factor (vWF) staining. Only 50% of Matrigel plugs from DiYF mice had a distinguishable vasculature and exhibited staining for vWF (, top). The vascular density was 4.2-fold lower in DiYF mice than in WT controls (P = 0.01) (, bottom). Thus, VEGF-induced angiogenesis was significantly impaired in DiYF mice.
Further, we assessed tumor-induced pathological angiogenesis in DiYF and WT mice. To this end, mouse melanoma cells (B16F10) were implanted subcutaneously into mice, and after 10 d tumors were excised. The average weight of tumors formed in DiYF mice was at least twofold lower than average tumor weight in WT mice (0.14 g vs. 0.29 g) (). Tissue section analysis revealed the presence of well-developed blood vessels that stained positively for CD31, and laminin in tumors from WT mice. In contrast, in tumors formed in DiYF mice, blood vessels were sparse and thin walled based on laminin staining (, top). The vascular density in tumors grown in DiYF mice was sixfold lower than that in WT mice (P = 0.009) (, bottom). Similar defects in tumor growth and neovascularization were observed using alternative model of tumor growth in vivo. Mouse prostate cancer cells (RM-1) were implanted into WT and DiYF mice, and tumor was excised after 10 d. Tumors formed in DiYF mice were smaller than in WT mice (1.17 g vs. 0.8 g) (). Tissue section analysis indicated the presence of substantially larger number of vWF-positive blood vessels in tumor developed in WT mice compared with DiYF mice (). Collectively, our in vivo studies demonstrate that tumor angiogenesis is impaired in DiYF mice, resulting in reduced tumor growth. Thus, it appears that β integrin cytoplasmic tyrosine motifs play crucial role in the regulation of pathological angiogenesis.
Knowing that monocytes/macrophages use β integrin for their arrest and extravasation at sites of inflammation, we assessed whether DiYF mutation affects macrophage recruitment into tumors using anti-F4/80 antibody staining. As shown in , considerable amounts of macrophages were found to be present in tumors implanted in both WT and DiYF mice. No substantial reductions in macrophage recruitment were found in DiYF mice. Similar results were observed in Matrigel model of VEGF-induced angiogenesis (). Thus, DiYF mutations within β integrin do not impair the process of macrophage recruitment in tumors.
Homozygous WT and DiYF mice were bred to obtain homozygous littermates. The average number of embryos per litter was the same. Size and weight of pups were not substantially different between WT and DiYF mice. In contrast to β integrin knockout mice, no embryonic mortality caused by placental defects was observed in DiYF mice. DiYF mutation did not affected survival of embryos at early stages of development. DiYF embryos did not exhibit any broad vascular defects (). Prenatal DiYF mutant mothers did not suffer from intrauterine hemorrhage and placental defects () as observed in β integrin knockout mice. To examine the survival of DiYF mutant mice after birth, the numbers of pups per litter from homozygous crosses were monitored. Even after 7 d of postpartum there was no significant difference in mortality of pups between these two groups. The DiYF mutant embryos (P = 12) or pups did not show any vasculature defects and hemorrhage in skin or in gastrointestinal tract as observed in β integrin knockout mice (). Development of large blood vessels exemplified by aorta also appeared to be normal because no significant differences (P = 0.445) in thickness and general histological structures of aorta were observed in DiYF mice compared with WT counterparts ().
To further investigate the effect of DiYF mutation on adult vasculature, tissue sections of liver () and kidney () were stained for vWF and laminin. No substantial differences in blood vessel density of vascular maturation were observed in DiYF mice. Vascular density was assessed in skin sections from WT and DiYF mice using staining for vWF, and there were no significant differences between these two groups (P = 0.725, = 16; ). Since vascular maturation and pericyte recruitment are known to be essential for pathological angiogenesis (), staining for smooth muscle actin was performed to reveal mature blood vessels (). Quantitative results demonstrate the similar density of smooth muscle actin–positive blood vessels in skins of WT and DiYF mice (P = 0.127; = 11). Since β integrin is known to be expressed on circulating white blood cells and involved in immune response, we demonstrated that the numbers of circulating immune cells do not differ among DiYF and WT mice. The numbers of circulating lymphocytes were 86.5 ± 0.93 and 80.1 ± 2.7 × 10 cells/μl for WT and DiYF mice, respectively. Monocyte counts were also similar: 2.34 ± 0.5 and 2.32 ± 0.6 × 10 cells/μl for WT and DiYF mice, respectively. Collectively, these data clearly indicate that DiYF mutation did not affect the embryogenesis, organogenesis, postpartum developments, normal vascular development and maturation, and levels of circulating white blood cells.
Previous studies demonstrated that αβ integrin controls migration, invasive potential, and angiogenic phenotype of ECs (). Therefore, we assessed whether impaired β integrin tyrosine phosphorylation affected EC capillary and tube formation ex vivo. WT but not DiYF ECs were able to form well-assembled and complete capillary cord-like structures in the presence of VEGF-A (). In contrast, DiYF ECs remained randomly scattered without any signs of organization (). The number of cords formed by WT EC was 5.4-fold higher compared with DiYF cells ().
A major phenotypic characteristic of ECs is the ability to assemble into the interconnected network of tube-like structures when grown in a three-dimensional matrix (). WT but not DiYF ECs formed clearly defined and well-connected networks of EC tubes (). These structures were relatively stable and remained well organized for at least 18 h. As evident from , DiYF ECs were not able to complete tube formation, and no obvious patterns were formed even in the presence of VEGF. The extent of tube formation was quantified by measuring the length of tubes; the mean values of three independent experiments are represented graphically in . VEGF treatment induced approximately threefold increase in the length of the tubes formed by WT ECs but had a little effect on DiYF ECs.
We next assessed whether DiYF mutations affected an outgrowth of vascular sprouts from aortic segments isolated from mice. First, an ex vivo angiogenic assay was performed in Matrigel enriched with growth factors. The aortic rings from WT mice produced an extensive network of vascular sprouts, whereas DiYF aortic rings failed to do so (). To elucidate the role of β integrin in VEGF-induced responses, the aortic ring assay was performed in the presence or absence of VEGF using growth factor–reduced Matrigel. Under these conditions, aortic rings from WT mice produced a significantly higher number of vascular sprouts both in the absence and presence of VEGF than did aortic rings from DiYF mice (). Quantification of the number of aortic ring sprouts indicated that DiYF cells formed fourfold fewer vascular sprouts ex vivo, regardless of stimulation, than did WT aortic rings (). VEGF produced a small increase in capillary formation of DiYF rings; however, the number of sprouts was only 20–25% of that observed in WT aortic rings ().
To further analyze the capillary growth from aortic rings, a detailed kinetic study was undertaken. The time curves of vascular growth are presented in . In the absence of stimulation, very few microvessels were detected in either WT or DiYF implants even after a prolonged incubation, whereas serum-induced neovascularization was considerably higher in WT implants than in DiYF implants (). The peak values of capillary growth were observed 8 d after implantation and were 10 and 45 microvessels per ring for DiYF and WT, respectively. VEGF with endothelial growth supplement was a stronger stimulus than endothelial growth supplement alone, but both produced extensive formation of capillaries in WT but not in DiYF aortic rings (). Collectively, these results indicate that the impaired pathological angiogenesis in DiYF mice was caused by the defective functional responses of endothelial cells.
To further define the nature of the angiogenic defect observed in DiYF mice, we compared angiogenesis-relevant functions of ECs isolated from WT and DiYF mice. We first assessed whether the mutation in β integrin cytoplasmic domain had any effect on EC adhesion and subsequent cell spreading on extracellular matrix substrates. WT and DiYF ECs were plated on various integrin ligands and numbers of attached and spread cells per field were counted. WT and DiYF ECs adhered and spread equally well on fibronectin, laminin-1, and collagen-coated plates (). In contrast, a significant difference (P < 0.001) in the behavior of WT and DiYF ECs was found using the αβ ligands vitronectin and entactin. On these substrates, DiYF ECs showed a twofold reduction in adhesion and a fourfold decrease in the number of spread cells on vitronectin and threefolds on entactin (). To further examine the role of β integrin cytoplasmic tyrosine motifs in regulation of receptor ligand-binding strength, we examined cell adhesion under conditions of shear stress. WT and excessive DiYF endothelial cells were plated on vitronectin-coated glass coverslips to achieve equal number of adherent cells. Then, adherent cells were subjected to perfusion with shear rates of 5–30 dyn/cm for 1 min. As shown in , shear stress revealed a dramatic difference in strength of adhesion between WT and DiYF endothelial cells. Shear stress of 10 to 15 dyne/cm almost completely abolishes DiYF endothelial cell adhesion to vitronectin, whereas >60% of WT endothelial cells remained tightly adhered to the matrix. Shear rate sufficient to wash away 50% of adherent cells was 23 and 8 dyne/cm for WT and DiYF endothelial cells, respectively (). This finding demonstrates that β integrin cytoplasmic tyrosine motifs regulates ligand-binding strength.
When the migratory activity of WT and DiYF ECs toward various extracellular matrix proteins known to be recognized by integrins was compared, results were similar to those of the adhesion assays: WT and DiYF ECs migrated equally well toward fibronectin, laminin, and collagen, but not toward vitronectin and entactin, where a 2.5-fold reduction in migration of DiYF ECs compared with WT was observed ().
We then compared the VEGF-induced migratory activity of WT and DiYF ECs. Stimulation of WT ECs with VEGF at 5, 10, and 20 ng/ml induced 1.5-, 2.5-, and 2.9-fold increases of migration compared with untreated ECs (). DiYF ECs also responded to VEGF stimulation; however, the rate of migration was substantially reduced when compared with WT cells (). Thus, tyrosine phosphorylation of αβ integrin appears to play an important role in VEGF-induced EC migration to extracellular matrix. To further confirm these results, we used an alternative and more physiologically relevant method to assess EC migration. WT and DiYF ECs were plated on various integrin ligands and were allowed to form a confluent monolayer. Then, a wound in the monolayer was created and the healing process was monitored at different time points. The quantitative aspects of wound recovery and representative images of ECs are presented in , respectively. Whereas WT and DiYF EC migrated equally well on fibronectin, laminin, and collagen, a threefold reduction in migration on vitronectin was observed in DiYF ECs compared with WT (). The DiYF mutation impairs αβ integrin-dependent and VEGF-stimulated responses of ECs, underscoring the role of phosphorylation of tyrosines in the cytoplasmic domain in the regulation of a cross-talk between αβ and VEGFRs.
Next, we sought to identify a molecular mechanism responsible for abnormalities observed in DiYF ECs. Previous studies using β-null mice demonstrated that the absence of β leads to up-regulation of VEGFR-2 and consequently to augmentation of angiogenic responses of ECs (). However, no differences in VEGFR-2 levels were observed between DiYF and WT ECs from lung or aortic origin (unpublished data). It was previously shown that integrin β forms a complex with VEGFR-2 immediately upon stimulation with VEGF, and this association was proposed to be necessary for the activation of angiogenic program in ECs (). Therefore, we sought to determine whether the DiYF mutations impaired the interaction of β integrin with VEGFR-2. Low levels of β–VEGFR-2 interaction were observed in unstimulated WT ECs in suspension or upon adhesion to extracellular matrix. VEGF stimulated a dramatic increase in formation of the complex between β and VEGFR-2 in WT ECs plated on vitronectin, but not in suspension or on laminin, demonstrating the ligand specificity of this phenomenon (). In contrast, no interaction between β and VEGR-2 was observed in DiYF ECs under any conditions (). Thus, β tyrosine phosphorylation was essential for an interaction between VEGFR-2 and αβ integrin.
To investigate the mechanism of β integrin-dependent VEGF signaling, we analyzed the VEGFR-2 phosphorylation status in WT and DiYF ECs after VEGF stimulation. The time courses of VEGR-2 phosphorylation are shown in . In WT ECs, VEGF induced a bell- shaped response with a maximum sixfold increase in VEGFR-2 phosphorylation relative to unstimulated cells. After 45 min, VEGFR-2 phosphorylation returned to the control level. In contrast, the maximum VEGFR-2 phosphorylation in DiYF ECs upon VEGF stimulation was only 2.5-fold over control. Importantly, VEGFR-2 remained phosphorylated three times longer in WT ECs than in DiYF ECs (). Thus, the lack of integrin phosphorylation in DiYF ECs resulted in reduced phosphorylation, and therefore activation, of VEGFR-2 in response to VEGF, which in turn affected all the signaling events downstream of VEGFR-2. To further examine this phenomenon WT and DiYF cells were induced with VEGF for various time periods and cell lysates were analyzed for phosphorylation of extracellular signal–regulated kinase (ERK)-1/2 as downstream target molecule of VEGFR-2. In WT ECs, VEGF induced strong bell-shaped response with a 3.5-fold increase in ERK-1/2 phosphorylation relative to unstimulated cells within 2.5 to 15 min. In contrast, the maximum ERK-1/2 phosphorylation in DiYF ECs upon VEGF stimulation was only 2.5-fold over control and appeared only after 15 min of VEGF stimulation. Importantly, VEGFR-2–mediated ERK-1/2 phosphorylation was immediate and longer in WT ECs than in DiYF ECs ().
An intrinsic property of integrin is an increase in soluble ligand binding in response to stimulation, a process referred to as integrin activation. We and others previously reported that VEGF activates αβ integrin on ECs in response to VEGF (). We sought to determine whether impaired activation of VEGFR-2 in DiYF ECs resulted in defective αβ activation by VEGF. VEGF induced a sixfold increase of fibrinogen binding to WT ECs, but only a threefold increase in binding to DiYF ECs (). MnCl, an agonist known to activate integrins and stimulate β integrin tyrosine phosphorylation (), produced at least a 12-fold increase in fibrinogen binding to WT ECs compared with a 5-fold increase observed in DiYF ECs (). The specificity of ligand binding was confirmed by addition of 10-fold excess of unlabeled fibrinogen. Similar results were observed when integrin activation was monitored using a monovalent activation-dependent ligand WOW-1 Fab. VEGF and MnCl stimulated 9- and 11-fold increases, respectively, in WOW-1 binding to WT ECs and 3.5- and 4-fold increases, respectively, to DiYF ECs ( and ). Thus, it is apparent that the mutations within the cytoplasmic domain of β integrin in the DiYF mice significantly impair the process of integrin activation, resulting in defective cell adhesion and migration.
To investigate the role played by β integrin in pathological angiogenesis, we replaced the WT β integrin with a mutated form unable to undergo phosphorylation, creating the knock-in mouse DiYF. The mutant β integrin is expressed and is present on cell surfaces at the normal levels, but is defective in signaling. The major advantage of this model is that it allows monitoring of αβ function in primary endothelial cells as opposed to model cell lines lacking appropriate receptors and signaling intermediates. The ex vivo and in vitro results from this model provided novel information that is different from the previously published results from in vitro studies and β knockout mice experiments (). A unique aspect of our experimental system is that compensatory or over-compensatory responses were not observed; these responses often occur in knockout models when a protein of interest is absent during development (). Using DiYF knock-in mice, we assessed pathological neovascularization in vivo and performed an extensive analysis of the underlying mechanisms of angiogenesis using ex vivo models. The major findings of this paper are the following. (a) Phosphorylation of β integrin in WT ECs occurred in response to integrin ligation and VEGF stimulation. (b) Lack of β integrin phosphorylation in DiYF knock-in mice resulted in impaired angiogenic response in three distinct in vivo models. As a result of reduced vascularization, tumor growth was significantly inhibited in DiYF mice compared with WT. (c) At the same time, DiYF mutation did not affect embryogenesis, organogenesis, and overall vascular development. In contrast to the phenotype of β integrin knockout mice, DiYF pups were broadly normal and did not show any hemorrhage in any organ. Vascular density and maturation in adult DiYF animal was also normal, indicating that integrin phosphorylation is crucial for pathological but not normal vascularization. (d) In ECs from DiYF mice, VEGF-induced functional responses (cell adhesion, spreading, migration, and capillary tube formation) were defective compared with WT. (e) Lack of β integrin phosphorylation in DiYF ECs lead to disruption of the VEGFR-2–β integrin complex and lack of VEGFR-2 phosphorylation in response to VEGF. (f) VEGF-induced integrin activation (inside-out signaling) was suppressed in DiYF ECs compared with WT ECs.
Our data show that, in WT ECs, phosphorylation of β integrin occurs in response to integrin ligation and VEGF stimulation. The finding that β phosphorylation occurs not only in response to adhesion to extracellular matrix (i.e., as a result of outside-in integrin signaling), but also upon treatment with VEGF, indicates that β might play an important regulatory role in outside-in integrin signaling, also known as integrin activation. In DiYF ECs, β cannot be phosphorylated, and integrin activation, as measured by soluble ligand binding to αβ, was considerably reduced compared with WT. Previous studies demonstrated that ligation or the treatment with the agonist Mn induces β integrin phosphorylation in cells other than ECs (). At the molecular level, it was also shown that β integrin phosphorylation controls the strength of receptor–ligand interaction (), and might contribute to the overall functional activity of the integrin.
In three distinct models, one that measured capillary formation in Matrigel and two others for vascularity of implanted tumors, we observed that pathological angiogenesis was severely impaired in DiYF mice. Our findings are consistent with the results of studies using αβ blocking antibodies and demonstrate a key role for αβ integrin in angiogenesis, suggesting that β integrin is a positive regulator of angiogenesis and its phosphorylation is a critical step in EC responses during VEGF-induced neovascularization. Further, our findings provide an additional argument that increased angiogenesis observed in β knockout mice () is a result of molecular compensation via VEGR-2; this emphasizes the physiological importance of VEGR-2–αβ cross-regulation in ECs, but does not reflect the true function of αβ integrin in angiogenesis in an organism expressing normal amounts of this receptor. Since our experimental approach does not involve a complete deletion of β integrin from the cell surface, it does not trigger compensatory responses (i.e., up-regulation of VEGR-2). The phenomenon of molecular compensation—in many cases, over-compensation—in knockout mice has been described in numerous reports and represents a major drawback of the knockout approach. Frequently, the loss of a particular protein results in increased expression of other members of the same family, revealing the functional redundancy (). It has been shown, for example, that αβ integrin can compensate for the loss of αβ integrin (). Analysis of the substitute molecule, which is structurally and functionally distinct from the targeted protein (, ), might reveal new connections within the molecular network. It is intriguing that VEGR-2, a tyrosine kinase receptor, can compensate for the loss of a member of integrin family of cell adhesion molecules, as VEGR-2 is distinct in regard to ligand repertoire and other functional aspects.
In the present study, impaired angiogenesis in DiYF knock-in mice resulted from the decreased adhesion, spreading, and migration of ECs in response to VEGF. Interestingly, impaired cell adhesion and migration was observed only on vitronectin as a substrate, but not on fibronectin or collagen. Nevertheless, ex vivo angiogenesis in Matrigel, and in vivo angiogenesis, was defective, emphasizing a role for αβ in this process. At the molecular level, this study demonstrated that impaired tyrosine phosphorylation of β integrin in DiYF ECs abrogated αβ–VEGFR-2 complex formation and resulted in dramatically reduced phosphorylation of VEGFR-2 upon VEGF stimulation. Since VEGF (via VEGFR-2 as its major functional receptor on ECs) induces phosphorylation of αβ and phosphorylation of αβ, in turn, is required for complete and sustained phosphorylation of VEGFR-2, it can be concluded that these two receptors are able to cross-activate each other in ECs, therefore forming a functional partnership that is essential for successful angiogenesis.
Collectively, our findings demonstrate that αβ and its inside-out and outside-in signaling is essential for pathological angiogenesis and undoubtedly represents a promising target for pharmaceutical approaches. However, when developing new inhibitors for angiogenesis, one should take into consideration the complexity of αβ regulation, its interactions with other receptors, and possible compensatory changes resulting from its suppression.
DiYF mice were generated in the laboratory of Dr. David R. Phillips and maintained on a C57/Bl6 background (seven generations of backcrossing). 6–8-wk-old WT and DiYF mice were used in our study. We performed all procedures according to protocols approved by Cleveland Clinic Foundation Institutional Animal Care and Use Committee.
WT and DiYF animals received an abdominal subcutaneous injection of 500 μl Matrigel mixed with VEGF (60 ng/ml) and heparin (60 units/ml). After 7 d, the animals were killed and dissected. Matrigel plugs were removed and digested using 5 ml Drabkin reagent, and quantification of neovascularization was assessed using a hemoglobin assay as per the manufacturer's protocol.
WT and DiYF mice were subcutaneously injected with freshly harvested 10 B16F10 mouse melanoma or RM-1 mouse prostate cancer cells. Tumors were collected 10 d after injection and tumor weights were measured. Tumors were also photographed and processed for immunohistochemical staining.
Immunohistochemistry and image analysis was performed as described previously (). Matrigel plugs or tumor tissues sections were stained with polyclonal rabbit anti-vWF antibody (Abcam, Inc.) or rat anti-F4/80 (Serotec, Inc). Sections were counterstained with hematoxylin. WT and DiYF skin, liver, and kidney tissue samples were processed and stained for vWF or laminin. B16F10 tumor sections were stained with polyclonal anti-CD31 (Fitzgerald Industries Intl.) and anti-laminin (Sigma-Aldrich) antibody.
The mouse aortic ring assay was performed as described previously (). WT and DiYF mouse lungs were excised, minced, and digested using collagenase-dispase reagent (3 mg/ml). Digests were strained and the resulting cell suspension was plated on flasks coated with 1 mg/ml fibronectin. Endothelial cells were isolated and characterized as described previously ().
The cell adhesion assay was performed as described previously (). Mouse lung endothelial cell suspensions were added to ligand-coated wells and placed in humidified incubator for 45 min. The wells were gently washed three times with DMEM and photographs were taken. The numbers of attached and spread cells per field were counted. Adhesion assay under shear stress conditions was performed as described previously using flow chamber (). Prior to the beginning of the experiment WT and 2.25-fold excess DiYF endothelial cells were added on vitronectin-coated coverslips to achieve approximately equal number of adherent cell population and allowed to interact with the matrix for 15 min. These coverslips were gently washed and introduced into the bottom of the flow chamber. Shear stress of 5–30 dyne/cm was applied using syringe pump for 1 min. Number of WT endothelial cells adhered to vitronectin under static condition per field was assigned the value of 100% and relative adhesion was calculated.
Endothelial cell migration assay was performed as described previously (). WT and DiYF mouse lung endothelial cells were grown to confluence in 12-well plates precoated with various integrin ligands. Cells were serum starved and then a wound was created. Images were recorded immediately after wounding (time zero) and 12 h later. Cell migration was quantified using image analysis of five randomly selected fields of denuded area. The mean wound area is expressed as percent of recovery (% R) from three identically treated plates using the equation % R = [1− (T/T)] × 100, where T is the wounded area at 0 h and T is the wounded area after 12 h.
WOW-1 Fab binding assay was performed as described previously (). To assess fibrinogen binding, WT and DiYF mouse lung endothelial cells were serum starved for 4 h and further induced with 20 ng/ml VEGF-165 or 1 mm MnCl. FITC-labeled fibrinogen was added at a final concentration of 200 nM for 45 min. Cells were fixed and washed twice with ice-cold PBS. FACS was performed using a FACS Calibur (Becton Dickinson), and data were analyzed using CellQuest software program.
The formation of vascular tube-like structures by WT and DiYF mouse lung endothelial cells were assessed on the basement membrane matrix preparation. WT and DiYF mouse lung endothelial cells were seeded on Matrigel-coated plates. Medium with or without 20 ng/ml VEGF was added, and cells were further incubated at 37°C for 8 h. The tube formation was observed using an inverted phase–contrast microscope, and photographs were taken from each well. Using ImagePro software, the degree of tube formation was quantified by measuring the length of tubes in random fields. The precapillary cord formation assay was performed as described previously ().
Immunoprecipitation was performed as described previously (). To analyze the β integrin tyrosine phosphorylation of WT and DiYF mouse lung microvascular endothelial cells, ECs were added to various integrin ligand-coated plates and incubated at 37°C for 60 min. Cell lysates containing equal amount of proteins were subjected to Western blot analysis using rabbit anti–integrin β [pY] and [pY] antibody (Biosource International, Inc). Cell lysates were also analyzed for β integrin expression as a loading control. Serum-starved WT and DiYF ECs were also treated with 20 ng/ml VEGF for 0–60 min. Cell lysates were analyzed by Western blot using anti–integrin β [pY], anti–integrin β [pY], anti–ERK-1/2 and anti–p-ERK-1/2 (Cell Signaling Technology) antibodies.
Values were expressed as mean ± SDs. p values were based on the paired test. Results were considered statically significant with P < 0.05. |
When SW B cells are challenged with either high affinity (HEL-SRBC) or low affinity (HEL-SRBC) antigen in CD45.1 congenic recipient mice, similar frequencies of donor-derived (CD45.2) GC B cells are produced at over the first 15 d of the response (), and these cells undergo equivalent rates of class switch recombination to IgG1 (). The extent of SHM measured during the early stages of the GC response (day 5) also does not differ (). However, as the responses progress, GC B cells responding to the lower affinity HEL-SRBC accumulate somatic mutations faster and by day 15 contain significantly more mutations per Ig heavy chain variable region gene than GC B cells responding to HEL-SRBC (). These observations confirm previous analyses of TD antihapten responses showing similar rates of SHM when initial antigen affinity is high or low but enhanced selection for mutated variable regions in B cells with low initial antigen affinity ().
SW B cells challenged with HEL-SRBC do not produce the burst of extrafollicular plasma cells that typically peaks around day 5 of responses to higher affinity antigens such as HEL-SRBC (). As a result, the levels of both total anti-HEL antibody () and anti-HEL IgG1 () present at day 5 are ∼100-fold lower when SW B cells are challenged with HEL-SRBC compared with HEL-SRBC. Nevertheless, the concentration of anti-HEL IgG1 in recipient serum increases progressively from days 5 to 20 of the response to HEL-SRBC (). This antibody is derived from SW donor B cells, because it is not detected in recipients receiving HEL-SRBC alone (unpublished data). To examine whether the antibodies elicited in response to HEL undergo affinity maturation, serum anti-HEL IgG1 present at day 15 of the two responses was tested by ELISA for binding to HEL and HEL. As expected, the IgG1 produced in response to HEL-SRBC bound efficiently to HEL but showed negligible binding to HEL (). In contrast, the serum anti-HEL IgG1 from recipients challenged with HEL-SRBC showed almost equivalent binding to HEL and HEL (), indicating that affinity maturation to HEL had indeed occurred.
To track the appearance and ultimate fate of GC B cells acquiring increased affinity for HEL, donor-derived B cells within the spleens of mice immunized with HEL-SRBC were stained directly with fluorescently labeled HEL at days 5, 10, and 15 of the response (). Analysis of CD45.2 donor cells revealed a progressive increase in HEL binding in mice challenged with HEL-SRBC but not HEL-SRBC (). When HEL binding was counterstained for surface IgG1 so that HEL binding by IgG1 responders could be visualized independently of BCR density, a distinct population of IgG1 B cells with uniformly increased affinity for HEL was evident by day 10 of the HEL-SRBC response and subsequently dominated the IgG1 donor population by day 15 (). This population was not apparent at any stage of the response to HEL-SRBC (), indicating that the cells were specifically selected because of their increased affinity for HEL.
To determine whether B cells with high affinity for HEL carry specific somatic mutations that increase their affinity for HEL, single donor-derived GC B cells were sorted from immunized mice, and their Ig heavy chain variable region genes were sequenced. By day 15 of the response to HEL-SRBC, 82% (23 out of 28) of the clones analyzed carried a specific point mutation in the tyrosine 53 codon encoding its substitution with aspartate (Y53D; ). No selection of any heavy chain mutation was evident in day 15 GC B cells produced in response to HEL-SRBC (), which was consistent with the proposition that the affinity of the HEL–HyHEL10 interaction is too high to permit further affinity maturation (). None of the 24 clones analyzed from the day 15 HEL-SRBC GC response contained a mutation in the Y53 codon, indicating that the Y53D mutation is selected specifically in response to HEL-SRBC and is therefore likely to increase the affinity of HyHEL10 for HEL. This was confirmed by sorting high affinity anti-HEL IgG1 donor B cells (see gate in ), as subsequent sequence analysis revealed that 96% (23 out of 24) of these clones carried the Y53D mutation (unpublished data).
Analysis of the binding of HEL to recombinant wild-type and Y53D-mutated HyHEL10 IgG1 antibodies showed that the Y53D mutation increases the affinity of HyHEL10 for HEL by ∼85-fold (). This affinity increase was also evident from the ability of the mutated form of HyHEL10 to recognize plate-bound HEL efficiently in ELISA under conditions in which binding of wild-type HyHEL10 to HEL was virtually undetectable (). Computer modeling revealed that the arginine side chain introduced at position 101 of HEL (D101R) to produce HEL () is likely to cause a major steric conflict with the phenol group of Y53 and that this conflict is resolved by the Y53D substitution (Fig. S1, available at ).
To examine the selection of the Y53D mutation over the course of the response to HEL-SRBC, Ig heavy chain gene sequence analysis of single GC B cells and plasma cells was performed on days 5, 10, and 15. On day 5, the Y53D mutation was not detectable in either the GC or the small plasma cell compartment (), which was consistent with the absence of high affinity anti-HEL B cells at this time point (). By day 10, however, this mutation was detectable in some GC B cells (22% of sequences) but was already present in the great majority of splenic plasma cells (86% of sequences). Similar domination of the GC B cell population by this mutation was not evident until day 15 (). Because hypermutated plasma cells must have derived from GC B cell precursors, this result shows that GC B cells generated in response to HEL-SRBC do not undergo stochastic differentiation into plasma cells but instead differentiate upon acquisition of the high-affinity Y53D mutation. Accordingly, Y53D-mutated clones that had left the GC and differentiated into plasma cells by day 10 of the response had a significantly lower overall rate of SHM than the Y53D-mutated clones that remained within the GC compartment (2.2 vs. 4.6 mutations/clone, respectively; P = 0.02).
To monitor affinity-based regulation of post-GC plasma cell differentiation more directly, we next challenged donor B cells from SW ×
mice with HEL-SRBC.
mice express GFP under the control of the promoter and, thus, can be detected via intrinsic green fluorescence (). By using SW ×
donor B cells in conjunction with our method for identifying somatically mutated B cells with high affinity for HEL (), we directly assessed the antigen affinity of post-GC IgG1 plasma cells in the spleen. Analysis of GFP expression by low and high affinity donor-derived IgG1 B cells on day 10 of the response to HEL-SRBC clearly demonstrated that GFP-expressing plasma cells originated almost exclusively from GC precursors that had acquired a high affinity anti-HEL BCR (2.54% of high affinity compared with 0.27% of low affinity cells; ). These GFP cells also had low but detectable levels of surface CD45.2 and IgG1 ( and not depicted), as is typical of plasma cells (). Because >95% of high affinity clones detected by HEL binding have the Y53D mutation, most of these GFP plasma cells are likely to secrete antibodies carrying this amino acid change. Indeed, the serum IgG1 found in mice challenged with HEL-SRBC shows almost identical antigen-binding characteristics to Y53D-mutated HyHEL10 ( and ).
The model system described in this report provides a unique window into the GC reaction by allowing the appearance, selection, and differentiation of high affinity somatically mutated B cells to be followed throughout the response. We have used this system to demonstrate that high affinity B cell specificities generated within the GC are harnessed to drive affinity maturation of the antibody response by a mechanism that ensures their rapid and selective differentiation into plasma cells.
Because low affinity B cells survive within GCs without undergoing plasma cell differentiation (), it is apparent that the affinity-dependent mechanism that regulates plasma cell differentiation from GC B cell precursors operates independently of the processes that govern GC B cell survival. The existence of this mechanism was not predicted from in vitro experiments, because these show that B cells can undergo stochastic plasma cell differentiation without requiring a BCR signal (). This would suggest that specific controls exist within the GC microenvironment that suppress plasma cell differentiation in the absence of signals from high affinity antigen.
The requirement for antigen-dependent signals to drive plasma cell differentiation from GC B cells presents an interesting parallel with the regulation of the extrafollicular plasma cell response. These early plasma cells arise independently of the GC reaction () but are similarly biased toward high affinity specificities or epitopes present at high density (). Therefore, the BCR–antigen interaction appears to play a key role in regulating TD plasma cell differentiation both before and after GC formation. It is possible that BCR signaling could facilitate plasma cell differentiation via the induction of Bcl-6 degradation () and subsequent lifting of Blimp-1 repression (). Alternatively, responding B cells may stochastically commence plasma cell differentiation but require BCR signals to survive beyond the very earliest stages of this process. Whatever the precise mechanism, it is apparent that the immune system places a high priority on ensuring that it devotes resources primarily to the production of relatively high affinity antibodies that are most likely to be biologically effective. The importance of this stringent regulation of plasma cell differentiation is perhaps underscored by the relatively permissive affinity requirements for GC B cells to enter the memory B cell compartment (, ). The emphasis on quality control of in vivo plasma cell differentiation may have been a critical development during the evolution of the immune system. Because the body is known to have only a relatively limited capacity within the specialized microenvironments that sustain plasma cells (, ), it can be seen that tight control over the specificities that enter the plasma cell compartment would be essential for ensuring that the antibodies that are produced provide effective immune protection.
SW mice (),
mice (), mutant HEL proteins (), conjugation of HEL to SRBC, and the adoptive transfer system () have been previously described. SW spleen cells were not purified before transfer. Mice were housed in a specific pathogen-free environment at Centenary Institute, and experiments were approved by the University of Sydney Animal Ethics Committee.
Serum anti-HEL antibody levels were analyzed by ELISA as previously described (). The specificity of serum IgG1 antibodies for HEL and HEL was determined by coating the ELISA plates with the respective antigens. The relative affinities of HyHEL10 and HyHEL10 for HEL were determined by capture ELISA. 5 μg/ml each of soluble HyHEL10 and HyHEL10 IgG1 antibodies was captured by plate-bound anti–mouse IgG1. Subsequent binding of HEL was detected with biotinylated HyHEL9, which recognizes an epitope on HEL distinct from that bound by HyHEL10. Nonlinear regression based on a sigmoidal binding curve was performed using software (Prism; GraphPad) to find the curve-fit and calculate the half-maximal binding concentration and relative affinity of HyHEL10 and HyHEL10 for HEL.
Splenocytes were prepared, stained for surface molecules with monoclonal antibodies, and analyzed on a FACSCalibur (BD Biosciences) as previously described (). To track affinity maturation, cells were stained with recombinant HEL conjugated to Alexa Fluor 647 (Invitrogen). For analysis of -GFP expression, CD45.2 was conjugated to Pacific blue (Invitrogen) to allow identification of donor-derived cells and data acquired on a flow cytometer (LSR II; BD Biosciences). Single cells were sorted on a FACSAria (BD Biosciences) as previously described (). The gates used for single cell sorting of GC B cells (CD45.2, syndecan-1) and plasma cells (CD45.2, syndecan-1) were the same as those previously described (). These gates have been verified through localization of antibody secreting activity () and high levels of intracellular Ig staining () to cells in the plasma cell gate and demonstration of high levels of GL7, PNA, and Fas on cells in the GC gate (, ).
The HyHEL10 Ig heavy chain variable region gene was amplified from single-responding SW donor B cells and sequenced as previously described (). Translated sequences were aligned with the original HyHEL10 protein sequence to determine the position and significance of the mutations, as previously described (). An unpaired test was used to calculate the probability (p-value) when comparing SHM frequency per clone in different responding populations.
The canonical T to G mutation encoding the Y53D substitution was introduced by PCR mutagenesis into a pcDNA3 vector (Invitrogen) encoding the HyHEL10 γ1-secreted Ig heavy chain. Wild-type and mutant heavy chain constructs were transiently expressed in Chinese hamster ovary cells along with wild-type HyHEL10 κ light chain construct, and culture supernatants were collected and concentrated.
Fig. S1 shows Rasmol wire frame representations based on the HEL–HyHEL10 complex that model the effects of the D101R and Y53D substitutions on the interaction. Online supplemental material is available at . |
We investigated Dok-2 activation after CD3 and/or CD28 ligation. In normal T cells and transformed T cell lines, Dok-2 appeared as a doublet of ∼53–56 kD (). Dok-2 was found weakly tyrosine phosphorylated in unstimulated CD4 T cells, T cell blasts, a CD8 cytotoxic T cell clone (CTL) (), and Hut-78 cells () but strongly phosphorylated in unstimulated J77 Jurkat cells (). TCR/CD3 stimulation led to substantial enhancement of Dok-2 phosphorylation in normal T cells () and Hut-78 cells () but little augmentation in J77 cells (). Phospho–Dok-2 migrated diffusely at and above 55 kD (, brackets), presumably because of the high content of phosphorylatable tyrosine motifs (). Importantly, superantigen (SAg)-pulsed Raji cells (antigen-presenting cells not expressing Dok-2) induced a substantial increase in Dok-2 tyrosine phosphorylation in T cell blasts (, middle, lane 4), demonstrating that physiological TCR engagement induces activation of Dok-2. T cell contact with unpulsed Raji cells also induced weak Dok-2 phosphorylation (, middle, lane 3), possibly caused by engagement of a receptor distinct from TCR. Similar observations were made with unpulsed and SAg-pulsed autologous monocyte-derived dendritic cells (unpublished data). Dok-2 tyrosine phosphorylation was detected as early as 20 s after TCR stimulation, returning to background levels after 20–30 min (), a kinetics similar to that of Zap-70 activation and of other phosphoproteins (unpublished data).
When T cells were stimulated with anti-CD28 antibody or B7-1–expressing cells (531-B7), Dok-2 tyrosine phosphorylation was only weakly induced above background level, whereas Vav-1, an effector of CD28 signaling, was more efficiently tyrosine phosphorylated (). In contrast, SAg-pulsed 531-B7 cells induced a substantial increment of Dok-2 tyrosine phosphorylation, confirming that TCR is the stronger inducer of this event (). Comprehensively, these data suggested that in normal T cells TCR engagement, rather than CD28, efficiently activated Dok-2. These data sharply contrasted with the behavior of Dok-1, the close Dok-2 homologue, which was reported to be inducibly phosphorylated in Jurkat cells by CD2 and CD28 but not by TCR triggering (, , ). One explanation for these opposite results may be the use of Jurkat cells, which also showed an unusually high basal phosphorylation of Dok-2 (). Jurkat cells lack expression of the lipid phosphatases, phosphatase and tensin homologue deleted in chromosome 10 (PTEN) and SHIP-1 (), which causes dysregulated accumulation of phosphatidylinositol (PI) 3,4,5-trisphosphate and PI 3,4-bisphosphate and basal activation of certain PH domain–containing proteins (, ). Consistent with this idea, in Hut-78 cells, which express both PTEN and SHIP-1 at levels comparable with primary T cells () (), TCR/CD3 stimulation led to strong tyrosine phosphorylation of Dok-2 (). Interestingly, Dok-1 was also tyrosine phosphorylated in Hut-78 after TCR/CD3 stimulation (). Moreover, similarly to Dok-2, its phosphorylation was detected as early as 20 s after TCR triggering in primary T cells (Fig. S1, available at ). Overall, these observations suggest a rapid activation of both Dok-2 and Dok-1 adaptors concomitant with TCR signal development, and may explain opposite results because of the use of Jurkat cells versus normal T cells.
Because TCR ligation induced a very rapid phosphorylation of Dok-2, we investigated whether it physically engaged with TCR signaling effectors. T cell blasts were stimulated with anti-CD3 (, lanes 2 and 4) or Raji cells pulsed or not with SAgs (, last four lanes) and subjected to Dok-2 immunoprecipitation followed by antiphosphotyrosine immunoblot (, first panel). A major phosphoprotein of ∼145 kD coprecipitated with Dok-2 after TCR stimulation, comigrated with phosphorylated SHIP-1 (lanes 2 and 6 with 4 and 8) and was identified as SHIP-1 by immunoblotting (, second panel). Kinetic assays further confirmed its rapid and relatively prolonged coprecipitation with Dok-2 after TCR engagement (Fig. S2, right, available at ). This data is consistent with the reported Dok-2–SHIP-1 interaction upon SLAM stimulation (). Similarly, tyrosine-phosphorylated SHIP-1 was detected in Dok-1 immunoprecipitates upon TCR stimulation (Fig. S3 A, available at ). A weak Dok-2–SHIP-1 association was seen in unstimulated cells and augmented after TCR stimulation (, anti–SHIP-1 immunoblot). Conversely, Dok-2 protein and its phosphorylated form were found in SHIP-1 immunoprecipitates (, fourth panel and Fig. S3 B). In accordance with the described interaction of SHIP-1 with one SH3 domain of Grb-2 (), substantial amounts of Grb-2 were constitutively associated with SHIP-1 (, third panel, lanes 3, 4, 7, and 8) that slightly increased upon TCR stimulation. A much lower amount of Grb-2 was also observed constitutively in Dok-2 immunoprecipitates that weakly augmented after TCR triggering. This augmentation paralleled the increased binding of SHIP-1 with Dok-2 and suggested stabilization of Grb-2–SHIP-1–Dok-2. These findings were confirmed by probing Grb-2 immunoprecipitates with anti–SHIP-1 and anti–Dok-2 antibodies (). As demonstrated in previous studies (), Grb-2 also bound to LAT upon TCR stimulation. Noteworthily, comparison of signal intensity of SHIP-1, Dok-2, and LAT coimmunoprecipitated with Grb-2 (, lanes 1 and 2) with their signals in the cell lysate (, lane 4) revealed the preferential association of SHIP-1 to Grb-2 with respect to Dok-2 and LAT, even after TCR stimulation.
Notably, pp36, pp47, and pp64 species were detected more strongly in SHIP-1 than Dok-2 immunoprecipitates, and an additional phosphoprotein of ∼116–118 kD was associated with SHIP-1 (, arrows). The broad appearance of the 36-kD molecular species was reminiscent of LAT. Indeed, immunoblotting of SHIP-1 immunoprecipitates with anti-LAT antibody confirmed that pp36 was LAT (). Moreover, tyrosine phosphorylation of SHIP-1 and coprecipitated LAT exhibited similar kinetic profiles, suggesting a causal link between these events (Fig. S2, left). Reciprocally, SHIP-1 and Dok-2 were observed in LAT immunoprecipitates upon TCR stimulation (unpublished data). Collectively, these data indicated that TCR triggering induced association of Dok-2–SHIP-1–Grb-2 to LAT, together with other phosphoproteins, suggesting that a bone fide negative signaling pathway is initiated coincidentally on the very same scaffold (i.e., LAT) that directs positive signal propagation.
The association of Dok-2–SHIP-1–Grb-2 with LAT suggested that LAT may play a role in the activation of Dok-2 and SHIP-1. To answer this question, we inhibited LAT expression by small interfering (si)RNA and asked whether this modified SHIP-1 and Dok-2 phosphorylation. LAT expression was reduced in Hut-78 cells by 80–90% in comparison with cells transfected with irrelevant siRNA (siNeg or siScr), whereas expression of other proteins such as Dok-2, SHIP-1, PLC-γ1, Zap-70, and Vav-1 remained unaffected ( and unpublished data). Upon TCR/CD3 stimulation, Hut-78 cells with reduced LAT expression exhibited impaired tyrosine phosphorylation of Dok-2 at 5 and 10 min (35–50% inhibition) as revealed with anti-pTyr351–Dok-2 antibody (). Consistent with previous findings in LAT-deficient Jurkat (), activation of PLC-γ1, as estimated by anti-pTyr783–PLC-γ1 antibody, was also partially (50–60%) affected. Thus, when these data were compared with PLC-γ1 activation, the reduction of TCR-induced Dok-2 phosphorylation was substantial after LAT knockdown (80–90%). In contrast, TCR-induced Zap-70 activation, known to occur upstream of LAT (), was not affected by LAT knockdown. Hut-78 cells with decreased LAT expression stimulated by CD3 also exhibited inhibition of SHIP-1 tyrosine phosphorylation (38–56% inhibition; ). Importantly, impaired TCR- mediated phospho–Dok-2 was reproduced in primary T cells (30–47% reduction) in which LAT expression was inhibited by siRNA (50%) () and with another siRNA against LAT in Hut-78 cells (unpublished data). The stronger association of LAT with SHIP-1 than with Dok-2 () suggested that Dok-2 tyrosine phosphorylation may depend on SHIP-1. Consistent with this hypothesis, we found that reduced expression of SHIP-1 (90% by siRNA) substantially affected TCR-mediated tyrosine phosphorylation of Dok-2 (∼60%; ) and of Dok-1 (∼40–50%; Fig. S4, available at ). Conversely, double knockdown of Dok-2 and Dok-1 expression did not impair TCR-induced tyrosine phosphorylation of SHIP-1 (unpublished data). Collectively, these data supported the idea that TCR stimulation induces association of the Dok-2–SHIP-1–Grb-2 complex with LAT followed by SHIP-1 and Dok-2 tyrosine phosphorylation.
To assay the role of Dok-2 in TCR signaling, we measured TCR-mediated IL-2 secretion in Hut-78 cells knocked down for Dok-2 by siRNA. Inhibition of Dok-2 expression (∼90%; , middle blot) resulted in marginal or no increase in IL-2 secretion (, histograms). Similar to Dok-2, knockdown of Dok-1 alone (∼80%; , top blot) had minor or no impact on TCR-mediated IL-2 production (, histograms). In contrast, combined inhibition of Dok-2 and Dok-1 expression (∼90% and 80%, respectively) resulted in clear enhancement of TCR-induced IL-2 secretion in comparison with cells transfected with control siNeg or siScr RNA (). Collectively, these data indicated that Dok-1 and Dok-2 adaptors act together to negatively regulate TCR-mediated IL-2 gene activation. To examine the possibility of potential RNAi off-target effects, Dok-2 expression was restored in siDok-1/2–transfected cells, under the assumption that this would also substitute for Dok-1 function. To this aim, Hut-78 cells were cotransfected with a plasmid encoding human Dok-2 together with siDok-1/2 and IL-2 promoter–dependent luciferase reporter plasmid. Substantial levels of Dok-2 expression were detected in transfected cells (, blot panel). Complementation of siDok-1/2–transfected cells by Dok-2 cDNA alone was sufficient to repress TCR-mediated IL-2 gene expression and reverse the enhancement caused by siDok-1/2 (, histograms).
To begin understanding the molecular mechanism wherein the signaling cascade Dok-2 and Dok-1 influence TCR-mediated activation, we analyzed these adaptor effects on Akt/PKB and Erk-1/2. These kinases have been found to be more effectively activated upon cytokine stimulation of Dok-1/Dok-2 myeloid cells (, ). We found that TCR-induced Erk-1/2 activation was poorly enhanced in Hut-78 doubly knocked down for Dok-2 and Dok-1 expression (unpublished data). However, double knockdown resulted in marked basal and prolonged TCR-mediated Akt activation as detected with an anti–pSer473-Akt antibody compared with siNeg-transfected Hut-78 cells (). These data are consistent with Dok1 and Dok-2 adaptors negatively controlling Akt activation () and extend this effect to the TCR signaling machinery.
Interestingly, by examining other TCR signal effectors in Hut-78 knocked down for Dok-2 and Dok-1, we consistently detected an increased activation of Zap-70 in stimulated cells as revealed by an anti-pTyr319–Zap-70 antibody (). This effect was particularly observed at early time points after TCR stimulation (and at 20 s; unpublished data). Enhanced Akt and Zap-70 activations were more pronounced in double than in single knockdown of Dok-2 and Dok-1 (unpublished data). Moreover, these effects appeared to be selective since p38 kinase activation was not enhanced and even decreased in unstimulated cells (). Alterations of the TCR-induced tyrosine phosphorylation profile were observed upon knockdown of Dok-2 and Dok-1. In particular, proteins of ∼36, 70, and 76 kD were more phosphorylated after TCR ligation (Fig. S5, ). In accordance with the enhancement of Zap-70 activation, LAT and SLP-76, as well as PLC-γ1, were found to be more phosphorylated (Y191, Y128 and Y783, respectively) (). Importantly, this was also the case for CD3ζ and its coprecipitated partner Zap-70 (). Overall, these data support the existence of a molecular mechanism that finely regulates TCR signal initiation by a negative feedback loop involving Dok-2 and Dok-1 recruitment, via SHIP-1, on LAT which reduces Zap-70 activation.
In this work we demonstrate that the adaptors Dok-2 and Dok-1 are key effectors of a TCR proximal negative feedback control mechanism. This notion stands on two novel sets of findings. First, TCR ligation induced rapid binding to LAT and tyrosine phosphorylation of Dok-2 and Dok-1 in complex with SHIP-1. The latter appeared to act as a carrier for these adaptors. Second, reduced expression of both Dok-2 and Dok-1 by siRNA resulted in increased amplitude and duration of TCR-induced Zap-70 activation and of other tyrosine-phosphorylated proteins and basal- and TCR-induced Akt activation with consequent augmentation of IL-2 gene expression. Individual knockdown of Dok-2 or Dok-1 had minimal or undetectable effects, suggesting that they exert redundant negative signaling functions, in agreement with previous data in cells of myeloid origin (, ). Comprehensively, our data demonstrate that LAT physically integrates, in apparent temporal coincidence, two opposing signals. A dominant signal promotes TCR-controlled activation (), whereas the second, implicating Dok adaptors and probably other modulatory proteins (), attenuates it. We propose that this type of mechanism may contribute to control the TCR activation threshold (“signal gatekeeper”) and/or to shape TCR signal form in intensity and duration (“signal tailoring”).
Triggering of BCR or FcɛRI was also reported to induce tyrosine phosphorylation of Dok adaptors and SHIP-1 (, , ). In light of our results, it will be of interest to determine whether antigen receptors use similar or distinct stratagems to recruit and activate Dok-dependent negative signals.
Previous studies in Jurkat cells showed that CD2 and CD28 receptors, but not TCR, induced potent tyrosine phosphorylation of Dok-1 and Dok-2, suggesting their participation in costimulatory signaling pathways (, , ; unpublished data). Our present results do not support this view in primary T cells because little or no tyrosine phosphorylation of Dok-2 was observed upon CD28 ligation. A possible explanation for this difference may be that in Jurkat cells, similar to Akt, activation of PH domain–containing Dok-1, 2 may be deregulated because of altered phosphatidylinositol metabolism, consequent to the absence of PTEN and SHIP-1 (, ).
In accordance with previous data (), SHIP-1 was constitutively bound to Grb-2. We also found some basal SHIP-1–Dok-2 association that likely resulted from weak basal tyrosine phosphorylation of both proteins (, , and S2). However, TCR stimulation led to a marked increase in SHIP-1 and Dok-2 phosphorylations and their prolonged association. In support of phosphotyrosine-dependent interactions, Dok-2 and Dok-1 PTB domains were shown to bind to tyrosine-phosphorylated SHIP-1 (, ). We examined the reciprocal role of Dok-1, Dok-2 and SHIP-1 in their activation by knocking down their expression in Hut-78 cells. Knockdown of SHIP-1 revealed that it primarily controls TCR-induced tyrosine phosphorylation of Dok-2 and Dok-1. Conversely, a decrease in TCR-mediated SHIP-1 tyrosine phosphorylation was not observed in T cells knocked down for Dok-2 and Dok-1 (unpublished data). Moreover, knockdown of LAT resulted in a marked alteration of TCR-mediated SHIP-1, Dok-2, and Dok-1 tyrosine phosphorylation. Altogether, our data support the idea that in T cells, SHIP-1 mediates recruitment and activation of Dok-2 and Dok-1 to LAT. In accordance with a SHIP-1 “carrier” function, SHIP-1–deficient thymocytes do not exhibit Dok-2 tyrosine phosphorylation upon SLAM stimulation (). A similar defect was observed for Dok-1 in SHIP-1–deficient B cells stimulated by BCR and FcγRIIB (). How SHIP-1 is connected to LAT remains to be determined as we did not observe binding of the SHIP-1 SH2 domain to LAT in glutathione -transferase pull-down assays (unpublished data). LAT contains several Grb-2 binding sites, and recent reports have indirectly suggested that LAT may be a source of negative signaling (, ). Thus, one scenario would involve recruitment to LAT of Grb-2/SHIP-1–Dok adaptors in direct competition with other positive signaling complexes containing Grb-2. It remains possible that other players may control Dok-2, Dok-1, and SHIP-1 recruitment/tyrosine phosphorylation because the latter was not completely abolished in LAT knocked down cells. A candidate may be, for instance, the inhibitory membrane adaptor LAX possessing several Grb-2 binding sites (). This possibility may, however, be less likely because the inhibition of SHIP-1 and Dok-2 phosphorylation was comparable to that of PLCγ-1 in LAT knocked down T cells. Overall, our findings support the notion that LAT drives concomitantly negative and positive signals with potential consequences on T cell fate.
Our data also reveal a surprising negative control exerted by Dok-2 and Dok-1 on TCR-induced activation of Zap-70 and on substrates whose phosphorylation depend on Zap-70 (e.g., LAT, SLP-76, and PLC-γ1) (). Because TCR-induced tyrosine phosphorylation of Dok-2 and Dok-1 is very rapid (as early as 20 s), these data imply that these adaptors counteract TCR signal development at its initiation step. This finding can be related to a work proposing that Dok-2 binds to c-Src and attenuates its activity upon epidermal growth factor stimulation by recruiting Csk, the Src kinase family inhibitor kinase (). Interestingly, Dok-1 was also recently demonstrated to control Csk localization at the plasma membrane and inhibit Src activation in a Csk-dependent manner upon platelet-derived growth factor stimulation (). Similarly, in B cells, Dok-3 may dampen B cell activation through Csk association in addition to its binding to SHIP-1 (, ). Optimal activation of Zap-70 is tightly dependent on Lck binding and catalytic activity (). Thus, the negative control of Dok-2 and Dok-1 on Zap-70 may be explained by their upstream control on Lck. In accordance with this proposition, we observed increased TCR-mediated tyrosine phosphorylation of CD3ζ upon knockdown of Dok-2 and Dok-1. However, the fact that knockdown of Dok-2 and Dok-1 did not dramatically affect these events suggests that these adaptors act as attenuators of TCR signal. This would also explain why we did not succeed in reproducibly detecting an increase in Zap-70 activation in siLAT-treated cells. In fact, LAT knockdown did not mimic the effect of Dok-2 and Dok-1 knockdown on Zap-70. Indeed, a 1.5– 2-fold increase in TCR-induced Zap-70 activation was detected, whereas Dok-2 and Dok-1 expressions were markedly decreased (90–80%). In contrast, LAT knockdown led to the clear reduced, but not abolished, Dok-2 and Dok-1 phosphorylation. The remaining phosphorylated Dok may still negatively control TCR-induced Zap-70 activation. Moreover, we observed a correlation between the levels of residual LAT expression and T cell activation. For instance, PLC-γ1 phosphorylation and IL-2 production were still noticeable (though strongly decreased) when residual LAT expression was around 10–15% or when the strength of TCR engagement was high (unpublished data).
A substantial enhancement of basal and prolonged TCR-induced activation of Akt was observed in Hut-78 cells knocked down for Dok-2 and Dok-1, in agreement with Akt activation increase in Dok-1/Dok-2 mice (). Our data of Dok-2 and Dok-1 knockdown in Hut-78 cells suggest that enhanced Akt activation may be a consequence of Zap-70 or, for the same matter, Src-PTK up-regulation. Indeed, although the precise mechanism of this regulation has yet to be determined, decreased Akt activation has been described upon overexpression of Dok-2 and epidermal growth factor receptor stimulation (). What is then the role of SHIP-1 in this mechanism? One possibility is that SHIP-1 needs to be correctly localized in the TCR signalosome to exert, together with Dok-bound partners, its phosphatase activity on PI 3,4,5-trisphosphate, and contribute to this negative feedback loop. Indeed, SHIP and Dok-2/Dok-1 mice present close phenotypes characterized by enhanced Akt activation, survival, and expansion of myeloid cells (, , ). The negative control of SHIP-1 on Akt activation has also been demonstrated in T cells with an inducible expression system (). However, because SHIP-1 appears to be mandatory for Dok-1 and Dok-2 recruitment (and phosphorylation), one appealing possibility is that SHIP-1 serves mostly as a carrier to facilitate the action of Dok proteins on signal attenuation. Since Akt plays an important role in T cell survival, IL-2 gene activation, and cell cycle progression (, ), Dok-2 and Dok-1 are therefore likely to play a role in these processes.
Several mechanisms that counteract signal onset and propagation have been proposed. These “negative” signals can emanate from membrane receptors (e.g., CD5, CTLA-4) or from phosphatases that constantly oppose activating phosphorylations on signaling components, or are activated as a consequence of receptor engagement (e.g., SHP-1, SHP-2). Our data show that Dok-2 and Dok-1 are involved in an early negative feedback mechanism acting through LAT. This molecular device may represent a mechanism to adapt toward homeostatic/tonic TCR signal intensity and/or attenuate incoming TCR signal. T cells indeed appear to require control mechanisms of their signaling machinery for maintaining a basal tonic signal and repress gene expression () while avoiding response to self-antigen and allowing survival and proliferative homeostasis (, ). Such a control implicating Dok-2 and Dok-1 is suggested by their slight basal tyrosine phosphorylation that we observed in primary T cells. Thus, Dok-1, 2 may play a gatekeeper function in setting the threshold of T cell activation and contributing to maintain peripheral T cell tolerance and homeostasis. Alternatively, but not exclusively, by shaping the intensity and duration of antigen-induced signals, Dok-2 and Dok-1 may influence naive T cell differentiation and effector functions.
Hut-78, Jurkat J77cl20 T cell, and Raji B cell lymphoma were cultured in RPMI 1640 containing 10% heat-inactivated FCS, 2 mM -glutamine, penicillin, and streptomycin. L cells expressing DRB1*0101 and B7-1 (531-B7) were cultured in complete DMEM with 50 μg/ml hygromycin B (). Primary T cells were maintained overnight in complete RPMI supplemented with 1 mM sodium pyruvate and nonessential amino acids (GIBCO BRL). Blood samples from healthy donors were obtained from Etablissement Français du Sang (EFS), Paris, France in accordance with a convention signed between the Institut Pasteur and EFS. Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient, and peripheral blood lymphocytes were obtained after PBMC adhesion (10 × 10/ml) in RPMI 2% FCS for 1 h at 37°C. Phytohemagglutinin/IL-2 T cell blasts were harvested at days 6–13 of culture established with PBMCs (3 × 10/ml) that were stimulated by phytohemagglutinin (2 μg/ml) for 2–3 d and then expanded with IL-2 (50 ng/ml; Chiron). Flow cytometric analysis showed that >97% cells were CD3 and 95% CD28 at days 6–8. Naive CD4 T cells were purified by negative selection (90–98%) and an AutoMACS apparatus (Miltenyi Biotec). The human CD8 CTL clone specific of CMV has been previously described ().
Mouse mAbs were against CD3 (UCHT1; Serotec), CD28 (CD28.2; Immunotech), Vav-1 (Vav-30; a gift from J. Griffin, Dana Farber Cancer Institute, Boston, MA), Dok-2 (E10; Santa-Cruz Biotechnology, Inc.), LAT (2E9; Upstate), SHIP-1 (P1C1; Santa-Cruz Biotechnology, Inc.), PTEN (A2B1; Santa-Cruz Biotechnology, Inc.), a cocktail of antiphosphotyrosine mAbs including 4G10 (Upstate), PY20 (Transduction Lab.), and PY99 (Santa-Cruz Biotechnology, Inc.). Rabbit antibodies were against Dok-2 (H-192 [Santa-Cruz Biotechnology, Inc.] and serum 157–2[]), Dok-1 (13602 [Abcam] and serum A.V.[]), SHIP-1 165.1 3′ (), CD3ζ chain (8.73 and 448 sera[]), Grb2 (C23) and Vav-1 (H211) (Santa-Cruz Biotechnology, Inc.), phospho-Tyr351-Dok-2, Ser473-Akt, Tyr319Zap-70, Tyr783-PLC-γ1, and Thr180-Tyr182-p38 kinase (Cell Signaling). mAb against phosphorylated SLP-76 Y128 was from BD Biosciences. Goat anti–mouse IgG and IgG were from Southern Biotechnology Associates, Inc.
siRNA duplexes containing 19 nucleotides with 2 thymidine 3′ overhangs were purchased from Eurogentec, Qiagen, and Proligo-Sigma. The sequences were as follows: 5′-GAAUGCUGCACCCGCUACA-3′ for siDok-2, 5′-GGUCAUGUUCUCUUUCGAG-3′ for siDok-1, 5′-GCUAAGUGCUUUACGAACA-3′ for siSHIP-1, 5′-ACGCAUGUACACACUCGCG-3′ for a randomly Dok-2 scrambled sequence (siScr), and two negative control RNA duplexes (siNeg) from Qiagen and Ambion, respectively. SiRNA used for silencing LAT gene expression was from Santa-Cruz Biotechnology, Inc.
To achieve maximal RNAi, Hut-78 cells (12 × 10/0.4 ml) were electroporated two to three times at 24 h intervals with siRNAs (200–300 nM) at 260 V, 950 μF (Bio Rad Laboratories). Cells were harvested 24–48 h after the last electroporation. For IL-2 gene activation, Hut-78 cells were first transfected with siDok-1/2 or siScr (300 nM). After 24 h, these cells were cotransfected with the same siRNA together with pCMV5 encoding HA-tagged human Dok-2 () and IL-2 promoter luciferase reporter plasmid (phIL-2-Luc, 10 μg) at 230 V, 950 μF. The total amount of DNA was adjusted to 25 μg with empty vector. Luciferase activity was measured in triplicate 24 h after the second transfection using a microplate luminometer (LB96V, Berthold; PerkinElmer). Primary T cells (6 × 10) were transfected with siRNA (1–1.5 μM) by nucleofection (Amaxa) according to the supplier's instructions and incubated in p24-well plates in complete RPMI medium for 48 h.
Primary T cells (6–10 × 10) were stimulated in RPMI (1.5 × 10/ml) at a ratio of 5:1 with APC (5 × 10/ml) previously pulsed for 1 h with a mixture of superantigens (Staphylococcal enterotoxin A, B, C at 0.2 μg/ml; Toxin Technology) at 37°C. Jurkat, Hut-78, and primary T cells (1.2 × 10/ml) were stimulated by cross-linking of CD3 (5 μg/ml) with anti-IgG antibodies (20 μg/ml) as previously described () or with soluble anti-CD3 (5 μg/ml). CD28 stimulation with antibodies was performed by cross-linking (10 μg/ml) and with 531 cells expressing B7.1 (). Cells were lysed at 4°C for 10 min in buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl, 1 mM EGTA, 50 mM NaF, 10 mM NaPO, 1 mM NaVO) containing 1% NP-40 and 1% -dodecyl-β--maltoside (Sigma-Aldrich) and inhibitors of proteases. Lysates were cleared by centrifugation and equal amounts of protein, determined by Bradford assay (Bio Rad Laboratories), were immunoprecipitated for 2 h at 4°C with the indicated antibodies preadsorbed to protein A– or protein G–Sepharose (Amersham Biosciences). Immunoprecipitates and whole cell lysates (15 μg proteins) were separated by SDS-PAGE and transferred to nitrocellulose membrane. Immunoblots were analyzed by enhanced chemiluminescence (Western Lightning; PerkinElmer) or by near-infrared fluorescence using secondary goat anti–mouse or goat anti–rabbit IgG labeled with AlexaFluor 680 (Invitrogen) and IRDye 800 (Rockland Immunochemicals), respectively. Signals were acquired and quantified with a Kodak Image Station 440 cf or an Odyssey scanner and the Odyssey 1.2 software (Li-Cor Biosciences), or after scanning films in the linear range of exposure. Phosphoprotein signals and RNAi efficiency were normalized to the protein loading charge evaluated by immunoblot.
SiRNA-treated Hut-78 cells were stimulated in triplicate in p96-well round plates with immobilized anti-CD3 for 4 h at 37°C. Goat anti–mouse IgG (4 μg/ml) were coated in wells in PBS overnight at 4°C and washed twice. UCHT1 was added at different concentrations for 1 h at 37°C in PBS and washed twice. After saturation of wells in RPMI 10% FCS, T cells were added (5 × 10/ml). TCR-independent activation was induced with PMA (5 ng/ml) and A23187 (0.1 μg/ml). IL-2 production was measured from 4 h cell culture supernatants by ELISA (R&D Systems). A sample of siRNA-treated cells was kept to check for RNAi efficiency by immunoblot.
In addition to Dok-2, Dok-1 was tyrosine phosphorylated (Fig. S1) and interacted with SHIP-1 (Fig. S3) after TCR stimulation of primary T cells. TCR-mediated Dok-1 tyrosine phosphorylation was dependent on SHIP-1 expression in Hut-78 cells (Fig. S4). Kinetics of TCR-induced SHIP-1–LAT and Dok-2–SHIP-1 complex formation are shown in Fig. S2. Fig. S5 shows TCR-induced tyrosine phosphorylation profile in Hut-78 cells knocked down for Dok-2 and Dok-1 expression in which enhanced phosphorylation of ∼36, 70, and 76 kD proteins was detected. Online supplemental material is available at . |
Prior to 1967, it was known that bacterial phagocytosis results in rapid oxygen consumption by the cell (), and that this “respiratory burst” produces large amounts of hydrogen peroxide (HO) (). This HO is a potential substrate for the enzyme myeloperoxidase (MPO), which catalyzes oxidation of substances by the decomposition of HO, and is particularly abundant in neutrophils (). This possible link led Klebanoff to investigate the microbicidal effects of MPO.
Klebanoff mixed live bacteria with MPO and low levels of HO (higher levels would kill bacteria directly), but saw no effect on viability. Reasoning that MPO might act indirectly, converting a harmless substance into something toxic, Klebanoff added iodide to the mix. The cellular halides chloride and iodide, when oxidized, become the potent germicides hypochlorous and iodine. MPO quickly oxidized iodide to iodine and the bacteria were killed.
Using traceable iodide, Klebanoff then showed that bacteria-containing neutrophils converted the iodide to iodine, whereas resting (nonphagocytosing) neutrophils or those treated with an MPO inhibitor, did not. This indicated that bacterial phagocytosis and the resulting MPO activity lead to and are required for iodine incorporation. These results, published in the , suggested that neutrophils use MPO-catalyzed iodination as a bactericidal mechanism (). The report was quickly followed with another paper showing that chloride is equally bactericidal (). Importantly, normal cellular levels of chloride provide sufficient substrate for MPO to kill ingested microorganisms, proving the system's physiological relevance.
Klebanoff's findings neatly tied together the respiratory burst, the formation of HO, and the presence of MPO.
So what's the controversy? “It sounded great, but it's wrong,” says Anthony Segal (University College, London, UK). This provocative stance is based on a number of observations. Segal found that only a small amount of the oxygen consumed in the respiratory burst is used for iodination and that the majority of proteins that get iodinated belong to the host not the bacteria (, ). Iodination fallout affecting host proteins is to be expected, but Segal argues, “if the object of the exercise is iodinate bacteria, then you would see it—it would be gross.” Additionally, Segal notes that one in a thousand people are MPO deficient but don't succumb to infections.
Segal instead believes that proteases, also found in the granules, are the neutrophils' bacteria-killing machines. His team made mice that lacked two of the proteases, cathepsin G and elastase, and showed that their neutrophils could no longer kill bacteria, even though iodination appeared normal (). He goes so far as to suggest that the MPO system is not involved in killing inside the phagosome at all but merely disposes of HO, which is itself just a byproduct of the respiratory burst.
In addition to HO, superoxide (O
) is also produced during the respiratory burst. In Segal's model, superoxide is important for readily mopping up free protons in the phagosome, thus raising the pH to levels at which the proteases work best. HO, on the other hand, just needs to be got rid of. Thus, far from iodination (or chlorination) being a bactericidal mechanism, Segal believes it is instead a readout of MPO's clean-up job.
Although people lacking MPO are healthy, MPO-deficient neutrophils in culture are slow to kill microbes, suggesting MPO is an early killing mechanism and that later-acting mechanisms such as the proteases are able to compensate in in vivo (). This is precisely Klebanoff's view. He believes that, “in normal cells the MPO system is probably the predominant killing mechanism, but there are others.” He agreed that proteases do contribute but qualifies this, adding, “especially when the MPO is not functioning.”
Since there is good evidence that both mechanisms, MPO and proteases, are microbicidal, “it's not either, or,” says Klebanoff. “It's and.” However, the final verdict as to whether MPO or protease is the predominant killing mechanism, or whether it's somewhere in the middle, is still not in. |
Analysis of LTβR-mediated signal transduction in mouse embryonic fibroblasts (MEFs) has provided a powerful system for the characterization of signaling components required for noncanonical NF-κB activation ().
MEFs were treated with agonistic anti-LTβR antibody, and processing of the p100 precursor to p52, the hallmark of noncanonical NF-κB activation, was assessed by immunoblot.
MEFs displayed constitutive and total processing of the p100 precursor protein without stimulation ().
MEFs, and processing of p100 to p52 was again assessed by immunoblot.
MEFs, indicating that the constitutive processing of p100 to p52 is caused by the loss of the gene.
In addition to the central role of the noncanonical NF-κB pathway in LTβR orchestration of secondary lymphoid tissues, noncanonical NF-κB activity also provides vital cues for B cells in several processes, including B cell development, peripheral maintenance, and antibody production (, ).
B cells.
B cells, lethally irradiated C57BL/6 mice were reconstituted with fetal liver cells derived from WT or
embryos. 8 wk after reconstitution, spleens from reconstituted mice were harvested for B cell purification and analysis (the fidelity of reconstitution and B cell purification was monitored by tracking the donor allele, Ly9.1; Fig. S1, available at ).Initially, to determine whether TRAF3 was also required for suppression of p100 processing in B lymphocytes, WT and
B cells were stimulated with agonistic anti-CD40 antibody, BAFF, or LPS, and the processing of p100 to p52 was assessed by immunoblot. As expected, ligation of either CD40 or BAFF-R, but not of TLR4, resulted in the processing of p100 to p52 in WT B cells (). In
B cells, however, generation of the p52 product occurred without stimulation (), again clearly defining TRAF3 as a negative regulator of noncanonical NF-κB activation.
B cells resulted in substantial induction of p100 protein, no further accumulation of p52 was observed in comparison with unstimulated
cells. Similarly, BAFF- and CD40-stimulated WT cells exhibit similar levels of p52 production even though ligation of CD40 maintains high levels of p100, whereas ligation of BAFF-R does not. This suggests that loss of TRAF3 results in the maximal rate of p100 processing.
B cells were cultured for 4 d in media alone or in the presence of either BAFF or agonistic anti-CD40 antibody, and viability was determined by propidium iodide exclusion. Cultured B cells undergo spontaneous apoptosis unless provided with an appropriate survival signal, including such factors as CD40L or BAFF, the latter of which provides a vital survival signal for the maintenance of peripheral B cells in vivo (). As shown in ,
B cells showed cell survival in the media control comparable with that seen in BAFF-treated WT cells.
B cells displayed high basal levels of intracellular adhesion molecule 1 (ICAM-1) expression, similar to that seen in BAFF-stimulated WT cells (), as well as the spontaneous formation of homotypic B cell aggregates (), a phenomenon that requires ICAM-1–LFA1 interaction (). Previous experiments indicated that certain B cell functions such as homotypic aggregation require both the canonical and noncanonical NF-κB activity ().
B cells described in this report may not be a sole consequence of constitutive noncanonical NF-κB activity. Although BAFF-R signaling is important for proper maintenance of B cell populations, excessive BAFF signaling also contributes to deleterious B cell responses by rescuing self-reactive B cells from peripheral deletion and promoting their entry into marginal zone niches, thereby increasing the opportunity for polyclonal stimulation and enhanced autoantibody production ().
B cells develop autoimmune phenotypes.
A recent study examining the regulation of NIK protein levels demonstrated that inhibition of TRAF3 resulted in the marked accumulation of NIK, which suggests that TRAF3 negatively regulates NIK stability (). To test whether or not TRAF3 is required for suppression of NIK protein levels, immunoblot analysis of NIK was performed on multiple TRAF3-deficient cell types, including immortalized B cells, 3T3s, and MEFs. As shown in , in which NIK was undetectable in WT cells, profound accumulation of NIK was observed in all cells lacking TRAF3, which correlated well with increased processing of p100 to p52 (, bottom). Importantly, several groups have previously reported the difficulty of detecting endogenous NIK (, ).
3T3s were treated with the proteosome inhibitor MG132 for 2 h to serve as a control (, right lanes).
cells, we performed siRNA-mediated knockdown of NIK in
MEFs. As shown in ,
MEFs treated with siRNA against NIK, but not with a scrambled control sequence, showed considerable reduction in NIK and p52 protein levels. Collectively, these findings indicate that TRAF3 indeed regulates activation of the noncanonical NF-κB pathway through inhibition of NIK protein levels.
At the present, it remains unclear how TRAF3 affects NIK protein stability. Quantitative PCR analysis of mRNA levels in MEFs and B cells demonstrated that loss of TRAF3 does not affect transcription of , which agrees with a published model that TRAF3 regulates NIK post-transcriptionally (Fig. S2, available at ) (). TRAF3 contains a zinc RING finger motif characteristic of molecules with ubiquitin ligase activity. In the Liao et al. study, the authors showed that direct interaction between NIK and TRAF3 was required for the suppressive function of TRAF3 toward NIK, suggesting that TRAF3 may lead to the ubiquitination and degradation of NIK ().
MEFs with a TRAF3 RING finger point mutant showed defect in suppression of NIK protein levels (unpublished data). However, we and other groups have been unable to see TRAF3-mediated ubiquitination of NIK in an overexpression assay (). Moreover, TRAF proteins have only been shown to mediate lysine 63 ubiquitin linkages, which are not associated with protein degradation (, ) but rather the promotion of positive signaling complexes. To add to the complexity of noncanonical NF-κB activation, Grech et al. recently reported that loss of TRAF2 also results in constitutive activation of the noncanonical NF-κB pathway (). As such, in-depth studies are required to determine how TRAF2 and TRAF3 are involved in controlling the levels of NIK before or after receiving signals from upstream receptors. Lessons learned from the characterization of the role of TRAF6-dependent ubiquitination in activation of the canonical NF-κB pathway suggest that additional players remain to be identified to complete our understanding of TRAF-dependent suppression of NIK ().
A previous study showed that constitutive activation of p52 in mice lacking the C terminus of p100 (the IκBδ portion) leads to early lethality 3–5 wk after birth (). Interestingly, TRAF3-deficient mice cannot survive beyond 2 wk of life (). Therefore, we hypothesized that constitutive noncanonical NF-κB activity may have contributed to the TRAF3-null phenotype.
/
mice in an attempt to generate double knockout animals.
mice from postnatal lethality.
mice die by postnatal day 12,
/
mice grow at normal rates into adulthood (PCR and Western blot analyses were performed to confirm the
/
genotype; ; and not depicted).
mice with one disrupted copy of the gene could survive as long as 18 d ().
mice, including drastically reduced spleen size and lymphocyte count, greatly reduced serum glucose levels, and elevated serum corticosterone ().
phenotypes are rescued with compound loss of the gene. Collectively, these data demonstrate that constitutive activation of the noncanonical NF-κB pathway is an essential contributor to the TRAF3-null phenotype.
Efforts to define the physiological role of TRAF3 began more than 10 yr ago. Unlike other TRAF family members, overexpression of TRAF3 did not result in the activation of the NF-κB pathway. As TRAF3-null mice died soon after birth, identification of the function of TRAF3 had remained a mystery. In this report, we provide genetic data that TRAF3 functions as a critical negative regulator of the noncanonical NF-κB pathway through inhibition of NIK protein levels. With our improved understanding of the bifurcation of the NF-κB signaling pathways, we are now able to reassess the role of TRAF3 function in B lymphocytes.
B cells receive a de facto BAFF signal suggests that TRAF3 plays an important role in the inhibition of inappropriate B cell activity and, therefore, may play a pivotal role in the suppression of B cell–mediated autoimmune disease. Most strikingly, we demonstrate that the lethal phenotype caused by TRAF3 deficiency results from constitutive processing of p100. Ultimately, the generation of a tissue-specific strategy in the targeted disruption of TRAF3 may be required to define the mechanism of the early postnatal lethality that comes with loss of TRAF3.
mice may result from deregulation of additional pathways.
C57BL/6 (The Jackson Laboratory) mice aged 6–12 wk were used as recipients in fetal liver transplant experiments. Targeted disruption of the allele and the allele has been described previously (, ). The TRAF3 mice colony is maintained by mating TRAF3 mice, which are in a mixed C57BL/6-129 background. All mice were maintained and bred under specific pathogen-free conditions in the University of California, Los Angeles Life Sciences mouse facility, and experiments were conducted within the parameters of our approved protocol by the Animal Research Committee.
The anti-p100/p52 and anti-NIK antibodies were purchased from Cell Signaling Technology, the anti-TRAF3 (M-20) antibody was purchased from Santa Cruz Biotechnology, Inc., and the anti-LTβR antibody was purchased from Qbiogene. The anti–β actin antibody, propidium iodide, and LPS were obtained from Sigma-Aldrich.
Fetal livers were isolated from E14.5–15.5 embryos in a mixed C57BL/6-129 background.
and
fetal livers were disrupted by passing cells through an 18-gauge needle multiple times. After filtration with a 70-μM cell strainer, 10 cells were injected intravenously into irradiated C57BL/6 mice. 4–6 wk after reconstitution, cells from the blood of recipient mice were genotyped and analyzed by FACS for the presence of the Ly9.1 marker, which is expressed on the C57BL/6-129 donor cells but not on the C57BL/6 recipient cells.
Splenocytes were harvested from fetal liver–reconstituted mice 6–8 wk after reconstitution. To obtain pure naive B cells, total splenocytes were stained with a biotin-conjugated anti-CD43 antibody (BD Biosciences), followed by streptavidin-conjugated magnetic microbeads (Miltenyi Biotec), and passed through a depletion-type magnetic sorting column (Miltenyi Biotec). Unbound cells were collected as the purified naive B cell sample and analyzed by FACS for Ly9.1, B220, and IgM markers. B cells from the reconstituted mice were retrovirally transformed using v-ABL.
B cells were cultured in RPMI medium 1640 supplemented with 10% FBS, 50 μM β-mercaptoethanol, 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies) at 37°C under 10% CO. MEFs isolated from E14.5–15.5 embryos were cultured in DMEM (Mediatech Inc.), supplemented with 5% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. TRAF3-null MEFs were infected with the Moloney murine leukemia virus ΨA-MLV, containing either the pBABEpuro-tandem affinity purification tag (TAP) or pBABEpuro-TAP-TRAF3 vector, and selected with 2.5 μg/ml puromycin.
Purified splenic B cells were stimulated with 15 μg/ml anti-CD40 mAb (FgK-45), 5 μg/ml LPS, or 100 ng /ml human BAFF (Amgen Biologicals). For the B cell survival assay, 2 × 10 B cells were stimulated for 96 h in a flatbottom 96-well plate, and cell death was measured after staining with propidium iodide and analyzed using a flow cytometer (FACScan; Becton Dickinson). In the homotypic aggregation assay, cells were cultured in a flatbottom 96-well microtiter plate at 5 × 10 cells/ml for 48 h, and cell images were taken using a microscope (Eclipse TE300; Nikon) with a 10× objective.
Sera were isolated from 8-d-old mice. The glucose level was determined by applying serum to a one-touch strip and measured with a glucometer (Active; ACCU-CHEK). The corticosterone amount was determined by using the Corticosterone Immunoassay kit (R&D Systems).
Fig. S1 shows FACScan flow cytometry analysis of B cell markers B220 and IgM and the C57BL/6-129 donor cell marker Ly9.1 on purified WT and
B cells from fetal liver–reconstituted mice. Fig.
MEFs and v-ABL–transformed B cells. Online supplemental material is available at . |
As a first approach, we sought to determine if t cells in HI are indeed naive B cells by assessing their class-switch recombination (CSR) status. CSR is a hallmark of the germinal center (GC) reaction and is absent in naive cells. To characterize rare t cells out of the bulk of normal cells, we took advantage of their unique BCL2/IGH translocation signature. Indeed, the BCL2/J fusion does not prevent CSR occurring on the downstream constant region of the IGH locus (, ), and frequency above 10 (., BCL2/J) were selected from our previous cohort of HI (), and two separate LR-PCR reactions were conducted: one designed to amplify the unswitched BCL2/Sμ region, and the other to amplify switched BCL2/Sγ regions (). Results show that only two out of six individuals were found positive for BCL2/Sμ, whereas five out of six were positive for BCL2/Sγ (, left, and ). Accordingly, individual and cumulative ratios of amplicons were significantly higher for BCL2/Sγ (9:50) than for BCL2/Sμ (2:55) (P = 0.02). In addition, identical BCL2/J junction signatures were found between BCL2/J and BCL2/Sγ amplicons, but not with BCL2/Sμ amplicons (, asterisks), indicating that most translocations previously detected in the blood of HI (–) are actually switched. Furthermore, clonal filiations between switched and unswitched cells were found in one sample (#102), supporting the presence of an active CSR process in the t clones. Altogether, and contrary to previous assumptions, these data clearly show that most peripheral t cells already underwent CSR, and therefore such cells are not naive B cells.
We next assessed if these long-lived t-bearing B cells in HI already transited through the GCs. Circulating naive and memory B cells can be distinguished on the basis of the CD27 cell surface marker and sIg isotypes (–). Fresh PBMCs from 10 healthy donors were collected, and IgD/CD27 naive, IgD/CD27 memory, and IgD/CD27 switched memory B cell subsets isolated by cell sorting (). The occurrence of t was then assessed in total PBMCs and in each fraction by the short-range BCL2/J PCR assay ( and ). As a first approach, we pooled data from the two CD27 memory subsets and examined the overall contribution of naive (CD27) and memory (CD27) B cells to the total t frequency calculated as a fraction of CD19 B cells (). t frequencies of CD19 B cells ranged from <1/10 to the unexpectedly high rate of ∼1/3.500 B cells in some individuals (, squares). Strikingly, although the level of naive t cells constantly remained at baseline (, circles), CD27 B cells accounted in a large part for the amplitude of t frequencies (, triangles). This clearly indicates that circulating t clones in HI are indeed predominantly B cells which transited through the GCs. To determine if the presence of high levels of t in some individuals was caused by a higher incidence of distinct translocations or to the clonal expansion of a given t B cell, we cloned and sequenced 55 out of 61 BCL2/J fragments from the CD27 subset. One major BCL2/J junction was observed in most individuals (, black bars), indicating that only one clone mainly accounted for t frequencies. Remarkably, this data demonstrate that the wide modulation of t frequency in HI is not caused by the accumulation of clonally unrelated t naive B cells in some individuals, but rather to the clonal expansion in the GCs of t B cells.
To define further the t cells, we next examined the repartition of the translocation in the two memory B cell subsets: the IgD/CD27 switched memory and the IgD/CD27 so-called “IgM memory” () (–). Unexpectedly, the IgD/CD27 subset contained significantly higher rates of translocation, both in terms of prevalence (nine out of nine samples versus six out of nine, P = 0.05) and frequency (35 versus 14%, P = 0.001). This predominance of a surface IgD/M and not IgG in t cells was in apparent contradiction with our previous LR-PCR results, in which switch to Cγ was prevalent on the translocated allele (). To investigate reasons underlying this paradox, we performed the BCL2/Sμ and BCL2/Sγ LR-PCR assays on the fractionated cells from four healthy donor samples (). As expected, CSR happened on both alleles in the IgD/CD27 subset (). More surprisingly, CSR also frequently occurred on the translocated allele in the predominant IgD/CD27 subset. This is in sharp contrast with the features of the peripheral IgDCD27 IgM memory B cell subset, which is entirely devoid of CSR both on the productive and the nonproductive alleles (Fig. S1, available at ) (, ). It is therefore very likely that the t cells are distinct from this subset, albeit superimposed on the basis of the CD27 and IgD markers. Most importantly, the presence of CSR on the nonfunctional, but not on the productive allele is atypical among normal circulating memory B cells. However, this “allelic paradox” stands as a hallmark of FL (, ). FL derives from follicle center B cells that encountered antigen and are undergoing somatic hypermutation and CSR (, ). In FL, CSR occurs as frequently on both alleles, but “downstream switch” (e.g., Sγ-to-Sα) happens at unusually high frequency on the productive allele, specifically sparing the Cμ region from deletion and allowing sIgM/D expression (). Consequently, despite CSR occurring on both alleles in >80% of cases, most FL cases still express a sIgM and only a minority expresses sIgG, sIgA, or no sIg () (, , ). This allelic paradox indicates the presence of a selective pressure in favor of sIgM expression on a B cell population that is at the same time permanently driven to switch (). Together with the presence of sIgM B cell follicular hyperplasia in transgenic mice (, ), this atypical feature of t cells both in FL and HI suggests a direct role of BCL2 ectopic expression in the GCs for this selection. Data from Tg mice indicate that long-term survival of follicular B lymphocytes in the GC is of key importance for the acquisition of further genetic changes, which in turn favor further cell transformation and progression to disease (). It is likely that in HI most t cells were similarly rescued by BCL2 from apoptosis and “frozen” at a differentiation stage in which constitutive activation-induced cytidine deaminase expression drives continuous somatic hypermutation and CSR activity (), two mechanisms conferring a high propensity for further oncogenic aberrations in the context of accumulating genomic instability (e.g., BCL6/p53) (, , ). In this scenario, t cells in HI would constitute much more advanced precursors of the FL pathogenesis pathway than previously thought.
Based on our data, we propose a new model in which long-lived t cells in peripheral blood of HI are mainly constituted by an atypical population of BCL2-rescued and incompletely maturated FL-like B cells released from the GCs (Fig. S2, ). Interestingly, this incomplete maturation phenotype does not prevent FL to exit from the GC nor its spreading to other secondary organs, including lymph nodes and bone marrow (, ). Contrary to normal GC-derived B cells, FL clones are prone to intense trafficking between follicles (, ). Do the similar features of FL-like cells in HI confer the same migration properties, and similarly to -Tg mice (), allow the seeding of premalignant niches? The persistence and clonal expansion of t follicular-like B cells over years in blood from HI (, ) goes along with the existence of niches, where follicular microenvironment might provide support for maintenance/proliferation in the context of polyclonal/chronic antigenic stimulation (, , , ). In support of this, hepatitus C virus patients, who display increased prevalence of t, show parallel regression of viral load and t frequency in blood after antiviral therapy ().
It remains fundamental to know if the rate of expanded FL-like cells in the peripheral blood of HI translates their activation status. Epidemiological studies have reported an increased prevalence of the t associated with environmental exposures that might contribute to lymphomagenesis (,). Further studies on the origin and status of the circulating FL-like B cells will thus be of prime importance, both to gain insights into disease progression and for the proper handling of t as an early biomarker of lymphoma.
Human samples were derived from three origins: 6 DNA samples from a cohort of HI (), for which the presence of t translocation had been previously assessed on total PBMCs (, ); 10 consecutive anonymous blood samples (200–300 ml) from the local blood bank; 30 FL DNA samples from two independent sources previously described (). Samples were collected after informed consent and approval by the local ethical committee (CCPPRB# 99–07, France).
The following antibodies were used: IgD biotinylated, CD19-APC (BD PharMingen), CD27-PE, CD20-FITC (Immunotech), and IgD-FITC (Caltag Laboratories). Primary biotinylated antibody was revealed with Streptavidin–PerCP-Cy5.5 conjugate (BD PharMingen).
PBMCs were isolated from peripheral blood by Ficoll-Hypaque density centrifugation (BD Biosciences); B cells were enriched to >95% using the B cell negative isolation kit (Dynal). B-enriched cells were preincubated for 15 min with PBS-BSA-1%–Azide-5% normal mouse serum and stained with either IgD biotinylated or IgD-FITC, CD27-PE, and CD19-APC for 20 min at +4°C. After washing with PBS-1% BSA-Azide and 15-min incubation with Streptavidin–PercP-Cy5.5, cells were analyzed on a FACScalibur (BD Biosciences). Cell sorting of (a) IgD/CD27 naive B cells, (b) IgD/CD27 IgM memory B cells, and (c) IgD/CD27 switched memory B cell subsets () was performed on a FACSAria (BD Biosciences). For all individuals, IgDCD27 and IgDCD27 B cell subsets were sorted to >98% purity, and the IgDCD27 B cell subset was sorted to >95% purity. Isolated cells were then cryopreserved at −80°C until DNA extraction. IgM- and IgD-only cells that represent only minor components (1–3%) of the memory B cell pool were not sorted as single cells (, ).
Total genomic DNA was prepared from PBMCs and/or sorted B cell subpopulations by the Qiagen DNA Blood Mini kit according to the manufacturer's instructions including RNase treatment (Qiagen). A two-step double-nested fluctuation PCR assay () was used to amplify BCL2/J junctions ( and Table S1, available at . , for mbr20A/21A and JHCo-B/Coint-B primers). As the frequency of t junctions is low in PBMCs from HI, 5–10 reactions were performed in parallel using 100 ng of DNA (per reaction) from each isolated B cell subpopulation. In the fluctuation range, at most one target molecule is present per PCR replicate, and if so will give rise to a detectable amplicon. The detection threshold of the assay is the function of the number of replicates performed with a constant amount of DNA per replicate (here, 2 × 10 for 100 ng). The frequency of the event can then be calculated from the number of positive BCL2/J amplicons using a Poisson's assumption (). The primary PCR conditions were as follows: 30 s at 94°C, 30 s at 62°C, and 1 min at 72°C, 40 cycles. 1 μl of each primary PCR reaction was used in the nested secondary PCR in the same conditions, except for 20 cycles.
For the amplification of BCL2/Cμ, the mbr20A/21A pair of nested forward primers was used in combination with a pair of nested reverse primers located in the Cμ and Sμ regions, respectively (3′Cμ-1B/3′Sμ-2B). For the amplification of BCL2/Cγ, the same forward primers were used in combination with a pair of reverse nested consensus primers designed to hybridize to the 3′ flanking sequences of all Cγ and Sγ regions (3′CγCons-1B/3′SγCons-2B). The same fluctuation PCR approach as for the short-range PCR assay was used (100 ng/replicate). Given the clonal nature of the tumor cells, a one-step standard LR-PCR approach was performed for FL patients. DNA extracted from all samples was controlled to be suitable for LR-PCR by performing the amplification of a 7.5-kb fragment β-globin gene and an 11-kb germline fragment of the TCR-β locus. The following PCR conditions were used: 3 min at 95°C, 35 cycles (1 min at 95°C, 12 min at 68°C) and 10 min at 68°C. 1 μl of the primary LR-PCR was used in the double-nested LR-PCR performed in the same conditions, except for 20 cycles.
PCR fragments were purified and cloned into the pGEM-T Easy Vector (Promega). Blunt-ended fragments generated by LR-PCR were purified and submitted to the A-tailing procedure before cloning. The resulting clones were sequenced and analyzed using the BLASTn shareware () and Vector NTI 9.0.0 sequence analysis software (Informax).
p values for t-associated switch junctions analysis and analysis of t cell distributions in peripheral B cell compartments were calculated by Fisher's exact test or chi-square test using STATA software (STATA corporation release 7.0). p values for t frequency were calculated by two-tailed Student's test.
Fig.S1 illustrates the set of LR-PCR data showing absence of CSR on the nonfunctional allele of the peripheral blood sIgM/DCD27 B cell subset. Fig. S2 illustrates a reappraisal of the current model of FL pathogenesis. Table S1 describes the sequence of the PCR primers. Online supplemental material is available at . |
Proper immunity against pathogenic infection depends on the well-orchestrated cooperation of innate and adaptive arms of the immune system. In the optimal situation, this leads to eradication of the pathogen and long-term memory that protects the host against future infection. Toll-like receptors, antigen receptors, costimulatory molecules, soluble mediators, and many other molecules are all important for the initiation, sustenance, and regulation of this intricate process. However, millions of years of evolutionary pressure have also enabled pathogens to develop their own sophisticated tools to manipulate the mammalian defense system to potentiate their survival and reproduction in the host. One example of this, described in an article by Matter et al. in a recent issue of the , suggests a new role for the tumor necrosis factor (TNF) receptor superfamily member CD27 in the clearance of persistent LCMV infections ().
The authors report that CD27 signaling is not beneficial for the course of the immune response against LCMV, but rather has a detrimental effect on protective antiviral immunity. This is a new and unexpected finding, as CD27 signaling has so far been regarded as an important positive immune regulator for the formation and function of effector and memory T cells. In the absence of CD27, for example, the magnitude of T and B cell effector responses is reduced compared with responses in wild-type animals (). Likewise, transgenic overexpression of CD70 augments the formation of effector T cells leading to enhanced protection against nonimmunogenic tumors (). In the new study, however, CD27-deficient mice were better protected against infection with a high dose of LCMV than were wild-type mice, because they were able to generate virus-specific neutralizing antibodies (nAbs). As shown previously by this group, the primary cytotoxic T cell–dependent response to this virus is normal in CD27 mice (). The intriguing finding that CD27 mice developed protective nAbs is explained by the fact that signals transmitted through CD27 during LCMV infection in wild-type mice lead to the destruction of splenic lymphoid architecture. The authors show that LCMV infection in wild-type mice, but not in CD27 mice, causes the destruction of germinal centers and the marginal zone, which are required for the development of nAbs. This destruction is attributed to the production of interferon (IFN)-γ and TNF-α by activated CD4 T cells, which depends on the expression of CD27 on these cells (). Blocking CD70 in wild-type mice also resulted in the production of nAbs and resistance against an otherwise persistent strain of LCMV (). Based on these findings, the authors suggest that blocking CD27 signaling could be a novel approach for treating clinically relevant chronic infections such as HIV and hepatitus C virus.
The splenic destruction wreaked by LCMV infection in mice fits well with previous studies on the effects of CD27–CD70 signaling. Chronic activation of CD27 through constitutive expression of CD70, for example, leads to the demise of the B cells both in the bone marrow and secondary lymphoid organs (). Consistent with this, a conspicuous finding in these CD70 transgenic mice is the early loss of the splenic marginal zone (), a structure comprising a tight organization of specialized macrophages and B cells that is required for a protective immune response against encapsulated bacteria and viruses (–).
The spleen is both a major site of early LCMV replication and the compartment in which cytotoxic T cell and antibody responses against the virus are initiated—both responses that contribute to viral clearance (–). Although it is interesting to speculate that marginal zone B cells and macrophages are uniquely important for the development of LCMV-specific nAbs, it is important to note that LCMV infection is also accompanied by loss of follicular structure and germinal center formation (, ). An intriguing interdependency seems to exist between macrophages in the marginal zone and B cells, as B cell depletion leads to gradual loss of macrophages in the marginal zone (). The organization of marginal zone B cells in turn depends on the presence of marginal zone macrophages (). During LCMV infection, follicular B cells remain present, but both marginal zone macrophages and marginal zone B cells disappear, although the cause of their mutual disappearance is not yet known.
During the initial phase of the immune response, antigen presenting cells (APCs) up-regulate a large number of costimulatory ligands belonging to both the immunoglobulin superfamily (most prominently the B7 proteins) and the TNF family. Taking into account the potential plethora of available coactivating ligands, it seems surprising that during LCMV infection elimination of only the CD27–CD70 interaction has such a drastic effect on B cell maintenance. Several features of CD27 expression and function may contribute to this apparently dominant role for CD27 during LCMV infection. First, as pointed out by Croft (), members of the TNF-R family may function in a sequential fashion with CD27 being activated early in the immune response. This role of CD27 early after infection might explain why its deletion has such a dramatic effect on the course of the antiviral immune response. Second, both in vitro and in vivo CD27 signaling has a strong effect on the differentiation of naive T cells into IFN-γ–secreting T helper (Th)1 effector cells. This effect appears to be due to the ability of CD27 to sensitize naive T cells to Th1 differentiation-inducing signals such as interleukin-12 (unpublished data). As mentioned previously, IFN-γ is known to contribute to the destruction of splenic architecture. Third, although induction of CD70 expression in vivo is hard to demonstrate using pathogens such as influenza virus, LCMV was found to induce CD70 expression on a substantial number of both T and B cells during the course of the infection (). It could well be that CD70 expression is differently regulated in various infections. This could depend on the tropism of the virus for cells of the immune system and/or recognition of the virus by immune cells. Moreover, it remains to be clarified whether other costimulatory systems, especially of the TNF–TNF receptor pathway, contribute to LCMV-induced B cell depletion. Of special interest is the 4-1BB system, as mice that chronically overexpress 4-1BB ligand display a B cell depletion phenotype similar to that seen in CD70 transgenic mice ().
Protective immunity against LCMV and other noncytolytic viruses depends on rapid induction of strong Th1/Tc1 responses and the production of nAbs, but these immune defense mechanisms are actively circumvented by many viruses. Some viruses, such as HIV and measles, induce a permanent or transient immunodepression, preventing Th1 induction, for example, by suppressing interleukin-12 production (). The induction of Th1/Tc1 immune responses promotes immunity against these viruses, probably at least in part by activating CD27–CD70 signaling. However, the study by Matter et al. raises the possibility that this signaling could also negatively affect the development of essential nAbs ().
Infection with the parasite provides an interesting parallel with LCMV infection in that infections are also associated with the destruction of the splenic marginal zone. In this system, marginal zone destruction is caused by a TNF-α–mediated decrease in the production of the chemokines CCL19 and CCL21, which are important for marginal zone maintenance (, ). CD70 has been shown to be up-regulated on dendritic cells from -infected mice (). However, whether CD70 expression and marginal zone destruction during infection are causally linked is not yet known. It is also unclear whether the production of nAbs against is precluded by the destruction of the marginal zone. Conspicuously, patients with visceral Leishmaniasis are known to suffer from secondary bacterial infections () and it would be interesting to determine if this could be caused by defects in the formation of nAbs against these bacteria.
Th1 responses are required for the successful eradication of the pathogen, whereas Th2 responses are detrimental and lead to nonhealing disease. It will therefore be interesting to investigate whether CD27-mediated costimulation, which is beneficial for immunity against as it propagates Th1/Tc1 responses, can also be detrimental caused by inhibition of nAb production.
The B cell defects in studies of LCMV infection bear a striking resemblance to the B cell dysfunctions observed in HIV-infected people. Architecture of both germinal centers (, ) and splenic marginal zones is disturbed in individuals infected with HIV (). Moreover, the ability to produce nAbs is rapidly lost in most people after HIV infection, but is maintained in chimpanzees that harbor the virus but do not develop disease (). Intriguingly, HIV-infected people, but not monkeys, display a chronic activation of the immune system, including increased expression of CD70 on CD8 T cells (–). Combining the mouse, monkey, and human data, it is tempting to suggest that the CD27–CD70 costimulatory pathway is instrumental in generating B cell dysfunction and in causing the absence of sustained nAb production after HIV infection. It will be interesting to investigate if people with long-term asymptomatic HIV infections who have stable nAb titers differ in CD27–CD70 expression and/or function compared with patients that progress to AIDS. Moreover, for persistent infections in humans, such as HIV and hepatitus C virus, specific blockade of the CD27–CD70 pathway may tip the balance, as in chronic LCMV, to favor a protective humoral immune response.
#text |
We initially examined whether –induced innate immune typhlocolitis was associated with excessive production of IL-12 or IL-23 using real-time quantitative PCR (Q-PCR) to assay the expression of these cytokines in the normal and inflamed intestine. As illustrated in , significantly increased expression of IL-23p19 and IL-12p40 mRNA, but not of IL-12p35, was observed in the cecum and colon of –infected (
) 129SvEvRAG mice. The increased expression of IL-23p19 was restricted to the intestine, as it was not observed in either the mesenteric LN (MLN) or spleen ().
As increased IL-17 expression has been associated with several IL-23–driven autoimmune T cell–mediated pathologies (), we analyzed whether increased IL-17 expression was also a feature of T cell–independent innate intestinal pathology.
129SvEvRAG mice (), which was again restricted to the intestine, as it was not increased in spleen ().
or control uninfected 129SvEvRAG mice overnight and quantified cytokine release into the medium.
129SvEvRAG mice, but there was no increase in IL-12 secretion.
129SvEvRAG mice. Three major subpopulations could be readily identified within the LPLs: Gr1CD11b (predominantly granulocytes), Gr1CD11b (monocytic cells), and Gr1CD11b cells (). Q-PCR analysis also revealed that all leukocyte subpopulations isolated from the intestine expressed high levels of IL-17 mRNA ().
129SvEvRAG mice expressed only very low levels of IL-17, around 100–1,000-fold lower than their intestinal counterparts (). Collectively, these results indicated that increased local production of IL-23 and IL-17 correlated with innate intestinal pathology.
To formally assess the requirement for IL-23 in innate intestinal pathology, we used an anti–IL-23p19 monoclonal antibody to neutralize IL-23 in vivo.
129SvEvRAG mice exhibited extensive inflammation in both the cecum and colon, treatment with anti-p19 throughout the course of the experiment resulted in highly attenuated intestinal pathology (). Anti-p19–treated mice had markedly reduced levels of inflammatory infiltrates and epithelial hyperplasia in the cecum and colon (). These results demonstrate that IL-23 plays an essential role in –induced innate immune typhlocolitis.
As
129SvEvRAG mice also exhibit marked systemic inflammatory responses (), we next examined the effect of anti–IL-23p19 treatment on these disease parameters. As shown in , anti–IL-23p19 treatment significantly reduced –induced splenomegaly in terms of both spleen weights and total spleen cell numbers.
129SvEvRAG mice ().
129SvEvRAG mice, these were not observed in mice that received anti–IL-23p19 (). These results demonstrate that the systemic innate immune activation triggered by infection is also dependent on IL-23.
As the main producers of IL-23 are activated DCs and macrophages(), we reasoned that high levels of IL-23 production might trigger a cascade of inflammatory cytokines that drives chronic intestinal inflammation. We therefore measured the levels of several proinflammatory cytokines in intestinal tissue homogenates.
129SvEvRAG mice contained markedly elevated levels of IL-6, monocyte chemoattractant protein–1 (MCP-1), IFN-γ, TNF-α, mouse chemokine CXCL1 (KCs), and IL-1β, samples isolated from
129SvEvRAG mice that had received anti–IL-23p19 expressed similar low levels of proinflammatory cytokines to uninfected controls ().
129SvEvRAG mice with anti–IL-23p19 also led to a similar reduction in IL-17 levels in the colon (), indicating that innate IL-17 production is also controlled by IL-23 in vivo. To confirm that anti–IL-23p19 was preventing intestinal pathology by inhibiting host innate immune activation and not through antibacterial effects, we used Q-PCR to assess infection levels. As shown in , treatment with anti–IL-23p19 had no effect on the level of colonization in 129SvEvRAG mice. These results indicate that neutralization of IL-23 prevents intestinal inflammation by inhibiting innate immune responses and suggest that high production of IL-23 in the intestine triggers a proinflammatory cytokine cascade that mediates local and systemic pathology.
To determine whether IL-23 was a general mediator of intestinal inflammation, we next evaluated the roles of IL-12 and IL-23 in a well-established T cell–dependent model of IBD (, ). Thus, naive CD4CD45RB T cells isolated from C57BL/6 mice were adoptively transferred into age-matched cohorts of syngeneic RAG recipient mice, p40RAG mice (lacking both IL-12 and IL-23), p35RAG mice (lacking only IL-12), or p19RAG mice (lacking only IL-23), and development of intestinal inflammation was monitored. As illustrated in , the severe colitis induced by naive CD4CD45RB T cell transfer into RAG recipients was highly attenuated in p40RAG recipients, confirming that IL-12p40 was essential for disease. Strikingly, although p35RAG recipients developed colitis of similar severity to that found in control RAG recipients, intestinal inflammation was highly attenuated in p19RAG recipients (). These results clearly indicate that IL-23, but not IL-12, is required for the development of T cell–mediated colitis.
In addition to colitis, naive CD4 T cell reconstitution of RAG recipients also leads to systemic immune pathology, including splenomegaly and hepatic inflammation (, ). We therefore analyzed these parameters to examine the role of IL-23 in T cell–mediated systemic immune pathology. T cell–mediated colitis was accompanied by marked splenomegaly in RAG recipients and, even though this was significantly decreased in p40RAG recipients, there was no significant decrease in splenomegaly in either p35RAG or p19RAG recipients (). FACS analysis confirmed that the adoptively transferred CD4 T cells had reconstituted the spleens of all groups of RAG mice and, even though this was reduced in p40RAG recipients, there was no significant reduction in CD4 T cells in p35RAG or p19RAG recipients (). A similar pattern was observed with respect to liver pathology, with numerous prominent inflammatory foci present in RAG, p35RAG, and p19RAG recipients but virtually absent in p40RAG recipients (). Collectively, these results show that IL-23 is not required for CD4 T cell–mediated systemic inflammatory responses and further indicate that either IL-12 or IL-23 alone is able to facilitate CD4 T cell–dependent inflammatory responses in the liver and spleen.
As the T cell transfer colitis model has been associated with increased local Th1 responses (), we also examined the levels of several proinflammatory cytokines in intestinal tissue homogenates from the various groups of RAG recipients. Although colon homogenates isolated from control RAG recipients contained elevated levels of TNF-α, IFN-γ, IL-6, MCP-1, IL-1β, and KC, these levels were markedly decreased in p40RAG recipients (). As expected, p35RAG recipients with colitis expressed similarly elevated levels of proinflammatory cytokines as control RAG recipients, whereas these were again attenuated in p19RAG recipients (). These results demonstrate that IL-23, but not IL-12, is required for the efficient expression of proinflammatory cytokine cascades in the intestine.
As IL-23 has been strongly associated with proinflammatory IL-17–secreting CD4 T cells (Th17), we also examined the levels of IL-17 in the colon homogenates and assayed for Th17 cells using intracellular FACS analysis. The highest levels of IL-17 were detected in colon homogenates isolated from p35RAG recipients (). In contrast, only low levels of IL-17 were present in colon homogenates from p40RAG or p19RAG recipients (). CD4 T cells isolated from the MLN and LPLs of RAG or p35RAG recipients contained a high proportion of IFN-γ cells and a small population of IL-17 cells ( and not depicted). Interestingly, CD4 T cells isolated from p40RAG or p19RAG recipients showed a marked decrease in the frequency of IFN-γ cells but still contained a small population of IL-17 cells (). These results show that T cell–mediated colitis correlates with increased frequencies of IFN-γ–secreting Th1 cells in intestinal lymphoid tissue and increased levels of IL-17 in the colon, suggesting that both Th1 and Th17 cells may contribute to pathogenesis. They further show that neither IL-12 nor IL-23 is absolutely required for the differentiation of Th17 cells from naive CD4 T cells in vivo.
Until recently, it had been widely accepted that the chronic intestinal inflammation found in human CD patients, as well as in many animal models of IBD, was caused by IL-12–driven excessive CD4 Th1 responses. However, the results presented in this paper clearly demonstrate that IL-23, but not IL-12, plays an essential role in the induction of chronic intestinal inflammation.
129RAG mice highlights a novel role for IL-23 in innate immune pathology in vivo.
Attenuation of intestinal inflammation by blockade or genetic ablation of IL-23 was accompanied by decreased production of many proinflammatory cytokines, including TNF-α, IFN-γ, MCP-1, IL-6, IL-1β, and KC, several of which have been implicated in the pathogenesis of IBD. As IL-23 is produced rapidly by DCs and macrophages after exposure to pathogen-derived molecules (, ), the most straightforward interpretation of our data is that IL-23, produced in response to intestinal bacteria, triggers a proinflammatory cytokine cascade that, if left unchecked, can lead to the development of chronic intestinal inflammation. As IL-23R is also expressed by DCs and macrophage populations, it has been proposed that IL-23 secretion can drive an autocrine feedback loop that amplifies local expression of cytokines like IL-1β and TNF-α (), which in turn stimulate release of additional proinflammatory mediators by stromal, epithelial, and endothelial cells.
Our experiments also highlight some novel aspects of the regulation of IL-17 expression in intestinal inflammation in vivo. To date, IL-23 has been proposed to trigger IL-17 production through the induction/expansion of a novel subset of CD4 Th17 cells, which have been associated with autoimmune pathology (, , , ).
129RAG mice showed that, concomitant with the increased IL-23 expression, there was a striking increase in both IL-17 mRNA expression and protein release in inflamed intestine, indicating that IL-23 also induces the secretion of IL-17 by non–T cells in an inflammatory environment. FACS sorting revealed that several subpopulations of LPLs expressed high levels of IL-17, including granulocytes and monocytes.
129RAG mice did not correlate with an increase in splenic IL-17 expression, suggesting that additional local signals in the inflamed intestine are required. A previous study noted that neutrophils may produce IL-17 in response to LPS, suggesting that bacteria may provide one such signal (). Increased expression of IL-17 has been reported in the intestinal mucosa of IBD patients, and histological analysis suggested that both T cells and monocytes may act as sources of IL-17 in the inflamed gut (). The IL-17R is widely expressed, and IL-17 binding promotes stromal, endothelial, and epithelial cells to secrete proinflammatory mediators that recruit neutrophils to sites of inflammation (). Together with our previous finding that a prominent granulocytic infiltrate is characteristic of –induced innate typhlocolitis (), this supports a role for IL-17 in the induction of innate intestinal pathology and suggests that several cell types may contribute to IL-17 production in the gut.
A very recent study reported that the T cell–mediated colitis that develops in IL-10 mice or in RAG recipients of IL-10 CD4 T cells was also dependent on IL-23 (). Although this was associated with increased development of pathogenic Th17 cells, anti–IL-17 treatment had little impact on colitis and had to be combined with anti–IL-6 treatment to attenuate disease (). These results indicate that even though IL-23–driven Th17 responses may play an important role in colitis, they constitute one of several potential innate and adaptive immune mechanisms that can contribute to intestinal pathology. Support for this hypothesis was obtained in a parallel study (see Kullberg et al. [] on p. of this issue) that examined the role of IL-23 in two models of –triggered T cell–dependent colitis. Though again highlighting a critical role for IL-23, these studies additionally identified a pathogenic role for IFN-γ, indicating that Th1 and Th17 responses may synergize to elicit maximal pathology during bacterially induced colitis (). Similarly, in our T cell–mediated colitis model the correlation between IL-17 and intestinal inflammation was not completely straightforward. Although elevated levels of IL-17 were found in colon homogenates from colitic RAG and p35RAG recipients, disease severity correlated with high frequencies of IFN-γ–secreting T cells, whereas only low proportions of IL-17–secreting T cells were present. These results again suggest that both Th1 and Th17 cells contribute to IL-23–dependent colitis. In addition, our observations that small populations of IL-17–secreting T cells were present in both p40RAG and p19RAG recipients clearly indicate that neither IL-12 nor IL-23 is required for differentiation of IL-17–secreting T cells in vivo. These findings are in accord with those of a very recent study in which infection with the intestinal bacterial pathogen elicited potent Th17 responses in both wild-type and p19 mice (). However, despite mounting strong Th17 responses, p19 mice had less colonic inflammation and failed to clear the infection, indicating that IL-23–driven inflammatory responses were an integral component of the protective response ().
Interestingly, a study using the dextran sulfate sodium model of acute colitis reported that neutralization of IL-17 exacerbated intestinal inflammation, suggesting an inhibitory role for IL-17 in this disease (). Furthermore, in vitro experiments using intestinal epithelial cell monolayers suggested that IL-17 could enhance mucosal barrier function (). Several recent studies have clearly shown that TGF-β, in the presence of proinflammatory cytokines such as IL-6, induces the differentiation of Th17 cells (–). Conversely, in the absence of inflammatory mediators, TGF-β promotes the development of Foxp3 T reg cells associated with suppression of inflammatory responses(, , –). These paradoxical observations illustrate the complexity of immune regulation in the intestine and indicate that understanding how interactions between pleiotropic factors such as IL-17 and TGF-β influence intestinal homeostasis presents an important future challenge.
As increasing scrutiny is given to the role of IL-23 in inflammatory responses, it is becoming apparent that it is a gross oversimplification to consider the IL-23–IL-17 and IL-12–IFN-γ pathways as two independent (and often mutually exclusive) axes of immune pathology. Instead, we favor the idea that IL-23 is a central conductor of a range of innate and adaptive inflammatory responses and that IL-23 itself may be regulated at several levels. Although bacterial stimuli may be a major inducer of IL-23 secretion, adaptive immune processes may also modulate its production. In support of this, we have recently observed that injection of agonistic anti-CD40 monoclonal antibody induces an IL-23–dependent acute inflammatory response in RAG mice that was accompanied by intestinal inflammation (). In contrast to most other models, anti-CD40–induced colitis was independent of the presence of a bacterial microflora (), indicating that there is an alternative route of IL-23 induction. The anti-CD40 treatment most likely mimicked strong T cell activation, characterized by marked up-regulation of CD40L that can then signal through CD40 on APCs. Thus, during sustained immune responses, activated T cells may provide a positive feedback loop for inducing further production of IL-23, thereby perpetuating inflammation. It would also seem important to have means of inhibiting IL-23 production and, because T reg cells and IL-10 have been implicated in down-modulation of innate and adaptive inflammatory responses, these seem obvious candidates. In fact, macrophages isolated from IL-10 mice show elevated secretion of both IL-12 and IL-23 in response to bacterial stimuli ().
Another important observation in these studies was that even though IL-23 was essential for local tissue inflammation in the intestine, it was not required for systemic inflammatory responses. This was clearly shown in the T cell transfer model of disease in which, even though colitis was highly attenuated in p19RAG mice, there was no inhibition of splenomegaly or in development of inflammatory foci in the liver. This suggests that IL-23 may be especially important for inflammatory responses within peripheral tissues, which is consistent with previous experimental observations in models of autoimmune inflammation in the brain () and joints () and in inflammation induced by bacterial pathogens in the lung () and intestine (). Together with our results, these studies are consistent with the hypothesis that the natural function of IL-23 in host defense may be in coordinating inflammatory responses against bacterial infection in peripheral tissues, but that dysregulated expression of IL-23 may promote harmful immune pathology in these sites.
RAG mice, where up-regulated IL-23 was present only in the cecum and colon but not in peripheral lymphoid tissues.
RAG mice were also attenuated after treatment with anti-p19 may reflect the sequential activation of local and systemic inflammatory responses in this model. In this case, we postulate that disease follows an “outside-in” sequence by which infection with the bacteria triggers local inflammation in the intestine, resulting in increased host cytokines and bacterial proinflammatory molecules reaching the systemic circulation. These in turn feed the systemic cytokine cascade that drives splenomegaly and liver pathology; therefore, preventing the initial inflammation in the intestine also shuts down the downstream systemic sequelae. In contrast, the T cell transfer model may represent an “inside-out” sequence in which naive T cell reconstitution is followed by a rapid T cell expansion in systemic lymphoid tissues. In the absence of T reg cells, this expansion proceeds in a dysregulated manner, allowing the excessive accumulation of both autoaggressive T cells that can mediate systemic inflammatory responses as well as bacterially reactive T cells that mediate intestinal inflammation. In this case, although IL-23 deficiency prevents the bacterially reactive T cells from causing colitis, it does not inhibit systemic T cell expansion and associated inflammation.
One additional point to note is that even though IL-23 is clearly a central mediator of intestinal inflammation, there may be additional IL-23–independent inflammatory pathways that also contribute to disease.
129RAG mice, mainly in the cecum, where the highest levels of colonization occur (). This indicates that pathogenic bacteria can also activate alternative innate immune mechanisms that synergize with IL-23 to drive severe pathology. It is clear that excessive immune responses of almost any variety, Th1 cell, Th2 cell, or innate immunity, can mediate intestinal inflammation (, ).
The increasing clinical use of biological therapies such as infliximab (anti–TNF-α) in human IBD illustrates the potential benefits that may be derived through molecular analysis of immune pathogenesis. However, the long-term effects of such therapies are still unknown and, given the essential role of TNF-α in host defense, concerns have been voiced over possible increased incidences of infections, such as , or tumors (). Although some encouraging results have been obtained in initial clinical trials of anti–IL-12p40 in CD (), the central role of IL-12 in resistance against many pathogenic infections () suggests that long-term administration may similarly depress systemic immune function. Agents that target IL-23 may have the advantage of selectively decreasing local immune responses in afflicted tissues while sparing systemic immune protective mechanisms, a highly desirable property for efficient therapeutic agents for IBD.
129SvEvRAG2 mice and wild-type B6, control B6RAG1, and IL-12/23–deficient strains on a B6RAG1 background (IL-12p40RAG, IL-12p35RAG, and IL-23p19RAG mice) were bred and maintained under specific pathogen-free conditions in accredited animal facilities at the University of Oxford. Experiments were conducted in accordance with the UK Scientific Procedures Act of 1986. Mice were routinely screened for the presence of spp. and were >6 wk old when first used.
NCI-Frederick isolate 1A (strain 51449; American Type Culture Collection) was grown on blood agar plates containing trimethoprim, vancomycin, and polymixin B (all obtained from Oxoid) under microaerophilic conditions as previously described (, ). For infections, bacterial viability was confirmed using fluorescent microscopy with a bacterial live/dead kit (BacLight; Invitrogen), and 129SvEvRAG2 mice were fed three times on alternate days with ∼5 × 10–2 × 10 CFU .
Anti–IL-23p19 monoclonal antibody (PAB1106) was produced and characterized as previously described (). Antibody treatment was commenced on the day of the first inoculation with , and mice received weekly i.p. injections of 1 mg anti–IL-23p19 or isotype control for the duration of the experiment.
Naive CD4CD45RB T cells were isolated from spleens of C57BL/6 mice using FACS sorting as previously described (). In brief, single cell suspensions were depleted of CD8, MHC class II, Mac-1, and B220 cells by negative selection using a panel of rat monoclonal antibodies, followed by sheep anti–rat–coated Dynabeads (Dynal). After staining with Cy-Chrome–conjugated anti-CD4, PE-conjugated anti-CD25, and FITC–anti-CD45RB (all obtained from BD Biosciences), naive CD4CD25CD45RB T cells were purified (∼99%) by cell sorting with a cell sorter (MoFlo; DakoCytomation). Sex-matched control or IL-12/23–deficient B6RAG mice received 4 × 10 CD4CD45RB T cells by i.p. injection, and development of intestinal inflammation was monitored as described in the next paragraph.
Mice were killed when symptoms of clinical disease (weight loss or diarrhea) became apparent in control groups, usually 6–8 wk after initiation of experiments. Samples of liver, cecum, and proximal, mid-, and distal colon were immediately fixed in buffered 10% formalin. 4–5 μm of paraffin-embedded sections was stained with hematoxylin and eosin, and inflammation was assessed as previously described (, ). Each sample was graded semiquantitatively from 0 to 4, and typical features of each grade are as follows: 0 = normal; 1 = mild epithelial hyperplasia; 2 = pronounced hyperplasia with substantial inflammatory infiltrates; 3 = severe hyperplasia and infiltration with marked decrease in goblet cells; and 4 = severe hyperplasia, severe transmural inflammation, ulceration, crypt abscesses, and severe depletion of goblet cells. Ceca and colons were assessed separately, and three separate sections from each sample were examined. The total colonic score was obtained by adding the individual scores from the sections of proximal, mid-, and distal colon.
Aliquots of 1–5 × 10 cells were stained in FACS buffer (HBSS, 0.1% BSA, 5 mM EDTA; both obtained from Sigma-Aldrich) using the following panel of monoclonal antibody to mouse cell surface molecules (all obtained from BD Biosciences): biotinylated anti–NK cells (DX5), biotinylated anti-Gr1 (Ly6G), allophycocyanin-conjugated anti-CD11b, PE–anti-CD11c, PerCP-conjugated anti-CD4, and PE–anti–mouse TCRβ. Biotinylated antibodies were detected using PE-, allophycocyanin-, or FITC-conjugated streptavidin (all obtained from BD Biosciences). Samples were first incubated for 10 min with Fc block (eBioscience) to block any nonspecific binding, and subsequent staining steps were performed for 20 min on ice, followed by washing with FACS buffer. Samples were fixed in FACS buffer containing 2% paraformaldehyde (Biolab) acquired using a FACSCalibur (Becton Dickinson) and analyzed with software (FlowJo; Tree Star, Inc.). For intracellular cytokine analysis, cells were incubated in complete RPMI 1640 (10% heat-inactivated FCS, 2 mM -glutamine, 100 U/ml penicillin and streptomycin [obtained from Invitrogen], and 0.05 mM 2-mercaptoethanol [obtained from Sigma-Aldrich]) containing 0.1 μM PMA and 1 μM ionomycin (both obtained from Sigma-Aldrich), plus 10 μg/ml Brefeldin A (Sigma-Aldrich), for 4 h at 37°C in a humidified incubator with 5% CO. Cell surface staining was performed as described previously in this paper, and, after fixing, the samples were incubated in permeabilization buffer (PBS, 0.1% BSA, 0.5% saponin; Sigma-Aldrich) containing allophycocyanin–anti–mouse IFN-γ, PE–anti–mouse IL-17 or appropriate isotype controls, allophycocyanin–rat IgG1, and PE–rat IgG2b (all obtained from BD Biosciences) for 30 min at room temperature, washed in permeabilization buffer, and analyzed as described in this paper.
129SvEvRAG2 mice as previously described (). Cells were stained with fluorescent antibodies, and leukocyte subpopulations were purified using FACS sorting as outlined previously in this paper. Cells were sorted into distinct subpopulations on the basis of size (forward scatter [FSC]) and granularity (side scatter [SSC]) in combination with surface marker expression (). Characteristics of subpopulations were as follows: granulocytes, FSCSSCGr1CD11bCD11c; monocytes, FSCSSCGr1CD11bCD11c; DCs, FSCSSCGr1CD11c; and NK cells, FSCSSCDX-5.
cDNA samples were assayed in triplicate using a detection system (Chromo4; GRI), and cytokine gene expression levels for each individual sample were normalized relative to HPRT using ΔCt calculations ().
In initial experiments, small pieces of colon (∼5 mm of mid-colon) were isolated and rinsed in HBSS/BSA and weighed. Colon explants were cultured overnight in 24-well tissue culture plates (Costar) in 500 μl complete RPMI 1640 at 37°C in an atmosphere containing 5% CO. After centrifugation at 10,000 to pellet debris, culture supernatants were transferred to fresh tubes and stored at −20°C. Cytokine concentrations were measured using specific sandwich ELISAs (IL-23, R&D Systems; IL-17, Bio-Rad Laboratories) and were normalized to the weight of the colon explant.
For more comprehensive analysis, frozen intestinal tissue samples were homogenized in PBS containing a cocktail of protease inhibitors (Protease Inhibitor Cocktail Tablets; Roche) using a Polytron Homogenizer. After centrifugation at 10,000 to pellet debris, concentrations of a panel of proinflammatory cytokines in supernatants were measured either using the cytometric bead assay (BD Biosciences) or the Luminex 100 assay (Bio-Rad Laboratories). In all cases, cytokine levels were normalized to the total protein level in each sample, as measured using the Bradford assay (Bio- Rad Laboratories).
DNA was purified from cecal contents taken from –infected mice using the DNA Stool kit (QIAGEN). DNA was determined using a Q-PCR method based on the gene and performed with a Chromo4 detection system, as previously described (, ).
The nonparametric Mann-Whitney test was used for comparing pathology scores and Q-PCR data, and an unpaired test was used to examine spleen weights and cell counts. Differences were considered statistically significant when P < 0.05. |
We isolated natural T reg cells and conventional CD4 LN T cells by staining and sorting CD425GITR and CD425GITR subsets (). An aliquot was stained for Foxp3, confirming expression in the CD425GITR (87% Foxp3) but not the CD425GITR subset (1% Foxp3; ). Low molecular weight RNA was extracted from conventional T cells and T reg cells from three independent sorts. Each set of samples was hybridized to microarrays containing oligonucleotide probes complementary to 173 known miRNAs (). Two hybridizations (chip 1 and chip 2 in ) were performed for each biological replicate () using reciprocal labeling with Cy3/Cy5 and Cy5/Cy3 to offset possible detection bias. This dataset was subjected to significance analysis of microarrays (SAM), in which each miRNA is assigned a score on the basis of its change in expression relative to the standard deviation of repeated measurements (). SAM identified 68 miRNAs that were differentially expressed between natural T reg cells and conventional CD4CD25 T cells (). 35 miRNAs were preferentially expressed in T reg cells (including miR-223, miR-146, miR-21, miR-22, miR-23a and b, miR-24, miR-214, miR-155, and others) and 33 were down-regulated in T reg cells (including miR-142-5p and -3p, miR-30b, c, e, and members of the Let-7 family). Differential miRNA expression was validated by real-time PCR and Northern blotting ().
From the dataset described in , we selected the 40 miRNAs to which SAM analysis had assigned the lowest q values, indicative of false discovery rates of 0–0.5%. 20 of these miRNAs were overexpressed in T reg cells and 20 were down-regulated in T reg cells compared with conventional CD4 T cells. We then used miRNA microarrays to track the expression of these miRNAs during the activation of conventional CD4 T cells (). Strikingly, the miRNA profile of conventional CD4 T cells began to resemble that of T reg cells so that 3 d after activation, 9 of the 20 most T reg cell–specific miRNAs were up-regulated (, yellow) and 11 of the 20 miRNAs most underexpressed in T reg cells were down-regulated (, blue). This pattern was highly nonrandom because none of 20 miRNAs overexpressed in T reg cells became down-regulated, and none of 20 miRNAs underexpressed in T reg cells became up-regulated in activated conventional T cells (). miRNA expression by activated T cells was dynamic so that miRNAs that had been selected for differential expression between T reg cells and naive CD4 T cells began to show a positive correlation between T reg cells and activated T cells 24 h after activation (R = 0.28). By day 3 of activation, this positive correlation strengthened to R = 0.6 and then declined again (day 10, R = 0.03; ). Hence, conventional CD4 T cells transiently adopt a T reg cell–like miRNA profile during activation.
To determine whether Foxp3, the signature transcription factor of T reg cells, plays a role in defining the T reg cell miRNA expression profile, we activated conventional CD4 T cells and transduced them with retroviruses encoding Foxp3-IRES-GFP or IRES-GFP alone. GFP cells were sorted 3–4 d later and, as expected, intracellular staining showed the presence of Foxp3 protein in Foxp3-IRES-GFP–transduced cells but not in cells transduced with the control vector (). We then compared miRNA expression between Foxp3 and control vector–transduced cells after 72 () and 96 h (not depicted) and found that 9 of the 10 miRNAs that were up-regulated in Foxp3-expressing cells at both time points were among the top 20 miRNAs preferentially expressed in T reg cells (, yellow). Among the miRNAs overexpressed in Foxp3-transduced cells was miR-146, which is overexpressed by T reg cells but not by activated T cells (see above). Conversely, 6 of the 10 miRNAs down-regulated in Foxp3-transduced cells were among the 20 most underexpressed miRNAs in T reg cells (, blue). This analysis shows that Foxp3 directly or indirectly contributes to the profile of miRNA expression in T reg cells.
The RNase III enzyme Dicer is essential for the processing of pre-miRNAs into mature, functional miRNAs; therefore, its deletion provides a genetic test for the relevance of miRs to T reg cell biology. In the conditional lckCre Dicer deletion model we had analyzed previously (), thymocyte numbers are reduced 10-fold and there are very few peripheral T cells (not depicted), precluding an analysis of the involvement of Dicer-generated RNAs in T reg cell development. We therefore crossed our conditional Dicer allele with CD4Cre, which deletes during the DN/DP transition (, ) significantly later during T cell development than lckCre (, ).
mice (19 and not depicted).
SP thymocytes and mature miRNAs were reduced ∼10-fold in naive CD4 peripheral T cells (19 and not depicted).
mice have moderately reduced numbers of peripheral CD4 T cells (19 and not depicted). Among these CD4 T cells, we found a substantial reduction in the frequency of natural T reg cells (2.7 ± 0.3% in CD4Cre
CD4 spleen cells, 7.5 ± 2.5% in
CD4 spleen, = 15, ratio = 2.8; 3.3 ± 0.8% in CD4Cre
CD4 LN cells, 7.6 ± 0.8% in
CD4 LN, = 13, ratio = 2.3; 0.8 ± 0.2% in CD4Cre
CD4 SP thymocytes, 3.7 ± 0.5% in
CD4 SP thymocytes, = 4, ratio = 4.6) and in the expression of Foxp3 mRNA compared with
controls (). The introduction of a Bcl-2 transgene failed to correct this deficiency in natural T reg cells (not depicted).
mice aged between 3 and 4 mo. Histopathological examination revealed immune pathology affecting the colon, lung, and liver. 5 of 11 4-mo-old CD4Cre
mice examined were affected by colitis, characterized by a diffuse infiltrate of inflammatory cells in the lamina propria and focal formation of crypt abscesses (). There also was focal portal and lobular inflammation in the liver in three mice (not depicted).
mice examined ( = 5) and in all
controls ( = 10).
The data presented above suggest that Dicer plays a role in T reg cell biology, but they do not distinguish between an involvement in T reg cell differentiation on the one hand and T reg cell maintenance or homeostasis on the other. It could be that Dicer-deficient T reg cells differentiate in normal numbers but are prone to apoptosis (), for example in response to the recognition of self-antigen (, ). Alternatively, homeostatic control () could partially compensate for a more serious defect in T reg cell differentiation than is apparent by their frequency at steady-state. We therefore examined the first wave of natural T reg cell development in the thymus. To exclude exchange between the thymic and the peripheral T cell pool, we used thymic organ culture initiated at embryonic day 15 (E15), when all thymocytes are still CD4CD8 DN.
and
controls were cultured for 10 d, and the frequency of T reg cells was evaluated (). In
control cultures, 2.4 ± 0.3% of CD4 SP cells were CD25 CD69 ( = 6), whereas in lckCre
culturesthe frequency of CD4 SP CD25 CD69 cells was reduced fourfold to 0.6 ± 0.2% ( = 11).
fetal thymic lobe ( = 6) versus 48 ± 22 per lckCre
lobe ( = 11), a difference of 22-fold.
thymi (). We conclude that the thymic differentiation of natural T reg cells is compromised in the absence of Dicer and mature miRNAs.
Because T reg cell differentiation can be driven by extrinsic signals such as TGF-β (), we asked whether T reg cell differentiation of Dicer-deficient T cell precursors could be rescued by a wild-type environment. To this end, we constructed mixed thymus chimeras () consisting of a wild-type component marked by the Thy1.1 alloantigen and a Thy1.2 component of either
controls or lckCre
(see Materials and methods). Embryonic day 15–17 thymi were dissociated by proteolysis, mixed as indicated, reaggregated, and cultured for 7–10 d. CD4 SP thymocytes that developed in these chimeras were analyzed for the presence of natural T reg cells, identified by CD25 and GITR. Mixed chimeras containing wild-type Thy1.1 and
Thy1.2 thymi generated distinct populations of CD25 GITR CD4 SP thymocytes within both the Thy1.1 (wild-type) and the Thy1.1 (
) subset (4.2 and 4.3%, respectively; ). In contrast, mixed chimeras containing wild-type Thy1.1 and lckCre
Thy1.2 thymi generated a distinct CD25 GITR population only in the Thy1.1 (wild-type) but not the Thy1.1 (lckCre
) CD4 SP subset (2.7 and 0.2%, respectively; ). Hence, the impaired thymic development of Dicer-deficient natural T reg cells is not rescued by the provision of an environment in which wild-type natural T reg cells develop normally.
T cell activation in the presence of TGF-β induces Foxp3 expression and T reg cell function (), providing a model system for postthymic T reg cell differentiation.
or control
CD4CD25 LN T cells with 200 ng/ml of plate-bound anti-TCR (H57) and anti-CD28. 2 d after exposure to 1 ng/ml TGF-β1 (Sigma-Aldrich), 48.5 ± 15.7% of
but only 13.5 ± 4.7% of CD4Cre
cells expressed Foxp3 ( = 6; ). This demonstrates a role for Dicer in the induction of Foxp3 expression by environmental signals. Inflammatory signals such as IL-6 have been shown to abrogate Foxp3 induction by TGF-β () and to induce IL-17 expression instead (, ). To address the possibility that T cell differentiation was diverted toward the IL-17 lineage in the absence of Dicer, we restimulated the cells 5 d after activation.
or
control cells after exposure to TGF-β, whereas IL-17 was readily induced by the combination of TGF-β and IL-6 ().
miRNAs control the expression of a large proportion of protein-coding genes at the posttranscriptional level (–), and Dicer is essential for embryonic development (). It was therefore surprising when recent studies showed that many aspects of T cell differentiation are relatively normal in the absence of (, ). Here we show that the deletion of results in a specific defect at a relatively late stage of T cell development. We find that is required, in a cell-autonomous fashion, for the development of natural T reg cells in the thymus, for normal T reg cell numbers in peripheral lymphoid organs, and for the efficient induction of Foxp3 in naive CD4 T cells by TGF-β.
mice develop immune pathology, in particular inflammatory bowel disease.
mice.
T cells are predisposed to Th1 responses (), which may contribute to the observed immune pathology.
Consistent with the importance of Dicer for T reg cell biology, we show that T reg cells express a characteristic set of miRNAs distinct from that of naive CD4 T cells, including 7 of a set of 21 miRNAs commonly overexpressed in solid tumors (miR-223, miR-214, miR-146, miR-21, miR-24, miR-155, and miR-191; reference ), which can affect the growth and/or the survival of tumor cells (, ). In contrast, Let-7, which negatively regulates Ras, is down-regulated in some human tumors () and in T reg cells (this study). miRNA 21 is encoded in the 3′ UTR of the gene (EMBL: AJ459711; MMU459711). Despite sixfold overexpression of miR-21 in T reg cells, real-time PCR primers in the coding region and the 3′ UTR showed no difference in mature Tmem49 mRNA levels between T reg cells and conventional T cells (not depicted). Intronic primers demonstrated slightly (1.6-fold) higher levels of Tmem49 primary transcript in T reg cells, consistent with the fact that only nuclear transcripts are potential targets for processing by the nuclear RNase III Drosha (). Similarly, mir-155 resides in the noncoding BIC transcript (EMBL: AY096003). BIC and miR-155 accumulate in B cell lymphomas, but the abundance of BIC transcript does not predict the amount of mature miR-155 (). Hence, the levels of conventional transcripts do not predict the expression of miRNAs encoded at the same location, ruling out the use of cDNA expression data as indicators of miRNA levels. Expression of miR-146 is low in naive T cells and selectively up-regulated in Th1 cells () and T reg cells (this study), but not in Th2 cells (), whereas miR-150 is expressed in naive T cells but down-regulated after activation in Th1 and Th2 cells (, ) as well as in T reg cells (this study). miR-142 and members of the Let7 family are also down-regulated in Th1 and Th2 cells () as well as in T reg cells.
The emerging picture is that T reg cells express an miRNA profile similar to that of acutely activated CD4 T cells. This brings into focus the knowledge that T reg cells constitutively express CD25, CTLA4, and GTIR, markers that are also induced by the activation of conventional CD4 T cells (, ), even though most T reg cells in peripheral LNs are CD69 CD62L and not actively dividing (). From this perspective, one could argue that T reg cells may be locked in a partially activated state. Understanding the molecular mechanisms that maintain this state in natural T reg cells will be key to their biology. Interestingly, our data show that the ectopic expression of the T reg cell signature transcription factor Foxp3 can confer a partial T reg cell miRNA profile. Hence, aspects of the T reg cell–specific miRNA profile may be under the direct or indirect control of Foxp3. It remains to be investigated whether Foxp3 is under miRNA control. In addition to the extensive overlap between the miRNA profile of T reg cells and activated T cells, our analysis has identified miRNAs that are overexpressed by T reg cells but not by activated T cells, for example miR-223 and miR-146. Detailed studies on the mRNA targets of these and other T reg cell–expressed miRNAs may provide further clues to how T reg cells develop and are maintained over time. The systemic manipulation of miRNA function () may open new avenues for the control of T reg cell development and function in vivo.
Animal work was performed according to the Animals (Scientific Procedures) Act, UK.
mice () on a mixed C57BL/129 background were crossed with LckCre or CD4Cre transgenic mice () to generate lckCre
or CD4Cre
mice and held in a conventional facility where they encountered and but no other identified pathogens, such as MHV or Sendai virus. Cells were stained, analyzed, and sorted by flow cytometry as described previously (). The following antibodies were used: CD25-PE, CD25-APC, CD69-FITC, and Thy1.1-biotin (BD Biosciences); CD4-TC and CD8-PE (Caltag); GITR-FITC (R&D Systems); Streptavidin-Alexa-405 (Invitrogen); and Foxp3-PE and Foxp3-APC (eBioscience). Cells were analyzed or sorted on Becton Dickinson Calibur, DIVA, or Aria flow cytometers.
LN T cells were activated at 1–3 × 10/ml with 200 ng/ml of plate-bound anti–TCR-β (H57; BD Biosciences) and 2 μg/ml anti-CD28 (BD Biosciences). For induction of Foxp3 expression, we added 1 ng/ml TGF-β (Sigma-Aldrich). Retroviral gene transfer was performed by spin infection of overnight-activated T cells (90 min, 2,000 rpm, 37°C, without polybrene) using mouse stem cell virus vectors as described previously (). Fetal thymic organ culture, reaggregate culture, and mixed thymic chimeras were established and cultured as described previously ().
Probes for 173 miRNAs referenced in miRBase () were synthesized in sense orientation (Sigma-Aldrich) and spotted on glass slides in 16 replicates. Low molecular weight RNA was isolated using the MirVana kit (Ambion) and miRNAs were reverse transcribed using the 3DNA Array Detection 900 miRNA RT kit (Genisphere). 100 ng of small RNAs were tailed with poly(A) polymerase and reverse transcribed using a poly(dT) primer and a unique capture sequence. The tagged cDNAs were concentrated with Microcon YM-10 columns (Millipore), resuspended in 60 μl hybridization buffer (18.5% formamide, 5xSSC, 5x Denhardt's solution, 0.5% SDS, 5mM KHPO), denatured at 95°C for 5 min, and hybridized to the array at 42°C overnight. Arrays were developed with Cy3- and Cy5-coupled DNA oligonucleotides with reverse complementary to the capture sequences and scanned with a GenePix 4000B scanner using Genepix Pro 5.0 (Axon). Data analysis was performed in Acuity (Molecular Dynamics). Data was filtered by removing spots with <55% of pixels one standard deviation above background after subtracting median background values. Cy3/Cy5 ratios were log() transformed and normalized by Lowess, and in some cases by median centering, and the average ratio of replicates was calculated for each miRNA that passed the filter criteria. To eliminate dye bias, each experiment was hybridized to two separate arrays, swapping the dye of each sample. Data were subjected to SAM as described previously (), accepting a false positive rate of 0.068.
Total RNA was isolated using RNAbee (Tel-Test) and reverse transcribed. Real-time PCR analysis was performed on an Opticon DNA engine (95°C for 15 min followed by 40 cycles at 94°C for 15 s, 60°C for 30 s, and 72°C for 30 s with a plate read at 72°C; MJ Research Inc.) and normalized to the geometric mean of Ywhaz (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) and Ube2L3 (ubiquitin conjugating enzyme E2L3) as described previously (). Primer sequences (5′ to 3′) are as follows: Ywhaz forward: CGTTGTAGGAGCCCGTAGGTCAT, Ywhaz reverse: TCTGGTTGCGAAGCATTGGG; Ube2L3 forward: AGGAGGCTGATGAAGGAGCTTGA, Ube2L3 reverse: TGGTTTGAATGGATACTCTGCTGGA; Foxp3 forward: ACTCGCATGTTCGCCTACTTCAG, Foxp3 reverse: GGCGGATGGCATTCTTCCAGGT; Tmem49 forward: GCCTGTGCTTCTATTCCAAACC, Tmem49 reverse: GAAAGTCACCATCTGCTCCA; Tmem49 3′UTR forward: GTTGAATCTCATGGCAACAGCAGTC, Tmem49 3′UTR reverse: AAGGGCTCCAAGTCTCACAAGACA; and Tmem49 intron 11 forward: AGAACCAGCAGATGTGTAGGCAGC, Tmem49 intron 11 reverse: GGGAAGAGGACCTAAACTCTGAGAGC.
For quantitative real-time RT-PCR of miRNAs, gene-specific reverse transcription was performed for each miRNA using 10 ng of low molecular weight RNA, 1 mM dNTPs, 50 U MutliScribe reverse transcriptase, 3.8 U RNase inhibitor, and 50 nM of gene-specific RT primer samples using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems). 15-μl reactions were incubated for 30 min at 16°C, 30 min at 42°C, and 5 min at 85°C to inactivate the reverse transcriptase. Real time RT-PCR reactions (1.35 μl of RT product, 10 μl TaqMan 2x Universal PCR master Mix, No AmpErase UNG [Applied Biosystems], and 10 μl TaqMan MicroRNA Assay Mix containing PCR primers and TaqMan probes) were run in triplicates at 95°C for 10 min followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Expression values were normalized to miR-17-5p. Gene-specific RT primers and TaqMan MicroRNA Assay Mix were from the TaqMan MircoRNA Assays Human Panel Early Access kit (Applied Biosystems). Northern blots were performed as described previously ().
Tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin and with periodic acid-Schiff for microscopic examination.
Fig. S1 shows the kinetics of miRNA expression after the activation of naive CD4 T cells. It is available at . |
To test whether CD40 has any role in the development of mammary carcinomas, we have introduced the rat HER2/neu oncogene into the CD40-KO background and evaluated tumor onset and progression. The analysis of >50 CD40-KO/NeuT mice showed slower tumor onset, reduced multiplicity (, left), and decreased total tumor volume (, right) compared with BALB/NeuT mice. Whole mount analysis of mammary glands from BALB/NeuT and CD40-KO/NeuT mice from 6 wk, when atypical hyperplasia is evident, until 17 wk, when palpable invasive carcinomas appear, confirmed the delayed carcinogenesis in the KO strain (). Comparison of whole mount samples from wild-type and CD40-deficient mice at different time points excluded any macroscopic difference in normal mammary gland development between the two strains, and therefore the possibility that a slower mammary gland development could account for the reduced tumor growth in CD40-KO/NeuT mice (Fig. S1, available at ).
To explain the reduced tumorigenicity of CD40-KO/NeuT mice, we tested two hypotheses: a weaker tolerance to tumor-associated antigens because of the reduced number of T reg cells characterizing CD40-KO mice (), and an impaired tumor angiogenesis because of the lack of CD40 on ECs. A single experimental approach that allows discrimination between the two hypotheses is BM transplantation (BMT). CD40-null donors, transferred into BALB/NeuT recipients, were expected to reproduce the CD40-KO/NeuT tumor phenotype if cells of BM origin, including T reg cells, are actively involved in the process. Complementary results could be obtained by the transfer of BALB/c BM into CD40-KO/NeuT mice to restore the BALB/NeuT morbidity. 5–6-wk-old mice, which have completed mammary gland development, were transplanted. At this time point, no sign of carcinomas are detectable. An earlier time point has been avoided because of radiation-induced impairment of normal mammary tree development.
Although CD40-KO > BALB/NeuT chimeras showed a reduced number of T reg cells, similar to that of CD40-null and CD40-KO/NeuT mice (), tumor onset and multiplicity remained identical to that of BALB/NeuT mice (, left). Accordingly, BALB/c > CD40-KO/NeuT chimeras showed the same number of T reg cells present in the periphery of BALB/c and BALB/NeuT mice (), without any effect on tumor onset and multiplicity (, right). Because donor BM did not modify the host tumor phenotype, we excluded a preeminent immunological role of CD40 in this tumor model and therefore focused on the second hypothesis testing whether CD40 is involved in tumor angiogenesis.
Despite the fact that the contribution of BM-derived endothelial progenitor cells to tumor endothelium is still debated, the most accepted theory is that tumor vessels originate mainly from host, rather than BM-derived, cells (). Indeed, (FVBTieβ-galxBALB/NeuT)F1 double transgenic mice and (FVBTieβ-galxBALB/c)F1 > BALB/NeuT chimeras show all blood vessels, or only vessels of BM origin, respectively, stained in blue because the β-galactosidase gene is placed under the endothelial Tie2 promoter (). Comparison of their tumor vasculature indicates that, in our tumor model, tumor-associated vessels derive mostly from host ECs rather than from donor BM-derived EC precursors (Fig. S2, available at ).
We first assessed CD40 expression on mouse ECs in vitro using the 1G11 EC clone isolated from mouse lung tissue (), which presents a typical endothelial phenotype, forms contact-inhibited monolayers on gelatin and capillary-like “tubes” in Matrigel, and can be kept in culture for several passages without loosing their endothelial characteristics (). 1G11 cells express low level of CD40 (25–28% positive cells) that is up-regulated by TNF-α plus IFN-γ treatment (50–55% positive cells). Triggering of CD40 receptor on 1G11 cells by recombinant sCD40L is able to up-regulate VCAM-1 expression on their surface and induce the production of vascular endothelial growth factor (VEGF; Fig. S3, available at ).
To verify the expression of CD40 on ECs in vivo, we have used a recently developed, very sensitive method, the dual radiolabeled mAb technique, which allows the quantification of EC-associated proteins with a precision and sensitivity not previously possible using immunohistochemical procedures (, ). Indeed, although immunohistochemistry failed to detect CD40 expression on mouse ECs, the dual radiolabeled mAb technique demonstrated the presence of CD40 on vessels of different organs, mainly the kidney, lung, pancreas, and in the tumor masses collected from BALB/NeuT mice (). Although the abnormal, irregular, and leaky vessels characterizing tumor vasculature could allow a small fraction of CD40 Ab to extravasate and bind to some CD40-expressing tumor-infiltrating cells, such as macrophages, the short pulse with the Ab and the extensive washing minimize such contribution. Indeed, the ratio of CD40/platelet EC adhesion molecule (PECAM)-1 in tumor vasculature was one of the highest, confirming CD40 expression on tumor vessels as part of their activate/inflamed state. As expected, no CD40 expression was detected on either normal or tumor vessels from CD40-KO/NeuT mice (not depicted).
sCD40L and Ab to CD40, but not isotype control Ab, are able to recruit ECs in Matrigel plugs implanted into CD40-sufficient, but not CD40-deficient, mice. On the contrary, bFGF recruited ECs into Matrigel plugs regardless of the CD40 genotype of recipient mice (). Immunostaining with Ab to CD31 confirmed the endothelial nature of some cells infiltrating the Matrigel plugs (). The number of CD40-recruited CD31 cells was significantly different in Matrigel plugs collected from CD40-sufficient and -deficient mice ().
A role of CD40 in tumor vessel formation and/or organization was confirmed by immunohistochemical analysis of tumors collected from the two strains at different sizes and at different time points. Staining with Ab to CD31 showed a different organization of tumor-associated vessels (, top). Tumors from BALB/NeuT mice present large vessels surrounding the tumor lobular structures and a few tiny vessels inside the parenchyma, whereas those from CD40-KO/NeuT mice lack all large vessels while numerous tiny vessels are dispersed in the tumor parenchyma. Accordingly, tumors from BALB/NeuT mice show dense collagen type IV (, bottom) and laminin (not depicted) around the large vessels and in the stromal septa defining the tumor lobules. This feature is much less evident in CD40-KO/NeuT tumors, which are characterized by undefined lobules with thin stromal septa.
Aside from the well-known expression of CD40L on activated CD4 T cells (), this molecule has also recently been described on human platelets, which release its soluble form (sCD40L) upon activation (, ). Because of the paucity of CD4 T cells infiltrating BALB/NeuT tumors (), and their virtual absence in early lesions (not depicted), we tested whether platelet-released sCD40L may contribute to the development of BALB/NeuT tumors.
First, we confirmed the expression of CD40L on activated mouse platelets because this evidence is lacking in the current literature. shows the presence of CD40L on the surface of thrombin-activated, but not resting, platelets. The adenosine diphosphate (ADP) receptor antagonist, clopidogrel (), is an anti-platelet drug with anti-aggregating properties. Among its actions, it inhibits ADP-induced CD40L expression on the platelet surface (, ). Therefore, to support the hypothesis that platelets might provide CD40L for interaction with CD40-expressing tumor ECs, we treated BALB/NeuT mice from an early age (4 wk) with clopidogrel, given chronically in the drinking water. The inhibitory activity of clopidogrel on platelet activation was confirmed by FACS analysis showing the inhibition of CD62P up-regulation on platelets stimulated in vitro with thrombin (Fig. S4, available at ). In addition, immunostaining of tumor vasculature from clopidogrel-treated and -untreated mice with Ab to CD41 shows dispersed or in blood clot–aggregated platelets, respectively ().
Clopidogrel treatment effectively decreased tumor multiplicity and size in BALB/NeuT mice () to the level of CD40-KO/NeuT mice. Because clopidogrel has additional effects other than inhibiting CD40L expression, we also treated CD40-KO/NeuT mice to evaluate any effects of clopidogrel other than inhibition of CD40L expression and therefore of CD40 triggering. Results show that clopidogrel had no significant effect on tumor growth in CD40-KO/NeuT mice (), indicating that, in our tumor system, its effect is mainly through the inhibition of platelet CD40L.
To study the role of CD40 in mammary carcinogenesis, we introduced the mutated rat oncogene, driven by mouse mammary tumor virus promoter, into a CD40-null background. Because CD40 is crucial in mounting an immune response (, ), CD40-KO/NeuT mice might have been expected to be more tumor prone than their CD40-sufficient counterpart. On the contrary, CD40-KO/NeuT mice showed delayed tumor onset and reduced multiplicity compared with BALB/NeuT mice, data confirmed by whole mount analysis of mammary glands from the two strains.
Two hypotheses were formulated to explain such an unexpected phenotype: a weaker tolerance to tumor-associated antigens because of the reduced T reg cell number in CD40-KO mice (), or an impaired tumor angiogenesis because of the lack of CD40 on ECs. Any possible direct role of CD40 on tumor cells was excluded because these tumors do not express it, neither in vitro nor in vivo (not depicted).
The spleen of CD40-KO mice has such a reduced number of CD4CD25 T reg cells to induce autoimmunity if transferred into nu/nu mice (). Indeed, we found a 50–80% reduction of T reg cells in the peripheral blood, spleen, and thymus of CD40-KO mice compared with normal BALB/c mice of the same age. Although CD40-KO T reg cells were equally capable of suppressing CD4CD25 cell proliferation as their wild-type counterpart (), it might be possible that their low number might allow a response to tumor-associated antigen, otherwise ignored by the immune system, thus explaining the milder tumor phenotype of CD40-KO/NeuT mice.
Supporting the second hypothesis are several reports involving CD40 in EC activation and proliferation (, –), at least in humans. It has been shown that CD40 engagement on human ECs induces the expression of several angiogenic factors in vitro and promotes angiogenesis in vivo (, ). Interestingly, up-regulation of CD40 has been observed in tumor vessels of renal carcinomas and Kaposi's sarcoma (). A Kaposi's sarcoma cell line engineered to release a soluble form of CD40, as decoy receptor, when injected s.c. into SCID mice develops significantly smaller tumors with reduced vascularization compared with the nontransduced counterpart ().
Both hypotheses have been challenged at the same time using BMT experiments in which CD40-KO BM was transferred into BALB/NeuT recipients and BALB/c BM into CD40-KO/NeuT mice. If CD40 expressed on cells of BM origin was involved, BM replacement was expected to modify host tumor outgrowth. Despite the fact that BMT brought the number of recipient T reg cells to the donor level, mammary carcinogenesis remained unchanged, indicating that CD40 on BM cells did not have any relevant role in tumor development in BALB/NeuT mice. Rather, BMT data are consistent with the second hypothesis, suggesting that, in this experimental tumor model, the main role of CD40 is nonimmunological but likely associated to the angiogenic phenotype that fosters malignancy.
A large body of literature reports on the expression and function of CD40 on human ECs (, –), whereas no clear data are available in the mouse system, except for two reports that—although suggesting a functional role of CD40 in mouse angiogenesis—do not show CD40 expression on ECs (, ). Here, the dual radiolabeled mAb technique showed, in vivo, CD40 expression on normal and tumor blood vessels, and the functional activity of CD40 on mouse ECs has been demonstrated, both in vitro and in vivo, using a primary EC line and Matrigel implantation assay, respectively.
Accordingly, tumors from BALB/NeuT mice have large and well-structured vessels delimiting the tumor lobular structures and a few small vessels inside the parenchyma, whereas those from CD40-KO/NeuT mice have only numerous tiny vessels dispersed in the tumor parenchyma, while lacking all main large vessels. Thus, BMT experiments, Matrigel assay, in vitro and vivo detection of CD40 on both normal and tumor-associated ECs, and immunohistological analysis of the tumor vasculature concur to demonstrate a role of CD40 in tumor angiogenesis.
Such an effect of CD40 on tumor vasculature and, consequently, on tumor growth, is relevant in the BALB/NeuT tumor model, in which tumor development is a very slow process. On the other hand, in transplantable models of tumor cell lines derived from the same BALB/NeuT mice, such an effect is undetectable, and tumors, which develop very rapidly in 2–3 wk, are even more aggressive in CD40-deficient than in wild-type mice (not depicted). This evidence suggests that in very rapid transplantable models, probably, is the immunological, antitumoral role of CD40 that prevails over the pro-tumoral angiogenic effect we see in the slow and more “physiological” transgenic model. Moreover, considering the redundancy of the factors involved in tumor angiogenesis, it is very likely that EC activation by CD40 triggering is an early event, whereas subsequent angiogenic stimuli can render its effect dispensable. This view is consistent with the delayed onset but late progression of some tumors in the CD40-null background.
The search of the cells providing the ligand for CD40 triggering is quite restricted. CD40L is mainly expressed on activated CD4 T cells () but is also present on human platelets, which upon activation release its soluble form (sCD40L; references and ). Human platelets up-regulate CD40L expression very rapidly after activation in vitro and during thrombus formation in vivo (). Platelet-derived CD40L induces ECs to express tissue factor () and adhesion molecules, as well as the secretion of chemokines, all molecules responsible for the recruitment and extravasation of leukocytes at the site of injury. Moreover, an increased serum level of sCD40L has been detected in patients with lung cancers () and bone tumors ().
There is increasing evidence that platelets may participate to tumor growth by contributing to the metastatic process (, ), protecting tumor cells from immune surveillance (, ) and regulating tumor cell invasion and angiogenesis (–). Platelet granules contain a variety of factors, such as VEGF, TGF-β, thrombin, and fibrinogen, which are secreted upon platelet activation. Many of these factors have been implicated in various steps of tumor progression and metastasis; indeed, being the tumor vasculature leaky, platelets may come in contact with the tumor and deposit in situ several of these angiogenic factors, which in turn can promote tumor vascularization.
In light of this evidence, and considering that BALB/NeuT tumors are virtually lacking infiltrating CD4 T cells, especially at early phases of transformation (21 and unpublished data), we have focused our attention on platelets as the likely source of CD40L. Once confirmed that CD40L is expressed on activated mouse platelets, and that platelets are present in the vessels of BALB/NeuT tumors, we functionally tested their involvement in the tumorigenic process. The ADP receptor antagonist clopidogrel () is an anti-platelet drug that, among its actions on platelet activation, has been found to specifically inhibit ADP-induced CD40L expression on the platelet surface (). We used clopidogrel to test whether platelets, and likely CD40L expressed by them, may have any role in tumor development in our model by treating young BALB/NeuT mice chronically. We hypothesized that clopidogrel, by inhibiting CD40L expression on the platelet surface, could delay/reduce tumor outgrowth in BALB/NeuT mice to the rate shown by CD40-KO/NeuT animals.
Because the clopidogrel effect on platelets is wider than inhibiting CD40L expression, we also treated CD40-KO/NeuT mice to distinguish the effect due to CD40 engagement from all other CD40-independent effects. CD40-KO/NeuT mice treated or not with clopidogrel did not show any statistically significant difference, whereas a highly significant difference was observed between treated and untreated BALB/NeuT mice. These results indicate that the major effect of clopidogrel is on the contribution of CD40L, whereas other effects on platelets are less involved in our system, especially if affecting other players that are redundant in the angiogenic process. For example, VEGF, one of the key molecules in the angiogenic process, is already released by the mammary tumors ().
The BM origin of CD40L donors was confirmed with BMT experiments using CD40L-KO (C57BL/6 background) donors to transplant lethally irradiated transgenic (C57BL/6×BALB/NeuT)F1 mice. In the CD40LKO > NeuT chimeras, tumor development is delayed compared with C57BL/6 > NeuT control chimeras (Fig. S5, available at ).
In conclusion, our results sustain the hypothesis of using anticoagulants in cancer therapy to prevent platelet interaction with tumor vasculature (), and highlights CD40/CD40L interaction as an additional mechanism that platelets use to promote neo-angiogenesis. At the same time, our findings, revealing an underestimated role of CD40 in fostering tumor neo-angiogenesis, raise a concern with using Abs to CD40 to enhance antitumor immune responses. Stimulation of CD40 may be a double-edged sword, and therefore a careful evaluation of the pros (antitumor effect by means of APC activation) and cons (angiogenesis promotion through EC activation) of CD40 triggering is needed in the design of an immunotherapeutic approach if prophylactic () or directed to incipient tumors.
BALB/cAnNCrl mice were purchased from Charles River Laboratories. CD40-KO mice () were provided on a BALB/c background by L. Adorini (Bioxell, Milan, Italy). Congenic, female BALB/NeuT mice have been described elsewhere (, ). The CD40-KO/NeuT strain has been obtained by introducing the HER2/neu transgene into CD40-KO mice. CD40 deficiency has been tested by FACS analysis on PBMCs using an anti-CD40 FITC-conjugated mAb (clone 3/23; BD Biosciences). The presence of the HER2/neu transgene has been checked by PCR on tail DNA as described previously (). Mice were bred and maintained at the Istituto Nazionale Tumori and treated according to the European Union guidelines. Animal studies were approved by the Animal Ethical Committee appointed by the Istituto Nazionale Tumori.
Whole mount preparations were performed as described at . Digital photos were acquired with a Nikon Coolpix 995 (Nital SpA) mounted on a stereoscopic microscope (MZ6; Leica).
5–6-wk-old mice were lethally γ-irradiated with 900 cGy, and BMT was performed as described previously ().
The analysis of T reg cell numbers was performed by cytofluorimetry with anti–CD4-FITC and anti–CD25-PE Ab (both from BD Biosciences). Percentage of T reg cells was calculated on the total number of CD4 cells.
The mAbs used for in vivo assessment of CD40 and PECAM-1 expression were 3/23 for mouse CD40, MEC 13.3 against mouse PECAM-1 (both from BD Biosciences), and P-23, a nonspecific, nonbinding murine IgG1 directed against human P-selectin (provided by D.C. Anderson, Pharmacia-Upjohn, Kalamazoo, MI). The specific binding (3/23 and MEC 13.3) and nonbinding (P23) mAbs were labeled with I and I, respectively (Du Pont-New England Nuclear). Mice were anesthetized intramuscularly with 150 mg/kg ketamine and 7.5 mg/kg xylazine. The right jugular vein and right carotid artery were cannulated with polyethylene tubing (PE-10). To measure CD40 or PECAM-1 expression, a mixture of I-labeled binding mAb (either 20 μg anti-CD40 or 10 μg anti–PECAM-1) and 0.5–5 μg of nonbinding I mAb (adjusted to ensure a total 131I injected activity of 500,000 ± 100,000 cpm), was injected through the jugular vein catheter (total volume 200 μl). Blood samples (200 μl) were obtained from the carotid artery catheter 5 min after injection of the mAb mixture for measurement of plasma I and I activity. Thereafter, an isovolemic blood exchange was rapidly performed by perfusion with 6 ml of bicarbonate-buffered saline through the jugular vein catheter with simultaneous blood withdrawal through the carotid artery catheter. This was followed by perfusion of 15 ml of bicarbonate-buffered saline through the carotid artery catheter after severing the inferior vena cava at the thoracic level. Organs were harvested and weighed before radioactivity measurements. The method for calculating CD40 and PECAM-1 expression has been described previously ().
Matrigel plug assay has been performed in BALB/c and CD40-KO mice by s.c. injection of growth factor–reduced Matrigel (BD Biosciences) containing 25 ng/ml bFGF (R&D Systems), 40 μg/ml anti-CD40 mAb (clone FGK; Qbiogene), isotype-matched control mAb (rat IgG2a; BD Biosciences), 500 ng/ml of msCD40L plus enhancer (CD40L soluble Set; Qbiogene) or without any additive. 6–10 d later, animals were killed and Matrigel plugs were recovered in OCT for immunostaining with rat anti–mouse mAb CD31 (see below), or fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned (5 μm), and stained with hematoxylin and eosin. The percentage of CD31 cells is calculated over the total number of cells recruited in the external area of the plugs (very few cells are present in the most inner part of the plug), and the mean of 10 fields per each sample is calculated.
Tumor samples were embedded in OCT compound, snap frozen, and stored at −80°C. Immunohistochemical analysis on 5-μm cryostat sections was performed as described previously (). The following Abs have been used: rat anti–mouse mAb CD31, rabbit polyclonal Ab anti–mouse collagen type IV (Chemicon), rat anti–mouse mAb Laminin (Chemicon), rat anti–mouse CD41 (Integrin IIb chain; BD Biosciences), and biotinylated goat anti–rat or anti–rabbit IgG as secondary Ab. Avidin–peroxidase complex (Sigma-Aldrich) was used and antigens were revealed with 3,3′-diaminobenzidine (Sigma-Aldrich) according to the manufacturer's instructions.
BALB/NeuT and CD40-KO/NeuT mice have been treated chronically, from 4 wk of age, with clopidogrel (Plavix) given continuously via the drinking water at a concentration of 0.25 mg/ml (equivalent to an oral dose of ∼30 mg/kg/day). The inhibitory effect of clopidogrel treatment on platelet activation was assessed by FACS analysis with anti-CD62P Ab on PRP obtained from treated and untreated mice, restimulated in vitro with thrombin (Fig. S4).
Data were expressed as the mean plus SD or SE. Differences between groups were analyzed for statistical significance by means of an unpaired test, with P < 0.05 as significant cutoff.
Fig. S1 illustrates comparative whole mount analysis of mammary glands from BALB/c and CD40-KO mice at different time points. Fig. S2 shows X-gal staining of tumor vessels in double transgenic (FVBTieβ-galxBALB/NeuT)F1 mice in comparison with BALB/NeuT mice transplanted with (FVBTieβ-galxBALB/c)F1 BM cells (FVBTieβ-galxB/c > BALB/NeuT). Fig. S3 shows functional CD40 expression on mouse 1G11 ECs. Fig. S4 shows the efficacy of clopidogrel treatment in inhibiting platelet activation. Figs. S1–S4 are available at . |
To address the question of whether or not platelets express TF, we first determined if platelets contained TF mRNA using highly purified platelet preparations that were isolated from healthy volunteers (reference ; Supplemental Materials and methods, available at ). The leukocyte-depleted preparations did not express CD45, PSGL-1, or CD14 (Fig. S1, available at , and unpublished data). Unexpectedly, we found that stimulated, but not quiescent, human platelets contain TF mRNA (); this was confirmed by amplification of the entire TF message and subsequent cloning of the PCR product (Fig. S2, available at , and unpublished data). We did not detect the alternatively spliced variant of TF () in stimulated platelets under the conditions of these experiments; the variant was detected, however, in HL60 myeloid leukocytes or resting primary human monocytes ( and unpublished data).
We surmised that unstimulated platelets contain intronic-rich TF transcripts, a feature that can prevent translation of the corresponding protein until an appropriate signal induces splicing and assembly of the mature mRNA (). We designed primer sets that flank intron four and found that freshly isolated platelets predominantly contain TF pre-mRNA (, see , and Fig. S3 A, ), a finding that was consistent in >40 subjects (unpublished data).
We next determined if platelets activated by fibrinogen and thrombin splice TF pre-mRNA into a mature transcript. Splicing of TF pre-mRNA was detected at 5 min, neared completion by 1 h, and was sustained for at least 4 h after the platelets were activated (). Other agonists such as ADP, collagen, or thrombin also induce TF pre-mRNA splicing in suspended platelets (Fig. S3). We have recently demonstrated that anucleate platelets possess a functional spliceosome and can splice pre-mRNAs when activated (), establishing a mechanism for this sequence of events (). Cloning and sequencing of the unspliced and spliced PCR products confirmed that they were TF (unpublished data). To unequivocally demonstrate the cell source of pre- and mature TF mRNA species, we screened for the transcripts in individual megakaryocytes, proplatelets, and mature platelets. TF pre-mRNA was present in the cytoplasm of hematopoietic stem cell–derived human megakaryocytes and proplatelets (). Consistent with detection of intronic-rich message in platelet precursors, we found TF pre-mRNA in freshly isolated platelets from circulating human blood (, top left). We also found that activated platelets express TF mRNA (, top right), confirming that the mature message is derived from platelets.
Since stimulated platelets use their splicing machinery to produce mature TF mRNA, we asked if TF-dependent procoagulant activity increased in activated cells. Freshly isolated platelets, at cell numbers (2 × 10 total) that approximate those found in 5–10 ml of whole blood, did not possess significant levels (P < 0.05) of procoagulant activity (). In contrast, procoagulant activity was markedly increased as early as 5 min after platelets adhered to fibrinogen in the presence of thrombin () and continued to accumulate in platelets and platelet-derived microparticles in a time-dependent fashion (). Platelets activated in suspension with ADP, collagen, or thrombin also accumulated TF-dependent procoagulant activity (unpublished data). We consistently observed that activated platelets possessed higher procoagulant activity than quiescent platelets in samples from different donors, but the magnitude was variable among subjects, ranging from a 2.8- to a 15.3-fold increase over baseline (mean increase over baseline 7.7 ± 2.0). In contrast to activated platelets, monocytes (5 × 10) stimulated with fibrinogen and thrombin did not generate appreciable procoagulant activity, although they did respond to lipopolysaccharide (unpublished data).
Next, we analyzed the protein by immunocytochemistry and observed TF on the surfaces of activated platelets that had adhered and spread on immobilized fibrinogen in the presence of thrombin but not on quiescent platelets (). Staining was detected on the surface of all the platelets, consistent with the detection of TF mRNA in every cell (, top right).
Deletion of factor VIIa from the activity assay or incubation of platelets with a neutralizing anti-TF antibody significantly (P < 0.05) reduced procoagulant activity in stimulated platelets (). This activity assay, however, evaluates factor Xa generation in the presence of supraphysiologic levels of exogenous factor VIIa (). Therefore, we determined if platelet-derived TF decreased plasma clotting times.
). Clotting was significantly delayed in the presence of an inhibitory anti-TF antibody (), indicating that TF generated by activated platelets is capable of accelerating in vitro clot formation in humans. Collectively, the studies depicted in demonstrate that bioactive TF protein accumulates in platelets adherent to fibrinogen in the presence of thrombin. The role of platelet-derived TF in the propagation and stabilization of platelet clots in vitro and in vivo will require further studies using both human and murine thrombosis models.
The intracellular signaling pathways that control TF pre-mRNA splicing and activity in platelets are not known (). In nucleated cells, serine-arginine (SR)–rich proteins regulate splicing, and we recently found that human platelets contain the SR family member SF2/ASF (). Thus, we considered that platelets possess critical upstream kinases that regulate SF2/ASF activity and focused on Clk family members because one of them, Clk1, contains an N-terminal region enriched in arginine-serine dipeptides (RS) that interacts with SF2/ASF (). We found Clk1 protein in the cytoplasm of mature megakaryocytes (unpublished data), in proplatelets that extend from the megakaryocytes (, arrows), and in quiescent circulating platelets from human blood (, arrows). In activated platelets, Clk1 was distributed to the tips of F-actin stress cables (). Intracellular redistribution of Clk1 resembles the accumulation of vinculin in focal adhesion complexes of platelets that are spread and activated on immobilized fibrinogen (unpublished data and reference ). Preliminary screens for other family members (Clk2, 3, and 4) were negative (unpublished data), suggesting that Clk1 is the primary Clk in mature, circulating platelets.
In nucleated cells, Clk1 directly phosphorylates SF2/ASF and alters the intracellular localization patterns of SR proteins (). Therefore, we captured endogenous platelet Clk1 by immunoprecipitation and determined if it regulates SF2/ASF phosphorylation. Clk1 from activated platelets markedly increased SF2/ASF phosphorylation (). Increased SF2/ASF phosphorylation was not seen when control IgG was used as the immunoprecipitating reagent (Fig. S4 A, available at ).
A benzothiazole compound, Tg003, suppresses Clk1-catalyzed phosphorylation and thereby inhibits SF2/ASF-dependent splicing of in vitro–transcribed pre-mRNAs in immortalized cell lines (). In stimulated platelets Tg003, but not its vehicle, suppressed Clk1-dependent SF2/ASF phosphorylation (), consistent with previous characterization of the inhibitor in other cells ().
We next asked if interruption of signaling from Clk1 to SF2/ASF modulates activation-dependent splicing and found that Tg003 prevented processing of TF pre-mRNA in activated platelets (). This Clk1-dependent splicing inhibitor also blocked the expression of TF protein in activated platelets and platelet-derived microparticles (). Consistent with its effect on protein, Tg003 significantly (P < 0.05) reduced TF-dependent procoagulant activity in stimulated platelets () and delayed the onset of plasma clot formation (). Puromycin, an inhibitor of mRNA translation, also significantly (P < 0.05) reduced TF-dependent procoagulant activity demonstrating the increases were caused by de novo protein synthesis (Fig. S5, available at ).
Although Tg003 blocked SF2/ASF phosphorylation () and the expression of TF protein in activated platelets (), it had no effect on other platelet functional responses that included cellular adherence and spreading, actin polymerization, organization of β-tubulin, or the redistribution of Clk1 to focal adhesion complexes (Fig. S6, available at ). These data suggest that the Clk1 signaling pathway primarily interfaces with the splicing machinery in platelets.
Pre-mRNA splicing and regulated translation of processed mRNAs are novel functions that allow activated platelets to alter their transcriptome and proteome in response to stimulation (, , ). IL-1β was the first platelet product discovered to be synthesized in this fashion (). In this report, we demonstrate that platelets also use their splicing machinery to control the expression of TF and identify a new intermediate, Clk1, in the signaling pathway leading to TF synthesis. Our results indicate that quiescent platelets contain TF pre-mRNA but do not express significant levels (P < 0.05) of TF protein or activity under basal conditions. In contrast, activated platelets express both TF mRNA and bioactive TF protein. Pre-mRNA splicing and translation of TF message into protein is observed as early as 5 min after activation and is sustained for at least 4 h. The time scale of this response suggests that platelet-derived TF sustains the growth of the thrombus and increases its stability by enhancing fibrin deposition (). Formation of a stable thrombus is essential for hemostasis and promotes wound healing at the site of vascular injury. Recent studies have found that mice with deficiencies in TF or fibrinogen form unstable thrombi (, ). Tissue factor also modulates inflammation and angiogenesis (, ), indicating that it affects prolonged functional responses. At present the relative contributions of platelet-derived TF and TF-positive microparticles to thrombus formation under different pathologic conditions are not known. Although on a per-cell basis lipopolysaccheride-stimulated monocytes generate greater amounts of TF than platelets (unpublished observations), the number of circulating platelets far exceed (i.e., ∼500–1,000-fold greater) the number of monocytes per volume of blood. In the case of platelets, our studies are the first to demonstrate that Clk1 modulates TF gene expression and suggest that splicing of TF pre-mRNA may be a potential therapeutic target in syndromes of disordered coagulation.
CD34 stem cells were isolated from human umbilical cord blood and were differentiated into megakaryocytes that produce proplatelets using methods that we have previously described (). Leukocyte-depleted human platelets were isolated from healthy volunteers using previously described methods (, ). The human studies were approved by the University of Utah Internal Review Board (IRB approval numbers 392 and 11919).
For most of the studies, primers that targeted sequences in exon four (5′-CTCGGACAGCCAACAATTCAG-3′) and five (5′-CGGGCTGTCTGTACTCTTCC-3′), and thus spanned intron four, were used to determine endogenous splicing of TF pre-mRNA in platelets. Indirect in situ hybridization or direct in situ PCR was used to detect TF pre-mRNA in megakaryocytes and platelets as previously described ().
Detailed strategies for protein detection by flow cytometry, Western blot analysis, and immunocytochemistry have been previously published (, ).
TF-dependent procoagulant activity was calculated with an Actichrome TF assay (American Diagnostica) as previously described ().
Platelets were left quiescent or activated in the presence or absence of Tg003. Platelet membranes were isolated and added to human plasma (37°C), and clotting was initiated with CaCl as previously described ().
Clk1 activity in platelets was determined using an immune complex kinase assay. An antibody against Clk1 was used for immunoprecipitation of the protein. Nonimmune rabbit IgG was used as a control, and in select experiments recombinant SF2/ASF was removed from the assays to screen for nonspecific incorporation of radiolabeled phosphate (Fig. S4, A and B). Kinase assays were performed by addition of recombinant SF2/ASF (Protein One) in the presence of γ-[P]ATP (MP Biomedicals). At the end of this incubation period, the agarose beads and immune complexes were removed by centrifugation, and the unbound sample, which contained SF2/ASF, was resolved by SDS-PAGE.
ANOVA was conducted to identify differences that existed among multiple experimental groups. If significant differences were found, a Student-Newman-Keuls post-hoc procedure was used to determine the location of the difference. For all of the analyses, P < 0.05 was considered statistically significant.
Supplemental Materials and methods details methods for cellular activation, in situ hybridization, and protein detection. Fig. S1 provides data from flow cytometric analysis for CD45 or control IgG in leukocyte-depleted platelets or monocytes. Fig. S2 shows that activated platelets express full-length, mature (mHTF) human TF mRNA. Fig. S3 shows TF pre-mRNA splicing in human platelets due to activation with ADP, collagen, and thrombin in suspension. Fig. S4 provides additional data regarding the specific Clk1-mediated SF2/ASF phosphorylation. Fig. S5 shows that the inhibition of translation prevents activated platelets from generating bioactive tissue factor. Fig. S6 shows that inhibition of Clk1 does not adversely affect platelet functional responses. Online supplemental material is available at . |
Though we observed no gross differences in the tissue composition of Alox15 mice compared with wild-type controls at 6–8 wk of age (Fig. S1, available at ), we noted a small but distinct increase in the death rate of homozygous knockout animals as they aged compared with wild-type controls (). Furthermore, examination of outwardly healthy Alox15 mice older than 8 wk revealed varying degrees of splenomegaly with 100% penetrance compared with wild-type controls (). Early morbidity and mortality was associated with increasingly severe splenomegaly ().
Analysis of splenic architecture revealed a remarkable decrease in the number of follicles and disrupted compartmentalization of the red and white pulp in the asymptomatic Alox15 compared with wild-type mice (). Strikingly, severe splenomegaly in moribund Alox15 mice was characterized by complete loss of splenic compartmentalization (). Phenotypic analysis of wild-type and Alox15 splenocytes by flow cytometry revealed a selective increase in the Mac-1/GR-1 (myeloid) population (), whereas differential counts of cytospin preparations of splenocytes from Alox15 mice versus wild-type controls demonstrated no expansion of other cell types (). Bone marrow was also affected, where the numbers of myeloid cells and their CD34/Gr-1 progenitors (), as well as megakaryocytes (), were increased at the expense of erythropoiesis ( and Fig. S2, available at ). Moribund animals regularly demonstrated more than 25% myeloblast cells in the bone marrow by morphology and CD34 expression (thus, these animals are herein referred to as being in blast crisis stage).
Asymptomatic Alox15 mice, whose spleens were at least twice the weight of wild-type controls, as well as all of those in the crisis stage, also displayed blood leukocytosis and basophilia, with myeloblasts apparent in the moribund mice (). Furthermore, though the lymph nodes were not grossly enlarged, they exhibited progressive hypercellularity and morphological changes, with pseudo-Gaucher cells present () (). Immunohistochemistry revealed a marked increase in the proportion of GR-1 cells in lymph node in both asymptomatic and moribund compared with wild-type animals (). We also detected myeloid infiltrates in the skin of most moribund Alox15 mice that correlated with the development of dermatitis (Fig. S3, available at ), a poor prognostic sign in human CML (). In contrast, other tissues (liver, kidney, lungs, and heart) displayed no evidence of pathology (unpublished data).
We observed significantly increased proliferative capacity in splenic Alox15 myeloid cells based on cell cycle analysis (). In addition, the percentage of apoptotic cells was decreased in Alox15 compared with wild-type cells, indicating enhanced survival of Alox15 cells (). We then investigated the relevance of 12/15-LO to human MPD. Consistent with the potential role of 12/15-LO in suppressing human disease, we found that human Bcr-Abl leukemia K562 cells (), despite being derived from the myeloid lineage (which generally expresses 12/15-LO), do not express detectable levels of 12/15-LO protein (). However, forced expression resulted in levels of 12/15-LO similar to those found in mouse macrophages () and led to a decrease in cell survival and proliferation that could be overcome by treatment with the 12/15-LO inhibitor PD146176 (). Thus, 12/15-LO can suppress human as well as mouse MPD.
To assess whether the MPD in Alox15 tissues fit the classification of a leukemia (), we tested whether the Alox15 phenotype was cell autonomous by transplanting Alox15 cells into syngeneic, nonirradiated wild-type mice. The splenic enlargement and disrupted compartmentalization, as well as the myeloid cell expansion, we observed in Alox15 mice was uniformly recapitulated in wild-type recipients transplanted with crisis stage splenocytes or bone marrow cells as early as 4 wk after transfer (). Though transplant of cells from 10–12-wk-old chronic stage Alox15 mice did not result in overt pathology in wild-type recipients, the cells from these mice were transplantable to mice with severe combined immunodeficiency in 4 out of 6 cases (). Moreover, though it was not possible to assess clonality of the transferred cells, persistence of Alox15 cells in the donor mice was confirmed by PCR for the neomycin cassette introduced by the Alox15 targeting vector (). We conclude from these data that Alox15 mice develop a MPD that transitions to a transplantable leukemia in a subset of animals over time.
To address the potential role of lipid mediators in 12/15-LO's impact on myeloid proliferation, we measured the lipid products released ex vivo during whole organ culture of Alox15 versus wild-type spleens. We found that Alox15 mice produced comparable amounts of 15-HETE, lipoxin A, and 5-HETE (a 5-lipoxygenase product) but reduced levels of 12-HETE and 13-HODE compared with wild-type controls (), indicating that 12-HETE and 13-HODE or their intermediates are the most likely lipid products to be involved in 12/15-LO–mediated suppression of leukemogenesis. This is consistent with the observation that a stable analogue of the 12-HpETE derivative, hepoxilin A, can suppress the growth of K562 cells in in vivo models (). Indeed, we found that 12-HpETE displayed a modest ability to suppress viability in Alox15 and K562 cells (). The effects of 12HpETE on viability may, however, be minimized in these studies because of its lability and potential to act in synergy with other 12/15-LO products in vivo.
Others have demonstrated that Abl hyperactivity in myeloid cells, associated with v-Abl or Bcr-Abl, can induce MPD in mice (, ). These studies compelled us to explore whether 12/15-LO expression may prevent MPD by suppressing endogenous Abl activity. To do this, we used concentrations of the Abl inhibitor STI571 sufficient to inhibit endogenous Abl activity. We found that, in contrast to the K562 cells (), Alox15 splenocytes did not undergo cell cycle arrest or apoptosis upon treatment with the Abl inhibitor STI571/imatinib (). We also used the endogenous Abl substrate Crk (), rather than the pathologic Bcr-Abl substrate CrkL, to assess whether endogenous Abl activity was increased, as endogenous Abl may not be capable of phosphorylating CrkL to the degree that Bcr-Abl can (). Nonetheless, we detected levels of phosphorylation of the major Abl substrate Crk that were comparable to the wild type (). Thus, at least in the ex vivo environment, the Alox15 MPD is independent of Abl activity.
Given the importance of ICSBP in MPD (), as well as our previous data demonstrating the ability of 12/15-LO to regulate ICSBP in other contexts (), we hypothesized that 12/15-LO affects hematopoiesis by regulating ICSBP. Consistent with this hypothesis, we found that ICSBP nuclear levels were reduced in splenocytes from Alox15 mice and were barely detectable in splenocytes from mice that had progressed to blast crisis (). Real-time PCR for ICSBP revealed that its transcript levels in chronic stage Alox15 mice were comparable to the wild type, suggesting posttranslational regulation of ICSBP in this phase of the disease. On the other hand, expression of the ICSBP gene was nearly ablated in splenocytes isolated from mice in crisis (). This suggests that ICSBP is differentially regulated between the chronic and crisis stages of Alox15 MPD and that ICSBP expression inversely correlates with disease progression in Alox15 mice, as in the case of CML (). As the decreased ICSBP expression in blast crisis mice was on an RNA level and, thus, caused by the gain of genetic mutations or transcriptional dysregulation that may complicate the interpretation of our experiments, we have focused our efforts in this study on elucidating the posttranslational mechanisms by which 12/15-LO regulates ICSBP in chronic stage Alox15 cells.
To investigate chronic stage Alox15 MPD, it was important to confirm that the decrease in nuclear levels of ICSBP in unfractionated chronic stage Alox15 splenocytes was a reflection of ICSBP dysregulation in chronic stage Alox15 myeloid cells. To do this, we examined the levels of ICSBP in enriched populations. Consistent with the RNA levels we observed in unfractionated spleens, chronic stage myeloid cells expressed similar ICSBP transcripts to wild-type controls (). However, we detected considerably lower nuclear levels of ICSBP protein in the Mac-1/GR-1 splenocyte populations isolated from Alox15 mice compared with those isolated from wild-type mice (). In contrast, the cytoplasmic levels of ICSBP in Alox15 myeloid cells were comparable to the wild type, indicating a nuclear accumulation defect (). The selectively decreased nuclear levels of ICSBP in the myeloid population, compared with the Mac-1/GR-1 population, reinforce the conclusion that the aberrant cells in Alox15 mice are contained within the myeloid compartment, which is consistent with the selective expression of 12/15-LO in the myeloid lineage ().
To ascertain whether the regulation of ICSBP nuclear accumulation by 12/15-LO corresponds with changes in the expression of ICSBP target genes, we measured levels of Bcl-2, an oncoprotein known to be suppressed by ICSBP (, ), in myeloid cells. We observed a dramatic up-regulation of Bcl-2 in the Alox15 myeloid population () that correlated inversely with the nuclear accumulation of ICSBP.
Given the impact of tyrosine phosphorylation events on the ability of ICSBP to serve as a repressor of gene expression (), we postulated that 12/15-LO regulates the nuclear accumulation and function of this transcription factor by influencing its phosphorylation status. Indeed, immunoprecipitation studies revealed an increase in tyrosine phosphorylation of ICSBP in Alox15 spleen (), indicating an important role for 12/15-LO in suppressing ICSBP phosphorylation.
Because the PI3-K pathway can promote Abl-independent CML, and in view of the capacity of 12/15- LO to regulate PI3-K in other settings (), we explored the possibility that the dysregulation of ICSBP in Alox15 cells is PI3-K dependent. Indeed, we found increased levels of phosphorylation of the PI3-K pathway product Akt in bone marrow myeloid cells from Alox15 mice (). Conversely, treatment with the PI3-K inhibitor Ly294002 suppressed ICSBP phosphorylation and restored nuclear levels of ICSBP to that of the wild type (). This led to a correction in Bcl-2 transcript levels in Alox15 myeloid cells and induction of apoptosis in these cells within 24 h (). These data establish a mechanistic link between PI3-K and ICSBP and indicate that 12/15-LO regulates myeloid leukemogenesis by affecting the PI3-K signaling pathway.
This study establishes 12/15-LO as a critical and somewhat unexpected regulator in the suppression of MPD. We also place 12/15-LO in mechanistic context by demonstrating its impact on the phosphorylation and nuclear localization of ICSBP, as well as that the ICSBP dysregulation in and survival of aberrant Alox15 cells is PI3-K dependent.
We found that Alox15 mice older than 10 wk develop MPD with 100% penetrance. In considering a novel mouse model for leukemic disease, it is important to evaluate the preleukemic state, pathology in knockouts as well as controls, and to assess the transferability of the leukemic syndrome (), all of which we have done. However, one should, if possible, also investigate whether the disease is clonal in nature using antigen receptor rearrangement or retroviral insertion sites. Unfortunately, given the strictly myeloid nature of the syndrome in our mice and the fact that these animals are free of retroviral insertions, none of the conventional methods were available to us to establish clonality (). Therefore, though the persistence of transplanted Alox15 cells 10 wk after transfer to wild-type recipients strongly suggests the clonal derivation of these cells, we have opted to make the more conservative conclusion that Alox15 mice develop a MPD that may become leukemic. Further studies, perhaps using a nontransforming retrovirus, will be required to completely elucidate this issue.
Given the indolent nature of the MPD that occurs in Alox15 mice, these animals provide a relatively unique opportunity to study the mechanisms underlying the chronic stage of MPD and its transition to blast crisis. In fact, as with humans with CML, we have found in this study that ICSBP is regulated differently in chronic and crisis stage cells, which appears to affect downstream targets of this transcription factor. In the future, it will be important to distinguish whether the switch to crisis is a result of transcriptional regulation of ICSBP by 12/15-LO or is caused by mutations in this gene. Indeed, it is possible that the occurrence of genetic aberrancies in ICSBP are a prerequisite of blastic transformation given the near uniform reduction in ICSBP transcripts in patients with crisis stage CML (). Thus, further studies to define mechanisms of blastic transformation in indolent models of CML such as Alox15 mice are critical to developing new therapies. Moreover, as we have shown that 12/15-LO is expressed selectively in the myeloid lineage among hematopoietic cells, this pathway may present a selective target for treating MPD.
Curiously, though Alox15 mice were generated nearly a decade ago, the MPD we describe in this manuscript was not previously reported. There are several possible explanations for this. One possibility is that the syndrome in Alox15 may have become more pronounced on the pure C57BL/6 genetic background described in this study, as opposed to the F = 7 animals previously studied. Additionally, the majority of Alox15 mice manifests a degree of splenomegaly that might not be obvious to investigators handling the mice for other purposes and, therefore, who are not focused on the integrity of lymphoid organs. This is less likely in the case of the more dramatic splenomegaly observed in mice in blast crisis, but the incidence of these mice is relatively low (∼15%). Importantly, scientists at the Jackson Laboratory, the vendor of Alox15 mice (see Materials and methods), have recently examined Alox15 mice versus wild-type controls and have observed a similar phenotype of splenomegaly and flow cytometric abnormalities, confirming our findings in an independent facility (Nicholson, A., personal communication). The history of Alox15 mice mirrors the plight of human CML patients who, because of their initially mild symptoms, are often considered healthy for some time before being diagnosed.
We demonstrated that 12/15-LO–mediated suppression of leukemic cell growth is dependent on its enzymatic activity, and our ex vivo data suggest that 12/15-LO is responsible for producing several lipid mediators in vivo, including 12-HETE. Interestingly, others have shown decreased levels of 12-HETE in the bone marrow of humans with CML (). This effect may be caused in part by reduced levels of platelet 12-lipoxygenase RNA levels in CML patients (). The common products between leukocyte and platelet 12-lipoxygenases likely explain the fact that these enzymes are often jointly implicated in disease pathology, as exemplified in the case of osteoporosis (, ). Another reason for the overlapping functions between these two enzymes may be transcellular oxidation products formed between platelets and leukocytes, such as lipoxins and their intermediates (–). Alternatively, other consequences of lipoxygenase activity, such as direct membrane oxidation and reactive oxygen species, may be involved ().
Consistent with the mechanistic links between them, the ICSBP and 12/15-LO–deficient mouse models for human MPD share several characteristics. Indeed, the decreased survival in ICSBP heterozygotes (9%) () is similar to what we observed in 12/15-LO–null mice, which would be expected in the context of the reduced (but not completely ablated) nuclear levels of ICSBP observed in 12/15-LO–deficient animals. Both models also display an initial MPD that transitions to a transplantable leukemia in a subset of animals over time. Additionally, it is important to note that both ICSBP and 12/15-LO are involved in inflammation (–), which may mediate in part their effects on leukemic disease, as many contemporary studies indicate that there may be a stronger connection between inflammation and carcinogenesis than previously recognized (). Finally, mouse ICSBP and 12/15-LO share a high degree of homology with their human counterparts (, ), each are down-regulated in cells from CML patients (, ), and both can suppress human CML growth; therefore, both of these molecules are mechanistically relevant to human pathology. The lack of requirement for Abl activation in the 12/15-LO and ICSBP-deficient models is of particular interest considering the resistance to Abl inhibitors in a substantial proportion of myeloid leukemia patients and the role of the PI3-K pathway in many of these cases ().
PI3-K is activated by cellular stress and various cytokine pathways, promoting cell survival, metabolism, and growth (). Mice deficient in PTEN, a suppressor of PI3-K–mediated Akt activation, succumb to myeloid leukemias (). Moreover, PI3-K inhibitors have been shown to kill imatinib-resistant leukemic cells, underscoring the importance of the PI3-K pathway in CML. However, because activation of PI3-K can be both Abl-dependent and -independent (, ), the PI3-K–dependent nature of the Alox15 syndrome does not readily elucidate whether 12/15-LO may be acting downstream of or in parallel to the Abl signaling cascade.
The role of 12/15-LO in regulating PI3-K signaling, especially in myeloid cells, was previously unknown. However, others have shown that the 12/15-LO products 13()-HODE and 15()-HETE up- and down-regulate, respectively, Akt phosphorylation in prostate cells (), whereas 12/15-LO products uniformly up-regulate Akt phosphorylation in vascular smooth muscle cells (). Thus, a cell-type selective effect of 12/15-LO on PI3-K signaling is apparent and will be of great interest to study in myeloid cells.
We have demonstrated decreased nuclear accumulation of ICSBP in Alox15 myeloid cells, which may be the result of PI3-K–mediated aberrant phosphorylation of ICSBP. In agreement with our study, it has been reported that tyrosine phosphorylation of ICSBP can decrease its direct binding to DNA, thereby limiting its ability to repress gene transcription (). However, this phosphorylation also increases its binding to other transcription factors (), enabling ICSBP to induce gene expression. Indeed, others have shown that phosphorylation of ICSBP on tyrosines 92 and 95 promotes its positive regulation of neurofibromin 1 expression without affecting ICSBP suppressor function, thereby promoting myeloid cell differentiation in vitro (, ). On the other hand, our data suggest that increased tyrosine phosphorylation of ICSBP can also promote MPD, likely through acting on distinct tyrosines. Thus, it will be important in future studies to characterize which tyrosine residues are phosphorylated as a result of 12/15-LO–mediated PI3-K pathway activity. In either case, the increased phosphorylation of ICSBP in our model may either subject it to a more rapid nuclear turnover secondary to decreased DNA binding or directly suppress its nuclear translocation. Our working model of the 12/15-LO–dependent regulation of ICSBP is presented in .
In addition to mouse MPD, several observations signify a potentially important role for 12/15-LO in human CML. 12/15-LO is expressed in human hematopoietic tissues and myeloid cells, its products are both diminished in and can suppress human myeloid leukemia (), and the Alox15 locus maps to a region of chromosome 17p13 implicated in human myeloid leukemogenesis (). Though a few other candidate genes are being considered on chromosome 17, our study represents the first direct evidence of the involvement of a particular locus in this susceptibility region in myeloid leukemia. Thus, it is of great interest to determine whether, as our data and that of others suggest, the Alox15 gene is mutated in human myeloid leukemias. We are currently investigating this possibility.
Further dissecting the mechanism by which 12/15-LO suppresses MPD will uncover novel regulatory pathways in hematopoiesis and may pave the way for the development of additional targeted therapies for the treatment of myeloid leukemias and other cancers.
C57BL/6 and Alox15 mice on a C57BL/6 background (backcrossed 11 generations) were purchased from The Jackson Laboratory and housed and bred in the Wistar Institute Animal Facility. Severe combined immunodeficiency mice were purchased from the Wistar Institute Animal Facility. All animals were treated in accordance with a Wistar Institute protocol approved by the institutional animal care and use committee. K562 cells were purchased from the American Type Culture Collection. Ly294002 and PD146176 were purchased from Calbiochem and Sigma-Aldrich, respectively. STI571 was supplied by Novartis. K562 cells were nucleofected (Amaxa) according to the manufacturer's optimized protocol.
Unperfused fresh spleens were cut into sections in duplicate and stimulated for 20 min with 1 μM ionomycin and 200 nM PMA in serum-free media. Some of the supernatant was used for lipoxin A analysis by ELISA (Oxford Biomedical Research, Inc.). The rest of the supernatant was extracted for analysis by stable isotope dilution normal phase chiral liquid chromatography (LC) coupled with electron capture atmospheric pressure chemical ionization (APCI)/mass spectrometry (MS) as described previously (). In brief, samples were spiked with deuterium-labeled internal standards, adjusted to pH 3 with 2.5 N hydrochloric acid, and extracted with 2 × 4 ml diethyl ether. The organic layer was evaporated to dryness under nitrogen, derivatized with 2,3,4,5,6-pentafluorobenzyl bromide in the presence of diisopropylethylamine, and evaporated to dryness under a stream of nitrogen. Derivatized samples were reconstituted in 100 μl of hexane/ethanol (97:3, vol/vol) and 20 μl was analyzed by LC/electron capture APCI/MS system for analysis. Quantitation was performed by comparison of peak area ratios of the analytes to their relevant stable isotope internal standard and interpolation of area ratios from a standard curve ().
8–10-wk-old wild-type mice were injected via the tail vein with 0.5 × 10 (Alox15 crisis) or 10 (Alox15 chronic and C57BL/6) splenocytes or 2 × 10 bone marrow cells, monitored every other day, and killed at 6 or 10 wk after transfer. Tumor transfer was assessed by blinded morphological characterization of the spleens by hematoxylin and eosin (H&E). The presence of Alox15 donor cells in the transplanted mice was confirmed by PCR of the spleen DNA with primers specific for the neomycin cassette introduced by the Alox15 targeting construct (see Primer sequences).
Blood samples were collected from the inferior vena cava, anticoagulated with EDTA, and kept on ice before slide preparation. Bone marrow cells were collected by flushing the femurs of wild-type and Alox15 mice. Cytospin slides were stained using Kwik-Diff reagents for Wright stains (Thermo Electron). Tissue samples were frozen in Tissue-Tek OCT and stored at −80°C. Morphology was analyzed in 8-μm sections stained with Gill's H&E. Tdt-mediated dUTP-biotin nick-end labeling staining for apoptotic cells was performed on three sections of spleen per mouse using a staining kit (Roche Applied Science) according to the manufacturer's instructions. The number of positive cells was quantified using Image-Pro software (Media Cybernetics).
Single cell suspensions of splenocytes, bone marrow cells, and Histopaque 1083–purified (Sigma-Aldrich) peripheral mononuclear cells were prepared and depleted of red blood cells by lysis in ammonium chloride buffer, and cells were stained using 2 μg/ml directly labeled specific antibody or isotype-matched antibodies as controls. Compensation was performed using anti-CD4 or Mac-1 (FITC) and anti-CD8 or Mac-1 (PE) staining. All flow cytometric antibodies were purchased from BD Biosciences. Cells were washed and analyzed using a BD FACSCalibur machine (BD Biosciences). For sorting, cells were stained as described earlier in this paragraph and sorted for the populations indicated in the figures at the Wistar Institute Flow Cytometry Core facility.
Single cell suspensions were fixed in ice-cold 70% ethanol at 4C° for 1 h, washed with PBS, and resuspended in 50 μg/ml propidium iodide containing 1.5 mM sodium citrate and 5 μg/ml RNase A in PBS. Samples were analyzed by flow cytometry.
RBC-depleted splenocytes were prepared as in Flow cytometry, and nuclear lysates were prepared using the NucBuster kit (EMD Biosciences) according to the manufacturer's instructions. For cytoplasmic lysates, extracts were prepared from isolated cells using 1% NP-40 lysis buffer with phosphatase and protease inhibitors, whereas total extracts were prepared using M-PER (Bio-Rad Laboratories) according to manufacturer's instructions. For immunoprecipitation, extracts were normalized to total protein concentration using a Bradford assay (Bio-Rad Laboratories), according to manufacturer's instructions, and immunoprecipitated overnight with 1 μg of antibody, captured with protein G beads, and washed five times with 0.1% SDS buffer before electrophoresis. For Western analysis of unprecipitated extracts, samples were normalized to total protein using Bradford assay according to manufacturer's instructions, resolved by 10% SDS page gel electrophoresis, transferred to polyvinylidene difluoride membranes, and immunoblotted with specific antibodies, as described in Results. All immunoblotting antibodies were obtained from Santa Cruz Biotechnology, Inc., except the anti-Crk antibody, which was purchased from BD Biosciences, and anti-phosphoAkt (Ser473), which was purchased from Cell Signaling.
Total RNA was extracted from splenocytes using TRI (Invitrogen) according to manufacturer's instructions. RNA was treated with Turbo DNase (Ambion) according to the manufacturer's instructions to remove any contaminating genomic DNA, and the absence of appreciable genomic DNA was confirmed by real-time PCR of the treated RNA. RNA was normalized by OD, and reverse transcription reaction was performed using a cDNA synthesis kit (Applied Biosystems) according to the manufacturer's instructions. Quantitative real-time PCR analysis was performed using Sybr green Master Mix (Applied Biosystems) and analyzed using the ABI 7000 machine (Applied Biosystems). Gene-specific primers (see the next paragraph) were designed using Primer Express (Applied Biosystems), and gene expression levels were normalized using β-actin as an internal control.
The following primer sequences were used: β-actin, (forward) 5′−TCAGCAAGCAGGAGTACGATG-3′ and (reverse) 5′−AACAGTCCGCCTAGAAGCACTT-3′; ICSBP (forward) 5′-TGGGCAGTTTTTAAAGGGAAGTT-3′ and (reverse) 5′-ACAGCGTAACCTCGTCTTCCA-3′; 12/15-LO (forward) 5′-ACCCCACCGCCGATTTT and (reverse) 5′-AGCTTCGGACCCAGCATTT; and neomycin cassette (forward) 5′-TTGGGTGGAGAGGCTATTCG-3′ and (reverse) 5′-AACACGGCGGCATCAGA-3′.
One-way analysis of variance (for comparing more than two groups) and -tests (for comparing two groups) were calculated using Prism software (GraphPad) according to the program's designation of the most statistically valid test for each experiment. The α level was set at 0.05 for all tests; p-values below this were considered statistically significant. All error bars represent means ± SD.
Fig. S1 shows comparable hematopoietic compartments to wild-type controls in young Alox15 mice. Fig. S2 depicts increased megakaryocytes and decreased red blood cell numbers in Alox15 mice. Fig. S3 shows myeloid infiltrates in the skin of moribund Alox15 mice. Online supplemental material is available at . |
One function of peritoneal B1 cells is to survey the abdominal cavity and to join forces with innate cells, such as macrophages (MΦ), for a rapid and efficient bacterial clearance. We mimicked an acute infection from the gut by administration of high doses of indomethacin, an antiinflammatory drug that disrupts the gut epithelial barrier (). Translocation of bacteria, predominantly from the small intestine (as indicated by 16SrRNA sequences, with being the predominant bacteria in the small intestine in our mouse colony) into the peritoneum-associated tissues (omentum and mesenterium; ) induced dramatic changes in the cellular composition of the peritoneal cavity. These changes consisted of a depletion of resident B1 cells and a massive influx of monocytes/MΦ or granulocytes from the blood (). To monitor the kinetics of B1 cell egress from the peritoneal cavity, we injected fluorescence-labeled live bacteria i.p. We found that 3 h after injection, similar percentages and numbers of B cells (B220IgM) and MΦ (B220IgM) were loaded with bacteria (). However, 6 h after peritonitis, the number of bacteria B cells as well as the total number of B1 cells (B220IgM Mac-1) present in the peritoneal cavity drastically decreased, whereas the number of B220Mac-1 cells containing bacteria increased ().
A similar egress of B1 cells from the peritoneal cavity was not only associated with bacteria phagocytosis, but also with cell stimulation by pure bacterial components. As shown in , i.p. injection of lipid A induced a significant depletion of B1 cells from the peritoneal cavity. We suspected that the omentum, an entry site for B1 cells (), might also represent an exit route. In agreement with this, we found that at 3 and 5 h after lipid A stimulation the numbers of B1 cells in the omentum increased, which were probably in transit out from the peritoneal cavity (). Target organs for B1 cell migration outside the peritoneum-associated tissues were evaluated 14 h after i.p. injection of GFP peritoneal cells into RAG2 mice. Stimulation with lipid A or peptidoglycan by i.p injection induced a significant increase in the numbers of B1 cells that migrated to the spleen or small intestine lamina propria ( and not depicted). Thus, the presence of bacteria or bacterial components in the abdominal cavity drives a large fraction of B1 cells out of the peritoneal cavity and thereby facilitates their migration to effector sites.
We next asked if the egress of peritoneal B1 cells induced by lipid A or peptidoglycan required direct signals through TLRs. The migration properties of peritoneal B1 cells isolated from TLR4 mice were compared with those from normal mice in competitive transfer experiments after stimulation with lipid A. Although equal numbers of B1 cells from GFP transgenic (Tg) and TLR4 mice were injected into the peritoneal cavity of RAG2 mice, much less TLR4-derived B1 cells could be detected outside the peritoneal cavity 14 h after transfer (). The majority of B1 cells that migrated to the omentum, mesenterium, spleen, or small intestine were derived from GFP Tg mice, and their homing index was three to four times higher as compared with that of B1 derived from TLR4 mice (). To ensure that TLR4 B1 cells were not innately deficient in their ability to home to peripheral tissues, we performed competitive transfer experiments in the absence of stimulation. As shown in , similar percentages of B1 cells from GFP Tg and TLR4 mice could be detected outside the peritoneal cavity, clearly indicating that the impaired egress of TLR4-deficient B1 cells after acute stimulation was due to their unresponsiveness to TLR ligands.
We next wanted to elucidate the mechanisms involved in TLR-induced B1 cell egress. We found that peritoneal B1 cells express much higher levels of α4, α6, and β1 integrins as compared with freely recirculating spleen B2 cells (). This observation raised the possibility that a high expression level of integrins is responsible for retention of B1 cells in the peritoneal cavity and would make pertinent the prediction that TLR-induced egress involves integrin down-regulation on B1 cells. Indeed, analyses of integrin levels after LPS stimulation revealed a striking and selective down-regulation of surface integrins on B1 cells (). Stimulation with LPS i.p. reduced the mean fluorescence intensity level of α4, α6, and β1 integrins on B1 cells to <50% by 6 h after injection, in parallel with cell activation, as assessed by CD69 expression (). In contrast, it did not affect the expression level of Mac-1 (α) on B1 cells nor of any integrins expressed on spleen B2 cells (). i.v. injection of LPS, although strongly activated spleen B2 cells, as assessed by CD69 up-regulation, failed to induce changes of integrin levels, whereas B1 cells responded by a similar down-regulation as seen after i.p. challenge (Fig. S1 A, available at ). It is therefore unlikely that the nonresponsiveness of spleen B2 cells is attributed to a lack of sufficient activation by LPS injection i.p.
Integrin modulation on B1 cells was the consequence of direct signaling through TLRs and not of engagement of B cell receptors because injection of lipid A had no effect on peritoneal cells from TLR4 mice, whereas it induced a clear integrin down-regulation in WT mice (). We further investigated whether the adaptor molecule myeloid differentiation primary response gene 88 (MyD88) was critical for TLR-dependent down-modulation of surface integrins on B1 cells. Consistent with complete MyD88 dependency for TLR2 but not TLR4 signaling described for other aspects of innate and adaptive immune responses (–), we found that MyD88 mice did not respond to synthetic TLR2 ligand (Pam2CSK4) but clearly down-regulated integrins on peritoneal B1 cells after TLR4 ligand (lipid A) stimulation (). We further confirmed that integrin down-modulation on peritoneal B1 cells was a direct effect of TLRs on B cells (rather than an indirect one through cytokines secreted by activated peritoneal MΦ) because purified B cells stimulated in vitro had an identical response with those from nonsorted cultures (Fig. S1 B).
To further prove the involvement of integrins in B1 cell migration, we examined the effect of blocking integrin function with antibodies. Thus, we performed short-term transfer experiments with peritoneal cells preincubated in vitro with integrin-blocking antibodies in the absence of any stimulation. Anti-α4 antibody treatment resulted in a modest decrease in B1 cell number in the peritoneal cavity and a slight increase in B1 cells that migrated to the spleen (). Similar results were obtained when peritoneal cells were preincubated with a combination of anti-α4, α6, and β1 antibodies (not depicted). Thus, down-modulation of integrins alone cannot explain the efficient egress of peritoneal B1 cells seen after TLR stimulation.
CD9 is known to be selectively expressed by B1 cells () and to regulate cell motility (). We found that CD9 expression on the surface of peritoneal B1 cells is correlative with α6 and β1 although not with α4 integrins (). These observations led us to examine if TLR stimulation induces modifications in CD9 expression that can be related with B1 cell migration out of the peritoneal cavity or peritoneum-associated tissues. As shown in , i.p. stimulation with LPS induced a considerable down-regulation of CD9 expression on B1 cells 5 h after activation in WT mice but not in TLR2,4 mice. LPS-induced CD9 changes on B1 cells paralleled modulation of α4, α6, and β1 integrin expression, including a coordinated up-regulation 14 h after stimulation (). Furthermore, similar to α4, α6, and β1 integrins, CD9 down-regulation after TLR4 stimulation with lipid A was MyD88 independent, whereas that induced through TLR2 after PAM2CSK4 stimulation was dependent on MyD88 (). Strikingly, in vitro preincubation of peritoneal cells with both anti-α4 and anti-CD9 antibodies induced a significant depletion of B1 cells from the peritoneal cavity and a parallel increase in the number of B1 cells that migrated to the omentum, mesenterium, or spleen 3 h after transfer (). Anti-CD9 antibody treatment alone exerted a strong effect on peritoneal cell migration, with increased numbers of B1 cells being either in transit or already homed to the spleen.
The enhanced egress of B1 cells observed after CD9 blocking was not due to the down-regulation of surface integrins (not depicted) but most likely to increased cell motility. This is based on the observations that both anti-CD9 antibody–treated cells or peritoneal cells from CD9 mice migrated much faster than control or WT cells on fibronectin-coated microchannels in response to CXCL13 (, and Videos S1 and S2, which are available at ). To further confirm a direct involvement of CD9 in B1 cell migration, we performed in vivo competitive transfer experiments between normal and CD9 B1 cells. Cells (1:1 ratio of CD9/GFP Tg B1) were preincubated in vitro with anti-α4 alone or anti-α4 and anti-CD9 antibodies, and B1 cell migration was evaluated 3 h after injection into the peritoneal cavity of RAG2 mice. As shown in , the majority of B1 cells that migrated to the spleen after integrin α4 blocking were derived from CD9 mice. In contrast, similar percentages of CD9 and GFP B1 cells were detected in the spleen after preincubation with both anti-α4 and anti-CD9 antibodies. This result clearly indicates that anti-CD9 antibodies enhanced the migration of B1 cells from GFP Tg mice by blocking the CD9 function, and that modulation of integrins and CD9 together is required to induce B1 cell mobilization. Thus, coordinated integrin-CD9 down-regulations induced by TLR stimulation cause efficient emigration of B1 cells from the peritoneal cavity.
Because the B1 cell migration under steady-state conditions was shown to require intact signaling through chemokine receptors (), we next inquired if chemokines and their receptors are also involved in B1 cell egress induced by TLR stimulation. Therefore, we tested the effect of pertussis toxin (PTX) treatment on the TLR-induced egress of B1 cells. Peritoneal cavity cells were incubated in vitro for 2 h with PTX, an enzyme that ADP ribosylates and inactivates Gαi proteins, or, as a control, with the non-ADP–ribosylating oligomer B subunit of PTX, and then transferred into the peritoneal cavity of RAG2 mice with lipid A. As shown in , PTX treatment caused a substantial (∼85–90%) inhibition of B1 cell migration to the omentum, mesenterium, and spleen, with their accumulation in the peritoneal cavity 8 h after cell transfer.
Several lymphoid chemokines were constitutively produced by omentum and mesothelial cells of the peritoneal wall (). Among these, the most abundantly expressed were CXCL13 and CCL19, and these were also the only chemokines of which we detected up-regulation at mRNA levels after stimulation (). Furthermore, CXCL13-producing cells were mainly located in the lymphoid aggregates of omentum (), and B1 cells showed robust chemotactic response to CXCL13, both in vivo and in vitro (, –). Consistent with published results (), except for the CXCL13, the mRNA levels for CXCL12, CCL19, and CCL21 were very low to undetectable in peritoneal MΦ. To determine the contribution of omentum-derived CXCL13 for emigration of peritoneal B1 cells, an equal number of GFP peritoneal cells were injected without or with lipid A into the peritoneal cavity of WT and CXCL13 mice. The efficiency of GFP B1 cell egress was evaluated 14 h after the transfer. As shown in , fewer B1 cells migrated after lipid A stimulation to the omentum in CXCL13 mice than in WT mice, and the number of GFP B1 cells recovered from the peritoneal cavity of CXCL13 mice was significantly higher than the number of B1 cells recovered from WT mice. In contrast, similar numbers of B1 cells were recovered from the peritoneal cavity of both CXCL13 and WT mice in the absence of stimulation (). CCR7-CCL19 receptor–ligand pair might also be involved in peritoneal B1 cell egress because CCR7 B1 cells tend to accumulate more than WT B1 cells in the peritoneal cavity after transfer into RAG2 mice (Fig. S2 A, available at ) and CCR7 mice accumulate much more peritoneal cells as compared with WT mice ( and unpublished data). Collectively, these results clearly indicate that G protein–coupled receptors are required for TLR-induced peritoneal B1 cell egress and that CXCL13 is an important chemokine involved in this emigration process.
We demonstrate here that independent from antigenic recognition through specific B cell receptors, direct signals through TLRs induce B1 cell egress from the peritoneal cavity. The sensing of bacteria or bacterial components triggers a series of coordinated events on B1 cells, consisting of rapid, transient, and parallel down-regulation of integrins and CD9. These changes are required for B1 cell egress because the lack of integrin-CD9 down-modulation is associated with a significant impairment in B1 cell egress in TLR4 mice after lipid A stimulation (). Involvement of integrins and CD9 on regulation of retention versus migration of peritoneal B1 cells was confirmed by antibody treatment as well as in competitive migration studies with WT and CD9-deficient B1 cells (). Furthermore, peritoneal B1 cells appear to use similar molecular machinery with classic innate cells to transduce signals from TLRs. Thus, the TLR4 signaling cascade involves both MyD88-dependent and -independent pathways, whereas signals through TLR2 are strictly dependent on MyD88 function ().
Besides changes in integrin-CD9 expression, the exit of B1 cells from their compartment requires chemokine receptors. This is clearly demonstrated by the fact that treatment with PTX almost completely inhibits migration out of peritoneal B1 cells, even under activation conditions. CXCR5-CXCL13 appears to be the receptor–ligand pair used by B1 cells not only for homing into the peritoneum, but also for their egress out of the peritoneal cavity. This conclusion is supported by the observation that a much larger fraction of B1 cells (injected directly into the peritoneal cavity, thus circumventing the involvement of CXCL13 for homing) remained in the peritoneal cavity of CXCL13 mice than in WT mice, even after stimulation through TLR ().
The responsiveness to CXCL13, abundantly produced in the omentum, appears to be influenced by modulation of CD9 because CD9-blocked peritoneal cells as well as those from CD9 mice migrated both faster and more efficiently to CXCL13 (Videos S1 and S2, and ).
Sphingosine-1-phosphate receptor 1 (S1P) was shown to be involved in lymphocyte egress from the lymphoid organs (). Peritoneal B1 cells express S1P and show a moderate chemotactic response to S1P ligand (S1P) under nonactivated conditions (Fig. S2, B and C). However, after TLR stimulation, both S1P expression and the chemotactic response of B1 cells to S1P decreased (Fig. S2, B and C), making it unlikely that S1P-S1P plays a significant role for peritoneal B1 cell egress.
TLR signals may control B1 cell recruitment and participation not only in acute responses, but also in steady-state conditions for the maintenance of immune system homeostasis. Indeed, we found that germ-free mice accumulate significantly more numbers of B1 cells in the peritoneal cavity or peritoneum-associated tissues compared with mice kept under specific pathogen-free conditions harboring a diverse gut microbiota (Fig. S3, available at ). We speculate that a significant fraction of B1 cells that survey the abdominal cavity are sensing the gut microbiota and are constantly induced to migrate out of the peritoneal cavity. They are likely to participate in maintenance of homeostasis of the gut barrier by producing secretory IgAs against commensal bacteria. The B1-derived IgAs are generated through a primitive T-independent and follicular-independent pathway and play an important role in the regulation of bacterial communities in the intestine (–). Our findings fully support the conclusion that B1 cells are innate-like B cells with specialized functions that are poised to react rapidly to gut-associated antigens and pathogens ().
Thus, the egress of B1 cells from the peritoneal cavity is a complex, multistep process governed by interplay between integrins, tetraspanins, and chemokines that would allow these innate-like B cells to coordinate efficient immune responses against infections.
The results presented here reveal an unexpected requirement for TLR signaling in the down-modulation of integrin and tetraspanin expression on innate-like B1 cells, which is critical for their rapid mobilization and participation in immune responses. TLRs are thus involved in the control of antibody responses in multiple steps: activation and recruitment of B1 cells to effector sites of immune responses; relocalization of splenic MZ B cells from the MZ to white pulp cords (, , ); and proliferation and differentiation of B1 MZ B cells into antibody-secreting cells (). These sequential TLR-orchestrated events lead to efficient removal of pathogens soon after infection and facilitate optimal transition from innate to adaptive immune responses.
Mice, all on a C57BL/6 background, were bred and maintained in specific pathogen-free conditions at the animal facility of the Research Center for Allergy and Immunology and used at 8–16 wk of age. Germ-free mice were obtained from Yakult. For cell transfer, WT, RAG2, and CXCL13 mice were injected i.p. with 10 peritoneal cells obtained from age- and sex-matched GFP Tg mice. For competitive transfer experiments, peritoneal cavity cells obtained from GFP Tg mice and TLR4 or CD9 mice were counted, stained for surface markers, mixed to a 1:1 ratio of B1 cells (∼10 B220IgM cells), and injected into RAG2 mice. All animal experiments were performed in accordance with approved protocols from the Institutional Animal Care at RIKEN.
Mice were stimulated with lipid A and endotoxin-free PAM2CSK4, both purchased from InvivoGen, and LPS ( O26:B6) from Sigma-Aldrich. For bacteria-induced peritonitis, the DH5α strain was transformed with pGex-4T-1 (GE Healthcare) vector expressing DsRed2 (original vector pDsRed2 was from CLONTECH Laboratories, Inc.). After 10–12 h of aerobic growth at 37°C, the cultures were maintained overnight under micro-aerophilic conditions at room temperature. After this period, 70–80% of the bacteria expressed detectable levels of DsRed2. After two washes with PBS, DsRed2 bacteria were quantified using a fluorescence microscope (Axioplan2; Carl Zeiss MicroImaging, Inc.), and 1–10 × 10 fluorescent units were injected into mice i.p. For disruption of the gut barrier, 10-wk-old BALB/c mice (Clea) were subcutaneously injected twice at 12-h intervals with 25 mg/kg indomethacin (Sigma-Aldrich). Mice were killed and analyzed after 1 d. DNA was extracted from cell suspensions obtained after the digestion of omentum and mesenterium, and the 16S rRNA was amplified by universal primers described previously ().
Peritoneal cells, spleen, and mesenteric lymph node cell suspensions were prepared as described previously (). Cells from parietal peritoneum, omentum, mesenterium, and small intestine lamina propria were isolated after stirring for 45 min with 1.5 mg/ml collagenase (Wako) and 0.5 U/ml dispase at 37°C. Cells were washed twice in RPMI medium with 2% FCS and stained for flow cytometry. Allophycocyanin (APC) anti-B220 (RA3-6B2), PE anti-CD49d (integrin α4, R1-2), PE anti-CD49f (integrin α6, GoR3), biotin anti-CD29 (integrin β1, Ha2-5), biotin anti-CD9 (KMC8), and FITC anti-CD21 were from BD Biosciences. APC anti-CD11b (Mac-1) and PE anti-CD69 (H1.2F3) were from eBioscience, and FITC or PE anti-IgM was from SouthernBiotech.
Peritoneal B1 cells (B220IgMMac-1) and spleen B cells (B220IgM) were sorted, (FACSAria; Becton Dickinson) or run through the nozzle without sorting, after staining with APC anti-B220, FITC anti-IgM, and PE anti–Mac-1. Cells were incubated with RPMI medium supplemented with 10% FCS and 20 μM 2-ME without or with 20 μg/ml LPS. After 12 h, cells were recovered, washed, and stained for surface integrins.
For integrin or/and CD9 blocking, cells were treated before i.p. injection for 20–30 min with 100 μg/ml of purified anti-integrin α4 antibody (clone R1-2) and 200 μg/ml anti-CD9 antibody (clone KMC8). All antibodies were obtained from BD Biosciences. For G protein inactivation, peritoneal cells were treated with 100 ng/ml oligomer B or PTX (Sigma-Aldrich) for 2 h in RPMI medium supplemented with 10% FCS and 20 μM 2-ME, washed, and injected i.p. into RAG-2 mice together with 10 μg lipid A.
Omenta were dissected from animals, placed in 24-well plates, fixed with Cytofix/Cytoperm solution (BD Biosciences) for 30 min at 4°C, and incubated for 60 min with the following antibodies: goat IgG anti–mouse CXCL13 (R&D Systems), rabbit IgG anti–Lyve-1 (Abcam), FITC-conjugated donkey anti–goat IgG (H+L), and Cy3-conjugated donkey anti–rabbit IgG (Jackson ImmunoResearch Laboratories). Stained tissues were placed on slides, mounted (Vectashield; Vector Laboratories), and analyzed with a confocal laser-scanning microscope (model DMRXA2; Leica).
The KK chamber (Effector Cell Institute) was used to detect real-time chemotaxis of peritoneal lymphocytes (). Peritoneal cells were incubated in the RPMI media with 10% FCS for 1.5 h at 37°C to remove the MΦ, harvested, and incubated for an additional 20 min with 200 μg/ml anti-CD9 (KMC8) or isotype control antibodies. Cells were washed in warm RPMI 1640, 2% FCS, and 10 mM Hepes, and then loaded (1 μl of 10 cells/ml) into the top wells. CXCL13 (PeproTech) was loaded into the bottom well (10 μg/ml). The micro cover glass was coated with 40 μg/ml fibronectin (Biogenesis). A charge-coupled device camera was used to record the migration of peritoneal B cells for 1.5 h at 37°C. The migration velocity of peritoneal lymphocytes on the KK chamber was analyzed using Volocity software (Improvision Inc.). Frames of video images were captured and digitized by the computer. To measure the cell displacement, the screen x and y coordinates of individual cells were tracked over the time, followed by the calculation of cell velocity. For each sample, ∼40 cells were tracked over a 30-min period. The frame-by-frame velocity data were used to calculate the mean velocity and the variance of velocity. Chemotactic response to S1P (Sigma-Aldrich) was performed using Transwell membranes as described previously ().
Total RNA was isolated from cells using the RNeasy mini-kit (QIAGEN). After the spectrometric analysis, equal amounts of RNA were used for cDNA synthesis. After DNase treatment, oligo dT primers were used for first-strand cDNA synthesis (RT). All procedures were performed according to the manufacturer's instructions (Invitrogen). Quantitative PCR was performed on an iCycler thermal cycler using SYBR Green Supermix according to instructions and analyzed by software (all Bio-Rad Laboratories). All primers were determined by BEACON DESIGNER (v2.1; Premier Biosoft International). Sequences were as follows: 36B4 forward: 5′-CACTGGTCTAGGACCCGAGAAG, reverse: 5′-GGTGCCTCTGGAGATTTTCG; CXCL13 forward: 5′-TGAAGTTGTGATCTGGACCAAGA, reverse: 5′-ACAGACTTTTGCTTTGGACATGTC; CXCL12 forward: 5′-AAGGTCGTCGCCGTGCTG, reverse: 5′-GATGCTTGACGTTGGCTCTGG; CCL21 forward: 5′-GGCTATAGGAAGCAAGAACCAAGT, reverse: 5′-TCCTCAGGGTTTGCACATAGCT; CCL19 forward: 5′-CCTGGGAACATCGTGAAAGC, reverse: 5′-GCACAGAGCTGATAGCCCCTTA; and S1P forward: 5′-TTCCGCAAGAACATCTCCAAGG, reverse: 5′-CAGCCCACATCTAACAGTAGTAGG.
Fig. S1 shows the down-regulation of integrin α4 and β1 on peritoneal B1 cells after in vivo (i.v.) and in vitro LPS stimulation. Fig. S2 shows possible involvement of CCR7 but not S1P for B1 cell egress from the peritoneal cavity. Fig. S3 presents the total B1 cell numbers in the peritoneal cavity and omentum in germ-free and specific pathogen-free mice. Videos S1 and S2 show real-time chemotaxis to CXCL13 of peritoneal B cells from WT and CD9 mice, respectively. The online supplemental material is available at . |
Previously, we have shown that a rapid influx of CD4CD25 nTreg cells occurs in infected dermal sites during the silent phase of disease, which is characterized by peak parasite numbers in the site of infection before the onset of adaptive immunity (). The early and preferential recruitment of nTreg cells to infected sites might be critical to the ultimate establishment of a state of immunosuppression that dampens anti-parasite immunity and favors the parasite persistence (, ). As CCR5 has been proposed to influence nTreg activity in graft-versus-host disease and tumor immunity, we hypothesized that CCR5 may drive the recruitment of nTreg cells to infectious sites.
To validate the CCR5 expression on nTreg cells, we performed quantitative real-time PCR analysis on total RNA isolated from resting or TCR-stimulated CD4CD25 or CD4CD25 T cells (). In freshly isolated, unstimulated CD4CD25 T cells, CCR5 mRNA levels were approximately fivefold greater than their CD4CD25 T cell counterparts. By 12 h after TCR stimulation, CCR5 mRNA levels in CD4CD25 nTreg cells were ∼20-fold greater than their CD4CD25 T cell counterparts. After 48 h of TCR engagement, CCR5 expression levels on CD4CD25 T cells, albeit fivefold lower than the 12-h time point, were nonetheless significantly greater (fourfold increase) than their CD4CD25 counterparts. This preferential CCR5 gene expression on nTreg cells is also confirmed by our finding that 1% of resting and 15% of activated CD4CD25 nTreg cells preferentially express cell surface CCR5 (). Thus, although the expression of CCR5 on nTreg cells is detectable in the absence of activation, its expression is significantly increased upon antigen stimulation, consistent with a recent study from Bystry et al. ().
To directly determine whether the CCR5 mRNA is translated into the functional protein in nTreg cells, we explored the chemotactic response profile of highly purified CD4CD25 and CD4CD25 T cells to CCR5 ligands MIP-1α, MIP-1β, or RANTES (100 ng/ml) in transwell chambers (). CD4CD25 T cells exhibited almost a 2.5-fold increased migratory capacity in the absence of chemokine stimulation compared with CD4CD25 T cells (not depicted). Our results show that both resting and activated CD4CD25 T cells migrated very poorly to every chemokine (). In stark contrast, 26, 36, and 21% of resting, unstimulated CD4CD25 T cells migrated in response to MIP-1α, MIP-1β, or RANTES, respectively (P < 0.003). Similarly, 81, 61, and 67% of anti-CD3–activated CD4CD25 T cells showed preferential chemotactic activity to MIP-1α, MIP-1β, or RANTES, respectively (P < 0.007). The addition of neutralizing antibodies to these chemokines completely abrogated the migration of nTreg cells in this assay (not depicted). Using total CD4 T cells, we observed that MIP-1α, MIP-1β, or RANTES strongly induced the migration of a substantial population of Foxp3CD4 T cells (). This confirms that nTreg cells, in contrast to CD4CD25 T cells, preferentially express CCR5 and respond to its ligands.
As nTreg cell suppression occurs via a contact-dependent and cytokine-independent manner in vitro, we postulated that these cells could use CCR5 to localize in physical proximity to target cells and engage in suppression. To this end, we isolated nTreg cells from WT or CCR5 mice and analyzed their ability to suppress proliferation of WT (, left) and CCR5 (, right) responder T cells. In all cases, CD4CD25 nTreg cells were equally capable of suppressing anti-CD3–induced proliferation regardless of nTreg cell/responder ratios. In vitro neutralization of each chemokine failed to abrogate the suppressive activity of CD4CD25 T cells (not depicted). Collectively, these results show that CCR5 is not required for nTreg cell suppressive function in vitro but does promote nTreg cell chemoattraction to CCR5 ligands.
We next sought to examine the relative contribution of CCR5 expression on CD4CD25 nTreg cells in a low-dose intradermal (i.d.) model of infection in C57BL/6 mice. WT and CCR5 C57BL/6 mice were infected with 10
metacyclics in one ear, and the parasite burden and disease severity were monitored 6 wk after infection. In WT mice, infection resulted in a transient pathology that coincided with an increase in the parasite burden (, left and right). Interestingly, CCR5 mice were readily able to control infection since the parasite burden in infectious sites was significantly reduced compared with WT (P < 0.009) and correlated with a reduced pathology throughout the course of infection (, left and right). These findings show that CCR5 is essential for parasite persistence and the establishment of infection in genetically resistant mice, consistent with the observation made by Sato et al. (, ).
In our low-dose murine model of infection, the long-term persistence of parasites in infected sites is exquisitely determined by an equilibrium that is established between IFN-γ–producing CD4 effector T cells and IL-10–producing CD4CD25 nTreg cells (). In this model, parasite growth or elimination is rapidly triggered if the activities of IFN-γ or IL-10 are, respectively, impaired. To determine if the increased resistance in CCR5 mice occurred as a result of a functional imbalance in the IFN-γ/IL-10 regulatory axis, WT and CCR5 mice were infected i.d. with , and the degree of anti-pathogen Th1 immunity was determined at 3 and 12 wk after infection (). At 3 wk after infection, CCR5 mice displayed a 2.7-fold increase in i.d. CD4 T cells compared with infected WT mice (6.3 × 10 vs. 2.3 × 10 CD4CD3). Surprisingly, this increased accumulation of T cells in CCR5 mice also correlated with a 3.6-fold increased number of IFN-γ–producing T cells in infected dermal sites compared with WT mice (79 × 10 vs. 22 × 10 TCRβ cells) at 3 wk after infection. CCR5 mice were highly resistant to infection as they had a significant reduction in the number of parasites in dermal sites and surprisingly developed little or no overt pathology in contrast to WT mice (). This increased ability to control the parasite load in infected CCR5 mice compared with WT mice at 12 wk was associated with a twofold increased accumulation of T cells in i.d. sites, which paralleled the kinetics of accumulation of IFN-γ–producing T cells in the site (48 × 10 vs. 23 × 10 TCRβ cells) before parasite killing (). Interestingly, CCR5 mice also had a 6.2-fold decrease in the frequency of IL-10–producing T cells in dermal sites compared with WT mice (8.7 vs. 1.4% of CD3 T cells), consistent with the decreased susceptibility to typically seen in WT mice devoid of nTreg cells (). Thus, CCR5 deficiency perturbs the equilibrium between effector and nTreg cells within sites of infection, amplifies local immune responses in favor of anti-pathogen immunity, and promotes parasite clearance.
To directly assess the relative contribution of CCR5 expressed by nTreg cells in host immunity and parasite persistence, freshly isolated WT or CCR5 CD4CD25 effector T cells were transferred into RAG recipients either alone or in combination with CD4CD25 T cells from naive WT or CCR5 C57BL/6 mice, and the severity of disease was monitored at various time points after infection (). At 3.5 wk after infection, the approximate time of peak parasite load establishment in C57BL/6 mice, recipients of either WT or CCR5 CD4CD25 T cells alone harbored very few parasites in the site and developed minimal or no dermal pathology (not depicted). At the same time point, RAG recipient mice reconstituted with either CD4CD25/CCR5 CD4CD25 or CD4CD25/WT CD4CD25 cell populations displayed a similar ability to control infection and developed a transient dermal pathology, which in each case was greater than in recipients of either WT or CCR5 CD4CD25 alone ( and not depicted). At 7 wk after infection, recipients reconstituted with CD4CD25/WT CD4CD25 T cells developed a lesion similar to that seen in chronically infected WT mice with >1,000 parasites persisting in the site (). In contrast, in the presence of CCR5 CD4CD25 T cells, RAG recipient mice healed faster and completely cleared the parasites from the site of infection to an extent similar to WT mice devoid of nTreg cells (). Thus, CCR5 expression on nTreg cells contributes directly to controlling anti-parasite immunity within infected sites.
Our studies clearly show that CCR5 deficiency alters the ability of nTreg cells to control immunity. To directly address whether CCR5 dictates their migration from peripheral lymphoid compartments to sites of persistent infection, we used a Ly5 congenic transfer system to independently monitor nTreg and effector T cells from WT and CCR5 mice at various time points after infection. We cotransferred at a 1:10 ratio CD4CD25 nTreg cells from naive Ly5.1 WT or Ly5.2 CCR5 mice and CD4CD25 T cells from naive Ly5.1 or Ly5.2 WT mice into RAG recipients, and the numbers of each subset were analyzed during the accumulation of peak parasite numbers in infected dermal sites. By 3 wk after infection, the frequency of WT Ly5.1CD4CD25 nTreg cells represented 44% of total CD4 T cells in the infected dermis (). In contrast, the frequency of CCR5 Ly5.2CD4CD25 T cells decreased to 6%, representing a 7.3-fold decrease compared with the WT condition (), suggesting that CCR5 may be directing the homing of nTreg cells in –infected sites during the silent phase of disease.
To examine the evolution of nTreg cell recruitment into inflamed sites, we monitored the number of nTreg cells at different time points in the spleen, draining LN, and infected dermal sites (). At 1.5 wk after infection, during the silent phase of disease characterized by parasite growth, WT nTreg cells (Ly5.1) represented 30% of CD4 T cells in dermal sites, although they were undetectable in draining LN or spleen. The frequency of WT nTreg cells steadily increased to ∼30–40% of CD4 T cells in the dermis and draining LN at 3 wk and remained at these levels at 5.5 wk after infection, during the stage of lesion formation and immune clearance of parasites, with the exception of the spleen where the percentage of WT nTreg cells represented 50% of T cells. At 10 wk, around the onset of the chronic phase, the Ly5.1 cells (WT nTreg) represented ∼15% of the CD4TCRβ T cells in the dermis and ∼25% in the LN. In addition, WT nTreg cells continued to accumulate efficiently in the spleen during acute and chronic phases of disease (50 vs. 15% at 10 wk). These quantitative changes were greatly due to the expansion or recruitment of Ly5.2 CD4 effector T cells in the site and not to a decrease in the absolute number of Ly5.1 CD4 nTreg cells (not depicted). In stark contrast, CCR5 nTreg cells (Ly5.2), while being undetectable in the LN and spleen, represented 15–20% of CD4TCRβ T cells in dermal sites at 1.5 wk, with the frequency drastically declining to ∼5–7% of total CD4 T cells at 3 and 5.5 wk after infection. During the chronic phase (10 wk), CCR5 nTreg cells (Ly5.2) represented <5% of the CD4TCRβ T cells in the site of infection. Surprisingly, CCR5 nTreg cells accumulated more efficiently than WT cells in the draining LN of infected mice at 3 wk (40 vs. 30%, respectively) and 5.5 wk (70 vs. 30%, respectively) after infection. At 10 wk after infection, CCR5 nTreg cells migrated less efficiently than their WT counterparts in the spleen during acute and chronic phases of disease (10 vs. 50% at 5.5 wk, and 1 vs. 15% at 10 wk, respectively). These frequency changes correlated with a decrease in the absolute number of Ly5.2 T cells in these sites, particularly during the chronic phase of infection (10 wk; ). Thus, WT nTreg cells home initially to the infected site where they maintain themselves until the chronic phase of the infection, whereas CCR5 nTreg cells have a reduced capacity to home to similar environments and appear to accumulate in the draining LN. In summary, these studies show that CCR5 is directly responsible for the recruitment of nTreg cells to –infected dermal sites where they promote infection and parasite persistence.
Our results show that CCR5 expression on CD4CD25 nTreg cells is essential for their trafficking from peripheral lymphoid tissues into infected dermal sites. We next wished to demonstrate that CCR5 ligands, such as MIP-1α, MIP-1β, or RANTES, are produced within the dermis after infection and are responsible for the chemoattraction of CD4CD25 T cells to these sites. To this end, we transferred WT CD4CD25 T cells from naive mice to infected RAG recipients either alone or in the presence of WT CD4CD25 nTreg cells at a 1:10 ratio. To assess chemokine production within infected sites, we isolated the infected dermis at 14 and 28 d after infection and performed RT-PCR analysis for CCR5, MIP-1α, MIP-1β, or RANTES gene expression (). In contrast to uninfected RAG recipients, which never expressed any chemokines, infection of similar hosts resulted in a significant induction of MIP-1α, MIP-1β, or RANTES with gene expression levels being, respectively, 15-, 40-, and 45-fold higher than uninfected controls and peaking at 28 d after infection. Transfer of CD4CD25 T cells resulted in a more rapid induction of MIP-1α, MIP-1β, or RANTES with gene expression levels being, respectively, 7-, 50-, and 45-fold higher than uninfected controls and peaking at 14 d after infection. Interestingly, cotransfer of CD4CD25/CD4CD25 T cells resulted in a significantly greater induction of MIP-1α, MIP-1β, or RANTES gene expression levels (respectively, 17-, 70-, and 75-fold higher than uninfected controls), peaking at 14 d after infection and correlating temporally with the increase in parasite burden and loss of disease control. Strikingly, RAG recipient mice transferred with CD4CD25 nTreg cells alone also resulted in significant expression of each chemokine at every time point and paralleled the expression of CCR5 () and Foxp3 (not depicted) in these sites, suggesting that nTreg cells themselves may paradoxically be responsible for the induction of CCR5 chemokines. Overall, these studies demonstrate that expression of the CCR5 ligands are actively induced after infection and are temporally correlated with the recruitment of nTreg cells in infected skin where they promote the establishment of infection.
CD4 nTreg cells represent a critical peripheral switch in the control of immune responses to self-antigens as well as to a variety of pathogenic microorganisms (, –). Previously, we showed that the establishment of pathogen persistence within –infected sites is dependent on a tight equilibrium between effector and nTreg cells (). The abrogation of nTreg cell function or IL-10 production promotes clearance of the parasite, whereas depletion of effector cells or inflammatory cytokines like IFN-γ promotes disease reactivation. Interestingly, nTreg cells preferentially accumulate in sites of infection during phases of parasite expansion and establishment of chronicity, in contrast to effector T cells, which increase during the acute phase of disease characterized by increased anti-pathogen immunity. Thus, differential chemokine receptor expression profiles between different T cell subsets could explain potential trafficking differences in vivo, as shown in other models (, , , , ).
In this study, we establish a novel relationship between CCR5-dependent chemotaxis and the development of immunity in infection, and show that this receptor regulates critical aspects of the disease. First, we show that CD4CD25 nTreg cells preferentially express CCR5 and respond very efficiently in vitro to its ligands MIP-1α, MIP-1β, or RANTES. Consistent with the enhanced control of systemic infection and dermal infection, we then show that CCR5 mice are resistant to infection in contrast to their WT counterparts (, ). We also show that this increased resistance correlates with a significant reduction in the production of IL-10 by dermal CD4 T cells and a predominant Th1 response in CCR5 mice. More importantly, CCR5 nTreg cells, in contrast to WT nTreg cells, migrated less efficiently to infected dermal sites and failed to suppress parasite-specific, IFN-γ–producing CD4 T cells, thus resulting in potent resistance to infection. Collectively, this study shows that CCR5 directs the recruitment of nTreg cells to –infected dermal sites where they promote the establishment of chronic infection.
We establish CCR5 as a critical checkpoint influencing the balance between regulatory and effector T cells in infectious sites. The rapid control of parasite load in CCR5 mice was associated with a significantly increased infiltration of CD4 effector T cells in infected dermal sites as well as an enhanced magnitude of IFN-γ–producing CD4 T cells in response to in CCR5 mice compared with control mice. IFN-γ could contribute indirectly to the resolution of infection in CCR5 mice by sustaining the inflammatory response in these mice. Although CCR5 may contribute to the pathogenesis of infectious diseases by promoting IFN-γ–producing Th1 responses, CCR5 deficiency actually augmented IFN-γ production by dermal CD4 T cells, further supporting the notion that disease resistance is likely caused by a lack of nTreg cell–mediated regulation in infected sites. These results are consistent with the predominant Th1 response seen in a high-dose model reported by Sato et al. (), but are contrasted by the apparent reduction of early antigen-specific IFN-γ responses in the spleen of outbred CCR5 mice infected i.v. with a high dose of . Our results show no impairment in the accumulation of nTreg cells in the LN; however, we did see a reduction of parasite numbers in this site, without any apparent alterations in nTreg cell numbers in the LN (not depicted). This may suggest that the primary site is necessary for parasite maintenance in the LN, possibly by the creation of specific priming conditions or favorable cytokine environment. The ability of CCR5 nTreg cells to migrate efficiently to the LN but not to the infected dermal site suggests that CCR5 ligands produced in infected sites are needed to fully engage nTreg cells in these sites. Overall, CCR5 signals during primary challenge dampen anti-pathogen effector mechanisms and control the intensity of the inflammatory response within –infected sites.
Our data indicates that CCR5 deficiency results in a drastic reduction in the production of IL-10 by dermal CD4 T cells, an observation consistent with a reduction of nTreg cell function in infectious sites. A function for IL-10 in promoting parasite persistence and susceptibility to infection in both susceptible and resistant strains is well documented (–). We have previously shown that IL-10, which is produced by nTreg cells, contributes directly to parasite persistence and concomitant immunity (). The mechanism by which IL-10 allows parasite growth and survival is not fully defined, but it is likely due to its potent deactivating role of infected APCs that would become unresponsive to activation by IFN-γ. These results support the notion that during the chronic phase of disease, IL-10–producing nTreg cells dominate over effector mechanisms producing IFN-γ. This immunosuppressive role for IL-10 is also applicable to other animal models of infection, in which the levels of IL-10 are highly predictive of the outcome of the clinical course infection (). The development of nTreg cells in CCR5 mice is similar to WT mice, thus excluding the possibility that inherent differences in nTreg cell development or maturation in WT and CCR5 mice underlie our findings. Our results do not exclude the possibility that CCR5 may have a direct impact on the expansion, survival, or function of nTreg cells in inflammatory settings. Although CCR5 deficiency decreases the cellular frequency of nTreg cells and consequential IL-10 production in dermal sites, CCR5 signals may be required for the terminal differentiation of IL-10–producing nTreg cells within infected sites, as recently suggested for conventional CD4 T cell effector functions ().
We also show that the CCR5 chemokines MIP-1α, MIP-1β, or RANTES are actively induced after a low-dose infection, consistent with those made in other studies using high-dose footpad infection models of other Leishmania strains (–). We also make the novel observation that this expression is temporally correlated with the recruitment of nTreg cells in infected dermal sites. The cellular sources of these chemokines are unknown and may be produced by a variety of cell types throughout the course of an immune response, including activated effector T cells, infected macrophages, and dendritic cells (). It is plausible that the initial inflammatory events after pathogen infection result in chemokine production by these cell types, which subsequently attracts nTreg cells to dampen local immune responses. Strikingly, the presence of nTreg cells alone or in the presence of effector T cells appeared to significantly increase the expression of each chemokine in infected sites, suggesting that nTreg cells themselves may paradoxically produce or induce the synthesis of the chemokines. Thus, CD4 nTreg cells may represent a functionally diversified population consisting of different subsets, such that some are responsible for the initial chemokine release whereas others are endowed with chemokine responsiveness.
Persistent infections such as AIDS, tuberculosis, and leishmaniasis may represent a compromise between pathogen and host, such that the former subverts host immune responses to promote pathogen replication, whereas the latter maintains strong effector immunity against reinfection while limiting potential immunopathology (). The cellular and molecular mechanisms underlying the establishment, maintenance, and disruption of the homeostatic balance between host and pathogen remain poorly understood. Many studies indicate that pathogens may establish chronic infections in immunocompetent hosts by engaging various regulatory T cells to promote immunosuppression and pathogen persistence. Our findings show that CCR5 expression on nTreg cells is a critical requirement for the establishment of chronicity in an immune host and shed light into the overall dynamics of T cell responses in infected sites. On the basis of these results, it can be envisaged that transient CCR5 blockade may lead to the development of novel immunotherapeutic strategies for various infectious diseases.
WT, CCR5, Ly5.1 congenic C57BL/6, and C57BL/10 RAG2 mice were obtained from the National Cancer Institute and The Jackson Laboratory. All mice were bred and maintained in a specific pathogen-free animal facility. All mice used were generally 6–8 wk of age. All procedures conformed to the norms of the McGill University Animal Care Committee.
Infective, metacyclic promastigotes (clone V1 MHOM/IL/80/Friedlin) were grown and isolated from 4–5-d-old stationary cultures by negative selection of infective forms using peanut agglutinin (Vector Laboratories) as described previously (). Mice were infected in the ear dermis with 10
metacyclic promastigotes in a volume of 10 μl, and parasitic loads in the ears were determined as described previously (, ). In brief, the ventral and dorsal sheets of the infected ears were separated and deposited dermal side down in DMEM containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μg/ml of liberase CI enzyme blend (Boehringer). Ears were incubated for 2 h at 37°C. The sheets were cut into small pieces using a Medimachine (Becton Dickinson). The tissue homogenates were filtered using a 70-μm cell strainer (Falcon Products Inc.) and serially diluted (increment 2) in a 96-well flat-bottom microtiter plate containing biphasic medium prepared using 50 μl NNN medium containing 20% of defibrinated rabbit blood overlaid with 100 μl M199/S. The number of viable parasites in each ear was determined from the highest dilution at which promastigotes could be grown out after 7 d of incubation at 26°C. The number of parasites was also determined in the local draining LNs (retromaxilar). The LNs were mechanically dissociated and the parasite load in LN cells was determined by limiting dilution as described above.
For the analysis of surface markers and intracytoplasmic staining for IFN-γ or IL-10, dermal single cell suspensions were stimulated with –infected or soluble leishmania antigen–stimulated bone marrow–derived dendritic cells (BMDCs) for 12–18 h as described previously (). During the last 4–6 h, cells were cultured with 10 μg/ml brefeldin A, fixed in 4% paraformaldehyde, and incubated with an anti–Fcγ III/II receptor in staining buffer as described previously (). Cells were permeabilized and stained with a variety of antibodies: CD3 (145-2C11, FITC), CD4 (RM4-5, PE), IFN-γ (XMG1.2, PE), IL-10 (JES5-16E3, PE), TCRβ (H57, APC), or rat IgG2b (A95-1) and rat IgG2a (R35-95; all from BD Biosciences) isotype controls. Staining for the nuclear marker Foxp3 was performed as per the manufacturer's protocol (eBioscience). Cell acquisition was performed using a FACSCalibur flow cytometer (Becton Dickinson) and CELLQuest software.
CD4CD25 or CD4CD25 T cells from appropriate mice were purified from a pool of LN cells on a FACSVantage cell sorter to a final purity of >98% as described previously (). Irradiated (3,000 R), T-depleted spleen cells were used as APCs and were prepared by negative selection of Thy1.2 on the AutoMACS magnetic separation system (Miltenyi Biotec). Responder T cells were purified by negative depletion of CD8, DX5, B220, and I-Ab cells from LNs by AutoMACS.
Proliferation assays were performed by culturing 5 × 10 CD4 T cells in 96-well flat-bottom microtiter plates in complete RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated FCS, with 1–2 × 10 irradiated T-depleted spleen cells and 0.5 μg/ml of soluble anti-CD3 for 72 h at 37°C in 7% CO. Cell cultures were pulsed with 1 μCi H-TdR for the last 6–12 h. All data represent the average counts per minute of triplicate determinations. All proliferation experiments were repeated at least three times.
CD4CD25 or CD4CD25 T cells (2–3 × 10/mouse) were transferred i.v. to RAG mice at various time points after i.d. infection as described previously ().
Evaluation of CCR5 gene expression was performed as follows. Total RNA was prepared from ear tissues or T cell cultures with the Trizol RNA extraction reagent, and cDNA was made using Superscript II with random hexamer primers (Invitrogen). Real-time PCR was performed using primers and a 6-carboxyfluorescein (FAM)-labeled probe for CCR5: forward primer, 5′-TGACGTCACTGGAGTTGTACGG-3′; reverse primer, 5′-GGTTCATGTCATGGATGGTGC-3′; probe, 5′-FAM-TTCAGCGCTCACTGCTCTTGTGACAG-TAMRA-3′ (Applied Biosystems). All PCR reactions were performed in triplicate using a TaqMan universal PCR master mix amplified with an ABI Prism 7700 Sequence Detection System for 40 cycles and quantified using standard curves for CCR5 and TaqMan Ribosomal RNA Control Reagents (Applied Biosystems). RT-PCR primers for MIP-1α, MIP-1β, RANTES, CCR5, Foxp3, and G3PDH were obtained from BD Biosciences. PCR products were electrophoresed, and semiquantitative analysis of gene expression was achieved by normalizing the test amplicon densitometric value with the intensity of the G3PDH amplicon for each sample by the Quantity One 4,4,1 software (Bio-Rad Laboratories) as described previously ().
Migration assays were done in 6.5-mm diameter, 5.0-μm pore size polycarbonate membrane filter transwell plates (Costar Corning). The lower well chamber contained 0.6 ml RPMI medium with respective chemokines (100 ng/ml). All chemokines were purchased from PeproTech. T cell suspensions (0.1 ml at 10 cells/ml) were added to the upper chamber. After incubation for 3 h at 37°C and 10% CO, the cells migrated into the lower chamber, were stained with anti-CD4 (L3T4, FITC) and anti-CD25 (PC61, PE) or anti-Foxp3 (PE), and analyzed by FACS.
Results are expressed as mean ± SD. To determine whether differences were statistically significant, the Student's test was performed using a two-tailed distribution with unpaired samples. In some instances, group comparisons were made by ANOVA or Kruskal-Wallis, followed by Dunnet's test to determine differences between genotypes. |
In the last 25 years, 25 million people have died of AIDS, and approximately 40 million now live with HIV. Every day, an estimated 11,200 people are infected and 8,000 die. Although 1.65 million people are now receiving antiretroviral treatment, 70% of those in need still lack drugs. The epidemics that are most out of control are among intravenous drug users (especially in Eurasia), men who have sex with men (everywhere), and the generalized epidemic in sub-Saharan Africa.
The AIDS meeting is one response to this epidemic. From its humble beginnings in 1985 as a small scientific gathering, the conference has grown to a complex mixture of ∼24,000 participants, including basic scientists, clinicians, epidemiologists, behavioral scientists, politicians, activists, and many who implement prevention and treatment programs in both developed and developing countries. Also present are 3,000 journalists from over 100 countries (see ). At any one time there can be 12 concurrent sessions, official press conferences (three per hour, every hour), “global village” events put on by nongovernmental organizations (NGOs), and activist events.
The activists in particular can get creative. The threat of protests kept representatives of the drug company Abbott from manning (or even constructing) their intended booth at the conference. Activists were more than happy to take over the prime real estate, with a banner declaring “Abbott: Your booth is as empty as your promises.” The group was protesting Abbott's pricing policy for Kaletra (lopinavir/ritonavir), a two-in-one, once-a-day pill that can be stored at room temperature and is thus perfect for developing country conditions.
Less raucous groups tried to get attention by forming Global Coalitions for Just About Anything You Can Imagine. (Right now, someone is preparing a PowerPoint slide in which they incorporate GCJAAYCI, which they will use happily without explaining what the acronym stands for. The only people who like jargon more than scientists are, it turns out, international policy professionals.) These groups vie with each other for more attention using seminars, press conferences, voluminous reports, and declarations.
This circus-like atmosphere was one reason that the International AIDS Society (IAS) created a second conference: the IAS Conference on HIV Pathogenesis, Treatment, and Prevention. This science- and clinical-only event is now held in odd years (the fourth will be next year in Sydney, Australia), whereas the original all-inclusive AIDS conference is held in the even years. The latter conference remains unique, as only here does science meet policy. Basic science is needed at both conferences, said IAS president Pedro Cahn, because it progresses more rapidly than the social science and field implementation.
But the adoption of a research-only conference may have accelerated the exodus of basic science from the original AIDS conference. Even outside of conferences, HIV scientists are constantly bombarded by interview and public speaking requests, and the opportunity to escape from the policy babble has been irresistible for some. By the 2004 Bangkok AIDS meeting, the reporter Laurie Garrett was declaring in the that the conference content was “the worst science ever presented at an AIDS meeting” with “top HIV laboratory researchers…finding it irrelevant.” David Ho, director of the Aaron Diamond AIDS Research Center (New York, NY) chose not to attend this year because of the “general lack of new science” and a vaccine meeting occurring soon afterwards. “True or not,” he said by email, “the general perception is that the meeting now serves a different purpose, which is to address policy and social sciences in the field. It is not regarded as a scientific or clinical meeting any more.”
Even some of the researchers who attended agreed with this statement. Kelly MacDonald (University of Toronto, Canada) points out that the largest number of basic researchers are based in the United States, and yet the AIDS conference is never held in the US because US immigration policies restrict entry of HIV-positive individuals. That makes any AIDS meeting an expensive international meeting for US researchers. Adding that factor to all the others, she says, “This is dying as a basic science meeting.”
It is certainly easy, with policy posturing getting more of the headlines than science, for the conference to be maligned. The local newspaper welcomed delegates with a cover story about the “AIDSerati” (Bill Clinton, Bill Gates, and friends), and then featured a piece by right-wing think-tanker Michael Fumento. According to Fumento's finely reasoned article, the whole thing is overblown. After all, a very early prediction of 50 million AIDS deaths in Africa by 2005 had never come to pass—it turned out to be only 20 million. Perhaps this is why Canadian Prime Minister Stephen Harper declined the conference invitation, choosing instead to tour a military base north of the Arctic Circle.
But as the and the rest of us observed the conference in action, it was hard not to be inspired, and on several levels. First, there was the fascinating assortment process by which issues and agendas were prioritized. The most obvious case of this prioritization was the consensus on the number one message from the conference. Like a jealous gaggle of fashion designers who somehow all end up showing aubergine-colored outfits for their Spring collection, the 24,000 participants interpreted this conference's generic official theme of “Time to Deliver” as requiring a renewed emphasis on prevention and new prevention technologies (see ).
This process is not only intriguing in its own right but clearly relevant for the researcher wondering where the funding tides will turn to next. Is his or her general area now the hit of the year, and, if not, why not? Policy debates also address more nuanced questions, such as which drug classes are most in need of further research, based on feedback from the field on resistance, prices, licensing difficulties, or dosing difficulties.
The emphasis on new prevention technologies has also resulted in stronger connections between researchers and field workers, as the researchers rely on the field workers to explain how communication strategies can be best used in prevention campaigns to increase compliance. “There is a new engagement, and I think it is very encouraging,” said David Cooper (University of New South Wales, Sydney, Australia). “It's very different from clinical trials where the science of clinical trials was well-known before HIV.”
Interaction is also fostered by the shift from in vitro to patient studies, many of which are based in developing countries where NGOs may be important actors in the healthcare system. The early years of HIV research were concentrated on understanding the actions of individual HIV genes as revealed by in vitro assays. The newer research is focused on the immunological response to the virus. It was not always clear whether the conference was helping funnel community concerns from the field to those carrying out these studies in developing countries (see ), but at least the potential for interaction was there.
The connections to policy debates may be more obvious for clinical researchers, but there are other benefits for basic researchers. The conference gives everyone license to think not just about details of data but also more broadly about strategy. “It's getting so focused doing basic science,” says Tremblay. “When you are always working in the same strain…maybe you are missing something.”
The presence of activists and scientists in one place results in a dialogue that “is very helpful both ways,” said Sharon Lewin (Monash University, Melbourne, Australia). “Basic scientists need to know what is important globally. What we probably don't do well is to make [scientists and activists] intelligible to each other.”
Ideally, those who wander into the universe of basic science can bring a refreshing change in perspective. One researcher presented a study on virus entry into (and inactivation by) oral epithelial cells. It was a nice piece of science but somewhat shrouded in talk of monolayer cultures and markers of transcytosis pathways. Then came the questions—most from scientists and some from nonscientists. An audience member who appeared to fall into the latter camp asked, “What happens when you add virus-infected breast milk?” The researcher seemed amused by the naïveté of the question. But the facts in the field are simple: we know that HIV does not appreciably infect via the oral route except in babies receiving infected breast milk. In this context, the simple question was the only relevant question.
Other questioners were more direct. After a presentation by a student whose ample enthusiasm was not matched by his clarity, an audience member observed: “You mentioned several times that this was very exciting. Why?” For Killian O'Brien of AIDS Calgary (a service organization), his impatience with impenetrable science in a vaccine session was phrased more urgently. “I'm sure all this science stuff is very important,” he said, shaking with nervousness. “But I'd like something to bring back to my clients. Sometimes we need straight English.”
Some jargon will always be needed to communicate ideas that are buried deeply in a complex technological landscape of ideas. But that jargon need not preclude understanding by others. The AIDS conference featured perhaps the most science-literate nonscientists in the world—activists who tossed around names of cytokines and cell surface markers without a second thought. In O'Brien's defense, I was completely stumped by most of the talks in the session that prompted his comment.
“We adopt jargon far too much,” agreed Kelly MacDonald, who chaired the symposium in question. With fewer big name scientists attending, the talks are often left to students, and many of them had clearly had insufficient guidance from their supervisors. “The [conference organizers] have set up for people to get tips on presentation but the uptake has been very low,” said MacDonald. “We're making this field completely inaccessible.”
Restoring that access is vital both for the HIV/AIDS community and for the research community in general. Marilyn Chase of the captured very well the unique nature and social impact of what is now undeniably an HIV/AIDS industry:
This remaking is one justification for the size of the AIDS industry. Other diseases are also important, but it is on the back of AIDS interventions that the possibilities of healthcare in developing countries are being redefined. Surely the scientific community should contribute to (or at least be aware of) this process?
The AIDS meeting is a place where researchers can witness, in one place, the winding path from basic discovery through clinical research to clinical impact in both familiar and less familiar environments. For those who are perceptive, it can give clues about how society prioritizes or fails to prioritize a particular research direction. At any one time there is at least one session that is purely basic science (see ). The rest—the surrounding chaos of human contention—may not change the experiment that gets done tomorrow, but its long-term impact on personal motivation and research strategy should be profound. |
Clamping the hepatic triad of WT C57BL/6 mice for various times and reperfusing for 24 h induces considerable time-dependent liver damage. Deletion of the AR exacerbates reperfusion injury, implicating endogenous adenosine in liver protection (). Protection, as manifested by reduced serum alanine aminotransferase (ALT) levels and lessened necrotic area, is produced in WT mice by administration of the synthetic AR agonist ATL146e immediately after the initiation of reperfusion. Serum ALT levels in ATL146e-treated mice are reduced by ∼58% versus vehicle-treated controls, and the necrotic area is 6.1 ± 0.8% as opposed to 79.3 ± 3% in vehicle-treated animals (lightly stained areas are necrotic; ). RAG-1 KO mice, which lack mature lymphocytes, also exhibit reduced ALT and necrosis when compared with age- and sex-matched WT C57BL/6 mice (63% reduction in serum ALT levels and 4.5 ± 1% reduced necrotic area; ). ATL146e treatment of C57BL/6 mice and lymphocyte deficiency in RAG-1 KO mice result in similar reductions in serum ALT levels and liver necrosis. To test the hypothesis that NKT cell activity contributes to liver IRI, we examined the effects of depleting these cells or blocking their CD1d-dependent activation. Treatment of WT C57BL/6 mice with anti-NK1.1 (PK136) 2 d before liver IRI substantially depletes NKT and NK cells in the spleen and liver as assessed by FACS analysis () while leaving conventional CD4 and CD8 T cell number intact (not depicted). This depletion results in ∼60% reduction in serum ALT levels 24 h after reperfusion and a large reduction in necrotic area (8.2 ± 2% necrotic area; ). The administration of a CD1d-blocking antibody 24 h before injury elicits a similar reduction in serum ALT levels and necrosis as does PK136 treatment (). Co-treatment with either antibody in conjunction with ATL146e affords no additional protection beyond that achieved by NK1.1 cell depletion or CD1d blockade alone. These results are consistent with CD1d-restricted NKT cell involvement in hepatic IRI.
Adoptive transfer of CD4NK1.1 NKT cells collected from the spleens of WT C57BL/6 mice into RAG-1 KO mice 4 d before surgery was found to reconstitute hepatic injury after IRI. This effect is cell number–dependent, with WT levels of injury restored by the adoptive transfer of 250,000 NKT cells and intermediate injury by 150,000 cells (). Approximately 75% of the CD4NK1.1 cells transferred expressed the invariant Vα14Jα18 TCR, as indicated by binding of an α-Gal-Cer–loaded CD1d tetramer (not depicted), and FACS analysis confirmed that the adoptively transferred NKT cells reach the livers of reconstituted animals (). Although the adoptive transfer of WT NKT cells reconstitutes liver injury after IRI, the transfer of 250,000 NKT cells collected from IFN-γ KO mice fails to do so; serum ALT levels are not significantly different from RAG-1 KO controls (). The adoptive transfer of 250,000 NKT cells from AR KO mice restores injury to RAG-1 KO mice to an extent similar to transfer of WT NKT cells, but treatment with ATL146e protects from tissue damage only when WT cells are transferred; AR deletion on the NKT cells abolishes the effect of agonist administration (). These findings suggest that NKT cells play a pivotal role in hepatic reperfusion injury, that this activity is dependent on the production of IFN-γ, and that the protection elicited by ATL146e treatment is dependent on the expression of functional ARs on NKT cells.
NKT cells isolated from postischemic mouse liver and liver- draining lymph nodes after 2 h of reperfusion display an activated phenotype, as indicated by an increase in intracellular IFN-γ expression as compared with sham surgery controls. Treatment with ATL146e at the initiation of reperfusion significantly inhibits this activation. (). Because activated NKT cells are known to release large amounts of IFN-γ and to stimulate IFN-γ release from bystander cells, we also examined plasma levels of IFN-γ 24 h after reperfusion injury. IRI substantially increased plasma IFN-γ concentrations at 24 h, and treatment with ATL146e, PK136, or anti-CD1d antibodies all diminished this elevation to a similar extent (). The large accumulation of neutrophils that is observed in the postischemic liver of WT C57BL/6 mice after 24 h of reperfusion was also reduced substantially in RAG-1 KO mice and to a similar extent in mice pretreated with PK136 or CD1d blocking antibody (). These findings indicate that NKT cells are activated rapidly after the initiation of reperfusion, that ATL146e inhibits this activation, and that the large accumulation of both serum IFN-γ and hepatic neutrophils that occurs 24 h after liver reperfusion is secondary to NKT cell activation.
CD4NK1.1 NKT cells purified from spleens of WT C57BL/6 mice were activated on immobilized anti-CD3 mAb to stimulate the release of IFN-γ, as measured in cell supernatants after 24 h of incubation. TCR-stimulated IFN-γ production is inhibited by ∼73% by coincubation with 100 nM ATL146e (). iNKT cells in a mixed splenocyte culture were selectively activated in a dose-dependent manner by α-Gal- Cer, and this activation stimulated the production of IFN-γ, which is inhibited competently by 100 nM ATL146e (). The iNKT-mediated production of IFN-γ that is stimulated by 1 μM α -Gal-Cer is inhibited by ATL146e with an EC value of 0.58 nM. The addition of 100 nM of the selective AR antagonist 4-(2-[7-amino-2-[2-furyl]triazolo [2,3-]triazin-5-yl-amino]ethyl)phenol (ZM241385) causes a right shift in the ATL146e dose-response curve that is characteristic of competitive AR blockade (). Co-treatment with 1 μM of the charged sulfonic acid adenosine receptor antagonist 8-sulfophenyltheophylline (8-SPT) also blocks the inhibitory effects of ATL146e on α-Gal-Cer–mediated IFN-γ production by a mixed splenocyte culture (). Because 8-SPT cannot cross the cell membrane, this indicates that the effects of ATL146e are mediated by ARs expressed on the cell surface. It is possible that some of the IFN-γ produced by mixed splenocytes might be derived from the transactivation of conventional lymphocytes or NK cells secondary to NKT cell activation. To eliminate these possible sources of IFN-γ, we also measured the release of IFN-γ from purified CD4 NK1.1 NKT cells activated with 1 μM α-Gal-Cer in the presence of lymphocyte-deficient, NK cell–depleted RAG-1 KO splenocytes as a source of APCs. IFN-γ derived from NKT cell activation in this experiment was reduced 93% by 100 nM ATL146e, and this effect was blocked by co-treatment with 100 nM ZM241385 (). These findings demonstrate for the first time that the production of IFN-γ by NKT cells in response to CD1d-dependent activation is inhibited by activation of the AR. Blockade of ATL146e activity by ZM241385 and 8-SPT indicate that this activity is dependent on functional cell surface expression of the AR.
IRI is characterized by initial tissue damage during the ischemic period followed by progressive injury during the reperfusion period. Reperfusion is a trigger for the generation of reactive oxygen species (ROS), release of cytokines, induction of adhesion molecules on vascular endothelial cells, and the adhesion and extravasation of leukocytes into postischemic tissue. We and others have found that treatment with agonists of ARs or depletion of CD4 lymphocytes effectively reduces inflammatory processes and the amount of tissue damage that occurs during reperfusion (, –). Of the total tissue necrosis that occurs in models of heart, kidney, skin, and liver IRI, 30–75% of the tissue injury occurs during reperfusion and can be prevented by treatment with AR agonists (). In this study we show that the activation of NKT cells by a CD1d-dependent mechanism plays a central role in initiating the inflammatory cascade responsible for reperfusion injury in the liver and that these cells are key targets of AR agonists (). Based on adoptive transfer experiments of NKT cells into RAG-1 KO mice, we show that NKT cells are sufficient to cause reperfusion injury even in the absence of other lymphocytes. Additionally, we show that the activity of NKT cells to mediate liver reperfusion injury is dependent on the production of IFN-γ and that activation of the Gs-coupled AR markedly inhibits the production of IFN-γ by NKT cells both in vitro and in vivo. Although cAMP elevation has been found to inhibit CD8 NKT cell cytotoxic activity (), the current study is the first to demonstrate inhibition of CD4 NKT cell cytokine production by a cAMP-elevating AR agonist.
Liver-resident NKT cells are known to play a role in tumor surveillance and protection from hepatitis B viral infection (, –). The selective activation of NKT cells with i.p. or i.v. injection of α-Gal-Cer results in an elevation of serum IFN-γ and ALT levels and induces liver tissue damage (). The involvement of TCR activation in reperfusion injury is supported by previous work demonstrating that blockade of TCR signaling with cyclosporine treatment reduces hepatic reperfusion injury (, ). Additionally, CD1d mice demonstrate considerably reduced liver reperfusion injury as compared with WT controls (). The activity of CD1d to activate NKT cells during reperfusion implicates host glycolipid antigens, possibly derived or released from necrotic cells, in the rapid activation of the innate immune system. When activated, NKT cells rapidly release large amounts of both IL-4 and IFN-γ, which has been demonstrated to act via a STAT-1–dependent mechanism to activate Kupffer cells, as well as hepatocytes and sinusoidal endothelial cells, to produce chemokines and up-regulate adhesion molecules responsible for promoting the infiltration of leukocytes (). IFN-γ also induces the generation of ROS and endoplasmic reticulum stress proteins in hepatocytes (). Although mediators such as FasL have been shown to play a role in lymphocyte-mediated liver injury (, ), we show that NKT cell–initiated reperfusion injury is dependent on the production of IFN-γ. Although it is unlikely that conventional CD4 T lymphocytes release large amounts of IFN-γ rapidly after exposure to activating stimuli, this is a characteristic response of CD4NK1.1 NKT cells (, ), and we show that NKT cells in the liver and liver draining lymph nodes have been stimulated to produce IFN-γ by 2 h after the initiation of reperfusion. Moreover, the mouse liver contains more NKT cells than any other immune organ (), and based on these considerations and the data shown in this study, we propose that liver reperfusion injury results from an inflammatory cascade initiated by the release of IFN-γ from NKT cells. This in turn may stimulate the release of TNF-α and other cytokines from Kupffer cells, driving chemotaxis and activation of neutrophils and culminating in secondary liver injury ().
The C-type lectin receptor, NK1.1, is expressed on NKT cells and NK cells (), and both cell types can be depleted competently by anti-NK1.1 antibodies as assessed by FACS analysis of splenocytes and liver leukocytes. The protective effect of PK136, therefore, indicates that NK cells, NKT cells, or both are involved in tissue damage after IRI. CD1d, however, acts specifically to prevent glycolipid antigen presentation to NKT cells (), so the observation that the blockade of CD1d protects from hepatic IRI to a similar extent as does PK136 treatment indicates that NKT cells are the NK1.1-expressing cell type predominantly responsible for the induction of reperfusion injury. It is possible, however, that NK cells are involved in the later stages of injury, owing to their transactivation by NKT cell–released cytokines (, ). The role of NK cells in hepatic IRI warrants further investigation. Previous studies have implicated T cells in reperfusion injury (–), but a T cell– activating stimulus has not previously been clearly identified. Our data implicate CD1d-dependent antigen presentation as a key early event in the inflammatory cascade, but it is probably not the only stimulus. HO derived from ROS is produced early during reperfusion and is known to facilitate activation of T cells through the oxidation of cysteine residues on protein tyrosine phosphatases that dephosphorylate activated TCRs (, ). In addition, HO directly activates NF-κB (), resulting in widespread activation of inflammatory cells. Thus, NKT cell activation and ROS may collaborate to trigger reperfusion injury.
The results of this study implicate NKT cells as predominant mediators of hepatic reperfusion injury that are sensitive to regulation by AR activation. Residual injury that is observed after blockade of NKT activation may be due to damage caused in an inflammatory cell–independent manner during the ischemic period. The majority of, but not all, mouse CD4NK1.1 NKT cells express an invariant Vα14Jα18 TCR, and we show that these cells are activated to produce IFN-γ early after the initiation of reperfusion. Moreover, this activation is inhibited by ATL146e treatment, resulting in substantial protection from injury. These data suggest that Vα14Jα18 iNKT cells play a pivotal role in reperfusion injury. Nevertheless, there are CD1d-dependent mouse NKT cells with diverse TCRs that may also be activated during reperfusion injury if CD1d-dependent ligands for these cells are generated. Protection from iNKT cell–mediated injury by AR activation may be relevant in humans because an analogous Vα24 NKT cell population exists (), and these and similar cells in other mammalian species are activated by glypolipid antigens (). It is notable that NKT cells with the invariant TCR are considerably less abundant in human than in mouse liver, and it remains to be seen whether this reduced cell number diminishes the contribution of iNKT cells to human liver IRI. Interestingly, a subpopulation of CD1d-reactive, non-iNKT cells has been identified in human liver (). These intrahepatic cells are Th1 cell polarized and display similar activity as their invariant counterparts. It is feasible that if the reduced numbers of iNKT cells found in human liver are insufficient to induce hepatic injury after reperfusion, the specialized subset of CD1d-restricted non-iNKT cells may be poised to act in their stead or in addition to invariant cells; this possibility merits further investigation. Human NKT cells have been implicated in the pathophysiology of primary biliary cirrhosis, suggesting that these cells are physiologically important in man ().
The results of this work suggest a paradigm shift in the way we view the role of T lymphocytes in IRI. Whereas myeloid cells have previously been thought of as the major facilitators of reperfusion injury, this study indicates that the initiation of the reperfusion-induced inflammatory cascade is dependent on CD1d-mediated IFN-γ production by NKT cells. Furthermore, profound protection is imparted when this early event in the inflammatory cascade is inhibited by AR activation; through this mechanism, the release of adenosine from injured tissue may serve as an endogenous regulator of NKT cell activity. Therapeutic agents that inhibit the activity of NKT cells may therefore hold promise in the treatment of IRI. Clinicians have historically attempted to limit the by-products of reperfusion-induced inflammation via the use of neutralizing antibodies to cytokines or free radical scavengers, but it may be possible to reduce the production of these mediators more substantially by targeting an upstream event in the cascade (i.e., NKT cell activation). The activities of ATL146e to potently inhibit the production of IFN-γ by CD1d-activated NKT cells and to dramatically protect the liver from reperfusion injury indicate that AR-selective agonists may be useful tools in the treatment of IRI. Moreover, there is no evidence of severe toxicity evoked by the use of AR agonists as antiinflammatory agents. It will be interesting to see if the inhibition of NKT cell activity by AR activation proves to be a clinically viable treatment for hepatic IRI or transplantation. It also will be of interest in future studies to define the role of the NKT cells in reperfusion injury in other tissue where NKT cells are less abundant.
WT, RAG-1 KO, and IFN-γ KO C57BL/6 mice were purchased from the Jackson Laboratory. AR KO mice on a mixed genetic background were provided by J.-F. Chen (Boston University, Boston, MA). All animal studies were approved by the University of Virginia Animal Care and Use Committee.
The KO locus of B6;129P-adora2a mice with an ablated AR gene on a mixed genetic background () was moved onto a C57BL/6 background by monitoring 96 microsatellites for five generations of marker-assisted breeding. In the resulting mouse line, DNA derived from the 129 strain can be detected only in an 8-cM region between D10Mit31 and D10Mit42 surrounding the locus on chromosome 10.
WT, AR KO, or IFN-γ KO C57BL/6 mice were killed, and spleens were removed. Splenocytes were passed through a 40-mm nylon cell strainer (BD Biosciences) and collected in phosphate-buffered saline. Red blood cells were lysed, and CD4 T lymphocytes were isolated with mouse CD4 subset column kits (R&D Systems), resulting in >92% pure CD4 T cells. The column-purified cells were stained for 30 min with FITC-conjugated anti–mouse CD4 and PE-conjugated anti–mouse NK1.1 (eBioscience) and sorted using a FACSVantage SE Turbo Sorter (Becton Dickinson) to produce cell populations of ≥99.8% pure CD4NK1.1 T lymphocytes.
Cells were washed and resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotic-antimycotic (Invitrogen). In vitro activation of NKT cells was achieved by co-culture for 48 h with splenocytes and 1 nM-1 μM α-Gal-Cer (KRN7000; obtained from K. Miyayama, Kirin Brewery Company, Tokyo, Japan) at 37°C in 5% CO. Alternately, NKT cells were activated by incubation for 24 h in 96-well plates coated with 2–10 μg/ml of immobilized anti-CD3 mAb (BD Biosciences) at 37°C in 5% CO. All in vitro T cell activation experiments were performed with the addition of 1 U/ml adenosine deaminase (ADA; Roche) to remove endogenous adenosine produced by the cells that may partially activate the AR. For select experiments, cells were co-cultured with ATL146e (obtained from J. Rieger, Adenosine Therapeutics, Charlottesville, VA) in the presence or absence of 100 nM of the selective AR antagonist ZM241385 (Tocris) or 1 μM of the cell-impermeable AR antagonist 8-SPT (Research Biochemicals International).
Mice were anesthetized by i.p. injection of 100 mg/kg ketamine and 10 mg/kg xylazine. Ambient temperature was controlled in the range of 24–26°C, and mice were placed on a heating pad at 37°C. The core body temperature of selected mice was monitored with a monitoring thermometer (TH-8 Thermalert; Physitemp) and ranged from 35–36°C. After midline laparotomy, a microaneurysm clip was applied to the hepatic triad above the bifurcation to clamp the flow of the hepatic artery, portal vein, and bile duct. After superfusion of the liver with warm saline, the peritoneum was closed during 72 min of ischemia. The peritoneum was then reopened, and the microaneurysm clip was removed. For select experiments, animals received an i.p. loading dose of 1 μg/kg ATL146e or vehicle control immediately after the onset of reperfusion, and a primed Alzet osmotic minipump was implanted i.p. 10 ng/kg/min ATL146e or vehicle was placed in the pumps and delivered until the experiment was terminated. The peritoneum was sutured, and the surgical wound was closed with metal staples. Animals were killed by cervical dislocation at various time points after the initiation of reperfusion, and blood was collected via retro-orbital bleed. Additionally, livers were perfused, and left liver lobes were collected.
NK1.1-expressing cells were depleted via a single i.p. injection of 200 μg PK136 (a gift from M. Brown, University of Virginia, Charlottesville, VA) () 2 d before hepatic IRI. Successful depletion was confirmed by FACS analysis of splenocytes and liver leukocytes collected at the termination of reperfusion. CD1d was blocked by a single i.p. injection of 300 μg of anti–mouse CD1d mAb clone 1B1 (a gift from M. Kronenberg, La Jolla Institute for Allergy and Immunology, San Diego, CA) () 24 h before hepatic IRI. Anti-NK1.1 (PK136) and anti-CD1d (clone 1B1) were purified from hybridomas in the University of Virginia hybridoma core.
CD4NK1.1 NKT cells were purified from WT, AR KO, or IFN-γ KO C57BL/6 mice and adoptively transferred into RAG-1 KO mice via jugular vein injection 4 d before hepatic IRI. Successful reconstitution was confirmed by FACS analysis. Control animals received vehicle injections.
After liver ischemia, blood was collected via retro-orbital bleed 24 h after the initiation of reperfusion. Serum ALT was measured with a transaminase kit according to the manufacture's protocol (Pointe Scientific). In brief, 20 μl of undiluted or 10×-diluted serum was added to 200 μl of a preheated (37°C) mix of 500 mM -alanine and 15 mM α-ketoglutaric acid in a 96-well plate. The plate was placed in a spectrophotometer preheated to 37°C, and the absorbance at 304 nm was measured every minute for 10 min. The slope of the linear portion of the change in absorbance over time was used to calculate IU/L of ALT.
Spleens were harvested, passed through a 40-mm nylon cell strainer (BD Biosciences) and collected in phosphate-buffered saline. Red blood cells were lysed. Alternately, livers were harvested, passed through a 40-μm cell strainer, and leukocyte fractions were isolated via Percol density gradient. Cells were washed and resuspended at 5 × 10 cells/ml in PBS supplemented with 5% FBS and 0.1% NaN. 0.1-ml aliquots were placed on ice and labeled for 30 min in the dark with anti–mouse CD45, anti–mouse CD3, anti–mouse CD4, anti–mouse CD8, anti–mouse NK1.1, anti–mouse DX5 (eBioscience), and/or α-Gal-Cer–loaded CD1d tetramer (National Institute of Allergy and Infectious Disease; Tetramer Facility). Control samples were labeled with isotype-matched control antibodies. Stained cells were washed with 1 ml iced PBS and resuspended in PBS containing 1% paraformaldehyde. The fluorescence intensity was measured with a dual laser benchtop flow cytometer (FACSCalibur; Becton Dickinson) with a minimum of 10,000 events being collected. An excitation wavelength of 488 nm and an emission wavelength of 530 nm were used for FITC-stained cells; an excitation wavelength of 488 nm and an emission wavelength of 585 nm were used for PE-stained cells; an excitation wavelength of 635 nm and an emission wavelength of 661 nm were used for APCs and Alexa 647–stained cells; and an excitation wavelength of 488 nm and an emission wavelength of 670 nm were used for PE-Cy5.5–stained cells. Analysis was performed with FlowJo software (Tree Star, Inc.), and CD45 cells were gated on for analysis.
Intracellular IFN-γ was detected in liver NKT cells by FACS analysis using FIX & PERM cell permeabilization reagents (Caltag Laboratories) according to the manufacturer's protocol.
Mice were killed, and livers were perfused with saline via the portal vein at various times after the initiation of reperfusion. Left liver lobes were harvested, fixed in 4% paraformaldehyde in PBS, pH 7.4, and embedded in paraffin. 4-μm sections were subjected to standard hematoxylin and eosin (H&E) staining. Necrotic area was quantified using Photoshop software (Adobe).
IFN-γ concentrations in cell culture supernatants or serum samples were measured by ELISA according to the manufacturer's protocol (eBioscience).
Prism software (GraphPad) was used for all statistical analyses. Unpaired tests or one-way analysis of variance (ANOVA) with post-hoc Dunnett's multiple comparison were used to compare experimental groups with a control group. |
IL-23p19 mRNA expression was increased in lesional psoriatic skin compared with nonlesional skin from psoriatic and normal donors (, left), in agreement with a previous report (). Similar results were observed for IL-12p40, including increased expression in nonlesional psoriatic skin compared with normal controls (, middle). In contrast, IL-12p35 expression was not increased but actually decreased in lesional psoriatic skin compared with nonlesional and normal skin (, right).
Because IL-23 is elevated in human psoriasis, we injected IL-23 protein intradermally into mice to explore the downstream consequences of aberrant cutaneous IL-23 exposure. Wild-type mice treated daily with IL-23, but not saline, developed visually apparent erythema and induration () and associated prominent dermal papillary blood vessels (). Induration was apparent by day 2 and persisted to day 7. Erythema was also apparent at day 2 but began to recede at day 5. Histological evaluation of IL-23–treated skin showed epidermal (and follicular) hyperplasia accompanied by parakeratosis at day 4 (, middle, red arrows). No rete ridges were observed, although hair follicles cut in the appropriate plane may falsely suggest otherwise. Focal neutrophilic exudates, reminiscent of Munro microabscesses, were often observed (, middle, green arrows). Markedly less parakeratosis and fewer focal neutrophilic exudates were observed by day 7 (unpublished data).
The specificity of this observation was confirmed using IL-23R–deficient mice (Fig. S1, available at ), which did not respond to intradermal IL-23 treatment (, right). IL-23–induced epidermal thickening was maximal by day 4 and began to decrease by day 7 (). IL-12 treatment, in comparison, did not induce significant changes in epidermal thickness. No changes in epidermal thickness were observed with TNF treatment either (), in agreement with a previous report (). Bioactivity of these cytokines was confirmed by examining expression of IL-12–dependent (IFN-γ, CXCL9, and CXCL10) and TNF-dependent (VCAM-1) genes in skin samples (Fig. S2). Immunohistochemical analysis demonstrated a mixed dermal infiltrate consisting of CD4 T cells, CD11c dendritic cells, F4/80 macrophages, and neutrophils that began 1 d after IL-23 treatment and was markedly increased by day 4 (Fig. S3). No CD8 T cells or increased numbers of mast cells were detected at either time point, in contrast to psoriasis (unpublished data). Aside from the obvious local changes in skin appearance, the IL-23 treatment regimen was well tolerated because the mice appeared healthy and did not lose weight during the course of the experiment. Serum cytokine levels, including GM-CSF, IL-1β, IL-2, IL-4, IL-5, IL-6, TNF, and IFN-γ, remained unchanged compared with saline-treated controls (unpublished data).
Epidermal thickening associated with IL-23 exposure was examined in detail using transmission electron microscopy. Hyperplastic skin was selected from animals without histopathologic evidence of suppurative dermatitis (day 4) for the purpose of defining ultrastructural features of the model in the absence of cellular infiltrates. Acanthosis (increased thickness of the stratum spinosum) was a predominant feature and accounted for the majority of the epidermal thickening and was characterized by spinous cell hyperplasia, hypertrophy, and spongiosis (intercellular edema) (). Spinous cells, although enlarged, maintained normal cytoplasmic contents and membrane structure. The granular cell layer (stratum granulosum) was more prominent in IL-23–treated treated skin when compared with untreated control skin. However, it was still very sparse, even in severely thickened skin, and often showed retained “end-stage” nuclei (i.e., nuclei with condensed and marginated chromatin). Nuclear fragmentation was not observed within this layer. The stratum corneum was characterized by altering areas of hyperparakeratosis (cells with retained epithelioid nuclei, keratohyalin granules, and compacted keratin filaments) and orthohyperkeratosis (). These results are discussed in the context of psoriasis keratinocyte biology in the Discussion. Additional transmission electron microscopy pictures are available at .
IL-17A is a proinflammatory cytokine that lies downstream of IL-23 in the pathogenesis of experimental autoimmune encephalomyelitis and whose presence is correlated with the progression of collagen-induced arthritis (, ). Human psoriatic skin has elevated IL-17A gene expression in lesions relative to nonlesional skin and normal controls (). IL-17A mRNA expression was increased in IL-23–treated mouse skin at day 1 and remained elevated at day 4 (). We used a blocking monoclonal antibody to IL-17A to determine whether IL-17A is required for IL-23–dependent epidermal thickening. Anti–IL-17A did not inhibit IL-23– stimulated acanthosis (), nor did it affect IL-23–dependent erythema, induration, and parakeratosis (unpublished data). Anti–IL-17A treatment inhibited IL-23–stimulated G-CSF () and MMP-13 expression (unpublished data), genes previously shown to be IL-17A regulated (, ). This anti–IL-17A antibody is efficacious at inhibiting experimental autoimmune encephalomyelitis, where IL-17A expressed in the central nervous system is a key pathogenic factor that contributes to paralysis (), and also in inhibiting collagen-induced joint inflammation models, where IL-17A is a key pathogenic factor driving cartilage and bone destruction (unpublished data). Intradermal IL-17A delivery caused significant keratinocyte hypertrophy, but minimal if any hyperplasia by day 4 (unpublished data).
TNF is a validated target for various inflammatory diseases, including psoriasis (, ). TNF mRNA was elevated in human psoriatic skin relative to normal controls () and was also induced in mouse skin by intradermal IL-23 treatment at day 1, but not at day 4 (). TNF protein was also detected by ELISA in IL-23–treated skin (unpublished data). We used a blocking monoclonal antibody to TNF to determine whether TNF is involved in IL-23–dependent epidermal thickening. In contrast to anti–IL-17A, anti-TNF treatment partially inhibited IL-23–dependent acanthosis (), and the degree of erythema and induration was decreased.
Immunohistochemical analysis of IL-23–treated skin at day 1 showed increased Ki67 cells in the epidermis, suggesting that keratinocytes had received an IL-23–stimulated proliferative signal by day 1 (). This observation was confirmed by increased keratin 16 (K16) gene expression, which is associated with keratinocyte hyperplasia in psoriasis (). Elevated K16 was evident at day 1, before changes in epidermal thickness, and was further increased at day 4 (). Increased expression of K16 was induced by IL-23, but not IL-12 or TNF (). Gene expression analysis of skin samples at day 1 suggested that the effects of IL-23 treatment on epidermal hyperproliferation were not due to increased gene expression of a panel of keratinocyte growth factors (). Furthermore, in vitro assays indicated that IL-23 did not have a direct effect on keratinocyte proliferation ().
Because IL-23 did not stimulate the expression of any growth factors tested at day 1 (a time point before visual epidermal thickening, but after a keratinocyte proliferative signal had been received based on Ki67 staining and K16 expression), we examined the novel IL-19 family of cytokines, which consists of IL-19, IL-20, and IL-24. These cytokines all bind the heterodimeric receptor IL-20R1/IL-20R2. IL-20 and IL-24 can also bind IL-22R1/IL-20R2 (). These receptors are highly expressed in epithelial cells, including keratinocytes (, ), and are all elevated in human psoriatic skin (), suggesting a possible role in disease pathology. IL-23 delivery to mouse skin elevated IL-19 and IL-24, but not IL-20, gene expression (, left). IL-23 did not increase IL-20R1, IL-20R2, or IL-22R1 gene expression in mouse skin (unpublished data). IL-19, IL-20, and IL-24 mRNA was also increased in human psoriatic skin relative to normal controls (, right). IL-20R1 and IL-22R1 were elevated in psoriatic skin compared with normal controls, whereas no significant changes were observed for IL-20R2 (Fig. S4, available at ).
IL-23 was delivered into the skin of IL-20R2 mice (Fig. S5, available at ) to simultaneously examine the role of all three IL-19 family cytokines. Epidermal thickening was significantly inhibited in IL-23–treated IL-20R2 mice compared with IL-23–treated wild-type controls (), despite the induction of IL-19 and IL-24 gene expression (). IL-23 was delivered into the skin of IL-19 and IL-24 mice (Fig. S6 and S7, respectively) to determine if IL-19 or IL-24 was required for IL-23–dependent epidermal hyperplasia. IL-23–stimulated epidermal thickening was observed in both IL-19 and IL-24 mice (). These data suggest that these cytokines are redundant in function because IL-24 and IL-19 gene expression was induced in IL-19 and IL-24 mice, respectively (). IL-20 gene expression was not significantly altered by IL-23 in skin from either IL-19 or IL-24 mice compared with wild-type controls (unpublished data). Importantly, in vitro stimulation of keratinocytes with increasing quantities of IL-19, IL-20, or IL-24 failed to stimulate proliferation, suggesting that IL-20R2 ligands are required but not sufficient for IL-23–stimulated epidermal hyperplasia (unpublished data).
We performed immunohistochemical analysis on IL-23–treated IL-20R2 skin to determine whether impaired signaling through IL-20R2 affected the cellular composition of the dermal infiltrate. Although epidermal thickening was not apparent in IL-23–treated IL-20R2 mice, there were elevated numbers of CD4 T cells and CD11c dendritic cells compared with saline-treated control mice, which was similar to IL-23–treated wild-type mice (, respectively). In contrast, there were reduced numbers of dermal F4/80 macrophages and neutrophils in IL-20R2 mice (). summarizes the differences between human psoriasis and wild-type or IL-20R2 mice in their responses to intradermal IL-23 treatment.
Because IL-23p19 and IL-12p40 gene expression was elevated in lesional psoriatic skin compared with nonlesional psoriatic skin and normal controls, we tested the hypothesis that IL-23 was involved in disease pathogenesis by injecting IL-23 intradermally into mice. IL-23 induced changes in mouse skin that share many features with human psoriasis, as summarized in . The most striking similarity to psoriasis microscopically was the acanthosis (epithelial hyperplasia) and parakeratosis (keratinocyte nuclei in the stratum corneum).
Histological analysis of psoriatic skin reveals the absence of the normal granular layer where normal keratinocyte differentiation starts to give rise to a highly cross-linked, lipid-rich anuclear structure that provides the first barrier to the environment (i.e., stratum corneum). In normal skin, the granular layer is defined by numerous keratinocyte nuclei undergoing condensation and disintegration. Psoriatic skin, however, has little to no keratinocyte nuclei disintegration, which ultimately results in a modified stratum corneum with retained keratinocyte nuclei (parakeratosis). One difference between the acute IL-23–induced skin hyperplasia response and the chronic psoriatic lesional skin, however, is that a modified granular layer is present. IL-23–stimulated granular layer keratinocytes have nuclei condensation but no evidence of nuclei disintegration. This gives rise to parakeratosis, similar to psoriasis, by a slightly different mechanism than observed in psoriasis. Whether this is due to the IL-23–stimulated hyperplasia being an acute self-limiting condition versus the chronic nature of psoriatic lesions is under investigation. Reversible edema in the epidermis (spongiosis) was also observed in the acute model, which is not commonly seen in chronic psoriatic skin lesions. A rare disease, granular parakeratosis, is characterized by parakeratosis but in combination with a granular layer. IL-23 may also play a role in this disease due to the similarities between it and our observations in this mouse model.
An IL-12p40 antagonist that blocks IL-23 and IL-12p70 simultaneously was successful in a phase I psoriasis clinical trial (), and IL-23p19, but not IL-12p35, gene expression was associated with human psoriasis (). These data plus the observation that intradermal injections of IL-12 did not cause psoriasiform lesions in mice are consistent with IL-23 being the relevant target of anti–IL-12p40 therapy in psoriasis. It is noteworthy that IL-12 treatment did not induce psoriasiform lesions despite enhanced IFN-γ expression because human psoriatic lesions have a distinct signature of IFN-γ–regulated genes (). This raises the possibility that IL-23 (dys)regulation may trigger psoriatic lesion formation and that IFN-γ–dependent processes may contribute to the chronicity of disease. This notion is consistent with IL-23 production early in an innate immune response to bacteria (–) that may initiate psoriatic lesions and with the multigenic nature of this disease ().
IL-23 is a potent regulator of memory T cell development and/or expansion that is skewed to IL-17A secretion (), and IL-23 has been implicated as a key disease driver in autoimmune disease models (, ). The human psoriatic data demonstrate that IL-17A gene expression is also associated with disease. Intradermal IL-23 injections stimulated elevated IL-17A expression; however, pretreatment with an antagonistic antibody did not ameliorate psoriasiform lesion formation. These data and the observation that direct IL-17A delivery has minimal effect on epidermal hyperplasia suggest that IL-17A, although downstream of IL-23 and associated with human psoriasis, is not required for disease pathogenesis. It is possible that IL-17F, whose expression is also skewed by IL-23 during memory T cell development () and is driven by IL-23 in this model (unpublished data), may compensate in some way for IL-17A because they both can bind IL-17RC (). Interestingly, psoriatic patients are relatively resistant to cutaneous infections (). IL-17A and IL-17F may play key roles in host defense to cutaneous infections because they stimulate neutrophil recruitment (, ), and IL-17A directly stimulates the anti-microbial gene β-defensin 2 in epithelial cells ().
IL-23 stimulates macrophage TNF production () and IL-23p19 transgenic mice have elevated serum TNF levels (). Neutralizing TNF partially inhibited epidermal hyperplasia induced by IL-23 in our study. This observation is consistent with the efficacy of anti-TNF–directed therapies in psoriasis (, ). TNF inhibition breaks the self-sustaining nature of psoriatic lesions by rapidly down-regulating several proinflammatory genes, including IL-23p19 and IL-19 (). TNF alone, however, was not sufficient to induce epidermal hyperplasia, indicating that IL-23 stimulates other signaling pathways that synergize with TNF for disease pathogenesis.
The physiologic functions of the structurally related cytokines IL-19, IL-20, and IL-24 appear to be quite diverse despite their shared receptor profile. IL-19 may be involved in the induction of Th2 responses (). IL-20 appears to play a key role in skin biology because IL-20 transgenic mice have aberrant epidermal differentiation with no immune cell infiltrates (). Adenoviral delivery of IL-24 (MDA-7/mob-5/c49a) induces apoptosis in tumor cells (), and IL-24 gene expression is increased during wound healing in the rat (). The common link between IL-19, IL-20, and IL-24 is their use of heterodimeric receptors that share the IL-20R2 subunit: IL-20R1/IL-20R2 heterodimer and IL-22R1/IL-20R2 heterodimer (). These receptors are primarily expressed in epithelial cells such as keratinocytes (, ) and are elevated in human psoriatic lesions (, ). Our human psoriatic data confirm that IL-20R1 and IL-22R2 are increased in psoriatic skin, although we did not detect a statistically significant increase in IL-20R2 mRNA compared with normal skin. Administration of IL-23 to IL-20R2 mice, but not IL-19 or IL-24 mice, resulted in significantly decreased acanthosis and parakeratosis. This suggests that either IL-19 or IL-24 may be sufficient to mediate epidermal hyperplasia via IL-20R2–containing receptors, although it is unclear whether these cytokines directly stimulate keratinocyte proliferation or alter the keratinocyte differentiation program.
Interestingly, cutaneous IL-23 exposure also drives high-level expression of IL-22, a cytokine structurally related to IL-19, IL-20, and IL-24 (unpublished data). IL-22 binds the heterodimeric receptor IL-22R1/IL-10RB, but it does not use IL-20R2. IL-22 activation of keratinocytes stimulates a variety of antimicrobial activities, such as psoriasin (S100A7), calgranulin A (S100A8), calgranulin B (S100A9), β-defensin 2, and β-defensin 3 (–). Therefore, cutaneous IL-23 expression in psoriatic skin drives both IL-22–dependent and IL-17A/IL-17F–dependent antimicrobial responses that protect the host.
The cellular mechanisms by which IL-23 drives IL-20R2–dependent epidermal hyperplasia and psoriasiform lesions remain to be investigated. Analysis of the inflammatory cell infiltrate in IL-23–treated IL-20R2 mice revealed decreased numbers of neutrophils and F4/80 macrophages in the dermis, suggesting that these recruited cells may contribute to the lesion development in our model. In support of this possibility, neutrophil depletion inhibits acanthosis in the fsn/fsn mouse, which spontaneously develops flaky skin (). Infiltrating myeloid cells can produce several factors associated with psoriasis, including iNOS, IL-19, MMP-12, and IL-23 ().
T cells, monocytes, and NK cells express IL-23R (), which raises the possibility that IL-23 may act directly on these cells to produce IL-19 and IL-24. Published data show that the major hematopoietic sources of IL-19 and IL-24 are monocytes and monocytes and T cells, respectively (). IL-23–treated bone marrow macrophages do not express IL-19, IL-20, or IL-24 (unpublished data), suggesting that IL-19 and IL-24 expression may be indirectly stimulated by IL-23. This notion is supported by our data that keratinocytes do not respond to IL-23 and that IL-19 is expressed by keratinocytes in psoriatic lesions (). TNF may be important here as IL-23 stimulates TNF expression in macrophages () and TNF directly stimulates IL-19 expression in normal human epidermal keratinocytes (unpublished data).
In summary, our data propose a molecular mechanism by which IL-23 dysregulation is one of the causative factors in psoriasis pathogenesis. Dysregulated cutaneous IL-23 production sets into motion several independent pathways. IL-23 contributes to the antimicrobial nature of psoriatic lesions by stimulating IL-17A (and IL-17F) and neutrophil recruitment. In parallel, IL-23 stimulates IL-19 and IL-24, which may directly act on keratinocytes in a TNF-regulated manner resulting in epidermal hyperplasia and/or altered keratinocyte differentiation. These functionally different arms suggest that IL-23 may have evolved as a “response to danger” cytokine invoking the body to protect itself by rapidly mobilizing antimicrobial components and instructing the epidermis to proliferate to provide additional protection from the environment. Given that IL-23–dependent epidermal hyperplasia was inhibited in IL-20R2 mice, targeting IL-20R2 and/or its associated receptors may be a novel therapeutic strategy for the treatment of this disease.
Psoriasis patients ( = 45) and normal volunteers ( = 30) consented under a protocol approved by the Stanford Panel on Human Subjects. Psoriasis patients needed to have a Psoriasis Area Severity Index of at least 8 and a typical lesion at least 1 cm in size suitable for biopsy. The target lesion and the surrounding 5-cm area could not have been treated with any medicated topical formulation for at least 2 wk before obtaining the biopsy. Patients treated with systemic immunosuppressives including corticosteroids were excluded. One 4-mm biopsy from a lesional and an adjacent nonlesional site was collected from psoriatic patients. One 4-mm biopsy was obtained from each normal volunteer. Specimens were flash frozen in liquid nitrogen.
Generation of IL-23R, IL-20R2, IL-19, and IL-24 mice are described in Online supplemented material. Recombinant human and murine IL-23 have been described (). Recombinant murine IL-12 was generated using approaches described previously (). Murine TNF and human keratinocyte growth factor 1 (KGF) were purchased (PeproTech). Rat anti–IL-17A (JL7.1D10) (), rat anti-TNF (MP6-XT22) (), and isotype control antibody (25D2) were purified from hybridoma culture supernatants. Rat anti-Ki67 (TEC-3; DakoCytomation), rat anti-neutrophils (7/4; Serotec), rat anti-CD4 (L3T4; BD Biosciences), rat anti-F4/80 (A3-1; Serotec), hamster anti-CD11c (HL3; BD Biosciences), and rat anti-CD31 (MEC 13.3; BD Biosciences) were purchased.
All animal protocols were approved by DNAX/Schering-Plough Biopharma's Institutional Animal Care and Use Committee. Hair was removed from the back of mice with electric clippers and a cream depilatory (Nair). 3 d later, mice were injected intradermally with IL-23, vehicle control, or other cytokines (IL-12 or TNF) in two locations on either side of the back for a total of 1 μg protein per mouse using a 29.5-gauge needle. Sterile saline was used as a vehicle control. Injections were performed daily until mice were killed as per the objective of the experiment. For antibody blocking studies, mice were treated subcutaneously with 0.2–1.0 mg anti–IL-17A, anti-TNF, or isotype control 2 d before the first cytokine injection. Mice were killed using carbon dioxide and blood was collected by cardiac puncture. Skin samples were removed from the prepared area, keeping at least 5 mm away from the hair boundary. Skin samples were frozen directly in liquid nitrogen for mRNA extraction, fixed in 10% neutral buffered formalin for histology, or embedded and frozen in OCT for immunohistochemistry.
Skin samples were fixed in a solution containing 2.5% glutaraldehyde and 2% formaldehyde with 0.12 M sodium cacodylate followed by secondary fixation in buffered 1% OsO. The samples were then dehydrated and embedded in epoxy resin by standard methods. Thin sections were stained with 5% uranyl acetate in 50% methanol and 1% aqueous lead citrate, and images were captured using an electron microscope (CM10; Philips).
RNA isolation was performed by standard techniques, and gene expression was calculated using the Δ-ΔCt method (using the mean cycle threshold value for ubiquitin and the gene of interest for each sample. The equation 1.8e (Ct ubiquitin − Ct gene of interest) × 10 was used to obtain the normalized values.
Skin sections were stained with hematoxylin and eosin, and epidermal thickness was determined by measuring the average interfollicular distance from the basal lamina to the bottom of the stratum corneum in a blinded manner. Similar results were obtained for wild-type mice on 129SvEv, C57BL/6, or mixed B6 × 129 genetic backgrounds. For immunohistochemistry, 8-μm sections were fixed with 75% acetone/25% ethanol. Endogenous peroxidase was quenched with Peroxidase Blocking Reagent (DakoCytomation) and endogenous biotin was blocked with SP-2001 (Vector Laboratories). Primary antibodies were incubated for 30 min at room temperature and visualized using VECTASTAIN elite ABC kit (Vector Laboratories). Positive staining developed as a brown reaction precipitate.
In vitro keratinocyte proliferation assays were performed as described previously (), with minor modification. Normal human epidermal keratinocytes (Cambrex) were seeded at 2,000/well in a 24-well plate in keratinocyte basal medium (Cambrex) supplemented with hydrocortisone (Cambrex) and 5 μg/ml human recombinant insulin (Roche Diagnostics). During seeding, cells were treated with vehicle control, 1–100 ng/ml human IL-23, or 10 ng/ml KGF as a control. After 6 d, cells were washed and incubated with 0.2% crystal violet (Sigma-Aldrich). Cells were lysed with 1% SDS and absorbance was read at 565 nm. The total number of cells per well was determined by reference to a standard curve. All conditions were performed in triplicate.
Human data was analyzed by Kruskal-Wallis with Dunn's Multiple Comparison after test. One-way or two-way ANOVA with Bonferroni after test were used where appropriate. P < 0.05 was considered to be statistically significant.
Fig. S1 is the gene deletion strategy for the IL-23R knockout mouse. Fig. S2 shows the in vivo bioactivity of intradermally injected IL-12 and TNF. Fig. S3 shows the IL-23–dependent inflammatory cell infiltrate into mouse skin. Fig. S4 shows the IL-19 family receptor subunits in human psoriasis. Figs. S5–S7 show the gene deletion strategy for IL-20R2, IL-19, and IL-24 knockout mice, respectively. Fig. S8 is a high magnification hematoxylin and eosin–stained section showing general features of an IL-23–mediated model. Fig. S9 shows a high magnification hematoxylin and eosin–stained section illustrating an epidermal microabscess. Fig. S10 is a high magnification electron micrograph (EM) of spinous (bottom) and granular (top) layers (note presence of intercellular edema). Fig. S11 is a medium-high EM of junction between spinous and granular layers (note the presence of nuclear degeneration [condensed chromatin] in a granular cell [red arrow] vs. a more normal nucleus in a spinous cell [blue arrow]). Fig. S12 is a low magnification EM from a control animal illustrating the “normal” appearance of the epidermis (note that the granular layer is present, but not prominent, and there is little keratin). Fig. S13 is a high magnification EM from a control animal illustrating the “normal” appearance of the corneum (note the absence of nucleated keratinocytes and sparse keratohyalin granules). Table S1 is genotyping primers for analyzing knockout mice. The online supplemental material is available at . |
Subsets and Splits