Interleukin 10- and Fc Receptor-Deficient Mice Resolve Leishmania mexicana Lesions
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感染与免疫杂志 2005年第4期
VA Medical Center
Department of Medicine, Division of Infectious Diseases, School of Medicine
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
ABSTRACT
Infection of C57BL/6 (B6) mice with Leishmania mexicana is associated with a minimal immune response and chronic disease. Here we show that B6 interleukin 10–/– (IL-10–/–) mice resolve their lesions and exhibit increased gamma interferon (IFN-), nitric oxide production, and delayed-type hypersensitivity. This enhanced resistance was dependent upon IL-12p40, since treatment of L. mexicana-infected IL-10–/– mice with anti-IL-12p40 monoclonal antibody abrogated healing. Antibody-opsonized L. mexicana induced IL-10 production by B6 macrophages in vitro, implicating antibody binding to Fc receptors as a mechanism involved in IL-10 production in this infection. Furthermore, B6 FcR–/– mice resolve L. mexicana lesions, and lymph node cells from these mice produced less IL-10 and more IFN- than cells from infected wild-type mice. These data demonstrate that removal of IL-10 or FcR leads to resolution of L. mexicana disease and support a model in which ligation of FcR by L. mexicana-bound immunoglobulin G promotes IL-10 production, leading to chronic disease.
INTRODUCTION
The protozoan parasite Leishmania mexicana causes chronic cutaneous disease in C57BL/6 (B6) mice, which are able to resolve infections with Leishmania major (5). Human L. mexicana infections often mirror this with more frequent chronic disease, an example being nonhealing ear infections, known as Chiclero's ulcer (40). While the immunologic responses involved in susceptibility to L. major have been described in depth, those contributing to the chronic disease seen during infection with American species of Leishmania are less well understood. The two immunosuppressive cytokines most associated with nonhealing forms of leishmaniasis are interleukin 4 (IL-4) and IL-10. For example, L. major infection in BALB/c mice results in nonhealing disease associated with high levels of IL-4, and neutralizing IL-4 can promote resistance (21). IL-10 also contributes to susceptibility in BALB/c mice and also modulates protective immune responses in B6 mice infected with L. major (8, 27, 37). In contrast, Leishmania amazonensis, a species closely related to L. mexicana that also causes chronic disease in B6 mice, still causes chronic infection of IL-4- and IL-10-deficient mice (24, 25). However, while previous studies indicate that IL-4 and IL-13 may contribute to the chronicity of L. mexicana infections (3, 45), the role of IL-10 in the chronic disease induced by L. mexicana in B6 mice has not been investigated.
The immune responses associated with L. mexicana infection are distinct from those observed following infection with L. major. L. major infection of B6 mice induces a significant immune response, characterized by increased cell migration to the draining lymph node (LN), proliferation of Leishmania-specific T cells, and production of gamma interferon (IFN-) (reviewed in references 41 and 43). In contrast, the magnitude of the immune response to L. mexicana or L. amazonensis is limited. Notably, mouse strains resistant to L. major not only fail to develop a dominant Th1 response when infected with these New World parasite species, they also show no evidence of a strong Th2 response (1, 45). Furthermore, in contrast to L. major lesions, lesions from L. mexicana-infected mice are characterized by a limited lymphocytic infiltrate. These observations suggest that the chronic nature of L. mexicana infections may not be linked with a Th2 response but rather is more likely the result of generalized immunosuppression mediated by IL-10. Studies with infectious diseases and models of autoimmunity indicate that the regulatory role of IL-10 can be vital to blocking immunopathologic responses. For example, infections of IL-10-deficient mice with Toxoplasma and Plasmodium result in fatal inflammatory responses (15, 32), and similarly, IL-10-deficient mice have spontaneous autoimmune colitis (31) and more severe experimental autoimmune encephalomyelitis (10, 44). These models demonstrate that IL-10 can play a critical protective role against immunopathology. Interestingly, however, there is no evidence that IL-10–/– mice infected with Leishmania—either L. major, L. amazonensis, or Leishmania donovani—develop an uncontrolled inflammatory response. Rather, in all cases the absence of IL-10 has been associated with enhanced, but not uncontrolled, immune responses (24, 27, 36).
IL-10 can be produced by several different cell types, including macrophages, dendritic cells, B cells, and T cells (35). Recently CD4+ CD25+ regulatory T (Treg) cells have been shown to suppress immune responses in L. major-infected mice, including Th1 responses in B6 mice and the initial development of Th2 responses in BALB/c mice (6, 9). Critically, these Treg cells may function through the production of IL-10; CD4+ CD25+ T cells from L. major lesions produced high levels of IL-10 when stimulated in vitro with L. major-infected dendritic cells, and Treg cells from IL-10-deficient mice were unable to suppress effector-T-cell responses following adoptive transfer to RAG–/– mice. However, other studies indicate that IL-10 from macrophages might also be crucial in promoting susceptibility to leishmaniasis, since overexpression of IL-10 in major histocompatibility complex class II+ cells, but not T cells, promoted susceptibility (19, 20). One pathway that leads to IL-10 production involves ligation of the macrophage FcR by immune complexes (51). In fact, L. major parasites opsonized with antibody were capable of augmenting lipopolysaccharide (LPS)-induced IL-10 production by macrophages in an FcR-dependent manner (27).
The role of IL-10, FcR, and immunoglobulin G (IgG) in leading to susceptibility to infections of B6 mice by New World Leishmania species, such as L. mexicana and L. amazonensis, is not clear. IL-10 does play a role in preventing the complete clearance of L. major in B6 mice (8) and is required for progressive disease in the much more susceptible BALB/c strain (27). In BALB/c mice, which are also much more susceptible than B6 mice to L. mexicana and other New World Leishmania strains, IL-10 contributes to susceptibility to L. mexicana, but both IL-10 and IL-4 must be blocked to achieve resistance (39). FcR and IgG have been shown to contribute to susceptibility of BALB/c mice to the related New World parasites Leishmania pifanoi and L. amazonensis (29), but this has not been linked with IL-10 production. In addition, B6 IL-10–/– mice do not heal L. amazonensis infections despite an increase in IFN- at early time points (24). Thus, the roles of IL-10, FcR, and IgG in L. mexicana infection are by no means understood, even in BALB/c mice, and have not been investigated in the relatively more resistant B6 mice, whose disease with both L. major and L. mexicana more closely resembles human leishmaniasis.
In the present study, we demonstrate that in the absence of IL-10, B6 mice are able to resolve L. mexicana infections. Resolution of infection was also observed in mice lacking FcR and circulating IgG, which implicates macrophages—stimulated through the FcR by IgG-opsonized parasites—as a critical source of IL-10. Consistent with this hypothesis, we demonstrate that IgG bound to the surface of L. mexicana promotes the induction of IL-10 from B6 macrophages in vitro. We further show that LN cells from L. mexicana-infected FcR–/– mice produce less IL-10 on antigen restimulation than cells of control mice. These studies reveal that IL-10 is required to suppress a healing immune response to L. mexicana in B6 mice and also to uncover a critical role for antibodies, through FcR binding, in this suppression.
MATERIALS AND METHODS
Mice. B6, B6 IL-4–/–, B6 IL-10–/–, and B6 2-microglobulin (2m)–/– mice were purchased from Jackson Laboratory (Bar Harbor, Maine). B6 FcR–/– and B6 control mice were purchased from Taconic (Germantown, N.Y.). Courses of infection consisted of groups of five mice per experiment, and rechallenge was performed on two to five mice per group. Mice were purchased at 4 to 6 weeks and were sex and age matched for all experiments. Animals were maintained in a specific-pathogen-free environment, and the animal colony was screened regularly, and tested negative, for the presence of murine pathogens.
Parasites and antigens. L. mexicana (MNYC/BZ/62/M379) promastigotes, provided by J. Alexander (University of Strathclyde, Glasgow, United Kingdom), were grown at 27°C in Grace's medium (pH 6.3; Life Technologies, Grand Island, N.Y.) supplemented with 20% heat-inactivated fetal bovine serum (FBS) (HyClone Labs, Logan, Utah), 2 mM L-glutamine, 100 U of penicillin/ml, and 100 μg of streptomycin/ml. Stationary-phase promastigotes (day 7 of culture) were washed three times in phosphate-buffered saline (PBS) and 5 x 106 parasites were injected into the hind footpad of mice. Lesions were monitored using a metric dial caliper. To assess delayed-type hypersensitivity (DTH), mice were injected with 5 x 106 parasites in the opposite footpad, and the baseline uninjected footpad thickness was subtracted from the injected footpad thickness at 48 h. Lesion-derived amastigotes were obtained from footpad lesions of mice chronically infected with L. mexicana. Axenic amastigotes were prepared by placing stationary-phase cultures of L. mexicana promastigotes at 32°C for 2 days with passage every 7 to 10 days at 1/100 in acidic Grace's medium (pH 5.5) supplemented as described above. Freeze-thaw antigen (FTAg) was prepared from L. mexicana stationary-phase promastigotes that were washed four times in PBS, resuspended at 109/ml (yielding 1 mg/ml of protein), and frozen (–80°C) and thawed rapidly (37°C) for five cycles.
Assay of cytokines and NO from in vitro restimulation. Single-cell suspensions were prepared from draining LNs, and 200-μl samples (8 x 105 cells) were cultured in 96-well tissue culture plates in Dulbecco's modified Eagle's medium (Mediatech, Herndon, Va.) supplemented with 10% heat-inactivated FBS, 25 mM HEPES (pH 7.4), 50 μM 2-mercaptoethanol, 2 mM L-glutamine, 100 U of penicillin/ml, and 100 μg of streptomycin/ml. Cells were stimulated with 10 μg of L. mexicana FTAg/ml (107 cell equivalents/ml) for 3 days at 37°C in a 5% CO2 incubator, and supernatants were assayed by enzyme-linked immunosorbent assay (ELISA) for IFN- as previously described (46) and for IL-10 using commercial antibodies as recommended by the manufacturer (BD Bioscience, San Diego, Calif.). Uninfected mice had no detectable IL-10 or IFN- production with antigen stimulation in these experiments. NO production was assayed by measuring nitrite (NO2) in 3-day supernatants with the Griess reagent as previously described (18). Cytokine and NO data shown are for groups of three to five individual mice.
In vitro infection of BMM. BMM were grown from B6 mice on petri dishes in 10 ml of complete macrophage medium (Dulbecco's modified Eagle's medium containing 10% heat-inactivated FBS, 100 U of penicillin/ml, 100 μg of streptomycin/ml, 2 mM L-glutamine, 25 mM HEPES, 30% L929 cell-conditioned medium) for 7 days (7.5 x 106 cells per petri dish) with an additional 10 ml of complete macrophage medium added on day 3 (27, 52, 53). Macrophages were harvested by gentle scraping in cold PBS (4°C), washed, and replated at 2 x 105/0.5 ml in 24-well plates in macrophage medium lacking L929 cell-conditioned medium. After resting overnight and being washed with fresh medium, LPS from E. coli O128:B12 (Sigma-Aldrich, St. Louis, Mo.) was added at a 100-ng/ml concentration, and macrophages were infected at a 10:1 multiplicity of infection with lesion amastigotes, axenic amastigotes, or axenic amastigotes opsonized for 30 min at 4°C with 50 μl of a 1/40 dilution of serum from B6 mice chronically infected long term with L. mexicana. Supernatants were collected, frozen at –20°C, and assayed for IL-10 by ELISA as described above.
Flow cytometry. Amastigotes (106) were washed in fluorescence-activated cell sorter (FACS) buffer (PBS with 0.1% sodium azide, 0.1% bovine serum albumin [Sigma-Aldrich]), and labeled with fluorescein isothiocyanate-conjugated goat antimouse IgG (Fc specific; BD Biosciences) on ice for 30 min, washed in FACS buffer, fixed with 2% formaldehyde in PBS, and acquired and analyzed on a FACSCaliber flow cytometer with CellQuest Pro software (BD Biosciences). As a control, tubes with amastigotes and an irrelevant fluorescein isothiocyanate-conjugated antibody were used. Lesion-derived amastigotes but not untreated axenic amastigotes had cell surface IgG. When axenic amastigotes were opsonized as described above, surface IgG was indistinguishable from that of lesion-derived amastigotes.
Measurement of Leishmania-specific serum IgG. Serum from infected mice was assayed for parasite-specific IgG1 and IgG2a by ELISA using soluble leishmanial antigen for capture, prepared as described previously (48), biotin-conjugated antimouse IgG1 and IgG2a (BD Biosciences), and peroxidase-conjugated streptavidin (Jackson ImmunoResearch, West Grove, Pa.). IgG quantitation shows mean and standard error of the mean (SEM) for three to five mice per group.
Anti-IL-12p40 treatment. Mice were injected intraperitoneally with 1 mg of anti-IL-12p40 monoclonal antibody (C17.8) prepared from an ammonium sulfate cut of ascites or 1 mg of rat IgG (Sigma-Aldrich) in 200 μl of PBS on days 0, 7, 14, and 21 postinfection.
Parasite quantitation. Parasite quantitation was performed by limiting dilution as described previously for three to five mice per group (11). For LN parasite quantitation, LN cell suspensions were brought to a 2-ml volume with tissue culture medium, and 20-μl samples were withdrawn for limiting dilution before wash steps for in vitro restimulation assays.
Statistical analysis. A two-tailed, unequal-variance Student t test was used to compare means of lesion sizes, log parasite burdens, NO production, and cytokine production from different groups of mice. Data are presented as means ± SEM, and differences were considered significant at P values of 0.05.
RESULTS
IL-10–/– mice resolve L. mexicana lesions. We investigated the role of the immunosuppressive cytokine IL-10 in the nonhealing phenotype of B6 mice with L. mexicana infection by studying the course of infection in IL-10–/– mice. Lesions of B6 IL-10–/– mice grew slightly more rapidly than those of B6 mice for the first 8 weeks (Fig. 1A). However, by 10 weeks postinfection, IL-10–/– mice began to resolve their lesions. Lesion parasite burdens were identical for IL-10–/– and B6 mice at 7 weeks postinfection (Fig. 1B); by 12 weeks, IL-10–/– mice, unlike B6 control mice, had significantly reduced parasite burdens. In the draining LN, parasite numbers were also reduced by 12 weeks postinfection for IL-10–/– mice versus results for B6 mice (Fig. 1B). By 28 weeks postinfection, there were more than 4 orders of magnitude fewer parasites in the lesions of IL-10–/– mice than in wild-type mice. In contrast to IL-10–/– mice, IL-4–/– mice failed to fully resolve L. mexicana lesions but did develop smaller lesions and have fewer parasites than controls (Fig. 1C and D). Taken together, these data indicate that IL-10 strongly contributes to the inability of L. mexicana-infected B6 mice to control parasites and to resolve lesions, while IL-4 only partly contributes to susceptibility.
In order to determine if the ability of IL-10–/– mice to heal was associated with increased immunity, we compared the IFN- and NO responses of draining LN cells from IL-10–/– and B6 mice infected with L. mexicana and also measured their ability to exhibit a DTH reaction. LN cells from IL-10–/– mice produced significantly more IFN- (Fig. 2A) and NO (Fig. 2B) than cells from B6 mice. Healed IL-10–/– mice also exhibited a DTH response when rechallenged with L. mexicana in the opposite footpad (Fig. 2C).
Healing in L. mexicana-infected IL-10–/– mice is mediated by IL-12p40. We next tested whether healing of L. mexicana infection in IL-10–/– mice is dependent upon IL-12 or IL-23 by blocking IL-12p40 in these mice for the first 3 weeks of infection. We found that lesion progression was unchanged for the first 8 weeks but that thereafter, anti-IL-12p40-treated IL-10–/– mice had continued lesion progression, while the control IgG-treated IL-10–/– mice had lesions that were beginning to resolve (Fig. 3A). Parasite burdens reflect the differences seen in lesion progression, with anti-IL-12p40 treatment increasing parasite burdens by nearly 5 orders of magnitude in IL-10–/– mice (Fig. 3B). L. mexicana-infected IL-12p40–/– mice have chronic disease indistinguishable from that of infected B6 mice (12), but as we show here, blocking IL-12p40 in the absence of IL-10 induces progression of disease. When taken together, these data demonstrate that in addition to possible direct suppression of inducible nitric oxide synthase, IL-10 also helps to suppress a protective Th1 pathway that is dependent on IL-12 and/or IL-23.
IgG is required for IL-10 production and chronic L. mexicana infection. Macrophages may be a critical source of the IL-10 that suppresses L. mexicana lesion resolution. Previous findings demonstrate that antibody-opsonized L. major and L. amazonensis induce IL-10 production from LPS-stimulated BALB/c macrophages (27). We extended these results to infection of B6 macrophages by L. mexicana. Lesion-derived amastigotes and antibody-opsonized axenic amastigotes stimulated IL-10 production from LPS-primed B6 macrophages in vitro, whereas unopsonized axenic amastigotes had little effect on IL-10 production (Fig. 4A). These data suggest that antibodies might contribute to the production of IL-10 by opsonizing parasites that are subsequently taken up by macrophages. Consistent with this idea, by 10 weeks IgG was detected in the sera of L. mexicana-infected mice (Fig. 4B).
In order to determine if the absence of antibodies might lead to resistance, we investigated the course of L. mexicana infection in B6 2m–/– mice. While not usually used to evaluate the contribution of antibody to an immune response, 2m–/– mice have very low antibody levels because 2m is a component of FcRn, a major histocompatibility complex class I homolog that protects IgG from catabolism (16, 26). We were unable to detect any circulating Leishmania-specific antibodies in L. mexicana-infected 2m–/– mice, even late in infection (Fig. 4C). We found that 2m–/– mice resolve L. mexicana lesions (Fig. 4D) and significantly reduce parasites loads (Fig. 4E). Since B6 CD8–/– mice exhibit a course of L. mexicana infection and cytokine profile identical to that with control mice (12), the healing of 2m–/– mice cannot be attributed to the lack of CD8+ T cells but rather is likely due to low IgG levels (although an effect from the lack of NKT cells cannot be ruled out). Thus, the healing of 2m–/– mice with L. mexicana infection is consistent with a requirement for IgG ligation of FcR for IL-10-induced susceptibility. Similarly, BALB/c mice lacking IgG are less susceptible to infections with L. pifanoi and L. amazonensis (29). Experiments to examine the role of antibody, using μMT mice, were inconclusive due to the development of a wasting disease that has previously been described in these mice (34).
FcR–/– mice resolve L. mexicana lesions. Taken together, the results described above implicate a mechanism of IL-10 production by macrophages that involves ligation of the FcR by antibody-opsonized parasites. To directly test whether IgG binding to FcR is critical for the production of IL-10 that promotes chronic disease, B6 FcR–/– and control mice were infected with L. mexicana, and the course of infection was monitored. Similar to the case with IL-10–/– mice, FcR–/– mice, which lack the common chain of FcRI, FcRIII, and FcRI, were able to resolve L. mexicana lesions (Fig. 5A). By 10 weeks of infection, parasite burdens were nearly 5 orders of magnitude lower in FcR–/– mice than in control mice, with greater differences seen at 32 weeks postinfection (Fig. 5B). Thus, similarly to IL-10–/– mice, FcR–/– mice resolved L. mexicana lesions and controlled parasite burdens. In addition, we found that at 10 weeks postinfection, draining LN cells from L. mexicana-infected FcR–/– mice produced fivefold less IL-10 than cells from infected B6 mice (Fig. 6A). We also found that FcR–/– mice exhibited increased IFN- responses in comparison to the previously observed weak responses from B6 mice (Fig. 6B) (12), as well as DTH responses upon rechallenge (Fig. 6C). Furthermore, at 22 weeks of infection, L. mexicana-infected FcR–/– mice had similar IgG2a responses but diminished (and undetectable) IgG1 responses compared with B6 mice (data not shown), demonstrating a more polarized Th1 response.
DISCUSSION
B6 mice develop chronic, nonhealing lesions following infection with L. mexicana, and the factors contributing to this susceptibility are not defined. Consistent with previous reports implicating a role for IL-4 in L. mexicana infections (45), IL-4–/– mice had smaller lesions than wild-type mice, although they were unable to fully resolve lesions and had more than 105 parasites remaining. More-striking results were obtained with IL-10–/– mice, which were able to completely resolve L. mexicana lesions and reduced the parasite burden to less than 104. One source of IL-10 may be Treg cells, since it has already been demonstrated that these cells produce IL-10 after infection with L. major and are important in maintaining parasite persistence after lesion resolution (8, 9). However, our results suggest that Treg cells are not the only source of IL-10 that has in vivo relevance in Leishmania infection. We found that opsonized L. mexicana amastigotes promoted enhanced IL-10 production by macrophages, and both FcR and circulating IgG were required for chronic disease; taken together, the data best fit a mechanism where IgG ligation of FcR on macrophages induces increased production of IL-10, leading to suppression of protective immune responses. On the other hand, our data do not rule out a role for Treg cells in L. mexicana infection, and these cells likely play a role in L. mexicana infection similar to that reported for L. major infection.
These results differ from infection with the closely related New World parasite, L. amazonensis; L. amazonensis-infected B6 IL-10–/– mice have higher levels of IFN- than controls but do not resolve their lesions (24). Consistent with this disparity, B6 2m–/– mice do not resolve L. amazonensis infection (49), while we show here that B6 2m–/– mice do heal following L. mexicana infection. Thus, important differences may exist between these closely related Leishmania species. Although we do not know the nature of these differences, they are not surprising, since even among strains of L. major substantial differences exist. For example, infection of BALB/c IL-4–/– mice with one strain of L. major leads to healing, while another is associated with chronic disease (30, 38). Identifying the molecular basis for such differences will be important in understanding the pathogenesis of difference species and strains of Leishmania.
Our findings are also different from L. mexicana infection of BALB/c IL-10–/– mice, in which lesion progression is identical to that with wild-type BALB/c infection but parasite burdens are somewhat diminished, and both IL-4 and IL-10 must be removed to achieve healing (39). This is consistent with previous findings that IL-4 plays a more significant role in the immune responses of BALB/c mice than with B6 mice (25, 39). Previous findings demonstrated that infection of BALB/c mice lacking FcR or IgG with another New World parasite, L. pifanoi, is associated with enhanced resistance (29). Our data extend those findings, not only by demonstrating similar resistance in FcR- and IgG-deficient mice to another Leishmania species but also by showing that FcR and IgG contribute to susceptibility in B6 mice, which develop much lower-level antibody responses following Leishmania infection than BALB/c mice. While we favor the hypothesis that FcR contributes to susceptibility due to enhanced IL-10 production, another possibility is that FcR facilitates parasite entry into macrophages. However, our finding that at 12 weeks postinfection, L. mexicana-infected 2m–/– mice, which have undetectable circulating anti-Leishmania IgG, have parasite burdens at least as high as those of infected B6 mice suggests that IgG opsonization is not required for infection of macrophages. Another possibility is that opsonized parasites contribute to local inflammatory responses, critical for lesion development or maintenance (13).
IL-10 has recently been linked with the persistence of L. major following resolution of a primary infection in B6 mice (8). However, here we report that while B6 IL-10–/– mice infected with L. mexicana resolve their lesions, the mice maintain a low number of parasites at the site of infection. Why L. mexicana persists in IL-10–/– mice while L. major does not is unclear. It may be that the requirement for IL-10 for parasite persistence is species specific. These differences may stem from the more chronic nature of L. mexicana infection, perhaps caused by induction of IL-10-independent pathways that prevent complete parasite clearance. Alternatively, in experiments reported here, IL-10–/– mice were infected with a different dose and by a different route than in the previous studies showing sterile clearance of parasites, and these factors may also influence the outcome of infection in IL-10–/– mice.
Both IL-10–/– and FcR–/– mice resolve lesions and effectively control parasite numbers with associated Th1 responses. However, FcR–/– mice have lower numbers of parasites than IL-10–/– mice, and the IFN- responses by cells from FcR–/– mice were greater than those seen in IL-10–/– mice. This suggests that signaling through the FcR may promote both IL-10-dependent and IL-10-independent mechanisms that reduce the ability of the host to control L. mexicana infection. The IL-10-independent pathway may be mediated by transforming growth factor or PGE2, both of which have been shown to increase susceptibility to Leishmania (7, 14, 17, 33) and are produced by monocytes in response to immune complex binding of FcR (28, 42). However, this IL-10-independent pathway alone appears insufficient to suppress healing in IL-10–/– mice.
IL-10 may act by inhibiting NO production, blocking IL-12 production, and/or downregulating antigen presentation (35); our studies indicate that IL-10 may promote susceptibility in L. mexicana-infected mice by all three pathways. We found that there was less NO production in B6 mice than in IL-10–/– mice, possibly from direct IL-10 suppression of inducible nitric oxide synthase. However, LN cells from L. mexicana-infected IL-10–/– and FcR–/– mice also produced more IFN- than cells from infected B6 mice, indicating that IL-10 also suppresses Th1 cell development. While B6 mice do not resolve L. mexicana infections, the lesions are controlled. In contrast to observations of others (2), we found that L. mexicana-infected IL-12p40–/– mice (and IL-12p35–/– mice [L. U. Buxbaum, unpublished data]) show no increased susceptibility to infection, suggesting that an IL-12-independent pathway prevents L. mexicana infection from becoming progressive (12). This prompted us to determine if healing in IL-10–/– mice was IL-12 or IL-23 dependent, and our present finding—that blockade of IL-12p40 in IL-10–/– mice leads to increased susceptibility—indicates that IL-10 must suppress the development of an IL-12- or IL-23-dependent immune response in B6 mice. This could be due to a direct effect on IL-12 or IL-23 production by dendritic cells or macrophages or to an indirect effect by decreasing CD4+-T-cell activation through suppression of antigen-presenting-cell function. The lack of a strong Th2 response induced by L. mexicana (12) and the lack of a large increase in draining LN cell numbers in this infection compared with that of L. major (data not shown) favors the latter possibility. In addition, the inability to promote healing by exogenous administration of IL-12 further suggests that a deficit in IL-12 production, by itself, is not the reason animals are unable to heal (12). Thus, in addition to decreasing NO and IL-12 or IL-23 production, it is likely that IL-10 has a role in suppression of T-cell priming, leading to decreased expansion of antigen-specific T cells and consequently less IFN- production, resulting in a decrease in macrophage activation.
It is clear that the development of a Th1 response, initiated by IL-12, is critical for resistance to all of the species of Leishmania. However, numerous factors can modulate the development of a Th1 immune response to Leishmania (47). For example, studies with L. major indicate that IL-4 can prevent Th1 cell development, in part by downregulating expression of the IL-12 receptor (22, 23). Nevertheless, the production of IL-4 by itself may not be sufficient to promote a susceptible phenotype, since IL-4 production is sometimes observed in L. major-infected mice that eventually heal (47). This suggests that in addition to IL-4, other factors regulate Th1-cell development. IL-10 is one of these factors, since in the absence of IL-10, BALB/c mice are resistant to L. major (27). Our results and those of others (4, 50) indicate that IL-4 is produced during L. mexicana infection, but we would argue that IL-4 by itself is insufficient to induce a susceptible phenotype. Rather, the production of IL-10 may be required to enforce a susceptible phenotype in L. mexicana infections. Thus, we show here that IL-10 is critical for maintaining chronic disease following L. mexicana infections and moreover that the production of IL-10 can be promoted by antibody-opsonized parasites. These studies offer a more complex view of how susceptibility to Leishmania develops, indicating that initial events (such as early IL-4 production) may contribute to susceptibility, but later events (such as antibody production) may be required to maintain a susceptible phenotype.
Our findings demonstrate that IL-10, rather than a classical IL-4-driven Th2 pathway, is primarily responsible for the lack of healing of chronic infections caused by L. mexicana and suggest that blockade of the IL-10 pathway with intralesional injection of anti-IL-10 or anti-IL-10 receptor antibodies may have potential therapeutic effects. In addition, the data indicate that caution should be exercised in the development of leishmanial vaccines, since those that induce strong antibody responses to parasite surface molecules may exacerbate subsequent infection by induction of IL-10 through an FcR-immune complex pathway.
ACKNOWLEDGMENTS
We thank Karen Joyce and Andrea Rosso for their technical support and David Artis and Jay Farrell for critical reading of the manuscript.
This work was supported by National Institutes of Health grants K08 AI01805 (L.U.B.) and R01 AI35914 (P.S.) and a Veterans Affairs Merit Review grant (L.U.B.).
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Department of Medicine, Division of Infectious Diseases, School of Medicine
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
ABSTRACT
Infection of C57BL/6 (B6) mice with Leishmania mexicana is associated with a minimal immune response and chronic disease. Here we show that B6 interleukin 10–/– (IL-10–/–) mice resolve their lesions and exhibit increased gamma interferon (IFN-), nitric oxide production, and delayed-type hypersensitivity. This enhanced resistance was dependent upon IL-12p40, since treatment of L. mexicana-infected IL-10–/– mice with anti-IL-12p40 monoclonal antibody abrogated healing. Antibody-opsonized L. mexicana induced IL-10 production by B6 macrophages in vitro, implicating antibody binding to Fc receptors as a mechanism involved in IL-10 production in this infection. Furthermore, B6 FcR–/– mice resolve L. mexicana lesions, and lymph node cells from these mice produced less IL-10 and more IFN- than cells from infected wild-type mice. These data demonstrate that removal of IL-10 or FcR leads to resolution of L. mexicana disease and support a model in which ligation of FcR by L. mexicana-bound immunoglobulin G promotes IL-10 production, leading to chronic disease.
INTRODUCTION
The protozoan parasite Leishmania mexicana causes chronic cutaneous disease in C57BL/6 (B6) mice, which are able to resolve infections with Leishmania major (5). Human L. mexicana infections often mirror this with more frequent chronic disease, an example being nonhealing ear infections, known as Chiclero's ulcer (40). While the immunologic responses involved in susceptibility to L. major have been described in depth, those contributing to the chronic disease seen during infection with American species of Leishmania are less well understood. The two immunosuppressive cytokines most associated with nonhealing forms of leishmaniasis are interleukin 4 (IL-4) and IL-10. For example, L. major infection in BALB/c mice results in nonhealing disease associated with high levels of IL-4, and neutralizing IL-4 can promote resistance (21). IL-10 also contributes to susceptibility in BALB/c mice and also modulates protective immune responses in B6 mice infected with L. major (8, 27, 37). In contrast, Leishmania amazonensis, a species closely related to L. mexicana that also causes chronic disease in B6 mice, still causes chronic infection of IL-4- and IL-10-deficient mice (24, 25). However, while previous studies indicate that IL-4 and IL-13 may contribute to the chronicity of L. mexicana infections (3, 45), the role of IL-10 in the chronic disease induced by L. mexicana in B6 mice has not been investigated.
The immune responses associated with L. mexicana infection are distinct from those observed following infection with L. major. L. major infection of B6 mice induces a significant immune response, characterized by increased cell migration to the draining lymph node (LN), proliferation of Leishmania-specific T cells, and production of gamma interferon (IFN-) (reviewed in references 41 and 43). In contrast, the magnitude of the immune response to L. mexicana or L. amazonensis is limited. Notably, mouse strains resistant to L. major not only fail to develop a dominant Th1 response when infected with these New World parasite species, they also show no evidence of a strong Th2 response (1, 45). Furthermore, in contrast to L. major lesions, lesions from L. mexicana-infected mice are characterized by a limited lymphocytic infiltrate. These observations suggest that the chronic nature of L. mexicana infections may not be linked with a Th2 response but rather is more likely the result of generalized immunosuppression mediated by IL-10. Studies with infectious diseases and models of autoimmunity indicate that the regulatory role of IL-10 can be vital to blocking immunopathologic responses. For example, infections of IL-10-deficient mice with Toxoplasma and Plasmodium result in fatal inflammatory responses (15, 32), and similarly, IL-10-deficient mice have spontaneous autoimmune colitis (31) and more severe experimental autoimmune encephalomyelitis (10, 44). These models demonstrate that IL-10 can play a critical protective role against immunopathology. Interestingly, however, there is no evidence that IL-10–/– mice infected with Leishmania—either L. major, L. amazonensis, or Leishmania donovani—develop an uncontrolled inflammatory response. Rather, in all cases the absence of IL-10 has been associated with enhanced, but not uncontrolled, immune responses (24, 27, 36).
IL-10 can be produced by several different cell types, including macrophages, dendritic cells, B cells, and T cells (35). Recently CD4+ CD25+ regulatory T (Treg) cells have been shown to suppress immune responses in L. major-infected mice, including Th1 responses in B6 mice and the initial development of Th2 responses in BALB/c mice (6, 9). Critically, these Treg cells may function through the production of IL-10; CD4+ CD25+ T cells from L. major lesions produced high levels of IL-10 when stimulated in vitro with L. major-infected dendritic cells, and Treg cells from IL-10-deficient mice were unable to suppress effector-T-cell responses following adoptive transfer to RAG–/– mice. However, other studies indicate that IL-10 from macrophages might also be crucial in promoting susceptibility to leishmaniasis, since overexpression of IL-10 in major histocompatibility complex class II+ cells, but not T cells, promoted susceptibility (19, 20). One pathway that leads to IL-10 production involves ligation of the macrophage FcR by immune complexes (51). In fact, L. major parasites opsonized with antibody were capable of augmenting lipopolysaccharide (LPS)-induced IL-10 production by macrophages in an FcR-dependent manner (27).
The role of IL-10, FcR, and immunoglobulin G (IgG) in leading to susceptibility to infections of B6 mice by New World Leishmania species, such as L. mexicana and L. amazonensis, is not clear. IL-10 does play a role in preventing the complete clearance of L. major in B6 mice (8) and is required for progressive disease in the much more susceptible BALB/c strain (27). In BALB/c mice, which are also much more susceptible than B6 mice to L. mexicana and other New World Leishmania strains, IL-10 contributes to susceptibility to L. mexicana, but both IL-10 and IL-4 must be blocked to achieve resistance (39). FcR and IgG have been shown to contribute to susceptibility of BALB/c mice to the related New World parasites Leishmania pifanoi and L. amazonensis (29), but this has not been linked with IL-10 production. In addition, B6 IL-10–/– mice do not heal L. amazonensis infections despite an increase in IFN- at early time points (24). Thus, the roles of IL-10, FcR, and IgG in L. mexicana infection are by no means understood, even in BALB/c mice, and have not been investigated in the relatively more resistant B6 mice, whose disease with both L. major and L. mexicana more closely resembles human leishmaniasis.
In the present study, we demonstrate that in the absence of IL-10, B6 mice are able to resolve L. mexicana infections. Resolution of infection was also observed in mice lacking FcR and circulating IgG, which implicates macrophages—stimulated through the FcR by IgG-opsonized parasites—as a critical source of IL-10. Consistent with this hypothesis, we demonstrate that IgG bound to the surface of L. mexicana promotes the induction of IL-10 from B6 macrophages in vitro. We further show that LN cells from L. mexicana-infected FcR–/– mice produce less IL-10 on antigen restimulation than cells of control mice. These studies reveal that IL-10 is required to suppress a healing immune response to L. mexicana in B6 mice and also to uncover a critical role for antibodies, through FcR binding, in this suppression.
MATERIALS AND METHODS
Mice. B6, B6 IL-4–/–, B6 IL-10–/–, and B6 2-microglobulin (2m)–/– mice were purchased from Jackson Laboratory (Bar Harbor, Maine). B6 FcR–/– and B6 control mice were purchased from Taconic (Germantown, N.Y.). Courses of infection consisted of groups of five mice per experiment, and rechallenge was performed on two to five mice per group. Mice were purchased at 4 to 6 weeks and were sex and age matched for all experiments. Animals were maintained in a specific-pathogen-free environment, and the animal colony was screened regularly, and tested negative, for the presence of murine pathogens.
Parasites and antigens. L. mexicana (MNYC/BZ/62/M379) promastigotes, provided by J. Alexander (University of Strathclyde, Glasgow, United Kingdom), were grown at 27°C in Grace's medium (pH 6.3; Life Technologies, Grand Island, N.Y.) supplemented with 20% heat-inactivated fetal bovine serum (FBS) (HyClone Labs, Logan, Utah), 2 mM L-glutamine, 100 U of penicillin/ml, and 100 μg of streptomycin/ml. Stationary-phase promastigotes (day 7 of culture) were washed three times in phosphate-buffered saline (PBS) and 5 x 106 parasites were injected into the hind footpad of mice. Lesions were monitored using a metric dial caliper. To assess delayed-type hypersensitivity (DTH), mice were injected with 5 x 106 parasites in the opposite footpad, and the baseline uninjected footpad thickness was subtracted from the injected footpad thickness at 48 h. Lesion-derived amastigotes were obtained from footpad lesions of mice chronically infected with L. mexicana. Axenic amastigotes were prepared by placing stationary-phase cultures of L. mexicana promastigotes at 32°C for 2 days with passage every 7 to 10 days at 1/100 in acidic Grace's medium (pH 5.5) supplemented as described above. Freeze-thaw antigen (FTAg) was prepared from L. mexicana stationary-phase promastigotes that were washed four times in PBS, resuspended at 109/ml (yielding 1 mg/ml of protein), and frozen (–80°C) and thawed rapidly (37°C) for five cycles.
Assay of cytokines and NO from in vitro restimulation. Single-cell suspensions were prepared from draining LNs, and 200-μl samples (8 x 105 cells) were cultured in 96-well tissue culture plates in Dulbecco's modified Eagle's medium (Mediatech, Herndon, Va.) supplemented with 10% heat-inactivated FBS, 25 mM HEPES (pH 7.4), 50 μM 2-mercaptoethanol, 2 mM L-glutamine, 100 U of penicillin/ml, and 100 μg of streptomycin/ml. Cells were stimulated with 10 μg of L. mexicana FTAg/ml (107 cell equivalents/ml) for 3 days at 37°C in a 5% CO2 incubator, and supernatants were assayed by enzyme-linked immunosorbent assay (ELISA) for IFN- as previously described (46) and for IL-10 using commercial antibodies as recommended by the manufacturer (BD Bioscience, San Diego, Calif.). Uninfected mice had no detectable IL-10 or IFN- production with antigen stimulation in these experiments. NO production was assayed by measuring nitrite (NO2) in 3-day supernatants with the Griess reagent as previously described (18). Cytokine and NO data shown are for groups of three to five individual mice.
In vitro infection of BMM. BMM were grown from B6 mice on petri dishes in 10 ml of complete macrophage medium (Dulbecco's modified Eagle's medium containing 10% heat-inactivated FBS, 100 U of penicillin/ml, 100 μg of streptomycin/ml, 2 mM L-glutamine, 25 mM HEPES, 30% L929 cell-conditioned medium) for 7 days (7.5 x 106 cells per petri dish) with an additional 10 ml of complete macrophage medium added on day 3 (27, 52, 53). Macrophages were harvested by gentle scraping in cold PBS (4°C), washed, and replated at 2 x 105/0.5 ml in 24-well plates in macrophage medium lacking L929 cell-conditioned medium. After resting overnight and being washed with fresh medium, LPS from E. coli O128:B12 (Sigma-Aldrich, St. Louis, Mo.) was added at a 100-ng/ml concentration, and macrophages were infected at a 10:1 multiplicity of infection with lesion amastigotes, axenic amastigotes, or axenic amastigotes opsonized for 30 min at 4°C with 50 μl of a 1/40 dilution of serum from B6 mice chronically infected long term with L. mexicana. Supernatants were collected, frozen at –20°C, and assayed for IL-10 by ELISA as described above.
Flow cytometry. Amastigotes (106) were washed in fluorescence-activated cell sorter (FACS) buffer (PBS with 0.1% sodium azide, 0.1% bovine serum albumin [Sigma-Aldrich]), and labeled with fluorescein isothiocyanate-conjugated goat antimouse IgG (Fc specific; BD Biosciences) on ice for 30 min, washed in FACS buffer, fixed with 2% formaldehyde in PBS, and acquired and analyzed on a FACSCaliber flow cytometer with CellQuest Pro software (BD Biosciences). As a control, tubes with amastigotes and an irrelevant fluorescein isothiocyanate-conjugated antibody were used. Lesion-derived amastigotes but not untreated axenic amastigotes had cell surface IgG. When axenic amastigotes were opsonized as described above, surface IgG was indistinguishable from that of lesion-derived amastigotes.
Measurement of Leishmania-specific serum IgG. Serum from infected mice was assayed for parasite-specific IgG1 and IgG2a by ELISA using soluble leishmanial antigen for capture, prepared as described previously (48), biotin-conjugated antimouse IgG1 and IgG2a (BD Biosciences), and peroxidase-conjugated streptavidin (Jackson ImmunoResearch, West Grove, Pa.). IgG quantitation shows mean and standard error of the mean (SEM) for three to five mice per group.
Anti-IL-12p40 treatment. Mice were injected intraperitoneally with 1 mg of anti-IL-12p40 monoclonal antibody (C17.8) prepared from an ammonium sulfate cut of ascites or 1 mg of rat IgG (Sigma-Aldrich) in 200 μl of PBS on days 0, 7, 14, and 21 postinfection.
Parasite quantitation. Parasite quantitation was performed by limiting dilution as described previously for three to five mice per group (11). For LN parasite quantitation, LN cell suspensions were brought to a 2-ml volume with tissue culture medium, and 20-μl samples were withdrawn for limiting dilution before wash steps for in vitro restimulation assays.
Statistical analysis. A two-tailed, unequal-variance Student t test was used to compare means of lesion sizes, log parasite burdens, NO production, and cytokine production from different groups of mice. Data are presented as means ± SEM, and differences were considered significant at P values of 0.05.
RESULTS
IL-10–/– mice resolve L. mexicana lesions. We investigated the role of the immunosuppressive cytokine IL-10 in the nonhealing phenotype of B6 mice with L. mexicana infection by studying the course of infection in IL-10–/– mice. Lesions of B6 IL-10–/– mice grew slightly more rapidly than those of B6 mice for the first 8 weeks (Fig. 1A). However, by 10 weeks postinfection, IL-10–/– mice began to resolve their lesions. Lesion parasite burdens were identical for IL-10–/– and B6 mice at 7 weeks postinfection (Fig. 1B); by 12 weeks, IL-10–/– mice, unlike B6 control mice, had significantly reduced parasite burdens. In the draining LN, parasite numbers were also reduced by 12 weeks postinfection for IL-10–/– mice versus results for B6 mice (Fig. 1B). By 28 weeks postinfection, there were more than 4 orders of magnitude fewer parasites in the lesions of IL-10–/– mice than in wild-type mice. In contrast to IL-10–/– mice, IL-4–/– mice failed to fully resolve L. mexicana lesions but did develop smaller lesions and have fewer parasites than controls (Fig. 1C and D). Taken together, these data indicate that IL-10 strongly contributes to the inability of L. mexicana-infected B6 mice to control parasites and to resolve lesions, while IL-4 only partly contributes to susceptibility.
In order to determine if the ability of IL-10–/– mice to heal was associated with increased immunity, we compared the IFN- and NO responses of draining LN cells from IL-10–/– and B6 mice infected with L. mexicana and also measured their ability to exhibit a DTH reaction. LN cells from IL-10–/– mice produced significantly more IFN- (Fig. 2A) and NO (Fig. 2B) than cells from B6 mice. Healed IL-10–/– mice also exhibited a DTH response when rechallenged with L. mexicana in the opposite footpad (Fig. 2C).
Healing in L. mexicana-infected IL-10–/– mice is mediated by IL-12p40. We next tested whether healing of L. mexicana infection in IL-10–/– mice is dependent upon IL-12 or IL-23 by blocking IL-12p40 in these mice for the first 3 weeks of infection. We found that lesion progression was unchanged for the first 8 weeks but that thereafter, anti-IL-12p40-treated IL-10–/– mice had continued lesion progression, while the control IgG-treated IL-10–/– mice had lesions that were beginning to resolve (Fig. 3A). Parasite burdens reflect the differences seen in lesion progression, with anti-IL-12p40 treatment increasing parasite burdens by nearly 5 orders of magnitude in IL-10–/– mice (Fig. 3B). L. mexicana-infected IL-12p40–/– mice have chronic disease indistinguishable from that of infected B6 mice (12), but as we show here, blocking IL-12p40 in the absence of IL-10 induces progression of disease. When taken together, these data demonstrate that in addition to possible direct suppression of inducible nitric oxide synthase, IL-10 also helps to suppress a protective Th1 pathway that is dependent on IL-12 and/or IL-23.
IgG is required for IL-10 production and chronic L. mexicana infection. Macrophages may be a critical source of the IL-10 that suppresses L. mexicana lesion resolution. Previous findings demonstrate that antibody-opsonized L. major and L. amazonensis induce IL-10 production from LPS-stimulated BALB/c macrophages (27). We extended these results to infection of B6 macrophages by L. mexicana. Lesion-derived amastigotes and antibody-opsonized axenic amastigotes stimulated IL-10 production from LPS-primed B6 macrophages in vitro, whereas unopsonized axenic amastigotes had little effect on IL-10 production (Fig. 4A). These data suggest that antibodies might contribute to the production of IL-10 by opsonizing parasites that are subsequently taken up by macrophages. Consistent with this idea, by 10 weeks IgG was detected in the sera of L. mexicana-infected mice (Fig. 4B).
In order to determine if the absence of antibodies might lead to resistance, we investigated the course of L. mexicana infection in B6 2m–/– mice. While not usually used to evaluate the contribution of antibody to an immune response, 2m–/– mice have very low antibody levels because 2m is a component of FcRn, a major histocompatibility complex class I homolog that protects IgG from catabolism (16, 26). We were unable to detect any circulating Leishmania-specific antibodies in L. mexicana-infected 2m–/– mice, even late in infection (Fig. 4C). We found that 2m–/– mice resolve L. mexicana lesions (Fig. 4D) and significantly reduce parasites loads (Fig. 4E). Since B6 CD8–/– mice exhibit a course of L. mexicana infection and cytokine profile identical to that with control mice (12), the healing of 2m–/– mice cannot be attributed to the lack of CD8+ T cells but rather is likely due to low IgG levels (although an effect from the lack of NKT cells cannot be ruled out). Thus, the healing of 2m–/– mice with L. mexicana infection is consistent with a requirement for IgG ligation of FcR for IL-10-induced susceptibility. Similarly, BALB/c mice lacking IgG are less susceptible to infections with L. pifanoi and L. amazonensis (29). Experiments to examine the role of antibody, using μMT mice, were inconclusive due to the development of a wasting disease that has previously been described in these mice (34).
FcR–/– mice resolve L. mexicana lesions. Taken together, the results described above implicate a mechanism of IL-10 production by macrophages that involves ligation of the FcR by antibody-opsonized parasites. To directly test whether IgG binding to FcR is critical for the production of IL-10 that promotes chronic disease, B6 FcR–/– and control mice were infected with L. mexicana, and the course of infection was monitored. Similar to the case with IL-10–/– mice, FcR–/– mice, which lack the common chain of FcRI, FcRIII, and FcRI, were able to resolve L. mexicana lesions (Fig. 5A). By 10 weeks of infection, parasite burdens were nearly 5 orders of magnitude lower in FcR–/– mice than in control mice, with greater differences seen at 32 weeks postinfection (Fig. 5B). Thus, similarly to IL-10–/– mice, FcR–/– mice resolved L. mexicana lesions and controlled parasite burdens. In addition, we found that at 10 weeks postinfection, draining LN cells from L. mexicana-infected FcR–/– mice produced fivefold less IL-10 than cells from infected B6 mice (Fig. 6A). We also found that FcR–/– mice exhibited increased IFN- responses in comparison to the previously observed weak responses from B6 mice (Fig. 6B) (12), as well as DTH responses upon rechallenge (Fig. 6C). Furthermore, at 22 weeks of infection, L. mexicana-infected FcR–/– mice had similar IgG2a responses but diminished (and undetectable) IgG1 responses compared with B6 mice (data not shown), demonstrating a more polarized Th1 response.
DISCUSSION
B6 mice develop chronic, nonhealing lesions following infection with L. mexicana, and the factors contributing to this susceptibility are not defined. Consistent with previous reports implicating a role for IL-4 in L. mexicana infections (45), IL-4–/– mice had smaller lesions than wild-type mice, although they were unable to fully resolve lesions and had more than 105 parasites remaining. More-striking results were obtained with IL-10–/– mice, which were able to completely resolve L. mexicana lesions and reduced the parasite burden to less than 104. One source of IL-10 may be Treg cells, since it has already been demonstrated that these cells produce IL-10 after infection with L. major and are important in maintaining parasite persistence after lesion resolution (8, 9). However, our results suggest that Treg cells are not the only source of IL-10 that has in vivo relevance in Leishmania infection. We found that opsonized L. mexicana amastigotes promoted enhanced IL-10 production by macrophages, and both FcR and circulating IgG were required for chronic disease; taken together, the data best fit a mechanism where IgG ligation of FcR on macrophages induces increased production of IL-10, leading to suppression of protective immune responses. On the other hand, our data do not rule out a role for Treg cells in L. mexicana infection, and these cells likely play a role in L. mexicana infection similar to that reported for L. major infection.
These results differ from infection with the closely related New World parasite, L. amazonensis; L. amazonensis-infected B6 IL-10–/– mice have higher levels of IFN- than controls but do not resolve their lesions (24). Consistent with this disparity, B6 2m–/– mice do not resolve L. amazonensis infection (49), while we show here that B6 2m–/– mice do heal following L. mexicana infection. Thus, important differences may exist between these closely related Leishmania species. Although we do not know the nature of these differences, they are not surprising, since even among strains of L. major substantial differences exist. For example, infection of BALB/c IL-4–/– mice with one strain of L. major leads to healing, while another is associated with chronic disease (30, 38). Identifying the molecular basis for such differences will be important in understanding the pathogenesis of difference species and strains of Leishmania.
Our findings are also different from L. mexicana infection of BALB/c IL-10–/– mice, in which lesion progression is identical to that with wild-type BALB/c infection but parasite burdens are somewhat diminished, and both IL-4 and IL-10 must be removed to achieve healing (39). This is consistent with previous findings that IL-4 plays a more significant role in the immune responses of BALB/c mice than with B6 mice (25, 39). Previous findings demonstrated that infection of BALB/c mice lacking FcR or IgG with another New World parasite, L. pifanoi, is associated with enhanced resistance (29). Our data extend those findings, not only by demonstrating similar resistance in FcR- and IgG-deficient mice to another Leishmania species but also by showing that FcR and IgG contribute to susceptibility in B6 mice, which develop much lower-level antibody responses following Leishmania infection than BALB/c mice. While we favor the hypothesis that FcR contributes to susceptibility due to enhanced IL-10 production, another possibility is that FcR facilitates parasite entry into macrophages. However, our finding that at 12 weeks postinfection, L. mexicana-infected 2m–/– mice, which have undetectable circulating anti-Leishmania IgG, have parasite burdens at least as high as those of infected B6 mice suggests that IgG opsonization is not required for infection of macrophages. Another possibility is that opsonized parasites contribute to local inflammatory responses, critical for lesion development or maintenance (13).
IL-10 has recently been linked with the persistence of L. major following resolution of a primary infection in B6 mice (8). However, here we report that while B6 IL-10–/– mice infected with L. mexicana resolve their lesions, the mice maintain a low number of parasites at the site of infection. Why L. mexicana persists in IL-10–/– mice while L. major does not is unclear. It may be that the requirement for IL-10 for parasite persistence is species specific. These differences may stem from the more chronic nature of L. mexicana infection, perhaps caused by induction of IL-10-independent pathways that prevent complete parasite clearance. Alternatively, in experiments reported here, IL-10–/– mice were infected with a different dose and by a different route than in the previous studies showing sterile clearance of parasites, and these factors may also influence the outcome of infection in IL-10–/– mice.
Both IL-10–/– and FcR–/– mice resolve lesions and effectively control parasite numbers with associated Th1 responses. However, FcR–/– mice have lower numbers of parasites than IL-10–/– mice, and the IFN- responses by cells from FcR–/– mice were greater than those seen in IL-10–/– mice. This suggests that signaling through the FcR may promote both IL-10-dependent and IL-10-independent mechanisms that reduce the ability of the host to control L. mexicana infection. The IL-10-independent pathway may be mediated by transforming growth factor or PGE2, both of which have been shown to increase susceptibility to Leishmania (7, 14, 17, 33) and are produced by monocytes in response to immune complex binding of FcR (28, 42). However, this IL-10-independent pathway alone appears insufficient to suppress healing in IL-10–/– mice.
IL-10 may act by inhibiting NO production, blocking IL-12 production, and/or downregulating antigen presentation (35); our studies indicate that IL-10 may promote susceptibility in L. mexicana-infected mice by all three pathways. We found that there was less NO production in B6 mice than in IL-10–/– mice, possibly from direct IL-10 suppression of inducible nitric oxide synthase. However, LN cells from L. mexicana-infected IL-10–/– and FcR–/– mice also produced more IFN- than cells from infected B6 mice, indicating that IL-10 also suppresses Th1 cell development. While B6 mice do not resolve L. mexicana infections, the lesions are controlled. In contrast to observations of others (2), we found that L. mexicana-infected IL-12p40–/– mice (and IL-12p35–/– mice [L. U. Buxbaum, unpublished data]) show no increased susceptibility to infection, suggesting that an IL-12-independent pathway prevents L. mexicana infection from becoming progressive (12). This prompted us to determine if healing in IL-10–/– mice was IL-12 or IL-23 dependent, and our present finding—that blockade of IL-12p40 in IL-10–/– mice leads to increased susceptibility—indicates that IL-10 must suppress the development of an IL-12- or IL-23-dependent immune response in B6 mice. This could be due to a direct effect on IL-12 or IL-23 production by dendritic cells or macrophages or to an indirect effect by decreasing CD4+-T-cell activation through suppression of antigen-presenting-cell function. The lack of a strong Th2 response induced by L. mexicana (12) and the lack of a large increase in draining LN cell numbers in this infection compared with that of L. major (data not shown) favors the latter possibility. In addition, the inability to promote healing by exogenous administration of IL-12 further suggests that a deficit in IL-12 production, by itself, is not the reason animals are unable to heal (12). Thus, in addition to decreasing NO and IL-12 or IL-23 production, it is likely that IL-10 has a role in suppression of T-cell priming, leading to decreased expansion of antigen-specific T cells and consequently less IFN- production, resulting in a decrease in macrophage activation.
It is clear that the development of a Th1 response, initiated by IL-12, is critical for resistance to all of the species of Leishmania. However, numerous factors can modulate the development of a Th1 immune response to Leishmania (47). For example, studies with L. major indicate that IL-4 can prevent Th1 cell development, in part by downregulating expression of the IL-12 receptor (22, 23). Nevertheless, the production of IL-4 by itself may not be sufficient to promote a susceptible phenotype, since IL-4 production is sometimes observed in L. major-infected mice that eventually heal (47). This suggests that in addition to IL-4, other factors regulate Th1-cell development. IL-10 is one of these factors, since in the absence of IL-10, BALB/c mice are resistant to L. major (27). Our results and those of others (4, 50) indicate that IL-4 is produced during L. mexicana infection, but we would argue that IL-4 by itself is insufficient to induce a susceptible phenotype. Rather, the production of IL-10 may be required to enforce a susceptible phenotype in L. mexicana infections. Thus, we show here that IL-10 is critical for maintaining chronic disease following L. mexicana infections and moreover that the production of IL-10 can be promoted by antibody-opsonized parasites. These studies offer a more complex view of how susceptibility to Leishmania develops, indicating that initial events (such as early IL-4 production) may contribute to susceptibility, but later events (such as antibody production) may be required to maintain a susceptible phenotype.
Our findings demonstrate that IL-10, rather than a classical IL-4-driven Th2 pathway, is primarily responsible for the lack of healing of chronic infections caused by L. mexicana and suggest that blockade of the IL-10 pathway with intralesional injection of anti-IL-10 or anti-IL-10 receptor antibodies may have potential therapeutic effects. In addition, the data indicate that caution should be exercised in the development of leishmanial vaccines, since those that induce strong antibody responses to parasite surface molecules may exacerbate subsequent infection by induction of IL-10 through an FcR-immune complex pathway.
ACKNOWLEDGMENTS
We thank Karen Joyce and Andrea Rosso for their technical support and David Artis and Jay Farrell for critical reading of the manuscript.
This work was supported by National Institutes of Health grants K08 AI01805 (L.U.B.) and R01 AI35914 (P.S.) and a Veterans Affairs Merit Review grant (L.U.B.).
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