The relative contribution of IL-4 receptor signaling and IL-10 to susceptibility to Leishmania major

The roles of IL-10 and IL-4 receptor signaling were evaluated in a murine model of Leishmania major infection. In previous studies the L. major substrain LV39 caused progressive, nonhealing lesions in BALB/c mice deficient for IL-4R alpha-chain (IL-4R alpha), while substrain IR173 was highly controlled. To explore whether IL-10 is responsible for inducing susceptibility to LV39, wild-type and IL-4R alpha(-/-) mice were treated with anti-IL-10R mAb, and in a genetic approach, the IL-4R alpha(-/-) mice were crossed with BALB/c IL-10(-/-) mice. In contrast to the lack of resistance conferred by IL-4R alpha gene deletion, partial resistance to LV39 was conferred by IL-10 gene deletion or treatment of BALB/c mice with anti-IL-10R mAb. Lesion sizes and LV39 parasite numbers were further and dramatically reduced in both anti-IL-10R-treated IL-4R alpha(-/-) mice and IL-4R alpha x IL-10 double knockouts. Anti-IL-10R mAb treatment further suppressed parasite growth in IL-4R alpha(-/-) mice infected with L. major IR173. Production of IFN-gamma was only increased relative to wild-type or littermate controls in IL-4R alpha(-/-) mice with complementary defects in IL-10. Comparisons of IFN-gamma-treated infected macrophages in vitro indicated that LV39 required 25- to 500-fold greater concentrations of IFN-gamma than IR173-infected macrophages to achieve a similar efficiency of parasite killing. These studies suggest that regardless of parasite substrain, IL-10 is as important as IL-4/IL-13 in promoting susceptibility to L. major and even more so for those substrains that are relatively resistant to IFN-gamma mediated killing.     

Severe CD4 T cell-mediated immunopathology in murine schistosomiasis is dependent on IL-12p40 and correlates with high levels of IL-17

C57BL/6 mice infected with the helminth Schistosoma mansoni develop small hepatic granulomas around parasite eggs, but concomitant immunization with soluble schistosome egg Ags (SEA) in CFA (SEA/CFA) causes marked exacerbation of the lesions in a Th1-dominated environment characterized by high levels of IFN-gamma. We explored the cause of the severe immunopathology by using IL-12p40(-/-) and IL-12p35(-/-) mice. SEA/CFA-immunized IL-12p40(-/-) mice, incapable of making IL-12 or IL-23, were completely resistant to high pathology, and their SEA-stimulated lymphoid cells failed to secrete significant IFN-gamma or IL-17. In contrast, SEA/CFA-immunized IL-12p35(-/-) mice, able to make IL-23 but not IL-12, developed severe lesions that correlated with high levels of IL-17, low IFN-gamma, and an expansion of activated CD4 T cells with a CD44(high)/CD62L(low) memory phenotype. In vivo administration of neutralizing anti-IL-17 mAb markedly inhibited hepatic granulomatous inflammation. Importantly, CBA mice, a naturally high pathology strain, also displayed elevated IL-17 levels comparable to those seen in the SEA/CFA-immunized BL/6 mice, and their lesions were similarly reduced by in vivo treatment with anti-IL-17. Our findings indicate that an IL-17-producing T cell population, likely driven by IL-23, significantly contributes to severe immunopathology in schistosomiasis.     

Acquired resistance and granuloma formation in experimental visceral leishmaniasis. Differential T cell and lymphokine roles in initial versus established immunity.

In naive BALB/c mice, acquisition of resistance to Leishmania donovani and formation of antileishmanial tissue granulomas are linked expressions that require both L3T4+ and Lyt 2+ cells as well as both IL-2 and IFN-gamma. To determine the mechanisms of established resistance to L. donovani, rechallenged immune BALB/c mice were treated with T cell- and lymphokine-depleting mAb or cyclosporin A. In the liver, resistance to rechallenge was inhibited by treatment with anti-Lyt 2 but not anti-L3T4 mAb. Resistance was also impaired by anti-IL-2 treatment but not by anti-IFN-gamma mAb. The hepatic granulomatous response to rechallenge, however, was not impaired by either anti-Lyt 2 or anti-IL-2 mAb nor by anti-L3T4 or anti-IFN-gamma treatment. In contrast, cyclosporin A suppressed granuloma formation but not antileishmanial activity. These results indicate a particularly important antileishmanial host defense role for Lyt 2+ cells and IL-2 in sensitized animals, and when compared to prior observations in L. donovani-infected naive mice, suggest that 1) discrete T cell- and lymphokine-dependent mechanisms are involved in initial acquisition of resistance vs established immunity, 2) more than one mechanism can mediate the development of tissue granulomas, and 3) granuloma formation by itself may not be required nor necessarily sufficient to confer antimicrobial activity.     

Th17 cells induce colitis and promote Th1 cell responses through IL-17 induction of innate IL-12 and IL-23 production

Both Th1 and Th17 cells have been implicated in the pathogenesis of inflammatory bowel disease and experimental colitis. However, the complex relationship between Th1 and Th17 cells and their relative contributions to the pathogenesis of inflammatory bowel disease have not been completely analyzed. Although it has been recently shown that Th17 cells can convert into Th1 cells, the underlying in vivo mechanisms and the role of Th1 cells converted from Th17 cells in the pathogenesis of colitis are still largely unknown. In this study, we report that Th17 cells from CBir1 TCR transgenic mice, which are specific for an immunodominant microbiota Ag, are more potent than Th1 cells in the induction of colitis, as Th17 cells induced severe colitis, whereas Th1 cells induced mild colitis when transferred into TCRβxδ(-/-) mice. High levels of IL-12 and IL-23 and substantial numbers of IFN-γ(+) Th1 cells emerged in the colons of Th17 cell recipients. Administration of anti-IL-17 mAb abrogated Th17 cell-induced colitis development, blocked colonic IL-12 and IL-23 production, and inhibited IFN-γ(+) Th1 cell induction. IL-17 promoted dendritic cell production of IL-12 and IL-23. Furthermore, conditioned media from colonic tissues of colitic Th17 cell recipients induced IFN-γ production by Th17 cells, which was inhibited by blockade of IL-12 and IL-23. Collectively, these data indicate that Th17 cells convert to Th1 cells through IL-17 induction of mucosal innate IL-12 and IL-23 production.     

IL-6 is required for the development of Th1 cell-mediated murine colitis

Proinflammatory cytokines have been demonstrated to play a crucial role in the pathogenesis of Crohn's disease. Among those cytokines, strong expression of IL-6 has been repeatedly demonstrated. To examine the role for IL-6 in the pathogenesis of Crohn's disease, we introduced anti-IL-6R mAb to a murine model of colitis. Colitis was induced in C.B-17-scid mice transferred with CD45RBhigh CD4+ T cells from BALB/c mice. Anti-IL-6R mAb or rat IgG was administered weekly after T cell transfer. ICAM-1 and VCAM-1 expression were analyzed by immunohistochemistry. Colonic cytokine expression was determined by RT-PCR. Mice treated with mAb showed normal growth, whereas controls lost weight. The average colitis score was 0.64 for mAb-treated mice and 1.80 for controls. T cell expansion in treated mice was less remarkable than in the controls. Colonic ICAM-1 and VCAM-1 expression were markedly suppressed by mAb. IFN-gamma, TNF-alpha, and IL-1beta mRNA were reduced by the treatment. The results presented here show a crucial role for IL-6 in the pathogenesis of murine colitis and suggest a therapeutic potential of anti-IL-6R mAb for treatment of human Crohn's disease.     

Role of interleukin-4 (IL-4), IL-12, and gamma interferon in primary and vaccine-primed immune responses to Friend retrovirus infection

The immunological resistance of a host to viral infections may be strongly influenced by cytokines such as interleukin-12 (IL-12) and gamma interferon (IFN-gamma), which promote T helper type 1 responses, and IL-4, which promotes T helper type 2 responses. We studied the role of these cytokines during primary and secondary immune responses against Friend retrovirus infections in mice. IL-4- and IL-12-deficient mice were comparable to wild-type B6 mice in the ability to control acute and persistent Friend virus infections. In contrast, more than one-third of the IFN-gamma-deficient mice were unable to maintain long-term control of Friend virus and developed gross splenomegaly with high virus loads. Immunization with a live attenuated vaccine virus prior to challenge protected all three types of cytokine-deficient mice from viremia and high levels of spleen virus despite the finding that the vaccinated IFN-gamma-deficient mice were unable to class switch from immunoglobulin M (IgM) to IgG virus-neutralizing antibodies. The results indicate that IFN-gamma plays an important role during primary immune responses against Friend virus but is dispensable during vaccine-primed secondary responses.     

IL-10 is required for prevention of necrosis in the small intestine and mortality in both genetically resistant BALB/c and susceptible C57BL/6 mice following peroral infection with Toxoplasma gondii

The role for IL-10 in the immunopathogenesis of acute toxoplasmosis following peroral infection was examined in both genetically susceptible C57BL/6 and resistant BALB/c mice. C57BL/6-background IL-10-targeted mutant (IL-10-/-) mice all died in 2 wk after infection with 20 cysts of the ME49 strain, whereas only 20% of control mice succumbed. Histological studies revealed necrosis in the small and large intestines and livers of infected IL-10-/- mice. The necrosis in the small intestine was the most severe pathologic response and was not observed in control mice. Treatment of infected IL-10-/- mice with either anti-CD4 or anti-IFN-gamma mAb prevented intestinal pathology and significantly prolonged time to death. Treatment of these animals with anti-IL-12 mAb also prevented the pathology. Significantly greater amounts of IFN-gamma mRNA were detected in the lamina propria lymphocytes obtained from the small intestine of infected IL-10-/- mice than those from infected control mice. In common with C57BL/6-background IL-10-/- mice, BALB/c-background IL-10-/- mice all died developing intestinal pathology after infection. Control BALB/c mice all survived even after infection with 100 cysts and did not develop the intestinal lesions. Treatment with anti-IFN-gamma mAb prevented the pathology and prolonged time to death of the infected IL-10-/- mice. These results strongly suggest that IL-10 plays a critical role in down-regulating IFN-gamma production in the small intestine following sublethal peroral infection with Toxoplasma gondii and that this down-regulatory effect of IL-10 is required for prevention of development of IFN-gamma-mediated intestinal pathology and mortality in both genetically resistant BALB/c and susceptible C57BL/6 mice.     

IL-23 enhances the inflammatory cell response in Cryptococcus neoformans infection and induces a cytokine pattern distinct from IL-12

IL-23, a heterodimeric cytokine composed of the p40 subunit of IL-12 and a novel p19 subunit, has been shown to be a key player in models of autoimmune chronic inflammation. To investigate the role of IL-23 in host resistance during chronic fungal infection, wild-type, IL-12- (IL-12p35-/-), IL-23- (IL-23p19-/-), and IL-12/IL-23- (p40-deficient) deficient mice on a C57BL/6 background were infected with Cryptococcus neoformans. Following infection, p40-deficient mice demonstrated higher mortality than IL-12p35-/- mice. Reconstitution of p40-deficient mice with rIL-23 prolonged their survival to levels similar to IL-12p35-/- mice. IL-23p19-/- mice showed a moderately reduced survival time and delayed fungal clearance in the liver. Although IFN-gamma production was similar in wild-type and IL-23p19-/- mice, production of IL-17 was strongly impaired in the latter. IL-23p19-/- mice produced fewer hepatic granulomata relative to organ burden and showed defective recruitment of mononuclear cells to the brain. Moreover, activation of microglia cells and expression of IL-1beta, IL-6, and MCP-1 in the brain was impaired. These results show that IL-23 complements the more dominant role of IL-12 in protection against a chronic fungal infection by an enhanced inflammatory cell response and distinct cytokine regulation.     

Rapid IL-4 production by Leishmania homolog of mammalian RACK1-reactive CD4(+) T cells in resistant mice treated once with anti-IL-12 or -IFN-gamma antibodies at the onset of infection with Leishmania major instructs Th2 cell development, resulting in nonhealing lesions

Rapid production of IL-4 by Leishmania homolog of mammalian RACK1 (LACK)-reactive CD4(+) T cells expressing the V beta 4-V alpha 8 TCR chains has been shown to drive aberrant Th2 cell development and susceptibility to Leishmania major in BALB/c mice. In contrast, mice from resistant strains fail to express this early IL-4 response. However, administration of either anti-IL-12 or -IFN-gamma at the initiation of infection allows the expression of this early IL-4 response in resistant mice. In this work we show that Leishmania homolog of mammalian RACK1-reactive CD4(+) T cells also expressing the V beta 4-V alpha 8 TCR chains are the source of the early IL-4 response to L. major in resistant mice given anti-IL-12 or -IFN-gamma Abs only at the onset of infection. Strikingly, these cells were found to be required for the reversal of the natural resistance of C57BL/6 mice following a single administration of anti-IL-12 or -IFN-gamma Abs. Together these results suggest that a deficiency in mechanisms capable of down-regulating the early IL-4 response to L. major contributes to the exquisite susceptibility of BALB/c mice to L. major.     

The roles of IL-4, IL-5 and mast cells in the accumulation of eosinophils during allergic cutaneous late phase reaction in mice

Late phase allergic response has been implicated in the pathogenesis of allergic diseases. In the current study, we investigated the role of IL-4, IL-5 and mast cells in the development of cutaneous late phase reaction (LPR) in mice. Antigenic challenge of ears of ovalbumin (OVA)-immunized BALB/c mice caused a biphasic ear swelling peaking at 1 hr (immediate phase reaction; IPR) and 24 hr (LPR). Ear swelling in LPR was significantly suppressed by the treatment with anti-IL-4 monoclonal antibody (mAb) before antigen challenge. Local eosinophil accumulation during LPR, however, was not inhibited by anti-IL-4 mAb. Moreover, anti-IL-5 mAb had no effect on the swelling response though it significantly suppressed the local accumulation of eosinophils. Interestingly, mast cell-deficient mice (WBB6F1-W/Wv) developed LPR without exhibiting IPR, while the magnitude of ear swelling and local eosinophilia was significantly lower than in normal congenic mice (+/+ mice). The present findings show that IL-4 and IL-5 differently regulate the development of LPR, and that IgE-mediated mast cell activation is required for full response.     

IL-12-assisted immunization generates CD4+ T cell-mediated immunity to Listeria monocytogenes

Mice infected with virulent Listeria monocytogenes develop long-lived acquired immunity. We previously reported that acquired immunity to Listeria could also be elicited by immunizing mice with non-viable Listeria or listerial proteins/peptides in combination with IL-12. Here we show that this IL-12-assisted immunization strategy was effective in class I but not in class II MHC-deficient mice, suggesting that antigen-specific CD4(+) T cells are selectively generated using this adjuvant system. We have also evaluated the importance of endogenous production of IFN-gamma and IL-12 for the efficacy of IL-12-assisted immunization. IFN-gamma-deficient mice immunized with HKLM and IL-12 failed to produce effective Listeria-specific responses. In contrast, IL-12-deficient mice were able to generate protective antigen-specific T cell responses in response to immunization with HKLM and IL-12, indicating that exogenous IL-12 is sufficient to initiate a cytokine cascade that results in a potent T(H)1 response. IL-12-assisted immunization provides a model in which both the generation and effector mechanisms of anti-bacterial antigen-specific CD4(+) effector cells can be analyzed.     

IL-9 is a susceptibility factor in Leishmania major infection by promoting detrimental Th2/type 2 responses

IL-9 is a cytokine produced by Th2 cells, induced during Leishmania major infection. Because the role of IL-9 in leishmaniasis is currently unknown, IL-9-deficient mice were generated by immunization with mouse IL-9 coupled to OVA. This produced strong and long-lasting neutralizing anti-IL-9 Abs in vivo. Anti-IL-9 vaccination showed protective effects, because it enabled L. major-infected nonhealer BALB/c mice to better resist to leishmaniasis with doubling the time span until pathological disease progression occurred. Increased resistance was also demonstrated by moderate footpad swelling and histopathology due to reduced parasite burden compared with sham-immunized BALB/c mice. Mechanistically, IL-9 neutralization in BALB/c mice resulted in a reduction of detrimental Th2/type 2 responses with an observed shift toward protective Th1 immune responses. This led to an alteration from alternative to classical macrophage activation with subsequent enhanced killing effector functions, as demonstrated by increased NO production but reduced arginase 1-mediated macrophage responses. Conclusively, the data show that IL-9 is a susceptible factor in leishmaniasis. They further suggest that IL-9 is able to influence Th dichotomy in leishmaniasis by promoting detrimental Th2/type 2 responses in BALB/c mice. The results extend efforts made to generate autoantibodies capable of regulating biological processes, with IL-9 a potential drug target against leishmaniasis.     

Complement C3a-induced IL-17 plays a critical role in an IgE-mediated late-phase asthmatic response and airway hyperresponsiveness via neutrophilic inflammation in mice

Allergen-specific IgE plays an essential role in the pathogenesis of allergic asthma. Although there has been increasing evidence suggesting the involvement of IL-17 in the disease, the relationship between IL-17 and IgE-mediated asthmatic responses has not yet been defined. In this study, we attempted to elucidate the contribution of IL-17 to an IgE-mediated late-phase asthmatic response and airway hyperresponsiveness (AHR). BALB/c mice passively sensitized with an OVA-specific IgE mAb were challenged with OVA intratracheally four times. The fourth challenge caused a late-phase increase in airway resistance associated with elevated levels of IL-17(+)CD4(+) cells in the lungs. Multiple treatments with a C3a receptor antagonist or anti-C3a mAb during the challenges inhibited the increase in IL-17(+)CD4(+) cells. Meanwhile, a single treatment with the antagonist or the mAb at the fourth challenge suppressed the late-phase increase in airway resistance, AHR, and infiltration by neutrophils in bronchoalveolar lavage fluid. Because IL-17 production in the lungs was significantly repressed by both treatments, the effect of an anti-IL-17 mAb was examined. The late-phase increase in airway resistance, AHR, and infiltration by neutrophils in bronchoalveolar lavage fluid was inhibited. Furthermore, an anti-Gr-1 mAb had a similar effect. Collectively, we found that IgE mediated the increase of IL-17(+)CD4(+) cells in the lungs caused by repeated Ag challenges via C3a. The mechanisms leading to the IgE-mediated late-phase asthmatic response and AHR are closely associated with neutrophilic inflammation through the production of IL-17 induced by C3a.     

Despite increased CD4+Foxp3+ cells within the infection site, BALB/c IL-4 receptor-deficient mice reveal CD4+Foxp3-negative T cells as a source of IL-10 in Leishmania major susceptibility

BALB/c IL-4Ralpha(-/-) mice, despite the absence of IL-4/IL-13 signaling and potent Th2 responses, remain highly susceptible to Leishmania major substain LV39 due exclusively to residual levels of IL-10. To address the contribution of CD4(+)CD25(+) T regulatory (Treg) cells to IL-10-mediated susceptibility, we depleted CD4(+)CD25(+) cells in vivo and reconstituted IL-4Ralpha x RAG2 recipients with purified CD4(+)CD25(-) T cells. Although anti-CD25 mAb treatment significantly decreased parasite numbers in IL-4Ralpha(-/-) mice, treatment with anti-IL-10R mAb virtually eliminated L. major parasites in both footpad and dermal infection sites. In addition, IL-4Ralpha x RAG2 mice reconstituted with CD4(+) cells depleted of CD25(+) Treg cells remained highly susceptible to infection. Analysis of L. major-infected BALB/c and IL-4Ralpha(-/-) inflammatory sites revealed that the majority of IL-10 was secreted by the CD4(+)Foxp3(-) population, with a fraction of IL-10 coming from CD4(+)Foxp3(+) Treg cells. All T cell IFN-gamma production was also derived from the CD4(+)Foxp3(-) population. Nevertheless, the IL-4Ralpha(-/-)-infected ear dermis, but not draining lymph nodes, consistently displayed 1.5- to 2-fold greater percentages of CD4(+)CD25(+) and CD4(+)Foxp3(+) Treg cells compared with the BALB/c-infected dermis. Thus, CD4(+)Foxp3(-) T cells are a major source of IL-10 that disrupts IFN-gamma activity in L. major-susceptible BALB/c mice. However, the increase in CD4(+)Foxp3(+) T cells within the IL-4Ralpha(-/-) dermis implies a possible IL-10-independent role for Treg cells within the infection site, and may indicate a novel immune escape mechanism used by L. major parasites in the absence of IL-4/IL-13 signaling.     

Epstein Barr Virus-Induced 3 (EBI3) Together with IL-12 Negatively Regulates T Helper 17-Mediated Immunity to Listeria monocytogenes Infection

Although the protective functions by T helper 17 (Th17) cytokines against extracellular bacterial and fungal infection have been well documented, their importance against intracellular bacterial infection remains unclear. Here, we investigated the contribution of Th17 responses to host defense against intracellular bacteria Listeria monocytogenes and found that Th17 cell generation was suppressed in this model. Unexpectedly, mice lacking both p35 and EBI3 cleared L. monocytogenes as efficiently as wild-type mice, whereas p35-deficient mice failed to do so. Furthermore, both innate cells and pathogen-specific T cells from double-deficient mice produced significantly higher IL-17 and IL-22 compared to wild-type mice. The bacterial burden in the liver of double-deficient mice treated with anti-IL-17 was significantly increased compared to those receiving a control Ab. Transfer of Th17 cells specific for listeriolysin O as well as administration of IL-17 and IL-22 significantly suppressed bacterial growth in p35-deficient mice, indicating the critical contribution of Th17 responses to host defense against the intracellular pathogen in the absence of IL-12 and proper Th1 responses. Our findings unveil a novel immune evasion mechanism whereby the intracellular bacteria exploit IL-27EBI3 to suppress Th17-mediated protective immunity.     

The role of interleukin-10 in susceptibility of BALB/c mice to infection with Leishmania mexicana and Leishmania amazonensis

Recent studies have demonstrated the critical role of IL-10 in susceptibility to cutaneous and visceral leishmaniasis caused by Leishmania major and Leishmania donovani, respectively. To determine whether IL-10 also plays a similar role in the susceptibility and pathogenesis of cutaneous leishmaniasis caused by the New World species, L. mexicana and L. amazonensis, we analyzed their course of infection in IL-10-deficient BALB/c mice and their wild-type counterparts. Although IL-10-deficient mice infected with either L. mexicana or L. amazonensis failed to control the lesion progression, we did observe consistently lower levels of infection in IL-10(-/-) mice compared with wild-type BALB/c mice. We also observed increased IFN-gamma and NO production and higher levels for IL-12p40 and IL-12Rbeta(2) mRNA in cells from IL-10(-/-) mice compared with cells from BALB/c mice. The mRNA levels for IL-4, which increased significantly in both IL-10(-/-) and BALB/c mice, were comparable. When treated with anti-IL-4 mAb, IL-10(-/-) mice resolved the infection more effectively and had significantly fewer parasites in their lesions compared with similarly treated BALB/c mice. These findings suggest that IL-10, although not the dominant mediator of susceptibility of BALB/c mice to infection with L. mexicana and L. amazonensis, does play a significant role in regulating the development of a protective Th1-type response. However, effective resolution of infection with these New World parasites requires neutralization of both IL-4 and IL-10.     

Compromised humoral and delayed-type hypersensitivity responses in IL-23-deficient mice

The heterodimeric cytokine IL-23 consists of a private cytokine-like p19 subunit and a cytokine receptor-like subunit, p40, which is shared with IL-12. Previously reported IL-12p40-deficient mice have profound immune defects resulting from combined deficiency in both IL-12 and IL-23. To address the effects of specific IL-23 deficiency, we generated mice lacking p19 by gene targeting. These mice display no overt abnormalities but mount severely compromised T-dependent humoral immune responses. IL-23p19(-/-) mice produce strongly reduced levels of Ag-specific Igs of all isotypes, but mount normal T-independent B cell responses. In addition, delayed type hypersensitivity responses are strongly impaired in the absence of IL-23, indicating a defect at the level of memory T cells. T cells stimulated with IL-23-deficient APCs secrete significantly reduced amounts of the proinflammatory cytokine IL-17, and IL-23-deficient mice phenotypically resemble IL-17-deficient animals. Thus, IL-23 plays a critical role in T cell-dependent immune responses, and our data provide further support for the existence of an IL-23/IL-17 axis of communication between the adaptive and innate parts of the immune system.     

IL-18 contributes to host resistance against infection with Pseudomonas aeruginosa through induction of IFN-gamma production

Pseudomonas aeruginosa keratitis destroys the cornea in susceptible (B6), but not resistant (BALB/c) mice. To determine mechanisms mediating resistance, the role of IFN-gamma, IL-12, and IL-18 was tested in BALB/c mice. RT-PCR analysis detected IFN-gamma mRNA expression levels in cornea that were significantly increased at 1-7 days postinfection. IL-18 mRNA was detected constitutively in cornea and, at 1-7 days postinfection, levels were elevated significantly, while no IL-12 mRNA was similarly detected. To test whether IL-18 contributed to IFN-gamma production, mice were treated with anti-IL-18 mAb. Treatment decreased corneal IFN-gamma mRNA levels, and bacterial load and disease increased/worsened, compared with IgG-treated mice. To stringently examine the role of IFN-gamma in bacterial killing, knockout (-/-) vs wild-type (wt) mice also were tested. All corneas perforated, and bacterial load was increased significantly in -/- vs wt mice. Because disease severity was increased in IFN-gamma(-/-) vs IL-18-neutralized mice, and since IL-18 also induces production of TNF, we tested for TNF-alpha in both groups. ELISA analysis demonstrated significantly elevated corneal TNF-alpha protein levels in IFN-gamma(-/-) vs wt mice after infection. In contrast, RT-PCR analysis of IL-18-neutralized vs IgG-treated infected mice revealed decreased corneal TNF-alpha mRNA expression. Next, to resolve whether TNF was required for bacterial killing, TNF-alpha was neutralized in BALB/c mice. No difference in corneal bacterial load was detected in neutralized vs IgG-treated mice. These data provide evidence that IL-18 contributes to the resistance response by induction of IFN-gamma and that IFN-gamma is required for bacterial killing.