Protective and memory immunity to Histoplasma capsulatum in the absence of IL-10

We determined whether the absence of IL-10 in mice influenced protective and memory immunity to Histoplasma capsulatum. IL-10(-/-) mice cleared primary and secondary infection more rapidly than wild-type controls. Administration of mAb to TNF-alpha or IFN-gamma, but not GM-CSF, abrogated protection in naive IL-10(-/-) mice; mAb to TNF-alpha, but not IFN-gamma or GM-CSF, subverted protective immunity in secondary histoplasmosis. The inflammatory cell composition in IL-10(-/-) mice was altered in those given mAb to IFN-gamma or TNF-alpha. More Gr-1(+) and Mac-3(+) cells were present in lungs of IL-10(-/-) mice given mAb to IFN-gamma, and treatment with mAb to TNF-alpha sharply reduced the number of CD8(+) cells in lungs of IL-10(-/-) mice. We ascertained whether the lack of IL-10 modulated memory T cell generation or the protective function of cells. The percentage of CD3(+), CD44(high), CD62(low), and IFN-gamma(+) cells in IL-10(-/-) mice was higher than that of wild-type at day 7 but not day 21 or 49 after immunization. Fewer splenocytes from immunized IL-10(-/-) mice were required to mediate protection upon adoptive transfer into infected TCR alphabeta(-/-) mice. Hence, deficiency of IL-10 confers a salutary effect on the course of histoplasmosis, and the beneficial effects of IL-10 deficiency require endogenous TNF-alpha and/or IFN-gamma. Memory cell generation was transiently increased in IL-10(-/-) mice, but the protective function conferred by cells from these mice following immunization is strikingly more vigorous than that of wild-type.     

TNF-alpha antagonism generates a population of antigen-specific CD4+CD25+ T cells that inhibit protective immunity in murine histoplasmosis

In both humans and mice, treatment with TNF-alpha antagonists is associated with serious infectious complications including disseminated histoplasmosis. The mechanisms by which inhibition of endogenous TNF-alpha alter protective immunity remain obscure. Herein, we tested the possibility that neutralization of this cytokine triggered the emergence of T cells that dampen immunity. The lungs of mice given mAb to TNF-alpha contained a higher proportion and number of CD4+CD25+ cells than controls. This elevation was not observed in IFN-gamma- or GM-CSF-deficient mice or in those given a high inoculum. Phenotypic analysis revealed that these cells lacked many of the characteristics of natural regulatory T cells, including Foxp3. CD4+CD25+ cells from TNF-alpha-neutralized mice suppressed Ag-specific, but not nonspecific, responses in vitro. Elimination of CD25+ cells in vivo restored protective immunity in mice given mAb to TNF-alpha and adoptive transfer of CD4+CD25+ cells inhibited immunity. In vitro and in vivo, the suppressive effect was reversed by mAb to IL-10. Thus, neutralization of TNF-alpha is associated with the induction of a population of regulatory T cells that alter protective immunity in an Ag-specific manner to Histoplasma capsulatum.     

Complex requirements for nascent and memory immunity in pulmonary histoplasmosis.

The presence of functional T cells is often required for successful resolution of infections with intracellular pathogens, yet the mechanisms by which they contribute to elimination of the invading pathogen in primary and secondary immunity are only partly understood. We report that increased mortality of naive alpha/beta TCR+ or CD4+ T cell-depleted mice infected with the fungus Histoplasma capsulatum is associated with impairment of IFN-gamma production. Upon secondary infection, mice concomitantly depleted of CD4+ and CD8+ cells exhibit decreased survival beyond day 25 of rechallenge, whereas elimination of either T cell subset or B cell deficiency does not result in accelerated mortality compared with controls. Remarkably, despite a decrease of H. capsulatum CFU in lungs of CD4+ plus CD8+-deficient mice, a progressive increase in spleen CFU is observed. The ability to control fungus growth in lungs is associated with vigorous TNF-alpha, but not IFN-gamma, production by bronchoalveolar lavage cells. In contrast, spleen cells from CD4+ plus CD8+-deficient mice are unable to produce TNF-alpha. Thus, the cellular and molecular requirements for protective immunity vary between primary and secondary infection. Furthermore, in secondary histoplasmosis, a sharp contrast can be drawn between lungs and spleens in their reliance upon T cells to control fungal replication. The opposing activities of these organs can be ascribed in part to differential production of TNF-alpha.     

IL-12 is required for antibody-mediated protective immunity against blood-stage Plasmodium chabaudi AS malaria infection in mice.

In this study, we investigated the role of endogenous IL-12 in protective immunity against blood-stage P. chabaudi AS malaria using IL-12 p40 gene knockout (KO) and wild-type (WT) C57BL/6 mice. Following infection, KO mice developed significantly higher levels of primary parasitemia than WT mice and were unable to rapidly resolve primary infection and control challenge infection. Infected KO mice had severely impaired IFN-gamma production in vivo and in vitro by NK cells and splenocytes compared with WT mice. Production of TNF-alpha and IL-4 was not compromised in infected KO mice. KO mice produced significantly lower levels of Th1-dependent IgG2a and IgG3 but a higher level of Th2-dependent IgG1 than WT mice during primary and challenge infections. Treatment of KO mice with murine rIL-12 during the early stage of primary infection corrected the altered IgG2a, IgG3, and IgG1 responses and restored the ability to rapidly resolve primary and control challenge infections. Transfer of immune serum from WT mice to P. chabaudi AS-infected susceptible A/J mice completely protected the recipients, whereas immune serum from KO mice did not, as evidenced by high levels of parasitemia and 100% mortality in recipient mice. Furthermore, depletion of IgG2a from WT immune serum significantly reduced the protective effect of the serum while IgG1 depletion had no significant effect. Taken together, these results demonstrate the protective role of a Th1-immune response during both acute and chronic phases of blood-stage malaria and extend the immunoregulatory role of IL-12 to Ab-mediated immunity against Plasmodium parasites.     

Regulation of infection with Histoplasma capsulatum by TNFR1 and -2

The concerted action of several cytokines is necessary for resolution of both primary and secondary infection with Histoplasma capsulatum. Among the soluble factors that contribute to tissue sterilization, TNF-alpha stands as a central mediator of protective immunity to this fungus. In this study, we explored the regulation of protective immunity by TNFR1 and -2. In primary pulmonary infection, both TNFR1-/- and -2-/- mice manifested a high mortality after infection with H. capsulatum, although TNFR1-/- mice were more susceptible than TNFR2 -/- mice. Overwhelming infection in the former was associated with a pronounced decrement in the number of inflammatory cells in the lungs and elevated IFN-gamma and TNF-alpha levels in the lungs. In contrast, IFN-gamma levels were markedly decreased in TNFR2-/- mice, and treatment with this cytokine restored protective immunity. Lung macrophages from both groups of knockout mice released substantial amounts of NO. Upon secondary infection, TNFR2-/- mice survived rechallenge and cleared infection as efficiently as C57BL/6 animals. In contrast, mice given mAb to TNFR1 succumbed to reexposure, and the high mortality was accompanied by a significant increase in fungal burden in the lungs. Both IL-4 and IL-10 were elevated in the lungs of these mice. The results demonstrate the pivotal influence of TNFR1 and -2 in controlling primary infection and highlight the differences between these receptors for regulation reexposure histoplasmosis.     

Adoptive transfer of CD8alpha+ dendritic cells (DC) isolated from mice infected with Chlamydia muridarum are more potent in inducing protective immunity than CD8alpha- DC

Chlamydial infections are serious public health concerns worldwide. In this study, we examined the role of dendritic cell (DC) subsets in inducing protective immunity against chlamydial infection using an adoptive transfer approach. We found that CD11c+CD8alpha+ (double-positive, DP) DC, compared with CD11c+CD8alpha- (single-positive, SP) DC isolated from infected mice, are more potent inducers of protective immunity. Specifically, mice pretreated with DPDC from infected mice, upon infection with Chlamydia trachomatis mouse pneumonitis (MoPn), experienced significantly less severe body weight loss and in vivo chlamydial growth. Analysis of MoPn-driven cytokine production by immune cells revealed that mice that were treated with DPDC produced significantly higher levels of Th1 (TNF-alpha, IFN-gamma, and IL-12) but lower levels of Th2 (IL-4, IL-5, and IL-13)-related cytokines than the recipients of SPDC following infection challenge. Moreover, DPDC-treated mice displayed significantly higher levels of MoPn-specific IgG2a production and delayed-type hypersensitivity responses compared with SPDC-treated mice. Furthermore, DPDC isolated from infected mice produced higher amounts of IL-12 and IL-10 in vitro in comparison with SPDC. These data indicate that CD8alpha+ DC have a significantly higher capacity in inducing protective immunity compared with CD8alpha- DC, demonstrating the crucial role of DC1-like cells in eliciting protection against C. trachomatis infection.     

Protection against Nippostrongylus brasiliensis infection in mice is independent of GM-CSF

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a cytokine with the capacity to promote inflammation in a wide variety of infectious and inflammatory diseases. These conditions include allergic airway inflammation, which is driven by T-helper 2 (Th2) cells. Because of the importance of Th2 cells in parasite infections, we have investigated the role of GM-CSF in mice infected with the nematode Nippostrongylus brasiliensis. The effect of primary and secondary infection was investigated in mice lacking functional genes for GM-CSF (CSF2 genes) (ΔGM-CSF mice), and in mice lacking the cytokine receptor common β chain (Δβ mice), the latter being unable to signal in response to GM-CSF and interleukin (IL)-5. ΔGM-CSF mice showed no significant defect in parasite immunity, measured by larval numbers in the lungs, worm numbers in the intestine or egg numbers in the faeces, in either primary or secondary infection. By contrast, the Δβ mice showed increased parasite burden, with higher numbers of lung larvae after secondary infection and higher numbers of intestinal worms and faecal eggs after both primary and secondary infection. Unexpectedly, there were increased numbers of circulating eosinophils in the ΔGM-CSF mice, associated with significantly reduced larval numbers in the lungs. These results indicate that GM-CSF is redundant in protection against N. brasiliensis infection, and that the increased susceptibility of Δβ mice to infection is likely to be attributed to the lack of IL-5 signalling in these mice. The results suggest that clinical use of agents that neutralise GM-CSF may not be associated with increased risk of parasite infection.     

IL-17-mediated regulation of innate and acquired immune response against pulmonary Mycobacterium bovis bacille Calmette-Guerin infection

IL-17 is a cytokine that induces neutrophil-mediated inflammation, but its role in protective immunity against intracellular bacterial infection remains unclear. In the present study, we demonstrate that IL-17 is an important cytokine not only in the early neutrophil-mediated inflammatory response, but also in T cell-mediated IFN-gamma production and granuloma formation in response to pulmonary infection by Mycobacterium bovis bacille Calmette-Guérin (BCG). IL-17 expression in the BCG-infected lung was detected from the first day after infection and the expression depended on IL-23. Our observations indicated that gammadelta T cells are a primary source of IL-17. Lung-infiltrating T cells of IL-17-deficient mice produced less IFN-gamma in comparison to those from wild-type mice 4 wk after BCG infection. Impaired granuloma formation was also observed in the infected lungs of IL-17-deficient mice, which is consistent with the decreased delayed-type hypersensitivity response of the infected mice against mycobacterial Ag. These data suggest that IL-17 is an important cytokine in the induction of optimal Th1 response and protective immunity against mycobacterial infection.     

B cells and CD4-CD8- T cells are key regulators of the severity of reactivation histoplasmosis

The fungus, Histoplasma capsulatum, produces a persistent infection. Reactivation histoplasmosis is largely a result of impaired immunity, but the perturbations associated with escape of the fungus from host defenses remain ill-defined. We analyzed a murine model of reactivation to elucidate the host defects that permit reactivation. C57BL/6 mice were infected intranasally and, 42 days later, they were depleted of CD4(+) and CD8(+) cells. Elimination of these cells, but not either alone, produced a persistent infection over several weeks. Neutralization of IFN-gamma, TNF-alpha, or both did not induce reactivation. Endogenous IL-10 exacerbated reactivation. Depletion of T cells in B cell(-/-) mice induced a markedly higher burden in organs when compared with wild type. However, the infection remained persistent. Elimination of CD4(+) cells alone or neutralization of cytokines increased the fungal load. The persistent infection was not dependent on gammadelta T cells or NK cells. Elimination of Thy-1.2(+) cells in mice given mAb to CD4 and CD8 transformed reactivation into a progressive, lethal infection in B cell(-/-) and wild-type mice, but the tempo of progression was accelerated in the former. The data reveal the complex control by the host to prevent reactivation of this fungus.     

Interleukin-4 causes delayed virus clearance in influenza virus-infected mice.

Two different subsets of T cells, Th1 and Th2 cells, have been demonstrated to secrete different profiles of cytokines and to influence various infections in different ways. Whereas cytokines secreted by Th1 cells, particularly gamma interferon, promote the generation of cell-mediated immunity, Th2 cells and their cytokines (interleukin-4 [IL-4], IL-5, IL-10, and IL-13) have been shown to function in recovery from parasitic infections and in antibody responses. In this study, we analyzed the effects of the dominant Th2 cytokine, IL-4, on immunity to virus infection. We assessed the effects of IL-4 on both secondary immune responses by an adoptive transfer assay and primary immune responses by in vivo treatment of influenza virus-infected mice with IL-4. The results demonstrated that IL-4 can function to inhibit antiviral immunity at both stages. We found that IL-4 treatment of sensitized cells during secondary stimulation in vitro had little effect on their ability to lyse virus-infected target cells in a 51Cr release assay. Nevertheless, the clearance of influenza A/PR/8/34 (H1N1) virus from the lungs of infected BALB/c mice was significantly delayed after the transfer of virus-specific T cells secondarily stimulated in the presence of IL-4 in comparison to virus clearance in recipients of cells stimulated in the absence of IL-4. In contrast to the adoptive transfer results, the treatment of PR8 virus-infected mice with IL-4 during primary infection greatly suppressed the generation of cytotoxic T-cell precursors, as assessed by secondary stimulation in vitro. In addition, culture supernatants of secondarily stimulated spleen cells from IL-4-treated mice contained significantly less gamma interferon and more IL-4 than did spleen cells from controls. More importantly, the treatment of mice with IL-4 resulted in an extremely significant delay in virus clearance. Thus, IL-4 can inhibit both primary and secondary antiviral immune responses.     

IL-10 is required for optimal CD8 T cell memory following Listeria monocytogenes infection

IL-10 is an important immunoregulatory cytokine that plays a central role in maintaining a balance between protective immunity against infection and limiting proinflammatory responses to self or cross-reactive Ags. We examined the full effects of IL-10 deficiency on the establishment and quality of T cell memory using murine listeriosis as a model system. IL-10(-/-) mice had reduced bacterial loads and a shorter duration of primary infection than did wild-type mice. However, the number of Ag-specific T cells in secondary lymphoid and nonlymphoid organs was diminished in IL-10(-/-) mice, compared with wild-type mice, at the peak of the effector response. Moreover, the frequency and protective capacity of memory T cells also were reduced in IL-10(-/-) mice when assessed up to 100 days postinfection. Remarkably, this effect was more pronounced for CD8 T cells than CD4 T cells. To address whether differences in the number of bacteria and duration of primary infection could explain these findings, both strains of mice were treated with ampicillin 24 hours after primary infection. Despite there being more comparable bacterial loads during primary infection, IL-10(-/-) mice still generated fewer memory CD8 T cells and were less protected against secondary infection than were wild-type mice. Finally, the adoptive transfer of purified CD8 T cells from previously infected wild-type mice into naive recipients conferred better protection than the transfer of CD8 T cells from immune IL-10(-/-) mice. Overall, these data show that IL-10 plays an unexpected role in promoting and/or sustaining CD8 T cell memory following Listeria monocytogenes infection.     

Th1-Th17 Cells Mediate Protective Adaptive Immunity against Staphylococcus aureus and Candida albicans Infection in Mice

We sought to define protective mechanisms of immunity to Staphylococcus aureus and Candida albicans bloodstream infections in mice immunized with the recombinant N-terminus of Als3p (rAls3p-N) vaccine plus aluminum hydroxide (Al(OH₃) adjuvant, or adjuvant controls. Deficiency of IFN-γ but not IL-17A enhanced susceptibility of control mice to both infections. However, vaccine-induced protective immunity against both infections required CD4+ T-cell-derived IFN-γ and IL-17A, and functional phagocytic effectors. Vaccination primed Th1, Th17, and Th1/17 lymphocytes, which produced pro-inflammatory cytokines that enhanced phagocytic killing of both organisms. Vaccinated, infected mice had increased IFN-γ, IL-17, and KC, increased neutrophil influx, and decreased organism burden in tissues. In summary, rAls3p-N vaccination induced a Th1/Th17 response, resulting in recruitment and activation of phagocytes at sites of infection, and more effective clearance of S. aureus and C. albicans from tissues. Thus, vaccine-mediated adaptive immunity can protect against both infections by targeting microbes for destruction by innate effectors.     

Production of IL-13 in spleen cells by IL-18 and IL-12 through generation of NK-like cells

Treatment of Nylon wool-passed cells (NWC) prepared from the spleen of C57BL/6 mice with IL-18 and IL-12, but not with IL-18 alone, resulted in induction of IFN-gamma, a Th1 cytokine, and GM-CSF at 24 h, and IL-13, a Th2 cytokine at 72 h. The induction of IL-13 was suppressed by anti-GM-CSF antibody, indicating involvement of GM-CSF in IL-13 production. When NWC incubated with IL-18 and IL-12 for 72 h ("primary treatment") were treated again with the same cytokines ("secondary treatment"), IL-13 was induced much more quickly than observed in the primary treatment. Flow cytometric analysis of NWC after the primary treatment showed marked increases in the CD4(-)CD8(-) non-T cell population bearing CD25(+), CD45RB(super high) and CD122(+). These cells were positive for CD49b but negative for NK1.1, indicating that they were not typical but NK-like cells. The NK-like cells produced IL-13 in response to the treatment with IL-18 alone, indicating that the generation of these cells in the primary treatment likely accounts for the quick production of IL-13 in the secondary treatment. These results show that IL-18 and IL-12 generates the NK-like cells in NWC by a process mediated by GM-CSF that are ready for producing IL-13.     

IL-17A produced by gammadelta T cells plays a critical role in innate immunity against listeria monocytogenes infection in the liver

IL-17A is originally identified as a proinflammatory cytokine that induces neutrophils. Although IL-17A production by CD4(+) Th17 T cells is well documented, it is not clear whether IL-17A is produced and participates in the innate immune response against infections. In the present report, we demonstrate that IL-17A is expressed in the liver of mice infected with Listeria monocytogenes from an early stage of infection. IL-17A is important in protective immunity at an early stage of listerial infection in the liver because IL-17A-deficient mice showed aggravation of the protective response. The major IL-17A-producing cells at the early stage were TCR gammadelta T cells expressing TCR Vgamma4 or Vgamma6. Interestingly, TCR gammadelta T cells expressing both IFN-gamma and IL-17A were hardly detected, indicating that the IL-17A-producing TCR gammadelta T cells are distinct from IFN-gamma-producing gammadelta T cells, similar to the distinction between Th17 and Th1 in CD4(+) T cells. All the results suggest that IL-17A is a newly discovered effector molecule produced by TCR gammadelta T cells, which is important in innate immunity in the liver.     

A predominant role for antibody in acquired immunity to chlamydial genital tract reinfection

Acquired immunity to murine Chlamydia trachomatis genital tract reinfection has long been assumed to be solely dependent on cell-mediated immunity. However, in this study, we identify a previously unrecognized protective role for Ab. Immunity develops in Ab-deficient mice following the resolution of primary chlamydial genital infection. Subsequent depletion of CD4+ T cells, but not CD8+ T cells, in those immune Ab-deficient mice before secondary infectious challenge, resulted in an infection that did not resolve. Passive immunization with immune (convalescent) serum conferred a marked level of protective immunity to reinfection, which was characterized by a striking decrease in bacterial shedding, from >100,000 inclusion forming units to fewer than 10 inclusion forming units, and a shortened duration of infection. Furthermore, mAbs to the chlamydial major outer membrane protein and LPS conferred significant levels of immunity to reinfection and reduced chlamydial shedding by >100-fold. Anti-heat shock protein 60 mAb had no protective effect. In contrast to the marked protective efficacy of immune serum on reinfection, the course of primary infection was essentially unaltered by the passive transfer of immune serum. Our results convincingly demonstrate that Abs contribute importantly to immunity to chlamydial genital tract reinfection, and that Ab-mediated protection is highly dependent on CD4+ T cell-mediated adaptive changes that occur in the local genital tract tissues during primary infection. These results impact our understanding of immunity to chlamydial genital infection and may provide important insight into vaccine development.     

Impaired protection against Mycobacterium bovis bacillus Calmette-Guerin infection in IL-15-deficient mice

To investigate the potential role of endogenous IL-15 in mycobacterial infection, we examined protective immunity in IL-15-deficient (IL-15(-/-)) mice after infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG) or recombinant OVA-expressing BCG (rBCG-OVA). IL-15(-/-) mice exhibited an impaired protection in the lung on day 120 after BCG infection as assessed by bacterial growth. CD4(+) Th1 response capable of producing IFN-gamma was normally detected in spleen and lung of IL-15(-/-) mice on day 120 after infection. Although Ag-specific CD8 responses capable of producing IFN-gamma and exhibiting cytotoxic activity were detected in the lung on day 21 after infection with rBCG-OVA, the responses were severely impaired on days 70 and 120 in IL-15(-/-) mice. The degree of proliferation of Ag-specific CD8(+) T cells in IL-15(-/-) mice was similar to that in wild-type mice during the course of infection with rBCG-OVA, whereas sensitivity to apoptosis of Ag-specific CD8(+) T cells significantly increased in IL-15(-/-) mice. These results suggest that IL-15 plays an important role in the development of long-lasting protective immunity to BCG infection via sustaining CD8 responses in the lung.     

Blocking the receptor for IL-10 improves antimycobacterial chemotherapy and vaccination

Novel approaches are required for the prevention and therapy of mycobacterial infections since the only vaccine in use, bacillus Calmette-Guérin, is poorly effective and chemotherapy is long and often ineffective in sterilizing the infection. We used a mouse model of Mycobacterium avium infection to address the usefulness of a mAb able to block IL-10R both in treatment of primary infections and in conventional multidrug therapy and subunit vaccination. Treatment of infected mice with this mAb during the entire period of experimental infection had little impact on the course of M. avium infection, with a slight improvement in the resistance of infected mice observed in the liver and spleen at day 30 of infection, which was associated with increased macrophage activation and priming of CD4(+) T cells for IFN-gamma production. Administration of this mAb later in infection had no effect on its course, but improved the effectiveness of chemotherapy when the latter was started in a chronic phase of infection. Also, the anti-IL-10R mAb acted as an adjuvant in the induction of protective immunity upon vaccination with a mycobacterial subunit preparation.     

Role of the interleukin 5 receptor system in hematopoiesis: molecular basis for overlapping function of cytokines.

Interleukin 5 (IL-5) is a kind of peptide hormone released from T lymphocytes of mammals infected with microorganisms or parasites. It is an acidic glycoprotein with a molecular mass of 40 to 50 kDa that consists of a homodimer of polypeptides. It controls hematopoiesis so that it increases natural immunity. In the mouse, IL-5 acts on committed B cells to induce differentiation into Ig-producing cells and on common progenitors for CD5+ pre-B cells and CD5+ macrophages to support their survival. The antibodies secreted by CD5+ B cells seem to be responsible for the primary protection against the infection with microorganisms or parasites. It also supports the growth and/or differentiation of eosinophil precursor and mature eosinophils, which can be effective for the removal of parasites in combination with the antibodies against them. Murine IL-5 receptor (IL-5R) consists of two different polypeptide chains; alpha chain and beta chain. The IL-5R alpha chain is 60 kDa protein that binds IL-5 with low affinity. The IL-5R beta chain is a 130 kDa protein which does not bind IL-5 by itself but is necessary to form the high affinity IL-5R. The beta chain was identified by using one of the anti-IL-5R mAb and anti-IL-3R mAb as the IL-3R homologue. This beta chain is also used as the beta chain of GM-CSF receptor. This fact suggests that there is a common signaling mechanism among these cytokines and efficient cooperation among them. At the same time, these findings may explain the overlapping role of these cytokines in the development of granulocytes.