IMMUNE REGULATION AND VACCINE DEVELOPMENT IN LEISHMANIASIS

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Endogenous IL-10 is a central mediator of immune homeostasis, necessary to keep in check the strong inflammatory reactions that can accompany the expression of anti-microbial immunity in local tissues. A consequence of the balance struck between host immunity and pathology can be chronic or persistent infection, and the absence of IL-10 has been shown to result in more efficient clearance of a variety of pathogens, including Leishmania major. We have addressed the dominant source of IL-10 dependent immune suppression induced by a strain of L. major that produces non-healing dermal lesions in the face of a strong Th1 response in C57BL/6 mice. The infection induced localized recruitment and production of IL-10 from innate cells, as well as from CD4+CD25+Foxp3+ natural Treg and CD4+CD25-Foxp3- inducible Treg. The latter cells appear to be closely linked to the Th1 effector response, in that they also produced high amounts of IFN-gamma. In Rag-/- reconstituted infected mice, the IL-10-producing Th1 cells were generated in the absence of either natural Treg or IL-10 from innate sources, and most importantly, were found to play a necessary and sufficient role in the suppression of protective immunity. Using IL-10 reporter mice, IFNg+IL-10-CD4+ T cells from chronically infected mice could be further stimulated in vitro with antigen to produce IL-10, suggesting that persistant stimulation with antigen drives Th1 cells through a program of development that includes IL-10 secretion as a mechanism of feedback control. IL-12 and especially IL-27 were found to enhance IL-10 induction in committed Th1 cells. Visceral leishmaniasis (VL) is a life threatening disease characterized by uncontrolled parasitization of spleen, liver, and bone marrow. The mechanisms underlying the failure to control the growth and systemic spread of Leishmania parasites in human visceral leishmaniasis (VL) are not well understood. While the absence of antigen-specific Th1 responses in PBMC from VL patients is thought to be causally related to disease progression, the finding that these patients also express elevated IFNgamma mRNA in lesional tissue, as well as elevated serum levels of pro-inflammatory cytokines, suggests that their immunologic defect cannot be simply explained by immune tolerance or Th2 polarization. As a possible homeostatic mechanism to control persistent infection-induced inflammation, elevated levels of the regulatory cytokine IL-10 have repeatedly been reported in clinical studies of VL. The main aim of our clinical studies in India has been to identify the cellular source of IL-10 in human VL, and determine if CD4+CD25+(Foxp3high) regulatory T cells are associated with active disease. We analyzed surface marker and gene expression in PBMC and splenic aspirates from Indian VL patients pre- and 3-4 weeks post-treatment with Amphotericin B. The results did not point to an important role for natural CD4+CD25+(Foxp3high) Treg cells in human VL: they did not accumulate in and were not a major source of IL-10 in the spleen, and their removal did not rescue antigen-specific IFN-gamma responses. By contrast, splenic T cells depleted of CD25+ cells expressed the highest levels of IL-10 mRNA, and were the predominant lymphocyte population in the VL spleen. The elevated levels of IL-10 in VL plasma significantly enhanced the growth of L. donovani amastigotes in human macrophages. CD4+ T cells in PBMC from VL patients could be stimulated by antigen followed by PMA/ionomycin to produce both IL-10 and IFN-gamma. Finally, analysis of PBMC indicated that on a per cell basis CD8+ T cells expressed the highest levels of IL-10 mRNA during active disease, with significant down-regulation observed post treatment. Our results suggests that Leishmania driven, IL-10 producing Foxp3- CD4+ and CD8+ T cells may play an important role in suppression of protective immune responses in VL patients. Leishmania tropica is the causative agent of old-world anthroponotic cutaneous leishmaniasis (ACL), which is characterized by lesions that take an extended period of time to heal, often resulting in disfiguring scars, and are more refractory to treatment than leishmaniasis caused by L. major. Immunologic studies involving experimental animal models of L. tropica infection are virtually non-existent. In the current study, infectious-stage, metacyclic L. tropica were used to establish dermal infections in C57Bl/6 and BALB/c mice. In both strains the lesions were slow to develop and showed minimal pathology. They nonetheless contained a stable number of between 10,000 and 100,000 parasites for over one year, which were efficiently picked up by a natural sand fly vector, Phlebotomus sergenti. Control of parasite growth depended on the development of a Th1 response, as C57Bl/6 mice genetically deficient in IL-12, IFN-gamma, or inducible nitric oxide synthase, or BALB/c mice treated with antibodies to IFN-gamma, harbored significantly more parasites. By contrast, IL-10 deficient mice harbored significantly fewer parasites throughout the course of infection. To further study the immunologic mechanisms that may prevent efficient clearance of the parasites, IL-10 and TGF-beta signaling were abrogated during the chronic phase of infection in wild type C57Bl/6 mice. Distinct from chronic L. major infection, IL-10 blockade alone had no effect on L. tropica, but required simultaneous treatment with anti-TGF-beta antibodies to promote efficient parasite clearance from the infection site. Thus, L. tropica appears to exploit multiple suppressive factors to maintain chronicity, and to establish the host as a long-term reservoir of infection for vector sand flies.