Regulation of the immune response in Trypanosoma cruzi and Leishmania sp. infections

Abstract

This research project encompasses four individual projects that address the study of immune response regulation by cytokines and resistance to infection by Trypanosoma cruzi and Leishmania major and for Leishmania amazonensis (Leishmania sp). These projects further advance our previous investigations on regulatory mechanisms of cytokines synthesis that are important to the resistance of the host against these infections and/or control potentially tissue-damaging inflammatory or autoimmune reactions, as may occur in T.cruzi infection. Project 1 investigates the regulation of the immune response by cytokines in the experimental murine model of T.cruzi infecfion. It focus on the synthesis of IL-12 and IFN-y: cellular origin and reciprocal regulation between IFN-y and IL-12 as well as the regulation by other cytokines as IL-10, IL-4, TGF-B, IL-18 and IFN-a,B and non-cytokine mediators as prostaglandins (PG) and nitric oxide (NO). The experimental protocols analyze cytokine production in the supernatants of spleen cell cultures from infected mice or cytokine message determination by RT-PCR. The use of neutralizing anti-cytokine mAbs, of recombinant cytokines or of specific inhibitors of PG or NO synthesis coupled to selective cellular depletion experiments should bring information as to the regulation of cytokine production during the infection. The role of IFN a,B as a cytokine of the innate immunity involved in the early resistance to infection and regulation of macrophage cytokine and NO synthesis will also be specialty focussed. Project 2 further extends our investigation on the regulation of cellular immune responses in patients with Chagas' disease. We have shown that antigen-specific T cell proliferation and activation are negatively regulated by IL-10 and PG in patients with the cardiac form of the disease, whereas in patients with the indeterminate form, this control is exerted only by PG. Production of IL-10, IL-12 and PG by PBMC, but not II-4 or IFN-y, where higher in patients with the cardiac form as compared with patients the indeterminate form of the disease. We want to investigate how the levels of these cytokines and also TNF--A and PG, synthesized by PBMC, are regulated in b" types of patients. Project 3 aims at understanding which is the biological role of immunesuppression during the acute phase of Chagas disease. The working hypothesis is that suppression of T cell responses is a regulatory mechanism that acts to limit potentially damaging autoreactivity that would be otherwise triggered by the intense T cell clonal activation seen during T.cruzi infection. Cross-reactivity between the parasite's antigens and a variety of self antigens has been frequently reported. The experimental approach consists at investigating whether infected mice that have had the suppression abrogated by treatment with the NO inhibitor aminoguanidine or iNOS KO mice show evidence of autoreactivity in the form of autologous MLR or by reactivity to self antigens tike myosin, actin or collagen. Besides this approach, we want to investigate whether mice transgenic TCR a,B for a peptide from myelin basic protein (MBP) will have their latent EAE triggered by the infection or by parasite antigens. Activation of latent EAE could occur by direct activation of T cells or by the intense Thl-type cytokine response that occurs in T.cruzi infection. Project 4 aims at studying the synthesis of IL-12 and other macrophage-derived cytokines, and their reciprocal regulation by distinct populations of macrophages infected with Leishmania major or with Leishmania amazonensis. Mononuclear cell populations grown from bone-marrow in the presence of M-CSF or GM-CSF will be studied. Cells with dendritic phenotype or macrophages differentiated by longer exposure to these cytokines, as well as two macrophage cell lines will be infected with leishmania amastigotes or promastigotes. The putative inhibitors of IL-12 synthesis ( TGF-B, IL-13, IFN-a,B, IL-10, PG) will be screened by using neutralizing mAbs in culture or by inhibiting the synthesis of PG. The cytokines produced by the distinct macrophage populations with be quantified by ELISA and by RNAse protection assays in different experimental conditions, that include the selective blocking of macrophage receptors involved in Leishmania invasion. (AU)