Unit: Early responses to parasites and Immunopathology

Director: LOUIS Jacques

A major goal of our Unit is to study in vivo, in various genetically defined mice, the early immunological events leading to resistance or susceptibility to intracellular parasites such as Lesihmania and Toxoplasma. The main focus of one group is to understand the overwhelming Th1 like response that results in the development of a lethal ileitis in C57BL/6 mice after oral infection with Toxoplasma gondii (T. gondii). Another group is studying the mechanisms that would lead either to the initiation of a Th1 like immune response in C57BL/6 resistant mice or to a Th2 like pattern in susceptible BALB/c mice after infection with Leishmania major (L. major).

The third group analyses how the immune response against malaria is influenced by the early mast cell activation mediated by Anopheles saliva.

The group of D Buzoni-Gatel investigates the intestinal Th1 and Th2 immune responses after oral infection with T. gondii. In B6 mice, the oral inoculation with the parasite induces a lethal ileitis that shares striking histological and immuno-pathological features with those observed in human inflammatory bowel diseases (IBD) such as Crohn or Coeliac diseases. Despite the increasing incidence of IBD in developed countries, there is yet no relevant animal model to mimic these diseases, the etiology of which is still uncertain. However the T. gondii driven ileitis model is quite useful to analyse 1) the immunological events that would lead to the complete damage of the epithelial barrier and 2) the lack of efficiency of the regulatory mechanisms. Therefore we use this model to determine in vivo the immunological disorders responsible for these pathologies. We found that intestinal NKT cells, the development and homeostasis of which require IL-15, are key players to initiate the inflammatory response after oral infection with the parasite. In collaboration with Dr Nadine Cerf-Bensussan (Hôpital Necker), we investigated the importance of IL-15 in the inflammatory process. Based on our observations made in both infected IL-15 deficient mice, and different chimeric mice that solely express IL-15 within hematopoietic cells or exclusively within non-hematopoietic cells, it has been demonstrated that intestinal epithelial cells were mandatory to produce the IL-15. Beside its importance for NKT cells, IL-15 can also be implicated in activation of non antigen specific cytotoxicity towards enterocytes that can account for epithelium damages. Thus we showed that intestinal intraepithelial lymphocytes (IELs) exhibit cytotoxic activity early after infection (15 hours) that is characterized by the over production of granzyme B and perforin. This cytotoxic activity, that is linked to the presence of IL-15, is further investigated in IL-15 transgenic mice over-expressing IL-15 under an epithelial cell specific promoter.

Mice deficient for TLR9 expression fail to develop lethal ileitis after infection with T. gondii. The lack of histological lesions in TLR9-/- mice is correlated with a significant decrease in IFN-g production by the CD4 T cells from the lamina propria. In collaboration with the Dartmouth Medical School (Hanover, USA), we are presently identifying the cell(s) expressing TLR9. We also observed that TLR9 activation was accompanied by type I IFN production. We are currently studying the role of these type I IFN in the intestinal inflammatory process. In collaboration with André Ouellette (University of California), we observed that Paneth cells over expressed TLR9 and discharged defensins in response to infection. The infection of mice deficient for defensin (matrilysin -/-) will allow to characterize the implication of these molecules into the inflammatory process.

The importance of the innate immunity for the shaping of the adaptative immune response is now well admitted. In this context, we (Noëlle Doyen and Nicolas Rachinel) are studying the role of Toll like receptors signaling in the innate response triggered by infection with Leishmania major. Several results indicate that some members of the TLRs family are involved in the response to L. major. Indeed, resistant C57BL/6 mice deficient for the MyD88 gene become susceptible to infection with L. major. The MyD88 protein is an adaptator molecule implicated in the signaling through TLRs. We have studied the response to infection in resistant mice deficient for the TLR2, 4 and 9 gene and the results indicate that a deficiency in TLR9 delays the resolution of lesions in resistant C57BL/6 mice. Analysis of the cell populations in draining Lymph nodes early following infection with L. major has revealed an increase in phagocytic, in conventional and plasmacytoïdes dendritic cells and in NK cells (DX5+, CD3-). Activation of these cells was reflected by increased cytokines (IL-12, IFN type I) expression and differences between wild type and TLR9 deficient mice are observed. Differences in the maturation of Th1, and Th2 cells between wild type and TLR9 deficient mice are also observed.

Our goal is to determine whether or not TLR signaling is involved in the activation of DCs following infection with L.major and whether or not this activation requires cellular interactions. The observed correlation between DC and NK cells activation argues in favor of an interaction between these cells.

An in vitro system is being developed to be able to study the process, including the cellular interaction required, leading to activation of DCs. Using Dcs from mice deficient in some TLR genes, such system should allow evaluating the relative contribution of TLRs signaling in activation of DCs by L.major.

The group of S. Mécheri investigates the role of mast cells in the elicitation of local (skin tissue) and distal (lymph nodes) immune responses caused by mosquito bites. The skin is one of the preferred sites of entry for various pathogens including Plasmodium parasites. We made the hypothesis that mosquito saliva, which contain a myriad of pharmacologically active compounds as well as IgE-inducing allergens, may alter the immune system in favor of the Plasmodium transmission. We recently reported that mosquito bites induce immunosuppressive cytokines resulting in downregulation of antigen-specific responses. This led us to explore the role of histamine, a characteristic mast cell mediator, in regulating the immune response against the Plasmodium Parasite. Recently, we have addressed two objectives ; 1) to study the role of histamine, a major mast cell product, and histamine receptors in the transmission of Plasmodium infection through mosquito bites, and 2) to test the capacity of histamine receptor antagonists to prevent or/and tu cure Plasmodium infection. Two complementary approaches have been used to delineate the mechanisms by which distinct histamine receptors may control Plasmodium infections ; 1) the pharmacologic approach which consists in using histamine receptor antagonists, and 2) the genetic approach in which H1R, H2R, and histidine decarboxylase (HDC) gene-deficient mice were used. HDC gene product converts histidine into histamine and these mice do not produce histamine and represent complementary tools to histamine receptor-deficient mice. Our results indicate that histamine receptor antagonists provide some protection against Plasmodium infection and prolong survival of infected mice. On the other hand, HR1- and HR2-KO mice are more resistant to Plasmodium infection than wild-type mice. Finally, HDC-KO mice were completely refractory to Plasmodium infection. These data indicate that histamine represents a susceptibility inflammatory product which may facilitate parasite transmission and development.

The use of the second or third generation of safe HR antogonists, which are widely administered to human population to cure allergic diseases, may now offer a new possibility to prevent or cure malaria disease. What will be done next is to identify the immunological mechanisms by which HDC-KO mice control Plasmodium infection.

Keywords: parasites, innate immune response, Th1/Th2 balance, mastocytes

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