Unit: Immunophysiopathology of infections
Director: Pierre-André CAZENAVE
The scientific activities of the Infectious Immunophysiopathology Unit are centered on the study of mechanisms controlling the physiology of the host immune system and those leading to immunopathologies following parasitic infections and diseases, namely Chagas' disease and Malaria. These studies are particularly grounded on more fundamental research on immune repertoires.
TcPRAC : Trypanosoma cruzi eukaryotic proline racemase B cell mitogen (P. Minoprio). We had previously shown that TcPRAC genes are essential to T. cruzi survival since functional TcPRAC knock down' parasites are not viable. We have studied the role of different TcPRAC isoforms using molecular approaches and parasite transgenesis. We have shown that non infective parasite forms that had integrated vectors allowing the hyper expression of TcPRAC genes improve metacyclogenesis and differentiation into infective forms. Mutant parasites also increase their infectious potential towards host cells suggesting that TcPRAC may be involved in virulence mechanisms. One possibility is that the racemization of free L- into D-proline by intracellular proline racemase would allow the post translational addition of D-residues into peptide chains expressed by the parasites upon infection. This hypothesis is attractive and, as suggested by our recently data revealing the presence of D-proline linked to soluble and membrane parasite protein extracts, may contribute to increase parasite resistance to lysosomal proteolytic enzymes and therefore to explain the T. cruzi ability to escape the parasitophorous vacuole and its further replication in the cytosol. These studies corroborate the significance of TcPRAC as a chemotherapeutic target. Taking the advantage of our functional, molecular and crystallographic studies, our present strategies are focused on a) the chemical modification of pyrrole carboxylic acid, the known inhibitor of proline racemases, in order to increase its solubility; b) the identification of new putative inhibitors of TcPRAC by detailed analysis of its catalytic site (pharmacodynamics, modelization and virtual screening of thousands of molecules; c) the identification of parasite proteins bearing D-proline and d) the development of an inducible system of genetic inactivation in T. cruzi to better explore new therapeutic targets.
Studies on the diversity and plasticity of the Immune system (A. Six). One important activity of the laboratory concerns the improvement of new technologies to explore the diversity and the plasticity of T- and B- cell repertoires represented by antigen specific Ig- and T-cell receptors. In collaboration with the groups of S. Pied and P.A. Cazenave, these studies focus on the study of repertoire alterations in the context of mouse and human Leishmaniasis and Malaria. We have continued to improve our ISEApeaks strategy by refining our experimental procedures and developing new statistical analysis representations of repertoires. We pursue our TCRBV repertoire analyses in mouse experimental malaria models: protection against P.yoelii infection in C57BL/6 mice immunized with irradiated P.yoelii sporozoites; cerebral malaria in B10.D2 infected by P. berghei. We are currently exploring the perturbation of brain lymphocytes of P.berghei-infected B10.D2 mice by comparison to spleen and blood lymphocytes in order to identify pathogenic or protective clones. In parallel, in collaboration with Petter Höglund's group at the Karolinska Institutet in Stockholm, we have investigated TCR usage of the T cell population isolated from pancreatic lymph nodes (PLN) of NOD mice. We reveal a possible preferential usage of particular TCRBV in the PLN when compared with inguinal lymph nodes. We also collaborated to a study lead by Pierre Boudinot at INRA that shows that in the trout, the TCRBV repertoire of intestinal intra-epithelial lymphocytes is as diverse as that of splenic T cells.
A novel peritoneal B cell population in mice (D. Rueff-Juy). In common laboratory strains, which are derived from only few ancestors, peritoneal B cells are enriched in a Mac-1+B220lowIgMhigh population of B-1 cells which encompasses 2 subsets: B-1a (CD5+) and B-1b (CD5-). Intringuingly, Mac-1+CD5+ or CD5- B cells have never been found in specific anatomic sites in other vertebrates analyzed so far. Therefore, in order to ascertain the peculiarity of the CD5+ peritoneal B cells in laboratory mice, we have revisited the peritoneal B cell populations in 9 inbred and 39 outbred wild-derived mouse strains belonging to several species and subspecies.Our study reveals a novel population of B cells present in the peritoneal cavity of all inbred and outbred wild strains and that we have named Bw cells, "w" for wild mice. Careful analysis indicates that this population is also present in variable proportions in common laboratory strains. In contrast, B-1a cells are present almost exclusively in the Mus musculus domesticus subspecies. Like B-1, Bw cells localize preferentially in the peritoneal cavity, and transfer experiments establish that Bw differentiation is dependent on intrinsic B cell control. Serum Ig levels are similar in laboratory and wild-derived strains, even in 2 wild strains whose MZ B cells show an unusual phenotype and a non classical structure of the splenic white pulp. Furthermore, Bw stimulated by TLR ligands produce higher amounts of anti-phosphorylcholine Abs than B2 cells in vitro. Altogether, these data show that the B-1a population is not representative of the peritoneal B cell populations of the whole genus Mus and strongly suggest that the evolutionary conserved Bw population may play an essential role in Ig secretion and antibody responses to pathogens. We propose that Bw cells serve as a link between innate and adaptive immunity.
Immune responses associated to malaria physiopathology (S. Pied). We aim to identify the protective and pathological responses associated to Plasmodium infection and their mutual effects. These studies are done in parallel in murine experimental models infected either by P. berghei ANKA (pathological responses) or P. yoelii (protective responses) and in control or P. falciparum infected patients developing various clinical forms of the disease (asymptomatic, mild, severe non cerebral and cerebral malaria).
1. Studies of NKT and NK cells response to P. yoelii infection (S. Pied, J. Roland). We observed a compartmentalized response of NK and NKT cells elicited during P. yoelii infection. Liver NKT cells are heterogeneous and composed of CD1d-dependent invariant CD4+ NKT cells, which represented the major subset, and DN NKT cells comprising both CD1d-dependent invariant and non-invariant cells and CD1d-independent cells. These activated hepatic NK and NKT cells secreted IFN-γ and TNF-α and inhibited the intra-hepatic development of the parasite in vitro. Although not essential to cure P. yoelii infection, NK and NKT cells are involved in the early control of primary infection. We also found an increase of NK1.1+ CD4high splenic T cells representing 40% of the total CD4 cells during infection. Contrary to the liver NKT cells, the NK1.1+ CD4high splenic T cells are independant of the CD1d molecule and expresse a diverse TCRVβ chain repertoire. The functional role of this NK1.1+ CD4high splenic T subpopulation during infection remains to be established. In parallel, the immunomodulatory effects of Anopheles stephensi saliva on P. yoelii-induced protective immune responses are studied in the frame of the GPH "Anophele".
2. Immune responses associated to cerebral malaria induced by P. berghei ANKA (S. Pied, P. A. Cazenave). We have previously showed that pathogenic CD8+ T cells are implicated in the physiopathological mechanisms leading to experimental cerebral malaria (CM) in P. berghei ANKA infected mice. The exact role of these cells is at present unknown. We first examine the role played by CD4+CD25+ regulatory T cells (Treg) in the control of pathogenic effector CD8 T cells during CM. Although the number of CD4+CD25high T cells expressing Foxp3 exhibiting in vitro suppressive function is increased during the course of infection, they do not protect mice from CM in vivo. In addition, we used an in vitro model of Brain Blood Barrier (BBB) to analyse changes in astrocytes, endothelial and microglial cells following exposure to CD8+ T cells recruited in the brain of P. berghei ANKA infected mice developing CM. We investigated functional modifications in gene expression using the GeneChip Mouse microarray (Affymetrix). This strategy allow us to define new factors involved in malaria pathophysiology.
3. IgG and IgE self-reactivities to brain antigens are associated to cerebral malaria or protection, respectively, in P. falciparum infected patients (S. Pied). We used a global approach based on quantitative immunoblotting and multivariate statistics to study the reactivity of sera from controls and P. falciparum infected patients developing asymptomatic, uncomplicated and severe non-cerebral or cerebral malaria against a human brain extract. We observed an increase of P. falciparum -infected patients plasmatic IgG reactivity to brain antigens with disease severity. Interestingly, we found in CM patients a strong reactivity with high molecular weight human brain proteins that has been identified by mass spectrometry. This was significantly correlated with high TNF-α plasma levels. In addition, IgE reactivities to two brain antigens (under identification) are associated with protection and plasmatic IL-10 levels in asymptomatic patients. These data strongly suggest the involvement of autoreactive immune response in P. falciparum malaria pathophysiology.
Genetic determinism of resistance for severe forms of experimental malaria induced by P. berghei ANKA ( P. A. Cazenave, S. Pied). We have shown genetic association of the resistant phenotype to cerebral malaria with two distinct regions respectively on chromosome 1 and 11. During this study, we observed an unusual phenotype caracterized by clearance of parasitaemia and establishment of protective immunity. This phenotype is associated to three chromosomal regions, namely 1, 4 and 9. C57Bl/6 congenic mice for either chomosome 1 region or chomosome 11 region have been derived. Congenics for chomosome 9 and double congenic for chromosome 1 and 11 will be obtained in few time (collaboration with Prof. D. Holmberg Laboratory, Umea University, Sweden).
New pathogenic mechanisms of experimental leishmaniasis due to L. major (P. A. Cazenave). We have shown that mice of the PWK inbred strain are susceptible to experimental leishmaniasis due to L. major. They develop a protracted but self-healing disease, characterized by a mixed Th1 plus Th2 pattern of immune responses in which IL-10 plays an aggravating role, and acquire resistance to a secondary challenge. This features are closed to those observed in human cutaneous leishmaniasis due to L. major and make PWK mice a suitable model for the human disease (collaboration with Prs. K. Dellagi and H. Louzir Laboratories, Institut Pasteur of Tunis, Tunisia, in the frame of a PTR programme).
Keywords: Immune system, immunopathology, B- and T- cell repertoires, proline racemase, Chagas’ disease, mitogen, cerebral malaria, TLR, genetic