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 of innate and adaptive immunity.
TcPRAC : a eukaryote proline racemase released by T. cruzi (P. Minoprio). We have pursued the molecular, biochemical, functional and structural analyses of TcPRAC, the first eukaryotic proline racemase that is expressed by the parasite Trypanosoma cruzi. One isoform of the enzyme is intracellular in the parasite non infective forms found in the insect vector and plays a role in the mechanisms of parasite differentiation into metacyclic infective forms and consequently in the acquisition of virulence. TcPRACA, the secreted version of the enzyme is released by the parasite upon infection and implicated in the mechanism of parasite escape by its mitogenic activity towards B- lymphocytes and the ensuing generation of non-specific antibodies. Using a molecular strategy to interfere with protein expression, we demonstrated that TcPRAC is an essential gene for parasite viability. Structural and functional studies of the secreted enzyme revealed that TcPRACA possesses two active sites and that interaction of the enzyme with its catalytic inhibitor is responsible for conformational changes of critical protein epitopes and abrogation of TcPRACA B-cellmitogenic property. The association of a highly sensitive microplate test to detect D-amino acids, developped in the laboratory, and bi-dimensional chromatography with chiral plates allowed the identification of D-proline within polypeptide chains expressed by the parasite, which would fairly contribute to parasite immune evasion and persistance inside the host. Presently, our studies are focused on the identification of those proteins in parasite extracts and also on the synthesis of soluble enzyme inhibitors to elaborate an immuno(chemo)therapy against Chagas' infection and disease.
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 TCRBV5.1 and TCRBV5.2 in the PLN when compared with inguinal lymph nodes. A molecular analysis of TCRBV-BJ segments highlights four TCR candidates for the initiation of the autoimmune diabetes.
Role of TLRs in B cell development and functions (D.Rueff-Juy). In the aim of studying the role of TLRs in B cell development and functions we have sought for a functional polymorphism of TLRs or of TLR-dependent pathways. To this end, we have studied the B cell responses of nine feral-derived mouse strains to LPS (TLR4) and CpG (TLR9). Two strains show a low proliferative or an unresponsiveness to LPS by either 3HT incorporation assays or CFSE analysis, and one strain is low responder to both ligands. Nevertheless, LPS and CpG are recognized by B cells of both strains unasmuch as up-regulation of CD69 expression is similar to that of C57BL/6. However their splenic B cells show an unusual profile of CD21/CD23 expression and failed to express high level of CD1d and CD9 markers suggesting that they lack of regular MZB cells. This assumption is reinforced by the structural disruption of the white pulp observed by section stained with anti-IgM and MOMA-1 antibodies. Interestingly, splenic B cells from strains displaying no and low proliferative responses to LPS secrete high level of IgM antibodies when cultured in vitro in the presence of LPS. Moreover, both mice do mount a high antibody response to PC-thyroglobulin. Experiments are in progress to assess the functionality of these antibodies in protection assays and to understand the molecular basis of the proliferative defect. FACS analysis of peritoneal B cells reveals that the B1a population found in all laboratory is absent in 24 feral strains tested so far. However these mice present an unknown peritoneal B cell population characterized by B220hi CD5- Mac1+ phenotype. This population (temporary named Bw) is co-expressed with B1-a in the peritoneal cavity of F1 (feral x laboratory mice) suggesting that this population is independent from B1a. These data show clearly that laboratory mice are not fully representative of the genus Mus. Transfer experiments with bone marrow and fetal liver cells in various recipients are in progress to get informations on the origin and the commitment of this new population.
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).
Studies of NKT and NK cells response to P. yoelii infection (S. Pied, J. Roland). We observed that the NKT cells elicited in the liver during P. yoelii infection 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 NKT cells were Th1-biaised and secreted IFN-γ and TNF-α known for their anti-plasmodial activity. Hepatic DN NKT cells isolated from infected mice inhibited the intra-hepatic development of the parasite in vitro in a CD1d-dependent manner. In vivo, B6.CD1d-deficient mice developed a higher liver parasite burden than B6 mice. These results suggest that, although not essential to cure P. yoelii infection, NKT cells and CD1d may be involved in the early control of primary infection. We also found organ-specific differences in the behaviour of NK cells in P. yoelii infected mice. They are activated and increased in the liver in contrast to the spleen. Liver NK cells from infected mice are IFN-γ-producing cells and exhibit a cytotoxic activity in vitro against P. yoelii liver stage. This liver NK cells parasitocidal activity is confirmed in vivo by comparing the outcome of P.yoelii infection in RAG-/- and RAGγ c-/- mice.
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 hypothesised that in CM susceptible mice the neuropathology could be, at least in part, the result of an inefficient control of pathogenic effector T cells by CD4+CD25+ regulatory T cells (Treg). 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. This absence of protection is not due to a blockade of Treg cells suppressive function in vivo by the production of IL-6 during the infection.
IgG self-reactivities to brain antigens are associated to cerebral malaria 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. This was significantly correlated with high TNF-a plasma levels. Experiment are in progress to identify these proteins.
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 4, 9 and 11. 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 close 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