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  Director : Salah Mécheri (smecheri@pasteur.fr)


  abstract

 

Our main objectives are a) to study the interactions, cell-to-cell- or exosome-mediated, between mast cells (MCs) and dendritic cells (DCs) and their implications in immune responses against soluble antigens as well as infectious agents, and b) since MCs are recognized as the major players in IgE-mediated allergic responses, we investigate the molecular mechanisms by which MC exocytosis occurs with a special focus on the identification of molecules involved in membrane fusion.



  report

cale

Mast cells induce phenotypic and functional dendritic cell maturation through the release of exosomes (D. Skokos, H. Goubran Botros, M. Roa, R. Peronet, C. Demeure, S. Mécheri)

MCs are considered as major players in IgE-mediated allergic responses but have also recently been recognized as active participants in innate as well as specific immune responses. Recent work provided evidence that MCs are able to activate B and T lymphocytes through the release of vesicles called exosomes. Here, we demonstrate that exosomes, which are located in the endocytic pathway, harbor exogenous antigens which associate with other molecules endowed with immunomodulatory functions including hsp60 and hsc70. Administration to naive mice of antigen-containing exosomes in the absence of conventional adjuvants elicits specific antibody responses across the MHC II haplotype barrier. We demonstrate that MC-exosomes induce immature DCs to up-regulate MHC class II, CD80, CD86, and CD40 molecules and to acquire potent antigen presenting capacity to T cells. Uptake and processing of antigen-associated exosomes by endogenous DCs was also demonstrated. Finally, exosome-associated hsps are critical for the acquisition by DCs of the antigen presenting function. In a collaborative work with the Proteomic technical platform, a project of proteomic analysis of murine and human MC exosomes has been initiated. In parallel, the molecular mechanisms involved in the biogenesis of exosomes and the regulation of their release in the external milieu are aspects presently developped in our laboratory. This work demonstrates a heretofore unrecognized collaborative interaction between MCs and DCs leading to the elicitation of specific immune responses.

Role of mast cells in bordetella infection (H. Goubran Botros, R. Peronet, S. Mécheri)

In colaboration with I. Tchou and N. Guiso-Maclouf (Bordetella Unit), we have investigated the role of MCs in bordetella infection. We have demonstrated that MCs, in contrast to B cells and macrophages, are resistant to cell lysis upon contact with Bordetella pertussis (B.P). Dependent on the genetic background (C3H/HeJ and DBA/2 strains) of mice from which MCs are generated, susceptibility to bacterial invasion (B. p. strain 81.32) is different. Accordingly, we found that DL50 induced by bronchiseptica (LapRem strain) is higher in C3H/HeJ than in DBA/2 mice. Using several mutants of B.P. for different toxins : filamentous hemagglutinin antigen (FHA), pertussis toxin, pertactin and adénylate cyclase, we found that only FHA was mandatory for bacterial entry into the cells.

Development of a mouse model to demonstrate the effect of salivary molecules-induced mast cell activation on malaria infection (C. Demeure, F. Marchand, S. Mécheri)

Christian Demeure studies the role of MCs in the development of the adaptative immune response, with a special focus on MC-DC cross-talk. Using a mouse model of malaria, we found that the bite of the mosquito vector anopheles causes mouse cutaneous MCs to degranulate and to recruit neutrophils at the inflammatory site. This recruitment does not occur in MC-deficient mice. These mice also maintain a lower blood parasitemia following Plasmodium berghei transmission by mosquitoes, although a similar death rate is seen. In-vitro, DCs stimulated by coculture with bone-marrow-derived MCs (BMMC) produce TNF-a and undergo a phenotypic maturation. These studies indicate that, at least in the skin which is a priviledged site for pathogen entry, MCwhich are able to react instantly to many stimuli can modulate DC function, thereby altering immune response to pathogens.

Analysis of the molecular machinery of inflammatory mediator release in immunologically stimulated mast cells (C. Marchand, C. Gasse, C. Brochetta, B. Iannascoli, U. Blank )

Following their stimulation through high affinity IgE receptors (FceRI), MCs massively release inflammatory mediators prestored in their intracytoplasmic granules by degranulation. The objectif of our group is to identify the molecular actors and molecular signaling events of this process. By developing a panoply of antibodies we have demonstrated the expression of multiple isoforms of SNARE proteins known to be involved in membrane fusion such as syntaxin 4. We have started to analyze their role using biochemical and cell biological approaches. We have also defined regulators such as the small GTPase Rab3D and an associated kinase (Rak3D) that is regulated by elevated calcium levels. Recently we have described the role of the SM (Sec/Munc) proteins Munc18-2 and Munc18-3 in MCs localised, respectively, on the plasma membrane and on secretory granules. Using a molecular cloning approach based on the double hybrid system we are now trying to identify new interacting partners that establish a link between the membrane fusion machinery and the signaling events initiated by the receptor. We also try to set up a functional test that allows to determine the functional implication of the various molecular players by specifically inhibiting their expression using RNAi technology. In collaboration with the imaging platform at the Pasteur Institute we have started a project aimed to analyse the highly dynamic degranulation process by videomicroscopy using fluorescent-tagged proteins. In particular, we will study intragranular soluble markers as well as membrane markers of the different granular compartments present in MCs. We also examine the interaction of these compartments with the cytoskeleton. These projects should yield a better understanding of the MC granule compartment and its biogenesis. It may also allow to conceive new molecular approaches for interference with the degranulation process, which can be harmful in the case of allergies, but also in other inflammatory pathologies.

Keywords: Mast cells, Dendritic cells, Exosomes, Immunoregulation, IgE, Exocytosis, Immunology, Allergy



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Laurette Lecarpentier (llecarp@pasteur.fr) Dr. Salah Mécheri, Chef de laboratoire IP (smecheri@pasteur.fr)

Dr. Ulrich Blank DR2 (CNRS ) (ublank@pasteur.fr)

Dr. Claudine Guérin-Marchand CR1 (CNRS) (clobert@pasteur.fr)

Dr M. Roa CR IP (mroa@pasteur.fr)

Dr C. Demeure CR IP (cdemeure@pasteur.fr)

Dimitris Skokos: 3e année de thèse

Cécile Gasse : 1ere année thèse

Christiana Brochetta : 1ere année de thèse

Hany Goubran-Botros: ingénieur

Bruno Iannascoli: technicien

Françoise Marchand: technicienne

Roger Peronet: technicien animalier


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