Projet ANR-08-GENO-0020 – HLH-genepathé : Bases moléculaires, mécanismes fonctionnels et thérapeutique alternative des syndromes lymphohistiocytaires
Programme : Du gène à la physiopathologie, des maladies rares aux maladies communes (GENOPAT) 2008
Année de financement : 2008
Coordinateur du projet : INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - DÉLÉGATION RÉGIONALE PARIS V (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - Délégation régionale Paris V)
Partenaires : Institut national de la santé et de la recherche médicale
Montant : 420000.0 euros
Durée : 48 mois
Résumé : The characterization and understanding of the molecular and functional bases leading to the occurrence of Hemophagocytic Lymphohistiocytic syndromes provide a way to approach critical mechanisms controlling T lymphocyte homeostasis. Through the study of human natural mutants of these conditions, critical effectors of the cytotoxic activity have been identified. The genetic and functional studies of the inherited forms that are yet molecularly uncharacterized should allow the identification of new effectors of this lytic machinery and their role in the cytotoxic function of lymphocytes. Some inherited forms may not result from T lymphocytes cytotoxic defect. Their molecular characterization would highlight new components and pathway critically involved in the regulation of lymphocytes homeostasis. Murine models of these conditions  will allow to better analyze the precise mechanisms leading of HLH onset and to test a new therapeutic approach based on IFNg neutralisation. First aim of this project will focus on the identification of new effector(s) involved in the cytotoxic function of lymphocytes and on a better characterization of the molecular interactions allowing the various components of this machinery to finely regulate cytotoxic granules secretion at the immunological synapse. Identification of new effectors will be based on a human genetic approach to characterize the molecular basis of inherited HLH with defective lymphocyte cytotoxicity. Linkage analysis using Affymetrix microarrays, biochemical and imaging approaches will be used for that purpose. 
Second aim of this project will focus on the characterization of the mechanisms and molecules leading to HLH in patients with apparently normal T lymphocyte cytotoxic activity. Understanding the role of syntaxin 11 in immune cells and in the pathophysiology leading to the occurrence of HLH in syntaxin 11 deficient patients will be a main objective. Subcellular expression and localization of various syntaxin 11-tagged constructs and search of syntaxin 11 partners through a two-hybrids approach will be conducted. The obtention of syntaxin 11 knock-out (KO) mice, with complete or conditioning gene targeting, should allow to identify which hematopoietic lineages (lymphoid and/or myeloid) deficient in syntaxin 11 account for the occurrence of an HLH. In addition, identification of new proteins regulating lymphocyte homeostasis independently of the cytotoxic function of lymphocytes will be searched through the genetic analysis of patients with inherited form of HLH, associated with preserved T lymphocyte cytotoxic activity in the absence of syntaxin 11mutations. 
Third aim of this project  will evaluate new therapeutic tools for HLH. We will use mouse models of inherited HLH (perforin KO mice, Rab27a-/- ashen mice, syntaxin 11 KO mice) to reproduce the feature of HLH using LCMV infection, and then to try to treat the disease by IFNg neutralisation.  The identification of novel FHL genes, in addition to their utility for patients clinical care and genetic counseling, will also point to additional unexpected molecules playing an important role in the cytotoxic activity of lymphocytes and in the regulation of lymphocytes homeostasis. The various approaches leading to the molecular and functional characterization of syntaxin 11  should help to determine whether the mechanisms leading to the development of the HLH associated with this molecular defect, are dependent directly or indirectly to the cytotoxic function of lymphocytes, as it is the case for the other inherited forms of HLH so far identified. Alternatively other mechanism regulating lymphocyte homeostasis should be characterized through this approach. 
If efficacy of the INFg neutralization can be demonstrated in the various models of murine HLH, this therapeutic approach will be considered as a preclinical step in the treatment of patients with primary and secondary HLH.