Blanc – Accords bilatéraux 2013 - SVSE 6 - Blanc – Accords bilatéraux 2013 - SVSE 6 - Génomique, Génétique, Bioinformatique et Biologie systémique

Candidate genes and pathways related to Autoimmune lymphoproliferative syndrome in a novel mouse strain deficient for Fas and P2X7 receptors – P2X7RFAS

Submission summary

The main goal of this french-taïwanese project is to identify genes and biochemical pathways involved in the development of lymphoproliferation leading to auto-immune responses in mice. The french coordinator of this project has produced, in collaboration with his french partner, a novel strain of C57Bl/6 (B6) mice bearing the lpr mutation (deficiency of death receptor Fas) and knock-out for the purinergic receptor P2X7 (P2X7R). On B6 genetic background, the lpr mutation induces a mild lymphoproliferation with limited autoimmune responses. In contrast, MRL/lpr mice develop a systemic autoimmune disease resembling systemic lupus erythematosus. The lack of P2X7R, in C57Bl/6, does not lead to any apparent disease. Interestingly, in B6/lpr-P2X7R KO mice, a strong lymphoproliferation appears with auto-immune responses currently under study. To determine which cell sub-populations are contributing to the disease found in B6/lpr, P2X7R KO, the french partners will produce mice which do not express P2X7R in T lymphocytes or dendritic cells (DC). Thus, CD4-Cre recombinase and CD11c-Cre recombinase mice will be mated with B6/lpr-P2X7R KO animals. In these new strains of mice, the disease severity will be evaluated by quantifying lymphoproliferation, measuring antibody and cytokine concentrations and assessing tissue damages. Thus, the respective roles of T lymphocytes and DC in the disease will be tested in these mouse strains. Recently, deep-sequencing technologies of cDNAs in order to determine a sample's RNA content, named RNA-Seq, have been developed as an approach for analyses of gene expression. By obtaining millions of reads of transcribed sequences, an RNA-Seq experiment can provide a comprehensive survey of the population of transcribed genes and their isoforms, revealing the molecular components of normal or pathological tissues. Statistical methods and bioinformatics for RNA sequence analyses have helped in the interpretation of this large amount data. The 2 taiwanese partners have a strong expertise in RNA seq and bioinformatics to analyze the levels of transcripts which are amplified or decreased in lymphoid organs or DC or sub-populations of T lymphocytes. These methods will be used to compare the transcriptomes of B6, B6/lpr, B6 P2X7R KO and B6 /lpr-P2X7R KO allowing us to identify the transcribed genes which are up- or down-modulated in the "autoimmune" B6/lpr-P2X7R KO mice. The same methods will be used to determine which genes, among those already identified, are preferentially up- or down-regulated in T lymphocytes or DC. Furthermore, the analyses performed in B6/lpr lacking P2X7R in T lymphocytes or DC only should help in defining which biological pathways are deregulated in cells lacking P2X7R and may unravel cellular synergies. Demonstrating that the identified genes ("driver genes") are involved in the autoimmune responses found in B6/lpr-P2X7R KO mice will require in vivo validation. To this end, several strategies may be used to determine whether these genes are involved in lymphoproliferation and/or autoimmunity :1) if a KO mouse for one of the identified genes exists, it will be mated with B6/lpr-P2X7R KO mice and the autoimmune responses will be evaluated in the B6 lpr/lpr, P2X7R KO carrying a novel nul allele; 2) injection into B6 lpr/lpr, P2X7R KO mice of lentiviruses encoding specific shRNA able to silence the expression of identified "driver gene"; 3) injection into B6 lpr/lpr, P2X7R KO mice of neutralizing monoclonal antibodies specific for the driver gene product(s). This ambitious collaborative project between taiwanese and french partners with complementary and multidisciplinary expertises should bring important information on the biological pathways involved in auto-immune responses and pathologies.

Project coordination

Pierre BOBÉ (Laboratoire Signalisation Calcique et Interactions Cellulaires dans le Foie) – pierre.bobe@u-psud.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

Chuang Genang University Department of Medical Biotechnology and Laboratory Science, Research Center for Emerging Viral Infections
National Chung Cheng University Department of Computer Science and Information Engineering
CNRS-UMR 8619, Université Paris Sud Institut de Biochimie et Biophysique Moléculaire et Cellulaire
UMR S 757 INSERM-Université Paris Sud Laboratoire Signalisation Calcique et Interactions Cellulaires dans le Foie

Help of the ANR 107,120 euros
Beginning and duration of the scientific project: December 2013 - 36 Months

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