Anales RANF

S6-04 IMMUNE CELLS FROM P2X4KO MICE EXHIBIT ALTERED P2X7 EXPRESSION AND FUNCTION B. Rissiek 1 , M. Lukowiak 1 , F. Rassendren 2 , A. Nicke 3 , F. Koch-Nolte 4 , Tim Magnus 1 1 . Department of Neurology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany, 2 . Institut de Génomique Fonctionnelle, CNRS UMR5203, Université de Montpellier, Montpellier, France, 3 .Walther Straub Institute for Pharmacology and Toxicology, Ludwig-Maximilians-Universität München, Munich, Germany; 4 . Institute of Immunology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany. P2rx4 and P2rx7 are neighboring genes encoding for the ATP-gated ion channels P2X4 and P2X7. The first P2X4ko mice were generated more than 10 years ago by targeting P2rx4 in 129 embryonic stem cells. However, this might potentially lead to the introduction of P2rx7 passenger mutations upon backcrossing to the C57BL/6 (B6) background. By analyzing the naturally occurring P451L polymorphism in P2X7 we could demonstrate that B6-P2X4ko mice (P2rx4 tm1Rass ) express a P2X7 variant (P2X7- 451P) which differs from the B6-WT P2X7 variant (P2X7-451L). We investigated the impact of this passenger mutation on B6-P2X4ko immune cells and observed an increased P2X7 expression on T cells when compared to B6-WT. This affected T cell sensitivity towards adenosine triphosphate (ATP). Further, B6-P2X4ko T cells were more susceptible towards nicotinamide adenine dinucleotide induced cell death (NICD) occurring after cell preparation, which influences the results of functional assays. When backcrossing B6-P2X4ko (P2X7-451P) to the Balb/c background (P2X7-451P), the passenger mutation and T cell related differences in P2X7 expression and function were neutralized. On innate immune cells we found that P2X4-deficiency in B6 mice leads to a lower P2X7 protein and mRNA expression. This was most prominent in peritoneal mast cells and macrophages whereas brain microglia exhibited almost unaltered P2X7 level. Interestingly, backcrossing of B6-P2X4ko to the Balb/c background did not normalize these differences. P2X4-deficiency in B6 and Balb/c mice influences P2X7- mediated calcium flux and pore formation in macrophages and P2X7-dependent mast cell degranulation. In conclusion, P2X7 expression and function can be mouse strain and cell type-specifically altered in P2X4ko mice. Our findings need to be considered when using these P2X4ko mice for complex immunological in vivo studies.