125 Protein Tyrosine Phosphatase Non-Receptor Type 22 Dephosphorylates NLRP3 to Enable Efficient Inflammasome Activation

Background and aim: Inflammasomes are large, multi-protein complexes in the cytosol, which form upon intracellular presence of danger-associated molecular patterns. Inflammasome assembly results in auto-cleavage and activation of the protease caspase-1, which in turn cleaves pro-IL-1β into its active form, and mediates its secretion. Excessive inflammasome activation results in severe inflammatory conditions, but in the intestine physiological IL1β secretion is important for immune homeostasis. Loss of function variants in the gene encoding the inflammasome receptor Nod-like receptor protein (NLRP)3 are associated with increased risk for Crohn's disease (CD). Here, we addressed if loss of protein tyrosine phosphatase non-receptor 22 (PTPN22) which is associated with several inflammatory disorders, including CD influences inflammasome activation. Methods: human cell lines, as wella as murine and human primary cells were treated with different inflammasome inducing agents and analysed by ELISA, qPCR and Western blot. Colitis in mice was induced by application of 2% DSS in the drinking water for 7 days. Intestinal biopsies and serum from CD and control patients were analysed by qPCR and ELISA. Results: Knockdown of PTPN22 in human cell lines as well as loss of PTPN22 in primary murine dendritic cells resulted in markedly reduced caspase-1 cleavage and IL-1 β secretion upon activation of the NLRP3 inflammasome. In contrast, IL-1 β secretion upon activation of AIM2 or NLRC4 inflammasomes was not affected. Immunoprecipitation of NLRP3 revealed tyrosine phosphorylation of NLRP3 and a direct interaction between NLRP3 and PTPN22. Loss of PTPN22 enhanced NLRP3 tyrosine phosphorylation, while a targeted mutation of tyrosine 861 in NLRP3 completely abolished its phosphorylation, and resulted in increased caspase-1 cleavage and IL-1β secretion. In acute DDS-induced colitis, PTPN22 knockout resulted in pronounced weight loss and aggravated macroscopic and microscopic colitis scores. Loss of PTPN22 enhanced NLRP3 phosphorylation together with reduced levels of mature IL-1 β in lamina propria mononuclear cells, but not in epithelial cells. In IBD patients, presence of a disease-associated gain-of-function variant within the gene locus encoding PTPN22 was accompanied with increased IL-1β mRNA and serum levels. Summary: we describe a novel and important regulatory mechanism of NLRP3 by tyrosine phosphorylation, which prevents aberrant inflammasome activation. We demonstrate that PTPN22 dephosphorylates NLRP3 to allow efficient inflammasome activation upon inflammatory insults. This helps to explain, how variants in PTPN22 contribute to the pathogenesis of CD as well as other inflammatory disorders.