284: Caspase-8-independent role of c-FLIP in Inflammasome Activation

Abstract

Cellular FLICE inhibitory protein (c-FLIP) is an inhibitor of caspase-8 and is required for macrophage development. Recent studies reveal a selective role of caspase-8 in non-canonical IL-1β production that is independent of caspase-1 or inflammasome. Here we demonstrated that c-FLIP is an unexpected contributor to canonical inflammasome activation for the generation of caspase-1 and active IL-1β. Lipopolysaccharides (LPS)-, R848-, and heat-killed Listeria monocytogenes (HKLM)-induced IL-1β production in macrophages was attenuated in the absence of c-FLIP. Decreased IL-1β expression was attributed to a reduced activation of caspase-1 in c-FLIP-deficient cells. In contrast, the production of TNF- α was not affected by deficiency in c-FLIP. c-FLIP interacted with NLRP3 or procaspase-1. Proximity ligation assay revealed that c-FLIP is required for the full NLRP3 inflammasome assembly, and c-FLIP associates with NLRP3 inflammasome components in situ . c-FLIP-deficiency also reduced AIM2 inflammasome activation. In contrast, c-FLIP inhibited SMAC mimetic- or FasL-induced IL-1β generation that is caspase-8-mediated. Our results demonstrate a caspase-8-independent role of c-FLIP in the optimal activation of the NLRP3 and AIM2 inflammasomes, and suggest that c-FLIP could be a valid target for treatment of inflammatory diseases caused by over-activation of inflammasomes. The aim of this study was to investigate how T-helper (Th) 17 cells and interleukin (IL) −17 contribute to root resorption during orthodontic tooth movement. Experiment 1: Rheumatoid arthritis (RA) model mice and wild type mice were subjected to the orthodontic force (OF) to induce root resorption (heavy OF) of the upper first molars. The double-immunofluorescence analysis for IL-17/CD4 was performed to detect Th17 cells in the periodontal ligament (PDL). Experiment 2: Atopic dermatitis (AD) model mice and wild type mice were subjected to the heavy OF to induce movement of the upper first molars. The expression levels of the tartrate-resistant acid phosphatase (TRAP), IL-17, IL-6, and receptor activator of nuclear factor kappaB ligand (RANKL) proteins were determined in the PDL by an immunohistochemical analysis. Moreover, the fluorescent localization immunoassay was performed to detect Th17 cells. Furthermore, the effects of the compression force on co-cultures of CD4 + cells from AD patients or healthy individuals and human PDL cells were investigated with regard to the levels of secretion and mRNA expression of IL-17, IL-6, RANKL, and osteoprotegerin (OPG). Results: Experiment 1: The double-immunofluorescence analysis for IL-17/CD4 detected immunoreaction. Experiment 2: The immunoreactivities for TRAP, IL-17, IL-6, and RANKL in the AD group were found to be significantly increased. The double-immunofluorescence analysis for IL-17/CD4 detected immunoreaction. The secretion of IL-17, IL-6 and RANKL, and the mRNA levels of IL-6 and RANKL in the AD patients were increased compared with those in healthy individuals. Th17-producing IL-17 may be associated with the deterioration of orthodontic root resorption.