DESCRIPTION (provided by applicant): We reported that dietary cholesterol induces progression from simple steatosis to NASH. We subsequently discovered that cholesterol crystals were present within the lipid droplets of steatotic hepatocytes in both patients with NASH and in mouse models of NASH induced by high-fat, high-cholesterol (HFHC) diet, but not in patients or mice with simple steatosis. We described that Kupffer cells (KCs) selectively surrounded and processed steatotic hepatocytes containing cholesterol crystals forming characteristic "crown-like structures" (CLS). Thus, both hepatocytes and KCs become exposed to cholesterol crystals, which have recently been shown to activate the NLRP3 inflammasome in animal models of atherosclerosis. We demonstrated NLRP3 activation in the KCs that form CLSs thus providing a mechanism by which exposure of hepatocytes and KCs to cholesterol crystals can lead to the chronic "sterile inflammation" of NASH. Collectively these results support our novel hypothesis that hepatic cholesterol crystallization causes NASH via activation of the NLRP3 inflammasome. We will pursue this hypothesis in both mouse models and humans with the following specific aims: SA1. Determine whether development of hepatocyte cholesterol crystals and KC-CLSs is the trigger that induces NLRP3 activation and progression from simple steatosis to NASH. a. In vitro experiments: HepG2 (human hepatoma) cells will be induced to develop large LDs either with or without cholesterol crystals and then co-cultured with THP1 macrophages or primary mouse KCs in contact or non- contact cocultures. We hypothesize that THP1 cells or KCs will demonstrate uptake of cholesterol crystals and NLRP3 activation only when co-cultured with HepG2 cells that have cholesterol crystals within their LDs. We will delineate the molecular mechanisms by which cholesterol crystals activate NLRP3 and induce IL-1β. b. In vivo experiments: Wild-type (Wt) C57BL/6J mice and hypercholesterolemic mice (Ldlr (-/-) and APOE2ki) will be exposed to high-fat diets ranging in cholesterol concentration from 0% to 1%. We hypothesize that all three mouse models will develop NLRP3 activation and histological NASH at that particular threshold dietary cholesterol concentration that leads to hepatic cholesterol crystals and CLSs in each mouse model, irrespective of other cholesterol-related factors. This will demonstrate that cholesterol crystallization is the critical switch that initiats NASH via NLRP3 activation. SA2. Determine whether genetic inactivation (global, KC-specific, or hepatocyte-specific) or pharmacological inhibition of the NLRP3 inflammasome prevent the development of cholesterol crystal-induced NASH. We will use global Casp1(-/-) and Nlrp3(-/-) KO mice to demonstrate that inactivation of the NLRP3 inflammasome inhibits cholesterol crystal-induced NASH. We will compare conditional KOs in which the Nlrp3 gene is selectively deleted either in KCs (Nlrp3...