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-1?. 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 (Nlrp3KC(-/-)) or hepatocytes (Nlrp3Hep(-/-)) to distinguish whether NLRP3 activation in hepatocytes (causing pyroptosis) or KCs (causing release of cytokines, chemokines and inflammatory recruitment) or both is contributing to cholesterol crystal-induced NASH. We will determine whether a novel, oral, pharmacological NLRP3 inhibitor can prevent or reverse cholesterol crystal-induced NASH in Wt mice on a HFHC diet, with potential therapeutic implications for human NASH. SA3. Determine the prevalence and risk factors of hepatic cholesterol crystallization in human NAFLD/NASH and evaluate whether hepatic cholesterol crystallization independently predicts NLRP3 activation and development of NASH vs simple steatosis. We will use data and stored frozen liver tissue specimens from an existing VA biorepository of well- characterized patients with NAFLD/NASH (n=166).
There is a worldwide epidemic of NAFLD related to the epidemics of obesity and diabetes. Obesity, diabetes and NAFLD/NASH are also extremely common problems in the VA population. From the liver standpoint the most important unanswered question is what causes the development of progressive NASH in a subset of patients with NAFLD that can ultimately lead to cirrhosis. Treatments for NASH cannot be designed rationally until the pathogenesis of NASH is better understood. Our research will demonstrate that hepatic cholesterol crystals are a hitherto unrecognized cause of NASH, leading to NLRP3 activation in hepatocytes and KCs and aggregation of KCs into CLSs and propagating the sterile inflammation and fibrosis that characterize NASH. We propose studies that span the translational spectrum from cell cultures and mouse models to human NASH. In addition to improving our understanding of NASH pathogenesis, our research will test novel therapeutic approaches targeting the NLRP3 inflammasome.
|Haczeyni, Fahrettin; Poekes, Laurence; Wang, Hans et al. (2017) Obeticholic acid improves adipose morphometry and inflammation and reduces steatosis in dietary but not metabolic obesity in mice. Obesity (Silver Spring) 25:155-165|
|Ioannou, George N; Subramanian, Savitha; Chait, Alan et al. (2017) Cholesterol crystallization within hepatocyte lipid droplets and its role in murine NASH. J Lipid Res 58:1067-1079|