Cholesterol lights the fire of NASH

 描述（由申请人提供）： 我们报道了膳食胆固醇诱导单纯性脂肪变性进展为NASH。我们随后发现，在NASH患者和高脂肪、高胆固醇（HFHC）饮食诱导的NASH小鼠模型中，脂肪变性肝细胞的脂滴内均存在胆固醇晶体，但在单纯性脂肪变性患者或小鼠中则不存在。我们描述了枯否细胞（KCs）选择性地包围和处理含有胆固醇晶体的脂肪变性肝细胞，形成特征性的“冠状结构”（CLS）。因此，肝细胞和KC都暴露于胆固醇晶体，最近已显示胆固醇晶体在动脉粥样硬化的动物模型中激活NLRP 3炎性体。我们证明了形成CLS的KC中的NLRP 3活化，从而提供了肝细胞和KC暴露于胆固醇晶体可导致NASH的慢性“无菌炎症”的机制。总的来说，这些结果支持我们的新假设，即肝脏胆固醇结晶通过激活NLRP 3炎性体引起NASH。我们将在小鼠模型和人类中追求这一假设，具体目标如下：SA 1。确定肝细胞胆固醇晶体和KC-CLS的形成是否是诱导NLRP 3活化和从单纯性脂肪变性进展为NASH的触发因素。a.体外实验：将诱导HepG 2（人肝细胞瘤）细胞在有或没有胆固醇晶体的情况下形成大LD，然后与THP 1巨噬细胞或原代小鼠KC在接触或非接触共培养物中共培养。我们假设THP 1细胞或KC只有在与LD内有胆固醇晶体的HepG 2细胞共培养时才能显示胆固醇晶体的摄取和NLRP 3的激活。我们将描述胆固醇晶体激活NLRP 3和诱导IL-1β的分子机制。B.体内实验：将野生型（Wt）C57 BL/6 J小鼠和高胆固醇血症小鼠（Ldlr（-/-）和APOE 2ki）暴露于胆固醇浓度范围为0%至1%的高脂肪饮食。我们假设，所有三种小鼠模型将在特定阈值饮食胆固醇浓度下发展NLRP 3活化和组织学NASH，这导致每个小鼠模型中的肝胆固醇晶体和CLS，而不考虑其他胆固醇相关因素。这将证明胆固醇结晶是通过NLRP 3激活启动NASH的关键开关。 SA 2.确定NLRP 3炎性体的遗传失活（全局、KC特异性或肝细胞特异性）或药理学抑制是否可预防胆固醇晶体诱导的NASH的发展。我们将使用全局Casp 1（-/-）和Nlrp 3（-/-）KO小鼠来证明NLRP 3炎性体的失活抑制胆固醇晶体诱导的NASH。我们将比较条件性科斯，其中Nlrp 3基因在KC（Nlrp 3 KC（-/-））或肝细胞（Nlrp 3 Hep（-/-））中选择性缺失，以区分肝细胞（引起焦亡）或KC（引起细胞因子、趋化因子和炎性募集的释放）或两者中的NLRP 3活化是否有助于胆固醇晶体诱导的NASH。我们将确定一种新型的口服药理学NLRP 3抑制剂是否可以预防或逆转HFHC饮食的Wt小鼠中胆固醇晶体诱导的NASH，对人类NASH具有潜在的治疗意义。 SA 3.确定人类NAFLD/NASH中肝脏胆固醇结晶的患病率和风险因素，并评估肝脏胆固醇结晶是否独立预测NLRP 3激活和NASH与单纯脂肪变性的发展。我们将使用来自现有VA生物储存库的数据和储存的冷冻肝组织标本，这些生物储存库具有良好的NAFLD/NASH患者特征（n=166）。

 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 (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).