Cholesterol lights the fire of NASH

 描述(由申请人提供)： 我们报告说，饮食中的胆固醇会导致从单纯性脂肪变性到NASH的进展。我们随后发现，在NASH患者和高脂高胆固醇(HFHC)饮食诱导的NASH小鼠模型中，脂肪变性肝细胞的脂滴中都存在胆固醇晶体，但在单纯性脂肪变性的患者或小鼠中没有。我们描述了Kupffer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，形成了特有的“冠状结构”(CLS)。因此，肝细胞和KCs都暴露在胆固醇晶体中，最近发现胆固醇晶体可以激活动脉粥样硬化动物模型中的NLRP3炎症体。我们证明了NLRP3在形成CLSS的KCs中的激活，从而提供了一种机制，通过该机制，肝细胞和KCs暴露于胆固醇晶体可以导致NASH的慢性“无菌炎症”。总之，这些结果支持我们的新假设，即肝脏胆固醇结晶通过激活NLRP3炎症体而导致NASH。我们将在小鼠模型和人类模型中探索这一假说，具体目标如下：SA1。确定肝细胞胆固醇晶体和KC-CLSS的发展是否是诱导NLRP3激活和从单纯性脂肪变性进展为NASH的触发因素。A.体外实验：无论有无胆固醇晶体，均可诱导人肝癌细胞形成较大的低密度脂蛋白，然后与THP1巨噬细胞或原代小鼠KCs在接触或非接触条件下共培养。我们假设，THP1细胞或KCs只有与LDs中含有胆固醇晶体的HepG2细胞共同培养时，才会显示出摄取胆固醇晶体和NLRP3激活的情况。我们将描述胆固醇晶体激活NLRP3和诱导IL-1β的分子机制。B.体内实验：野生型(Wt)C57BL/6J小鼠和高胆固醇血症小鼠(Ldlr(-/-)和APOE2ki)将暴露于胆固醇浓度从0%到1%的高脂饮食中。我们假设，无论其他胆固醇相关因素如何，所有三种小鼠模型都将在特定的饮食胆固醇浓度下发生NLRP3激活和组织学NASH，从而导致每个小鼠的肝脏胆固醇结晶和CLSS。这将证明胆固醇结晶是通过NLRP3激活启动NASH的关键开关。SA2.确定NLRP3炎症体的遗传失活(全局的、KC特异性的或肝细胞特异性的)或药物抑制是否可以防止胆固醇晶体诱导的NASH的发展。我们将使用全球CASP1(-/-)和Nlrp3(-/-)KO小鼠来证明NLRP3炎症体的失活抑制胆固醇晶体诱导的NASH。我们将比较KCs(Nlrp3KC(-/-))或肝细胞(Nlrp3Hep(-/-))中Nlrp3基因选择性缺失的条件性KO，以区分NLRP3在肝细胞(导致下垂)或KCs(导致细胞因子、趋化因子和炎性募集)中的激活，还是两者都参与了胆固醇晶体诱导的NASH。我们将确定一种新型的口服药理NLRP3抑制剂是否可以预防或逆转服用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。SA3.确定肝脏胆固醇结晶在人NAFLD/NASH中的患病率和危险因素，并评估肝脏胆固醇结晶是否独立预测NASH和单纯性脂肪变性NLRP3的激活和发展。我们将使用来自现有VA生物信息库的数据和存储的冷冻肝组织样本，这些样本具有良好的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).