Innate Immunity, Cholesterol, and NASH Pathogenesis

项目摘要/摘要 非酒精性脂肪性肝病(NAFLD)是慢性肝病的最常见原因，至少影响 占世界人口的四分之一，因此与美国国立卫生研究院的任务高度相关。从脂肪变性到纤维化， 肝脏炎症推动疾病进展，这项研究将阐明胆固醇的核心作用。 缺乏以证据为基础的FDA批准的NASH治疗方案，突显了进一步 了解疾病发病机制和确定潜在靶点的研究。我们引人入胜的预赛 最近发表的研究结果表明，先天免疫细胞(中性粒细胞和巨噬细胞)是 NAFLD的发病机制及进展。这项提议的首要假设是，监管失调 先天性免疫和炎症是NAFLD的主要、不可或缺的特征，在很大程度上由 胆固醇。我们将通过同时执行三个特定目标来检验这一假设 小鼠NAFLD模型和不同严重程度的NASH患者的样本。在SA1中，PI将 综合分析不同阶段NASH患者外周血中的新鲜中性粒细胞。这将是 包括NETsis，胆固醇诱导NETs如何许可巨噬细胞的反应，以及 肝内皮细胞上的中性粒细胞。小鼠模型将专注于靶向Net以减轻肝损伤 在纳什。我们将使用富含果糖、棕榈酸酯、反式脂肪和胆固醇(按重量计为1.2%)的FPC饮食，其中 已被证明在16周内诱导肝纤维化相关参数。SA2将使用喂食的活小鼠 不同的基于FPC的饮食与超声引导下的细针抽吸肝组织分离，随后 通过单细胞RNA测序(scRNA-seq)检测巨噬细胞全基因组表达 进行性或退化性饮食诱导的NASH。我们的数据表明，高胆固醇在 饮食最早在4周时下调巨噬细胞特异性转录。拟议的试验计划将 对饮食胆固醇如何影响不同的肝脏巨噬细胞群提供了前所未有的见解。 我们还将描述不同的肝脏巨噬细胞对共培养肝脏的功能影响。 星状细胞(HSC)。此外，我们将使用巨噬细胞基因启动子区域的定向测序 以确定表观遗传调控中的关键适应性变化。最后，在SA3中，PI将专注于集成 这些稳健的分析和利用Cre-Lox系统产生髓系特异性靶向突变的小鼠 基因。我们已经发现，编码骨桥蛋白(OPN)的SPP1在 在NASH的巨噬细胞中，我们将测试一种OPN纳米颗粒在预防和逆转饮食诱导方面的研究 纳什。携带Col1a1-GFP转基因小鼠将被用于直接评估不同治疗方法的效果 肝星状细胞的激活、分化和纤维化反应策略。总而言之，我们的多学科 专家的方法和出色的协同作用有可能从根本上改变我们对 胆固醇如何塑造和失调NASH患者的先天免疫，并确定新的治疗方法。

PROJECT SUMMARY /ABSTRACT Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease, affecting at least a quarter of the world’s population, and therefore highly relevant to the NIH mission. From steatosis to fibrosis, hepatic inflammation drives disease progression, and this study will elucidate the central roles of cholesterol. The lack of evidence-based, FDA-approved treatment options for NASH underscores the need for further research into understanding disease pathogenesis and identifying potential targets. Our compelling preliminary and recently-published findings demonstrate innate immune cells (neutrophils and macrophages) are central to the pathogenesis and progression of NAFLD. The overarching hypothesis of this proposal is that dysregulation of innate immunity and inflammation is a dominant, indispensable feature of NAFLD, largely mediated by cholesterol. We will test this hypothesis through completion of three Specific Aims performed in parallel using murine NAFLD models and samples from patients with varying severity of NASH. In SA1, the PI will comprehensively characterize fresh peripheral neutrophils from patients with different stages of NASH. This will include NETosis, how cholesterol-induced NETs license responses from macrophages and differential effect of neutrophils on liver endothelial cells. Murine models will focus on targeting of NETs to attenuate hepatic injury in NASH. We will use the FPC diet, rich in fructose, palmitate, trans-fat, and cholesterol (1.2% by wt), which has been shown to induce hepatic fibrosis-associated parameters within 16 weeks. SA2 will use live mice fed different FPC-based diets with isolation of hepatic tissue by ultrasound-guided fine-needle aspiration, followed by single-cell RNA sequencing (scRNA-seq) to measure genome-wide expression of macrophages from progressing or regressing diet-induced NASH. Our data demonstrate that withdrawal of high cholesterol in the diet downregulates macrophage-specific transcription as early as 4 weeks. The proposed experimental plan will provide unprecedented insights into how dietary cholesterol affects different hepatic macrophage populations. We will also characterize the functional consequences of different hepatic macrophages on co-cultured hepatic stellate cells (HSCs). Moreover, we will use targeted sequencing of promoter regions in macrophage genes in order to define key adaptive changes in epigenetic regulation. Finally, in SA3, the PI will focus on integrating these robust analyses and utilizing Cre-Lox system to generate mice with myeloid-specific targeted mutants of genes. As we have found that osteopontin (OPN)-encoding SPP1 expression was strongly induced in macrophages in NASH, we will test an OPN nanoparticle in prevention and reversal studies of diet-induced NASH. Mice carrying the Col1a1-GFP transgene will be used to directly assess the effect of different therapeutic strategies on activation, differentiation, and fibrogenic responses of HSCs. Taken together, our multidisciplinary approach and outstanding synergism of experts has the potential to fundamentally change our understanding of how cholesterol shapes and dysregulates innate immunity in NASH and identify novel treatments.