Innate Immunity, Cholesterol, and NASH Pathogenesis

项目概要/摘要 非酒精性脂肪性肝病（NAFLD）是慢性肝病最常见的原因， 占世界人口的四分之一，因此与NIH的使命高度相关。从脂肪变性到纤维化， 肝脏炎症驱动疾病进展，这项研究将阐明胆固醇的中心作用。 缺乏基于证据的FDA批准的NASH治疗方案强调了进一步研究NASH的必要性。 研究了解疾病的发病机制和确定潜在的目标。我们令人信服的初步证据 最近发表的研究结果表明，先天性免疫细胞（中性粒细胞和巨噬细胞）是 NAFLD的发病机制和进展。这一提议的首要假设是， 先天免疫和炎症是NAFLD的主要，不可或缺的特征，主要由 胆固醇我们将通过完成三个并行执行的特定目标来测试这一假设， 小鼠NAFLD模型和来自具有不同严重程度NASH的患者的样品。在SA 1中，PI将 全面表征来自不同阶段NASH患者的新鲜外周中性粒细胞。这将 包括NETosis、胆固醇诱导NET如何许可来自巨噬细胞的应答以及 中性粒细胞对肝内皮细胞的影响。鼠模型将集中于靶向NET以减轻肝损伤 在NASH。我们将使用FPC饮食，富含果糖，棕榈酸，反式脂肪和胆固醇（1.2%重量）， 已显示在16周内诱导肝纤维化相关参数。SA 2将使用喂食 通过超声引导细针抽吸分离肝组织， 通过单细胞RNA测序（scRNA-seq）测量巨噬细胞的全基因组表达， 进展或消退饮食诱导的NASH。我们的数据表明，在高胆固醇人群中， 饮食下调巨噬细胞特异性转录早在4周。拟议的实验计划将 为膳食胆固醇如何影响不同的肝脏巨噬细胞群体提供了前所未有的见解。 我们还将描述不同的肝巨噬细胞对共培养的肝巨噬细胞的功能影响。 星状细胞（HSC）。此外，我们将使用巨噬细胞基因启动子区域的靶向测序， 以确定表观遗传调控中的关键适应性变化。最后，在SA 3中，PI将重点关注集成 这些强大的分析和利用Cre-Lox系统产生具有骨髓特异性靶向突变体的小鼠， 基因.我们发现，骨桥蛋白（OPN）编码的SPP 1表达被强烈诱导， 我们将测试OPN纳米颗粒在预防和逆转研究饮食诱导的NASH巨噬细胞， 纳什携带Col 1a 1-GFP转基因的小鼠将用于直接评估不同治疗剂的作用。 HSC的活化、分化和纤维化反应的策略。综合起来，我们的多学科 方法和专家的杰出协同作用有可能从根本上改变我们对 胆固醇如何影响NASH的先天免疫并使其失调，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.