Pathobiology of Liver Injury

项目摘要 我的长期职业目标是明确非酒精性肝炎患者的肝脏炎症机制。 脂肪性肝炎(NASH)，美国最常见的慢性肝病。纳什是 以内质网(ER)应激为特征，导致内质网应激传感器肌醇激活 由于肝细胞内有毒脂质的积累，需要酶-1α(IRE1α)。巨噬细胞- 与循环髓系细胞重新进入肝脏相关的介导性肝脏炎症也是关键。 在纳什。目前的建议将肝细胞衍生的脂质介质与巨噬细胞介导的炎症联系起来。 通过提出来自脂毒性肝细胞的细胞外小泡(EV)将巨噬细胞招募到肝脏， 导致肝脏损伤和炎症。在初步实验中，我们观察到脂毒性 肝细胞(用游离脂肪酸棕榈酸酯处理)释放神经酰胺富含的促炎EV IRE1α依赖方式。神经酰胺衍生的1-磷酸鞘氨醇(S1P)在这些EVS上激活其 巨噬细胞上的受体S1P1可能促进巨噬细胞向肝脏的趋化作用。这导致了 肝细胞IRE1α调控PA诱导EV的生物发生、释放和脂质运输的中心假说 (神经酰胺和神经酰胺衍生的S1P)积聚，进而将巨噬细胞吸引到肝脏 促进NASH发病机制。因此，这项建议的目标是理解：i)IRE1α如何 介导富含神经酰胺的EVS的释放；II)神经酰胺衍生的脂质介质S1P如何在PA- 受刺激的EVS将巨噬细胞招募到肝脏；以及III)脂毒性EV的产生和信号是否具有靶向性 在体内能减少肝脏炎症吗？拟议中的实验将在体外和体内使用互补的 脂肪毒性和NASH的活体模型；以及药理学、分子和遗传学方法 提出三个综合假设。首先，我们将直接检验棕榈酸酯诱导ER的假设 应激通过a)IRE1α激活的转录因子X-BOX驱动神经酰胺的生物合成导致EV的释放 结合蛋白-1上调神经酰胺生物合成调节酶丝氨酸 棕榈酰基转移酶1(SPT1)，以及b)神经酰胺通过神经酰胺向多囊体转移 运输蛋白STARD11。其次，我们将直接检验脂毒性电动汽车上的S1P激活的假设 巨噬细胞趋化作用：a)鞘氨醇激酶2在PA-1上形成S1P，隔室产生S1P； B)EVS上的S1P通过S1P1受体激活巨噬细胞的趋化作用。第三，我们将直接 在NASH小鼠模型中测试阻断EV释放或信号转导在体内有益的假设 IRE1α肝细胞特异性基因敲除小鼠减少肠道病毒释放，以及b)遗传和药理学 S1P信号对巨噬细胞的抑制作用。这份R01拨款申请由目前的K08提前批出 阶段性研究人员将对NASH中巨噬细胞募集的过程产生机械性见解，从而 确定潜在的可用药靶点，例如IRE1α、Spt或S1P1受体的抑制剂。

PROJECT ABSTRACT My long term career objective is to define the mechanisms of liver inflammation in nonalcoholic steatohepatitis (NASH), the most-prevalent chronic liver disease in the United States of America. NASH is characterized by endoplasmic reticulum (ER) stress, which results in activation of the ER stress sensor Inositol Requiring Enzyme-1 alpha (IRE1α), due to the accumulation of toxic lipids within hepatocytes. Macrophage- mediated liver inflammation associated with recruitment of circulating myeloid cells into the liver is also pivotal in NASH. The current proposal links hepatocyte-derived lipid mediators to macrophage-mediated inflammation by proposing that extracellular vesicles (EVs) from lipotoxic hepatocytes recruit macrophages to the liver, resulting in liver injury and inflammation. In preliminary experiments we have observed that lipotoxic hepatocytes (treated with the free fatty acid palmitate) release ceramide-enriched proinflammatory EVs in an IRE1α-dependent manner. Sphingosine 1-phosphate (S1P), derived from ceramide, on these EVs activates its receptor S1P1 on macrophages, which may promote macrophage chemotaxis into the liver. This has led to the central hypothesis that hepatocyte IRE1α regulates PA-induced EV biogenesis, release and lipid cargo (ceramide and ceramide-derived S1P) accumulation, which in turn attracts macrophages into the liver promoting NASH pathogenesis. Therefore, the goals of this proposal are to understand: i) how IRE1α mediates release of ceramide-enriched EVs; ii) how the ceramide-derived lipid mediator, S1P, on PA- stimulated EVs recruits macrophages to the liver; and iii) can lipotoxic EV production and signaling be targeted in vivo to decrease liver inflammation? The proposed experiments will employ complementary in vitro and in vivo models of lipotoxicity and NASH, respectively; and pharmacological, molecular and genetic approaches to address three integrated hypotheses. First we will directly test the hypothesis that palmitate-induced ER stress drives ceramide biosynthesis leading to EV release by a) the IRE1α-activated transcription factor, X-box binding protein-1 (XBP1) upregulation of the ceramide biosynthesis regulating enzyme serine palmitoyltransferase 1 (SPT1), and b) the transfer of ceramide to multivesicular bodies via the ceramide transport protein STARD11. Second we will directly test the hypothesis that S1P on lipotoxic EVs activates macrophage chemotaxis by a) compartmental generation of S1P by sphingosine kinase 2 forming S1P on PA- induced EVs, and b) S1P on EVs activates macrophage chemotaxis via S1P1 receptor. Third we will directly test the hypothesis that interrupting EV release or signaling is salutary in vivo in a NASH mouse model by a) reduction of EV release by IRE1α hepatocyte-specific knockout mice, and b) genetic and pharmacologic inhibition of S1P signaling on macrophages. This R01 grant application by a current K08 awarded early stage investigator will yield mechanistic insights into the processes of macrophage recruitment in NASH, thus identifying potentially druggable targets, e.g., inhibitors of IRE1α, SPT or S1P1 receptor.