Pathobiology of Liver Injury

项目摘要 我的长期职业目标是确定非酒精性肝病患者肝脏炎症的机制。 脂肪性肝炎（NASH）是美国最流行的慢性肝病。nash是 其特征在于内质网（ER）应激，其导致ER应激传感器肌醇的激活 需要酶-1 α（IRE1 α），因为肝细胞内毒性脂质蓄积。巨噬细胞- 介导的肝脏炎症与循环髓样细胞进入肝脏的募集有关， 在NASH。目前的提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囊泡（EV）向肝脏募集巨噬细胞， 导致肝损伤和炎症。在初步的实验中，我们观察到， 肝细胞（用游离脂肪酸棕榈酸酯处理）在一个细胞内释放富含神经酰胺的促炎性EV。 IRE1 α依赖性方式。来自神经酰胺的鞘氨醇1-磷酸（S1P）在这些EV上激活其 受体S1P1，其可促进巨噬细胞趋化性进入肝脏。这导致 肝细胞IRE1 α调节PA诱导的EV生物合成、释放和脂质负荷的中心假设 （神经酰胺和神经酰胺衍生的S1P）积累，进而吸引巨噬细胞进入肝脏 促进NASH发病。因此，本提案的目标是了解：i）IRE1 α 介导富含神经酰胺的EV的释放; ii）神经酰胺衍生的脂质介体S1P如何在PA- 受刺激的EV将巨噬细胞募集到肝脏;以及iii）脂毒性EV产生和信号传导是否可以被靶向 在体内减少肝脏炎症？拟议的实验将采用互补的体外和体内 分别为脂毒性和NASH的体内模型;以及药理学、分子和遗传学方法， 提出三个综合假设。首先，我们将直接测试棕榈酸诱导的ER 应激驱动神经酰胺的生物合成，导致EV通过a）IRE1 α激活的转录因子X-box释放 结合蛋白-1（XBP1）上调神经酰胺生物合成调节酶丝氨酸 棕榈酰转移酶1（SPT 1），和B）通过神经酰胺将神经酰胺转移到多泡体 转运蛋白STARD 11。其次，我们将直接测试脂毒性EV上的S1P激活的假设， a）通过鞘氨醇激酶2在PA上形成S1P而隔室产生S1P， 诱导的EV，和B）EV上的S1 P通过S1 P1受体激活巨噬细胞趋化性。第三，直接 通过a）在NASH小鼠模型中测试中断EV释放或信号传导在体内是有益的假设 IRE1 α肝细胞特异性敲除小鼠EV释放的减少，和B）遗传和药理学 抑制巨噬细胞上的S1P信号传导。这R01赠款申请由目前的K08授予早期 阶段研究者将产生对NASH中巨噬细胞募集过程的机制性见解， 识别潜在的可用药靶点，例如，IRE1 α、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.