Pathobiology of Liver Injury

肝损伤的病理学

• 批准号: 9208922
• 负责人: Harmeet Malhi
• 金额: $ 35.78万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-23 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9208922
• 关键词: Address; Americas; Anabolism; Applications Grants; Attenuated; Award; Binding Proteins; Biogenesis; Biological Assay; Boxing; Carrier Proteins; Ceramides; Characteristics; Chemotaxis; Data; Development; Disease; Disease Progression; Enzymes; Factor X; Fatty Liver; Generations; Genetic; Genetic Transcription; Goals; Grant; Hepatic; Hepatocyte; Immune; In Vitro; Inflammation; Inflammatory; Inositol; Knockout Mice; Lead; Leukocytes; Link; Lipids; Liver; Liver diseases; Mass Spectrum Analysis; Mediating; Modeling; Molecular Genetics; Multivesicular Body; Mus; Myeloid Cells; Nonesterified Fatty Acids; Obesity; Palmitates; Pathogenesis; Pathway interactions; Process; Production; Proteins; Public Health; Recruitment Activity; Research; Research Personnel; Role; Signal Transduction; Sphingosine-1-Phosphate Receptor; Staging; Testing; United States; Up-Regulation; Vesicle; activating transcription factor; base; career; chronic liver disease; endoplasmic reticulum stress; exosome; extracellular vesicles; genetic approach; immune activation; in vitro Model; in vivo; in vivo Model; inhibitor/antagonist; insight; intercellular communication; lipid mediator; liver inflammation; liver injury; macrophage; mouse model; nonalcoholic steatohepatitis; novel therapeutic intervention; receptor; research study; sensor; serine palmitoyltransferase; sphingosine 1-phosphate; sphingosine kinase; steroidogenic acute regulatory protein; vesicular release

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.

项目摘要 我的长期职业目标是定义非酒精性肝注射机制 脂肪性肝炎（NASH），美利坚合众国最先进的慢性肝病。纳什是 内质网（ER）应力的特征，这导致ER应力传感器肌醇激活 由于肝细胞内有毒脂质的积累，需要酶-1α（IRE1α）。巨噬细胞 与募集循环髓样细胞募集到肝脏有关的介导的肝炎症也是关键 在纳什。当前的提案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 通过提出从脂肪毒性肝细胞募集巨噬细胞到肝脏的细胞外蔬菜（EV）， 导致肝损伤和感染。在初步实验中，我们观察到脂毒 肝细胞（用游离脂肪酸棕榈酸酯处理）释放富含神经酰胺的促炎性电动汽车 IRE1α依赖性方式。源自神经酰胺的1-磷酸盐（S1P）在这些EV上激活其 巨噬细胞上的受体S1P1，这可能会促进巨噬细胞趋化性。这导致了 肝细胞IRE1α调节PA诱导的EV生物发生，释放和脂质货物的中心假设 （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 促进纳什发病机理。因此，该提议的目标是理解：i）IRE1α如何 调解富含神经酰胺的电动汽车的释放； ii）pa-上如何使用神经酰胺衍生的脂质介质S1P 刺激的电动汽车将巨噬细胞招募到肝脏； iii）可以针对脂肪毒性EV的产生和信号 体内减少肝脏炎症？提出的实验将在体外和 脂肪毒性和NASH的体内模型；以及药物，分子和遗传学方法 解决三个综合假设。首先，我们将直接检验棕榈酸酯诱导的ER的假设 应力驱动神经酰胺的生物合成，从而导致A）IRE1α激活转录因子X-Box释放EV 结合蛋白-1（XBP1）神经酰胺生物合成调节酶序列的上调 棕榈酰转移酶1（SPT1），b）通过神经酰胺将神经酰胺转移到多层体内 传输蛋白星11。其次，我们将直接检验脂肪毒性EV上S1P激活的假设 通过a）通过鞘氨酸激酶2在Pa-上形成S1P的巨噬细胞趋化性 诱导的电动汽车和电动汽车上的b）S1P通过S1P1受体激活巨噬细胞趋化性。第三我们将直接 测试一个假设，即通过A中断EV释放或信号传导在NASH小鼠模型中是有益的。 减少IRE1α肝细胞特异性敲除小鼠的EV释放，b）遗传和药理 S1P信号在巨噬细胞上的抑制。当前K08提早授予的R01赠款申请 阶段研究者将对纳什巨噬细胞募集过程产生机械洞察力，因此 鉴定潜在的可药物靶标，例如IRE1α，SPT或S1P1受体的抑制剂。