Organelle Dysfunction in Liver Inflammation

肝脏炎症中的细胞器功能障碍

• 批准号: 9204405
• 负责人: Harmeet Malhi
• 金额: $ 7.95万
• 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: 2016-01-15 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9204405
• 关键词: Address; Anabolism; Award; Biogenesis; Biological Assay; Cartoons; Cell Communication; Cells; Ceramides; Chemicals; Chemotaxis; Clinical Investigator Award; Data; Development; Disease Progression; Endosomes; Enzymes; Evolution; Factor X; Fatty Liver; Foundations; Functional disorder; Funding; Future; Genetic Transcription; Goals; Grant; Hepatic; Hepatocyte; Immune; In Vitro; Inflammation; Inflammatory; Injury; Inositol; Lead; Link; Lipids; Liver; Liver diseases; Macrophage Activation; Mass Spectrum Analysis; Measurement; Mediating; Mediator of activation protein; Mentors; Modeling; Molecular Genetics; Motion; Mus; Nonesterified Fatty Acids; Obesity; Organelles; Palmitates; Pathogenesis; Pathway interactions; Pharmacology; Process; Production; Proteins; Public Health; Recruitment Activity; Regulation; Research; Research Proposals; Rodent Model; Role; Signal Transduction; Specificity; Sphingolipids; Stress; Systems Biology; Testing; Transcriptional Regulation; Up-Regulation; Vesicle; Work; base; bindin; career; endoplasmic reticulum stress; experimental study; extracellular vesicles; feeding; genetic approach; immune activation; in vitro Model; in vivo; in vivo Model; inhibitor/antagonist; injured; insight; liver inflammation; macrophage; metabolomics; nonalcoholic steatohepatitis; novel therapeutic intervention; public health relevance; response; sensor; targeted treatment; transcription factor; vesicular release

 DESCRIPTION (provided by applicant): My long term career objective is to define the mechanisms of liver inflammation in nonalcoholic steatohepatitis (NASH). The current proposal views this via a systems biology approach, focusing on defining the cross-talk between lipotoxic hepatocytes and proinflammatory macrophages at a cellular level; and the endoplasmic reticulum stress sensor Inositol Requiring Enzyme-1 alpha (IRE1α) and extracellular vesicle (EV) biogenesis at a sub-cellular level. Signals from injured hepatocytes serve to recruit macrophages to the liver, activate proinflammatory pathways in resident and recruited macrophages and thus set in motion an inflammation-injury feed-forward loop. In preliminary experiments we have observed that lipotoxic hepatocytes (treated with the toxic free fatty acid palmitate) release proinflammatory extracellular vesicles; these vesicles are released in an IRE1α-dependent manner; and are enriched in ceramides. Furthermore, the ceramide enrichment also occurs in an IRE1α-dependent manner. Our preliminary observations have led to the central hypothesis that IRE1α regulates palmitate-induced extracellular vesicle biogenesis and cargo, thereby leading to the release of proinflammatory extracellular vesicles, which in turn activate macrophages thus promoting NASH pathogenesis. Therefore, the goals of this proposal are to understand: i) how IRE1 mediates release of lipotoxic EVs; ii) what imparts specificity t lipotoxic EV cargo, and iii) how are lipotoxic EVs proinflammatory? The proposed experiments will employ complementary in vitro and in vivo models of lipotoxicity and NASH, respectively; and chemical, pharmacological, molecular and genetic approaches to address the specific aims to test the hypotheses that: i) IRE1α and the IRE1α-activated transcription factor, X- box bindin protein-1 (XBP-1) drive the release of lipotoxic hepatocyte EVs via increased biogenesis of ceramides, and ii) IRE1α-mediated ceramide transport is necessary for EV release, and iii) IRE1α-dependent proinflammatory EV release in vivo leads to macrophage recruitment and liver inflammation. To address these hypotheses the applicant has become adept at EV isolation and characterization, macrophage isolation, assays of macrophage activation and chemotaxis, transcriptional regulation, metabolomics by mass spectrometry and in vivo rodent models of conditional deletion of IRE1. With funding through this R03 small grant program for K08 awardees the applicant hopes to gain additional preliminary data to propel this research proposal to an R01 proposal in the near future. This work represents a coherent and logical extension of the applicant's earlier work on endoplasmic reticulum stress in the pathogenesis of NASH and represents a significant step forward in independence from previous research mentors. The applicant has established a network consisting of Dr. Gregory J. Gores as her primary mentor, and Dr. Vijay Shah and Dr. Nicholas LaRusso as collaborators. Our results will yield mechanistic insights into the processes of macrophage activation and recruitment in NASH, thus identifying potentially druggable targets.

 描述（由申请人提供）：我的长期职业目标是确定非酒精性脂肪性肝炎（NASH）肝脏炎症的机制。目前的提案通过系统生物学方法来看待这一点，重点是在细胞水平上定义脂毒性肝细胞和促炎巨噬细胞之间的串扰;以及在亚细胞水平上定义内质网应激传感器肌醇需要酶-1 α（IRE 1 α）和细胞外囊泡（EV）生物发生。来自受损肝细胞的信号用于将巨噬细胞募集到肝脏，激活驻留和募集的巨噬细胞中的促炎途径，从而启动炎症-损伤前馈回路。在初步实验中，我们观察到脂毒性肝细胞（用毒性游离脂肪酸棕榈酸酯处理）释放促炎细胞外囊泡;这些囊泡以IRE 1 α依赖性方式释放;并富含神经酰胺。此外，神经酰胺富集也以IRE 1 α依赖性方式发生。我们的初步观察导致了一个中心假设，即IRE 1 α调节棕榈酸诱导的细胞外囊泡生物发生和货物，从而导致促炎细胞外囊泡的释放，这反过来又激活巨噬细胞，从而促进NASH发病机制。因此，本提案的目标是理解：i）IRE 1如何介导脂毒性EV的释放; ii）是什么赋予脂毒性EV货物特异性，以及iii）脂毒性EV如何促炎？拟议的实验将分别采用脂毒性和NASH的补充体外和体内模型;以及化学、药理学、分子和遗传学方法来解决特定目标，以测试以下假设：i）IRE 1 α和IRE 1 α激活的转录因子X盒结合蛋白-1（XBP-1）通过增加神经酰胺的生物合成来驱动脂毒性肝细胞EV的释放，和ii）IRE 1 α介导的神经酰胺转运是EV释放所必需的，和iii）体内IRE 1 α依赖性促炎EV释放导致巨噬细胞募集和肝脏炎症。为了解决这些假设，申请人已经变得擅长EV分离和表征、巨噬细胞分离、巨噬细胞活化和趋化性测定、转录调节、通过质谱法的代谢组学和IRE 1 β条件性缺失的体内啮齿动物模型。通过为K 08获奖者提供的R 03小额资助计划，申请人希望获得更多的初步数据，以推动这项研究提案在不久的将来成为R 01提案。这项工作代表了申请人早期关于NASH发病机制中内质网应激的工作的连贯和逻辑延伸，并代表了独立于先前研究导师的重要一步。申请人已经建立了一个网络，由Gregory J. Gores博士作为她的主要导师，Vijay Shah博士和Nicholas LaRusso博士作为合作者。我们的研究结果将产生对NASH中巨噬细胞激活和募集过程的机制性见解，从而确定潜在的药物靶点。