Project 4 Title: Innate immunity and cell death in ALD

项目 4 名称：ALD 中的先天免疫和细胞死亡

• 批准号: 10056025
• 负责人: LAURA E. NAGY
• 金额: $ 27.75万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10056025
• 关键词: Affect; Alcohol abuse; Alcoholic Liver Diseases; Alcohols; American; Apoptosis; Bone Marrow Transplantation; Breeding; Caspase; Cell Culture Techniques; Cell Death; Cells; Cessation of life; Clinical; Complex; Coupled; Data; Development; Differential Diagnosis; Disease; Environment; Enzyme-Linked Immunosorbent Assay; Ethanol; Family member; Functional disorder; Gap Junctions; Hepatic; Hepatocyte; High Fat Diet; Ice; Immune; Inflammasome; Inflammation; Inflammatory; Inflammatory Response; Injury; Knock-out; Lead; Liver; Liver diseases; Mediating; Messenger RNA; Methods; MicroRNAs; Mitogen-Activated Protein Kinases; Modeling; Molecular; Mus; Myeloid Cells; Natural Immunity; Organelles; Pathway interactions; Patients; Phosphorylation; Phosphotransferases; Plasma; Prevention; Process; Proteins; Proteome; Proteomics; RIPK1 gene; RIPK3 gene; Receptor-Interacting Serine/Threonine Protein Kinase 2; Regulation; Regulatory Pathway; Resolution; Sampling; Signal Transduction; Stress; Therapeutic; Tweens; Ubiquitin; Ubiquitination; alcohol exposure; base; biomarker identification; circulating biomarkers; in vivo; inhibition of autophagy; insight; liver biopsy; liver injury; mixed lineage kinase 3; mouse model; nonalcoholic steatohepatitis; novel; predictive modeling; protein protein interaction; receptor function; therapeutic development; tissue injury; treatment strategy; ubiquitin-protein ligase

ABSTRACT Eighteen million Americans abuse alcohol, with alcohol-associated liver disease (ALD) affecting over 10 million people. The development of ALD is a complex process involving both parenchymal and non-parenchymal cells in liver. The impact of ethanol on hepatocytes is characterized as a condition of “organelle stress” with multi- factorial changes in hepatocellular function, ultimately leading to hepatocellular death, including multiple path- ways of programmed cell death (apoptosis, necroptosis, pyroptosis, and ferroptosis). Enhanced hepatic inflam- mation during ethanol exposure is also an important contributor to injury and there is a strong relationship be- tween inflammation, cell death, and progression of ALD. Inflammation and cell death are both “double-edged” swords: depending on the pathophysiologic situation, they have deleterious and/or protective functions. Hepato- cyte death via caspase-dependent apoptosis is associated with progression of ALD. However, we discovered that receptor-interacting protein 3 (RIP3), a key protein in the necroptotic pathway of programmed cell death, drives ethanol-induced liver injury in a murine model (mALD) and that RIP3 expression is increased in liver of ALD patients. These novel findings indicate that the RIP3-dependent signaling network is critical to the progres- sion of ALD. Interestingly, we now find that mixed lineage kinase like (MLKL), the canonical downstream effector of RIP3, is not involved in mALD, suggesting that non-canonical functions of RIP3 mediate mALD. We also find that the RIP3-MLKL pathway differentially contributes to mALD vs. high-fat diet-induced liver injury (HFD). In HFD, RIP3 does not contribute to injury. Instead, MLKL mediates injury via an inhibition of autophagy in hepato- cytes. In order to leverage these new mechanistic insights into the pathophysiology of ALD into therapeutic strategies for treatment or prevention of ALD, we propose to 1) Leverage the differential contributions of RIP3 to mALD and HFD models to develop circulating biomarkers to distinguish ALD and NASH in patients, 2) RIP3 is a multifunctional protein that contains a kinase domain that phosphorylates MLKL and a RHIM domain involved in protein-protein interactions. We will use kinase-dead and RHIM-deleted mice, as well as cell specific knock- outs to identify the non-canonical and cell-specific mechanisms of RIP3 action in mALD and 3) Discover the post-translational mechanisms by which ethanol disrupts the RIP3 signaling network using non-targeted prote- omics, coupled with mouse and cell based targeted mechanistic approaches to determine if ethanol-induced changes in phosphorylation and ubiquitination lead to dysregulation of signaling in the RIP3 network. Impact: By understanding the regulation and interplay between inflammation and specific programmed cell death path- ways within the hepatocellular environment, our results will inform development of therapeutic strategies for the treatment of ALD.

摘要 1800万美国人酗酒，与酒精相关的肝病(ALD)影响着1000多万人 人民。肌萎缩侧索硬化症的发生是一个涉及实质和非实质细胞的复杂过程 在肝脏里。乙醇对肝细胞的影响是一种细胞器应激状态，具有多细胞毒性。 肝细胞功能的因子变化，最终导致肝细胞死亡，包括多途径- 程序性细胞死亡的方式(细胞凋亡、坏死性下垂、下垂和铁性下垂)。增生性肝炎- 酒精暴露期间的信息也是导致伤害的一个重要因素，与 炎症、细胞死亡和ALD进展之间的关系。炎症和细胞死亡都是“双刃剑” 剑：根据病理生理状况，它们具有有害和/或保护功能。希帕托- Caspase依赖的细胞死亡与ALD的进展有关。然而，我们发现， 受体相互作用蛋白3(RIP3)是细胞程序性死亡的坏死性途径中的关键蛋白， 在小鼠模型(MALD)中驱动乙醇诱导的肝损伤，RIP3在小鼠肝脏中的表达增加 ALD患者。这些新的发现表明，依赖于RIP3的信号网络对这一进程至关重要。 阿尔德的锡恩。有趣的是，我们现在发现典型的下游效应器(MLKL)之类的混合谱系激酶 RIP3的功能不参与MALD，提示RIP3的非规范功能介导了MALD。我们还发现 RIP3-MLKL通路在MALD和高脂饮食诱导的肝损伤(HFD)中的作用不同。在……里面 HFD、RIP3不会导致损伤。取而代之的是，MLKL通过抑制肝脏中的自噬来调节损伤。 细胞。为了将这些对ALD的病理生理学的新的机械学见解转化为治疗 对于ALD的治疗或预防策略，我们建议1)利用RIP3的不同贡献来 MALD和HFD模型开发循环生物标记物来区分ALD和NASH患者，2)RIP3是 一种多功能蛋白质，含有使MLKL磷酸化的激酶域和参与其中的Rhim结构域 在蛋白质-蛋白质相互作用中。我们将使用激酶死亡和Rhim缺失的小鼠，以及细胞特异的敲打- OUTS以确定MALD中RIP3作用的非规范和细胞特异性机制和3)发现 乙醇通过非靶向蛋白干扰RIP3信号网络的翻译后机制 组学，结合基于小鼠和细胞的靶向机制方法来确定乙醇诱导的 磷酸化和泛素化的变化导致RIP3网络中信号的失调。影响： 通过了解炎症和特定的程序性细胞死亡路径之间的调节和相互作用- 在肝细胞环境中，我们的结果将为开发治疗策略提供信息 ALD的治疗。