Project 4 Title: Innate immunity and cell death in ALD

摘要 1800万美国人滥用酒精，酒精相关性肝病（ALD）影响超过1000万人 人ALD的发生发展是一个复杂的过程，涉及实质细胞和非实质细胞 在肝脏中。乙醇对肝细胞的影响被描述为一种具有多方面影响的“细胞器应激”状态。 肝细胞功能的因素变化，最终导致肝细胞死亡，包括多途径- 程序性细胞死亡的方式（凋亡、坏死性凋亡、焦亡和铁亡）。增强的肝炎症- 乙醇暴露期间的信息也是损伤的重要因素，并且与以下因素有很强的关系： 炎症、细胞死亡和ALD的进展。炎症和细胞死亡都是“双刃剑” 剑：根据病理生理情况，它们具有有害和/或保护功能。肝- 通过半胱天冬酶依赖性凋亡的细胞死亡与ALD的进展相关。然而，我们发现， 受体相互作用蛋白3（RIP 3）是程序性细胞死亡的坏死性凋亡途径中的关键蛋白， 在小鼠模型（mALD）中驱动乙醇诱导的肝损伤，并且RIP 3表达在小鼠肝脏中增加。 酒精性痴呆患者。这些新的发现表明，RIP 3依赖的信号网络是至关重要的进展， 锡永ALD。有趣的是，我们现在发现混合谱系激酶样（MLKL），典型的下游效应子， 的RIP 3，是不参与mALD，这表明RIP 3的非典型功能介导mALD。我们还发现 RIP 3-MLKL通路对mALD与高脂饮食诱导的肝损伤（HFD）的贡献不同。在 HFD，RIP 3不会导致损伤。相反，MLKL通过抑制肝细胞自噬介导损伤。 细胞。为了将这些对ALD病理生理学的新机制见解用于治疗， 治疗或预防ALD的策略，我们建议1）利用RIP 3的差异贡献， mALD和HFD模型，以开发循环生物标志物来区分患者中的ALD和NASH，2）RIP 3是 一种多功能蛋白质，含有磷酸化MLKL的激酶结构域和相关的RHIM结构域 在蛋白质相互作用中。我们将使用激酶死亡和RHIM缺失的小鼠，以及细胞特异性敲除- 找出RIP 3在mALD中作用的非经典和细胞特异性机制，以及3）发现 翻译后机制，乙醇破坏RIP 3信号网络使用非靶向蛋白， 组学，再加上小鼠和细胞为基础的靶向机制的方法，以确定是否乙醇诱导 磷酸化和泛素化的变化导致RIP 3网络中信号传导的失调。影响： 通过了解炎症和特定程序性细胞死亡途径之间的调节和相互作用- 在肝细胞环境中的方式，我们的研究结果将为治疗策略的发展提供信息。 治疗ALD。

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.