Project 4 Title: Innate immunity and cell death in ALD

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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.