Investigating the Role of the C5 complosome in Ethanol-induced Immunometabolic Dysregulation of Kupffer Cells in Alcohol-associated Liver Disease - PROJECT SUMMARY Alcohol misuse is a leading cause of liver-related mortality. Incidence and mortality rates of alcohol-associated liver disease (ALD) are increasing, yet there are no effective therapeutics to prevent disease progression and identification of new targets for intervention is essential. Alcohol misuse causes leaky gut. Translocation of gut- derived microbial byproducts [lipopolysaccharide (LPS)] to the liver are important contributors to chronic inflammation and associated liver damage during ALD. Alcohol misuse impacts innate immunity, including complement, and chronic exposure can dysregulate homeostatic immune processes leading to pronounced hepatic inflammation. The resident macrophage of the liver, Kupffer Cells (KCs) are recognized as initiators of ethanol-induced inflammation and key drivers of ALD progression yet the mechanisms underlying KC dysfunction in the liver are unclear. Indeed, ethanol can directly impact KC function by enhancing LPS-induced pro-inflammatory responses (i.e. cytokine IL-1β), leading to hyperactivated phenotypes that perpetuate non- resolving hepatic inflammation. Our preliminary data suggests that ethanol impacts intracellular complement activation (complosome) and increases the localization of a GPCR, complement component 5a receptor 1 (C5aR1) to the mitochondria in KCs. Further, we show that mitochondrial C5aR1 (mtC5aR1) contributes to LPS- and ATP-induced NLRP3 inflammasome activation and IL-1β secretion in vitro. Despite these findings, there remain gaps in our understanding related to the specific role of the complosome as a critical driver of chronic inflammation in the liver. The goal of this proposal is to define novel mechanisms of alcohol-induced metabolic reprogramming of KCs to identify specific, targetable factors amplifying pathogen sensing in ALD. While the complosome has been implicated in cellular metabolism in immune cells, there are no current studies exploring complosome activation in the liver disease of any etiology. It is our working hypothesis that ethanol-induced mtC5aR1 localization is an important mechanism of ethanol priming and hyperactivation in KCs. Specifically, we hypothesize that the C5 complosome mediates ethanol-induced mitochondrial reactive species (mtROS) production and suppression of oxidative phosphorylation, leading to activation of the NLRP3 inflammasome and enhanced IL-1β secretion. Making use of CRISPR-generated C5aR1-/- and C5-/- immortalized mouse KCs (ImKCs) and several C5aR1 antagonists, in Aim 1 we will define if the C5 complosome is required for ethanol-induced mtROS production and immunometabolic reprogramming using in vitro and ex vivo exposure paradigms. In Aim 2, we will determine if KC-mediated liver injury and hepatic NLRP3 inflammasome activation is C5aR1-dependent using a mouse model of ALD and cell-specific C5aR1 deficient mice. In vitro we will elucidate if mtC5aR1 activation is specifically required for KC secretion of IL-1β. Taken together, these studies will reveal whether the complosome coordinates alcohol-mediated chronic inflammation and associated hepatic damage and potentially identify new therapeutic targets to prevent/or treat liver disease.
