ABSTRACT Liver immune cell activation is central to alcohol associated liver disease (ALD) and is poorly understood. The protein homeostasis network/pathways are crucial in safeguarding the cellular proteome from cellular stress. Amongst these, proteostasis mediator HSP90 is widely studied and extensively explored as a therapeutic target in cancer. We reported two isoforms of HSP90, HSP90AA1 (stress inducible cytoplasmic HSP90) and HSP90B1/GP96 (Endoplasmic reticulum paralog of HSP90) are important in ALD. These studies point to a pathophysiological role for HSP90 in ALD. Studies during the last funding period of this project established that liver targeting of HSP90 inhibitor 17-DMAG using nanoparticles in ALD can prevent and reverse liver injury, GP96/HSP90B1 deficiency in macrophages facilitates resolution of liver inflammation and ALD, M-TLR4 deficient mice are not protected from ALD, inhibition of HSP90 reduced NLRP3 inflammasome activity decreasing IL-1β protein and HSF1 partially contributes to reduced pro-inflammatory signaling, whereas deficiency of HSF1 did not protect from ALD, suggesting that HSF1 does not contribute to resolution of ALD. In the next project period we will investigate mechanisms mediated by HSP90AA1 and HSP90B1/GP96 in alcohol induced liver macrophage activation resulting in ALD. We propose that HSP90 induces inflammation by alternate mechanisms (independent of HSP90/TLR4 axis), for instance macrophage aerobic glycolysis via epigenetic mechanisms or HIF1α. In our preliminary data, we found that HSP90AA1 is detected in the nucleus in ALD and its inhibition can modulate expression of chromatin-modifying enzymes. Pilot data also reveal that inhibition of HSP90AA1 reduces alcohol mediated induction of glucose transporter (SLC2a1/GLUT1) and hexokinase II, two important mediators of glycolysis, suggesting a role for HSP90 in metabolic programming of M1 macrophages in ALD. On the other hand, HSP90B1/GP96 induced preferably in liver macrophages promotes inflammation via glycolysis and its inhibition induces GRP78+ATF3+Trem2+ restorative/reparative macrophages resolving ALD. We hypothesize that alcohol induced HSP90AA1 facilitates macrophage aerobic glycolysis via epigenetic mechanisms whereas HSP90B1/GP96 and ER stress facilitate macrophage glycolysis via HIF1α in ALD. Inhibition of HSP90 in ALD induces ATF3+Trem2+ reparative macrophages using OXPHOS facilitating protection. The specific Aims of this proposal are - 1) To identify HSP90AA1 mediated epigenetic and metabolic programming of macrophages in murine ALD in vivo and human AH PBMCs in vitro. 2) To characterize the significance of macrophage-specific HSP90B1/GP96 in macrophage metabolism and assess significance of ATF3 and TREM2 in macrophages conferring protection from liver injury. Collectively, during the next project period our studies will identify novel metabolic and epigenetic mechanisms regulated by HSP90 and characterize reparative macrophages crucial in resol...