Background and aims: Alcohol-associated hepatitis (AH) is the most life-threatening form of alcohol-associated liver disease (ALD). AH is characterized by severe inflammation attributed to increased levels of ethanol, microbes or microbial components, and damage-associated molecular pattern (DAMP) molecules in the liver. HSPB1 (Heat Shock Protein Family B (Small) Member 1; also known as Hsp25/27) is a DAMP that is rapidly increased in and released from cells experiencing stress, including hepatocytes. The goal of this study was to define the role of HSPB1 in AH pathophysiology. Methods: Serum HSPB1 was measured in a retrospective study of 184 heathy controls (HC), heavy alcohol consumers (HA), patients with alcohol-associated cirrhosis (AC), and patients with AH recruited from major hospital centers. HSPB1 was also retrospectively evaluated in liver tissue from 10 HC and AH patients and an existing liver RNA-seq dataset. Finally, HSPB1 was investigated in a murine Lieber-DeCarli diet model of early ALD as well as cellular models of ethanol stress in hepatocytes and hepatocyte-macrophage communication during ethanol stress. Results: Circulating HSPB1 was significantly increased in AH patients and levels positively correlated with disease-severity scores. Likewise, HSPB1 was increased in the liver of patients with severe AH and in the liver of ethanol-fed mice. In vitro, ethanol-stressed hepatocytes released HSPB1, which then triggered TNF α-mediated inflammation in macrophages. Anti-HSPB1 antibody prevented TNF α release from macrophages exposed to media conditioned by ethanol-stressed hepatocytes. Conclusions: Our findings support investigation of HSPB1 as both a biomarker and therapeutic target in ALD. Furthermore, this work demonstrates that anti-HSPB1 antibody is a rational approach to targeting HSPB1 with the potential to block inflammation and protect hepatocytes, without inactivating host defense.

The authors have declared no competing interest.

This study was funded by NIDDK 1K08DK114713 (JSM), NIAAA Northeast Ohio Alcohol Research Center Pilot and Feasibility Award (JSM), NIAAA Northeast Ohio Alcohol Research Center 5P50AA024333 (LEN), NIAAA Johns Hopkins Clinical Resource for Alcoholic Hepatitis Investigation 5R24AA025017.

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Study protocols were approved by the Institutional Review Boards (IRB) at the Cleveland Clinic and MetroHealth Hospitals. The IRB of Cleveland Clinic and MetroHealth Hospitals gave ethical approval for this work. All procedures were performed according to the IRB guidelines and all subjects provided written informed consent.

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