Society: AASLD

Background: Alcoholic hepatitis (AH) is a life-threatening form of alcohol-associated liver disease (ALD), with no effective therapy. Liver neutrophil infiltration is a hallmark of AH, yet the mechanisms by which alcohol promotes neutrophil recruitment and disrupts neutrophil functions remain elusive. Identifying new therapeutic targets to reduce neutrophil-mediated liver damage is essential. Bruton’s tyrosine kinase (BTK), a non-receptor tyrosine kinase, plays an important role in neutrophil development and function, however, the mechanism by which BTK regulates neutrophil-mediated liver damage, specifically in AH, is not yet studied.
Methods: Female, C57BL6 mice aged 10-12 weeks received Lieber-deCarli diet with alcohol for 10 days followed by one binge (alcoholic hepatitis-AH-model) or calorie-matched control diet. Some mice received oral gavage of Evobrutinib, a BTK inhibitor (BTKi) or DMSO from day7 to day10. Liver damage was assessed by measuring serum ALT. Serum levels of proinflammatory cytokines were measured by ELISA. BTK phosphorylation (pBTK) was assessed in human or mouse livers by western blotting, and in bone marrow (BM) progenitors or liver neutrophils by flow cytometry. Circulating neutrophils from healthy controls (HC) and AH patients were analyzed for BTK expression by RNA-seq. Tandem mass spectrometry (LC-MS/MS) was performed in mouse liver lysates to identify BTK substrates.
Results: Our results from unbiased RNA-seq in circulating neutrophils showed a significant increase in BTK in AH patients compared to HC. Consistently, pBTK was significantly increased in neutrophils from AH patients compared to HC. We found that pBTK (Y223 and Y551) was upregulated in humans as well as in mouse livers with ALD. We further dissected that acute alcohol binge rather than chronic moderate alcohol use triggers pBTK in mice. In vitro, physiologically relevant doses of alcohol induced rapid pBTK induction in neutrophils that was partially TLR4-mediated. In a preclinical model of AH, administration of Evobrutinib, a small molecule BTK inhibitor, significantly decreased pro-inflammatory cytokines levels, and attenuated liver damage. Importantly, we discovered that alcohol-induced liver neutrophil infiltration correlated with the expansion of BM granulopoiesis, and that pBTK in BM progenitors was essential for alcohol-induced granulopoiesis. In vivo, BTK inhibition significantly reduced granulopoiesis, circulating neutrophils, and liver infiltration in the presence of alcohol administration. Mechanistically, using LC-MS/MS we discovered CD84 as a novel kinase target of BTK which is involved in granulopoiesis.
Conclusion: Our findings define a novel role of BTK in regulating CD84 phosphorylation and granulopoiesis that could be harnessed to address pressing therapeutic needs in AH.

John Asara

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