Innate immune dysregulation in alcohol-associated liver disease - ABSTRACT Alcohol-associated hepatitis (AH) is the most severe manifestation of alcohol-associated liver disease that is characterized by hepatocyte dysfunction, systemic inflammation and poor clinical outcomes. We recently reported heterogeneity in neutrophils in AH with over-activated, high-density (HDN), and functionally exhausted, low density (LDN) neutrophil populations that contribute to liver damage. We hypothesize that neutrophil dysfunctions in AH are related to neutrophil heterogeneity and functional differences between neutrophil populations. We found that the alcohol-induced LDNs are generated in vivo in AH patients from HDNs after extracellular traps (NETs) release. However, the phenotype and function of these LDNs are yet to be delineated in AH. Efferocytosis, that is a process for removal of NETs, is decreased by alcohol and our preliminary data suggest that LDNs express high levels of CD47, a “do not eat me” signal and CD24, a signal that prevents cells to undergo apoptosis, permitting longevity of LDNs in AH. We hypothesize that alcohol-induced CD47 contributes to neutrophil’s longevity and immunosuppressive effects. Our preliminary results suggest that alcohol-induced neutrophil activation and NET release are associated with activation and phosphorylation (pBTK) of the Bruton’s tyrosine kinase (BTK). We postulate that pBTK is a molecular regulator of alcohol-induced NET release and LDN generation. Finally, we propose that neutrophil-specific inhibition of BTK will ameliorate neutrophil-mediated inflammation and liver damage. The Aims are #1: To explore neutrophil phenotypes through characterization of their transcriptomic profiles and functional roles in severe alcohol-associated hepatitis by a) single cell RNAseq in patients with AH; b) characterizing surface marker expression and function of neutrophil populations in AH; c) determine whether LDNs are a causal factor in AH or a consequence of AH. Aim#2 will test the role of CD47, a “do not eat me” signal, and CD24, a signal to prevent apoptosis, on neutrophil dysfunction, inflammation and liver damage in AH by assessing the effects of alcohol on CD47 and CD24 expression and function in circulating neutrophil populations and in the liver and by testing the effect of CD47 deficiency or administration of anti- CD47 and/or anti-CD24 blocking antibodies on neutrophil heterogeneity, function and liver damage in vivo in a preclinical model of AH. Aim#3 will evaluate the role of alcohol-induced BTK phosphorylation in neutrophil dysfunction and explore BTK-targeting therapeutic interventions in a preclinical model of AH by a) defining the role of BTK activation and its interactions with STING/IRF3 in neutrophil heterogeneity, NET induction and LDN generation; b) testing the effect of neutrophil-specific deletion of BTK in a mouse model of AH. Our results will reveal unique heterogeneity of neutrophils in AH and provide better understanding of the controversial roles of neutrophils in AH. The proposed studies will provide novel insights into the role of BTK at in neutrophils and AH and define its interactions with other key signal transduction pathways that may identify BTK, CD47 or CD24 as new therapeutic targets for amelioration of AH.
