Characterization and Function of Neutrophils in Alcoholic Hepatitis in Humans and Mice: Roles for Neutrophilic p47phox in Disease Progression

Intrahepatic neutrophil infiltration has been implicated in the pathogenesis of severe alcoholic hepatitis (SAH), a disease with high short-term morality; however, how neutrophils contribute to SAH progression remain obscure. This study aimed to characterize intrahepatic neutrophil infiltration and its involvement in AH pathogenesis. We found that hepatic expression of neutrophil cytosolic factor 1 (NCF1), a key factor in controlling neutrophilic ROS production, was upregulated and correlated with neutrophil number, inflammation and ROS-associated genes in SAH patients. Ncf1 floxed mice were generated using CRISPR/Cas9 technology by inserting two LoxP sequences into intron 1 and intron 8 of the mouse Ncf1 gene, then crossed with Lyz Cre mice to generated myeloid cell-specific Ncf1 knockout (Ncf1Lyz-/-) mice. The LyzCre negative Ncf1 floxed mice were used as corresponding WT littermate control. The chronic-plus-binge ethanol feeding model was used in this study. The total RNAs were extracted from ETOH-fed 5 WT and 4 Ncf1Lyz-/- mouse livers and submitted for a Poly(A) RNA sequencing. Genetic deletion of the Ncf1 gene in neutrophils abolished hepatic ROS, inflammation, and fibrosis induced by ethanol feeding. RNA-sequencing analysis and the data from experimental models revealed that neutrophilic NCF1-dependent ROS promoted alcoholic liver injury by inhibiting AMP-activated protein kinase (a key regulator of lipid metabolism) and microRNA-223 (a key anti-inflammatory and anti-fibrotic microRNA). Our data suggest that divergent pathogeneses exist in SAH and neutrophilic NCF1-dependent ROS promotes alcoholic liver injury by inhibiting AMPK and microRNA-223. Mouse liver mRNA profiles of Etoh-fed wild type (WT) and myeloid cell-specific Ncf1 knockout (Ncf1-Lyz-/-; Ncf1-MKO) mice