Interleukin-6 Trans Signaling and STAT-3 Activation Promote Neutrophilic Inflammation in Alcohol-Associated Hepatitis
Alcohol associated hepatitis (AH) is characterized by neutrophilic inflammation leading to liver injury. Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates hepatic regeneration and hepatic acute phase responses. In the liver IL-6 can signal through either membrane-bound or soluble IL-6 receptors (IL-6R).Aims:to determine the role of IL-6 trans signaling in the pathogenesis of alcohol-associated hepatitis.Methods:Using human liver RNA-Seq, we compared expression of IL-6 signaling pathway components in 51 patients with no liver disease (N=10), early alcohol-related steatohepatitis (early ASH, N=12), non-severe AH (N=11) and severe AH (N=18). Ingenuity Pathway Analysis was used to determine upstream regulators of IL-6 receptor expression. For in vitro studies, HepG2 cells were cultured overnight with either TGF-ß1 (5 ng/ml) or 1% FBS, and STAT3 was activated with either 1) IL-6 to simulate classical signaling or 2) hyper-IL-6, a recombinant IL-6/IL-6Ra peptide that selectively activates trans-IL-6 signaling. RNA-Seq was performed on stimulated cells to define an IL-6 trans-signaling gene signature. This signature was used to define a patient subset with high IL-6TS activity (IL-6TS high). For in vivo studies, female mice 14-16 years old were treated with a 10 day chronic-plus binge ethanol liver injury model.Results:Human liver RNA-Seq data demonstrated declining IL-6R expression with progressively increasing severity, and pathway analysis of 3,513 differentially expressed genes identified TGF-ß1 as the strongest negative upstream regulator of IL-6R expression in AH. Despite reduced IL-6R expression, STAT3-dependent gene expression was enhanced in severe AH. In vitro, TGF-ß1 suppressed STAT3 phosphorylation by both classical and trans IL-6 signaling. However, only high-dose hyper-IL-6 (20 ng/ml) restored STAT3 activation, while TGF-ß1-mediated suppression of STAT3 persisted despite high-dose IL-6 (20 ng/ml) treatment. RNA-Seq of hyper-IL-6-stimulated hepatocytes was enriched in genes associated with neutrophil recruitment, and we identified a set of 11 co-clustered genes that characterized a subset of AH patients with enhanced expression of these IL-6TS-inducible transcripts (IL-6TS high). These patients exhibited increased intrahepatic neutrophilic infiltration on liver biopsies and enrichment of pathways associated with leukocyte migration on IPA analysis. In mice, ethanol treatment increased hepatic STAT3 activation despite reduced IL-6R and gp130 expression, and ethanol enhanced expression of neutrophil chemoattractants, includingIcam1,Lbp, andLcn2.Conclusion:Together, these findings suggest that trans IL-6 signaling preserves hepatocyte STAT3-dependent expression of neutrophil chemoattractants in AH. Future studies will examine if inhibiting IL-6 trans signaling abrogates neutrophilic inflammation in AH. Overall design: Using human liver RNA-Seq, we compared expression of IL-6 signaling pathway components in 51 patients no liver disease early alcohol-related steatohepatitis (early ASH, N=12), non-severe AH (N=11) and severe AH (N=18). Ingenuity Pathway Analysis was used to determine upstream regulators of IL-6 receptor expression. For in vitro studies, HepG2 cells were cultured overnight with either TGF-ß1 (5 ng/ml) or 1% FBS, and STAT3 was activated with either 1) IL-6 to simulate classical signaling or 2) hyper-IL-6, a recombinant IL-6/IL-6Ra peptide that selectively activates trans-IL-6 signaling. RNA-Seq was performed on stimulated cells to define an IL-6 trans-signaling gene signature.  This signature was then used to define a subset of patients with high IL-6TS activity (IL-6TS high). For in vivo studies, female mice 14-16 years old were treated with a 10 day chronic-plus binge ethanol model of alcohol-related liver injury, and liver tissues was assessed with Western Blot and qPCR.