Plasma multi-omics outlines association of urobilinogen with corticosteroid non-response, inflammation and leaky gut in Sever Alcoholic Hepatitis

Abstract

ABSTRACTBackground and AimsSevere alcoholic hepatitis (SAH) has a high mortality and corticosteroid therapy is effective in 60% patients. Reliable indicators of response to therapy and mortality in SAH are needed. A total of 223 SAH patients, 70 in derivative [50 responders (R) and 20 non-responders (NR)] and 153 in validation cohort [136R, 17NR] were subjected to plasma metabolic/meta-proteomic analysis using UHPLC-HRMS and validated using Machine-Learning (ML). Temporal metabolic changes were assessed using Weighted Metabolome Correlation Network Analysis (WMCNA). Functionality (inflammatory-nature, effect on membrane integrity and glucocorticoid receptor) of non-response indicator was assessedin-vitroon primary healthy neutrophils or mice enterocytes. Baseline plasma metabolomics and meta-proteomics clearly discriminated NR and showed significant increase in urobilinogen (3.6-fold), cholesterol sulfate (6.9-fold), Adenosine monophosphate (4.7-fold) and others (p<0.05, FC>1.5, FDR<0.01). Increase in alpha/beta diversity, biosynthesis of secondary metabolites was a characteristic feature of NR (p<0.05). NR were metabolically inactive however R showed temporal change in the metabolite expression post-corticosteroid therapy (p<0.05). Plasma urobilinogen predicted non-response [AUC=0.94] with a hazard-ratio of 1.5(1.2-1.6) and cut-off >0.07mg/ml segregated non-survivors (p<0.01) and showed >98% accuracy using ML. Plasma urobilinogen directly correlated with circulating bacterial peptides linked to bilirubin to urobilinogen metabolising bacteria (r2>0.7;p<0.05). Urobilinogen inducedneutrophil activation,oxidative-stressandpro-inflammatory cytokines (CXCR1, NGAL, NOXO1, NOX4, IL15, TNFα and others, p<0.05), promotedcorticosteroid resistanceby increasing the expression of GR-Beta and trans-repression genes under GR-alpha (inflammatory-NFkB, MAPK-MAP) and reducing GR-alpha, and transactivation (anti-inflammatory) gene levels. Urobilinogen also promoted leaky gut by deregulating intestinal membrane junction proteins.ConclusionPlasma metabolome/meta-proteome can stratify pre-therapy steroid response. Increase in plasma Urobilinogen pedals a vicious cycle of bacterial translocation and increase in inflammation and corticosteroid non-response in SAH patients.