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

Background and Aims Severe 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 assessed in-vitro on 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 induced neutrophil activation , oxidative-stress and pro-inflammatory cytokine s (CXCR1, NGAL, NOXO1, NOX4, IL15, TNFα and others, p<0.05), promoted corticosteroid resistance by 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. Conclusion Plasma 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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement Financial Support: The work was supported from project DST (DST-SERB) (EMR/1016/004819) ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: institute of liver and biliary science ethical committee provided ethical clearance I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data associated to the manuscript could be obtained from Dr jaswinder singh maras on request * SAH : Severe Alcoholic Hepatitis R : Responders NR : Non-responders DF : Discriminant Factor DEMs : Differentially Expressed Metabolites GR : Glucocorticoid Receptor H2DCFDA : 1’,7’-dichlorodihydrofluorescein diacetate MFI : Mean fluorescence intensity ML : Machine Learning MS : Mass Spectrometry PLS-DA : Partial Least Square Discriminant Analysis PHN : Primary Human Neutrophils PME : Primary Mice Enterocytes Pred : Prednisolone U : Urobilinogen WMCNA : Weighted Metabolome Correlation Network Analysis