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

Abstract

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 cytokines (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

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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).

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I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

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Data Availability

Data associated to the manuscript could be obtained from Dr jaswinder singh maras on request

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