Katyal, A. | Search bioRxiv

bioRxiv2025

A Triple-Hit Alcoholic Liver Disease Model Linking High-Fat Diet, Endotoxins, and Neo-Antigen Formation

BACKGROUNDExisting rodent models of alcoholic liver disease (ALD) often fail to replicate its full clinical progression--from steatosis to alcoholic hepatitis and ultimately cirrhosis--and overlook key metabolic contributors. Clinically, high-fat diets and gut-derived endotoxins (from enteric dysbiosis) act as additional "hits" that exacerbate alcohol-induced liver injury. Moreover, alcohol metabolism generates oxidative stress and modifies self-proteins into neo-antigens, potentially triggering immune responses. A model that incorporates these elements is critical for studying ALD pathogenesis and its immunological aspects. METHODSMale Wistar rats were randomized into four groups. The AL group received alcohol (36% caloric equivalent) and a single LPS dose (1 {micro}g/kg) 24 hours before sacrifice. The AF group was fed a high-fat diet (35% caloric equivalent) for 15 days, then maintained on alcohol and fat. The ALF group was similarly primed with fat, maintained on alcohol and fat, and given LPS prior to sacrifice. The ICC group served as isocaloric controls. Liver injury was assessed via histology, biochemical markers (AST, ALT, MDA), and serum endotoxin levels. Antibody titers against liver proteins were analyzed by ELISA; neo-antigens were identified using proteomics. RESULTSAll treated groups showed elevated liver enzymes, MDA, and endotoxin levels, indicating progressive liver damage. The ALF group exhibited the most severe pathology within six weeks, progressing from steatosis to steatohepatitis and fibrosis. In contrast, the AF group showed steatosis alone, and the AL group displayed moderate inflammation. Neo-antigens were most abundant in the ALF group. CONCLUSIONWe present a novel, rapid-onset triple-hit model of ALD that mimics the diseases full spectrum. This model enables mechanistic studies on how diet and gut-derived endotoxins contribute to ALD and highlights the immunogenic potential of alcohol-induced neo-antigens.

immunology↗

bioRxiv2025

Chenodeoxycholic Acid Modulation via Bacteroides intestinalis AM1 underscores a Novel Approach in Acute Liver Failure

BackgroundAcute liver failure (ALF) is associated with rapid and progressive hepatocellular injury, and severe metabolic-microbial derangements. We investigated early metabolic markers of non-survival, and a potential microbial intervention using Bacteroides intestinalis-AM1, to improve outcomes in ALF. MethodPlasma metabolomics and meta-proteomics were performed in 40 ALF patients and 5 healthy controls (training cohort). A non-survival marker panel was identified and validated in 270 ALF patients (test cohort) using high resolution mass spectrometry and machine learning. It was functionally validated in acetaminophen-induced ALF mouse model. B. intestinalis-AM1 was used to study alteration of gut bacteria and amelioration of liver injury. ResultsALF non-survivors showed a distinct metabolomic signature with elevated primary bile acids {chenodeoxycholic acid (CDCA), cholic acid (CA)}, tryptophan, tyrosine, and enrichment of pathways linked to inflammation, cell death, and stress response (p<0.01, FDR<0.01, FC>1.5). Non-survivors had higher alpha/beta diversity (p<0.05) with increase in Proteobacteria, Firmicutes, Actinobacteria (p<0.05); functionally associated with energy, amino acid and xenobiotic metabolism (p<0.05). A gut microbiota derangement in converting primary to secondary bile acids was evident as CDCA and cytotoxic metabolites (4-(2-Amino phenyl)-2,4-dioxobutanoate, L-Tyrosine) were higher. Elevated CDCA (logFC>10) levels correlated with mortality in ALF patients as well as in mouse model. In the later, administration of B. intestinalis-AM1 bacteria, (10^9) reduced CDCA and CA levels by enhancing FXR, FGF15, SLC10A1 gene expression, attenuating inflammation (IL-1beta, TLR4-signalling), necroptosis, and modulating glutathione(oxidative-repair), tryptophan(inflammation), and histidine (tissue repair) metabolism. ConclusionHigh levels of chenodeoxycholic acid (CDCA) represent a poor prognostic indicator in ALF patients. B. intestinalis-AM1, a primary-to-secondary bile acid converter, effectively reduce CDCA levels, activated FXR, reduced inflammation and protected hepatocytes, highlighting its therapeutic potential in ALF.

microbiology↗