Microbial Monotherapy With Leuconostoc sp LB-P8 Improves Inflammation and Fibrosis in Mouse Primary Sclerosing Cholangitis by Inhibiting Transforming Growth Factor β1/SMAD Signaling

Background & aims: Primary sclerosing cholangitis (PSC) is a prototypical disease with an impaired gut-liver axis, frequently associated with inflammatory bowel disease, and linked to microbial dysbiosis. However, how microbes influence PSC progression remains unclear. We identified a novel L sp. (LB-P8) with potential anti-fibrotic properties via inhibition of transforming growth factor beta-1 (TGF-β)/SMAD signaling and investigated its therapeutic efficacy and mechanisms in mouse PSC models.

Methods: Human intestinal cells were cultured with or without LB-P8 to assess metabolite profiles (untargeted ultra-performance liquid chromatography-tandem mass spectrometry) and gene expression in co-cultured myofibroblasts and macrophages (reverse transcription-quantitative polymerase chain reaction). LB-P8 was orally gavaged following disease onset in three cholestatic models: bile duct ligation (BDL), 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding, and the Mdr2^-/- mouse. Hepatic injury, inflammation, and fibrosis were assessed by serum alanine aminotransferase and Alkaline Phosphatase, histology, immunofluorescence, Picrosirius red staining, and reverse transcription-quantitative polymerase chain reaction. Nanostring GeoMX spatial transcriptomic analysis was performed on DDC-fed liver. LB-P8 was also tested in dextran sodium sulfate (DSS)-induced colitis.

Results: Metabolomic profiling of LB-P8 cultures revealed increased detection of 10 metabolites involved in anti-fibrosis and anti-inflammation. RNA Sequencing analysis of hepatic stellate cells showed downregulation of TGF-β, epithelial-mesenchymal transition (EMT,) and Integrin signaling pathways. LB-P8 reduced liver fibrosis in BDL, DDC-fed, and Mdr2^-/- mouse models as evident by ∼50% lower Picrosirius red staining and decreased expression of Col1a1 and Timp1. In Mdr2^-/- and DDC-fed mice, LB-P8 reduced periportal macrophage number (F4/80; ∼30%). GeoMX spatial transcriptomics revealed reduced TGF-β1 in cholangiocyte and myofibroblast regions. Finally, LB-P8 suppressed expression of colonic markers of inflammation and fibrosis in the DSS model of bowel injury.

Conclusions: LB-P8 ameliorates cholestatic liver disease progression by reducing TGF-β-mediated fibroblast activation, and periportal accumulation of macrophages. Targeting the gut-liver axis with LB-P8 may represent a novel therapeutic strategy for PSC.

Cholestatic Liver Disease; Gut Microbiota; LB-P8; Live Biotherapeutic Product; Liver Fibrosis; Primary Sclerosing Cholangitis; TGF-β1/SMAD Signaling.