Abstract

Background: PSC is an archetypical example of a disease with an impaired gut-liver axis and PSC patients with or without inflammatory bowel disease exhibit enteric microbial dysbiosis. Gut microbiota produce diverse metabolic products but how microbes influence PSC disease progression is unclear. By screening bacterial species (sp.) in a transwell gut-liver axis model, we identified novel Leuconostoc sp. (LB-P8) that may have anti-fibrotic properties via inhibition of TGF-β/SMAD signaling. Here we investigated whether oral dosing of LB-P8 improved inflammation and fibrosis in mouse models of PSC.

Methods: We used two models: chronic feeding (7 weeks) of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and the Mdr2-/- mouse. Live culture or freeze-dried preparations of LB-P8 were orally gavaged daily for 4 weeks following disease onset. Peribiliary injury, inflammation, fibrosis, and cholangiocyte senescence were measured biochemically (ALT & ALP), and by histology, immunofluorescence, picrosirius red staining, hydroxyproline content, and qPCR. LB-P8-derived metabolites were analyzed using untargeted UPLC-MS/MS. Finally, RNA-seq was performed on hepatic stellate cells (HSCs) cultured in the presence or absence of LB-P8.

Results: LB-P8 reduced DDC-fed and Mdr2-/- mouse model-induced elevations of serum ALT (~50 %), and ALP (20-50%). Fibrosis was reduced in live culture LB-P8 treated mice as evident by reduced picrosirius red staining (~50%), and hepatic hydroxyproline content. In Mdr2-/- mice, live culture LB-P8 treatment reduced hepatic mRNA expression of the fibrosis marker, Col1a1 (>50%), inflammatory markers, Tnf-α and Mcp-1 (~50%), and senescence markers, p16 and p21 (~80%). In DDC-fed mice, LB-P8 reduced macrophage number (F4/80; ~30%) and senescent cholangiocytes (p21 immunofluorescence) (~40%). Metabolomic profiling of LB-P8 cultures revealed increased detection of 38 metabolites involved in anti-fibrosis/inflammation compared to control and negative strains. RNA seq analysis of HSC further showed that LB-P8 decreased TGF-β, EMT, and integrin signaling pathways.

Conclusion: LB-P8 ameliorates cholestatic liver disease progression likely by reducing cholangiocyte senescence, TGF-β-mediated fibroblast activation, and periportal accumulation of macrophages. These data suggest that LB-P8 targeting of the gut-liver axis is a potential novel therapeutic strategy for the treatment of PSC.