A subset of hepatic fibroblasts marked by Gli1 expression generates portal fibrosis and promotes ductular reaction.

BACKGROUND & AIMS: Gli1+ cells were previously described as myofibroblast precursors. We aimed to delineate the identity of Gli1+ cells and their contribution to wound healing in liver.

METHODS

Gli1+ cells were analyzed in normal mouse liver by single-cell RNA sequencing (n = 3). Genetic cell fate tracing and depletion of Gli1+ cells were achieved in male mice, undergoing cholestatic injury induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or bile duct ligation (BDL), and metabolic dysfunction-associated steatohepatitis (MASH). Gli1 expression was examined by fluorescence in-situ hybridization in human liver (n = 16). Cell interactions were assessed using organoids.

RESULTS

In normal liver, Gli1 demarcated a periductal subpopulation (11.8%) of portal fibroblasts, which transcriptome pertains to extracellular matrix and structure organization. In liver injury, Gli1+ fibroblasts accumulated with fibrosis as multilayers around bile ducts and discontinuously along the inner border of the ductular reaction, predominantly in biliary models and less markedly in MASH. In humans, periductal Gli1+ cells were present in normal liver and accumulated in primary sclerosing cholangitis and, to a lesser extent, in MASH, notably alongside the ductular reaction in late-stage diseases. Co-culture of cholangiocyte organoids with Gli1+ fibroblasts showed bidirectional interactions fostering proliferation of both cell types. Genetic depletion of Gli1+ cells (n ≥4/group) caused a marked reduction of fibrosis, assessed by Sirius Red area, in DDC-fed and BDL mice after 1 week (p <0.01) and of ductular reaction, assessed by cytokeratin-19+ cell count in DDC (p <0.01), although cholestatic injury persisted or even increased.

CONCLUSIONS

Gli1 demarcates a subpopulation of fibroblasts that play a key role in the wound healing process combining portal fibrosis and ductular reaction notably in biliary diseases.

IMPACT AND IMPLICATIONS: The heterogeneity and the functions of hepatic fibroblasts in liver fibrosis remain poorly defined. Gli1+ fibroblasts are a transcriptionally distinct subset of hepatic fibroblasts, mainly located around bile ducts. Gli1+ fibroblasts develop bidirectional interactions with cholangiocytes, underlying portal fibrosis and ductular reaction formation, as they typically occur in biliary diseases. The therapeutic targeting of Gli1+ hepatic fibroblasts is of potential interest in biliary diseases.