Hedgehog Signaling Demarcates a Niche of Fibrogenic Peribiliary Mesenchymal Cells

In response to tissue injury, stromal cells secrete extracellular matrix (ECM) components that remodel the tissue and lead to fibrosis. Parenchymal stellate cells are the primary contributors to fibrosis in models of hepatocellular and cholestatic injury. The liver comprises different, heterogenous compartments; stromal cells within those compartments might have unique identities and regional functions. The portal tract contains the bile duct, which is surrounded by stromal cells often called portal fibroblasts. We investigated the contributions of these cells to hepatic injury.

We performed studies with Gli1:Cre^ERT2;Rosa26:lox-STOP-lox-tdTomato mice. Mice underwent bile duct ligation or were fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine to induce cholestatic injury or were given carbon tetrachloride to induce liver fibrosis. Liver tissues were collected and analyzed by histology and immunofluorescence, and mesenchymal cells were isolated. We performed lineage tracing experiments to determine the fates of peribiliary mesenchymal cells (PMCs) that surround the bile duct after cholestatic and hepatocellular injury. We used cell sorting combined with RNA sequencing to isolate stellate cells and PMCs, and we identified determinants of cell identity within each population. Liver tissues were obtained from patients with primary sclerosing cholangitis, alcoholic liver disease, or nonalcoholic steatohepatitis or individuals without disease and were analyzed by quantitative reverse transcription polymerase chain reaction.

Gli1 was a marker of mesenchymal cells that surround the biliary tree but not epithelial cells of the canals of Hering. Lineage-traced Gli1⁺ PMCs proliferated and acquired a myofibroblast phenotype after cholestatic injury; Gli1⁺ PMCs were found only surrounding the main duct of a portal tract but not the epithelial cells of the ductular reaction, which were instead encased by stellate cells. Compared with stellate cells, Gli1⁺ PMCs expressed a different subset of genes, including genes that are markers of active hedgehog signaling, Osr1 (encodes a transcription factor), and ECM-related genes. Loss of hedgehog signaling reduced expression of Osr1 and PMC-specific ECM genes. Liver tissues from patients with liver disease had increased expression of genes that define PMC identity compared with control liver tissues.

In lineage-tracing studies of mice, we found that Gli1⁺ PMCs are a subset of stromal cells characterized by active hedgehog signaling that proliferate, acquire a myofibroblast phenotype, and surround the biliary tree in response to cholestatic injury.