Activated hepatic stellate cells and portal fibroblasts contribute to cholestatic liver fibrosis in MDR2 knockout mice

Background & Aims: Chronic liver injury often results in the activation of hepatic myofibroblasts and the development of liver fibrosis. Hepatic myofibroblasts may originate from 3 major sources: hepatic stellate cells (HSCs), portal fibroblasts (PFs), and fibrocytes, with varying contributions depending on the etiology of liver injury. Here, we assessed the composition of hepatic myofibroblasts in multidrug resistance gene 2 knockout (Mdr2^−/−) mice, a genetic model that resembles primary sclerosing cholangitis in patients. Methods: Mdr2^−/− mice expressing a collagen-GFP reporter were analyzed at different ages. Hepatic non-parenchymal cells isolated from collagen-GFP Mdr2^−/− mice were sorted based on collagen-GFP and vitamin A. An NADPH oxidase (NOX) 1/4 inhibitor was administrated to Mdr2^−/− mice aged 12–16 weeks old to assess the therapeutic approach of targeting oxidative stress in cholestatic injury. Results: Thy1⁺ activated PFs accounted for 26%, 51%, and 54% of collagen-GFP⁺ myofibroblasts in Mdr2^−/− mice at 4, 8, and 16 weeks of age, respectively. The remaining collagen-GFP⁺ myofibroblasts were composed of activated HSCs, suggesting that PFs and HSCs are both activated in Mdr2^−/− mice. Bone-marrow-derived fibrocytes minimally contributed to liver fibrosis in Mdr2^−/− mice. The development of cholestatic liver fibrosis in Mdr2^−/− mice was associated with early recruitment of Gr1⁺ myeloid cells and upregulation of pro-inflammatory cytokines (4 weeks). Administration of a NOX inhibitor to 12-week-old Mdr2^−/− mice suppressed the activation of myofibroblasts and attenuated the development of cholestatic fibrosis. Conclusions: Activated PFs and activated HSCs contribute to cholestatic fibrosis in Mdr2^−/− mice, and serve as targets for antifibrotic therapy. Lay summary: Activated portal fibroblasts and hepatic stellate cells, but not fibrocytes, contributed to the production of the fibrous scar in livers of Mdr2^−/− mice, and these cells can serve as targets for antifibrotic therapy in cholestatic injury. Therapeutic inhibition of the enzyme NADPH oxidase (NOX) in Mdr2^−/− mice reversed cholestatic fibrosis, suggesting that targeting NOXs may be an effective strategy for the treatment of cholestatic fibrosis.