Single-cell transcriptomics reveals anti-inflammatory effects of prednisolone on the hepatic niche in biliary atresia.

Biliary atresia (BA) is a life-threatening paediatric liver disease, characterized by bile duct obstruction, progressive inflammation and fibrosis. Treatment options are limited, often necessitating surgery or liver transplantation to prevent irreversible liver damage. Prednisolone has shown promise in treating BA; however, there is currently no consensus on its effectiveness, and the mechanism of action remains unknown. We investigated the effects of prednisolone in a BA model induced by Rhesus Rotavirus (RRV). Liver samples were collected from day-21 and day-28 post-RRV infected mice injected with PBS or steroid, and subjected to single-cell RNA sequencing (scRNA-seq) and immunohistochemistry. We identified subpopulations of hepatic stellate cells (HSCs) and macrophages that serve as key effectors of prednisolone-mediated disease amelioration. Prednisolone treatment at 28-days post RRV-infection suppressed pro-inflammatory Ly6c-high macrophages, increased immunosuppressive M2 Kupffer cells, and promoted deactivation of HSCs into the quiescent state. Computational reconstruction of HSC differentiation pathways revealed that the Nr3c1-Klf7 gene regulatory network (GRN) modulates the transition of HSCs between quiescence and activation, and we show that HSCs differentiate into three myofibroblast (MyoFbs) subpopulations, each exhibiting different pathogenic potential. We identified a pro-inflammatory MyoFB subpopulation (MFB-2) characterized by the Fosl1-Hnf1b GRN. MFB-2 secretes elevated levels of macrophage-activating cytokines, exhibits resistance to apoptotic signals, and displayed impaired responsiveness to prednisolone. The HSC and MyoFb subtypes were also identified in BA patient livers with highly conserved gene expression and functional profiles, supporting their importance in BA progression. Prednisolone treatment transforms the liver immune-mesenchymal niche from an inflammatory to regenerative state in BA and modulation of GRNs in HSCs plays a pivotal role.