PROJECT SUMMARY: Obstructive cholestatic liver diseases carry a high medical burden as there is no medical therapy to prevent disease progression and thus they remain a leading indication for liver transplantation. While the initial target in obstructive cholangiopathies is the bile duct, the immune response is the major cause for ongoing liver injury. Macrophages are known to play a significant role in the mechanism of cholestatic liver injury, however, disease- modulating immunotherapies have not been established and represent an unmet medical need. We have been the first to perform single-cell RNA sequencing (scRNA-seq) on cholestatic liver samples and will use this preliminary data in the current study to overcome the gap in medical therapy. We have identified a subset of RORA-expressing macrophages in cholestatic liver samples that is at the interface between cholestatic and normal macrophages on pseudotime trajectory analysis. RORA encodes a retinoic acid receptor-related orphan receptor alpha (ROR) that is known to promote anti-inflammatory polarization of human macrophages. Our data suggests that RORA+ macrophages may emerge in cholestatic liver injury and thus be a novel therapeutic target. While ROR-agonism has shown improvement in hepatic injury in a murine model of non-alcoholic steatohepatitis, the role of RORA in cholestatic liver disease has not been investigated. We hypothesize that RORA+ hepatic macrophages are necessary for the reparative response after cholestatic injury; thus, ROR agonism will promote repair through the conversion of pro-inflammatory macrophages into this critical pro-restorative subset. We will investigate our hypothesis through: 1) correlation between clinical parameters of liver injury and the transcriptional prolife of RORA+ hepatic macrophages in cholestatic and non-cholestatic human liver diseases using scRNA-seq, 2) identification of the reparative macrophage immune response after alleviation of biliary obstruction using an innovative murine model of reversible bile duct ligation, and 3) evaluation of changes in disease phenotype in our murine model upon ROR-agonism. Transcriptional correlation between human and murine macrophage subsets will provide the foundation to translate findings from the current study into future human immune-modulatory therapeutic trials. In addition, data obtained from this proposal will enable further studies on ROR-agonism in disease-specific murine models of cholestasis as well as fate-mapping experiments to determine the origin (bone-marrow derived versus tissue-resident) of the ROR-responsive macrophages.