Primary sclerosing cholangitis (PSC) is characterized by chronic inflammation and progressive fibrosis of the biliary tree. The bile acid receptor TGR5 (GPBAR1) is found on biliary epithelial cells (BECs), where it promotes secretion, proliferation and tight junction integrity. Thus, we speculated that changes in TGR5-expression in BECs may contribute to PSC pathogenesis. TGR5-expression and -localization were analyzed in PSC livers and liver tissue, isolated bile ducts and BECs from Abcb4^-/-, Abcb4^-/-/Tgr5^Tg and ursodeoxycholic acid (UDCA)- or 24-norursodeoxycholic acid (norUDCA)-fed Abcb4^-/- mice. The effects of IL8/IL8 homologues on TGR5 mRNA and protein levels were studied. BEC gene expression was analyzed by single-cell transcriptomics (scRNA-seq) from distinct mouse models. TGR5 mRNA expression and immunofluorescence staining intensity were reduced in BECs of PSC and Abcb4^-/- livers, in Abcb4^-/- extrahepatic bile ducts, but not in intrahepatic macrophages. No changes in TGR5 BEC fluorescence intensity were detected in liver tissue of other liver diseases, including primary biliary cholangitis. Incubation of BECs with IL8/IL8 homologues, but not with other cytokines, reduced TGR5 mRNA and protein levels. BECs from Abcb4^-/- mice had lower levels of phosphorylated Erk and higher expression levels of Icam1, Vcam1 and Tgfβ2. Overexpression of Tgr5 abolished the activated inflammatory phenotype characteristic of Abcb4^-/- BECs. NorUDCA-feeding restored TGR5-expression levels in BECs in Abcb4^-/- livers. Reduced TGR5 levels in BECs from patients with PSC and Abcb4^-/- mice promote development of a reactive BEC phenotype, aggravate biliary injury and thus contribute to the pathogenesis of sclerosing cholangitis. Restoration of biliary TGR5-expression levels represents a previously unknown mechanism of action of norUDCA. Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease-associated with progressive inflammation of the bile duct, leading to fibrosis and end-stage liver disease. Bile acid (BA) toxicity may contribute to the development and disease progression of PSC. TGR5 is a membrane-bound receptor for BAs, which is found on bile ducts and protects bile ducts from BA toxicity. In this study, we show that TGR5 levels were reduced in bile ducts from PSC livers and in bile ducts from a genetic mouse model of PSC. Our investigations indicate that lower levels of TGR5 in bile ducts may contribute to PSC development and progression. Furthermore, treatment with norUDCA, a drug currently being tested in a phase III trial for PSC, restored TGR5 levels in biliary epithelial cells.

TGR5 (Gpbar1) is a G protein-coupled receptor responsive to bile acids (BAs), which is expressed in different non-parenchymal cells of the liver, including biliary epithelial cells, liver-resident macrophages, sinusoidal endothelial cells (LSECs), and activated hepatic stellate cells (HSCs). Mice with targeted deletion of TGR5 are more susceptible towards cholestatic liver injury induced by cholic acid-feeding and bile duct ligation, resulting in a reduced proliferative response and increased liver injury. Conjugated lithocholic acid (LCA) represents the most potent TGR5 BA ligand and LCA-feeding has been used as a model to rapidly induce severe cholestatic liver injury in mice. Thus, TGR5 knockout (KO) mice and wildtype (WT) littermates were fed a diet supplemented with 1% LCA for 84 h. Liver injury and gene expression changes induced by the LCA diet revealed an enrichment of pathways associated with inflammation, proliferation, and matrix remodeling. Knockout of TGR5 in mice caused upregulation of endothelin-1 (ET-1) expression in the livers. Analysis of TGR5-dependent ET-1 signaling in isolated LSECs and HSCs demonstrated that TGR5 activation reduces ET-1 expression and secretion from LSECs and triggers internalization of the ET-1 receptor in HSCs, dampening ET-1 responsiveness. Thus, we identified two independent mechanisms by which TGR5 inhibits ET-1 signaling and modulates portal pressure.

Background and Aims: A recent multicenter genetic exploration of the biliary atresia splenic malformation syndrome identified mutations in the ciliary gene PKD1L1 as candidate etiologic contributors. We hypothesized that deletion of Pkd1l1 in developing hepatoblasts would lead to cholangiopathy in mice. Approach and Results: CRISPR-based genome editing inserted loxP sites flanking exon 8 of the murine Pkd1l1 gene. Pkd1l1 ^Fl/Fl cross-bred with alpha-fetoprotein-Cre expressing mice to generate a liver-specific intrahepatic Pkd1l1-deficient model (LKO). From embryonic day 18 through week 30, control (Fl/Fl) and LKO mice were evaluated with standard serum chemistries and liver histology. At select ages, tissues were analyzed using RNA sequencing, immunofluorescence, and electron microscopy with a focus on biliary structures, peribiliary inflammation, and fibrosis. Bile duct ligation for 5 days of Fl/Fl and LKO mice was followed by standard serum and liver analytics. Histological analyses from perinatal ages revealed delayed biliary maturation and reduced primary cilia, with progressive cholangiocyte proliferation, peribiliary fibroinflammation, and arterial hypertrophy evident in 7- to 16-week-old LKO versus Fl/Fl livers. Following bile duct ligation, cholangiocyte proliferation, peribiliary fibroinflammation, and necrosis were increased in LKO compared with Fl/Fl livers. Conclusions: Bile duct ligation of the Pkd1l1-deficient mouse model mirrors several aspects of the intrahepatic pathophysiology of biliary atresia in humans including bile duct dysmorphogenesis, peribiliary fibroinflammation, hepatic arteriopathy, and ciliopathy. This first genetically linked model of biliary atresia, the Pkd1l1 LKO mouse, may allow researchers a means to develop a deeper understanding of the pathophysiology of this serious and perplexing disorder, including the opportunity to identify rational therapeutic targets.

Introduction: TGR5 (Gpbar-1) is a G-Protein-coupled bile acid (BA) receptor (Kawamata et al. 2003) expressed on nonparenchymal liver cells such as hepatic stellate cells (HSCs) and liver sinusoidal endothelial cells (LSECs) (Keitel et al. 2006, 2008). Lithocholic acid (LCA) is a hydrophobic bile acid. Feeding mice with a diet containing 1%LCA leads to the development of a severe toxic liver injury (Fickert et al. 2006). The most relevant complication of liver damage is the development of portal hypertension (PH). It has been demonstrated previously that activated HSCs contribute to PH by Endothelin-1 (ET-1)-induced cell-contraction in a cAMP-dependent manner (Rockey et al 1993, Reinehr et al. 2002). If TGR5 plays a role during this process was unknown. Methods: 8 – 12 week old wildtype (WT) and TGR5 knockout (KO) mice were sacrificed after receiving a diet containing 1%LCA for 84 hrs. Liver damage was determined via analysis of liver enzymes in serum and HE-staining. Portal pressure was measured by cannulation of the portal vein. Hepatic mRNA-expression of different genes was determined by RT-PCR analysis. HSC contraction was tested using an HSC-contraction assay. Localization of the ETA-receptor on HSCs was analyzed by immunofluorescence (IF) and colocalization-analysis. ET-1 levels in LSECs were measured via ELISA-immunoassay and RT-PCR. Results: TGR5 KO mice suffered from a more severe liver damage as compared to WT animals when fed a 1%LCA diet as determined by the area of necrosis seen in liver tissue as well as increased serum AST-levels. Measurement of the portal pressure in these mice revealed an induction of PH in LCA-fed KO mice but in none of the control groups. RT-PCR-analysis confirmed an increased expression of genes associated with PH, e.g. ET-1 in livers of LCA-fed TGR5 KO mice. Challenge of LSECs as the most important source of ET-1 in the liver with a TGR5 agonist caused a significant reduction of ET-1 secretion. Moreover, treatment of mice and isolated HSCs with a TGR5 agonist was able to attenuate ET-1-induced PH as well as HSC contraction, respectively. By means of IF of the ETA-receptor on the surface of HSCs and colocalization analysis we were able to demonstrate internalization of this receptor after treatment with a TGR5 agonist. Conclusion: In conclusion, TGR5 plays a protective role against the development of PH after induction of liver injury by two synergistically acting mechanisms.