Inhibiting acute injury–induced senescence mediated by TGFβ signaling in regenerative epithelium improves liver regeneration.

Senescent renal epithelia promote fibrosis and functional loss after injury, whereas depletion improves regeneration in aged or irradiated kidneys.

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.

Acute-on-chronic liver failure (ACLF) is characterised by high short-term mortality, systemic inflammation, and failure of hepatic regeneration. Its treatment is a major unmet medical need. This study was conducted to explore whether combining TAK-242, a Toll-like receptor-4 (TLR4) antagonist, with granulocyte-colony stimulating factor (G-CSF), could reduce inflammation whilst enhancing liver regeneration. Two mouse models of ACLF were investigated. Chronic liver injury was induced by carbon tetrachloride; lipopolysaccharide (LPS) or galactosamine (GalN) were then administered as extrahepatic or hepatic insults, respectively. G-CSF and/or TAK-242 were administered daily. Treatment durations were 24 hours and 5 days in the LPS model and 48 hours in the GalN model. In a mouse model of LPS-induced ACLF, treatment with G-CSF was associated with significant mortality (66% after 48 hours vs. 0% without G-CSF). Addition of TAK-242 to G-CSF abrogated mortality (0%) and significantly reduced liver cell death, macrophage infiltration and inflammation. In the GalN model, both G-CSF and TAK-242, when used individually, reduced liver injury but their combination was significantly more effective. G-CSF treatment, with or without TAK-242, was associated with activation of the pro-regenerative and anti-apoptotic STAT3 pathway. LPS-driven ACLF was characterised by p21 overexpression, which is indicative of hepatic senescence and inhibition of hepatocyte regeneration. While TAK-242 treatment mitigated the effect on senescence, G-CSF, when co-administered with TAK-242, resulted in a significant increase in markers of hepatocyte regeneration. The combination of TAK-242 and G-CSF inhibits inflammation, promotes hepatic regeneration and prevents mortality in models of ACLF; thus, this combination could be a potential treatment option for ACLF. Acute-on-chronic liver failure is associated with severe liver inflammation and poor short-term survival. Therefore, effective treatments are urgently needed. Herein, we have shown, using mouse models, that the combination of granulocyte-colony stimulating factor (which can promote liver regeneration) and TAK-242 (which inhibits a receptor that plays a key role in inflammation) could be effective for the treatment of acute-on-chronic liver failure.

Biliary diseases can cause inflammation, fibrosis, bile duct destruction, and eventually liver failure. There are no curative treatments for biliary disease except for liver transplantation. New therapies are urgently required. We have therefore purified human biliary epithelial cells (hBECs) from human livers that were not used for liver transplantation. hBECs were tested as a cell therapy in a mouse model of biliary disease in which the conditional deletion of Mdm2 in cholangiocytes causes senescence, biliary strictures, and fibrosis. hBECs are expandable and phenotypically stable and help restore biliary structure and function, highlighting their regenerative capacity and a potential alternative to liver transplantation for biliary disease.

In the adult liver, a population of facultative progenitor cells called biliary epithelial cells (BECs) proliferate and differentiate into cholangiocytes and hepatocytes after injury, thereby restoring liver function. In mammalian models of chronic liver injury, Notch signaling is essential for bile duct formation from these cells. However, the continual proliferation of BECs and differentiation of hepatocytes in these models have limited their use for determining whether Notch signaling is required for BECs to replenish hepatocytes after injury in the mammalian liver. Here, we used a temporally restricted model of hepatic repair in which large-scale hepatocyte injury and regeneration are initiated through the acute loss of Mdm2 in hepatocytes, resulting in the rapid, coordinated proliferation of BECs. We found that transient, early activation of Notch1- and Notch3-mediated signaling and entrance into the cell cycle preceded the phenotypic expansion of BECs into hepatocytes. Notch inhibition reduced BEC proliferation, which resulted in failure of BECs to differentiate into hepatocytes, indicating that Notch-dependent expansion of BECs is essential for hepatocyte regeneration. Notch signaling increased the abundance of the insulin-like growth factor 1 receptor (IGF1R) in BECs, and activating IGFR signaling increased BEC numbers but suppressed BEC differentiation into hepatocytes. These results suggest that different signaling mechanisms control BEC expansion and hepatocyte differentiation.