Biliary atresia is a neonatal liver disease with extrahepatic bile duct obstruction and progressive liver fibrosis. The etiology and pathogenesis of the disease are unknown. We previously identified a plant toxin, biliatresone, responsible for biliary atresia in naturally-occurring animal models, that causes cholangiocyte destruction inin-vitromodels. Decreases in reduced glutathione (GSH) mimic the effects of biliatresone, and agents that replenish cellular GSH ameliorate the effects of the toxin. The goals of this study were to define signaling pathways downstream of biliatresone that lead to cholangiocyte destruction and to determine their relationship to GSH. Using cholangiocyte culture and 3D cholangiocyte spheroid cultures, we found that biliatresone and decreases in GSH upregulated RhoU/Wrch1, a Wnt signaling family member, which then mediated an increase in Hey2 in the NOTCH signaling pathway, causing downregulation of the transcription factor Sox17. When these genes were up- or down-regulated, the biliatresone effect on spheroids was phenocopied, resulting in lumen obstruction. Biopsies of patients with biliary atresia demonstrated increased RhoU/Wrch1 and Hey2 expression in cholangiocytes. We present a novel pathway of cholangiocyte injury in a model of biliary atresia, which is relevant to human BA and may suggest potential future therapeutics.

Biliary atresia (BA) is a neonatal liver disease characterized by progressive obstruction and fibrosis of the extrahepatic biliary tree as well as fibrosis and inflammation of the liver parenchyma. Recent studies found that infants who will go on to develop BA have elevated direct bilirubin levels in the first few days of life, suggesting that the disease starts in utero. The etiology and pathogenesis of BA, however, remain unknown. Here, we discuss recent studies examining potential pathogenetic mechanisms of BA, including genetic susceptibility, involvement of the immune system, and environmental insults such as viruses and toxins, although it is possible that there is not a single etiological agent but rather a large group of injurious insults that result in a final common pathway of extrahepatic bile duct obstruction and liver fibrosis. The management and diagnosis of BA have not advanced significantly in the past decade, but given recent advances in understanding the timing and potential pathogenesis of BA, we are hopeful that the next decade will bring early diagnostics and novel therapeutics.

Introduction: Bile duct integrity is essential for the maintenance of the structure and function of the biliary tree. We previously showed that cholangiocyte injury in a toxic model of biliary atresia leads to increased monolayer permeability. Increased epithelial permeability was also shown in other cholangiopathies. We hypothesized that after initial cholangiocyte injury, leakage of bile acids into the duct submucosa propagates cholangiocyte damage and fibrosis. We thus aimed to determine the impact of bile acid exposure on cholangiocytes and the potential therapeutic effect of a non-toxic bile acid. Materials and methods: Extrahepatic bile duct explants were isolated from adult and neonatal BALB/c mice. Explants were cultured with or without glycochenodeoxycholic acid and ursodeoxycholic acid. They were then fixed and stained. Results: Explants treated with glycochenodeoxycholic acid demonstrated cholangiocyte injury with monolayer disruption and partial lumen obstruction compared to control ducts. Masson’s trichrome stains revealed increased collagen fibers. Myofibroblast marker α-SMA stains were significantly elevated in the periductal region. The addition of ursodeoxycholic acid resulted in decreased cholangiocyte injury and reduced fibrosis. Conclusions: Bile acid leakage into the submucosa after initial cholangiocyte injury may serve as a possible mechanism of disease propagation and progressive fibrosis in cholangiopathies.

Background: Mechanisms underlying bile duct injury in biliary atresia (BA) remain unclear and mechanisms of bile duct repair are unknown. This study aimed to explore the roles of microtubule instability and Wnt and Hippo signaling pathways in a biliatresone-induced BA model. Methods: Using primary murine neonatal cholangiocytes in both 2D and 3D cultures, and ex-vivo extra hepatic bile ducts (EHBD) which also has peri-cholangiocyte area, we analyzed injury and recovery processes. Injury was induced by the toxin biliatresone and recovery was induced by toxin wash-out. Results: Microtubule stabilizer paclitaxel prevented biliatresone-induced injury, both to cholangiocytes as well as reduced periductal αSMA stain, this process is mediated by decreased glutathione levels. RhoU and Wnt11 (Wnt signaling) and Pard6g and Amotl1 (Hippo signaling) are involved in both injury and recovery processes, with the latter acting upstream to Wnt signaling. Conclusions: Early stages of biliatresone-induced EHBD injury in cholangiocytes and periductal structures are reversible. Wnt and Hippo signaling pathways play crucial roles in injury and recovery, providing insights into BA injury mechanisms and potential recovery avenues. Impact: Microtubule stabilization prevents cholangiocyte injury and lumen obstruction in a toxic model of biliary atresia (biliatresone induced). Early stages of biliatresone-induced injury, affecting both cholangiocytes and periductal structures, are reversible. Both Wnt and Hippo signaling pathways play a crucial role in bile duct injury and recovery, with a noted interplay between the two. Understanding mechanisms of cholangiocyte recovery is imperative to unveil potential therapeutic avenues.

Objective: Hepatic and biliary tract diseases are common in sickle cell disease (SCD) patients, likely due to sickling, hemosiderosis, viral hepatitis, or cholelithiasis. Literature is lacking on associations between SCD, autoimmune hepatitis (AIH), and/or sclerosing cholangitis (SC)-together, autoimmune liver disease (AILD). We aimed to better understand the relationship of these diseases in pediatric patients. Materials and Methods: A retrospective analysis of patients with SCD and AILD at the Children’s Hospital of Philadelphia (January 2008 to August 2015). Results: Seven patients, ages 8 to 23 years (3 males), were identified. Three had AIH, 2 SC, and 2 AIH/SC overlap, known as autoimmune SC. All patients with AIH treated with azathioprine significantly improved their liver enzymes. One patient with SC and inflammatory bowel disease underwent successful bone marrow transplant. Two SC patients died from SCD complications. Conclusions: In this cohort, there seems to be an association between SCD and AILD; SC in this population was severe. Physicians should be aware of this and evaluate patients with SCD and elevated liver enzymes for AILD.

Cholangiopathies are poorly understood disorders with no effective therapy. The extrahepatic biliary tree phenotype is less studied compared to the intrahepatic biliary injury in both human disease and Mdr2^−/− mice, the established cholestatic mouse model. This study aimed to characterize the extra hepatic biliary tree of Mdr2^−/− mice at various ages and to determine if injury can be repaired with the antioxidant and glutathione precursor N-acetyl-L-Cysteine treatment (NAC). We characterized extra hepatic bile ducts (EHBD)s at various ages from 2 to 40 weeks old FVB/N and Mdr2^−/− mice. We examined the therapeutic potential of local NAC ex vivo using EHBD explants at early and late stages of injury; and systematic therapy by in vivo oral administration for 3 weeks. EHBD and liver sections were assessed by histology and immunofluorescent stains. Serum liver enzyme activities were analyzed, and liver spatial protein expression analysis was performed. Mdr2^−/− mice developed progressive EHBD injury, similar to extrahepatic PSC. NAC treatment of ex vivo EHBD explants led to improved duct morphology. In vivo, oral administration of NAC improved liver fibrosis, and decreased liver enzyme activities. Spatial protein analysis revealed cell-type specific differential response to NAC, collectively indicating a transition from pro-apoptotic into proliferative state. NAC treatment should be further investigated as a potential therapeutic option for human cholangiopathies.