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Extrahepatic cholangiocyte obstruction is mediated by decreased glutathione, Wnt and Notch signaling pathways in a toxic model of biliary atresia.

ABSTRACT: 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 in in-vitro models. 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.

SUBMITTER: Fried S

PROVIDER: S-EPMC7200694 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Extrahepatic cholangiocyte obstruction is mediated by decreased glutathione, Wnt and Notch signaling pathways in a toxic model of biliary atresia.

Fried Sophia S Gilboa Dafna D Har-Zahav Adi A Lavrut Pierre-Marie PM Du Yu Y Karjoo Sara S Russo Pierre P Shamir Raanan R Wells Rebecca G RG Waisbourd-Zinman Orith O

Scientific reports 20200505 1

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 in in-vitro models. Decreases in reduced glutathione (GSH) mimic the effects of biliatresone, and agents that replenish cellular GSH ameliorate the effects of the t ...[more]

PMID: 32371929

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Project description:UnlabelledBiliary atresia, the most common indication for pediatric liver transplantation, is a fibrotic disease of unknown etiology affecting the extrahepatic bile ducts of newborns. The recently described toxin biliatresone causes lumen obstruction in mouse cholangiocyte spheroids and represents a new model of biliary atresia. The goal of this study was to determine the cellular changes caused by biliatresone in mammalian cells that ultimately lead to biliary atresia and extrahepatic fibrosis. We treated mouse cholangiocytes in three-dimensional (3D) spheroid culture and neonatal extrahepatic duct explants with biliatresone and compounds that regulate glutathione (GSH). We examined the effects of biliatresone on SOX17 levels and determined the effects of Sox17 knockdown on cholangiocytes in 3D culture. We found that biliatresone caused disruption of cholangiocyte apical polarity and loss of monolayer integrity. Spheroids treated with biliatresone had increased permeability as shown by rhodamine efflux within 5 hours compared with untreated spheroids, which retained rhodamine for longer than 12 hours. Neonatal bile duct explants treated with the toxin showed lumen obstruction with increased subepithelial staining for α-smooth muscle actin and collagen, consistent with fibrosis. Biliatresone caused a rapid and transient decrease in GSH, which was both necessary and sufficient to mediate its effects in cholangiocyte spheroid and bile duct explant systems. It also caused a significant decrease in cholangiocyte levels of SOX17, and Sox17 knockdown in cholangiocyte spheroids mimicked the effects of biliatresone.ConclusionBiliatresone decreases GSH and SOX17 in mouse cholangiocytes. In 3D cell systems, this leads to cholangiocyte monolayer damage and increased permeability; in extrahepatic bile duct explants, it leads to disruption of the extrahepatic biliary tree and subepithelial fibrosis. This mechanism may be important in understanding human biliary atresia. (Hepatology 2016;64:880-893).

Project description:Background and aimsThe etiology of biliary atresia (BA) is not known and is likely multifactorial, including a genetic predisposition, a viral or environmental trigger, an aberrant autoimmune response targeting cholangiocytes, and unique susceptibilities of the neonatal bile ducts to injury. Damaged cholangiocytes may express neo self-antigens and elicit autoreactive T-cell-mediated inflammation and B-cell production of autoantibodies. The aim of this study was to discover autoantibodies in BA that correlated with outcomes.Approach and resultsAn autoantigen microarray encompassing approximately 9,500 autoantigens was used to screen for serum immunoglobulin M (IgM) and immunoglobulin G (IgG) autoantibodies in patients with BA or other liver disease controls. Validation of candidate autoantibodies by enzyme-linked immunosorbent assay on a second cohort of subjects (6-12 months following Kasai portoenterostomy) and correlations of autoantibodies with outcomes were performed (serum bilirubin levels and need for liver transplant in first 2 years of life). Mean anti-chitinase 3-like 1 (CHI3L1), anti-delta-like ligand (DLL-4), and antisurfactant protein D (SFTPD) IgM autoantibodies in BA were significantly higher compared with controls, and IgM autoantibody levels positively correlated with worse outcomes. Immunofluorescence revealed cholangiocyte-predominant expression of CHI3L1, DLL-4, and SFTPD. The humoral autoantibody response was associated with C3d complement activation and T-cell autoimmunity, based on detection of cholangiocyte-predominant C3d co-staining and peripheral blood autoreactive T cells specific to CHI3L1, DLL-4 and SFTPD, respectively.ConclusionsBA is associated with cholangiocyte-predominant IgM autoantibodies in the first year after Kasai portoenterostomy. Anti-CHI3L1, anti-DLL-4, and anti-SFTPD IgM autoantibody correlations with worse outcomes and the detection of C3d on cholangioctyes and antigen-specific autoreactive T cells suggest that autoimmunity plays a role in the ongoing bile duct injury and progression of disease.

Dataset Information

Extrahepatic cholangiocyte obstruction is mediated by decreased glutathione, Wnt and Notch signaling pathways in a toxic model of biliary atresia.

Publications

Extrahepatic cholangiocyte obstruction is mediated by decreased glutathione, Wnt and Notch signaling pathways in a toxic model of biliary atresia.

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