O’Hara, Steven P.

Cholangiocytes are specialized epithelial cells that line the intrahepatic and extrahepatic biliary tree and play a critical role in bile modification, liver homeostasis, and response to injury. Cholangiocytes exhibit notable heterogeneity and plasticity, and their dysfunction is central to a spectrum of diseases targeting the bile ducts, collectively called cholangiopathies. These disorders include genetic, infectious, immune-mediated, and malignant diseases, with primary sclerosing cholangitis (PSC) representing one of the most complex and enigmatic of these disorders. PSC is a progressive, fibro-inflammatory disease of the bile ducts that is closely linked to inflammatory bowel disease, carries a heightened risk of cancer, and lacks any approved therapies. This review explores the biology of cholangiocytes, including their development, functional plasticity, and roles in secretion, absorption, and cellular signaling. We provide a detailed examination of cholangiopathies, particularly PSC, a complex cholangiopathy characterized by a paradoxical state of cholangiocyte senescence and hyperproliferation. We describe how immune cell dysfunction, the gut microbiome, genetic predispositions, and environmental factors converge to mediate PSC pathogenesis. We revisit the foundational technologies that empowered early discoveries and shaped the field as we know it today. We also explore how newer techniques such as organoid cultures, single-cell transcriptomics, epigenomics, and spatialomics have transformed our modern understanding of biliary pathophysiology. Finally, we provide an overview of existing rodent models of cholangiopathies and discuss their relevance to human disease. PSC remains therapeutically unaddressed, and thus ongoing multidisciplinary efforts are essential to developing targeted interventions. This review serves as a comprehensive resource for researchers and clinicians navigating the rapidly evolving landscape of cholangiocyte-centered liver disease research.

ABSTRACT Background and Aims Cellular senescence is a hallmark of several liver diseases, including primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC). Senescent cholangiocytes exhibit a senescence‐associated secretory phenotype (SASP), characterized by profibroinflammatory mediator release. Current cost‐effective biomarkers predicting disease progression, particularly for PSC, are limited and often lack mechanistic relevance. We sought to define a plasma biomarker signature for PSC and PBC. Methods Plasma from early‐ and late‐stage PSC and PBC, alcoholic liver disease (ALD), inflammatory bowel disease (IBD) patients, and healthy controls was analyzed. Seventy‐one analytes were quantified using Luminex Multiplex Immunoassay or enzyme‐linked immunosorbent assay (ELISA). Principal component analysis (PCA) identified key patterns. Findings from the PSC cohort were then applied to additional cohorts. Results Second principal component (PC2) (17 analytes, 17.1% variability) best separated PSC from controls. ANOVA showed significant differences in PC2 between early PSC vs. controls ( p = 0.0001), late PSC vs. controls ( p < 0.0001), and early vs. late PSC ( p < 0.0001). PC2 analytes also distinguished early PBC vs. controls ( p < 0.0332), late PBC ( p < 0.0001), and ALD ( p < 0.0001), and early vs. late PBC ( p < 0.0001), but not IBD vs. controls ( p = 0.119). Logistic regression using PC2 demonstrated strong discrimination of early‐ and late‐stage PSC (AUC = 0.86) and control vs. early‐stage PSC (AUC = 0.83). Conclusion This is the first study to define a plasma SASP biomarker signature associated with cholestatic liver disease. These analytes track disease stage and represent both mechanistic indicators and potential clinical trial endpoints.

Abstract Cholangiocytes are epithelial cells that line the intra‐ and extrahepatic ducts of the biliary tree. The main physiologic function of cholangiocytes is modification of hepatocyte‐derived bile, an intricate process regulated by hormones, peptides, nucleotides, neurotransmitters, and other molecules through intracellular signaling pathways and cascades. The mechanisms and regulation of bile modification are reviewed herein. © 2013 American Physiological Society. Compr Physiol 3:541‐565, 2013.

Cholangiopathies comprise a spectrum of chronic intrahepatic and extrahepatic biliary tract disorders culminating in progressive cholestatic liver injury, fibrosis, and often cirrhosis and its sequela. Treatment for these diseases is limited, and collectively, they are one of the therapeutic “black boxes” in clinical hepatology. The etiopathogenesis of the cholangiopathies likely includes disease-specific mediators but also common cellular and molecular events driving disease progression (eg, cholestatic fibrogenesis, inflammation, and duct damage). The common pathways involve cholangiocytes, the epithelial cells lining the intrahepatic and extrahepatic bile ducts, which are central to the pathogenesis of these disorders. Current information suggests that cholangiocytes function as a signaling “hub” in biliary tract-associated injury. Herein, we review the pivotal role of cholangiocytes in cholestatic fibrogenesis, focusing on the crosstalk between cholangiocytes and portal fibroblasts and HSCs. The proclivity of these cells to undergo a senescence-associated secretory phenotype, which is proinflammatory and profibrogenic, and the intrinsic intracellular activation pathways resulting in the secretion of cytokines and chemokines are reviewed. The crosstalk between cholangiocytes and cells of the innate (neutrophils and macrophages) and adaptive (T cells and B cells) immune systems is also examined in detail. The information will help consolidate information on this topic and guide further research and potential therapeutic strategies for these diseases.

Abstract Senescent cells secrete proinflammatory factors known as the senescence-associated secretory phenotype (SASP), contributing to tissue dysfunction and aging. Mitochondrial dysfunction is a key feature of senescence, influencing SASP via mitochondrial DNA (mtDNA) release and cGAS/STING pathway activation. Here, we demonstrate that mitochondrial RNA (mtRNA) also accumulates in the cytosol of senescent cells, activating RNA sensors RIG-I and MDA5, leading to MAVS aggregation and SASP induction. Inhibition of these RNA sensors significantly reduces SASP factors. Furthermore, BAX and BAK plays a key role in mtRNA leakage during senescence, and their deletion diminishes SASP expression in vitro and in a mouse model of Metabolic Dysfunction Associated Steatohepatitis (MASH). These findings highlight mtRNA's role in SASP regulation and its potential as a therapeutic target for mitigating age-related inflammation.

Primary sclerosing cholangitis (PSC) is a chronic fibroinflammatory disease of the biliary tract characterized by cellular senescence and periportal fibrogenesis. Specific disease features that are cell intrinsic and either genetically or epigenetically mediated remain unclear due in part to a lack of appropriate, patient‐specific, in vitro models. Recently, our group developed systems to create induced pluripotent stem cell (iPSC)‐derived cholangiocytes (iDCs) and biliary epithelial organoids (cholangioids). We use these models to investigate whether PSC cholangiocytes are intrinsically predisposed to cellular senescence. Skin fibroblasts from healthy controls and subjects with PSC were reprogrammed to pluripotency, differentiated to cholangiocytes, and subsequently grown in three‐dimensional matrigel‐based culture to induce formation of cholangioids. RNA sequencing (RNA‐seq) on iDCs showed significant differences in gene expression patterns, including enrichment of pathways associated with cell cycle, senescence, and hepatic fibrosis, that correlate with PSC. These pathways also overlapped with RNA‐seq analysis on isolated cholangiocytes from subjects with PSC. Exome sequencing on the subjects with PSC revealed genetic variants of unknown significance in the genes identified in these pathways. Three‐dimensional culture revealed smaller size, lack of a central lumen, and increased cellular senescence in PSC‐derived cholangioids. Congruent with this, PSC‐derived iDCs showed increased secretion of the extracellular matrix molecule fibronectin as well as the inflammatory cytokines interleukin‐6, and chemokine (C‐C motif) ligand 2. Conditioned media (CM) from PSC‐derived iDCs more potently activated hepatic stellate cells compared to control CM. Conclusion: We demonstrated efficient generation of iDCs and cholangioids from patients with PSC that show disease‐specific features. PSC cholangiocytes are intrinsically predisposed to cellular senescence. These features are unmasked following biliary differentiation of pluripotent stem cells and have functional consequences in epithelial organoids.

AbstractBackground & AimsPrimary sclerosing cholangitis (PSC) is a chronic liver disease characterized by peribiliary inflammation and fibrosis. Cholangiocyte senescence is a prominent feature of PSC. Here, we hypothesize that extracellular vesicles (EVs) from senescent cholangiocytes influence the phenotype of target cells.MethodsEVs were isolated from normal human cholangiocytes (NHCs), cholangiocytes from PSC patients and NHCs experimentally induced to senescence. NHCs, malignant human cholangiocytes (MHCs) and monocytes were exposed to 108 EVs from each donor cell population and assessed for proliferation, MAPK activation and migration. Additionally, we isolated EVs from plasma of wild‐type and Mdr2−/− mice (a murine model of PSC), and assessed mouse monocyte activation.ResultsEVs exhibited the size and protein markers of exosomes. The number of EVs released from senescent human cholangiocytes was increased; similarly, the EVs in plasma from Mdr2−/− mice were increased. Additionally, EVs from senescent cholangiocytes were enriched in multiple growth factors, including EGF. NHCs exposed to EVs from senescent cholangiocytes showed increased NRAS and ERK1/2 activation. Moreover, EVs from senescent cholangiocytes promoted proliferation of NHCs and MHCs, findings that were blocked by erlotinib, an EGF receptor inhibitor. Furthermore, EVs from senescent cholangiocytes induced EGF‐dependent Interleukin 1‐beta and Tumour necrosis factor expression and migration of human monocytes; similarly, Mdr2−/− mouse plasma EVs induced activation of mouse monocytes.ConclusionsThe data continue to support the importance of cholangiocyte senescence in PSC pathogenesis, directly implicate EVs in cholangiocyte proliferation, malignant progression and immune cell activation and migration, and identify novel therapeutic approaches for PSC.

Primary sclerosing cholangitis (PSC) is a pathogenically complex, chronic, fibroinflammatory disorder of the bile ducts without known etiology or effective pharmacotherapy. Emerging in vitro and in vivo evidence support fundamental pathophysiologic mechanisms in PSC centered on enterohepatic circulation. To date, no studies have specifically interrogated the chemical footprint of enterohepatic circulation in PSC. Herein, we evaluated the metabolome and lipidome of portal venous blood and bile obtained at the time of liver transplantation in patients with PSC (n = 7) as compared to individuals with noncholestatic, end-stage liver disease (viral, metabolic, etc. (disease control, DC, n = 19)) and to nondisease controls (NC, living donors, n = 12). Global metabolomic and lipidomic profiling was performed on serum derived from portal venous blood (portal serum) and bile using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and differential mobility spectroscopy-mass spectroscopy (DMS-MS; complex lipid platform). The Mann–Whitney U test was used to identify metabolites that significantly differed between groups. Principal-component analysis (PCA) showed significant separation of both PSC and DC from NC for both portal serum and bile. Metabolite set enrichment analysis of portal serum and bile demonstrated that the liver-disease cohorts (PSC and DC) exhibited similar enrichment in several metabolite categories compared to NC. Interestingly, the bile in PSC was uniquely enriched for dipeptide and polyamine metabolites. Finally, analysis of patient-matched portal serum and biliary metabolome revealed that these biological fluids were more homogeneous in PSC than in DC or NC, suggesting aberrant bile formation and enterohepatic circulation. In summary, PSC and DC patients exhibited alterations in several metabolites in portal serum and bile, while PSC patients exhibited a unique bile metabolome. These specific alterations in PSC are amenable to hypothesis testing and, potentially, therapeutic pharmacologic manipulation.