The Epigenetic Reader, Bromodomain Containing 2, Mediates Cholangiocyte Senescence via Interaction With ETS Proto-Oncogene 1.

BACKGROUND & AIMS: We reported that cholangiocyte senescence, regulated by the transcription factor ETS proto-oncogene 1 (ETS1), is a pathogenic feature of primary sclerosing cholangitis (PSC). Furthermore, histone 3 lysine 27 is acetylated at senescence-associated loci. The epigenetic readers, bromodomain and extra-terminal domain (BET) proteins, bind acetylated histones, recruit transcription factors, and drive gene expression. Thus, we tested the hypothesis that BET proteins interact with ETS1 to drive gene expression and cholangiocyte senescence. METHODS: We performed immunofluorescence for BET proteins (BRD2 and 4) in liver tissue from liver tissue from PSC patients and a mouse PSC model. Using normal human cholangiocytes (NHCs), NHCs experimentally induced to senescence (NHCsen), and PSC patient-derived cholangiocytes (PSCDCs), we assessed senescence, fibroinflammatory secretome, and apoptosis after BET inhibition or RNA interference depletion. We assessed BET interaction with ETS1 in NHCsen and tissues from PSC patient, and the effects of BET inhibitors on liver fibrosis, senescence, and inflammatory gene expression in mouse models. RESULTS: Tissue from patients with PSC and a mouse PSC model exhibited increased cholangiocyte BRD2 and 4 protein (∼5×) compared with controls without disease. NHCsen exhibited increased BRD2 and 4 (∼2×), whereas PSCDCs exhibited increased BRD2 protein (∼2×) relative to NHC. BET inhibition in NHCsen and PSCDCs reduced senescence markers and inhibited the fibroinflammatory secretome. ETS1 interacted with BRD2 in NHCsen, and BRD2 depletion diminished NHCsen p21 expression. BET inhibitors reduced senescence, fibroinflammatory gene expression, and fibrosis in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine-fed and Mdr2-/- mouse models. CONCLUSION: Our data suggest that BRD2 is an essential mediator of the senescent cholangiocyte phenotype and is a potential therapeutic target for patients with PSC.

Cellular senescence in the cholangiopathies.

PURPOSE OF REVIEW: Cellular senescence (i.e. permanent withdrawal from the cell cycle) is increasingly recognized as a pathologic feature in a variety of inflammatory liver diseases, including primary sclerosing cholangitis (PSC), primary biliary cholangitis (PBC) and additional cholangiopathies. Herein, we provide an update on the interplay between cholangiocytes, cellular senescence and the cholangiopathies. RECENT FINDINGS: The themes covered by this review include novel models for studying the role of senescent cholangiocytes and the cholangiopathies, identification and modulation of key pathways or molecules regulating cholangiocyte senescence, and discovery of druggable targets to advance therapeutic options for the cholangiopathies. Most recent studies focused on PSC; however, the concepts and findings may be applied to additional cholangiopathies. SUMMARY: Cholangiopathies present unique and divergent clinicopathological features, causes and genetic backgrounds, but share several common disease processes. Cholangiocyte senescence in the cholestatic cholangiopathies, primarily PSC and PBC, is regarded as a key pathogenetic process. Importantly, senescent cholangiocytes exhibit phenotypic features including the senescence-associated secretory phenotype (SASP) and resistance to apoptosis that provide new directions for basic research and new prognostic and therapeutic approaches for clinical practice.

The transcription factor ETS1 promotes apoptosis resistance of senescent cholangiocytes by epigenetically up-regulating the apoptosis suppressor BCL2L1.

Primary sclerosing cholangitis (PSC) is an idiopathic, progressive cholangiopathy. Cholangiocyte senescence is important in PSC pathogenesis, and we have previously reported that senescence is regulated by the transcription factor ETS proto-oncogene 1 (ETS1) and associated with overexpression of BCL2 like 1 (BCL2L1 or BCL-xL), an anti-apoptotic BCL2-family member. Here, we further explored the mechanisms regulating BCL-xL-mediated, apoptosis resistance in senescent cholangiocytes and uncovered that ETS1 and the histone acetyltransferase E1A-binding protein P300 (EP300 or p300) both promote BCL-xL transcription. Using immunofluorescence, we found that BCL-xL protein expression is increased both in cholangiocytes of livers from individuals with PSC and a mouse model of PSC. Using an in vitro model of lipopolysaccharide-induced senescence in normal human cholangiocytes (NHCs), we found increased BCL-xL mRNA and protein levels, and ChIP-PCRs indicated increased occupancy of ETS1, p300, and histone 3 Lys-27 acetylation (H3K27Ac) at the BCL-xL promoter. Using co-immunoprecipitation and proximity ligation assays, we further demonstrate that ETS1 and p300 physically interact in senescent but not control NHCs. Additionally, mutagenesis of predicted ETS1-binding sites within the BCL-xL promoter blocked luciferase reporter activity, and CRISPR/Cas9-mediated genetic deletion of ETS1 reduced senescence-associated BCL-xL expression. In senescent NHCs, TRAIL-mediated apoptosis was reduced ∼70%, and ETS1 deletion or RNAi-mediated BCL-xL suppression increased apoptosis. Overall, our results suggest that ETS1 and p300 promote senescent cholangiocyte resistance to apoptosis by modifying chromatin and inducing BCL-xL expression. These findings reveal ETS1 as a central regulator of both cholangiocyte senescence and the associated apoptosis-resistant phenotype.

Senescent cholangiocytes release extracellular vesicles that alter target cell phenotype via the epidermal growth factor receptor.

BACKGROUND & AIMS: Primary 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. METHODS: EVs 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. RESULTS: EVs 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. CONCLUSIONS: The 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.

Targeting senescent cholangiocytes and activated fibroblasts with B-cell lymphoma-extra large inhibitors ameliorates fibrosis in multidrug resistance 2 gene knockout (Mdr2-/- ) mice.

Cholangiocyte senescence has been linked to primary sclerosing cholangitis (PSC). Persistent secretion of growth factors by senescent cholangiocytes leads to the activation of stromal fibroblasts (ASFs), which are drivers of fibrosis. The activated phenotype of ASFs is characterized by an increased sensitivity to apoptotic stimuli. Here, we examined the mechanisms of apoptotic priming in ASFs and explored a combined targeting strategy to deplete senescent cholangiocytes and ASFs from fibrotic tissue to ameliorate liver fibrosis. Using a coculture system, we determined that senescent cholangiocytes promoted quiescent mesenchymal cell activation in a platelet-derived growth factor (PDGF)-dependent manner. We also identified B-cell lymphoma-extra large (Bcl-xL) as a key survival factor in PDGF-activated human and mouse fibroblasts. Bcl-xL was also up-regulated in senescent cholangiocytes. In vitro, inhibition of Bcl-xL by the small molecule Bcl-2 homology domain 3 mimetic, A-1331852, or Bcl-xL-specific small interfering RNA induced apoptosis in PDGF-activated fibroblasts, but not in quiescent fibroblasts. Likewise, inhibition of Bcl-xL reduced the survival and increased apoptosis of senescent cholangiocytes, compared to nonsenescent cells. Treatment of multidrug resistance 2 gene knockout (Mdr2-/- ) mice with A-1331852 resulted in an 80% decrease in senescent cholangiocytes, a reduction of fibrosis-inducing growth factors and cytokines, decrease of α-smooth muscle actin-positive ASFs, and finally in a significant reduction of liver fibrosis. CONCLUSION: Bcl-xL is a key survival factor in ASFs as well as in senescent cholangiocytes. Treatment with the Bcl-xL-specific inhibitor, A-1331852, reduces liver fibrosis, possibly by a dual effect on activated fibroblasts and senescent cholangiocytes. This mechanism represents an attractive therapeutic strategy in biliary fibrosis. (Hepatology 2018;67:247-259).

ETS Proto-oncogene 1 Transcriptionally Up-regulates the Cholangiocyte Senescence-associated Protein Cyclin-dependent Kinase Inhibitor 2A.

Primary sclerosing cholangitis (PSC) is a chronic, fibroinflammatory cholangiopathy (disease of the bile ducts) of unknown pathogenesis. We reported that cholangiocyte senescence features prominently in PSC and that neuroblastoma RAS viral oncogene homolog (NRAS) is activated in PSC cholangiocytes. Additionally, persistent microbial insult (e.g. LPSs) induces cyclin-dependent kinase inhibitor 2A (CDKN2A/p16INK4a) expression and senescence in cultured cholangiocytes in an NRAS-dependent manner. However, the molecular mechanisms involved in LPS-induced cholangiocyte senescence and NRAS-dependent regulation of CDKN2A remain unclear. Using our in vitro senescence model, we found that LPS-induced CDKN2A expression coincided with a 4.5-fold increase in ETS1 (ETS proto-oncogene 1) mRNA, suggesting that ETS1 is involved in regulating CDKN2A This idea was confirmed by RNAi-mediated suppression or genetic deletion of ETS1, which blocked CDKN2A expression and reduced cholangiocyte senescence. Furthermore, site-directed mutagenesis of a predicted ETS-binding site within the CDKN2A promoter abolished luciferase reporter activity. Pharmacological inhibition of RAS/MAPK reduced ETS1 and CDKN2A protein expression and CDKN2A promoter-driven luciferase activity by ∼50%. In contrast, constitutively active NRAS expression induced ETS1 and CDKN2A protein expression, whereas ETS1 RNAi blocked this increase. Chromatin immunoprecipitation-PCR detected increased ETS1 and histone 3 lysine 4 trimethylation (H3K4Me3) at the CDKN2A promoter following LPS-induced senescence. Additionally, phospho-ETS1 expression was increased in cholangiocytes of human PSC livers and in the Abcb4 (Mdr2)-/- mouse model of PSC. These data pinpoint ETS1 and H3K4Me3 as key transcriptional regulators in NRAS-induced expression of CDKN2A, and this regulatory axis may therefore represent a potential therapeutic target for PSC treatment.

Macrophages contribute to the pathogenesis of sclerosing cholangitis in mice.

BACKGROUND & AIMS: Macrophages contribute to liver disease, but their role in cholestatic liver injury, including primary sclerosing cholangitis (PSC), is unclear. We tested the hypothesis that macrophages contribute to the pathogenesis of, and are therapeutic targets for, PSC. METHODS: Immune cell profile, hepatic macrophage number, localization and polarization, fibrosis, and serum markers of liver injury and cholestasis were measured in an acute (intrabiliary injection of the inhibitor of apoptosis antagonist BV6) and chronic (Mdr2-/- mice) mouse model of sclerosing cholangitis (SC). Selected observations were confirmed in liver specimens from patients with PSC. Because of the known role of the CCR2/CCL2 axis in monocyte/macrophage chemotaxis, therapeutic effects of the CCR2/5 antagonist cenicriviroc (CVC), or genetic deletion of CCR2 (Ccr2-/- mice) were determined in BV6-injected mice. RESULTS: We found increased peribiliary pro-inflammatory (M1-like) and alternatively-activated (M2-like) monocyte-derived macrophages in PSC compared to normal livers. In both SC models, genetic profiling of liver immune cells identified a predominance of monocytes/macrophages; immunohistochemistry confirmed peribiliary monocyte-derived macrophage recruitment (M1>M2-polarized), which paralleled injury onset and was reversed upon resolution in acute SC mice. PSC, senescent and BV6-treated human cholangiocytes released monocyte chemoattractants (CCL2, IL-8) and macrophage-activating factors in vitro. Pharmacological inhibition of monocyte recruitment by CVC treatment or CCR2 genetic deletion attenuated macrophage accumulation, liver injury and fibrosis in acute SC. CONCLUSIONS: Peribiliary recruited macrophages are a feature of both PSC and acute and chronic murine SC models. Pharmacologic and genetic inhibition of peribiliary macrophage recruitment decreases liver injury and fibrosis in mouse SC. These observations suggest monocyte-derived macrophages contribute to the development of SC in mice and in PSC pathogenesis, and support their potential as a therapeutic target. LAY SUMMARY: Primary sclerosing cholangitis (PSC) is an inflammatory liver disease which often progresses to liver failure. The cause of the disease is unclear and therapeutic options are limited. Therefore, we explored the role of white blood cells termed macrophages in PSC given their frequent contribution to other human inflammatory diseases. Our results implicate macrophages in PSC and PSC-like diseases in mice. More importantly, we found that pharmacologic inhibition of macrophage recruitment to the liver reduces PSC-like liver injury in the mouse. These exciting observations highlight potential new strategies to treat PSC.

Metabolomic Profiling of Portal Blood and Bile Reveals Metabolic Signatures of Primary Sclerosing Cholangitis.

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.

Absence of the intestinal microbiota exacerbates hepatobiliary disease in a murine model of primary sclerosing cholangitis.

UNLABELLED: Primary sclerosing cholangitis (PSC) is a chronic, idiopathic, fibroinflammatory cholangiopathy. The role of the microbiota in PSC etiopathogenesis may be fundamentally important, yet remains obscure. We tested the hypothesis that germ-free (GF) mutltidrug resistance 2 knockout (mdr2(-/-) ) mice develop a distinct PSC phenotype, compared to conventionally housed (CV) mdr2(-/-) mice. Mdr2(-/-) mice (n = 12) were rederived as GF by embryo transfer, maintained in isolators, and sacrificed at 60 days in parallel with age-matched CV mdr2(-/-) mice. Serum biochemistries, gallbladder bile acids, and liver sections were examined. Histological findings were validated morphometrically, biochemically, and by immunofluorescence microscopy (IFM). Cholangiocyte senescence was assessed by p16(INK4a) in situ hybridization in liver tissue and by senescence-associated ß-galactosidase staining in a culture-based model of insult-induced senescence. Serum biochemistries, including alkaline phosphatase, aspartate aminotransferase, and bilirubin, were significantly higher in GF mdr2(-/-) (P < 0.01). Primary bile acids were similar, whereas secondary bile acids were absent, in GF mdr2(-/-) mice. Fibrosis, ductular reaction, and ductopenia were significantly more severe histopathologically in GF mdr2(-/-) mice (P < 0.01) and were confirmed by hepatic morphometry, hydroxyproline assay, and IFM. Cholangiocyte senescence was significantly increased in GF mdr2(-/-) mice and abrogated in vitro by ursodeoxycholic acid (UDCA) treatment. CONCLUSIONS: GF mdr2(-/-) mice exhibit exacerbated biochemical and histological features of PSC and increased cholangiocyte senescence, a characteristic and potential mediator of progressive biliary disease. UDCA, a commensal microbial metabolite, abrogates senescence in vitro. These findings demonstrate the importance of the commensal microbiota and its metabolites in protecting against biliary injury and suggest avenues for future studies of biomarkers and therapeutic interventions in PSC.

Role of the Intestinal Microbiome in Cholestatic Liver Disease.

Hepatobiliary health and disease is influenced by multiple factors including genetics, epigenetics, and the environment. Recently, multiple lines of evidence suggest that the microbiome also plays a central role in the initiation and/or progression of several liver diseases. Our current understanding of the dynamic interplay between microbes, microbial products and liver health and pathophysiology is incomplete. However, exciting insights are continually being made that support both a central role of the microbiome and a need for further interrogation of the microbes or microbe-associated molecules involved in the initiation and progression of select liver diseases.

The enteric microbiome in hepatobiliary health and disease.

Increasing evidence points to the contribution of the intestinal microbiome as a potentially key determinant in the initiation and/or progression of hepatobiliary disease. While current understanding of this dynamic is incomplete, exciting insights are continually being made and more are expected given the developments in molecular and high-throughput omics techniques. In this brief review, we provide a practical and updated synopsis of the interaction of the intestinal microbiome with the liver and its downstream impact on the initiation, progression and complications of hepatobiliary disease.

MicroRNAs and benign biliary tract diseases.

Cholangiocytes, the epithelial cells lining the biliary tree, represent only a small portion of the total liver cell population (3-5%), but they are responsible for the secretion of up to 40% of total daily bile volume. In addition, cholangiocytes are the target of a diverse group of liver diseases affecting the biliary tract, the cholangiopathies; for most of these conditions, the pathological mechanisms are unclear. MicroRNAs (miRNAs) are small, noncoding RNAs that posttranscriptionally regulate gene expression. Thus, it is not surprising that altered miRNA profiles underlie the dysregulation of several proteins involved in the pathobiology of the cholangiopathies, as well as showing promise as diagnostic and prognostic tools. Here the authors review recent work relevant to the role of miRNAs in the etiopathogenesis of several of the cholangiopathies (i.e., fibroinflammatory cholangiopathies and polycystic liver diseases), discuss their value as prognostic and diagnostic tools, and provide suggestions for further research.

Development and characterization of human-induced pluripotent stem cell-derived cholangiocytes.

Cholangiocytes are the target of a heterogeneous group of liver diseases known as the cholangiopathies. An evolving understanding of the mechanisms driving biliary development provides the theoretical underpinnings for rational development of induced pluripotent stem cell (iPSC)-derived cholangiocytes (iDCs). Therefore, the aims of this study were to develop an approach to generate iDCs and to fully characterize the cells in vitro and in vivo. Human iPSC lines were generated by forced expression of the Yamanaka pluripotency factors. We then pursued a stepwise differentiation strategy toward iDCs, using precise temporal exposure to key biliary morphogens, and we characterized the cells, using a variety of morphologic, molecular, cell biologic, functional, and in vivo approaches. Morphology shows a stepwise phenotypic change toward an epithelial monolayer. Molecular analysis during differentiation shows appropriate enrichment in markers of iPSC, definitive endoderm, hepatic specification, hepatic progenitors, and ultimately cholangiocytes. Immunostaining, western blotting, and flow cytometry demonstrate enrichment of multiple functionally relevant biliary proteins. RNA sequencing reveals that the transcriptome moves progressively toward that of human cholangiocytes. iDCs generate intracellular calcium signaling in response to ATP, form intact primary cilia, and self-assemble into duct-like structures in three-dimensional culture. In vivo, the cells engraft within mouse liver, following retrograde intrabiliary infusion. In summary, we have developed a novel approach to generate mature cholangiocytes from iPSCs. In addition to providing a model of biliary differentiation, iDCs represent a platform for in vitro disease modeling, pharmacologic testing, and individualized, cell-based, regenerative therapies for the cholangiopathies.

Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.

UNLABELLED: Primary sclerosing cholangitis (PSC) is an incurable cholangiopathy of unknown etiopathogenesis. Here we tested the hypothesis that cholangiocyte senescence is a pathophysiologically important phenotype in PSC. We assessed markers of cellular senescence and senescence-associated secretory phenotype (SASP) in livers of patients with PSC, primary biliary cirrhosis, hepatitis C, and in normals by fluorescent in situ hybridization (FISH) and immunofluorescence microscopy (IFM). We tested whether endogenous and exogenous biliary constituents affect senescence and SASP in cultured human cholangiocytes. We determined in coculture whether senescent cholangiocytes induce senescence in bystander cholangiocytes. Finally, we explored signaling mechanisms involved in cholangiocyte senescence and SASP. In vivo, PSC cholangiocytes expressed significantly more senescence-associated p16(INK4a) and γH2A.x compared to the other three conditions; expression of profibroinflammatory SASP components (i.e., IL-6, IL-8, CCL2, PAI-1) was also highest in PSC cholangiocytes. In vitro, several biologically relevant endogenous (e.g., cholestane 3,5,6 oxysterol) and exogenous (e.g., lipopolysaccharide) molecules normally present in bile induced cholangiocyte senescence and SASP. Furthermore, experimentally induced senescent human cholangiocytes caused senescence in bystander cholangiocytes. N-Ras, a known inducer of senescence, was increased in PSC cholangiocytes and in experimentally induced senescent cultured cholangiocytes; inhibition of Ras abrogated experimentally induced senescence and SASP. CONCLUSION: Cholangiocyte senescence induced by biliary constituents by way of N-Ras activation is an important pathogenic mechanism in PSC. Pharmacologic inhibition of N-Ras with a resultant reduction in cholangiocyte senescence and SASP is a new therapeutic approach for PSC.

Characterization of cultured cholangiocytes isolated from livers of patients with primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic, idiopathic cholangiopathy. The role of cholangiocytes (biliary epithelial cells) in PSC pathogenesis is unknown and remains an active area of research. Here, through cellular, molecular and next-generation sequencing (NGS) methods, we characterize and identify phenotypic and signaling features of isolated PSC patient-derived cholangiocytes. We isolated cholangiocytes from stage 4 PSC patient liver explants by dissection, differential filtration and immune-magnetic bead separation. We maintained cholangiocytes in culture and assessed for: (i) cholangiocyte, cell adhesion and inflammatory markers; (ii) proliferation rate; (iii) transepithelial electrical resistance (TEER); (iv) cellular senescence; and (v) transcriptomic profiles by NGS. We used two well-established normal human cholangiocyte cell lines (H69 and NHC) as controls. Isolated PSC cells expressed cholangiocyte (eg, cytokeratin 7 and 19) and epithelial cell adhesion markers (EPCAM, ICAM) and were negative for hepatocyte and myofibroblast markers (albumin, α-actin). Proliferation rate was lower for PSC compared with normal cholangiocytes (4 vs 2 days, respectively, P<0.01). Maximum TEER was also lower in PSC compared with normal cholangiocytes (100 vs 145 Ωcm(2), P<0.05). Interleukin-6 (IL-6) and IL-8 (protein and mRNA) were both increased compared with NHCs and H69s (all P<0.01). The proportion of cholangiocytes staining positive for senescence-associated ß-galactosidase was higher in PSC cholangiocytes compared with NHCs (48% vs 5%, P<0.01). Finally, NGS confirmed cholangiocyte marker expression in isolated PSC cholangiocytes and extended our findings regarding pro-inflammatory and senescence-associated signaling. In conclusion, we have demonstrated that high-purity cholangiocytes can be isolated from human PSC liver and grown in primary culture. Isolated PSC cholangiocytes exhibit a phenotype that may reflect their in vivo contribution to disease and serve as a vital tool for in vitro investigation of biliary pathobiology and identification of new therapeutic targets in PSC.

Primary sclerosing cholangitis and the microbiota: current knowledge and perspectives on etiopathogenesis and emerging therapies.

Primary sclerosing cholangitis (PSC) is a chronic, fibroinflammatory, cholestatic liver disease of unknown etiopathogenesis. PSC generally progresses to liver cirrhosis, is a major risk factor for hepatobiliary and colonic neoplasia, and confers a median survival to death or liver transplantation of only 12 years. Although it is well recognized that approximately 75% of patients with PSC also have inflammatory bowel disease (IBD), the significance of this association remains elusive. Accumulating evidence now suggests a potentially important role for the intestinal microbiota, and enterohepatic circulation of molecules derived therefrom, as a putative mechanistic link between PSC and IBD and a central pathobiological driver of PSC. In this concise review, we provide a summary of and perspectives regarding the relevant basic, translational, and clinical data, which, taken together, encourage further investigation of the role of the microbiota and microbial metabolites in the etiopathogenesis of PSC and as a potential target for novel pharmacotherapies.

Micro-computed tomography and nuclear magnetic resonance imaging for noninvasive, live-mouse cholangiography.

The cholangiopathies are a diverse group of biliary tract disorders, many of which lack effective treatment. Murine models are an important tool for studying their pathogenesis, but existing noninvasive methods for assessing biliary disease in vivo are not optimal. Here we report our experience with using micro-computed tomography (microCT) and nuclear magnetic resonance (MR) imaging to develop a technique for live-mouse cholangiography. Using mdr2 knockout (mdr2KO, a model for primary sclerosing cholangitis (PSC)), bile duct-ligated (BDL), and normal mice, we performed in vivo: (1) microCT on a Siemens Inveon PET/CT scanner and (2) MR on a Bruker Avance 16.4 T spectrometer, using Turbo Rapid Acquisition with Relaxation Enhancement, IntraGate Fast Low Angle Shot, and Half-Fourier Acquisition Single-shot Turbo Spin Echo methods. Anesthesia was with 1.5-2.5% isoflurane. Scans were performed with and without contrast agents (iodipamide meglumine (microCT), gadoxetate disodium (MR)). Dissection and liver histology were performed for validation. With microCT, only the gallbladder and extrahepatic bile ducts were visualized despite attempts to optimize timing, route, and dose of contrast. With MR, the gallbladder, extra-, and intrahepatic bile ducts were well-visualized in mdr2KO mice; the cholangiographic appearance was similar to that of PSC (eg, multifocal strictures) and could be improved with contrast administration. In BDL mice, MR revealed cholangiographically distinct progressive dilation of the biliary tree without ductal irregularity. In normal mice, MR allowed visualization of the gallbladder and extrahepatic ducts, but only marginal visualization of the diminutive intrahepatic ducts. One mouse died during microCT and MR imaging, respectively. Both microCT and MR scans could be obtained in ≤20 min. We, therefore, demonstrate that MR cholangiography can be a useful tool for longitudinal studies of the biliary tree in live mice, whereas microCT yields suboptimal duct visualization despite requiring contrast administration. These findings support further development and application of MR cholangiography to the study of mouse models of PSC and other cholangiopathies.

The dynamic biliary epithelia: molecules, pathways, and disease.

Cholangiocytes, the cells lining bile ducts, are a heterogenous, highly dynamic population of epithelial cells. While these cells comprise a small fraction of the total cellular component of the liver, they perform the essential role of bile modification and transport of biliary and blood constituents. From a pathophysiological standpoint, cholangiocytes are the target of a diverse group of biliary disorders, collectively referred to as the cholangiopathies. To date, the cause of most cholangiopathies remains obscure. It is known, however, that cholangiocytes exist in an environment rich in potential mediators of cellular injury, express receptors that recognize potential injurious insults, and participate in portal tract repair processes following hepatic injury. As such, cholangiocytes may not be only a passive target, but are likely directly and actively involved in the pathogenesis of cholangiopathies. Here, we briefly summarize the characteristics of the reactive cholangiocyte and cholangiocyte responses to potentially injurious endogenous and exogenous molecules, and in addition, present emerging concepts in our understanding of the etiopathogenesis of several cholangiopathies.