Search

Arctic populations are amongst the highest exposed populations to long-range transported contaminants globally, with the main exposure pathway being through the diet. Dietary advice is an important immediate means to address potential exposure and help minimize adverse health effects. The objective of this work is to enable easier access to dietary advice and communication guidance on contaminants with a focus on the Arctic. This manuscript is part of a special issue summarizing the Arctic Monitoring and Assessment Programme's Assessment 2021: Human Health in the Arctic. The information was derived with internet searches, and by contacting relevant experts directly. Results include risk communication efforts in European Arctic countries, effectiveness evaluation studies for several Arctic countries, experience of social media use, and the advantages and challenges of using social media in risk communication. We found that current risk communication activities in most Arctic countries emphasize the importance of a nutritious diet. Contaminant-related restrictions are mostly based on mercury; a limited amount of dietary advice is based on other contaminants. While more information on effectiveness evaluation was available, specific information, particularly from Arctic countries other than Canada, is still very limited.

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., (Copyright © 2024 American Association for the Study of Liver Diseases.)

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.

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., (Copyright © 2023 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Vascular smooth muscle cells (VSMCs) help to maintain the normal physiological contractility of arterial vessels to control blood pressure; they can also contribute to vascular disease such as atherosclerosis. Ca 2+ /calmodulin-dependent kinase II (CaMKII), a multifunctional enzyme with four isoforms and multiple alternative splice variants, contributes to numerous functions within VSMCs. The role of these isoforms has been widely studied across numerous tissue types; however, their functions are still largely unknown within the vasculature. Even more understudied is the role of the different splice variants of each isoform in such signaling pathways. This review evaluates the role of the different CaMKII splice variants in vascular pathological and physiological mechanisms, aiming to show the need for more research to highlight both the deleterious and protective functions of the various splice variants.

The cholangiopathies are a group of liver diseases that affect cholangiocytes, the epithelial cells that line the bile ducts. Biliary atresia (BA), primary biliary cholangitis (PBC), and primary sclerosing cholangitis (PSC) are three cholangiopathies with significant immune-mediated pathogenesis where chronic inflammation and fibrosis lead to obliteration of bile ducts and eventual liver cirrhosis. Cellular senescence is a state of cell cycle arrest in which cells become resistant to apoptosis and profusely secrete a bioactive secretome. Recent evidence indicates that cholangiocyte senescence contributes to the pathogenesis of BA, PBC, and PSC. This review explores the role of cholangiocyte senescence in BA, PBC, and PSC, ascertains how cholangiocyte senescence may promote a senescence-associated immunopathology in these cholangiopathies, and provides the rationale for therapeutically targeting senescence as a treatment option for BA, PBC, and PSC., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

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., (© 2021 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Background & Aims: Cholangiocyte senescence is important in the pathogenesis of primary sclerosing cholangitis (PSC). We found that CDKN2A (p16), a cyclin-dependent kinase inhibitor and mediator of senescence, was increased in cholangiocytes of patients with PSC and from a PSC mouse model (multidrug resistance 2; Mdr2 -/- ). Given that recent data suggest that a reduction of senescent cells is beneficial in different diseases, we hypothesised that inhibition of cholangiocyte senescence would ameliorate disease in Mdr2 -/- mice., Methods: We used 2 novel genetic murine models to reduce cholangiocyte senescence: (i) p16 Ink4a apoptosis through targeted activation of caspase (INK-ATTAC)x Mdr2 -/- , in which the dimerizing molecule AP20187 promotes selective apoptotic removal of p16 -expressing cells; and (ii) mice deficient in both p16 and Mdr2 . Mdr2 -/- mice were also treated with fisetin, a flavonoid molecule that selectively kills senescent cells. p16, p21, and inflammatory markers (tumour necrosis factor [TNF]-α, IL-1β, and monocyte chemoattractant protein-1 [MCP-1]) were measured by PCR, and hepatic fibrosis via a hydroxyproline assay and Sirius red staining., Results: AP20187 treatment reduced p16 and p21 expression by ~35% and ~70% ( p >0.05), respectively. Expression of inflammatory markers (TNF-α, IL-1β, and MCP-1) decreased (by 60%, 40%, and 60%, respectively), and fibrosis was reduced by ~60% ( p >0.05). Similarly, p16 -/- xMdr2 -/- mice exhibited reduced p21 expression (70%), decreased expression of TNF-α, IL-1β (60%), and MCP-1 (65%) and reduced fibrosis (~50%) ( p >0.05) compared with Mdr2 -/- mice. Fisetin treatment reduced expression of p16 and p21 (80% and 90%, respectively), TNF-α (50%), IL-1β (50%), MCP-1 (70%), and fibrosis (60%) ( p >0.05)., Conclusions: Our data support a pathophysiological role of cholangiocyte senescence in the progression of PSC, and that targeted removal of senescent cholangiocytes is a plausible therapeutic approach., Lay Summary: Primary sclerosing cholangitis is a fibroinflammatory, incurable biliary disease. We previously reported that biliary epithelial cell senescence (cell-cycle arrest and hypersecretion of profibrotic molecules) is an important phenotype in primary sclerosing cholangitis. Herein, we demonstrate that reducing the number of senescent cholangiocytes leads to a reduction in the expression of inflammatory, fibrotic, and senescence markers associated with the disease., Competing Interests: The authors have no conflict of interest related to the manuscript. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2021 The Authors.)

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 10 8 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., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (© 2019 O'Hara et al.)

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.

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., (Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

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)., (© 2017 by the American Association for the Study of Liver Diseases.)

Competing Interests: Competing interests: None declared.

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/p16 INK4a ) 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., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (© 2017 S. Karger AG, Basel.)

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., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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 β-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., (© 2015 by the American Association for the Study of Liver Diseases.)

The healthspan of mice is enhanced by killing senescent cells using a transgenic suicide gene. Achieving the same using small molecules would have a tremendous impact on quality of life and the burden of age-related chronic diseases. Here, we describe the rationale for identification and validation of a new class of drugs termed senolytics, which selectively kill senescent cells. By transcript analysis, we discovered increased expression of pro-survival networks in senescent cells, consistent with their established resistance to apoptosis. Using siRNA to silence expression of key nodes of this network, including ephrins (EFNB1 or 3), PI3Kδ, p21, BCL-xL, or plasminogen-activated inhibitor-2, killed senescent cells, but not proliferating or quiescent, differentiated cells. Drugs targeting these same factors selectively killed senescent cells. Dasatinib eliminated senescent human fat cell progenitors, while quercetin was more effective against senescent human endothelial cells and mouse BM-MSCs. The combination of dasatinib and quercetin was effective in eliminating senescent MEFs. In vivo, this combination reduced senescent cell burden in chronologically aged, radiation-exposed, and progeroid Ercc1(-/Δ) mice. In old mice, cardiac function and carotid vascular reactivity were improved 5 days after a single dose. Following irradiation of one limb in mice, a single dose led to improved exercise capacity for at least 7 months following drug treatment. Periodic drug administration extended healthspan in Ercc1(-/∆) mice, delaying age-related symptoms and pathology, osteoporosis, and loss of intervertebral disk proteoglycans. These results demonstrate the feasibility of selectively ablating senescent cells and the efficacy of senolytics for alleviating symptoms of frailty and extending healthspan., (© 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

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.

Cholangiocytes (biliary epithelial cells) actively participate in microbe-induced proinflammatory responses in the liver and contribute to inflammatory and infectious cholangiopathies. We previously demonstrated that cholangiocyte TLR-dependent NRas activation contributes to proinflammatory/ proliferative responses. We test the hypothesis that LPS-induced activation of NRas requires the EGFR. SV40-transformed human cholangiocytes (H69 cells), or low passage normal human cholangiocytes (NHC), were treated with LPS in the presence or absence of EGFR or ADAM metallopeptidase domain 17 (TACE) inhibitors. Ras activation assays, quantitative RT-PCR, and proliferation assays were performed in cells cultured with or without inhibitors or an siRNA to Grb2. Immunofluorescence for phospho-EGFR was performed on LPS-treated mouse samples and specimens from patients with primary sclerosing cholangitis, primary biliary cirrhosis, hepatitis C, and normal livers. LPS-treatment induced an association between the TLR/MyD88 and EGFR/Grb2 signaling apparatus, NRas activation, and EGFR phosphorylation. NRas activation was sensitive to EGFR and TACE inhibitors and correlated with EGFR phosphorylation. The TACE inhibitor and Grb2 depletion prevented LPS-induced IL6 expression (p<0.05) and proliferation (p<0.01). Additionally, cholangiocytes from LPS-treated mouse livers and human primary sclerosing cholangitis (PSC) livers exhibited increased phospho-EGFR (p<0.01). Moreover, LPS-induced mouse cholangiocyte proliferation was inhibited by concurrent treatment with the EGFR inhibitor, Erlotinib. Our results suggest that EGFR is essential for LPS-induced, TLR4/MyD88-mediated NRas activation and induction of a robust proinflammatory cholangiocyte response. These findings have implications not only for revealing the signaling potential of TLRs, but also implicate EGFR as an integral component of cholangiocyte TLR-induced proinflammatory processes.

Competing Interests: Potential conflict of interest: Nothing to report.

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., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

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 β-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.

Cholangiocytes, the cells lining bile ducts, comprise a small fraction of the total cellular component of the liver, yet perform the essential role of bile modification and transport of biliary and blood constituents. Cholangiopathies are a diverse group of biliary disorders with the cholangiocyte as the target cell; the etiopathogenesis of most cholangiopathies remains obscure. MicroRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression. These small RNAs may not only be involved in the etiopathogenesis of disease, but are showing promise as diagnostic and prognostic tools. In this brief review, we summarize recent work regarding the role of microRNAs in the etiopathogenesis of several cholangiopathies, and discuss their utility as prognostic and diagnostic tools.

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.

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., (© 2014 by the American Association for the Study of Liver Diseases.)

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.

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., (Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

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.

Cholangiocytes, the epithelial cells lining intrahepatic bile ducts, express multiple toll-like receptors (TLRs) and, thus, have the capacity to recognize and respond to microbial pathogens. In previous work, we demonstrated that TLR4, which is activated by gram-negative lipopolysaccharide (LPS), is upregulated in cholangiocytes in response to infection with Cryptosporidium parvum in vitro and contributes to nuclear factor-kappaB (NF-kB) activation. Here, using an in vivo model of biliary cryptosporidiosis, we addressed the functional role of TLR4 in C. parvum infection dynamics and hepatobiliary pathophysiology. We observed that C57BL mice clear the infection by 3 wk post-infection (PI). In contrast, parasites were detected in bile and stool in TLR4-deficient mice at 4 wk PI. The liver enzymes alanine transaminase (ALT) and aspartate transaminase (AST), and the proinflammatory cytokines tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-6 peaked at 1 to 2 wk PI and normalized by 4 wk in infected C57BL mice. C57BL mice also demonstrated increased cholangiocyte proliferation (PCNA staining) at 1 wk PI that was resolved by 2 wk PI. In contrast, TLR4-deficient mice showed persistently elevated serum ALT and AST, elevated hepatic IL-6 levels, and histological evidence of hepatocyte necrosis, increased inflammatory cell infiltration, and cholangiocyte proliferation through 4 wk PI. These data suggest that a TLR4-mediated response is required for efficient eradication of biliary C. parvum infection in vivo, and lack of this pattern-recognition receptor contributes to an altered inflammatory response and an increase in hepatobiliary pathology.

Cholangiocytes, the epithelial cells lining the bile ducts in the liver, are periodically exposed to potentially injurious microbes and/or microbial products. As a result, cholangiocytes actively participate in microbe-associated, hepatic proinflammatory responses. We previously showed that infection of cultured human cholangiocytes with the protozoan parasite, Cryptosporidium parvum, or treatment with gram-negative bacteria-derived LPS, activates NFκB in a myeloid differentiation 88 (MyD88)-dependent manner. Here, we describe a novel signaling pathway initiated by Toll-like receptors (TLRs) involving the small GTPase, Ras, that mediates cholangiocyte proinflammatory cytokine production and induction of cholangiocyte proliferation. Using cultured human cholangiocytes and a Ras activation assay, we found that agonists of plasma membrane TLRs (TLR 1, 2, 4, 5, and 6) rapidly (<10 min) activated N-Ras, but not other p21 Ras isoforms, resulting in the rapid (<15 min) phosphorylation of the downstream Ras effector, ERK1/2. RNA interference-induced depletion of TRAF6, a downstream effector of MyD88 and known activator of MAPK signaling, had no effect on N-Ras activation. Following N-Ras activation the proinflammatory cytokine, IL6, is rapidly secreted. Using a luciferase reporter, we demonstrated that LPS treatment induced IL6 promoter-driven luciferase which was suppressed using MEK/ERK pharmacologic inhibitors (PD98059 or U0126) and RNAi-induced depletion of N-Ras. Finally, we showed that LPS increased cholangiocyte proliferation (1.5-fold), which was inhibited by depletion of N-Ras; TLR agonist-induced proliferation was also inhibited following pretreatment with an IL6 receptor-blocking antibody. Together, our results support a novel signaling axis involving microbial activation of N-Ras likely involved in the cholangiocyte pathogen-induced proinflammatory response.

Mucosal surface epithelial cells are equipped with several defense mechanisms that guard against pathogens. Recent studies indicate that microRNAs (miRNAs) mediate post-transcriptional gene suppression and may be a critical component of the complex regulatory networks in epithelial immune responses. Transcription of miRNA genes in epithelial cells can be elaborately controlled through pathogen recognition receptors, such as Toll-like receptors (TLRs), and associated nuclear factor kappaB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, and ultimately nuclear transcription factor associated-transactivation and transrepression. Activation of these intracellular signaling pathways may also modulate the process of miRNA maturation. Functionally, miRNAs may modulate epithelial immune responses at every step of the innate immune network, including production and release of cytokines/chemokines, expression of adhesion and costimulatory molecules, shuttling of miRNAs through release of exosomes and feedback regulation of immune homeostasis. Therefore, miRNAs act as critical regulators to the fine-tuning of epithelial immune responses.

Cryptosporidiosis remains a significant cause of enteric disease worldwide. Basic investigations of host: pathogen interactions have revealed the intricate processes mediating infection. The following summarizes the interactions that mediate infection and the host responses that both permit and ultimately clear the infection., (Copyright © 2011 Institut Pasteur. Published by Elsevier SAS. All rights reserved.)

In the eyes of many, the steel city of Gary, Indiana, entered a period of decline in the middle of the twentieth century. The once great city was seemingly racked by job loss, crime, racial division, or moral decay. Which of these caused the decline of the city depended upon the perspective of the story's teller. Each narrative of decline contained a different moment where the city went wrong and it began to decay. For some it was the moral decay of the 1950s, for others it was the rise of black power and politics in the 1960s, for still others it was the white backlash against civil rights in the 1970s. Some saw a microcosm of America, some saw a dangerous cauldron of race and ethnicity. The source of decline and the origins of the urban crisis were largely in the eye of the beholder. The stories people chose to tell about Gary mattered because for much of the twentieth century, Gary was at the center of American narratives about industrialism. These were outsider narratives of decline read onto the Indiana steel city because Gary represented larger debates. People read onto Gary their changing expectations and anxieties about industry and industrial spaces. This article traces the changing attitudes outsiders held toward Gary from the middle of the twentieth century through the period of deindustrialization at the end of the century and examines American narratives about deindustrialization and urban decline.

Biliary tract infection with the Group I carcinogenic liver fluke Opisthorchis viverrini is associated with severe inflammation leading to cholangiocarcinoma--a major biliary cancer in Southeast Asia. However, mechanism(s) by which the liver fluke induces host mucosal immune/inflammatory responses is unclear. In the present study we address whether a normal immortalized human cholangiocyte cell line (H69 cells) recognizes and responds to O. viverrini excretory/secretory products (OVES). Expression of multiple TLRs, activation of NF-κB, and expression of pro-inflammatory cytokines were monitored in the presence and absence of OVES. Our results showed that OVES induced increased cholangiocyte TLR4 mRNA expression, induced IκB-α degradation in a MyD88-dependent manner, and activated NF-κB nuclear translocation. Moreover, OVES induced expression and secretion of the strong chemoattractant chemokine interleukin 8 (IL-8) and pro-inflammatory cytokine IL-6. These results demonstrate that secreted/excreted products of O. viverrini are recognized by human cholangiocytes and initiate innate mucosal immunity/inflammatory cascades, a primary event in the pathogenesis of opisthorchiasis and cholangiocarcinoma., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Unlabelled: Angiogenesis defines the growth of new blood vessels from preexisting vascular endothelial networks and corresponds to the wound healing process that is typified by the process of liver fibrosis. Liver fibrosis is also associated with increased endotoxin within the gut lumen and its associated portal circulation. However, the interrelationship of gut endotoxin and its receptor, toll-like receptor 4 (TLR4), with liver fibrosis and associated angiogenesis remains incompletely defined. Here, using complementary genetic, molecular, and pharmacological approaches, we provide evidence that the pattern recognition receptor that recognizes endotoxin, TLR4, which is expressed on liver endothelial cells (LECs), regulates angiogenic responses both in vitro and in vivo. Mechanistic studies have revealed a key role for a cognate TLR4 effector protein, myeloid differentiation protein 88 (MyD88), in this process, which culminates in extracellular protease production that regulates the invasive capacity of LECs, a key step in angiogenesis. Furthermore, TLR4-dependent angiogenesis in vivo corresponds to fibrosis in complementary liver models of fibrosis., Conclusion: These studies provide evidence that the TLR4 pathway in LECs regulates angiogenesis through its MyD88 effector protein by regulating extracellular protease production and that this process is linked to the development of liver fibrosis.

Internalization of the obligate intracellular apicomplexan parasite, Cryptosporidium parvum, results in the formation of a unique intramembranous yet extracytoplasmic niche on the apical surfaces of host epithelial cells, a process that depends on host cell membrane extension. We previously demonstrated that efficient C. parvum invasion of biliary epithelial cells (cholangiocytes) requires host cell actin polymerization and localized membrane translocation/insertion of Na(+)/glucose cotransporter 1 (SGLT1) and of aquaporin 1 (Aqp1), a water channel, at the attachment site. The resultant localized water influx facilitates parasite cellular invasion by promoting host-cell membrane protrusion. However, the molecular mechanisms by which C. parvum induces membrane translocation/insertion of SGLT1/Aqp1 are obscure. We report here that cultured human cholangiocytes express several nonmuscle myosins, including myosins IIA and IIB. Moreover, C. parvum infection of cultured cholangiocytes results in the localized selective aggregation of myosin IIB but not myosin IIA at the region of parasite attachment, as assessed by dual-label immunofluorescence confocal microscopy. Concordantly, treatment of cells with the myosin light chain kinase inhibitor ML-7 or the myosin II-specific inhibitor blebbistatin or selective RNA-mediated repression of myosin IIB significantly inhibits (P < 0.05) C. parvum cellular invasion (by 60 to 80%). Furthermore ML-7 and blebbistatin significantly decrease (P < 0.02) C. parvum-induced accumulation of SGLT1 at infection sites (by approximately 80%). Thus, localized actomyosin-dependent membrane translocation of transporters/channels initiated by C. parvum is essential for membrane extension and parasite internalization, a phenomenon that may also be relevant to the mechanisms of cell membrane protrusion in general.

MicroRNAs, central players of numerous cellular processes, regulate mRNA stability or translational efficiency. Although these molecular events are established, the mechanisms regulating microRNA function and expression remain largely unknown. The microRNA let-7i regulates Toll-like receptor 4 expression. Here, we identify a novel transcriptional mechanism induced by the protozoan parasite Cryptosporidium parvum and Gram(-) bacteria-derived lipopolysaccharide (LPS) mediating let-7i promoter silencing in human biliary epithelial cells (cholangiocytes). Using cultured cholangiocytes, we show that microbial stimulus decreased let-7i expression, and promoter activity. Analysis of the mechanism revealed that microbial infection promotes the formation of a NFkappaB p50-C/EBPbeta silencer complex in the regulatory sequence. Chromatin immunoprecipitation assays (ChIP) demonstrated that the repressor complex binds to the let-7i promoter following microbial stimulus and promotes histone-H3 deacetylation. Our results provide a novel mechanism of transcriptional regulation of cholangiocyte let-7i expression following microbial insult, a process with potential implications for epithelial innate immune responses in general.

Biliary cryptosporidiosis is associated with acquired immunodeficiency syndrome (AIDS) cholangiopathy and occurs almost exclusively in adult patients with AIDS. Infection of biliary epithelial cells (cholangiocytes) with Cryptosporidium parvum induces Toll-like receptor (TLR) 4 expression and stimulates a TLR-dependent response against infection. Here, we tested whether human immunodeficiency virus type 1 (HIV-1) Tat affects TLR expression and, hence, anti-C. parvum defense responses. Using an in vitro model of human biliary cryptosporidiosis, we found that recombinant Tat protein increased TLR4 mRNA expression in both uninfected and C. parvum-infected cholangiocytes. Conversely, Tat decreased TLR4 protein levels and suppressed C. parvum-induced TLR4 protein expression. Using actinomycin to inhibit transcription, we found that Tat increased the half-life of TLR4 mRNA from approximately 25 to 60 min, and RNA gel-shift assays demonstrated direct binding of Tat to TLR4 mRNA. In vitro transcription/translation studies suggested that Tat does not affect transcription but does decrease TLR4 translation. Importantly, more parasites were found in Tat-treated cells than in control cells 48 h after infection. These findings suggest that Tat inhibits cholangiocyte TLR4 protein expression through translational inhibition. These events appear to diminish the ability of cholangiocytes to initiate an innate immune response to C. parvum. We suggest that these findings may contribute to the unusual susceptibility of HIV-infected individuals to biliary cryptosporidiosis.

Biliary epithelial cells (cholangiocytes) respond to proinflammatory cytokines such as IFN-gamma and actively participate in the regulation of biliary inflammatory response in the liver. B7-H1 (also known as CD274 or PD-L1) is a member of the B7 costimulatory molecules and plays a critical immunoregulatory role in cell-mediated immune responses. In this study, we show that resting human cholangiocytes in culture express B7-H1 mRNA, but not B7-H1 protein. IFN-gamma induces B7-H1 protein expression and alters the microRNA (miRNA) expression profile in cholangiocytes. Of those IFN-gamma-down-regulated miRNAs, we identified microRNA-513 (miR-513) with complementarity to the 3'-untranslated region of B7-H1 mRNA. Targeting of the B7-H1 3'-untranslated region by miR-513 results in translational repression. Transfection of cholangiocytes with an antisense oligonucleotide to miR-513 induces B7-H1 protein expression. Additionally, transfection of miR-513 precursor decreases IFN-gamma-induced B7-H1 protein expression and consequently influences B7-H1-associated apoptotic cell death in cocultured Jurkat cells. Thus, miR-513 regulates B7-H1 translation and is involved in IFN-gamma-induced B7-H1 expression in human cholangiocytes, suggesting a role for miRNA-mediated gene silencing in the regulation of cholangiocyte response to IFN-gamma.