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Taurocholate Induces Biliary Differentiation of Liver Progenitor Cells Causing Hepatic Stellate Cell Chemotaxis in the Ductular Reaction

Role in Pediatric Cystic Fibrosis Liver Disease
Open ArchivePublished:September 18, 2017DOI:https://doi.org/10.1016/j.ajpath.2017.08.024
      Cystic fibrosis liver disease (CFLD) in children causes progressive fibrosis leading to biliary cirrhosis; however, its cause(s) and early pathogenesis are unclear. We hypothesized that a bile acid–induced ductular reaction (DR) drives fibrogenesis. The DR was evaluated by cytokeratin-7 immunohistochemistry in liver biopsies, staged for fibrosis, from 60 children with CFLD, and it demonstrated that the DR was significantly correlated with hepatic fibrosis stage and biliary taurocholate levels. To examine the mechanisms involved in DR induction, liver progenitor cells (LPCs) were treated with taurocholate, and key events in DR evolution were assessed: LPC proliferation, LPC biliary differentiation, and hepatic stellate cell (HSC) chemotaxis. Taurocholate induced a time-dependent increase in LPC proliferation and expression of genes associated with cholangiocyte differentiation (cytokeratin 19, connexin 43, integrin β4, and γ-glutamyltranspeptidase), whereas the hepatocyte specification marker HNF4α was suppressed. Functional cholangiocyte differentiation was demonstrated via increased acetylated α-tubulin and SOX9 proteins, the number of primary cilia+ LPCs, and increased active γ-glutamyltranspeptidase enzyme secretion. Taurocholate induced LPCs to release MCP-1, MIP1α, and RANTES into conditioned medium causing HSC chemotaxis, which was inhibited by anti-MIP1α. Immunofluorescence confirmed chemokine expression localized to CK7+ DR and LPCs in CFLD liver biopsies. This study suggests that taurocholate is involved in initiating functional LPC biliary differentiation and the development of the DR, with subsequent induction of chemokines that drive HSC recruitment in CFLD.
      Cystic fibrosis–associated liver disease (CFLD) is characterized by indolent intrahepatic cholestasis that usually manifests by early adolescence.
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      This has led to the hypothesis that the two processes of LPC expansion and HSC activation with the resultant DR and fibrosis, respectively, are closely related. Although histological evidence of a DR has previously been demonstrated in children with extrahepatic biliary atresia,
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      the potential role of LPCs driving HSC activation and fibrosis via development of the DR has not previously been assessed in CFLD.
      Bile acids are synthesized in hepatocytes from cholesterol and are usually conjugated to taurine or glycine to form bile salts and secreted into the bile canaliculi. These bile secretions flow into bile ducts, where they are stabilized by bicarbonate ions and water secreted by cholangiocytes as they are excreted from the liver into the gall bladder. They aid lipid digestion in the duodenum by forming micelles. However, their partial hydrophobic nature, which is essential in forming micelles, also makes them toxic to the liver in higher concentrations.
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      Taurocholate and other bile salts are able to up-regulate the expression of inflammatory genes in the liver.
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      Taurocholate has also been shown to stimulate cytokine secretion from cholangiocytes
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      which induce transdifferentiation of portal fibroblasts
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      Secretion of MCP-1/CCL2 by bile duct epithelia induces myofibroblastic transdifferentiation of portal fibroblasts.
      as well as facilitating HSC migration.
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      We previously demonstrated that taurocholate induced the expression of monocyte chemotaxis protein-1 (MCP-1) in normal hepatocytes, and that MCP-1 produced by cholestatic hepatocytes from bile duct-ligated rats induced the chemotaxis of HSCs.
      • Ramm G.A.
      • Shepherd R.W.
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      • Ney A.D.
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      • Lewindon P.J.
      Fibrogenesis in pediatric cholestatic liver disease: role of taurocholate and hepatocyte-derived monocyte chemotaxis protein-1 in hepatic stellate cell recruitment.
      Markedly increased expression of MCP-1 was observed in hepatocytes and cholangiocytes in the liver of children with cholestatic liver diseases.
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      • Turlin B.
      • Lewindon P.J.
      Fibrogenesis in pediatric cholestatic liver disease: role of taurocholate and hepatocyte-derived monocyte chemotaxis protein-1 in hepatic stellate cell recruitment.
      In this study, we investigated whether the DR is present in the liver of children with CFLD and examined mechanisms associated with its induction and role in fibrogenesis. We compared the severity of the DR in CFLD liver biopsies with those from other, more aggressively cholestatic pediatric liver disease and then specifically examined a potential mechanism of DR induction in CFLD through investigating the relationship between biliary taurocholate concentrations and the DR in liver biopsies from children with CFLD in vivo. We then used in vitro cell culture models and ex vivo liver slices to investigate the in vivo observed effects of elevated taurocholate on LPC differentiation, the secretion of chemokines by LPCs, and their chemotactic effects on HSCs in driving hepatic fibrogenesis.

      Materials and Methods

      Patient Specimens

      This study is part of a long-term prospective cohort study of patients with CFLD, identified and referred from the CF Clinic, Royal Children's Hospital, Brisbane, that is previously well documented.
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      • Setchell K.D.
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      • Ramm G.A.
      Endogenous ursodeoxycholic acid and cholic acid in liver disease due to cystic fibrosis.
      • Ramm G.A.
      • Shepherd R.W.
      • Hoskins A.C.
      • Greco S.A.
      • Ney A.D.
      • Pereira T.N.
      • Bridle K.R.
      • Doecke J.D.
      • Meikle P.J.
      • Turlin B.
      • Lewindon P.J.
      Fibrogenesis in pediatric cholestatic liver disease: role of taurocholate and hepatocyte-derived monocyte chemotaxis protein-1 in hepatic stellate cell recruitment.
      • Lewindon P.J.
      • Pereira T.N.
      • Hoskins A.C.
      • Bridle K.R.
      • Williamson R.M.
      • Shepherd R.W.
      • Ramm G.A.
      The role of hepatic stellate cells and transforming growth factor-beta(1) in cystic fibrosis liver disease.
      • Lewindon P.J.
      • Shepherd R.W.
      • Walsh M.J.
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      • Williamson R.
      • Pereira T.N.
      • Frawley K.
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      • Smith J.L.
      • Ramm G.A.
      Importance of hepatic fibrosis in cystic fibrosis and the predictive value of liver biopsy.
      • Pereira T.N.
      • Lewindon P.J.
      • Smith J.L.
      • Murphy T.L.
      • Lincoln D.J.
      • Shepherd R.W.
      • Ramm G.A.
      Serum markers of hepatic fibrogenesis in cystic fibrosis liver disease.
      CFLD was defined as two of the following: i) hepatomegaly with or without splenomegaly; ii) persistent (>6 months) elevation of serum alanine aminotransferase (>1.5× upper limit normal); and iii) abnormal liver ultrasound (abnormal echogenicity or nodular edge). Those with liver synthetic dysfunction or a history of hepatobiliary surgery were excluded. The study conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the ethics committees of the Royal Children's Hospital, Brisbane, and the QIMR Berghofer Medical Research Institute. Informed consent was obtained from parents or patients.
      Liver tissue was available from 60 children with CFLD (age, 11.8 ± 4.5 years). For comparison with other pediatric cholestatic liver diseases, liver tissue was also available from three infants with biliary atresia (age, 1.2 ± 1.4 years), five with idiopathic neonatal hepatitis (age, 3.4 ± 3.6 years), as well as four pediatric donor liver controls (age, 6.8 ± 4.5 years). Bile was available from 7 children with CFLD.

      Immunohistochemistry

      Formalin-fixed, paraffin-embedded liver sections were subjected to heat antigen retrieval, before exposure to 3% hydrogen peroxide in phosphate-buffered saline and Background Sniper (Biocare Medical, Pacheco, CA). Sections were then stained with primary antibody: mouse anti-human CK7 monoclonal antibody (dilution 1:100; Dako, Glostrup, Denmark), secondary antibody: rabbit anti-mouse biotinylated IgG (dilution 1:400, Dako), with detection using a streptavidin-biotin/horseradish peroxidase kit (Dako) and chromogenic substrate 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St. Louis, MO), and counterstained with hematoxylin. Digital images were captured using Aperio ScanScope XT slide scanner (Leica Biosystems, Buffalo Grove, IL) under ×20 magnification at 0.5 μm/pixel. Image analysis was performed using Aperio Positive Pixel Count algorithm software version 9.1. Positively stained cells were counted, normalized to the total section area, and results correlated with Scheuer's hepatic fibrosis staging.

      Biliary Taurocholate Measurement

      Taurocholate concentration was measured in endoscopic bile samples available from seven patients with CFLD, as previously described.
      • Ramm G.A.
      • Shepherd R.W.
      • Hoskins A.C.
      • Greco S.A.
      • Ney A.D.
      • Pereira T.N.
      • Bridle K.R.
      • Doecke J.D.
      • Meikle P.J.
      • Turlin B.
      • Lewindon P.J.
      Fibrogenesis in pediatric cholestatic liver disease: role of taurocholate and hepatocyte-derived monocyte chemotaxis protein-1 in hepatic stellate cell recruitment.
      Liver biopsies were processed for histochemical CK7 quantification as above. All patients had histological evidence of hepatic fibrosis. Biliary taurocholate levels were correlated with CK7 quantification.

      Culture of Cell Lines and in Vitro Studies

      The murine LPC line PIL-2
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      • Yeoh G.C.
      • Quail E.A.
      Generation and characterization of p53 null transformed hepatic progenitor cells: oval cells give rise to hepatocellular carcinoma.
      was cultured in Williams' E medium containing 2 mmol/L glutamine, penicillin (100 U/mL), streptomycin (100 μg/mL), insulin (10 μg/mL), epidermal growth factor (10 ng/mL), insulin-like growth factor II (30 ng/mL), and 10% fetal bovine serum (FBS). The human HSC line LX-2 was cultured in Dulbecco's modified Eagle's medium with 2 mmol/L glutamine, penicillin (100 U/mL), streptomycin (100 μg/mL), and 2% FBS. PIL-2 cells were treated with taurocholate at 0 to 300 μmol/L concentration to establish a dose-response, with an optimal concentration of 150 μmol/L taurocholate chosen for all in vitro and ex vivo experiments, as previously described.
      • Ramm G.A.
      • Shepherd R.W.
      • Hoskins A.C.
      • Greco S.A.
      • Ney A.D.
      • Pereira T.N.
      • Bridle K.R.
      • Doecke J.D.
      • Meikle P.J.
      • Turlin B.
      • Lewindon P.J.
      Fibrogenesis in pediatric cholestatic liver disease: role of taurocholate and hepatocyte-derived monocyte chemotaxis protein-1 in hepatic stellate cell recruitment.
      For differentiation assays, cells were lysed 1 to 4 days after taurocholate treatment. LPC differentiation was assessed using real-time quantitative PCR (qPCR) and/or Western blot analysis evaluating markers for cholangiocyte (connexin 43, Cx43; cytokeratin 19, Ck19; integrin β 4, Itgb4; γ-glutamyl transpeptidase 1, Ggt1; epithelial cell adhesion molecule, EpCam; SYR-related HMG box transcription factor 9, Sox9) and hepatocyte (albumin; hepatocyte nuclear factor 4α, Hnf4α) phenotype.

      qPCR for LPC Differentiation Marker and Chemokine mRNA Expression

      RNA was extracted from both untreated and taurocholate-treated PIL-2 cells using the RNeasy kit (Qiagen, Hilden, Germany), and 1 μg was reverse transcribed into cDNA using SensiFast (Bioline, Taunton, MA). qPCR was performed on a Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA) with HPRT used as the reference gene. Primers are listed in Table 1.
      Table 1List of qPCR Primers
      PrimersNCBI accessionForwardReverse
      Tgf-β1NM_0215785′-GCTTCAGCTCCACAGAGAAGA-3′5′-CCAGGCTCCAAATGTAGGGG-3′
      Cx43NM_0102885′-AGCTAGGCGGCAAAAGTAGG-3′5′-ACTCACTCATGTATACAGAACCAT-3′
      Ck19NM_0084715′-GTCGAGGGAGGGGTTAGAGT-3′5′-CCATCTGAGCTACCAGCGAG-3′
      Ggt1NM_0081165′-CAGCACCACAGGAAAAGTTGAG-3′5′-ACGGATTTCACCAGGGACAG-3′
      Itgb4NM_0010056085′-GACCTATGAAGAAGGTGCTC-3′5′-GGCTCAGATGCGTGCCATAG-3′
      Sox9NM_0114485′-AGTACCCGCATCTGCACAAC-3′5′-TACTTGTAATCGGGGTGGTCT-3′
      EpCamNM_0085325′-CTGGGAGGAGGATAAAGC-3′5′-AGAAGAATGGAACAGGGAC-3′
      Hprt1NM_0135565′-CAAACTTTGCTTTCCCTGGT-3′5′-TCTGGCCTGTATCCAACACTTC-3′
      RantesNM_0311165′-TGCCCACGTGAAGGAGTATTTTTA-3′5′-TGGCGGTTCCTTCGAGTGACAA-3′
      PdgfbNM_0110575′-ATCCGCTCCTTTGATGATCT-3′5′-GAGCTTTCCAACTCGACTCC-3′
      Mip1αNM_0113375′-CTGCCCTTGCTGTTCTTCTC-3′5′-CTTGGACCCAGGTCTCTTTG-3′
      Mcp-1NM_0113335′-GCTGACCCCAAGAAGGAATG-3′5′-GTGCTTGAGGTGGTTGTGGA-3′
      hCcr1NM_0012955′-CCTGCTGACGATTGACAGGTA-3′5′-TTGGAAAAGTATAAGCCTGGCAT-3′
      hCcr5NM_0005795′-TGCTACTCGGGAATCCTAAAAACT-3′5′-TTCTGAACTTCTCCCCGACAAA-3′
      Hnf4αNM_008261Qiagen QuantiTect cat.no. QT00144739
      AlbNM_009654Qiagen QuantiTect cat.no. QT00115570
      Information on the primers can be found by their ascension numbers at the NCBI Nucleotide database: https://www.ncbi.nlm.nih.gov/nuccore.
      qPCR, real-time quantitative PCR.

      MTT Cell Proliferation Assay

      Cell proliferation was measured over a 2-hour period at 1 to 4 days following treatment of PIL-2 cells with 150 μmol/L sodium taurocholate using the method of Alley et al.
      • Alley M.C.
      • Scudiero D.A.
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      • Mayo J.G.
      • Shoemaker R.H.
      • Boyd M.R.
      Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay.
      Absorbance was measured at 570 nm with a 620-nm reference filter.

      Western Blot Analysis

      Cell lysates were prepared from PIL-2 cells disrupted in RIPA buffer with Complete EDTA-free protease inhibitors (Roche, Basel, Switzerland). Lysates were resolved on 10% SDS-PAGE gels as described in Laemmli et al
      • Laemmli U.K.
      Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
      and transferred to polyvinyl difluoride membranes (Immobilon-FL; Merck Millipore, Billerica, MA). Membranes were probed with anti-CK19 (ab15463; dilution 1:2000; Abcam, Cambridge, MA); anti-SOX9 (AB5535; dilution 1:1000; Merck Millipore); anti-HNF4α (Santa Cruz sc6556; dilution 1:1000; Santa Cruz Biotechnology, Dallas, TX); anti–acetylated α-tubulin (T7451; dilution 1:1000; Sigma-Aldrich); with anti–β-actin used to detect the β-actin loading control (dilution 1:2000; Cell Signaling Technology, Danvers, MA), in TBST LI-COR Odyssey blocking buffer (LI-COR, Lincoln, NE). Blots were probed with an anti-mouse 680RD or anti-rabbit 800CW secondary at dilution 1:20,000 in TBST LI-COR Odyssey blocking buffer. Secondary localization was detected with the LI-COR Odyssey CLx Infrared Imaging System. Band quantification was performed using Image Studio Lite version 5.2 (LI-COR).

      Immunofluorescence

      To examine primary cilia, PIL-2 cells were grown on glass coverslips to 60% to 80% confluence and treated for 3 days with and without 150 μmol/L taurocholate, and were then serum-starved overnight before fixation. Cells were placed at 4°C for 30 minutes to destabilize microtubules and then fixed in 4% paraformaldehyde for 10 minutes. Cells were permeabilized with 0.5% Triton X-100, blocked with 5% bovine serum albumin, and probed with mouse anti–acetylated α-tubulin (dilution 1:600; Sigma-Aldrich) and rabbit anti–γ-tubulin (dilution 1:400; Sigma-Aldrich) antibodies overnight in phosphate-buffered saline with 0.05% Tween 20 with 1% bovine serum albumin. Primary binding was probed with AlexaFluor 594 donkey anti-rabbit (Invitrogen, Carlsbad, CA) and AlexaFluor 594 goat anti-mouse (Invitrogen) at dilution 1:500 for 1 hour in phosphate-buffered saline with 0.05% Tween 20 with 1% bovine serum albumin. Images were captured with a Zeiss AxioScop2 (Carl Zeiss, Oberkochen, Germany). Quantification of acetylated α-tubulin spots was performed using Particle Analysis in ImageJ software version 1.50i (NIH, Bethesda, MD; http://imagej.nih.gov/ij).
      To identify the cellular source of HSC chemokines in liver biopsies from children with CFLD, we performed dual immunofluorescence for chemokines MCP-1; regulated upon activation, normal T cell expressed and secreted (RANTES); or macrophage inflammatory protein 1α (MIP1α), together with the LPC/DR marker, CK7. Primary antibodies used were: mouse anti-CK7 monoclonal antibody (dilution 1:150; Abcam), goat anti-human MCP-1 polyclonal antibody (dilution 1:8; R&D Systems, Minneapolis, MN), goat anti-human CCL5/RANTES polyclonal antibody (dilution 1:83; R&D Systems), goat anti-human MIP1α (dilution 1:50; R&D Systems), incubated overnight at 4°C followed by a 30-minute incubation step at room temperature with donkey anti-mouse IgG AlexaFluor 555 (dilution 1:300; Invitrogen) and donkey anti-goat IgG AlexaFluor 488 (dilution 1:300; Molecular Probes, Eugene, OR). Sections were mounted in Prolong Gold (Invitrogen) with DAPI and examined by confocal microscopy (Zeiss 780NLO).

      Ex Vivo Liver Slice Studies

      Precision-cut liver slices were made as previously described.
      • Gobert G.N.
      • Nawaratna S.K.
      • Harvie M.
      • Ramm G.A.
      • McManus D.P.
      An ex vivo model for studying hepatic schistosomiasis and the effect of released protein from dying eggs.
      A minimum of three liver slices each from three separate C57BL6 WT mice for each time point and each treatment group, were incubated in Williams' E medium containing 2 mmol/L glutamine, penicillin (100 U/mL), streptomycin (100 μg/mL), and 10% FBS at 37°C for 2 and 4 days with and without 150 μmol/L taurocholate. Conditioned medium was examined for GGT enzyme activity. Slices were harvested for mRNA quantitation using qPCR.

      γ-Glutamyl Transpeptidase Activity Assay

      GGT activity was determined from in vitro cell cultures and ex vivo liver slice conditioned media using the GGT activity colorimetric assay kit (Sigma-Aldrich). Protein for normalization was measured using the Pierce BCA protein assay (Thermo Fisher Scientific, Waltham, MA).

      Quantification of Cytokine Protein Secretion from LPCs with or without Taurocholate

      Chemokines MCP-1, MIP1α, and RANTES, released from LPCs, were quantified using the Cytokine Bead Array (BD Biosciences, San Jose, CA), according per manufacturer's protocol. The concentrations of transforming growth factor-1β (TGF-β1) and platelet-derived growth factor-BB (PDGFB) secreted by LPCs were determined by enzyme-linked immunosorbent assay (R&D Systems).

      Chemotaxis Assays

      Induction of HSC chemotaxis via taurocholate treatment of LPCs was examined by measuring cell migration through 8-μm 24-well cell culture inserts (Greiner Bio-One, Kremsmünster, Austria) in modified Boyden chambers. Assays were performed using 0.5 × 106 LX-2 cells (serum starved overnight) per mL within the insert, and the inserts were placed suspended within test medium. Test medium was either normal nonconditioned serum-free Williams' E medium or conditioned medium from LPCs with and without taurocholate. Chemokine neutralization was performed by pretreating conditioned medium with 2.5 μg/mL anti-mouse MCP-1 (AF-479-NA), 1 μg/mL anti-mouse MIP1α (AF-450-NA), or 1 μg/mL anti-mouse RANTES (AF478) polyclonal neutralization antibodies (R&D Systems), 1 hour before the assay. Testing of MIP1α as chemokine used murine (40-337; ProSci, Poway, CA) or human (40-364; ProSci) recombinant purified MIP1α proteins. Blocking of the CCR5 receptor used the neutralization/blocking antibody MAB182 (R&D Systems) applied to the LX-2 cells within the Boyden chamber insert for 1 hour before the assay at an insert concentration of 20 μg/mL. Chemotaxis assay were performed for 24 hours, and migrated cells on the underside of the insert membrane were then stained with Cell Stain (90144; Merck Millipore), treated with Extraction Buffer (90145; Merck Millipore) and absorbance measured at 560 nm. Results from separate experimental replicates were normalized to the mean absorbance of control samples (nonconditioned serum-free assay medium and FBS positive control).

      Statistical Analysis

      Results are presented as means ± SEM. Statistical analysis was performed with GraphPad Prism software version 6 (GraphPad Software, La Jolla, CA). The following tests were performed: For immunohistochemical analysis, the U-test and one-way nonparametric analysis of variance with Kruskal-Wallis and Dunn's multiple comparison tests. For chemotaxis assay, one-way analysis of variance with Sidak's multiple comparison. For qPCR time course, analysis of variance with Sidak's post hoc test and two-way analysis of variance to examine the overall treatment effect. For qPCR chemokine expression, unpaired t-test was used. For cytokine bead array assay, unpaired t-test with Welch's correction was used. Differences were considered significant for P < 0.05.

      Results

      Ductular Reaction Correlates with Fibrosis and Biliary Taurocholate in CFLD

      The DR, as assessed by CK7 expression, was markedly increased in CFLD and contained expanded reactive bile ducts, and both single LPCs and strings of biliary cells/LPCs (Figure 1). The DR was mainly present in the periphery of portal tracts as irregular strands/chords of CK7+ cells and as isolated single CK7+ LPCs in adjacent areas. This DR was increased in CFLD (P < 0.05), as well as in biliary atresia (P = 0.114) and idiopathic neonatal hepatitis (P = 0.016), versus controls (Figure 2A). When stratified for fibrosis stage, there was a highly significant positive correlation between CK7 staining and increasing fibrosis stage in CFLD (Spearman r = 0.59, P < 0.0001) (Figure 2B). Bile samples were available from 7 of the 60 children with CFLD. There was a positive statistical correlation between biliary taurocholate concentrations and the extent of the DR as assessed via CK7 quantification (r = 0.528, P = 0.0321) (Figure 2C).
      Figure thumbnail gr1
      Figure 1Evidence of the ductular reaction (DR) in cystic fibrosis liver disease (CFLD). Immunohistochemistry for CK7 expression in the liver of a donor control subject (A) and a child with CFLD (B and C). The CFLD patient shows a marked DR (B) and numerous single CK7+ liver progenitor cells adjacent to areas of DR (C). Scale bars: 200 μm (A); 160 μm (B); 60 μm (C). Original magnification: ×10 (A); ×8 (B); ×40 (C).
      Figure thumbnail gr2
      Figure 2Assessment of the ductular reaction in cystic fibrosis liver disease (CFLD) and other pediatric cholestatic liver diseases. A: CK7 expression in three pediatric cholestatic liver diseases: CFLD, biliary atresia (BA), and idiopathic neonatal hepatitis (NH) versus donor control livers. Statistical calculations were performed using an analysis of variance with Kruskal-Wallis test. B: Correlation between CK7 expression and hepatic fibrosis stage in livers of children with CFLD (Spearman r = 0.59, P < 0.0001). Statistical calculations were performed using an analysis of variance with Kruskal-Wallis test. C: Correlation between hepatic CK7 expression and biliary taurocholate levels in children with CFLD (Pearson r2 = 0.528; one-tailed P = 0.0283). Data are expressed as means ± SEM. n = 7 (C). P < 0.05, ∗∗P < 0.01.

      Taurocholate Induces Biliary Differentiation of LPCs

      Because in CFLD, the extent of the DR was demonstrated to correlate with both fibrosis severity and biliary taurocholate levels, it was hypothesized that taurocholate may induce LPC biliary differentiation and the DR. To interrogate this hypothesis, the murine LPC line PIL-2 was treated with 0 to 300 μmol/L taurocholate for 4 days to establish the optimal conditions for all in vitro and ex vivo studies. Taurocholate induced a dose-dependent increase in mRNA expression of the cholangiocyte markers Cx43 (analysis of variance, P < 0.0001) (Figure 3A) and Ck19 (analysis of variance, P < 0.0001) (Figure 3B). A taurocholate concentration of 150 μmol/L was chosen for all subsequent experiments as has previously been described in in vitro studies,
      • Ramm G.A.
      • Shepherd R.W.
      • Hoskins A.C.
      • Greco S.A.
      • Ney A.D.
      • Pereira T.N.
      • Bridle K.R.
      • Doecke J.D.
      • Meikle P.J.
      • Turlin B.
      • Lewindon P.J.
      Fibrogenesis in pediatric cholestatic liver disease: role of taurocholate and hepatocyte-derived monocyte chemotaxis protein-1 in hepatic stellate cell recruitment.
      • Lamireau T.
      • Zoltowska M.
      • Levy E.
      • Yousef I.
      • Rosenbaum J.
      • Tuchweber B.
      • Desmouliere A.
      Effects of bile acids on biliary epithelial cells: proliferation, cytotoxicity, and cytokine secretion.
      and also being consistent with previous observations in children with CFLD that displayed a mean biliary taurocholate concentration of 108 μmol/L versus 41 μmol/L in healthy controls.
      • Ramm G.A.
      • Shepherd R.W.
      • Hoskins A.C.
      • Greco S.A.
      • Ney A.D.
      • Pereira T.N.
      • Bridle K.R.
      • Doecke J.D.
      • Meikle P.J.
      • Turlin B.
      • Lewindon P.J.
      Fibrogenesis in pediatric cholestatic liver disease: role of taurocholate and hepatocyte-derived monocyte chemotaxis protein-1 in hepatic stellate cell recruitment.
      Figure thumbnail gr3
      Figure 3Taurocholate induces markers of cholangiocyte differentiation in liver progenitor cells. Taurocholate (0 to 300 μmol/L) induced a dose-dependent increase in mRNA expression of cholangiocyte markers Cx43 (analysis of variance, P < 0.0001) (A) and Ck19 (analysis of variance, P < 0.0001) (B) in PIL-2 cells (n = 4). Dunnett's multiple comparisons test was performed relative to untreated cells. PIL-2 cells treated with 150 μmol/L taurocholate for up to 4 days induced a time-dependent increase in the mRNA expression of cholangiocyte markers Cx43 (C) (analysis of variance, P < 0.0001), Ck19 (D) (analysis of variance, P < 0.0001), Itgb4 (E) (analysis of variance, P = 0.029), and Ggt1 (F) (analysis of variance, P = 0.001), expressed relative to vehicle control at each time point. Sidak's multiple comparisons test was used for comparison at individual time points of taurocholate treatment. Data are expressed as means ± SEM. n = 4. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001. versus control
      PIL-2 cells were treated with 150 μmol/L taurocholate for 0 to 4 days, and markers of cholangiocyte differentiation were further assessed. Taurocholate induced a time-dependent increase in mRNA expression of cholangiocyte markers Cx43 (analysis of variance, P < 0.0001) (Figure 3C), Ck19 (analysis of variance, P < 0.0001) (Figure 3D), Itgb4 (analysis of variance, P < 0.029) (Figure 3E), and Ggt1 (analysis of variance, P < 0.001) (Figure 3F). Taurocholate treatment did not significantly alter expression of two other cholangiocyte markers, EpCam and Sox9 (data not shown).
      Of interest, a concomitant decrease in mRNA expression of the master regulator of hepatic differentiation, Hnf4α was observed (analysis of variance, P = 0.0255) (Figure 4A). A second hepatocyte marker, albumin (Alb) showed no significant difference with taurocholate treatment (Figure 4B). Taken together, these results suggest that taurocholate augments the cholangiocytic differentiation of the LPC line PIL-2.
      Figure thumbnail gr4
      Figure 4The effect of taurocholate on markers of hepatocyte differentiation in liver progenitor cells. PIL-2 cells treated with 150 μmol/L taurocholate for up to 4 days resulted in a time-dependent decrease in mRNA expression for the hepatocyte marker hepatocyte nuclear factor 4α (Hnf4α; analysis of variance, P = 0.0255) (A), expressed relative to vehicle control at each time point, although there was no apparent effect on albumin (Alb) expression (B). Data are expressed as means ± SEM. n = 4.

      LPC Proliferation Is Induced by Taurocholate

      Integral to the formation of the DR is the expansion of LPCs in chronic liver disease. To assess the potential effect of taurocholate on LPC proliferation, PIL-2 cells with and without 150 μmol/L taurocholate were subjected to an MTT cell proliferation assay every 24 hours for 1 to 4 days. Taurocholate induced a time-dependent increase in LPC proliferation (analysis of variance, P = 0.0003) (Figure 5), which was most notable early, that is, after 1 to 2 days.
      Figure thumbnail gr5
      Figure 5Liver progenitor cell proliferation is induced by taurocholate. Taurocholate (150 μmol/L) induced a time-dependent increase in cell proliferation (analysis of variance, P = 0.0003) in PIL-2 cells treated for 0 to 4 days, as assessed using an MTT assay. Sidak's multiple comparisons test was performed for treatment comparison at individual time points. Data represent means ± SEM. n = 4. ∗∗P < 0.01. A570 nm, absorbance at 570 nm; A620 nm, absorbance at 620 nm.

      Taurocholate Induces Differentiation of LPCs into Functional Cholangiocytes

      Although Figure 3 demonstrates that taurocholate induced the expression of cholangiocyte-associated genes in LPCs, an assessment of actual cholangiocyte function remained to be confirmed. Thus, we examined expression of proteins involved in various aspects of biliary cell function. We observed that CK19, a protein responsible for the structural integrity of cholangiocytes is significantly induced by taurocholate (P = 0.0006) (Figure 6, A and B). Taurocholate also induced a significant >2.5-fold increase in SOX9 protein, a key transcriptional regulator of cholangiocyte differentiation and bile duct formation (P = 0.001) (Figure 6, A and C). Conversely, HNF4α protein expression, a marker and transcriptional regulator of hepatocyte specification, was significantly decreased by taurocholate (P = 0.019) (Figure 6, A and D).
      Figure thumbnail gr6
      Figure 6Taurocholate induces functional characteristics of biliary differentiation in liver progenitor cells in vitro at the protein level. PIL-2 cells were treated with 150 μmol/L taurocholate for 4 days. A: Western blot analysis revealed an increased expression of CK19 and SOX9 proteins, whereas HNF4α protein expression was decreased, relative to the β-actin loading control. Increased expression of acetylated α-tubulin protein relative β-actin was also observed. B–E: Band quantification from infrared imaging demonstrated that taurocholate induced statistically significant increases in CK19 (B), SOX9 (C), and acetylated α-tubulin (E), whereas conversely, HNF4α protein expression was significantly decreased (D). Data are expressed as means ± SEM. n = 4 (A–E). P < 0.05, ∗∗∗P < 0.001 (t-test). AC, acetylated; kDa, kilodalton.
      One key morphological characteristic of cholangiocytes is the presence of primary cilia with acetylated α-tubulin being a key structural component. Western blot analysis revealed a significant induction of acetylated α-tubulin protein expression in taurocholate-treated LPCs (P = 0.026) (Figure 6, A and E). Because acetylated α-tubulin can also be found within other cellular structures such as microtubule networks, the cellular localization of acetylated α-tubulin was further investigated using immunofluorescence of LPCs with and without taurocholate. Expression of acetylated α-tubulin causes primary cilia to appear as discrete dots or lines, whereas microtubule networks form thin filament-meshed patterns.
      • Grzelak C.A.
      • Martelotto L.G.
      • Sigglekow N.D.
      • Patkunanathan B.
      • Ajami K.
      • Calabro S.R.
      • Dwyer B.J.
      • Tirnitz-Parker J.E.
      • Watkins D.N.
      • Warner F.J.
      • Shackel N.A.
      • McCaughan G.W.
      The intrahepatic signalling niche of hedgehog is defined by primary cilia positive cells during chronic liver injury.
      As can be seen in Figure 7A, taurocholate induced a marked increase in the number of acetylated α-tubulin+ primary cilia when compared to controls, with quantitation of the relative fluorescence showing a 2.4-fold increase (P = 0.022) (Figure 7B).
      Figure thumbnail gr7
      Figure 7Taurocholate induces morphological and biochemical characteristics of functional biliary differentiation in liver progenitor cells in vitro. A and B: PIL-2 cells grown on coverslips were treated with 150 μmol/L taurocholate for 3 days versus untreated controls, and serum starved overnight to enhance cilia production. Cells were examined for the presence of primary cilia via expression of acetylated α-tubulin (green). Cells were also labelled with the nuclear strain (DAPI). These images show an increase in the number of primary cilia in response to taurocholate (A, right panel) versus control (A, left panel), which when the individually stained spots/lines of fluorescence were quantified, show a statistically significant 2.4-fold increase (B) using the t-test. C: Taurocholate (150 μmol/L, 1 to 4 days) induced a time-dependent increase in GGT enzyme activity (represented as nmole of p-nitroanilide per hour/mL) in conditioned media from PIL-2 cells (analysis of variance, P < 0.0001). Sidak's multiple comparisons test was performed for comparison at individual time points of taurocholate treatment. Data are expressed as means ± SEM. n = 3 (A and B); n = 4 (C). ∗∗P < 0.01, ∗∗∗∗P < 0.0001 versus control. Original magnification, ×100.
      Another measure of functional LPC-cholangiocyte differentiation is the production of enzymatically active GGT, which is expressed and released by cholangiocytes within bile ducts. PIL-2 cells were treated with and without 150 μmol/L taurocholate for 1 to 4 days with conditioned medium removed for analysis every 24 hours. Taurocholate induced a significant time-dependent increase in GGT activity (analysis of variance, P < 0.0001), with maximal production between days 3 to 4 (Figure 7C). At the conclusion of the experiment (day 4), this result remained highly significant when corrected for cellular protein concentration (Supplemental Figure S1).

      The Effect of Taurocholate in ex Vivo Precision-Cut Liver Slices

      To demonstrate potential physiological relevance of our in vitro observations, precision-cut liver slices from normal control mice, treated with and without 150 μmol/L taurocholate for up to 4 days, were examined. The hepatic mRNA expression of Cx43, as a representative cholangiocyte marker, was significantly increased in a time-dependent manner (analysis of variance, P = 0.0354) (Figure 8A). Conditioned medium collected was assayed for GGT activity and normalized to liver slice wet weight. Taurocholate again induced a significant increase in GGT activity, peaking at day 2 (analysis of variance, P = 0.0021) (Figure 8B).
      Figure thumbnail gr8
      Figure 8Effect of taurocholate in an ex vivo liver model. To demonstrate the potential physiological relevance of our in vitro observation, we used precision-cut liver slices treated with 150 μmol/L taurocholate for 4 days to demonstrate a time-dependent increase in mRNA expression of the cholangiocyte marker Cx43 (analysis of variance, P = 0.0354) (A), as well as GGT enzyme activity normalized to tissue weight (analysis of variance, P = 0.0021) (B). Data are expressed means ± SEM. n = 3 replicates from 3 mice (A); n = 6 replicates from 3 mice (B).

      Chemokine Expression Induced by Taurocholate in LPCs

      PIL-2 cells treated with 150 μmol/L taurocholate showed significantly increased expression of MCP-1, MIP1α, and RANTES mRNA at 6 hours (Figure 9A). We also examined the gene expression of the profibrotic cytokine TGF-β1 and HSC mitogen PDGFB, which both have HSC chemotactic potential. Although taurocholate induced increased expression of both TGF-β1 and PDGFB, this did not reach statistical significance (Figure 9A).
      Figure thumbnail gr9
      Figure 9Profiling of hepatic chemokines expressed and secreted by liver progenitor cells after taurocholate treatment. A: Taurocholate induced a significant increase in Mcp-1, Mip1α, and Rantes mRNA expression in PIL-2 cells at 6 hours. Statistical significance for each gene transcript was assessed using the t-test. B: Taurocholate treatment induced a significant increase in MIP1α and RANTES protein release into the culture medium at 6 hours. MCP-1 and PDGFB were also increased but this did not reach statistical significance. The effect of taurocholate was lost after 24 hours, except for the effect on PDGFB (P = 0.0153). TGF-β protein release was not affected by taurocholate treatment. Data are expressed as means ± SEM. P < 0.05, ∗∗∗P < 0.001 versus control.
      Conditioned medium from PIL-2 cells with and without 150 μmol/L taurocholate for 6 hours or 24 hours was assessed for protein secretion of key hepatic chemokines. Rantes, Mcp-1, Mip1α, Tgf-β1, and Pdgfb were all expressed constitutively by PIL-2 cells. After 6 hours, taurocholate treatment resulted in a twofold increase in MIP1α protein release (P < 0.0001), whereas RANTES and PDGFB increased by approximately 1.5-fold (Figure 9B). Taurocholate induced an increase in MCP-1 secretion; however, this did not reach statistical significance. By 24 hours, chemokine levels were indistinguishable from controls, except for PDGFB, which remained elevated in response to taurocholate (P < 0.05). No effect was observed on TGF-β1 release.

      HSCs Migrate in Response to Chemokines Secreted by Taurocholate-Treated LPCs

      Chemotaxis of the LX-2 HSC line was significantly induced by conditioned medium from PIL-2 cells treated with taurocholate for either 6 hours (P < 0.001) or 24 hours (P < 0.01) (Figure 10A). Similar levels of chemotaxis were demonstrated using primary rat HSCs (data not shown). To determine the identity of chemokines responsible for this chemotaxis, conditioned medium was pretreated with neutralizing antibodies to various different chemokines. Chemotaxis was completely inhibited by anti-MIP1α neutralizing antibody (nAb); however, anti-RANTES nAb had no effect (Figure 10B). Although chemotaxis of LX-2 cells was partially suppressed using anti-MCP-1 nAb, this did not reach statistical significance.
      Figure thumbnail gr10
      Figure 10Treatment of liver progenitor cells with taurocholate induces hepatic stellate cell (HSC) chemotaxis. PIL-2 cells were treated with 150 μmol/L taurocholate for 6 or 24 hours, and conditioned medium from these cells was used in modified Boyden chambers to examine LX-2 chemotaxis after 24 hours. A: Treatment of LX-2 HSCs with conditioned medium from either 6 or 24 hours taurocholate-treated PIL-2 cells resulted in significant HSC chemotaxis across the Boyden chamber membranes. B: Chemotaxis of LX-2 cells induced by conditioned medium from 24 hours taurocholate-treated PIL-2 cells was inhibited by anti-MIP1α antibody. Statistical significance was determined using analysis of variance with Sidak's multiple comparisons test performed for specific comparisons. Data are expressed as means ± SEM. n > 4 (A); n = 3 (B). ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001. A560, absorbance at 560 nm; CM, conditioned medium; FBS, fetal bovine serum; TCA, taurocholate.

      HSCs Migrate in Response to MIP1α Mediated by CCR5

      As shown in Figure 10B, chemotaxis of HSCs in response to conditioned medium from PIL-2 cells treated with taurocholate was inhibited by anti-MIP1α nAb. To strengthen the case for the involvement of MIP1α as a HSC chemotactic agent, a chemotaxis assay was performed on LX2 cells using either murine or human recombinant purified MIP1α. Both murine (analysis of variance, P = 0.022) (Figure 11A) and human (analysis of variance, P = 0.0009) (Figure 11B) MIP1α produced a significant dose-dependent increase in HSC migration, approaching the effect observed with the positive control (FBS).
      Figure thumbnail gr11
      Figure 11MIP1α induces hepatic stellate cell (HSC) chemotaxis. A and B: LX-2 cells were placed in modified Boyden chambers with physiologically relevant concentrations of either murine recombinant MIP1α (A) or human recombinant MIP1α (B) and assessed for chemotactic potential. MIP1α produced a significant dose-dependent increase in HSC chemotaxis (analysis of variance, P = 0.022 and P = 0.0009, respectively). Sidak's multiple comparisons test was performed for treatment comparison, C: The chemoattractive effect of murine recombinant MIP1α was attenuated by pre-treating LX-2 cells with an anti-CCR5 neutralizing antibody. D: Pre-treatment of LX-2 cells with anti-CCR5 also attenuated chemotaxis in response to conditioned medium from 24-hour taurocholate-treated PIL-2 cells. Analysis of variance with Sidak's multiple comparisons test was performed for specific comparisons. Data are expressed as means ± SEM. n = 3 (A and B); n = 4 (C and D). P < 0.05, ∗∗P < 0.01, and ∗∗∗∗P < 0.0001. A560, absorbance at 560 nm; FBS, fetal bovine serum; TCA, taurocholate.
      LX-2 cells were examined for the expression of both known major hepatic MIP1α receptors, C-C chemokine receptor type 1 and 5 (CCR1 and CCR5). Using qPCR, expression of both Ccr1 and Ccr5 was demonstrated, with Ccr5 showing higher mRNA levels versus Ccr1 (Supplemental Figure S2). To investigate whether CCR5 was the major contributing receptor in HSC chemotaxis, LX-2 cells were pretreated with an anti-CCR5 nAb. This antibody effectively attenuated the migration of LX-2 HSCs in response to both recombinant murine MIP1α (Figure 11C) and conditioned medium from PIL-2 cells treated with 150 μmol/L taurocholate (Figure 11D).

      DR Cells and LPCs Express Chemokines in Liver from Children with CFLD

      To assess the potential clinical relevance of taurocholate-induced chemokine expression demonstrated in in vitro and ex vivo studies, liver sections from patients with CFLD (n = 22) were examined for the cellular source of chemokines. Dual immunofluorescence confirmed that CK7+ DR cells, and single CK7+ LPCs, express MCP-1 (Figure 12A), RANTES (Figure 12B), and MIP1α (Figure 13).
      Figure thumbnail gr12
      Figure 12Cellular localization of MCP-1 and RANTES expression in liver from children with cystic fibrosis liver disease (CFLD). Dual immunofluorescence was performed on CFLD patients to demonstrate the colocalization of CK7+ ductular reaction cells with MCP-1 protein expression (A) and RANTES protein expression (B). Sections are shown with DAPI in blue, CK7 in red, and either MCP-1 or RANTES in green, with colocalization (merge). Scale bars: 20 μm (A); 50 μm (B). Original magnification: ×63 (A); ×40 (B).
      Figure thumbnail gr13
      Figure 13Cellular localization of MIP1α expression in liver from children with cystic fibrosis liver disease (CFLD). Dual immunofluorescence was performed on CFLD patients for the association of MIP1α protein expression with clusters of CK7+ ductular reaction cells (A) and individual CK7+ liver progenitor cells (B). Sections are shown with DAPI in blue, CK7 in red, and M1Pα in green with colocalization (merge). Scale bars = 20 μm. Original magnification: ×40, 1.4 zoom (A); ×63 (B).

      Discussion

      The DR is proposed to be associated with the initiation of hepatic fibrosis in a number of chronic liver diseases.
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      Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers.
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      Fibrosis correlates with a ductular reaction in hepatitis C: roles of impaired replication, progenitor cells and steatosis.
      • Lowes K.N.
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      Oval cell numbers in human chronic liver diseases are directly related to disease severity.
      • Richardson M.M.
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      Progressive fibrosis in nonalcoholic steatohepatitis: association with altered regeneration and a ductular reaction.
      However, the precise mechanisms involved in its induction and indeed in the interactions between LPCs and HSCs, which may drive both the DR and fibrogenesis, remain to be elucidated. This histological process has been reported in many adult liver diseases, including chronic hepatitis C,
      • Clouston A.D.
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      have elucidated a role for many different soluble mediators in hepatic fibrosis. Understanding the interaction between LPCs and HSCs is of great importance for clarifying the contribution of these cells in mediating fibrogenesis and for designing therapeutic strategies aimed at reduction of hepatic fibrosis.
      Bile salt retention is part of the proposed mechanism of injury and biliary fibrosis in CFLD, and we previously demonstrated that biliary/serum levels of the conjugated bile acid taurocholate are elevated in children with CFLD.
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      In the present study, it was demonstrated that the extent of the DR correlates with biliary taurocholate concentration in children with CFLD and thus speculate a potential mechanistic link. To determine whether taurocholate plays a role in inducing the DR, both an LPC in vitro cell culture model and ex vivo precision-cut liver slices were used, and a reductionist strategy was employed to examine the impact of taurocholate on three key events in the evolution of the DR: LPC proliferation, biliary differentiation of LPCs, and HSC chemotaxis.
      In this study, it was demonstrated that taurocholate increased LPC proliferation in vitro, suggesting that taurocholate may play a role in enhancing LPC expansion as part of the DR in CFLD. This observation is supported by studies from Clouzeau-Girard et al
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      showing that taurocholate leads to uncharacterized cell proliferation around the portal tracts in ex vivo liver slices. Our data are also consistent with the observation that feeding taurocholate to rats increases cholangiocyte numbers,
      • Alpini G.
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      although this study did not distinguish whether these were mature adult cholangiocytes or derived from LPCs.
      The biliary differentiation of LPCs and subsequent formation of reactive bile ductular structures appear to be an important component of DR development. A significant increase in expression of the cholangiocyte genes Ck19, Cx43, Itgb4, and Ggt1 was demonstrated in response to taurocholate treatment of LPCs. Conversely, the hepatocyte lineage marker Hnf4α was decreased, with albumin expression unchanged. This in vitro result may be consistent with LPC differentiation that occurs during the DR in vivo and provides one potential mechanism for induction of the DR in pediatric cholestatic liver diseases such as CFLD. Further supporting this concept, it was demonstrated that taurocholate induced functional cholangiocyte differentiation, evidenced through the increased expression of acetylated α-tubulin, CK19, and SOX9 protein, the increased numbers of LPCs elaborating primary cilia, as well as the secretion of enzymatically active GGT in vitro. The effect of taurocholate on cholangiocyte function was further confirmed in ex vivo precision-cut liver slices, showing induced expression of Cx43 mRNA and secretion of active GGT.
      In addition, this study has demonstrated that taurocholate-exposed LPCs secrete a number of chemokines including MCP-1, MIP1α, RANTES, and PDGFB; however, HSC chemotaxis in response to taurocholate-treated LPC conditioned medium appears to be principally due to MIP1α. Neutralizing antibodies to either MIP1α in LPC conditioned medium or the MIP1α receptor CCR5 on HSCs attenuated taurocholate-treated LPC-induced HSC chemotaxis. Thus, we propose that MIP1α derived from bile acid–exposed LPCs may recruit HSCs (via CCR5) during the DR and aid in the initiation of hepatic fibrosis in CFLD. We did not demonstrate that RANTES aided in the chemoattraction of HSCs. Neutralization of MCP-1 resulted in a limited inhibition of HSC chemotaxis, however this did not reach statistical significance due to biological variability of the data. RANTES and MCP-1 may induce recruitment of other cells to the LPC niche as part of the fibrogenic process such as additional LPCs or inflammatory cells, as we previously described.
      • Ramm G.A.
      • Shepherd R.W.
      • Hoskins A.C.
      • Greco S.A.
      • Ney A.D.
      • Pereira T.N.
      • Bridle K.R.
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      • Meikle P.J.
      • Turlin B.
      • Lewindon P.J.
      Fibrogenesis in pediatric cholestatic liver disease: role of taurocholate and hepatocyte-derived monocyte chemotaxis protein-1 in hepatic stellate cell recruitment.
      • Ruddell R.G.
      • Knight B.
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      • Akhurst B.
      • Summerville L.
      • Subramaniam V.N.
      • Olynyk J.K.
      • Ramm G.A.
      Lymphotoxin-beta receptor signaling regulates hepatic stellate cell function and wound healing in a murine model of chronic liver injury.
      Migration of HSCs toward LPCs, as well as HSC activation, could explain why fibrosis begins in periportal regions of the liver, where there is decreased bile flow through bile ducts, and thus increased accumulation of hydrophobic bile acids.
      • Smith J.L.
      • Lewindon P.J.
      • Hoskins A.C.
      • Pereira T.N.
      • Setchell K.D.
      • O'Connell N.C.
      • Shepherd R.W.
      • Ramm G.A.
      Endogenous ursodeoxycholic acid and cholic acid in liver disease due to cystic fibrosis.
      This pattern of chemokine expression upon in vitro taurocholate treatment may be indicative of the specific role of LPCs in generating a microenvironment that aids in the activation of HSCs. In the current study, immunofluorescence revealed that MCP-1, RANTES, and MIP1α are all expressed by DR cells and CK7+ LPCs in liver from children with CFLD, providing in vivo evidence validating our in vitro observations.
      HSC activation is also influenced by other cell types present during chronic liver injury and inflammation, because the LPC niche harbors immune cells including liver macrophages, which also secrete MCP-1, MMPs, TIMPS,
      • Duffield J.S.
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      Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair.
      • Olynyk J.K.
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      • Ramm G.A.
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      • Bacon B.R.
      • Tracy T.F.
      Gadolinium chloride suppresses hepatic oval cell proliferation in rats with biliary obstruction.
      and chemokines. The downstream chemoattractive effect caused by exposure of LPCs to increased levels of taurocholate appears likely to recruit HSCs into the LPC niche and places these two cell types within close proximity and potentially in direct contact. Supporting this, activated HSCs have been observed adjacent to LPCs in various mouse model of fibrosis,
      • Grzelak C.A.
      • Martelotto L.G.
      • Sigglekow N.D.
      • Patkunanathan B.
      • Ajami K.
      • Calabro S.R.
      • Dwyer B.J.
      • Tirnitz-Parker J.E.
      • Watkins D.N.
      • Warner F.J.
      • Shackel N.A.
      • McCaughan G.W.
      The intrahepatic signalling niche of hedgehog is defined by primary cilia positive cells during chronic liver injury.
      • Ruddell R.G.
      • Knight B.
      • Tirnitz-Parker J.E.
      • Akhurst B.
      • Summerville L.
      • Subramaniam V.N.
      • Olynyk J.K.
      • Ramm G.A.
      Lymphotoxin-beta receptor signaling regulates hepatic stellate cell function and wound healing in a murine model of chronic liver injury.
      • Köhn-Gaone J.
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      • Tirnitz-Parker J.E.
      Divergent inflammatory, fibrogenic, and liver progenitor cell dynamics in two common mouse models of chronic liver injury.
      as well as in human liver diseases,
      • Wood M.J.
      • Gadd V.L.
      • Powell L.W.
      • Ramm G.A.
      • Clouston A.D.
      Ductular reaction in hereditary hemochromatosis: the link between hepatocyte senescence and fibrosis progression.
      • Gadd V.L.
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      • Clouston A.D.
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      Portal, but not lobular, macrophages express matrix metalloproteinase-9: association with the ductular reaction and fibrosis in chronic hepatitis C.
      demonstrating recruitment of each cell type toward the other during fibrosis. Various studies have also suggested that membrane contact (and cross talk) between LPCs and HSCs is a key mediator of fibrogenesis.
      • Ruddell R.G.
      • Knight B.
      • Tirnitz-Parker J.E.
      • Akhurst B.
      • Summerville L.
      • Subramaniam V.N.
      • Olynyk J.K.
      • Ramm G.A.
      Lymphotoxin-beta receptor signaling regulates hepatic stellate cell function and wound healing in a murine model of chronic liver injury.
      • Dwyer B.J.
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      • Ramm G.A.
      • Tirnitz-Parker J.E.
      TWEAK and LTβ signaling during chronic liver disease.
      • Tirnitz-Parker J.E.
      • Olynyk J.K.
      • Ramm G.A.
      Role of TWEAK in coregulating liver progenitor cell and fibrogenic responses.
      In this context, our previous work showed that HSCs treated with the TNF superfamily member, lymphotoxin-β, induces NF-κB signaling, up-regulating chemotaxis-associated factors such as RANTES and ICAM-1, and aiding in LPC and inflammatory cell recruitment.
      • Ruddell R.G.
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      • Akhurst B.
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      • Ramm G.A.
      Lymphotoxin-beta receptor signaling regulates hepatic stellate cell function and wound healing in a murine model of chronic liver injury.
      Because HSCs express the lymphotoxin-β receptor on their cell surface, and lymphotoxin-β is a membrane-bound ligand on LPCs, this suggests that direct cell–cell contact between LPCs and HSCs may be required to initiate chemokine expression.
      • Ruddell R.G.
      • Knight B.
      • Tirnitz-Parker J.E.
      • Akhurst B.
      • Summerville L.
      • Subramaniam V.N.
      • Olynyk J.K.
      • Ramm G.A.
      Lymphotoxin-beta receptor signaling regulates hepatic stellate cell function and wound healing in a murine model of chronic liver injury.
      Adding to the complexity of this system, high concentrations of taurocholate have also been shown to induce inflammatory gene expression in hepatocytes, as well as cytokine/chemokine secretion, that may also lead to HSC recruitment and activation.
      • Ramm G.A.
      • Shepherd R.W.
      • Hoskins A.C.
      • Greco S.A.
      • Ney A.D.
      • Pereira T.N.
      • Bridle K.R.
      • Doecke J.D.
      • Meikle P.J.
      • Turlin B.
      • Lewindon P.J.
      Fibrogenesis in pediatric cholestatic liver disease: role of taurocholate and hepatocyte-derived monocyte chemotaxis protein-1 in hepatic stellate cell recruitment.
      • Allen K.
      • Jaeschke H.
      • Copple B.L.
      Bile acids induce inflammatory genes in hepatocytes: a novel mechanism of inflammation during obstructive cholestasis.
      The present study suggests that the DR precedes fibrosis. However, there is an alternate hypothesis stating that fibrosis precedes the DR, and there is some evidence to support this in animal studies.
      • Van Hul N.K.
      • Abarca-Quinones J.
      • Sempoux C.
      • Horsmans Y.
      • Leclercq I.A.
      Relation between liver progenitor cell expansion and extracellular matrix deposition in a CDE-induced murine model of chronic liver injury.
      It is also possible that the two processes may be injury context-specific thus further investigations are warranted to fully elucidate the temporal nature of LPC niche development. In addition to HSCs, portal fibroblasts may also contribute to CFLD. However, whereas previous studies have shown that both activated HSCs and myofibroblasts produce collagen in this disease,
      • Lewindon P.J.
      • Pereira T.N.
      • Hoskins A.C.
      • Bridle K.R.
      • Williamson R.M.
      • Shepherd R.W.
      • Ramm G.A.
      The role of hepatic stellate cells and transforming growth factor-beta(1) in cystic fibrosis liver disease.
      the role of myofibroblasts derived from portal fibroblasts and indeed the potential recruitment by, and interaction between, these cells and LPCs has not been previously investigated.

      Conclusions

      In conclusion, this study has shown that the DR is present in CFLD and is correlated with biliary taurocholate levels, as well as increasing hepatic fibrosis severity. Thus, we propose that the following model may in part help to explain the pathogenesis of CFLD (Figure 14). In this model, the CFTR defect causes thickened mucous secretions and obstruction of the intrahepatic bile ducts. This obstruction elevates biliary levels of bile salts such as taurocholate in the vicinity of LPCs, induced as a result of hepatic injury and potentially compromised hepatocyte replication associated with the chronic nature of liver injury in CFLD. In response to supraphysiological levels of bile salts, LPCs first undergo proliferation followed by differentiation toward cholangiocytes as part of the DR. Also, in response to altered bile salt levels, LPCs secrete chemokines, including MCP-1, MIP1α, and RANTES, and this likely attracts HSCs and other immune/inflammatory cells to the LPC niche. Cell–cell contact between LPCs and recruited HSCs may result in activation of HSCs and subsequent fibrogenesis, at least in part via interaction between the HSC lymphotoxin-β receptor via cognate ligands on LPCs, with further inflammatory niche chemotaxis, as we previously described.
      • Ruddell R.G.
      • Knight B.
      • Tirnitz-Parker J.E.
      • Akhurst B.
      • Summerville L.
      • Subramaniam V.N.
      • Olynyk J.K.
      • Ramm G.A.
      Lymphotoxin-beta receptor signaling regulates hepatic stellate cell function and wound healing in a murine model of chronic liver injury.
      Increased understanding of the role of hydrophobic bile acids in CFLD and their impact on the LPC niche, cellular cross talk with HSCs, and induction of the DR will provide important new knowledge of clinical relevance, which may aid in the development of targeted therapeutic strategies to reduce fibrosis in CFLD and potentially in other pediatric cholestatic disorders.
      Figure thumbnail gr14
      Figure 14Proposed model for taurocholate-induced ductular reaction (DR) and fibrogenesis in children with cystic fibrosis liver disease. The cystic fibrosis transmembrane conductance regulator (CFTR) defect results in obstruction of the intrahepatic bile ducts (red X). This obstruction elevates biliary taurocholate levels, which drives liver progenitor cell (LPC) proliferation and biliary/cholangiocyte differentiation as part of the DR. In response to increased taurocholate, LPCs secrete chemokines that attract hepatic stellate cells (HSCs)/myofibroblasts (and likely other immune/inflammatory cells) to the LPC niche and/or the region of DR development. Cell–cell contact between LPCs and recruited HSCs may result in activation of HSCs and subsequent fibrogenesis.

      Acknowledgments

      We thank Clay Winterford, Nigel Waterhouse, Tam Hong Nguyen, and Fernando Guimaraes (QIMR Berghofer Medical Research Institute) for technical support, and Prof. George Yeoh (University of Western Australia) for his gift of the PIL-2 murine liver progenitor cell line.

      Supplemental Data

      Figure thumbnail figs1
      Supplemental Figure S1GGT enzyme activity in conditioned medium from PIL-2 cells treated ±150 μmol/L taurocholate (D), normalized to protein concentration at the conclusion of the experiment (day 4). Statistical significance was assessed using the t-test. Data are expressed as means ± SEM. ∗∗∗P < 0.001.
      Figure thumbnail figs2
      Supplemental Figure S2Expression levels of Ccr1 and Ccr5. LX-2 cells were examined for Ccr1 and Ccr5 mRNA expression relative to the abundant housekeeping gene Hprt1. Statistical significance was assessed using the t-test. Data are expressed as means ± SEM. ∗∗∗∗P < 0.0001.

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