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Protective potential of the gallbladder in primary sclerosing cholangitis

  • Author Footnotes
    † These authors share first authorship
    Nora Cazzagon
    Correspondence
    Corresponding author: Nora Cazzagon, , Dept. of Surgery, Oncology and Gastroenterology, University of Padova, Via N. Giustiniani 2, 35128, Padova, Italy, Phone: +39 049 8212894
    Footnotes
    † These authors share first authorship
    Affiliations
    Sorbonne Université, INSERM, Centre de Recherche Saint-Antoine (CRSA), Paris, France

    Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy

    European Reference Network on Hepatological Diseases (ERN RARE-LIVER), Azienda Ospedale-Università Padova, Padova, Italy
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  • Author Footnotes
    † These authors share first authorship
    Ester Gonzalez-Sanchez
    Footnotes
    † These authors share first authorship
    Affiliations
    Sorbonne Université, INSERM, Centre de Recherche Saint-Antoine (CRSA), Paris, France

    TGF-β and Cancer Group. Oncobell Program, Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet De Llobregat, Barcelona, Spain

    Oncology Program, Ciberehd, National Biomedical Research Institute on Liver and Gastrointestinal Diseases, Instituto De Salud Carlos III, Spain

    Department of Physiological Sciences, Faculty of Medicine and Health Sciences, University of Barcelona, Spain
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  • Haquima El-Mourabit
    Affiliations
    Sorbonne Université, INSERM, Centre de Recherche Saint-Antoine (CRSA), Paris, France
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  • Dominique Wendum
    Affiliations
    Sorbonne Université, INSERM, Centre de Recherche Saint-Antoine (CRSA), Paris, France

    Assistance Publique-Hôpitaux de Paris (AP-HP). Sorbonne Université, Department of Pathology, Saint-Antoine Hospital, Paris, France
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  • Dominique Rainteau
    Affiliations
    Sorbonne Université, INSERM, Centre de Recherche Saint-Antoine (CRSA), Paris, France

    Assistance Publique-Hôpitaux de Paris (AP-HP). Sorbonne Université, Department of Clinical Metabolomics, Saint Antoine Hospital, Paris, France
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  • Lydie Humbert
    Affiliations
    Sorbonne Université, INSERM, Centre de Recherche Saint-Antoine (CRSA), Paris, France

    Assistance Publique-Hôpitaux de Paris (AP-HP). Sorbonne Université, Department of Clinical Metabolomics, Saint Antoine Hospital, Paris, France
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  • Christophe Corpechot
    Affiliations
    Sorbonne Université, INSERM, Centre de Recherche Saint-Antoine (CRSA), Paris, France

    Assistance Publique-Hôpitaux de Paris (AP-HP). Sorbonne Université, Department of Hepatology, Reference Center for Inflammatory Biliary Diseases and Autoimmune Hepatitis (CRMR MIVB-H), ERN RARE-LIVER, Saint-Antoine Hospital, Paris, France
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  • Olivier Chazouillères
    Affiliations
    Sorbonne Université, INSERM, Centre de Recherche Saint-Antoine (CRSA), Paris, France

    Assistance Publique-Hôpitaux de Paris (AP-HP). Sorbonne Université, Department of Hepatology, Reference Center for Inflammatory Biliary Diseases and Autoimmune Hepatitis (CRMR MIVB-H), ERN RARE-LIVER, Saint-Antoine Hospital, Paris, France
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  • Author Footnotes
    # These authors share senior authorship.
    Lionel Arrivé
    Footnotes
    # These authors share senior authorship.
    Affiliations
    Assistance Publique-Hôpitaux de Paris (AP-HP). Sorbonne Université, Department of Radiology, Saint-Antoine Hospital, Paris, France
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    # These authors share senior authorship.
    Chantal Housset
    Footnotes
    # These authors share senior authorship.
    Affiliations
    Sorbonne Université, INSERM, Centre de Recherche Saint-Antoine (CRSA), Paris, France

    Assistance Publique-Hôpitaux de Paris (AP-HP). Sorbonne Université, Department of Hepatology, Reference Center for Inflammatory Biliary Diseases and Autoimmune Hepatitis (CRMR MIVB-H), ERN RARE-LIVER, Saint-Antoine Hospital, Paris, France
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  • Author Footnotes
    # These authors share senior authorship.
    Sara Lemoinne
    Correspondence
    Corresponding author: Sara Lemoinne, , Assistance Publique – Hôpitaux de Paris, Sorbonne University, Department of Hepatology, Saint-Antoine Hospital, 184 rue Faubourg Saint Antoine, 75012 Paris, France, Phone: +33 1 49282923 Fax: +33 149282107
    Footnotes
    # These authors share senior authorship.
    Affiliations
    Sorbonne Université, INSERM, Centre de Recherche Saint-Antoine (CRSA), Paris, France

    Assistance Publique-Hôpitaux de Paris (AP-HP). Sorbonne Université, Department of Hepatology, Reference Center for Inflammatory Biliary Diseases and Autoimmune Hepatitis (CRMR MIVB-H), ERN RARE-LIVER, Saint-Antoine Hospital, Paris, France
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  • Author Footnotes
    † These authors share first authorship
    # These authors share senior authorship.
Open AccessPublished:December 16, 2022DOI:https://doi.org/10.1016/j.jhepr.2022.100649

      Highlights

      • Gallbladder enlargement is associated with alleviated cholestasis in PSC patients
      • Gallbladder volume is negatively correlated with bile acid hydrophobicity in PSC
      • Cholecystectomized PSC patients display more severe cholangiographic features
      • Cholecystectomy aggravates cholangitis and liver fibrosis in Abcb4 knockout mice

      Abstract

      Background & Aims

      Gallbladder enlargement is common in patients with primary sclerosing cholangitis (PSC). The gallbladder may confer hepatoprotection against bile acid overload, through the sequestration and cholecystohepatic shunt of bile acids. The aim of this study was to assess the potential impact of gallbladder on disease features and bile acid homeostasis in PSC.

      Methods

      PSC patients from a single tertiary center who underwent liver magnetic resonance imaging (MRI) with three-dimensional cholangiography and concomitant analyses of serum bile acids were included. Gallbladder volume was measured by MRI and a cut-off of 50 mL was used to define gallbladder enlargement. Bile acid profiles and PSC severity as assessed by blood tests and MRI features, were compared among patients according to the gallbladder size (enlarged vs. normal-sized) or presence (removed vs. conserved). The impact of cholecystectomy was also assessed in the Abcb4 knockout mouse model of PSC.

      Results

      Sixty-one PSC patients, all treated with ursodeoxycholic acid (UDCA), were included. The gallbladder was found enlarged in 30 patients, whereas 11 patients had been previously cholecystectomized. Patients with enlarged gallbladders compared to those with normal-sized gallbladders, had significantly lower alkaline phosphatase, lower tauro- vs. glycoconjugates ratio and higher UDCA vs. total bile acids ratio. In addition, the gallbladder volume was negatively correlated with the hydrophobicity index of bile acids. Cholecystectomized patients compared to others displayed significantly higher aspartate aminotransferase and more severe bile duct strictures and dilatations. In the Abcb4 knockout mice, cholecystectomy caused an increase in hepatic bile acid content and in circulating secondary bile acids, and an aggravation in cholangitis, inflammation and liver fibrosis.

      Conclusion

      Altogether, our findings indicate that the gallbladder fulfills protective functions in PSC.

      LAY SUMMARY

      In PSC patients, the gallbladder status impacts on bile acid homeostasis and disease features. We found evidence of lessened bile acid toxicity in PSC patients with enlarged gallbladders and of increased disease severity in those who were previously cholecystectomized. In the Abcb4 knockout mouse model of PSC, cholecystectomy causes an aggravation of cholangitis and liver fibrosis. Overall, our results infer a protective role of the gallbladder in PSC.

      Graphical abstract

      Keywords

      Conflict of interest:

      Nora Cazzagon, Ester Gonzalez-Sanchez, Haquima El-Mourabit, Dominique Wendum, Dominique Rainteau, Lydie Humbert, Olivier Chazouillères, Lionel Arrivé, Chantal Housset and Sara Lemoinne declare no conflict of interest related to this paper.
      Christophe Corpechot declares the following conflicts of interest : Intercept, Arrow, Cymabay, Ipsen, Calliditas, Gilead.

      Financial support statement:

      This work was supported by funding from the Microbiome Foundation, PSC Partners Seeking a Cure, Agence Nationale de la Recherche (ANR) grant # 15-CE14-0007-01 and Fondation pour la Recherche Médicale (FRM) EQU202003010517. E.G-S. received post-doctoral fellowships from the Spanish Association for the Study of the Liver (AEEH), from the Alfonso Martin Escudero Foundation (FAME), and a post-doctoral contract from CIBEREHD, Spanish National Biomedical Research Institute on Liver and Gastrointestinal Diseases, Instituto de Salud Carlos III.

      Authors contributions:

      NC, LA, CH and SL designed the study. NC, EGS, SL, HEM, DW, DR, LH, LA generated the data. NC, EGS, CH and SL analyzed the data. All authors critically revised and approved the final version of the manuscript.

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      Patients and methods

      Patients study design

      PSC patients followed at the reference center for inflammatory biliary diseases and auto-immune hepatitis in Saint-Antoine Hospital, Paris, France, between January 2004 and December 2013, who underwent both MRI with a three-dimensional magnetic resonance cholangiography (3D-MRC), and blood sampling allowing mass spectrometry analysis of bile acids, within less than 15 days apart , were included and their records retrospectively reviewed. The date of blood sampling was used to define the inclusion date. The research was conducted in accordance with the 2013 Declaration of Helsinki and the 2018 Declaration of Istanbul and was approved by the Saint-Antoine Hospital institutional review committee. Written informed consent was provided by all subjects. The diagnosis of PSC was based on typical macroscopic imaging (i.e., multiple strictures of the large intra- and/or extrahepatic bile ducts on 3D-MRC)
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      ,
      European Association for the Study of the Liver
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      . Exclusion criteria were a diagnosis of small-duct PSC, secondary sclerosing cholangitis or immunoglobin G4-associated cholangitis and a history of liver transplantation before inclusion. Sixty-one patients, including 11 patients who were previously cholecystectomized, were thus included. Fasting gallbladder volume was calculated by the analysis of MRI as described below. It was previously reported that median fasting gallbladder volume measured by MRI was 67 mL in PSC patients and 32 mL in healthy controls and the threshold of 50 mL represented a clear cut between PSC patients and healthy controls (75% of PSC patients displayed a gallbladder volume above 50mL and 75% of healthy controls displayed a gallbladder volume below 50mL)
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      . Therefore, gallbladders which volume reached or exceeded 50 mL were considered unambiguously enlarged, and patients whose gallbladder volume was < or ≥ 50 mL, were separated in two groups. Patients’ data at the time of inclusion were compared between the two groups. The Mayo and Amsterdam - Oxford risk scores were calculated as previously described
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      . Percutaneous liver biopsy was performed in 38 patients, mostly before the inclusion MRI, and liver fibrosis was staged on a 0-to-4 scale, according to a METAVIR-derived scoring system
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      Baseline values and changes in liver stiffness measured by transient elastography are associated with severity of fibrosis and outcomes of patients with primary sclerosing cholangitis.
      . The diagnosis of cirrhosis required one of the following criteria: fibrosis stage F4 on liver biopsy, liver stiffness measured by vibration controlled transient elastography (VCTE) ≥ 14.4 kPa
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      Baseline values and changes in liver stiffness measured by transient elastography are associated with severity of fibrosis and outcomes of patients with primary sclerosing cholangitis.
      or presence of liver dysmorphy associated with signs of portal hypertension (i.e., platelet count < 150.109/L, presence of esophageal or gastric varices).

      MRI

      MRI with 3D-MRC was performed as previously described by our group and in compliance with the International PSC study group (IPSCSG) recommendations
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      ,
      • Schramm C.
      • Eaton J.
      • Ringe K.I.
      • Venkatesh S.
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      . The procedure was preceded by fasting for at least four hours. An axial T2-weighted single-shot fast spin-echo was performed. A 3D fat-suppressed T1-weighted gradient-echo sequence was performed before and after the administration of 20 mL of Gd-DOTA (Dotarem, Guerbet, Aulnay-sous-Bois, France), for hepatic arterial, portal venous, and equilibrium phase acquisition (at 30 seconds, 80 seconds and 3 minutes, respectively). MRC was performed with a free-breathing 3D high-spatial-resolution fast spin echo sequence. Native images and 3D maximum intensity projection reconstructions were analyzed on a workstation, using the Carestream Picture Archiving and Communication System (version 11.32; Carestream Health, Rochester, NY). Maximum intensity projections were analyzed on thick slabs of 10 or 20 mm, orientated in the acquisition plane. In patients with conserved gallbladders, the fasting gallbladder volume was calculated as previously described
      • Said K.
      • Edsborg N.
      • Albiin N.
      • Bergquist A.
      Gallbladder emptying in patients with primary sclerosing cholangitis.
      with modifications, integrating the area of gallbladder cross-sections obtained at 5-mm interval on T2-weighted images (Fig. S1). Moreover, in order to assess the stability of gallbladder volume over time in the same patient, we repeated the measurements of gallbladder volume in at least three subsequent annual MRI sessions. The presence of gallstones, gallbladder polyps or cholecystitis was also assessed. Other MRC features including bile duct strictures or dilatations, cystic duct stenosis, parenchymal enhancement heterogeneity or dysmorphy, and portal hypertension were scored in all patients as previously described
      • Ruiz A.
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      . The two ANALI scores, with and without gadolinium administration, which were associated with radiologic progression and clinical outcome in our previous studies
      • Lemoinne S.
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      • Dohan A.
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      • et al.
      Simple Magnetic Resonance Scores Associate With Outcomes of Patients With Primary Sclerosing Cholangitis.
      ,
      • Ruiz A.
      • Lemoinne S.
      • Carrat F.
      • Corpechot C.
      • Chazouillères O.
      • Arrivé L.
      Radiologic course of primary sclerosing cholangitis: assessment by three-dimensional magnetic resonance cholangiography and predictive features of progression.
      , were also calculated.

      Mouse study design

      Abcb4-/- mice and Abcb4+/+ littermates were bred, using Abcb4+/- heterozygous mice on an FVB/N genetic background (FVB·129P2-Abcb4tm1Bor/J) provided by Sanofi R&D (Chilly-Mazarin, France). Mice were housed at CRSA animal facility (Institutional Animal Care and Use Direction, DDPP agreement No. C 75-12-01), in a temperature-controlled, specific pathogen-free environment, on a 12-hour light-dark cycle, with free access to a standard chow (LASQCdiet® Rod16-R, Genobios, Laval, France) and water. All animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” and experiments were approved by the Ethics Committee in Animal Experiments Charles Darwin No.5 (No. APAFIS#1600-201509021149154 v3).
      Gallbladder volume was assessed in 4-week-old male Abcb4+/+ and Abcb4−/− mice, who underwent cholecystectomy under isoflurane anaesthesia, following 4-hour fasting. After cholecystectomy, gallbladders were weighted and their volume determined gravimetrically, assuming a density of 1 g/mL. The impact of gallbladder removal on PSC phenotypic traits was assessed in 7-week-old male Abcb4−/− mice, who underwent cholecystectomy or sham operation, as previously described
      • Debray D.
      • Rainteau D.
      • Barbu V.
      • Rouahi M.
      • El Mourabit H.
      • Lerondel S.
      • et al.
      Defects in gallbladder emptying and bile Acid homeostasis in mice with cystic fibrosis transmembrane conductance regulator deficiencies.
      . Cholangiopathy features are indeed well established in Abcb4-/- mice at the age of 7 weeks
      • Gonzalez-Sanchez E.
      • El Mourabit H.
      • Jager M.
      • Clavel M.
      • Moog S.
      • Vaquero J.
      • et al.
      Cholangiopathy aggravation is caused by VDR ablation and alleviated by VDR-independent vitamin D signaling in ABCB4 knockout mice.
      . Five weeks after surgery, mice were sacrificed under isoflurane anesthesia, blood was drawn from the vena cava and the liver and spleen were collected and weighed. Liver samples were harvested following recommendations from the IPSCSG
      • Fickert P.
      • Pollheimer M.J.
      • Beuers U.
      • Lackner C.
      • Hirschfield G.
      • Housset C.
      • et al.
      Characterization of animal models for primary sclerosing cholangitis (PSC).
      .

      Bile acid analyses

      Bile acids were analyzed using high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS), in the serum from fasted patients and in the liver and plasma from unfasted mice, as previously described
      • Debray D.
      • Rainteau D.
      • Barbu V.
      • Rouahi M.
      • El Mourabit H.
      • Lerondel S.
      • et al.
      Defects in gallbladder emptying and bile Acid homeostasis in mice with cystic fibrosis transmembrane conductance regulator deficiencies.
      ,
      • Humbert L.
      • Maubert M.A.
      • Wolf C.
      • Duboc H.
      • Mahé M.
      • Farabos D.
      • et al.
      Bile acid profiling in human biological samples: comparison of extraction procedures and application to normal and cholestatic patients.
      , using a Q-Trap 5500. All chemicals and solvents were of the highest purity available. Cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), UDCA, lithocholic acid (LCA), hyocholic acid (HCA), hyodeoxycholic acid (HDCA) and their glyco and tauro derivatives were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France); 3-sulfate derivatives were a generous gift from J Goto (Niigita University of Pharmacy and Applied Life Science, Niigata, Japan); 23-Nor-5β-cholanoic acid-3α,12β diol, all muricholic acids, and their glyco and tauro derivatives were purchased from Steraloids Inc (Newport, USA). Acetic acid, ammonium carbonate, ammonium acetate and methanol, all of HPLC grade, were purchased from Sigma-Aldrich. The hydrophobicity index was determined based on the concentration and retention time of the different bile acids along a methanol gradient on a C18 column, LCA and tauroursodeoxycholic acid-3S, displaying the highest and lowest retention time, respectively.

      Fibroblast growth factor (FGF) 19 and C4 assays

      Concentrations of FGF19 were measured in the serum samples collected from PSC patients for bile acid analysis, using a sandwich ELISA (FGF19 Quantikine® ELISA kit, R&D Systems, Minneapolis, MN, USA). C4 was measured using HPLC-MS/MS.

      Mouse liver (immuno)histology

      Formalin (4%)-fixed, paraffin-embedded mouse liver tissue samples were cut into 4-μm-thick sections. Ductular reaction, liver inflammation and fibrosis, the hallmarks of cholangiopathy in the Abcb4-/- mice, were assessed using cytokeratin 19 (CK19), F4/80 immunostaining and Sirius red staining, respectively. Liver tissue sections were stained with hematoxylin and eosin or Sirius red or immunostained for cytokeratin 19 (CK19) or F4/80, using an anti-CK19 antibody (TROMA III, Developmental Studies Hybridoma Bank, Iowa University, IA, USA) and an anti-F4/80 antibody (SP115, Abcam, Cambridge, UK), respectively.
      Stained sections were scanned on a virtual slide scanner (Hamamatsu, Tokyo, Japan) 2.0 HT, using a 3-charge-coupled device, time-delay integration camera with a resolution of 1.84 μm/pixel (x20 objective) and 0.92 μm/pixel (x40 objective). Morphometric analyses were performed blinded, using Image J analysis software (National Institutes of Health, Bethesda, MD, USA). Hematoxylin and eosin-stained liver tissue sections were blindly analyzed by a senior pathologist expert in liver histopathology (D.W.) following the Nakanuma scoring system, which was previously shown to have a strong predictive value in PSC
      • de Vries E.M.G.
      • de Krijger M.
      • Färkkilä M.
      • Arola J.
      • Schirmacher P.
      • Gotthardt D.
      • et al.
      Validation of the prognostic value of histologic scoring systems in primary sclerosing cholangitis: An international cohort study.
      . The following histopathologic features were assessed in 10 portal spaces semi-quantitatively on a 0-3 scale: cholangitis and hepatitis activities, fibrosis stage and bile duct loss. Due to the specific characteristics of liver damage in Abcb4-/- mice, grading criteria for cholangitis activity were adapted as previously described
      • Gonzalez-Sanchez E.
      • El Mourabit H.
      • Jager M.
      • Clavel M.
      • Moog S.
      • Vaquero J.
      • et al.
      Cholangiopathy aggravation is caused by VDR ablation and alleviated by VDR-independent vitamin D signaling in ABCB4 knockout mice.
      . Briefly: grade 0: no activity; grade 1: one bile duct with polymorphonuclear cell infiltrate; grade 2: 2 or more bile ducts with polymorphonuclear cell infiltrate; grade 3: presence of fibro-obliterative cholangitis.

      Reverse transcription-quantitative PCR (RT-qPCR)

      Total RNA was extracted from mouse liver tissue using RNeasy columns (Qiagen, Courtaboeuf, France). Complementary DNA was synthesized from total RNA (1 μg), using the SuperScript II Reverse Transcriptase (Thermo Fisher Scientific), and qPCR was performed on a Light-Cycler 96 (Roche Diagnostics, Basel, Switzerland), using Sybr Green Master Mix (Roche Diagnostics). The mRNA levels of target genes were normalized for those of hypoxanthine phosphoribosyltransferase 1 (Hprt1) and expressed as relative levels (2-ΔΔCt). Primer sequences are provided in Table S1. The following genes were evaluated: Tnfα, a pro-inflammatory cytokine typically produced by reactive cholangiocytes in PSC
      • Guicciardi M.E.
      • Trussoni C.E.
      • LaRusso N.F.
      • Gores G.J.
      The Spectrum of Reactive Cholangiocytes in Primary Sclerosing Cholangitis.
      , α-smooth muscle actin (Acta2) and collagen 1a1 (Col1a1), which are fibrosis markers, and the genes encoding the main proteins involved in bile acid homeostasis, including cytochrome P450 7A1 (Cyp7a1) and sodium taurocholate cotransporting polypeptide Ntcp (Slc10a1), which are down-regulated in cholestasis
      • Wagner M.
      • Zollner G.
      • Trauner M.
      Nuclear receptor regulation of the adaptive response of bile acid transporters in cholestasis.
      ,
      • Fuchs C.D.
      • Trauner M.
      Role of bile acids and their receptors in gastrointestinal and hepatic pathophysiology.
      .

      Statistical analyses

      Patients’ characteristics were provided either as median and interquartile range (IQR), or as absolute number and percentage. Graphically, they were presented as box-whisker plots. To overcome potential assay variability, biochemical variables were expressed as ratios of upper limits of normal (ULN). Comparisons between two groups of patients defined according to gallbladder size (≥ vs. < 50 mL) or gallbladder presence (vs. cholecystectomy) were performed using chi-square test or Fisher test for categorical variables and Mann-Whitney test or Student t-test for continuous variables, as appropriate. Potential correlations between gallbladder volume and any other continuous parameter were assessed using Spearman coefficient. Mice quantitative data were presented as box-whisker plots, and compared between cholecystectomized and sham-operated mice, using student’s t-test. In mouse studies, survival rates were calculated based on the Kaplan-Meier estimates and Log-rank test was used to assess if survival differed between groups. Statistical analyses of patients’ data were performed using IBM Statistical Package for the Social Sciences (SPSS), Statistics v. 24 and subsequent versions. Mice data were analyzed using GraphPad Prism software version 5.0 (GraphPad Software San Diego, CA, USA). The graphical abstract was designed using BioRender. p values < 0.05 were considered significant.

      Results

      Characteristics of the PSC study population

      The study population comprised 61 patients (42 males and 19 females) with PSC. Eleven of them had been cholecystectomized before the date of inclusion for complications of gallstone disease and a suspicion of gallbladder cancer in five and two cases, respectively, whereas in the remaining four patients, the justification of cholecystectomy was unknown. The MRI at inclusion was used to measure gallbladder volume in the patients whose gallbladders were conserved at that time. It was performed for diagnostic purposes in five (8.2%) patients, and during follow-up in the remaining 56 (91.8%). All patients were taking UDCA (10 to 25 mg.kg-1.day-1) at the time of inclusion. Patients were aged 33
      • Debray D.
      • Rainteau D.
      • Barbu V.
      • Rouahi M.
      • El Mourabit H.
      • Lerondel S.
      • et al.
      Defects in gallbladder emptying and bile Acid homeostasis in mice with cystic fibrosis transmembrane conductance regulator deficiencies.
      • Pomare E.W.
      • Heaton K.W.
      The effect of cholecystectomy on bile salt metabolism.
      • Bidault-Jourdainne V.
      • Merlen G.
      • Glénisson M.
      • Doignon I.
      • Garcin I.
      • Péan N.
      • et al.
      TGR5 controls bile acid composition and gallbladder function to protect the liver from bile acid overload.
      • Mauad T.H.
      • van Nieuwkerk C.M.
      • Dingemans K.P.
      • Smit J.J.
      • Schinkel A.H.
      • Notenboom R.G.
      • et al.
      Mice with homozygous disruption of the mdr2 P-glycoprotein gene. A novel animal model for studies of nonsuppurative inflammatory cholangitis and hepatocarcinogenesis.
      • Fickert P.
      • Fuchsbichler A.
      • Wagner M.
      • Zollner G.
      • Kaser A.
      • Tilg H.
      • et al.
      Regurgitation of bile acids from leaky bile ducts causes sclerosing cholangitis in Mdr2 (Abcb4) knockout mice.
      • Gonzalez-Sanchez E.
      • El Mourabit H.
      • Jager M.
      • Clavel M.
      • Moog S.
      • Vaquero J.
      • et al.
      Cholangiopathy aggravation is caused by VDR ablation and alleviated by VDR-independent vitamin D signaling in ABCB4 knockout mice.
      European Association for the Study of the Liver
      EASL Clinical Practice Guidelines: management of cholestatic liver diseases.
      • Kim W.R.
      • Therneau T.M.
      • Wiesner R.H.
      • Poterucha J.J.
      • Benson J.T.
      • Malinchoc M.
      • et al.
      A revised natural history model for primary sclerosing cholangitis.
      • de Vries E.M.
      • Wang J.
      • Williamson K.D.
      • Leeflang M.M.
      • Boonstra K.
      • Weersma R.K.
      • et al.
      A novel prognostic model for transplant-free survival in primary sclerosing cholangitis.
      • Bedossa P.
      • Poynard T.
      An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group.
      • Corpechot C.
      • Gaouar F.
      • El Naggar A.
      • Kemgang A.
      • Wendum D.
      • Poupon R.
      • et al.
      Baseline values and changes in liver stiffness measured by transient elastography are associated with severity of fibrosis and outcomes of patients with primary sclerosing cholangitis.
      • Ruiz A.
      • Lemoinne S.
      • Carrat F.
      • Corpechot C.
      • Chazouillères O.
      • Arrivé L.
      Radiologic course of primary sclerosing cholangitis: assessment by three-dimensional magnetic resonance cholangiography and predictive features of progression.
      • Schramm C.
      • Eaton J.
      • Ringe K.I.
      • Venkatesh S.
      • Yamamura J.
      MRI working group of the IPSCSG. Recommendations on the use of magnetic resonance imaging in PSC-A position statement from the International PSC Study Group.
      • Fickert P.
      • Pollheimer M.J.
      • Beuers U.
      • Lackner C.
      • Hirschfield G.
      • Housset C.
      • et al.
      Characterization of animal models for primary sclerosing cholangitis (PSC).
      • Humbert L.
      • Maubert M.A.
      • Wolf C.
      • Duboc H.
      • Mahé M.
      • Farabos D.
      • et al.
      Bile acid profiling in human biological samples: comparison of extraction procedures and application to normal and cholestatic patients.
      • de Vries E.M.G.
      • de Krijger M.
      • Färkkilä M.
      • Arola J.
      • Schirmacher P.
      • Gotthardt D.
      • et al.
      Validation of the prognostic value of histologic scoring systems in primary sclerosing cholangitis: An international cohort study.
      • Guicciardi M.E.
      • Trussoni C.E.
      • LaRusso N.F.
      • Gores G.J.
      The Spectrum of Reactive Cholangiocytes in Primary Sclerosing Cholangitis.
      • Wagner M.
      • Zollner G.
      • Trauner M.
      Nuclear receptor regulation of the adaptive response of bile acid transporters in cholestasis.
      • Fuchs C.D.
      • Trauner M.
      Role of bile acids and their receptors in gastrointestinal and hepatic pathophysiology.
      • Choi M.
      • Moschetta A.
      • Bookout A.L.
      • Peng L.
      • Umetani M.
      • Holmstrom S.R.
      • et al.
      Identification of a hormonal basis for gallbladder filling.
      • Mousa O.Y.
      • Juran B.D.
      • McCauley B.M.
      • Vesterhus M.N.
      • Folseraas T.
      • Turgeon C.T.
      • et al.
      Bile Acid Profiles in Primary Sclerosing Cholangitis and Their Ability to Predict Hepatic Decompensation.
      • van Nieuwerk C.M.
      • Groen A.K.
      • Ottenhoff R.
      • van Wijland M.
      • van den Bergh Weerman M.A.
      • Tytgat G.N.
      • et al.
      The role of bile salt composition in liver pathology of mdr2 (-/-) mice: differences between males and females.
      • Li Y.
      • Tang R.
      • Leung P.S.C.
      • Gershwin M.E.
      • Ma X.
      Bile acids and intestinal microbiota in autoimmune cholestatic liver diseases.
      years at the time of PSC diagnosis, and 36 (25-52) years at the time of inclusion. IBD was present in 50 (82.0%) patients, including 31 with ulcerative colitis, 16 with Crohn’s disease and 3 with undetermined IBD. No patient had active ileal inflammation at the time of inclusion. Additional disease features are shown in Table 1.
      Table 1Disease features in the PSC study population
      PSC patients n=61
      Features at diagnosis
      Age, years33(20-42)
      Male gender42(69)
      IBD50(82)
      PSC localization
      • Intrahepatic only
      15(24.6)
      • Intra + extrahepatic
      45(73.8)
      • Extrahepatic only
      1(1.6)
      Liver biopsy40(65.6)
      Fibrosis according METAVIR
      • F0
      7(17.5)
      • F1
      12(30)
      • F2
      11(27.5)
      • F3
      8(20)
      • F4
      2(5)
      Features at inclusion
      Age, years36(25-52)
      Time between diagnosis and inclusion, years5.8(2.2-10.0)
      Total bilirubin, μmol/L15.4(11.0-36.3)
      AST, x ULN1.4(0.8-2.5)
      γGT, x ULN2.9(1.4-5.7)
      ALP, x ULN1.5(1.0-2.8)
      PT, %91(78-100)
      Serum albumin, g/L40.3(36.5-42.6)
      Platelets, x 109/L283(190-348)
      Liver stiffness, kPa10.8(7.4-17.4)
      Mayo risk score-0.24(-0.72-0.44)
      Amsterdam - Oxford score at inclusion1.72(1.16-2.11)
      Quantitative variables are expressed as median (interquartile range). Nominal variables are expressed as absolute number (percentage). Abbreviations: AST, aspartate aminotransferase; ULN, upper limit of normal; γGT, gamma-glutamyl transpeptidase; ALP, alkaline phosphatase; PT: prothrombin time.

      Evaluation of gallbladder volume in the PSC study population

      The gallbladder was present in 50 PSC patients of the study population, at the time of inclusion. Fasting gallbladder volumes measured by MRI analysis, ranged between 7 mL and 174 mL in these patients. The median gallbladder volume was 62 (37-106) mL, which is consistent with the values of 67 mL and 55 mL, reported in previous studies of gallbladder volume in PSC
      • van de Meeberg P.C.
      • Portincasa P.
      • Wolfhagen F.H.
      • van Erpecum K.J.
      • VanBerge-Henegouwen G.P.
      Increased gall bladder volume in primary sclerosing cholangitis.
      ,
      • Said K.
      • Edsborg N.
      • Albiin N.
      • Bergquist A.
      Gallbladder emptying in patients with primary sclerosing cholangitis.
      . Using a cut-off of 50 mL, we separated the 50 patients in two groups, i.e., 20 and 30 patients whose fasting gallbladder volume was < and ≥ 50 mL respectively, as illustrated in Fig. 1A. The median values of gallbladder volume in the two groups were 31 (18-45) mL and 87 (65-128) mL, respectively (Fig. 1B). It should be noted that no cholecystitis, stricture of the cystic duct or obstructive gallbladder mass was detected among the patients with enlarged gallbladders (Table S2).
      Figure thumbnail gr1
      Figure 1Gallbladder enlargement and related cholestatic profile in PSC patients. A) Gallbladder volume was measured on MRI in PSC patients with conserved gallbladders, who were subsequently separated into two groups with enlarged (≥ 50ml, n=30) or normal-sized (<50 mL, n=20) gallbladders, here illustrated by gallbladder volumes of 11 and 103 mL, respectively B-D) Graphs showing the distribution of gallbladder volume (B), serum alkaline phosphatase levels (C), tauroconjugates/ total BA ratios, Tauro-/Glycoconjugates ratios and UDCA/ total BA ratios (D), in the two groups. Levels of significance are indicated in the figure (Mann-Whitney test). A p value of less than 0.05 was considered significant.
      In addition, to evaluate the variability of gallbladder volume over time in an individual patient, we examined gallbladder volume on subsequent annual MRI in a subgroup of 36 (72%) patients, including 19 with enlarged gallbladders. This analysis showed that, within a range of 3 to 7 years, the gallbladder volume was constantly inferior or superior to 50 mL in an individual patient, with no overlap between the two groups (Fig. S2).

      Disease features in PSC patients with enlarged gallbladders compared to those with normal-sized gallbladders

      Whereas the clinical characteristics of PSC patients with enlarged and normal-sized gallbladders were mostly similar, the serum levels of alkaline phosphatase were significantly lower in patients with enlarged gallbladders than in those with normal-sized gallbladders (Fig. 1C and Table S3). Except for gallbladder volume, we found no significant difference in the MRI features of patients whose gallbladders were enlarged or not (Table S2). We looked for a potential influence of gallbladder volume on the metabolism of bile acids, as assessed by their serum profile, and found that among the 50 PSC patients of the study population whose gallbladders were conserved at the time of inclusion, the gallbladder volume was negatively correlated with the hydrophobicity index of circulating bile acids (ρ = -0.399, p < 0.01). Moreover, gallbladder volume was positively correlated with the ratio of UDCA (ρ = 0.43, p < 0.01) and of sulfoconjugates (ρ = 0.31, p = 0.03) vs. total bile acids. Analyses of the serum bile acid profiles in patients with enlarged gallbladders compared to those with normal-sized gallbladders, showed a lower ratio of tauroconjugates vs. glycoconjugates and vs. total bile acids and a higher ratio of UDCA vs. total bile acids (Fig. 1D and Table 2). We also observed trends towards lower cholic acid concentration and ratio vs. chenodeoxycholic acid, and towards a lower hydrophobicity index in patients with enlarged gallbladders (Table 2). FGF19 and C4 serum levels were available in only half of the patients of both groups and showed no difference between the two (Table 2). Importantly, potential confounding factors that could have affected bile acid metabolism were evenly distributed in the two groups. Notably, comparisons between the two groups revealed no difference in the prevalence of patients with IBD (all in clinical and endoscopic remission), or with previous intestinal resection for IBD nor in UDCA dosage at the time of inclusion (Table S4). We found no significant difference in adverse outcome-free survival (defined by survival without liver transplantation or cirrhosis decompensation) between the two groups (data not shown).
      Table 2Serum bile acids in patients with normal-sized vs. enlarged gallbladders
      Normal-sized gallbladder (n=20)Enlarged gallbladder (n=30)p
      Total bile acids, μmol/L35.41(13.54-198.00)33.49(12.10-149.5)0.64
      Primary BA, μmol/L5.76(3.14-73.69)4.48(1.12-50.53)0.10
      • CA, μmol/L
      3.09(1.02-31.37)2.11(0.28-19.86)0.09
      • CDCA, μmol/L
      3.29(1.83-35.52)2.61(0.86-28.57)0.21
      • CA/CDCA
      0.74(0.49-1.60)0.62(0.28-1.07)0.07
      Secondary BA, μmol/L0.55(0.08-1.65)0.54(0.09-1.27)0.81
      • DCA, μmol/L
      0.43(0.06-1.48)0.25(0.09-1.16)0.69
      • LCA, μmol/L
      0.04(0.01-0.29)0.09(0.00-0.29)0.89
      UDCA, μmol/L18.09(4.59-68.00)28.4(10.20-90.39)0.38
      Primary/secondary BA13.19(2.79-156.10)10.95(2.83-33.20)0.48
      Glycoconjugates, μmol/L26.82(11.91-151.43)24.63(8.87-120.08)0.45
      Tauroconjugates, μmol/L2.40(0.90-34.83)0.86(0.25-20.93)0.14
      Tauro-/glycoconjugates0.13(0.06-0.38)0.06(0.02-0.19)0.04
      UDCA/total BA0.65(0.49-0.73)0.80(0.65-0.90)<0.01
      Glycoconjugates/total BA0.78(0.70-0.88)0.78(0.68-0.87)0.94
      Tauroconjugates/total BA0.12(0.05-0.27)0.04(0.02-0.16)0.04
      Hydrophobicity index0.82(0.74-0.90)0.77(0.69-0.83)0.09
      FGF-19132.84(79.73-1120.44)129.76(70.88-182.90)0.44
      C40.70(0.20-1.34)1.45(0.20-2.38)0.26
      Quantitative variables are expressed as median (interquartile range). Abbreviations: BA, bile acid; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, Deoxycholic acid; LCA, lithocholic acid; UDCA, Ursodeoxycholic acid; FGF-19, fibroblast growth factor-19; C4, 7α-hydroxy-4-cholesten-3-one. Levels of significance are indicated in the table (Mann-Whitney test). A p value of less than 0.05 was considered significant.

      Disease features in cholecystectomized PSC patients compared to patients with conserved gallbladders

      Eleven patients of the PSC study population had been cholecystectomized previous to the time of inclusion. Cholecystectomy had been performed 3.4 (1.3-4.6) years before the date of inclusion. Compared to the non-cholecystectomized patients, patients who had been previously cholecystectomized showed similar clinical and biochemical characteristics, except for aspartate aminotransferase levels, which were significantly higher in these patients at inclusion (Fig. 2A and Table S5). In addition, severe MRI features were significantly more frequent at inclusion in these patients than in those with conserved gallbladders (Fig. 2B and Table 3). Thus, severe strictures (p = 0.02) and dilatations (p < 0.01) of the common bile duct, severe dilatations of the right (p = 0.01) and left (p = 0.04) hepatic ducts and marked intrahepatic bile duct dilatations (p=0.05) were all more frequent in cholecystectomized patients (Fig. 2C and Table 3). We also observed a trend towards a higher frequency of marked intrahepatic bile duct involvement (p=0.09) and left hepatic duct enhancement (p=0.09) in these patients (Table 3). Yet, no significant difference in serum bile acids was observed between the two groups, except for a trend towards higher levels of total bile acids and towards a lower ratio of UDCA vs. total bile acids in the group of cholecystectomized patients (Table S6). No difference in the adverse outcome-free survival was found between the two groups (data not shown).
      Figure thumbnail gr2
      Figure 2Impact of cholecystectomy on PSC features in patients. Comparison of A) Aspartate aminotransferase, B-C) cholangiographic features at MRI including representative images (B) and quantitative analyses (C), in PSC patients whose gallbladders were conserved (n=50) or previously removed (n=11). Levels of significance are indicated in the figure (Mann-Whitney test for A; Chi-squared test for C). A p value of less than 0.05 was considered significant.
      Table 3MRI features in non-cholecystectomized vs. cholecystectomized patients
      Non-cholecystectomized (n=50)Cholecystectomized (n=11)p
      CBD strictures0.02
      • Absent
      15(30.0)1(9.1)
      • ≤ 75%
      13(26.0)0(0)
      • > 75%
      22(44.0)10(90.9)
      CBD stricture length0.21
      • ≤ 2 mm
      0(0)0(0)
      • 3-10 mm
      3(6.0)0(0)
      • > 10 mm
      32(64)10(90.9)
      CBD dilatation<0.01
      • ≤ 10 mm
      49(98.0)8(72.7)
      • 11-14 mm
      1(2.0)2(18.2)
      • ≥ 15 mm
      0(0)1(9.1)
      CBD enhancement0.60
      • Absent
      34(79.0)6(60.0)
      • Thickness <2 mm
      5(11.6)2(20.0)
      • Thickness 2-6 mm
      4(9.3)2(20.0)
      • Thickness >6 mm
      0(0)0(0)
      RHD strictures0.29
      • Absent
      10(20.0)1(9.1)
      • ≤ 75%
      8(16.0)0(0)
      • > 75%
      31(62.0)10(90.9)
      RHD stricture length0.65
      • Absent
      11(2.0)1(9.1)
      • ≤ 2 mm
      3(6.0)0(0)
      • 3-10 mm
      9(18.0)2(18.2)
      • > 10 mm
      26(52.0)8(72.7)
      RHD dilatation0.01
      • ≤ 6 mm
      47(94.0)7(63.6)
      • 7-8 mm
      1(2.0)2(18.2)
      • ≥ 9 mm
      1(2.0)2(18.2)
      RHD enhancement0.58
      • Absent
      34(79.1)6(60)
      • Thickness < 2mm
      5(11.6)1(10)
      • Thickness 2-6 mm
      3(7.0)2(20)
      • Thickness > 6 mm
      0(0)1(10)
      LHD strictures0.33
      • Absent
      13(26.0)1(9.1)
      • ≤ 75%
      8(16.0)1(9.1)
      • > 75%
      29(58.0)9(81.8)
      LHD stricture length0.45
      • Absent
      14(28.0)1(9.1)
      • ≤ 2 mm
      4(8.0)1(9.1)
      • 3-10 mm
      8(16.0)1(9.1)
      • > 10 mm
      25(48.0)8(72.7)
      LHD dilatation0.04
      • ≤ 6 mm
      46(92.0)7(63.6)
      • 7-8 mm
      2(4.0)2(18.2)
      • ≥ 9 mm
      2(4.0)2(18.2)
      LHD enhancement0.09
      • Absent
      35(81.4)5(50)
      • Thickness < 2mm
      6(14.0)1(10)
      • Thickness 2-6 mm
      2(4.6)3(30)
      • Thickness > 6 mm
      0(0)1(10)
      IHBD Stricture1.00
      • Absent
      0(0)0(0)
      • ≤ 75%
      3(6.0)0(0)
      • > 75%
      47(94.0)11(100)
      IHBD involvement0.09
      • absent
      0(0)1(9.1)
      • ≤ 25%
      1(2.0)0(0)
      • > 25%
      49(98.0)10(90.9)
      IHBD dilatation0.05
      • None (≤ 3 mm)
      15(30.0)1(9.1)
      • Mild (4 mm)
      14(28.0)1(9.1)
      • Marked (≥ 5 mm)
      21(42.0)9(81.8)
      IHBD enhancement0.27
      • Absent
      31(72.0)5(50)
      • Thickness < 2mm
      6(13.9)2(20)
      • Thickness 2-6 mm
      6(13.9)1(10)
      • Thickness > 6 mm
      0(0)1(10)
      Intraductal stones1.00
      • Absent
      34(68.0)7(63.6)
      • Present
      16(32.0)4(36.3)
      Dysmorphy0.32
      • Absent
      23(46.0)3(27.3)
      • Present
      27(54.0)8(72.7)
      Splenomegaly (splenic index > 480 cm3)0.73
      • Absent
      32(61.6)8(72.7)
      • Present
      20(38.4)3(27.3
      Portal Hypertension0.73
      • Absent
      34(64.0)8(72.7)
      • Present
      18(36.0)3(27.3)
      Parenchymal enhancement heterogeneity after GBCA0.32
      • Absent
      14(32.6)1(10)
      • Present
      29(67.4)9(90)
      ANALI score without gadolinium0.61
      • 0
      14(28.0)1(9.1)
      • 1
      4(8.0)1(9.1)
      • 2
      5(10.0)1(9.1)
      • 3
      4(8.0)0(0)
      • 4
      13(26.0)5(45.4)
      • 5
      10(20.0)3(27.3)
      ANALI score with gadolinium0.32
      • 0
      13(30.2)1(10)
      • 1
      5(11.6)2(20)
      • 2
      25(58.0)7(70)
      Nominal variables are expressed as absolute number (percentage). Abbreviations: CBD, common bile duct; RHD, right hepatic duct; LHD, left hepatic duct; IHBD, intrahepatic bile duct; GBCA, Gadolinium based contrast agent. Levels of significance are indicated in the table (Chi-squared test or Fisher test according the frequencies, as appropriate). A p value of less than 0.05 was considered significant.
      Overall, these findings suggest that to some extent, the gallbladder exerted protective functions in PSC patients, and even more so when the gallbladder is enlarged, whereas cholecystectomy was associated with more severe disease features. To address this latter possibility experimentally, we examined the direct impact of cholecystectomy on PSC phenotypic phenotypic traits in the Abcb4 knockout mouse model.

      Impact of cholecystectomy on PSC phenotypic traits in the Abcb4 knockout mice

      Abcb4-/- mice showed no evidence of gallbladder enlargement (data not shown). Cholecystectomy was well tolerated and did not significantly affect animals’ survival or body weight gain as compared to sham operation, until the end of experiment (Fig. S3). Histological analysis by an expert pathologist showed that cholecystectomized mice developed more severe cholangitis, fibrosis and bile duct loss (Fig. 3A). Immunostaining analyses revealed that ductular reaction, liver inflammation and fibrosis were significantly more severe in cholecystectomized Abcb4-/- mice than in sham-operated Abcb4-/- mice (Fig. 3B). In keeping with these results, the hepatic expression of Tnfα was significantly higher in cholecystectomized vs. sham-operated Abcb4-/- mice (Fig. 3C). Likewise, the hepatic expression of fibrosis markers, i.e., α-smooth muscle actin (Acta2) and collagen 1a1 (Col1a1), was significantly higher in cholecystectomized Abcb4-/- mice (Fig. 3C). The hepatic expressions of cytochrome P450 7A1 (Cyp7a1) and of sodium taurocholate cotransporting polypeptide Ntcp (Slc10a1) were both significantly decreased in cholecystectomized as compared to sham-operated Abcb4-/- mice (Fig. 4A). This implied that cholestasis was aggravated as a result of cholecystectomy in Abcb4-/- mice, which was confirmed by a significant increase in total bile acid content in the liver of cholecystectomized vs. sham-operated Abcb4-/- mice (Fig. 4B). The analyses of bile acid species showed that the concentrations of taurocholic acid (TCA) were significantly increased both in the liver and plasma of cholecystectomized Abcb4-/- mice as compared to sham-operated mice (Fig. 4C). In addition, although both primary and secondary bile acids were increased in the plasma of cholecystectomized mice as compared to sham-operated mice, only the elevation of secondary bile acids was statistically significant (Fig. 4C). Overall, these results clearly demonstrated that cholecystectomy caused an increase in bile acid overload and an aggravation of cholangiopathy in the Abcb4 knockout mouse model of PSC.
      Figure thumbnail gr3
      Figure 3Impact of cholecystectomy on PSC phenotypic traits in Abcb4-/- mice. Liver samples were collected from Sham-operated (n=7) or cholecystectomized (n=6) Abcb4-/- mice, five weeks after surgery, for A) histological analysis of hematoxylin and eosin-stained tissue sections (left panel, cholangitis characterized by periductal inflammation (arrows) and onion-skin fibrosis (arrowheads)), according to an adaptation of Nakanuma score (cholangitis activity, fibrosis score and bile duct loss were evaluated using a 0 to 3 scale (right panel, hepatitis activity=0 in all samples, not shown); B) analysis of ductular reaction, inflammation and fibrosis, assessed by cytokeratin (CK)-19, F4/80 and Sirius red staining, respectively; C) RT-qPCR analysis of proinflammatory (Tnfα) and profibrotic factors (Col1a1, Acta2) expression. Data were normalized for Hprt1 and expressed relative to a pool of hepatic mRNA from wildtype (WT) mice. Levels of significance are indicated in the figure (Student’s t test). A p value of less than 0.05 was considered significant. Scale bars = 250 μm for hematoxylin and eosin and F4/80; 500 μm for CK-19, 1mm for Sirius red.
      Figure thumbnail gr4
      Figure 4Impact of cholecystectomy on bile acid metabolism in Abcb4-/- mice. Liver and plasma samples were collected from Sham-operated (n=7) or cholecystectomized (n=6) Abcb4-/- mice, five weeks after surgery, for A) RT-qPCR analysis of hepatic expression of genes involved in bile acid homeostasis (Data were normalized for Hprt1 and expressed relative to a pool of hepatic mRNA from wildtype (WT) mice); B-C) HPLC-MS/MS analysis of total bile acids (B) and individual bile acid species (C) in liver and plasma. Levels of significance are indicated in the figure (Student’s t test). A p value of less than 0.05 was considered significant.

      Discussion

      In the present study, we provide several pieces of evidence indicating that the gallbladder promotes hepatobiliary protection in PSC. First, we show that, compared to PSC patients with normal gallbladder volume, PSC patients with enlarged gallbladders have significantly lower alkaline phosphatase, which is associated with a favorable prognosis in PSC
      • de Vries E.M.G.
      • Wang J.
      • Leeflang M.M.G.
      • Boonstra K.
      • Weersma R.K.
      • Beuers U.H.
      • et al.
      Alkaline phosphatase at diagnosis of primary sclerosing cholangitis and 1 year later: evaluation of prognostic value.
      . We also show that, compared to PSC patients with conserved gallbladders, PSC patients who underwent a cholecystectomy display more severe cholangiographic features and higher serum aspartate aminotransferase, which are both associated with adverse outcome in PSC
      • Lemoinne S.
      • Cazzagon N.
      • El Mouhadi S.
      • Trivedi P.J.
      • Dohan A.
      • Kemgang A.
      • et al.
      Simple Magnetic Resonance Scores Associate With Outcomes of Patients With Primary Sclerosing Cholangitis.
      ,
      • Kim W.R.
      • Therneau T.M.
      • Wiesner R.H.
      • Poterucha J.J.
      • Benson J.T.
      • Malinchoc M.
      • et al.
      A revised natural history model for primary sclerosing cholangitis.
      . Furthermore, we demonstrate that cholecystectomy causes an aggravation of cholangiopathy features in the Abcb4 knockout mouse model of PSC, in support of a causal relationship between gallbladder removal and increased disease severity. Two main mechanisms may account for the protective functions of the gallbladder in PSC, i.e. a release of hyperpressure in the biliary tract
      • Bidault-Jourdainne V.
      • Merlen G.
      • Glénisson M.
      • Doignon I.
      • Garcin I.
      • Péan N.
      • et al.
      TGR5 controls bile acid composition and gallbladder function to protect the liver from bile acid overload.
      , and a modulation of the enterohepatic circulation and composition of the bile acid pool
      • Housset C.
      • Chrétien Y.
      • Debray D.
      • Chignard N.
      Functions of the Gallbladder.
      • Debray D.
      • Rainteau D.
      • Barbu V.
      • Rouahi M.
      • El Mourabit H.
      • Lerondel S.
      • et al.
      Defects in gallbladder emptying and bile Acid homeostasis in mice with cystic fibrosis transmembrane conductance regulator deficiencies.
      • Pomare E.W.
      • Heaton K.W.
      The effect of cholecystectomy on bile salt metabolism.
      , which may ultimately restrict bile acid cytotoxicity. Both mechanisms can be amplified by gallbladder enlargement, and suppressed by cholecystectomy.
      To address the impact of gallbladder enlargement in PSC, we first measured fasting gallbladder volume using MRI in the largest number of PSC patients so far. The threshold was set at 50 mL to define gallbladder enlargement on the basis of a previous MRI study, which included PSC patients and healthy subjects. Based on these criteria, we found that the gallbladder was enlarged in more than half of the PSC patients. In none of them did we detect a local cause of gallbladder distension such as stenosis of the cystic duct. We suspect that gallbladder enlargement is caused by hyperpressure in the biliary tract of PSC patients, as recently shown to explain gallbladder enlargement in bile duct-ligated mice
      • Bidault-Jourdainne V.
      • Merlen G.
      • Glénisson M.
      • Doignon I.
      • Garcin I.
      • Péan N.
      • et al.
      TGR5 controls bile acid composition and gallbladder function to protect the liver from bile acid overload.
      . However, the reason why some PSC patients develop gallbladder enlargement whereas others do not is yet unknown. As in previous studies
      • van de Meeberg P.C.
      • Portincasa P.
      • Wolfhagen F.H.
      • van Erpecum K.J.
      • VanBerge-Henegouwen G.P.
      Increased gall bladder volume in primary sclerosing cholangitis.
      ,
      • Said K.
      • Edsborg N.
      • Albiin N.
      • Bergquist A.
      Gallbladder emptying in patients with primary sclerosing cholangitis.
      , we found no relationship between gallbladder enlargement on the one hand, and the severity of bile duct strictures, the stage of disease or the association with IBD, on the other hand. We found even so no relationship with the serum levels of FGF19, the hormone that stimulates gallbladder filling
      • Choi M.
      • Moschetta A.
      • Bookout A.L.
      • Peng L.
      • Umetani M.
      • Holmstrom S.R.
      • et al.
      Identification of a hormonal basis for gallbladder filling.
      . However, this data was available in only half of the patients. Of particular importance regarding potential long-term effects of gallbladder enlargement, gallbladder volume showed little or no variation over time in the same patient. It was previously shown that both fasting and postprandial gallbladder volumes were enlarged in PSC subjects, whereas the ejection fraction in response to a non-physiological test meal was normal
      • van de Meeberg P.C.
      • Portincasa P.
      • Wolfhagen F.H.
      • van Erpecum K.J.
      • VanBerge-Henegouwen G.P.
      Increased gall bladder volume in primary sclerosing cholangitis.
      ,
      • Said K.
      • Edsborg N.
      • Albiin N.
      • Bergquist A.
      Gallbladder emptying in patients with primary sclerosing cholangitis.
      . In a similar situation, i.e., Cftr-deficient mice, in which both fasting and postprandial gallbladder volumes were enlarged, whereas the ejection fraction in response to a non-physiological stimulus (cholecystokinin-8) was normal, we previously demonstrated that the enterohepatic circulation of bile acids and the formation of secondary bile acids were decreased
      • Debray D.
      • Rainteau D.
      • Barbu V.
      • Rouahi M.
      • El Mourabit H.
      • Lerondel S.
      • et al.
      Defects in gallbladder emptying and bile Acid homeostasis in mice with cystic fibrosis transmembrane conductance regulator deficiencies.
      . Likewise, in PSC patients, we found that the gallbladder volume was negatively correlated with bile acid hydrophobicity. On the contrary, gallbladder volume was positively correlated with the ratio of UDCA to total bile acids. Therefore, decreased bile acid toxicity may have contributed to lessen cholestatic liver injury as attested by lower alkaline phosphatase in the patients with enlarged gallbladders as compared to those with normal-sized gallbladders. In addition, PSC patients with enlarged gallbladders compared to those with normal-sized gallbladders, displayed lower tauro- vs. glycoconjugates ratios. Previous work demonstrated the prognostic value of circulating bile acid profiles in PSC patients. Notably, lower tauro- vs. glycoconjugates ratios were shown to predict a lower risk of hepatic decompensation
      • Mousa O.Y.
      • Juran B.D.
      • McCauley B.M.
      • Vesterhus M.N.
      • Folseraas T.
      • Turgeon C.T.
      • et al.
      Bile Acid Profiles in Primary Sclerosing Cholangitis and Their Ability to Predict Hepatic Decompensation.
      .
      We evaluated the impact of cholecystectomy, as another approach to assess gallbladder-mediated protection in PSC. PSC patients whose gallbladders had been removed were characterized by an increased severity of cholangiographic features as compared to patients whose gallbladders were conserved. Severe dilatations in particular were more frequent in the different portions of the biliary tract of cholecystectomized patients as compared to others. This finding strongly suggests that cholecystectomy triggers an aggravation of hyperpressure in the biliary tract in PSC, as previously reported in bile duct-ligated mice
      • Bidault-Jourdainne V.
      • Merlen G.
      • Glénisson M.
      • Doignon I.
      • Garcin I.
      • Péan N.
      • et al.
      TGR5 controls bile acid composition and gallbladder function to protect the liver from bile acid overload.
      . In keeping with this scenario, the serum levels of aspartate aminotransferase were significantly higher in cholecystectomized patients as compared to others, as one would expect from an increase in the biliary infarcts that develop upstream of biliary obstructions. Serum bile acid analyses revealed no significant difference between cholecystectomized and non-cholecystectomized patients. However, we observed a trend towards higher levels of total bile acids in the group of cholecystectomized patients. On a small number of subjects, we also observed trends towards lower C4 and higher FGF19 concentrations in this group, reflecting lower and higher rates of bile acid synthesis and ileal reabsorption, respectively. This is consistent with post-cholecystectomy acceleration of bile acid enterohepatic circulation
      • Housset C.
      • Chrétien Y.
      • Debray D.
      • Chignard N.
      Functions of the Gallbladder.
      • Debray D.
      • Rainteau D.
      • Barbu V.
      • Rouahi M.
      • El Mourabit H.
      • Lerondel S.
      • et al.
      Defects in gallbladder emptying and bile Acid homeostasis in mice with cystic fibrosis transmembrane conductance regulator deficiencies.
      • Pomare E.W.
      • Heaton K.W.
      The effect of cholecystectomy on bile salt metabolism.
      . We confirmed the deleterious effects of cholecystectomy on PSC phenotypic traits in the Abcb4 knockout mice. All histopathological features of PSC, i.e. cholangitis, inflammation and liver fibrosis, were aggravated by cholecystectomy in this model. We showed that the deleterious effect of cholecystectomy involved an increase in the overload of bile acids most notably taurocholate, which was previously shown to trigger biliary damage in different murine models, including the Abcb4 knockout mice
      • van Nieuwerk C.M.
      • Groen A.K.
      • Ottenhoff R.
      • van Wijland M.
      • van den Bergh Weerman M.A.
      • Tytgat G.N.
      • et al.
      The role of bile salt composition in liver pathology of mdr2 (-/-) mice: differences between males and females.
      ,
      • Li Y.
      • Tang R.
      • Leung P.S.C.
      • Gershwin M.E.
      • Ma X.
      Bile acids and intestinal microbiota in autoimmune cholestatic liver diseases.
      . The enterohepatic circulation of bile acids was also likely accelerated as attested by an increased proportion of secondary bile acids in the circulation of Abcb4 knockout mice post-cholecystectomy.
      Thus, our present results infer that cholecystectomy may cause disease aggravation in PSC patients. The deleterious effects of cholecystectomy on liver disease also likely apply to other pathological settings. A very large cohort study previously showed that independent of cholelithiasis, patients who underwent cholecystectomy were twice more likely to develop cirrhosis and to have elevated serum aminotransferase, than those without previous cholecystectomy
      • Ioannou G.N.
      Cholelithiasis, cholecystectomy, and liver disease.
      .
      We recognize that our study has some limitations. The main limitation relates to the restricted size of the PSC study population. Moreover, the significant differences we observed between patient groups were limited to surrogate markers, such as alkaline phosphatase or cholangiographic features. Larger studies are required to confirm our findings and to investigate the potential impact of gallbladder presence and size on the natural history of PSC.
      The major strength of our study is to confront data obtained from PSC patients with those obtained in Abcb4-/- mice. Together, these data suggest that the gallbladder could provide hepatobiliary protection in PSC through the sequestration of bile and the modification of bile acid molecular species composition. These protective mechanisms are suppressed by cholecystectomy and to some extent amplified by gallbladder enlargement. More work remains to be done to fully elucidate the complex role of the gallbladder in PSC and in other chronic liver diseases.

      Data Availability Statement

      The data that support the findings of this study are available on request from the corresponding author [SL, NC]. The data are not publicly available due to privacy restrictions.

      Acknowledgements

      The authors gratefully acknowledge T. Coulais, L. Dinard, A. Guyomard, T. Ledent, Q. Pointout from the CRSA animal facility, F. Merabtene, B. Solhonne, Y.Chrétien (CRSA), S. Fouquet, R. and M-L. Niepon (Institut de la Vision) from Sorbonne Université Imaging and Cytometry (LUMIC) network for their assistance. The authors also acknowledge the CRI Biochemistry Platform (Université Paris Diderot) where plasma biochemical analyses were performed.

      ABBREVIATIONS

      ABC
      ATP-binding cassette transporter
      BA
      bile acid
      CA
      cholic acid
      CCT
      cholecystectomy
      CDCA
      chenodeoxycholic acid
      CFTR
      cystic fibrosis transmembrane conductance regulator
      CK19
      cytokeratin 19
      DCA
      deoxycholic acid
      FGF
      fibroblast growth factor
      HCA
      hyocholic acid
      HDCA
      hyodeoxycholic acid
      IBD
      inflammatory bowel disease
      LCA
      lithocolic acid
      HPLC-MS/MS
      high-performance liquid chromatography coupled to tandem mass spectrometry
      MRC
      magnetic resonance cholangiography
      MRI
      magnetic resonance imaging
      PSC
      primary sclerosing cholangitis
      UDCA
      ursodeoxycholic acid
      ULN
      upper limit of normal
      VCTE
      vibration controlled transient elastography

      Appendix A. Supplementary data

      The following is/are the supplementary data to this article:

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