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Bile from Patients with Primary Sclerosing Cholangitis Contains Mucosal-Associated Invariant T-Cell Antigens

  • Laura Valestrand
    Affiliations
    Norwegian PSC Research Center, Division of Surgery, Inflammatory Diseases and Transplantation, Oslo University Hospital, Oslo, Norway

    Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway

    Institute of Clinical Medicine, University of Oslo, Oslo, Norway

    Section of Gastroenterology, Division of Surgery, Inflammatory Diseases and Transplantation, Oslo University Hospital, Oslo, Norway
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  • Fei Zheng
    Affiliations
    Norwegian PSC Research Center, Division of Surgery, Inflammatory Diseases and Transplantation, Oslo University Hospital, Oslo, Norway

    Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway

    Institute of Clinical Medicine, University of Oslo, Oslo, Norway
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  • Simen H. Hansen
    Affiliations
    Norwegian PSC Research Center, Division of Surgery, Inflammatory Diseases and Transplantation, Oslo University Hospital, Oslo, Norway

    Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway
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  • Jonas Øgaard
    Affiliations
    Norwegian PSC Research Center, Division of Surgery, Inflammatory Diseases and Transplantation, Oslo University Hospital, Oslo, Norway

    Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway

    Institute of Clinical Medicine, University of Oslo, Oslo, Norway
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  • Johannes R. Hov
    Affiliations
    Norwegian PSC Research Center, Division of Surgery, Inflammatory Diseases and Transplantation, Oslo University Hospital, Oslo, Norway

    Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway

    Institute of Clinical Medicine, University of Oslo, Oslo, Norway

    Section of Gastroenterology, Division of Surgery, Inflammatory Diseases and Transplantation, Oslo University Hospital, Oslo, Norway
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  • Niklas K. Björkström
    Affiliations
    Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden
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  • Tom H. Karlsen
    Affiliations
    Norwegian PSC Research Center, Division of Surgery, Inflammatory Diseases and Transplantation, Oslo University Hospital, Oslo, Norway

    Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway

    Institute of Clinical Medicine, University of Oslo, Oslo, Norway

    Section of Gastroenterology, Division of Surgery, Inflammatory Diseases and Transplantation, Oslo University Hospital, Oslo, Norway
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  • Xiaojun Jiang
    Affiliations
    Norwegian PSC Research Center, Division of Surgery, Inflammatory Diseases and Transplantation, Oslo University Hospital, Oslo, Norway

    Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway

    Institute of Clinical Medicine, University of Oslo, Oslo, Norway
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  • Espen Melum
    Correspondence
    Address correspondence to Espen Melum, M.D., Ph.D., Norwegian PSC Research Center, Division of Surgery, Inflammatory Diseases and Transplantation, Oslo University Hospital, Rikshospitalet, Postboks 4950 Nydalen, 0424 Oslo, Norway.
    Affiliations
    Norwegian PSC Research Center, Division of Surgery, Inflammatory Diseases and Transplantation, Oslo University Hospital, Oslo, Norway

    Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway

    Institute of Clinical Medicine, University of Oslo, Oslo, Norway

    Section of Gastroenterology, Division of Surgery, Inflammatory Diseases and Transplantation, Oslo University Hospital, Oslo, Norway

    Hybrid Technology Hub-Centre of Excellence, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo, Norway
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Open AccessPublished:January 18, 2022DOI:https://doi.org/10.1016/j.ajpath.2021.12.008
      Primary sclerosing cholangitis (PSC) is associated with altered microbiota of the gut and bile. Mucosal-associated invariant T (MAIT) cells, enriched in human liver, uniquely recognize microbial-derived metabolites. This study aimed to determine whether bile from patients with PSC contains antigens activating MAIT cells. Bile was collected at the time of liver transplantation from patients with PSC (n = 28). The bile samples were either directly incubated with peripheral blood mononuclear cells from healthy donors or with antigen-presenting cells followed by co-culture with peripheral blood mononuclear cells. MAIT cell activation was assessed by flow cytometry. An anti-MR1 antibody was used to determine whether the activation was major histocompatibility complex class I–related protein (MR1) restricted. Biliary microbiota profiles were generated using 16S rRNA amplicon sequencing, and the abundance of the bacterial gene ribD was predicted. Eight of 28 bile samples could activate MAIT cells. This activation was partly MR1-dependent in five of eight bile samples. Microbial DNA was detected in 15 of 28 bile samples, including the five bile samples leading to MR1-dependent activation. A higher abundance of the ribD gene expression in the group of bile samples that could activate MAIT cells was predicted on the basis of the 16S sequencing. In co-culture experiments, cholangiocytes could take up and present biliary antigens to MAIT cells. These findings suggest a pathophysiological pathway in PSC connecting the immune system and the microbiome.
      Primary sclerosing cholangitis (PSC) is a chronic liver disease, characterized by bile duct inflammation, progressive fibrosis, and a strong association with inflammatory bowel disease (IBD), seen in up to 80% of the patients.
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      Biliary epithelium and liver B cells exposed to bacteria activate intrahepatic MAIT cells through MR1.
      We therefore hypothesized that bile contains potential MAIT antigens that can be processed by cholangiocytes and presented to MAIT cells to initiate or modulate the immune response in inflammatory bile duct diseases.

      Materials and Methods

      Patient Bile Samples and Peripheral Blood from Healthy Donors

      Bile was collected from the gallbladder of patients directly after liver transplantation due to PSC (n = 28), alcohol-related liver disease (n = 4), autoimmune hepatitis (n = 2), or hemochromatosis (n = 1). A sterile scalpel was used to cut a small opening in the gallbladder, and a minimum of 3 mL of bile was aspirated into sterile tubes with a sterile 20-mL syringe, stored, and aliquoted on ice until longtime storage at −80°C. Written informed consent was obtained from all study participants. In accordance with the Declaration of Helsinki, ethical approval was obtained from the Regional Committees for Medical and Health Research Ethics of South East Norway (reference numbers 2012-286 and 2016-1540). Buffy coats from healthy donors were obtained from Oslo University blood bank, and the usage was approved by Regional Committee for Medical and Health Research Ethics of South East Norway (reference number S-05172).

      Clinical Characterization

      For clinicopathologic characterizations of included patients, the following indexes were calculated:
      Model for End-Stage Liver Disease Sodium score
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      Cell Isolation and Cell Culture

      Peripheral blood mononuclear cells (PBMCs) were separated from buffy coats using Ficoll Paque Plus (GE Healthcare Life Sciences, Uppsala, Sweden). The collected PBMCs were aliquoted in a cell cryopreservation media (Merck KGaA, Darmstadt, Germany) and stored in liquid nitrogen. For experiments, PBMCs were thawed, washed, and maintained in RPMI 1640 medium (Lonza, Basel, Switzerland) supplemented with 2 mmol/L l-glutamine (Merck KGaA), 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA), 1% Gibco Antibiotic-Antimycotic (10,000 units/mL of penicillin, 10,000 μg/mL of streptomycin, and 25 μg/mL of Amphotericin B; Thermo Fisher Scientific). A human monocyte cell line (THP1) was maintained in supplemented media as described for the PBMCs, and a previously immortalized human cholangiocyte cell line (H69) was cultured in conditioned medium as previously described.
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      • Lee D.W.
      • Murray S.L.
      • Rogers L.C.
      • Wolkoff L.I.
      • Mulberg A.E.
      • Cherington V.
      • Jefferson D.M.
      Regulation of intracellular pH by immortalized human intrahepatic biliary epithelial cell lines.
      Both cell lines were maintained in 37°C incubators with 5% CO2, and the PBMCs were cultivated for at least 1 hour before application in experiments.

      Escherichia coli Culture and Fixation

      Escherichia coli (DH5α; Thermo Fisher Scientific) was cultured overnight at 37°C in Luria-Bertani broth (Sigma-Aldrich, St. Louis, MO), then counted by standard plate-counting methods, aliquoted in a solution made by 50% glycerol and 50% fetal calf serum, and stored at −80°C. To fixate the E. coli, aliquots were thawed and washed in phosphate-buffered saline (PBS), then incubated for 5 minutes at room temperature in 1% formaldehyde (HistoLab, Gothenburg, Sweden) and vortexed before washing and resuspension in PBS in different dilutions.

      5-OP-RU Preparation

      5-Amino-6-d-ribitylaminouracil (5-A-RU; MuseChem, Fairfield, NJ) was stored at 4°C in solid form until dissolved in sterile and distilled water, and frozen at −80°C in 5 mmol/L stock solutions. Fifteen minutes before application in experiments, the solid chemical was mixed with methylglyoxal (Sigma-Aldrich) and used in the indicated doses.

      Immunofluorescence Staining of MR1 on H69 Cells

      A total of 100,000 H69 cells in 500 μL conditioned medium were seeded on eight-chamber slides (Thermo Fisher Scientific) before overnight incubation in a 37°C incubator with 5% CO2. The slides were then washed 2 × 1 minute in cold PBS before 15-minute fixation with formaldehyde 4% (Sigma-Aldrich) and permeabilized for 5 minutes with 0.2% Triton X-100 (Sigma-Aldrich) at room temperature. The slides were then blocked with 1% bovine serum albumin and 0.1% Tween 20 in PBS for 15 minutes before washings and staining with primary anti-MR1 antibody (catalog number 13260-1-AP; Proteintech, Rosemont, IL) or an isotype control antibody (catalog number 30000-0-AP; Proteintech) for 1 hour at room temperature. The slides were then washed and incubated for 1 hour with Alexa Fluor 488 goat anti-rabbit secondary antibody (catalog number A11008; Thermo Fisher Scientific) before washings and mounting with ProLong Gold antifade with DAPI (Thermo Fisher Scientific) and coverslips. After an overnight incubation at room temperature in the dark, images were acquired with an Eclipse e400 Fluorescent Microscope (Nikon, Tokyo, Japan) with a DS-Fi1 camera (Nikon) controlled by NIS-Element BR 3.10 software (Nikon) with identical exposure times and camera settings.

      MAIT Cell Activation Assay

      PBMCs were plated in 96 round-bottom plates (5 × 105 cells in 200 μL medium per well in triplicates) and incubated in supplemented medium with bile (diluted 1:200), fixed E. coli (250 colony-forming units/cell), or PBS for 24 hours. In experiments with multiple PBMC donors, the PBMCs were plated as singlets instead of triplicates and they were incubated with bile for 48 hours; otherwise the protocol was identical to experiments with one PBMC donor. In experiments with blocking of MR1-mediated antigen presentation, 20 μg/mL of the monoclonal anti-MR1 antibody clone 26.5 (BioLegend, San Diego, CA) was added 30 minutes before incubation with bile or fixed E. coli. Intracellular cytokines were analyzed by flow cytometry after 6 hours of culture with brefeldin A and monensin (eBioscience, San Diego, CA).

      Co-Culture Activation Assay

      In co-culture assays, THP1 or H69 cells were used as antigen-presenting cells (APCs) and seeded in 96-well flat-bottomed plates for H69 cells and round-bottomed plates for THP1 cells (8 × 104 cells per well) followed by overnight incubation. Bile (diluted 1:200), fixed E. coli (250 colony-forming units/cell), or PBS was then added, followed by 24 hours of incubation. Antigens not taken up by the APCs were washed off with cell culture medium, followed by addition of PBMCs and 24 hours of incubation. In experiments with blocking of MR1-mediated antigen presentation, 20 μg/mL of an MR1-blocking antibody (clone 26.5) was added 30 minutes before incubation with PBMCs. Intracellular cytokines were analyzed by flow cytometry after 6 hours of culture with brefeldin A and monensin (eBioscience).

      Antibodies and Flow Cytometry

      The following antibodies against human epitopes were used: CD3 [1:200; clone HIT3a; phycoerythrin (PE); catalog number 300308], TCR Vα7.2 (1:100; 3C10; APC; catalog number 351708), CD161 [1:100; HP-3G10; fluorescein isothiocyanate (FITC); catalog number 339906], CD19 (1:200; HIB19; APCCY7; catalog number 302218), CD69 (1:200; FN50; PECY7; catalog number 310912), granzyme B (1:200; QA16A02; PercpCy5.5; catalog number 372212), and viability dye (1:100; Zombie NIR; catalog number 423106), all purchased from BioLegend. Human MR1 5-OP-RU (1:800, PE) and MR1 6-formylpterin (6-FP, PE) (1:800) tetramers were kindly provided by the NIH Tetramer Core. Cell surface markers were stained using directly conjugated antibodies for 30 minutes at 4°C in PBS with 2% fetal calf serum, and dead cells were excluded using a live/dead fixable viability dye (Zombie NIR). For staining of intracellular markers, cells were fixed for 45 minutes using BD Fixation/Permeabilization Kit (BD Biosciences, San Jose, CA) and washed once in Perm/Wash before 45 minutes of staining with intracellular monoclonal antibodies in Perm/Wash followed by the last wash with Perm/Wash and data acquisition. Flow cytometric analysis was performed using a BD FACS Verse flow cytometer (BD Biosciences), and results were analyzed in Flow Jo version 10.1 (BD Life Science, San Jose, CA).

      16S rRNA Gene Sequencing and Analysis

      Microbial DNA from bile samples was extracted using the QIAamp DNA mini kit (Qiagen, Hilden, Germany), as previously described.
      • Shimamura M.
      • Yamamura M.
      • Nabeshima T.
      • Kitano N.
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      • Andrew P.
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      • Sköld M.
      • Xiong X.
      • Illarionov P.A.
      • Besra G.S.
      • Behar S.M.
      • Fadrosh D.W.
      • Ma B.
      • Gajer P.
      • Sengamalay N.
      • Ott S.
      • Brotman R.M.
      • Ravel J.
      • Moen A.E.F.
      • Tannæs T.M.
      • Vatn S.
      • Ricanek P.
      • Vatn M.H.
      • Jahnsen J.
      Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy.
      ,
      • Valestrand L.
      • Lie Berntsen N.
      • Zheng F.
      • Schrumpf E.
      • Hyll Hansen S.
      • Hemming Karlsen T.
      • Steven Blumberg R.
      • Roksund Hov J.
      • Jiang X.
      • Melum E.
      Lipid antigens in bile from patients with chronic liver diseases activate natural killer T cells.
      The hypervariable regions V3 and V4 of the prokaryotic 16S rRNA gene were amplified using the 319F-806R primer pair and a dual-indexing approach with barcoded primers and Phusion High-Fidelity PCR master mix with HF buffer (Thermo Fisher Scientific).
      • Fadrosh D.W.
      • Ma P.G.B.
      • Sengamalay N.
      • Ott S.
      • Brotman R.M.
      • Ravel J.
      An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform.
      16S rRNA sequencing on the Illumina MiSeq (Illumina Inc., San Diego, CA) platform and data analysis were performed as described previously.
      • Valestrand L.
      • Lie Berntsen N.
      • Zheng F.
      • Schrumpf E.
      • Hyll Hansen S.
      • Hemming Karlsen T.
      • Steven Blumberg R.
      • Roksund Hov J.
      • Jiang X.
      • Melum E.
      Lipid antigens in bile from patients with chronic liver diseases activate natural killer T cells.
      In short, paired-end reads were filtered for Illumina Universal Adapters and PhiX, demultiplexed, quality trimmed, and merged. Denoising to amplicon sequence variants and taxonomic classification were performed using the Deblur Plugin
      • Amir A.
      • Daniel M.
      • Navas-Molina J.
      • Kopylova E.
      • Morton J.
      • Xu Z.Z.
      • Eric K.
      • Thompson L.
      • Hyde E.
      • Gonzalez A.
      • Knight R.
      Deblur rapidly resolves single-nucleotide community sequence patterns.
      in the Quantitative Insights Into Microbial Ecology 2 platform version 2019.7.
      • Bolyen E.
      • Rideout J.R.
      • Dillon M.R.
      • Bokulich N.A.
      • Abnet C.C.
      • Al-Ghalith G.A.
      • et al.
      Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2.
      In each bile sample, taxa with number of reads of <100 were discarded. There were no detectable levels of bacteria in the negative controls, and so no identified contaminants were removed from the data set before further analyses were performed. PICRUSt2 analysis was run on unfiltered bile samples to predict the abundance of the ribD gene in the sequenced bacteria compared against a published gene database.
      • Markowitz V.M.
      • Chen I.M.A.
      • Palaniappan K.
      • Chu K.
      • Szeto E.
      • Grechkin Y.
      • Ratner A.
      • Jacob B.
      • Huang J.
      • Williams P.
      • Huntemann M.
      • Anderson I.
      • Mavromatis K.
      • Ivanova N.N.
      • Kyrpides N.C.
      IMG: the integrated microbial genomes database and comparative analysis system.

      Statistical Analysis

      All values are presented as means ± SEM unless otherwise stated. Statistical significance was evaluated using t-test for variables meeting criteria of normal distribution. For experiments where multiple comparisons were included, one-way analysis of variance was used followed by correction for multiple testing using the Bonferroni method. To evaluate statistical significance for categorical data, a Fisher exact test was applied. For evaluating correlations between clinicopathologic scores and presence of MAIT antigens or ribD abundance, a Pearson r test was performed. P < 0.05 was considered statistically significant. Statistical tests were performed using the Prism GraphPad software version 8.0 (Graphpad Software Inc., La Jolla, CA).

      Results

      Bile from Patients with PSC Activates MAIT Cells

      To investigate the presence of potential MAIT cell antigens, bile from 28 patients with PSC (Table 1) was screened using PBMCs from healthy donors (Figure 1A, two additional donors are shown in Supplemental Figure S1). The percentages of GrB+ and CD69+ MAIT cells were analyzed by flow cytometry (gating strategies in Figure 1, A and B, and Supplemental Figure S1) with E. coli as a positive control. Incubation with eight of the bile samples led to the activation of MAIT cells, as measured by increased CD69 and GrB expression (Figure 1, B and D, and Supplemental Figure S1). To evaluate the potency of the antigens, a serial-dilution experiment was performed for two of the bile samples demonstrating a clear dose-response relationship (Figure 1C). Because 20 of 28 bile samples did not activate MAIT cells and because MAIT cell response induced by the different bile samples was highly variable, it is unlikely that the activation of MAIT cells results from a physiological constituent in bile.
      Table 1Clinical Characteristics of Bile Sample Donors with PSC
      Variables at time of transplantation
      BileSample no.SexDiagnosisMELD-Na scoreLiver cirrhosisUDCA/ABComorbiditiesERCBilirubin,mg/dLALT,U/LALP,U/LCRP,mg/L
      1FPSC18YesNo/yesCDNo5.03714124
      2MPSC8NoNo/yesYes1.44124010
      3MPSC10YesNo/noHT, adrenal insufficiency, UCNo1.410423815
      4MPSC13NoYes/yesHypothyroidism, UCNo4.612050550
      5MPSC9NoYes/noUC, celiac disease, arthralgiaNo2.22645284.6
      6FPSC12YesNo/noYes3.2823251.7
      7FPSC22YesYes/noUCNo14.58928123
      8MPSC7YesNo/noNo0.52616712
      9FPSC, HCC6YesYes/noUC, colectomyNo0.9441422
      10FPSC6NoNo/noUC, colectomyYes0.431691.1
      11MPSC17NoNo/yesNo4.28711322
      12MPSC17YesNo/noIleoanal pouch, UC, colectomyNo2.417147311
      13MPSC22YesNo/yesUCYes17.710372415
      14FPSC7NoYes/noAsthmaYes0.9641935.7
      15MPSC7YesYes/noUCNo1.21283443.1
      16MPSC19YesYes/noIndetermined colitisNo5.310066546
      17MPSC15YesNo/noUCYes5.112772214
      18MPSC6YesYes/noWPW syndrome, UCYes0.62293373.5
      19FPSC6NoNo/noUCYes0.435712.8
      20MPSC8NoNo/noUCYes0.81192551.1
      21MPSC13YesNo/noUCNo2.13231627.8
      22MPSC15NoNo/noUCYes10.633956517
      23MPSC6NoNo/yesUCYes0.66140772
      24MPSC7NoNo/noNo0.436681.2
      25MPSC6YesNo/yesHCMYes0.6973921.9
      26MPSC, HCC18YesNo/noUCNo4.04417773
      27MPSC15YesYes/yesCD, gastric ulcerNo4.27220322
      28MPSC15YesYes/noIgA nephritis, UC, HCMYes7.124231220
      The presence of liver cirrhosis in the explanted liver was evaluated by a liver pathologist. Biochemistry values represent the values just before liver transplantation, and whether ERC had been performed the last 6 months before liver transplantation was registered.
      F, female; M, male; AB, antibiotics; ALP, alkaline phosphatase; ALT, alanine aminotransferase; CD, Crohn disease; CRP, C-reactive protein; ERC, endoscopic retrograde cholangiography; HCC, hepatocellular carcinoma; HCM, hemochromatosis; HT, hypertension; MELD-Na, Model for End-Stage Liver Disease Sodium; PSC, primary sclerosing cholangitis; UC, ulcerative colitis; UDCA, ursodeoxycholic acid; WPW, Wolff-Parkinson-White.
      Figure thumbnail gr1
      Figure 1Mucosal-associated invariant T (MAIT) cells are activated by antigens in bile from patients with primary sclerosing cholangitis (PSC). A: Representative flow plots show the gating strategy for identifying human MAIT cells; CD161+ T-cell receptor Vα7.2+ T cells within peripheral blood mononuclear cells (PBMCs) after excluding B cells and dead cells (CD19 and Zombie NIR) and duplicates. Gating strategy for Vα7.2 T cells is also shown. B: Representative flow plots of CD69+ granzyme B+ (GrB+) MAIT cells, after incubation with phosphate-buffered saline (PBS), fixed Escherichia coli, or bile and PBMCs. C: Bar plots showing the percentage of CD69+ GrB+ MAIT cells within PBMCs after incubation with bile sample numbers 2 and 4 in serial dilutions. D and E: Bar plots showing the percentage of CD69+ GrB+ MAIT cells (D) or CD69+ GrB+ Vα7.2 T cells (E) after stimulation with 28 bile samples (diluted 1:200) that were collected from the gallbladder from patients with PSC at the time of liver transplantation. Fixed E. coli is used as positive control, and PBS is used as negative control. CE: Experiments in were performed with triplicates. D and E: Representative results from one of three independent experiments with three healthy donors are shown. C: Representative results from one of two experiments with two healthy donors for bile 2 and one donor for bile 4 are shown. CE: Statistical significance was evaluated by t-test (D and E) and one-way analysis of variance, followed by correction for multiple testing using the Bonferroni method within each bile sample in serial dilutions, including the PBS control (C). CE: P indicates the results from significance testing between individual bile samples compared with PBS. All data are presented as means ± SEM (CE). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001. FSC, forward scatter; SSC, side scatter.
      Expression levels of CD69 and GrB on the Vα7.2 T cells (ie, non-MAIT cells) were considerably lower than those observed for MAIT cells, indicating that the activation of MAIT cells was specific and not due to a general immune cell activation (Figure 1E). Patients with PSC with MAIT cell activating bile demonstrated a higher Model for End-Stage Liver Disease Sodium score than patients with PSC without MAIT cell activating bile (P = 0.016) and a trend towards higher scores for three other clinicopathologic indexes: Aspartate Aminotransaminase to Platelet Ratio Index test (P = 0.072), Child Pugh Score (P = 0.056), and Fibrosis-4 Index (P = 0.23) (Supplemental Figure S2).

      Professional Antigen-Presenting Cells and Cholangiocytes Take Up Antigens and Activate MAIT Cells

      To investigate whether MAIT antigens in bile could be taken up and presented by APCs and subsequently activate MAIT cells, a co-culture assay with THP1 cells, a cell type that has previously been shown to take up and present MR1 antigens, was used.
      • Ussher J.E.
      • Bilton M.
      • Attwod E.
      • Shadwell J.
      • Richardson R.
      • de Lara C.
      • Mettke E.
      • Kurioka A.
      • Hansen T.H.
      • Klenerman P.
      • Willberg C.B.
      CD161++CD8+ T cells, including the MAIT cell subset, are specifically activated by IL-12+IL-18 in a TCR-independent manner.
      ,
      • Ussher J.E.
      • van Wilgenburg B.
      • Hannaway R.F.
      • Ruustal K.
      • Phalora P.
      • Kurioka A.
      • Hansen T.H.
      • Willberg C.B.
      • Phillips R.E.
      • Klenerman P.
      TLR signaling in human antigen-presenting cells regulates MR1-dependent activation of MAIT cells.
      Eight bile samples were used, which were able to activate MAIT cells in the aforementioned experiments. Of these, five bile samples led to activation of MAIT cells after incubation with APCs followed by addition of MAIT cells within PBMCs (Figure 2A). Next, whether human cholangiocytes could take up these potential antigens from bile and activate MAIT cells was examined. Studies first confirmed that the cholangiocyte cell line H69 expressed MR1 by immunofluorescence staining (Supplemental Figure S3) and that the cells were capable of presenting 5-OP-RU, leading to activation of MAIT cells in a dose-dependent manner (Supplemental Figure S4). Five bile samples that activated MAIT cells after preincubation with the THP1 cell line were used, and four of these samples led to activation of MAIT cells in the assay using H69 cells as APCs (Figure 2B).
      Figure thumbnail gr2
      Figure 2Antigen-presenting cells take up biliary antigens and activate mucosal-associated invariant T (MAIT) cells. A: Percentages of CD69+ granzyme B+ (GrB+) MAIT cells within peripheral blood mononuclear cells after co-culture with THP1 as antigen-presenting cells that were preloaded with biliary antigen from the eight bile samples activating MAIT cells. B: Bar plots showing the percentages of CD69+ GrB+ MAIT cells in co-culture with a human cholangiocyte cell line H69 preloaded with the five bile samples that the THP1 cells in A could present to and activate MAIT cells. All experiments were performed in triplicates. A: Representative results from one of three independent experiments using three healthy donors are shown. B: Representative results from one of two independent experiments with the same healthy donor are shown. Statistical significance was evaluated by t-test. P indicates the results from significance testing between individual bile samples compared with phosphate-buffered saline (PBS). All data are presented as means ± SEM (A and B). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

      MAIT Cell Activation by Antigens in Bile Is Partially MR1-Dependent

      To investigate whether the observed MAIT cell activation was dependent on MR1 antigen–TCR interaction, TCR-mediated activation was blocked with a monoclonal anti-MR1 antibody (clone 26.5) which has previously been shown to interfere with MR1-TCR interaction.
      • Huang S.
      • Gilfillan S.
      • Cella M.
      • Miley M.J.
      • Lantz O.
      • Lybarger L.
      • Fremont D.H.
      • Hansen T.H.
      Evidence for MR1 antigen presentation to mucosal-associated invariant T cells.
      In experiments with direct incubation of bile and PBMCs, the expression of the activation markers CD69 and GrB was reduced in three of eight bile samples after blocking of the TCR with the anti-MR1 antibody (Figure 3, A and D ). The MR1 restriction was not restricted to a single donor because similar blocking was seen in an experiment using one of the activating bile samples and 10 different healthy donors (Figure 3B). The MR1-independent MAIT cell activation observed for the five remaining bile samples could be explained by cytokine stimulation with multiple possible sources due to the mix of mononuclear cells in the PBMCs used in the experiments.
      • Ussher J.E.
      • Bilton M.
      • Attwod E.
      • Shadwell J.
      • Richardson R.
      • de Lara C.
      • Mettke E.
      • Kurioka A.
      • Hansen T.H.
      • Klenerman P.
      • Willberg C.B.
      CD161++CD8+ T cells, including the MAIT cell subset, are specifically activated by IL-12+IL-18 in a TCR-independent manner.
      ,
      • Sakala I.G.
      • Kjer-Nielsen L.
      • Eickhoff C.S.
      • Wang X.
      • Blazevic A.
      • Liu L.
      • Fairlie D.P.
      • Rossjohn J.
      • McCluskey J.
      • Fremont D.H.
      • Hansen T.H.
      • Hoft D.F.
      Functional heterogeneity and antimycobacterial effects of mouse mucosal-associated invariant T cells specific for riboflavin metabolites.
      • van Wilgenburg B.
      • Scherwitzl I.
      • Hutchinson E.C.
      • Leng T.
      • Kurioka A.
      • Kulicke C.
      • de Lara C.
      • Cole S.
      • Vasanawathana S.
      • Limpitikul W.
      • Malasit P.
      • Young D.
      • Denney L.
      • Moore M.D.
      • Fabris P.
      • Giordani M.T.
      • Oo Y.H.
      • Laidlaw S.M.
      • Dustin L.B.
      • Ho L.-P.
      • Thompson F.M.
      • Ramamurthy N.
      • Mongkolsapaya J.
      • Willberg C.B.
      • Screaton G.R.
      • Klenerman P.
      STOP-HCV Consortium
      MAIT cells are activated during human viral infections.
      • Sattler A.
      • Dang-Heine C.
      • Reinke P.
      • Babel N.
      IL-15 dependent induction of IL-18 secretion as a feedback mechanism controlling human MAIT-cell effector functions.
      In the cell-based assay, where APCs were preincubated with bile followed by washings before adding the PBMCs, reduction of CD69 and GrB expression was seen for all five bile samples that activated the MAIT cells (Figure 3, C and E), implying involvement of the MR1-TCR pathway for all five bile samples.
      Figure thumbnail gr3
      Figure 3Mucosal-associated invariant T (MAIT) cell activation is partially major histocompatibility complex class I–related protein (MR1) restricted. A: Representative flow plots showing CD69+ granzyme B+ (GrB+) activated MAIT cells within peripheral blood mononuclear cells (PBMCs) after incubation with Escherichia coli, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), or bile and an anti-MR1 antibody or an isotype control. B: Graph showing percentage of CD69+ GrB+ activated MAIT cells incubated with an anti-MR1 antibody or an isotype control incubated together with PBMCs from 10 different healthy donors and E. coli or bile sample 4. C: Representative flow plots showing CD69+ GrB+ activated MAIT cells after incubation with THP1 cells as antigen-presenting cells that had been preincubated with bile, fixed E. coli, or 5-OP-RU and an anti-MR1 antibody or an isotype control. D: Bar plots showing the percentage of CD69+ GrB+ activated MAIT cells within PBMCs incubated with an anti-MR1 antibody or an isotype control. E: Bar plots showing the percentage of CD69+ GrB+ activated MAIT cells within PBMCs after incubation with THP1 cells preincubated with bile, fixed E. coli, or 5-OP-RU and the anti-MR1 antibody or an isotype control. All experiments were performed in triplicates, except in B. AE: Representative results from two independent experiment (A, B, and D) and three independent experiments (C and E) are shown. Statistical significance was evaluated by t-test. P indicates the results from significance testing between the individual bile samples, fixed E. coli, or 5-OP-RU with the isotype control compared with the corresponding samples with the anti-MR1 antibody. All data are presented as means ± SEM (D and E). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001. PBS, phosphate-buffered saline.

      Bile from Patients with Non-PSC Chronic Liver Diseases Activates MAIT Cells

      After having demonstrated that bile from patients with PSC contained antigens capable of activating MAIT cells, the study explored whether the presence of MAIT antigens in bile was specific to patients with PSC. Bile from seven patients was included with other chronic liver diseases, alcohol-related liver disease (n = 4), hemochromatosis (n = 1), and autoimmune hepatitis (n = 2) (Table 2), and screened for MAIT cell activating antigens in a plate assay with PBMCs. Two of the seven bile samples activated MAIT cells, as measured by increased CD69 and GrB expression: one from a patient with alcohol-related liver disease and one from a patient with autoimmune hepatitis (Figure 4A). However, the activation was more potent in the group of bile samples from patients with PSC compared with patients not with PSC. None of the potential MAIT cell activating antigens in the two MAIT cell activating bile samples could be presented by antigen-presenting cells (THP1) (data not shown), and the activation observed in the assay with PBMCs was not MR1-dependent (Figure 4B).
      Table 2Clinical Characteristics of Bile Sample Donors with Other Chronic Liver Diseases than Primary Sclerosing Cholangitis
      Variables at time of transplantation
      BileSample no.SexDiagnosisMELD-Na scoreLiver cirrhosisABComorbiditiesERCBilirubin,mg/dLALT,U/LALP,U/LCRP,mg/L
      29MHCM27YesNoNo20.34013017
      30FAIH18YesNoNo3.15610613
      31FAIH, HCC8YesYesSplenectomyNo1.0239512
      32MALD12YesNoDM, pancreatitis, endocarditisNo1.3482558.9
      33MALD17YesNoDM, MI, AF, renal insufficiencyNo0.92520537
      34MALD15YesNoNo1.9138635
      35MALD17YesYesMINo1.82211136
      The presence of liver cirrhosis in the explanted liver was evaluated by a liver pathologist. Biochemistry values represent the values just before liver transplantation, and whether ERC had been performed the last 6 months before liver transplantation was registered.
      F, female; M, male; AB, antibiotics; AF, atrial fibrillation; AIH, autoimmune hepatitis; ALD, alcohol-related liver disease; ALP, alkaline phosphatase; ALT, alanine aminotransferase; CRP, C-reactive protein; DM, diabetes mellitus; ERC, endoscopic retrograde cholangiography; HCC, hepatocellular carcinoma; HCM, hemochromatosis; MELD-Na, Model for End-Stage Liver Disease Sodium; MI, myocardial infarction.
      Figure thumbnail gr4
      Figure 4Mucosal-associated invariant T (MAIT) cells are activated by antigens in bile from patients with other chronic liver diseases than primary sclerosing cholangitis (PSC). Bile from seven patients with other chronic liver diseases than PSC was used to evaluate if activation of MAIT cells by biliary antigens was specific to bile from patients with PSC. A: Bar plots showing the percentage of CD69+ granzyme B+ (GrB+) MAIT cells after incubation with the seven bile samples (diluted 1:200) that were collected from the gallbladder of the patients, at the time of liver transplantation. Fixed Escherichia coli is used as positive control, and phosphate-buffered saline (PBS) is used as negative control. B: Bar plots showing the percentage of CD69+ GrB+ activated MAIT cells after incubation with an anti–major histocompatibility complex class I–related protein (MR1) antibody (26.5) or an isotype control within peripheral blood mononuclear cells. All experiments were performed in triplicates. A and B: Representative results from one of three independent experiments with three different healthy donors are shown. Statistical significance was evaluated by t-test. P indicates the results from significance testing between individual bile samples compared with PBS. All data are presented as means ± SEM (A and B). A and B: n = 4 patients with alcohol-related liver disease; n = 1 patient with hemochromatosis; n = 2 patients with autoimmune hepatitis. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. 5-OP-RU, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil.

      Microbiome Profiling Reveals Bacteria with the Ability to Synthesize MAIT Antigens

      To examine whether the MAIT cell-activating antigens were of microbial origin, microbial DNA was extracted and 16S rRNA sequencing of the 35 bile samples included in the study was performed. In total, microbial DNA was detected in 15 of 35 (42.9%) bile samples, as demonstrated by the 16S rRNA sequencing (Figure 5A). Of note, all of the 15 bile samples with bacterial colonization were from patients with PSC, and the frequency of colonization of bile (53.6%) was in line with the previously reported percentages among patients with PSC.
      • Liwinski T.
      • Zenouzi R.
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      • Kantowski M.
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      • Schachschal G.
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      Of the eight MAIT cell-activating bile samples, microbial DNA was detected in five, which represented all the bile samples that activated MAIT cells in an MR1-dependent pathway (Figure 5A). To investigate whether the sequenced bacteria in the MAIT cell-activating samples were capable of producing MAIT cell antigens (ie, vitamin B metabolites), abundance of the ribD gene, encoding one of the key enzymes in the vitamin B metabolism, was investigated. By comparing the sequenced bacteria against a published gene database,
      • Markowitz V.M.
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      a higher abundance of the ribD gene expression in the group of MAIT cell activating bile samples compared with the group of bile samples unable to activate the MAIT cells (Figure 5B) was predicted. As expected, among the patients with PSC included in the study, 75% had concomitant IBD,
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      Primary sclerosing cholangitis – a comprehensive review.
      but no correlation was found between IBD status and ribD levels or between IBD and MAIT cell-activating antigens in bile (data not shown). There were no associations between ribD abundance and disease severity, as measured with the clinicopathologic scores: Model for End-Stage Liver Disease Sodium score, Aspartate Aminotransaminase to Platelet Ratio Index test, and Fibrosis-4 Index (Supplemental Figure S5).
      Figure thumbnail gr5
      Figure 5Microbiome profiling reveals bacteria with the ability to synthesize mucosal-associated invariant T (MAIT) cell antigens. A: The number of sequenced reads of microbial DNA at phylum level is shown for each of the 15 bile samples where microbial DNA could be detected by 16S rRNA sequencing. B: Prediction of the ribD gene abundance arranged according to MAIT cell activation in bile samples, obtained with PICRUSt2. B: Statistical significance was evaluated by a two-sided Fisher exact test, and the threshold for presence of ribD encoding bacteria was set at 0.0005 (dashed line). P indicates the results from significance testing between the presence of ribD encoding bacteria in the group of MAIT cell-activating and non-MAIT cell-activating bile samples. ∗P < 0.05.

      Discussion

      The combination of clinical observations and genetic studies has established PSC as a disease with features of autoimmunity, and increasing amounts of evidence point toward a role for the microbiota in the pathophysiology in PSC. However, mechanistic understanding of the interaction between the microbiota and immune system is largely lacking. This study proposes the interaction of MAIT cells with bacterial metabolites in bile as a novel pathophysiological pathway linking PSC development with the biliary microbiota.
      The portal vein draining the intestine supplies the liver with antigen-rich blood and microbial products. The bile ducts represent another possible entry route for microbial products to the liver as they are in direct connection with the intestine and hence its bacterial flora. The portal tracts thus represent a seat of interaction between the microbiome and the immune system as this is where the bile ducts colocalize with the portal veins that drain into the sinusoids that are rich in different immune subsets. MAIT cells have preference to localize around the bile ducts in the portal tracts
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      ,
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      MAIT cells are chronically activated in patients with autoimmune liver disease and promote profibrogenic hepatic stellate cell activation.
      and are likely to interact with the microbiome at this location. In line with this, a recent publication investigating the immune cell populations in brush samples from the bile ducts detected a mucosal MAIT cell population in both PSC patients and non-PSC patients.
      • Zimmer C.L.
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      A biliary immune landscape map of primary sclerosing cholangitis reveals a dominant network of neutrophils and tissue-resident T cells.
      In the present study, MAIT cells were activated by 8 of 28 bile samples from patients with PSC, suggesting a role in regulating the immune response against bile-derived pathogens. This function was not exclusive to patients with PSC as bile from patients with other end-stage liver diseases also could activate MAIT cells. The overall activation induced by non-PSC bile samples was less potent and, more importantly, the activation was not dependent on MR1-restricted antigens. These findings are in line with previous studies indicating that bile from patients with PSC is colonized with bacteria and therefore more likely contains MAIT cell antigens.
      • Liwinski T.
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      • Lohse A.W.
      • Heeren J.
      • Franke A.
      • Schramm C.
      Alterations of the bile microbiome in primary sclerosing cholangitis.
      IBD status was not associated with ribD levels or the presence of MAIT cell antigens.
      The role of bile in the pathophysiology of PSC has previously been studied in the context of the toxic-bile theory, with high levels of bile salts contributing either directly or indirectly in the pathophysiology.
      • Chazouillères O.
      Primary sclerosing cholangitis and bile acids.
      Bile is secreted by hepatocytes with subsequent modification by cholangiocytes and consists of endogenous components such as bile salts, bilirubin, and phospholipids, as well as exogenous drugs, xenobiotics, and environmental toxins.
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      Bile formation and secretion.
      A report indicates that bile can also contain antigens that activate natural killer T cells.
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      • Melum E.
      Lipid antigens in bile from patients with chronic liver diseases activate natural killer T cells.
      The present study expands on this and shows that other major type of unconventional T cells are also activated by antigens in bile. TCR-independent modes of activation of the immune system by bile has been reported (for instance, by hepatocyte-derived IL-7 production).
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      The immunobiology of mucosal-associated invariant T cell (MAIT) function in primary biliary cholangitis: regulation by cholic acid-induced interleukin-7.
      This mechanism could account for the partial MR1-independent activation observed for five of the eight activating bile samples. Taken together, these results establish bile as an immune active compartment of the human liver with broad ramifications.
      MAIT cells possess a highly conserved semi-invariant T-cell receptor. Three different healthy donors largely showing similar results were used, suggesting that the effects observed were ubiquitous and not related to a specific donor. A MAIT cell-specific up-regulation of CD69 and GrB was observed compared with the remaining T-cell population, and blocking with an anti-MR1 antibody significantly reduced the activation. Together, these two observations strongly suggest that the effects observed are MAIT-specific and MR1-restricted. Because remaining activation was also seen after blocking MR1, it is likely that other immune-activating compounds exists in the bile, as previously reported.
      • Valestrand L.
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      Lipid antigens in bile from patients with chronic liver diseases activate natural killer T cells.
      A healthy biliary tract has generally been considered a sterile environment, but recent evidence points toward a healthy bile microbiome.
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      The human gallbladder microbiome is related to the physiological state and the biliary metabolic profile.
      Bacteria has been detected in 40.5% to 46% of bile samples from patients with PSC, including ribD gene containing bacteria, such as Klebsiella species.
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      ,
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      In line with these previous findings, the study detected microbial DNA in 15 of 28 bile samples from PSC patients, including species belonging to the phyla Actinobacteriota, Bacteroidota, Firmicutes, Fusobacteriota, Campilobacterota, and Proteobacteria.
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      Alterations of the bile microbiome in primary sclerosing cholangitis.
      Bacteria described to synthesize riboflavin-derived metabolites and potently activate MAIT cells through TCR-mediated activation
      • Tastan C.
      • Karhan E.
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      overlapped with the detection of Streptococcus, Klebsiella, and Enterobacteriaceae in the bile samples that elicited MAIT cell activation. This was supported by a predicted higher abundance of the ribD gene in the group of MAIT cell activating bile samples, together providing strong evidence for the existence of microbial-derived MAIT cell activating antigens in bile. Some of these bacteria can potentially be altered with antibiotics selectively affecting some of the species known to produce MAIT antigens.
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      Impact of oral vancomycin on gut microbiota, bile acid metabolism, and insulin sensitivity.
      These data provide a mechanistic link between the immune system and the microbiome. The MR1-dependent mechanism found in the group of PSC bile samples suggests that MAIT cells could play an important role in the pathophysiology of PSC and have implications for treatment.

      Acknowledgments

      We thank the following members at the Norwegian PSC Research Center: Anne Pharo for technical assistance; Liv Wenche Thorbjørnsen for biobank management; Merete Tysdal for administrative support; Alexandra Goetz for technical assistance and methodological discussions; and Brian K. Chung and Georg Schneditz for methodological discussions.

      Supplemental Data

      Figure thumbnail figs1
      Supplemental Figure S1Activation of mucosal-associated invariant T (MAIT) cells by biliary antigens. A and B: A total of 28 bile samples were incubated with peripheral blood mononuclear cells (PBMCs) from two different healthy donors, and the bar plots show percentage of CD69+ and granzyme B+ (GrB+) MAIT cells. C: PBMCs from six different healthy donors were incubated with 10 bile samples or fixed Escherichia coli. The graphs show the percentage of CD69+ and granzyme B+ MAIT cells. D: Confirmation of the antibody-based gating strategy for MAIT cells using the 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU) tetramer (1:800). All experiments were performed with triplicates, except in C. Statistical significance was evaluated by t-test. P indicates the results from significance testing between individual bile samples compared with phosphate-buffered saline (PBS). Results are presented as means ± SEM (AC). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.
      Figure thumbnail figs2
      Supplemental Figure S2Difference in clinicopathologic scores between primary sclerosing cholangitis patients with mucosal-associated invariant T (MAIT) cell activating and nonactivating bile samples. Model for End-Stage Liver Disease Sodium (MELD-Na) score distribution (A), Child Pugh Score (B), Aspartate Aminotransaminase to Platelet Ratio Index (APRI) test distribution (C), and Fibrosis-4 (Fib4) Index for liver fibrosis distribution (D). Statistical significance was evaluated by t-test. ∗P < 0.05.
      Figure thumbnail figs3
      Supplemental Figure S3Major histocompatibility complex class I–related protein (MR1) immunofluorescence staining of H69 cells. A: Staining with an anti-MR1 antibody. B: Control staining with an isotype antibody. Representative images of from two independent experiments are shown. Scale bar = 100 μm (A and B).
      Figure thumbnail figs4
      Supplemental Figure S4Serial dilutions of 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU) incubated with the human cholangiocyte cell line H69 and peripheral blood mononuclear cells (PBMCs). The percentage of CD69+ and granzyme B+ (GrB+) mucosal-associated invariant T (MAIT) cells among PBMCs is shown. HD, healthy donor; PBS, phosphate-buffered saline.
      Figure thumbnail figs5
      Supplemental Figure S5Correlation between ribD gene abundance and primary sclerosing cholangitis clinicopathologic staging represented by three different indexes. A: Model for End-Stage Liver Disease Sodium (MELD-Na) score distribution. B: Fibrosis-4 (Fib4) Index for liver fibrosis distribution. C: Aspartate Aminotransaminase to Platelet Ratio Index (APRI). Statistical significance was evaluated by Pearson r test. Black lines indicate linear regression.

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