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Gut microbiota depletion exacerbates cholestatic liver injury via loss of FXR signalling

Nature Metabolism volume 3, pages 1228–1241 (2021)Cite this article

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Abstract

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease of unknown aetiology for which there are no approved therapeutic options. Patients with PSC display changes in gut microbiota and in bile acid (BA) composition; however, the contribution of these alterations to disease pathogenesis remains controversial. Here we identify a role for microbiota-dependent changes in BA synthesis that modulates PSC pathophysiology. In a genetic mouse model of PSC, we show that loss of microbiota-mediated negative feedback control of BA synthesis results in increased hepatic BA concentrations, disruption of bile duct barrier function and, consequently, fatal liver injury. We further show that these changes are dependent on decreased BA signalling to the farnesoid X receptor, which modulates the activity of the rate-limiting enzyme in BA synthesis, CYP7A1. Moreover, patients with advanced stages of PSC show suppressed BA synthesis as measured by serum C4 levels, which is associated with poor disease prognosis. Our preclinical data highlight the microbiota-dependent dynamics of BA metabolism in cholestatic liver disease, which could be important for future therapies targeting BA and gut microbiome interactions, and identify C4 as a potential biomarker to functionally stratify patients with PSC and predict disease outcomes.

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Fig. 1: Depletion of microbiota induces bile infarcts and fatal liver injury in Mdr2−/− mice.
Fig. 2: Depletion of microbial BA metabolism by ABx abrogates ileal FXR activation.
Fig. 3: Increased BA synthesis after ABx treatment and hepatic BA accumulation in Mdr2−/− mice.
Fig. 4: Depletion of gut microbiota in Mdr2−/− mice causes a breach of the cholangiocyte barrier and leakage of bile into the periductular fields.
Fig. 5: Therapeutic reconstitution of FXR activation rescues Mdr2−/− mice from cholangiocyte barrier breach and lethal cholestasis.
Fig. 6: Suppressed BA synthesis in patients with PSC is associated with poor prognosis.

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Data availability

Raw sequence reads of 16S rRNA amplicon sequencing are available via the NCBI BioProject database (BioProjectID PRJNA613068). Source data are provided with this paper.

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Acknowledgements

We thank S. Strauch and B. Jansen (University Hospital RWTH Aachen); B. Begher-Tibbe and Z. Hobloss (Leibniz Research Centre for Working Environment and Human Factors, Technical University Dortmund) for their excellent technical assistance. This study was supported by the German Research Foundation (DFG) (grants CRC1382, TR285/10-2, GRK 2375 to C.T.), the Federal Ministry of Education and Research (ObiHep grant no. 01KU1214A to C.T.), the Liver-LiSyM grant (BMBF) to C.T. (031L0041), A.G. (FKZ 031L0052) and J.G.H. (031L0045), the HDHL-INTIMIC Di-Mi-Liv to C.T., K.M.S. and H.U.M., the BMBF Knowledge Platform on Food, Diet, Intestinal Microbiomics and Human Health to C.T. and K.M.S., the SFB 985 project C3 to C.T., the Interdisciplinary Centre for Clinical Research (START grant no. 691438) within the Faculty of Medicine at RWTH Aachen University, the Helmholtz Association (to T.S.), the Swedish Research Council to H.U.M., and the German National Academic Foundation (to C.E. and K.M.S.). C.V.S. is supported by a Walter-Benjamin Fellowship from the DFG (SCHN 1640/1-1). K.M.S. is supported by the DFG consortium (SCHN 1626/1-1).

Author information

Author notes

  1. These authors contributed equally: Kai Markus Schneider, Lena Susanna Candels.

  2. These authors jointly supervised this work: Ahmed Ghallab, Christian Trautwein.

Authors and Affiliations

  1. Department of Medicine III, University Hospital RWTH Aachen, Aachen, Germany

    Kai Markus Schneider, Lena Susanna Candels, Carolin Victoria Schneider, Antje Mohs, Lijun Liao, Carsten Elfers, Konrad Kilic, Maximilian Hatting, Mick Frissen & Christian Trautwein

  2. Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Kai Markus Schneider

  3. Norwegian PSC Research Center, Section of Gastroenterology and Research Institute of Internal Medicine, Division of Surgery, Inflammatory Diseases and Transplantation, Oslo University Hospital and University of Oslo, Oslo, Norway

    Johannes R. Hov & Tom H. Karlsen

  4. Leibniz Research Centre for Working Environment and Human Factors at the Technical University Dortmund, Dortmund, Germany

    Maiju Myllys, Reham Hassan, Ayham Zaza, Jan G. Hengstler & Ahmed Ghallab

  5. Department of Molecular and Clinical Medicine/Wallenberg Laboratory, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden

    Annika Wahlström, Marcus Henricsson, Antonio Molinaro & Hanns-Ulrich Marschall

  6. Center for Mass Spectrometry (CMS), Faculty of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, Germany

    Sebastian Zühlke

  7. Department of Anesthesiology and Pain Management, Shanghai East Hospital, Tongji University, Shanghai, China

    Lijun Liao

  8. Institute National de Recherche en Informatique et en Automatique (INRIA), Le Chesnay, France

    Dirk Drasdo

  9. Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    A. Sloan Devlin

  10. Helmholtz Centre for Infection Research, Braunschweig, Germany and Hannover Medical School, Hannover, Germany

    Eric J. C. Gálvez & Till Strowig

  11. Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt

    Reham Hassan & Ahmed Ghallab

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Contributions

K.M.S., L.S.C. and A.G. performed most of the experiments, analysed most of the data and drafted the manuscript. A.G. designed and performed intravital imaging and intervention experiments, supervised MALDI imaging experiments. K.M.S. designed and supervised the study. H.U.M. gave critical input on study design, analysed the data and revised the manuscript. M.M. and R.H. performed tissue staining and helped with animal experiments. C.V.S. analysed clinical data. A.W., M. Henricsson and A.M. performed BA, C4 and FG19 analyses. A.M. organized experimental mice and helped with the animal experiments. S.Z. performed MALDI imaging. L.L., C.E., K.K., M. Hatting and M.F. were involved in animal experiments. A.Z. and D.D. performed two-photon imaging analysis; E.J.C.G. performed microbiota analysis. T.S. supervised the microbiota analysis and reviewed the manuscript. T.H.K. provided clinical data and patient samples, gave important intellectual input and reviewed the manuscript. J.H. supervised the study, gave critical input on study design and contributed parts of the manuscript. J.R.H. provided clinical data and patient samples, supervised interpretation and analysis of clinical data, reviewed and discussed the manuscript. A.S.D. provided intellectual input and contributed to experiments for revisions. C.T. designed the study, supervised the experiments and data analyses, drafted the manuscript and provided funds.

Corresponding author

Correspondence to Christian Trautwein.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Metabolism thanks James Tabibian and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary handling editor: Dr Ashley Castellanos-Jankiewicz.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Mdr2−/− mice display the typical features of sclerosing cholangitis.

(a) Sirius red and (b) CK-19 staining on liver tissue sections of WT and Mdr2-/- mice showing development of periportal fibrosis and ductular reaction in the Mdr2-/- mice. (c) Whole slide scans of CK19-immunostained liver tissue showing periportal ductular reaction in the Mdr2-/- mice. The stainings were done in 3-4 mice per experimental group.

Source data

Extended Data Fig. 2 Depletion of microbiota induces bile infarcts and fatal liver injury in Mdr2-/- mice.

(a) Serum ratios primary/secondary BA in Mdr2-/- mice (n = 22) and WT mice (n = 11);(unpaired two-tailed t test; p = 0.0001). (b) Massive loss of body weight and (WT n = 4; WT + ABx n = 4; Mdr2-/- n = 3; Mdr2-/- + ABx n = 5; One way ANOVA and Dunn’s Multiple Comparison Test Mdr2-/- vs. Mdr2-/- + ABx for T1 p = 0.0264; for T3 p = 0.0117; for T4 p = 0.019; for T5 p = 0.0057; for T6 p = 0.0053; for T7 p = 0.015). (c) Formation of bile infarcts in the liver tissue of Mdr2-/- mice following antibiotic treatment (ABx, n = 10) compared to Mdr2-/- mice (n = 5);(One sample t Test, two tailed; p = 0.0328). (d) Treatment with ABx (n = 16) results in a significant increase of the levels of GLDH compared to untreated Mdr2-/- mice (n = 12);(WT n = 10; WT ABx n = 8); One-way ANOVA and Sidak’s multiple Comparison Test; WT vs. Mdr2-/-: 95% CI -1904 to – 36.16; p = 0.0396; Mdr2-/- vs. Mdr2-/- + ABx: 95% CI -1714 to -48.89; p = 0.035) (e) Whole slide scans of H&E stained liver tissue bile infarcts in the Mdr2-/- Abx mice. The staining was done in 3-4 mice per experimental group. All Data are presented as the mean ± standard deviation (SD) and considered significant at p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***), p < 0.0001 (****).

Source data

Extended Data Fig. 3 ABx treatment of Mdr2-/- induces severe periductal inflammation.

(a + b) Immunohistochemistry (IF) staining performed on liver tissue sections using antibodies against CD11b and Ly6G, and the corresponding quantifications showing significantly increased numbers of CD11b + (Mdr2-/- mice + ABx n = 7; Mdr2-/- mice Ctrl. n = 9; WT Ctrl. n = 4; WT + ABx n = 4; one-way ANOVA with Sidak’s multiple comparison test: Mdr2-/- vs. Mdr2-/- + ABx: 95% CI -25.09 to -7.728; p = 0.0002; WT + ABx vs. Mdr2-/- + ABx: 95% CI -37.51 to -15.92; p < 0.0001) and Ly6G+ immune cells in Mdr2-/- mice following ABx treatment compared to untreated Mdr2-/- controls (Mdr2-/- mice + ABx n = 7; Mdr2-/- mice Ctrl. n = 9; WT Ctrl. n = 4; WT + ABx n = 4; one-way ANOVA with Sidak’s multiple comparison test: Mdr2-/- vs. Mdr2-/- + ABx: 95% CI -23.17 to -4.156; p = 0.0038; WT + ABx vs. Mdr2-/- + ABx: 95% CI -30.29 to -6.641; p = 0.0018). (c) Representative images of IHC staining using antibodies against F4/80 and FSP1 on liver tissue sections of WT as well as Mdr2-/- mice with and without ABx treatment; The staining was done in 3-4 mice per experimental group. (d) Gating strategy for neutrophils (defined as CD45 + , CD11b + , Ly6G+) and MoMFs (defined as CD45 + , CD11bhi, F4/80low) in flow cytometry. All Data are presented as the mean ± standard deviation (SD) and considered significant at p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****) respectively (One-way ANOVA, Sidak’s multiple comparison test).

Source data

Extended Data Fig. 4 Microbiota depletion by a broad-spectrum antibiotic cocktail.

(a) Enlarged caecum of Mdr2-/- mouse upon ABx treatment and Caecum length in Mdr2-/- mice with (n = 3) or without ABx treatment(n = 4),(unpaired t test; two tailed; p = 0.0002;). (b) Stool pellets of Mdr2-/- with and without ABx treatment were diluted, plated on LB-medium and cultured under aerobic conditions for CFU quantification. (c) Serum concentration of the FXR-antagonistic primary BA Tauro-β-muricholic acid in WT mice (n = 11) and Mdr2-/- mice (n = 22); (Unpaired t test; two tailed; p = 0.0001). (d) Loss of the secondary bile acids (DCA, LCA) upon microbiota depletion by ABx treatment; (Mdr2-/- mice + ABx n = 11; Mdr2-/- mice Ctrl. n = 17; WT Ctrl. n = 11; WT + ABx n = 8; one-way ANOVA with Dunn’s Multiple Comparison Test; DCA: Mdr2-/- vs. Mdr2-/- + ABx: p = 0.0002; WT vs. WT + ABx: p = 0.0002; LCA: Mdr2-/- vs. Mdr2-/- + ABx: p < 0.0001; WT vs. WT + ABx: p = 0.0003). (e) Western blot quantification of relative FGF15 protein levels in WT and Mdr2-/- mice. One way ANOVA with Sidak’s multiple comparison test; WT vs. Mdr2-/- p < 0.0004; WT vs. WT + ABx: p = 0.0001; All Data are presented as the mean ± standard deviation (SD) and considered significant at p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***), p < 0.0001 (****).

Source data

Extended Data Fig. 5 Whole slide scans of CK-10 and MALDI MSI signal.

Whole slide scans of individual CK-19-immunostained liver tissue and MALDI MSI signal of taurocholic acid as well as the superimposed image in WT and Mdr2-/- mice treated with and without ABx.

Extended Data Fig. 6 Correlation of total BA with liver function tests.

Correlation of Total BA in liver tissue with (a) serum ALT and (b) serum GLDH. Number of values 10; for ALT 95% CI: 0.09906 to 1.167; R squared 0.4830; p = 0.0257; for GLDH: 95% CI: 0.9751 to 3.418; R squared 0.6825; p = 0.0032. (c + d) Intravital imaging of BA transport in livers of Mdr2-/- mice with and without ABx treatment using the green-fluorescent bile acid analogue cholyl-lysyl-fluorescein (CLF); showing cell death and bile leakage in the periductular fields in ABx-treated Mdr2-/- mice. Green: CLF; red: tetramethylrhodamine ethyl ester (TMRE); blue: Hoechst 33258. The stills correspond to Supplementary videos 1A-D; scale bars: 50 µm; The intravital imaging was done in 3-6 mice per experimental group.

Source data

Extended Data Fig. 7 Impaired barrier function of cholangiocytes in Mdr2-/- mice after ABx treatment.

Co-staining of CK-19 and ZO-1 showing impaired barrier function of cholangiocytes in livers of ABx treated Mdr2-/- mice; the staining was done in 3 mice per experimental group.

Extended Data Fig. 8 Treatment with the FXR agonist GW4064 attenuates BA synthesis in ABx-treated Mdr2-/- mice.

(a) Total BA concentrations are reduced in the serum of ABx-treated mice following GW4064 treatment. (WT n = 11; WT ABx n = 6; Mdr2-/- n = 22; Mdr2-/- + ABx n = 15; Mdr2-/- + ABx + GW4064 n = 5); One-way ANOVA with Sidak’s Multiple Comparison Test: Mdr2-/- vs. Mdr2-/- + ABx p = 0.0013; Mdr2-/- + ABx vs. Mdr2-/- + ABx + GW4064; p = 0.0168. (b) Total BA concentrations in feces are not significantly altered by GW4064 treatment (WT n = 11; WT ABx n = 8; Mdr2-/- n = 20; Mdr2-/- + ABx n = 14; Mdr2-/- + ABx + GW4064 n = 5); One-way ANOVA with Sidak’s Multiple Comparison Test: Mdr2-/- + ABx vs. Mdr2-/- + ABx + GW4064; p = 0.0198); as well as concentrations of TβMCA (c) in stool are not significantly altered by GW4064 treatment. (WT n = 11; WT ABx n = 8; Mdr2-/- n = 20; Mdr2-/- + ABx n = 14; Mdr2-/- + ABx + GW4064 n = 5); One-way ANOVA with Sidak’s Multiple Comparison Test: Mdr2-/- vs. Mdr2-/- + ABx p < 0.0001; Mdr2-/- + ABx vs. Mdr2-/- + ABx + GW4064; p = 0.5607; WT vs. WT + ABx p < 0.0001. (d + e) Serum levels of liver enzymes (AST and GLDH) of ABx-treated Mdr2-/- mice with or without additional treatment with GW4064 (WT n = 10; WT + ABx n = 8; Mdr2-/- mice n = 13; Mdr2-/- mice + ABx n = 14; Mdr2-/- mice + ABx + GW4064 n = 5; for AST: one-way ANOVA with Sidak’s Multiple Comparison Test: Mdr2-/- vs. Mdr2-/- + ABx: 95%CI: -1504 to -575.8 p < 0.0001; Mdr2-/- + ABx vs. Mdr2-/- + ABx + GW4064 95%CI: -560.8 to -706.9 p = 0.0001; for GLDH: one-way ANOVA with Sidak’s Multiple Comparison Test: WT vs. WT + ABx 95%CI: -1901 to -39.04; p = 0.038; Mdr2-/- vs. Mdr2-/- + ABx: 95% CI -1712 to -51.47; p = 0.0333; Mdr2-/- + ABx vs. Mdr2-/- + ABx + GW4064: 95% CI 602.7 to 2830; p = 0.0009). All Data are presented as the mean ± standard deviation (SD) and considered significant at p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****) respectively (One-way ANOVA, Sidak’s multiple comparison test).

Source data

Extended Data Fig. 9 Reconstitution of FXR activation rescues Mdr2-/- mice from cholangiocyte death and tight-junction leakiness.

Intravital imaging of ABx-treated Mdr2-/- liver showing rescue of the massive periportal damage in ABx-treated Mdr2-/- mice by GW4064 treatment; the intravital imaging was done in 3-6 mice per experimental group.

Extended Data Fig. 10 Impaired BA conversion correlates with disease progression and dismal prognosis of PSC patients.

(a) Spearman-rank correlation of Conjugated/deconjugated BA with serum AP and gGT levels for C4 levels >6.05 nM (p < 0.0001/p = 0.9013 for 67/46 pairs) and (b) < 6.05 nM (p < 0.015/p = 0.0002 for 67/46 pairs). (c) Significantly higher serum concentrations of total BAs (Ctrl n = 100;PSC C4 > 6.05 n = 73; PSC C4 < 6.05 n = 47); Ctrl. vs. PSC C4 > 6.05 p = 0.0021; Ctrl vs. PSC C4 < 6.05 and PSC C4 > 6.05 PSC C4 < 6.05 p < 0.0001) and (d) primary BAs in PSC patients compared to controls (Ctrl n = 100;PSC C4 > 6.05 n = 73; PSC C4 < 6.05 n = 47); all groups p < 0.0001). (e) Secondary BAs do not differ between PSC patients and controls (Ctrl n = 100;PSC C4 > 6.05 n = 73; PSC C4 < 6.05 n = 47); Ctrl- vs. PSC C4 > 6.05 p < 0.001. (f) Serum FGF19 levels measured by ELISA in PSC patients (n = 120) and controls (n = 48); p = 0.0289. (g) Spearman-rank correlation of FGF19 levels with Mayo risk score. (h) Kaplan-Meier survival curves of PSC patients according to the quartiles of serum ratios of conjugated vs. deconjugated BAs. P = 0.0001, two-sided Log-rank (Mantel-Cox) test. (i) Kaplan-Meier survival curve according to the ratio of conjugated vs deconjugated BAs (Cut-off 62,39 – based on Youden’s index; n = 30 for each group; p < 0.0001). All Data are presented as the mean ± standard deviation (SD) and considered significant at p < 0.01 (**) and p < 0.001 (***), p < 0.0001 (****) respectively (One-way ANOVA, Sidak’s multiple comparison test).

Source data

Supplementary information

Reporting Summary

Supplementary Video 1

Intravital imaging of CLF transport in the livers of WT mice. After tail vein bolus injection, CLF appears in the liver sinusoids within few seconds, followed by rapid uptake by hepatocytes and secretion into the bile canaliculi. After approximately 2 h CLF is cleared from the bile canaliculi. The video corresponds to Fig. 3f,g (WT) of the main manuscript. Green: CLF; red: TMRE; blue: Hoechst 33258.

Supplementary Video 2

Intravital imaging of CLF transport in the livers of ABx-treated WT mice. The video shows fast clearance of CLF from the bile canaliculi of WT mice after depletion of gut microbiota by ABx treatment. The video corresponds to Fig. 3f,g (WT + ABx) of the main manuscript. Green: CLF; red: TMRE; blue: Hoechst 33258.

Supplementary Video 3

Intravital imaging of CLF transport in the livers of Mdr2−/− mice. The video shows delayed clearance of CLF from bile canaliculi of Mdr2−/− mice in comparison to WT mice. The video corresponds to Fig. 3f,g (Mdr2−/−) of the main manuscript. Green: CLF; red: TMRE; blue: Hoechst 33258.

Supplementary Video 4

Intravital imaging of CLF transport in the livers ABx-treated Mdr2−/− mice. The video shows delayed clearance of CLF from bile canaliculi ABx-treated Mdr2−/− mice and leakage into the periductular fields. The video corresponds to Fig. 3f,g (Mdr2−/− + ABx) of the main manuscript. Green: CLF; red: TMRE; blue: Hoechst 33258.

Supplementary Video 5

Depletion of gut microbiota in Mdr2−/− mice leads to bile leakage from interlobular bile ducts into periductular fields. Close-up of the interlobular bile ducts and periductular fields of Mdr2−/− mice. The video shows no detectable leakage of CLF into the periductular fields. The video corresponds to Fig. 4a,b (Mdr2−/−) of the main manuscript. Green: CLF; red: TMRE; blue: Hoechst 33258; pink: PI.

Supplementary Video 6

Depletion of gut microbiota in Mdr2−/− mice leads to bile leakage from interlobular bile ducts into periductular fields. Close-up at the interlobular bile ducts and periductular fields of ABx-treated Mdr2−/− mice. The video shows leakage of CLF into the periductular fields as soon as it is transported to the interlobular bile ducts; CLF leakage coincides with death of some hepatocytes neighboring the periductular fields (circles). The video corresponds to Fig. 4a,c (Mdr2−/− + ABx) of the main manuscript. Green: CLF; red: TMRE; blue: Hoechst 33258; pink: PI.

Supplementary Video 7

Cholangiocyte death and shunting of CLF into the periductular fields after depletion of gut microbiota in Mdr2−/− mice. Green: CLF; red: TMRE; blue: Hoechst 33258. The video corresponds to Fig. 4d of the main manuscript.

Supplementary Video 8

Reconstitution of FXR activation rescues antibiotic-treated Mdr2−/− mice from cholangiocyte damage and bile leakage. Green: CLF; red: TMRE; blue: Hoechst 33258. The video corresponds to Fig. 5c,d (+GW4064) of the main manuscript.

Supplementary Tables 1–10

Source data

Source Data Fig. 1

Statistical source data.

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H&E staining (Fig. 1b); CD45 IHC staining (Fig. 1f).

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Unprocessed blots (Fig. 2j).

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Unprocessed blots (Fig. 3a).

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Unprocessed blots (Fig. 5b).

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Sirius red staining (Extended Data Fig. 1d).

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Immunofluorescence staining for CD11b (Extended Data Fig. 3a) and Ly6G (Extended Data Fig. 3b).

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Schneider, K.M., Candels, L.S., Hov, J.R. et al. Gut microbiota depletion exacerbates cholestatic liver injury via loss of FXR signalling. Nat Metab 3, 1228–1241 (2021). https://doi.org/10.1038/s42255-021-00452-1

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