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Hepatology
Original research
TGF-β2 silencing to target biliary-derived liver diseases
  1. Anne Dropmann1,
  2. Steven Dooley1,
  3. Bedair Dewidar1,2,
  4. Seddik Hammad1,3,
  5. Tatjana Dediulia1,
  6. Julia Werle1,
  7. Vanessa Hartwig1,
  8. Shahrouz Ghafoory4,
  9. Stefan Woelfl4,
  10. Hanna Korhonen5,
  11. Michel Janicot5,
  12. Katja Wosikowski5,
  13. Timo Itzel6,
  14. Andreas Teufel6,
  15. Detlef Schuppan7,8,9,
  16. Ana Stojanovic10,
  17. Adelheid Cerwenka10,
  18. Stefanie Nittka11,
  19. Albrecht Piiper12,
  20. Timo Gaiser13,
  21. Naiara Beraza14,15,
  22. Malgorzata Milkiewicz16,
  23. Piotr Milkiewicz17,
  24. John G Brain18,
  25. David E J Jones18,
  26. Thomas S Weiss19,
  27. Ulrich M Zanger20,21,
  28. Matthias Ebert22,
  29. Nadja M Meindl-Beinker1
  1. 1 Molecular Hepatology-Alcohol Associated Diseases, Department of Medicine II, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
  2. 2 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tanta University, Tanta, Egypt
  3. 3 Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt
  4. 4 Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany
  5. 5 Isarna Therapeutics GmbH, Munchen, Germany
  6. 6 Hepatology and Clinical Bioinformatics, Department of Medicine II, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany
  7. 7 Research Center for Immunotherapy, University Medical Center, Johannes Gutenberg University, Mainz, Germany
  8. 8 Institute of Translational Immunology, University Medical Center, Johannes Gutenberg University Mainz, Mainz, Germany
  9. 9 Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA
  10. 10 Department of Immunobiochemistry, Centre for Biomedicine and Medical Technology (CBTM) and European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
  11. 11 Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany
  12. 12 Medizinische Klinik 1, Klinikum der Goethe-Universität, Frankfurt am Main, Germany
  13. 13 Institute of Pathology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
  14. 14 Gut Microbes and Health Institute Strategic Programme, Quadram Institute, Norwich, UK
  15. 15 CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Derio, Spain
  16. 16 Department of Medical Biology, Pomeranian Medical University, Szczecin, Poland
  17. 17 Liver and Internal Medicine Unit, Department of General, Transplant and Liver Surgery, Medical University of Warsaw, Warsaw, Poland
  18. 18 NIHR Applied Immunobiology and Transplant Research Group, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK
  19. 19 Department of Pediatrics and Juvenile Medicine, Center for Liver Cell Research, University of Regensburg Hospital, Regensburg, Germany
  20. 20 Department of Molecular and Cell Biology, Dr Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany
  21. 21 Eberhard-Karls-University Tübingen, Tübingen, Germany
  22. 22 Department of Medicine II, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
  1. Correspondence to Steven Dooley, Department of Medicine II, Section Molecular Hepatology, Medical Faculty Mannheim, University of Heidelberg, Mannheim 68167, Germany; steven.dooley{at}medma.uni-heidelberg.de; Nadja M Meindl-Beinker; Nadja.Meindl-Beinker{at}medma.uni-heidelberg.de

Abstract

Objective TGF-β2 (TGF-β, transforming growth factor beta), the less-investigated sibling of TGF-β1, is deregulated in rodent and human liver diseases. Former data from bile duct ligated and MDR2 knockout (KO) mouse models for human cholestatic liver disease suggested an involvement of TGF-β2 in biliary-derived liver diseases.

Design As we also found upregulated TGFB2 in liver tissue of patients with primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC), we now fathomed the positive prospects of targeting TGF-β2 in early stage biliary liver disease using the MDR2-KO mice. Specifically, the influence of TgfB2 silencing on the fibrotic and inflammatory niche was analysed on molecular, cellular and tissue levels.

Results TgfB2-induced expression of fibrotic genes in cholangiocytes and hepatic stellate cellswas detected. TgfB2 expression in MDR2-KO mice was blunted using TgfB2-directed antisense oligonucleotides (AON). Upon AON treatment, reduced collagen deposition, hydroxyproline content and αSMA expression as well as induced PparG expression reflected a significant reduction of fibrogenesis without adverse effects on healthy livers. Expression analyses of fibrotic and inflammatory genes revealed AON-specific regulatory effects on Ccl3, Ccl4, Ccl5, Mki67 and Notch3 expression. Further, AON treatment of MDR2-KO mice increased tissue infiltration by F4/80-positive cells including eosinophils, whereas the number of CD45-positive inflammatory cells decreased. In line, TGFB2 and CD45 expression correlated positively in PSC/PBC patients and localised in similar areas of the diseased liver tissue.

Conclusions Taken together, our data suggest a new mechanistic explanation for amelioration of fibrogenesis by TGF-β2 silencing and provide a direct rationale for TGF-β2-directed drug development.

  • cholestasis
  • TGF-beta
  • fibrosis
  • primary biliary cirrhosis
  • primary sclerosing cholangitis
http://creativecommons.org/licenses/by-nc/4.0/

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.

https://doi.org/10.1136/gutjnl-2019-319091

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    Supplementary materials

    Footnotes

    • AD and SD are joint first authors.

    • Correction notice This article has been corrected since it published Online First. Figures 5-8 have been replaced for clarity.

    • Contributors AD performed mouse model experiments, immunofluorescence staining, statistical analysis and figure preparation. AD, BD, TD, JW and VH performed the experiments including stainings, immunohistochemistry, qPCR and Western Blot. SN supported determination of plasma parameters. UMZ supported the fluidigm-platform based analyses. AP, TG and TSW provided PSC and PBC patient samples for further analyses by AD. JB and DEJJ contributed clinical data to GSE79850. NB, MM and PM shared the Polish patient cohort including analysis of TGFB2 expression. SH provided the data on non-biliary derived animal models and performed analysis of ductular reaction markers with AD. TD supported statistical analyses; AT and TI supported graphical presentation of fluidigm data. SG and SW performed the in situ hybridisation. AS and AC performed, analysed and interpreted the fluidigm analysis. DS provided the mouse cholangiocyte cell line and supported the interpretation of the data. KW, HK and MJ supported the study with their knowledge about the PK/PD characteristics and dosing regimens of the AON in mice and contributed to the discussion about the data. AD, NMB and SD designed the experiments; NMB and SD organised sample collection; AD, NMB and SD interpreted the data; AD, SD and NMB wrote this manuscript. ME provided infrastructure. All authors contributed with productive discussions and knowledge to the final version of this manuscript.

    • Funding This work was supported by the BMBF [Liver Systems Medicine, LiSyM, grant number no. PTJ-031L0043; to SD], Isarna Therapeutics GmbH (to SD and AD), SFB1366 [Project number 394046768-SFB 1366; C02; to AC] and by SPP 1937 [CE 140/2-1 to AC]. NMB was supported by the ESF Baden Württemberg (www.esf-bw.de) and the Ministerium für Wissenschaft, Forschung und Kunst, Baden Württemberg [Margarete von Wrangell Programme], TD was funded by the EU [IT-Liver consortium, Marie Curie Training Network], UMZ by the Robert Bosch Foundation, Stuttgart and AD by Isarna Therapeutics; sponsors did not play a role in study design, data collection, analysis and interpretation of the data.

    • Competing interests Isarna Therapeutics GmbH supported this study financially. The company develops TGF-β isoform specific antisense oligonucleotides for therapeutic approaches. However, the company did not influence experimental design and data interpretation. KW, HK and MJ were employed by Isarna Therapeutics at the time of contribution. The position of AD was funded by Isarna Therapeutics.

    • Patient consent for publication Informed consent was obtained from all patients.

    • Ethics approval Government’s Animal Care Committee: 35-9185.81/G-138/13, Regierungspräsidium Karlsruhe. Tissue procurement was approved by the local Medical Ethics Committees (15-101-0318 Ethikkomission der Universität Regensburg; 2007-011N-MA and 2012-293N-MA Ethikkomission II der Universität Heidelberg, KB/58/A/2016 Bioethical Committee of Medical University of Warsaw).

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Data availability statement Data are available in public, open access repositories, directly included in the article, uploaded as supplementary iformation or available upon reasonable request from first or last authors. Data sets were obtained from the following public, open access repositories: https://www.ebi.ac.uk/arrayexpress/, www.ncbi.nlm.nih.gov, GEO datasets.