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Original research
Neutrophils prevent rectal bleeding in ulcerative colitis by peptidyl-arginine deiminase-4-dependent immunothrombosis
  1. Moritz Leppkes1,2,
  2. Aylin Lindemann1,
  3. Stefanie Gößwein1,
  4. Susanne Paulus1,
  5. Dominik Roth1,
  6. Anne Hartung1,
  7. Eva Liebing1,
  8. Sebastian Zundler1,2,
  9. Miguel Gonzalez-Acera1,
  10. Jay V Patankar1,
  11. Fabrizio Mascia1,
  12. Kristina Scheibe1,
  13. Markus Hoffmann3,
  14. Stefan Uderhardt2,3,
  15. Christine Schauer3,
  16. Sebastian Foersch4,
  17. Clemens Neufert1,2,
  18. Michael Vieth5,
  19. Georg Schett2,3,
  20. Raja Atreya1,2,
  21. Anja A Kühl6,
  22. Andre Bleich7,
  23. Christoph Becker1,
  24. Martin Herrmann3,
  25. Markus F Neurath1,2
  1. 1Medical Clinic 1, University Clinic, Friedrich Alexander University, Erlangen, Germany
  2. 2Deutsches Zentrum Immuntherapie, Erlangen, Germany
  3. 3Medical Clinic 3, University Clinic, Friedrich Alexander University, Erlangen, Germany
  4. 4Institute of Pathology, University Medical Center, Mainz, Germany
  5. 5Friedrich Alexander University, Institute of Pathology, Klinikum Bayreuth, Erlangen, Germany
  6. 6Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
  7. 7Institute for Laboratory Animal Science, Hannover Medical School, Hannover, Germany
  1. Correspondence to Dr Moritz Leppkes, Department of Medicine 1, University Clinic, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany; moritz.leppkes{at}uk-erlangen.de

Abstract

Objective Bleeding ulcers and erosions are hallmarks of active ulcerative colitis (UC). However, the mechanisms controlling bleeding and mucosal haemostasis remain elusive.

Design We used high-resolution endoscopy and colon tissue samples of active UC (n = 36) as well as experimental models of physical and chemical mucosal damage in mice deficient for peptidyl-arginine deiminase-4 (PAD4), gnotobiotic mice and controls. We employed endoscopy, histochemistry, live-cell microscopy and flow cytometry to study eroded mucosal surfaces during mucosal haemostasis.

Results Erosions and ulcerations in UC were covered by fresh blood, haematin or fibrin visible by endoscopy. Fibrin layers rather than fresh blood or haematin on erosions were inversely correlated with rectal bleeding in UC. Fibrin layers contained ample amounts of neutrophils coaggregated with neutrophil extracellular traps (NETs) with detectable activity of PAD. Transcriptome analyses showed significantly elevated PAD4 expression in active UC. In experimentally inflicted wounds, we found that neutrophils underwent NET formation in a PAD4-dependent manner hours after formation of primary blood clots, and remodelled clots to immunothrombi containing citrullinated histones, even in the absence of microbiota. PAD4-deficient mice experienced an exacerbated course of dextrane sodium sulfate-induced colitis with markedly increased rectal bleeding (96 % vs 10 %) as compared with controls. PAD4-deficient mice failed to remodel blood clots on mucosal wounds eliciting impaired healing. Thus, NET-associated immunothrombi are protective in acute colitis, while insufficient immunothrombosis is associated with rectal bleeding.

Conclusion Our findings uncover that neutrophils induce secondary immunothrombosis by PAD4-dependent mechanisms. Insufficient immunothrombosis may favour rectal bleeding in UC.

  • IBD
  • mucosal injury
  • leukocytes
  • ulcerative colitis
  • bleeding

Data availability statement

Data are available in a public, open access repository. Data sharing not applicable as no datasets generated and/or analysed for this study. Data are available upon reasonable request. All data relevant to the study are included in the article or uploaded as supplementary information. All data relevant to the study are included in the article or uploaded as supplementary information.RNAseq studies are made available in a public, open access repository (Accession: E-MTAB-10824). Additional data are available upon reasonable request.

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Data availability statement

Data are available in a public, open access repository. Data sharing not applicable as no datasets generated and/or analysed for this study. Data are available upon reasonable request. All data relevant to the study are included in the article or uploaded as supplementary information. All data relevant to the study are included in the article or uploaded as supplementary information.RNAseq studies are made available in a public, open access repository (Accession: E-MTAB-10824). Additional data are available upon reasonable request.

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Footnotes

  • ML and AL are joint first authors.

  • Contributors ML, AL, SG, SP, DR, CS, FM and EL performed experiments. ML drafted the manuscript. SF, SZ, RA, AAK, AB and MV provided valuable samples and advice for this study. ML, MFN and MH designed the study and all authors edited the manuscript. ML serves as the guarantor responsible for the overall content of this manuscript.

  • Funding This work has been supported by the Deutsche Forschungsgemeinschaft (BE3686/2-1, KFO 257 CEDER, FOR 2438, TRR241 (A03, B04, C04, INF), CRC 1181 (B02, C02, C03, C05), SPP1656, SCHA 2040/1-1), the German Bundeswirtschaftsministerium (Grant-No. ZF4010106MD9) and the Interdisciplinary Center for Clinical Research (IZKF) of the University Erlangen-Nuremberg (J63, J68).

  • Competing interests None declared.

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

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

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