Article Text
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.
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.
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