Figure 3.

Mucosal damage leads to the formation of red blood clots subject to remodelling to neutrophil-rich fibrin layers characterised by marked PAD-activity. Colonoscopy was performed in mice and mucosal wounds were induced using an endoscopic forceps. (A) Top Left: forceps during endoscopy; top right: forceps with biopsy; bottom right: red blood clot on the mucosal wound directly after wounding; bottom left: whitish fibrin covers the mucosal wound 6 hours after wounding. The red blood clot was remodelled. (B) H&E staining of sections of mucosal ulcers (18 hours after injury) display an amorphous layer rich in granulocytes at the surface of the mucosal ulceration. An overview of a cross-section (left) and a high-power magnification (centre) is presented, as well as an image of healthy mucosa (right). (C) The remodelled layer covering the wound bed is positive for MPO as evidenced by immunofluorescence. MPO staining shows fiber-like constitution, indicative of a partially extracellular localisation, colocalised to DNA. (D) The remodelled layer is also characterised by fibrin deposition, as evidenced by fibrinogen immunofluorescence (top) as compared with control (bottom) (scale bars = 100 µm). (E) H3cit is preferentially detected in the wound surface, also featuring (F) cleaved complement C3d. (G) Flow cytometric analysis of healthy and wound tissue 18 hours after wounding. Both the LPL fractions and the IEL fractions of wounds were analysed. In wound tissues, CD11b⁺Ly6G⁺ neutrophils are the dominant cell type in the IEL fraction, whereas the lamina propria features large populations of both CD11b⁺Ly6G⁺ neutrophils and CD11b⁺Ly6C^hi monocytes. (H) Quantification of the cellular composition of colon wounds and adjacent healthy colon tissue (n = 18 wounds studied, *** p < 0.001, Student’s t-test, ** p < 0.01, Student’s t-test, * p < 0.05, Student’s t-test). (I) Colonic wounds were mounted on glass slides as a whole and subjected to epifluorescence analysis after immunostaining of H3cit (in red) and Hoechst (in green) staining. The wound surface of two different wounds both derived from wild-type mice is presented. Left: a wound with persistent presence of the primary blood clot. A cell-rich layer in proximity to the blood clot characterised by H3cit-positive chromatin threads separates the blood clot from the adjacent mucosa. Centre: a remodelled colonic ulcer surface, covered by H3cit⁺ chromatin. Right: no H3cit is detectable on healthy mucosa. Images are representative of > 10 colonic wounds studied. (J) Western blot analyses of colonic wounds and healthy mucosa collected 18 hours after wounding were performed. (K) Densitometry of Western blots as in (J) shows increased presence of H3cit in the wound bed as compared with healthy control tissues (** p < 0.001, Student’s t-test, 3 independent experiments performed). Loading control: β-Actin. All scale bars = 100 µm. H3cit, citrullinated histone H3; IEL, intraepithelial lymphocyte; LPL, lamina propria leucocyte; MPO, myeloperoxidase; PAD, peptidyl-arginine deiminase.