Figure 7.

PAD4-mediated immunothrombosis controls mucosal haemostasis in acute DSS-induced colitis. Acute colitis was induced in mice by 4 % DSS in the drinking water for 7 days. (A) H&E staining and (B) immunofluorescence of MPO of murine colonic wounds at day 9 shows a massive destruction of the epithelial cell layer with abundant neutrophils in the colon lumen (scale bars = 100 µm). (C) Colon tissue lysates from day 9 of acute DSS-induced colitis and untreated controls were analysed by an immuno-dot blot technique. The amount of citrullinated histone H3 and hence PAD activity is markedly increased under inflammatory conditions. β−actin immunoblot serves as a loading control (representative of 3 independent experiments with a total of n = 12 samples). (D) Semiquantitative image density analyses of immuno-dot blots as in (C) reveal a significantly higher H3cit expression in DSS-colitis tissue compared with healthy tissue (* p < 0.05, Student’s t test). (E) Immunofluorescence of MPO (top), EpCAM and H3cit (bottom) performed on cryosections of colonic tissue samples derived from day 9 of acute DSS-induced colitis (4 %) shows the increased presence of MPO in the inflamed mucosa with H3cit restricted to the eroded area with a breached epithelial lining (as seen by lack of EpCAM staining (top part of the lower micrograph)) (representative of n > 10 samples, scale bar = 100 µm). (F) Weight (in %) of mice subjected to 4 % DSS in the drinking water was measured during the course of the experiments comparing PAD4-deficient (PAD4^−/−) mice and PAD4-proficient littermates (WT). Please appreciate the accelerated weight loss in PAD4^−/− mice. (G) Lethality of mice, interpreted by the number of mice suffering a weight loss of > 20 %, was strongly increased in the PAD4^−/− group (*** p < 0.001, Fisher’s two-tailed exact test). (H) At the time of sacrifice, colon length of mice was measured. A reduced length is typical of increased inflammation. A reduced colon length was observed in PAD4^−/− mice as compared with WT littermates after DSS treatment. (I) Rectal bleeding is a typical hallmark of severe acute DSS-induced colitis, detectable on both clinical assessment (left) and on analyses by endoscopy of affected mice (right). This was evident more often in PAD4^−/− mice. (J) The number of mice suffering from persistent rectal bleeding was strongly increased in PAD4^−/− mice subjected to DSS as compared with wild-type littermates (*** p < 0.001, Fisher’s two tailed exact test, n: number of mice suffering from rectal bleeding, N: total number of mice studied). Complete blood count was performed on day 10 of DSS-induced colitis. (K) Haematocrit and (L) haemoglobin were measured in the blood of WT and PAD4^−/− mice subjected to drinking water containing DSS or not (H₂O). when subjected to DSS, PAD4^−/− mice exhibited significantly more blood loss than WT mice, whereas untreated mice showed no differences (** p < 0.01, Student’s t test). DSS, dextrane sodium sulfate; MPO, myeloperoxidase; PAD, peptidyl-arginine deiminase; WT, wild-type.