Figure 5. Wound-infiltrating PMNs promote inflammation and accumulation of DSBs in injured IECs.

(A–D) Superficial wounds were introduced to the colonic mucosa with or without Ab-mediated PMN depletion, as described in Methods. (A) At day 4 after injury, colonic wounds were extracted and either OCT-fixed, sectioned, and analyzed for DSBs (γH2AX, green, left panel), or the nuclei of wound IECs were analyzed for DSBs by COMET assay (right panel). (B) Quantification of DSBs in tissue and isolated IECs, as shown by representative images in A. More than 400 and 1000 nuclei were analyzed for γH2AX and COMET assay, respectively (n = 3, ***P < 0.001). (C) Colonic wound tissue was analyzed by immunoblotting for γH2AX, LB1, and RAD51 protein expression at day 4 after wounding in PMN intact and depleted mice. (D) OCT-fixed colonic wounds were sectioned and immunostained for LB1 (red). (E–I) Epithelial injury was induced by introduction of DSS 3% (wt/vol) to drinking water. (E) Relative expression analysis for GBP (a marker of inflamed IECs), MPO (a tissue PMN marker), and (F) miRNAs in distal colon tissue 4 and 12 days following initiation of DSS. Data were normalized to nontreated control tissue, using GAPDH and U-6 as reference genes for protein and miRNA expression, respectively (n = 3, **P < 0.01). (G) DSB formation in nuclei of epithelial cells isolated from DSS-injured distal colons (days 4 and 12 following DSS initiation) were analyzed by the COMET assay (n = 3, ***P < 0.001). (H) Gene expression and (I) immunoblotting analyses of LB1, RAD51, and DSB marker H2AX (n = 3, *P < 0.05). Two-tailed Student’s t test and 1-way ANOVA were used for statistical analyses (P values). Data are mean ± SD from at least 3 independent experiments.