Neutrophil-induced genomic instability impedes resolution of inflammation and wound healing
Veronika Butin-Israeli, … , Stephen B. Hanauer, Ronen Sumagin
Veronika Butin-Israeli, … , Stephen B. Hanauer, Ronen Sumagin
Published January 14, 2019
Citation Information: J Clin Invest. 2019;129(2):712-726. https://doi.org/10.1172/JCI122085.
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Research Article Gastroenterology Inflammation

Neutrophil-induced genomic instability impedes resolution of inflammation and wound healing

Abstract

Neutrophil (PMN) infiltration of the intestinal mucosa is a hallmark of tissue injury associated with inflammatory bowel diseases (IBDs). The pathological effects of PMNs are largely attributed to the release of soluble mediators and reactive oxygen species (ROS). We identified what we believe is a new, ROS-independent mechanism whereby activated tissue-infiltrating PMNs release microparticles armed with proinflammatory microRNAs (miR-23a and miR-155). Using IBD clinical samples, and in vitro and in vivo injury models, we show that PMN-derived miR-23a and miR-155 promote accumulation of double-strand breaks (DSBs) by inducing lamin B1–dependent replication fork collapse and inhibition of homologous recombination (HR) by targeting HR-regulator RAD51. DSB accumulation in injured epithelium led to impaired colonic healing and genomic instability. Targeted inhibition of miR-23a and miR-155 in cultured intestinal epithelial cells and in acutely injured mucosa decreased the detrimental effects of PMNs and enhanced tissue healing responses, suggesting that this approach can be used in therapies aimed at resolution of inflammation, in wound healing, and potentially to prevent neoplasia.

Authors

Veronika Butin-Israeli, Triet M. Bui, Hannah L. Wiesolek, Lorraine Mascarenhas, Joseph J. Lee, Lindsey C. Mehl, Kaitlyn R. Knutson, Stephen A. Adam, Robert D. Goldman, Arthur Beyder, Lisa Wiesmuller, Stephen B. Hanauer, Ronen Sumagin

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Figure 5

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

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Wound-infiltrating PMNs promote inflammation and accumulation of DSBs in...
(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.