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 1

Tissue-infiltrating PMNs promote accumulation of DSBs.

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Tissue-infiltrating PMNs promote accumulation of DSBs. (A) Healthy and I...
(A) Healthy and IBD colon tissue sections were stained by immunofluorescence for LA/C (green) and CD11b (red) (upper panels), or LB1 (red) and γH2AX (green) (middle and lower panels). Images in lower panels depict representative normal and IBD cell nuclei. (B) Quantitation of immune cell infiltrate (CD11b-positive cells) and nuclear DNA damage (cells positive for γH2AX foci). A total of more than 400 nuclei were examined (n = 3; *P < 0.05). (C) Relative mRNA expression of the indicated genes was determined by qRT-PCR analysis of freshly obtained tissue biopsies from healthy controls and patients with IBD. Data shown as active IBD relative to nonactive controls using GAPDH as a reference gene (n = 9 patients, *P < 0.05). (D) Relative fluorescence intensity in confocal images (as shown in A) was quantified as an index of LA/C and LB1 expression. For each condition, 12 random fields were analyzed (n = 3; *P < 0.05). (E) Relative mRNA expression analysis of human clinical samples. Data are shown as active/nonactive controls using GAPDH as a reference gene (n = 9 patients, *P < 0.05). (F–K) IECs were cocultured for 24 hours with either freshly isolated PMNs or PMN-MPs (ratio of 2 PMNs, or MPs derived from 4 PMNs, to 1 IEC). (F) Representative immunofluorescence images show γH2AX foci (red) and LB1 (green) following treatment for 24 hours with human PMN/PMN-MP. (G) Representative transmission electron microscopy image depicts isolated human PMN-MPs. (H) Quantitation of IECs positive for nuclear γH2AX foci. Total of more than 800 nuclei were analyzed (n = 5; *P < 0.05). (I) IECs were stained for LB1 (red) and LA/C (green) following 24 hours of PMN-MP treatment. (J) mRNA and (K) protein analysis of IECs following 24 hours of coculture with human PMN or PMN-MPs (n = 5, *P < 0.05). One-way ANOVA was used for statistical analyses (P values). Data are mean ± SD from at least 3 independent experiments.