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 7

Induction of genomic instability by PMN-MP–derived miR-23a and miR-155.

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Induction of genomic instability by PMN-MP–derived miR-23a and miR-155. ...
(A–D) Long-term coculture of IECs and PMN-MPs was performed as described in Methods. (A and B) Immunofluorescence detection of BrdU (replication marker; green) and γH2AX (DSB marker; red). Approximately 600 cells were analyzed for each PD per marker, representative images are shown in (A) and quantification is shown in (B) (n = 3; ***P < 0.001). (C) Induction of apoptosis and aneuploidy during long-term IEC and PMN-MP cocultures was determined. At the defined PD time, cells were harvested, stained for Annexin V and propidium iodide (PI), and analyzed by FACS (n = 3, *P < 0.05; **P < 0.01). (D) The effect of miR-23a and miR-155 inhibition by ASOs (1 nM each, replaced every 48 hours) during long-term IEC and PMN-MP coculture on the indicated processes. Approximately 500 cells per PD per marker were analyzed (n = 3, ***P < 0.001, ##P < 0.01). (E–K) Patient-derived colonoids grown in 3D cultures were treated with PMN-MPs (24 hours, derived from fMLF-stimulated PMNs). (E) Representative phase contrast image of a mature colonoid (~10 days in culture) used in experiments. (F) Relative expression analysis of miRNAs and genes of interest following PMN-MP treatment. Data shown relative to nontreated, control colonoids, using U-6 and GAPDH as reference genes, respectively (n = 3, *P < 0.05). (G) Representative immunoblots and (H) quantification of lysed colonoids following PMN-MP treatment. (I–K) 3D colonoids were serially sectioned and processed for immunofluorescence analyses. (I) Representative images of γH2AX (a DSB marker, green) induction, quantified in panel J (left bars). For DSB analysis (J, middle bars), COMET assay was performed on dissociated colonoid epithelial cell nuclei. (K) LB1 downregulation (red) and formation of micronuclei was determined by staining for LB1 (red) and DNA (blue). Micronuclei are indicated by white arrows and quantified in panel J (right bars). For quantification, at least 20 colonoids were analyzed per condition (n = 3, ***P < 0.001). 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.