Abstract

ABSTRACT Accumulation of neutrophil extracellular traps (NETs) in ulcerative colitis (UC) is associated with impaired intestinal epithelial barrier integrity. However, little is known about how NETs affect intestinal epithelial repair. This study sheds light on the molecular mechanisms through which excess NETs cause intestinal epithelial damage in UC mice. We found that UC mice had elevated levels of circulating cell‐free DNA (cfDNA), mainly from NET byproducts (e.g., NET‐DNA). NET‐DNA in the intestine worsened UC symptoms, while DNase I treatment to eliminate it alleviated these symptoms. RNA‐seq analysis revealed significant changes in IL‐22 mRNA between wild‐type and peptidylarginine deiminase 4 knockout (PAD4−/−) mice. Flow cytometry results indicated that NET‐DNA mainly affected IL‐22 secretion by group 3 innate lymphoid cells (ILC3s), while other forms of DNA had little influence on IL‐22 expression. The IL‐22+ILC3s ratio was restored in both DNase I‐treated and PAD4−/− mice; moreover, levels of mucin, tight junction proteins, and Ki67 were significantly increased. Co‐incubating ILC3s or the mouse lymphocyte cell line MNK3 with NET‐DNA decreased IL‐22 levels. ILC3s expressed the NET‐DNA receptor coiled‐coil domain containing protein 25 (CCDC25); however, NET‐DNA did not affect IL‐22 secretion in shCCDC25‐MNK3 cells. Additionally, inhibiting ILK‐HIF‐1α proteins, downstream of CCDC25, increased IL‐22 production in MNK3 cells. Finally, we established an in vitro culture system using MNK3 and Caco‐2 cells. The supernatant from NET‐DNA‐treated MNK3 cells increased FITC‐dextran permeability and reduced ZO‐1 expression in Caco‐2 cells. Thus, CCDC25 in ILC3s responds to NET‐DNA by reducing IL‐22 levels in UC mice, negatively impacting mucosal healing.