Histones of Neutrophil Extracellular Traps Directly Disrupt the Permeability and Integrity of the Intestinal Epithelial Barrier

Background Increased neutrophil extracellular trap (NET) formation and abundant NET-associated proteins are frequently found in the inflamed colon of patients with inflammatory bowel disease. Peptidyl arginine deiminase 4 (PAD4) activation is essential for the generation of NET and NET-mediated pathogenesis. However, the role of PAD4-dependent NET formation in murine inflammatory bowel disease models and the molecular mechanisms responsible for the altered gut barrier function are unknown. Methods Wild-type and Pad4 knockout (Pad4(-/-)) mice were administrated 3% dextran sulfate sodium (DSS) in their drinking water. Caco-2 monolayers were used to test the effect of NETs on intestinal barrier function and cytotoxicity. Histones were intrarectally administrated to wild-type mice to determine their effects on intestinal barrier function and cytotoxicity in vivo. Results PAD4 deficiency reduced the severity of DSS-induced colitis with decreased intestinal NET formation and enhanced gut barrier function and integrity in mice. NETs disrupted the barrier function in intestinal epithelial Caco-2 monolayers through their protein, rather than DNA, components. Pretreatment of NETs with histone inhibitors abrogated the effects on epithelial permeability. Consistent with these observations, adding purified histone proteins to Caco-2 monolayers significantly damaged epithelial barrier function, which was associated with the abnormal distribution and integrity of tight junctions as well as with increased cell death. Furthermore, intrarectal administration of histones damaged the intestinal barrier integrity and induced cytotoxicity in the mouse colon epithelium. Conclusions PAD4-mediated NET formation has a detrimental role in acute colitis. NET-associated histones directly inhibit intestinal barrier function, resulting in cytotoxicity in vitro and in vivo. Lay Summary Peptidyl arginine deiminase 4-dependent neutrophil extracellular trap formation is detrimental to intestinal barrier function in acute colitis. Neutrophil extracellular trap-associated histones altered the integrity of tight junction and adherens junction proteins as well as induced intestinal epithelial cell death that resulted in increased gut epithelium permeability.

Automated summary

This study found that in the inflamed colon of patients with inflammatory bowel disease, there is increased formation of neutrophil extracellular traps (NETs) and abundant NET-associated proteins. Using a mouse model of inflammatory bowel disease, it was discovered that PAD4-dependent NET formation has detrimental effects in acute colitis. Furthermore, histones associated with NETs directly affect intestinal barrier function and induce cell death in the intestinal epithelium, leading to increased permeability of the gut epithelium.