Neutrophil extracellular trap formation and its implications in nonsteroidal anti‐inflammatory drug‐induced small intestinal injury

Abstract Background and Aim Nonsteroidal anti‐inflammatory drugs (NSAIDs) damage the small intestine via neutrophil infiltration driven by the mucosal invasion of enterobacteria. The antimicrobial function of neutrophils is partially dependent on neutrophil extracellular traps (NETs). Excessive NET formation has been associated with several inflammatory diseases. Here, we aimed to investigate the role of NETs in NSAID‐induced small intestinal damage using human samples and an experimental mouse model. Methods Human small intestine specimens were obtained from NSAID users during double‐balloon enteroscopy. Wild‐type, protein arginine deiminase 4 (PAD4) knockout, and antibiotic‐treated mice were administered indomethacin to induce small intestinal injury. The expression of NET‐associated proteins, including PAD4, citrullinated histone H3 (CitH3), cell‐free DNA, and myeloperoxidase (MPO), was evaluated. Results The double‐positive stained area with CitH3 and MPO, which is specific for neutrophil‐derived extracellular traps, was significantly high in the injured small intestinal mucosa of NSAID users. In a mouse model, small intestinal damage developed at 6 h after indomethacin administration, accompanied by increased mRNA levels of interleukin‐1β and keratinocyte chemoattractant and elevated NET‐associated protein levels of PAD4, CitH3, and MPO in small intestine and serum levels of cell‐free DNA. Both genetic deletion and pharmacological inhibition of PAD4 attenuated this damage by reducing the mRNA expression of inflammatory cytokines and NET‐associated proteins. Furthermore, mice pretreated with antibiotics showed resistance to indomethacin‐induced small intestinal damage, with less NET formation. Conclusion These results suggest that NETs aggravate NSAID‐induced small intestinal injury. Therefore, NET inhibition could be a potential treatment for NSAID‐induced small intestinal injury.