outer membrane vesicles increase ICAM-1 neutrophils exhibiting elevated NET formation via ROS-PAD4 signaling.

Background: (), as an anaerobic Gram-negative coccus, produces outer membrane vesicles (OMVs) to promote bacterial colonization and periodontitis progress. Neutrophils are the first immune cells during bacterial stimulation, while neutrophil extracellular traps (NETs) are the early triggers in the progress of periodontitis. However, the interactions between neutrophils and are still unknown. Therefore, this study investigated the effects and underlying mechanisms of OMVs on neutrophils.

Methods: Neutrophil migration, apoptosis, and phagocytosis and reactive oxygen species (ROS) production were assessed following stimulation with OMVs. The differential gene expression in neutrophils was characterized using RNA sequencing. The correlation between intercellular adhesion molecule 1 (ICAM-1) expression and NET formation was verified through flow cytometry and immunofluorescence. The NET formation-associated components, including cell-free DNA, neutrophil elastase, myeloperoxidase, and the PAD4 expression were analyzed. The frequencies of the ICAM-1 neutrophils and NET formation were evaluated in neutrophils pretreated with CYBB or PAD4 inhibitors.

Results: Neutrophils exhibited robust migration, increased apoptosis, and ROS production following exposure to OMVs. No significant differences were observed in neutrophil phagocytosis. RNA sequencing analysis revealed a significant increase in the expression of mRNA. And higher ICAM-1 neutrophils in the OMVs group enhanced the levels of NET formation via activation of ROS-PAD4 signaling pathway.

Conclusions: OMVs were initially found to increase the population of ICAM-1 neutrophils, which subsequently exhibited elevated NET formation via the ROS-PAD4 signaling pathway. This study elucidates a novel pathogenic mechanism of OMVs and highlights the potential of targeting ICAM-1 neutrophils as a therapeutic approach for chronic periodontitis.