Veillonella parvula Outer Membrane Vesicles Increase ICAM-1 + Neutrophils Exhibiting Elevated NETs Formation via ROS-PAD4 Signaling

Lina Xu^1,2,3Yiting Jiang^1,2,3Xuri Zhao⁴Jiabao Zhuang^1,2,3Huiwen Chen^1,2,3Teng Li^2,3,5Zhiyan He^2,3,5Wei Zhou^2,3,5*Zhongchen Song^1,2,3*

• ¹Department of Periodontology, Shanghai Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
• ²College of Stomatology, School of Medicine, Shanghai Jiao Tong University, Pudong, Shanghai Municipality, China
• ³Shanghai Key Laboratory of Stomatology, Shanghai Jiao Tong University, Shanghai, Shanghai Municipality, China
• ⁴Department of Stomatology, Taizhou Hospital of Zhejiang Province affiliated to Wenzhou Medical University, Zhejiang, China
• ⁵Laboratory of Oral Microbiota and Systemic Diseases, Shanghai Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

Background: Veillonella parvula (V. parvula) as an anaerobic gram-negative coccus produces outer membrane vesicles (OMVs) to promote bacteria colonization and periodontitis progress. During bacteria stimulation, neutrophils are the first immune cells and neutrophil extracellular traps (NETs) are early triggers in the progress of periodontitis. However, the interactions between neutrophils and V. parvula for progress in periodontitis are still unknown. Therefore, this study investigates the effects and underlying mechanisms of V. parvula OMVs on neutrophils.Methods: Neutrophil migration, apoptosis, phagocytosis and reactive oxygen species (ROS) production were assessed following V. parvula OMVs stimulation. Differential gene expression in neutrophils was characterized using RNA sequencing. The correlation between ICAM-1 expression and NETs formation was verified through flow cytometry and immunofluorescence. NETs formation-associated components, including cell-free DNA, neutrophil elastase, and myeloperoxidase, and PAD4 expression were analyzed. The frequencies of ICAM-1+ neutrophils and NETs formation were evaluated in neutrophils pre-treated with CYBB or PAD4 inhibitors.Results: Neutrophils exhibited robust migration, increased apoptosis and ROS production following V. parvula OMVs exposure. No significant differences were observed in neutrophil phagocytosis. RNA sequencing analysis revealed a significant increase in ICAM-1 mRNA expression and in the levels of NETs formation along with higher ICAM-1+ neutrophils in the V. parvula OMVs group. Moreover, we confirmed ROS-PAD4 signaling pathway activation.Conclusions: V. parvula OMVs were firstly found to increase the population of ICAM-1+ neutrophils, which subsequently exhibited elevated NETs formation via the ROS-PAD4 signaling pathway. This study elucidates a novel pathogenic mechanism of V. parvula OMVs and highlights the potential of targeting ICAM-1+ neutrophils as a therapeutic approach for chronic periodontitis.