Differential activation of neutrophil extracellular traps by specific periodontal bacteria

Periodontitis is a chronic inflammatory disease initiated by periodontal pathogens. Neutrophils play a pivotal role within the periodontal lesion, where they have a cytotoxic arsenal at their disposal, including Reactive oxygen species (ROS) and release of neutrophil extracellular trap (NETs). ROS production is essential for NET release and neutrophils in periodontitis patients are hyperactive and hyper-reactive with regard to ROS release (Matthews 2007), which may lead to disease progression. Here we characterise NET and ROS release in response to specific periodontal bacteria. Peripheral blood neutrophils were incubated with a panel of bacteria, including F. nucleatum and C. gingivalis. Neutrophil total ROS and superoxide release were measured (2hours) by luminol and lucigenin-enhanced chemiluminescence respectively. NET release was assayed by incubating the cells with bacteria for 4hours and fluorometrically quantifying NET DNA with sytox green (Palmer 2012). In all assays phorbol myristate acetate [PMA] was used as a positive control. Relative to unstimulated neutrophils S. Gordinii, F. nucleatum, V. parvula and A. viscosus stimulated significantly greater total ROS release (n=5, p=<0.05). S. Gordinii and V. parvula (n=5, p=<0.05) also stimulated significantly higher levels of superoxide.Bacterial stimulation of NETs (n=5) showed marked heterogeneity, with F. nucleatum and S. Gordinii stimulating higher levels of NETs than other species. Collectively, these data demonstrate variability between periodontal bacteria in their ability to stimulate neutrophil total ROS production, superoxide and NET responses. This variability may contribute to altered NET production in-vivo by specific periodontal bacteria and may contribute to the pathogenesis of periodontitis. Mechanisms may include bacterial avoidance of NET stimulation and thus persistence of infection, or excess NET release with associated autoimmunity.