Aggregatibacter actinomycetemcomitans – Induced Release of NETs from Human Neutrophils under Anaerobic Conditions

The periopathogen Aggregatibacter actinomycetemcomitans is associated with aggressive periodontitis, a severe disease that causes rapid tooth loss among young adolescents. A. actinomycetemcomitans produce and secrete leukotoxin a pore forming protein which causes death of human polymorphonuclear leukocytes (PMNs) such as neutrophils. PMNs are the most abundant leukocyte and have important defence mechanisms such as formation of neutrophil extracellular traps (NETs) used to trap pathogens and the release of chemokines, such as IL-8. The aim of the study was to investigate the host-pathogen interaction between PMNs and A. actinomycetemcomitans focusing on the role of leukotoxin-induced NET-formation under anaerobic conditions. This study has examined the impact of high-leukotoxic JP2-clones and low-leukotoxic non-JP2 clones of A. actinomycetemcomitans as well as purified leukotoxin. Based on previous studies performed under aerobic conditions, we hypothesized that the highleukotoxic clones would induce greater production of NETs than low-leukotoxic clones. A second hypothesis was that PMNs stimulated with purified leukotoxin would produce more NETs than unstimulated controls. PMNs from healthy donors were infected with A. actinomycetemcomitansstrains or stimulated with leukotoxin isolated from high-leukotoxic JP2-clones collected from patient samples. NETs were quantified using immunofluorescent microscopy and IL-8 release was measured using ELISA. Our results showed a trend of increased NET-formation during A. actinomycetemcomitans infection and an increased NET-formation upon leukotoxin stimulation under both aerobic and anaerobic conditions. Untreated PMNs released low basal levels of IL-8 while A. actinomycetemcomitans infected PMNs released higher levels under aerobic conditions, whereas under anaerobic conditions the results were more uncertain.