The bactericidal and thrombotic potentials of extracellular TRAP-enclosed neutrophil vesicles released in response to <i>Staphylococcus aureus</i> at high glucose concentration

The ejection of neutrophil extracellular traps (NETs) is one of the characteristic features of neutrophils. Rapid release of NET-containing extracellular vesicles (NET-EVs) to the extracellular space has been reported as a novel NET release mechanism in response to Staphylococcus aureus. An increased number of these NET-EVs are released from neutrophils maintained at high-glucose conditions. Though NET is released to fight infection, unregulated NET release observed in inflammatory diseases causes thrombosis. This study was planned to analyze the antibacterial and thrombotic potentials of NET-EVs. Freshly isolated human neutrophils were treated with different glucose concentrations along with S. aureus culture supernatant to stimulate NET release. Increased release of NET-EVs was observed from 15 mM glucose concentration onward. Neutrophil origin of these NET-EVs was established using anti-CD63 antibody and the presence of NET components were confirmed. Then, NET-EVs were analyzed for antimicrobial and antibiofilm activities, clotting time, thrombin time, platelet aggregation and fibrinolysis. The NET-EVs possessed both antibacterial and antibiofilm activities, which were diminished in NET-EVs released at higher glucose concentrations. Clotting and platelet aggregation in the presence of NET-EVs were considerably less up to glucose concentrations of 15 and 20 mM, respectively, after which both increased drastically. Fibrinolysis was slower at glucose concentrations higher than 15 mM. In conclusion, NET-EVs shared many characteristics of suicidal NETs such as antibacterial, antibiofilm, and thrombotic properties. The formation of NET-EVs seems to be an adaptation of neutrophils to reduce local inflammation and to extend their antimicrobial activity to distant areas.