Nitric oxide dependent increase in free radical generation mediates release of extracellular traps from human neutrophils

High availability of NO at the inflammatory/infection site is noticed often with oxidative stress and neutrophil extracellular traps (NETs) contents, but role of NO remains unexplored in NETs formation. In the present study incubation of adhered human neutrophils with DETA-NONOate led to NETs release in a time and concentration dependent manner, as assessed by confocal microscopy and by measuring extracellular DNA and NET-bound elastase, which was blocked by N-acetyl cysteine, suggesting role of free radicals. A time and concentration dependent augmentation in free radical formation by NO donors was measured by using DCF-DA, NBT, and by p47 phox migration to the neutrophils membrane. NO mediated formation of NETs and free radicals was significantly attenuated by pretreatment of neutrophils with diphenyleneiodonium, a dual inhibitor of NADPH-oxidase/NO synthase (NOS), 4-aminobenzoic acid hydrazide, a myeloperoxidase inhibitor and 7-nitroindazole, a NOS inhibitor, suggesting enzymatic free radical generation. We thus provide first experimental evidence that NO by augmenting free radical formation in human neutrophils mediates NETs release