Neutrophil NET Formation with Microbial Stimuli Requires Late Stage NADPH Oxidase Activity

Abstract

Neutrophils respond to a range of stimuli by releasing extracellular traps (NETs), a mesh consisting of chromatin plus granule and cytoplasmic proteins. We have investigated NET release in response to phorbol myristate acetate (PMA), Pseudomonas aeruginosa (PAO1), Staphylococcus aureus and Candida albicans, and the involvement of NADPH oxidase (NOX2) and myeloperoxidase (MPO) activities. An oxidative mechanism was involved with each stimulus, and the NOX2 inhibitor diphenylene iodonium (DPI) gave almost total inhibition. Notably, DPI added up to 60–90 min after stimulation still gave significant inhibition of subsequent NET formation. As most of the NOX2 activity had already occurred by that time, this indicates a requirement for late-stage low-level oxidant production. Inhibition of histone citrullination did not suppress NET formation, indicating that this was not the essential oxidant-dependent step. With PMA and P. aeruginosa PAO1, MPO activity played an important role in the induction of NETs and MPO inhibitors added up to 30–90 min after stimulation suppressed NET formation. NET formation with S. aureus and C. albicans was insensitive to MPO inhibition. Thus, MPO products are important with some stimuli but not others. Our results extend earlier observations with PMA and show that induction of NETs by microbial stimuli requires late stage oxidant production. Others have shown that NET formation involves NOX2-dependent elastase release from granules. As this is an early event, we conclude from our results that there is more than one oxidant-dependent step.

Neutrophils extracellular traps and ferroptosis in diabetic wounds

“…It has been reported that MPO played a synergistic role, but the formation of NETs was independent of MPO activity 32 . However, another study suggested that PMA‐stimulated NETs formation is strongly dependent on MPO, but this dependence is not obvious for NETs induced by S. aureus and C. albicans , which is contrary to the above argument 28 . Kathryn Akong‐Moore et al showed that 4‐amino benzoic acid hydrazide (ABAH), an inhibitor of MPO enzyme activity, significantly inhibited Pseudomonas aeruginosa‐induced NETosis in human peripheral blood.…”

“…form NETs in an NADPH oxidase-dependent manner, while Staphylococcus aureus can not only form NETs in an NADPH oxidase-dependent manner, its derived lipoprotein was found to act on Toll-like receptor 2 (TLR2) on neutrophils to activate PAD4 and induce NETs formation. 27,28 It has been suggested that PMA, an activator of protein kinase C (PKC), activates the (Raf)/MEK (MAPK/ ERK kinase)/extracellular signal-regulated kinase (ERK) kinase cascade. This promotes the production of reactive oxygen species (ROS) by NADPH oxidase, which leads to NETosis.…”

“…32 However, another study suggested that PMA-stimulated NETs formation is strongly dependent on MPO, but this dependence is not obvious for NETs induced by S. aureus and C. albicans, which is contrary to the above argument. 28 Kathryn Akong-Moore et al showed that 4-amino benzoic acid hydrazide (ABAH), an inhibitor of MPO enzyme activity, significantly inhibited Pseudomonas aeruginosa-induced NETosis in human peripheral blood. However, there was little effect on murine bone marrow-derived and murine skin pouch-derived neutrophils by ABAH.…”

High-Throughput Analysis of Neutrophil Extracellular Trap Levels in Subtypes of People with Type 1 Diabetes

“…After outlining a processing definition specific to identify, analyze, and quantify NETosis, we studied NET formation over a 6 h time course in HC donors in response to different doses of two NET-inducing stimuli known to employ distinct mechanisms. While PMA acts through the activation of NADPH and the release of granular proteins such as MPO and NE, ionomycin induces NET formation by the mobilization of calcium [ 10 , 28 ]. While the former was shown to produce a ‘suicidal’ form of NETosis, involving cell membrane rupture, calcium ionophores such as ionomycin mainly induce ‘vital’ NETosis, characterized by the externalization of chromatin and granular proteins through vesicular transport [ 29 ].…”