Figure 5. The NADPH oxidase is the source of extracellular superoxide mediating NET release in pyocyanin-stimulated human neutrophils.

A) Pyocyanin (50 µM) oxidizes β-NADH (20 µM) and β-NADPH (20 µM) in a cell-free system and produces superoxide anions as detected by the Diogenes assay. DPI (10 µM) and DMSO (solvent of DPI) have no effect (data are mean+/− S.E.M., n = 3). B) DPI inhibits basal, PMA- and pyocyanin-stimulated superoxide production in human neutrophils. PMNs were pretreated with 10 µM DPI, stimulated by 20 µM pyocyanin and superoxide production was measured by the Diogenes assay for 60 min. Concentrations of PMNs were: 10⁶/mL for PMA stimulation and 5×10⁶/mL for pyocyanin. C) Pyocyanin produces low-level intracellular ROS in the absence of the NADPH oxidase. DCFDA-loaded healthy and X-CGD neutrophils (patient 1) were exposed to 20 µM pyocyanin or 100 nM PMA and intracellular production of reactive oxygen species was measured by flow cytometry. Similar results were obtained with patient 2′s cells. D) Low mitochondrial superoxide levels are detected in both, healthy and X-CGD neutrophils stimulated by pyocyanin. Healthy or CGD neutrophils (patient #2) loaded with MitoSox Red were exposed to 30 µM pyocyanin for 30 min and mitochondrial superoxide production was measured by flow cytometry. Data were only obtained from one CGD patient. Antimycin A was used as positive control. E) Dose-dependence of pyocyanin-stimulated superoxide production in normal and X-CGD neutrophils (Lucigenin) (patient 2 and 3). F) Scavenging extracellular ROS (catalase and SOD) eliminates pyocyanin-induced ROS production and NET formation in human neutrophils. Human neutrophils were exposed to 30 µM pyocyanin in the presence or absence of 1500 U/mL catalase and 12.5 µg/mL SOD. Superoxide production was followed with Lucigenin for 60 min; NET formation was followed for 4 hrs. Data represent mean+/− S.E.M. of two independent experiments. G) Similarity of chemical structures of pyocyanin and flavin adenine dinucleotide (FAD) and their redox reactions involved in superoxide generation. DMSO, dimethyl sulfoxide; DPI, diphenylene iodonium; RLU, relative luminescence unit; DCF-DA, 2′-7′-Dichlorodihydrofluorescein diacetate; CGD, chronic granulomatous disease; PMA, phorbol 12-myristate-13-acetate; PMN, polymorphonuclear neutrophil; FAD, flavin adenine dinucleotide; cat, catalase; SOD, superoxide dismutase.