Singlet Oxygen Is Essential for Neutrophil Extracellular Trap Formation,

Abstract 3209

Neutrophil extracellular traps (NETs), which capture microbes in extracellular structures consisting of DNA fibers and antimicrobial granule proteins, are pivotal for innate host defense, but the mechanism of NET formation remains unclear. Previous observation in neutrophils of chronic granulomatous disease (CGD) patients, which defect NADPH oxidase (Nox) and fail to produce reactive oxygen species (ROS), demonstrated that ROS are important for NET formation. However, the active species were not identified. Neutrophils first generate superoxide by Nox activation, and this superoxide is converted to hydrogen peroxide (H₂O₂) by superoxide dismutase. Then, hypochlorous acid is produced from H₂O₂ by myeloperoxidase (MPO), and reacts with H₂O₂ to form singlet oxygen (¹O₂). In the current study, we revealed that ¹O₂, one of the ROS, is essential for NET formation. We first examined the effect of ¹O₂ scavengers, a-phenyl-N-tert-butyl nitrone (PBN), 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone), or uric acid (UA), on Nox-dependent NET formation upon stimulation with phorbol myristate acetate (PMA). We previously showed that PBN and edaravone suppressed ¹O₂ release from activated neutrophils, but did not affect superoxide release (Sommani et al. J Pharmacol Sci 2007, Kawai et al. J Pharmacol Sci 2008). We also showed that PBN is a more specific ¹O₂ scavenger, since it neither affected MPO activity, nor reacted with hypochlorous acid (Kawai et al. J Pharmacol Sci 2008). UA is a well-known ¹O₂ scavenger. Human neutrophils were isolated from peripheral blood by sedimentation through two-step Percoll gradients. The CGD patient was a 24-year-old male with gp91-phox deficiency with a G-to-A point mutation at nucleotide 389 in exon 10. Healthy volunteers and the patient provided written informed consent for participation in an institutional review board-approved protocol at Kyoto University Hospital. NET formation by PMA was visualized by fluorescence microscopy with Sytox green, a cell-impermeable DNA dye, and scanning electron microscopy (SEM). NET formation was observed at 3 h after PMA stimulation (100 ng/ml) in healthy neutrophils. The treatment of edaravone (10 μM), PBN (4 mM), or UA (5 μg/ml) significantly suppressed NET formation, suggesting that ¹O₂ is involved in NET formation. In contrast, CGD neutrophils did not exhibit NET formation, which is consistent with previous reports (Fuchs et al. J Cell Biol 2007. Bianchi et al. Blood 2009). We next explored the direct effect ¹O₂ in NET formation. First, we detected ¹O₂ generation by the most specific method with irradiation of porfimer sodium (Photofrin) in a cell-free system, by direct analysis of near-infrared luminescence at 1270 nm using Raman spectroscopy. The treatment of edaravone (400 μM) almost completely suppressed the ¹O₂ spectrum, as well as azide (400 μM), a well-known ¹O₂ scavenger. Next, we observed the accumulation of Photofrin in neutrophils by fluorescence confocal microscopy after incubation with Photofrin (10 μg/ml) for 1 h. These findings indicated that ¹O₂ was generated in neutrophils by Photofrin with light irradiation. NET formation by Photofrin (10 μg/ml, for 1 h) with irradiation (LED lamp, λ_max=660 nm, for 5 min) was analyzed by fluorescence microscopy and SEM. Interestingly, NET formation was observed in CGD neutrophils, as well as healthy neutrophils. It is noteworthy that CGD neutrophils did not exhibit Nox-dependent NET formation by PMA stimulation. This was suppressed by the treatment of ¹O₂ scavengers, edaravone (10 μM) or PBN (4 mM). These results suggested that ¹O₂ directly induced NET formation in both healthy and CGD neutrophils, independent of Nox activation. Taken together, our findings demonstrated that ¹O₂ is essential for NET formation, which supported a recent report that MPO is required for NET formation (Metzler et al. Blood, 2011), since ¹O₂ is generated downstream of MPO. The modulation of NET formation should be effective in the control of infectious or inflammatory disorders. From the current study, the suppression of ¹O₂ could be an optimal target for regulating NET formation, minimizing impairment of innate host defense.