Singlet Oxygen Is Essential for Neutrophil Extracellular Trap Formation,
Authors: Nishinaka Yoko , Morita Makiko , Arai Toshiyuki , Adachi Souichi , Takaori-Kondo Akifumi , Yamashita Kouhei 
Published: October 2019

Abstract
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, λmax=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.

Disclosures:
No relevant conflicts of interest to declare.