Singlet oxygen is essential for neutrophil extracellular trap formation

https://doi.org/10.1016/j.bbrc.2011.08.052Get rights and content

Abstract

Neutrophil extracellular traps (NETs) that bind invading microbes are pivotal for innate host defense. There is a growing body of evidence for the significance of NETs in the pathogenesis of infectious and inflammatory diseases, 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), revealed that ROS contributed to the formation of NETs. However, the active species were not identified. In this study, we discovered that singlet oxygen, one of the ROS, mediated Nox-dependent NET formation upon stimulation with phorbol myristate acetate. We also revealed that singlet oxygen itself could induce NET formation by a distinct system generating singlet oxygen with porfimer sodium (Photofrin) in CGD neutrophils, as well as healthy neutrophils. This was independent of Nox activation. These results show that singlet oxygen is essential for NET formation, and provide novel insights into the pathogenesis of infectious and inflammatory diseases.

Highlights

► Neutrophil extracellular traps (NETs) are pivotal for innate host defense. ► ROS contribute to NET formation but the active species are not identified. ► Singlet oxygen (1O2) scavengers inhibited Nox-dependent NET formation. ► 1O2 itself could make NETs independent of Nox activation. ► These results indicate that 1O2 is essential for NET formation.

Introduction

Recent investigations highlighted a novel killing mechanism of neutrophils, called neutrophil extracellular traps (NETs), which capture microbes in extracellular structures consisting of DNA fibers and antimicrobial granule proteins [1], [2]. There have been many reports on the antimicrobial effects and pro-inflammatory roles of NETs [3], but the mechanism of NET formation remains unclear. Previous observation revealed that reactive oxygen species (ROS) generated by activated neutrophils contributed to the formation of NETs [4]. However, the active species have not been identified.

Neutrophils first generate superoxide anion (O2radical dot) by NADPH oxidase (Nox) activation, and this O2radical dot is converted to hydrogen peroxide (H2O2) by superoxide dismutase. Hypochlorous acid (HOCl) is produced from H2O2 by myeloperoxidase (MPO), and reacts with H2O2 to form singlet oxygen (1O2) [5]. The role of 1O2 in microbicidal activity is not fully understood. It was recently reported that the ROS with chemical signature of ozone, which is converted from 1O2 by immunoglobulin or several amino acids, contributes to killing of bacteria [6], [7]. A recent study showed that MPO is required for NET formation [8], indicative of the involvement of 1O2 in NET formation.

We previously showed that edaravone, (3-methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger, and α-phenyl-N-tert-butyl nitrone (PBN), a spin trap agent, suppressed 1O2 release from activated neutrophils, but did not affect O2radical dot release [9], [10]. Furthermore, we demonstrated that PBN neither affects MPO activity, nor reacts with HOCl [10].

In this study, we first examined the effect of edaravone or PBN on Nox-dependent NET formation by phorbol myristate acetate (PMA) stimulation, to elucidate the involvement of 1O2 in NET formation. We next studied whether NETs are formed by a distinct system generating 1O2 with porfimer sodium (Photofrin) in neutrophils of a patient with chronic granulomatous disease (CGD), which is an inherited immunodeficiency with Nox defect, leading to recurrent life-threatening infections. These results should uncover an essential role of 1O2 in NET formation.

Section snippets

Reagents

Hanks’ balanced salt solution (HBSS) was purchased from Invitrogen (Carlsbad, CA); trans-1-(2′-methoxyvinyl)pyrene (MVP) and Sytox green were from Molecular Probes (Eugene, OR); and 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-α]pyrazin-3-one (CLA) was from Tokyo Kasei Kogyo (Tokyo, Japan). PBN was obtained from Radical Research Ltd. (Hino, Tokyo, Japan) and dissolved in phosphate-buffered saline (PBS) to a final concentration 100 mM (pH 7.4). Edaravone was a kind gift from Tanabe–Mitsubishi Pharma

Results and discussion

ROS production of healthy or CGD neutrophils upon stimulation with PMA was detected by chemiluminescence. The production of both 1O2 and O2radical dot of healthy neutrophils was observed. The administration of edaravone (10 μM) or PBN (4 mM), a scavenger of singlet oxygen [9], [10], suppressed 1O2, but not O2radical dot production (Fig. 1A). Neither 1O2 nor O2radical dot release from CGD neutrophils was observed (Fig. 1B).

NET formation by PMA was visualized by fluorescence microscopy and SEM. NET formation was observed at 3 h

Author contributions

Y.N., T.A., and K.Y. designed and performed experiments, analyzed the data, and prepared the paper. S.A. and A.T.-K. supervised the study.

Acknowledgments

The authors thank Tomoko Numata, Yasushi Nakata (HORIBA, Ltd.), Keiko Furuta, and Haruyasu Kohda (Division of Electron Microscopic Study, Center for Anatomical Studies, Graduate School of Medicine, Kyoto University) for excellent technical assistance. This work was supported by the Ministry of Education, Science, Sports, and Culture of Japan.

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