Mitochondrial ROS and base excision repair steps leading to DNA nick formation drive ultraviolet induced-NETosis

Abstract

Reactive oxygen species (ROS) is essential for neutrophil extracellular trap formation (NETosis), and generated either by NADPH oxidases (e.g., during infections) or mitochondria (e.g., sterile injury) in neutrophils. We recently showed that ultraviolet (UV) radiation, a sterile injury-inducing agent, dose-dependently induced mitochondrial ROS generation, and increasing levels of ROS shifted the neutrophil death from apoptosis to NETosis. Nevertheless, how ROS executes UV-induced NETosis is unknown. In this study, we first confirmed that UV doses used in our experiments generated mitochondrial ROS, and the inhibition of mitochondrial ROS suppressed NETosis (Mitosox, SYTOX, immunocytochemistry, imaging). Next, we showed that UV irradiation extensively oxidized DNA, by confocal imaging of 8-oxyguanine (8-oxoG) in NETs. Immunofluorescence microscopy further showed that a DNA repair protein, proliferating cell nuclear antigen, was widely distributed throughout the DNA, indicating that the DNA repair machinery was active throughout the genome during UV-induced NETosis. Inhibition of specific steps of base excision repair (BER) pathway showed that steps leading up to DNA nick formation, but not the later steps, suppressed UV-induced NETosis. In summary, this study shows that (i) high levels of mitochondrial ROS produced following UV irradiation induces extensive oxidative DNA damage, and (ii) early steps of the BER pathway leading to DNA nicking results in chromatin decondensation and NETosis. Collectively, these findings reveal how ROS induces NOX-independent NETosis, and also a novel biological mechanism for UV irradiation- and -mitochondrial ROS-mediated NETosis.

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“…MitoSOX is a mitochondria-specific dye that fluoresces red upon oxidation ( Kim et al., 2009 ). The fluorescence resulting from the oxidation of MitoSOX was measured using a Molecular Devices SpectraMax i3 plate reader, employing excitation at 510 nm and emission detection at 580 nm, following cell activation ( Azzouz and Palaniyar, 2023 ).…”

A metabolic perspective of the neutrophil life cycle: new avenues in immunometabolism

“…However, the origin and mechanism of the DNA scaffold in in vivo NETs in bacterial infections remains debated. Transcription factor and kinase (p38 MAPK) activity, peptidylarginine deiminase 4 (PAD4) mediated histone citrullination or ROS-mediated DNA oxidation and repair machinery and resulting chromatin decondensation for NET formation may be important in homeostasis and inflammatory disease states (146)(147)(148)(149). In cancer settings, NET formation drives metastases and during sterile conditions, increased NETs are indicative of autoimmune disease states (80,150).…”