Abstract
Neutrophils are the first line of defense of the human immune system against pathogens. Photobiomodulation, mediated by mitochondrial photoacceptors such as cytochrome c oxidase, has emerged as a method to modulate neutrophil function through targeted light exposure. Despite the extensive characterization of neutrophil extracellular traps (NETs) formation (NETosis), the wavelength-specific modulation of neutrophil photoactivation and the involvement of redox pathways remain poorly defined. In this study, the effects of monochromatic (365 nm, 415 nm, 437 nm, and 625 nm) and dichromatic LED-light irradiation on NETs formation were systematically examined. The highest netotic responses were elicited by UV-A (365 nm) and violet-blue light (415 nm), whereas 437 nm showed the lowest induction and 625 nm stimulated a moderate netotic response. The pharmacological inhibition of NETosis induced by 365 nm and 415 nm irradiation with specific NADPH oxidase inhibitor, apocynin, and mitochondrial reactive oxygen species (mtROS) scavenger, MitoTEMPO, attenuated NETs formation by engaging both enzymatic and mitochondrial oxidative sources. Notably, mtROS played a dominant role under 415 nm stimulation in contrast to 365 nm-induced NETosis as demonstrated by higher sensitivity to MitoTEMPO. Importantly, combined simultaneous irradiation with 415 nm and 625 nm LEDs resulted in a significant suppression of NETs formation by more than 50%, highlighting a potent inhibitory synergy observed for the first time and suggesting a new approach of wavelength pairing to modulate neutrophil activation. These results were further supported by measurements of ROS production using a luminol-amplified chemiluminescence assay. Collectively, these findings delineate a wavelength- and ROS-dependent framework for light-induced neutrophil activation, with mitochondrial pathways exerting central control particularly under short-wavelength irradiation.