Near infrared laser irradiation induces NETosis via oxidative stress and autophagy

NETosis is a novel immune defense strategy in which neutrophil activation results in the formation of extracellular DNA/protein network which is able to kill microbial populations. NETosis can be induced in vitro by lipopolysaccharide (LPS) or phorbol myristate acetate (PMA). Due to the importance of NETosis in different physiological and pathological processes, photobiostimulation effect on this neutrophil activation mechanism has been investigated. Human granulocytes, isolated from venous blood of healthy donors, were stimulated with a diode laser emitting at 980nm with an energy intensity ranging from 0 to 75 joules. After 3h of laser stimulation, granulocytes were fixed and colored with crystal violet in order to assess the NETosis morphology while extracellular DNA produced has been quantified using Sytox Green fluorescent dye. To evaluate ROS production and autophagy role in photobiostimulation-induced NETosis, granulocytes were pre-treated with ROS scavengers (vitamin C, sodium pyruvate, l-NAME, sodium azide), and an autophagy inhibitor (wortmannin). Laser stimulation induced an energy-dependent neutrophil extracellular trap (NET) production in human granulocytes starting from 50-J laser intensity. ROS scavengers and the autophagy inhibitor were able to abrogate both morphological features of NETosis and extracellular DNA production without modifying the basal level of NETosis. Photobiostimulation induced an increase in NET production due to an increase in ROS levels and autophagy activation.