254 - Reactive oxygen species quantification by electron paramagnetic resonance and neutrophil NETs formation

Abstract

Polymorphonuclear leukocytes are the most abundant cells of the innate immune of human. Neutrophils are the first line defense cells to confront infectious pathogens such as bacteria and fungi by phagocytosis. Therefore, neutrophils may participate in the sterile inflammation. To kill the pathogen, neutrophils release their granule, produce reactive oxygen species (ROS) and Neutrophil Extracelular Traps (NETs) formation. However, NET´s are also harmful to host cell. They are associated with the progression of several diseases. ROS participate in the formation of NETs in the canonical pathway. This study aimed to quantify the production of ROS and the impact in the NETs formation by neutrophils. Neutrophils were activated by PMA. ROS production was inhibited by apocynin for 60 minutes. Electron Paramagnetic Resonance (EPR) enabled evaluation of cell activation through the detection of superoxide anion and NETs were quantified by Sytox green staining. EPR results showed that neutrophils in contact with the activator are able to produce ROS in the first minutes. It has been observed that this rate of production increases overtime. Neutrophils in contact with the NADPH oxidase inhibitor, the complex capable of producing ROS, inhibited its production in the first minutes. Therefore, ROS inhibitor was able of inhibit basal ROS production. NETs formation decreases with the production of ROS. EPR is able to quantify initial ROS production. Interaction of neutrophils with PMA promoted time dependent increase of ROS production, while apocynin inhibited ROS even as basal rate production. Therefore, inhibition of ROS decreased the formation of NETs.