Abstract
To prevent the spread of pathogens neutrophils as the first line of defense are able to release Neutrophil Extracellular Traps (NETs), a recently discovered form of immune response. Reactive oxygen species (ROS) have been shown to be essential for many different induction routes of NET formation. Therefore, pharmacological inhibition of ROS generation has implications for research and medicine related to NETs. The application of diphenylene iodonium (DPI), an inhibitor of NADPH oxidase activity, is limited due to its toxicity to host cells as well as microbes. Therefore, we investigated the effect of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) a membrane-permeable radical scavenger on NET formation triggered by phorbol esters and Candida albicans. We quantified the amount of NETs with two complementary methods, using a microscopic analysis and an online fluorescence-based assay. In line with removal of ROS, Tempol reduced the amount of NET formation by neutrophils challenged with those stimuli significantly. Since Tempol efficiently blocks NET formation in vitro, it might be promising to test the effect of Tempol in experimental models of disorders in which NETs probably have hazardous effects.
Highlights
Polymorphonuclear neutrophils (PMNs) are circulating leukocytes and serve as a first line of defense against microbial infections, but are at the same time a main contributor to hazardous effects during inflammation (Nathan, 2006)
We showed that Tempol removed neutrophil-produced Reactive Oxygen Species (ROS) effectively in a dose-dependent manner upon Phorbol Myristate Acetate (PMA) and C. albicans stimulation
Tempol reduced the amount of Neutrophil Extracellular Traps (NETs) release upon PMA and C. albicans stimulation
Summary
Polymorphonuclear neutrophils (PMNs) are circulating leukocytes and serve as a first line of defense against microbial infections, but are at the same time a main contributor to hazardous effects during inflammation (Nathan, 2006). Approximately 2 × 1011 neutrophils are produced per day (Borregaard, 2010). These professional phagocytes contain a large reservoir of antimicrobial proteins, which they secrete or release into the phagosome containing engulfed microbes (Amulic et al, 2012). The cells express the phagocyte NADPH oxidase complex which enables them to produce superoxide (Segal et al, 2012). The superoxide anion is converted spontaneously or enzymatically driven to other Reactive Oxygen Species (ROS). The exposure to antimicrobial proteins and ROS concertedly kills the phagocytosed microbe (Nathan, 2006)
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