Quantitative analysis of hemin-induced neutrophil extracellular trap formation and effects of hydrogen peroxide on this phenomenon

Formation of neutrophil extracellular traps (NETs) can perpetuate sterile inflammation; thus, it is important to clarify their pathophysiological characteristics. Free heme, derived via hemolysis, is a major contributor to organ damage, and reportedly induces neutrophil activation as well as reactive oxygen species (ROS) production and NET formation. For this study, we examined hemin (Fe³⁺ -protoporphyrin IX)-induced NET formation quantitatively in vitro as well as the effects of oxidative stress.NETs formed in vitro from cultured neutrophils were quantitatively detected by using nuclease treatment and Sytox Green, a nucleic acid stain. Hemin-induced NET production was found to be in a dose-dependent manner, NADPH oxidase-dependent and toll-like receptor (TLR)-4 independent. Additionally, the iron molecule in the porphyrin ring was considered essential for the formation of NETs. In the presence of low concentrations of hydrogen peroxide, low concentrations of hemin-induced NETs were enhanced, unlike those of phorbol myristate acetate (PMA)-induced NETs.Quantitative analysis of NET formation may prove to be a useful tool for investigating NET physiology, and hemin could function as a possible therapeutic target for hemolysis-related events.