Neutrophil Extracellular Traps Are Generated By Reactive Oxygen Species Through Toll-Like Receptor 4

Abstract

Introduction: Neutrophil extracellular traps (NETs) are complexes of DNA, granular enzymes and histone proteins released by activated neutrophils during sepsis. NETs function to bind, disarm and destroy infectious microbes extracellulary following stimulation by lipopolysaccharide. However, the role of NET formation in non-infectious stimuli, such as oxidative stress, is unknown. Therefore, we sought to determine if neutrophils activated by reactive oxygen species (ROS) release NETs. Methods: Neutrophils were isolated from femurs and tibias of rats and HeJ (TLR4 Mutant) and HeOUJ (WT) mice using a percol gradient. Cells were treated at varying time points with xanthine oxidase and its substrate hypoxanthine (XO/HX), hydrogen peroxide, phorbol-myristate-acetate (PMA, positive control) and cell culture media (negative control). Slides were fixed and stained for DNA and histones. NETs release was verified by immunofluoresence and quantified by calculating the percentage of extranuclear histones with high field microscopy analysis. Results: NETs stained positively for DNA and histone proteins, and appeared as web-like structures extending extracellularly from neutrophils (Image 1). Neutrophils treated with hydrogen peroxide or XO/HX released NETs in a time dependant manner with peak release at 12 hrs post treatment, whereas neutrophils with media treatment alone released no NETs. Furthermore TLR4 WT neutrophils co-treated with XO/HX released NETs while TLR4 Mutant neutrophils released significantly less NETs. NETs stimulated with PMA, the positive control, appeared as extracellular histones and DNA in close proximity to the neutrophil of origin while oxidative-stressed NETs extended multiple diameters further and overlapped with other cells, suggesting that NETs released due to oxidative stress may be more intense than NETs release due to PMA in murine neutrophils. Conclusions: This study demonstrates that oxidative stress can induce the release of NETs from activated neutrophils. Further, NET generation involves TLR4, a receptor found to play a critical role in both infectious and non-infectious inflammatory conditions. These results provide evidence that NETs may play a previously unrecognized role in sterile inflammation.