In vitro induction of NETosis: Comprehensive live imaging comparison and systematic review

Abstract

BackgroundMultiple inducers of in vitro Neutrophil Extracellular Trap (NET) formation (NETosis) have been described. Since there is much variation in study design and results, our aim was to create a systematic review of NETosis inducers and perform a standardized in vitro study of NETosis inducers important in (cardiac) wound healing.MethodsIn vitro NETosis was studied by incubating neutrophils with PMA, living and dead bacteria (S. aureus and E. coli), LPS, (activated) platelets (supernatant), glucose and calcium ionophore Ionomycin using 3-hour periods of time-lapse confocal imaging.ResultsPMA is a consistent and potent inducer of NETosis. Ionomycin also consistently resulted in extrusion of DNA, albeit with a process that differs from the NETosis process induced by PMA. In our standardized experiments, living bacteria were also potent inducers of NETosis, but dead bacteria, LPS, (activated) platelets (supernatant) and glucose did not induce NETosis.ConclusionOur systematic review confirms that there is much variation in study design and results of NETosis induction. Our experimental results confirm that under standardized conditions, PMA, living bacteria and Ionomycin all strongly induce NETosis, but real-time confocal imaging reveal different courses of events.

Root extracellular traps versus neutrophil extracellular traps in host defence, a case of functional convergence?

“…Kenny et al, 2017). A comprehensive review of these pathways has been published recently (Hoppenbrouwers et al, 2017) and is beyond the scope of this review. However, pathways relevant to comparative discussions will be highlighted in later sections.…”

Neutrophil extracellular traps in nasal secretions of patients with stable and exacerbated chronic rhinosinusitis and their contribution to induce chemokine secretion and strengthen the epithelial barrier

“…In light of these previous results, we investigated whether NETs may contribute to increased epithelial permeability in CRS. 45 Therefore, NETs can mediate inflammatory responses by increasing the neutrophil migration through the increase of the endothelial cells surface expression of ICAM-1 in CRS.NETs formation is triggered by different physiological and nonphysiological compounds 46. Therefore, the present study suggests that NETs may have beneficial effects on the epithelial permeability, possibly contributing to the maintenance of epithelial integrity.…”

“…NETs formation is triggered by different physiological and nonphysiological compounds. 46 It is also suggested that antibiotics could be non-physiological compounds that influence NETs release.…”

Anti‐β2GPI/β2GPI induces human neutrophils to generate NETs by relying on ROS

“…21,22 NETosis stimulated via the PKC stimulator, PMA leads to phosphorylation of PKC, p38 MAPKs, and ERK1/2. 23,24 However, no stimulation of these kinases was detected in the reaction to anti-β2GPI/β2GPI. Zn 2+ is crucial for NETosis triggered via PMA.…”

“…Although numerous studies have identified reactions linked with NET delivery, the understanding of the reactions inside cells that participate in NET generation is insufficient regarding the various NET stimulators.Although numerous candidate targets of anti-β 2 GPI/β 2 GPI have been identified in signal pathways to cause NETosis, most promising mechanisms were shown to be irrelevant to NETosis. NETosis stimulated via the PKC stimulator, PMA leads to phosphorylation of PKC, p38 MAPKs, and ERK1/2 23,24. NETosis stimulated via the PKC stimulator, PMA leads to phosphorylation of PKC, p38 MAPKs, and ERK1/2 23,24.…”