NETosis markers: Quest for specific, objective, and quantitative markers

Abstract

More than 10years have passed since the discovery of neutrophil extracellular traps (NETs) in 2004. NETs are extracellular web-like DNA decorated with antimicrobial proteins, which are released from activated neutrophils. The state of neutrophils with NET formation is called NETosis. It has been realized that NETosis includes suicidal NETosis and vital NETosis. The former state means cell death of neutrophils, whereas the latter state preserves living neutrophilic functions. Although both suicidal and vital NETosis play essential roles in elimination of microorganisms, excessive formation of NETs, especially the ones derived from suicidal NETosis, can harm the hosts. Therefore, the discovery of NETosis markers and development of evaluation methods are important. In this review, we compare the methods for evaluating NETosis, including immunocytological and immunohistological detection of co-localized neutrophil-derived proteins and extracellular DNA, and citrullinated histones, detection of NET remnants in fluid samples, and flow cytometric detection of cell-appendant NET components, with focus on the specificity, objectivity, and quantitativity. Since the gold standard marker of NETosis or method of NET detection has not been established yet, researchers should choose the most appropriate marker or method in each situation based on the knowledge of the respective virtues and faults.

Interactions between Neutrophils and Pseudomonas aeruginosa in Cystic Fibrosis

“…Lack of flagellar motility, not flagellum expression per se, is responsible for impaired NET release induced by flagellated P. aeruginosa [205]. This is consistent with previous data showing that early CF isolates of P. aeruginosa induce larger amounts of NETs and are more resistant against NET-mediated attacks than late isolates obtained from the same CF patients [128,170] (Figure 1). Whether loss of swimming motility alone is responsible for reduction in P. aeruginosa -initiated NET release or loss of other NET-inducing or appearance of NET-inhibitory factors also contribute, remains to be studied.…”

“…Several microorganisms including P. aeruginosa were shown to trigger NET release in PMNs. Only NET-forming PMNs release protein-DNA complexes (myeloperoxidase-DNA or neutrophil elastase-DNA); not apoptotic or necrotic PMNs [120,125,126,127,128]. Despite the vast amount of literature published in the last 12 years on NETs, signaling pathways leading to NET formation are still largely unknown, and only the involvement of a few molecules is known.…”

“…ecDNA is a main component of P. aeruginosa aggregates, and ecDNA obtained from necrotic PMNs has been shown to support aggregate growth of P. aeruginosa [214]. NET formation can likely be the source of PMN DNA in CF airways, since P. aeruginosa elicits robust NET release in PMNs [122,128,170,205]. Pyocyanin, a redox pigment and toxin of P. aeruginosa , is present in chronic CF airways and promotes the biofilm-forming ability of ecDNA [64,221].…”

The definition of neutrophil extracellular traps and the concentration of short-chain fatty acids in salmonella-induced inflammation of the intestine against the background of vancomycin and Bacteroides fragilis

“…Thus, non-nuclear neutrophils retain the ability to phagocytosis pathogenic microorganisms, regardless of the loss of DNA. The third, ROS-dependent mechanism of NETosis, is the release of mitochondrial DNA, instead of nuclear, by recognizing the lipopolysaccharide (LPS) of bacteria or C5a, resulting in the formation of NETs for 15 min [35][36][37][38].…”

The source of cell-free mitochondrial DNA in trauma and potential therapeutic strategies

“…However, as a potential clinical biomarker, this is not quantitative, is laborious and is prone to observer bias. A number of studies have described methods for quantitating NETs with flow cytometry using a similar staining approach [59]. A major limitation of NET measurement in either case is the inability to specifically measure mtDNA within the structure (Table 1).…”