The Role of Reactive Oxygen Species (ROS) in the Formation of Extracellular Traps (ETs) in Humans

Extracellular traps (ETs) are reticulate structures of extracellular DNA associated with antimicrobial molecules. Their formation by phagocytes (mainly by neutrophils: NETs) has been identified as an essential element of vertebrate innate immune defense. However, as ETs are also toxic to host cells and potent triggers of autoimmunity, their role between pathogen defense and human pathogenesis is ambiguous, and they contribute to a variety of acute and chronic inflammatory diseases. Since the discovery of ET formation (ETosis) a decade ago, evidence has accumulated that most reaction cascades leading to ET release involve ROS. An important new facet was added when it became apparent that ETosis might be directly linked to, or be a variant of, the autophagy cell death pathway. The present review analyzes the evidence to date on the interplay between ROS, autophagy and ETosis, and highlights and discusses several further aspects of the ROS-ET relationship that are incompletely understood. These aspects include the role of NADPH oxidase-derived ROS, the molecular requirements of NADPH oxidase-dependent ETosis, the roles of NADPH oxidase subtypes, extracellular ROS and of ROS from sources other than NADPH oxidase, and the present evidence for ROS-independent ETosis. We conclude that ROS interact with ETosis in a multidimensional manner, with influence on whether ETosis shows beneficial or detrimental effects

Biomolecules / The role of reactive oxygen species (ROS) in the formation of extracellular traps (ETs) in humans

Extracellular traps (ETs) are reticulate structures of extracellular DNA associated with antimicrobial molecules. Their formation by phagocytes (mainly by neutrophils: NETs) has been identified as an essential element of vertebrate innate immune defense. However, as ETs are also toxic to host cells and potent triggers of autoimmunity, their role between pathogen defense and human pathogenesis is ambiguous, and they contribute to a variety of acute and chronic inflammatory diseases. Since the discovery of ET formation (ETosis) a decade ago, evidence has accumulated that most reaction cascades leading to ET release involve ROS. An important new facet was added when it became apparent that ETosis might be directly linked to, or be a variant of, the autophagy cell death pathway. The present review analyzes the evidence to date on the interplay between ROS, autophagy and ETosis, and highlights and discusses several further aspects of the ROS-ET relationship that are incompletely understood. These aspects include the role of NADPH oxidase-derived ROS, the molecular requirements of NADPH oxidase-dependent ETosis, the roles of NADPH oxidase subtypes, extracellular ROS and of ROS from sources other than NADPH oxidase, and the present evidence for ROS-independent ETosis. We conclude that ROS interact with ETosis in a multidimensional manner, with influence on whether ETosis shows beneficial or detrimental effects

The Initial Inflammatory Response to Bioactive Implants Is Characterized by NETosis

<div><p>Implants trigger an inflammatory response, which is important for osseointegration. Here we studied neutrophil extracellular trap (NET) release of human neutrophils in response to sandblasted large-grit acid etched (SLA) implants using fluorescent, confocal laser scanning and scanning electron microscopy. Our studies demonstrate that human neutrophils rapidly adhered to SLA surfaces, which triggered histone citrullination and NET release. Further studies showed that albumin or acetylsalicylic acid had no significant effects on the inflammatory response to SLA surfaces. In contrast to bioinert materials, which do not osseointegrate, the bioactivity of SLA surfaces is coupled with the ability to release NETs. Further investigations are necessary for clarifying the role of NETosis for osseointegration.</p></div

Neutrophil extracellular trap (NET) formation characterises stable and exacerbated COPD and correlates with airflow limitation

<span>Background: COPD is a progressive disease of the airways that is characterized by </span><span class="searchterm">neutrophilic</span><span> inflammation, a condition known to promote the excessive formation of </span><span class="searchterm">neutrophil</span><span> extracellular traps (NETs). The presence of large amounts of NETs has recently been demonstrated for a variety of inflammatory lung diseases including cystic fibrosis, asthma and exacerbated COPD. Objective: We test whether excessive NET generation is restricted to exacerbation of COPD or whether it also occurs during stable periods of the disease, and whether NET presence and amount correlates with the severity of airflow limitation. Patients, materials and methods: Sputum samples from four study groups were examined: COPD patients during acute exacerbation, patients with stable disease, and smoking and non-smoking controls without airflow limitation. Sputum induction followed the ECLIPSE protocol. Confocal laser microscopy (CLSM) and electron microscopy were used to analyse samples. Immunolabelling and fluorescent DNA staining were applied to trace NETs and related marker proteins. CLSM specimens served for quantitative evaluation. Results: Sputum of COPD patients is clearly characterised by NETs and NET-forming </span><span class="searchterm">neutrophils</span><span>. The presence of large amounts of NET is associated with disease severity ( p < 0.001): over 90 % in exacerbated COPD, 45 % in stable COPD, and 25 % in smoking controls, but less than 5 % in non-smokers. Quantification of NET-covered areas in sputum preparations confirms these results. Conclusions: NET formation is not confined to exacerbation but also present in stable COPD and correlates with the severity of airflow limitation. We infer that NETs are a major contributor to chronic inflammatory and lung tissue damage in COPD.</span

Characteristics of cells adhered to SLA surface from whole peripheral blood as detected by immunofluorescence.

<p>(A) NE-postitive PMNs with their typically lobulated nuclei, and other NE-negative nucleated cells. (B) A single PMN committed to the NETotic cascade as clearly shown by decompensated chromatin, the swollen, partly disrupted nucleus and NE and citH3 staining co-located with chromatin and at cytoplasmic locations. (C) Chromatin extrusion from PMN. (D) Fully spread NETs between PMNs of different activation states. Scale bars: 10μm.</p

Surface treatment on SLA titanium plate sets.

<p>Surface treatment on SLA titanium plate sets.</p

Immunostaining of neutrophils with NETs.

<p>4h SLA sample. Staining as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121359#pone.0121359.g003" target="_blank">Fig. 3</a>. Overview: ripe NETs (arrows) between the adhered cells. Scale bars: 50μm.</p

Cell adhesion to SLA surfaces from whole peripheral blood with different pre-treatments.

<p>(A) Representative images of the three pre-treatment groups (blue: DAPI, green: NE, red: CitH3), scale bar: 20μm. (B-F) Boxplots (interquartile range; line: median, whiskers: 1.5 x interquartile range) showing absolute (B-D) and relative (E) cell numbers and the areas covered by NETs (F) for the three groups. Superscript letters indicate groups of statistically significant differences. The three types of treatment were compared by univariate analysis of variance and by Tukey HSD post hoc test with subject and sampling session as covariates,. Superscript letters (a, b) indicate groups of statistically significant differences at the <i>P</i><0.05 level (similar letters: no significant differences, different letters: significant difference).</p