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

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

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

New Aspects on the Structure of Neutrophil Extracellular Traps from Chronic Obstructive Pulmonary Disease and <i>In Vitro</i> Generation

<div><p>Polymorphonuclear neutrophils have in recent years attracted new attention due to their ability to release neutrophil extracellular traps (NETs). These web-like extracellular structures deriving from nuclear chromatin have been depicted in ambiguous roles between antimicrobial defence and host tissue damage. NETs consist of DNA strands of varying thickness and are decorated with microbicidal and cytotoxic proteins. Their principal structure has in recent years been characterised at molecular and ultrastructural levels but many features that are of direct relevance to cytotoxicity are still incompletely understood. These include the extent of chromatin decondensation during NET formation and the relative amounts and spatial distribution of the microbicidal components within the NET. In the present work, we analyse the structure of NETs found in induced sputum of patients with acutely exacerbated chronic obstructive pulmonary disease (COPD) using confocal laser microscopy and electron microscopy. <i>In vitro</i> induced NETs from human neutrophils serve for purposes of comparison and extended analysis of NET structure. Results demonstrate that COPD sputa are characterised by the pronounced presence of NETs and NETotic neutrophils. We provide new evidence that chromatin decondensation during NETosis is most extensive and generates substantial amounts of double-helix DNA in ‘beads-on-a-string’ conformation. New information is also presented on the abundance and location of neutrophil elastase (NE) and citrullinated histone H3 (citH3). NE occurs in high densities in nearly all non-fibrous constituents of the NETs while citH3 is much less abundant. We conclude from the results that (i) NETosis is an integral part of COPD pathology; this is relevant to all future research on the etiology and therapy of the disease; and that (ii) release of ‘beads-on-a-string’ DNA studded with non-citrullinated histones is a common feature of in vivo NETosis; this is of relevance to both the antimicrobial and the cytotoxic effects of NETs.</p></div

Electron microscopic characterisation of NET components.

<p>SEM (A,C) and TEM (B,D–H) micrographs from COPD sputum (A–E,H) and <i>in vitro</i> induced NETs (F,G). A: Fibrous strands diverge from a PMN cell body with a sculptured surface and attached granulae and merge into loosely structured texture. B: Motif corresponding to that of A. Thinly spread areas (inset) exhibit a fibrous texture with attached globules (arrows). C: Bacterium (open arrow) entrapped by NET fibres with globular protrusions. D,E: Sections of COPD sputum. D: Bacterium (open arrow) surrounded by a fibrous network that embeds spherical granules with amorphous content (arrow). E: Undisturbed PMN with normal nuclear chromatin and amorphous vesicular inclusions (arrows). F–H: Sections of <i>in vitro</i> induced NETs. F: NETs attached to the remnants of their cell of origin. NET fibres exhibit less accretions than those from COPD sputum NETs (cf. E); arrows indicate neutrophilic granula. G: Fibres and neutrophilic granula of the NETs shown in F. H: Membrane coated in COPD sputum vesicle containing NET-like structures.</p

Individual PMNs in various stages of NETosis.

<p>COPD sputa immunolabelled for NE (green: A,B), citH3 (green: C–F), and PAD4 (red: F). DNA stained with either PI (red: A–D) or DAPI (blue: F). A: Non-activated stage with still lobulated nucleus, NE confined to cytoplasm. B: advanced stage with highly swollen nucleus, NE colocalised with chromatin (staining to the right extracellular). C–E: Stages during nuclear swelling. C: Nuclear lobulation still visible, part of chromatin stains for citH3. D: Staining for citH3 most intense in perinuclear cytoplasm. E: Chromatin extrusion. F: CitH3 and PAD4 colocalised in cytoplasm and nucleus. Scale bars are all at 5 µm and valid for each photograph within the individual combinations A–F.</p

CLSM images of NETs in COPD sputum.

<p>Immunolabelling for neutrophil elastase (NE, green) or citrullinated Histone H3 (citH3, green), DNA stained with propidium iodide (PI, red). A–C: The sputum matrix contains PMNs (open arrows), citH3-positive granules and areas of condensed NETs (asterisks). D–F: Less condensed matrix traversed by thin NET strands associated with bacteria (arrowheads), non-activated PMNs (open arrow) and NETotic PMNs (solid arrow); asterisks indicate fully spread NETs. G–I: COPD sputum treated with DNAse. The DNA meshwork is dissolved while PMNs and citH3 positive granules persist.</p