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

Abstract

Background: COPD is a progressive disease of the airways that is characterized by neutrophilic inflammation, a condition known to promote the excessive formation of neutrophil 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 neutrophils. 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.

Fig. 1

NET-forming neutrophils and NETs in sputum samples of COPD subjects stained with anti-NE (green) and PI (red). a non-activated neutrophil with lobulated nucleus and cytoplasmic localisation of NE. (B-D) activated neutrophils at different stages of NET formation. b nucleus swollen, NE staining still confined to cytoplasm. c NE present in both, cytoplasm and nucleus. d ruptured cell in early phase of NET release. e, f Representative examples of NET morphology. NETs are characterised by extensive colocalisation of DNA and NE (the lack of absolute DNA-NE overlap is explainable by the irregular molecular structure of NETs [24], and by secondary alteration this structure during sputum transport). e Long stretch of NET-DNA (arrow) extending between two dense aggregates. In part of the motif, overlap of DNA stain and NE stain is illustrated by stippled lines. f Clusters of NET-DNA, cell debris and an intact neutrophil (asterisk) connected by thin NET trajectories (arrows)

Fig. 2

Identification of NET-forming neutrophils and NETs in COPD sputum by additional methods of analysis. a-c CLSM images. a activated/NET-forming neutrophil stained for citH3 (green) and PAD4 (red), DNA blue. b activated/NET-forming neutrophil stained for citH3 (green) and DNA (red). c Overview image of citH3-stained specimen showing large trajectories of NET DNA intermingled with numerous activated/NET-forming and non-activated neutrophils. The presence of citH3 and PAD4 in both the cytoplasm and the nuclei of the neutrophils conforms with the seminal study on histone deimination in NETosis by Neeli et al. [24, 68] and with our own previous fndings on NET micromorphology [24, 68]. d-e TEM images of ultrathin sections. d Tight attachment of NETs (arrows) to the surface of a bronchiolar epithelial cell (arrowhead) from COPD sputum; NET fibres are also wrapped around an apparently intact (non-NET-forming) neutrophil. e Tangential section through an activated/NET-forming neutrophil outside the nuclear region. The cell is embedded in a mass of NETs clotted with amorphous sputum substance (arrow) and contains various granulae (g), a presumably autophagic vacuole (v), indication of vesicular traffic (arrowheads), and NET-like fibres (asterisk). f-g TEM images of on-grid immunogold stained sputum NETs. f NE epitopes are abundant in the aggregations of organic matter along the NET fibres. g Labelling for citH3 is far less abundant than NE stain and clustered at distinct sites of the NET meshwork. h SEM image of sputum NETs with an entangled bacterium (arrowhead)

Fig. 3

Quantification of neutrophils and NETs. a, b Evaluation in categories, bars represent percentages of total individuals sampled per study group (a) and per COPD severity stage (b). c, d Percentages of NET coverage in sputum preparations. c Bar chart showing means ± standard errors. d Boxplots with medians and interquartile ranges, whiskers have maximum 1.5 interquartile range. Supercript asterisks indicate significant differences between groups (P < 0.05). e Scatter plot illustrating the relation between FEV1 and the percentage of NET coverage, study groups represented by different symbols (Spearman’s rank correlation coefficient ρ = −0.562, P < 0.001)