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)