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

et al. (2014) New Aspects on the Structure of Neutrophil Extracellular Traps from Chronic Obstructive Pulmonary Disease and In Vitro Generation. PLoS ONE 9(5): e97784. doi:10.1371/journal.pone.0097784 New Aspects on the Structure of Neutrophil Extracellular Traps from Chronic Obstructive Pulmonary Disease and In Vitro Generation Astrid Obermayer 0 Walter Stoiber 0 Wolf-Dietrich Krautgartner 0 Michaela Klappacher 0 Barbara Kofler 0 Peter Steinbacher 0 Ljubomir Vitkov 0 Fikreta Grabcanovic-Musija 0 Michael Studnicka 0 Bernhard Ryffel, French National Centre for Scientific Research, France 0 1 Department of Cell Biology, University of Salzburg , Salzburg , Austria , 2 Department of Pediatrics, Paracelsus Medical University , Salzburg , Austria , 3 Clinic of Operative Dentistry, Periodontology and Preventive Dentistry, Saarland University , Homburg, Germany , 4 University Clinic of Pneumology, Paracelsus Medical University , Salzburg , Austria 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. In vitro 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. - . These authors contributed equally to this work. Neutrophil extracellular traps (NETs) are web-like extracellular structures generated by activated polymorphonuclear neutrophils (PMNs) in a distinct process of cell death termed NETosis, involving the extrusion of nuclear DNA after chromatin decondensation. They consist of a scaffold of DNA strands of varying thickness laced with histones, neutrophil elastase (NE) and other antimicrobial, and potentially cytotoxic, molecules from the neutrophil cytoplasm [13]. Their fine structural morphology is characterised by two main components: smooth stretches with diameters of 1517 nm and globular domains with diameters of 2550 nm [46]. According to present evidence, NETosis can be initiated by a variety of molecular signals that bind to neutrophil surface receptors, among them bacterial breakdown products and endogenous pro-inflammatory inducers such as the chemokine IL8 [2,7]. A prominent feature of the reaction cascade leading to NETosis is the citrullination of core histones (mainly reported for histone H3) mediated by peptidyl arginine deiminase 4 (PAD4), likely a requisite for sufficient chromatin decondensation [2,8,9]. NETs have been shown to be effective in killing bacterial and fungal pathogens in vitro (eg. [10,11] and in vivo on the lung mucosa, thereby interacting with surfactant protein D [12]. The antimicrobial activity appears to depend not only on the enzymes and histones, but also on the integrity of the DNA network, as it can be inhibited by DNase treatment [1,13]. This is also a mechanism devised by bacteria as part of their strategies for escaping from NETs [14]. Knowledge on the effects and interactions of NETs in vivo is still fragmentary. Recent evidence from a bacterial infection model in mouse skin suggests that NET release in vivo occurs while the PMNs still move around by pseudopodial crawling, thereby casting large areas of NETs [15]. Evidence is accumulating that exaggerated NETosis and/or slowed NET clearance inflict tissue damage and organ function impairment in a broad variety of inflammatory diseases, including such affecting the human airways. It has been established that NETosis contributes to the pathology of allergic asthma [16] and especially of cystic fibrosis (CF) [5,1719], - and there is indication that NETs also play a role in chronic obstructive pulmonary disease (COPD). COPD is an inflammatory disorder of the human airways which alternates between flare-ups (exacerbations) and periods of stable condition [20,21]. It is characterised by recurrent bacterial infection [22,23], neutrophil infiltration and emphysematous alveolar wall destruction [2427]. The PMN-derived protease NE (a main constituent of NETs, see above) has been assigned a proinflammatory role in COPD, specifically by stimulating the secretion of IL-8 [28], the neutrophil chemoattractant which has been shown to drive NETosis in CF [17]. COPD is thus a prominent candidate for NET formation and NETosis-mediated tissue damage (cf. [1,2,6]), although there is as yet no clear morphological evidence. The present study uses confocal laser scanning microscopy (CLSM) and electron microscopy (SEM, TEM) to establish the presence of NETs in sputum samples from patients with exacerbated COPD. Comparison with in vitro induced NETs from human neutrophils permits our results to expand the present knowledge on the micromorphology of these effector structures of innate host defence and inflammatory response. Materials and Methods Investigations were carried out within the framework of a larger study on airway inflammation in patients with COPD at the University Clinic of Pneumology in Salzburg, Austria. Patients with COPD in acute exacerbation (thus their probability of having airway NETs is very high, n = 16) were recruited from the inpatient population of the clinic; COPD was diagnosed according to the GOLD guidelines [29]. Non-smoking control persons without airflow limitation (thus t (...truncated)