Enhanced neutrophil extracellular trap (NET) formation in sputum of stable COPD patients

COPD is characterized by neutrophilic airway inflammation. Neutrophil extracellular trap formation (NETosis), which consists of extracellular neutrophilic DNA, histones and granule enzymes, such as neutrophil elastase may regulate innate immune responses and potentially contribute to neutrophil-associated diseases. The presence of NETosis in sputum of stable COPD patients remains unknown. The aim of this study was to determine the level of NET formation in induced sputum from patients with COPD and to examine its relationship with sputum neutrophilia and airway obstruction. We evaluated induced sputum samples of 23 COPD patients (mean age, 67.0 ± 8.1 years; mean FEV1, 57.8 ± 16.6% pred.) and 10 healthy controls (mean age 55.0 ± 16.6 years) in whom airway obstruction were assessed using lung function testing. Sputum cells were harvested and stained with anti-histone H1, anti-neutrophil-elastase and DAPI. Concentration of extracellular DNA in sputum supernatants was quantified using a NanoDrop 2000 spectrophotometer. NETosis was markedly upregulated in COPD sputum compared with healthy controls as evaluated by immunofluorescence microscopy. NETosis was associated with significantly higher concentration of extracelluar DNA in sputum supernatant (mean 656.0 ± 894.6 ng/µL in COPD versus 300.2 ± 216.0 ng/µl in controls, p< 0.0001). Extracellular DNA correlated with absolute neutrophil numbers in sputum (r= 0.698; p< 0.0001) and airway obstruction (r=0.512; p=0.001). These results indicate that NETosis is markedly increased in the airways of stable COPD patients, suggesting that failure of timely clearance of NETosis may contribute to the unresolving neutrophilic airway inflammation in COPD.