Effects of cigarette smoke and smoke condensate on neutrophil extracellular trap formation

Abstract

Background: Neutrophil extracellular traps (NETs) constitute a network of chromatin fibres containing histone and antimicrobial peptides that are released by activated neutrophils. NETs protect the host against infection by trapping and facilitating phagocytosis of potentially harmful pathogens. Objectives: The aim of the current study was to investigate the effects cigarette smoke condensate (CSC) on phorbol-ester (PMA)-mediated NETosis in vitro, as well as the effects of cigarette. Methods: Isolated human blood neutrophils were exposed to PMA (6.25 ng/ml) in the presence or absence of CSC (40-80 μg/ml) for 90 min at 37oC. Alternatively neutrophils of non-smokers and smokers were activated with PMA (6.25 ng/ml) for 90 min at 37oC. NET formation was measured using a spectrofluorimetric procedure to detect extracellular DNA and fluorescence microscopy was used to visualize nets. Oxygen consumption by PMA-activated neutrophils was measured using an oxygen sensitive electrode. Cotinine levels were measured in smokers and non-smokers for objective confirmation of smoking status Results: Activation of neutrophils with PMA was associated with induction of NETosis that was significantly attenuated in the presence of CSC (40 and 80 μg/ml), with mean fluorescence intensities of 65% and 66% of that observed with untreated cells, respectively, and confirmed by fluorescence microscopy. The rate and magnitude of oxygen consumption by activated neutrophils pre-treated with CSC (80 μg/ml) was significantly less than that observed with untreated cells (73% of the control system), indicative of decreased production of reactive oxidant species in the presence of CSC. When comparing smokers and non- smokers, neutrophils from smokers showed a decrease in both oxygen consumption and the number of NET-forming cells consistent with attenuation of NET formation due to inhalation of cigarette smoke. Conclusion: The inhibition of NETosis observed in the presence of CSC and CS (in smokers) correlated with attenuation of oxygen consumption by PMA-activated neutrophils suggesting a mechanistic relationship between these events. Smoking-related attenuation of NETosis may impair host immune responses and increase the risk of respiratory infections, in vivo.