Neutrophil extracellular traps released in response of cigarette smoke extract increase the secretion of proinflammatory cytokines by human alveolar epithelial cells

SABBIONE, FLORENCIA; KEITELMAN, IRENE; GUZMÁN, MAURICIO; GALLETTI, JEREMÍAS; FERRERO, M; BALDI, PABLO; TOWSTYKA, NADIA YASMÍN; GIORDANO, MIRTA N.; JANCIC, CAROLINA CRISTINA; TREVANI, ANALÍA S.

Mar del Plata

Congreso; 64 Reunión Anual de la Sociedad Argentina de Inmunología; 2016

Cigarette smoking is a major cause of chronic obstructive pulmonary disease. Gas phase of cigarette smoke (CS) can reach the alveolar epithelium inducing the secretion of chemokines and cytokines that contribute to recruit neutrophils (PMN) into the airway lumen. In response to diverse stimuli, PMN release neutrophil extracellular traps (NET) through a process called NETosis. Previous studies determined that CS and uric acid (UA), a major DAMP found at high concentrations in lungs of patients with acute lung injury, can induce NETosis. The aim of this study was to determine if a short exposure to CS is able to induce NETosis, and if these NET induce proinflammatory cytokine secretion by alveolar epithelial cells, comparing their effects with those produced by NET released in response to UA (8 mg/dl). CS extract (CSE) prepared according to a standard method from cigarettescontaining an equivalent of 13.6 mg/ml tar was used at 10%. PMN were incubated with or without stimuli for 1h, then washed and cultured for 3 more hours at 37°C. NET were identified by confocal microscopy by colocalization of DNA with myeloperoxidase. Short exposure to both stimuli induced NETosis. NET released by 106 PMN were isolated and added to A549 epithelial cell monolayers. Both CSE- and UA-induced NET significantly increased the secretion of IL-8 and IL-6 (p<0.05; n=4), and IL-1b (n=2) by A549 as compared to that induced by supernatants from unstimulated PMN and basal conditions. These effects were not observed with supernatants from CS-stimulated PMN pretreated with the NETosis inhibitor CL-amidine (200 μM; n=2). These findings suggest that CSE might also promote alveolar inflammation by triggering NETosis. Together with our previous findings indicating similar properties of NET induced by monosodium urate crystals, these results alsosuggest that NET exert proinflammatory effects on epithelial cells independently of the sterile stimulus that induced their release.