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A3564 - Electronic (E)-Cigarette Vapor Extract Impairs Neutrophil Chemotaxis, Reactive Oxygen Species Production and Neutrophil Extracellular Trap Formation
Author Block: C. M. Bojanowski1, R. Corriden2, A. Meier3, L. E. Crotty Alexander4; 1Pulmonary, Critical Care and Sleep Medicine, University of California, San Diego, La Jolla, CA, United States, 2Department of Pediatrics and Skaggs School of Pharmacy, University of California, San Diego, La Jolla, CA, United States, 3Anesthesiology, University of California, San Diego, La Jolla, CA, United States, 4Medicine, UCSD and VASDHS, San Diego, CA, United States.
Rationale: Electronic (e)-cigarettes are promoted as a “safer” alternative to conventional cigarette smoke and their use has substantially increased over recent years. However, little is know about the potential cytotoxicity of e-cigarette vapor on human innate immunity. As part of the innate immune system, neutrophils are the first line of defense against environmental insults. We hypothesized that exposure of circulating human neutrophils to e-cigarette vapor extract (EVE) would negatively impact their host defense functions ex vivo.
Methods: Fresh human neutrophils were isolated from peripheral blood via the polymorphoprep technique and were incubated with a range of EVE dilutions, for 20 minutes at 37C and 5% CO2. To determine the effect on chemotaxis, neutrophils were incubated in the top chambers of transwell inserts, with and without EVE, with the bacterial chemoattractant fMLP in the lower chambers. Migration of neutrophils to the lower chambers was quantified using an elastase-based method. For neutrophil extracellular trap (NET) assays, neutrophils were stimulated with phorbol myristate acetate (PMA) after EVE exposure, and NET production was quantified using the fluorescent DNA dye PicoGreen. For assessment of reactive oxidant species (ROS) production, neutrophils were loaded with Dichloro-dihydro-fluorescein diacetate (DCFH-DA) for 20 minutes prior to stimulation with various concentrations of PMA. Fluorescence (ROS production) was read every 15 minutes for 2 hours.
Results: Neutrophils exposed to EVE displayed impaired chemotaxis when exposed to fMLP. Normal F-actin polarization in the setting of a chemokine gradient was lost with exposure to EVE, and the chemotactic index was significantly reduced at all levels of exposure. EVE exposure also inhibited PMA-induced ROS production. Finally, with this brief ex vivo exposure to EVE, NET formation was completely inhibited.
Conclusions: Acute, short-term EVE exposure impaired human neutrophil functionality. This suggests that following EVE exposure, neutrophils may not be successfully recruited to sites of insult in vivo, likely due to disruption of their intracellular polarity and ablation of the F-actin leading edge required for neutrophil mobilization. Furthermore, EVE exposure also decreased neutrophil functions required at the site of insult by decreasing NET formation and ROS production. These findings provide further evidence that e-cigarette vapor negatively impacts host innate immune responses. Further studies are needed to fully elucidate potential short-term and long-term toxicities associated with these widely used products.