Vaping /E‐liquid Exposure Causes Dysregulation of Neutrophil Extracellular Trap formation

Abstract

IntroductionElectronic cigarettes (e‐cigarettes) vaporize e‐liquids containing propylene glycol and vegetable glycerine (PG/VG), nicotine, and flavors. The inhaled aerosol from e‐liquids may be potentially toxic or harmful. Although the short and long‐term health consequences of e‐cigarettes use are poorly understood, their use has risen steeply among adolescents and young adults. Neutrophils play an essential role in innate immune and are the first responders during infections. Thus, this study aims to test whether vaping can affect neutrophil function. We hypothesized that vaping e‐cigarettes and exposure to e‐liquids activate neutrophils, cause degranulation, and induce extracellular traps (NETs) through altered calcium signaling.Method and MaterialsNeutrophils were isolated from human blood samples of non‐vapor control (n=9) and e‐cigarette users (n=5) and studied within 2 hours. To measure changes in cytosolic calcium, neutrophils were loaded with Fluo‐4. Intracellular calcium responses were monitored on a Tecan multi‐plate reader following stimulation by 1 µM thapsigargin and 1 µM fMLP/100ng per ml LPS. In addition, since elevations in intracellular calcium trigger neutrophil activation and degranulation, elastase activity in the media samples was measured using a specific fluorogenic substrate (MAA‐3133, © vivitide, LLC.) at excitation/emission wavelengths 380/460 nm. To study NET formation, neutrophils were labeled with SYTOX Green dye and then exposed to different concentrations of Juul e‐liquids (menthol, classic tobacco flavors) and Bidi Stick flavors (Arctic, Gold, and Solar). Data acquisition was performed with Cytation‐5 Cell Imaging Plate Reader, then analysis and representative images were captured by confocal microscopy.ResultsNeutrophils from e‐cigarette users (n=5 vapers) showed significantly (P < 0.001) increased intracellular calcium responses to thapsigargin and fMLP/LPS treatments compared to the non‐user controls (n=9). Additionally, vapers' neutrophils also secreted more elastase (P < 0.001) compared to the control non‐users, indicating that e‐cigarette users may be susceptible to increased systemic protease activity. Our data also indicated that Juul and Bidi Stick e‐liquids stimulated neutrophils to induce NETs in a dose‐dependent manner. Furthermore, the study revealed that Juul e‐liquid menthol flavor with 5% nicotine could significantly (P < 0.001) release NETs than equivalent nicotine ± benzoic acid alone.SummaryCirculating neutrophils are normally quiescent. However, our data indicated that circulating neutrophils from vapers were abnormally active and degranulated by released elastase. Additionally, e‐cigarette liquids dysregulated neutrophils by increasing NETs production. Overall, we speculate that vaping imbalanced innate immune system and may lead to abnormal responses to the infection, inappropriate inflammatory responses, and adverse health effects.