PROJECT SUMMARY/ABSTRACT E-cigarettes are a significant public health concern, with over 7 million adults and more than 5 million youth reporting current e-cigarette use. The recent outbreak of e-cigarette and vaping product associated lung injury (EVALI) further highlights the need for research on the effects of e-cigarette use. Pod e-cigarettes, such as JUUL, have rapidly gained popularity since their release in 2016 and now represent over 70% of the e-cigarette market. However, relatively little is known about their effects on the human respiratory system. Specifically, whether and how they affect respiratory mucosal immune defenses, and how these responses compare to those elicited by previous generation e-cigarettes (vape pens, tanks, box mods), represents a critical knowledge gap that will be the focus of this application. We will use tightly linked mechanistic human in vivo and in vitro studies to determine how pod e-cigarettes affect respiratory macrophages, a critical respiratory immune cell type. Preliminary data from our lab and other groups has demonstrated that previous generation e-cigarettes and their components (flavoring chemicals, propylene glycol, vegetable glycerin) have the potential to impair respiratory mucosal immune defenses, including phagocytosis, ciliary beating, oxidative burst, glycolysis, mitochondrial respiration, and immune-related gene expression. Because of JUUL’s unique chemical composition, aerosolization parameters, and puff topography, we anticipate observing additional or differential effects in JUUL users in comparison with previous generation e-cigarette users. We will test this hypothesis in two specific aims: SA1 will determine if users of pod e-cigarette devices have unique innate immune responses in the central airways and SA2 will investigate the effects of aerosolized pod e-liquid and its components on macrophage function ex vivo. To achieve SA1, induced sputum samples from e-cigarette users (both previous generation and pod cohorts), smokers, and nonsmokers will be analyzed for differences in immune cell composition, phagocytic function, mitochondrial energy, morphology, and immunohistochemical staining. Induced sputum will also be analyzed for biomarkers of airway disease, including inflammation, acute lung injury, and vascular damage. To achieve SA2, primary human pulmonary macrophages obtained from bronchoscopies will be adhered to transwells and exposed at air-liquid interface to aerosols from JUUL pods (tobacco & mint flavor), nicotine salts, and benzoic acid alone using a novel in vitro exposure system. The cells will then be assayed for cytotoxicity, inflammatory response, phagocytic function, and bioenergetic capacity to determine the effects of each component of the e- liquid on these endpoints. Data derived from these aims will be integrated to provide highly translational, mechanistic insight into the effects of pod e-cigarette devices on respiratory immune dysfunction, which will addres...