Impact of Acid, Base, and Salt Components of E-cigarettes in Pregnancy - PROJECT SUMMARY Electronic cigarette (e-cig) use during pregnancy has become a major health concern in recent years and is perpetuated by the perception that e-cigs are less harmful than traditional combustible cigarettes. Recent estimates show 7% of women use e-cigs in pregnancy, and 45% view them as less harmful and may help them quit or reduce combustible cigarettes in pregnancy. Recent reports indicate e-cig vaping leads to heightened risk to respiratory infection in nonpregnant adults, but there is limited understanding in the neonatal population. We performed preliminary RNA-seq analysis following exposure to freebase nicotine, the most addictive component of a 3rd gen e-cig, during pregnancy in our preclinical pregnant rat model using a state-of-the-art vaping system with a commercial e-cig unit that accurately replicates human exposure and use. Our novel data showed a major separation of immune-related genes between male and female fetal lungs, with sex-specific deficits in innate immune pathways, including the neutrophil system. Flow cytometry analysis of fetal lungs confirmed sex specific changes in innate immune cell populations following freebase nicotine exposure, primarily in neutrophils. Importantly, our preliminary in vivo experiments show sex-specific enhanced susceptibility to neonatal viral infection in the freebase nicotine-exposed lung. Our compelling preliminary evidence demonstrated that nicotine aerosols alters both neonatal lung development and function in a sex-specific manner. However, the newer 4th gen e-cigs also have an acidic component in addition to the nicotine (base). Although freebase nicotine is the most studied component, we tested the impact of aerosolizing the acidic component, benzoic acid, the acid used by JUUL, a popular manufacturer. Preliminary RNA-seq data showed both viral-sensing pathways, and type I interferon (IFN) signaling pathways, were downregulated in a sex-specific manner following exposure to both the acidic (benzoic acid) and the basic (nicotine) components in the latest 4th gen e-cigs. We hypothesize that fetal exposure to aerosolization of benzoic acid (acid), freebase nicotine (base), or in combination as nicotine benzoate (salt) alters the developing lung immune environment in a sex- dependent manner, resulting in deficient antiviral innate immune responses and increased risk to respiratory viral infections. To test this hypothesis, we will (1) Identify sex-dependent alterations in fetal lung immune cell function, distribution and inflammatory profiles following gestational e-cig exposure (carrier, benzoic acid, freebase nicotine, & nicotine benzoate salt), (2) Define the sex-dependent impact of acid, base, and salt components of e-cigs on fetal lung antiviral innate pathways using single-cell RNA seq, stochastic modeling and reinforcement algorithms, and (3) Assess the sex-dependent efficacy of postnatal IFN treatment in enhancing antiviral respiratory responses after prenatal exposure to e-cig’s acid, base, and salt components. These findings will help managing neonatal immune alterations that may differ based on sex, and directly address several “Research Areas of Emphasis” of NIEHS Strategic Plan 2025-2029.
