Transcriptomic Profiling of Lung Environment Changes Due to E-cigarette Exposure in Mice

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Abstract Introduction: The landscape of electronic (e)-cigarettes has rapidly evolved and many flavored 4th generation devices are available. E-liquids contain humectants, propylene glycol and glycerin, flavorant chemicals, and nicotine. While flavoring chemicals are generally recognized as safe for gastrointestinal consumption, flavors have been shown to have detrimental effect on cellular functions following inhalation. We previously conducted bulk RNAseq on mouse lungs across different types of e-cigarette exposures. However, transcriptional changes to specific cell types are difficult to assess using bulk RNAseq. Deconvolution is a computational method that uses a reference gene matrix to estimate the relative proportions of different cell types in a bulk sample. We applied this method to our bulk RNAseq data to obtain further insight into cellular responses to e-cigarettes. Methods: Female C57BL/6 mice (8-weeks-old) were exposed to tobacco, menthol, mint, or mango-flavored JUUL aerosols, or room air for 1 hour/d, 5 d/week, x4 weeks using the InExpose System (Scireq, Emka). Lung RNA was isolated for bulk RNAseq. Differential gene expression (DGE) analysis was performed using DESeq2. Ingenuity Pathway Analysis was performed on significant differentially expressed genes (DEGs). A reference single-cell RNAseq dataset from postnatal day 28 murine lungs was used to construct a gene signature matrix for deconvolution of bulk RNAseq data in CIBERSORTx using default settings. Results: Tobacco and menthol e-cigarette exposures had the greatest number of DEGs. Out of the different flavors, 3 out of the 4 groups (menthol, tobacco, and mango) showed overlap in transcriptional changes such as downregulation of the alpha and beta globin chain genes (HBA1/HBA2 and HBB) and upregulation of Neutral cholesterol ester hydrolase 1 (Nceh1) gene. Mint exposure was associated with 1 DEG, Heat shock protein 1B (Hspa1b). Based on the deconvolution, the inferred proportion of alveolar type 2 cells and arterial endothelial cells were decreased in the tobacco and menthol groups compared to air controls. Conversely, the proportion of alveolar type 1 cells and B cells were estimated to be higher in the tobacco and menthol groups compared to air controls. Conclusion: Daily tobacco and menthol flavored e-cigarette exposures were associated with many transcriptomic changes within the lungs. These transcriptomic changes translated into inferred alterations in cell proportions of alveolar type 1 and 2 cells, arterial endothelial cells, and B cells. These data suggest that these cell types are either exhibiting an altered transcriptional state or have changed in proportions. Further studies are needed for validation of these findings.

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