Unflavored electronic cigarette exposure induces alterations in airway ciliary structure and function

Abstract

Electronic cigarettes (e-cigs) have been introduced as a safer alternative to traditional combustible cigarettes and have been growing in popularity. E-cig e-liquids all contain the carrier compounds, vegetable glycerin (VG), propylene glycol (PG), and nicotine, together with different flavors, but the effects of inhalation of these compounds on the airway are not well understood. This study investigates the effects of e-cig exposure on primary human airway epithelial cells grown in air-liquid interface (ALI) cultures, specifically focusing on mucociliary clearance, the lung's primary host defense mechanism whereby pathogens and particles trapped by mucus are cleared by unidirectional beating by ciliated cells. We developed a microcontroller-based exposure system to reproducibly examine cellular and molecular changes in ALI cultures from e-cig exposure. Here we show heterogeneous, donor-dependent effects of different e-cig flavors on airway epithelial cells. Examining the effects of the unflavored carrier compounds common to all e-cigs, we found that ALI airway cultures exposed to PG:VG (30:70 ratio) with 5% nicotine unflavored e-cigs show a reduction in ciliary beat frequency. Moreover, using transmission electron microscopy, we identified defects in ciliary ultrastructure induced by unflavored e-cigs. Phosphoproteomic analysis uncovered changes in phosphorylation of proteins involved in cadherin and actin binding and the Rho GTPase signaling pathway, which are all involved in cytoskeletal remodeling that may influence ciliary structure and function. Altogether, our findings suggest that exposure to all e-cigs reduces mucociliary clearance.

Keywords: Ciliary beating frequency; Cytoskeletal remodeling; E-cigarettes; Motile cilia; Phosphoproteomics; Rho GTPase.

Fig. 1

Donor and flavor-specific responses to e-cig aerosols in human bronchial epithelial cells grown in ALI. A Schematic illustrating ABSC isolation from donor tissue, seeding into tissue culture transwell inserts and initiating air–liquid interface (ALI). B Schematic showing basic components of the exposure system. C Schematic outlining the timeline of ABSC seeding and ALI initiation followed by e-cig aerosol exposure. D Representative IF images of Keratin 5 (KRT5)-expressing (magenta) ABSCs in ALI cultures across the six exposure conditions of room air control, unflavored PG:VG (30:70 ratio) with 5% nicotine, and mint flavored, red energy flavored, strawberry flavored, and tobacco flavored e-cigs. E Representative IF images of acetylated alpha tubulin (Ac-Tub) (yellow) ciliated cells in ALI cultures across the six exposure conditions. F Representative IF images of Muc5AC (cyan) mucus cells in ALI cultures across the six exposure conditions. G Quantification of number of Keratin 5 + across the six exposure groups. H Quantification of number of Ac-Tub + across the six exposure groups. I Quantification of number of Muc5AC + mucus cells across the six exposure groups. Graphs represent mean ± SEM, n = 3 biological replicates (3 different donors), each with 3 technical replicates (3 ALI transwell cultures derived from each of the 3 different donors) for a total of 9 replicates per exposure condition. Each dot represents a replicate. P values are calculated from all technical replicates across the biological replicates as described above. Scale bar = 100 μm

Fig. 2

Unflavored e-cig aerosols induce reduction in the relative beat frequency of ciliated cells and disruption of ciliary ultrastructure. A Representative movement maps of field of views (FOVs) from microscopy videos of ALI cultures exposed to unflavored e-cig aerosols or air control. B Quantification of percent ciliary movements from microscopy videos of ALI cultures exposed to unflavored e-cig aerosols or air control. C Representative ciliary beat frequency (CBF) maps of FOVs from microscopy videos of ALI cultures exposed to unflavored e-cig aerosols or air control. D Quantification of average CBF from microscopy videos of ALI cultures exposed to unflavored e-cig aerosols or air control. E p = 0.0153 (n = 2 biological replicates, 20 technical replicates). Each dot represents one video per condition. TEM images of cilia axonemes from ALI cultures that were exposed to unflavored e-cig aerosols or air control. Scale bar 200 nm. (n = 1 biological replicate, 2 technical replicates) Red arrows point to ultrastructural defects

Fig. 3

Unflavored e-cigarette aerosols induce alterations in the phosphoproteomic profile of bronchial epithelial cells grown in ALI. A Comparison of significantly altered molecules in airway cells following exposure to unflavored e-cig aerosols. The number of differentially expressed genes (RNA), proteins, and phosphopeptides are shown. (RNA: n = 2 biological replicates, 2 technical replicates. Protein; n = 2 biological replicate, 3 technical replicates, Phosphopeptides: n = 1 biological replicate, 3 technical replicates) A molecule was considered significantly altered if it exhibited a fold change > 1 or < −1 and an adjusted p-value ≤ 0.05. B Volcano plots depicting differential phosphorylation of phosphorylated sites in airway cells following exposure to unflavored e-cig aerosols compared to room air control. Each point represents a unique phosphorylation site. The x-axis shows the log2 fold change (log2FC) in phosphorylation, while the y-axis shows the -log10 of the adjusted p-value. Cyan points indicate decreased phosphorylation (log₂ fold change <—1), magenta points indicate increased phosphorylation (log₂ fold change > 1), and grey points indicate phosphorylation sites that did not meet those criteria. Point size corresponds to significance, with larger points representing phosphorylation sites with an adjusted p-value ≤ 0.05. C-E Heatmaps depicting pathway analysis for the proteins with dysregulated phosphorylation that belong to Gene Ontology (GO) Biological Processes terms (C), GO Molecular Function ontology (D), and Reactome Pathways Database (E). For all heatmaps, the color intensity of each box represents the degree of enrichment for a specific GO term calculated as -log10(adjusted p-value). A gradient from white (low enrichment) to purple (high enrichment) indicates the strength of the association. Numbers within each box show the number of genes from the input list associated with that term that have statistically significant enrichment (p-value < 0.05). Red boxes highlight terms of interest. (H) STRING network of the enriched proteins belonging to the GO terms “gomf cadherin binding” (purple) and “gomf actin binding” (yellow) and REACTOM term “rho GTPase cycle” (pink) were analyzed using STRING interaction network analysis. Interaction confidence was displayed as the edge thickness. Medium confidence interactions (combined score > 0.4) were used for the analysis. Phosphopeptides: n = 1 biological replicate, 3 technical replicates)