Transcriptomic response of primary human airway epithelial cells to flavoring chemicals in electronic cigarettes

Abstract

The widespread use of electronic cigarettes (e-cigarettes or e-cig) is a growing public health concern. Diacetyl and its chemical cousin 2,3-pentanedione are commonly used to add flavors to e-cig; however, little is known about how the flavoring chemicals may impair lung function. Here we report that the flavoring chemicals induce transcriptomic changes and perturb cilia function in the airway epithelium. Using RNA-Seq, we identified a total of 163 and 568 differentially expressed genes in primary normal human bronchial epithelial (NHBE) cells that were exposed to diacetyl and 2,3-pentanedione, respectively. DAVID pathway analysis revealed an enrichment of cellular pathways involved in cytoskeletal and cilia processes among the set of common genes (142 genes) perturbed by both diacetyl and 2,3-pentanedione. Consistent with this, qRT-PCR confirmed that the expression of multiple genes involved in cilia biogenesis was significantly downregulated by diacetyl and 2,3-pentanedione in NHBE cells. Furthermore, immunofluorescence staining showed that the number of ciliated cells was significantly decreased by the flavoring chemicals. Our study indicates that the two widely used e-cig flavoring chemicals impair the cilia function in airway epithelium and likely contribute to the adverse effects of e-cig in the lung.

Figure 1

Identification of differential gene expression in NHBE cells exposed to flavoring chemicals by RNA-seq. (A) Schematic workflow of the study. (B,C) Volcano plot of RNA-seq results with top 10 genes annotated in diacetyl or 2,3-pentanedione-exposed NHBE cells for 24 h. Black dots represent gene with Padj < 0.05. Grey dots represent genes that do not meet the significance threshold. (D,E) qPCR validation of top 10 genes identified by RNA-seq with diacetyl or 2,3-pentanedione. *P < 0.05 compared to control. N = 3 subjects.

Figure 2

The transcriptomes of diacetyl and 2,3-pentanedione-exposed NHBE cells are highly overlapped. (A) The Venn diagram shows the overlap of differentially expressed gene sets (Padj < 0.05). (B) qPCR validation of the top 10 overlapped gene set with diacetyl and 2, 3-pentanedione treatment. *P < 0.05 compared to control. N = 3 subjects. (C) Expression of genes involved in cilia processes measured by qPCR. *P < 0.05 compared to control. N = 3 subjects. (D) Expression of cilia-involved genes in HNBE cells exposed to diacetyl or 2,3-pentanedione at varying concentrations. P < 0.05 compared to control.

Figure 3

Immunofluorescence staining. NHBE cells at ALI day 14 were exposed to diacetyl (25 ppm) or 2,3-pentanedione (100 ppm) for 48 h and stained for β-tubulin IV (a marker for ciliated cells) or MUC5AC (a marker for goblet cells). (A) The first panel shows nuclei visualized by DAPI staining. The second panel shows ciliated cells stained for β-tubulin IV. Representative images are shown. (B) The number of ciliated cells was normalized to the number of total cells in the field of view. Each dot represents a single image. *P < 0.05 compared to control. N = 1–3 subjects. (C) The first panel shows nuclei visualized by DAPI staining. The second panel shows goblet cells stained for MUC5AC. Representative images are shown. (D) The number of goblet cells was normalized to the number of total cells in the field of view. Each dot represents a single image. *P < 0.05 compared to control. N = 3 subjects.