Human bronchial epithelial cells exposed in vitro to cigarette smoke at the air-liquid interface resemble bronchial epithelium from human smokers

Abstract

Organotypic culture of human primary bronchial epithelial cells is a useful in vitro system to study normal biological processes and lung disease mechanisms, to develop new therapies, and to assess the biological perturbations induced by environmental pollutants. Herein, we investigate whether the perturbations induced by cigarette smoke (CS) and observed in the epithelium of smokers' airways are reproducible in this in vitro system (AIR-100 tissue), which has been shown to recapitulate most of the characteristics of the human bronchial epithelium. Human AIR-100 tissues were exposed to mainstream CS for 7, 14, 21, or 28 min at the air-liquid interface, and we investigated various biological endpoints [e.g., gene expression and microRNA profiles, matrix metalloproteinase 1 (MMP-1) release] at multiple postexposure time points (0.5, 2, 4, 24, 48 h). By performing a Gene Set Enrichment Analysis, we observed a significant enrichment of human smokers' bronchial epithelium gene signatures derived from different public transcriptomics datasets in CS-exposed AIR-100 tissue. Comparison of in vitro microRNA profiles with microRNA data from healthy smokers highlighted various highly translatable microRNAs associated with inflammation or with cell cycle processes that are known to be perturbed by CS in lung tissue. We also found a dose-dependent increase of MMP-1 release by AIR-100 tissue 48 h after CS exposure in agreement with the known effect of CS on this collagenase expression in smokers' tissues. In conclusion, a similar biological perturbation than the one observed in vivo in smokers' airway epithelium could be induced after a single CS exposure of a human organotypic bronchial epithelium-like tissue culture.

Fig. 1.

Schematic representation of the whole cigarette smoke (CS) exposure system and the exposure chamber. In the Vitrocell VC10 smoking robot (A), 3R4F cigarettes produce discontinuously whole CS (15% CS vol/vol with synthetic air) at the rate of 1 puff/min per cigarette. CS then enters the dilution chamber (B) (35 ml/8 s), where it is mixed continuously with synthetic air (0.2 to 0.5 l/min). The flow of diluted CS passes then in the exposure chamber (C), where the cell culture inserts are placed just underneath the aerosol inlet (D) during the defined length of exposure. In a cell culture insert, normal human bronchial epithelial (NHBE) cells are grown and differentiated on a porous membrane support, allowing an air-liquid interface with warmed culture medium below the cells and a gaseous test atmosphere above them.

Fig. 2.

Morphology of primary organotypic culture of human bronchial epithelial cells showed after Hematoxylin/Eosin staining (A), Alcian Blue-Periodic Acid Schiff staining (B), and p63 immunostaining (C). As observed in vivo, this in vitro culture model forms a multilayered pseudo-stratified epithelium composed of ciliated cells (A, arrows indicating cilia), nonciliated cells (C, arrows indicating p63-negative nonciliated epithelial cells), goblet cells located on the apical side (B, asterisks), and basal cells (C, arrow heads indicating p63-positive basal cells). Scale bar equals 30 μm.

Fig. 3.

Venn diagrams representing the overlapping upregulated (A) and downregulated (B) genes present in the 4 in vivo smoking gene signatures used in the Gene Set Enrichment Analysis (GSEA). C: Venn diagram representing the overlapping samples (S, smokers; NS, nonsmokers) from 3 of the 4 in vivo datasets used in this study. No overlapping samples were found between the dataset GSE7895 and the other in vivo datasets. The following authors used the following genes: Shaykhiev et al. (56), GSE20257; Harvey et al. (19), GSE4498; Beane et al. (3), GSE7895; and Strulovici-Barel et al. (61), GSE19667.

Fig. 4.

In vitro/in vivo side-by-side heatmap for the leading edge (LE) genes. Heatmap of the fold changes for in vitro up- and downregulated LE genes are plotted side by side with extracted fold changes from in vivo studies [Shaykhiev et al. (56), Harvey et al. (19), Beane et al. (3)]. Fold changes from in vivo studies are obtained by taking the log2 of the gene expression ratio between smokers' samples (column of the matrix) against the average expression level from nonsmokers within the same study. AIR-100 (MatTek) fold changes are obtained similarly for each exposure time.

Fig. 5.

In vivo smoking gene signatures derived from airway epithelium transcriptomes are significantly enriched in CS-exposed AIR-100 tissue. The GSEA approach was used to assess the enrichment of genes derived from in vivo smoking gene signatures in the transcriptomic profiles of CS-exposed AIR-100 tissue after different postexposure time. Bar plots in A–J exhibit the normalized enrichment score computed for each postexposure time point. The gene set (GS) used to perform GSEA included smoking gene signatures from Harvey et al. (19) (A, B), Beane et al. (3) (C, D), Shaykhiev et al. (56) (E, F), and Strulovici-Barel et al. (61) (G, H). I and J correspond to GSEA performed with the low-CS-exposure signature. Low exposure (LE) vs. nonsmoker (NS). Each signature was split into upregulated (GS UP) and downregulated GS (GS DN). They were tested for enrichment independently against ranked gene expression profiles of CS-exposed AIR-100 tissue. Enrichments observed among the most significantly up- or downregulated genes in AIR-100 tissue were indicated by “AIR100 UP” and “AIR100 DN”. ***False discovery rate (FDR) ≤ 0.0002; **FDR ≤ 0.01; *FDR ≤ 0.05.

Fig. 6.

In vivo smoking cessation downregulated gene signature is found significantly enriched in the upregulated leading edge of CS-exposed AIR-100 tissue. The GSEA approach was again used to assess the enrichment of genes derived from in vivo smoking cessation gene signatures (former smokers vs. current smokers) in the transcriptomic profiles of CS-exposed AIR-100 tissue after different postexposure time. Two in vivo smoking cessation gene signatures [Beane et al. (3) (A, B) and Zhang et al. (71) (C, D)] were tested for enrichment independently against ranked gene expression profiles from AIR-100. Normalized enrichment score computed for each postexposure time points are represented in A–D bar plots. Both gene signatures were split into upregulated and downregulated GS (GS UP and GS DN). ***FDR ≤ 0.0002; **FDR ≤ 0.01; *FDR ≤ 0.05.

Fig. 7.

Comparison between in vivo human smoker miRNA signature [from Schembri et al. (54)] and CS-exposed AIR-100 in vitro miRNA dataset. A: vertical axis of the bar plot represents the best t-statistics (i.e., lowest negative or highest positive t-scores) obtained for the coefficients β_t or β_i of the linear model. The 14 miRNAs detected by both platforms and contributing to the in vivo signature (highlighted in green) are all downregulated upon CS exposure. B: heatmap showing the different response kinetics of 8 CS-induced miRNAs expressed in AIR-100 tissue that correspond to the most significant element of the in vivo miRNA smoking signature. Vertical color gradients, responses dependent on exposure time; black, absence of response; green, downregulation in CS-exposed AIR-100 tissue vs. control.

Fig. 8.

Measurement of the basolateral secretion of pro-matrix metalloproteinase 1 (MMP-1) protein 48 h after various CS or air exposure times (7, 14, 21, 28 min) of AIR-100 tissue. A significant dose-dependent increase in MMP-1 secretion was seen after 14 min, 21 min, and 28 min of exposure to whole CS (up to 14-fold for 28-min exposure time) compared with sham. *P < 0.01, 1-way ANOVA and Tukey's multiple-comparison post hoc test, means ± SE, N = 3 replicates.