Smoking-Associated Disordering of the Airway Basal Stem/Progenitor Cell Metabotype

Abstract

The airway epithelium is a complex pseudostratified multicellular layer lining the tracheobronchial tree, functioning as the primary defense against inhaled environmental contaminants. The major cell types of the airway epithelium include basal, intermediate columnar, ciliated, and secretory. Basal cells (BCs) are the proliferating stem/progenitor population that differentiate into the other specialized cell types of the airway epithelium during normal turnover and repair. Given that cigarette smoke delivers thousands of xenobiotics and high levels of reactive molecules to the lung epithelial surface, we hypothesized that cigarette smoke broadly perturbs BC metabolism. To test this hypothesis, primary airway BCs were isolated from healthy nonsmokers (n = 11) and healthy smokers (n = 7) and assessed by global metabolic profiling by liquid chromatography-mass spectrometry. The analysis identified 52 significant metabolites in BCs differentially expressed between smokers and nonsmokers (P < 0.05). These changes included metabolites associated with redox pathways, energy production, and inflammatory processes. Notably, BCs from smokers exhibited altered levels of the key enzyme cofactors/substrates nicotinamide adenine dinucleotide, flavin adenine dinucleotide, acetyl coenzyme A, and membrane phospholipid levels. Consistent with the high burden of oxidants in cigarette smoke, glutathione levels were diminished, whereas 3-nitrotyrosine levels were increased, suggesting that protection of airway epithelial cells against oxidative and nitrosative stress is significantly compromised in smoker BCs. It is likely that this altered metabotype is a reflection of, and likely contributes to, the disordered biology of airway BCs consequent to the stress cigarette smoking puts on the airway epithelium.

Keywords: airway basal cells; cigarette smoke; metabolic profiling; oxidative stress; progenitor cells.

Figure 1.

Characterization of primary human nonsmoker and smoker airway basal cells. Immunohistochemical characterization of cytopreps of primary human airway basal cells isolated using selective culture methods from large airway epithelial samples obtained by bronchoscopy from healthy nonsmokers and healthy smokers. Shown are cytokeratin 5 (KRT5; basal cell), tetraspanin-24 (CD151; basal cell), secretoglobin family 1A member 1 (SCGB1A1; secretory cell), mucin 5AC (MUC5AC; secretory cell), β-tubulin IV (ciliated cell), and chromogranin A (CHGA; neuroendocrine cell). Scale bar, 20 μm. Data shown are representative images from a single primary donor sample obtained for each phenotype.

Figure 2.

Untargeted liquid chromatography–mass spectrometry metabolite profiling of basal cells from nonsmokers and smokers. (A) Retention time versus mass depicting 2,920 aligned features detected with positive ion monitoring and quantified across all basal cell (BC) samples. (B) Retention time versus mass depicting 952 features observed in at least 50% of BC samples from at least one group. (C) Principal component analysis (PCA) showing that BCs from the nonsmoking group (green) cluster separately from the smoking group (red). Each point corresponds to a single sample. Each sample is connected by a vector to the groups’ centroid, representing the geometric center of each axis for all samples in that group. (D) Unsupervised hierarchical clustering analysis, with samples color-coded by phenotype, displays expression patterns and clustering of 952 quantified BC metabolites. Each column represents a single subject BC sample; horizontal bars represent feature intensity depicted as a heat map, ranging from cold (blue) to hot (red).

Figure 3.

Smoking-affected metabolic pathways. Schematic diagram based on metabolic pathway analysis of differentially expressed metabolites between smokers and nonsmokers using Metaboanalyst software. Dominant pathways affected include redox reactions, cell membrane metabolism, energy metabolism, and enzyme cofactors and cofactor metabolites. CoA, coenzyme A; FAD, flavin adenine dinucleotide; NAD, nicotinamide adenine dinucleotide.

Figure 4.

Basal cell redox status expressed as glutathione/oxidized glutathione (GSH/GSSG) and 3-nitrotyrosine/tyrosine (3-NT/Tyr) ratios. (A) Relative amounts of GSH accumulated in BCs from smokers compared with nonsmokers. (B) Relative amounts of GSSG accumulated in BCs from smokers compared with nonsmokers. (C) GSH/GSSG ratio comparing smokers with nonsmokers was calculated on a subject-by-subject basis by measuring relative ion counts for each molecule in each subject. (D) Relative amounts of 3-NT accumulated in BCs from smokers compared with nonsmokers. (E) Relative amounts of unmodified tyrosine accumulated in BCs from smokers compared with nonsmokers. (F) The 3-NT/Tyr ratio comparing smokers to nonsmokers was calculated on a subject-by-subject basis by measuring relative ion counts for each molecule in each subject. For all panels, data are presented as mean ± SEM, with statistics calculated by an unpaired Student’s t test.