Integration of transcriptome analysis with pathophysiological endpoints to evaluate cigarette smoke toxicity in an in vitro human airway tissue model

Abstract

Exposure to cigarette smoke (CS) is a known risk factor in the pathogenesis of smoking-caused diseases, such as chronic obstructive pulmonary diseases (COPD) and lung cancer. To assess the effects of CS on the function and phenotype of airway epithelial cells, we developed a novel repeated treatment protocol and comprehensively evaluated the progression of key molecular, functional, and structural abnormalities induced by CS in a human in vitro air–liquid-interface (ALI) airway tissue model. Cultures were exposed to CS (diluted with 0.5 L/min, 1.0 L/min, and 4.0 L/min clean air) generated from smoking five 3R4F University of Kentucky reference cigarettes under the International Organization for Standardization (ISO) machine smoking regimen, every other day for 4 weeks (3 days per week, 40 min/day). By integrating the transcriptomics-based approach with the in vitro pathophysiological measurements, we demonstrated CS-mediated effects on oxidative stress, pro-inflammatory cytokines and matrix metalloproteinases (MMPs), ciliary function, expression and secretion of mucins, and squamous cell differentiation that are highly consistent with abnormalities observed in airways of smokers. Enrichment analysis on the transcriptomic profiles of the ALI cultures revealed key molecular pathways, such as xenobiotic metabolism, oxidative stress, and inflammatory responses that were perturbed in response to CS exposure. These responses, in turn, may trigger aberrant tissue remodeling, eventually leading to the onset of respiratory diseases. Furthermore, changes of a panel of genes known to be disturbed in smokers with COPD were successfully reproduced in the ALI cultures exposed to CS. In summary, findings from this study suggest that such an integrative approach may be a useful tool for identifying genes and adverse cellular events caused by inhaled toxicants, like CS.

Cellular and Molecular Signatures of Oxidative Stress in Bronchial Epithelial Cell Models Injured by Cigarette Smoke Extract

“…The oxidative status of the cell can also be evaluated by measuring reduced glutathione (GSH) levels, which are decreased under oxidative stress conditions. GSH is measured in cell lysates using spectrophotometry [25,26,28,31,33,41,53,84,85,89,97], liquid chromatography coupled to mass spectrometry (LC-MS) [97], enzymatic methods [51] and fluorescence [87]. Custom-made or commercially available kits can be used [42,44].…”

“…Oxidative stress is also responsible for protein carbonylation. The intracellular level of protein carbonyls can be detected either by ELISA [97] or immunoblotting assays [10]. Other products of oxidative stress (3-nitrotyrosine (3-NT), 8-isoprostane and 8-hydroxydeoxyguanosine) can be detected using enzymatic immunoassays [44,88,90].…”

“…In addition, 16HBE cells can be grown in an ALI model where they are able to form cilia [67]. The complexity of ALI cultures can be increased by including human fibroblasts, immunocompetent cells or extracellular matrix components such as fibronectin or collagen [7][8][9]39,[97][98][99][100][101][102][103].…”

“…In bronchial epithelial cells, CSE reduces short-and long-term cell proliferation, increases β-galactosidase activity and decreases the expression and activation of anti-aging Sirt1, a NAD + -dependent histone deacetylase [82]. Sirt1 protects against senescence via regulating FoxO3 activity, p53, p-21, NF-κB, histones and a variety of proteins involved in DNA damage repair [29,97]. Deacetylation of FoxO3 by Sirt1 favors the expression of antioxidant genes and negatively regulates pro-survival genes, such as survivin.…”

The Current Status of Cigarette Smoking and Air Pollution Associated Lung Cancer

“…A recent study reported that ACE2 was significantly overexpressed in the bronchial and alveolar epithelial cells in smokers, independent of age or gender, indicating that CS related ACE2 overexpression may assist the SARS-CoV-2 infection, and smoking cessation may potentially attenuate the risk of COVID-19 transmission (173). An in vitro air-liquid-interface (ALI) human airway tissue model was successfully developed to assess the effects of CS on the function and phenotype of airway epithelial cells in smokers, including xenobiotic metabolism, oxidative stress, and inflammatory responses (174).…”