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

Rui Xiong,Ying Chen,Yue Wu,Xuefei Cao,Sheila M Healy,Priya Tripathi,Qiangen Wu,Hans Rosenfeldt,Kelly Davis,Matthew Bryant,Levan Muskhelishvili,Tao Chen

doi:10.1007/s00204-021-03008-0

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.

Highlights

Cigarette smoke (CS) is a complex mixture of over 7000 chemicals (USDHHS 2014)
Deposited particle mass on the Quartz Crystal Microbalance (QCM) for the low, mid, and high CS concentrations were recorded as 0.681 ± 29.37% relative standard deviation (RSD), 3.478 ± 19.24%RSD, and 10.54 ± 8.11%RSD μg/cm2 for each 40-min exposure, which is equivalent to a particle mass of 0.14–2.11 μg/cm2 per 3R4F cigarette
We integrated transcriptome analysis with pathophysiological measurements and conducted a comprehensive evaluation of the toxicity responses produced by whole CS in an in vitro human ALI airway tissue model

Summary

Introduction

Cigarette smoke (CS) is a complex mixture of over 7000 chemicals (USDHHS 2014). Of these chemicals, 93 have been established by FDA as Harmful or Potentially Harmful Constituents (HPHCs) in tobacco products and tobacco smoke, being identified as carcinogens, respiratory toxicants, cardiovascular toxicants, reproductive or developmental toxicants, and addictive agents (FDA 2012). Cigarette smoking adversely affects nearly every tissue in the body, the respiratory system, contributing to the initiation and progression of both malignant and nonmalignant lung diseases, such as lung cancer, asthma, and chronic obstructive pulmonary disease (COPD) (USDHHS 2014). Long-term CS exposure is the primary risk factor for developing the chronic obstructive pulmonary disease (COPD). Reactive oxygen/nitrogen species released by the epithelial and immune cells result in further oxidative damage, leading to perpetual oxidative stress and chronic inflammatory reactions in the lung. These molecular initiating events eventually cause the modification of tissue structure and function that are characteristic of COPD (Kirkham and Barnes 2013)

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Conclusion