Intermittent exposure to whole cigarette smoke alters the differentiation of primary small airway epithelial cells in the air-liquid interface culture

Abstract

Cigarette smoke (CS) is the leading risk factor to develop COPD. Therefore, the pathologic effects of whole CS on the differentiation of primary small airway epithelial cells (SAEC) were investigated, using cells from three healthy donors and three COPD patients, cultured under ALI (air-liquid interface) conditions. The analysis of the epithelial physiology demonstrated that CS impaired barrier formation and reduced cilia beat activity. Although, COPD-derived ALI cultures preserved some features known from COPD patients, CS-induced effects were similarly pronounced in ALI cultures from patients compared to healthy controls. RNA sequencing analyses revealed the deregulation of marker genes for basal and secretory cells upon CS exposure. The comparison between gene signatures obtained from the in vitro model (CS vs. air) with a published data set from human epithelial brushes (smoker vs. nonsmoker) revealed a high degree of similarity between deregulated genes and pathways induced by CS. Taken together, whole cigarette smoke alters the differentiation of small airway basal cells in vitro. the established model showed a good translatability to the situation in vivo. Thus, the model can help to identify and test novel therapeutic approaches to restore the impaired epithelial repair mechanisms in COPD, which is still a high medical need. Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide and its prevalence continues to rise 1. The main risk factor to develop COPD is cigarette smoke 2,3. Smoking induces epithelial injury and this repeated injury of the epithelium triggers a pathophysiologic response, which leads to tissue remodeling of the airways that is characteristic for COPD 4,5. These changes of the small airway epithelium in COPD include: Goblet cell metaplasia 6-8 , reduced cilia function 9-13 , reduced club cell numbers 7,14,15 , basal membrane thickening 16,17 , epithelial barrier dysfunction 18-20 and squamous metaplasia 8,21-23. Furthermore, the epithelial defense mechanisms against inhaled particles and pathogens are impaired enabling sub-epithelial penetration of pathogens that increases the risk of COPD patients suffering from bacterial and viral infections and subsequent exacerbations 24-26. To address cigarette smoke (CS)-induced damage on epithelial cells in vitro, previous studies used primary epithelial cells or cell lines that were mostly exposed to cigarette smoke extract (CSE) or to whole CS. These studies demonstrate smoke effects e.g. on epithelial barrier integrity, mucus production and cilia toxicity 27-34. The majority of these studies focus on the pathophysiology of large airways, i.e. bronchial or tracheal epithelial cells.

Effect of sub-chronic exposure to cigarette smoke, electronic cigarette and waterpipe on human lung epithelial barrier function

“…There is increasing recognition that CS can disrupt the epithelial barrier due to the toxicity of the mixture of chemicals acting through several molecular mechanisms that include MAPK (mitogen-activated protein kinase), TGFβ (transforming growth factor beta-1), and reactive oxygen species [ 25 – 29 ]. EC aerosol produced from e-liquid (propylene-glycol and glycerol) and smoke from TW contain many of the same toxicants found in CS; therefore, it is not surprising that similar pathways may be activated by exposure to EC and TW [ 10 , 30 – 33 ].…”

Small Airways Disease, Biomarkers and COPD: Where are We?

“…Further, cigarette smoke exposure appears to promote the expression of MUC5AC , and differentiation of goblet cells, as well as reduced expression of SCGB1A1 in club cells. 55 …”

Development of a miniaturized 96-Transwell air–liquid interface human small airway epithelial model

“…The ciliary beating of the respective hSAE cells was investigated after four weeks of ALI culture based on a recently published method 40 . Prior to cell imaging, cells were washed at the apical site three times with pre-warmed 1 × DPBS to remove mucus.…”

Influence of Hypoxia on the Epithelial-Pathogen Interactions in the Lung: Implications for Respiratory Disease

“…The epithelial barrier is critical in the innate host defense as a loss of barrier function increases the susceptibility of the host to infection and injury by pathogens and proteases (103). In addition, a pathological hallmark of several chronic inflammatory respiratory diseases, including asthma and COPD, is impaired epithelial barrier function and increased epithelial permeability, which may permit access for pathogens to the underlying submucosa (5,7,(104)(105)(106)(107). A number of factors have been implicated in epithelial barrier dysfunction including infection (108)(109)(110) and inhalation of noxious particles such as cigarette smoke (111).…”

Preclinical Development of Orally Inhaled Drugs (OIDs)—Are Animal Models Predictive or Shall We Move Towards In Vitro Non-Animal Models?

“…Also, they can replicate some of the key features that need to be kept into account when developing an inhalation therapy, namely (i) the constitution and thickness of the pulmonary lining fluid [67] and (ii) mucociliary clearance [60][61][62]. For example, ALI cultures have been used to model the effects of smoke exposure on epithelial cells [104] and the authors have created a complex, diseased ALI culture model capable of reproducing the chemoresistance mechanisms observed in patients affected by non-small-cell lung cancer [105,106]. Also, culturing human airway epithelial cells isolated from patients, makes it possible to conduct patient-specific research and drug-screening, for example in cystic fibrosis, asthma and COPD [107][108][109][110].…”