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

Julia A Gindele,Ronald G Crystal,Jürgen Schymeinsky,Karsten Quast,Michael Brendel,Haijun Zhang,Yael Strulovici-Barel,Matthew J Thomas,Christian T Wohnhaas,Kerstin Benediktus,Gerald Birk,Fabian Heinemann,Michelle Leblanc,Birgit Stierstorfer,Tobias Kiechle

doi:10.1038/s41598-020-63345-5

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. non-smoker) 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.

Highlights

Cigarette smoke (CS) is the leading risk factor to develop Chronic obstructive pulmonary disease (COPD)
To further understand the molecular mechanisms underlying small airway disease and airway remodeling in COPD a new in vitro protocol was established for the cultivation of primary small airway epithelial cells (SAEC) at air-liquid interface (ALI)
MUC5AC+ goblet cells are normally absent in the human small airways[35], an intermediate cell population of SCGB1A1+/MUC5AC+ double positive secretory cells and a few SCGB1A1−/MUC5AC+ goblet cells were detected in the ALI cultures (Fig. 1a)

Summary

Introduction

Cigarette smoke (CS) is the leading risk factor to develop COPD. 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. 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 COPD4,5 These changes of the small airway epithelium in COPD include: Goblet cell metaplasia[6,7,8], reduced cilia function[9,10,11,12,13], reduced club cell numbers[7,14,15], basal membrane thickening[16,17], epithelial barrier dysfunction[18,19,20] and squamous metaplasia[8,21,22,23]. 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[27,28,29,30,31,32,33,34]. How the in vitro model translates to the situation in vivo, the gene expression profiles of the ALI cultures were compared to the profiles of human small airway samples from healthy non-smokers and healthy smokers

Methods

Results

Conclusion