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.
Background: The human small airway epithelium (SAE) plays a central role in the early events in the pathogenesis of most inherited and acquired lung disorders. Little is known about the molecular phenotypes of the specific cell populations comprising the SAE in humans, and the contribution of SAE specific cell populations to the risk for lung diseases. Methods: Drop-seq single-cell RNA-sequencing was used to characterize the transcriptome of single cells from human SAE of nonsmokers and smokers by bronchoscopic brushing. Results: Eleven distinct cell populations were identified, including major and rare epithelial cells, and immune/inflammatory cells. There was cell type-specific expression of genes relevant to the risk of the inherited pulmonary disorders, genes associated with risk of chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis and (non-mutated) driver genes for lung cancers. Cigarette smoking significantly altered the cell type-specific transcriptomes and disease risk-related genes. Conclusions: This data provides new insights into the possible contribution of specific lung cells to the pathogenesis of lung disorders.
Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic fibrotic lung disease with an irreversible decline of lung function. “Bronchiolization”, characterized by ectopic appearance of airway epithelial cells in the alveolar regions, is one of the characteristic features in the IPF lung. Based on the knowledge that club cells are the major epithelial secretory cells in human small airways, and their major secretory product uteroglobin (SCGB1A1) is significantly increased in both serum and epithelial lining fluid of IPF lung, we hypothesize that human airway club cells contribute to the pathogenesis of IPF. By assessing the transcriptomes of the single cells from human lung of control donors and IPF patients, we identified two SCGB1A1 + club cell subpopulations, highly expressing MUC5B, a significant genetic risk factor strongly associated with IPF, and SCGB3A2, a marker heterogeneously expressed in the club cells, respectively. Interestingly, the cellular proportion of SCGB1A1 + MUC5B + club cells was significantly increased in IPF patients, and this club cell subpopulation highly expressed genes related to mucous production and immune cell chemotaxis. In contrast, though the cellular proportion did not change, the molecular phenotype of the SCGB1A1 + SCGB3A2 high club cell subpopulation was significantly altered in IPF lung, with increased expression of mucins, cytokine and extracellular matrix genes. The single cell transcriptomic analysis reveals the cellular and molecular heterogeneity of club cells, and provide novel insights into the biological functions of club cells in the pathogenesis of IPF.
The club cell, a small airway epithelial (SAE) cell, plays a central role in human lung host defense. We hypothesized that subpopulations of club cells with distinct functions may exist. The SAE of healthy nonsmokers and healthy cigarette smokers were evaluated by single-cell RNA sequencing, and unsupervised clustering revealed subpopulations of SCGCB1A1+KRT5loMUC5AC− club cells. Club cell heterogeneity was supported by evaluations of SAE tissue sections, brushed SAE cells, and in vitro air–liquid interface cultures. Three subpopulations included: (1) progenitor; (2) proliferating; and (3) effector club cells. The progenitor club cell population expressed high levels of mitochondrial, ribosomal proteins, and KRT5 relative to other club cell populations and included a differentiation branch point leading to mucous cell production. The small proliferating population expressed high levels of cyclins and proliferation markers. The effector club cell cluster expressed genes related to host defense, xenobiotic metabolism, and barrier functions associated with club cell function. Comparison of smokers vs. nonsmokers demonstrated that smoking limited the extent of differentiation of all three subclusters and altered SAM pointed domain-containing Ets transcription factor (SPDEF)-regulated transcription in the effector cell population leading to a change in the location of the branch point for mucous cell production, a potential explanation for the concomitant reduction in effector club cells and increase in mucous cells in smokers. These observations provide insights into both the makeup of human SAE club cell subpopulations and the smoking-induced changes in club cell biology.
Rationale. Club cells, a small airway epithelium (SAE) secretory cell that expresses SCGB1A1 but not KRT5 (basal cell marker) or MUC5AC (goblet cell marker), play a central role in host defense of the human lung. In healthy, non-smoker SAE, club cell differentiation begins with basal stem/progenitor cells and proceeds through an intermediate cell phase before arriving at the mature club cell. In addition to their role in expressing host defense genes, club cells give rise to MUC5AC-positive goblet cells. Based on data demonstrating that ∼50% club cells express MUC5B, a secretory mucin critical for mucociliary clearance, we hypothesized that there may be subpopulations of club cells, each with distinct functions. Methods. The SAE was sampled from normal nonsmokers and healthy smokers by bronchoscopy and brushing. Using single cell
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