Abstract
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.
Highlights
The small airway epithelium (SAE) plays a central role in the early events in the pathogenesis of smoking-related lung disorders and is the first site of smoking-induced alterations in the lung[1,2,3,4,5,6]
In order to determine whether club cells in the small airway constitute a homogenous or heterogenous population and to determine whether cigarette smoking caused changes to one or more club cell subtypes, SAE of normal nonsmoker and healthy smoker lungs were sampled by bronchoscopy and brushing, and single cell transcriptomics was utilized to identify and characterize heterogeneity in the SCGB1A1+ club cell population
Expressed gene analysis led to the identification of three club cell categories: progenitor, effector, and proliferating club cells, with a proposed differentiation pathway of the progenitor club cells → effector club cells → proliferating club cells
Summary
The small airway epithelium (SAE) plays a central role in the early events in the pathogenesis of smoking-related lung disorders and is the first site of smoking-induced alterations in the lung[1,2,3,4,5,6]. Smoking causes changes in SAE basal cell differentiation with increased goblet cell numbers and decreased club and ciliated cells[30]. In order to determine whether club cells in the small airway constitute a homogenous or heterogenous population and to determine whether cigarette smoking caused changes to one or more club cell subtypes, SAE of normal nonsmoker and healthy smoker lungs were sampled by bronchoscopy and brushing, and single cell transcriptomics was utilized to identify and characterize heterogeneity in the SCGB1A1+ club cell population. The proportion of the differentiated club cells of the effector subpopulation was decreased and the proportion of mucous cells was increased with cigarette smoking, a finding that was linked to disrupted SAM pointed domain-containing Ets transcription factor (SPDEF) signaling in the effector cell population. The presence of club cell heterogeneity suggests that specific pharmacologic targeting could be possible, leading to direct therapies to increase effector club cell numbers and overall club cell function in cigarette smokers
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