AbstractBackground and ObjectiveSmoking disturbs the bronchial‐mucus‐barrier. This study assesses the cellular composition and gene expression shifts of the bronchial‐mucus‐barrier with smoking to understand the mechanism of mucosal damage by cigarette smoke exposure. We explore whether single‐cell‐RNA‐sequencing (scRNA‐seq) based cellular deconvolution (CD) can predict cell‐type composition in RNA‐seq data.MethodsRNA‐seq data of bronchial biopsies from three cohorts were analysed using CD. The cohorts included 56 participants with chronic obstructive pulmonary disease [COPD] (38 smokers; 18 ex‐smokers), 77 participants without COPD (40 never‐smokers; 37 smokers) and 16 participants who stopped smoking for 1 year (11 COPD and 5 non‐COPD‐smokers). Differential gene expression was used to investigate gene expression shifts. The CD‐derived goblet cell ratios were validated by correlating with staining‐derived goblet cell ratios from the COPD cohort. Statistics were done in the R software (false discovery rate p‐value < 0.05).ResultsBoth CD methods indicate a shift in bronchial‐mucus‐barrier cell composition towards goblet cells in COPD and non‐COPD‐smokers compared to ex‐ and never‐smokers. It shows that the effect was reversible within a year of smoking cessation. A reduction of ciliated and basal cells was observed with current smoking, which resolved following smoking cessation. The expression of mucin and sodium channel (ENaC) genes, but not chloride channel genes, were altered in COPD and current smokers compared to never smokers or ex‐smokers. The goblet cell‐derived staining scores correlate with CD‐derived goblet cell ratios.ConclusionSmoking alters bronchial‐mucus‐barrier cell composition, transcriptome and increases mucus production. This effect is partly reversible within a year of smoking cessation. CD methodology can predict goblet‐cell percentages from RNA‐seq.