Epigenetic Dysregulation of the NKX2 ‐1/ SPDEF Axis Drives Persistent Goblet Cell Differentiation and Epithelial Barrier Dysfunction in Chronic Obstructive Pulmonary Disease

BACKGROUND AND OBJECTIVE: Despite improved respiratory symptoms after smoking cessation, patients with chronic obstructive pulmonary disease (COPD) remain susceptible to exacerbations and persistent airway inflammation, wherein the underlying mechanisms for sustained inflammation remain unclear. To address this knowledge gap, we investigated the persistence of airway epithelial barrier dysfunction in ex-smokers with COPD and examined the relationship between goblet cell hyperplasia and barrier dysfunction. METHODS: We analysed differentiated primary bronchial epithelial cells from never smokers with normal lung function, ex-smokers (> 10-year cessation), and current smokers with COPD using RNA sequencing, ATAC sequencing, and single-cell analyses to examine barrier function and cell differentiation. RESULTS: Genes associated with ciliary formation and motility were progressively downregulated from never smokers to ex-smokers to current smokers with COPD. The expression of junction-associated molecules was decreased in both ex-smokers and current smokers, showing a significant inverse correlation with the proportion of MUC5AC-positive cells. Single-cell analyses revealed distinct alterations in cell differentiation trajectories, particularly persistent goblet cell hyperplasia associated with increased expression of the transcription factor SPDEF, linked to epigenetic changes in the NKX2-1 gene regulatory regions. CONCLUSION: Epigenetic mechanisms maintain persistent alterations in airway epithelial differentiation after smoking cessation, leading to mucus-producing cell hyperplasia through dysregulation of the NKX2-1/SPDEF axis. This hyperplasia correlates with reduced junction-associated molecule expression and subsequent barrier dysfunction. Therapeutic strategies targeting epithelial barrier restoration and/or normalisation of epigenetic dysregulation may benefit patients with COPD, even after smoking cessation.