Early versus delayed dupilumab differentially restores IL-13–induced airway barrier dysfunction

BACKGROUND: Interleukin (IL)-13 promotes airway epithelial remodeling in asthma; however, the mechanisms by which IL-4/IL-13 blockade restores airway barrier function and how treatment timing alters epithelial cell states remain unclear. METHODS: Primary human bronchial epithelial cells were differentiated under air-liquid interface conditions and stimulated with IL-13. Dupilumab was added either concurrently or after 4 days. Airway barrier function was assessed using transepithelial electrical resistance (TEER), fluorescein isothiocyanate-dextran permeability, immunofluorescence, RT-qPCR, and ciliary beat frequency. Single-cell RNA sequencing with RNA velocity and cell type-stratified preranked gene set enrichment analyses were performed. RESULTS: IL-13 treatment triggered epithelial phenotypic remodeling, characterized by goblet cell expansion, and subsequently reduced ciliary activity, decreased TEER, and increased permeability. Early dupilumab treatment preserved TEER and attenuated permeability, whereas delayed dupilumab treatment resulted in partial TEER recovery with increased permeability. Single-cell analyses identified mucus-producing goblet and mucous ciliated states, and inflammatory parabasal clusters expressing alarmins and periostin. Delayed dupilumab treatment was associated with goblet cell subsets consistent with dedifferentiation-like transition toward a club cell-like state. TEER inversely correlated with MUC5AC expression, and asthmatic airway epithelium showed reduced TEER and disrupted occludin localization around goblet cells. Gene ontology analysis showed enrichment of cilium programs with early dupilumab treatment, whereas delayed dupilumab treatment favored mitochondrial respiration, ATP production, and protein homeostasis. CONCLUSIONS: Dupilumab ameliorates IL-13-induced airway barrier dysfunction in a timing-dependent manner. Single-cell analyses highlight epithelial plasticity and persistent inflammatory programs as key determinants of differential barrier recovery, providing mechanistic insights into variable clinical responses to dupilumab in asthma.