Combined Exposure to Ragweed and House Dust Mite Exacerbates Airway Epithelial Barrier Dysfunction: A Multimodal...

Background and Objectives: Dysfunction of the airway epithelial barrier is increasingly recognized as an early pathogenic mechanism in allergic respiratory diseases. Although individual aeroallergens such as ragweed (RW) pollen and house dust mite (HDM) are known to impair epithelial integrity, the effects of combined exposure, more reflective of real-world conditions, remain insufficiently characterized. This study aimed to evaluate the impact of single versus combined allergen exposure on airway epithelial barrier function using a multimodal experimental approach. Materials and Methods: Differentiated normal human bronchial epithelial (NHBE) cells were exposed to RW (100 µg/mL), HDM (100 µg/mL), or a combined extract (RW + HDM; total 100 µg/mL). Barrier function under air-liquid interface conditions was assessed by transepithelial electrical resistance (TEER), while real-time cellular responses were evaluated using xCELLigence impedance monitoring. Structural alterations were examined by occludin-based immunofluorescence imaging, and transcriptional changes associated with epithelial stress and inflammation were analyzed by RT-qPCR. Results: Allergen exposure induced time- and concentration-dependent impairment of epithelial barrier function. Combined exposure resulted in the most pronounced and sustained reduction in TEER and impedance measurements. These functional changes were accompanied by disruption of tight junction organization and coordinated transcriptional modulation of genes involved in inflammatory and stress responses. Conclusions: Combined exposure to RW and HDM extracts induced more severe and persistent epithelial barrier dysfunction than individual allergens. These findings support the role of the airway epithelium as a central regulator of allergic airway disease and highlight barrier disruption as an early pathogenic event. The multimodal framework applied in this study provides an integrated platform for investigating epithelial responses to complex environmental exposures.