## 2 Materials and Methods
### Culture of Differentiated Bronchial Epithelium at Air-Liquid Interface
The cell cultures were exposed to the following allergen extracts or molecules and at different concentrations: RW extract 200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL; HDM extract 200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL; combined RW + HDM extracts in equal parts at 200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL (HR200), 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL (HR100), 50 \(\mathrm{\SIUnitSymbolMicro g}\)/mL (HR50) and 25 \(\mathrm{\SIUnitSymbolMicro g}\)/mL (HR25); combined Amb a allergen mixture corresponding to 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL of allergen extract (AM100; Amb a 1.01 at 10 \(\mathrm{\SISymbolMicro g}\)/mL, Amb a 11 at 5 \(\mathrm{\SISymbolMicro g}\)/mL and Amb a 12 at 2 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) [40]; combined Der p 1 + Amb a allergen mixture corresponding to 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL of allergen extract (DAM100; combined Der p 1 at 10 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, Amb a 1.01 at 10 \(\mathrm{\SISymbolMicro g}\)/mL, Amb a 11 at 5 \(\mathrm{\SIUnitSymbolMicro g}\)/mL and Amb a 12 at 2 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) [41]; combined Der p 1 + Amb a allergen mixture corresponding to 50 \(\mathrm{\SIUnitSymbolMicro g}\)/mL of allergen extract (DAM50). Allergens and allergen extracts were diluted in cell medium and medium alone was used as a negative control, while 5% Triton-X (Sigma Aldrich, St. Louis, MO, USA) served as a positive control. TEER values were recorded before allergen exposure, 30 min post-exposure, hourly for the first 4 h and daily for up to 72 h (Figure 1). Each condition was tested in triplicate through two sets of experiments, using only technical replicates, as the epithelial cells were sourced from the same batch. The sample size was determined based on standard practices in similar experimental designs to ensure reliable and reproducible measurements. The TEER measurement points prior to allergen addition served as the baseline for normalization. The normalized TEER was set at this point and subsequent changes in cell responses were measured relative to this baseline. Normalized TEER = (TEER\({}_{\mathrm{T}}\)/TEER\({}_{0}\)) \(\times\) 100  
where TEER\({}_{\mathrm{T}}\) is the transepithelial electrical resistance measured at each time point for each treatment and TEER\({}_{0}\) is the base transepithelial electrical resistance (measured before addition of allergens or control). The data are presented as the mean \(\pm\) standard deviation (SD). This approach provided valuable insights into barrier function dynamics and cellular responses under varying experimental conditions. The mechanisms underlying epithelial barrier dysfunction and the immune responses triggered in allergic airway inflammation are illustrated in Figure 1.  
Figure 1: The mechanisms of epithelial barrier dysfunction and immune response in allergic airway inflammation. This schematic illustrates the impact of allergens, such as house dust mites and rag-weed pollen, on airway epithelial integrity and the subsequent immune response. The organization of epithelial tight junctions, including ZO-1, occluding, claudins, junctional adhesion molecules (JAMs) and adherens junctions. Exposure to allergens leads to tight junction disruption, reducing epithelial barrier function. The central diagram illustrates allergen-induced epithelial activation and epithelial-derived cytokines (TSLP, IL-25, IL-33), promoting type 2 inflammation. This cascade leads to the activation of dendritic cells, innate lymphoid cells (ILC2) and Th2 cells, which secrete IL-4, IL-5, IL-6 and IL-13, driving eosinophilic inflammation, mucus hypersecretion and increased IgE production by plasma cells. The lower section illustrates immune cell trafficking and neuro-immune interactions, where sensory neurons release neuropeptides, like substance P (SP), calcitonin gene-related peptide (CGRP) and neuromodin U (NMU), that further amplify inflammation.