## 2 Materials and Methods
### Culture of Differentiated Bronchial Epithelium at Air-Liquid Interface  
Normal human bronchial epithelial cells (NHBE; Bronchial Epi Cells for B-ALI; Lonza Walkersville Inc., Walkersville, MD, USA) were used to establish an in vitro model of a differentiated bronchial epithelium. This model aimed to replicate the properties of a normal bronchial epithelium, including tight junctions, apical microvilli and regulated ion transport. Cells were cultured according to the manufacturer's protocol with adjustments based on previous studies. Initially, NHBE cells were expanded in T75-treated cell culture flasks (Corning, New York, NY, USA) and were incubated at 37 \({}^{\circ}\)C in a humidified atmosphere containing 5% CO\({}_{2}\). The cells were cultured in PneumaCultTM-ALI culture media (StemcellTM Technologies, Vancouver, BC, Canada), supplemented with the necessary growth factors. Upon reaching 70-90% confluence, cells were trypsinized, counted and seeded in specific plates at densities of 1000 cells/mm\({}^{2}\) on 12-well Transwell inserts (CostarB), Corning, New York, NY, USA) with 0.4 \(\upmu\)m pore membranes. The inserts were pre-coated with 100 \(\upmu\)L of fibronectin (20 \(\upmu\)g/mL) before seeding. In the Transwell system, NHBE cells underwent a three-phase culture process over 24 days: (1) expansion at a liquid-liquid interface to establish a confluent monolayer (the first three days), (2) transition to an air-liquid interface to simulate physiological bronchial conditions and (3) differentiation into a pseudostratified, ciliated epithelium. Medium in the basal chamber was changed every 2 days, while the apical chamber remained exposed to air to mimic in vivo respiratory conditions. From the second week onward, excess mucus was gently removed using periodic PBS washes. This protocol enabled the formation of a structurally and functionally intact respiratory epithelium. Throughout the culture period,cell morphology, confluence and epithelial barrier integrity were carefully monitored to ensure successful differentiation. Transepithelial Electrical Resistance (TEER) Measurement for the Assessment of Epithelial Barrier Integrity and Allergen-Induced Responses  
The successful establishment of the air-liquid interface facilitated the development of a structurally and functionally differentiated bronchial epithelium which was subsequently exposed to either allergen fragments or allergen extracts. Transepithelial electrical resistance measurements were performed at regular intervals using an epithelial Ohmvoltmeter (Evom2, World Precision Instruments, Sarasota, FL, USA) by placing electrodes in both the apical and basal compartments of the Transwell system. A low-voltage alternating current was applied to determine the resistance across the epithelial monolayer, with values expressed in Ohm\(\cdot\)cm\({}^{2}\) (\(\Omega\cdot\)cm\({}^{2}\)) to account for membrane surface area. This method provided a real-time, non-invasive evaluation of barrier function dynamics under different experimental conditions. Allergen exposures included different concentrations of HDM and ragweed pollen extracts, as well as mixtures containing purified allergen molecules (Amb a 1, Amb a 11, Amb a 12 and Der p 1).