## 4 Discussion
We adhered to the MIRTA (Minimum Information for Reporting on the TEER Assay) recommendations, which provide guidelines for standardizing TEER assay procedures to ensure data consistency and reproducibility across studies. To comply with these standards, we maintained proper calibration of volt-ohm meters and electrodes and recorded the temperature throughout the experiment [56, 57]. Our findings demonstrate that combined RW and HDM exposure had the most pronounced impact on barrier disruption, followed by HDMs alone, with RW showing a slightly less severe but still significant effect. These outcomes highlight the importance of studying multi-allergen interactions, as they more accurately reflect real-world exposure scenarios and provide critical insights into asthma pathogenesis. The central outcome of this study was the synergistic effect of combined RW and HDM exposure on epithelial barrier integrity. While both allergen sources individually disrupted barrier function, their combined exposure resulted in significantly greater TEER reduction and disorganization of TJ proteins, particularly ZO-1, compared to either allergen alone. This synergistic effect aligns with recent studies, for instance, a study demonstratedthat co-exposure to multiple allergens significantly exacerbates airway inflammation and barrier dysfunction compared to single-allergen exposure [11]. Similarly, another study emphasized that multi-allergen exposure can lead to more severe asthma phenotypes due to overlapping inflammatory and barrier-disrupting mechanisms [58]. Also, [PERSON] et al. (2012) reinforced the concept that simultaneous exposure to multiple allergens can amplify inflammatory responses and compromise epithelial barrier integrity, potentially through interconnected yet mechanistically distinct pathways, suggesting a multifaceted interplay between immune activation and structural barrier dysfunction [9]. These studies support our observation that combined RW pollen and HDM exposure results in greater TEER reduction and TJ disorganization compared to individual allergens. Another study by [PERSON] et al. (2020) investigated the effects of combined grass pollen and HDM exposure on nasal epithelial cells and found that combined exposure led to greater barrier disruption and inflammatory cytokine release than either allergen alone [36]. While their study focused on the nasal epithelium, the mechanisms of barrier disruption are similar to those in the bronchial epithelium due to common TJ proteins and immune regulatory pathways. Our findings extend these observations to the bronchial epithelium, emphasizing the broader significance of multi-allergen interactions in allergic airway diseases. Specifically, we investigated the combined exposure to two of the most common allergens implicated in allergic sensitization: house dust mites (HDMs) from indoor environments and ragweet (RW) pollen from outdoor environments. The significant barrier disruption caused by HDMs alone in our study is consistent with numerous previous investigations. For example, studies demonstrated that HDM proteases, particularly Der p 1, Der p 2 and Der f 1, directly degrade TJ proteins such as ZO-1 and occludin, leading to increased paracellular permeability [24]. Similarly, [PERSON] et al. (2020) showed that HDM exposure disrupts epithelial barrier function by activating protease-activated receptor 2 (PAR-2), which leads to impaired TJ assembly [10]. Our TEER measurements and immunofluorescence staining for ZO-1 corroborate these findings, confirming that HDM exposure rapidly and severely compromises barrier integrity. HDMs are among the most significant allergens, producing cysteine and serine proteases that may alter the epithelial barrier [16]. The ability of HDM extracts to disrupt epithelial immune and barrier functions is closely linked to allergic sensitization. However, this effect does not appear to be reliant on only serine and cysteine protease activity, suggesting that other components or mechanisms within the extract contribute to epithelial dysfunction.