## 4 Discussion
Understanding these alternative pathways may provide new insights into how HDM exposure promotes allergic responses and barrier impairment [44]. Der p1 is the primary allergenic protease, disrupting the barrier by cleaving occludin and claudin, degrading ZO-1, reducing TEER and increasing permeability [18; 59; 60]. On the other hand, another study found that HDM-induced barrier disruption is partially reversible upon allergen removal, suggesting that epithelial cells retain some capacity for repair [59; 61]. Our study found that RW alone caused a smaller TEER reduction compared to HDMs but still significantly impaired barrier integrity. The combination of RW and HDM allergens, however, resulted in the most severe disruption, suggesting that the distinct mechanisms of action of these allergens interact synergistically to exacerbate barrier dysfunction. Unlike HDMs, RW may not directly degrade TJ proteins but induces oxidative stress and inflammatory responses that compromise barrier integrity over time [62]. This is consistent with another study which compared the effects of seasonal (pollen) and perennial (HDM) allergens on epithelial barrier function and found that pollen-induced barrier disruption is generally less severe but more prolonged compared to HDM-induced disruption [63]. RW is known to induce oxidative stress and inflammatory cytokine release, which can weaken TJ integrity indirectly. In contrast, HDMs contain proteases that directly degrade TJ proteins such as ZO-1 and occludin. When combined, these mechanisms likely interact to amplify barrier dysfunction. Our IF staining revealed extensive fragmentation and redistribution of ZO-1 in cells exposed to both allergens, suggesting that the combined exposure not only degrades TJ proteins but also impairs their ability to reorganize and maintain barrier function. This finding is consistent with studies who demonstrated that HDM proteases disrupt TJ assembly, while oxidative stress from RW further inhibits repair mechanisms [36]. The synergistic effects of combined RW and HDM exposure have important implications for asthma pathogenesis. Epithelial barrier dysfunction is a critical early event in asthma development, as it facilitates the penetration of allergens and pathogens, leading to heightened immune activation and chronic inflammation [10; 64]. Our findings suggest that individuals exposed to multiple allergens, such as RW pollen and HDMs, may be at greater risk of developing severe asthma due to compounded barrier disruption. This is particularly relevant in urban environments, where individuals are often exposed to a combination of seasonal and perennial allergens, as well as to pollution, which serves as an exacerbating factor that amplifies allergic responses [12]. Moreover, the differential effects of RW and HDMs on barrier integrity may explain the heterogeneity observed in asthma phenotypes. For example, patients with perennial asthma, often associated with HDM sensitivity, may experience more severe barrier dysfunction compared to those with seasonal asthma linked to ragweed pollen exposure [37; 64; 65]. However, the combined exposure scenario, which is more reflective of real-world conditions, likely contributes to the development of mixed or severe asthma phenotypes characterized by persistent inflammation and remodeling. Our findings on TJ remodeling following allergen exposure are consistent with several studies investigating the molecular mechanisms of TJ disruption. HDM proteases induce the redistribution of ZO-1 from the cell membrane to the cytoplasm, leading to TJ disassembly [24]. Similarly, oxidative stress from pollen exposure disrupts the cytoskeletal organization necessary for TJ maintenance [36]. IF staining for ZO-1 revealed similar patterns of TJ disorganization following RW and HDM exposure, supporting these mechanisms.