## 4 Discussion
Interestingly, TJ remodeling in response to allergen exposure is partially mediated by the activation of mitogen-activated protein kinase (MAPK) signaling pathways, which regulate TJ protein expression and assembly [62]. This suggests that targeting MAPK signaling may be a potential therapeutic strategy for restoring barrier integrity in asthma. Our findings showing extensive TJ disorganization following combined RW pollen and HDM exposure highlight the need for further research into the signaling pathways underlying TJ remodeling. Our results highlight the need for therapeutic strategies that target epithelial barrier restoration in asthma. Current treatments, such as corticosteroids and bronchodilators, primarily address inflammation and airway hyperresponsiveness but do not directly repair barrier dysfunction [2; 4; 66]. The therapeutic implications of our findings are supported by several studies investigating strategies to restore epithelial barrier function in asthma. For example, antioxidants can attenuate allergen-induced oxidative stress and improve barrier integrity [36; 67; 68]. Protease inhibitors can block HDM-mediated TJ degradation and restore barrier function [16; 26; 27]. These studies suggest that combination therapies targeting both oxidative stress and protease activity could offer a more effective approach to ameliorating allergen-induced barrier disruption and preventing asthma exacerbations in individuals exposed to multiple allergens [69]. Additionally, the potential of TJ stabilizers, such as synthetic peptides that mimic TJ proteins, has been described for enhancing barrier integrity and preventing allergen penetration [6; 62]. Our findings showing severe barrier disruption following combined RW pollen and HDM exposure emphasize the importance of developing such targeted therapies to address the complex mechanisms underlying multi-allergen-induced barrier dysfunction. While our study provides novel insights into the combined effects of RW and HDM allergens on epithelial barrier integrity, several limitations should be acknowledged. Firstly, the ALI model, while physiologically relevant, does not fully replicate the complexity of the in vivo airway environment, including the presence of immune cells and stromal interactions [49]. Future studies incorporating co-culture systems or animal models may provide a more comprehensive understanding of allergen-induced barrier dysfunction. Secondly, our study focuses on acute allergen exposure, whereas chronic exposure may have different effects on barrier function and repair mechanisms. Longitudinal studies are needed to investigate the long-term impact of combined allergen exposure on epithelial integrity and asthma progression. While our study effectively assesses barrier integrity through TEER measurements and ZO-1 immunostaining, due to technical constraints, it does not include an analysis of downstream cytokine or alarm responses, such as TSLP, IL-33 or IL-6. The inclusion of cytokine and alarm profiling could have broadened the scope of our analysis and we plan to address these markers in future studies to better elucidate the relationship between barrier dysfunction and subsequent inflammatory signaling. Additionally, although endotoxin levels were not directly measured, dialysis of the allergen extracts in DPBS using a 3.5 kDa membrane likely facilitated the removal of small endotoxin fragments, thereby reducing the likelihood of endotoxin contamination influencing the results. Despite these limitations, our findings provide important insights into epithelial integrity in response to allergen exposure and lay the groundwork for further mechanistic investigations. Finally, while we identified synergistic effects between ragweed pollen and HDMs, further studies are needed to advance our understanding of the specific molecular mechanisms and their broader implications, as well as to identify potential therapeutic targets.