PROJECT SUMMARY Severe allergic asthma is marked by airway inflammation, remodeling, and hyperresponsiveness, which correlates with decreased lung function and increased mortality. We have identified the endoplasmic reticulum (ER) based unfolded protein response (UPR) as a critical mediator of lung epithelial inflammatory and fibrotic responses to allergens. Recent reports highlight that increases in the UPR pathways are potentially classified as an endotype of severe asthma. However, the mechanisms whereby the UPR response is initiated and perpetuated in settings of severe asthma remain unaddressed, and will be the focus of this application. Our novel preliminary results now suggest that allergen challenge increases reactive oxygen species (ROS) in the ER and oxidative modification of cysteines in GRP78. Oxidation of GRP78 resultes in dissociation of GRP78 and causes initial activation of the UPR transducer, ATF6. We observed that the ATF6 target, PDIA5, was also significantly upregulated in both asthmatics and a mouse asthma model in association with an enhanced allergic responses in lungs. Intriguingly, PDIA5 reduces cysteines disulfides to (-S-S-) to sulfhydryls (-SH) of ATF6, which is suggestive of a feed-forward regulatory mechanism to sustain the UPR. Based on these observations, we hypothesize that allergen-induced oxidation of GRP78 initiates the UPR signaling, with subsequent increases in PDIA5 prolong the UPR and thereby increasing allergic airway responses. To examine this hypothesis, we propose the following specific aims: In Specific Aim #1 we will determine the functional role of allergen-induced reactive oxygen species and subsequent oxidation of GRP78 in initiation of the UPR, expression of cytokines/chemokines, and subsequent induction of pro-inflammatory response and development of lung remodeling. The specific Aim #2 seeks to dissect the critical requirement of allergen-induced PDIA5 in disulfide mediated processing of a transducer of UPR ATF6 in severe allergic airway responses. In both aims we will use transgenic mouse models, cell culture and sensitive redox and biochemical assays. Most importantly we will examine the efficacy of specific inhibitors of ATF6α (Ceapin-A7) and PDIA5 (LOC14) in attenuating allergen-induced UPR, decreasing subsequent pro- inflammatory responses, and ultimately resulting in resolution of allergen-induced lung pathology. These studies will shed light on the importance of the allergen-induced lung epithelial UPR in pro-inflammatory responses, lung remodeling and offers insight into new and highly needed treatment modalities for severe allergic airway disease beyond supportive care.