PROJECT SUMMARY/ABSTRACT Wildfires continue to adversely impact public health in the United States and across the world, with increasing prevalence of wildfires due to climate change. Evidence demonstrates that wildfire smoke is associated with asthma exacerbation, decreased lung function, and altered immune responses, in addition to other pulmonary health outcomes. However, the molecular mechanisms driving wildfire health effects, including those that underly susceptibility of asthmatics, remain understudied. We have evidence that extraceullar vesicles (EVs), an emerging mechanism of toxicity, likely play significant roles in mediating the health effects of wildfire smoke. These changes have been observed alongside alterations in proteins associated with the the hypoxia inducible factor 1 subunit alpha (HIF-1⍺) pathway in the lung, representing a key pathway underlying ashtma pathophysiology. However, the role of EVs in influencing preexisting disease susceptibility factors to wildfire smoke exposure health effects remains to be studied. This study is specifically designed to test the novel hypothesis that canonical HIF-1⍺ pathway proteins will be enhanced in EVs both at baseline in asthmatics and following wildfire smoke exposure, with inhibition of HIF-1⍺ resulting in attenuation of inflammatory effects. We will test this hypothesis through two specific aims (SA): SA1 (in vitro) will examine whether EV release and content associated with activation of the HIF-1⍺ hypoxia pathway differentiate responsivity of epithelial cells from asthmatics and non-asthmatics to wildfire-relevant biomass smoke condensate and SA2 (in vivo) will examine whether EV release and content associated with activation of the HIF-1⍺ hypoxia pathway differentiate responsivity of asthmatics and non-asthmatic human subjects exposed to wildfire-relevant biomass smoke. To investigate this mechanism in vitro, we will expose differentiated primary human lung cells from asthmatic and non-asthmatic donors to wildfire-relevant biomass condensate with and without HIF-1⍺ inhibition in SA1. We will then analyze cellular transcriptomic and secreted extracellular vesicle proteomic profiles to identify significant differences between disease groups and treatment conditions. Leveraging human clinical samples, we will isolate EVs from induced sputum from human subjects (asthmatic and non-asthmatic) exposed to biomass smoke and assess EV proteomic signatures from bulk and cell-type-specific EVs in SA2. In both aims, data will be analyzed using both standard between group statistical tests and advanced predictive modeling through machine learning to uncover critical mediators of wildfire smoke susceptibility. Data from the two aims will be integrated to provide highly translational, innovative insight to understand mechanisms of wildfire smoke health effects, highlighting therapeutic targets and exposure biomarkers to leverage in the alleviation of wildfire-associated morbidity and mortality...