Project Summary Abstract Macrophages contribute to ozone toxicity by regulating both the initiation and resolution of lung inflammation; these processes are mediated by distinct macrophage subpopulations broadly classified as proinflammatory/cytotoxic (M1) and anti-inflammatory/wound repair (M2) macrophages. M1 and M2 macrophage activation is controlled, in part, by intracellular metabolism; thus, while high glycolytic capacity is associated with M1 activity, increases in fatty acid oxidation and mitochondrial oxidative phosphorylation are required for M2 macrophage activation. New data suggest that ozone toxicity is due to impaired M2 activation and a failure to resolve inflammation, however, mechanisms are not known. A preliminary RNA-seq analysis of lung macrophages collected after ozone exposure revealed significant enrichment of the estrogen receptor signaling pathway among differentially expressed genes. This was associated with down-regulation of PPARγ expression, a transcription factor known to promote M2 phenotype and resolution of inflammation by shifting intracellular metabolism to fatty acid oxidation. Estrogen has been shown to regulate PPARγ expression by activating estrogen receptor alpha (ESR1) located at the cell membrane, and to promote macrophage anti- inflammatory activity by shifting metabolism to fatty acid oxidation; we speculate that this pathway is important in macrophage responses to ozone. We hypothesize that ozone interferes with extra-nuclear ESR1 signaling in macrophages and downstream activation of PPARγ; this leads to impaired fatty acid oxidation and M2 macrophage activation resulting in aberrant resolution of inflammation and increased tissue injury. Three aims are proposed to test this hypothesis. In the first two aims (K99 period of this award), we will analyze the effects of ozone on macrophage bioenergetics and the role for ESR1 signaling in macrophage immunometabolism and phenotypic activation. Aim 3 (R00 award period) will focus on 1) elucidation of mechanisms by which ozone interferes with extra-nuclear ESR1 signaling, 2) the role of extra- nuclear ESR1 in macrophage bioenergetics and phenotype in response to ozone and how this influences lung injury, and 3) developing strategies to mitigate ozone-induced lung injury by rescuing extra-nuclear ESR1 signaling. Results of these studies will provide novel data on mechanisms underlying macrophage responses to inhaled ozone and represent a significant departure from the co-primary mentor’s areas of emphasis. The career development plan will be performed at Rutgers University, a first-class institution with a strong training environment and will consist of technical training and professional development activities. These will provide the candidate with conceptual knowledge and training required to achieve his long-term goal of becoming an established investigator at an academic research institution directing an independent research program focused on elucidating ...