PROJECT SUMMARY Significance. Pulmonary inflammation, which contributes toward the severity of lung diseases, is markedly exacerbated by obesity. Thus, there is a need for novel therapeutic approaches for targeting pulmonary inflammation in obesity. One promising strategy involves increased dietary consumption of docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid, which exerts anti-inflammatory properties. The first step toward developing DHA as a therapeutic for pulmonary inflammation in obesity is to establish the cellular targets and mechanisms of this unique fatty acid. One key target of DHA is alveolar macrophages (AMs), which are critical for maintaining respiratory homeostasis and drive pulmonary inflammation in obesity. Based on strong preliminary data, we propose the central hypothesis that DHA targets AMs to improve the inflammatory response through two key mechanisms. The first mechanism, tested in Aim 1, involves DHA esterification into plasma membrane phospholipids of AMs and thereby controlling the biophysical organization of sphingolipid/cholesterol-enriched lipid rafts to lower inflammatory signaling in obese mice. The second mechanism, tested in Aim 2, involves DHA displacing arachidonic acid (AA) in the phospholipidome. AA is an omega-6 PUFA which exerts pro-inflammatory effects through its conversion to hydroxylated derivatives such as prostaglandins. Displacing AA with DHA would shift the balance from pro-inflammatory AA derivatives in obesity to anti-inflammatory and pro-resolution DHA derivatives. One such DHA derivative is resolvin D1 (RvD1). RvD1 binds the G-protein coupled receptor ALX/FPR2 and decreases inflammation by driving macrophages to have an anti-inflammatory phenotype. To test the central hypothesis, the applicant will rely on advanced imaging tools, biochemical assays, and knockout mouse models including the innovative use of a newly generated myeloid specific DHA-deficient mouse. Impact: The proposed studies will establish mechanisms by which DHA improves pulmonary inflammation in obese mice and inform future clinical studies. This research will provide the applicant with skills in developing and implementing rigorous study design, use of advanced laboratory techniques (cutting-edge biophysical microscopy methods, flow cytometry, and biochemical assays), foster interdisciplinary collaboration, and enhance leadership skills through writing, project management, and mentoring. Ultimately, this will build the foundation for a successful biomedical research career.