Project Summary Bacterial endophthalmitis is a vision-threatening complication of eye surgeries and ocular trauma. The vision loss in endophthalmitis occurs due to uncontrolled inflammation-mediated retinal tissue damage. The long-term goal of our research has been to study the pathobiology of endophthalmitis and identify potential therapeutic targets for treatment. Our recent work using transcriptomics and metabolomics has uncovered the importance of cellular metabolism in regulating the innate immune response during experimental Staphylococcus aureus (SA) endophthalmitis. Notably, we observed significant impairment in the antioxidant glutathione peroxidase 4 (GPX4) signaling, which plays a crucial role in reducing lipid peroxide accumulation and preventing ferroptosis cell death. Ferroptosis, a newly discovered form of cell death linked to iron overload, is regulated by GPX4. Surprisingly, the role of the GPX4/Ferroptosis axis in ocular infections has remained unexplored. Based on our findings, we hypothesize that reduced GPX4 levels, combined with elevated iron levels during endophthalmitis, contribute to ferroptotic cell death in the retina. In support, our preliminary data show, downregulation of GPX4, an increased labile iron pool, excessive lipid peroxidation, and induced expression of ACSL4 in SA-infected retina and cultured cells. Here, we will employ mouse genetic tools and pharmacological interventions to elucidate the mechanisms underlying impaired GPX4 signaling (Aim 1), investigate the role of ACSL4 as the final executor of ferroptosis (Aim 2), and test potential of nanoformulations to enhance GPX4 levels and reduce ferroptosis as a novel approach to treat bacterial endophthalmitis (Aim 3). The knowledge gained from this study into the regulation of ferroptosis during endophthalmitis and the development of therapeutic strategies could have a significant impact not only on ocular infections but also on other eye diseases involving ferroptosis. Ultimately, our work aims to contribute to the advancement of therapeutic interventions in the field, offering new hope for preserving vision and improving outcomes in patients affected by these conditions.