A new thiol-independent mechanism of epithelial host defense against Pseudomonas aeruginosa: iNOS/NO^• sabotage of theft-ferroptosis

Ferroptosis is a redox-driven type of regulated cell death program arising from maladaptation of three metabolic pathways: glutathione homeostasis, iron handling and lipid peroxidation. Though GSH/Gpx4 is the predominant system detoxifying phospholipid hydroperoxides (PLOOH) in mammalian cells, recently Gpx4-independent regulators of ferroptosis like ferroptosis suppressor protein 1 (FSP1) in resistant cancer lines and iNOS/NO^• in M1 macrophages have been discovered. We previously reported that Pseudomonas aeruginosa (PA) utilizes its 15- lipoxygenase (pLoxA) to trigger ferroptotic death in epithelial cells by oxidizing the host arachidonoyl-phosphatidylethanolamine (ETE-PE) into pro-ferroptotic 15-hydroperoxy- arachidonyl-PE (15-HpETE-PE). Here we demonstrate that PA degrades the host GPx4 defense by activating the lysosomal chaperone-mediated autophagy (CMA). In response, the host stimulates the iNOS/NO^•-driven anti-ferroptotic mechanism to stymie lipid peroxidation and protect GPx4/GSH-deficient cells. By using a co-culture model system, we showed that macrophage-produced NO^• can distantly prevent PA stimulated ferroptosis in epithelial cells as an inter-cellular mechanism. We further established that suppression of ferroptosis in epithelial cells by NO^• is enabled through the suppression of phospholipid peroxidation, particularly the production of pro-ferroptotic 15-HpETE-PE signals. Pharmacological targeting of iNOS (NO^• generation) attenuated its anti-ferroptotic function. In conclusion, our findings define a new inter-cellular ferroptosis suppression mechanism which may represent a new strategy of the host against P. aeruginosa induced theft-ferroptosis.