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.

Redox lipid reprogramming commands susceptibility of macrophages and microglia to ferroptotic death.

Ferroptotic death is the penalty for losing control over three processes-iron metabolism, lipid peroxidation and thiol regulation-that are common in the pro-inflammatory environment where professional phagocytes fulfill their functions and yet survive. We hypothesized that redox reprogramming of 15-lipoxygenase (15-LOX) during the generation of pro-ferroptotic signal 15-hydroperoxy-eicosa-tetra-enoyl-phosphatidylethanolamine (15-HpETE-PE) modulates ferroptotic endurance. Here, we have discovered that inducible nitric oxide synthase (iNOS)/NO•-enrichment of activated M1 (but not alternatively activated M2) macrophages/microglia modulates susceptibility to ferroptosis. Genetic or pharmacologic depletion/inactivation of iNOS confers sensitivity on M1 cells, whereas NO• donors empower resistance of M2 cells to ferroptosis. In vivo, M1 phagocytes, in comparison to M2 phagocytes, exert higher resistance to pharmacologically induced ferroptosis. This resistance is diminished in iNOS-deficient cells in the pro-inflammatory conditions of brain trauma or the tumour microenvironment. The nitroxygenation of eicosatetraenoyl (ETE)-PE intermediates and oxidatively truncated species by NO• donors and/or suppression of NO• production by iNOS inhibitors represent a novel redox mechanism of regulation of ferroptosis in pro-inflammatory conditions.