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

Abstract

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.

P. aeruginosa augments irradiation injury via 15-lipoxygenase–catalyzed generation of 15-HpETE-PE and induction of theft-ferroptosis

“…Further, we performed experiments in which intestinal epithelial cells (HIEC6 and Caco2) were coincubated with PAO1 strains expressing different levels of pLoxA: (a) wild type (PAO1-WT), (b) pLoxA overexpressing strain (PAO1-Δ wsp F) and (c) pLoxA-deficient strain (PAO1- loxA:Tn ) ( 12 , 27 ). We found that only pLoxA-containing strains of PAO1 (PAO1-WT and PAO1-Δ wsp F) induced ferroptosis, in sharp contrast to the pLoxA-deficient strain (PAO1- loxA:Tn ) ( Supplemental Figure 10 ) ( 12 , 27 , 28 ).…”

“…The selenoenzyme glutathione peroxidase 4 (GPx4) is the main endogenous antiferroptotic shield of the host that regulates ferroptosis by eliminating the accumulation of proferroptotic signals 1-SA-2-15-HpETE-PE ( 10 ). In our previous studies, we demonstrated that PAO1 triggers chaperone-mediated autophagy and elimination of GPx4 as a part of its proferroptotic strategy ( 28 ). Indeed, we found that ileal GPx4 expression and enzymatic activity were significantly lower in PAO1-treated mice versus control on day 4 ( Figure 4, B and D ).…”

“…Execution of ferroptosis requires sufficient amounts of substrates, phospholipids with PUFA residues regulated by phospholipid remodeling enzymes like acyl-coenzyme A synthetase long chain family member 4 (ACSL4) and lysophosphosphatidylcholine acyltransferase 3 (LPCAT3) ( 10 , 46 , 47 ). In our previous studies, we demonstrated that PAO1-induced theft-ferroptosis executed by the secreted pLoxA (a) hijacks the host phospholipid peroxidation pathway whereby ACSL4 and LPCAT3 act as important regulators of the ferroptotic response and (b) targets the host GPx4 degradation by activating chaperone-mediated autophagy pathway ( 12 , 28 ). Of note, the concept of theft-ferroptosis exploited as a pathogenic mechanism by P .…”

Relevance of Ferroptosis to Cardiotoxicity Caused by Anthracyclines: Mechanisms to Target Treatments

“…Using a co-culture model system, iNOS/NO ( • ) in M1 macrophages has been confirmed to inhibit the effect of NO ( • ) on epithelial cells by inhibiting phospholipid peroxidation, especially the generation of 15-HpETE-PE signal that promotes ferroptosis. It is an intercellular mechanism that distantly prevents the ferroptosis of epithelial cells stimulated by Pseudomonas aeruginosa ( 60 ).…”

Targeting Ferroptosis for Lung Diseases: Exploring Novel Strategies in Ferroptosis‐Associated Mechanisms

“…Further study suggested that PA can activate the lysosomal chaperone-mediated autophagy (CMA) to decrease the host GPX4 defense. Meanwhile, the host can stymie lipid peroxidation and protect GPX4/GSH-deficient cells by stimulating the inducible nitric oxide synthase (iNOS)/NO-driven antiferroptotic mechanism [ 195 ]. Therefore, promoting the iNOS/NO-driven antiferroptotic mechanism may provide a new strategy for the host against PA-induced ferroptosis.…”