ABSTRACT
Ferroptosis is a form of programmed cell death that is pathogenic to several acute and chronic diseases and executed via oxygenation of polyunsaturated phosphatidylethanolamines (PE) by 15-lipoxygenases (15-LO) that normally use free polyunsaturated fatty acids as substrates. Mechanisms of the altered 15-LO substrate specificity are enigmatic. We sought a common ferroptosis regulator for 15LO. We discovered that PEBP1, a scaffold protein inhibitor of protein kinase cascades, complexes with two 15LO isoforms, 15LO1 and 15LO2, and changes their substrate competence to generate hydroperoxy-PE. Inadequate reduction of hydroperoxy-PE due to insufficiency or dysfunction of a selenoperoxidase, GPX4, leads to ferroptosis. We demonstrated the importance of PEBP1-dependent regulatory mechanisms of ferroptotic death in airway epithelial cells in asthma, kidney epithelial cells in renal failure, and cortical and hippocampal neurons in brain trauma. As master regulators of ferroptotic cell death with profound implications for human disease, PEBP1/15LO complexes represent a new target for drug discovery.
Subject(s)
Acute Kidney Injury/pathology , Asthma/pathology , Brain Injuries, Traumatic/pathology , Cell Death , Phosphatidylethanolamine Binding Protein/metabolism , Acute Kidney Injury/metabolism , Animals , Apoptosis , Asthma/metabolism , Brain Injuries, Traumatic/metabolism , Cell Death/drug effects , Cell Line , Humans , Isoenzymes/metabolism , Lipoxygenase/chemistry , Lipoxygenase/metabolism , Mice , Models, Molecular , Oxazolidinones/pharmacology , Oxidation-Reduction , Phosphatidylethanolamine Binding Protein/chemistryABSTRACT
Ferroptosis is a non-apoptotic form of regulated cell death that is triggered by the discoordination of regulatory redox mechanisms culminating in massive peroxidation of polyunsaturated phospholipids. Ferroptosis inducers have shown considerable effectiveness in killing tumour cells in vitro, yet there has been no obvious success in experimental animal models, with the notable exception of immunodeficient mice1,2. This suggests that the effect of ferroptosis on immune cells remains poorly understood. Pathologically activated neutrophils (PMNs), termed myeloid-derived suppressor cells (PMN-MDSCs), are major negative regulators of anti-tumour immunity3-5. Here we found that PMN-MDSCs in the tumour microenvironment spontaneously die by ferroptosis. Although decreasing the presence of PMN-MDSCs, ferroptosis induces the release of oxygenated lipids and limits the activity of human and mouse T cells. In immunocompetent mice, genetic and pharmacological inhibition of ferroptosis abrogates suppressive activity of PMN-MDSCs, reduces tumour progression and synergizes with immune checkpoint blockade to suppress the tumour growth. By contrast, induction of ferroptosis in immunocompetent mice promotes tumour growth. Thus, ferroptosis is a unique and targetable immunosuppressive mechanism of PMN-MDSCs in the tumour microenvironment that can be pharmacologically modulated to limit tumour progression.
Subject(s)
Neoplasms , Humans , Mice , Animals , Tumor MicroenvironmentABSTRACT
Programmed ferroptotic death eliminates cells in all major organs and tissues with imbalanced redox metabolism due to overwhelming iron-catalyzed lipid peroxidation under insufficient control by thiols (Glutathione (GSH)). Ferroptosis has been associated with the pathogenesis of major chronic degenerative diseases and acute injuries of the brain, cardiovascular system, liver, kidneys, and other organs, and its manipulation offers a promising new strategy for anticancer therapy. This explains the high interest in designing new small-molecule-specific inhibitors against ferroptosis. Given the role of 15-lipoxygenase (15LOX) association with phosphatidylethanolamine (PE)-binding protein 1 (PEBP1) in initiating ferroptosis-specific peroxidation of polyunsaturated PE, we propose a strategy of discovering antiferroptotic agents as inhibitors of the 15LOX/PEBP1 catalytic complex rather than 15LOX alone. Here we designed, synthesized, and tested a customized library of 26 compounds using biochemical, molecular, and cell biology models along with redox lipidomic and computational analyses. We selected two lead compounds, FerroLOXIN-1 and 2, which effectively suppressed ferroptosis in vitro and in vivo without affecting the biosynthesis of pro-/anti-inflammatory lipid mediators in vivo. The effectiveness of these lead compounds is not due to radical scavenging or iron-chelation but results from their specific mechanisms of interaction with the 15LOX-2/PEBP1 complex, which either alters the binding pose of the substrate [eicosatetraenoyl-PE (ETE-PE)] in a nonproductive way or blocks the predominant oxygen channel thus preventing the catalysis of ETE-PE peroxidation. Our successful strategy may be adapted to the design of additional chemical libraries to reveal new ferroptosis-targeting therapeutic modalities.
Subject(s)
Ferroptosis , Phosphatidylethanolamine Binding Protein , Glutathione/metabolism , Iron/metabolism , Lipid Peroxidation , Lipids , Oxidation-Reduction , Phosphatidylethanolamine Binding Protein/antagonists & inhibitorsABSTRACT
Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are pathologically activated neutrophils that are crucial for the regulation of immune responses in cancer. These cells contribute to the failure of cancer therapies and are associated with poor clinical outcomes. Despite recent advances in the understanding of PMN-MDSC biology, the mechanisms responsible for the pathological activation of neutrophils are not well defined, and this limits the selective targeting of these cells. Here we report that mouse and human PMN-MDSCs exclusively upregulate fatty acid transport protein 2 (FATP2). Overexpression of FATP2 in PMN-MDSCs was controlled by granulocyte-macrophage colony-stimulating factor, through the activation of the STAT5 transcription factor. Deletion of FATP2 abrogated the suppressive activity of PMN-MDSCs. The main mechanism of FATP2-mediated suppressive activity involved the uptake of arachidonic acid and the synthesis of prostaglandin E2. The selective pharmacological inhibition of FATP2 abrogated the activity of PMN-MDSCs and substantially delayed tumour progression. In combination with checkpoint inhibitors, FATP2 inhibition blocked tumour progression in mice. Thus, FATP2 mediates the acquisition of immunosuppressive activity by PMN-MDSCs and represents a target to inhibit the functions of PMN-MDSCs selectively and to improve the efficiency of cancer therapy.
Subject(s)
Fatty Acid Transport Proteins/metabolism , Fatty Acids/metabolism , Neoplasms/metabolism , Neoplasms/pathology , Neutrophils/metabolism , Aged , Animals , Arachidonic Acid/metabolism , Dinoprostone/metabolism , Fatty Acid Transport Proteins/antagonists & inhibitors , Female , Humans , Lipid Metabolism , Lipids , Male , Mice , Middle Aged , Neutrophils/pathology , STAT5 Transcription Factor/metabolismABSTRACT
Ischemia reperfusion injury represents a common pathological condition that is triggered by the release of endogenous ligands. While neutrophils are known to play a critical role in its pathogenesis, the tissue-specific spatiotemporal regulation of ischemia-reperfusion injury is not understood. Here, using oxidative lipidomics and intravital imaging of transplanted mouse lungs that are subjected to severe ischemia reperfusion injury, we discovered that necroptosis, a nonapoptotic form of cell death, triggers the recruitment of neutrophils. During the initial stages of inflammation, neutrophils traffic predominantly to subpleural vessels, where their aggregation is directed by chemoattractants produced by nonclassical monocytes that are spatially restricted in this vascular compartment. Subsequent neutrophilic disruption of capillaries resulting in vascular leakage is associated with impaired graft function. We found that TLR4 signaling in vascular endothelial cells and downstream NADPH oxidase 4 expression mediate the arrest of neutrophils, a step upstream of their extravasation. Neutrophil extracellular traps formed in injured lungs and their disruption with DNase prevented vascular leakage and ameliorated primary graft dysfunction. Thus, we have uncovered mechanisms that regulate the initial recruitment of neutrophils to injured lungs, which result in selective damage to subpleural pulmonary vessels and primary graft dysfunction. Our findings could lead to the development of new therapeutics that protect lungs from ischemia reperfusion injury.
Subject(s)
Endothelium, Vascular/metabolism , Lung/metabolism , Necroptosis , Neutrophil Infiltration , Neutrophils/metabolism , Reperfusion Injury/metabolism , Animals , Endothelium, Vascular/injuries , Humans , Lung/blood supply , Mice , Mice, Knockout , Reperfusion Injury/genetics , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolismABSTRACT
The vast majority of membrane phospholipids (PLs) include two asymmetrically positioned fatty acyls: oxidizable polyunsaturated fatty acids (PUFA) attached predominantly at the sn2 position, and non-oxidizable saturated/monounsaturated acids (SFA/MUFA) localized at the sn1 position. The peroxidation of PUFA-PLs, particularly sn2-arachidonoyl(AA)- and sn2-adrenoyl(AdA)-containing phosphatidylethanolamines (PE), has been associated with the execution of ferroptosis, a program of regulated cell death. There is a minor subpopulation (≈1-2â mol %) of doubly PUFA-acylated phospholipids (di-PUFA-PLs) whose role in ferroptosis remains enigmatic. Here we report that 15-lipoxygenase (15LOX) exhibits unexpectedly high pro-ferroptotic peroxidation activity towards di-PUFA-PEs. We revealed that peroxidation of several molecular species of di-PUFA-PEs occurred early in ferroptosis. Ferrostatin-1, a typical ferroptosis inhibitor, effectively prevented peroxidation of di-PUFA-PEs. Furthermore, co-incubation of cells with di-AA-PE and 15LOX produced PUFA-PE peroxidation and induced ferroptotic death. The decreased contents of di-PUFA-PEs in ACSL4 KO A375 cells was associated with lower levels of di-PUFA-PE peroxidation and enhanced resistance to ferroptosis. Thus, di-PUFA-PE species are newly identified phospholipid peroxidation substrates and regulators of ferroptosis, representing a promising therapeutic target for many diseases related to ferroptotic death.
Subject(s)
Arachidonate 15-Lipoxygenase , Phosphatidylethanolamines , Phosphatidylethanolamines/metabolism , Arachidonate 15-Lipoxygenase/metabolism , Cell Death , Phospholipids/metabolism , Fatty Acids, Unsaturated/metabolism , Lipid PeroxidationABSTRACT
Ferroptosis, triggered by discoordination of iron, thiols and lipids, leads to the accumulation of 15-hydroperoxy (Hp)-arachidonoyl-phosphatidylethanolamine (15-HpETE-PE), generated by complexes of 15-lipoxygenase (15-LOX) and a scaffold protein, phosphatidylethanolamine (PE)-binding protein (PEBP)1. As the Ca2+-independent phospholipase A2ß (iPLA2ß, PLA2G6 or PNPLA9 gene) can preferentially hydrolyze peroxidized phospholipids, it may eliminate the ferroptotic 15-HpETE-PE death signal. Here, we demonstrate that by hydrolyzing 15-HpETE-PE, iPLA2ß averts ferroptosis, whereas its genetic or pharmacological inactivation sensitizes cells to ferroptosis. Given that PLA2G6 mutations relate to neurodegeneration, we examined fibroblasts from a patient with a Parkinson's disease (PD)-associated mutation (fPDR747W) and found selectively decreased 15-HpETE-PE-hydrolyzing activity, 15-HpETE-PE accumulation and elevated sensitivity to ferroptosis. CRISPR-Cas9-engineered Pnpla9R748W/R748W mice exhibited progressive parkinsonian motor deficits and 15-HpETE-PE accumulation. Elevated 15-HpETE-PE levels were also detected in midbrains of rotenone-infused parkinsonian rats and α-synuclein-mutant SncaA53T mice, with decreased iPLA2ß expression and a PD-relevant phenotype. Thus, iPLA2ß is a new ferroptosis regulator, and its mutations may be implicated in PD pathogenesis.
Subject(s)
Ferroptosis/physiology , Group VI Phospholipases A2/metabolism , Animals , Arachidonate 15-Lipoxygenase/metabolism , Disease Models, Animal , Female , Group VI Phospholipases A2/physiology , Humans , Iron/metabolism , Leukotrienes/metabolism , Lipid Metabolism/physiology , Lipid Peroxides/metabolism , Lipids/physiology , Male , Mice , Mice, Inbred C57BL , Oxidation-Reduction , Parkinson Disease/metabolism , Phosphatidylethanolamine Binding Protein/metabolism , Phospholipases/metabolism , Phospholipids/metabolism , Rats , Rats, Inbred LewABSTRACT
The recently identified ferroptotic cell death is characterized by excessive accumulation of hydroperoxy-arachidonoyl (C20:4)- or adrenoyl (C22:4)- phosphatidylethanolamine (Hp-PE). The selenium-dependent glutathione peroxidase 4 (GPX4) inhibits ferroptosis, converting unstable ferroptotic lipid hydroperoxides to nontoxic lipid alcohols in a tissue-specific manner. While placental oxidative stress and lipotoxicity are hallmarks of placental dysfunction, the possible role of ferroptosis in placental dysfunction is largely unknown. We found that spontaneous preterm birth is associated with ferroptosis and that inhibition of GPX4 causes ferroptotic injury in primary human trophoblasts and during mouse pregnancy. Importantly, we uncovered a role for the phospholipase PLA2G6 (PNPLA9, iPLA2beta), known to metabolize Hp-PE to lyso-PE and oxidized fatty acid, in mitigating ferroptosis induced by GPX4 inhibition in vitro or by hypoxia/reoxygenation injury in vivo. Together, we identified ferroptosis signaling in the human and mouse placenta, established a role for PLA2G6 in attenuating trophoblastic ferroptosis, and provided mechanistic insights into the ill-defined placental lipotoxicity that may inspire PLA2G6-targeted therapeutic strategies.
Subject(s)
Ferroptosis/physiology , Group VI Phospholipases A2/metabolism , Trophoblasts/metabolism , Animals , Female , Glutathione Peroxidase/metabolism , Group VI Phospholipases A2/genetics , Group VI Phospholipases A2/physiology , Humans , Iron/metabolism , Lipid Peroxides/metabolism , Mice , Mice, Knockout , Phosphatidylethanolamines/metabolism , Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism , Placenta/metabolism , Pregnancy , Premature Birth/metabolism , Signal TransductionABSTRACT
BACKGROUND AND AIMS: Iron is essential yet also highly chemically reactive and potentially toxic. The mechanisms that allow cells to use iron safely are not clear; defects in iron management are a causative factor in the cell-death pathway known as ferroptosis. Poly rC binding protein 1 (PCBP1) is a multifunctional protein that serves as a cytosolic iron chaperone, binding and transferring iron to recipient proteins in mammalian cells. Although PCBP1 distributes iron in cells, its role in managing iron in mammalian tissues remains open for study. The liver is highly specialized for iron uptake, utilization, storage, and secretion. APPROACH AND RESULTS: Mice lacking PCBP1 in hepatocytes exhibited defects in liver iron homeostasis with low levels of liver iron, reduced activity of iron enzymes, and misregulation of the cell-autonomous iron regulatory system. These mice spontaneously developed liver disease with hepatic steatosis, inflammation, and degeneration. Transcriptome analysis indicated activation of lipid biosynthetic and oxidative-stress response pathways, including the antiferroptotic mediator, glutathione peroxidase type 4. Although PCBP1-deleted livers were iron deficient, dietary iron supplementation did not prevent steatosis; instead, dietary iron restriction and antioxidant therapy with vitamin E prevented liver disease. PCBP1-deleted hepatocytes exhibited increased labile iron and production of reactive oxygen species (ROS), were hypersensitive to iron and pro-oxidants, and accumulated oxidatively damaged lipids because of the reactivity of unchaperoned iron. CONCLUSIONS: Unchaperoned iron in PCBP1-deleted mouse hepatocytes leads to production of ROS, resulting in lipid peroxidation (LPO) and steatosis in the absence of iron overload. The iron chaperone activity of PCBP1 is therefore critical for limiting the toxicity of cytosolic iron and may be a key factor in preventing the LPO that triggers the ferroptotic cell-death pathway.
Subject(s)
DNA-Binding Proteins/metabolism , Fatty Liver/etiology , Iron Compounds/metabolism , Lipid Peroxidation , Metallochaperones/metabolism , RNA-Binding Proteins/metabolism , Animals , Fatty Liver/metabolism , Fatty Liver/pathology , Female , Hepatocytes/metabolism , Humans , Liver/metabolism , Liver/pathology , Male , Mice, Knockout , Oxidative StressABSTRACT
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)/NOâ¢-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.
Subject(s)
Ferroptosis/physiology , Macrophages/metabolism , Nitric Oxide Synthase Type II/metabolism , Animals , Arachidonate 15-Lipoxygenase/metabolism , Arachidonate 15-Lipoxygenase/physiology , Cell Death , Female , Iron/metabolism , Iron/physiology , Leukotrienes/metabolism , Lipid Peroxidation/physiology , Lipid Peroxides/metabolism , Male , Mice , Mice, Inbred C57BL , Microglia/metabolism , Nitric Oxide Synthase Type II/physiology , Oxidation-Reduction , Reactive Oxygen Species/metabolismABSTRACT
Enigmatic lipid peroxidation products have been claimed as the proximate executioners of ferroptosis-a specialized death program triggered by insufficiency of glutathione peroxidase 4 (GPX4). Using quantitative redox lipidomics, reverse genetics, bioinformatics and systems biology, we discovered that ferroptosis involves a highly organized oxygenation center, wherein oxidation in endoplasmic-reticulum-associated compartments occurs on only one class of phospholipids (phosphatidylethanolamines (PEs)) and is specific toward two fatty acyls-arachidonoyl (AA) and adrenoyl (AdA). Suppression of AA or AdA esterification into PE by genetic or pharmacological inhibition of acyl-CoA synthase 4 (ACSL4) acts as a specific antiferroptotic rescue pathway. Lipoxygenase (LOX) generates doubly and triply-oxygenated (15-hydroperoxy)-diacylated PE species, which act as death signals, and tocopherols and tocotrienols (vitamin E) suppress LOX and protect against ferroptosis, suggesting a homeostatic physiological role for vitamin E. This oxidative PE death pathway may also represent a target for drug discovery.
Subject(s)
Arachidonic Acid/metabolism , Fatty Acids, Unsaturated/metabolism , Phospholipids/metabolism , Animals , Arachidonic Acid/antagonists & inhibitors , Cell Death/drug effects , Cell Line , Coenzyme A Ligases/antagonists & inhibitors , Coenzyme A Ligases/deficiency , Coenzyme A Ligases/metabolism , Fatty Acids, Unsaturated/antagonists & inhibitors , Female , Male , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
sn2-15-Hydroperoxy-eicasotetraenoyl-phosphatidylethanolamines ( sn2-15-HpETE-PE) generated by mammalian 15-lipoxygenase/phosphatidylethanolamine binding protein-1 (15-LO/PEBP1) complex is a death signal in a recently identified type of programmed cell demise, ferroptosis. How the enzymatic complex selects sn2-ETE-PE as the substrate among 1 of â¼100 total oxidizable membrane PUFA phospholipids is a central, yet unresolved question. To unearth the highly selective and specific mechanisms of catalytic competence, we used a combination of redox lipidomics, mutational and computational structural analysis to show they stem from (i) reactivity toward readily accessible hexagonally organized membrane sn2-ETE-PEs, (ii) relative preponderance of sn2-ETE-PE species vs other sn2-ETE-PLs, and (iii) allosteric modification of the enzyme in the complex with PEBP1. This emphasizes the role of enzymatic vs random stochastic free radical reactions in ferroptotic death signaling.
Subject(s)
Arachidonate 15-Lipoxygenase/metabolism , Cell Death/physiology , Phosphatidylethanolamines/metabolism , Animals , Arachidonate 15-Lipoxygenase/chemistry , Catalysis , Cell Line , Mice , Mutation , Oxidation-Reduction , Phosphatidylethanolamine Binding Protein/genetics , Phosphatidylethanolamine Binding Protein/metabolism , Phosphatidylethanolamines/chemistry , Substrate SpecificityABSTRACT
Since its discovery 75years ago, a wealth of knowledge has accumulated on the role of cardiolipin, the hallmark phospholipid of mitochondria, in bioenergetics and particularly on the structural organization of the inner mitochondrial membrane. A surge of interest in this anionic doubly-charged tetra-acylated lipid found in both prokaryotes and mitochondria has emerged based on its newly discovered signaling functions. Cardiolipin displays organ, tissue, cellular and transmembrane distribution asymmetries. A collapse of the membrane asymmetry represents a pro-mitophageal mechanism whereby externalized cardiolipin acts as an "eat-me" signal. Oxidation of cardiolipin's polyunsaturated acyl chains - catalyzed by cardiolipin complexes with cytochrome c. - is a pro-apoptotic signal. The messaging functions of myriads of cardiolipin species and their oxidation products are now being recognized as important intracellular and extracellular signals for innate and adaptive immune systems. This newly developing field of research exploring cardiolipin signaling is the main subject of this review. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.
Subject(s)
Cardiolipins/metabolism , Signal Transduction/physiology , Animals , Cytochromes c/metabolism , Humans , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Oxidation-Reduction , Phospholipids/metabolismABSTRACT
We (66) have previously described an NSAID-insensitive intramitochondrial biosynthetic pathway involving oxidation of the polyunsaturated mitochondrial phospholipid, cardiolipin (CL), followed by hydrolysis [by calcium-independent mitochondrial calcium-independent phospholipase A2-γ (iPLA2γ)] of oxidized CL (CLox), leading to the formation of lysoCL and oxygenated octadecadienoic metabolites. We now describe a model system utilizing oxidative lipidomics/mass spectrometry and bioassays on cultured bovine pulmonary artery endothelial cells (BPAECs) to assess the impact of CLox that we show, in vivo, can be released to the extracellular space and may be hydrolyzed by lipoprotein-associated PLA2 (Lp-PLA2). Chemically oxidized liposomes containing bovine heart CL produced multiple oxygenated species. Addition of Lp-PLA2 hydrolyzed CLox and produced (oxygenated) monolysoCL and dilysoCL and oxidized octadecadienoic metabolites including 9- and 13-hydroxyoctadecadienoic (HODE) acids. CLox caused BPAEC necrosis that was exacerbated by Lp-PLA2 Lower doses of nonlethal CLox increased permeability of BPAEC monolayers. This effect was exacerbated by Lp-PLA2 and partially mimicked by authentic monolysoCL or 9- or 13-HODE. Control mice plasma contained virtually no detectable CLox; in contrast, 4 h after Pseudomonas aeruginosa (P. aeruginosa) infection, 34 ± 8 mol% (n = 6; P < 0.02) of circulating CL was oxidized. In addition, molar percentage of monolysoCL increased twofold after P. aeruginosa in a subgroup analyzed for these changes. Collectively, these studies suggest an important role for 1) oxidation of CL in proinflammatory environments and 2) possible hydrolysis of CLox in extracellular spaces producing lysoCL and oxidized octadecadienoic acid metabolites that may lead to impairment of pulmonary endothelial barrier function and necrosis.
Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/physiology , Cardiolipins/biosynthesis , Endothelial Cells/physiology , Animals , Cardiolipins/blood , Cattle , Cells, Cultured , Electric Impedance , Hydrolysis , Mice, Inbred C57BL , Oxidation-Reduction , Pseudomonas Infections/blood , Signal TransductionABSTRACT
Cross-presentation is one of the main features of dendritic cells (DCs), which is critically important for the development of spontaneous and therapy-inducible antitumor immune responses. Patients, at early stages of cancer, have normal presence of DCs. However, the difficulties in the development of antitumor responses in patients with low tumor burden raised the question of the mechanisms of DC dysfunction. In this study, we found that, in differentiated DCs, tumor-derived factors blocked the cross-presentation of exogenous Ags without inhibiting the Ag presentation of endogenous protein or peptides. This effect was caused by intracellular accumulation of different types of oxidized neutral lipids: triglycerides, cholesterol esters, and fatty acids. In contrast, the accumulation of nonoxidized lipids did not affect cross-presentation. Oxidized lipids blocked cross-presentation by reducing the expression of peptide-MHC class I complexes on the cell surface. Thus, this study suggests the novel role of oxidized lipids in the regulation of cross-presentation.
Subject(s)
Antigen Presentation/immunology , Cross-Priming/immunology , Dendritic Cells/immunology , Lipids/immunology , Neoplasms/immunology , Acetylcysteine/analogs & derivatives , Acetylcysteine/pharmacology , Animals , Cell Line, Tumor , Cells, Cultured , Culture Media, Conditioned/metabolism , Culture Media, Conditioned/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Flow Cytometry , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class II/immunology , Humans , Interferon-gamma/pharmacology , Lipids/chemistry , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Confocal , Neoplasms/metabolism , Neoplasms/pathology , Ovalbumin/immunology , Oxidation-Reduction , Peptide Fragments/immunologyABSTRACT
Macrophages play a fundamental role in innate immunity and the pathogenesis of silicosis. Phagocytosis of silica particles is associated with the generation of reactive oxygen species (ROS), secretion of cytokines, such as TNF, and cell death that contribute to silica-induced lung disease. In macrophages, ROS production is executed primarily by activation of the NADPH oxidase (Phox) and by generation of mitochondrial ROS (mtROS); however, the relative contribution is unclear, and the effects on macrophage function and fate are unknown. In this study, we used primary human and mouse macrophages (C57BL/6, BALB/c, and p47(phox-/-)) and macrophage cell lines (RAW 264.7 and IC21) to investigate the contribution of Phox and mtROS to silica-induced lung injury. We demonstrate that reduced p47(phox) expression in IC21 macrophages is linked to enhanced mtROS generation, cardiolipin oxidation, and accumulation of cardiolipin hydrolysis products, culminating in cell death. mtROS production is also observed in p47(phox-/-) macrophages, and p47(phox-/-) mice exhibit increased inflammation and fibrosis in the lung following silica exposure. Silica induces interaction between TNFR1 and Phox in RAW 264.7 macrophages. Moreover, TNFR1 expression in mitochondria decreased mtROS production and increased RAW 264.7 macrophage survival to silica. These results identify TNFR1/Phox interaction as a key event in the pathogenesis of silicosis that prevents mtROS formation and reduces macrophage apoptosis.
Subject(s)
Mitochondria/metabolism , NADPH Oxidases/metabolism , Receptors, Tumor Necrosis Factor, Type I/metabolism , Silicosis/metabolism , Animals , Cell Death , Cell Line , Disease Models, Animal , Female , Gene Expression Regulation , Lung Injury/etiology , Lung Injury/metabolism , Lung Injury/pathology , Macrophages/metabolism , Mice , Mice, Knockout , NADPH Oxidases/genetics , Protein Binding , Protein Transport , Reactive Oxygen Species/metabolism , Silicon Dioxide/adverse effects , Silicon Dioxide/metabolism , Silicosis/geneticsABSTRACT
Although the role of ferroptosis in killing tumor cells is well established, recent studies indicate that ferroptosis inducers also sabotage anti-tumor immunity by killing neutrophils and thus unexpectedly stimulate tumor growth, raising a serious issue about whether ferroptosis effectively suppresses tumor development in vivo. Through genome-wide CRISPR-Cas9 screenings, we discover a pleckstrin homology-like domain family A member 2 (PHLDA2)-mediated ferroptosis pathway that is neither ACSL4-dependent nor requires common ferroptosis inducers. PHLDA2-mediated ferroptosis acts through the peroxidation of phosphatidic acid (PA) upon high levels of reactive oxygen species (ROS). ROS-induced ferroptosis is critical for tumor growth in the absence of common ferroptosis inducers; strikingly, loss of PHLDA2 abrogates ROS-induced ferroptosis and promotes tumor growth but has no obvious effect in normal tissues in both immunodeficient and immunocompetent mouse tumor models. These data demonstrate that PHLDA2-mediated PA peroxidation triggers a distinct ferroptosis response critical for tumor suppression and reveal that PHLDA2-mediated ferroptosis occurs naturally in vivo without any treatment from ferroptosis inducers.
Subject(s)
Neoplasms , Animals , Mice , Disease Models, Animal , Lipid Peroxidation/physiology , Reactive Oxygen Species/metabolismABSTRACT
Mitochondrial trifunctional protein (TFP) deficiency is an inherited metabolic disorder leading to a block in long-chain fatty acid ß-oxidation. Mutations in either HADHA and HADHB, which encode the TFPα and ß subunits, respectively, usually result in combined TFP deficiency. A single common mutation, HADHA c.1528G>C (p.E510Q), leads to isolated 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency. TFP also catalyzes a step in the remodeling of cardiolipin (CL), a phospholipid critical to mitochondrial membrane stability and function. We explored the effect of mutations in TFP subunits on CL and other phospholipid content and composition and the consequences of these changes on mitochondrial bioenergetics in patient-derived fibroblasts. Abnormalities in these parameters varied extensively among different fibroblasts, and some cells were able to maintain basal oxygen consumption rates similar to controls. Although CL reduction was universally identified, a simultaneous increase in monolysocardiolipins was discrepant among cells. A similar profile was seen in liver mitochondria isolates from a TFP-deficient mouse model. Response to new potential drugs targeting cardiolipin metabolism might be dependent on patient genotype.
ABSTRACT
A stable mitochondrial pool is crucial for healthy cell function and survival. Altered redox biology can adversely affect mitochondria through induction of a variety of cell death and survival pathways, yet the understanding of mitochondria and their dysfunction in primary human cells and in specific disease states, including asthma, is modest. Ferroptosis is traditionally considered an iron dependent, hydroperoxy-phospholipid executed process, which induces cytosolic and mitochondrial damage to drive programmed cell death. However, in this report we identify a lipoxygenase orchestrated, compartmentally-targeted ferroptosis-associated peroxidation process which occurs in a subpopulation of dysfunctional mitochondria, without promoting cell death. Rather, this mitochondrial peroxidation process tightly couples with PTEN-induced kinase (PINK)-1(PINK1)-Parkin-Optineurin mediated mitophagy in an effort to preserve the pool of functional mitochondria and prevent cell death. These combined peroxidation processes lead to altered epithelial cell phenotypes and loss of ciliated cells which associate with worsened asthma severity. Ferroptosis-targeted interventions of this process could preserve healthy mitochondria, reverse cell phenotypic changes and improve disease outcomes.
Subject(s)
Asthma , Cell Cycle Proteins , Epithelial Cells , Ferroptosis , Membrane Transport Proteins , Mitochondria , Mitophagy , Phenotype , Transcription Factor TFIIIA , Humans , Mitochondria/metabolism , Asthma/metabolism , Asthma/pathology , Epithelial Cells/metabolism , Membrane Transport Proteins/metabolism , Transcription Factor TFIIIA/metabolism , Transcription Factor TFIIIA/genetics , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Male , Protein Kinases/metabolism , Female , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Animals , Lipid Peroxidation , Mice , Middle AgedABSTRACT
Ovarian cancer remains a major health threat with limited treatment options available. It is characterized by immunosuppressive tumor microenvironment (TME) maintained by tumor- associated macrophages (TAMs) hindering anti-tumor responses and immunotherapy efficacy. Here we show that targeting retinoblastoma protein (Rb) by disruption of its LxCxE cleft pocket, causes cell death in TAMs by induction of ER stress, p53 and mitochondria-related cell death pathways. A reduction of pro-tumor Rb high M2-type macrophages from TME in vivo enhanced T cell infiltration and inhibited cancer progression. We demonstrate an increased Rb expression in TAMs in women with ovarian cancer is associated with poorer prognosis. Ex vivo, we show analogous cell death induction by therapeutic Rb targeting in TAMs in post-surgery ascites from ovarian cancer patients. Overall, our data elucidates therapeutic targeting of the Rb LxCxE cleft pocket as a novel promising approach for ovarian cancer treatment through depletion of TAMs and re-shaping TME immune landscape. Statement of significance: Currently, targeting immunosuppressive myeloid cells in ovarian cancer microenvironment is the first priority need to enable successful immunotherapy, but no effective solutions are clinically available. We show that targeting LxCxE cleft pocket of Retinoblastoma protein unexpectedly induces preferential cell death in M2 tumor-associated macrophages. Depletion of immunosuppressive M2 tumor-associated macrophages reshapes tumor microenvironment, enhances anti-tumor T cell responses, and inhibits ovarian cancer. Thus, we identify a novel paradoxical function of Retinoblastoma protein in regulating macrophage viability as well as a promising target to enhance immunotherapy efficacy in ovarian cancer.