Ferroptosis as a Death Mechanism in Lung Injury - Project 2

铁死亡作为肺损伤的死亡机制 - 项目 2

• 批准号: 10399560
• 负责人: Valerian E Kagan
• 金额: $ 36.74万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-03 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10399560
• 关键词: Acute; Acute Lung Injury; Acute Respiratory Distress Syndrome; Alveolar Macrophages; Animals; Apoptosis; Apoptotic; Arachidonate 15-Lipoxygenase; Bacterial Pneumonia; Biological Markers; Brain Diseases; Cardiolipins; Cell Death; Cell Death Signaling Process; Cells; Cessation of life; Chronic; Clinical; Complex; Cytoprotective Agent; Development; Environment; Epithelial; Epithelial Cells; Event; Evolution; Exposure to; Human; Hydrogen Peroxide; Immune; Immune response; Immunocompromised Host; Immunosuppression; Impairment; Individual; Inflammation; Innate Immune Response; Kidney Diseases; Lead; Lipid Peroxidation; Lipids; Liver diseases; Lung; Membrane; Mitochondria; Modeling; Molecular; Mus; Oxidation-Reduction; Oxidative Stress; Oxides; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Phosphatidylethanolamine; Phospholipids; Play; Property; Protein Isoforms; Pseudomonas aeruginosa; Pseudomonas aeruginosa infection; Reaction; Reactive Oxygen Species; Reporting; Respiratory Tract Infections; Role; Sampling; Scaffolding Protein; Signal Transduction; Signaling Molecule; Testing; Virulence Factors; Virulent; Work; base; cell injury; design; epithelial injury; glutathione peroxidase; in vivo; inhibitor; lipidomics; lung injury; macrophage; mortality; novel; novel therapeutic intervention; novel therapeutics; oxidation; pathogen; pathogenic bacteria; peroxidation; predictive marker; prevent; programs; selenoenzyme; small molecule

In ARDS, bacterial pathogens damage host cells, activate innate immune responses, and create a pro-oxidant environment leading to cell death via one of the death programs. This Project will focus on a recently described death program, ferroptosis, realized via Fe-dependent activation of lipid peroxidation under conditions of deficiency of glutathione peroxidase 4 (GPX4), a seleno-enzyme uniquely capable of reducing phospholipid hydroperoxides. We identified 15-hydroperoxy-arachidonoyl-phosphatidylethanolamines (15-HOO-AA-PE) as specific lipid biomarkers of ferroptosis. We also discovered that complexes of 15-lipoxygenase (15LOX) with a scaffold protein, PEBP1, play the major role in generating 15-HOO-AA-PE signals. We divulged ferroptosis as a death program of the human pulmonary epithelium. Ferroptosis occurs in alternatively activated macrophages with low levels of NO•/iNOS, thus causing immuno-suppression. Unexpectedly, we discovered that a common Gram-negative pathogen, P. aeruginosa – that does not contain polyunsaturated phospholipid oxidation substrates– expresses 15LOX (pLoxA) which oxidizes host polyunsaturated PE, generates 15-HOO-AA-PE and causes ferroptosis in epithelial cells and macrophages independently of the endogenous host 15-LOX. Ferroptosis-inducing pLoxA was detected in clinical P. aeruginosa isolates from ARDS patients. 15-HOO-AA- PE were identified in the lung samples from severely immuno-compromised patients with ARDS. Thus, we postulate the existence of a vicious cycle whereby inflammation/oxidative stress driven ferroptosis supported by endogenous 15-LOX acts as the major contributor to immunosuppression that sets the stage for the secondary P. aeruginosa infection of immune-impaired lung and further enhancement of ferroptosis by exogenous bacterial pLoxA. We propose to design and use selective small molecule pLoxA inhibitors, which will act as anti-ferroptotic agents thus representing new classes of pulmonary protectors. Aim 1 will reveal and decipher pathogenic mechanisms through which reactions of phospholipid peroxidation catalyzed by isoforms of endogenous mammalian 15-LOX or exogenous bacterial pLoxA – in conditions of GPX4/GSH deficiency – lead to accumulation of hydroperoxy-phospholipids in murine lung epithelial cells (MLE) and alveolar macrophages and establish molecular identity and ferroptotic properties of these products. By using redox lipidomics we will identify and quantify 15-HOO-AA-PE biomarkers of ferroptosis in vivo using a two-hit model of immunosuppression. We will also employ iNOS KO animals exposed to P. aeruginosa to reveal the role of NO• as a regulator of pLoxA-driven AA-PE oxidation and ferroptotic death in the mouse lung vivo. In Aim 2, we will design and develop selective inhibitors of pLoxA regulating P. aeruginosa-driven ferroptosis in epithelia and macrophages as a new class of small-molecule cytoprotective agents preventing breach of the barrier and immunosuppression.

在ARDS中，细菌病原体破坏宿主细胞，激活先天免疫反应，并产生促氧化剂 环境通过死亡程序之一导致细胞死亡。该项目将集中在最近描述的 死亡程序，铁凋亡，通过铁依赖性激活的脂质过氧化作用的条件下实现， 谷胱甘肽过氧化物酶4（GPX 4）缺乏，这是一种独特的能够减少磷脂的硒酶 氢过氧化物。我们鉴定了15-过氧化氢-花生四烯酸-磷脂酰乙醇胺（15-HOO-AA-PE）， 铁凋亡的特异性脂质生物标志物。我们还发现，15-脂氧合酶（15 LOX）与一种 支架蛋白PEBP 1在产生15-HOO-AA-PE信号中起主要作用。我们泄露了铁性下垂 人类肺上皮细胞的死亡程序。铁凋亡发生在交替激活的巨噬细胞中 NO·/iNOS水平低，从而引起免疫抑制。出乎意料的是，我们发现，一个共同的 革兰氏阴性病原体，铜绿假单胞菌-不含多不饱和磷脂氧化 底物-表达15 LOX（pLoxA），其氧化宿主多不饱和PE，产生15-HOO-AA-PE， 引起上皮细胞和巨噬细胞中的铁凋亡，与内源性宿主15-LOX无关。 在ARDS患者的临床铜绿假单胞菌分离株中检测到亚铁中毒诱导pLoxA。15-HOO-AA- 在严重免疫功能低下的ARDS患者的肺样本中鉴定出PE。因此我们 假设存在一个恶性循环，炎症/氧化应激驱动的铁凋亡支持 内源性15-LOX作为免疫抑制的主要贡献者， 继发性铜绿假单胞菌感染免疫受损的肺和进一步加强铁凋亡 外源细菌pLoxA。我们建议设计和使用选择性小分子pLoxA抑制剂， 将作为抗铁中毒剂，因此代表了新型的肺保护剂。目标1将揭示和 破译亚型催化的磷脂过氧化反应的致病机制 内源性哺乳动物15-LOX或外源性细菌pLoxA -在GPX 4/GSH缺乏的条件下- 导致过氧化氢磷脂在鼠肺上皮细胞（MLE）和肺泡 巨噬细胞，并建立这些产物的分子身份和铁毒性。通过使用氧化还原 脂质组学，我们将确定和定量15-HOO-AA-PE生物标志物的铁凋亡在体内使用两次击中模型 免疫抑制。我们还将采用暴露于铜绿假单胞菌的iNOS KO动物来揭示 NO·作为pLoxA驱动的AA-PE氧化和小鼠肺内铁中毒死亡的调节剂在目标2中， 将设计和开发pLoxA的选择性抑制剂，调节上皮中铜绿假单胞菌驱动的铁凋亡， 巨噬细胞作为一类新的小分子细胞保护剂，防止屏障破坏， 免疫抑制