Ferroptosis as a Death Mechanism in Lung Injury - Project 2

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 通过其中一个死亡程序导致细胞死亡的环境。该项目将集中在最近描述的 死亡程序，铁下垂，通过铁依赖的激活脂质过氧化在以下条件下实现 谷胱甘肽过氧化物酶4(Gpx4)缺乏症--一种唯一能还原磷脂的硒酶 过氧化氢。我们鉴定了15-hydroperoxy-arachidonoyl-phosphatidylethanolamines(15-HOO-AA-PE)为 铁性上睑下垂的特异性脂质生物标志物。我们还发现，15-脂氧合酶(15LOX)与A 支架蛋白PEBP1在产生15-HoO-AA-PE信号中起主要作用。我们透露了铁性下垂是 人类肺上皮细胞的死亡程序。铁下垂发生在交替激活的巨噬细胞中 低水平的NO·/iNOS，从而引起免疫抑制。出乎意料的是，我们发现一种常见的 不含多不饱和磷脂氧化的革兰氏阴性病原菌铜绿假单胞菌 底物-表达15LOX(PLoxA)，氧化宿主多不饱和PE，产生15-HoO-AA-PE和 引起上皮细胞和巨噬细胞铁性下垂，不依赖内源性宿主15-LOX。 在ARDS患者的铜绿假单胞菌临床分离株中检测到铁性下垂诱导的pLoxA。15-呼呼-AA- 在免疫功能严重受损的ARDS患者的肺样本中发现了PE。因此，我们 假设存在炎症/氧化应激导致的铁性下垂支持的恶性循环 通过内源性15-LOX作为免疫抑制的主要贡献者，为 免疫受损肺继发铜绿假单胞菌感染及铁性下垂的进一步加重 外源细菌pLoxA。我们建议设计和使用选择性小分子pLoxA抑制剂， 将起到抗铁下垂的作用，因此代表了新的肺保护剂类别。目标1将揭示和 破译异构体催化磷脂过氧化反应的致病机制 在Gpx4/GSH缺乏的情况下，内源性哺乳动物15-LOX或外源细菌pLoxA- 导致小鼠肺上皮细胞和肺泡内过氧化氢磷脂蓄积 巨噬细胞，并确定这些产物的分子特性和铁链性质。通过使用氧化还原 脂质组学我们将使用两次打击模型在活体内识别和量化铁下垂的15-HoO-AA-PE生物标志物 免疫抑制。我们还将使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氧化和小鼠肺内铁链死亡的调节因子。在目标2中，我们 将设计和开发选择性的pLoxA抑制剂来调节铜绿假单胞菌引起的上皮细胞铁下垂 巨噬细胞作为一类新型的小分子细胞保护剂，防止屏障的突破和 免疫抑制。

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.