Therapeutic efficacies of neutrophil elastase and RIP1 inhibitors in acute lung injury

中性粒细胞弹性蛋白酶和RIP1抑制剂治疗急性肺损伤的疗效

• 批准号: 10311120
• 负责人: HASEM HABELHAH
• 金额: $ 23.18万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-02 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10311120
• 关键词: Ablation; Acute Lung Injury; Adverse effects; Affect; Alveolar; American; Animal Model; Apoptosis; Attenuated; Bacteria; Biochemical; Breathing; CASP8 gene; Cell Death; Cell Survival; Cessation of life; Clinical Research; Clinical Trials; Consensus; Development; Effectiveness; Endothelium; Enzymes; Event; FDA approved; Genetic; Goals; Human; Human body; Hypoxemia; Incidence; Inflammation; Injury; Invaded; Japanese; Knock-out; Knockout Mice; Lethal Dose 50; Leukocyte Elastase; Leukocytes; Lipopolysaccharides; Lung; Measures; Mediating; Metalloproteases; Modeling; Molecular; Multicenter Studies; Mus; Neutrophil Activation; Pathway interactions; Peptide Hydrolases; Perinatal; Pharmacology; Phase; Phase III Clinical Trials; Phosphotransferases; Play; Proteins; Pulmonary Edema; Pulmonary Inflammation; RIPK1 gene; RIPK3 gene; Reactive Oxygen Species; Receptor Inhibition; Respiratory Failure; Role; Stains; Study models; Syndrome; TNF gene; Tissues; Treatment Efficacy; Treatment Protocols; Ubiquitination; United States; Ventilator; experimental study; improved; in vivo; inflammatory milieu; inhibitor; insight; interstitial; kinase inhibitor; lung injury; mortality; mouse model; neutrophil; neutrophil elastase inhibitor; novel; novel therapeutic intervention; pathogen; prevent; public health relevance; pulmonary function; small molecular inhibitor; ventilation

5. Project Summary/Abstract Acute lung injury (ALI) is a syndrome of respiratory failure characterized by protein-rich pulmonary edema that causes severe hypoxemia and labored breathing. In the United States, the annual incidence of ALI is about 200,000 with a mortality rate of 30-40%; yet, there is no FDA-approved pharmacological therapy for ALI. Neutrophils are the most abundant circulating white blood cells in the human body and provide the first line of defense against invading pathogens. In the lung, while neutrophils are essential for pathogen elimination, exuberant accumulation and prolonged survival of neutrophils in the interstitial and alveolar spaces contribute to ALI by releasing a variety of injurious molecules, including neutrophil elastase (NE), metalloproteases, reactive oxygen species (ROS), and other proteolytic enzymes. Although all these injurious molecules contribute to ALI, an increasing body of studies have demonstrated that NE is the most potent hydrolytic enzyme that plays a key role in lung alveolar injury, and that inhibition of NE with small molecular inhibitors [e.g. Sivelestat (Siv)] reduces measures of lung injury and inflammation in animal models of ALI. However, clinical studies have not yet provided a clear consensus. Whereas in Japanese phase III clinical trials, Siv improved pulmonary functions, in North American multicenter studies, Siv rather increased long-term mortality by unknown mechanisms. Thus, identification of molecular events associated with adverse effects of Siv in ALI is an important task for the development of novel and combination treatment regimens. Recently, in unbiased functional protease purification experiments, we discovered that NE cleaves receptor interacting protein 1 (RIP1), and that inhibition of NE with Siv leads to RIP1-dependent necroptosis in mouse and human neutrophils following treatment with lipopolysaccharide (LPS) or TNFα. Such RIP1-induced necroptosis can be inhibited by RIP1 kinase inhibitors (e.g. Nec-1s) or knockout (KO) of RIP3. We found that LPS/Siv-induced necroptosis was significantly inhibited in RIP3 KO neutrophils. Importantly, in a mouse model of ALI induced by 50% lethal dose of LPS, Siv in combination with Nec-1s resulted in 90% survival of the mice (P=0.001), whereas neither Siv nor Nec-1s alone afforded significant protection against the death. Thus, we hypothesize that inhibition of NE with Siv in ALI leads to RIP1-mediated necroptosis in neutrophils, which causes secondary inflammation and lung injury, and that inhibition of RIP1-mediated necroptosis significantly improves the effectiveness of NE inhibitors in ALI. Aim-1. Determine the therapeutic efficacies of RIP1 and NE inhibitors in lung injury and inflammation using mouse models of ALI. Aim-2. Determine how genetic ablation of NE or RIP3 affects lung injury and inflammation in ALI mice.

5.项目总结/摘要 急性肺损伤（ALI）是一种以富含蛋白质的肺水肿为特征的呼吸衰竭综合征， 会导致严重的低氧血症和呼吸困难在美国，ALI的年发病率约为 20万例，死亡率为30-40%;然而，没有FDA批准的药物治疗ALI。 嗜中性粒细胞是人体中最丰富的循环白色血细胞，并提供了第一道免疫屏障。 防御入侵的病原体。在肺中，虽然中性粒细胞对病原体的清除至关重要， 中性粒细胞在间质和肺泡腔中的大量积聚和延长的存活有助于 通过释放多种有害分子，包括中性粒细胞弹性蛋白酶（NE），金属蛋白酶， 活性氧（ROS）和其它蛋白水解酶。尽管所有这些有害分子 由于NE对ALI的作用，越来越多的研究表明NE是最有效的水解酶， 在肺泡损伤中起关键作用的酶，以及用小分子抑制剂抑制NE [e.g. Sivelestat（Siv）]减少ALI动物模型中肺损伤和炎症的测量。然而，在这方面， 临床研究尚未提供明确的共识。而在日本的III期临床试验中， 改善肺功能，在北美多中心研究中，Siv反而增加了长期死亡率 未知的机制。因此，鉴定与Siv在ALI中的不良反应相关的分子事件， 是开发新的联合治疗方案的重要任务。最近，在无偏见的 功能蛋白酶纯化实验中，我们发现NE切割受体相互作用蛋白1 （RIP 1），并且用Siv抑制NE导致小鼠和人中RIP 1依赖性坏死性凋亡 用脂多糖（LPS）或TNFα处理后的中性粒细胞。这种RIP 1诱导的坏死性凋亡可以是 受RIP 1激酶抑制剂（例如Nec-1 s）或RIP 3敲除（KO）抑制。我们发现LPS/Siv诱导的 在RIP 3 KO中性粒细胞中，坏死性凋亡被显著抑制。更重要的是，在小鼠ALI模型中， 50%致死剂量LPS、Siv联合Nec-1 s组小鼠存活率为90%（P=0.001）， 而Siv和Nec-1 s都不能单独对死亡提供显著的保护。因此，我们假设 在ALI中用Siv抑制NE导致RIP 1介导的中性粒细胞坏死性凋亡，这引起继发性 炎症和肺损伤，抑制RIP 1介导坏死性凋亡显著改善了 NE抑制剂在ALI中的有效性。目标一确定RIP 1和NE抑制剂在 肺损伤和炎症。目标二确定NE或RIP 3的基因消融 影响ALI小鼠的肺损伤和炎症。