Role of Neutrophil Extracellular Traps in AAA Pathogenesis

中性粒细胞胞外陷阱在 AAA 发病机制中的作用

• 批准号: 9700321
• 负责人: Gilbert Rivers Upchurch
• 金额: $ 26.86万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9700321
• 关键词: Role; Neutrophil; Extracellular; Traps; AAA

DESCRIPTION (provided by applicant): Neutrophils are the early responders to acute inflammation during vessel wall injury or insult. Apart from phagocytosis and degranulation, NETosis (formation of Neutrophil Extracellular Trap or NET) is another aspect of neutrophil-mediated defense mechanism. Multiple proteins/enzymes such as histones, neutrophil elastase and myeloperoxidase are released along with NETs. Therefore, induction of NETosis in sterile inflammation is deleterious for many chronic diseases such as atherosclerosis. Abdominal aortic aneurysms (AAAs) remain a life-threatening disease in US adults with no available nonsurgical therapies highlighting a great need to develop a treatment strategy. During AAA formation, aortic wall undergoes major pathological changes with degradation of extracellular matrix and marked infiltration of inflammatory cells, such as neutrophils and lymphocytes. In murine experimental models, neutrophils have been detected in early stages of AAA growth. Recently, we have shown that genetic (IL-1ß and IL-1R1 deficiency in mice) and pharmacological (anakinra treatment) inhibition of IL-1ß signaling protect mice from AAA formation with significant reduction in aortic infiltration of inflammatory cells including neutrophils. Our preliminary result and published literatures suggest that IL-1ß induces NETosis in neutrophils. Therefore, we hypothesize that aortic IL-1ß induces NETosis and exacerbates inflammation in AAA. To test this hypothesis, we are proposing three specific aims. First we will test if genetic and pharmacological inhibition of IL-1ß signaling suppress NETosis and the AAA growth in mice. For these aims, we will use C57BL/6 (wild-type, WT), IL-1ß knockout (KO) and IL-1R1 KO mice, mouse AAA models such as elastase perfusion and angiotensin II infusion, knockdown IL-1ß or IL-1R1 using AMAXA Nucleofector, adoptive transfer of IL-1R1 KO neutrophils to neutrophil depleted WT mice. To examine and quantify NETosis, we will use the state-of-the-arts immunohistochemistry, confocal and intravital microscopy, and imaging flow cytometry. We will show that NET formation promotes inflammation and AAA growths, and, pharmacological inhibition of NETosis by (i) impairing IL-1ß signaling (using anakinra) and (ii) degrading NETs (using DNase), suppresses AAA growth in mice. We will further test the hypothesis that human neutrophils exhibit IL-1ß-dependent NETosis and induce inflammation in human aortic wall ex vivo. We will use proteomics approaches to identify proinflammatory proteins in human NETs, use human aortic explants to examine inflammatory response of IL-1b-induced NETs and parallel flow chambers to examine IL-1ß-induced NET-mediated immune cell recruitment from blood. Altogether, this proposal includes innovative concept that IL-1ß synthesized in aortic wall induces NETosis leading to increased tissue inflammation and AAA growth, and innovative methods to reduce NETosis and suppress AAA growth. These aims when achieved will provide the foundation for developing novel therapies for AAAs in human.

描述（由申请人提供）：中性粒细胞是血管壁损伤或损伤期间急性炎症的早期反应者。除了吞噬作用和脱粒作用之外，NETosis（中性粒细胞胞外陷阱或NET的形成）是中性粒细胞介导的防御机制的另一个方面。组蛋白、中性粒细胞弹性蛋白酶和髓过氧化物酶等多种蛋白质/酶与 NET 一起释放。因此，在无菌炎症中诱导NETosis对于许多慢性疾病如动脉粥样硬化是有害的。腹主动脉瘤（AAA）对于美国成年人来说仍然是一种危及生命的疾病，没有可用的非手术疗法，这突出表明非常需要制定治疗策略。在AAA形成过程中，主动脉壁发生重大病理变化，细胞外基质降解，中性粒细胞和淋巴细胞等炎性细胞明显浸润。在小鼠实验模型中，已在 AAA 生长的早期阶段检测到中性粒细胞。最近，我们发现，通过基因（小鼠中 IL-1ß 和 IL-1R1 缺陷）和药理学（阿那白滞素治疗）抑制 IL-1ß 信号可保护小鼠免受 AAA 形成，并显着减少包括中性粒细胞在内的炎症细胞的主动脉浸润。我们的初步结果和已发表的文献表明 IL-1ß 在中性粒细胞中诱导 NETosis。因此，我们假设主动脉 IL-1ß 诱导 NETosis 并加剧 AAA 中的炎症。为了检验这一假设，我们提出了三个具体目标。首先，我们将测试 IL-1ß 信号传导的遗传和药理学抑制是否会抑制小鼠中的 NETosis 和 AAA 生长。为了实现这些目标，我们将使用 C57BL/6（野生型，WT）、IL-1ß 敲除 (KO) 和 IL-1R1 KO 小鼠、小鼠 AAA 模型（例如弹性蛋白酶灌注和血管紧张素 II 输注）、使用 AMAXA Nucleofector 敲低 IL-1ß 或 IL-1R1、将 IL-1R1 KO 中性粒细胞过继转移至中性粒细胞耗尽的 WT 小鼠。为了检查和量化 NETosis，我们将使用最先进的免疫组织化学、共聚焦和活体显微镜以及成像流式细胞术。我们将证明 NET 的形成会促进炎症和 AAA 的生长，并且通过 (i) 损害 IL-1ß 信号传导（使用阿那白滞素）和 (ii) 降解 NET（使用 DNase）对 NETosis 进行药理学抑制，从而抑制小鼠的 AAA 生长。我们将进一步检验以下假设：人类中性粒细胞表现出 IL-1ß 依赖性 NETosis 并在体外诱导人主动脉壁炎症。我们将使用蛋白质组学方法来识别人类 NET 中的促炎蛋白，使用人类主动脉外植体来检查 IL-1b 诱导的 NET 的炎症反应，并使用平行流室来检查 IL-1ß 诱导的 NET 介导的免疫细胞从血液中的募集。总而言之，该提案包括主动脉壁中合成的 IL-1ß 诱导 NETosis 导致组织炎症增加和 AAA 生长的创新概念，以及减少 NETosis 和抑制 AAA 生长的创新方法。这些目标一旦实现，将为开发人类 AAA 的新疗法奠定基础。