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: 9111039
• 负责人: Gilbert Rivers Upchurch
• 金额: $ 39.5万
• 依托单位: UNIVERSITY OF VIRGINIA
• 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 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9111039
• 关键词: Abdomen; Abdominal Aortic Aneurysm; Acute; Adoptive Transfer; Adult; Aneurysm; Angiotensin II; Aorta; ApoE knockout mouse; Atherosclerosis; Blood; Blood Cells; Cells; Cellular Infiltration; Cessation of life; Chronic; Chronic Disease; Coculture Techniques; Confocal Microscopy; DNA; Data; Defense Mechanisms; Deoxyribonucleases; Development; Disease; Elastases; Enzymes; Exhibits; Experimental Models; Extracellular Matrix Degradation; Flow Cytometry; Foundations; Gelatinase A; Genetic; Genetic screening method; Gout; Growth; Health; Hemorrhage; High Mobility Group Proteins; Histones; Human; Human Volunteers; ITGAM gene; Image; Immune; Immune system; Immunofluorescence Immunologic; Immunohistochemistry; In Vitro; Infiltration; Inflammation; Inflammatory; Inflammatory Response; Infusion procedures; Injury; Interleukin-1 beta; Knock-out; Knockout Mice; Leukocyte Elastase; Life; Literature; Lymphocyte; Medial; Mediating; Medical; Methods; Modeling; Mus; Necrosis; Neutrophil Activation; Operative Surgical Procedures; Pathogenesis; Patients; Perfusion; Peroxidases; Phagocytosis; Pharmacological Treatment; Plasma; Play; Postoperative Period; Process; Proteins; Proteomics; Publishing; Recruitment Activity; Research; Role; Rupture; Ruptured Abdominal Aortic Aneurysm; Scanning Electron Microscopy; Sepsis; Shock; Signal Transduction; Staging; Staining method; Stains; Sterility; Testing; Thrombosis; Tissues; Trauma; Vasculitis; Western Blotting; Wild Type Mouse; Work; abdominal aorta; anakinra; cell type; extracellular; immunocytochemistry; inflammatory marker; inhibitor/antagonist; innovation; intravital microscopy; knock-down; microscopic imaging; neutrophil; novel therapeutics; peripheral blood; recombinant human DNase; repaired; response; response to injury; treatment strategy

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（由申请方提供）：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（形成神经细胞外陷阱或NET）是嗜中性粒细胞介导的防御机制的另一个方面。多种蛋白质/酶如组蛋白、嗜中性粒细胞弹性蛋白酶和髓过氧化物酶与NET一起沿着释放。因此，在无菌性炎症中诱导NETosis对许多慢性疾病如动脉粥样硬化是有害的。腹主动脉瘤（AAA）在美国成人中仍然是一种危及生命的疾病，没有可用的非手术治疗方法，这突出了制定治疗策略的迫切需要。在AAA形成过程中，主动脉壁经历了主要的病理变化，细胞外基质降解和显著的炎性细胞（如中性粒细胞和淋巴细胞）浸润。在鼠实验模型中，在AAA生长的早期阶段检测到中性粒细胞。最近，我们已经表明，遗传（小鼠中的IL-1 β和IL-1 R1缺乏）和药理学（阿那白滞素治疗）抑制IL-1 β信号传导可保护小鼠免于AAA形成，并显著减少炎性细胞（包括中性粒细胞）的主动脉浸润。我们的初步结果和已发表的文献表明，IL-1 β诱导中性粒细胞NETosis。因此，我们假设主动脉IL-1 β诱导NETosis并加重AAA中的炎症。为了验证这一假设，我们提出了三个具体目标。首先，我们将测试是否遗传和药理学抑制IL-1 β信号转导抑制NETosis和AAA生长的小鼠。为了这些目的，我们将使用C57 BL/6（野生型，WT）、IL-1 R1敲除（KO）和IL-1 R1 KO小鼠、小鼠AAA模型（如弹性蛋白酶灌注和血管紧张素II输注）、使用AMAXA Nucleofector敲低IL-1 R1或IL-1 R1、将IL-1 R1 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形成促进炎症和AAA生长，并且通过（i）损害IL-1 β信号传导（使用阿那白滞素）和（ii）降解NET（使用DNA酶）药理学抑制NETosis，抑制小鼠AAA生长。我们将进一步检验人中性粒细胞表现出IL-1 β依赖性NETosis并诱导离体人主动脉壁炎症的假设。我们将使用蛋白质组学的方法来确定促炎蛋白在人类NET，使用人主动脉外植体检查IL-1b诱导的NET和平行流动室的炎症反应，检查IL-1b诱导的NET介导的免疫细胞从血液中招募。总而言之，该提案包括主动脉壁中合成的IL-1 β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，将为开发治疗人类AAAs的新疗法提供基础。