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（由申请人提供）：中性粒细胞是血管壁损伤或损伤时急性炎症的早期反应者。除了吞噬和脱粒，NETosis（形成中性粒细胞胞外陷阱或NET）是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、中性粒细胞弹性酶和髓过氧化物酶与NETs一起释放。因此，无菌炎症诱导NETosis对许多慢性疾病（如动脉粥样硬化）是有害的。腹主动脉瘤（AAAs）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，因此非常需要制定治疗策略。在AAA形成过程中，主动脉壁发生了主要的病理改变，细胞外基质降解，炎症细胞（如中性粒细胞和淋巴细胞）明显浸润。在小鼠实验模型中，在AAA生长的早期阶段检测到中性粒细胞。最近，我们发现遗传（小鼠IL-1ß和IL-1R1缺乏）和药理学（阿那白素治疗）抑制IL-1ß信号可以保护小鼠免受AAA的形成，并显著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诱导中性粒细胞NETosis。因此，我们假设主动脉IL-1ß诱导NETosis并加剧AAA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IL-1ß信号的遗传和药理抑制是否会抑制小鼠NETosis和AAA的生长。为了达到这些目的，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，如弹性蛋白酶灌注和血管紧张素II输注，使用AMAXA核因子敲除IL-1ß或IL-1R1，过性转移IL-1R1 KO中性粒细胞到中性粒细胞枯竭的WT小鼠。为了检验和量化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的形成促进炎症和AAA的生长，并且，通过(i)损害IL-1ß信号（使用安纳白素）和（ii）降解NET（使用dna酶）的药物抑制NETosis，抑制小鼠AAA的生长。我们将进一步验证人中性粒细胞在体外表现出il -1ß依赖性NETosis并诱导人主动脉壁炎症的假设。我们将使用蛋白质组学方法鉴定人类NETs中的促炎蛋白，使用人主动脉外植体检测il -1b诱导的NETs的炎症反应，平行流室检测il -1ß诱导的net介导的血液免疫细胞募集。综上所述，该提案包括了在主动脉壁合成IL-1ß诱导NETosis导致组织炎症和AAA生长增加的创新概念，以及减少NETosis和抑制AAA生长的创新方法。这些目标的实现将为开发人类AAAs的新疗法提供基础。