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（由适用提供）：中性粒细胞是血管壁损伤或损伤期间对急性炎症的早期反应。除了吞噬作用和脱粒外，Netosis（中性粒细胞外陷阱或净的形成）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，例如组蛋白，中性粒细胞弹性酶和骨髓过氧化物酶与网络释放。因此，对于许多慢性疾病，例如动脉粥样硬化，无菌感染中肠病的诱导是无菌感染的。在美国成年人中，腹部主动脉瘤（AAA）仍然是一种威胁生命的疾病，没有可用的非手术疗法，强调了制定治疗策略的巨大需求。在AAA形成期间，主动脉壁随着细胞外基质降解和明显的炎症细胞（例如中性粒细胞和淋巴细胞）的明显浸润而经历重大病理变化。在AAA生长的早期阶段，已经检测到鼠实验模型，中性粒细胞。最近，我们已经表明，IL-1ß信号传导对IL-1ß信号传导抑制遗传（IL-1ß和IL-1R1缺乏症）和药物（Anakinra治疗）抑制可保护小鼠免受AAA形成，并显着降低包括中性粒细胞在内的炎性细胞的主动脉浸润。我们的初步结果和发表的文献表明，IL-1ß在中性粒细胞中诱导了肠病。因此，我们假设主动脉IL-1ß诱导Netosis并加剧AAA中的感染。为了检验这一假设，我们提出了三个具体目标。首先，我们将测试遗传和药物抑制IL-1ß信号传导抑制小鼠Netosis和AAA的生长。 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, adaptive transfer of IL-1R1 KO neutrophils to neutrophil depleted WT老鼠。为了检查和量化Netosis，我们将使用最新的免疫组织化学，共聚焦和内内显微镜以及成像流式细胞仪。我们将表明净形成促进注射和AAA生长，并将进一步检验以下假设：人类嗜中性粒细胞暴露了IL-1ß依赖性肠病并诱导人主动脉壁的炎症。我们将使用蛋白质组学方法来鉴定人类网中的促炎蛋白，使用人主动脉植体检查IL-1B诱导的网和平行流室的炎症反应，以检查IL-1ß诱导的网络介导的Immunocell从血液中募集。总而言之，该提案包括创新的概念，即主动脉壁中的IL-1ß合成会影响净生命，从而导致组织感染和AAA生长增加，以及减少Netosis并抑制AAA生长的创新方法。实现这些目标将为为人类的AAA开发新疗法提供基础。