Variability in neutrophil activation

中性粒细胞激活的变异性

• 批准号: 10246517
• 负责人: Grace Ming Lee
• 金额: $ 8.05万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10246517
• 关键词: Adherence; Affect; Agonist; Antibodies; Antigen-Antibody Complex; Antiphospholipid Syndrome; Award; Biological; Biological Assay; Biology; Blood Coagulation Factor; Cardiopulmonary Bypass; Cell surface; Characteristics; Complication; Coupled; Cytoplasmic Granules; Data; Disease; Disease Outcome; Fc Receptor; Fostering; Future; G-Protein-Coupled Receptors; GTP-Binding Proteins; Generations; Heparin; Immune; Immunology; Individual; Knowledge; Laboratories; Learning; Leukocytes; Lipids; Lipopolysaccharides; Measures; Mediating; Mediator of activation protein; Metalloproteases; Myeloproliferative disease; Neutrophil Activation; Operative Surgical Procedures; Pathology; Pathway interactions; Patients; Phenotype; Phosphotransferases; Physiological; Platelet Activation; Play; Population; Predisposition; Protamines; Publications; Publishing; Reporting; Research; Research Personnel; Research Support; Resistance; Resolution; Risk; Risk Assessment; Role; Sickle Cell Anemia; Signal Pathway; Signal Transduction; Techniques; Testing; Thrombin; Thrombosis; Thrombus; Time; Toll-like receptors; Translating; Tumor Antibodies; Variant; Vascular Endothelial Cell; Whole Blood; Work; adverse outcome; base; cytokine; density; granulocyte; leucyl-phenylalanine; leucylmethionine; neutrophil; prevent; receptor; receptor-mediated signaling; response; skills; stem; success; therapeutic target; thrombotic

Project Summary/Abstract I am currently supported by a K08 (5K08-HL127183, awarded 9/4/2015) to study the biological activity of protamine/heparin immune complexes (IC), a newly described immune complication in the cardiopulmonary bypass population. In the course of my work, I developed a whole blood assay to measure IC-mediated neutrophil activation, as measured by matrix metalloprotease 9 granule release. Using this assay, in a recent publication, we report that the neutrophil response to ICs is highly variable among healthy subjects, and this variability represents a fixed response: some individuals have neutrophils which always activate readily in the presence of ICs, while others have neutrophils which are always minimally responsive. We have termed this susceptibility to neutrophil degranulation, the “neutrophil activation phenotype.” In new preliminary data, we now show that the neutrophil activation phenotype: 1) is not associated with Fc receptor variants, 2) is not limited to IC-Fc receptor interactions and is more broadly reflective of susceptibility to neutrophil activation by a variety of receptor- mediated agonists, 3) is determined at the cellular level and is retained with isolated neutrophils, and 4) is correlated with propensity for NET release. Building on this strong new preliminary data, I will test the hypothesis that the neutrophil activation phenotype is determined by differences in receptor-mediated signaling responses and is correlated with differences in procoagulant activity. In this proposal, I will determine the cellular factors which contribute to the neutrophil activation phenotype. In Subaim 1, I will determine if the phenotype is associated with differences in neutrophil effector function beyond degranulation, and I will examine ROS generation, neutrophil adherence, and ability to release proinflammatory lipid and cytokine mediators. In this subaim, I will also quantify low density granulocytes, a subpopulation with enhanced effector functions. In Subaim 2, I will build on preliminary data demonstrating that the response to ICs is highly correlated with the response to fMLP and to LPS, and I will determine if differences in signaling through the PI3K pathway (a point of convergence for neutrophil Fcɣ, G-protein coupled, and Toll-like receptors) contribute to the phenotype. In Subaim 3, I will build on preliminary data demonstrating that “high” subjects have a greater tendency to undergo NETosis, and I will determine if this translates into differences in procoagulant activity. Specifically, I will determine if the phenotype correlates with NET and NET component release, resistance to NET dissolution, and thrombin generation. In completing the proposed work, I will expand on my current K08-supported research objectives and investigate the heterogeneous neutrophil response in healthy subjects. In doing so I will continue to develop new knowledge/skills in immunology and neutrophil biology, learn new laboratory techniques, and foster new collaborative relationships. With the support of this R03, I will generate sufficient preliminary data for a R01 application to identify strong predictors of disease outcome and to identify therapeutic targets in disease. All of these skills will be essential to my long-term success as I transition to an independent investigator.

项目概要/摘要 我目前得到 K08（5K08-HL127183，于 2015 年 9 月 4 日授予）的支持，研究 鱼精蛋白/肝素免疫复合物（IC），一种新描述的心肺免疫并发症 绕过人口。在工作过程中，我开发了一种全血检测方法来测量 IC 介导的中性粒细胞 活化，通过基质金属蛋白酶 9 颗粒释放来测量。在最近的出版物中使用这种测定法， 我们报告说，中性粒细胞对 IC 的反应在健康受试者中存在很大差异，并且这种差异 代表固定的反应：有些人的中性粒细胞在存在时总是很容易激活 IC，而其他人则具有中性粒细胞，其反应始终最低。我们将这种敏感性称为 中性粒细胞脱颗粒，“中性粒细胞激活表型”。在新的初步数据中，我们现在表明 中性粒细胞激活表型：1) 与 Fc 受体变异无关，2) 不限于 IC-Fc 受体 相互作用，更广泛地反映了各种受体对中性粒细胞激活的易感性 介导的激动剂，3) 在细胞水平上测定并与分离的中性粒细胞一起保留，4) 是 与 NET 释放倾向相关。基于这个强有力的新初步数据，我将检验这个假设 中性粒细胞激活表型是由受体介导的信号反应的差异决定的 并且与促凝血活性的差异相关。在这个提案中，我将确定细胞因素 这有助于中性粒细胞激活表型。在 Subaim 1 中，我将确定表型是否为 与脱粒之外的中性粒细胞效应功能差异相关，我将检查 ROS 生成、中性粒细胞粘附以及释放促炎脂质和细胞因子介质的能力。在这个 subaim，我还将量化低密度粒细胞，这是一种具有增强效应功能的亚群。位于苏拜姆 2、我将根据初步数据证明对 IC 的反应与反应高度相关 fMLP 和 LPS，我将确定通过 PI3K 途径的信号传导是否存在差异（这一点 中性粒细胞 Fcɣ、G 蛋白偶联和 Toll 样受体的收敛）对表型有贡献。在 Subaim 3，我将根据初步数据证明“高”受试者更有可能经历 NETosis，我将确定这是否会转化为促凝血活性的差异。具体来说，我将 确定表型是否与 NET 和 NET 成分释放、对 NET 溶解的抵抗力相关，以及 凝血酶的产生。在完成拟议的工作时，我将扩展我当前 K08 支持的研究 目标并研究健康受试者的异质性中性粒细胞反应。这样做我将继续 发展免疫学和中性粒细胞生物学方面的新知识/技能，学习新的实验室技术，以及 培养新的合作关系。有了这个R03的支持，我会生成足够的初步数据 R01 应用程序可识别疾病结果的强预测因素并确定疾病的治疗靶点。 当我转变为独立调查员时，所有这些技能对于我的长期成功至关重要。