Variability in neutrophil activation

项目摘要/摘要 我目前得到了K08(5K08-HL127183,2015年9月4日获奖)的支持，以研究 鱼精蛋白/肝素免疫复合体(IC)--一种新的心肺免疫并发症 绕过人口。在我工作的过程中，我开发了一种全血检测方法来测量IC介导的中性粒细胞 激活，通过基质金属蛋白酶9颗粒释放来衡量。在最近的一份出版物中，使用这种分析方法， 我们报告说，中性粒细胞对ICS的反应在健康受试者中是高度可变的，并且这种变异性 代表一种固定的反应：一些人有中性粒细胞，在病毒存在时总是很容易激活 ICS，而其他人的中性粒细胞总是最小的反应。我们将这种易感性称为 中性粒细胞脱颗粒，即“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受体变异体无关，2)不限于IC-Fc受体 更广泛地反映了多种受体激活中性粒细胞的敏感性。 介导的激动剂，3)在细胞水平上确定，并与分离的中性粒细胞一起保留，以及4)是 与净释放倾向相关。基于这一强劲的新初步数据，我将检验这一假设 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 并与促凝血剂活性的差异相关。在这项提案中，我将确定细胞因素 这与中性粒细胞激活表型有关。在Subaim 1中，我将确定表型是否为 与脱颗粒后中性粒细胞效应器功能的差异有关，我将检查ROS 产生、中性粒细胞黏附，以及释放促炎脂质和细胞因子介质的能力。在这 子目标，我还将量化低密度粒细胞，这是一个具有增强效应功能的亚群。在苏巴伊姆 2，我将以初步数据为基础，证明对IC的反应与反应高度相关 到fMLP和内毒素，我将确定通过PI3K途径的信号差异(一点 中性粒细胞Fcɣ、G蛋白偶联和Toll样受体的融合对表型有贡献。在……里面 Subaim 3，我将建立在初步数据的基础上，证明“高”的受试者更有可能经历 NETsis，我将确定这是否会转化为促凝血活性的差异。具体来说，我会 确定表型是否与净释放和净成分释放、对净溶出的抵抗力相关，以及 凝血酶生成。在完成拟议的工作时，我将对我目前由K08支持的研究进行扩展 目的探讨健康人中性粒细胞异质性反应。在这样做的同时，我将继续 发展免疫学和中性粒细胞生物学的新知识/技能，学习新的实验室技术，以及 培养新的协作关系。在R03的支持下，我将为以下内容生成足够的初步数据 R01应用程序，用于确定疾病结局的强大预测因素，并确定疾病的治疗靶点。 当我过渡到一名独立的调查员时，所有这些技能对我的长期成功都是必不可少的。

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.