Variability in neutrophil activation

项目总结/摘要 我目前得到K 08（5 K 08-HL 127183，2015年9月4日授予）的支持，研究以下物质的生物活性： 鱼精蛋白/肝素免疫复合物（IC），一种新的心肺免疫并发症 分流人口。在我的工作过程中，我开发了一种全血测定法来测量IC介导的中性粒细胞 活化，如通过基质金属蛋白酶9颗粒释放测量的。在最近的出版物中， 我们报告说，中性粒细胞对IC的反应在健康受试者中是高度可变的， 代表了一种固定的反应：有些人有中性粒细胞，在存在的情况下总是容易激活。 IC，而其他人有中性粒细胞，总是最低限度的反应。我们把这种易感性称为 中性粒细胞脱颗粒，即“中性粒细胞活化表型”。在新的初步数据中，我们现在表明， 中性粒细胞活化表型：1）与Fc受体变体无关，2）不限于IC-Fc受体 相互作用，并更广泛地反映了各种受体对中性粒细胞活化的敏感性， 介导的激动剂，3）在细胞水平测定并保留在分离的嗜中性粒细胞中，和4） 与NET释放倾向相关。基于这些强有力的新的初步数据， 中性粒细胞活化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。在这个建议中，我将确定细胞因素 其有助于嗜中性粒细胞活化表型。在Subaim 1中，我将确定表型是否是 与中性粒细胞效应器功能的差异有关，除脱粒外，我将检查ROS 生成、嗜中性粒细胞粘附和释放促炎脂质和细胞因子介质的能力。在这 子目标，我也将量化低密度粒细胞，增强效应功能的亚群。因苏拜姆 2，我将以初步数据为基础，证明对IC的反应与对IC的反应高度相关。 fMLP和LPS，我将确定是否通过PI 3 K途径的信号传导差异（点 中性粒细胞Fc γ、G蛋白偶联受体和Toll样受体的会聚）有助于表型。在 Subaim 3，我将建立在初步数据表明，“高”的主题有更大的倾向， NETosis，我将确定这是否会转化为促凝血活性的差异。具体来说，我会 确定表型是否与NET和NET组分释放、NET溶解抗性相关，以及 凝血酶生成。在完成建议的工作，我将扩大我目前的K 08支持的研究 目的探讨健康人中性粒细胞反应的异质性。为此，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， 培养新的合作关系。在此R 03的支持下，我将生成足够的初步数据， R 01应用程序，用于确定疾病结局的强预测因子，并确定疾病中的治疗靶点。 所有这些技能将是必不可少的，我的长期成功，因为我过渡到一个独立的调查员。

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.