Neutrophil Activation Phenotypes in Healthy Subjects and Implications for Bacterial Clearance

项目总结/摘要 在我目前的K 08（5 K 08-HL 127183，“鱼精蛋白/肝素的生物学意义”）衍生的工作中 抗体”），我们开发了全血测定来测量中性粒细胞活化/脱粒，如定量的 通过基质金属蛋白酶9（MMP 9）颗粒释放。在最近发表的工作（Duarte ME等人，Blood Adv. 2019），我们发现健康受试者对免疫复合物激活中性粒细胞的易感性不同， (ICs)导致不同程度的MMP 9释放。通过纵向测试，我们发现这种易感性 对于给定个体来说，脱颗粒代表固定的表型：一些受试者具有中性粒细胞， 总是很容易激活，而其他人有中性粒细胞持续最低限度的反应。我们有 将这种对中性粒细胞脱粒的易感性称为“中性粒细胞活化表型”。因为专注 在我的K 08中，我最初的研究集中在中性粒细胞对各种抗原/抗体IC的反应上。 然而，在新的初步数据中，我们现在表明中性粒细胞活化表型：1）不限于 IC/Fcγ受体相互作用，而是更广泛地反映了对中性粒细胞活化的易感性， 多种激动剂，包括细菌肽N-甲酰基-甲硫氨酸-亮氨酸-苯丙氨酸（fMLP）、脂多糖（LPS）和 细菌上清液，2）与对PI 3 K抑制的不同敏感性相关，这是 许多中性粒细胞活化途径，3）是家族性的，4）并且与NET释放和ROS产生相关。 基于这一强有力的初步数据，我们将检验中性粒细胞活化表型是 由受体介导的信号传导反应的遗传差异决定，导致 杀菌活性在目标1中，我们将确定有助于中性粒细胞增殖的细胞和遗传因素。 活化表型我们将确定是否信号差异有助于中性粒细胞表型。我们将 检查沿着PI 3 K通路多个位点的激活模式，并测试特定激酶的作用 抑制剂的在这个目标中，我们也将建立在我们的新的观察，即中性粒细胞表型是家族性的， 我们将对来自表型受试者的幼稚和活化中性粒细胞进行RNA测序，以确定遗传学特征。 嗜中性粒细胞表型的决定因素。在目标2中，我们将确定中性粒细胞活化表型是否具有 与感染性疾病和细菌清除的相关性。我们将使用金黄色葡萄球菌作为模型 病原体，以确定是否其他中性粒细胞效应功能，除了脱粒，与 中性粒细胞活化表型并且，我们将确定表型是否转化为杀菌能力的差异， 活动在完成拟议的工作，我们将确定异质性中性粒细胞反应的决定因素， 在健康受试者中，我们将确定中性粒细胞反应性的这种变异性是否导致了 杀死细菌。在此R21的支持下，我们将为R 01应用程序生成足够的初步数据， 确定中性粒细胞活化表型是否有助于在以下患者中观察到的异质性： 感染和确定疾病结果的强预测因子。

Project Summary/Abstract In work derived from my current K08 (5K08-HL127183, “Biological Significance of Protamine/Heparin Antibodies”), we developed a whole blood assay to measure neutrophil activation/degranulation, as quantified by matrix metalloprotease 9 (MMP9) granule release. In recently published work (Duarte ME, et al. Blood Adv. 2019), we showed that healthy subjects differ in their susceptibility to neutrophil activation by immunecomplexes (ICs), resulting in varying degrees of MMP9 release. With longitudinal testing, we showed that this susceptibility to degranulation represents a fixed phenotype for a given individual: some subjects have neutrophils which always activate readily, while others have neutrophils which are persistently minimally responsive. We have termed this susceptibility to neutrophil degranulation, the “neutrophil activation phenotype”. Because of the focus of my K08, my initial studies were focused on the neutrophil response to a variety of antigen/antibody ICs. However, in new preliminary data, we now show that the neutrophil activation phenotype: 1) is not limited to IC/Fcγ receptor interactions and instead, is more broadly reflective of susceptibility to neutrophil activation by a variety of agonists including the bacterial peptide N-formyl-met-leu-phe (fMLP), lipopolysaccharide (LPS), and bacterial supernatant, 2) is associated with differential sensitivity to PI3K inhibition, a point of convergence for many neutrophil activation pathways, 3) is familial, 4) and is correlated with NET release and ROS generation. Building on this strong preliminary data, we will test the hypothesis that the neutrophil activation phenotype is determined by genetic differences in receptor-mediated signaling responses, resulting in differences in bactericidal activity. In Aim 1, we will determine the cellular and genetic factors which contribute to the neutrophil activation phenotype. Wewill determine if differences in signaling contribute to the neutrophil phenotype. We will examine activation patterns at multiple sites along the PI3K pathway and test the effect of specific kinase inhibitors. In this aim, we will also build on our novel observation that the neutrophil phenotype is familial, and we will perform RNA sequencing of naïve and activated neutrophils from phenotyped subjects to identify genetic determinants of the neutrophil phenotype. In Aim 2, we will determine if the neutrophil activation phenotype has relevance to infectious disease and bacterial clearance. We will use Staphylococcus aureus as a model pathogen to determine if other neutrophil effector functions, beyond degranulation, are associated with the neutrophil activation phenotype. And, we will determine if the phenotype translates into differences in bactericidal activity. In completing the proposed work, we will identify determinants of the heterogeneous neutrophil response in healthy subjects, and we will determine if this variability in neutrophil reactivity results in differential ability to kill bacteria. With the support of this R21, we will generate sufficient preliminary data for a R01 application to determine if the neutrophil activation phenotype contributes to the heterogeneity seen among patients with infection and identify strong predictors of disease outcome.