Neutrophil Development During Inflammation and Atherosclerosis

项目摘要 中性粒细胞对组织中的炎症信号有急性反应，并在心血管疾病中发挥作用 （CAD）。在被胆固醇和氧化低密度脂蛋白激活后，中性粒细胞产生细胞因子， 去甲肾上腺素释放髓过氧化物酶、杀天青素和弹性蛋白酶，产生中性粒细胞胞外陷阱 （NET），并触发裂解性细胞死亡。中性粒细胞由骨髓中的干细胞祖细胞产生 一旦成熟，就能排出到血液和组织中对抗炎症。我们最近发现了一个早期的 定向中性粒细胞祖干细胞（NeP）存在于人类和小鼠骨髓中。这里我们 目前的新数据表明，NeP存在于小鼠和人类的外周血中， CAD，表明NeP调节骨髓外的粒细胞生成和炎症。在这 项目，我们将集中在炎症通过激活Nlrp 3炎性体，因为我们发现，Nlrp 3和 lllb在NeP中高度表达。炎性小体传感器Nlrp 3的激活触发了一个强大的 炎症反应，包括IL-1β的产生。在项目1中，我们假设， NeP内的Nlrp 3炎性体触发粒细胞生成和中性粒细胞异质性， 促进动脉粥样硬化进展。我们将通过研究炎性小体来验证我们的假设 在患有心血管疾病（CAD）的小鼠和人类中激活。在具体目标1中，我们将测试 冠心病患者中性粒细胞亚群的表型和功能变化 与健康受试者相比。这些受试者正在接受医学上必要的冠状动脉 血管造影术作为正在进行的弗吉尼亚州冠状动脉评估（CAVA）研究的一部分，以及其 动脉粥样硬化是临床测量的。我们将分析NeP、中性粒细胞异质性以及 这些受试者血液中的嗜中性Nlrp 3炎性小体，并将这些发现与临床参数联系起来。 在具体目标2中，我们将使用动脉粥样硬化小鼠模型来测试Nlrp 3的内在作用如何。 中性粒细胞中的炎性小体影响粒细胞生成和动脉粥样硬化。我们将使用混合 使用Nlrp 3-/-、Gsdmd-/-和Gsdme-/-小鼠的嵌合小鼠模型，以评估炎性体途径如何 NeP调节粒细胞生成。新的嗜中性粒细胞特异性和巨噬细胞特异性Nlrp 3敲除小鼠 与动脉粥样硬化易感的载脂蛋白E缺陷（apoE-/-）小鼠杂交，将用于直接 比较中性粒细胞与巨噬细胞Nlrp 3炎性小体的作用如何影响中性粒细胞异质性 和动脉粥样硬化。我们将使用一种新的小鼠模型来评估Nlrp 3在NeP中的作用， 中性粒细胞对动脉粥样硬化的影响我们将定义Nlrp 3炎性小体如何在中性粒细胞祖细胞中活化， 影响粒细胞生成和动脉粥样硬化，这反过来又可能导致新的治疗方法 靶向中性粒细胞中的Nlrp 3组分用于动脉粥样硬化和其它炎性疾病。

PROJECT SUMMARY Neutrophils acutely respond to inflammatory signals in tissues and play a role in cardiovascular disease (CAD). After activation by cholesterol and oxidized low density lipoproteins, neutrophils produce cytokines, degranulate to release myeloperoxidase, azurocidin, and elastase, generate neutrophil extracellular traps (NETs), and trigger lytic cell death. Neutrophils are produced in the bone marrow by stem cell progenitors and, once mature, egress to blood and tissues to fight inflammation. We recently discovered an early committed neutrophil progenitor stem cell (NeP) present in human and mouse bone marrow. Here, we present new data demonstrating the presence of NeP in the peripheral blood of mice and humans with CAD, suggesting that NeP modulate granulopoiesis and inflammation outside of the bone marrow. In this project, we will focus on inflammation via activation of the Nlrp3 inflammasome, as we find that Nlrp3 and ll1b are highly expressed in NeP. Activation of the inflammasome sensor Nlrp3 triggers a robust inflammatory response, including IL-1β production. In Project 1, we hypothesize that activation of the Nlrp3 inflammasome within NeP triggers granulopoiesis and neutrophil heterogeneity which promotes atherosclerosis progression. We will test our hypothesis by studying inflammasome activation in both mice and humans with cardiovascular disease (CAD). In Specific Aim 1 we will test how neutrophil subsets are phenotypically and functionally changed in human CAD patients compared to healthy subjects. These subjects are undergoing medically necessary coronary angiography as part of the ongoing Coronary Assessment in Virginia (CAVA) study, and their extent of atherosclerosis is measured clinically. We will analyze NeP, neutrophil heterogeneity, and the role of the neutrophilic Nlrp3 inflammasome in blood of these subjects and link these findings to clinical parameters. In Specific Aim 2 we will use atherosclerotic mouse models to test how intrinsic action of the Nlrp3 inflammasome in neutrophils impacts granulopoiesis and atherosclerosis. We will use mixed chimeric mouse models with Nlrp3-/-, Gsdmd-/-, and Gsdme-/- mice to assess how inflammasome pathways in NeP regulate granulopoiesis. New neutrophil-specific and macrophage-specific Nlrp3 knock-out mice crossed to atherosclerosis-susceptible, apolipoprotein E-deficient (apoE-/-) mice will be used to directly compare how action of neutrophil vs macrophage Nlrp3 inflammasomes impact neutrophil heterogeneity and atherosclerosis. We will use a new mouse model to assess Nlrp3 action in NeP versus mature neutrophils on atherosclerosis. We will define how Nlrp3 inflammasome activation in neutrophil progenitors impacts granulopoiesis and atherosclerosis, which in turn could lead to new therapeutic approaches targeting Nlrp3 components in neutrophils for atherosclerosis and other inflammatory diseases.