Brain endothelium and innate immune responses after stroke

中风后的脑内皮和先天免疫反应

基本信息

批准号：
10303327
负责人：
William Taylor Kimberly
金额：
$ 45.14万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-06-01 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10303327
关键词：
Activation Analysis Acute Acute Brain Injuries Address Age Animals Astrocytes Attenuated Binding Biological Assay Biological Models Biological Response Modifiers Blood Blood - brain barrier anatomy Blood Circulation Brain Brain Edema Brain Injuries CRISPR/Cas technology Cells Cerebral Edema Cerebral hemisphere Cessation of life Communication Complement Contralateral Data Development Dissociation Edema Endothelial Cells Endothelium Exploratory/Developmental Grant Exposure to Female Filament Flow Cytometry Fluorescence-Activated Cell Sorting Functional disorder Future Gatekeeping Human Immune Immune response Immunomodulators In Situ Hybridization Inflammation Inflammatory Inflammatory Response Injury Innate Immune Response Investigation Ischemic Stroke Knock-out Laboratories Ligands Location Macrophage Activation Measurement Measures Mediating Microglia Middle Cerebral Artery Occlusion Modeling Mononuclear Morbidity - disease rate Myelogenous Myeloid Cells Nervous System Physiology Neurological outcome Neurological status Neurons Nuclear Translocation Oligodendroglia PECAM1 gene PTPRC gene Pathway interactions Patients Pericytes Peripheral Phase Plasma Play Population Production Rattus Role Sentinel Signal Transduction Source Stroke Testing Time Tissues Transcript Water Work aged attenuation brain cell brain endothelial cell brain tissue cell type cytokine experimental study high reward high risk immune activation in vivo knock-down macrophage male monocyte mortality neurovascular injury novel p65 post stroke promoter receptor recruit response selective expression small hairpin RNA stroke model stroke patient therapeutic target tool

项目摘要

ABSTRACT Endothelial cells reside at the interface of the blood-brain barrier, making them ideally situated to act as a gatekeeper of the systemic immune response to acute brain injury. Recent work in our laboratory has identified a circulating immune regulator, soluble ST2 (sST2), which we propose as a key signal that bridges brain injury and the inflammatory response. We have shown that sST2 is expressed by brain endothelial cells and elevated in both brain tissue and systemic circulation after experimental stroke in rats. Our pilot rat studies demonstrate that sST2 expression peaks at 3 days after stroke during the acute pro-inflammatory phase, and rapidly subsides by day 7 during the transition to the reparative phase of the immune response. In human patients, sST2 plasma level in the first 3 days after stroke is associated with cerebral edema, inflammatory peripheral monocytes, and long-term neurologic outcome after acute neurovascular injury. We hypothesize that the sST2 pathway exemplifies the principle that following acute injury, the brain endothelium secretes signals both locally and into circulation that orchestrate a biphasic innate immune response. We predict that excessive activation of local microglia and blood-derived macrophages due to acutely increased sST2 level augment acute inflammation, increase damage to the blood-brain barrier, and enhance edema formation. We further predict that the rapid decline in sST2 level by day 7 facilitates the transition to reparative inflammation. However, our understanding of the signaling that regulates communication between the ischemic brain and acute myeloid inflammatory cells is incomplete. In Aim 1, we will validate the brain sST2 source by using fluorescence activated cell sorting of brain cells and in situ hybridization. We will functionally characterize the activation profile of tissue resident microglia/macrophages and peripheral myeloid cells through flow cytometry, qPCR array, and cytokine analysis. We will directly perturb the activation state by ex vivo exposure to sST2 or its neuroprotective ligand, IL-33. We will also examine how these findings vary in the setting of aged animals, which has an important impact on the inflammatory response. In Aim 2, we will directly test our hypothesis that sST2 plays a causal role by studying an sST2 knockout rat that we generated via CRISPR-Cas9. We will complement these studies by knocking down sST2 level in vivo using AAV9-shRNA delivered to the brain. In each model system, we will assess the effect of reduced or absent sST2 on brain-resident and circulating innate immune cells, anticipating an attenuation of the acute pro-inflammatory response and an augmentation of the reparative response. We will further examine how reducing sST2 level alters brain edema and brain water content. Taken together, this study provides a critical opportunity to answer fundamental questions about the signaling role of endothelium in the pathophysiology of brain edema and determine the role of sST2.

抽象的内皮细胞位于血脑屏障的界面上，使其理想地充当对急性脑损伤的全身免疫反应的看门人。我们实验室的最新工作已经确定循环免疫调节剂可溶性ST2（SST2），我们建议将其作为桥接脑损伤的关键信号和炎症反应。我们已经表明，SST2由脑内皮细胞表达并升高在大鼠实验性中风后脑组织和全身循环中。我们的飞行员大鼠研究表明 SST2表达在急性促炎阶段的中风后3天达到峰值，并迅速消退到第7天，在过渡到免疫反应的修复阶段。在人类患者中，SST2血浆中风后的前三天水平与脑水肿，炎症性周围单核细胞和急性神经血管损伤后的长期神经系统结局。我们假设SST2途径例证了急性损伤后的原则，脑内皮分泌在本地和进入精心策划双相先天免疫反应的循环。我们预测局部过度激活小胶质细胞和血液衍生的巨噬细胞由于SST2水平急性增加急性炎症，增加对血脑屏障的损害，并增强水肿的形成。我们进一步预测了快速第7天，SST2水平的下降促进了对修复性炎症的过渡。但是，我们的理解调节缺血性脑和急性髓样炎症细胞之间通信的信号传导是不完整的。在AIM 1中，我们将使用荧光激活的细胞排序来验证脑SST2源脑细胞和原位杂交。我们将在功能上表征组织驻留的激活曲线小胶质细胞/巨噬细胞和周围髓样细胞通过流式细胞仪，QPCR阵列和细胞因子分析。我们将通过离体暴露于SST2或其神经保护配体IL-33来直接扰动激活状态。我们还将研究这些发现在老年动物的环境中如何有所不同，这对炎症反应。在AIM 2中，我们将直接检验我们的假设，即SST2通过研究起着因果作用我们通过CRISPR-CAS9生成的SST2敲除老鼠。我们将通过击倒来补充这些研究 SST2在体内使用AAV9-shRNA的体内水平。在每个模型系统中，我们将评估脑居住和循环的先天免疫细胞减少或不存在SST2，预计会减弱急性促炎反应和修复反应的增强。我们将进一步研究如何降低SST2水平会改变脑水肿和脑水含量。综上所述，这项研究提供了关键回答有关内皮在病理生理学中的信号传导作用的基本问题的机会脑水肿并确定SST2的作用。