Brain endothelium and innate immune responses after stroke

摘要 内皮细胞位于血脑屏障的界面，使它们理想地位于充当脑屏障的位置。 急性脑损伤的全身免疫反应的守门人。我们实验室最近的工作已经确定 一种循环免疫调节剂，可溶性ST 2（sST 2），我们认为它是连接脑损伤的关键信号 和炎症反应。我们已经证明sST 2由脑内皮细胞表达，并且在脑血管内皮细胞中升高。 在大鼠实验性中风后的脑组织和体循环中。我们的试点大鼠研究表明， sST 2表达在中风后3天的急性促炎期达到峰值，并迅速消退， 在向免疫应答的修复阶段过渡期间的第7天。在人类患者中，sST 2血浆 中风后前3天的水平与脑水肿、炎性外周单核细胞和 急性神经血管损伤后的长期神经学结局。我们假设sST 2通路 阐明了急性损伤后，脑内皮细胞在局部和脑内分泌信号的原理。 血液循环，协调双相先天免疫反应。我们预测，过度激活局部 由于sST 2水平的急性增加而导致的小胶质细胞和血液来源的巨噬细胞增加急性炎症， 增加对血脑屏障的损害，并促进水肿形成。我们进一步预测， 到第7天sST 2水平的下降促进向修复性炎症的转变。然而，我们的理解是， 调节缺血性脑和急性骨髓炎性细胞之间通讯的信号 不完整在目标1中，我们将通过使用荧光激活细胞分选来验证脑sST 2来源。 脑细胞和原位杂交。我们将从功能上表征组织驻留的激活概况， 通过流式细胞术、qPCR阵列和细胞因子分析，对小胶质细胞/巨噬细胞和外周髓样细胞进行细胞因子分析。 我们将通过离体暴露于sST 2或其神经保护性配体IL-33来直接扰乱激活状态。我们 还将研究这些发现在老年动物的环境中是如何变化的，这对 炎症反应。在目标2中，我们将通过研究直接检验sST 2发挥因果作用的假设 我们通过CRISPR-Cas9产生的sST 2敲除大鼠。我们将通过拆除 使用递送至脑的AAV 9-shRNA的体内sST 2水平。在每个模型系统中，我们将评估 减少或缺乏sST 2对脑驻留和循环先天免疫细胞，预计衰减的免疫反应。 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sST 2水平改变脑水肿和脑含水量。总之，这项研究提供了一个关键的 有机会回答关于内皮细胞在血管内皮细胞病理生理学中的信号作用的基本问题， 脑水肿，并确定sST 2的作用。

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.