Nanocrystal Quantum Dot Biomimetics of SARS-CoV-2 to Interrogate Neutrophil-Mediated Neuroinflammation at the Blood-Brain Barrier

项目概要/摘要 公共/卫生/相关性：60%的COVID-19康复患者存在慢性或复发性神经功能缺损 现在是治疗SARS-CoV-2感染中枢神经系统后遗症的未满足的医疗需求 （CNS）。德国最近的一项研究表明，这些症状持续超过一年，类似于患者 因感染SARS-CoV-1而出现慢性症状。因此，显然需要采取干预措施， 针对新冠肺炎后的慢性神经系统症状。阐明SARS-CoV-2影响 中枢神经系统对于设计这种干预措施至关重要。 目的：本研究旨在确定SARS-CoV-2对中枢神经系统的作用途径， 血脑屏障（BBB）失调，由缓激肽（BK）的嗜中性粒细胞依赖性“风暴”介导。我们 假设这场风暴引起神经炎症，最终破坏正常的神经信号， 为神经系统症状的持久性提供了基质。 研究计划：最近的研究报告了血脑屏障的完整性改变，以响应刺突（S）蛋白 因此提示SARS-CoV-2或炎性免疫细胞的神经侵入途径 通过BBB。根据这些观察结果，本提案将研究促炎介质 与COVID感染相关的激活嗜中性粒细胞介导的BK上调;这导致 由于紧密连接（TJ）失调，通过BBB的细胞旁间隙的渗透性。这样的模型 与从COVID-19患者中采集的支气管肺泡液中观察到的BK上调水平一致， 在急性炎症中，中性粒细胞能够与激肽系统结合，重塑内皮屏障。 作为天然SARS-CoV-2的替代物，我们将构建S蛋白包被的量子点作为高保真仿生物 研究调节SARS-CoV-2渗透性的大小和结构限制， 的BBB。这些构建体将用于在存在相关炎症因子的情况下使中性粒细胞偏向于促炎状态。 激肽释放酶-激肽因子增加培养的bEnd.3单层的渗透性，一种高保真的体外模型 用于鼠BBB系统。我们的荧光SARS-CoV-2生物模拟物的渗透性增加将表明BBB泄漏，并与全球屏障健康的补充测量相证实，如通过 跨内皮电阻（TEER）。最后，我们将构建一个相关的扫描离子电导 和共聚焦显微镜系统，以检查失调的屏障功能的异质性和特异性 TJ表达和定位的纳米级变化来调节它。

PROJECT SUMMARY/ABSTRACT Public/health/relevance: Chronic, or recurring, neurological deficits in 60% of recovered COVID-19 patients are now an unmet medical need to treat the aftermath of SARS-CoV-2 infection of the central nervous system (CNS). A recent study from Germany suggests that these symptoms persist beyond a year, similarly to patients suffering from chronic symptoms due to SARS-CoV-1 infection. Thus, there is clear need for interventions against chronic neurologic symptoms after COVID. Elucidating the mechanism for SARS-CoV-2 impact on the CNS is essential to inform the design of such interventions. Objective: This proposal aims to identify a pathway for SARS-CoV-2’s effects on the CNS through a dysregulated blood-brain barrier (BBB) mediated by a neutrophil-dependent “storm” of bradykinin (BK). We hypothesize that this storm induces neuroinflammation that ultimately disrupts normal neuronal signaling, providing the substrate for enduring neurological symptoms. Research Plan: Recent studies have reported altered integrity of the BBB in response to the spike (S) protein of SARS-CoV-2, thereby suggesting a neuroinvasive pathway for SARS-CoV-2 or inflammatory immune cells through the BBB. In line with these observations, this proposal will investigate how pro-inflammatory mediators associated with COVID infection activate neutrophil-mediated upregulation of BK; this leads to an increased permeability through paracellular gaps across the BBB due to dysregulated tight junctions (TJs). Such a model aligns with the upregulated levels of BK observed in bronchoalveolar fluid taken from COVID-19 patients coupled with the ability of neutrophils to engage the kinin system to remodel endothelial barriers in acute inflammation. As a proxy for native SARS-CoV-2, we will construct S protein coated quantum dots as high fidelity biomimetics of SARS-CoV-2 to investigate the size and structural constraints regulating SARS-CoV-2 permeability across the BBB. These constructs will be used to bias neutrophils to a pro-inflammatory state in the presence of relevant kallikrein-kinin factors to increase the permeability of cultured bEnd.3 monolayers, a high-fidelity in vitro model system for murine BBB. A leakier BBB will be indicated by increased permeability of our fluorescent SARS-CoV-2 biomimetic and corroborated with complementary measurements of global barrier health, as measured by transendothelial electrical resistance (TEER). Lastly, we will construct a correlated scanning ion conductance and confocal microscope system to examine the heterogeneity of dysregulated barrier function and the specific nanoscale changes in TJ expression and localization that regulate it.