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

项目摘要/摘要 公共/健康/相关性：60%新冠肺炎康复者的慢性或复发性神经功能障碍 现在是治疗SARS-CoV-2感染中枢神经系统的后遗症的一个未得到满足的医疗需求 (CNS)。德国最近的一项研究表明，这些症状会持续一年以上，与患者类似 因感染SARS-CoV-1而出现慢性症状。因此，显然有必要进行干预。 抗COVID后的慢性神经系统症状。SARS-CoV-2对人类免疫功能影响机制的研究 中枢神经系统对于指导此类干预措施的设计至关重要。 目的：探讨SARS冠状病毒S对中枢神经系统的作用途径。 由中性粒细胞依赖的缓激肽(BK)“风暴”介导的血脑屏障(BBB)失调。我们 假设这场风暴引发了神经炎症，最终扰乱了正常的神经元信号， 为持久的神经症状提供底物。 研究计划：最近的研究报告了血脑屏障的完整性因SPEKE(S)蛋白的改变而改变 SARS-CoV-2，从而提示SARS-CoV-2或炎性免疫细胞的神经侵袭途径 通过BBB。根据这些观察结果，这项提案将调查促炎介质如何 与COVID感染相关的激活中性粒细胞介导的BK上调；这导致 由于调节失调的紧密连接(TJ)，通过细胞旁间隙穿过血脑屏障的渗透性。这样一种模式 与新冠肺炎患者支气管肺泡液中观察到的BK上调水平一致 中性粒细胞参与激肽系统在急性炎症中重塑内皮屏障的能力。 作为天然SARS-CoV-2的替代物，我们将构建S蛋白包裹的量子点作为高保真仿生 以研究调节SARS-CoV-2跨膜通透性的大小和结构限制 BBB。这些构造将被用来在相关的存在的情况下将中性粒细胞偏向促炎状态 激肽释放酶-激肽释放酶因子对体外培养的bEnd.3单层细胞通透性的影响 小鼠血脑屏障系统。我们的荧光SARS-CoV-2仿生药物的渗透性增加将表明血脑屏障发生泄漏，并与全球屏障健康的补充测量相印证，如 跨内皮细胞电阻(TEER)。最后，我们将构造一个相关的扫描离子电导 并用共聚焦显微镜系统检测屏障功能紊乱的异质性和特异性 TJ表达的纳米级变化和调节它的定位。

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.