Neuroimmune modulation across the BBB: Regulation of ILS trafficking by TNF

描述（由申请人提供）：拟议研究的目的是确定TNF（神经炎症的重要组成部分）上调血脑屏障（BBB）内皮细胞中il - 15的产生、il - 15在血脑屏障上的运输以及由此产生的神经炎症和自身免疫性疾病的病理生理意义的机制。促炎细胞因子TNF不仅可以穿过血脑屏障发挥其多能性作用，还可以调节内皮信号传导和其他细胞因子的渗透。我们的初步结果通过芯片、qPCR和免疫组织化学显示，il - 15及其受体在TNF或脂多糖刺激下的脑微血管内皮细胞中表现出特异性和强劲的上调。我们将从四个方面阐述il - 15增强肿瘤坏死因子在中枢神经系统中的作用的总体假设。(1)验证TNF上调il - 15转运的假说。我们将通过受体敲除和野生型小鼠的原位脑灌注研究来确定il - 15受体在转运il - 15中的作用，确定TNF对Transwell系统培养的原代脑微血管内皮细胞中il - 15的跨细胞通透性的影响，并通过免疫荧光运输研究确定il - 15r1、IL2R2和IL2R3受体在TNF促进的il - 15转运中的差异作用和相互作用。(2)验证TNF刺激血脑屏障内皮细胞蛋白合成和基底外侧分泌il - 15的假说。这将通过蛋白质翻译和周转分析、il - 15、其受体和胞内细胞器的时间依赖共聚焦分析和酶联免疫吸附测定定量来实现。我们还将测试前列腺素在tnf诱导的基底外侧il - 15分泌增加中的介导作用。(3)验证内皮细胞il - 15系统上调可增强tnf诱导的神经炎症的假设，表现为发热、疾病行为、反应性胶质瘤和大脑产生促炎细胞因子。除了在小鼠身上研究可溶性IL15受体阻断这些作用的作用外，还将确定敲除小鼠和野生型小鼠培养的脑微血管内皮细胞的炎症变化。(4)验证il - 15系统上调会加剧实验性自身免疫性脑脊髓炎（EAE）的假设，EAE是一种神经免疫调节障碍，也是多发性硬化症的一种模型。研究il - 15在EAE小鼠血脑屏障中渗透的调控变化，以及可溶性受体减轻EAE症状和疾病负担的作用。初步结果、我们过去的经验和文献支持了研究的可行性。通过完成这些研究，我们将获得对脑内皮细胞中il - 15和il - 15受体运输的全新和必要的理解，并确定干预神经炎症和中枢神经系统自身免疫性疾病的新治疗靶点。神经炎症是许多中枢神经系统疾病的常见病因，包括感染（细菌、真菌、病毒和寄生虫）、神经艾滋病、中风、全身缺氧、自身免疫性疾病（如多发性硬化症、横切性脊髓炎、急性弥漫性脑脊髓病）、肿瘤、各种神经退行性疾病（如阿尔茨海默病、帕金森病、肌萎缩侧索硬化症），甚至糖尿病和代谢综合征。多发性硬化症（MS）是一种破坏性和进行性疾病，发病年龄通常在20至40岁之间。在美国，大约有25万到35万人被医生诊断为多发性硬化症。这一估计表明，每周约有200个新病例被诊断出来。促炎细胞因子TNF和IL15在中枢神经系统的神经炎症和自身免疫性疾病的发生、进展和消退中发挥重要作用。它们与血脑屏障的相互作用在很大程度上决定了这些血源性细胞因子的最终结果。

DESCRIPTION (provided by applicant): The goals of the proposed study are to determine the mechanisms by which TNF, an important component of neuroinflammation, upregulates IL15 production in the endothelial cells of the blood-brain barrier (BBB), the transport of IL15 across the BBB, and the resulting pathophysiological implications for neuroinflammatory and autoimmune disorders. The proinflammatory cytokine TNF not only crosses the BBB to exert its multipotent effects, but it also modulates the endothelial signaling and permeation of other cytokines. IL15 and its receptors show specific and robust upregulation in cerebral microvessel endothelial cells challenged by TNF or lipopolysaccharide, shown by microarray, qPCR and immunohistochemistry in our preliminary results. We will address the overall hypothesis that IL15 potentiates the effects of TNF in the CNS with four aims. (1) Test the hypothesis that TNF upregulates IL15 transport. We will identify the roles of IL15 receptors in transporting IL15 by use of in-situ brain perfusion studies in receptor knockout and wildtype mice, determine the effects of TNF on the transcellular permeability of IL15 in primary cerebral microvessel endothelial cells cultured in the Transwell system, and determine the differential roles and interactions of IL15R1, IL2R2, and IL2R3 receptors in TNF-facilitated IL15 transcytosis by immunofluorescent trafficking studies. (2) Test the hypothesis that TNF stimulates protein synthesis and basolateral secretion of IL15 from the endothelial cells of the BBB. This will be achieved by protein translation and turnover assays, confocal analyses of time-dependent colocalization of IL15, its receptors, and intracellular organelles, and quantification by enzyme-linked immunosorbent assay. We will also test the mediating effect of prostaglandins in the TNF-induced increase of the basolateral secretion of IL15. (3) Test the hypothesis that an upregulated endothelial IL15 system enhances TNF-induced neuroinflammation, shown by fever, sickness behavior, reactive gliosis, and cerebral production of proinflammatory cytokines. Apart from studies on mice to test the effects of soluble IL15 receptors in blocking these effects, the inflammatory changes in cultured cerebral microvessel endothelial cells obtained from knockout and wildtype mice will also be identified. (4) Test the hypothesis that an upregulated IL15 system exacerbates experimental autoimmune encephalomyelitis (EAE), a disorder of neuroimmune modulation and a model for multiple sclerosis. The regulatory changes of IL15 permeation across the BBB of the EAE mice will be determined, and the effects of soluble receptors in reducing the symptoms and disease burden of EAE will be determined. The feasibility of the studies is supported by preliminary results, our past experience, and the literature. By completion of the proposed studies, we will acquire novel and essential understanding of IL15 and IL15 receptor trafficking in cerebral endothelial cells, and identify new therapeutic targets for intervention in neuroinflammation and CNS autoimmune diseases. PUBLIC HEALTH RELEVANCE Neuroinflammation is a common etiology in many CNS disorders, including infection (bacterial, fungal, viral, and parasitic), neuroAIDS, stroke, global hypoxia, autoimmune diseases (e.g., multiple sclerosis, transverse myelitis, acute disseminating encephalomyelopathy), tumor, a variety of neurodegenerative disorders (e.g., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), and even diabetes and the metabolic syndrome. Multiple sclerosis (MS) is a devastating and progressive disorder with an age of onset typically between 20 and 40. There are about 250,000 - 350,000 people in the United States with MS diagnosed by a physician. This estimate suggests that about 200 new cases are diagnosed each week. The proinflammatory cytokines TNF and IL15 play important roles in the initiation, progression, and resolution of neuroinflammation and autoimmune disorders of the CNS. Their interactions with the blood-brain barrier largely determine the final outcome of these blood-borne cytokines.