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

跨 BBB 的神经免疫调节：TNF 对 ILS 运输的调节

• 批准号: 8322735
• 负责人: Weihong Pan
• 金额: $ 31.51万
• 依托单位: LSU PENNINGTON BIOMEDICAL RESEARCH CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8322735
• 关键词: AIDS neuropathy; Acute Transverse Myelitis; Address; Age of Onset; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Autoimmune Diseases; Bacterial Infections; Behavior; Biological Assay; Blood; Blood - brain barrier anatomy; Brain; Brain Pathology; CNS autoimmune disease; Cell Culture Techniques; Central Nervous System Diseases; Cerebrum; Clinical; Cytokine Signaling; Data; Diabetes Mellitus; Diagnosis; Disease; Endothelial Cells; Endothelium; Enzyme-Linked Immunosorbent Assay; Etiology; Exhibits; Experimental Autoimmune Encephalomyelitis; Feasibility Studies; Feline Panleukopenia; Gliosis; Goals; Health; Hypoxia; IL2RA gene; Immunohistochemistry; In Situ; Inflammatory; Interleukin-15; Intervention; Knock-out; Knockout Mice; Ligands; Lipopolysaccharides; Literature; Mediating; Mediator of activation protein; Metabolic syndrome; Minor; Modeling; Multiple Sclerosis; Mus; Neurodegenerative Disorders; Organelles; Outcome; Parkinson Disease; Pathway interactions; Perfusion; Peripheral; Permeability; Physicians; Play; Production; Prostaglandins; Protein Biosynthesis; Proteins; Regulation; Resolution; Role; Signal Transduction; Sorting - Cell Movement; Stroke; Surface; Symptoms; System; TNF gene; Testing; Time; Transcriptional Activation; Translations; Treatment Efficacy; Tumor Necrosis Factor-alpha; United States; Up-Regulation; Viral; Work; burden of illness; cyclooxygenase 2; cytokine; encephalomyelopathy; experience; neuroinflammation; new therapeutic target; novel; protein degradation; receptor; trafficking; transcytosis; tumor

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.

描述（由申请人提供）：本研究的目的是确定TNF（神经炎症的重要组分）上调血脑屏障（BBB）内皮细胞中IL 15产生、IL 15跨BBB转运的机制，以及由此产生的神经炎症和自身免疫性疾病的病理生理学意义。促炎细胞因子TNF不仅穿过BBB发挥其多能效应，而且还调节内皮信号传导和其他细胞因子的渗透。在我们的初步结果中，通过微阵列、qPCR和免疫组织化学显示，IL 15及其受体在TNF或脂多糖攻击的脑微血管内皮细胞中显示出特异性和稳健的上调。我们将讨论IL 15增强TNF在CNS中的作用的总体假设，其目的有四个。(1)检验TNF上调IL 15转运的假设。我们将通过在受体敲除小鼠和野生型小鼠中进行原位脑灌注研究来鉴定IL 15受体在转运IL 15中的作用，确定TNF对在Transwell系统中培养的原代脑微血管内皮细胞中IL 15的跨细胞渗透性的影响，并确定IL 15 R1、IL 2 R2、IL 15 R3和IL 15 R4的不同作用和相互作用。和IL 2 R3受体在TNF促进的IL 15转胞吞作用中的作用。(2)检验TNF刺激BBB内皮细胞的蛋白质合成和基底侧分泌IL 15的假设。这将通过蛋白质翻译和周转试验，共聚焦分析的时间依赖性共定位的IL 15，其受体，和细胞内细胞器，并通过酶联免疫吸附测定定量。我们还将测试在TNF-诱导的基底侧IL 15分泌增加中，野牡丹素的介导作用。(3)检验上调的内皮细胞IL 15系统增强TNF诱导的神经炎症的假设，表现为发热、病态行为、反应性神经胶质增生和促炎细胞因子的大脑产生。除了对小鼠进行研究以测试可溶性IL 15受体在阻断这些作用中的作用之外，还将鉴定从敲除和野生型小鼠获得的培养的脑微血管内皮细胞中的炎性变化。(4)测试上调的IL 15系统加重实验性自身免疫性脑脊髓炎（EAE）的假设，EAE是神经免疫调节的紊乱和多发性硬化症的模型。将确定IL 15渗透穿过EAE小鼠的BBB的调节变化，并将确定可溶性受体在减轻EAE的症状和疾病负担中的作用。研究的可行性得到了初步结果、我们过去的经验和文献的支持。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受体运输的新的和必要的理解，并确定新的治疗靶点，用于干预神经炎症和CNS自身免疫性疾病。神经炎症是许多CNS病症的常见病因，包括感染（细菌、真菌、病毒和寄生虫）、神经AIDS、中风、全身缺氧、自身免疫性疾病（例如，多发性硬化、横肌萎缩、急性播散性脑脊髓病），肿瘤，各种神经变性疾病（例如，阿尔茨海默氏症、帕金森氏症、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25万至35万人被医生诊断为MS。这一估计表明，每周约有200例新病例被诊断出来。促炎细胞因子TNF和IL 15在CNS的神经炎症和自身免疫性疾病的发生、发展和消退中起重要作用。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。

项目成果

Mass spectrometric quantification of MIF-1 in mouse brain by multiple reaction monitoring.

通过多反应监测对小鼠脑中 MIF-1 进行质谱定量。

• DOI: 10.1016/j.peptides.2009.04.004
• 发表时间: 2009
• 期刊: Peptides
• 影响因子: 3
• 作者: Kheterpal,Indu;Kastin,AbbaJ;Mollah,Sahana;Yu,Chuanhui;Hsuchou,Hung;Pan,Weihong
• 通讯作者: Pan,Weihong