Pathogenesis of viral meningitis

病毒性脑膜炎的发病机制

基本信息

批准号：
7969712
负责人：
Dorian McGavern
金额：
$ 172.33万
依托单位：
NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7969712
关键词：
Accounting Acute Adult Age Age-Years Arboviruses Biological Models Blood Blood Vessels Brain CCL2 gene CCL3 gene CCL4 gene CCL7 gene CXCL2 gene Cells Central Nervous System Diseases Central Nervous System Viral Diseases Chemotactic Factors Child Climate Data Disease Disorder by Site Encephalitis Enterovirus Extravasation Fever Flow Cytometry Fluorescent Dyes Genes Genetic Goals Granzyme Green Fluorescent Proteins HIV-1 Hand Headache Herpesviridae Human Image Immune response Immune system Immunity Immunocompetent Immunocompromised Host Incidence Infection Inflammation Injury Interferon Type II Knock-out Label Laboratories Laboratory Study Left Link Lymphocyte Meningeal Meninges Meningitis Microscopy Molecular Motor Seizures Mumps virus Mus Myelitis Names Natural Immunity Neck Neurologic Onset of illness Pathogenesis Pathology Pathway interactions Persons Play Population Process RANTES RNA Viruses Recruitment Activity Role Route Seizures Shapes Site Stromal Cells Subarachnoid Space Symptoms System Testing Time Time Study Translating Tumor Necrosis Factor-alpha Upper arm Vascular Endothelium Vascular blood supply Viral Pathogenesis Viral meningitis Virus Virus Diseases cell injury cell killing chemokine cranium cytotoxic disability human TNF protein immunopathology in vivo injured insight interest monocyte mortality neutrophil novel pathogen perforin prevent stem two-photon

项目摘要

Acute viral infection of the central nervous system can induce a variety of disease states. A disease state of particular interest to our group stems from the ability of viruses to induce meningitis (or inflammation of the lining of the brain). It is estimated that viral meningitis is induced with a peak monthly incidence of 1 in 100,000 persons, particularly in temperate climates. The disease is associated with symptoms that include fever, headache, stiffness of the neck, and seizures. Enteroviruses are the most common cause of viral meningitis, accounting for approximately 75-90% of the cases. Other common meningitis-inducing viruses in humans include herpesviruses, human immunodeficiency virus-1, arbovirus, mumps virus, and lymphocytic choroimeningitis virus (LCMV). While complications associated with enterovirus-induced meningitis (the most common viral meningitis) in adults are rare, and are often seen in the immunocompromised, studies have shown that infection of children less that one year of age can result in mild to moderate neurological disability by the age of 5. On the other end of the spectrum, herpesviruses induce an array of CNS disorders that include encephalitis, myelitis, and meningitis, and these disorders have a very high mortality rate if left untreated. Because so many viruses have the capacity to induce meningitis and injure the CNS, it is important to uncover potential routes to pathogenesis in murine model systems. To gain novel mechanistic insights into CNS viral pathogenesis, our laboratory studies the LCMV model system. LCMV is an ambisense negative stranded RNA virus that is both a murine and human pathogen. Intracerebral inoculation of immunocompetent mice with LCMV induces a fatal choriomeningitis and is the disease for which LCMV is named. Importantly, LCMV does not kill the cells it infects in vivo, meaning that all cellular injury observed following infection is caused by the host immune system. The immune system is absolutely required for the convulsive seizures and fatalities observed during LCMV-induced meningitis. In fact, this disease is considered a prototypic cytotoxic lymphocyte (CTL)-dependent disorder because depletion of CTL converts LCMV infected mice to asymptomatic carriers. However, at the outset of our studies, the mechanism by which CTL caused fatal meningitis in the LCMV system was unknown. Over the past year, our laboratory set out to gain real time mechanistic insights into this disease process with the hope of better understanding the pathogenesis of viral meningitis in general. To accomplish this aim we utilized intravital two photon microscopy to watch viral meningitis unfold in real time. By injecting a fluorescent dye in the blood supply and labeling LCMV-specific CTL with green fluorescent protein (GFP), we were able to visualize through a surgically thinned skull the activities of the cells thought to be responsible for the disease. Our real time studies revealed a surprising and unexpected twist in the pathogenesis of this classic CTL-dependent disorder. Imaging of meningeal vasculature revealed obvious evidence of immunopathology. Many vessels were observed to be leaking blood-derived material into the subarachnoid space, which can cause fatal seizures. However, the GFP-labeled CTL were not associated in any way with the vascular injury. Moreover, genetic knockout of all the pathways known to be involved in CTL effector function (i.e., perforin, TNF-alpha, interferon-gamma, granzymes, Fas, degranulation) did not have any effect on the disease. These data suggested that CTL were not directly responsible for disease pathogenesis and might be operating through an indirect mechanism. To test this supposition, we examined the composition of the CNS infiltrate in LCMV-infected mice and observed an influx of monocytes and neutrophils (referred to as myelomonocytic cells) that coincided with the arrival of CTL. To define the activities of these cells at the site of disease, we examined GFP-tagged myelomonocytic cells at the peak of meningitis using two photon microscopy. Our real time studies revealed that the activities of myelomonocytic cells caused significant injury to meningeal blood vessels resulting in the leakage of blood-derived material into the subarachnoid space. Interestingly, neutrophils and monocytes appeared to accomplish this through non-overlapping mechanisms. Neutrophils caused sustained leakage of meningeal blood vessels by adhering to vascular endothelium and synchronously extravasating. Monocytes, on the other hand, caused transient breaches in blood vessel integrity by localizing perivascularly. We proved that both of these mechanisms of vascular injury were required for fatal convulsive seizures through depletion studies. Depletion of neutrophils or monocytes alone had no impact on disease; however, depletion of both populations simultaneously prevented fatal convulsive seizures and preserved vascular integrity. These data indicated that myelomonocytic cells rather than CTL were directly responsible for rapid onset disease observed during LCMV-induced meningitis. To examine the mechanistic link between CTL (adaptive immunity) and myelomonocytic cells (innate immunity), we used gene arrays to quantify chemoattractants that might draw myelomonocytic cells into the CNS following infection. Our results revealed that six chemokines (CCL2, CCL3, CCL4, CCL5, CCL7, and CXCL2) were upregulated at the peak of disease, and flow cytometry studies revealed that three of these chemokines (CCL3, 4, and 5) were directly produced by CNS-infiltrating virus-specific CTL. These data suggested that CTL caused disease by recruiting a massive number of myelomonocytic cells into the meningeal space, which then induced fatal vascular injury. It is presently unclear why CTL recruit myelomonocytic cells to a site of viral infection, but we predict that disconnecting (or at least reducing) the pathogenic link between the innate and adaptive immune system might represent a viable treatment option for viral meningitis as well as other infections of the CNS. We also predict that this novel mode of pathogenesis revealed in the LCMV model system exists in other disease states involving CTL. We are presently examining whether it is possible to decouple these two arms of the immune system to limit pathology and are trying to determine precisely how myelomonocytic cells cause such severe injury to meningeal vasculature. We are also examining interactions between CTL and the meningeal stromal network. During our studies we made the exciting observation that LCMV primarily infects stromal cells in the meninges. From an immunological perspective, the meningeal stromal network remains completely undefined, and these cells likely play a major role in shaping immune responses within the subarachnoid space. The overall goal of this project is to use a combination virological, molecular, immunological, and intravital imaging approaches to provide a comprehensive mechanistic understanding of how the innate and adaptive immune systems operate in the meninges following acute viral infection. Our long term hope is that these studies will translate into therapies to better manage acute CNS viral infections in humans.

中枢神经系统的急性病毒感染可以诱导多种疾病状态。我们组特别感兴趣的疾病状态源于病毒诱导脑膜炎的能力（或大脑内膜炎症）。据估计，病毒性脑膜炎是在每月100,000人中有1人的峰值诱导的，尤其是在温带气候中。该疾病与包括发烧，头痛，颈部僵硬和癫痫发作有关的症状有关。肠病毒是病毒性脑膜炎的最常见原因，约占病例的75-90％。人类中其他常见的脑膜炎病毒包括疱疹病毒，人类免疫缺陷病毒-1，Arbovirus，腮腺炎病毒和淋巴细胞蛋白粘膜肾上腺炎病毒（LCMV）。尽管成年人中与肠肠病毒诱发的脑膜炎（最常见的病毒性脑膜炎）相关的并发症很少见，并且在免疫功能低下的人中经常会出现，但研究表明，较小一岁的儿童感染可能会导致5岁时轻度至中度的神经性残疾，而在5岁时，疱疹病毒造成了疾病和疾病的疾病，这些疾病是群体造成的。如果未治疗，脑膜炎和这些疾病的死亡率很高。因为如此多的病毒具有诱导脑膜炎并损害中枢神经系统的能力，因此在鼠模型系统中发现潜在的发病机理非常重要。为了获得对中枢神经系统病毒发病机理的新机械洞察力，我们的实验室研究LCMV模型系统。 LCMV是一种Ambisense负链RNA病毒，既是鼠类又是人类病原体。脑内接种与LCMV的免疫能力小鼠会诱导致命的绒毛膜炎，并且是LCMV命名的疾病。重要的是，LCMV不会杀死其体内感染的细胞，这意味着感染后观察到的所有细胞损伤都是由宿主免疫系统引起的。在LCMV诱导的脑膜炎期间观察到的抽搐性癫痫发作和死亡绝对需要免疫系统。实际上，该疾病被认为是一种原型的细胞毒性淋巴细胞（CTL）依赖性疾病，因为CTL的耗竭将被LCMV感染的小鼠转化为无症状的载体。但是，在我们的研究开始时，CTL在LCMV系统中引起致命脑膜炎的机制尚不清楚。在过去的一年中，我们的实验室着手获得对这种疾病过程的实时机理洞察力，希望更好地了解一般的病毒性脑膜炎的发病机理。为了实现这一目的，我们利用插入的两个光子显微镜实时观察病毒性脑膜炎。通过在血液供应中注入荧光染料并将LCMV特异性CTL标记为绿色荧光蛋白（GFP），我们能够通过手术稀薄的头骨可视化细胞的活性，这些细胞的活动被认为是疾病。我们的实时研究表明，这种经典CTL依赖性疾病的发病机理发生了令人惊讶和意外的扭曲。脑膜脉管系统的成像揭示了免疫病理学的明显证据。观察到许多血管将血液来源的物质泄漏到蛛网膜下腔中，这可能导致致命的癫痫发作。但是，GFP标记的CTL与血管损伤无关。此外，所有已知参与CTL效应函数的途径（即perforin，tnf-alpha，Interferon-gamma，Granzymes，Fas，Fas，脱粒）对疾病没有任何影响。这些数据表明，CTL并不直接负责疾病发病机理，并且可能通过间接机制运行。为了测试这种假设，我们检查了CNS浸润在LCMV感染的小鼠中的组成，并观察到与CTL的到来相吻合的单核细胞和中性粒细胞（称为骨髓细胞细胞）的涌入。为了定义这些细胞在疾病部位的活性，我们使用两个光子显微镜检查了脑膜炎峰值GFP标记的脊髓细胞细胞。我们的实时研究表明，脊髓细胞细胞的活性对脑膜血管造成了重大损伤，导致血管泄漏到蛛网膜下腔中。有趣的是，中性粒细胞和单核细胞似乎通过非重叠的机制来实现这一目标。嗜中性粒细胞通过粘附在血管内皮和同步散发性的情况下导致脑膜血管的持续泄漏。另一方面，单核细胞通过血管周围定位引起血管完整性的短暂漏洞。我们证明，通过耗尽研究，这两种死亡的抽搐癫痫发作都需要这两种机制。仅嗜中性粒细胞或单核细胞的耗竭对疾病没有影响。然而，两个人群的耗尽同时阻止了致命的抽搐性癫痫发作并保留了血管完整性。这些数据表明，脊髓细胞细胞而不是CTL直接导致在LCMV诱导的脑膜炎期间观察到的快速发作疾病。为了检查CTL（自适应免疫）和脊髓细胞细胞（先天免疫）之间的机械联系，我们使用基因阵列来量化可能在感染后可能将骨髓细胞细胞吸引到中枢神经系统中的趋化剂。我们的结果表明，在疾病峰值下，六种趋化因子（CCL2，CCL3，CCL4，CCL7和CXCL2）上调，流式细胞仪研究表明，这些趋化因子（CCL3、4和5）中的三个是由CNS浸润的病毒特异性病毒特异性CTL直接产生的。这些数据表明，CTL通过募集大量骨髓形成细胞进入脑膜空间来引起疾病，然后诱导致命的血管损伤。目前尚不清楚CTL为什么将脊髓细胞细胞募集到病毒感染部位，但我们预测，与先天性和适应性免疫系统之间的致病联系（或至少降低）可能代表病毒脑膜炎以及其他CNS感染的可行治疗选择。我们还预测，在涉及CTL的其他疾病状态中，LCMV模型系统中揭示了这种新型的发病机理模式。我们目前正在研究是否有可能将免疫系统的这两个臂解矛，以限制病理学，并试图确切确定骨髓细胞细胞如何对脑膜脉管系统造成如此严重的损伤。我们还正在研究CTL和脑膜基质网络之间的相互作用。在我们的研究中，我们进行了令人兴奋的观察结果，即LCMV主要感染脑膜中的基质细胞。从免疫学的角度来看，脑膜基质网络仍然完全不确定，并且这些细胞可能在塑造亚蛛网膜下腔内的免疫反应中起主要作用。该项目的总体目标是使用病毒学，分子，免疫和浸润成像方法的组合，以对急性病毒感染后的先天和适应性免疫系统在脑膜中如何运作，提供全面的机械理解。我们的长期希望是，这些研究将转化为更好地管理人类急性中枢神经系统病毒感染的疗法。