Immune Privilege, CNS Autoimmunity, and Clostridium perfringens Epsilon Toxin

免疫特权、中枢神经系统自身免疫和产气荚膜梭菌 Epsilon 毒​​素

• 批准号: 10754021
• 负责人: TIMOTHY VARTANIAN
• 金额: $ 67.97万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10754021
• 关键词: Active Immunization; Animal Model; Autoimmunity; Binding; Bioinformatics; Biological Assay; Blood; Blood - brain barrier anatomy; Blood Vessels; Brain; Brain Stem; CNS autoimmunity; Candidate Disease Gene; Catalogs; Cell Separation; Cell Surface Receptors; Cells; Cerebellum; Clinical; Clostridium perfringens; Clostridium perfringens epsilon toxin; Confocal Microscopy; DNA Sequence Alteration; Demyelinations; Disease; Endothelial Cells; Endothelium; Endotoxins; Environmental Risk Factor; Event; Experimental Autoimmune Encephalomyelitis; Experimental Models; Flow Cytometry; Frequencies; Functional disorder; Gene Deletion; Gene Transfer; Genes; Genetic Risk; Goals; Human; Immune; Immunohistochemistry; Individual; Inflammatory; Knockout Mice; Lesion; Leukocyte Trafficking; Leukocytes; Localized Lesion; Location; Lymphocyte; Mediating; Meningeal; Messenger RNA; Modeling; Molecular; Molecular Target; Multiple Sclerosis; Mus; Mutant Strains Mice; Myelin; Neuroanatomy; Neurologic; Pathogenicity; Pathology; Pathway interactions; Persons; Pertussis Toxin; Phenotype; Prevalence; Process; Prosencephalon; Proteins; RNA; Role; Sampling; Spinal Cord; T-Lymphocyte; Techniques; Testing; Time; Tissues; Toxin; Transcriptional Regulation; Wild Type Mouse; Work; adaptive immune response; autoreactivity; cellular targeting; clinical phenotype; clinically relevant; diagnostic tool; disability; gain of function; gene function; gene induction; gut microbiome; high dimensionality; immune cell infiltrate; immunopathology; innovation; loss of function; loss of function mutation; migration; multiple sclerosis patient; neuropathology; novel diagnostics; postcapillary venule; receptor; single-cell RNA sequencing; therapeutic evaluation; therapeutic target; transcriptome sequencing; young adult

Why some people develop Multiple Sclerosis and others do not, despite similar genetic risk and quantities of circulating autoreactive lymphocytes, is not known. Our long-term goal is to identify environmental triggers of MS, define the molecular and cellular basis of their action, and in doing so, propose new diagnostic tools and therapeutic targets. The objectives of this proposal are to determine mechanistically how Clostridium perfringens epsilon toxin (ETX) and Bordetella pertussis toxin (PTX) overcome CNS immune privilege to trigger autoimmunity in the context of myelin autoreactive lymphocytes and to understand why ETX causes lesions to develop in the forebrain, cerebellum, brainstem, and spinal cord in contrast to PTX where lesions are more commonly localized to the spinal cord. The central hypothesis of this project is that ETX and PTX trigger CNS autoimmunity by inducing critical dysfunction at CNS barriers necessary for entry of pathogenic lymphocytes. The central hypothesis will be tested by pursuing two aims: 1) Determining the effect of cell specific deletion or introduction of the ETX receptor MAL (Myelin and Lymphocyte Protein) in active immunization models of experimental autoimmune encephalomyelitis (EAE), compare the neuroanatomical location, phenotype, and activation state of immune infiltrates between PTX- and ETX-induced EAE, and explore the effect of ETX on human lymphocytes, and 2) Determine the genes induced and suppressed in CNS-endothelial cells by ETX and PTX and define their function in overcoming CNS immune privilege through loss-of-function strategies. We will pursue these aims using an innovative combination of targeted genetic mutations to isolate cellular and molecular targets of ETX required to induce disease. We will use confocal microscopy, immunohistochemistry, high dimensional flow cytometry, and unbiased sampling of the entire CNS to compare the effects of ETX with PTX on immune phenotype, demyelination, and neuroanatomic localization of lesions. To determine toxin induced genes functioning to overcome CNS immune privilege, we will apply a combination of unbiased mRNA profiling techniques to CNS endothelial cells isolated from different neuroanatomic regions, advanced bioinformatics to define relevant gene modules, immunohistochemistry to validate localization of these induced proteins within individual post-capillary venules, and conditional loss-of-function mutations in endothelial cells to determine function. The rationale underlying this proposal is that completion will define the role by which a toxin, clinically associated with MS, functions in the multi-step process of autoimmunity, and will identify key molecular targets that can be tested therapeutically. This work will also help establish an experimental model that has greater clinical relevance to MS and more closely resembles MS neuropathology than experimental autoimmune encephalomyelitis models reliant on pertussis toxin.

尽管遗传风险和多发性硬化症数量相似，但为什么有些人会患上多发性硬化症，而另一些人却不会？ 循环自身反应性淋巴细胞，尚不清楚。我们的长期目标是确定环境因素 MS，定义其作用的分子和细胞基础，并在此过程中提出新的诊断工具和 治疗目标。该提案的目标是从机制上确定梭状芽胞杆菌如何 产气荚膜杆菌毒素 (ETX) 和百日咳博德特氏菌毒素 (PTX) 克服中枢神经系统免疫特权以触发 髓磷脂自身反应性淋巴细胞背景下的自身免疫，并了解为什么 ETX 会导致病变 与 PTX 相比，前脑、小脑、脑干和脊髓中的病变更常见 通常定位于脊髓。该项目的中心假设是ETX和PTX触发CNS 通过诱导致病性淋巴细胞进入所必需的中枢神经系统屏障的关键功能障碍来产生自身免疫。 将通过追求两个目标来检验中心假设：1）确定细胞特异性缺失或 在主动免疫模型中引入 ETX 受体 MAL（髓磷脂和淋巴细胞蛋白） 实验性自身免疫性脑脊髓炎 (EAE)，比较神经解剖位置、表型和 PTX 和 ETX 诱导的 EAE 之间免疫浸润的激活状态，并探讨 ETX 对 人淋巴细胞，2) 确定 ETX 和中枢神经系统内皮细胞中诱导和抑制的基因 PTX 并定义其通过功能丧失策略克服 CNS 免疫特权的功能。我们将 使用靶向基因突变的创新组合来分离细胞和 诱发疾病所需的 ETX 分子靶标。我们将使用共聚焦显微镜、免疫组织化学、 高维流式细胞术，并对整个中枢神经系统进行无偏采样，以比较 ETX 与 PTX 对免疫表型、脱髓鞘和病变神经解剖学定位的影响。确定毒素 诱导基因发挥作用以克服 CNS 免疫特权，我们将应用无偏 mRNA 的组合 对从不同神经解剖区域分离的中枢神经系统内皮细胞进行分析技术，先进 生物信息学来定义相关基因模块，免疫组织化学来验证这些诱导的定位 单个毛细血管后微静脉内的蛋白质，以及内皮细胞中的条件性功能丧失突变 来确定功能。该提案的基本原理是，完成后将定义一个角色 临床上与多发性硬化症相关的毒素在自身免疫的多步骤过程中发挥作用，并将识别关键的 可以进行治疗测试的分子靶点。这项工作还将有助于建立实验模型 与实验相比，与多发性硬化症具有更大的临床相关性，并且更类似于多发性硬化症的神经病理学 依赖百日咳毒素的自身免疫性脑脊髓炎模型。