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.

为什么有些人发展多发性硬化症和其他人没有，尽管类似的遗传风险和数量 循环的自身反应性淋巴细胞是未知的。我们的长期目标是确定环境触发因素 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， 治疗目标本提案的目的是确定梭菌如何在机械上 产气荚膜梭菌毒素（ETX）和百日咳杆菌毒素（PTX）克服CNS免疫豁免， 髓鞘自身反应性淋巴细胞背景下的自身免疫，并了解为什么ETX导致病变， 在前脑、小脑、脑干和脊髓中发展，而PTX的病变更多 通常局限于脊髓。该项目的中心假设是ETX和PTX触发CNS 通过在致病性淋巴细胞进入所必需的CNS屏障处诱导严重功能障碍而引起自身免疫。 将通过追求两个目标来测试中心假设：1）确定细胞特异性缺失的影响，或 将ETX受体MAL（髓鞘和淋巴细胞蛋白）引入主动免疫模型， 实验性自身免疫性脑脊髓炎（EAE），比较神经解剖位置，表型， 探讨PTX和ETX诱导EAE的免疫浸润激活状态，探讨ETX对EAE免疫浸润的影响。 人淋巴细胞，和2）确定在CNS-内皮细胞中由ETX诱导和抑制的基因，和 PTX和定义其在通过功能丧失策略克服CNS免疫赦免中的功能。我们将 利用靶向基因突变的创新组合来实现这些目标， 诱导疾病所需的ETX分子靶点。我们将使用共聚焦显微镜，免疫组织化学， 高维流式细胞术，以及整个CNS的无偏采样，以比较ETX与 紫杉醇对免疫表型、脱髓鞘和病变神经解剖定位的影响。来测定毒素 诱导基因的功能，以克服中枢神经系统免疫豁免，我们将应用无偏见的mRNA的组合， 从不同神经解剖区域分离的CNS内皮细胞的分析技术， 生物信息学定义相关基因模块，免疫组织化学验证这些诱导的 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 以确定功能。这一建议的基本理由是，完成工作将确定一个组织的作用， 毒素，临床上与MS相关，在自身免疫的多步骤过程中发挥作用，并将确定关键的 可以进行治疗测试的分子靶点。这项工作也将有助于建立一个实验模型 与MS具有更大的临床相关性，并且与实验性MS神经病理学更相似， 依赖百日咳毒素的自身免疫性脑脊髓炎模型。