Utilizing Single Cell Biological Approaches to Understand CNS TB

项目总结 结核分枝杆菌(Mtb)是引起结核病的病原体。结核病是导致 在全球范围内死于传染病，在感染艾滋病毒的个人中尤其普遍。而当 结核病通常被认为是一种呼吸系统疾病，它还会感染其他器官系统。中枢性感染 神经系统(CNS-TB)是这种疾病最严重的形式，死亡率接近50%， 尽管进行了积极的临床干预。中枢神经系统结核与严重的神经功能障碍有关，包括 脑神经麻痹、认知障碍、中风和癫痫。大脑的分子、细胞和网络 对CNS-TB的水平反应几乎完全未知。我们假设CNS-TB导致显著的 神经炎症信号的激活，以及神经胶质和神经元功能障碍。 在免疫抑制的情况下，患中枢神经系统结核的风险显著增加，这在艾滋病毒中是明显的。 受感染的人，结核病脑膜炎(TBM)的发生率较高。一种受调控的肿瘤坏死因子 (肿瘤坏死因子)反应是预防结核病所必需的免疫能力的关键特征，在 进行性艾滋病毒感染。适当的肿瘤坏死因子反应的重要性通过重新激活在临床上得到了验证。 对自身免疫性疾病进行抗肿瘤坏死因子治疗的患者中的结核病。我们最近报道了肿瘤坏死因子缺乏(a 免疫抑制模型)在小鼠(肿瘤坏死因子-/-)，促进中枢神经系统结核感染，一种高炎症反应， 脑部大体病理和死亡率。我们将利用一个真实的CNS-TB小鼠模型，在该模型中， 将结核分枝杆菌(Mtb)注射到对照组和免疫低下组(肿瘤坏死因子-/-)的大脑中 研究常驻中枢神经系统细胞如何反应。虽然结核分枝杆菌主要感染小胶质细胞和星形胶质细胞，但人类 小鼠神经元也是结核分枝杆菌的宿主细胞。因此，多个中枢神经系统细胞直接和 间接导致脑部感染，产生复杂的细胞和组织水平的反应。来处理这件事 复杂性，我们将利用单细胞rna-seq，这是一种先进的基因组方法，它允许转录 逐个细胞分析对CNS-TB的反应。这种方法使得能够识别 不同类型的细胞，例如星形胶质细胞，以及它们独特的转录反应的分析。在……里面 此外，单细胞rna-seq可以通过以下方式研究细胞对CNS-TB反应的异质性 分析单个细胞。我们将利用单细胞rna-seq来确定缺乏适当的免疫 反应调节神经炎性信号转导并导致中枢神经系统驻留细胞的广泛破坏 中枢神经系统结核小鼠模型的建立。通过这样做，我们将在开普敦大学之间建立伙伴关系 城镇和塔夫茨大学。重点将放在培训南非科学家的神经学- 免疫相互作用、单细胞rna-seq和高级基因组分析方法，从而 帮助南非发展强大的研究基础设施。

PROJECT SUMMARY Mycobacterium tuberculosis (Mtb) is the causative pathogen in tuberculosis (TB). TB is the leading cause of death from infectious disease globally and is especially prevalent in individuals infected with HIV. While normally thought of as a respiratory disease, TB also infects other organ systems. Infection of the central nervous system (CNS-TB) is the most severe form of the disease, and has a mortality rate of nearly 50%, despite aggressive clinical intervention. CNS-TB is associated with severe neurological dysfunction including cranial nerve palsies, cognitive impairment, stroke, and seizures. The brain’s molecular, cellular, and network level response to CNS-TB is almost completely unknown. We hypothesize CNS-TB leads to significant activation of neuroinflammatory signaling, as well as glial and neuronal dysfunction. The risk of developing CNS-TB is markedly increased under conditions of immune suppression, evident in HIV infected individuals, who have a higher occurrence of TB meningitis (TBM). A regulated tumor necrosis factor (TNF) response is a critical feature of immune competence necessary for protection against TB, and is lost in progressive HIV infection. The importance of a proper TNF response is clinically validated by the reactivation of TB in patients on anti-TNF treatment for autoimmune diseases. We recently reported that TNF deficiency (a model of immune suppression) in mice (TNF-/-), promotes CNS-TB infection, a hyper-inflammatory response, gross brain pathology, and mortality. We will utilize a realistic mouse model of CNS-TB in which active Mycobacterium tuberculosis (Mtb) will be injected into the brains of control and immune compromised (TNF-/-) mice to study how resident CNS cell respond. Although Mtb primarily infects microglia and astrocytes, human and mouse neurons also act as host cells for Mtb. Therefore, multiple CNS cells react both directly and indirectly to brain infection, creating a complex cellular- and tissue-level response. To deal with this complexity, we will utilize single cell RNA-seq, a cutting edge genomic approach, which allows transcriptional analysis of the response to CNS-TB on a cell-by-cell basis. This approach enables the identification of individual types of cells, for example astrocytes, and analysis of their unique transcriptional response. In addition, single cell RNA-seq allows investigation of the heterogeneity of the cellular response to CNS-TB by analyzing individual cells. We will utilize single cell RNA-seq to determine how the lack of a proper immune response regulates neuroinflammatory signaling and leads to broad-scale disruption of CNS resident cells in a mouse model of CNS-TB. In doing so, we will establish a partnership between The University of Cape Town and Tufts University. Emphasis will be placed on training South African scientists in neuro- immune interactions, single cell RNA-seq, and advanced genomic analysis approaches, thereby helping develop robust research infrastructure in South Africa.