Utilizing Single Cell Biological Approaches to Understand CNS TB

项目摘要 结核分枝杆菌（Mycobacterium tuberculosis，Mtb）是结核病（TB）的致病菌。结核病是导致 在全球范围内，传染病导致的死亡在感染艾滋病毒的个体中尤为普遍。而 结核病通常被认为是一种呼吸道疾病，但它也会感染其他器官系统。中央感染 神经系统结核（CNS-TB）是该疾病的最严重形式，并且具有近50%的死亡率， 尽管积极的临床干预。CNS-TB与严重的神经功能障碍有关，包括 颅神经麻痹、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 神经炎性信号传导的激活以及神经胶质和神经元功能障碍。 在免疫抑制条件下，发生CNS-TB的风险显著增加，在HIV中很明显。 受感染的个体，结核性脑膜炎（TBM）的发生率较高。调节性肿瘤坏死因子 (TNF)免疫应答是抵抗结核病所必需的免疫能力的关键特征， 进行性HIV感染。适当的TNF应答的重要性在临床上通过重新激活 抗TNF治疗自身免疫性疾病患者的结核病。我们最近报道，TNF缺乏（a 免疫抑制模型）在小鼠（TNF-/-）中，促进CNS-TB感染，高度炎症反应， 脑病理学和死亡率我们将利用一个真实的CNS-TB小鼠模型， 将结核分枝杆菌（Mtb）注射到对照组和免疫受损（TNF-/-）组的脑中。 小鼠来研究驻留的CNS细胞如何反应。虽然Mtb主要感染小胶质细胞和星形胶质细胞，但人类 并且小鼠神经元也充当Mtb的宿主细胞。因此，多种CNS细胞直接反应， 间接导致大脑感染，产生复杂的细胞和组织水平的反应。处理这个 复杂性，我们将利用单细胞RNA-seq，一种尖端的基因组方法，它允许转录 在逐个细胞的基础上分析对CNS-TB的反应。这种方法能够识别 单个类型的细胞，例如星形胶质细胞，以及它们独特的转录应答的分析。在 此外，单细胞RNA-seq允许通过以下方式研究对CNS-TB的细胞应答的异质性： 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系统是如何导致 反应调节神经炎症信号传导，并导致CNS驻留细胞的大规模破坏， CNS-TB小鼠模型。在这样做的过程中，我们将建立一个合作伙伴关系，开普敦大学之间 镇和塔夫茨大学。重点将放在培训南非科学家在神经， 免疫相互作用，单细胞RNA-seq和先进的基因组分析方法，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.