Identifying human neuron IFN-γ dependent anti-Toxoplasma gondii responses

项目摘要： 弓形虫是一种细胞内寄生虫，潜伏感染许多宿主，包括人类。 成功的潜伏感染需要T.弓形虫逃避精氨酸诱导的细胞内在反应， 从快速生长的形式转变为缓慢生长的被囊形式。在人类中，这种终身感染 发生在T.在获得性免疫缺陷的情况下，弓形虫可以重新激活。在 艾滋病患者，弓形体脑炎是最常见的局灶性脑病变的原因，可以 即使经过适当的治疗，也会导致长期的神经功能缺损。最近的研究还表明， 即使在接受有效抗逆转录病毒治疗的HIV+患者中，持续T.弓形虫感染可能对 影响认知和整体免疫反应。尽管它在临床上很重要，但我们缺乏一种机制， 了解T的真正原因。弓形虫在中枢神经系统的发病机制，包括什么 可以长期潜伏感染。这种理解对于最终预防症状性 艾滋病病毒/艾滋病患者。在CNS中，囊肿主要存在于神经元中。基于有限的 体外和体内研究中，已经推测，虽然感染的星形胶质细胞清除细胞内 寄生虫，神经元没有，因此是持续感染的事实上的宿主细胞。在过去 十年来，我们的开创性工作对这一模型提出了质疑，包括表明IFN-γ刺激 神经元建立了抗寄生虫的防御机制。由于大多数这项工作是在鼠模型中进行的， 神经元，它将错过人类神经元特异性通路（例如，人类缺乏末端IFN-γ 依赖于鼠细胞用来杀死细胞内寄生虫的蛋白质）。该补助金的目标是 通过建立稳健的干细胞衍生的人神经元模型（hPSC神经元）来解决这一差距， 可以用来机械地定义人类神经元-T。弓形虫相互作用使免疫逃避 和坚持。为了实现这一目标，我们将利用我们在CNS方面的协同专业知识， 弓形虫病（Koshy博士）和干细胞生物学（Churko博士）来产生Cre报告基因hPSC神经元 和表达CRISPRi的hPSC神经元（Aim 1），并开始定义 细胞因子刺激的hPSC-神经元对照T.弓形虫（Aim 2）。随着这些目标的实现，我们将 已经建立并验证了将构成长期计划的重要基础的工具， 在分子上定义了T神经元。弓形虫相互作用，使控制和持久的T。弓形虫 人类神经元这里提出的工作代表了发展人类的重要的第一步。 神经元特异性治疗急性和慢性弓形虫病。这样的疗法将大有裨益 为有弓形体脑炎风险的艾滋病毒/艾滋病人群提供治疗。

Project Summary: Toxoplasma gondii is an intracellular parasite that latently infects many hosts, including humans. Successful latent infection requires T. gondii to evade cytokine-induced, cell intrinsic responses and switch from a fast-growing form to a slow-growing encysted form. In humans, this life-long infection occurs in the brain where T. gondii can reactivate in the setting of acquired immune deficiencies. In AIDS patients, toxoplasmic encephalitis is the most common cause of focal brain lesions and can cause prolonged neurologic deficits even after appropriate treatment. Recent studies also suggest that, even in HIV+ patients on effective antiretroviral therapy, persistent T. gondii infection may adversely affect cognition and global immune responses. Despite its clinical importance, we lack a mechanistic understanding of what is actually responsible for T. gondii’s pathogenesis in the CNS, including what enables long-term latent infection. Such understanding is crucial to eventually preventing symptomatic disease in HIV/AIDs patients. Within the CNS, cysts are primarily found in neurons. Based on limited in vitro and in vivo studies, it has been presumed that while infected astrocytes cleared intracellular parasites, neurons did not and thus were the de facto host cell for persistent infection. In the last decade, our pioneering work has questioned this model, including showing that IFN-γ-stimulated neurons mount anti-parasitic defenses. As most of this work has occurred in murine models and neurons, it will have missed human neuron-specific pathways (e.g., humans lack the terminal IFN-γ dependent proteins used by murine cells to kill intracellular parasites). The goal of this grant is to address this gap by establishing robust stem cell derived human neuron models (hPSC neurons) which can be used to mechanistically define human neuron-T. gondii interactions that enable immune evasion and persistence. To accomplish this goal, our will leverage our synergistic expertise in CNS toxoplasmosis (Dr. Koshy) and stem cell biology (Dr. Churko) to generate Cre reporter hPSC neurons and CRISPRi-expressing hPSC neurons (Aim 1) and to begin to define the mechanisms by which cytokine stimulated hPSC-neurons control T. gondii (Aim 2). With the completion of these aims, we will have established and validated tools that will form the essential foundation of a long-term program to molecularly define the neuron-T. gondii interactions that enable control and persistence of T. gondii in human neurons. The work proposed here represents an important first step toward developing human neuron-specific therapies for acute and chronic toxoplasmosis. Such therapies will be of great benefit for the HIV/AIDS population at risk for toxoplasmic encephalitis.