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

项目总结： 弓形虫是一种细胞内寄生虫，潜伏感染包括人类在内的许多宿主。 成功的潜伏感染要求弓形虫逃避细胞因子诱导的细胞内源性反应和 从快速增长的形式切换到缓慢增长的包围式形式。在人类身上，这种终生感染 发生在大脑中，弓形虫可以在获得性免疫缺陷的背景下重新激活。在……里面 艾滋病患者中，弓形体脑炎是脑局灶性病变的最常见原因，并可 即使在适当的治疗后，也会导致长期的神经功能障碍。最近的研究还表明， 即使在接受有效抗逆转录病毒治疗的艾滋病毒患者中，持续的弓形虫感染也可能产生不利影响。 影响认知和全球免疫反应。尽管它在临床上很重要，但我们缺乏一种 了解弓形虫在中枢神经系统中的实际致病原因，包括 使长期潜伏感染成为可能。这样的理解对于最终预防症状至关重要 艾滋病毒/艾滋病患者的疾病。在中枢神经系统内，囊泡主要存在于神经元中。基于受限制的 体外和体内研究表明，当被感染的星形胶质细胞清除细胞内时 寄生虫，神经元没有，因此是持续感染的事实上的宿主细胞。在最后一次 十年来，我们的开创性工作对这一模型提出了质疑，包括显示干扰素-γ刺激 神经元建立起抗寄生虫的防御系统。由于这些研究大多发生在小鼠模型和 神经元，它将错过人类神经元特有的通路(例如，人类缺少末端干扰素-γ 小鼠细胞用来杀死细胞内寄生虫的依赖蛋白)。这笔赠款的目标是 通过建立强大的干细胞来源的人类神经元模型(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。使免疫逃避成为可能的弓形虫相互作用 和坚持不懈。为了实现这一目标，我们将利用我们在CNS的协同专业知识 弓形虫病(Koshy博士)和干细胞生物学(Churko博士)产生Cre报告hPSC神经元 和表达CRISPRi的hPSC神经元(目标1)，并开始定义 细胞因子刺激的hPSC神经元控制弓形虫(目标2)。随着这些目标的实现，我们将 已建立并验证工具，这些工具将构成长期计划的重要基础 从分子上定义神经元-T。弓形虫相互作用使弓形虫能够控制和持续存在于 人类神经元。这里提出的工作代表着人类发展的重要的第一步 急性和慢性弓形虫病的神经元特异性治疗。这样的疗法将会有很大的好处 针对有弓形虫脑炎风险的艾滋病毒/艾滋病人群。

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.