RNAseq analyses in immunocompromised and immune competent mice infected with the AIDS pathogen Cryptosporidium

 描述（由申请人提供）：隐孢子虫属是世界上最普遍的引起绦虫的病原体之一。虽然艾滋病患者的隐孢子虫病可能很严重，有时甚至致命，但没有一种药物可用于治疗这种脆弱人群的隐孢子虫病。免疫疗法被认为是一种有前途的方法，但艾滋病毒造成的免疫缺陷使得T辅助细胞的数量和功能都减少，特别是在肠道中。在致力于解决这一治疗空白的过程中，我们发现用可溶性弓形虫抗原（STAg）刺激先天免疫应答是IFNγ缺陷小鼠中隐孢子虫感染的快速有效的治疗方法。由于STAg以不依赖T细胞的方式刺激先天免疫，因此了解STAg显示抗隐孢子虫活性的机制将为免疫功能低下患者隐孢子虫病的新型免疫疗法的开发提供信息。STAg的抗隐孢子虫活性可能是由于刺激先天性免疫应答，因为作用迅速，在治疗的前两天内减少卵囊脱落。重要的是，我们的初步数据表明，STAg治疗在IFNγ不存在的情况下是有效的。本研究首次全面分析了导致隐孢子虫生物体清除的IFNγ非依赖性先天免疫机制。此外，这些研究将是第一个表征早期宿主隐孢子虫体内转录组的研究，这一数据集将成为隐孢子虫研究界的宝贵资源。本研究的目的是：目的#1：表征野生型和IFNγ缺陷型小鼠中清除隐孢子虫感染所需的早期免疫应答。在这个目标中，我们将优化我们的模型的时间和剂量的STAg治疗，并确定是否可以概括的STAg的效果与纯化的TgPRF治疗。目的#2：鉴定与STAg药物保护作用相关的宿主和寄生虫基因表达的变化，以对抗隐孢子虫感染。使用RNA测序，我们将表征从感染C的野生型和IFNγ缺陷小鼠获得的回肠组织的转录组。用STAg治疗。对这一综合性时间进程的分析将产生关于STAg对C.将使用免疫细胞耗竭或敲除小鼠测试的细小病毒。这些研究将确定有效对抗艾滋病机会病原体隐孢子虫的关键先天免疫应答，并为开发IFNγ依赖性和独立性免疫治疗目前无法治疗的隐孢子虫病提供必要的数据。

 DESCRIPTION (provided by applicant): Cryptosporidium species are among the world's most ubiquitous diarrhea-causing pathogens. Although cryptosporidiosis can be severe and sometimes lethal in AIDS patients, not a single drug is available for the treatment of cryptosporidiosis in this vulnerable population. Immunotherapy is regarded as a promising approach but the immunodeficiency imposed by HIV is such that T helper cells are reduced both in number and function, particularly in the gut. In working to address this therapeutic gap, we discovered that stimulation of innate immune responses with soluble Toxoplasma antigen (STAg) is a rapid and effective treatment for Cryptosporidium parvum infection in IFNγ deficient mice. As STAg stimulates innate immunity in a T-cell independent manner, understanding the mechanisms by which STAg displays anti-Cryptosporidium activity would inform development of a novel immunotherapy for cryptosporidiosis in immunocompromised patients. The anti-Cryptosporidium activity of STAg is likely due to the stimulation of innate immune responses because the effect is rapid, reducing oocyst shedding within the first two days of treatment. Importantly, our preliminary data show that STAg treatment is effective in the absence of IFNγ. The present studies constitute the first comprehensive analysis of IFNγ independent innate immune mechanisms that lead to clearance of Cryptosporidium organisms. Moreover, these studies will be the first to characterize the early host-Cryptosporidium transcriptome in vivo, a data set that will be an invaluable resource to the Cryptosporidium research community. The aims of this study are: Aim #1: To characterize the early immune response required for clearance of Cryptosporidium infection in wild type and IFNγ deficient mice. In this aim, we will optimize our model with respect to timing and dosage of STAg treatment, and determine if the effect of STAg can be recapitulated by treatment with purified TgPRF. Aim #2: To identify changes in host and parasite gene expression associated with STAg medicated protection against Cryptosporidium infection. Using RNA seq, we will characterize the transcriptome of ileum tissues obtained from wild type and IFNγ deficient mice infected with C. parvum and treated with STAg. Analyses of this comprehensive time course will generate hypotheses on the mechanism of STAg action against C. parvum that will be tested using immune cell depletion or knock-out mice. These studies will identify the key innate immune responses effective against the AIDS opportunistic pathogen Cryptosporidium, and provide data necessary for the development of both IFNγ dependent and independent immunotherapeutics for currently untreatable cryptosporidiosis.