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

 描述(申请人提供)：隐孢子虫是世界上最普遍的腹泻病原体之一。虽然隐孢子虫病在艾滋病患者中可能很严重，有时甚至是致命的，但在这一脆弱的人群中，没有一种药物可用于治疗隐孢子虫病。免疫治疗被认为是一种很有前途的方法，但艾滋病毒造成的免疫缺陷导致T辅助细胞在数量和功能上都减少了，特别是在肠道。在努力解决这一治疗缺口的过程中，我们发现用可溶性弓形虫抗原(STAg)刺激先天免疫反应是治疗干扰素γ缺陷小鼠微小隐孢子虫感染的一种快速有效的方法。由于STAg以非T细胞依赖的方式刺激天然免疫，了解STAg显示抗隐孢子虫活性的机制将有助于开发一种新的免疫治疗方法来治疗免疫低下患者的隐孢子虫病。STAG的抗隐孢子虫活性可能是由于刺激了天然免疫反应，因为作用迅速，在治疗的头两天内减少了卵囊的脱落。重要的是，我们的初步数据显示，在没有干扰素γ的情况下，STAG治疗是有效的。本研究首次全面分析了干扰素γ非依赖于隐孢子虫生物清除的先天免疫机制。此外，这些研究将是第一次描述体内早期宿主-隐孢子虫转录组的特征，这一数据集将是隐孢子虫研究界的宝贵资源。本研究的目的是：目的1：研究野生型和干扰素γ缺陷小鼠清除隐孢子虫感染所需的早期免疫反应。为此，我们将在STAG治疗的时机和剂量方面对我们的模型进行优化，并确定是否可以用纯化的TgPRF处理STAG的效果。目的#2：确定与STAG药物保护隐孢子虫感染相关的宿主和寄生虫基因表达的变化。利用RNAseq技术，我们将对野生型和干扰素γ缺陷小鼠的回肠组织转录组进行鉴定，这些小鼠感染微小隐孢子虫并经STAG处理。对这一综合时间过程的分析将产生关于STAG对微小弧菌作用机制的假设，这将通过免疫细胞耗尽或基因敲除小鼠进行测试。这些研究将确定对艾滋病机会性病原体隐孢子虫有效的关键先天免疫反应，并为开发目前无法治疗的隐孢子虫病的干扰素γ依赖和独立免疫疗法提供必要的数据。

 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.