Innate Immune Recognition of Cryptosporidium parvum

描述（由申请人提供）：艾滋病相关的机会性寄生虫隐孢子虫是世界范围内导致疟疾的重要原因。在免疫功能低下的宿主，如艾滋病患者，这种寄生虫可能会导致严重的，长期的，最终致命的疾病。目前还没有批准用于艾滋病患者隐孢子虫病的特异性治疗方法，也没有疫苗可用于预防隐孢子虫感染。因此，继续寻找新的和有效的干预和预防方式至关重要。本项目的总体目标是研究先天免疫系统识别和应答C。小的虽然先天性免疫显然是重要的，但宿主细胞受体、寄生虫配体和信号通路参与启动和介导对C. parvum未知。关于这些受体、配体和途径的知识对于开发新的和有效的预防和干预方式是必不可少的。TLR介导的途径（特别是TLR 2、4和9）已被证明在对几种寄生虫的先天免疫应答中是重要的。然而，这些途径在先天免疫中的作用对C。parvum未知。初步研究表明，这些途径可能参与抵抗这种寄生虫的感染。假设C.寄生虫的先天性免疫应答和随后对该寄生虫的先天性免疫应答的起始由特异性TLR，特别是TLR 2、4和9介导。具体的目的是确定哪些TLR参与了对C.在体外人和鼠细胞系中以及在体内小鼠中的parvum。在第一部分的具体目的中，研究了TLR介导的C.将在人和鼠细胞系中研究parvum，其中使用基于siRNA的方法将转录后沉默个体TLR和相关共受体的表达。该策略将通过对C. parvum在已引入特异性TLR（和共受体）基因的人细胞系中以及在源自TLR缺陷小鼠的细胞系中诱导促炎反应。在第二部分中，对C.通过将粪便卵囊脱落和肠道寄生虫负荷与WT小鼠进行比较，确定TLR 2、4和9缺陷小鼠中的细小病毒感染。此外，将在C.细小病毒感染和未感染的WT和TLR缺陷小鼠。这将使未来的研究，以确定配体/s和阐明所涉及的特定信号通路。长期目标是确定是否可以靶向特定分子或途径以增强对寄生虫的先天免疫或预防由先天免疫应答失调诱导的免疫病理学。

DESCRIPTION (provided by applicant): The AIDS-associated opportunistic parasite Cryptosporidium is a significant cause of diarrheal disease worldwide. In immunocompromised hosts, such as AIDS patients, this parasite may cause severe, protracted and ultimately fatal disease. There is no specific treatment approved for cryptosporidiosis in AIDS patients and there is no vaccine available to prevent Cryptosporidium infection. Thus, the continued search for novel and effective interventional and preventive modalities is crucial. The overall goal of this project is to investigate the mechanisms by which the innate immune system recognizes and responds to C. parvum. While innate immunity is clearly important, the host cell receptors, parasite ligands and signaling pathways involved in initiating and mediating innate immune responses to C. parvum are unknown. Knowledge about these receptors, ligands and pathways is essential for development of novel and effective preventive and interventional modalities. TLR-mediated pathways (particularly TLRs 2, 4 and 9) have been shown to be important in innate immune responses to several parasites. However, the role of these pathways in innate immunity to C. parvum is unknown. Preliminary studies indicate that these pathways may be involved in resistance to infection with this parasite. The hypothesis is that recognition of C. parvum and consequent initiation of innate immune responses to this parasite is mediated by specific TLRs, in particular TLRs 2, 4 and 9. The specific aim is to determine which TLRs are involved in innate immune responses to C. parvum in human and murine cell lines in vitro and in mice in vivo. In the first part of the specific aim, TLR-mediated pro-inflammatory responses to C. parvum will be investigated in human and murine cell lines in which the expression of individual TLRs and associated co-receptors will be post-transcriptionally silenced using siRNA-based approaches. This strategy will be complemented by assessment of C. parvum induced proinflammatory responses in human cell lines into which specific TLR (and co-receptor) genes have been introduced as well as in cell lines derived from TLR-deficient mice. In the second part of the aim, resistance to C. parvum infection in mice deficient in TLRs 2, 4 and 9 will be determined by comparing fecal oocyst shedding and intestinal parasite burden to that in WT mice. In addition, expression and secretion of proinflammatory cytokines and chemokines will be evaluated in C. parvum-infected and uninfected WT and TLR-deficient mice. This will enable future studies to identify the ligand/s and elucidate the specific signaling pathways involved. The long-term goal is to determine whether specific molecules or pathways can be targeted to enhance innate immunity to the parasite or to prevent immunopathology induced by dysregulated innate immune responses.