Signal Transduction During Toxoplasma Infection

描述（由申请人提供）：本申请的长期目标是了解细胞内弓形虫感染的分子发病机制。寄生虫是免疫抑制人群中的重要病原体，被NIAID分类为B类生物防御生物。然而，在大多数情况下，这种广泛传播的寄生虫感染是无症状的。宿主在感染期间的存活需要稳健的1型细胞因子产生，然而过度旺盛的应答可导致宿主病理和死亡。因此，寄生虫和宿主都受益于受调节的促炎细胞因子应答。待检验的基本假设是T.弓形虫具有破坏导致促炎细胞因子产生的宿主信号传导途径的分子机制。最近的工作揭示了弓形虫能够从感染的细胞内下调细胞因子如TNF-1。越来越多的证据表明，这是通过复杂的操纵宿主细胞内信号级联。该提案的目标是阐明弓形虫破坏先天免疫系统中促炎信号级联的机制，特别是在体内感染靶向的细胞如巨噬细胞和树突状细胞内。将采用细胞和生物化学方法来确定弓形虫干扰宿主信号转导的点。在这方面，寄生虫诱导的信号分子STAT 3的激活，已知参与促炎细胞因子IL-12和TNF-1的下调，将被检查。在感染的巨噬细胞中TNF-1抑制的性质是深刻的。因此，我们将采用分子方法来确定寄生虫如何影响TNF-1基因的启动子活性。本提案的最后一部分将集中精力研究寄生虫感染的巨噬细胞和树突状细胞在小鼠体内感染期间是否表现出抑制表型。这将使用细胞和免疫学方法来完成，以确定体内感染细胞中IL-12和TNF-1的产生及其体内应答确定的Toll样受体配体的能力。此外，还将分析巨噬细胞和树突状细胞的细胞表面活化标志物表达。从这些研究中，我们期望获得重要的信息，T。弓形虫与宿主的先天免疫系统相互作用，使人们能够更深入地了解这种重要的机会致病菌的感染。 项目叙述/相关性：该项目与公共卫生的相关性在于，弓形虫感染了全球30-80%的人口。虽然通常是无症状的感染，但由于免疫功能不佳，寄生虫会成为一种毁灭性的，有时是致命的感染。该项目将提高我们对寄生虫免疫力的理解，从而改进治疗策略。

DESCRIPTION (provided by applicant): The long-term objective of this application is to understand the molecular pathogenesis of infection with the intracellular protozoan Toxoplasma gondii. The parasite is an important pathogen in immunosuppressed populations and is classified by the NIAID as a category B biodefense organism. Nevertheless, in most cases infection with this widespread parasite is asymptomatic. Host survival during infection requires robust Type 1 cytokine production, yet overly exuberant responses can result in host pathology and death. Thus, both parasite and host benefit from regulated pro-inflammatory cytokine responses. The underlying hypothesis to be tested is that T. gondii possesses molecular mechanisms to subvert host signaling pathways leading to pro-inflammatory cytokine production. Recent work has revealed the ability of Toxoplasma to down-regulate cytokines such as TNF-1 from within infected cells. Increasing evidence indicates this is achieved through sophisticated manipulation of host intracellular signaling cascades. The goal of this proposal is to elucidate mechanisms by which Toxoplasma subverts pro-inflammatory signaling cascades in the innate immune system, in particular within cells such as macrophages and dendritic cells that are targeted for in vivo infection. Cellular and biochemical methods will be employed to define the points at which Toxoplasma interferes with host signal transduction. In this regard, parasite-induced activation of signaling molecule STAT3, known to be involved in down-regulation of pro-inflammatory cytokines IL-12 and TNF-1, will be examined. The nature of TNF-1 suppression in infected macrophages is profound. Therefore, we will employ molecular methods to determine how the parasite influences promoter activity of the TNF-1 gene. The last part of this proposal will focus effort on examining if parasite-infected macrophages and dendritic cells display a suppression phenotype during in vivo mouse infections. This will be accomplished using cellular and immunological methods to determine IL-12 and TNF-1 production in infected cells in vivo and their in vivo ability to respond to defined Toll-like receptor ligands. In addition, macrophage and dendritic cell expression of cell surface activation markers will be analyzed. From these studies, we expect to gain important information on how T. gondii interacts with the host innate immune system, allowing a deeper understanding of infection with this important opportunistic pathogen. PROJECT NARRATIVE / RELEVANCE: The relevance of the project to public health is that Toxoplasma infects between 30-80% of the human population worldwide. While normally an asymptomatic infection, with suboptimal immune function the parasite emerges as a devastating and sometimes lethal infection. This project will enhance our understanding of immunity to the parasite, leading to improved treatment strategies.