Modeling Entamoeba histolytica host-parasite interactions

溶组织内阿米巴（Eh）是一种结肠原生动物寄生虫，99%的感染是非侵入性的。先天免疫系统如何决定Eh的致病性和风险水平是宿主-寄生虫相互作用中尚未解决的问题。当Eh入侵时，它对寄生虫来说是一个死胡同，因为囊肿的产生停止并可能杀死宿主。Eh入侵刺激白细胞介素-1ß炎症反应。尽管Eh成分也可以通过toll样受体激活诱导炎症，但它并不能重现活性Eh的侵袭性炎症IL-1ß产生。这表明宿主感知Eh和其他病原体感知途径被激活以诱导炎症。我们最近发现NLRP3炎性体是控制IL-1ß对Eh快速反应的分子传感器。炎性小体是可被病原体激活的胞质多聚体复合物。由于炎性小体激活仅在直接接触Eh后发生，我们假设炎性小体激活是先天免疫系统用来区分入侵寄生虫威胁的中心机制。具体目的是：1)确定Eh激活炎性小体的分子机制；2)量化炎性小体在调节炎症反应强度方面的功能。为了研究这一点，我们将使用NLRP3途径的药理学抑制剂评估巨噬细胞（mo）中的炎症小体激活情况，并对炎症小体成分基因缺陷的mo进行评估。机制研究将阐明其在吞噬作用和吞噬体成熟中的作用。其他研究将调查危险信号ATP是否可以通过阻断P2X7受体和酶促细胞外ATP的消耗来触发炎性体。炎性体的激活将使用caspase-1探针进行共聚焦显微镜实时观察。研究将在WT和基因缺陷小鼠中验证炎症小体成分或炎症小体途径的抑制。这些研究将量化一种机制，以确定Eh如何能够成功定植而不引起不必要的宿主炎症反应。

Entamoeba histolytica (Eh) is a colonic protozoan parasite where 99% of infections are non-invasive. How the innate immune system determines the pathogenicity and level of risk posed by Eh are unanswered questions in this host-parasite interaction. When Eh invades, it is a dead end for the parasite as cysts production ceases and may kill the host. Eh invasion stimulates interleukin IL-1ß inflammatory responses. Even though Eh components can also induce inflammation through Toll-like receptor activation, it does not recapitulate the aggressive inflammatory IL-1ß production towards live Eh. This indicates that the host senses Eh and that other pathogen sensing pathways are activated to induce inflammation. We recently identified that NLRP3 inflammasome is the molecular sensor controlling the rapid IL-1ß response to Eh. Inflammasomes are cytosolic multimeric complexes that can be activated by pathogens. As inflammasome activation only occurs following direct contact with Eh, we hypothesize that inflammasome activation is a central mechanism used by the innate immune system to discriminate the threat posed by invading parasites. The specific aims are: 1) to determine the molecular mechanism of inflammasome activation by Eh and, 2) to quantify the function of inflammasomes in regulating the magnitude of the inflammatory response. To investigate this, we will assess inflammasome activation in macrophages (mo) using defined pharmacological inhibitors of the NLRP3 pathway and in mo that are genetically deficient for inflammasome components. Mechanistic studies will be done to elucidate a role for phagocytosis and phagosome maturation. Other studies will investigate if the danger signal ATP can trigger the inflammasome by blocking the P2X7 receptor and by enzymatic depletion of extracellular ATP. Inflammasome activation will be visualized in real-time using confocal microcopy with caspase-1 probes. Studies will be verified in WT and genetically deficient mice for inflammasome components or following inhibition of the inflammasome pathway. These studies will quantify a mechanism to define how Eh is able to successfully colonize without evoking unwanted host inflammatory responses.