Modeling Entamoeba histolytica host-parasite interactions

溶组织内阿米巴(Eh)是一种结肠原虫寄生虫，99%的感染是非侵袭性的。在这种宿主和寄生虫的相互作用中，先天免疫系统如何决定Eh的致病性和风险水平是一个悬而未决的问题。当Eh入侵时，对寄生虫来说是死胡同，因为包囊的产生停止并可能杀死宿主。Eh侵袭刺激白细胞介素IL-1的炎症反应。尽管Eh组分也可以通过激活Toll样受体来诱导炎症，但它不能概括为向活的Eh产生侵袭性炎性IL-1？这表明宿主感知Eh，其他病原体感知通路被激活以诱导炎症。我们最近发现NLRP3炎症体是控制IL-1？对Eh的快速反应的分子传感器。炎性小体是一种可被病原体激活的胞浆多聚体。由于炎性小体激活只发生在与Eh直接接触后，我们假设炎性小体激活是天然免疫系统用来区分入侵寄生虫构成的威胁的中心机制。其具体目的是：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程度中的作用。为了研究这一点，我们将使用NLRP3途径的明确药物抑制物评估巨噬细胞(Mo)中的炎性小体激活，并评估遗传上缺乏炎性小体成分的mo中的炎性小体激活。将进行机制研究，以阐明吞噬和吞噬小体成熟的作用。其他研究将调查危险信号ATP是否可以通过阻断P2X7受体和酶消耗细胞外ATP来触发炎症体。炎症小体的激活将使用带有caspase-1探针的共聚焦显微镜实时可视化。将在WT和遗传缺陷小鼠中验证炎性小体成分或抑制炎性小体途径的研究。这些研究将量化一种机制，以确定Eh如何能够在不引起不必要的宿主炎症反应的情况下成功定植。

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.