Modeling Entamoeba histolytica host-parasite interactions

溶组织内阿米巴(Eh)是一种寄生虫，它要么无害地定植在粘液层，要么在入侵结肠时引发疾病。在感知到寄生虫后，先天免疫细胞必须确定对宿主的危险程度，并启动适当的反应。区分寄生虫威胁程度的一种方法是区分与寄生虫表面的直接接触和远距离分泌/释放的成分。对于阿米巴来说，通过直接接触天然免疫细胞选择性地激活传感器和细胞反应是未知的。与释放的Eh组分引起的反应相比，活体Eh与宿主组织/细胞的直接接触引起的反应在幅度和质量上都不同。我们最近发现，Eh通过一种接触性事件激活NLRP3炎症体，以调节生物活性IL-1？的释放，这与体内IL-1？选择性地释放以响应寄生虫入侵有关。这表明NLRP3炎症体1)是Eh与先天免疫细胞直接结合的感觉，2)是区分完整的侵袭性寄生虫的中心途径，只有在需要的时候/在需要的地方才集中某些宿主防御。在动物疾病模型中的研究表明，主要的Eh表面粘附素，半乳糖凝集素，是激活抗Eh防御的关键。半乳糖凝集素通过分别与粘蛋白和未知宿主细胞受体结合半乳糖/半乳糖蛋白残基，介导Eh在黏蛋白和侵袭过程中与MUC2黏蛋白的黏附。我们发现NLRP3的激活需要通过表面相关的半乳糖凝集素与Eh结合。天然可溶性半乳糖凝集素具有免疫刺激作用，可诱导强大的肿瘤坏死因子-α，但不能激活NLRP3炎症体。因此，作用于宿主细胞的特定寄生虫分子传递的信号传递关于寄生虫遭遇性质的关键信息，这些信息被区分并转化为与寄生虫威胁类型匹配的反应。我们的数据表明，接触通过涉及半乳糖凝集素的黏附机制直接引发免疫激活，并假设含有半乳糖凝集素的接触依赖的信号突触参与了导致NLRP3炎症体激活的信号级联反应。研究内容包括：1，确定接触依赖与接触非依赖的先天免疫细胞反应，确定由表面相关半乳糖凝集素和天然可溶性半乳糖凝集素介导的先天免疫细胞反应，以及哪些先天免疫细胞反应需要激活NLRP3炎症体，以及2确定接触依赖半乳糖凝集素信号突触激活NLRP3炎症体的分子机制。对于目标1，我们将使用多重细胞因子阵列来评估多达60种感染相关细胞因子的分泌，以确定反映接触与非接触模式的质量/大小的先天免疫细胞特征。对于目标2，我们已经确定a5？1整合素是Eh诱导的NLRP3激活的表面受体，需要EhCP5的结合和激活。然而，尽管EhCP5与a5？1整合素结合，但纯化的EhCP5诱导微弱的激活，单独不能触发NLRP3。完整的a5？1整合素激活和NLRP3诱导同时需要表面结合的半乳糖凝集素。共聚焦显微镜显示5？1整合素、p-paxlin(活性整合素信号)和NLRP3迅速定位于Eh接触部位，提示Gal-lectin调节5？1整合素的募集。我们将测试固定在Eh表面的半乳糖凝集素是否将a5？1整合素招募到含有半乳糖凝集素、EhCP5、a5？1整合素和NLRP3的活性信号复合体中。我们将确定NLRP3是否在5？1整合素激活后被招募，以及5？1整合素及其活性信号成分是否直接与NLRP3相互作用来调节其活性。

Entamoeba histolytica (Eh) is a parasite that either harmlessly colonizes the mucus layer or trigger disease when it invades the colon. Upon sensing parasites, innate immune cells must determine the level of danger to the host and initiate appropriate responses. One way to discriminate the degree of parasite threat is to distinguish between direct contact with parasite surfaces vs components that are secreted/released at a distance. The sensors and cellular responses selectively activated by direct contact with innate immune cells are unknown for ameba. Direct contact of live Eh with host tissue/cells elicits a response that is different in both magnitude and quality compared to responses elicited by released Eh components. We recently identified Eh activates the NLRP3 inflammasome through a contact-dependent event to regulate release of bioactive IL-1ß, which correlates in vivo where IL-1ß is selectively released in response to invading parasites. This indicates NLRP3 inflammasome 1), senses direct binding of Eh to innate immune cells and 2), is a central pathway for distinguishing intact invasive parasites to focus certain host defenses only when/where they are required. Studies in animal models of disease have demonstrated the major Eh surface adhesin, the Gal-lectin, is critical for activating anti-Eh defenses. The Gal-lectin mediates Eh attachment to MUC2 mucin during colonization and host cells during invasion by binding Gal/GalNAc residues on mucin and unknown host cell receptors, respectively. We identified NLRP3 activation requires Eh binding via surface-associated Gal-lectin. Native soluble Gal-lectin is immunostimulatory and induces robust TNF-a but it does not activate the NLRP3 inflammasome. Thus, signals conveyed by specific parasite molecules acting upon the host cell deliver critical information about the nature of the parasite encounter, which are distinguished and translated into responses that match the type of parasite threat. Our data suggest contact directly provokes immune activation via an adherence mechanism involving the Gal-lectin and hypothesize that a contact-dependent signaling synapse containing the Gal-lectin engages a signaling cascade leading to NLRP3 inflammasome activation. The study aima are 1, to determine innate immune cell responses that are contact-dependent vs contact-independent, define those that are mediated by surface-associated Gal-lectin vs native soluble Gal-lectin, and which ones require activation of the NLRP3 inflammasome and, 2 to determine the molecular mechanism of the contact-dependent-Gal-lectin-signaling synapse that activates NLRP3 inflammasome. For aim 1 we will use multiplex cytokine array to assess secretion of up to 60 infection-associated cytokines to define innate immune cell signatures reflecting the quality/magnitude of contact vs. non-contact modes. For aim 2 we have already identified a5ß1 integrin is a surface receptor for Eh-induced NLRP3 activation that requires binding and activation by EhCP5. However, despite binding to a5ß1 integrin, purified EhCP5 induces weak activation and alone does not trigger NLRP3. Full a5ß1 integrin activation and NLRP3 induction simultaneously requires surface bound Gal-lectin. Confocal microscopy revealed a5ß1 integrin, p-paxillin (active integrin signaling) and NLRP3 rapidly localize to the site of Eh contact, suggesting Gal-lectin regulates a5ß1 integrin recruitment. We will test if Gal-lectin immobilize on the surface of Eh recruits a5ß1 integrin into an active signaling complex containing Gal-lectin, EhCP5, a5ß1 integrin and NLRP3. We will determine whether NLRP3 is recruited following a5ß1 integrin activation and whether a5ß1 integrin and its active signaling components interact directly with NLRP3 to regulate its activity.