Mechanistic study of human placental infection by Listeria monocytogenes

描述（由申请人提供）：胎盘的一个关键功能是保护发育中的胎儿。绒毛外细胞滋养层和合体滋养层在与母体组织的界面处以及绒毛外细胞滋养层和合体滋养层在与胎儿血液的界面处提供屏障功能。 胎盘内皮细胞对这种保护至关重要。这种保护是如此有效，以至于少数病原体可以绕过它并感染胎儿。其中一种病原体是单核细胞增生李斯特菌，它是威胁生命的食源性疾病-李斯特菌病的病原体。孕妇极易感染胎盘病毒，并可将这种病原体传播给胎盘和胎儿，导致胎儿和新生儿死亡（25%）和长期神经系统后遗症（13%）。两项研究令人信服地证明，L。单核细胞增多症通过侵入绒毛外细胞滋养层和合体滋养层进入人胎盘。然而，L.滋养层细胞内外的单核细胞增多症还不确定。我们的初步数据使我们观察到L.单核细胞增多症在原代人合体滋养层中显示非典型的细胞内生命周期。的确，L.单核细胞增多症被内化到那些细胞中，并且最初在胞质溶胶中增殖。然而，细菌负荷在感染后10-20小时严重降低。这些数据支持了这一提议的中心假设，即合胞体滋养层可以建立抗菌反应。这种反应不足以完全阻止某些病原体，如L。单核细胞增多症，其增殖足够的时间以将感染传递到其他胎盘细胞并到达胎儿。为了扩展这一发现，目的1将建立原代人胎盘细胞（合体滋养层细胞、细胞滋养层细胞、成纤维细胞、巨噬细胞（Hofbauer细胞）和内皮细胞）对L.单核细胞增多症在感染的经胎盘进展。原代人胎盘细胞的培养将用于L。单核细胞增生细胞内生命周期（Ai 1A）。将使用足月胎盘绒毛外植体研究组织感染的连续阶段（目的1B）。目的二是探讨合胞体滋养层细胞对L.单核细胞增多症这将涉及下一代测序平台，以进行暴露于或不暴露于野生型L的细胞的比较转录谱分析。单核细胞增多症在确认结果后，将选择最相关的途径，以确定其抗菌功能。该建议的重点是建立相对易感性感染的各种原代细胞组成的人胎盘，并确定先天性免疫反应的人合体滋养层。从长远来看，在提案过程中获得的数据将确定毒力机制和宿主途径，这些机制和宿主途径可以被治疗剂靶向，以降低妊娠相关感染的发病率和死亡率。

DESCRIPTION (provided by applicant): A key function of the placenta is to protect the developing fetus. The barrier functions provided at the interface with maternal tissues by extravillous cytotrophoblasts and the syncytiotrophoblast, and at the interface with fetal blood by placental endothelial cells, are crucial for this protection. This protection is so efficient that nly a few pathogens can circumvent it and infect the fetus. One of these pathogens is Listeria monocytogenes, the etiologic agent of the life-threatening foodborne illness listeriosis. Pregnant women are highly susceptible to listeriosis and can transmit this pathogen to the placenta and fetus, leading to fetal and neonatal death (25%) and long-term neurologic sequelae (13%). Two studies convincingly argued that L. monocytogenes enters the human placenta by invading extravillous cytotrophoblasts and the syncytiotrophoblast. However, the fate of L. monocytogenes within and beyond those trophoblastic cells is unsettled. Our preliminary data led to the observation that L. monocytogenes displays an atypical intracellular lifecycle in primary human syncytiotrophoblasts. Indeed, L. monocytogenes is internalized into those cells and initially proliferates in the cytosol. However, the bacterial burden severely decreases 10-20 hours post-infection. These data support the central hypothesis of this proposal that the syncytiotrophoblast can establish an antimicrobial response. This response is not sufficient to completely halt some pathogens, such as L. monocytogenes, which proliferates for enough time to pass on infection to other placental cells and reaches the fetus. To expand on this finding, Aim 1 will establish the susceptibility of primary human placental cells (syncytiotrophoblasts, cytotrophoblasts, fibroblasts, macrophages (Hofbauer cells), and endothelial cells) to L. monocytogenes during the transplacental progression of infection. Culture of primary human placental cells will be used for detailed study of the L. monocytogenes intracellular lifecycle (Ai 1A). Term placental villous explants will be used for study of the successive stages of the infection of the tissue (Aim 1B). Aim 2 will identify innate defense mechanisms activated in syncytiotrophoblastic cells in response to L. monocytogenes infection. This will involve next- generation sequencing platforms to perform comparative transcriptional profiling of cells exposed, or not, to wild type L. monocytogenes. After confirmation of the results, the most pertinent pathways will be selected in order to establish their antimicrobial functions. This proposal focuses on establishing the relative susceptibility to infection of the various primary cells that compose the human placenta, and on identifying the innate immune responses of the human syncytiotrophoblast. In the long term, the data acquired in the course of the proposal will identify virulence mechanisms and host pathways that can be targeted by therapeutic agents to decrease the morbidity and mortality of pregnancy-associated infections.