Mechanistic study of human placental infection by Listeria monocytogenes

描述(申请人提供)：胎盘的一个重要功能是保护发育中的胎儿。绒毛外细胞滋养细胞和合体滋养细胞在与母体组织的界面上以及在与胎儿血液的界面上所起的屏障作用 胎盘内皮细胞对这种保护至关重要。这种保护是如此有效，以至于只有几种病原体可以绕过它，感染胎儿。其中一种病原体是单核细胞增多性李斯特氏菌，它是威胁生命的食源性疾病李斯特菌病的病原体。孕妇对李斯特菌病高度易感，并可将这种病原体传播到胎盘和胎儿，导致胎儿和新生儿死亡(25%)和长期的神经后遗症(13%)。两项研究令人信服地证明，单核细胞增多性李斯特氏菌通过侵入绒毛外细胞滋养层细胞和合体滋养层细胞进入人类胎盘。然而，这些滋养层细胞内外的单核细胞增多性李斯特菌的命运尚未确定。我们的初步数据导致观察到，在原代人类合体滋养层细胞中，单核细胞增多性李斯特氏菌表现出非典型的细胞内生命周期。事实上，单核细胞增多性李斯特菌被内化到这些细胞中，最初在胞浆中增殖。然而，细菌负荷在感染后10-20小时内严重减少。这些数据支持这一建议的中心假设，即合体滋养层细胞可以建立抗菌反应。这种反应不足以完全阻止一些病原体，如单核细胞增多症李氏杆菌，它会在足够长的时间内增殖，将感染传递给其他胎盘细胞并到达胎儿。为了扩大这一发现，目标1将确定原代人类胎盘细胞(合体滋养层细胞、细胞滋养层细胞、成纤维细胞、巨噬细胞(霍夫鲍尔细胞)和内皮细胞)在经胎盘感染过程中对单核细胞增多性乳杆菌的易感性。原代培养的人胎盘细胞将用于详细研究单核细胞增多性李斯特菌的细胞内生命周期(Ai1a)。足月胎盘绒毛外植体将用于研究组织感染的连续阶段(目标1B)。目的2鉴定合体滋养层细胞对单核细胞增多性李斯特菌感染的天然防御机制。这将涉及下一代测序平台，以对暴露于或未暴露于野生型单核细胞增多性乳杆菌的细胞进行比较转录图谱分析。在确认结果后，将选择最相关的途径来确定它们的抗菌功能。这项建议侧重于确定构成人类胎盘的各种初级细胞对感染的相对易感性，并识别人类合体滋养层细胞的先天免疫反应。从长远来看，提案过程中获得的数据将确定治疗药物可以针对的毒力机制和宿主途径，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.