Mechanisms of placental infection by food-borne pathogens

描述(申请人提供)：孕妇是侵袭性沙门氏菌感染的高危人群，这可能会导致母婴并发症。我们发现，孕鼠感染沙门氏菌后，胎盘滋养层细胞(TbCs)细菌大量增殖，导致胎儿和母体迅速死亡。在体外，沙门氏菌在人滋养层细胞来源的绒毛膜癌细胞中生长旺盛。然而，关于孕期沙门氏菌感染易感性增加的机制，我们的认识存在差距。我们的长期目标是开发能够控制孕妇细胞内感染结局的策略。这项应用的总体目标是确定使脐带血对沙门氏菌高度敏感的分子机制。我们的中心假设是胎盘TBCs可以有效地感染沙门氏菌，并提供一个独特的细胞内生态位，允许由于无效的免疫反应而导致的不受控制的毒力沙门氏菌复制，导致胎盘死亡。我们的中心假设将通过三个特定的目标来检验，这将确定：1)沙门氏菌在TBCs中不受控制的细胞内生长的机制，2)干扰素-β？3)沙门氏菌引起胎盘炎症的机制。研究将使用原代分离的人类滋养层细胞和胎盘外植体进行体外研究，并使用怀孕的小鼠模型进行体内研究。具体目标1将检验沙门氏菌在滋养层细胞的独特细胞内环境中茁壮成长的假设。我们将阐明不同胎龄的不同滋养细胞亚群是否由于特定的细胞-细胞相互作用而特异性地促进沙门氏菌的复制。我们还将确定细菌注射毒力因子在促进进入TBCs方面的作用，以及滋养层细胞吞噬的相对作用。最后，我们将描述沙门氏菌增殖的细胞内环境(亚细胞空泡)的分子特征。特定目标2将检验滋养层对干扰素？？低反应性的假设。导致对沙门氏菌的敏感性。我们将检测干扰素-？沙门氏菌是如何在TBCs中调节JAK-STAT-1信号通路的，以及是否能抑制干扰素的负反馈抑制？SOCS等信号转导途径有助于TBC-沙门氏菌感染的解决。具体目标3将测试工作假设，即ST诱导的TBC死亡的机制不同于在其他感染细胞中遇到的机制，并有助于明显的炎症。我们将确定不同的炎症(IL-1？、IL-18、caspase)和/或细胞死亡信号(细胞凋亡、下垂或坏死性下垂)是TBC对沙门氏菌的敏感性的原因。总体而言，我们希望确定独特的滋养细胞特征与沙门氏菌毒力因子在破坏胎盘方面的相对重要性。这一贡献将极大地重塑我们对怀孕期间感染风险的理解。这项研究的成功完成将代表着开发合理的治疗方案来管理妊娠期胎盘感染的研究要求的连续研究的关键的第一步。

DESCRIPTION (provided by applicant): Pregnant women are at high risk for invasive Salmonella infection, which can cause both maternal and fetal complications. We found that Salmonella infection in pregnant mice caused rapid fetal and maternal death due to massive bacterial proliferation in placental trophoblast cells (TBCs). In vitro, Salmonella flourished in human trophoblast-derived choriocarcinoma cells. However, there is a gap in our knowledge regarding the mechanism of increased susceptibility to Salmonella infection during pregnancy. Our long-term objective is to develop strategies that can control the outcome of intracellular infections in pregnant women. The overall objective of this application is to identify the molecular mechanisms that render TBCs highly susceptible to Salmonella. Our central hypothesis is that placental TBCs are productively infected by Salmonella and provide a unique intra- cellular niche that permits uncontrolled virulent Salmonella replication due to an ineffectie immune response, resulting in placental death. Our central hypothesis will be tested by three Specific Aims which will identify: 1) the mechanism of uncontrolled intracellular growth of Salmonella in TBCs, 2) the role(s) of IFN-?? in susceptibility of TBCs to Salmonella, and 3) the mechanism of Salmonella-induced placental inflammation. Studies will be performed in vitro using primary isolated human trophoblast cells and placental explants, and in vivo using pregnant mouse models. Specific Aim 1 will test the hypothesis that Salmonella thrive within a unique intracellular environment of trophoblast cells. We will elucidate whether different trophoblast subpopulations of differing gestational age specifically promote Salmonella replication due to specific cell-cell interactions. We will also identify the role of bacterial injectisome virulence factors in facilitating entry into TBCs and the relative role of trophoblast phagocytosis. Lastly, we will characterize the molecular features of the intracellular environment (sub-cellular vacuole) in which Salmonella proliferates. Specific Aim 2 will test the hypothesis that trophoblastic hypo-responsiveness to IFN-?? contributes to susceptibility to Salmonella. We will test whether IFN-? pre- treatment of TBCs can contain Salmonella infection, how Salmonella modulates the JAK-STAT-1 signaling pathway in TBCs, and if knocking down negative-feedback inhibitors of IFN-?? signaling such as SOCS expression can aid resolution of TBC-Salmonella infection. Specific Aim 3 will test the working hypothesis that the mechanism of ST-induced TBC death is distinct from that encountered in other infected cells and contributes to overt inflammation. We will identify differential inflammation (IL-1?, IL-18, caspase) and/or cell death signaling (apoptosis, pyroptosis or necroptosis) as a cause of TBC susceptibility to Salmonella. Overall we expect to identify the relative importance of unique trophoblast features versus Salmonella virulence factors in destruction of the placenta. This contribution will significantly reshape our understanding of infection risk during pregnancy. Successful completion of this study will represent the critical first step in the continuum of research requird to develop rational treatment regimens for management of placental infections during pregnancy.