Establishing the roles of lncRNAs in placental infection by Listeria monocytogenes

项目摘要 胎盘屏障是保护胎儿免受大多数感染的有效细胞屏障。但也有 病原体如革兰氏阳性细菌单核细胞增生李斯特菌（Lm）已经进化出毒力 打破这一屏障的机制。感染和炎症的胎儿胎盘单位具有毁灭性的 后果包括早产、宫内生长受限、胎儿死亡或严重感染 患有长期神经系统后遗症的新生儿。细胞滋养层（CYT）和合体滋养层（SYN）是 形成胎盘屏障的胎儿上皮细胞。多核的SYN是一个真正的合胞体， 在潜在的CYT融合时产生，并且已知对微生物具有抗性。然而，我们缺乏 了解胎盘屏障的防御机制，对胎盘屏障的防御机制知之甚少。 使得一些病原体能够克服这一障碍。为了填补我们知识上的这些空白，我们研究了 Lm与分离自健康足月人胎盘的原代CYT/SYN的相互作用。RNA测序（RNA- seq，n=3）的感染与未感染的CYT/SYN原代培养物的比较，证实了在CYT/SYN原代培养物中存在转录调控因子。 感染后5小时炎症反应。通路分析揭示了几个炎症通路 在Lm的存在下上调，细胞因子阵列证实转录反应是 与细胞因子的生产显着增加。重要的是，一些长的非编码RNA 在感染Lm后，lncRNA转录物显著上调。然而，这些角色 lncRNA仍然未知。LncRNA在调节免疫和细胞因子的表达中起关键作用， 感染的巨噬细胞中的炎症基因，但它们在一般情况下，特别是在 胎盘细胞感染的背景。这项工作将测试4种选择的lncRNA影响 胎盘细胞对Lm感染的易感性和/或它们对Lm的转录和炎症反应。要求1 将确定lncRNA在胎盘细胞对Lm感染易感性中的作用。的表达 将调节lncRNA，并监测对Lm感染的敏感性长达24小时。 目的2将确定lncRNAS在胎盘细胞转录组和胎盘细胞分泌中的作用。 感染期间的细胞因子。选择的lncRNA的表达将被沉默，细胞培养上清液将被纯化。 将对细胞裂解物进行细胞因子阵列和RNA-seq。这项工作完成后， 我们将建立lncRNA在形成人类胎盘的细胞易感性中的作用， Lm感染的屏障、细胞因子的产生以及细胞对感染的转录应答。这些 数据将使我们能够开发一个新的项目，该项目将侧重于建立作用和行动机制， 胎盘免疫防御感染中已鉴定的lncRNA。从长远来看，lncRNA 有前途的诊断工具和治疗目标，可用于降低发病率和死亡率， 妊娠相关感染。

PROJECT SUMMARY The placental barrier is an efficient cell barrier that protects the fetus from most infections. However, some pathogens such as the Gram-positive bacterium Listeria monocytogenes (Lm) have evolved virulence mechanisms to breach this barrier. Infection and inflammation of the fetoplacental unit have devastating consequences including preterm birth, intrauterine growth restriction, fetal death, or severe infection of the neonate with long-term neurological sequelae. Cytotrophoblasts (CYT) and the syncytiotrophoblast (SYN) are fetal epithelial cells that form the placental barrier. The multinucleated SYN is a true syncytium that is generated upon fusion of underlying CYT and is known to be resistant to microbes. However, we lack knowledge regarding the defense mechanisms of the placental barrier and know little about the mechanisms that allow some pathogens to overcome this barrier. To address these gaps in our knowledge, we study the interaction of Lm with primary CYT/SYN isolated from healthy, term human placentas. RNA sequencing (RNA- seq, n=3) of infected versus non-infected primary culture of CYT/SYN established that there is a transcriptional inflammatory response 5 h post-infection. Pathway analysis revealed several inflammatory pathways upregulated in the presence of Lm and a cytokine array confirmed that the transcriptional response was paralleled with a significant increase in the production of cytokines. Importantly, several long non-coding RNAs (lncRNAs) transcripts were significantly upregulated after infection with Lm. However, the roles of these lncRNAs remain unknown. LncRNAs play a critical role in regulating the expression of immune and inflammatory genes in infected macrophages, but they have been understudied in general and in particular in the context of infection of placental cells. This work will test the hypothesis that 4 selected lncRNAs affect placental cell susceptibility to Lm infection and/or their transcriptional and inflammatory responses to Lm. Aim 1 will establish the role of the lncRNAs in placental cell susceptibility to Lm infection. The expression of the lncRNAs will be modulated and the resulting susceptibility to infection by Lm will be monitored for up to 24 h. Aim 2 will establish the roles of the lncRNAS in the placental cell transcriptome and in the production of cytokines during infection. The expression of selected lncRNAs will be silenced, cell culture supernatants will be subjected to a cytokine array and RNA-seq will be performed on cell lysates. At the completion of this work, we will have established the roles of the lncRNAs in the susceptibility of cells that form the human placental barrier to Lm infection, the production of cytokines, and the cell transcriptional response to infection. These data will allow us to develop a new project that will focus on establishing the roles and mechanisms of action of the identified lncRNAs in the placental immune defense against infections. In the long-term, lncRNAs are promising diagnostic tools and therapeutic targets that can be used to decrease the morbidity and mortality of pregnancy-associated infections.