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.
Infections of the placenta have devastating consequences on the developing fetus. Using the bacterium Listeria monocytogenes as a model pathogen, this study will establish the role of newly identified long non- coding RNAs (lncRNAs) in the antimicrobial responses of human placental cells. LncRNAs are promising diagnostic tools and therapeutic targets that can be used to decrease the morbidity and mortality of pregnancy- associated infections.
