Listeria monocytogenes (Lm) is a bacterial pathogen that utilizes an intracellular lifecycle to spread throughout the body, including the placenta in pregnant individuals. Placental infection and disease can lead to negative fetal outcomes including spontaneous abortion, birth defects, and stillbirths. Extracellular vesicles (EVs) are tiny particles secreted by nearly every cell type in the body and serve as a cellular signaling mechanism. EVs have been implicated in many cellular functions and diseases throughout the body, including those involving the placenta. Placental EVs can have immunomodulatory effects, but during placental disease they can also act in a pro-inflammatory manner, leading to disease progression. EVs can also be proinflammatory during intracellular bacterial infection, where they can communicate the infection and coordinate an immune response. In this dissertation, I investigated how Lm infection of trophoblasts alters the EVs produced by the infected cells, and how they can activate an immune response. Chapter 1 of the dissertation details the current literature on the role that EVs play during bacterial infections and placental development and disease. Chapter 2 focuses on establishing a trophoblast stem cell model (TSC) to study placental infections. TSCs are the source of trophoblasts in the placenta, and cultivation of these cells allow for the continual study of placental disease. Here, I found that TSCs are susceptible to Lm infection, although it requires a higher bacterial load and longer time course compared to other cell types. This chapter details ways to model placenta-pathogen interactions in vitro, allowing for the study of these interactions in a laboratory setting. Chapter 3 investigated how Lm infection of TSCs altered the cargo of the tEVs produced. Previous studies into EVs from infected cells found components from the bacterial cells loaded into the EVs, including bacterial DNA, RNA, and proteins. We found many more unique proteins in the tEVs from infected cells. The infection tEVs had a substantial increase in the number of peptides identified of ribosomal, histone, and tubulin proteins, among others. Gene ontology (GO) analysis showed that the proteins seen in the tEVs from infected TSCs primarily belonged to RNA-binding pathways. This result piqued our curiosity as to if Lm infection also changed the RNA loaded into the tEVs. We performed RNA sequencing to determine the host RNA profiles found in the tEVs. We found different RNA profiles in the tEVs from uninfected and Lm-infected cells. GO analysis on the mRNAs overrepresented in the infection tEVs found that they represent genes from vasculogenesis and placental development pathways. Our results in this chapter show that Lm infection can alter the production and contents of tEVs from TSCs. Chapter 4 of this dissertation aimed to determine how tEVs from Lm-infected TSCs affect immune cells. We found that macrophages treated with infection tEVs produced TNF-α, a pro-inflammatory cytokine. Surprisingly, when we subsequently infected tEV treated cells with Lm, some of the cells became more susceptible to Lm infection. Similar results were seen with treatment with macrophage EVs, where infection EVs made the macrophages susceptible to Lm infection. The work in this chapter suggests that tEVs from Lm-infected TSCs can indeed induce a pro-inflammatory response in macrophages, although this makes the cells more susceptible to infection. Overall, the work presented here explores potential mechanisms as to how the placenta communicates bacterial infections.
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