(204) Pretreatment of trophoblast stem cells and syncytiotrophoblast cells with syncytin proteins increases immune stimulated genes in immune stimulated cells

Victoria Caravaggio¹, Cristine R. Camp¹, Rachel C. West¹

¹Department of Anatomy, Physiology, and Pharmacology, Auburn University, Auburn, United States

Abstract Text:

The placenta plays a critical role in pregnancy protecting the growing fetus from maternal inflammation or infection. Within the placenta, the syncytiotrophoblast layer (STB) provides the first line of defense against infection, as it comes in direct contact with the maternal blood supply. The placenta is able to mount a robust type-1 interferon response, by upregulating the expression of interferon-induced transmembrane proteins (IFITMs), and interferon stimulated genes (ISGs) to protect against viral infection. These genes are known to be highly protective, producing responses that work to prevent viral entry into the host cell, specifically working against the entry of enveloped viruses, or viral replication. They also can have embryotoxic effects, depending on the duration and severity of the infection and subsequent immune response. IFITM proteins inhibit the cell fusion necessary to form the STB, leading to placental dysfunction and fetal demise. Two of the most important genes related to STB cell fusion are the endogenous retroviral envelope proteins Syncytin-1 and Syncytin-2. These proteins have also been implicated in modulation of the innate immune response. Based on the relative abundance and immune modulatory activity of these proteins in the placenta, we hypothesized that Syncytin-1 and -2 influence the levels of IFITMs and ISGs in immune stimulated trophoblast cells. To test this hypothesis, trophoblast stem cells (TSCs) were grown in either stem or STB differentiation culture conditions for 24 or 72 hours, respectively. TSCs and STBs were pretreated with 7.4 µg/mL of recombinant syncytin-1 or syncytin-2 for 2 hours, then treated with 250 U/mL of Interferon-beta (IFNB) for 6 hours before RNA was collected. RT-qPCR was done to quantify relative levels of interferon stimulated genes, and a one-way ANOVA followed by a Tukey’s multiple comparison test was used to determine statistical significance (p < 0.05). We evaluated the relative expression of IFITM1, IFITM2, IFITM3, ISG15 and ISG20. In the TSCs pretreated with Syncytin-1 or -2, IFITM1 (p < 0.01) and IFITM2 (p < 0.01) were both significantly higher in pre-treated cells compared to IFNB treatment alone, whereas IFITM3 was only significantly higher in TSCs pretreated with syncytin-2. In the STB cells, only pre-treatment with syncytin-2 resulted in higher levels of IFITM2 (p < 0.05) and IFITM3 (p < 0.0001) compared to IFNB treated cells. ISG20 levels were higher in both syncytin pretreated TSCs (p < 0.01) and STBs (p < 0.05) compared to IFNB only cells; whereas there were no significant differences in ISG15 levels between pretreated groups and IFN only TSCs or STBs. These data suggest that syncytin proteins can create a more robust interferon stimulated gene response. This is an interesting phenomenon as upregulation of IFITM genes and proteins are directly linked to poor cell fusion and differentiation towards the syncytiotrophoblast layer of the placenta. It is well understood that interferons have cytotoxic effects on the embryo during pregnancy and that IFITM proteins contribute to this toxicity. In this study, we demonstrate that the immune modulatory effects of syncytin proteins make trophoblast cells further susceptible to IFN-related cytotoxicity.