O77. Functional screening of toll-like receptors in seven trophoblast cell lines

Abstract

Introduction Pregnancy is considered a natural inflammatory condition, but pregnancy complications such as preeclampsia and miscarriage are linked to excessive inflammation in the placenta. Toll-like receptors (TLRs) serve as sensors for danger signals and are crucial for initiating inflammation. A successful pregnancy depends on proper function of fetal trophoblasts, the main cell type in the placenta. We have shown broad functional TLR expression in primary first trimester trophoblasts (Tangeras et al., J. Reprod. Immunol., 2014), and trophoblast TLR activation may contribute substantially to placental inflammation. Trophoblast cell lines are commonly used as surrogates for primary trophoblasts for in vitro research. With respect to TLR mediated inflammation, the translatability of trophoblast cell lines warrants examination. Objectives This study aimed to assess TLR1-10 gene expression and activation in seven trophoblast cell lines of different origins and compare to primary first trimester trophoblasts. Methods The choriocarcinoma trophoblast cell lines BeWo, JAR, JEG-3, AC1M-32 and ACH-3P and the SV40 transformed extravillous trophoblast cell lines HTR-8/SVneo and SGHPL-5 were included and compared to primary first trimester trophoblasts ( n =6).Gene expression of TLR1-10 was analyzed using RT-qPCR. Following specific TLR ligand activation for 24 hours, trophoblast release of interleukin (IL)-1 β , IL-6, IL-8, IL-9, IL-10, IL-12 (p70), interferon (IFN)- γ -inducible protein (IP)-10, tumor necrosis factor (TNF)- α , IFN- γ , and vascular endothelial growth factor (VEGF)-A was measured by multiplex immunoassay. Results All choriocarcinoma cell lines demonstrated broad TLR gene expression, but lacked functional cytokine response to TLR ligand activation. On the contrary, the SV40 transformed cell lines showed restricted TLR gene expression, and responded to activation of TLR2, TLR3 and/or TLR4 by significantly upregulated production of inflammatory cytokines such as IL-6, IL-8 and/or IFN- γ . The primary first trimester trophoblasts demonstrated both a broad TLR gene expression profile and prominent cytokine response to TLR ligand activation. Only SGHPL-5 showed a TLR activated cytokine response comparable to primary first trimester trophoblasts. Conclusion Most of the trophoblast cell lines tested showed markedly lower inflammatory TLR properties compared to primary first trimester trophoblasts, and SGHPL-5 and HTR8/SVneo were most TLR responsive. This warrants caution when translating trophoblast immune function from cell line studies.