Placental inflammation by HMGB1 activation of TLR4 at the syncytium.

INTRODUCTION: Normal pregnancy is characterized by an elevated inflammatory state involving the placenta. The placental inflammation is further increased in preeclampsia, resulting in release of harmful danger signals to the maternal circulation. Activation of toll-like receptors (TLR)2 and TLR4 by endogenous danger signals plays a role in inflammatory diseases. Placental TLR2 and TLR4 expression has been reported, and high mobility group box 1 (HMGB1) is a likely endogenous activator of these receptors. We aimed to examine HMGB1 activation of TLR2 and TLR4 as mechanisms of placental inflammation in normal and preeclamptic pregnancies, by combined analysis of expression and function of the ligand HMGB1, the receptors TLR2 and TLR4, and the cytokine responder interleukin (IL)-8. METHODS: Protein expression was analyzed in placental tissue from normal and preeclamptic pregnancies, and cytokine responses to two distinct HMGB1 isoforms were examined in placental explants and trophoblasts. Inflammatory and anti-angiogenic markers were measured in maternal serum. RESULTS: We demonstrated strong co-localized expression of HMGB1, TLR4 and IL-8 in the syncytium layer of the placenta. Syncytium TLR4 expression and maternal serum levels of IL-8 were significantly increased in preeclamptic compared to normal pregnancies. Functionality was confirmed by TLR4-dependent release of IL-8 from placental explants and trophoblasts in response to the inflammatory isoform of HMGB1. DISCUSSION: This demonstrates a role for the HMGB1-TLR4 pathway at the syncytium layer and suggests involvement in placental inflammation and preeclampsia.

Functional Toll-like receptors in primary first-trimester trophoblasts.

Toll-like receptors (TLRs) are an important part of the body's danger response system and crucial for initiating inflammation in response to cellular stress, tissue damage, and infections. Proper placental development is sensitive to inflammatory activation, and a role for TLRs in trophoblast immune activation has been suggested, but no overall examination has been performed in primary trophoblasts of early pregnancy. This study aimed to broadly examine cell surface and endosomal TLR gene expression and activation in first-trimester trophoblasts. Gene expression of all ten TLRs was examined by quantitative RT-PCR (RT-qPCR) in primary first-trimester trophoblasts (n = 6) and the trophoblast cell line BeWo, and cytokine responses to TLR ligands were detected by quantitative multiplex immunoassay. Primary first-trimester trophoblasts broadly expressed all ten TLR mRNAs; TLR1, TLR2, TLR3, TLR4, and TLR6 mRNA were expressed by all primary trophoblast populations, while TLR5, TLR7, TLR8, TLR9, and TLR10 mRNA expression was more restricted. Functional response to ligand activation of cell surface TLR2/1, TLR4, and TLR5 increased IL-6 and/or IL-8 release (P < 0.01) from primary trophoblasts. For endosomal TLRs, TLR3 and TLR9 ligand exposure increased receptor-specific production of IL-8 (P < 0.01) and IFN-γ-induced protein 10 (IP-10; P < 0.001) or vascular endothelial growth factor A (VEGFA; P < 0.01). In contrast, BeWo cells expressed lower TLR mRNA levels and did not respond to TLR activation. In conclusion, primary first-trimester trophoblasts broadly express functional TLRs, with inter-individual variation, suggesting that trophoblast TLR2, TLR3, TLR4, TLR5, and TLR9 might play a role in early placental inflammation.