Cholesterol Crystals and NLRP3 Mediated Inflammation in the Uterine Wall Decidua in Normal and Preeclamptic Pregnancies.

Preeclampsia is a hypertensive and inflammatory pregnancy disorder associated with cholesterol accumulation and inflammation at the maternal-fetal interface. Preeclampsia can be complicated with fetal growth restriction (FGR) and shares risk factors and pathophysiological mechanisms with cardiovascular disease. Cholesterol crystal mediated NLRP3 inflammasome activation is central to cardiovascular disease and the pathway has been implicated in placental inflammation in preeclampsia. Direct maternal-fetal interaction occurs both in the uterine wall decidua and at the placental surface and these aligned sites constitute the maternal-fetal interface. This study aimed to investigate cholesterol crystal accumulation and NLRP3 inflammasome expression by maternal and fetal cells in the uterine wall decidua of normal and preeclamptic pregnancies. Pregnant women with normal (n = 43) and preeclamptic pregnancies with (n = 28) and without (n = 19) FGR were included at delivery. Cholesterol crystals were imaged in decidual tissue by both second harmonic generation microscopy and polarization filter reflected light microscopy. Quantitative expression analysis of NLRP3, IL-1ß and cell markers was performed by immunohistochemistry and automated image processing. Functional NLRP3 activation was assessed in cultured decidual explants. Cholesterol crystals were identified in decidual tissue, both in the tissue stroma and near uterine vessels. The cholesterol crystals in decidua varied between pregnancies in distribution and cluster size. Decidual expression of the inflammasome components NLRP3 and IL-1ß was located to fetal trophoblasts and maternal leukocytes and was strongest in areas of proximity between these cell types. Pathway functionality was confirmed by cholesterol crystal activation of IL-1ß in cultured decidual explants. Preeclampsia without FGR was associated with increased trophoblast dependent NLRP3 and IL-1ß expression, particularly in the decidual areas of trophoblast and leukocyte proximity. Our findings suggest that decidual accumulation of cholesterol crystals may activate the NLRP3 inflammasome and contribute to decidual inflammation and that this pathway is strengthened in areas with close maternal-fetal interaction in preeclampsia without FGR.

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.

PP040. Activation of endosomal toll-like receptors in first trimester trophoblasts.

INTRODUCTION: A mild systemic inflammation may be beneficial to normal pregnancy, however exaggerated inflammation may contribute to pregnancy complications. Infections and cell stress or damage may evoke placental inflammation by activation of Toll-like receptors (TLRs). TLR3, TLR7, TLR8 and TLR9 are located intracellulary on endosomes and are activated by nucleic acids from microbes and damaged cells. Trophoblasts play a crucial role during placentation, and the role of TLRs in first trimester trophoblasts needs to be determined. OBJECTIVES: To characterize endosomal TLR gene expression and activation in first trimester trophoblasts, to extend knowledge of endosomal TLR involvement in placental inflammation. METHODS: Primary trophoblasts were isolated from six first trimester placentas by enzyme degradation and gradient centrifugation. Gene expression of TLR3, TLR7, TLR8 and TLR9 in primary first trimester trophoblasts and the trophoblast cell line BeWo was quantified by RT-qPCR. The trophoblasts were stimulated with ligands for endosomal TLRs, and release of pro-inflammatory cytokines was analyzed by multiplex. RESULTS: Primary first trimester trophoblasts showed gene expression of all endosomal TLRs, and endosomal TLR activation gave increased production of the pro-inflammatory cytokines IL-6, IL-8, and IP-10. CONCLUSION: Primary first trimester trophoblasts express functional endosomal TLRs, indicating TLR-mediated trophoblast involvement in early placental inflammation.

PP042. Cell surface toll-like receptors in primary first trimester trophoblasts.

INTRODUCTION: The first trimester of pregnancy is characterised by a mild pro-inflammatory environment, however excessive inflammation threatens placental development and function. Toll-like receptors (TLRs) are crucial in initiating inflammation. TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10 are expressed on the cell surface, and respond to microbial infection and cell damage and stress signals. Recent findings of TLRs in trophoblasts indicate a role in inflammation during pregnancy, but further studies are warranted. OBJECTIVES: To investigate gene expression and function of cell surface TLRs in first trimester trophoblasts, to extend knowledge on the role of trophoblast TLRs during placental development. METHODS: Primary trophoblasts were isolated from first trimester placentas (n=6) by enzyme degradation and density gradient centrifugation. Gene expression of TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10 was quantified by RT-qPCR in primary first trimester trophoblasts and the trophoblast cell line BeWo. Trophoblasts were stimulated with cell surface TLR ligands and pro-inflammatory cytokine release was analysed by multiplex immunoassay. RESULTS: Primary first trimester trophoblasts expressed all cell surface TLR mRNAs, and activation of TLR2/1, TLR4 and TLR5 induced IL-6 and/or IL-8. CONCLUSION: The broad expression of functional cell surface TLRs in primary first trimester trophoblasts suggests a central role for trophoblasts in placental inflammation and immune activation.