Pre-eclampsia is associated with increased levels of cholesterol and uric acid and an inflamed placenta expressing danger-sensing pattern recognition receptors (PRRs). Crystalline cholesterol and uric acid activate the PRR Nod-like receptor protein (NLRP)3 inflammasome to release interleukin (IL)-1β and result in vigorous inflammation. We aimed to characterize crystal-induced NLRP3 activation in placental inflammation and examine its role in pre-eclampsia. We confirmed that serum total cholesterol and uric acid were elevated in pre-eclamptic compared to healthy pregnancies and correlated positively to high sensitivity C-reactive protein (hsCRP) and the pre-eclampsia marker soluble fms-like tyrosine kinase-1 (sFlt-1). The NLRP3 inflammasome pathway components (NLRP3, caspase-1, IL-1β) and priming factors [complement component 5a (C5a) and terminal complement complex (TCC)] were co-expressed by the syncytiotrophoblast layer which covers the placental surface and interacts with maternal blood. The expression of IL-1β and TCC was increased significantly and C5a-positive regions in the syncytiotrophoblast layer appeared more frequent in pre-eclamptic compared to normal pregnancies. In-vitro activation of placental explants and trophoblasts confirmed NLRP3 inflammasome pathway functionality by complement-primed crystal-induced release of IL-1β. This study confirms crystal-induced NLRP3 inflammasome activation located at the syncytiotrophoblast layer as a mechanism of placental inflammation and suggests contribution of enhanced NLRP3 activation to the harmful placental inflammation in pre-eclampsia.

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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 antiangiogenic 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.

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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-1β 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-1β 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-1β in cultured decidual explants. Preeclampsia without FGR was associated with increased trophoblast dependent NLRP3 and IL-1β 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.

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