Pattern recognition receptor mediated inflammation in placental trophoblasts

Studie av mekanismer for betennelse i morkaken Betennelse (inflammasjon) er en immunrespons som kroppen setter i gang for å beskytte seg mot infeksjoner og vevsskade. Alle mennesker er født med "mønster-gjenkjennende" pattern recognition reseptorer (PRRer) som en del av immunforsvaret. Disse reseptorene er avgjørende for igangsettelse av betennelsesreaksjoner, blant annet gjennom produksjon av signalmolekyler kalt cytokiner. En betennelsesreaksjon er som regel både nødvendig og gunstig for kroppen, men hvis responsen blir for kraftig eller langvarig kan den føre til stor skade og sykdom. Et normalt svangerskap er assosiert med en mild betennelsestilstand, mens hos kvinner som utvikler svangerskapsforgiftning (preeklampsi) vil nivået av betennelsesmarkører, som cytokiner og C-reaktivt protein (CRP), være forhøyet. Man tror svangerskapsforgiftning begynner tidlig i svangerskapet med en ufullstendig utvikling av morkaken. En mangelfull utvikling av morkaken vil gi redusert blodtilførsel fra mor til morkake, og kan resultere i «stressede» celler og betennelse i morkaken. En slik stresset morkake produserer faresignaler som kan aktivere PRRer på f.eks trofoblaster - fosterceller som utgjør den største delen av morkaken. Aktivering av PRRer på trofoblaster kan igangsette overdreven immunrespons i morkaken, som igjen vil kunne bidra til betennelsen man observerer ved svangerskapsforgiftning, men kunnskapen om de underliggende sykdomsmekanismene er mangelfull. Formålet med denne avhandlingen var å bidra til økt kunnskap om betennelse igangsatt av PRRer i morkaken ved friske svangerskap og svangerskapsforgiftning. Vi studerte PRR-aktivering av trofoblaster isolert fra morkaker tidlig i svangerskapet, og sammenliknet resultatene med syv trofoblast-cellelinjer (modifiserte celler med forlenget levetid). De isolerte trofoblastene uttrykte et bredt repertoar av fungerende PRRer, men kun én av syv cellelinjer viste seg å ha et liknende uttrykk av funksjonelle PRRer. Det brede uttrykket av PRRer vi påviste i de isolerte trofoblastene tyder på at disse cellene spiller en viktig rolle ved betennelse i morkaken. Videre viser våre resultater at det er viktig å velge riktig trofoblast-cellelinje når man skal studere disse betennelsesreaksjonene og tilgangen til isolerte trofoblaster er begrenset. For å avklare om kjente PRR-aktiverende faresignaler som kolesterol, urinsyre og HMGB1 spiller en rolle ved utvikling av svangerskapsforgiftning, ble morkakevev og blodprøver fra kvinner med og uten svangerskapsforgiftning undersøkt. Vi avdekket høyere nivåer av kolesterol og urinsyre, men ikke HMGB1, i blodprøver fra kvinner med svangerskapsforgiftning sammenlignet med friske gravide kvinner. I morkaken fant vi at HMGB1 og PRRen som gjenkjenner HMGB1, TLR4, var høyest uttrykt i morkaker fra kvinner med svangerskapsforgiftning. HMGB1 aktivering av TLR4 økte produksjonen av cytokinet interleukin (IL)-8, og vi målte forhøyet IL-8 nivå både i morkakevev og blodprøver fra kvinner med svangerskapsforgiftning. PRRen NLRP3, som aktiveres av kolesterol og urinsyre, var spesielt høyt uttrykt av trofoblaster i morkaken, og vi målte et høyere uttrykk av det NLRP3 responsive cytokinet IL-1 i trofoblaster ved svangerskapsforgiftning. Videre viste vi at de aktuelle PRR mekanismene lar seg aktivere av kolesterol og HMGB1 i morkakevev og en trofoblast-cellelinje. Studiene av PRR mekanismene TLR4 og NLRP3 påviste forhøyede nivåer av kolesterol, urinsyre og/eller HMGB1 som mulige underliggende årsaker til betennelse i morkaken og utvikling av svangerskapsforgiftning. Resultatene i denne avhandlingen indikerer at trofoblaster i morkaken og bestemte PRR mekanismer er involvert i betennelsen som observeres i både normale svangerskap og ved svangerskapsforgiftning. Det langsiktige målet med arbeidet er å kunne identifisere kvinner med økt risiko for svangerskapsforgiftning, samt kartlegge nye muligheter for behandling av sykdommen

Toll-like receptor profiling of seven trophoblast cell lines warrants caution for translation to primary trophoblasts

Introduction Excessive placental inflammation is associated with pregnancy complications. Toll-like receptors (TLRs) are sensors for danger signals from infections and damaged tissue and initiate inflammation. Trophoblasts in the placenta broadly express TLRs. Trophoblast cell lines are used as surrogates for primary trophoblasts for in vitro studies, but the inflammatory translatability of trophoblast cell lines warrants examination. We aimed to assess TLR1-10 gene expression and activation in seven trophoblast cell lines and compare this to primary trophoblasts. Methods The five choriocarcinoma trophoblast cell lines BeWo, JAR, JEG-3, AC1M-32 and ACH-3P, and the two SV40 transfected trophoblast cell lines HTR-8/SVneo and SGHPL-5 were included and compared to primary first trimester trophoblasts (n = 6). TLR1-10 gene expression was analyzed by RT-qPCR. Cells were stimulated by specific TLR1-9 ligands for 24 h and cytokine release was measured by a 10-plex immunoassay. Results All choriocarcinoma cell lines demonstrated broad TLR gene expression, but lacked functional cytokine response to TLR ligand activation. In contrast, SV40 transfected cell lines showed restricted TLR gene expression, but SGHPL-5 cells displayed significantly increased levels of interleukin (IL)-6, IL-8, IL-12 and vascular endothelial growth factor A after TLR3 and/or TLR4 activation (P < 0.01), while TLR2 activation increased IL-6 and IL-8 levels (P < 0.05). HTR8/SVneo cells responded to TLR3 activation by increased IL-6 and interferon (IFN)-γ (P < 0.05). The SGHPL-5 TLR profile most closely resembled primary trophoblast. Discussion The characterized trophoblast cell line TLR profiles serve as a reference and warrant caution when selecting trophoblast cell lines as in vitro models for immune responses in primary trophoblasts

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

Placental inflammation in pre-eclampsia by Nod-like receptor protein (NLRP)3 inflammasome activation in trophoblasts

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

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 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

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 3 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 tophoblasts 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

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

Placental inflammation by HMGB1 activation of TLR4 at the syncytium

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

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-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