[Show abstract][Hide abstract] ABSTRACT: Objective: Toll-like receptors (TLRs) recognize microbial ligands and host products released during tissue damage, the so-called "danger signals." This study was conducted to determine whether changes in TLR-4 and TLR-2 expression can be detected in the trophoblasts at the placental bed of women with and without preeclampsia. Study design: Placental bed biopsies were obtained from women with: (1) normal term pregnancies with and without labor (each n = 20); (2) preeclampsia who delivered preterm (n = 15); and (3) preterm labor and intact membranes (PTL) with and without chorioamnionitis (each n = 15). The expression pattern of TLR-4 and TLR-2 in the trophoblasts was analyzed by double immunohistochemistry. Results: (1) The median percentage of TLR-4 positive interstitial trophoblasts was significantly higher in preeclamptic patients than in patients with PTL without or with histologic chorioamnionitis (P =.0002 and P =.02, respectively). (2) The median percentage of TLR-2 positive interstitial trophoblasts was not different among study groups (P >.05). (3) TLR-4 positive trophoblasts were also frequently immunoreactive to activated nuclear factor-kappa B, tumor. necrosis factor-alpha, and M30 (a specific apoptosis antigen for trophoblast). (4) Lipopolysaccharide treatment inhibited migration of trophoblast cell lines in vitro. Conclusion: TLR-4 protein expression is increased in interstitial trophoblasts of patients with preeclampsia. We propose that "danger signals" at the feto-maternal interface, recognized by trophoblasts through TLR-4, may play a key role in creating a local abnormal cytokine milieu. This suggests a novel mechanism that links activation of the innate immune system through TLR-4 and preeclampsia.
[Show abstract][Hide abstract] ABSTRACT: ProblemThrombophilia is associated with pregnancy complications. Treatment with low molecular weight heparin (LMWH) improves pregnancy outcome, but the underlying mechanisms are not clear.Methods of studyWe analyzed Treg frequency in blood from thrombophilic pregnancies treated with LMWH (n = 32) or untreated (n = 33) and from healthy pregnancies (n = 39) at all trimesters. Additionally, we treated pregnant wild-type, heterozygous and homozygous factor-V-Leiden (FVL) mice with LMWH or PBS and determined Treg frequency, pro-/anti-inflammatory cytokine levels and Caspase-3-activity in placenta and decidua.ResultsTreg frequencies were increased in second and third trimester in LMWH-treated thrombophilic pregnancies compared to controls. Treg levels were comparable to those of normal pregnancies. Homozygous FVL mice had decreased decidual Tregs compared to wild-type mice. LMWH treatment normalized Tregs and was associated with increased decidual IL-10 mRNA. LMWH diminished Caspase-3-activity in mice of all genotypes.Conclusion
We demonstrated anti-apoptotic and anti-inflammatory effects of LMWH in pregnant FVL mice. LMWH increased Treg levels in mice and humans, which suggests benefits of LMWH treatment for thrombophilic women during pregnancy.
American Journal Of Reproductive Immunology 12/2014; 73(5). DOI:10.1111/aji.12348 · 3.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is growing interest in the role of viral infections and their association with adverse pregnancy outcomes. While the trophoblast is permissive to viruses, little is still known about their impact on the placenta. We previously established that viral single stranded RNA (ssRNA), a Toll-like receptor 8 (TLR8) agonist, induces a restricted pro-inflammatory cytokine/chemokine response by upregulating the secretion of IL-6 and IL-8. In parallel the type I interferon, IFNbeta, is produced and acts back on the cell in an autocrine/paracrine manner to trigger caspase-3-dependent apoptosis. In this current study, we sought to extend these findings by determining the mechanisms involved, whether viral ssRNA could induce a trophoblast antiviral response, and to evaluate the influence of viral ssRNA on pregnancy outcome using a mouse model. Herein we report that viral ssRNA induced human first trimester trophoblast inflammation, type I IFN production, an antiviral response and apoptosis in both a TLR8/MyD88-dependent and -independent manner. Furthermore, administration of viral ssRNA to pregnant mice induced placental caspase-3 activation, a pro-inflammatory cytokine/chemokine, type I IFN, and antiviral response, as well as immune cell infiltration. Thus, ssRNA viral infections may compromise pregnancy by altering placental trophoblast survival and function through both TLR8 and non-TLR8 signaling pathways, leading to immune changes at the maternal-fetal interface.
Copyright 2014 by The Society for the Study of Reproduction.
Biology of Reproduction 11/2014; 92(1). DOI:10.1095/biolreprod.114.124032 · 3.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ProblemDiabetes confers an increased risk of preeclampsia, but its pathogenic role in preeclampsia is poorly understood. The objective of this study was to elucidate the effects of excess glucose on trophoblast function and whether any changes could be reversed by metformin.Method of studyThe human first trimester trophoblast cell line (Sw.71) was treated with glucose at 5, 10, 25, and 50 mm, in the presence and absence of metformin. Trophoblast migration was quantified and supernatant cytokine, chemokine, and angiogenic factors measured.ResultsIncreasing concentrations of glucose significantly increased trophoblast secretion of the inflammatory cytokines/chemokines: IL-1β, IL-6, IL-8, GRO-, RANTES, and G-CSF; significantly increased trophoblast secretion of the anti-angiogenic factors sFlt-1 and sEndoglin; and significantly decreased trophoblast migration. Excess glucose-induced trophoblast IL-1β production was inhibited by disabling the Nalp3/ASC inflammasome. Metformin partially reduced the glucose-induced inflammatory response, but had no effect on the anti-angiogenic or antimigratory response.Conclusion
Excess glucose induced a pro-inflammatory, anti-angiogenic, and antimigratory state in first trimester trophoblast cells. Glucose-induced trophoblast IL-1β secretion was mediated by the inflammasome. Glucose-induced inflammation was partially reversed by metformin. These findings demonstrate the pleiotropic effects of hyperglycaemia on the trophoblast, providing potential explanations for the strong link between diabetes and preeclampsia.
American Journal Of Reproductive Immunology 11/2014; 73(4). DOI:10.1111/aji.12339 · 3.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective
Antiphospholipid antibodies (aPL) interfere with several physiologic functions of human trophoblasts, including reducing their ability to migrate, decreasing their production of angiogenic factors, and inducing an inflammatory response. This may provide the underlying mechanism by which aPL responses lead to recurrent pregnancy loss or preeclampsia in women with obstetric antiphospholipid syndrome (APS). Although treatment with heparin may reduce the rate of recurrent pregnancy loss, the risk of preeclampsia remains high. Therefore, alternative treatments are needed for the management of pregnant patients with APS. Since aspirin-triggered lipoxins (ATLs) have immune and angiogenic modulatory properties, the objective of this study was to determine the effects of the ATL 15-epi-lipoxin A4 on the function of aPL-altered human trophoblasts in the first trimester of pregnancy.MethodsA first-trimester human trophoblast cell line (HTR8) was treated with mouse anti-human β2-glycoprotein I monoclonal antibodies (aPL) in the presence or absence of the ATL 15-epi-lipoxin A4. Trophoblast migration and interactions with endometrial endothelial cells were measured using Transwell and coculture assays. Trophoblast secretion of cytokines and angiogenic factors was measured by enzyme-linked immunosorbent assay.ResultsTreatment of HTR8 cells with ATL reversed the aPL-induced decrease in trophoblast migration, an effect that appeared to be regulated through restoration of interleukin-6 production. Using a model of spiral artery transformation, aPL and sera from APS patients with pregnancy morbidity disrupted trophoblast-endothelial cell interactions, and treatment with ATL restored the stability of the cocultures. In contrast, ATL treatment did not resolve the proinflammatory and antiangiogenic responses of trophoblasts induced by aPL.Conclusion
These findings indicate that ATLs may have some benefits in terms of preventing the effects of aPL on trophoblast function, which raises the possibility of the use of ATLs as an adjuvant therapy in women with aPL.
Arthritis and Rheumatology 10/2014; 67(2). DOI:10.1002/art.38934
[Show abstract][Hide abstract] ABSTRACT: ProblemMicrobial-driven responses in placenta are linked with adverse pregnancy outcomes. The role of Toll-like receptor (TLR) function in Hofbauer cells (HBCs) and fetal macrophages of the placental villous core remains understudied.Method of StudyFlow cytometry and immunohistochemistry (IHC) were used to establish the phenotype of HBCs. Regulation of cytokine secretion in response to treatment with TLR agonists and expression levels of TLRs and co-activators were compared in HBCs, placental fibroblasts (FIBs), and human umbilical vein endothelial cells (HUVECs) using ELISA and qPCR.ResultsAlthough flow cytometry and IHC revealed HBCs to be M2 (anti-inflammatory) macrophages, LPS and polyinosinic: polycytidylic acid [poly (I:C)] treatments markedly enhanced IL-6 secretion by HBCs, and expression of TLR-2, TLR-3, TLR-4, CD14, and MD-2 was the highest in HBCs.Conclusion
These results indicate that although HBCs are M2 macrophages, inflammatory responses are induced through TLR-3 and TLR-4 in this cell type, suggesting a role in microbial-driven placental/fetal inflammation.
American Journal Of Reproductive Immunology 10/2014; 73(1). DOI:10.1111/aji.12336 · 3.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ProblemWomen with antiphospholipid syndrome (APS) are at increased risk of recurrent pregnancy loss (RPL) and preeclampsia. Antiphospholipid antibodies (aPL) directly alter trophoblast function. Treatment with low molecular weight heparin (LMWH) reduces the risk of RPL but not preeclampsia. Moreover, LMWH stimulates trophoblast sFlt-1 release, an anti-angiogenic factor associated with preeclampsia. Since vitamin D deficiency is associated with APS and preeclampsia, this study sought to determine the effect of vitamin D on trophoblast function in the setting of aPL and LMWH.Method of studyA human first trimester trophoblast cell line (HTR8) and primary trophoblast cultures were treated with or without aPL in the presence and absence of vitamin D, LMWH or both. Trophoblast secretion of inflammatory cytokines and angiogenic factors were measured by ELISA.ResultsVitamin D alone or in combination with LMWH attenuated the aPL-induced trophoblast inflammatory response in the HTR8 cells and primary cultures. While vitamin D did not have any impact on aPL-mediated modulation of angiogenic factors in the primary trophoblast, it significantly inhibited LMWH-induced sFlt-1 release.ConclusionLMWH in combination with vitamin D may be more beneficial than single-agent therapy by preventing aPL-induced trophoblast inflammation and reversing LMWH-induced sFlt-1 secretion.
American Journal Of Reproductive Immunology 07/2014; 73(3). DOI:10.1111/aji.12301 · 3.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Problem Inflammation during pregnancy has devastating consequences for the placenta and fetus. These events are incompletely understood, thereby hampering screening and treatment. Method of studyThe inflammatory profile of villous tissue was studied in pregnancies at high-risk of placental dysfunction and compared to uncomplicated pregnancies. The systemic inflammatory profile was assessed in matched maternal serum samples in cases of reduced fetal movements (RFM). ResultsPlacentas from RFM pregnancies had a unique inflammatory profile characterized by increased interleukin (IL)-1 receptor antagonist and decreased IL-10 expression, concomitant with increased numbers of placental macrophages. This aberrant cytokine profile was evident in maternal serum in RFM, as were increased levels of alarmins (uric acid, HMGB1, cell-free fetal DNA). Conclusion
This distinct inflammatory profile at the maternal-fetal interface, mirrored in maternal serum, could represent biomarkers of placental inflammation and could offer novel therapeutic options to protect the placenta and fetus from an adverse maternal environment.
American journal of reproductive immunology (New York, N.Y.: 1989) 05/2014; 72(4). DOI:10.1111/aji.12274 · 2.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There has been growing interest in the role of viral infections and their association with adverse pregnancy outcomes. However, little is known about the impact viral infections have on the fetal membranes (FM). Toll-like receptors (TLR) are thought to play a role in infection-associated inflammation at the maternal-fetal interface. Therefore, the objective of this study was to characterize the cytokine profile and antiviral response in human FMs exposed to viral dsRNA, which activates TLR3, and viral ssRNA, which activates TLR8; and to determine the mechanisms involved. The viral dsRNA analog, Poly(I:C), induced upregulated secretion of MIP-1α, MIP-1β, RANTES and TNF-α, and downregulated IL-2 and VEGF secretion. In contrast, viral ssRNA induced a broader panel of cytokines in the FMs by upregulating the secretion of IL-1β, IL-2, IL-6, G-CSF, MCP-1, MIP-1α, MIP-1β, RANTES, TNF-α and GRO-α. Using inhibitory peptides against TLR adapter proteins, FM secretion of MIP-1β and RANTES in response to Poly(I:C) was MyD88-dependent; MIP-1α secretion was dependent on MyD88 and TRIF; and TNF-α production was independent of MyD88 and TRIF. Viral ssRNA-induced FM secretion of IL-1β, IL-2, IL-6, G-CSF, MIP-1α, RANTES and GRO-α was dependent on MyD88 and TRIF; MIP-1β was dependent upon TRIF, but not MyD88; and TNF-α and MCP-1 secretion was dependent on neither. Poly(I:C), but not ssRNA, induced a FM antiviral response by upregulating the expression of inflammasome components, IFNβ, MxA, OAS and APOBEC3G. These findings demonstrate that human FMs respond to two viral signatures by generating distinct inflammatory cytokine/chemokine profiles and antiviral responses through different mechanisms.
Molecular Human Reproduction 04/2014; 20(7). DOI:10.1093/molehr/gau028 · 3.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bacterial infection-associated inflammation is thought to be a major cause of preterm premature rupture of membranes (PPROM). Pro-inflammatory cytokines, such as IL1B, can weaken the fetal membranes (FM) by upregulating matrix metalloproteinases and inducing apoptosis. The mechanism by which infection leads to inflammation at the maternal-fetal interface and subsequent preterm birth is thought to involve innate immune pattern recognition receptors (PRR), such as the Toll-like receptors (TLR) and Nod-like receptors (NLR) which recognize pathogen-associated molecular patterns (PAMPs). The objective of this study was to determine the cytokine profile generated by FMs in response to the following bacterial TLR and NLR agonists: peptidoglycan (PDG; TLR2); lipopolysaccharide (LPS; TLR4); flagellin (TLR5); CpG ODN (TLR9); iE-DAP (Nod1) and MDP (Nod2). PDG, LPS, Flagellin, iE-DAP and MDP triggered FMs to generate an inflammatory response, however, the cytokine profiles were distinct for each TLR and NLR agonist; and only IL1B and RANTES were commonly upregulated in response to all five PAMPs. CpG ODN, in contrast, had a mild stimulatory effect only on MCP-1, and primarily downregulated basal FM cytokine production. IL1B secretion induced by PDG, LPS, flagellin, iE-DAP and MDP was associated with its processing. Furthermore, FM IL1B secretion in response to TLR2, TLR4 and TLR5 activation was caspase-1 dependent, while Nod1 and Nod2 induced IL1B secretion independent of caspase-1. These findings demonstrate that FMs respond to different bacterial TLR and NLR PAMPs by generating distinct inflammatory cytokine profiles through distinct mechanisms that are specific to the innate immune PRR activated.
Biology of Reproduction 01/2014; 90(2). DOI:10.1095/biolreprod.113.115428 · 3.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Women with antiphospholipid syndrome (APS) are at risk for pregnancy complications. Antiphospholipid antibodies (aPL) alter trophoblast function by triggering an inflammatory cytokine response; modulating angiogenic factor secretion; and inhibiting migration. While patients with APS are often treated with hydroxychloroquine (HCQ), its effect on trophoblast function is poorly understood.
A human first trimester trophoblast cell line was treated with or without antihuman β2 GPI mAbs in the presence or absence of HCQ. Supernatants were analyzed by ELISA. Cell migration was measured using a colormetric assay.
Antiphospholipid antibodies-induced trophoblast IL-8, IL-1 β, PlGF, and sEndoglin secretion were not altered by HCQ. aPL-induced inhibition of trophoblast migration was partially reversed by HCQ, even though HCQ significantly increased secretion of pro-migratory IL-6 to greater than baseline. aPL-induced upregulation of TIMP2 appears to inhibit trophoblast migration; the inability of HCQ to prevent aPL-induced TIMP2 may explain why migration was only partially restored.
Hydroxychloroquine reversed the aPL-inhibition of trophoblast IL-6 secretion and partially limited aPL-inhibition of cell migration. Thus, some form of combination therapy that includes HCQ may be beneficial to pregnant APS patients.
American Journal Of Reproductive Immunology 12/2013; 71(2). DOI:10.1111/aji.12184 · 3.32 Impact Factor