Single- and double-stranded viral RNA generate distinct cytokine and antiviral responses in human fetal membranes.

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 up-regulated secretion of MIP-1α, MIP-1ß, RANTES and TNF-α, and down-regulated interleukin (IL)-2 and VEGF secretion. In contrast, viral ssRNA induced a broader panel of cytokines in the FMs by up-regulating the secretion of IL-1ß, IL-2, IL-6, G-CSF, MCP-1, MIP-1α, MIP-1ß, RANTES, TNF-α and GRO-α. Using inhibitory peptides against TLR adapter proteins, FM secretion of MIP-1ß 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-1ß, IL-2, IL-6, G-CSF, MIP-1α, RANTES and GRO-α was dependent on MyD88 and TRIF; MIP-1ß 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 an FM antiviral response by up-regulating the expression of IFNß, myxovirus-resistance A, 2',5'-oligoadenylate synthetase and apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G. These findings demonstrate that human FMs respond to two viral signatures by generating distinct inflammatory cytokine/chemokine profiles and antiviral responses through different mechanisms.

Nod1, but not the ASC inflammasome, contributes to induction of IL-1ß secretion in human trophoblasts after sensing of Chlamydia trachomatis.

Chlamydia trachomatis (Ct) is an obligate intracellular bacterial pathogen. Previously, we showed that infection of human trophoblast cells by Ct triggers the secretion of the pro-inflammatory cytokine, interleukin (IL)-1ß. The aim of this study was to understand the innate immune pathways involved in trophoblast production of IL-1ß after Ct infection. The approach we took was to inhibit the expression or function of the key Toll-like receptors (TLRs), Nod-like receptors, and inflammasome components that have been associated with chlamydia infection. In this study, we report that Ct-induced trophoblast IL-1ß secretion is associated with the transcription of IL-1ß mRNA, the translation and processing of pro-IL-1ß, and the activation of caspase-1. In addition, we demonstrate that Ct-induced IL-1ß production and secretion by the trophoblast is independent of TLR2, TLR4, MyD88, and the Nalp3/ASC inflammasome. Instead we report, for the first time, the importance of Nod1 for mediating trophoblast IL-1ß secretion in response to a Ct infection.

Syncytin 1 in the human placenta.

This study characterises HERV-W (syncytin 1) expression in normal and pathologic placenta and in BeWo cells. HERV-W mRNA levels were higher in the first trimester than at term, and similar patterns were observed with another retrovirally-derived mRNA species, ERV-3. N-glycosylated syncytin 1 precursor (73 kDa) is cleaved to surface-associated (SU) and transmembrane (TM) subunits. Both were evident in villous trophoblast, where perinuclear and punctate cytoplasmic deposits were observed, and linear TM subunit immunoreactivity was seen at the syncytial microvillous membrane. Punctate immunoreactivity was seen in BeWo cells with antibodies to SU and TM, and the two were co-localised. SU immunoreactivity was observed in association with fetal endothelium, and this effect was increased in tissue from pre-eclamptic placentas, which also showed a higher level of total SU protein. Absence of the TM subunit from endothelium suggests it is not a biosynthetic source. We suggest that SU is released from trophoblast into fetal circulation where it may bind vascular endothelium.

The isolation and characterization of a novel telomerase immortalized first trimester trophoblast cell line, Swan 71.

Studies using first trimester trophoblast cells may be limited by the inability to obtain patient samples and/or adequate cell numbers. First trimester trophoblast cell lines have been generated by SV40 transformation or similar methods, however, this approach is known to induce phenotypic and karyotypic abnormalities. The introduction of telomerase has been proposed to be a viable alternative for the immortalization of primary human cells. To investigate whether telomerase-induced immortalization might be a more feasible approach for the generation of first trimester trophoblast cell lines, we isolated primary trophoblast cells from a 7-week normal placenta and infected the cells with human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase. Although this hTERT-infected first trimester trophoblast cell line, which we have named Swan 71, has been propagated for more than 100 passages, it still has attributes that are characteristic of primary first trimester trophoblast cells. The Swan 71 cells are positive for the expression of cytokeratin 7, vimentin and HLA-G, but do not express CD45, CD68 or the Fibroblast Specific Antigen (FSA), CD90/Thy-1. In addition, we also demonstrated that the Swan 71 cells secrete fetal fibronectin (FFN) as well as low levels of human Chorionic Gonadotrophin (hCG). Moreover, the Swan 71 cells exhibit a cytokine and growth factor profile that is similar to primary trophoblast cells and are resistant to Fas, but not TNF-alpha-induced apoptosis. This suggests that the Swan 71 cells may represent a valuable model for future in vitro trophoblast studies.

Utero-placental haemodynamics in the pathogenesis of pre-eclampsia.

Pre-eclampsia is associated with insufficient adaptations of spiral arteries which theoretically alter haemodynamics within the intervillous space. Such changes could damage the syncytiotrophoblast and release factors which instigate maternal endothelial dysfunction. We tested this hypothesis using an in vitro dual perfusion model of the human placenta, representing putative changes in flow arising from these spiral artery maladaptations. Whilst fetal-side flow rates remained constant (6 ml/min) perfusion rates on the maternal side were increased from 14 ml/min to 45 ml/min. As well as increasing placental derived intervillous hydrostatic pressures, and changes in flow dynamics observed by colour Doppler, these elevated flow rates resulted in morphologic damage, vacuolation and shedding of the syncytiotrophoblast, focal features previously defined in pre-eclampsia. The collected maternal perfusates recovered under high flow conditions also contained significantly elevated levels of biochemical markers of syncytial damage, including lactate dehydrogenase, alkaline phosphatase and human chorionic gonadotrophin. There were also significant elevations in chemokines GROalpha and RANTES, compared with the low flow perfusions. The soluble components of the maternal high flow rate perfusions decreased the number and proliferation of HUVECs after 24h exposure. These results could not be attributed to GROalpha or RANTES alone or in combination. This study provides evidence that alterations in intervillous flow have the potential to influence both the integrity of the syncytiotrophoblast and the liberation of potentially pathogenic soluble factors. This therefore offers a putative link between utero-placental maladaptations in pregnancy and the vascular endothelial complications of pre-eclampsia.

Pre-eclampsia is associated with dendritic cell recruitment into the uterine decidua.

Pre-eclampsia is a leading cause of fetal and maternal morbidity and mortality that preferentially affects primiparous patients. It is associated with systemic inflammation and impaired trophoblast invasion of the decidua. Decidual cells are the major cell type of the pregnant endometrium. Macrophages and dendritic cells are major specialized antigen-presenting cells that promote both innate immunity and immune tolerance. Macrophage infiltration is implicated in impaired trophoblast invasion that leads to pre-eclampsia. By contrast, the potential modulating role of decidual dendritic cells in the genesis of pre-eclampsia has not been investigated. Interleukin-1beta (IL-1beta), a pro-inflammatory cytokine, has been implicated in the genesis of pre-eclampsia. Thus, we postulate that pre-eclampsia would be associated with enhanced decidual dendritic cells infiltration and that IL-1beta would enhance the production of relevant dendritic cell-recruiting chemokines. We used immunohistochemistry to demonstrate a marked infiltrate of immature and mature dendritic cells in pre-eclamptic decidua. Further, immunohistochemistry and immunoassays of placental bed biopsies revealed that pre-eclamptic decidua displays elevated levels of several monocyte- and dendritic cell-recruiting chemokines. Leukocyte-free first-trimester decidual cells were then treated with IL-1beta, which enhanced the mRNA and protein expression of these chemokines. The current study also confirmed previous reports that macrophages directly impaired trophoblast invasion and that this inhibitory effect is augmented by the conditioned medium of IL-1beta-treated first-trimester decidual cells. However, unlike macrophages, dendritic cells did not directly impede trophoblast invasion. This study demonstrates that the inflammatory milieu of pre-eclampsia induces decidual cells to promote dendritic cell infiltration. Given their unusual versatility in mediating both immunity and tolerance, these novel findings suggest that dendritic cells may play a critical role either in the pathogenesis of pre-eclampsia or its prevention in subsequent pregnancies.

Cell type-specific expression and function of toll-like receptors 2 and 4 in human placenta: implications in fetal infection.

Placental infection is associated with adverse fetal outcomes. Toll-like receptors (TLRs) are critical regulators of the innate immune response based on their ability to recognize and respond to pathogen-associated molecular patterns expressed by microbes. To date, cell-type specific expression and regulation of TLR function in human term placenta remains largely unelucidated. The goal of the current study was to examine the in vivo and in vitro patterns of TLR expression and function in major cell types of term placenta. Immunohistochemical analysis of terminal and stem villi localized TLR-2, which recognizes peptidoglycan (PG) from Gram-positive bacteria, to endothelial cells and macrophages, and to a lesser extent to syncytiotrophoblast (SCTs) and fibroblasts (FIBs). Staining for TLR-4, the receptor for Gram-negative bacterial lipopolysaccharide (LPS), was most prominent in SCTs and endothelial cells. Results from Western blotting, conventional, and quantitative PCR (qRTPCR) analyses using protein and mRNA isolated from cultures of SCTs and myofibroblasts (mFIBs) revealed that SCTs expressed TLR-2 and TLR-4, whereas mFIBs expressed only TLR-4. In addition, qRTPCR showed that LPS treatment increased TLR-2 expression in SCTs, indicating that infection with Gram-negative bacteria may enhance innate immune responses in placenta toward a broad range of microorganisms. In addition, treatment with LPS increased IL-8 levels in both SCTs and mFIBs, whereas PG treatment only stimulated IL-8 levels in SCTs. Our results indicate that there exist cell type-specific patterns of TLR function in placenta which likely regulate innate immune response at the maternal-fetal interface.

Expression of Toll-like receptors in the human decidua.

BACKGROUND: Successful trophoblast invasion and transformation of the maternal spiral arteries requires that the pregnant endometrium (i.e., decidua) act in an immunologically paradoxical fashion, accepting the semi-allogenic placenta, while maintaining host defenses against an array of microbial pathogens. In contrast to the growing evidence that the immune surveillance molecules known as Toll-like receptors (TLRs) are expressed by trophoblasts and fetal membranes, to date, no studies have been conducted on the decidua. METHODS: Decidual tissues and cells were obtained from women undergoing first trimester elective terminations or repeat Cesarean sections and analyzed at both the protein and mRNA level. RESULTS: We now demonstrate for the first time that human decidua differentially express TLRs and their downstream signaling molecules as well as TLR stimulated induction of cytokine production in the first and third trimester of pregnancy. CONCLUSIONS: These findings suggest that the decidua is a critical component of the innate immune response in pregnancy. Moreover, the results have implications for the success or failure of compromised pregnancies in early or late gestation.

Trophoblast-macrophage interactions: a regulatory network for the protection of pregnancy.

PROBLEM: Macrophages are one of the first immune cells observed at the implantation site. Their presence has been explained as the result of an immune response toward paternal antigens. The mechanisms regulating monocyte migration and differentiation at the implantation site are largely unknown. In the present study, we demonstrate that trophoblast cells regulate monocyte migration and differentiation. We propose that trophoblast cells 'educate' monocytes/macrophages to create an adequate environment that promote trophoblast survival. METHOD OF STUDY: CD14(+) monocytes were isolated from peripheral blood using magnetic beads. Co-culture experiments were conducted using a two-chamber system. Monocytes were stimulated with lipopolysaccharide (LPS) and cytokine levels were determined using multiplex cytokine detecting assay. RESULTS: Trophoblast cells increase monocyte migration and induce a significant increase in the secretion and production of the pro-inflammatory cytokines [interleukin-6 (IL-6), IL-8, tumor necrosis factor-alpha] and chemokines (growth-related oncogen-alpha, monocyte chemoattractant protein-1, macrophage inflammatory protein-1beta, RANTES). Furthermore, the response of monocytes to LPS was different in monocytes pre-exposed to trophoblast cells. CONCLUSION: The results of this study suggest that trophoblast cells are able to recruit and successfully educate monocytes to produce and secrete a pro-inflammatory cytokine and chemokine profile supporting its growth and survival. Furthermore we demonstrate that trophoblast cells can modulate monocytes response to bacterial stimuli.

Regulation of chemokine production in response to pro-inflammatory cytokines in first trimester decidual cells.

OBJECTIVE: Chemokines initiate the immune response by controlling leukocyte migration and lymphocyte development. Macrophage infiltration of the decidua has been implicated in the genesis of recurrent miscarriage and preeclampsia. Therefore, we determined whether cultured human decidual cells produce monocyte/macrophage-recruiting chemokines in response to a potent pro-inflammatory cytokine, interleukin-1beta (IL-1beta), and whether decidual cell-conditioned medium contains monocyte- and macrophage-chemoattractant activity. METHODS: Leukocyte-free first trimester decidual cells were treated for 6h with estradiol (E(2)) and medroxyprogesterone acetate (MPA) to mimic the steroidal milieu of pregnancy, or E(2) and MPA and IL-1beta (1 ng/ml) to mimic inflamed decidua. Total RNA was used for cDNA synthesis. Biotinylated cRNAs were generated and chemically fragmented for hybridization on Affymetrix HG_U133 Plus 2.0 chips followed by fluorescence labeling and optical scanning. Raw data generated from Affymetrix GCOS 1.2 (GeneChip Operating Software) were analyzed by GeneSpring 7.2 software. Subsequently microarray results were validated by real time RT-PCR and Western blotting. A functional study of monocyte migration was carried out also using conditioned media from culture. RESULTS: Five chemokines responsible for monocyte/macrophage chemoattraction and activation, including C-C motif ligand 2 (CCL2), CCL5, C-X-C motif ligand 2 (CXCL2), CXCL3 and CXCL8, were markedly elevated from 29- to 975-fold after exposure to IL-1beta in cultured first trimester decidual cells. The results of real-time RT-PCR (up-regulation from 43- to 3069-fold) and Western blotting (up-regulation from 15- to 300-fold) confirmed the microarray findings. Monocyte migration was significantly induced by the conditioned medium from IL-1beta-treated decidual cells. CONCLUSIONS: Treatment of first trimester decidual cells with IL-1beta induces secretion of monocyte/macrophage recruiting-chemokines and promotes monocyte migration. Extrapolation of these in vitro results to the milieu of implantation site suggests a mechanism whereby IL-1beta could mediate excessive macrophage infiltration of the decidua.

Toll-like receptors and their role in the trophoblast.

The aim of this review is to discuss the role of Toll-like receptors at the maternal-fetal interface and the capacity of trophoblast cells to initiate innate immune responses towards infection. The maternal-fetal interface represents an immunologically unique site that must promote tolerance to the allogenic fetus, whilst maintaining host defense against a diverse array of possible pathogens. Clinical studies have shown a strong association between certain complications of pregnancy and intrauterine infections. Therefore, innate immune responses against microorganisms at the maternal-fetal interface may have a significant impact on the success of a pregnancy. There is growing evidence that trophoblast cells are able to recognize and respond to pathogens through the expression of Toll-like receptors, an important part of innate immunity. This review will discuss the role of Toll-like receptors at the maternal-fetal interface, the potential for trophoblast cells to function as components of the innate immune system and the impact TLR-mediated trophoblast responses may have on pregnancy outcome.

Induction of tumor necrosis factor alpha production by adhered human monocytes: a key role for Fcgamma receptor type IIIa in rheumatoid arthritis.

OBJECTIVE: Small IgG rheumatoid factor immune complexes may provide the trigger for macrophage-derived tumor necrosis factor alpha (TNFalpha) production in rheumatoid arthritis. Immune complexes may bind to any of 3 IgG Fc receptors (FcgammaR). Therefore, the ability of monocyte-derived macrophages to produce TNFalpha was examined following ligation of each of the 3 human FcgammaR, using murine monoclonal antibodies (mAb) to each receptor as a model for small immune complexes. METHODS: Adhered human monocytes expressing all 3 FcgammaR were incubated with murine anti-FcgammaR mAb directed against FcgammaRI, FcgammaRII, or FcgammaRIII. Supernatants were collected at various time points and tested for the presence of TNFalpha and interleukin-1alpha (IL-1alpha) by enzyme-linked immunosorbent assay. RESULTS: The anti-FcgammaRIII mAb induced adhered human monocytes to release TNFalpha. However, F(ab)2 and Fab fragments of the anti-FcgammaRIII mAb failed to induce TNFalpha production. TNFalpha was undetectable following incubation with the anti-FcgammaRI or anti-FcgammaRII mAb. Furthermore, blocking FcgammaRI or FcgammaRII had no effect on the levels of TNFalpha released in response to the anti-FcgammaRIII mAb. Of the 3 anti-FcgammaR mAb, only anti-FcgammaRIII induced IL-1alpha production from adhered human monocytes, and this was inhibited by the presence of a neutralizing anti-TNFalpha mAb. CONCLUSION: This study suggests a dominant role for FcgammaRIIIA in the induction of both TNFalpha and IL-1alpha production by human macrophages in rheumatoid arthritis following receptor ligation by small immune complexes. The signaling of TNFalpha production may require the ligation of either 3 FcgammaRIIIA receptors or only 2 FcgammaRIIIA receptors, where one interaction must involve binding via an Fc domain. In addition, IL-1alpha production following FcgammaRIIIA ligation appears to be dependent on the presence of TNFalpha.

Do self-perpetuating B lymphocytes drive human autoimmune disease?

Normal immunological memory is thought to be underpinned by T lymphocytes. However, in rheumatoid arthritis there are indications that T-lymphocyte control has been subverted by self-perpetuating B lymphocytes. Potential mechanisms in other autoimmune states are less clear, but a number of observations suggest that misappropriation of immunological memory by B lymphocytes may be a common feature of human autoantibody-associated disease. Put simply, autoantibodies drive their own production. If so, the availability of safe B-lymphocyte-depleting agents provides a potential means for reversal of autoimmunity.