Jennifer H. Southcombe

The immunomodulatory role of syncytiotrophoblast microvesicles.

Immune adaptation is a critical component of successful pregnancy. Of primary importance is the modification of cytokine production upon immune activation. With the discovery that normal pregnancy itself is a pro-inflammatory state, it was recognised that the classical Th1/Th2 cytokine paradigm, with a shift towards ‘type 2’ cytokine production (important for antibody production), and away from ‘type 1’ immunity (associated with cell mediated immunity and graft rejection), is too simplistic. It is now generally agreed that both arms of cytokine immunity are activated, but with a bias towards ‘type 2’ immunity. Many factors are released from the placenta that can influence the maternal cytokine balance. Here we focus on syncytiotrophoblast microvesicles (STBM) which are shed from the placenta into the maternal circulation. We show that STBM can bind to monocytes and B cells and induce cytokine release (TNFα, MIP-1α, IL-1α, IL-1β, IL-6, IL-8). Other cytokines are down-modulated, such as IP-10 which is associated with ‘type 1’ immunity. Therefore STBM may aid the ‘type 2’ skewed nature of normal pregnancy. We also observed that PBMC from third trimester normal pregnant women produce more TNFα and IL-6 in response to STBM than PBMC from non-pregnant women, confirming that maternal immune cells are primed by pregnancy, possibly through their interaction with STBM.

Extracellular vesicles and reproduction–promotion of successful pregnancy

Extracellular vesicles (EVs) are membrane-bound complexes secreted from cells under both physiological and pathological conditions. They contain proteins, nucleic acids and lipids and act as messengers for cell–cell communication and signalling, particularly between immune cells. EV research is a rapidly evolving and expanding field, and it appears that all biological fluids contain very large numbers of EVs; they are produced from all cells that have been studied to date, and are known to have roles in several reproductive processes. This review analyses the evidence for the role of EVs throughout human reproduction, starting with the paternal and maternal gametes, followed by the establishment and continuation of successful pregnancies, with specific focus, where possible, on the interaction of EVs with the maternal immune system. Importantly, variations within the EV populations are identified in various reproductive disorders, such as pre-term labour and pre-eclampsia.

Multicolor Flow Cytometry and Nanoparticle Tracking Analysis of Extracellular Vesicles in the Plasma of Normal Pregnant and Pre-eclamptic Women

ABSTRACT Excessive release of syncytiotrophoblast extracellular vesicles (STBMs) from the placenta into the maternal circulation may contribute to the systemic inflammation that is characteristic of pre-eclampsia (PE). Other intravascular cells types (platelets, leukocytes, red blood cells [RBCs], and endothelium) may also be activated and release extracellular vesicles (EVs). We developed a multicolor flow cytometry antibody panel to enumerate and phenotype STBMs in relation to other EVs in plasma from nonpregnant (NonP) and normal pregnant (NormP) women, and women with late-onset PE. Nanoparticle tracking analysis (NTA) was used to determine EV size and concentration. In vitro-derived STBMs and EVs from platelets, leukocytes, RBCs, and endothelial cells were examined to select suitable antibodies to analyze the corresponding plasma EVs. Flow cytometry analysis of plasma from NonP, NormP, and PE showed that STBMs comprised the smallest group of circulating EVs, whereas most were derived from platelets. The next most abundant group comprised unidentified orphan EVs (which did not label with any of the antibodies in the panel), followed by EVs from RBCs and leukocytes. NTA showed that the total number of EVs in plasma was significantly elevated in NormP and late-onset PE women compared to NonP controls, and that EVs were smaller in size. In general, EVs were elevated in pregnancy plasma apart from platelet EVs, which were reduced. These studies did not show any differences in EVs between NormP and PE, probably because late-onset PE was studied.

ST2 and IL-33 in Pregnancy and Pre-Eclampsia

Normal pregnancy is associated with a mild systemic inflammatory response and an immune bias towards type 2 cytokine production, whereas pre-eclampsia is characterized by a more intense inflammatory response, associated with endothelial dysfunction and a type 1 cytokine dominance. Interleukin (IL)-33 is a newly described member of the IL-1 family, which binds its receptor ST2L to induce type 2 cytokines. A soluble variant of ST2 (sST2) acts as a decoy receptor to regulate the activity of IL-33. In this study circulating IL-33 and sST2 were measured in each trimester of normal pregnancy and in women with pre-eclampsia. While IL-33 did not change throughout normal pregnancy, or between non-pregnant, normal pregnant or pre-eclamptic women, sST2 was significantly altered. sST2 was increased in the third trimester of normal pregnancy (p<0.001) and was further increased in pre-eclampsia (p<0.001). This increase was seen prior to the onset of disease (p<0.01). Pre-eclampsia is a disease caused by placental derived factors, and we show that IL-33 and ST2 can be detected in lysates from both normal and pre-eclampsia placentas. ST2, but not IL-33, was identified on the syncytiotrophoblast layer, whereas IL-33 was expressed on perivascular tissue. In an in vitro placental perfusion model, sST2 was secreted by the placenta into the ‘maternal’ eluate, and placental explants treated with pro-inflammatory cytokines or subjected to hypoxia/reperfusion injury release more sST2, suggesting the origin of at least some of the increased amounts of circulating sST2 in pre-eclamptic women is the placenta. These results suggest that sST2 may play a significant role in pregnancies complicated by pre-eclampsia and increased sST2 could contribute to the type 1 bias seen in this disorder.

A unique secreted adenovirus E3 protein binds to the leukocyte common antigen CD45 and modulates leukocyte functions

Significance Human adenoviruses encode Early region 3 (E3) proteins that manipulate the host immune response to establish an infection or to persist longer. To date, only a few E3 functions from a single adenovirus species (C) have been characterized, all of which act directly on infected cells. Here we describe a secreted E3 protein that is uniquely expressed by species D adenoviruses. This protein targets noninfected leukocytes using a cell surface phosphatase as a receptor. We provide evidence that this interaction suppresses leukocyte activation and effector functions, implying that species D adenoviruses can affect the host distant from the site of infection. The E3 transcription unit of human adenoviruses (Ads) encodes immunomodulatory proteins. Interestingly, the size and composition of the E3 region differs considerably among Ad species, suggesting that distinct sets of immunomodulatory E3 proteins may influence their interaction with the human host and the disease pattern. However, to date, only common immune evasion functions of species C E3 proteins have been described. Here we report on the immunomodulatory activity of a species D-specific E3 protein, E3/49K. Unlike all other E3 proteins that act on infected cells, E3/49K seems to target uninfected cells. Initially synthesized as an 80- to 100-kDa type I transmembrane protein, E3/49K is subsequently cleaved, with the large ectodomain (sec49K) secreted. We found that purified sec49K exhibits specific binding to lymphoid cell lines and all primary leukocytes, but not to fibroblasts or epithelial cells. Consistent with this binding profile and the molecular mass, the sec49K receptor was identified as the cell surface protein tyrosine phosphatase CD45. Antibody-blocking studies suggested that sec49K binds to the membrane proximal domains present in all CD45 isoforms. Functional studies showed that sec49K can suppress the activation and cytotoxicity of natural killer cells as well as the activation, signaling, and cytokine production of T cells. Thus, we have discovered an adenovirus protein that is actively secreted and describe immunomodulatory activities of an E3 protein uniquely expressed by a single Ad species.

Interleukin-1 family cytokines and their regulatory proteins in normal pregnancy and pre-eclampsia

Maternal systemic inflammation is a feature of pre‐eclampsia, a condition in pregnancy characterized by hypertension and proteinuria. Pre‐eclampsia is caused by the placenta; many placental factors contribute to the syndromes progression, and proinflammatory cytokines have been identified previously as one such mediator. The interleukin (IL)‐1 family of cytokines are key regulators of the inflammatory network, and two naturally occurring regulatory molecules for IL‐1 family cytokines, IL‐1RA and sST2, have been found previously to be elevated in maternal blood from women with pre‐eclampsia. Here we investigate more recently identified IL‐1 family cytokines and regulatory molecules, IL‐1RAcP, IL‐37, IL‐18BP, IL‐36α/β/γ/Ra and IL‐38 in pre‐eclampsia. Pregnant women have more circulating IL‐18BP and IL‐36Ra than non‐pregnant women, and sIL‐1RAcP is elevated from women with pre‐eclampsia compared to normal pregnancies. The placenta expresses all the molecules, and IL‐37 and IL‐18BP are up‐regulated significantly in pre‐eclampsia placentas compared to those from normal pregnancies. Together, these changes contribute to the required inhibition of maternal systemic cytotoxic immunity in normal pregnancy; however, in pre‐eclampsia the same profile is not seen. Interestingly, the increased circulating levels of sIL‐1RAcP and increased placental IL‐18BP and IL‐37, the latter of which we show to be induced by hypoxic damage to the placenta, are all factors which are anti‐inflammatory. While the placenta is often held responsible for the damage and clinical symptoms of pre‐eclampsia by the research community, here we show that the pre‐eclampsia placenta is also trying to prevent inflammatory damage to the mother.

Peripheral blood invariant natural killer T cells throughout pregnancy and in preeclamptic women.

Invariant natural killer T (iNKT) cells are implicated in the pathogenesis of several diseases. They influence both innate and adaptive immune responses through their capacity to rapidly produce large quantities of cytokines upon activation. During pregnancy maternal immunity is biased towards type 2 cytokine production to regulate type 1 cytokines that could be harmful for the developing fetus. This shift to type 2 cytokines does not occur in preeclamptic women and there is an exaggerated maternal inflammatory response which is dangerous for both mother and baby. We have therefore investigated the numbers, phenotype and functional activity of iNKT cells throughout pregnancy and in women diagnosed with preeclampsia. We demonstrate that the numbers of iNKT cells in the peripheral blood do not change between the first, second and third trimesters of pregnancy, but the cells become activated and less able to produce the type 1 cytokine IFNγ. However, iNKT cells are unchanged in preeclamptic women, when compared to normal pregnancy, suggesting that these cells are not primary players in the pathogenesis of the disease.

IFPA Meeting 2011 workshop report III: Placental immunology; epigenetic and microRNA-dependent gene regulation; comparative placentation; trophoblast differentiation; stem cells

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2011 there were twelve themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology: 1) immunology; 2) epigenetics; 3) comparative placentation; 4) trophoblast differentiation; 5) stem cells.

Detection of Soluble ST2 in Human Follicular Fluid and Luteinized Granulosa Cells

Follicular fluid (FF) contains various cytokines that are involved with folliculogenesis, some of which have been shown to be associated with oocyte quality and the implantation potential of a resulting embryo. Several IL-1 family members have previously been identified in FF. This study investigates a newly identified member of the family, IL-33, and its receptor ST2, comparing values to those of FF Granulocyte-Colony Stimulating Factor (G-CSF) – a known predictor of Assisted Reproductive Technology (ART) success. FF was collected from patients undergoing in vitro fertilisation/intra-cytoplasmic sperm injection (IVF/ICSI) at oocyte retrieval to analyse IL-33 and sST2 expression in human follicles. sST2, but not IL-33, is highly increased in the FF compared to plasma levels (up to 7.9-fold), with higher levels in larger follicles (p<0.05). Furthermore, we identify that human luteinised granulosa cells are one possible source of the FF sST2, as these cells express and secrete sST2 when cultured ex vivo. FF associated with oocytes which when fertilised develop into good quality embryos have higher sST2 levels than those which are graded average (p<0.01). These embryos were transferred to the patient and levels of FF sST2 compared between successful and unsuccessful ICSI cycles. However unlike G-CSF, sST2 levels cannot be used to predict cycle outcome.

Measurement of sST2 is comparable to PlGF in the diagnosis of early-onset pre-eclampsia.

A diagnostic test to confirm pre-eclampsia would be beneficial for the clinical management of the syndrome. The Triage PlGF test is able to confirm pre-eclampsia with high accuracy, with the greatest efficacy at <35weeks gestation. We recently found that the anti-inflammatory protein sST2 is elevated in the plasma of pre-eclamptic women compared to normal controls. Here sST2 and PlGF are compared in early-onset and late-onset pre-eclamptic women. sST2 was found to be an equally good diagnostic tool for early-onset (sST2 AUC 0.944 versus PlGF AUC 0.995; not significant) but not late-onset disease.