Classification of preeclampsia according to molecular clusters with the goal of achieving personalized prevention.

The prevention of pre-eclampsia is difficult due to the syndromic nature and multiple underlying mechanisms of this severe complication of pregnancy. The current clinical distinction between early- and late-onset disease, although clinically useful, does not reflect the true nature and complexity of the pathologic processes leading to pre-eclampsia. The current gaps in knowledge on the heterogeneous molecular pathways of this syndrome and the lack of adequate, specific diagnostic methods are major obstacles to early screening and tailored preventive strategies. The development of novel diagnostic tools for detecting the activation of the identified disease pathways would enable early, accurate screening and personalized preventive therapies. We implemented a holistic approach that includes the utilization of different proteomic profiling methods of maternal plasma samples collected from various ethnic populations and the application of systems biology analysis to plasma proteomic, maternal demographic, clinical characteristic, and placental histopathologic data. This approach enabled the identification of four molecular subclasses of pre-eclampsia in which distinct and shared disease mechanisms are activated. The current review summarizes the results and conclusions from these studies and the research and clinical implications of our findings.

Molecular subclasses of preeclampsia characterized by a longitudinal maternal proteomics study: distinct biomarkers, disease pathways and options for prevention.

OBJECTIVES: The heterogeneous nature of preeclampsia is a major obstacle to early screening and prevention, and a molecular taxonomy of disease is needed. We have previously identified four subclasses of preeclampsia based on first-trimester plasma proteomic profiles. Herein, we expanded this approach by using a more comprehensive panel of proteins profiled in longitudinal samples. METHODS: Proteomic data collected longitudinally from plasma samples of women who developed preeclampsia (n=109) and of controls (n=90) were available from our previous report on 1,125 proteins. Consensus clustering was performed to identify subgroups of patients with preeclampsia based on data from five gestational-age intervals by using select interval-specific features. Demographic, clinical, and proteomic differences among clusters were determined. Differentially abundant proteins were used to identify cluster-specific perturbed KEGG pathways. RESULTS: Four molecular clusters with different clinical phenotypes were discovered by longitudinal proteomic profiling. Cluster 1 involves metabolic and prothrombotic changes with high rates of early-onset preeclampsia and small-for-gestational-age neonates; Cluster 2 includes maternal anti-fetal rejection mechanisms and recurrent preeclampsia cases; Cluster 3 is associated with extracellular matrix regulation and comprises cases of mostly mild, late-onset preeclampsia; and Cluster 4 is characterized by angiogenic imbalance and a high prevalence of early-onset disease. CONCLUSIONS: This study is an independent validation and further refining of molecular subclasses of preeclampsia identified by a different proteomic platform and study population. The results lay the groundwork for novel diagnostic and personalized tools of prevention.

Early pathways, biomarkers, and four distinct molecular subclasses of preeclampsia: The intersection of clinical, pathological, and high-dimensional biology studies.

Preeclampsia is a syndromic disease of the mother, fetus, and placenta. The main limitation in early and accurate diagnosis of preeclampsia is rooted in the heterogeneity of this syndrome as reflected by diverse molecular pathways, symptoms, and clinical outcomes. Gaps in our knowledge preclude successful early diagnosis, personalized treatment, and prevention. The advent of "omics" technologies and systems biology approaches addresses this problem by identifying the molecular pathways associated with the underlying mechanisms and clinical phenotypes of preeclampsia. Here, we provide a brief overview on how the field has progressed, focusing on studies utilizing state-of-the-art transcriptomics and proteomics methods. Moreover, we summarize our systems biology studies involving maternal blood proteomics and placental transcriptomics, which identified early maternal and placental disease pathways and showed that their interaction influences the clinical presentation of preeclampsia. We also present an analysis of maternal blood proteomics data which revealed distinct molecular subclasses of preeclampsia and their molecular mechanisms. Maternal and placental disease pathways behind these subclasses are similar to those recently reported in studies on the placental transcriptome. These findings may promote the development of novel diagnostic tools for the distinct subtypes of preeclampsia syndrome, enabling early detection and personalized follow-up and tailored care of patients.

Placental galectins regulate innate and adaptive immune responses in pregnancy.

Introduction: Galectins are master regulators of maternal immune responses and placentation in pregnancy. Galectin-13 (gal-13) and galectin-14 (gal-14) are expressed solely by the placenta and contribute to maternal-fetal immune tolerance by inducing the apoptosis of activated T lymphocytes and the polarization of neutrophils toward an immune-regulatory phenotype.Furthermore, their decreased placental expression is associated with pregnancy complications, such as preeclampsia and miscarriage. Yet, our knowledge of the immunoregulatory role of placental galectins is incomplete. Methods: This study aimed to investigate the effects of recombinant gal-13 and gal-14 on cell viability, apoptosis, and cytokine production of peripheral blood mononuclear cells (PBMCs) and the signaling pathways involved. Results: Herein, we show that gal-13 and gal-14 bind to the surface of non-activated PBMCs (monocytes, natural killer cells, B cells, and T cells) and increase their viability while decreasing the rate of their apoptosis without promoting cell proliferation. We also demonstrate that gal-13 and gal-14 induce the production of interleukin (IL)-8, IL-10, and interferon-gamma cytokines in a concentration-dependent manner in PBMCs. The parallel activation of Erk1/2, p38, and NF-ĸB signaling evidenced by kinase phosphorylation in PBMCs suggests the involvement of these pathways in the regulation of the galectin-affected immune cell functions. Discussion: These findings provide further evidence on how placenta-specific galectins assist in the establishment and maintenance of a proper immune environment during a healthy pregnancy.

G-CSF Infusion for Stem Cell Mobilization Transiently Increases Serum Cell-Free DNA and Protease Concentrations.

G-CSF for stem cell mobilization increases circulating levels of myeloid cells at different stages of maturation. Polymorphonuclear cells (PMNs) are also mobilized in high numbers. It was previously reported that G-CSF primes PMNs toward the release of neutrophils extracellular traps (NETs). Since NETs are often involved in thrombotic events, we hypothesized that high G-CSF blood concentrations could enhance PMN priming toward NET formation in healthy hematopoietic stem cell donors, predisposing them to thrombotic events. However, we found that G-CSF does not prime PMNs toward NETs formation, but increases the serum concentration of cell-free DNA, proteases like neutrophils elastase and myeloperoxidase, and reactive oxygen species. This could possibly create an environment disposed to induce thrombotic events in the presence of additional predisposing factors.

Placental Protein 13 (Galectin-13) Polarizes Neutrophils Toward an Immune Regulatory Phenotype.

Termed as galectin-13, placental protein 13 (PP13) is exclusively expressed in the placenta of anthropoid primates. Research on PP13 in normal and pathologic pregnancies show alteration of PP13 concentrations in pregnancy affected by preeclampsia or gestational diabetes. Galectins are also described as potent immunomodulators, and PP13 regulates T cell function in the placenta. Therefore, this study aims to investigate the effects of PP13 on neutrophils; a cell type often ignored in pregnancy, but present in the uterus and placenta from the early stages of pregnancy. Since neutrophil function is dysregulated during pathologic pregnancies, a link between PP13 and neutrophil activity is possible. We determined that PP13 reduces the apoptosis rate in neutrophils. Also, PP13 increases the expression of PD-L1 and production of HGF, TNF-α, reactive oxygen species (ROS), and MMP-9 in these cells. This phenotype resembles one observed in permissive tumor neutrophils; able to sustain tissue and vessel growth, and inhibit T cell activation. At the same time, PP13 does not alter all neutrophil functions, i.e., extrusion of neutrophil extracellular traps, degranulation, phagocytosis, and ROS production following bacterial exposure. PP13 seems to play an essential role in regulating the activity of neutrophils in the placenta by polarizing them toward a placental-growth-permissive phenotype.

Skin Transplantation and Lymphoid Organ Analysis in Mice.

Skin transplantation in mice is an important procedure to evaluate immune responses generated against heterologous grafts, especially given its highly immunogenic nature. In fact, skin is one of the most challenging organs in terms of allograft retention. In this protocol, we provide a detailed procedure for skin grafting using the tail skin as donor organ that is grafted on the dorsal site of thoracic cage in a recipient mouse. We also provide protocols for the systematic analysis of lymphoid organ analysis in transplanted mice. Together these protocols may be valuable for evaluation of parameters that affect skin grafting, including genetic factors, immune cell activation as well as the analysis of compounds that may be useful in allowing graft tolerance.

Placental Galectins Are Key Players in Regulating the Maternal Adaptive Immune Response.

Galectins are potent immunomodulators that regulate maternal immune responses in pregnancy and prevent the rejection of the semi-allogeneic fetus that also occurs in miscarriages. We previously identified a gene cluster on Chromosome 19 that expresses a subfamily of galectins, including galectin-13 (Gal-13) and galectin-14 (Gal-14), which emerged in anthropoid primates. These galectins are expressed only by the placenta and induce the apoptosis of activated T lymphocytes, possibly contributing to a shifted maternal immune balance in pregnancy. The placental expression of Gal-13 and Gal-14 is decreased in preeclampsia, a life-threatening obstetrical syndrome partly attributed to maternal anti-fetal rejection. This study is aimed at revealing the effects of Gal-13 and Gal-14 on T cell functions and comparing the expression of these galectins in placentas from healthy pregnancies and miscarriages. First-trimester placentas were collected from miscarriages and elective termination of pregnancies, tissue microarrays were constructed, and then the expression of Gal-13 and Gal-14 was analyzed by immunohistochemistry and immunoscoring. Recombinant Gal-13 and Gal-14 were expressed and purified, and their effects were investigated on primary peripheral blood T cells. The binding of Gal-13 and Gal-14 to T cells and the effects of these galectins on apoptosis, activation marker (CD25, CD71, CD95, HLA-DR) expression and cytokine (IL-1ß, IL-6, IL-8, IL-10, IFNγ) production of T cells were examined by flow cytometry. Gal-13 and Gal-14 are primarily expressed by the syncytiotrophoblast at the maternal-fetal interface in the first trimester, and their placental expression is decreased in miscarriages compared to first-trimester controls. Recombinant Gal-13 and Gal-14 bind to T cells in a population- and activation-dependent manner. Gal-13 and Gal-14 induce apoptosis of Th and Tc cell populations, regardless of their activation status. Out of the investigated activation markers, Gal-14 decreases the cell surface expression of CD71, Gal-13 increases the expression of CD25, and both galectins increase the expression of CD95 on T cells. Non-activated T cells produce larger amounts of IL-8 in the presence of Gal-13 or Gal-14. In conclusion, these results show that Gal-13 and Gal-14 already provide an immunoprivileged environment at the maternal-fetal interface during early pregnancy, and their reduced expression is related to miscarriages.

Computational Methodologies for the in vitro and in situ Quantification of Neutrophil Extracellular Traps.

Neutrophil extracellular traps (NETs) are a neutrophil defensive mechanism where chromatin is expelled together with antimicrobial proteins in response to a number of stimuli. Even though beneficial in many cases, their dysfunction has been implicated in many diseases, such as rheumatoid arthritis and cancer. Accurate quantification of NETs is of utmost importance for correctly studying their role in various diseases, especially when considering them as therapeutic targets. Unfortunately, NET quantification has a number of limitations. However, recent developments in computational methodologies for quantifying NETs have vastly improved the ability to study NETs. Methods range from using ImageJ to user friendly applications and to more sophisticated machine-learning approaches. These various methods are reviewed and discussed in this review.

Prevention of pre-eclampsia after infertility treatment: Preconceptional minimalisation of risk factors.

Although an increased risk of pre-eclampsia in pregnancies conceived after infertility treatment has been reported, it remains unknown whether preconceptional minimalisation of known risk factors would help in preventing pre-eclamsia. Obesity and preconceptional blood pressure are discussed as major risks for the development of pre-eclampsia and low doses of aspirins, folic acid, statins and metformin are discussed as potential preventive treatments to decrease the risk of pre-eclampsia. In the present review we discuss whether present-day reproductive medicine could progress towards complication-free pregnancy.

Feto-Maternal Microchimerism: The Pre-eclampsia Conundrum.

Feto-maternal microchimerism (FMM) involves bidirectional cross-placental trafficking during pregnancy, leading to a micro-chimeric state that can persist for decades. In this manner a pregnant woman will harbor cells from her mother, as well as, cells from her child. Historically, eclampsia, a severe disorder of pregnancy provided the basis for FMM following the detection of trophoblast cells in the lungs of deceased women. Bi-directional cell trafficking between mother and fetus is also altered in pre-eclampsia and has been suggested to contribute to the underlying etiology. FMM has been implicated in tolerance promotion, remission of auto-inflammatory disorders during pregnancy, or the development of autoimmune conditions post-partum. The underlying mechanism whereby the host immune system is modulated is unclear but appears to involve HLA class II molecules, in that incompatibility between mother and fetus promotes remission of rheumatoid arthritis, whereas feto-maternal HLA compatibility may assist in the post-partum initiation of scleroderma. Couples having a high degree of HLA class II compatibility have an increased risk for pre-eclampsia, while the occurrence of scleroderma and rheumatoid arthritis is greater in pre-eclamptic cases than in women with normal pregnancies, suggesting a long term autoimmune predisposition. Since pregnant women with pre-eclampsia exhibit significantly lower levels of maternally-derived micro-chimerism, the question arises whether pre-eclampsia and post-partum development of autoimmune conditions occur due to the failure of the grandmothers cells to adequately regulate an inappropriate micro-chimeric constellation.

Disruption of Coronin 1 Signaling in T Cells Promotes Allograft Tolerance while Maintaining Anti-Pathogen Immunity.

The ability of the immune system to discriminate self from non-self is essential for eradicating microbial pathogens but is also responsible for allograft rejection. Whether it is possible to selectively suppress alloresponses while maintaining anti-pathogen immunity remains unknown. We found that mice deficient in coronin 1, a regulator of naive T cell homeostasis, fully retained allografts while maintaining T cell-specific responses against microbial pathogens. Mechanistically, coronin 1-deficiency increased cyclic adenosine monophosphate (cAMP) concentrations to suppress allo-specific T cell responses. Costimulation induced on microbe-infected antigen presenting cells was able to overcome cAMP-mediated immunosuppression to maintain anti-pathogen immunity. In vivo pharmacological modulation of this pathway or a prior transfer of coronin 1-deficient T cells actively suppressed allograft rejection. These results define a coronin 1-dependent regulatory axis in T cells important for allograft rejection and suggest that modulation of this pathway may be a promising approach to achieve long-term acceptance of mismatched allografts.

Controversies in Screening and Diagnostic Criteria for Gestational Diabetes in Early and Late Pregnancy.

This review serves to evaluate the screening and diagnostic strategies for gestational diabetes and overt diabetes in pregnancy. We focus on the different early screening and diagnostic approaches in first trimester including fasting plasma glucose, random plasma glucose, oral glucose tolerance test, hemoglobin A1c, risk prediction models and biomarkers. Early screening for gestational diabetes is currently not recommended since the potential benefits and harms of early detection and subsequent treatment need to be further evaluated in randomized controlled trials.

Excessive Neutrophil Activity in Gestational Diabetes Mellitus: Could It Contribute to the Development of Preeclampsia?

Gestational diabetes mellitus is a transient form of glucose intolerance occurring during pregnancy. Pregnancies affected by gestational diabetes mellitus are at risk for the development of preeclampsia, a severe life threatening condition, associated with significant feto-maternal morbidity and mortality. It is a risk factor for long-term health in women and their offspring. Pregnancy has been shown to be associated with a subliminal degree of neutrophil activation and tightly regulated generation of neutrophil extracellular traps (NETs). This response is excessive in cases with preeclampsia, leading to the presence of large numbers of NETs in affected placentae. We have recently observed that circulatory neutrophils in cases with gestational diabetes mellitus similarly exhibit an excessive pro-NETotic phenotype, and pronounced placental presence, as detected by expression of neutrophil elastase. Furthermore, exogenous neutrophil elastase liberated by degranulating neutrophils was demonstrated to alter trophoblast physiology and glucose metabolism by interfering with key signal transduction components. In this review we examine whether additional evidence exists suggesting that altered neutrophil activity in gestational diabetes mellitus may contribute to the development of preeclampsia.

Gestational Diabetes Mellitus Is Associated with Altered Neutrophil Activity.

Gestational diabetes mellitus (GDM) is a unique form of glucose intolerance, in that it is transient and solely occurs in pregnancy. Pregnancies with GDM are at high risk of developing preeclampsia (PE), a leading cause of fetal and maternal morbidity or mortality. Since PE is associated with excessive activation of circulatory neutrophils and occurrence of neutrophil extracellular traps (NETs) in affected placentae, we examined these features in cases with GDM, as this could be a feature linking the two conditions. Our data indicate that neutrophil activity is indeed altered in GDM, exhibiting pronounced activation and spontaneous generation of NETs by isolated neutrophils in in vitro culture. In this manner, GDM may similarly affect neutrophil behavior and NET formation as witnessed in other forms of diabetes, with the addition of the physiological changes mediated by pregnancy. Since circulatory TNF-α levels are elevated in cases with GDM, a feature also observed in this study, we examined whether this pro-inflammatory cytokine contributed to neutrophil activation. By using infliximab, a clinically utilized TNF-α antagonist, we observed that the pro-NETotic effect of GDM sera was significantly reduced. We also detected pronounced neutrophil infiltrates in placentae from GDM cases. The occurrence of NETs in these tissues is suggested by the extracellular co-localization of citrullinated histones and myeloperoxidase. In addition, elevated neutrophil elastase (NE) mRNA and active enzymatic protein were also detected in such placentae. This latter finding could be important in the context of previous studies in cancer or diabetes model systems, which indicated that NE liberated from infiltrating neutrophils enters surrounding cells, altering cell signaling by the degradation of IRS1. These findings could potentiate the underlying inflammatory response process in GDM and possibly open an avenue for the therapeutic interventions in gestational hyperglycemia.

Multimodal Regulation of NET Formation in Pregnancy: Progesterone Antagonizes the Pro-NETotic Effect of Estrogen and G-CSF.

Human pregnancy is associated with a mild pro-inflammatory state, characterized by circulatory neutrophil activation. In order to explore the mechanism underlying this alteration, we examined NETosis during normal gestation. Our data indicate that neutrophils exhibit a pro-NETotic state, modulated in a multimodal manner during pregnancy. In general, circulatory granulocyte colony-stimulating factor, the levels of which increase during gestation, promotes neutrophil extracellular trap (NET) formation. Early in pregnancy, NETosis is enhanced by chorionic gonadotropin, whereas toward term is stimulated by estrogen. A complex interaction between estrogen and progesterone arises, wherein progesterone restrains the NETotic process. In this state, extensive histone citrullination is evident, yet full NETosis is inhibited. This coincides with the inability of neutrophil elastase to translocate from the cytoplasm to the nucleus and is regulated by progesterone. Our findings provide new insight concerning gestational and hormone-driven pathologies, since neutrophil recruitment, activation, and NET release could be associated with excessive endothelial and placental injury.

Monoclonal regulatory T cells provide insights into T cell suppression.

Regulatory T cells (Tregs) have a crucial role in maintaining lymphocyte homeostasis. However an understanding of how Tregs function at a cellular and molecular level has not yet been fully elucidated. Here, we make use of a T cell receptor (TCR) transgenic, Rag(-/-) mouse expressing a Forkhead-Box-Protein P3 (Foxp3) transgene. This mouse provides a source of monoclonal CD4(+) Foxp3(+) T cells with a defined specificity. Here we show that monoclonal B3K506 Tregs are functional in vitro and in vivo and clearly require cognate antigen to be suppressive. We further show that the strength of Treg stimulation determines the strength of Treg mediated suppression. Finally we analysed various suppressive mechanisms used by monoclonal Tregs and found that Treg-Tconv proximity is a parameter, which correlates with enhanced suppression.

IL-7R signaling in regulatory T cells maintains peripheral and allograft tolerance in mice.

Foxp3(+)CD4(+) regulatory T cells (Treg) have a crucial role in controlling CD4(+) T-cell activation, proliferation, and effector function. However, the molecular mechanisms regulating Treg function remain poorly understood. Here we assessed the role of IL-7, a key cytokine regulating T-cell homeostasis, in suppressor capacity of Treg. Using a skin allograft model in which transplant acceptance is controlled by the number of transferred Treg, we find that Treg impair the proliferation of allogeneic CD4(+) T cells, decrease production of IFNγ by effector T cells, and prevent early and increase late IL-7 induction by lymph node stromal cells. Increased IL-7 availability enhanced Treg survival, stabilized Treg molecular signature, enhanced surface IL-2Rα expression, and improved IL-2 binding of Treg, which diminished proliferation of alloreactive CD4(+) T cells. Sequestration of IL-7 or impairment of IL-7R signaling after allograft transplantation abolished Treg-mediated tolerance by limiting their suppressive capacity. Aged Il7rα-ΔTreg mice displayed mild symptoms of autoimmunity correlating with impaired expansion of effector Treg in response to IL-2. Thus, IL-7R signaling on Treg supports the functional activity of effector Treg by increasing their IL-2 sensitivity in the lymph node during peripheral and allograft tolerance.

Isolation of murine lymph node stromal cells.

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.