Placental Related Disorders of Pregnancy 2.0.

Following our first Special Issue, we are pleased to present this Special Issue in the International Journal of Molecular Sciences entitled 'Placental Related Disorders of Pregnancy 2 [...].

The differential placental expression of ERp44 and pre-eclampsia; association with placental zinc, the ERAP1 and the renin-angiotensin-system.

INTRODUCTION: Endoplasmic reticulum resident protein 44 (ERp44) is a zinc-metalloprotein, regulating Endoplasmic reticulum aminopeptidase 1 (ERAP1) and Angiotensin II (Ang II). We explored placental ERp44 expression and components of the renin-angiotensin-system (RAS) in pre-eclampsia (PE), correlating these to ERAP1 expression and placental zinc concentrations. METHODS: Placental tissue, taken at time of delivery in normotensive women or women with PE (n = 12/group), were analysed for ERp44, AT1R, AT2R and AT4R by qPCR. Protein ERp44 expression was measured by immunohistochemistry and compared to previously measured ERAP1 expression. Placental zinc was measured by inductively-coupled-mass-spectrometry. RESULTS: ERp44 gene/protein expression were increased in PE (P < 0.05). AT1R expression was increased (P = 0.02) but AT4R decreased (P = 0.01) in PE, compared to normotensive controls. A positive association between ERp44 and AT2R expression was observed in all groups. ERp44 was negatively correlated with ERAP1 protein expression in all samples. Placental zinc concentrations were lower in women with PE (P = 0.001) and negatively associated with ERp44 gene expression. DISCUSSION: Increased placental ERp44 could further reduce ERAP1 release in PE, potentially preventing release of Ang IV and thus lowering levels of Ang IV which consequently diminishes the possibility of counterbalancing the activity of vasoconstrictive, Ang II. The lower placental zinc may contribute to dysfunction of the ERp44/ERAP1 complex, exacerbating the hypertension in PE.

A Comparative Review of Pregnancy and Cancer and Their Association with Endoplasmic Reticulum Aminopeptidase 1 and 2.

The fundamental basis of pregnancy and cancer is to determine the fate of the survival or the death of humanity. However, the development of fetuses and tumors share many similarities and differences, making them two sides of the same coin. This review presents an overview of the similarities and differences between pregnancy and cancer. In addition, we will also discuss the critical roles that Endoplasmic Reticulum Aminopeptidase (ERAP) 1 and 2 may play in the immune system, cell migration, and angiogenesis, all of which are essential for fetal and tumor development. Even though the comprehensive understanding of ERAP2 lags that of ERAP1 due to the lack of an animal model, recent studies have shown that both enzymes are associated with an increased risk of several diseases, including pregnancy disorder pre-eclampsia (PE), recurrent miscarriages, and cancer. The exact mechanisms in both pregnancy and cancer need to be elucidated. Therefore, a deeper understanding of ERAP's role in diseases can make it a potential therapeutic target for pregnancy complications and cancer and offer greater insight into its impact on the immune system.

Macrophages and Wnts in Tissue Injury and Repair.

Macrophages are important players in the immune system that sense various tissue challenges and trigger inflammation. Tissue injuries are followed by inflammation, which is tightly coordinated with tissue repair processes. Dysregulation of these processes leads to chronic inflammation or tissue fibrosis. Wnt ligands are present both in homeostatic and pathological conditions. However, their roles and mechanisms regulating inflammation and tissue repair are being investigated. Here we aim to provide an overview of overarching themes regarding Wnt and macrophages by reviewing the previous literature. We aim to gain future insights into how tissue inflammation, repair, regeneration, and fibrosis events are regulated by macrophages.

Placental Related Disorders of Pregnancy.

We are pleased to present this Special Issue of International Journal of Molecular Sciences, entitled 'Placental Related Disorders of Pregnancy' [...].

Cellular immune responses in the pathophysiology of preeclampsia.

Preeclampsia, defined as new-onset hypertension accompanied by proteinuria occurring at 20 weeks of gestation or later, is a leading cause of perinatal morbidity and mortality worldwide. The pathophysiology of this major multi-systemic syndrome includes defective deep placentation, oxidative stress, endothelial dysfunction, the presence of an anti-angiogenic state, and intravascular inflammation, among others. In this review, we provide a comprehensive overview of the cellular immune responses involved in the pathogenesis of preeclampsia. Specifically, we summarize the role of innate and adaptive immune cells in the maternal circulation, reproductive tissues, and at the maternal-fetal interface of women affected by this pregnancy complication. The major cellular subsets involved in the pathogenesis of preeclampsia are regulatory T cells, effector T cells, NK cells, monocytes, macrophages, and neutrophils. We also summarize the literature on those immune cells that have been less characterized in this clinical condition, such as γδ T cells, invariant natural killer T cells, dendritic cells, mast cells, and B cells. Moreover, we discuss in vivo studies utilizing a variety of animal models of preeclampsia to further support the role of immune cells in this disease. Finally, we highlight the existing gaps in knowledge of the immunobiology of preeclampsia that require further investigation. The goal of this review is to promote translational research leading to clinically relevant strategies that can improve adverse perinatal outcomes resulting from the obstetrical syndrome of preeclampsia.

Overexpression of ERAP2N in Human Trophoblast Cells Promotes Cell Death.

The genes involved in implantation and placentation are tightly regulated to ensure a healthy pregnancy. The endoplasmic reticulum aminopeptidase 2 (ERAP2) gene is associated with preeclampsia (PE). Our studies have determined that an isoform of ERAP2-arginine (N), expressed in trophoblast cells (TC), significantly activates immune cells, and ERAP2N-expressing TCs are preferentially killed by both cytotoxic T lymphocytes (CTLs) and Natural Killer cells (NKCs). To understand the cause of this phenomenon, we surveyed differentially expressed genes (DEGs) between ERAP2N expressing and non-expressing TCs. Our RNAseq data revealed 581 total DEGs between the two groups. 289 genes were up-regulated, and 292 genes were down-regulated. Interestingly, most of the down-regulated genes of significance were pro-survival genes that play a crucial role in cell survival (LDHA, EGLN1, HLA-C, ITGB5, WNT7A, FN1). However, the down-regulation of these genes in ERAP2N-expressing TCs translates into a propensity for cell death. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that 64 DEGs were significantly enriched in nine pathways, including "Protein processing in endoplasmic reticulum" and "Antigen processing and presentation", suggesting that the genes may be associated with peptide processes involved in immune recognition during the reproductive cycle.

The Differential Expression of ERAP1/ERAP2 and Immune Cell Activation in Pre-eclampsia.

Pre-eclampsia (PE) is a disorder of pregnancy, often leading to serious and fatal complications. Endoplasmic reticulum aminopeptidase 1 and 2 (ERAP1/ERAP2) are present in the placenta. They are involved in processes regulating blood pressure, angiogenesis, cytokine receptor shedding, and immune recognition. Previous studies have associated both ERAP1/ERAP2 genetic variants with PE, although the underlying mechanisms remain unknown. Less is known about the roles for these enzymes in early placentation, which could be a contributory factor to PE. To ascertain whether ERAP1/ERAP2 change in PE and whether such a change is present before PE is clinically diagnosed, we analyzed mRNA and ERAP1/2 protein expression in the placenta in the early first trimester (8-14 weeks) and at delivery in normotensive or PE women (n = 12/group). Gene expression was analyzed using qPCR, and protein expression and localization were assessed by immunohistochemistry. Additionally, we profiled peripheral immune cells from normotensive and PE (n = 5/group) women for activation and expression of cytotoxic markers using flow cytometry to investigate a possible correlation with placental expression of ERAP1/2. Finally, we characterized the cytokines released from immune cells isolated from normotensive women and those with PE, stimulated ex vivo by JEG-3 trophoblast cells. The ERAP1 protein was significantly upregulated in first trimester placentae compared to placentae at delivery from both normotensive and PE women (p < 0.05): expression of placental ERAP1 protein was also relatively higher in normotensive than PE women. Although the protein expression of both ERAP1/ERAP2 was significantly lower in women with PE compared to normotensive controls (p < 0.05), ERAP2 protein expression remained unchanged in normotensive women at delivery compared to expression in the first trimester. Flow cytometry analysis revealed an increase in activation and cytotoxic natural killer (NK) cells in peripheral blood of PE compared to normotensive women. Intriguingly, there was a notable difference in cytokine release from the activated immune cells when further stimulated by trophoblast cells. The immune cells from PE released elevated expressions of interleukin (IL)-2, IL-4, and most notably, pro-inflammatory IL-13 and IL-17α, inflammatory cytokines tumor necrosis factor (TNF)-α and interferon (IFN)-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF) compared to normal peripheral blood mononuclear cells (PBMCs). Taken together, these findings suggest that differential lymphocyte activation could be associated with altered ERAP1/ERAP2 expression.

Transient Receptor Potential Melastatin 4 Induces Astrocyte Swelling But Not Death after Diffuse Traumatic Brain Injury.

Traumatic brain injury (TBI) is a prevalent disease with significant costs. Although progress has been made in understanding the complex pathobiology of focal lesions associated with TBI, questions remain regarding the diffuse responses to injury. Expression of the transient receptor potential melastatin 4 (Trpm4) channel is linked to cytotoxic edema during hemorrhagic contusion expansion. However, little is known about Trpm4 following diffuse TBI. To explore Trpm4 expression in diffuse TBI, rats were subjected to a diffuse central fluid percussion injury (CFPI) and survived for 1.5 h to 8 weeks. The total number of Trpm4+ cells, as well as individual cellular intensity/expression of Trpm4, were assessed. Hemotoxylin and eosin (H&E) labeling was performed to evaluate cell damage/death potentially associated with Trpm4 expression following diffuse TBI. Finally, ultrastructural assessments were performed to evaluate the integrity of Trpm4+ cells and the potential for swelling associated with Trpm4 expression. Trpm4 was primarily restricted to astrocytes within the hippocampus and peaked at 6 h post-injury. While the number of Trpm4+ astrocytes returned to sham levels by 8 weeks post-CFPI, cellular intensity occurred in region-specific waves following injury. Correlative H&E assessments demonstrated little evidence of hippocampal damage, suggesting that Trpm4 expression by astrocytes does not precipitate cell death following diffuse TBI. Additionally, ultrastructural assessments showed Trpm4+ astrocytes exhibited twice the soma size compared with Trpm4- astrocytes, indicating that astrocyte swelling is associated with Trpm4 expression. This study provides a foundation for future investigations into the role of Trpm4 in astrocyte swelling and edema following diffuse TBI.

The role of endoplasmic reticulum aminopeptidase 2 in modulating immune detection of choriocarcinoma.

Gestational choriocarcinomas are derived from placental trophoblast cells, with HLA-C being the only class I polymorphic molecule expressed. However, choriocarcinomas have not been profiled for endoplasmic reticulum aminopeptidase 2 (ERAP2) expression. ERAP2 trims peptides presented by human leukocyte antigens (HLA) that have shown to modulate immune response. Over 50% of choriocarcinomas we screened lack ERAP2 expression, which suggests that the absence of ERAP2 expression allows immune evasion of choriocarcinoma cells. We demonstrate that the ability of choriocarcinoma cells to activate lymphocytes was lowest with cells lacking ERAP2 (JEG-3) or HLA-C (JAr). This observation suggests that activation is dependent on expression of both ERAP2 and HLA-C molecules. In addition, an ERAP2 variant in which lysine is changed to asparagine (K392N) results in increased trimming activity (165-fold) for hydrophobic peptides and biologically never been detected. We hypothesize that homozygosity for the N392 ERAP2 variant is prohibited because it modulates the immune recognition of placental trophoblasts. We demonstrate that NK-cell activation and killing were significantly dependent on forced expression of the N392 ERAP2 isoform in JEG-3 cells. Cytotoxicity was confirmed by 7AAD killing assays showing that N392 ERAP2-isoform expressing JEG-3 cells had the highest percentage of apoptotic cells independent of the expression level of CD11a on lymphocytes. This is the first report showing that N392 ERAP2 promotes an immune clearance pathway for choriocarcinoma cells, and provides an explanation for why embryonic homozygosity for the N392 ERAP2 variant is not detected in any population.

Endoplasmic Reticulum Aminopeptidase 2, a common immunological link to adverse pregnancy outcomes and cancer clearance?

Endoplasmic Reticulum Aminopeptidase 2 (ERAP2) trims HLA class I-binding peptides, determining the peptide repertoire presented for immune recognition. Variation in the ERAP2 amino acid sequence could affect the ability of some fetuses and tumors to achieve immune evasion. For example, homozygosity for an ERAP2 variant that has increased trimming efficiency for hydrophobic molecules has never been detected in mothers and fetuses. Thus, it is possible that this single nucleotide polymorphism (SNP) in the ERAP2 gene has been selected against in order to prevent alteration of the immune privileged uterine environment, and to allow tumors to escape immune recognition. Currently, there are no immunological treatments or prophylactic approaches to ensure a healthy pregnancy outcome, and the success of cancer immunotherapies is variable. Understanding the role of ERAP2 in immune evasion mechanisms in pregnancy and cancer may improve fetal survival and tumor clearance. This review summarizes current knowledge about ERAP2 and its N392 variant, and their relationship to pregnancy outcomes and cancer immune evasion/recognition.

A Novel ERAP2 Haplotype Structure in a Chilean Population: Implications for ERAP2 Protein Expression and Preeclampsia Risk.

Single nucleotide polymorphisms (SNPs) in the endoplasmic reticulum aminopeptidase 2 (ERAP2) gene are associated with preeclampsia (PE) in different populations. rs2549782, a coding variant (N392K) that significantly affects substrate specificity, is in linkage disequilibrium (LD) with rs2248374, a marker SNP associated with ERAP2 protein expression in previously studied populations. As a result of non-sense mediated RNA decay, ERAP2 protein is not expressed from the rs2248374 G allele. We previously reported that the fetal rs2549782 minor G allele is associated with PE in African-Americans, but not Chileans. In this study, we found that rs2549782 was in LD with rs2248374 in African-Americans, but not in Chileans. The unexpected lack of strong LD in Chileans raised the possibility that rs2248374 could be associated with PE in the absence of an association with rs2549782. However, we found no significant association for this allele with PE in Chileans. Chileans homozygous for the rs2248374 G allele did not express 110 kDa ERAP2 protein, consistent with non-sense mediated RNA decay, and carriers of the rs2248374 A allele did. We conclude that the Chilean ERAP2 haplotype structure allows for the expression of the major T allele of rs2549782 encoding 392N, which could impact peptide trimming and antigen presentation. Our discovery of racial differences in genetic structure and association with PE reveal here-to-fore unrecognized complexity of the ERAP2 locus.

Expansion of effector memory TCR Vbeta4+ CD8+ T cells is associated with latent infection-mediated resistance to transplantation tolerance.

Therapies that control largely T cell-dependent allograft rejection in humans also possess the undesirable effect of impairing T cell function, leaving transplant recipients susceptible to opportunistic viruses. Prime among these opportunists are the ubiquitous herpesviruses. To date, studies are lacking that address the effect of viruses that establish a true latent state on allograft tolerance or the effect of tolerance protocols on the immune control of latent viruses. By using a mixed chimerism-based tolerance-induction protocol, we found that mice undergoing latent infection with gammaHV68, a murine gamma-herpesvirus closely related to human gamma-herpesviruses such as EBV and Kaposi's sarcoma-associated herpesvirus, significantly resist tolerance to allografts. Limiting the degree of virus reactivation or innate immune response did not reconstitute chimerism in latently infected mice. However, gammaHV68-infected mice showed increased frequency of CD8+ T cell alloreactivity and, interestingly, expansion of virus-induced, alloreactive, "effector/effector memory" TCR Vbeta4+CD8+ T cells driven by the gammaHV68-M1 gene was associated with resistance to tolerance induction in studies using gammaHV68-M1 mutant virus. These results define the viral gene and immune cell types involved in latent infection-mediated resistance to allograft tolerance and underscore the influence of latent herpesviruses on allograft survival.

Immunosuppressive and trafficking properties of donor splenic and bone marrow dendritic cells.

BACKGROUND: Infusion of donor dendritic cells (DC) has been shown to prolong allograft survival in a number of models. However, many regimens that utilize donor DC do not consistently produced tolerance or long-term allograft survival. We hypothesized that one factor limiting the therapeutic effect of donor DC is their relative inability to traffic to recipient peripheral lymph nodes and inhibit the function of resident alloreactive T cells. METHODS: Donor strain DC isolated from the spleens or bone marrow of Flt3L-treated mice were transferred intravenously into recipients at the time of skin grafting. Where indicated, recipients were treated with an anti-CD40L antibody and CTLA4-Ig. RESULTS: Infusion of donor DC together with costimulatory blockade promoted donor-specific prolongation of skin allograft survival in mice. Perhaps due to their more immature phenotype, bone marrow DC trafficked more effectively to the spleen, bone marrow, and thymus and were associated with significantly longer allograft survival than were splenic DC. Neither population of DC trafficked well to peripheral lymph nodes. Consistent with our hypothesis, splenic but not lymph node T cells from DC-treated recipients displayed donor-specific hyporesponsiveness in vitro. CONCLUSION: These data suggest that one factor contributing to rejection following treatment with donor DC plus costimulation blockade is the persistence of donor-reactive T cells within the recipient's secondary lymphoid structures. Strategies to improve DC trafficking to these structures may enhance their therapeutic effect.

Costimulation requirements for antiviral CD8+ T cells differ for acute and persistent phases of polyoma virus infection.

The requirement for costimulation in antiviral CD8+ T cell responses has been actively investigated for acutely resolved viral infections, but it is less defined for CD8+ T cell responses to persistent virus infection. Using mouse polyoma virus (PyV) as a model of low-level persistent virus infection, we asked whether blockade of the CD40 ligand (CD40L) and CD28 costimulatory pathways impacts the magnitude and function of the PyV-specific CD8+ T response, as well as the humoral response and viral control during acute and persistent phases of infection. Costimulation blockade or gene knockout of either CD28 or CD40L substantially dampened the magnitude of the acute CD8+ T cell response; simultaneous CD28 and CD40L blockade severely depressed the acute T cell response, altered the cell surface phenotype of PyV-specific CD8+ T cells, decreased PyV VP1-specific serum IgG titers, and resulted in an increase in viral DNA levels in multiple organs. CD28 and CD40L costimulation blockade during acute infection also diminished the memory PyV-specific CD8+ T cell response and serum IgG titer, but control of viral persistence varied between mouse strains and among organs. Interestingly, we found that CD28 and CD40L costimulation is dispensable for generating and/or maintaining PyV-specific CD8+ T cells during persistent infection; however, blockade of CD27 and CD28 costimulation in persistently infected mice caused a reduction in PyV-specific CD8+ T cells. Taken together, these data indicate that CD8+ T cells primed within the distinct microenvironments of acute vs persistent virus infection differ in their costimulation requirements.

Late priming and variability of epitope-specific CD8+ T cell responses during a persistent virus infection.

Control of persistently infecting viruses requires that antiviral CD8(+) T cells sustain their numbers and effector function. In this study, we monitored epitope-specific CD8(+) T cells during acute and persistent phases of infection by polyoma virus, a mouse pathogen that is capable of potent oncogenicity. We identified several novel polyoma-specific CD8(+) T cell epitopes in C57BL/6 mice, a mouse strain highly resistant to polyoma virus-induced tumors. Each of these epitopes is derived from the viral T proteins, nonstructural proteins produced by both productively and nonproductively (and potentially transformed) infected cells. In contrast to CD8(+) T cell responses described in other microbial infection mouse models, we found substantial variability between epitope-specific CD8(+) T cell responses in their kinetics of expansion and contraction during acute infection, maintenance during persistent infection, as well as their expression of cytokine receptors and cytokine profiles. This epitope-dependent variability also extended to differences in maturation of functional avidity from acute to persistent infection, despite a narrowing in TCR repertoire across all three specificities. Using a novel minimal myeloablation-bone marrow chimera approach, we visualized priming of epitope-specific CD8(+) T cells during persistent virus infection. Interestingly, epitope-specific CD8(+) T cells differed in CD62L-selectin expression profiles when primed in acute or persistent phases of infection, indicating that the context of priming affects CD8(+) T cell heterogeneity. In summary, persistent polyoma virus infection both quantitatively and qualitatively shapes the antiviral CD8(+) T cell response.

Approaches toward reversal of increased vascular permeability in C1 inhibitor deficient mice.

C1 inhibitor (C1INH) deficient mice have increased vascular permeability that can be demonstrated by the extravasation of Evans Blue dye. This vascular leak is reversed with protease inhibitors, such as C1INH itself, DX88 (a recombinant variant Kunitz domain plasma kallikrein inhibitor), and the bradykinin receptor type 2 antagonist, Hoe140. The studies described here were undertaken for the following reasons: (1) To provide a more quantitative analysis of the effects of these interventions; (2) to provide data to further test the hypothesis that increased vascular permeability results from contact system activation with kallikrein-mediated release of bradykinin; (3) to test the hypothesis that the amino terminal non-serpin domain of C1INH modulates access to complex proteases, such as kallikrein complexed with high molecular weight kininogen (HK); and (4) to determine whether attenuated androgens or estrogens exert a direct effect on C1INH synthesis. To characterize the differences in these reagents, the dose-response and the rate of reappearance of increased vascular permeability in C1INH(-/-) mice were determined for the following agents: human plasma-derived C1INH, a recombinant Kunitz domain plasma kallikrein inhibitor (DX88), a bradykinin receptor antagonist (Hoe140), and a recombinant C1INH with an amino terminal truncation at amino acid 98 and substitution of the P2 Ala with a Val (Cserp98,A443V). C1INH and Cserp98,A443V were equivalent in activity, which provides further support for the hypothesis that the vascular leak is mediated by bradykinin and suggests that the amino terminal domain neither enhances nor interferes with access to kallikrein within the kallikrein-HK complex. DX88 was effective at very low doses, as was Hoe140. The duration of action of Hoe140 was quite prolonged. The data indicate that, in the mouse, neither danazol nor estrogens have a significant effect on C1INH synthesis.