Autophagy genes and signaling pathways in endometrial decidualization and pregnancy complications.

Autophagy is a process that occurs in almost all eukaryotic cells and this process is controlled by several molecular processes. Its biological roles include the provision of energy, the maintenance of cell homeostasis, and the promotion of aberrant cell death. The importance of autophagy in pregnancy is gradually becoming recognized. In literature, it has been indicated that autophagy has three different effects on the onset and maintenance of pregnancy: embryo (embryonic development), feto-maternal immune crosstalk, and maternal (decidualization). In humans, proper decidualization is a major predictor of pregnancy accomplishment and it can be influenced by different factors. This review highlights the genes, pathways, regulation, and function of autophagy in endometrial decidualization and other involved factors in this process.

Impact of Antithyroperoxidase Antibodies (Anti-TPO) on Ovarian Reserve and Early Embryo Development in Assisted Reproductive Technology Cycles.

Autoimmune thyroid disease (AITD) is one of the most common endocrinopathies and is more prevalent in women. It becomes evident that the circulating antithyroid antibodies that often follow AITD have effects on many tissues, including ovaries, and therefore that this common morbidity might have an impact on female fertility, the investigation of which is the aim of the present research. Ovarian reserve, ovarian response to stimulation and early embryo development in infertile patients with thyroid autoimmunity were assessed in 45 women with thyroid autoimmunity and 45 age-matched control patients undergoing infertility treatment. It was demonstrated that the presence of anti-thyroid peroxidase antibodies is associated with lower serum anti-Müllerian hormone levels and antral follicle count. Further investigation revealed the higher prevalence of sub-optimal response to ovarian stimulation in TAI-positive women, lower fertilization rate and lower number of high-quality embryos in this group of patients. The cut-off value for follicular fluid anti-thyroid peroxidase antibody affecting the above-mentioned parameters was determined to be 105.0 IU/mL, highlighting the necessity of closer monitoring in couples seeking infertility treatment with ART.

Uterine glands impact embryo survival and stromal cell decidualization in mice.

Uterine glands are essential for the establishment of pregnancy and have critical roles in endometrial receptivity to blastocyst implantation, stromal cell decidualization, and placentation. Uterine gland dysfunction is considered a major contributing factor to pregnancy loss, however our understanding of how glands impact embryo survival and stromal cell decidualization is incomplete. Forkhead box A2 (FOXA2) is expressed only in the glandular epithelium and regulates its development and function. Mice with a conditional deletion of FOXA2 in the uterus are infertile due to defective embryo implantation arising from a lack of leukemia inhibitory factor (LIF), a critical factor of uterine gland origin. Here, a glandless FOXA2-deficient mouse model, coupled with LIF repletion to rescue the implantation defect, was used to investigate the roles of uterine glands in embryo survival and decidualization. Studies found that embryo survival and decidualization were compromised in glandless FOXA2-deficient mice on gestational day 6.5, resulting in abrupt pregnancy loss by day 7.5. These findings strongly support the hypothesis that uterine glands secrete factors other than LIF that impact embryo survival and stromal cell decidualization for pregnancy success.

B1 lymphocytes develop independently of Notch signaling during mouse embryonic development.

B1 lymphocytes are a small but unique component of the innate immune-like cells. However, their ontogenic origin is still a matter of debate. Although it is widely accepted that B1 cells originate early in fetal life, whether or not they arise from hematopoietic stem cells (HSCs) is still unclear. In order to shed light on the B1 cell origin, we set out to determine whether their lineage specification is dependent on Notch signaling, which is essential for the HSC generation and, therefore, all derivatives lineages. Using mouse embryonic stem cells (mESCs) to recapitulate murine embryonic development, we have studied the requirement for Notch signaling during the earliest B-cell lymphopoiesis and found that Rbpj-deficient mESCs are able to generate B1 cells. Their Notch independence was confirmed in ex vivo experiments using Rbpj-deficient embryos. In addition, we found that upregulation of Notch signaling induced the emergence of B2 lymphoid cells. Taken together, these findings indicate that control of Notch signaling dose is crucial for different B-cell lineage specification from endothelial cells and provides pivotal information for their in vitro generation from PSCs for therapeutic applications. This article has an associated 'The people behind the papers' interview.

Ovalbumin causes impairment of preimplantation embryonic growth in asthma-induced mice.

Insufficient experimental studies have reported the effect of ovalbumin (OVA) as an allergen towards embryonic growth in asthma mouse model. The impact of 10 µg/200 µL OVA on maternal inflammatory and oxidative stress (OS) responses, and preimplantation embryonic development was investigated in this study. We first established OVA-induced asthma mouse model, and following superovulation, mated the females and challenged them with Methacholine (Mch) test. Upon embryo retrieval, only those with the highest implantation potential were cultured in vitro. Significant reduction in the number of embryos at each preimplantation stage was noted in the treated group. Uneven sized blastomeres at 2-, 4- and 8-cell stages were also evident in this group. Embryo fragmentation was significant at only 2-, 4- and 8-cell stages. We also found that OVA tended to raise maternal inflammatory and OS biomarker levels as well as to cause inappropriate levels of pregnancy hormones progesterone (P4) and estrogen (E2) although insignificant. The combined results indicate that 10 µg/200 µL OVA had altered both quality and quantity of the embryos in asthma mouse model although its effect on pregnancy hormones, inflammatory and OS responses were non-pathological.

Transcriptional intermediary factor 1 (TIF1) and anti-TIF1γ antibody-positive dermatomyositis.

Recently, great advancements have been made towards understanding the mechanisms underlying dermatomyositis (DM). Many novel autoantibodies, such as anti-MDA5, anti-TIF1γ, anti-NXP2, and anti-SAE, have been reported to be involved in DM. DM is now classified based on these myositis-specific autoantibodies. Anti-TIF1γ antibodies are closely associated with juvenile DM and adult cancer-associated DM. Anti-TIF1γ antibody-positive DM tends to present severe cutaneous manifestations, mild myositis, and dysphagia. TIF1γ (also known as TRIM33) plays a role in transcriptional elongation, DNA repair, differentiation of cells, embryonic development, and mitosis. Moreover, TIF1γ has been shown to suppress various tumors via the TGF-ß/Smad and the Wnt/ß-Catenin signaling pathways. In this review, we explore the relationship between TIF1γ, cancer, and DM. We also discuss the pathogenesis of anti-TIF1γ antibody-positive DM.

Roles of HLA-G in the Maternal-Fetal Immune Microenvironment.

During pregnancy, the maternal uterus and fetus form a special microenvironment at the maternal-fetal interface to support fetal development. Extravillous trophoblasts (EVTs), differentiated from the fetus, invade into the decidua and interact with maternal cells. Human leukocyte antigen (HLA)-G is a non-classical MHC-I molecule that is expressed abundantly and specifically on EVTs in physiological conditions. Soluble HLA-G (sHLA-G) is also found in maternal blood, amniotic fluid, and cord blood. The abnormal expression and polymorphisms of HLA-G are related to adverse pregnancy outcomes such as preeclampsia (PE) and recurrent spontaneous abortion (RSA). Here we summarize current findings about three main roles of HLA-G during pregnancy, namely its promotion of spiral artery remodeling, immune tolerance, and fetal growth, all resulting from its interaction with immune cells. These findings are not only of great significance for the treatment of pregnancy-related diseases but also provide clues to tumor immunology research since HLA-G functions as a checkpoint in tumors.

ASGR1 and Its Enigmatic Relative, CLEC10A.

The large family of C-type lectin (CLEC) receptors comprises carbohydrate-binding proteins that require Ca2+ to bind a ligand. The prototypic receptor is the asialoglycoprotein receptor-1 (ASGR1, CLEC4H1) that is expressed primarily by hepatocytes. The early work on ASGR1, which is highly specific for N-acetylgalactosamine (GalNAc), established the foundation for understanding the overall function of CLEC receptors. Cells of the immune system generally express more than one CLEC receptor that serve diverse functions such as pathogen-recognition, initiation of cellular signaling, cellular adhesion, glycoprotein turnover, inflammation and immune responses. The receptor CLEC10A (C-type lectin domain family 10 member A, CD301; also called the macrophage galactose-type lectin, MGL) contains a carbohydrate-recognition domain (CRD) that is homologous to the CRD of ASGR1, and thus, is also specific for GalNAc. CLEC10A is most highly expressed on immature DCs, monocyte-derived DCs, and alternatively activated macrophages (subtype M2a) as well as oocytes and progenitor cells at several stages of embryonic development. This receptor is involved in initiation of TH1, TH2, and TH17 immune responses and induction of tolerance in naïve T cells. Ligand-mediated endocytosis of CLEC receptors initiates a Ca2+ signal that interestingly has different outcomes depending on ligand properties, concentration, and frequency of administration. This review summarizes studies that have been carried out on these receptors.

Early Life Glucocorticoid Exposure Modulates Immune Function in Zebrafish (Danio rerio) Larvae.

In this study we have assessed the effects of increased cortisol levels during early embryonic development on immune function in zebrafish (Danio rerio) larvae. Fertilized eggs were exposed to either a cortisol-containing, a dexamethasone-containing (to stimulate the glucocorticoid receptor selectively) or a control medium for 6 h post-fertilization (0-6 hpf). First, we measured baseline expression of a number of immune-related genes (socs3a, mpeg1.1, mpeg1.2, and irg1l) 5 days post-fertilization (dpf) in larvae of the AB and TL strain to assess the effectiveness of our exposure procedure and potential strain differences. Cortisol and dexamethasone strongly up-regulated baseline expression of these genes independent of strain. The next series of experiments were therefore carried out in larvae of the AB strain only. We measured neutrophil/macrophage recruitment following tail fin amputation (performed at 3 dpf) and phenotypical changes as well as survival following LPS-induced sepsis (150 µg/ml; 4-5 dpf). Dexamethasone, but not cortisol, exposure at 0-6 hpf enhanced neutrophil recruitment 4 h post tail fin amputation. Cortisol and dexamethasone exposure at 0-6 hpf led to a milder phenotype (e.g., less tail fin damage) and enhanced survival following LPS challenge compared to control exposure. Gene-expression analysis showed accompanying differences in transcript abundance of tlr4bb, cxcr4a, myd88, il1ß, and il10. These data show that early-life exposure to cortisol, which may be considered to be a model or proxy of maternal stress, induces an adaptive response to immune challenges, which seems mediated via the glucocorticoid receptor.

Long-Term Programming of CD8 T Cell Immunity by Perinatal Exposure to Glucocorticoids.

Early life environmental exposure, particularly during perinatal period, can have a life-long impact on organismal development and physiology. The biological rationale for this phenomenon is to promote physiological adaptations to the anticipated environment based on early life experience. However, perinatal exposure to adverse environments can also be associated with adult-onset disorders. Multiple environmental stressors induce glucocorticoids, which prompted us to investigate their role in developmental programming. Here, we report that perinatal glucocorticoid exposure had long-term consequences and resulted in diminished CD8 T cell response in adulthood and impaired control of tumor growth and bacterial infection. We found that perinatal glucocorticoid exposure resulted in persistent alteration of the hypothalamic-pituitary-adrenal (HPA) axis. Consequently, the level of the hormone in adults was significantly reduced, resulting in decreased CD8 T cell function. Our study thus demonstrates that perinatal stress can have long-term consequences on CD8 T cell immunity by altering HPA axis activity.

Rac2 Regulates the Migration of T Lymphoid Progenitors to the Thymus during Zebrafish Embryogenesis.

The caudal hematopoietic tissue in zebrafish, the equivalent to the fetal liver in mammals, is an intermediate hematopoietic niche for the maintenance and differentiation of hematopoietic stem and progenitor cells before homing to the thymus and kidney marrow. As one of the ultimate hematopoietic organs, the thymus sustains T lymphopoiesis, which is essential for adaptive immune system. However, the mechanism of prethymic T lymphoid progenitors migrating to the thymus remains elusive. In this study, we identify an Rho GTPase Rac2 as a modulator of T lymphoid progenitor homing to the thymus in zebrafish. rac2-Deficient embryos show the inability of T lymphoid progenitors homing to the thymus because of defective cell-autonomous motility. Mechanistically, we demonstrate that Rac2 regulates homing of T lymphoid progenitor through Pak1-mediated AKT pathway. Taken together, our work reveals an important function of Rac2 in directing T lymphoid progenitor migration to the thymus during zebrafish embryogenesis.

Effects of norfloxacin nicotinate on the early life stage of zebrafish (Danio rerio): Developmental toxicity, oxidative stress and immunotoxicity.

Norfloxacin nicotinate (NOR-N), an adduct of norfloxacin (NOR) and nicotinic acid, has been widely used for replacing NOR in animal husbandry and fishery industry. Nowadays, increasing evidences showed that NOR could pose toxic effects on fish and other aquatic organisms, but as its adduct, whether NOR-N could cause adverse effects on aquatic organisms is still unclear. To evaluate the toxic effects of NOR-N on the early life stage of zebrafish, we determined the changes in embryonic development (hatching rate, body length, malformation rate and mortality), antioxidant enzyme (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (Gpx)) activities, malondialdehyde (MDA) content and gene expression levels related to antioxidant enzymes (Cu/Zn-sod, Mn-sod, CAT and Gpx) and innate immune system (tumor necrosis factor α (TNFα), interferon (IFN), Interleukin-1 beta (IL-1ß), IL-8, CXCL-clc, CC-chemokine, lysozyme (Lzy) and complement factors (C3)) after embryonic exposure to NOR-N till 96 hpf. The results showed that NOR-N exposure could decreased the hatching rate and body length, and increased abnormality and mortality as concentration-dependent during embryonic development process. NOR-N induced oxidative stress in zebrafish larvae through increasing the contents of MDA and the activities of SOD, CAT and Gpx, as well as the mRNA levels of genes related to these antioxidant enzymes. Moreover, the expression of TNFα, IFN, IL-1ß, IL-8, CXCL-clc, CC-chemokine, Lzy and C3 genes were significantly up-regulated after exposure to high concentration (5 and/or 25 mg/L) of NOR-N till 96 hpf, indicating that the innate immune system in zebrafish larvae was disturbed by NOR-N. Overall, our results suggested that NOR-N caused development toxicity, oxidative stress and immunotoxicity on the early life stage of zebrafish. Thus, widespread application of NOR-N might pose potential ecotoxicological risk on aquatic ecosystems.

Developmental and Immune Role of a Novel Multiple Cysteine Cluster TLR From Eisenia andrei Earthworms.

Earthworms are not endowed with adaptive immunity and they are rely on the tools of innate immunity. Cells of the innate immune system utilize pattern recognition receptors, such as Toll-like receptors, to detect the pathogen-associated molecular patterns (PAMPs). The first earthworm TLR was isolated from Eisenia andrei earthworms (EaTLR), which belongs to the single cysteine cluster TLR (sccTLR). Here, we identified a new multiple cysteine cluster TLR (mccTLR) in E. andrei earthworms. Phylogenetic DNA analysis revealed that it has no variability within one earthworm as well as in the population. By screening of the tissue expression profile, the TLR was expressed primarily in earthworm seminal vesicles and receptacles suggesting a connection to sperm cells. Seminal vesicles are often heavily infected by gregarine parasites. As a sign of immune response, a strong melanization reaction is visible around parasites. Stimulation experiments with profilin from related parasite Toxoplasma gondii, led to the upregulation of mccEaTLR in the earthworm seminal vesicles. Also, profilin activated prophenoloxidase cascade, the efficient mechanism of innate immunity. However, its involvement in the NF-κB signaling was not proven. Further, we provide evidence that the antibiotics metronidazole and griseofulvin destroyed the developing spermatocytes. The observed decrease in the mccEaTLR mRNA levels after the antibiotic treatment of parasites is caused by the decline of sperm cells numbers rather than by diminution of the parasites. Since earthworms with extensively reduced parasite load had a similar amount of mccEaTLR mRNA, presumably, earthworm sperm cells have a certain level of mccEaTLR expressed as a standard, which can be augmented by particular antigenic stimulation. Also, mccEaTLR was expressed mainly in the early stages of earthworm development and presumably is primarily involved in early embryonic development. Expression of mccEaTLR in seminal vesicles correlates with the expression of endothelial monocyte-activation polypeptide II. High-throughput sequencing of gregarine DNA from seminal vesicles of individual earthworms resulted in great diversity of the observed genotypes. Phylogenetically, all observed OTUs belong to the clade of earthworm gregarines suggesting host specificity. Overall, mccEaTLR is supposed to play a function role in early embryonic development and potentially it participates in immune response against parasites.

Expression of avian ß-defensin mRNA in the chicken yolk sac.

The chicken yolk sac (YS) plays an important role in nutrient absorption and immune function for the developing embryo. The avian ß-defensins (AvBD) are cationic peptides that are important members of the innate immune system. The objective of this study was to profile AvBD mRNA expression patterns and distribution of cells expressing AvBD mRNA in the chicken YS. Expression of AvBD1, 2, 7, and 10 mRNA was low at embryonic day 7 (e7), increased to e9 through e13 and then declined to e19. Using in situ hybridization, AvBD10 mRNA was found to be expressed in endodermal epithelial cells, while AvBD1, 2, and 7 mRNA were expressed in heterophils. The developmental expression pattern and distribution of AvBD mRNA in the YS reveals the importance of these genes to protection of the developing chick embryo.

An insight into the role of the death receptor CD95 throughout pregnancy: Guardian, facilitator, or foe.

The prototype death receptor CD95 (Fas) and its ligand, CD95L (FasL), have been thoroughly studied due to their role in immune homeostasis and elimination of infected and transformed cells. The fact that CD95 is present in female reproductive cells and modulated during embryogenesis and pregnancy has raised interest in its role in immune tolerance to the fetoplacental unit. CD95 has been shown to be critical for proper embryonic formation and survival. Moreover, altered expression of CD95 or its ligand causes autoimmunity and has also been directly involved in recurrent pregnancy losses and pregnancy disorders. The objective of this review is to summarize studies that evaluate the mechanisms involved in the activation of CD95 to provide an updated global view of its effect on the regulation of the maternal immune system. Modulation of the CD95 system components may be the immune basis of several common pregnancy disorders.

Enniatin B1 exerts embryotoxic effects on mouse blastocysts and induces oxidative stress and immunotoxicity during embryo development.

Enniatins are mycotoxins of Fusarium fungi that naturally exist as mixtures of cyclic depsipeptides. Previous reports have documented hazardous effects of enniatins on cells, such as apoptosis. However, their effects on pre- and post-implantation embryonic development require further clarification. Here, we showed for the first time that enniatin B1 (ENN B1) exerts cytotoxic effects on mouse blastocyst-stage embryos and induces intracellular oxidative stress and immunotoxicity in mouse fetuses. Co-incubation of blastocysts with ENN B1 triggered significant apoptosis and led to a decrease in total cell number predominantly through loss of inner cell mass. In addition, ENN B1 appeared to exert hazardous effects on pre and postimplantation embryo development potential in an in vitro development assay. Treatment of blastocysts with 1-10 µM ENN B1 led to increased resorption of post-implantation embryos and decreased fetal weight in the embryo transfer assay in a dose-dependent manner. Importantly, in an in vivo model, intravenous injection with ENN B1 (1, 3, and 5 mg/kg body weight/d) for 4 days resulted in apoptosis of blastocyst-stage embryos and impairment of embryonic development from the zygote to blastocyst stage, subsequent degradation of embryos, and further decrease in fetal weight. Intravenous injection with 5 mg/kg body weight/d ENN B1 additionally induced a significant increase in total reactive oxygen species (ROS) content and transcription levels of genes encoding antioxidant proteins in mouse fetal liver. Moreover, ENN B1 triggered apoptosis through ROS generation and strategies to prevent apoptotic processes effectively rescued ENN B1-mediated hazardous effects on embryonic development. Transcription levels of CXCL1, IL-1ß, and IL-8 related to innate immunity were downregulated after intravenous injection of ENN B1. These results collectively highlight the potential of ENN B1 to exert cytotoxic effects on embryos as well as oxidative stress and immunotoxicity during mouse embryo development.

New Insights Into the Role of Qa-2 and HLA-G Non-classical MHC-I Complexes in Malignancy.

It is well established that the immune system can identify and destroy neoplastic transformed cells in a process known as immunosurveillance. Most studies have focused on the classical major histocompatibility complex (MHC) class Ia molecules, which are known to play an important role on the presentation of tumor antigens to the immune system in order to activate a response against tumor cells. However, a larger family of non-classical MHC class Ib-related molecules has received less attention. In this mini-review, we discuss the role of class Ib murine Qa-2 and its proposed human HLA-G homolog on immunosurveillance during embryogenesis and cancer. Whereas, both HLA-G and Qa-2 are involved in immune tolerance in pregnancy, the current evidence suggests that they play opposite roles in cancer. HLA-G appears to promote tumor progression while Qa-2 acts as a tumor suppressor awaking the immune system to reject tumor cells, as suggested by studies on different cancer cell models, such as melanoma, lymphoma, lung carcinoma, and our own results in mammary carcinoma.

Dynamic changes in intrathymic ILC populations during murine neonatal development.

Members of the innate lymphoid cell (ILC) family have been implicated in the development of thymic microenvironments and the recovery of this architecture after damage. However, a detailed characterization of this family in the thymus is lacking. To better understand the thymic ILC compartment, we have utilized multiple in vivo models including the fate mapping of inhibitor of DNA binding-2 (Id2) expression and the use of Id2 reporter mice. Our data demonstrate that ILCs are more prominent immediately after birth, but were rapidly diluted as the T-cell development program increased. As observed in the embryonic thymus, CCR6+ NKp46- lymphoid tissue inducer (LTi) cells were the main ILC3 population present, but numbers of these cells swiftly declined in the neonate and ILC3 were barely detectable in adult thymus. This loss of ILC3 means ILC2 are the dominant ILC population in the thymus. Thymic ILC2 were able to produce IL-5 and IL-13, were located within the medulla, and did not result from ILC3 plasticity. Furthermore, in WT mice, thymic ILC2 express little RANKL (receptor activator of nuclear factor kappa-B ligand) arguing that functionally, these cells provide different signals to LTi cells in the thymus. Collectively, these data reveal a dynamic switch in the ILC populations of the thymus during neonatal development.

Embryotoxic cytokines-Potential roles in embryo loss and fetal programming.

Cytokines in the reproductive tract environment at conception mediate a dialogue between the embryo and maternal tissues to profoundly influence embryo development and implantation success. Through effects on gene expression and the cell stress response, cytokines elicit an epigenetic impact with consequences for placental development and fetal growth, which in turn affect metabolic phenotype and long-term health of offspring. There is substantial evidence demonstrating that pro-survival cytokines, such as GM-CSF, CSF1, LIF, HB-EGF and IGFII, support embryos to develop optimally. Less attention has been paid to cytokines that adversely impact embryo development, including the pro-inflammatory cytokines TNF, TRAIL and IFNG. These agents elicit cell stress, impair cell survival and retard blastocyst development, and at sufficiently high concentrations, can cause embryo demise. Experiments in mice suggest these so-called 'embryotoxic' cytokines can harm embryos through pro-apoptotic and adverse programming effects, as well as indirectly suppressing uterine receptivity through the maternal immune response. Embryotrophic factors may mitigate against and protect from these adverse effects. Thus, the balance between embryotrophic and embryotoxic cytokines can impart effects on embryo development and implantation, and has the potential to contribute to endometrial 'biosensor' function to mediate embryo selection. Embryotoxic cytokines can be elevated in plasma and reproductive tract tissues in inflammatory conditions including infection, diabetes, obesity, PCOS and endometriosis. Studies are therefore warranted to investigate whether excessive embryotoxic cytokines contribute to infertility and recurrent implantation failure in women, and compromised reproductive performance in livestock animals.