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.

ARIH2 is essential for embryogenesis, and its hematopoietic deficiency causes lethal activation of the immune system.

The E3 ligase ARIH2 has an unusual structure and mechanism of elongating ubiquitin chains. To understand its physiological role, we generated gene-targeted mice deficient in ARIH2. ARIH2 deficiency resulted in the embryonic death of C57BL/6 mice. On a mixed genetic background, the lethality was attenuated, with some mice surviving beyond weaning and then succumbing to an aggressive multiorgan inflammatory response. We found that in dendritic cells (DCs), ARIH2 caused degradation of the inhibitor IκBß in the nucleus, which abrogated its ability to sequester, protect and transcriptionally coactivate the transcription factor subunit p65 in the nucleus. Loss of ARIH2 caused dysregulated activation of the transcription factor NF-κB in DCs, which led to lethal activation of the immune system in ARIH2-sufficent mice reconstituted with ARIH2-deficient hematopoietic stem cells. Our data have therapeutic implications for targeting ARIH2 function.

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.

OCT4 induces embryonic pluripotency via STAT3 signaling and metabolic mechanisms.

OCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 establishes and protects the pluripotent lineage in the embryo, we used comparative single-cell transcriptomics and quantitative immunofluorescence on control and OCT4 null blastocyst inner cell masses at two developmental stages. Surprisingly, activation of most pluripotency-associated transcription factors in the early mouse embryo occurs independently of OCT4, with the exception of the JAK/STAT signaling machinery. Concurrently, OCT4 null inner cell masses ectopically activate a subset of trophectoderm-associated genes. Inspection of metabolic pathways implicates the regulation of rate-limiting glycolytic enzymes by OCT4, consistent with a role in sustaining glycolysis. Furthermore, up-regulation of the lysosomal pathway was specifically detected in OCT4 null embryos. This finding implicates a requirement for OCT4 in the production of normal trophectoderm. Collectively, our findings uncover regulation of cellular metabolism and biophysical properties as mechanisms by which OCT4 instructs pluripotency.

Mechanical forces in avian embryo development.

Research using avian embryos has led to major conceptual advances in developmental biology, virology, immunology, genetics and cell biology. The avian embryo has several significant advantages, including ready availability and ease of accessibility, rapid development with marked similarities to mammals and a high amenability to manipulation. As mechanical forces are increasingly recognised as key drivers of morphogenesis, this powerful model system is shedding new light on the mechanobiology of embryonic development. Here, we highlight progress in understanding how mechanical forces direct key morphogenetic processes in the early avian embryo. Recent advances in quantitative live imaging and modelling are elaborating upon traditional work using physical models and embryo manipulations to reveal cell dynamics and tissue forces in ever greater detail. The recent application of transgenic technologies further increases the strength of the avian model and is providing important insights about previously intractable developmental processes.

NLR functions beyond pathogen recognition.

The last 10 years have witnessed the identification of a new class of intracellular pattern-recognition molecules--the nucleotide-binding domain and leucine-rich repeat-containing family (NLR). Members of this family garnered interest as pattern-recognition receptors able to trigger inflammatory responses against pathogens. Many studies support a pathogen-recognition function for human NLR proteins and shed light on their role in the broader control of adaptive immunity and various disease states. Other evidence suggests that NLRs function in processes unrelated to pathogen detection. Here we discuss recent advances in our understanding of the biology of the human NLR proteins and their non-pathogen-recognition function in tissue homeostasis, apoptosis, graft-versus-host disease and early development.

Structure, function, and evolution of Gga-AvBD11, the archetype of the structural avian-double-ß-defensin family.

Out of the 14 avian ß-defensins identified in the Gallus gallus genome, only 3 are present in the chicken egg, including the egg-specific avian ß-defensin 11 (Gga-AvBD11). Given its specific localization and its established antibacterial activity, Gga-AvBD11 appears to play a protective role in embryonic development. Gga-AvBD11 is an atypical double-sized defensin, predicted to possess 2 motifs related to ß-defensins and 6 disulfide bridges. The 3-dimensional NMR structure of the purified Gga-AvBD11 is a compact fold composed of 2 packed ß-defensin domains. This fold is the archetype of a structural family, dubbed herein as avian-double-ß-defensins (Av-DBD). We speculate that AvBD11 emanated from a monodomain gene ancestor and that similar events might have occurred in arthropods, leading to another structural family of less compact DBDs. We show that Gga-AvBD11 displays antimicrobial activities against gram-positive and gram-negative bacterial pathogens, the avian protozoan Eimeria tenella, and avian influenza virus. Gga-AvBD11 also shows cytotoxic and antiinvasive activities, suggesting that it may not only be involved in innate protection of the chicken embryo, but also in the (re)modeling of embryonic tissues. Finally, the contribution of either of the 2 Gga-AvBD11 domains to these biological activities was assessed, using chemically synthesized peptides. Our results point to a critical importance of the cationic N-terminal domain in mediating antibacterial, antiparasitic, and antiinvasive activities, with the C-terminal domain potentiating the 2 latter activities. Strikingly, antiviral activity in infected chicken cells, accompanied by marked cytotoxicity, requires the full-length protein.

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.

Multiomics uncovers developing immunological lineages in human.

The development of the human immune system during embryonic and fetal life has historically been difficult to research due to limited access to human tissue. Experimental animal models have been widely used to study development but cellular and molecular programmes may not be conserved across species. The advent of multiomic single-cell technologies and an increase in human developmental tissue biobank resources have facilitated single-cell multiomic studies focused on human immune development. A critical question in the near future is "How do we best reconcile scientific findings across multiple omic modalities, developmental time, and organismic space?" In this review, we discuss the application of single-cell multiomic technologies to unravel the major cellular lineages in the prenatal human immune system. We also identify key areas where the combined power of multiomics technologies can be leveraged to address specific immunological gaps in our current knowledge and explore new research horizons in human development.

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.

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.

The Effects of Sperm and Seminal Fluid of Immunized Male Mice on In Vitro Fertilization and Surrogate Mother-Embryo Interaction.

The latest vaccination campaign has actualized the potential impact of antigenic stimuli on reproductive functions. To address this, we mimicked vaccination's effects by administering keyhole limpet hemocyanin (KLH ) to CD1 male mice and used their sperm for in vitro fertilization (IVF). Two-cell embryos after IVF with spermatozoa from control (C) or KLH-treated (Im) male mice were transferred to surrogate mothers mated with vasectomized control (C) or KLH-treated (Im) male mice, resulting in four experimental groups: C-C, Im-C, C-Im, and Im-Im. The pre-implantation losses were significantly lower in the Im-C group than in the C-Im group. At the same time, the resorption rates reduced markedly in the C-Im compared to the Im-C group. Embryo and placenta weights were significantly higher in the Im-Im group. Although the GM-CSF levels were lower in the amniotic fluid of the gestating surrogate mothers in the Im-Im group, they were strongly correlated with embryo mass. The number-size trade-off was only significant in the Im-Im group. This suggests a positive, cooperative effect of spermatozoa and seminal fluid from immune-primed males on embryo growth and the optimal distribution of surrogate mother maternal resources despite the negative impact of males' antigenic challenge on the IVF success rate.

The developing immune network in human prenatal skin.

Establishment of a well-functioning immune network in skin is crucial for its barrier function. This begins in utero alongside the structural differentiation and maturation of skin, and continues to expand and diversify across the human lifespan. The microenvironment of the developing human skin supports immune cell differentiation and has an overall anti-inflammatory profile. Immunologically inert and skewed immune populations found in developing human skin promote wound healing, and as such may play a crucial role in the structural changes occurring during skin development.

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.

Upregulation of interferon-alpha gene in bovine embryos produced in vitro in response to experimental infection with noncytophatic bovine-viral-diarrhea virus.

In-vitro fertilization is a routine livestock-breeding technique widely used around the world. Several studies have reported the interaction of bovine viral-diarrhea virus (BVDV) with gametes and in-vitro-produced (IVP) bovine embryos. Since, gene expression in BVDV-infected IVP bovine embryos is scarcely addressed. The aim of this work was to evaluate the differential expression of genes involved in immune and inflammatory response. Groups of 20-25 embryos on Day 6 (morula stage) were exposed (infected) or not (control) to an NCP-BVDV strain in SOF medium. After 24 h, embryos that reached expanded blastocyst stage were washed. Total RNA of each embryo group was extracted to determine the transcription levels of 9 specific transcripts related with antiviral and inflammatory response by SYBR Green real time quantitative (RT-qPCR). Culture media and an aliquot of the last embryos wash on Day 7 were analyzed by titration and virus isolation, respectively. A conventional PCR confirmed BVDV presence in IVP embryos. A significantly higher expression of interferon-α was observed in blastocysts exposed to NCP-BVDV compared to the controls (p < 0.05). In this study, the upregulation of INFα and TLR7 genes involved in inflammatory and immune response in BVDV-infected IVP bovine embryos is a new finding in this field. This differential expression suggest that embryonic cells could function in a manner like immune cells by recognizing and responding early to interaction with viral pathogens. These results provide new insights into the action of BVDV on the complex molecular pathways controlling bovine early embryonic development.

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.

SMN deficiency negatively impacts red pulp macrophages and spleen development in mouse models of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease that is the leading genetic cause of infantile death. It is caused by a severe deficiency of the ubiquitously expressed Survival Motor Neuron (SMN) protein. SMA is characterized by α-lower motor neuron loss and muscle atrophy, however, there is a growing list of tissues impacted by a SMN deficiency beyond motor neurons. The non-neuronal defects are observed in the most severe Type I SMA patients and most of the widely used SMA mouse models, however, as effective therapeutics are developed, it is unclear whether additional symptoms will be uncovered in longer lived patients. Recently, the immune system and inflammation has been identified as a contributor to neurodegenerative diseases such as ALS. To determine whether the immune system is comprised in SMA, we analyzed the spleen and immunological components in SMA mice. In this report, we identify: a significant reduction in spleen size in multiple SMA mouse models and a pathological reduction in red pulp and extramedullary hematopoiesis. Additionally, red pulp macrophages, a discrete subset of yolk sac-derived macrophages, were found to be altered in SMA spleens even in pre-symptomatic post-natal day 2 animals. These cells, which are involved in iron metabolism and the phagocytosis of erythrocytes and blood-borne pathogens are significantly reduced prior to the development of the neurodegenerative hallmarks of SMA, implying a differential role of SMN in myeloid cell ontogeny. Collectively, these results demonstrate that SMN deficiency impacts spleen development and suggests a potential role for immunological development in SMA.

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.

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.

Difference in Developmental Kinetics of Y-Specific Monoclonal Antibody Sorted Male and Female In Vitro Produced Bovine Embryos.

Sex-related growth differences between male and female embryos remain an attractive subject for reproductive biologists. This study aimed to investigate the endogenous factors that play a crucial role in the pace of early development between male and female bovine embryos. Using sex pre-selected semen by Y-specific monoclonal antibodies for the production of bovine embryos, we characterized the critical endogenous factors that are responsible for creating the development differences, especially during the pre-implantation period between male and female embryos. Our results showed that at day seven, (57.8%) Y-sperm sorted in vitro cultured embryos reached the expanded blastocyst (BL) stage, whereas the X-sperm sorted group were only 25%. Y-BLs showed higher mRNA abundance of pluripotency and developmental competency regulators, such as Oct4 and IGF1-R. Interestingly, Y-sperm sorted BLs had a homogeneous mitochondrial distribution pattern, higher mitochondrial membrane potential (∆Ñ°m), efficient OXPHOS (oxidative phosphorylation) system and well-encountered production of ROS (reactive oxygen species) level. Moreover, Y-blastocysts (BLs) showed less utilization of glucose metabolism relative to the X-BLs group. Importantly, both sexes showed differences in the timing of epigenetic events. All these factors directly or indirectly orchestrate the whole embryonic progression and may help in the faster and better quality yield of BL in the Y-sperm sorted group compared to the X counterpart group.