Primary infection of Toxoplasma gondii can cause serious abnormal pregnancy outcomes such as miscarriage and stillbirth. Inhibitory molecule B7-H4 is abundantly expressed in dendritic cells (DCs) and plays an important role in maintaining immune tolerance. However, the role of B7-H4 in decidual DCs (dDCs) in T. gondii-induced abnormal pregnancy outcomes is not clear.

Women in early pregnancy infected by Toxoplasma gondii may have severe adverse pregnancy outcomes, such as spontaneous abortion and fetal malformation. The inhibitory molecule T cell immunoglobulin and mucin domain 3 (Tim-3) is highly expressed on decidual dendritic cells (dDCs) and plays an important role in maintaining immune tolerance. However, whether T. gondii infection can cause dDC dysfunction by influencing the expression of Tim-3 and further participate in adverse pregnancy outcomes is still unclear.

Vertical transmission of Toxoplasma gondii (T. gondii) infection during gestation can result in severe complications such as abortion, congenital malformation, fetal teratogenesis, etc. Immune inhibitory molecule Tim-3 was discovered to be expressed on some decidual immune cells and participates in the maintenance of maternal-fetal tolerance. Dysregulation of Tim-3 expression on decidual NK (dNK) cells was observed in several cases of pregnancy complications, whereas the role of Tim-3 on dNK cells during T. gondii infection remains unclear. In the present study, T. gondii infected Tim-3-/- pregnant mice, and anti-Tim-3 neutralizing antibody treated and infected human dNK cells were successfully established to explore the role of Tim-3 in dysfunction of dNK cells during abnormal pregnancy. Our results illustrated that Tim-3-/- pregnant mice displayed more worse pregnancy outcomes with T. gondii infection compared to infected WT pregnant mice. Also, it demonstrated that Tim-3 expression on dNK cells was significantly down-regulated following T. gondii infection. Data suggested a remarkable activation of dNK cells in Tim-3-/- mice and anti-Tim-3 neutralizing antibody treated and infected groups, with higher ratios of activating receptor NKG2D to inhibitory receptor NKG2A or KIR2DL4, IFN-γ/IL-10, and increased granule production compared with that of the infected group. Mechanism analysis proved that T. gondii-induced Tim-3 down-regulation significantly activated the phosphatidylinositol-3-kinase (PI3K)-AKT and JAK-STAT signaling pathway, by which the GranzymeB, Perforin, IFN-γ, and IL-10 production were further up-regulated. Our research demonstrated that the decrease of Tim-3 on dNK cells caused by T. gondii infection further led to dNK cells function disorder, which finally contributed to the development of abnormal pregnancy outcomes.

Toxoplasma gondii infection during pregnancy can lead to fetal defect(s) or congenital complications. The inhibitory molecule B7-H4 expressed on decidual macrophages (dMφ) plays an important role in maternal-fetal tolerance. However, the effect of B7-H4 on the function of dMφ during T. gondii infection remains unclear.

Toxoplasma gondii infection can cause adverse pregnancy outcomes, such as recurrent abortion, fetal growth restriction and infants with malformations, among others. Decidual myeloid-derived suppressor cells (dMDSCs) are a novel immunosuppressive cell type at the fetal-maternal interface which play an important role in sustaining normal pregnancy that is related to their high expression of the inhibitory molecule leukocyte immunoglobulin-like receptor B4 (LILRB4). It has been reported that the expression of LILRB4 is downregulated on decidual macrophages after T. gondii infection, but it remains unknown whether T. gondii infection can induce dMDSC dysfunction resulting from the change in LILRB4 expression.

Toxoplasma gondii is a ubiquitous, obligate intracellular parasite capable of crossing the placental barrier and causing spontaneous abortion, preterm labor, or significant disease in the surviving neonate. To better understand molecular mechanisms underlying abnormal pregnancy outcomes caused by T. gondii, placental proteins extracted from T. gondii-infected and -uninfected mice were comparatively analyzed using label-free liquid chromatography-tandem mass spectrometry. Significant difference was observed in the expression of 58 out of 792 proteins in infected placentas (p<0.05) compared with that in uninfected placentas. Quantitative real-time polymerase chain reaction, western blotting, and immunohistochemical staining were used to validate the results of the proteomic analysis. Some placental proteins differentially expressed in infected and uninfected mice were found to be associated with several different biological processes of pregnancy, particularly with trophoblast invasion and placental development. The results provide possible novel insights into the molecular mechanisms for abnormal pregnancy outcomes associated with T. gondii infection.

Inhibitory receptors and activating receptor expressed on decidual natural killer (dNK) cells are generally believed to be important in abnormal pregnancy outcomes and induced adverse pregnancy. However, if Toxoplasma gondii (T. gondii) infection induced abnormal pregnancy was related to dNK cells changes is not clear. In this study, we used human dNK cells co-cultured with human extravillous cytotrophoblast (EVT) cells following YFP-Toxoplasma gondii (YFP-T. gondii) infection in vitro and established animal pregnant infection model. Levels of inhibitory receptors KIR2DL4 and ILT-2, their ligand HLA-G, and activating receptor NKG2D in human decidua, and NKG2A and its ligand Qa-1 and NKG2D in mice uterine were analyzed by real-time PCR and flow cytometry with levels of NKG2D significantly higher than those of KIR2DL4 and ILT-2 in vitro and in invo. The level of NKG2D was positively correlated with cytotoxic activity of dNK cells in vitro. Numbers of abnormal pregnancies were significantly greater in the infected group than in the control group. This result demonstrated that the increased NKG2D expression and imbalance between inhibitory receptors of dNK cells and HLA-G may contribute to abnormal pregnancy outcomes observed upon maternal infection with T. gondii.

Vertical transmission of the intracellular parasite Toxoplasma gondii (T. gondii) can lead to devastating consequences during gestation. Tim-3, a negative immune regulator, is constitutively expressed on decidual macrophages, but its specific role during T. gondii infection has not yet been explored. In the present study, we discovered that Tim-3 plays an important role in the abnormal pregnancy due to T. gondii infection using Tim-3-/- pregnant mice and anti-Tim-3 neutralizing antibody treated human decidual macrophages. The results showed that abnormal pregnancy outcomes were more prevalent in Tim-3-/- infected pregnant mice than in wild-type infected pregnant mice. Tim-3 expression in decidual macrophages was significantly down-regulated after T. gondii infection both in vitro and in vivo. Tim-3 down-regulation by T.gondii infection could strengthen M1 activation and weaken M2 tolerance by changing the M1 and M2 membrane molecule expression, arginine metabolic enzymes synthesis, and cytokine secretion profiles of decidual macrophages. Moreover, Tim-3 down-regulation by T.gondii infection led to PI3K-AKT phosphorylation inhibition, downstream transcription factor C/EBPβ expression, and SOCS1 activation, which resulted in enzymes synthesis regulation and cytokines secretion. Our study demonstrates that Tim-3 plays an indispensable role in the adverse pregnancy outcomes caused by T. gondii infection.

Toxoplasma gondii (T. gondii) infection in early pregnancy can result in miscarriage, dead fetus, and other abnormalities. The LILRB4 is a central inhibitory receptor in uterine dendritic cells (uDCs) that plays essential immune-regulatory roles at the maternal-fetal interface. In this study, T. gondii-infected human primary uDCs and T. gondii-infected LILRB4-/- pregnant mice were utilized. The immune mechanisms underlying the role of LILRB4 on uDCs were explored in the development of abnormal pregnancy outcomes following T. gondii infection in vitro and in vivo. Our results showed that the expression levels of LILRB4 on uDCs from normal pregnant mice were obviously higher than non-pregnant mice, and peaked in mid-gestation. The LILRB4 expression on uDC subsets, especially tolerogenic subsets, from mid-gestation was obviously down-regulated after T. gondii infection and LILRB4 decrease could further regulate the expression of functional molecules (CD80, CD86, and HLA-DR or MHC II) on uDCs, contributing to abnormal pregnancy outcomes. Our results will shed light on the molecular immune mechanisms of uDCs in abnormal pregnancy outcomes by T. gondii infection.

Toxoplasma gondii (T. gondii) is a ubiquitous intracellular protozoan parasite that causes adverse pregnancy outcomes. Its infection downregulates the Treg cell population and TGF-β level, which may contribute to adverse pregnancy outcomes. TGF-β plays a critical role in Treg cell development, but whether TGF-β treatment can affect the number and function of Treg cells and hence alleviate adverse pregnancy outcomes caused by T. gondii infection remains elusive. In this study, T. gondii-infected pregnant mice were treated with TGF-β or TGF-β-neutralizing antibody. The pregnancy outcomes were observed on gestational day 14. The numbers of Treg cells and pSmad3, programmed death 1 (PD-1), and Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) expression of Treg cells were analyzed by flow cytometry. Histological changes were assessed using HE staining, while IL-10 and TNF-α levels were measured using ELISA. The results indicated that TGF-β treatment improved the T. gondii-induced adverse pregnancy outcomes, with alleviation of hemorrhage, restoration of uterine spiral arteries of the placenta, and increased Treg cell numbers; meanwhile, TGF-β neutralization resulted in more serious adverse pregnancy outcomes, with serious hemorrhage, more dilated uterine spiral arteries, and decreased Treg cell numbers. pSmad3 expression in CD4+ cells and CTLA-4 and PD-1 levels on Treg cells were upregulated by TGF-β treatment, but downregulated by TGF-β neutralization. The ratio of IL-10/TNF-α also increased after TGF-β treatment, but decreased after TGF-β neutralization. Our data indicate that TGF-β treatment could improve adverse pregnancy outcomes caused by T. gondii infection by upregulating Treg cell differentiation and function via the TGF-β/Smad3 signaling pathway, but not the proliferation of Treg cells.

Our current aim was to investigate whether injection of TGF-β1 played an important role in improving abnormal pregnancy outcomes with T. gondii infection and how the TGF-β1 regulated. Results showed that TGF-β1 exhibited improved pregnancy outcomes induced by T. gondii infection. dNK cytotoxicity was increased with T. gondii infection while decreased with TGF-β1 treatment. dNK cytotoxicity related NKG2D/DAP10 expression, perforin, granzyme, IFN-γ and killer subsets were all increased with T. gondii infection while decreased after TGF-β1 treatment. In addition, anti-TGF-β1 antibodies could aggregate the cytotoxicity of dNK cells and the levels of molecules above. These results indicated that TGF-β1 treatment could improve the abnormal pregnancy outcomes with T. gondii infection by decreasing the cytotoxicity of dNK cells mediated by NKG2D/DAP10 pathway and killer subset. These results suggested that TGF-β1 might be a potential immunoprotective method for the treatment of abnormal pregnancy outcomes following T. gondii infection.

During gestation, Toxoplasma gondii infection produces a series of complications including stillbirths, abortions, and congenital malformations. The inhibitory receptor, LILRB4, which is mainly expressed by professional antigen-presenting cells (especially macrophages and dendritic cells) may play an important immune-regulatory role at the maternal-fetal interface. To assess the role of LILRB4 during T. gondii infection, LILRB4-/- and T. gondii infected pregnant mouse models were established. Further, human primary-decidual macrophages were treated with anti-LILRB4 neutralizing antibody and then infected with T. gondii. These in vivo and in vitro models were used to explore the role of LILRB4 in T. gondii-mediated abnormal pregnancy outcomes. The results showed that abnormal pregnancy outcomes were more prevalent in LILRB4-/- infected pregnant mice than in wild-type infected pregnant mice. In subsequent experiments, expression levels of LILRB4, M1, and M2 membrane-functional molecules, arginine metabolic enzymes, and related cytokines were assessed in uninfected, infected, LILRB4-neutralized infected, and LILRB4-/- infected models. The results demonstrated T. gondii infection to downregulate LILRB4 on decidual macrophages, which strengthened M1 activation functions and weakened M2 tolerance functions by changing M1 and M2 membrane molecule expression, synthesis of arginine metabolic enzymes, and cytokine secretion profiles. These changes contributed to abnormal pregnancy outcomes. The results of this study provide not only a deeper understanding of the immune mechanisms operational during abnormal pregnancy, induced by T. gondii infection, but also identify potential avenues for therapeutic and preventive treatment of congenital toxoplasmosis.

This study aims to investigate whether IL-10 regulate decidual Treg cells apoptosis to reverse the abnormal pregnancy outcomes with Toxoplasma gondii (T. gondii) infection. Recombinant mouse IL-10 (rIL-10) treatment and IL-10 deficiency (IL-10(-/-)) abnormal pregnancy animal models with T. gondii infection were established. Apoptosis related molecules cleaved Caspase-3 and Caspase-8 in decidual Treg cells were examined using flow cytometry. The levels of cleaved Caspase-3 and Caspase-8 in decidual Treg cells were up-regulated with T. gondii infection. Compared to infected group, the expressions of cleaved Caspase-3 and Caspase-8 in decidual Treg cells were down-regulated in rIL-10-treated group, while up-regulated in infected IL-10(-/-) group. In addition, pregnant outcomes were improved in rIL-10-treated group, while worse in IL-10(-/-) group compared to infected group. These findings revealed that IL-10 reduced the decidual Treg cells apoptosis contributing to improving adverse pregnant outcomes following T. gondii infection.

Myeloid-derived suppressor cells (MDSCs) play a key role in maintaining maternal-fetal tolerance for a successful pregnancy, but the role of MDSCs in abnormal pregnancy caused by Toxoplasma gondii infection is unknown. Herein, we revealed a distinct mechanism by which T-cell immunoglobulin domain and mucin domain containing protein-3 (Tim-3), an immune checkpoint receptor that balances maternal-fetal tolerance during pregnancy, contributes to the immunosuppressive function of MDSCs during T. gondii infection. The expression of Tim-3 in decidual MDSCs was significantly downregulated following T. gondii infection. The proportion of monocytic MDSCs population, the inhibitory effect of MDSCs on T-cell proliferation, the levels of STAT3 phosphorylation, and the expression of functional molecules (Arg-1 and IL-10) in MDSCs were all decreased in T. gondii-infected pregnant Tim-3 gene knockout (Tim-3KO) mice compared with infected pregnant WT mice. After treatment with Tim-3-neutralizing Ab in vitro, the expression levels of Arg-1, IL-10, C/EBPβ, and p-STAT3 were decreased, the interaction between Fyn and Tim-3 or between Fyn and STAT3 was weakened, and the binding ability of C/EBPβ to the promoters of ARG1 and IL10 was decreased in human decidual MDSCs with T. gondii infection, while opposite results were observed following treatment with galectin-9 (a ligand for Tim-3). Inhibitors of Fyn and STAT3 also downregulated the expression of Arg-1 and IL-10 in decidual MDSCs and exacerbated adverse pregnancy outcomes caused by T. gondii infection in mice. Therefore, our studies discovered that the decrease of Tim-3 after T. gondii infection could downregulate the functional molecules of Arg-1 and IL-10 expression in decidual MDSCs through the Fyn-STAT3-C/EBPβ signaling pathway and weaken their immunosuppressive function, which eventually contribute to the development of adverse pregnancy outcomes.