Ultrasound-switchable fluorescence thermometry with dual detection channels using temperature-sensitive liposomes.

Temperature measurements in biological tissues play a crucial role in studying metabolic activities. In this study, we introduce a noninvasive thermometry technique based on two-color ultrasound-switchable fluorescence (USF). This innovative method allows for a local temperature mapping within a microtube filled with temperature-sensitive liposomes as nano imaging agents. By measuring the temperature-dependent fluorescence emission of the liposomes using a spectrometer, we identify four characteristic temperatures. The local background temperature can be estimated by analyzing the corresponding appearance time of these four characteristic temperatures in the dynamic USF signals captured by a camera-based USF system with two detection channels. Simultaneous measurements with an infrared (IR) camera showed a 0.38°C ± 0.27°C difference between USF thermometry and IR thermography in a physiological temperature range of 36.48°C-40.14°C. This shows that the two-color USF thermometry technique is a reliable, noninvasive tool with excellent spatial and thermal resolution.

In vivo tumor ultrasound-switchable fluorescence imaging via intravenous injections of size-controlled thermosensitive nanoparticles.

Near-infrared fluorescence imaging has emerged as a noninvasive, inexpensive, and ionizing-radiation-free monitoring tool for assessing tumor growth and treatment efficacy. In particular, ultrasound switchable fluorescence (USF) imaging has been explored with improved imaging sensitivity and spatial resolution in centimeter-deep tissues. This study achieved size control of polymer-based and indocyanine green (ICG) encapsulated USF contrast agents, capable of accumulating at the tumor after intravenous injections. These nanoprobes varied in size from 58 nm to 321 nm. The bioimaging profiles demonstrated that the proposed nanoparticles can efficiently eliminate the background light from normal tissue and show a tumor-specific fluorescence enhancement in the BxPC-3 tumor-bearing mice models possibly via the enhanced permeability and retention effect. In vivo tumor USF imaging further proved that these nanoprobes can effectively be switched 'ON' with enhanced fluorescence in response to a focused ultrasound stimulation in the tumor microenvironment, contributing to the high-resolution USF images. Therefore, our findings suggest that ICG-encapsulated nanoparticles are good candidates for USF imaging of tumors in living animals, indicating their great potential in optical tumor imaging in deep tissue.

Noninvasive measurement of local temperature using ultrasound-switchable fluorescence.

Measuring the local background temperature in diseased and inflamed tissues is highly desirable, especially in a non-invasive way. In this work, ultrasound-switchable fluorescence (USF) technique was utilized to estimate the local background temperature for the first time by analyzing the temperature dependence of fluorescence emission from USF contrast agents induced by a focused ultrasound (FU) beam. First, temperature-sensitive USF agents with distinct temperature switching-on thresholds were synthesized, and their thermal switching characteristics were quantified using an independent spectrometer system. Second, the USF contrast agent suspension was injected into a microtube that was embedded into a phantom and the dynamic USF signal was acquired using a camera-based USF system. The differential profile of the measured dynamic USF signal was computed and compared with the thermal switching characteristics. This allowed for the calculation of the local background temperature of the sample in the FU focal volume based on the estimation of heating speed. An infrared (IR) camera was used to acquire the surface temperature of the sample and further compare it with the USF system. The results showed that the difference between the temperatures acquired from the USF thermometry and the IR thermography was 0.64 ± 0.43 °C when operating at the physiological temperature range from 35.27 to 39.31 °C. These results indicated the potential use of the USF system for measuring the local temperature in diseased tissues non-invasively. The designed USF-based thermometry shows a broad application prospect in high spatial resolution temperature imaging with a tunable measurement range in deep tissue.

Effect of B7-H4 downregulation induced by Toxoplasma gondii infection on dysfunction of decidual macrophages contributes to adverse pregnancy outcomes.

BACKGROUND: 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. METHODS: Changes in B7-H4 expression on dMφ after T. gondii infection were explored both in vivo and in vitro. B7-H4-/- pregnant mice (pregnant mice with B7-H4 gene knockout) and purified primary human dMφ treated with B7-H4 neutralizing antibody were used to explore the role of B7-H4 signaling on regulating the membrane molecules, synthesis of arginine metabolic enzymes and cytokine production by dMφ with T. gondii infection. Also, adoptive transfer of dMφ from wild-type (WT) pregnant mice or B7-H4-/- pregnant mice to infected B7-H4-/- pregnant mice was used to examine the effect of B7-H4 on adverse pregnancy outcomes induced by T. gondii infection. RESULTS: The results illustrated that B7-H4-/- pregnant mice infected by T. gondii had poorer pregnancy outcomes than their wild-type counterparts. The expression of B7-H4 on dMφ significantly decreased after T. gondii infection, which resulted in the polarization of dMφ from the M2 toward the M1 phenotype by changing the expression of membrane molecules (CD80, CD86, CD163, CD206), synthesis of arginine metabolic enzymes (Arg-1, iNOS) and production of cytokines (IL-10, TNF-α) production. Also, we found that the B7-H4 downregulation after T. gondii infection increased iNOS and TNF-α expression mediated through the JAK2/STAT1 signaling pathway. In addition, adoptive transfer of dMφ from a WT pregnant mouse donor rather than from a B7-H4-/- pregnant mouse donor was able to improve adverse pregnancy outcomes induced by T. gondii infection. CONCLUSIONS: The results demonstrated that the downregulation of B7-H4 induced by T. gondii infection led to the dysfunction of decidual macrophages and contributed to abnormal pregnancy outcomes. Moreover, adoptive transfer of B7-H4+ dMφ could improve adverse pregnancy outcomes induced by T. gondii infection.

Size effect of liposomes on centimeter-deep ultrasound-switchable fluorescence imaging and ultrasound-controlled release.

Liposomes have been widely used in both medical imaging and drug delivery fields due to their excellent biocompatibility and easy surface modification. Recently our lab reported for the first-time the implementation of temperature-sensitive and indocyanine green (ICG)-encapsulated liposome microparticles for in vivo ultrasound-switchable fluorescence (USF) imaging. A previous study showed that liposome microparticles achieved USF imaging in centimeter-deep tissue. This study aimed to control the size of liposomes at the nanoscale and study the size effect on the USF imaging depth. Also, we explored the feasibility of combining USF imaging with ultrasound-controlled release. Liposomes were synthesized via the hydration method and the size was controlled by an extruding process. Characterization parameters, including fluorescence profile, spectra, size, stability, encapsulation efficiency, and ultrasound-controlled release, were evaluated. USF imaging in blood serum was conducted successfully in a phantom model, and an imaging depth study was conducted at 1.0 cm and 2.5 cm and confirmed that nano-sized liposomes had a stronger USF signal than micron-sized liposomes. Additionally, releasing tests indicated that both ultrasound power and exposure time affected the release efficiency in that increasing the power and extending the exposure time led to higher release efficiency. Above all, this study shows the potential for using liposomes for USF imaging and ultrasound-controlled release.

B7-H4 reduction induced by Toxoplasma gondii infection results in dysfunction of decidual dendritic cells by regulating the JAK2/STAT3 pathway.

BACKGROUND: 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. METHODS: We established T. gondii-infected abnormal pregnancy model in wild-type (WT) and B7-H4 knockout (B7-H4-/-) pregnant mice in vivo and cultured primary human dDCs in vitro. The abnormal pregnancy outcomes were observed and the expression of B7-H4, functional molecules (CD80, CD86, and MHC-II or HLA-DR), indoleamine 2,3-dioxygenase (IDO), cytokines (IL-10 and IL-12), and signaling molecules JAK2/STAT3 in dDCs was detected by flow cytometry and Western blot. RESULTS: Our results showed that T. gondii infection significantly decreased B7-H4 expression in dDCs. In addition, B7-H4-/- infected pregnant mice showed much more severe abnormal pregnancy outcomes than their counterparts. Importantly, B7-H4-/- infection further regulated the expression of molecules (CD80, CD86, and MHC-II or HLA-DR), enzyme IDO, and cytokines (IL-10 and IL-12) in dDCs. We further discovered that B7-H4-/- infection impairs the JAK2/STAT3 pathway, contributing to dDC dysfunction. CONCLUSIONS: Taken together, the results show that reduction of B7-H4 by T. gondii infection significantly modulates the decrease in cytokine IL-10 and enzyme IDO and the increase in cytokine IL-12, contributing to dDC dysfunction. Moreover, the JAK2/STAT3 pathway is involved in the regulation of B7-H4 by T. gondii infection and in the subsequent IDO and cytokine production, which ultimately contributes to abnormal pregnancy outcomes.

Magnetic carbon nanotubes-molecularly imprinted polymer coupled with HPLC for selective enrichment and determination of ferulic acid in traditional Chinese medicine and biological samples.

A molecularly imprinted polymer (MIP) with magnetic carbon nanotubes (MCNTs) as carrier was synthesized and used for the enrichment and determination of ferulic acid (FA) by high-performance liquid chromatography (HPLC). The morphology and structure of the FA magnetic carbon nanotubes-molecularly imprinted polymers (MCNTs@FA-MIPs) were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, and X-ray diffraction. The results demonstrated that the prepared MCNTs@FA-MIPs had excellent magnetic properties and uniform appearance. The adsorption properties of the novel MIP were studied by kinetic, and isothermal adsorption experiments. The results showed that the MCNTs@FA-MIPs exhibited relatively good uptake kinetics (equilibrium time, 2 h), high adsorption capacity (50 mg⋅g-1), fast separation, and good selectivity for the template molecule with a separation factor α of 1.73. The MCNTs@FA-MIPs combined with HPLC were successfully applied to the separation, enrichment, and determination of FA in a Ligusticum chuanxiong extract and in plasma of rats which had been administered with Taitai beauty essence. The recoveries for FA were 95.53-100.03 % with relative standard deviations (RSDs) less than 5.5%. The results confirmed that the proposed MCNTs@FA-MIPs offered efficient extraction of FA from traditional Chinese medicinal preparations and blood samples and with high specificity.

The Role of Tim-3 on dNK Cells Dysfunction During Abnormal Pregnancy With Toxoplasma gondii Infection.

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.

Improving sensitivity and imaging depth of ultrasound-switchable fluorescence via an EMCCD-gain-controlled system and a liposome-based contrast agent.

BACKGROUND: The ultrasound-switchable fluorescence (USF) technique was recently developed to achieve high-resolution fluorescence imaging in centimeters-deep tissue. This study introduced strategies to significantly improve imaging sensitivity and depth using an electron multiplying charge-coupled device (EMCCD) camera-based USF imaging system and a newly developed USF contrast agent of indocyanine green (ICG)-encapsulated liposomes. For a quantitative study, a phantom of a sub-millimeter silicone tube embedded in centimeter-thick chicken breast tissue was adopted in this study as a model. METHODS: The synthesized ICG-liposome was characterized and compared with the previously reported ICG-nanogel. The exposure of the EMCCD camera was controlled via the MATLAB (The MathWorks, Inc. USA), instead of an external hardware trigger. The stability of the electron multiplying (EM) gain of the EMCCD camera was compared between two trigger modes: the MATLAB trigger mode and the external hardware trigger mode. The signal-to-noise ratio (SNR) of the USF imaging with different EM gain in various thick tissue was studied. RESULTS: The hydrodynamic size of the ICG-liposome was ~181 nm. The ICG-liposome had a sharper temperature switching curve and a better USF performance than the previously reported ICG-nanogel. The EM gain was more stable in MATLAB trigger mode than the external hardware trigger mode. Although, as usual, the SNR decreased quickly with the increase of the tissue thickness, the proposed strategies improved the SNR and the imaging depth significantly by adopting the novel contrast agent and controlling the EM gain. CONCLUSIONS: We successfully imaged the sub-millimeter silicone tube with an inner diameter of 0.76 mm and an outer diameter of 1.65 mm in 5.5 cm-thick chicken breast tissue using 808 nm excitation light with a low intensity of 28.35 mW/cm2, the improved EMCCD camera-based USF imaging system and the novel ICG-liposomes.

The Role of Decidual PD-1+ Treg Cells in Adverse Pregnancy Outcomes due to Toxoplasma gondii Infection.

Toxoplasma gondii infection during pregnancy can result in adverse pregnancy outcomes. Previously, we have reported that these outcomes are associated with the impaired function of decidual Treg cells; however, the detailed mechanisms involved were unclear. It has been reported that the suppressive capacity of Treg cells is dependent on PD-1 expression. The present study explored the role of decidual PD-1+ Treg cell function in adverse pregnancy outcomes due to T. gondii infection. Toxoplasma gondii-infected pregnant mice were sacrificed on gestational day 14 and their pregnancy outcomes were observed. The expression of PD-1 on decidual Treg cells and expressions of Foxp3, CTLA-4, TGF-ß, and IL-10 on decidual PD-1+ and PD-1- Treg cells were determined using flow cytometry. The results showed that the expression of PD-1 on decidual Treg cells was clearly higher in the T. gondii-infected mice than in the normal mice. Meanwhile, the expressions of Foxp3, CTLA-4, TGF-ß, and IL-10 on decidual PD-1+ Treg cells were higher in the infected mice than in the normal mice. The expressions were higher in decidual PD1+ Treg cells than in PD-1- Treg cells in the infected mice. However, these expressions on PD-1- Treg cells did not significantly differ between the infected and normal mice. Nonetheless, the absolute percentages of decidual PD-1+ Treg cells decreased significantly in the infected mice compared with those in the normal mice. These results suggest that T. gondii infection mainly influences the function of decidual PD-1+ Treg cells, which would result in an insufficiently immunotolerant microenvironment and consequently in adverse pregnancy outcomes.

Role of Tim-3 in Decidual Macrophage Functional Polarization During Abnormal Pregnancy With Toxoplasma gondii Infection.

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/EBPß 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.

Decidual macrophage M1 polarization contributes to adverse pregnancy induced by Toxoplasma gondii PRU strain infection.

Recent evidence indicates that macrophages at the maternal-fetal interface adapt to a phenotype characterized by alternative activation (M2 polarization) and exhibit immunosuppressive functions that favor the maintenance of pregnancy. The bias of M2 decidual macrophages toward M1 has been clinically linked to pregnancy-related complications, such as preeclampsia and preterm delivery. The aim of this study was to investigate the effect of Toxoplasma gondii PRU strain infection on the bias of decidual macrophage polarization and its contribution to adverse pregnancy outcomes. A mouse model with adverse pregnancy outcome was established by infection with T. gondii PRU strain and the expression levels of functional molecules in decidual macrophages of mice were measured. The results showed that T. gondii infection caused seriously adverse pregnancy outcome in mice. The placentae of infected mice showed obvious congestion and inflammatory cell infiltration. The expression of CD206, MHC-II, and arginase-1 considered as M2 markers was decreased in decidual macrophages after T. gondii infection, whereas the expression of CD80, CD86, iNOS, and cytokines TNF-α and IL-12 considered as M1 markers was increased. Furthermore, iNOS-positive expression was observed in the decidua basalis of infected mice. Our results indicated that T. gondii infection was responsible for the bias of M2 decidual macrophages toward M1, which changes the immunosuppressive microenvironment at the maternal-fetal interface and contributes to adverse pregnancy outcomes.

Proteomic profiling of human decidual immune proteins during Toxoplasma gondii infection.

A Toxoplasma gondii infection during pregnancy can result in spontaneous abortion, preterm labor, or congenital fetal defects. The decidual immune system plays a critical role in regulating the immune micro-environment and in the induction of immune tolerance. To better understand the factors that mediate the decidual immune response associated with the T. gondii infection, a large-scale study employing TMT proteomics was conducted to characterize the differential decidual immune proteomes from infected and uninfected human decidual immune cells samples. The decidual immune cells from 105 human voluntary abortion tissues were purified, and of the 5510 unique proteins identified, 181 proteins were found to be differentially abundant (>1.2-fold cutoff, p < 0.05) in the T. gondii-infected decidual immune cells. 11 proteins of 181 differentially expressed proteins associated with trophoblast invasion, placental development, intrauterine fetal growth, and immune tolerance were verified using a quantitative real-time polymerase chain reaction and western blotting. This systematic analysis for the proteomics of decidual immune cells identified a broad range of immune factors in human decidual immune cells, shedding a new insight into the decidual immune molecular mechanism for abnormal pregnancy outcomes associated with T. gondii infection.

Inhibition of autophagy enhanced cobalt chloride­induced apoptosis in rat alveolar type II epithelial cells.

Hypoxia is a type of cellular stress that may result in apoptosis and autophagy. The molecular mechanisms underlying the association between autophagy and apoptosis remain unclear, particularly in hypoxic conditions. Transmission electron microscope, AO­PI staining, flow cytometry and western blot were used to examine the crosstalk between autophagy and apoptosis in hypoxic conditions. Rat alveolar type II epithelial RLE­6TN cells were cultured in a long­term hypoxic environment established by cobalt (II) chloride. It was demonstrated that autophagy and apoptosis occurred in RLE­6TN cells under hypoxic conditions. Treatment of RLE­6TN cells with the autophagy inhibitor 3­methyladenine increased the generation of reactive oxygen species, mitochondrial damage and hypoxia­induced apoptosis. The expression of caspases, particularly caspase­9, increased and may have participated in these processes. The data indicated that the inhibition of autophagy enhanced apoptosis through the mitochondria­mediated intrinsic pathway. These findings provide important insight into the molecular mechanism of autophagy and apoptosis crosstalk. This may provide new insights into pulmonary disease surveillance, diagnosis and treatment.

LILRB4 Decrease on uDCs Exacerbate Abnormal Pregnancy Outcomes Following Toxoplasma 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.

MiR-541-5p regulates lung fibrosis by targeting cyclic nucleotide phosphodiesterase 1A.

AIM OF THE STUDY: Idiopathic pulmonary fibrosis (IPF) is a lethal human disease with short survival time and few treatment options. In this study, we aim to demonstrate that cyclic nucleotide phosphodiesterase 1A (PDE1A), a Ca2+/calmodulin-stimulating PDE family member, plays a critical role in the induction of fibrosis and angiogenesis in the lung. MATERIALS AND METHODS: To induce pulmonary damage, adult male SD rats were treated with bleomycin in a dose of 6 mg/kg body weight by a single intratracheal instillation. For in vivo silencing of PDE1A in rat lung, a nonspecific control siRNA or PDE1A-specific siRNA was used to treat rat through nasal instillation. Human normal pulmonary fibroblasts MRC-5 and hFL1 and rat lung fibroblasts were used as in vitro model. Immunohistochemistry and immunoflurescence staining were performed to detect PDE1A and α-SMA expression. Reverse transcription-qPCR was performed to detect microRNA and mRNA expression. In vitro wound healing assay was performed to detect pulmonary fibroblasts'mortality ability. RESULTS: In vitro studies showed that PDE1A can stimulate lung fibroblasts to undergo myofibroblastic changes. This led to the identification of miR-541-5p as one of the miRNA candidates associated with bleomycin response. We found that miR-541-5p expression is downregulated in TGF-ß-treated lung fibroblasts and the rat pulmonary fibrosis model. Overexpression of miR-541-5p in lung fibroblasts inhibited mortality of human lung fibroblasts. CONCLUSIONS: MiR-541-5p is a key effector in lung fibroblastsby by regulating PDE1A expression at protein translation level and its overexpression is protective against bleomycin-induced lung fibrosis.

The Effect of TGF-ß on Treg Cells in Adverse Pregnancy Outcome upon Toxoplasma 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-ß level, which may contribute to adverse pregnancy outcomes. TGF-ß plays a critical role in Treg cell development, but whether TGF-ß 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-ß or TGF-ß-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-ß 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-ß 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-ß treatment, but downregulated by TGF-ß neutralization. The ratio of IL-10/TNF-α also increased after TGF-ß treatment, but decreased after TGF-ß neutralization. Our data indicate that TGF-ß treatment could improve adverse pregnancy outcomes caused by T. gondii infection by upregulating Treg cell differentiation and function via the TGF-ß/Smad3 signaling pathway, but not the proliferation of Treg cells.