The effect of Toxoplasma gondii infection on Galectin-9 expression in decidual macrophages contributing to dysfunction of decidual NK cells during pregnancy

doi:10.21203/rs.3.rs-4436558/v1

Download PDF

Research Article

The effect of Toxoplasma gondii infection on Galectin-9 expression in decidual macrophages contributing to dysfunction of decidual NK cells during pregnancy

https://doi.org/10.21203/rs.3.rs-4436558/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Background: Toxoplasma gondii infection causes adverse pregnancy outcomes by affecting the expression of immunotolerant molecules in decidual immune cells. Galectin-9 (Gal-9) is widely expressed in decidual macrophages (dMφ) and is crucial for maintaining normal pregnancy by interacting with the immunomodulatory protein Tim-3. However, the effects of T. gondii infection on Gal-9 expression levels in dMφ during pregnancy, as well as the impact of altered Gal-9 expression levels on the maternal-fetal tolerance function of dNK cells, are still unknown.

Methods: Pregnancy outcomes of T. gondii infected C57BL/6 and Lgals9^-/- pregnant mice models were recorded. The pathological change was evaluated by HE staining. The expression of Gal-9 in T. gondii infected dMφ were examined using flow cytometry and immunofluorescence. Expressions of Gal-9, JNK, p-JNK, FOXO1 were detected by Western Blot. The binding of FOXO1 to the promoter of Lgals9 was determined by ChIP-PCR. Supernatants from control or infected dMφ was co-cultured with purified human dNK cells with or without recombinant human Gal-9 protein (rhGal-9) or neutralized antibody of Tim-3, the expression of ERK, p-ERK, CREB, p-CREB, T-bet, IL-10 and IFN-γ were assayed by Western Blot.

Results: T. gondii infection promoted JNK phosphorylation and increased FOXO1 expression in dMφ, resulting in reduction of Gal-9 due to the elevated binding of FOXO1 with Lgals9 promoter. Downregulation of Gal-9 enhanced the phosphorylation of ERK and inhibited phosphorylation of CREB and expression of IL-10, and promoted the expression of T-bet and IFN-γ. In mice model, knockout of Gal-9 aggravated adverse pregnancy outcomes caused by T. gondii infection during pregnancy.

Conclusions: T. gondii infection suppressed Gal-9 expression in dMφ by activating the JNK/FOXO1 signaling pathway, and reduction of Gal-9 resulted in dysfunction of dNK via Gal-9/Tim-3 interaction. This study provided new insights for the molecular mechanisms of the adverse pregnancy outcomes caused by T. gondii.

Toxoplasma gondii

Gal-9

dMφ

dNK

adverse pregnancy outcomes

Toxoplasma gondii, an intracellular parasite, is the causative pathogen of toxoplasmosis which is a widespread zoonotic disease [1, 2]. T. gondii is an opportunistic pathogenic parasite, especially in immunocompromised individuals. When pregnant women are infected with T. gondii, vertical transmission of this parasite results in congenital toxoplasmosis in newborns including congenital defects, damage to nerves, eyes or other organs due to disruption of maternal-fetal tolerance balance [3]. The maternal-fetal interface comprises the maternal derived decidua and the fetal derived placenta [4]. Various immune cells are resident at the maternal-fetal interface and important for the maintenance of normal pregnancy [5]. It is reported that decidual immune cells in early human pregnancy are predominantly consisted of natural killer (NK) cells (approximately 70%) and macrophages (approximately 20%), with variable proportions of T-cells (approximately 10–20%) and scarce presence of dendritic cells (DC), B cells and natural killer T (NKT) cells [6, 7]. These decidual immune cells, along with their cytokines, play important roles in maintaining normal pregnancy outcomes. Decidual macrophages (dMφ), as one of the main decidual immune cells, contribute to maternal fetal tolerance maintenance, trophoblast invasion and spiral artery remodeling during early pregnancy [8–10]. The gene expression profiling of dMφ tends to be M2 phenotype during early pregnancy [11]. In humans, the dMφ expresses CD163, CD209, CD206 and DC-SIGN on their surfaces and predominantly secretes IL-10 [12]. DMφ also produces other immune tolerance factor e.g indoleamine 2,3-dioxygenase (IDO) to sustain its biological function [13].

Galectin-9 (Gal-9) is involved in the modulation of the endometrial immune system, which is an essential member of the galectin family encoded by the Lgals9 gene and expressed in the nucleus, cell membrane and cytoplasm of cells [14, 15]. It is expressed by multiple cells at the maternal-fetal interface including trophoblasts, stromal cells and dMφ. The biological functions of Gal-9 are related to immunomodulation, the reproductive function maintenance and the facilitation of pregnancy establishment [16]. It is reported that, compared with non-pregnant women, plasma Gal-9 concentration is significantly elevated in normal pregnant women with the trophoblast cells of full-term placental tissues expressing the highest Gal-9 protein levels [17]. However, Gal-9 expression levels in trophoblast cells of villous tissue as well as in plasma are reduced in patients with miscarriage [18]. These findings highlight Gal-9’s crucial role in maintaining maternal-fetal immune tolerance during normal pregnancy. Due to T. gondii infection causing abortion by inhibiting the expression of immune negative molecules such as Tim-3, LILRB4, B7-H4 on various decidual immune cells [19–21], the impact of T. gondii infection on Gal-9 expression in dMφ is researched in this study.

Forkhead box transcription factor O1 (FOXO1) has multiple biological functions. It is reported that FOXO1 might repress the transcription of Lgals9 by binding to the promoter [22]. And the bioactivity of FOXO1 could be manipulated by JNK [23]. Moreover, JNK signalling pathway could be efficiently activated during T. gondii infection [24]. Hence, it is required to explore whether T. gondii infection alters Gal-9 expression in dMφ through the JNK/FOXO1 signalling pathway.

At the maternal-fetal interface, dNK cells represent the predominant leukocyte population, characterised by surface markers CD56⁺ and CD16⁻ in humans, and CD122⁺ and CD3⁻ in mice [25]. A significant portion of dNK cells express T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) which is a receptor of Gal-9 and regulates dNK cell function at the maternal-fetal interface in early pregnancy [26]. However, whether Gal-9 from dMφ binding with Tim-3 to influence dNK cell function during T. gondii infection remains unclear and need to be elucidated. And this research reveals a reduction of Gal-9 expression in dMφ following T. gondii infection, mediated by the activation of the JNK/FOXO1 signalling pathway. Diminished Gal-9 level in dMφ results in elevation of ERK phosphorylation by binding to Tim-3, which further inhibits IL-10 expression and increases IFN-γ expression in dNK cells. This study aims to elucidate the underlying molecular mechanisms that T. gondii infection caused dysfunction of dNK cells due to Gal-9 downregulation in dMφ, which offers novel insights into the immune molecular pathways contributing to adverse pregnancy outcomes associated with T. gondii infection.

Toxoplasma gondii RH strain

T. gondii RH tachyzoites were propagated and maintained in HFF cells cultured in DMEM medium supplemented with 10% FBS according to the previous study. After co-culturing HFF cells with Toxoplasma tachyzoites for 48 hours, the cultures were collected. The cells were then removed by centrifugation at 5000 g for 5 minutes, and the tachyzoites remaining in the supernatant were purified by centrifugation at 4000 g for 7 minutes.

Mice

6 to 8-week-old C57BL/6 wild-type mice were purchased from Ji’nan Pengyue Laboratory Animal Breeding Co., Ltd. (Jinan, China). Lgals9-knockout (Lgals9^−/−) C57BL/6 mice were obtained from GemPharmatech Co., Ltd. (Nanjing, China). All mice were housed in the specific pathogen-free animal facility of Binzhou Medical University under controlled conditions, with a temperature maintained at 23 ± 2°C and a 12/12-hour light/dark cycle. The experimental protocols were approved by the Animal Ethics Committee of Binzhou Medical University.

Mouse experiments

For the T. gondii infected pregnant mouse model, 8-week-old female C57BL/6 WT mice and Lgals9^−/− mice were mated with corresponding male mice at a ratio of 2:1. The appearance of the copulatory plug was designated as day 0 of gestation. In the infected group, pregnant mice were intraperitoneally infected with 300 T. gondii tachyzoites on gestation day (GD) 8. Pregnant mice in the control group were inoculated with PBS.

Cell preparation of mice

The uteri and placentas of the mice were carefully separated and extensively washed with cold PBS. Subsequently, the tissues were finely minced into small fragments and enzymatically digested using 0.1% collagenase type IV (Sigma-Aldrich, MO, USA) and 25 IU/ml DNase I (Sigma-Aldrich) for 1 hour at 37°C with agitation. Following digestion, the tissue pieces were filtered through a 48-µm sterile mesh to obtain a cell suspension. Mononuclear cells were isolated from the white film layer after Ficoll density gradient centrifugation performed in mouse lymphocyte separation medium (TBD Science, Tianjin, China). Finally, the collected cells were suspended in cold PBS and utilized for subsequent flow cytometry analysis.

Human sample collection

This study was approved by the Ethics Committee of Binzhou Medical University, Yantai, China and in accordance with the Declaration of Helsinki. Decidual tissues were collected from the placentas of healthy pregnant women who had undergone termination of pregnancy due to social reasons between six and eight weeks of gestation. The patients have all read and signed the informed consent form.

Isolation and purification of human dMφ and dNK

The decidual tissues were washed with cold PBS, cut into small fragments using ophthalmic scissors and digested with 0.1% collagenase IV (Sigma-Aldrich) and 25 IU/ml DNase I (Sigma-Aldrich) in an incubator at 37°C for 60 min. The resulting suspension was filtered through 48-µm mesh and washed twice in cold PBS. The decidual mononuclear cells were isolated and purified by Ficoll density gradient centrifugation. These cells were then employed for flow cytometry analysis. For further purification, the dMφ cells were isolated using a human CD14 positive isolation kit (Stemcell, Toronto, Canada) according to the manufacturer’s instructions. The dNK cells were purified using a human NK cell isolation kit (Stemcell) according to the manufacturer’s instructions. The purified dMφ cells were analyzed using Western blotting, immunofluorescence and chromatin immunoprecipitation (ChIP) -PCR methods. The purified dNK cells were used for subsequent co-culture experiments. The specific culture and treatment conditions of the purified dMφ and dNK were explained for each experiment.

Human decidual mononuclear cell culture and treatment

The mononuclear cells were evenly divided into two groups: the uninfected group and the infected group. T. gondii tachyzoites were added to the cells of the infected group at a ratio of 1:3 (T. gondii tachyzoites: cells). All cells were cultured in RPMI-1640 medium supplemented with 10% FBS (Gibco, MA, USA), 100 IU/ml streptomycin, and 100 IU/ml penicillin for 24 hours at 37 ℃ in a humidified 5% CO2 incubator. Subsequently, all cells were prepared for flow cytometry staining and analysis.

Flow cytometry staining and analysis

The prepared human and murine decidual mononuclear cell (1×10⁶ cells of each group) suspensions were first stained with antibodies against membrane molecules and then with antibody of intracellular molecule Gal-9, after the cells were treated with intracellular fixation & permeabilization buffer set according to the manufacturer’s instructions (eBioscience, CA, USA). The experiments were replicated at least 3 times. Data was analyzed using a BD FACSCanto™ TM II Flow Cytometer (BD Biosciences, NJ, USA) and FlowJo analysis software (Version 10.0, FlowJo LLC, OR, USA).

M2 macrophage differentiation of THP-1 cells

THP-1 cells were added to 100 mm cell culture dishes (8×10⁶ cells/dish) and supplemented with 50 nM phorbol 12-myristate 13-acetate (PMA) to induce differentiation into macrophages. After 24 hours, the supernatant was discarded, and fresh medium was added, supplemented with 0.1 µM medroxyprogesterone acetate (MPA). After 72 hours, the cells were polarized into M2 macrophages. Flow cytometry was used to detect the phenotype of M2 macrophages. Antibodies against CD14, CD206, CD209 were used for surface staining according to the manufacturer's instructions. The induced THP-1 cells were transfected with pcDNA3.1-FOXO1 using jetPRIME® reagent (Polyplus, Illkirch, France), followed by T. gondii infection, then the cells were harvest and used for detecting Gal-9 by western blot.

Western blot analysis

Purified human dMφ cells were infected with T. gondii tachyzoites at 1:3 ratio (T. gondii tachyzoites: cells) with or without 10 µM SP600125 (JNK inhibitor [JNKi]), 10 µg/ml AS1842856 (FOXO1 inhibitor [FOXO1i]), 10 µg FOXO1 overexpression plasmid. The supernatant of T. gondii infected dMφ cells was collected and added into the purified dNK cells, supplemented with 10 µg of recombinant human Gal-9 protein (rhGal-9), 10 µg/ml Tim-3 neutralizing antibody (α-Tim-3), 10 µM PD98059 (p-ERK inhibitor [p-ERKi]). The dMφ and dNK cells were washed with PBS and lysed using RIPA cell lysis buffer. The total protein concentration was determined using a BCA kit (Solarbio, Beijing, China). Subsequently, 30 µg of protein was separated by SDS-PAGE electrophoresis using a 12% Bis-Tris gel (Solarbio) and then transferred onto a PVDF membrane (Millipore, MA, USA). After blocking with 5% non-fat milk, the blots were probed with primary antibodies, including rabbit anti-Gal-9 (1: 2000, Proteintech, Wuhan, China), rabbit anti-JNK, rabbit anti-phosphorylated JNK (p-JNK, 1:2000, Proteintech), rabbit anti-FOXO1 (1:2000, Proteintech), rabbit anti-ERK (1:2000, Proteintech), rabbit anti-phosphorylated ERK (p-ERK, 1:2000, Proteintech), rabbit anti-CREB (1:2000, Proteintech), rabbit anti-phosphorylated CREB (p-CREB, 1:2000, Proteintech), rabbit anti-IL-10 (1:2000, Wanleibio, Shenyang, China), rabbit anti-T-bet (1:2000, Wanleibio), rabbit anti-IFN-γ (1:2000, Bioss, Beijing, China), and rabbit anti-GAPDH antibodies (1:40000, Proteintech). Subsequently, the PVDF membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000, Proteintech). The images were captured using the Bio-Rad ChemiDoc XRS + imaging system (BioRad, CA, USA).

Immunofluorescent analysis

The purified dMφ cells were cultured in 60 mm cell culture dishes, divided into an uninfected group and an RH strain infected group. In the RH strain infection group, T. gondii parasites were added at a ratio of 1:3. After 24 hours, all cells were fixed in 4% paraformaldehyde (PFA) at room temperature (RT) for 20 minutes, followed by cell permeabilization with 0.2% Triton X-100 for 10 minutes. After blocking with normal goat serum for 1 h at RT, the samples were then incubated with rabbit anti-Gal-9 antibody (Proteintech, 1:100 dilution) and rabbit anti-FOXO1 antibody (Proteintech, 1:100 dilution) overnight in a humid chamber at 4°C followed by Alexa Fluor 549- or 488- conjugated IgG incubation. After PBS washing, 4’,6-diamidino-2-phenylindole (DAPI, Sigma) was used to stain nuclei. Fluorescence signals were analyzed using an inverted fluorescence microscope (Olympus, Tokyo,,Janpan). For detecting expression of Gal-9 in mouse placental tissue, placenta were prepared into frozen sections and stained with rabbit anti-Gal-9 antibody and Alexa Fluor 488–conjugated IgG.

ChIP-PCR assay

ChIP assays were conducted using the Pierce Magnetic ChIP Kit (ThermoFisher, USA). The dMφ cells were fixed with 1% formaldehyde for 10 minutes at room temperature, with the fixation reaction quenched using glycine. Nuclei were isolated via cell lysis, followed by digestion with micrococcal nuclease and sonication to achieve a fragment length of approximately 150–900 bp. After purification, 5 µg of chromatin was utilized for each immunoprecipitation. For each immunoprecipitation, 5 µg of anti-FOXO1 antibody or control IgG was added to the digested, cross-linked chromatin. After eluting chromatin from antibody/protein G magnetic beads, the immunoprecipitated chromatin was purified and subjected to quantitative real-time PCR detection using specific primers. The primers used for ChIP-qPCR were synthesized by Sangon Biotech (Shanghai, China) and are listed in: Table S1.

Reagents and antibodies

The reagents and antibodies used in the study are listed in: Table S2.

Statistical analysis

Statistical analysis was conducted using GraphPad Prism 9.0 software (GraphPad Software, CA, USA). Data are presented as the mean ± standard error (SE). Unpaired and paired t-tests were utilized to identify differences. One-way analysis of variance (ANOVA) was applied with 95% confidence intervals. Significance was defined as p < 0.05.

The abnormal pregnancy outcomes were associated with Gal-9 in T. gondii infected mice

To investigate the impact of Gal-9 on the adverse pregnancy outcomes caused by T. gondii, both WT (wild-type) and Lgals9^−/− (gal-9 knockout) mice models infected with T. gondii were established, and the pregnancy outcomes were evaluated. For the T. gondii-infected pregnant mouse model, 8-week-old female C57BL/6 WT mice and Lgals9^−/− mice were mated with corresponding male mice at a ratio of 2:1. The appearance of the copulatory plug was designated as day 0 of gestation. In the infected group, pregnant mice were intraperitoneally infected with 300 T. gondii tachyzoites on gestation day (GD) 8. Pregnant mice in the control group were inoculated with PBS. Pregnancy outcomes were observed on GD 14. The experimental results indicated that adverse pregnancy outcomes in Lgals9^−/− mice were significantly more severe than those in WT mice under infection conditions (Fig. 1a). The placental weight of Lgals9^−/− mice was noticeably reduced compared to WT mice (Fig. 1b), along with a significant decrease in fetal weight (Fig. 1c). Lgals9^−/− mice exhibited a higher rate of fetal resorption (Fig. 1d). Hematoxylin and eosin (HE) staining revealed that, following T. gondii infection, Lgals9^−/− mice exhibited more pronounced disruption of placental tissue integrity, increased infiltration of inflammatory cells, and severe hemorrhaging compared to WT mice (Fig. 1e). Immunofluorescence was employed to detect the expression of Gal-9 in frozen sections of mouse placenta. The results revealed a significant decrease in Gal-9 expression in the placenta of the infected group compared with the uninfected group (Fig. 1f). These results indicated that Lgals9^−/− mice experience more severe pregnancy outcomes following T. gondii infection, suggesting that deficiency of Gal-9 exacerbates adverse pregnancy outcomes.

Gal-9 was decreased in dMφ after T. gondii infection

To explore the impact of T. gondii infection on Gal-9 expression in various immune cells, we isolated the human decidual immune cells at the maternal-fetal interface during early pregnancy. It was found that the expression of Gal-9 was significantly decreased in dMφ infected by T. gondii (Fig. 2a, b). Simultaneously, we utilized flow cytometry to assess the expression of Gal-9 in THP-1-derived M2-type macrophages (dMφ like THP-1). Firstly, phenotype of dMφ like THP-1 was assayed by staining CD206, CD209 through flow cytometry detection. The expression of CD206 and CD209 was high in dMφ like THP-1 cells as well as in human dMφ (Fig. 2c, d), which suggested that dMφ like THP-1 could be used for the mechanism research in the subsequent experiments. Then it was found that Gal-9 expression was also reduced in these dMφ like THP-1 cells after T. gondii infection (Fig. 2e, f). The western blot results also indicated that T. gondii infection caused a decrease in Gal-9 expression in dMφ (Fig. 2g). Additionally, in vivo, T. gondii infection also resulted in reduction of Gal-9 in dMφ of WT mice (Fig. 2h, i). These data collectively confirmed that T. gondii infection contributed to a significant decrease of Gal-9 expression within dMφ.

T. gondii infection attenuated Gal-9 expression in dMφ via the JNK/FOXO1 signaling pathway

Previous studies have reported FOXO1 acting as a transcriptional repressor to inhibit Gal-9 expression [22]. To elucidate the mechanism underlying the impact of T. gondii infection on Gal-9 expression, western blot analysis was performed to assess the expression levels of p-JNK and FOXO1 in dMφ infected with T. gondii. The results revealed an increase in both p-JNK and FOXO1 levels following T. gondii infection, accompanied by a decrease in Gal-9 expression (Fig. 3a, b). To investigate whether the expression level of Gal-9 in dMφ was regulated by JNK/FOXO1, during T. gondii infection in vitro, SP600125, a JNK inhibitor, was introduced to suppress phosphorylation of JNK in human dMφ. Additionally, a FOXO1 inhibitor, AS1842856 was used to inhibit FOXO1, while a FOXO1 recombined plasmid pcDNA3.1-FOXO1 was transfected into dMφ to upregulate FOXO1 expression levels in dMφ like THP-1. Then western blot assay was performed to analyze the expression of JNK, phosphorylated JNK (p-JNK), FOXO1 and Gal-9. The results suggested that SP600125 resulted in a decrease in the expression levels of p-JNK and FOXO1 in human dMφ or dMφ like THP-1, however, there was a significant increase observed in Gal-9 expression (Fig. 3c, d). Inhibition of FOXO1 by AS1842856 resulted in an increase in Gal-9 expression, whereas overexpression of FOXO1 caused a decrease in Gal-9 expression (Fig. 3e, f). To determine the mechanism by which FOXO1 regulates Gal-9 expression, ChIP-PCR assay was performed in T. gondii-infected dMφ cells. Primers targeting the Gal-9 promoter and anti-FOXO1 antibody were used for ChIP-PCR. The results demonstrated that FOXO1 could directly bind to the promoter region of Lgals9 ( -1185 to -1171 bp, -1009 to -1022 bp, -574 to -561 bp, -141 to -128 bp), and this binding ability was enhanced following T. gondii infection (Fig. 3g, h). Immunofluorescence analysis similarly demonstrated an upregulation of FOXO1 expression and a downregulation of Gal-9 expression in T. gondii-infected dMφ cells (Fig. 3i). These findings suggested that the expression levels of Gal-9 following T. gondii infection were regulated by the JNK/FOXO1 signaling pathway.

The reduction of Gal-9 derived from dMφ activated ERK phosphorylation via the Gal-9/Tim-3 interaction, consequently impacting the function of dNK cells

To elucidate the immune function of human dMφ under T. gondii infection conditions, we prepared a conditioned medium (CM) containing secreted products from dMφ. To assess the impact of Gal-9 derived from dMφ on maternal immunity, we treated dNK cells with CM from human dMφ infected with or without T. gondii. We observed that CM from T. gondii-infected dMφ (CM^Inf) significantly suppressed IL-10 expression in dNK cells while enhancing IFN-γ expression compared with CM from uninfected dMφ (CM^Uninf) (Fig. 4a, b). To validate whether the reduction of Gal-9 affects human dNK cell function, we added recombinant human Gal-9 (rhGal-9) protein into the CM^Inf. Following treatment with rhGal-9, there was an observed elevation in IL-10 expression and decrease in IFN-γ expression in dNK (Fig. 4a, b). It was also found that p-CREB and T-bet, downstream molecules of ERK signaling pathway, was respectively decreased and increased in dNK cells under CM^inf co-culturing. Conversely, addition of rhGal-9 could efficiently promote phosphorylation of CREB and repress the expression of T-bet (Fig. 4a, b). It is reported that p-CREB is a transcription factor of IL-10, and T-bet is a transcription factor of IFN-γ [27, 28].

Tim-3 has been reported to serve as a receptor for Gal-9, regulating immune cell functions. To validate whether Gal-9 secreted by dMφ affects function of dNK cells through interacting with Tim-3 on dNK cells, we utilized a Tim-3 neutralizing antibody (α-Tim-3) to disturb their binding. The results showed that there was an increase in p-ERK expression, a decrease in IL-10 expression along with its transcription factor p-CREB, and an elevation in expression of IFN-γ and T-bet which is a classical transcriptional factor of IFN-γ when α-Tim-3 was added into CM^inf (Fig. 4c, d). To further confirm whether the Gal-9/Tim-3 signaling pathway impacts dNK cell function by activating ERK phosphorylation, PD98059 (a p-ERK inhibitor) was employed to suppress phosphorylation of ERK. The results showed that inhibition of ERK phosphorylation could increase the expression of IL-10 and the phosphorylation of CREB, but decrease the expression of IFN-γ and T-bet (Fig. 4e, f). These results suggest that the reduction of Gal-9 in T. gondii-infected dMφ might impact the function of dNK cells through enhancing ERK phosphorylation via repressing the interaction of Gal-9 and Tim-3.

DMφ, characterised by M2-like macrophages, are integral components of decidual immune cells at the maternal-fetal interface, crucial for maintaining maternal-fetal tolerance and trophoblast invasion [29]. The removal of macrophages in pregnant rats results in abortions, highlighting their significance in sustaining normal pregnancies [16, 30]. Under normal pregnancy conditions, dMφ exhibits a high expression of immunosuppressive molecules such as IL-10, IDO, Tim-3 and LILRB4, contributing to their tolerogenic effect [31–33]. However, in pregnancy complications, such as pre-eclampsia (PE), premature birth and recurrent pregnancy loss (RPL), and miscarriages, dMφ displays an abnormal polarization with high expression of inflammatory factors [32, 34]. T. gondii, the pathogen associated with Torch Syndrome, contributes to adverse pregnancy outcomes including preterm labour, miscarriage, teratology or stillbirth. A previous study reported a reduction in IL-10, Tim-3 or LILRB4 expression in dMφ in mouse model of T. gondii-induced miscarriage [31, 33]. Nevertheless, Gal-9 is an important ligand of Tim-3, and there is no report about that T. gondii infection affects the expression level of the Gal-9 in dMφ.

Our investigation found that Gal-9 levels were significantly decreased in decidual tissue of mice following T. gondii infection, accompanied by serious adverse pregnancy outcomes in mice lacking Gal-9. Especially, T. gondii infection resulted in reduced placental and fetal mice weights in Lgals9 knock out (Lgals9^−/−) pregnant mice compared with wild-type pregnant mice. Flow cytometry analysis showed that lower Gal-9 expression in dMφ of T. gondii-infected pregnant mice was obtained, compared with uninfected pregnant mice. Similarly, in vitro, FCM result exhibited a significant reduction of Gal-9 expression in human primary dMφ or dMφ like THP-1 following T. gondii infection, while Gal-9 levels in other immune cells such as dNK and decidual myeloid-derived suppressor cells (MDSC) remained unaffected after infection (Data was not shown). Additionally, western blot assay also revealed that Gal-9 expression was significantly declined after infection in human dMφ. These results suggest that T. gondii infection caused reduced Gal-9 expression, indicating its important role in abnormal pregnancy outcomes induced by T. gondii infection.

However, the underlying molecular mechanisms of that T. gondii infection reduced Gal-9 expression remain unclear. Previous studies have indicated that the upstream transcriptionally activity of Lgals9 can be downregulated by FOXO1 [22]. Additionally, it is reported that T. gondii infection is involved in increasing phosphorylation of JNK, which induces an inflammatory response [24]. Moreover, JNK phosphorylation in turn induces the activity of FOXO1, which mediates multiple signaling pathways [35]. Our experiments demonstrated a significant elevation in phosphorylation of JNK and expression of FOXO1 in human dMφ following T. gondii infection in vitro, suggesting a potential relationship between T. gondii infection and increased p-JNK and FOXO1 levels. However, it need to be further verified whether T. gondii affecting expression of Gal-9 is through promoting JNK phosphorylation and activating FOXO1 in dMφ. To explore whether the decrease in Gal-9, which is caused by T. gondii infection in human dMφ, was regulated by affecting the activation of JNK/FOXO1 signal pathway, a JNK inhibitor, SP600125, was added to inhibit phosphorylation of JNK in the presence of T. gondii infection. The expression levels of FOXO1 and Gal-9 were detected using western blot assay in the infection group with or without JNK inhibitor. The results showed that JNK inhibitor decreased FOXO1 levels and increased Gal-9 levels under T. gondii infection, suggesting that in the presence of T. gondii, JNK phosphorylation affected the expression of FOXO1 and Gal-9 in dMφ. Furthermore, dMφ infected with T. gondii was respectively treated with FOXO1 inhibitor or transfected with a plasmid pcDNA3.1-FOXO1. The expression levels of Gal-9 in the infected group with or without FOXO1 inhibitor or pcDNA3.1-FOXO1 were detected using western blot assay, wherein the expression level of Gal-9 was increased in the infected dMφ treated with FOXO1 inhibitor and decreased in the infected dMφ with FOXO1 overexpression, suggesting that T. gondii infection inhibited the expression of Gal-9 through the activation of FOXO1. To examine whether T. gondii affects the binding of FOXO1 to Lgals9 promoter, we conducted ChIP-PCR using the dMφ like THP-1 cell line, which was divided into uninfected and infected groups. The results revealed that binding of FOXO1 to the upstream promoter of Lgals9 was enhanced post-infection, suggesting that FOXO1 could bind to the Gal-9 promoter and suppressed its transcriptional expression following T. gondii infection.

IL-10, crucial for maintaining normal maternal-fetal tolerance, is produced by dNK cells and dMφ in decidual immune cells [36, 37]. Our findings suggested that decreased Gal-9 expression in dMφ due to T. gondii infection further influenced the function of dNK cell via binding to Tim-3. This is evidenced by reduced IL-10 expression and increased IFN-γ expression in dNK cells. It is reported that Tim-3, a receptor for Gal-9, is downregulated after T. gondii infection, increasing IFN-γ expression through the Fyn/ERK signaling pathway, enhancing dNK cytotoxicity and disrupting the balance of maternal-fetal tolerance [38, 39]. Activation of Tim-3 by Gal-9 inhibits the ERK signaling pathway and promotes IL-10 production [40, 41]. To investigate whether T. gondii-caused reduction of Gal-9 in dMφ affected function of dNK cell by binding to Tim-3 and promoted ERK phosphorylation, we collected conditioned medium (CM) of uninfected or infected dMφ in vitro. Co-culture of infected (CM^Inf) and uninfected CM (CM^Uninf) with purified human dNK cells, along with the addition of rhGal-9 protein, Tim-3 neutralizing antibody or p-ERK inhibitor, revealed that phosphorylation of ERK was increased in co-cultured dNK cells treated with CM^Inf compared with CM^Uninf, accompanied by decrease of p-CREB and IL-10 and increase of T-bet and IFN-γ. P-CREB and T-bet is correspondingly classic transcription factors of IL-10 and IFN-γ [27, 28]. RhGal-9 protein treatment increased the expression of p-CREB, IL-10 and decreased the expression of T-bet, IFN-γ, which could be reversed by adding Tim3 neutralizing antibody, indicating that Gal-9 could affect the function of dNK by Tim-3. CM^Inf co-cultured dNK cells treated with rhGal-9 and p-ERK inhibitor could decrease expression of T-bet and IFN-γ, and increase expression of p-CREB and IL-10. These results suggested that the T. gondii-caused the reduction of Gal-9 in dMφ promoted ERK phosphorylation by binding to Tim-3 on dNK, which, in turn, induced an upregulation of IFN-γ and downregulation of IL-10, thereby affecting the function of dNK.

In summary, T. gondii infected dMφ promotes the phosphorylation of JNK and activates FOXO1, which binds to the upstream promoter of Lgals9, inhibiting the expression of Gal-9. Within the maternal-fetal microenvironment, interaction of decidual immune cells maintains maternal-fetal immune tolerance. The dNK cells predominant at the maternal-fetal interface. These dNK cells are impacted by decreased Gal-9 expression in dMφ. Reduced Gal-9 levels lead to the phosphorylation of ERK via binding with Tim-3 on dNK cells, resulting in elevated IFN-γ levels and decreased IL-10 expression, thereby affecting dNK cell function and disrupting maternal-fetal homeostasis, ultimately resulting in adverse pregnancies (Fig. 5). This study provided new insights for the molecular mechanisms of the adverse pregnancy outcomes caused by T. gondii.

dMφ

decidual Macrophage

dNK

decidual natural killer cell

Tim-3

T cell immunoglobulin domain and mucin domain-3

FBS

Foetal bovine serum

FOXO1

Forkhead box protein O1

JNK

c-Jun N-terminal kinase

ERK

extracellular regulated protein kinases

CREB

Cyclic AMP Response Element-binding Protein

T-bet

T-box brain protein 1

IL-10

Interleukin-10

IFN-γ

Interferon gamma.

Supplementary information

The online version contains supplementary material available at xxx.

Acknowledgements

We thank Aiqun Xu, Zhihua Lv and Jing Zheng for their help in collecting clinical specimens.

Ethics approval and consent to participate

This study was conducted in accordance with the Helsinki Declaration and its subsequent amendments, and received approval from the Ethics Committee of Binzhou Medical University (Approval No: 2016-029). The female participants, diagnosed with early pregnancy deemed normal by professional obstetricians and gynecologists, devoid of complications, medication, or a history of toxoplasmosis infection, opted voluntarily for pregnancy termination. All sample collections were conducted with participants' consent, and all participants signed informed consent forms.

Authors’ contributions

YSR, XMH designed the experiments and wrote the manuscript; SYW, XYX and YZJcontributed to sample collection; XW, YSR and SYW did experiments; XW, YSR and LQR analyzed the data; ZDL, HXZ and XBL revised the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by funds from the National Natural Science Foundation of China (NO.82201958), Natural Science Foundation of Shandong province (NO. ZR2020MH164). National Natural Science Foundation of China (NO. 81871680), Taishan Scholar Foundation of Shandong province (NO. ts201712066).

Availability of data and materials

All data generated in this study are presented within the published article.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Author details

¹Department of Immunology, Binzhou Medical University, Yantai, Shandong, P. R. China 264003. ²Department of Microbiology, Binzhou Medical University, Yantai, Shandong, P. R. China 264003.

Balasundaram P, Avulakunta ID. Human Growth and Development. StatPearls. 2024.
Norwitz ER, Schust DJ, Fisher SJ. Implantation and the survival of early pregnancy. N Engl J Med. 2001;345:1400–1408.
Kaye A. Toxoplasmosis: diagnosis, treatment, and prevention in congenitally exposed infants. J Pediatr Health Care. 2011;25:355–364.
Ander SE, Diamond MS, Coyne CB. Immune responses at the maternal-fetal interface. Sci Immunol. 2019;4:eaat6114.
Erlebacher A. Immunology of the maternal-fetal interface. Annu Rev Immunol. 2013;31:387–411.
Trundley A, Moffett A. Human uterine leukocytes and pregnancy. Tissue Antigens. 2004;63:1–12.
Bulmer JN, Williams PJ, Lash GE. Immune cells in the placental bed. Int J Dev Biol. 2010;54:281–294.
Ding J, Zhang Y, Cai X, Zhang Y, Yan S, Wang J, et al. Extracellular vesicles derived from M1 acrophages deliver miR-146a-5p and miR-146b-5p to suppress trophoblast migration and invasion by targeting TRAF6 in recurrent spontaneous abortion. Theranostics. 2021;11:5813–5830.
Renaud SJ, Graham CH. The role of macrophages in utero-placental interactions during normal and pathological pregnancy. Immunol Invest. 2008;37:535–564.
Smith SD, Dunk CE, Aplin JD, Harris LK, Jones RL. Evidence for immune cell involvement in decidual spiral arteriole remodeling in early human pregnancy. Am J Pathol. 2009;174:1959–1971.
Zhang Y, Ma L, Hu X, Ji J, Mor G, Liao A. The role of the PD-1/PD-L1 axis in macrophage differentiation and function during pregnancy. Hum Reprod. 2019;34:25–36.
Svensson J, Jenmalm MC, Matussek A, Geffers R, Berg G, Ernerudh J. Macrophages at the fetal-maternal interface express markers of alternative activation and are induced by M-CSF and IL-10. J Immunol. 2011;187:3671–3682.
Huang HL, Yang HL, Lai ZZ, Yang SL, Li MQ, Li DJ. Decidual IDO⁺ macrophage promotes the proliferation and restricts the apoptosis of trophoblasts. J Reprod Immunol. 2021;148:103364.
Lv Y, Ma X, Ma Y, Du Y, Feng J. A new emerging target in cancer immunotherapy: Galectin-9 (LGALS9). Genes Dis. 2022;10:2366–2382.
Popovici RM, Krause MS, Germeyer A, Strowitzki T, von Wolff M. Galectin-9: a new endometrial epithelial marker for the mid- and late-secretory and decidual phases in humans. J Clin Endocrinol Metab. 2005;90:6170–6176.
Greenbaum S, Averbukh I, Soon E, Rizzuto G, Baranski A, Greenwald N, et al. A spatially resolved timeline of the human maternal-fetal interface. Nature. 2023;619:595–605.
Enninga EAL, Harrington SM, Creedon DJ, Ruano R, Markovic SN, Dong H, et al. Immune checkpoint molecules soluble program death ligand 1 and galectin-9 are increased in pregnancy. Am J Reprod Immunol. 2018;79:e12795.
Wu M, Zhu Y, Zhao J, Ai H, Gong Q, Zhang J, et al. Soluble costimulatory molecule sTim3 regulates the differentiation of Th1 and Th2 in patients with unexplained recurrent spontaneous abortion. Int J Clin Exp Med. 2015;8:8812–8819.
Xie H, Li Z, Zheng G, Yang C, Liu X, Xu X, et al. Tim-3 downregulation by Toxoplasma gondii infection contributes to decidual dendritic cell dysfunction. Parasit Vectors. 2022;15:393.
Cui L, Wang Y, Ren L, Li Z, Jiang Y, Wang C, et al. Effect of B7-H4 downregulation induced by Toxoplasma gondii infection on dysfunction of decidual macrophages contributes to adverse pregnancy outcomes. Parasit Vectors. 2022;15:464.
Li Y, Guo J, Zhang H, Li Z, Ren Y, Jiang Y, et al. LILRB4 regulates the function of decidual MDSCs via the SHP-2/STAT6 pathway during Toxoplasma gondii infection. Parasit Vectors. 2023;16:237.
Murata H, Tanaka S, Hisamatsu Y, Tsubokura H, Hashimoto Y, Kitada M, et al. Transcriptional regulation of LGALS9 by HAND2 and FOXO1 in human endometrial stromal cells in women with regular cycles. Mol Hum Reprod. 2021;27:gaab063.
Weng Q, Liu Z, Li B, Liu K, Wu W, Liu H. Oxidative Stress Induces Mouse Follicular Granulosa Cells Apoptosis via JNK/FoxO1 Pathway. PLoS One. 2016;11:e0167869.
Li Y, Xiu F, Mou Z, Xue Z, Du H, Zhou C, et al. Exosomes derived from Toxoplasma gondii stimulate an inflammatory response through JNK signaling pathway. Nanomedicine (Lond). 2018;13:1157–1168.
Manaster I, Mandelboim O. The unique properties of uterine NK cells. Am J Reprod Immunol. 2010;63:434–444.
Li YH, Zhou WH, Tao Y, Wang SC, Jiang YL, Zhang D, et al. The Galectin-9/Tim-3 pathway is involved in the regulation of NK cell function at the maternal-fetal interface in early pregnancy. Cell Mol Immunol. 2016;13:73–81.
Lu X, Oh-Hora M, Takeda K, Yamasaki S. Selective suppression of IL-10 transcription by calcineurin in dendritic cells through inactivation of CREB. Int Immunol. 2022;34:197–206.
Lazarevic V, Szabo S, Glimcher LH. T-bet Runs INTERFERence. Immunity.2017;46:968–970.
Zhao QY, Li QH, Fu YY, Ren CE, Jiang AF, Meng YH. Decidual macrophages in recurrent spontaneous abortion. Front Immunol. 2022;13:994888.
Meng YH, Zhou WJ, Jin LP, Liu LB, Chang KK, Mei J, et al. RANKL-mediated harmonious dialoguebetween fetus and mother guarantees smooth gestation by inducing decidualM2 macrophage polarization. Cell Death Dis. 2017;8:e3105.
Li Z, Zhao M, Li T, Zheng J, Liu X, Jiang Y, et al. Decidual Macrophage Functional Polarization during Abnormal Pregnancy due to Toxoplasma gondii: Role for LILRB4. Front Immunol. 2017;8:1013.
Ning F, Liu H, Lash GE. The Role of Decidual Macrophages During Normal and Pathological Pregnancy. Am J Reprod Immunol. 2016;75:298–309.
Zhang D, Ren L, Zhao M, Yang C, Liu X, Zhang H, et al. Role of Tim-3 in Decidual Macrophage Functional Polarization During Abnormal Pregnancy With Toxoplasma gondii Infection. Front Immunol. 2019;10:1550.
Brown MB, von Chamier M, Allam AB, Reyes L. M1/M2 macrophage polarity in normal and complicated pregnancy. Front Immunol. 2014;5:606.
Ju Y, Xu T, Zhang H, Yu A. FOXO1-dependent DNA damage repair is regulated by JNK in lung cancer cells. Int J Oncol. 2014;44:1284–1292.
Vondra S, Höbler AL, Lackner AI, Raffetseder J, Mihalic ZN, Vogel A, et al. The human placenta shapes the phenotype of decidual macrophages. Cell Rep. 2023;42:111977.
Murphy SP, Fast LD, Hanna NN, Sharma S. Uterine NK cells mediate inflammation-induced fetal demise in IL-10-null mice. J Immunol. 2005;175:4084–4090.
Lee J, Su EW, Zhu C, Hainline S, Phuah J, Moroco JA, et al. Phosphotyrosine-dependent coupling of Tim-3 to T-cell receptor signaling pathways. Mol Cell Biol. 2011;31:3963–3974.
Ko HM, Lee SH, Bang M, Kim KC, Jeon SJ, Park YM, et al. Tyrosine kinase Fyn regulates iNOS expression in LPS-stimulated astrocytes via modulation of ERK phosphorylation. Biochem Biophys Res Commun. 2018;495:1214–1220.
Yu X, Lang B, Chen X, Tian Y, Qian S, Zhang Z, et al. The inhibitory receptor Tim-3 fails to suppress IFN-γ production via the NFAT pathway in NK-cell, unlike that in CD4⁺ T cells. BMC Immunol. 2021;22:25.
Qiu Z, Lu P, Wang K, Zhao X, Li Q, Wen J, et al. Dexmedetomidine Inhibits Neuroinflammation by Altering Microglial M1/M2 Polarization Through MAPK/ERK Pathway. Neurochem Res. 2020;45:345–353.

No competing interests reported.

GraphicalAbstract.png
TableS1Lgals9Promoter.docx
Additional file 1: Table S1. The primers used for ChIP-PCR.
TableS2Reagentsandantibodys.docx
Additional file 2: Table S2. Reagents and antibodies used in this study.

Download PDF

Editorial decision: Revision requested
04 Jun, 2024
Reviews received at journal
02 Jun, 2024
Reviews received at journal
28 May, 2024
Reviewers agreed at journal
25 May, 2024
Reviewers agreed at journal
23 May, 2024
Reviewers invited by journal
23 May, 2024
Editor assigned by journal
21 May, 2024
Submission checks completed at journal
20 May, 2024
First submitted to journal
17 May, 2024

You are reading this latest preprint version

The effect of Toxoplasma gondii infection on Galectin-9 expression in decidual macrophages contributing to dysfunction of decidual NK cells during pregnancy

Status:

Version 1

Abstract

Figures

Background

Methods

Mice

Mouse experiments

Cell preparation of mice

Human sample collection

Isolation and purification of human dMφ and dNK

Human decidual mononuclear cell culture and treatment

Flow cytometry staining and analysis

M2 macrophage differentiation of THP-1 cells

Western blot analysis

Immunofluorescent analysis

ChIP-PCR assay

Reagents and antibodies

Statistical analysis

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1