Abstract
The protozoan parasite Toxoplasma gondii causes severe pathologies in the infected fetus following vertical transmission during pregnancy. Primary T. gondii infection increases the risk of miscarriage during the first trimester of gestation; however, the cellular and molecular mechanisms are not fully understood. Extravillous trophoblasts (EVTs) are fetal cells that migrate and invade the maternal decidua to allow placenta formation and embryo implantation. The transcription factors Forkhead box O3a (FOXO3a) and FOXO1 play a central role in the regulation of EVT migration and invasion. Hence, impairment of EVT functions is associated with FOXO3a/FOXO1 dysregulation and poor pregnancy outcomes. Interestingly, T. gondii-driven inactivation of FOXO3a and FOXO1 was reported in fibroblasts and macrophages. Using a combination of cell imaging, reverse genetics and biochemical approaches in the human trophoblast cell line HTR-8/SVneo, herein we provide evidence that infection with type I RH T. gondii strain inhibits invasiveness and migratory activities in EVTs by repressing FOXO3a-and FOXO1-dependent and independent gene expression. Indeed, either T. gondii infection or single knockdown of FOXO3a and FOXO1 reduced invasion and migration properties in EVTs. Selective chemical blockade of parasite motility and host cell entry indicates that active infection is indispensable for reduced EVT migration but is only partially required for T. gondii-driven repression of EVT invasiveness. Mechanistically, T. gondii infection led to AKT-sensitive phosphorylation and nuclear exclusion of FOXO3a and FOXO1 in EVTs. An RT-qPCR-based screening identified a subset of invasion-and migration-associated genes downregulated in T. gondii-infected EVTs (MMP2, MMP3, MMP14, TIMP2, MUC1, and ITGB3). Transcription of genes encoding MMP3 and integrin β3 decreased in FOXO3a KD and FOXO1 KD EVT cell lines, respectively. These data along with pharmacological inhibition of AKT in infected cells provide evidence that T. gondii downregulates MMP3 and ITGB3 gene expression in EVTs in an AKT-FOXO3a/FOXO1-dependent fashion. In all, we have uncovered a novel regulatory mechanism involved in the repression of EVT migration and invasion properties during T. gondii infection. Further investigation using in vivo and ex vivo models of placental infection is required to determine whether T. gondii-driven dysregulation of EVT functions contributes to pregnancy complications associated with congenital toxoplasmosis.
Introduction
The obligate intracellular parasite Toxoplasma gondii (T. gondii) belongs to a group of human pathogens collectively known as TORCH (i.e., T. gondii, rubella virus, cytomegalovirus, and herpes virus), which have the capacity to cross the placental barrier and cause severe congenital pathologies associated with primary infection acquired during pregnancy (Ostrander and Bale, 2019). The consequences of congenital toxoplasmosis range from early-trimester miscarriage to late-trimester neurological and ocular fetal and perinatal defects (e.g., retinochoroiditis, hydrocephalus, mental retardation, etc.) (). It is estimated that the global annual incidence of congenital toxoplasmosis is ~190,000 cases, accounting for 1.2 million disability-adjusted life years (DALYS) per year (Rostami et al., 2019). Unfortunately, there are no effective vaccines approved for human use and current treatments are teratogenic (Zhang et al., 2022b; ). Hence, there is an urgent need to better understand the molecular mechanisms that regulate interactions between T. gondii and the host within the placental microenvironment to identify safer and more efficient treatments.
At the maternal-fetal interface, T. gondii infects extravillous trophoblasts (EVTs) (), a fetal cell population that anchors the placenta to the uterine implantation site (i.e., decidua) and is therefore essential for normal placentation and embryo development (; ; ). In addition to EVTs, T. gondii is also able to infect other subpopulations of trophoblasts (i.e., villous cytotrophoblasts and syncytiotrophoblast); however, EVTs appear to be particularly susceptible to the infection (Robbins et al., 2012; ). Accumulating evidence indicates that T. gondii modulates host cell signaling pathways and biological functions to establish a safe replicative niche within EVTs (Milian et al., 2019; ; ; ). For instance, it was shown that T. gondii intracellular proliferation relies on the sequential expression of macrophage inhibitory factor (MIF) and ERK1/2-dependent synthesis of COX-2 in the human trophoblast cell line HTR-8/SVneo (Milian et al., 2019). Consistent with this, it was described that T. gondii triggers a COX-2-PGE2-mediated anti-inflammatory cytokine response that promotes parasite growth in human EVTs (). In parallel, enhanced transcription of the gene encoding the transferrin receptor (TFRC) was reported in T. gondii-infected HTR-8/SVneo cells (), suggesting that the parasite hijacks iron metabolism in EVTs. Conversely, EVTs deploy defense mechanisms to control T. gondii infection, including reactive oxygen species (ROS) production, inflammasome activation, and pyroptosis (Quan et al., 2023).
The ability of EVTs to migrate and invade the maternal decidua is required for (i) reshaping decidual spiral arteries to ensure sufficient blood flow and oxygen concentration (), (ii) allowing nutrient exchange between mother and fetus, and (iii) promoting immune cell interactions necessary for an immunotolerant environment (; ). Dysregulation of these biological processes is known to contribute to pregnancy-related pathologies, such as preeclampsia or placenta accreta spectrum (). EVT migration and invasion properties require the activity of diverse transcription factors (e.g., FOXO3a, FOXO1, SNAIL, STAT3, β-catenin, etc.) (, , ; ; ; Li et al., 2015), adhesion molecules (e.g., ICAM, MUC-1, integrin β3, integrin β1, etc.) (Thirkill et al., 2007; Shyu et al., 2011; ), and metalloproteinases (MMPs) that degrade the extracellular matrix (e.g., MMP-2, MMP-3, MMP-9, pappalysin-1, etc.) (Zhang et al., 2020a; ; ; Li et al., 2015; Suman et al., 2013; ; Onogi et al., 2011).
The transcription factors Forkhead box O3a (FOXO3a) and FOXO1 play crucial roles in decidualization and embryo implantation. In addition, FOXO3a and FOXO1 are central regulators of EVT functions, including invasion, migration, and related gene expression programs (e.g., ITGB3, ICAM1, MMP9, etc.) (, ; Long et al., 2019; Vasquez et al., 2018; ). Hence, dysregulation of FOXO3a and FOXO1 has been associated with poor pregnancy outcomes, such as preeclampsia and gestational diabetes mellitus (Zhang et al., 2020b; ; Zhang et al., 2022a; ; ; ). In contrast to FOXO3a and FOXO1, activation of the TGF-β1-SMAD2/3 signaling pathway has been shown to inhibit HTR-8/SVneo cell invasiveness by preventing transcription of invasion-related genes (i.e., connective tissue growth factor and kisspeptin) (; ). Similarly, the transcription factor GATA2 was described as a repressor of invasion and associated genes in human EVTs (Ogoyama et al., 2021).
Interestingly, T. gondii infection has been reported to downregulate adhesion molecule expression (e.g., integrin β1) in human monocytes () and genes encoding MMPs (e.g., MMP1, MMP3, etc.) in human decidual macrophages (). Moreover, inactivation of FOXO3a- and FOXO1-dependent host autophagic responses were described in T. gondii-infected human fibroblasts () and murine macrophages (), respectively. Conversely, increased SMAD2 phosphorylation and TGF-β1 secretion were detected in T. gondii-infected macrophages () and in human trophoblast cell line HTR-8/SVneo (). In all, these studies indicate that T. gondii alters migration- and invasion-related signaling pathways, transcription factors, and gene expression programs in different host cell types. However, the ability of T. gondii to regulate EVT migratory and invasive properties and its molecular basis have yet to be investigated. Here, we show that T. gondii infection leads to reduced migration and invasiveness of HTR-8/SVneo, an EVT model. Mechanistically, these events involve parasite-driven AKT-sensitive nuclear exclusion of FOXO3a and FOXO1 and repression of FOXO3a/FOXO1-dependent and -independent transcription of invasion- and migration-related genes.
Materials and methods
Reagents
Culture media and supplements were purchased from Wisent and Gibco; MK-2206 was obtained from Cayman Chemical; SB431542 was purchased from Selleck Chemicals; mycalolide B was obtained from Fujifilm; cOmplete EDTA-free protease inhibitor and PhosSTOP phosphatase inhibitor tablets were obtained from Roche; DAPI was purchased from Invitrogen; human TGF-β1 recombinant protein was obtained from Cell Signaling Technologies; rat tail collagen type-I was purchased from Gibco.
Parasite maintenance and harvest
T. gondii RH and ME49 strains were maintained by serial passage in Vero cells, as previously described (). Cultures were grown in DMEM with 5% heat-inactivated FBS, 2 mM L-glutamate, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 µg/mL streptomycin, and incubated at 37 °C, 5% CO2. For experimental infections, freshly egressed tachyzoites were harvested, pelleted by centrifugation (1,300 × g, 7 min, 4 °C), resuspended in ice-cold PBS (pH 7.2-7.4), and passed through a syringe fitted with a 27 G needle. After removing large debris and intact host cells via low-speed centrifugation (200 × g, 3 min, 4 °C), the supernatant containing parasites was filtered through a 3-µm polycarbonate filter (Millipore). Tachyzoites were washed twice in PBS and finally resuspended in the appropriate culture medium, according to the experiment.
Soluble T. gondii antigens and heat-killed parasites
STAgs and HK parasites were prepared from freshly egressed tachyzoites, as previously described (). For STAgs, parasites were resuspended in ice-cold PBS, subjected to three 5-min cycles of freezing/thawing using liquid nitrogen and a 37 °C water bath, and then sonicated on ice for 5 min (1 s on/off pulses, 30% duty cycle) using a Sonic Dismembrator FB505 (ThermoFisher). After centrifugation (21,000 × g, 15 min, 4 °C), soluble material containing STAg was collected and used for downstream experiments. HK parasites were prepared by incubation at 56 °C for 10 min. Parasites were then pelleted by centrifugation (1,300 × g, 7 min, RT) and resuspended in the appropriate culture medium according to the experiment.
Parasite treatment with mycalolide B
T. gondii cultures were treated with mycalolide B prior to infection, as described (Rosowski et al., 2014). Briefly, freshly egressed tachyzoites were incubated with 3 μM mycalolide B or vehicle (i.e., DMSO) for 15 min at 37 °C and washed twice in warm PBS (pH 7.2-7.4). Parasites were then pelleted by centrifugation (1,300 × g, 7 min, RT) and resuspended in cell culture media for downstream experiments.
Infection and treatment of HTR-8/SVneo cells
HTR-8/SVneo cells (ATCC # CRL-3271) were plated one day before infection in DMEM supplemented with 10% FBS, 2 mM L-glutamate, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C, 5% CO2. Cultures were serum-starved for 1 h, then inoculated with live or HK parasites (multiplicity of infection [MOI] of 1:1, 2:1, 5:1), treated with 50 μg/mL STAgs, or left uninfected in fresh medium without FBS. Any remaining extracellular parasites were rinsed away with warm PBS (pH 7.2-7.4) 1 h following inoculation, fresh medium without FBS was added with inhibitors or vehicle when applicable, and cells were incubated until experiment completion.
Viability assays
Viability of HTR-8/SVneo cells was determined by the resazurin assay as described (). Briefly, cells were treated with increasing concentrations of MK-2206 (0.0625–32 µM), SB431542 (0.195-200 µM) or an equivalent volume of DMSO (vehicle) for 24 h at 37 °C, 5% CO2. The medium was removed and replaced with fresh culture medium supplemented with 0.025% resazurin. Cultures were incubated for 4 h in the presence of the inhibitors or DMSO for 4 h at 37 °C, 5% CO2. Optical density was measured using a Multiskan GO (ThermoFisher) at 600 and 570 nm. Absorbance at 600 nm was subtracted from readings at 570 nm. Experiments were performed in three biological replicates (n = 3); each sample was analyzed in a technical triplicate.
Invasion assays
Trophoblast cell invasiveness was monitored by a collagen-based matrix invasion assays as described (, ). Briefly, the upper chamber of 8-μm pore size membrane inserts (Corning) was coated with 5 μg/cm² rat tail collagen-I (Gibco ThermoFisher Scientific) diluted in sterile PBS and incubated at room temperature at least 4 h prior to invasion assays. HTR-8/SVneo cells were infected or treated with different stimuli for 4 h and were collected by centrifugation (200 × g, 7 min, 4 °C). Approximately 7.5 × 104 cells were resuspended in serum-free DMEM and were seeded in the insert upper chamber. DMEM containing 10% FBS was added to the insert lower chamber to induce cell invasion. Following a 16 h incubation period, non-invasive cells that remained in the upper chamber were removed with a cotton swab. Cells that had migrated through the matrigel towards the bottom chamber were gently rinsed with PBS. Inserts membranes were then fixed in 2% paraformaldehyde (PFA) in PBS for 10 min at RT and stained with Hema 3 staining (ThermoFisher Scientific). Cell invasion was visualized by light microscopy with a 20x objective of a Nikon Eclipse microscope. Images were acquired from four different fields of view per treatment with the NIS-element software and analyzed with ImageJ (NIH, USA). Briefly, the area of invasion was analyzed by applying a manual threshold to detect the Hema 3 staining. Cell viability was determined prior to cell seeding in the matrigel and following invasion assays by cell counting using trypan blue staining.
Cell migration assays
In vitro wound-healing assays were carried out to measure trophoblast cell migration as described (, ). Briefly, HTR-8/SVneo cells were seeded in 6-well tissue culture plates at a starting density of 1 × 106 cells/well and were cultured overnight to reach 90% confluence. Cells were infected or treated with different stimuli for 1 h and monolayers were then scratched with a 1,000 μL pipette tip to create a cross-shaped wound (). Cultures were washed with PBS at RT and fresh serum-free DMEM was added. Images were acquired at 0 and 16 h post-scratch wound by light microscopy with a 4x objective of a Nikon Eclipse microscope using the NIS-element software. To measure the area of wound closure, images were taken from the same area and cells bordering the wounds were traced using ImageJ software “wound-healing assay size tool”. The area of migration was calculated from the equation ((Wi-Wz)/Wi) × 100; where Wi is the area of wound at t = 0 h and Wz is the area of wound closure after 16 h. Percentage of migration was normalized to uninfected control cultures or scrambled shRNA-transduced cell lines according to the experiment. In parallel, cells were collected at 0 and 16 post-scratch wound and stained with trypan blue to count live cells at each time point and calculate the proliferation ratio.
Western blot analysis
Following infection and other treatments, cells were lysed directly in RIPA lysis buffer (25 mM Tris (pH 7.6), 150 mM NaCl, 1% Triton-X 100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with cOmplete EDTA-free protease inhibitor, PhosSTOP phosphatase inhibitor tablets and 10 mM 1,10-phenanthroline monohydrate (Sigma-Aldrich). Lysates were stored at -80 °C or diluted with a solution of Laemmli buffer, heated at 95 °C for 5 min and processed immediately for SDS-PAGE. Resolved proteins were transferred onto PVDF membranes. Membranes were blocked for 1 h at RT in TBS 0.1% Tween-20 (TBS-T), 5% skim milk, then probed with the following primary antibodies: anti-phospho-AKT (S473) (clone D9E, #4060), anti-phospho-AKT (T308) (clone C31E5E, #2965), anti-AKT (clone C67E7, #4691), anti-phospho-FOXO3a (S253) (clone D18H8, #13129), anti-phospho-FOXO3a/FOXO1 (T32/T24) (polyclonal, #9464), anti-FOXO1 (clone C29H4, #2880), anti-phospho-FOXO3a (S253) (clone D18H8, #13129), anti-FOXO3a (clone 75D8, #2497), anti-phospho-SMAD2 (S465/S467) (clone E8F3R, #18338), anti-SMAD2 (clone D43B4) #5339, anti-Lamin B1 (clone D4Q4Z, #12586), anti-α-tubulin (clone DM1A, #3873), anti-GATA2 (clone 79802S, #79802S), and β-actin (clone 8H10D10, #3700) were obtained from Cell Signaling Technologies; anti-T. gondii profilin-like protein (polyclonal, #AF3860) was purchased from R&D Systems. Membranes were then probed with the following horseradish peroxidase (HRP)-conjugated antibodies: goat anti-rabbit IgG (#A0545), goat anti-mouse IgG (#A4416) (Sigma-Aldrich) or rabbit anti-goat (R&D Systems). Subsequently, proteins were visualized using the Clarity ECL Western blotting substrate (Bio-Rad) and exposing the membranes to autoradiography film (Denville Scientific).
Preparation of HTR-8/SVneo nuclear and cytoplasmic extracts
Following infection and other treatments, cells were gently scraped in ice-cold PBS 5 mM EDTA (supplemented with cOmplete EDTA-free protease inhibitor, PhosSTOP phosphatase inhibitor tablets, and 10 mM 1,10-phenanthroline [Sigma-Aldrich]). A fraction of the cell suspensions was kept aside to prepare Whole Cell Lysates (WCL); these samples were lysed and processed for western blotting as detailed above. Samples were centrifuged (500 × g, 5 min at 4 °C), and the pellets were resuspended in 300 μL of ice-cold Hypotonic Lysis Buffer (HBL) (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, cOmplete EDTA-free protease inhibitor, PhosSTOP phosphatase inhibitor tablets, and 10 mM 1,10-phenanthroline). Samples were incubated for 15 min on ice with regular mixing, then 30 μL of 10% IGEPAL CA-630 was added to suspensions. After centrifugation (16,000 × g, 1 min,4 °C), supernatants (containing the cytoplasmic contents) were transferred to new tubes. Pellets were washed twice by adding 500 μL HBL (without IGEPAL CA-63) and centrifuged (16,000 × g, 1 min,4 °C). Pellets were then lysed in 60 μL of Nuclear Lysis Buffer (NLB) (20 mM HEPES, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 5% glycerol, cOmplete EDTA-free protease inhibitor, PhosSTOP phosphatase inhibitor tablets, and 10 mM 1,10-phenanthroline). After 15 min of incubation on ice with occasional vortexing, lysates were centrifuged, and supernatants (containing the nuclear material) were transferred to new tubes. Samples were next processed for western blot analyses.
Immunofluorescence and confocal microscopy
HTR-8/SVneo cultures were seeded onto glass coverslips in 24-well plates overnight. At the indicated time points, cells were rinsed with PBS three times and then fixed with 3.7% PFA in PBS for 15 min at RT. Cells were permeabilized with 0.2% Triton X-100 (in PBS) for 5 min at RT for total FOXO1 and FOXO3a staining. Cells were permeabilized with 100% methanol for 20 min at -20 °C for phospho-SMAD2 (S465/467) staining. Samples were kept in blocking solution (5% skim milk, 1% BSA, 5% normal goat serum in PBS) for 1 h at RT and incubated for 2 h at RT with the following primary antibodies diluted in PBS with 1% BSA: anti-FOXO3a (clone 75D8, #2497), anti-FOXO1 (clone C29H4, #2880), and anti-phospho-SMAD2 (S465/S467) (clone E8F3R, #18338) were obtained from Cell Signaling Technologies. Samples were then incubated with the following fluorochrome-conjugated secondary antibody for 2 h at RT: donkey anti-rabbit IgG (H+L) Alexa Fluor 647 (#A-31573) was purchased from Invitrogen. Nuclei were stained with 300 nM DAPI (Invitrogen, #D21490) for 5 min at RT. Coverslips were mounted onto slides with Fluoromount G (#00-4958-02, Invitrogen). Samples were visualized with a 40x objective of a LSM780 Zeiss confocal microscope, image acquisition was carried out using ZEN software, and image processing was performed with ImageJ.
Purification of RNA and quantitative RT-PCR
Total cellular RNA was extracted with QIAzol (Qiagen) according to the manufacturer’s specifications. RNA concentration and purity were assessed spectrophotometrically by using a NanoDrop One instrument (Thermo Fisher Scientific). Approximately 1 μg of RNA was reverse transcribed using LunaScript® RT SuperMix Kit (New England Biolabs). Real-time quantitative PCR (RT-qPCR) was performed with Luna® qPCR Master Mix (New England Biolabs) using a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). Relative quantification was calculated using the comparative Ct method (ΔΔCt) (Taylor et al., 2010) and relative expression was normalized to human HPRT1. Primers used for RT-qPCR were designed using NCBI Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) and are listed in Supplementary Table S1.
Lentivirus production and HTR-8/SVneo transduction
Lentiviruses were produced in HEK-293T cells using Lenti-Pac HIV Expression Packaging Kit, as per manufacturer’s guidelines (GeneCopoeia; #LT001). Lentivirus titers were measured using Lenti-Pac HIV qRT-PCR Titration Kit (GeneCopoeia; #LT005) according to the manufacturer’s protocol. To generate FOXO1 and FOXO3a KD cells, HTR-8/SVneo cells were transduced with the different lentivirus preparations for 3 days in the culture medium supplemented with 5 µg/mL polybrene (hexadimethrine bromide). Transduced cultures were selected for 6 days using 2 µg/mL puromycin to generate stable cell lines used immediately for downstream experiments. Plasmids and shRNA clones were purchased from GeneCopoeia (Supplementary Table S2).
Statistical analysis
Data are presented as the mean (standard deviation) (SD) of the mean. Statistical significance was determined by using one-way or two-way ANOVA followed by a Tukey post hoc test or a two-tailed independent Student’s t-test; calculations were performed by using Prism 7 software package (GraphPad). Differences were considered significant when *P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.
Results
Toxoplasma gondii inhibits invasion and migration properties, and related genes in human trophoblasts
HTR-8/SVneo is an immortalized human EVT cell line widely used as a model system to study invasion and migration (), two essential processes for placenta implantation, development and healthy pregnancy (). Given that EVTs are preferentially infected by T. gondii in an in vitro placental model (Robbins et al., 2012), we set out to elucidate whether the parasite interferes with two of their main biological functions; namely, invasive and migratory capacities. To test this, HTR-8/SVneo cells were infected with RH T. gondii tachyzoites at multiplicity of infection (MOI) 5:1 and carried out collagen-based matrix invasion and “wound-healing” migration in vitro assays. A significant decrease in EVT invasion and migration was detected at 16 hours post-infection (h.p.i) (Figures 1A, B, respectively). These effects were not attributed to reduced cell viability nor to changes in proliferation rate, as no differences were observed between infected and uninfected control cells (Supplementary Figures S1A, B, respectively). HTR-8/SVneo infection with increasing parasite – host cell ratios revealed that T. gondii-driven downregulation of EVT invasion and migration is detectable at MOI 2:1 but is more pronounced at MOI 5:1 (Supplementary Figure S2).
Figure 1
Alteration of biological functions in T. gondii-infected cells at the maternal-fetal interface has been linked to dysregulation of host gene expression (; ). Hence, we postulated that T. gondii downregulates transcriptional programs associated with invasion and migration in human EVTs. Our RT-qPCR analyses revealed decreased expression of a subset of transcripts encoding proteins related to invasion and migration (i.e., MMP2, MMP3, MMP14, TIMP2, MUC1, and ITGB3) in T. gondii-infected HTR-8/SVneo cells (Figure 1C). However, our screening also identified invasion- and migration-related genes that were not significantly modulated upon infection (i.e., PAPPA, SNAIL, MMP9, MMP11, ICAM1, CTNNB1, and CDH2) (Figure 1D). These data provide evidence that repression of EVT invasion and migration by T. gondii correlates with dysregulation of specific gene expression programs.
We next sought to determine whether live infection was required for the dysregulation of invasion and migration in EVTs. In contrast to infection by live tachyzoites, heat-killed (HK) parasites and soluble T. gondii antigens (STAgs) had no effect on the invasive and migration properties of HTR-8/SVneo cells (Figures 2A, B, respectively). To further investigate the requirement of live infection for T. gondii-driven modulation of EVT functions, we pretreated freshly isolated parasite cultures with mycalolide B, which prevents host cell entry without blocking rhoptry- and microneme-dependent protein secretion (; Rosowski et al., 2014). We initially assessed the efficacy of mycalolide B treatment by performing RT-qPCR experiments. Consistent with reduced parasite internalization, we observed a marked decrease in T. gondii TUB1A mRNA abundance in HTR-8/SVneo cultures incubated with mycalolide B-pretreated parasites compared to cells infected with T. gondii tachyzoites pretreated with vehicle (i.e., DMSO) (Supplementary Figure S3A). We then tested the effect of mycalolide B treatment in EVT invasion and migration impairment by T. gondii. Incubation with mycalolide B-pretreated tachyzoites or infection with untreated parasite cultures led to a marked decrease in HTR-8/SVneo cell invasiveness (Figure 2C); however, the inhibitory effect was significantly attenuated upon mycalolide B pretreatment. In contrast to control T. gondii cultures, mycalolide B-pretreated parasites failed to inhibit HTR-8/SVneo cell migration (Figure 2D). Of note, no changes were detected in EVT viability and proliferation upon HK and STAg treatment (Supplementary Figures S3B, C, respectively) or incubation with mycalolide B-pretreated parasites (Supplementary Figures S3D, E, respectively). Taken together, this set of experiments indicates that reduced EVT invasion by T. gondii is partially dependent on host cell entry whereas parasite-driven impairment of EVT migration requires active live infection.
Figure 2
Downregulation of invasion-related genes in T. gondii-infected human trophoblasts is SMAD2- and GATA2-independent
The TGF-β1-SMAD2/3 signaling pathway has been shown to repress EVT invasiveness through the inhibition of several transcription factors and invasion-associated genes (; ). Interestingly, phosphorylation and subsequent nuclear translocation of SMAD2 was reported in T. gondii-infected macrophages (). Hence, we sought to investigate whether TGF-β1-SMAD2/3 signaling played a role in reduced invasion and expression of associated genes in human EVTs during T. gondii infection. To begin testing this hypothesis, we monitored changes in the phosphorylation status of SMAD2 upon infection. As expected, stimulation with recombinant TGF-β1 induced SMAD2 phosphorylation at residues S465/S467 in HTR-8/SVneo cultures (Figure 3A), and was blocked upon treatment with the TGF-β1 receptor (ALK4/5/7) inhibitor SB431542 () without affecting cell viability (Supplementary Figure S4A). In stark contrast, RH T. gondii tachyzoites failed to upregulate SMAD2 phosphorylation in human EVTs (Figure 3A). Consistent with this, nuclear accumulation of SMAD2 was detected in cells stimulated with TGF-β1 and was sensitive to pharmacological blockade of ALK4/5/7 (Figure 3B). However, no changes in the subcellular localization of SMAD2 were detected in T. gondii-infected HTR-8/SVneo cells.
Figure 3
The transcription factor GATA2 was identified as a negative regulator of the invasion activity and related genes in HTR-8/SVneo cells (Ogoyama et al., 2021). According to our screening, one of the genes negatively targeted by GATA2 was also downregulated in T. gondii-infected HTR-8/SVneo cultures (i.e., MMP14) (Figure 1C). Thus, we set out to elucidate whether T. gondii-driven inhibition of MMP14 in human EVTs was GATA2-dependent. However, western blotting experiments showed that T. gondii infection does not increase GATA2 protein levels in HTR-8/SVneo cells compared to uninfected control cultures (Figure 3C). Parasite extracts (i.e., isolated from their host cells [“Tg only”]) were probed in parallel to rule out potential cross-reactivity of the antibodies tested against parasite proteins (Figure 3C). In all, these experiments suggest that T. gondii-driven inhibition of invasion in the human trophoblast cell line HTR-8/SVneo occurs through SMAD2- and GATA2-independent mechanisms.
Toxoplasma gondii infection leads to AKT-dependent phosphorylation and nuclear exclusion of FOXO3a and FOXO1 in human trophoblasts
In addition to SMAD2 and GATA2, the regulation of gene expression by FOXO3a- and FOXO1 has been associated with EVT invasion and migration (, , ). Of note, T. gondii has been reported to downregulate FOXO3a and FOXO1 transcriptional activity through their AKT-dependent nuclear exclusion in human fibroblasts and murine macrophages, respectively (; ). Thus, we postulated that repression of FOXO3a- and FOXO1-inducible genes and functions also occurred during T. gondii infection in human EVTs. To address this, we first monitored changes in the phosphorylation status of AKT, FOXO3a, and FOXO1 by western blotting. Increased and sustained phosphorylation of AKT at residues S473 and T308 was detected in T. gondii-infected HTR-8/SVneo cells compared to uninfected control cultures (Figure 4A). Accordingly, heightened phosphorylation levels of FOXO3a at AKT-sensitive residues S253 and T32 as well as FOXO1 at residue T24 were observed in infected samples. Of note, the kinetics of FOXO3a and FOXO1 phosphorylation matched closely those observed for AKT. To confirm that T. gondii-induced phosphorylation of FOXO3a and FOXO1 was mediated by AKT, HTR-8/SVneo cells were treated with MK-2206, a pan-AKT inhibitor (), or with DMSO (vehicle). AKT phosphorylation at S473 and T308 was abrogated in the presence of MK-2206 regardless of infection status (Figure 4B) without affecting cell viability (Supplementary Figure S4B). Importantly, pharmacological inhibition of AKT prevented FOXO3a phosphorylation at S253 and T32 and FOXO1 phosphorylation at T24 in T. gondii-infected HTR-8/SVneo cultures (Figure 4B) (). Furthermore, modulation of FOXO3a and FOXO1 phosphorylation required live parasite infection as no changes were observed following treatment with HK parasites or STAgs (Figure 4C).
Figure 4
Based on our data showing T. gondii-inducible AKT-sensitive phosphorylation of FOXO3a and FOXO1 in EVTs (Figures 4A–C), we assessed the subcellular localization of these transcription factors in HTR-8/SVneo cells following T. gondii infection. Under serum-deprived conditions, FOXO3a and FOXO1 were detected mostly in the nucleus in uninfected control cells (Figures 5A–D), consistent with low phosphorylation of FOXO3a and FOXO1 at AKT-sensitive residues (Figures 4A–C). Conversely, FOXO3a and FOXO1 were exported from the host cell nucleus upon T. gondii-infection (Figures 5A, C, respectively) in an MOI-dependent fashion (Supplementary Figures S5A, B, respectively). Significant differences in FOXO3a and FOXO1 subcellular localization were highlighted by their decreased co-localization coefficients with nuclear staining (Figures 5B, D, respectively). As expected, treatment of HTR-8/SVneo cultures with the pan-AKT inhibitor MK-2206 prevented T. gondii-driven changes in FOXO3a and FOXO1 subcellular localization (Figures 5A–D, respectively). In line with immunofluorescence and confocal microscopy data, western blot analyses of nuclear and cytoplasmic protein extracts confirmed AKT-sensitive nuclear exclusion of FOXO3a and FOXO1 in T. gondii-infected HTR-8/SVneo cells (Figure 5E). Taken together, these results indicate that T. gondii induces AKT-dependent phosphorylation and nuclear export of FOXO3a and FOXO1 in human EVTs.
Figure 5
FOXO3a and FOXO1 inactivation is involved in the inhibition of invasion and migration in T. gondii-infected trophoblasts
Next, we set out to elucidate whether parasite-driven repression of FOXO3a and FOXO1 contributed to reduced invasion and migration activities in T. gondii-infected human trophoblasts. To address this, we first carried out invasion and migration assays in HTR-8/SVneo cell lines knocked down (KD) in FOXO3a and FOXO1 (Figures 6A, B, respectively) generated by lentiviral transduction of shRNAs. Consistent with previous reports (, ), invasion and migration properties were significantly downregulated in FOXO3a KD EVTs compared with Scrambled control cultures (Figures 6C, E, respectively). Unlike FOXO3a, FOXO1 appeared to be dispensable for HTR-8/SVneo invasiveness, as no differences were detected between Scrambled and FOXO1 KD cell lines (Figure 6D). However, migration activity was significantly diminished in HTR-8/SVneo cells expressing reduced levels of FOXO1 (Figure 6F), in line with a previous report (). Importantly, differences in invasion and migration detected between Scrambled and FOXO3a or FOXO1 KD HTR-8/SVneo cell lines were not due to defects in cell viability and proliferation rates (Supplementary Figures S6A-D).
Figure 6
After having confirmed that FOXO3a and FOXO1 are required for HTR-8/SVneo cell invasiveness and/or migration, we then monitored whether T. gondii-driven inhibition of EVT invasion- and migration-related genes (Figure 1C) was dependent on FOXO3a and/or FOXO1 repression. RT-qPCR experiments showed reduced MMP3 mRNA expression in FOXO3a KD HTR-8/SVneo cells compared with Scrambled control cultures (Figure 6G). Similarly, accumulation of ITGB3 transcripts was diminished in an HTR-8/SVneo cell line expressing reduced levels of FOXO1 (Figure 6H). Based on our data from reverse-genetics experiments (Figures 6A–H) along with results showing that pharmacological blockade of AKT prevented FOXO3a and FOXO1 nuclear exclusion despite T. gondii infection (Figures 5A–D, respectively), we formulated the hypothesis that parasite-driven AKT-sensitive inhibition of FOXO3a and FOXO1 activity contributes to repression of invasion- and migration-associated genes MMP3 and ITGB3 in T. gondii-infected EVTs. In line with this notion, RH T. gondii tachyzoites failed to downregulate MMP3 and ITGB3 mRNA levels in HTR-8/SVneo cells treated with the pan-AKT inhibitor MK-2206 (Figures 7A, B, respectively). Taken together, these data suggest that T. gondii inhibits invasion and migration properties in the human trophoblast cell line HTR-8/SVneo through FOXO3a/FOXO1-dependent and -independent mechanisms (Figure 8).
Figure 7
Figure 8
Discussion
Primary T. gondii infection during the first trimester of gestation is associated with high risk of spontaneous abortion, stillbirth, premature birth, and fetal growth restriction (). It is well-documented that T. gondii-driven dysregulation of maternal immune responses in the placental microenvironment contributes to such poor pregnancy outcomes (; Liu et al., 2018; Zhan et al., 2018). However, the cellular and molecular mechanisms involved are not fully understood. An ex vivo study using human placental explants from first trimester pregnancies revealed that EVTs are quite permissive to T. gondii infection in contrast to syncytiotrophoblast (Robbins et al., 2012). Similar results were observed in vitro following infection of human EVT and villous trophoblast cell lines with T. gondii (). Interestingly, Wang and colleagues recently reported a decrease in EVT invasion and migration activities when co-cultured with human macrophages pre-treated with T. gondii antigens (Wang et al., 2024), suggesting indirect parasite-driven EVT dysregulation. Aberrant EVT migration and invasion of the decidua have deleterious consequences during the first trimester of pregnancy (); however, no direct link had been established between T. gondii infection and alteration of EVT functions required for normal placentation and embryo implantation. Herein, using cell imaging in combination with pharmacological and reverse genetics approaches in the human trophoblast cell line HTR-8/SVneo, we demonstrate that T. gondii infection directly downregulates invasiveness and migration of HTR-8/SVneo cells by repressing FOXO1- and FOXO3a-dependent and -independent transcriptional programs. Our data show that T. gondii infection decreases the expression of genes encoding several MMPs (i.e., MMP2, MMP14, and MMP3) in HTR-8/SVneo cells. These enzymes are essential for extracellular matrix degradation, a key step in EVT invasiveness and migration activities (Zhu et al., 2012). We also confirmed T. gondii-driven inhibition of transcripts responsible for the synthesis of integrin β3 and Mucin 1 (ITGB3 and MUC1, respectively) two proteins required for EVT adhesion and migration to the maternal decidua (; Thirkill et al., 2007). Interestingly, in addition to type I RH T. gondii strain, we observed that a type II strain (i.e., ME49) is able to reduce migration of HTR-8/SVneo cells without affecting their invasiveness (Supplementary Figure S7). These data warrant further investigation to determine whether T. gondii-driven modulation of human EVT functions is strain-dependent and identify potential strain-specific regulatory mechanisms.
In vivo studies using mice harboring a specific deletion for Foxo3 or Foxo1 in the uterine compartment have demonstrated that these the transcription factors are essential for successful endometrial decidualization and embryo implantation during early pregnancy (Long et al., 2019; Vasquez et al., 2018). Moreover, genetic ablation of FOXO3 or FOXO1 showed their requirement for invasion and migration activities and associated transcriptional programs in human EVTs (, , ). Of note, alterations in FOXO3a and FOXO1 expression and activity have been associated with preeclampsia (, ; Zhang et al., 2022a; ; ), a pregnancy-related complication characterized by impaired EVT invasion and migration (, , ). For instance, lower FOXO3a expression and increased phosphorylation at the AKT-sensitive residue S253 were detected in placental tissues of preeclampsia patients when compared to samples isolated from healthy pregnant women (). Similarly, FOXO1 was reported to be poorly expressed in the placenta of preeclampsia patients in comparison to the control group (; Sheridan et al., 2015; Zhang et al., 2022a; ). Furthermore, a significant decrease in FOXO1 serum concentrations was correlated with the onset of preeclampsia (). In line with these findings, we observed that T. gondii infection induces AKT-sensitive phosphorylation and nuclear exclusion of FOXO3a and FOXO1 in HTR-8/SVneo cells. In addition, we detected a marked reduction in FOXO3a and FOXO1 protein expression upon infection. These data agree with previous work showing that AKT-dependent FOXO3a and FOXO1 phosphorylation and translocation to the cytosolic compartment induces binding of the chaperone protein 14-3–3 and proteasomal degradation (; ). Future investigation using co-immunoprecipitation assays and proteasome inhibitors will provide insight into the mechanistic basis for decreased FOXO3a and FOXO1 levels in T. gondii-infected EVTs.
Our reverse genetics experiments combined with pharmacological blockade of AKT indicate that T. gondii downregulates the expression of migration- and -invasion associated genes MMP3 and ITGB3 via AKT-dependent phosphorylation and nuclear exclusion of FOXO3a and FOXO1, respectively. Even though a potential link between congenital toxoplasmosis and preeclampsia has been explored, a clear correlation remains to be established (Liu et al., 2018; ). Thus, based on our data along with previous reports, it is tempting to speculate that parasite-driven repression of FOXO3a- and FOXO1-regulated transcriptional programs and biological processes in EVTs contribute to pregnancy-related pathologies, including preeclampsia. Further investigation using ex vivo human placental models combined with experimental models of congenital toxoplasmosis in mice deficient for Foxo3 and Foxo1 in the uterine compartment will help shed light on this matter.
Among TORCH pathogens, only T. gondii and human cytomegalovirus (HCMV) have been shown to disrupt host cell functions by modulating the activity of FOXO family members (; ; ; ). For instance, HCMV infection induces FOXO3a nuclear translocation which is crucial for immediate early viral gene expression and HCMV reactivation in fibroblasts (; ). In stark contrast, T. gondii inhibits FOXO3a-regulated transcriptional programs related to autophagy via AKT-dependent nuclear export of FOXO3a in human fibroblasts (). Similarly, AKT-inducible phosphorylation and cytoplasmic accumulation of FOXO1 were reported in T. gondii-infected macrophages (). Despite this evidence, dysregulation of FOXO transcriptional activity by TORCH pathogens, including T. gondii, had not been investigated in trophoblasts. Confirming and extending previous studies in fibroblasts and macrophages (; ), we observed that T. gondii inhibits transcription of invasion and migration-related genes associated FOXO3a and FOXO1 activity in an AKT-dependent fashion in the human trophoblast cell line HTR-8/SVneo. Similar to T. gondii, other TORCH pathogens, such as Listeria monocytogenes, preferentially infect EVTs (Robbins et al., 2010). Interestingly, upregulation of AKT phosphorylation was reported in HCMV-infected human EVTs (Lin et al., 2020). Hence, future investigation is required to determine whether in addition to T. gondii, inactivation of FOXO3a and/or FOXO1 via AKT-sensitive phosphorylation by other TORCH pathogens, contributes to dysregulation of EVT functions and poor pregnancy outcomes.
Reverse genetics approaches combined with transcriptional profiling studies carried out in the human cell line HTR-8/SVneo identified FOXO3a as a central regulator of EVT migration and invasion activities (, ). More specifically, RNA-seq experiments in WT and FOXO3a KD cells identified ICAM1, PTGS2, and MMP9 among the top migration- and invasion-associated genes under transcriptional control of FOXO3a in human EVTs (, ). Surprisingly, we did not detect a significant reduction in ICAM1, PTGS2, and MMP9 mRNA levels in FOXO3a KD versus control WT HTR-8/SVneo cells. This discrepancy might be related to differences in the experimental design utilized in both studies, including target identification and validation methods (i.e., RNA-seq versus RT-qPCR), cell culture conditions, time of incubation, lysis buffer for sample collection, etc. Even though MMP3 had not been previously reported as a transcriptional target of FOXO3a, bioinformatics approaches identified three consensus binding sites for FOXO3a in the MMP3 promoter, suggesting a functional interaction (Samatar et al., 2002). In line with this in silico analysis, our RT-qPCR experiments revealed a significant decrease in MMP3 mRNA abundance in FOXO3a KD HTR-8/SVneo cultures when compared to WT control cells. Of note, inhibition of MMP3 gene expression was phenocopied in WT HTR-8/SVneo cells infected with T. gondii and was reversed by pharmacological blockade of AKT. These findings along with our microscopy data showing that T. gondii drives AKT-dependent FOXO3a phosphorylation and nuclear exclusion, suggest that MMP3 downregulation in T. gondii-infected EVTs is due to the inhibition of FOXO3a transcriptional activity via AKT.
It is well-documented that MMP3 levels increase in various types of cancer which facilitates tumor cell invasion and metastasis (Phromnoi et al., 2009; Liang et al., 2021; Seccareccia et al., 2014). Although less investigated, a growing body of work suggests that MMP3 is also involved in EVT invasion and normal pregnancy outcomes (; ; Reister et al., 2006; Williams et al., 2011). Immunofluorescence analyses of first trimester placental tissues combined with western blot data of isolated primary placental cells revealed that MMP3 is mostly expressed and secreted by villous trophoblasts and EVTs (). In addition, MMP3 was detected in EVTs of the basal plate in human term placental samples, specifically in EVTs that are in direct contact with maternal cells, suggesting a role for MMP3 during labor (). Importantly, a reduction in MMP3 expression in EVTs surrounding spiral arteries was reported in patients with severe preeclampsia as compared to healthy controls (Reister et al., 2006). Furthermore, treatment with folic acid was shown to enhance human EVT invasion, an effect that was associated with increased secretion of MMP3 and MMP2 in human placental explants (Williams et al., 2011). Thus, based on previous reports and our findings, it is conceivable that T. gondii-driven repression of MMP3 is partially responsible for reduced EVT invasion activity and pregnancy complications associated with congenital toxoplasmosis. Future work will shed light on this matter.
The cell surface glycoprotein integrin β3 binds to diverse extracellular matrix molecules (e.g., fibrinogen, collagen, fibronectin, etc.) and triggers intracellular signals involved in cell invasion and migration (). Reduced integrin β3 () and FOXO1 (; Sheridan et al., 2015; Zhang et al., 2022a; ) levels were detected in placental samples isolated from preeclampsia patients when compared to healthy controls. Furthermore, mechanistic studies revealed that FOXO1-dependent transcription of the integrin β3-encoding gene ITGB3 is required for HTR-8/SVneo cell migration and adhesion activities (). The authors also reported that FOXO1 transcriptional activity, integrin β3 expression, and migration properties in EVTs are negatively regulated by the kinase AKT (). In line with these findings, we observed that either T. gondii infection or genetic ablation of FOXO1 reduces ITGB3 mRNA accumulation and migratory activity in HTR-8/SVneo cells. Moreover, we demonstrated that T. gondii-driven activation of AKT triggers FOXO1 nuclear exclusion and downregulates ITGB3 mRNA levels in infected HTR-8/SVneo cultures. This set of experiments along with previous reports support the notion that inhibition of FOXO1 transcriptional activity via AKT leads to a decrease in ITGB3 gene expression and contributes to reduced migration properties in T. gondii-infected EVTs. Further investigation is required to confirm these findings in vivo.
Our reverse genetics experiments provide evidence that reduced expression of FOXO3a or FOXO1 downregulates but does not completely abrogate the invasive and migratory properties of human EVTs. Consistent with this, among the invasion- and migration-associated transcripts whose expression diminished in T. gondii-infected EVTs (i.e., MMP2, MMP3, MMP14, TIMP2, MUC1, and ITGB3), we only identified MMP3 and ITGB3 as transcriptional targets of FOXO3a and FOXO1, respectively. These data indicate that T. gondii also alters migration, invasion, and related transcriptional programs thorough FOXO3a- and FOXO1-independent mechanisms. For instance, the TGF-β1-SMAD2/3 signaling pathway, which is activated by T. gondii infection in macrophages (), was reported to reduce invasiveness by preventing invasion-related gene expression (i.e., connective tissue growth factor and kisspeptin) in of HTR-8/SVneo cells (; ). Similarly, the transcription factor GATA2 was described as a negative regulator of trophoblast invasion through MMP14 repression (Ogoyama et al., 2021). Surprisingly, our data revealed that the phosphorylated form of SMAD2 is not translocated to the nucleus and GATA2 expression does not increase in T. gondii-infected HTR-8/SVneo cells. Therefore, it is unlikely that T. gondii-driven repression of HTR-8/SVneo invasion is controlled by the TGF-β1-SMAD2/3 or GATA2 signaling pathways.
MMP2 is one of the most critical MMPs involved in EVTs invasion (Staun-Ram et al., 2004; Seval et al., 2004; Li et al., 2015). The MMP2 gene was identified as a FOXO3a transcriptional target in smooth muscle cells in response to NOX4 and urotensin-II signaling (). However, in our HTR-8/SVneo FOXO3a KD model, MMP2 mRNA accumulation did not appear to be regulated via FOXO3a. Nevertheless, we cannot exclude the possibility that T. gondii infection inhibits MMP2 expression through alternative mechanisms. Changes in mRNA levels observed in our screening could also stem from post-transcriptional regulatory mechanisms such as miRNA activity, which has been reported during T. gondii infection in human placental explants (Medina et al., 2020). Of note, miR-346 and miR-582-3p have been shown to reduce MMP2 mRNA expression and MMP2 activity as well as invasion and migration in the HTR-8/SVneo cell line (Su et al., 2017). Hence, miRNA-dependent repression of migration and invasion activities in T. gondii-infected EVTs, including but not limited to the downregulation of MMP2, deserves further investigation.
In addition to MMP2, we identified other FOXO3a-/FOXO1-independent genes whose downregulation during T. gondii infection may also contribute to impaired invasion and migration activities in EVTs (i.e., MMP14, TIMP2, and MUC1). In support of this notion, MMP14 and TIMP2 are known to form a complex that facilitates the activation of pro-MMP2 (Nishida et al., 2008). Accordingly, a decrease in both MMP14 and TIMP2 expression has been associated with impaired EVT invasion (Wang et al., 2014; Ogoyama et al., 2021; Onogi et al., 2011). Furthermore, genetic ablation of MMP3 was shown to inhibit the enzymatic activity of MMP2 in human umbilical vein endothelial cells (). These previous studies along with our findings warrant further investigation to determine whether in addition to transcriptional repression, T. gondii indirectly inhibits MMP2 by blocking MMP3 gene expression, as well as the activity of other migration- and invasion-related proteins in EVTs, through post-translational regulatory mechanisms.
In sum, we report a novel mechanism whereby T. gondii prevents migration and invasion activities in infected human trophoblast cell line HTR-8/SVneo that involves the inhibition of FOXO3a/1-dependent and -independent transcriptional mechanisms (Figure 8). Given that we only carried out a screening of a subset of invasion- and migration associated genes, future work using reverse and forward genetics combined with multi-omics approaches (i.e., RNA-seq, miR-seq, ChIP-seq, and proteomics) is likely to provide further insights into EVT dysregulation during T. gondii infection via FOXO3a/1-dependent- and independent transcriptional repression and alternative mechanisms, such as miRNAs and post-translational modifications. Importantly, this study highlights the disruption of central EVT functions during T. gondii infection in vitro and warrant further investigation using ex vivo and in vivo models, and primary cells to determine whether defects in EVT migration and invasion contribute to the pathogenesis of congenital toxoplasmosis.
Statements
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.
Ethics statement
Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.
Author contributions
AL: Conceptualization, Formal Analysis, Writing – review & editing, Investigation, Writing – original draft. L-PL: Investigation, Supervision, Writing – review & editing, Conceptualization, Formal Analysis, Writing – original draft. VC: Formal Analysis, Investigation, Writing – review & editing. SC: Conceptualization, Formal Analysis, Writing – review & editing, Investigation. CV: Conceptualization, Formal Analysis, Writing – review & editing, Methodology. MJ: Project administration, Funding acquisition, Conceptualization, Writing – original draft, Writing – review & editing, Supervision, Formal Analysis.
Funding
The author(s) declare financial support was received for the research and/or publication of this article. This work was supported by an operating grant from the Canadian Institutes of Health Research (CIHR) to MJ (PJT166017). MJ holds a salary award Senior from Fonds de Recherche du Québec en santé (FRQS). AL was supported by a doctoral scholarship Calmette Yersin from the Pasteur Network. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Acknowledgments
We are grateful to Jessie Tremblay for assistance with FACS, fluorescence microscopy experiments, and data analysis.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb.2025.1651142/full#supplementary-material
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Summary
Keywords
Toxoplasma gondii, extravillous trophoblasts, invasion, migration, FOXO3a, FOXO1, AKT
Citation
Lebourg A, Leroux L-P, Chaparro V, Chagneau S, Vaillancourt C and Jaramillo M (2025) Toxoplasma gondii infection inhibits invasion and migration of human extravillous trophoblasts through dysregulation of FOXO1- and FOXO3a-dependent and -independent mechanisms. Front. Cell. Infect. Microbiol. 15:1651142. doi: 10.3389/fcimb.2025.1651142
Received
20 June 2025
Revised
20 November 2025
Accepted
24 November 2025
Published
10 December 2025
Volume
15 - 2025
Edited by
Mario Alberto Rodriguez, Center for Research and Advanced Studies, National Polytechnic Institute of Mexico (CINVESTAV), Mexico
Reviewed by
Ji-Hun Shin, University of California, Irvine, United States
Aaron Martinez, University of Sonora, Mexico
Updates
Copyright
© 2025 Lebourg, Leroux, Chaparro, Chagneau, Vaillancourt and Jaramillo.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Maritza Jaramillo, maritza.jaramillo@inrs.ca
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