Novel 3D human trophoblast culture to explore T. cruzi infection in the placenta.

Introduction: Human trophoblastic cell lines, such as BeWo, are commonly used in 2D models to study placental Trypanosoma cruzi infections. However, these models do not accurately represent natural infections. Three-dimensional (3D) microtissue cultures offer a more physiologically relevant in vitro model, mimicking tissue microarchitecture and providing an environment closer to natural infections. These 3D cultures exhibit functions such as cell proliferation, differentiation, morphogenesis, and gene expression that resemble in vivo conditions. Methods: We developed a 3D culture model using the human trophoblastic cell line BeWo and nonadherent agarose molds from the MicroTissues® 3D Petri Dish® system. Both small (12-256) and large (12-81) models were tested with varying initial cell numbers. We measured the diameter of the 3D cultures and evaluated cell viability using Trypan Blue dye. Trophoblast functionality was assessed by measuring ß-hCG production via ELISA. Cell fusion was evaluated using confocal microscopy, with Phalloidin or ZO-1 marking cell edges and DAPI staining nuclei. T. cruzi infection was assessed by microscopy and quantitative PCR, targeting the EF1-α gene for T. cruzi and GAPDH for BeWo cells, using three parasite strains: VD (isolated from a congenital Chagas disease infant and classified as Tc VI), and K98 and Pan4 (unrelated to congenital infection and classified as Tc I). Results: Seeding 1000 BeWo cells per microwell in the large model resulted in comparable cellular viability to 2D cultures, with a theoretical diameter of 408.68 ± 12.65 µm observed at 5 days. Functionality, assessed through ß-hCG production, exceeded levels in 2D cultures at both 3 and 5 days. T. cruzi infection was confirmed by qPCR and microscopy, showing parasite presence inside the cells for all three tested strains. The distribution and progression of the infection varied with each strain. Discussion: This innovative 3D model offers a simple yet effective approach for generating viable and functional cultures susceptible to T. cruzi infection, presenting significant potential for studying the placental microenvironment.

Plasmodium falciparum alters the trophoblastic barrier and stroma villi organization of human placental villi explants.

BACKGROUND: The sequestration of Plasmodium falciparum infected erythrocytes in the placenta, and the resulting inflammatory response affects maternal and child health. Despite existing information, little is known about the direct impact of P. falciparum on the placental barrier formed by trophoblast and villous stroma. This study aimed to assess placental tissue damage caused by P. falciparum in human placental explants (HPEs). METHODS: HPEs from chorionic villi obtained of human term placentas (n = 9) from normal pregnancies were exposed to P. falciparum-infected erythrocytes (IE) for 24 h. HPEs were embedded in paraffin blocks and used to study tissue damage through histopathological and histochemical analysis and apoptosis using TUNEL staining. Culture supernatants were collected to measure cytokine and angiogenic factors and to determine LDH activity as a marker of cytotoxicity. A subset of archived human term placenta paraffin-embedded blocks from pregnant women with malaria were used to confirm ex vivo findings. RESULTS: Plasmodium falciparum-IE significantly damages the trophoblast layer and the villous stroma of the chorionic villi. The increased LDH activity and pathological findings such as syncytial knots, fibrin deposits, infarction, trophoblast detachment, and collagen disorganization supported these findings. The specific damage to the trophoblast and the thickening of the subjacent basal lamina were more pronounced in the ex vivo infection. In contrast, apoptosis was higher in the in vivo infection. This disparity could be attributed to the duration of exposure to the infection, which significantly varied between individuals naturally exposed over time and the 24-h exposure in the ex vivo HPE model. CONCLUSION: Exposure to P. falciparum-IE induces a detachment of the syncytiotrophoblast, disorganization of the stroma villi, and an increase in apoptosis, alterations that may be associated with adverse results such as intrauterine growth restriction and low birth weight.

The trophoblast surface becomes refractory to adhesion by congenitally transmitted Toxoplasma gondii and Listeria monocytogenes during cytotrophoblast to syncytiotrophoblast development.

The placenta is a critical barrier against viral, bacterial, and eukaryotic pathogens. For most teratogenic pathogens, the precise molecular mechanisms of placental resistance are still being unraveled. Given the importance of understanding these mechanisms and challenges in replicating trophoblast-pathogen interactions using in vitro models, we tested an existing stem-cell-derived model of trophoblast development for its relevance to infection with Toxoplasma gondii. We grew human trophoblast stem cells (TSCT) under conditions leading to either syncytiotrophoblast (TSSYN) or cytotrophoblast (TSCYT) and infected them with T. gondii. We evaluated T. gondii proliferation and invasion, cell ultrastructure, as well as for transcriptome changes after infection. TSSYNs cells showed similar ultrastructure compared to primary cells and villous explants when analyzed by transmission electron microscopy and scanning electron microscopy (SEM), a resistance to T. gondii adhesion could be visualized on the SEM level. Furthermore, TSSYNs were highly refractory to parasite adhesion and replication, while TSCYTs were not. RNA-seq data on mock-treated and infected cells identified differences between cell types as well as how they responded to T. gondii infection. We also evaluated if TSSC-derived SYNs and CYTs had distinct resistance profiles to another vertically transmitted facultative intracellular pathogen, Listeria monocytogenes. We demonstrate that TSSYNs are highly resistant to L. monocytogenes, while TSCYTs are not. Like T. gondii, TSSYN resistance to L. monocytogenes was at the level of bacterial adhesion. Altogether, our data indicate that stem-cell-derived trophoblasts recapitulate resistance profiles of primary cells to T. gondii and highlight the critical importance of the placental surface in cell-autonomous resistance to teratogens.IMPORTANCECongenital toxoplasmosis can cause a devastating consequence to the fetus. To reach the fetus's tissues, Toxoplasma gondii must cross the placenta barrier. However, how this parasite crosses the placenta and the precise molecular mechanisms of placental resistance to this parasite are still unknown. In this study, we aimed to characterize a new cellular model of human trophoblast stem cells to determine their resistance, susceptibility, and response to T. gondii. Syncytiotrophoblast derived from trophoblast stem cells recapitulate the resistance profile similarly to placenta cells. We also showed that these cells are highly resistant to Listeria monocytogenes, at the level of bacterial adhesion. Our results suggest that resisting pathogen adhesion/attachment may be a generalized mechanism of syncytiotrophoblast resistance, and trophoblast stem cells represent a promising model to investigate cell-intrinsic mechanisms of resistance to pathogen adhesion and replication.

In vitro regulation of gene expression of pregnancy-associated proteins and cytokines in bovine endometrial epithelial cells and bovine trophoblastic cells by infection with Neospora caninum.

Abortion caused by the parasite Neospora caninum is an important threat to the livestock industry worldwide. Trophoblasts and caruncular cells play major roles in initiating innate immune responses and controlling parasite infection at the fetal-maternal interface. In the present study, bovine uterine epithelial cells (BUECs) and bovine trophoblastic (BT) cells treated with bovine interferon-gamma (IFN-γ), IFN-alpha (IFN-α) and IFN-tau (IFN-τ) followed by infection with N. caninum were examined by measuring the mRNA expression levels of numerous pregnancy-associated proteins and observing parasite growth to elucidate the host-parasite interaction at the uteroplacental region. N. caninum infection increased the expression of prolactin-related protein 1 (PRP1), pregnancy-associated glycoprotein 1 (PAG1), and cytokines (TNF-α, IL-8 and IL-10) in BUECs and of IL-8 in BT cells. Bovine IFN-γ inhibited IL-8 and TNF-α expression in BUECs and IL-8 in BT cells. In contrast, the expression of the interferon-stimulated gene OAS1 was significantly increased by treatment of the infected BT cells with IFN-γ. However, treatment with bovine IFNs did not inhibit N. caninum growth in either cell line. In conclusion, our results suggest that bovine IFN-γ plays a crucial role in control of pathogenesis in uterus and induction of inflammatory response in the placental region following N. caninum infection, rather than growth inhibition of the parasites.

BjussuLAAO-II, an l-amino acid oxidase from Bothrops jararacussu snake venom, impairs Toxoplasma gondii infection in human trophoblast cells and villous explants from the third trimester of pregnancy.

One-third of the world's population is estimated to be affected by toxoplasmosis. Pregnancy-related Toxoplasma gondii infection can cause vertical transmission, infect the fetus, and cause miscarriage, stillbirth, and fetal death. The current study showed that both human trophoblast cells (BeWo lineage) and human explant villous were resistant to T. gondii infection after incubation with BjussuLAAO-II, an l-amino acid oxidase isolated from Bothrops jararacussu. Almost 90% of the parasite's ability to proliferate in BeWo cells was decreased by the toxin at 1.56 µg/mL and showed an irreversible anti-T. gondii effect. Also, BjussuLAAO-II impaired the key events of adhesion and invasion of T. gondii tachyzoites in BeWo cells. BjussuLAAO-II antiparasitic properties were associated with the intracellular production of reactive oxygen species and hydrogen peroxide, since the presence of catalase restored the parasite's growth and invasion. In addition, T. gondii growth in human villous explants was decreased to approximately 51% by the toxin treatment at 12.5 µg/mL. Furthermore, BjussuLAAO-II treatment altered IL-6, IL-8, IL-10 and MIF cytokines levels, assuming a pro-inflammatory profile in the control of T. gondii infection. This study contributes to the potential use of a snake venom l-amino acid oxidase for the development of agents against congenital toxoplasmosis and the discovery of new targets in parasites and host cells.

BEWO trophoblast cells and Toxoplasma gondii infection modulate cell death mechanisms in THP-1 monocyte cells by interference in the expression of death receptor and intracellular proteins.

Crosstalk between trophoblast and monocytes is essential for gestational success, and it can be compromised in congenital toxoplasmosis. Cell death is one of the mechanisms involved in the maintenance of pregnancy, and this study aimed to evaluate the role of trophoblast in the modulation of monocyte cell death in the presence or absence of Toxoplasma gondii infection. THP-1 cells were stimulated with supernatants of BeWo cells and then infected or not with T. gondii. The supernatants were collected and analyzed for the secretion of human Fas ligand, and cells were used to determine cell death and apoptosis, cell death receptor, and intracellular proteins expression. Cell death and apoptosis index were higher in uninfected THP-1 cells stimulated with supernatants of BeWo cells; however, apoptosis index was reduced by T. gondii infection. Macrophage migration inhibitory factor (MIF) and transforming growth factor (TGF)-ß1, secreted by BeWo cells, altered the cell death and apoptosis rates in THP-1 cells. In infected THP-1 cells, the expression of Fas/CD95 and secretion of FasL was significantly higher; however, caspase 3 and phosphorylated extracellular-signal-regulated kinase (ERK1/2) were downregulated. Results suggest that soluble factors secreted by BeWo cells induce cell death and apoptosis in THP-1 cells, and Fas/CD95 can be involved in this process. On the other hand, T. gondii interferes in the mechanism of cell death and inhibits THP-1 cell apoptosis, which can be associated with active caspase 3 and phosphorylated ERK1/2. In conclusion, our results showed that human BeWo trophoblast cells and T. gondii infection modulate cell death in human THP-1 monocyte cells.

Cyclooxygenase (COX)-2 modulates Toxoplasma gondii infection, immune response and lipid droplets formation in human trophoblast cells and villous explants.

Congenital toxoplasmosis is represented by the transplacental passage of Toxoplasma gondii from the mother to the fetus. Our studies demonstrated that T. gondii developed mechanisms to evade of the host immune response, such as cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2) induction, and these mediators can be produced/stored in lipid droplets (LDs). The aim of this study was to evaluate the role of COX-2 and LDs during T. gondii infection in human trophoblast cells and villous explants. Our data demonstrated that COX-2 inhibitors decreased T. gondii replication in trophoblast cells and villous. In BeWo cells, the COX-2 inhibitors induced an increase of pro-inflammatory cytokines (IL-6 and MIF), and a decrease in anti-inflammatory cytokines (IL-4 and IL-10). In HTR-8/SVneo cells, the COX-2 inhibitors induced an increase of IL-6 and nitrite and decreased IL-4 and TGF-ß1. In villous explants, the COX-2 inhibitors increased MIF and decreased TNF-α and IL-10. Furthermore, T. gondii induced an increase in LDs in BeWo and HTR-8/SVneo, but COX-2 inhibitors reduced LDs in both cells type. We highlighted that COX-2 is a key factor to T. gondii proliferation in human trophoblast cells, since its inhibition induced a pro-inflammatory response capable of controlling parasitism and leading to a decrease in the availability of LDs, which are essentials for parasite growth.

ERK1/2 phosphorylation and IL-6 production are involved in the differential susceptibility to Toxoplasma gondii infection in three types of human (cyto/ syncytio/ extravillous) trophoblast cells.

During pregnancy, Toxoplasma gondii can triggers serious manifestations and potentially affect the fetal development. In this scenario, differences in susceptibility of trophoblast cells to T. gondii infection might be evaluated in order to establish new therapeutic approaches capable of interfering in the control of fetal infection by T. gondii. This study aimed to evaluate the susceptibility of cytotrophoblast, syncytiotrophoblast and extravillous trophoblast cells to T. gondii infection. Our data demonstrate that HTR-8/SVneo cells (extravillous trophoblast cells) present higher susceptibility to T. gondii infection when compared to syncytiotrophoblast and cytotrophoblast cells, whereas syncytiotrophoblast was the cell type more resistant to the parasite infection. Also, cytotrophoblast and syncytiotrophoblast cells produced significantly more IL-6 than HTR-8/SVneo cells. On the other hand, HTR-8/SVneo cells showed higher ERK1/2 phosphorylation than cytotrophoblast and syncytiotrophoblast cells. ERK1/2 inhibition reduced T. gondii infection and increased IL-6 production in HTR-8/SVneo cells. Thus, it is plausible to conclude that the greater susceptibility of HTR-8/SVneo cells to infection by T. gondii is related to a higher ERK1/2 phosphorylation and lower levels of IL-6 in these cells compared to other cells, suggesting that these mediators may be important to favor the parasite infection in this type of trophoblastic population.

Evaluation of Apoptosis, Proliferation, and Adhesion Molecule Expression in Trophoblastic Tissue of Women With Recurrent Spontaneous Abortion and Infected With Toxoplasma gondii.

Recurrent spontaneous abortion is an obstetric complication with undefined causes. Apoptosis, proliferation, and adhesion are considered important factors in the pathogenesis of abortion. This work aimed to determine Bax and Bcl-2 as a proapoptotic and antiapoptotic protein, Ki67 and P27kip as proliferative and antiproliferative proteins, and E-cadherin and CD44 as adhesion molecules in the trophoblastic tissues in cases with recurrent miscarriage. Immunohistochemistry and quantitative polymerase chain reaction analysis of Bax, Bcl-2, Ki67, P27kip, E-cadherin, and CD44 in paraffin-embedded sections of placental tissues obtained from 108 women were divided into 3 categories: 66 Toxoplasma gondii-positive women with recurrent abortion, 22 T. gondii-negative women with recurrent abortion, and 20 women with no history of abortion as a control group. The mean ratio of the expression of Bax and P27kip proteins was 35.3% and 36.1%, which is significantly higher than that of the second group (19.88 and 20.02%), and the third group (12.3% and 10.98%), while the mean ratio of the expression of Bcl-2, Ki67, E-cadherin, and CD44 proteins was 12.35%, 11.23%, 10.32%, and 9.97%, which is significantly lower than that of the second group (33.75%, 13.18%, 21.88%, and 23.29%) and that of the third group (38.58%, 39.27%, 37.98%, and 35.79%). The presence of proapoptotic protein (Bax) and antiproliferative protein (P27kip) at high levels and the presence of antiapoptotic protein (Bcl-2), proliferative protein (Ki67), and adhesion molecules (E-cadherin and CD44) in lower levels in the T. gondii-positive group clarify the mechanism involved in the induction of abortion and loss of pregnancy.

Copaifera spp. oleoresins impair Toxoplasma gondii infection in both human trophoblastic cells and human placental explants.

The combination of pyrimethamine and sulfadiazine is the standard care in cases of congenital toxoplasmosis. However, therapy with these drugs is associated with severe and sometimes life-threatening side effects. The investigation of phytotherapeutic alternatives to treat parasitic diseases without acute toxicity is essential for the advancement of current therapeutic practices. The present study investigates the antiparasitic effects of oleoresins from different species of Copaifera genus against T. gondii. Oleoresins from C. reticulata, C. duckei, C. paupera, and C. pubiflora were used to treat human trophoblastic cells (BeWo cells) and human villous explants infected with T. gondii. Our results demonstrated that oleoresins were able to reduce T. gondii intracellular proliferation, adhesion, and invasion. We observed an irreversible concentration-dependent antiparasitic action in infected BeWo cells, as well as parasite cell cycle arrest in the S/M phase. The oleoresins altered the host cell environment by modulation of ROS, IL-6, and MIF production in BeWo cells. Also, Copaifera oleoresins reduced parasite replication and TNF-α release in villous explants. Anti-T. gondii effects triggered by the oleoresins are associated with immunomodulation of the host cells, as well as, direct action on parasites.

Adenosine A3 Receptor Mediates ERK1/2- and JNK-Dependent TNF-α Production in Toxoplasma gondii-Infected HTR8/SVneo Human Extravillous Trophoblast Cells.

Toxoplasma gondii is an intracellular parasite that causes severe disease when the infection occurs during pregnancy. Adenosine is a purine nucleoside involved in numerous physiological processes; however, the role of adenosine receptors in T. gondii-induced trophoblast cell function has not been investigated until now. The goal of the present study was to evaluate the intracellular signaling pathways regulated by adenosine receptors using a HTR-8/SVneo trophoblast cell model of T. gondii infection. HTR8/SVneo human extravillous trophoblast cells were infected with or without T. gondii and then evaluated for cell morphology, intracellular proliferation of the parasite, adenosine receptor expression, TNF-α production and mitogen-activated protein (MAP) kinase signaling pathways triggered by adenosine A3 receptor (A3AR). HTR8/SVneo cells infected with T. gondii exhibited an altered cytoskeletal changes, an increased infection rate and reduced viability in an infection time-dependent manner. T. gondii significantly promoted increased TNF-α production, A3AR protein levels and p38, ERK1/2 and JNK phosphorylation compared to those observed in uninfected control cells. Moreover, the inhibition of A3AR by A3AR siRNA transfection apparently suppressed the T. gondii infection-mediated upregulation of TNF-α, A3AR production and MAPK activation. In addition, T. gondii-promoted TNF-α secretion was dramatically attenuated by pretreatment with PD098059 or SP600125. These results indicate that A3AR-mediated activation of ERK1/2 and JNK positively regulates TNF-α secretion in T. gondii-infected HTR8/SVneo cells.

Inflammasome activation and IL-1 signaling during placental malaria induce poor pregnancy outcomes.

Placental malaria (PM) is associated with severe inflammation leading to abortion, preterm delivery, and intrauterine growth restriction. Innate immunity responses play critical roles, but the mechanisms underlying placental immunopathology are still unclear. Here, we investigated the role of inflammasome activation in PM by scrutinizing human placenta samples from an endemic area and ablating inflammasome components in a PM mouse model. The reduction in birth weight in babies from infected mothers is paralleled by increased placental expression of AIM2 and NLRP3 inflammasomes. Using genetic dissection, we reveal that inflammasome activation pathways are involved in the production and detrimental action of interleukin-1ß (IL-1ß) in the infected placenta. The IL-1R pharmacological antagonist Anakinra improved pregnancy outcomes by restoring fetal growth and reducing resorption in an experimental model. These findings unveil that IL-1ß-mediated signaling is a determinant of PM pathogenesis, suggesting that IL-1R antagonists can improve clinical outcomes of malaria infection in pregnancy.

Measuring mitochondrial respiration in adherent cells infected with Trypanosoma cruzi Chagas, 1909 using Seahorse extracellular flux analyser.

Infection with Trypanosoma cruzi Chagas, 1909 is reported to increase the production of reactive oxygen species in patients with Chagas disease. Mitochondria dysfunction, host inflammatory response and inadequate antioxidant response are described as the main factors leading to oxidative stress during acute and chronic stages of the disease. The Seahorse XFe24 extracellular flux platform allows energy metabolism determination through mitochondrial respiration and glycolysis measurements. XFe24 platform can be used in in vitro models of T. cruzi-infected cells, which allow the assessment and even modulation of endogenous conditions of infected cells, generating readouts of real-time cellular bioenergetics changes. In this protocol, we standardised the use of XFe24 technology in T. cruzi infected AC16 cardiomyocytes and SGHPL-5 trophoblasts. In addition, we provide a list of optimised assay specifications, advantages and critical steps to be considered during the process. Cardiomyocytes and trophoblasts are attractive target cells to evaluate the metabolic environment in acute, chronic and congenital Chagas transmission scenarios.

Immune response profile of caruncular and trophoblast cell lines infected by high- (Nc-Spain7) and low-virulence (Nc-Spain1H) isolates of Neospora caninum.

BACKGROUND: Bovine neosporosis, one of the main causes of reproductive failure in cattle worldwide, poses a challenge for the immune system of pregnant cows. Changes in the Th-1/Th-2 balance in the placenta during gestation have been associated with abortion. Cotyledon and caruncle cell layers form the maternal-foetal interface in the placenta and are able to recognize and induce immune responses against Neospora caninum among other pathogens. The objective of the present work was to elucidate the immunomodulation produced by high- (Nc-Spain7) and low-virulence (Nc-Spain1H) isolates of N. caninum in bovine trophoblast (F3) and caruncular cells (BCEC-1) at early and late points after infection. Variations in the mRNA expression levels of toll-like receptor-2 (TLR-2), Th1 and Th2 cytokines (IL-4, IL-10, IL-8, IL-6, IL-12p40, IL-17, IFN-γ, TGF-ß1, TNF-α), and endothelial adhesion molecules (ICAM-1 and VCAM-1) were investigated by RT-qPCR, and protein variations in culture supernatants were investigated by ELISA. RESULTS: A similar pattern of modulation was found in both cell lines. The most upregulated cytokines in infected cells were pro-inflammatory TNF-α (P < 0.05-0.0001) and IL-8 (P < 0.05-0.001) whereas regulatory IL-6 (P < 0.05-0.001) and TGF-ß1 (P < 0.05-0.001) were downregulated in both cell lines. The measurement of secreted IL-6, IL-8 and TNF-α confirmed the mRNA expression level results. Differences between isolates were found in the mRNA expression levels of TLR-2 (P < 0.05) in both cell lines and in the mRNA expression levels (P < 0.05) and protein secretion of TNF-α (P < 0.05), which were higher in the trophoblast cell line (F3) infected with the low-virulence isolate Nc-Spain1H. CONCLUSIONS: Neospora caninum infection is shown to favor a pro-inflammatory response in placental target cells in vitro. In addition, significant immunomodulation differences were observed between high- and low-virulence isolates, which would partially explain the differences in virulence.

Susceptibility of human villous (BeWo) and extravillous (HTR-8/SVneo) trophoblast cells to Toxoplasma gondii infection is modulated by intracellular iron availability.

Congenital toxoplasmosis is a serious health problem that can lead to miscarriage. HTR-8/SVneo is a first trimester extravillous trophoblast, while BeWo is a choriocarcinoma with properties of villous trophoblast cells. In the placenta, iron is taken up from Fe-transferrin through the transferrin receptor being the ion an important nutrient during pregnancy and also for Toxoplasma gondii proliferation. The aim of this study was to evaluate the role of iron in T. gondii proliferation in BeWo and HTR-8/SVneo cells and in human chorionic villous explants. The cells were infected with T. gondii, iron supplemented or deprived by holo-transferrin or deferoxamine, respectively, and parasite proliferation and genes related to iron balance were analyzed. It was verified that the addition of holo-transferrin increased, and DFO decreased the parasite multiplication in both trophoblastic cells, however, in a more expressive manner in HTR-8/SVneo, indicating that the parasite depends on iron storage in trophoblastic cells for its growth. Also, tachyzoites pretread with DFO proliferate normally in trophoblastic cells demonstrating that DFO itself does not interfere with parasite proliferation. Additionally, T. gondii infection induced enhancement in transferrin receptor mRNA expression levels in trophoblastic cells, and the expression was higher in HTR-8/SVneo compared with BeWo. Finally, DFO-treatment was able to reduce the parasite replication in villous explants. Thus, the iron supplementation can be a double-edged sword; in one hand, it could improve the supplement of an essential ion to embryo/fetus development, and on the other hand, could improve the parasite proliferation enhancing the risk of congenital infection.

Human Placental Syncytiotrophoblasts Restrict Toxoplasma gondii Attachment and Replication and Respond to Infection by Producing Immunomodulatory Chemokines.

Toxoplasma gondii is a major source of congenital disease worldwide, but the cellular and molecular factors associated with its vertical transmission are largely unknown. In humans, the placenta forms the key interface between the maternal and fetal compartments and forms the primary barrier that restricts the hematogenous spread of microorganisms. Here, we utilized primary human trophoblast (PHT) cells isolated from full-term placentas and human midgestation chorionic villous explants to determine the mechanisms by which human trophoblasts restrict and respond to T. gondii infection. We show that placental syncytiotrophoblasts, multinucleated cells that are in direct contact with maternal blood, restrict T. gondii infection at two distinct stages of the parasite lytic cycle-at the time of attachment and also during intracellular replication. Utilizing comparative transcriptome sequencing (RNA-seq) transcriptional profiling, we also show that human placental trophoblasts from both the second and third trimesters respond uniquely to T. gondii infection compared to trophoblast cell lines, typified by the upregulation of several immunity-related genes. One of the most differentially induced genes was the chemokine CCL22, which relies on the secretion of a parasite effector(s) either during or after invasion for its induction. Collectively, our findings provide new insights into the mechanisms by which the human placenta restricts the vertical transmission of T. gondii at early and late stages of human pregnancy and demonstrate the existence of at least two interferon-independent pathways that restrict T. gondii access to the fetal compartment.IMPORTANCEToxoplasma gondii is a major source of congenital disease worldwide and must breach the placental barrier to be transmitted from maternal blood to the developing fetus. The events associated with the vertical transmission of T. gondii are largely unknown. Here, we show that primary human syncytiotrophoblasts, the fetus-derived cells that comprise the primary placental barrier, restrict T. gondii infection at two distinct stages of the parasite life cycle and respond to infection by inducing a unique immunomodulatory transcriptional profile. Collectively, our findings provide important insights into the mechanisms by which human syncytiotrophoblasts restrict T. gondii infection at early and late stages of human pregnancy, identify both permissive and resistant human placental cell types, and identify the placenta-enriched signaling pathways induced in response to infection.

Differential susceptibility of bovine caruncular and trophoblast cell lines to infection with high and low virulence isolates of Neospora caninum.

BACKGROUND: Neospora caninum, one of the main causes of abortion in cattle, is very effective at crossing the placental barrier and placental damage is crucial in the pathogenesis of abortion. Bovine trophoblast and caruncular cell layers are key cellular components in the maternal-foetal interface in placentomes, playing a fundamental role in placental functionality. METHODS: We studied tachyzoite adhesion, invasion, proliferation and egress of high- (Nc-Spain7) and low- (Nc-Spain1H) virulence N. caninum isolates in established cultures of bovine caruncular epithelial (BCEC-1) and trophoblast (F3) cells. The parasite invasion rate (pInvR) and the cell infection rate (cInfR) were determined by immunostaining plaque assay at different time points and multiplicities of infection (MOIs), respectively. In addition, tachyzoite growth kinetics were investigated using real-time PCR (qPCR) analysis and immunostaining plaque assay at different times. RESULTS: Neospora caninum invaded and proliferated in both cell lines. The pInvR was higher in F3 compared to BCEC-1 cells for the Nc-Spain7 isolate (P < 0.05), and higher for the Nc-Spain7 than the Nc-Spain1H in F3 cells (P < 0.01). The cInfR was also higher in F3 cells than in BCEC-1 cells for both isolates (P < 0.0001), and the cInfR for the Nc-Spain7 isolate was higher than for the Nc-Spain1H isolate in both cell lines (P < 0.05). Tachyzoite growth kinetics showed tachyzoite exponential growth until egress at 58 hpi for both isolates in F3, whereas Nc-Spain1H showed a non-exponential growth pattern in BCEC-1. Asynchronous egress of both isolates was observed from 22 h post-infection onwards in BCEC-1. In addition, the tachyzoite yield (TY58h) was higher in F3 than in BCEC-1 infected by both isolates (P < 0.0001), highlighting better replication abilities of both parasites in F3. Nc-Spain7 showed shorter doubling times and higher TY58h compared to Nc-Spain1H in F3 cells; adhesion, invasion and proliferation mechanisms were very similar for both isolates in BCEC-1. CONCLUSIONS: Our results indicate a highly similar behavior of high- and low-virulence isolates in their interactions with maternal caruncular cells and suggest an important role of foetal trophoblasts in the pathogenesis of N. caninum infection.

Transcriptome modulation of bovine trophoblast cells in vitro by Neospora caninum.

Neospora caninum is one of the most efficient transplacentally transmitted pathogens in cattle and is a cause of abortion in this domestic species. The invasion and proliferation of Neospora caninum in the placenta and its dissemination to the foetus are crucial events in the outcome of an infection. In the bovine placenta, the placentomes are formed by maternal caruncles, which are delimited by a maternal epithelium and foetal cotyledons, which are delimited by an epithelial layer named the trophoblast. These epithelia form a physical barrier against foetal infection. Furthermore, trophoblast cells act as an innate immune defence at the foetal-maternal interface. Neospora caninum invades and proliferates in trophoblast cells in vitro, but it is unknown whether host cell modulation events, which affect the immune response and other processes in the trophoblast, occur. In this work, we investigated the transcriptomic modulation by Neospora caninum infection in the bovine trophoblast cell line F3. In addition, two Neospora caninum isolates with marked differences in virulence, Nc-Spain1H and the Nc-Spain7, were used in this study to investigate the influence of these isolates in F3 modulation. The results showed a clear influence on extracellular matrix reorganisation, cholesterol biosynthesis and the transcription factor AP-1 network. Interestingly, although differences in the transcriptome profiles induced by each isolate were observed, specific isolate-modulated processes were not identified, suggesting very similar regulation in both isolates. Differential expression of the N. caninum genes between both isolates was also investigated. Genes involved in host cell attachment and invasion (SAG-related and microneme proteins), glideosome, rhoptries, metabolic processes, cell cycle and stress response were differentially expressed between the isolates, which could explain their variability. This study provides a global view of Neospora caninum interactions with bovine trophoblast cells and of the intra-specific differences between two Neospora caninum isolates with biological differences.

Enrofloxacin and Toltrazuril Are Able to Reduce Toxoplasma gondii Growth in Human BeWo Trophoblastic Cells and Villous Explants from Human Third Trimester Pregnancy.

Classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic strategies is indispensable to minimize the side effects and improve the control of infection. Previous studies demonstrated that enrofloxacin and toltrazuril reduced the incidence of Neospora caninum and Toxoplasma gondii infection. The aim of the present study was to evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii infection in human trophoblast cells (BeWo line) and in human villous explants from the third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine, or combination of sulfadiazine+pyrimethamine, and the cellular or tissue viability was verified. Next, BeWo cells were infected by T. gondii (2F1 clone or the ME49 strain), whereas villous samples were only infected by the 2F1 clone. Then, infected cells and villous were treated with all antibiotics and the T. gondii intracellular proliferation as well as the cytokine production were analyzed. Finally, we evaluated the direct effect of enrofloxacin and toltrazuril in tachyzoites to verify possible changes in parasite structure. Enrofloxacin and toltrazuril did not decrease the viability of cells and villous in lower concentrations. Both drugs were able to significantly reduce the parasite intracellular proliferation in BeWo cells and villous explants when compared to untreated conditions. Regardless of the T. gondii strain, BeWo cells infected and treated with enrofloxacin or toltrazuril induced high levels of IL-6 and MIF. In villous explants, enrofloxacin induced high MIF production. Finally, the drugs increased the number of unviable parasites and triggered damage to tachyzoite structure. Taken together, it can be concluded that enrofloxacin and toltrazuril are able to control T. gondii infection in BeWo cells and villous explants, probably by a direct action on the host cells and parasites, which leads to modifications of cytokine release and tachyzoite structure.

Azithromycin treatment is able to control the infection by two genotypes of Toxoplasma gondii in human trophoblast BeWo cells.

Trophoblast infection by Toxoplasma gondii plays a pivotal role in the vertical transmission of toxoplasmosis. Here, we investigate whether the antibiotic therapy with azithromycin, spiramycin and sulfadiazine/pyrimethamine are effective to control trophoblast infection by two Brazilian T. gondii genotypes, TgChBrUD1 or TgChBrUD2. Two antibiotic protocols were evaluated, as follow: i) pre-treatment of T. gondii-tachyzoites with selected antibiotics prior trophoblast infection and ii) post-treatment of infected trophoblasts. The infection index/replication and the impact of the antibiotic therapy on the cytokine milieu were characterized. It was observed that TgChBrUD2 infection induced lower infection index/replication as compared to TgChBrUD1. Regardless the therapeutic protocol, azithromycin was more effective to control the trophoblast infection with both genotypes when compared to conventional antibiotics. Azithromycin induced higher IL-12 production in TgChBrUD1-infected cells that may synergize the anti-parasitic effect. In contrast, the effectiveness of azithromycin to control the TgChBrUD2-infection was not associated with the IL-12 production. BeWo-trophoblasts display distinct susceptibility to T. gondii genotypes and the azithromycin treatment showed to be more effective than conventional antibiotics to control the T. gondii infection/replication regardless the parasite genotype.