Trophoblast-macrophage crosstalk on human extravillous under Toxoplasma gondii infection.

INTRODUCTION: The interaction between human extravillous trophoblasts and macrophages has an important role in implantation and placentation. However, any dysfunction in this communication system is associated with pregnancy pitfalls, and a Toxoplasma gondii infection can be a potential problem in this crosstalk. Therefore, the aim of this study was to assess the influence of infected macrophages on cytokine production and the incidence of apoptosis in T. gondii-infected extravillous trophoblast cells. METHODS: HTR-8/SVneo cells were treated with supernatant from macrophages infected or not by T. gondii (conditioned medium) in order to analyze apoptosis and cytokine production in comparison to uninfected control conditions. RESULTS: The IL-6 secretion by HTR-8/SVneo cells increased synergistically by treatment with conditioned medium and T. gondii infection. The apoptosis index of HTR-8/SVneo cells was also upregulated by treatment with conditioned medium and infection. In addition, a low expression of Fas/CD95 and a high soluble FasL release were observed during infection, although no significant change was observed in the proliferation of T. gondii. DISCUSSION: The parasite modulates the high apoptosis index in HTR-8/SVneo cells in order to favor its establishment inside its host cells. On the other hand, the conditioned medium from uninfected macrophages restores the apoptosis rates, although the effect of the infection seems to be stronger. In conclusion, our results showed that T. gondii infection in human extravillous trophoblasts is able to modulate the trophoblast-macrophage crosstalk.

Susceptibility to Toxoplasma gondii proliferation in BeWo human trophoblast cells is dose-dependent of macrophage migration inhibitory factor (MIF), via ERK1/2 phosphorylation and prostaglandin E2 production.

INTRODUCTION: Macrophage migration inhibitory factor (MIF) participates in the immune response to Toxoplasma gondii, triggers ERK1/2 and prostaglandin E2 (PGE2) activation, but there is limited information on these mechanisms in human trophoblast. The present study aimed to verify the role of MIF in the ERK1/2 phosphorylation and PGE2 production, as well as its effect on the susceptibility to T. gondii in BeWo cells. METHODS: BeWo cells were treated with increasing concentrations of recombinant MIF (rMIF) and/or T. gondii-soluble tachyzoite antigen (STAg) and analyzed for ERK1/2 phosphorylation and PGE2 production by Western blotting and ELISA, respectively. Cells were also treated with increasing concentrations of rMIF, rPGE2, or ERK1/2 inhibitor and tested for T. gondii proliferation. The supernatants of cells treated with rPGE2 were assayed for cytokine production by ELISA or CBA. RESULTS: ERK1/2 phosphorylation and PGE2 production increased when the cells were treated with low MIF concentrations while the parasitism control occurred only at high MIF concentrations. STAg was unable to change ERK1/2 phosphorylation or PGE2 release. BeWo cells demonstrated increased T. gondii proliferation and reduced production of pro-inflammatory cytokines when treated with PGE2, while PD98059 diminished the parasite proliferation. DISCUSSION: The intracellular mechanisms triggered by MIF are dose-dependent in BeWo cells, and PGE2 is an important factor for the persistence of T. gondii at the maternal fetal interface. CONCLUSION: MIF was unable to control T. gondii infection in BeWo cells at low concentrations since ERK1/2 and PGE2 expression were activated, demonstrating a critical effect of these mediators favoring parasite proliferation.

Differential apoptosis in BeWo cells after infection with highly (RH) or moderately (ME49) virulent strains of Toxoplasma gondii is related to the cytokine profile secreted, the death receptor Fas expression and phosphorylated ERK1/2 expression.

INTRODUCTION: Alterations of apoptosis are commonly associated with pregnancy complications and abortion. Modulation of apoptosis is a relevant feature of Toxoplasma gondii infection and it is related to parasite strain types. The aim of the present study was to evaluate the possible factors that are involved in the differential apoptosis of BeWo cells infected with distinct T. gondii strain types. METHODS: Human trophoblastic cells (BeWo cell line) were infected with RH or ME49 strains, the cytokine production was measured and the phosphorylation of anti-apoptotic ERK1/2 protein was analyzed. Also, cells were treated with different cytokines, infected with RH or ME49 strain, and analyzed for apoptosis index and Fas/CD95 death receptor expression. RESULTS: ME49-infected BeWo cells exhibited a predominantly pro-inflammatory cytokine profile, whereas cells infected with RH strain had a higher production of anti-inflammatory cytokines. Also, the incidence of apoptosis was higher in ME49-infected cells, which have been treated with pro-inflammatory cytokines compared to cells infected with RH and treated with anti-inflammatory cytokines. Moreover, Fas/CD95 expression was higher in cells infected with either ME49 or RH strain and treated with pro-inflammatory cytokines compared to anti-inflammatory cytokine treatment. The phosphorylation of ERK1/2 protein increased after 24 h of infection only with the RH strain. CONCLUSION: These results suggest that opposing mechanisms of interference in apoptosis of BeWo cells after infection with RH or ME49 strains of T. gondii can be associated with the differential cytokine profile secreted, the Fas/CD95 expression and the phosphorylated ERK1/2 expression.

Trophoblast cells are able to regulate monocyte activity to control Toxoplasma gondii infection.

INTRODUCTION: Toxoplasma gondii is an intracellular parasite that causes severe disease when the infection occurs during pregnancy. Trophoblast cells constitute an important maternal-fetal barrier, with monocytes concentrating around them. Thus, interactions between trophoblasts and monocytes are important for maintaining a successful pregnancy, especially in cases of infection. This study aimed to evaluate the role of trophoblast cells (BeWo line) on monocyte (THP-1 line) activity in the presence or absence of T. gondii infection. METHODS: THP-1 cells were stimulated with supernatants of BeWo cells, previously infected or not with T. gondii, and then infected with parasites. The supernatant of both cells were collected and analyzed for cytokine production and T. gondii proliferation in THP-1 cells was determined. RESULTS: The results showed that after infection, the pattern of cytokines secreted by THP-1 and BeWo cells was characterized as a pro-inflammatory profile. Furthermore, supernatant of BeWo cells infected or not, was able to change the cytokine profile secreted by infected THP-1 cells, and this supernatant became THP-1 cells more able to control T. gondii proliferation than those that had not been stimulated. DISCUSSION: This effect was associated with secretion of interleukin (IL)-6 by the THP-1 cells and soluble factors secreted by BeWo cells, such as IL-6 and MIF. CONCLUSION: Together, these results suggest that trophoblast cells are able to modulate monocyte activity, resulting in the control of T. gondii infection and subsequent maintenance of pregnancy.

Azithromycin and spiramycin induce anti-inflammatory response in human trophoblastic (BeWo) cells infected by Toxoplasma gondii but are able to control infection.

Toxoplasma gondii is an important pathogen which may cause fetal infection if primary infection. Our previous studies have used human choriocarcinoma trophoblastic cells (BeWo cell line) as experimental model of T. gondii infection involving placental microenvironment. This study aimed to examine the effects of azithromycin and spiramycin against T. gondii infection in BeWo cells. Cells were treated with different concentrations of the macrolide antibiotics and analyzed first for cell viability using thiazolyl blue tetrazole (MTT) assay. As cell viability was significantly decreased with drug concentrations higher than 400 µg/mL, the concentration range used in further experiments was from 50 to 400 µg/mL. The number of infected cells and intracellular replication of T. gondii decreased after treatment with each drug. The infection induced up-regulation of the macrophage migration inhibitory factor (MIF), which was also enhanced in infected cells after treatment with azithromycin, but not with spiramycin. Analysis of the cytokine profile showed increase TNF-α, IL-10 and IL-4 production, but decreased IFN-γ levels, were detected in infected cells and treated with each drug. In conclusion, treatment of human trophoblastic BeWo cells with with azithromycin or spiramycin is able to control the infection and replication of T. gondii. In addition, treatment with these macrolides, especially with azityromycin induces an anti-inflammatory response and high MIF production, which can be important for the establishment and maintenance of a viable pregnancy during T. gondii infection.

Evaluation of vertical transmission of Toxoplasma gondii in Calomys callosus model after reinfection with heterologous and virulent strain.

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes a variety of clinical syndromes, but the infection is severe in immunocompromised individuals and during pregnancy due to the possibility of transplacental transmission of the parasite causing congenital toxoplasmosis. Vertical transmission of the parasite usually occurs when females are primarily infected during pregnancy. Calomys callosus is resistant to T. gondii ME49 strain, which presents a moderate virulence and congenital disease occurs only during the acute phase of infection. The aim of this study was to determine whether vertical transmission occurs when females of C. callosus chronically infected with ME49 strain of T. gondii are reinfected with a highly virulent strain (RH, type I). Females were infected with cysts of the ME49 strain. On the 1st day of pregnancy, animals were reinfected with tachyzoites of the RH strain. In the 19th day of pregnancy, placentas and embryos were processed for morphological analysis, immunohistochemistry and for detection of the parasite by PCR and mouse bioassay. Morphological and immunohistochemical analyses revealed the presence of parasites only in placental tissues. Mouse bioassay results showed seroconversion only in mice that were inoculated with placental tissues. Also, T. gondii DNA was detected only in placental samples. Congenital toxoplasmosis does not occur in C. callosus females chronically infected with the moderately virulent ME49 strain of T. gondii and reinfected with the highly virulent RH strain, thus indicating that primary T. gondii infection before pregnancy leads to an effective long-term immunity preventing transplacental transmission to the fetus.

Action of neuwiedase, a metalloproteinase isolated from bothrops neuwiedi venom, on skeletal muscle: an ultrastructural and immunocytochemistry study

The damaging effects of neuwiedase, a non-hemorrhagic snake venom metalloproteinase from P-I class, on gastrocnemius muscle are studied herein. Following neuwiedase injection, ultrastructural alterations were detected early showing disarrangement of skeletal muscle fibers (characterized by discontinuity of Z lines), mitochondrial swelling, and disruption of plasma membrane and basal lamina. Degradation of skeletal muscle and the appearance of an amorphous substance, primarily composed of cellular debris, were noted after 24 hours. The presence of neuwiedase at the injection site (detected by immunocytochemistry) revealed highly specific labeling of myofibril components of damaged myocytes. In addition, proteolysis of muscle proteins assayed through myofibrils extracted from gastrocnemius muscle indicated that neuwiedase provoked degradation of myofibrils, especially myosin. These results suggest that skeletal muscle damage, induced by neuwiedase, is probably due to its proteolytic action on myofibrils, which are responsible for the maintenance of the cellular architecture.

Apoptosis and S phase of the cell cycle in BeWo trophoblastic and HeLa cells are differentially modulated by Toxoplasma gondii strain types.

Transplacental transmission of Toxoplasma gondii causes congenital toxoplasmosis, one of the most severe forms of infection. The ability of the parasite to survive intracellularly largely depends on the blocking of different proapoptotic signaling cascades of the host cells. During pregnancy, however, alterations in the incidence of apoptosis are associated with abnormal placental morphology and function. The aim of this study was to evaluate the incidence of apoptosis and cell proliferation in trophoblastic (BeWo cell line) and uterine cervical (HeLa cell line) cells infected with a highly virulent RH strain or a moderately virulent ME49 strain of T. gondii. BeWo and HeLa cells were infected with RH or ME49 tachyzoites (2:1 and 5:1; parasite:cell) or medium alone (control). After 2 h, 6 h and 12 h of incubation, cells were fixed in 10% formalin and analyzed by immunohistochemistry to determine the apoptosis (expression of cytokeratin 18 neo-epitope--clone M30) and cell in S phase (expression of proliferating cell nuclear antigen--PCNA) indices. RH strain-infected BeWo and HeLa cells showed a lower apoptosis index than non-infected controls, whereas a higher apoptosis index was found in ME49 strain-infected cells compared to controls. In addition, RH-infected cells displayed lower apoptosis index than ME49-infected cells, even though active caspase-3 was detected in both cell types infected with either RH or ME49 strains as well in non-infected cells in all analyzed times of infection. Also, the cell S phase indices were higher in ME49 strain-infected BeWo and HeLa cells as compared to non-infected controls and RH strain-infected cells. These results indicate that RH and ME49 strains of T. gondii possess opposing mechanism of interference in apoptosis and cell cycle S phase of both BeWo and HeLa cells and these differences can be associated to evasion strategies of the parasite to survive inside the host cells.