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BeWo Trophoblasts are Unable to Control Replication of Toxoplasma gondii, Even in the Presence of Exogenous IFN-γ
Placenta (2006), 27, 691e698 doi:10.1016/j.placenta.2005.06.006
BeWo Trophoblasts are Unable to Control Replication of Toxoplasma gondii, Even in the Presence of Exogenous IFN-g J. G. Oliveiraa, N. M. Silvab, A. A. D. Santosa, M. A. Souzab, G. L. S. Ferreiraa, J. R. Mineob,* and E. A. V. Ferroa a Laboratory of Histology and Embriology, Instituto de Cieˆncias Biome´dicas, Universidade Federal de Uberlaˆndia, Av. Para´ 1720, Uberlaˆndia, Minas Gerais, Brazil 38400-902; b Laboratory of Immunoparasitology, Instituto de Cieˆncias Biome´dicas, Universidade Federal de Uberlaˆndia, Av. Para´ 1720, Uberlaˆndia, Minas Gerais, Brazil 38400-902 Paper accepted 13 June 2005
The ability of RH strain of Toxoplasma gondii to invade and grow into BeWo cells was investigated in the present study using IFN-g, L-tryptophan, or a-methyl-tryptophan treatments. HeLa cells were used in the same conditions for comparison purposes. It was demonstrated that BeWo cells are more permissive to T. gondii infection, making them more susceptible to this pathogen when compared to HeLa cells. Infection rates of BeWo cells do not show any significant alteration in different protocols using IFN-g. In addition, BeWo treated with L-tryptophan was unable to significantly increase parasite growth. In contrast, HeLa cells treated with IFN-g or IFN-g plus L-tryptophan are able to impair or increase, respectively, parasite replication, providing evidence that this indoleamine-2,3-dioxygenase-dependent phenomenon is operant in these cells, whereas it is inactive in BeWo. Therefore, our data support the hypothesis that the immunological mechanisms controlling infection at the maternalefetal interface are different from those occurring in the periphery. At the same time that operating regulatory mechanisms work inside and outside the cells located at that microenvironment to prevent maternal rejection of the concept, these events might facilitate the progression of infection caused by intracellular pathogens, as T. gondii. Placenta (2006), 27, 691e698 Ó 2005 Elsevier Ltd. All rights reserved. Keywords: T. gondii; BeWo; HeLa; Infection rates; Maternalefetal interface
INTRODUCTION Toxoplasma gondii is an obligated intracellular coccidian and an important opportunistic pathogen for a wide range of host including human beings. In human fetus, toxoplasmosis is associated with severe congenital defects when the primary infection is acquired during the first trimester of pregnancy [1]. Control for this infection is a result of complex and compartmented immunological mechanisms. Cellular immunity is considered the key component of the host immune response, because it is responsible to control T. gondii replication [2,3]. In mice, the activation of macrophages by IFN-g in the presence of costimulators, such as LPS or TNF-a, is necessary to trigger the cytotoxic activity of the macrophages against T. gondii [4]. The inhibition of Toxoplasma replication or its destruction is the result of various mechanisms such as: (i) oxidative mechanism [5,6]; (ii) non-oxidative mechanism, represented by production of nitrogen monoxide (NO) by * Corresponding author. Tel.: C55 34 3218 2195; fax: C55 34 3218 2333. E-mail address: [email protected] (J.R. Mineo). 0143e4004/$esee front matter
macrophages activated by IFN-g [7e9]; (iii) non-oxygendependent mechanisms may also be toxoplasmicidal, such as the induction by IFN-g of indoleamine-2,3-dioxygenase (IDO), which degrade the tryptophan required for parasite growth [10]. When IFN-g is administered in vivo, significant protection against T. gondii is observed in a mouse model of toxoplasmosis [11,12]. These observations suggest that IFN-g is of primary importance in host resistance against T. gondii. However, the production of pro-inflammatory cytokines is harmful to the success of pregnancy [13,14]. During pregnancy occurs immune modulation, resulting in tolerance of the fetal allograft. This is manifested by a decreased production of IFN-g leading towards a Th2-type immune response by secretion of regulatory cytokines in the placenta. Also, there is evidence for induction of maternal T-cell tolerance to the fetal allograft by tryptophan deprivation as a result of constitutive expression of the tryptophan-degrading enzyme IDO in placental trophoblast cell [15]. IDO expression has been demonstrated in human placental trophoblast [16]. The aim of this work was to verify the BeWo cells susceptibility to T. gondii, and the role played by IFN-g to control the infection in this trophoblastic cell line. Attempting to answer these questions, an in vitro model of infection from Ó 2005 Elsevier Ltd. All rights reserved.
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a lineage of trophoblastic BeWo cells was performed. BeWo cells originate from a naturally occurring human choriocarcinoma and they express HLA-G transcripts and express functional IFN-g receptors [17,18].
0.1 M phosphate buffer (pH 7.4), dehydrated in ethanol, and embedded in Epon resin. Ultrathin sections were stained with lead citrate and uranyl acetate, and they were then observed in a Zeiss EM 109 electron microscope.
MATERIALS AND METHODS
Immunocytochemical studies
Parasites
Immunolocalization of the parasites in infected BeWo and HeLa cells cultured on glass coverslips was carried out in accordance with the following protocol: (i) the samples were incubated for 10 min at room temperature with 5% acetic acid to block endogenous alkaline phosphatase; (ii) in order to block nonspecific binding sites, the samples were also treated with 2% normal goat serum diluted in PBS with 0.05 M Tris, pH 7.4 (TBS) for 30 min at 37 (C; (iii) the preparations were incubated for 12 h at 4 (C with rabbit anti-T. gondii serum. Negative controls were carried out by irrelevant primary antibodies (anti-collagen type IV); (iv) the preparations were then rinsed in TBS and incubated with biotinylated goat antirabbit immunoglobulin G (Sigma Chemical CO., St. Louis, MO) for 30 min at 37 (C; (v) the reaction signal was amplified by using the ABC system (Biomeda, Foster City, USA), developed with fast redenaphthol (Sigma Chemical CO., St. Louis, USA), and counterstained with Mayer’s hematoxylin.
The highly virulent type I RH strain of T. gondii was used in all experiments and was maintained by serial passage in Swiss mice by a standard procedure as previously described [19]. Cell culture and infection BeWo (American Type Culture Collection, USA) (2 ! 104 cells/mL) and HeLa (American Type Culture Collection, USA) (2 ! 104 cells/mL) cells were seeded on round glass coverslips placed into 24-well plates in a volume of 300 mL of RPMI-1640 supplemented with 10% FBS, 0.37% of sodium bicarbonate, 10,000 U/mL of penicillin and 10 mg/mL of streptomycin (Cultilab, Brazil), L-glutamine, essential aminoacids, sodium pyruvate and 2-b-mercaptoethanol (Sigma, CO, USA). After allowed to adhere for 72 h, the cells were washed and infected by T. gondii at a proportion of 10 parasites per cell and incubated for 3 h at 37 (C in 5% CO2. Extracellular non-adherent parasites were removed by washing. After 24 h, the adherent cells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), washed in water and stained with Giemsa (Labssynth, Diadema-S.P., Brazil), 5 drops/mL of distillated water for 40 min, washed, and mounted on glass slides and analyzed by light microscope. The results were expressed as infection rates under light microscopy by the following parameters: (a) percentage of infected cells or with attached parasite per 100 cells; (b) the average number of parasites per cell; and (c) the average of total number of parasites per 100 cells. Two independent experiments were performed in triplicate and the infection rates were determined by two observers. Treatment with anti-TgSAG-1 (p30) monoclonal antibody To determine if the TgSAG-1 (p30) antigen may interfere with the infection of BeWo cells by T. gondii, as observed for human fibroblast and murine enterocytes [19], monoclonal antibody to TgSAG-1 was used in the present study. BeWo and HeLa cells were cultured for 72 h, when the medium was removed and the cells were fixed or not with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at 4 (C. Prior to the infection, parasites were treated for 30 min with antiTgSAG-1 (p30) monoclonal antibody (E9 clone, Laboratory of Immunoparasitology from Universidade Federal de Uberlaˆndia, MG, Brazil) or medium only. Fixed or unfixed cells were incubated with T. gondii at concentrations of 10 parasites per cell for 3 or 24 h, respectively, at 37 (C, and analyzed by immunocytochemistry to measure attachment and/or invasion. Alternatively, live cells were fixed in 2.5% glutaraldehyde in
Treatment of BeWo and HeLa cells with IFN-g, L-tryptophan or a-methyl-tryptophan After 72 h of culture, BeWo and HeLa cells were submitted to different conditions. In a first set of experiments, BeWo and HeLa cells were treated or not with increasing concentrations of human recombinant IFN-g (PeproTech Inc, Rocky Hill, USA) from 100 to 500 U/mL and incubated for additional 24 h. The medium was then removed from the wells, and the cells were infected with T. gondii at a proportion of 10 parasites/cell for 3 h at 37 (C in 5% CO2 and again treated or not with human recombinant IFN-g. The extracellular and unadherent parasites were removed and the culture was incubated for a further 24 h in each one of the following conditions: (i) treated with medium only; (ii) treated with 1 mM of L-tryptophan; or (iii) treated with 1 mM of a-methyl-tryptophan. After the incubation, the cells were fixed and stained with Giemsa (Labssynth, Diadema-S.P., Brazil), 5 drops/mL of distillated water for 40 min, washed, and mounted on glass slides. The infection rates were analyzed by light microscopy. Nitrite determination The nitrite concentrations in the cell culture supernatants were determined by Griess assay [20]. Briefly, 100 ml of samples were added to each well of a 96-well plate, and 100 ml of 1:1 mixture for 1% sulfanilamide dihydrochloride in 2.5% and 0.1% naphthylenediamide dihydrochloride in H3PO4 was added to the samples. The A550 was determined with a microplate reader (Titertek Multiskan plus MK 11, Labsystems, Finlaˆndia) with reference to a standard curve for concentrations of sodium nitrite (Sigma chemical CO., St.
Oliveira et al.: Interaction Between BeWo Trophoblast and T. gondii
Louis, USA) from 0.1 to 50 nmol. Each experiment was conducted in triplicate and repeated at least twice. Statistical analysis In all experiments data were expressed as mean G SD and the statistical differences between groups were determined using the unpaired Student’s t-test (Graph Pad Software, v. 4.0, San Diego, USA). Differences were considered statistically significant when P ! 0.05. RESULTS BeWo and HeLa cells do not present any differential morphological alteration after being infected by T. gondii As shown in Figure 1, both BeWo and HeLa cells did not show any significant morphological alterations on their general cellular architecture or differences in the patterns of organelle distribution after being infected by T. gondii. BeWo cells are characterized for showing threadlike projection in the membrane, cytoplasm with large deposits of glycogen, free polysomes, prominent granular endoplasmic reticulum, decondensed chromatin nucleus and evident nucleolus. BeWo cells with intracellular parasites showed numerous mitochondria next to the membrane of the parasitophorous vacuole (Figure 1G). Infection rates of BeWo and HeLa cells are equally affected by parasites treated with anti-TgSAG-1 antibody After 24 h of infection with T. gondii previously treated with anti-TgSAG-1 mAb, BeWo cells showed significant reduction in the percentage of infected cells, the average number of parasites per cell, and the total number of parasites, when compared with untreated parasites (Figure 2AeC). Similarly, HeLa cells showed significant decrease in all analyzed infection rates, when they were infected with parasites previously treated with anti-TgSAG-1 mAb (Figure 2AeC). Both BeWo and HeLa fixed cells were allowed to interact for 3 h with T. gondii previously treated with anti-TgSAG-1 mAb. It was observed that the percentages of cells harboring attached parasites and the average numbers of parasites per cell were considerably reduced when compared with untreated parasites, but the degree of inhibition was indistinguishable between both cell lineages (Figure 2D, E). BeWo cells fail to control replication of T. gondii in the presence of exogenous IFN-g The infection rates analyzed after 24 h of T. gondii interaction showed that BeWo presented higher susceptibility to the parasites when compared with HeLa cells. In spite of treatment from 100 to 500 U/mL of IFN-g (data not shown), BeWo presented a higher number of parasites per cell and parasite growth than HeLa cells in the same condition (Figure 3), showing that BeWo cells failed to control T. gondii growth
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in response to IFN-g treatment. IFN-g concentration of 100 U/mL was chosen for all other experiments. When BeWo cells were treated with IFN-g before addition of T. gondii and after L-tryptophan supplementation, the same pattern of infection of cells treated with IFN-g only was observed. In contrast, the addition of L-tryptophan to HeLa cells pre-treated with IFN-g antagonized the protective effect of this cytokine (Figure 3AeC). Interestingly, when both lineages of cells were treated with IFN-g plus a-methyl-tryptophan, a competitive inhibitor of IDO, a smaller parasitism was observed compared with cells treated with IFN-g plus Ltryptophan. When both cells were treated twice with IFN-g, before and after infection, it was observed that BeWo cells did not get control of the parasite growth, while HeLa cells presented an efficient control of the parasite replication (Table 1). These differences were due to the unresponsiveness of BeWo cells and the responsiveness of HeLa cells to IFN-g treatment, generating a reduction of almost 50% of parasite growth in the latter cell lineage. Moreover, BeWo cells receiving double treatment with IFN-g plus L-tryptophan showed no significant alteration in infection rates, whereas parasite growth was entirely restored in HeLa cells submitted to the same condition (Table 1). Under effect of double treatment with IFN-g plus amethyl-tryptophan, however, both BeWo and HeLa cells responded with a decrease in the number of parasites per cell and in the total number of parasites when compared with L-tryptophan treatment or even with medium only (Table 1). The effect of a-methyl-tryptophan treatment in both cell lines probably may be due to direct interference in the mechanisms of T. gondii replication, since it will overplus the tryptophan up-take, a critical aminoacid for parasite metabolism. Even though the production of nitrite was not observed on IFN-g unstimulated or stimulated BeWo and HeLa cells infected by T. gondii (data not shown), taken as a whole, the present results suggest that BeWo are more susceptible to T. gondii infection than HeLa cells. DISCUSSION Experimental models show that T. gondii can attach to and penetrate in any nucleated cells [21], including trophoblast cells of rodents [22,23] and human villous trophoblast [24]. Trophoblast cells are key components on the hemochorial human placenta, since they have been an interface between the mother and the baby. There are intracellular mechanisms in these cells that regulate their patterns of responses when exposed to various stimuli, leading to secretion of a variety of cytokines. These mechanisms are especially important since critical balance between deciduous invasion versus operating mechanisms prevents fetal rejection. In vitro and in vivo evidences suggest that IFN-g is unable to induce MHC class II expression in trophoblastic cells. Recent findings obtained with JEG 3 (a subclone of BeWo) show that this phenomenon does not take place because of receptor loss to IFN-g, which is
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Figure 1. Photomicrographs showing BeWo and HeLa cells in culture and its interaction with T. gondii: (A) HeLa cells after 72 h of culture and (B) after 24 h of infection with T. gondii (arrowhead), stained with Giemsa. Bar, 23 mm. (C) HeLa cells after 72 h of culture and 3 h of interaction with T. gondii stained by alkaline phosphatase substrate in the immunocytochemistry assay (arrowhead). Bar, 9 mm. (D) BeWo cells after 72 h of culture and (E) after 24 h of infection with T. gondii (arrowhead), stained with Giemsa. Bar, 23 mm. (F) BeWo cells after 72 h of culture and 3 h of infection with T. gondii stained by alkaline phosphatase substrate in the immunocytochemistry assay (arrowhead). Bar, 9 mm. (G) Electromicrograph from BeWo cells after 3 h of co-culture with T. gondii, showing tachyzoite inside a parasitophorous vacuole (arrows) surrounded by numerous mitochondrias (m) of the host cell. Bar scale: 1 mm.
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B
A P = 0.0051 P = 0.0002
50
25
0 Host cells BeWo HeLa Parasites Untreated Anti-TgSAG-1
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D 750
Percentage of cells with attached parasites (mean ± SD)
Total number of parasites per 100 cells (mean ± SD)
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0 Host cells BeWo HeLa Parasites Untreated Anti-TgSAG-1
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75
P = 0.0028 P = 0.0043
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0 Fixed host cells BeWo HeLa Parasites Untreated Anti-TgSAG-1
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P = 0.0023
E Number of attached parasites per cell (mean ± SD)
P = 0.0007
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Number of parasites per cell (mean ± SD)
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75
P = 0.0008
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Figure 2. Effect of anti-TgSAG-1 (p30) mAb treatment on T. gondii attachment and invasion in BeWo (closed bar) and HeLa (open bar) cells. Anti-TgSAG-1 treated or untreated parasites were incubated with both fixed or live host cells for 3 or 24 h, respectively. The following parameters were measured: A: percentage of infected cells; B: average number of parasites per live cell; C: total number of parasites per 100 live cells; D: percentage of fixed cells harboring attached parasites; E: number of attached parasites per fixed cell.
constitutively present in these cells, but due to regulatory events that follow the binding of this cytokine to its receptor, either causing specifically a negative regulation or silencing MHC class II transactivators [25].
In the present investigation, the progression of BeWo cells infection by T. gondii was studied. We have found that BeWo cells are more permissive to infection to this parasite than HeLa cells.
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Percentage of infected cells (mean ± SD)
A
P = 0.0002
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P = 0.0385
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Number of parasites per infected cell (mean ± SD)
B
P = 0.0007
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Total number of parasites per 100 cells (mean ± SD)
C 1000
P = 0.0006
P < 0.0001 P = 0.0093
P = 0.0247
P = 0.0055
P = 0.0020
750 500 250 0
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Figure 3. Infection rates due to T. gondii in BeWo and HeLa cells: effect of single treatment with IFN-g (100 U/mL) plus L-tryptophan or a-methyltryptophan during parasiteehost cell interactions. Cells were infected with T. gondii tachyzoites at a ratio of 10:1 parasites per cell. After 24 h of infection, they were fixed, stained by Giemsa stain, and analyzed. The following infection rates were measured: A: percentage of infected cells per 100 cells; B: average number of parasites per cell; C: total number of parasites per 100 cells.
Possible innate resistance and mechanisms available to trophoblast include the induction of oxygen reactive [26], nitrogen intermediates [7,27,28], and the enzyme indoleamine2,3-dioxygenase (IDO) that converts tryptophan to kynurenine [29] or alternative mechanisms [30]. Our results showed no difference of nitrite production in BeWo and HeLa cell culture supernatants and it means that the difference found for T. gondii growth in both cells is not related with nitric oxide. Without any treatment, HeLa cells were more resistant to T. gondii infection. T. gondii is probably able to evade defense parasiticidal mechanisms of BeWo cells, making them more susceptible to this pathogen. In the present study, treatment with recombinant IFN-g resulted in an inhibitory effect of intracellular tachyzoite growth only in HeLa cells. Treatment of BeWo and HeLa cells with IFN-g did not affect the percentage of infected cells, although in HeLa cells it induced a smaller number of parasites per cell, suggesting a control role to the parasitic growth. The failure of BeWo cells to control the number of T. gondii per cell in response to IFN-g treatment is unusual, since this is a common host defense mechanism against this pathogen. Indeed, the failure of IFN-g to control parasite growth in BeWo cells was not limited to T. gondii but also to other microorganisms [29]. Probably, the mechanisms of response to intracellular pathogens involve alternative pathways that do not involve the IFN-g in BeWo cells. We and others have demonstrated that one of the mechanisms to control T. gondii replication induced by IFN-g is L-tryptophan degradation, through induction of IDO, either in vivo or in vitro [10,31e36]. In addition, the mechanisms of IDO induction dependent on IFN-g were already investigated in many cell types, including HeLa cells [29,32,37e40]. Thus, in the present study, HeLa cells were utilized to compare with BeWo cells. We observed that HeLa cells submitted to IFN-g and L-tryptophan treatments showed different behavior when compared to BeWo cells under the same condition. In HeLa cells, the L-tryptophan treatment was capable to increase the average number of parasite and therefore, restore completely the inhibition given by IFN-g demonstrating that the antitoxoplasmic mechanism in HeLa cells is dependent on IFN-g. The compound a-methyl-tryptophan has been reported to be a potent competitive inhibitor of IDO activity when tested in vitro using purified enzyme [41], as well as in vivo at the maternalefetal interface [15]. The addition of this inhibitor did not significantly change the percentage of infected cells for BeWo and HeLa cells. However, when both cells were treated with IFN-g followed by a-methyl-tryptophan, a meaningful decrease was observed in the number of parasites per cell and in the total number of parasites compared with IFN-g plus L-tryptophan treatment or medium only. These results were more remarkable in double treatment schedule and might be due to the fact that a-methyl-tryptophan is used by the parasite instead of L-tryptophan in its process of protein synthesis. It is already known that L-tryptophan is an essential residue for some parasite components, where a single substitution is enough to produce a considerable disturbance in parasite cytoskeleton [42].
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Table 1. Effect of double treatment with IFN-g plus L-tryptophan or a-methyl-tryptophan in the infection rates due to T. gondii tachyzoites in BeWo and HeLa cells BeWo
HeLa
Host cells
Infection rates
Experimental conditions
Percentage of infected cells
Number of parasites per cell
Total number of parasites
Percentage of infected cells
Number of parasites per cell
Total number of parasites
Medium only Double treatment with IFN-g only Double treatment with IFN-g plus L-trp Double treatment with IFN-g plus a-m-trp
47.7 G 4.2 51.7 G 11.0 41.0 G 7.8 38.0 G 6.0
14.5 G 3.4 11.9 G 1.5 15.1 G 2.8e 10.3 G 1.4i
657.0 G 127.3 606.3 G 99.9 604.0 G 39.6f 401.7 G 27.5j
36.7 G 9.5 31.0 G 4.6 35.3 G 4.9 36.0 G 3.0
11.0 G 1.1a 6.9 G 1.0c 14.5 G 1.3g 8.2 G 0.9k
408.6 G 37.6b 212.3 G 24.5d 507.0 G 29.5h 292.7 G 6.5l
Results are expressed as mean G SD of two independent experiments in triplicate. Statistically significant results were obtained with the following comparisons: a versus c (P Z 0.0091); c versus g (P Z 0.0013); g versus k (P Z 0.0020); e versus i (P Z 0.0055); f versus j (P Z 0.0018); b versus d (P Z 0.0108); d versus h (P Z 0.0002); h versus l (P Z 0.0003).
The intracellular response seen in BeWo cells is quite intriguing and apparently not dependent on mechanisms directly linked to IFN-g, but relate to pathways unrelated to IFN-g stimuli. As intracellular tryptophan concentration probably achieves its optimal values for the parasite in this system, yielding an intracellular beneficial environment to T. gondii, no augment of replication was observed after further addition of this aminoacid. BeWo cells do not express IDO constitutively or in response to IFN-g treatment [29]. This feature makes these cells particularly attractive to different species of intracellular pathogens. In spite of the differences found for BeWo and HeLa cells concerning the intracellular parasite replication, in the present study we observed similar mechanism of invasion of both cells by the parasite, since TgSAG-1 was effectively blocked by specific antibodies, as seen in other host cells [19]. It was possible to observe formation of the parasitophorous vacuole (PV) inside the BeWo and HeLa cells with mitochondrias associated with the PV membrane after interaction with T. gondii. Vacuoles containing T. gondii completely avoid fusion with normal host endocytic and exocytic vesicles, and the parasite replicates within this protected intracellular niche [43]. Our data support the idea that immunological control of pathogens in the placental trophoblast should be different from that occurring in the periphery. Regulatory mechanisms operating inside and outside the cell allow that at the same time that they prevent maternal rejection to the concept, they turn this microenvironment particularly susceptible to intracellular pathogens as T. gondii. Indeed, the increase in
susceptibility of the BeWo cells to T. gondii infection in our model may be explained by the hypothesis that claims the existence of areas potentially susceptible to the parasite infection. In this context, recent studies have elegantly shown that IDO is expressed in different cell populations from both human [44,45] and murine [46] placenta. Considering that the IDO expression is not uniform in placenta, the areas with small expression of this enzyme turn out to be potentially more susceptible to infection by intracellular parasites [16]. Abortions may be caused by fetal infection as a result of different regulatory mechanisms and reflecting a strong connection between hormonal levels and patterns of cytokine secretion [47]. High levels of inflammatory cytokines, such as TNF-a, IL-2 and IFN-g may have abortive potential [14]. Even though the present work presented results of an in vitro system, further studies to be carried out on placenta pathologies or abortions induced by T. gondii can elucidate some of the immunoregulatory processes involved in the maintenance of a normal pregnancy. This is the first report describing that T. gondii tachyzoites continues to replicate inside the trophoblast cells of the cell line BeWo even in the presence of IFN-g and also that there was no inhibitory effect to reverse when L-tryptophan was added in IFN-g-treated cells. These data lead to the conclusion that, even though the unresponsiveness to IFN-g in trophoblastic cells is a worthy event to the host to prevent allogeneic fetal rejection, it may also be considered detrimental to the host by allowing the replication of parasites that can achieve fetal tissues.
ACKNOWLEDGMENTS This work was supported by the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), FAPEMIG and CAPES. REFERENCES [1] Remington JS, McLeod R, Thulliez P, Desmonts G. In: Remington JS, Klein OJ, editors. Infectious diseases of the fetus and newborn infant, 5th ed. Philadelphia: W. B. Saunders; 2001, p. 205e356.
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characterisation of Toxoplasma antigen, infiltrates and major histocompatibility complex gene products. J Neuroimmunol 2001;31:185e98. Sibley LD, Adams LB, Krahenbuhl JL. Macrophage interactions in toxoplasmosis. Res Immunol 1993;144:38e40. Hughes HPA. Oxidative killing of intracellular parasites mediated by macrophages. Parasitol Today 1988;4:340e7. Murray HW, Cohn ZA. Macrophage oxygen-dependent anti-microbial activity. I. Susceptibility of Toxoplasma gondii to oxygen intermediates. J Exp Med 1979;150:938e49. Adams LB, Hibbs JB, Taintor Jr RR, Krahenbuhl JL. Microbiostatic effect of murine-activates macrophages for Toxoplasma gondii. Role for synthesis of nitrogen oxides from L arginine. J Immunol 1990;144: 2725e9. Ding A, Nathan C, Stuher D. Release of reactives nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. J Immunol 1988;141:2407e12. Drapier J, Wietzerbin J, Hibbs J. Interferon-gamma and tumor necrosis factor induce the L-arginine-dependent cytotoxic effector mechanism in murine macrophages. Eur J Immunol 1988;18:1587e92. Pfefferkorn ER, Rebhun S, Eckel M. Characterization of an indoleamine2,3-dioxygenase induced by gamma-interferon in cultures human fibroblasts. J Interferon Res 1986;6:267e79. McCabe RE, Luft BJ, Remington JS. Effect of murine interferon gamma on murine toxoplasmosis. J Infect Dis 1984;150:961e2. Suzuki Y, Orellana MA, Robbert DS, Jack SR. Interferon-g: the major mediator of resistance against Toxoplasma gondii. Science 1988;240: 516e8. Dealtry GB, O’Farrell MK, Fernandez N. The Th2 cytokine environment of the placenta. Int Arch Allergy Immunol 2000;123:107e19. Ragupathy R. Th1-type immunity is incompatible with successful pregnancy. Immunol Today 1997;18:478e82. Munn DH, Zhou M, Attwood JT, Bondarev I, Conway SJ, Marshall B, et al. Prevention of allogeneic fetal rejection by tryptophan catabolism. Science 1998;281:1191e3. Kudo Y, Boyd CAR. Human placental indoleamine 2,3-dioxigenase: cellular localization and characterization of an enzyme preventing fetal infection. Biochim Biophys Acta 2000;1500:119e24. Ellis SA, Palmer MS, McMichael AJ. Human trophoblast and the choriocarcinoma cell line BeWo express a truncated HLA class I molecule. J Immunol 1990;144:731e5. Fulop V, Steller MA, Berkowitz RS, Anderson DJ. Interferon-gamma receptors on human gestational choriocarcinoma cell lines: quantitative and functional studies. Am J Obstet Gynecol 1992;167:524e30. Mineo JR, Kasper LH. Attachment of Toxoplasma gondii to host cells involves major surface protein, SAG-1 (P30). Exp Parasitol 1994;79:11e20. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal Biochem 1982;126:131e8. Tenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii from animals to humans. Int J Parasitol 2000;30:1217e58. Ferro EAV, Bevilacqua E, Favoreto-Junior S, Silva DAO, Mortara RA, Mineo JR. Calomys callosus (Rodentia: Cricetidae) trophoblast cells as host cells to Toxoplasma gondii in early pregnancy. Parasitol Res 1999;85: 647e54. Ferro EAV, Silva DA, Bevilacqua E, Mineo JR. Effect of Toxoplasma gondii infection kinetics on trophoblast cell population in Calomys callosus, a model of congenital toxoplasmosis. Infect Immun 2002;70: 7089e94. Abbasi M, Kowalewska-Grochowska K, Bahar MA, Kilani RT, WinklerLowen B, Guilbert LJ. Infection of placental trophoblasts by Toxoplasma gondii. J Infect Dis 2003;188:608e16. Entrican G. Immune regulation during pregnancy and hoste pathogen interactions in infectious abortion. J Comp Pathol 2002;126: 79e94. Arsenijevic D, Onuma H, Pecqueur C, Raimbault S, Manning BS, Miroux B. Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production. Nat Genet 2000;26: 435e9. Alexander I, Scharton-Kersten TM, Yap G, Roberts CW, Liew FY, Sher A. Mechanisms of innate resistance to Toxoplasma gondii infection. Philos Trans R Soc Lond B Biol Sci 1997;352:1355e9.
[28] Smith SC, Guilbert LJ, Yui J, Baker PN, Davidge ST. The role of reactive nitrogen/oxygen intermediates in cytokine-induced trophoblast apoptosis. Placenta 1999;20:309e15. [29] Entrican G, Wattegedera S, Chui M, Oemar L, Rocchi M, McInnes C. Gamma interferon fails to induce expression of indoleamine 2,3dioxigenase and does not control the growth of Chlamydophila abortus in BeWo trophoblast cells. Infect Immun 2002;70:2690e3. [30] Dimier IH, Bout DT. Inhibition of Toxoplasma gondii replication in IFNgamma-activated human intestinal epithelial cells. Immunol Cell Biol 1997;75:511e4. [31] Silva NM, Rodrigues CV, Santoro MM, Reis LFL, Alvarez-Leite JI, Gazzinelli RT. Expression of indoleamine 2,3-dioxygenase, tryptophan degradation and kynurenine formation during in vivo infection with Toxoplasma gondii: induction by endogenous IFN-g and requirement of IRF-1. Infect Immun 2002;70:859e68. [32] Da¨ubener W, Sports B, Hucke C, Adam R, Stins M, Kim KS, et al. Restriction of Toxoplasma gondii growth in human brain microvascular endothelial cells by activation of indoleamine 2,3-dioxygenase. Infect Immun 2001;69:6527e31. [33] Cera´volo IP, Chaves ACL, Bonjardim CA, Sibley D, Romanha AJ, Gazzinelli RT. Replication of Toxoplasma gondii, but not Trypanosoma cruzi, is regulated in human fibroblasts activated with gamma interferon: requirement of a functional JAK/STAT pathway. Infect Immun 1999;67: 2233e40. [34] Nagineni CN, Pardhasaradhi K, Martins MC, Detrick B, Hooks JJ. Mechanisms of interferon-induced inhibition of Toxoplasma gondii replication in human retinal pigment epithelial cells. Infect Immun 1996; 64:4188e96. [35] Murray HW, Szuro-Sudol A, Wellner D, Oca MJ, Granger AM, Libby DM, et al. Role of tryptophan degradation in respiratory burstindependent antimicrobial activity of gamma interferon-stimulated human macrophages. Infect Immun 1989;57:845e9. [36] Schmitz JL, Carlin JM, Borden EC, Byrne GI. Beta interferon inhibits Toxoplasma gondii growth in human monocyte-derived macrophages. Infect Immun 1989;57:3254e6. [37] Frumento G, Rotondo R, Tonetti M, Ferrara GB. T cell proliferation is blocked by indoleamine 2,3-dioxygenase. Transplant Proc 2001;33: 428e30. [38] Grohmann U, Fallarino F, Bianchi R, Belladonna ML, Vacca C, Orabona C, et al. IL-6 inhibits the tolerogenic function of CD8 alphaC dendritic cells expressing indoleamine 2,3-dioxygenase. J Immunol 2001; 167:708e14. [39] Hwu P, Du MX, Lapointe R, Do M, Taylor MW, Young HA. Indoleamine 2,3-dioxygenase production by human dendritic cells results in the inhibition of T cell proliferation. J Immunol 2000;164:3596e9. [40] Werner-Felmayer G, Werner ER, Fuchs D, Hausen A, Reibnegger G, Wachter H. Characteristics of interferon induced tryptophan metabolism in human cells in vitro. Biochim Biophys Acta 1989;1012:140e7. [41] Cady SG, Sono M. 1-Methyl-DL-tryptophan, beta-(3-benzofuranyl)-D-Lalanine (the oxygen analog of tryptophan), and beta-[3-benzo(b)thienyl]D-L-alanine (the sulfur anolog of tryptophan) are competitive inhibitors for indoleamine 2,3-dioxigenase. Arch Biochem Biophys 1991;291: 326e33. [42] Jewett TJ, Sibley LD. Aldolase forms a bridge between cell surface adhesins and the actin cytoskeleton in apicomplexan parasites. Mol Cell 2003;11:885e94. [43] Sibley LD. Intracellular parasite invasion strategies. Science 2004;304: 248e53. [44] Kudo Y, Hara T, Katsuki T, Toyofuku A, Katsura Y, Takikawa O, et al. Mechanisms regulating the expression of indoleamine 2,3-dioxygenase during decidualization of human endometrium. Hum Reprod 2004;19: 1222e30. [45] Honig A, Rieger L, Dietl J, Kammerer U. Mechanisms regulating the expression of indoleamine 2,3-dioxygenase during decidualization of human endometrium. Hum Reprod 2004;19:2683e4. [46] Baban B, Chandler P, McCool D, Marshall B, Munn DH, Mellor AL. Indoleamine 2,3-dioxygenase expression is restricted to fetal trophoblast giant cells during murine gestation and is maternal genome specific. J Reprod Immunol 2004;6:67e77. [47] Entrican G, Buxton D, Longbottom D. Chlamydial infection in sheep: immune control versus fetal pathology. J R Soc Med 2001;94: 273e7.
BeWo Trophoblasts are Unable to Control Replication of Toxoplasma gondii, Even in the Presence of Exogenous IFN-g J. G. Oliveiraa, N. M. Silvab, A. A. D. Santosa, M. A. Souzab, G. L. S. Ferreiraa, J. R. Mineob,* and E. A. V. Ferroa a Laboratory of Histology and Embriology, Instituto de Cieˆncias Biome´dicas, Universidade Federal de Uberlaˆndia, Av. Para´ 1720, Uberlaˆndia, Minas Gerais, Brazil 38400-902; b Laboratory of Immunoparasitology, Instituto de Cieˆncias Biome´dicas, Universidade Federal de Uberlaˆndia, Av. Para´ 1720, Uberlaˆndia, Minas Gerais, Brazil 38400-902 Paper accepted 13 June 2005
The ability of RH strain of Toxoplasma gondii to invade and grow into BeWo cells was investigated in the present study using IFN-g, L-tryptophan, or a-methyl-tryptophan treatments. HeLa cells were used in the same conditions for comparison purposes. It was demonstrated that BeWo cells are more permissive to T. gondii infection, making them more susceptible to this pathogen when compared to HeLa cells. Infection rates of BeWo cells do not show any significant alteration in different protocols using IFN-g. In addition, BeWo treated with L-tryptophan was unable to significantly increase parasite growth. In contrast, HeLa cells treated with IFN-g or IFN-g plus L-tryptophan are able to impair or increase, respectively, parasite replication, providing evidence that this indoleamine-2,3-dioxygenase-dependent phenomenon is operant in these cells, whereas it is inactive in BeWo. Therefore, our data support the hypothesis that the immunological mechanisms controlling infection at the maternalefetal interface are different from those occurring in the periphery. At the same time that operating regulatory mechanisms work inside and outside the cells located at that microenvironment to prevent maternal rejection of the concept, these events might facilitate the progression of infection caused by intracellular pathogens, as T. gondii. Placenta (2006), 27, 691e698 Ó 2005 Elsevier Ltd. All rights reserved. Keywords: T. gondii; BeWo; HeLa; Infection rates; Maternalefetal interface
INTRODUCTION Toxoplasma gondii is an obligated intracellular coccidian and an important opportunistic pathogen for a wide range of host including human beings. In human fetus, toxoplasmosis is associated with severe congenital defects when the primary infection is acquired during the first trimester of pregnancy [1]. Control for this infection is a result of complex and compartmented immunological mechanisms. Cellular immunity is considered the key component of the host immune response, because it is responsible to control T. gondii replication [2,3]. In mice, the activation of macrophages by IFN-g in the presence of costimulators, such as LPS or TNF-a, is necessary to trigger the cytotoxic activity of the macrophages against T. gondii [4]. The inhibition of Toxoplasma replication or its destruction is the result of various mechanisms such as: (i) oxidative mechanism [5,6]; (ii) non-oxidative mechanism, represented by production of nitrogen monoxide (NO) by * Corresponding author. Tel.: C55 34 3218 2195; fax: C55 34 3218 2333. E-mail address: [email protected] (J.R. Mineo). 0143e4004/$esee front matter
macrophages activated by IFN-g [7e9]; (iii) non-oxygendependent mechanisms may also be toxoplasmicidal, such as the induction by IFN-g of indoleamine-2,3-dioxygenase (IDO), which degrade the tryptophan required for parasite growth [10]. When IFN-g is administered in vivo, significant protection against T. gondii is observed in a mouse model of toxoplasmosis [11,12]. These observations suggest that IFN-g is of primary importance in host resistance against T. gondii. However, the production of pro-inflammatory cytokines is harmful to the success of pregnancy [13,14]. During pregnancy occurs immune modulation, resulting in tolerance of the fetal allograft. This is manifested by a decreased production of IFN-g leading towards a Th2-type immune response by secretion of regulatory cytokines in the placenta. Also, there is evidence for induction of maternal T-cell tolerance to the fetal allograft by tryptophan deprivation as a result of constitutive expression of the tryptophan-degrading enzyme IDO in placental trophoblast cell [15]. IDO expression has been demonstrated in human placental trophoblast [16]. The aim of this work was to verify the BeWo cells susceptibility to T. gondii, and the role played by IFN-g to control the infection in this trophoblastic cell line. Attempting to answer these questions, an in vitro model of infection from Ó 2005 Elsevier Ltd. All rights reserved.
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a lineage of trophoblastic BeWo cells was performed. BeWo cells originate from a naturally occurring human choriocarcinoma and they express HLA-G transcripts and express functional IFN-g receptors [17,18].
0.1 M phosphate buffer (pH 7.4), dehydrated in ethanol, and embedded in Epon resin. Ultrathin sections were stained with lead citrate and uranyl acetate, and they were then observed in a Zeiss EM 109 electron microscope.
MATERIALS AND METHODS
Immunocytochemical studies
Parasites
Immunolocalization of the parasites in infected BeWo and HeLa cells cultured on glass coverslips was carried out in accordance with the following protocol: (i) the samples were incubated for 10 min at room temperature with 5% acetic acid to block endogenous alkaline phosphatase; (ii) in order to block nonspecific binding sites, the samples were also treated with 2% normal goat serum diluted in PBS with 0.05 M Tris, pH 7.4 (TBS) for 30 min at 37 (C; (iii) the preparations were incubated for 12 h at 4 (C with rabbit anti-T. gondii serum. Negative controls were carried out by irrelevant primary antibodies (anti-collagen type IV); (iv) the preparations were then rinsed in TBS and incubated with biotinylated goat antirabbit immunoglobulin G (Sigma Chemical CO., St. Louis, MO) for 30 min at 37 (C; (v) the reaction signal was amplified by using the ABC system (Biomeda, Foster City, USA), developed with fast redenaphthol (Sigma Chemical CO., St. Louis, USA), and counterstained with Mayer’s hematoxylin.
The highly virulent type I RH strain of T. gondii was used in all experiments and was maintained by serial passage in Swiss mice by a standard procedure as previously described [19]. Cell culture and infection BeWo (American Type Culture Collection, USA) (2 ! 104 cells/mL) and HeLa (American Type Culture Collection, USA) (2 ! 104 cells/mL) cells were seeded on round glass coverslips placed into 24-well plates in a volume of 300 mL of RPMI-1640 supplemented with 10% FBS, 0.37% of sodium bicarbonate, 10,000 U/mL of penicillin and 10 mg/mL of streptomycin (Cultilab, Brazil), L-glutamine, essential aminoacids, sodium pyruvate and 2-b-mercaptoethanol (Sigma, CO, USA). After allowed to adhere for 72 h, the cells were washed and infected by T. gondii at a proportion of 10 parasites per cell and incubated for 3 h at 37 (C in 5% CO2. Extracellular non-adherent parasites were removed by washing. After 24 h, the adherent cells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), washed in water and stained with Giemsa (Labssynth, Diadema-S.P., Brazil), 5 drops/mL of distillated water for 40 min, washed, and mounted on glass slides and analyzed by light microscope. The results were expressed as infection rates under light microscopy by the following parameters: (a) percentage of infected cells or with attached parasite per 100 cells; (b) the average number of parasites per cell; and (c) the average of total number of parasites per 100 cells. Two independent experiments were performed in triplicate and the infection rates were determined by two observers. Treatment with anti-TgSAG-1 (p30) monoclonal antibody To determine if the TgSAG-1 (p30) antigen may interfere with the infection of BeWo cells by T. gondii, as observed for human fibroblast and murine enterocytes [19], monoclonal antibody to TgSAG-1 was used in the present study. BeWo and HeLa cells were cultured for 72 h, when the medium was removed and the cells were fixed or not with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at 4 (C. Prior to the infection, parasites were treated for 30 min with antiTgSAG-1 (p30) monoclonal antibody (E9 clone, Laboratory of Immunoparasitology from Universidade Federal de Uberlaˆndia, MG, Brazil) or medium only. Fixed or unfixed cells were incubated with T. gondii at concentrations of 10 parasites per cell for 3 or 24 h, respectively, at 37 (C, and analyzed by immunocytochemistry to measure attachment and/or invasion. Alternatively, live cells were fixed in 2.5% glutaraldehyde in
Treatment of BeWo and HeLa cells with IFN-g, L-tryptophan or a-methyl-tryptophan After 72 h of culture, BeWo and HeLa cells were submitted to different conditions. In a first set of experiments, BeWo and HeLa cells were treated or not with increasing concentrations of human recombinant IFN-g (PeproTech Inc, Rocky Hill, USA) from 100 to 500 U/mL and incubated for additional 24 h. The medium was then removed from the wells, and the cells were infected with T. gondii at a proportion of 10 parasites/cell for 3 h at 37 (C in 5% CO2 and again treated or not with human recombinant IFN-g. The extracellular and unadherent parasites were removed and the culture was incubated for a further 24 h in each one of the following conditions: (i) treated with medium only; (ii) treated with 1 mM of L-tryptophan; or (iii) treated with 1 mM of a-methyl-tryptophan. After the incubation, the cells were fixed and stained with Giemsa (Labssynth, Diadema-S.P., Brazil), 5 drops/mL of distillated water for 40 min, washed, and mounted on glass slides. The infection rates were analyzed by light microscopy. Nitrite determination The nitrite concentrations in the cell culture supernatants were determined by Griess assay [20]. Briefly, 100 ml of samples were added to each well of a 96-well plate, and 100 ml of 1:1 mixture for 1% sulfanilamide dihydrochloride in 2.5% and 0.1% naphthylenediamide dihydrochloride in H3PO4 was added to the samples. The A550 was determined with a microplate reader (Titertek Multiskan plus MK 11, Labsystems, Finlaˆndia) with reference to a standard curve for concentrations of sodium nitrite (Sigma chemical CO., St.
Oliveira et al.: Interaction Between BeWo Trophoblast and T. gondii
Louis, USA) from 0.1 to 50 nmol. Each experiment was conducted in triplicate and repeated at least twice. Statistical analysis In all experiments data were expressed as mean G SD and the statistical differences between groups were determined using the unpaired Student’s t-test (Graph Pad Software, v. 4.0, San Diego, USA). Differences were considered statistically significant when P ! 0.05. RESULTS BeWo and HeLa cells do not present any differential morphological alteration after being infected by T. gondii As shown in Figure 1, both BeWo and HeLa cells did not show any significant morphological alterations on their general cellular architecture or differences in the patterns of organelle distribution after being infected by T. gondii. BeWo cells are characterized for showing threadlike projection in the membrane, cytoplasm with large deposits of glycogen, free polysomes, prominent granular endoplasmic reticulum, decondensed chromatin nucleus and evident nucleolus. BeWo cells with intracellular parasites showed numerous mitochondria next to the membrane of the parasitophorous vacuole (Figure 1G). Infection rates of BeWo and HeLa cells are equally affected by parasites treated with anti-TgSAG-1 antibody After 24 h of infection with T. gondii previously treated with anti-TgSAG-1 mAb, BeWo cells showed significant reduction in the percentage of infected cells, the average number of parasites per cell, and the total number of parasites, when compared with untreated parasites (Figure 2AeC). Similarly, HeLa cells showed significant decrease in all analyzed infection rates, when they were infected with parasites previously treated with anti-TgSAG-1 mAb (Figure 2AeC). Both BeWo and HeLa fixed cells were allowed to interact for 3 h with T. gondii previously treated with anti-TgSAG-1 mAb. It was observed that the percentages of cells harboring attached parasites and the average numbers of parasites per cell were considerably reduced when compared with untreated parasites, but the degree of inhibition was indistinguishable between both cell lineages (Figure 2D, E). BeWo cells fail to control replication of T. gondii in the presence of exogenous IFN-g The infection rates analyzed after 24 h of T. gondii interaction showed that BeWo presented higher susceptibility to the parasites when compared with HeLa cells. In spite of treatment from 100 to 500 U/mL of IFN-g (data not shown), BeWo presented a higher number of parasites per cell and parasite growth than HeLa cells in the same condition (Figure 3), showing that BeWo cells failed to control T. gondii growth
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in response to IFN-g treatment. IFN-g concentration of 100 U/mL was chosen for all other experiments. When BeWo cells were treated with IFN-g before addition of T. gondii and after L-tryptophan supplementation, the same pattern of infection of cells treated with IFN-g only was observed. In contrast, the addition of L-tryptophan to HeLa cells pre-treated with IFN-g antagonized the protective effect of this cytokine (Figure 3AeC). Interestingly, when both lineages of cells were treated with IFN-g plus a-methyl-tryptophan, a competitive inhibitor of IDO, a smaller parasitism was observed compared with cells treated with IFN-g plus Ltryptophan. When both cells were treated twice with IFN-g, before and after infection, it was observed that BeWo cells did not get control of the parasite growth, while HeLa cells presented an efficient control of the parasite replication (Table 1). These differences were due to the unresponsiveness of BeWo cells and the responsiveness of HeLa cells to IFN-g treatment, generating a reduction of almost 50% of parasite growth in the latter cell lineage. Moreover, BeWo cells receiving double treatment with IFN-g plus L-tryptophan showed no significant alteration in infection rates, whereas parasite growth was entirely restored in HeLa cells submitted to the same condition (Table 1). Under effect of double treatment with IFN-g plus amethyl-tryptophan, however, both BeWo and HeLa cells responded with a decrease in the number of parasites per cell and in the total number of parasites when compared with L-tryptophan treatment or even with medium only (Table 1). The effect of a-methyl-tryptophan treatment in both cell lines probably may be due to direct interference in the mechanisms of T. gondii replication, since it will overplus the tryptophan up-take, a critical aminoacid for parasite metabolism. Even though the production of nitrite was not observed on IFN-g unstimulated or stimulated BeWo and HeLa cells infected by T. gondii (data not shown), taken as a whole, the present results suggest that BeWo are more susceptible to T. gondii infection than HeLa cells. DISCUSSION Experimental models show that T. gondii can attach to and penetrate in any nucleated cells [21], including trophoblast cells of rodents [22,23] and human villous trophoblast [24]. Trophoblast cells are key components on the hemochorial human placenta, since they have been an interface between the mother and the baby. There are intracellular mechanisms in these cells that regulate their patterns of responses when exposed to various stimuli, leading to secretion of a variety of cytokines. These mechanisms are especially important since critical balance between deciduous invasion versus operating mechanisms prevents fetal rejection. In vitro and in vivo evidences suggest that IFN-g is unable to induce MHC class II expression in trophoblastic cells. Recent findings obtained with JEG 3 (a subclone of BeWo) show that this phenomenon does not take place because of receptor loss to IFN-g, which is
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Figure 1. Photomicrographs showing BeWo and HeLa cells in culture and its interaction with T. gondii: (A) HeLa cells after 72 h of culture and (B) after 24 h of infection with T. gondii (arrowhead), stained with Giemsa. Bar, 23 mm. (C) HeLa cells after 72 h of culture and 3 h of interaction with T. gondii stained by alkaline phosphatase substrate in the immunocytochemistry assay (arrowhead). Bar, 9 mm. (D) BeWo cells after 72 h of culture and (E) after 24 h of infection with T. gondii (arrowhead), stained with Giemsa. Bar, 23 mm. (F) BeWo cells after 72 h of culture and 3 h of infection with T. gondii stained by alkaline phosphatase substrate in the immunocytochemistry assay (arrowhead). Bar, 9 mm. (G) Electromicrograph from BeWo cells after 3 h of co-culture with T. gondii, showing tachyzoite inside a parasitophorous vacuole (arrows) surrounded by numerous mitochondrias (m) of the host cell. Bar scale: 1 mm.
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B
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Figure 2. Effect of anti-TgSAG-1 (p30) mAb treatment on T. gondii attachment and invasion in BeWo (closed bar) and HeLa (open bar) cells. Anti-TgSAG-1 treated or untreated parasites were incubated with both fixed or live host cells for 3 or 24 h, respectively. The following parameters were measured: A: percentage of infected cells; B: average number of parasites per live cell; C: total number of parasites per 100 live cells; D: percentage of fixed cells harboring attached parasites; E: number of attached parasites per fixed cell.
constitutively present in these cells, but due to regulatory events that follow the binding of this cytokine to its receptor, either causing specifically a negative regulation or silencing MHC class II transactivators [25].
In the present investigation, the progression of BeWo cells infection by T. gondii was studied. We have found that BeWo cells are more permissive to infection to this parasite than HeLa cells.
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Percentage of infected cells (mean ± SD)
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Figure 3. Infection rates due to T. gondii in BeWo and HeLa cells: effect of single treatment with IFN-g (100 U/mL) plus L-tryptophan or a-methyltryptophan during parasiteehost cell interactions. Cells were infected with T. gondii tachyzoites at a ratio of 10:1 parasites per cell. After 24 h of infection, they were fixed, stained by Giemsa stain, and analyzed. The following infection rates were measured: A: percentage of infected cells per 100 cells; B: average number of parasites per cell; C: total number of parasites per 100 cells.
Possible innate resistance and mechanisms available to trophoblast include the induction of oxygen reactive [26], nitrogen intermediates [7,27,28], and the enzyme indoleamine2,3-dioxygenase (IDO) that converts tryptophan to kynurenine [29] or alternative mechanisms [30]. Our results showed no difference of nitrite production in BeWo and HeLa cell culture supernatants and it means that the difference found for T. gondii growth in both cells is not related with nitric oxide. Without any treatment, HeLa cells were more resistant to T. gondii infection. T. gondii is probably able to evade defense parasiticidal mechanisms of BeWo cells, making them more susceptible to this pathogen. In the present study, treatment with recombinant IFN-g resulted in an inhibitory effect of intracellular tachyzoite growth only in HeLa cells. Treatment of BeWo and HeLa cells with IFN-g did not affect the percentage of infected cells, although in HeLa cells it induced a smaller number of parasites per cell, suggesting a control role to the parasitic growth. The failure of BeWo cells to control the number of T. gondii per cell in response to IFN-g treatment is unusual, since this is a common host defense mechanism against this pathogen. Indeed, the failure of IFN-g to control parasite growth in BeWo cells was not limited to T. gondii but also to other microorganisms [29]. Probably, the mechanisms of response to intracellular pathogens involve alternative pathways that do not involve the IFN-g in BeWo cells. We and others have demonstrated that one of the mechanisms to control T. gondii replication induced by IFN-g is L-tryptophan degradation, through induction of IDO, either in vivo or in vitro [10,31e36]. In addition, the mechanisms of IDO induction dependent on IFN-g were already investigated in many cell types, including HeLa cells [29,32,37e40]. Thus, in the present study, HeLa cells were utilized to compare with BeWo cells. We observed that HeLa cells submitted to IFN-g and L-tryptophan treatments showed different behavior when compared to BeWo cells under the same condition. In HeLa cells, the L-tryptophan treatment was capable to increase the average number of parasite and therefore, restore completely the inhibition given by IFN-g demonstrating that the antitoxoplasmic mechanism in HeLa cells is dependent on IFN-g. The compound a-methyl-tryptophan has been reported to be a potent competitive inhibitor of IDO activity when tested in vitro using purified enzyme [41], as well as in vivo at the maternalefetal interface [15]. The addition of this inhibitor did not significantly change the percentage of infected cells for BeWo and HeLa cells. However, when both cells were treated with IFN-g followed by a-methyl-tryptophan, a meaningful decrease was observed in the number of parasites per cell and in the total number of parasites compared with IFN-g plus L-tryptophan treatment or medium only. These results were more remarkable in double treatment schedule and might be due to the fact that a-methyl-tryptophan is used by the parasite instead of L-tryptophan in its process of protein synthesis. It is already known that L-tryptophan is an essential residue for some parasite components, where a single substitution is enough to produce a considerable disturbance in parasite cytoskeleton [42].
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Table 1. Effect of double treatment with IFN-g plus L-tryptophan or a-methyl-tryptophan in the infection rates due to T. gondii tachyzoites in BeWo and HeLa cells BeWo
HeLa
Host cells
Infection rates
Experimental conditions
Percentage of infected cells
Number of parasites per cell
Total number of parasites
Percentage of infected cells
Number of parasites per cell
Total number of parasites
Medium only Double treatment with IFN-g only Double treatment with IFN-g plus L-trp Double treatment with IFN-g plus a-m-trp
47.7 G 4.2 51.7 G 11.0 41.0 G 7.8 38.0 G 6.0
14.5 G 3.4 11.9 G 1.5 15.1 G 2.8e 10.3 G 1.4i
657.0 G 127.3 606.3 G 99.9 604.0 G 39.6f 401.7 G 27.5j
36.7 G 9.5 31.0 G 4.6 35.3 G 4.9 36.0 G 3.0
11.0 G 1.1a 6.9 G 1.0c 14.5 G 1.3g 8.2 G 0.9k
408.6 G 37.6b 212.3 G 24.5d 507.0 G 29.5h 292.7 G 6.5l
Results are expressed as mean G SD of two independent experiments in triplicate. Statistically significant results were obtained with the following comparisons: a versus c (P Z 0.0091); c versus g (P Z 0.0013); g versus k (P Z 0.0020); e versus i (P Z 0.0055); f versus j (P Z 0.0018); b versus d (P Z 0.0108); d versus h (P Z 0.0002); h versus l (P Z 0.0003).
The intracellular response seen in BeWo cells is quite intriguing and apparently not dependent on mechanisms directly linked to IFN-g, but relate to pathways unrelated to IFN-g stimuli. As intracellular tryptophan concentration probably achieves its optimal values for the parasite in this system, yielding an intracellular beneficial environment to T. gondii, no augment of replication was observed after further addition of this aminoacid. BeWo cells do not express IDO constitutively or in response to IFN-g treatment [29]. This feature makes these cells particularly attractive to different species of intracellular pathogens. In spite of the differences found for BeWo and HeLa cells concerning the intracellular parasite replication, in the present study we observed similar mechanism of invasion of both cells by the parasite, since TgSAG-1 was effectively blocked by specific antibodies, as seen in other host cells [19]. It was possible to observe formation of the parasitophorous vacuole (PV) inside the BeWo and HeLa cells with mitochondrias associated with the PV membrane after interaction with T. gondii. Vacuoles containing T. gondii completely avoid fusion with normal host endocytic and exocytic vesicles, and the parasite replicates within this protected intracellular niche [43]. Our data support the idea that immunological control of pathogens in the placental trophoblast should be different from that occurring in the periphery. Regulatory mechanisms operating inside and outside the cell allow that at the same time that they prevent maternal rejection to the concept, they turn this microenvironment particularly susceptible to intracellular pathogens as T. gondii. Indeed, the increase in
susceptibility of the BeWo cells to T. gondii infection in our model may be explained by the hypothesis that claims the existence of areas potentially susceptible to the parasite infection. In this context, recent studies have elegantly shown that IDO is expressed in different cell populations from both human [44,45] and murine [46] placenta. Considering that the IDO expression is not uniform in placenta, the areas with small expression of this enzyme turn out to be potentially more susceptible to infection by intracellular parasites [16]. Abortions may be caused by fetal infection as a result of different regulatory mechanisms and reflecting a strong connection between hormonal levels and patterns of cytokine secretion [47]. High levels of inflammatory cytokines, such as TNF-a, IL-2 and IFN-g may have abortive potential [14]. Even though the present work presented results of an in vitro system, further studies to be carried out on placenta pathologies or abortions induced by T. gondii can elucidate some of the immunoregulatory processes involved in the maintenance of a normal pregnancy. This is the first report describing that T. gondii tachyzoites continues to replicate inside the trophoblast cells of the cell line BeWo even in the presence of IFN-g and also that there was no inhibitory effect to reverse when L-tryptophan was added in IFN-g-treated cells. These data lead to the conclusion that, even though the unresponsiveness to IFN-g in trophoblastic cells is a worthy event to the host to prevent allogeneic fetal rejection, it may also be considered detrimental to the host by allowing the replication of parasites that can achieve fetal tissues.
ACKNOWLEDGMENTS This work was supported by the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), FAPEMIG and CAPES. REFERENCES [1] Remington JS, McLeod R, Thulliez P, Desmonts G. In: Remington JS, Klein OJ, editors. Infectious diseases of the fetus and newborn infant, 5th ed. Philadelphia: W. B. Saunders; 2001, p. 205e356.
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