Effect of 17 β-Estradiol and Progesterone on Astrocytes Infected with Toxoplasma

| Toxoplasma gondii is an opportunistic parasite that invade microglia, astrocytes, and neurons. In guinea pigs infected with Toxoplasma, brain cysts increased significantly in males subjected to gonadectomy and treated with hexoestrol, compared with control. The effect of 17β-estradiol and progesterone on astrocytes infected with Toxoplasma is unknown. The objective of the study was to evaluate the effect of 17β-estradiol and progesterone on Toxoplasma infection in astrocytes in vitro. Astrocytes were pre-treated with 17β-estradiol or progesterone at concentrations of 10, 20, 40, 80, or 160 nM for 48 hours and infected with Toxoplasma tachyzoites. The percentage of infected astrocytes was evaluated by immunocytochemistry and parasite replication by MTT. ANOVA and a Dunnett’s T3 post-hoc test were used. Pretreatment with 17β-estradiol and 17β-estradiol + tamoxifen (1μM) resulted in a significant reduction in parasite replication at 48 hours post-infection. Infected astrocytes were gradually reduced whit 17β-estradiol plus tamoxifen. Progesterone reduced the parasite replication at 48 hours, and this effect was reversed by mifepristone. The percentage of infected astrocytes at 24 hours was reduced with progesterone at all doses. 17β-estradiol plus progesterone had a synergistic effect, increasing Toxoplasma infection in astrocytes at 24 hours however, at 48 hours the infection was reduced. The 17β-estradiol + progesterone at 160 nM, as well as PPT and DPN, reduced Toxoplasma replication and percentage of infected astrocytes. Toxoplasma infection in astrocytes was reduced by the effect of 17β-estradiol and its agonist PPT as well as progesterone. These results suggest that ERα and PRs activation diminishes Toxoplasma-infection in astrocytes.

mune response in the central nervous system (Appel et al., 2001).However, more studies on the T. gondii mechanism of invasion and replication process in astrocytes are necessary.T. gondii infection in astrocytes significantly increases monocyte chemotactic protein-(MCP-1) secretion.This protein may contribute to the cell clustering seen during human cerebral reactivation of T. gondii (Brenier-Pinchart et al., 2004).Pro-inflammatory protein expression differs between type I and II strains and among different human nervous system cells.For example, the parasite burden declines in microglial cells and neurons over the course of infection, but remains high in endothelial cells (Mammari et al., 2014).This differential effect on early parasite multiplication may be correlated with a higher production of immune mediators by neurons and microglial cells compared with endothelial cells, and suggests that the different protein expression profiles depend on the parasitic strain and on the human nervous cell type (Contreras-Ochoa et al., 2013;Brenier-Pinchart et al., 2004).
Tamoxifen, a derivative of triphenylethylene, is a selective estrogen receptor modulator (SERM).Tamoxifen has been used as an antagonist for ERβ in the nervous system.This drug competes with estrogens for binding to estrogen receptors (ERs) at high affinity, ranging from 100 to 1000 times that of estrogen.Mice treated with tamoxifen citrate and 17β-estradiol, at a dose of 1.2 µmole, decreases the number of T. gondii cysts compared with E2 alone (Pung and Luster, 1986).
In guinea pigs infected with the Beverley Strain of T. gondii, the number of brain cysts of T. gondii increased significantly (p<0.001) in males that were castrated and treated with hexoestrol, as well as in females, compared with control guinea pigs (Kittas and Henry, 1979).In other experiment performed on mice, the number of T. gondii brain cysts increased significantly in infected mice treated with hexoestrol (Kittas and Henry, 1980).
In infected female mice treated with pharmacological doses (0.1-10 µmoles) of 17β-estradiol, the number of T. gondii cysts (T45 strain) increased in a dose-dependent way; however, the administration of progesterone did not affect cyst formation (Pung and Luster, 1986).
The role of E2 and progesterone receptors in toxoplasmosis is currently unknown.The objective of the present study was to evaluate the effect of 17β-estradiol and progesterone on T. gondii infection in astrocytes in vitro.

PArAsItes
Six-week-old male and female Swiss mice weighing 20-25 g were intraperitoneally injected with 1 x 10 5 T. gondii tachyzoites (strain RH) and sacrificed at 7 days; the infection was maintained by injecting new mice every three

MIxed cortIcAl cells
Cortical cell isolation for astrocyte culture can be performed using 1 to 3 newborn rats, not more than 4 days old.Three newborn Wistar rats were sacrificed by decapitation.Brain tissue was obtained by craniotomy and mechanically dissociated in OPTI-MEM 51985 medium (Invitrogen, Carlsbad, CA) supplemented with 10% horse serum (HS) and a 1% solution of penicillin streptomycin.Cells were counted using a 0.3% trypan blue solution for determining survival percentage.The cells were plated (50,000) on 0.1% poly-L-lysine pre-treated cover slips placed in 24-well plates and maintained at 37 o C in a 95% air and 5% CO2 atmosphere.The medium was changed every two days until 90% confluence was obtained.

Astrocyte sePArAtIon
After 7 or 8 days of culture, 90% confluent cells were shaken at 180 rpm for 30 min on an orbital shaker to remove microglia.The supernatant containing the microglia was discarded.The plate was shaken again at 240 rpm for 6 hr to eliminate oligodendrocyte precursor cells (OPC).The resulting astrocytes were obtained with 95% purity.

exPerIMentAl treAtMents In Astrocytes in vitro
On the fifteenth day of culture, the astrocytes (90% confluence) were hormonally pre-treated for 48 hours with 10, 20, 40, 80, or 160 nM/mL of E2 or P4, or the combination of E2 and P4 at a concentration of 160 nM/mL.Tamoxifen was used at a concentration of 1 µM/mL plus E2 10, 20, 40, 80, or 160 nM/mL.PPT or DPN was added to the cultured astrocytes at a concentration of 1 nM/mL, mifepristone at a dose of 1 µM/mL plus P4 at doses of 10, 20, 40, 80, and 160 nM/mL.; then, tachyzoites of T. gondii were added (4 x 10 3 parasites per well) and allowed to infect the astrocytes for a period of 24 and 48 hours.
Each hormone dose was evaluated independently in three replicates, repeating the experiment three times.For the immunocytochemical experiment, infected and uninfected cells were fixed with 3.7% paraformaldehyde for 5 minutes at room temperature and stored at 4°C.

IMMunocytocheMIcAl Method
T. gondii infection in cultured astrocytes was detected by the immunocytochemical method, in consecutive order, as follows.To identify astrocytes, cells were washed twice with phosphate buffer solution (0.1 M PBS) for 5 minutes and permeabilized with 0.2% Triton X-100 in PBS (TPBS) for 1 hour.Cells were incubated with 10% goat serum in PBS containing 0.01% sodium azide (blocking solution) for 2 hours at 4 o C.After washing, the astrocytes were incubated overnight in mouse anti-glial fibrillary acidic protein (GFAP) antibody (1:500, Dako Corporation, Carpinteria, CA) and rabbit anti-Toxoplasma antibody (1:1500, Gen Way Biotech, San Diego, CA) diluted in a blocking solution and incubated at 4 o C for 16 hours in a humidity chamber.After washing with TPBS, the cells were incubated in 10% pre-immune goat serum in a PBS buffer for 1 hour at room temperature and then were incubated with Alexa Fluor 594 antibody-labelled anti-mouse IgG (1:1000, Abcam 150116) and Alexa Flour 488 antibody-labelled anti-rabbit IgG (1:1000, Abcam 150073) for 1 hour at room temperature in darkness in a humidity chamber.
Finally, the cells were washed two times with PBS and incubated 5 minutes with Nucleic Acid Stain (DAPI, Invitrogen), diluted 1:3000 in PBS.

MIcroscoPIc AnAlysIs
Astrocytes were evaluated a total of nine times for each hormone dose as well as for their agonists and antagonists.One hundred cells were analysed for each dose: The number of infected astrocytes was determined using digital images from an Olympus IX71 microscope (40x magnification), using Image Pro-Plus software 6.0 of Media Cybernetics 2.6 to merge the images.

T. gondii rePlIcAtIon
The MTT assay is based on the presence of the enzyme mitochondrial succinate dehydrogenase in live cells.Only viable and early apoptotic cells are capable of reducing the tetrazolium salt MTT (yellow), resulting in the formation of water-insoluble formazan crystals (purple); dead cells will therefore retain the yellow colour of the medium (Mossman, 1983).
T. gondii replication in infected and treated astrocytes was evaluated by the MTT assay.Using the method described above, 96-well plates were used to culture astrocytes (5 x 10 4 cells/1.7 cm 2 ).To began the assay, 40 µL of MTT solution (5 mg/ml, Sigma) was added for 2 h and incubated at 37°C, in 5% CO2 and 95% air for 2 h.Subsequently, 100 µL of 50% dimethyl formamide and 20% sodium dodecyl sulfate (SDS) was added for dissolving the formazan crys

cytotoxIcIty AssAy
To evaluate the cytotoxicity on astrocytes, the MTT assay was used.Progesterone and 17B-estradiol, along with their antagonists or agonists, were added to the astrocytes in vitro.In one assay, cells were treated with E2 or P4, at doses of 10, 20, 40, 80, and 160 nM/mL.In another assay, the astrocytes were treated with E2 plus P4 both at 160 nM/mL or E2 at 10, 20, 40, 80, and 160 nM/mL plus 1 µM/mL tamoxifen.To test the action of progesterone, this hormone was added at doses of 10, 20, 40, 80, and 160 nM/mL plus mifepristone at a concentration of 1 µM/mL.PPT or DPN were added to the cells at a dose of 1 nM/ mL.All assays were conducted at 24 and 48 hours.

stAtIstIcAl AnAlysIs
Quantitative variables were expressed as mean ± SEM.
Analysis of experimental groups was made by ANOVA and a Dunnett's T3 post-hoc test.
The control group consisted of uninfected cells that were 100% viable.Data of cytotoxicity of E2, P4, tamoxifen, mifepristone, DPN and PPT on astrocytes was determined using ANOVA and a Dunnett's T3 post-hoc test.

RESULTS
Representative T. gondii uninfected astrocytes are shown in Figure 1A. Figure 1B (control; T. gondii-infected astrocytes without hormones) shows the presence of T. gondii tachyzoites, which had replicated approximately four times at this point.Figure 1C (T.gondii-infected astrocytes treated with E2 at 160 nM) shows clear morphological preservation at 24 hours.At 48 hours post-infection, however, the astrocytes were entirely destroyed, with clearly ruptured membranes (Figure 1D).
The addition of 17β-estradiol at 160 nM significantly increased T. gondii replication at 24 hours post-infection in astrocytes in vitro compared with the untreated control (p<0.05, Figure 2A).However, at 48 hours post-infection,

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Academic Publishers The  C) At 24 hours post-infection, the percentage of infected astrocytes treated with E2 was significantly reduced at doses 10, 40, 80, and 160 nM with respect to the control; the combination of E2 + tamoxifen (orange line) also significantly reduced infection, at all doses, compared with the control; D) However, at 48 hours post-infection, the percentage of infected astrocytes was significantly increased at doses of 80 nM E2, and combined E2 + tamoxifen at 80 and 160 nM showed a pattern similar to the control.
the pre-treatment with E2 and E2 + tamoxifen resulted in a significant reduction in parasite replication at all doses, compared with the untreated control (p<0.001, Figure 2B).
The percentage of infected astrocytes treated with E2 was reduced compared with the control at all doses from 20 to 160 nM (p<0.001, Figure 2C).This effect was reversed by the action of tamoxifen, mainly at 10, 20, 40, and 160 nM doses at 24 hours post-infection (Figure 2C).The percentage of infected astrocytes was also reduced when they were treated with E2 at doses from 10 to 40 nM for 48 hours.However, an 80 nM dose resulted in a significant increase in the number of infected astrocytes (p<0.001, Figure 2D).In contrast, the combination of E2 + tamoxifen reversed these effects, resulting in a significant reduction in the percentage of infected astrocytes at 10, 40, and 80 nM doses (p>0.05),but an increase at 20 nM, compared with E2 alone (Figure 2D).
Progesterone did not affect T. gondii replication at 24 hours at any experimental concentration (Figure 3A).However, at 48 hours, P4 significantly reduced parasite replication compared with untreated controls (p< 0.001), and this effect was reversed by mifepristone at 20 and 80 nM (Figure 3B).
The percentage of infected astrocytes 24 hours after treatment with P4 was significantly reduced, compared with untreated controls (p<0.001), at all doses (Figure 3C).Mifepristone exerted an antagonistic effect on progesterone at doses from 20 to 160 nM, resulting in significantly fewer infected astrocytes (p<0.001) compared with the control (Figure 3C).A significant reduction in infected astrocytes treated with P4 was observed at doses of 10, 20, 80, and 160 nM, compared with untreated controls p<0.001), at 48 hours (Figure 3D).Mifepristone exhibited an antagonistic effect on P4, significantly increasing the percentage of  infected cells (p<0.001) at doses of 10, 80, and 160 nM at 48 hours p.i.
At 24 hours, T. gondii replication in astrocytes was not affected by PPT or DPN, nor was there any difference between combined E2+P4 and controls (Figure 4A and 5A).However, at 48 hours, the replication of T. gondii was significantly reduced by PPT and DPN compared with untreated controls p<0.001(Figure 4B).The percentage of infected astrocytes was reduced by the effect of PPT and DPN with a significant difference (p<0.001)versus controls at 24 hours (Figure 4C).However, at 48 hours, DPN induced a moderate reduction of infected astrocytes compared with the control (Figure 4D).
Intracellular tachyzoites are also known to manipulate a variety of signal transduction pathways related to apoptosis, antimicrobial effector mechanisms, and immune cell maturation (Fagard et al., 1999;Li et al., 2010).In the present study, most astrocytes died at 48 hours post-infection, possibly as a result of high Toxoplasma replication or a low number of E2 or P4 receptors.However, the number of parasites in the remaining infected astrocytes increased.

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The   T. gondii may control the apoptotic machinery in the astrocytes, through inhibition of caspases 3/7 or PUMA gene expression at the beginning of the infection, to promote replication (Appel et al., 2001).Li et al. (2010) observed that tachyzoites of the T. gon-dii RH strain had entered astrocytes at 1 h post-infection, and that autophagosomes, which appeared at 4 h, were pronouncedly increased.However, after 12 h, their number was considerably decreased and damage to the cells occurred 48 h later; autophagosomes disappeared and more astrocytes were destroyed.In our study, the astrocytes were almost totally destroyed at 48 hours, when T. gondii had replicated eight times.This might be due to the disappearance of the astrocyte autophagosomes; however, further studies would be required to test this hypothesis (Li et al., 2010).
Astrocytes are cells of the central nervous system that are sensitive to steroid hormones because they have receptors for 17β-estradiol and progesterone, among others.In the present study, pre-treatment of astrocytes with 17β-estradiol reduced T. gondii replication at 48 hours; however, this pattern changed with high doses.E2 may exert a protective action against T. gondii infection, because a reduction in the percentage of infected astrocytes was observed at 24 hours post-infection.E2 can regulate immune mediators (cytokines) in the infection process (Halonen et al., 1996;Fagard et al., 1999;Mammari et al., 2014).
Pung and Luster have shown that tamoxifen inhibits T. gondii infection susceptibility in mice (Pung and Luster, 1986).We confirmed in our results that tamoxifen reversed the effect of E2 on the susceptibility to infection at 48 hours, possibly because E2 receptors are antagonized by tamoxifen in infected astrocytes.
Progesterone at 48 hours p.i., combined with mifepristone, reduced the replication of the parasite.Mifepristone may be a progesterone receptor antagonist in infected astrocytes (Khan et al., 2013;Bouchard et al., 2011;Farina et al., 2005;Melcangi et al., 2014), possibly affected by dose used or time of exposure to mifepristone.Since the combination of P4 with mifepristone further reduced the percentage of infected astrocytes at 24 hours post-infection, this result suggests that they act at low doses modulating the infection.
The percentage of infected astrocytes treated with E2 plus P4 reduced T. gondii replication at 48 hours p.i., but not at 24 hours.This result is probably due to a synergistic effect, with both hormones activating the same cellular signaling pathway (De Marinis et al., 2013).However, more studies are necessary to confirm this.
T. gondii replication was reduced by PPT and DPN at 48 hours p.i.These results suggest that an activation of α and β estradiol receptors in astrocytes is induced.PPT and DPN reduced the number of infected astrocytes at 24 hours.Paradoxically, at 48 hours, PPT produced an increase in infected astrocytes.This effect could be due to a reduction in estradiol receptor α response; however, this must be confirmed.
DPN produced a moderate reduction in the percentage of infected astrocytes, compared with controls, at 24 hours and 48 hours p.i.These results suggest that ERβ partici-pated less in the control of the infection process than ERα.
In the present study, the RH (Type I) T. gondii strain was used.This is the most virulent strain, but it has been shown that virulence depends on target cell type; specific types of cells can be more or less sensitive to infection (Contreras-Ochoa et al., 2013).

CONCLUSIONS
Toxoplasma infection in astrocytes is reduced through the effect of 17β-estradiol.The 17β-estradiol agonists DPN and PPT also decrease T. gondii infection.The combination of 17β-estradiol plus progesterone had a synergistic effect on T. gondii infection that was dependent on exposure time.Mifepristone reversed the effect of progesterone on the T. gondii infection process.

Figure 1 :
Figure 1: Representative photographs of Toxoplasma gondii infection in cultured astrocytes detected by immunocytochemistry.A) Uninfected astrocytes, morphologically preserved; B) Untreated astrocytes infected with tachyzoites of T. gondii Rh strain (control) at 24 hours; C) Hormone treated astrocytes were morphologically preserved at 24 hours post-infection; D) Hormone treated astrocytes at 48 hours post-infection.The astrocytes have been destroyed by Toxoplasma infection, with clear membrane rupture photomicrographs were taken at 40X.als, and the solution was incubated overnight.Absorbance was measured at 570 nm in an ELISA plate reader (Biolab-System, USA).The percentage of viability was calculated from the optical density (OD) of each well, as follows: [(mean OD of treated, infected astrocytes in well with drug) / (mean OD of non-treated and infected astrocytes)] x 100.Results were given as mean viability ± SEM.

Figure 2 :
Figure 2: T. gondii replication in astrocytes, as evaluated by MTT Replication is expressed as percent of the control value.Untreated astrocytes (control), astrocytes pretreated with 17β-estradiol (E2) (blue line), and astrocytes treated with E2+tamoxifen (orange line), were measured for T. gondii replication and infected astrocytes at 24 and 48 hours.A) T. gondii replication increased significantly in astrocytes treated with 160 nM E2, compared with all other E2 doses and the control (p<0.05;ANOVA and post-hoc Dunnett´s T3); B) At 48 hours, T. gondii replication decreased significantly after treatment with E2 (blue line) and E2 + tamoxifen (orange line) (*p<0.001),compared with the untreated control (green line);

Figure 3 :
Figure3: T. gondii replication in astrocytes, as evaluated by MTT.Replication is expressed as percent of the control value Untreated astrocytes (control), astrocytes pretreated with P4 (purple line), and astrocytes treated with P4+mifeprestone (red line), were measured for T. gondii replication and percentage of infected astrocytes at 24 and 48 hours.At 24 hours (A) the percentage of T. gondii replication treated with all doses of progesterone (P4) (purple line) and P4 + mifepristone (red line), exhibited no statistical differences compared with the control (green line); (B) Percentage of T. gondii replication was significantly reduced in all doses of P4 and P4 + mifepristone (p<0.05).The percentage of infected astrocytes was reduced by P4 and P4 + mifepristone at all doses, compared with the control, at 24 hours (C) and 48 hours (D), with the exception of P4-40nM and P4+Mif-10nM, post-infection (p<0.001).Mifepristone reversed the effect of P4 at 20 and 40 nM, at 24 hours p.i.

Figure 4 :
Figure 4: T. gondii replication (expressed as % of control) at (A) 24 hours and (B) 48 hours.C, D) T. gondii infected astrocytes at 24 (C) and 48 (D) hours after treatment (expressed as % of control) with 4, 4', 4''-(4-propyl-(1H)-pyrazole-1, 3, 5 triyl) trisphenol (PPT, brown bar) or hydroxy-phenyl-propionitrile (DPN, beige bar).A) T. gondii replication was similar in both groups and the control, with no statistically significant differences observed; B) The percentage of replication of T. gondii in astrocytes decreased significantly in both the PPT and the DPN groups, compared with the control (p<0.001);C) At 24 hours, the percentage of infected astrocytes treated with PPT and DPN was significantly reduced, compared with the control (p<0.001);D) At 48 hours, the percentage of infected astrocytes in the group treated with PPT was similar to the control.In contrast, in the DPN-treated group, infection was significantly reduced compared with the control (p<0.05).

Figure 5 :
Figure 5: T. gondii replication (expressed as % of control) after treatment with E2 combined with progesterone (E2+P4) (purple and blue bar) at 24 hours and 48 hours (A) and percentage of infected astrocytes (B)A) The replication of T. gondii in astrocytes decreased with E2 + P4, compared with the control, at 48 hours post-infection (p<0.001);B) The percentage of infected astrocytes treated with E2+P4 increased compared with the control at 24 hours (p<0.001) and at 48 hours (p<0.05).