Effect of 17β-Estradiol, Progesterone, and Tamoxifen on Neurons Infected with Toxoplasma gondii In Vitro

Toxoplasma gondii (T. gondii) is the causal agent of toxoplasmosis, which produces damage in the central nervous system (CNS). Toxoplasma–CNS interaction is critical for the development of disease symptoms. T. gondii can form cysts in the CNS; however, neurons are more resistant to this infection than astrocytes. The probable mechanism for neuron resistance is a permanent state of neurons in the interface, avoiding the replication of intracellular parasites. Steroids regulate the formation of Toxoplasma cysts in mice brains. 17β-estradiol and progesterone also participate in the control of Toxoplasma infection in glial cells in vitro. The aim of this study was to evaluate the effects of 17β-estradiol, progesterone, and their specific agonists–antagonists on Toxoplasma infection in neurons in vitro. Neurons cultured were pretreated for 48 h with 17β-estradiol or progesterone at 10, 20, 40, 80, or 160 nM/mL or tamoxifen 1 μM/mL plus 17β-estradiol at 10, 20, 40, 80, and 160 nM/mL. In other conditions, the neurons were pretreated during 48 h with 4,4′,4″-(4-propyl-[1H] pyrozole-1,3,5-triyl) trisphenol or 23-bis(4-hydroxyphenyl) propionitrile at 1 nM/mL, and mifepristone 1 µM/mL plus progesterone at 10, 20, 40, 80, and 160 nM/mL. Neurons were infected with 5000 tachyzoites of the T. gondii strain RH. The effect of 17β estradiol, progesterone, their agonists, or antagonists on Toxoplasma infection in neurons was evaluated at 24 and 48 h by immunocytochemistry. T. gondii replication was measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. 17β-Estradiol alone or plus tamoxifen reduced infected neurons (50%) compared to the control at 48 h. Progesterone plus estradiol decreased the number of intracellular parasites at 48 h of treatment compared to the control (p < 0.001). 4,4′,4″-(4-propyl-[1H] pyrozole-1,3,5-triyl) trisphenol and 23-bis(4-hydroxyphenyl) propionitrile reduced infected neurons at 48 h of treatment significantly compared to the control (p < 0.05 and p < 0.001, respectively). The Toxoplasma infection process was decreased by the effect of 17β-estradiol alone or combined with tamoxifen or progesterone in neurons in vitro. These results suggest the essential participation of progesterone and estradiol and their classical receptors in the regulation of T. gondii neuron infection.


Introduction
Toxoplasma gondii is an intracellular obligate protozoan. It is the causal agent of toxoplasmosis, which induces damage in the central nervous system. Its success as an invasive organism is that it possesses a great capacity to migrate transepithelial [1]. When decreases PRLR and ERβ and increases ERα; E2 decreases PRLR, and prolactin (PRL) reduces ERα and ERβ expression [39]. However, the effect of estradiol and progesterone on cultured neurons infected with Toxoplasma is unknown. This study aimed to determine the role of 17β-estradiol and progesterone as well as agonists (i.e., DPN and PPT) and antagonists (i.e., tamoxifen and mifepristone) on neurons infected by Toxoplasma gondii in vitro.

Ethical Aspects
This study was carried out from January 2019 to March 2020 in strict accordance with the recommendations of Official Mexican Standards NOM-067. The protocol was approved with registration number CI-07618. All animal experiments were approved by the biosafety, research, and ethics committees of the University Center of Health Sciences of the University of Guadalajara.

Parasites
We used tachyzoites of the virulent RH strain of Toxoplasma gondii for our experiments. They were obtained by intraperitoneal (i.p.) passage in Swiss-Webster female mice of six-week-olds, injected with 100,000 tachyzoites. After three days, parasites were harvested from peritoneal exudates to infect neurons. The concentration of tachyzoites was determined by counting in a hemocytometer using a light microscope [39].

Cortical Neuron Culture
For the culture of neurons, we sacrificed one to three newborn rats not more than four days old. We used the brain cortex. The brain tissue was obtained by craniotomy and mechanically dissociated in OPTI-MEM 51985 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% horse serum (HS) and a 1% solution of penicillin-streptomycin. Cells were counted using a 0.3% Trypan Blue solution to determine the percentage of survival. The cells were plated (50,000) onto 0.1% poly-L-lysine pretreated coverslips placed in 24-well plates and maintained at 37 • C in a 95% air and 5% CO 2 atmosphere. The medium was changed every two days until obtaining 90% confluence.

Neuron Separation
After seven or eight days of culture, 90% of confluent cells were separated (oligodendrocytes, microglia, and astrocytes). Then neurons were incubated in Neurobasal ® medium-plus B27 supplement plus cytosine arabinoside 5 µM for 24 h. The medium was replaced every three days. The neurons obtained had 95% purity.

Experimental Treatments in Neurons In Vitro
We add to neurons 10, 20, 40, 80, or 160 nM/mL of E2 or P4 for 48 h or the combination of E2 and P4 at a concentration of 160 nM/mL. Tam was utilized at a concentration of 1 µM/mL plus E2 10, 20, 40, 80, or 160 nM/mL. PPT or DPN was administrated to the cultured neurons at a concentration of 1 nM/mL. Mif was added at a dose of 1 µM/mL plus P4 at doses of 10, 20, 40, 80, and 160 nM/mL; then, T. gondii tachyzoites were added (5 × 10 3 parasites per well), and they were allowed to infect the neurons for periods of 24 and 48 h. Each hormone dose was evaluated independently in three replications, repeating the experiment three times. For the immunocytochemical experiment, infected and uninfected cells were fixed with 3.7% paraformaldehyde, for 5 min at room temperature and stored at 4 • C.

Immunocytochemical Method
We detected T. gondii infection in cultured neurons by immunocytochemistry. Neurons were washed with phosphate-buffered solution (0.1 M PBS), and they were permeabilized with Triton X-100 in PBS (TPBS) for 1 h. Next, the neurons were incubated with mouse anti-β-Tubulin (β-Tubulin) antibody (1:450, MAB1637); Millipore, Carpinteria, CA, USA) and rabbit anti-Toxoplasma antibody (1:1500; GenWay Biotech, San Diego, CA, USA) diluted and incubated at 4 • C for 16 h in a humidity chamber. Then, cells were incubated with Alexa Fluor 594 antibody-labeled anti-mouse IgG (1:1000, Abcam 150116) and Alexa Flour 488 antibody-labeled anti-rabbit IgG (1:1000, Abcam 150073) for one hour at room temperature under conditions of darkness in a humidity chamber. Finally, the cells were washed twice with PBS and incubated for 5 min with a nucleic acid stain (DAPI, Invitrogen) diluted 1:3000 in PBS.

Microscopic Analysis
The neurons were quantified a total of nine times for each hormone dose and their agonists and antagonists. We counted one hundred cells for each dose: the number of infected neurons was evaluated using digital images from an Olympus IX71 microscope (40× magnification) employing Image-Pro Plus ver.6.0 software (Media Cybernetics 2.6) to merge the images.

T. gondii Replication
The enzyme mitochondrial succinate dehydrogenase develops the MTT Tetrazolium reduction assay in live cells. Only viable and early apoptotic cells can reduce the tetrazolium salt MTT (yellow), resulting in the formation of water-insoluble formazan crystals (purple); dead cells will therefore retain the yellow color of the medium [40][41][42].
T. gondii replication in infected and treated neurons was evaluated by the MTT assay. We used 96-well plates to culture neurons (5 × 10 4 cells/1.7 cm 2 ) using the method described previously. Forty microliters of MTT solution (5 mg/mL; Sigma, St. Louis, MO, USA) were added for two h and incubated at 37 • C in 5% CO 2 and 95% air. Subsequently, we added 100 µL of 50% dimethylformamide and 20% sodium dodecyl sulfate (SDS) to dissolve the formazan crystals, and the solution was incubated overnight. Absorbance was measured at 570 nm in an enzyme-linked immunosorbent assay (ELISA) plate reader (Biolab-System,Los Angeles, CA, USA). The percentage of replication was calculated from the optical density (OD) of each well as follows: ((mean OD of treated, infected neurons in well with drug)/(mean OD of non-treated and infected neurons)) × 100. Results are presented as the mean viability ± standard error of the mean (SEM).

Cytotoxicity Assay
To evaluate the cytotoxicity of neurons, we used the MTT assay. Progesterone and 17B-estradiol, along with their antagonists or agonists, were added to the neurons in vitro. The neurons were treated with E2 or P4 at 10, 20, 40, 80, and 160 nM/mL doses. In another assay, we added to the neurons E2 plus P4 at 160 nM/mL or E2 at 10, 20, 40, 80, and 160 nM/mL plus one µM/mL tamoxifen. We add progesterone at doses of 10, 20, 40, 80, and 160 nM/mL plus mifepristone at a 1 µM/mL concentration. PPT or DPN was added to the cells at a 1 nM/mL dose for 48 h.

Statistical Analysis
Quantitative variables are represented as the mean ± SEM. All the experimental groups were analyzed by analysis of variance (ANOVA) and a Dunnett T3 post hoc test. The control group consisted of uninfected cells that were 100% viable.

Results
Step and Displacement Analysis All data were compared vs. the control (100%). At 24 h, 17β-estradiol reduced approximately 10% T. gondii replication at doses of 10 and 80 nM, with a statistically significant difference (p < 0.001 and p < 0.005, respectively). The combination of E2 (10 nM) plus Tam reduced T. gondii replication by 20% with a statistically significant difference of (p < 0.001). We observed a reduction in T. gondii replication of 10% only with E2 at 10 nM and with E2 plus tamoxifen at a similar percentage with a significant difference compared to the control (p < 0.05) at 48 h ( Figure 1A,B). The percentage of infected neurons treated with E2 was markedly reduced (58%) at 10 nM at 24 h. The combination of E2 at 10 nM plus Tam at one µM/mL decreased 50% the percentage of infected neurons at 48 h with a statistically significant difference of p < 0.001 ( Figure 1C,D).

Results
Step and Displacement Analysis All data were compared vs. the control (100%). At 24 h, 17β-estradiol reduced approximately 10% T. gondii replication at doses of 10 and 80 nM, with a statistically significant difference (p < 0.001 and p < 0.005, respectively). The combination of E2 (10 nM) plus Tam reduced T. gondii replication by 20% with a statistically significant difference of (p < 0.001). We observed a reduction in T. gondii replication of 10% only with E2 at 10 nM and with E2 plus tamoxifen at a similar percentage with a significant difference compared to the control (p < 0.05) at 48 h ( Figure 1A,B). The percentage of infected neurons treated with E2 was markedly reduced (58%) at 10 nM at 24 h. The combination of E2 at 10 nM plus Tam at one μM/mL decreased 50% the percentage of infected neurons at 48 h with a statistically significant difference of p < 0.001 ( Figure 1C,D).     In Figure 3A,B, the neurons infected with Toxoplasma gondii under the effect of DPN (1 nM/mL) at 24 and 48 h exhibited parasites intra-and extracellularly. In (C) and (D), the neurons in the presence of PPT (1 nM/mL) at 24 h and 48 h were more infected concerning DPN. The neurons were damaged, and a remarkable percent of them were destroyed. In Figure 3A,B, the neurons infected with Toxoplasma gondii under the effect of DPN (1 nM/mL) at 24 and 48 h exhibited parasites intra-and extracellularly. In (C) and (D), the neurons in the presence of PPT (1 nM/mL) at 24 h and 48 h were more infected concerning DPN. The neurons were damaged, and a remarkable percent of them were destroyed. Figure 4A,B show neurons in the presence of E2 plus tamoxifen exhibiting a minor intracellular infection compared to E2 or PPT at 48 h. However, the neurons disintegrated and their cytoplasmic membrane was broken at 24 h of treatment.
PPT and DPN decreased T. gondii replication by approximately 20% compared to the control with a statistically significant difference of (p < 0.001) at 24 and 48 h p.i. (Figure 5A,B). The agonist of ERβ DPN reduced the percentage of infected neurons at 24 and 48 h (22% and 16%, respectively) versus the control with a statistically significant difference of (p < 0.001) ( Figure 5C,D).
Toxoplasma gondii replication decreased by progesterone, only at a dose of 20 nM at 24 h. The combination of progesterone plus Mif (1 µM/mL) did not affect T. gondii replication ( Figure 6A,B). There was a significant reduction in the percent of infected neurons by progesterone or by its combination with Mif (p < 0.001) both at 24 and at 48 h p.i. (Figure 6 C,D). Figure 7A,B, show that the neurons treated with progesterone at 24 and 48 h had more damage by Toxoplasma than E2 plus Tam or PPT or DPN. In Figure 7C,D, neurons infected and treated with progesterone and Mifepristone were better preserved than those treated only with progesterone. Microorganisms 2021, 9,           The effects of E2 plus P4 on Toxoplasma gondii replication and the percentage of infected neurons are shown in Figure 8A,B. The pretreatments reduced the percentage of replication and neurons infected with E2 plus P4 (25-30%) at 24 and 48 h with a statistically significant difference of * p < 0.05 and ** p < 0.001, respectively.  Figure 7A,B, show that the neurons treated with progesterone at 24 and 48 h had more damage by Toxoplasma than E2 plus Tam or PPT or DPN. In Figure 7C,D, neurons infected and treated with progesterone and Mifepristone were better preserved than those treated only with progesterone. The effects of E2 plus P4 on Toxoplasma gondii replication and the percentage of infected neurons are shown in Figure 8A,B. The pretreatments reduced the percentage of replication and neurons infected with E2 plus P4 (25-30%) at 24 and 48 h with a statistically significant difference of * p < 0.05 and ** p < 0.001, respectively.

Discussion
To our knowledge, this is a pioneer study in the analysis of the effect of hormonal steroids (i.e., 17β-estradiol and progesterone) as well as its agonists and antagonists regarding Toxoplasma gondii infection in cultured cortex neurons.
In brain mice, Toxoplasma gondii tachyzoites invade the microglia and the neurons [42,43]. In primary cultures of human fetal cells, T. gondii tachyzoites were replicated in astrocytes and neurons; the highest T. gondii infection was observed in astrocytes [44,45]. In the present study, neurons treated with E2 at a physiological dose (10 nM) decreased T. gondii replication at 24 h p.i. The highest concentration of E2 (160 nM) did not control the Toxoplasma infection. A saturation process could be taking place in the estradiol receptors. However, the percentage of infected neurons decreased at all doses of estradiol utilized in this study. 17β-Estradiol regulates the invasion of Toxoplasma gondii into neurons and may avoid the entry of parasites. However, a greater number of studies are necessary to corroborate this hypothesis. Tamoxifen in combination with estradiol reduced T. gondii replication in neurons even with a lower E2 dose; maybe the mechanism involves preferment binding of tamoxifen to ERβ compared to ERα, similar to what occurs with the DPN agonist. Tamoxifen, as a SERM member, competes with estradiol: however, its affinity for classical estrogen receptors is minor to E2, and it could act as a partial agonist [26,46]. Pung and Luster 1986 [36] demonstrated that tamoxifen could reduce Toxoplasma gondii cysts in mouse brain. In this study, we observed a reduction in Toxoplasma tachyzoites inside neurons by tamoxifen; this effect was similar in astrocytes infected by Toxoplasma gondii [47]. On the other hand, it has been reported that tamoxifen induces autophagy in infected cells, and this may be another mechanism involved in reducing Toxoplasma gondii burden in neurons [48,49].
The interaction of E2 and Tam also decreased the percentage of infected neurons. Our results are similar to those found by Zhang in 2017, who showed that tamoxifen reduces the number of parasites by approximately 50% in host cells [50].
The mechanism by which tamoxifen affects Toxoplasma infection is unknown. However, in Figure 9, we discuss the possible interactions of sex hormones with T. gondii infections.

Discussion
To our knowledge, this is a pioneer study in the analysis of the effect of hormonal steroids (i.e., 17β-estradiol and progesterone) as well as its agonists and antagonists regarding Toxoplasma gondii infection in cultured cortex neurons.
In brain mice, Toxoplasma gondii tachyzoites invade the microglia and the neurons [42,43]. In primary cultures of human fetal cells, T. gondii tachyzoites were replicated in astrocytes and neurons; the highest T. gondii infection was observed in astrocytes [44,45]. In the present study, neurons treated with E2 at a physiological dose (10 nM) decreased T. gondii replication at 24 h p.i. The highest concentration of E2 (160 nM) did not control the Toxoplasma infection. A saturation process could be taking place in the estradiol receptors. However, the percentage of infected neurons decreased at all doses of estradiol utilized in this study. 17β-Estradiol regulates the invasion of Toxoplasma gondii into neurons and may avoid the entry of parasites. However, a greater number of studies are necessary to corroborate this hypothesis. Tamoxifen in combination with estradiol reduced T. gondii replication in neurons even with a lower E2 dose; maybe the mechanism involves preferment binding of tamoxifen to ERβ compared to ERα, similar to what occurs with the DPN agonist. Tamoxifen, as a SERM member, competes with estradiol: however, its affinity for classical estrogen receptors is minor to E2, and it could act as a partial agonist [26,46]. Pung and Luster 1986 [36] demonstrated that tamoxifen could reduce Toxoplasma gondii cysts in mouse brain. In this study, we observed a reduction in Toxoplasma tachyzoites inside neurons by tamoxifen; this effect was similar in astrocytes infected by Toxoplasma gondii [47]. On the other hand, it has been reported that tamoxifen induces autophagy in infected cells, and this may be another mechanism involved in reducing Toxoplasma gondii burden in neurons [48,49].
The interaction of E2 and Tam also decreased the percentage of infected neurons. Our results are similar to those found by Zhang in 2017, who showed that tamoxifen reduces the number of parasites by approximately 50% in host cells [50].
The mechanism by which tamoxifen affects Toxoplasma infection is unknown. However, in Figure 9, we discuss the possible interactions of sex hormones with T. gondii infections. Microorganisms 2021, 9, x FOR PEER REVIEW 12 of 15 Observe that tamoxifen can induce autophagy and the steroid hormones can avoid the entrance of parasites to neurons through the membrane. The mechanism of this last process is unknown. Figure 9 shows the interactions between hormones, and their receptors are shown on its specific receptor. Tamoxifen binds to ERβ (blue and purple), and it is possible that this complex, induces the formation of an autophagosome. With the presence of proteins (in green) necessary for this process, in consequence, it will active a lysosome and will form an autolysosome. Progesterone (P4, pink), receptor of progesterone (PRA, light green), E2 (estradiol, gray), estradiol receptor beta (ER-beta-E2, gray and purple), selective agonist for receptor β of E2, 23-bis(4-hydroxyphenyl) propionitrile (DPN, green triangle). Estrogen receptor α bind to estradiol (ER-alfa-E2, orange and gray), selective agonist of receptor α, E2 4,4',4"-(4-propyl-[1H] pyrozole-1,3,5-triyl) trisphenol (PPT, brown triangle ), selective estrogen receptor modulator, tamoxifen (Tam, blue rhombus).
In the present study, DPN produced an essential reduction in Toxoplasma gondii infection in neurons in vitro. The response of the neurons to this agonist maybe is due to fact of its high specificity for ERβ, affording better neuronal protection [51][52][53][54][55].
Progesterone did not reduce Toxoplasma gondii replication despite; it decreased the percentage of infected neurons. Progesterone could modulate the number of their receptors in neurons in vitro [55]. In current studies of our group of work, we reported that progesterone could reduce ERβ and increase ERα in THP1 cells, maybe in neurons could be a similar regulation and thus modulate the parasite invasion [39].
The antagonist of progesterone, Mif, did not revert the action of progesterone on T. gondii replication at any of the experimental dosages; this could be due to the dose and time of exposure. However, there are no previous studies in this regard; thus, it is imperative to acquire a more significant number of investigations on this topic. The combination of progesterone and Mif exerted a lesser effect on Toxoplasma infected neurons compared Observe that tamoxifen can induce autophagy and the steroid hormones can avoid the entrance of parasites to neurons through the membrane. The mechanism of this last process is unknown. Figure 9 shows the interactions between hormones, and their receptors are shown on its specific receptor. Tamoxifen binds to ERβ (blue and purple), and it is possible that this complex, induces the formation of an autophagosome. With the presence of proteins (in green) necessary for this process, in consequence, it will active a lysosome and will form an autolysosome. Progesterone (P4, pink), receptor of progesterone (PRA, light green), E2 (estradiol, gray), estradiol receptor beta (ER-beta-E2, gray and purple), selective agonist for receptor β of E2, 23-bis(4-hydroxyphenyl) propionitrile (DPN, green triangle). Estrogen receptor α bind to estradiol (ER-alfa-E2, orange and gray), selective agonist of receptor α, E2 4,4 ,4 -(4-propyl-[1H] pyrozole-1,3,5-triyl) trisphenol (PPT, brown triangle ), selective estrogen receptor modulator, tamoxifen (Tam, blue rhombus).
In the present study, DPN produced an essential reduction in Toxoplasma gondii infection in neurons in vitro. The response of the neurons to this agonist maybe is due to fact of its high specificity for ERβ, affording better neuronal protection [51][52][53][54][55].
Progesterone did not reduce Toxoplasma gondii replication despite; it decreased the percentage of infected neurons. Progesterone could modulate the number of their receptors in neurons in vitro [55]. In current studies of our group of work, we reported that progesterone could reduce ERβ and increase ERα in THP1 cells, maybe in neurons could be a similar regulation and thus modulate the parasite invasion [39].
The antagonist of progesterone, Mif, did not revert the action of progesterone on T. gondii replication at any of the experimental dosages; this could be due to the dose and time of exposure. However, there are no previous studies in this regard; thus, it is imperative to acquire a more significant number of investigations on this topic. The combination of progesterone and Mif exerted a lesser effect on Toxoplasma infected neurons compared with progesterone alone. One explanation could be a change in progesterone membrane receptors as well as in their affinity [13,[30][31][32].
The combination of 17β-estradiol and progesterone significantly reduced Toxoplasma gondii replication and the percentage of infected neurons; this may be due to the major expression of classical estrogen receptors [39], E2 plus progesterone modulating the viability of extracellular parasites and the passive invasion to neurons. This result is similar to host cells infected with tachyzoites treated with dehydroepiandrosterone (DHEA) [13,56,57].

Conclusions
E2 and tamoxifen as well as PPT and DPN regulated Toxoplasma gondii infection in neurons.
The neuronal response to these agonists is clear evidence of the essential participation of classical estrogen receptors in the process of Toxoplasma gondii infection.
The combination of E2 and P4 decreased Toxoplasma gondii infected neurons. This action could be considered as a neuro-protector effect on T. gondii infection.