T. gondii Wh6 strain (avirulent strain) with genotype Chinese 1 (ToxoDB#9) was isolated as previously described (20). Cysts were maintained in the brain of chronically infected mice for in vivo infection. To collect cysts, brains from infected mice were mechanically homogenized in 1-ml sterile phosphate-buffered saline (PBS). Cyst numbers were counted in a 10-μl brain suspension using a light microscope (21). Tachyzoites of T. gondii were passaged in human foreskin fibroblast (HFF) monolayers for in vitro experiments. Mouse primary astrocytes were purchased from FenghuiShengwu (Changsha, China) and cultured in Dulbecco's modified Eagle medium (DMEM) medium supplemented with 10% fetal bovine serum.

Mice were divided into three groups (three mice/group): control group (non-infection group), chronic group (chronic infection without cyclophosphamide treatment), and TE group (chronic infection with cyclophosphamide treatment). The Wh6 strain cysts were prepared by homogenization of the brain tissues in phosphate-buffered saline (PBS). Seven-week-old female BALB/c mice were intragastrically administered with 30 cysts. After 6 weeks, mice with latent infection were intraperitoneally injected with cyclophosphamide (50 mg/kg; Baxter Oncology GmbH, Germany) to induce recurrence of toxoplasmosis as previously described (11). Seven days later, all mice of the three groups were euthanized for collection of the brain tissues for further experiments. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Anhui Medical University.

ESAs from T. gondii were prepared as described previously (12). Tachyzoites of T. gondii were harvested as described above.

After resuspension with serum-free DMEM, 2 × 10⁷ freshly collected tachyzoites were added into HFF monolayers. T. gondii-infected HFFs were further cultured in the serum-free DMEM medium at 37°C in 5% CO₂ for another 48 h. The supernatants of the infected HFFs were collected by centrifugation at 12,000 g for 10 min at 4°C and then filtered through a 0.22-mm membrane filter. Protein concentration in the supernatants was determined by BCA kits according to the manufacturer's instructions (Thermo-Fisher, Boston, MA). Protein samples were stored at −80°C until use. Non-infected HFFs in serum-free DMEM were used as a negative control.

Mouse primary astrocytes were seeded in six-well cell culture plates. Cells were treated with different doses of TgESAs (0, 0.10, 0.15, and 0.30 mg/ml) for 24 h when the cell confluence reached approximately 70%. Then, C3 protein expression level and A1-specific gene transcription levels were detected by Western blotting and quantitative real-time PCR (qRT-PCR), respectively. In some experiments, astrocytes were pretreated with NFκB inhibitor BAY11-7082 (1 and 5 μM) (22). After 12 h, TgESAs (0.30 mg/ml) was added, and cells were co-cultured for further 24 h.

Mice were anesthetized with 1% pentobarbital and transcardially perfused with 20 ml ice-cold 4% paraformaldehyde after an initial flush with 20 ml ice-cold 0.01 M PBS. Brains were removed and post-fixed with 4% paraformaldehyde for 12 h. Brain tissues were subsequently dehydrated in 30% sucrose in 0.01 M PBS for 48 h. Tissues were embedded in optimal cutting temperature compound (OCT Compound, SAKURA, USA) and then sliced coronally (10–20 μm) on a cryostat microtome (CM3050S, Leica, Germany). For immunofluorescence staining, the samples were blocked with 5% bovine serum albumin (BSA) and 0.5% Triton X-100 (containing 0.02% normal goat serum) for 2 h at room temperature. The samples were incubated with primary antibodies overnight at 4°C and then with the appropriate fluorescent secondary antibodies for 2 h at room temperature. Primary antibodies included anti-GFAP (1:50, Abcam) and anti-C3 (1:400, Abcam). Fluorescent images (astrocytes in mouse cortex) were captured using an Olympus BX53 fluorescence microscope (Olympus, Tokyo, Japan) and processed using ImageJ software (ImageJ, National Institutes of Health, Bethesda, MD) for quantification of florescence intensity.

Mouse brain tissues (mainly from cortex, 100 mg) were homogenized intensively and centrifuged at 12,000 g for 15 min at 4°C. Concentrations of tumor necrosis factor (TNF)-α and interleukin (IL)-1α in the mouse brain were evaluated using commercial kits according to the manufacturer's instructions (BioLegend, USA).

Proteins extracted from mouse brains (mainly from cortex) or astrocytes were separated using SDS-PAGE electrophoresis and then transferred to a PVDF membrane (Millipore, USA). After blocking with 5% BSA for 1 h at room temperature, PVDF membrane was incubated with primary antibodies overnight at 4°C and then with the fluorescent secondary antibodies for 1 h at room temperature. Primary antibodies included anti-GFAP (1:1,000, Abcam), anti-C3 (1:2,000, Abcam), anti-β-actin (1:5,000, Abcam), and anti-Neu-N (1:1,000, Cell Signaling Technology). Fluorescent images were captured by the Tacon 5200 (Biotanon, China) and analyzed using ImageJ software.

Total RNA was extracted from astrocytes using Trizol reagent (Tiangen Biotech, China) according to the manufacturer's protocols. The concentrations of the extracted RNA were measured using NanoDrop 2000c (ThermoFisher, USA) (23–25). Total RNA (1 μg) was reverse-transcribed to cDNA using a reverse transcription kit (TaKaRa, Japan). QRT-PCR was performed on the QuantStudio® 6 Flex real-time PCR instrument (Applied Biosystems, USA) using SYBR™ Green qPCR Master Mix (ThermoFisher, USA). Gene expression levels were normalized to β-tubulin levels using the 2^−ΔΔCt method. All primers for A1 astrocyte-specific genes used in the present study are listed in Table 1 (18).

All statistical analyses were performed using SPSS (Version 24, IBM, USA). All data are expressed as mean ± SEM (standard error of the mean). Differences between groups were assessed by one-way ANOVA followed by Student-Newman-Keuls (SNK) multiple comparison posttest or Student's t test. Differences were considered statistically significant when P < 0.05.

To establish a mouse model of TE, BALB/c mice were orally infected with Wh6 tissue cysts. Six weeks later, Toxoplasma-infected mice were immunosuppressed by intraperitoneal injection with cyclophosphamide to reactivate chronic T. gondii infection. As we can see in Figure 1A, mice with TE showed piloerection and hunching posture, which are typical physical characteristics of acute toxoplasmosis. Western blotting results showed that expression levels of neuron marker Neu-N in mice with TE decreased significantly compared to those in the control group (Figures 1B,C, Ctrl group vs. TE group, 1.00 ± 0.00 vs. 0.52 ± 0.09, P < 0.05). These results indicated that neurons in the central nervous system (CNS) were damaged when the mouse brains were infected with tachyzoites of T. gondii.

The proportion of A1 astrocyte (GFAP⁺C3⁺) was significantly higher in the TE group compared to the controls (Figure 2A) as detected by immunofluorescent assay (IFA).

The expression level of A1-specific protein C3 was evaluated using Western blotting. Consistent with IFA result, the expression level of C3 in brains of the TE group increased dramatically (Figures 2B,C, Ctrl group vs. TE group, 1.00 ± 0.00 vs. 6.98±1.06, P < 0.05), although the expression level of C3 in the chronic group was apparently higher than that of the control group. The concentrations of A1 cytokine inducers (TNF-α and IL-1α) were subsequently measured in the mouse brain using ELISA. The results indicated that TNF-α (Ctrl group vs. TE group, 56.20 ± 19.49 vs. 616.4 ± 104.8, P < 0.05) and IL-1α (Ctrl group vs. TE group, 198.2 ± 75.34 vs. 3,291 ± 260.0, P < 0.05) expression levels were remarkably enhanced in mice with TE (Figures 2D,E).

Since T. gondii tachyzoites can manipulate cells in the mouse brain that they do not productively invade (26), the non-colocalization of tachyzoites and A1 astrocytes prompted us to hypothesize that TgESAs induced astrocyte polarization. To test this hypothesis, the mouse primary astrocyte was incubated in vitro with TgESAs. Western blotting results showed that the expression level of C3 was robustly elevated in the TgESA group (Ctrl group vs. 0.30 mg/ml TgESA group, 1.00 ± 0.00 vs. 11.83 ± 1.32, P < 0.05) (Figures 3A,B). Then, the transcription levels of A1-specific genes were evaluated using qRT-PCR (18). As shown in Figure 3C, after incubation with TgESAs, the transcription levels of Amigo2, Ggta1, H2-D1, H2-T23, and Psmb8 genes were enhanced apparently, while there was no statistical difference in the transcription levels of Fbln5, Iigp1, and Serping1 genes between the control and TgESA groups. Interestingly, when primary astrocytes were pretreated with NFκB inhibitor BAY11-7082, the C3 expression level decreased significantly comparing to the non-inhibitor treatment group (TgESA group). Differences in the C3 expression level between the control and BAY11-7082 groups (TgESA group vs. TgESA + 5 μM BAY group, 9.87 ± 1.60 vs. 2.30 ± 0.70, P < 0.05) suggested that TgESAs polarized astrocytes to A1 subtype via NFκB signaling pathway (Figures 3D,E). These results indicated that TgESAs induced mouse primary astrocyte polarization to A1 subtype in vitro through activation of NFκB signaling pathway.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.