Eight-week-old male C57BL/6 mice were purchased from the Animal Center of Sun Yat-sen University (Guangzhou, China), maintained in specific-pathogen-free environment, and had free access to a commercial basal diet and tap water ad libtum. Animals were provided with humane care and healthful conditions. All individuals who are involved in animal experiments received instruction in experimental methods and in the care, maintenance, and handling of mice; and all efforts were made to minimize animal suffering. Animals were sacrificed using CO₂ asphyxiation and the appropriate organs were harvested. The protocol used in this study was approved by the Committee on the Ethics of Animal Experiments of Sun Yat-sen University [Permit Numbers: SCXK (Guangdong) 2016–0029].

Tachyzoites of T. gondii RH strain were propagated by serial passage in human foreskin fibroblast-1 (HFF-1) cell (BNCC100406, BeNa Culture Collection, Beijing, China) monolayer grown in Dulbecco’s modified eagle’s medium (11995065, Gibco, Thermo Fisher Scientific Inc., Rockford, USA) supplemented with 10% (V/V) fetal bovine serum (10099141, Gibco, Thermo Fisher Scientific Inc.). After lysis of T. gondii-infected cells, tachyzoites were harvested by centrifugation at 2 000 × g for 5 min. The tachyzoites were enumerated using manual counting with a hemocytometer, and experimental T. gondii-infected mice were intraperitoneally (i.p.) injected with 1×10² tachyzoites.

Galectins was blockaded by α-lactose to limit binding to their receptors as described previously (25, 26). A total of 42 C57BL/6 mice were divided into 4 groups: 11 mice were infected with T. gondii tachyzoites (Tg-infected mice); 11 mice were infected with T. gondii tachyzoites and injected i.p. with 27 mM of α-lactose (L8040-100, Sigma-Aldrich, St. Louis, MO, USA) solution in PBS (Tg+lact mice) twice daily starting from day 0 post infection (p.i.) until death of the mice; 10 mice were injected i.p. with α-lactose solution twice daily as α-lactose control (lact mice), while 10 mice were injected with equal volume of PBS twice daily as uninfected control (uninfected mice). Mortality was monitored daily. Another 16 mice were equally divided into four groups. They were sacrificed by CO₂ asphyxiation on day 7 p.i., and the liver and spleen of each mouse were harvested for further analysis.

Antibodies used for in vitro analysis include anti-CD86 mAb (GB13585) and anti-CD206 pAb (GB113497) (Wuhan Servicebio Biotechnologies Co., Ltd., Wuhan, China), anti-β-actin mAb (BM0627) (Wuhan Boster Biological Engineering Co., Ltd., Wuhan, China), anti-β-tubulin mAb (3G6) (Abbkine Inc., Wuhan, China), anti-38 kDa protein of T. gondii mAb (K97A) (Thermo Fisher Scientific Inc.), and anti-CD137 pAb (ab203391) (Abcam Plc., Waltham, USA).

Total RNA was extracted from about 100 mg of liver or spleen from each mouse using the MiniBEST Universal RNA Extraction Kit (9767, TaKaRa Bio, Inc., Shiga, Japan) according to the manufacturer’s protocol. The quality of total RNA was analyzed by running 5 μl of each RNA sample on a 1.0% agarose gel stained with ethidium bromide. The quantity of total RNA was estimated by measuring the ratio of absorbance at 260 and 280 nm using a NanoDrop One spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA). First-strand cDNA was constructed from 1.5 μg of total RNA with oligo (dT) as primers using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (6210A, TaKaRa Bio Inc.). The cDNA products were stored at −80°C for use. To determine the mRNA levels of T. gondii major surface antigen 1 (SAG1), IFNγ, TNFα, IL-12p40, IL-4, IL-10, Gal-1, Gal-3, Gal-8, Gal-9, CD44, CD137, PDI, and Tim-3, qRT-PCR was performed using the SYBR^® Premix Ex Taq™ II Kit (Tli RNaseH Plus) (RR820A, TaKaRa Bio Inc.) according to the manufacturer’s instructions. The primer sequences are listed in Table 1. Briefly, a total of 20 μl reaction mixture contained 10 μl of SYBR^® Premix Ex Taq™ II (2×), 1 μl of each primer (8 μM), 6 μl of dH₂O, and 2 μl of cDNA (0.2 μg/μl). Amplification was pre-denaturized for 30 s at 95°C, followed by 40 cycles of 5 s at 95°C and 30 s at 60°C using a CFX96 Touch^® Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Specific mRNA expression levels were normalized to that of the housekeeping gene, GAPDH, and the results are expressed as fold change compared to uninfected mice.

Hepatic tissues were treated with RIPA lysis buffer (P0013B, Beyotime, Haimen, China), and lysate was incubated on ice for 30 min. After centrifugation at 10, 000 × g for 5 min at 4°C, the protein concentration of each lysate was determined using a BCA Protein Assay Kit (P0010S, Beyotime). Total protein (20 μg) from each sample was boiled in SDS-PAGE loading buffer under reducing conditions, run on a NuPAGE^™ 4-12% Bis-Tris Protein Gel (NP0322BOX, Thermo Fisher Scientific Inc.), and transferred to PVDF membranes (FFP24, Beyotime). The membranes were blocked with QuickBlock^™ blocking buffer (P0222, Beyotime) in PBSTw, probed with primary antibodies, and followed by application of horseradish peroxidase-conjugated secondary antibodies [goat anti-mouse (BA1050, Wuhan Boster Biological Engineering Co., Ltd.) or goat anti-rabbit (BA1054, Wuhan Boster Biological Engineering Co., Ltd.)]. The signal was revealed by BeyoECL Plus Kit (P0018S, Wuhan Boster Biological Engineering Co., Ltd.) and imaged using an Odyssey FC imaging system (LI-COR Biosciences, Lincoln, NE, USA).

For histological examination, the liver and spleen tissues were harvested and immediately fixed in 10% buffered formaldehyde (Guangzhou Chemical Reagent Factory, China) for 48 h. Five-micrometer-thick sections (50 or 100 μm distance between sections) of the organs from mice were stained with hematoxylin (1051740500, Sigma-Aldrich) and eosin (318906, Sigma-Aldrich) solution to evaluate the histological changes. Sections, blinded for groups, were histopathologically evaluated under 20× objective lens in five noncontiguous sections from 4 mice of each group. The semi-quantitative histopathological scores were determined based on previously described criteria (20, 27) with modification. In brief, the histological changes were scored as 0, 1, 2, and 3 (absent, mild inflammation, moderate inflammation and necrosis, and severe inflammation and necrosis, respectively).

Tissue samples of livers and spleens of mice from different groups were fixed in 3% glutaraldehyde and 1% osmium tetroxide [both in 100 mM PBS, pH 7.2] before being dehydrated through a series of graded ethanol solutions. The fixed tissues were then embedded in SPI Pon 812 resin as instructed by the manufacture (02660-AB, Structure Probe Inc., West Chester, PA, USA). Ultrathin sections (70 nm) were cut from the embedded tissues using the Leica EM UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany) and mounted on formvar-coated grids. The sections were then stained for 15 min in aqueous 1% uranyl acetate followed by 0.2% lead citrate. Finally, samples were analyzed under a JEM100CX-II transmission electron microscope (JEOL Ltd., Tokyo, Japan) at an accelerating voltage of 100 kV.

Immunohistochemistry analysis was carried out using a Strept Avidin Biotin Complex (SABC)-alkaline phosphatase (AP) kit (SA1052, Wuhan Boster Biological Engineering Co., Ltd.). Liver sections (5 μm) of mice from different groups were deparaffinized and rehydrated in distilled water, followed by inactivating endogenous peroxidases with 3% hydrogen peroxide for 10 min at 37°C. Heat-induced antigen retrieval was carried out in an 800 W microwave oven (Media, Shunde, China) for 30 min. Then, nonspecific binding was blocked by incubation in 5% bovine serum albumin in PBS (pH 7.4) for 20 min at room temperature. The sections were incubated with primary antibodies in a dilution of 1:200 overnight at 4°C, and then incubated with the goat anti-rabbit IgG secondary antibody in the kit. Sections incubated with secondary antibodies were only used as isotype controls. Signals were detected with SABC-AP reagent, and developed using BCIP/NBT reagent. Finally, the sections were counterstained with hematoxylin (1051740500, Sigma-Aldrich) and imaged under a M8 slice scanner (Precipoint, Jena, Germany). Positive cells were identified by dark-brown staining, and quantified by using Image-Pro Plus (Image Z1 software, Media Cybernetics, MD, USA). The number of cells in each field was determined under high power field as well as the area of each field (0.177 mm²). The density of positive cells was expressed as cell number per square millimeter.

Liver sections (5 μm) were dewaxed firstly, and treated with antigen retrieval solution (P0088, Beyotime) according to the manufacture’s protocol. Then the sections were blocked using Immunol Staining Blocking Buffer (P0102, Beyotime) at room temperature for 60 min. The fluorescence sections were incubated with primary antibody at room temperature for 60 min in a dark chamber, incubated with fluorescence labeling secondary antibody at room temperature for 60 min in a dark chamber, and then incubated with DAPI (ab228549, Abcam) for 30 min in a dark chamber. CD137⁺ cells were marked by green fluorescent signals. The sections were observed under an EVOS FL autofluorescence microscope (Thermo Fisher Scientific Inc.), and positive cells were automatically counted by Image-pro Plus 6.0 (Media Cybernetics).

Statistical analysis of the data was performed using Log-rank test, Wilcoxon rank sum test, Student’s t-test, and one-way ANOVA followed by Bonferoni’s multiple comparison tests using IBM SPSS Statistics version 22.0 (IBM Corp., Armonk, NY, USA). Pearson’s correlation coefficient was used to analyze correlations between the levels of Gal-9, CD137, and IFNγ. All graphs were generated using GraphPad Prism 8 (GraphPad software, San Diego, CA, USA). Data are presented as mean ± standard deviation (SD) at least three independent biological replicates. A value of P lower than 0.05 was considered statistically significant.

Mice were monitored daily after T. gondii infection. Tg-infected mice died between 7 and 9 days p.i., whereas Tg+lact mice died between 7 and 10 days p.i. Log-rank test showed that compared with Tg-infected mice, there was a significant increase of survival rate of Tg+lact mice (P < 0.01, Figure 1A). Compared with Tg-infected mice, T. gondii SAG1 mRNA expression level in the liver of Tg+lact mice was significantly decreased (P < 0.01) (Figure 1B). Western blot assay confirmed that T. gondii p38 protein level was decreased in the liver tissue of Tg+lact mice in comparison of that of Tg-infected mice (Figure 1C). The results indicated that blockage of galectin-receptor interactions may contribute to host defense against acute T. gondii infection.

To evaluate the effects of blockage of galectin-receptor interactions on tissue pathological changes, the liver and spleen tissues of mice from different groups were examined histologically (Figure 2A). No histological changes were observed in the liver and spleen tissues of uninfected mice injected with PBS or α-lactose. After T. gondii infection, severe damage with a great number of inflammatory foci of cell infiltrates including neutrophils, lymphocytes, and mononuclear cells were observed in the liver tissue of Tg-infected mice. In comparison, attenuated histological damage was observed in the liver tissue of Tg+lact mice. Compared with Tg-infected mice, semi-quantitative histopathological score based on pathological changes in the liver was significantly decreased in Tg+lact mice (P < 0.05, Figure 2B). There were a large amount of infiltrated inflammatory cells and tachyzoites observed in the spleens of Tg-infected mice and Tg+lact mice, and lots of lymphocytes were intruded into the red pulps, which led to the boundary between the white pulp and red pulp blurred (Figure 2A). However, the semi-quantitative histopathological scores in the spleens between Tg-infected mice and Tg+lact mice had no significant difference (P > 0.05, Figure 2B).

Transmission electron microscopy (TEM) was used to reveal the subcellular structures of the liver and spleen tissues from the two T. gondii-infected groups ( Figure 3). Cellular organelles, such as mitochondria and endoplasmic reticulum (ER) in the hepatocytes of Tg-infected mice, became swollen and deformed. In addition, neutrophils, Kupffer cells (KCs), and numerous T. gondii tachyzoites were observed in the liver tissue of this group. Similar subcellular structure changes of hepatocytes were observed in the liver of Tg+lact mice, however, the lesion was less severe and fewer T. gondii tachyzoites were observed in the liver tissue. In the necrosis area of spleen tissue of Tg-infected mice, cell membranes and intercellular connections were damaged, and normal host cells were hardly observed. In addition, there were numerous extracellular T. gondii tachyzoites observed by TEM. However, the damage of ultrastructure of the spleen tissue of Tg+lact mice was less severe. The number of tachyzoites was fewer, and most of them were inside intracellular parasitophorous vacuoles (PVs) of the spleen tissue cells rather than egressed into extracellular space of the tissue.

The mRNA expression levels of Gal-1, Gal-3, Gal-8, and Gal-9 (Figure 4A), and Gal-9 receptors (CD44, CD137, PDI, and Tim-3) (Figure 4B) in the livers of Tg-infected mice and Tg+lact mice were examined by using qRT-PCR and presented as heatmaps. The most conspicuous upregulation values were found for Gal-9 and its receptor CD137. Compared with uninfected mice, the mRNA levels of Gal-9 (P < 0.01) and CD137 (P < 0.001 and P < 0.05, respectively) were significantly increased in the liver and spleen of Tg-infected mice, and the levels of Gal-9 (P < 0.05 and P < 0.01, respectively) and CD137 (P < 0.01) were significantly increased in the liver and spleen of Tg+lact mice. Compared with Tg-infected mice, the mRNA levels of CD137 in the liver and Gal-9 in the spleen of Tg+lact mice were significantly increased (P < 0.01, Figure 4C).

Immunofluorescence assay showed that there were only a few of CD137⁺ cells observed in the liver tissue of uninfected mice (Figure 5A). However, compared with uninfected mice, the numbers of CD137⁺ cells in the livers of both Tg-infected mice and Tg+lact mice were significantly increased (P < 0.001). Compared with Tg-infected mice, the number of CD137⁺ cells in the liver of Tg+lact mice was significantly increased (P < 0.01, Figure 5B). Western blot assay confirmed that the protein level of CD137 in the liver tissue of Tg+lact mice was increased compared with that of Tg-infected mice (Figure 5C).

The effects of α-lactose treatment on Th1 and Th2 cytokine responses during T. gondii infection were evaluated by measuring the mRNA expression levels of IFNγ, TNFα, IL-12p40, IL-4, and IL-10 in the livers and spleens of mice from different groups. Compared with uninfected mice, there were significantly increased mRNA expression levels of IFNγ (P < 0.001 and P < 0.05, respectively), TNFα (P < 0.05), IL-12p40 (P < 0.01), and IL-4 (P < 0.05) in the liver and spleen of Tg-infected mice, and significantly increased IL-10 level (P < 0.001) in the liver of Tg-infected mice on day 7 p.i.; significantly increased levels of IFNγ (P < 0.01), TNFα (P < 0.01 and P < 0.05, respectively), IL-12p40 (P < 0.01 and P < 0.05, respectively), IL-4 (P < 0.001), and IL-10 (P < 0.01 and P < 0.001, respectively) in the liver and spleen of Tg+lact mice on day 7 p.i. However, compared with Tg-infected mice, there were significantly increased IFNγ level (P < 0.01) in the liver, and significantly increased levels of IL-4 (P < 0.001) and IL-10 (P < 0.01) in the liver and spleen of Tg+lact mice on day 7 p.i. The results indicated that blockage of galectin-receptor interactions may increase both Th1 and Th2 responses in the liver of T. gondii-infected mice (Figure 6).

The correlations between the mRNA levels of Gal-9 and CD137, Gal-9 and IFNγ, and CD137 and IFNγ in the liver and spleen of Tg-infected mice or Tg+lact mice were evaluated, herein only significant correlations were presented. In Tg-infected mice, there were significant correlations between the mRNA levels of Gal-9 and CD137 (r = 0.986, P = 0.014), Gal-9 and IFNγ (r = 0.991, P = 0.009), and CD137 and IFNγ (r = 0.998, P = 0.002) in the liver (Figure 7A), and significant positive correlations between the mRNA levels of Gal-9 and CD137 (r = 0.990, P = 0.010), Gal-9 and IFNγ (r = 0.997, P = 0.003), and CD137 and IFNγ (r = 0.976, P = 0.024) in the spleen (Figure 7B). In Tg+lact mice, there was significant correlation between the mRNA levels of CD137 and IFNγ (r = 0.955, P = 0.045) in the liver (Figure 7C).

Macrophages are a major player in defense against pathogens in the liver. In uninfected mice, only a few macrophages (stained brown) were observed in the liver tissue when using CD86 and CD206 as the immunohistochemical labels for M1 and M2 macrophages, respectively (Figure 8A). Compared with uninfected mice, the numbers of CD86⁺ macrophages in the livers of Tg-infected and Tg+lact mice were significantly increased (P < 0.001). However, there was no significant difference between the two T. gondii-infected groups (P > 0.05). Compared with uninfected mice, the numbers of CD206⁺ macrophages in the livers of Tg-infected mice and Tg+lact mice were significantly increased (P < 0.001). Compared with Tg-infected mice, the number of CD206⁺ macrophages in the liver of Tg+lact mice was significantly increased (P < 0.001, Figure 8B). The results indicated that blockage of galectin-receptor interactions may induce M2 macrophage polarization in the liver of T. gondii-infected mice.