Blockage of Galectin-Receptor Interactions Attenuates Mouse Hepatic Pathology Induced by Toxoplasma gondii Infection

Abstract

Toxoplasma gondii (T. gondii), one of the most important Apicomplexan protozoa, causes toxoplasmosis in human throughout the world. Galectin (Gal)-9 triggers a series of immune events via binding to its receptors, including T cell immunoglobulin and mucin-containing molecule 3, CD137, CD44, and protein disulfide isomerase. To examine the regulatory role of galectin-receptor interactions in anti-toxoplasmic activities, C57BL/6 mice were infected with T. gondii RH strain and intraperitoneally injected with alpha (α)-lactose to block the interactions of galectins and their receptors. Heatmaps showed upregulated values for Gal-9 and CD137 in the livers of T. gondii-infected mice and T. gondii-infected mice treated with α-lactose. Compared with T. gondii-infected mice, T. gondii-infected mice treated with α-lactose showed significantly increased survival rate, decreased tissue parasite burden, attenuated liver histopathology, increased mRNA expression levels of CD137, IFNγ, IL-4, and IL-10 in the liver, and increased Gal-9 mRNA expression level in the spleen. Correlation analysis showed that significant positive correlations existed between the mRNA expression levels of Gal-9 and CD137, Gal-9 and IFNγ, as well as between CD137 and IFNγ in the liver and spleen of T. gondii-infected mice; between CD137 and IFNγ in the liver of T. gondii-infected mice treated with α-lactose. In addition, blockage of galectin-receptor interactions showed enhanced M2 macrophage polarization in the liver of T. gondii-infected mice. Our data indicate that Gal-9-CD137 interaction may play an important role in T. gondii proliferation and liver inflammation in mice during acute T. gondii infection, through regulating T cell and macrophage immune responses.

Keywords: C57BL/6 mice; CD137; Gal-9; T. gondii; hepatic pathology.

Figure 1

Survival rate and parasite burden. (A) Survival rate. Tg-infected mice (n = 11) died between days 7 and 9 p.i., while Tg+lact mice (n = 11) died between days 7 and 10 p.i. (B) Parasite burden in the liver tissues was estimated based on SAG1 mRNA level measured by qRT-PCR. Values are means from triplicate measurements, and data are shown as − ΔΔCt values; two independent experiments were performed with four mice per group. **P < 0.01. Y axis represents the mRNA relative expression level (REL) of different genes. (C) Parasite burden in the liver tissues was estimated based on the protein level of p38 from T. gondii detected by Western blot assay.

Figure 2

The liver and spleen histopathology of mice from different groups. Mice were i.p. inoculated with 10² tachyzoites of T. gondii and killed at day 7 p.i. (A) Representative microscopic pictures showed tissue sections stained with H&E from different groups. Original magnification 50× (scale bar = 200 µm) of the large image; the small image is 1000× (scale bar = 20 μm). Yellow arrows indicate T. gondii tachyzoites. (B) Histopathological score analysis of the liver and spleen tissues. Data are represented as mean ± SD. There were 4 mice per group, and the data are representative of two experiments. *P < 0.05.

Figure 3

The ultrastructure of the livers and spleens in T. gondii-infected mice with or without α-lactose treatment. Blue stars indicate T. gondii tachyzoites, blue rectangles indicate the accumulation areas of tachyzoites, and red circles indicate the large necrosis areas. N, nucleus; ER, endoplasmic reticulum; Mt, mitochondrion; Ne, neutrophil; KC, Kupffer cell; PV, parasitophorous vacuole.

Figure 4

Gal-9 and its receptor CD137 were upregulated during T. gondii infection. Heatmap visualization showed the mRNA expressions of Gal-1, Gal-3, Gal-8, and Gal-9 (A) and the receptors of Gal-9 (CD44, CD137, PDI, and Tim-3) (B) in the livers of Tg-infected mice and Tg+lact mice, while histograms showed the expression levels of Gal-9 and CD137 in the livers and spleens of mice from different groups (C). Liver and spleen tissues were collected on day 7 p.i. for evaluating mRNA expression levels by using qRT-PCR. Values are means from triplicate measurements, and data are presented as means ± SD; two independent experiments were performed with four mice per group. *P < 0.05, **P < 0.01, and ***P < 0.001. Y axis represents the mRNA relative expression level (REL) of different genes.

Figure 5

CD137⁺ cells in the liver tissues of mice from different groups. (A) Immunofluorescence staining showed CD137⁺ cells (labeled green) in the livers of mice. Original magnification 200× (scale bar = 100 µm). (B) Morphometric analysis in liver tissues by showing the number of CD137⁺ cells per square millimeter. Data are presented as means ± SD; experiments were performed with four mice per group. **P < 0.01, and ***P < 0.001. (C) The protein levels of CD137 in the livers of mice were detected by Western blot assay.

Figure 6

The mRNA expression levels of Th1 and Th2 cytokines in the livers (A) and spleens (B) of mice from different groups on day 7 p.i. Values are means from triplicate measurements, and data are presented as means ± SD; two independent experiments were performed with four mice per group. *P < 0.05, **P < 0.01, and ***P < 0.001. Y axis represents the mRNA relative expression level (REL) of different genes.

Figure 7

Correlation analysis between the mRNA expression levels detected in the livers and spleens of Tg-infected mice or Tg+lact mice (n = 4). (A) Significant correlations between Gal-9 and CD137, Gal-9 and IFNγ, and CD137 and IFNγ in the liver, and (B) Significant correlations between Gal-9 and CD137, Gal-9 and IFNγ, and CD137 and IFNγ in the spleen of Tg-infected mice. (C) Significant correlation between CD137 and IFNγ in the liver of Tg+lact mice. The r value generates for the theoretical line of best fit, while the P value indicates the significance of the correlation.

Figure 8

Immunohistochemical staining for CD86⁺ and CD206⁺ cells in the livers of mice from different groups. (A) Immunohistochemical staining of CD86⁺ and CD206⁺ cells were labeled by CD86 mAb and CD206 pAb, respectively. Red arrows indicate positive cells and black arrows indicate tachyzoites. Original magnification 200× (scale bar = 100 µm). (B) Morphometric analysis showed the number of CD86⁺ or CD206⁺ cells per square millimeter in the liver tissues. Data are presented as means ± SD; experiments were performed with four mice per group. ***P < 0.001.