The CXCL10/CXCR3 Axis Promotes Disease Pathogenesis in Mice upon CVA2 Infection

Figures

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  FIG 1. Visualization of inflammation-related genes during CVA2 infection. (A) Expression profiles of inflammation-related genes in organs and tissues (brains, lungs, hearts, and skeletal muscles) of control and CVA2-infected mice. (B) Transcription levels of the most significantly upregulated gene (CXCL10), its structural homologous molecules (CXCL9 and CXCL11), and its receptor (CXCR3) in the organs and tissues. n = 4 per group. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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  FIG 2. IHC staining of CXCL10 in mice infected with CVA2 at 3 dpi and 5 dpi. Black arrows indicate CXCL10 (brown). The number of CXCL10-positive cells per mm2 was quantified by ImageJ software. Scale bar = 50 μm. (A, B) Brains. (C, D) Lungs. (E, F) Hearts. (G, H) Skeletal muscles. n = 3 per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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  FIG 3. IHC staining of CXCR3 in tissue specimens of mice infected with CVA2 at 3 dpi and 5 dpi. Black arrows indicate CXCR3 (brown). The number of CXCR3-positive cells per mm2 was quantified by ImageJ software. Scale bar = 50 μm. (A, B) Brains. (C, D) Lungs. (E, F) Hearts. (G, H) Skeletal muscles. n = 3 per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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  FIG 4. The CXCL10/CXCR3 axis plays a crucial role in disease pathogenesis upon CVA2 infection. (A) Experimental scheme showing infection with CVA2 and treatment with αCXCR3 or IgG isotype. (B) Representative clinical signs induced by CVA2 infection in IgG isotype-treated mice included weight loss, ataxia, and single or double hind limb paralysis. CVA2-infected, αCXCR3-treated mice appeared to be in relatively good health. (C to E) Body weights (C), mean clinical scores (D), and survival rates (E) of IgG-treated groups after CVA2 infection, αCXCR3-treated groups after infection, and the control group. The IgG-treated mice did not survive longer than 5 dpi, and the clinical scores on the days afterwards were carryovers from 5 dpi. n = 8 to 10 per group. **, P < 0.01; ****, P < 0.0001. (F) (a) Histopathological changes of the organs and tissues (brains, lungs, hearts, and skeletal muscles) in IgG isotype and αCXCR3 groups. Scale bar = 1 mm. (b to e) Histology scores for brains (b), lungs (c), hearts (d), and skeletal muscles (e) were evaluated by a person blinded to the treatment of groups. n = 3 per group. *, P < 0.05; **, P < 0.01.

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  FIG 5. Viral distribution in the organs and tissues of IgG isotype- or αCXCR3-treated mice after CVA2 infection. (A) (a) Immunohistochemical staining of viral antigen was conducted in the slices of brains, lungs, hearts, and skeletal muscles. Black arrows indicate the locations of viral antigen. Bar = 50 μm. The number of VP1-positive cells per mm2 was quantified by ImageJ software. n = 3 per group. (b) Brains. (c) Lungs. (d) Hearts. (e) Skeletal muscles. (B to E) Relative expression of VP1 gene in brains (B), lungs (C), hearts (D), and skeletal muscles (E). n = 4 per group. ns, not significant; *, P < 0.05; **, P < 0.01.

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  FIG 6. Blockade of CXCR3 alleviates apoptosis and proinflammatory cytokine expression induced by CVA2. At 5 dpi, the proteins related to apoptosis and genes related to proinflammatory cytokines were determined in IgG isotype and αCXCR3 groups. (A) Immunofluorescence staining of Cl-Caspase-3 (red) was conducted in the organs and tissues. Scale bar = 50 μm. The number of Cl-Caspase-3-positive cells per mm2 was quantified with ImageJ software. n = 3 per group. (A) (b) Brains. (c) Lungs. (d) Hearts. (e) Skeletal muscles. (B) Protein levels of Cl-Caspase-3, Cl-PARP1, and β-actin were measured by Western blotting. n = 2. (C) Densitometry measures for Western blots using ImageJ software in specimens from (a) brains, (b) lungs, (c) hearts, and (d) skeletal muscles. n = 4. (D to G) Relative expression levels of inflammatory cytokines (IL-1β, IL-6, TNF-α, and IFN-γ) were tested in brains (D), lungs (E), hearts (F), and skeletal muscles (G). n = 4 per group. (H) Expression levels of inflammatory cytokines in serum. n = 8. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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  FIG 7. Blockade of CXCR3 causes alteration of inflammatory cell infiltration. (A) IHC staining of CD11b, F4/80, and Ly6G in organs and tissues from IgG isotype- and αCXCR3-treated mice. Black arrows mark CD11b+ cells (mononuclear leucocytes), red arrows mark F4/80+ cells (macrophages), and green arrows mark Ly6G+ cells (neutrophils). (B) Numbers of CD11b+ cells, F4/80+ cells, and Ly6G+ cells per mm2 in the organs and tissues of mice.