Involvement of chemokine receptor CXCR3 in the defense mechanism against Neospora caninum infection in C57BL/6 mice

Abstract

C-X-C motif chemokine receptor 3 (CXCR3) is an important receptor controlling the migration of leukocytes, although there is no report regarding its role in Neospora caninum infection. Herein, we investigated the relevance of CXCR3 in the resistance mechanism to N. caninum infection in mice. Wild-type (WT) C57BL/6 mice and CXCR3-knockout (CXCR3KO) mice were used in all experiments. WT mice displayed a high survival rate (100%), while 80% of CXCR3KO mice succumbed to N. caninum infection within 50 days. Compared with WT mice, CXCR3KO mice exhibited significantly lower body weights and higher clinical scores at the subacute stage of infection. Flow cytometric analysis revealed CXCR3KO mice as having significantly increased proportions and numbers of CD11c-positive cells compared with WT mice at 5 days post infection (dpi). However, levels of interleukin-6 and interferon-γ in serum and ascites were similar in all groups at 5 dpi. Furthermore, no differences in parasite load were detected in brain, spleen, lungs or liver tissue of CXCR3KO and WT mice at 5 and 21 dpi. mRNA analysis of brain tissue collected from infected mice at 30 dpi revealed no changes in expression levels of inflammatory response genes. Nevertheless, the brain tissue of infected CXCR3KO mice displayed significant necrosis and microglial activation compared with that of WT mice at 21 dpi. Interestingly, the brain tissue of CXCR3KO mice displayed significantly lower numbers of FoxP3⁺ cells compared with the brain tissue of WT mice at 30 dpi. Accordingly, our study suggests that the lack of active regulatory T cells in brain tissue of infected CXCR3KO mice is the main cause of these mice having severe necrosis and lower survival compared with WT mice. Thus, CXCR3⁺ regulatory T cells may play a crucial role in control of neosporosis.

Keywords: CXCR3; CXCR3KO; IFN-γ; IL-6; Neospora caninum; neosporosis; regulatory T-cells.

Figure 1

Entire experimental design in this study. (A) Survival experiment, 10 C57BL/6 mice (WT) and 10 CXCR3KO mice (KO) were intraperitoneally (i.p) infected with Neospora caninum tachyzoites (1 × 10⁶). Mice were monitored daily for survival rate, body weight, and clinical scores. All mice were sacrificed at 48 days post infection (dpi). (B) WT (n = 7) and KO mice (n = 7) were i.p infected with N. caninum tachyzoites (1 × 10⁶). Four uninfected mice from each strain were used as control groups. All mice were sacrificed at 5 dpi. Ascitic fluid, blood and different organs (brain, spleen, lung, and liver) were collected for examination of parasite burden, flow cytometry and cytokine ELISAs. (C) mRNA expression analysis of selective host genes in brain samples. Although six WT mice and six KO mice were infected with N. caninum, euthanasia was performed due to human endpoints for two CXCR3KO mice before sampling. Therefore, N. caninum-infected WT (n = 6) and KO mice (n = 4) were sacrificed at 30 dpi. Brains of infected mice were collected and subjected to RNA extraction. (D) Eight WT mice and eight KO mice were infected with 1 × 10⁶ N. caninum tachyzoites and sacrificed at 21 dpi. Three uninfected WT mice and three uninfected KO mice were used as control groups. Different organs (brain, spleen, lung, and liver) were collected for examination of parasite burden (right half) and histopathological analysis (left half of brain).

Figure 2

Clinical parameters of mice infected with Neospora caninum. Wild-type (WT; n = 10) and CXCR3-knockout (CXCR3KO) mice (n = 10) were intraperitoneally infected with N. caninum tachyzoites. Alterations of body weight (A), clinical scores (B), and survival rate (C) of infected mice for 50 days post infection (dpi) were measured. *Significant differences (p < 0.05) between groups were analyzed with log-rank test (C).

Figure 3

Recruitment of immune cells in ascitic fluid of mice following Neospora caninum infection. Wild-type (WT) and CXCR3-knockout (CXCR3KO) mice were infected intraperitoneally with N. caninum tachyzoites. After 5 days, peritoneal exudate cells of infected mice (n = 7) and uninfected mice (n = 4) were collected. Cells were analyzed by flow cytometry to determine absolute numbers of peritoneal cells (A), CD11b⁺ cells (B), CD11c⁺ cells (C), NK cells (D), NKT cells (E), CD4⁺ cells (F), and CD8⁺ cells (G). Values per individual (symbols) and mean levels (horizontal lines) are shown. Data were analyzed with two-way ANOVA followed by Tukey–Kramer multiple-comparison test. *p < 0.05.

Figure 4

Concentrations of IL-6 and IFN-γ in sera and ascites of mice following Neospora caninum infection. Wild-type (WT) and CXCR3-knockout (CXCR3KO) mice were infected intraperitoneally with N. caninum tachyzoites. After 5 days, the sera and ascites of infected mice (n = 7) and uninfected mice (n = 4) were collected. Concentrations of IL-6 (A) and IFN-γ (B) were measured. Values per individual (symbols) and mean levels (horizontal lines) are shown. Data were analyzed with two-way ANOVA followed by Tukey–Kramer multiple-comparison test. *p < 0.05.

Figure 5

mRNA expression analysis of selective host genes in brain samples. Brain samples were collected from Neospora caninum-infected wild-type (WT) and CXCR3-knockout (KO) mice 30 days after the infection. All collected samples were subjected to mRNA expression analysis using specific primers for genes related to brain pathology. Expression levels of marker genes related to brain pathology were compared. Fold expression per individual (symbols) and mean levels (horizontal lines) against WT mice are shown (WT, n = 6, KO, n = 4). Expression of IL-10 mRNA was not detected. Data were analyzed with the Mann–Whitney U-test or t-test. *p < 0.05.

Figure 6

T cell, astrocyte, and microglia populations in brain (frontal lobes, striatum, and diencephalon) of mice. Brains from wild-type (WT) and CXCR3-knockout (CXCR3KO) mice surviving at 21 days after infection (n = 8), as well as brains from uninfected mice (n = 3), were subjected to histopathological processing. T cell, astrocyte, and microglia populations were identified using immunohistochemical staining for CD3, ionized calcium-binding adapter molecule 1 (Iba1), and glial fibrillary acidic protein (GFAP), respectively. (A) Representative immunohistochemical images were taken from serial tissue sections containing T cells, astrocytes, and microglia. Glial nodules and necrosis are shown as arrowheads and arrows, respectively. Scale bars, 50 μm. (B) Positive areas were compared between WT and CXCR3KO or infected and uninfected mice. Horizontal bars represent the mean value. Data were analyzed with two-way ANOVA followed by the Tukey–Kramer multiple-comparison test. *p < 0.05.

Figure 7

Population of FoxP3⁺ cells in mouse brains. (A) Brain regulatory T cells were identified by immunohistochemistry for FoxP3 and counterstaining with Mayer’s hematoxylin. Nuclear positivity for FoxP3 was detected in the brains of infected wild-type (WT) and CXCR3-knockout (CXCR3KO) mice (arrowheads). (B) To clarify the proportion of Treg cells to other infiltrating cells, proportions of nuclear-positive area for FoxP3 to hematoxylin were analyzed with two-way ANOVA followed by the Tukey–Kramer multiple-comparison test. *p < 0.05.