CCR2 receptor is essential to activate microbicidal mechanisms to control Toxoplasma gondii infection in the central nervous system

Abstract

Chemokines comprise a structurally related family of cytokines that regulate leukocyte trafficking. Because infection with Toxoplasma gondii can induce an important inflammatory reaction that, if left uncontrolled, can lead to death, we investigated the role of the chemokine receptor CCR2 in T. gondii infection. We orally infected CCR2(-/-) mice with five ME-49 T. gondii cysts and monitored morbidity, survival, and immune response thereafter. The CCR2(-/-) mice displayed higher susceptibility to infection as all mice died on day 28 after infection. Despite similar Th1 responses, a more evident anti-inflammatory response was induced in the peripheral organs of CCR2(-/-) mice compared with wild-type C57BL/6 mice. Additionally, CCR2(-/-) mice presented greater parasitism and a milder inflammatory reaction in their peripheral organs with lesser CD4(+) and MAC-1(+) and greater CD8(+) cell migration. The parasite load decreased in these organs in CCR2(-/-) mice but remained uncontrolled in the central nervous system. Additionally, we observed down-regulated inducible nitric oxide synthase expression in peripheral organs from CCR2(-/-) mice that was associated with a small nitric oxide production by spleen macrophages. In conclusion, in the absence of CCR2, another mechanism is activated to control tissue parasitism in peripheral organs. Nevertheless, CCR2 is essential for the activation of microbicidal mediators that control T. gondii replication in the central nervous system.

Figure 1

Immunohistochemical staining for CCR2 and CCL2 and quantification of positive cells in the small intestine of CCR2^−/− and WT mice infected with five ME-49 T. gondii cysts by oral route. The CCR2 positive cells were found in the small intestine from WT noninfected mice (A), and the expression was higher on day 8 after infection (B). WT (C) and CCR2^−/− (E) uninfected mice presented immunohistochemistry staining for CCL2 in the small intestine, and the expression was higher in the organ in WT (D) and CCR2^−/− (F) on day 8 after infection. Number of infiltrating CCR2⁺ cells (G) in the small intestine from WT mice and CCL2⁺ cells (H) in the small intestine from WT and CCR2^−/− mice. The positive cells were counted by immunohistochemistry in 40 microscopic fields. Data are representative of at least two independent experiments with three mice per group and two noncontiguous sections from each mouse. Original magnification, ×40. *, significantly different (P < 0.05).

Figure 2

Mortality rates and tissue parasitism of CCR2^−/− and WT mice orally infected with five ME-49 T. gondii cysts. The mortality rate for eight mice from each group was determined (A). CCR2^−/− mice were significantly more susceptible to toxoplasmosis than were WT mice (χ² = 12.47; P = 0.0004; df = 1). The tissue parasitism in the small intestine (B), liver (C), lung (D), and CNS (E, F) were detected by immunohistochemistry staining and scored by counting the number of parasitophorous vacuoles per 40 microscopic fields in the peripheral organs and number of parasitophorous vacuoles and cyst-like structures per section in the CNS. Data are representative of at least two independent experiments of three mice per group that provided similar results (*P < 0.05).

Figure 3

Histological changes in the small intestine, liver, lung, and CNS of CCR2^−/− and WT mice infected perorally with T. gondii. WT (A, C, E) and CCR2^−/− (B, D, F) mice were inoculated with T. gondii and the peripheral organs were collected on day 8 after infection. Observed were inflammatory infiltrates in the lamina propria, epithelium, and submucosa (asterisks) in the small intestine (A, B); inflammatory foci (arrows) in the parenchyma of the liver (C, D); and inflammatory cell infiltration within the alveolar walls (asterisks) in the lung (E, F). The inflammatory reaction was milder in the peripheral organs of CCR2^−/− (B, D, F) compared to WT mice (A, C, E). The CNS from WT (G) and CCR2^−/− mice (H) was analyzed on day 23 after infection. The lesions characterized by glial nodules (small arrows), vascular cuffing (arrowheads) and inflammatory cells in the meninges (large arrows) were similar in both lineages of mice in this period of infection. Slides were stained with hematoxylin and eosin. Original magnification, ×10.

Figure 4

Immunohistochemical staining, number of CD4⁺, CD8⁺, and MAC-1⁺ cells into the small intestine and CNS, and flow cytometry analysis of small intestine of CCR2^−/− and WT mice after oral T. gondii infection with five cysts. The small intestine (A) and CNS (F) were collected on days 8 and 23 after infection, respectively, and CD4⁺, CD8⁺, and MAC-1⁺ cells were detected by immunohistochemistry. Original magnifications: small intestine, ×40; CNS, ×10. The positive MAC-1⁺ (B), CD4⁺ (C,) and CD8⁺ (D) cells were counted in 40 microscopic fields in the small intestine and per sagittal section in the CNS (G). Original magnification ×40. E: Flow cytometry analysis of the intraepithelial lymphocytes in the small intestine on day 8 after infection. The data represent the mean ± SD and are representative of at least two independent experiments with three mice per group that provided similar results. *, significantly different from values obtained from WT mice (P < 0.05).

Figure 5

Levels of IFN-γ, TNF-α, IL-4, IL-10, IL-12p70, and TGF-β in the serum samples (A–D) and intestinal homogenate (E–J) from CCR2^−/− and WT mice on day 8 after T. gondii infection with five cysts by oral route. The cytokine levels were measured by ELISA. The values shown were the mean and SD of five mice per data point. The experiments were repeated twice and provided similar results. *, significantly different from values obtained from WT mice (P < 0.05); nd, not detected.

Figure 6

Immunohistochemical staining for iNOS in the small intestine and in the CNS of WT (A, C) and CCR2^−/− (B, D) mice infected with five ME-49 T. gondii cysts by oral route. The animals were infected and the small intestine were collected on day 8 (A, B) and the CNS on day 23 (C, D) after infection for iNOS immunostaining. Original magnification, ×40.