Chemokine receptor CXCR3 and its ligands CXCL9 and CXCL10 are required for the development of murine cerebral malaria

Abstract

Cerebral malaria is a significant cause of global mortality, causing an estimated two million deaths per year, mainly in children. The pathogenesis of this disease remains incompletely understood. Chemokines have been implicated in the development of cerebral malaria, and the IFN-inducible CXCR3 chemokine ligand IP-10 (CXCL10) was recently found to be the only serum biomarker that predicted cerebral malaria mortality in Ghanaian children. We show that the CXCR3 chemokine ligands IP-10 and Mig (CXCL9) were highly induced in the brains of mice with murine cerebral malaria caused by Plasmodium berghei ANKA. Mice deficient in CXCR3 were markedly protected against cerebral malaria and had far fewer T cells in the brain compared with wild-type mice. In competitive transfer experiments, CXCR3-deficient CD8(+) T cells were 7-fold less efficient at migrating into the infected brains than wild-type CD8(+) T cells. Adoptive transfer of wild-type CD8(+) effector T cells restored susceptibility of CXCR3-deficient mice to cerebral malaria and also restored brain proinflammatory cytokine and chemokine production and recruitment of T cells, independent of CXCR3. Mice deficient in IP-10 or Mig were both partially protected against cerebral malaria mortality when infected with P. berghei ANKA. Brain immunohistochemistry revealed Mig staining of endothelial cells, whereas IP-10 staining was mainly found in neurons. These data demonstrate that CXCR3 on CD8(+) T cells is required for T cell recruitment into the brain and the development of murine cerebral malaria and suggest that the CXCR3 ligands Mig and IP-10 play distinct, nonredundant roles in the pathogenesis of this disease.

Fig. 1.

Chemokine mRNA and protein levels in brain and spleen during murine CM. C57BL/6 mice were infected with P. berghei ANKA (2 × 10⁶ parasitized red blood cells), and their brains (A–C) and spleens (D–F) were harvested at the indicated time points. mRNA levels were analyzed by qPCR and are shown normalized to GAPDH (A and D) and as fold induction compared with uninfected mice (B, C, E, and F). Brain and spleen IP-10 and Mig protein levels were determined by ELISA (C and F, Inset). Results are representative of two independent experiments with two or three mice per group.

Fig. 2.

CXCR3 KO mice are protected from murine CM. Wild-type and CXCR3 KO C57BL/6 mice were infected with P. berghei ANKA. (A) Mortality was checked twice daily. The experiment is representative of four separate infections, each with at least eight mice per group. (B) Parasitemia was monitored every other day by Giemsa-stained thin blood smears. The experiment is representative of two independent infections. (C) Brain histology was assessed by H&E staining. Brains were removed after heart perfusion with 10% formalin of uninfected mice or mice 8 days after infection, as indicated. (Scale bars: 20 μm.)

Fig. 3.

CD3⁺CD8⁺ T cell sequestration is reduced in the brains of CXCR3 KO mice infected with P. berghei ANKA. Flow cytometric analysis of brain-sequestered T lymphocytes and NK cells is shown. Wild-type and CXCR3 KO mice were infected with P. berghei ANKA, and brains were harvested at different days after infection. (Left) Number of brain sequestered CD3⁺ (Top), CD3⁺CD8⁺ (Middle), and NK (Bottom) cells were averaged over four independent experiments and are shown ± SD. (Right) Representative primary flow cytometric dot plots are shown after lymphocyte gating for T cells (Top and Middle) and NK/NKT cells (Bottom). The percentage of lymphocyte gate is shown for the indicated cell populations.

Fig. 4.

CXCR3 is required for efficient CD8 T cell trafficking to the brain. Wild-type (Thy1.1) and CXCR3 KO (Thy1.2) OT-I cells were activated in vitro for 5 days and were adoptively cotransferred (5 × 10⁶ each) into the same Thy1.1×Thy1.2 wild-type mouse by i.v. injection. Mice were infected 5 days before adoptive transfer with P. berghei ANKA. Eight days after infection, when mice developed signs of murine CM, spleens (A) and brains (B) were harvested, and Thy1.1 and Thy1.2 single positive cells were analyzed by flow cytometry as shown. Results are representative of three independent experiments. *, P < 0.05 compared with wild-type OT-I cells. (Right) Representative primary flow cytometric dot plots are shown after lymphocyte gating. The percentage of lymphocyte gate is shown for the indicated cell populations.

Fig. 5.

Wild-type CD8 T cells restore CXCR3 KO murine CM mortality. (A) Wild-type or CXCR3 KO splenocytes (Left) or CD8⁺ T cells (Right) were prepared from P. berghei ANKA-infected mice 5 days after infection and i.v. transferred into CXCR3 KO mice 2 h before infection with P. berghei ANKA. (B) Brains of wild-type or CXCR3 mice with or without CD8⁺ T cell transfer were harvested on day 8 after infection, and brain-sequestered recipient CD8⁺ T (Thy1.2) cells were analyzed by flow cytometry. (C) Representative primary flow cytometric dot plots are shown after lymphocyte gating. The percentage of lymphocyte gate is shown for the indicated cell populations. (D) Chemokine and IFN-γ expression in the brain was analyzed for mice from experiments shown in C.

Fig. 6.

IP-10 and Mig KO are protected from murine CM and are expressed on different cells in the brain. (A and B) Wild-type and Mig KO (A) or IP-10 KO (B) in the C57BL/6 background were infected with P. berghei ANKA, and mortality from murine CM was monitored twice daily. One representative experiment from three independent infections is shown with at least eight mice per group. (C–E) Representative immunohistochemistry (C and D) and immunofluorescence (E) of mouse brains harvested day 0 or day 8 after infection, staining for Mig (C) or IP-10 (D and E). Arrows show endothelial cells staining, arrowheads show neuronal staining. (Scale bars: 20 μm.)