Experimental cerebral malaria develops independently of caspase recruitment domain-containing protein 9 signaling - PubMed (original) (raw)

Experimental cerebral malaria develops independently of caspase recruitment domain-containing protein 9 signaling

Julius Clemence R Hafalla et al. Infect Immun. 2012 Mar.

Abstract

The outcome of infection depends on multiple layers of immune regulation, with innate immunity playing a decisive role in shaping protection or pathogenic sequelae of acquired immunity. The contribution of pattern recognition receptors and adaptor molecules in immunity to malaria remains poorly understood. Here, we interrogate the role of the caspase recruitment domain-containing protein 9 (CARD9) signaling pathway in the development of experimental cerebral malaria (ECM) using the murine Plasmodium berghei ANKA infection model. CARD9 expression was upregulated in the brains of infected wild-type (WT) mice, suggesting a potential role for this pathway in ECM pathogenesis. However, P. berghei ANKA-infected Card9(-/-) mice succumbed to neurological signs and presented with disrupted blood-brain barriers similar to WT mice. Furthermore, consistent with the immunological features associated with ECM in WT mice, Card9(-/-) mice revealed (i) elevated levels of proinflammatory responses, (ii) high frequencies of activated T cells, and (iii) CD8(+) T cell arrest in the cerebral microvasculature. We conclude that ECM develops independently of the CARD9 signaling pathway.

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Figures

Fig 1

Fig 1

CARD9 expression during a Plasmodium berghei (strain ANKA) infection. RT-PCR of CARD9 expression was performed using total RNA isolated from brains, spleens, and livers of uninfected mice and on days 5 and 7 from P. berghei ANKA-infected mice and using primers specific for the CARD9 coding sequence. Results were normalized to amplification products using GAPDH primers. (A) Relative expression levels from two to three experiments. *, P < 0.05 following Kruskal-Wallis test and Dunn's multiple comparison test. (B) Mean fold change shown as cumulative data from two experiments.

Fig 2

Fig 2

_Card9_−/− mice develop ECM similar to WT mice. (A) ECM development after sporozoite-induced infection by intravenous injection of 104P. berghei ANKA sporozoites. Survival curves are based on cumulative data from two independent experiments, with five mice per group. (B) Time to blood stage infection (prepatency) after intravenous injection of 104P. berghei ANKA sporozoites. (C) Parasitemia levels (for panel A) determined by Giemsa-stained blood smears. (D) ECM development after transfusion-mediated infection by intravenous injection of 104P. berghei ANKA-infected red blood cells (iRBCs). Survival curves are based on cumulative data from three independent experiments, with four to six mice per group. (E) Parasitemia levels (for panel D) determined by Giemsa-stained blood smears.

Fig 3

Fig 3

_Card9_−/− mice display systemic cytokine responses similar to those of WT mice. Cytokine concentrations in plasma samples from infected mice. Cytokines were determined using the BD cytometric bead assay inflammation kit. Data shown are for IL-17 (A), IL-2 (B), IL-4 (C), IL-6 (D), IL-10 (E), IFN-γ (F), MCP-1 (G), and TNF-α (H). Day 0 represent data from uninfected mice. Figures show representative data from two to three experiments, four mice per group.

Fig 4

Fig 4

Systemic cellular responses and leukocyte sequestration to the brain in WT and _Card9_−/− mice infected with P. berghei ANKA. (A to D) High proportion of antigen-experienced CD8+ and CD4+ T cells in spleens after infection. Splenic leukocytes were isolated from uninfected mice and infected WT and _Card9_−/− mice on day 7 and surface stained for CD11a (A, B) or CD62L (C, D) and CD8+ or CD4+. (A, C) Shown are representative fluorescence-activated cell sorting (FACS) plots displaying the proportions of CD11ahi (A) or CD62Llo (C) CD8+ (left) and CD4+(right) T cells before and 7 days after infection with 104P. berghei ANKA-infected iRBCs. (B, D) Graphs show the percentage (mean ± standard error of the mean [SEM]) of CD11ahi or CD62Llo cells of total splenic CD8+ (left) and CD4+ (right) T cells before and 7 days after infection. Data are from one representative out of two experiments, with at least four mice per group. (E, F) High proportion of IFN-γ-producing cells in spleens after infection. Following splenic leukocyte isolation, cells were cultured with anti-CD3 and anti-CD28 antibodies for 5 h in the presence of brefeldin A. Cells were subsequently stained for surface CD8+ or CD4+ and intracellular IFN-γ. (E) Shown are representative FACS plots displaying the proportions of IFN-γ+ CD8+ (left) and CD4+(right) T cells before and 7 days after infection with 104P. berghei ANKA-infected iRBCs. (F) Graphs show the percentage (mean ± SEM) of IFN-γ+ cells of total splenic CD8+ (left) and CD4+ (right) T cells before and 7 days after infection. Data are from one representative out of two experiments, with at least four mice per group. (G, H) High proportion of infiltrating leukocytes in brains after infection. Brain-sequestered lymphocytes were isolated from uninfected and day 7 infected WT and _Card9_−/− mice. Cells were surface stained for CD8 and CD4. (G) Representative FACS plots show the proportions of CD8+ leukocytes before and 7 days after infection with 104P. berghei ANKA-infected iRBCs. (H) Graphs show the percentage (mean ± SEM) of CD8+ leukocytes before and 7 days after infection. Data are from one representative out of three experiments, with at least four mice per group.

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