Microglia exhibit a dynamic response, modulating inducible nitric oxide synthase expression and the production of pro-inflammatory cytokines during experimental cerebral malaria

Abstract

Microglia play a fundamental role in maintaining central nervous system homeostasis by monitoring brain tissue for physical, structural, and biochemical alterations. Its involvement in the pathogenesis of various neurological disorders is well documented. However, the role of microglia in cerebral malaria, a disease associated with high mortality and long-term neurological sequelae, remains poorly understood. In this study, we utilized the classical model of experimental cerebral malaria (Plasmodium berghei ANKA-infected C57BL/6 mice) to investigate the dynamics and response of resident brain cell populations, particularly microglia, and the influx of other leukocytes during the development of experimental cerebral malaria. By employing flow cytometry and established markers for different leukocyte populations, we were able to discern and document an increase in the number of Ly6C⁺ T cells (CD45^hiCD11b^-CD3⁺ cells), inflammatory monocytes (CD45^hiCD11b⁺TMEM119^-CD206^- cells), resident macrophages (CD45^hiCD11b⁺TMEM119^-CD206⁺ cells), and microglia (CD45^lowCD11b⁺ TMEM119⁺CD206^- cells) following infection. Moreover, our ex vivo analysis demonstrated an increment in the overall number of inflammatory monocytes, resident macrophages and microglia expressing inducible nitric oxide synthase (iNOS), in addition to those producing interleukin-1β or TNF. These findings highlight the pronounced reactivity of microglia in experimental cerebral malaria and provide valuable information on cell dynamics and immune responses in the brain.

Keywords: IL-1β; TNF; experimental cerebral malaria; iNOS; macrophages; malaria; microglia; nitric oxide.

Figure 1

Progression of cerebral malaria in C57BL/6 mice infected with Plasmodium berghei ANKA. C57BL/6 mice were inoculated with 1x10⁶ iRBCs. Parasitemia (percentage of GFP⁺ cells) (A), body temperature (B), extravasation of Evans Blue dye to the brain parenchyma (C), and survival rate (D) were assessed in naive and infected C57BL/6 mice at various time points. Significant differences between groups were analyzed using the following statistical tests: unpaired t-test (A), one-way ANOVA (B, C), and the Long-Rank test (D). The results were statistically significant at p < 0.0006 (***) and p < 0.0001 (****). Graphs A and B: 10 mice per group. Graph C: 4–5 mice per group. Graph D: 10–22 mice per group. The data were pooled from two independent experiments (A, B, D) or are representative of two independent experiments (C).

Figure 2

Plasmodium berghei ANKA infection alters the dynamics of cell populations in the mouse brain during ECM. C57BL/6 mice were inoculated with 1x10⁶ iRBCs. Representative dot plot of CD45 and CD11b cells, gated on live cells, in naive animal and at different stages of infection (A). Absolute numbers of CD45^hiCD11b^-, CD45^hiCD11b⁺ and CD45^lowCD11b⁺ cells (B). Representative histograms illustrating the expression of CD3 on CD45^hiCD11b^- cells (C) and Ly6C on CD45^hiCD11b⁺ cells (D) on day 6 post-infection. Median fluorescence intensity (MFI) of F4/80 on the CD45^hiCD11b⁺ and CD45^lowCD11b⁺ cells (E). Significant differences between the groups were analyzed by one-way ANOVA with results indicated for p = 0.0002 (***) and p < 0.0001 (****). The number of mice per group ranged from 9 to 11. The data were obtained from a pool of two independent experiments.

Figure 3

Expression of TMEM119 and CD206 by monocytes/macrophages and microglia. C57BL/6 mice were inoculated with 1x10⁶ iRBCs. Percentage of TMEM119⁺ cells within CD45^hiCD11b⁺F4/80⁺ (A) and CD45^lowCD11b⁺F4/80⁺ (B) populations. Representative histogram and bar plots showing the median fluorescence intensity (MFI) of TMEM119 on the CD45^hiCD11b⁺ and CD45^lowCD11b⁺ cells (C). Percentage of CD206⁺ cells within CD45^hiCD11b⁺F4/80⁺ (D) and CD45^lowCD11b⁺F4/80⁺ (E) populations. Representative histogram and bar plots showing the MFI of CD206 on the CD45^hiCD11b⁺ and CD45^lowCD11b⁺ cells (F). The dashed line in the histograms represents the FMO (fluorescence minus one) control. Significant differences between groups were analyzed by one-way ANOVA, with the results indicated for p = 0.03 (*), p = 0.007 (**), p = 0.0006 (***) and p < 0.0001 (****). The number of mice per group ranged from 9 to 11. The data were obtained from two independent experiments.

Figure 4

Total number and percentage of inflammatory monocytes, resident macrophages and microglia in the mouse brain following infection with Plasmodium berghei ANKA. C57BL/6 mice were inoculated with 1x10⁶ iRBCs. The absolute number (A-C) and percentage (D-F) of inflammatory monocytes (CD45^hiCD11b⁺TMEM119^-CD206^-), resident macrophages (CD45^hiCD11b⁺TMEM119^-CD206⁺), and microglia (CD45^lowCD11b⁺TMEM119⁺CD206^-). Significant differences between groups were analyzed by one-way ANOVA, with results indicated for p < 0.04 (*), p < 0.001 (**), p < 0.0005 (***), and p < 0.0001 (****). The number of mice per group ranged from 8 to 11. The data were obtained from a pool of two independent experiments.

Figure 5

Total number of inflammatory monocytes, resident macrophages and microglia expressing iNOS, and producing IL-1β or TNF during ECM. C57BL/6 mice were inoculated with 1x10⁶ iRBCs. The absolute number of iNOS⁺ (A-C), IL-1β⁺ (D-F), and TNF⁺ (G-I) cells among inflammatory monocytes, resident macrophages and microglia. Significant differences between groups were analyzed by one-way ANOVA, with results indicated for p < 0.03 (*), p < 0.002 (**), and p < 0.0001 (****). The number of mice per group ranged from 4 to 6. Representative data from two independent experiments.