Kinetics of monocyte subpopulations during experimental cerebral malaria and its resolution in a model of late chloroquine treatment

Abstract

Cerebral malaria (CM) is one of the most severe forms of malaria and is a neuropathology that can lead to death. Monocytes have been shown to accumulate in the brain microvasculature at the onset of neurological symptoms during CM. Monocytes have a remarkable ability to adapt their function to their microenvironment from pro-inflammatory to resolving activities. This study aimed to describe the behavior of monocyte subpopulations during infection and its resolution. C57BL/6 mice were infected with the Plasmodium berghei ANKA strain and treated or not with chloroquine (CQ) on the first day of the onset of neurological symptoms (day 6) for 4 days and followed until day 12 to mimic neuroinflammation and its resolution during experimental CM. Ly6C monocyte subpopulations were identified by flow cytometry of cells from the spleen, peripheral blood, and brain and then quantified and characterized at different time points. In the brain, the Ly6C^int and Ly6C^low monocytes were associated with neuroinflammation, while Ly6C^hi and Ly6C^int were mobilized from the peripheral blood to the brain for resolution. During neuroinflammation, CD36 and CD163 were both involved via splenic monocytes, whereas our results suggest that the low CD36 expression in the brain during the neuroinflammation phase was due to degradation. The resolution phase was characterized by increased expressions of CD36 and CD163 in blood Ly6C^low monocytes, a higher expression of CD36 in the microglia, and restored high expression levels of CD163 in Ly6C^hi monocytes localized in the brain. Thus, our results suggest that increasing the expressions of CD36 and CD163 specifically in the brain during the neuroinflammatory phase contributes to its resolution.

Keywords: blood; brain; cerebral malaria (CM); chloroquine; mice; monocyte; spleen.

Figure 1

Late treatment with chloroquine (CQ) prolongs survival and limits parasitemia and neurological damage. (A) Plasmodium berghei ANKA-infected C57BL/6 mice treated intraperitoneally from day 6 (D6) to D9 post-infection with 25 mg kg⁻¹ day⁻¹ CQ and followed by clinical and parasitological examination, organ sampling, and flow cytometry analysis. (B) Survival curve in treated and untreated mice. (C) Course of parasitemia in treated and untreated mice. (D) Rapid murine cerebral behavior scale (RMCBS) scores during the course of infection in treated and untreated mice. (E) Weight of treated and untreated mice. Treated and untreated mice were compared for all parameters on D7, D8, and D9 using the Mann–Whitney U test. *p < 0.05, ***p < 0.0005.

Figure 2

Monocyte subpopulations evolve differently in the spleen, peripheral blood, and brain during infection and resolution in response to chloroquine (CQ) treatment. The spleen, blood, and the brain were sampled at different time points of infection with the Plasmodium berghei ANKA strain in CQ-treated and untreated mice. Monocyte subpopulations, as well as the microglia, were identified and quantified from live cells by flow cytometry using FlowJo. (A, B) Percentages of Ly6C^hi (A) and Ly6C^low (B) monocytes in the spleen in treated and untreated mice. (C–H) Three percentages of the monocyte subpopulations in the peripheral blood (C–E) and in the brain (F–H) during kinetics in treated and untreated mice. (I) Percentage of the microglia during kinetics. The results are presented as curves and bars for better visualization of the kinetics and the significant differences. For each subpopulation, one-way ANOVA first confirmed that the means were not equal before two-by-two comparisons using an unpaired two-tailed Students t-test. Significant differences between consecutive time points are shown with an asterisk, while differences from day 0 (D0) are shown with a section symbol. One symbol denotes p < 0.05, two symbols p < 0.005, and three symbols p < 0.0005.

Figure 3

CD36 and CD163 receptors are differentially expressed in terms of expression level and evolution during the kinetics of infection and its resolution by the monocyte subpopulations from one compartment to another. (A, B) Mean fluorescence intensity (MFI) of CD36 (A) and CD163 (B) by the different Ly6C monocyte subpopulations and the microglia characterized in the spleen, blood, and brain were analyzed using FlowJo during Plasmodium berghei ANKA infection in chloroquine (CQ)-treated and untreated mice. To obtain comparable data during kinetics between the different subpopulations of each compartment studied (i.e., the spleen, blood, brain), the heatmap presents the data as a ratio to the highest MFI value obtained during the kinetics ×100 for each compartment.

Figure 4

Schematic illustration of the main results obtained for the evolution of the percentages of the monocyte subpopulations in the three compartments (i.e., the spleen, blood and brain) and their CD36 and CD163 receptor expression phenotypes during the two major phases of the infection kinetics (neuroinflammation and its resolution) in a model of experimental cerebral malaria.