Iron Prevents the Development of Experimental Cerebral Malaria by Attenuating CXCR3-Mediated T Cell Chemotaxis

<div><p>Cerebral malaria is a severe neurological complication of <i>Plasmodium falciparum</i> infection. Previous studies have suggested that iron overload can suppress the generation of a cytotoxic immune response; however, the effect of iron on experimental cerebral malaria (ECM) is yet unknown. Here we determined that the incidence of ECM was markedly reduced in mice treated with iron dextran. Protection was concomitant with a significant decrease in the sequestration of CD4⁺ and CD8⁺ T cells within the brain. CD4⁺ T cells demonstrated markedly decreased CXCR3 expression and had reduced IFNγ-responsiveness, as indicated by mitigated expression of IFNγR2 and T-bet. Additional analysis of the splenic cell populations indicated that parenteral iron supplementation was also associated with a decrease in NK cells and increase in regulatory T cells. Altogether, these results suggest that iron is able to inhibit ECM pathology by attenuating the capacity of T cells to migrate to the brain.</p></div

The Expression of CXCR3 on Splenic CD4⁺ T Cells is Decreased by Parenteral Iron Supplementation.

<p>Representative flow cytometric dot plots of CXCR3⁺ CD4⁺ and CD8⁺ T cells (<b>a</b>) and the percentage of CXCR3⁺ cells after gating on CD4⁺ or CD8⁺ T cells (<b>b</b>). Representative flow cytometric histograms of CXCR3 (<b>c</b>) and the MFI of CXCR3 (<b>d</b>) after gating on CD4⁺CD44^hi or CD8⁺CD44^hi T cells. All experiments were performed on day 7 post-infection. The numbers shown on the dot plots indicate the mean percentage of cells inside the gate ± S.E.M. <i>n</i> = 13 for all groups. The average of two individual experiments is shown. FeD = iron dextran, PBS = control. Statistically significant differences, shown by asterisks (** <i>P</i> < 0.01 and *** <i>P</i> < 0.001), were determined by unpaired Student’s t-test.</p

Systemic Inflammation is Augmented in FeD Mice.

<p>Concentration of IFNγ (<b>a</b>), TNFα (<b>b</b>), IL-10 (<b>c</b>), IL-1β (<b>d</b>) and IL-6 (<b>e</b>) in the serum on day 7 post-infection. <i>n</i> = 5 for the control, uninfected group mice and <i>n</i> = 6 for all other groups. Levels of TNFα are below the limit of detection in the uninfected groups. FeD = iron dextran, PBS = control. Statistically significant differences, shown by asterisks (* <i>P</i> < 0.05 and ** <i>P</i> < 0.01), were determined by unpaired Student’s t-test.</p

Sequestration of CD4⁺ and CD8⁺ T Cells in the Brain is Reduced in FeD Mice.

<p>The total number of cells recovered after isolation (<b>a</b>), the total number (<b>b</b>) and percentage (<b>c</b>) of CD8⁺ T cells after gating on infiltrating leukocytes (CD45⁺CD11b^lo-hi), representative flow cytometric dot plots of CD4⁺ and CD8⁺ T cells after gating on infiltrating leukocytes (<b>d</b>), and the total number (<b>e</b>) and percentage (<b>f</b>) of CD4⁺ T cells after gating on infiltrating leukocytes, on day 7 post-infection. The numbers shown on the dot plots indicate the mean percentage of cells inside the gate ± S.E.M. <i>n</i> = 5 mice were used for each group. UI = uninfected, I = infected, FeD = iron dextran, PBS = control. Statistically significant differences, shown by asterisks (* <i>P</i> < 0.05 and *** <i>P</i> < 0.001), were determined by unpaired Student’s t-test.</p

Iron Dextran Prevents the Development of ECM.

<p>Survival (<b>a</b>) and mean parasitemia (<b>b</b>) of infected mice treated with iron dextran, dextran M_w 5kDa and dextran M_w 70kDa. PBS-treated mice were used as a control. BBB disruption was assessed using Evans blue (EB). EB quantification (<b>c</b>) is shown as mean μg of EB per g of brain tissue normalized to mean μg of EB per g of heart tissue. Representative picture of EB-stained brains (<b>d</b>). For survival: <i>n</i> = 29 for control mice, <i>n</i> = 31 for FeD mice and <i>n</i> = 5 for Dex5 and Dex70 mice. The average of four individual experiments is shown for the control and FeD mice. For parasitemia: <i>n</i> = 20 for control and FeD mice and <i>n</i> = 5 for Dex5 and Dex70. The average of three individual experiments is shown for the control and FeD mice. For EB: <i>n</i> = 5 for control, uninfected and infected mice, and <i>n</i> = 6 for iron dextran-treated, uninfected and infected mice. PBS = control, Dex5 = dextran M_w 5kDa, Dex70 = dextran M_w 70kDa, FeD = iron dextran, UI = uninfected, I = infected. Statistically significant differences, shown by asterisks (* <i>P</i> < 0.05, ** <i>P</i> < 0.01, and *** <i>P</i> < 0.001), were determined by log-rank test (survival) and unpaired Student’s t-test (BBB disruption).</p

The Expression of Genes Involved in T Cell Chemotaxis are Attenuated by Parenteral Iron Supplementation.

<p>The expression of genes involved in T cell chemotaxis is shown for the brain (<b>a</b>) and the spleen (<b>b</b>) on day 7 post-infection. mRNA levels were normalized to <i>Gusb</i>. 2 samples pooled from 6 mice (3 mice per sample) were used for each group. UI = uninfected, I = infected, FeD = iron dextran, PBS = control.</p

FeD Mice have Modulated Frequencies of Splenic NK Cells and Tregs Early During the Infection.

<p>Representative flow cytometric dot plots of NK cells (<b>a</b>) and the percentage of NK cells (<b>b</b>) on day 3 and day 7 post-infection. Representative flow cytometric dot plots of Tregs (<b>c</b>) and the percentage of Tregs after gating on CD4⁺ T cells (<b>d</b>) on day 3 and day 7 post-infection. The numbers shown on the dot plots indicate the mean percentage of cells inside the gate ± S.E.M. On day 3 post-infection, <i>n</i> = 6 for control mice and <i>n</i> = 5 for FeD mice, except for Tregs, where <i>n</i> = 5 for the control mice. On day 7 post-infection, <i>n</i> = 6 for control mice and <i>n</i> = 6 for FeD mice. FeD = iron dextran, PBS = control. Statistically significant differences, shown by asterisks (* <i>P</i> < 0.05 ** <i>P</i> < 0.01), were determined by unpaired Student’s t-test.</p

Iron Dextran Mitigates the Upregulation of IFNγR2 and T-bet on CD4⁺ T Cells in the Spleen.

<p>Representative flow cytometric dot plots for IFNγR2⁺ CD4⁺ T cells (<b>a</b>) and the percentage of IFNγR2⁺ cells (<b>b</b>) after gating on CD4⁺ T cells on day 3 and day 7 post-infection. Representative flow cytometric dot plots for T-bet⁺ CD4⁺ T cells (<b>c</b>) and the percentage of T-bet⁺ cells (<b>d</b>) after gating on CD4⁺ T cells on day 3 and 7 post-infection. The numbers shown on the dot plots indicate the mean percentage of cells inside the gate ± S.E.M. On day 3 post-infection, <i>n</i> = 6 for control mice and <i>n</i> = 5 for FeD mice. On day 7 post-infection, <i>n</i> = 6 for control mice and <i>n</i> = 6 for FeD mice. FeD = iron dextran, PBS = control. Statistically significant differences, shown by asterisks (*** <i>P</i> < 0.001), were determined by unpaired Student’s t-test.</p

Tissue parasite sequestration is inhibited by parenteral iron supplementation.

<p>Parasite levels in the brain (<b>a</b>), spleen (<b>b</b>) and liver (<b>c</b>) on day 7 post-infection. Luciferase activity is shown as the total RLU per organ normalized to the total RLU in the control mice before symptoms. <i>n</i> = 8 for the control mice before symptoms, <i>n</i> = 8 for the control, symptomatic mice and <i>n</i> = 12 for the FeD mice. FeD = iron dextran, PBS = control. Statistically significant differences, shown by asterisks (** <i>P</i> < 0.01 and *** <i>P</i> < 0.001), were determined by unpaired Student’s t-test.</p