Figure A.

Iron Dextran Administered after Infection can Prevent the Development of ECM. Survival of mice treated with PBS or iron dextran starting 4 or 5 days post-infection. The average of two individual experiments is shown. n = 17 for PBS mice; n = 9 for FeD mice, 4 days post-infection; and n = 8 for FeD mice, 5 days post-infection. PBS = control, FeD = iron dextran. Statistically significant differences, shown by asterisks (* P < 0.05), were determined by log-rank test. Figure B. RLU Measured in the Blood, Brain, Spleen and Liver. Parasitemia was determined by measuring relative luminescence units (RLU) per μL of blood in infected FeD mice and control mice (a). Parasite levels in the brain (b), spleen (c) and liver (d) on day 7 post-infection were determined by measuring RLU. For parasitemia: n = 10 for the control and FeD mice. For tissue parasite burden: n = 6 for all groups, except for the control, unsymptomatic mice (n = 4) and the control, symptomatic mice (n = 4). Shown on the graphs are the average ± S.E.M. FeD = iron dextran, PBS = control. Statistically significant differences, shown by asterisks (** P < 0.01 and *** P < 0.001), were determined by unpaired Student’s t-test. Figure C. Systemic Inflammation in FeD Mice is Increased Only Late during the Infection. Concentration of IFNγ (a), TNFα (b), IL-10 (c), IL-1β (d) and IL-6 (e) in the serum. n = 2 for all groups. FeD = iron dextran, PBS = control. Table A. Immune Response-related Gene Expression in the Brain is Mostly Reduced or Unchanged in FeD Mice. The mRNA expression of immune related genes was determined in the brain on day 7 post-infection. mRNA levels were normalized to Gusb. 2 samples pooled from 6 mice (3 mice per sample) were used for each group. Shown in the table are the averages UI = uninfected, I = infected, FeD = iron dextran, PBS = control. Table B. Immune Response-related Gene Expression in the Spleen is Mostly Reduced or Unchanged in FeD Mice. The mRNA expression of immune related genes was determined in the spleen on day 7 post-infection. mRNA levels were normalized to Gusb. 2 samples pooled from 6 mice (3 mice per sample) were used for each group. Shown in the table are the averages. UI = uninfected, I = infected, FeD = iron dextran, PBS = control. Table C. Immune Response-related Gene Expression in the Liver is Mostly Reduced or Unchanged in FeD Mice. The mRNA expression of immune related genes was determined in the liver on day 7 post-infection. mRNA levels were normalized to Hsp90. 2 samples pooled from 6 mice (3 mice per sample) were used for each group. Shown in the table are the averages. UI = uninfected, I = infected, FeD = iron dextran, PBS = control. Figure D. Iron Dextran Causes a Minor Delay in the Proliferation of Splenic CD4+ T Cells. Representative flow cytometric histograms of CSFE after gating on CD4+ or CD8+ T cells on day 1 (a) and day 4 (b) after ex vivo stimulation. Splenic cells were isolated on day 7 post-infection. n = 6 for control mice and n = 7 for FeD mice. FeD = iron dextran, PBS = control. Figure E. Percentage of Activated CD4+ and CD8+ T Cells in the Spleen is Slightly Decreased in the FeD Mice. Representative flow cytometric dot plots and the percentage of CD62LloCD25+ cells (a) and representative flow cytometric dot plots and the percentage of CD62LloCD44+ cells (b) after gating on CD4+ or CD8+ 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. Shown on the graphs are the average ± S.E.M n = 20 for all groups. The average of three individual experiments is shown. FeD = iron dextran, PBS = control. Statistically significant differences, shown by asterisks (*** P < 0.001), were determined by unpaired Student’s t-test. Figure F. Iron Supplementation Results in a Slight Increase in the Priming Capacity of Splenic cDCs. The percentage of CD11b+CD11c+ cells, and the MFI of CXCL10, CD40 and MHCII after gating on CD11b+CD11c+ cells on day 3 (a) and day 7 post-infection (b). On day 3 post-infection, n = 6 for control mice and n = 5 for FeD mice. On day 7 post-infection, n = 6 for control mice and n = 6 for FeD mice. Shown on the graphs are the average ± S.E.M FeD = iron dextran, PBS = control. Statistically significant differences, shown by asterisks (* P < 0.05 and ** P < 0.01), were determined by unpaired Student’s t-test. Figure G. A Greater Percentage of Splenic CD4+ T Cells in FeD Mice Show an Activated Phenotype and Express CXCR3 on Day 3. Representative flow cytometric dot plots of CD62LloCD25+ and CD62LloCD44+ cells (a) and the percentage of CD62LloCD25+ and CD62LloCD44+ cells (b) after gating on CD4+ T cells. Representative flow cytometric dot plots of CXCR3+ CD4+ T cells (c), and the percentage of CXCR3+ cells and the MFI of CXCR3 (d) after gating on CD4+ T cells. All experiments were performed on day 3 post-infection. The numbers shown on the dot plots indicate the mean percentage of cells inside the gate ± S.E.M. Shown on the graphs are the average ± S.E.M n = 6 for control mice and n = 5 for FeD mice. FeD = iron dextran, PBS = control. Statistically significant differences, shown by asterisks (** P < 0.01 and *** P < 0.001), were determined by unpaired Student’s t-test. Figure H. Attenuated Expression of IFNγR2 and T-bet does not Result in Decreased Expression of CXCR3 on Splenic CD8+ T Cells. Representative flow cytometric dot plots for IFNγR2+ CD8+ T cells (a) and the percentage of IFNγR2+ cells (b) after gating on CD8+ T cells on day 3 and day 7 post-infection. Representative flow cytometric dot plots for T-bet+ CD8+ T cells (c) and the percentage of T-bet+ cells (d) after gating on CD8+ T cells 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, n = 6 for control mice and n = 5 for FeD mice. On day 7, n = 6 for control mice and n = 6 for FeD mice. FeD = iron dextran, PBS = control. Statistically significant differences, shown by asterisks (** P < 0.01), were determined by unpaired Student’s t-test. Figure I. Iron Supplementation Decreases STAT1 Phosphorylation in Splenic CD4+ T cells in FeD Mice. Representative western blot of pSTAT1 and STAT1. n = 2 for all groups. FeD = iron dextran, PBS = control.
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