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. 2007 Aug;171(2):548-59.
doi: 10.2353/ajpath.2007.061033. Epub 2007 Jun 28.

A role for natural regulatory T cells in the pathogenesis of experimental cerebral malaria

Fiona H Amante  1 , Amanda C Stanley, Louise M Randall, Yonghong Zhou, Ashraful Haque, Karli McSweeney, Andrew P Waters, Chris J Janse, Michael F Good, Geoff R Hill, Christian R Engwerda
Affiliations

A role for natural regulatory T cells in the pathogenesis of experimental cerebral malaria

Fiona H Amante et al. Am J Pathol. 2007 Aug.

Abstract

Cerebral malaria (CM) is a serious complication of Plasmodium falciparum infection that is responsible for a significant number of deaths in children and nonimmune adults. A failure to control blood parasitemia and subsequent sequestration of parasites to brain microvasculature are thought to be key events in many CM cases. Here, we show for the first time, to our knowledge, that CD4(+)CD25(+)Foxp3(+) natural regulatory T (Treg) cells contribute to pathogenesis by modulating immune responses in P. berghei ANKA (PbA)-infected mice. Depletion of Treg cells with anti-CD25 monoclonal antibody protected mice from experimental CM. The accumulation of parasites in the vasculature and brain was reduced in these animals, resulting in significantly lower parasite burdens compared with control animals. Mice lacking Treg cells had increased numbers of activated CD4(+) and CD8(+) T cells in the spleen and lymph nodes, but CD8(+) T-cell recruitment to the brain was selectively reduced in these mice. Importantly, a non-Treg-cell source of interleukin-10 was critical in preventing experimental CM. Finally, we show that therapeutic administration of anti-CD25 monoclonal antibody, even when blood parasitemia is established, can prevent disease, confirming a critical and paradoxical role for Treg cells in experimental CM pathogenesis.

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Figures

Figure 1
Figure 1
ECM is prevented in C57BL/6 mice lacking Treg cells. Mice were injected i.p. with 0.5 mg of anti-CD25 mAb or 0.5 mg of control mAb 1 day before infection with PbA. FACS profiles of CD25 and Foxp3 expression on splenic leukocytes gated on CD4+ T cells from control mice or Treg-depleted mice, as indicated, are shown 1 day after antibody administration (A). Analysis of splenic CD4+ T cell, CD8+ T cell, NK cell, NK T cell, and B cell numbers from control mice (closed bars) or Treg-depleted mice (open bars) at the same time are also shown (B). Data are single representative samples (A) or mean ± SEM (B) of individual samples from five mice per treatment group. Results are from one representative experiment of three performed. Survival (C), parasitemia (mean ± SEM; n = 5 mice per group) (D), and clinical disease (mean score ± SEM; n = 5 mice per group) (E) were monitored in mice that received anti-CD25 mAb (open triangles) or control mAb (closed circles). Results in C represent data pooled from four separate experiments yielding similar results (n = 20 mice per treatment group), and the open box indicates the time when mice displayed ECM symptoms. Results in D and E are one representative experiment of four performed. Statistical differences of *P < 0.05 are indicated.
Figure 2
Figure 2
Anti-CD25 mAb treatment 14 days before PbA infection prevents ECM in C57BL/6 mice. Mice were injected i.p. with 0.5 mg of anti-CD25 mAb (open bars or open triangles) or 0.5 mg of control mAb (closed bars or closed circles) 14 days before infection with PbA. FACS analysis of splenic CD4+, CD8+, NK, NK T, and B cell numbers (A) as well as CD4+CD25+Foxp3+ T cells (B) on the day of infection are shown. Data represent the mean ± SEM of individual samples from three mice per treatment group. Survival (C), parasitemia (mean ± SEM) (D), and clinical disease (mean score ± SEM) (E) were monitored. The open box in C–E indicates the time when mice displayed ECM symptoms. Data are from one representative experiment of four performed. Statistical differences of *P < 0.05 are indicated.
Figure 3
Figure 3
Reduced PbA accumulation in the vasculature and brain of mice lacking Treg cells. Mice were injected i.p. with 0.5 mg of anti-CD25 mAb or 0.5 mg of control mAb 14 days before infection with transgenic PbA expressing luciferase as indicated. Mice were injected with luciferin on day 5 p.i., and whole-body images recorded for 1 minute, 5 minutes after luciferin injection (A). Parasite burden determined by microscopic examination of blood smears (B; n = 5 mice per group) and bioluminescence (C; n = 5 mice per group) are shown. After whole-body imaging, mice were sacrificed and perfused, and brains were removed for recording images for 5 minutes, 1 hour after luciferin injection (D). Bioluminescence of brain tissue was also recorded (E; n = 3 mice per group). Data are from one representative experiment of two performed. Statistical differences of *P < 0.05, **P < 0.01, and ***P < 0.001 are indicated.
Figure 4
Figure 4
Changes in leukocyte recruitment to the brain of mice lacking Treg cells. A: VCAM-1 and ICAM-1 staining was performed on brain sections at ECM in control mice as indicated (antibodies administered 14 day before PbA infection). Arrows indicate cerebral vessels expressing adhesion molecules. B: The number of VCAM-1- and ICAM-1-positive vessels in the brains of mice injected i.p. with 0.5 mg of anti-CD25 mAb (closed bars) or 0.5 mg of control mAb (hashed bars) 14 days before PbA infection, as indicated, were determined when the latter group developed ECM and compared with expression in the brains of naïve mice (open bars). C: The total number of leukocytes in brain tissue was enumerated microscopically at the time of ECM in control-treated mice. FACS analysis of brain CD8+ T cell (D) and macrophage/monocytes (E) at the same time are shown. Numbers above FACS gates indicate the percentage of gated cells. Data represent the mean ± SEM of individual samples in each group (n = 3–5 mice/group) from one representative experiment of three performed. Statistical differences of *P < 0.01 and **P < 0.001 are indicated.
Figure 5
Figure 5
Increased T-cell activation in the absence of Treg cells during PbA infection. Mice were injected i.p. with 0.5 mg of anti-CD25 mAb (closed bars) or 0.5 mg of control mAb (hashed bars) 14 days before infection with PbA. The number (mean ± SEM) of CD4+ T (A) and CD8+ T (B) cells expressing CD25 and CD69 as indicated in the spleen, lymph nodes (LN), blood and brain were measured by FACS on the day of PbA infection (day 0) and days 5 and 7 p.i. These data are from three individual mice per group and are one representative experiment of two performed. Statistical differences of *P < 0.05, **P < 0.01, and ***P < 0.001 are indicated.
Figure 6
Figure 6
CD4+ T-cell activation is enhanced in the absence of Treg cells resulting in improved IFNγ-dependent control of PbA. Mice were injected i.p. with 0.5 mg of anti-CD25 mAb (closed bars) or 0.5 mg of control mAb (hashed bars) 14 days before infection with PbA. The number (mean ± SEM) (A) of IFNγ-producing splenic CD4+ and CD8+ T cells were determined by ELISPOT 5 days after PbA infection and compared with naïve mice (open bars). These data are from three individual mice per group and are one representative experiment of two performed. Serum cytokine levels were measured 5 days p.i. (mean ± SEM of individual serum samples from five mice per treatment group) in naive mice, control mice and Treg cell-depleted mice, as indicated (B). Data are from one representative experiment of two performed. C: IFNγ and IL-10 mRNA levels were determined in spleen tissue taken from naive mice, control mice, and Treg cell-depleted mice at 5 days p.i. as indicated. IFNγ and IL-10 mRNA levels are presented relative to 1000 HPRT mRNA molecules (normalized) (mean ± SEM of individual serum samples from five mice per group). Statistical differences of *P < 0.05, **P < 0.01, and ***P < 0.001 are indicated.
Figure 7
Figure 7
IL-10 is required for protection from ECM in the absence of Treg cells. B6.IL-10−/− mice (A) and C57BL/6 mice (B) were injected i.p. with 0.5 mg of control mAb (closed circle) or 0.5 mg of anti-CD25 mAb (open triangle) 1 day before infection with PbA and survival was monitored. Results represent data pooled from two separate experiments yielding similar results (n = 10 mice per treatment group), and the open box indicates the time when mice displayed ECM symptoms.
Figure 8
Figure 8
Depletion of Treg cells before onset of neurological symptoms prevents ECM in C57BL/6 mice. Mice were injected i.p. with 0.5 mg of anti-CD25 mAb (open triangle) or 0.5 mg of control mAb (closed circle) 4 days after infection with PbA (n = 5 mice per group). A survival curve is shown, and the open box indicates the time when mice exhibited ECM symptoms. Data are from one representative experiment of two performed.
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