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Infection and Immunity, May 2007, p. 2275-2282, Vol. 75, No. 5
0019-9567/07/$08.00+0     doi:10.1128/IAI.01783-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

CD4+ CD25+ Regulatory T Cells Suppress CD4+ T-Cell Function and Inhibit the Development of Plasmodium berghei-Specific TH1 Responses Involved in Cerebral Malaria Pathogenesis{triangledown}

Catherine Q. Nie, Nicholas J. Bernard, Louis Schofield, and Diana S. Hansen*

The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia

Received 8 November 2006/ Returned for modification 1 January 2007/ Accepted 12 February 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The infection of mice with Plasmodium berghei ANKA constitutes the best available mouse model for human Plasmodium falciparum-mediated cerebral malaria, a devastating neurological syndrome that kills nearly 2.5 million people every year. Experimental data suggest that cerebral disease results from the sequestration of parasitized erythrocytes within brain blood vessels, which is exacerbated by host proinflammatory responses mediated by cytokines and effector cells including T lymphocytes. Here, T cell responses to P. berghei ANKA were analyzed in cerebral malaria-resistant and -susceptible mouse strains. CD4+ T-cell proliferation and interleukin-2 (IL-2) production in response to parasite-specific and polyclonal stimuli were strongly inhibited in cerebral malaria-resistant mice. In vitro and in vivo depletion of CD4+ CD25+ regulatory T (Treg) cells significantly reversed the inhibition of CD4+ T-cell proliferation and IL-2 production, indicating that this cell population contributes to the suppression of T-cell function during malaria. Moreover, in vivo depletion of Treg cells prevented the development of parasite-specific TH1 cells involved in the induction of cerebral malaria during a secondary parasitic challenge, demonstrating a regulatory role for this cell population in the control of pathogenic responses leading to fatal disease.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasmodium falciparum-mediated cerebral malaria is responsible for about 2.5 million deaths each year (48). This neurological syndrome is characterized by the occurrence of convulsions, seizures, and coma (47). Plasmodium berghei ANKA murine malaria has many features in common with human disease and is thus the best available model for certain important aspects of clinical malaria (7, 29, 42). Despite extensive research, the precise mechanisms leading to pathogenesis during cerebral malaria are not fully understood. Current views support the idea that cerebral disease results from the combined effect of sequestration of parasitized red blood cells (pRBC) within blood vessels in the brain and a strong host proinflammatory response mediated by cytokines and effector cells (4, 9, 13, 34, 36). In fact, a large body of work indicates that immune responses mediated by activated T cells are involved in disease induction. Both CD8+ as well as CD4+ subsets of T cells have been shown to contribute to cerebral pathogenesis. It has been shown that ß2-microglobulin knockout mice are resistant to P. berghei-mediated cerebral malaria (49), and the depletion of CD8+ T cells with specific antibody protects susceptible mice against disease (14, 49). More recently, CD8+ lymphocytes were found to be sequestered in brains of mice affected by cerebral malaria (4, 34), and it has been suggested that the accumulation of these cytotoxic effector cells within brain blood vessels contributes to disease induction (34).

Like CD8+ T cells, CD4+ lymphocytes were also found to be sequestered in brains of mice affected by cerebral malaria (4). In fact, the in vivo depletion of CD4+ T cells with anti-CD4 antibody (11) or the genetic deletion of major histocompatibility complex class II molecules (49) also results in the protection of susceptible mice from P. berghei-mediated cerebral malaria. The mechanism by which CD4+ T cells mediate cerebral disease induction has not been fully elucidated but is thought to involve the production of TH1 cytokines, such as gamma interferon (IFN-{gamma}), which exacerbate the inflammatory cascade responsible for local and systemic inflammation and severe disease induction. Consistent with this view, C57BL/6 mice, usually prone to developing TH1-dominated responses (43), are susceptible to murine cerebral malaria syndrome, whereas BALB/c mice, with a TH2-biased response, are resistant (21). Furthermore, anti-inflammatory cytokines such as transforming growth factor ß (TGF-ß) and interleukin-10 (IL-10) have been found to regulate type 1 responses during infection (35) and to play a protective role against P. berghei-mediated cerebral malaria (22), respectively. However, whether resistance to P. berghei-mediated cerebral malaria is associated with the development of suppressive mechanisms aimed at directly inhibiting pathogenic T-cell responses is not fully understood. Therefore, in this study, we analyzed the level of T-cell responses in cerebral malaria-susceptible and -resistant mouse strains. We found that during malaria, CD4+ T cells from cerebral disease-resistant mice display a profound inhibition of cell proliferation and IL-2 secretion in response to both parasite-specific and polyclonal stimuli. Interestingly, CD4+ CD25+ regulatory T (Treg) cells appeared to play a role in the inhibition of T-cell function occurring during infection. Moreover, cell depletion experiments revealed that Treg cells also prevent the development of parasite-specific TH1 memory cells involved in the induction of cerebral malaria during a secondary parasitic challenge, demonstrating a regulatory role for this cell population in the control of pathogenic responses leading to fatal disease.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice and infections. Eight- to 10-week-old BALB/c and C57BL/6 mice were used throughout the experiments. Groups of 10 to 15 mice were injected intraperitoneally with 1 x 106 P. berghei ANKA pRBC. In some experiments, mice were injected intravenously with 250 µg of anti-CD25 antibody (clone PC61) 2 days and 6 h before challenge in order to deplete CD4+ CD25+ T cells. CD4+ CD25+ T-cell depletion was confirmed by flow cytometry. In drug cure experiments, mice were treated with chloroquine (10 mg/kg) and pyrimethamine (10 mg/kg) for 5 to 7 days starting at day 5 postinfection (p.i). Parasitemia was assessed from Giemsa-stained smears of tail blood prepared every 2 to 3 days. Mortality was checked daily. Mice were coded and scored "blind" for neurological symptoms (40). The following parameters were used: reaction and self-righting reflexes (score of 0 to 5), grip strength (score of 0 to 2), fur appearance (score of 0 to 3), and posture (score of 0 to 5). All experiments were performed in compliance with local animal ethics committee requirements.

Proliferation assays. Spleen cells from BALB/c or C57BL/6 mice were collected at different times after infection with P. berghei ANKA. Splenic CD4+ cells were purified by negative selection with Dynabeads according to the manufacturer's instructions (98 to 99% purity) (Dynal Biotech, Oslo, Norway). CD25+ cell depletion was performed by incubation with biotinylated anti-CD25 antibody (7D4; BD Pharmingen, San Diego, CA) for 20 min at 4°C, followed by incubation with CELLection biotin binder magnetic beads (Dynal Biotech, Oslo, Norway) according to the manufacturer's instructions. For proliferation assays, total splenocyte suspensions, diluted in complete RPMI 1640 medium-5% fetal calf serum, were seeded in 96-well plates at a density of 2 x 106 cells/ml. Purified CD4+ cells were added to plates at a density of 5 x 105 cells/ml. Naïve syngeneic spleen cells irradiated to 3,000 rads were added as antigen-presenting cells (APCs) at a density of 2 x 106 cells/ml. All cells were then stimulated in triplicate for 3 days with P. berghei ANKA total lysate (50 µg/ml) or anti-CD3 (5 µg/ml) (BD Pharmingen, San Diego, CA). Cells cultured in medium alone were used as background controls. [methyl-3H]thymidine (2 µCi/well, 5 Ci/mmol; Amersham Biosciences, United Kingdom) was added 16 h before harvesting, and radioactivity was measured using a Betaplate counter. The cell culture supernatants were collected to determine cytokine content by capture enzyme-linked immunosorbent assay (ELISA).

Flow cytometry. Spleen cells from BALB/c or C57BL/6 mice were incubated with anti-CD16 antibody (Fc block), washed, and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD8 (53-6.7) and FITC- or phycoerythrin-conjugated anti-CD4 (L3T4) antibodies. To assess CD25+ cell depletion, some samples were simultaneously stained with FITC- or phycoerythrin-conjugated anti-CD25 (clones PC61 and 7D4, respectively), (all antibodies were from BD Pharmingen, San Diego, CA). The cells were then washed two times with phosphate-buffered saline containing 1% fetal calf serum and suspended in 200 µl of phosphate-buffered saline. The cells were then analyzed in a FACScalibur cytofluorometer (BD Bioscience) using CellQuest software. Viable cells were gated by forward and side scattering.

ELISA for IL-2, IL-4, IL-10, and IFN-{gamma} detection. The following pairs of antibodies were used: JES6-1A12 for capture and JES6-5H4 for detection of IL-2, 11B11 for capture and BVD6-24G2 for detection of IL-4, JES5-2A5 for capture and JES5-16E3 for detection of IL-10, and R4-6A2 for capture and XMG1-2 for detection of IFN-{gamma} (all antibodies were from BD Pharmingen, San Diego, CA). Antibodies used for detection were biotinylated. Ninety-six-well plates were coated with capture antibody by overnight incubation at 4°C in phosphate buffer at pH 9 for IL-4 and IFN-{gamma} and phosphate buffer at pH 7 for IL-2 and IL-10. Plates were then blocked with 1% bovine serum albumin for 1 h at 37°C. Splenocyte culture supernatants were tested in duplicate by overnight incubation at 4°C under mild agitation. The plates were then incubated for 3 h at 20°C with the respective biotinylated antibody followed by a 2-h incubation at 20°C with streptavidin-peroxidase conjugate (Pierce, Rockford, IL). Bound complexes were detected by reaction with tetramethylbenzidine (KBL, MD) and H2O2. Absorbance was read at 450 nm. The cytokine concentration in samples was calculated as pg/ml using recombinant murine cytokines (BD Pharmingen, San Diego, CA) for the preparation of standard curves.

Statistical analysis. A two-tailed Student's t test was used for data evaluation. Differences in mortality rates of P. berghei-infected mice during the period of susceptibility were assessed by Cox-Mantel log rank analysis.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition of T-cell proliferative responses during P. berghei infection. The proliferative responses to P. berghei lysate and anti-CD3 antibody were analyzed in splenocytes from cerebral malaria-susceptible (C57BL/6) and cerebral malaria-resistant (BALB/c) mice collected at different times p.i. with P. berghei ANKA. In BALB/c splenocytes, parasite-specific responses peaked at day 3 p.i. and then rapidly declined to background levels for the rest of the infection period (Fig. 1A). Polyclonal responses to anti-CD3 antibody were markedly lower in splenocytes from BALB/c malaria-infected mice than those obtained with naïve control splenocytes (day 0). The reduction of cell proliferation was evident as early as day 3 p.i. and continued to develop as the infection progressed (Fig. 1B). In contrast, in cerebral malaria-susceptible C57BL/6 mice, parasite-specific proliferative responses were also detectable at day 3 p.i. but then increased, reaching a peak at day 5 p.i (Fig. 1E). Splenocytes from infected C57BL/6 mice also showed a normal proliferative response to anti-CD3 antibody up to day 5 p.i. (Fig. 1F). Both polyclonal as well as parasite-specific cell proliferation only decreased at day 7 p.i., when animals showed severe disease symptoms. Taken together, these results suggest that during malaria, disease-resistant mice mount a short-lived parasite-specific response and that T cells from infected animals become unresponsive to stimulation through the T-cell receptor.


Figure 1
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  		FIG. 1. T-cell proliferative responses during P. berghei infection. Splenocytes from P. berghei ANKA-infected BALB/c (A, B) or C57BL/6 (E, F) mice were stimulated for 3 days with P. berghei ANKA total lysate (A, E) or anti-CD3 (B, F). Cells cultured in medium alone were used as background controls. Cell proliferation was determined by [methyl-3H]thymidine incorporation. Each point represents the mean of three samples ± standard error (SE). (C, D, G, H) Spleen cells from BALB/c (C, D) or C57BL/6 (G, H) mice were stained with anti-CD4 and anti-CD8 antibodies. The absolute numbers of CD4+ T cells (C, G) and CD8+ T cells (D, H) were calculated. Each bar represents the mean of three samples ± SE. *, P < 0.05; **, P < 0.005 (between day 0 and days p.i.). Each experiment is representative of three separate infections.

 
It has been shown that murine malaria infection is accompanied by a significant deletion of parasite-specific T cells (15). Therefore, to determine whether the reduced proliferative capacity of splenocytes from infected mice reflected a reduction in cellularity in response to infection, spleen cells from P. berghei-infected mice were stained with anti-CD4 and anti-CD8 antibodies, and the absolute number of splenic T cells was determined by flow cytometry. Unlike proliferative responses, the absolute number of splenic CD4+ and CD8+ T cells increased shortly after infection in both mouse strains (Fig. 1C, D, G, and H). In C57BL/6 mice, total T-cell numbers decreased slightly by day 7 p.i., coinciding with assays of the kinetic of proliferation (Fig. 1G and H). In contrast, in BALB/c mice, both CD4+ and CD8+ lymphocyte numbers significantly decreased only after day 10 p.i. (Fig. 1C and D), suggesting that the inhibition of cell proliferation observed during the first week of infection does not reflect a reduction in the total T-cell numbers.

Inhibition of CD4+ T-cell proliferation and IL-2 production during P. berghei infection. Figure 1 indicates that during malaria, T cells from disease-resistant BALB/c mice develop a stronger inability to proliferate in response to parasite-specific and polyclonal stimuli than cells from fully susceptible C57BL/6 mice. Therefore, to further analyze the mechanisms underlying the inhibition of cell proliferation, BALB/c mice were chosen as an experimental model. In the first series of experiments, we sought to investigate whether the nonresponsiveness observed in total splenocytes was attributable to CD4+ T lymphocytes. To that end, CD4+ T cells from malaria-infected BALB/c mice were isolated as described in Materials Methods and stimulated in vitro with P. berghei lysate and anti-CD3 antibodies in the presence of irradiated APCs. As observed with total splenocytes, parasite-specific CD4+ T-cell proliferation was detectable only early after infection (day 4) and then markedly decreased (Fig. 2A). Proliferative responses to anti-CD3 displayed a 50% reduction compared to noninfected controls as early as day 4 p.i. (Fig. 2D). Polyclonal proliferative responses remained suppressed up to day 10 p.i.


Figure 2
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  		FIG. 2. Inhibition of CD4+ T-cell proliferative responses during P. berghei infection. CD4+ T cells from P. berghei ANKA-infected BALB/c mice were stimulated for 3 days with P. berghei ANKA total lysate (A, B, C) or anti-CD3 (D, E, F). Cells cultured in medium alone were used as background controls. Cell proliferation was determined by [methyl-3H]thymidine incorporation (A, D), and IL-2 (B, E) and IL-10 (C, F) levels in cell culture supernatant were assessed by capture ELISA. Each experiment is representative of at least three separate infections. Each point represents the mean of three samples ± SE.

 
IL-2, IL-10, and TGF-ß are cytokines that were reported to be involved in the control of T-cell proliferation. Whereas IL-2 stimulates proliferative responses (8, 45), both IL-10 (32) and TGF-ß (10) exert immunosuppressive activity, and they have also been shown to play anti-inflammatory roles during malaria infection (22, 35). To investigate whether the inhibition of CD4+ T-cell proliferation observed during P. berghei infection was associated with changes in the levels of these regulatory cytokines, cell culture supernatants from CD4+ T-cell proliferation assays were used to determine cytokine content by capture ELISA. Like proliferative responses, IL-2 levels in supernatants of P. berghei lysate-stimulated CD4+ T cells were detectable only at day 4 p.i. (Fig. 2B). Consistent with the kinetics of cell proliferation, IL-2 production by CD4+ T cells in response to anti-CD3 stimulation was significantly inhibited from day 4 p.i. onwards (Fig. 2E). Parasite-specific IL-10 production peaked at day 4 p.i. and remained elevated at days 7 to 10 p.i. (Fig. 2C). In spite of the low proliferative and IL-2 responses, IL-10 production in response to anti-CD3 antibody was high at early times p.i. (Fig. 2F). No TGF-ß above background levels could be detected (data not shown). Thus, in malaria, the inhibition of CD4+ T-cell proliferation is associated with decreased IL-2 production and enhanced IL-10 levels.

CD4+ CD25+ Treg cell depletion reverses suppression of proliferation in CD4+ T cells from malaria-infected mice. CD4+ CD25+ Treg cells constitute a subset of immunosuppressive lymphocytes that have been shown to inhibit both the IL-2 production and cell proliferation of conventional CD4+ T lymphocytes (44). To determine whether Treg cells were involved in the suppression of CD4+ T-cell proliferation observed during malaria, total CD4+ T lymphocytes were isolated from malaria-infected BALB/c mice, and CD25+ cells were depleted using magnetic beads as described in Materials and Methods. CD25 cell depletion was confirmed by flow cytometry (Fig. 3A). Total CD4+ T cells and CD25-depleted cells were then stimulated with anti-CD3, and levels of cell proliferation and cytokine production were determined. CD25 depletion did not significantly affect the proliferative responses or cytokine production of naïve lymphocytes (day 0) in response to anti-CD3 (Fig. 3B to D). To the contrary, CD25 depletion significantly reversed the inhibition of both cell proliferation and IL-2 production in CD4+ T cells from malaria-infected mice (Fig. 3B and C). In contrast, IL-10 production by CD4+ T cells from malaria-infected mice was significantly reduced after the depletion of CD4+ CD25+ cells, suggesting that during malaria infection, this inhibitory cytokine is produced largely by Treg cells (Fig. 3D).


Figure 3
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  		FIG. 3. In vitro depletion of CD25+ CD4+ cells reverses inhibition of CD4+ T-cell proliferation during malaria infection. Total CD4+ T lymphocytes were isolated from malaria-infected BALB/c mice, and CD25+ cells were depleted using magnetic beads (A). Total CD4+ T cells or CD25-depleted CD4+ T cells from P. berghei ANKA-infected BALB/c mice were stimulated for 3 days with anti-CD3. Cells cultured in medium alone were used as background controls. Cell proliferation was determined by [methyl-3H]thymidine incorporation (B), and IL-2 (C) and IL-10 (D) levels in cell culture supernatant were assessed by capture ELISA. Each experiment is representative of three separate infections. Each point represents the mean of three samples ± SE. *, P < 0.05; **, P < 0.01; ***, P < 0.005.

 
Although antibody-mediated depletion of CD25+ cells is a standard procedure to effectively remove Treg cells, one disadvantage of this approach is that CD25 becomes up-regulated in activated T cells, thus making it difficult to specifically target Treg cells in pools of cells containing activated lymphocytes. Therefore, to assess whether Treg cells were also involved in the inhibition of proliferation affecting parasite-specific cells and to minimize the depletion of parasite-specific activated T cells, CD25+ CD4+ cells were depleted in vivo by injection with the anti-CD25 monoclonal antibody PC61 prior to infection with P. berghei ANKA. Preliminary experiments indicated that 2 doses of PC61 antibody were enough to deplete splenic Treg cells for a period of 2 weeks (Fig. 4A). Groups of Treg-depleted and control BALB/c mice were then infected with P. berghei. Purified CD4+ T cells from infected animals were then stimulated at different times p.i. with P. berghei lysate and anti-CD3 antibody, and levels of cell proliferation and cytokine production were determined. As observed in in vitro depletion experiments, in vivo depletion of Treg cells prior to parasitic challenge significantly reversed the inhibition of cell proliferation and IL-2 production in response to anti-CD3 in cells from malaria-infected mice (Fig. 4D and E). Parasite-specific proliferation rates as well as IL-2 secretion were significantly higher in CD4+ T-cell cultures from Treg-depleted mice than in those from nondepleted control mice (Fig. 4B and C). No consistent differences in IL-10 levels were detected after in vivo depletion of CD25+ cells (data not shown). Taken together, these experiments indicate that during murine malaria, Treg cells appear to mediate the inhibition of conventional CD4+ T-cell function in cerebral malaria-resistant mice.


Figure 4
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  		FIG. 4. In vivo depletion of Treg cells reverses inhibition of CD4+ T-cell proliferation during malaria infection. BALB/c mice were injected twice with anti-CD25 monoclonal antibody (2 days apart). Splenocytes were isolated at different times postinjection, and Treg depletion was assessed by flow cytometry on gated CD4+ cells (A). CD4+ T cells from malaria-infected Treg-depleted or control mice were stimulated for 3 days with P. berghei ANKA total lysate (B, C) or anti-CD3 (D, E). Cells cultured in medium alone were used as background controls. Cell proliferation was determined by [methyl-3H]thymidine incorporation (B, D), and IL-2 (C, E) levels in cell culture supernatant were assessed by capture ELISA. Each experiment is representative of at least three separate infections. Each point represents the mean of three samples ± SE. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

 
In vivo depletion of Treg cells does not increase fatality rates in BALB/c mice during a primary infection with P. berghei ANKA. To assess the relevance of Treg cell-mediated immunosuppression during malarial pathogenesis, BALB/c mice were depleted of CD4+ CD25+ cells as described in the legend of Fig. 4 and subsequently challenged with P. berghei. The effect of Treg depletion on disease severity was then examined. As shown in Fig. 5A, in vivo Treg depletion resulted in exacerbated disease symptoms compared to control mice. In spite of this disease aggravation, only 20% of Treg-depleted mice succumbed to cerebral malaria during a primary parasitic challenge (Fig. 5B). No significant differences in parasitemia levels were found between control and depleted mice (Fig. 5C). Taken together, these results suggest that during malaria, Treg cells might alleviate disease severity by inhibiting pathogenic T-cell responses in cerebral malaria-resistant BALB/c mice, but they do not appear to have a major role in the control of cerebral disease during a primary parasitic challenge.


Figure 5
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  		FIG. 5. Treg cell depletion does not increase fatality rates in BALB/c mice during a primary infection with P. berghei ANKA. BALB/c mice were injected twice with anti-CD25 monoclonal antibody (2 days apart). Mice were then challenged with P. berghei ANKA (1 x 106 pRBC). (A) Mice were coded and scored "blind" for neurological symptoms on days 5, 6, and 7 p.i. *, P < 0.001. (B) The percent survival was monitored daily. (C) Parasitemia was assessed from Giemsa-stained blood films. Each point represents the mean parasitemia ± SE of the surviving animals. This infection is representative of three separate experiments.

 
Treg cells inhibit the development of parasite-specific TH1 memory cells involved in malarial pathogenesis. Cerebral malaria pathogenesis has been associated with the development of TH1 immune responses. Therefore, we sought to investigate whether Treg cells played a role in the control of TH1/TH2 responses to malaria parasites. To that end, BALB/c mice were injected with PC61 monoclonal antibody and then challenged with P. berghei ANKA. At day 5 p.i., the animals were drug cured as described in Materials and Methods, and 2 weeks later, we examined cell proliferation and cytokine production by splenocytes from malaria-primed mice. Spleen cells from control as well as Treg-depleted mice displayed similar proliferative responses to polyclonal stimulation with anti-CD3 (Fig. 6A). However, parasite-specific cell proliferation was two times higher in PC61-injected animals than in control mice (Fig. 6A). Cells from both groups of mice produced similar levels of IL-4 and IFN-{gamma} in response to anti-CD3 antibody (Fig. 6B and C). Cells from Treg-depleted and control animals also produced similar levels of IL-4 in response to P. berghei lysate (Fig. 6B). In contrast, parasite-specific IFN-{gamma} production was nearly six times higher in animals that were challenged in the absence of Treg cells than in controls (Fig. 6C), indicating that this cell population inhibits the development of P. berghei-specific TH1 responses during malaria infection. Similar results were obtained when CD4+ cells from infected animals were cultured in the presence of irradiated APCs (data not shown). To further assess the relevance of these findings in the context of cerebral malaria pathogenesis, mice were depleted of Treg cells, challenged with P. berghei ANKA, and then drug cured as described above. Two weeks later, control and Treg-depleted mice were reinfected with P. berghei ANKA, and the course of disease was monitored. Control BALB/c mice were highly resistant to P. berghei-mediated cerebral malaria during reinfection (Fig. 7B). In contrast, mice that were primed in the absence of Treg cells displayed more severe disease signs (Fig. 7A), and 80% of the animals succumbed to fatal disease (Fig. 7B). Parasitemia levels were significantly lower in the more susceptible Treg-depleted mice than in control animals (Fig. 7C). The small proportion of Treg-depleted mice that survived the parasitic challenge had no patent parasitemia at day 14 p.i. These mice did not show any signs of cerebral disease, suggesting that the reduction in parasitemia observed does not reflect increased sequestration of pRBC and that these animals have been able to completely control the infection. Taken together, these results indicate that Treg cells prevent the development of TH1 memory cells mediating both protective immunity and malarial pathogenesis.


Figure 6
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  		FIG. 6. Treg cells inhibit the development of parasite-specific TH1 responses. Spleen cells from malaria-infected Treg-depleted or control mice were stimulated for 3 days with P. berghei ANKA total lysate or anti-CD3 antibody. Cells cultured in medium alone were used as background controls. Cell proliferation was determined by [methyl-3H]thymidine incorporation (A), and IL-4 (B) and IFN-{gamma} (C) production in cell culture supernatant was measured by capture ELISA. The experiment is representative of two separate infections. Each bar represents the mean of three samples ± SE. *, P < 0.01; **, P < 0.005.

 

Figure 7
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  		FIG. 7. Treg cells inhibit the development of parasite-specific memory cells involved in the induction of malarial pathogenesis. BALB/c mice (n = 10) were injected twice (2 days apart) with anti-CD25 monoclonal antibody. Treg-depleted and control mice were then challenged with P. berghei ANKA. Mice were then drug cured, rested for 2 weeks, and then rechallenged with P. berghei ANKA (1 x 106 pRBC). (A) Mice were coded and scored "blind" for neurological symptoms on days 6 to 10 p.i. *, P < 0.05; **, P < 0.005; ***, P < 0.0005. (B) The percent survival was monitored daily (P = 0.01). (C) Parasitemia was assessed from Giemsa-stained blood films. Each point represents the mean parasitemia ± SE of the surviving animals. *, P < 0.05; **, P < 0.001.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Control of infection is one of the main functions of the immune system. Some infectious agents are difficult to eradicate and therefore require a potent immune response, which in some cases can result in adverse side effects and immunopathology. Lymphoid populations exerting immunosuppressive activity, such as CD4+ CD25+ Treg cells, have been shown to play a critical role in balancing protective immune responses and immune-mediated pathology. Numerous examples demonstrating a role for Treg cells in the control of pathogenic immune responses including gut flora-mediated inflammation and severe colitis (1), intestinal inflammation produced by infection with Helicobacter hepaticus (23, 27) and Helicobacter pylori (38), lung inflammation and acute wasting disease caused by the opportunistic fungus Pneumocystis carinii (17), and severe disease after Candida albicans infection (31), among others, have been reported. The present study shows that Treg cells are also involved in the inhibition of proliferative responses and IL-2 production during rodent malaria. Moreover, our data have also established that Treg cells prevent the development of P. berghei-specific TH1 memory cells involved in the induction of cerebral malaria, demonstrating a regulatory role for this cell population in the control of fatal pathogenesis.

Treg cell involvement during malaria infection has been previously reported. Human studies indicated that the up-regulation of the X-linked forkhead/winged-helix transcription factor (FOXP3) and Treg cells correlates with increased parasite growth rates (46). In mice, the role of Treg cells was studied after infection with Plasmodium yoelii (16) and Plasmodium berghei NK65 (25). Similar to our findings here, Treg cell depletion in those infections resulted in decreased or delayed parasitemia, suggesting that Treg cell activation could constitute a mechanism utilized by the parasite to evade host immunity. Although infection of mice with P. yoelii and P. berghei NK65 results in hyperparasitemia and the death of susceptible mice due to hemolytic anemia, it does not induce T-cell-mediated cerebral immunopathology. Therefore, a contribution of Treg cells to the control of pathogenic immune responses cannot be accurately assessed in those infection models. To the contrary, infection of mice with P. berghei ANKA used here induces cerebral malaria, which results from the sequestration of pRBC and an exacerbated host proinflammatory response (42). Using this disease model, we found that Treg-mediated inhibition of parasite-specific TH1 responses results in both reduced protective immunity (higher parasitemia) and protection of mice from lethal cerebral pathogenesis. Thus, our results support the notion that although TH1 responses could be beneficial to control malaria infection, they should be carefully regulated in order to prevent harmful pathogenesis.

Treg cells have been shown to exert immunosuppressive activity by cell-contact-dependent mechanisms (44) and through the secretion of suppressive cytokines such as TGF-ß (33) and IL-10 (2). In the present study, Treg depletion alleviated malaria-induced inhibition of T-cell proliferation and IL-2 secretion and correlated with significantly reduced IL-10 responses, suggesting that Treg cells could be mediating their suppressive activity during infection through the production of this cytokine. In addition, it has been shown that in vivo neutralization of IL-10 in mice enhances TH1-driven pathogenesis and fatality rates during P. berghei-mediated cerebral malaria (22). Further work is still required to elucidate whether the Treg-mediated inhibition of TH1 polarization observed here also occurs through an IL-10-dependent mechanism. On the other hand, anergic T cells (5) and Treg cells (41) have been found to express high levels of the negative regulator cytolytic-T-lymphocyte-associated antigen 4 (CTLA-4). In addition, anti-CTLA-4 antibody treatment has been found to abrogate the ability of Treg cells to inhibit colitis (39). Interestingly, CTLA-4 blockade also induces increased liver injury in P. berghei-infected mice (19) and exacerbates the induction of cerebral malaria (18). Therefore, these observations together raise the possibility that in malaria infection, the expression of CTLA-4 molecules in Treg cells might be responsible for the inhibition of T-cell function and TH1-driven cerebral pathogenesis.

In many other infection models investigated, Treg cells appeared to play a role during chronic rather than acute stages of infection (3, 30). Interestingly, although Treg depletion enhanced disease severity and increased proinflammatory cytokine levels in peripheral blood (data not shown), it did not result in increased fatality rates after a primary infection with P. berghei ANKA. In contrast, a higher proportion of animals primed in the absence of Treg cells developed cerebral malaria and succumbed to disease during a secondary parasitic challenge. This increased disease susceptibility correlated with the acquisition of parasite-specific IFN-{gamma}-producing cells, indicating that during murine malaria, Treg cells inhibit TH1 polarization. Treg cells have been found to suppress TH1 responses and contribute to TH2 polarization in other parasitic infections such as Schistosoma mansoni (28) in murine models of tissue injury (6) and during the prevention of inflammatory bowel disease (37).

In addition to an enhanced TH1 response, Treg depletion in malaria-infected, drug-cured, and rested mice resulted in higher proliferative responses to P. berghei ANKA lysate than control animals, presumably reflecting a higher frequency of memory cells developing in the absence of Treg cells. Although TH1 responses appeared to play a role in the control of parasitemia, they also contributed to disease induction, suggesting that the Treg cell-mediated inhibition of memory formation reported here could somehow be advantageous for the host. Treg cells were found to control the development of immunological memory in other experimental models such as mouse autoimmune colitis (20) and Listeria monocytogenes infection (24) and in H. pylori-infected individuals (26). Interestingly, it is well established that immunity against human malaria is difficult to achieve and takes several years to develop for individuals living in areas of endemicity (12). The reasons for this are still unknown. Further work is still required to elucidate whether Treg cells can also contribute to this process in human malaria.


   ACKNOWLEDGMENTS
 
This work was supported by the NH&MRC project grant 356239 and program grant 215201. L.S. is an International Research Scholar of the Howard Hughes Medical Institute.


   FOOTNOTES
 
* Corresponding author. Mailing address: The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia. Phone: 61 3 93452644. Fax: 61 3 93470852. E-mail: hansen{at}wehi.edu.au Back

{triangledown} Published ahead of print on 26 February 2007. Back

Editor: W. A. Petri, Jr.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Infection and Immunity, May 2007, p. 2275-2282, Vol. 75, No. 5
0019-9567/07/$08.00+0     doi:10.1128/IAI.01783-06
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