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The Journal of Immunology, 2007, 179, 2748 -2757
Copyright © 2007 by The American Association of Immunologists, Inc.
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African Trypanosomiasis: Naturally Occurring Regulatory T Cells Favor Trypanotolerance by Limiting Pathology Associated with Sustained Type 1 Inflammation1

Martin Guilliams*,{dagger}, Guillaume Oldenhove{ddagger}, Wim Noel*,{dagger}, Michel Hérin, Lea Brys*,{dagger}, Patrizia Loi§, Véronique Flamand§, Muriel Moser{ddagger}, Patrick De Baetselier*,{dagger} and Alain Beschin2,*,{dagger}

* Department of Molecular and Cellular Interactions, Vlaams Instituut voor Biotechnologie, and {dagger} Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussel, Belgium; {ddagger} Institut de Biologie et Médecine Moléculaires, and § Institute for Medical Immunology, Université Libre de Bruxelles, Gosselies, Belgium; and Cell and Tissue Laboratory, Unité de Recherche en Physiologie Moléculaire, Facultés Universitaires Notre-Dame de la Paix, Namur, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Tolerance to African trypanosomes requires the production of IFN-{gamma} in the early stage of infection that triggers the development of classically activated macrophages controlling parasite growth. However, once the first peak of parasitemia has been controlled, down-regulation of the type 1 immune response has been described. In this study, we have evaluated whether regulatory T cells (Tregs) contribute to the limitation of the immune response occurring during Trypanosoma congolense infection and hereby influence the outcome of the disease in trypanotolerant C57BL/6 host. Our data show that Foxp3+ Tregs originating from the naturally occurring Treg pool expanded in the spleen and the liver of infected mice. These cells produced IL-10 and limited the production of IFN-{gamma} by CD4+ and CD8+ effector T cells. Tregs also down-regulated classical activation of macrophages resulting in reduced TNF-{alpha} production. The Treg-mediated suppression of the type 1 inflammatory immune response did not hamper parasite clearance, but was beneficial for the host survival by limiting the tissue damages, including liver injury. Collectively, these data suggest a cardinal role for naturally occurring Tregs in the development of a trypanotolerant phenotype during African trypanosomiasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
To efficiently deal with infections, the host must appropriately regulate the pathogen-triggered immune response to achieve effective parasite control while avoiding collateral tissue damage. Tissue damage can be limited by mechanisms controlling the magnitude of immune effector responses toward the pathogen. Such mechanisms involve the generation of immunosuppressive cells, including macrophages (1) and T cells (2).

Suppressive T cells have been divided into naturally occurring regulatory T cells (Tregs)3 and inducible Tregs, such as TR1 or TH3 cells (2). In normal conditions, naturally occurring CD4+CD25+Foxp3+ Tregs play an essential role in avoiding autoimmunity through inhibition of autoreactive T cells and maintenance of peripheral self-tolerance (3). This Treg population constitutes 10% of peripheral CD4+ T cells in mice and humans and exhibits a broad TCR repertoire recognizing various self and non-self Ags (4, 5). Recently, it was proposed that activation of naturally occurring Tregs by pathogens may be a crucial strategy to down-regulate the immune response and to establish a chronic infection (6, 7). However, Tregs can also provide advantages to the host: by limiting pathological immune responses they can reduce collateral tissue damage (8, 9), and by preventing complete parasite clearance they can sustain immunity to re-infection (10).

African trypanosomes are protozoan parasites that cause sleeping sickness in humans and nagana disease in livestock in sub-Saharan Africa. Despite the hostile environment of the host’s bloodstream, these extracellular parasites evade the immune response and establish a chronic infection through a combination of Ag variation of their variant-specific glycoprotein surface coat and induction of a generalized state of immunosuppression (11). Trypanosoma congolense-induced immunosuppression has been described in natural cattle infections as well as in experimental rodent infections (12, 13, 14). This suppression affects both cytokine production and T cell proliferation. Macrophages (15) as well as T cells (16) have been reported to contribute to the down-regulation of the immune response during African trypanosome infection. However, though most investigations have focused on the phenotype and the function of suppressive macrophages elicited during African trypanosomiasis (17, 18), the nature of the suppressive T cells remains poorly documented. Moreover, the influence of immunosuppression on the outcome of the disease remains a matter of debate.

C57BL/6 mice when infected with T. congolense are considered as relatively tolerant to the disease, being able to control the first peak of parasitamia and to develop a chronic and systemic infection lasting for ~5 mo (18). In the present work, we have addressed whether Tregs play a role in the immunosuppression induced during T. congolense infection and influence the outcome of the disease. Our data show that naturally occurring Foxp3+ Tregs expand in the spleen and the liver of infected C57BL/6 mice. These cells play an essential role in the down-regulation of the type 1 immune response during the chronic stage of infection. Hereby, Tregs avoid injury to the liver, preserving its parasite clearance function, and contribute to the development of the trypanotolerant phenotype in T. congolense-infected mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Parasites, mice, and infections

T. congolense variant Ag type 13 (Tc13) organisms (19) were provided by Dr. H. Tabel (University of Saskatchewan, Saskatchewan, Saskatchewan, Canada). Female wild-type mice (Harlan), IL-10 knockout (KO) mice (B & K Universal), Ly5.1 congenic mice (Charles River Breeding Laboratories), and nu/nu C57BL/6 mice (Taconic Farms) were kept under filter barrier and used at 8–12 wk of age. Parasites stored at –80°C were used to infect cyclophosphamide-treated F1 (C57BL/6 x BALB/c) mice (bred in house) by i.p. inoculation. At day 4 postinfection, mice were bled, and parasites were purified by DEAE-cellulose chromatography (20). C57BL/6 mice were then infected with 2000 purified parasites. Parasitemia was monitored by tail-blood puncture. Experiments were performed in compliance with the relevant laws and institutional guidelines.

In vivo depletion of CD25+ T cells

Mice were i.p. injected with 1 mg of PC61 anti-CD25 Ab provided by S. Sakaguchi (Kyoto University, Kyoto, Japan) 3 days before infection. Efficiency of the Treg depletion was assessed by flow cytometry using the 7D4 Ab (BD Biosciences) recognizing a distinct epitope on CD25 than PC61, and the FJK-16s Ab (eBioscience) for Foxp3.

Reconstitution experiments

Ly5.2+CD8+ T cells, Ly5.2+CD4+CD25– T cells, and Ly5.1+CD4+CD25+ Tregs were isolated from the spleen of noninfected mice by combination of MACS purification and FACS sorting. CD8+ and CD4+ T cells were positively selected on magnetic separation columns according to the manufacturer’s protocol (Miltenyi Biotec). The isolated CD4+ T cells were then incubated with anti-CD4-FITC and anti-CD25-PE Ab, and CD4+CD25– and CD4+CD25+ T cells were sorted on a FACSVantage SE flow cytometer (BD Biosciences) with a purity ranging from 95 to 98%. Ly5.2+CD4+CD25– T cells (106) and Ly5.2+CD8+ T cells (2 x 105) in 0.2 ml of LPS-free PBS were i.v. injected alone (mice without Tregs) or together with Ly5.1+CD4+CD25+ T cells (2 x 105, mice with Tregs) in nu/nu Ly5.2 C57BL/6 mice. Mice were infected 6 days after the adoptive cell transfer. For parameter analyses in the reconstitution study, control mice were noninfected reconstituted mice taken with mice at day 21 postinfection, i.e., 27 days after adoptive transfer.

Isolation of liver and spleen cells

Liver nonparenchymal cells were isolated as follows. Animals were euthanized (CO2) and livers were perfused through the portal vein with 10 ml of 100 U/ml collagenase type III (Worthington Biochemical) in HBSS. Then, the liver was minced and incubated in 10 ml of 100 U/ml collagenase III (20 min, 37°C). The resulting cell suspension was passed through a 100-µm nylon mesh filter and then centrifuged at 300 x g (10 min, 4°C). After erythrocyte lysis, the pellet was resuspended in 10 ml of HBSS supplemented with 2 mM EDTA and 10% FCS and overlayed on 10 ml of LymphoPrep (Lucron Bioproducts). After centrifugation at 430 x g (30 min, 17°C), the layer of low-density cells at the interface containing nonparenchymal cells was harvested. Spleen cells were isolated following mechanical disruption of the spleen and erythrocyte lysis.

Quantification of cytokines

To evaluate IFN-{gamma} and IL-10 production by T cells, spleen and liver cells resuspended at 2 x 106/ml in RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.1 mM non-essential amino acids, 2 mM L-glutamine, and 5 x 10–5 M 2-ME (all from Invitrogen Life Technologies) were incubated in the presence of 1 µg/ml anti-CD3 Ab (clone 145-2C11). In parallel, spontaneous TNF-{alpha} production by macrophages was assessed. Cytokines were quantified in culture supernatants collected after 3 days with specific sandwich ELISAs for IFN-{gamma}, IL-10 (BD Pharmingen), or TNF-{alpha} (R&D Systems), in accordance to the manufacturers’ protocols.

FACS analysis

For surface markers, cells were stained for 30 min at 4°C using conventional protocols. Cells were incubated with anti-Fc{gamma}R Ab (clone 2.4G2) before adding (1 µg/106 cells) the following: FITC-conjugated anti-CD8a (clone Ly-2), FITC-conjugated anti-CD45.1 (clone A20), PE-Cy5-conjugated anti-CD4 (clone L3T4), or FITC- or PE-conjugated anti-CD25 (clone PC61 or clone 7D4) Abs. Intracellular stainings were performed in accordance with the manufacturer’s protocols using PE-conjugated anti-IFN-{gamma} (clone XMG1.2) and FITC- or PE-conjugated anti-Foxp3 (FJK-16s) Abs. All Abs were purchased from BD Biosciences with the exception of anti-Foxp3 and anti-CD45.1 (eBioscience). For intracellular IFN-{gamma} staining, total liver and spleen cells were cultured 2 h on anti-CD3-coated plates (clone 145-2C11) before adding brefeldin A (BD Biosciences). Four hours later, cells were fixed and permeabilized (Fix/Perm kit; BD Biosciences). Cells were analyzed on FACSVantage SE flow cytometer or FACSCanto II using the CellQuest program (BD Biosciences) or FlowJo, respectively.

Gene expression analysis

CD4+ and CD4– cells were isolated from spleen and liver of reconstituted mice by positive CD4 selection on magnetic separation columns, according to the manufacturer’s protocol (Miltenyi Biotec). The CD4+ T cell population was then sorted in a CD4+Ly5.1+ and a CD4+Ly5.2+ T cell fraction using a FACSVantage SE flow cytometer (BD Biosciences) with a purity ranging from 95 to 98%. Gene expression in the isolated populations was analyzed by quantitative real-time PCR using the conditions described (21). Results of the PCR analyses were normalized against the housekeeping gene S12. Primers used were: IL-10 (forward) 5'-ACTCAATACACACTGCAGGTG-3' (reverse) 5'-GGACTTTAAGGGTTACTTGG-3'); Foxp3 (forward) 5'-CCTGGCCTGCCACCTGGGATCAA-3' (reverse) 5'-TTCTCACAACCAGGCCACTTG-3'; TGF-β (forward) 5'-TTGCTTCAGCTCCACAGAGA-3' (reverse) 5'-TGGTTGTAGAGGGCAAGGAC-3'; and S12 (forward) 5'-GGAAGGCATAGCTGCTGGAGGTGT-3' (reverse) 5'-CCTCGATGACATCCTTGGCCTGAG-3'.

Microscopy and aminotransferase levels

Spleen and liver were fixed in Bouin solution (Sigma-Aldrich). Histologic sections embedded in paraffin were stained with H&E saffron for microscopic evaluations. Liver alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured in serum samples, using commercially available kits (Boehringer Mannheim).

Statistical analysis

All comparisons were tested for statistical significance using the unpaired t test with Welch’s correction from GraphPad Prism 4.0 software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Expansion of Tregs correlates with limitation of the type 1 immune response in T. congolense-infected C57BL/6 mice

To evaluate whether Tregs are elicited during T. congolense infection in trypanotolerant C57BL/6 mice, the expression of the Treg-specific transcription factor Foxp3 was investigated. The experiments were performed in the spleen but also in the liver because the latter organ has been suggested as the major site for trypanosome clearance (22, 23). A transient increase in the percentage of Foxp3+ cells within the CD4+ T cell population was observed in the spleen and the liver during the first month of infection (Fig. 1, A and B). This resulted in a gradual expansion of the total number of Foxp3+CD4+ T cells in both organs (Fig. 1, C and D) after the control of the first peak of parasitemia (acute phase) and in the chronic stage of infection (Fig. 1E). The expansion of Foxp3+CD4+ T cells was not linked to the peaks of parasitemia and was higher in the liver than in the spleen.


Figure 1
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  		FIGURE 1. Tregs expand in the spleen and the liver of T. congolense-infected C57BL/6 mice. CD4+ T cells from the spleen and the liver were analyzed for Foxp3 expression at different time points postinfection by flow cytometry. Percentage (A and B) and total number (C and D) of CD4+Foxp3+ T cells were determined in wild-type mice ({square}) and IL-10 KO mice ({blacksquare}). Data are shown as mean ± SEM of three individual mice in each group. *, p < 0.05 comparing infected and noninfected mice (day 0 postinfection). **, p < 0.05 comparing wild-type and IL-10 KO mice. E, Parasitemia (mean ± SEM of 10 mice) in the acute and chronic stage of infection is illustrated. IL-10 KO mice died within 10 ± 0.4 days postinfection. Waves of parasitemia similar as in the chronic stage of infection were observed until the wild-type animals died (148 ± 21 days). #, p < 0.01 comparing survival of wild-type and IL-10 KO mice. At least three experiments were performed with similar results.

 
We and others have previously shown that trypanotolerance (i.e., the ability of the host to control the first peak of parasitemia and to develop a chronic infection) requires the sequential development of an IFN-{gamma}-dependent immune response in the acute stage of infection followed by an IL-10-dependent immune response at later stage that counterbalances the infection-associated immunopathology, including liver necrosis (18, 22, 23, 24, 25, 26). We thus hypothesized that the expansion of Tregs coincided with the switch from an IFN-{gamma} to an IL-10 immune environment in African trypanosome-infected hosts. In agreement, we observed that the secretion of IFN-{gamma} by spleen and liver immune cells from T. congolense-infected mice, as well as the production of TNF-{alpha}, peaked during the first week of infection and returned to levels observed in noninfected animals by day 14 postinfection (Fig. 2), i.e., when Tregs were significantly expanding (Fig. 1). In parallel, IL-10 production increased from day 7 postinfection and was maintained during the chronic stage of infection (Fig. 2). Moreover, liver damage seemed limited, as marginal liver transaminase (ALT/AST) levels were found in the serum during the first month of infection (Fig. 2). Thus, the kinetic of expansion of Tregs parallels the suppression of the type 1 immune response and the induction IL-10 secretion, suggesting the involvement of these cells in the immunoregulatory network elicited during T. congolense infection.


Figure 2
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  		FIGURE 2. Type 1 immune mediators are down-regulated in the chronic stage of T. congolense infection in wild-type mice but not IL-10 KO C57BL/6 mice. Spleen (A, C, and E) and liver (B, D, and F) cells harvested at various time points postinfection in wild-type mice ({square}) and IL-10 KO mice ({blacksquare}) were cultured in vitro. IFN-{gamma} (A and B) and IL-10 (E and F) were quantified in supernatants of cells activated with anti-CD3 Ab. C and D, Spontaneous TNF-{alpha} release was quantified. ALT and AST levels (G and H) were measured in the serum from wild-type mice ({square}) and IL-10 KO mice ({blacksquare}). Data are shown as mean ± SEM of three individual mice. *, p < 0.04 comparing infected and noninfected mice. **, p < 0.05 comparing wild-type and IL-10 KO mice. Three experiments were performed with similar results.

 
Limited expansion of Tregs associates with susceptibility of IL-10 KO mice to T. congolense infection

Because the induction of IL-10 secretion coincided with the expansion of Tregs, we addressed whether Tregs could develop in T. congolense-infected IL-10 KO C57BL/6 mice. In contrast to wild-type mice, CD4+Foxp3+ T cells expanded to lower levels in the liver of infected IL-10 KO mice (Fig. 1). IL-10 KO mice controlled the first peak of parasitemia as efficiently as wild-type mice (Fig. 1E), but died within 10 ± 0.4 days postinfection. The increased susceptibility of IL-10 KO mice was associated with the loss of parasite control (from day 8 postinfection) (Fig. 1) and absence of down-regulation of IFN-{gamma} and TNF-{alpha} production, but enhanced ALT/AST levels at day 10 postinfection (Fig. 2). These data suggest that the expansion of Tregs in an IL-10 environment favors trypanotolerance by down-regulating the secretion of inflammatory mediators, hereby protecting the integrity of the liver that preserves its parasite clearance capacity. Thus, the sudden burst of parasitemia observed in IL-10 KO mice might be the result of liver damage.

Anti-CD25 Ab treatment inefficiently depletes Tregs in T. congolense-infected mice

Anti-CD25 (IL-2R{alpha}) treatment is commonly used for depleting Tregs. However, although CD25 expression was enhanced on CD4+ T cells during T. congolense infection, not all CD25+ T cells expressed Foxp3 (Fig. 3). The CD4+CD25+Foxp3– T cells most probably represents an effector population, as CD25 can be up-regulated on conventional T cells upon activation (28). Vice versa, not all Foxp3+CD4+ cells were CD25+, in particular in the liver (Fig. 3). Thus, as in other chronic infection models (9, 27), CD25 is unsuitable for the monitoring or the depletion of Tregs in T. congolense-infected hosts.


Figure 3
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  		FIGURE 3. Partial depletion of Tregs following anti-CD25 treatment in T. congolense-infected C57BL/6 mice. At day 10 posttreatment with anti-CD25, spleen and liver cells from noninfected or infected mice were stained with anti-CD4, anti-CD25, and anti-Foxp3 Abs. A, The percentage of CD25+ and Foxp3+ cells was evaluated within the gated CD4+ T cells. FACS profiles are representative of one of six animals tested in two independent experiments. Data are shown as mean percentage ± SEM of three individual mice. Spleen (B and D) and liver (C and E) cells harvested at day 10 postinfection in mice treated with control ({square}) or anti-CD25 Ab ({blacksquare}) were cultured in vitro. IFN-{gamma} (B and C) was quantified in supernatants of cells activated with anti-CD3 Ab. Spontaneous TNF-{alpha} release (D and E) was quantified. Three experiments were performed with similar results. Data are shown as mean ± SEM of three individual mice. *, p < 0.05 comparing mice treated with control or anti-CD25 Ab.

 
As far as depletion of CD25+ cells before infection is concerned, we observed that the elimination of Foxp3+ or CD25+ T cells within the CD4+ population from the spleen and the liver was 1) partial at day 3 (data not shown) and 10 posttreatment in noninfected mice (Fig. 3) and 2) transient, Foxp3+ and CD25+ cells returning to normal values within 21 days posttreatment (data not shown). In infected animals, depletion was as inefficient as in noninfected animals and still allowed the expansion of Foxp3+ cells in the liver and the spleen as compared with anti-CD25-treated noninfected animals (Fig. 3). The depletion of Tregs resulted in a significant increase of IFN-{gamma} and TNF-{alpha} production at day 10 postinfection (Fig. 3) but did not affect control of the first peak of parasitemia (data not shown). However, considering that the modulation of type 1 cytokine production and the depletion of Tregs were transient (returning to normal levels by day 21 posttreatment), it was not surprising that the anti-CD25 treatment did not affect the survival and liver pathology during this chronic infection lasting for 5 mo (data not shown). The partial and transient depletion of Foxp3+CD4+ T cells observed after anti-CD25 treatment, which is in agreement with recent reports (29, 30, 31, 32, 33), could lead to a misinterpretation on the possible role of Treg in our infection model. Therefore, we used reconstitution experiments in nude mice as an alternative to investigate the activity of Tregs during T. congolense infection.

CD4+Foxp3+ T cells expanding during T. congolense infection originate from the naturally occurring Treg population

To investigate whether the CD4+Foxp3+ T cell population emerging in T. congolense-infected mice are naturally occurring Tregs or induced Tregs (i.e., CD4+ T cells that would have gained Foxp3 expression during the infection), two groups of nu/nu C57BL/6 mice reconstituted with T cells from noninfected C57BL/6 mice were used. The first group was reconstituted with CD8+ and CD4+CD25– T cells coming from wild-type (Ly5.2) C57BL/6 mice (mice reconstituted without Tregs). The second group received CD8+ and CD4+CD25– T cells from wild-type (Ly5.2) C57BL/6 mice together with CD4+CD25+ T cells (as source of naturally occurring Tregs) from congenic Ly5.1 C57BL/6 mice (mice reconstituted with Tregs). In this experimental setup, naturally occurring Tregs (CD4+Ly5.1+Foxp3+) could be distinguished from induced Tregs (CD4+Ly5.2+Foxp3+).

Upon T. congolense infection, marginal levels of CD4+Foxp3+ T cells were found in the spleen and the liver of mice reconstituted without Tregs (Fig. 4, A and B). In contrast, in infected mice reconstituted with Tregs, the expansion of Foxp3+ Tregs within the CD4+ T cell population was similar as in wild-type mice (Fig. 4, A and B, and Fig. 1). These data suggest that CD4+Foxp3+ T cells expanding during T. congolense infection originate from the naturally occurring Treg pool.


Figure 4
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  		FIGURE 4. Foxp3+CD4+ Tregs expanding during T. congolense infection are naturally occurring Tregs. The nu/nu C57BL/6 mice were reconstituted with CD8+ and CD4+CD25– T cells from Ly5.2 mice in the absence or presence of CD4+CD25+ Tregs from Ly5.1 mice before infection. A and B, CD4+ T cells from the spleen and liver of mice with Tregs ({square}) or without Tregs ({blacksquare}) were analyzed for Foxp3 expression at different time points postinfection by flow cytometry. Data are shown as mean percentage ± SEM of three individual mice. *, p < 0.04 comparing infected and noninfected mice. **, p < 0.05 comparing mice reconstituted with or without Tregs. At day 14 postinfection, liver cells were stained with CD4, Ly5.1, and Foxp3 Abs. C, Foxp3 expression was evaluated on the gated CD4+ fraction (left). Using indicated gates (middle), Foxp3 expression was analyzed on CD4+Ly5.1+ and CD4+Ly5.1– T cells (right). FACS profiles are representative of one of six animals tested in two independent experiments. Data are shown as mean percentage ± SEM of three individual mice.

 
In mice reconstituted with Tregs chronically infected with T. congolense, <1.5% of the CD4+Ly5.1– T cells in the spleen (data not shown) and the liver (Fig. 4C) expressed a significant level of Foxp3. In contrast, ~85% of the CD4+Foxp3+ T cells expressed the Ly5.1 marker. The increase of CD4+Foxp3+ T cells observed in T. congolense-infected mice can therefore be assigned to the expansion of naturally occurring Tregs and not to CD4+CD25– T cells acquiring Foxp3 expression, even in the presence of naturally occurring Tregs.

Tregs suppress IFN-{gamma} production and limit immunopathology during T. congolense infection

Using reconstituted nu/nu mice, we also addressed whether the expanding Tregs influence the outcome of the disease. Mice reconstituted without Tregs and mice reconstituted with Tregs controlled the first peak of parasitemia as efficiently as wild-type animals (Figs. 5 and 1A). However, from day 12 postinfection, a gradual rise of parasitemia occurred in both groups of animals until the mice died. Yet, mice reconstituted without Tregs died significantly earlier than mice reconstituted with Tregs (survival time: 21.8 ± 0.6 days vs 29.0 ± 0.7 days).


Figure 5
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  		FIGURE 5. T. congolense-infected mice die earlier in the absence of Tregs. Parasitemia and survival were determined in mice reconstituted as described in Fig. 4 with ({circ}) or without Tregs ({blacksquare}). Data are shown as mean ± SEM of six individual mice per group. Three experiments were performed with similar results. *, p < 0.05 comparing parasitemia in mice with or without Tregs. #, p < 0.03 comparing survival of mice with or without Tregs (21.8 ± 0.6 days vs 29.0 ± 0.7 days).

 
In mice reconstituted with Tregs, IFN-{gamma} and TNF-{alpha} modulations in the spleen and the liver were comparable with the ones observed in wild-type animals (Figs. 6 and 2). The production of these type 1 mediators was up-regulated during the first peak of parasitemia (day 7), but returned to values observed in noninfected reconstituted mice in the chronic stage of infection (days 14 and 21). Conversely, in mice reconstituted without Tregs, the IFN-{gamma} and TNF-{alpha} production was sustained at high levels throughout the infection period. Thus, Tregs may play a major role in the down-regulation of the type 1 immune response during the chronic stage of T. congolense infection. Intracellular stainings performed on cells isolated from the spleen (data not shown) and the liver of infected reconstituted mice showed that only a marginal percentage (<2%) of CD4+Ly5.1+ Tregs produced IFN-{gamma} (Fig. 7). IFN-{gamma} was mainly produced by CD4+Ly5.2+ effector T cells and by CD8+ T cells. Moreover, by comparing mice reconstituted with and without Tregs, it was found that the presence of Tregs significantly diminished the IFN-{gamma} secretion by CD4+ as well as CD8+ effector T cells.


Figure 6
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  		FIGURE 6. Tregs contribute to the down-regulation of type 1 immune response during the chronic stage of T. congolense infection. Spleen (A, C, and E) and liver (B, D, and F) cells harvested at various time points postinfection in mice reconstituted as described in Fig. 4 with Tregs ({square}) or without Tregs ({blacksquare}) were cultured in vitro. IFN-{gamma}, (A and B) and IL-10 (E and F) were quantified in supernatants of cells activated with anti-CD3 Ab. Spontaneous TNF-{alpha} release (C and D) was quantified. Three experiments were performed with similar results. Data are shown as mean ± SEM of three individual mice. *, p < 0.03 comparing mice with or without Tregs.

 

Figure 7
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  		FIGURE 7. Tregs limit IFN-{gamma} production by CD4+ and CD8+ T cells during T. congolense infection. At day 14 postinfection, liver cells from mice reconstituted as described in Fig. 4 with or without Tregs were stimulated in vitro with anti-CD3 Abs. The percentage of cells producing IFN-{gamma} was evaluated by intracellular FACS staining, gating on CD4+Ly5.1– nonregulatory T cells, CD4+Ly5.1+ Tregs, and CD8+ T cells. FACS profiles are representative of one of six animals tested in two independent experiments. Data are shown as mean percentage ± SEM of three individual mice. *, p < 0.03 comparing mice with or without Tregs.

 
The continuous high production of type 1 mediators in infected mice without Tregs was associated with a dramatic increase of ALT and AST levels in the serum at day 21 postinfection, indicating liver pathology in these mice (Fig. 8, A and B). At that time, the livers from infected mice both with and without Tregs were enlarged to similar extent. However, only the liver surface from infected mice without Tregs showed extensive pale geographic areas highly suggestive of anoxic infarcts (Fig. 8, C and D). Microscopic examination of the livers from infected mice with and without Tregs revealed intense lobular and portal mononuclear infiltrates mainly composed of lymphocytes in portal tracts and of lymphocytes mixed with monocytes in sinusoidal spaces. However, the pale zones in the liver from infected mice without Tregs proved to be infarcts because coagulative necrosis was histologically demonstrated: cellular outline and tissue architecture could still be discerned, whereas hepatocytes were characterized by deeply eosinophilic cytoplasm and nucleus undergoing karyolysis, pyknosis, or karyorrhexis (Fig. 8E). In contrast, no infarct was present in the liver from infected mice with Tregs, but single cell necrosis (Councilman body) was occasionally observed (Fig. 8F). It should be mentioned that no pathological difference was observed in the spleen of infected mice with and without Tregs. Yet, similar and massive infiltration of inflammatory cells occurred in the spleens of both experimental groups (data not shown). The liver (Fig. 8, G–J) and the spleen (data not shown) from noninfected mice with and without Tregs had normal macroscopic and microscopic appearances, indicating that the damage to the liver resulted from T. congolense infection and not from autoimmune reactions.


Figure 8
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  		FIGURE 8. Tregs avoid liver injury during T. congolense infection. ALT and AST levels (A and B) were measured in the serum from mice reconstituted as described in Fig. 4 with Tregs (noninfected) (Figure 8) and (infected) ({square}), or without Tregs (noninfected) (dark gray) and (infected) ({blacksquare}) at various time points. Data are shown as mean ± SEM of three individual mice. Two experiments were performed with similar results. *, p < 0.03 comparing noninfected and infected mice without Tregs. **, p < 0.03 comparing infected mice with or without Tregs. Macroscopic examination of liver at day 21 postinfection revealed the presence of anoxic infarcts (arrow) in mice without Tregs (C) but not in mice with Tregs (D). E, Microscopic analysis (H&E staining at original magnification of x40) of liver sections from infected mice without Tregs indicate abrupt border (dotted line) between an infarct (bottom right corner) and viable tissue (top left corner). F, In infected mice with Tregs, only single cell necrosis (arrow) was observed. The livers from noninfected mice reconstituted with (G) and without (H) Tregs showed normal macroscopic (G and H) and microscopic (I and J) appearances. Macroscopic and microscopic analyses are representative of four animals tested in two independent experiments.

 
In conclusion, these reconstitution experiments indicate that Tregs play an essential role in the suppression of the type 1 immune response and as such delayed the onset of immunopathology, including liver injury, in the chronic stage of T. congolense infection. Consequently, parasite clearance capacity of the host liver is prolonged, allowing longer survival.

Tregs are not the sole source of IL-10 during T. congolense infection

Infections of IL-10 KO mice with T. congolense confirmed that IL-10 is essential for trypanotolerance (34). However, although mice reconstituted without Tregs produced IL-10 to similar levels as mice reconstituted with Tregs at any time point postinfection with T. congolense (Fig. 6), they were more trypanosusceptible as evidenced by shorter survival and increased pathology. To analyze which cells other than Tregs contributed to IL-10 secretion, CD4+Ly5.2+ T cells, CD4+Ly5.1+ T cells and CD4– cells were isolated at day 21 postinfection from the spleen and the liver of mice reconstituted with or without Tregs. IL-10 and Foxp3 gene expression levels were then quantified by real-time PCR analyses. Foxp3 expression in both organs was only found in the CD4+Ly5.1+ fraction confirming their Treg identity (Fig. 9A). IL-10 expression was induced to similar levels in the CD4+Ly5.1+ Treg and the CD4+Ly5.2+ nonregulatory T cell fractions (Fig. 9B). Thus, this suggests that CD4+Foxp3– effector T cells as well as CD4+Foxp3+ Tregs produce IL-10 upon T. congolense infection. In addition, IL-10 expression was also induced in the CD4– fraction (Fig. 9C). Because the percentage of Tregs within the CD4+ T cell population never exceeded 20% (Figs. 4 and 1), the nonregulatory T cells and the CD4– cells probably represented the major sources of IL-10 in the spleen and the liver of T. congolense-infected hosts.


Figure 9
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  		FIGURE 9. Tregs and nonregulatory T cells contribute to IL-10 production during T. congolense infection. CD4+Ly5.1+ Tregs, CD4+Ly5.1– nonregulatory T cells and CD4– cells were isolated at day 21 postinfection from the spleen and the liver of mice reconstituted as described in Fig. 4 with and without Tregs. Relative Foxp3 (A), IL-10 (B and C), and TGF-β (D and E) mRNA expression was determined by real-time PCR and normalized with the ribosomal protein S12 gene in CD4+Ly5.1+ Tregs (Figure 9) (A, B, and D), CD4+Ly5.1– nonregulatory T cells isolated from mice with Tregs ({square}) (A, B, and D) or without Tregs ({blacksquare}) (A, B, and D) and CD4– cells isolated from mice reconstituted with Tregs ({square}) (C and E) or without Tregs ({blacksquare}) (C and E). Two experiments were performed with similar results. Data are shown as mean ± SEM of three individual mice. *, p < 0.05 comparing infected (Inf) and noninfected mice (Non-Inf).

 
Beside IL-10, TGF-β has been implicated in the immunosuppressive effect of Tregs (35). Because IL-10 expression was induced to similar levels in Tregs and nonregulatory T cells, TGF-β gene expression level was quantified in the CD4+Ly5.1+ Treg and the CD4+Ly5.2+ nonregulatory T cell fractions of mice reconstituted with or without Tregs. TGF-β mRNA expression was higher in Tregs than in nonregulatory T cells but was not affected by T. congolense infection (Fig. 9D). In CD4– cells, TGF-β mRNA expression tended to be induced only in the liver of infected mice, yet to similar level in mice reconstituted with and without Tregs (Fig. 9E). These data suggest that TGF-β is not the major Treg effector molecule in this infection model, although they do not exclude a role of TGF-β in African trypanosomiasis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Previous studies, as well as the present work, reveal that trypanotolerance requires a sequential switch from an IFN-{gamma}-dependent to an IL-10-dependent immune response. Suppression of the type 1 immune response, including the secretion of the trypanotoxic compound TNF-{alpha} by classically activated macrophages, once passed the first peak of parasitemia, is essential to preserve host tissues from injury and to favor the establishment of a chronic infection (18, 24, 34). Although alternatively activated macrophages have been proposed to contribute to the down-regulation of the type 1 immune response in trypanotolerant hosts (1, 36), the potential role of Tregs in this immune regulation is not known. Therefore, we have investigated whether Tregs influence the outcome of T. congolense infection in trypanotolerant C57BL/6 mice.

We have observed that CD4+Foxp3+ Tregs expand during the first month of infection in the spleen and the liver of T. congolense-infected animals, suggesting a role for these cells in the immunoregulatory network elicited during African trypanosomiasis. This expansion occurs (right) after the peak production of IFN-{gamma} and TNF-{alpha}, suggesting as reported (37), that the generation of Tregs and induction of their suppressive activity depend on their earlier activation by inflammatory signals (released by activated effector T cells or APCs).

If Tregs play a major role in trypanotolerance, one could expect that their expansion would be defective in trypanosusceptible BALB/c mice. We observed that the percentages of Foxp3+CD4+ T cells in T. congolense-infected BALB/c and C57BL/6 mice were increased to similar levels at day 9 postinfection (14.2 ± 1.6% vs 12.1 ± 1.3% in the spleen and 8.1 ± 1.4% vs 7.4 ± 1.1% in the liver of BALB/c vs C57BL/6 mice). This finding indicates that the susceptibility of BALB/c mice is not due to the absence of Tregs. However, as BALB/c mice do not control parasite growth, die within 10 ± 1 days postinfection, and thus do not evolve from an acute to a chronic stage of infection (11), a role for Tregs is not excluded in the chronic stage of T. congolense infection in C57BL/6 mice. Indeed, using a reconstitution model based on nu/nu mice, we have shown that Tregs limit the IFN-{gamma} production by CD4+ as well as CD8+ T cells, hereby circumventing prolonged TNF-{alpha} production by macrophages. This indicates that Tregs are key immune regulators during the chronic stage of African trypanosomiasis, and confirms that these cells can efficiently limit CD4+ and CD8+ type 1 immune effector responses (6, 8, 9). As such, Tregs may be the suppressive T cell population described in African trypanosome-infected hosts many years ago (16). In addition, CD4+Foxp3– cells producing high levels of IFN-{gamma} may represent the "pathogenic T cells" described by Shi et al. (38) in T. congolense-infected mice.

In contrast to what has been described in Leishmania infection, the absence of Tregs is not beneficial to the host during African trypanosomiasis. Indeed, the absence of Tregs in reconstituted mice does not lead to sterile immunity despite the longer (i.e., lasting during the chronic stage) type 1 immune response, which is known to be important for parasite control (6, 10). Similarly, the lower expansion of Tregs and prolonged type 1 immune response in the liver of IL-10 KO mice does not associate with enhanced parasite clearance. Thus, systemic extracellular African trypanosomes, at the difference of localized intracellular parasites, cannot be fully eliminated by increasing the type 1 immune response. However, the absence of Tregs in T. congolense-infected reconstituted mice dramatically exacerbates the immunopathological damages, leading to liver destruction. Similar pathological symptoms were observed in the livers of infected IL-10 KO animals. Moreover, in both experimental groups, the accumulated liver damages correlate with a sudden burst in parasitemia. Together, the present data sustain the idea that the liver is the main organ responsible for parasite elimination during African trypanosome infection (23). Therefore, limiting the type 1 inflammation by Tregs and/or IL-10 may delay the onset of immunopathology, protect the integrity of the liver, and ensure its long-lasting parasite clearance capacity, hereby favoring trypanotolerance.

Reconstitution experiments have shown that Tregs are essential for trypanotolerance. However, T. congolense-infected IL-10 KO mice that have low levels of Tregs exhibit a shorter survival than mice reconstituted without Tregs. Thus, the role of IL-10 in trypanotolerance goes beyond the sole activation of Treg suppressive activity. In this context, the prolonged survival of infected mice reconstituted without Tregs as compared with IL-10 KO mice may result from the IL-10 produced by cells other than Tregs. These include 1) CD4+Foxp3– T cells as evidenced in this study, that may encompass TR1 cells (39) and/or conventional Th1 cells similar to those described in Leishmania- and Toxoplasma-infected mice (40, 41), but also 2) CD4– cells possibly including macrophages (42) and/or regulatory B cells (43). Thus, Tregs and IL-10 seem to be two integrated but nonredundant elements of the immunoregulatory network required to avoid inflammation induced tissue damage and to favor trypanotolerance. The relative importance of IL-10 produced by Tregs and nonregulatory T cells during T. congolense infection could be addressed using wild-type or IL-10 KO nu/nu mice reconstituted with Tregs and conventional T cells from wild-type or IL-10 KO mice.

On the other hand, the secretion of IL-10 by spleen and liver cells from T. congolense-infected mice reconstituted with and without Tregs was similar. Thus, the nonregulatory T cell sources of IL-10 can compensate for the absence of naturally occurring Tregs in African trypanosome-infected hosts. However, this was not sufficient to suppress IFN-{gamma} production by effector T cells, suggesting that Treg function involves more than mere IL-10 production. TGF-β does not likely contribute to the suppressive activity of Tregs because mRNA expression was not induced upon T. congolense infection in that population. Other suppressive mechanisms used by Tregs include inhibitory receptors like CTLA-4 (44, 45), glucocorticoid-induced TNFR (46, 47), and LAG-3 (48) or the production of granzyme B (49).

Collectively, our data demonstrate that Foxp3+ Tregs are elicited during T. congolense infection. Although they limit the magnitude of effector type 1 immune responses against this parasite, they do not directly affect parasite control. Yet, Treg activity is beneficial to the host and contributes to trypanotolerance in this chronic and systemic infection by avoiding collateral tissue damage caused by a long-lasting type 1 immune environment.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported, in frame of an Interuniversity Attraction Pole Program, by grants from the Institute for Promotion of Innovation by Science and Technology in Flanders-Vlaanderen for Generisch Basisonderzoek aan de Universiteiten and by the fund for Scientific Research in Flanders-Vlaanderen. M.G., and G.O. are recipients of a fellowship from Scientific Research in Flanders-Vlaanderen, the Institute for Promotion of Innovation by Science and Technology in Flanders-Vlaanderen, and Fonds National de la Recherche Scientifique-Télévie. M.M. is a senior research associate from Fonds National de la Recherche Scientifique. Back

2 Address correspondence and reprint requests to Dr. Alain Beschin, Vlaams Instituut voor Biotechnologie, Department of Molecular and Cellular Interactions, Vrije Universiteit Brussel, Laboratory of Cellular and Molecular Immunology, 1050 Brussel, Belgium. E-mail address: abeschin{at}vub.ac.be Back

3 Abbreviations used in this paper: Treg, regulatory T cell; KO, knockout; ALT, alanine aminotransferase; AST, aspartate aminotransferase. Back

Received for publication June 20, 2007. Accepted for publication June 20, 2007.


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 Materials and Methods
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