Depletion of regulatory T cells decreases cardiac parasitosis and inflammation in experimental Chagas disease

Abstract

Infection with the protozoan parasite Trypanosoma cruzi may lead to a potentially fatal cardiomyopathy known as Chagas heart disease. This disease is characterized by infiltration of the myocardium by mononuclear cells, including CD4+ T cells, together with edema, myofibrillary destruction and fibrosis. A multifaceted systemic immune response develops that ultimately keeps parasitemia and tissue parasitosis low. T helper 1 and other pro-inflammatory T cell responses are effective at keeping levels of T. cruzi low in tissues and blood, but they may also lead to tissue inflammation when present chronically. The mechanism by which the inflammatory response is regulated in T. cruzi infected individuals is complex, and the specific roles that Th17 and T regulatory (Treg) cells may play in that regulation are beginning to be elucidated. In this study, we found that depletion of Treg cells in T. cruzi-infected mice leads to reduced cardiac parasitosis and inflammation, accompanied by an augmented Th1 response early in the course of infection. This is followed by a down-regulation of the Th1 response and increased Th17 response late in infection. The effect of Treg cell depletion on the Th1 and Th17 cells is not observed in mice immunized with T. cruzi in adjuvant. This suggests that Treg cells specifically regulate Th1 and Th17 cell responses during T. cruzi infection, and may also be important for modulating parasite clearance and inflammation in the myocardium of T. cruzi-infected individuals.

Introduction

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, constitutes a major public health burden in Latin America, and is the leading cause of infectious myocarditis in the world (Kirchhoff et al. 2004). Infected individuals may develop acute myocarditis characterized by myocyte destruction, myocardial lymphocytic infiltration and up-regulation of inflammatory mediators including cytokines, chemokines, and nitric oxide. Chronic Chagas heart disease is a cardiomyopathy resulting from longstanding inflammation and fibrosis and sustained expression of pro-inflammatory mediators (Huang et al. 1999; Machado et al. 2008; Parada et al. 1997; Rodriguez-Salas et al. 1998; Teixeira et al. 1978). Both acute and chronic Chagas heart disease may lead to heart failure and death (Tanowitz et al. 2009). The etiology of Chagas disease is multifaceted, and may involve an autoimmune component in addition to protracted damage to myocytes and cardiac neuronal and vascular networks caused by parasites persisting in host tissues (Bonney et al. 2011; Cummings and Tarleton 2003; Davila et al. 1989; Girones et al. 2007; Leon et al. 2004; Ribeiro dos Santos et al. 1992; Rossi 1990; Teixeira et al. 2011; Zhang and Tarleton 1999). Both cytotoxic CD8+ effector T lymphocytes and IFN-γ-producing CD4+ T cells (Th1 cells), are important for controlling T. cruzi parasitosis (Hoft et al. 2000; Hunter et al. 1997; Kumar and Tarleton 2001; Rodrigues et al. 2000; Tzelepis et al. 2007), yet these T cells may also contribute to the development of pathogenic inflammation during T. cruzi infection (Bonney et al. 2011; Gomes et al. 2003; Laucella et al. 2004; Minoprio 2001; Ribeiro dos Santos et al. 1992; Rizzo et al. 1989; Rocha Rodrigues et al. 2012; Soares et al. 2001; Tarleton et al. 1996; Tarleton et al. 1994). IL-17, which is produced by Th17 cells and other cell types, has been associated with both pro- and anti-inflammatory functions in other disease models, and may play an anti-inflammatory role during T. cruzi infection by indirectly down-regulating the functions of pro-inflammatory Th1 cells, without interfering with parasite clearance (Guedes et al. 2012; Soares et al. 2012; Tosello Boari et al. 2012). Conversely, anti-inflammatory cytokines produced by Th2 lymphocytes and other cell types, including IL-4, IL-10, and TGF-β, may help control inflammatory T cell responses and prevent secondary tissue damage during T. cruzi infection (Hunter et al. 1997; Jacysyn et al. 2003; Mariano et al. 2008; Soares et al. 2001). Despite this level of regulation, pathogenic inflammation often, but not always, develops and persists in T. cruzi infected individuals, leading to the question of what other cell types and cytokines may be involved in controlling the inflammatory response and pathogenesis induced during Chagas heart disease, as well as what is the possible cause of defective resolution of inflammation. Robust immune responses to both parasite antigens and host proteins, as well as indications of cardiac damage, have been widely observed following exposure to T. cruzi antigens, indicating that persistence of T. cruzi antigens or even DNA is sufficient to trigger some of these immune responses even in the absence of large numbers of live parasites (Bonney et al. 2011; Giordanengo et al. 2000; Leon et al. 2004; Motran et al. 2000; Schnapp et al. 2002; Sterin-Borda et al. 2003). Although the relevance of these autoimmune response to Chagas pathogenesis is unclear, this may help explain the lack of a clear direct correlation between disease severity and parasitemia in T. cruzi-infected individuals, especially during chronic infection.

CD4+CD25+Foxp3+ regulatory T (Treg) cells modulate pro-inflammatory immune responses to both infectious agents and self-antigens by suppressing proliferation and IFN-γ production by other subsets of CD4+ T cells, as well as CD8+ T cells (Belkaid et al. 2002; Miyara et al. 2011; Piccirillo and Shevach 2004; Shevach and Suri-Payer 1998; Zheng et al. 2009). However, the role of Tregs and the interplay between Tregs and other cells in naturally occurring T. cruzi infection is not clear (Araujo et al. 2007; de Araujo et al. 2012; de Araujo et al. 2011; Kotner and Tarleton 2007; Mariano et al. 2008; Sales et al. 2008; Sathler-Avelar et al. 2009; Vitelli-Avelar et al. 2006). T. cruzi infection has been associated with decreased frequency of Tregs in children with the indeterminate form of the disease compared to uninfected children, while adults with chronic Chagas disease have increased levels of Tregs circulating in their peripheral blood (Vitelli-Avelar et al. 2005; Vitelli-Avelar et al. 2006). Collectively, these findings suggest that Tregs may be down-regulated during acute T. cruzi infection, when parasitemia is highest, in order to promote the expansion of effector and helper T cells, and later up-regulated to limit tissue damage during chronic infection by controlling the magnitude of potentially harmful immune responses. This latter function would simultaneously allow a low level of prolonged parasite persistence by limiting the parasite-specific immune response, which is consistent with widely reported observations.

Several groups have utilized experimental models of Chagas disease to more closely examine the role of Tregs. A limited role for Tregs in controlling T. cruzi infection and subsequent development of cardiac pathology in mice has been reported by at least two independent groups, yet depletion of Tregs during the acute phase of T. cruzi infection caused increased expression of inflammatory mediators, more severe myocarditis, increased tissue parasitism and hastened mortality by a third group (Kotner and Tarleton 2007; Mariano et al. 2008; Sales et al. 2008). Recently, treatment of T. cruzi-infected mice with a non-depleting anti-CD25 monoclonal antibody was found to cause increased expression of inflammatory mediators, but this was accompanied by reduced parasitemia and decreased disease severity (Nihei et al. 2014). Anti-CD25 treatment had not previously been observed to significantly impact the development of myocarditis in T. cruzi–infected mice, even though Treg depletion with anti-GITR was shown to cause increased inflammation and parasitosis in the myocardium (Mariano et al. 2008). As an alternative approach, the effect of Treg recruitment on the pathogenesis of experimental Chagas disease was examined using treatment with granulocyte colony-stimulatory factor, and found to reduce both parasite load and the severity of myocarditis (Vasconcelos et al. 2013). Transfer of Tregs from mice immunized with a recombinant T. cruzi protein (rSSP4) into mice that were infected with T. cruzi reduced cardiac inflammation and prolonged survival, but increased blood parasitemia and cardiac parasitosis (Flores-Garcia et al. 2013). Variable results have been reported for the effect of Treg cell depletion on serum and cardiac tissue cytokine levels, although no significant effect of Treg depletion on the expansion or function of CD8+ effector T cells or antibody production in T. cruzi-infected mice has been reported (Couper et al. 2007; Fontenot et al. 2005; Hori et al. 2003; Kohm et al. 2006; Kotner and Tarleton 2007; Liu et al. ; Mariano et al. 2008; Sales et al. 2008; Shevach et al. 2003; von Boehmer 2005).

A number of questions regarding the role of Treg cells in T. cruzi infection remain unanswered, including what effect depletion of these cells has on the development of cardiac damage and potentially pathogenic Th1 and Th17 cell, independent of damage caused by live parasites. Interplay between these two cell populations may be influential in determining disease outcomes, as they cell types have been widely associated with both protective immunity against pathogens and the development of deleterious immune responses, including autoimmunity. To test the hypothesis that Treg cells indirectly control cardiac parasitism and inflammation in T. cruzi-infected mice by modulating Th1 and Th17 cell responses, we functionally depleted CD4+CD25+ Treg cells using anti-CD25 antibodies prior to and during infection, and assessed the development of Th1 and Th17 cells responses and cardiac pathology. We similarly depleted Treg cells in mice immunized with T. cruzi antigens in the form of heat-killed T. cruzi (HKTC) to test the hypothesis that Treg cell responses are necessary for preventing the development of pathogenic inflammation driven by Th1 and Th17 cell responses triggered by T. cruzi antigens, independent of other damage incurred during active infection. This line of investigation followed up on our earlier work in which we found that immunization with HKTC induces cardiac damage in mice, as evidenced by an increase serum cardiac troponin I, accompanied by the development of T and B cell responses with similar antigen-specificity as the immune response that develops during T. cruzi infection, but lacking (Bonney and Engman 2008) the robust Th1 and Th17 responses observed during T. cruzi infection (Bonney and Engman 2008).

Here we report new findings that depletion of Treg cells with anti-CD25 antibody leads to a reduction in myocardial parasitosis and inflammation in T. cruzi infected mice, as well as down-regulation of Th1 cells and an up-regulation of Th17 cells. Although immunization with T. cruzi antigens has been previously shown to induce cardiac damage and polyantigenic immunity, Treg cell depletion had no significant effect on the development of myocarditis or Th17 cell responses in immunized mice. These results provide evidence that Treg cells are important for controlling cardiac damage during T. cruzi infection likely by modulating pro-inflammatory Th1 and Th17 cell responses.

Materials and methods

Experimental animals, infections and immunizations

Male A/J mice (Jackson Laboratories, Bar Harbor, ME) were 4 to 6 wk of age upon initiation of experiments. Mice were anesthetized by a single intraperitoneal (i.p.) injection of Avertin®, 240mg/kg of body weight for each experimental manipulation. A cardiotropic substrain of the Brazil strain of T. cruzi (Brazil Heart) generated in our laboratory (Hyland et al. 2007) was propagated in H9C2 rat myoblasts to generate trypomastigotes for infection. Mice were infected by i.p. injection of 1 × 10⁴ Brazil heart strain trypomastigotes in Dulbecco's phosphate-buffered saline (PBS; GibcoBRL, Grand Island, NY) or injected with PBS alone. Mice were immunized with HKTC or PBS in an emulsion of CFA in a 1:1 ratio. A total volume of 150 μl was distributed in three subcutaneous injections in the dorsal flank. Seven days later, mice were given a boost immunization in an identical manner. Mice were housed in pathogen-free animal facilities, and the use and care of mice were in accordance with the guidelines of the Center for Comparative Medicine at Northwestern University.

Preparation of antigens

HKTC was prepared by washing T. cruzi epimastigotes or tissue culture trypomastigotes three times in PBS, then resuspending in PBS at a concentration of 6.67 × 10⁸ parasites/ml unless otherwise noted, and incubating in an 80°C waterbath for 10 minutes. Complete loss of viability was verified by culture of treated parasites for 30 days in both trypomastigote and epimastigote culture medium (DMEM and a liver digest-neutralized, tryptose medium, LDNT). Parasite antigen preparations were stored at −20°C until use.

Histopathology

Hearts were removed, washed in PBS, and fixed in 10% buffered formalin for at least 24 hours. Fixed hearts were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined by light microscopy. Each section was examined microscopically for evidence of inflammation (mononuclear and polymorphonuclear cellular infiltration; fibrosis; edema) and was assigned a score between 0 and 4, with 0 representing no evidence of inflammation; 1 representing evidence of inflammation in 1-25% of the cardiac sections examined; and 2, 3, and 4 representing inflammation in 26-50%, 51-75%, and 76-100% of cardiac sections, respectively, as previously described (Godsel et al. 2003; Leon et al. 2003). Six sections per heart were analyzed at 20X magnification to determine the histopathological score. To quantify the number of amastigote nests in the cardiac tissue, the heart was sectioned transversely and at least three hematoxylin and eosin-stained sections cut 25 to 150 μm apart about the center of the heart were scanned in entirety under 40X magnification while nests were manually counted. The mean number of nests per section of each heart was used for group analysis. All histologic assessment was conducted blindly on coded slides.

In vivo Treg depletion with anti-CD25 antibodies

Purified anti-CD25 (PC-61.5.3) was purchased from BioXCell (West Lebanon, NH). Mice were depleted of CD25+ cells by i.p. injection of 400 μg antibody diluted in sterile PBS at -3, 0, 4, and 8 d.p.i. Controls for each treatment received matching doses of normal rat IgG1.

Intracellular cytokine staining and flow cytometric analysis

RBC-free single cell suspensions were prepared from mouse spleens as described previously (Daniels et al. 2008) or isolated from cardiac tissue using the Miltenyi Biotec gentleMACS Dissociator and manufacturer's protocol. Briefly, hearts were rinsed with Hanks's Buffered Salt Solution (HBSS), quartered, and incubated with collaganase II and DNase I. The mixture was then mechanically dissociated using the gentleMACS Dissociator, incubated for 30 minutes at 37°C, and mechanically dissociated for a second time to produce a single cell suspension that was then processed using the same RBS lysis, rinsing, and counting procedures as described for splenocytes in the aforementioned source. The cells were washed and resuspended in complete DMEM. Cells were plated at 5 × 10⁵ cells per well and cultured in 96-well plates for 72 hours while being stimulated with anti-CD3 and anti-CD28 (Ebioscience) or BSA (Sigma-Aldrich). For intracellular cytokine staining, cells were washed and re-stimulated with 5 ng/ml phorbol 12-myristate 13-acetate (Sigma-Aldrich) and 500 ng/ml ionomycin (Fisher Scientific) for 3 hours, then with 1:1000 Golgi Plug and 1:1000 Golgi Stop (BD Biosciences) for 3 hours. Cells were washed and blocked for 10 minutes with 1:100 anti-CD16/32 (Ebioscience) then stained with anti-CD90-PE-Cy7 and anti-CD4-PE-Cy5.5 (Ebioscience) for 20 minutes and washed. Cells were then fixed with 4% paraformaldehyde at 4°C for 20 minutes and permeabilized using the Perm/Wash buffer from the BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit (BD Biosciences). Intracellular cytokine staining was performed using anti-IFN-γ-FITC and anti-IL-17-PE (BD Biosciences). For staining of Tregs, the Ebioscience Mouse Regulatory T Cell Staining Kit #1 was used, following the manufacturer's protocol to stain for CD4, CD25, and Foxp3. Flow cytometry was performed using an LSRII flow cytometer with FACS Diva software (Becton Dickinson, Mountain View, CA), and analyzed with FlowJo 7.6 Software.

Statistical analysis

For comparison of two groups, significance was determined by Student's t test. For comparison of multiple groups and a control, the significance was assessed by one-way analysis of variance, followed by adjustment for multiple comparisons by the Dunnett test (post hoc analysis). The control group for comparison is specified in each figure legend. P values of <0.05 were considered significant.

Results

Anti-CD25 treatment depletes Treg cells from T. cruzi–infected and HKTC-immunized mice

Depletion of Treg cells was accomplished prior to exposure to infection with live or immunization with heat-killed T. cruzi. Mice were administered anti-CD25 every four days, with the first two treatments given prior to infection or immunization and two more treatments given at four and eight days after initial infection or immunization. Treg depletion was assess by analyzing splenic CD4+ T cell populations via flow cytometry for expression of CD25 and Foxp3 at 10 d.p.i., the midpoint of the infection course, and at 21 d.p.i., when infected mice experience peak cardiac inflammation and parasitosis. At 10 d.p.i., a greater than 40-fold reduction in CD4+CD25+Foxp3+Treg cells was observed in T. cruzi-infected and HKTC-immunized mice (Fig. 1). Although there was partial recovery of this cell population by 21 d.p.i., a significant reduction of Treg cells was maintained after the cessation of anti-CD25 treatment (Fig. 1).

Fig. 1.

Anti-CD25 treatment results in sustained reduction of CD4+CD25+Foxp3+ cells. Mice were infected with T. cruzi (INF), or immunized with HKTC or PBS in CFA, and treated with IgG or anti-CD25 (PC61). At 10 d.p.i. and 21 d.p.i., splenocytes were isolated and analyzed for expression of CD4, CD25, and Foxp3. (A) Representative flow plots of CD4+ cells gated for Foxp3 and CD25 expression are shown. (B) The mean percentages of CD4+ cells that were also CD25+Foxp3+ are shown for the three groups of mice at 10 and 21 d.p.i. Error bars indicate SEM (n=5 for INF and HKTC; n=3 for PBS). Data represent two independent experiments. * P < 0.05.

To address whether anti-CD25 treatment effectively eliminates Treg cell populations or leaves them extant but functionally inactive due to down-regulation of CD25, we specifically assessed the effect of treatment on the numbers of CD4+CD25+ and CD4+Foxp3+ cells recovered (Fig. 2). A significant reduction of CD4+CD25+ cells was observed in T. cruzi-infected and HKTC-immunized mice at both 10 d.p.i. and 21 d.p.i. (Fig. 2A). A reduction in the proportion of CD4+ cells that was Foxp3+ was also observed in both groups at 10 d.p.i.; however, by 21 d.p.i. there was an approximately two-fold increase in the percent of CD4+Foxp3+ in T. cruzi-infected mice (Fig. 2B). These data indicate that down-regulation of CD25 expression, which has been demonstrated to be necessary for Treg function, was maintained throughout the course of this experiment, even though there was an increase in the proportion of CD4+Foxp3+ cells in T. cruzi-infected mice during the course of infection.

Fig. 2.

Anti-CD25 treatment causes sustained down-regulation of CD25 on CD4+ cells and depletion of CD4+ Foxp3+ cells. Mice were infected with T. cruzi (INF), or immunized with HKTC or PBS in CFA, and treated with IgG or anti-CD25 (PC61). At 10 d.p.i. and 21 d.p.i., splenocytes were isolated and analyzed for expression of CD4, CD25, and Foxp3. The mean percentage of (A) CD4+CD25+ and (B) CD4+Foxp3+ cells is shown. Error bars indicate SEM (n=5 for INF and HKTC; n=3 for PBS). Data represent two independent experiments. * P < 0.05.

Depletion of Treg cells reduces myocardial parasitosis and inflammation in T. cruzi–infected mice

To determine the role of Treg cells in controlling the development of cardiac inflammation and parasitosis, we infected mice with T. cruzi for 10 or 21 days and treated with control IgG or anti-CD25, then assessed cardiac sections for signs of histopathology (Fig. 3). At 10 d.p.i., no or very little cardiac parasitosis (Fig. 3A) and low levels of myocarditis (Fig. 3B) were observed in both IgG and anti-CD25 treated groups, and there was no significant difference between the two groups. By 21 d.p.i., however, Treg cell-depleted mice had significantly lower cardiac parasitosis (Fig. 3A) than control treated mice, as well as lower levels of cardiac inflammation (Fig. 3B).

Fig. 3.

Depletion of Treg cells reduces myocardial parasitosis and inflammation in T. cruzi–infected mice. Mice were infected with T. cruzi, or immunized with HKTC or PBS for 10 or 21 days and treated with IgG (open circles) or anti-CD25 (PC61, filled circles); only data for infected groups are shown because all other mice showed no signs of cardiac inflammation or parasitosis. Hematoxylin and eosin-stained cardiac sections were examined for (A) parasite pseudocysts or (B) inflammation. The degree of inflammation was scored based on a scale of 0 to 4 where 0 represents <1% of heart affected by inflammatory infiltrate, 1 = 1-25%, 2 = 26-50%, 3 = 51-75%, and 4 = 76-100%.

We previously found that immunization with HKTC in the absence of live T. cruzi is sufficient to induce acute cardiac damage and humoral and cell-mediated immune responses specific for a number of T. cruzi and cardiac antigens, but immunization with HKTC alone is not sufficient to induce the significant myocarditis that is observed following T. cruzi infection (Bonney et al. 2013; Bonney et al. 2011). Therefore, we also wanted to test the hypothesis that induction of a robust regulatory T cell response is necessary to limit the potentially harmful expansion of inflammatory mediators such as Th1 and Th17 cells induced by exposure to T. cruzi antigens, and that HKTC-immunized mice would develop myocarditis following Treg cell depletion. Mice were immunized with HKTC or PBS, treated with control IgG or anti-CD25, and development of myocarditis was assessed histologically. None of the mice developed significant myocarditis by 10 or 21 d.p.i. (data not shown), indicating that some factor that is independent of the normal Treg cell response is important for preventing HKTC-induced polyantigenic immune responses and cardiomyocyte damage from progressing into myocarditis. The lack of detectable cardiac injury or inflammation in the PBS-immunized control group also indicates that use of the adjuvant CFA to promote antigen uptake and presentation by macrophages does not result in non-specific cardiac damage or inflammation, and suggests that any immunopathology observed in HKTC-immunized mice is triggered by antigen exposure.

Depletion of Treg cells leads to down-regulation of Th1 cells and up-regulation of Th17 cells in T. cruzi-infected mice

To test the hypothesis that Treg cell depletion affects Th1 and Th17 cell response during T. cruzi infection, mice were treated with IgG or anti-CD25 and infected with T. cruzi. At 10 and 21 d.p.i., CD4+ splenocytes were isolated and analyzed via flow cytometry for expression of IFN-γ and IL-17, as indicators of Th1 and Th17 cells, respectively (Fig. 4). By 10 d.p.i., both the proportion of CD4+ cells identified as Th1 cells and the total number of Th1 cells were significantly higher in T. cruzi-infected that had been depleted of Treg cells than in infected mice that had been treated with control IgG (Fig. 4A). A statistically significant elevation in the total number of Th17 cells in infected mice that had been depleted of Treg cells compared to control mice was also observed (Fig. 4A). By 21 d.p.i., the proportion of CD4+ cells identified as Th17 cells in T. cruzi-infected mice that had been depleted of Treg cells had increased dramatically and was significantly higher than in mice treated with control IgG (Fig. 4B and C). Conversely, T. cruzi infected mice that had been depleted of Treg cells had significantly fewer Th1 cells than those in the control group (Fig. 4B).

Fig. 4.

Depletion of Tregs leads to differential modulation of Th1 and Th17 responses in HKTC-immunized and T. cruzi–infected mice. Mice were infected with T. cruzi (INF), or immunized with HKTC or PBS in CFA, and treated with IgG (solid bars) or anti-CD25 (PC61). At 10 or 21 d.p.i., splenocytes were isolated and analyzed for expression of CD4, IFN-γ and IL-17 via flow cytometry. The mean percentage of cytokine-positive and total numbers of cells expressing cytokine are shown for (A) 10 d.p.i. and (B) 21 d.p.i. Error bars indicate SEM (n=5 for INF and HKTC; n=3 for PBS). Data represent two independent experiments. * P < 0.05. (C) Representative flow plots depicting IFN-γ and IL-17 expression at 21 d.p.i. are shown (quantified in the bottom of Panel of A).

Previously we reported that HKTC immunization induces acute cardiac damage in mice, as evidenced by an increase in serum cardiac troponin I, as well as polyantigenic T and B cell responses specific for a similar panel of parasite and host antigens as those observed in T. cruzi -infected mice. However, HKTC-immunized mice did not develop robust Th1 immunity or histological indications of myocarditis, leading us to propose that exposure to T. cruzi antigens, such as in the form of HKTC, may be sufficient to induce myocarditis if presented in the context of a Th1-dominant immune response, such as that which naturally exists during active T. cruzi infection. Here we tested the hypothesis that Treg cell responses are necessary for preventing immunization with HKTC from inducing robust Th1 immunity leading to pathogenic cardiac inflammation. HKTC-immunized were treated with anti-CD25 antibodies and assessed for the development of myocarditis and Th1 and Th17 cell responses following the same protocol described for T. cruzi -infected mice. No significant differences in Th1 or Th17 cell responses between Treg cell-depleted mice and mice treated with control IgG were observed in HKTC-immunized or control mice immunized with PBS, with the exception of a modest but statistically significant increase in the proportion of Th1 cells observed at 21 d.p.i. in Treg-depleted mice immunized with HKTC (Fig. 4). This did not correlate to a significant elevation in the total number of Th1 cells in these groups, yet this result is interesting because it contrasts with the significantly lower proportion of Th1 cells observed in T. cruzi infected mice that were depleted of Tregs compared to those that were treated with control IgG (Fig. 4B and C). This indicates that the magnitude and temporal progression of the Th1 response may determine whether T. cruzi-induced immunity contributes to the development of myocarditis.

Discussion

Treg cells are known to prevent protective pro-inflammatory immune responses initiated against pathogens or host antigens from over-expanding and causing deleterious bystander tissue damage (Belkaid et al. 2002; Piccirillo et al. 2008; Yamaguchi and Sakaguchi 2006). Treg cells have an important and complex relationship with Th1 and Th17 cells, as Treg cells and Th17 cells develop from naïve CD4+ T cells under the influences of some of the same factors, yet have largely opposing functions, and both modulate the function of Th1 cells during the development of potentially pathogenic inflammatory responses(Le Cabec et al. 2005; Xu et al. 2007) (Dardalhon et al. 2008; Le Cabec et al. 2005; Li et al. 2007; Speert and Silverstein 1985; Xu et al. 2007).

IFN-γ production by Th1 cells and other cell types is crucial for control of T. cruzi parasitosis, but Th1 cells are also linked to the development of a number of inflammatory and autoimmune diseases (Bonney et al. 2011; Gomes et al. 2003; Hoft et al. 2000; Hunter et al. 1997; Kumar and Tarleton 2001; Laucella et al. 2004; Minoprio 2001; Ribeiro dos Santos et al. 1992; Rizzo et al. 1989; Rodrigues et al. 2000; Soares et al. 2001; Tarleton et al. 1996; Tarleton et al. 1994; Tzelepis et al. 2007). Similarly, Th17 cells have been linked to the pathogenesis of a number of inflammatory and autoimmune diseases, yet there is evidence that IL-17 plays an anti-inflammatory role during T. cruzi infection by indirectly down-regulating the functions of pro-inflammatory Th1 cells (Cobb and Smeltz 2012; Cooke 2006; Da Matta Guedes et al. ; Daniels et al. 2008; Dardalhon et al. 2008; Valaperti et al. 2008).

The current body of literature reports mixed findings regarding the extent to which Treg cells are responsible for controlling T. cruzi infection and concomitant inflammatory T cell responses. At least one group has reported no effect of Treg cell depletion on mortality, parasite clearance, production of pro-inflammatory cytokines, or development of myocarditis in T. cruzi-infected mice, while at least one other group has demonstrated that Treg cell depletion leads to reduction of survival, down-regulation of certain cytokines, and, with the use of a certain Treg cell agonist, increased cardiac parasitosis and inflammation (Kotner and Tarleton 2007; Mariano et al. 2008; Sales et al. 2008). Anti-CD25 treatment has not previously been observed to significantly impact the development of myocarditis in T. cruzi-infected mice; however, Treg depletion with anti-GITR causes increased inflammation and parasitosis in the myocardium, indicating that the method or efficacy of Treg depletion may affect disease outcome (Mariano et al. 2008). Due to the lack of consensus in earlier reports, a number of questions regarding the role of Treg cells in T. cruzi infection remain unanswered, including what effect depletion of these cells has on the development of Th1 and Th17 cell responses, and how these responses modulate Chagas disease pathogenesis independent of the cellular damage caused by live parasites.

In this study, depletion of Treg cells led to decreased cardiac parasitosis and inflammation in T. cruzi–infected mice. These findings indicate that Treg cells are important for controlling the inflammatory and parasite-specific immune responses generated in response to T. cruzi infection. We hypothesized that Treg cells indirectly control pathogenesis during T. cruzi infection by modulating Th1 and Th17 responses, and that efficacious depletion of Treg cells would have a significant impact on both pathogenesis and the development of Th1 and Th17 cell responses. At an early point in the course of T. cruzi infection, mice undergoing active Treg cell depletion developed a significantly increased Th1 cell response compared to control mice, accompanied by a smaller, but significant increase in the number of Th17 cells. Late in the course of infection, however, T. cruzi–infected mice that had received anti-CD25 developed a markedly reduced Th1 response, which was accompanied by an increase in the percentage of Th17 cells. This suggests that, Treg cells control the development of Th cell responses during T. cruzi infection and that, in the absence of Treg cell-mediated immunoregulation, the Th1 population expands early on, resulting in more effective early parasite clearance and a reduction in cardiac parasitosis. Decreased Treg cell-mediated regulation also results in increased Th17 cell responses later in the course of infection, which may act to further down-regulate inflammatory Th1 responses, resulting in a reduction of cardiac inflammation. The reduction in cardiac inflammation could also be explained by a secondary effect in which reduced cardiac parasitosis leads to less parasite-induced tissue injury and decreased infiltration of parasite-specific lymphocytes into the myocardium. Because it has been reported that a combination of IL-12 and IFN-γ signaling can convert Th17 cells into a subset of cells termed Th1/Th17, which produce both IFN-γ and IL-17, we examined the possibility that the increase in Th17 cells observed in Treg-depleted T. cruzi-infected mice could be completely or partially attributed to development of a population of Th1/Th17 cells (Speert and Silverstein 1985). However, no notable increase in CD4+ cells co-expressing IFN-γ and IL-17 was observed (Fig. 4C).

In conclusion, our findings provide evidence that Treg cells are, in fact, important for controlling parasite clearance and inflammation during T. cruzi infection. In the future we plan on utilizing more quantitative methods such as qPCR to determine the effect of Treg depletion on parasite levels in cardiac and other tissues throughout the course of infection, and to examine changes in Treg, Th1, and Th17 cell populations at more time points to give a more complete picture of the temporal effects of Treg cell depletion. What role, if any, Treg cells have in controlling the development of potentially pathogenic autoreactive T and B cell responses following T. cruzi infection or exposure to T. cruzi antigens, which has been reported by a number of groups, also remains to be elucidated (Bonney and Engman 2008; Bonney et al. 2011; Ribeiro dos Santos et al. 1992; Santos-Buch and Teixeira 1974; Soares et al. 2001; Tarleton et al. 1997). Thorough understanding of how to safely promote the development of protective immunity will be crucial to the success of ongoing efforts to develop vaccines against T. cruzi, and vaccination protocols and antigenic targets may have to be specifically engineered to minimize inadvertently disrupting the balance of Treg cell responses.