Is the adaptive immune response in murine Trypanosoma cruzi infection influenced by zinc supplementation?

European Journal of Pharmaceutical Sciences 111 (2018) 330–336

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Is the adaptive immune response in murine Trypanosoma cruzi infection influenced by zinc supplementation?

MARK

Cássia Mariana Bronzon da Costa, Marina Del Vecchio Filipin, Fabrícia Helena Santello, Luiz Miguel Pereira, Miriam Paula Alonso Toldo, José Clóvis do Prado Júnior, Ana Amélia Carraro Abrahão⁎ School of Pharmaceutical Sciences of Ribeirão Preto (FCFRP), University of São Paulo, Av. do Café s/n, 14040-903, Ribeirão Preto, São Paulo, Brazil

A R T I C L E I N F O

A B S T R A C T

Keywords: Chagas disease Zinc Immune response Rat Pregnancy

Chagas disease afflicts 7 to 8 million people worldwide and congenital Chagas' disease usually leads to changes in the maternal environment, culminating in fetal adaptations. Several articles have described the importance of micronutrients on pregnancy, which is sensitive to infections. In Trypanosoma cruzi endemic regions, the Chagas disease is aggravated by the lack of micronutrients in an average diet, to which pregnant women are more susceptible. The aim of this study was to evaluate distinct T cells phenotypes and intracellular cytokines by flow cytometry in pregnant Wistar rats under zinc therapy during experimental Chagas' disease. Twenty female Wistar rats were infected with 1 × 105 blood trypomastigotes (Y strain) and 30 days after infection the animals were mated and grouped: pregnant infected (PI–n = 5), pregnant infected/zinc supplied (PIZ–n = 5), pregnant control (PC–n = 5), control/zinc supplied (PCZ–n = 5). Zinc supplementation: 20 mg of zinc/Kg/day (gavage) for 18 days followed by euthanasia. The immune parameters showed: decreased percentages of CD62LlowCD44high surface marker for infected and treated group (PIZ) when compared to PI (p < 0.05). Concerning to T regulatory cells (Treg cells), a significantly lower percentage of splenic Treg cells was found in the infected and treated group (PIZ) as compared to the PI group (p < 0.05). The expression of the co-stimulatory molecule CD28+ displayed a significant reduced percentage in TCD8+ for infected and zinc treated group (PIZ) as compared to (PI). The percentages of CD4+/CD11a+ T cells subsets were lower on PIZ as compared to PI. Concerning to CD45RA+ (B lymphocytes) analysis, infected pregnant and treated group (PIZ) showed a significant decrease in CD45RA percentage when compared to (PI) (p < 0.05). The intracellular cytokine profiles for TCD4+ and TCD8+ producing IL-4 and IFN-γ revealed that zinc treated and untreated infected pregnant group (PI and PIZ) displayed increased cytokines concentrations as compared to zinc treated and untreated pregnant controls (PC and PCZ). Our data revealed the involvement of zinc as a signaling molecule in the modulation of the inflammatory process and immune response which occurs during pregnancy of T. cruzi infected rats. Zinc acted in a dual fashion, modulating the host's immune response in a way to protect the organism against the deleterious effects of the infection and an overwhelming pro-inflammatory response during pregnancy.

1. Introduction Chagas disease is a potentially life-threatening illness caused by the protozoan parasite Trypanosoma cruzi. About 6 million to 7 million people worldwide are estimated to be infected with T. cruzi. In Latin America vector-borne transmission is the main way of infection where humans come in contact with faeces or urine of triatomine bugs (Steverding, 2014). The importance of micronutrients in human health is unquestionable and zinc (Zn) is an essential trace element with a wide range of actions such as antioxidant activity, cellular metabolism ⁎

and as a component of proteins involved in cell structures. It also plays an important role in immune function, protein and DNA synthesis, cell division, energy metabolism and growth (Bonaventura et al., 2015; Yasuno et al., 2011). The immunomodulatory role of zinc during pregnancy in the acute phase of Chagas' disease has been demonstrated by our group (da Costa et al., 2013). Although pregnancy is a normal physiological state, the maternal environment depends on an adequate nutrition, and is closely related to an appropriate fetal growth and development (King, 2006). Maternal nutritional deficiency is associated with poor pregnancy outcomes, including low birth weight, fetal

Corresponding author at: Department of Clinical Analysis, Toxicology and Food Science, Brazil. E-mail address: [email protected] (A.A.C. Abrahão).

http://dx.doi.org/10.1016/j.ejps.2017.10.014 Received 30 January 2017; Received in revised form 9 October 2017; Accepted 10 October 2017 Available online 12 October 2017 0928-0987/ © 2017 Elsevier B.V. All rights reserved.

European Journal of Pharmaceutical Sciences 111 (2018) 330–336

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2.3. Treatment schedule

growth restriction and prematurity (Black et al., 2008; Christian, 2010). (Beach et al., 1982) described that marginal zinc deficiency in mice during gestation exerted both short- and long-term detrimental effects in their offspring, with reduced lymphoid organ size and immunoglobulin concentrations. (Dardenne, 2002) studied zinc-deficient mice and showed that several immuno-deficiencies observed at birth persisted through adulthood, even when offspring were fed with a zincadequate diet. Depending on the different sub-populations of T lymphocytes a distinct cytokine profile is present leading to a successful pregnancy or abortion. T helper type 1 lymphocytes (Th1) produce interleukin-2 (IL-2), interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), which is a characteristic pro-inflammatory response, enhancing host's immunity against alloantigen. T helper type 2 lymphocytes (Th2) cells produce IL-3, IL-4 and IL-10, promoting the production of antibodies with anti-cytotoxic activity (Carlier et al., 2011). Transmitting chagasic mothers have a higher parasite load, reduced ability to produce IFN-γ, IL-2 and TNF-α by T cells, although they are able to synthesize enhanced levels of IL-10. On the other hand, nontransmitting chagasic mothers show high monocyte activation and enhanced concentrations of circulating TNF-α (Alonso-Vega et al., 2005; Carlier, 2005). During pregnancy, several factors, including regulatory T cells, are involved in the physiological changes responsible for maternal-fetal tolerance (Leber et al., 2010). The importance of cytotoxic CD8+ T cells (CTLs) in host defense against T. cruzi has already been described (Padilla et al., 2009). Several studies using different experimental models for Chagas' disease described that these cells also play an essential role enhancing effectors mechanisms of the immune response such as activating phagocytes, cytotoxic CD8+ T lymphocytes and the production of cytokines (Martin and Tarleton, 2005). Zinc supplementation has a positive effect on immune system of male rats during Chagas disease chronic phase (Brazão et al., 2011). Since pregnancy displays a distinct immune response, we investigated the immunomodulatory role of this element during the chronic phase of T. cruzi infection in pregnant Wistar rats. The aims of this work were to evaluate the importance of zinc as a possible immunomodulatory element in order to improve the homeostasis during pregnancy as well as the rat's immune response against T. cruzi. For that the phenotypic profile of distinct T cell subsets involved in the adaptive immune response during pregnancy associated with zinc supplementation was evaluated.

Zinc-treated groups were orally administered with zinc sulfate (via gavage) (Sigma Chemical Co.MO, USA) dissolved in 0.1 ml of distilled water at a dose of 20 mg/kg body weight (da Costa et al., 2013), once a day, starting the day after the pregnancy confirmation until the end of the experiment. 2.4. Euthanasia Each animal was anesthetized with 2.5% tribromoethanol (SigmaAldrich, USA) administered intraperitoneally (dose of 25 mg/kg/animal) and later euthanized by decapitation. Blood samples were collected to obtain plasma and serum. 2.5. Splenic and peritoneal cell suspension After euthanasia, cells were harvested from peritoneal cavity and washed with RPMI 1640 medium (Sigma-Aldrich, USA) and resuspended (2 × 107 cells/ml). Spleens were aseptically removed and the splenic tissue was mechanically disaggregated by extrusion through a 100 μm nylon cell strainer (Falcon, USA) and washed in a hypotonic buffer (160 mM NH4Cl, 10 mM Tris-HCl, pH = 7.4) for red blood cell lysis. Cells were homogenized in RPMI 1640 medium to produce a single cell suspension and the concentration was adjusted to 2 × 107 cells/ml. Splenic and peritoneal cell viability was assessed by 0.4% trypan blue solution (Sigma-Aldrich, USA) (da Costa et al., 2017). 2.6. Cell surface phenotype analysis The cells (2 × 106 cells/well) were resuspended in staining buffer (BSA) (BD-Pharmingen, San Diego, USA) and dispensed into 12 × 75 mm round-bottomed polystyrene tubes (Falcon, USA) for cytofluorometric analysis. Following Fc receptor blocking (anti-CD32), cells were incubated with specific monoclonal antibodies for 30 min at 4 °C in the dark (da Costa et al., 2017). Fluorescence analysis was performed using a FACS Canto flow cytometer (BD Biosciences, California, USA) and the percentages of positive cells for each labeling were determined using the FACSDiva software. All conjugated monoclonal antibodies were obtained from BD Biosciences Pharmingen (CA, USA). Peritoneal cells phenotype and splenic T-cell subsets were identified according to the surface expression of the following CD markers: antiTCD3+ (APC), anti-TCD4+ (PE-Cy7), anti-TCD8+ (PerCP), anti-TCD62L (PE), anti-TCD44 (FITC), anti-TCD4+/TCD28+ (PE-Cy7/PE) anti-TCD8+/TCD28 (PerCP/PE), anti-TCD4+/CD11a+ (PE-Cy7/FITC), anti-TCD8+/CD11a+ (PerCP/FITC). For regulatory T cells analysis, membranes were marked with monoclonal antibodies anti-CD3 (FITC), anti-CD4 (PE-Cy7), and anti-CD25 (PE). Cells were also permeabilized and marked with an antibody to the forkhead box transcription factor (FoxP3) labeled with Alexa Fluor 647 (Biolegend, Inc. San Diego, USA).

2. Material and methods 2.1. Animals Four-week old female Wistar rats (100 g) from Facility House of the São Paulo University Campus of Ribeirão Preto were used. Animals were randomized into the following groups: pregnant control (PC), pregnant control with zinc supplementation (PCZ), pregnant infected (PI), pregnant infected supplied with zinc (PIZ). The experimental protocols were approved by the local Ethics Committee (Protocol No 11.1.1210.53.2).

2.7. CFSE labeling The CFSE labeling was performed using Cell Trace CFSE Cell Proliferation kit, for flow cytometry (Molecular Probes, Life Technologies USA). Briefly, splenocytes cells were washed in warm phosphate-buffered saline (PBS) and labeled with CFSE (carboxyfluorescein succinimidyl ester). The samples were cultured at 2 × 106/ ml cells in RPMI1640 culture medium with or without Concanavalin A 5 μg/ml (ConA, Sigma-Aldrich, USA) for 10 min at 37 °C. Dye was then quenched by washing in ice-cold RPMI-1640 medium (Sigma Aldrich, USA). Splenocytes were cultured in a humidified atmosphere of 5% CO2 at 37 °C for 5 days. Cells were collected and incubated with monoclonal antibody, stained with anti-CD3-Allophycocyanin (APC) monoclonal antibody. The data were acquired by gating on CD3+ T cell population, assessed by flow cytometry using FACSCan and FACSDiva software (BD

2.2. Experimental infection/pregnancy Each group (n = 5) was inoculated intraperitoneally (i.p.) with 1 × 105 trypomastigote forms of the Y strain of T. cruzi. Thirty days post infection one male Wistar rat was introduced into each cage and was allowed to mate with the females. Pregnancy confirmation was made by the vaginal plug. The assays were performed on day 18 of gestation, corresponding to 48th day of T. cruzi infection (chronic phase).

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Biosciences). The sequential loss of CFSE fluorescence (CFSElow) was used to measure cell division.

%CD4+CD62Llow CD44high

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2.8. Intracellular cytokine staining Splenocytes were placed in 96-well plates (Corning Inc., USA) and stimulated, in vitro, using ionomycin 500 ng/ml (Sigma-Aldrich, USA) and phorbol 12-myristate 13-acetate 50 ng/ml (PMA). The cells were incubated for 4 h at 37 °C in 5% CO2. After, cells were cultured in the presence of brefeldin A (10 μg/ml), which has an inhibitory effect on protein secretion for 2 h. Cells were washed in Stain Buffer BSA and blocked for nonspecific binding using Fc block (anti-CD32). Surface staining was performed with fluorochrome-conjugated monoclonal antibodies directed against rat membrane markers: anti-CD3-FITC, antiCD4-PECy7 and anti-CD8-PerCP. Briefly, cells were fixed and permeabilized with Cytofix/Cytoperm solution followed by washing in Perm/ Wash solution and then, stained intracellularly with mAbs against cytokines: anti-IL4 PE, anti-IFN-γ PE, anti-IL10 PE, anti-TNF-α PE. Finally, they were washed in Perm/Wash solution, fixed in 1% paraformaldehyde in PBS and analyzed by flow cytometry in a FACS Canto flow cytometer (Becton Dickinson Immunocytometry Systems), using FACSDiva software. GolgiPlug reagent, containing Brefeldin A and reagents for cell fixation/permeabilization (Cytofix/Cytoperm and Perm/ Wash) were purchased from BD Biosciences, as well as all antibodies and appropriate isotype controls (Brazão et al., 2015).

b

PC low

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Fig. 2. Percentage of CD62L and CD44 T cells subsets from spleen of pregnant Wistar rats infected with the Y strain of T. cruzi. Animals were grouped in: Pregnant control (PC); Pregnant uninfected and treated with zinc (PCZ); pregnant infected (PI) and pregnant infected and zinc treated group (PIZ). The groups not sharing the same superscript differ significantly by One-way ANOVA and Bonferroni's multiple comparison posttest (p < 0.05). ND: non detected.

3.1. Flow cytometry analysis of splenic TCD4+ and TCD8+ subsets from chronically T. cruzi pregnant rats Pregnant Wistar rats infected and treated with zinc (PIZ) exhibited a significant reduction in lymphocytes percentages of TCD3+ TCD4+ and TCD3+ TCD8+ as compared to PI (p < 0.05) (Fig. 1). 3.2. T cell memory markers TCD62Llow and CD44high Zinc supplementation did not interfere on T cell memory cells in healthy animals (PCZ), contrasting with the elevation observed in the infected group (PI). When infected animals were treated with zinc (PIZ), the percentage of CD62LlowCD44high surface marker significantly decreased compared to the infected (Pl) counterpart (p < 0.05) (Fig. 2).

0.5 0.4

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Fig. 3. Percentage of the specific phenotypic marker identified by labeling of CD4+ CD25+ FOXP3high (Treg cells) in pregnant Wistar rats infected with the Y strain of T. cruzi. Animals were grouped in: Pregnant control (PC); Pregnant uninfected and treated with zinc (PCZ); pregnant infected (PI) and pregnant infected and zinc treated group (PIZ). The groups not sharing the same superscript differ significantly by One-way ANOVA and Bonferroni's multiple comparison posttest (p < 0.05). ND: non detected.

3.3. Analysis of TCD4+ CD25+ FoxP3high regulatory T cells in splenic lymphocytes populations

cells) from splenocytes. T regulatory cells were non detected in PC group. Significantly lower percentage of splenic Treg cells was found in the infected and treated group (PIZ) as compared to the PI group (p < 0.05) (Fig. 3).

Using intracellular Foxp3+ expressed in CD4+/CD25+ cells, we evaluated the percentage of regulatory (or suppressor) T cells (Treg 25

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3.4. CD28 and CD11a molecular expression from T lymphocytes populations

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The co-stimulatory molecules CD28+ and CD11a+ were particularly responsive to zinc supplementation and T. cruzi infection (Fig. 4). Infected group (PI) displayed increased TCD8+/CD28+ populations as compared to control (PC). Decreased CD28+ in TCD8+ subset was found in zinc treated animals (PIZ) when compared to PI (Fig. 4, B). Infected group (PI) significantly increased the CD4+/CD11a+ population when compared to control group (PC), (Fig. 4, C). Zinc supplementation displayed low percentages of CD4+/CD11a+ in comparison to PI, as well as for PC and PCZ (Fig. 4, C). Zinc significantly increased CD8+/CD11a+ in uninfected/treated control (PCZ) (Fig. 4, D).

a

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Fig. 1. Percentage of CD3 CD4 and CD3 CD8 subsets in pregnant Wistar rats infected with the Y strain of T. cruzi. Animals were grouped in: Pregnant control (PC); Pregnant uninfected and treated with zinc (PCZ); pregnant infected (PI) and pregnant infected and zinc treated group (PIZ). The groups not sharing the same superscript differ significantly by One-way ANOVA and Bonferroni's multiple comparison posttest (p < 0.05).

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PC PCZ

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PI PIZ

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%CD8+ CD28+

%CD4+ CD28+

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0.4

Fig. 4. Percentage of expression of CD4+ CD28+ (A), CD8+ CD28+ (B) CD4+ CD11a+ (C) and CD8+ CD11a+ (D) in pregnant Wistar rats infected with the Y strain of T. cruzi. Animals were grouped in: Pregnant control (PC); Pregnant uninfected and treated with zinc (PCZ); pregnant infected (PI) and pregnant infected and zinc treated group (PIZ). The groups not sharing the same superscript differ significantly by One-way ANOVA and Bonferroni's multiple comparison posttest (p < 0.05).

B

A

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Experimental groups

3.5. CD45RA+ (B lymphocytes) analysis

lymphocytes as compared to infected and untreated groups (Figs. 6 and 7).

CD45RA+ is one of the CD45+ isoforms and is expressed exclusively on B cells in rats. Increased percentages of CD45RA+ subsets were observed in infected group (PI) when compared to control (PC). Pregnant infected and zinc treated (PIZ) showed a significant decrease in CD45RA+ percentage in relation to (PI) (p < 0.05) (Fig. 5).

4. Discussion The main goal of this study was to verify the possible immunomodulatory properties of zinc in pregnant Wistar rats during the chronic phase of the experimental Chagas' disease. The immune-stimulatory properties of zinc have been described for > 50 years, playing an essential role on TH1/TH2 balance (Haase and Rink, 2014). TCD4+ and TCD8+ are pivotal tools to mediate immunity, usually observed in animals and human viral, bacterial, and others pathogenic infections (Tarleton, 2015). In order to protect the organism from parasitic aggression, the immune system generates CD4+ and CD8+ lymphocytes that migrate to lymph nodes and lymphoid organs to induce an adequate immune response (Basso, 2013). However, the maternal shift from TH1 to TH2 response pattern is a critical step for pregnancy success (La Rocca et al., 2014). Moreover, during Chagas disease, high frequency CTLs cells are associated to tissue damage in a long term period of infection (Sathler-Avelar et al., 2009). On 48th day of infection, significant alterations in the percentages of CD4+ and CD8+ T cells were observed when compared to infected and untreated counterparts. It seems that other mechanisms must be involved in the attempt to promote an adequate balance of the immune response during pregnancy. T. cruzi infection is characterized by a spontaneous apoptosis of B cells. The work of (Zuniga et al., 2000) analyzed the expression of Fas ligand, an apoptotic transmembrane protein, expressed in B lymphocytes from mice infected with T. cruzi. According to the authors, enhanced Fas expression explains the decrease in B lymphocytes proliferation in T. cruzi infected animals. In contrast to the beneficial role of antibodies against external agents, auto-antibodies have a negative effect on gestation course and are related to intrauterine fetal death, miscarriages, and pre-eclampsia (Ayres and Sulak, 1991; Harris, 1990; Muzzio et al., 2013). Concerning to the role of B cells, our results showed significant decreased percentages of CD45RA subpopulation for

3.6. Intracellular cytokine staining Intracellular cytokine profiles for TCD4+ and TCD8+ were assessed (Figs. 6 and 7). A significant reduction in TNF-α production was observed in infected and zinc treated group (PIZ) when compared to (PI) in TCD4+ population. No alterations were observed for TNF-α produced by TCD8+ population when compared to PI group. There was no significant change in IL-4, IL-10 and IFN-γ for PIZ in CD4+ and CD8+ T

%CD45RA+

40 30

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20 10 0

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Fig. 5. Percentage of expression of CD45RA subsets in pregnant Wistar rats infected with the Y strain of T. cruzi. Animals were grouped in: Pregnant control (PC); Pregnant uninfected and treated with zinc (PCZ); pregnant infected (PI) and pregnant infected and zinc treated group (PIZ). The groups not sharing the same superscript differ significantly by One-way ANOVA and Bonferroni's multiple comparison posttest (p < 0.05).

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IL-4 20

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Fig. 6. Percentage of expression of CD4+ IL-4+, CD4+ IL-10+, CD4+ IFN-γ+ and CD4+ TNF-α+ in pregnant Wistar rats infected with the Y strain of T. cruzi. Animals were grouped in: Pregnant control (PC); Pregnant uninfected and treated with zinc (PCZ); pregnant infected (PI) and pregnant infected and zinc treated group (PIZ). The groups not sharing the same superscript differ significantly by One-way ANOVA and Bonferroni's multiple comparison posttest (p < 0.05).

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Fig. 7. Percentage of expression of TCD8+ IL-4+, TCD8+ IL-10+, TCD8+ IFN-γ+ and T CD8+ TNF-α+ in pregnant Wistar rats. Animals were grouped in: Pregnant control (PC); Pregnant uninfected and treated with zinc (PCZ); pregnant infected (PI) and pregnant infected and zinc treated group (PIZ). The groups not sharing the same superscript differ significantly by One-way ANOVA and Bonferroni's multiple comparison posttest (p < 0.05).

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an unsuccessful pregnancy followed by miscarriage. On the other hand, the anti-inflammatory Th2 immune response produces characteristic cytokines such as IL-3, IL-4 and IL-10, which promote antibody production, block cytotoxic activity and inhibit decidual NK cells. Therefore, Th2 immune response is closely related to a flourishing pregnancy (Carlier, 2005). Our results are in agreement with the previous studies, as the analysis of intracellular cytokines in CD4+ T cells from zinc treated and infected rats demonstrated a reduced TNF-α concentrations when compared to infected and untreated counterparts. In addition, no statistically significant changes were observed in the expression of IL4 and IL-10 in CD4+ and CD8+ T lymphocytes as compared to infected and untreated groups. In T. cruzi infection, two lymphocyte subsets are responsible for the persistence of a memory immune response. One of them displays low proliferative ability and reduced cytokine production (TEM CD62Llow) while the other lymphocyte subpopulation shows enhanced proliferative ability and enhanced cytokine production (TCM CD62Lhigh) (Martin and Tarleton, 2005). With viral infection, some studies showed that 95% of T lymphocyte activation is not antigen specific dependent (Bell et al., 1992). Other articles, covering the detection of the MHC class I tetramer, proved that about 50% of CD8+ lymphocyte activation is related to a single epitope. To better understand the specificity of the memory immune response against T. cruzi, (Tarleton, 2015) showed that CD8+ T cells are responsible for the identification and detection of parasite epitopes. The heterogeneity of the immune response mediated by T cells was also studied with Leishmania major, through the analysis of mice cultured cells stimulated with L. major antigen (SLA - soluble leishmanial antigen). The authors found an increase in IFN-γ production related to CD4+ T cells CD62Llow (Zaph et al., 2004). Similar to the previous study, our results showed that T. cruzi infection elevated the CD62Llow population and IFN-γ production. However the treatment with zinc decreased CD62Llow population and maintained the IFN-γ levels. Elevated CD62Llow cells and INF-γ levels are related to infection resistance and tissue damage (Canavaci et al., 2014). Finally we conclude that the analysis of our data points in the direction that zinc exerted a dual role. For the fetuses, zinc played a protective role that can be evidenced by decreased percentages of TCD4+, TCD8+, TCD8+ CD28+ and Treg cells, thus contributing to a successful pregnancy. A bulk of evidence shows that zinc is considered a positive immunomodulatory agent for the host's immune response (da Costa et al., 2013; da Costa et al., 2017). In our data, zinc acted to down modulate the host's immune response, which is not helpful to contain parasite replication. However, several articles concerning to humans and experimental models have shown that during the chronic T. cruzi infection, an exacerbated immune response could trigger tissue damage (Silverio et al., 2012; Arguello et al., 2014). We want to emphasize that in this case, zinc actually exerted a down modulation, which is necessary and adequate for both pregnancy and T. cruzi chronic phase. Our results, for the first time on rats, clearly demonstrated that zinc modulated the host's immune response in a way to protect the organism against the deleterious effects of the infection during pregnancy. Thus, supplementary studies are needed to correlate the immune effects of zinc supplementation with the pathogeny of Chagas disease, which will allow an embracing comprehension about the supplementation during pregnancy.

pregnant and zinc treated animals when compared to infected and untreated counterparts. We can infer that probably zinc exerted a down modulation action protecting the fetuses from an exacerbated humoral immune response. Several infections require the activation of CD28 molecule which participates in the B cell differentiation, antibody production and the recruitment of cells to sites of inflammation under induction of chemokines (Bour-Jordan and Blueston, 2002). CD28 is also related to the expansion of regulatory T cells and some studies have been reported that knockout CD28−/− mice displayed a reduced number of CD4+ CD25+ FOXP3high Treg cells (Salomon, 2012; Tang et al., 2003). Indeed, our results indicated a reduction in the percentage of CD8+/CD28+ cells in infected and zinc treated females, compared to infected and untreated animals, thus confirming the down modulation pattern of zinc during pregnancy. Maternal-fetal interface is organized in order to modulate the maternal immune response against the embryo. Additional immune mechanisms are generated to protect the fetus from maternal immune rejection, which includes T-cell proliferation inhibition. This mechanism is guaranteed by the deprivation of tryptophan via 2,3-dioxygenase (IDO), an enzyme expressed by trophoblasts and macrophages that catabolizes tryptophan in kynurenine, a toxic product to T and dendritic cells (La Rocca et al., 2014; Munn et al., 1998). Our data revealed a decreased T cell proliferation in zinc treated infected rats as compared to untreated counterparts. These results suggest a possible immunomodulatory pattern for zinc, as well as its protective role during pregnancy. For the characterization of regulatory T cells, operative mechanisms were evaluated during T. cruzi infection. Studies have demonstrated the involvement of regulatory T cells in the immune response to infections caused by viruses, bacteria, fungi and protozoa (Sakaguchi et al., 2008). Treg cells may act in a dual fashion, triggering a massive suppressive activity of an effective immune response, causing unregulated deleterious effects to the host, or just controlling an exacerbated pro-inflammatory reaction, consequently preventing the development of tissue damage (Galgani et al., 2009). Although the specific mechanisms of Treg immunomodulatory action during pregnancy are still poorly understood, some studies have shown a key role of these cells in the maternal-fetal tolerance mechanism. During early gestational period, there is an increased percentage of Treg cells in the peripheral blood, reaching a peak in the third trimester and declining in the postpartum period (Sasaki et al., 2004). In our studies we observed that pregnant females treated with zinc triggered a reduction in the percentage of these cells as compared to untreated counterparts. At first glance this result seems uncommon but it can be explained based on two facts. First, for the recruitment of Treg cells a specific CD28 molecule is fundamental for the cellular migration (Liu et al., 2006). Our results revealed a drop in the percentage of CD8+/CD28+, consequently less Treg cells are requested. Moreover, our experiment was performed after 18 days of pregnancy (48 days of infection), the final stage of rat pregnancy. Therefore, less Treg cells are needed, triggering a pro-inflammatory immune response. This phenomenon coincides with enhanced concentrations of oxytocin which helps in the uterus muscle contraction, necessary to the birth process. The role of TNF-α during infection is rather complex: On one hand this pro-inflammatory cytokine plays a favorable role in the host during the acute phase of infection, controlling parasite replication. While on the other hand, an overproduction of this cytokine contributes to the development of the pathology processes which will take place in the late chronic phase of the disease (Araújo-Jorge et al., 2012). During pregnancy, the cytokine profile is altered with a predominance of a Th2 immune response, enabling an adequate microenvironment and immune tolerance to the fetal-placental tissues, which is pivotal for the maintenance of a successful gestation. The duality of actions of the immune response can lead to different fates. A suited Th1 response leads to the production of several cytokines such as IL-2, IFN-γ and TNF-α, which are essential to contain parasite dissemination throughout the host's body. However, this kind of response can induce

Financial support This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (Grant number: 2013/04205-6). Acknowledgements We would like to thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). 335

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