J. Pineal Res. 2015; 58:210–218

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Molecular, Biological, Physiological and Clinical Aspects of Melatonin

Doi:10.1111/jpi.12207

Journal of Pineal Research

Immunoregulatory actions of melatonin and zinc during chronic Trypanosoma cruzi infection Abstract: After one century of the discovery of Chagas’ disease and the development of an efficient drug with amplitude of actions both in the acute and chronic phase is still a challenge. Alternative immune modulators have been exhaustively used. For that purpose, melatonin and zinc were administered during chronic Trypanosoma cruzi-infected Wistar rats and several endpoints were assessed. Melatonin has a remarkable functional versatility, being associated with important antioxidant, anti-inflammatory, and anti-apoptotic effects. The cross-talk between zinc and the immune system includes its ability to influence the production and signaling of numerous inflammatory cytokines in a variety of cell types. Our study showed that zinc triggered a decrease in the generation of IFN-c for TCD4+ cells. Reduced percentage of CD4+T cells producing TNF-a was observed in control melatonin or zinc-and-melatonin-treated animals as compared with untreated rats. On the other hand, a significant increase in the percentage of IL-4 from CD4+ and CD8+ T lymphocytes producers was observed 60 days after infection, for all zinc-treated animals, whether infected or not. Melatonin and zinc therapies increased the percentages of CD4+ and CD8+ T lymphocytes IL-10 producers. CD4+CD25highFoxp3+ T cells were also elevated in zincand melatonin-treated animals. The modulation of the immune system influenced by these molecules affected cytokine production and the inflammatory process during chronic T. cruzi infection. Elucidation of the interplay between cytokine balance and the pathogenesis of Chagas’ disease is extremely relevant not only for the comprehension of the immune mechanisms and clinical forms but, most importantly, also for the implementation of efficient and adequate therapies.

Introduction Melatonin, an ancient amphiphilic molecule ubiquitously present in nature. It is an important chronobiological regulatory molecule that is released from the pineal gland during the nighttime [1]. Melatonin production has now been documented in a wide range of vertebrate and invertebrate tissues out of the pineal gland [2,3] where it functions as an autocrine and paracrine agent. This indoleamine exerts profound effects on a wide range of tissues, through receptor-mediated and receptor-independent mechanisms [4]. Melatonin acts as a radical scavenger [5–7], activator of antioxidant enzymes [8], oncostatic [9], anti-aging [10], and immunomodulatory properties [11–13] that counteract inflammatory insults [14–17]. Due to the fact of its multiplicity of actions, melatonin has been widely tested in distinct experimental and clinical studies showing a variety of benefits. Melatonin has been shown to have efficacy in different infections. Galley et al. [18] studying the actions of oral melatonin administration in patients with sepsis described its beneficial effects on sepsis-induced mitochondrial dys210

^nia Braza ~o, Fabricia Helena Va Santello, Marina Del Vecchio Filipin, Angela Palamin Azevedo, Mıriam Paula Alonso Toldo, Fabiana Rossetto de Morais and  Clo  vis do Prado Jr Jose rio de Parasitologia, Departamento de Laborato lises Clınicas, Toxicolo gicas e Ana gicas, Faculdade de Cie ^ncias Bromatolo ^uticas de Ribeira ~o Preto, Universidade Farmace ~o Paulo, Ribeira ~o Preto, SP, Brasil de Sa

Key words: immune response, melatonin, Trypanosoma cruzi, zinc ^nia Braza ~o, Address reprint requests to Va lises Clınicas, Departamento de Ana gicas e Bromatolo gicas, Faculdade de Toxicolo ^ncias Farmace ^uticas de Ribeira ~o Preto Cie ~o Paulo, FCFRP-USP, Universidade de Sa  s/no, Ribeira ~o Preto, Avenida do Cafe SP14040-903, Brasil. E-mail: [email protected] Received October 10, 2014; Accepted January 15, 2015.

function. Our group has studied the actions of melatonin during Trypanosoma cruzi infection [19, 20] and noted a protective effect of this indolamine in animals with cardiac inflammatory disease which is a hallmark of the chronic chagasic disease [21]. Other studies have also noticed the actions of melatonin in modulating the host’s immune response during acute T. cruzi infection, consequently leading to reduced parasitemia [22]. Zinc is a highly effective anti-oxidant [23, 24] and antiinflammatory agent [25], with catalytic, structural, and regulatory functions that make this element essential for the development and function of the immune system [26]. Several studies have now shown benefits of zinc supplementation on infections [27–29]. In therapeutic dosages, zinc displays a wide range of actions. It can be used for the treatment of acute diarrhea in infants and children, the common cold, Wilson’s disease, sickle cell disease, and for the prevention of blindness in patients with age-related macular degeneration [30]. Chagas’ disease or American tripanosomiasis, caused by the flagellate protozoan Trypanosoma cruzi, is still a major public health problem in Latin America, causing

Influence of melatonin and zinc treatment on the immune response approximately 50,000 deaths a year, with approximately eight million infected people in 18 endemic countries [31, 32]; also, there are approximately 90 million people at risk to be infected [33]. Upon exposure to the parasite, adult individuals undergo an acute phase that lasts for 4–8 wk, usually asymptomatic or oligosymptomatic. Without specific treatment, the acute phase continues and is followed by a chronic phase, with several different clinical profiles. Although the exact mechanisms associated with the establishment or maintenance of the distinct clinical outcomes of Chagas’ disease are undoubtedly very complex and still not completely clarified, several authors have shown that the balance between pro-inflammatory (IFN-c and TNF-a) versus anti-inflammatory cytokines (IL-10 profile) is likely to be a determining factor in the type of response established in T. cruzi-infected patients, as well as the intensity of parasite burden and the effectiveness of the host’s immune response in limiting peripheral damage [34]. After one century of the discovery of Chagas’ disease and the development of an efficient drug with amplitude of actions both in the acute and chronic phase is still a challenge. In the shadow of other neglected diseases, South American trypanosomiasis has not received much attention from the pharmaceutical industry and the available therapy using benznidazole is not effective for all parasite strains, displays toxic side effects, in addition to being ineffective during human chronic phase [35]. Based on the critical lack of evidence about the actions of the current available drug and the absence of an effective and feasible treatment for Chagas’ disease, this study proposes the design of rational therapeutic strategy using melatonin and zinc as a new approach targeting to protect animals against the harmful actions of T. cruzi infection. The use of these agents is based on the hypothesis regarding to the immunomodulating actions of these drugs. We believe that the dynamics of these effects depends on the recognition and cooperation of the immune and endocrine systems in a way that melatonin and zinc alone or together regulate the inflammatory and immunopathological processes as well as the neuroendocrine network through peripheral receptors. Although several studies have attempted to explain the clinical results of immune modulating substances, few trials have evaluated the effect of different treatment schemes with melatonin and zinc. For that we used T. cruzi-infected Wistar rats, and several immune parameters were evaluated, including the overall frequency of lymphocytes producing intracellular cytokines (IFN-c, TNF-a, IL-4 and IL-10) determined for the CD4+ and CD8+ T lymphocytes. Flow cytometry analysis was used to assess the percentages of NK (CD161+), NKT (CD3+CD161+), and CD4+CD25+Foxp3 high (Treg). The choice of these immune markers is based on the involvement of these cells in the control of the immune response, either protecting such as CD4+CD25+Foxp3 high (Treg) or sometimes leading to tissue damage as CD8 and NK cells.

Materials and methods Animals Male Wistar rats (40 animals) weighing 90–100 g were used in all experiments. The rats were obtained from the Facility House of the University Campus of Ribeir~ ao Preto. Animals were randomized into the following groups: control (C), zinc control (ZC), melatonin control (MC), melatonin and zinc control (MZC), infected (I), zinc infected (ZI), melatonin infected (MI), and melatonin and zinc infected (MZI). A total of five animals were used per group per experiment day. The rats were housed in groups of five in plastic cages, and commercial rodent diet and water were available ad libitum. Rat bedding was changed 3 times/wk to avoid ammonia concentration from urine. The protocol of this study was approved by the local Ethics Committee (protocol number 08.1.835.53.5). Parasites and experimental infection Rats were intraperitoneally (i.p.) inoculated with 1 9 105 blood trypomastigotes of the Y strain of T. cruzi [36]. Parasitemia was determined using Brener’s method [37]. The assays were performed 60 days after infection. It is important to emphasize that because Wistar rats are normally resistant to most T. cruzi strains, we found it necessary to use relatively high inoculums (1 9 105 blood trypomastigotes). Treatment scheme Rats were orally treated with zinc sulfate (Sigma Chemical Co., St Louis, MO, USA) dissolved in 0.1 mL of distilled water at a dose of 20 mg/kg body weight and with 0.1 mL of melatonin (Sigma Chemical Co.), which had been dissolved in polyethylene glycol 400 (PEG 400), at 5 mg/kg of body weight once a day. Melatonin was administered at the same time each day, beginning the day after inoculation and then every day until the end of the experiment [38]. Euthanasia Animals were euthanized according to previous methods [38]. Tribromoethanol (2.5%) was administered intraperitoneally at a dose of 0.1 mL per 10 g of body weight. Cell phenotyping Cells from spleen tissues were dispersed by extrusion through a 70-lm Nylon Cell Strainer and macerated in RPMI 1640 medium to produce a single-cell suspension. Cell number was estimated using a Neubauer chamber. Cells (2 9 106) from the cell suspension of each organ from each experimental group were placed in 96-well round-bottom plates for cytofluorometric analysis. Following Fc receptor blocking, the cells were incubated with monoclonal antibodies: anti-CD161-FITC and antiCD3APC from BD Biosciences Pharmingen, San Jose, 211

Braz~ ao et al. CA, USA. Stained cells were stored in sealed tubes in the dark in PBS containing 1% paraformaldehyde. All steps were performed at 4°C. Cell analysis was performed using a Becton Dickinson FACScan flow cytometer with DIVA-BD software (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). Intracellular staining of Foxp3 Spleen cells (2 9 106) were surface-stained with fluorochrome-labeled anti-CD3-FITC, anti-CD4 (PECy7), and anti-CD25 (PE) mAbs. The cells were permeabilized and intracellularly stained for Foxp3 according to the manufacturer guidelines (eBioscience, San Diego, CA, USA), washed and analyzed by flow cytometry.

Immunocytometry Systems). The results are expressed as the percentage of cytokines producing CD4+ and CD8+T cells. Statistical analysis The results were expressed as means/standard error of mean. A value of P < 0.05 was considered statistically significant. The methods used for statistical analysis were chosen considering the small sample size. Differences among groups were determined by one-way ANOVA with Bonferroni’s post-test. All statistical analyses were performed using Graph Pad Prism version 5.0 (GraphPad Software, Inc., San Diego, CA, USA).

Results Intracellular staining of IL-4, IL-10, IFN-c, and TNF-a CD4+- and CD8+-specific intracellular cytokines were measured using a flow cytometry assay. Cells (2 9 106) from the suspension of the spleen from each experimental group were placed in 96-well round-bottom plates. The spleen cells from Wistar rats were stimulated with 5 lg/mL de phorbol 12-myristate 13-acetate (PMA) and 1 lg/mL ionomycin for 4 hr at 37°C under 5% CO2, and cytokine secretion was inhibited with 1 lM Brefeldin A (GolgiPlugTM; Pharmingen, San Diego, CA, USA), a protein transport inhibitor that was added during the final 4 hr of incubation. The cells were washed, and after blocking with anti-CD16/32 mAb, the cells were incubated with anti-CD3, CD4, and CD8 antibodies (30 min, 4°C in the dark) (BD Biosciences, San Jose, CA, USA) to detect surface expression. After surface staining, the cells were permeabilized by incubation with Cyto fix/Cyto perm buffer (BD Biosciences Pharmingen) for 20 min. After resuspension in perm/wash buffer (BD Biosciences Pharmingen), the cells were incubated with anti-IL-10, IFN-c, and TNF-a (PE) diluted in Perm/Wash buffer (BD Biosciences Pharmingen) at 4°C for 30 min, washed, resuspended in PBS containing 1% paraformaldehyde, and then stored in sealed tubes in the dark. Analysis of these cells was performed using a Becton Dickinson FACScan flow cytometer with DIVA-BD software (Becton Dickinson (A) 5

Our first objective in this study was to determine whether the melatonin and zinc administration in rats was associated with changes in the production of some cytokines, such as IFN-c, TNF-a, IL-4, and IL-10 in CD4+ and CD8+ T lymphocytes. Sixty days after the T. cruzi infection, zinc-treated animals presented reduced (P < 0.01) percentage of CD4+ T lymphocytes IFN-c producers (Fig. 1A). For control animals, treated or not, no difference was observed in the percentage of CD4+ T cells producing IFN-c. Zinc-treated control animals displayed a reduction in the percentage of IFN-c-producing CD8+ T cells (P < 0.001) (Fig. 1B). Reduced percentage of CD4+T cells producing TNF-a in melatonin- or melatonin–and-zinc-treated rats were observed (P < 0.001) comparing control and untreated animals (Fig. 2A). The same treatment did not affect the percentage of CD8+ T cells producing TNF-a for any treated group, whether infected or not (Fig. 2B). A significant increase in the percentage of CD4+ T lymphocytes IL-4 producers was observed 60 days after infection, for any zinc-treated group, whether infected (P < 0.01) or not (P < 0.001) (Fig. 3A). A similar increase in the percentages of IL-4 in CD8+ T cells was observed in infected and zinc-treated animals (P < 0.01) compared with untreated and noninfected counterparts (Fig. 3B). (B) 3.0 CD8+IFNγ + %

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Fig. 1. Intracellular IFN-c expression in CD4+ (A) and CD8+ (B) T cells following PMA (5 lg/mL) and ionomycin (1 lg/mL) stimulation. Lymphocytes were obtained from male Wistar rats i.p. infected with 1.0 9 105 blood trypomastigotes of the Y strain of Trypanosoma cruzi on the 60th day post-infection of experimental disease, for the following groups: control (C), zinc control (ZC), melatonin control (MC), zinc and melatonin control (ZMC), infected (I), zinc infected (ZI), melatonin infected (MI), and zinc and melatonin infected (ZMI). One-way ANOVA with Bonferroni’s multiple comparison test was used to compare groups (**P < 0.01; and ***P < 0.001). All data are expressed as mean  S.E.M. n = 5/group/day of experiment.

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Fig. 2. Intracellular TNF-a expression in CD4+ (A) and CD8+ (B) T cells following PMA-ionomycin stimulation. Lymphocytes were obtained from male Wistar rats i.p. infected with 1.0 9 105 blood trypomastigotes of the Y strain of Trypanosoma cruzi on the 60th day post-infection of experimental disease, for the following groups: control (C), zinc control (ZC), melatonin control (MC), zinc and melatonin control (ZMC), infected (I), zinc infected (ZI), melatonin infected (MI), and zinc and melatonin infected (ZMI). One-way ANOVA with Bonferroni’s multiple comparison test was used to compare groups (***P < 0.001). All data are expressed as mean  S.E.M. n = 5/ group/day of experiment.

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Fig. 3. Intracellular IL-4 expression in CD4+ (A) and CD8+ (B) T cells following PMA-ionomycin stimulation. Lymphocytes were obtained from male Wistar rats i.p. infected with 1.0 9 105 blood trypomastigotes of the Y strain of Trypanosoma cruzi on the 60th day post-infection of experimental disease, for the following groups: control (C), zinc control (ZC), melatonin control (MC), zinc and melatonin control (ZMC), infected (I), zinc infected (ZI), melatonin infected (MI), and zinc and melatonin infected (ZMI). One-way ANOVA with Bonferroni’s multiple comparison test was used to compare groups (*P < 0.05; **P < 0.01 and ***P < 0.001). All data are expressed as mean  S.E.M. n = 5/group/day of experiment.

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Fig. 4. Intracellular IL-10 expression in CD4+ (A) and CD8+ (B) T cells following PMA-ionomycin stimulation. Lymphocytes were obtained from male Wistar rats i.p. infected with 1.0 9 105 blood trypomastigotes of the Y strain of Trypanosoma cruzi on the 60th day post-infection of experimental disease, for the following groups: control (C), zinc control (ZC), melatonin control (MC), zinc and melatonin control (ZMC), infected (I), zinc infected (ZI), melatonin infected (MI), and zinc and melatonin infected (ZMI). One-way ANOVA with Bonferroni’s multiple comparison test was used to compare groups (**P < 0.01; and ***P < 0.001). All data are expressed as mean  S.E.M. n = 5/group/day of experiment.

Increased percentages of CD4+ and CD8+ T lymphocytes IL-10 producers (P < 0.001) were observed for infected treated groups as compared with untreated counterparts (Fig. 4A,B). For control animals under zinc therapy, an increase in the percentages of IL-10 in CD4+ and CD8+ T cells (P < 0.01) was observed (Fig. 4A,B). To determine the actual contribution of melatonin and zinc during the chronic T. cruzi infection, we examined

whether the oral administration of these substances affect the innate immune response. As shown in Fig. 5A, we observed that the percentages of NK cells in melatoninor zinc-treated rats were diminished (P < 0.001) compared with the infected and untreated animals. Our study also showed that melatonin and zinc treatment did not affect the percentage of spleen NKT cells for any group, regardless of the infection status (Fig. 5B). On 213

Braz~ ao et al. Additionally, to determine whether these cells are actually Treg or not, the expression of a transcription factor characteristic of the regulatory T cells, Foxp3, was evaluated in splenic CD4+CD25high T cells from zinc- and melatonin-treated groups. In noninfected rats, the great majority of CD4+CD25high T cells from zinc or melatonin plus zinc expressed Foxp3 (P < 0.05), a phenotype characteristic of Treg cells (Fig. 6B). On day 60 postinfection, CD4+CD25highFoxp3+ T cells were increased (P < 0.01) in zinc- and melatonin-treated animals (Fig. 6B).

the other hand, on the 60th day postinfection, an increase (P < 0.001) in both NK and NKT cells was noted in the infected and untreated animals, as compared to noninfected animals. Next, we investigate the ability of melatonin and zinc in modulating T regulatory cells in chronic T. cruzi infection. It is important to emphasize that upon activation all T cells express CD25, an a chain of the high-affinity IL-2 receptor (IL-2R), both regulatory cells and the activated ones [39, 40]. Because regulatory cells were first described as a subset of CD4+ T lymphocytes that express the highest levels of CD25 [41], we first estimated the proportion of such CD4+CD25 high T cell subpopulation among lymphocytes. We observed that the percentages of CD4+CD25+ as well as CD4+CD25highT cells in zinctreated rats were diminished compared with the infected and untreated animals. However, these CD4+CD25highT cells increased (P < 0.05) at day 60 in melatonin-treated animals (Fig. 6A).

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The request for new more effective agents to treat Chagas’ disease has not been matched by drug discovery efforts. An estimated 8 million people have this chronic infection and the only two treatments (benznidazole and nifurtimox) are available; both have significant limitations due to the side effects besides displaying uncertain efficacy for

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Fig. 6. Representative FACS stain for CD25+ (A) and CD25high (B) on gated CD4+ T cells and Foxp3 expression by CD4+CD25high T cells (C) from the spleen of male Wistar rats i.p. infected with 1.0 9 105 blood trypomastigotes of the Y strain of Trypanosoma cruzi on the 60th day post-infection of experimental disease, for the following groups: control (C), zinc control (ZC), melatonin control (MC), melatonin and zinc control (MZC), infected (I), zinc infected (ZI), melatonin infected (MI), and melatonin and zinc infected (MZI). One-way ANOVA with Bonferroni’s multiple comparison test was used to compare groups (*P < 0.05; **P < 0.01, and ***P < 0.001). All data are expressed as mean  S.E.M. n = 5/ group/day of experiment.

(A) CD4+ CD25+ %

Fig. 5. Phenotypic characterization of splenic NK (A) and NKT (B) cells obtained from male Wistar rats i.p. infected with 1.0 9 105 blood trypomastigotes of the Y strain of Trypanosoma cruzi on the 60th day post-infection of experimental disease, for the following groups: control (C), zinc control (ZC), melatonin control (MC), zinc and melatonin control (ZMC), infected (I), zinc infected (ZI), melatonin infected (MI), and zinc and melatonin infected (ZMI). One-way ANOVA with Bonferroni’s multiple comparison test was used to compare groups (***P < 0.001). All data are expressed as mean  S.E.M. n = 5/group/day of experiment.

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Influence of melatonin and zinc treatment on the immune response curing chronic infection [35, 42]. For economic reasons, the pharmaceutical industry has not invested substantially in tropical diseases such as Chagas’ disease, despite the recognized expansion of this illness into wealthier parts of the world [43]. To address this question, we combined in vivo follow up of compounds with proven immunomodulatory effects in a murine model of chronic T. cruzi infection. Our first experimental study using the melatonin and zinc combinations was published in 2011; in that study, oral administration of these substances protected infected animals against the harmful pathogenic effects induced by Chagas’ disease [38]. In the current investigation, several immune parameters were evaluated to confirm the immunomodulatory effect of melatonin and zinc on the host’s immune response during the experimental chronic Chagas’ disease. Studies have also demonstrated that during T. cruzi infection, NKT-cell actions can minimize the intensity of the chronic inflammatory responses against the pathogen, preventing unwanted self-damaging immune responses. NKT cells are unprecedented effector cells that share surface receptors with both classical T cells (TCR and CD4) and natural killer (NK) cells (NK1.1) representing a connection between the innate and the adaptive immune responses [44], displaying both cytotoxic abilities as well as providing signals required for driving the adaptive immune response. On the other hand, although NK cells are absolutely required for the clearance of the pathogen, the strong and uncontrolled activation of NK cells as well as pro-inflammatory monocytes may also lead to tissue damage [45, 46]. Vitelli-Avelar et al. [47,48] emphasized the importance of NKT subsets in Chagas’ disease where it was demonstrated that patients in the late indeterminate clinical form of Chagas’ disease display a high frequency of circulating NKT cells, whereas patients with cardiac disease show basal levels of NKT cells and a negative association with the high frequency of activated CD8+ T cells. As NKT cells display a strong association with decreased inflammatory response, a reasonable hypothesis would be that the enhanced frequency of NKT cells found in patients bearing the indeterminate form would contribute to control an exacerbated immune response that culminates in a strong cytotoxic response and subsequent tissue damage. NKT cells have also been shown to play an important role in modulating the activation of CD8+ T cells via apoptosis, as well as throughout the secretion of regulatory cytokines [49]. It is well established that zinc can improve NKT cell development, maturation, and function. Zinc has critical effects in homeostasis, immune actions, oxidative stress, and in apoptosis [50]. Regarding to the effects of melatonin on the innate cells, including NK cells, several reports have been published, with contradictory results. Some have demonstrated that melatonin administration increases the number of NK cells [51–53]. Conversely, one in vivo study claimed that melatonin administration is ineffective in stimulating an increase in the number of NK cells in rats with mammary tumors [54]. In another study, Provinciali et al. [55] demonstrated that the long-term melatonin administration in old mice had no effect on the NK

cell number. Our data indirectly are in accordance with the statements above. In our study, we demonstrated that the percentages of NK cells in melatonin- or zinc-treated rats were diminished (P < 0.05) compared with the infected and untreated ones. Additionally, our study showed that melatonin and zinc treatment did not affect the percentage of spleen NKT cells for any groups, regardless of infection status. Regulatory T cells, termed Treg cells, play a critical role in the immune system homeostasis and in the modulation of the immune response, mediating peripheral tolerance, preventing autoimmune diseases [56], and controlling inflammation. Treg cells may also play a role in controlling the adverse events triggered by the massive antigen release induced by trypanosomicidal agents during Chagas disease [47, 57–59]. A recent study [59] showed that the ratio of Foxp3 gene expression in Treg cells in patients with the late indeterminate clinical form of Chagas disease was significantly higher than that in cardiac patients. Using the peroxidase immunohistochemistry technique, Da Silveira et al. [58] found that the distribution of Foxp3 in patients with digestive disorders closely related to prognosis. The higher Foxp3 expression in the chagasic patients was associated with better prognosis. In line with this, according to Vitelli-Avelar et al. [47], the reduced frequency of CD4+CD25+ T cell expressing Foxp3 is correlated with the severity of the Chagas’ disease cardiomyopathy. The relationship between melatonin and Treg cells remains unclear. However, the Foxp3-inducing activity of zinc has been demonstrated [60]. These data indirectly confirm ours, in which Foxp3 expression in CD4+CD25+T lymphocytes was increased in infected zinc- and melatonin-treated groups. Presently, we have hypothesized that the induction of Foxp3 expression by zinc and melatonin contributes to minimize the effects of inflammation and the strong host’s immune response through the up-regulation of Treg cells. The immunosuppressive functions carried out by Treg cells are based on several mechanisms. One of them is the secretion of anti-inflammatory cytokines including IL-10, TGF-b, and IL-35 [61]. Numerous investigators have demonstrated a correlation between the anti-inflammatory/ pro-inflammatory cytokine profiles and disease progression [62, 63]. In line with this finding, cytokine analysis has shown a positive relationship between TNF-a and IFN-c expressions and the development of cardiac lesions [45, 64]. In fact, Abel et al. [65] and Gomes et al. [64] described that peripheral blood mononuclear cells from chronic chagasic cardiomyopathy patients produce more IFN-c and less IL-10 when compared to the chronic patients in the indeterminate phase of the disease, suggesting that Th1-cell-mediated process plays an important role in the pathogenesis of Chagas’ disease. The production of IL-10 by chronic T. cruzi-infected mice has been implicated in the suppression of inflammation and thereby minimizes pathology [66]. It is also well recognized that the secretion and function of cytokines are adversely affected by zinc deficiency. Some reports indicate that zinc supplementation may lead to down-regulation of pro-inflammatory cytokines [24, 67] 215

Braz~ ao et al. through up-regulation of A20, a zinc finger protein that inhibited NF-jB activation [29]. Studies conducted by Prasad and coworkers [68] also showed that zinc decreased the generation of cytokines, including TNF-a and IL-1b, indicating the protective role of this element in some chronic diseases where oxidative stress and chronic inflammation are involved. Consistent with these statements, our results confirm and expand on the finding that zinc therapy triggered a reduction in IFN-c levels (from CD4+ T cells) as compared to infected and untreated group. A lessen in the percentage of IFN-c-producing CD8+ T cells was also observed in the control animals under zinc therapy. As described above, the relationship between zinc and Chagas disease has been described previously by our research group [38]. Different from this, the previous assays were performed in adult male Wistar rats (100 days), in which IL-10 levels were markedly increased on 60th day postinfection in zinc- and melatonin-treated animals. In the present study, performed using Wistar rats with 4 wk, enhanced concentrations of IL-10 were also observed in infected zinc- and melatonin-treated groups when compared to untreated counterparts. Recent research revealed that melatonin exhibits multiple anti-inflammatory effects through the regulation of different molecular pathways [17, 69]. There is persuasive evidence that melatonin inhibit the effects of pro-inflammatory transcription factors as well as reduces the up-regulation of a variety of pro-inflammatory mediators [70], such as nitric oxide, prostanoids, leukotrienes, cytokines, inflammatory enzymes, and adhesion molecules [17, 71]. The wide involvement of this multifunctional indolamine in the immune system includes its ability to inhibit the production of TNF-a, by blocking transcriptional factors, consequently ameliorating the inflammation [11]. In accordance with this, our study showed that melatonin or melatonin plus zinc therapy decreased the percentages of CD4+T cells producing TNF-a. Although the molecular mechanism mediating the pathogenesis of Chagas’ disease, remains poorly understood, a direct correlation has been reported between the severity of infection and increased concentrations of Th1 cytokines. On the other hand, some studies describe a protective role of IL-4 in chronic Chagas’ disease, presumably by providing a negative IFN-c-producing response, preventing the establishment of heart tissue lesions [72]. Our study is congruent with these previous findings, emphasizing the important role for IL-4 during chronic Chagas’ disease, as a significant increase in the percentage of IL-4 producers CD4+ T lymphocytes were observed 60 days after infection, for all zinc-treated groups, whether infected or not. A similar increase in the percentages of IL-4 in CD8+ T cells was observed in infected and zinc-treated animals as compared to untreated and uninfected counterparts. The ultimate goal of this work is that the concentrations of several cytokines are modified through the actions of the therapy with melatonin, zinc, or combined. In fact, the modulation of the immune system by these molecules seems to affect the Th1/Th2 balance during chronic T. cruzi infection, besides being more effective, better tolerated, 216

and simpler to administer than the current regimens for treating Chagas’ disease. Elucidation of the interplay between cytokine balance and pathogenesis of Chagas’ disease is of major importance not only for the comprehension of the immune mechanisms and clinical forms but, most importantly, also for the implementation of efficient and adequate therapies.

Acknowledgements We thank Inara Fernanda Lage Gallo and Cristiana Goncßalez for technical support. This study was supported by fellowships from FAPESP.

Author contributions We hereby certify that it is an original publication and the manuscript has not been previously submitted or published elsewhere. FS, MF, AA, and MT conceived the study design and conducted the studies. FM performed the flow cytometric analysis. VB and JP performed the statistical analyses, participated in the study design, and helped in drafting of the manuscript. All authors have made substantial contributions and final approval of the conceptions, drafting, and final version.

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