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During the course of Trypanosma cruzi infection, the immunological system of the host is involved in distinct complex interactions with the endocrine system, and the prolactin (PRL) is one among several other hormones involved in immunoregulation. Although intensive studies in trying to understand the mechanisms which underlie Chagas disease, there are some pieces still missing in this complex puzzle. Since the scarcity of data concerning to the role of PRL involvement in Chagas disease and taking into account the existence of a crosstalk between neuroendocrine hormones and the immune system, the purpose of the current study was to evaluate a possible up-regulation triggered by PRL on the cellular immune response of T. cruzi-infected rats and the aptness of PRL in reversing the immunosuppression caused by the parasitic infection. The data shown herein demonstrate that PRL strongly induced the proliferation of T lymphocytes coupled with an activation of macrophages and the production of enhanced concentrations of nitric oxide, leading to a reduction of blood trypomastigotes during the peak of parasitemia. Regarding to phenotypic analysis of T cell populations during the acute phase of T. cruzi infection, an enhancement of both CD3+CD4+ and CD3+CD8+ subsets were observed in infected groups, being the highest numbers of these T cells subsets found in the PRL treated and infected group. Since NO is a central signaling molecule, involved in a number of cellular interactions with some components of the immune system and those of the neuroendocrine system, PRL can be considered as an alternative hormone able to up-regulate the host's immune system consequently lowering the pathological effects of T. cruzi infection.
The interaction between the endocrine and the immune system is well established and it has been clearly demonstrated by several researchers that certain hormones are capable of modulating cellular immune functions (Santos et al., 2008; Filipin et al., 2008; Caetano et al., 2009). Additionally, these mechanisms of interaction may be beneficial or detrimental for the host resistance during a variety of infections, including those caused by parasites (Santello et al., 2007, Escobedo et al., 2005).
Trypanosoma cruzi, the etiological agent of Chagas' disease, is an intracellular protozoan that causes a serious immune imbalance during the acute phase, characterized by severe thymocyte depletion and myocardial inflammation (Pérez et al., 2007). The control of T. cruzi infection is achieved through the cooperative interactions of a number of cells including CD3+CD4+ and CD3+CD8+ T, B lymphocytes (Tarleton, 1990), NK cells, macrophages (Sacks and Sher, 2002), and the production of inflammatory cytokines, such as IFN-γ and/or TNF-α which play a central role in regulating parasite replication (Umekita and Mota, 2000; Pascutti et al., 2003), by activating macrophages to synthesize NO, which, in the mouse, is considered the major effective molecule for intracellular amastigotes killing (Vespa et al., 1994; Silva et al., 2003).
Several studies have demonstrated the importance of CD4+ and CD8+ T cell compartments. Mice lacking these T cell subsets display an enhanced susceptibility to infection followed by increased parasitemia and shortened survival time in (Tarleton et al., 1994).
The course of Chagas´ disease can be profoundly affected by the endocrine host's response (Roggero et al., 2006). PRL is a peptide hormone secreted by the anterior pituitary gland and it is involved in the regulation of the physiological processes of growth and reproduction in a wide range of vertebrate species (Quintanar et al., 2007). Furthermore, evidences have demonstrated that this hormone is also produced at sites outside the pituitary gland (extrapituitary PRL), such as placenta, brain, uterus, dermal fibroblasts, NK, T and B cells. (Bole-Feysot et al., 1998).
The main step of PRL action is its binding capacity to specific PRL receptors (Bole-Feysot et al., 1998; Yu-Lee, 2002), that are found in cells and tissues of central nervous system, pituitary gland, adrenal cortex, thymus, spleen, lymphocytes T and B, macrophages, kidney, etc, acting among other functions as an immunoregulator (Bole-Feysot et al., 1998).
Research dating from the 1930s had suggested a role of PRL in modulating the immune system (Smith, 1930; Kooijman et al., 1996). Later, several other papers demonstrated that PRL exhibited immunostimulatory properties (Yu-Lee, 1997; Clevenger et al., 1998), up-regulates cytokine secretion and lymphocyte differentiation, especially in response to antigen and mitogens (Biswas et al., 2006), besides its ability to reverse the glucocorticoid-induced immunosuppression (Kelley and Dantzer, 1991).
Based on the fact that T. cruzi infections cause severe and long-lasting suppression of the immune responses during the acute phase of infection, the evaluation of potential therapeutics in up-regulating the cellular immune responsiveness of T.cruzi-infected rats receiving PRL was performed, using as tools thymocytes proliferation, peritoneal macrophage counts, flow cytometry analysis of T cell subpopulations (CD3+CD4+ and CD3+CD8+) and NO production.
MATERIAL AND METHODS
Male Wistar rats 4 weeks old, weighing 90 to 100g were used in all experiments. Rats were obtained from the Facility House of the Universitary Campus of Ribeirão Preto. Animals were randomized into one of the following groups: Control (C); Control PRL-treated (CP); Infected (I); Infected PRL-treated (IP). A number of 5 animals were used per group/per day of experiment. They were separated in number of 5 in plastic cages and commercial rodent diet and water were available ad libitum. Rat pad was changed 3 times/week to avoid concentration of ammonia from urine. The protocol of this study was approved by the local Ethics Committee (protocol number 09.1.279.53.6). Experiments were done twice and samples of each parameter were done in triplicate. Unfortunately, it was impossible to use more animals due to the orientations of the local Ethics Committee. In the total it was used 120 animals (the experiments were performed in duplicate), what seems to be a reasonable number of animals for an experiment enabling conditions to draw conclusions.
Parasites and Experimental Infection
Rats were intraperitoneally inoculated with 1 - 105 blood trypomastigotes of the Y strain of T. cruzi (Silva and Nussenzweig, 1953). The studies were performed on 7, 14 and 21 days post-infection. Parasitemia was determined by Brener's method (Brener, 1969). It is important to emphasize that since Wistar rats are normally resistant to most T.cruzi strains, we found it necessary to use relatively high inoculums (1 - 105 blood trypomastigotes), which resulted in a more intense pathological response such as enhanced parasitemia.
Each animal received subcutaneously, 40µg (Ryníková et al., 1988; Zellweger et al., 1996; Oberbeck et al., 2003) of ovine PRL (Sigma-Aldrich Chemicals) diluted in 0.1ml of saline solution (0.9%), once a day at the same time and during the course of the experiment (7, 14 and 21 days). Treatment schedule started at the same day of infection.
Animals were decapitated with prior anesthesia using thribromoethanol (2.5%), administrated intraperitoneally at a dose of 0.1 ml/10g body weight.
Peritoneal macrophage counts
Peritoneal cells were harvested by the injection of 10ml of cold RPMI 1640 medium into the peritoneal cavity. The cells were centrifuged at 410g for 15 min, the pellet was resuspended in RPMI 1640 medium, diluted with Turkey solution (15ml glacial acetic acid; 0.2ml gentian violet 2%; 500 ml distilled water) and the macrophages were counted in a Neubauer chamber.
Preparation of peritoneal cell suspensions and measurement of nitrite production
NO production was measured according to Dost et al. (2006), as accumulated supernatant nitrite (a stable breakdown product of NO), determined by a spectrophotometric method using the Griess reaction.
Macrophage cells, harvested from peritoneal cavity, were adjusted to a concentration of 5 - 106 cells/ml and cultured in 96-well flat-bottomed plates, with or without LPS (10 μg/ml) (Escherichia coli, Sigma, USA), at 37°C for 48 h in 5% CO2 atmosphere. Subsequently, the supernatants were collected, transferred to a new 96-well flat-bottom culture plates and incubated with Griess reagent, prepared by mixing equal volumes of sulfanilamide 1% in phosphoric acid solution 5% and naphthylethylene diamine dihydrochloride 0.1%, at room temperature for 5 min. Samples of each animal were done in triplicate, and the absorbance determined at 540 nm. The concentration of nitrite was obtained by comparison with a standard curve of serially diluted sodium nitrite (Ding et al., 1988) and expressed in micromoles.
Thymocytes proliferation assay
Thymi were aseptically removed. To prepare a single-cell suspension, the cells were teased out in serum-free RPMI-1640 medium. After centrifugation for 10 min at 300g at 4 °C, pelleted cells were resuspended in RPMI-1640 containing 5% FBS and added to 96-well flat-bottomed plates (0.1 ml/well) at a cell density of 5 - 106/ml. The cells were subsequently stimulated with Concanavalin A (mitogenic concentration: 4 μg/ml; Sigma) and incubated at 37 °C in humidified 5% CO2 atmosphere for 72 h. The experiments were performed in triplicate with a final volume of 0.2 ml/well. Cellular proliferation was determined by MTT assay. After incubation of the cells with the MTT reagent for approximately 4 h, acidified isopropanol was added to lyse the cells and solubilize the purple formazan salt crystals. Samples of each animal were done in triplicate, and the absorbance determined by a spectrophotometric method at a wavelength of 570 nm. An increase in cells proliferation results in an elevated amount of MTT formazan and consequently increased absorbance values are measured (Gieni et al., 1995). Experiments were conducted in triplicate.
The splenic tissue was mechanically disrupted by extrusion through a 70µm nylon cell strainer and homogenized in RPMI 1640 medium to produce a single cell suspension. The staining steps were performed in PBS containing 10% heat inactivated normal rat serum, and the cells were washed with PBS containing 1% FBS and 0.01 mol/l sodium azide. Stained cells were stored for analysis in PBS containing 0.01 mol/l sodium azide and 1% paraformaldehyde, in sealed tubes held in the dark. All steps were performed at 4°C. Analysis of these cells was performed using a Becton Dickinson FACScan flow cytometer with DIVA-BD software (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). All conjugated monoclonal antibodies were obtained from BD Biosciences PharMingen (CA, USA): anti-CD3+PE, anti-CD4+FITC and anti-CD8+PerCP.
The methods used for statistical analysis were chosen considering the small sample of animals. Differences among groups were determinate by One-way ANOVA with Bonferroni's post test (Figure 2 and Figure 3) and by Kruskall-Wallis test followed by Dunn's multiple comparisons test (Figure 4, Figure 5 and Table 1). The difference between parasitemia values (Figure 1) was analyzed by the Mann-Whitney nonparametric test.
The results were expressed as means/Standard Error of Mean and as Median and Range. A value of p<0.05 was considered statistically significant. All statistical analysis were made using Graph Pad Prism version 4.0 (GraphPad Software, Inc., San Diego, CA, USA).
Parasitemia (Figure 1)
The peak of parasitemia occurred on 7th day post infection and all groups showed a total absence of trypomastigotes after the 21 days post infection.
On the peak of parasitemia, PRL-treated animals displayed reduced number of parasites when compared to untreated counterpart (p < 0.05).
Peritoneal macrophage counts (Table 1)
On 14th day post inoculum, the infected and PRL-treated group mediated a statistically significant increase in the number of peritoneal macrophages as compared to its infected counterpart (I). However, on 7th and 21st days of the experiment, any significant alteration in the number of macrophages between non-treated or PRL-treated infected animals was observed.
Nitric Oxide (Figure 2)
Without LPS stimulation
For the LPS non-stimulated groups, significant enhanced concentrations of nitrite were observed in infected and PRL-treated group when compared with all other groups, including the infected and non-supplemented counterpart, during the 7th and 21st days of experiments (p < 0.05).
Concerning the non-infected groups (C and CP), no significant alterations in nitrite levels were observed.
With LPS stimulation
On 7 and 14 days of experiments, nitrite concentration reached the highest values for infected and PRL-treated animals when compared to all other groups (p < 0.05).
Thymocytes proliferation assay (Figure 3)
For Con-A non-stimulated infected groups (I and IP), the highest thymocyte proliferation occurred in PRL-treated animals when compared to infected counterpart, during all days of experiments (p < 0.05).
On 14 day post infection, Con A-stimulated cells from infected and PRL-treated animals displayed enhanced levels of thymocyte proliferation when compared to its untreated and infected counterpart (p < 0.05).
Concerning the non-infected groups (C and CP), no significant thymic alterations were observed between them.
Flow cytometry (Figures 4 and 5)
On 21 st day post infection an increase of both CD3+CD4+ and CD3+CD8+ subsets was noted in infected groups when compared to uninfected counterparts.
In addition, on 14th day post infection, enhanced percentage of T CD3+CD4+ lymphocytes was found for PRL treated animals as compared to infected and non-treated animals (p < 0.05).
For CD3+CD8+ splenocytes, the expansion was also more remarkable in infected animals subjected to PRL treatment, when compared with infected and non-treated counterpart, on 21st day post infection (p < 0.05).
In the latest decades, renewed attention has been given to the important actions of PRL outside of the reproductive system, especially on the immunity (Yu-Lee, 2002; Carreño et al., 2005).
Clinical, animal, and in vitro studies suggest that PRL exhibits immune stimulatory properties (Yu-Lee, 1997). This hormone is able to stimulate T, B and NK cells, macrophages, neutrophils, CD34 hematopoietic cells, and antigen-presenting dendritic cells (Matera et al., 2001). However, controversies are still described and discussed by several authors, such as Oberbeck and coworkers (2003) that observed a decreased survival and a profound suppression of cellular immune functions in septic mice, after the administration of prolactin, during systemic inflammation. Furthermore, studies of Matera et al. (1992 and 1997) indicated a double-faceted regulatory role of PRL and concluded that the hormone concentrations, the variety of isotypes, the existence of multiple receptor subunits, and the complexity of their intracellular signaling may explain the specificity of PRL action on different target cells.
Regarding to T.cruzi infection, some papers demonstrate an immuno-stimulatory effect of the pituitary hormone PRL (Pearson, 2007; Corrêa-de-Santana et al., 2009). Several other papers describe a protection from a variety of normally lethal infections, in mice which received PRL, including Salmonella typhimurium (Di Carlo et al., 1993), and Toxoplasma gondii (Benedetto et al., 1995). Furthermore, to complement the in vivo studies Corrêa-de-Santana and coworkers (2009) have shown that in rat cells the PRL production was down-regulated following T. cruzi infection. Our data seem to be complementary to these observations, since PRL treatment induced an enhancement of the immune response, as indicated by the significant reduction in parasitemia levels, on the peak of parasitemia (7 day post infection).
In our experiments, besides the significant reduction of blood parasites cited above, it was also observed enhanced number of peritoneal macrophages as well as increased concentrations of NO compared to untreated groups. Several other papers indirectly confirm our results, demonstrating that peritoneal macrophages activated by cytokines produce large quantities of reactive nitrogen intermediates, such NO, which plays an important role in the intracellular killing of several pathogens, including T. cruzi (Liew et al., 1990; Adams et al., 1990; Murray et al., 1992; Vespa et al., 1994).
Bolander (2001 and 2002), has also reported that PRL leads to a transient elevation in the production of NO in mammary epithelial cells and this NO could be responsible for the enhancement in DNA synthesis thus modulating PRL effects and actions in the cell system. Additionally, studies of Tripathi and Sodhi (2007) also demonstrated that PRL treatment can induce NO production in murine peritoneal macrophages and peripheral neutrophils.
Regarding to the actions of PRL on lymphoproliferation, the data presented in our paper describe enhanced levels of Con A-stimulated thymocytes from infected and PRL-treated animals when compared to its infected and untreated counterparts. For Con-A non-stimulated and infected groups, the highest thymocyte proliferation occurred in PRL-treated animals when compared to infected ones.
The PRL role in maintenance of thymocytes integrity is supported by the mitogenic mechanisms of this hormone. Studies suggest that PRL treatment can induce expression of the antiapoptotic protein (Biswas et al., 2006), Bcl-2, which play an important role in regulating the development, maturation, and activation of lymphocytes. Some researchers describe an increasing effect of PRL in inducing enhanced antigen-specific peripheral T cells proliferation in vitro (Clevenger et al., 1991; Sabharwal et al., 1992). A number of possible factors provide support for the expression of PRL receptor on thymocytes (De Mello-Coelho et al.,1998; Kooijman et al., 2000) and thymic dendritic cells (Carreño et al., 2004) coupling with the regulation of cytokine secretion demonstrated in vitro (Biswas and Chattopadhyay, 1992; Majumder et al., 2002).
According to Abrahamsohn and Coffman (1995) and Brazão et al. (2008), T. cruzi infected cells loose progressively their ability of lymphoproliferative responses to parasite antigens or mitogens, and this fact is a characteristic of the acute phase of Chagas disease (Kierszenbaum and Sztein, 1994). We also observed that infected and untreated animals displayed a significant reduction in the proliferative response when compared to its uninfected counterparts.
Phenotypic analysis to assess the composition and activation status of T lymphocytes during the acute phase of T. cruzi infection was performed, and an enhancement of both CD3+CD4+ and CD3+CD8+ subsets were observed in infected groups. However, it is noteworthy to emphasize that the highest numbers of these T cells subsets where found in the PRL treated and infected group. These results indirectly support some other studies which describe that under stressful conditions, like infective challenges, PRL is important to maintain a steady-state homeostasis of the lymphocytes sub-populations (Dorshkind and Horseman, 2000) balancing the negative effects of immunosuppression mediated by glucocorticoids and other inflammatory mediators (Dorshkind and Horseman, 2001), such as those induced during the acute phase of T. cruzi infection
Our data reveal an evident role of PRL affecting at multiple levels the regulation of the components of the host's immune response, through an integrated cooperation of the immune and neuroendocrine systems. Future experiments in this field need to be directed at improving our understanding of the key mechanisms underlying the involvement of PRL and the immune response during infectious diseases.