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Chagas disease, a tropical disease caused by the hemoflagellate protozoan Trypanosoma cruzi, affects around 10 million people in South America with 14.000 heart failure-induced deaths per year (World Healthy Organization 2010). Chagas heart disease (CHD) is characterised by a complex immune response that begins during the acute phase and persists during all chronic stages, triggered by the presence of parasites or their antigens in cardiac tissue. Leucocyte activation and recruitment characterise CHD; the release of pro-inflammatory cytokines such as IFN-gamma and TNF-alpha, as well as chemokines, potentiates the inflammatory process. The inflammation contributes to vasculopathy and fibrosis of the cardiac tissue, culminating in loss of heart function (Brener & Gazzinelli 1997; Teixeira et al. 2002; Talvani & Teixeira 2011).
Nutritional status is an important modulator of the immune response, determining risk and prognosis of infectious diseases; it is also directly influenced by the infection (Werneck et al. 2011). Malnutrition is the biggest cause of immunodeficiency worldwide. The biological mechanisms of interaction between infections and various nutritional deficiencies, involving both macronutrients (protein, carbohydrate and fat) and micronutrients (electrolytes, vitamins and minerals), have been described in various experimental studies with T. cruzi (Carlomagno et al. 1996; de Souza et al. 2003; Carvalho et al. 2006; Malafaia & Talvani 2011). Protein deficiency reduces production of inflammatory mediators, such as IFN-gamma and TNF-alpha, both essential to activate immune mechanisms against trypanosomatids (De Andrade & Zicker 1995; Malafaia 2009; Serafim et al. 2010). Although cytokine-related protein malnutrition has been reported (Gomes et al. 1994; Carlomagno et al. 1996), the role of chemokines and endothelin-1 in protein nutritional deficiency in T. cruzi infection remains unclear.
Chemokines (CCL2/MCP-1, CCL5/RANTES, CCL3/MIP-1alpha and CCL4/MIP-1beta) and their receptors in particular are essential inflammatory molecules in human Chagas disease; they are associated with high production of IFN-gamma and TNF-alpha in patients with the cardiac form of the disease (Talvani et al. 2004; Gomes et al. 2005; Cunha-Neto et al. 2009; Talvani & Teixeira 2011). Chemokines can also be released by macrophages activated by T. cruzi antigens in vitro (Aliberti et al. 2001; Coelho et al. 2002; Talvani et al. 2009) and by leucocyte recruitment into heart tissue, worsening CHD in mice (Talvani et al. 2000; Teixeira et al. 2002). However, to our knowledge, there is no investigation into CX3CL1's (Fractalkine), a particular chemokine with chemoattractant and adhesion roles, involvement in T. cruzi infection. This chemokine is expressed in endothelial cells activated by pro-inflammatory cytokines (Umehara et al. 2004) and soluble CX3CL1 induces migration of T CD8+, NK cells and macrophages. Its receptor (CXCR1) is also found in these cellular populations (Umehara & Imai 2001). Inappropriately elevated expression of CX3CL1 may result in extensive tissue damage caused by activated leucocytes in chronic inflammatory disease.
On the other hand, endothelin (ET-1), a protease-sensitive vasoconstrictor substance synthesized by cardiomyocytes and endothelial cells, is an inflammatory mediator with potent vasocontractile effects (Yanagisawa et al. 1988; Abraham & Distler 2007) but with an a described role in T. cruzi heart infection (Tanowitz et al. 2005). ET-1, together with bradykinin pathway, seems to possess an oedematogenic property, rendering cardiomyocytes more susceptible to T. cruzi infection (Andrade et al. 2011). Circulating levels of ET-1 rise in association with the pathogenesis of human and experimental CHD (Petkova et al. 2000; Salomone et al. 2001; Camargos et al. 2004; Tanowitz et al. 2005). Dietary intake can also interfere with the ET-1 system. This was demonstrated by a study in rats with induced focal glomerular sclerosis, where animals fed with a 22% of protein expressed more ET-1, ETA and ETB receptors than animals fed with 6% of protein (Nakamura et al. 1995).
As alterations in dietary intake of micronutrients in individuals living in endemic areas might affect immune functions and the risk of distinct infections, as reviewed to other microorganisms (Malafaia & Talvani 2011), the aim of this investigation was to evaluate the effect of a low protein diet on the production of both inflammatory mediators, CX3CL1 and ET-1, and thus on the pathological course during acute and chronic experimental T. cruzi infection.
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Underfeeding, particularly a protein-deficient diet, is accompanied by significant impairment of immune functions and resistance to many infectious diseases in both humans and experimental animals (Fillol et al. 2009; Malafaia 2009; Serafim et al. 2010; Malafaia & Talvani 2011). The precise mechanisms by which protein deficiency interferes with the immune response are essentially unknown, but this metabolic disturbance seems to evolve as an extension of the immune system to help the host survive widespread life-threatening infectious diseases, especially severe infections such as malaria, tuberculosis and trypanosomiasis (Roth et al. 2011). We detected of changes of the inflammatory parameters in the serum and heart of T. cruzi-infected rats fed with a low protein (LP) diet during both acute and chronic phases of experimental infection. A LP diet coupled with T. cruzi infection increased the levels of two inflammatory vascular mediators (ET-1 and CX3CL1) in serum and heart homogenate during the acute and chronic phases of experimental Chagas disease.
Malnutrition and infections, especially among people living in poor and neglected areas, are major obstacles for healthy development and survival worldwide (Koethe et al. 2009). In Latin America, an insufficient diet could change the generation and release of essential inflammatory mediators that would interfere in the course of Chagas heart disease. Cardiac and vascular clinical disturbances observed in individuals with Chagas disease are driven by an inflammatory environment, assuming parasites or their antigenic molecules as triggers for the release and recruitment of a set of inflammatory mediators and activated leucocytes, respectively (Talvani & Teixeira 2011).
ET-1 and its receptors belong to the inflammatory mediators whose function has been studied in experimental models of T. cruzi infection. ET-1 mediates cardiac structural alterations during chronic infection such as heart remodelling and hypertrophy (Shubeita et al. 1990; Tanowitz et al. 2005). In rats, treatment with ET-1 antagonists boosted parasitemia and myocardial parasite load, suggesting that ET-1 receptors may also play a role in the defence against T. cruzi (Petkova et al. 2000; Camargos et al. 2004; Rachid et al. 2006).
Based on other inflammatory disease studies, ET-1 mediates the release of pro-inflammatory cytokines and chemokines, including TNF-alpha, CCL2/MCP-1, CCL3/MIP-1 and CCL5/RANTES (Molet et al. 2000; Simonson & Ismail-Beigi 2011), all of them already described during pathogenesis of experimental and human Chagas disease (Talvani et al. 2000, 2004; Cunha-Neto et al. 2009; Paiva et al. 2009; Guedes et al. 2010). In our investigation, ET-1 was elevated in plasma and in the homogenate of cardiac tissue, but its level was twice higher under the LP diet during the initial phase of T. cruzi infection. A previous study also associated LP diet with ET-1 in rats, showing that a conventional 22% protein diet increased glomerular mRNA levels for ET-1 and its receptors A and B in animals with glomerular sclerosis, in comparison with those receiving (6%) LP diet (Nakamura et al. 1995). In the presence of T. cruzi, at the initial stage of infection, the LP diet interfered with the rodent immune system, which prevents efficient control of parasites at the peak of parasitemia, even in the presence of high levels of serum ET-1 (data not shown). CX3CL1 is the unique chemokine with dual function: that of an adhesion molecule and that of a chemoattractant (Umehara & Imai 2001). For our knowledge, it is the first time that CX3CL1 level was analysed during T. cruzi infection, and we observed high serum levels of this chemokine in infected animals after 15 days. However, in the presence of the LP diet, there were high levels of CX3CL1 in serum and cardiac homogenate in both phases of infection. These observations reinforce the idea that increasing of ET-1 results in initial invasion of the cardiovascular system (Petkova et al. 2000), and the establishment of this new pathological condition might induce a rising CX3CL1 production. Both mediators may participate in the release of CCL2, CCL5, CCL3 and other pro-inflammatory cytokines (Umehara et al. 2004; Popovic et al. 2008; Simonson & Ismail-Beigi 2011) and, in the particular case of T. cruzi infection, may intensify myocardial dysfunction in experimental models (Petkova et al. 2000; Maldonado et al. 2004).
Coincidentally, CX3CL1 also facilitates the recruitment of circulating leucocytes, especially those CD14+ and CD16+ monocytes expressing or co-expressing CX3CR1, by mediating cell adhesion in transmigration pathways (Bazan et al. 1997; Umehara & Imai 2001; Ancuta et al. 2003). In this study, rats fed with NP presented greater inflammatory infiltration with resident (CD163) and recruited (CD68) macrophages in cardiac tissue. After cardiac muscle damage, monocytes/macrophage influx into inflammatory tissues in experimental models with T. cruzi infection rose. These monocytes/macrophages are, in part, over-stimulated by IFN-gamma and become the most important sources for the release of other inflammatory mediators aiming to kill T. cruzi (Talvani et al. 2000). CX3CL1 also participates in the attraction and activation of NK cells through CX3CR1, according to studies on non-parasitic diseases (Yoneda et al. 2000; Morris & Ley 2004), which would lead us to believe in a positive feedback towards parasite control, because this cell is the primary source of IFN-gamma in the Chagas disease. Still enriching this hypothesis, T CD8+ and CD4+ cells can also express CX3CR1, and an increasing number of T CD8+, NK and CD68 cells have been observed around 12 days after T. cruzi infection in the rat model, coincidentally at the peak of parasitemia (Maldonado et al. 2004). Our colleagues also observed a significant increasing in the CD163 population at day 20 after infection.
The involvement of ET-1 and CX3CL1 as inflammatory mediators in the immunopathogenesis of experimental Chagas disease appears to be partially dependent on the nutritional status of the mammalian host. It is now widely accepted that a low protein diet causes atrophy of thymus and other lymphoid tissues, reduced B-cell activation, alterations in the cytokine and cellular profile in rodents (Prestes-Carneiro et al. 2006; Fock et al. 2008; Silva et al. 2010) and that these alterations of acquired immune response appear to be partially dependent on the stimulus.
In summary, this study reinforces the conception that protein restriction alters the immune system of infected animals, reflected (1) in a high load of circulating parasites and increasing of ET-1 and CX3CL1 production in serum and tissue during the initial phase of infection, and (2) leucocyte influx control into heart tissue. This study provides insight into possible mechanisms by which a low protein diet may affect experimental CHD and also presents the chemokine CX3CL1 as a potential inflammatory mediator contributing to the pathogenesis of T. cruzi infection.