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Keywords:

  • CX3CL1/Fractalkine;
  • endothelin-1;
  • low protein diet;
  • inflammation;
  • Trypanosoma cruzi ;
  • Chagas heart disease

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Objective

Chagas heart disease is developed as a result of the infection with Trypanosoma cruzi. Protein malnutrition contributes to secondary immunodeficiency. The aim of this study was to investigate the role of a low protein diet on the production of endothelin-1 and CX3CL1 in blood and cardiac tissue samples in an experimental model with T. cruzi infection.

Methods

Fisher rats were submitted to low protein (6%) and normal protein (15%) diets and then infected with the Y strain of T. cruzi. At days 15 and 120, parasites and immune cells were evaluated.

Results

The low protein diet reduced body weight and circulating serum proteins, but promoted elevation of CX3CL1 and endothelin-1 levels in infected animals, which were unable to control blood parasitemia replication. In heart tissue, the low protein diet reduced cardiac CX3CL1, endothelin-1 and leucocyte infiltration in the acute phase, in particular CD68 and CD163 macrophage phenotypes.

Conclusion

Together, these results highlight the participation of endothelin-1 and CX3CL1 in the inflammatory process of Chagas diesease, both being mediators partially controlled by the host nutritional status.

Objectif

La maladie cardiaque de Chagas se développe à la suite d'une infection par Trypanosoma cruzi. La malnutrition protéique contribue à une immunodéficience secondaire. Le but de cette étude était d’étudier le rôle d'un régime pauvre en protéines sur la production d'endothéline-1 et de CX3CL1 dans des échantillons de sang et de tissus cardiaques dans un modèle expérimental d'infection par T. cruzi.

Méthodes

Des rats Fisher ont été soumis à des régimes de teneur faible (6%) et normal (15%) en protéines et ont ensuite été infectés avec la souche Y de T. cruzi. Aux jours 15 et 120, les parasites et les cellules immunitaires ont été évalués.

Résultats

Le régime alimentaire faible en protéines a réduit le poids corporel et les protéines sériques, mais a promu l’élévation de CX3CL1 et de l'endothéline-1 chez les animaux infectés, qui étaient incapables de contrôler la réplication parasitémique. Dans le tissu cardiaque, le régime alimentaire faible en protéines a réduit le CX3CL1 cardiaque, l'endothéline-1 et l'infiltration leucocytaire dans la phase aiguë, en particulier les phénotypes macrophages CD68 et CD163.

Conclusion

L'ensemble de ces résultats met en évidence la participation de l'endothéline-1 et du CX3CL1 dans le processus inflammatoire de la maladie de Chagas, les deux étant des médiateurs partiellement contrôlés par l’état nutritionnel de l'hôte.

Objetivo

La enfermedad cardiaca por Chagas se desarrolla como resultado de una infección por Trypanosoma cruzi. La desnutrición protéica contribuye a un estado de inmunodeficiencia secundaria. El objetivo de este estudio era investigar el papel de una dieta baja en proteínas sobre la producción de la Endotelina-1 y la CX3CL1 en sangre y muestras de tejido cardiaco, en un modelo experimental con infección por T. cruzi.

Métodos

Se sometieron ratas de Fisher a una de dos dietas: baja en proteínas (6%) y con contenido protéico normal (15%), y después se les infectó con la cepa Y de T. cruzi. En los días 15 y 120 se evaluaron parásitos y células del sistema inmune.

Resultados

La dieta pobre en proteínas redujo el peso corporal y las proteínas circulantes en suero, pero promovió la elevación de los niveles de CX3CL1 y endotelina-1 en animales infectados, que no fueron capaces de controlar la replicación de la parasitemia en sangre. En tejido cardiaco, la dieta pobre en proteínas redujo la CX3CL1 cardiaca, la endotelina-1 y la infiltración de leucocitos en la fase aguda, en particular los fenotipos de macrófagos CD68 y CD163.

Conclusión

Juntos, estos resultados evidencian la participación de la endotelina-1 y la CX3CL1 en el proceso inflamatorio de la enfermedad de Chagas, siendo ambos mediadores parcialmente controlados por el estatus nutricional del hospedero.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Parasites

Blood trypomastigote forms of Trypanosoma cruzi Y strain were maintained through successive passages in Swiss mice at the Laboratory of Chagas disease, Federal University of Ouro Preto (UFOP), Minas Gerais State, Brazil, and used to infect Fisher rats in this study.

Experimental model and infection

Male Fisher rats aged 21 days from the Animal Facility at the Nutrition School, Universidade Federal de Ouro Preto, Minas Gerais, Brazil, were used in this study. Rats were fed low protein – LP (6%) and normal protein – NP (15%) diets for 14 days after weaning and divided into four groups with similar weight according to their respective diets. Ten animals each from the LP and NP groups were infected with 4, 2 × 105 trypomastigotes/70 g of body weight of T. cruzi Y strain (Camargos et al. 2000), while the respective control groups (10 each LP and NP) were not infected with parasites. After infection, groups of animals were maintained in cages with a 12 h light–dark cycle with food (each group with its specific diet ‘LP or NP’) and water ad libitum for the duration of the experiment.

Parasitemia was assessed by collecting fresh blood from the tail of the animal, from the day 3 post-infection until the day when parasites were no longer detectable. The infected groups were euthanised by overdose of ketamine and xylasine on day 15 (acute phase) and on day 120 (chronic phase) post-infection. Experiments were repeated twice to obtain ten animals/group (acute and chronic phases). Experiments adhered to the guidelines issued by the Brazilian College of Animal Experimentation (COBEA) and the Ethics Committee on Animal Research at UFOP - CEUA.

Diets

Low protein and normal protein diets were formulated in the Laboratory of Experimental Nutrition, Nutrition School, UFOP, as shown in Table 1.

Table 1. Nutrients used in each 3000 g of diet (g/Kg)
NutrientsNP dietLP diet
  1. a

    Casein used in normal protein (NP) and low protein (NP) diets were 6% and 15%, respectively.

  2. b

    Quantity in grams was based on protocols of the Laboratory of Experimental Nutrition, ENUT/UFOP and contents of minerals and vitamins were based on Reeves et al. 1993.

Corn starch1597.5 g1987.5 g
Caseina600 g210 g
Sacarose300 g300 g
Oil210 g210 g
Fiber (cellulose)150 g150 g
Mix of saltsb105 g105 g
Mix of vitaminsb30 g30 g
Choline7.5 g7.5 g

Biochemical evaluation

Animals were weighed weekly after weaning to evaluate nutritional parameters. A blood sample was collected by cardiac puncture to evaluate CX3CL1 and ET-1 on day 15 and 120 under deep terminal anaesthesia. Total protein and albumin were also measured in serum using commercial kits (Labtest Diagnóstica S.A, Lagoa Santa, MG, Brazil).

CX3CL1 and endothelin-1 immunoassays

Circulating levels of CX3CL1 and endothelin-1 (ET-1) from rats submitted to a low protein and normal diet were detected in serum obtained from cardiac puncture (2 ml), at the time of euthanasia. For analysis, samples were thawed, and excess of proteins was removed by acid/salt precipitations as routinely performed in our laboratory (Lula et al. 2009). Briefly, equal volumes of plasma and 1.2% trifluoracetic acid/1.35m NaCl were mixed and left at room temperature for 10 min. Afterwards, the samples were centrifuged for 5 min at 10 000 rpm, and supernatants adjusted for salt content (0.14m sodium chloride and 0.01m sodium phosphate) and pH 7.4 to determinate the concentrations of rodent inflammatory mediators. CX3CL1 and endothelin-1 were quantified by ELISA using kits and protocols from the manufacturers (R&D System, UK and Cayman Chemical's Co., respectively). All samples were measured simultaneously in duplicate.

Histological analysis

Rats were euthanised 15 and 120 days post-infection, and fragments of heart (half right atrium and left ventricle) as previously described by Novaes et al. 2011 were taken from each animal for histopathology analysis. Tissue fragments were fixed in 10% buffered formalin solution, dehydrated, cleared and embedded in paraffin. Blocks were cutted into 6-μm-thick sections and stained with haematoxylin–eosin (H&E) for inflammation assessment and amastigote nest evaluation. Twenty fields (74 931 μm2 each) from each H&E stained section were randomly chosen at 40× magnification, giving a total area of 1.49×106 μm2 of analysed myocardium area. Images were obtained through a Leica DM 5000 B microchamber (Leica Application Suite, UK, version 2.4.0 R1) and processed by Leica Qwin (V3) image analyzer software. Cell nuclei were counted in the heart tissue (nuclei from leucocyte + cardiac cells) from non-infected and infected animals fed with LP and NP diets; the degree of inflammation was considered as the quantity of nuclei over the value found in those data from non-infected group, as previously described by Caldas et al. 2008.

CX3CR1 and macrophages immunolabeling in cardiac tissue

Immunolabeling assays were processed in the Laboratory ‘Profa Conceição Machado’, Department of Morphology, UFMG, Brazil. For antigen retrieval, after deparaffinization and rehydration, sections were placed in 0.01m citrate buffer and heated in a microwave oven at 95°C for 10 min (Ramos-Vara 2011). Sections were then washed in phosphate buffer standard (PBS) and submitted to sequential incubations with: PBS containing 0.1m azide and 0.1% peroxide, for endogenous peroxidase activity blockade; PBS-2% BSA solution, for inhibition of unspecific reactions; monoclonal antibodies CX3CR1 (R&D System – 1:100), CD68 (clone ED1) and CD163 (clone ED2) (Serotec – 1:400); in PBS-2% BSA for specific immunostaining; HRP (horseradish peroxidase)-conjugated secondary antibodies and DAB (3,3′Diaminobenzidine) solution for development of the enzymatic reaction. Among all incubations, samples were washed with PBS for 10 min. For morphometric analysis, the software KS-400 Zeiss coupled with an Axioplan 2-Zeiss microscope was used. Normal serum was used instead of primary antibodies in the negative controls.

Statistical analysis

The results of biochemical, immune and immunohistochemistry assays were analysed by nonparametric Newman–Keuls multiple comparison and Tukey. The level of statistical significance was set at  0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Biochemical and physiological parameters

Before the LP diet started, after weaning and at 21 days of life, all animals presented similar body weight, but there was a clear reduction in this physiological parameter after the introduction of the LP diet, independently of the presence or absence of T. cruzi infection (Figure 1a). In consequence, as presented in Table 2, total protein and albumin were always reduced in rats on the LP diet, independently of the acute (15 days) or chronic (120 days) phase of T. cruzi infection. Even without interference of the parasites on the biochemical or physiological parameters, the LP diet exerted partial influence in the parasitemia curve (Figure 1b). Circulating parasites increased around days 4 and 5 of infection, considered the peak of parasites in blood, in animals on a LP diet for 2 weeks after weaning.

Table 2. Serum proteins in mice infected with Trypanosoma cruzi under normal protein (NP) or low protein (LP) diet
 Total protein (mg/dL)
NPLPNPLP
  1. Values are shown as mean ± SD (n = 10/group). *P < 0.05 when LP was compared to NP in distict phases of infection #P < 0.05 when chronic phase was compared with respective acute phase.

15 days0.346 ± 0.0060.291 ± 0.012*0.362 ± 0.0050.289 ± 0.012*
120 days0.381 ± 0.009#0.331 ± 0.003*,#0.408 ± 0.009#0.302 ± 0.008*
 Albumin (mg/dL)
NPLPNPLP
15 days0.248 ± 0.0090.186 ± 0.005*0.239 ± 0.0080.185 ± 0.001*
120 days0.321 ± 0.012#0.222 ± 0.009*,#0.344 ± 0.006#0.252 ± 0.013*,#
 uninfectedinfected with T. cruzi
image

Figure 1. Body weight and profile of circulating Trypanosoma cruzi in rats under low protein diet. After the end of animals weaning, at 21 days of life, healthy rats were weight and a low protein (LP) or a normal protein (NP) diet started and maintained during 14 days before T. cruzi infection (a). Body weight was evaluated during 2 weeks, and during this time, daily tail blood collection was used to quantify circulating parasites in those animals submitted to LP or NP diet (b). Results are given as mean (n = 10/group) ± SEM and * P < 0.05, when compared with NP diet.

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Early levels of serum and cardiac endothelin-1 (ET-1) and CX3CL1 in Trypanosoma cruzi-infected animals

Non-infected rats fed the LP diet for 29 days (see Figure 1a – ‘14 days + 15 days’) presented high levels of serum ET-1, but when this diet was administrated to T. cruzi-infected animals, there was an increase in this inflammatory mediator 15 days post-infection (Figure 2a). The infection with T. cruzi or LP diet per se increased ET-1 levels in short- and long-term infection or feeding, compared with age-matched uninfected animals receiving an NP diet. Coincident with the stabilization of the inflammatory process observed in the late chronic phase of the infection (120 days post-infection), we saw a significant reduction in systemic ET-1 levels in infected animals receiving both LP and NP diets (Figure 2b) in comparison with the acute phase of infection.

image

Figure 2. Concentrations of endothelin-1 in Trypanosoma cruzi-infected rats fed with low and normal protein diets. The concentration of endothelin-1 in serum of T. cruzi-infected rats (n = 10) and in the respective control group (non-infected animals) was measured by ELISA in the acute (a) and chronic (b) phases of experimental infection. In parallel, heart homogenate was also used to evaluate endothelin-1 after 15 days (c) and 120 (d) of disease. * < 0.05 when animals fed with low protein (LP) diet were compared with those fed with normal protein (NP) diet; & P < 0.05 when infected LP group was compared with LP uninfected group.

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Serum levels of ET-1 were analysed in parallel with those from homogenates of cardiac tissue. In the acute phase, ET-1 levels in the animals receiving an LP diet were higher than in those receiving a NP diet (Figure 2c). However, the association of the infection plus LP diet resulted in the highest level of this inflammatory mediator in cardiac tissue homogenate. Similar to serum data, in the chronic phase, ET-1 measured in cardiac tissue homogenate was lower than during the acute phase (Figure 2d). Again, the LP diet increased ET-1 levels in infected and uninfected animals.

Together with ET-1, CX3CL1 was also evaluated in association with T. cruzi infection and with LP diet. There was an elevation in serum levels of CX3CL1 in rats infected with T. cruzi during the acute phase (Figure 3a). However, in chronic infection, serum CX3CL1 remained similar to acute phase levels (Figure 3b). Interestingly and distinctly from ET-1, LP diet did not change the levels of CX3CL1 in homogenates of cardiac tissue among uninfected rats, only in infected ones in both acute and chronic phases under the NP diet (Figure 3c, d).

image

Figure 3. Concentrations of CX3CL1/Fractalkine in Trypanosoma cruzi-infected rats fed with low and normal protein diets. The detection of the chemokine CX3CL1 in serum of T. cruzi-infected rats (n = 10) and respective control group (uninfected animals) was measured by ELISA in the acute (a) and chronic (b) phases of experimental infection. Hearts were also processed and their homogenate used to evaluate CX3CL1 after 15 days (c) and 120 (d) of disease. *P < 0.05 when animals fed with low protein (LP) diet were compared with those fed with normal protein (NP) diet; & P < 0.05 when infected LP group was compared with LP uninfected group.

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Inflammation during infection

Inflammatory infiltration in cardiac tissue in infected animals fed with NP (Figure 4a,e) and LP (Figure 4b,e) was higher than in uninfected rats (small square inserted in Figure 4a,b). After 4 months of infection, there was a reduction in infiltration of leucocytes into the heart tissue with both NP (Figure 4c,f) and LP (Figure 4d,f) diets.

image

Figure 4. Low protein diet reduces leucocyte infiltration during experimental Trypanosoma cruzi infection. Inflammatory infiltration is shown in cardiac tissues from rats fed with normal protein (NP) diet after infection with T. cruzi during acute (a) and chronic (c) phases. Similar histological evaluation was performed in animals fed with low protein (LP) diet in concomitance during the acute- (b) and chronic- (d) T. cruzi infection. Uninfected rats fed with NP or LP diets were also represented by small squares (inserted in a) and (in b), respectively. Quantification of this inflammatory infiltration at 15 days (e) and at 120 days (f) was performed in according to the total number of nuclei in the section less the nuclei from myocardium cells (from unifected groups). Results are given as the mean ± SEM of nuclei in 74 931 μm2 per section of cardiac tissue and * P < 0.05 when animals fed with LP diet were compared with those fed with NP diet; & P < 0.05 when infected LP and NP groups were compared with their respective uninfected groups. Tissues were analysed in sections with a 40× magnification using Leica Application Suite version 2.4.0 R1.

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There were no recruited (CD68) and resident (CD163) stained macrophages in cardiac tissue from those uninfected groups fed with NP and LP diets (Figure 5a). However, the presence of parasites triggers the increase in CD68 (Figure 5b) and of CD163 cells (Figure 5c) in cardiac tissue from rats fed with NP diet. Once more, the LP diet induced a partial reduction in CD68 (Figure 5d) and CD163 (Figure 5e) cells as shown in Figure 5h. Both CD68 and CD163 macrophage analyses were performed during the acute phase (15 days) of infection, when inflammatory process and these macrophages were clearly predominant in heart tissue of T. cruzi-infected rats (Camargos et al. 2000; Maldonado et al. 2004; Rachid et al. 2006).

image

Figure 5. Low protein diet reduces resident and recruited macrophages in cardiac tissue during acute experimental Trypanosoma cruzi infection. Immunohistochemistry using anti-CD68 (clone ED1) ‘recruited macrophages’ (b and d) and anti-CD163 (clone ED2) ‘resident macrophages’ (c and e) were quantified in cardiac tissues (f) during acute phase in those animals receiving NP and LP diets, respectively. Uninfected rats fed with NP and LP diets are represented in (a). Results are given as the mean ± SEM of nuclei in 74931 μm2 per section cardiac tissue and * P < 0.05 when animals fed with LP diet were compared with those fed with NP diet. Small black arrows indicate CD163 (b, c) and CD68 macrophages (d, e). Tissues were analysed in sections with 40× magnification using the software KS-400 Zeiss coupled with an Axioplan 2-Zeiss microscope.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study was supported by Universidade Federal de Ouro Preto (UFOP), Rede Mineira de Bioterismo (FAPEMIG) and research fellowships from Conselho Nacional de Desenvolvimento Científico e Tecnológico (Bahia MT and Talvani A). We also want to thank Carlos Henrique da Silva, from the Laboratory ‘Profa Conceição Machado’ by his technical support.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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