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. Author manuscript; available in PMC: 2021 Jun 1.
Published in final edited form as: Parasitol Res. 2020 Mar 24;119(6):1829–1843. doi: 10.1007/s00436-020-06645-z

The role of fat on cardiomyopathy outcome in mice models of chronic Trypanosoma cruzi infection

Paul Zaki 1,*, Elisa LBC Domingues 2,*, Farhad M Amjad 1, Maiara B Narde 2, Karolina R Gonçalves 3, Mirelle L Viana 2, Heberth de Paula 2, Wanderson G de Lima 4, Huan Huang 1, Maria T Bahia 3,4, Phillip E Scherer 5, Fabiane M dos Santos 2,**, Louis M Weiss 1,6,**, Herbert B Tanowitz 1,6
PMCID: PMC7263952  NIHMSID: NIHMS1579132  PMID: 32206887

Abstract

The underlying pathogenic mechanisms of cardiomyopathy in Chagas Disease are still unsolved. In order to better clarify the role of fat on the evolution of cardiomyopathy, the present study employed three murine models of chronic Trypanosoma cruzi infection: (1) aP2-RIDα/β transgenic mice (RID mice; an adipose tissue model which express a gain-of function potent anti-inflammatory activity), (2) allograft inflammatory factor-1 knockout mice (Aif1−/−) and (3) a Swiss outbred mice. RID mice and non-transgenic mice (wild type – WT) were infected with blood trypomastigotes of Brazil strain. During acute stage RID showed lower parasitemia, lower heart inflammation and a decrease in the relative distribution of parasite load from cardiac muscle tissue towards epididymal fat. Nevertheless, comparable profiles of myocardial inflammatory infiltrates and relative distribution of parasite load were observed amongst RID and WT at chronic stage. Aif1−/− and Aif1+/+ mice were infected with bloodstream trypomastigotes of Tulahuen strain and fed with high fat diet (HFD) or regular diet (RD). Interestingly, Aif1+/+ HFD infected mice showed the highest mortality. Swiss mice infected with blood trypomastigotes of Berenice-78 strain on a HFD had higher levels of TNFα and more inflammation in their heart tissue than infected mice fed a RD. These various murine models implicate adipocytes in the pathogenesis of chronic Chagas Disease and suggest that HFD can lead to a significant increase in the severity of parasite-induced chronic cardiac damage. Furthermore, these data implicate adipocyte TLR4-, TNFα- and IL-1β- mediated signaling in pro-inflammatory pathways and Aif-1 gene expression in the development of chronic Chagas disease.

Keywords: Trypanosoma cruzi, adipose tissue, fat pro-inflammatory pathway, Aif-1, high fat diet

INTRODUCTION

Chagas disease is a parasitic zoonosis caused by Trypanosoma cruzi, a flagellate protozoan transmitted to humans primarily by vectors (Chagas 1909). Recognized as one of the 20 neglected tropical diseases of the world, Chagas disease shows considerable morbidity and mortality mainly in endemic Latin American countries. It remains a major public health problem, even more than 100 years after the first description of T. cruzi. The emergence of Chagas disease transmitted to humans through blood transfusions and other non-vector mechanisms has further spread the infection even to non-endemic areas (Schmunis 2007). In addition, emigration from endemic countries has increased the number of infected patients residing in non-endemic regions. For example, the United States has ~300,000 infected individuals and Spain ~42,000 infected individuals presenting important public health challenges (Briceno and Mendez 2007). Furthermore, there are also over 5,500 individuals infected with T. cruzi in Canada, over 80,000 in the rest of Europe and in the Western Pacific region, over 3,000 in Japan, and ~1,500 in Australia (Schmunis and Yadon 2010; Coura and Viñas 2010).

Clinical manifestations can occur in two different time-points of the infection previously defined as acute and chronic stages of Chagas disease, but the most important features of morbidity are related to the dilated congestive chronic cardiomyopathy that typically develops years or even decades after the primary infection (Tanowitz et al. 2009; Coura 2007). Based on pathology and clinical findings, chronic Chagas’ disease is usually characterized into three major clinical syndromes: indeterminate, cardiac and gastrointestinal. These syndromes are shaped by genetic and biological variability from both the parasite strain and the host (Macedo and Pena 1998; del Puerto et al. 2012; Caldas et al. 2008). Approximately 20 to 30% of infected people develop signs of cardiac pathology, with progressive damage of the myocardium. Another 10% of infected people develop gastrointestinal manifestations characterized by dilatation of the esophagus and/or colon (Coura and Viñas 2010; Moolani et al. 2012). Approximately 60% of people have the indeterminate stage, where chronic infection can be detected, but symptoms have not developed.

Although the pathogenesis of Chagas heart disease is still unsolved, chronic heart failure is related to the persistence of the parasite in the heart, leading to a cascade of tissue destruction, myocarditis, fibrosis, and ventricular dilatation (Benvenuti et al. 2008; Santos et al. 2012; Caldas et al. 2013). The transition from acute to chronic phase, which is accompanied by a marked decrease in parasitemia due to the mounting of a relatively effective immune response to keep parasites below detectable levels in the host. The cause-effect relationship of the acute phase parasitemia with the outcome leading to chronic disease is still poorly understood (Sanches et al. 2014). Variations in the number of circulating parasites, including high peaks of parasitemia during the acute stage, have been shown to be dependent on the parasite strain, inoculation dose and also associated with an inadequate protective host immune response (Kayama and Takeda 2010; Vago et al. 2000). In this context, the role of additional host factors in the development of chronic chagasic cardiomyopathy has been investigated. Amongst these host factors is the physiological state of adipose tissue.

Adipocytes are a safe long-term reservoir for T. cruzi and contribute to the pathogenesis of Chagas chronic cardiomyopathy (Tanowitz et al. 2017). Previous findings examined the contributions of a high fat diet in an acute in vivo model of T. cruzi infection. Decreased levels of parasitemia, mortality and myocardium parasite load were observed in infected CD1-mice fed with high fat diet (Nagajyothi et al. 2014). In the current manuscript we examined the role of the inflammatory response of adipocytes during acute and chronic T. cruzi infection. In addition, we evaluated the effects of high fat diet in a model of Aif-1 gene expression and in a Swiss outbred model of chronic T. cruzi infection. Since increased Aif-1 expression has been identified in clinically important inflammatory conditions, e.g. rheumatoid arthritis, systemic sclerosis, endometriosis, and transplant-associated arteriosclerosis (Casimiro et al. 2013), we investigated the role of Aif-1 in the inflammatory response and outcome during T. cruzi infection.

METHODS

Ethical approval

All animal procedures and experimental protocols concerning experiments with transgenic aP2-RID α/β mice model and Aif-1 target inactivated mice model were approved by the Institutional Animal Care and Use Committees (IACUC) of Albert Einstein College of Medicine (No. 20151206) and conducted in accordance with the guidelines of the National Research Council (Guide for Care and Use of Laboratory Animals: Eight Edition, Washington, DC: The National Academic Press, 2011).

All procedures or experimental protocols conducted in the Swiss mice model were performed according to CONCEA (National Council for Control of Animal Experimentation) and behavior instructions for the use of animals in research from Brazil. The experiment with Swiss mice model was also previously approved by the Ethics Committee on the Use of Animals of the Federal University of Espírito Santo (protocol number 43/2015).

Transgenic aP2-RID α/β mice model

In order to examine the effect of the absence of an inflammatory response in adipocytes, the present work employed the aP2-RID α/β transgenic mice (RID) and a control model consisting of non-transgenic mice in the same background (wild type – WT). This transgenic model was developed by Dr. Sherer at UT Southwestern to selectively inhibit pro-inflammatory signaling pathways in the adipocyte. RID α/β is an adenoviral protein complex that suppresses the local host immune response by potently inhibiting a number of pro-inflammatory signaling pathways (e.g. TLR4-, TNFα- and IL-1β- mediated signaling) (Chin and Horwitz 2005). A total of 22 six-week old RID and 22 six-week old WT male mice were inoculated via the intraperitoneal route with 1.0 x 104 Brazil strain bloodstream trypomastigotes. Another eight age-matched uninfected RID male mice and eight age-matched uninfected WT male mice were used as negative controls. Animals were maintained on a 12-hour light/dark cycle, fed with commercial chow and water ad libitum.

The role of fat pro-inflammatory pathways TLR4-, TNFα- and IL-1β- mediated signaling under the levels of T. cruzi in the vertebrate host were followed in blood, heart, epididymal fat and subcutaneous fat during the acute and chronic stages of infection.

The level of circulating parasites was followed during acute stage of infection at two day intervals until 10 days post-infection (dpi), then at five day intervals until 30 dpi and thereafter weekly until 60 dpi. Ten microliters of fresh blood were collected from mouse tail and the number of circulating parasites was evaluated by counting in a Neubauer hemocytometer as described previously (Morris et al. 1989). Parasitemia curves were plotted considering the average of parasites per milliliter of peripheral blood obtained from infected animals. Mortality was followed in parallel with the parasitemia evaluation until 60 dpi.

Quantitative assessment of parasite load was determined in DNA samples from heart tissue, white epidydimal fat and beige anterior subcutaneous fat collected from mice at 15 dpi and 60 dpi, respectively at acute and chronic stages of infection. Fresh tissues were stored at −80°C until DNA isolation using Trizol DNA extraction protocol (Invitrogen) following the manufacturer's instruction. A standard curve in the range of 0.01 pg to 1 ng for the quantification of T. cruzi DNA by real time polymerase chain reaction (qPCR) was developed using the T. cruzi 195-bp repeat DNA-specific primers TCZ-F (5′-GCTCTTGCCCACAAGGGTGC-3′) and TCZ-R (5′-CCAAGCAGCGGATAGTTCAGG-3′) and genomic DNA purified from T. cruzi epimastigotes. A quantitative real-time polymerase chain reaction (qPCR) was used to quantify parasite load employing PCR SYBR Green Master Mix (Roche Applied Science, CT) containing MgCl2 employing an iQ5 LightCycler (Bio-Rad). The mouse microglobulin primers β2F2 (5′-TGGGAAGCCGAACATACTG-3′) and β2R2 (5′-GCAGGCGTATGTATCAGTCTCA-3′) were included as an internal control. Melting curve analysis was used to assess primer specificity. The number of parasites per 100 ng of host DNA was calculated by dividing the number of parasites (copies of T. cruzi DNA obtained by real time PCR) by number of host cells (copies of β2-microglobulin obtained by real time PCR).

Blood samples were collected before infection, at 15 dpi, 30 dpi and 60 dpi by retro-orbital bleeding from the mice venous sinus for cytokines multiplex immunoassay. Samples were collected at 10AM. Mice were not fasted prior to collection. Food was provided continuously to the mice during the entire experiment using routine husbandry practices. Serum samples obtained after blood centrifugation at 4°C were stored at − 80°C and sent to the UT Southwestern Metabolic Phenotyping Core facility for cytokine measurements. A Millipore (Billerica, MA) mouse cytokine/chemokine magnetic bead panel immunology multiplex assay kit (Cat# MCYTOMAG-70k-06 IFN-gamma, IL-1-beta, TNF-alpha, IL-4, IL-6, and IL-10) and a MAGPIX® multiplex analyzer were used to the cytokines immunoassay. Spline-curve and 5 PL curve fitting models were used to calculate the different cytokine levels from the samples.

Half of the mice from each group were euthanized at 15 dpi and the survivors were euthanized at 60 dpi for histopathology and morphometric analysis. Heart tissue fragments collected after euthanasia were fixed in a 10% buffered formalin solution, dehydrated, cleared and embedded in paraffin. The blocks were cut into 4 μm-thick sections and stained with hematoxylin and eosin (H&E) for inflammation assessment. Each slide was evaluated and scored in a blinded fashion by an experienced cardiac pathologist (Dr. Stephen M. Factor, Jacobi Medical Center/ Bronx Municipal Hospital, Bronx, NY) using a Nikon optical microscope. Images were also captured and analyzed using NIH-Image J. The inflammatory infiltrate and parasite nests were evaluated based on a semiquantitative approach and the scoring system. For each myocardial sample, histologic evidence of myocarditis and the inflammatory process was classified in terms of degree of degenerating cardiac muscle fibers, level of inflammatory infiltrate, fibrosis and was graded on a three-point scale ranging from 0 to 3+. A zero-score indicated lowest or negligible changes and 3 the most damaged state. According to semiquantitative inflammatory infiltrate classification, 0 was absent; 1, focal or mild myocarditis (lymphocytes seen in 2% to 15% of the entire section); 2, moderate (lymphocytes seen in 20% to 60% of the section); and 3, severe myocarditis (lymphocytes seen in >70% of the section). Mild myocarditis was focal; moderate and severe inflammation was either multifocal or diffuse (Chapadeiro et al. 1988). The quantification of parasites was based on the mean number of amastigotes seen in the entirety of the two examined sections: absent (0 parasites seen), rare (one amastigote nest), moderate (2 to 10 nests), or abundant to more than 10 nests (Castro-Sesquen et al. 2011).

Aif-1 target inactivated mice model

To probe the role of Aif-1 on a model of T. cruzi infection administered a commercial high fat diet (HFD; D12492 Research Diets, Inc., New Brunswick, NJ) or a regular diet (RD; D12450 Research Diets, Inc., New Brunswick, NJ), we employed a mice model with target inactivate Aif-1 gene, the knockout mice (Aif-1−/−) and a control wild type mouse in the same background with preserved Aif-1 gene expression (Aif-1+/+). This knockout model in C57Bl/6 mice was developed by Casimiro et al. (2013) to selectively delete the coding regions of the Aif-1 gene. Developing spermatids and cells of monocyte/macrophage lineage have been found to express Aif-1 in inflammatory conditions.

A total of 10 post-weaning Aif-1−/− male mice and 10 post-weaning Aif-1+/+ male mice were fed either RD or HFD for 50 days before infecting them via intraperitoneal route with 1.0 x 104 Tulahuen strain bloodstream trypomastigotes. The HFD consisted of (by kcal) 60% fat with added cholesterol, 20% protein and 20% carbohydrate; the RD consisted of 10% fat, 20% protein and 70% carbohydrate. All mice were fed the assigned diets for the duration of the experiment. Animals were maintained on a 12-hour light/dark cycle and water ad libitum. Weight gain was weekly evaluated since baseline feeding at 50 days before infection until 50th day post-infection. Mortality data was daily notified until 50th day post-infection.

Swiss outbred chronic mice model

Twelve outbred Swiss female mice (30 days old, 18–20 g body weight) were infected via intraperitoneal administration with 5 x 103 blood trypomastigotes of Berenice-78 strain (T. cruzi II): 6 were fed with HFD (452 Kcal% and 20% fat) and 6 were fed with RD, a standard commercial food for rodents (332 Kcal% and 4% fat). Twelve other female mice were maintained as uninfected/healthy controls, of which 6 were fed with HFD and 6 with RD. The HFD was based on the HFD of Estadella et al. (2004) and consisted of 15g standard feed, 10g roasted peanuts, 10g milk chocolate and 5g corn starch biscuit. This HFD had 20% fat, 20% protein, 48% carbohydrate, 4% fibers and 8% humidity, totalizing 452Kcal% (HFD; Pragsoluções Biociências - Domeneghetti & Correa®, Inc., São Paulo, SP); whereas the composition of the regular diet (RD) was 4% fat, 23% protein, 51% carbohydrate, 4.5% fibers and 17.5% humidity, totalizing 332Kcal% (RD; InVivo®, Inc., Três Coraçõoes, MG). The animals received HFD and RD starting at the time of infection and continuing until day 90. The body weight of each animal and the average food intake of all groups were monitored weekly.

Parasitemia was determined daily from 4th day post infection until there were no detectable parasites. Fresh blood was collected from the mice’s tail and the number of parasites was quantified in accordance with the methodology of Brener (1962). Parasitemia curves were plotted considering the average parasitemia obtained from six mice. The area under the individual parasitemia curve was measured to evaluate the parasitemia levels in T. cruzi-infected groups.

Serum levels of total cholesterol, glucose and low-density lipoprotein cholesterol (LDL-cholesterol) were evaluated in the morning (10AM) before inoculation, and at day 30, 60 and 90 post-infection. Data were obtained via spectrophotometry in a SINNOWA® automatic analyzer, using Bioclin commercial kits. Serum levels of cytokine tumor necrosis factor-alpha (TNF-alpha) were quantified at day 90 by ELISA according to the manufacturer’s instructions (Invitrogen, Camarillo, CA, USA).

Heart and retroperitoneal white adipose tissue (WAT) fragments were collected on the day 90 post-infection for the histopathology. Tissues were fixed in 10% buffered formalin, dehydrated, cleared, embedded in paraffin, 4 μm-thick sections were obtained, and sections were stained with hematoxylin and eosin (H&E). Tissues were assessed for myocardial inflammation and for adipose cell size in WAT. Twenty fields from each H&E section of the heart were randomly chosen with 40x magnification of images obtained from a Leica DM 5000 micro-camera and the Leica Applications Suite software built into the Leica Q-Win plus VS image analyzer. The inflammatory process was evaluated based on the number of cell nuclei quantified in the myocardial muscle of non-infected and infected mice. Infected animals were considered to have a relevant inflammatory process when the cell nucleus numbers were greater than the median value plus one standard deviation quantified in the non-infected group. Twenty photos of each H&E slide of WAT were taken with 40x magnification. The size of five adipose cells (one at each end and one central) of each photo was measured with the aid of the Image-J analyzer in the Leica Applications Suite software.

Statistical analysis

Analysis of areas under the parasitemia curves was performed by individually calculating the area of parasitemia curve from each animal, followed by the unpaired T test for comparison between infected groups. The means of parasite load, cytokine levels, body mass, food intake, total cholesterol, glucose, LDL-cholesterol, white adipose cell size and myocardium inflammatory cells were analyzed via analysis of variance, followed respectively by Tukey’s multiple comparison tests and Dunn’s non-parametric test. Mortality data were analyzed using Gehan-Breslow-Wilcoxon test. In all cases, differences were considered as significant when p < 0.05. Statistical calculations were performed using Graph-Pad Prism software.

RESULTS

Transgenic aP2-RID α/β mice model

Parasites were detected in the peripheral blood of all 22 RID and 22 WT infected mice during the acute stage of infection and a lower area under the curve of parasitemia was observed in RID mice with one distinct peak of 2.25 x 105 trypomastigotes/1mL of blood at 18 dpi (Fig. 1a and Fig. 1b). Both RID and WT infected groups had similar rates of survival during the 60 days of infection (Fig. 1c). The levels of parasite load in tissue during infection was assessed by PCR. T. cruzi DNA from heart, white epididymal fat and beige subcutaneous fat were assessed during both the acute and chronic stages of infection, on days 15 and 60 post-infection respectively. The number of parasites resident in cardiac muscle tissue, epididymal fat and subcutaneous fat were similar between RID and WT mice in both acute and chronic stages of infection (Fig. 2). The mean parasitism detected at 15 dpi inside epididymal fat DNA from RID and WT mice were higher than the parasite load seen in heart muscle tissue (p < 0.05) (Fig. 3), further underlining the importance of adipose tissue as a primary target for infection. To better evaluate the possibility of earlier evasion of circulating parasites preferentially to white epididymal fat instead of cardiac muscle tissue, the ratio of T. cruzi DNA in epididymal fat was compared to T. cruzi DNA in cardiac muscle tissue at 15 dpi and at 60dpi. Only RID mice presented a significant increase in the ratio of parasite load in eFAT compared to cardiac muscle tissue (p < 0.01) at 15 dpi (Fig. 4a), suggesting that the inflammatory response in adipocytes from WT mice was important in limiting parasite load in adipose tissue during acute T. cruzi infection.

Figure 1: Influence of fat pro-inflammatory pathways ablation on circulating trypomastigotes and host survival.

Figure 1:

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(A) Parasitaemia curves during infection (acute stage) in RID and WT mice inoculated with 1.0 x 104 Brazil strain bloodstream trypomastigotes via an intraperitoneal route.

(B) Areas under parasitemia curves at the acute stage in RID and WT mice inoculated with 1.0 x 104 Brazil strain bloodstream trypomastigotes via an intraperitoneal route.

(C) Survival cure during acute and chronic stages of Trypanosoma cruzi infection with Brazil strain.

RID: transgenic mice with adipose tissue ablation/or dysfunction in fat pro-inflammatory pathways TLR4-, TNFα- and IL-1β- mediated signaling named aP2-RID α/β transgenic mouse. WT: wild type control model of non-transgenic mice.

Figure 2: Effect of fat pro-inflammatory pathways ablation on parasite load.

Figure 2:

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Quantitative analysis of the parasite load in RID and WT mice from samples of heart, epididymal fat and subcutaneous fat obtained at 15 and 60 days post-infection with 1.0 x 104 Brazil strain bloodstream trypomastigotes via an intraperitoneal route.

RID: transgenic mice with adipose tissue ablation/or dysfunction in fat pro-inflammatory pathways TLR4-, TNFα- and IL-1β- mediated signaling named aP2-RID α/β transgenic mouse. WT: wild type control model of non-transgenic mice.

Figure 3: Influence of adipose tissue inflammatory pathways ablation on the evolution of parasitic load distribution in different tissues.

Figure 3:

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Quantitative analysis of parasite load using DNA from heart, white epididymal fat and subcutaneous fat obtained at 15 dpi (acute infection) and 60 dpi (chronic infection) in RID and WT mice inoculated via an intraperitoneal route with 1.0 x 104 T. cruzi Brazil strain bloodstream trypomastigotes.

RID: transgenic mice with adipose tissue ablation/or dysfunction in fat pro-inflammatory pathways TLR4-, TNFα- and IL-1β- mediated signaling named aP2-RID α/β transgenic mouse. WT: wild type control model of non-transgenic mice.

Figure 4: The influence of fat pro-inflammatory pathways ablation on the outcome of cardiac damage in a Trypanosoma cruzi experimental infection.

Figure 4:

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(A) Quantitative analysis of the ratio of T. cruzi DNA in epididymal fat compared to T. cruzi DNA in cardiac muscle tissue obtained from mice at 15 and 60 dpi with 1.0 x 104 Brazil strain bloodstream trypomastigotes.

(B) Cardiac inflammatory infiltrate score observed at 15 and 60 dpi with 1.0 x 104 Brazil strain bloodstream trypomastigotes. The graph represents the results obtained from animals included in the non-infected RID (RID NI) and infected RID (RID IF) as well as control uninfected (WT NI) and infected WT (WT IF) at 15 and 60 dpi.

(C to H): Hematoxylin and eosin staining for inflammation assessment. (C) RID infected at 15 days post-infection; (D) WT infected at 15 days post-infection; (E) RID infected at 60 days post-infection; (F) WT infected at 60 days post-infection; (G) RID uninfected, and (H) WT uninfected. *Arrows point to amastigote nests, Images taken at 40x, Bar is 10μm RID: transgenic mice with adipose tissue ablation/or dysfunction in fat pro-inflammatory pathways TLR4-, TNFα- and IL-1β- mediated signaling named aP2-RID α/β transgenic mouse. WT: wild type control model of non-transgenic mice.

Amastigote nests were seen in 80% of WT infected mice at day 15 post infection. In contrast, amastigote nests were not found in cardiac muscle tissue from RID mice on day 15 post-infection. Amastigotes were not seen in cardiac fibers from either RID or WT infected mice at day 60 post-infection. Inflammatory infiltrates (predominantly lymphocytes with polymorphonuclear cells) were more prominent in both atria and ventricles of WT infected mice compared to infected RID mice at day 15 post-infection. Hearts obtained from infected RID mice had fewer parasites and a reduction in inflammation and necrosis in the acute stage of infection, with histological scores ranging from 1 to 2+. Infected WT animals presented higher scores (from 2 to 3+) for inflammation and necrosis than infected RID mice on day 15 post-infection (Fig. 4b). Inflammation in the connective tissue of the atria and epicardial adipose tissue inflammation was also seen in 20 to 40% of WT infected mice on day 15 post-infection.

In contrast, there were no statistical differences in the histology of hearts from RID and WT infected mice at day 60 post-infection. The histological inflammatory score of hearts were ~0.5+ in RID infected mice and 0.5 to 1.5+ in WT infected mice. The majority of RID and WT infected animals presented mild or focal inflammation in the right ventricle, and mild inflammation in the atria. Mild inflammation in epicardial fat was also seen in about 50% of WT infected mice (Fig. 4b). All the control uninfected RID and WT mice had heart histological scores of 0 and infiltrated immune cells were absent in cardiac muscle fibers.

Plasma levels of IFN-γ, IL-1β, TNF-α, IL-4, IL-6, and IL-10 were assessed before infection, and at 15 dpi, 30 dpi and 60 dpi. A significant increase in the serum levels of the inflammatory cytokine IFN-γ was observed after 15 and 30 days of infection in both RID and WT infected mice (p < 0.05) (Fig. 5a). TNF-α was more elevated in WT infected mice in relation to non-infected mice at 15 days post-infection (p < 0.05) (Fig. 5a). IL-1β was similar in both RID and WT infected mice at all time points (Fig. 5a). Serum levels of IL-4 and IL-6 were induced in both RID and WT infected mice compared to baseline (p < 0.05). A significant increase of IL-10 on day 15, 30 and 60 post-infection in relation to baseline (p < 0.05) was detected only in RID infected mice (Fig. 5b). As elevated IL-10 is associated with a better outcome in chronic Chagas disease, this suggests that the absence of inflammation in adipose tissue may have a beneficial effect.

Figure 5: Influence of fat pro-inflammatory pathways TLR4-, TNFα- and IL-1β- mediated signaling on the serum levels of cytokines in Chagas experimental disease.

Figure 5:

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(A) Quantification of pro-inflammatory cytokines IFN-γ, Il-1β and TNF-α in serum obtained from mice before infection with Trypanosoma cruzi (white circles) and at 15, 30 and 60 dpi (black circles).

(B) Quantification of regulatory cytokines IL-4, IL-6 and IL-10 in serum obtained from mice at baseline (white circles) and 15, 30 and 60 dpi (black circles).

The results (mean ± SD) represent those obtained from animals from control uninfected aP2-RID α/β transgenic mice (RID NI), control uninfected wild type non-transgenic mice (WT NI), infected aP2-RID α/β transgenic mice (RID IF) and infected wild type non-transgenic mice (WT IF).

*P < 0.05, as assessed using Dunn’s test.

Aif-1 target inactivated mice model

In order to further investigate the role of adipose tissue inflammation on the evolution of T. cruzi infection Aif-1 knockout mice were administered a HFD or RD. Infection was more severe in mice with Aif-1 gene expression fed a HFD. The survival curve of Aif-1+/+ HFD mice was significantly worse than that of Aif-1+/+ RD mice and Aif-1−/− HFD mice after 50 days of T. cruzi infection (p < 0.05) (Fig. 6).

Figure 6: Effect of allograft inflammatory factor 1 (Aif1) plus high fat diet (HFD) on survial in experimental murine Chagas disease.

Figure 6:

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Survival observed in mice during 50 days of infection with 1.0 x 104 Tulahuen strain blood trypomastigotes. The graph represents the results obtained from animals included in the Allograft inflammatory factor 1 knockout (Aif1−/−) and wild type (Aif1+/+) mice infected with T. cruzi and fed with high fat diet (HFD) or regular diet (RD).

Aif1−/− HFD: allograft inflammatory factor 1 knockout mice fed with high fat diet; Aif1−/− RD: allograft inflammatory factor 1 knockout mice fed with regular diet; Aif1+/+ HFD: wild type mice fed with high fat diet; Aif1+/+ RD: wild type mice fed with regular diet.

* P < 0.05, as assessed using Gehan-Breslow-Wilcoxon test.

Swiss outbred chronic mice model

The evaluation of biochemical levels of total cholesterol, glucose and LDL-cholesterol revealed a significant elevation of total cholesterol in mice fed with HFD. After the 1st month of infection (30 days of HFD feeding), both groups of animals fed with HFD (infected and control) had higher levels of total cholesterol compared to the animals fed with RD, independently of the presence or absence of T. cruzi infection (Fig. 7a). Furthermore, there was no difference in the levels of LDL-cholesterol and glucose kinetically evaluated during three months of infection, as indicated respectively in Fig. 7b and Fig. 7c.

Figure 7: Effect of a high fat diet (HFD) on biochemical parameters in a Swiss mouse model of chronic Trypanosoma cruzi infection.

Figure 7:

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Evaluation of serum levels of total-cholesterol (A), LDL-cholesterol (B) and glucose (C) kinetically measured before inoculation, 1, 2- and 3 months after inoculation of the Berenice-78 Trypanosoma cruzi strain into Swiss mice, as well as for non-infected animals fed with a high-fat diet or with a regular diet.

NI-RD: uninfected animals fed with a regular diet. NI-HFD: uninfected animals fed with a hypercaloric high-fat diet. IF-RD: infected animals fed with a regular diet. IF-HFD: infected animals fed with a hypercaloric high-fat diet.

Differences considered significant at P < 0.05, as assessed using Tukey’s test, #indicating significance in relation to NI-RD, &indicating significance in relation to IF-RD, *indicating significance in relation to IF-HFD.

All of the Swiss mice developed parasitemia during the acute stage of infection, confirming 100% of infectivity in mice on both the HFD and RD. However, the HFD had an effect on the parasitemia, as the infected HFD group showed two smaller peaks corresponding to 45000 and 20000 trypomastigotes/0.1mL of blood, respectively, on the day 11 and 22 post infection (Fig. 8a). The mean trypomastigote level in the infected RD group had two distinct peaks corresponding to 65000 and 90000 trypomastigotes/0.1mL of blood respectively at 11 and 24 days post infection (Fig. 8a). Furthermore, the area under the parasitemia curve was smaller (p < 0.05) amongst the infected Swiss mice fed with HFD compared to those fed with RD (Fig. 8b).

Figure 8: Influence of a high fat diet on circulating parasites in a chronic Swiss mouse model of Trypanosoma cruzi infection.

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(A) Parasitemia curves in Swiss mice inoculated with 5000 trypomastigotes of the Berenice-78 T. cruzi strain via intraperitoneal administration.

(B) Areas under the parasitemia curves in Swiss mice inoculated with 5000 trypomastigotes of the Berenice-78 T. cruzi strain via intraperitoneal administration.

Regular diet: animals fed daily with a regular diet throughout all the infection’s stages.

High fat diet: animals fed daily with a hypercaloric high-fat diet throughout all the infection’s stages.

* P < 0.05, as assessed using unpaired T-test.

To assess the effects of HFD on myocardial injury, a quantitative analysis of heart muscle tissue inflammation was performed on the day 90 post infection and the serum levels of TNF-α were measured. The HFD infected Swiss mice had a higher serum TNF-α level (35.9 ± 33.2 pg/mL) after 90 days of infection compared to the healthy control groups (p < 0.05). There were no differences in the levels of TNF-α RD (13.9 ± 3.9 pg/mL) compared to the uninfected mice fed with RD (6.2 ± 4.4 pg/mL) or HFD (9.1 ± 5.5 pg/mL) (Fig. 9).

Figure 9: Effect of a high fat diet on serum inflammatory response in a chronic Swiss mouse model of Trypanosoma cruzi infection.

Figure 9:

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TNF-α serum levels measured at 90 dpi (5000 trypomastigotes of the Berenice-78 T. cruzi strain). Animals fed either a RD or HFD. NI-RD: uninfected animals fed with a regular diet. NI-HFD: uninfected animals fed with a hypercaloric high-fat diet. IF-RD: infected animals fed with a regular diet. IF-HFD: infected animals fed with a hypercaloric high-fat diet. '

*P < 0.05, as assessed using Tukey’s test.

The analysis of heart tissue damage corroborated the findings of TNF-α, the HFD infected Swiss mice had the highest amounts of mononuclear inflammatory cells (298.9 ± 13.8) compared to the other groups of mice: infected RD (286.8 ± 17.4), uninfected HFD (276.2 ± 10.4) and non infected RD (228 ± 11.6) (p < 0.001). Additionally, the heart muscle tissue of both infected groups (RD and HFD) and uninfected animals fed with HFD had more inflammatory cells than that of the healthy control mice fed with RD (Fig. 10). Figure 11 demonstrates an absence of inflammation in RD uninfected mice (Fig. 11a), and a pattern of focal myocardium inflammation with infiltrates of mononuclear inflammatory cells in the presence of T. cruzi infection (Fig. 11b and Fig. 11 d) and a generalized inflammation profile in uninfected HFD mice (Fig. 11c).

Figure 10: Effect of a high fat diet on the outcome of cardiac damage in a chronic Swiss mouse model of Trypanosoma cruzi infection.

Figure 10:

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Quantitative analysis of the myocardium inflammatory process expressed by the number of nuclei cells observed in samples of heart tissue of Swiss mice at 90 dpi with 5x103 blood trypomastigotes of the Berenice-78 Trypanosoma cruzi strain. The graph represents the mean ± SD of the results obtained from animals included in the control non-infected regular diet group (NI-RD), non-infected hypercaloric high-fat diet group (NI-HFD), infected regular diet group (IF-RD) and infected hypercaloric high-fat diet group (IF-HFD).

*P < 0.05, as assessed using Tukey’s test.

Figure 11: Photomicrography of the effect of high fat diet (HFD) and Trypanosoma cruzi infection on the infiltrates of mononuclear inflammatory cells in the myocardium.

Figure 11:

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Hematoxylin and eosin staining for inflammation assessment (40x magnification, scale bar 50μm) in myocardial sections of a non-infected animal fed with a regular diet (RD) (A), infected animal fed with a RD (B), non-infected animal fed with a hypercaloric HFD (C) and infected animal fed with a hypercaloric HFD (D).

To analyze the impact of HFD on adipogenesis in white adipose tissue (WAT), which is considered a secretory organ with inflammatory factors related to obesity and metabolic syndrome, the size of the adipose cells of WAT fragments were evaluated after 90 days of T. cruzi infection. The occurrence of hypertrophy in WAT was more often observed in animals nourished with HFD, mainly in the absence of T. cruzi infection (Fig. 12 and Fig. 13). The size of the adipose cells of infected and uninfected Swiss mice fed with HFD were respectively greater than the size of adipose cells of infected and uninfected Swiss mice fed with RD (Fig. 12). Additionally, it was possible to verify that T. cruzi infection led to a smaller size of the adipose cells, since uninfected groups had greater sizes of adipose cells compared to infected groups fed the same diet (HFD or RD) (Fig. 12). This is consistent with inflammation in the WAT due to T. cruzi infection and previously observed lipolysis in WAT with T. cruzi infection.

Figure 12: Effect of a high fat diet (HFD) on the size of adipose cells of a chronic Swiss mouse model of Trypanosoma cruzi infection.

Figure 12:

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Analysis of the size of adipose cells observed in samples of retroperitoneal white adipose tissue (WAT) of Swiss mice at 90 dpi with 5x103 blood trypomastigotes of the Berenice-78 Trypanosoma cruzi strain. The graph represents the mean ± SD of the results obtained from animals included in the control non-infected regular diet (NI-RD), non-infected hypercaloric high-fat diet (NI-HFD), infected regular diet (IF-RD) and infected hypercaloric high-fat diet (IF-HFD) groups.

* P < 0.05, as assessed using Tukey’s test.

Figure 13: Photomicrography of the effect of a high fat diet (HFD) and Trypanosoma cruzi infection on the size of adipose cells in white adipose tissue.

Figure 13:

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Hematoxylin and eosin staining for size assessment (40x magnification, scale bar 50μm) in retroperitoneal white adipose tissue sections of a non-infected animal fed with a regular diet (RD) (A), of an infected animal fed with a RD (B), of a non-infected animal fed with a hypercaloric HFD (C) and of an infected animal fed with a hypercaloric HFD (D).

DISCUSSION

Given the lack of an effective therapeutic approach for chronic T. cruzi infection (Morillo et al. 2015; Santos et al. 2016) and the invoked role of parasites in causing progression to cardiomyopathy (Garcia et al. 2005; Santos et al. 2012), there is a need to better define the specific pathogenic steps in different tissues over the course of this disease. Previous studies have shown that adipose tissue is an early target of T. cruzi infection (Nagajyothi et al. 2012) and the high affinity of T. cruzi for lipoproteins and adipose tissue could explain how myocardial damage is limited during the acute stage of Chagas’ disease (Nagajyothi et al. 2014).

In mice with a selective reduction of pro-inflammatory pathways involving TLR4-, TNFα- and IL-1β- mediated signaling the observed lower levels of circulating parasites during the acute stage of infection is consistent with T. cruzi trafficking to the adipose tissue. These data suggest that inflammatory pathways in the adipocyte are important to transiently target T. cruzi into white epididymal adipocytes (WAT) rather than cardiac muscle tissue during acute infection.

Benzanidazol and nifurtimox have been successful for cure in acute infection, but have been ineffective during the chronic stage of infection (Morillo et al. 2015; Bahia et al. 2014; Santos et al. 2016). It is possible that the efficacy of trypanocidal drugs currently available for treatment of Chagas disease, Nifurtimox and benznidazole (Guedes et al. 2006; Bern 2015), could be improved by improving the activity of these compounds in adipose tissue. Alternatively, decreases in WAT due to diet and exercise, or pharmacological manipulation of WAT inflammation may be useful adjunctive therapies when combined with trypanocidal agents. Bustamante et al. (2013) demonstrated that successful elimination of the parasite is a function of accessibility of cells and tissues to the drug.

The search for effective therapies for chronic infection is still a major challenge. Although the exact mechanisms that lead to cardiac derangements in chronic Chagas disease are incompletely understood, the involvement of immunopathological mechanisms and the parasite persistence have been linked to the upregulation of many pro-inflammatory pathways (Marin-Neto et al. 2007; Rassi Jr et al. 2007; Gutierrez et al. 2009). The data in the current study suggest that a selective reduction of the contributions of the adipocyte to pro-inflammatory events mediated a relative sequestration of T. cruzi in WAT associated with an amelioration of heart inflammatory changes, especially during acute infection. In addition, the current study revealed that the ratio of T. cruzi DNA from WAT and cardiac muscle tissue are not stable over the course of the infection; specifically, a decrease in this ratio was observed in the progression from acute to chronic infection. Some studies using sensitive detection methods, such as qPCR assays, have revealed clear associations between the presence of the parasite in the heart and evolution of pathological process involved with chronic Chagas cardiomyopathy (Santos et al. 2016). The data presented demonstrate that, in addition, parasites in adipose tissue also play a critical role in the pathogenesis of Chagas heart disease. Notably, there is a lack of a decrease of parasites in beige anterior subcutaneous fat at the chronic stage of infection in both transgenic and non-transgenic fat.

The up-regulation of many pro-inflammatory pathways, activated by the presence of parasite, can underlie the pathological progression of chronic Chagas’ disease (Marin-Neto et al. 2007; Urbina 2009). Herein, we measured plasma levels of inflammatory and regulatory cytokines prior to infection until the chronic stage of the infection to better evaluate the role of the inflammatory response of the adipocyte. Interestingly, an enhancement in TNF-α was observed only in non-transgenic infected mice on day 15 post-infection, and a significant increase in the regulatory cytokine IL-10 were detected only in transgenic (RID) infected mice during all time-points of infection. Other studies have demonstrated that the development of cardiomyopathy in Chagas’ disease correlated with the production of high levels of pro-inflammatory cytokines, such as TNF-α and IFN-γ, and low levels of regulatory cytokines, such as IL-10 and IL-4 (Samudio et al. 1998; D’Ávila et al. 2009; Guedes et al., 2009). Elevated levels of TNF-α, in particular, have been strongly associated with the development of cardiac lesions representing a marker of left ventricular dysfunction in patients with Chagas’ cardiomyopathy and also with systolic ventricular dysfunction in the dog model of experimental T. cruzi infection (Lula et al. 2009; Santos et al. 2012). The data presented here demonstrating an increase in TNF-α levels in inflammation-compromised adipocytes match the immune responses and cardiac alterations linked with higher levels of circulating parasites during the acute stage of infection. The elevation of TNF-α synthesis was maintained after infection in wild type mice.

Various changes in the balance in pro-inflammatory M1 macrophages and regulatory M2 macrophages, or M2 macrophages with a gene expressing profile more consistent with a role in remodeling, may be found among the vast number of immune cells highly enriched in adipose tissue during different disease processes (Tanowitz et al. 2017). During experimental T. cruzi murine infections there is a shift toward M2 macrophages and this has been linked to promoting parasite survival (Cabalén et al. 2016). It, however, is unclear if the M1 response is muted or the M2 population expanded as a consequence of this infection in adipose tissue. Aif-1 is a 17kDa EF hand motif-bearing protein expressed in cells of monocyte/macrophage lineages and related with the increase of inflammatory response in other clinical conditions, e.g. rheumatoid arthritis and systemic sclerosis (Casimiro et al. 2013). Herein we found that T. cruzi infection in mice knockout for Aif-1 had a more aggressive when these mice were administered a HFD, suggesting that the Aif-1 expression in macrophages lineages of adipose tissue may be a key role to maintain homeostasis or promote parasite survival, as Aif-1 gene expression may be exacerbated by host factors such as a HFD (which is also known to pro-inflammatory in adipose tissue). The expression of Aif-1 primarily in immune cells high enriched in adipose tissue may be a key role for the progression toward a more aggressive outcome of T. cruzi infection, mainly in the presence of HFD, a host factor condition which may modulate and enhance the long-term expression of Aif-1 gene.

To evaluate the effects of HFD on the cardiac inflammatory process in a Swiss outbred chronic mouse model of T. cruzi infection, the amount of mononuclear inflammatory cells were assessed in heart muscle tissue at 90 days post infection along with the serum of TNF-α. Interestingly, higher serum levels of TNF-α and greater amounts of inflammatory cells were simultaneously detected in the myocardium tissue of infected HFD mice. The synthesis of the inflammatory TNF-α was stimulated by HFD alone, consistently with the pro-inflammatory role of HFD in obesity and its consequent endothelial trauma (Fernandes et al. 2006; Arslan et al. 2010). High levels of TNF-α are known to lead to a number of inflammatory changes in vascular tissue that increase the likelihood of cardiovascular disease (Winkler et al. 2003). It is worth noting that the present study demonstrates greater amounts of mononuclear inflammatory cells in the heart muscle tissue of infected Swiss outbred mice fed with HFD, suggesting that the host’s immune response pattern together with the T. cruzi infection may contribute to the pathogenesis of Chagas cardiomyopathy.

An overall analysis of our results in these various murine models that alter adipocyte biology is that these data are consistent with the concept that the early invasion of WAT by T. cruzi during acute infection is a critical event ant that the balance of inflammation in the WAT is important in the development of this disease.

ACKNOWLEDGEMENT

This work was supported by the Brazilian institutions Fundação de Amparo à Pesquisa e Inovação do Espírito Santo (FAPES, grant term number 565/2015), Federal University of Espírito Santo (UFES), and by a grant from the United States of America National Institute of Allergy and Infectious Diseases AI124000.

Footnotes

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REFERENCES

  1. Arslan N, Erdur B, Aydin A (2010) Hormones and cytokines in childhood obesity. Indian Pediatr 47:829–839. [DOI] [PubMed] [Google Scholar]
  2. Bahia MT, Diniz Lde F, Mosqueira VC (2014) Therapeutical approaches under investigation for treatment of Chagas disease. Expert Opin Investig Drugs 23(9): 1225–37 [DOI] [PubMed] [Google Scholar]
  3. Benvenuti LA, Rogério A, Freitas HFG et al. (2008) Chronic American trypanosomiasis: parasite persistence in endomyocardial biopsies is associated with high-grade myocarditis. Ann Trop Med Parasitol 102(6):481–487 [DOI] [PubMed] [Google Scholar]
  4. Bern C (2015) Chagas’ disease. N Engl J Med 373:456–466 [DOI] [PubMed] [Google Scholar]
  5. Brener Z (1962) Therapeutic activity and criterion of cure in mice experimentally infected with Trypanosoma cruzi. Rev Inst Med Trop 4: 389–396 [PubMed] [Google Scholar]
  6. Briceno-Leon R, Mendez Galvan J (2007) The social determinants of Chagas disease and the transformations of Latin America. Mem Inst Oswaldo Cruz 102(1): 109–121 [DOI] [PubMed] [Google Scholar]
  7. Bustamante JM, Craft JM, Crowe BD, Ketchie SA, Tarleton RL (2013) New, combined, and reduced dosing treatment protocols cure Trypanosoma cruzi infection in mice. J Infect Dis 209(1):150–162 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cabalén ME, Cabral MF, Sanmarco LM et al. (2016) Chronic Trypanosoma cruzi infection potentiates adipose tissue macrophage polarization toward an anti-inflammatory M2 phenotype and contributes to diabetes progression in a diet-induced obesity model. Oncotarget 7(12):13400–13415 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Caldas IS, Talvani A, Caldas S et al. (2008) Benznidazole therapy during acute phase of Chagas disease reduces parasite load but does not prevent chronic cardiac lesions. Parasitol Res 103:413–421 [DOI] [PubMed] [Google Scholar]
  10. Caldas IS, Guedes PMM, Santos FM et al. (2013) Myocardial scars correlate with eletrocardiographic changes in chronic Trypanosoma cruzi infection for dogs treated with benznidazole. Trop Med Int Health 18(1):75–84 [DOI] [PubMed] [Google Scholar]
  11. Casimiro I, Chinnasamy P, Sibinga NES (2013) Genetic inactivation of the allograft inflammatory factor-1 locus. Genesis 51(10): 1–13 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Castro-Sesquen YE, Gilman RH, Yauri, V et al. (2011) Cavia porcellus as a Model for Experimental Infection by Trypanosoma cruzi. Am J Pathol 179(1):281–288 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chagas C (1909) Nova tripanozomiaze humana. Estudos sobre a morfologia e o ciclo evolutivo do Schizotrypanum cruzi n. gen., n. sp. Agente etiológico de uma nova entidade morbida para o homem. Mem Inst Oswaldo Cruz 1:159–218 [Google Scholar]
  14. Chapadeiro E, Beraldo PS, Jesus PC et al. (1988) Cardiac lesions in Wistar rats inoculated with various strains of Trypanosoma cruzi. [Article in Portuguese.] Rev Soc Bras Med Trop 21:95–103 [DOI] [PubMed] [Google Scholar]
  15. Chin YR, Horwitz MS (2005) Mechanism for removal of tumor necrosis factor receptor 1 from the cell surface by the adenovirus RIDα/β complex. J Virol 79(21):13606–13617 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Coura JR (2007) Chagas disease: what is known and what is needed—a background article. Mem Inst Oswaldo Cruz 102(1): 113–22 [DOI] [PubMed] [Google Scholar]
  17. Coura JR, Viñas PA (2010) Chagas disease: a new worldwide challenge. Nature 465(7301):S6–S7 [DOI] [PubMed] [Google Scholar]
  18. D’Ávila DA, Guedes PM, Castro AM et al. (2009) Immunological imbalance between IFN-g and IL-10 levels in the sera of patients with the cardiac form of Chagas disease. Mem Inst Oswaldo Cruz 104:100–105 [DOI] [PubMed] [Google Scholar]
  19. del Puerto F, Nishizawa JE, Kikuchi M et al. (2012) Protective human leucocyte antigen haplotype, HLA-DRB1*01-B*14, against chronic Chagas disease in Bolivia Neglected Tropical Diseases 6(3):e1587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Estadella D, Oyama LM, Dâmaso AR, Ribeiro EB, Oller do Nascimento CM (2004) Effect of palatable hyperlipidic diet on lipid metabolism of sedentary and exercised rats. Nutrition 20:218–224 [DOI] [PubMed] [Google Scholar]
  21. Fernandes JL, Soeiro A, Ferreira CB et al. (2006) Acute coronary syndromes and inflammation. Rev Soc Cardiol 3:178–187 [Google Scholar]
  22. Garcia S, Ramos CO, Senra JFV et al. (2005) Treatment with benznidazole during the chronic phase of experimental Chagas’ disease decreases cardiac alterations. Antimicrob Agent and Chem 49(4): 1521–1528 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Guedes PM, Fietto JLR, Lana M, Bahia MT (2006) Advances in Chagas disease chemotherapy. Antiinfect Agents Med Chem 5:175–186 [Google Scholar]
  24. Guedes PMM, Veloso VM, Afonso LCC et al. (2009) Development of chronic cardiomyopathy in canine Chagas disease correlates with high IFN-g, TNF-a, and low IL-10 production during the acute infection phase. Vet Immunol Immunophatol 130:43–52. [DOI] [PubMed] [Google Scholar]
  25. Gutierrez FR, Guedes PM, Gazinelli RT et al. (2009) The role of parasite persistence in pathogenesis of Chagas heart disease. Parasite Immunol 31:673–685 [DOI] [PubMed] [Google Scholar]
  26. Kayama H, Takeda K (2010) The innate immune response to Trypanosoma cruzi infection. Microbes Infect 12: 511–517 [DOI] [PubMed] [Google Scholar]
  27. Lula JF, Rocha MO, Nunes MC et al. (2009) Plasma concentrations of tumor necrosis factor-a, tumor necrosis factor-related apoptosis-inducing ligand, and FasLigand/CD95L in patients with Chagas cardiomyopathy correlate with left ventricular dysfunction. Eur J Heart Fail 11: 825–831. [DOI] [PubMed] [Google Scholar]
  28. Macedo AM, Pena SDJ (1998) Genetic variability of Trypanosoma cruzi: implications for the pathogenesis of Chagas disease. Parasitol Today 14(3): 119–124 [DOI] [PubMed] [Google Scholar]
  29. Marin-Neto JA, Cunha-Neto E, Maciel BC et al. (2007) Pathogenesis of chronic Chagas heart disease. Circulation 115:1109–1123 [DOI] [PubMed] [Google Scholar]
  30. Moolani Y, Bukhman G, Hotez PJ (2012) Neglected tropical diseases as hidden causes of cardiovascular disease, PLoS Neglected Tropical Diseases 6(6):e1499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Morris SA, Weiss LM, Factor S, Bilezikian JP, Tanowitz H, Wittner M (1989) Verapamil ameliorates clinical, pathologic and biochemical manifestations of experimental chagasic cardiomyopathy in mice. J Am Coll Cardiol 14(3):782–789 [DOI] [PubMed] [Google Scholar]
  32. Morillo CA, Marin-Neto JA, Avezum A et al. (2015) Randomized trial of benznidazole for Chronic Chagas´ cardiomyopathy. N Engl J Med 373 (14):1925–1306 [DOI] [PubMed] [Google Scholar]
  33. Nagajyothi F, Desruisseaux MS, Machado FS et al. (2012) Response of adipose tissue to early infection with Trypanosoma cruzi (Brazil Strain). J Infect Dis 205(5):830–840 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nagajyothi F, Weiss LM, Zhao D et al. (2014) High Fat Diet Modulates Trypanosoma cruzi Infection Associated Myocarditis. PLoS Neglected Tropical Diseases 8(10):e3118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rassi A Jr, Rassi A, Rassi SG (2007) Predictors of mortality in chronic Chagas disease: a systematic review of observational studies. Circulation 115:1101–1108 [DOI] [PubMed] [Google Scholar]
  36. Samudio M, Montenegro-James S, Cabral M et al. (1998) Cytokine responses in Trypanosoma cruzi-infected children in Paraguay. Am J Trop Med Hyg 58:119–121 [DOI] [PubMed] [Google Scholar]
  37. Sanches TLM, Cunha LD, Silva GK et al. (2014) The Use of a Heterogeneously Controlled Mouse Population Reveals a Significant Correlation of Acute Phase Parasitemia with Mortality in Chagas Disease. PLoS ONE 9(3):e91640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Santos FM, Lima WG, Gravel AS et al. (2012) Cardiomyopathy prognosis after benznidazole treatment in chronic canine Chagas’ disease. J Antimicrob Chemother 67:1987–1995 [DOI] [PubMed] [Google Scholar]
  39. Santos FM, Mazzeti AL, Caldas S et al. (2016) Chagas cardiomyopathy: The potential effect of benznidazole treatment on diastolic dysfunction and cardiac damage in dogs chronically infected with Trypanosoma cruzi. Acta Trop 161:44–54 [DOI] [PubMed] [Google Scholar]
  40. Schmunis GA (2007) Epidemiology of Chagas disease in non-endemic countries: the role of international migration. Mem Inst Oswaldo Cruz 102(1):75–85 [DOI] [PubMed] [Google Scholar]
  41. Schmunis GA, Yadon ZE (2010) Chagas disease: a Latin American health problem becoming a world health problem. Acta Trop 115(1-2): 14–21 [DOI] [PubMed] [Google Scholar]
  42. Tanowitz HB, Machado FS, Jelicks LA et al. (2009) Perspectives on Trypanosoma cruzi-induced heart disease (Chagas disease). Prog Cardiovasc Dis 51:524–539 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tanowitz HB, Scherer PE, Mota MM, Figueiredo LM (2017) Adipose Tissue: A Safe Haven for Parasites? Trends Parasitol 33(4):276–284 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Urbina JÁ (2009) Ergosterol biosynthesis and drug development for Chagas disease. Mem Inst Oswaldo Cruz 104(1):311–318 [DOI] [PubMed] [Google Scholar]
  45. Vago AR, Andrade LO, Leite AA et al. (2000) Genetic characterization of Trypanosoma cruzi directly from tissues of patients with chronic Chagas disease: differential distribution of genetic types into diverse organs. Am J Pathol 156: 1805–1809 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Winkler G, Kiss S, Keszthelyi L et al. (2003) Expression of tumor necrosis factor (TNF-alpha) protein in the subcutaneous and visceral adipose tissue in correlation with adipocyte cell volume, serum TNF-alpha, soluble serum TNF-receptor-2 concentrations and C- peptide level. Eur J Endrocrinol 149(2): 129–135. [DOI] [PubMed] [Google Scholar]

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