All the methodologies performed are in accordance with the standards of the National Council for Control of Animal Experimentation (CONCEA), and were previously approved by the Animal Research Ethics Committee of the Federal University of Ouro Preto (CEUA-UFOP), under the protocol number 36/2015. The experiments comply with the standards of animal research explicit in Law 11.749, of 2008, regulated by Decree No. 6.899, of July 15, 2009.

C57BL/6 male mice aged 21-days were used. The animals were divided into groups according to the composition of diet they received: normolipidic diet, monounsaturated fatty acid diet with olive oil (olive oil diet) and saturated fatty acid diet with lard (lard diet) (Table 1). The diets administration was initiated after weaning and, after 60 days of diet, mice were infected with T. cruzi. The analyses were performed at the day 30 post infection (30 dpi), except in three independent experiments in which the survival rate and the blood parasitemia were followed by 60 days, the period when the number of parasites in the blood decreased.

The food intake was calculated by weighting the food provided every other day divided by the number of mice in the cage. The food intake (g) multiplied by the calorie provided, according to the offered diet) indicates the calorie intake. The Lee's index, indicator of obesity in rodents, proposed by Lee (14) and described by Bernardis and Petterson (15), was calculated by dividing the cubic root of body weight (g) by the naso-anal length (cm) and multiplied the result by 10.

Total cholesterol and triglycerides were determined using commercial kits from Bioclin® (Belo Horizonte, MG, Brazil) according to protocols available by the manufacturer.

The total lipids in the liver were quantified according to Folch method. Briefly, 100 mg liver tissue was macerated in 400 mL of chloroform/methanol (2:1) and centrifuged at 3,000 g, for 10 min at 22°C. Following this, 800 mL of chloroform and 640 mL of NaCl (0.73%) were added to the supernatant, and samples were centrifuged at 3,000 rpm, for 10 min at 22°C. The lower phase was washed three times with 600 mL of Folch solution (a solution of distilled water containing 48% methanol, 3% chloroform, and 2% NaCl at 0.29%) and the extracted lipids were dried overnight at 50°C. The amount of lipid of each sample was calculated by the difference between the weight of samples before and after they were dried.

Mice were inoculated intraperitoneally with 50 blood trypomastigote forms of the Colombian strain of T. cruzi, obtained after two consecutive passages of the strain in swiss mice. After the infection, the blood parasitemia levels were evaluated daily by counting the number of parasites in 5 ml tail-vein blood samples using an optical microscope. Mortality rate among the groups of animals was updated daily. In addition, the body weight was assessed daily by weighing the animals on a digital scale.

The genomic DNA was extracted from 10 mg of heart or adipose tissue using the Wizard® SV Genomic DNA Purification System kit (Promega) according to the manufacturer's instructions. Real-time polymerase chain reaction (PCR) was performed to quantify the heart parasitism as previously described (16).

To determine the number of cells infiltrated in the heart and epididymal adipose tissue, small pieces of the tissues were fixed in dimethyl sulfoxide (DMSO)-Methanol (1:5) for 30 days, dehydrated through successive incubations in crescent concentrations of ethanol, cleared in xylol and fixed in plastic paraffin (Paraplast^®). The paraffin-fixed tissues were cut into sections with a size of 4 μm, stained with hematoxylin and eosin (HE) and the cell nuclei present in the fragments were quantified in 20 images (random fields). The images visualized by the 40X objective were scanned through the Leica DM 5000 B (Leica Application Suite, version 2.4.0R1) and processed through the Leica Qwin V3 image analyzer program.

Total RNA from the heart and epididymal adipose tissue was extracted using the TRIzol reagent (Invitrogen) and the SV Total RNA Isolation System kit (Promega) according to the manufacturer's instructions. Complementary DNA was synthesized from 500 ng of RNA through a High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Real-time PCR assays were performed in a StepOnePlus Real-Time PCR System (Applied Biosystems) using SYBR Green Mix reagents (Invitrogen). The mean cycle threshold (Ct) values from duplicate measurements were used to calculate the expression of the two target genes, which were normalized to the housekeeping genes GAPDH or 18S. The sequences of all primers used are described in Table 2.

Levels of CCL2 were detected in the supernatant of the homogenized cardiac and adipose tissues. For sample preparation, 20 mg of heart and 40 mg of epididymal adipose tissues were macerated in 200 mL of phosphate buffered saline (PBS) and, after centrifugation at 1,500 g, for 10 min at 4°C, the supernatant was collected. Inflammatory mediators were measured by enzyme-linked immunosorbent assay (ELISA) using a specific kit (Peprotech^®) and were performed according to the manufacturer's information. The absorbance values were measured using the eMax ELISA reader (Molecular Devices) at 450 nm.

Catalase activity was determined as described by Aebi (17) based on its ability to convert hydrogen peroxide (H₂O₂) into water and molecular oxygen. Briefly, 100 mg liver tissue was macerated in 1 mL of 0.1 M phosphate buffer, pH 7.2, centrifuged at 10,000 g, 10 min at 4°C. For the assay, 10 μL of the supernatant was added in 50 μL of K₂HPO₄, 40 μL of milli-Q water (Millipore, Bedford, MA) and 900 μL of 2.5 mmol/L H₂O₂. The enzyme activity was measured at 240 nm at 30 s, 2 and 3 min by decaying the absorbances. One-unit (U) of catalase is equivalent to the hydrolysis of 1 mmol of H₂O₂ per minute.

Pyrogallol undergoes autoxidation producing the superoxide anion (O⁻). The SOD enzyme competes for the O⁻ by decreasing the 3-(4,5-Dimethylthiazol-2-yl) (MTT) reduction. For the assay, the supernatant of 100 mg of liver tissue was mixed with MTT and pyrogallol and incubated at 37°C for 5 min. The reaction was stopped with DMSO and the plate was read at 570 nm. The results were expressed as U of SOD per mg of protein in the supernatant of the liver tissue. One unit of SOD is defined as the amount of enzyme required for 50% inhibition of MTT reduction.

For the dosage of oxidized glutathione (GSSG), we used a standard solution of 10 mM oxidized glutathione for the samples and a standard solution of 50 mM oxidized glutathione in 5% sulfosalicylic acid (SSA) for the curve. The samples were obtained from the supernatant of 100 mg of liver homogenized in 1 mL of 5% sulfosalicylic acid buffer - SSA. Following this, 100 μL of the samples were pipetted in 50 mL tubes and pipetted between 0.5 and 2.0 μL of vinylpyridine and 1–5 μL of triethanolamine (TEA) was added to maintain the pH between 6.0 and 7.0. The tubes were filled with distilled water to a volume of 15 mL, homogenized and kept at room temperature for 1 h. After the incubation, 10 μL of samples were measured at 412 nm. The samples were incubated for additional 5 min at room temperature and, afterwards, 50 μL of nicotinamide adenine dinucleotide phosphate (NADPH) was added. The plate was read each 1 min during the 5 min incubation. Oxidized glutathione in the samples was calculated based on pre-defined concentrations for the calibration curve (p1: 0.25, p2: 0.125, p3: 0.062, p4: 0.0312, p5: 0.0156). For the measurement of total glutathione, 10 μL of samples were pipetted in a 96-well plate and immediately after the addition of 50 μL of NADPH, the absorbances were read each 1 min during the 5 min incubation. The values for reduced glutathione (GSH) were obtained from the difference between total and oxidized glutathiones.

Data were expressed as means ± SEM. Multiple groups were compared using one-way analysis of variance (ANOVA) followed by Tukey-Kramer post-test. The survival rate was compared by log-rank test (Mentel-Cox) and the student's t-test was used to compare differences among two experimental groups. All analyzes were performed using the Prism 8 software (GraphPad Software). Groups were considered statistically different when p-values < 0.05.

Firstly, we evaluated if the intake of normal and hyperlipidic diets, and the calories provided by diets were similar among the groups. To address this point, newly weaned mice were fed with either a normolipidic diet, monounsaturated fatty acid diet (olive oil) or with a saturated fatty acid diet (lard) for 90 days. None of them modified mice's body weight (Figure 1A). Although mice fed with normolipidic diet consumed a higher quantity of food than groups fed with olive oil or lard diets (Figure 1B), the calorie intake was similar among all animals (Figure 1C). The effect of hyperlipidic diets on the biochemical and body parameters of mice were also evaluated. The liver/body weight ratio was not different among animals receiving distinct diets (Figure 1D) and mice fed a lard diet presented higher amount of liver lipids when compared to those fed with normolipidic and olive oil diets (Figure 1E). We observed a decreased plasma levels of total cholesterol in mice fed with olive oil and lard diets (Figure 1F) while triglycerides level was similar among all animals (Figure 1G). The Lee's index, an indicative degree of obesity in mice (18), was similar among all animals (Figure 1H), but the weight of the epididymal and subcutaneous adipose tissues was higher in association with the lard diet (Figure 1I).

To evaluate if the olive oil diet or lard diet would affect the response against the Colombian strain of T. cruzi, mice were infected after 60 days of diet. The number of parasites circulating in the mice blood was similar between the infected animals in all days evaluated (Figure 2A); the difference was not evident even during the peak of the parasitemia, at day 27 post infection (Figure 2B). Interestingly, at the day 30 post infection, mice fed with the lard diet presented higher parasitism in the heart (Figure 2C) and adipose tissue (Figure 2D) compared to the mice fed with normolipidic diet or olive oil diet. Despite the high number of parasites among the group fed with lard diet, the percentage of survival was not different between the groups that received the different types of diet (Figure 2E).

Since mice fed with lard diets presented with high tissue parasitism, we investigated whether the diets interfere with the migration of inflammatory cells to the heart and adipose tissue following infection. For this purpose, after 60 days of either normolipidic, olive oil or lard diets followed by 30 dpi with T. cruzi Colombian strain, the production of the chemokine CCL2 and the tissue inflammation were evaluated. The infection increased the CCL2 production in the heart tissue (Figure 3A) and the number of inflammatory cells infiltrating the tissue (Figures 3B,C) independently of the diet administered. Although the lard diet increases the heart CCL2 production after infection compared with infected normolipidic group, the number of inflammatory cells in the tissue is similar among the infected groups independent of the dietary type (Figures 3B,C). Mice fed with the normolipidic diet presented with an increased production of CCL2 in adipose tissue after infection compared to all other dietary group (Figure 4A) despite this, the tissue inflammation increased similarly for all groups (Figures 4A–C).

Since TLRs are particularly important for parasite recognition by immune cells, we investigated whether the hyperlipidic diets could interfere with the TLR expression in the cardiac and adipose tissue cells after T. cruzi infection. After receiving either normolipidic, olive oil or lard diets for 60 days, mice were infected by Colombian strain of T. cruzi and the mRNA expression levels of Tlr2 and Tlr9 were evaluated in the cardiac and adipose tissues of infected and non-infected mice. Infected mice fed with olive oil diet presented higher Tlr2 and Tlr9 expression in the heart (Figures 5A,B) while those fed with lard diet presented higher Tlr2 and Tlr9 expression in the adipose tissue (Figures 5C,D).

The T. cruzi infection-caused oxidative stress, resulting in the accumulation of free radicals, which altered the expression and/or activity of antioxidant enzymes such as oxidized glutathione (GSSG), catalase (CAT), and superoxide dismutase (SOD). Activities of GSSG, CAT and SOD were evaluated in the liver of mice fed with either a control, olive oil, or lard diet for 60 days, followed by 30 dpi with T. cruzi. The infection significantly downregulated the GSH/GSSG ratio in a diet-independent manner (Figure 6A). In response to the parasite-induced oxidative stress, we observed a slight decreased in the CAT activity in mice under normolipidic or olive oil diets, while infected mice under lard diet presented with intense reduction of catalase activity (Figure 6B). In addition, SOD is important for the protection of cells from oxidative insults, and we observed increases in SOD activity after 30 dpi. This increase was independent of the dietary type (Figure 6C).