This study was carried out strictly in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Brazilian National Council of Animal Experimentation (http://www.cobea.org.br/) and the Federal Law 11.794 (October 8, 2008). The institutional Committee for Animal Ethics of the Federal University of Minas Gerais (License protocol number 92/2009) approved all the procedures used in the present study.

Trypomastigotes of the Arequipa T. cruzi strain and the CL Brener clone were cultured in rat L6 myoblast cells using RPMI-1640 medium supplemented with antibiotics (penicillin 60 mg/L and streptomycin 10 mg/L) and 10% fetal bovine serum (FBS), at 37°C in 5% CO₂ atmosphere (Contreras et al., 1985). The Arequipa strain was donated by Gilman’s research group at the Laboratory of Infectious Diseases Research, Universidad Peruana Cayetano Heredia (UPCH), Peru, and the CL Brener clone was donated by Bartholomeu’s group at the Laboratory of Immunology and Genomics of Parasites, Universidade Federal de Minas Gerais (UFMG), Brazil. Previous experimental infections using the Arequipa strain in mice consistently failed to produce detectable parasitemia, and the strain was thus classified as non-virulent (AQP300). Subsequently, this strain was reactivated (AQP-RE) and used to infect mice and evaluate antioxidant activity in macrophages.

To reactivate the non-virulent strain (AQP300), fifth-instar nymphs of Triatoma infestans were artificially fed with blood containing 10⁶ epimastigotes/ml (Kollien and Schaub, 2000). Between days 25 and 28 post-feeding, the contents of the intestinal and rectal ampulla were collected in sterile PBS (pH 7.2, 4°C). The suspension was sterile gas-filtered and subjected to differential centrifugation: 200 g for 5 min to remove macroscopic debris (discard the pellet), and subsequently 3500 g for 10 min at 4°C to recover the parasites (Bonaldo et al., 1988; Contreras et al., 1985). The pellet was resuspended in sterile PBS, and the metacyclic trypomastigotes were quantified in a Neubauer chamber. The percentage of transformation of epimastigotes to metacyclics in triatomines was 35–45% (reported range between 30–50%), and a typical yield of 1 - 5 × 10⁶ metacyclics was obtained per gut group, depending on the initial load and time of collection (Kollien and Schaub, 2000). For in vivo experiments, purified metacyclics were adjusted to 10⁵ parasites/mouse in sterile PBS and inoculated i.p. into C57BL/6 mice. Weight, parasitemia, and animal welfare were monitored to continue with the following experiments.

Peritoneal cells were elicited in C57BL/6 mice by intraperitoneal injection of 2 mL thioglycolate broth per animal. After 72 hours, mice were euthanized by CO₂ inhalation into an unoccupied and clean chamber, followed by cervical dislocation (Johnson and Patterson-Kane, 2020). Peritoneal cells were harvested using 5 mL of sterile cold PBS containing 1% sodium heparin (5000 IU/mL) (Layoun et al., 2015). Cells were kept on ice and centrifuged at 260 g for 10 minutes at 4 °C. The cell pellet was resuspended in RPMI medium with 10% FBS. Cells were then incubated with fluorochrome-conjugated antibodies for 20 minutes at room temperature, protected from light (Supplementary Table 1). After incubation, the cells were centrifuged again at 260 g for 10 minutes and resuspended in 200 μL of PBS for immediate analysis by flow cytometry (Perfetto et al., 2004), using a Cytek Northern Lights (Cytek Biosciences Inc., China). Data were analyzed using FlowJo 10.0 software (Tree Star, Ashland, OR, USA).

Trypomastigotes from CL Brener, AQP300, and AQP-RE strains, along with Zymosan A particles (Saccharomyces cerevisiae; Sigma-Aldrich, was used as a positive control for phagocytosis and ROS/NO production), were labeled with 5 mM carboxyfluorescein succinimidyl ester (CFSE, Invitrogen) for 10 minutes at 37 °C in PBS (Koo et al., 2016) followed by two washes in PBS containing 1% BSA to remove unbound dye. Labeled Zymosan A was resuspended in RPMI and adjusted to 20 mg/mL (stock). Next, peritoneal macrophages (2 x 10⁶ cells/mL) were infected with the labeled trypomastigotes at a 5:1 parasite-to-macrophage ratio or incubated with CFSE-Zymosan A at a 10:1 particle-to-cell ratio (corresponding to ~200 µg/mL final concentration) for 3 hours at 37°C, under gentle orbital agitation (80 rpm) to ensure uniform suspension contact without adherence to plastic surfaces. Non-internalized parasites or excess Zymosan A were removed by washing twice with sterile PBS. Cells were then centrifuged at 260 g for 10 minutes and resuspended in 200 μL of PBS for flow cytometry analysis (Harboe et al., 2012), with 50,000 events acquired within the macrophage gate. For ROS detection, macrophages were treated with 20 μM of carboxy-H₂DCFDA (5- or 6-carboxy-2’,7’-dichlorodihydrofluorescein diacetate) (Duanghathaipornsuk et al., 2021). For NO detection, macrophages were treated with 2 mM of DAF-2DA (4,5-diaminofluorescein diacetate). To evaluate ROS and NO inhibition, 10 mM diphenyleneiodonium chloride (DPI) and 10 mM aminoguanidine (AG) were used to inhibit NADPH oxidase and iNOS activity, respectively (Dhiman and Garg, 2011).

To investigate genetic variation in antioxidant genes, genomic sequencing reads of the Arequipa strain were retrieved from the NCBI Sequence Read Archive (SRA; accession number PRJNA274442) and compared to the CL Brener reference genome. Quality control of the reads was performed using Trimmomatic, with a minimum Phred score of 20 and read length cutoff of 50 bp (Bolger et al., 2014). Cleaned reads were mapped to the CL Brener non-Esmo-like haplotype (version 20), obtained from the TriTrypDB database, using Bowtie 2 (Langmead and Salzberg, 2012). Reads corresponding to annotated antioxidant genes, ascorbate peroxidase, iron superoxide dismutase, trypanothione synthetase, trypanothione reductase, and tryparedoxin peroxidase, were extracted and assembled using the CAP3 Contig Assembly Program (Huang and Madan, 1999). Resulting contigs were aligned to the reference genome using BLASTn (Altschul et al., 1990) and refined using the Contig Editor Program (GeneStudio, USA) until scaffolds for each antioxidant gene were obtained (Supplementary Information). Highly conserved regions were selected for primer design using EPrimer3 (http://bioinfo.nhri.org.tw/cgi-in/emboss/eprimer3), a tool based on the EMBOSS suite (Rice et al., 2000). These primers (Supplementary Table 2) were subsequently used for qPCR analysis of gene expression.

Peritoneal macrophages (1×10⁶ cells/mL) were infected with trypomastigotes from the CL Brener, AQP300 and AQP-RE strains at a parasite-to-cell ratio of 5:1. Samples were collected at 3- and 24-hours post-infection. Total RNA was extracted using TRIzol reagent (Invitrogen, USA) following the manufacturer’s instructions. cDNA was synthesized using reverse transcription with oligo(dT) primers and M-MLV reverse transcriptase (Promega, USA) (Green and Sambrook, 2012). Quantitative PCR was performed in 15 μL reactions using SYBR Green Master Mix (Applied Biosystems, USA), 0.1 μM of each primer, and 2 μL of cDNA. The amplification conditions were: 95 °C for 10 min, followed by 40 cycles at 95 °C for 30 s and 60 °C for 1 min. Expression levels were normalized to TcGAPDH (XM_814806) as a constitutive control. Relative expression was calculated using the 2^–ΔCt method (Livak and Schmittgen, 2001). Data acquisition was done on a StepOne Plus Real-Time PCR System (Applied Biosystems-USA).

C57BL/6 female mice (6–8 weeks old) were maintained at the animal facility of the Universidade Federal de Minas Gerais (UFMG), Brazil. All procedures were conducted under protocols approved by the UFMG Animal Ethics Committee (Protocol No. 92/2009). Mice (n=12 per group) were infected with 1×10⁴; blood trypomastigotes of the AQP-RE strain or CL Brener clone suspended in 200 µL sterile phosphate-buffered saline (PBS, pH 7.4). An uninfected control group (n=4) was included. Parasitemia was monitored from days 5 to 30 post-infection via tail vein blood sampling (5 μL), counted in 50 microscopic fields at 400× magnification (Brener, 1965). Parasite load in cardiac tissue was determined by real-time PCR using DNA extracted from 25 mg of homogenized heart tissue via the phenol-chloroform method (Vago et al., 2000). qPCR reactions were prepared using TaqMan 2X Master Mix with uracil-n-glycosylase (Applied Biosystems), 1 μM primers (cruzi1: 5’-ASTCGGCTGATCGTTTTCGA-3’ and cruzi2: 5’-AATTCCTCCAAGCAGCGGATA-3’), and 0.2 μM probe (cruzi3: 5’-CACACACTGGACACCAA-3’, labeled 5’ with FAM (6-carboxyfluorescein) and 3’ with MGB (minor groove binder)) in a 20 μL volume with 100 ng of DNA (Piron et al., 2007). Amplification was performed at 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 58 °C for 1 min, and 72 °C for 1 min. Data acquisition was done on a StepOne Plus Real-Time PCR System (Applied Biosystems-USA). Quantitative PCR (qPCR) was performed using a standard curve that was generated from 10-fold serial dilutions of genomic T. cruzi DNA (ranging from 10^6 to 10^0 parasite equivalents per reaction) mixed with 100 ng of uninfected mouse DNA to simulate tissue matrix effects. Parasite equivalents were interpolated from the standard curve and expressed as equivalent parasites per mg of tissue (Eq-p/mg) (Cummings and Tarleton, 2003; Duffy et al., 2009; Piron et al., 2007).

To evaluate host immune responses, the expression of IFN-γ, TNF-α, and IL-10 in cardiac tissue was assessed via reverse transcription and qPCR, following protocols described above for antioxidant gene expression. Total RNA was extracted from 25 mg of heart tissue using TRIzol. Mouse β-actin (Actb, NM_007393.5) served as the endogenous reference gene. Relative gene expression was calculated using the 2^–ΔCt method (Livak and Schmittgen, 2001). The oligonucleotide sequences used for amplification were as follows: β-actin (BaF, 5′-GCTTCTTTGCAGCTCCTTCGT-3′; BaR, 5′-CGTCATCCATGGCGAACTG-3′), IL-10 (10F, 5′-CATTTGAATTCCCTGGGTGAGA-3′;10R, 5′-TGCTCCACTGCCTTGCTCTT-3′), TNF-α (TFF,5′-CATCTTCTCAAAATTCGAGTGACAA-3′;TFR,5′-CCTCCACTTGGTGGTTTGCT-3′), and INF-γ (IFF, 5′-TCAGCAACAGCAAGGCGAAA-3′; IFR, 5′-CCGCTTCCTGAGGCTGGAT-3′).

The experiments were performed in triplicate with two repeats. In this study, the results were evaluated by the Mann Whitney test for non-parametric data, using the Graph Prism Instat program, with a 95% confidence interval (p <0.05).

To establish a robust foundation for downstream analyses, we first conducted preliminary validation steps to confirm the experimental model and strain behavior. A reactivation protocol using Triatoma infestans successfully restored the infectivity of the originally non-virulent Arequipa strain (AQP300), yielding the reactivated strain (AQP-RE), which exhibited parasitemia levels comparable to the virulent CL Brener clone (Supplementary Figure 1). Genomic and Bioinformatic analysis confirmed the conservation of seven key antioxidant genes in the Arequipa strain genome, indicating that observed phenotypic differences in antioxidant responses likely stem from transcriptional regulation rather than gene loss (Supplementary Figure 2). Additionally, flow cytometric immunophenotyping of peritoneal exudate cells from C57BL/6 mice validated macrophages with the MHCII⁺Ly6C⁻ phenotype as the predominant cell population, supporting their selection for subsequent infection assays (Supplementary Figure 3). Functional assays using these validated macrophages revealed comparable phagocytosis rates for CL Brener, AQP-RE, and AQP300 strains (46.4 ± 16.1%, 42.5 ± 13.9%, and 44.0 ± 11.7%, respectively; Figure 1A). However, significant differences (p < 0.05) were observed in intracellular effector responses such as NO production. In this sense, macrophages infected with AQP-RE and AQP300 exhibited significantly higher nitric oxide (NO) production (5.7 ± 1.6% and 6.2 ± 1.8%, respectively) than those infected with CL Brener (3.9 ± 1.5%) (Figure 1B). On the other hand, the production of reactive oxygen species (ROS) did not have significant differences, but showed some increase in macrophages infected with AQP-RE (9.8 ± 2.9%) and AQP300 (9.5 ± 2.9%) compared to CL Brener (7.0 ± 2.5%) (Figure 1C). These results suggest that, although phagocytic uptake was similar, the lower production of NO and ROS in response to CL Brener infection could reflect a more effective antioxidant response by this virulent strain, facilitating intracellular survival.

To investigate the early antioxidant response of T. cruzi strains with differing virulence profiles, the expression of key antioxidant enzymes was quantified in macrophages infected with CL Brener, AQP-RE, or AQP300 at 3- and 24-hours post-infection. Macrophages infected with the virulent CL Brener clone exhibited significantly elevated transcript levels (p < 0.05) for multiple redox-regulatory genes, including TcAPX (ascorbate peroxidase), TcCPX (cytosolic tryparedoxin peroxidase), TcMPX (mitochondrial tryparedoxin peroxidase), TcTR (trypanothione reductase), TcTS (trypanothione synthetase), and both cytosolic and mitochondrial TcSODs (superoxide dismutases) (Figures 2A–B) (Supplementary Table 3). These enzymes form the core of the parasite’s antioxidant defense system and are critical for neutralizing host-derived reactive oxygen and nitrogen species. In contrast, the AQP-RE strain displayed a moderate but statistically significant upregulation of select antioxidant genes (TcAPX, TcCPX, TcMPX, and TcSOD-B) relative to the avirulent AQP300 strain (p < 0.05), suggesting partial activation of antioxidant mechanisms. The lowest expression levels were consistently observed in AQP300-infected macrophages, aligning with its inability to establish systemic infection in vivo. These results support the hypothesis that early overexpression of antioxidant enzymes facilitates parasite survival under oxidative stress and is a key feature of virulence in T. cruzi. Reduced antioxidant gene expression in the AQP300 strain likely contributes to its attenuated phenotype and limited replicative capacity within host cells.

In this study, in vivo infection assays using C57BL/6 mice revealed that systemic infection was established exclusively by the CL Brener and AQP-RE strains. In contrast, the parental AQP300 strain failed to induce detectable bloodstream trypomastigotes. Parasitemia became detectable as early as 5 days post-infection (dpi) in both groups. The CL Brener clone displayed a biphasic parasitemia profile, with prominent peaks observed at 15 and 21 dpi (443.2 ± 105.8 and 1328.4 ± 187.4 parasites/μL, respectively). In contrast, AQP-RE exhibited a delayed onset of parasitemia and a single peak at 21 dpi (1328.4 ± 187.4 parasites/μL) and maintaining detectable parasitemia levels for approximately six days before declining. Notably, parasitemia at day 15 was significantly higher in mice infected with CL Brener (1410.4 ± 631.6 parasites/μL) compared to those infected with AQP-RE (443.2 ± 105.8 parasites/μL) (p < 0.05) (Figure 3A). During CL Brener infection, parasitemia peaked earlier and showed a more pronounced rise and fall in blood parasite count. Therefore, while overall parasitemia values for AQP-RE were lower compared to CL Brener, the peak was more prolonged and occurred later. In the same way, the quantitative PCR analysis of cardiac tissue revealed that the CL Brener clone exhibited markedly higher tissue tropism and parasite proliferation at all evaluated time points (p < 0.05). Parasite equivalents per milligram (Eq-P/mg) of cardiac tissue reached 52.8 ± 39.8 at 5 dpi, sharply increasing to 28,037.8 ± 11,659.5 at 15 dpi, then decreasing to 8,826.7 ± 1,275.6 at 30 dpi and 331.5 ± 117.9 at 60 dpi. In contrast, AQP-RE-infected mice showed significantly lower parasite loads: 2.7 ± 1.3, 322.8 ± 75.6, 829.3 ± 385.9, and 63.5 ± 22.5 Eq-P/mg at the same respective time points (Figure 3B). These results confirm the enhanced virulence and persistence of the CL Brener clone in host tissues and highlight the attenuated phenotype of the AQP-RE strain, despite its partial capacity to infect in vivo.

Temporal analysis of cytokine transcript levels in cardiac tissue revealed distinct immunological profiles associated with T. cruzi strain virulence. At 5 days post-infection (dpi), mice infected with the moderately virulent AQP-RE strain exhibited significantly higher expression of pro-inflammatory cytokines IFN-γ and TNF-α compared to those infected with the virulent CL Brener clone (p < 0.05) (Figures 4A, B). This early cytokine response likely reflects increased innate immune activation in response to the parasite’s reduced antioxidant defenses. However, this pattern was reversed at later time points (15, 30, and 60 dpi), when CL Brener-infected mice displayed significantly elevated IFN-γ and TNF-α expression (p < 0.05), consistent with ongoing parasite replication and chronic tissue inflammation. In contrast, expression of the anti-inflammatory cytokine IL-10 followed an inverse pattern. AQP-RE-infected mice showed higher IL-10 transcript levels at 5 and 15 dpi, suggesting early immunoregulatory activity that may contribute to limited tissue pathology. At 30 and 60 dpi, however, IL-10 expression was significantly higher in CL Brener-infected animals (p < 0.05) (Figure 4C) (Supplementary Table 4), indicative of a delayed compensatory immune regulation in response to persistent inflammation and parasitemia. These findings highlight a time-dependent shift in host cytokine responses shaped by parasite virulence. The ability of the CL Brener clone to sustain both pro-inflammatory and regulatory cytokine expression over time reflects its capacity to modulate the host immune environment, thereby enabling chronic infection and promoting cardiac pathology, a hallmark of Chagas disease progression.