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In the heart tissue of acutely Trypanosoma cruzi -infected mice miR-145-5p and miR-146b-5p are, respectively, downregulated and upregulated. Here, we used the H9C2 rat cardiomyoblast cell line infected with the Colombian T. cruzi strain to investigate the parasite-host cell interplay, focusing on the regulation of miR-145-5p and miR-146b-5p expression. Next, we explored the effects of interventions with the trypanosomicidal drug Benznidazole (Bz) alone or combined with Pentoxifylline (PTX), a methylxanthine derivative shown to modulate immunological and cardiac abnormalities in a model of chronic chagasic cardiomyopathy, on parasite load and expression of miR-145-5p and miR-146b-5p. The infection of H9C2 cells with trypomastigote forms allowed parasite cycle with intracellular forms multiplication and trypomastigote release. After 48 and 144 h of infection, upregulation of miR-145-5p (24 h: 2.38 ± 0.26; 48 h: 3.15 ± 0.9-fold change) and miR-146b-5b (24 h: 2.60 ± 0.46; 48 h: 2.97 ± 0.23-fold change) was detected. The peak of both miRNA levels paralleled with release of trypomastigote forms. Addition of 3 µM and 10 µM of Bz 48 h after infection reduced parasite load but did not interfere with miR-145-5p and miR-146b-5p levels. Addition of PTX did not interfere with Bz-induced parasite control efficacy. Conversely, combined Bz + PTX treatment decreased the levels of both microRNAs, resembling the expression levels detected in non-infected H9C2 cells. Moreover, the use of miR-145-5p and miR-146b-5p mimic/inhibitor systems before infection of H9C2 cells decreased parasite load, 72 h postinfection. When H9C2 cells were treated with miR-145-5p and miR-146b-5p mimic/inhibitor 48 h after infection, all the used systems, except the miR-146b-5p inhibitor, reduced parasite load. Altogether, our data indicate that these microRNAs putatively control signaling pathways crucial for parasite–host cell interaction. Thus, miR-145-5p and miR-146b-5p deserve to be further investigated as biomarkers of parasite control and tools to identify therapeutic adjuvants to etiological treatment in Chagas disease.

The Apicomplexa protozoan Toxoplasma gondii is a mandatory intracellular parasite and the causative agent of toxoplasmosis. This illness is of medical importance due to its high prevalence worldwide and may cause neurological alterations in immunocompromised persons. In chronically infected immunocompetent individuals, this parasite forms tissue cysts mainly in the brain. In addition, T . gondii infection has been related to mental illnesses such as schizophrenia, bipolar disorder, depression, obsessive-compulsive disorder, as well as mood, personality, and other behavioral changes. In the present study, we evaluated the kinetics of behavioral alterations in a model of chronic infection, assessing anxiety, depression and exploratory behavior, and their relationship with neuroinflammation and parasite cysts in brain tissue areas, blood-brain-barrier (BBB) integrity, and cytokine status in the brain and serum. Adult female C57BL/6 mice were infected by gavage with 5 cysts of the ME-49 type II T . gondii strain, and analyzed as independent groups at 30, 60 and 90 days postinfection (dpi). Anxiety, depressive-like behavior, and hyperactivity were detected in the early (30 dpi) and long-term (60 and 90 dpi) chronic T . gondii infection, in a direct association with the presence of parasite cysts and neuroinflammation, independently of the brain tissue areas, and linked to BBB disruption. These behavioral alterations paralleled the upregulation of expression of tumor necrosis factor (TNF) and CC-chemokines (CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β and CCL5/RANTES) in the brain tissue. In addition, increased levels of interferon-gamma (IFNγ), TNF and CCL2/MCP-1 were detected in the peripheral blood, at 30 and 60 dpi. Our data suggest that the persistence of parasite cysts induces sustained neuroinflammation, and BBB disruption, thus allowing leakage of cytokines of circulating plasma into the brain tissue. Therefore, all these factors may contribute to behavioral changes (anxiety, depressive-like behavior, and hyperactivity) in chronic T . gondii infection.

High blood pressure is one of the leading global causes of cardiovascular diseases. The chronic action of high concentrations of angiotensin II (Ang II) promotes arterial hypertension. Ang II acts via AT 1 and AT 2 receptors. Acting via AT 1 R, Ang II can induce the production of inflammatory cytokines and reactive oxygen species, promoting oxidative stress, which may influence cardiac electrical traits. In hypertensive patients, a dispersed QTc interval may predict cardiovascular events and mortality. Benznidazole (Bz), an antiprotozoal prodrug, also has immunomodulatory properties. Here, we tested the idea that in a model of Ang II-induced BP overload, cardiomyopathy will be associated with a prolonged QTc interval. Then, we investigated the effects of Bz therapy on BP overload, electrical changes, and oxidant/antioxidant imbalance. C57BL/6 mice were implanted with an osmotic minipump containing Ang II or saline as a control. At 7 days post-surgery (dps), Ang II infusion increased mean BP, which was sustained until 28 dps. Further, the Ang II-infused group had prolonged QTc interval and QRS complex. Bz or the AT 1 R antagonist losartan (Los) were administered from 7 to 28 dps. Compared with the vehicle-treated group, Los therapy restored mean BP to normal but did not affect long-QTc. At 14 and 28 dps, Bz therapy improved BP, and restored QTc dispersion to normal, while improving RR interval and QRS complex changes. Ang II infusion increased IL-6 concentrations and oxidant/antioxidant imbalance in cardiac tissue. Bz therapy showed a beneficial effect, tending to restore the IL-6 concentrations and oxidant/antioxidant balance to physiological levels, which was correlated with reversal of the dispersed QTc interval. Altogether, our data support that Bz therapy deserves further evaluation as an anti-inflammatory and antioxidant adjuvant tool to improve BP overload and long-QTc syndrome underlying cardiovascular diseases.

CCL3, a member of the CC-chemokine family, has been associated with macrophage recruitment to heart tissue and parasite control in the acute infection of mouse with , the causative agent of Chagas disease. Here, we approached the participation of CCL3 in chronic chagasic cardiomyopathy (CCC), the main clinical form of Chagas disease. We induced CCC in C57BL/6 ( 3 ) and CCL3-deficient ( 3 ) mice by infection with the Colombian Type I strain. In 3 mice, high levels of CCL3 mRNA and protein were detected in the heart tissue during the acute and chronic infection. Survival was not affected by CCL3 deficiency. In comparison with 3 , chronically infected 3 mice presented reduced cardiac parasitism and inflammation due to CD8 cells and macrophages. Leukocytosis was decreased in infected 3 mice, paralleling the accumulation of CD8 T cells devoid of activated CCR5 LFA-1 cells in the spleen. Further, -infected 3 mice presented reduced frequency of interferon-gamma (IFNγ) cells and numbers of parasite-specific IFNγ-producing cells, while the antigen-specific cytotoxic activity was increased. Stimulation of CCL3-deficient macrophages with IFNγ improved parasite control, in a milieu with reduced nitric oxide (NO ) and tumor necrosis factor (TNF), but similar interleukin-10 (IL-10), concentrations. In comparison with chronically -infected 3 counterparts, 3 mice did not show enlarged heart, loss of left ventricular ejection fraction, QTc prolongation and elevated CK-MB activity. Compared with 3 , infected 3 mice showed reduced concentrations of TNF, while IL-10 levels were not affected, in the heart milieu. In spleen of 3 NI controls, most of the CD8 T-cells expressing the CCL3 receptors CCR1 or CCR5 were IL-10 , while in infected mice these cells were mainly TNF . Lastly, selective blockage of CCR1/CCR5 (Met-RANTES therapy) in chronically infected 3 mice reversed pivotal electrical abnormalities (bradycardia, prolonged PR, and QTc interval), in correlation with reduced TNF and, mainly, CCL3 levels in the heart tissue. Therefore, in the chronic infection CCL3 takes part in parasite persistence and contributes to form a CD8 T-cell and macrophage-enriched cardiac inflammation. Further, increased levels of CCL3 create a scenario with abundant IFNγ and TNF, associated with cardiomyocyte injury, heart dysfunction and QTc prolongation, biomarkers of severity of Chagas' heart disease.

TGF-β involvement in Chagas disease cardiomyopathy has been clearly demonstrated. The TGF-β signaling pathway is activated in the cardiac tissue of chronic phase patients and is associated with an increase in extracellular matrix protein expression. The aim of this study was to investigate the effect of GW788388, a selective inhibitor of TβR1/ALK5, on cardiac function in an experimental model of chronic Chagas' heart disease. To this end, C57BL/6 mice were infected with Trypanosoma cruzi (102 parasites from the Colombian strain) and treated orally with 3mg/kg GW788388 starting at 120 days post-infection (dpi), when 100% of the infected mice show cardiac damage, and following three distinct treatment schedules: i) single dose; ii) one dose per week; or iii) three doses per week during 30 days. The treatment with GW788388 improved several cardiac parameters: reduced the prolonged PR and QTc intervals, increased heart rate, and reversed sinus arrhythmia, and atrial and atrioventricular conduction disorders. At 180 dpi, 30 days after treatment interruption, the GW3x-treated group remained in a better cardiac functional condition. Further, GW788388 treatment reversed the loss of connexin-43 enriched intercellular plaques and reduced fibrosis of the cardiac tissue. Inhibition of the TGF-β signaling pathway reduced TGF-β/pSmad2/3, increased MMP-9 and Sca-1, reduced TIMP-1/TIMP-2/TIMP-4, and partially restored GATA-6 and Tbox-5 transcription, supporting cardiac recovery. Moreover, GW788388 administration did not modify cardiac parasite load during the infection but reduced the migration of CD3+ cells to the heart tissue. Altogether, our data suggested that the single dose schedule was not as effective as the others and treatment three times per week during 30 days seems to be the most effective strategy. The therapeutic effects of GW788388 are promising and suggest a new possibility to treat cardiac fibrosis in the chronic phase of Chagas' heart disease by TGF-β inhibitors.

Chronically Trypanosoma cruzi -infected mice show signs of behavioral and cognitive changes, resembling aspects of Chagas disease patients. Inflammatory mediators, such as cytokines and nitric oxide (NO) have been linked to mental disorders. Preclinical studies showed the partial effects of the trypanossomicidal drug benznidazole (Bz) on mnemonic alterations. Here, we investigated the participation of the parasite and systemic inflammatory profile in behavioral and cognitive changes, using Bz combined with the immunoregulator pentoxifylline (PTX). Chronically T. cruzi -infected C57BL/6 mice were treated with Bz (25 mg/Kg/day) and PTX (20 mg/Kg/day) as mono or combined therapies, submitted to behavioral tests, and canonical biological stressors were analyzed. Bz therapy had no effects on anxiety, but partially ameliorated innate compulsive behavior, depression, and memory loss, while PTX and, mainly, Bz + PTX had a broader beneficial effect on these changes. Bz and Bz + PTX reduced parasitemia. The three therapies decreased the parasite burden in the brain. Bz and Bz + PTX therapies reduced oxidative stress in the brain tissue, while PTX and Bz + PTX therapies efficiently controlled the elevated concentrations of GABA/glutamate in the cerebral cortex. Even after parasite control, serum concentrations of NO and tumor necrosis factor (TNF) enhanced as the disease progressed. Bz and, mainly, Bz + PTX treatments reduced NO levels. The three therapeutic schemes hamper the progressive increase of TNF levels. Reanalysis of available data on the systemic miRNA transcriptome supports the beneficial role of Bz + PTX therapy on pivotal hubs involved in inflammation of the central nervous system and neurodegenerative disorders. Moreover, principal components analysis (PCA-2D and 3D projections) underlined the distinction between the noninfected and vehicle-treated infected groups, while Bz + PTX-treated infected mice were closer to noninfected controls. The combined Bz + PTX therapy reduced parasite load and regulated pivotal neurochemical changes in the brain and the systemic inflammatory profile, improving behavioral and cognitive changes in a model of Chagas disease.

Toxoplasma gondii infects one-third of the world population. For decades, it has been considered a silent lifelong infection. However, chronically T. gondii -infected persons may present psychiatric and neurocognitive changes as anxiety, depression, and memory loss. In a model of long-term chronic infection, behavioral alterations parallel neuroinflammation and systemic high cytokine levels, and may reflect brain cyst load. Recent findings support that in chronic infection an active parasite-host interplay involves an immune-mediated control of tissue cysts. Here, we tested the idea that etiological treatment in chronic phase may add advantage to intrinsic immune-mediated cyst control and impact behavioral changes. Thus, we combined sulfadiazine-plus-pyrimethamine (S+P), the first-choice therapy for toxoplasmosis, to study the association of brain cyst load and biological processes related to the immune response (neuroinflammation, blood-brain barrier -BBB- disruption and serum cytokine levels), with behavioral and neurocognitive changes of long-term chronic infection. Female C57BL/6 mice (H-2 b ) were infected (5 cysts, ME-49 strain) and treated with S+P from 30 to 60 days postinfection (dpi), compared with vehicle (Veh)-treated and noninfected controls. At endpoints (pre-therapy, 30 dpi; S+P therapy, 60 dpi; after ceased therapy, 90 dpi), independent groups were subjected to behavioral tests, and brain tissues and sera were collected. Multiple behavioral and neurocognitive changes were detected in the early (30 dpi) and long-term (60 and 90 dpi) chronic infection. S+P therapy resolved locomotor alterations, anxiety, and depressive-like behavior, partially or transiently ameliorated hyperactivity and habituation memory loss. Analysis after therapy cessation showed that S+P therapy reduced the number of stimuli required for aversive memory consolidation. S+P therapy resulted in reduced brain cyst load, neuroinflammation and BBB disruption, and lowered systemic Th1-cytokine levels. Correlation analysis revealed association between IFNγ, TNF and MCP-1/CCL2 serum levels, brain cyst load and behavioral and neurocognitive alterations. Moreover, principal-component analysis (PCA-2D and 3D projections) highlighted distinction between clusters (noninfected; Veh-treated and S+P-treated infected). Thus, our data suggest that S+P therapy added gain to intrinsic brain cyst control and, direct or indirectly, ameliorated inflammation-related alterations, traits associated with behavioral and neurocognitive alterations.

Memory impairment has been associated with chronic Chagas disease (CD), a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi . In degenerative diseases, memory loss has been associated with increased oxidative stress, revealed as enhanced lipid peroxidation, in the cerebral cortex. Benznidazole (Bz), a trypanocidal drug efficient to reduce blood parasite load in the acute and chronic phases of infection, showed controversial effects on heart disease progression, the main clinical manifestation of CD. Here, we evaluated whether C57BL/6 mice infected with the Colombian type I T . cruzi strain present memory deficit assessed by (i) the novel object recognition task, (ii) the open field test and (iii) the aversive shock evoked test, at 120 days post infection (dpi). Next, we tested the effects of Bz therapy (25mg/Kg/day, for 30 consecutive days) on memory evocation, and tried to establish a relation between memory loss, parasite load and oxidative stress in the central nervous system (CNS). At 120 dpi, T . cruzi -infected mice showed memory impairment, compared with age-matched non-infected controls. Bz therapy (from 120 to 150 dpi) hampered the progression of habituation and aversive memory loss and, moreover, reversed memory impairment in object recognition. In vehicle-administered infected mice, neuroinflammation was absent albeit rare perivascular mononuclear cells were found in meninges and choroid plexus. Bz therapy abrogated the infiltration of the CNS by inflammatory cells, and reduced parasite load in hippocampus and cerebral cortex. At 120 and 150 dpi, lipid peroxidation was increased in the hippocampus and cortex tissue extracts. Notably, Bz therapy reduced levels of lipid peroxidation in the cerebral cortex. Therefore, in experimental chronic T . cruzi infection Bz therapy improved memory loss, in association with reduction of parasite load and oxidative stress in the CNS, providing a new perspective to improve the quality of life of Chagas disease patients.

Chagas disease (CD), caused by infection with the protozoan parasite Trypanosoma cruzi, is a neglected tropical illness that affects 6-7 million individuals worldwide. CD may progress to chronic cardiac (CARD), digestive (DIG), or cardio-digestive (CARD/DIG) forms. Parasite genetic diversity, defined in discrete typing units (DTUs) TcI-TcVI and TcBat, may contribute to clinical forms. Here, we challenged this idea by studying a population born in the State of Pernambuco, Northeast of Brazil. Patients born in eco-geographically distinct mesoregions (Sertão, Agreste, Zona da Mata, and Metropolitan Region) attending the referral PROCAPE hospital were serologically diagnosed for CD and characterized as non-CARD (16.5%), CARD (67%), DIG (12.1%), and CARD/DIG (4.3%). Out of 346 CD patients not subjected to etiological treatment, 128 (37%) were positive for conventional PCR targeting T. cruzi kDNA. DTU genotyping using pre-established primers and algorithms revealed 85/128 (66%) samples amplified for at least one target, and of these, 49 (58%) were classified into DTUs, showing a higher distribution of DTUs TcV (16) and TcIII (13) but also TcIV (7), TcI (3), TcVI (3), and TcII (1). Mixed infections by TcI + TcV (2), TcIII + TcIV (3), and TcIII + TcV (1) were also found. Out of the 49 classified samples, 36 were CARD patients (73.5%), infected mainly by TcV (13) and TcIII (10). Considering patients' birthplace, in the Agreste, all DTUs were detected, with prevailing TcV and TcIII; in Zona da Mata, DTU characterization reflected the input of samples (21/49, 43%), again mainly TcV and TcIII. DTUs TcI-TcVI were only detected in the blood of patients born in the Agreste, area of ecological transition. Although not frequently found in human infections in Brazil, the prevailing TcV, TcIII, and TcIV were detected in patients born in Pernambuco. These data could not disclose an identifiable association of T. cruzi DTU circulating in the blood of patients with clinical forms of CD.

Oxidative stress promotes T . cruzi growth and development of chronic Chagas heart dysfunction. However, the literature contains gaps that must be fulfilled, largely due to variations in parasite DTU sources, cell types, mouse strains, and tools to manipulate redox status. We assessed the impact of oxidative environment on parasite burden in cardiomyoblasts and the effects of the Nrf2-inducer COPP on heart function in BALB/c mice infected with either DTU-II Y or DTU-I Colombian T . cruzi strains. Treatment with antioxidants CoPP, apocynin, resveratrol, and tempol reduced parasite burden in cardiomyoblasts H9C2 for both DTUI- and II-strains, while H 2 O 2 increased it. CoPP treatment improved electrical heart function when administered during acute stage of Y-strain infection, coinciding with an overall trend towards increased survival and reduced heart parasite burden. These beneficial effects surpassed those of trypanocidal benznidazole, implying that CoPP directly affects heart physiology. CoPP treatment had beneficial impact on heart systolic function when performed during acute and evaluated during chronic stage. No impact of CoPP on heart parasite burden, electrical, or mechanical function was observed during the chronic stage of Colombian-strain infection, despite previous demonstrations of improvement with other antioxidants. Treatment with CoPP also did not improve heart function of mice chronically infected with Y-strain. Our findings indicate that amastigote growth is responsive to changes in oxidative environment within heart cells regardless of the DTU source, but CoPP influence on heart parasite burden in vivo and heart function is mostly confined to the acute phase. The nature of the antioxidant employed, T . cruzi DTU, and the stage of disease, emerge as crucial factors to consider in heart function studies.