Correlation of blood-based immune molecules with cardiac gene expression profiles reveals insights into Chagas cardiomyopathy pathogenesis.

Introduction: Understanding compartmentalized immune responses in target organs is crucial for elucidating the pathogenesis of various diseases. However, obtaining samples from affected vital organs often poses safety challenges. In this study, we aimed to investigate potential correlations between the levels of disease-associated immune molecules in the bloodstream with their gene expression profiles in the hearts of patients suffering from Chagas Cardiomyopathy (CCC). This debilitating and often fatal condition is caused by infection with the protozoan Trypanosoma cruzi. Methods: Blood samples were analyzed using the Bio-Plex platform. Gene Expression Omnibus (GEO) database was used to determine gene expression profile in heart tissue from CCC and non-Chagas controls (CTRL). Results: Elevated levels of inflammatory cytokines were detected in the plasma of CCC patients, and these levels correlated with clinical indicators of deteriorating cardiac function. Notably, 75% of the soluble factors assessed in the plasma exhibited a consistent relationship with their gene expression levels in the cardiac tissue of CCC patients. Analysis of interactions and signaling pathways related to these molecules revealed an overrepresentation of inflammatory pathways in both blood and heart compartments. Moreover, we identified that differentially expressed genes in CCC cardiac tissue were primarily associated with T-cell signaling pathways and correlated with the presence of CD8+ T cells in the myocardium. Discussion: Our findings establish a strong correlation between relevant immune molecules and their signaling pathways in both the blood and heart tissue in CCC. This validates the use of blood as a non-invasive medium for understanding immunopathology and identifying markers for cardiac dysfunction in Chagas disease.

T-Cell Subpopulations Exhibit Distinct Recruitment Potential, Immunoregulatory Profile and Functional Characteristics in Chagas versus Idiopathic Dilated Cardiomyopathies.

Chronic Chagas cardiomyopathy (CCC) is one of the deadliest cardiomyopathies known and the most severe manifestation of Chagas disease, which is caused by infection with the parasite Trypanosoma cruzi. Idiopathic dilated cardiomyopathies (IDC) are a diverse group of inflammatory heart diseases that affect the myocardium and are clinically similar to CCC, often causing heart failure and death. While T-cells are critical for mediating cardiac pathology in CCC and IDC, the mechanisms underlying T-cell function in these cardiomyopathies are not well-defined. In this study, we sought to investigate the phenotypic and functional characteristics of T-cell subpopulations in CCC and IDC, aiming to clarify whether the inflammatory response is similar or distinct in these cardiomyopathies. We evaluated the expression of systemic cytokines, determined the sources of the different cytokines, the expression of their receptors, of cytotoxic molecules, and of molecules associated with recruitment to the heart by circulating CD4+, CD8+, and CD4-CD8- T-cells from CCC and IDC patients, using multiparameter flow cytometry combined with conventional and unsupervised machine-learning strategies. We also used an in silico approach to identify the expression of genes that code for key molecules related to T-cell function in hearts of patient with CCC and IDC. Our data demonstrated that CCC patients displayed a more robust systemic inflammatory cytokine production as compared to IDC. While CD8+ T-cells were highly activated in CCC as compared to IDC, CD4+ T-cells were more activated in IDC. In addition to differential expression of functional molecules, these cells also displayed distinct expression of molecules associated with recruitment to the heart. In silico analysis of gene transcripts in the cardiac tissue demonstrated a significant correlation between CD8 and inflammatory, cytotoxic and cardiotropic molecules in CCC transcripts, while no correlation with CD4 was observed. A positive correlation was observed between CD4 and perforin transcripts in hearts from IDC but not CCC, as compared to normal tissue. These data show a clearly distinct systemic and local cellular response in CCC and IDC, despite their similar cardiac impairment, which may contribute to identifying specific immunotherapeutic targets in these diseases.

Impact of diminazene aceturate on renin-angiotensin system, infectious myocarditis and skeletal myositis in mice: An in vitro and in vivo study.

Although renin-angiotensin system (RAS) imbalance is manifested in cardiomyopathies with different etiologies, the impact of RAS effectors on Chagas cardiomyopathy and skeletal myositis is poorly understood. Given that diminazene aceturate (DMZ) shares trypanocidal, angiotensin-converting enzyme 2 (ACE2) and angiotensin-(1-7) stimulatory effects, we investigated the impact of DMZ on cardiomyocytes infection in vitro, renin-angiotensin system, Chagas cardiomyopathy and skeletal myositis in vivo. Cardiomyocytes and T. cruzi were used to evaluate DMZ toxicity in vitro. The impact of 20-days DMZ treatment (1 mg/kg) was also investigated in uninfected and T. cruzi-infected mice as follows: control uninfected and untreated, uninfected treated with DMZ, infected untreated and infected treated with DMZ. DMZ had low toxicity on cardiomyocytes, induced dose-dependent antiparasitic activity on T. cruzi trypomastigotes, and reduced parasite load but not infection rates in cardiomyocytes. DMZ increased ACE2 activity and angiotensin-(1-7) plasma levels but exerted no interference on angiotensin-converting enzyme (ACE) activity, ACE, ACE2 and angiotensin II levels in uninfected and infected mice. DMZ treatment also reduced IFN-γ and IL-2 circulating levels but was ineffective in attenuating parasitemia, MCP-1, IL-10, anti-T. cruzi IgG, nitrite/nitrate and malondialdehyde production, myocarditis and skeletal myositis compared to infected untreated animals. As the antiparasitic effect of DMZ in vitro did not manifest in vivo, this drug exhibited limited relevance to the treatment of Chagas disease. Although DMZ is effective in upregulating angiotensin-(1-7) levels, this molecule does not act as a potent modulator of T. cruzi infection, which can establish heart and skeletal muscle parasitism, lipid oxidation and inflammatory damage, even in the presence of high concentrations of this RAS effector.

Thioridazine aggravates skeletal myositis, systemic and liver inflammation in Trypanosoma cruzi-infected and benznidazole-treated mice.

While thioridazine (Tio) inhibits the antioxidant defenses of Trypanosoma cruzi, the gold standard antitrypanosomal drug benznidazole (Bz) has potent anti-inflammatory and pro-oxidant properties. The combination of these drugs has never been tested to determine the effect on T. cruzi infection. Thus, we compared the impact of Tio and Bz, administered alone and in combination, on the development of skeletal myositis and liver inflammation in T. cruzi-infected mice. Swiss mice were randomized into six groups: uninfected untreated, infected untreated, treated with Tio (80 mg/kg) alone, Bz (50 or 100 mg/kg) alone, or a combination of Tio and Bz. Infected animals were inoculated with a virulent T. cruzi strain (Y) and treated by gavage for 20 days. Mice untreated or treated with Tio alone developed the most intense parasitemia, highest parasitic load, elevated IL-10, IL-17, IFN-γ, and TNF-α plasma levels, increased N-acetylglucosaminidase and myeloperoxidase activity in the liver and skeletal muscle, as well as severe myositis and liver inflammation (P < 0.05). All parameters were markedly attenuated in animals receiving Bz alone (P < 0.05). However, the co-administration of Tio impaired the response to Bz chemotherapy, causing a decrease in parasitological control (parasitemia and parasite load), skeletal muscle and liver inflammation, and increased microstructural damage, when compared to the group receiving Bz alone (P < 0.05). Altogether, our findings indicated that Tio aggravates systemic inflammation, skeletal myositis and hepatic inflammatory damage in T. cruzi-infected mice. By antagonizing the antiparasitic potential of Bz, Tio limits the anti-inflammatory, myoprotectant and hepatoprotective effects of the reference chemotherapy, aggravating the pathological remodeling of both organs. As the interaction of T. cruzi infection, Bz and Tio is potentially toxic to the liver, inducing inflammation and microvesicular steatosis; this drug combination represents a worrying pharmacological risk factor in Chagas disease.

Could phenothiazine-benznidazole combined chemotherapy be effective in controlling heart parasitism and acute infectious myocarditis?

Phenothiazines inhibit major antioxidant defense mechanisms in trypanosomatids and exhibit potent cytotoxic effects in vitro. However, the relevance of these drugs in the treatment of Trypanosoma cruzi-induced acute myocarditis is poorly explored, especially in combination with reference trypanocidal drugs. Thus, we compared the antiparasitic and cardioprotective potential of thioridazine (TDZ) and benznidazole (Bz) administered in monotherapy and combined in a murine model of T. cruzi-induced acute myocarditis. Female mice were randomized into six groups: (i) uninfected untreated, (ii) infected untreated, or infected treated with (iii) Bz (100 mg/kg), (iv) TDZ (80 mg/kg), (v) Bz (100 mg/kg) + TDZ (80 mg/kg), or (vi) Bz (50 mg/kg) + TDZ (80 mg/kg). Infected animals were inoculated with 2000 T. cruzi trypomastigotes and treated by gavage for 20 days. Animals that received TDZ alone presented the highest levels of parasitemia, parasitic load and anti-T. cruzi immunoglobulin G titers; cardiac upregulation of N-acetyl-ß-D-glucosaminidase activity, nitric oxide, malondialdehyde and cytokines (IFN-γ, TNF-α, IL-10 and IL-17); as well as microstructural damage compared to the other groups (p < 0.05). These parameters were reduced in groups receiving Bz monotherapy compared to the other groups (p < 0.05). The combination of TDZ and Bz attenuated the response to treatment, worsening parasitological control, oxidative heart damage and myocarditis compared to the group treated with Bz alone (p < 0.05). Our results indicate that when administered alone, TDZ potentiated the pathological outcomes in animals infected with T. cruzi. Moreover, TDZ attenuated the antiparasitic effect of Bz when administered together, impairing parasitological control, potentiating inflammation, molecular oxidation and pathological microstructural remodeling of the heart. Thus, our findings indicate that TDZ acts as a pharmacological risk factor and Bz-based monotherapy remains a better cardioprotective drug against Trypanosoma cruzi-induced acute myocarditis.

Could pre-infection exercise training improve the efficacy of specific antiparasitic chemotherapy for Chagas disease?

Considering a potential exercise-drug interaction, we investigated whether exercise training could improve the efficacy of specific antiparasitic chemotherapy in a rodent model of Chagas disease. Wistar rats were randomized into five groups: sedentary and uninfected (CT); sedentary and infected (SI); sedentary, infected and treated (SIT); trained and infected (TI); trained, infected and treated (TIT). After 9-weeks running training, the animals were infected with T. cruzi and followed up for 4 weeks, receiving 100 mg kg-1 day-1 benznidazole. No evidence of myocarditis was observed in CT animals. TI animals exhibited reduced parasitemia, myocarditis, and reactive tissue damage compared to SI animals, in addition to increased IFN-γ, IL-4, IL-10, heart non-protein antioxidant (NPA) levels and glutathione-s transferase activity (P < 0.05). The CT, SIT and TIT groups presented similar reductions in parasitemia, cytokines (IFN-γ, TNF-α, IL-4, IL-10, IL-17 and MCP-1), inflammatory infiltrate, oxidative heart damage and antioxidant enzymes activity compared to SI and TI animals, as well as reduced heart microstructural remodeling (P < 0.05). By modulating heart inflammation and redox metabolism, exercise training exerts a protective effect against T. cruzi infection in rats. However, the antiparasitic and cardioprotective effects of benznidazole chemotherapy are more pronounced, determining similar endpoints in sedentary and trained T. cruzi-infected rats.

Trans-fatty acids aggravate anabolic steroid-induced metabolic disturbances and differential gene expression in muscle, pancreas and adipose tissue.

AIMS: Although anabolic steroids (AS) and trans-fatty acids overload exerts systemic toxicity and are independent risk factors for metabolic and cardiovascular disorders, their interaction remains poorly understood. Thus, we investigated the impact of a diet rich in trans-fatty acids (HFD) combined with AS on glycemic control, lipid profile, adipose tissue, skeletal muscle and pancreas microstructure and expression of genes involved in energy metabolism. MAIN METHODS: Forty-eight C57BL/6 mice were randomized into 6 groups treated for 12 weeks with a standard diet (SD) or a diet rich in C18:1 trans-fatty isomers (HFD), alone or combined with 10 or 20 mg/kg testosterone cypionate (AS). KEY FINDINGS: Our results indicated that AS improved glycemic control, upregulated gene expression of Glut-4 and CPT-1 in skeletal muscle, FAS, ACC and UCP-1 in adipose tissue. AS also reduced total and LDL cholesterol in mice fed a SD. When combined with the HFD, AS was unable to induce microstructural adaptations in adipose tissue, pancreatic islets and ß-cells, but potentiated GCK and Glut-2 (pancreas) and Glut-4 and CPT-1 (skeletal muscle) upregulation. HFD plus AS also downregulated FAS and ACC gene expression in adipose tissue. Combined with HFD, AS increased triacylglycerol circulating levels, improved insulin sensitivity and glycemic control in mice. SIGNIFICANCE: Our findings indicated that HFD and AS can interact to modulates glycemic control and lipid profile by a mechanism potentially related with a reprogramming of genes expression in organs such as the pancreas, adipose tissue and skeletal muscle.

Concomitant exercise training attenuates the cardioprotective effects of pharmacological therapy in a murine model of acute infectious myocarditis.

When administered alone, preinfection exercise training and benznidazole-based chemotherapy induce cardioprotection in Chagas disease. However, the effect of concomitant exercise and benznidazole treatment is unknown. We investigated whether exercise and specific chemotherapy could interact to modulate parasitemia, inflammation, redox status and heart damage in a murine model of T. cruzi infection. Wistar rats were randomized into an uninfected control group (CNT) and four groups infected with T. cruzi: sedentary untreated (SUN) and treated (STR), and trained untreated (TUN) and treated (TTR). Running training was administered 5 days/week for 4 weeks. Treated animals concomitantly received 100 mg/kg/day benznidazole. Heart inflammation and reactive damage were not detected in CNT animals. Compared to SUN, TUN animals presented increased levels of parasitemia, myocarditis, nitric oxide, hydrogen peroxide, protein carbonyl, malondialdehyde, cytokines (IFN-γ, TNF-α, IL-4, IL-6, IL-10 and IL-17), catalase, superoxide dismutase and glutathione reductase activity, as well as reduced heart non-protein antioxidant levels (P < 0.05). TTR animals exhibited higher levels of parasitemia, myocarditis, hydrogen peroxide, malondialdehyde, IFN-γ, TNF-α and IL-6 than STR animals (P < 0.05), which showed the lowest levels of all analyzed parameters compared to the other groups (P < 0.05). Our findings indicate that exercise aggravates acute infection. When concomitantly administered with benznidazole, exercise training impaired parasitic control and chemotherapy-induced cardioprotection in T. cruzi-infected rats. Considering that exercise training and T. cruzi infection constitute independent metabolic challenges, the negative effects of concomitant treatment are potentially related to the overlapping oxidative and immunoinflammatory demands of exercise and the infection itself.

Could angiotensin-modulating drugs be relevant for the treatment of Trypanosoma cruzi infection? A systematic review of preclinical and clinical evidence.

Although leucocytes are targets of renin-angiotensin system (RAS) effector molecules and RAS-modulating drugs exert immunomodulatory effects, their impact on Trypanosoma cruzi infection remains poorly understood. By using the framework of a systematic review, we integrated the preclinical and clinical evidence to investigate the relevance of angiotensin-inhibiting drugs on T. cruzi infections. From a comprehensive and structured search in biomedical databases, only original studies were analysed. In preclinical and clinical studies, captopril, enalapril and losartan were RAS-modulating drugs used. The main in vitro findings indicated that these drugs increased parasite uptake per host cells, IL-12 expression by infected dendritic cells and IFN-γ by T lymphocytes, in addition to attenuating IL-10 and IL-17 production by CD8 + T cells. In animal models, reduced parasitaemia, tissue parasitism, leucocytes infiltration and mortality were often observed in T. cruzi-infected animals receiving RAS-modulating drugs. In patients with Chagas' disease, these drugs exerted a controversial impact on cytokine and hormone levels, and a limited effect on cardiovascular function. Considering a detailed evaluation of reporting and methodological quality, the current preclinical and clinical evidence is at high risk of bias, and we hope that our critical analysis will be useful in mitigating the risk of bias in further studies.