The Therapeutic Potential of Angeli's Salt in Mitigating Acute Trypanosoma cruzi Infection in Mice.

Chagas disease (CD), caused by Trypanosoma cruzi, is a neglected tropical disease prevalent in Latin America. Infected patients are treated to eliminate the parasite, reduce the cardiomyopathy risk, and interrupt the disease transmission cycle. The World Health Organization recognizes benznidazole (BZ) and nifurtimox as effective drugs for CD treatment. In the chronic phase, both drugs have low cure rates and serious side effects. T. cruzi infection causes intense tissue inflammation that controls parasite proliferation and CD evolution. Compounds that liberate nitric oxide (NO) (NO donors) have been used as anti-T. cruzi therapeutics. Currently, there is no evidence that nitroxyl (HNO) affects T. cruzi infection outcomes. This study investigated the effects of the HNO donor Angeli's salt (AS) on C57BL/6 mice infected with T. cruzi (Y strain, 5 × 103 trypomastigotes, intraperitoneally). AS reduced the number of parasites in the bloodstream and heart nests and increased the protective antioxidant capacity of erythrocytes in infected animals, reducing disease severity. Furthermore, in vitro experiments showed that AS treatment reduced parasite uptake and trypomastigote release by macrophages. Taken together, these findings from the murine model and in vitro testing suggest that AS could be a promising therapy for CD.

Beneficial effects of acetylsalicylic acid (aspirin) on the actions of extracellular vesicles shed by Trypanosoma cruzi in macrophages.

Trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas disease, shed extracellular vesicles (EVs) that promote the susceptibility of host cells to infection. During T. cruzi infection, the immune response of the host is important for controlling parasitism, which is necessary for survival. Macrophages produce inflammatory mediators, such as eicosanoids and nitric oxide (NO), with trypanocidal effects that control the parasite load in the early stages of the disease. In this study, we evaluated the contribution of host cyclooxygenase (COX) to the actions of EVs shed by T. cruzi strain Y (EVs-Y) in infected macrophages. RAW 264.7 macrophages exposed to EVs-Y and then infected with trypomastigote forms of T. cruzi produced less NO, and an increased number of trypomastigote forms were internalized in the cell compared to the controls, indicating that the effects exerted by EVs-Y favor the parasite. Interestingly, when macrophages were pretreated with acetylsalicylic acid, a dual COX inhibitor, before exposure to EVs-Y and subsequent infection with trypomastigote forms, there was an increase in NO production and a decrease in trypomastigote uptake compared to the controls. These results suggest that EVs-Y modulates the macrophage response in favor of T. cruzi and indicate a role for COX in the effects of EVs.

Metabolic syndrome improves cardiovascular dysfunction and survival during cecal ligation and puncture-induced mild sepsis in mice.

AIMS: Sepsis is a potentially fatal systemic inflammatory response and its underlying pathophysiology is still poorly understood. Studies suggest that obesity, a component of metabolic syndrome (MS), is associated with sepsis survival. Therefore, this study focused on investigating the influence of MS on mortality and cardiovascular dysfunction induced by sublethal cecal ligation and puncture (SL-CLP). MAIN METHODS: Newborn Swiss mice received monosodium glutamate (MSG) (4 mg kg-1 day-1, s.c.) during the first 5 d of life for MS induction, while the control pups received equimolar saline solution. On the 75th day, SL-CLP was used to induce mild sepsis (M-CLP) in the MS (MS-M-CLP) and control (SAL-M-CLP) mice. The effect of MS on sepsis in mice was assessed by determining the survival rate and quantification of nitric oxide (NO) in the plasma, and associating this data with hematological and cardiovascular parameters. KEY FINDINGS: MS improved the survival of septic mice, preventing impairment to hematological and cardiovascular parameters. In addition, MS attenuated plasmatic NO increase, which is a typical feature of sepsis. SIGNIFICANCE: These findings provide new insights into the relationship between obesity and mild sepsis in mice, thus revealing an approach in favor of the "obesity paradox."

Concanavalin-A stimulates IL-17 and nitric oxide production and induces macrophage polarization and resistance to Trypanosoma cruzi infection.

AIMS: Chagas disease is a neglected tropical disease. The ability of Trypanosoma cruzi to survive within phagocytes is likely a critical factor for T. cruzi dissemination in the host. For control of the parasite load and host survival, macrophage action is required. Concanavalin-A (Con-A) presents properties that modulate immune functions and protect hosts from several experimental infectious diseases. Here, we evaluated the effects of Con-A on peritoneal macrophages as well as on the course of experimental infection by T. cruzi. MAIN METHODS: BALB/c mice, a susceptible model for T. cruzi infection, were treated with Con-A via the intraperitoneal route and 3 days later infected with T. cruzi. We quantified parasitemia, cytokines and nitric oxide (NO). Peritoneal exudate and macrophages were collected for macrophage phenotyping and cell viability, NO and cytokine detection, as well as for T. cruzi internalization and release index determination. KEY FINDINGS: Con-A treatment induced IL-17a and NO production by cells from the peritoneal cavity, and M1 marker expression predominated on peritoneal macrophages. These cells are also more prone to producing TNF-α, IL-6 and NO when infected by T. cruzi and show high trypanocidal capacity. Due to a hostile peritoneal microenvironment caused by Con-A, which induces macrophage cNOS and iNOS expression, infected BALB/c mice showed reduced parasitemia and an increased survival rate. SIGNIFICANCE: We conclude that Con-A can induce peritoneal M1 macrophage polarization to increase trypanocidal activity, resulting in ameliorated systemic infection in a susceptible experimental model.

Combination Therapy Using Benznidazole and Aspirin during the Acute Phase of Experimental Chagas Disease Prevents Cardiovascular Dysfunction and Decreases Typical Cardiac Lesions in the Chronic Phase.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is one of the main causes of death due to cardiomyopathy and heart failure in Latin American countries. The treatment of Chagas disease is directed at eliminating the parasite, decreasing the probability of cardiomyopathy and disrupting the disease transmission cycle. Benznidazole (BZ) and nifurtimox (Nfx) are recognized as effective drugs for the treatment of Chagas disease by the World Health Organization, but both have high toxicity and limited efficacy, especially in the chronic disease phase. At low doses, aspirin (ASA) has been reported to protect against T. cruzi infection. We evaluated the effectiveness of BZ in combination with ASA at low doses during the acute disease phase and evaluated cardiovascular aspects and cardiac lesions in the chronic phase. ASA treatment prevented the cardiovascular dysfunction (hypertension and tachycardia) and typical cardiac lesions. Moreover, BZ+ASA-treated mice had a smaller cardiac fibrotic area than BZ-treated mice. These results were associated with an increase in numbers of eosinophils and reticulocytes and levels of nitric oxide in the plasma and cardiac tissue of ASA-treated mice relative to respective controls. These effects of ASA and BZ+ASA in chronically infected mice were inhibited by pretreatment with the lipoxin A4 (LXA4) receptor antagonist Boc-2, indicating that the protective effects of ASA are mediated by ASA-triggered lipoxin. These results emphasize the importance of exploring new drug combinations for treatments of the acute phase of Chagas disease that are beneficial for patients with chronic disease.

Metabolic syndrome agravates cardiovascular, oxidative and inflammatory dysfunction during the acute phase of Trypanosoma cruzi infection in mice.

We evaluated the influence of metabolic syndrome (MS) on acute Trypanosoma cruzi infection. Obese Swiss mice, 70 days of age, were subjected to intraperitoneal infection with 5 × 102 trypomastigotes of the Y strain. Cardiovascular, oxidative, inflammatory, and metabolic parameters were evaluated in infected and non-infected mice. We observed higher parasitaemia in the infected obese group (IOG) than in the infected control group (ICG) 13 and 15 days post-infection. All IOG animals died by 19 days post-infection (dpi), whereas 87.5% of the ICG survived to 30 days. Increased plasma nitrite levels in adipose tissue and the aorta were observed in the IOG. Higher INF-γ and MCP-1 concentrations and lower IL-10 concentrations were observed in the IOG compared to those in the ICG. Decreased insulin sensitivity was observed in obese animals, which was accentuated after infection. Higher parasitic loads were found in adipose and hepatic tissue, and increases in oxidative stress in cardiac, hepatic, and adipose tissues were characteristics of the IOG group. Thus, MS exacerbates experimental Chagas disease, resulting in greater damage and decreased survival in infected animals, and might be a warning sign that MS can influence other pathologies.

Extracellular Vesicles Shed By Trypanosoma cruzi Potentiate Infection and Elicit Lipid Body Formation and PGE2 Production in Murine Macrophages.

During the onset of Trypanosoma cruzi infection, an effective immune response is necessary to control parasite replication and ensure host survival. Macrophages have a central role in innate immunity, acting as an important trypanocidal cell and triggering the adaptive immune response through antigen presentation and cytokine production. However, T. cruzi displays immune evasion mechanisms that allow infection and replication in macrophages, favoring its chronic persistence. One potential mechanism is the release of T. cruzi strain Y extracellular vesicle (EV Y), which participate in intracellular communication by carrying functional molecules that signal host cells and can modulate the immune response. The present work aimed to evaluate immune modulation by EV Y in C57BL/6 mice, a prototype resistant to infection by T. cruzi strain Y, and the effects of direct EV Y stimulation of macrophages in vitro. EV Y inoculation in mice prior to T. cruzi infection resulted in increased parasitemia, elevated cardiac parasitism, decreased plasma nitric oxide (NO), reduced NO production by spleen cells, and modulation of cytokine production, with a reduction in TNF-α in plasma and decreased production of TNF-α and IL-6 by spleen cells from infected animals. In vitro assays using bone marrow-derived macrophages showed that stimulation with EV Y prior to infection by T. cruzi increased the parasite internalization rate and release of infective trypomastigotes by these cells. In this same scenario, EV Y induced lipid body formation and prostaglandin E2 (PGE2) production by macrophages even in the absence of T. cruzi. In infected macrophages, EV Y decreased production of PGE2 and cytokines TNF-α and IL-6 24 h after infection. These results suggest that EV Y modulates the host response in favor of the parasite and indicates a role for lipid bodies and PGE2 in immune modulation exerted by EVs.

Trypanosoma cruzi: Inhibition of infection of human monocytes by aspirin.

Cell invasion by Trypanosoma cruzi and its intracellular replication are essential for progression of the parasite life cycle and development of Chagas disease. Prostaglandin E2 (PGE2) and other eicosanoids potently modulate host response and contribute to Chagas disease progression. In this study, we evaluated the effect of aspirin (ASA), a non-selective cyclooxygenase (COX) inhibitor on the T. cruzi invasion and its influence on nitric oxide and cytokine production in human monocytes. The pretreatment of monocytes with ASA or SQ 22536 (adenylate-cyclase inhibitor) induced a marked inhibition of T. cruzi infection. On the other hand, the treatment of monocytes with SQ 22536 after ASA restored the invasiveness of T. cruzi. This reestablishment was associated with a decrease in nitric oxide and PGE2 production, and also an increase of interleukin-10 and interleukin-12 by cells pre-treated with ASA. Altogether, these results reinforce the idea that the cyclooxygenase pathway plays a fundamental role in the process of parasite invasion in an in vitro model of T. cruzi infection.

Nitric oxide-releasing indomethacin enhances susceptibility to Trypanosoma cruzi infection acting in the cell invasion and oxidative stress associated with anemia.

Trypanosoma cruzi is the causative agent of Chagas disease. Approximately 8 million people are thought to be affected with this disease worldwide. T. cruzi infection causes an intense inflammatory response, which is critical for the control of parasite proliferation and disease development. Nitric oxide-donating nonsteroidal anti-inflammatory drugs (NO-NSAIDs) are an emergent class of pharmaceutical derivatives with promising utility as chemopreventive agents. In this study, we investigated the effect of NO-indomethacin on parasite burden, cell invasion, and oxidative stress in erythrocytes during the acute phase of infection. NO-indomethacin was dissolved in dimethyl formamide followed by i.p. administration of 50 ppm into mice 30 min after infection with 5×10(3) blood trypomastigote forms (Y strain). The drug was administered every day until the animals died. Control animals received 100 µL of drug vehicle via the same route. Within the NO-indomethacin-treatment group, parasitemia and mortality (100%) were higher and oxidative stress in erythrocytes, anemia, and entry of parasites into macrophages were significantly greater than that seen in controls. Increase in the entry and survival of intracellular T. cruzi was associated with inhibition of nitric oxide production by macrophages treated with NO-indomethacin (2.5 µM). The results of this study provide strong evidence that NO-NSAIDs potently inhibit nitric oxide production, suggesting that NO-NSAID-based therapies against infections would be difficult to design and would require caution.

Aspirin modulates innate inflammatory response and inhibits the entry of Trypanosoma cruzi in mouse peritoneal macrophages.

The intracellular protozoan parasite Trypanosoma cruzi causes Chagas disease, a serious disorder that affects millions of people in Latin America. Cell invasion by T. cruzi and its intracellular replication are essential to the parasite's life cycle and for the development of Chagas disease. Here, we present evidence suggesting the involvement of the host's cyclooxygenase (COX) enzyme during T. cruzi invasion. Pharmacological antagonist for COX-1, aspirin (ASA), caused marked inhibition of T. cruzi infection when peritoneal macrophages were pretreated with ASA for 30 min at 37°C before inoculation. This inhibition was associated with increased production of IL-1ß and nitric oxide (NO(∙)) by macrophages. The treatment of macrophages with either NOS inhibitors or prostaglandin E2 (PGE2) restored the invasive action of T. cruzi in macrophages previously treated with ASA. Lipoxin ALX-receptor antagonist Boc2 reversed the inhibitory effect of ASA on trypomastigote invasion. Our results indicate that PGE2, NO(∙), and lipoxins are involved in the regulation of anti-T. cruzi activity by macrophages, providing a better understanding of the role of prostaglandins in innate inflammatory response to T. cruzi infection as well as adding a new perspective to specific immune interventions.

Trypanosoma cruzi: in vivo evaluation of iron in skin employing X-ray fluorescence (XRF) in mouse strains that differ in their susceptibility to infection.

Trypanosoma cruzi, the causative agent of Chagas' disease (CD), is a substantial public health concern in Latin America. Laboratory mice inoculated with T. cruzi have served as important animal models of acute CD. Host hypoferremic responses occur during T. cruzi infection; therefore, it has been hypothesized that T. cruzi requires iron for optimal growth in host cells and, unlike extracellular pathogens, may benefit from host hypoferremic responses. Recent technological improvements of X-ray fluorescence are useful for diagnostics or monitoring in biomedical applications. The goal of our study was to determine whether the iron availabilities in Swiss and C57BL/6 mice differ during the acute phase of T. cruzi infection and whether the availability correlates with oxidative stress in the susceptible and resistant phenotypes identified in these mice. Our results showed that the decrease in iron levels in the skin of resistant infected mice correlated with the increase in oxidative stress associated with anemia and the reduction in parasite burden.

5-Lipoxygenase plays a role in the control of parasite burden and contributes to oxidative damage of erythrocytes in murine Chagas' disease.

Chagas' disease is accompanied by severe anemia and oxidative stress, which may contribute to mortality. In this study, we investigated the role of 5-lipoxygenase (5-LO) in the control of parasitism and anemia associated with oxidative damage of erythrocytes in experimental Trypanosoma cruzi infection. Wild-type C57BL/6, 129Sv mice treated or not with nordihydroguaiaretic acid (NDGA, 5-LO inhibitor), mice lacking the 5-LO enzyme gene (5-LO(-/-)) and inducible nitric oxide synthase gene (iNOS(-/-)) were infected with the Y strain of T. cruzi. Impairment of 5-LO resulted in increased numbers of trypomastigote forms in the blood and amastigote forms in the heart of infected mice. We assessed oxidative stress in erythrocytes by measuring oxygen uptake, induction time and chemiluminescence following treatment with tert-butyl hydroperoxide (TBH). Our results show that 5-LO metabolites increased lipid peroxidation levels in erythrocytes during the early phase of murine T. cruzi infection. NDGA treatment reduced oxidative damage of erythrocytes in C57BL/6 T. cruzi-infected mice but not in C57BL/6 iNOS(-/-) infected mice, showing that the action of NDGA is dependent on endogenous nitric oxide (NO). In addition, our results show that 5-LO metabolites do not participate directly in the development of anemia in infected mice. We conclude that 5-LO products may not only play a major role in controlling heart tissue parasitism, i.e., host resistance to acute infection with T. cruziin vivo, but in the event of an infection also play an important part in erythrocyte oxidative stress, an NO-dependent effect.

Involvement of nitric oxide (NO) and TNF-alpha in the oxidative stress associated with anemia in experimental Trypanosoma cruzi infection.

Trypanosoma cruzi infection in mice is associated with severe hematological changes, including anemia, which may contribute to mortality. TNF-alpha and nitric oxide (NO) play a critical role in establishing host resistance to this pathogen. We hypothesized that phagocyte-derived NO damages erythrocytes and contributes to the anemia observed during T. cruzi infection. To test this hypothesis, two strains of mice that differed in susceptibility and NO response to T. cruzi infection were used in these studies. We also blocked endogenous NO production by aminoguanidine (AG) treatment or blocked TNF-alpha with a neutralizing antibody and used mice that cannot produce phagocyte-derived NO (C57BL/6 iNOS(-/-)). Following infection with T. cruzi, resistant (C57BL/6) and susceptible (Swiss) mice displayed a parasitemia that peaked at the same time (i.e., day 9), yet parasitemia was 3-fold higher in Swiss mice (P < 0.05). All Swiss mice were dead by day 23 post-infection, while no C57BL/6 mice died during the study. At 14 days post-infection anemia in C57BL/6 mice was more severe than in Swiss mice. Treatment of both strains with the NO inhibitor, AG (50 mg/kg), and the use of iNOS(-/-) mice, revealed that the anemia in T. cruzi-infected mice is not caused by NO. However, the reticulocytosis that occurs during infection was significantly reduced after treatment with AG in both Swiss and C57BL/6 mice (P < 0.05). In addition, we showed that neutralization of TNF-alpha in vivo induced a significant increase in circulating reticulocytes in T. cruzi-infected C57BL/6 mice (P < 0.05), but did not modify other hematologic parameters in these mice. The evaluation of the oxidative stress after induction by t-butyl hydroperoxide (t-BHT) revealed that the treatment with AG completely protected against NO-mediated haemoglobin oxidation. Further, treatment with AG, but not with anti-TNF-alpha, protected against the infection-induced reduction of antioxidant capacity of erythrocytes as assessed by oxygen uptake and induction time. In summary, this is the first report showing the participation of NO and TNF-alpha in the oxidative stress to erythrocytes in acute T. cruzi infection. Further, our data suggest that NO does not play a direct role in development of the anemia. However, NO may contribute to other hematological changes noted during T. cruzi infection, such as the elevation of circulating reticulocytes and the reduction in circulating leukocytes and neutrophils.