Severity of chronic experimental Chagas' heart disease parallels tumour necrosis factor and nitric oxide levels in the serum: models of mild and severe disease

Heart tissue inflammation, progressive fibrosis and electrocardiographic alterations occur in approximately 30% of patients infected by Trypanosoma cruzi, 10-30 years after infection. Further, plasma levels of tumour necrosis factor (TNF) and nitric oxide (NO) are associated with the degree of heart dysfunction in chronic chagasic cardiomyopathy (CCC). Thus, our aim was to establish experimental models that mimic a range of parasitological, pathological and cardiac alterations described in patients with chronic Chagas’ heart disease and evaluate whether heart disease severity was associated with increased TNF and NO levels in the serum. Our results show that C3H/He mice chronically infected with the Colombian T. cruzi strain have more severe cardiac parasitism and inflammation than C57BL/6 mice. In addition, connexin 43 disorganisation and fibronectin deposition in the heart tissue, increased levels of creatine kinase cardiac MB isoenzyme activity in the serum and more severe electrical abnormalities were observed in T. cruzi-infected C3H/He mice compared to C57BL/6 mice. Therefore, T. cruzi-infected C3H/He and C57BL/6 mice represent severe and mild models of CCC, respectively. Moreover, the CCC severity paralleled the TNF and NO levels in the serum. Therefore, these models are appropriate for studying the pathophysiology and biomarkers of CCC progression, as well as for testing therapeutic agents for patients with Chagas’ heart disease.     

Treatment of chronically Trypanosoma cruzi-infected mice with a CCR1/CCR5 antagonist (Met-RANTES) results in amelioration of cardiac tissue damage

The comprehension of the molecular mechanisms leading to Trypanosoma cruzi-elicited heart dysfunction might contribute to design novel therapeutic strategies aiming to ameliorate chronic Chagas disease cardiomyopathy. In C3H/He mice infected with the low virulence T. cruzi Colombian strain, the persistent cardiac inflammation composed mainly of CCR5⁺ T lymphocytes parallels the expression of CC-chemokines in a pro-inflammatory IFN-γ and TNF-α milieu. The chronic myocarditis is accompanied by increased frequency of peripheral CCR5⁺LFA-1⁺ T lymphocytes. The treatment of chronically T. cruzi-infected mice with Met-RANTES, a selective CCR1/CCR5 antagonist, led to a 20–30% decrease in CD4⁺ cell numbers as well as IL-10, IL-13 and TNF-α expression. Further, Met-RANTES administration impaired the re-compartmentalization of the activated CD4⁺CCR5⁺ lymphocytes. Importantly, Met-RANTES treatment resulted in significant reduction in parasite load and fibronectin deposition in the heart tissue. Moreover, Met-RANTES treatment significantly protected T. cruzi-infected mice against connexin 43 loss in heart tissue and CK-MB level enhancement, markers of heart dysfunction. Thus, our results corroborate that therapeutic strategies based on the modulation of CCR1/CCR5-mediated cell migration and/or effector function may contribute to cardiac tissue damage limitation during chronic Chagas disease.     

Interferon-Gamma Promotes Infection of Astrocytes by Trypanosoma cruzi

The inflammatory cytokine interferon-gamma (IFNγ) is crucial for immunity against intracellular pathogens such as the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (CD). IFNγ is a pleiotropic cytokine which regulates activation of immune and non-immune cells; however, the effect of IFNγ in the central nervous system (CNS) and astrocytes during CD is unknown. Here we show that parasite persists in the CNS of C3H/He mice chronically infected with the Colombian T. cruzi strain despite the increased expression of IFNγ mRNA. Furthermore, most of the T. cruzi-bearing cells were astrocytes located near IFNγ⁺ cells. Surprisingly, in vitro experiments revealed that pretreatment with IFNγ promoted the infection of astrocytes by T. cruzi increasing uptake and proliferation of intracellular forms, despite inducing increased production of nitric oxide (NO). Importantly, the effect of IFNγ on T. cruzi uptake and growth is completely blocked by the anti-tumor necrosis factor (TNF) antibody Infliximab and partially blocked by the inhibitor of nitric oxide synthesis L-NAME. These data support that IFNγ fuels astrocyte infection by T. cruzi and critically implicate IFNγ-stimulated T. cruzi-infected astrocytes as sources of TNF and NO, which may contribute to parasite persistence and CNS pathology in CD.     

Metallothionein-1 and nitric oxide expression are inversely correlated in a murine model of Chagas disease

Chagas disease, caused by Trypanosoma cruzi, represents an endemic among Latin America countries. The participation of free radicals, especially nitric oxide (NO), has been demonstrated in the pathophysiology of seropositive individuals with T. cruzi. In Chagas disease, increased NO contributes to the development of cardiomyopathy and megacolon. Metallothioneins (MTs) are efficient free radicals scavengers of NO in vitro and in vivo. Here, we developed a murine model of the chronic phase of Chagas disease using endemic T. cruzi RyCH1 in BALB/c mice, which were divided into four groups: infected non-treated (Inf), infected N-monomethyl-L-arginine treated (Inf L-NAME), non-infected L-NAME treated and non-infected vehicle-treated. We determined blood parasitaemia and NO levels, the extent of parasite nests in tissues and liver MT-I expression levels. It was observed that NO levels were increasing in Inf mice in a time-dependent manner. Inf L-NAME mice had fewer T. cruzi nests in cardiac and skeletal muscle with decreased blood NO levels at day 135 post infection. This affect was negatively correlated with an increase of MT-I expression (r = -0.8462, p < 0.0001). In conclusion, we determined that in Chagas disease, an unknown inhibitory mechanism reduces MT-I expression, allowing augmented NO levels.     

Behavioural alterations are independent of sickness behaviour in chronic experimental Chagas disease

The existence of the nervous form of Chagas disease is a matter of discussion since Carlos Chagas described neurological disorders, learning and behavioural alterations in Trypanosoma cruzi-infected individuals. In most patients, the clinical manifestations of the acute phase, including neurological abnormalities, resolve spontaneously without apparent consequence in the chronic phase of infection. However, chronic Chagas disease patients have behavioural changes such as psychomotor alterations, attention and memory deficits, and depression. In the present study, we tested whether or not behavioural alterations are reproducible in experimental models. We show that C57BL/6 mice chronically infected with the Colombian strain of T. cruzi (150 days post-infection) exhibit behavioural changes as (i) depression in the tail suspension and forced swim tests, (ii) anxiety analysed by elevated plus maze and open field test sand and (iii) motor coordination in the rotarod test. These alterations are neither associated with neuromuscular disorders assessed by the grip strength test nor with sickness behaviour analysed by temperature variation sand weight loss. Therefore, chronically T. cruzi-infected mice replicate behavioural alterations (depression and anxiety) detected in Chagas disease patients opening an opportunity to study the interconnection and the physiopathology of these two biological processes in an infectious scenario.     

Elevated Serum Levels of Macrophage Migration Inhibitory Factor Are Associated with Progressive Chronic Cardiomyopathy in Patients with Chagas Disease

Clinical symptoms of chronic Chagas disease occur in around 30% of the individuals infected with Trypanosoma cruzi and are characterized by heart inflammation and dysfunction. The pathogenesis of chronic chagasic cardiomyopathy (CCC) is not completely understood yet, partially because disease evolution depends on complex host-parasite interactions. Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine that promotes numerous pathophysiological processes. In the current study, we investigated the link between MIF and CCC progression.Immunohistochemical analysis demonstrated MIF overexpression in the hearts from chronically T. cruzi-infected mice, particularly those showing intense inflammatory infiltration. We also found that MIF exogenously added to parasite-infected murine macrophage cultures is capable of enhancing the production of TNF-α and reactive oxygen species, both with pathogenic roles in CCC. Thus, the integrated action of MIF and other cytokines and chemokines may account for leukocyte influx to the infected myocardium, accompanied by enhanced local production of multiple inflammatory mediators. We further examined by ELISA the level of MIF in the sera from chronic indeterminate and cardiomyopathic chagasic patients, and healthy subjects. CCC patients displayed significantly higher MIF concentrations than those recorded in asymptomatic T. cruzi-infected and uninfected individuals. Interestingly, increased MIF levels were associated with severe progressive Chagas heart disease, in correlation with elevated serum concentration of high sensitivity C-reactive protein and also with several echocardiographic indicators of left ventricular dysfunction, one of the hallmarks of CCC. Our present findings represent the first evidence that enhanced MIF production is associated with progressive cardiac impairment in chronic human infection with T. cruzi, strengthening the relationship between inflammatory response and parasite-driven pathology. These observations contribute to unravel the elements involved in the pathogenesis of CCC and may also be helpful for the design of novel therapies aimed to control long-term morbidity in chagasic patients.     

High fat diet aggravates cardiomyopathy in murine chronic Chagas disease

Infection with Trypanosoma cruzi, the etiologic agent in Chagas disease, may result in heart disease. Over the last decades, Chagas disease endemic areas in Latin America have seen a dietary transition from the traditional regional diet to a Western style, fat rich diet. Previously, we demonstrated that during acute infection high fat diet (HFD) protects mice from the consequences of infection-induced myocardial damage through effects on adipogenesis in adipose tissue and reduced cardiac lipidopathy. However, the effect of HFD on the subsequent stages of infection – the indeterminate and chronic stages – has not been investigated. To address this gap in knowledge, we studied the effect of HFD during indeterminate and chronic stages of Chagas disease in the mouse model. We report, for the first time, the effect of HFD on myocardial inflammation, vasculopathy, and other types of dysfunction observed during chronic T. cruzi infection. Our results show that HFD perturbs lipid metabolism and induces oxidative stress to exacerbate late chronic Chagas disease cardiac pathology.     

Inducible nitric oxide synthase in heart tissue and nitric oxide in serum of Trypanosoma cruzi-infected rhesus monkeys: association with heart injury

Background

The factors contributing to chronic Chagas'' heart disease remain unknown. High nitric oxide (NO) levels have been shown to be associated with cardiomyopathy severity in patients. Further, NO produced via inducible nitric oxide synthase (iNOS/NOS2) is proposed to play a role in Trypanosoma cruzi control. However, the participation of iNOS/NOS2 and NO in T. cruzi control and heart injury has been questioned. Here, using chronically infected rhesus monkeys and iNOS/NOS2-deficient (Nos2 ^−/−) mice we explored the participation of iNOS/NOS2-derived NO in heart injury in T. cruzi infection.

Methodology

Rhesus monkeys and C57BL/6 and Nos2 ^−/− mice were infected with the Colombian T. cruzi strain. Parasite DNA was detected by polymerase chain reaction, T. cruzi antigens and iNOS/NOS2⁺ cells were immunohistochemically detected in heart sections and NO levels in serum were determined by Griess reagent. Heart injury was assessed by electrocardiogram (ECG), echocardiogram (ECHO), creatine kinase heart isoenzyme (CK-MB) activity levels in serum and connexin 43 (Cx43) expression in the cardiac tissue.

Results

Chronically infected monkeys presented conduction abnormalities, cardiac inflammation and fibrosis, which resembled the spectrum of human chronic chagasic cardiomyopathy (CCC). Importantly, chronic myocarditis was associated with parasite persistence. Moreover, Cx43 loss and increased CK-MB activity levels were primarily correlated with iNOS/NOS2⁺ cells infiltrating the cardiac tissue and NO levels in serum. Studies in Nos2 ^−/− mice reinforced that the iNOS/NOS2-NO pathway plays a pivotal role in T. cruzi-elicited cardiomyocyte injury and in conduction abnormalities that were associated with Cx43 loss in the cardiac tissue.

Conclusion

T. cruzi-infected rhesus monkeys reproduce features of CCC. Moreover, our data support that in T. cruzi infection persistent parasite-triggered iNOS/NOS2 in the cardiac tissue and NO overproduction might contribute to CCC severity, mainly disturbing of the molecular pathway involved in electrical synchrony. These findings open a new avenue for therapeutic tools in Chagas'' heart disease.     

Long non-coding RNA BDNF-AS modulates osteogenic differentiation of bone marrow-derived mesenchymal stem cells

Chagas disease is an acute or chronic illness that causes severe inflammatory response, and consequently, it may activate the inflammatory cholinergic pathway, which is regulated by cholinesterases, including the acetylcholinesterase. This enzyme is responsible for the regulation of acetylcholine levels, an anti-inflammatory molecule linked to the inflammatory response during parasitic diseases. Thus, the aim of this study was to investigate whether Trypanosoma cruzi infection can alter the activity of acetylcholinesterase and acetylcholine levels in mice, and whether these alterations are linked to the inflammatory cholinergic signaling pathway. Twenty-four mice were divided into two groups: uninfected (control group, n = 12) and infected by T. cruzi, Y strain (n = 12). The animals developed acute disease with a peak of parasitemia on day 7 post-infection (PI). Blood, lymphocytes, and brain were analyzed on days 6 and 12 post-infection. In the brain, acetylcholine and nitric oxide levels, myeloperoxidase activity, and histopathology were analyzed. In total blood and brain, acetylcholinesterase activity decreased at both times. On the other hand, acetylcholinesterase activity in lymphocytes increased on day 6 PI compared with the control group. Infection by T. cruzi increased acetylcholine and nitric oxide levels and histopathological damage in the brain of mice associated to increased myeloperoxidase activity. Therefore, an intense inflammatory response in mice with acute Chagas disease in the central nervous system caused an anti-inflammatory response by the activation of the cholinergic inflammatory pathway.     

Bioluminescent imaging of Trypanosoma cruzi infection

Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is a major public health problem in Central and South America. The pathogenesis of Chagas disease is complex and the natural course of infection is not completely understood. The recent development of bioluminescence imaging technology has facilitated studies of a number of infectious and non-infectious diseases. We developed luminescent T. cruzi to facilitate similar studies of Chagas disease pathogenesis. Luminescent T. cruzi trypomastigotes and amastigotes were imaged in infections of rat myoblast cultures, which demonstrated a clear correlation of photon emission signal strength to the number of parasites used. This was also observed in mice infected with different numbers of luminescent parasites, where a stringent correlation of photon emission to parasite number was observed early at the site of inoculation, followed by dissemination of parasites to different sites over the course of a 25-day infection. Whole animal imaging from ventral, dorsal and lateral perspectives provided clear evidence of parasite dissemination. The tissue distribution of T. cruzi was further determined by imaging heart, spleen, skeletal muscle, lungs, kidneys, liver and intestines ex vivo. These results illustrate the natural dissemination of T. cruzi during infection and unveil a new tool for studying a number of aspects of Chagas disease, including rapid in vitro screening of potential therapeutical agents, roles of parasite and host factors in the outcome of infection, and analysis of differential tissue tropism in various parasite-host strain combinations.     

Detection of Soluble Antigen and DNA of Trypanosoma cruzi in Urine Is Independent of Renal Injury in the Guinea Pig Model

The diagnosis of Chagas disease in humans is generally limited to the detection of specific antibodies. Detection of T. cruzi antigens in urine has been reported previously, but is not used in the diagnosis. In this study, soluble T. cruzi antigens and DNA were detected in urine samples and were associated with kidney injury and systemic detection of the parasite. We used 72 guinea pigs infected with T. cruzi Y strain and 18 non-infected guinea pigs. Blood, kidney, heart and urine samples were collected during the acute phase and chronic phase. Urine samples were concentrated by ultrafiltration. Antigens were detected by Western Blot using a polyclonal antibody against trypomastigote excretory-secretory antigen (TESA). T. cruzi DNA was detected by PCR using primers 121/122 and TcZ1/TcZ2. Levels of T. cruzi DNA in blood, heart and kidney were determined by quantitative PCR. T. cruzi antigens (75 kDa, 80 kDa, 120 kDa, 150 kDa) were detected in the acute phase (67.5%) and the chronic phase (45%). Parasite DNA in urine was detected only in the acute phase (45%). Kidney injury was characterized by high levels of proteinuria, kidney injury molecule-1 (KIM-1) and urea, and some histopathological changes such as inflammation, necrosis, fibrosis and scarce parasites. The detection of antigens and DNA in urine was associated with the presence of parasite DNA in blood and heart and with high levels of parasite DNA in blood, but not with the presence of parasite in kidney or kidney injury. These results suggest that the detection of T. cruzi in urine could be improved to be a valuable method for the diagnosis of Chagas disease, particularly in congenital Chagas disease and in immunocompromised patients.     

Global Metabolomic Profiling of Acute Myocarditis Caused by Trypanosoma cruzi Infection

Chagas disease is caused by Trypanosoma cruzi infection, being cardiomyopathy the more frequent manifestation. New chemotherapeutic drugs are needed but there are no good biomarkers for monitoring treatment efficacy. There is growing evidence linking immune response and metabolism in inflammatory processes and specifically in Chagas disease. Thus, some metabolites are able to enhance and/or inhibit the immune response. Metabolite levels found in the host during an ongoing infection could provide valuable information on the pathogenesis and/or identify deregulated metabolic pathway that can be potential candidates for treatment and being potential specific biomarkers of the disease. To gain more insight into those aspects in Chagas disease, we performed an unprecedented metabolomic analysis in heart and plasma of mice infected with T. cruzi. Many metabolic pathways were profoundly affected by T. cruzi infection, such as glucose uptake, sorbitol pathway, fatty acid and phospholipid synthesis that were increased in heart tissue but decreased in plasma. Tricarboxylic acid cycle was decreased in heart tissue and plasma whereas reactive oxygen species production and uric acid formation were also deeply increased in infected hearts suggesting a stressful condition in the heart. While specific metabolites allantoin, kynurenine and p-cresol sulfate, resulting from nucleotide, tryptophan and phenylalanine/tyrosine metabolism, respectively, were increased in heart tissue and also in plasma. These results provide new valuable information on the pathogenesis of acute Chagas disease, unravel several new metabolic pathways susceptible of clinical management and identify metabolites useful as potential specific biomarkers for monitoring treatment and clinical severity in patients.     

Trypanosoma cruzi Coaxes Cardiac Fibroblasts into Preventing Cardiomyocyte Death by Activating Nerve Growth Factor Receptor TrkA

Rationale

Cardiomyocytes express neurotrophin receptor TrkA that promotes survival following nerve growth factor (NGF) ligation. Whether TrkA also resides in cardiac fibroblasts (CFs) and underlies cardioprotection is unknown.

Objective

To test whether CFs express TrkA that conveys paracrine signals to neighbor cardiomyocytes using, as probe, the Chagas disease parasite Trypanosoma cruzi, which expresses a TrkA-binding neurotrophin mimetic, named PDNF. T cruzi targets the heart, causing chronic debilitating cardiomyopathy in ∼30% patients.

Methods and Results

Basal levels of TrkA and TrkC in primary CFs are comparable to those in cardiomyocytes. However, in the myocardium, TrkA expression is significantly lower in fibroblasts than myocytes, and vice versa for TrkC. Yet T cruzi recognition of TrkA on fibroblasts, preferentially over cardiomyocytes, triggers a sharp and sustained increase in NGF, including in the heart of infected mice or of mice administered PDNF intravenously, as early as 3-h post-administration. Further, NGF-containing T cruzi- or PDNF-induced fibroblast-conditioned medium averts cardiomyocyte damage by H₂O₂, in agreement with the previously recognized cardioprotective role of NGF.

Conclusions

TrkA residing in CFs induces an exuberant NGF production in response to T cruzi infection, enabling, in a paracrine fashion, myocytes to resist oxidative stress, a leading Chagas cardiomyopathy trigger. Thus, PDNF-TrkA interaction on CFs may be a mechanism orchestrated by T cruzi to protect its heart habitat, in concert with the long-term (decades) asymptomatic heart parasitism that characterizes Chagas disease. Moreover, as a potent booster of cardioprotective NGF in vivo, PDNF may offer a novel therapeutic opportunity against cardiomyopathies.     

Mesenchymal Bone Marrow Cell Therapy in a Mouse Model of Chagas Disease. Where Do the Cells Go?

Background

Chagas disease, resulting from infection with the parasite Trypanosoma cruzi (T. cruzi), is a major cause of cardiomyopathy in Latin America. Drug therapy for acute and chronic disease is limited. Stem cell therapy with bone marrow mesenchymal cells (MSCs) has emerged as a novel therapeutic option for cell death-related heart diseases, but efficacy of MSC has not been tested in Chagas disease.

Methods and Results

We now report the use of cell-tracking strategies with nanoparticle labeled MSC to investigate migration of transplanted MSC in a murine model of Chagas disease, and correlate MSC biodistribution with glucose metabolism and morphology of heart in chagasic mice by small animal positron emission tomography (microPET). Mice were infected intraperitoneally with trypomastigotes of the Brazil strain of T. cruzi and treated by tail vein injection with MSC one month after infection. MSCs were labeled with near infrared fluorescent nanoparticles and tracked by an in vivo imaging system (IVIS). Our IVIS results two days after transplant revealed that a small, but significant, number of cells migrated to chagasic hearts when compared with control animals, whereas the vast majority of labeled MSC migrated to liver, lungs and spleen. Additionally, the microPET technique demonstrated that therapy with MSC reduced right ventricular dilation, a phenotype of the chagasic mouse model.

Conclusions

We conclude that the beneficial effects of MSC therapy in chagasic mice arise from an indirect action of the cells in the heart rather than a direct action due to incorporation of large numbers of transplanted MSC into working myocardium.     

High Fat Diet Modulates Trypanosoma cruzi Infection Associated Myocarditis

Background

Trypanosoma cruzi, the causative agent of Chagas disease, has high affinity for lipoproteins and adipose tissue. Infection results in myocarditis, fat loss and alterations in lipid homeostasis. This study was aimed at analyzing the effect of high fat diet (HFD) on regulating acute T. cruzi infection-induced myocarditis and to evaluate the effect of HFD on lipid metabolism in adipose tissue and heart during acute T. cruzi infection.

Methodology/Principal Findings

CD1 mice were infected with T. cruzi (Brazil strain) and fed either a regular control diet (RD) or HFD for 35 days following infection. Serum lipid profile, tissue cholesterol levels, blood parasitemia, and tissue parasite load were analyzed to evaluate the effect of diet on infection. MicroPET and MRI analysis were performed to examine the morphological and functional status of the heart during acute infection. qPCR and immunoblot analysis were carried out to analyze the effect of diet on the genes involved in the host lipid metabolism during infection. Oil red O staining of the adipose tissue demonstrated reduced lipolysis in HFD compared to RD fed mice. HFD reduced mortality, parasitemia and cardiac parasite load, but increased parasite load in adipocytes. HFD decreased lipolysis during acute infection. Both qPCR and protein analysis demonstrated alterations in lipid metabolic pathways in adipose tissue and heart in RD fed mice, which were further modulated by HFD. Both microPET and MRI analyses demonstrated changes in infected RD murine hearts which were ameliorated by HFD.

Conclusion/Significance

These studies indicate that Chagasic cardiomyopathy is associated with a cardiac lipidpathy and that both cardiac lipotoxicity and adipose tissue play a role in the pathogenesis of Chagas disease. HFD protected mice from T. cruzi infection-induced myocardial damage most likely due to the effects of HFD on both adipogenesis and T. cruzi infection-induced cardiac lipidopathy.     

Modulation of inflammatory response and parasitism by 15-Deoxy-Δ(12,14) prostaglandin J(2) in Trypanosoma cruzi-infected cardiomyocytes

Trypanosoma cruzi infection produces an intense inflammatory response in diverse tissues including the heart. The inflammatory reaction is critical for the control of the parasites’ proliferation and evolution of Chagas disease. 15-Deoxy-Δ^12,14 prostaglandin J₂ (15dPGJ2) can repress the inflammatory response in many experimental models. However, the precise role of peroxisome proliferator-activated receptor γ (PPARγ) ligands in T. cruzi infection or in Chagas disease is poorly understood. This work reports the first evidence that 15dPGJ2 treatment increases the number of intracellular parasites as shown by fluorescence microscopy and it is also able to inhibit the expression and activity of different inflammatory enzymes such as inducible nitric oxide synthase (NOS-2), matrix metalloproteinases 2 and 9 (MMP-2, MMP-9), as well as pro-inflammatory cytokine (TNF-α and IL-6) mRNA expression in neonatal mouse cardiomyocytes after T. cruzi infection. Transfection of cardiomyocytes with small interfering RNA (siRNA) induces silencing of PPARγ and impairs the effects of 15dPGJ2 on the modulation of pro-inflammatory enzymes. Moreover, transfection restores the ability of these cells to control the intracellular growth of T. cruzi. We also found that PPARγ-independent pathways are involved, since 15dPGJ2 also exerts its effect through extracellular signal-regulated kinases-mitogen-activated protein kinase (Erk-MAPK) and nuclear factor-κB (NF-κB). The use of specific pharmacological inhibitors confirmed these findings. Our data point out that 15dPGJ2 is a potent modulator of the inflammatory process and regulator of parasites growth through PPARγ-dependent and independent (Erk-MAPK- and NF-κB) pathways in T. cruzi infected neonatal cardiac cells.     

Role of CCL3/MIP-1α and CCL5/RANTES during acute Trypanosoma cruzi infection in rats

Chagas’ disease is caused by Trypanosoma cruzi infection and is characterized by chronic fibrogenic inflammation and heart dysfunction. Chemokines are produced during infection and drive tissue inflammation. In rats, acute infection is characterized by intense myocarditis and regression of inflammation after control of parasitism. We investigated the role of CCL3 and CCL5 during infection by using DNA vaccination encoding for each chemokine separately or simultaneously. MetRANTES treatment was used to evaluate the role of CCR1 and CCR5, the receptors for CCL3 and CCL5. Vaccination with CCL3 or CCL5 increased heart parasitism and decreased local IFN-γ production, but did not influence intensity of inflammation. Simultaneous treatment with both plasmids or treatment with MetRANTES enhanced cardiac inflammation, fibrosis and parasitism. In conclusion, chemokines CCL3 and CCL5 are relevant, but not essential, for control of T. cruzi infection in rats. On the other hand, combined blockade of these chemokines or their receptors enhanced tissue inflammation and fibrosis, clearly contrasting with available data in murine models of T. cruzi infection. These data reinforce the important role of chemokines during T. cruzi infection but suggest that caution must be taken when expanding the therapeutic modulation of the chemokine system in mice to the human infection.     

Paradoxical effects of vitamin C in Chagas disease

Trypanosoma cruzi infection stimulates inflammatory mediators which cause oxidative stress, and the use of antioxidants can minimize the sequelae of Chagas disease. In order to evaluate the efficacy of vitamin C in minimizing oxidative damage in Chagas disease, we orally administered ascorbic acid to Swiss mice infected with 5.0?×?10⁴ trypomastigote forms of T. cruzi QM2 strain. These animals were treated for 60?days to investigate the acute phase and 180?days for the chronic phase. During the acute phase, the animals in the infected and treated groups demonstrated lower parasitemia and inflammatory processes were seen in more mice in these groups, probably due to the higher concentration of nitric oxide, which led to the formation of peroxynitrite. The decrease in reduced glutathione concentration in this group showed a circulating oxidant state, and this antioxidant was used to regenerate vitamin C. During the chronic phase, the animals in the infected and treated group showed a decrease in ferric reducing ability of plasma and uric acid concentrations as well as mobilization of bilirubin (which had higher plasma concentration), demonstrating cooperation between endogenous non-enzymatic antioxidants to combat increased oxidative stress. However, lower ferrous oxidation in xylenol orange concentrations was found in the infected and treated group, suggesting that vitamin C provided biological protection by clearing the peroxynitrite, attenuating the chronic inflammatory process in the tissues and favoring greater survival in these animals. Complex interactions were observed between the antioxidant systems of the host and parasite, with paradoxical actions of vitamin C.     

Mir-190b negatively contributes to the Trypanosoma cruzi- infected cell survival by repressing PTEN protein expression

Chagas disease, which is caused by the intracellular protozoanTrypanosoma cruzi, is a serious health problem in Latin America. The heart is one of the major organs affected by this parasitic infection. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection, and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. Previous studies have reported that the establishment of parasitism is connected to the activation of the phosphatidylinositol-3 kinase (PI3K), which controls important steps in cellular metabolism by regulating the production of the second messenger phosphatidylinositol-3,4,5-trisphosphate. Particularly, the tumour suppressor PTEN is a negative regulator of PI3K signalling. However, mechanistic details of the modulatory activity of PTEN on Chagas disease have not been elucidated. To address this question, H9c2 cells were infected with T. cruzi Berenice 62 strain and the expression of a specific set of microRNAs (miRNAs) were investigated. Our cellular model demonstrated that miRNA-190b is correlated to the decrease of cellular viability rates by negatively modulating PTEN protein expression in T. cruzi-infected cells.     

Haeme oxygenase activity protects the host against excessive cardiac inflammation during experimental Trypanosoma cruzi infection

The infection with Trypanosoma cruzi induces a robust cardiac inflammation that plays a pathogenic role in the development of Chagas heart disease. In this study, we aimed at investigating the effects of Haem Oxygenase (HO) during experimental infection by T. cruzi in BALB/c and C57BL/6 mice. HO has recently emerged as a key factor modulating the immune response in diverse models of inflammatory diseases. In mice with two different genetic backgrounds, the pharmacologic inhibition of HO activity with zinc-protoporphyrin IX (ZnPPIX) induced enhanced myocarditis and reduced parasitaemia, which was accompanied by an amplified production of nitric oxide and increased influx of CD4⁺, CD8⁺ and IFN-γ⁺ cells to the myocardium in comparison with the control group. Conversely, treatment with haemin (an activator of HO) lead to a decreased number of intracardiac CD4⁺ (but not CD8⁺) cells compared to the control group. The mechanism involved in these observations is a modulation of the induction of regulatory T cells, because the stimulation or inhibition of HO was parallelled by an enhanced or reduced frequency of regulatory T cells, respectively. Hence, HO may be involved in the regulation of heart tissue inflammation and could be a potential target in conceiving future therapeutic approaches for Chagas disease.