Trypanosoma cruzi Produces the Specialized Proresolving Mediators Resolvin D1, Resolvin D5, and Resolvin E2.

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease (CD). CD is a persistent, lifelong infection affecting many organs, most notably the heart, where it may result in acute myocarditis and chronic cardiomyopathy. The pathological features include myocardial inflammation and fibrosis. In the Brazil strain-infected CD-1 mouse, which recapitulates many of the features of human infection, we found increased plasma levels of resolvin D1 (RvD1), a specialized proresolving mediator of inflammation, during both the acute and chronic phases of infection (>100 days postinfection) as determined by enzyme-linked immunosorbent assay (ELISA). Additionally, ELISA on lysates of trypomastigotes of both strains Tulahuen and Brazil revealed elevated levels of RvD1 compared with lysates of cultured epimastigotes of T. cruzi, tachyzoites of Toxoplasma gondii, trypomastigotes of Trypanosoma brucei, cultured L6E9 myoblasts, and culture medium containing no cells. Lysates of T. cruzi-infected myoblasts also displayed increased levels of RvD1. Lipid mediator metabolomics confirmed that the trypomastigotes of T. cruzi produced RvD1, RvD5, and RvE2, which have been demonstrated to modulate the host response to bacterial infections. Plasma RvD1 levels may be both host and parasite derived. Since T. cruzi synthesizes specialized proresolving mediators of inflammation, as well as proinflammatory eicosanoids, such as thromboxane A2, one may speculate that by using these lipid mediators to modulate its microenvironment, the parasite is able to survive.

Thromboxane A2 is a key regulator of pathogenesis during Trypanosoma cruzi infection.

Chagas' disease is caused by infection with the parasite Trypanosoma cruzi. We report that infected, but not uninfected, human endothelial cells (ECs) released thromboxane A(2) (TXA(2)). Physical chromatography and liquid chromatography-tandem mass spectrometry revealed that TXA(2) is the predominant eicosanoid present in all life stages of T. cruzi. Parasite-derived TXA(2) accounts for up to 90% of the circulating levels of TXA(2) in infected wild-type mice, and perturbs host physiology. Mice in which the gene for the TXA(2) receptor (TP) has been deleted, exhibited higher mortality and more severe cardiac pathology and parasitism (fourfold) than WT mice after infection. Conversely, deletion of the TXA(2) synthase gene had no effect on survival or disease severity. TP expression on somatic cells, but not cells involved in either acquired or innate immunity, was the primary determinant of disease progression. The higher intracellular parasitism observed in TP-null ECs was ablated upon restoration of TP expression. We conclude that the host response to parasite-derived TXA(2) in T. cruzi infection is possibly an important determinant of mortality and parasitism. A deeper understanding of the role of TXA(2) may result in novel therapeutic targets for a disease with limited treatment options.

Role of SOCS2 in modulating heart damage and function in a murine model of acute Chagas disease.

Infection with Trypanosoma cruzi induces inflammation, which limits parasite proliferation but may result in chagasic heart disease. Suppressor of cytokine signaling 2 (SOCS2) is a regulator of immune responses and may therefore participate in the pathogenesis of T. cruzi infection. SOCS2 is expressed during T. cruzi infection, and its expression is partially reduced in infected 5-lipoxygenase-deficient [knockout (KO)] mice. In SOCS2 KO mice, there was a reduction in both parasitemia and the expression of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), IL-6, IL-10, SOCS1, and SOCS3 in the spleen. Expression of IFN-γ, TNF-α, SOCS1, and SOCS3 was also reduced in the hearts of infected SOCS2 KO mice. There was an increase in the generation and expansion of T regulatory (Treg) cells and a decrease in the number of memory cells in T. cruzi-infected SOCS2 KO mice. Levels of lipoxinA(4) (LXA(4)) increased in these mice. Echocardiography studies demonstrated an impairment of cardiac function in T. cruzi-infected SOCS2 KO mice. There were also changes in calcium handling and in action potential waveforms, and reduced outward potassium currents in isolated cardiac myocytes. Our data suggest that reductions of inflammation and parasitemia in infected SOCS2-deficient mice may be secondary to the increases in Treg cells and LXA(4) levels. This occurs at the cost of greater infection-associated heart dysfunction, highlighting the relevance of balanced inflammatory and immune responses in preventing severe T. cruzi-induced disease.

Mechanisms of Trypanosoma cruzi persistence in Chagas disease.

Trypanosoma cruzi infection leads to development of chronic Chagas disease. In this article, we provide an update on the current knowledge of the mechanisms employed by the parasite to gain entry into the host cells and establish persistent infection despite activation of a potent immune response by the host. Recent studies point to a number of T. cruzi molecules that interact with host cell receptors to promote parasite invasion of the diverse host cells. T. cruzi expresses an antioxidant system and thromboxane A(2) to evade phagosomal oxidative assault and suppress the host's ability to clear parasites. Additional studies suggest that besides cardiac and smooth muscle cells that are the major target of T. cruzi infection, adipocytes and adipose tissue serve as reservoirs from where T. cruzi can recrudesce and cause disease decades later. Further, T. cruzi employs at least four strategies to maintain a symbiotic-like relationship with the host, and ensure consistent supply of nutrients for its own survival and long-term persistence. Ongoing and future research will continue to help refining the models of T. cruzi invasion and persistence in diverse tissues and organs in the host.

Identification of a functional prostanoid-like receptor in the protozoan parasite, Trypanosoma cruzi.

Trypanosoma cruzi infection in humans and experimental animals causes Chagas disease which is often accompanied by myocarditis, cardiomyopathy, and vasculopathy. T. cruzi-derived thromboxane A2 (TXA2) modulates vasculopathy and other pathophysiological features of Chagasic cardiomyopathy. Here, we provide evidence that epimastigotes, trypomastigotes, and amastigotes of T. cruzi (Brazil and Tulahuen strains) express a biologically active prostanoid receptor (PR) that is responsive to TXA2 mimetics, e.g. IBOP. This putative receptor, TcPR, is mainly localized in the flagellar membrane of the parasites and shows a similar glycosylation pattern to that of bona fide thromboxane prostanoid (TP) receptors obtained from human platelets. Furthermore, TXA2-PR signal transduction activates T. cruzi-specific MAPK pathways. While mammalian TP is a G-protein coupled receptor (GPCR); T. cruzi genome sequencing has not demonstrated any confirmed GPCRs in these parasites. Based on this genome sequencing it is likely that TcPR is unique in these protists with no counterpart in mammals. TXA2 is a potent vasoconstrictor which contributes to the pathogenesis of Chagasic cardiovascular disease. It may, however, also control parasite differentiation and proliferation in the infected host allowing the infection to progress to a chronic state.

Perspectives on the Trypanosoma cruzi-host cell receptor interactions.

Chagas disease is caused by the parasite Trypanosoma cruzi. The critical initial event is the interaction of the trypomastigote form of the parasite with host receptors. This review highlights recent observations concerning these interactions. Some of the key receptors considered are those for thromboxane, bradykinin, and for the nerve growth factor TrKA. Other important receptors such as galectin-3, thrombospondin, and laminin are also discussed. Investigation into the molecular biology and cell biology of host receptors for T. cruzi may provide novel therapeutic targets.

Alterations in myocardial gene expression associated with experimental Trypanosoma cruzi infection.

Chagas disease, characterized by acute myocarditis and chronic cardiomyopathy, is caused by infection with the protozoan parasite Trypanosoma cruzi. We sought to identify genes altered during the development of parasite-induced cardiomyopathy. Microarrays containing 27,400 sequence-verified mouse cDNAs were used to analyze global gene expression changes in the myocardium of a murine model of chagasic cardiomyopathy. Changes in gene expression were determined as the acute stage of infection developed into the chronic stage. This analysis was performed on the hearts of male CD-1 mice infected with trypomastigotes of T. cruzi (Brazil strain). At each interval we compared infected and uninfected mice and confirmed the microarray data with dye reversal. We identified eight distinct categories of mRNAs that were differentially regulated during infection and identified dysregulation of several key genes. These data may provide insight into the pathogenesis of chagasic cardiomyopathy and provide new targets for intervention.

Microarray technology in the investigation of diseases of myocardium with special reference to infection.

Microarrays are now routinely employed to characterize gene expression of thousands of genes from a single hybridization. The genome wide gene expression profile aids in the understanding of genes that may be regulated in a particular pathological condition. This paper provides an overview of microarray technology and its recent developments followed by its usage in studies of cardiovascular disease and how it pertains to viral and parasitic infections of the heart.

Trypanosoma cruzi invasion is associated with trogocytosis.

Trogocytosis was originally thought to be restricted to the interaction of cells of the immune system with cancer cells. Such membrane exchanges are probably a general process in cell biology, and membrane exchange has been demonstrated to occur between non-immune cells within an organism. Herein, we report that membrane and protein exchange, consistent with trogocytosis, between Trypanosoma cruzi (both the Brazil and Tulahuen strains) and the mammalian cells it infects. Transfer of labeled membrane patches was monitored by labeling of either parasites or host cells, i.e. human foreskin fibroblasts and rat myoblasts. Trypomastigotes and amastigotes transferred specific surface glycoproteins to the host cells along with membranes. Exchange of membranes between the parasite and host cells occurred during successful invasion. Extracellular amastigotes did not transfer membrane patches and were did not transfer either membranes or proteins to the host cells. Membrane exchange was also found to occur between interacting epimastigotes in cell-free culture and may be important in parasite-parasite interactions as well. Further studies should provide new insights into pathogenesis and provide targets for therapeutic intervention.

Role of vasoactive mediators in the pathogenesis of Chagas' disease.

This review focuses on the vascular pathogenesis of Chagas' disease, the cardiomyopathy caused by infection with the parasitic protozoa Trypanosoma cruzi. Recent studies strongly suggests that T. cruzi infection is linked to functional changes in the activity of two potent vasoactive peptidergic mediators, endothelin-1, a vasoconstrictor, and kinins, a group of vasodilator and pro-inflammatory peptides related to bradykinin. Understanding the molecular mechanisms underlying disturbances of vascular homoeostasis? induced by T. cruzi may provide opportunities for therapeutic intervention and amelioration of heart pathology.

Cardioprotective effects of phosphoramidon on myocardial structure and function in murine Chagas' disease.

Chagas' disease is an important cause of cardiomyopathy. Endothelin-1, a vasoactive peptide has been implicated in the pathogenesis of chagasic cardiomyopathy. C57BL/6 x 129sv and CD1 mice were thus, infected with trypomastigotes of Trypanosoma cruzi (Brazil strain) and these infected mice were compared with infected mice treated with phosphoramidon. This compound inhibits endothelin-converting enzyme and neutral endopeptidases and does not affect the growth of the parasite in culture. Phosphoramidon was given in a dose of 10mg/kg for the initial 15 days post-infection None of the C57Bl/6 x 129sv mice died as a result of infection. However, there was marked myocardial inflammation and fibrosis in infected, untreated mice. The hearts of the infected, phosphoramidon-treated mice showed significantly less pathology. Cardiac magnetic resonance imaging of infected mice revealed right ventricular dilation that was less severe in those treated with phosphoramidon. Phosphoramidon-treated CD1 mice survived the acute infection. Transthoracic echocardiography demonstrated left ventricular dilation and reduced percent fractional shortening and relative wall thickness. These alterations were also attenuated as a result of phosphoramidon treatment. These data suggest that endothelin-1 contributes to the pathogenesis of chagasic cardiomyopathy and interventions that inhibit the synthesis of endothelin-1 and/or neutral endopeptidase might have a protective effect on myocardial structure and function in murine Chagas' disease.

Chagas heart disease: report on recent developments.

Chagas disease, caused by the parasite Trypanosoma cruzi, is an important cause of cardiac disease in endemic areas of Latin America. It is now being diagnosed in nonendemic areas because of immigration. Typical cardiac manifestations of Chagas disease include dilated cardiomyopathy, congestive heart failure, arrhythmias, cardioembolism, and stroke. Clinical and laboratory-based research to define the pathology resulting from T. cruzi infection has shed light on many of the cellular and molecular mechanisms leading to these manifestations. Antiparasitic treatment may not be appropriate for patients with advanced cardiac disease. Clinical management of Chagas heart disease is similar to that used for cardiomyopathies caused by other processes. Cardiac transplantation has been successfully performed in a small number of patients with Chagas heart disease.

Bioactive lipids in Trypanosoma cruzi infection.

Chagas disease is caused by Trypanosoma cruzi, a protozoan parasite. Chagas disease remains a serious health problem in large parts of Mexico and Central and South America, where it is a major cause of morbidity and mortality. This disease is being increasingly recognized in non-endemic regions due to immigration. Heart disease develops in 10-30% of infected individuals. It is increasingly clear that parasite- and host-derived bioactive lipids potently modulate disease progression. Many of the changes that occur during acute and chronic Chagas disease can be accounted for by the effects of arachidonic acid (AA)-derived lipids such as leukotrienes, lipoxins, H(P)ETEs, prostaglandins (PGs) and thromboxane. During the course of infection with T. cruzi, changes in circulating levels of AA metabolites are observed. Antagonism of PG synthesis with cyclooxygenase (COX) inhibitors has both beneficial and adverse effects. Treatment with COX inhibitors during acute infection may result in increased parasite load and mortality. However, treatment instituted during chronic infection may be beneficial with no increase in mortality and substantial improvement with cardiac function. Recently, T. cruzi infection of mice deficient in AA biosynthetic enzymes for various pathways has yielded more insightful data than pharmacological inhibition and has highlighted the potential deleterious effects of inhibitors due to "off-target" actions. Using COX-1 null mice, it was observed that parasite biosynthesis is dependent upon host metabolism, that the majority of TXA(2) liberated during T. cruzi infection is derived from the parasite and that this molecule may act as a quorum sensor to control parasite growth/differentiation. Thus, eicosanoids present during acute infection may act as immunomodulators aiding the transition to, and maintenance of, the chronic stage of the disease. It is also likely that the same mediators that initially function to ensure host survival may later contribute to cardiovascular damage. Collectively, the eicosanoids represent a new series of targets for therapy in Chagas disease with defined potential therapeutic windows in which to apply these agents for greatest effect. A deeper understanding of the mechanism of action of non-steroidal anti-inflammatory drugs may provide clues to the differences between host responses in acute and chronic T. cruzi infection.

Microarray analysis of the mammalian thromboxane receptor-Trypanosoma cruzi interaction.

Trypanosoma cruzi, the etiological agent of Chagas disease, causes vasculopathy and cardiomyopathy in humans and is associated with elevated levels of several vasoactive molecules such as nitric oxide, endothelin-1 and thromboxane A 2 (TXA 2). Parasite derived TXA 2 modulates vasculopathy and other pathophysiological features of Chagasic cardiomyopathy. Previously, we demonstrated that in response to infection with T. cruzi, TXA 2 receptor (TP) null mice displayed increased parasitemia; mortality and cardiac pathology compared with wild type (WT) and TXA 2 synthase null mice. In order to further study the role of TXA 2-TP signaling in the development of Chagas disease, GeneChip microarrays were used to detect transcriptome changes in rat fat pad endothelial cells (RFP-ECs) which is incapable of TXA 2 signaling (TP null) to that of control (wild type) and RFP-EC with reconstituted TP expression. Genes that were significantly regulated due to infection were identified using a time course of 2, 18 and 48 hrs post infection. We identified several key genes such as suppressor of cytokine signaling (SOCS-5), several cytokines (CSF-1, CXCF ligands), and MAP kinases (MAPK-1, Janus kinase) that were upregulated in the absence of TP signaling. These data underscore the importance of the interaction of the parasite with mammalian TP and may explain the increased mortality and cardiovascular pathology observed in infected TP null mice.

Aspirin treatment of mice infected with Trypanosoma cruzi and implications for the pathogenesis of Chagas disease.

Chagas disease, caused by infection with Trypanosoma cruzi, is an important cause of cardiovascular disease. It is increasingly clear that parasite-derived prostaglandins potently modulate host response and disease progression. Here, we report that treatment of experimental T. cruzi infection (Brazil strain) beginning 5 days post infection (dpi) with aspirin (ASA) increased mortality (2-fold) and parasitemia (12-fold). However, there were no differences regarding histopathology or cardiac structure or function. Delayed treatment with ASA (20 mg/kg) beginning 60 dpi did not increase parasitemia or mortality but improved ejection fraction. ASA treatment diminished the profile of parasite- and host-derived circulating prostaglandins in infected mice. To distinguish the effects of ASA on the parasite and host bio-synthetic pathways we infected cyclooxygenase-1 (COX-1) null mice with the Brazil-strain of T. cruzi. Infected COX-1 null mice displayed a reduction in circulating levels of thromboxane (TX)A(2) and prostaglandin (PG)F(2α). Parasitemia was increased in COX-1 null mice compared with parasitemia and mortality in ASA-treated infected mice indicating the effects of ASA on mortality potentially had little to do with inhibition of prostaglandin metabolism. Expression of SOCS-2 was enhanced, and TRAF6 and TNFα reduced, in the spleens of infected ASA-treated mice. Ablation of the initial innate response to infection may cause the increased mortality in ASA-treated mice as the host likely succumbs more quickly without the initiation of the "cytokine storm" during acute infection. We conclude that ASA, through both COX inhibition and other "off-target" effects, modulates the progression of acute and chronic Chagas disease. Thus, eicosanoids present during acute infection may act as immunomodulators aiding the transition to and maintenance of the chronic phase of the disease. A deeper understanding of the mechanism of ASA action may provide clues to the differences between host response in the acute and chronic T. cruzi infection.

Alterations in the Brain Transcriptome in Plasmodium Berghei ANKA Infected Mice.

We have used cDNA microarrays to compare gene expression profiles in brains from normal mice to those infected with the ANKA strain of Plasmodium berghei, a model of cerebral malaria. For each of three brains in each group, we computed ratios of all quantifiable genes with a composite reference sample and then computed ratios of gene expression in infected brains compared to untreated controls. Of the almost 12,000 unigenes adequately quantified in all arrays, approximately 3% were significantly downregulated (P < 0.05, ≥ 50% fold change) and about 7% were upregulated. Upon inspection of the lists of regulated genes, we identified a high number encoding proteins of importance to normal brain function or associated with neuropathology, including genes that encode for synaptic proteins or genes involved in cerebellar function as well as genes important in certain neurological diseases such as Alzheimer's disease or autism. These results emphasize the important impact of malarial infection on gene expression in the brain and provide potential biomarkers that may provide novel therapeutic targets to ameliorate the neurological sequelae of this infection.

Role of NO synthase in the development of Trypanosoma cruzi-induced cardiomyopathy in mice.

Trypanosoma cruzi infection results in an increase in myocardial NO and intense inflammation. NO modulates the T. cruzi-induced myocardial inflammatory reaction. NO synthase (NOS)1-, NOS2-, and NOS3-null mice were infected with T. cruzi (Brazil strain). Infected NOS1-null mice had increased parasitemia, mortality, and left ventricular inner diameter (LVID). Chronically infected NOS1- and NOS2-null and wild-type mice (WT) exhibited increased right ventricular internal diameter (RVID), although the fold increase in the NOS2-null mice was smaller. Infected NOS3-null mice exhibited a significant reduction both in LVID and RVID. Reverse transcriptase-polymerase chain reaction showed expression of NOS2 and NOS3 in hearts of infected NOS1-null and WT mice, whereas infected NOS2-null hearts showed little change in expression of other NOS isoforms. Infected NOS3-null hearts showed an increase only in NOS1 expression. These results may indicate different roles for NOS isoforms in T. cruzi-induced cardiomyopathy.

Regulation of host cell cyclin D1 by Trypanosoma cruzi in myoblasts.

Infection with the parasite Trypanosoma cruzi causes Chagas disease. In this study we demonstrated that there was an increase in cyclin D1 expression in T. cruzi (Tulahuen strain)-infected myoblasts. To examine a possible mechanism for the increased cyclin D1 expression we transfected L(6)E(9) myoblasts with cyclin D1 luciferase reporter constructs and infected with T. cruzi. There was no evidence of an increase in promoter activity. Additionally, quantitative PCR did not demonstrate any change in cyclin D1 message during infection. Moreover, we demonstrated that the cyclin D1 protein was significantly stabilized after infection. Collectively, these data indicate that infection with T. cruzi increases cyclin D1 protein abundance post-translationally.

A magnetic resonance imaging study of intestinal dilation in Trypanosoma cruzi-infected mice deficient in nitric oxide synthase.

Infection with Trypanosoma cruzi causes megasyndromes of the gastrointestinal (GI) tract. We used magnetic resonance imaging (MRI) to monitor alterations in the GI tract of T. cruzi-infected mice, and to assess the role of nitric oxide (NO) in the development of intestinal dilation. Brazil strain-infected C57BL/6 wild-type (WT) mice exhibited dilatation of the intestines by 30 days post-infection. Average intestine lumen diameter increased by 72%. Levels of intestinal NO synthase (NOS) isoforms, NOS2 and NOS3, were elevated in infected WT mice. Inflammation and ganglionitis were observed in all infected mice. Intestinal dilation was observed in infected WT, NOS1, NOS2, and NOS3 null mice. This study demonstrates that MRI is a useful tool to monitor intestinal dilation in living mice and that these alterations may begin during acute infection. Furthermore, our data strongly suggests that NO may not be the sole contributor to intestinal dysfunction resulting from this infection.

Trypanosoma cruzi infection induces proliferation of vascular smooth muscle cells.

Trypanosoma cruzi infection causes cardiomyopathy and vasculopathy. Previous studies have demonstrated that infection of human umbilical vein endothelial and smooth muscle cells resulted in activation of extracellular signal-regulated kinase (ERK). In the present study, smooth muscle cells were infected with trypomastigotes, and immunoblot analysis revealed an increase in the expression of cyclin D1 and proliferating cell nuclear antigen (PCNA), important mediators of smooth muscle cell proliferation. Interestingly, after infection, the expression of caveolin-1 was reduced in both human umbilical vein endothelial cells and smooth muscle cells. Immunoblot and immunohistochemical analyses of lysates of carotid arteries obtained from infected mice revealed increased expression of PCNA, cyclin D1, its substrate, phospho-Rb (Ser780), and phospho-ERK1/2. The expression of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1), caveolin-1, and caveolin-3 was reduced in carotid arteries obtained from infected mice. There was an increase in the abundance of pre-pro-endothelin-1 mRNA in the carotid artery and aorta from infected mice. The ET(A) receptor was also elevated in infected arteries. ERK activates endothelin-1, which in turn exerts positive feedback activating ERK, and cyclin D1 is a downstream target of both endothelin-1 and ERK. There was significant incorporation of bromodeoxyuridine into smooth muscle cell DNA when treatment was with conditioned medium obtained from infected endothelial cells. Taken together, these data suggest that T. cruzi infection stimulates smooth muscle cell proliferation and is likely a result of the upregulation of the ERK-cyclin D1-endothelin-1 pathway.