Immunopathology of cardiomyopathy in the experimental Chagas disease

The mechanisms by which Trypanosoma cruzi causes cardiomyopathy and induces neuronal destruction are discussed in this paper. The results suggest that autoimmunity in the chronic phase is the main cause of the progressive cardiac destruction, and that autoreactivity is restricted to the CD4+ T cell compartment. During the acute phase, the neuronal and cardiac fiber destruction occurs when ruptured parasite nests release T. cruzi antigens that bind to the cell surface in the vicinity which become targets for the cellular and humoral immune response against T. cruzi. The various factors involved in the genesis of autoimmunity in chronic T. cruzi infection include molecular mimicry, presentation of self-antigens and imbalance of immune regulation.     

Heat-killed Trypanosoma cruzi induces acute cardiac damage and polyantigenic autoimmunity

Chagas heart disease, caused by the protozoan parasite Trypanosoma cruzi, is a potentially fatal cardiomyopathy often associated with cardiac autoimmunity. T. cruzi infection induces the development of autoimmunity to a number of antigens via molecular mimicry and other mechanisms, but the genesis and pathogenic potential of this autoimmune response has not been fully elucidated. To determine whether exposure to T. cruzi antigens alone in the absence of active infection is sufficient to induce autoimmunity, we immunized A/J mice with heat-killed T. cruzi (HKTC) emulsified in complete Freund's adjuvant, and compared the resulting immune response to that induced by infection with live T. cruzi. We found that HKTC immunization is capable of inducing acute cardiac damage, as evidenced by elevated serum cardiac troponin I, and that this damage is associated with the generation of polyantigenic humoral and cell-mediated autoimmunity with similar antigen specificity to that induced by infection with T. cruzi. However, while significant and preferential production of Th1 and Th17-associated cytokines, accompanied by myocarditis, develops in T. cruzi-infected mice, HKTC-immunized mice produce lower levels of these cytokines, do not develop Th1-skewed immunity, and lack tissue inflammation. These results demonstrate that exposure to parasite antigen alone is sufficient to induce autoimmunity and cardiac damage, yet additional immune factors, including a dominant Th1/Th17 immune response, are likely required to induce cardiac inflammation.     

Behavior of atrial natriuretic factor in an experimental model of Trypanosoma cruzi infection in rats

Enhanced atrial natriuretic factor (ANF) production by the heart is related to hemodynamic overload, cardiac growth, and hypertrophy. The heart is one of the most affected organs during Trypanosoma cruzi infection. We tested the hypothesis that myocarditis produced by parasite infection alters the natriuretic peptide system by investigating the behavior of plasma ANF during the acute and chronic stages of T. cruzi infection in rats. Sprague-Dawley rats were infected with T. cruzi clone Sylvio-X10/7. Cardiac morphology showed damage to myocardial cells and lymphocyte infiltration in the acute phase; and fibrosis and cell atrophy in the chronic period. Plasma ANF levels (radioimmunoassay) were significantly higher in acute (348 +/- 40 vs. 195 +/- 36 pg/ml, P < 0.05, n = 17) and chronic T. cruzi myocarditis (545 +/- 81 vs. 229 +/- 38 pg/ml, P < 0.001, n = 11) than in their respective controls (n = 10 and 14). Rats in the chronic phase also showed higher levels than rats in the acute phase (P < 0.01). The damage of myocardial cells produced by the parasite and the subsequent inflammatory response could be responsible for the elevation of plasma ANF during the acute period of T. cruzi infection. The highest plasma ANF levels found in chronically infected rats could be derived from the progressive failure of cardiac function.     

CTLA-4 blockage increases resistance to infection with the intracellular protozoan Trypanosoma cruzi

Recent studies have revealed an important role for CTLA-4 as a negative regulator of T cell activation. In the present study, we evaluated the importance of CTLA-4 to the immune response against the intracellular protozoan, Trypanosoma cruzi, the causative agent of Chagas' disease. We observed that the expression of CTLA-4 in spleen cells from naive mice cultured in the presence of live trypomastigote forms of T. cruzi increases over time of exposure. Furthermore, spleen cells harvested from recently infected mice showed a significant increase in the expression of CTLA-4 when compared with spleen cells from noninfected mice. Blockage of CTLA-4 in vitro and/or in vivo did not restore the lymphoproliferative response decreased during the acute phase of infection, but it resulted in a significant increase of NO production in vivo and in vitro. Moreover, the production of IFN-gamma in response to parasite Ags was significantly increased in spleen cells from anti-CTLA-4-treated infected mice when compared with the production found in cells from IgG-treated infected mice. CTLA-4 blockade in vivo also resulted in increased resistance to infection with the Y and Colombian strains of T. cruzi. Taken together these results indicate that CTLA-4 engagement is implicated in the modulation of the immune response against T. cruzi by acting in the mechanisms that control IFN-gamma and NO production during the acute phase of the infection.     

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.     

IL-10-IFN-γ double producers CD4+ T cells are induced by immunization with an amastigote stage specific derived recombinant protein of Trypanosoma cruzi

During the acute phase of infection, T. cruzi replicates extensively and releases immunomodulatory molecules that delay parasite-specific responses mediated by effector T cells. This mechanism of evasion allows the parasite to spread in the host. Parasite molecules that regulate the host immune response during Chagas'disease have not been fully identified. GPI-anchored mucins, glycoinositolphospholipids, and glycoproteins comprise some of the most abundant T. cruzi surface molecules. IL-10 IFN-γ-secreting CD4+ T cells are activated during chronic infections and are responsible for prolonged persistence of parasite and for host protection against severe inflammatory responses. In this work we evaluated the role of rMBP::SSP4 protein of T. cruzi, a recombinant protein derived from a GPI anchored antigen, SSP4, as an immunomodulator molecule, finding that it was able to induce high concentrations of IL-10 and IFN-γ both in vivo and in vitro; during this last condition, both cytokines were produced by IL-10-IFN-γ-secreting CD4+ T cells.     

Trypanosoma cruzi in a caviomorph rodent: parasitological and pathological features of the experimental infection of Trichomys apereoides (Rodentia, Echimyidae)

To understand the interaction of Trypanosoma cruzi with caviomorph rodents, which supposedly have an ancient co-evolutionary history with this parasite, experimental infection of laboratory reared Trichomys apereoides with several isolates of both genotypes of the parasite was studied. Parasitemia, pattern of hematic cells, specific humoral immune response, histopathological features and parasite clearance were appraised. T. apereoides maintained stable infections independent of the T. cruzi genotype as demonstrated by positive PCR results in analyses of several tissues after a 5 months follow-up. The acute phase was characterized by abundant and disseminated presence of amastigotes, vacuolization and/or myocytolysis. Lymphocytosis was a common feature. The chronic phase was characterized mainly by lymphomacroeosinophilic infiltrates independent of the inoculated T. cruzi isolate. T. cruzi of different genotypes did not show any tissular preference in T. apereoides.     

Fas/Fas-L expression, apoptosis and low proliferative response are associated with heart failure in patients with chronic Chagas' disease

Chagas' disease affects 16-18 million patients in South America and heart involvement is the major cause of morbidity and mortality. Heart failure is the most severe clinical manifestation of the chronic phase of infection with Trypanosoma cruzi. The intensity and nature of the immune response is associated with the clinical outcome of the disease. In murine models, a low proliferative response and T-cell apoptosis have been observed during acute infection. In the present study the immune response of patients in the chronic phase of infection was analyzed. Patients were divided into: (a) asymptomatic, i.e., without involvement of the heart or digestive system; and (b) with heart failure. Patients with heart failure presented a significantly lower peripheral blood mononuclear cell (PBMC) proliferative response to T. cruzi antigens compared to asymptomatic patients. This low response was associated with antigen-induced apoptosis. Apoptosis of PBMC and a low proliferative response were also associated with double Fas/Fas-L expression and high production of TNF-alpha, a cytokine known to induce programmed cell death. These results suggest that apoptosis of PBMC, probably triggered by double expression of Fas/Fas-L and TNF-alpha, is implicated in the immune regulatory mechanism during the chronic phase of Chagas' disease.     

Different infective forms trigger distinct immune response in experimental Chagas disease

Although metacyclic and blood trypomastigotes are completely functional in relation to parasite-host interaction and/or target cell invasion, they differ in the molecules present on the surface. Thus, aspects related to the variability that the forms of T. cruzi interacts with host cells may lead to fundamental implications on the immune response against this parasite and, consequently, the clinical evolution of Chagas disease. We have shown that BT infected mice presented higher levels of parasitemia during all the acute phase of infection. Moreover, the infection with either MT or BT forms resulted in increased levels of total leukocytes, monocytes and lymphocytes, specifically later for MT and earlier for BT. The infection with BT forms presented earlier production of proinflammatory cytokine TNF-α and later of IFN-γ by both T cells subpopulations. This event was accompanied by an early cardiac inflammation with an exacerbation of this process at the end of the acute phase. On the other hand, infection with MT forms result in an early production of IFN-γ, with subsequent control in the production of this cytokine by IL-10, which provided to these animals an immunomodulatory profile in the end of the acute phase. These results are in agreement with what was found for cardiac inflammation where animals infected with MT forms showed intense cardiac inflammation later at infection, with a decrease in the same at the end of this phase. In summary, our findings emphasize the importance of taking into account the inoculums source of T. cruzi, since vectorial or transfusional routes of T. cruzi infection may trigger distinct parasite-host interactions during the acute phase that may influence relevant biological aspects of chronic Chagas disease.     

Suppression of cell-mediated immunity in experimental Chagas' disease.

The effect of acute infection with the Tulahuén strain of Trypanosoma cruzi on the cellular immune response in Swiss mice was studied. Mice were immunized with either Freund's complete adjuvant or oxazolone, a skin sensitizing agent, and subsequently skin-tested with either BCG protoplasm or oxazolone to detect delayed hypersensitivity. Depression of the response to these antigens was observed in infected mice during the stage of marked parasitemia. Mice which were responsive to oxazolone before infection lost their ability to respond as the infection progressed. When immunized with live attenuated T. cruzi before infection with virulent organisms, mice developed a greater than normal sensitivity to oxazolone and survived infection. These experiments do not conclude whether immunosuppression due to infection with T. cruzi is directed toward induction or expression of the cell-mediated immune response to the antigens employed.     

V gamma 1 gammadelta T cells regulate type-1/type-2 immune responses and participate in the resistance to infection and development of heart inflammation in Trypanosoma cruzi-infected BALB/c mice

Many different cell populations or lineages participate in the resistance to Trypanosoma cruzi infection. gammadelta T cells may also take part in a network of interactions that lead to control of T. cruzi infection with minimal tissue damage by controlling alphabeta T cell activation, as was previously suggested. However, the gammadelta T cell population is not homogeneous and its functions might vary, depending on T cell receptor usage or distinct stimulatory conditions. In this study, we show that the in vivo depletion of V gamma 1-bearing gammadelta T cells, prior to the infection of BALB/c mice with the Y strain of T. cruzi, induces an increased susceptibility to the infection with lower amounts of IFN-gamma being produced by conventional CD4+ or CD8+ T cells. In addition, the production of IL-4 by spleen T cells in V gamma 1-depleted mice was increased and the production of IL-10 remained unchanged. Since V gamma 1(+) gammadelta T cell depletion diminished the conversion of naive to memory/activated CD4 T cells and the production of IFN-gamma during the acute infection, these cells appear to function as helper cells for conventional CD4+ Th1 cells. Depletion of V gamma 1(+) cells also reduced the infection-induced inflammatory infiltrate in the heart and skeletal muscle. More importantly, V gamma 1(+) cells were required for up-regulation of CD40L in CD4+ and CD8+ T cells during infection. These results show that a subset of gammadelta T cells (V gamma 1(+)), which is an important component of the innate immune response, up-regulates the type 1 arm of the adaptative immune response, during T. cruzi infection.     

A DNA vaccine encoding for TcSSP4 induces protection against acute and chronic infection in experimental Chagas disease

Immunization of mice with plasmids containing genes of Trypanosoma cruzi induces protective immunity in the murine model of Chagas disease. A cDNA clone that codes for an amastigote-specific surface protein (TcSSP4) was used as a candidate to develop a DNA vaccine. Mice were immunized with the recombinant protein rTcSSP4 and with cDNA for TcSSP4, and challenged with bloodstream trypomastigotes. Immunization with rTcSSP4 protein makes mice more susceptible to trypomastigote infection, with high mortality rates, whereas mice immunized with a eukaryotic expression plasmid containing the TcSSP4 cDNA were able to control the acute phase of infection. Heart tissue of gene-vaccinated animals did not show myocarditis and tissue damage at 365 days following infection, as compared with control animals. INF-γ was detected in sera of DNA vaccinated mice shortly after immunization, suggesting the development of a Th1 response. The TcSSP4 gene is a promising candidate for the development of an anti-T. cruzi DNA vaccine.     

Trypanosoma cruzi immune evasion mediated by host cell-derived microvesicles

The innate immune system is the first mechanism of vertebrate defense against pathogen infection. In this study, we present evidence for a novel immune evasion mechanism of Trypanosoma cruzi, mediated by host cell plasma membrane-derived vesicles. We found that T. cruzi metacyclic trypomastigotes induced microvesicle release from blood cells early in infection. Upon their release, microvesicles formed a complex on the T. cruzi surface with the complement C3 convertase, leading to its stabilization and inhibition, and ultimately resulting in increased parasite survival. Furthermore, we found that TGF-β-bearing microvesicles released from monocytes and lymphocytes promoted rapid cell invasion by T. cruzi, which also contributed to parasites escaping the complement attack. In addition, in vivo infection with T. cruzi showed a rapid increase of microvesicle levels in mouse plasma, and infection with exogenous microvesicles resulted in increased T. cruzi parasitemia. Altogether, these data support a role for microvesicles contributing to T. cruzi evasion of innate immunity.     

Circulating levels of cyclooxygenase metabolites in experimental Trypanosoma cruzi infections

Trypanosoma cruzi induces inflammatory reactions in several tissues. The production of prostaglandin F2alpha, 6-keto-prostaglandin F1alpha and thromboxane B2, known to regulate the immune response and to participate in inflammatory reactions, was studied in mice experimentally infected with T. cruzi. The generation of nitric oxide (NO), which could be regulated by cyclooxygenase metabolites, was also evaluated. In the acute infection the extension of inflammatory infiltrates in skeletal muscle as well as the circulating levels of cyclooxygenase metabolites and NO were higher in resistant C3H mice than in susceptible BALB/c mice. In addition, the spontaneous release of NO by spleen cells increased earlier in the C3H mouse strain. In the chronic infections, the tissue inflammatory reaction was still prominent in both groups of mice, but a moderate increase of thromboxane B2 concentration and in NO released by spleen cells was observed only in C3H mice. This comparative study shows that these mediators could be mainly related to protective mechanisms in the acute phase, but seem not to be involved in its maintenance in the chronic T. cruzi infections.     

Antigen-specific T cells maintain an effector memory phenotype during persistent Trypanosoma cruzi infection

Infection with the protozoan parasite Trypanosoma cruzi is a major cause of morbidity and mortality in Central and South America. Control of acute experimental infection with T. cruzi is dependent on a robust T cell and type 1 cytokine response. However, little evidence exists demonstrating the development and persistence of a potent antiparasite T cell memory response, and there has been much speculation that the majority of the immune response to T. cruzi infection is not directed against the parasite. In this study, we used an experimental mouse model of T. cruzi infection to test both the Ag specificity and the functional and phenotypic characteristics of the responding T cell population. We observed a vigorous antiparasite response from both CD4(+) and CD8(+) T cells that was maintained in the face of persistent infection. T cells from infected mice also proliferated in response to re-exposure to Ag, and CD8(+) T cells underwent spontaneous proliferation when transferred to naive congenic mice, both characteristic of central memory T cells. Interestingly, T cells from infected mice showed significant down-regulation of CD62L, a characteristic associated with an effector memory phenotype. These results suggest that T cells maintained in mice with chronic T. cruzi infection are fully functional memory cells that cannot be easily categorized in the current central/effector memory paradigm.     

Trypanosoma cruzi: influence of predominant bacteria from indigenous digestive microbiota on experimental infection in mice

To verify the influence of some predominant components from indigenous microbiota on systemic immunological responses during experimental Chagas disease, germ-free NIH Swiss mice were mono-associated with Escherichia coli, Enterococcus faecalis, Bacteroides vulgatus or Peptostreptococcus sp. and then infected with the Y strain of Trypanosoma cruzi. All the mono-associations predominantly induced a Th1 type of specific immune response to the infection by T. cruzi. A direct correlation was observed between a higher survival rate and increased IFN-gamma and TNF-alpha production (P<0.05) in E. faecalis-, B. vulgatus-, and Peptostreptococcus-associated mice. Moreover, higher levels of anti-T. cruzi IgG1 and anti-T. cruzi IgG2a were also found in mono-associated animals after infection. On the other hand, with the exception of E. faecalis-associated mice, mono-association induced a lower IL-10 production after infection (P<0.05) when compared with germ-free animals. Interestingly, spleen cell cultures from non-infected germ-free and mono-associated mice spontaneously produced higher levels (P<0.05) of IL-10 than cultures from infected mono-associated mice, except again for E. faecalis-associated animals. In conclusion, the presence of the components of the indigenous microbiota skews the immune response towards production of inflammatory cytokines during experimental infection with T. cruzi in gnotobiotic mice. However, the degree of increase in production of cytokines depends on each bacterial component.     

Evaluation of immune responses raised against Tc13 antigens of Trypanosoma cruzi in the outcome of murine experimental infection

We have previously reported that genetic immunization with Tc13Tul antigen of Trypanosoma cruzi, the aetiological agent of Chagas' disease, triggers harmful effects and non-protective immune responses. In order to confirm the role of Tc13 antigens during T. cruzi infection, herein we studied the humoral and cellular immune responses to the Tc13Tul molecule and its EPKSA C-terminal portion in BALB/c T. cruzi-infected mice or mice immunized with recombinant Tc13Tul. Analysis of the antibody response showed that B-cell epitopes that stimulate a sustained IgM production along the infection and high levels of IgG in the acute phase are mainly located at the Tc13 N- and C-terminal domains, respectively. DTH assays showed that T-cell epitopes are mainly at the Tc13 N-terminal segment and that they do not elicit an efficient memory response. Recombinant Tc13Tul did not induce IFN-gamma secretion in either infected or immunized mice. However, a putative CD8+Tc13Tul-derived peptide was found to elicit IFN-gamma production in chronically infected animals. Immunization with recombinant Tc13Tul did not induce pathology in tissues and neither did it protect against the infection. Our results show that in the outcome of T. cruzi infection the Tc13 family protein mainly triggers non-protective immune responses.     

Trypanosoma cruzi: acute infection affects expression of alpha-2-macroglobulin and A2MR/LRP receptor differently in C3H and C57BL/6 mice

Although a complete cellular and humoral immune response is elicited in Chagas' disease, recent data suggest that other natural elements of innate immunity may also contribute to the initial host primary defense. alpha-Macroglobulins are a family of plasma proteinase inhibitors that are acute-phase reactants in Trypanosoma cruzi-infected mice and humans. Mice contain a tetrameric alpha-2-macroglobulin (MAM) and a monomeric murinoglobulin (MUG). Heterogeneity in their reactions was observed in murine T. cruzi-infected plasma A2M levels despite an overall increase. In addition, up-regulation of the A2M receptor (A2MR/LRP) was observed in peritoneal macrophages during T. cruzi infection. Here, we show that during T. cruzi infection (Y strain), the MAM and MUG hepatic mRNA levels and the corresponding plasma protein levels were up-regulated in C3H and C57BL/6 (B6) mice, but with different kinetics. On the contrary, A2MR/LRP mRNA levels increased in acutely infected C3H mice, but decreased in B6 mice, in both liver and heart. Immunocytochemistry of infected B6 heart cryosections confirmed a less intense endothelium labeling by the fluoresceinated ligand for A2MR/LRP. On the other hand, infected B6 spleen cells displayed higher F-A2M-FITC binding and MAC1 expression, confirming higher A2MR/LRP expression in macrophages. In uninfected mice, as well as after T. cruzi infection, higher A2M plasma levels were measured in C3H mice than in B6 mice. The lower tissue T. cruzi parasitism found in C3H-infected mice could reflect an inhibitory effect of A2M on parasite invasion. Our present data further contribute to clarifying aspects of the role of A2MR/LRP in a model of acute Chagas' disease in different mouse strains.     

CD28 is required for T cell activation and IFN-gamma production by CD4+ and CD8+ T cells in response to Trypanosoma cruzi infection

In the present study we evaluated the mechanisms behind the implication of the costimulatory molecule CD28 for the immune response against the intracellular protozoan parasite Trypanosma cruzi. Our results reveal a critical role for CD28 in the activation of both CD4+ and CD8+ T cells and induction of the effector mechanisms that ultimately mediate the control of parasite growth and pathogenesis in infected mice. CD28-deficient (CD28-/-) mice are highly susceptible to T. cruzi infection, presenting higher parasitemia and tissue parasitism, but less inflammatory cell infiltrate in the heart than C57Bl/6 wild-type (WT) mice. All the infected WT mice survived acute infection, whereas 100% of CD28-/- mice succumbed to it. The increased susceptibility of the CD28-/- mice was associated with a dramatic decrease in the production of IFN-gamma by both CD4+ and CD8+ T cells resulting in a diminished capacity to produce nitric oxide (NO) and mediate parasite killing. T cell activation was also profoundly impaired in CD28-/- mice, which presented decreased lymphoproliferative response after the infection compared to WT mice. Together, these data represent the first evidence that CD28 is critical for efficient CD4+ T cell activation in response to T. cruzi infection in mice.