An exacerbated metabolism and mitochondrial reactive oxygen species contribute to mitochondrial alterations and apoptosis in CD4 T cells during the acute phase of Trypanosoma cruzi infection.

Chagas disease caused by Trypanosoma cruzi parasite is an endemic infection in America. It is well known that T. cruzi causes a strong immunosuppression during the acute phase of infection. However, it is not clear whether T. cruzi infection is related to metabolic alterations in CD4 T cells that prevent downstream effector function. Here, we evaluated the CD4 T cell metabolic and mitochondrial profiles from non-infected (NI), acute phase (AP) and chronic phase (CP) T. cruzi infected mice. CD4 T cells from all groups showed increased glucose uptake after stimulation. Moreover, the bioenergetic analysis revealed a rise in glycolysis and a higher oxidative metabolism in CD4 T cells from the AP. These cells showed increased proton leak and uncoupling protein 3 (UCP3) expression that correlated with mitochondrial ROS (mROS) accumulation, mitochondrial membrane potential (MMP) depolarization and expression of PD-1. In addition, CD4 T cells with mitochondrial alteration displayed an activated phenotype, and were less functional and more prone to apoptosis. In contrast, mitochondrial alterations were not observed during in vivo activation of CD4 T cells in a model of OVA-immunization. The Mn-superoxide dismutase (SOD2) expression, which is involved in mROS detoxification, was increased during the AP and CP of infection. Remarkably, the apoptosis observed in CD4 T cells with MMP depolarization was prevented by incubation with N-acetyl cysteine (NAC). Thus, our results showed that infection triggered an exacerbated metabolism together with mROS production in CD4 T cells from the AP of infection. However, antioxidant availability may not be sufficient to avoid mitochondrial alterations rendering these cells more susceptible to apoptosis. Our investigation is the first to demonstrate an association between a disturbed metabolism and an impaired CD4 T cell response during T. cruzi infection.

Involvement of the beta-adrenergic system in the cardiac chronic form of experimental Trypanosoma cruzi infection.

Changes in the cardiac beta-adrenergic system in early stages of Trypanosoma cruzi infection have been described. Here, we studied an early (135 days post-infection-p.i.) and a late stage (365 days p.i.) of the cardiac chronic form of the experimental infection (Tulahuen or SGO-Z12 strains), determining plasma epinephrine and norepinephrine levels, beta-receptor density, affinity and function, cardiac cAMP concentration and phosphodiesterase activity, cardiac contractility, and the presence of beta-receptor autoantibodies. Tulahuen-infected mice presented lower epinephrine and norepinephrine levels; lower beta-receptor affinity and density; a diminished norepinephrine response and higher cAMP levels in the early stage, and a basal contractility similar to non-infected controls in the early and augmented in the late stage. The Tulahuen strain induced autoantibodies with weak beta-receptor interaction. SGO-Z12-infected mice presented lower norepinephrine levels and epinephrine levels that diminished with the evolution of the infection; lower beta-receptor affinity and an increased density; unchanged epinephrine and norepinephrine response in the early and a diminished response in the late stage; higher cAMP levels and unchanged basal contractility. The SGO-Z12 isolate induced beta-receptor autoantibodies with strong interaction with the beta-receptors. None of the antibodies, however, acted a as beta-receptor agonist. The present results demonstrate that this system is seriously compromised in the cardiac chronic stage of T. cruzi infection.

Immunization with the C-terminal region of Trypanosoma cruzi ribosomal P1 and P2 proteins induces long-term duration cross-reactive antibodies with heart functional and structural alterations in young and aged mice.

The R13 peptide sequence (EEEDDDMGFGLFD) that corresponds to the C-terminal region of Trypanosoma cruzi ribosomal P1 and P2 proteins differs from the eukariotic P concensus sequence EESDDDMGFGLFD (H13) only in a nonconservative amino acid substitution. The immunization of BALB/c mice with R13 synthetic peptide coupled to a carrier protein (OVA) induces specific (anti-R13) and autoreactive (anti-H13 and anti-heart) antibodies as well as heart functional alterations. Since aged human and experimental animals are impaired in their responses to most foreign antigens but they produce greater amounts of autoantibodies, in this work we used aged mice as an experimental model able to exaggerate the autoimmune component of the R13-induced response in case it was present. We studied whether these antibodies generated in the absence of the parasite would induce pathological changes in heart tissues. The levels of antibodies against R13 (foreign antigen) and H13 (autoantigen) studied comparatively in 2- and 12-month-old mice 10 days after the third immunization with R13 coupled to OVA were, as we expected for a foreign antigen, higher in almost all sera from 2-month-old mice tested than in sera from 12-month-old mice. Besides, these specific and cross-reactive antibody response remain elevated as long as 150 days post third immunization. In addition, the isotype pattern that recognizes R13 and the self-sequence H13 showed no differences between sera from young and aged mice. Moreover, when ECG traces were obtained from immunized mice, the heart functional alterations observed at 10 days continued at 80 and 150 days after the third immunization, showing an association with the levels of antibodies. In addition, despite the fact that the heart tissue morphology showed no alterations 10 days post third immunization, several abnormalities in the tissue architecture were revealed at 80 and 150 days post third immunization. This report demonstrates the biological relevance of R13-induced cross-reactive antibodies in some of the electrophysiologic and histological changes found in T. cruzi-infected mammalians.

Characterization of autoantibodies generated in mice by immunization with the C-terminal region of Trypanosoma cruzi ribosomal P1 and P2 proteins.

The R13 peptide sequence (EEEDDDMGFGLFD) that corresponds to the C-terminal region of Trypanosoma cruzi ribosomal P1 and P2 proteins differs from the eukariotic P consensus sequence EESDDDMGFGLFD (H13) only in a nonconservative amino acid substitution. Since the immunization with R13 peptide coupled to a carrier protein like OVA would break the tolerance to a self-sequence and generate autoantibodies, we characterized the antibodies induced in mice by R13 immunization, analyzing by ELISA their capacity to bind to R13 and the self-sequence H13. Besides, we studied the course of these reactivities a long time after immunization. It was found that all R13-immunized mice had antibodies against H13 and this reactivity was always lower than R13 reactivity. The anti-H13 reactivity evaluated by competitive ELISA demonstrated that the H13 peptide is able to inhibit the binding of immune sera to R13 at high doses. When the levels and the avidity of anti-R13 and anti-H13 were evaluated at 10 and 80 days post third immunization, it was observed that anti-R13 levels were higher than anti-H13 levels in all sera from 10 days after the third immunization. However, avidity of both antibodies was high. In sera from 80 days post third immunization, anti-R13 and anti-H13 levels and avidity either remained elevated or showed a rise, whereas anti-OVA levels declined. Moreover, when ECG traces were obtained from immunized mice, the heart functional alterations observed at 10 days continued at 80 days after the third immunization, showing an association with the levels of the antibodies. In addition, the isotype pattern that recognizes R13 and the self-sequence H13 is different. For anti-R13 response, IgG1 reactivity was higher than IgG2; meanwhile, for anti-H13 response IgG2 reactivity was higher than IgG1. These results indicate that sera from R13-immunized mice bind the H13 sequence and this autoreactivity may be self-perpetuating.

Trypanosoma cruzi: immune response and functional heart damage induced in mice by the main linear B-cell epitope of parasite ribosomal P proteins.

We report herein on the specific and autoimmune humoral response generated by the immunization of mice with the R13 synthetic peptide coupled to a carrier protein, OVA. This peptide corresponds to the C-terminal region of Trypanosoma cruzi ribosomal P1 and P2 proteins (EEEDDDMGFGLFD), a sequence that differs from the other eukariotic P consensus sequence (EESDDDMGFGLFD) only in a nonconservative amino acid substitution. The antibody response studied by ELISA revealed that all R13-immunized mice had antibodies against R13, consisting mainly of IgG1 and IgG2 isotypes, even though IgG3 and IgE isotypes were also observed. The self-reactivity of anti-R13 sera assayed by immunoblot, revealed that all sera contained IgG antibodies binding to mouse and human 38-kDa heart antigen. This antigenic band binds several immunoglobulin isotypes (IgG2 > IgG3 > IgE > IgG1). The specificity of anti-R13 antibodies analyzed by competitive inhibition of R13 ELISA using R13 and R7 (MGFGLFD) peptides revealed that the reactivity of the induced anti-P antibodies was not absorbed by R7. Therefore, the main immunogenic region of R13 for mouse would be EEEDDD, which contains the amino acid substitution. In parallel with this humoral response, both partial protection and heart damage were observed in R13-immunized mice. In fact, the R13-immunized mice showed significantly lower parasitemia and longer survival than the control animals. In addition, all R13-immunized mice showed electrocardiographic changes (bradycardia, prolonged PQ segment, and intraventricular conduction disturbances), which are typical findings in Chagas disease patients. This study represents the first definitive report in which one defined B-cell epitope, the single peptide R13 from T. cruzi, coupled to a carrier protein was able to induce specific and autoreactive antibodies as well as to generate heart functional alterations.

Exoantigens from trypanosoma cruzi contain cruzipain.

This paper shows that human antibodies specific for exoantigens of pI 4.5 (Eas 4.5), released by the blood forms of the parasite, obtained from chagasic patients sera by immunoabsorption react with cruzipain, the major cysteinyl proteinase of Trypanosoma cruzi. Sera from mice immunized with Eas 4.5 also recognize cruzipain. In addition, mouse antisera to cruzipain were reactive with Eas 4.5 as well as with total antigens excreted by culture-trypomastigotes. This reactivity was inhibited by cruzipain as revealed by enzyme-linked immunosorbent assay (ELISA). Furthermore, it was observed by immunoblot that the exoantigens recognized by mouse antisera to cruzipain have molecular weights between 50 and 60 kDa and human antibodies specific for Eas 4.5 recognize cruzipain with apparent molecular weight of 50 kDa. These findings suggest the presence of cruzipain in Eas and the subsequent release of this enzyme by the parasite.

Trypanosoma cruzi: immunological cross-reactivity of the major cysteinyl proteinase (cruzipain) with a parasite cytosol acidic antigen (FIV).

This paper shows that a polyclonal monospecific rabbit antiserum to cruzipain, the major T. cruzi cystein proteinase, cross-reacts with a cytosol acidic antigen (F IV) isolated from the epimastigote stage of the same parasite. In addition, antibodies specific for F IV purified from chagasic patient sera or murine anti F IV sera, also react with cruzipain. This was demonstrated by ELISA, DOT-ELISA, native and electrophoretic Immunoblot. These findings suggest that F IV contains an antigen immunologically cross-reactive with cruzipain.

Chagas' disease: IgG isotypes against Trypanosoma cruzi cytosol acidic antigens in patients with different degrees of heart damage.

We studied the reactivity of IgG isotypes detected in sera from chronic Chagas' disease patients with a Trypanosoma cruzi cytosol acidic antigenic fraction (F IV) and parasite epimastigote forms (EPI). All patients studied had positive serology for Chagas' disease, with normal electrocardiogram (Group I), abnormal ECG without cardiomegaly (Group II), and abnormal ECG with cardiomegaly (Group III). The highest levels of antibodies were observed in sera from Group II patients. A high prevalence of IgG1 and IgG3, low levels of IgG2, and IgG4 isotypes against EPI were found in sera from all groups by ELISA. When the F IV was used as antigen, IgG1 was the main antibody isotype detected by ELISA in all groups of patients. The antigenic recognition patterns by IgG1 among the different clinical groups by immunoblotting of F IV revealed some differences. The sera from Group I recognized antigens of F IV of 80, 53, and 43 kDa. Sera from Group III recognized mainly one antigenic band of 43 kDa. Finally, sera from Group II showed greater diversity of binding by IgG1, detecting between one and six bands in the 80 and 30 kDa ranges.