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.

Proteolytic cleavage of chemokines by Trypanosoma cruzi's cruzipain inhibits chemokine functions by promoting the generation of antagonists.

Chagas disease is a chronic inflammatory disease caused by infection with Trypanosoma cruzi. Although it had a decline in recent years, it still affects millions of people in Latin America. The host immune response against this parasite is complex and relies on the development of an efficient T cell-mediated response; however, T. cruzi displays a number of evasion mechanisms allowing it to remain undetected even for years. One of these is the secretion of anti-inflammatory molecules such as proteases and the modulation of biological functions of chemokines. Our objective was to analyze the effect of a major cysteine protease, cruzipain, on a number of critical functions of several CC chemokines, both in vitro and in vivo. Initially, using a murine model of T. cruzi infection, we demonstrated that CCL-2 and CCL-12 chemokines are highly expressed at different stages and correlated with an increase in the expression of cruzipain. In addition, we demonstrated that cruzipain is capable of differentially cleaving CCL-2 and CCL-12 chemokines, as well as CCL-13. Analysis of the proteolysed products identified unique cleavage sites in these chemokines. These cruzipain-modified chemokine products were tested in chemotaxis assays using monocytic cells. We found that cruzipain treated-CCL-2 maintained its biological activity, in contrast to the closely related CCL-12 and CCL-13 chemokines, which showed little or null agonist activity after treatment. Furthermore, based on this analysis, a 14-mer cruzipain-derived chemokine peptide (CDCP-1) was chemically synthesized and tested for agonist activity using in vitro chemotaxis assays. Interestingly, CDCP-1 showed antagonist activity affecting in vitro migration of monocytic cells and calcium flux release. In conclusion, our results demonstrate that cruzipain modulates biological functions of chemokines through proteolytic cleavage, by generating chemokine-derived peptides with antagonist activities. This event could play a role during the latest phases of Chagas disease, when the parasite may differentially modulate chemokine-mediated inflammatory responses.

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: the major cysteinyl proteinase (cruzipain) is a relevant immunogen of parasite acidic antigens (FIII).

This study examined the immune responses induced by cruzipain, a well-characterized T. cruzi antigen, to determine whether it is a relevant immunogen among the parasite acidic antigens (FIII), for which some biological properties have been studied previously. Humoral and cellular immune responses were investigated in BALB/C mice after immunization with cruzipain or FIII. Skin tests revealed immediate type-hypersensitivity (ITH) and delayed-type hypersensitivity (DTH) reactions to cruzipain in both groups of immunized mice. IgG1 and IgE isotypes against cruzipain were detected by ELISA in both groups and immunoblot studies showed that these antibodies recognized a major protein band of 50 kDa, cruzipain. The antigen-specific proliferative responses of spleen lymphocytes from both groups of immunized mice were also increased. Immunization with cruzipain of FIII antigen significantly enhanced the percentage survival of mice challenged with 10(3) trypomastigotes. The results revealed high cross-reactivity between cruzipain and FIII, suggesting the cruzipain is a relevant immunogen among the parasite acidic antigens.

Trypanosoma cruzi: transfer of protection by lymph node cells obtained from mice immunized with exoantigens of pI 4.5.

Exoantigens of pI 4.5 (Ea 4.5) of T. cruzi released to the circulation of infected mice are able to induce partial protective immune response in mice (F. Cerbán et al. 1991, International Archives of Allergy and Applied Immunology 96, 35-40). In order to analyze the participation of cellular immunity in the parasitemia control, we i.d. immunized mice with Ea 4.5 plus Bordetella pertussis as adjuvant. The role of immune cells in protective immunity was examined by adoptive transfer experiments. The immune lymph node cells (LNC) transferred the capacity to control the parasitemia, since it was observed that the normal recipients of immune LNC, which were afterward infected, presented a significant decrease in parasite levels with respect to the animals receiving LNC from control mice. This capacity was absent in the spleen cells. In addition, polystyrene nonadherent cells from immune LNC transferred the capacity to control T. cruzi infection. It was observed that Ig+ cells and enriched T cells from immunized mice are able to control the parasitemia. To define epitopes of Ea 4.5 able to stimulate protective immunity, the levels of parasitemia were examined in mice immunized with Ea 4.5 untreated or treated with sodium metaperiodate. These animals presented similar levels of parasitemia and in both cases they are significantly lower than the parasitemias of the control animals, suggesting that the most relevant epitopes for the protective immune response that control the beginning of the infection are not carbohydrates. Later, on Day 30 postinfection only the animals immunized with untreated Ea 4.5 maintained a significant decrease in parasite levels.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Enhancement of natural antibodies in mice immunized with exoantigens of pI 4.5 from Trypanosoma cruzi.

This paper deals with the enhancement of natural antibodies in mice immunized with a previously purified exoantigen of Trypanosoma cruzi from infected mouse plasma by isoelectric focusing, called Ea 4.5. A simultaneous rinse of IgG antibodies recognizing acidic sciatic nerve antigen (SNA) and other conserved antigens such as myoglobin, actin, thyroglobulin, and tubulin was observed. The highest level of antibodies was revealed when myoglobin was used as antigen in the ELISA test. Good correlation was found between the level of antibodies reactive with SNA and with highly conserved antigens. Furthermore, absorption experiments showed that a fraction of antibodies binding SNA are polyreactive and also react with the highly conserved antigens. The histological studies of sciatic nerve, heart and skeletal muscle performed 1 month after the last immunization revealed no modifications with respect to the control animals. Based on these and a previous result [1], indicating that injection of Ea 4.5 induced in mice a partial protection against T. cruzi, the possibility exists that a percentage of antibodies induced by Ea 4.5 may correspond to the natural autoantibody type and take part in protective and/or pathogenic effects.

Chagas' disease cardioneuropathy: association of anti-Trypanosoma cruzi and anti-sciatic nerve antibodies.

The aim of this work was to study whether Trypanosoma cruzi infection could elicit humoral immune response to the well-defined parasite antigen acidic fraction separated from T. cruzi cytosol by isoelectric focusing and designated fraction IV (FIV) and whether this response could account for some of the autoreactive immune response against peripheral nerve components. Chagasic patients with positive serology for Chagas' disease were classified as group I (n = 12) with normal electrocardiograms (ECG) and no signs of disease, group II (n = 12) with ECG abnormalities but without cardiomegaly, and group III (n = 12) with cardiomegaly and congestive heart failure. Sera from patients in group II showed the highest frequency of positive reactivity against FIV. Ninety-two percent had titers higher than 1/400 while the percentage for groups I and III was 50%. The autoreactive response against human sciatic nerve saline extract (SNS) was studied. The binding of IgG to SNS was positive in groups I (58%), II (66%), and III (75%) patients. The treatment of SNS with periodate diminished the ability of antigens to fix IgG from these chagasic patients. Absorption studies were performed to investigate whether FIV and SNS could have cross-reactive epitopes. Preabsorption of positive sera with FIV inhibited 48-69% of samples' reactivity against antigen. In contrast, preabsorption of positive sera with SNS inhibited only 12-23% of samples' reactivity against antigen. Overall, these results suggest that FIV-T. cruzi and sciatic nerve components possess some epitopes, possibly of a carbohydrate nature, in common. Thus, infection in Chagas' disease could overcome the tolerance to self components and could lead to autoimmunity.

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.

Immune response in mice immunized with acidic antigenic fractions from Trypanosoma cruzi cytosol.

The humoral and cellular immune responses as well as the resistance to infection with bloodstream forms of T. cruzi were studied in mice immunized with acidic antigenic fractions from parasite cytosol, F III and F IV, plus Bordetella pertussis as adjuvant. The immunization with F III induced positive ITH and DTH responses to homologous antigens. In mice immunized with F IV, the ITH was negative and four out of six animals presented positive DTH reactions. In both groups of mice the analysis of IgG against T. cruzi showed that the major isotype elicited was IgG1. Specific IgE was also detected in sera from F III immunized mice, thus confirming the presence of homocytotropic antibodies. The parasitemias reached by F III and F IV immunized mice after challenge were lower than those of the controls showing in this way a partial protection against the acute infection. The histological studies of heart and skeletal muscle performed two months after the infection revealed variable mononuclear infiltration in all infected mice despite immunization.

Trypanosoma cruzi: involvement of IgG isotypes in the parasitemia control of mice immunized with parasite exoantigens of isoelectric point 4.5.

In a previous work we demonstrated that Trypanosoma cruzi exoantigens of pI 4.5 (Ea 4.5), whose most important epitopes are glucidic, are able to induce a partially protective immune response in mice. To ascertain the involvement of antibody isotypes in this protection, we immunized mice with Ea 4.5 plus Bordetella pertussis as adjuvant. The analysis of immune response by skin test revealed the occurrence of specific immediate type hypersensitivity on Day 15 after the last immunization. By ELISA and using Ea 4.5 as antigen, specific IgG1 antibody was detected. When formaldehyde-fixed epimastigotes were used as antigen, binding of IgG1 and IgG2 was observed. Trypomastigotes incubated for 1 hr at 33 degrees C with the immune sera and then injected in normal syngeneic mice produced a significantly lower parasitemia than trypomastigotes incubated with the control sera. This capacity of anti-Ea 4.5 sera was resistant to 56 degrees C for 2 hr and was diminished after the absorption of immune sera with the carbohydrate moiety of Ea 4.5. The assay with the immune IgG1 and IgG2, separated through protein A-Sepharose affinity chromatography, showed that IgG1 retains most of this capacity. Purified immune IgG1 revealed two antigenic bands of molecular weight between 50 and 55 kDa in SDS-PAGE of Ea 4.5.

Trypanosoma cruzi exoantigens: can those recognized by sera from chagasic patients trigger a protective immune response in mice?

Exoantigens (Ea) of Trypanosoma cruzi released in blood during the acute phase of experimental murine infection and recognized as antigens by sera from chagasic patients were grouped into two zones: one zone of pI 4-5 (Ea4-5), which had components of 35 kDa, 50 kDa and slightly higher than 100 kDa, MW, and another zone, of pI 6-7 (Ea 6-7), with Ea of 50 kDa, 66-80 kDa and higher than 100 kDa. Immunization of mice with Ea4-5 or Ea6-7 prior to infection induced a protective immune response, as judged by levels of parasitaemia which were significantly lower than those of controls. Analysis of the immune response by skin test revealed that both groups of Ea induced immediate type hypersensitivity, the values of which were higher in animals immunized with Ea4-5. These antigens also induced specific cellular immunity (delayed-type hypersensitivity). There was a direct correlation between intensity of reactivity and the drop in the number of blood forms of parasites in these animals. Antibodies able to fix the epimastigote surface were also detected by ELISA and the immunofluorescence test in mice immunized with Ea4-5 or Ea6-7. There were no qualitative or quantitative differences in the antibody induced by the two groups of Ea; the main isotypes of these antibodies which recognized Ea expressed on the parasite surface were IgG1 and IgG2.

IgG isotype profiles induced in mice by two Trypanosoma cruzi electronegative antigens.

In this work we studied the IgG isotypes induced in mice immunized with two Trypanosoma cruzi acidic antigenic fractions (F IV and Eas 4.5) and the level of protection to a later infection with parasites. F IV is a cytosolic antigen from epimastigotes, and Eas 4.5 is an exoantigen released by trypomastigotes. The most relevant epitopes of Eas 4.5 are carbohydrates. A high prevalence of IgG1, low levels of IgG3 and no IgG2 antibodies against F IV and Eas 4.5 were found in sera obtained 2 weeks after the last antigen dose from animals immunized with F IV (group I) or Eas 4.5 (group II). Immunized mice from both groups were infected with trypomastigotes, and the parasitemias detected later on were significantly lower than in control groups (p less than 0.01, group I; p less than 0.001, group II). The amount of IgG2-specific antibodies, which was only detected using epimastigotes as antigen in ELISA, was significantly increased after the infection, but no major changes were seen in the profiles of other isotypes.