Cell-mediated immunity in experimental Trypanosoma cruzi infection

Infection of humans with the protozoan Trypanosoma cruzi leads to Chagas disease, or American trypanosomiasis, a disease that affects nearly 20 million people, and constitutes one of the largest socioeconomic burdens in Latin America. Much of the present knowledge on pathogenic mechanisms underlying T. cruzi infection comes from experimental murine models. Here, George A. DosReis reviews recent findings about the features of host cell-mediated immunity against the parasite and possible mechanisms leading to chronic infection.     

Induction of B- and T-cell responses to cruzipain in the murine model of Trypanosoma cruzi infection

Trypanosoma cruzi, the causative agent of Chagas' disease, is an important cause of heart disease in Latin America. The parasite is transmitted mucosally, with both intra- and extracellular life stages in the human host. Cruzipain, the major cysteinyl proteinase of T. cruzi, has been shown to be antigenic in both humans and mice during infection with the parasite. We extend these observations, showing here that multiple murine immune subsets of potential importance for vaccine-induced protection can be induced by cruzipain. Cruzipain-specific serum IgG responses were induced during chronic infection with T. cruzi. In addition, T. cruzi mucosal infection stimulated the development of cruzipain-specific secretory IgA detectable in fecal extracts from infected mice. Cruzipain-specific type 1 cytokine responses characterized by the production of IFN-gamma but not IL-4 were also detectable during murine infection. Furthermore, immunization of mice with a DNA vaccine encoding cruzipain was shown to stimulate cytotoxic T lymphocyte (CTL) responses capable of recognizing and lysing T. cruzi-infected cells. The induction of serum antibody, mucosal IgA, Th1 cytokine and CTL responses by cruzipain in mice supports the use of this parasite protein for further efforts in T. cruzi vaccine development.     

Trypanosoma cruzi: immune response in mice immunized with parasite antigens

The humoral and cellular immune responses were studied in mice immunized with flagellar fraction (F), F plus Bordetella pertussis as adjuvant (F-Bp), and microsomal (Mc) subcellular fractions from the epimastigote forms of Trypanosoma cruzi. The immune response was studied before and after the challenge with 50 bloodstream forms of T. cruzi, Tulahuén strain. The immunization with F-Bp, but not with Mc or F and Bp separately, protected mice, in terms of parasitemia and mortality, from the challenge with the parasite. Before the challenge, levels of specific antibodies in mice immunized with F-Bp were higher than in mice immunized with F or Mc. Antibody levels 17 days after the infection were similar in the three groups of mice while nonimmunized mice reached lower levels. Early during the infection nonimmunized infected mice lacked delayed-type hypersensitivity (DTH) responses to parasite antigens and to concanavalin A (Con A). Mice immunized with F-Bp, however, presented positive DTH responses to parasite antigens and Con A both, before and after the challenge with T. cruzi. DTH reaction was transferred with spleen cells. Mice immunized with Mc behaved similarly to infected nonimmunized animals in their reactivity to parasite antigens. These results indicated striking differences between protected and nonprotected mice in humoral and cellular immune responses during experimental T. cruzi infection.     

Prevalence of anti-R-13 antibodies in human Trypanosoma cruzi infection

Abstract Infection with Trypanosoma cruzi develops in three phases: acute, indeterminate or asymptomatic, and chronic phase (with cardiac or digestive manifestations). Moreover, transmission may occur from infected mothers to newborn, the so-called congenital form. In the present study, humoral responses against T. cruzi total extract and against the 13 amino acid peptide named R-13 derived from the parasite ribosomal P protein, previously described as a possible marker of chronic Chagas heart disease, were determined pateints and in blood bank donors from endemic areas. While in sera from acute phase, only IgM anti- T.cruzi response was observed, both IgM and IgG anti- T. cruzi antibodies were detected in sera from congenitally infected newborns. The percentage of positive response in sera from blood bank donors was relatively high in endemic regions. Antibodies against the R-13 peptide were present in a large proportion of cardiac chagasic patients but were totally lacking in patients with digestive form of Chagas disease. Furthermore, anti-R-13 positive responses were detected in congenitally infected newborns.     

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

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

Trypanosoma cruzi: effect of parasite subpopulation on murine pregnancy outcome

C3H/HeN female mice infected with distinct Trypanosoma cruzi subpopulations (RA strain [pantropic/reticulotropic] and K98 clone of the CA-I strain [myotropic]) show differences both in inflammatory compromise of the genital tract and in the outcome of pregnancy. The group of mice infected with the K98 clone show lymphomononuclear infiltrates in pelvian fat and in uterus interstitium, coexisting with the presence of T. cruzi DNA, and show moderate oophoritis, perioophoritis, and vasculitis. However, neither parasite DNA nor inflammatory foci were detected in the uterus, and only mild oophoritis was observed among RA-infected mice at mating time. Independently from the parasite subpopulation, females developed estrous 30 days postinoculation (PI), and at the same time, parasite counts were similar for K98 and for RA-infected mice. However, fertility was significantly diminished in K98-infected females. On day 14 of gestation, fetal resorptions increased in this group and cannot be attributed to hormonal disbalance because similar serum progesterone levels were found in all groups. At this time (44 days PI), parasitemia was higher in K98- than in RA-infected mice. However, resorptions were not triggered by massive infection because polymerase chain reaction failed to prove parasite DNA in resorbing fetuses. In contrast with K98 females, RA-infected mice delivered T. cruzi-infected newborns.     

Isolation of cardiac mast cells in experimental Trypanosoma cruzi infection

Mast cells (MC) secrete diverse pre-stored chemical mediators that are pivotal in inflammatory and fibrotic etiologies, such as Trypanosoma cruzi-induced myocardiopathy. However, due to reduced number of cardiac MC, in situ and in vitro identification, and difficult tissue isolation, these cells are rarely addressed. In this work we optimized the identification of cardiac and peritoneal MC and developed an enzymatic method for MC isolation using control and T. cruzi-infected mice. MC were identified by: toluidine blue (TB); alcian blue (AB)/safranin (S); AB or a mixed solution composed by AB/S/TB. Previous evaluations of cardiac MC in T. cruzi infection were based on TB staining and our results using AB/S/TB solution showed an increase in, at least, five times the detection of MC. This mixed solution may improve the identification of MC populations also from skin, mucosa and tissues that are infected by other pathogens or under the influence of chronic inflammation, leading to more precise results. Furthermore, the appropriate combination of samples (frozen/unfixed/thick slices) and staining protocols can assure the best evaluation of MC. We have also isolated cardiac MC using collagenase and developed a highly efficient 60%/70% Percoll-graded protocol that enriched in, at least, 95% the population of cardiac MC.     

Trypanosoma cruzi: the role of PGE2 in immune response during the acute phase of experimental infection

Chagas disease is characterized by cardiac lesions and a high level of PGE2. Our objective was to investigate the role of PGE2 in cardiac lesions. BALB/c mice were infected with Trypanosoma cruzi (1x10(3) trypomastigote forms) and were treated daily with PBS, meloxicam (0.5 mg/kg) or etoricoxib (0.6 mg/kg). The animals were sacrificed on the 21st day of infection and we collected the cardiac tissue and spleen cells for tissue culture. We observed that treatment with COX-2 inhibitors was able to decrease synthesis of PGE2 by spleen cells. This reduction was accompanied by reduction of the inflammatory infiltrate, parasite nets, cardiac fibrosis and fewer COX-2 positive cells in cardiac tissue obtained from these animals. In conclusion, treatment with COX-2 inhibitors, and consequent inhibition of PGE2 synthesis, was able to reduce the cardiac damage observed during the acute phase of experimental Chagas disease, thus demonstrating the involvement of this mediator in the cardiac lesion.     

Trypanosoma cruzi: identification of specific epimastigote antigens by human immune sera

Soluble antigens from epimastigotes of Trypanosoma cruzi were analyzed by western blot in terms of their reactivity with sera from patients with Chagas' disease. In addition, sera from patients with visceral (AVL) and tegumental leishmaniasis (ATL) were also tested in order to identify cross-reactivities with Trypanosoma cruzi antigens. Twenty eight polypeptides with molecular weights ranging from 14 kDa to 113 kDa were identified with sera from Chagas' disease patients. An extensive cross-reactivity was observed when sera from human visceral leishmaniasis were used, while only a slight cross-reaction was observed with sera from tegumental leishmaniasis. On the other hand, 10 polypeptides specifically reacting with sera from Chagas' disease patients were identified. Among them, the antigens with molecular weights of 46 kDa and 25 kDa reacted with all sera tested and may be good candidates for specific immunodiagnosis of Chagas' disease.     

Trypanosoma cruzi: the effects of zinc supplementation during experimental infection

It is well recognized that zinc is an essential trace element, influencing growth and affecting the development and integrity of the immune system. The use of oligoelements as zinc can be considered a tool in modulating the effectiveness of the immune response. In this work zinc was daily and orally supplied in male Wistar rats infected with the Y strain of Trypanosoma cruzi. Parasitemia was evaluated and a significant reduction on blood parasites was observed. In order to check some immunological parameters peritoneal macrophages were counted revealing higher percentages for zinc supplied group. Consequently enhanced concentrations of IFN-gamma was found and for the first time NO was evaluated in T. cruzi infected animals under the influence of zinc therapy, revealing enhanced concentrations when compared to unsupplied counterparts. We conclude that zinc is able to up-regulate the host's immune response against parasite replication.     

Absence of Bim sensitizes mice to experimental Trypanosoma cruzi infection

Chagas disease is a life-threatening disorder caused by the protozoan parasite Trypanosoma cruzi. Parasite-specific antibodies, CD8⁺ T cells, as well as IFN-γ and nitric oxide (NO) are key elements of the adaptive and innate immunity against the extracellular and intracellular forms of the parasite. Bim is a potent pro-apoptotic member of the Bcl-2 family implicated in different aspects of the immune regulation, such as negative selection of self-reactive thymocytes and elimination of antigen-specific T cells at the end of an immune response. Interestingly, the role of Bim during infections remains largely unidentified. To explore the role of Bim in Chagas disease, we infected WT, Bim^+/−, Bim^−/− mice with trypomastigotes forms of the Y strain of T. cruzi. Strikingly, our data revealed that Bim^−/− mice exhibit a delay in the development of parasitemia followed by a deficiency in the control of parasite load in the bloodstream and a decreased survival compared to WT and Bim^+/− mice. At the peak of parasitemia, peritoneal macrophages of Bim^−/− mice exhibit decreased NO production, which correlated with a decrease in the pro-inflammatory Small Peritoneal Macrophage (SPM) subset. A similar reduction in NO secretion, as well as in the pro-inflammatory cytokines IFN-γ and IL-6, was also observed in Bim^−/− splenocytes. Moreover, an impaired anti-T. cruzi CD8⁺ T-cell response was found in Bim^−/− mice at this time point. Taken together, our results suggest that these alterations may contribute to the establishment of a delayed yet enlarged parasitic load observed at day 9 after infection of Bim^−/− mice and place Bim as an important protein in the control of T. cruzi infections.Subject terms: Cell death and immune response, Infectious diseases     

Indiscriminate representation of VH-gene families in the murine B lymphocyte responses to Trypanosoma cruzi

The utilization of the nine major homology families of VH-genes was quantitated in the B lymphocyte response to Trypanosoma cruzi infection of C57BL/6 mice. Normal and infected mice at various times after parasite inoculation were compared for VH-gene distribution of CFU-B produced by activated blasts recovered from spleen and lymph nodes, and for relative hybridization of total spleen RNA with each of the family probes. T. cruzi infection results in large increases of splenic RNA in the various homology families, and the numbers of activated CFU-B, reflecting the massive B lymphocyte responses. In acute phase, all nine families are expressed in roughly the same proportions as in normal mice, whereas in chronic infection, B cells expressing S107 and 7183 VH-genes might be preferentially stimulated. These results establish the polyclonal nature of the host response to T. cruzi infection.     

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.     

Gastrointestinal infection with Mexican TcI Trypanosoma cruzi strains: different degrees of colonization and diverse immune responses

Mexican Ninoa and Queretaro (Qro) TcI strains of Trypanosoma cruzi have shown different degrees of virulence, and the two strains produce heterogeneous immune responses in the hearts of infected mice. This work shows that the same strains can invade the intestine by an intraperitoneal route and establish an infection, mainly in the colon. The three segments of the small intestine (duodenum, jejunum and ileum) were infected to a lesser degree than the colon. Despite the fact that parasites were predominantly found in the colon, an obvious inflammatory reaction was observed in the submucosal layer along the entire intestinal tract, with the virulent Qro strain causing significantly more areas of higher immune infiltration. A clear recruitment of CD4+ and CD8+ T lymphocytes to the mesenteric ganglia was observed during infection with the virulent strain. Macrophages were also differentially distributed in the gastrointestinal tract. These later cells infiltrated fewer amastigote nests in the mice infected with the Qro strain than in the mice infected with the Ninoa strain. When IFN-γ, TNF-α, and IL-4 levels were measured, an increase in these cytokines was observed compared with the uninfected mice. The role of these inflammatory reactions in the pathogenesis of Chagas enteropathy is also discussed in this paper.     

Efficacy of novel benznidazole solutions during the experimental infection with Trypanosoma cruzi

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi. About 8 million people throughout Latin America are infected causing approximately 10,000 deaths annually. Benznidazole, available as unique 100 mg tablets in many of the endemic countries, is currently the drug of choice for the specific treatment of this condition. Despite of the large number of pediatric patients infected, there are no commercial liquid dosage forms available to treat this trypanosomiasis. This work showed that novel benznidazole–water–polyethylene glycol 400 solutions are active against T. cruzi in a murine model of Chagas' disease. Present results constitute the first demonstration on the usefulness of benznidazole solutions in infected mice.     

Isotype specific immune responses in murine experimental toxocariasis

In this work, a murine experimental model of toxocariasis has been developed in BALB/c, C57BL/10 and C3H murine strains orally inoculated with 4,000 Toxocara canis embryonated eggs, in order to investigate the isotype-specific immune responses against excretory-secretory antigens from larvae. T. canis specific IgG+M, IgM, IgG, IgA, IgG1, IgG2a and IgG3 were tested by ELISA. The dynamics of the specific immunoglobulins (IgG+IgM) production showed a contrasting profile regarding the murine strain. Conversely to the results obtained with the IgM isotype, the IgG antibody class showed similar patterns to those obtained with IgG+IgM antibodies, only in the case of the BALB/c strain, being different and much higher than the obtained with IgG+IgM antibodies, when the C3H murine strain was used. The antibodies IgG+IgM tested in BALB/c and C57BL/10 were both of the IgM and IgG isotypes. Conversely, in the C3H strain only IgG specific antibody levels were detected. The IgG1 subclass responses showed a similar profile in the three murine strains studied, with high values in BALB/c, as in the case of the IgG responses.