CTLA-4 regulates the murine immune response to Trypanosoma cruzi infection

Infection with Trypanosoma cruzi causes a profound suppression of T cell responsiveness to polyclonal or antigenic stimuli. In this study, we quantified expression of the negative T cell regulatory molecule CTLA-4 in T. cruzi infected mice and analysed its influence on the immune suppression. Levels of splenic CTLA-4 expression were highest around day 10 after infection, reaching 5% in resistant B6D2F1 mice, but exceeding 10% of CD4(+) T cells in C57BL/6 mice that were susceptible to mortal disease. The proliferative response of explanted splenocytes to CD3-mediated stimulation was strongly suppressed in both the susceptible and the resistant strains. Blockade of CTLA-4 in vitro with a monoclonal antibody affected neither proliferative response nor cytokine production (IFN-gamma, IL-4 and IL-2) by splenic T cells from infected C57BL/6 mice. Treatment of mice with anti-CTLA-4 antibody on the day of infection decreased IFN-gamma production and reduced mortality by about 50%. We conclude that high CTLA-4 expression is a hallmark of severe disease in murine T. cruzi infection, and that CTLA-4 has a regulative influence at the early stages during priming of the immune reaction to the parasite, augmenting a strong Th1-biased response.     

Specific suppression of delayed hypersensitivity expression to Trypanosoma cruzi in mice

The absence of cutaneous delayed type hypersensitivity (DTH) expression was investigated in Trypanosoma cruzi infected mice. Neither spleen cells nor peritoneal exudate cells from infected mice transferred DTH to normal recipients in local or systemic adoptive transfer experiments. Expression of DTH in T. cruzi immunized mice was suppressed specifically by Thy-1 negative spleen cells from acutely and subacutely infected animals. Suppression was observed only upon systemic transfer, but not when infected mice spleen cells were added to DTH effector cells and transferred to normal recipients. These results suggest that cutaneous DTH expression in acutely infected mice, might be blocked by mechanisms other than those described for suppression of lymphocyte proliferation and of DTH induction to T. cruzi.     

Trypanosoma cruzi: deficient lymphocyte reactivity during experimental acute Chagas'' disease in the absence of suppressor T cells

Infection of mice with Trypanosoma cruzi has been shown to lead to an impaired ability of lymphocytes to proliferate in response to mitogenic stimulation which is manifested during the acute period of the disease. A possible involvement of suppressor T lymphocytes has been postulated by other authors and was investigated in this work as a part of our efforts to disclose the mechanisms underlying the immunologic deficiency. Spleen cells from acutely infected CBA/J mice readily exhibited unresponsiveness to stimulation with concanavalin A, phytohaemagglutinin or a bacterial lipopolysaccharide. However, these cells were unable to reduce the responses that normal syngeneic-mouse spleen cells mounted to these mitogens when cultured together in equal proportions. Furthermore, removal of the Lyt 2.1-bearing cells, known to include the suppressor T cell subpopulation, from infected mouse splenocyte suspensions, did not alter the deficient responsive status of the remaining cells. These results, together with the severe depletion of the T-cell compartment which occurs in the spleens of animals acutely infected with T. cruzi, do not support an important role of suppressor T lymphocytes in the noted deficiency in lymphoid cell reactivity to mitogens. Reduced numbers of responder cells, intrinsic lymphocyte alterations or suppression by cells other than T lymphocytes remain plausible explanations to be explored.     

Immunoregulatory actions of melatonin and zinc during chronic Trypanosoma cruzi infection

After one century of the discovery of Chagas' disease and the development of an efficient drug with amplitude of actions both in the acute and chronic phase is still a challenge. Alternative immune modulators have been exhaustively used. For that purpose, melatonin and zinc were administered during chronic Trypanosoma cruzi‐infected Wistar rats and several endpoints were assessed. Melatonin has a remarkable functional versatility, being associated with important antioxidant, anti‐inflammatory, and anti‐apoptotic effects. The cross‐talk between zinc and the immune system includes its ability to influence the production and signaling of numerous inflammatory cytokines in a variety of cell types. Our study showed that zinc triggered a decrease in the generation of IFN‐γ for TCD4⁺ cells. Reduced percentage of CD4⁺T cells producing TNF‐α was observed in control melatonin or zinc‐and‐melatonin‐treated animals as compared with untreated rats. On the other hand, a significant increase in the percentage of IL‐4 from CD4⁺ and CD8⁺ T lymphocytes producers was observed 60 days after infection, for all zinc‐treated animals, whether infected or not. Melatonin and zinc therapies increased the percentages of CD4⁺ and CD8⁺ T lymphocytes IL‐10 producers. CD4⁺CD25^highFoxp3⁺ T cells were also elevated in zinc‐ and melatonin‐treated animals. The modulation of the immune system influenced by these molecules affected cytokine production and the inflammatory process during chronic T. cruzi infection. Elucidation of the interplay between cytokine balance and the pathogenesis of Chagas’ disease is extremely relevant not only for the comprehension of the immune mechanisms and clinical forms but, most importantly, also for the implementation of efficient and adequate therapies.     

Role of placental alkaline phosphatase in the internalization of trypomatigotes of Trypanosoma cruzi into HEp2 cells

BACKGROUND: In vitro, Trypanosoma cruzi invades a wide variety of mammalian cells by an unique process that is still poorly understood. Trypomastigotes adhere to specific receptors on the outer membrane of host cells before intracellular invasion, causing calcium ion mobilization and rearrangement of host cell microfilaments. OBJECTIVE: To test if placental alkaline phosphatase (PLAP), a trophoblast plasma membrane protein anchored by a glycosylphosphatidylinositol molecule, is involved in the transplacental transmission of this parasite. METHOD: We cultured HEp2 cells with the parasite and studied PLAP and actin microfilaments. The results were correlated with invasion rate. RESULTS: Human HEp2 tumour cells express PLAP. HEp2 cells infected with trypomastigotes showed alteration in their alkaline phosphatase activity and a different pattern of actin organization, compared to control cells. Perturbation of PLAP from HEp2 cells before infection with T. cruzi trypomastigotes decreased the invasion rate. CONCLUSION: Placental alkaline phosphatase could be involved in the internalization of T. cruzi into HEp2 cells, via activation of tyrosine kinase and rearrangement of actin microfilaments.     

Evaluation of IFN-gamma production by CD8 T lymphocytes in response to the K1 peptide from KMP-11 protein in patients infected with Trypanosoma cruzi

The cellular response mediated by MHC class I restricted CD8+ T cells has been shown to be crucial in the control of Chagas disease. The K1 peptide derived from T. cruzi KMP-11 protein has a high binding affinity to the HLA-A*0201 molecule. Nevertheless, it is not known whether this peptide is processed and displayed as an MHC class I epitope during natural infection by T. cruzi. The aim of this study was to evaluate, by ELISPOT assay, the ability of K1 peptide to activate CD8+ T lymphocytes to produce IFN-gamma. Therefore, CD8+ T lymphocytes from 22 HLA-A*0201+ individuals, 12 chronic chagasic patients and 10 uninfected controls, were analysed. The results revealed that two of the chagasic patients had IFN-gamma-secreting CD8+ T cells that were able to respond to K1 peptide with a relative frequency of 110 and 230 per million CD8+ T cells. In contrast, none of HLA-A*0201+ uninfected controls responded to K1 peptide. Responses to HLA-A*0201 restricted peptide from the influenza matrix protein were found in six chagasic patients and four uninfected controls with an average frequency of 175 and 111 cells per million CD8+ T cells, respectively. Moreover, a flow cytometric assay for degranulation showed that chagasic responders had K1-specific cytotoxic CD8+ T cells. It is shown here for the first time that the K1 peptide is efficiently processed, presented and recognized by CD8+ T lymphocytes during the natural course of Chagas disease.     

Modulation of T-cell responsiveness during Trypanosoma cruzi infection: analysis in different lymphoid compartments

Spleen and lymph node cells of Trypanosoma cruzi-infected mice were studied for mitogen-induced responsiveness in terms of proliferation and lymphokine production (IL-2, IFN-gamma). Splenocyte (SP) as well as lymph node cell (LN) proliferation and IL-2 production were depressed during the acute phase of the infection. Proliferative capacity of LN cells recovered completely and that of SP partially during the chronic phase. In contrast to these suppressive effects, the mitogen-induced IFN-gamma response was enhanced. In vitro co-incubation of normal SP or LN cells with trypomastigotes resulted in a reduced mitogen-induced cell proliferation and IL-2 secretion, similar to those seen with cells taken from infected mice. In contrast, trypomastigotes exerted a stimulatory activity on the mitogen-induced IFN-gamma response of both SP and LN cells. Addition of lymph node cells from T. cruzi-infected mice (LN-I) to lymph node cells of control mice (LN-C) suppressed strongly the mitogen-induced responsiveness of such cocultures. A marginal level of suppression was recorded in cocultures of spleen cells from infected mice (SP-I) and control spleen cells (SP-C). The potent suppressive cells within LN-I populations were identified as macrophage-like and such cells were absent in SP-C and peritoneal exudate cells from T. cruzi infected animals.     

Immunopathological aspects of experimental Trypanosoma cruzi infection: correlation of immune complexes and other serological features with muscle lesions during the infection

The pathogenesis of the tissue lesions observed in American trypanosomiasis (Chagas' disease) seems to depend on a variety of mechanisms. The present study was dedicated to the investigation of the evolution of muscle lesions and its relationship with immunological and parasitological parameters. Mice were infected i.p. with 10(5) blood forms of Trypanosoma cruzi (Columbian strain). A progressive increase in parasitaemia was observed which correlated with an increase in serum levels of IgG and IgM Trypanosoma-specific antibodies. Immune complex like material was detected in the serum (125I-C1q binding assay) on day 8 and reached maximum level between day 21 and 28. A concomitant fall in C3 levels was also observed. Inflammatory lesions and parasites were first observed in the striated muscle 2 weeks after infection. The cellular infiltrates involved macrophages and lymphoid cells; later on (week 3 onwards), intense necrosis appears and at the same time immunoglobulin and complement deposition were observed. These observations may indicate that, in addition to the cellular immune response, humoral mechanisms could contribute to the worsening of tissue lesions.     

Initial induction of immunity, followed by suppression of responses to parasite antigens during Trypanosoma cruzi infection of mice

Infection of a relatively resistant strain of mice (C57BL/6J) with the protozoan parasite Trypanosoma cruzi results in both the induction of parasite-specific T-helper cells and nonspecific suppressor cells. A time course study of the activation of help and suppression revealed that parasite-specific T-helper cell activity increases very early in infection (less than 12 days) at a time when suppression of non-parasite-specific responses and suppressor cell activity is increasing. Between 12 and 14 days of infection, the T-helper cell response to T. cruzi, as measured by the antibody response to hapten-T. cruzi in vitro, is suddenly and dramatically regulated. As reported previously, plastic and G-10 adherent cells appear to be responsible for the regulation of antibody responses to heterologous antigen during T. cruzi infection. These adherent suppressor cells are also responsible for the suppression of antibody responses to hapten-T. cruzi following the first 2 weeks of infection. Suppressor cells continue to regulate the parasite-specific response well into chronic infection even though the response to hapten-T. cruzi appears to return to normal levels. These results are the first to directly implicate nonspecific suppressor cells in the regulation of anti-T. cruzi humoral immune responses.     

Alterations induced by Trypanosoma cruzi in activated mouse lymphocytes

Although a number of immunological anomalies have been shown to occur during the acute period of Trypanosoma cruzi infection, the contribution of the parasite has not been clarified. In this work, we co-cultured activated splenic mononuclear cells (SMC) from normal oulbred (CD1) or inbred (CBA/J) mice with purified T. cruzi trypomastigotes and studied ensuing T- and B- lymphocyte alterations. In the presence of parasites, phytohaemagglutinin-stimulated SMC from either mouse background manifested a marked reduction in both lymphoproliferative capacity (i.e., ³H-thymidine incorporation) and cell membrane levels of interleukin-2 receptors (H.-2R; determined by flow cytomet.y) relative to SMC from parasite-free cultures. Thus, substantial proportions of activated SMC either became unable to express detectable levels of IL-2R or expressed this receptor in significantly lower numbers than control SMC. Supernatants from T. cruzi suspensions reproduced these suppressive effects on phytohaemagglutinin-stimulated SMC from normal or chronically infected CD1 or CBA/J mice. Similar results were obtained with SMC activated with a bacterial lipopolysaccharide. Since IL-2R expression is required for activated lymphocytes to progress through the cell cycle and multiply to mount effective immune responses, impaired IL-2R expression by T. cruzi provides a plausible hypothesis for the wide-ranged immunosuppression that occurs in the infected host.     

Susceptibility to acute Trypanosoma cruzi infection in autoimmune strains of mice

We previously observed that mice bearing the autoimmune associated lpr gene exhibit increased susceptibility to challenge with Trypanosoma cruzi, the causative agent of Chagas' disease. We have now tested two other autoimmune prone strains of mice, BXSB and NZB, and found that these animals also show increased sensitivity to acute T. cruzi infection. When challenged with a standard dose (10(2)) of Y strain trypomastigotes, BXSB males (BXSB-YSB), which develop early onset autoimmune disease, suffered high mortality, while late onset autoimmune BXSB females and minimally autoimmune male BXSB mice whose Y chromosome was derived from C57BL/6J mice (BXSB-YB/6) also recover. NZB mice were found to be highly susceptible to challenge while NZW and NZB/W were resistant. A finding common to all groups of susceptible autoimmune mice was increased plasma levels of T. cruzi specific antibody, especially IgM. The data indicate that in two of the three autoimmune prone strains examined, increased T. cruzi susceptibility appears to be linked to restricted genetic elements (i.e. lpr gene and the YSB associated factor) which also influence the rapidity of onset of autoimmunity.     

Influence of melatonin therapy and orchiectomy on T cell subsets in male Wistar rats infected with Trypanosoma cruzi

Abstract:  Gonadal steroids exert an important influence on the host immune response during infection. Changes resulting from the absence or replacement of gonadal hormones may represent a distinct evolution of a particular parasite. Taking into account the greater susceptibility of males to parasites, the magnitude of the immune response seems to depend on the interaction of many hormones that will act synergistically with other immune cells. The aims of this research were to evaluate the effects of the luck of male sex hormones due to orchiectomy, and the influence of oral administration of melatonin on the immune response of male Wistar rats infected with the Y strain of Trypanosoma cruzi . The percentage of CD3⁺ CD4⁺ and CD3⁺ CD8⁺ lymphocyte T cell subsets were evaluated using flow cytometry and the measurement of IL-2 and IL-12. For all parameters examined, a synergistic action of melatonin and orchiectomy on the host's immune response was observed, promoting an effective response against the parasite during the acute phase of infection. These results offer insight into other possibilities for possibly controlling T. cruzi proliferation through melatonin therapy and also the stimulatory effects on host's immune response triggered by the absence of male gonadal steroids during the acute phase of infection.     

Programmed death 1 ligand signaling regulates the generation of adaptive Foxp3+CD4+ regulatory T cells

Although mature dendritic cells (DCs) are potent initiators of adaptive immune response, immature steady-state DCs contribute to immune tolerance. In this study, we show that ex vivo splenic DCs are capable of inducing conversion of naïve CD4⁺ T cells to adaptive Foxp3⁺CD4⁺ regulatory T cells (aTreg) in the presence of TGF-β. In particular, when compared with splenic CD8α⁻ DCs, the CD8α⁺ DC subset were superior in inducing higher frequencies of conversion. This was not attributable to the difference in basal level of costimulation, because deficiency of CD40 or CD80/86 signaling did not diminish the differential induction of Foxp3. Conversion was regulated by DC maturation status. Further insights into the molecular mechanisms of conversion were gained by analyzing the contribution of several costimulatory and coinhibitory receptors. Costimulatory signals through GITR suppressed conversion, whereas coinhibitory signaling via programmed death 1 ligand (PD-L1) but not PD-L2 was required for conversion. Ex vivo PD-L1^−/− DCs failed to support Foxp3 induction in the presence of TGF-β. In vivo blocking PD-L1 signaling abolished conversion in a tumor-induced aTreg conversion model. Collectively, this study highlights the cellular and molecular parameters that might be exploited to control the de novo generation of aTregs and peripheral tolerance.     

Role of cellular immunity in protection against Trypanosoma cruzi in mice

The importance of antibodies in the maintenance of immunity against Trypanosoma cruzi in mice is emphasized by the failure of either immunization or transfer of immune T cells to afford B cell suppressed mice complete protection against a lethal infection. Both procedures did, however, significantly prolong survival indicating a contributory role for T cells other than simply as helper cells in antibody production. The complete protection afforded intact mice following transfer of immune T cells can be attributed to a significant T cell-mediated augmentation of IgG antibody production.     

Release of nitric oxide during the experimental infection with Trypanosoma cruzi

We analysed the production of nitric oxide (NO) intermediates by cells from BALB/c mice infected with either virulent (Tulahuén or RA) or avirulent (CA-1) strains of Trypanosoma cruzi. Peritoneal or spleen cells from mice infected with T. cruzi released NO when incubated without further stimuli. Cells from mice during the acute stage of infection accumulated higher levels of inducible NO synthase mRNA and produced both, before and after lypopolysaccharide stimulation, higher amounts of NO than cells from mice chronically infected with T. cruzi. NO synthesis showed similar kinetics in connection with all three strains ofT. cruzi, but cells from mice inbred with the Tulahuén or RA strains released higher levels of IFN-γ, an activator of the NO pathways, than cells from mice infected with the CA-1 strain. In vivo administration of L-Ng-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO synthase, increased the susceptibility of mice to T. cruzi. We conclude that infection with T. cruzi induces NO production, and suggest that NO plays a role in the resistance against the parasite.     

Effect of protective and non-protective antibodies in the phagocytosis rate of Trypanosoma cruzi blood forms by mouse peritoneal macrophages

The phagocytosis of Trypanosoma cruzi blood forms by mouse peritoneal macrophages is significantly enhanced by sera from chronic chagasic patients, rabbits and mice presenting 'lytic antibodies' (LA) which are associated with resistance and active infections as well as 'conventional serology antibodies' (CSA) which are immunoglobulins involved in the positivity of serological diagnostic tests. The phagocytosis rate, however, is not influenced by sera from mice immunized with T. cruzi antigen or chagasic patients submitted to specific treatment, both displaying only CSA but not LA. The efficacy of LA in increasing phagocytosis is related to their ability to bind to epitopes of living trypomastigotes, a property lacking in CSA that bind only to fixed parasites. This phenomenon is apparently the reason for the low effectiveness of antigens used for vaccination in Chagas' disease which only induce CSA, immunoglobulins apparently unable to mediate a number of regular effector immune mechanisms such as complement-mediated lysis, antibody-dependent cell cytotoxicity and phagocytosis.     

Cytokine mRNA levels in the hearts of inbred mice that develop different degrees of cardiomyopathy during infection with Trypanosoma cruzi

Profiles of cytokine mRNA expression were examined by semiquantitative RT-PCR in the hearts of DBA/2 (pathopermissive) and B10.D2 (pathoresistant) mice during infection with the Brazil strain of Trypanosoma cruzi . The levels and time-course profiles of IFNγ, IL-1β and IL-10 mRNA expression were similar in each strain. TNFα, iNOs, and IL-13 mRNA expression peaked at comparable levels and times after infection in each strain, but declined more rapidly in B10.D2 than in DBA/2 mice. Peak IL-2 mRNA levels were also similar between the two strains, but occurred earlier in DBA/2 than in B10.D2 mice. Levels of IL-4, IL-6 and IL-12 mRNA were significantly higher in DBA/2 than in B10.D2 mice from day 10 through day 50 of infection. With the exception of IL-1β, which was expressed constitutively in both strains, the levels of mRNA of all other cytokines examined reached their peak no later than day 20 and declined significantly by day 50 after infection. The inflammatory infiltrate paralleled the latter cytokines; starting at day 10 in DBA/2 mice and at day 15 in the B10.D2 s, peaking between days 20 and 30 in both strains, decreasing to minimal levels by day 50 in the pathoresistant mice, but maintaining a mild amount through day 70 in the pathopermissive strain. The inflammation was composed mostly of lymphocytes and histiocytes throughout the entire process. These data demonstrate differences in the profiles of cytokine mRNA that may be related to the differential degree of cardiac pathology that develops in these two strains of mice upon infection with T. cruzi.     

Seroprevalence of Trypanosoma cruzi in kidney transplant donors and recipients in Mexico City

Infectious diseases are common causes of morbidity and mortality among kidney transplant recipients. Chagas disease (CD) has been recognized as an emerging infectious complication of transplantation caused by the parasite Trypanosoma cruzi. CD is prevalent in Mexico, particularly in the southern coastal region. The impact on Mexican kidney transplant programs has not been previously studied prospectively. From 2009 through 2010, serum samples from 59 kidney transplant donors and 405 renal transplant recipients were screened for antibodies against T. cruzi. Serum was initially screened using a locally developed ELISA test; positive results were confirmed by an indirect immunofluorescense test, in accordance with Panamerican Health Organization/World Health Organization guidelines. None of the donors were seropositive for T. cruzi, while 8 (1.97%) kidney transplant recipients were confirmed to be seropositive for T. cruzi. None of them have developed clinical manifestations of CD, although specific screening of recipients was not performed. A prospective study is planned to define the epidemiology and outcome of CD among kidney transplant donors and recipients in Mexico more thoroughly.     

Trypanosoma cruzi infection in mice induces a polyisotypic hypergammaglobulinaemia and parasite-specific response involving high lgG2a concentrations and highly avid lgG1 antibodies

Trypanosoma cruzi infection in BALB/c mice induced a reversible polyisotypic hypergammaglobulinaemia, with particularly high levels of IgG2a, IgM and IgE. Hypergammaglobulinaemia started during the acute phase of infection and persisted during chronic disease until 11–13 weeks post-infection (w.p.i.), when immunoglobulin levels, with the exception of IgE, returned near normal values. Parasite-specific antibodies counted for 14 to 23% of gammaglobulinaemia, in acute and chronic infection respectively. The titres of IgM antibodies rose from two w.p.i. IgA, IgE and IgG subclass antibodies built up gradually over the time of parasite clearance (i.e., between three and six w.p.i.). All antibody isotypes, including IgM reached significant and stable titres throughout chronic infection. IgG2a, IgG1 and IgM antibodies had constantly higher titres than the other antibody isotypes. The dominance of IgG2a antibodies was due to their high plasma concentrations, around 70% of all antibodies available in the chronic infection. IgG1 had the highest functional avidity, whereas its concentration corresponded to only 10% of the whole antibody fraction. These results indicate that T. cruzi infection in mice induces a polyisotypic humoral immune response, dominated by some antibody isotypes, with major differences in concentrations and functional avidities. This could be of crucial importance in determining the outcome of infection.