Immunoregulatory mechanisms in Chagas disease: modulation of apoptosis in T-cell mediated immune responses.

BACKGROUND: Chronic Chagas disease presents different clinical manifestations ranging from asymptomatic (namely indeterminate) to severe cardiac and/or digestive. Previous results have shown that the immune response plays an important role, although no all mechanisms are understood. Immunoregulatory mechanisms such as apoptosis are important for the control of Chagas disease, possibly affecting the morbidity in chronic clinical forms. Apoptosis has been suggested to be an important mechanism of cellular response during T. cruzi infection. We aimed to further understand the putative role of apoptosis in Chagas disease and its relation to the clinical forms of the disease. METHODS: Apoptosis of lymphocytes, under antigenic stimuli (soluble T. cruzi antigens - TcAg) where compared to that of non-stimulated cells. Apoptosis was evaluated using the expression of annexin and caspase 3(+) by T cells and the percentage of cells positive evaluated by flow cytometry. In addition activation and T cell markers were used for the identification of TCD4(+) and TCD8(+) subpopulations. The presence of intracellular and plasma cytokines were also evaluated. Analysis of the activation status of the peripheral blood cells showed that patients with Chagas disease presented higher levels of activation determined by the expression of activation markers, after TcAg stimulation. PCR array were used to evaluate the contribution of this mechanism in specific cell populations from patients with different clinical forms of human Chagas disease. RESULTS: Our results showed a reduced proliferative response associated a high expression of T CD4(+)CD62L(-) cells in CARD patients when compared with IND group and NI individuals. We also observed that both groups of patients presented a significant increase of CD4(+) and CD8(+) T cell subsets in undergoing apoptosis after in vitro stimulation with T. cruzi antigens. In CARD patients, both CD4(+) and CD8(+) T cells expressing TNF-α were highly susceptible to undergo apoptosis after in vitro stimulation. Interestingly, the in vitro TcAg stimulation increased considerably the expression of cell death TNF/TNFR superfamily and Caspase family receptors genes in CARD patients. CONCLUSIONS: Taken together, our results suggest that apoptosis may be an important mechanism for the control of morbidity in T. cruzi infection by modulating the expression of apoptosis genes, the cytokine environment and/or killing of effector cells.

Matrix metalloproteinases 2 and 9 are differentially expressed in patients with indeterminate and cardiac clinical forms of Chagas disease.

Dilated chronic cardiomyopathy (DCC) from Chagas disease is associated with myocardial remodeling and interstitial fibrosis, resulting in extracellular matrix (ECM) changes. In this study, we characterized for the first time the serum matrix metalloproteinase 2 (MMP-2) and MMP-9 levels, as well as their main cell sources in peripheral blood mononuclear cells from patients presenting with the indeterminate (IND) or cardiac (CARD) clinical form of Chagas disease. Our results showed that serum levels of MMP-9 are associated with the severity of Chagas disease. The analysis of MMP production by T lymphocytes showed that CD8(+) T cells are the main mononuclear leukocyte source of both MMP-2 and MMP-9 molecules. Using a new 3-dimensional model of fibrosis, we observed that sera from patients with Chagas disease induced an increase in the extracellular matrix components in cardiac spheroids. Furthermore, MMP-2 and MMP-9 showed different correlations with matrix proteins and inflammatory cytokines in patients with Chagas disease. Our results suggest that MMP-2 and MMP-9 show distinct activities in Chagas disease pathogenesis. While MMP-9 seems to be involved in the inflammation and cardiac remodeling of Chagas disease, MMP-2 does not correlate with inflammatory molecules.

Cytokine profile, proliferation and phosphorylation of ERK1/2 and Akt in circulating mononuclear cells from individuals during the chronic intestinal phase of Schistosomiasis mansoni infection.

BACKGROUND: The immune response to Schistosoma mansoni is characterized by a granulomatous reaction around the parasite eggs that are trapped in the host liver, and this reaction modulates the immune response during the chronic phase of the disease. The typical peripheral blood mononuclear cell (PBMC) response of patients during the chronic intestinal phase of infection is characterized by a decreased response to an S. mansoni soluble egg antigen. To obtain a greater understanding of Schistosoma infections, this study investigated the effects of the soluble egg antigen (SEA) and soluble adult worm antigen (SWAP) of S. mansoni on cellular proliferation, cytokine production, and ERK1/2 and Akt phosphorylation in PBMCs from infected (XTO) and egg-negative (NI) individuals living in the same endemic area. METHODS: The activation status was evaluated by cell immunophenotypic staining (cytometry). The cell proliferation assay was by CFSE method. Cytokine detection assay (Th1 and Th2) was by Cytometric Bead and Array phosphorylation status was by ELISA. RESULTS: The XTO, NI and BD (blood donor) individuals from an area not endemic for schistosomiasis were compared. The CD4(+) T lymphocyte proliferation rate was lower in the XTO group, but not the NI group, after SEA stimulation compared to the BD group. The CD8(+) T cell proliferation rate was lower in the XTO group in the unstimulated cultures and after both SEA and SWAP stimulation compared to the BD group. Cytokine analysis after either SEA or SWAP stimulation showed a balanced cytokine pattern in the XTO and NI groups. ERK1/2 and Akt phosphorylation were only marginally detected in all groups; however, a decrease in ERK 1/2 phosphorylation was observed in the SWAP-stimulated XTO group compared to both the NI and BD groups. CONCLUSIONS: The data indicate that SEA-stimulated CD4(+) T cells from infected patients have a lower proliferation rate than the same cells from the NI group. Furthermore, we observed that SWAP stimulation influences ERK1/2 phosphorylation in the XTO group.

Inflammatory mediators from monocytes down-regulate cellular proliferation and enhance cytokines production in patients with polar clinical forms of Chagas disease.

Exposure to Trypanosoma cruzi parasites induces monocytes and macrophages to produce various endogenous mediators, including prostaglandins and cytokines. To clarify the involvement of monocytes as an important source of inflammatory mediators in Chagas disease patients, we evaluated PBMC before and after depletion of adherent cells (monocytes) from patients with indeterminate (IND) and cardiac (CARD) clinical forms and from non-infected individuals (NI). We demonstrated that after the partial depletion of adherent cells, production of PGE2 was slightly decreased in patients with Chagas disease. Inhibition of the cells by indomethacin increased the proliferation in PBMC cells from patients after antigen stimulation. Pro-inflammatory cytokines as IL-2 and IFN-γ also had a greater decrease after partial depletion of adherent cells in both clinical forms of Chagas disease. IL-10 and IL-5 levels were also reduced after partial depletion of adherent cells both in IND and CARD patients. In addition, we evaluated the APC potential of B cells and observed that the MHCII and CD80 molecules had an increased expression after partial depletion of most monocytes in all groups. Thus, inflammatory mediators produced by monocytes seem to be important to modulate immune responses in Chagas disease by regulating the processes of inflammation and antigen presentation.

Plasma cytokine expression is associated with cardiac morbidity in chagas disease.

The expression of immune response appears to be associated with morbidity in Chagas disease. However, the studies in this field have usually employed small samples of patients and statistical analyses that do not consider the wide dispersion of cytokine production observed in these patients. The aim of this study was to evaluate the plasma cytokine levels in well-defined clinical polar groups of chagasic patients divided into categories that better reflect the wide cytokine profile and its relationship with morbidity. Patients infected with Trypanosoma cruzi (T. cruzi) were grouped as indeterminate (IND) and cardiac (CARD) forms ranging from 23 to 69 years of age (mean of 45.6±11.25). The IND group included 82 individuals, ranging from 24 to 66 years of age (mean of 39.6±10.3). The CARD group included 94 patients ranging from 23 to 69 years of age (mean of 48±12.52) presenting dilated cardiomyopathy. None of the patients have undergone chemotherapeutic treatment, nor had been previously treated for T. cruzi infection. Healthy non-chagasic individuals, ranging from 29 to 55 years of age (mean of 42.6±8.8) were included as a control group (NI). IND patients have a higher intensity of interleukin 10 (IL-10) expression when compared with individuals in the other groups. By contrast, inflammatory cytokine expression, such as interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interleukin 1 beta (IL-1ß), proved to be the highest in the CARD group. Correlation analysis showed that higher IL-10 expression was associated with better cardiac function, as determined by left ventricular ejection fraction and left ventricular diastolic diameter values. Altogether, these findings reinforce the concept that a fine balance between regulatory and inflammatory cytokines represents a key element in the establishment of distinct forms of chronic Chagas disease.

Profile of central and effector memory T cells in the progression of chronic human chagas disease.

BACKGROUND: Chronic Chagas disease presents several different clinical manifestations ranging from asymptomatic to severe cardiac and/or digestive clinical forms. Several studies have demonstrated that immunoregulatory mechanisms are important processes for the control of the intense immune activity observed in the chronic phase. T cells play a critical role in parasite specific and non-specific immune response elicited by the host against Trypanosoma cruzi. Specifically, memory T cells, which are basically classified as central and effector memory cells, might have a distinct migratory activity, role and function during the human Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: Based on the hypothesis that the disease severity in humans is correlated to the quality of immune responses against T. cruzi, we evaluated the memory profile of peripheral CD4(+) and CD8(+) T lymphocytes as well as its cytokine secretion before and after in vitro antigenic stimulation. We evaluated cellular response from non-infected individuals (NI), patients with indeterminate (IND) or cardiac (CARD) clinical forms of Chagas disease. The expression of CD45RA, CD45RO and CCR7 surface molecules was determined on CD4(+) and CD8(+) T lymphocytes; the pattern of intracellular cytokines (IFN-gamma, IL-10) synthesized by naive and memory cells was determined by flow cytometry. Our results revealed that IND and CARD patients have relatively lower percentages of naive (CD45RA(high)) CD4(+) and CD8(+) T cells. However, statistical analysis of ex-vivo profiles of CD4(+) T cells showed that IND have lower percentage of CD45RA(high) in relation to non-infected individuals, but not in relation to CARD. Elevated percentages of memory (CD45RO(high)) CD4(+) T cells were also demonstrated in infected individuals, although statistically significant differences were only observed between IND and NI groups. Furthermore, when we analyzed the profile of secreted cytokines, we observed that CARD patients presented a significantly higher percentage of CD8(+)CD45RA(high) IFN-gamma-producing cells in control cultures and after antigen pulsing with soluble epimastigote antigens. CONCLUSIONS: Based on a correlation between the frequency of IFN-gamma producing CD8+ T cells in the T cell memory compartment and the chronic chagasic myocarditis, we propose that memory T cells can be involved in the induction of the development of the severe clinical forms of the Chagas disease by mechanisms modulated by IFN-gamma. Furthermore, we showed that individuals from IND group presented more T(CM) CD4(+) T cells, which may induce a regulatory mechanism to protect the host against the exacerbated inflammatory response elicited by the infection.

Avaliação do perfil fenotípico das células B reguladoras nas diferentes formas clínicas da Doença de Chagas / Evaluation of phenotypic profile of regulatory B cells in different clinical forms of Chagas Disease

Vários mecanismos imunorreguladores têm sido propostos na infecção causada pelo protozoário T. cruzi como imunossupressão, apoptose e citocinas reguladoras. Recentemente, foi identificada uma nova subpopulação de células B com caracteríticas reguladoras, capazes de suprimir a progressão e/ou melhorar a recuperação de inflamações mediadas pela imunidade adquirida, principalmente pela produção de IL-10 e TGF-. Apesar de existirem relatos sobre alguns marcadores para Bregs em camundongos, não há uma definição clara de uma combinação específica de marcadores para determinar a população das células B reguladoras humana. Atualmente, ainda não existem relatos sobre o papel ou mesmo a presença de células B reguladoras em pacientes portadores da doença de Chagas. Diante disso, nosso objetivo nesse trabalho foi avaliar o perfil fenotípico das células B reguladoras em indivíduos portadores das formas clínicas indeterminada (IND) ou cardíaca (CARD) da doença de Chagas. Nossos resultados mostraram que pacientes apresentando a forma clínica IND possuem maior percentual de células B CD19+CD21+CD1d+, uma das subpopulação de células B reguladoras descrita em modelo murino, no sangue periférico. Adicionalmente, nossos dados mostraram que os pacientes do grupo IND também possuem maior percentual de células B CD19+CD43-CD1d+, outra subpopulação de células B reguladoras, assim como os pacientes do grupo CARD, após estimulação in vitro com antígenos do T. cruzi.

Nossos resultados mostraram ainda que as subpopulações de células B CD19+CD21+ e CD19+CD43- dos pacientes com a forma clínica CARD apresentaram maior expressão intracitoplasmática de IL-10. Por outro lado, a expressão intracitoplasmática de TGF- foi maior nos linfócitos B reguladores dos indivíduos do grupo NI. Além disso, as células B CD19+ dos pacientes portadores da doença de Chagas apresentam maior expressão de marcadores de ativação e de apoptose. De fato, ainda são necessários estudos adicionais para melhor compreender a atividade funcional das células B reguladoras durante a infecção por T cruzi. No entanto, nossos dados reforçam a importância dos mecanismos de imunorregulação no desenvolvimento da doença de Chagas, sugerindo que a resposta imune contra o T. cruzi pode ser regulada por vários mecanismos supressores do hospedeiro e que os mecanismos reguladores desenvolvidos pelos indivíduos portadores das diferentes formas clínicas são distintos

Avaliação do perfil fenotípico das células B reguladoras nas diferentes formas clínicas da Doença de Chagas

Vários mecanismos imunorreguladores têm sido propostos na infecção causada pelo protozoário T. cruzi como imunossupressão, apoptose e citocinas reguladoras. Recentemente, foi identificada uma nova subpopulação de células B com caracteríticas reguladoras, capazes de suprimir a progressão e/ou melhorar a recuperação de inflamações mediadas pela imunidade adquirida, principalmente pela produção de IL-10 e TGF-. Apesar de existirem relatos sobre alguns marcadores para Bregs em camundongos, não há uma definição clara de uma combinação específica de marcadores para determinar a população das células B reguladoras humana. Atualmente, ainda não existem relatos sobre o papel ou mesmo a presença de células B reguladoras em pacientes portadores da doença de Chagas. Diante disso, nosso objetivo nesse trabalho foi avaliar o perfil fenotípico das células B reguladoras em indivíduos portadores das formas clínicas indeterminada (IND) ou cardíaca (CARD) da doença de Chagas. Nossos resultados mostraram que pacientes apresentando a forma clínica IND possuem maior percentual de células B CD19+CD21+CD1d+, uma das subpopulação de células B reguladoras descrita em modelo murino, no sangue periférico. Adicionalmente, nossos dados mostraram que os pacientes do grupo IND também possuem maior percentual de células B CD19+CD43-CD1d+, outra subpopulação de células B reguladoras, assim como os pacientes do grupo CARD, após estimulação in vitro com antígenos do T. cruzi.

Nossos resultados mostraram ainda que as subpopulações de células B CD19+CD21+ e CD19+CD43- dos pacientes com a forma clínica CARD apresentaram maior expressão intracitoplasmática de IL-10. Por outro lado, a expressão intracitoplasmática de TGF- foi maior nos linfócitos B reguladores dos indivíduos do grupo NI. Além disso, as células B CD19+ dos pacientes portadores da doença de Chagas apresentam maior expressão de marcadores de ativação e de apoptose. De fato, ainda são necessários estudos adicionais para melhor compreender a atividade funcional das células B reguladoras durante a infecção por T cruzi. No entanto, nossos dados reforçam a importância dos mecanismos de imunorregulação no desenvolvimento da doença de Chagas, sugerindo que a resposta imune contra o T. cruzi pode ser regulada por vários mecanismos supressores do hospedeiro e que os mecanismos reguladores desenvolvidos pelos indivíduos portadores das diferentes formas clínicas são distintos

Avaliação do perfil fenotípico das células B reguladoras nas diferentes formas clínicas da Doença de Chagas / Evaluation of phenotypic profile of regulatory B cells in different clinical forms of Chagas Disease

Vários mecanismos imunorreguladores têm sido propostos na infecção causada pelo protozoário T. cruzi como imunossupressão, apoptose e citocinas reguladoras. Recentemente, foi identificada uma nova subpopulação de células B com caracteríticas reguladoras, capazes de suprimir a progressão e/ou melhorar a recuperação de inflamações mediadas pela imunidade adquirida, principalmente pela produção de IL-10 e TGF-. Apesar de existirem relatos sobre alguns marcadores para Bregs em camundongos, não há uma definição clara de uma combinação específica de marcadores para determinar a população das células B reguladoras humana. Atualmente, ainda não existem relatos sobre o papel ou mesmo a presença de células B reguladoras em pacientes portadores da doença de Chagas. Diante disso, nosso objetivo nesse trabalho foi avaliar o perfil fenotípico das células B reguladoras em indivíduos portadores das formas clínicas indeterminada (IND) ou cardíaca (CARD) da doença de Chagas. Nossos resultados mostraram que pacientes apresentando a forma clínica IND possuem maior percentual de células B CD19+CD21+CD1d+, uma das subpopulação de células B reguladoras descrita em modelo murino, no sangue periférico. Adicionalmente, nossos dados mostraram que os pacientes do grupo IND também possuem maior percentual de células B CD19+CD43-CD1d+, outra subpopulação de células B reguladoras, assim como os pacientes do grupo CARD, após estimulação in vitro com antígenos do T. cruzi. Nossos resultados mostraram ainda que as subpopulações de células B CD19+CD21+ e CD19+CD43- dos pacientes com a forma clínica CARD apresentaram maior expressão intracitoplasmática de IL-10. Por outro lado, a expressão intracitoplasmática de TGF- foi maior nos linfócitos B reguladores dos indivíduos do grupo NI. Além disso, as células B CD19+ dos pacientes portadores da doença de Chagas apresentam maior expressão de marcadores de ativação e de apoptose. De fato, ainda são necessários estudos adicionais para melhor compreender a atividade funcional das células B reguladoras durante a infecção por T cruzi. No entanto, nossos dados reforçam a importância dos mecanismos de imunorregulação no desenvolvimento da doença de Chagas, sugerindo que a resposta imune contra o T. cruzi pode ser regulada por vários mecanismos supressores do hospedeiro e que os mecanismos reguladores desenvolvidos pelos indivíduos portadores das diferentes formas clínicas são distintos.