The role of interleukin 10 (IL-10) and gamma interferon (IFN-γ) on the development of pathology in human Chagas' disease was investigated. Two categories of patients, low and high producers of IFN-γ, were identified based on the levels of secretion of this cytokine in the supernatant of peripheral blood mononuclear cell (PBMC) cultures. Eighty-three percent of the patients presenting with cardiac disease (CARD) of different degrees and 59% of the patients with the indeterminate form of disease (IND) were identified as high IFN-γ producers. PBMC from IND patients classified as low IFN-γ producers secreted significantly higher amounts of IL-10 than did those from other groups. Flow cytometry analysis demonstrated that in PBMC from the IND group, the majority of the IL-10-producing cells were monocytes (CD14^High+cells), whereas in the CARD group, the major sources of IFN-γ were T lymphocytes (CD3⁺CD4⁺cells). These results suggest an association between the production of IFN-γ by CD3⁺CD4⁺cells and morbidity in Chagas' disease, whereas the production of IL-10 by macrophages/monocytes leads to regulation of the immune response in IND patients. We hypothesize that an exacerbated production of IFN-γ againstTrypanosoma cruziantigens favors the development of a strong Th1 response in CARD patients, which leads to progression of heart 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-1β), 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.

Chemokines and chemokine receptors (CKRs) control the migration of leukocytes during the inflammatory process and are important immunological markers of type 1 (CCR5 and CXCR3) and type 2 (CCR3 and CCR4) responses. The coexpression of CKRs (CCR2, CCR3, CCR5, CXCR3, and CXCR4) and intracellular cytokines (interleukin-10 [IL-10], IL-4, tumor necrosis factor alpha [TNF-α], and gamma interferon [IFN-γ]) on T CD4⁺and CD8⁺peripheral cells from individuals with indeterminate (IND) or cardiac (CARD) clinical forms of Chagas' disease after in vitro stimulation withTrypanosoma cruziantigens, were evaluated in this study. The percentage of T CD4⁺and CD8⁺cells coexpressing CCR5 and IFN-γ, CXCR3 and IFN-γ, and CXCR3 and TNF-α were higher in CARD than in IND individuals; on the other hand, the percentage of T CD4⁺or CD8⁺cells coexpressing CCR3 and IL-10 or coexpressing CCR3 and IL-4 were lower in CARD individuals than in IND individuals. In addition, a significant positive correlation between the expression of CCR5 or CXCR3 and IFN-γ was observed in CARD individuals contrasting with a significant positive correlation between the expression of CCR3 and IL-4 and of CCR3 and IL-10 in IND patients. These results reinforce the hypothesis that aT. cruzi-exacerbated specific type 1 immune response developed by CARD chagasic patients is associated with the development of heart pathology.

Cytokines are involved in the immunopathogenesis of nonalcoholic fatty liver disease (NAFLD), but the relationship between them and clinical parameters of NAFLD progression is still unknown. Using flow cytometry, we evaluated the plasma levels of IL-1β, IL-6, IL-12, TNF and IL-10 and their association with clinical and biochemical parameters of liver function during simple steatosis (NAFL) and nonalcoholic steatohepatitis (NASH) in biopsy-proven patients. The NASH patients showed higher levels of IL-6 associated with a lower IL-10/IL-6 ratio. Besides heatmaps were similar in the NAFL and NASH groups, the same did not occur in signature curves, the NASH patients were high producers to IL-12 and IL-6 while the NAFL patients were not high producers of any cytokines evaluated. Integrative biomarker network analysis revealed that cytokines are differently correlated with clinical parameters, while IL-12, IL-10 presented moderate and negative correlations with glycemic and lipid profile in the NAFL group. The NASH group IL-12 and TNF revealed stronger and positive correlations with transient elastography parameters and NAFLD liver fibrosis score. These data suggest that IL-6 and IL-10 might act in chronic inflammation and insulin resistance whereas IL-12 and TNF may be involved in promoting liver damage and NAFLD progression. Plasma concentration analysis of these molecules and their association with clinical parameters can be used as new biomarkers to monitoring NAFLD progression and to reflect NASH development.

CD25^High CD4⁺ regulatory T cells (Treg cells) have been described as key players in immune regulation, preventing infection-induced immune pathology and limiting collateral tissue damage caused by vigorous anti-parasite immune response. In this review, we summarize data obtained by the investigation of Treg cells in different clinical forms of Chagas' disease. Ex vivo immunophenotyping of whole blood, as well as after stimulation with Trypanosoma cruzi antigens, demonstrated that individuals in the indeterminate (IND) clinical form of the disease have a higher frequency of Treg cells, suggesting that an expansion of those cells could be beneficial, possibly by limiting strong cytotoxic activity and tissue damage. Additional analysis demonstrated an activated status of Treg cells based on low expression of CD62L and high expression of CD40L, CD69, and CD54 by cells from all chagasic patients after T. cruzi antigenic stimulation. Moreover, there was an increase in the frequency of the population of Foxp3⁺ CD25^HighCD4⁺ cells that was also IL-10⁺ in the IND group, whereas in the cardiac (CARD) group, there was an increase in the percentage of Foxp3⁺ CD25^High CD4⁺ cells that expressed CTLA-4. These data suggest that IL-10 produced by Treg cells is effective in controlling disease development in IND patients. However, in CARD patients, the same regulatory mechanism, mediated by IL-10 and CTLA-4 expression is unlikely to be sufficient to control the progression of the disease. These data suggest that Treg cells may play an important role in controlling the immune response in Chagas' disease and the balance between regulatory and effector T cells may be important for the progression and development of the disease. Additional detailed analysis of the mechanisms on how these cells are activated and exert their function will certainly give insights for the rational design of procedure to achieve the appropriate balance between protection and pathology during parasite infections.