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. 2012;6(4):e1630.
doi: 10.1371/journal.pntd.0001630. Epub 2012 Apr 24.

Deficient regulatory T cell activity and low frequency of IL-17-producing T cells correlate with the extent of cardiomyopathy in human Chagas' disease

Affiliations

Deficient regulatory T cell activity and low frequency of IL-17-producing T cells correlate with the extent of cardiomyopathy in human Chagas' disease

Paulo Marcos Matta Guedes et al. PLoS Negl Trop Dis. 2012.

Abstract

Background: Myocardium damage during Chagas' disease results from the immunological imbalance between pro- and production of anti-inflammatory cytokines and has been explained based on the Th1-Th2 dichotomy and regulatory T cell activity. Recently, we demonstrated that IL-17 produced during experimental T. cruzi infection regulates Th1 cells differentiation and parasite induced myocarditis. Here, we investigated the role of IL-17 and regulatory T cell during human Chagas' disease.

Methodology/principal findings: First, we observed CD4(+)IL-17(+) T cells in culture of peripheral blood mononuclear cells (PBMC) from Chagas' disease patients and we evaluated Th1, Th2, Th17 cytokine profile production in the PBMC cells from Chagas' disease patients (cardiomyopathy-free, and with mild, moderate or severe cardiomyopathy) cultured with T. cruzi antigen. Cultures of PBMC from patients with moderate and severe cardiomyopathy produced high levels of TNF-α, IFN-γ and low levels of IL-10, when compared to mild cardiomyopathy or cardiomyopathy-free patients. Flow cytometry analysis showed higher CD4(+)IL-17(+) cells in PBMC cultured from patients without or with mild cardiomyopathy, in comparison to patients with moderate or severe cardiomyopathy. We then analyzed the presence and function of regulatory T cells in all patients. All groups of Chagas' disease patients presented the same frequency of CD4(+)CD25(+) regulatory T cells. However, CD4(+)CD25(+) T cells from patients with mild cardiomyopathy or cardiomyopathy-free showed higher suppressive activity than those with moderate and severe cardiomyopathy. IFN-γ levels during chronic Chagas' disease are inversely correlated to the LVEF (P = 0.007, r = -0.614), while regulatory T cell activity is directly correlated with LVEF (P = 0.022, r = 0.500).

Conclusion/significance: These results indicate that reduced production of the cytokines IL-10 and IL-17 in association with high levels of IFN-γ and TNF-α is correlated with the severity of the Chagas' disease cardiomyopathy, and the immunological imbalance observed may be causally related with deficient suppressor activity of regulatory T cells that controls myocardial inflammation.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Higher IL-10 and lower TNF-α and IFN-γ secretion in free/mild vs. moderate/severe cardiomyopathy patients' PBMC.
Levels of cytokines IL-17 (A), IL-10 (B), TNF-α (C) and IFN-γ (D) as examined by enzyme-linked immunosorbent assay in PBMC culture supernatants (×ばつ106 cells/mL in a 48 plate well) from patients, after 48 h of antigenic stimulation with trypomastigote antigen (100ηg/well) and independent of the stimuli (Medium). The Chagas' disease patients were grouped as: Group 1 (n = 10): Patients not treated with benznidazole and free/mild cardiomyopathy, group 2 (n = 11): Patients not treated with benznidazole but with moderate/severe cardiomyopathy, group 3 (n = 8): Patients previously treated with benznidazole free/mild cardiomyopathy. Healthy Individuals (n = 10) from the same endemic areas were included in this study as controls, composing the group 4, as described in Materials and Methods. The results are expressed in picograms per milliliter. Statistical differences are represented by letters: a and b, P<0.05 (Spearman).
Figure 2
Figure 2. Increased frequency of CD4+IL-17+ Tcells in PBMC from free/mild cardiomyopathy patients.
To examine the existence of Th17 lymphocytes in chronic Chagas' disease patients, PBMC (×ばつ106 cells/ml in a 48 plate well) from patients were cultured by 48 h with trypomastigote antigen (10 μg/mL) and the intracellular expression of IL-17 determined in CD3+CD4+ T cells by flow cytometry. PBMC from control and Chagas' disease patients in these analyses were gated on lymphocytes via their forward (FSC) and side scatter (SSC) properties, and CD3+CD4+IL-17+ were analyzed to determine the Th17 population. Representatives flow cytometry analysis of CD3+CD4+IL-17+ T cells gated lymphocytes from, Healthy individuals, Free/Mild cardiomyopathy patients, Moderate/Severe cardiomyopathy patients and Bz-treated patients are shown in A., while B shows the grouped analyses of all subjects in each group. Chagas' disease patients were grouped as: Group 1 (n = 10): Patients not treated with benznidazole and free/mild cardiomyopathy, group 2 (n = 11): Patients not treated with benznidazole but with moderate/severe cardiomyopathy, group 3 (n = 8): Patients previously treated with benznidazole free/mild cardiomyopathy. Healthy Individuals (n = 10) from the same endemic areas were included in this study as controls, composing the group 4, as described in Materials and Methods.
Figure 3
Figure 3. Characterization of CD4+CD25+ Treg in patients with different clinical manifestations of Chagas disease.
Representative flow cytometry analysis of CD4+CD25high, CD4+CD25+Low, CD4+CD25− gated lymphocytes is shown in A. PBMC from control and Chagas' disease patients in these analyses were gated on lymphocytes via their forward (FSC) and side scatter (SSC) properties, and CD4+CD25high (B), CD4+CD25+Low (C), CD4+CD25− (D) were performed to determine the regulatory T cell population. CD4+CD25high (E), CD4+CD25+Low (F), CD4+CD25− (G) cells were analyzed for their expression of membrane CTLA-4, GITR, CD103, and intracellular Foxp3. PBMC (×ばつ106 cells/mL in a 48 plate well) from Chagas' disease patients were cultured with trypomastigote antigen (10 μg/mL) after 48 h of antigenic stimulation the expression of surface markers (CD4, CD25, CD103, CTLA-4, GITR) and transcriptional factor (Foxp3) were determined. The results are expressed as means ± standard errors. a and b indicate statistical differences with P<0.05.
Figure 4
Figure 4. Patients with severe Chagas' disease cardiomyopathy exhibit deficient suppressor activity of Treg.
PBMC (×ばつ106 cells/mL in a 48 plate well) from Chagas' disease patients were cultured with trypomastigote antigen (10 μg/mL) after 48 h of antigenic stimulation the mean intensity of fluorescence of CTLA-4 in CD4+CD25high T cells (A) were performed. Free/Mild cardiomyopathy patients displayed high levels of CTLA-4 in CD4+CD25high T cells (free/mild cardiomyopathy vs. moderate/severe P = 0.0352). For functional characterization of CD4+CD25+ regulatory T cells in Chagas' disease patients magnetic bead-sorted CD4+CD25+ T cells purified from PBMC from free/mild cardiomyopathy patients (n = 5), severe cardiomyopathy (n = 5), and healthy individuals (n = 5), were tested for their ability to suppress the proliferation of allogeneic PBMC. The CD4+CD25+ T cells were harvested and suppressor activity determined as % of proliferation inhibition in culture from PBMC/CD4+CD25+ T cells 1∶5 (B) and 1∶10 (C) proportion. Allogeneic PBMCs (×ばつ105 cells/well in a 96 plate well) CFSE stained were cultured during 72 h with medium only, CD4+CD25+ (×ばつ104 and ×ばつ104 cells/well, ratio of 1∶5), PHA (10 ηg/well), PHA plus CD4+CD25+ (ratio of 1∶10 and 1∶5) from Chagas' disease patients (free/mild cardiomyopathy patients and severe cardiomyopathy patients) or healthy controls. a and b indicate statistical differences with P<0.05 (healthy vs. free/mild cardiomyopathy P = 0.547; healthy patients vs. moderate/severe P = 0.0159; free/mild cardiomyopathy vs. moderate/severe P = 0.0189).
Figure 5
Figure 5. High Treg activity and reduced levels of IFN-γ correlate with normal LVEF.
Correlation analyses were performed considering the values of LVEF and the levels IFN-γ in PBMC culture of patients (A) or in vitro suppressive activity of Treg (B). In C is showed the correlation between the frequency of CD4+CD25highFoxp3+ and CD3+CD4+IL-17+ T cells in PBMC from patients with the different clinical forms of Chagas' disease (free/mild cardiomyopathy patients, patients with moderate/severe cardiomyopathy and chagasic chronic patients treated with benznidazol after in vitro stimulation with T. cruzi antigens. The p values as long as the correlation coefficient are shown for each graph.

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