Background
Chagas disease, a neglected disease caused by
Trypanosoma cruzi, remains a serious public health problem and affects about 10 million people in Latin America [
1]. Chronic cardiomyopathy represents the most important and severe manifestation of human Chagas disease, eventually affecting approximately 20–30 % of individuals. The majority of the chronically affected individuals present the indeterminate (IND) form of the disease, with an apparent absence of morbidity [
2,
3]. Epidemiological studies in endemic areas have shown that 2–5 % of patients will evolve each year from the indeterminate to the cardiac clinical form of the disease [
4].
Although the pathophysiology of Chagas disease is not completely understood, it is widely accepted that the involvement of the immune response is critical in determining the disease outcome [
5‐
8]. While the balance between inflammatory and anti-inflammatory cytokines produced by circulating cells in patients with IND form leans towards to an anti-inflammatory profile, patients with chagasic cardiomyopathy seem to display a predominantly inflammatory pattern [
6,
9,
10]. The type of immune response induced in these individuals seems to be critical for the maintenance of a “healthy” balance between the parasite and the host [
11]. In fact, several studies have demonstrated that immunoregulatory mechanisms are important for the control of infection, possibly affecting disease morbidity in chronic clinical forms [
11,
12] such as T-cell suppression, polyclonal lymphocyte activation, and regulatory cytokines [
13,
14]. Infection with
T. cruzi leads to polyclonal lymphocyte activation [
15], which, by itself, promotes T-cell apoptosis [
16,
17]. In addition, antigens released by
T. cruzi, such as
trans-sialidase and HSP70, induce lymphocyte apoptosis [
18,
19]. Therefore, it is possible that the parasite exploits host cell apoptosis to evade the immune response. Evidences also indicate that apoptosis plays a role in the resolution of inflammation [
20].
In the present work, we evaluated the contribution of apoptosis in specific T cell populations on the development/maintenance of different clinical manifestations of human Chagas disease. Our findings demonstrated that in vitro stimulation with T. cruzi antigens induce lymphocyte apoptosis by continued cell activation, modulation of the expression of apoptosis genes and cytokine secretion profile. These findings may contribute to the regulation of immune response during human Chagas disease.
Methods
Study population
The patients that agreed to participate in this study were identified and selected from those being attended at the Referral Outpatient Center for Chagas Disease, which is located at the Clinics Hospital of the Federal University of Minas Gerais (UFMG), Brazil, under the medical care of one of us (MOCR). These patients were enrolled in a prospective cohort study initiated 20 years ago as previously described [
13]. Patients infected with
T. cruzi were grouped as indeterminate (IND) or with cardiomyopathy (CARD). The IND group included 15 asymptomatic individuals with age ranging from 24 to 66 years (mean of 39.6 ± 10.3), with no significant alterations in electrocardiography, chest X-ray and echocardiogram. The CARD group included 15 patients with age ranging from 23 to 69 years (mean of 48 ± 12.52) presenting dilated cardiomyopathy, characterized by the echocardiographic finding of a dilated left ventricle with impaired ventricular systolic function. Left ventricular ejection fraction (LVEF) and left ventricular diastolic diameter (LVDD) were used as clinical parameters of the ventricular function for Chagas disease patients, where LVEF <55 % and LVDD/body surface area ≥31 mm were used to define Chagas disease dilated cardiomyopathy [
3]. None of the patients had undergone chemotherapeutic treatment, nor been previously treated for
T. cruzi infection. Healthy individuals with age ranging from 29 to 55 years [mean of 42.6 ± 8.8), from a non-endemic area for Chagas disease with negative serological tests for the infection were included in the control group (non-infected NI).
Ethics statement
This study was carried out in full accordance with all international and Brazilian accepted guidelines and was approved by the Ethics Committee of the René Rachou Research Center – FIOCRUZ (14/2006 CEPSH-IRR) and UFMG protocol COEP-ETHIC 001/79). All enrolled patients gave written informed consent prior to the inclusion in the study.
Trypanosoma cruzi soluble antigen preparations
The CL strain of
T. cruzi was used for antigenic preparation as described elsewhere [
21]. After preparation, the protein concentration was determined, aliquoted and stored at - 70 °C prior use.
Short-term in vitro whole blood cultures with T. cruzi antigens
Whole blood samples (final concentration of 1 × 106 cells/mL) were treated with staurosporine (Sigma, St. Louis, MO, USA) (4 μM), soluble T. cruzi antigens (TcAg) (25 μg/mL) or untreated (stimulated with medium alone – RPMI 1640 supplemented with 1.6 % L-glutamine, 3 % antibiotic-antimycotic, 5 % of AB Rh-positive heat-inactivated normal human serum), and incubated for approximately 24 h at 37 °C in 5 % CO2. Following incubation, the cultures were treated with 220 μL of EDTA at 20 mM and maintained at room temperature for 15 min prior immunophenotypic staining for apoptosis assay, cell surface markers, and intracellular cytokine analysis.
Cell preparation and proliferation assay
PBMC from Chagas patients and healthy individuals were isolated by Ficolldiatriazoate density gradient centrifugation (LSM; Organon Teknica, Charlesnton, S.C.) as previously described (Gomes, 2003). The cells were washed in RPMI 1640 medium and cultured in flat-bottom 96-well plates (Nunc Brand Products). Proliferative responses were evaluated by incubating 2.5 ×105cells/well for TcAg (25 μg/mL) or 1.5 ×105cell/well for mitogen stimulation (PHA, 10 μg/mL), respectively, in a final volume of 200 μL of complete RPMI-1640. Incubation was carried out in a humidified 5 % CO2 incubator at 37 °C for 3 days for PHA-stimulated cultures and 6 days for antigen-stimulated cultures. Cells were pulsed for the last 6 h of incubation with 1 μCi of [3H] methyl thymidine (PerkinElmer LAS, Shelton, CT, USA), and harvested onto glass fiber filters (Printed Filtermat A, Wallac, Finland). Radioactive incorporation was determined by liquid scintillation spectrometry (MicroBeta JET, PerkinElmer Inc., USA).
Analysis of apoptosis profile
Short-term in vitro whole blood cultures were washed with 6 mL of FACS buffer (PBS supplemented with 0.5 % bovine serum albumin-BSA and 0.1 % sodium azide) by centrifugation at 600xg for 7 min at room temperature and resuspended in 5 mL of FACS buffer.
For the annexin V analysis, aliquots of 150 μL were transferred to polystyrene tubes and incubated for 30 min at room temperature (RT) with 2 μL of allophycocyanin (APC) – labeled anti-CD4 (RPA-T4) (BD Pharmingen) or anti-CD8 (RPA-T8) (BD Pharmingen) monoclonal antibodies. Following incubation, the red blood cells were lysed by the addition of 3 mL of FACS lysing solution (Becton Dickinson, CA, USA) for 10 min, and cells washed with 2 mL of PBS by centrifugation at 600 xg for 7 min at room temperature. The cells were resuspended in annexin V binding buffer (0.1 M Hepes/NaOH (pH 7.4) 1.4 M NaCl, 25 mM CaCl2 - Biosciences, San Jose, CA), for working solution (1X), then incubated for 15 min at RT (25 °C) in the dark with 5 μL annexin V-PE and 5 μL 7AAD. The reaction was stopped by the addition of 100 μL of 1x binding buffer for each tube.
For caspase-3 analysis, aliquots of 150 μL of whole blood cultures were transferred to polystyrene tubes and incubated for 30 min at room temperature with 2 μL of fluorescein isothiocyanate (FITC)-labeled anti-CD45 (2D1) (BD Biosciences) and 2 μL of peridinin chlorophyll-a protein (PerCP)-labeled anti-CD14 (M5E2) (BD Biosciences) monoclonal antibodies. The tubes were incubated in the dark for 10 min at RT. The cells were permeabilized in saponin buffer (0.5 %) (Sigma) for 15 min at RT in the dark. Finally, the cells were incubated with PE-conjugated rabbit anti-active caspase-3 mAb (C92-605) (BD Pharmingem) using 20 μL/1×106 cells for 60 min at RT in the dark. Phenotypic analyses were performed by flow cytometry using a Becton Dickinson FACScalibur flow cytometer Analysis was performed on 7 × 104 lymphocytes (gated according to their forward and side scatter properties. The sample acquisition and data analysis were performed using CellQuest software (BD Biosciences, USA).
Analysis of cell surface markers and intracytoplasmic cytokines
Cultured cells were washed twice in PBS containing 1 % BSA and stained with monoclonal antibodies specific for cell-surface markers. Antibodies to CD4 (RPA-T4), CD8 (RPA-T8) and CD62L (DREG-56) (all from BD Pharmingen) were used. The cells were then fixed in formaldehyde (4 %) and permeabilized in saponin buffer (0.5 %) (Sigma, USA) for 15 min. Finally, the cells were incubated with anti-TNF-α (PE) (L293) (BD Biosciences) washed and ressuspended in FACS buffer prior acquisition in flow cytometer.
Phenotypic analyses were performed by flow cytometry using a Becton Dickinson FACScalibur flow cytometer, collecting data on 7 × 104 lymphocytes gated according to their forward and side scatter properties. The sample acquisition and data analysis were performed using CellQuest software (BD Biosciences, USA).
Detection of plasmatic cytokine levels by Cytometric Bead Array (CBA)
A cytometric beads array (CBA) immunoassay kit (BD Biosciences, USA) was used to measure cytokine levels (IFN-γ, TNF-α, IL-2 and IL-10) in plasma as described in previous studies [
6]. The data were acquired in a Becton Dickinson FACScalibur flow cytometer and analyzed using BD CBA software (BD Biosciences, USA). The results were expressed by mean intensity of fluorescence (MIF).
Apoptotic pathways triggered by T. cruzi infection in different clinical forms of Chagas disease
In order to determine putative apoptotic pathways triggered by
T. cruzi infection, PBMC from infected patients IND (
n = 2) and CARD (
n = 2) were incubated only with culture medium- culture non-stimulated; or in the presence of antigen- culture stimulated with TcAg at a final concentration of 25 μg/mL. After 18 h of incubation at 37 °C and 5 % CO
2 air, cells were recovered and washed with PBS. Subsequently, cells were submitted to a total RNA extraction protocol using NucleoSpin® RNA II kit (Macherey-Nagel, Germany). The total RNA was quantified according to standard procedures using spectrophotometer (Thermo Scientific, USA) and evaluated in agarose gel to confirm its integrity [
22]. The cDNAs were obtained with Superscript II kit (Invitrogen, USA) using 120 ng of total RNA, according to manufacturer’s instructions. Afterwards, a RT-PCR reaction was performed using previously established constitutive human primers to confirm the cDNA synthesis.
The apoptotic transcripts were evaluated using a Human Apoptosis RT2 Profiler™ PCR Array kit (SABiosciences, USA) in a qPCR machine (7500, Applied Biosystem, USA), according to manufacturer’s instructions. Thirty one transcripts involved with pro-apoptotic activity were evaluated. Out of thirty one genes, twenty belong to TNF and TNF receptor superfamily, cell death domains and inductors of apoptosis; and eleven to the caspases family, which are also involved with pro-apoptotic activity. The qPCR data were analyzed by PCR Array Data Analysis Web Portal (SABiosciences, USA), and the results were expressed using the method of 2-ΔΔC.
Statistical analyses
Statistical analyses were conducted using the R 2.15.0 software. Initially, the Anderson-Darling test was applied to verify whether the obtained data represent a normal distribution. Statistical comparative analyses were performed using the non-parametric: Mann–Whitney test to compare two groups (NI x IND or NI x CARD or IND x CARD); Kruskal-Wallis test to compare three groups (NI x IND x CARD) and, together with the Bonferroni correction (significance level, 0.05/3 = 0.0167). All tests were performed considering a significance level of 5 % (α = 0.05).
Discussion
In the present work, we have shown that apoptosis is associated with reduced proliferative response, a high expression of T CD4+CD62L− cells, an increase TNF-α intracellular production and expression of genes of cell death TNF/TNFR superfamily and caspase family in CARD patients.
In this context, some studies have been shown that CARD patients presented low proliferation of T cells when compared with healthy and non-chagasic cardiomyopathy donors [
29‐
32]. Although, the mechanisms of proliferative dysfunction in Chagas disease need further investigation, studies suggest that they could be related to the decrease of co-stimulatory molecules expression, receptor cytokine starvation or expression of inhibitory receptors as PD-1 [
12,
32,
33].
Some studies suggest that activated cells are susceptible to apoptosis, which may represent a mechanism of immunoregulation [
34,
35]. To assess the activation status of T cell subsets, we evaluated whether CD62L is downregulated in these cell subpopulations. Our data showed that CARD patients presented a significantly higher percentage of TCD4
+CD62L
− cells suggesting that a putative involvement of this cell type in the exacerbation of the immune response to the parasite and, consequently, on the development of myocarditis by cell death induced by activation. Dos Santos et al
. [
36] showed that the majority of TCD4 and TCD8 lymphocytes in the inflammatory foci from the heart of chagasic patients did not express or slightly expressed CD62L and T CD8
+ cells are the majority of the activated cells in the tissue when compared with CD4
+ T cells. The inflammatory process occurring in Chagas’ disease mainly consists of CD8 T lymphocytes, CD4 T lymphocytes and macrophages. The extent of inflammatory reaction and the tissue damage caused may contribute to loss of myocardial cells, and to the heart failure that is observed on more severe cases of chronic Chagas’ disease. Tostes Jr et al
. [
37] showed that myocardial cell loss by apoptosis and fibrosis contributes to heart failure in the chronic phase of Chagas’ disease. In fact, apoptosis has been considered a cause of heart failure in other diseases such as myocardial infarction and heart hypertrophy [
38,
39], as well as a mechanism involved in the control of the immune response in experimental models [
26]. On the other hand, heart failure by itself may induce apoptosis [
40].
In this work, high expression of annexin by lymphocytes from CARD group was observed when compared with IND and NI groups, as well as an increase on the expression of annexin by CD4
+ T cells and caspase by both CD4
+ and CD8
+ T cells by chagasic patients when compared with NI group after
in vitro stimulation with
T. cruzi antigens. In Chagas’ disease, the occurrence of apoptosis in T lymphocytes was observed after antigen stimulation in experimental models [
26] although its significance in terms of clinical form or outcome of the disease was not clear. Apoptosis-like death has been reported in amastigote nests and trypomastigotes forms from
T. cruzi, and this mechanism has been associated with control of parasite burden regulated by the parasite itself or by the host, parasite evasion of the host’s immune response and clonal selection [
41‐
44]. Together, these findings suggest that although the lymphocytes from CARD patients presented lower proliferative response upon antigenic recall, lymphocytes from IND and CARD patients presented a singular ability to undergo apoptosis that may reflect different regulatory mechanism.
Rodrigues et al. [
45] demonstrated a high percentage of lymphocyte apoptosis in patients with a severe cardiomyopathy, associating this event to activation of programmed death pathways, by Fas/Fas-L or TNF-α receptors, leading to parasite escape, and consequently, to a continuous stimulation of the immune system. In this context, T cells apoptosis in experimental model leads to an increase of parasite growth [
46]. Indeed, apoptosis has been suggested to be an important mechanism to control the immune response and heart damages [
47].
In order to determine whether TNF-α contributes to T cell apoptosis, the plasmatic and intracellular production by T cell subsets was evaluated. Our data demonstrated that CARD group showed increased levels of circulating and intracytoplasmic TNF-α, and up-regulation of the TNF receptor gene superfamily. Lula et al. [
48] have shown correlation among soluble ligands of TNF superfamily (TNF-α, TRAIL and FasL/CD95L) and functional disorders of the left ventricle in chronic chagasic patients with cardiomyopathy. These results are associated with ligand receptors associated with programmed cell death, suggesting that apoptotic mechanisms are involved with the development of miocardiopathy in Chagas disease.
Ferreira et al. [
49] showed a correlation between high serum levels of TNF-α and the occurrence of severe Chagas cardiomyopathy. Also, it has been shown that there is an inverted correlation between high levels of TNF-α with the lowest left ventricular ejection fraction seen in patients with chronic Chagas cardiomyopathy [
50]. Moreover, patients with heart failure are shown to have a significantly lower PBMC proliferative response but higher levels of apoptosis and Fas and Fas-L expression [
45]. TNF-α may play a role on the high levels apoptosis and in the low proliferative response observed in patients with heart failure. TNF-α may contribute to the induction of apoptosis by the interaction with its receptor or by induction of Fas and Fas-L expression [
51]. Our results suggest that the high levels of TNF-α detected in plasma from CARD group may contribute to the heart condition. Additionally, our data support the hypothesis that high levels of TNF-α and up-regulation of the TNF receptor gene superfamily lead to an increase in apoptosis, and consequently the exacerbation of the pathology.
Conclusions
Here, we showed that apoptosis is associated with low proliferative response, intense T cell activation, high TNF-α production and up-regulation of genes associated with TNF receptors superfamily and caspase family in CARD ’patients. These results suggest that apoptosis could interfere on the development and/or maintenance of the different clinical forms of Chagas disease. Assuming that the immunological regulation in the IND group, may control the development of Chagas cardiomyopathy, the absence of this mechanism in the CARD group, may be one of the factors associated with sustained inflammation which would, consequently, lead to a higher morbidity in the latter group. The association of lymphocyte apoptosis, induced by the constant activation of the immunological system, with high levels of inflammatory cytokines and associated pathological events (fibrosis and apoptosis) may contribute with the development and progression of heart injuries in the chronic phase of the human Chagas disease.
Acknowledgements
This work financially supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Grant # 474887/2004–9; Grant # 404151/2012–4; Grant # 474796/2012–4, Grant # 470304/2011–1) and Fundação de Amparo Pesquisa do Estado de Minas Gerais (FAPEMIG) (Grant # CBB-1322/05; Grant # PPM-00501-13); Programa de Apoio à Pesquisa Estratégica em Saúde/FIOCRUZ (PAPES/FIOCRUZ) (PAPES IV – n° 400266/2006–7, PAPES V – n° Grant# 407692/2012–6). Ana Thereza Chaves is supported by a doctoral degree fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) – PROSUP (n° 078/2006–9). Rodrigo Correa-Oliveira, Manoel Otávio da Costa Rocha, Juliana de Assis Silva Gomes Estanislau, Andréa Teixeira Carvalho, Ricardo Toshio Fujiwara and Elaine Maria de Souza Fagundes are supported by CNPq fellowships (Bolsa de produtividade em Pesquisa).
We thank Adriana Bozzi of the Stanford Cardiovascular Institute, Stanford University for comments that greatly improved the manuscript.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Conception and design of the experiments: ATC, JASG, ACT, EMSF and RCO. Performed the experiments: ATC, JAF, KFS, RCCF, PHGG. Analyzed the data: ATC, JASG, ACT, MJFM, PHGG. Contributed reagents/materials, analysis tools: JASG, RCO, RTF. Wrote the paper: ATC, JASG, JAF, ROC. Assisted with patient care and case identification: MOCR. All authors read and approved the final manuscript.