Advertisement
Research Article

Polymorphic Sites at the Immunoregulatory CTLA-4 Gene Are Associated with Chronic Chagas Disease and Its Clinical Manifestations

  • Fabrício C. Dias equal contributor,

    equal contributor Contributed equally to this work with: Fabrício C. Dias, Tiago da S. Medina

    Affiliations: Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil, CEA, Institute of Emerging Diseases and Innovative Therapies, Research Division in Hematology and Immunology, Saint-Louis Hospital, Paris, France

    X
  • Tiago da S. Medina equal contributor,

    equal contributor Contributed equally to this work with: Fabrício C. Dias, Tiago da S. Medina

    Affiliation: Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil

    X
  • Celso T. Mendes-Junior,

    Affiliation: Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil

    X
  • Roberto O. Dantas,

    Affiliation: Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil

    X
  • Cristina W. Pissetti,

    Affiliation: Laboratório de Imunologia, Universidade do Triângulo Mineiro, Uberaba, Minas Gerais, Brazil

    X
  • Virmondes Rodrigues Junior,

    Affiliation: Laboratório de Imunologia, Universidade do Triângulo Mineiro, Uberaba, Minas Gerais, Brazil

    X
  • Renata Dellalibera-Joviliano,

    Affiliation: Departamento de Cirurgia e Anatomia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil

    X
  • José A. Marin-Neto,

    Affiliation: Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil

    X
  • Fredy R. S. Gutierrez,

    Affiliation: School of Medicine, University Antonio Nariño, Bogotá, Colombia

    X
  • Philippe Moreau,

    Affiliation: CEA, Institute of Emerging Diseases and Innovative Therapies, Research Division in Hematology and Immunology, Saint-Louis Hospital, Paris, France

    X
  • João S. Silva,

    Affiliation: Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil

    X
  • Eduardo A. Donadi mail

    eadonadi@fmrp.usp.br

    Affiliations: Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil, Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil

    X
  • Published: October 24, 2013
  • DOI: 10.1371/journal.pone.0078367

Abstract

Background

Chagas disease affects approximately 10 million people mainly in Latin America. The immune regulation by the host seems to be an essential factor for disease evolution, and immune system inhibitory molecules such as CTLA-4 and PD-1 favor the maintenance of peripheral tolerance. Considering that polymorphisms at the immunoregulatory CTLA-4 and PDCD1 genes may alter their inhibitory function, we investigated the association of alleles, genotypes and haplotypes of polymorphic sites observed at the CTLA-4 and PDCD1 genes with different clinical manifestations of chronic Chagas disease (indeterminate, cardiac, digestive and mixed).

Methods

The polymorphisms at the CTLA-4 (-1722T/C, -318C/T and +49A/G) and PDCD1 (PD-1.3G/A) genes were typed using TaqMan methodology in 277 chronic Chagas disease patients classified into four groups, according to clinical characteristics, and 326 non-infected controls.

Results

Our results showed that CTLA-4 -1722CC genotype (22%), -1722C allele (27%) and CTLA-4 TCG (8.6%), TCA (26%) and CCA (15%) haplotypes were strongly associated with the indeterminate form, while the CTLA-4 -318CT genotype (82%) and CTLA-4 -318T allele (47%) were found mainly in patients with the mixed form of the disease. The CTLA-4 TCG haplotype (10.2%) was associated with the digestive form. On the other hand, the PD-1.3G/A polymorphism was not associated with chronic Chagas disease and its clinical manifestations.

Conclusions

Here, we showed that alleles, genotypes and haplotypes reported to increase the expression of the regulatory molecule CTLA-4 were associated with the indeterminate form of the disease. Taken together, our data support the idea that polymorphic sites at immunoregulatory genes may influence the development of Chagas disease variants.

Introduction

The parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, which affects mainly Latin American populations. An estimated population of 25 million is living at risk of infection, representing a prevalence of 10 million cases all over the world. It was estimated that this illness killed more than 10,000 people worldwide in 2008 [1]. Chagas disease presents acute and chronic phases. The acute phase, which lasts 1 to 2 months, is characterized by an asymptomatic period in most cases, although sometimes it may present acute myocarditis accompanied by cardiomegaly or meningoencephalitis, which can be lethal [2]. In the chronic phase, 60-70% of cases are asymptomatic and 30-40% of patients develop cardiac, digestive or cardiodigestive forms [3]. The symptoms of the cardiac form involve abnormalities of the heart conduction system, bradyarrhythmias and tachyarrhythmias, apical aneurysms, cardiac failure, thromboembolism, and sudden death [4-6]. The digestive manifestations are mainly megaesophagus and megacolon [7]; the main symptoms of megaesophagus are dysphagia with odynophagia, epigastric pain, regurgitation and ptyalism, while megacolon causes prolonged obstipation, abdominal distention, and large bowel obstruction due to fecaloma or sigmoid volvulus [3,8].

The resistance to T. cruzi infection is characterized by an increased production of INF-γ by CD4+ T cells, which leads to CD8+ T cell activation and differentiation [9]. However, CD8+ T cells are responsible for cytotoxicity against host infected cells leading to extensive fibrosis and cytolysis and contributing to heart damage [10]. The immune regulation by the host seems to be an essential factor for disease evolution, and inhibitory molecules such as CTLA-4 (Cytotoxic T Lymphocyte-Associated Antigen-4) and PD-1 (Programmed Death Receptor-1) favor the maintenance of peripheral tolerance, restraining T cell activation and proliferation.

The CTLA-4 molecule is expressed on activated T cell surface and the interaction of CTLA-4 with its ligands B7.1 and B7.2 generates a negative signal that regulates T cell activation and proliferation [11-13]. Moreover, regulatory T cells (Tregs) express high levels of CTLA-4 on their surface, indicating that this molecule may play an important role in their function [14]. The CTLA-4 gene, located at chromosomal region 2q33, contains more than 100 polymorphic sites [15], and distinct polymorphisms have been associated with autoimmune and infectious diseases [16-20]. The CTLA-4 +49A/G single nucleotide polymorphism (SNP) is located in exon 1 and promotes a Threonine (A) to Alanine (G) substitution in the protein leader sequence at amino acid position 17 [21]. Threonine in this position results in a stronger interaction of CTLA-4 with B7.1 molecules, inducing a higher inhibitory effect on T-cell activation when compared to the presence of Alanine [22]. Moreover, the presence of Alanine at position 17 of the CTLA-4 gene results in inefficient glycosylation and decreased molecule expression on the cell surface [23]. The CTLA-4 -1722T/C and CTLA-4 -318C/T SNPs were identified at the promoter region of the CTLA-4 gene and some particular alleles may affect CTLA-4 mRNA and protein levels [24,25].

The PD-1 molecule is expressed on the cell surface of activated T and B cells and myeloid lineage cells [26], and interacts with the ligands PD-L1 and PD-L2 [27,28]. Interaction of PD-1 with its ligands activates a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM), inducing a negative signal that inhibits T and B cells activation and proliferation, leading to peripheral tolerance [28-30]. The PD-1 molecule is encoded by the PDCD1 gene, which is located at chromosomal region 2q37.3 [31] and contains approximately 230 SNPs [32]. The PD-1.3G/A SNP is located at position +7146 at intron 4, and the presence of an A allele disrupts the binding affinity of the Runt-Related Transcription Factor (RUNX1/AML1), altering mRNA expression level, mRNA stability or both [31].

In this study, we investigated the association of alleles, genotypes and haplotypes at CTLA-4 (-1722T/C rs733618, -318C/T rs5742909, +49A/G rs231775) and PD-1.3G/A (+7146 rs11568821) polymorphic sites with the diverse clinical manifestations of chronic Chagas disease. In summary, we observed that polymorphisms related to augmented expression of CTLA-4 molecule contributed to the development of the mild indeterminate chagasic form.

Materials and Methods

Ethics statement

The protocol was approved by the Institutional Review Board of the School of Medicine of Ribeirão Preto, University of São Paulo (Protocol number 11237/2009) and written informed consent was obtained from the patients.

Subjects

A total of 277 chronic Chagas disease patients exhibiting positive serology for T. cruzi antigens were studied. Patients were classified into four groups according to clinical characteristics: cardiac, presenting or not congestive heart failure (n = 90), digestive (n = 67), mixed or cardiodigestive (n = 39) and indeterminate (asymptomatic) (n = 81). T. cruzi-infected patients were submitted to clinical examination, electrocardiography and chest, esophagus and colon contrast X-ray exams, thus classifying them into the cardiac, digestive, mixed or indeterminate form. Only patients exhibiting well defined clinical variants of Chagas disease were included in the study, and patients aged less than 18 years old and those exhibiting chronic infectious disorders were excluded. The total number of patients was determined after considering the exclusion criteria. Indeterminate patients exhibited this clinical variant for at least 20 years. A total of 326 unrelated healthy bone marrow donors of both sexes from the same geographic region of the patients exhibiting negative serology for T. cruzi antigens were randomly selected to constitute the control group. The demographic characteristics of patients and healthy controls are shown in Table 1.

Clinical forms
CharacteristicINDCDDGMXWGHC
n81906739277326
Sex (Male/Female)48/3347/4338/2913/26146/131192/134
Age (Year)55.0 ± 17.164.9 ± 15.361.2 ± 17.462.0 ± 15.360.7 ± 16.749.5 ± 13.8

Table 1. Demographic characteristics of patients with Chagas disease and healthy controls.

IND (Indeterminate). CD (Cardiac). DG (Digestive). MX (Mixed). WG (Whole Group). HC (Healthy Control). Variables are expressed as mean ± SEM.

DNA extraction and genotyping

Genomic DNA was extracted from peripheral blood leucocytes using a standard salting out procedure [33]. Genotyping of the CTLA-4 -1722T/C (rs733618; Assay ID: C_2415791_10), CTLA-4 -318C/T (rs5742909; Assay ID: C_27834180_10), CTLA-4 +49A/G (rs231775; Assay ID: C_2415786_20) and PD-1.3G/A (+7146) (rs11568821; Assay ID: C_57931290_10) single nucleotide polymorphisms was performed by real-time PCR using the TaqMan SNP (Single Nucleotide Polymorphism) Genotyping Assay and the StepOnePlus automatic instrument (Applied Biosystems, Foster City, CA), according to the manufacturer’s instructions.

Statistical analysis

Allele and genotype frequencies were estimated by the direct counting method, and adherences of phenotypical proportions to expectations under Hardy-Weinberg equilibrium (HWE) were tested by the complete enumeration method using the GENEPOP 3.4 software [34]. Linkage Disequilibrium (LD) between CTLA-4 SNPs was evaluated by means of Lewontin’s standardized coefficient D’ and by a likelihood ratio test of linkage disequilibrium implemented at the ARLEQUIN software [35]. CTLA-4 haplotype frequencies were estimated in each population sample by a coalescence-based method implemented in the PHASE v2 software [36]. The frequency of each allele, genotype or haplotype was compared between patients and controls by the two-sided Fisher exact test, with the aid of the GraphPad Instat 3.05 software, which was also used to estimate the Odds Ratio (OR) and its 95% Confidence Interval (CI). The Bonferroni correction was used to adjust the significance levels for multiple testing, resulting in α = 0.0011 (i.e., 0.05/44, where 44 indicates the number of tests involving 4 alleles in 11 group comparisons), α = 0.0004 (i.e., 0.05/132, where 132 indicates the number of tests involving 12 genotypes in 11 group comparisons), and α = 0.0006 (i.e., 0.05/88, where 88 indicates the number of tests involving 8 haplotypes in 11 group comparisons).

Results

Genotype and allele frequencies of CTLA-4 -1722T/C, -318C/T and +49A/G SNPs

To understand how polymorphic sites observed at genes related to immune regulation can be associated with clinical forms of Chagas disease, we firstly analyzed whether the frequencies of three polymorphisms at the CTLA-4 gene were associated with clinical features. Genotype and allele frequencies of the CTLA-4 gene polymorphisms in patients with chronic Chagas disease and controls are shown in Table 2. The -1722CC genotype frequency was increased in patients exhibiting the indeterminate form compared to controls (p = 0.0001), and also with patients presenting the cardiac (p<0.0001) and digestive (p<0.0001) variants. This genotype was not observed in patients with the cardiac, digestive and mixed forms. As a consequence, the indeterminate group exhibited an increased frequency of the -1722C allele frequency compared to controls (p<0.0001) and to patients with the cardiac (p<0.0001), digestive (p<0.0001) and mixed (p = 0.0001) forms.

Clinical forms
INDCDDGMX WG HC
PolymorphismnFreq.nFreq.nFreq.nFreq.nFreq.nFreq.
CTLA-4 -1722(n = 69)(n = 75)(n = 64)(n = 33)(n = 241)(n = 310)
C/C150.22*00.0000.0000.00150.06180.06
C/T70.1070.0960.0930.09230.10230.07
T/T470.68680.91580.91300.912030.842690.87
C Allele370.27*70.0560.0530.05530.11590.10
T Allele1010.73*1430.951220.95630.954290.895610.90
CTLA-4 -318(n = 70)(n = 73)(n = 64)(n = 34)(n = 241)(n = 306)
C/C250.36300.41350.5540.12*940.39*1680.55
C/T350.50310.43180.28280.82*1120.471190.39
T/T100.14120.16110.1720.06350.14190.06
C Allele850.61910.62880.69360.53*3000.62*4550.74
T Allele550.39550.38400.31320.47*1820.38*1570.26
CTLA-4 +49(n = 77)(n = 84)(n = 67)(n = 36)(n = 264)(n = 324)
A/A400.52350.42420.63220.611390.531390.43
A/G330.43390.46230.34120.331070.401500.46
G/G40.05100.1220.0320.06180.07350.11
A Allele1130.731090.651070.80560.783850.734280.66
G Allele410.27590.35270.20160.221430.272200.34
PD-1.3(n = 81)(n = 90)(n = 66)(n = 39)(n = 276)(n = 326)
G/G680.84720.80590.89340.872330.842660.82
G/A130.16130.1450.0850.13360.13550.17
A/A00.0050.0620.0300.0070.0350.01
G Allele1490.921570.871230.93730.945020.915870.90
A Allele130.08230.1390.0750.06500.09650.10

Table 2. Genotype and allele frequencies of polymorphisms at the CTLA-4 and PDCD1 genes in patients with Chagas disease and healthy controls.

IND (Indeterminate). CD (Cardiac). DG (Digestive). MX (Mixed). WG (Whole Group). HC (Healthy Control).
*frequencies that show statistical differences.

The -1722T allele frequency was decreased in the indeterminate group compared to controls (p<0.0001) and to patients with the cardiac (p<0.0001), digestive (p<0.0001) and mixed (p = 0.0001) forms.

The -318CC genotype was decreased in the whole group of patients (p = 0.0003) and in the group with the mixed form (p<0.0001) compared to controls. A decreased frequency of the -318CC genotype was also observed in patients with the mixed form compared to digestive form (p<0.0001). The -318CT genotype frequency was increased in the group with the mixed form of the disease compared to controls (p<0.0001) and to patients with the cardiac (p = 0.0001) and digestive (p<0.0001) forms.

An increased frequency of the -318T allele was observed in the whole group (p<0.0001) and in the group with the mixed form (p = 0.0005) compared to controls. The -318C allele presented the opposite association pattern.

None of the genotype or allele frequencies of the CTLA-4 +49A/G polymorphism showed statistically significant difference between the different clinical forms of Chagas disease and healthy controls. The Odds Ratio and 95% Confidence Interval values obtained for the comparisons exhibiting significant differences are shown in Table 3.

Genotypes and AllelesComparisonOR(95%CI)ComparisonOR(95%CI)ComparisonOR(95%CI)
-1722CCIND vs. HC4.51(2.1-9.5)IND vs. CD42.95(2.5-733.3)IND vs. DG36.69(2.2-627.4)
-1722CIND vs. HC3.48(2.2-5.5)IND vs. CD7.48(3.2-17.5)IND vs. DG7.45(3.0-18.4)
IND vs. MX7.69(2.3-26.0)
-1722TIND vs. HC0.29(0.2-0.5)IND vs. CD0.13(0.05-0.3)IND vs. DG0.13(0.06-0.3)
IND vs. MX0.13(0.04-0.4)
-318CCWG vs. HC0.53(0.4-0.7)MX vs. HC0.11(0.04-0.3)MX vs. DG0.11(0.04-0.4)
-318CTMX vs. HC7.33(3.0-18.2)MX vs. CD6.32(2.3-17.1)MX vs. DG11.93(4.2-33.6)
-318TWG vs. HC1.76(1.4-2.3)MX vs. HC2.58(1.6-4.3)
-318CWG vs. HC0.57(0.4-0.7)MX vs. HC0.39(0.2-0.7)

Table 3. Comparisons of genotype and allele frequencies of polymorphisms at the CTLA-4 gene between different presentation forms of Chagas disease.

IND (Indeterminate). CD (Cardiac). DG (Digestive). MX (Mixed). WG (Whole Group). HC (Healthy Control). OR, odds ratio. 95%CI, 95% confidence interval. Statistically significant values at a 5% significance level after Bonferroni correction.

Genotype and Allele Frequencies of PD-1.3G/A SNP

We also analyzed the association of an important polymorphism at the PDCD1 gene in individuals infected with T. cruzi. Genotype and allele frequencies of the PD-1.3G/A polymorphism in patients with chronic Chagas disease and healthy controls are shown in Table 2. Neither the genotype nor allele frequencies of the PD-1.3G/A polymorphism showed statistically significant difference between the different clinical forms of Chagas disease and healthy controls.

The majority of SNP genotype distributions evaluated in this study adhered to the Hardy-Weinberg Equilibrium, only the CTLA-4 -1722C/T SNP of the control group was not in equilibrium (p<0.05), due to an excess of the CC genotype.

Haplotype frequency of polymorphisms at the CTLA-4 (-1722T/C; -318C/T; +49A/G) gene

To further understand how the ensemble of polymorphisms participates in the resistance/susceptibility to Chagas disease, we analyzed the haplotypes of SNPs observed at the CTLA-4 gene. The Lewontin’s D’ coefficient of Linkage Disequilibrium (LD) was evaluated between all three possible pairs of CTLA-4 SNPs in both chagasic patients and healthy controls and failed to reveal significant LD only between SNPs -318 and +49 among chagasic patients (D’ = 0.1166; χ2 = 1.5825; p = 0.2084). Moreover, the likelihood ratio test of linkage disequilibrium indicated the existence of significant LD between all three possible pairs of CTLA-4 SNPs (p = 0.0000 ± 0.0000 for each pair) in both groups (Table 4). Given the significant LD between all three possible pairs of CTLA-4 SNPs, CTLA-4 haplotype frequencies were estimated. Haplotype frequencies of polymorphisms at the CTLA-4 gene of patients with chronic Chagas disease and controls are shown in Table 5. The CTLA-4 -1722T/-318C/+49G haplotype frequency was underrepresented in the whole group of patients (p<0.0001), as well as in the groups with the digestive (p = 0.0005) and indeterminate (p<0.0001) forms compared to controls.

Sample group (SNPs)Dχ2p-value
HC (CTLA-4 -1722 vs. CTLA-4 -318)0.804714.85050.0001
HC (CTLA-4 -1722 vs. CTLA-4 +49)0.582941.13110.0000
HC (CTLA-4 -318 vs. CTLA-4 +49)0.473926.63260.0000
WG (CTLA-4 -1722 vs. CTLA-4 -318)0.848225.64870.0000
WG (CTLA-4 -1722 vs. CTLA-4 +49)0.307014.20430.0002
WG (CTLA-4 -318 vs. CTLA-4 +49)0.11661.58250.2084

Table 4. Linkage Disequilibrium standardized coefficient (D’) between CTLA-4 SNPs among chagasic patients and healthy controls.

HC (Healthy Control). WG (Whole Group).
Clinical forms
HaplotypesINDCDDGMXWGHC
nFreq.nFreq.nFreq.nFreq.nFreq.nFreq.
-1722/-318/+49
TTG110.079170.112100.07860.083440.089290.046
CTA00.00000.00000.00010.01410.00220 0.003
TTA440.314400.263300.234260.3611400.2851310.210
CTG00.00010.00700.00010.01420.00410.002
TCG120.086*340.224130.102*80.111670.136*1470.235
TCA360.257*550.361690.539290.4031890.3842600.415
CCG160.11450.03320.01610.014240.049420.067
CCA210.150*00.00040.03100.000250.051140.022

Table 5. Haplotype frequencies of polymorphisms at the CTLA-4 gene in patients with Chagas disease and healthy controls.

IND (Indeterminate). CD (Cardiac). DG (Digestive). MX (Mixed). WG (Whole Group). HC (Healthy Control). * frequencies that show statistical differences.

The indeterminate group exhibited a decreased frequency of the CTLA-4 TCA haplotype (p = 0.0005) compared to controls. The CTLA-4 TCA haplotype was also overrepresented in patients with the digestive form compared to the indeterminate (p<0.0001) group. An increased frequency of the CTLA-4 CCA haplotype was observed in the indeterminate patients (p<0.0001) compared to controls, and also with patients presenting the cardiac (p<0.0001) and mixed (p = 0.0001) forms of the disease.

The Odds Ratio and 95% Confidence Interval values obtained for the comparisons exhibiting significant differences are shown in Table 6. Table 7 summarizes the major results obtained in this study.

HaplotypesComparisonOR(95%CI)ComparisonOR(95%CI)
TCGWG vs. HC0.51(0.4-0.7)DG vs. HC0.37(0.2-0.7)
IND vs. HC0.31(0.2-0.6)
TCAIND vs. HC0.49(0.3-0.7)DG vs. IND3.58(2.0-5.7)
CCAIND vs. HC7.71(3.8-15.6)IND vs. CD54.87(3.3-915.2)
IND vs. MX26.09(1.6-437.2)

Table 6. Comparisons of haplotype frequencies of -1722/-318/+49 polymorphisms at the CTLA-4 gene between different presentation forms of Chagas disease.

IND (Indeterminate). CD (Cardiac). DG (Digestive). MX (Mixed). WG (Whole Group). HC (Healthy Control). OR, odds ratio. 95%CI, 95% confidence interval. Statistically significant values at a 5% significance level after Bonferroni correction.
Genotypes, alleles and haplotypesClinical forms
IndeterminateDigestiveMixed
CTLA-4 genotypes↑ -1722CC-318CC
-318CT
CTLA-4 alleles↑ -1722C-318C
↓ -1722T-318T
CTLA-4 -1722/-318/+49 haplotypes↓ TCG↓ TCG
↓ TCA
↑ CCA

Table 7. Summary of the associations between genotypes, alleles and haplotypes of CTLA-4 polymorphisms with Chagas disease and its clinical forms, showing increased or decreased frequencies of genotypes, alleles and haplotypes.

Discussion

Chagas disease can be manifested under different clinical forms. In humans, the clinical pattern is tightly regulated by the magnitude of the immune response. Approximately 60% of patients remain asymptomatic throughout their lives after T. cruzi infection, characterizing the indeterminate form, probably by inducing a functional regulatory response accompanied by increased IL-10 production [37], which is able to efficiently control CD8+ T cells, promoting an anti-inflammatory milieu. These patients are also able to control the parasite burden after inducing cytotoxic NK cells [38]. On the other hand, Tregs play a pivotal role in the pathogenesis of Chagas disease, since Tregs from cardiac and digestive patients are not functional or are decreased in number and function, contributing to a pro-inflammatory microenvironment induced by IFN-γ-producing CD4+ T cells and TNF-producing monocytes [37]. In concert, these events promote parasite control and tissue damage [39].

CTLA-4 and PD-1 molecules are normally expressed on the Treg surface producing a negative signal and thus preventing T cell activation [40]. Polymorphic sites at genes encoding these regulatory molecules can unbalance the immune regulation during T. cruzi infection, altering Treg function and contributing to the appearance of distinct clinical forms in Chagas disease. In this study, we analyzed three polymorphic sites at the CTLA-4 gene. Overall, the CTLA-4 -1722 polymorphic site is less studied [41,42], whereas the CTLA-4 -318 and CTLA-4 +49 polymorphic sites have been more frequently explored in several diseases [16,18,41-44]. We showed that the CTLA-4 -1722CC genotype is strongly associated with the indeterminate form, i. e., this genotype is not observed in other clinical variants of Chagas disease, suggesting that the presence of this genotype protects against the development of the cardiac, digestive and mixed variants. Considering that this allele has been associated with an increased expression of CTLA-4 [19], it is possible that the increased expression of CTLA-4 on the Treg surface may ameliorate its function, controlling disease-induced damage. The absence of the CTLA-4 -1722CC genotype in other Chagas disease variants may indicate that it can be a marker of the indeterminate form, suggesting that this genotype may protect against the development of the major chronic Chagas disease clinical manifestations. This finding supports the idea that patients exhibiting the indeterminate form have an increased immunoregulatory response.

Overall, the frequency of the CTLA-4 -318T allele is increased in patients presenting the mixed form of the disease in relation to controls. In addition, the CTLA-4 -318CT genotype is increased in the mixed form compared to cardiac and digestive groups as well as compared to controls, indicating that the CTLA-4 -318T allele and the CTLA-4 -318CT genotype are primarily associated with the mixed form of Chagas disease.

Besides the CTLA-4 -1722 and CTLA-4 -318 polymorphic sites that are located at the CTLA-4 promoter region, we also investigated a polymorphic site located at position +49 in exon 1 of the CTLA-4 gene. It was previously reported that the CTLA-4 +49AA genotype is associated with impaired T cell proliferation by increasing CTLA-4 function, whereas the CTLA-4 +49GG genotype impairs Treg function by increasing T cell proliferation and consequently causing a pro-inflammatory response [45]. However, no association was observed between the CTLA-4 +49 polymorphic site and the clinical forms of Chagas disease.

When we evaluated CTLA-4 polymorphic sites as haplotypes (-1722T/C; -318C/T; +49A/G), we observed that the indeterminate patients are strongly associated with a reduced frequency of TCG and TCA haplotypes (probably associated with decreased expression/function of CTLA-4) and a higher frequency of CCA haplotype (probably associated with increased expression/function of CTLA-4), suggesting that these haplotypes confer an up-regulation of the CTLA-4 molecule. Thus, the expression of a specific CTLA-4 haplotype can confer protection against or susceptibility to Chagas disease by modulating CTLA-4 function. It was previously shown that patients with the indeterminate form of the disease have a higher amount of IL-10-producing Treg cells [46] and increased number of CTLA-4+ CD8+ T cells compared to non-infected individuals and to cardiac patients [47].

Regarding PD-1, our group showed an up-regulation of PD-1 and its ligands on lymphocytes and APCs, respectively, after T. cruzi infection in murine models. After blocking PD-1 and its ligands, we observed a high inflammatory migration to the heart tissue, suggesting that this molecule is crucial to control inflammatory processes [48]. We also analyzed one polymorphic site at the PDCD1 gene that encodes the PD-1 protein. Here, genotype or allele frequencies of the PD-1.3G/A polymorphism are not associated to the development of a specific clinical form of Chagas disease. Our data provide information of the importance of the CTLA-4 gene as a genetic marker associated with different manifestation forms of chronic Chagas disease.

Taken together, our data show that polymorphic sites observed at genes encoding regulatory molecules might modulate the appearance of different clinical forms of Chagas disease. Overall, alleles, genotypes and haplotypes reported to increase the expression of the regulatory molecule CTLA-4 were associated with a milder form of the disease (indeterminate), suggesting that regulatory mechanisms play a crucial role during the course of Chagas disease.

Author Contributions

Conceived and designed the experiments: FCD TSM JSS EAD. Performed the experiments: FCD TSM. Analyzed the data: FCD TSM CTMJ. Contributed reagents/materials/analysis tools: FCD TSM CTMJ ROD CWP VRJ RDJ JAMN FRSG PM JSS EAD. Wrote the manuscript: FCD TSM EAD.

References

  1. 1. WHO (2012) Chagas Disease (American trypanosomiasis). Available: http://www.who.int/mediacentre/factsheet​s/fs340/en/index.html. Accessed 2013 January 03.
  2. 2. Moncayo A, Ortiz Yanine MI (2006) An update on Chagas disease (human American trypanosomiasis). Ann Trop Med Parasitol 100: 663-677. doi:10.1179/136485906X112248. PubMed: 17227647.
  3. 3. Rassi A Jr., Rassi A, Marin-Neto JA (2010) Chagas disease. Lancet 375: 1388-1402. doi:10.1016/S0140-6736(10)60061-X. PubMed: 20399979.
  4. 4. Rassi A Jr., Rassi A, Little WC (2000) Chagas' heart disease. Clin Cardiol 23: 883-889. doi:10.1002/clc.4960231205. PubMed: 11129673.
  5. 5. Marin Neto JA, Simões MV, Sarabanda AV (1999) Chagas' heart disease. Arq Bras Cardiol 72: 247-280. PubMed: 10513039.
  6. 6. Marin-Neto JA, Rassi A Jr., Maciel BC, Simoes MV, Schmidt A (2010) Chagas heart disease. In: S. YusufJA CairnsAJ CammEL FallenBJ Gersh. Evidence-based cardiology. 3rd ed. Oxford, UK: BMJ Publishing Group Books. pp. 823-841.
  7. 7. de Rezende JM, Luquetti AO (1994) Chagasic megavisceras. In: Chagas' disease and the nervous system. Washington, DC: Pan American Health Organization. pp. 149-171.
  8. 8. de Oliveira RB, Troncon LE, Dantas RO, Menghelli UG (1998) Gastrointestinal manifestations of Chagas' disease. Am J Gastroenterol 93: 884-889. doi:10.1111/j.1572-0241.1998.270_r.x. PubMed: 9647012.
  9. 9. Brener Z, Gazzinelli RT (1997) Immunological control of Trypanosoma cruzi infection and pathogenesis of Chagas' disease. Int Arch Allergy Immunol 114: 103-110. doi:10.1159/000237653. PubMed: 9338602.
  10. 10. Abel LC, Rizzo LV, Ianni B, Albuquerque F, Bacal F et al. (2001) Chronic Chagas' disease cardiomyopathy patients display an increased IFN-gamma response to Trypanosoma cruzi infection. J Autoimmun 17: 99-107. doi:10.1006/jaut.2001.0523. PubMed: 11488642.
  11. 11. Brunner MC, Chambers CA, Chan FK, Hanke J, Winoto A et al. (1999) CTLA-4-Mediated inhibition of early events of T cell proliferation. J Immunol 162: 5813-5820. PubMed: 10229815.
  12. 12. Chambers CA, Allison JP (1999) Costimulatory regulation of T cell function. Curr Opin Cell Biol 11: 203-210. doi:10.1016/S0955-0674(99)80027-1. PubMed: 10209159.
  13. 13. Alegre ML, Frauwirth KA, Thompson CB (2001) T-cell regulation by CD28 and CTLA-4. Nat Rev Immunol 1: 220-228. doi:10.1038/35105024. PubMed: 11905831.
  14. 14. Gavin M, Rudensky A (2003) Control of immune homeostasis by naturally arising regulatory CD4+ T cells. Curr Opin Immunol 15: 690-696. doi:10.1016/j.coi.2003.09.011. PubMed: 14630204.
  15. 15. Ueda H, Howson JM, Esposito L, Heward J, Snook H et al. (2003) Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature 423: 506-511. doi:10.1038/nature01621. PubMed: 12724780.
  16. 16. Duan S, Zhang G, Han Q, Li Z, Liu Z et al. (2011) CTLA-4 exon 1 +49 polymorphism alone and in a haplotype with -318 promoter polymorphism may confer susceptibility to chronic HBV infection in Chinese Han patients. Mol Biol Rep 38: 5125-5132. doi:10.1007/s11033-010-0660-7. PubMed: 21161390.
  17. 17. Slot MC, Sokolowska MG, Savelkouls KG, Janssen RG, Damoiseaux JG et al. (2008) Immunoregulatory gene polymorphisms are associated with ANCA-related vasculitis. Clin Immunol 128: 39-45. doi:10.1016/j.clim.2008.03.506. PubMed: 18448390.
  18. 18. Khalilzadeh O, Mojazi Amiri H, Tahvildari M, Anvari M, Esteghamati A et al. (2009) Pretibial myxedema is associated with polymorphism in exon 1 of CTLA-4 gene in patients with Graves' ophthalmopathy. Arch Dermatol Res 301: 719-723. doi:10.1007/s00403-008-0919-1. PubMed: 19037649.
  19. 19. Hudson LL, Rocca K, Song YW, Pandey JP (2002) CTLA-4 gene polymorphisms in systemic lupus erythematosus: a highly significant association with a determinant in the promoter region. Hum Genet 111: 452-455. doi:10.1007/s00439-002-0807-2. PubMed: 12384790.
  20. 20. Fernández-Mestre M, Sánchez K, Balbás O, Gendzekhzadze K, Ogando V et al. (2009) Influence of CTLA-4 gene polymorphism in autoimmune and infectious diseases. Hum Immunol 70: 532-535. doi:10.1016/j.humimm.2009.03.016. PubMed: 19345707.
  21. 21. Sun T, Hu Z, Shen H, Lin D (2009) Genetic polymorphisms in cytotoxic T-lymphocyte antigen 4 and cancer: the dialectical nature of subtle human immune dysregulation. Cancer Res 69: 6011-6014. doi:10.1158/0008-5472.SABCS-6011. PubMed: 19638588.
  22. 22. Sun T, Zhou Y, Yang M, Hu Z, Tan W et al. (2008) Functional genetic variations in cytotoxic T-lymphocyte antigen 4 and susceptibility to multiple types of cancer. Cancer Res 68: 7025-7034. doi:10.1158/0008-5472.CAN-08-0806. PubMed: 18757416.
  23. 23. Anjos S, Nguyen A, Ounissi-Benkalha H, Tessier MC, Polychronakos C (2002) A common autoimmunity predisposing signal peptide variant of the cytotoxic T-lymphocyte antigen 4 results in inefficient glycosylation of the susceptibility allele. J Biol Chem 277: 46478-46486. doi:10.1074/jbc.M206894200. PubMed: 12244107.
  24. 24. Shastry BS (2002) SNP alleles in human disease and evolution. J Hum Genet 47: 561-566. doi:10.1007/s100380200086. PubMed: 12436191.
  25. 25. Wang XB, Zhao X, Giscombe R, Lefvert AK (2002) A CTLA-4 gene polymorphism at position -318 in the promoter region affects the expression of protein. Genes Immun 3: 233-234. doi:10.1038/sj.gene.6363869. PubMed: 12058260.
  26. 26. Agata Y, Kawasaki A, Nishimura H, Ishida Y, Tsubata T et al. (1996) Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes. Int Immunol 8: 765-772. doi:10.1093/intimm/8.5.765. PubMed: 8671665.
  27. 27. Dong H, Zhu G, Tamada K, Chen L (1999) B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med 5: 1365-1369. doi:10.1038/70932. PubMed: 10581077.
  28. 28. Latchman Y, Wood CR, Chernova T, Chaudhary D, Borde M et al. (2001) PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat Immunol 2: 261-268. doi:10.1038/85330. PubMed: 11224527.
  29. 29. Freeman GJ, Long AJ, Iwai Y, Bourque K, Chernova T et al. (2000) Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med 192: 1027-1034. doi:10.1084/jem.192.7.1027. PubMed: 11015443.
  30. 30. Nishimura H, Honjo T (2001) PD-1: an inhibitory immunoreceptor involved in peripheral tolerance. Trends Immunol 22: 265-268. doi:10.1016/S1471-4906(01)01888-9. PubMed: 11323285.
  31. 31. Prokunina L, Castillejo-López C, Oberg F, Gunnarsson I, Berg L et al. (2002) A regulatory polymorphism in PDCD1 is associated with susceptibility to systemic lupus erythematosus in humans. Nat Genet 32: 666-669. doi:10.1038/ng1020. PubMed: 12402038.
  32. 32. GeneCards (2012) Programmed Cell Death 1. Available: http://www.genecards.org/cgi-bin/carddis​p.pl?gene=PDCD1&search=pdcd1. Accessed 2013 January 03.
  33. 33. Miller SA, Dykes DD, Polesky HF (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16: 1215. doi:10.1093/nar/16.3.1215. PubMed: 3344216.
  34. 34. Raymond M, Rousset F (1995) Genepop (version 1.2): population-genetics software for exact tests and ecumenicism. J Hered 86: 248-249.
  35. 35. Excoffier L, Lischer HE (2010) Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour 10: 564-567. doi:10.1111/j.1755-0998.2010.02847.x. PubMed: 21565059.
  36. 36. Stephens M, Smith NJ, Donnelly P (2001) A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 68: 978-989. doi:10.1086/319501. PubMed: 11254454.
  37. 37. Gomes JA, Bahia-Oliveira LM, Rocha MO, Martins-Filho OA, Gazzinelli G et al. (2003) Evidence that development of severe cardiomyopathy in human Chagas' disease is due to a Th1-specific immune response. Infect Immun 71: 1185-1193. doi:10.1128/IAI.71.3.1185-1193.2003. PubMed: 12595431.
  38. 38. Vitelli-Avelar DM, Sathler-Avelar R, Massara RL, Borges JD, Lage PS et al. (2006) Are increased frequency of macrophage-like and natural killer (NK) cells, together with high levels of NKT and CD4+CD25high T cells balancing activated CD8+ T cells, the key to control Chagas' disease morbidity? Clin Exp Immunol 145: 81-92. doi:10.1111/j.1365-2249.2006.03123.x. PubMed: 16792677.
  39. 39. Souza PE, Rocha MO, Rocha-Vieira E, Menezes CA, Chaves AC et al. (2004) Monocytes from patients with indeterminate and cardiac forms of Chagas' disease display distinct phenotypic and functional characteristics associated with morbidity. Infect Immun 72: 5283-5291. doi:10.1128/IAI.72.9.5283-5291.2004. PubMed: 15322024.
  40. 40. Carreno BM, Collins M (2002) The B7 family of ligands and its receptors: new pathways for costimulation and inhibition of immune responses. Annu Rev Immunol 20: 29-53. doi:10.1146/annurev.immunol.20.091101.091806. PubMed: 11861596.
  41. 41. Rahimifar S, Erfani N, Sarraf Z, Ghaderi A (2010) ctla-4 gene variations may influence cervical cancer susceptibility. Gynecol Oncol 119: 136-139. doi:10.1016/j.ygyno.2010.06.006. PubMed: 20615526.
  42. 42. Pincerati MR, Dalla-Costa R, Pavoni DP, Petzl-Erler ML (2010) Genetic polymorphisms of the T-cell coreceptors CD28 and CTLA-4 in Afro- and Euro-Brazilians. Int J Immunogenet 37: 253-261. doi:10.1111/j.1744-313X.2010.00917.x. PubMed: 20477885.
  43. 43. Kuo HC, Liang CD, Yu HR, Wang CL, Lin IC et al. (2011) CTLA-4, position 49 A/G polymorphism associated with coronary artery lesions in Kawasaki disease. J Clin Immunol 31: 240-244. doi:10.1007/s10875-010-9484-4. PubMed: 21082224.
  44. 44. Martin TM, Bye L, Modi N, Stanford MR, Vaughan R et al. (2009) Genotype analysis of polymorphisms in autoimmune susceptibility genes, CTLA-4 and PTPN22, in an acute anterior uveitis cohort. Mol Vis 15: 208-212. PubMed: 19180256.
  45. 45. Kouki T, Sawai Y, Gardine CA, Fisfalen ME, Alegre ML et al. (2000) CTLA-4 gene polymorphism at position 49 in exon 1 reduces the inhibitory function of CTLA-4 and contributes to the pathogenesis of Graves' disease. J Immunol 165: 6606-6611. PubMed: 11086105.
  46. 46. de Araujo FF, Vitelli-Avelar DM, Teixeira-Carvalho A, Antas PR, Silva Gomes Assis J, et al. (2011) Regulatory T cells phenotype in different clinical forms of Chagas' disease. PLoS Negl Trop. Drosophila Inf Serv 5: e992. doi: 10.1371/journal.pntd.0000992
  47. 47. Souza PE, Rocha MO, Menezes CA, Coelho JS, Chaves AC et al. (2007) Trypanosoma cruzi infection induces differential modulation of costimulatory molecules and cytokines by monocytes and T cells from patients with indeterminate and cardiac Chagas' disease. Infect Immun 75: 1886-1894. doi:10.1128/IAI.01931-06. PubMed: 17283096.
  48. 48. Gutierrez FR, Mariano FS, Oliveira CJ, Pavanelli WR, Guedes PM et al. (2011) Regulation of Trypanosoma cruzi-induced myocarditis by programmed death cell receptor 1. Infect Immun 79: 1873-1881. doi:10.1128/IAI.01047-10. PubMed: 21357717.