Association Between the Lymphotoxin-Alpha Gene Polymorphism and Chagasic Cardiopathy

Abstract

Lymphotoxin-alpha (LT-alpha or LTA) is an inflammatory cytokine that is involved in the organization and maintenance of the inflammatory process and in the arrangement of cells at the site of inflammation. These features suggest an important role in the development of chronic Chagas' disease, especially the cardiac form. The objective of this study was to evaluate LT-alpha genetics and its biological role in chronic Chagas' disease. A total of 284 subjects were studied. The LT-alpha single-nucleotide polymorphism (+252) was analyzed by the polymerase chain reaction–restriction fragment length polymorphism and expression by enzyme-linked immunosorbent assay in culture supernatants and in individual T lymphocytes by flow cytometry. The risk of developing the cardiac form was 2.8 times higher among carriers of genotype GG and 2.4 times among carriers of genotype GA when compared to subjects carrying genotype AA. Seropositive subjects carrying the G allele produced significantly higher levels of LT-alpha. The cytokine was mainly expressed by CD8⁺ T lymphocytes in the absence of any stimulus and after stimulation with the Trypanosoma cruzi antigen. This study provides genetic and biological evidence for an important role of LT-alpha in the development of the cardiac form of Chagas' disease.

Introduction

C hagas' disease is an important chronic infection caused by the protozoan Trypanosoma cruzi. The disease continues to be a major public health problem in most Latin American countries, affecting about 9 million people (Schofield and others 2006). The disease has an acute and a chronic phase. Chronic Chagas' disease can be divided into the indeterminate, cardiac, digestive, and mixed forms (Rassi and others 2010).

Despite a large number of studies, the reason why some individuals remain asymptomatic and others develop severe symptoms is unknown. In an attempt to better understand the clinical presentation of the disease, Chagas' disease studies have gone through 3 phases. The first phase emphasized the importance of the parasite for tissue injury. The target of the second phase was the immune system, focusing on the importance of autoimmunity for the genesis of lesions. We are currently in the third phase, the genomic phase, in which the disease is seen as an interaction between 2 variable and coevolving genomes: the genome of the parasite and the human genome (Macedo and others 2004). This view suggests that the different clinical forms of Chagas' disease can be explained by variations in tissue tropism, a process that is influenced by parasite and host genetic factors (Macedo and Pena 1998; Vago and others 2000; Andrade and others 2002).

Host factors have been studied using numerous approaches. One approach consists of the evaluation of the contribution of candidate genes and of the immune response of individuals infected with T. cruzi to the development of chronic lesions. Lymphotoxin-alpha (LT-alpha or LTA) is one of these candidate genes. This inflammatory cytokine is produced by activated T lymphocytes, B lymphocytes, and natural killer cells (Ware and others 1995) and plays an important role in the recruitment and induction of adhesion molecules such as vascular cell adhesion molecule (VCAM), intracelluar cell adhesion molecule (ICAM), E-selectin, and mucosal vascular addressin cell adhesion molecule (MadCAM)-1 (Kratz and others 1996; Cuff and others 1998, 1999). The biological role of LT-alpha has been studied in different infectious and parasitic diseases such as pulmonary tuberculosis (Roach and others 2001), cerebral malaria (Engwerda and others 2002), leishmaniasis (Engwerda and others 2004), experimental toxoplasmosis (Schlüter and others 2003), and infection with T. brucei (Magez and others 2002). However, few studies have investigated the role of LT-alpha in Chagas' disease. The first report on experimental Chagas' disease demonstrated that lymphocytes are able to control the infection through LT-alpha (Krassner and others 1982). The second report, an immunogenetic study, showed an association between the LTA +80C/+252G haplotype and susceptibility to the development of chronic chagasic cardiomyopathy (Ramasawmy and others 2007).

The objective of the present study was to investigate the role of the LT-alpha gene polymorphism at position +252 and the production of this cytokine in patients with the indeterminate and cardiac forms of Chagas' disease and uninfected control individuals from the same endemic area.

Materials and Methods

A case–control study was conducted in the municipality of Água Comprida, southern region of Triângulo Mineiro (Minas Gerais, Brazil). The region was endemic for T. cruzi and therefore continues to present a high prevalence of Chagas' disease. Epidemiological and entomological data demonstrate the interruption of vector transmission in 1999 (Vinhaes and Dias 2000; Villela and others 2009). A total of 482 unrelated individuals (mean age: 49.8±14.4; range: 25–91 years) were first selected. However, successful single-nucleotide polymorphism typing was only possible in 284 subjects. Subjects with complete serological data for T. cruzi infection were included in the study. In addition, only subjects aged 25 years or older were included in the analysis, since they corresponded to the youngest seropositive individuals of the sample studied. All subjects were from the same endemic area.

Infection with T. cruzi was evaluated by passive hemagglutination (Salck Laboratory) and enzyme-linked immunosorbent assay (ELISA; Abbott). A subject with 2 positive tests was defined as positive (Consenso Brasileiro em Doença de Chagas 2005). In addition, patients infected with T. cruzi were submitted to clinical examination, electrocardiography, and chest, esophagus, and colon contrast X-ray exams for classification into the cardiac, digestive, mixed, or indeterminate form (Anonymous 1985; Consenso Brasileiro em Doença de Chagas 2005). Patients with the cardiac form were classified according to the Criteria Committee of the New York Heart Association (1994). In the present study, only subjects with the cardiac and indeterminate forms and seronegative individuals were analyzed. Subjects younger than 25 years and HIV-positive individuals were excluded. The treatment criteria did not exclude any patient. The study was approved by the Research Ethics Committee of the Universidade Federal do Triângulo Mineiro, Brazil (protocol number 920).

Peripheral blood mononuclear cells (PBMC) were separated by density centrifugation on a Ficoll-Hypaque gradient (Pharmacia Biotech) according to the manufacturer's recommendations. After separation, the cells were washed and resuspended in an RPMI medium supplemented with 5% fetal bovine serum (Gibco), 2 mM l-glutamine (Gibco), 50 mM 2-mercaptoethanol (Merck), and 40 μg/mL gentamicin. PBMC (2×10⁶ cells/well) were cultured in the presence of 5 μg/mL T. cruzi (strain Y) antigens and 5 μg/mL phytohemagglutinin (PHA; Sigma). The latter was used as a nonspecific stimulus of T cells. The plates were incubated at 37°C in a 5% CO₂ atmosphere for 48 and 120 h. The culture supernatants were collected and stored at −70°C.

For flow cytometry, fluorescein isothiocyanate anti-CD8, PE-Cy5 anti-CD4, and phycoerythrin (PE) anti-LT-alpha antibodies (BD Pharmingen) and appropriate isotype controls were used according to the manufacturer's instructions. PBMC were recovered from 48-h cell cultures incubated in the presence of T. cruzi antigens or medium alone (without PHA). The cells (2×10⁵ cells/tubes) were transferred to 5-mL polystyrene tubes and washed once with cold buffer (phosphate-buffered saline/5% bovine serum albumin) by centrifugation at 400 g for 10 min at 20°C.

The cells were first labeled for surface markers (CD4 and CD8). After permeabilization using Fix and Perm reagent (BD Pharmingen), the cells were incubated with PE-conjugated anti-LT-alpha antibodies. A total of 20,000 events/tube were acquired in a FACSCalibur^® flow cytometer (BD). Cell Quest^™ software provided by the manufacturer was used for data acquisition and analysis.

LT-alpha titration was performed on culture supernatants by ELISA using commercial antibody sets according to the manufacturer's instructions (BD Pharmingen). Absorbance was read at 405 nm in a microplate reader (BioRad). The sensitivity of the test was 2 pg/mL.

Genomic DNA was obtained from white blood cells using the DNAzol reagent (Gibco). The presence of the LT-alpha gene polymorphism at position +252 was determined by polymerase chain reaction amplification as described previously (forward primer: CTC CTG CAC CTG CCT GGA TC; reverse primer: GAA GAG ACG TTC AGG TGG TGT CAT) (Invitrogen) (Warzocha and others 1998), followed by digestion with NcoI (10 U/μL) (Gibco). The digestion products were analyzed on a polyacrylamide gel. The LTA +252A allele produces one fragment of 368 bp, whereas the LTA +252G allele produces 2 fragments (235 and 133 bp).

The association of genotypes and allele frequencies with age, gender, serology, and clinical form was investigated by univariate analysis using the chi-square test. In addition, a multivariate logistic regression model was used, which permitted the calculation of odds ratios (ORs) adjusted for age and gender. Since the data showed no normal distribution (Shapiro–Wilk test) or variance heterogeneity (Levene test), the nonparametric Kruskal–Wallis and Mann–Whitney tests were used to evaluate LT-alpha levels according to genotype, allele frequency, serology, and clinical form.

Differences in the expression of LT-alpha between CD4⁺ and CD8⁺ T cells or between unstimulated cells and cells stimulated with T. cruzi antigen were evaluated using the paired t-test, since the data were normally distributed (Shapiro–Wilk test). T-cell subsets were compared between the cardiac and indeterminate clinical forms using the parametric Student t-test for independent samples. Statistical analysis was performed with the SPSS 17.0 (SPSS) or Statistica 8.0 program (Statsoft). A P-value of 0.05 was considered to indicate statistical significance.

Results

Genotyping

Analysis of the LT-alpha gene polymorphism showed significant association between T. cruzi seropositivity and the genotypes obtained (χ² , P=0.080). However, presence of allele G did not show a significant association (χ² , P=0.237). The seropositivity rate was 46.8% (66/141) for subjects carrying genotype AA, 43.9% (50/114) for subjects with genotype AG, and 24.1% (7/29) for subjects with genotype GG. The seropositivity rate was 39.9% (57/143) when subjects carrying allele G were combined.

The cardiac and indeterminate forms were not significantly associated with gender (χ² , P=0.177) or age class (χ² , P=0.197) (Table 1). However, analysis of the association between clinical form and LT-alpha genotypes showed a higher frequency of genotype AG between subjects with the cardiac form (χ² , P=0.025) (Table 1). The OR observed for carriers of this genotype was 2.40 [95% confidence interval (CI): 1.12–5.16, P=0.025] upon univariate analysis (Table 1). Since allele G has been shown to be associated with high production of LT-alpha (Messer and others 1991; Ozaki and others 2002), genotypes GG and AG were analyzed together as presence of allele G. The frequency of the G allele was higher among subjects with the cardiac form (68.4%) compared to AA homozygotes (47.0%) (χ² , P=0.017) (Table 1), indicating a higher risk of developing the cardiac form of Chagas' disease among carriers of the G allele (OR=2.45, 95% CI: 1.17–5.12, P=0.017) (Table 1). This association between the presence of allele G and the cardiac form was still observed in multivariate analysis adjusted for gender and age (OR=2.27, 95% CI: 1.06–4.85, P=0.035).

Table 1.

Genotypes and Allele Frequencies Obtained for Healthy Subjects and Patients with Chagas' Disease

	Clinical form of Chagas' disease
Parameter	Cardiac	Indeterminate	Univariate P-value ^a	OR (95% CI) ^b	Multivariate P-value ^c	Adjusted OR (95% CI)
Gender
Female	59/106 (55.7%)	47/106 (44.3%)	—	1.00	—
Male	63/97 (64.9%)	34/97 (35.1%)	0.177	1.48 (0.84–2.60)	0.754/0.702
Age
<50 years	25/49 (51.0%)	24/49 (49.0%)	—	1.00	—
≥50 years	88/143 (61.5%)	55/143 (38.5%)	0.197	1.54 (0.80–2.95)	0.650/0.641
Genotype
AA	31/66 (47.0%)	35/66 (53.0%)	—	1.00	—
AG	34/60 (68.0%)	16/50 (32.0%)	0.025	2.40 (1.12–5.16)	0.057
GG	5/7 (71.4%)	2/7 (28.6%)	0.234	2.82 (0.51–15.60)	0.577
G allele
Absent	31/66 (47.0%)	35/66 (53.0%)	—	1.00	—	1.00
Present	39/57 (68.4%)	18/57 (31.6%)	0.017	2.45 (1.17–5.12)	0.035	2.27 (1.06–4.85)

Univariate P-value obtained by the chi-square test.

OR (95% CI): odds ratio with 95% confidence interval. In comparisons showing P-value<0.05, the OR represents the chance of develop cardiac form of Chagas' disease in a specified category in relation to the reference category (indicated by value 1.00).

Multivariate P-value obtained by logistic regression. Two models were tested, one including the genotype and another including allele G. Thus, the 2 values reported for gender and age correspond to the P-value obtained for each logistic regression model.

Table 2 shows the association between the parameters studied and the presence of congestive heart failure. No significant differences were observed in terms of gender (χ² , P=0.566) or genotype (χ² , P=0.288) (Table 2). Multivariate analysis showed that subjects older than 50 years were less likely to develop heart failure than those younger than 50 years (OR=0.05, 95% CI: 0.06–0.55, P=0.014). With respect to the LT-alpha gene polymorphism, subjects carrying allele G were at a higher risk of developing heart failure (OR=4.74, 95% CI: 1.05–21.45, P=0.043).

Table 2.

Genotypes and Allele Frequencies Obtained for Subjects with Congestive Heart Failure

	Congestive heart failure
Parameter	Present	Absent	Univariate P-value ^a	OR (95% CI) ^b	Multivariate P-value ^c	Adjusted OR (95% CI)
Gender
Female	18/26 (69.2%)	8/26 (30.8%)	—	1.00	—
Male	31/41 (75.6%)	10/41 (24.4%)	0.566	1.38 (0.46–4.12)	0.765
Age
<50 years	17/18 (94.4%)	1/18 (5.6%)	—	1.00	—	1.00
≥50 years	29/46 (63.0%)	17/46 (37.0%)	0.012	0.10 (0.01–0.82)^d	0.014	0.05 (0.06–0.55)
Genotype
AA	12/23 (52.2%)	11/23 (47.8%)	—	1.00	—
AG	13/19 (68.4%)	6/19 (31.6%)	0.288	1.99 (0.56–7.05)
GG	2/2 (100.0%)	0/2 (0.0%)	0.341	3.67 (0.15–90.42)
G allele
Absent	12/23 (52.2%)	11/23 (47.8%)	—	1.00	—	1.00
Present	15/21 (71.4%)	6/21 (28.6%)	0.19	2.29 (0.66–8.01)	0.043	4.74 (1.05–21.45)

Univariate P-value obtained by the chi-square test.

OR (95% CI): In comparisons showing P-value<0.05, the OR represents the chance of develop congestive heart failure (CHF) in specified category in relation to the reference category (indicated by value 1.00).

Multivariate P-value obtained by logistic regression. Only the model including allele G was tested because of the small number of subjects with CHF data.

To facilitate interpretation of the association between age and CHF, the inverse value of the OR was calculated. The results showed that subjects <50 years have a 9.97 (1.22–81.69) times higher chance of developing CHF than those ≥50 years.

Production of LT-alpha by PBMC

LT-alpha levels in 48-h culture supernatants were compared between genotypes of the LT-alpha polymorphism at position +252. No significant differences in cytokine production were observed in the absence of any stimulus (Fig. 1A). Seropositive subjects produced more LT-alpha than seronegative individuals after stimulation with the T. cruzi antigen (P<0.001) (Fig. 1A). Comparison of the clinical forms showed higher production of LT-alpha in patients with the cardiac form compared to those with the indeterminate form (P=0.012) (Fig. 1B). Seropositive subjects carrying allele G produced higher levels of LT-alpha in the absence of any stimulus than seropositive subjects without this allele (P=0.011) (Fig. 1C).

FIG. 1.

Lymphotoxin-alpha (LT-alpha) expression in chagasic patients. (A) LT-alpha levels in culture supernatants. LT-alpha was assayed by enzyme-linked immunosorbent assay in supernatants of peripheral blood mononuclear cells cultured for 48 h in the absence of any stimulus and in the presence of the Trypanosoma cruzi antigen. Comparison of subjects according to positive and negative serology (P<0.001, Mann–Whitney test). LT-alpha medium: seronegative (n=85)×seropositive subjects (n=27); LT-alpha T. cruzi antigen: seronegative (n=84)×seropositive subjects (n=26). (B) Comparison of subjects according to cardiac or indeterminate form (P=0.012, Mann–Whitney test). LT-alpha medium: cardiac (n=12)×indeterminate patients (n=11); LT-alpha T. cruzi antigen: cardiac (n=12)×indeterminate patients (n=11). (C) Comparison of subjects according to the presence or absence of allele G (P=0.011, Mann–Whitney test). LT-alpha medium seropositive: absence (n=9)×presence (n=7); LT-alpha T. cruzi seropositive: absence (n=9)×presence (n=7). Results are expressed as pg/mL. The horizontal line indicates the median, bars the 25% and 75% percentiles, and vertical lines the 10% and 90% percentiles. (D) Comparison of the percentage of LT-alpha-positive CD4⁺ and CD8⁺ T cells in the absence of any stimulus and after stimulation with T. cruzi antigen between patients with the indeterminate and cardiac form. CD4 medium: cardiac (n=6)×indeterminate (n=3); CD8 medium: cardiac (n=13)×indeterminate (n=7); CD4 T. cruzi: cardiac (n=6)×indeterminate (n=3); CD8 T. cruzi: cardiac (n=13)×indeterminate (n=7); cardiac medium: CD4×CD8 (n=6); cardiac T. cruzi: CD4×CD8 (n=6); cardiac CD4: medium×T. cruzi (n=6); cardiac CD8: medium×T. cruzi (n=13); indeterminate medium: CD4×CD8 (n=3); indeterminate T. cruzi: CD4×CD8 (n=3); indeterminate CD4: medium×T. cruzi (n=3); indeterminate CD8: medium×T. cruzi (n=7).

Expression of LT-alpha by T lymphocytes

In patients with the indeterminate form, comparison of the expression of LT-alpha between CD4⁺ and CD8⁺ T lymphocytes showed higher expression of this cytokine by CD8⁺T lymphocytes. This difference was observed in the absence of any stimulus (P=0.010) and after stimulation with the T. cruzi antigen (P=0.022) (Fig. 1D). In patients with the cardiac form, CD4⁺ T lymphocytes expressed higher LT-alpha levels in the absence of any stimulus than CD4⁺ T cells after stimulation with T. cruzi antigen (P=0.007) (Fig. 1D).

Discussion

LT-alpha was chosen as a candidate gene in the present study because of its importance for the organization and maintenance of inflammatory responses (Ware and others 1995), its role in the induction of adhesion molecules such as VCAM, ICAM, and E-selectin (Kratz and others 1996; Cuff and others 1998; Hjelmström and others 2000), and its chromosome location on chromosome 6, close to the human leukocyte antigen genes (Makhatadze 1998). However, only 2 studies have investigated the importance of LT-alpha in Chagas' disease. The first study on experimental Chagas' disease demonstrated the role of this cytokine in the direct control of T. cruzi proliferation (Krassner and others 1982). Another immunogenetic study showed an association between the LTA +80A/+252G haplotype of the LT-alpha gene and development of the cardiac form of the disease (Ramasawmy and others 2007). However, this is the first study suggesting an association between the presence of the polymorphism and development of the cardiac form, and therefore confirms the results reported by Ramasawmy and others (2007). In addition, this study identified the main T lymphocyte subtype expressing LT-alpha and demonstrated that infected patients carrying allele G produced significantly more LT-alpha. As in the study of Ramasawmy and others (2007), the sample size of the present study is an obvious limitation. However, the choice of the endemic area, the subject selection criteria, and the multivariate approach, including variables such as gender, age, and the presence of heart failure, are strengths of the present study.

In the present study, the presence of the polymorphism was only associated with development of the chronic cardiac form. Allele G has been reported to be significantly more frequent among patients with the chronic cardiac form than among asymptomatic patients (16% versus 8%) (Ramasawmy and others 2007), in agreement with our results (55.7% versus 34.0%). The fact that the polymorphism was associated with serology could be explained by the importance of LT-alpha in the direct control of T. cruzi proliferation (Krassner and others 1982). Also, other cytokines produced by macrophages that are related to the innate immune response, such as tumor necrosis factor alpha (TNF-α), might be important to help parasite control (Pissetti and others 2011). In contrast, LT-alpha, a cytokine mainly produced by T lymphocytes (Ware and others 1995) and involved in adaptive immunity, would contribute more to the development of lesions observed in the chronic form.

The functional effect of this polymorphism was evaluated by determining the production of LT-alpha by PBMC in 48- and 120-h cultures. A higher production of LT-alpha in the absence of any stimulus was observed in subjects with positive T. cruzi serology carrying allele G. This finding complies with the results reported by Messer and others (1991) and Ozaki and others (2002). Messer and others (1991) evaluated the production of LT-alpha by ELISA and observed significant differences on days 2, 3, and 4. Ozaki and others (2002), using a luciferase reporter gene assay, showed an increased transcriptional activity in patients with myocardial infarction carrying allele G. Despite its location inside an intron, the LT-alpha gene polymorphism seems to exert a functional effect. The binding activity of some nuclear factors such as AP-1, Jun, and c-Fos is increased in the presence of allele G (Hjelmström and others 2000; Ozaki and others 2002).

Patients with chronic chagasic cardiopathy present an exacerbated inflammatory response when compared to patients with the indeterminate form whose immune response is apparently more regulated (Higuchi and others 1987; Souza and others 2004; Dutra and others 2005). As a consequence, a functional effect of the polymorphism is observed in one condition, but not in the other. In addition, the expression of other cytokines may exert regulatory effects on LT-alpha expression. There are no studies investigating the association between the presence of the polymorphism and production of LT-alpha in Chagas' disease. A recent report showed an association between the homozygous genotype of allele A at position +80 of the LT-alpha gene and lower plasma TNF-α levels (Ramasawmy and others 2007).

Analysis of the expression of LT-alpha by CD4⁺ and CD8⁺ T lymphocytes showed that the cytokine is expressed mainly by CD8⁺ T lymphocytes in the absence of any stimulus and after stimulation with T. cruzi antigen. This finding suggests that CD8⁺ cells are the main T lymphocyte subtype producing LT-alpha. Moreover, the production of LT-alpha by CD4⁺ cells is downregulated in patients with cardiopathy after the addition of T. cruzi antigen, suggesting the presence of regulatory mechanisms in this group of patients. These findings, together with the observation of a higher frequency of CD8⁺ T lymphocytes in the inflammatory exudate of patients with chronic chagasic cardiopathy (Reis and others 1993; Higuchi and others 1997), support the hypothesis that LT-alpha may be involved in the pathogenesis of heart lesions. Furthermore, LT-alpha and inflammatory infiltration predominantly consisting of CD8⁺ T lymphocytes may contribute to the loss of myocardiocytes by apoptosis, a phenomenon observed in patients with severe cardiac involvement in Chagas' disease (Tostes and others 2005).

Conclusion

The present results suggest that LT-alpha may play a role in the pathogenesis of chronic chagasic cardiopathy, contributing to the development of this chronic form. These findings may be useful for future identification of potential therapeutic targets and genetic risk factors.

Footnotes

Acknowledgments

This project was supported by CNPq, CAPES, FAPEMIG, and Universidade Federal do Triângulo Mineiro.

Author Disclosure Statement

No competing financial interests exist.

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