International Journal of Infectious Diseases (2009) 13, 493—498
http://intl.elsevierhealth.com/journals/ijid
Influence of TNF and IL10 gene polymorphisms in the immunopathogenesis of leprosy in the south of Brazil Danilo Santana Alessio Franceschi a, Priscila Saamara Mazini a, ˜o Chagas Rudnick a, Ana Maria Sell a, Cristiane Conceic¸a ´cia Ribas b, Paulo Roberto Peixoto b, Luiza Tamie Tsuneto a, Maria Lu Jeane Eliete Laguila Visentainer a,* a
´rio de Imunogene ´tica, Departamento de Ana ´lises Clı´nicas, Universidade Estadual de Maringa ´, Laborato ´, PR, CEP 87020-900, Brazil Av. Colombo, 5790, Maringa b ´rio da Sau ´, Parana ´, Brazil Centro de Especialidades do Ministe ´de, Maringa Received 14 May 2008; received in revised form 15 August 2008; accepted 18 August 2008 Corresponding Editor: Sunit K. Singh, Hyderabad, India
KEYWORDS Leprosy; Cytokine polymorphisms; SNPs; TNF308
Summary Objective: To determine whether cytokine polymorphisms are associated with leprosy and/or their subtypes in a Brazilian population. Methods: Genotyping using polymerase chain reaction with sequence-specific primers (PCR-SSP) was performed for: TNF308/238, IL2330/+166, IL6174, IFNG+874, TGFB1+869/+915, and IL10592/ 819/1082 in 240 healthy controls and 167 patients with leprosy. Results: For TNF308, a higher frequency of GG genotype (85.5% vs. 74.1% in healthy controls, p = 0.009), along with a decreased frequency of GA/AA genotypes was observed among leprosy patients as compared to the control group (14.5% vs. 25.9%, p = 0.009). The GG genotype was particularly higher in patients with tuberculoid (TT) and borderline (BB) leprosy (90.5% and 89.8%, respectively). Analysis of IL10 genotypes revealed a lower frequency of GCC/GCC haplotype in lepromatous leprosy (LL) patients (6.2%) in comparison to controls (15.4%). Conclusion: It is suggested that the G!A substitution at position 308 in the TNF promoter region plays an important role in leprosy patients. # 2008 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Introduction * Corresponding author. Tel.: +55 44 3261 4864; fax: +55 44 3261 4931. E-mail addresses:
[email protected],
[email protected] (J.E.L. Visentainer).
Leprosy, caused by the obligate intracellular microorganism Mycobacterium leprae, is a chronic infectious disease still considered a major health problem in some countries of Africa, Asia, and Latin America. According to the World
1201-9712/$36.00 # 2008 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijid.2008.08.019
494 Health Organization, approximately 260 000 new patients were affected in 2006.1 The disease causes skin lesions and neuropathy. Secondary complications of the neuropathy can result in deformity and disability. Damage development in leprosy individuals is normally caused by host immune responses driven to mycobacterial elimination.2 The Ridley—Jopling classification of leprosy is based on clinical and histopathological criteria, which suggest a disease spectrum with five clinical categories: tuberculoid (TT), borderline tuberculoid (BT), borderline borderline (BB), borderline lepromatous (BL), and lepromatous (LL) leprosy.3 At one pole, TT leprosy consists in few well-defined skin patches, few bacilli (paucibacillary), and vigorous cellmediated immunity (CMI). At the other pole, LL leprosy presents many skin lesions with uncontrolled proliferation of leprosy bacilli (multibacillary) and inefficient CMI.4 Borderline leprosy (BB) manifests clinical and immunological features with characteristics between the two forms.5 There is high Th1 cytokine production in skin lesions of paucibacillary patients (TT), like interferon-g (IFN-g), interleukin (IL)-2, IL-7, IL-12, IL-15, and IL-18, characteristic of intense CMI. Infected macrophages are activated by IFN-g, and IL-2 induces clonal expansion of activated Tcells, leading to an enhanced production of IFN-g,6 also seen in natural killer (NK) cells stimulated by IL-12.7 Autocrinous macrophage tumor necrosis factor-a (TNF-a) production acts synergically working for Th1 pattern maintenance.8 IFN-g, in synergy with TNF-a, activates infected macrophages, thereby initiating a major effector mechanism of CMI. Multibacillary patients (LL and BB) present a Th2 cytokine pattern (transforming growth factor-b1 (TGF-b1), IL-4, IL-5, and IL-10) in local skin lesions, indicating elevated antibody production, but inefficient CMI.9 An immunosuppressive effect conferred by IL-4 can lead to mycobacterial proliferation, inhibiting monocyte and T cell activation,10 and chronic local IL-10 release differentiates CD4+ Tcells in a regulatory T cell subpopulation, acting in antigen-specific immunosuppression.11 TGF-b, an activated monocyte product, inhibits IFN-g and IL-2 expression, permitting disease progression by the lack of macrophage cytotoxicity.12 Single nucleotide polymorphisms (SNPs) are considered the most abundant source of genomic variation in humans, and differences in coded protein structure can be caused when these are present in genes.13 Some SNPs have been described that may be important factors in leprosy susceptibility and outcome.14—22 The aim of this study was to investigate if some cytokine SNPs are involved in the pathogenesis of leprosy and its subtypes.
Methods Patients and controls A total of 167 leprosy patients (96 males, 71 females), of mixed ethnicity, undergoing treatment at Centro de Espe´ nico de Sau cialidades do Sistema U ´de (SUS) in the cities of Maringa ´, Sarandi, and Umuarama, state of Parana ´, Brazil, were enrolled in this study between 2005 and 2007. Approval for human study protocols was obtained from the human subject review boards at the Universidade Estadual de Maringa ´ in southern Brazil.
D.S.A. Franceschi et al. Patients were classified into four distinct groups according to clinical and laboratory observations for leprosy diagnosis. Of the total patients, 43 (25.7%) were diagnosed to have TT leprosy, 65 (38.9%) LL leprosy, and 50 (29.9%) BB leprosy; nine (5.4%) were diagnosed with an indeterminate form of the disease. Two hundred forty individuals (133 males, 107 females) without any history of mycobacterial infection and belonging to the same geographical region as the patients were included as healthy controls. These individuals were of mixed ethnic background. The mean age of patients was 46.0 14.8 years and of controls was 35.6 10.7 years. The population of Brazil is made up of genetically heterogeneous groups, formed mainly by European explorers, settlers, and immigrants, mixed with native Amerindians and African slaves.23 The sample was considered to be of mixed ethnic groups because, according to Parra et al. (2003),24 in Brazil, at an individual level, skin color determined by physical evaluation is a poor predictor of genomic African ancestry.
Genotyping of SNPs for cytokine genes Genomic DNA extraction was performed by EZ-DNA kit (Biological Industries, Beit Haemek, Israel) or Neoscience kit (One Lambda Inc., San Diego, CA, USA) using 150 ml of buffy-coat. DNA samples were stored in a freezer at 20 8C until use. After DNA concentration adjustment (25—200 ng/ml), sequence-specific primer PCR (PCR-SSP; One Lambda Cytokine Genotyping Primer Pack, One Lambda, CA, USA) was performed for genotyping of the following SNPs: TNF 308 (G!A), IL6174 (G!C), IFNG+874 (T!A), TGFB1+869 (T!C) and TGFB1+915 (G!C), and IL10592 (G!C), IL10819 (C!T), and IL101082 (G!A) according to manufacturer’s instructions. Genotyping of TNF 238 (G!A), IL2330 (T!G), and IL2+166 (G!T) was performed using a kit developed in house.25 Amplified product was transferred in 2.5% agarose gel and electrophoresed at 150 V for 9 min approximately.
Statistical analysis Allele frequencies were obtained through direct counting. Chi-square tests with Yate’s correction or Fisher’s exact tests were carried out to analyze disease association using GraphPad Software (San Diego, CA, USA; http://www.graphpad.com/quickcalcs/contingency1.cfm). Odds ratios (OR) with 95% confidence intervals (95% CI) were calculated using SISA statistics online (available at: http://www.quantitativeskills.com/sisa/). A p-value of 0.05 was considered to be statistically significant. Hardy—Weinberg equilibrium was tested for each SNP using Arlequin software version 3.1 (http://cmpg.unibe.ch/software/arlequin3/). Finally, the observed p-values were corrected by means of the Bonferroni correction to enable multiple comparisons. Since six independent regions were investigated (chromosome 1 IL10 region, chromosome 4 IL2 region, chromosome 6 TNF region, chromosome 7 IL6 region, chromosome 12 IFNG region, and chromosome 19 TGFB1 region), the corrected pvalue was six times the observed p-value.
Cytokine gene polymorphisms and leprosy, Brazil Table 1
495
Cytokine genotypes and frequencies in leprosy patients and controls in a population from the south of Brazil.
SNP
Genotype
Leprosy n (%)
Controls n (%)
p-Value
G/G G/A or A/A
N = 167 147 (88.0) 20 (12.0)
N = 219 192 (87.7) 27 (12.3)
0.96
T/T T/A A/A
N = 164 28 (17.1) 80 (48.8) 56 (34.1)
N = 240 33 (13.8) 131 (54.6) 76 (31.6)
C/C G/G or G/C
N = 164 18 (11.0) 146 (89.0)
N = 240 21 (8.8) 219 (91.3)
0.57
T/T T/G or G/G
N = 167 93 (55.7) 74 (44.3)
N = 219 108 (49.3) 111 (50.7)
0.25
G/G G/T or T/T
N = 167 77 (46.1) 90 (53.9)
N = 219 113 (51.6) 106 (48.4)
0.33
ACC/ACC or ACC/ATA or ATA/ATA GCC/ACC or GCC/ATA GCC/GCC
N = 165 78 (47.3) 67 (40.6) 20 (12.1)
N = 240 95 (39.6) 108 (45) 37 (15.4)
0.43 0.44 0.15
CG/CC or CC/CC or TC/TC or TC/CC TG/CC or CG/CG or TG/TC TG/TG or TG/CG
N = 164 13 (7.9) 37 (22.6) 114 (69.5)
N = 238 12 (5.0) 58 (24.4) 168 (70.6)
0.33 0.53 0.90
TNF238
IFNG+874
IL6174
IL2330
IL2+166
IL101082/819/592
TGFB1+869/+915
0.44 0.29 0.68
SNP, single nucleotide polymorphism.
Results Association between TNF308 GA/AA genotypes and leprosy The cytokine genotypes and frequencies in the leprosy patients and control group, sampled in the south of Brazil, are shown in Table 1. The genotype frequency distribution was in Hardy—Weinberg equilibrium. None of the cytokine genes showed a significant difference between leprosy patients and controls except for the TNF 308 polymorphism (Table 2). A significantly higher frequency of GG genotype was observed in leprosy patients per se (85.5%) compared to healthy controls (74.1%) ( p = 0.009, OR = 2.06, 95% CI 1.22—3.46). Similarly, a decreased frequency of TNF 308
GA/AA genotypes was observed among patients (14.5%) in comparison to the control group (25.9%) ( p = 0.009, OR = 0.48, 95% CI 0.29—0.82). After Bonferroni correction, these results remained statistically significant ( p = 0.05). Therefore, the allelic frequency of TNF 308A was significantly higher in the control group (13.4%) than in the leprosy patients (7.9%) ( p = 0.02, OR = 0.55, 95% CI 0.34—0.89), although this difference was not significant after correction. No significant differences were found in TNF 308 GG genotype frequencies among the leprosy subgroups (Table 3). The analysis revealed that TT, BB, and LL patients presented high frequencies of TNF 308 GG genotype (90.5%, 89.8%, and 81.5%, respectively) and decreased GA/AA frequencies (9.5% TT patients and 10.2% BB patients) in comparison to healthy individuals (74.1% GG and 25.9% GA/AA). However, the p-value for LL patients did not reach signifi-
Table 2 Genotype and allelic frequencies of TNF308 single nucleotide polymorphism in leprosy patients and control individuals in a population from the south of Brazil. TNF308
Leprosy N = 165
Controls N = 239
p-Value
OR
95% CI
Corrected p-value
G/G G/A or A/A G A
141 24 304 26
177 62 414 64
0.009 0.009 0.02 0.02
2.06 0.48 1.81 0.55
1.22—3.46 0.29—0.82 1.12—2.92 0.34—0.89
0.05 0.05 0.12 0.12
(85.5) (14.5) (92.1) (7.9)
(74.1) (25.9) (86.6) (13.4)
Results are n (%). OR, odds ratio; 95% CI, 95% confidence interval.
496 Table 3
D.S.A. Franceschi et al. TNF and IL10 gene frequencies for subgroups of leprosy and controls in a population from the south of Brazil.
SNP
LL
TT
BB
Controls
TNF308 GG GA or AA
N = 65 53 (81.5) 12 (18.5)
N = 42 38 (90.5) a 4 (9.5) b
N = 49 44 (89.8) a 5 (10.2) b
N = 239 177 (74.1) a 62 (25.9) b
IL101082/819/592 ACC/ACC or ACC/ATA or ATA/ATA GCC/ACC or GCC/ATA GCC/GCC
N = 65 33 (50.8) 28 (43.0) 4 (6.2) c
N = 42 15 (35.7) 21 (50) 6 (14.3)
N = 49 26 (53.1) 16 (32.6) 7 (14.3)
N = 240 95 (39.6) 108 (45) 37 (15.4) c
Results are n (%). SNP, single nucleotide polymorphism; LL, lepromatous leprosy, TT, tuberculoid leprosy; BB, borderline leprosy. a TT vs. controls, p-value = 0.009, OR = 3.33 (1.14—9.70), corrected p-value = 0.05; BB vs. controls, p-value = 0.03, OR = 3.08 (1.17—8.12), corrected p-value = 0.17. b TT vs. controls, p-value = 0.009, OR = 0.30 (0.10—0.88), corrected p-value = 0.05; BB vs. controls, p-value = 0.03 OR = 0.32 (0.12—0.86), corrected p-value = 0.17. c LL vs. controls, p-value = 0.02, corrected p-value = 0.12.
cance, and only the TT subgroup maintained a significant difference after Bonferroni correction (corrected pvalue = 0.05).
Association between IL10 genotypes and leprosy Analysis of IL10 genotypes did not show any significant differences between leprosy patients and controls. When each subgroup was considered separately (Table 3), a lower frequency of IL101082G/819C/592C haplotype was found in LL patients (6.2%) in comparison to controls (15.4%) ( p = 0.02). However, after accounting for multiple testing this result was no longer significant (corrected p-value = 0.12).
Association between other SNPs and leprosy No association was found between any other SNP and the leprosy subgroups. No significant differences were found in the comparison of genotypes according to gender (data not shown).
Discussion The present study shows a positive association between the TNF 308 GG genotype and leprosy per se, and a negative association with TNF 308 GA/AA. Previous studies have shown associations between susceptibility to leprosy or leprosy subtypes and polymorphisms in cytokine gene regions. An early report conducted by Shaw et al.15 in a Brazilian cohort suggested that TNF2/LTA2 haplotypes confer a protective effect against leprosy. Also in Brazilian patients, Santos et al.16 found that the presence of TNF2 had a protective effect in leprosy patients per se compared to a control group. However, contradictory results have been found in other studies. Roy et al.,14 studying Indian patients, found that the frequency of TNF2 was higher in lepromatous patients than in healthy individuals. This result was similar to the observations of Vejbaesya et al.22 in leprosy patients from Thailand, which showed this SNP to be involved in susceptibility to the multibacillary form of the infection. These contradictory findings are mainly due to ethnic differences. Differences in the allelic constitutions of these populations may be strongly involved in the direction of the
association. With regard to allelic frequencies in control subjects, it can be seen that the presence of the TNF 308A allele is lower in Asian studies (2.8% and 4.6%) while being constitutively elevated in the Brazilian control groups of the present study (13.4%) and the Santos study (16.3%). Such constitutive differences in frequencies may be able to explain why Brazilian studies showed negative associations of the A allele to leprosy and Asian studies observed positive associations. With regard to the issue of TNF-a production being affected by these polymorphisms, it is unclear at the moment if the TNF G!A substitution in serum levels of this cytokine plays a role. Some studies have failed to associate the presence of the A allele with different levels of TNF-a production, while other attempts have found that a G!A substitution at the 308 position is a much stronger transcriptional activator than the common GG allele, which is associated with lower production (reviewed by Bayley et al.).26 Moraes et al.,27 in a study on BT leprosy patients using the Mitsuda test, found that TNF 308 G!A patients showed greater induration sizes compared to TNF 308 GG patients, when neither group showed reverse reactions and had not been exposed to the BCG vaccine, suggesting higher levels of TNF-a in BT patients with G!A substitution. In addition, Vanderborght et al. (2007)28 found that patients with the presence of the A allele in the 308 TNF promoter region exhibited lower bacteriological indices. One hypothesis is that this direct effect on gene regulation probably results in dissemination of bacilli by weak macrophage activation, which is dependent on TNF-a, resulting in susceptibility to M. leprae infection. Elevated frequencies of the GG genotype could be a reflex of diminished GA/AA frequencies or could be related to a lower TNF-a release potential. This present study did not find an association of 238 G!A substitution with leprosy per se or subgroups, found in other studies.16,22 The exact influence of the TNF 238 G/A variant on the TNF-a function and expression is not yet fully understood, although the localization of this polymorphism is in the regulatory region.29,30 The issue is further complicated because these polymorphisms may be in linkage disequilibrium with themselves and as a part of extended haplotypes, including LTA and classes I and II HLA alleles, found in the same chromosome 6.15
Cytokine gene polymorphisms and leprosy, Brazil IL10 SNPs in the distal and the proximal regions are arranged to form haplotypes in the promoter gene, and these combinations have been associated with secretion of the cytokine in vitro.31—33 In this study, the occurrence of the IL101082G/819C/592C haplotype was not associated to an outcome of leprosy. Although lepromatous patients showed lower frequencies of this haplotype compared to control individuals, the statistical significance of this negative association was lost after correction for multiple comparisons. In Indian patients, the presence of the 3575T/2849G/ 2763C/1082A/819C/592C haplotype conferred resistance to leprosy per se and the 3575T/2849G/2763C/ 1082A/819T/592A haplotype was associated to development of the severe form of leprosy.19 In another Brazilian study, IL10819 TT was increased in leprosy patients per se as compared to controls,16 which could not be confirmed in this research. According to Moraes et al. (2004),18 the extended haplotype 3575T/2849A/2763C/1082G/819T is associated with susceptibility, although the three distal SNPs were sufficient to capture the information.18 There is probably a need to verify the interaction of more extended IL10 SNP haplotypes with the disease, and a search for new genes and SNPs that have interplay with IL10 SNPs. Although IL-2, IFN-g, IL-6, and TGF-b1 cytokines present proinflammatory and anti-inflammatory characteristics in leprosy immunopathogenesis,12,34 analysis of polymorphisms that were previously associated with production of these cytokines did not show differences, even in subgroups. In this study, however, the effect of these polymorphisms on cytokine production in patients and control subjects was not investigated. In summary, the present study provides consistent evidence for the involvement of TNF genes in susceptibility to leprosy. The G!A substitution at position 308 in the TNF promoter region may be involved in different CMI in leprosy patients due to its influence on the production of TNF-a. However, establishment of the infection and disease development may be conditioned by other immunological and genetic factors. Therefore, further studies of other polymorphic immunoregulatory genes are needed to understand the immune mechanism of susceptibility or resistance to leprosy.
Acknowledgments The authors would like to thank the individuals who took part in this study, Dr Marcelo Mira and Dr Narinder Mehra for revision of the manuscript, and Maria Lu ´cia Ribas and Maria da Silva for their cooperation in collecting patient data. This study was partly supported by CNPq (Ministry of Science and Technology), Fundac¸a ˜o Arauca ´ria (Government of Parana ´), and the Brazilian Ministry of Healthy. The manuscript was linguistically revised by Peter Grimshaw. Conflict of interest: No conflict of interest to declare.
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