Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 29, Num. 1, 2011, pp. 28-32

Indian Journal of Medical Microbiology, Vol. 29, No. 1, January-March, 2011, pp. 28-32

Original Article

Human leucocyte antigens and cytokine gene polymorphisms and tuberculosis

A Akgunes, AY Coban, B Durupinar

Ondokuz Mayis University, Medical School, Department Medical Microbiology, 55139, Samsun, Turkey

Correspondence Address: B Durupinar, Ondokuz Mayis University, Medical School, Department Medical Microbiology, 55139, Samsun, Turkey, belma.durupinar@att.net

Date of Submission: 21-May-2009
Date of Acceptance: 27-Nov-2010

Code Number: mb11007

PMID: 21304191

DOI: 10.4103/0255-0857.76520

Abstract

Keywords: Tuberculosis susceptibility, host genetics, HLA antigen, cytokine, polymorphism

Introduction

Tuberculosis (TB) continues to be a global health problem. In the recent decades, the number of reported cases has increased in both industrialized and developing countries. ^[1] Host factors, especially host genes, will influence susceptibility and severity to infection and the health outcome for the patient. ^[2] Therefore, the identification of host genes responsible for susceptibility and resistance to TB should provide a significant contribution for understanding the pathogenesis and may lead to the development of new prophylaxis and treatment strategies.

One important element of resistance and susceptibility to infections is the major histocompatibility complex (MHC), known as human leucocyte antigens (HLA) in humans. The highly polymorphic nature of the HLA allows it to bind a repertoire of peptides that are specific for each HLA molecule, thereby influencing T cell polarization and, hence, the profile of the cytokines secreted. ^[3] In humans, a significant difference in cytokine secretion profiles has been observed in peripheral blood mononuclear cells from HLA-B8 and -DR3-positive individuals. ^[4] Our earlier studies have shown that HLA-B7 and -Cw1 antigens can influence the immune responses to TB. ^[5] The immune mechanisms behind HLA-DR-associated susceptibility to TB remain unclear.

Cytokines, their genes and receptors have been implicated in the protective immunity, pathophysiology and development of TB. ^[6] Among these cytokines, tumour necrosis factor-α (TNF-α) has multiple roles in defence and pathological response to TB. ^[7] TNF-α can act synergistically with interferon-gamma (IFN-γ) to activate macrophages^[8] and, thus, impact on disease and resistance to TB. ^[9] Interleukin-10 (IL-10) is an important anti-inflammatory cytokine and is considered a macrophage-deactivating cytokine. IL-10 downregulates IFN-γ production ^[10] and the Th1-induced response to TB. ^[9]

Polymorphisms affecting antigen processing and presentation, and hence the profile of cytokines secreted, can influence the efficiency of the immune response to infection and can play a significant role in the host response. ^[11] However, how polymorphisms in HLA, cytokines and other genes interact to ultimately determine genetic susceptibility to TB remains an open question.

In this context, our objective was to investigate whether these genes might be associated with protection from or susceptibility to TB.

Materials and Methods

Study populations

The study group consisted of 30 pulmonary TB and 30 healthy subjects who were randomly selected. Diagnosis of the patients was based on the findings of acid-fast bacilli on microscopic examination of a specimen, such as an expectorated sputum smear. Chest X-ray (CXR) was also used as a definitive diagnostic procedure when the classic picture of TB-like, upper lobe infiltrations and cavities was present. A control group was composed of 30 healthy organ donors, matched for age and sex and ethnicity and from the same geographical area as the patients. The study protocol was approved by the ethics committee and written informed consent was obtained from the study participants.

Isolation of genomic DNA

Genomic DNA was extracted from blood samples by using a Puregene Genomic DNA isolation kit (Gentra Systems, Minneapolis, MN, USA) according to the manufacturer′ instructions.

HLA genotyping

HLA genotyping was performed for a total of 32 HLA DRB and DQB alleles using polymerase chain reaction (PCR) amplification with DRB and DQB low-resolution typing by the PCR-sequence-specific primer method. The PCR reaction mixtures contain 50 ng/μl genomic DNA, 10x PCR buffer, dNTP, taq polymerase (Promega, Fitchburg, Wisconsin, USA) and specific primers. PCR amplifications were carried out in a Gene Amp PCR system 9700 (Perkin-Elmer, Norwalk CT, USA). PCR mixtures were loaded in a 3-mm-wide slot in 2% agarose gels. Gels were examined under UV illumination and the results were documented by photography.

Cytokine genotyping

All genotypes were determined by using a PCR-sequence-specific oligonucletid method by a commercially available kit (Tepnel Lifecodes, Stamford, USA; Lot 03067F) in accordance with the manufacturer′s instructions. This kit contains specific probes to detect the polymorphisms of several cytokines, cytokine receptors and receptor antagonists mentioned above. The allele and genotype frequencies of the following cytokine genes were determined: IL-1α (C/T,C/C,T/T -889), IL-1β (C/T, C/C, T/T +3962), IL-12B (A/C, A/A, C/C -1188), IL-10 (A/A, A/G, G/G -1082, A/C, A/A, C/C -592, C/T, C/C, T/T -819), IL-6 (C/G, C/C, G/G -174), IL-1R 1970 (C/T, C/C, T/T), IL-1Ra 11100 (C/T, C/C, T/T), IL-1β (C/T, C/C, T/T -511, C/T, C/C, T/T +3962), TGF-β (C/T, C/C, T/T +869, C/G, G/G +915), TNF-α (A/G, G/G -238, A/G, G/G -308) and IFN-γ (A/T, A/A, T/T +874).

Statistical analysis

Allele and genotype frequencies of individual polymorphisms were estimated by direct counting. Frequencies of alleles and genotypes were compared between patients and controls by the Chi-square or Fisher′s exact test. In this study, our hypothesis was that the polymorphisms in HLA and cytokine genes might be associated with TB, based on preliminary work. The level of significance was set at 0.05; the P-values were corrected for multiple testing (pc), applying a correction factor (i.e., the total number of HLA and cytokine alleles defined) for new associations, and corrections for multiple comparisons were not necessary for the allele HLA-CwFNx0101, which we have previously identified as being increased in TB patients (13). Odds ratios (OR) with 95% confidence intervals were calculated using the Statcalc program (Epi Info version 6.0.4. CDC, Atlanta, GA, USA; July1996).

Results

A comparison of class I and class II allele distribution at the phenotypic level was carried out between TB patients and controls. [Table - 1] lists the allele frequencies that were significantly different between the two groups.

A comparison of TB patients with controls revealed a strong association with TB. HLA-CwFNx0101 [P = 0.05, OR (95% confidence interval) = 2.269 (1.702-3.027)] and HLA-CwFNx0112 [P = 0.05, OR (95% confidence interval) = 0.452 (0.269-1.677)] alleles were overrepresented in the TB group than in the controls [Table - 1]. Statistically significant differences for the HLA-CwFNx0112 allele, however, were lost after the Bonferroni correction of the P-values.

We also evaluated the frequencies of cytokine gene polymorphisms in TB patients and in healthy control subjects.

The frequency of IL-1α -889, IL-1β +3962, IL-12B -1188, IL-6 -174, IL-1R 1970, IL-1Ra 11100, IL-1β -511 and +3962, TGF-β +869 and +915, TNF-α -238 and -308 and IFN-γ +874 alleles did not show any significant difference between the TB patients and the healthy controls. However, the frequency of IL-10, -819 C and 592 C alleles varied significantly between the two groups studied (pc = 0.038 and 0.0172, respectively). This difference may be explained by a higher frequency of allele C in patients with TB compared with controls, in which the allele C had a lower frequency (0.80 vs. 0.57). Statistically significant differences were still maintained after the Bonferroni correction of the P-values (pc) [Table - 2].

Genotype distributions for all the cytokine genes studied were in equilibrium according to the Hardy-Weinberg distribution in TB patients and controls. The frequency of IL-1α (C/T,C/C,T/T -889), IL-1β (C/T, C/C, T/T +3962), IL-12B (A/C, A/A, C/C -1188), IL-6 (C/G, C/C, G/G -174), IL-1R 1970 (C/T, C/C, T/T), IL-1Ra 11100 (C/T, C/C, T/T), IL-1β (C/T, C/C, T/T -511, C/T, C/C, T/T +3962), TGF-β (C/T, C/C, T/T +869, C/G, G/G +915), TNF-α (A/G, G/G -238, A/G, G/G -308) and IFN-gamma (A/T, A/A, T/T +874) genotypes did not show any significant difference between the TB patients and the healthy controls (data not shown).

From the IL-10 cluster, a positive significant difference was found at position -1082, where the G/G genotype was overrepresented in TB patients (20%, pc = 0.027); also, the C/C genotype at the IL-10 position -592 was significantly overrepresented in the TB patients (63% vs. 35%, pc = 0.054). Although these differences could be explained by the highest frequency of C/C and G/G homozygous patients with TB, in contrast to the control group, statistically significant differences for the C/C genotype, however, were lost after a Bonferroni correction of the P-values (pc) [Table - 2].

It is noteworthy that the two IL-10 polymorphisms (-819 and -592) were in linkage disequilibrium in the TB and control groups. No differences were observed regarding the distribution of IL-10 haplotypes between controls and patients (data not shown).

Discussion

Host genes may affect every stage of infectious disease and responsiveness to antimicrobial agents or vaccines. Polymorphisms located within some regions of the host genes (e.g., HLA, cytokine genes), mostly single nucleotide polymorphisms (SNPs) or microsatellites, were described to affect gene transcription, causing inter-individual variations such as clinical outcome or cytokine production. For instance, the highly polymorphic nature of HLA antigens allows them to bind a repertoire of peptides that are specific for each HLA molecule, thereby influencing T cell polarization and, hence, the profile of cytokines secreted. The HLA/peptide complexes generated in response to infection determine the patterns of cytokines secreted and also influence the outcome of immune response. ^[3],[6],[7]

SNPs in several candidate genes have been linked to a relatively increased risk for TB. ^[6],[12] In this polymorphism-association study, we investigated the significance of the relationship between HLA and several cytokine gene polymorphisms and susceptibility to TB.

In the present study, we determined HLA class I and class II alleles in TB patients and compared the frequencies of HLA alleles between the TB patients and the healthy subjects. Consequently, we found that the HLA class I allele CwFNx0101 was significantly highly frequent in TB patients than in controls, suggesting that HLA-CwFNx0101 (P = 0.046, OR = 2.269, 95% confidence interval = 1.702-3.027) may be associated with susceptibility to TB. In our study, although HLA-BFNx0140 and -CwFNx0112 alleles were less frequent in TB patients than in controls, we did not find any significant correlation between the two groups. There were no statistically significant differences between the class II alleles. Discrepancies in candidate alleles between studies may be explained by co-segregation, sample size (most studies used low-sample size with low-statistical power) and the clinical features of the study participants (some studies used general TB patients, whereas some others used the cases with various clinical subsets). Alternatively, many earlier studies have focused on HLA class II, whereas only a few studies have investigated both HLA class I and class II. Also, indirect serological tests may fail to distinguish between closely related alleles. Furthermore, the nomenclature of MHC alleles is not uniform among the studies. ^[13]

Studies using population-based designs with candidate genes have implicated genes responsible for immunological pathways, such as IFN-γ, IL-10 or TNF. IL-10 is known to have macrophage-deactivating properties, and undermines the Th1 response. ^{[9],[14],[15]} In the present study, three SNPs (-1082, -819, -592) in the promoter of the IL-10 gene were investigated and statistically significant associations between individual cytokine SNPs and TB were found. There was an association of IL-10 -1082 G/G genotype with TB. This may be explained by a higher frequency of G/G homozygous at -1082. Although analysis of allele frequencies of IL-10 polymorphisms at position -1082 showed no association with susceptibility or resistance to TB, there was an association of the IL-10 -1082 G/G genotype with TB. G/G genotype may be associated with susceptibility to TB. IL-10 -819 and -592 C allele are also suggested to be associated with TB. IL-10 seems to influence the susceptibility of the host to TB infection, and, determining the polymorphisms in IL-10 genes may be beneficial for predicting the disease susceptibility.

Several case-control studies examined the association of IL-10 promoter polymorphisms in TB susceptibility and severity, where discrepancies were observed among these studies. In some studies, IL-10 -1082 allele and heterozygosity for the IL-10 -1082 polymorphism were associated with TB susceptibility, ^[6] while in other studies with large sample sizes, IL-10 1082 A/G polymorphism were not associated with TB. ^[16] One study showed that the IL-10 -1082 G allele frequency was found to be significantly more common in TB patients than in healthy controls. ^[17] . For IL-10, a previous report has shown that there was no difference in the IL-10 genotype frequencies in Spanish TB patients and that the IL-10 G/A genotype was more frequent in TB susceptibility in Cambodian and Sicilian patients. ^[17],[18] Association of the IL-10 gene promoter SNPs with TB is controversial, suggesting the possibility of ethnic-specific genetic variations and susceptibility to TB, and may involve more distal promoter elements of the IL-10 gene. ^{[6],[17],[18],[19]}

Genetic studies of infectious diseases are difficult to conduct owing to the multifactorial nature of the diseases that include host, pathogen and environmental variables in different proportions for each disease and for every subject studied. ^[20] Based on the accumulated knowledge on the role of cytokines and their receptors in the immune response and immunopathogenesis of TB, these molecules are good candidates to explain the development of different clinical manifestations of the infection. However, the multiple interrelations between cytokines and their receptors and other genes (e.g., HLA, chemokines) make the results of such studies difficult to interpret, and may require a very high number of subjects to be studied.

Altogether, our results suggest that the polymorphisms in HLA (class I) and cytokine (IL-10) genes may affect susceptibility to TB and increase the risk of developing the disease. These polymorphisms may be one of the many genetic factors affecting disease outcome. HLA and other closely linked cytokine genes might be crucial for protective immune response and may serve as biomarkers of protection or susceptibility to tuberculosis. To better understand and confirm the biological significance of these results, further studies with larger sample sizes are necessary to clearly elucidate the discrepancies.

References

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