Interferon gamma (IFN-γ) and interleukin 10 (IL-10) are believed to play opposing roles in host immunity against mycobacterial infection. IFN-γ activates macrophages, while IL-10 downregulates the expression of T helper type 1 cytokines, MHC class II antigens and costimulatory molecules on macrophages. Associations of IFN-γ −179 (G/T), +874 (A/T), +875 miscrosatellite CA repeats and +4766 (C/T), and IL-10 −1082 (A/G), −819 (C/T) and −592 (C/A) with tuberculosis (TB) were investigated in 385 HIV-negative patients and 451 controls in a Hong Kong Chinese population. The frequency of a low IFN-γ-producing +874 A/A genotype was significantly over-represented in the patient group (P<0.001, OR=3.79, 95% CI=1.93–7.45). We identified 10 alleles in the IFN-γ CA repeats and observed a significant difference in allele frequency distribution between patients and controls (P<0.001). By grouping alleles into 12 and non-12 CA repeats, the non-12/non-12 genotype yielded a similar significant result (P<0.001, OR=4.56, 95% CI=2.21–9.43) as observed in +874 A/A genotype. Weak associations of the IL-10 GCC/− genotype (P=0.04) and the low IFN-γ-producing A/A genotype (P=0.06) with TB relapse/extrapulmonary cases were found. This study suggests the possible role of interferon gamma in TB susceptibility.
Tuberculosis (TB) has been declared as a major global health threat by the World Health Organization (WHO) since 1993. According to WHO, approximately 32% of the world's population is infected with Mycobacterium tuberculosis (M. tuberculosis) and 5000 persons succumb to TB everyday. In Hong Kong, TB is still prevalent with about 7000 cases every year. As only 10% of the population infected by M. tuberculosis will develop clinical TB, differences in host immunity and genetic factors may account for the development of TB after infection. Host genetic susceptibility to mycobacterial infection in certain racial groups and familial cases has been reviewed.1
Interferon gamma (IFN-γ) is a key T helper (Th) type 1 cytokine produced primarily by natural killer cells and T cells. Its production plays a pivotal role in macrophage activation for controlling M. tuberculosis infection.2 Mice with a disrupted IFN-γ gene, when challenged with M. tuberculosis, fail to produce reactive nitrogen intermediates and restrict the growth of the bacilli.3 Humans with inherited complete or partial IFN-γ receptor deficiency are highly susceptible to infection by atypical mycobacteria.4 There is a single-nucleotide polymorphism +874 (A/T) located at the 5′-end of a CA repeat at the first intron of human IFN-γ. The +874 T allele is linked to the 12 CA repeats, whereas the A allele is linked to the non-12 CA repeats.5 The specific sequence of the T allele is found to provide a binding site for the transcription factor nuclear factor-κB (NF-κB). As NF-κB induces IFN-γ expression, this T allele correlates with high IFN-γ expression, whereas the A allele correlates with low expression.5 Apart from +874 (A/T), two potentially functional polymorphisms have also been reported at the promoter −179 (G/T)6 and 3′-untranslated region +4766 (C/T).7
Interleukin 10 (IL-10), an anti-inflammatory Th2 cytokine, downregulates the IFN-γ production of T cells, and the secretion of tumor necrosis factor, nitric oxide and the expression of costimulatory molecules and MHC class II of macrophages.8 IL-10 gene disrupted mice infected with M. tuberculosis show only a minor increase in resistance.9 Mice with overexpressed IL-10 show no increase in susceptibility, but confer a reactivation susceptible phenotype.10 Hence, IL-10 may play a potentially central role in promoting reactivation of the latent stage of TB. There are three biallelic polymorphisms in the IL-10 promoter including −1082 (G/A), −819 (C/T) and −592 (C/A) from the transcriptional start site. Eight haplotypes (GCC, GCA, GTA, GTC, ATA, ATC, ACC and ACA) could theoretically be generated. However, as these polymorphic sites are linked, only three haplotypes (GCC, ATA and ACC) are observed and each is correlated with different IL-10 production.11 The GCC haplotype is related to a higher IL-10 production by peripheral blood mononuclear cells (PBMCs) culture.11 A schematic figure illustrating the gene structure and localization of the functional polymorphisms of IFN-γ and IL-10 is shown in Figure 1. To test for the possible associations of IFN-γ and IL-10 with TB, a case–control association study was performed in the Hong Kong Chinese population.
Associations of IFN-γ +874 (A/T) and CA repeats with TB susceptibility
The frequency of the IFN-γ +874 (A/T) in 385 TB patients and 451 control is shown in Table 1. We found that the A/A genotype was significantly over-represented in TB patients (P<0.001, OR=3.79, 95% CI=1.93–7.45).
For the CA repeats, we identified 10 alleles in the CA repeats (ranged from 10 to 19 CA repeats) in our 345 TB patients and the same 451 controls (Table 2). The 12, 13 and 15 CA repeats were found to be the most common alleles in both patient and control groups. A significant difference in the allele frequency distribution between patients and controls was observed (P<0.001). As in vitro production of IFN-γ was reported to have a correlation with the presence and absence of the 12 CA repeats,12 all alleles were grouped into 12 and non-12 CA repeats to investigate the association of the CA repeats genotype with TB. We found similar genotype distribution of the CA repeats (non-12/12) with +874 (A/T). The homozygous non-12/non-12 CA repeats genotype yielded a similar significant result (P<0.001, OR=4.56, 95% CI=2.21–9.43) as observed in +874 A/A genotype (Table 1).
Absolute linkage of IFN-γ +874 T allele with +875 12 CA repeats allele
We found that the presence of the +875 12 CA repeats allele (genotyped by Genescan) was absolutely linked with the +874 T allele (genotyped by TaqMan) from analyzing the 345 TB patients and 451 controls (Table 2).
IFN-γ −179 (G/T) and +4766 (C/T) were nonpolymorphic in our population
By direct sequencing of the potentially functional −179 (G/T) (TB, n=41; controls, n=48) and +4766 (C/T) (TB, n=37; controls, n=56) of IFN-γ, we only found homozygous G/G at −179 and homozygous C/C at +4766. These two sites were nonpolymorphic in our population, although frequencies of the rare alleles are 4% at −179 and 6% at +4766 in African Americans6 and Caucasians,7 respectively.
No association of IL-10 with TB susceptibility
We did not find a significant association between IL-10 and TB (Table 3). As IL-10 GCC is a minor haplotype, no homozygous GCC could be observed in our sample.
Relationships of IFN-γ and IL-10 with TB clinical features
We also studied the relationship of IFN-γ and IL-10 with TB clinical features (relapse, extrapulmonary and pulmonary TB). Weak associations were found in IFN-γ A/A (P=0.06) and IL-10 GCC/− genotype (P=0.04) with the development of relapse/extrapulmonary TB (Table 4).
The genotype frequencies of the various IFN-γ and IL-10 polymorphisms were in Hardy–Weinberg equilibrium in both patient and control groups (P>0.05).
This is the first study demonstrating the association of IFN-γ with TB in the Chinese population. Our result showed that those carrying IFN-γ +874 A/A genotype had a 3.79-fold increased risk of developing TB. Similar genotype distribution and association were observed in the +875 CA repeats (non-12/12) as +874 (A/T) in our study, and the non-12 CA repeats allele was absolutely linked to the +874 A allele in our population as previously reported in the UK population.5 As +874 A allele corresponds to a lower IFN-γ expression, lower IFN-γ level may impair the activation of macrophages, resulting in TB development. Reduced IFN-γ production and mRNA expression are observed from stimulated PBMCs in TB patients.13, 14 This may be ascribed to the +874 A/A genotype,14 and also the reduced expression of nuclear cyclic adenosine 5′-monophosphate response element-binding protein and reduced IFN-γ promoter activity in TB patients.15 Moreover, IFN-γ production from PBMCs is depressed in TB patients with A/A genotype when compared with A/T or T/T genotype at the time of diagnosis and after completion of treatment.14 This suggests that A/A genotype may be related to TB severity or reactivation. However, only weak associations were found with the far advanced TB in Spanish (P<0.042)14 and the development of relapse/extrapulmonary TB in our study (P=0.06).
Positive association of IFN-γ +874 with TB was reported first in the Sicilian,16 then Spanish14 and South African populations.17 Comparison of A/A genotype frequency among these populations is summarized in Table 5. The frequency of the risk A/A genotype in our population (45.7%) was found to be a bit lower than in the South African one (47%), but much higher than in the Sicilian (26%) and Spanish populations (28%). Also, the allele frequency of the protective 12 CA repeats in our population (33.3%) was found to be lower than Caucasian (35%), but higher than African American (26%).18 According to the Global TB control report 2004 from the WHO, the annual notification rate per 100 000 in South Africa is 558, whereas the rate is 113 in China and below 20 in Spain. This trend of ranking in TB incidence rate and frequency of A/A genotype among populations suggests that the differences of incidence rate in different populations depend on the duration of ancestral selection for resistance to M. tuberculosis.19 The TB epidemic reached a peak in the early 1800s in Western Europe and peaked at about 1870–1888 in Eastern Europe. Later, the epidemic was spread to North America by European migrants at 1900. It was not until 1910 when Europeans moved into South Africa that the incidence of TB began to appear and with an increased frequency among South Africans. Although when Asia first experienced TB is unknown, the incidence started to gradually increase in 1951 and remains at its peak today in India and China.19 It is hence postulated that over successive generations of selective pressure from TB, those not carrying the risk A/A genotype resist TB and survive. As only the more resistant individuals survive and reproduce, the frequency of non-AA genotype increases, and eventually the TB epidemic in the white population decreases.
In this study, the association of IL-10 GCC/− genotype with the development of relapse/extrapulmonary TB (P=0.04) but not with TB susceptibility may suggest the role of GCC/− genotype in preventing TB reactivation in contrast to that observed in mice with overexpressed IL-10.10 More relapse/extrapulmonary cases should be recruited to verify this association. The GCC/− genotype corresponding to higher IL-10 level may suppress IFN-γ production and hence favors TB development. Higher stimulated IL-10 production from PBMCs in TB patients than controls is observed13, 14 and related to IL-10 −1082 (A/G).14 Association of IL-10 −1082 with TB was previously observed in the Cambodian20 but not in the Gambian21 and Spanish populations.14 In Table 5, the −1082 G allele frequency is found to be very low in our population compared with others. The frequency of G allele in our population (4%) was similar to that of Korean (7.4%)22 and Japanese (6.5%),23 but was significantly different from Caucasians (48%).24 The G allele frequency in Cambodians is more like that in Caucasians than in other Asian populations, however, there are significant departures from the Hardy–Weinberg equilibrium in both their cases and controls.20 Nevertheless, inconsistent results in association of IL-10 polymorphisms with TB may reflect ethnic-specific genetic variations, or suggest the possibility that other more distal promoter elements are involved.25
During M. tuberculosis infection, key cytokines regulate cells of the immune system and prompt efficient immune responses. Recently, we reported IL-12 to be associated with TB susceptibility.26 Hence, a unique profile of cytokines as determined by the respective functional polymorphisms of their genes may be crucial in controlling the development of clinical TB. Our results showed that individuals with low IFN-γ-producing genotypes had higher risk in developing TB. Further studies in other populations and on other cytokine genes should be performed to address the roles of cytokines in resisting TB, which can help develop a novel strategy in the treatment of TB and more efficacious vaccine.
Materials and methods
Patients and controls
All patients were recruited from territory-wide chest clinics under the Department of Health of Hong Kong Special Administrative Region (HKSAR) over a 2-year period from 2000 to 2002. All patients were either smear/culture positive or with clinical–radiological and histological evidences for TB. All patients responded to anti-mycobacterial treatment and none had HIV infection. Since 1952, the government of Hong Kong has implemented BCG vaccination to all newborns. The coverage rate reached 80% since 1962 and near 100% since 1979. There were 238 males and 147 females in our 385 patient's group, their mean age± standard deviation (s.d.) being 47±18 years. This patients group included 42 relapse, 54 extrapulmonary (lymph node, meninges, miliary, abdomen, bone and joint, spine, genitourinary tract, naso/oropharynx, larynx and pericardium) and 331 pulmonary TB cases. Ethnically matched Red Cross blood donors (279 males and 172 females; mean age± s.d. was 29±10 years) were recruited as control. Informed consents were obtained from all patients and the study was approved by the Ethics Committees of the Faculty of Medicine, the University of Hong Kong and the Department of Health, HKSAR.
Genotyping for IFN-γ polymorphisms
Genomic DNA was isolated from frozen whole blood-EDTA from patients and buffy-coat lymphocytes from controls using the standard salting-out technique or a DNA extraction kit (Qiagen, CA, USA). A region containing the IFN-γ CA repeats polymorphism was amplified with PCR condition as follows: 50 ng genomic DNA was mixed with 0.2 μmol/l primers, 1.5 mmol/l MgCl2, 0.2 mmol/l dNTPs and 0.5U Taq DNA polymerase (Amsersham Biosciences, England). The mixture was then initially subjected for 2 min at 95°C, followed by 35 cycles of denaturation for 1 min at 95°C, annealing for 1 min at 60°C and extension for 1 min at 72°C; final extension was for 5 min at 72°C. The labeled PCR products were electrophoresed on a 4% polyacrylamide gel with an internal size standard Genescan 400HD ROX (Applied Biosystems, CA, USA) on ABI 377 DNA sequencer (Applied Biosystems, CA, USA). The result was analyzed by Genescan Analysis software 3.1 (Applied Biosystems, CA, USA). Controls with homozygous 12/12 (n=7), homozygous 13/13 (n=6) and homozygous 15/15 (n=3) CA repeats were confirmed by sequencing. Direct sequencing was performed on IFN-γ −179 and IFN-γ +4766 on ABI 3730 DNA Analyzer (Applied Biosystems, CA, USA). For IFN-γ +874, alleles were amplified and distinguished with conditions stated by the manufacturer on ABI 7700 Sequence Detection System (Applied Biosystems, CA, USA). All the primers and probes used are listed in Table 6.
Genotyping for IL-10 promoter polymorphisms
Genotyping of IL-10 −1082 and −592 were performed as described previously.24 As −819 C allele and −592 C allele of IL-10 was absolutely linked, only −592 was genotyped.
Age and sex differences between the patients and controls were first investigated by unpaired t-test and χ2 test, respectively. The OR and P-values were next calculated by multiple logistic regression adjusted for sex and age between patients and controls (SAS version 8.2, SAS Institute, USA). The allele distribution of the IFN-γ CA repeats polymorphism between the patients and controls was evaluated by χ2 test with a 2 × 10 contingency table. All polymorphisms were tested for Hardy–Weinberg equilibrium using a χ2 test between observed and expected numbers separately in patients and controls.
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We thank HS Wong for assistance in statistical analyses. This study was funded by The Outstanding Researcher Award of the University of Hong Kong (YL Lau), Edward Sai Kim Ho Tung Paediatric Education and Research Fund (HW Tso), the Shun Tak District Min Yuen Tong and the Genome Research Centre of the University of Hong Kong.
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Tso, H., Ip, W., Chong, W. et al. Association of interferon gamma and interleukin 10 genes with tuberculosis in Hong Kong Chinese. Genes Immun 6, 358–363 (2005). https://doi.org/10.1038/sj.gene.6364189
- interferon gamma
- interleukin 10
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