Interleukin -10 (IL-10) is an extrusive TH-2 cell cytokine produced by lymphoid cells. IL 10 is one of the major anti inflammatory cytokine which regulates many proinflammatory cytokines including IFNg [D'Andrea A et al]. IL 10 plays an important role in the progression of many diseases [Nesrina Imami et al, Fengbo Sun et al, Ambreen Ansari et al, Hyoung Doo Shin et al]. It has been recently reported that IFNg/IL10 ratio shows a significant correlation with clinical severity in pulmonary and extra-pulmonary tuberculosis [Jamil B et al]. By overriding the anti-mycobacterial signals delivered by IFNg to macrophages, IL 10 helps in the maintenance of mycobacterial infection [PJ Murray et al]. IL-10 is a pleiotrophic cytokine that can both stimulate and suppress the immune response [Mocellin S et al]. IL 10 directly inhibits HIV-1 replication; on the other hand it may also promote viral persistence by inactivation of effector immune mechanisms [Dshanta D. Naicker et al].
The ability to produce IL 10 differs between individuals and a significant influence of hereditary factors including the SNPs has been established [J Eskdale et al, Ambreen Ansari et al]. Three single nucleotide polymorphism (SNPs) of IL-10 gene (positions -1082A/G, -819T/C, and -592A/C) have been reported which influence on its expression [Turner et al].In particular, there are many studies reporting the influence of SNP at -1082(A/G) position on the production levels of IL 10 in many diseases [Fengbo Sun et al, Kingo K et al]. In this study we have focussed on the effect of -1082A/G SNP on the levels of IL 10 produced in TB, HIV, and HIV-TB infected patient groups. Allelic frequencies of cytokine gene polymorphism has shown a considerable variations in different ethnic groups [Bagheri M et al] and many reports involving HIV and TB patient groups are in concurrence with this observation [Ambreen Ansari et al, Naimish R. Patel et al]. There are conflicting reports on association of 'A' and 'G' allele with high or low production of IL 10 in different disease groups. Many reports on TB have shown 'A' allele as high producer and 'G' allele as low producer of IL 10 [J Eskdale et al, Ambreen Ansari et al] where as in few other disease groups A/G polymorphism and 'G' allele has been associated with high production of IL 10 [ Fengbo Sun et al, Turner DM et al].
Materials and Methods:
The study was approved by the Institutional Ethical Committee, written informed consent was obtained from all the 436 participants. Participants included were categorised as: (1) HIV+ group (130): HIV-1 infected individuals as determined by ELISA and a confirmatory Western Blot or Immunoblot assay, asymptomatic with no history of tuberculosis; (2) Active tuberculosis group (108) (TB): HIV-1 seronegative patients with recently diagnosed active pulmonary or extra-pulmonary tuberculosis, based on clinical picture, sputum-positivity and radiological involvement suggestive of pulmonary tuberculosis and FNAC or biopsy from the infected sites in the case of extra-pulmonary TB; (3) HIV-TB co-infected group (89) (HT): Confirmed with HIV-1 and tuberculosis disease; (4) Healthy control group (109): Asymptomatic volunteers HIV-seronegative with no history of TB.
DNA Isolation:
DNA was isolated from the whole blood using Flexigene DNA kit according to the manufacturer instructions (QIAGEN, Hilden, Germany). DNA concentrations were estimated at 260 nm using ND-1000 spectrophotometer (Thermosceintifics, Wilmington DE, 19810 USA)
IL-10 -1082 (A-G) Polymorphism:
Typing of IL-10 gene polymorphisms were performed by amplification refractory mutation system - polymerase chain reaction (ARMS-PCR). Components of the reaction mix included template DNA, forward primer (F): 5'-GTA AGC TTC TGT GGC TGG AGT C-3' and reverse primers (R1): 5'-AAC ACT ACT AAG GCT TCT TTG GGT A-3'; (R2): 5'-AAC ACT ACT AAG GCT TCT TTG GGT G-3' (MWG- biotech AG, Hyderabad) (Table 1), Mgcl2, dNTPs and Taq DNA polymerase of standard concentrations. Reaction conditions were 95°C for 1 minute followed by 30 cycles of 95°C for 1.30 minutes, and at 63 °C annealing temperature for 1.30 minutes, 72 °C for 1 minute then 1 cycle at 72 °C for 10 minutes. The PCR products were resolved on a 2% agarose gel followed by ethidium bromide staining and visualized by UV transilluminator (BIO-RAD, U.S.A).
Stimulation Assay:
Peripheral blood mononuclear cells (PBMCs) were isolated from the patients and controls and wear cultured according to the protocol described previously (Anuradha B 2008 et al). Ag85A at a final concentration of 10μg/mL was used to stimulate the cells in culture.
IL-10 and IFN-g assay:
The cytokine levels were measured in culture supernatants by sandwich ELISA by using IL-10 and IFNg commercial kit (eBioscience Inc., San Diego, CA, USA). Briefly, to each well, 100μL of capture antibody (1x) diluted in coating buffer was added and incubated overnight at 4°C. After 5 washes, the wells were blocked with 200μL/well of 1X Assay diluent and incubated for 1 hour at room temperature (RT). An additional 5 washes were followed by the addition of 100μL/well of supernatants and standards, and then incubated for 2 hours at RT. After another 5 washes, 100 μL/well of detection antibody diluted in 1X Assay diluent was added, incubated for 1 hour at RT and washed 5 times. After addition of 100μL/well of Avidin-HRP diluted in 1X Assay Diluent and incubation for 30 minutes at RT and 7 washes, the wells were incubated with the substrate (100μL/well) for 15 minutes at RT. Stop solution (50μL/well) was added and absorbance was measured at 450 nm and subtracted with the absorbance values at 540nm. The concentrations were calculated using MPM software version 6.1.
Statistical analysis:
Polymorphism: Analysis of case-control data was performed with Open Epi: Open Source Epidemiologic Statistics for Public Health, version 2.3. Chi-Square (χ2) test was used for comparing genotype frequencies. A 2 x 2 cross-tabulation method was used to determine odds ratio (OR) with 95% confidence interval. A 'p' value of <0.05 was considered significant.
Cytokine assays: Mean and standard deviation values were determined. Statistical values were obtained using t-test for unpaired data. Grouped data was analysed using one-way ANOVA followed by Bonferroni test and Tukey's multiple comparison test for inter and intra group analysis using Graph Pad Prism software, Version 5.00.
Results
IL-10 (-1082 A-G) polymorphism:
In HIV-TB, A/A (56%) genotype is high when compared to other genotypes (A/G: 36%; G/G: 8%) but the G/G genotype was significantly (p<0.05) associated with increased risk towards the TB. While in TB the frequency of homozygous A/A (73%) genotype is significantly high (p<0.05) compared to other genotypes (A/G: 25%; G/G: 2%). But, in case of HIV the A/G genotype (73%) is highly frequent (A/A: 26%; G/G: 1%) and is also significantly (p<0.05) associated with HIV disease progression. (Figure: 1; Table: 1)
Association between IL-10 (-1082) gene polymorphism and its level in serum:
Highest mean level of IL-10 was observed in HIV-TB individuals with 'G' allele compared to lowest levels of IL-10 in 'A' allele with 117.47 pg/ml. While their IFNg levels were exactly opposite with 329.60 pg/ml in 'A' and 25.0 pg/ml in 'G' allele. IL-10 levels wear significantly high with A allele in TB (715.52) compared to G with 277.7 pg/ml. In the remaining groups, IL-10 levels wear more or less similar between the A and G. (Table: 2)
Figure 1: A bar graph illustrating the frequency distribution of the three genotypes in different groups.
Table 1: Frequency distribution of genotypes in IL-10(A-G)
A/A
Study groups
n(%)
p-value
OR
CI
Control(100)
56(56)
-
-
-
HIV (116)
30(25.9)
0.000
0.274
0.155-0.485
HT (89)
50(56.2)
0.98
1.007
0.568-1.787
TB (100)
73(73)
0.012
2.124
1.178-3.830
A/G
Control(100)
43(43)
-
-
-
HIV (116)
85(73.3)
0.000
3.635
2.058-6.419
HT (89)
32(36)
0.323
0.744
0.415-1.335
TB (100)
25(25)
0.007
0.442
0.243-0.804
G/G
Control(100)
1(1)
-
-
-
HIV (116)
1(0.8)
0.916
0.861
0.089- 8.351
HT (89)
7(7.8)
0.019
8.451
1.318-53.47
TB (100)
2(2)
0.561
2.02
0.259-15.633
Table 2: IL 10 Production with respect to alleles in different groups.
Mean ± SD
IL 10
IFNg
Allele
Control
HIV
HIV-TB
TB
Control
HIV
HIV-TB
TB
A
337.08±
154.43
794.85±
355.89
117.47±
203.22
715.52 ±
364.62
612.73±
642.48
329.60±
413.86
133.24±
18.67
21.04±
137.16
G
348.17±
22.96
698.89±
595.57
1209.26±
588.95
277.78±
266.36
669.93±
825.20
25.00±
6.96
8.08±
3.30
13.61±
6.05
Inference:
Our observations indicate positive association of G/G genotype with HIV-TB, where as it is A/G in case of HIV and A/A in case of TB. Accordingly, the IL-10 levels in relation with allele frequency, reports an association of 'A' allele with higher production of IL-10 in HIV and TB groups where as in HIV-TB 'G' allele was accounted for higher production.
The results of the present study indicated that the HIV positive individuals with G/G genotype in IL-10 gene at position -1082 (A-G) may develop co-infection with TB.
