Abstract
Objectives: This study investigated and analyzed the association of interferon-γ (IFN-γ) gene polymorphisms and Th1/Th2 balance in tuberculosis susceptibility. Methods: From January 2018 to January 2020, 121 tuberculosis patients that were hospitalized for initial treatment and were chosen as the pulmonary tuberculosis group, and another 80 healthy individuals with physical examination in the same period were selected as the control group. The genetic distribution of IFN-γ +874 sites in the two sets of subjects was detected by sequence-specific primer PCR (PCR-SSP), the serum IFN-γ level was detected by ELISA, and the ratio of Th1 and Th2 on cells in two sets of subjects were detected by flow cytometry. Subsequently, we compared the differences in IFN-γ degree and the changes in Th1/Th2 in patients with different genotypes. Results: The difference in the distribution of IFN-γ (+874 T/A) genotype frequencies between the two groups of subjects was significantly different (P<0.05), and the frequency of A allele in the tuberculosis group was clearly higher than that in the control group (P<0.05). Compared with the control group, the Th1 cells percentage in the pulmonary tuberculosis group decreased remarkably, the Th2 cells percentage increased significantly, while the Th1/Th2 ratio was notably decreased (P<0.05). In addition, the Th1/Th2 ratio in tuberculosis patients with an IFN-γ (+874 T/A) genotype AA was remarkably lower than those with AT+TT genotype (P<0.05). Conclusion: An AA genotype with IFN-γ (+874 T/A) is a susceptible genotype for tuberculosis. The mechanism may be related to the imbalance of Th1/Th2 which is more likely to occur in patients with AA genotype. This can lead to a decline in immune function of the body, making it more susceptible to infection with mycobacterium which induces tuberculosis.
Keywords: Polymorphism of IFN-γ gene, tuberculosis, susceptibility, Th1/Th2 balance
Introduction
Tuberculosis is a disease caused by the bacterium Mycobacterium tuberculosis that affects multiple organs [1]. Among which, pulmonary tuberculosis is the most common form of tuberculosis [2]. Interferon-γ (IFN-γ) is a Th1 cytokine produced by activated T cells and NK cells, it has various biological functions such as anti-viral replication, inhibition of cell division, immune regulation, anti-tumor effects, etc. Researchers have shown that IFN-γ is a key cytokine involved in the immune process of Tuberculosis cells. Whether produced by activation of an early immune response in tuberculosis, or as a product of T lymphocyte-specific antigen immune inductions; where IFN-γ plays a crucial role in the infection and pathogenesis of tuberculosis by activating macrophages to fight and kill Mycobacterium [3,4]. Studies have shown that individuals with defects in IFN-γ or its receptor are susceptible to infection with Mycobacterium tuberculosis. However, there has been a lack in reports of the relationship between IFN-γ gene polymorphisms and Th1/Th2 balance in pulmonary tuberculosis patients [5,6]. This study explored and analyzed the association of IFN-γ gene polymorphism and Th1/Th2 balance in tuberculosis susceptibility. The reports are as follows.
Material and methods
Clinical material
From January 2018 to January 2020, a total of 121 tuberculosis patients that were hospitalized for initial treatment were chosen as the pulmonary tuberculosis group. In addition, 80 healthy individuals who underwent physical examination during the same period were included as the control group. The study was conducted in line with approval by the hospital ethics committee.
Inclusive and exclusive criteria
Inclusive criteria: (1) Patients met the diagnostic criteria in the Guidelines for Diagnosis and Treatment of Tuberculosis [7]; (2) Age ≥18 years old; (3) Subjects in the control group did not have a history of tuberculosis exposure; and (4) Both groups voluntarily signed the informed consent.
Exclusive criteria: (1) Patients with severe heart, liver, kidney or dysfunction in vital organs; (2) Patients with HIV infection; (3) Patients who took drugs or alcohol, or had a history of drug abuse; (4) Patients with diabetes, hepatitis, malignant tumors or immune system diseases; or (5) Females during pregnancy or lactation.
Methods
Detection of IFN-γ gene polymorphism
2 ml of peripheral blood was drawn from both groups of subjects and EDTA-K2 was used for anticoagulation. We extracted the DNA from the peripheral blood by DNA extraction kit (purchased from Tiangen Biochemical Technology (Beijing) Co., LTD.), then it was dissolved in TE buffer and stored at -20°C. The sequence-specific primer polymerase chain reaction (PCR-SSP) was adopted, and the design of primer sequence is listed in the references (Table 1). Next, a 1.5% agarose gel containing Goldview nucleic acid dye and 1×TAE buffer was added into the 2 tubes of amplified PCR products. After 30 min of electrophoresis via 80V constant voltage and gel imaging, the amplified PCR products were observed. An internal reference (996 bp) positive band was treated as the effective amplification for each amplification. We judged the results according to the electrophoretogram of PCR products of each sample. When the product with type 1 specific forward primer tube appeared in the target band, it was assessed as 1/1 homozygous; when only adduct type 2 products were present, it was determined to be 2/2 homozygous; and 1/2 heterozygote was confirmed when the target band appeared in both tubes.
Table 1.
Primers and amplified product length of SNP at IFN-γ by PCR-SPP analysis
| Locus | Primer | Position (bp) |
|---|---|---|
| IFN-γ (+874 A/T) | ||
| Specific F1 | 5’-TTACAACACAAAATCAAATCT-3’ | 1414 |
| Specific F2 | 5’-TTACAACACAAAATCAAATCA-3’ | |
| Common R | 5’-TCATTCTCATTTTCTATTCTTT-3’ | |
| Control | ||
| Control F | 5’-AGAAAAAATGGCTAAGAAAT-3’ | 996 |
| Control R | 5’-AATGCACTGGGAGACAAT-3’ |
We selected the samples of +847 A/T, T/T, T/A and A/A genotypes, respectively. The obtained PCR amplification products were sequenced and analyzed with internal reference primers. After being confirmed by sequencing, the PCR amplification products were added as quality control materials to each batch of specimens to ensure the accuracy of PCR amplification results.
Measuring of Th1 and Th2 cells ratio by flow cytometry
The ratio of Th1/Th2 to CD4+ cells was detected by flow cytometry. Four ml of fasting peripheral blood in both groups of subjects were drawn and mononuclear cells (PBMC) were separated by Ficoll density gradient centrifugation. PBMC was suspended in 2 ml of RPMI 1640 culture medium that contained 10% fetal bovine serum, and the density of PBMC was settled by 1×106/ml. One ml of the above PBMC suspension was placed in two culture wells, and PMA (50 g/mL), Ionomycin (750 g/L) and BFA (1 ml/L) were added into each well to secrete tissue cytokines into cells, and cultured for 4 h in an incubator at 37°C. We collected the cells and divided them into control tubes and experimental tubes. FITC-mouse anti-human CD4 mAb IgG1 was added in both tubes, mixed and protected from light, and placed at room temperature for 20 minutes to stain the cell surface antigens. After 200 μl Cytofix/Cytoperm solution was added and placed at 4°C for 20 min, the cells were thoroughly fixed and washed for intracellular cytokine staining. We added the APC-anti-IL-4 and PE-cy7-anti-IFN-γ into the experimental tube, and the isotype control antibody of the above antibody into the control tube. We kept the tubes in the dark at 4°C for 20 min, then we resuspended with Perm/Wash solution, and analyzed by flow cytometry within 24 h.
Statistical analysis
Data processing and analysis were conducted via statistical software SPSS 22.0. The frequencies of genotypes and alleles were compared by χ2 test after the distribution of genotype and allele frequency conformed to Hardy-Weinberg equilibrium law. The comparison of measurement and enumeration data were conducted through t-test and χ2 test respectively, with P<0.05 the difference was considered to be significantly different.
Results
Clinical data
There were 73 males and 48 females in the observation group who were aged between 18-76 years old. The average age was (46.58±9.32) years old. There were 53 males and 37 females who were in control group that were aged between 18-74 years old. Their average age was (45.94±10.32). There was not statistically significant difference in gender or age between the two groups (P>0.05).
Detection of a Hardy Weinberg genetic balance
A Hardy-Weinberg balance test was carried out to measure the genotype frequencies in the tuberculosis and control groups. According to the test results, the measured genotype frequency had no significant difference with the expected genotype frequency, and each gene had reached genetic balance (χ2=0.394, 0.192, P>0.05) with the characteristics of group representation (Table 2).
Table 2.
The genotype at IFN-γ gene +874 of two groups of subjects via Hardy-Weinberg balance test
| Genotype | Pulmonary tuberculosis group | HWE χ2 | P | Control group | HWE χ2 | P | ||
|---|---|---|---|---|---|---|---|---|
|
|
|
|||||||
| Measured value (n) | Predictive value (n) | Measured value (n) | Predictive value (n) | |||||
| AA | 94 (77.69) | 96 (79.34) | 0.394 | 0.694 | 36 (45.00) | 33 (41.25) | 0.192 | 0.848 |
| AT | 19 (15.70) | 20 (16.53) | 28 (35.00) | 33 (41.25) | ||||
| TT | 8 (6.61) | 5 (4.13) | 16 (20.00) | 14 (17.50) | ||||
The distribution of IFN-γ (+874 T/A) polymorphisms
The frequency of the IFN-γ (+874 T/A) AA genotype in the pulmonary tuberculosis group was 77.69%, the AT genotypic frequency was 15.70%, and TT genotypic frequency was 6.61%. In the control-group subjects, the AA genotypic frequency was 45.00%, AT genotypic frequency was 35.00% and TT genotypic frequency was 20.00%. The difference in frequency distribution of IFN-γ (+874 T/A) between the two sets of subjects was statistically significant (P<0.05) (Table 3).
Table 3.
Comparison of genotypic frequency of IFN-γ (+874 T/A) polymorphism [n (%)]
| Group | Number of cases | Genotypic frequency of IFN-γ | ||
|---|---|---|---|---|
|
| ||||
| AA (%) | AT (%) | TT (%) | ||
| Pulmonary tuberculosis group | 121 | 94 (77.69) | 19 (15.70) | 8 (6.61) |
| Control group | 80 | 36 (45.00) | 28 (35.00) | 16 (20.00) |
| Z | - | -4.752 | ||
| P | - | 0.000 | ||
Comparison of allele frequencies of IFN-γ (+874 T/A) polymorphism
The A allele frequency of IFN-γ (+874 T/A) in the tuberculosis group was 89.67%, and the T allele frequency was 14.46%. For control-group patients, the A allele frequency was 62.50% and the T allele frequency was 37.50%. The frequency of A allele in the tuberculosis group was remarkably higher than that in the control group (P<0.05) (Table 4).
Table 4.
Comparison of allele frequencies of IFN-γ (+874 T/A) polymorphism
| Group | n | A | T |
|---|---|---|---|
| Pulmonary tuberculosis group | 242 | 217 (89.67) | 35 (14.46) |
| Control group | 160 | 100 (62.50) | 60 (37.50) |
| χ2 | - | 30.752 | |
| P | - | 0.000 | |
Comparison of Th1 and Th2 cell percentages
The percentage of Th1 cells in the pulmonary tuberculosis group was remarkably reduced, the percentage of Th2 was increased significantly, and Th1/Th2 ratio was significantly decreased compared to the control group (P<0.05) (Table 5 and Figure 1).
Table 5.
Comparison of Th1 and Th2 cell percentages between the two groups
| Group | Number of cases | Th1 cells (%) | Th2 cells (%) | Th1/Th2 |
|---|---|---|---|---|
| Pulmonary tuberculosis group | 121 | 3.14±0.97 | 3.27±1.16 | 1.09±0.25 |
| Control group | 80 | 4.89±1.46 | 1.18±0.23 | 3.95±1.52 |
| t | - | 10.215 | 15.897 | 20.311 |
| P | - | 0.000 | 0.000 | 0.000 |
Figure 1.
Th2 and Th2 were expressed in both groups. Note: A: Expression of Th1 cells in patients with pulmonary tuberculosis group. B: Expression of Th1 cells in patients with Control group. C: Expression of Th2 cells in patients with pulmonary tuberculosis group. D: Expression of Th2 cells in patients with Control group.
Comparison of Th1/Th2 ratio in subjects with different IFN-γ (+874 T/A) genotypes
The Th1/Th2 ratio of subjects with IFN-γ (+874 T/A) genotype AA tuberculosis was remarkably lower than in subjects with an AT+TT genotype (P<0.05) (Table 6).
Table 6.
Comparison of Th1/Th2 ratio in patients with different IFN-γ (+874 T/A) loci genotype
| Genotype | Number of cases | Th1/Th2 | t | P |
|---|---|---|---|---|
| AA | 94 | 0.85±0.17 | 11.555 | 0.000 |
| AT+TT | 27 | 1.20±0.26 |
Discussion
The interaction between pathogenic microorganisms and the hosts is a very complex process. Infection and its different clinical outcomes are affected by a variety of factors, including exposure to pathogenic microorganisms, the differences in virulence, individual health status as well as genetic factors of host [8,9]. Previous studies on infectious diseases were primarily focused on the description of disease epidemiology and etiology, while the host factors were ignored, especially the crucial impact of genetic factor on infection and progression of disease after infection [10].
The expression of cytokines is regulated by genes. SNPs that are located in the regulatory region of gene expression affect the expression of cytokines, and they play a crucial role in terms of individual differences in diseases [11]. The structure and function of genes are very complex, and any sequence changes can lead to a change in the entire function of the gene. The IFN-γ (+874 T/A) locus is located in the first intron region, and the change of intron sequence does not directly change the amino acids, but may change the alternative splicing of mRNA. This leads to a decrease in the mRNA products, as well as affecting its gene expression and the physiological function of the coding proteins, thus leading to the occurrence of diseases [12-14].
The research for the occurrence of tuberculosis is related to the imbalance of cellular immune function in the body, and the balance of Th1/Th2 plays a decisive role in the progression, deterioration, control and prevention of tuberculosis [15-17]. The main Th1 cytokines are IFN-γ and TNF-α, and the main Th2 cytokines are IL-4 and IL-10. However, there have been no studies on whether IFN-γ (+874 T/A) polymorphism affects the Th1/Th2 balance in tuberculosis patients. This study investigated and analyzed the association of IFN-γ gene polymorphisms in tuberculosis susceptibility and Th1/Th2 balance.
The tests showed that the difference in the distribution of IFN-γ (+874 T/A) genotype frequency between the two groups of subjects was statistically significant, and the frequency of an A allele in the tuberculosis group was clearly higher than that in the control group. The IFN-γ gene is closely related to the susceptibility of pulmonary tuberculosis. In particular, the A allele and the homozygous AA genotype can seriously increase the risk of tuberculosis, which is similar to the results reported by scholars [18,19]. Studies have shown that the polymorphism of the T/A gene at IFN-γ intron +874 can affect its expression in peripheral blood, of which TT is the highest expression genotype, AA is the lowest expression genotype, and TA is the medium expression genotype [20,21]. The expression of IFN-γ plays a crucial role in the immune response of the body. The concentration of IFN-γ mononuclear cells in the stimulated peripheral blood of tuberculosis patients is reduced, and the production of IFN-γ in tuberculosis patients with an AA genotype is remarkably inhibited [22,23].
Compared to the control group, the cell percentage with Th1 in the pulmonary tuberculosis group was decreased remarkably, Th2 increased significantly, and Th1/Th2 ratio had a notable decline. Similar to results by other scholars [24,25], when human body is infected with Mycobacterium tuberculosis, the function of Th1 cells is impaired, the effect of Th2 cells is enhanced, the function of various immune effector cells decreased, and a large number of cytokines participate in immune response, leading to the onset of disease. In addition, the Th1/Th2 ratio of tuberculosis subjects with IFN-γ (+874 T/A) genotype AA was remarkably lower than that of patients with AT+TT genotype, suggesting that the polymorphism of gene IFN-γ may influence the imbalance of Th1/Th2 cell ratios in patients with tuberculosis. Among them, patients with AA genotype had more obvious imbalance of Th1/Th2 ratio. It results in a decrease of immune function and decline in anti-infection ability, thus is more likely to support tuberculosis [26]. The results of this study are basically similar to those reported by scholars [27,28], the IFN-γ (+874 T/A) gene polymorphism affects the risk of tuberculosis infection. The mechanism may be that IFN-γ gene polymorphisms affects the immune mechanism of the body, so that patients with different genotypes have different resistance to tuberculosis, resulting in differences in susceptibility.
However, as the sample size included in the study is small, and no in-depth analysis of a more influential mechanisms is provided, there may existed certain deviations in the research results. We will further expand the sample capacity and analyze the different mechanisms to achieve more dependable clinical research results.
In summary, an AA genotype with IFN-γ (+874 T/A) is the susceptible genotype of tuberculosis. Its mechanism may be an imbalance of Th1/Th2 which is much more likely to occur in patients with an AA genotype. This will lead to a decline in immune function of body, making it more susceptible to mycobacterium infection and tuberculosis.
Disclosure of conflict of interest
None.
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