The Methylation in B7-H4 and BTLA Genes are Associated with the Risk of Pulmonary Tuberculosis

Introduction

Tuberculosis (TB) is a common, serious infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis) infection, which is still a major public health problem. Before the COVID-19 pandemic, TB was the leading cause of death from a single pathogen, and the most common type of this disease was pulmonary TB (PTB).¹ The COVID-19 pandemic resulted in reduced access to TB diagnosis and treatment. There were approximately 10.6 million new cases of TB worldwide and about 0.78 million new cases of TB in China in 2021 according to the Global Tuberculosis Report 2022.² Studies showed that about a quarter of the population was infected with M. tuberculosis globally, while less than 10% of those infected would develop active TB.³ This suggested that the pathogenesis of TB was influenced by multiple factors, including nutrition, immunity, genetic factors, smoke, alcohol, and HIV infection.^4–6

Genome-wide association studies and twin studies had confirmed the important influence of host genetics on TB susceptibility.^7,8 In addition, candidate gene association studies had found many important variants related to TB susceptibility, most of which were mainly located in immune-related genes, such as Toll-like receptor genes, vitamin D metabolic pathway genes and human leukocyte antigen genes.^9–11 However, these studies only could explain part of the heritability of TB. Epigenetics also showed the important roles in TB development. For instance, abnormal DNA methylation, which was known as an important epigenetic mode, of several genes was considered to affect the development of TB.¹²

The PTB development was affected by the host’s immune function, and host control of M. tuberculosis infection required innate and adaptive immune responses. B- and T-lymphocyte attenuator (BTLA) and B7 homologous body 4 (B7-H4) were two newly discovered immune checkpoint inhibitors.^13,14 BTLA was mainly expressed in dendritic cells (DC), T cells, B cells and some myeloid cells. The interaction between BTLA and its ligand herpesvirus entry mediator (HVEM) could inhibit the proliferation and activation of T cells, as well as the expression of a variety of cytokines in T cells.¹⁵ The function of BTLA in PTB had been investigated, and BTLA exerted immune memory on αβT cells in active PTB.¹⁶ B7-H4 was inductively expressed by immune cells such as T cells, B cells, DC cells and macrophages. The increased expression of B7-H4 in DC might be associated with the increase of Tregs, and could influence the differentiation of naive T cells, thereby affecting the identification of M. tuberculosis by the immune system and inducing anti-TB specific immune responses.¹⁷ BTLA and B7-H4 expression levels were abnormally increased in CD11c+ antigen presenting cells from peripheral blood among PTB patients, suggesting that these factors were closely involved in the pathogenesis of PTB.¹⁸

Previous studies revealed that genetic variation in B7-H4, BTLA was related the susceptibility to many diseases, including autoimmune thyroid diseases,¹⁹ ankylosing spondylitis (AS),²⁰ breast cancer.²¹ Nevertheless, the genetic effect of B7-H4, BTLA genes on the risk of PTB had not yet been explored. Therefore, we conducted this study to investigate the association among several single nucleotide polymorphisms (SNPs) in B7-H4, BTLA genes, as well as their methylation level, and PTB susceptibility in the Chinese Han population.

Materials and Methods

PTB Patients and Controls

We continuously collected the patients with PTB from Anhui Chest Hospital, and all patients were diagnosed by senior clinicians according to the diagnosis for PTB (WS 288–2017) and the classification of TB (WS 196–2017) from the Health Industry Standard of the People’s Republic of China. Exclusion criteria of PTB patients were: HIV-positive, other infectious diseases, autoimmune diseases and cancer. In this study, we selected healthy volunteers from the local health examination center as control group, and all of whom had no history of infectious diseases, pulmonary diseases, bacterial or viral infections.

The study was conducted in compliance with the provisions of the Declaration of Helsinki and approved by the Ethics Committee of Anhui Chest Hospital (K2023-011). Written informed consent was obtained from all participants, peripheral blood (3–5 mL), and clinical data were collected. Genomic DNA was extracted from peripheral blood using the Flexi Gene-DNA Kit (Qiagen, Valencia, CA). The required clinical data included fever, drug resistance, drug-induced liver injury (DILI), pulmonary infection, leukopenia, sputum smear, etc.

SNP Selection and Genotyping

A total of ten SNPs, including B7-H4 gene rs10754339, rs10801935, rs10923223, rs1937956, rs3738414 and BTLA gene rs9288952, rs1982809, rs2971205, rs75368388, rs9288953 were genotyped by SNPscan technique, with technical support from the Center for Genetic and Genomic Analysis, Genesky Biotechnologies Inc., Shanghai. Only participants with 100% genotyping success for the selected SNPs were included in the final analysis.

The selection of these SNPs was mainly based on the following principles of screening tag SNPs. The tag SNPs of B7-H4 and BTLA genes was selected using genetic data from Ensembl genome browser 85 and CHBS_1000g with Haploview 4.0 software (Cambridge, MA, USA) based on following strategy: (1) minor allele frequency ≥0.05; (2) an r² cutoff of 0.8 for linkage disequilibrium; (3) the flanking 2.0 kb regions of each gene.

Methylation Detecting

In this study, DNA samples were first treated with bisulfite, and then used for targeted region methylation sequencing to the promoter regions in B7-H4 and BTLA genes. The CpG islands of the target fragments in the promoter regions of these two genes were sequenced using the MethylTarget technique with the support of the Genesky Biotechnology (Shanghai) Center for Genetic and Genome Analysis. Only one specific target fragment was respectively detected on B7-H4 and BTLA genes, and the primer sequence of these fragments were shown in Table S1. The methylation level of every specific target fragment could be got through calculating the mean methylation level of all CpG sites on the fragment.

Statistical Analysis

We used the chi-squared test to assess whether the genotype distribution of 10 SNPs in B7-H4 and BTLA genes in the control group achieved Hardy–Weinberg Equilibrium (HWE). The distributions of allele and genotype between PTB patients and controls were compared by logistic regression model, and odds ratios (OR) with 95% confidence intervals (CI) was calculated. The association of B7-H4 and BTLA genes variation with PTB susceptibility was analyzed under two genetic models (dominant model and recessive model), and haplotype analysis was conducted using SHEsis software.²² The methylation levels of B7-H4 and BTLA genes were expressed by median (P₂₅, P₇₅). The differences of B7-H4 and BTLA genes methylation levels between different groups were analyzed by Mann–Whitney U-test and Kruskal–Wallis H-test. All statistical analyses were calculated with SPSS 23.0, and a P-value <0.05 was regarded as statistically significant. Bonferroni correction was used for multiple testing in SNP analysis, and P<0.0055 (0.05/9) was considered as statistically significant.

Results

Comparison of B7-H4 and BTLA Genes Polymorphisms Among PTB Patients and Controls

Ten SNPs in B7-H4 and BTLA genes was successfully genotyped in 496 patients with PTB and 502 controls. The differences regarding age (an average age of patients: 44.73 ± 18.04 years, an average age of controls: 44.42 ± 3.50 years) and gender (patients: 183 female/313 male; controls: 201 female/301 male) distribution between PTB patients and controls showed no significant. HWE test was performed for the genotype distribution of all SNPs in controls, and the results showed that nine SNPs exhibited HWE except BTLA gene rs9288952.

Our results revealed that GA genotype frequency of rs3738414 variant in B7-H4 gene was increased in PTB patients when compared to controls (P=0.030 after adjustment of gender and age), while the difference was not statistically significant after Bonferroni correction (P>0.0055) (Table 1). Similarly, there was no statistical association between the allele, genotype frequencies of other SNPs (rs10754339, rs1937956, rs10923223, rs10801935) in B7-H4 gene and PTB susceptibility. For BTLA gene, the frequencies of AA genotype in rs1982809 variant, GG genotype in rs2971205 variant, C allele in rs9288953 variant seemed to be increased in PTB patients than that in controls (P=0.039, P=0.018, P=0.045 after adjustment of gender and age), but no statistically significant association was found after Bonferroni correction (P>0.0055) (Table 1). The association among B7-H4, BTLA genes polymorphism and PTB susceptibility was also assessed under dominant model, recessive mode, and no association reached statistical significance after Bonferroni correction (P>0.0055).

Table 1 Association of B7-H4, BTLA Genes Polymorphisms with PTB Susceptibility

We constructed the haplotypes of B7-H4 and BTLA genes by SHEsis software, and only the haplotypes with a frequency more than 3% were included in Table 2. The results of haplotype analysis showed that when compared with controls, AATTG haplotype of B7-H4 gene and GATT haplotype of BTLA gene had significantly decreased frequencies (P=0.033, P=0.045) in PTB patients, while AGTC haplotype of BTLA gene had an increased frequency (P=0.002). Other haplotypes of B7-H4 and BTLA genes did not exhibit any significant association with PTB susceptibility.

Table 2 Haplotype Analysis of B7-H4, BTLA Genes in PTB Patients

Association Between B7-H4, BTLA Genes Polymorphisms and Several Clinical Manifestations in PTB Patients

We respectively divided PTB patients into two groups based on whether the patients had specific clinical features or not, such as fever, drug resistance, DILI, pulmonary infection, hypoproteinemia, sputum smear-positive. Then, we analyzed the association between the allele and genotype frequencies of all SNPs in B7-H4, BTLA genes and above clinical manifestations in PTB patients. The results demonstrated that the frequency of BTLA gene rs9288953 C allele was significantly increased in PTB patients with drug resistance in comparison with the patients without drug resistance (P=0.035) (Table 3). However, other SNPs in B7-H4 and BTLA genes were not associated with clinical manifestations of PTB.

Table 3 Relationship Between B7-H4, BTLA Genes Polymorphisms and the Clinical Manifestations in PTB Patients

Comparison of B7-H4, BTLA Genes Methylation Levels in PTB Patients and Controls

The methylation levels of B7-H4, BTLA genes were examined in 98 PTB patients and 97 controls. No difference in the distribution of age (an average age of patients: 43.52 ± 17.59 years, an average age of controls: 43.45 ± 4.49 years) or gender (patients: 45 female/53 male; controls: 46 female/51 male) between PTB patients and controls was found. In this study, only one specific target fragment was detected in B7-H4 and BTLA genes, respectively. Therefore, the methylation level of every target fragment was used to represent the methylation level of B7-H4 and BTLA genes in this study.

In PTB patients, the methylation levels of B7-H4, BTLA genes were 0,2966(0.2938-0.3003), 0.7735 (0.7171–0.8433). In the control group, the methylation level of B7-H4, BTLA genes were 0.2941(0.2914-0.7925), 0.7456 (0.7149–0.7925). As shown in Figures 1 and 2, we found that the methylation levels of B7-H4, BTLA genes in PTB patients were both higher than that in controls (P=0.001, P=0.005). We also analyzed the association among B7-H4, BTLA genes methylation levels and some clinical manifestations in PTB patients. This study did not obtain any significant association between B7-H4, BTLA genes methylation levels and the clinical manifestations, including fever, drug resistance, DILI, pulmonary infection, hypoproteinemia, sputum smear-positive (Table 4).

Figure 1 The methylation levels of B7-H4 genes in PTB patients and controls.

Figure 2 The methylation levels of BTLA genes in PTB patients and controls.

Table 4 Association Between B7-H4, BTLA Methylation Levels and Clinical Characteristics of PTB Patients

Association Between B7-H4, BTLA Genes Polymorphisms and Their Methylation Levels

This study conducted an association analysis to assessed whether the genotype of each SNP of B7-H4, BTLA genes affected their methylation levels in 98 PTB patients (Table 5). Our results demonstrated that rs10754339 variant in B7-H4 gene could affect its methylation level. In addition, no statistically significant association was found among BTLA gene polymorphism and its methylation level.

Table 5 Association between B7-H4, BTLA genes genotypes on their methylation levels

Discussion

As a complex infectious disease, genetic factors played an important role in the pathogenesis of PTB. Further elucidation of host genetic differences was an important way to understand the pathogenesis of PTB and formulate appropriate treatment, prognosis strategies. Recently, some studies had extensively studied the regulatory role of checkpoint inhibitory molecules BTLA and B7-H4 in a variety of immune cells, and revealed their influence on the pathogenesis of PTB.^16,23 The present study was the first to explore the potential association among BTLA, B7-H4 genes variation and PTB susceptibility from the perspective of gene polymorphisms and epigenetics, and provided several previously undescribed findings.

B7-H4, also known as B7S1 and B7x, was the newest member of the B7 family, and could modulate antigen-specific immune responses by inhibiting T cell activation and the production of cytokines.²⁴ One previous research demonstrated that B7-H4-expressing macrophages was significantly increased in the peripheral blood from lung cancer patients when compared to TB patients and healthy controls.²⁵ In addition, the high expression of B7-H4 was not only positively related to tumor progression, but also involved in tumor immune escape. Chen et al found that mature activated DCs expressed CD83 and several negative immune molecules, such as B7-H4, BTLA,²⁵ and an obvious proportion increase regarding the co-expression of BTLA and B7-H4 was found in mDC from TB patients.¹⁷ Moreover, this study also implied that the high expression of B7-H4 and BTLA might play a negative regulatory influence of the mDCs on anti-TB immunity through promoting the expression of CD83 and HLA-DR. Some studies were also conducted the potential connection and the polymorphism in B7-H4 gene and susceptibility to human disease. Zhang et al performed an association study of rs10754339, rs10801935, rs3738414 variants in B7-H4 gene in breast cancer, and found that these SNPs were connected with the risk of breast cancer.²⁶ The results by Chen et al indicated that two polymorphisms (rs10801935, rs3738414) in B7-H4 gene were significantly associated with AS susceptibility, and TAG haplotype was a risk factor for AS by haplotype analysis.²⁰ Therefore, we speculated that B7-H4 gene polymorphism might also be related to the susceptibility to PTB, and analyzed the association between five SNPs (rs10754339, rs10801935, rs10923223, rs1937956, rs3738414) in B7-H4 gene with PTB risk. The rs3738414 GA genotype was found to increase susceptibility to PTB, but this association was not statistically significant after Bonferroni correction. Furthermore, AATTG haplotype in the B7-H4 gene might be associated with an increased risk of PTB. This suggested that B7-H4 gene variation might be involved in the pathogenesis of PTB, but further verification was needed.

As a newly discovered inhibitory receptor, BTLA exerted important roles in suppressing the overreactive immune response and could play immunosuppressive effects on the host by activating B cells and T cells.²⁷ Recent evidences described the important function of BTLA in many diseases, including inflammatory disorder, autoimmune diseases and PTB.^23,28 Previous studies regarding the roles of BTLA on PTB mainly focused on its expression level in several specific cell.^18,23 Wang et al revealed the effect of BTLA in CD11c antigen presenting cells (APCs) in active PTB patients, and found that BTLA-expressing CD11c APCs showed a stronger ability to stimulate T cell proliferation.¹⁸ Another study demonstrated that the expression of CD11c⁺ BTLA was positively related to CRP, which might be correlated with inflammatory changes in PTB patients.²⁹ The present study mainly focused on studying the association of four SNPs (rs1982809, rs2971205, rs75368388, rs9288953) in BTLA gene with PTB risk, while no significant association was found between these SNPs and the risk of PTB. Contrary to our study, previous studies displayed significant correlation between BTLA gene polymorphisms and cancer, autoimmune diseases. Andrzejczak et al found that rs1982809 variant was significantly related to non-small cell lung cancer risk.³⁰ Karabon et al reported that rs1982809, rs9288953 variants would affect the susceptibility to chronic lymphocytic leukemia.³¹ It was worth noting that rs9288953 variant was found to be significantly associated with the development of drug resistance among PTB patients. Therefore, BTLA gene polymorphism could be used as a potential index to determine the clinical classification of PTB patients.

In addition to DNA sequences, important genetic information was also present in epigenetic variations.³² Gene polymorphism and epigenetic mechanisms might interact and together influenced the biological function and disease progression.³³ Therefore, the regulatory role of abnormal DNA methylation, which was considered as a common epigenetic trait, of certain key genes in human diseases had been reported.³⁴ DNA methylation had become an irreplaceable way to explore the pathogenesis of PTB. The results of Wang et al showed that abnormal methylation status of several key genes in the vitamin D metabolic pathway was closely connected with the risk and prognosis of PTB.¹² Considering the potential significance of B7-H4 and BTLA genes polymorphisms in PTB development, this study further analyzed the relationship among their methylation levels and PTB risk. Here, the present results demonstrated that the methylation levels of B7-H4 and BTLA genes were significantly increased in PTB patients. Interestingly, we found that B7-H4 methylation level was affected rs10754339 variants among PTB patients. This implied that abnormal methylation levels of B7-H4 and BTLA genes were involved in the pathogenesis of PTB, and there might be an interaction between B7-H4 gene polymorphism and its methylation in PTB patients. The above findings provided important clues to reveal the occurrence of PTB, while relevant results still needed to be further verified.

Some limitations of this study should be noted. First, our study only screened several tagSNPs in B7-H4 and BTLA genes, and some SNPs with important functions might be missed. Second, we compared methylation levels of B7-H4 and BTLA genes between PTB patients and controls in a cross-sectional study, and did not analyze their changes levels over the course of PTB development. Third, our study did not exclude the possible influence of environmental factors and ethnic background on the results.

In summary, B7-H4, BTLA genes polymorphism might not be affected PTB susceptibility in the Chinese population, while the abnormal methylation levels of B7-H4, BTLA genes were associated with PTB risk. These results firstly revealed the roles of B7-H4, BTLA genes methylation in the development of PTB, which was important for PTB control and prevention. Moreover, the further studies regarding the mechanism of B7-H4, BTLA genes variation on PTB development was still warranted in future.