Background
Pulmonary tuberculosis (TB) caused by
Mycobacterium tuberculosis (MTB), is one of the most contagious diseases in humans. A recent report (2014) from WHO estimated that one third of the global population has been latently infected without clinical symptoms. In 2012, approximately 8.6 million people worldwide were diagnosed with TB and 1.3 million died from TB [
1]. MTB could infect human by invading antigen presenting cell (Macrophages and Dendritic cells) in the lung. But only 5-10% of infected people developed clinical symptoms probably due to undermined immunity response [
1].
Toll-like receptors (TLRs) are pattern recognizing receptors (PRRs) and play an important role in regulating human’s immune system. The TLRs can recognize pathogen-associated molecular patterns (PAMPs) in both extracellular and intracellular environment. The TLRs have the ability to initiate signaling pathways that are responsible for activating both innate and acquired immune responses as well as the production of inflammatory cytokines. The members of TLRs family have been detected on human plasma membrane (TLR1, TLR2, TLR4-6) and in leukocyte endosome (TLR3, TLR7-9) [
2]. For the risk of MTB infection, the most important TLRs are TLR1, TLR2, TLR4, TLR6 and TLR9 [
3]. TLR1 combines with TLR2 as a heterodimer and recognizes triacylated lipopeptides of MTB. TLR2-TLR6 complex recognizes diacylated lipopeptides and bacterial LTA of Mycoplasmal compounds. TLR4 recognizes lipopolysaccharide (LPS) and TLR9 homodimer recognizes the exogenous MTB DNA as PAMPs [
4]. Studies focusing on TLR1, 2, 4 and 6 have found that single nucleotide polymorphisms (SNPs) of these TLRs genes might be associated with differential TB risk. However, neither systematic review nor meta-analysis has been reported for TLR9 [
3].
The gene of toll-like receptor 9 is located on chromosome 3p21.3. The total length of TLR9 gene is approximate 5 kb. Its coding gene has two exons, and the major coding region is in the second exon [
5]. Based on NCBI SNP database, twelve SNPs have been identified for TLR9 gene, in which NM_017442.3:c.-1486 T > C (rs187084) located at the upstream of promoter may be an important one [
6]. Previous study it has shown that C genotype was associated with reduced TLR9 transcription activity when compared with T genotype [
7], indicating population with C genotype may be susceptible to diseases related to TLR9 gene.
Researches have indicated certain race population with special genotype of TLR9 polymorphism might have higher risk for TB; however the findings are inconsistent and inconclusive. From public health perspective, given the widespread and severe nature of the disease, it is important to understand the association between TLR9 polymorphism and TB risk, so that individuals with higher risk genotype could be identified and receive targeted preventive care. In this study, we conducted a systematic review of current literature on this issue and analysis of the associations between TLR9 polymorphisms and TB risk.
Discussion
In this study, we performed a meta-analysis to assess the association between seven extensively studied TLR9 polymorphisms (rs187084, rs352165, rs5743836, rs5743842, rs352139, rs352140 and rs352167) and TB risk. The analysis revealed an association between certain TLR9 polymorphism and TB risk. In addition, 5 different genetic models (Allele, Heterozygote, Homozygote, Dominant and Recessive model) were analyzed in all polymorphisms. A subgroup analysis by race was also performed for rs187084, rs352139 and rs5743836 polymorphisms, the studies of which included Indians, Iranian and West African, Indonesians, Vietnamese, Chinese and Mexicans. The results showed that rs187084 and rs5743836 polymorphisms were not associated with TB risk, while the association between rs352139 polymorphism and TB risk may vary by race.
We chose TLR9 gene in our analysis because it’s critical role in the incidence of TB from previous studies. Chen et al. demonstrated that cell apoptosis induced by
Mycobacterium Tuberculosis required TLR9 in animal model [
24]. Another study reported that the deficiency in a major adapter of TLR9 signaling pathway significantly increased the lethality of TB. The myeloid differentiation factor 88 knock-out mice (MyD88 −/−) died within 4 weeks after MTB infection [
25]. All these evidences indicated the critical role of TLR9 in TB incidence and progression.
TLR9 is a recognition receptor in leukocytes endosome, especially in B cells, plasmacytoid dendritic cells (pDCs) and macrophages. It is localized in the endosomal and lysosomal compartments. It is one of the most important receptors for the initiation of innate immune response against intracellular pathogens. The function of TLR9 is to recognize the unmethylated Cytosine-phosphate-Guanine (CpG) sequence which usually appears in bacterial DNA [
5]. In recent study, hybrid RNA-DNA chains generated by viruses during reverse transcription were recognized by TLR9 [
26]. The Toll-IL-1 receptor (TIR) domain of TLR9 in intracellular space associates with myeloid differentiation factor 88 (MyD88), and becomes the initiator component of TLR9 signaling pathway. Once bacterial CpG-DNA in lysosome combines with the nucleic acid ligand of TLR9, the signaling pathway is activated. MyD88, once stimulated, recruits IL-1 receptor associated kinase 4 (IRAK-4) to TLR9 through interaction of the functional domains. Then, IRAK-1 is phosphorylated and recruits Tumor necrosis factor receptor associated factor 6 (TRAF6). Consequently the inhibitor of nuclear factor kappa-B kinase (IКK) complex is activated, leading to the activation of Mitogen-activated protein (MAP) kinases (JNK, p38 MAPK). The nuclear factor inhibiter (IкB) is then phosphorylated and the level of nuclear factor kappa-B (NF-кB) is elevated [
27]. NF-кBs translocate to the nucleus, and then bind to DNA sites to regulate relevant gene transcription. Finally, inflammatory cytokines are synthesized through host defense immune response by TLRs- NF-кB pathway. The signaling pathway of TLR9 was shown in Figure
2.
SNPs rs352165 and rs352167 were located in intron 5 of aminolevulinate delta-, synthase 1 (ALAS1) gene in the downstream of TLR9. Sanchez D et al. have reported that these two SNPs were in linkage disequilibrium (LD) with rs352139 and rs352140 SNPs of TLR9 [
15]. Thus they chose rs352165 and rs352167 as proxies for TLR9 gene. Lack of significant association with TB risk from our analysis indicated there was no relationship between rs352165/ rs352167 and this disease.
In our study, the association of rs352139 with TB risk varied by race. In Indonesians AA genotype is associated with lower risk of TB, while in Mexicans it is associated with higher risk of TB. The Mexicans with AA allele had 2.73 times higher risk of TB than Indonesians with the same genotype. There is no known explanation for the racial difference. Also, we observed a significant association between rs352139 polymorphism and TB risk in Indonesians for Allele, Heterozygote and Homozygote models. There was also a significant association between rs352139 polymorphism and TB risk in Mexicans for Allele, Dominant and Recessive models. Although 4 case-control studies of rs352139 were included in this analysis, there was only one study for each racial group. Therefore, our results should be interpreted with caution, and more studies with larger sample size should be considered to confirm this differential risk by race.
The SNP rs352139 is located in the intron of TLR9 gene (Figure
2). TIR domain consists of the end of exon 1, intron and a part of exon 2 which contains SNP rs352139. It is the only polymorphism located in the intron between two exons. The other polymorphisms are located in either the upstream of coding region or the exons. It is possible that the intron of TLR9 gene plays an important role in regulating the TLR-mediated immunologic response. However, it is currently unclear how this intronic SNP induces such a phenotype change. It is possible that it influences signaling by creating an alternative splicing site and thus, affecting the mRNA transcription and the final protein product. Similarly, the rs352139 SNP could be a likely marker in linkage disequilibrium with a polymorphic regulatory region that controls TLR9 expression or serve as a functional coding region SNP.
The SNP rs187084 is located in the promoter of TLR9 gene, which could create a Sp1 binding site [
28]. Variant alleles of this SNP can alter the function of TLR9, impact the response to bacterial pathogens, and thereby change individual’s disease risk [
29,
30]. In our analysis, several studies investigated the role of rs187084 in the development of tuberculosis, but none of them showed significant association.
The SNP rs5743836 is located in the promoter region of TLR9. This SNP was thought to be associated with increased transcriptional activity and function of TLR9 [
31]. In our study, rs5743836 polymorphism showed no significant association with TB risk. Nevertheless in a study of Caucasian patients, Digna Rosa Velez et al reported that the rs5743836 polymorphism had significant association with dominant and recessive models (TT vs. CT&CC, OR = 0.50, 95% CI 0.28–0.87, p = 0.015). However, we did not include these data in our analysis because only the detail of additive model was presented in the original article.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
ZC carried out the study design, data analysis. WW carried out the manuscript review. JL participated in the literature research. JW participated in the literature research. SF participated in the literature research. GZ conceived of the study. All authors read and approved the final manuscript.