Toll-Like Receptor 2 Arg677Trp Polymorphism Is Associated with Susceptibility to Tuberculosis in Tunisian Patients

Toll-like receptor 2 (TLR2) is critical in the immune response to mycobacteria. Herein, we report that the frequency of a human TLR2 Arg677Trp polymorphism (C2029T nucleotide substitution) in tuberculosis patients in Tunisia is significantly higher than in healthy controls (P < 0.0001). This finding suggests that this polymorphism could be a risk factor for tuberculosis.     

The rs5743708 gene polymorphism in the TLR2 gene contributes to the risk of tuberculosis disease

Background: It has been reported that one single nucleotide polymorphisms (SNPs) rs5743708 in TLR2 gene might be associated with the susceptibility to tuberculosis disease. Owing to mixed and inconclusive results, we conducted a meta-analysis to systematically summarize and clarify the association between the rs5743708 gene polymorphism in the TLR2 gene and the risk of tuberculosis disease. Methods: A systematic search of studies on the association of the rs5743708 gene polymorphism in the TLR2 gene with susceptibility to tuberculosis disease was conducted in PubMed. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were used to pool the effect size. Results: A total of nineteen case-control studies from 13 articles on rs2910164 and 3 studies on the rs5743708 gene polymorphism in the TLR2 gene and the risk of tuberculosis disease were included. A significant relationship between the rs5743708 gene polymorphism in the TLR2 gene and tuberculosis disease was discovered in an allelic genetic model (OR: 2.801, 95% CI: 2.130-3.683, P=0.000), a homozygote model (OR: 5.795, 95% CI: 1.982-16.941, P=0.001), a heterozygote model (OR: 2.628, 95% CI: 1.888-3.569, P=0.000), a dominant genetic model (OR: 2.786, 95% CI: 2.003-3.877, P=0.000) and a recessive genetic model OR: (5.568, 95% CI: 1.907-16.255, P=0.002). In sub-group analysis base on ethnicity, significance was observed between the Caucasian group and the Asian group. Conclusions: The rs5743708 gene polymorphism in the TLR2 gene contributes to the risk of tuberculosis disease. Individuals with the rs5743708 gene polymorphism in the TLR2 gene are under a higher risk for tuberculosis disease.     

The association between microsatellite polymorphisms in intron II of the human Toll-like receptor 2 gene and tuberculosis among Koreans

The observation that Toll-like receptor (TLR)2-deficient mice are highly susceptible to mycobacteria suggests that mutations altering TLR2 expression may impair host response to Mycobacterium tuberculosis. We evaluated the association between guanine-thymine (GT) repeat polymorphism in intron II of the TLR2 gene and the presence of tuberculosis (TB) in Koreans. The numbers of GT repeats were determined by PCR and gene scans for 176 TB patients and 196 controls. The recombinant TLR2 promoter/exonI/exonII/intronII/luciferase constructs including three representative repeats: (GT)13, (GT)20, and (GT)24 were transfected into K562 cells, and luciferase activities were estimated and compared. The expression of TLR2 on CD14+ peripheral blood mononuclear cells (PBMC) from healthy volunteers were measured with flow cytometry. Genotypes with shorter GT repeats were more common among TB patients (49.4 vs 37.7%, P=0.02). This observation was confirmed among 82 other TB patients as a validation cohort. Shorter GT repeats were associated with weaker promoter activities and lower TLR2 expression on CD14+ PBMCs. In conclusion, the development of TB disease in Koreans was associated with shorter GT repeats in intron II of the TLR2 gene. This association is correlated with lower expression of TLR2 through weaker promoter activity for genes with shorter GT repeats.     

A common human TLR1 polymorphism regulates the innate immune response to lipopeptides

Toll-like receptors (TLR) are critical mediators of the immune response to pathogens and human polymorphisms in this gene family regulate inflammatory pathways and are associated with susceptibility to infection. Lipopeptides are present in a wide variety of microbes and stimulate immune responses through TLR1/2 or TLR2/6 heterodimers. It is not currently known whether polymorphisms in TLR1 regulate the innate immune response. We stimulated human whole blood with triacylated lipopeptide, a ligand for TLR1/2 heterodimers, and found substantial inter-individual variation in the immune response. We sequenced the coding region of TLR1 and found a non-synonymous polymorphism, I602S (base pair T1805G), that regulated signalling. In comparison to TLR1_602S, the 602I variant mediated substantially greater basal and lipopeptide-induced NF-kappaB signalling in transfected HEK293 cells. These signalling differences among TLR1 variants were also found with stimulation by extracts of Mycobacterium tuberculosis. Furthermore, individuals with the 602II genotype produced substantially more IL-6 than those with the 602SS variant in a lipopeptide-stimulated whole-blood cytokine assay. Together, these observations demonstrate that variation in the inflammatory response to bacterial lipopeptides is regulated by a common TLR1 transmembrane domain polymorphism that could potentially impact the innate immune response and clinical susceptibility to a wide spectrum of pathogens.     

Genetic Polymorphisms in the Toll‐like Receptor 10, Interleukin (IL)17A and IL17F Genes Differently Affect the Risk for Tuberculosis in Croatian Population

Proinflammatory conditions leading to activation of macrophages via interferon‐γ bear an important role in host defence against intracellular bacteria such as Mycobacterium tuberculosis (Mt). Interleukin‐17 plays a similar role, as it appears to be also an activator of macrophages. Recently, the TLR‐10 was identified as an anti‐inflammatory factor that exerts its action via association with the TLR‐2 chain at the cell surface of macrophages, the latter being an Mt‐binding protein. We have previously found that gene polymorphisms that either inactivate the TLR2 gene product or have a dominant‐negative role are associated with tuberculosis (TB) in Croatian population. We have now extended our survey and found that single nucleotide polymorphism (SNP) in TLR10 (rs11096957) is associated with risk for TB. Homozygotes carrying the A allele are associated with predisposition to disease as analysed by the dominant model of inheritance. In contrast, SNPs in the proinflammatory IL17A and IL17F genes (rs2275913 and rs763780, respectively), found previously to correlate with the disease occurrence in Chinese population, were not significantly associated with tuberculosis in the Croatian population.     

Toll‐like receptor‐2 Arg753Gln and Arg677Trp polymorphisms and susceptibility to pulmonary and peritoneal tuberculosis

Recent evidence suggests that toll‐like receptor‐2 (TLR2) is important for host defense against Mycobacterium tuberculosis (MTB). TLR2 polymorphisms have shown significant impact on susceptibility or resistance to tuberculosis (TB). This case–control study aims to determine the influence of TLR2 (Arg753Gln and Arg677Trp) polymorphisms on the susceptibility to develop pulmonary or peritoneal TB. Genotyping of TLR2 (Arg753Gln and Arg677Trp) polymorphisms was carried out on 52 patients with pulmonary TB, 44 patients with peritoneal TB, and 50 healthy controls using polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP). There was a significant association between the GA genotype (heterozygous mutant) of TLR2 Arg753Gln polymorphism and the risk of infection with pulmonary TB (p = 0.003, OR = 4.83) and TB peritonitis (p = 0.003, OR = 6.2). Differences in the genotype frequencies of TLR2 Arg677Trp polymorphisms between patients with pulmonary or peritoneal TB and healthy controls were not detected. GA753 TLR2 polymorphism may play a role in the susceptibility to pulmonary and peritoneal TB infection. Further studies on a large number of ethnically diverse patient cohorts may help to confirm the possible effect of these polymorphisms on the susceptibility to pulmonary and peritoneal TB.     

Genetic polymorphisms of CCL1 rs2072069 G/A and TLR2 rs3804099 T/C in pulmonary or meningeal tuberculosis patients

CCL1, one of the members of the CC chemokine family, is an inflammatory mediator that stimulates the migration of human monocytes. CCL1 expression is induced by Mycobacterium tuberculosis and TLR ligands in macrophage. TLR2 plays critical role in host immune response against M. tuberculosis infection by regulating the macrophage activation and cytokine secretion. M. tuberculosis causes different clinical forms of tuberculosis (TB) disease. Single-nucleotide polymorphisms (SNPs) in the CCL1 gene and TLR2 gene may be associated with the development of different clinical forms of TB, depending on the different immune mechanisms. This study was to evaluate the possible association between CCL1 rs2072069 G/A or/and TLR2 rs3804099 T/C (T597C) polymorphisms and pulmonary tuberculosis (PTB) or/and tuberculous meningitis (TBM) in a sample of the Chinese adult population. A case-control study was designed to compare the allele frequency and genotype distribution between control (n=386) and TB (n=341) who had either PTB (n=230) or TBM (n=111). The genotype typing was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. TLR2 variant genotype 597CC was associated with susceptibility to PTB rather than to TBM. In the male PTB subgroup, 597CC genotype was identified in a higher rate, compared with male control subgroup. This study demonstrates that T597C polymorphism of TLR2 is a risk factor for susceptibility to PTB rather than to TBM in a sample of Chinese adult population. Patient gender may affect the outcome of M. tuberculosis infection. TLR2 gene may influence the development of PTB and TBM by different immune mechanisms.     

Expression of toll-like receptor 2 and plasma level of interleukin-10 are associated with outcome in tuberculosis

Toll-like receptor (TLR) 2-mediated innate immunity is an important defense system against Mycobacterium tuberculosis infection. Studies on TLR2 protein expression and downstream cytokines in tuberculosis patients are lacking. TLR2 expression in the peripheral blood monocytes of 87 tuberculosis patients and 94 healthy subjects was evaluated using flow cytometry. TLR2 expression and its downstream cytokines, including interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-alpha, and interferon-gamma, were correlated with the clinical manifestations and outcomes of tuberculosis. The TLR2 expression in peripheral blood monocytes was higher in tuberculosis patients than in healthy subjects. Among the tuberculosis patients, those aged ≥70 years with disseminated tuberculosis or aged <70 years with symptom duration ≥14?days had lower initial TLR2 expression. After two months of treatment, TLR2 expression decreased in most patients, except in those whose sputum samples remained culture-positive for M. tuberculosis. Proportional hazards regression analyses revealed that high initial TLR2 expression and IL-10 plasma level were associated with shorter survival. TLR2 may play an important role in the course of tuberculosis. Its expression on peripheral blood monocytes and the plasma level of the downstream anti-inflammatory cytokine IL-10 may be important outcome predictors and have potential use in the management of tuberculosis patients.     

Epiregulin (EREG) variation is associated with susceptibility to tuberculosis

Although host genetics influences susceptibility to Mycobacterium tuberculosis, the human genes regulating pathogenesis remain largely unknown. We used M. tuberculosis-stimulated macrophage gene expression profiling in conjunction with a case-control genetic association study to discover epiregulin (EREG), as a novel candidate tuberculosis (TB) susceptibility gene. Using a genome-wide association study dataset, we found that among the 21 genes with greater than 50-fold induction, EREG had the most polymorphisms associated with TB. We genotyped haplotype-tagging polymorphisms in discovery (N = 337 cases, N = 380 controls) and validation (N = 332 cases) datasets and an EREG polymorphism (rs7675690) was associated with susceptibility to TB (genotypic comparison; corrected P = 0.00007). rs7675690 was also associated more strongly with infections caused by the Beijing lineage of M. tuberculosis when compared with non-Beijing strains (controls vs Beijing, OR 7.81, P = 8.7 × 10(-5); non-Beijing, OR 3.13, P = 0.074). Furthermore, EREG expression was induced in monocytes and peripheral blood mononuclear cells stimulated with M. tuberculosis as well as TLR4 and TLR2/1/6 ligands. In murine macrophages, EREG expression induced by M. tuberculosis was MYD88- and TLR2-dependent. Together, these data provide the first evidence for an important role for EREG as a susceptibility gene for human TB.     

Role of Toll‐like receptor 2 (–196 to –174) polymorphism in susceptibility to pulmonary tuberculosis in Pakistani population

Tuberculosis (TB) is a global public health problem and a source of preventable deaths each year, with 8.8 million new cases of TB and 1.6 million deaths worldwide. Pakistan ranks sixth on the list of 22 high‐burden tuberculosis countries in the world. The transitions from infection to clinical disease are very few signifying that host‐defence factors could lead to the development of active disease. Toll‐like receptor (TLR) 2 polymorphisms have been associated with regulation of TLR expression and development of active TB. In this study, blood samples of 187 subjects including 100 healthy and 87 TB positive were collected from three districts of Pakistan. DNA was extracted from blood and TLR 2 (–196 to –174del) polymorphism was analysed by polymerase chain reaction (PCR). The results suggest that the frequency of –196 to –174del/del polymorphism of TLR2 was significantly higher in TB‐positive patients compared with healthy. Results revealed that (–196 to –174del) polymorphism may increase the susceptibility to TB in healthy population of Pakistan. Moreover, males with heterozygous genotype (I/D) are more prone to TB than females with the same genotype. The occurrence of TB infection has been found positively associated with the age, suggesting that the population within the range of 21–45 years is more susceptible to Mycobacterium tuberculosis than other age groups studied. A significant association is also observed between smoking and the chances of developing TB, confirming that smoking strongly promotes its incidence.     

Genetic modulation of TLR8 response following bacterial phagocytosis

Human Toll‐like receptors (TLRs) TLR7, TLR8, and TLR9 are important immune sensors of foreign nucleic acids encountered by phagocytes. Although there is growing evidence implicating TLR7 and TLR9 in the detection of intracellular pathogenic bacteria, characterization of such a role for TLR8 is currently lacking. A recent genetic study has correlated the presence of a TLR8 single nucleotide polymorphism (SNP) (rs3764880:A>G; p.Met1Val) with the development of active tuberculosis, suggesting a role for TLR8 in the detection of phagosomal bacteria. Here we provide the first direct evidence that TLR8 sensing is activated in human monocytic cells following Helicobacter pylori phagocytosis. In addition, we show that rs3764880 fine tunes translation of the two TLR8 main isoforms, without affecting protein function. Although we show that TLR8 variant 2 (TLR8v2) is the prevalent form of TLR8 contributing to TLR8 function, we also uncover a role for the TLR8 long isoform (TLR8v1) in the positive regulation of TLR8 function in CD16⁺CD14⁺ differentiated monocytes. Thus, TLR8 sensing can be activated following bacterial phagocytosis, and rs3764880 may play a role in the modulation of TLR8‐dependent microbicidal response of infected macrophages. Hum Mutat 31:1069–1079, 2010. © 2010 Wiley‐Liss, Inc.     

Toll‐Like Receptor 2 (P631H) Mutant Impairs Membrane Internalization and is a Dominant Negative Allele

We have sequenced 416 Toll‐like receptor‐2 (TLR2) alleles in 208 subjects in a tuberculosis case–control study in Croatian Caucasian population. We found ten single nucleotide polymorphisms (SNP) among which three were novel (S97S, T138I and L266F). The genotype containing TLR2‐P631H SNP was significantly overrepresented in patients with tuberculosis when compared to contact controls, suggesting a small yet increased risk to disease. The causative agent of tuberculosis is Mycobacterium tuberculosis, which can bind to TLR2 with its lipoprotein coat. The TLR2‐P631H mutant has a dominant negative effect on the wild type TLR2 signalling in transfected HEK293 kidney cells using the NF‐κB‐driven luciferase as a reporter gene with ligands like M. avium extracts, Pam3CysSK4 or FSL‐1 that bind TLR2/TLR1 or TLR2/TLR6 heterodimers, respectively. Studies on internalization from the Regular Madine Darby Canine Kidney cell surface into the early endosomal compartments showed a lower rate of the mutant compared to the wild type. Our data, in combination with a report by others show that the TLR2‐P631H allele could be associated with protection to meningococcal meningitis, suggest that by dominantly inhibiting the response of cells important in the immune response this mutant might confer either protection or susceptibility to meningitis or tuberculosis, respectively.     

Toll样受体在结核病免疫应答中的作用

结核病(Tuberculosis)主要是由结核分枝杆菌(Mycobacterium tuberculosis,MTB)引起的重大传染性疾病。Toll样受体(Toll-like receptors,TLRs)在宿主的抗结核免疫应答中发挥重要作用。研究表明,TLR1、2、4、6和9与宿主抗MTB感染有关,其中TLR2的作用更为突出。免疫应答的早期阶段,TLR2介导了巨噬细胞的活化,通过产生具有直接杀伤效应的分子或者诱导宿主细胞的凋亡抑制MTB的增殖。然而TLR2介导的信号通路也可通过降低MHC-Ⅱ分子的表达来削弱抗原递呈的能力,促进MTB在宿主内的存活。近几年临床研究发现TLRs多态性位点与结核病易感有关也从侧面证实了TLRs在抗MTB感染中发挥重要作用。     

Arg753Gln polymorphism of the human Toll-like receptor 2 gene from infection to disease in pediatric tuberculosis

The aim of this study is to examine the occurrence of the Arg753Gln polymorphism of the Toll-like receptor 2 (TLR2) gene in Turkish children with pulmonary and/or extrapulmonary tuberculosis (TB) disease compared with that in healthy children with latent TB infection (LTBI) and to assess the risk of progression from LTBI to active TB disease in children. The Arg753Gln polymorphism of the TLR2 gene was studied in 198 TB patients compared with 200 ethnically and age-matched children with LTBI. The culture confirmed TB patients were more frequently Arg753Gln heterozygous [odds ratio (OR) 5.05, 95% confidence interval (95% CI) 2.61-9.76, p = 0.00], and Gln allele frequency was significantly higher in the patient group (13.86% vs 3.5%, OR 4.40, 95% CI 2.34-8.30, p = 0.00). We also showed that the frequencies of the heterozygous Arg753Gln genotype and the Gln allele were significantly higher in patients with pulmonary TB alone and in patients with definitive pulmonary plus extrapulmonary TB than in children with LTBI. Our data suggest that the Arg753Gln polymorphism of the TLR-2 gene influences the speed of progression from infection to TB disease in children. Further investigations are needed to clarify whether this polymorphism has a strong impact on susceptibility to TB in children.     

Association of TLR polymorphisms with development of tuberculosis in Indonesian females

Tuberculosis (TB) is caused by Mycobacterium tuberculosis and is a major cause of morbidity and mortality worldwide. Many candidate genes have been investigated for a possible association with TB. Toll-like receptors (TLRs) are known to play important roles in human innate immune systems. Polymorphisms in and functions of TLRs have been investigated to identify associations with specific infectious diseases, including TB. Here, we examined whether single-nucleotide polymorphisms (SNPs) in TLRs and genes in TLR signaling were associated with TB susceptibility in Indonesian and Vietnamese populations. A statistically significant association was observed between TB susceptibility in a classified Indonesian female group and rs352139, an SNP located in the intron of TLR9, using the genotype (P = 2.76E-04) and recessive (AA vs AG+GG, P = 2.48E-04, odds ratio = 1.827, 95% confidence interval = 1.321-2.526) models. Meta-analysis of the Indonesian and Vietnamese populations showed that rs352139 was significantly associated with TB in the recessive model. This finding indicated that a TLR9 polymorphism might have an important role in the susceptibility to M. tuberculosis in Asian populations.     

Mycobacterium tuberculosis lipoproteins directly regulate human memory CD4(+) T cell activation via Toll-like receptors 1 and 2

The success of Mycobacterium tuberculosis as a pathogen relies on its ability to regulate the host immune response. M. tuberculosis can manipulate adaptive T cell responses indirectly by modulating antigen-presenting cell (APC) function or by directly interacting with T cells. Little is known about the role of M. tuberculosis molecules in direct regulation of T cell function. Using a biochemical approach, we identified lipoproteins LprG and LpqH as major molecules in M. tuberculosis lysate responsible for costimulation of primary human CD4(+) T cells. In the absence of APCs, activation of memory CD4(+) T cells with LprG or LpqH in combination with anti-CD3 antibody induces Th1 cytokine secretion and cellular proliferation. Lipoprotein-induced T cell costimulation was inhibited by blocking antibodies to Toll-like receptor 2 (TLR2) and TLR1, indicating that human CD4(+) T cells can use TLR2/TLR1 heterodimers to directly respond to M. tuberculosis products. M. tuberculosis lipoproteins induced NF-κB activation in CD4(+) T cells in the absence of TCR co-engagement. Thus, TLR2/TLR1 engagement alone by M. tuberculosis lipoprotein triggered intracellular signaling, but upregulation of cytokine production and proliferation required co-engagement of the TCR. In conclusion, our results demonstrate that M. tuberculosis lipoproteins LprG and LpqH participate in the regulation of adaptive immunity not only by inducing cytokine secretion and costimulatory molecules in innate immune cells but also through directly regulating the activation of memory T lymphocytes.     

A novel polymorphism in the toll-like receptor 2 gene and its potential association with staphylococcal infection

The toll-like receptor 2 (TLR2) has gained importance as a major mammalian receptor for lipoproteins derived from the cell wall of a variety of bacteria, such as Borrelia burgdorferi, Treponema pallidum, and Mycoplasma fermentans. We were interested in identifying mutations in the TLR2 gene that might prove to be associated with altered susceptibility to septic shock. We performed a mutation screen of the TLR2 gene using single-stranded conformational polymorphism in 110 normal, healthy study subjects and detected an Arg753Gln mutation in three individuals. No other missense mutations were detected in the TLR2 open reading frame. Functional studies demonstrate that the Arg753Gln polymorphism, in comparison to the wild-type TLR2 gene, is significantly less responsive to bacterial peptides derived from B. burgdorferi and T. pallidum. In a septic shock population, the Arg753Gln TLR2 polymorphism occurred in 2 out of 91 septic patients. More importantly, both of the subjects with the TLR2 Arg753Gln polymorphism had staphylococcal infections. These findings suggest that a mutation in the TLR2 gene may predispose individuals to life-threatening bacterial infections.     

Toll-like receptor 4 plays a protective role in pulmonary tuberculosis in mice

Toll-like receptors (TLR) play an essential role in the innate recognition of microorganisms by the host. To determine the role of TLR4 in host defense against lung tuberculosis, TLR4 mutant (C3H/HeJ) and wild-type (C3H/HeN) mice were intranasally infected with live Mycobacterium tuberculosis. TLR4 mutant mice were more susceptible to pulmonary tuberculosis, as indicated by a reduced survival and an enhanced mycobacterial outgrowth. Lung infiltrates were more profound in TLR4 mutant mice and contained more activated T cells. Splenocytes of infected TLR4 mutant mice demonstrated a reduced capacity to produce the protective type 1 cytokine IFN-gamma upon antigen-specific stimulation, indicating that TLR4 may be involved in the generation of acquired T cell-mediated immunity. These data suggest that TLR4 plays a protective role in host defense against lung infection by M. tuberculosis.     

TLR2 deficiency by compromising p19 (IL-23) expression limits Th 17 cell responses to Mycobacterium tuberculosis

CD4(+) T(h)1 cells producing IFN-γ are of extreme importance in controlling infections by Mycobacterium tuberculosis both in mice and in men. In addition to IFN-γ-producing T cells, IL-17-producing T cells (T(h)17) have been observed during mycobacterial infections. Nevertheless, their contribution for the host immune response to mycobacteria as well as the signals triggering M. tuberculosis -specific T(h)17 cell differentiation and maintenance are not fully understood. We show that signaling via Toll-like receptor (TLR) 2 has a major impact on the regulation of p19 (IL-23) expression in response to M. tuberculosis and therefore on the establishment of T(h)17 cell responses to M. tuberculosis infection. Diminished T(h)17 responses in the lung of M. tuberculosis -infected TLR2-deficient animals were not caused by defective cell differentiation in the draining lymph node (LN) but rather by reduced maintenance at the site of infection. Consistent with the decreased numbers of T(h)17 cells in the lungs of infected TLR2-deficient animals, we observed reduced expression of CXCL9, CXCL10 and CXCL11, chemokines involved in recall responses to M. tuberculosis. Our data provides insights into the TLR2 role in infection with M. tuberculosis, with implications in pathophysiology of the disease and vaccine design.