TLR4 Thr399Ile Research Articles

Study of disease-relevant polymorphisms in the TLR4 and TLR9 genes: A novel method applied to the analysis of the Portuguese population

Toll-like receptors (TLRs) are cellular receptors that mediate recognition of microbial challenges and the subsequent inflammatory response. Genetic variations within these inflammation-associated genes may alter host-pathogen defence mechanisms affecting susceptibility towards infectious diseases. Taking into account the significance of these genes, we developed a simple and rapid method based in the bi-directional PCR amplification of specific alleles (Bi-PASA) for genotyping known sequence variants in TLR4 (Asp299Gly and Thr399Ile) and TLR9 (T-1237C) genes. This method allows genotype determination in a single reaction and is amenable to large-scale analysis. We used Bi-PASA to characterize the distribution of these polymorphisms in the Portuguese population. A total of 388 randomly selected blood donors of Portuguese origin (203 females and 185 males) were genotyped and allele frequencies were determined. Among the tested individuals, 11.1% and 10.8% were heterozygous for Asp299Gly and Thr399Ile, respectively. In what concerns the T-1237C variation in TLR9, the variant allele was present in 19.4% of the individuals tested. Besides confirming the usefulness of the Bi-PASA in polymorphism analysis, the data presented provide valuable information on TLR polymorphisms in the Portuguese population that can be used to stratify risk patients with increased susceptibility to infection.

Toll-like receptor and antiphospholipid mediated thrombosis: in vivo studies

Objective:A study was undertaken to investigate the in vivo pathogenic role of Toll-like receptor 4 (TLR-4) in the antiphospholipid syndrome (APS) by studying the thrombogenic antiphospholipid (aPL) activity in lipopolysaccharide...

Genotypic analysis of Asp299Gly and Thr399Ile polymorphism of Toll-like receptor 4 in systemic autoimmune diseases of Korean population

Dear Editor, Systemic autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus are mainly characterized by chronic inXammation ampliWed by large quantities of proinXammatory cytokines released from immune cells including macrophages and dendritic cells. Although the pathogenesis of autoimmune diseases are largely unknown, adaptive immune responses in various systemic autoimmune diseases can be driven by innate immune system as exempliWed in pathogenic autoantibody production triggered by infectious agents in the pathogenesis of rheumatoid arthritis and systemic lupus erythematosus [1, 2]. Toll-like receptors (TLRs) are phylogenetically conserved receptors expressed on various cells, mainly on macrophages and dendritic cells that recognize pathogen-associated molecular patterns and are key players in cellular innate immunity with an emerging role as a bridge between innate and adaptive immune responses [3]. TLRs, especially TLR4, are predominantly activated by bacterial lipopolysaccharides [4] and by endogenous ligands such as heat shock proteins [5], fragments of hyaluronic acid [6] and Wbronectin [7], which are released by damaged cells. Activation of TLRs Wnally leads to activation of nuclear transcription factors such as NFB via signal transduction involving various intracellular signaling systems and to expression of various inXammatory cytokines. There are two variants of TLR4 gene, Asp299Gly and Thr399Ile; both are present in a putative coreceptor-binding region of TLR-4 and in linkage disequilibrium [8]. The polymorphisms at these two TLR4 variants appeared to be correlated with the susceptibility to gram-negative septic shock [9], the development of atherosclerosis [10] and ischemic stroke [11], and with the host susceptibility to autoimmune process in human [12, 13]. Therefore, aberrant TLR functions, especially TLR4 involved in host defense against Gram-negative bacteria, may alter the response of this receptor to stimulation with LPS and probably endogenous ligands and eventually may induce the loss of self-tolerance and triggering of autoimmune diseases. The expression and function of TLR4 in systemic autoimmune diseases have been studied by diVerent groups, but the frequencies of variant alleles and the clinical signiWcance were diVerent depending on diagnosis or on race [9–14]. The frequencies of TLR4 gene polymorphism in these two alleles among Korean population, is not known. Therefore, we report the frequencies of TLR4 Asp299Gly and Thr399Ile polymorphism in E.-S. Kang Division of Diagnostic Immunology, Department of Laboratory Medicine, Ewha Womans University Mokdong Hospital, College of Medicine, Ewha Womans University, Mokdong, Yangcheongu, Seoul 135-710, South Korea

Analysis of TLR4 Polymorphic Variants: New Insights into TLR4/MD-2/CD14 Stoichiometry, Structure, and Signaling

TLR4 is the signal-transducing receptor for structurally diverse microbial molecules such as bacterial LPS, respiratory syncytial virus fusion (F) protein, and chlamydial heat shock protein 60. Previous studies associated two polymorphic mutations in the extracellular domain of TLR4 (Asp(299)Gly and Thr(399)Ile) with decreased LPS responsiveness. To analyze the molecular basis for diminished responsiveness, site-specific mutations (singly or coexpressed) were introduced into untagged and epitope (Flag)-tagged wild-type (WT) TLR4 expression vectors to permit a direct comparison of WT and mutant signal transduction. Coexpression of WT TLR4, CD14, and MD-2 expression vectors in HEK293T cells was first optimized to achieve optimal LPS-induced NF-kappaB reporter gene expression. Surprisingly, transfection of cells with MD-2 at high input levels often used in the literature suppressed LPS-induced signaling, whereas supraoptimal CD14 levels did not. Under conditions where WT and polymorphic variants were comparably expressed, significant differences in NF-kappaB activation were observed in response to LPS and two structurally unrelated TLR4 agonists, chlamydial heat shock protein 60 and RSV F protein, with the double, cosegregating mutant TLR4 exhibiting the greatest deficiency. Overexpression of Flag-tagged WT and mutant vectors at input levels resulting in agonist-independent signaling led to equivalent NF-kappaB signaling, suggesting that these mutations in TLR4 affect appropriate interaction with agonist or coreceptor. These data provide new insights into the importance of stoichiometry among the components of the TLR4/MD-2/CD14 complex. A structural model that accounts for the diminished responsiveness of mutant TLR4 polymorphisms to structurally unrelated TLR4 agonists is proposed.

The Toll-like receptor 4 (Asp299Gly) polymorphism is a risk factor for Gram-negative and haematogenous osteomyelitis

Osteomyelitis is a bone infection caused mostly by Staphylococcus aureus but also by Gram-negative bacteria. Toll-like receptors (TLRs), after recognizing microbial products, induce a signal in neutrophils, leading to NF-kappaB activation and transcription of pro-inflammatory genes. Polymorphisms in TLR2 (Arg753Gln) and TLR4 (Asp299Gly, Thr399Ile) genes are associated with bacterial infections, we therefore studied these polymorphisms in osteomyelitis patients. Homozygotes for the TLR4 (Asp299Gly) polymorphism were significantly more frequent among the 80 osteomyelitis patients than in the 155 healthy controls (3/80, 3.8%versus 0/155, 0%; P = 0.038). Carriers of one or two G alleles of this tlr4 polymorphism were more likely to have Gram-negative, haematogenous and/or chronic osteomyelitis than those without this mutation (P < 0.031). Patients with the TLR4 (Thr399Ile) mutant, which cosegregates with the TLR4 (Asp299Gly), were also carriers of this second polymorphism. No differences for the TLR2 (Arg753Gln) genotypes were found between patients and controls. Neutrophils of patients homozygous for the TLR4 (Asp299Gly) polymorphism showed lower LPS-induced apoptosis reduction, phosphorylation of the inhibitor of NF-kappaB, and lower IL-6 and TNF-alpha levels (P < 0.05). We report here for the first time an association between this TLR4 polymorphism and susceptibility to Gram-negative bacteria and haematogenous osteomyelitis.

Adiposity and bone mineral density of Chilean elderly women in relation to toll-like receptor 4 gene polymorphisms

Background: It has been proposed that the toll-like receptor-4 gene (TLR4) may participate in the development of obesity and osteoporosis, in addition to its well-known role in the immune response. On the other hand, the adipose tissue of obese subjects shows an increased expression of the proinflammatory cytokine, tumour necrosis factor-alpha (TNF-α), which is released after lipopolysaccharide recognition by TLR4.Aim: To estimate the allele/genotype frequencies and linkage disequilibrium measures of Asp299Gly and Thr399Ile polymorphisms of the TLR4 gene in the Chilean elderly population, and to screen for their association with variables related to adiposity or bone mineral density.Subjects and methods: The study group included 227 unrelated Chilean elderly women (61–95 years) recruited from a population-based sample. Adiposity and bone mineral density measures were obtained using dual-energy X-ray absorptiometry.Results: The allele frequencies for TNF −308A, TLR4 299Gly and TLR4 −399Ile were 9.3%, 4.6% and 4.4%, respectively, with Asp299Gly and Thr399Ile being in strong linkage disequilibrium (D′ = 0.88). Although seriously restricted by the low frequency of the allele variants, no relevant association between genotypes and adiposity-related variables were found. Likewise, no significant association between osteoporosis status (categorized as osteoporosis, osteopenia or normal status) with TLR4 Asp299Gly or TNF −308G>A genotypes was found.Conclusion: It is unlikely that TLR4 Asp299Gly, TLR4 Thr399Ile or TNF −308G>A polymorphisms have a major influence on adiposity, bone mineral density or osteoporosis status in Chilean elderly women.

Mutations in Innate Immune System NOD2/CARD 15 and TLR-4 (Thr399Ile) Genes Influence the Risk for Severe Acute Graft-versus-Host Disease in Patients Who Underwent an Allogeneic Transplantation

NOD2 and TLR-4 genes belong to the innate immune system that detects invading pathogens through several pattern-recognition receptors. Here we analyzed 403 patients for NOD2 gene mutations and 307 patients for TLR-4 gene mutations (Thr399Ile) with their respective donors and correlated the results with the incidence of acute graft-versus-host disease (aGVHD), severe acute GVHD (saGVHD), the risk for transplant-related mortality (TRM), overall survival (OS) and incidence of infectious complications. We performed a retrospective single-center study. Genotyping of TLR-4 and NOD2 were evaluated by real-time polymerase chain reaction. Surprisingly, we found a significant reduced incidence of aGVHD, saGVHD, and intestinal GVHD for patients with NOD2 gene mutations on the donor side with 50%, 0% and 2% compared to patients with the wild-type NOD2 gene with 65%, 17%, and 26%, respectively (P<0.02). However, the incidence of saGVHD increased in patients with NOD2 mutations on the patient and donor (P/D) side with 44% versus 17% compared to patients with the wild-type gene (P<0.03). TLR-4 gene mutations at P/D side had an increased risk for saGVHD with 42% versus 15% of patients with wild-type gene (P<0.04). OS, TRM, and incidence of infectious complications were not influenced by the mutated genes. Multivariate analysis confirmed that NOD2 gene mutations on the donor side had a reduced risk for saGVHD (P<0.001), whereas mutations of the NOD2 gene on P/D side had an increased risk for saGVHD (P<0.01) in our analysis. These results suggest that NOD2 mutations have influence on the occurrence of acute GVHD after transplantation.

Monitoring of genetic polymorphisms in toll-like receptors 2, 4, 5, and 9 with real-time PCR assays

RATIONALE: Toll-like receptors (TLRs) constitute a family of transmembrane proteins that play a crucial role in the innate immune system by differentially recognizing pathogen-associated molecular patterns (PAMPs) through an extracellular domain and initiating inflammatory signaling pathways through an intracellular domain. Due to the central role of TLRs in the innate immune response, genetic variation in this gene family is predicted to alter susceptibility to infections in a PAMP-specific manner based on the ligand recognition of the individual TLR. METHODS: Real-time PCR assays were developed to enable rapid genotyping of the following TLR polymorphisms: TLR2 (Arg753Gln), TLR4 (Asp299Gly and Thr399Ile), TLR5 (Arg392STOP) and TLR9 (-1237). The distribution of these polymorphisms within a population of 40 healthy control subjects was determined based on melting peak analysis. RESULTS: The frequency of individuals in this study who were heterozygous for polymorphisms was observed to be 10% for TLR2 (Arg753Gln) and TLR4 (Asp299Gly and Thr399Ile), 18% for TLR5 (Arg392STOP) and 20% for TLR9 (-1237). The only variant homozygote was found for TLR9 (-1237) at a frequency of 2.5%. These polymorphic frequencies are in general concordance with other larger genetic studies. CONCLUSIONS: In this study, we developed real-time PCR assays to genotype polymorphisms in TLRs that have been associated with increased risks for staphylococcal infections (TLR2), a higher risk for sepsis from gram-negative bacteria (TLR4), susceptibility to infection from flagellated bacteria (TLR5) and an increased risk for asthma (TLR9). Monitoring of these TLR polymorphisms will be potentially important in the evaluation of individuals with unexplained or recurrent infections.

Polymorphisms of toll-like receptor 2 and 4 genes in rheumatoid arthritis and systemic lupus erythematosus.

Human toll-like receptors (TLRs) participate in the innate response and signal the activation of adaptive immunity. Therefore, these TLRs may be important in autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We investigated, by using a polymerase chain reaction restriction-fragment length polymorphism method, the possible association between the polymorphisms of TLR2 (Arg677Trp and Arg753Gln) and TLR4 (Asp299Gly and Thr399Ile) genes with the susceptibility or severity of RA and SLE. Our study population consisted of 122 patients with SLE, 224 patients with RA, and a control group of 199 healthy individuals. The TLR2 polymorphisms were very rare in our population; no individual carrying the TLR2-Arg677Trp polymorphism was observed, whereas the TLR2-Arg753Gln polymorphism was present in only 1% of the total population. We found no statistically significant differences in the TLR4-Asp299Gly and the TLR4-Thr399Ile genotype or allele distribution between SLE patients, RA patients, and control individuals. Similarly, no association was found with any of the demographic and clinical parameters tested either in RA or in SLE patients. In conclusion, a case-control study was used to analyze, for the first time, the influence of TLR2 and TLR4 gene polymorphism on the predisposition and clinical characteristics of SLE and RA but provided no evidence for association of TLR2 or TLR4 gene polymorphism with either disease in the population under study.

Variation in Toll-like receptor 4 and susceptibility to group A meningococcal meningitis in Gambian children.

Meningitis is an important cause of childhood mortality in sub-Saharan Africa. A case-control study investigating the role of the Toll-like receptor 4 gene in host susceptibility to Group A Neisseria meningitidis meningitis was conducted in 524 Gambian children. There was no association between a functional Toll-like receptor 4 gene polymorphism and meningitis, suggesting that this variant is not a major determinant of disease in this population.

Mutations in the gene for toll-like receptor 4 and multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system with heterogeneous pathological features, disease courses and genetical backgrounds. In this study we determined whether genetic variants of toll-like receptor (TLR) 4, which confer substantial differences in the inflammation elicited by bacterial lipopolysaccharide, are related to the development of MS. We found no differences in the frequencies of the cosegregating TLR4 Asp299Gly and Thr399Ile polymorphisms between Austrian MS patients (11.6%) and age-matched controls (13.7%). Furthermore, we could not detect any influence of these mutations on clinical parameters and serum levels of soluble adhesion molecules of MS patients. Our data indicate that these TLR4 polymorphisms have no influence on the incidence, progression and inflammatory parameters of MS.

Polymorphisms of TLR4: Rapid Genotyping and Reduced Response to Lipopolysaccharide of TLR4 Mutant Alleles

Pathogen recognition receptors such as Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns, lead to the activation of innate immunity. Genetic variations in these receptors may lead to an altered host immune response to pathogens. We developed homogeneous fluorescence-based PCR assays as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) genotyping assays to detect TLR4 polymorphisms. These assays were compared with restriction fragment length polymorphism (RFLP) analysis. Peripheral blood monocytes from donors with differing genotypes were prepared and exposed to bacterial products in vitro. The abundance of mRNAs of the proinflammatory cytokines interleukin (IL)-1 beta, IL-6, and tumor necrosis factor-alpha from these monocytes were monitored by real-time reverse transcription-PCR. By our homogeneous PCR method, the allele frequencies were 5.6% for the TLR4 Asp299Gly and 6.0% for the TLR4 Thr399Ile polymorphism in 116 healthy German Caucasians. Nine incorrect genotype calls were detected in the RFLP analysis and two in the TaqMan genotype analysis. MALDI-TOF-MS allowed clear detection of all TLR4 alleles. Monocytes from donors homozygous for the TLR4 mutant alleles Asp299Gly and Thr399Ile were lipopolysaccharide hyporesponsive and exhibited median effective concentrations (EC(50)s) approximately fourfold higher than those of monocytes carrying wild-type or heterozygous alleles. In contrast, a TLR2 agonist elicited similar responses in monocytes irrespective of the TLR4 genotype. Homogeneous fluorescence-based PCR assays provide a specific and sensitive method for high-throughput genotyping of TLR4 mutations. The newly developed PCR and MALDI-TOF-MS assays may be useful to evaluate the presence of TLR4 polymorphisms in patients to predict susceptibility to bacterial infection.