Toll-like Receptor 4 Thr399Ile Research Articles

ROLE OF TLR2, TLR4 GENE POLYMORPHISM IN DEVELOPING MICROVASCULAR COMPLICATIONS IN ADOLESCENTS WITH TYPE 1 DIABETES MELLITUS

Long-term complications of type 1 diabetes mellitus (T1DM) in children and adolescents are an important problem in modern medicine. Recently, the role of immune mechanisms, in particular, chronic inflammation, in the development of both T1DM and its microvascular complications has been actively discussed. Activation of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) leads to hyperproduction of proinflammatory cytokines, chemokines, adhesion molecules involved in the formation of diabetic microvascular complications. At the same time, TLR2 and TLR4 gene polymorphism alters the immune susceptibility to the endogenous ligands, which may increase the risk of diabetic microangiopathies. The aim of this study is to evaluate the frequency of genotypes and alleles of TLR2 and TLR4 genes distribution and to determine the content of TNFα, IL-1, VCAM-1, fractalkine, endothelin-1 in adolescents with T1DM with microvascular complications. We examined 139 adolescents with T1DM from 14 to 18 years old and 56 healthy teenagers. Patients with T1DM were divided into two groups: Group I – patients with poor glycemic control (HbA1C > 9.0%), (n = 64); Group II – patients with satisfactory glycemic control of T1DM (HbA1C ≤ 9.0%), (n = 75), including adolescents with optimal (HbA1C < 7.5%) and suboptimal glycemic control (7.5% ≤ HbA1C ≤ 9.0%) (ISPAD clinical practice consensus guidelines 2014). According to the presence of microvascular complications, the groups were subdivided into subgroups: Iа (n = 49), IIа (n = 38) – adolescents with verified microvascular disorders: diabetic retinopathy, nephropathy and neuropathy; Ib (n = 15), IIb (n = 37) – without microvascular complications. Allelic variants of TLR genes were determined using test systems GosNII genetics (Moscow). The content of cytokines in blood serum was carried out by the method of enzyme-linked immunosorbent assay “BIOSCIENCE”. Data were analyzed using software packages Statistica version 6.0. The assessment of TLR2 (Arg753Gln) and TLR4 (Thr399Ile) polymorphism distribution did not reveal significant differences between the observed subgroups and the control. In Ia and IIa subgroups (with complications) Asp299Gly variant was noted to be significantly less common when compared to subgroups Ib, IIb and controls. The presence of Gly allele in TLR4 gene was found to disrupt the expression of TNFα and VCAM-1 and can be considered protective for the development of microvascular complications.

Toll-like Receptor 4 Gene Polymorphisms in Chinese Population After Allogeneic Hematopoietic Stem Cell Transplantation

Objectives:: Graft-versus-host disease (GVHD) is the most common complication after hematopoietic stem cell transplantation (HSCT) and remains to be a major cause of mortality. Activation of toll-like receptor 4 (TLR-4) by lipopolysaccharide induces the NF-κB signaling pathway to release critical proinflammatory cytokines and increases the recipient response to GVHD. In order to clarify the role of TLR-4 in the occurrence of acute GVHD after HSCT, we collected 208 samples from HSCT recipients and their human lecucyte antigen identical donors to test the hypothesis that TLR-4polymorphism in the recipients or donors influence the risk of acute GVHD in allogeneic HSCT recipients. Methods:: TLR-4 Asp299Gly and Thr399Ile polymorphisms of each sample were examined by using DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism methods. Results:: No homozygous or heterozygous variant alleles of the Asp299Gly or Thr339Ile polymorphism were detected in any samples in our study. Our results demonstrate that TLR-4 Asp299Gly and Thr399Ile polymorphisms might be very rare in the Chinese population (Eastern China and Taiwan region). Conclusion:: The results of this study cannot confirm the role of TLR-4 mutations in the pathogenesis of GVHD in humans, yet we reach a definite conclusion by a TLR-4 knockout murine GVHD model in our ongoing project.

THE ROLE OF ADIPONECTIN AND TOLL-LIKE RECEPTOR 4 GENE POLYMORPHISMS ON NON-PROLIFERATIVE RETINOPATHY IN TYPE 2 DIABETES MELLITUS PATIENTS. A CASE-CONTROL STUDY IN ROMANIAN CAUCASIANS PATIENTS.

Persistent inflammation and impaired neovascularization are important contributors to the development of diabetic retinopathy (DR). Gene polymorphisms of adiponectin (APN) were demonstrated to have an important role on the plasma level and activity of adiponectin. APN has anti-inflammatory, anti-diabetic and anti-atherogenic properties. Toll-Like Receptor 4 (TLR4) is a critical mediator of innate immunity. Polymorphisms in TLR-4 gene were shown to be associated with impaired inflammatory response in diabetes. The aim of the study was to analyze the association of +276G>T variant of APN gene and Asp299Gly and Thr399Ile of TLR-4 gene variants in relationship with T2DM and DR in an Eastern European population group. The distribution of the mutant alleles in 198 T2DM patients with DR and 200 non-T2DM controls was examined. Genomic DNA from T2DM patients and healthy controls genotyped through the use of PCR-RFPL assay. Genotype and allele frequencies of the Asp299Gly and Thr399Ile polymorphisms differed between T2DM patients and non diabetic subjects (P<0.001). Moreover, the presence of the minor alleles of these polymorphisms were significantly identified as protective factors against T2DM, under a dominant model of Fisher's exact test (χ2=4.988, phi=0.745, OR=0.767, 95% CI=0.602-0.867, P<0.001; respectively χ2=5.254, phi=0.820, OR=0.487, 95% CI=0.211-0.648, P<0.001). Genotype analysis for the adiponectin 276G>T gene polymorphism yielded no significant association with T2DM, but revealed a borderline significance for the association with DR (χ2=5.632, phi=0.423, OR =1.101, 95% CI=0.887-1.203, P=0.009). We found an association between the TLR4 Asp299Gly and Thr399Ile polymorphisms and protection for DR. The APN genetic polymorphism is not associated with T2DM.

Association of Helicobacter pylori infection with Toll-like receptor-4 Thr399Ile polymorphism increased the risk of peptic ulcer development in North of Iran.

Toll-like receptor-4 (TLR4) polymorphisms may influence host immune response against Helicobacter pylori (H.pylori). This study aimed to investigate whether TLR4 polymorphisms are associated with H.pylori susceptibility and risk of peptic ulcer development or not. The TLR4+3725G/C polymorphism was studied using polymerase chain reaction with confronting two-pair primers (PCR-CTPP). In addition, TLR4 Asp299Gly and Thr399Ile polymorphisms were evaluated by PCR-restriction fragment length polymorphism (RFLP). There was no significant difference in TLR4+3725G/C and Asp299Gly genotype frequencies between non-peptic ulcer (NPUD) and peptic ulcer (PUD) individuals in the context of peptic ulcer development and susceptibility to infection with H.pylori. Nevertheless, a significant association with increased risk for PUD development was observed for polymorphism TLR4 Thr399Ile [odds ratio (OR)=4.2; 95% confidence interval (CI)=1.35-13.26; p=0.01]. Correspondingly, TLR4 Thr399Ile polymorphism was associated with H.pylori susceptibility (OR=0.27; 95% CI=0.08-0.88; p=0.04). In addition, TLR4 Thr399Ile polymorphism increased 4.2-fold, the risk of peptic ulcer development in individuals infected by H.pylori carrying CT+TT genotype. Our results showed that TLR4 Thr399Ile polymorphism along with H.pylori infection may play critical roles in peptic ulcer development in North of Iran.

Association of Toll-like receptor 4 single-nucleotide polymorphisms Asp299Gly and Thr399Ile with the risk of primary open angle glaucoma

Toll-like receptor 4 (TLR4) is a transmembrane receptor that mediates immune responses to exogenous and endogenous ligands. Previously, non-coding single nucleotide polymorphisms (SNPs) in the TLR4 gene were related to primary open angle glaucoma (POAG). This study was undertaken to investigate whether coding TLR4 Asp299Gly (rs4986790 A/G) and Thr399Ile (rs4986791 C/T) are associated with POAG in a Mexican population. One hundred and eighty-seven unrelated Mexican patients with POAG (94 men and 95 women; mean age 66.49 ± 14.3years) and 109 control subjects (40 men and 69 women; age, 63.28 ± 7.93years) were included. SNPs Asp299Gly (rs4986790 A/G) and Thr399Ile (rs4986791 C/T) were genotyped by a Taqman® Allelic Discrimination Assayand. Allelic, genotypic, haplotypic, and model-based (dominant, recessive, and codominant) associations of the SNPs with POAG were analyzed using Chi-squared tests or Fisher exact tests and logistic regression. Strong linkage disequilibrium was observed among the SNPs (D' = 0.8692), which were located in one haplotype block. With respect to allelic diversity, the minor allele of both SNPs generates a significantly increased risk of POAG. The minor allele of Asp299Gly conferred the highest increased risk of POAG (P = 0.0054, OR = 4.47, 95% CI = 1.46-13.70). With regard to genotypic diversity, individuals carrying the minor allele of Asp299Gly and Thr399Ile had a significant increased risk for POAG with OR of 4.47 (P = 0.054, 95% CI = 1.30-15.35) and 3.5, respectively (P = 0.012, 95% CI = 1.17-10.44). Haplotype analysis was non-significant. TLR4 coding SNPs Asp299Gly and Thr399Ile might be used as genetic susceptibility alleles for POAG in Mexican population. Our findings support the role of TLR4 in the pathophysiology of glaucoma.

Association between TLR2 and TLR4 Gene Polymorphisms and the Susceptibility to Inflammatory Bowel Disease: A Meta-Analysis.

BackgroundThe associations between toll-like receptor 2 (TLR2) and toll-like receptor 4(TLR4) polymorphisms and inflammatory bowel disease (IBD) susceptibility remain controversial. A meta-analysis was performed to assess these associations.MethodsA systematic search was performed to identify all relevant studies relating TLR2 and TLR4 polymorphisms and IBD susceptibility. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. Subgroup analyses were performed by ethnicity and publication quality.ResultsThirty-eight eligible studies, assessing 10970 cases and 7061 controls were included. No TLR2 Arg677Trp polymorphism was found. No significant association was observed between TLR2 Arg753Gln polymorphism and Crohn’s disease (CD) or ulcerative colitis (UC) in all genetic models. Interestingly, TLR4 Asp299Gly polymorphism was significantly associated with increased risk of CD and UC in all genetic models, except for the additive one in CD. In addition, a statistically significant association between TLR4 Asp299Gly polymorphism and IBD was observed among high quality studies evaluating Caucasians, but not Asians. Associations between TLR4 Thr399Ile polymorphisms and CD risk were found only in the allele and dominant models. The TLR4 Thr399Ile polymorphism was associated with UC risk in pooled results as well as subgroup analysis of high quality publications assessing Caucasians, in allele and dominant models.ConclusionsThe meta-analysis provides evidence that TLR2 Arg753Gln is not associated with CD and UC susceptibility in Asians; TLR4 Asp299Gly is associated with CD and UC susceptibility in Caucasians, but not Asians. TLR4 Thr399Ile may be associated with IBD susceptibility in Caucasians only. Additional well-powered studies of Asp299Gly and other TLR4 variants are warranted.

LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis.

Colorectal carcinoma (CRC) is characterized by unlimited proliferation and suppression of apoptosis, selective advantages for tumor survival, and chemoresistance. Lipopolysaccharide (LPS) signaling is involved in both epithelial homeostasis and tumorigenesis, but the relative roles had by LPS receptor subunits CD14 and Toll-like receptor 4 (TLR4) are poorly understood. Our study showed that normal human colonocytes were CD14(+)TLR4(-), whereas cancerous tissues were CD14(+)TLR4(+), by immunofluorescent staining. Using a chemical-induced CRC model, increased epithelial apoptosis and decreased tumor multiplicity and sizes were observed in TLR4-mutant mice compared with wild-type (WT) mice with CD14(+)TLR4(+) colonocytes. WT mice intracolonically administered a TLR4 antagonist displayed tumor reduction associated with enhanced apoptosis in cancerous tissues. Mucosa-associated LPS content was elevated in response to CRC induction. Epithelial apoptosis induced by LPS hypersensitivity in TLR4-mutant mice was prevented by intracolonic administration of neutralizing anti-CD14. Moreover, LPS-induced apoptosis was observed in primary colonic organoid cultures derived from TLR4 mutant but not WT murine crypts. Gene silencing of TLR4 increased cell apoptosis in WT organoids, whereas knockdown of CD14 ablated cell death in TLR4-mutant organoids. In vitro studies showed that LPS challenge caused apoptosis in Caco-2 cells (CD14(+)TLR4(-)) in a CD14-, phosphatidylcholine-specific phospholipase C-, sphingomyelinase-, and protein kinase C-ζ-dependent manner. Conversely, expression of functional but not mutant TLR4 (Asp299Gly, Thr399Ile, and Pro714His) rescued cells from LPS/CD14-induced apoptosis. In summary, CD14-mediated lipid signaling induced epithelial apoptosis, whereas TLR4 antagonistically promoted cell survival and cancer development. Our findings indicate that dysfunction in the CD14/TLR4 antagonism may contribute to normal epithelial transition to carcinogenesis, and provide novel strategies for intervention against colorectal cancer.

Genomic Analysis of Single Nucleotide Polymorphisms Asp299Gly and Thr399Ile in Japanese Patients with Invasive Aspergillosis.

Single nucleotide polymorphisms (SNPs) 1063A/G (Asp299Gly) and 1363C/T (Thr399Ile) in the gene encoding Toll-like receptor 4 (TLR4) increase susceptibility to invasive aspergillosis. However, limited information is available on the prevalence of these SNPs in Japan. Therefore, we surveyed these TLR4 SNPs by using formalin-fixed and paraffin-embedded tissue blocks obtained from autopsies of patients with invasive pulmonary aspergillosis. Tissue samples of approximately 30% patients were included in genomic analysis. However, none of these samples showed the presence of TLR4 Asp299Gly and Thr399Ile polymorphisms. Thus, the present study provided information on the prevalence of TLR4 SNPs in Japanese patients with invasive aspergillosis and indicated that these SNPs played a minor role in increasing the susceptibility of Japanese individuals to invasive aspergillosis.

Toll-like receptor 4 gene polymorphisms and susceptibility to colorectal cancer: a meta-analysis and review.

IntroductionMany case-control studies have investigated the association between toll-like receptor 4 (TLR4) Asp299Gly and Thr399Ile polymorphisms and risk of colorectal cancer (CRC). However, published data are still conflicting.Material and methodsA systematic search was conducted in the electronic databases of PubMed, MEDLINE, EMBASE, Web of Science and CNKI between 2000 and 2014. The associations between TLR4 polymorphisms and CRC susceptibility were assessed by pooled odds ratios (ORs) and 95% confidence intervals (95% CI) in fixed or random effects models.ResultsIn total nine case-control studies were identified in this meta-analysis. For TLR4 Asp299Gly polymorphism, 9 studies included 1198 cases and 1290 controls. The GG genotype carriers had higher risk for developing CRC than AA + GA genotype carriers (OR = 1.95, 95% CI: 1.00–3.77, p = 0.05). No association was found in other genetic models (p > 0.05). Analysis stratified by ethnicity showed no association in any genetic models among the Asian or Caucasian population. For TLR4 Thr399Ile polymorphism, 6 studies contained 619 cases and 632 controls. The overall analysis showed significantly increased risk in TT homozygote carriers compared to CC homozygote (OR = 4.99, 95% CI: 1.41–17.65, p = 0.01) and C carriers (TC + CC) (OR = 4.50, 95% CI: 1.27–15.87, p = 0.02). In terms of analyses stratified by race, a significant association was found in each genetic model among the Asian population, rather than the Caucasian group.ConclusionsThe GG homozygote carriers of TLR4 Asp299Gly and TT homozygote carriers of TLR4 Thr399Ile polymorphisms might be correlated with an increased risk of CRC, suggesting they may serve as genetic risk factors for CRC.

Role of TLR4 C&gt;1196T (Thr399Ile) and TLR4 A&gt;896G (Asp299Gly) polymorphisms in a North Indian population with asthma: a case‐control study

Toll-like receptor 4 (TLR4) is the most important TLR among the pattern recognition receptors which recognizes lipopolysaccharide of gram-negative bacteria. They identify a highly conserved structure of microbes called pathogen-associated molecular patterns and activate immune and inflammatory responses that have been shown to be involved in the pathogenesis of asthma. The role of TLR4 gene polymorphisms in asthma was detected in a total of 964 individuals, including 483 healthy controls and 481 asthma patients from a North Indian population. The genotyping was carried out using polymerase chain reaction-restriction fragment length polymorphism method. Statistical analysis revealed that the heterozygous genotype as well as the mutant (T) allele of the TLR4 C>1196T (Thr399Ile) polymorphism shows resistance towards asthma with OR = 0.70, 95% CI (0.49-0.99), P corrected value = 0.046 and OR = 0.72, 95% CI (0.52-0.98), P corrected value = 0.039, respectively. However, no association was found between the TLR4 A>896G (Asp299Gly) polymorphism and asthma patients (P > 0.05). This is the first study conducted in India conferring TLR4 (Thr399Ile) polymorphism resistance towards asthma, while lack of association was found between TLR4 (Asp299Gly) polymorphism and asthma in the studied North Indian population.

Toll-like receptor 4 polymorphisms to determine acute pancreatitis susceptibility and severity: a meta-analysis.

To investigate the correlation of toll-like receptor 4 (TLR4) gene Asp299Gly and Thr399Ile polymorphisms and acute pancreatitis (AP) risk and severity. To get a more precise estimation of the relationship, a comprehensive search was performed to examine all the eligible studies of TLR4 Asp299Gly and Thr399Ile polymorphisms and AP risk. The odds ratios with 95% confidence intervals were used to assess the strength of the association. Publication bias was analyzed by Begg's funnel plots. In total, six studies with 1255 cases and 998 controls were included in this meta-analysis. Totally, no significant associations were found between TLR4 Asp299Gly or Thr399Ile polymorphisms and AP risk using five models with high homogeneity (P > 0.05). Furthermore, stratification analysis by ethnicity or assay also found no significant association in these two polymorphisms (P > 0.05), and TLR4 Asp299Gly was not associated with AP severity (P > 0.05). In addition, no publication bias was found in these studies (P > 0.05). Our current meta-analysis suggests that TLR4 Asp299Gly and Thr399Ile polymorphisms may not be risk factors to AP susceptibility.

Associations Between the Four Toll-Like Receptor Polymorphisms and the Risk of Gastric Cancer: A Meta-Analysis

The association between Toll-like receptor 2 (TLR2) -196 to -174del polymorphism and Toll-like receptor 4 (TLR4) polymorphisms (Asp299Gly, Thr399Ile, and 3725G>C) and gastric cancer risk are still conflicting. For better understanding of the effects of these four polymorphisms on gastric cancer risk, a meta-analysis was performed. An extensive search was performed to identify all case-control studies investigating such associations. Odds ratios (ORs) with 95% confidence intervals (CIs) were estimated to assess the relationship. A total of 21 studies (3,436 cases and 4,239 controls) were found to be eligible for meta-analysis. In the overall analysis, a significantly increased risk was observed in TLR4 Asp299Gly polymorphism (G allele vs. A allele: OR=1.84, 95%CI: 1.41, 2.39; GA vs. AA: OR=1.89, 95%CI: 1.43, 2.48; Recessive model: OR=1.90, 95%CI: 1.44, 2.49) and TLR4 Thr399Ile polymorphism (T allele vs. C allele: OR=1.97, 95%CI: 1.22, 3.18; TC vs. CC: OR=1.94, 95%CI: 1.19, 3.15; Recessive model: OR=1.98, 95%CI: 1.21, 3.21), whereas no associations were found in any genetic models of TLR2 -196 to -174del and TLR4 3725G>C polymorphisms. Similar results were found in the subgroup analyses by ethnicity. However, we detected that A allele carriers of the TLR4 Asp299Gly polymorphism might have an increase risk of gastric cancer in the Helicobacter pylori-positive population (G allele vs. A allele: OR=2.01, 95%CI: 1.22, 3.31). The results of this meta-analysis indicate that the TLR4 Asp299Gly and Thr399Ile polymorphisms are risk factors for gastric cancer development.

Interleukin-17 serum levels and TLR4 polymorphisms in ulcerative colitis.

Inflammatory bowel disease, an autoimmune disease, has two clinical manifestations including Crohn's disease and ulcerative colitis (UC). IL-17 has been the target of intensive research in autoimmune diseases. The influence of Toll like receptor 4 (TLR-4) gene polymorphisms on IL-17 production has also been revealed in UC patients and tissue inflammation in mice. To investigate the association between the TLR-4 gene polymorphisms, Asp299Gly and Thr399Ile and IL-17 serum levels with ulcerative colitis. Additionally, we aimed to study modulation effects of forenamed gene polymorphisms on IL-17 serum levels in UC patients and controls. A total of 256 healthy controls and 85 UC patients enrolled in our study. DNA was extracted and PCR-RFLP technique was employed to determine Asp299Gly and Thr399Ile polymorphisms in TLR-4 gene and IL-17 serum levels were measured by ELISA method. There was no significant difference between the frequency of Asp299Gly A>G and Thr399Ile C>T in UC patients and controls. While IL-17 serum levels in UC patients were significantly higher than controls (p=0.003), no significant difference in IL-17 levels between different genotypes existed. Additionally, a significant inverse relationship was observed between hemoglobin level and IL-17 serum levels in UC patients (p=0.039). Increased IL-17 serum levels in our UC patients might be explained through the synergistic activity of IL-17/IL-23 axis and pro-inflammatory cytokines, causing severe clinical outcome in patients with IBD. The prolonged excretion of blood in stool driven by inflammatory process which causes iron metabolism disorder and anemia may elucidate the inverse correlation between hemoglobin and IL-17 serum levels in UC patients. Lack of association between the TLR-4 gene polymorphisms and UC in our study was consistent with the results from other Caucasian populations.

Toll-like receptor 4 polymorphisms and susceptibility to multiple autoimmune diseases: evidence based on pooled analysis

ENWEndNote BIBJabRef, Mendeley RISPapers, Reference Manager, RefWorks, Zotero AMA Wang X, Zhang K, Xiong D. Review paperToll-like receptor 4 polymorphisms and susceptibility to multiple autoimmune diseases: evidence based on pooled analysis. Central European Journal of Immunology. 2013;38(3):380-387. doi:10.5114/ceji.2013.37757. APA Wang, X., Zhang, K., & Xiong, D. (2013). Review paperToll-like receptor 4 polymorphisms and susceptibility to multiple autoimmune diseases: evidence based on pooled analysis. Central European Journal of Immunology, 38(3), 380-387. https://doi.org/10.5114/ceji.2013.37757 Chicago Wang, Xiao-qin, Kui Zhang, and Ding Xiong. 2013. "Review paperToll-like receptor 4 polymorphisms and susceptibility to multiple autoimmune diseases: evidence based on pooled analysis". Central European Journal of Immunology 38 (3): 380-387. doi:10.5114/ceji.2013.37757. Harvard Wang, X., Zhang, K., and Xiong, D. (2013). Review paperToll-like receptor 4 polymorphisms and susceptibility to multiple autoimmune diseases: evidence based on pooled analysis. Central European Journal of Immunology, 38(3), pp.380-387. https://doi.org/10.5114/ceji.2013.37757 MLA Wang, Xiao-qin et al. "Review paperToll-like receptor 4 polymorphisms and susceptibility to multiple autoimmune diseases: evidence based on pooled analysis." Central European Journal of Immunology, vol. 38, no. 3, 2013, pp. 380-387. doi:10.5114/ceji.2013.37757. Vancouver Wang X, Zhang K, Xiong D. Review paperToll-like receptor 4 polymorphisms and susceptibility to multiple autoimmune diseases: evidence based on pooled analysis. Central European Journal of Immunology. 2013;38(3):380-387. doi:10.5114/ceji.2013.37757.

Retraction Note: CD14 Promoter-159 Polymorphism Is Associated with Reduced Risk of Intestinal-Type Gastric Cancer in a Japanese Population

Host genetic factors may play a key role in determining the long-term outcome of the Helicobacter pylori infection. Toll-like receptor 4 (TLR4) and CD14-mediated recognition of lipo-polysaccharide (LPS) is required for efficient recognition of Gram-negative bacterial infections. We investigated the effects of common polymorphisms of TLR4 Asp299Gly, Thr399Ile, and CD14 promoter-C159T on the risk of gastric cancer including its subtypes and clinicopathologic features. We also investigated the effects of these polymorphisms on histologic degree of H. pylori-induced gastritis. The study was performed in 149 gastric cancer (GC) cases [mean age 64.0 ± 12.4, M:F = 109:40] and 94 patients without evidence of GC (mean age 64.1 ± 12.3, M:F = 65:25, Peptic ulcer diseases = 43.6%, gastritis = 56.4%) as the control group. TLR4 Asp299Gly, Thr399Ile, and CD14 promoter-C159T were determined by PCR-RFLP in all the patients. Gastritis scores of non-cancerous gastric mucosa were assessed according to the updated Sydney system in H. pylori-positive subjects (n = 179). The frequencies of CD14-260 TT and T carrier were significantly lower in patents with intestinal-type gastric cancer than in controls (OR = 0.31; 95%CI = 0.12–0.78, OR = 0.38; 95%CI = 0.18–0.81, respectively). Compared to patients older than 61 years, the atrophy score in antrum was significantly lower in TT and CT patients. TLR4 Asp299Gly and Thr399Ile were not detected in all the patients. Our data suggests that CD14 promoter-159TT and T carrier were associated with a lower risk of developing gastric mucosal atrophy in H. pylori-infected patients of more than 61 years of age, and these genotypes may reduce the risk of intestinal-type gastric cancer.

Increased NFk-B activity in HCT116 colorectal cancer cell line harboring TLR4 Asp299Gly variant.

Toll-Like Receptor 4 (TLR4), considered one of the most important TLR, recognizes lipopolysaccharide of Gram-negative bacteria. Recognition of ligands by TLRs induces signaling pathways resulting in activation of transcriptional factors such as NF-κB which are involved in the expression of inflammatory cytokines and chemokines. To prevent an inappropriate immune response, a complex network of molecules negatively regulates TLRs and their associated signaling pathways. Two cosegregating single nucleotide polymorphisms of the human TLR4 gene, namely Asp299Gly and Thr399Ile, have been associated with hyporesponsiveness to inhaled LPS. The purpose of this study was to determine the impact of TLR4 gene variant on NF-κB activity in colorectal cancer cell line. HCT116 cells were transfected with wild-type and mutants Flag-CMV1-TLR4 expression vectors. Western blot analysis was performed to evaluate selected molecules involved in TLR4 signaling. NF-κB activity was assessed by dual-luciferase reporter assay and cytokine profiles were evaluated byELISA and Cytometric Bead Array method. Results showed that the activity of pNF-κB was higher in cells harboring TLR4 D299G compared to the other cells. However, the activity of pAKT, pERK1 and pIRAK was higher in wild-type. The results of cytokine measurements showed about four fold higher level of IL-8 in cells with wild-type TLR4. This study suggest that TLR4 Asp299Gly gene variant has an impact on TLR4 signaling and potentially on intestinal homeostasis due to impaired control signals at the epithelial cell level which may lead to chronic intestinal inflammation and interrupted intestinal homeostasis and may eventually lead to colorectal cancer.

Lack of association between TLR4 Asp299Gly and Thr399Ile polymorphisms and sepsis susceptibility: A meta-analysis

Sepsis, a condition of systemic inappropriate inflammation response to the invasion of microorganisms, results in considerable morbidity and mortality in patients. Some, but not all, epidemiological studies have suggested that Toll-like receptor 4 (TLR4) polymorphisms, Asp299Gly and Thr399Ile, may influence the risk of at-risk patients for sepsis. Our work tried to further study the association of the two common polymorphisms with sepsis susceptibility by performing a meta-analysis of previous data. Electronic searches of MEDLINE, EMBASE and Web of Science databases were performed. Original observational studies dealing with the association between polymorphisms Asp299Gly and/or Thr399Ile and sepsis risk were selected. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated with random-effects model or fixed-effects model based on the heterogeneity analysis. Seventeen studies including 2212 cases and 3880 controls were included with most subjects of Caucasian ethnicity. The odds ratio for the association of Asp299Gly polymorphism with sepsis risk was 1.22 (95% CI: 0.90–1.65, P=0.21), and the association of Thr399Ile polymorphism was 1.16 (95%CI: 0.70–1.91, P=0.57). Subgroup analysis and sensitivity analysis did not change the results. Our meta-analysis revealed that the two common TLR4 polymorphisms, Asp299Gly and Thr399Ile, have no strong association with the likelihood of sepsis in Caucasian populations. Further studies are needed to investigate the effect of genetic networks and their mutual interactions in TLR4 signaling pathway on sepsis susceptibility.

Influence of Common Non-Synonymous Toll-like Receptor 4 Polymorphisms on Bronchopulmonary Dysplasia and Prematurity in Human Infants

Bronchopulmonary dysplasia (BPD) is a common chronic lung disease and major risk factor for severe respiratory syncytial virus (RSV) infection among preterm infants. The Toll-like receptor 4 (TLR4) is involved in oxidative injury responses in the lungs. Two non-synonymous single nucleotide polymorphisms in the TLR4 gene have been associated with RSV infection in children. However, it is unclear to what extent this association is confounded by BPD or prematurity. In this study, we analyzed two population-based cohorts of preterm infants at risk for BPD as well as ethnicity-matched infants born at term, to test whether the TLR4 polymorphisms Asp299Gly (rs4986790) and Thr399Ile (rs4986791) are independently associated with BPD or premature birth. In a Canadian cohort (n = 269) composed of a majority of Caucasian preterm infants (BPD incidence of 38%), the TLR4-299 heterozygous genotype was significantly under-represented in infants without BPD (1.6% of infants versus 12% in infants with severe BPD) after adjusting for twins, ethnicity, gestational age, birth weight and gender (p = 0.014). This association was not replicated in a Finnish cohort (n = 434) of premature singletons or first-born siblings of Caucasian descent, although the incidence of BPD was substantially lower in this latter population (15%). We did not detect a significant association (>2-fold) between TLR4 genotypes and prematurity (p>0.05). We conclude that these TLR4 genotypes may have, at best, a modest influence on BPD severity in some populations of high-risk preterm infants. Further studies are warranted to clarify how clinical heterogeneity may impact genetic susceptibility to BPD.

Analysis of TLR4 and TLR2 polymorphisms in inflammatory bowel disease in a Guangxi Zhuang population

To study the polymorphisms of toll-like receptor 4 (TLR4) gene Asp299Gly, Thr399Ile and TLR2 gene Arg753Gln, Arg677Trp and susceptibility to inflammatory bowel disease (IBD) in the Zhuang population from Guangxi, China. A case-control study was performed from February 2007 to October 2011 which included 146 Zhuang patients with IBD in the experimental group and 164 healthy Zhuang subjects who acted as the control group. All patients and healthy subjects were from the Guangxi Zhuang Autonomous Region of China. Genomic DNA was extracted from intestinal tissue by the phenol chloroform method. TLR4 gene Asp299Gly, Thr399Ile and TLR2 gene Arg753Gln, Arg677Trp were amplified by polymerase chain reaction (PCR), and then detected by PCR-restriction fragment length polymorphism (RFLP). The TLR4 gene Asp299Gly was digested using Nco I restriction enzyme, and a single band of 249 bp was observed which showed that it was a wild type (AA). The TLR4 gene Thr399Ile was digested using Hinf Irestriction enzyme and only the wild type (CC) was detected. In addition, the TLR2 gene Arg677Trp was digested using Aci I restriction enzyme and only the wild type (CC) was detected. The TLR2 gene Arg753Gln was digested using Pst I restriction enzyme. Only the wild type (GG) as a single band of 254 bp was observed during RFLP. Overall, no heterozygous or homozygous single nucleotide polymorphism mutations were found in patients with Crohn's disease and ulcerative colitis both in the TLR4 gene Asp299Gly, Thr399Ile and the TLR2 gene Arg677Trp, Arg753Gln in the Zhuang population from the Guangxi Zhuang Autonomous Region of China. The TLR4 gene Asp299Gly, Thr399Ile and TLR2 gene Arg753Gln, Arg677Trp polymorphisms may not be associated with IBD in the Zhuang population from the Guangxi Zhuang Autonomous Region of China.

The Evolutionary History of TLR4 Polymorphisms in Europe

Infections exert important evolutionary pressures shaping the human genome, especially on genes involved in host defense. A crucial step for host defense is recognition of pathogens by pattern recognition receptors on innate immune cells, among which Toll-like receptor 4 (TLR4) is one of the best known. Genetic variation in TLR4 (Asp299Gly, Thr399Ile) has been recently described. Haplotype frequencies of these polymorphisms differ among African, Asian and European populations, suggesting evolutionary pressures exerted by local infections. The TLR4 299Gly/399Ile haplotype, characteristic mainly of European populations, has relatively high frequency in the Iberian peninsula. This region is also described as refuge area during the last glacial maximum 20,000 years ago, from which repopulation of Europe took place. We speculate that a genetic bottleneck in the Iberian peninsula could have promoted the increased frequency of this haplotype by genetic drift. This hypothesis is supported by three arguments: (1) the West-East gradient of prevalence in the haplotype among European populations; (2) ancient DNA from Neolithic burials in the Iberian peninsula, dated 6,600–4,500 years before present, confirmed the relatively high frequency of this haplotype in the region, and (3) no functional differences between this haplotype and wild-type TLR4 have been found. In contrast, the disappearance of the 299Gly/399Thr haplotype in Europe is most likely due to negative selection due to sepsis. In conclusion, differences in distribution of TLR4 polymorphisms Asp299Gly and Thr399Ile in European populations are most likely due to a combination of population migration events combined with selection due to sepsis.