Toll-like Receptor 4 Asp299Gly Research Articles

Expression of toll-like receptor 4 gene polymorphism and inflammatory factors in peripheral blood on patients with sepsis

Objective: To observe the presence of toll-like receptor 4 (TLR4) gene polymorphism in the venous blood in patients with sepsis, meanwhile, to observe the expression and the diagnostic value of TLR4 mRNA, interferon-γ (IFN-γ), interleukin-23 (IL-23), procalcitonin (PCT) and C-reactive protein (CRP) in patients with different degrees of sepsis.Methods: The peripheral blood samples from the subjects were collected to extract genomic DNA, gene sequencing and enzyme digestion method were applied to the detection of TLR4 Asp299Gly loci polymorphism. The expression of TLR4 mRNA in the peripheral blood was detected by reverse transcriptase polymerase chain reaction (RT-PCR). The expression levels of IFN-γ, IL-23, PCT and CRP were measured by enzyme linked immunosorbent assay (ELISA).Results: (1) The gene polymorphism was not present in TLR4 Asp299Gly loci; (2) The expression of TLR4 mRNA in the peripheral blood in patients with sepsis: on d1, there were statistically significant differences between the normal group and the group (APACHE II ≤ 20), between the normal group and the group (APACHE II > 20), between the group (APACHE II ≤ 20) and the group (AAPACHE II > 20) (t = 5.741, 14.780 and 10.500, all p < .01). APACHE II stands for Acute Physiology and Chronic Health Evaluation II. On d7, there were statistically significant differences between the normal group and the group (APACHE II ≤ 20), between the normal group and the group (APACHE II > 20), between the group (APACHE II ≤ 20) and the group (APACHE II > 20) (t = 4.186, 13.830 and 9.645, all p < .01); (3) The expression levels of IFN-γ, IL-23, PCT and CRP were obviously upregulated (all p < .01), and TLR4 was positively correlated with IFN-γ and IL-23 (all p < .01); (4) The best cutoff value of TLR4 mRNA at baseline was 891.6 μg/L, with a sensitivity of 100% and a diagnostic specificity of 57%. The best cutoff value of IFN-γ at baseline was 84.5 μg/L, with a sensitivity of 100% and a diagnostic specificity of 57%. The best cutoff value of IL-23 at baseline was 861 μg/L, with a sensitivity of 100% and a diagnostic specificity of 97%.Conclusions: (1) The Asp299Gly polymorphism is not present in TLR4 gene; (2) The expression levels of TLR4 mRNA, IFN-γ and IL-23 are relatively high in patients with sepsis, and will be higher with the increased severity of sepsis; (3) TLR4 mRNA, IFN-γ and IL-23 can be used as molecular biomarkers for the early diagnosis of sepsis.

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.

P253 ASSOCIATION OF TLR4 GENE POLYMORPHISMS WITH GASTRODUODENAL DISEASES AMONG PATIENTS WITH HELICOBACTERIOSIS

Toll-like receptor-4 (TLR4) polymorphisms may influence host immune response against Helicobacter pylori. Toll-like receptor-4 Asp299Gly and Thr399Ileu substitutions reduce the responsiveness to H. pylorilipopolysaccharide. The aim of this study was to investigate the associations of TLR4 polymorphisms with H. pylori-associated gastroduodenal disease manifestations.

Association of chronic inflammation and accelerated atherosclerosis among an indigenous black population with chronic kidney disease.

IntroductionInflammation plays a major role in the development of atherosclerosis and cardiovascular morbidity and mortality in chronic kidney disease (CKD) patients. Toll-like receptor-4 (TLR4) is a major receptor for lipopolysaccharides (endotoxin) and other ligands involved in the pathogenesis of inflammation. We determined whether endotoxin levels and the presence of TLR4 polymorphisms are associated with markers of inflammation and atherosclerosis among South African CKD patients.Materials and methodsEndotoxin, lipopolysaccharide binding protein (LBP), serum CD14 (sCD14), interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and carotid intima media thickness (CIMT) were measured in 160 participants (120 CKD patients and 40 controls). Associations between endotoxins and CIMT in the presence of sCD14, IL-8 and MCP-1, were assessed using odds ratios. Participants were screened for the presence of Asp299Gly and Thr399Ile TLR4 polymorphisms, and CIMT and inflammatory markers were compared between subjects with and without TLR4 polymorphisms.ResultsEndotoxin levels correlated with sCD14 (r = 0.441, p<0.001) and MCP-1 (r = 0.388, p<0.001) levels while increased CIMT was associated with MCP-1 (r = 0.448, p<0.001), sCD14 levels (r = 0.476, p<0.001), LBP (r = 0.340, p<0.001), and IL-8 (r = 0.395, p<0.001). Atherosclerosis was associated with endotoxin levels (odds ratio: 4.95; 95% confidence interval: 2.52–9.73; p<0.001), and was predicted by higher serum levels of inflammatory markers. Analysis of patients with TLR4 polymorphisms showed reduced serum levels of inflammatory markers and CIMT values compared with the patients carrying the wild type TLR4 alleles.ConclusionThe study demonstrated associations between circulating endotoxaemia, systemic inflammation and accelerated atherosclerosis among South African CKD patients, and showed that the atherogenic predictive power of endotoxaemia was significantly increased by the presence of elevated levels of inflammatory markers. Additional findings, which must be confirmed, suggest that TLR4 polymorphisms are associated with low levels of inflammatory markers and CIMT values.

TOLL LIKE RECEPTOR TYPE FOUR GENE POLYMORPHISM IN NEONATAL SEPSIS IN ZAGAZIG UNIVERSITY CHILDREN’S HOSPITAL

Introduction: The immune response constitutes the first-line defense against microbial infections. Toll like receptors (TLRs) play a fundamental role in neonatal innate immunity, and one of them, toll like receptor 4 (TLR4) is considered a major pattern recognition receptor (PRR) in recognizing lipopolysaccharide (LPS) of gram-negative bacteria. Single nucleotide polymorphisms (SNPs) among various TLR genes have been identified and maybe related to susceptibility/resistance to certain infections and other inflammatory diseases.Objective: The purpose of this study was to find a relation between sepsis-related outcomes in neonates and TLR4 Asp299Gly gene polymorphism.Method: we carried out a case control study on 72 neonates. 36 neonates with culture proven gram-negative sepsis and 36 neonates as controls. Both groups were genotyped for TLR4 Asp299Gly gene polymorphism, by polymerase chain reaction (PCR), and restriction fragment length polymorphism analysis (RFLP) by using BccI enzyme.Results: No statistically significant association between TLR4 Asp299Gly gene polymorphism and severity of neonatal sepsis (P>.99).Conclusion: TLR4 Asp299Gly gene polymorphism is not associated with increased severity of gram-negative bacterial sepsis in neonates.

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.

Role of toll-like receptor 4 Asp299Gly polymorphism in the development of cardiovascular diseases in HIV-infected patients

Cardiovascular diseases (CVDs) are one of the main causes of morbimortality in HIV-infected patients on suppressive antiretroviral therapy. The objective of this work was to evaluate the role of single nucleotide polymorphisms (SNPs) in lipopolysaccharide (LPS) Toll-like receptor 4 (TLR4) and CVDs occurrence in HIV-infected patients. Additionally, the functional consequences of carrying these SNPs were analyzed. The association of TLR4 SNPs, Asp299Gly/Thr399Ile with CVDs occurrence was analyzed using multivariate logistic regression models. Clinical, immunological, and traditional cardiovascular risk factors were used as covariates. The monocyte phenotype and response were assessed by multiparametric flow cytometry comparing carriers with noncarriers of this SNP. Asp299Gly SNP, assayed in 253 HIV-infected patients, was independently associated with the occurrence of CVDs after adjusting for CD4+ T-cell nadir, HCV-coinfection, bacterial pneumonia, diabetes mellitus, and traditional cardiovascular risk factors [odds ratio (confidence interval 95%) = 3.672 (1.061-12.712), P = 0.04). Carriers of Asp299Gly SNP showed higher percentage of patrolling and intermediate monocytes producing a proinflammatory combination of cytokines compared with noncarriers (P = 0.037 and P = 0.046, respectively). Intermediate monocyte subset levels correlated with soluble interleukin-6 levels only in carriers (r = 0.89; P = 0.01). TLR4 Asp299Gly polymorphism is independently associated with the occurrence of CVDs in HIV-infected patients. The proinflammatory profile associated to this variant could be involved in the development of atherosclerotic pathologies.

Relationship between Arg753Gln Toll-like receptor 2 and Asp299Gly Toll-like receptor 4 genetic variations and susceptibility to colorectal cancer in Southern Iran

Variations in Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) encoding genes have been associated with tumorigenesis through the disruption of immune and inflammatory responses. The aims of this study were to evaluate the two single nucleotide polymorphisms (SNPs) of the genes Arg753Gln TLR2 (rs5743708) and Asp299Gly TLR4 (rs4986790) in colorectal cancer patients in southern Iran. Colorectal cancer patients and healthy controls were included in this study (150 Persian subjects in each group). Blood samples were used for genotyping by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The association between these SNPs and colorectal cancer and clinicopathological factors, including age, gender, tumor stage and differentiation were also investigated. A significant association was found between Arg753Gln TLR2 SNP and colorectal cancer. This SNP was significantly more frequent in male patients. However, there was no association between Asp299Gly TLR4 and colorectal cancer. Therefore, Arg753Gln TLR2 SNP can be considered as a risk factor for colorectal cancer incidence in southern Iran, especially in men. Further investigations in other populations are recommended in order to assess the association of this SNP with colorectal cancer.

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.

Functional variants in intercellular adhesion molecule-1 and toll-like receptor-4 genes are more frequent in children with febrile urinary tract infection with renal parenchymal involvement.

We studied the functional polymorphisms of intercellular adhesion molecule-1 (ICAM-1) and toll-like receptor-4 (TLR-4) genes and risk of acute pyelonephritis (APN) in children attending Assiut University Children's Hospitals, Egypt, from 2011 to 2015. Urinary tract infections (UTIs) were diagnosed in 380 children: 98 had APN and 282 had lower UTIs. Four single-nucleotide polymorphisms in ICAM-1 and TLR-4 genes were genotyped in all subjects: ICAM-1 rs1799969 Gly241Arg, ICAM-1 rs5498 Glu469Lys, TLR-4 rs4896791 Thr399Ile and TLR-4 rs4896790 Asp299Gly. Patients with APN were significantly more likely to have AA genotype of the ICAM-1 rs5498 (1462 A/G) polymorphism (p=0.04) than children with lower UTIs and the TLR-4 Asp299Gly GG genotype (p=0.002) and G allele (p=0.006) than healthy controls. The association with the ICAM-1 Glu469Lys (1462A/G) was less evident. The GG genotype was associated with a modest relative risk of 1.4 (p=0.1) of developing APN, but was not an independent odds ratio, at 1.2 (p=0.48). Functional variants in ICAM-1 and TLR-4 genes were increasingly common in children with febrile UTIs with renal parenchymal involvement, but the ICAM-1 Glu469Lys (1462A/G) association was less evident. TLR4 Asp299Gly might independently increase renal parenchymal infection rather than renal scarring.

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 Toll-like receptor 4 Asp299Gly polymorphism and coronary heart disease susceptibility.

Published data on the association between Toll-like receptor 4 (TLR4) Asp299Gly polymorphism and coronary heart disease (CHD) susceptibility are inconclusive. To derive a more precise estimation of the relationship, a meta-analysis was performed. English-language studies were identified by searching PubMed and Embase databases (up to November 2016). All epidemiological studies were regarding Caucasians because no TLR4 Asp/Gly and Gly/Gly genotypes have been detected in Asians. A total of 20 case-control studies involving 14,416 cases and 10,764 controls were included in the meta-analysis. Overall, no significant associations were found between TLR4 Asp299Gly polymorphism and CHD susceptibility in the dominant model (OR=0.89; 95%CI=0.74 to 1.06; P=0.20) pooled in the meta-analysis. In the subgroup analysis by CHD, non-significant associations were found in cases compared to controls. When stratified by control source, no significantly decreased risk was found in the additive model or dominant model. The present meta-analysis suggests that the TLR4 Asp299Gly polymorphism was not associated with decreased CHD risk in Caucasians.

TLR4 Asp299Gly (rs4986790) polymorphism and coronary artery disease: a meta-analysis.

Background. Previous studies have shown conflicting results on the association between toll-like receptor 4 (TLR4) Asp299Gly (rs4986790) polymorphism and coronary artery disease (CAD). The aim of this study was to evaluate the influence of TLR4 Asp299Gly polymorphism on CAD risk, CRP level and the number of stenotic coronary arteries, as well as to investigate whether G allele carriers would benefit more from statin treatment.Methods. PubMed, EMBASE, and CNKI databases were searched until May 2015. All the statistical tests were performed using R version 3.1.2. Odds ratio (OR) and 95% confidence interval (CI) were used to assess the association between TLR4 Asp299Gly polymorphism and CAD risk, the number of stenotic vessels, and the incidence of cardiovascular events according to statin-treated patients. Weighted mean difference (WMD) was calculated for the association between Asp299Gly and CRP level.Results. Overall, 12 case-control studies with 10,258 cases and 5,891 controls were included, and no association of TLR4Asp299Gly polymorphism with CAD was found (G allele vs. A allele: OR = 0.97, 95% CI [0.81–1.17], P = 0.75; AA vs. GG + AG: OR = 0.97, 95% CI [0.80–1.18], P = 0.76; GG vs. AG + AA: OR = 1.08, 95% CI [0.57–2.02], P = 0.82; AG vs. AA + GG: OR = 1.03, 95% CI [0.85–1.25], P = 0.74). Also, no association was noted between Asp299Gly and CRP level (WMD = −0.10, 95% CI [−0.62, 0.41], P = 0.69). Furthermore, no synergistic effect of statin and 299Gly was reported (Statin_AA vs. Statin_AG/GG: OR = 1.12, 95% CI [0.41–3.09], P = 0.82).Discussion. This meta-analysis suggests no association of TLR4 Asp299Gly polymorphism with CAD and CRP level. It is further indicated that the G allele carriers may not benefit more from statin treatment. Further studies should include large sample size and high-quality literature to understand this issue in depth.

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.

Single nucleotide polymorphism of toll-like receptor 4 (TLR4) is associated with juvenile spondyloarthritis in Croatian population.

Single nucleotide polymorphisms (SNP) of toll-like and NOD-like receptors have been associated with altered receptor activity and modified production of proinflammatory cytokines leading to a number of diseases. Our aim was to determine whether SNP of TLR2 (Arg753Gln), TLR4 (Asp299Gly, Thr399Ile), and NLRP3 (Q705K) influence susceptibility to juvenile spondyloarthrtis (jSpA) and juvenile idiopathic arthritis (JIA). After the DNA extraction, 26 patients with jSpA, 11 with oligoarticular, polyarticular, or systemic JIA, and 40 healthy controls were genotyped for Arg753Gln, Asp299Gly, Thr399Ile, and Q705K SNP using real-time PCR-SNP analysis. Statistically significant difference in genotype frequency for Thr399Ile SNP of TLR4 was observed in the jSpA (χ2 = 6.705, p = 0.035) and not in the JIA group (χ2 = 3005, p = 0.223). Regarding Asp299Gly SNP, no significant difference in genotype frequency was found; however, allele frequency was significant in both jSpA and JIA patients. No significant difference in genotype or allele frequency was observed for Arg735Gln and Q705K SNP. The399Ile polymorphism of TLR4 may be responsible for altered immune response to microbial infection in variant carriers and represent a mechanism of triggering overproduction of proinflammatory cytokines and long-term inflammation in jSpA. SNP of TLR2, NLRP3, and TLR4 (Asp299Gly) were not associated with jSpA or JIA.

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.

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.

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.

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.

The Toll-Like Receptor 4 Polymorphism Asp299Gly but Not Thr399Ile Influences TLR4 Signaling and Function

The common, co-segregating Toll-like receptor 4 (TLR4) non-synonymous single nucleotide polymorphisms (SNPs), Asp299Gly and Thr399Ile, are associated with hyporesponsiveness to inhaled lipopolysaccharide (LPS) and increased susceptibility to Gram negative pathogens in humans. The purpose of this study was to identify the relative contributions of the Asp299Gly and the Thr399Ile variants in inhibiting the function of TLR4. 293/hMD2-CD14 cell line was transfected with lentiviral constructs containing human wild type (WT) TLR4-EGFP or TLR4-EGFP with Asp299Gly, Thr399Ile or Asp299Gly/Thr399Ile complementary DNA (cDNA). Multiple stable cell lines were established for each construct: three for WT TLR4, Asp299Gly, and Thr399Ile, and only two for Asp299Gly/Thr399Ile mutants and EGFP control. We did not observe a significant effect of polymorphisms on cell surface and intracellular TLR4 expression nor were there any significant differences in TLR4 and EGFP protein levels assessed by Western blotting and confocal microscopy among the multiple cell lines of each of the constructs. All cell lines had a dose-dependent responsiveness to LPS stimulation. However, compared to the WT TLR4, cells expressing TLR4 with Asp299Gly but not Thr399Ile polymorphism produced significantly less (P<0.05) IL-8 following LPS stimulation. Similarly, cells expressing TLR4 Asp299Gly but not Thr399Ile allele had significantly lower percentage of phosphorylated and total NF-κB P65 following LPS stimulation. While we could not do statistics on the Asp299Gly/Thr399Ile group, we observed a reduced responsiveness to LPS compared to WT TLR4. Taken together, we observed that the TLR4 Asp299Gly variant, but not the Thr399Ile variant, is responsible for impaired responsiveness of TLR4 to LPS and corresponding activation of NF-κB.