Wild-type TLR4 Research Articles

PWE-177 The functional relevance of toll-like receptor 4 mutations in oesophageal adenocarcinoma

Introduction Altered innate immune signalling may disrupt host-microbe homeostasis and promote an inflammatory microenvironment that favours tumourigenesis. The aim of this project was to assess whether somatic mutations in oesophageal adenocarcinoma (OAC) could interfere with Toll-like receptor (TLR) signalling, with a specific focus on TLR4. Method We interrogated the mutational profiles of TLR genes in 170 OAC samples, which had undergone whole genome sequencing as part of the oesophageal ICGC study. We performed site-directed mutagenesis and created 13 plasmids expressing mutant TLR4 with a CMV promoter (12 single nucleotide variants (SNVs), and one plasmid combined two SNVs). Luciferase reporter gene assays were performed in HEK293 cells stimulated with synthetic monophosphoryl lipid A (MPLA) to assess how the mutants altered TLR4 signalling and downstream activation of nuclear factor kappa B (NF-κB). Two of the TLR4 mutants were taken forward for transfection into the OAC cell line OE33, and ELISA was used to measure cytokine secretion after stimulation with MPLA. Results The TLR signalling pathway appeared to be recurrently mutated with missense mutations in 23/170 (13.5%) of tumour samples, including mutations in TLR1 (1.2%), TLR2 (0.6%), TLR4 (4.7%), TLR5 (0.6%), TLR7 (1.8%), TLR9 (1.2%), and MYD88 (1.5%). Based on our data and another recent study by Dulak et al . 1 , TLR4 mutations were the most frequent (5.4% combined data). One tumour sample contained two TLR4 mutations, E439G and F703C. Interestingly, the mutation E439G was identified both in our dataset and in the study by Dulak et al . Furthermore, in silico modelling suggested that the E439G mutation disrupts hydrogen bonds in the binding site of LPS and MD2. The use of NF-κB luciferase reporter assays demonstrated a significant decrease in ligand-dependent signalling for 8/12 of the TLR4 mutations tested in HEK293 cells. A double mutation of E439G with F703C revealed a further decrease in TLR4 signalling. Similarly, the concentration of IL-8 was lower for TLR4 mutants E439G and E439G+F703C transfected into OE33 cells stimulated with MPLA, in comparison to wild-type TLR4. Conclusion TLR4 is recurrently mutated in OAC, and the majority of these mutations showed decreased TLR4 signalling in response to ligand stimulation, both in HEK293 and OE33 cells. The biological relevance of this in relation to host-microbe homeostasis and carcinogenesis is being explored. Disclosure of interest None Declared. Reference Dulak AM, et al . Exome and whole-genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity. Nat Genet. 2013;45(5):478–486.

Toll-like receptor 4 mediates the regenerative effects of bone grafts for calvarial bone repair.

Craniofacial trauma is difficult to repair and presents a significant burden to the healthcare system. The inflammatory response following bone trauma is critical to initiate healing, serving to recruit inflammatory and progenitor cells and to promote angiogenesis. A role for inflammation in graft-induced bone regeneration has been suggested, but is still not well understood. The current study assessed the impact of Toll-like receptor (TLR4) signaling on calvarial repair in the presence of morselized bone components. Calvarial defects in wild-type and global TLR4(-/-) knockout mouse strains were treated with fractionated bone components in the presence or absence of a TLR4 neutralizing peptide. Defect healing was subsequently evaluated over 28 days by microcomputed tomography and histology. The matrix-enriched fraction of morselized bone stimulated calvarial bone repair comparably with intact bone graft, although the capacity for grafts to induce calvarial bone repair was significantly diminished by inhibition or genetic ablation of TLR4. Overall, our findings suggest that the matrix component of bone graft stimulates calvarial bone repair in a TLR4-dependent manner. These results support the need to better understand the role of inflammation in the design and implementation of strategies to improve bone healing.

The TLR4 D299G and T399I SNPs are constitutively active to up-regulate expression of Trif-dependent genes.

Dysregulated Toll-Like Receptor (TLR) signalling and genetic polymorphisms in these proteins are linked to many human diseases. We investigated TLR4 functional variants D299G and T399I to assess the impact on LPS-induced responsiveness in comparison to wild-type TLR4. The mechanism by which this occurs in unclear as these SNPs do not lie within the lipid A binding domain or dimerisation sites of the LPS-TLR4/MD2 receptor complexes. Transfection of TLR4D299G, TLR4T399I or TLR4D299G. T399I into HEK cells resulted in constitutive activation of an NF-κB reporter gene and a blunting of the LPS-induced reporter activation compared to WT-TLR4. Unstimulated human monocyte/macrophages, from patients with the D299G and T399I SNPs demonstrated a downregulation of many genes, particularly Tram/Trif signalling pathway constitutents compared to the TLR4 wild-type subjects supporting the concept of basal receptor activity. Monocyte/macrophages from carriers of the TLR4 D299G and T399I polymorphisms stimulated with LPS showed >6 fold lower levels of NF-κB and ∼12 fold higher IFN-β gene expression levels compared to wild-type subjects (P<0.05; MWU test) and dramatically altered resultant cytokine profiles. We conclude that these TLR4 SNPs affect constitutive receptor activity which impacts on the hosts ability to respond to LPS challenge leading to a dysregulated sub-optimal immune response to infection.

Toll-like receptor 4 mutant and null mice retain morphine-induced tolerance, hyperalgesia, and physical dependence.

The innate immune system modulates opioid-induced effects within the central nervous system and one target that has received considerable attention is the toll-like receptor 4 (TLR4). Here, we examined the contribution of TLR4 in the development of morphine tolerance, hyperalgesia, and physical dependence in two inbred mouse strains: C3H/HeJ mice which have a dominant negative point mutation in the Tlr4 gene rendering the receptor non-functional, and B10ScNJ mice which are TLR4 null mutants. We found that neither acute antinociceptive response to a single dose of morphine, nor the development of analgesic tolerance to repeated morphine treatment, was affected by TLR4 genotype. Likewise, opioid induced hyperalgesia and opioid physical dependence (assessed by naloxone precipitated withdrawal) were not altered in TLR4 mutant or null mice. We also examined the behavioural consequence of two stereoisomers of naloxone: (−) naloxone, an opioid receptor antagonist, and (+) naloxone, a purported antagonist of TLR4. Both stereoisomers of naloxone suppressed opioid induced hyperalgesia in wild-type control, TLR4 mutant, and TLR4 null mice. Collectively, our data suggest that TLR4 is not required for opioid-induced analgesic tolerance, hyperalgesia, or physical dependence.

Relationship between toll-like receptor 2 Arg677Trp and Arg753Gln and toll-like receptor 4 Asp299Gly polymorphisms and cytomegalovirus infection

The association among specific single-nucleotide polymorphisms (SNPs) in TLR2 (Arg677Trp, Arg753Gln) and TLR4 (Asp299Gln) and human cytomegalovirus (CMV) infection was studied in infants and adults. The TLR2 and TLR4 polymorphisms were genotyped in 151 patients with CMV infections and in 78 unrelated healthy individuals. Genotyping was performed by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified fragments. The viral load was measured by quantitative real-time PCR. Almost all of the patients with CMV infections were wild-type homozygotes without TLR2 and TLR4 polymorphisms. No significant differences in TLR2 and TLR4 polymorphisms were observed between infants with or without CMV infection. Compared with adults with CMV infections, heterozygosity for the TLR2 Arg677Trp and TLR4 Asp299Gly SNPs was detected more frequently in healthy individuals (p<0.05). Logistic regression analysis showed that the wild-type TLR2 genotype was associated with an increased risk of CMV infection and that heterozygosity for TLR2 and TLR4 SNPs diminished the risk of CMV infection in adult patients. An association between CMV load and the TLR4 SNP was found. Our results suggest that the wild-type TLR2 genotype may be a risk factor for CMV replication in adult patients.

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.

TLR4 is required for the obesity-induced pancreatic beta cell dysfunction

Obesity is an important inducing factor for type 2 diabetes. However, the mechanism underlying high-fat-(HF) diet-induced obesity in pancreatic beta cell dysfunction is still unclear. Toll-like receptor-4 (TLR4) is a key mediator of innate immunity. To investigate the effects of TLR4 in obesity-induced pancreatic beta cell dysfunction, we used male diabetic (db/db), obese (ob/ob) mice, TLR4-wild type (WT), and TLR4-knockout mice that were fed with normal diet or HF diet for 24 weeks. Immunostaining of TLR4 and TLR4 mRNA level in pancreatic islet were assessed. The results from biological characteristics, glucose tolerance test, insulin tolerance test, and insulin release test showed that the function of pancreatic islet was impaired in HF-fed TLR4 WT mice, but was protected in HF-fed TLR4 deficient (TLR4(-/-)) mice. By electron microscope detection, we observed that beta cell insulin secretory vesicles increased in HF-fed TLR4 WT mice. Ultrastructure of beta cell in HF-fed TLR4(-/-) mice was similar to that in normal chow diet-fed TLR4 WT mice. Then, glucose-stimulated insulin secretion assay by using primary pancreatic islet showed that the secretion function of pancreatic islet in HF-fed TLR4(-/-) mice was better than that in HF-fed TLR4 WT mice. Furthermore, in HF-fed TLR4(-/-) mice, the mRNA levels of IL-6, TNF-α, and MCP-1 genes in pancreatic islet were significantly lower than those in HF-fed TLR4 WT mice. Consistent with the change in gene expression, HF-fed TLR4 WT mice but not HF-fed TLR4(-/-) mice exhibited macrophage invasion in pancreatic island. Taken together, our data indicated that HF diet-induced obesity can stimulate the up-regulation of TLR4 locating on the surface of pancreatic beta cell, and subsequently lead to the recruitment of macrophage into pancreatic islet, which finally results in pancreatic beta cell dysfunction. This process is a possible mechanism involved in obesity-induced pancreatic beta cell dysfunction.

Risk of Repeated Moraxella catarrhalis Colonization Is Increased in Children With Toll-like Receptor 4 Asp299Gly Polymorphism

Moraxella catarrhalis is a common causative agent of acute otitis media and other respiratory infections in children. Toll-like receptor (TLR) 4 is an important protein of human innate immunity. One polymorphic site Asp299Gly of TLR4 is proven to result in an impaired response to lipopolysaccharide from Gram-negative bacteria. We investigated whether Finnish children who carry Asp299Gly had increased risk of M. catarrhalis colonization during their first 2 years of life. This was a prospective cohort study carried out in Turku, Finland. We studied M. catarrhalis colonization in 161 Finnish children, whose nasopharyngeal specimens were taken at 2, 12 and 24 months of age. The semiquantitative culture method was used for identification of different bacterial species and the pyrosequencing-based method for detection of TLR4 Asp299Gly. Of 161 subjects, 126 (78%) were TLR4 A/A wild type and 35 (22%) were A/G heterozygote variants. The prevalence of M. catarrhalis increased from 24% at 2 months to 48% at 12 months and to 58% at 24 months of age. Of the 35 subjects with TLR4 variant, 15 (43%) were M. catarrhalis positive at all 3 time points, whereas only 11 (9%) subjects with TLR4 wild type were positive at these time points (relative risk 4.91, 95% confidence interval: 2.482-9.711, P=0.0001). Moreover, subjects with TLR4 variant had significantly higher bacterial load of M. catarrhalis in their nasopharynx than those with TLR4 wild type (P=0.0032). Our results indicate that children with TLR4 Asp299Gly polymorphism have an increased risk of repeated M. catarrhalis colonization.

Toll-like receptor 4 deficiency accelerates the development of insulin-deficient diabetes in non-obese diabetic mice.

Background/ObjectiveToll-like receptors (TLR) mediate the recognition of microbial constituents and stress-induced endogenous ligands by the immune system. They may also be involved in the maintenance or break down of tolerance against autologous antigens. The aim of our investigation was to study the consequence of TLR4 deficiency on the development of insulin-deficient diabetes in the NOD mouse.MethodsThe TLR4 defect of the C57BL/10ScN mouse was backcrossed onto the NOD background and the effect of TLR4 deficiency on diabetes development was analysed by in vivo and in vitro studies.ResultsCompared to animals with wildtype TLR4 expression (TLR4+/+), female NOD mice carrying a homozygous TLR4 defect (TLR4−/−), showed significant acceleration of diabetes development, with a younger age at diabetes onset (TLR4+/+ 177±22 d, TLR−/−: 118±21 d; p<0.01). Pancreata of 120 d old TLR4−/− NOD mice revealed increased proportions of islets with advanced stages of immune cell infiltration compared to TLR4+/+ mice (p<0.05). TLR4 deficiency did not affect the susceptibility of islet cells to the beta cell damaging mediators nitric oxide or the inflammatory cytokines tumor necrosis factor alpha, interleukin-1 beta and interferon gamma. The lack of TLR4 further had no effect on the frequency of regulatory T-cells but reduced their capacity to inhibit T-cell proliferation.ConclusionsOur findings demonstrate that TLR4 deficiency results in an acceleration of diabetes development and immune cell infiltration of islets in NOD mice. We conclude that TLR4 is involved in the progression of the insulitis process thereby controlling the development of insulin-deficient diabetes in NOD mice.

Molecular Cloning and Functional Analysis of the Duck TLR4 Gene

Toll-like receptor 4 (TLR4) recognizes pathogen-associated molecular patterns in some animals and has been shown to be closely associated with several diseases such as tumors, atherosclerosis, and asthma. However, its function in ducks is not clear. Alternative splicing of the TLR4 gene has been identified in pigs, sheep, mice, and other species, but has not yet been reported in the duck. In this study, alternative splicing of the duck TLR4 gene was investigated using reverse transcription-polymerase chain reaction (RT-PCR). Duck TLR4 gene (duTLR4, accession number: KF278109) was found to consist of 3367 nucleotides of coding sequence. An alternative splice form, TLR4-b, was identified and shown by alignment to retain the intron between exons 1 and 2. Real-time quantitative polymerase chain reaction (qPCR) analyses suggested that duTLR4-a (wild-type) mRNA is widely expressed in various healthy tissues, whereas TLR4-b is expressed at only low levels. Following stimulation of normal duck embryo fibroblasts with lipopolysaccharide, the expression of both isoforms initially increased and then decreased. Expression of the wild-type isoform subsequently increased again, while that of the variant remained low. The expression levels of wild-type TLR4 were further analyzed by transient transfection of a pcDNA3.1(+)-TLR4-a overexpression vector into duck embryo fibroblasts. qRT-PCR analyses showed that after stimulation with LPS and poly(I:C) the expression levels of IL-1β, IL6, and MHC II increased with a response-efficacy relationship. Our experimental results indicate that TLR4 plays an important role in resistance to both bacterial and viral infections in the duck.

Toll-Like Receptor 2 Regulates Intestinal Inflammation by Controlling Integrity of the Enteric Nervous System

In the intestines, Toll-like receptor 2 (TLR2) mediates immune responses to pathogens and regulates epithelial barrier function; polymorphisms in TLR2 have been associated with inflammatory bowel disease phenotype. We assessed the effects of TLR2 signaling on the enteric nervous system (ENS) in mice. TLR2 distribution and function in the ileal neuromuscular layer of mice were determined by immunofluorescence, cytofluorimetric analysis, immunoprecipitation, and immunoblot analyses. We assessed morphology and function of the ENS in Tlr2(-/-) mice and in mice with wild-type Tlr2 (wild-type mice) depleted of intestinal microbiota, using immunofluorescence, immunoblot, and gastrointestinal motility assays. Levels and signaling of glial cell line-derived neurotrophic factor (GDNF) were determined using quantitative reverse transcriptase polymerase chain reaction, immunohistochemistry, and immunoprecipitation analyses. Colitis was induced by administration of dextran sulfate sodium or 2,4 dinitrobenzensulfonic acid to Tlr2(-/-) mice after termination of GDNF administration. TLR2 was expressed in enteric neurons, glia, and smooth muscle cells of the intestinal wall. Tlr2(-/-) mice had alterations in ENS architecture and neurochemical profile, intestinal dysmotility, abnormal mucosal secretion, reduced levels of GDNF in smooth muscle cells, and impaired signaling via Ret-GFRα1. ENS structural and functional anomalies were completely corrected by administration of GDNF to Tlr2(-/-) mice. Wild-type mice depleted of intestinal microbiota had ENS defects and GDNF deficiency, similar to Tlr2(-/-) mice; these defects were partially restored by administration of a TLR2 agonist. Tlr2(-/-) mice developed more severe colitis than wild-type mice after administration of dextran sulfate sodium or 2,4 dinitrobenzensulfonic acid; colitis was not more severe if Tlr2(-/-) mice were given GDNF before dextran sulfate sodium or 2,4 dinitrobenzensulfonic acid. In mice, TLR2 signaling regulates intestinal inflammation by controlling ENS structure and neurochemical coding, along with intestinal neuromuscular function. These findings provide information as to how defective TLR2 signaling in the ENS affects inflammatory bowel disease phenotype in humans.

5-Fluorouracil Induce the Expression of TLR4 on HCT116 Colorectal Cancer Cell Line Expressing Different Variants of TLR4.

Two common single nucleotide polymorphisms (SNPs) of the human TLR4 gene, namely Asp299Gly (D299G) and Thr399Ile (T399I), have been shown to impair the ability of certain individuals to respond properly to TLR4 ligands. 5-Fluorouracil (5-FU) is widely used for the treatment of patients with advanced colon cancers. The present study examined the impact of two common polymorphisms of the TLR4 genes on the response of the HCT116 colorectal cancer cells to 5-FU. HCT116 was transfected with Flag-CMV1-TLR4 wild-type (WT) and D299G, T399I expression plasmids. The cytotoxic effect of 5-FU on transfected cells was assessed by MTT assay. FACS analysis was performed to show the effect of 5-FU and LPS on the expression of different variants of TLR4. The lowest IC50-value was measured in cells expressing the WT TLR4 and non-transfected cells were more resistance to the drug compared to the other cells. 5-FU significantly induced the expression of TLR4 protein in the presence and absence of LPS. 5-FU also induced HMGB1 secretion, Cas3 and PARP activity and these effects were stronger in cells expressing WT TLR4 than the other cells. In conclusion, 5-FU-induced TLR4 expression and LPS had synergistic effect with 5-FU to induced apoptosis in colorectal cancer cells.

Bronchoalveolar Lavage Fluid Utilized Ex Vivo to Validate In Vivo Findings: Inhibition of Gap Junction Activity in Lung Tumor Promotion is Toll-Like Receptor 4-Dependent.

TLR4 protects against lung tumor promotion and pulmonary inflammation in mice. Connexin 43 (Cx43), a gap junction gene, was increased in Tlr4 wildtype compared to Tlr4-mutant mice in response to promotion, which suggests gap junctional intercellular communication (GJIC) may be compromised. We hypothesized that the early tumor microenvironment, represented by Bronchoalveolar Lavage Fluid (BALF) from Butylated hydroxytoluene (BHT; promoter)-treated mice, would produce TLR4-dependent changes in pulmonary epithelium, including dysregulation of GJIC in the Tlr4-mutant (BALBLps-d) compared to the Tlr4-sufficient (BALB; wildtype) mice. BHT (4 weekly doses) was injected ip followed by BALF collection at 24 h. BALF total protein and total macrophages were significantly elevated in BHT-treated BALBLps-d over BALB mice, similar to previous findings. BALF was then utilized in an ex vivo manner to treat C10 cells, a murine alveolar type II cell line, followed by the scrape-load dye transfer assay (GJIC), Cx43 immunostaining, and quantitative RT-PCR (Mcp-1, monocyte chemotactic protein 1). GJIC was markedly reduced in C10 cells treated with BHT-treated BALBLps-d BALF for 4 and 24 h compared to BALB and control BALF from the respective mice (p < 0.05). Mcp-1, a chemokine, was also significantly increased in the BHT-treated BALBLps-d BALF compared to the BALB mice, and Cx43 protein expression in the cell membrane altered. These novel findings suggest signaling from the BALF milieu is involved in GJIC dysregulation associated with promotion and links gap junctions to pulmonary TLR4 protection in a novel ex vivo model that could assist in future potential tumor promoter screening.

Structural Analyses of Human Toll-like Receptor 4 Polymorphisms D299G and T399I

TLR4 polymorphism replacing Asp-299 with Gly and Thr-399 with Ile (D299G/T399I) causes LPS hyporesponsiveness. TLR4(SNPs)·MD-2·LPS exhibits an agonistic 2:2:2 architecture. Local structural differences were observed around D299G, but not around T399I, SNP site. These local differences cause the modulation of surface properties of TLR4, which may affect ligand binding. This study provides structural evidence of the functionality of the mutant TLR4 carrying the SNPs. Toll-like receptor 4 (TLR4) and its coreceptor MD-2 recognize bacterial lipopolysaccharide (LPS) and signal the innate immune response. Two single nucleotide polymorphisms (SNPs) of human TLR4, D299G and T399I, have been identified and suggested to be associated with LPS hyporesponsiveness. Moreover, the SNPs have been proposed to be associated with a variety of infectious and noninfectious diseases. However, how the SNPs affect the function of TLR4 remains largely unknown. Here, we report the crystal structure of the human TLR4 (D299G/T399I)·MD-2·LPS complex at 2.4 Å resolution. The ternary complex exhibited an agonistic "m"-shaped 2:2:2 architecture that was similar to that of the human wild type TLR4·MD-2·LPS complex. Local structural differences that might affect the binding of the ligands were observed around D299G, but not around T399I, SNP site.

Respiratory syncytial virus fusion protein-induced toll-like receptor 4 (TLR4) signaling is inhibited by the TLR4 antagonists Rhodobacter sphaeroides lipopolysaccharide and eritoran (E5564) and requires direct interaction with MD-2.

ABSTRACTRespiratory syncytial virus (RSV) is a leading cause of infant mortality worldwide. Toll-like receptor 4 (TLR4), a signaling receptor for structurally diverse microbe-associated molecular patterns, is activated by the RSV fusion (F) protein and by bacterial lipopolysaccharide (LPS) in a CD14-dependent manner. TLR4 signaling by LPS also requires the presence of an additional protein, MD-2. Thus, it is possible that F protein-mediated TLR4 activation relies on MD-2 as well, although this hypothesis has not been formally tested. LPS-free RSV F protein was found to activate NF-κB in HEK293T transfectants that express wild-type (WT) TLR4 and CD14, but only when MD-2 was coexpressed. These findings were confirmed by measuring F-protein-induced interleukin 1β (IL-1β) mRNA in WT versus MD-2−/− macrophages, where MD-2−/− macrophages failed to show IL-1β expression upon F-protein treatment, in contrast to the WT. Both Rhodobacter sphaeroides LPS and synthetic E5564 (eritoran), LPS antagonists that inhibit TLR4 signaling by binding a hydrophobic pocket in MD-2, significantly reduced RSV F-protein-mediated TLR4 activity in HEK293T-TLR4–CD14–MD-2 transfectants in a dose-dependent manner, while TLR4-independent NF-κB activation by tumor necrosis factor alpha (TNF-α) was unaffected. In vitro coimmunoprecipitation studies confirmed a physical interaction between native RSV F protein and MD-2. Further, we demonstrated that the N-terminal domain of the F1 segment of RSV F protein interacts with MD-2. These data provide new insights into the importance of MD-2 in RSV F-protein-mediated TLR4 activation. Thus, targeting the interaction between MD-2 and RSV F protein may potentially lead to novel therapeutic approaches to help control RSV-induced inflammation and pathology.

Variants of Toll-like Receptor 4 Predict Cardiac Recovery in Patients with Dilated Cardiomyopathy

The clinical course of patients with dilated cardiomyopathy (DCM) varies from cardiac recovery to end stage heart failure. The etiology of this variability is largely unknown. In this study, we investigated the impact of coding polymorphisms of the innate immune protein Toll-like receptor 4 (TLR4) on left ventricular performance in patients with DCM. Two variants of TLR4 (rs4986790, TLR4 c.1187A→G, p.299D→G and rs4986791,TLR4 c.1487C→T, p.T399I) were investigated in 158 patients with DCM. Other reasons for heart failure were excluded by coronary angiography, myocardial biopsy, and echocardiography. Risk factors, age, gender, or treatment did not differ among the groups. At the follow-up evaluation (median 4.0-5.4 months), patients carrying the TLR4 wild type gene displayed cardiac recovery under intense medical heart failure therapy indexed by reduced left ventricular dilation, improved left ventricular ejection fraction, and reduced NT-probrain natriuretic peptide blood level when compared with the initial evaluation. In contrast, patients carrying both the rs4986790 and the rs4986791 variant showed significantly reduced improvement of left ventricular ejection fraction (p = 0.006) and left ventricular dilation (p = 0.015) at the follow-up evaluation when compared with carriers of the wild type gene under the same treatment conditions. In addition, NT-probrain natriuretic peptide level in carriers of both TLR4 variants did not change significantly at the follow up when compared with the first evaluation. Among patients with DCM, the presence of the TLR4 variants rs4986790 and rs4986791 predicts impaired cardiac recovery independently of medical treatment or cardiac risk factors.

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.

R753Q polymorphism compromises TLR2 signaling functions by altering TLR6-TLR2 hetero-dimerization, TLR2 tyrosine phosphorylation and MyD88 recruitment (180.2)

Abstract TLR2 senses lipoproteins expressed by Gram positive bacteria, mycoplasma, mycobacteria and fungi. The Arg753Gln (R753Q) polymorphism in the TIR domain of TLR2 has been linked to increased incidence of tuberculosis, leprosy and other diseases. But the molecular mechanisms by which it affects TLR2 functions are unclear. We studied the impact of complementation of HEK293 cells and mouse TLR2-/- macrophages with wild-type (WT) vs. R753Q TLR2 on TLR2 expression, interactions with TLR6, tyrosine phosphorylation, recruitment of MyD88, and functional responses. Transfected WT and R753Q TLR2 variants had comparable expression, and even 100-fold increase in input amounts of R753Q vs. WT TLR2 did not overcome deficiency of the mutant receptor. R753Q TLR2 exhibited deficient agonist-induced tyrosine phosphorylation, hetero-dimerization with TLR6 and recruitment of MyD88. R753Q TLR2 mediated impaired activation of p38, NF-κB, and induction of IL-8 mRNA in response to inactivated Mycobacterium tuberculosis, mycobacteria cell wall, culture filtrate fractions, lipoarabinomannan and Pam3Cys. In contrast, stimulation with TNF-α led to similar NF-κB reporter activation and IL-8 gene expression in cells expressing wild-type or R753Q TLR2. These results revealed deficient TLR2 tyrosine phosphorylation, hetero-dimerization with TLR6 and recruitment of MyD88 as key features underscoring signaling deficiency of the R753Q TLR2.

The Asp299Gly Polymorphism Alters TLR4 Signaling by Interfering with Recruitment of MyD88 and TRIF

AbstractAsp299Gly (D299G) and, to a lesser extent, Thr399Ile (T399I) TLR4 polymorphisms have been associated with Gram-negative sepsis and other infectious diseases, but the mechanisms by which they affect TLR4 signaling are unclear. In this study, we determined the impact of the D299G and T399I polymorphisms on TLR4 expression, interactions with myeloid differentiation factor 2 (MD2), LPS binding, and LPS-mediated activation of the MyD88- and Toll/IL-1R resistance domain-containing adapter inducing IFN-β (TRIF) signaling pathways. Complementation of human embryonic kidney 293/CD14/MD2 transfectants with wild-type (WT) or mutant yellow fluorescent protein-tagged TLR4 variants revealed comparable total TLR4 expression, TLR4–MD2 interactions, and LPS binding. FACS analyses with anti-TLR4 Ab showed only minimal changes in the cell-surface levels of the D299G TLR4. Cells transfected with D299G TLR4 exhibited impaired LPS-induced phosphorylation of p38 and TANK-binding kinase 1, activation of NF-κB and IFN regulatory factor 3, and induction of IL-8 and IFN-β mRNA, whereas T399I TLR4 did not cause statistically significant inhibition. In contrast to WT TLR4, expression of the D299G mutants in TLR4−/− mouse macrophages failed to elicit LPS-mediated induction of TNF-α and IFN-β mRNA. Coimmunoprecipitation revealed diminished LPS-driven interaction of MyD88 and TRIF with the D299G TLR4 species, in contrast to robust adapter recruitment exhibited by WT TLR4. Thus, the D299G polymorphism compromises recruitment of MyD88 and TRIF to TLR4 without affecting TLR4 expression, TLR4–MD2 interaction, or LPS binding, suggesting that it interferes with TLR4 dimerization and assembly of intracellular docking platforms for adapter recruitment.

The Toll-like receptor (TLR) 4 variant D299G promotes inflammation-associated colon cancer progression in the intestinal epithelium

In active IBD colitis, TLR4 expression is significantly upregulated in intestinal epithelial cells (IEC). The variant TLR4-D299G has recently been associated with increased IBD risk. However, the phenotypic consequences of this mutation in IEC have not yet been resolved. The AIM OF THIS STUDY was to determine the effects of TLR4-D299G on IEC function. IEC lines (Caco-2) stably overexpressing HA-tagged wildtype TLR4, mutant TLR4-D299G or TLR4-T399I were generated. Gene expression profiling using DNA microarray analysis (Human Gene 1.0 ST; Affymetrix) was performed. Findings were confirmed by qRT-PCR and validated by immunoblot, ELISA, confocal immunofluorescence and functional analyses. In-vivo tumorigenicity was tested using the CD-1 nu/nu mice xenograft model. Human colon cancer specimens (n=214) were genotyped and assessed for disease stage. In contrast to control clones, TLR4-D299G IEC exhibited a mesenchymal phenotype with multipolar mitotic spindles. TLR4-D299G induced Wnt/ß-catenin activation, leading to IEC de-differentiation: a) phosphorylated ß-catenin translocated from the membrane to the cytoplasm and accumulated in the nucleus, b) E-cadherin redistributed to the cytoplasm which correlated with loss of cell polarity, and c) the transcriptional factor Snail2 anchored to the plasma membrane. The major TLR4-D299G targets were dominated by genes interconnected with “tumorigenesis” and “inflammation” (A2M, ALCAM, ANXA1, C5, CD47, CHI3L1, HPX, IL2RG, TF, TFPI, SERPINC1). TLR4-D299G IEC constitutively secreted large protein amounts of coagulation and complement factors (A2M, TFPI, C3a, C5a). TLR4-D299G induced Wnt-dependent STAT3 activation, which triggered IEC invasion. All of these effects were not evident in the control clones (TLR4-WT, TLR4-T399I, mock). Xenografts in mice revealed TLR4-D299G-mediated acceleration of intestinal tumor growth - which was blocked by a specific STAT3 inhibitor. At time of diagnosis human colon adenocarcinomas from patients with TLR4-D299G were more frequently of an advanced stage (UICC≥III: 70% vs. 46%; P=0.0142) with metastasis (UICC=IV: 42% vs. 19%; P=0.0065) than those with wild-type TLR4. Expression of STAT3 mRNA was significantly increased among human colonic adenocarcinomas with TLR4-D299G than those with wild-type TLR4. However, expression of TLR4 mRNA was elevated in all primary human colon cancers, regardless of genotype, when compared to normal tissues. Our findings show that TLR4-D299G drives inflammation-associated epithelial-mesenchymal transition and neoplastic progression in the IEC line Caco-2 in-vitro and in-vivo and associates with advanced colon cancer in humans. Future studies must determine whether TLR4-D299G puts IBD patients at increased risk to develop a more aggressive phenotype of colitis - associated neoplasia. [funded in part by the CCFA (SRA-1790)]