IL-10 release requires stronger toll-like receptor 4-triggering than TNF: a possible explanation for the selective effects of heterozygous TLR4 polymorphism Asp(299)Gly on IL-10 release

The toll-like receptor 4 Asp(299)Gly polymorphism results in an inactive receptor. Heterozygosis is associated with reduced LPS-inducible IL-10 protein and IL-10 mRNA from blood leukocytes and isolated monocytes, while numerous other mediators are not affected. We could exclude that this effect is due to the differences in the kinetics of IL-10 release, in the expression of total surface TLR4 or in LPS-binding to monocytes between subjects heterozygous for the Asp(299)Gly polymorphism or homozygous carriers of the wild-type allele. Furthermore, we could show that IL-10 induction in general requires stronger LPS-triggering than TNF and is more sensitive to LPS inhibitors. The lower number of responsive wild-type TLR4 receptors on monocytes of heterozygotes may explain why only IL-10 release is affected.     

Differential activation of human gingival epithelial cells and monocytes by Porphyromonas gingivalis fimbriae

Humans develop periodontitis in response to challenge by microbial dental plaque. Inflammation begins after perturbation of gingival epithelial cells by subgingival bacteria interacting through pattern-recognition receptors, including the Toll-like receptors (TLR). Porphyromonas gingivalis is a major periodontopathogen that interacts with epithelial cells through its cell surface fimbriae (FimA), leading to colonization and/or invasion. Previous work by our group has established membrane CD14 as an essential coreceptor for TLR2-mediated activation of transfected cell lines by P. gingivalis FimA. We have shown that gingival epithelial cells express TLR2 but not CD14 on their cell surfaces. We thus speculated that P. gingivalis FimA does not readily activate epithelial innate immune responses but rather functions to promote P. gingivalis colonization in the absence of a vigorous FimA-induced response. This hypothesis was verified by the findings that primary human gingival epithelial cells responded poorly to FimA in terms of interleukin (IL)-6, IL-8, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha responses, in stark contrast to the marked response to other TLR2 agonists (Pam3Cys, FSL-1) that are not strictly dependent on CD14. On the other hand, CD14-expressing human primary monocytes responded with high levels of the same cytokines to both FimA and the control TLR2 agonists. The gingival epithelial cells failed to respond to FimA even in the presence of exogenously added soluble CD14. These data indicate that the gingival epithelial cell hyporesponsiveness to FimA is attributable to the lack of membrane-expressed but not soluble CD14. In conclusion, P. gingivalis FimA differentially activates human monocytes and epithelial cells, perhaps reflecting different tactics used by P. gingivalis when interacting with different host cell types or a host strategy to limit inflammation.     

Common human Toll-like receptor 4 polymorphisms--role in susceptibility to respiratory syncytial virus infection and functional immunological relevance

Evidence suggests that Toll-like receptor 4 (TLR4) contributes to immune recognition of respiratory syncytial virus (RSV). The TLR4 gene harbours a polymorphism-Asp299Gly-previously associated with reduced TLR4 signalling. To understand of how host genetic variation influences the outcome of RSV infection in children, we examined the association between the TLR4 299Gly allele and severe RSV disease. By genotyping 236 children with RSV infection and 219 healthy controls we found no association between the risk of severe RSV infection and Asp299Gly polymorphisms (P>0.05), and we demonstrate that the TLR4 Asp299Gly genotype does not influence susceptibility to either RSV serotype A or B (P>0.05). Finally, examining the functional impact of the TLR4 Asp299Gly polymorphism (n=58), we demonstrate that proinflammatory cytokine production following TLR4 activation was indistinguishable between homozygous (Asp/Asp) and heterozygous (Asp/Gly) subjects. We conclude that the Asp299Gly TLR4 polymorphism does not alter receptor function and does not influence the risk of severe RSV infection.     

Interleukin-1beta modulates proinflammatory cytokine production in human epithelial cells

Periodontitis is a chronic human inflammatory disease initiated and sustained by dental plaque microorganisms. A major contributing pathogen is Porphyromonas gingivalis, a gram-negative bacterium recognized by Toll-like receptor 2 (TLR2) and TLR4, which are expressed by human gingival epithelial cells (HGECs). However, it is still unclear how these cells respond to P. gingivalis and initiate inflammatory and immune responses. We have reported previously that HGECs produce a wide range of proinflammatory cytokines, including interleukin-6 (IL-6), IL-8, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha (TNF-alpha), and IL-1beta. In this study, we show that IL-1beta has a special role in the modulation of other inflammatory cytokines in HGECs challenged with P. gingivalis. Our results show that the increased production of IL-1beta correlates with the cell surface expression of TLR4, and more specifically, TLR4-normal HGECs produce fourfold more IL-1beta than do TLR4-deficient HGECs after challenge. Moreover, blocking the IL-1beta receptor greatly reduces the production of "secondary" proinflammatory cytokines such as IL-8 or IL-6. Our data indicate that the induction of IL-1beta plays an important role in mediating the release of other proinflammatory cytokines from primary human epithelial cells following challenge with P. gingivalis, and this process may be an inflammatory enhancement mechanism adopted by epithelial cells.     

A TLR4 polymorphism is associated with asthma and reduced lipopolysaccharide-induced interleukin-12(p70) responses in Swedish children

BACKGROUND: Bacterial signals play an important role in the maturation of the immune system. Polymorphisms in genes coding for receptors to bacterial components can alter the immune responsiveness of the host to microbial agents and may indicate the development of aberrant immune responses that are associated with immune-mediated diseases such as atopic diseases. OBJECTIVE: The study's objective was to investigate the relationship between TLR4 and CD14 gene polymorphisms, the LPS responsiveness of PBMCs, and the presence of asthma and allergic rhinoconjunctivitis in children. METHODS: The TLR4 (Asp299Gly) and CD14/-159 polymorphisms were determined in 115 Swedish children aged 8 and 14 years. LPS-induced IL-12(p70), IL-10, and IFN-gamma responses of PBMCs from 69 of the children were analyzed by means of ELISA. The levels of soluble CD14 in serum samples were analyzed by means of ELISA, and the total IgE levels were analyzed by means of UniCAP Total IgE (Pharmacia Diagnostics, Uppsala, Sweden). RESULTS: Decreased LPS-induced IL-12(p70) and IL-10 responses were associated with the TLR4 (Asp299Gly) polymorphism and independently with asthma, especially atopic asthma. The TLR4 (Asp299Gly) polymorphism was associated with a 4-fold higher prevalence of asthma in school-aged children (adjusted odds ratio 4.5, 95% CI 1.1-17.4) but not to allergic rhinoconjunctivitis. CONCLUSION: A TLR4 polymorphism modifies innate immune responses in children and may be an important determinant for the development of asthma. This may influence the outcome of intervention studies that use microbial stimuli as immune modulators.     

Toll-like 4 receptor variant, Asp299Gly, and reduced risk of hemorrhagic cystitis after hematopoietic stem cell transplantation

Hemorrhagic cystitis (HC) is a major cause of morbidity after hematopoietic stem cell transplantation. Toll-like receptor 4 (TLR4) is a pattern recognition receptor of the innate immune system and induces inflammation. Individuals with the single nucleotide polymorphisms Thr399Ile (rs4986791) or Asp299Gly (rs4986790) of TLR4 show diminished inflammatory responsiveness to endotoxins. The genotype of TLR4 was determined in 166 children who underwent allogeneic hematopoietic stem cell transplantation and in their donors. Asp299Gly was present in 21 patients (13%) and 24 donors (14%). Thr399Ile was found in 22 patients (13%) and 25 donors (15%). The incidence of HC was significantly lower in patients with Asp299Gly (0% vs 23%; P = .009) and in patients who underwent transplantation from a donor with Asp299Gly (4% vs 23%; P = .05). The trend was the same for Thr399Ile-donor positive (8% vs 22%; P = .17), recipient positive (9% vs 22%; P = .25), donor or recipient positive (8% vs 23%; P = .04). Multivariate analysis revealed age, conditioning with busulfan, and absence of Asp299Gly as independent risk factors for HC. In conclusion, the TLR4 Asp299Gly variant seems to confer protection against hemorrhagic cystitis. This study provides the first indication that the innate immune system through TLR4 signaling pathway plays a role in the pathogenesis of HC after hematopoietic stem cell transplantation.     

Toll receptor 4 Asp299Gly polymorphism and its association with preterm birth and premature rupture of membranes in a South American population

Preterm birth (PTB) is a worldwide health problem and remains the leading cause of perinatal morbidity and mortality. Systemic and local intrauterine infections have been implicated in the pathogenesis of preterm labor and delivery. Common pathways between PTB, premature rupture of ovular membranes (PROM) and altered molecular routes of inflammation have been proposed. There is evidence to support a genetic component in these conditions. Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, is thought to play a key role in eliciting an inflammatory response. LPS is recognized by proteins of the innate immune system, including Toll-like receptor 4 (TLR4). Individuals from some European countries carrying the variant alleles resulting in an amino acid substitution (Asp299Gly) are at increased risk of Gram-negative infections and premature birth. The objective of this study was to determine if preterm newborns have different allele frequency of the Asp299Gly TLR4 variant from healthy term neonates in Uruguay. The impact of PROM was also examined. There was an increase in the risk for fetuses carrying the Asp299Gly substitution in TLR4 of being severely premature (<33 weeks) and to present PROM at the same time.     

Effect of the functional toll-like receptor 4 Asp299Gly polymorphism on susceptibility to late-onset Alzheimer's disease

Experimental data have shown an upregulated expression of toll-like receptors, particularly toll-like receptor 4 (TLR4), in neurodegeneration. The Asp299Gly polymorphism of the TLR4 gene has been associated with an attenuated receptor signalling and a blunted inflammatory response. In the present study, we sought to determine whether this common genetic variant could influence susceptibility to late-onset Alzheimer's disease (LOAD) in an Italian population sample. A cohort of 277 LOAD patients and 300 cognitively healthy controls were genotyped for the TLR4 Asp299Gly polymorphism using restriction isotyping. The frequency of the minor 299Gly allele was significantly higher in the controls than in the LOAD cases (7.2% versus 3.1%, respectively, P=0.003). Additionally, the frequency of the variant genotypes (Asp/Gly and Gly/Gly) was 13.0% in the controls and 5.4% in LOAD patients (P=0.002). After adjustment for age, gender, and the APOE varepsilon4 carrier status, the odds ratio for the development of LOAD associated with the Asp/Gly and Gly/Gly versus Asp/Asp genotype was 0.37 (95% CI: 0.20-0.69, P=0.002). Our data further support a role for innate immunity in neurodegeneration and give the first evidence that the TLR4 Asp299Gly variant may be protective toward the development of LOAD.     

Porphyromonas gingivalis invasion of gingival epithelial cells.

Porphyromonas gingivalis, a periodontal pathogen, can invade primary cultures of gingival epithelial cells. Optimal invasion occurred at a relatively low multiplicity of infection (i.e., 100) and demonstrated saturation at a higher multiplicity of infection. Following the lag phase, during which bacteria invaded poorly, invasion was independent of growth phase. P. gingivalis was capable of replicating within the epithelial cells. Invasion was an active process requiring both bacterial and epithelial cell energy production. Invasion was sensitive to inhibitors of microfilaments and microtubules, demonstrating that epithelial cell cytoskeletal rearrangements are involved in bacterial entry. P. gingivalis, but not epithelial cell, protein synthesis was necessary for invasion. Invasion within the epithelial cells was not blocked by inhibitors of protein kinase activity. Invasion was inhibited by protease inhibitors, suggesting that P. gingivalis proteases may be involved in the invasion process. Low-passage clinical isolates of P. gingivalis invaded with higher efficiency than the type strain. Serum inhibited invasion of the type strain but had no effect on the invasion of a clinical isolate. Invasion of gingival epithelial cells by P. gingivalis may contribute to the pathology of periodontal diseases.     

Single step high-throughput determination of Toll-like receptor 4 polymorphisms

BACKGROUND: Toll-like receptors are central components of host defence in humans, responsible for recognition of pathogen-associated molecular patterns and activation of innate immunity. Toll-like receptor 4 (TLR4) is activated by lipopolysaccharide (LPS) and other microbial components, thereby initiating the expression and release of pro-inflammatory cytokines. The common, frequently co-segregating allelic variants Asp299Gly and Thr399Ile have been related to susceptibility to gram-negative infections and sepsis and may be involved in the development of atherosclerosis. Identification of TLR4 Asp299Gly and Thr399Ile genotypes can be important for examination of genotype/phenotype relationships as well as for individual risk assessment of patients. METHODS: TLR4 Asp299Gly and Thr399Ile genotypes were detected by a single tube polymerase chain reaction (PCR), based on exonuclease degradation of dual labelled allele-specific oligonucleotides. The assay results were compared with conventional restriction fragment length polymorphism (RFLP) analysis. RESULTS: Genotypes of 345 individuals were determined simultaneously in a single PCR assay. Allele frequencies for our population were 6.8% for the TLR4 Asp299Gly polymorphism and 6.4% for the Thr399Ile polymorphism. Validation by RFLP analysis revealed a correct detection of all genotypes. CONCLUSIONS: We have developed a novel method for the detection of the TLR4 Asp299Gly and Thr399Ile mutations, permitting rapid genotyping which should be useful for large-scale population studies as well as applicable for routine clinical testing.     

Human Conjunctival Epithelial Cells Lack Lipopolysaccharide Responsiveness Due to Deficient Expression of MD2 but Respond After Interferon-γ Priming or Soluble MD2 Supplementation

Inflammatory responses to Gram-positive and Gram-negative bacterial cell wall components are initiated by Toll-like receptor 2 (TLR2) and TLR4, respectively. Therefore, the existence of functionally active TLR2 and TLR4 in human conjunctival epithelial cells (HCEC) are critical for the effective host defense against bacterial infections in the eye. We examined the ability of HCEC to respond to TLR4 ligand, lipopolysaccharide (LPS), or TLR2 ligands, lipoteichoic acid (LTA) and peptidoglycan (PGN) using the Chang conjunctival epithelial cell line and the primary conjunctival epithelial cell line (IOBA-NHC) as in vitro models. Incubation of Chang cells with LPS (1 to 1,000 ng/ ml) failed to stimulate IL-6 production where as stimulation with LTA or PGN resulted in marked increases in IL-6 production. Semi-quantitative RT–PCR and immunofluorescence analyses showed that Chang cells express TLR2 and TLR4 mRNA and proteins. However, these cells expressed little or no mRNA encoding MD2, an accessory molecule required for TLR4 signaling. Incubation of Chang epithelial cells with interferon-γ (IFNγ), but not TNF-α, stimulated MD2 mRNA expression and restored LPS responsiveness. In addition, when Chang cell cultures were supplemented with soluble MD2, LPS was able to stimulate IL-6 production. The lack of LPS response, deficient expression of MD2, and induction of MD2 expression and LPS response after IFNγ priming, were also evident in IOBA-NHC cells. These results demonstrate that HCEC lack LPS responsiveness due to deficient expression of MD2 and that the response can be restored by IFN-γ priming or MD2 supplementation.     

Toll-like receptor (TLR) 2 and 4 mutations in periodontal disease

Toll-like receptors (TLR) are signal molecules essential for the cellular response to bacterial cell wall components. Different functional effective polymorphisms for the TLR 4 gene (Asp299Gly; Thr399Ile) and for the TLR 2 gene (Arg677Trp, Arg753Gln) have recently been described that are associated with impaired lipopolysaccharide signal transduction. A total of 122 patients with chronic periodontal disease and 122 healthy unrelated controls were genotyped for the Asp299Gly and Thr399Ile polymorphism of the TLR 4 gene and the Arg677Trp and Arg753Gln mutation of the TLR 2 gene. The mutations were identified with polymerase chain reaction followed by restriction fragment length polymorphism (RFLP) analysis. The prevalence of the Asp299Gly and the Thr399Ile mutant allele was 4.1% (10/244) and 4.5% (11/244) among periodontitis patients. For the healthy controls the prevalence was 3.3% (8/244) for the Asp299Gly (P = 0.810) and 3.7% (9/244) for the Thr399Ile mutant allele (P = 0.819). The Arg753Gln mutant allele was found in 2.9% (7/244) of the periodontitis subjects as compared to 4.1% (10/244) in the control group (P = 0.622). The Arg677Trp mutant allele was not found in any of the study subjects. Unlike in ulcerative colitis there was not observed an association between chronic periodontitis and the various mutations of the TLR 2 and 4 gene.     

Bacterial fimbriae and their peptides activate human gingival epithelial cells through Toll-like receptor 2.

Gingival epithelial cells are a central component of the barrier between oral microflora and internal tissues. Host responses to periodontopathic bacteria and surface components containing fimbriae are thought to be important in the development and progression of periodontal diseases. To elucidate this mechanism, we established immortalized human gingival epithelial cells (HGEC) that were transfected with human papillomavirus. HGEC predominantly expressed Toll-like receptor (TLR) 2, but not TLR4 or CD14. They also induced interleukin-8 (IL-8) production when stimulated with Porphyromonas gingivalis fimbriae and Staphylococcus aureus peptidoglycan, but not Escherichia coli-type synthetic lipid A. Furthermore, an active synthetic peptide composed of residues 69 to 73 (ALTTE) of the fimbrial subunit protein, derived from P. gingivalis and similar to a common component of cell wall peptidoglycans in parasitic bacteria, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), significantly induced IL-8 production and NF-kappaB activation in HGEC, and these cytokine-producing activities were augmented by a complex of soluble CD14 and lipopolysaccharide-binding protein (LBP). IL-8 production in HGEC stimulated with these bacterial components was clearly inhibited by mouse monoclonal antibody to human TLR2. These findings suggest that P. gingivalis fimbrial protein and its active peptide are capable of activating HGEC through TLR2.     

Interleukin-8 and intercellular adhesion molecule 1 regulation in oral epithelial cells by selected periodontal bacteria: multiple effects of Porphyromonas gingivalis via antagonistic mechanisms

Interaction of bacteria with mucosal surfaces can modulate the production of proinflammatory cytokines and adhesion molecules produced by epithelial cells. Previously, we showed that expression of interleukin-8 (IL-8) and intercellular adhesion molecule 1 (ICAM-1) by gingival epithelial cells increases following interaction with several putative periodontal pathogens. In contrast, expression of IL-8 and ICAM-1 is reduced after Porphyromonas gingivalis ATCC 33277 challenge. In the present study, we investigated the mechanisms that govern the regulation of these two molecules in bacterially infected gingival epithelial cells. Experimental approaches included bacterial stimulation of gingival epithelial cells by either a brief challenge (1.5 to 2 h) or a continuous coculture throughout the incubation period. The kinetics of IL-8 and ICAM-1 expression following brief challenge were such that (i) secretion of IL-8 by gingival epithelial cells reached its peak 2 h following Fusobacterium nucleatum infection whereas it rapidly decreased within 2 h after P. gingivalis infection and remained decreased up to 30 h and (ii) IL-8 and ICAM-1 mRNA levels were up-regulated rapidly 2 to 4 h postinfection and then decreased to basal levels 8 to 20 h after infection with either Actinobacillus actinomycetemcomitans, F. nucleatum, or P. gingivalis. Attenuation of IL-8 secretion was facilitated by adherent P. gingivalis strains. The IL-8 secreted from epithelial cells after F. nucleatum stimulation could be down-regulated by subsequent infection with P. gingivalis or its culture supernatant. Although these results suggested that IL-8 attenuation at the protein level might be associated with P. gingivalis proteases, the Arg- and Lys-gingipain proteases did not appear to be solely responsible for IL-8 attenuation. In addition, while P. gingivalis up-regulated IL-8 mRNA expression, this effect was overridden when the bacteria were continuously cocultured with the epithelial cells. The IL-8 mRNA levels in epithelial cells following sequential challenge with P. gingivalis and F. nucleatum and vice versa were approximately identical and were lower than those following F. nucleatum challenge alone and higher than control levels or those following P. gingivalis challenge alone. Thus, together with the protease effect, P. gingivalis possesses a powerful strategy to ensure the down-regulation of IL-8 and ICAM-1.     

IFNs activate toll-like receptor gene expression in viral infections

Toll-like receptors (TLRs) mediate innate immune responses to microbes. TLR2, TLR5, TLR6, and TLR9 have been implicated in responses to bacterial components, and TLR4 is the receptor for Gram-negative bacteria. Recently, TLR4 was described to function in respiratory syncytial virus-induced NF-kappaB activation. Here we have analyzed TLR1-9 mRNA expression in human primary macrophages infected with influenza A and Sendai viruses. TLR1, TLR2, TLR4, TLR6, and TLR8 mRNAs were expressed at basal levels in macrophages. Viral infection enhanced TLR1, TLR2, TLR3, and TLR7 mRNA expression, and neutralizing anti-IFN-alpha/beta antibodies downregulated gene expression of these TLRs. Exogenously added IFN-alpha upregulated TLR1, TLR2, TLR3, and TLR7 mRNA expression in macrophages, as well as TLR3 mRNA expression in epithelial and endothelial cell lines. IFN-gamma enhanced the expression of TLR1 and TLR2 mRNA in macrophages, and TLR3 in epithelial and endothelial cells. The data suggests a novel role for IFNs in the activation of innate immunity.     

Toll-like receptor (TLR) 4 polymorphisms are associated with a chronic course of sarcoidosis

The aetiology of sarcoidosis, an inflammatory granulomatous multi-system disorder, is unclear. It is thought to be the product of an unknown exogenous antigenic stimulus and an endogenous genetic susceptibility. Toll-like receptors (TLR) are signal molecules essential for the cellular response to bacterial cell wall components. Lipopolysaccharide (LPS), for example, binds to TLR 4. Two different polymorphisms for the TLR4 gene (Asp299Gly and Thr399Ile) have been described recently. This leads to a change in the extracellular matrix function of TLR4 and to impaired LPS signal transduction. We genotyped a total of 141 Caucasian patients with sarcoidosis and 141 healthy unrelated controls for the Asp299Gly and Thr399Ile polymorphisms in the TLR4 gene. The mutations were identified with polymerase chain reaction followed by restriction fragment length polymorphism (RFLP) analysis. Among sarcoidosis patients the prevalence for each Asp299Gly and Thr399Ile mutant allele was 15.6% (22/141). In the control group the prevalence was 5.67% (8/141) (P = 0.07). In the subgroup of patients with acute sarcoidosis there was no difference in the control group (P = 0.93), but there was a highly significant association between patients with a chronic course of sarcoidosis and TLR4 gene polymorphisms (P = 0.01).     

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

Background: It has been proposed that the toll-like receptor-4 gene (TLR4) may participate in the development of obesity and osteoporosis, in addition to its well-known role in the immune response. On the other hand, the adipose tissue of obese subjects shows an increased expression of the proinflammatory cytokine, tumour necrosis factor-alpha (TNF-α), which is released after lipopolysaccharide recognition by TLR4.

Aim: To estimate the allele/genotype frequencies and linkage disequilibrium measures of Asp299Gly and Thr399Ile polymorphisms of the TLR4 gene in the Chilean elderly population, and to screen for their association with variables related to adiposity or bone mineral density.

Subjects and methods: The study group included 227 unrelated Chilean elderly women (61–95 years) recruited from a population-based sample. Adiposity and bone mineral density measures were obtained using dual-energy X-ray absorptiometry.

Results: The allele frequencies for TNF ?308A, TLR4 299Gly and TLR4 ?399Ile were 9.3%, 4.6% and 4.4%, respectively, with Asp299Gly and Thr399Ile being in strong linkage disequilibrium (D′?=?0.88). Although seriously restricted by the low frequency of the allele variants, no relevant association between genotypes and adiposity-related variables were found. Likewise, no significant association between osteoporosis status (categorized as osteoporosis, osteopenia or normal status) with TLR4 Asp299Gly or TNF ?308G>A genotypes was found.

Conclusion: It is unlikely that TLR4 Asp299Gly, TLR4 Thr399Ile or TNF ?308G>A polymorphisms have a major influence on adiposity, bone mineral density or osteoporosis status in Chilean elderly women.