• Animal cells and systems
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• v.13 no.1
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• pp.1-8
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• 2009

Toll-like receptor (TLR) family proteins, type I transmembrane proteins, play a central role in human innate immune response by recognizing common structural patterns in diverse molecules from bacteria, viruses and fungi. Recently four structures of the TLR and ligand complexes have been determined by high resolution x-ray crystallographic technique. In this review we summarize reported structures of TLRs and their proposed activation mechanisms. The structures demonstrate that binding of agonistic ligands to the extracellular domains of TLRs induces homo- or heterodimerization of the receptors. Dimerization of the TLR extracellular domains brings their two C-termini into close proximity. This suggests a plausible mechanism of TLR activation: ligand induces dimerization of the extracellular domains, which enforces juxtaposition of intracellular signaling domains for recruitment of intracellular adaptor proteins for signal initiation.

• Journal of Yeungnam Medical Science
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• v.30 no.1
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• pp.10-16
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• 2013

Background: Toll-like receptors (TLRs) are well-known pattern recognition receptors. Among the 13 TLRs, TLR2 is the most known receptor for immune response. It activates mitogen-activated protein kinases (MAPKs), which are counterbalanced by MAPK phosphatases [MKPs or dual-specificity phosphatases (DUSPs)]. However, the regulatory mechanism of DUSPs is still unclear. In this study, the effect of a TLR2 ligand (TLR2L, Pam3CSK4) on DUSP4 expression in Raw264.7 cells was demonstrated. Methods: A Raw264.7 mouse macrophage cell line was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% antibiotics (100 U/mL penicillin and 100 g/mL streptomycin) at $37^{\circ}C$ in 5% $CO_2$. TLR2L (Pam3CSK4)-mediated DUSP4 expressions were confirmed with RT-PCR and western blot analysis. In addition, the detection of reactive oxygen species (ROS) was measured with lucigenin assay. Results: Pam3CSK4 induced the expression of DUSP1, 2, 4, 5 and 16. The DUSP4 expression was also increased by TLR4 and 9 agonists (lipopolysaccharide and CpG ODN, respectively). Pam3CSK4 also induced ERK1/2 phosphorylation and ROS production, and the Pam3CSK4-induced DUSP4 expression was decreased by ERK1/2 (U0126) and ROS (DPI) inhibitors. U0126 suppressed the ROS production by Pam3CSK4. Conclusion: Pam3CSK4-mediated DUSP4 expression is regulated by ERK1/2 and ROS. This finding suggests the physiological importance of DUSP4 in TLR2-mediated immune response.

• Clinical and Experimental Reproductive Medicine
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• v.45 no.1
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• pp.1-9
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• 2018

Objective: To determine the localization, expression, and function of Toll-like receptors (TLRs) in fallopian tube epithelial cells. Methods: The localization of TLRs in fallopian tube epithelial cells was investigated by immunostaining. Surprisingly, the intensity of staining was not equal in the secretory and ciliated cells. After primary cell culture of fallopian tube epithelial cells, ring cloning was used to isolate colonies of ciliated epithelial cells, distinct from non-ciliated epithelial cells. The expression of TLRs 1-10 was examined by quantitative real-time polymerase chain reaction, and protein localization was confirmed by immunostaining. The function of the TLRs was determined by interleukin (IL)-6 and IL-8 production in response to TLR2, TLR3, TLR5, TLR7, and TLR9 ligands. Results: Fallopian tube epithelial cells expressed TLRs 1-10 in a cell-type-specific manner. Exposing fallopian tube epithelial cells to TLR2, TLR3, TLR5, TLR7, and TLR9 agonists induced the secretion of proinflammatory cytokines such as IL-6 and IL-8. Conclusion: Our findings suggest that TLR expression in the fallopian tubes is cell-type-specific. According to our results, ciliated cells may play more effective role than non-ciliated cells in the innate immune defense of the fallopian tubes, and in interactions with gametes and embryos.

• Clinical and Experimental Reproductive Medicine
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• v.45 no.4
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• pp.154-162
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• 2018

Objective: The fallopian tubes play a critical role in the early events of fertilization. The rapid innate immune defense is an important part of the fallopian tubes. Toll-like receptor 3 (TLR3), as a part of the innate immune system, plays an important role in detecting viral infections. In this basic and experimental study, the effect of sex hormones on the function of TLR3 in the OE-E6/E7 cell line was investigated. Methods: The functionality of TLR3 in this cell line was evaluated by cytokine measurements (interleukin [IL]-6 and IL-1b) and the effects of sex hormones on TLR3 were tested by an enzyme-linked immunosorbent assay kit. Additionally, TLR3 small interfering RNA (siRNA) and a TLR3 function-blocking antibody were used to confirm our findings. Results: The production of IL-6 significantly increased in the presence of polyinosinic-polycytidylic acid (poly(I:C)) as the TLR3 ligand. Using a TLR3-siRNA-ransfected OE-E6/E7 cell line and function-blocking antibody confirmed that cytokine production was due to TLR3. In addition, 17-${\beta}$ estradiol and progesterone suppressed the production of IL-6 in the presence and absence of poly(I:C). Conclusion: These results imply that sex hormones exerted a suppressive effect on the function of TLR3 in the fallopian tube cell line when different concentrations of sex hormones were present. The current results also suggest that estrogen receptor beta and nuclear progesterone receptor B are likely to mediate the hormonal regulation of TLR3, as these two receptors are the main estrogen and progesterone receptors in OEE6/E7 cell line.

• Korean Journal of Poultry Science
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• v.45 no.3
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• pp.209-217
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• 2018

The innate immune recognition is based on the detection of microbial products. Toll-like receptors (TLRs) located on the cell surface and the endosome senses microbial components and nucleic acids, respectively. Chicken TLRs mediate immune responses by sensing ligands from pathogens, have been studied as immune adjuvants to increase the efficacy of vaccines. Single nucleotide polymorphisms (SNPs) of TLR3 and TLR4 genes in chicken were associated with resistance and susceptibility to viral infection. In this study, SNPs of chTLR3 and chTLR4 genes were retrieved from public database and annotated with chicken reference genome. Three-dimensional models of the chTLR3 and chTLR4 proteins were built using a Swiss modeler. We identified 35 and 13 nsSNPs in chTLR3 and chTLR4 genes respectively. Sorting Intolerant from Tolerant (SIFT) and Polymorphism Phenotyping v2 (Polyphen-2) analyses, suggested that, out of 35 and 13 nsSNPs, 4 and 2 SNPs were identified to be deleterious in chTLR3 and chTLR4 gene respectively. In chTLR3, 1 deleterious SNP was located in ectodomain and 3 were located in the Toll / IL-1 receptor (TIR) domain. Further structural model of chTLR3-TIR domain suggested that 1 deleterious SNP be present in the B-B loop region, which is important for TIR-TIR domain interactions in the downstream signaling. In chTLR4, the deleterious SNPs were located both in the ectodomain and TIR domain. SNPs predicted for chTLR3 and chTLR4 in this study, might be related to resistance or susceptible to viral infection in chickens. Results from this study will be useful to develop the effective measures in chicken against infectious diseases.

• IMMUNE NETWORK
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• v.8 no.2
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• pp.59-65
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• 2008

Background: Toll-like receptors (TLRs) detect microbial infection and can directly induce innate host defense responses, which are thought to play critical roles in protecting the tubotympanum from infection. However, little is known about the relationship between TLRs, which are related to innate immunity, and immunoglobulins, which are related to adaptive immunity, in recurrent otitis media with effusion (OME). We therefore investigated the expression of TLR2 and TLR4 and immunoglobulin in children with OME. Methods: The study population consisted of 72 children with OME, 31 with more than 4 episodes in 12 months or more than 3 episodes in 6 months (otitis-prone group), and 41 with fewer than 3 episodes in 12 months (non-otitis prone group). The expression in middle ear effusion of TLR2 and TLR4 mRNA, as determined by Real time- -polymerase chain reaction (RT-PCR), and the concentrations of IgG, IgA, and IgM, as determined by Enzyme-linked immunosorbent assay(ELISA), were compared between the two groups. Results: Expression of TLR2 and TLR4 mRNA was lower in the otitis prone than in the non-otitis prone group, but the difference was not statistically significant (p>0.05). Between group differences in the concentrations of IgG, IgA and IgM in effusion fluid were not significant (p>0.05), and there were no correlations between immunoglobulin concentration and the expression of TLR2 and TLR4. Conclusion: Although there was a trend toward lower expression of TLR2 and TLR4 in the otitis-prone group, the differences, and those in immunoglobulin concentration, did not differ significantly between the otitis-prone and non-prone groups.

• BMB Reports
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• v.49 no.6
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• pp.305-310
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• 2016

Toll-like receptors (TLRs) play a critical role in the innate immune response against pathogens. Each TLR recognizes specific pathogen-associated molecular patterns, after which they activate the adaptor protein MyD88 or TRIF-assembled signaling complex to produce immune mediators, including inflammatory cytokines and type I IFNs. Although the activation of TLR is important for host defense, its uncontrolled activation can damage the host. During the past decade, numerous studies have demonstrated that GSK3β is a key regulator of inflammatory cytokine production in MyD88-mediated TLR signaling via TLR2 and TLR4. Recently, GSK3β has also been implicated in the TRIF-dependent signaling pathway via TLR3. In this review, we describe current advances on the regulatory role of GSK3β in immune responses associated with various TLRs. A better understanding of the role of GSK3β in TLR signaling might lead to more effective anti-inflammatory interventions.

• Korean Journal of Food Science and Technology
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• v.39 no.2
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• pp.175-180
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• 2007

Toll-like receptors induce innate immune responses recognizing conserved microbial structural molecules that are known as pathogen-associated molecular patterns (PAMPs). Ligand-induced homotypic oligomerization was found to proceed in LPS-induced activation of TLR4 signaling pathways. TLR2 is known to heterodimerize with TLR1 or TLR6 and recognize diacyl- or triacyl-lipopeptide, respectively. These results suggest that ligand-induced receptor dimerization of TLR4 and TLR2 is required for the activation of downstream signaling pathways. Therefore, receptor dimerization may be one of the first lines of regulation in the activation of TLR-mediated signaling pathways and induction of subsequent innate and adaptive immune responses. Here, we report biochemical evidence that curcumin from the plant Curcuma longa inhibits activation of $NF-{\kappa}B$, expression of COX-2, and dimerization of TLRs induced by TLR2, TLR3 and TLR4 agonists. These results imply that curcumin can modulate the activation of TLRs and subsequent immune/inflammatory responses induced by microbial pathogens.

• Journal of Microbiology and Biotechnology
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• v.17 no.11
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• pp.1862-1867
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• 2007

An individual's immune response is critical for host protection from many different pathogens, and the responsiveness can be assessed by the amount of cytokine production upon stimulating bacterial components such as lipopolysaccharide (LPS). The difference between individuals in their peripheral blood mononuclear cells (PBMC) responsiveness to LPS, a Gram-negative endotoxin, was investigated from 27 healthy individuals. We observed a large variation in $IFN{\gamma}$ production among different individuals. The PBMC of the consistently three highest and three lowest $IFN{\gamma}$ producers were investigated. Since previous studies described that a single point mutation in the coding region of TLR2 and TLR4 is linked to the individual responsiveness to pathogenic bacterial infections, we first examined the known point mutations in the coding region of $TLR2^{Pro681His}$, $TLR4^{Pro714His}$ located in the cytoplasmic regions of the Toll-like domain as well as $TLR4^{Asp299Gly}$ located in the extracellular region. None of these mutations were associated with an individual's responsiveness to LPS, despite the presence of $TLR4^{Asp299Gly}$ mutation. Further investigation revealed that the variation of PBMC responsiveness to LPS among healthy individuals was due to constitutive expression levels of TLR4 and TLR2. This result is consistent with an aging-related low expression of Toll-like receptors in the mouse model of LPS responsiveness. The present study therefore suggests that the constitutive expression levels of TLR2 and TLR4 may contribute to the individual response to LPS.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.36 no.6
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• pp.481-489
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• 2010

Introduction: TLR-5, a member of the toll-like receptor (TLR) family, is a element of the type I transmembrane receptors, which are characterized by an intracellular signaling domain homolog to the interleukin-1 receptor. These receptors recognize microbial components, particularly bacterial flagellin. All-trans retinoic acid (atRA, tretinoin), a natural metabolite of vitamin A, acts as a growth and differentiation factor in many tissues, and is also needed for immune functions. In this study, THP-1 human macrophage-monocytes were used to examine the mechanisms by which atRA regulated the expression of TLR-5. Because the molecular mechanism underlying this regulation at the transcriptional level is also unclear, this study examined which putative transcription factors are responsible for TLR-5 expression by atRA in immune cells. Materials and Methods: This study examined whether atRA induces the expression of TLR-5 in THP-1 cells using reverse transcription-polymerase chain reaction (RT-PCR), and which transcription factors are involved in regulating the TLR-5 promoter in RAW264.7 cells using a reporter assay system. Western blot analysis was used to determine which signal pathway is involved in the expression of TLR-5 in atRA-treated THP-1 cells. Results: atRA at a concentration of 10 nM greatly induced the expression of TLR-5 in THP-1 cells. Human TLR-5 promoter contains three Sp-1/GC binding sites around -50 bp and two NF-kB binding sites at -380 bp and -160 bp from the transcriptional start site of the TLR-5 gene. Sp-1/GC is primarily responsible for the constitutive TLR-5 expression, and may also contribute to NF-kB at -160 bp to induce TLR-5 after atRA stimulation in THP-1 cells. The role of NF-kB in TLR-5 expression was further confirmed by inhibitor pyrrolidine dithiocarbamate (PDTC) experiments, which greatly reduced the TLR-5 transcription by 70-80%. Conclusion: atRA induces the expression of the human TLR-5 gene and NF-kB is a critical transcription factor for the atRA-induced expression of TLR-5. Accordingly, it is conceivable that retinoids are required for adequate innate and adaptive immune responses to agents of infectious diseases. atRA and various synthetic retinoids have been used therapeutically in human diseases, such as leukemia and other cancers due to the antiproliferative and apoptosis inducing effects of retinoids. Therefore, understanding the molecular regulatory mechanism of TLR-5 may assist in the design of alternative strategies for the treatment of infectious diseases, leukemia and cancers.