• Journal of Ginseng Research
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• 제32권4호
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• pp.315-323
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• 2008

In the previous studies, we isolated the compound K rich fractions (CKRF) and showed that CKRF inhibited Toll-like receptor (TLR) 4- or TLR9-induced inflammatory signaling. To extend our previous studies,1) we investigated the molecular mechanisms of CKRF in the TLR4-associated signaling via nuclear factor (NF)-${\kappa}B$, and in vivo role of CKRF for induction of tolerance in lipopolysaccharide (LPS)-induced septic shock. In murine bone marrow-dervied macrophages, CKRF significantly inhibited the induction of mRNA expression of proinflammatory mediators such as tumor necrosis factor-${\alpha}$, interleukin-6, cyclooxygenase-2, and inducible nitric oxide synthase. In addition, CKRF significantly attenuated the transcriptional activities of TLR4/LPS-induced NF-${\kappa}B$. Nuclear translocation of NF-${\kappa}B$ in response to LPS stimulation was significantly abrogated by pre-treatment with CKRF. Furthermore, CKRF inhibited the recruitment of p65 to the interferon-sensitive response element flanking region in response to LPS. Finally, oral administration of CKRF significantly protected mice from Gram-negative bacterial LPS-induced lethal shock and inhibited systemic inflammatory cytokine levels. Together, these results demonstrate that CKRF modulates the TLR4-dependent NF-${\kappa}B$ activation, and suggest a therapeutic role for Gram-negative septic shock.

• Journal of Veterinary Science
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• 제23권2호
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• pp.27.1-27.16
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• 2022

Background: The role of Toll-like receptors (TLRs) in a feline infectious peritonitis virus (FIPV) infection is not completely understood. Objectives: This study examined the expression of TLR3, TLR7, TLR9, tumor necrosis factor-alpha (TNF-α), interferon (IFN)-β, and interleukin (IL)-10 upon an FIPV infection in Crandell-Reese feline kidney (CRFK) cells and feline monocytes. Methods: CRFK cells and monocytes from feline coronavirus (FCoV)-seronegative cats and FCoV-seropositive cats were infected with type II FIPV-79-1146. At four, 12, and 24 hours post-infection (hpi), the expression of TLR3, TLR7, TLR9, TNF-α, IFN-β, and IL-10, and the viral load were measured using reverse transcription quantitative polymerase chain reaction. Viral protein production was confirmed using immunofluorescence. Results: FIPV-infected CRFK showed the upregulation of TLR9, TNF-α, and IFN-β expression between 4 and 24 hpi. Uninfected monocytes from FCoV-seropositive cats showed lower TLR3 and TLR9 expression but higher TLR7 expression compared to uninfected monocytes from FCoV-seronegative cats. FIPV-infected monocytes from FCoV-seropositive cats downregulated TLR7 and TNF-α expression between 4 and 24 hpi, and 4 and 12 hpi, respectively. IFN-β was upregulated early in FIPV-infected monocytes from FCoV-seropositive cats, with a significant difference observed at 12 hpi compared to FCoV-seronegative cats. The viral load in the CRFK and FIPV-infected monocytes in both cohorts of cats was similar over time.ConclusionTLR7 may be the key TLR involved in evading the innate response against inhibiting TNF-α production. Distinct TLR expression profiles between FCoV-seronegative and FCoV-seropositive cats were observed. The associated TLR that plays a role in the induction of IFN-β needs to be explored further.

• International Journal of Oral Biology
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• 제41권1호
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• pp.17-23
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• 2016

Chronic/cyclic neutropenia, leukocyte adhesion deficiency syndrome, Papillon-$Lef{\grave{e}}vre$ syndrome, and $Ch{\grave{e}}diak$-Higashi syndrome are associated with severe periodontitis, suggesting the importance of neutrophils in the maintenance of periodontal health. Various Toll-like receptor (TLR) ligands are known to stimulate neutrophil function, including FcR-mediated phagocytosis. In the present study, the effect of TLR2 activation on the non-opsonic phagocytosis of oral bacteria and concomitant production of reactive oxygen species (ROS) by human neutrophils was evaluated. Neutrophils isolated from peripheral blood were incubated with Streptococcus sanguinis or Porphyromonas gingivalis in the presence of various concentrations of $Pam_3CSK_4$, a synthetic TLR2 ligand, and analyzed for phagocytosis and ROS production by flow cytometry and chemiluminescence, respectively. $Pam_3CSK_4$ significantly increased the phagocytosis of both bacterial species in a dose-dependent manner. However, the enhancing effect was greater for S. sanguinis than for P. gingivalis. $Pam_3CSK_4$ alone induced ROS production in neutrophils and also increased concomitant ROS production induced by bacteria. Interestingly, incubation with P. gingivalis and $Pam_3CSK_4$ decreased the amounts of ROS, as compared to $Pam_3CSK_4$ alone, indicating the possibility that P. gingivalis survives within neutrophils. However, neutrophils efficiently killed phagocytosed bacteria of both species despite the absence of $Pam_3CSK_4$. Although P. gingivalis is poorly phagocytosed even by the TLR2-activated neutrophils, TLR2 activation of neutrophils may help to reduce the colonization of P. gingivalis by efficiently eliminating S. sanguinis, an early colonizer, in subgingival biofilm.

• The Korean Journal of Physiology and Pharmacology
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• 제22권4호
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• pp.379-389
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• 2018

A nucleobase adenine is a fundamental component of nucleic acids and adenine nucleotides. Various biological roles of adenine have been discovered. It is not produced from degradation of adenine nucleotides in mammals but produced mainly during polyamine synthesis by dividing cells. Anti-inflammatory roles of adenine have been supported in IgE-mediated allergic reactions, immunological functions of lymphocytes and dextran sodium sulfate-induced colitis. However adenine effects on Toll-like receptor 4 (TLR4)-mediated inflammation by lipopolysaccharide (LPS), a cell wall component of Gram negative bacteria, is not examined. Here we investigated anti-inflammatory roles of adenine in LPS-stimulated immune cells, including a macrophage cell line RAW264.7 and bone marrow derived mast cells (BMMCs) and peritoneal cells in mice. In RAW264.7 cells stimulated with LPS, adenine inhibited production of pro-inflammatory cytokines $TNF-{\alpha}$ and IL-6 and inflammatory lipid mediators, prostaglandin $E_2$ and leukotriene $B_4$. Adenine impeded signaling pathways eliciting production of these inflammatory mediators. It suppressed $I{\kappa}B$ phosphorylation, nuclear translocation of nuclear factor ${\kappa}B$ ($NF-{\kappa}B$), phosphorylation of Akt and mitogen activated protein kinases (MAPKs) JNK and ERK. Although adenine raised cellular AMP which could activate AMP-dependent protein kinase (AMPK), the enzyme activity was not enhanced. In BMMCs, adenine inhibited the LPS-induced production of $TNF-{\alpha}$, IL-6 and IL-13 and also hindered phosphorylation of $NF-{\kappa}B$ and Akt. In peritoneal cavity, adenine suppressed the LPS-induced production of $TNF-{\alpha}$ and IL-6 by peritoneal cells in mice. These results show that adenine attenuates the LPS-induced inflammatory reactions.

• Molecules and Cells
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• 제38권1호
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• pp.58-64
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• 2015

Activating transcription factor-3 (ATF3) acts as a negative regulator of cytokine production during Gram-negative bacterial infection. A recent study reported that ATF3 provides protection from Streptococcus pneumoniae infection by activating cytokines. However, the mechanism by which S. pneumoniae induces ATF3 after infection is still unknown. In this study, we show that ATF3 was upregulated via Toll-like receptor (TLR) pathways in response to S. pneumoniae infection in vitro. Induction was mediated by TLR4 and TLR2, which are in the TLR family. The expression of ATF3 was induced by pneumolysin (PLY), a potent pneumococcal virulence factor, via the TLR4 pathway. Furthermore, ATF3 induction is mediated by p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Thus, this study reveals a potential role of PLY in modulating ATF3 expression, which is required for the regulation of immune responses against pneumococcal infection in macrophages.

• Molecules and Cells
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• 제38권12호
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• pp.1111-1117
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• 2015

Toll-like receptor 5 (TLR5) is a specific receptor for microbial flagellin and is one of the most well-known receptors in the TLR family. We reported previously that TLR5 signaling is well maintained during aging and that caveolin-1 may be involved in TLR5 signaling in aged macrophages through direct interactions. Therefore, it is important to clarify whether caveolin-1/TLR5 interactions affect TLR5 expression during aging. To assess the effect of caveolin-1 on TLR5, we analyzed TLR5 expression in senescent fibroblasts and aged tissues expressing high levels of caveolin-1. As expected, TLR5 mRNA and protein expression was well maintained in senescent fibroblasts and aged tissues, whereas TLR4 mRNA and protein were diminished in those cells and tissues. To determine the mechanism of caveolin-1-dependent TLR5 expression, we examined TLR5 expression in caveolin-1 deficient mice. Interestingly, TLR5 mRNA and protein levels were decreased dramatically in tissues from caveolin-1 knockout mice. Moreover, overexpressed caveolin-1 in vitro enhanced TLR5 mRNA through the MAPK pathway and prolonged TLR5 protein half-life through direct interaction. These results suggest that caveolin-1 may play a crucial role in maintaining of TLR5 by regulating transcription systems and increasing protein half-life.

• Journal of Microbiology and Biotechnology
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• 제26권7호
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• pp.1333-1340
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• 2016

The main objective of this study was to investigate whether Lactobacillus rhamnosus GG (LGG) ameliorated the effects of Citrobactor rodentium infection in Toll-like receptor 2 (TLR2) knockout (KO) and TLR4 KO mice, as well as in wild-type C57BL/6 (B6) mice. TLR2 KO, TLR4 KO, and B6 mice were divided into three groups per each strain. Each group had an uninfected control group (n = 5), C. rodentium-infected group (n = 8), and LGG-pretreated C. rodentium-infected group (n = 8). The survival rate of B6 mice infected with C. rodentium was higher when pretreated with LGG. Pretreatment with LGG ameliorated C. rodentium-induced mucosal hyperplasia in B6 and TLR4 KO mice. However, in C-rodentium-infected TLR2 KO mice, mucosal hyperplasia persisted, regardless of pretreatment with LGG. In addition, LGG-pretreated B6 and TLR4 KO mice showed a decrease in spleen weight and downregulation of tumor necrosis factor alpha, interferon gamma, and monocyte chemotactic protein 1 mRNA expression compared with the non-pretreated group. In contrast, such changes were not observed in TLR2 KO mice, regardless of pretreatment with LGG. From the above results, we conclude that pretreatment with LGG ameliorates C. rodentium-induced colitis in B6 and TLR4 KO mice, but not in TLR2 KO mice. Therefore, LGG protects mice from C. rodentium-induced colitis in a TLR2-dependent manner.

• 한국가금학회지
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• 제45권3호
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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.

• The Korean Journal of Physiology and Pharmacology
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• 제14권4호
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• pp.235-240
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• 2010

Toll-like receptors (TLRs) functionally expressed in salivary epithelial cells, but their roles remain elusive. Among TLRs family, TLR3 is activated by dsRNA, a byproduct of viral infection. The aim of this study was to investigate the role of TLR3 in the inflammatory immune responses using HSG cells. Reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR and ELISA were performed to identify expression of TLRs and TLR3-mediated chemokine inductions. The chemotaxis assay of activated T lymphocytes was also performed. Treatment of HSG cells with polyinosinic: polycytidylic acid (poly(I:C)) significantly increased interferon-$\gamma$-inducible protein 10 (IP-10), interferoninducible T-cell $\alpha$ chemoattractant (I-TAC), and regulated on activation, normal T-cells expressed and secreted (RANTES) gene expressions in a concentration-dependent manner. Anti-TLR3 antibody blocked the increases of IP-10 and I-TAC genes. Poly(I:C)-induced increases of IP-10 and I-TAC were also confirmed at protein levels from cell lysates, but their release into extracellular medium was detected only in IP-10. We found that the culture media from HSG cells stimulated with poly(I:C) significantly increases T lymphocyte migration. Our results suggest that TLR3 plays an important role in chemokine induction, particularly IP-10, in salivary epithelial cells.

• The Korean Journal of Physiology and Pharmacology
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• 제19권1호
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• pp.51-57
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• 2015

The etiology of periodontal disease is multifactorial. Exogenous stimuli such as bacterial pathogens can interact with toll-like receptors to activate intracellular calcium signaling in gingival epithelium and other tissues. The triggering of calcium signaling induces the secretion of pro-inflammatory cytokines such as interleukin-8 as part of the inflammatory response; however, the exact mechanism of calcium signaling induced by bacterial toxins when gingival epithelial cells are exposed to pathogens is unclear. Here, we investigate calcium signaling induced by bacteria and expression of inflammatory cytokines in human gingival epithelial cells. We found that peptidoglycan, a constituent of grampositive bacteria and an agonist of toll-like receptor 2, increases intracellular calcium in a concentration-dependent manner. Peptidoglycan-induced calcium signaling was abolished by treatment with blockers of phospholipase C (U73122), inositol 1,4,5-trisphosphate receptors, indicating the release of calcium from intracellular calcium stores. Peptidoglycan-mediated interleukin-8 expression was blocked by U73122 and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester). Moreover, interleukin-8 expression was induced by thapsigargin, a selective inhibitor of the sarco/endoplasmic reticulum calcium ATPase, when thapsigargin was treated alone or co-treated with peptidoglycan. These results suggest that the gram-positive bacterial toxin peptidoglycan induces calcium signaling via the phospholipase C/inositol 1,4,5-trisphosphate pathway, and that increased interleukin-8 expression is mediated by intracellular calcium levels in human gingival epithelial cells.