• BMB Reports
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• v.50 no.2
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• pp.55-57
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• 2017

Toll-like receptor 4 (TLR4) together with MD2, one of the key pattern recognition receptors for a pathogen-associated molecular pattern, activates innate immunity by recognizing lipopolysaccharide (LPS) of Gram-negative bacteria. Although LBP and CD14 catalyze LPS transfer to the TLR4/MD2 complex, the detail mechanisms underlying this dynamic LPS transfer remain elusive. Using negative-stain electron microscopy, we visualized the dynamic intermediate complexes during LPS transfer-LBP/LPS micelles and ternary CD14/LBP/LPS micelle complexes. We also reconstituted the entire cascade of LPS transfer to TLR4/MD2 in a total internal reflection fluorescence (TIRF) microscope for a single molecule fluorescence analysis. These analyses reveal longitudinal LBP binding to the surface of LPS micelles and multi-round binding/unbinding of CD14 to single LBP/LPS micelles via key charged residues on LBP and CD14. Finally, we reveal that a single LPS molecule bound to CD14 is transferred to TLR4/MD2 in a TLR4-dependent manner. These discoveries, which clarify the molecular mechanism of dynamic LPS transfer to TLR4/MD2 via LBP and CD14, provide novel insights into the initiation of innate immune responses.

• Molecules and Cells
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• v.24 no.1
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• pp.119-124
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• 2007

TLR4 together with CD14 and MD-2 forms a pattern recognition receptor that plays an initiating role in the innate immune response to Gram-negative bacteria. Here, we employed the surface plasmon resonance technique to investigate the kinetics of binding of LPS to recombinant CD14, MD-2 and TLR4 proteins produced in insect cells. The dissociation constants ($K_D$) of LPS for immobilized CD14 and MD-2 were $8.7{\mu}m$, and $2.3{\mu}m$, respectively. The association rate constant ($K_{on}$) of LPS for MD-2 was $5.61{\times}10^3M^{-1}S^{-1}$, and the dissociation rate constant ($K_{off}$) was $1.28{\times}10^2S^{-1}$, revealing slow association and fast dissociation with an affinity constant $K_D$ of $2.33{\times}10^6M$ at $25^{\circ}C$. These affinities are consistent with the current view that CD14 conveys LPS to the TLR4/MD-2 complex.

• Toxicological Research
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• v.31 no.1
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• pp.11-16
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• 2015

Our body's immune system has defense mechanisms against pathogens such as viruses and bacteria. Immune responses are primarily initiated by the activation of toll-like receptors (TLRs). In particular, TLR4 is well-characterized and is known to be activated by gram-negative bacteria and tissue damage signals. TLR4 requires myeloid differentiation factor 2 (MD2) as a co-receptor to recognize its ligand, lipopolysaccharides (LPS), which is an extracellular membrane component of gram-negative bacteria. Gambogic acid is a xanthonoid isolated from brownish or orange resin extracted from Garcinia hanburyi. Its primary effect is tumor suppression. Since inflammatory responses are related to the development of cancer, we hypothesized that gambogic acid may regulate TLR4 activation. Our results demonstrated that gambogic acid decreased the expression of pro-inflammatory cytokines ($TNF-{\alpha}$, IL-6, IL-12, and $IL-1{\beta}$) in both mRNA and protein levels in bone marrow-derived primary macrophages after stimulation with LPS. Gambogic acid did not inhibit the activation of Interferon regulatory factor 3 (IRF3) induced by TBK1 overexpression in a luciferase reporter gene assay using IFN-${\beta}$-PRD III-I-luc. An in vitro kinase assay using recombinant TBK1 revealed that gambogic acid did not directly inhibit TBK1 kinase activity, and instead suppressed the binding of LPS to MD2, as determined by an in vitro binding assay and confocal microscopy analysis. Together, our results demonstrate that gambogic acid disrupts LPS interaction with the TLR4/MD2 complex, the novel mechanism by which it suppresses TLR4 activation.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.10a
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• pp.92-92
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• 2019

Excessive or chronic inflammation contributes to the pathogenesis of many inflammatory diseases such as sepsis, rheumatoid arthritis, and ulcerative colitis. Hibiscus syriacus L. has been used as a medicinal plant in many Asian countries, even though its anti-inflammatory activity has been unclear. Therefore, we investigated the anti-inflammatory effect of anthocyanin fractions from the H. syriacus L. varieties Pulsae (PS) on the lipopolysaccharide (LPS)-induced expression of proinflammatory mediators and cytokines in RAW264.7 macrophages. PS suppressed LPS-induced nitric oxide (NO) and prostaglandin $E_2$ ($PGE_2$) secretion concomitant with downregulation of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. Furthermore, PS inhibited the production of proinflammatory cytokines such as tumor necrosis factor-alpha ($TNF-{\alpha}$), interleukin-6 (IL-6), and IL-12 in LPS-stimulated RAW264.7 macrophages. Further study showed that PS significantly decreased LPS-induced nuclear translocation of the nuclear $factor-{\kappa}B$ ($NF-{\kappa}B$) subunits, p65 and p50. Molecular docking data showed that many anthocyanins from PS fit into the hydrophobic pocket of MD2 and bound to Toll-like receptor 4 (TLR4), indicating that PS inhibits the TLR4-MD2-mediated inflammatory signaling pathway. Especially, apigenin-7-O-glucoside most powerfully bound to MD2 and TLR4 through LYS122, LYS122, and SER127 at a distance of $2.205{\AA}$, $3.098{\AA}$, and $2.844{\AA}$ and SER441 at a distance of $2.873{\AA}$ (docking score: -8.4) through hydrogen bonding, respectively. Additionally, PS inhibited LPS-induced TLR4 dimerization/expression on the cell surface, which consequently decreased MyD88 recruitment and IRAK4 phosphorylation. PS completely blocked LPS-mediated mortality in zebrafish larvae by diminishing the recruitment of neutrophil and macrophages accompanied by low levels of proinflammatory cytokines. Taken together, our results indicate that PS attenuates LPS-mediated inflammation in both in vitro and in vivo by blocking the TLR4/MD2-MyD88/IRAK4-$NF-{\kappa}B$ axis. Therefore, PS might be used as a novel modulatory candidate for effective treatment of LPS-mediated inflammatory diseases.

• Biomolecules & Therapeutics
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• v.22 no.4
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• pp.295-300
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• 2014

The actin cytoskeleton plays an important role in macrophage-mediated inflammatory responses by modulating the activation of Src and subsequently inducing nuclear factor (NF)-${\kappa}B$ translocation. In spite of its critical functions, few papers have examined how the actin cytoskeleton can be regulated by the activation of toll-like receptor (TLR). Therefore, in this study, we further characterized the biological value of the actin cytoskeleton in the functional activation of macrophages using an actin cytoskeleton disruptor, cytochalasin B (Cyto B), and explored the actin cytoskeleton's involvement in morphological changes, cellular attachment, and signaling events. Cyto B strongly suppressed the TLR4-mediated mRNA expression of inflammatory genes such as cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-${\alpha}$, and inducible nitric oxide (iNOS), without altering cell viability. This compound also strongly suppressed the morphological changes induced by lipopolysaccharide (LPS), a TLR4 ligand. Cyto B also remarkably suppressed NO production under non-adherent conditions but not in an adherent environment. Cyto B did not block the co-localization between surface glycoprotein myeloid differentiation protein-2 (MD2), a LPS signaling glycoprotein, and the actin cytoskeleton under LPS conditions. Interestingly, Cyto B and PP2, a Src inhibitor, enhanced the phagocytic uptake of fluorescein isothiocyanate (FITC)-dextran. Finally, it was found that Cyto B blocked the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at 1 min and the phosphorylation of heat shock protein 27 (HSP27) at 5 min. Therefore, our data suggest that the actin cytoskeleton may be one of the key components involved in the control of TLR4-mediated inflammatory responses in macrophages.

• Biomedical Science Letters
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• v.15 no.3
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• pp.257-260
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• 2009

Toll-like receptors (TLRs) recognize molecular structures derived from microbes including bacteria, viruses, yeast, and fungi. TLRs have emerged as a major signaling component of the mammalian host defense. TLR4 is a member of the Toll family that senses lipopolysaccharide (LPS), a cell wall component of gram negative bacteria. LPS recognition by TLR4 requires an additional accessory molecule, MD-2. LPS induces the activation of NF-${\kappa}B$ and IRF3 through MyD88 or TRIF-dependent pathways. The activation of NF-${\kappa}B$ leads to the induction of inflammatory gene products including cytokines and cyclooxygenase-2 (COX-2). This study was carried out to investigate the anti-inflammatory effects of oak wood vinegar. Oak wood vinegar inhibits the NF-${\kappa}B$ activation and COX-2 expression induced by LPS. These results provide new ideas to understand the mechanism of oak wood vinegar for its anti-bacterial and anti-inflammatory activities.

• Journal of Ginseng Research
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• v.46 no.1
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• pp.156-166
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• 2022

Background: Panax ginseng Meyer (P. ginseng), a herb distributed in Korea, China and Japan, exerts benefits on diverse inflammatory conditions. However, the underlying mechanism and active ingredients remains largely unclear. Herein, we aimed to explore the active ingredients of P. ginseng against inflammation and elucidate underlying mechanisms. Methods: Inflammation model was constructed by lipopolysaccharide (LPS) in C57BL/6 mice and RAW264.7 macrophages. Molecular docking, molecular dynamics, surface plasmon resonance imaging (SPRi) and immunofluorescence were utilized to predict active component. Results: P. ginseng significantly inhibited LPS-induced lung injury and the expression of proinflammatory factors, including TNF-α, IL-6 and IL-1β. Additionally, P. ginseng blocked fluorescencelabeled LPS (LPS488) binding to the membranes of RAW264.7 macrophages, the phosphorylation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs). Furthermore, molecular docking demonstrated that ginsenoside Ro (GRo) docked into the LPS binding site of toll like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) complex. Molecular dynamic simulations showed that the MD2-GRo binding conformation was stable. SPRi demonstrated an excellent interaction between TLR4/ MD2 complex and GRo (K_D value of 1.16 × 10^-9 M). GRo significantly inhibited LPS488 binding to cell membranes. Further studies showed that GRo markedly suppressed LPS-triggered lung injury, the transcription and secretion levels of TNF-α, IL-6 and IL-1β. Moreover, the phosphorylation of NF-κB and MAPKs as well as the p65 subunit nuclear translocation were inhibited by GRo dose-dependently. Conclusion: Our results suggest that GRo exerts anti-inflammation actions by direct inhibition of TLR4 signaling pathway.

• Molecules and Cells
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• v.27 no.2
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• pp.251-255
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• 2009

Sepsis is the leading cause of death in critically ill patients. Today, around 60% of all cases of sepsis are caused by Gram-negative bacteria. The cell wall component lipopolysaccharide (LPS) is the main initiator of the cascade of cellular reactions in Gram-negative infections. The core receptors for LPS are toll-like receptor 4 (TLR4), MD-2 and CD14. Attempts have been made to antagonize the toxic effect of endotoxin using monoclonal antibodies against CD14 and synthetic lipopolysaccharides but there is as yet no effective treatment for septic syndrome. Here, we describe an inhibitory effect of a phosphatidylethanolamine derivative, PE-DTPA (phosphatidylethanolamine diethylenetriaminepentaacetate) on LPS recognition. PE-DTPA bound strongly to CD14 ($K_d$, $9.52{\times}10^{-8}M$). It dose dependently inhibited LPS-mediated activation of human myeloid cells, mouse macrophage cells and human whole blood as measured by the production of tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) and nitric oxide, whereas other phospho-lipids including phosphatidylserine and phosphatidylethanolamine had little effect. PE-DTPA also inhibited transcription dependent on $NF-{\kappa}B$ activation when it was added together with LPS, and it rescued LPS-primed mice from septic death. These results suggest that PE-DTPA is a potent antagonist of LPS, and that it acts by competing for binding to CD14.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.56 no.4
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• pp.380-387
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• 2023

Stocking density is associated with Paralichthys olivaceus growth; thus, fish should be rapidly reared at high densities for commercial reasons. Studies have reported that high stocking density retards growth; however, few have investigated the malformations caused by stocking density stress. This study compared the growth and malformation rates of P. olivaceus at different densities and stress- and immune-related gene expression between malformed and normal fish. Forty days post-hatching, fish (total length, 1.49±0.02 cm) were reared at 800 (low density; LD), 1500 (medium density; MD), and 4000 (high density; HD) fish/m², and the growth rate was measured weekly. On day 30, RNA was extracted from the kidneys, and the expression of stress-, immune-, and malformation-related genes was analyzed using qRT-PCR. The malformation rate in the HD groups was approximately three times higher (62%) than that in the LD and MD groups (approximately 20%), and growth was lower regarding length and weight. The stress-related (HSP70 and GPX) and immune-related (PIR and IgM) genes showed higher mRNA expression in the HD group and malformed fish than in the LD group and normal fish. However, TLR3 showed the opposite results. In summary, high stocking density suppressed growth and increased malformation risk in P. olivaceus.