• Molecules and Cells
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• 제25권1호
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• pp.99-104
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• 2008

Gangliosides, sialic acid-containing glycosphingolipids, are implicated in many neuronal diseases, but the precise molecular mechanisms underlying their pathological activities are poorly understood. Here we report that TLR2 participates in the initiation of ganglioside-triggered inflammatory signaling responses. Using FACS analysis and immunofluorescence microscopy, we found that gangliosides rapidly enhanced the cell surface expression of TLR2 in microglia, while reducing that of TLR4. The ganglioside-dependent increase of TLR2 expression was also observed at the messenger and protein levels. We also showed that gangliosides stimulate the interaction of TLR2 with Myd88, an adaptor for TLRs, and obtained evidence that lipid raft formation is closely associated with the ganglioside-induced activation of TLR2 and subsequent inflammatory signaling. These results collectively suggest that TLR2 contributes to the ability of gangliosides to cause inflammatory conditions in the brain.

• Journal of Ginseng Research
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• 제31권4호
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• pp.181-190
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• 2007

본 연구에서는 고려홍삼으로부터 새로 분리한 CK 함유분획을 이용하여 마우스 대식세포에 대한 선천면역반응 조절에 미치는 영향을 조사하였다. 본 연구에서 사용된 농도의 CK 함유분획에서는 세포독성 효과가 관찰되지 않았으며 CK함유 분획의 전처리에 의하여 그람음성세균의 LPS, 또는 CpG-ODN에 의해 유도되는 NF-${\kappa}B$와 MAPK 활성 및 전염증성 사이토카인, NO의 분비가 TLR4 및 TLR9 특이적으로 억제되었다. 이와 같은 결과는 CK 함유분획이 TLR4을 매개로하는 염증반응뿐만 아니라 TLR9을 통한 염증반응에도 영향을 미치는 것으로 해석된다. 따라서 앞으로 CK 함유 분획에 포함된 개별 사포닌 등 시료 성분에 대한 면밀한 분석, 그리고 이들 개별 물질이 각각의 신호전달 체계에 미치는 영향과 그 기작에 대한 연구가 더욱 필요할 것으로 사료되며 염증억제제로서의 개발 가능성을 탐구하기 위한 생체 내에서의 효능 및 작용기전 분석이 요구된다.

• 생명과학회지
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• 제18권12호
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• pp.1637-1643
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• 2008

HSP90에 노출된 혈관평활근세포에서 IL-6 transcript가 증가하고, IL-6 단백질의 분비가 증가하며, 또한 IL-6 유전자의 promote가 활성화되었다. HSP90에 의한 IL-6 유전자의 promoter 활성화는 dominant negative 형태의 TLR-4와 MyD88에 의하여 크게 감소되었지만, dominant negative 형태의 TLR-3와 TRIF의 영향을 받지 않았다. 그리고 TLR-4의 이합체화(dimerization)를 저해하는 curcumin은 HSP90에 의한 IL-6의 분비 및 IL-6 유전자 promoter 활성화를 억제하였다. 그리고 IL-6 유전자의 promoter의 NF-${\kappa}B$- 또는 C/EBP-binding sequence에 변이는 HSP90에 의한 IL-6 유전자의 promoter 활성화 억제하였다. 이러한 결과는 혈관평활근세포에서 HSP90에 의한 IL-6 유전자 활성화에 TLR-4와 NF-${\kappa}B$B가 관여함을 의미한다.

• Journal of Ginseng Research
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• 제43권2호
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• pp.291-299
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• 2019

Background: Ginsenosides of Korean Red Ginseng extracts (RGE) and its saponin components suppress secretion of inflammasome-mediating cytokines, whereas the nonsaponin fraction (NS) of RGE oppositely stimulates cytokine secretion. Although direct exposure of NS to macrophages in mice induces cytokine production, oral administration of NS has not been studied in inflammasome-related disease in animal models. Methods: Mice were fed RGE or NS for 7 days and then developed peritonitis. Peritoneal cytokines were measured, and peritoneal exudate cells (PECs) were collected to assay expression levels of a set of toll-like receptors (TLRs) and cytokines in response to NS ingestion. In addition, the role of intestinal bacteria in NS-fed mice was assessed. The effect of preexposure to NS in bone marrow-derived macrophages (BMDMs) on cytokine production was further confirmed. Results: NS ingestion attenuated secretion of peritoneal cytokines resulting from peritonitis. In addition, the isolated PECs from NS-fed mice presented lower TLR transcription levels than PECs from control diet-fed mice. BMDMs treated with NS showed downregulation of TLR4 mRNA and protein expression, which was mediated by the $TLR4-MyD88-NF{\kappa}B$ signal pathway. BMDMs pretreated with NS produced less cytokines in response to TLR4 ligands. Conclusion: NS administration directly inhibits TLR4 expression in inflammatory cells such as macrophages, thereby reducing secretion of cytokines during peritonitis.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2020년도 춘계학술대회
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• pp.86-86
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• 2020

The leaves of Staphylea bumalda (S. bumalda) as a deciduous tree distributed in Korea, China and Japan are used to treat respiratory diseases or inflammation. However, there is no scientific research on the immune-enhancing activity of S. bumalda leaves. Thus, in this study, we investigated the effect of water extracts from S. bumalda leaves (SBL) on the macrophage activity using mouse macrophage cells, RAW264.7. SBL increased production of immunomodulators such as NO, iNOS, IL-1β, IL-6, TNF-α and MCP-1 in RAW264.7 cells and activated phagocytic activity of RAW264.7 cells. Inhibition of TLR2 and TLR4 blocked SBL-mediated production of immunomodulators in RAW264.7 cells. In addition, SBL-mediated production of immunomodulators was attenuated by JNK inhibition in RAW264.7 cells. SBL increased JNK phosphorylation, while Inhibition of TLR2 and TLR4 blocked SBL-mediated JNK phosphorylation in RAW264.7 cells. These results are thought to be evidence that SBL activates JNK through stimulation of TLR2 and TLR4 in macrophage to induce the production of immunomodulators. In LPS-stimulated RAW264.7 cells, SBL inhibited over-production of immunomodulators. Summarizing the results, SBL showed immunostimulatory activity under normal conditions and immunosuppressive activity under LPS-induced excessive immune response conditions.

• Asian Pacific Journal of Cancer Prevention
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• 제14권4호
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• pp.2295-2299
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• 2013

Background: To explore the role of caveolin-1(CAV-1) gene silencing on MAPK activation in lipopolysaccharide (LPS)-challenged human mammary epithelial cells. Methods: We established a MCF-10ACE of CAV-1 gene silencing from human mammary epithelial cell line MCF-10A by RNAi technology. DNA Microarray were used to detect the expression of inflammation-associated genes in MCF10ACE. Western blotting was used to examine the activation of MAPK in lipopolysaccharide(LPS)-challenged MCF-10A and MCF-10ACE. Moreover, immunofluorescence and Western bloting were performed to detect the co-localization of CAV-1 and toll-like receptor 4 (TLR4) in human mammary epithelial cells. Results: MCF-10ACE exhibited significant increases in inflammation-associated gene expression, especially IL-6 (~7-fold) and IL6R (~17-fold). In addition, LPS-induced p38 MAPK and JNK MAPK activation was significantly increased in MCF-10ACE. Furthermore, CAV-1 co-localized with TLR4 and appeared a negative correlation trend. Conclusion: CAV-1 gene silencing promotes MAPK activation via TLR4 signaling in human mammary epithelial cells response to LPS.

• Journal of Yeungnam Medical Science
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• 제30권1호
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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.

• Nutrition Research and Practice
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• 제8권5호
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• pp.516-520
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• 2014

BACKGROUND/OBJECTIVES: Carnosic acid (CA), found in rosemary (Rosemarinus officinalis) leaves, is known to exhibit anti-obesity and anti-inflammatory activities. However, whether its anti-inflammatory potency can contribute to the amelioration of obesity has not been elucidated. The aim of the current study was to investigate the effect of CA on Toll-like receptor 4 (TLR4) pathways in the presence of lipopolysaccharide (LPS) in 3T3-L1 adipocytes. MATERIALS/METHODS: 3T3-L1 adipocytes were treated with CA ($0-20{\mu}M$) for 1 h, followed by treatment with LPS for 30 min; mRNA expression of adipokines and protein expression of TLR4-related molecules were then measured. RESULTS: LPS-stimulated 3T3-L1 adipocytes showed elevated mRNA expression of tumor necrosis factor (TNF)-${\alpha}$, interleukin-6, and monocyte chemoattractant protein-1, and CA significantly inhibited the expression of these adipokine genes. LPS-induced up regulation of TLR4, myeloid differentiation factor 88, TNF receptor-associated factor 6, and nuclear factor-${\kappa}B$, as well as phosphorylated extracellular receptor-activated kinase were also suppressed by pre-treatment of 3T3-L1 adipocytes with CA. CONCLUSIONS: Results of this study suggest that CA directly inhibits TLR4-MyD88-dependent signaling pathways and decreases the inflammatory response in adipocytes.

• BMB Reports
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• 제49권6호
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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.

• Molecules and Cells
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• 제24권1호
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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.