• IMMUNE NETWORK
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• 제11권4호
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• pp.223-226
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• 2011

Although exercise-induced growth factors such as Insulin-like growth factor-I (IGF-I) are known to affect various aspects of physiology in skeletal muscle cells, the molecular mechanism by which IGF-I modulates anti-inflammatory effects in these cells is presently unknown. Here, we showed that IGF-I stimulation suppresses the expression of toll-like receptor 4 (TLR4), a key innate immune receptor. A pharmacological inhibitor study further showed that PI3K/Akt signaling pathway is required for IGF-I-mediated negative regulation of TLR4 expression. Furthermore, IGF-I treatment reduced the expression of various NF-${\kappa}B$-target genes such as TNF-${\alpha}$ and IL-6. Taken together, these findings indicate that the anti-inflammatory effect of exercise may be due, at least in part, to IGF-I-induced suppression of TLR4 and subsequent downregulation of the TLR4-dependent inflammatory signaling pathway.

• Molecules and Cells
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• 제27권2호
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• pp.211-215
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• 2009

Toll-like receptors (TLRs) play a critical role in sensing microbial components and inducing innate immune and inflammatory responses by recognizing invading microbial pathogens. Lipopolysaccharide-induced dimerization of TLR4 is required for the activation of downstream signaling pathways including nuclear factor-kappa B ($NF-{\kappa}B$). Therefore, TLR4 dimerization may be an early regulatory event in activating ligand-induced signaling pathways and induction of subsequent immune responses. Here, we report biochemical evidence that 6-shogaol, the most bioactive component of ginger, inhibits lipopolysaccharide-induced dimerization of TLR4 resulting in the inhibition of $NF-{\kappa}B$ activation and the expression of cyclooxygenase-2. Furthermore, we demonstrate that 6-shogaol can directly inhibit TLR-mediated signaling pathways at the receptor level. These results suggest that 6-shogaol can modulate TLR-mediated inflammatory responses, which may influence the risk of chronic inflammatory diseases.

• 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.

• 한국임상수의학회지
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• 제26권6호
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• pp.520-523
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• 2009

본 연구는 말의 정상안과 재발성 포도막염이 있는 안구에서의 TLR-2, -4, -9 mRNA 발현의 정량적 차이를 비교하기 위해 수행되었다. 정상 및 재발성 포도막염이 있는 말 각 6두 에서 안구를 적출하여 모양체, 홍채, 망막 및 맥락막을 수집하였다. Real-time PCR assay 통해 정상안과 재발성 포도막염이 있는 안구에서의 TLR-2, -4, -9의 mRNA 발현 차이를 정량적으로 비교하였다. 말의 재발성 포도막염 시에는 모양체, 홍채에서 정상인 경우에 비해 4-12 배의 TLR-2 와 TLR-9 mRNA 발현증가를 보였으며, 맥락막 및 망막에서는 2-6 배의 TLR-2, -4, -9 mRNA 발현 증가를 보였다. 본 연구 결과는 Toll-like receptor 2, -4, -9이 말의 재발성 포도막염의 병리기전에 영향을 미치고 있음을 시사한다. 하지만 재발성 포도막염 시의 Toll-like receptor 2, -4, -9의 구체적 역할을 밝히기 위해서는 다양한 후속 연구가 요구된다.

• Parasites, Hosts and Diseases
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• 제53권4호
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• pp.431-438
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• 2015

In Trichinella spiralis infection, type 2 helper T (Th2) cell-related and regulatory T ($T_{reg}$) cell-related immune responses are the most important immune events. In order to clarify which Toll-like receptors (TLRs) are closely associated with these responses, we analyzed the expression of mouse TLR genes in the small intestine and muscle tissue during T. spiralis infection. In addition, the expression of several chemokine- and cytokine-encoding genes, which are related to Th2 and $T_{reg}$ cell mediated immune responses, were analyzed in mouse embryonic fibroblasts (MEFs) isolated from myeloid differentiation factor 88 (MyD88)/TIR-associated proteins (TIRAP) and Toll receptor-associated activator of interferons (TRIF) adapter protein deficient and wild type (WT) mice. The results showed significantly increased TLR4 and TLR9 gene expression in the small intestine after 2 weeks of T. spiralis infection. In the muscle, TLR1, TLR2, TLR5, and TLR9 gene expression significantly increased after 4 weeks of infection. Only the expression of the TLR4 and TLR9 genes was significantly elevated in WT MEF cells after treatment with excretory-secretory (ES) proteins. Gene expression for Th2 chemokine genes were highly enhanced by ES proteins in WT MEF cells, while this elevation was slightly reduced in MyD88/$TIRAP^{-/-}$ MEF cells, and quite substantially decreased in $TRIF^{-/-}$ MEF cells. In contrast, IL-10 and $TGF-{\beta}$ expression levels were not elevated in MyD88/$TIRAP^{-/-}$ MEF cells. In conclusion, we suggest that TLR4 and TLR9 might be closely linked to Th2 cell and $T_{reg}$ cell mediated immune responses, although additional data are needed to convincingly prove this observation.

• Journal of Plant Biotechnology
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• 제32권4호
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• pp.269-273
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• 2005

Methylotrophic 효모 Pichia pastoris 발현시스템을 사용하여 인간 TLR9 단백질의 세포내 TIR 도메인을 발현하였다. TIR 단백질이 P. pastoris에서 발현되어 배지 속으로 분비되는 것을 SDS-PAGE로 확인하였고, 발현된 단백질을 western-blot, MALDI-TOF 질량분석으로 동정하였다. 이를 통하여 TIR 딘백질이 P. pastoris에서 안정적으로 발현됨을 알 수 있었다. 그리고 발현된 단백질을 니켈 친화, 양이온교환수지, 겔 투과 크로마토그라피를 사용하여 순수 분리 정제하였다. P. pastoris를 이용한 단백질의 발현과 정제방법은 대장균에서 잘 발현되지 않는 단백질의 발현에 응용될 수 있을 것이다.

• BMB Reports
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• 제47권7호
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• pp.361-368
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• 2014

Lipid components in biological membranes are essential for maintaining cellular function. Phosphoinositides, the phosphorylated derivatives of phosphatidylinositol (PI), regulate many critical cell processes involving membrane signaling, trafficking, and reorganization. Multiple metabolic pathways including phosphoinositide kinases and phosphatases and phospholipases tightly control spatio-temporal concentration of membrane phosphoinositides. Metabolizing enzymes responsible for PI 4,5-bisphosphate (PI(4,5)P2) production or degradation play a regulatory role in Toll-like receptor (TLR) signaling and trafficking. These enzymes include PI 4-phosphate 5-kinase, phosphatase and tensin homolog, PI 3-kinase, and phospholipase C. PI(4,5)P2 mediates the interaction with target cytosolic proteins to induce their membrane translocation, regulate vesicular trafficking, and serve as a precursor for other signaling lipids. TLR activation is important for the innate immune response and is implicated in diverse pathophysiological disorders. TLR signaling is controlled by specific interactions with distinct signaling and sorting adaptors. Importantly, TLR signaling machinery is differentially formed depending on a specific membrane compartment during signaling cascades. Although detailed mechanisms remain to be fully clarified, phosphoinositide metabolism is promising for a better understanding of such spatio-temporal regulation of TLR signaling and trafficking.

• Journal of Microbiology and Biotechnology
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• 제17권11호
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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 Microbiology and Biotechnology
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• 제27권8호
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• pp.1529-1538
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• 2017

Klebsiella pneumoniae is an opportunistic and clinically significant emerging pathogen. We investigated the relative roles of Toll-like receptor (TLR) 2 and TLR4 in initiating host defenses against K. pneumoniae. TLR2 knockout (KO), TLR4 KO, TLR2/4 double KO (DKO), and wild-type (WT) mice were inoculated with K. pneumoniae. Mice in each group were sacrificed after either 12 or 24h, and the lungs, liver, and blood were harvested to enumerate bacterial colony-forming units (CFU). Cytokine and chemokine levels were analyzed using enzyme-linked immunosorbent assay and real-time PCR, and pneumonia severity was determined by histopathological analysis. Survival was significantly shortened in TLR4 KO and TLR2/4 DKO mice compared with that of WT mice after infection with $5{\times}10^3CFU$. TLR2 KO mice were more susceptible to infection than WT mice after exposure to a higher infectious dose. Bacterial burdens in the lungs and liver were significantly higher in TLR2/4 DKO mice than in WT mice. Serum $TNF-{\alpha}$, MCP-1, MIP-2, and nitric oxide levels were significantly decreased in TLR2/4 DKO mice relative to those in WT mice, and TLR2/4 DKO mice showed significantly decreased levels of $TNF-{\alpha}$, IL-6, MCP-1, and inducible nitric oxide synthase mRNA in the lung compared with those in WT mice. Collectively, these data indicate that TLR2/4 DKO mice were more susceptible to K. pneumoniae infection than single TLR2 KO and TLR4 KO mice. These results suggest that TLR2 and TLR4 play cooperative roles in lung innate immune responses and bacterial dissemination, resulting in systemic inflammation during K. pneumoniae infection.

• Reproductive and Developmental Biology
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• 제39권4호
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• pp.105-115
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• 2015

Toll-like receptor 7 (TLR7) is critical for the triggering of innate immune response by recognizing the conserved molecular patterns of single-stranded RNA (ssRNA) viruses and mediated antigenic adaptive immunity. To understand how TLR7 distinguish pathogen-derived molecular patterns from the host self, it is essential to be able to identify TLR7 receptor interaction interfaces, such as active sites or R848-agonist binding sites. The functional interfaces of TLR7 can serve as targets for structure-based drug design in studying the TLR7 receptor's structure-function relationship. In contrast to mammalian TLR7, chicken TLR7 (chTLR7) is unknown for its important biological function. Therefore, it has been targeted to mediate contrasting evolutionary patterns of positive selection into non-synonymous SNPs across eleven species using TLR7 conservation patterns (evolutionary conserved and class-specific trace residues), where protein sequence differences to the TLR7 receptors of interest record mutation that have passed positive section across the species. In this study, we characterized the Lys609 residue on chTLR7-ECD homodimer interfaces to reflect the current tendency of evolving positive selection to be transfer into a stabilization direction of the R848-agonist/chTLR7-ECDs complex under the phylogenetically variable position across species and we suggest a potential indicator for contrasting evolutionary patterns of both the species TLR-ECDs.