• 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.

• Molecules and Cells
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• 제23권1호
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• pp.1-10
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• 2007

Invading pathogens are recognized by diverse germline-encoded pattern-recognition receptors (PRRs) which are distributed in three different cellular compartments: extracellular, membrane, and cytoplasmic. In mammals, the major extracellular PRRs such as complements may first encounter the invading pathogens and opsonize them for clearance by phagocytosis which is mediated by membrane-associated phagocytic receptors including complement receptors. The major membrane-associated PRRs, Toll-like receptors, recognize diverse pathogens and generate inflammatory signals to coordinate innate immune responses and shape adaptive immune responses. Furthemore, certain membrane-associated PRRs such as Dectin-1 can mediate phagocytosis and also induce inflammatory response. When these more forefront detection systems are avoided by the pathogens, cytoplasmic PRRs may play major roles. Cytoplasmic caspase-recruiting domain (CARD) helicases such as retinoic acid-inducible protein I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5), mediate antiviral immunity by inducing the production of type I interferons. Certain members of nucleotide-binding oligomerization domain (NOD)-like receptors such as NALP3 present in the cytosol form inflammasomes to induce inflammatory responses upon ligand recognition. Thus, diverse families of PRRs coordinately mediate immune responses against diverse types of pathogens.

• Molecules and Cells
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• 제24권2호
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• pp.247-254
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• 2007

Improvement in the pharmacokinetic properties of short interfering RNAs (siRNAs) is a prerequisite for the therapeutic application of RNA interference technology. When injected into mice as unmodified siRNAs complexed to DOTAP/Chol-based cationic liposomes, all 12 tested siRNA duplexes caused a strong induction of cytokines including interferon ${\alpha}$, indicating that the immune activation by siRNA duplexes is independent of sequence context. When modified by various combinations of 2'-OMe, 2'-F, and phosphorothioate substitutions, introduction of as little as three 2'-OMe substitutions into the sense strand was sufficient to suppress immune activation by siRNA duplexes, whereas the same modifications were much less efficient at inhibiting the immune response of single stranded siRNAs. It is unlikely that Toll-like receptor 3 (TLR3) signaling is involved in immune stimulation by siRNA/liposome complexes since potent immune activation by ds siRNAs was induced in TLR3 knockout mice. Together, our results indicate that chemical modification of siRNA provides an effective means to avoid unwanted immune activation by therapeutic siRNAs. This improvement in the in vivo properties of siRNAs should greatly facilitate successful development of siRNA therapeutics.

• 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.

• 고려인삼학회:학술대회논문집
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• 고려인삼학회 2008년도 춘계 학술대회
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• pp.76-76
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• 2008

• 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.

• Clinical and Experimental Pediatrics
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• 제48권3호
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• pp.315-320
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• 2005

목 적 : 본 연구에서는 가와사끼병에서 toll-like receptor(TLR)의 발현정도를 살펴 염증반응이 유발되기 시작하는 기전에 대해 접근하고자 하였다. 방 법 : 2003년 3월부터 8월까지 연세의료원에서 가와사끼병으로 진단 받은 환아 10명과 발열대조군 10명 및 정상대조군 10명의 말초혈액을 얻은 후 유세포분석기(flow cytometry)를 시행하여 CD14 양성인 단핵구에서의 TLR-2 발현정도를 측정하였다. 또한 말초 혈액 단핵구의 total RNA를 분리한 후 역전사중합효소 연쇄반응(RT-PCR)을 시행하여 TLR-2의 mRNA 발현을 살펴보았다. 결 과 : 환자군에서의 TLR-2 발현은 정상대조군보다 통계적으로 유의하게 증가되어 있었으나 임상경과에 따른 양상을 보면 급성기보다 아급성기에서 감소하였지만 통계적으로 유의한 차이는 보이지 않았고 환자군과 발열대조군의 TLR-2 발현도 의미있는 차이를 보이지 않았다. 또한 급성기 환자군의 말초혈액 단 핵구에서 TLR-2의 mRNA 발현이 증가되어 있었다. 결 론 : TLR-2의 발현은 가와사끼병 환자에서 정상대조군과 비교하여 증가되어 있었으며 이는 TLR 및 이를 통한 선천성 면역계(innate immunity)가 가와사끼병의 병인과 연관될 수 있음을 시사한다. 앞으로 TLR의 발현이 가와사끼병에서의 염증유발에 있어 구체적으로 어떤 역할을 하는지에 대한 연구가 더 필요할 것으로 사료된다.

• 고려인삼학회:학술대회논문집
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• 고려인삼학회 2007년도 춘계 학술대회
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• pp.10-10
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• 2007

• BMB Reports
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• 제48권12호
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• pp.696-701
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• 2015

Toll-like receptor 3 (TLR3) recognizes viral double-stranded RNA. It stimulates pro-inflammatory cytokine and interferon production. Here we reported the expression of a novel isoform of TLR3 in human astrocyte cell lines whose message is generated by alternative splicing. The isoform represents the N-terminus of the protein. It lacks many of the leucine-rich repeat domains, the transmembrane domain, and the intracellular Toll/interleukin-1 receptor domain of TLR3. Type I interferons (interferon-α and interferon-β) induced the expression of this isoform. Exogenous overexpression of this isoform inhibited interferon regulatory factor 3, signal transducers and activators of transcription 1, and Inhibitor of kappa B α signaling following stimulation. This isoform of TLR3 also inhibited the production of chemokine interferon-γ-inducible protein 10. Our study clearly demonstrated that the expression of this isoform of TLR3 was a negative regulator of signaling pathways and that it was inducible by type I interferons. We also found that this isoform could modulate inflammation in the brain.

• 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.