• The Korean Journal of Malacology
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• v.31 no.3
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• pp.233-241
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• 2015

Toll-like receptors (TLRs) are a major pattern recognition receptor that recognize the structure of invading pathogen and play key roles by triggering immune response. In this study, we identified a sequence of TLR homolog and characterized at molecular level from the abalone (Haliotis discus hannai). Multiple alignments and phylogenetic analysis of abalone TLR protein belongs to the TLR 2/6. Expression level of abalone TLR 2/6 in the tissue was comparatively high in the mantle, gill, digestive duct, and hemocytes, but lowest in the muscle. Expression level of abalone TLR 2/6 mRNA in the mantle, gill, digestive duct, and hemocytes was 20-fold, 60-fold, 115-fold, 112-fold higher than in the muscle, respectively. Expression level of abalone TLR 2/6 mRNA in the mantle was steadily increased until 12 h and decreased post-infection with Vibrio parahemolyticus. While the expression level of abalone TLR 2/6 mRNA in the gill and hemocytes was drastically increased at 6 and 9 h post-infection with Vibrio parahemolyticus, respectively. These results suggest that abalone TLR 2/6 is conserved through evolution and may play roles similar to its mammalian counterparts.

• IMMUNE NETWORK
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• v.24 no.4
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• pp.25.1-25.14
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• 2024

Lupus is characterized by the autoantibodies against nuclear Ags, underscoring the importance of identifying the B cell subsets driving autoimmunity. Our research focused on the mitochondrial activity and CXCR4 expression in CD11c⁺ B cells from lupus patients after ex vivo stimulation with a TLR9 agonist, CpG-oligodeoxyribonucleotide (ODN). We also evaluated the response of CD11c⁺ B cells in ODN-injected mice. Post-ex vivo ODN stimulation, we observed an increase in the proportion of CD11c^hi cells, with elevated mitochondrial activity and CXCR4 expression in CD11c⁺ B cells from lupus patients. In vivo experiments showed similar patterns, with TLR9 stimulation enhancing mitochondrial and CXCR4 activities in CD11c^hi B cells, leading to the generation of anti-dsDNA plasmablasts. The CXCR4 inhibitor AMD3100 and the mitochondrial complex I inhibitor IM156 significantly reduced the proportion of CD11c⁺ B cells and autoreactive plasmablasts. These results underscore the pivotal roles of mitochondria and CXCR4 in the production of autoreactive plasmablasts.

• Development and Reproduction
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• v.21 no.4
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• pp.361-370
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• 2017

Koi herpesvirus (KHV), also known as Cyprinid herpes virus 3 (Cyprinid 3) is lethal disease in common carp and koi (Cyprinus carpio). Two different groups (KK and RK) were infected KHV by intraperitoneal injection. Fish for gene expression analysis were sampled at 0 h, 12 h, 24 h, 48 h and 72 h post infection (p.i). The results showed that two immune related gene, Interferons (INFs) ${\alpha}{\beta}$ and Interleukin (IL)-12 p35 induced a high response in RK. The IL-12 p35 cytokine and Toll-like receptor (TLR) 9 were significantly high expressed on 48 h post infection (p.i) in RK as compared to the KK. The histopatological examination reveals focal necrosis in liver and infiltrate of lymphocytes in spleen of KK as compared to the RK. In immunohistochemistry analysis, the KHV protein high expressed in the infected kidney cell and slenocyte of KK. Therefore, the expression of IL-12 p35, IFN ${\alpha}{\beta}$ and TLR 9 may provide a potentially genes related with KHV resistance in Koi and red common carp ${\times}$ koi.

• Journal of Yeungnam Medical Science
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• v.30 no.1
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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.

• Bulletin of the Korean Chemical Society
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• v.33 no.9
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• pp.2878-2882
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• 2012

Amentoflavone is naturally occurring bioflavonoid that is found in a number of plants. In this paper, the anti-inflammatory activity of amentoflavone in LPS-stimulated macrophages and its mode of action were examined. Using LPS-stimulated RAW264.7 macrophage cells, we found that amentoflavone exerted anti-inflammatory activities through inhibition of nitric oxide (NO) production and tumor necrosis factor (TNF)-${\alpha}$ and macrophage inflammatory protein (MIP)-2 secretion. Amentoflavone (1.0-20 ${\mu}M$) gradually inhibited nitrite production without cytotoxicity. Amentoflavone (1.0 and 10 ${\mu}M$) effectively suppressed both TNF-${\alpha}$ and MIP-2 cytokine release from LPS-stimulated RAW264.7 cells. The expression of mIL-$1{\beta}$ and mMIP-2 cytokine mRNAs was completely inhibited while expression of mMIP-1 was effectively suppressed and mTNF-${\alpha}$ expression was slightly inhibited by 10 ${\mu}M$ amentoflavone. We also demonstrated that the innate immune response to amentoflavone involves the toll-like receptor (TLR) and mitogen-activated protein kinase (MAPK) pathways. LPS-induced upregulation of p38 MAPK phosphorylation was significantly reduced by 10 ${\mu}M$ amentoflavone. These results suggest that amentoflavone exhibits effective anti-inflammatory activities through regulation of TLR4 and phosphorylation of p38 MAPKs.

• Tuberculosis and Respiratory Diseases
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• v.58 no.4
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• pp.367-374
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• 2005

Background : The fact that only 10-20% of chronic cigarette smokers develop chronic obstructive pulmonary disease (COPD) reflects the presence of genetic factors associated with the susceptibility to COPD. Recently, it was reported that the surfactant protein A increases the secretion of matrix metalloprotease 9, which degrades extracellular matrices of the lung, through a Toll-like receptor 2 (TLR2). In this context, possible role of TLR2 in the pathogenesis of COPD was postulated, and a functional dinucleotide repeat polymorphism in intron II of TLR2 was evaluated for any association with COPD. Method : Male patients with COPD and male smokers with a normal pulmonary function were enrolled in this study. The number of Guanine-Thymine repeats in intron II of the TLR2 gene were counted. Because the distributions of the repeats were trimodal, the alleles were classified into three subclasses, 12-16 repeats: short (S) alleles; 17-22 repeats: medium length (M) alleles; and 23-27 repeats: long (L) alleles. Result : 125 male patients with COPD and 144 age- and gender-matched blood donors with a normal lung function were enrolled. There were no differences in the distribution of each allele subclass (S, M and L) between the COPD and control group (p=0.75). The frequencies of the genotypes with and without each allele subclass in the COPD and control group were similar. Conclusion : A microsatellite polymorphism in intron II of TLR2 gene was not associated with the development of COPD in Koreans.

• BMB Reports
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• v.56 no.6
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• pp.359-364
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• 2023

KAI1/CD82, a membrane tetraspanin protein, can prevent various cancers and retinal disorders through its anti-angiogenic and anti-metastatic capacity. However, little is known about its anti-inflammatory effect and molecular mechanism. Therefore, the present study aimed to inLPSvestigate effect of a recombinant protein of the large extracellular domain of human KAI1 (Gly 111-Leu 228, rhKAI1) on lipopolysaccharides (LPS)-stimulated RAW264.7 macrophage-like cells and mouse bone marrow-derived macrophages (BMDM) and to identify its underlying mechanism. Our data showed that rhKAI1 suppressed expression levels of classically macrophages (M1) phenotype-related surface markers F4/80+CD86+ in LPS-stimulated BMDM and RAW264.7 cells. In addition, LPS markedly increased mRNA expression and release levels of pro-inflammatory cytokines and mediators such as interleukin (IL)-1β, IL-6, tumor necrosis factor-α, cyclooxygenase-2, nitric oxide and prostaglandin E2, whereas these increases were substantially down-regulated by rhKAI1. Furthermore, LPS strongly increased expression of NF-κB p65 in the nuclei and phosphorylation of ERK, JNK, and p38 MAPK. However, nuclear translocation of NF-κB p65 and phosphorylation of JNK were greatly reversed in the presence of rhKAI1. Especially, rhKAI1 markedly suppressed expression of toll-like receptor (TLR4) and prevented binding of LPS with TLR4 through molecular docking predict analysis. Importantly, Glu 214 of rhKAI1 residue strongly interacted with Lys 360 of TLR4 residue, with a binding distance of 2.9 Å. Taken together, these findings suggest that rhKAI1 has an anti-inflammatory effect on LPS-polarized macrophages by interacting with TLR4 and down-regulating the JNK/NF-κB signaling pathway.

• Nutrition Research and Practice
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• v.9 no.4
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• pp.364-369
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• 2015

BACKGROUND/OBJECTIVES: Inflammation is associated with various types of acute and chronic alcohol liver diseases. In this study, we examined whether umbelliferone (7-hydroxycoumarin, UF) ameliorates chronic alcohol-induced liver damage by modulating inflammatory response and the antioxidant system. METHODS: Rats were fed a Liber-Decarli liquid diet containing 5% alcohol with or without UF (0.05 g/L) for 8 weeks, while normal rats received an isocaloric carbohydrate liquid diet. RESULTS: Chronic alcohol intake significantly increased serum tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) and interleukin 6 levels and decreased interleukin 10 level; however, UF supplementation reversed the cytokines related to liver damage. UF significantly suppressed hepatic lipopolysaccharide binding protein, toll-like receptor 4 (TLR4), nuclear factor kappa B, and TNF-${\alpha}$ gene expression increases in response to chronic alcohol intake. Masson's trichrome staining revealed that UF improved mild hepatic fibrosis caused by alcohol, and UF also significantly increased the mRNA expressions and activities of superoxide dismutase and catalase in liver, and thus, decreased lipid peroxide and mitochondrial hydrogen peroxide levels. CONCLUSIONS: The findings of this study indicate that UF protects against alcohol-induced liver damage by inhibiting the TLR4 signaling pathway and activating the antioxidant system.

• BMB Reports
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• v.44 no.9
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• pp.607-612
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• 2011

Several investigators have shown that CpG-DNA has outstanding effects as a Th1-responsive adjuvant and that its potent adjuvant effects are enhanced by encapsulation with a liposome of proper composition. In this study, we showed that encapsulation with phosphatidyl-${\beta}$-oleoyl-${\gamma}$-palmitoyl ethanolamine (DOPE): cholesterol hemisuccinate (CHEMS) complex enhances the immunostimulatory activity of CpG DNA and the binding of CpG-DNA to TLR9. We also examined involvement of myeloid differentiation protein (MyD88) and NF-${\kappa}B$ activation in liposome-encapsulated CpG-DNA-induced IL-8 promoter activation. In this manuscript, the natural phosphodiester bond CpG-DNA encapsulated by DOPE : CHEMS complex is designated as Lipoplex(O). Importantly, we successfully screened B cell epitopes of envelope protein (E protein) of hepatitis C virus (HCV-E) and attachment glycoprotein G of human respiratory syncytial virus (HRSV-G) by immunization with complexes of several peptides and Lipoplex(O) without carriers. Therefore, Lipoplex(O) is potentially applicable as a universal adjuvant for peptide-based epitope screening and antibody production.

• BMB Reports
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• v.52 no.2
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• pp.133-138
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• 2019

Upon viral infection, the 2', 5'-oligoadenylate synthetase (OAS)-ribonuclease L (RNaseL) system works to cleave viral RNA, thereby blocking viral replication. However, it is unclear whether OAS proteins have a role in regulating gene expression. Here, we show that OAS1 and OAS3 act as negative regulators of the expression of chemokines and interferon-responsive genes in human macrophages. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) technology was used to engineer human myeloid cell lines in which the OAS1 or OAS3 gene was deleted. Neither OAS1 nor OAS3 was exclusively responsible for the degradation of rRNA in macrophages stimulated with poly(I:C), a synthetic surrogate for viral double-stranded (ds)RNA. An mRNA sequencing analysis revealed that genes related to type I interferon signaling and chemokine activity were increased in $OAS1^{-/-}$ and $OAS3^{-/-}$ macrophages treated with intracellular poly(I:C). Indeed, retinoic-acid-inducible gene (RIG)-I- and interferon-induced helicase C domain-containing protein (IFIH1 or MDA5)-mediated induction of chemokines and interferon-stimulated genes was regulated by OAS3, but Toll-like receptor 3 (TLR3)- and TLR4-mediated induction of those genes was modulated by OAS1 in macrophages. However, stimulation of these cells with type I interferons had no effect on OAS1- or OAS3-mediated chemokine secretion. These data suggest that OAS1 and OAS3 negatively regulate the expression of chemokines and interferon-responsive genes in human macrophages.