• 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.

• Parasites, Hosts and Diseases
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• v.50 no.4
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• pp.281-286
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• 2012

Helminthic infections afflict over 1.5 billion people worldwide, while Mycobacterium tuberculosis infects one third of the world's population, resulting in 2 million deaths per year. Although tuberculosis and helminthic infections coexist in many parts of the world, and it has been demonstrated that the T-helper 2 and T-regulatory cell responses elicited by helminths can affect the ability of the host to control mycobacterial infection, it is still unclear whether helminth infections in fact affect tuberculosis disease. In this review article, current progress in the knowledge about the immunomodulation induced by helminths to diminish the protective immune responses to bacille Calmette-Guerin vaccination is reviewed, and the knowledge about the types of immune responses modulated by helminths and the consequences for tuberculosis are summarized. In addition, recent data supporting the significant reduction of both M. tuberculosis antigen-specific Toll-like receptor (TLR) 2 and TLR9 expression, and pro-inflammatory cytokine responses to TLR2 and TLR9 ligands in individuals with M. tuberculosis and helminth co-infection were discussed. This examination will allow to improve understanding of the immune responses to mycobacterial infection and also be of great relevance in combating human tuberculosis.

• Journal of Plant Biotechnology
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• v.32 no.4
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• pp.269-273
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• 2005

Toll-like receptors (TLR) are important components of innate immunity in the defense against pathogens. TLRs recognize pathogen-associated common molecular patterns. TLRs are similar to the receptors involved in defense responses in plants. TLR protein is a type 1 membrane protein, consisting of an extracellular domain containing leucine-rich repeats and a cytoplasmic domain. The cytoplasmic domain delivers ligand recognition signals that result in production of anti-microbial agents. The cytoplasmic domain (amino acid 858-1032) of toll-like receptor 9 has been expressed using methylotrophic yeast Pichia pastoris. The protein expression was confirmed by Western-blot, N-terminal sequencing and MALDl-TOF mass spectrometry. The proteins have been purified by nickel affinity, cation exchange and gel-filtration chromatography.

• BMB Reports
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• v.51 no.1
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• pp.27-32
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• 2018

Non-small-cell lung cancer (NSCLC) is commonly caused by a mutation in the epidermal growth factor receptor (EGFR) and subsequent aberrant EGFR signaling with uncontrolled kinase activity. A deletion mutation in EGFR exon 19 is frequently observed in EGFR gene mutations. We designed a DNAzyme to suppress the expression of mutant EGFR by cleaving the mutant EGFR mRNA. The DNAzyme (named Ex19del Dz) specifically cleaved target RNA and decreased cancer cell viability when transfected into gefitinib-resistant lung cancer cells harboring EGFR exon 19 deletions. The DNAzyme decreased EGFR expression and inhibited its downstream signaling pathway. In addition to EGFR downregulation, Ex19del Dz containing CpG sites activated Toll-like receptor 9 (TLR9) and its downstream signaling pathway via p38 kinase, causing an immunostimulatory effect on EGFR-mutated NSCLC cells. Thus, dual effects of this DNAzyme harboring the CpG site, such as TLR9 activation and EGFR downregulation, leads to apoptosis of EGFR-mutated NSCLC cells.

• International Journal of Oral Biology
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• v.33 no.3
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• pp.77-82
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• 2008

Innate immune response is initiated by the recognition of unique microbial molecular patterns through pattern recognition receptors (PRRs). The purpose of this study is to dissect the expression of various PRRs in gingival epithelial cells of differentiated versus undifferentiated states. Differentiation of immortalized human gingival epithelial HOK-16B cells was induced by culture in the presence of high $Ca^{2+}$ at increased cell density. The expression levels of various PRRs in HOK-16B cells were examined by realtime reverse transcription polymerase chain reaction (RTPCR) and flow cytometry. In addition, the expression of human beta defensins (HBDs) was examined by real time RT-PCR and the amounts of secreted cytokines were measured by enzyme linked immunosorbent assay. In undifferentiated HOK-16B cells, NACHT-LRR-PYDcontaining protein (NALP) 2 was expressed most abundantly, and toll like receptor (TLR) 2, TLR4, nucleotide-binding oligomerization domain (NOD) 1, and NOD2 were expressed in substantial levels. However, TLR3, TLR7, TLR8, TLR9, ICE protease-activating factor (IPAF), and NALP6 were hardly expressed. In differentiated cells, the levels of NOD2, NALP2, and TLR4 were different from those in undifferentiated cells at RNA but not at protein levels. Interestingly, differentiated cells expressed the increased levels of HBD-1 and -3 but secreted reduced amount of IL-8. In conclusion, the repertoire of PRRs expressed by gingival epithelial cells is limited, and undifferentiated and differentiated cells express similar levels of PRRs.

• IMMUNE NETWORK
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• v.12 no.1
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• pp.27-32
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• 2012

Background: Toll-like receptors (TLRs) have been extensively studied in recent years. However, functions of these molecules in murine B cell biology are largely unknown. A TLR4 stimulant, LPS is well known as a powerful polyclonal activator for murine B cells. Methods: In this study, we explored the effect of a murine TLR9 stimulant, M6-395 (a synthetic CpG ODNs) on B cell proliferation and Ig production. Results: First, M6-395 was much more potent than LPS in augmenting B cell proliferation. As for Ig expression, M6-395 facilitated the expression of both TGF-${\beta}1$-induced germ line transcript ${\alpha}$ ($GLT{\alpha}$) and IL-4-induced $GLT{\gamma}1$ as levels as those by LPS and Pam3CSK4 (TLR1/2 agonist) : a certain Ig GLT expression is regarded as an indicative of the corresponding isotype switching recombination. However, IgA and IgG1 secretion patterns were quite different--these Ig isotype secretions by M6-395 were much less than those by LPS and Pam3CSK4. Moreover, the increase of IgA and IgG1 production by LPS and Pam3CSK4 was virtually abrogated by M6-395. The same was true for the secretion of IgG3. We found that this unexpected phenomena provoked by M6-395 is attributed, at least in part, to its excessive mitogenic nature. Conclusion: Taken together, these results suggest that M6-395 can act as a murine polyclonal activator but its strong mitogenic activity is unfavorable to Ig isotype switching.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.33 no.6
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• pp.636-642
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• 2007

Background: CpG DNA plays an important role in immune cell function. This study examined whether the temporal control of toll-like receptor (TLR)9 by CpG DNA can regulate the expression of matrix metalloproteinase-9(MMP-9). Methods and materials: Macrophages were cultured in the presence of 10% FBS. For the various MMP genes analysis, RT-PCR and real-time PCR were performed. In addition, zymography assay performed for the MMP activity. The phosphorylation assay did for the ERK1/2 and NF${\kappa}B$ activation, and luciferase promoter assay was for the NF${\kappa}B$ activity. Results: CpG DNA induced the mRNA expression of MMP-2, MMP-9, and MMP-13, but not of MMP-7, MMP-8, and MMP-12, in a time-dependent manner. Especially, the mRNA expression of MMP-9 was strongly induced by CpG DNA using real-time RT-PCR. The TLR9 inhibitor, chloroquine, suppressed CpG DNA-induced MMP-9 expression and its activity. Moreover, CpG DNA induced the phosphorylation of ERK and the inhibition of ERK by U0126 suppressed CpG DNA-induced MMP-9 expression and its activity. CpG DNA stimulated $I{\kappa}B-{\alpha}$ degradation and luciferase activity. In addition, pretreatment of SN-50, the inhibitor of NF${\kappa}B$, strongly blocked the CpG DNA-induced MMP-9 expression and activity. Conclusion: These observations suggest that CpG DNA may play important roles in the activation of macrophages by regulating the production of MMP-9 via the sequential TLR9-ERK-NF${\kappa}B$ signaling pathway.

• 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.

• Clinical and Experimental Pediatrics
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• v.48 no.1
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• pp.6-12
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• 2005

In the last few years, a spectrum of dendritic cells(DCs), including toll like receptors(TLRs), might play a critical role in regulating allergy and asthma. DC plays a central role in initiating immune responses, linking innate and adaptive responses to pathogen. Human peripheral blood has three non-overlapping dendritic subset that expressed various 11 TLRs. These dendritic subsets and TLR contribute significant polarizing influences on T helper differentiation, but how this comes about is less clear. A better understanding of DC immunobiology may lead to the comprehension of allergy pathophysiology to prevent early stage allergic march.

• 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.