• Clinical and Experimental Reproductive Medicine
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• 제50권1호
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• pp.44-52
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• 2023

Objective: The DNA integrity of spermatozoa that attach to fallopian tube (FT) cells is higher than spermatozoa that do not attach. FT epithelial cells can distinguish normal and abnormal sperm chromatin. This study investigated the effects of sperm with a high-DNA fragmentation index (DFI) from men with unexplained repeated implantation failure (RIF) on the Toll-like receptor (TLR) signaling pathway in human FT cells in vitro. Methods: Ten men with a RIF history and high-DFI and 10 healthy donors with low-DFI comprised the high-DFI (>30%) and control (<30%) groups, respectively. After fresh semen preparation, sperm were co-cultured with a human FT epithelial cell line (OE-E6/E7) for 24 hours. RNA was extracted from the cell line and the human innate and adaptive immune responses were tested using an RT2 profiler polymerase chain reaction (PCR) array. Results: The PCR array data showed significantly higher TLR-1, TLR-2, TLR-3, TLR-6, interleukin 1α (IL-1α), IL-1β, IL-6, IL-12, interferon α (IFN-α), IFN-β, tumor necrosis factor α (TNF-α), CXCL8, GM-CSF, G-CSF, CD14, ELK1, IRAK1, IRAK2, IRAK4, IRF1, IRF3, LY96, MAP2K3, MAP2K4, MAP3K7, MAP4K4, MAPK8, MAPK8IP3, MYD88, NFKB1, NFKB2, REL, TIRAP, and TRAF6 expression in the high-DFI group than in the control group. These factors are all involved in the TLR-MyD88 signaling pathway. Conclusion: The MyD88-dependent pathway through TLR-1, TLR-2, and TLR-6 activation may be one of the main inflammatory pathways activated by high-DFI sperm from men with RIF. Following activation of this pathway, epithelial cells produce inflammatory cytokines, resulting in neutrophil infiltration, activation, phagocytosis, neutrophil extracellular trap formation, and apoptosis.

• Molecules and Cells
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• 제28권4호
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• pp.365-368
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• 2009

Toll-like receptors (TLRs) play an important role in host defense by sensing invading microbial pathogens and initiating innate immune responses. The stimulation of TLRs by microbial components triggers the activation of myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter inducing interferon-${\beta}$ (TRIF)-dependent downstream signaling pathways. Isoliquiritigenin (ILG), an active ingredient of Licorice, has been used for centuries to treat many chronic diseases. ILG inhibits the MyD88-dependent pathway by inhibiting the activity of inhibitor-${\kappa}B$ kinase. However, it is not known whether ILG inhibits the TRIF-dependent pathway. To evaluate the therapeutic potential of ILG, we examined its effect on signal transduction via the TRIF-dependent pathway of TLRs induced by several agonists. ILG inhibited nuclear factor-${\kappa}B$ and interferon regulatory factor 3 activation induced by lipopolysaccharide or polyinosinic-polycytidylic acid. ILG inhibited the lipopolysaccharide-induced phosphorylation of interferon regulatory factor 3 as well as interferon-inducible genes such as interferon inducible protein-10, and regulated activation of normal T-cell expressed and secreted (RANTES). These results suggest that ILG can modulate TRIF-dependent signaling pathways of TLRs, leading to decreased inflammatory gene expression.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제36권6호
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• pp.481-489
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• 2010

Introduction: TLR-5, a member of the toll-like receptor (TLR) family, is a element of the type I transmembrane receptors, which are characterized by an intracellular signaling domain homolog to the interleukin-1 receptor. These receptors recognize microbial components, particularly bacterial flagellin. All-trans retinoic acid (atRA, tretinoin), a natural metabolite of vitamin A, acts as a growth and differentiation factor in many tissues, and is also needed for immune functions. In this study, THP-1 human macrophage-monocytes were used to examine the mechanisms by which atRA regulated the expression of TLR-5. Because the molecular mechanism underlying this regulation at the transcriptional level is also unclear, this study examined which putative transcription factors are responsible for TLR-5 expression by atRA in immune cells. Materials and Methods: This study examined whether atRA induces the expression of TLR-5 in THP-1 cells using reverse transcription-polymerase chain reaction (RT-PCR), and which transcription factors are involved in regulating the TLR-5 promoter in RAW264.7 cells using a reporter assay system. Western blot analysis was used to determine which signal pathway is involved in the expression of TLR-5 in atRA-treated THP-1 cells. Results: atRA at a concentration of 10 nM greatly induced the expression of TLR-5 in THP-1 cells. Human TLR-5 promoter contains three Sp-1/GC binding sites around -50 bp and two NF-kB binding sites at -380 bp and -160 bp from the transcriptional start site of the TLR-5 gene. Sp-1/GC is primarily responsible for the constitutive TLR-5 expression, and may also contribute to NF-kB at -160 bp to induce TLR-5 after atRA stimulation in THP-1 cells. The role of NF-kB in TLR-5 expression was further confirmed by inhibitor pyrrolidine dithiocarbamate (PDTC) experiments, which greatly reduced the TLR-5 transcription by 70-80%. Conclusion: atRA induces the expression of the human TLR-5 gene and NF-kB is a critical transcription factor for the atRA-induced expression of TLR-5. Accordingly, it is conceivable that retinoids are required for adequate innate and adaptive immune responses to agents of infectious diseases. atRA and various synthetic retinoids have been used therapeutically in human diseases, such as leukemia and other cancers due to the antiproliferative and apoptosis inducing effects of retinoids. Therefore, understanding the molecular regulatory mechanism of TLR-5 may assist in the design of alternative strategies for the treatment of infectious diseases, leukemia and cancers.

• 한국재료학회:학술대회논문집
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• 한국재료학회 2010년도 춘계학술발표대회
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• pp.41.1-41.1
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• 2010

생체재료란 손상된 생체조직의 기능을 가능한 한 정상에 가까운 상태로 회복시킬 때 이용되는 재료이다. 생체재료는 보건 의료 분야 중 치료, 재활 및 예방의 수단으로 생체에 적용되기 때문에 인간 수명연장 및 사고, 질병의 증가 등에 따라 그 중요성이 점점 증가되고 있는 추세이다. 그럼에도 불구하고, 생체재료 이식에 따른 면역기전에 대해서는 명확하게 밝혀지지 않고 있다. 따라서 본 연구에서는 미생물의 특이 구조를 인식하여 염증반응을 유도하는 TLR (Toll like receptor)을 통한 면역학적 기전을 생체재료 이식시 나타나는 면역반응에 적용하고자 macrophage cell line인 RAW264.7을 이용하여 확인하였다. 생체재료(칼슘-인산계 뼈이식제, PCL microphere)의 특성은 electron microscope(SEM, TEM)을 통하여 관찰하였고 RAW264.7에 의한 세포 독성실험 (MTT와 LDH assay) 결과 세포독성은 없는 것으로 확인되었다. 생체재료가 TLR pathway에 미치는 영향을 Western blotting과 RT-PCR을 이용하여 확인하였으며, 면역반응 시 유도되는 cytokine을 RT-PCR과 ELISA를 이용하여 확인하였다.

• Animal Bioscience
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• 제34권4호
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• pp.489-498
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• 2021

Objective: Milk production is one of the most desirable traits in livestock. Recently, the toll-like receptor (TLR) has been identified as a candidate gene for milk traits in cows. So far, there is no information concerning the contribution of this gene in milk traits in sheep. This study was designed to investigate the TLR 4 gene polymorphisms in Barki ewes in Egypt and then correlate that with milk traits in order to identify potential single nucleotide polymorphisms (SNPs) for these traits in sheep. Methods: A part of the ovine TLR 4 gene was amplified in Barki ewes, to identify the SNPs. Consequently; Barki ewes were genotyped using polymerase chain reaction-single strand conformation polymorphism protocol. These genotypes were correlated with milk traits, which were the daily milk yield (DMY), protein percentage (PP), fat percentage (FP), lactose percentage, and total solid percentage (TSP). Results: Age and parity of the ewe had a significant effect (p<0.05 or p<0.01) on DMY, FP, and TSP. The direct sequencing identified a missense mutation located in the coding sequence of the gene (rs592076818; c.1710C>A) and was predicted to change the amino acid sequence of the resulted protein (p.Asn570Lys). The association analyses suggested a significant effect (p<0.05) of the TLR genotype on the FP and PP, while the DMY tended to be influenced as well (p = 0.07). Interestingly, the presence of the G allele tended to increase the DMY (+40.5 g/d) and significantly (p<0.05 or p<0.01) decreased the FP (-1.11%), PP (-1.21%), and TSP (-7.98%). Conclusion: The results of this study suggested the toll-like receptor 4 (TLR4) as a candidate gene to improve milk traits in sheep worldwide, which will enhance the ability to understand the genetic architecture of genes underlying SNPs that affect such traits.

• Molecular & Cellular Toxicology
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• 제5권3호
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• pp.187-192
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• 2009

Toll-like receptors (TLRs) play an important role in host defense by sensing invading microbial pathogens. The stimulation of TLRs by microbial components triggers the activation of the myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter inducing interferon-$\beta$ (TRIF)-dependent downstream signaling pathways. TLR/MyD88 signaling pathway induces the activation of nuclear factor-kappa B (NF-${\kappa}B$) and the expression of inflammatory cytokine genes, including tumor necrosis factor-alpha, interleukin (IL)-6, IL-12, and IL-$1{\beta}$. On the other hand, TLR/TRIF signaling pathway induces the delayed-activation of NF-${\kappa}B$ and interferon regulatory factor 3 (IRF3), and the expression of type I interferons (IFNs) and IFN-inducible genes. The divalent heavy metal cadmium (Cd) is clearly toxic to most mammalian organ systems, especially the immune system. Yet, the underlying toxic mechanism(s) remain unclear. Cd inhibits the MyD88-dependent pathway by ceasing the activity of inhibitor-${\kappa}B$ kinase. However, it is not known whether Cd inhibits the TRIF-dependent pathway. Presently, Cd inhibited NF-${\kappa}B$ and IRF3 activation induced by lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid. Cd inhibited LPS-induced IRF3 phosphorylation and IFN-inducible genes such as interferon inducible protein-10 and regulated on activation normal T-cell expressed and secreted (RANTES). These results suggest that Cd can modulate TRIF-dependent signaling pathways of TLRs.

• 한국자원식물학회지
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• 제25권5호
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• pp.561-567
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• 2012

본 연구는 EA에 의한 대식세포의 활성화를 매개로 한 항암효과와 항암작용과 관련된 대식세포의 면역조절효과를 확인하였다. 연구 결과 EA에 의해 RAW264.7세포 및 peritoneal macrophage 모두에서 항암효과가 증가하였으며, 증가된 대식세포의 항암효과는 TLR4 signaling을 blocking하는 CLI-095을 함께 처리하였을 때 일부 감소되었다. 이는 EA에 의한 항암 효과가 부분적으로 TLR4를 경유하는 기전으로 나타나는 것을 의미한다. 또한, EA에 대한 대식세포의 NO 분비조절효과를 측정하였으며, EA는 대식세포의 NO 생성을 증가시켰으나, 인위적으로 염증을 유발시켜 NO를 과도하게 분비한 상태에서는 NO 분비를 오히려 억제시키는 결과를 나타내었다. 이와 같이 EA에 의한 NO조절에 대한 이중 효과는 인체에 면역증강과 항염증 효과라는 긍정적인 효과를 나타내는 방향으로 조절하고 있으므로 EA를 이용한 항암요법의 보조제 및 면역보조제로써의 활용에 유익할 것으로 사료된다. 향후 EA에 대한 항암 작용 및 NO 조절에서 세포내 신호전달 작용기전에 대한 심도 있는 연구가 진행되어야 할 것으로 보인다.

• 생명과학회지
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• 제33권6호
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• pp.463-470
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• 2023

Desmarestia tabacoides Okamura는 전 세계적으로 널리 분포하는 갈조류 중 하나이다. 몇몇 산말류의 항종양, 멜라닌 생성 억제 및 광보호 활성에 대한 연구는 있었으나 D. tabacoides Okamura의 항염증 기전에 대해서는 보고되지 않아 본 연구에서는 LPS (lipopolysaccharide)로 자극된 RAW 264.7 세포에서 D. tabacoides Okamura 에탄올 추출물(DTEE)의 항염증 기전을 inducible nitric oxide synthase (iNOS)와 cyclooxygenase (COX)-2의 발현 및 이들의 상위신호전달물질인 nuclear factor (NF)-κB, mitogen-activated protein kinase (MAPK) 그리고 phosphoinositide-3-kinase (PI3K)/Akt의 인산화 조절 정도를 통해 분석하였다. DTEE의 처리는 세포 독성 없이 LPS로 유도된 NO와 prostaglandin (PG) E₂의 생성과 이들의 생성 효소인 iNOS 및 COX-2의 발현을 유의하게 억제하였다. 그리고 LPS에 의해 활성화된 NF-κB 및 상위 신호 전달 물질인 extracellular signal-regulated kinase (ERK), c-Jun NH₂-terminal kinase (JNK) 및 p38은 DTEE 처리에 의해 유의적으로 억제되었다. DTEE의 처리는 RAW 264.7 세포에서 LPS에 의해 활성화되는 adaptor molecule인 Toll-like receptor (TLR) 4 및 myeloid differentiation primary response (MyD) 88 또한 유의적으로 억제하였다. 이 결과를 통해 DTEE는 LPS에 의해 유도된 TLR4와 NF-κB 및 MAPK의 활성을 억제함으로써 염증 매개인자의 발현을 조절하였고, 이는 DTEE가 염증을 완화할 수 있는 기능성 식품의 소재로써 유용하게 사용될 수 있음을 시사한다.

• 한국축산식품학회지
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• 제43권5호
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• pp.938-947
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• 2023

In the present study, we aimed to examine the inhibition of genomic DNA from Lactiplantibacillus plantarum (LpDNA) on Porphyromonas gingivalis lipopolysaccharide (PgLPS)-induced inflammatory responses in RAW264.7 cells. Pretreatment with LpDNA for 15 h significantly inhibited PgLPS-induced mRNA expression and protein secretion of interleukin (IL)-1β, IL-6, and monocyte chemoattractant protein-1. LpDNA pretreatment also reduced the mRNA expression of Toll-like receptor (TLR)2 and TLR4. Furthermore, LpDNA inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs) and the activation of nuclear factor-κB (NF-κB) induced by PgLPS. Taken together, these findings demonstrate that LpDNA attenuates PgLPS-induced inflammatory responses by regulating MAPKs and NF-κB signaling pathways through the suppression of TLR2 and TLR4 expression.

• The Korean Journal of Physiology and Pharmacology
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• 제22권2호
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• pp.145-153
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• 2018

The subgranular zone (SGZ) of hippocampal dentate gyrus (HDG) is a primary site of adult neurogenesis. Toll-like receptors (TLRs), are involved in neural system development of Drosophila and innate immune response of mammals. TLR2 is expressed abundantly in neurogenic niches such as adult mammalian hippocampus. It regulates adult hippocampal neurogenesis. However, the role of TLR2 in adult neurogenesis is not well studied in global or focal cerebral ischemia. Therefore, this study aimed to investigate the role of TLR2 in adult neurogenesis after photochemically induced cerebral ischemia. At 7 days after photothrombotic ischemic injury, the number of bromodeoxyuridine (BrdU)-positive cells was increased in both TLR2 knock-out (KO) mice and wild-type (WT) mice. However, the increment rate of BrdU-positive cells was lower in TLR2 KO mice compared to that in WT mice. The number of doublecortin (DCX) and neuronal nuclei (NeuN)-positive cells in HDG was decreased after photothrombotic ischemia in TLR2 KO mice compared to that in WT mice. The survival rate of cells in HDG was decreased in TLR2 KO mice compared to that in WT mice. In contrast, the number of cleaved-caspase 3 (apoptotic marker) and the number of GFAP (glia marker)/BrdU double-positive cells in TLR2 KO mice were higher than that in WT mice. These results suggest that TLR2 can promote adult neurogenesis from neural stem cell of hippocampal dentate gyrus through increasing proliferation, differentiation, and survival from neural stem cells after ischemic injury of the brain.