• Korean Journal of Poultry Science
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• v.45 no.3
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• pp.209-217
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• 2018

The innate immune recognition is based on the detection of microbial products. Toll-like receptors (TLRs) located on the cell surface and the endosome senses microbial components and nucleic acids, respectively. Chicken TLRs mediate immune responses by sensing ligands from pathogens, have been studied as immune adjuvants to increase the efficacy of vaccines. Single nucleotide polymorphisms (SNPs) of TLR3 and TLR4 genes in chicken were associated with resistance and susceptibility to viral infection. In this study, SNPs of chTLR3 and chTLR4 genes were retrieved from public database and annotated with chicken reference genome. Three-dimensional models of the chTLR3 and chTLR4 proteins were built using a Swiss modeler. We identified 35 and 13 nsSNPs in chTLR3 and chTLR4 genes respectively. Sorting Intolerant from Tolerant (SIFT) and Polymorphism Phenotyping v2 (Polyphen-2) analyses, suggested that, out of 35 and 13 nsSNPs, 4 and 2 SNPs were identified to be deleterious in chTLR3 and chTLR4 gene respectively. In chTLR3, 1 deleterious SNP was located in ectodomain and 3 were located in the Toll / IL-1 receptor (TIR) domain. Further structural model of chTLR3-TIR domain suggested that 1 deleterious SNP be present in the B-B loop region, which is important for TIR-TIR domain interactions in the downstream signaling. In chTLR4, the deleterious SNPs were located both in the ectodomain and TIR domain. SNPs predicted for chTLR3 and chTLR4 in this study, might be related to resistance or susceptible to viral infection in chickens. Results from this study will be useful to develop the effective measures in chicken against infectious diseases.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.5
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• pp.2329-2334
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• 2014

Curcumin can inhibit proliferation of liver cancer cells by inducing apoptosis, but the specific signaling pathways involved are not completely clear. Here, we report that curcumin inhibited proliferation of MHCC97H liver cancer cells by induction of apoptosis in a concentration dependent manner via stimulating intracellular reactive oxygen species (ROS) generation. Also, we showed that increased intracellular ROS formation activated the TLR-4/MyD-88 signaling pathway, resulting in activation of caspase-8 and caspase-3, which eventually led to apoptosis in MHCC97H cells. These results showed that as an prooxidant, curcumin exerts anti-cancer effects by inducing apoptosis via the TLR-4/MyD-88 signaling pathway.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.7
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• pp.1019-1025
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• 2011

Toll-like receptors (TLRs) play an important role in the recognition of invading pathogens and the modulation of innate immune responses in mammals. The TLR4 and TLR7 are well known to recognize the bacterial lipopolysaccharide (LPS) and single stranded (ssRNA) ligands, respectively and play important role in host defense against Gram-negative bacteria and ssRNA viruses. In the present study, coding exon fragments of these two TLRs were identified, cloned, sequenced and analyzed in terms of insertion-deletion polymorphism, within bovine TLRs 4 and 7, thereby facilitating future TLR signaling and association studies relevant to bovine innate immunity. Comparative sequence analysis of TLR 4 exons revealed that this gene is more variable, particularly the coding frame (E3P1), while other parts showed percent identity of 95.7% to 100% at nucleotide and amino acid level, respectivley with other Bos indicus and Bos taurus breeds from different parts of the world. In comparison to TLR4, sequence analysis of TLR7 showed more conservation among different B. indicus and B. taurus breeds, except single point mutation at 324 nucleotide position (AAA to AAM) altering a single amino acid at 108 position (K to X). Percent identity of TLR7 sequences (all 3 exons) was between 99.2% to 100% at nucleotide and amino acid level, when compared with available sequence database of B. indicus and B. taurus. Simple Modular Architecture Research Tool (SMART) analysis showed variations in the exon fragments located in the Leucine Rich Repeat (LRR) region, which is responsible for binding with the microbial associated molecular patterns and further, downstream signaling to initiate anti-microbial response. Considering importance of TLR polymorphism in terms of innate immunity, further research is warranted.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.12
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• pp.1942-1949
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• 2019

Objective: Leukocyte cell-derived chemotaxin 2 (LECT2) is associated with several physiological processes including inflammation, tumorigenesis, and natural killer T cell generation. Chicken LECT2 (chLECT2) gene was originally identified as one of the differentially expressed genes in chicken kidney tissue, where the chickens were fed with different calcium doses. In this study, the molecular characteristics and gene expression of chLECT2 were analyzed under the stimulation of toll-like receptor 3 (TLR3) ligand to understand the involvement of chLECT2 expression in chicken metabolic disorders. Methods: Amino acid sequence of LECT2 proteins from various species including fowl, fish, and mammal were retrieved from the Ensembl database and subjected to Insilco analyses. In addition, the time- and dose-dependent expression of chLECT2 was examined in DF-1 cells which were stimulated with polyinosinic:polycytidylic acid (poly [I:C]), a TLR3 ligand. Further, to explore the transcription factors required for the transcription of chLECT2, DF-1 cells were treated with poly (I:C) in the presence or absence of the nuclear factor ${\kappa}B$ ($NF{\kappa}B$) and activated protein 1 (AP-1) inhibitors. Results: The amino acid sequence prediction of chLECT2 protein revealed that along with duck LECT2 (duLECT2), it has unique signal peptide different from other vertebrate orthologs, and only chLECT2 and duLECT2 have an additional 157 and 161 amino acids on their carboxyl terminus, respectively. Phylogenetic analysis suggested that chLECT2 is evolved from a common ancestor along with the actinopterygii hence, more closely related than to the mammals. Our quantitative polymerase chain reaction results showed that, the expression of chLECT2 was up-regulated significantly in DF-1 cells under the stimulation of poly (I:C) (p<0.05). However, in the presence of $NF{\kappa}B$ or AP-1 inhibitors, the expression of chLECT2 is suppressed suggesting that both $NF{\kappa}B$ and AP-1 transcription factors are required for the induction of chLECT2 expression. Conclusion: The present results suggest that chLECT2 gene might be a target gene of TLR3 signaling. For the future, the expression pattern or molecular mechanism of chLECT2 under stimulation of other innate immune receptors shall be studied. The protein function of chLECT2 will be more clearly understood if further investigation about the mechanism of LECT2 in TLR pathways is conducted.

• The Korean Journal of Physiology and Pharmacology
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• v.25 no.3
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• pp.217-225
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• 2021

Neuropathic pain (NP) that contributes to the comorbidity between pain and depression is a clinical dilemma. Neuroinflammatory responses are known to have potentially important roles in the initiation of NP and depressive mood. In this study, we aimed to investigate the effects of paeoniflorin (PF) on NP-induced depression-like behaviors by targeting the hippocampal neuroinflammation through the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling pathway. We used a murine model of NP caused by unilateral sciatic nerve cuffing (Cuff). PF was injected intraperitoneally once a day for a total of 14 days. Pain and depression-like behavior changes were evaluated via behavioral tests. Pathological changes in the hippocampus of mice were observed by H&E staining. The levels of proinflammatory cytokines in the hippocampus were detected using ELISA. Activated microglia were measured by immunohistochemical staining. The TLR4/NF-κB signaling pathway-associated protein expression in the hippocampus was detected by western blotting. We found that the PF could significantly alleviate Cuff-induced hyperalgesia and depressive behaviors, lessen the pathological damage to the hippocampal cell, reduce proinflammatory cytokines levels, and inhibit microglial over-activation. Furthermore, PF downregulated the expression levels of TLR4/NF-κB signaling pathway-related proteins in the hippocampus. These results indicate that PF is an effective drug for improving the comorbidity between NP and depression.

• Journal of Veterinary Science
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• v.24 no.2
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• pp.20.1-20.13
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• 2023

Background: As Actinobacillus pleuropneumonniae (APP) infection causes considerable losses in the pig industry, there is a growing need to develop effective therapeutic interventions that leverage host immune defense mechanisms to combat these pathogens. Objectives: To demonstrate the role of microRNA (miR)-127 in controlling bacterial infection against APP. Moreover, to investigate a signaling pathway in macrophages that controls the production of anti-microbial peptides. Methods: Firstly, we evaluated the effect of miR-127 on APP-infected pigs by cell count/enzyme-linked immunosorbent assay (ELISA). Then the impact of miR-127 on immune cells was detected. The cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 were evaluated by ELISA. The expression of cytokines (anti-microbial peptides [AMPs]) was assessed using quantitative polymerase chain reaction. The expression level of IL-6, TNF-α and p-P65 were analyzed by western blot. The expression of p65 in the immune cells was investigated by immunofluorescence. Results: miR-127 showed a protective effect on APP-infected macrophage. Moreover, the protective effect might depend on its regulation of macrophage bactericidal activity and the generation of IL-22, IL-17 and AMPs by targeting sphingosine-1-phosphate receptor3 (SIPR3), the element involved in the Toll-like receptor (TLR) cascades. Conclusions: Together, we identify that miR-127 is a regulator of S1PR3 and then regulates TLR/nuclear factor-κB signaling in macrophages with anti-bacterial acticity, and it might be a potential target for treating inflammatory diseases caused by APP.

• BMB Reports
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• v.52 no.6
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• pp.373-378
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• 2019

The nucleotide-binding and oligomerization domain (NOD) is an innate pattern recognition receptor that recognizes pathogen- and damage-associated molecular patterns. The 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) is a matrix degradation product found in the synovial fluids of patients with osteoarthritis (OA). We investigated whether NOD2 was involved in 29-kDa FN-f-induced pro-catabolic gene expression in human chondrocytes. The expression of mRNA and protein was measured using quantitative real-time polymerase chain reaction (qrt-PCR) and Western blot analysis. Small interfering RNAs were used for knockdown of NOD2 and toll-like receptor 2 (TLR-2). An immunoprecipitation assay was performed to examine protein interactions. The NOD2 levels in human OA cartilage were much higher than in normal cartilage. NOD1 and NOD2 expression, as well as pro-inflammatory cytokines, including interleukin-1beta (IL-$1{\beta}$) and tumor necrosis factor-alpha (TNF-${\alpha}$), were upregulated by 29-kDa FN-f in human chondrocytes. NOD2 silencing showed that NOD2 was involved in the 29-kDa FN-f-induced expression of TLR-2. Expressions of IL-6, IL-8, matrix metalloproteinase (MMP)-1, -3, and -13 were also suppressed by TLR-2 knockdown. Furthermore, NOD2 and TLR-2 knockdown data demonstrated that both NOD2 and TLR-2 modulated the expressions of their adaptors, receptorinteracting protein 2 (RIP2) and myeloid differentiation 88, in 29-kDa FN-f-treated chondrocytes. 29-kDa FN-f enhanced the interaction of NOD2, RIP2 and transforming growth factor beta-activated kinase 1 (TAK1), an indispensable signaling intermediate in the TLR-2 signaling pathway, and activated nuclear factor-${\kappa}B$ (NF-${\kappa}B$), subsequently leading to increased expressions of pro-inflammatory cytokines and cartilage-degrading enzymes. These results demonstrate that 29-kDa FN-f modulated pro-catabolic responses via cross-regulation of NOD2 and TLR-2 signaling pathways.

• The Korea Journal of Herbology
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• v.38 no.6
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• pp.73-91
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• 2023

Objectives : This study used a network pharmacology approach to elucidate the efficacy and molecular mechanisms of Daehwangmokdanpitang (DHMDPT) on Psoriasis. Methods : Using OASIS databases and PubChem database, compounds of DHMDPT and their target genes were collected. The putative target genes of DHMDPT and known target genes of psoriasis were compared and found the correlation. Then, the network was constructed using Cytoscape 3.10.1. The key target genes were screened by Analyzer network and their functional enrichment analysis was conducted based on the Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathways to predict the mechanisms. Results : The result showed that total 30 compounds and 439 related genes were gathered from DHMDPT. 264 genes were interacted with psoriasis gene set, suggesting that the effects of DHMDPT are closely related to psoriasis. Based on GO enrichment analysis and KEGG pathways, 'Binding', 'Cytokine Activity', 'Receptor Ligand Activity' 'HIF-1 signaling pathway', 'IL-17 signaling pathway', 'Toll-like receptor signaling pathway', and 'TNF signaling pathway' were predicted as functional pathways of 16 key target genes of DHMDPT on psoriasis. Among the target genes, IL6, IL1B, TNF, AKT1 showed high correlation with the results of KEGG pathways. Additionally, Emodin, Acetovanillone, Gallic acid, and Ferulic acid showed a high relevance with key genes and their mechanisms. Conclusion : Through a network pharmacological method, DHMDPT was predicted to have high relevance with psoriasis. This study could be used as a basis for studying therapeutic effects of DHMDPT on psoriasis.

• Journal of Veterinary Science
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• v.24 no.1
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• pp.2.1-2.14
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• 2023

Background: Hypothermia is a crucial environmental factor that elevates the risk of cardiovascular disease, but the underlying effect is unclear. Objectives: This study examined the role of cold stress (CS) in cardiac injury and its underlying mechanisms. Methods: In this study, a chronic CS-induced myocardial injury model was used; mice were subjected to chronic CS (4℃) for three hours per day for three weeks. Results: CS could result in myocardial injury by inducing the levels of heat shock proteins 70 (HSP70), enhancing the generation of creatine phosphokinase-isoenzyme (CKMB) and malondialdehyde (MDA), increasing the contents of tumor necrosis factor-α (TNF-α), high mobility group box 1 (HMGB1) interleukin1b (IL-1β), IL-18, IL-6, and triggering the depletion of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH). Multiple signaling pathways were activated by cold exposure, including pyroptosis-associated NOD-like receptor 3 (NLRP3)-regulated caspase-1-dependent/Gasdermin D (GSDMD), inflammation-related toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)-mediated nuclear factor kappa B (NF-κB), and mitogen-activated protein kinase (MAPK), as well as oxidative stressinvolved thioredoxin-1/thioredoxin-interacting protein (Txnip) signaling pathways, which play a pivotal role in myocardial injury resulting from hypothermia. Conclusions: These findings provide new insights into the increased risk of cardiovascular disease at extremely low temperatures.

• Molecules and Cells
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• v.43 no.3
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• pp.251-263
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• 2020

Flagellin, a major structural protein of the flagellum found in all motile bacteria, activates the TLR5- or NLRC4 inflammasome-dependent signaling pathway to induce innate immune responses. Flagellin can also serve as a specific antigen for the adaptive immune system and stimulate anti-flagellin antibody responses. Failure to recognize commensal-derived flagellin in TLR5-deficient mice leads to the reduction in anti-flagellin IgA antibodies at steady state and causes microbial dysbiosis and mucosal barrier breach by flagellated bacteria to promote chronic intestinal inflammation. Despite the important role of anti-flagellin antibodies in maintaining the intestinal homeostasis, regulatory mechanisms underlying the flagellin-specific antibody responses are not well understood. In this study, we show that flagellin induces interferon-β (IFN-β) production and subsequently activates type I IFN receptor signaling in a TLR5- and MyD88-dependent manner in vitro and in vivo. Internalization of TLR5 from the plasma membrane to the acidic environment of endolysosomes was required for the production of IFN-β, but not for other pro-inflammatory cytokines. In addition, we found that anti-flagellin IgG2c and IgA responses were severely impaired in interferon-alpha receptor 1 (IFNAR1)-deficient mice, suggesting that IFN-β produced by the flagellin stimulation regulates anti-flagellin antibody class switching. Our findings shed a new light on the regulation of flagellin-mediated immune activation and may help find new strategies to promote the intestinal health and develop mucosal vaccines.