• Journal of Animal Science and Technology
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• v.62 no.3
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• pp.385-395
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• 2020

Polyinosinic:polycytidylic acid (poly[I:C]) can stimulate Toll-like receptor 3 (TLR3) signaling pathways. In this study, DF-1 cells were treated with poly(I:C) at various concentrations and time points to examine the comparative expression patterns of innate immune response genes. The viability of DF-1 cells decreased from 77.41% to 38.68% when cells were treated different dose of poly(I:C) from 0.1 ㎍/mL to 100 ㎍/mL for 24 h respectively. The expressions of TLR3, TLR4, TLR7, TLR15, TLR21, IL1B, and IL10 were increased in dose- and time-dependent manners by poly(I:C) treatment. On the contrary, the expression patterns of interferon regulatory factors 7 (IRF7), Jun proto-oncogene, AP-1 transcription factor subunit (JUN), Nuclear Factor Kappa B Subunit 1 (NF-κB1), and IL8L2 were varied; IRF7 and IL8L2 were increasingly expressed whereas the expressions of JUN and NF-κB1 were decreased in a dose-dependent manner after they were early induced. In time-dependent analysis, IRF7 expression was significantly upregulated from 3 h to 24 h, whereas JUN and NF-κB1 expressions settled down from 6 h to 24 h after poly(I:C) treatment although they were induced at early time from 1 h to 3 h. Poly(I:C) treatment rapidly increased the expression of IL8L2 from 3 h to 6 h with a plateau at 6 h and then the expression of IL8L2 was dramatically decreased until 24 h after poly(I:C) treatment although the expression level was still higher than the non-treated control. These results may provide the basis for understanding host response to viral infection and its mimicry system in chickens.

• 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.

• Journal of Microbiology and Biotechnology
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• v.33 no.7
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• pp.934-940
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• 2023

Syneilesis palmata (SP) is a traditional medicinal plant. SP has been reported to have anti-inflammatory, anticancer, and anti-human immunodeficiency virus (HIV) activities. However, there is currently no research available on the immunostimulatory activity of SP. Therefore, in this study, we report that S. palmata leaves (SPL) activate macrophages. Increased secretion of both immunostimulatory mediators and phagocytic activity was observed in SPL-treated RAW264.7 cells. However, this effect was reversed by the inhibition of TLR2/4. In addition, inhibition of p38 decreased the secretion of immunostimulatory mediators induced by SPL, and inhibition of TLR2/4 decreased the phosphorylation of p38 induced by SPL. SPL augmented p62/SQSTM1 and LC3-II expression. The increase in protein levels of p62/SQSTM1 and LC3-II induced by SPL was decreased by the inhibition of TLR2/4. The results obtained from this study suggest that SPL activates macrophages via TLR2/4-dependent p38 activation and induces autophagy in macrophages via TLR2/4 stimulation.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2023.04a
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• pp.44-44
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• 2023

Syneilesis palmata (SP) has been used as a traditional medicinal plant and vegetable. SP was reported to exert pharmacological activities such as anti-inflammation, anti-cancer, and anti-HIV. However, there are no studies on the immunostimulatory activity of SP. Thus, in this study, we report that S. palmata leaves (SPL) induce the activation of macrophages. An increase in both secretions of immunostimulatory mediators and phagocytotic activity was observed in SPL-treated RAW264.7 cells. However, this was reversed by inhibition of TLR2/4. In addition, the p38 inhibition reduced the SPL-mediated secretion of immunostimulatory mediators, and the SPL-mediated p38 activation was blocked by the TLR2/4 inhibition. SPL augmented both p62/SQSTM1 and LC3-II. TLR2/4 inhibition blocked the SPL-mediated increase of p62/SQSTM1 and LC3-II. These findings indicate that SPL may activate macrophages through TLR2/4-dependent p38 activation and activate autophagy through TLR2/4 stimulation.

• The Korean Journal of Physiology and Pharmacology
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• v.18 no.6
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• pp.475-480
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• 2014

We investigated the question of whether cholesterol catabolite can influence expression of inflammatory cytokines via Toll-like receptors (TLR) in monocytic cells. Treatment of THP-1 monocytic cells with 27-hydroxycholesterol (27OHChol) resulted in induction of gene transcription of TLR6 and elevated level of cell surface TLR6. Addition of FSL-1, a TLR6 agonist, to 27OHChol-treated cells resulted in transcription of the $IL-1{\alpha}$ gene and enhanced secretion of the corresponding gene product. However, cholesterol did not affect TLR6 expression, and addition of FSL-1 to cholesterol-treated cells did not induce expression of $IL-1{\alpha}$. Using pharmacological inhibitors, we investigated molecular mechanisms underlying the expression of TLR6 and $IL-1{\alpha}$. Treatment with Akt inhibitor IV or U0126 resulted in significantly attenuated expression of TLR6 and $IL-1{\alpha}$ induced by 27OHChol and 27OHChol plus FSL-1, respectively. In addition, treatment with LY294002, SB202190, or SP600125 resulted in significantly attenuated secretion of $IL-1{\alpha}$. These results indicate that 27OHChol can induce inflammation by augmentation of TLR6-mediated production of $IL-1{\alpha}$ in monocytic cells via multiple signaling pathways.

• Biomedical Science Letters
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• v.27 no.3
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• pp.177-181
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• 2021

Allergy is an immune response that appears in certain people, and reactions such as coughing, shortness of breath, and hives occur. The immune system plays an important role in homeostasis and host defense, and allergies cause hypersensitivity reactions when an imbalance of immunity occurs. Mutations in the TLR genes are associated with autoimmune conditions such as allergies and asthma. It has been reported that a locus in the TLR1-TLR6-TLR10 region may be associated with atopic sensitization or allergy. Therefore, the purpose of this study was to select an allergy patient group and a healthy control group to determine how the genetic mutation of TLR1 affects the onset of disease. This study was conducted in 709 patients and 5,025 control groups out of 10,956 patients with data from KARE and HEXA cohorts. As a result of logistic regression analysis of 6 SNPs selected from the TLR1 gene, only rs117033348 showed a statistically significant correlation (P = 0.002356). The influence of rs117033348 was examined using PolyPhen-2, and a significant result was shown. Therefore, it can be predicted that the G base in rs117033348 will have an influence on the human body. In addition, Geography of Genetic Variants browser was used to confirm the geographical distribution of allele frequencies for the TLR1 gene. Although it was found that there was a large racial difference in the prevalence of TLR1 SNP, it could be confirmed that the polymorphism of rs117033348 conducted in this study was only specific in East Asia when compared with each race.

• Journal of Microbiology and Biotechnology
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• v.32 no.10
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• pp.1344-1354
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• 2022

Laryngeal cancer is one of the highest incidence, most prevalently diagnosed head and neck cancers, making it critically necessary to probe effective targets for laryngeal cancer treatment. Here, real-time quantitative reverse transcription PCR (qRT-PCR) and western blot analysis were used to detect gene expression levels in laryngeal cancer cell lines. Fluorescence in situ hybridization (FISH) and subcellular fractionation assays were used to detect the subcellular location. Functional assays encompassing Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), transwell and wound healing assays were performed to examine the effects of target genes on cell proliferation and migration in laryngeal cancer. The in vivo effects were proved by animal experiments. RNA-binding protein immunoprecipitation (RIP), RNA pulldown and luciferase reporter assays were used to investigate the underlying regulatory mechanisms. The results showed that KIF26B antisense RNA 1 (KIF26B-AS1) propels cell proliferation and migration in laryngeal cancer and regulates the toll-like receptor 4 (TLR4) signaling pathway. KIF26B-AS1 also recruits FUS to stabilize TLR4 mRNA, consequently activating the TLR4 signaling pathway. Furthermore, KIF26B-AS1 plays an oncogenic role in laryngeal cancer via upregulating TLR4 expression as well as the FUS/TLR4 pathway axis, findings which offer novel insight for targeted therapies in the treatment of laryngeal cancer patients.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.18
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• pp.8445-8450
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• 2016

Background: Breast cancer is the most common neoplasm in women and the most frequent cause of death in those between 35 and 55 years of age. All multicellular organisms have an innate immune system, whereas the adaptive or 'acquired' immune system is restricted to vertebrates. This study focused on the effect of conditioned medium isolated from cultured breast cancer cells on NB4 neutrophil-like cells. Materials and Methods: In the current study neutrophil-like NB4 cells were incubated with MCF-7 cell-conditioned medium. After 6 h incubation the intracellular receptor TLR2, was analyzed. Results: The results revealed that MCF-7 cell-conditioned medium elicited expression of TLR2 in NB4 cells. Conclusions: This treatment would result in the production of particular stimulants (i.e. soluble cytokines), eliciting the expression of immune system receptors. Furthermore, the flow cytometry results demonstrated that MCF-7 cell-conditioned medium elicited an effect on TLR2 intracellular receptors.

• Biomolecules & Therapeutics
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• v.24 no.4
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• pp.387-394
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• 2016

Sepsis, a serious clinical problem, is characterized by a systemic inflammatory response to infection and leads to organ failure. Toll-like receptor (TLR) signaling is intimately implicated in hyper-inflammatory responses and tissue injury during sepsis. Histone deacetylase (HDAC) inhibitors have been reported to exhibit anti-inflammatory properties. The aim of this study was to investigate the hepatoprotective mechanisms of trichostatin A (TSA), a HDAC inhibitor, associated with TLR signaling pathway during sepsis. The anti-inflammatory properties of TSA were assayed in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Polymicrobial sepsis was induced in mice by cecal ligation and puncture (CLP), a clinically relevant model of sepsis. The mice were intraperitoneally received TSA (1, 2 or 5 mg/kg) 30 min before CLP. The serum and liver samples were collected 6 and 24-h after CLP. TSA inhibited the increased production of tumor necrosis factor (TNF)-${\alpha}$ and interleukin (IL)-6 in LPS-stimulated RAW264.7 cells. TSA improved sepsis-induced mortality, attenuated liver injury and decreased serum TNF-${\alpha}$ and IL-6 levels. CLP increased the levels of TLR4, TLR2 and myeloid differentiation primary response protein 88 (MyD88) protein expression and association of MyD88 with TLR4 and TLR2, which were attenuated by TSA. CLP increased nuclear translocation of nuclear factor kappa B and decreased cytosolic inhibitor of kappa B ($I{\kappa}B$) protein expression, which were attenuated by TSA. Moreover, CLP decreased acetylation of $I{\kappa}B$ kinase (IKK) and increased association of IKK with $I{\kappa}B$ and TSA attenuated these alterations. Our findings suggest that TSA attenuates liver injury by inhibiting TLR-mediated inflammatory response during sepsis.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.14
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• pp.5867-5872
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• 2014

Background: Cervical cancer is listed as one of high-incidence endemic diseases in Xinjiang. Our study aimed to evaluate the expression of TLR9 in uterine cervical tissues of Uyghur women and examine associations with clinicopathological variables. We further characterized the direct effects of TLR9 upon the selective silencing of human papillomavirus (HPV) E6 and E7 oncoprotein expression in HPV 16-positive human cervical carcinoma cells treated with siRNA in vitro. Materials and Methods: Immunohistochemistry was applied to evaluate TLR9 expression in 97 formalin-fixed paraffin-embedded cervical samples from Uyghur women; 32 diagnosed with cervical squamous cell carcinomas (CSCC), 14 with low-grade cervical intraepithelial neoplasias (CINI), 10 medium-grade (CINII), 24 high-grade (CINIII), and 17 chronic cervicitis. $BLOCK-iT^{TM}$ U6 RNAi Entry Vector $pENTR^{TM}$/U6-E6 and E7 was constructed and transfected the entry clone directly into the mammalian cell line 293FT. Then the HPV 16-positive SiHa human cervical carcinoma cell line was infected with RNAi recombinant lentivirus. RT-PCR and Western blotting were used to determine the expression of TLR9 in both SiHa and HPV 16 E6 and E7 silenced SiHa cells. Results: Immunohistochemical staining showed that TLR9 expression was undetectable (88.2%) or weak (11.8%) in chronic cervicitis tissues. However, variable staining was observed in the basal layer of all normal endocervical glands. TLR9 expression, which was mainly observed as cytoplasmic staining, gradually increased in accordance with the histopathological grade in the following order: chronic cervicitis (2/17, 11.8%)