• Molecules and Cells
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• 제43권3호
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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.

• Journal of Animal Science and Technology
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• 제65권1호
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• pp.183-196
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• 2023

Interferon-alpha inducible protein 6 (IFI6) is an interferon-stimulated gene (ISG), belonging to the FAM14 family of proteins and is localized in the mitochondrial membrane, where it plays a role in apoptosis. Transcriptional regulation of this gene is poorly understood in the context of inflammation by intracellular nucleic acid-sensing receptors and pathological conditions caused by viral infection. In this study, chicken IFI6 (chIFI6) was identified and studied for its molecular features and transcriptional regulation in chicken cells and tissues, i.e., lungs, spleens, and tracheas from highly pathogenic avian influenza virus (HPAIV)-infected chickens. The chIFI6-coding sequences contained 1638 nucleotides encoding 107 amino acids in three exons, whereas the duck IFI6-coding sequences contained 495 nucleotides encoding 107 amino acids. IFI6 proteins from chickens, ducks, and quail contain an IF6/IF27-like superfamily domain. Expression of chIFI6 was higher in HPAIV-infected White Leghorn chicken lungs, spleens, and tracheas than in mock-infected controls. TLR3 signals regulate the transcription of chIFI6 in chicken DF-1 cells via the NF-κB and JNK signaling pathways, indicating that multiple signaling pathways differentially contribute to the transcription of chIFI6. Further research is needed to unravel the molecular mechanisms underlying IFI6 transcription, as well as the involvement of chIFI6 in the pathogenesis of HPAIV in chickens.

• International Journal of Oral Biology
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• 제31권3호
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• pp.87-92
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• 2006

Schwann cells play an important role in peripheral nerve regeneration. Upon nerve injury, Schwann cells are activated and produce various proinflammatory mediators including IL-6, LIF and MCP-1, which result in the recruitment of macrophages and phagocytosis of myelin debris. However, it is unclear how the nerve injury induces Schwann cell activation. Recently, it was reported that necrotic cells induce immune cell activation via toll-like receptors (TLRs). This suggests that the TLRs expressed on Schwann cells may recognize nerve damage by binding to the endogenous ligands secreted by the damaged nerve, thereby inducing Schwann cell activation. To explore the possibility, we stimulated iSC, a rat Schwann cell line, with damaged neuronal cell extracts (DNCE). The stimulation of iSC with DNCE induced the expression of various inflammatory mediators including IL-6, LIF, MCP-1 and iNOS. Studies on the signaling pathway indicate that $NF-{\kappa}B$, p38 and JNK activation are required for the DNCE-induced inflammatory gene expression. Furthermore, treatment of either anti-TLR3 neutralizing antibody or ribonuclease inhibited the DNCE-induced proinflammatory gene expression in iSC. In summary, these results suggest that damaged neuronal cells induce inflammatory Schwann cell activation via TLR3, which might be involved in the Wallerian degeneration after a peripheral nerve injury.

• Clinical and Experimental Reproductive Medicine
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• 제51권3호
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• pp.236-246
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• 2024

Objective: Nicotinamide mononucleotide (NMN) is extensively utilized as an anti-aging agent and possesses anti-inflammatory properties. Lipopolysaccharide (LPS) activates Toll-like receptor 4, a process modulated by intracellular signaling pathways such as the Wnt/β-catenin pathway. This study investigated the impact of NMN on osteogenesis in the presence of LPS. Methods: To elucidate the role of NMN in osteogenesis in the context of Gram-negative bacterial infection after LPS treatment, we cultured a mouse pre-osteoblast cell line (MC3T3-E1) and subsequently incubated it with NMN and/or LPS. We then evaluated osteogenic activity by measuring alkaline phosphatase activity, assessing gene expression and protein levels, and performing Alizarin Red S staining and immunocytochemistry. Results: MC3T3-E1 cells underwent successful differentiation into osteoblasts following treatment with osteogenic induction medium. LPS diminished features related to osteogenic differentiation, which were subsequently partially reversed by treatment with NMN. The restorative effects of NMN on LPS-exposed MC3T3-E1 cells were further substantiated by elucidating the role of Wnt/β-catenin signaling, as confirmed through immunocytochemistry. Conclusion: This study showed that infection with Gram-negative bacteria disrupted the osteogenic differentiation of MC3T3-E1 cells. This adverse effect was partially reversed by administering a high-dose of NMN. Drawing on these results, we propose that NMN could serve as a viable therapeutic strategy to preserve bone homeostasis in elderly and immunocompromised patients.

• Molecules and Cells
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• 제45권4호
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• pp.257-272
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• 2022

In addition to inducing apoptosis, caspase inhibition contributes to necroptosis and/or autophagy depending on the cell type and cellular context. In macrophages, necroptosis can be induced by co-treatment with Toll-like receptor (TLR) ligands (lipopolysaccharide [LPS] for TLR4 and polyinosinic-polycytidylic acid [poly I:C] for TLR3) and a cell-permeable pan-caspase inhibitor zVAD. Here, we elucidated the signaling pathways and molecular mechanisms of cell death. We showed that LPS/zVAD- and poly I:C/zVAD-induced cell death in bone marrow-derived macrophages (BMDMs) was inhibited by receptor-interacting protein kinase 1 (RIP1) inhibitor necrostatin-1 and autophagy inhibitor 3-methyladenine. Electron microscopic images displayed autophagosome/autolysosomes, and immunoblotting data revealed increased LC3II expression. Although zVAD did not affect LPS- or poly I:C-induced activation of IKK, JNK, and p38, it enhanced IRF3 and STAT1 activation as well as type I interferon (IFN) expression. In addition, zVAD inhibited ERK and Akt phosphorylation induced by LPS and poly I:C. Of note, zVAD-induced enhancement of the IRF3/IFN/STAT1 axis was abolished by necrostatin-1, while zVAD-induced inhibition of ERK and Akt was not. Our data further support the involvement of autocrine IFNs action in reactive oxygen species (ROS)-dependent necroptosis, LPS/zVAD-elicited ROS production was inhibited by necrostatin-1, neutralizing antibody of IFN receptor (IFNR) and JAK inhibitor AZD1480. Accordingly, both cell death and ROS production induced by TLR ligands plus zVAD were abrogated in STAT1 knockout macrophages. We conclude that enhanced TRIF-RIP1-dependent autocrine action of IFNβ, rather than inhibition of ERK or Akt, is involved in TLRs/zVAD-induced autophagic and necroptotic cell death via the JAK/STAT1/ROS pathway.

• Biomolecules & Therapeutics
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• 제20권5호
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• pp.433-445
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• 2012

During the course of evolution, animals encountered the harmful effects of fungi, which are strong pathogens. Therefore, they have developed powerful mechanisms to protect themselves against these fungal invaders. ${\beta}$-Glucans are glucose polymers of a linear ${\beta}$(1,3)-glucan backbone with ${\beta}$(1,6)-linked side chains. The immunostimulatory and antitumor activities of ${\beta}$-glucans have been reported; however, their mechanisms have only begun to be elucidated. Fungal and particulate ${\beta}$-glucans, despite their large size, can be taken up by the M cells of Peyer's patches, and interact with macrophages or dendritic cells (DCs) and activate systemic immune responses to overcome the fungal infection. The sampled ${\beta}$-glucans function as pathogen-associated molecular patterns (PAMPs) and are recognized by pattern recognition receptors (PRRs) on innate immune cells. Dectin-1 receptor systems have been incorporated as the PRRs of ${\beta}$-glucans in the innate immune cells of higher animal systems, which function on the front line against fungal infection, and have been exploited in cancer treatments to enhance systemic immune function. Dectin-1 on macrophages and DCs performs dual functions: internalization of ${\beta}$-glucan-containing particles and transmittance of its signals into the nucleus. This review will depict in detail how the physicochemical nature of ${\beta}$-glucan contributes to its immunostimulating effect in hosts and the potential uses of ${\beta}$-glucan by elucidating the dectin-1 signal transduction pathway. The elucidation of ${\beta}$-glucan and its signaling pathway will undoubtedly open a new research area on its potential therapeutic applications, including as immunostimulants for antifungal and anti-cancer regimens.

• Asian-Australasian Journal of Animal Sciences
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• 제31권8호
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• pp.1366-1372
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• 2018

Objective: A disintegrin and metallopeptidase with thrombospondin motifs type 8 (ADAMTS8) is crucial for diverse physiological processes, such as inflammation, tissue morphogenesis, and tumorigenesis. The chicken ADAMTS8 (chADAMTS8) gene was differentially expressed in the kidney following exposure to different calcium concentrations, suggesting a pathological role of this protein in metabolic diseases. We aimed to examine the molecular characteristics of chADAMTS8 and analyze the gene-expression differences in response to toll-like receptor 3 (TLR3) stimulation. Methods: The ADAMTS8 mRNA and amino acid sequences of various species (chicken, duck, cow, mouse, rat, human, chimpanzee, pig, and horse) were retrieved from the Ensembl database and subjected to bioinformatics analyses. Reverse-transcription polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR) experiments were performed with various chicken tissues and the chicken fibroblast DF-1 cell line, which was stimulated with polyinosinic-polycytidylic acid (poly[I:C]; a TLR3 ligand). Results: The chADAMTS8 gene was predicted to contain three thrombospondin type 1 (TSP1) domains, whose amino acid sequences shared homology among the different species, whereas sequences outside the TSP1 domains (especially the amino-terminal region) were very dif­ferent. Phylogenetic analysis revealed that chADAMTS8 is evolutionarily clustered in the same clade with that of the duck. chADAMTS8 mRNA was broadly expressed in chicken tissues, and the expression was significantly up-regulated in the DF-1 cells in response to poly(I:C) stimulation (p<0.05). These results showed that chADAMTS8 may be a target gene for TLR3 signaling. Conclusion: In this report, the genetic information of chADAMTS8 gene, its expression in chicken tissues, and chicken DF-1 cells under the stimulation of TLR3 were shown. The result suggests that chADAMTS8 expression may be induced by viral infection and correlated with TLR3-mediated signaling pathway. Further study of the function of chADAMTS8 during TLR3-dependent inflammation (which represents RNA viral infection) is needed and it will also be important to examine the molecular mechanisms during different regulation, depending on innate immune receptor activation.

• Asian Pacific Journal of Cancer Prevention
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• 제15권15호
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• pp.6145-6150
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• 2014

Toll-like receptors (TLRs) are expressed in immune and tumor cells and recognize pathogen-associated molecular patterns. Cervical cancer (CC) is directly linked to a persistent infection with high risk human papillomaviruses (HR-HPVs) and could be associated with alteration of TLRs expression. TLR9 plays a key role in the recognition of DNA viruses and better understanding of this signaling pathway in CC could lead to the development of novel immunotherapeutic approaches. The present study was undertaken to determine the level of TLR9 expression in cervical neoplasias from Tunisian women with 53 formalin-fixed and paraffin-embedded specimens, including 22 samples of invasive cervical carcinoma (ICC), 18 of cervical intraepithelial neoplasia (CIN), 7 of condyloma and 6 normal cervical tissues as control cases. Quantification of TLR9 expression was based on scoring four degrees of extent and intensity of immunostaining in squamous epithelial cells. TLR9 expression gradually increased from CIN1 (80% weak intensity) to CIN2 (83.3% moderate), CIN3 (57.1% strong) and ICC (100% very strong). It was absent in normal cervical tissue and weak in 71.4% of condyloma. The mean scores of TLR9 expression were compared using the Kruskall-Wallis test and there was a statistical significance between normal tissue and condyloma as well as between condyloma, CINs and ICC. These results suggest that TLR9 may play a role in progression of cervical neoplasia in Tunisian patients and could represent a useful biomarker for malignant transformation of cervical squamous cells.

• IMMUNE NETWORK
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• 제11권3호
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• pp.155-162
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• 2011

Background: Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA) and induces inflammation. In this study we attempted to ascertain if there are endogenous host molecules controlling the production of cytokines and chemokines. Two candidates, ribosomal protein L19 and L22, were analyzed to determine if they influence cytokine production followed by TLR3 activation. In this study we report that L19 acts upon production of IP-10 or IL-8 differently in glioblastoma cells. Methods: L19 or L22 was transfected into HEK293-TLR3, A549 or A172 cells. After treatment with several inhibitors of NF-${\kappa}B$, PI3K, p38 or ERK, production of IL-8 or IP-10 was measured by ELISA. siRNA was introduced to suppress expression of L19. After Vesicular stomatitis virus infection, viral multiplication was measured by western blot. Results: L19 increased ERK activation to produce IL-8. In A172 cells, in which TLR3 is expressed at endosomes, L19 inhibited interferon regulatory factor 3 (IRF3) activation and IP-10 production to facilitate viral multiplication, whereas L19 inhibited viral multiplication in A549 cells bearing TLR3 on their cell membrane. Conclusion: Our results suggest that L19 regulates TLR3 signaling, which is cell type specific and may be involved in pathogenesis of autoimmune diseases and chronic inflammatory diseases.

• 한국가금학회지
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• 제46권1호
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• pp.31-37
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• 2019

닭의 clathrin-associated adaptor protein $3-{\delta}$ subunit 2(AP3S2)는 clathrin-coated vesicle를 가진 표적 세포막으로 암 배양 단백질 수송에 관여한다. AP3S2는 C형 간염 바이러스 감염으로 간 섬유화를 매개하고, 2형 당뇨병과 관련이 있는 것으로 알려져 있다. 또한, AP3S2는 clathrin-dependent endocytosis를 통해 숙주 세포로의 바이러스 유입에 관련된 역할을 하는 것으로 알려져 있다. 본 연구는 기존 연구에서 닭 신장조직에서 차별 발현 유전자로 발굴된 닭 AP3S2 유전자의 분자유전학적 특성을 구명하고, 닭의 조직에서의 유전자 발현 양상을 조사하며, 톨-유사수용체 3 (Toll-like receptor 3; TLR3) 자극에 의한 전사 조절을 연구하였다. 닭 AP3S2 유전자가 코딩하는 단백질의 구조는 다른 종과 매우 보존적이고 진화적으로 제브라 피쉬와 가장 가깝고, 포유류와 가장 먼 것으로 추정되었다. 닭의 다양한 조직에서 닭 AP3S2 유전자의 전사 수준을 조사한 결과, 폐에서 가장 높게 발현되었으며, 그 다음은 비장 순이었다. 닭의 배아 섬유아세포 주인 DF-1세포에서 조사한 결과, AP3S2 유전자의 발현은 TLR3 신호자극에 의해 감소하였다. 전사조절인자인 $NF{\kappa}B$나 AP-1의 억제제를 이용하여 조사한 결과, $NF{\kappa}B$나 AP-1의 억제에 의해 유전자 발현이 영향을 받지 않았다. 이 결과는 DF-1 세포에서 닭 AP3S2 유전자의 발현은 적어도 이 두 전사조절인자와는 독립적인 경로에 의해 조절됨을 시사한다. 본 연구의 결과는 닭 AP3S2가 바이러스 감염에 역할을 하고, TLR3 신호에 관여함을 제시한다. 추가연구를 통해 닭 AP3S2의 전사 조절과 바이러스 침입 메커니즘을 구명할 필요가 있다고 사료된다.