• Molecules and Cells
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• v.45 no.4
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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.

• Journal of Animal Science and Technology
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• v.64 no.1
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• pp.123-134
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• 2022

Toll-like receptors (TLRs), as a part of innate immunity, plays an important role in detecting pathogenic molecular patterns (PAMPs) which are structural components or product of pathogens and initiate host defense systems or innate immunity. Precise negative feedback regulations of TLR signaling are important in maintaining homeostasis to prevent tissue damage by uncontrolled inflammation during innate immune responses. In this study, we identified and characterized the function of the pancreatic progenitor cell differentiation and proliferation factor (PPDPF) as a negative regulator for TLR signal-mediated inflammation in chicken. Bioinformatics analysis showed that the structure of chicken PPDPF evolutionarily conserved amino acid sequences with domains, i.e., SH3 binding sites and CDC-like kinase 2 (CLK2) binding sites, suggesting that relevant signaling pathways might contribute to suppression of inflammation. Our results showed that stimulation with polyinosinic:polycytidylic acids (Poly [I:C]), a synthetic agonist for TLR3 signaling, increased the mRNA expression of PPDPF in chicken fibroblasts DF-1 but not in chicken macrophage-like cells HD11. In addition, the expression of pro-inflammatory genes stimulated by Poly(I:C) were reduced in DF-1 cells which overexpress PPDPF. Future studies warrant to reveal the molecular mechanisms responsible for the anti-inflammatory capacity of PPDPF in chicken as well as a potential target for controlling viral resistance.

• Journal of Veterinary Science
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• v.24 no.1
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• pp.11.1-11.14
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• 2023

Background: Peripheral blood mononuclear cells (PBMCs) are commonly used to assess in vitro immune responses. However, PBMC isolation is a time-consuming procedure, introduces technical variability, and requires a relatively large volume of blood. By contrast, whole blood assay (WBA) is faster, cheaper, maintains more physiological conditions, and requires less sample volume, laboratory training, and equipment. Objectives: Herein, this study aimed to develop a porcine WBA for in vitro evaluation of immune responses. Methods: Heparinized whole blood (WB) was diluted (non-diluted, 1/2, 1/8, and 1/16) in RPMI-1640 media, followed by phorbol myristate acetate and ionomycin. After 24 h, cells were stained for interferon (IFN)-γ secreting T-cells followed by flow cytometry, and the supernatant was analyzed for tumor necrosis factor (TNF)-α. In addition, diluted WB was stimulated by lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C), reference strain KCTC3557 (RS), field isolate (FI), of heat-killed (HK) Streptococcus suis, and porcine reproductive and respiratory syndrome virus (PRRSV). Results: The frequency of IFN-γ⁺CD3⁺ T-cells and concentration of TNF-α in the supernatant of WB increased with increasing dilution factor and were optimal at 1/8. WB TNF-α and interleukin (IL)-10 cytokine levels increased significantly following stimulation with LPS or poly I:C. Further, FI and RS induced IL-10 production in WB. Additionally, PRRSV strains increased the frequency of IFN-γ⁺ CD4^-CD8⁺ cells, and IFN-γ was non-significantly induced in the supernatant of re-stimulated samples. Conclusions: We propose that the WBA is a rapid, reliable, and simple method to evaluate immune responses and WB should be diluted to trigger immune cells.

• Journal of Veterinary Science
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• v.24 no.3
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• pp.37.1-37.14
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• 2023

Background: Toll-like receptor (TLR) agonists have been used as adjuvants to modulate immune responses in both animals and humans. Objectives: The objective of this study was to evaluate the combined effects of the TLR 4 agonist monophosphoryl lipid A (MPL) and the TLR 3 agonist polyinosinic:polycytidylic acid (Poly I:C) on equine peripheral blood mononuclear cells (PBMCs), monocyte-derived dendritic cells (MoDCs), and bone marrow-derived mesenchymal stromal cells (BM-MSCs). Methods: The PBMCs, MoDCs, and BM-MSCs collected from three mixed breed horses were treated with MPL, Poly I:C, and their combination. The mRNA expression of interferon gamma (IFN-γ), interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-12p40, tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein-1 (MCP-1) was determined using real-time polymerase chain reaction. Results: The combination of MPL and Poly I:C significantly upregulated immunomodulatory responses in equine cells/ without cytotoxicity. The combination induced greater mRNA expression of pro-inflammatory cytokines IFN-γ and IL-6 than MPL or Poly I:C stimulation alone in PBMCs. In addition, the combination induced significantly higher mRNA expression of IL-1β, IL-6, and IL-12p40 in MoDCs, and IL-8, MCP-1, and VEGF in BM-MSCs compared to stimulation with a single TLR agonist. Conclusions: The combination of MPL and Poly I:C can be used as a potential adjuvant candidate for vaccines to aid in preventing infectious diseases in horses.

• Fisheries and Aquatic Sciences
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• v.25 no.11
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• pp.559-571
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• 2022

Galectins, a family of ß-galactoside-binding lectins, have emerged as soluble mediators in infected cells and pattern recognition receptors (PRRs) responsible for evoking and regulating innate immunity. The present study aimed to evaluate the role of galectin-1 in the host immune response of redlip mullet (Liza haematocheilia). We established a cDNA database for redlip mullet, and the cDNA sequence of galectin-1 (LhGal-1) was characterized. In silico analysis was performed, and the spatial and temporal expression patterns in gills and blood in response to lipopolysaccharide polyinosinic:polycytidylic acid, and Lactococcus garvieae were estimated via quantitative real-time PCR. Functional assays were conducted using recombinant protein to investigate carbohydrate binding, bacterial binding, and bacterial agglutination activity. LhGal-1 was composed of 135 amino acids. Conserved motifs (H-NPR, -N- and -W-E-R) within the carbohydrate recognition domain were found in LhGal-1. The tissue distribution revealed that the healthy stomach expressed high levels of LhGal-1. The temporal monitoring of LhGal-1 mRNA expression in the gill and blood showed its significant upregulation in response to immune challenges with different stimulants. rLhGal-1 exhibited binding activity in response to carbohydrates and bacteria. Moreover, the agglutination of rLhGal-1 against Escherichia coli was observed. Collectively, our findings suggest that LhGal-1 may function as a PRR in redlip mullet. Furthermore, LhGal-1 can be considered a significant gene to play a protective role in redlip mullet immune system.

• Journal of Applied Biological Chemistry
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• v.54 no.4
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• pp.279-283
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• 2011

Toll-like receptors (TLRs) play an important role in induction of innate immune responses. The activation of TLRs triggers inflammatory responses that are essential for host defense against invading pathogens. Phenethyl isothiocyanate (PEITC) extracted from cruciferous vegetables has an effect on anti-inflammatory therapy. Dysregulated activation of nuclear factor-${\kappa}$B (NF-${\kappa}$B), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) has been shown to play important roles in the development of certain inflammatory disease. To evaluate the therapeutic potential of PEITC, NF-${\kappa}$B activation and COX-2 and iNOS expression induced by lipopolysaccharide (LPS, TLR4 agonist), polyinosinic-polycytidylic acid (Poly[I:C], TLR3 agonist), 2 kDa macrophageactivating lipopeptide (MALP-2, TLR2 and TLR6 agonist) or oligodeoxynucleotide 1668 (ODN1668, TLR9 agonist) were examined. PEITC inhibits the activation of NF-${\kappa}$B induced by LPS or Poly[I:C] but not by MALP-2 or ODN1668. PEITC also suppressed the iNOS expression induced by LPS or Poly[I:C]. However, PEITC did not suppress COX-2 expression induced by LPS, Poly[I:C], MALP-2, or ODN1668. These results suggest that PEITC has the specific mechanism for antiinflammatory responses.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.8
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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-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.

• Korean Journal of Poultry Science
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• v.50 no.4
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• pp.283-291
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• 2023

Long-chain fatty acids (LCFAs) are vital in cellular compartments, primarily regulating lipid metabolism. Fatty Acid-Binding Proteins (FABPs) facilitate LCFA transport, lipid synthesis, storage, and act as signaling molecules influencing various pathways, including inflammation. FABP4, in particular, is linked to vascular and cardio-related diseases, and it plays a role in macrophage-mediated inflammatory responses. Previous studies have identified FABP4 as not only a representative biomarker for lipogenesis but also as having correlations with immune responses. This study aims to investigate the regulation of the chicken FABP4 (chFABP4) gene by toll-like receptor 3 (TLR3) activation and determine the signaling pathways that are involved in chFABP4 transcriptional regulation. We analyzed the transcriptional regulation of chFABP4 in TLR3-stimulated DF-1 cells. The results showed that chFABP4 was up-regulated upon stimulation with polyinosinic-polycytidylic acid (PIC), a TLR3 ligand. Notably, chFABP4 transcription was independently regulated in the NF-κB signaling pathway. It was up-regulated in p38 inhibition, demonstrating that the p38 signaling pathway might suppress the transcription of chFABP4 within TLR3-activated DF-1 cells. In contrast, chFABP4 expression was down-regulated in JNK signaling pathway inhibition, suggesting the positive regulation of JNK signaling pathway for chFABP4 transcription in DF-1 cells in response to TLR3 activation, consistent with findings in macrophages. MEK pathway inhibition resulted in a similar regulation to NF-κB signaling. These results suggest that each MAPK contributes differentially to the transcriptional regulation of chFABP4 by in DF-1 cells in response to TLR3 activation.

• Korean Journal of Poultry Science
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• v.46 no.3
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• pp.185-191
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• 2019

Nucleoporin 210 (Nup210) is associated with several physiological processes including muscle and neural cell differentiation, autoimmune diseases, and peripheral T cell homeostasis. Chicken Nup210 (chNup210) gene was originally identified as one of the differentially expressed genes (DEGs) in the kidney tissues of chicken. To elucidate the role of Nup210 in metabolic disease of chicken, we studied the molecular characteristics of chNup210 and analyzed its gene expression under the stimulation of Toll-like receptor 3 (TLR3) ligands. The Nup210 genomic DNA and amino acid sequences of various species including fowls, fishes, and mammals were retrieved from the Ensemble database and subjected to bioinformatics analyses. The expression of Nup210 from several chicken tissues was probed through qRT-PCR, and chicken fibroblast DF-1 cell line was used to determine the change in expression of chNup210 after stimulation with TLR3 ligand, polyinosinic-polycytidylic acid (poly (I:C)). The chNup210 gene was highly expressed in chicken lung and spleen tissues. Although highly conserved among the species, chNup210 was evolutionary clustered in the same clade as that of duck compared to other mammals. Furthermore, this study revealed that chNup210 is expressed in TLR3 signaling pathway and provides fundamental information on Nup210 expression in chicken. Future studies that offer insight into the involvement of chNup210 in the chicken innate immune response against viral infection are recommended.