Objective: To explore the effect and mechanism of activation of peripheral blood mononuclear cell (PBMC) Toll-like receptor (TLR3) signaling pathway in recombinant HBsAg (rHBsAg) immune response. Methods: White blood cells were collected from peripheral blood of 13 healthy donors in the preparation of blood products. PBMC was isolated and treated with Poly I:C (Poly I:C group) and PBS (control group) respectively. 48 h later, some cells were collected and the expressions of TLR3 signaling pathway proteins were detected by flow cytometry. After activating (Poly I:C group)/inactivating (control group) TLR3 signaling pathway, rHBsAg was given to both groups for 72 h, and the proportions of DC, T, B cells and their subsets in PBMC were detected by flow cytometry. Paired <i>ti>-test, paired samples wilcoxon signed-rank test and canonical correlation analyses were used for statistical analysis. Results: The percentage of TLR3 protein-positive cells (19.21%) and protein expression (8 983.95), NF-κB protein expression (26 193.13), the percentage of pNF-κB protein-positive cells (13.73%) and its proportion in NF-κB (16.03%), and the percentage of pIRF3 protein-positive cells (12.64%) and its proportion in IRF3 (21.80%) in Poly I:C group were higher than those in control group (11.54%, 8 086.00, 22 340.66, 8.72%, 9.71%, 9.57%, 19.12%) (<i>Pi><0.05), and the percentage of TRIF protein-positive cells (89.75%) and protein expression (304 219.54) were higher in Poly I:C group than in the control group (89.64%, 288 149.72) (<i>Pi>>0.05). After PBMC stimulation by rHBsAg, the proportions of mDC (2.90%), pDC (1.80%), B cell (5.31%) and plasma cell (67.71%) in Poly I:C group were significantly higher than those in the control group (1.83%, 0.81%, 4.23%, 58.82%) (<i>Pi><0.05). Results of canonical correlation analysis showed that the expression of TLR3 protein was positively correlated with the proportions of plasma cells, the expression of pIRF3 protein was positively correlated with the proportions of plasma cells and mDC, and the percentage of pNF-κB protein-positive cells and the percentage of pIRF3 protein-positive cells were positively correlated with the proportion of CD4⁺T cells. Conclusions: Poly I:C can activate TLR3/TRIF/NF-κB and TLR3/TRIF/IRF3 signaling pathway, promote the function of downstream signaling molecules, and then promote the maturation of DC, induce the immune responses of CD4⁺T cell, and promote the maturation and activation of B cells and the immune response of rHBsAg.
Adaptor Proteins, Vesicular Transport/pharmacology* ; Hepatitis B Surface Antigens ; Humans ; Immunity ; Leukocytes, Mononuclear/metabolism* ; NF-kappa B ; Poly I-C/pharmacology* ; Signal Transduction ; Toll-Like Receptor 3/metabolism* ; Toll-Like Receptors

Viperin is an IFN-stimulated gene (ISG)-encoded protein that was identified in human primary macrophages treated with IFN-γ and in human primary fibroblasts infected with cytomegalovirus (CMV). This protein plays multiple roles in various cell types. It inhibits viral replication, mediates signaling pathways, and regulates cellular metabolism. Recent studies have shown that viperin inhibits IFN expression in macrophages, while it enhances TLR7 and TLR9-mediated IFN production in plasmacytoid dendritic cells, suggesting that viperin can play different roles in activation of the same pathway in different cell types. Viperin also controls induction of ISGs in macrophages. However, the effect of viperin on induction of ISGs in cell types other than macrophages is unknown. Here, we show that viperin differentially induces ISGs in 2 distinct cell types, macrophages and fibroblasts isolated from wild type and viperin knockout mice. Unlike in bone marrow-derived macrophages (BMDMs), viperin downregulates the expression levels of ISGs such as bone marrow stromal cell antigen-2, Isg15, Isg54, myxovirus resistance dynamin like GTPase 2, and guanylate binding protein 2 in murine embryonic fibroblasts (MEFs) treated with type I or II IFN. However, viperin upregulates expression of these ISGs in both BMDMs and MEFs stimulated with polyinosinic-polycytidylic acid or CpG DNA and infected with murine CMV. The efficiency of viral entry is inversely proportional to the expression levels of ISGs in both cell types. The data indicate that viperin differentially regulates induction of ISGs in a cell type-dependent manner, which might provide different innate immune responses in distinct cell types against infections.
Animals ; Carrier Proteins ; Cytomegalovirus ; Dendritic Cells ; DNA ; Dynamins ; Fibroblasts ; GTP Phosphohydrolases ; Humans ; Immunity, Innate ; Interferons ; Macrophages ; Mesenchymal Stromal Cells ; Metabolism ; Mice ; Mice, Knockout ; Orthomyxoviridae ; Poly I-C

PURPOSE: Tripartite-motif-containing protein 56 (TRIM56) has been found to exhibit a broad antiviral activity, depending upon E3 ligase activity. Here, we attempted to evaluate the function of TRIM56 in multiple myeloma (MM) and its underlying molecular basis. MATERIALS AND METHODS: TRIM56 expression at the mRNA and protein level was measured by qRT PCR and western blot analysis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry analysis was performed to investigate the effect of TRIM56 on MM cell proliferation and apoptosis. The concentrations of interferon (IFN)-β, interleukin (IL)-6, and tumor necrosis factor-α in MM cell culture supernatants were detected with respective commercial ELISA kits. Western blot was employed to determine the effect of TRIM56 on toll-like receptor 3 (TLR3)/toll-IL-1 receptor (TIR) domain-containing adaptor inducing IFN-β (TRIF) signaling pathway. RESULTS: TRIM56 expression was prominently decreased in MM cells. Poly (dA:dT)-induced TRIM56 overexpression in U266 cells suppressed proliferation, induced apoptosis, and enhanced inflammatory cytokine production, while TRIM56 knockdown improved growth, diminished apoptosis, and inhibited inflammatory cytokine secretion in RPMI8226 cells. Moreover, TRIM56 knockdown blocked TLR3 signaling pathway. Furthermore, poly (I:C), a TLR3 agonist, markedly abolished TRIM56 depletion-induced increase of proliferation, decrease of apoptosis, and reduction of inflammatory factor in MM cells. CONCLUSION: TRIM56 may act as a tumor suppressor in MM through activation of TLR3/TRIF signaling pathway, contributing to a better understanding of the molecular mechanism of TRIM56 involvement in MM pathogenesis and providing a promising therapy strategy for patients with MM.
Adaptor Proteins, Vesicular Transport/metabolism ; Apoptosis/drug effects ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cytokines/secretion ; Disease Progression ; Down-Regulation/drug effects ; Gene Knockdown Techniques ; Humans ; Multiple Myeloma/metabolism ; Multiple Myeloma/pathology ; Poly I-C/pharmacology ; Signal Transduction/drug effects ; Toll-Like Receptor 3/metabolism ; Tripartite Motif Proteins/deficiency ; Tripartite Motif Proteins/metabolism ; Ubiquitin-Protein Ligases/deficiency ; Ubiquitin-Protein Ligases/metabolism

OBJECTIVE: The fallopian tubes play a critical role in the early events of fertilization. The rapid innate immune defense is an important part of the fallopian tubes. Toll-like receptor 3 (TLR3), as a part of the innate immune system, plays an important role in detecting viral infections. In this basic and experimental study, the effect of sex hormones on the function of TLR3 in the OE-E6/E7 cell line was investigated. METHODS: The functionality of TLR3 in this cell line was evaluated by cytokine measurements (interleukin [IL]-6 and IL-1b) and the effects of sex hormones on TLR3 were tested by an enzyme-linked immunosorbent assay kit. Additionally, TLR3 small interfering RNA (siRNA) and a TLR3 function-blocking antibody were used to confirm our findings. RESULTS: The production of IL-6 significantly increased in the presence of polyinosinic-polycytidylic acid (poly(I:C)) as the TLR3 ligand. Using a TLR3-siRNA-ransfected OE-E6/E7 cell line and function-blocking antibody confirmed that cytokine production was due to TLR3. In addition, 17-β estradiol and progesterone suppressed the production of IL-6 in the presence and absence of poly(I:C). CONCLUSION: These results imply that sex hormones exerted a suppressive effect on the function of TLR3 in the fallopian tube cell line when different concentrations of sex hormones were present. The current results also suggest that estrogen receptor beta and nuclear progesterone receptor B are likely to mediate the hormonal regulation of TLR3, as these two receptors are the main estrogen and progesterone receptors in OE-E6/E7 cell line.
Cell Line ; Enzyme-Linked Immunosorbent Assay ; Epithelial Cells* ; Estradiol ; Estrogen Receptor beta ; Estrogens ; Fallopian Tubes* ; Female ; Fertilization ; Gonadal Steroid Hormones* ; Immune System ; Immunity, Innate ; Interleukin-6 ; Poly I-C ; Progesterone ; Receptors, Progesterone ; RNA, Small Interfering ; Toll-Like Receptor 3* ; Toll-Like Receptors*

Interleukin (IL)-17 plays an important role in rhinitis and the level thereof correlates with the severity of disease. However, no mouse model for IL-17-dominant rhinitis has yet been developed. Our objective was to establish a mouse model of IL-17-dominant rhinitis via intranasal application of polyinosinic-polycytidylic acid (abbreviated as Poly(I:C)). Mice were divided into 6 groups (n=8 for each group); 1) 1 negative control group, 2) 1 positive control group (OVA/alum model), 3) 2 Poly(I:C) groups (10 or 100 µg), and 4) 2 OVA/Poly(I:C) groups (10 or 100 µg). The positive control group was treated with the conventional OVA/alum protocol. In the Poly(I:C) and OVA/Poly(I:C) groups, phosphate-buffered saline or an OVA solution plus Poly(I:C) were administered. The OVA/Poly(I:C) groups exhibited significantly greater neutrophil infiltration and increased IL-17/interferon γ expression compared with the other groups. However, the levels of total immunoglobulin E (IgE), OVA-specific IgE, eosinophil infiltration, IL-4, IL-5, IL-6, and IL-10 were significantly lower in the OVA/Poly(I:C) groups than in mice subjected to conventional Th2-dominant OVA/alum treatment (the positive control group). IL-17 and neutrophil measurement, chemokine (C-X-C motif) ligand 1 immunohistochemistry, and confocal microscopy revealed increased numbers of IL-17-secreting cells in the nasal mucosa of the OVA/Poly(I:C) groups, which included natural killer cells, CD4 T cells, and neutrophils. In conclusion, we developed a mouse model of IL-17-dominant rhinitis using OVA together with Poly(I:C). This model will be useful in research on neutrophil- or IL-17-dominant rhinitis.
Animals ; Chemokine CXCL1 ; Eosinophils ; Immunoglobulin E ; Immunoglobulins ; Immunohistochemistry ; Interleukin-10 ; Interleukin-17 ; Interleukin-4 ; Interleukin-5 ; Interleukin-6 ; Interleukins ; Killer Cells, Natural ; Mice* ; Microscopy, Confocal ; Nasal Mucosa ; Neutrophil Infiltration ; Neutrophils ; Ovum ; Poly I-C* ; Rhinitis* ; T-Lymphocytes

Galangin (3,5,7-trihydroxyflavone) is a polyphenolic compound abundant in honey and medicinal herbs, such as Alpinia officinarum. In this study, we investigated the anti-inflammatory effects of galangin under in vitro and in vivo neuroinflammatory conditions caused by polyinosinic-polycytidylic acid (poly(I:C)), a viral mimic dsRNA analog. Galangin suppressed the production of nitric oxide, reactive oxygen species, and pro-inflammatory cytokines in poly(I:C)-stimulated BV2 microglia. On the other hand, galangin enhanced anti-inflammatory interleukin (IL)-10 production. Galangin also suppressed the expression of pro-inflammatory markers in poly(I:C)-injected mouse brains. Further mechanistic studies showed that galangin inhibited poly(I:C)-induced nuclear factor (NF)-κB activity and phosphorylation of Akt without affecting MAP kinases. Interestingly, galangin increased the expression and transcriptional activity of peroxisome proliferator-activated receptor (PPAR)-γ, known to play an anti-inflammatory role. To investigate whether PPAR-γ is involved in the anti-inflammatory function of galangin, BV2 cells were pre-treated with PPAR-γ antagonist before treatment of galangin. We found that PPAR-γ antagonist significantly blocked galangin-mediated upregulation of IL-10 and attenuated the inhibition of tumor necrosis factor (TNF)-α and IL-6 in poly(I:C)-stimulated microglia. In conclusion, our data suggest that PI3K/Akt, NF-κB, and PPAR-γ play a pivotal role in mediating the anti-inflammatory effects of galangin in poly(I:C)-stimulated microglia.
Alpinia ; Animals ; Brain ; Cytokines ; Gene Expression* ; Hand ; Honey ; In Vitro Techniques ; Interleukin-10 ; Interleukin-6 ; Interleukins ; Mice ; Microglia ; Negotiating ; Nitric Oxide ; Peroxisomes ; Phosphorylation ; Phosphotransferases ; Plants, Medicinal ; Poly I-C ; Reactive Oxygen Species ; Tumor Necrosis Factor-alpha ; Up-Regulation

Galangin (3,5,7-trihydroxyflavone) is a polyphenolic compound abundant in honey and medicinal herbs, such as Alpinia officinarum. In this study, we investigated the anti-inflammatory effects of galangin under in vitro and in vivo neuroinflammatory conditions caused by polyinosinic-polycytidylic acid (poly(I:C)), a viral mimic dsRNA analog. Galangin suppressed the production of nitric oxide, reactive oxygen species, and pro-inflammatory cytokines in poly(I:C)-stimulated BV2 microglia. On the other hand, galangin enhanced anti-inflammatory interleukin (IL)-10 production. Galangin also suppressed the expression of pro-inflammatory markers in poly(I:C)-injected mouse brains. Further mechanistic studies showed that galangin inhibited poly(I:C)-induced nuclear factor (NF)-κB activity and phosphorylation of Akt without affecting MAP kinases. Interestingly, galangin increased the expression and transcriptional activity of peroxisome proliferator-activated receptor (PPAR)-γ, known to play an anti-inflammatory role. To investigate whether PPAR-γ is involved in the anti-inflammatory function of galangin, BV2 cells were pre-treated with PPAR-γ antagonist before treatment of galangin. We found that PPAR-γ antagonist significantly blocked galangin-mediated upregulation of IL-10 and attenuated the inhibition of tumor necrosis factor (TNF)-α and IL-6 in poly(I:C)-stimulated microglia. In conclusion, our data suggest that PI3K/Akt, NF-κB, and PPAR-γ play a pivotal role in mediating the anti-inflammatory effects of galangin in poly(I:C)-stimulated microglia.
Alpinia ; Animals ; Brain ; Cytokines ; Gene Expression* ; Hand ; Honey ; In Vitro Techniques ; Interleukin-10 ; Interleukin-6 ; Interleukins ; Mice ; Microglia ; Negotiating ; Nitric Oxide ; Peroxisomes ; Phosphorylation ; Phosphotransferases ; Plants, Medicinal ; Poly I-C ; Reactive Oxygen Species ; Tumor Necrosis Factor-alpha ; Up-Regulation

Mesenchymal stem cells (MSCs) have been used experimentally for treating inflammatory disorders, partly owing to their immunosuppressive properties. The goal of the study was to determine whether TLR ligands can enhance the therapeutic efficacy of bone marrow-derived MSCs for the treatment of inflammatory bowel disease. Mice (C57BL6) were administered with 4% dextran sulfate sodium (DSS) in drinking water for 7 days and injected with MSCs on days 1 and 3 following DSS ingestion. Our results demonstrated that among various TLR ligands, MSCs treated with polyinosinic-polycytidylic acid [poly(I:C)], which is a TLR3 ligand, more profoundly induced IDO, which is a therapeutically relevant immunosuppressive factor, without any observable phenotype change in vitro. The poly(I:C)-treated MSCs attenuated the pathologic severity of DSS-induced murine colitis when injected i.p. but not i.v. In summary, preconditioning MSCs with poly(I:C) might improve their efficacy in treating DSS-induced colitis, and this effect at least partly depends on the enhancement of their immunosuppressive activity through increasing their production of IDO.
Animals ; Colitis* ; Dextran Sulfate ; Drinking Water ; Eating ; In Vitro Techniques ; Indoleamine-Pyrrole 2,3,-Dioxygenase* ; Inflammatory Bowel Diseases ; Ligands ; Mesenchymal Stromal Cells ; Mice ; Phenotype ; Poly I-C ; Toll-Like Receptors

OBJECTIVETo investigate the pathogenic mechanisms of airway inflammation and recurrent wheezing induced by recurrent respiratory virus infection after respiratory syncytial virus (RSV) infection.

METHODSSixty-four female BALB/c mice (aged 6-8 weeks) were randomly divided into four groups: control, RSV, Poly(I:C), and RSV+Poly(I:C) (n=16 each). The bronchoalveolar lavage fluid (BALF) was collected on the 3rd day after Poly(I:C) administration, and the total cell number and differential counts in BALF were determined. Hematoxylin-eosin staining was used to observe pulmonary pathological changes. The airway responsiveness was detected. ELISA was used to measure the levels of interferon-γ (IFN-γ), interleukin-4 (IL-4), interleukin-13 (IL-13), matrix metallopeptidase-9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in BALF.

RESULTSCompared with the other three groups, the RSV+Poly(I:C) group had significant increases in the total number of inflammatory infiltrating cells in the airway, airway responsiveness, and MMP-9 level in BALF (P<0.05). The RSV+Poly(I:C) group showed more severe pulmonary tissue injuries compared with the control and RSV groups (P<0.01). Compared with the RSV group, the RSV+Poly(I:C) group showed significant reductions in the levels of IL-4 and TIMP-1 in BALF (P<0.01).

CONCLUSIONSViral re-infection in the late stage of RSV infection may cause an imbalance of MMP-9/TIMP-1 expression and thus contribute to aggravated airway inflammation.

Animals ; Asthma ; etiology ; Bronchoalveolar Lavage Fluid ; chemistry ; Female ; Lung ; pathology ; Matrix Metalloproteinase 9 ; analysis ; Mice ; Mice, Inbred BALB C ; Poly I-C ; pharmacology ; Respiratory Syncytial Virus Infections ; complications ; Tissue Inhibitor of Metalloproteinase-1 ; analysis

BACKGROUND: Several mechanisms for the pathogenesis of many liver diseases are related with oxidative stress, endotoxins, and infections by many microorganisms. These can lead to chronic hepatitis, cirrhosis, and even liver cancer. The aim of this study was to evaluate the effects of S-adenosylmethionine (SAMe) and its combinations with taurine and/or betaine against hepatotoxicites induced by lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (polyI:C). METHODS: RAW 264.7 macrophage cells and seven-week-old male C57BL/6 mice were pretreated with SAMe (SAM or AdoMet), taurine, and/or betaine. In order to mimic hepatic injury like endotoxemia or viral infection, cells and mice were treated with LPS or polyI:C. Concentrations of glutathione (GSH), mRNA expressions of GSH synthesizing enzymes, and inflammatory markers were measured by biochemical assays and quantitative real-time PCR. RESULTS: In RAW 264.7 cells and mice, pretreatment of SAMe alone or SAMe with taurine and/or betaine attenuated the decrease in GSH levels and mRNA expressions of GSH synthesizing enzymes. In addition, pretreatment of SAMe with taurine and/or betaine prevented the excessive increase in inflammatory mediators produced by LPS or polyI:C treatment. CONCLUSIONS: Treatment with SAMe in combination with taurine and betaine, would have anti-oxidant functions in addition to anti-inflammatory action against bacterial and/or viral inflammation.
Animals ; Betaine* ; Endotoxemia ; Endotoxins ; Fibrosis ; Glutathione ; Hepatitis, Chronic ; Humans ; Inflammation ; Lipopolysaccharides ; Liver Diseases ; Liver Neoplasms ; Macrophages ; Male ; Mice ; Oxidative Stress ; Poly I-C ; RAW 264.7 Cells ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; S-Adenosylmethionine* ; Taurine*