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1.
Viruses ; 16(4)2024 04 17.
Article in English | MEDLINE | ID: mdl-38675965

ABSTRACT

Epstein-Barr virus (EBV), a Herpesviridae family member, is associated with an increased risk of autoimmune disease development in the host. We previously demonstrated that EBV DNA elevates levels of the pro-inflammatory cytokine IL-17A and that inhibiting Toll-like receptor (TLR) 3, 7, or 9 reduces its levels. Moreover, this DNA exacerbated colitis in a mouse model of inflammatory bowel disease (IBD). In the study at hand, we examined whether inhibition of TLR3, 7, or 9 alleviates this exacerbation. Mice were fed 1.5% dextran sulfate sodium (DSS) water and administered EBV DNA. Then, they were treated with a TLR3, 7, or 9 inhibitor or left untreated. We also assessed the additive impact of combined inhibition of all three receptors. Mice that received DSS, EBV DNA, and each inhibitor alone, or a combination of inhibitors, showed significant improvement. They also had a decrease in the numbers of the pathogenic colonic IL-17A+IFN-γ+ foci. Inhibition of all three endosomal TLR receptors offered no additive benefit over administering a single inhibitor. Therefore, inhibition of endosomal TLRs reduces EBV DNA exacerbation of mouse colitis, offering a potential approach for managing IBD patients infected with EBV.


Subject(s)
DNA, Viral , Herpesvirus 4, Human , Inflammatory Bowel Diseases , Toll-Like Receptors , Animals , Female , Mice , Colitis/chemically induced , Colitis/drug therapy , Colitis/virology , Dextran Sulfate , Disease Models, Animal , DNA, Viral/adverse effects , DNA, Viral/pharmacology , Endosomes/drug effects , Endosomes/metabolism , Epstein-Barr Virus Infections/virology , Epstein-Barr Virus Infections/complications , Epstein-Barr Virus Infections/drug therapy , Inflammatory Bowel Diseases/chemically induced , Inflammatory Bowel Diseases/drug therapy , Inflammatory Bowel Diseases/virology , Interleukin-17/metabolism , Mice, Inbred C57BL , Toll-Like Receptor 3/antagonists & inhibitors , Toll-Like Receptor 3/metabolism , Toll-Like Receptor 7/antagonists & inhibitors , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 9/antagonists & inhibitors , Toll-Like Receptor 9/metabolism , Toll-Like Receptors/antagonists & inhibitors , Toll-Like Receptors/metabolism
2.
J Neurochem ; 163(6): 517-530, 2022 12.
Article in English | MEDLINE | ID: mdl-36321194

ABSTRACT

Inflammation associated with viral infection of the nervous system has been involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease (AD) and multiple sclerosis. Polyinosinic:polycytidylic acid (poly[I:C]) is a Toll-like receptor 3 (TLR3) agonist that mimics the inflammatory response to systemic viral infections. Despite growing recognition of the role of glial cells in AD pathology, their involvement in the accumulation and clearance of amyloid ß (Aß) in the brain of patients with AD is poorly understood. Neprilysin (NEP) and insulin-degrading enzyme (IDE) are the main Aß-degrading enzymes in the brain. This study investigated whether poly(I:C) regulated Aß degradation and neurotoxicity by modulating NEP and IDE protein levels through TLR3 in astrocytes. To this aim, primary rat primary astrocyte cultures were treated with poly(I:C) and inhibitors of the TLR3 signaling. Protein levels were assessed by Western blot. Aß toxicity to primary neurons was measured by lactate dehydrogenase release. Poly(I:C) induced a significant decrease in NEP levels on the membrane of astrocytes as well as in the culture medium. The degradation of exogenous Aß was markedly delayed in poly(I:C)-treated astrocytes. This delay significantly increased the neurotoxicity of exogenous Aß1-42. Altogether, these results suggest that viral infections induce Aß neurotoxicity by decreasing NEP levels in astrocytes and consequently preventing Aß degradation.


Subject(s)
Alzheimer Disease , Amyloid beta-Peptides , Astrocytes , Insulysin , Neprilysin , Virus Diseases , Animals , Rats , Alzheimer Disease/metabolism , Alzheimer Disease/virology , Amyloid beta-Peptides/metabolism , Astrocytes/metabolism , Astrocytes/virology , Insulysin/metabolism , Neprilysin/metabolism , Toll-Like Receptor 3/antagonists & inhibitors , Poly I-C/pharmacology , Virus Diseases/complications
3.
Int J Mol Sci ; 23(9)2022 Apr 23.
Article in English | MEDLINE | ID: mdl-35563088

ABSTRACT

Urolithin A is an active compound of gut-microbiota-derived metabolites of polyphenol ellagic acid that has anti-aging, antioxidative, and anti-inflammatory effects. However, the effects of urolithin A on polyinosinic acid-polycytidylic acid (poly(I:C))-induced inflammation remain unclear. Poly(I:C) is a double-stranded RNA (dsRNA) similar to a virus and is recognized by Toll-like receptor-3 (TLR3), inducing an inflammatory response in immune cells, such as macrophages. Inflammation is a natural defense process of the innate immune system. Therefore, we used poly(I:C)-induced RAW264.7 cells and attenuated the inflammation induced by urolithin A. First, our data suggested that 1-30 µM urolithin A does not reduce RAW264.7 cell viability, whereas 1 µM urolithin A is sufficient for antioxidation and the decreased production of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and C-C chemokine ligand 5. The inflammation-related proteins cyclooxygenase-2 and inducible nitric oxide synthase were also downregulated by urolithin A. Next, 1 µM urolithin A inhibited the levels of interferon (INF)-α and INF-ß. Urolithin A was applied to investigate the blockade of the TLR3 signaling pathway in poly(I:C)-induced RAW264.7 cells. Moreover, the TLR3 signaling pathway, subsequent inflammatory-related pathways, and antioxidation pathways showed changes in nuclear factor-κB (NF-κB) signaling and blocked ERK/mitogen-activated protein kinase (MAPK) signaling. Urolithin A enhanced catalase (CAT) and superoxide dismutase (SOD) activities, but decreased malondialdehyde (MDA) levels in poly(I:C)-induced RAW264.7 cells. Thus, our results suggest that urolithin A inhibits TLR3-activated inflammatory and oxidative-associated pathways in macrophages, and that this inhibition is induced by poly(I:C). Therefore, urolithin A may have antiviral effects and could be used to treat viral-infection-related diseases.


Subject(s)
Coumarins , NF-kappa B , Toll-Like Receptor 3 , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Antioxidants/pharmacology , Coumarins/pharmacology , Inflammation/chemically induced , Inflammation/drug therapy , Mice , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Poly I-C/pharmacology , RAW 264.7 Cells , RNA, Double-Stranded/pharmacology , STAT1 Transcription Factor/antagonists & inhibitors , STAT1 Transcription Factor/metabolism , Signal Transduction , Toll-Like Receptor 3/antagonists & inhibitors , Toll-Like Receptor 3/metabolism
4.
Front Immunol ; 12: 677905, 2021.
Article in English | MEDLINE | ID: mdl-34025676

ABSTRACT

Most blood transfusion-related adverse reactions involve the immunologic responses of recipients to exogenous blood components. Extracellular vesicles isolated from packed red cells can affect the recipient's immune system. Mast cells are traditionally known as effector cells for allergic transfusion reactions. However, growing evidence supports the notion that activated mast cells might disturb host innate immunologic responses. Exosomes are a type of extracellular vesicle. To determine the effect of exosomes on mast cells, we enriched exosomes derived from volunteer plasma (EXs-nor) and packed red cells (EXs-RBCs) using ultracentrifugation and incubated them with a human mast cell line (HMC-1). We found that EXs-RBC exposure increased the expression of tryptase-1 and prostaglandin D2, the production of multiple inflammatory mediators, and the levels of Toll-like receptor-3 (TLR-3) and phospho-mitogen-activated protein kinase (MAPK) in HMC-1 cells. MAPK inhibitors (SB203580, PD98059, and SP600125) and a TLR-3/dsRNA complex inhibitor reduced the EXs-RBC-stimulated production of inflammatory mediators in HMC-1 cells, whereas the TLR-3 agonist [poly (A:U)] elevated the production of these mediators. These results indicate that EXs-RBCs activate HMC-1 cells and elicit the production of multiple inflammatory mediators, partly via the TLR-3 and MAPK pathways. Mast cells activated by EXs-RBCs exhibit complex inflammatory properties and might play a potential role in transfusion-related adverse reactions.


Subject(s)
Erythrocytes/immunology , Exosomes/immunology , Immunity, Innate , Inflammation Mediators/metabolism , MAP Kinase Signaling System/immunology , Mast Cells/immunology , Adolescent , Adult , Anthracenes/pharmacology , Blood Donors , Blood Transfusion/methods , Cell Line , Cell Survival/drug effects , Flavonoids/pharmacology , Humans , Imidazoles/pharmacology , MAP Kinase Signaling System/drug effects , Male , Middle Aged , Mitogen-Activated Protein Kinases/metabolism , Pyridines/pharmacology , Toll-Like Receptor 3/agonists , Toll-Like Receptor 3/antagonists & inhibitors , Toll-Like Receptor 3/metabolism , Young Adult
5.
Nucleic Acids Res ; 49(11): 6082-6099, 2021 06 21.
Article in English | MEDLINE | ID: mdl-34057477

ABSTRACT

Oligonucleotide-based therapeutics have the capacity to engage with nucleic acid immune sensors to activate or block their response, but a detailed understanding of these immunomodulatory effects is currently lacking. We recently showed that 2'-O-methyl (2'OMe) gapmer antisense oligonucleotides (ASOs) exhibited sequence-dependent inhibition of sensing by the RNA sensor Toll-Like Receptor (TLR) 7. Here we discovered that 2'OMe ASOs can also display sequence-dependent inhibitory effects on two major sensors of DNA, namely cyclic GMP-AMP synthase (cGAS) and TLR9. Through a screen of 80 2'OMe ASOs and sequence mutants, we characterized key features within the 20-mer ASOs regulating cGAS and TLR9 inhibition, and identified a highly potent cGAS inhibitor. Importantly, we show that the features of ASOs inhibiting TLR9 differ from those inhibiting cGAS, with only a few sequences inhibiting both pathways. Together with our previous studies, our work reveals a complex pattern of immunomodulation where 95% of the ASOs tested inhibited at least one of TLR7, TLR9 or cGAS by ≥30%, which may confound interpretation of their in vivo functions. Our studies constitute the broadest analysis of the immunomodulatory effect of 2'OMe ASOs on nucleic acid sensing to date and will support refinement of their therapeutic development.


Subject(s)
Nucleotidyltransferases/antagonists & inhibitors , Oligonucleotides, Antisense/chemistry , Toll-Like Receptor 9/antagonists & inhibitors , Adult , Animals , Base Sequence , Cells, Cultured , DNA , Humans , Mice , Signal Transduction , Toll-Like Receptor 3/antagonists & inhibitors , Toll-Like Receptor 7/antagonists & inhibitors
6.
Eur J Pharmacol ; 890: 173654, 2021 Jan 05.
Article in English | MEDLINE | ID: mdl-33068589

ABSTRACT

Tobacco smoke is the major risk factor for developing chronic obstructive pulmonary disease (COPD). Viral infection is a major cause of COPD exacerbation, which lacks effective drug treatments. In the present study, to mimic the pathogenesis of COPD, we employed a TLR3 ligand [Poly (I:C), PIC] to mimic viral infection to determine whether it enhances the effects of cigarette smoke (CS)-induced airway inflammation and remodeling. Our results showed that PIC enhanced the effects of cigarette smoke extract (CSE)-induced inflammatory cytokine IL-1ß, TNF-α and IL-8 mRNA expression and remodeling factor E-cadherin, α-SMA and TGF-ß1 mRNA expression with TLR3 upregulation and EGFR phosphorylation in pulmonary epithelial NCI-H292 cells. These responses were inhibited by a TLR3/dsRNA complex inhibitor (TLR3i) or a tyrosine kinase inhibitor icotinib (Ico). Similarly, in the PIC-enhanced CS-induced airway inflammation and remodeling mouse model, treatment with TLR3i or Ico reduced the mRNA and protein expression of the inflammatory cytokines IL-1ß and TNF-α and keratinocyte chemoattractant (KC) and the remodeling factors α-SMA, TGF-ß1, MMP-9 and MUC5AC, while increasing E-cadherin mRNA and protein expression. Furthermore, we found that TLRi and Ico can attenuate the airway hyperreactivity induced by PIC, which is enhanced by CS. Finally, PIC enhanced the effects of CS on TLR3 upregulation and EGFR phosphorylation and significantly increased Erk1/2 and P38 phosphorylation, whereas TLR3i and Ico markedly suppressed TLR3 upregulation and EGFR, Erk1/2 and P38 phosphorylation in the model. Our findings suggest that TLR3/EGFR may be a potential target for the treatment of airway inflammation and remodeling in COPD.


Subject(s)
Airway Remodeling/drug effects , Cigarette Smoking/adverse effects , Pneumonia/prevention & control , Poly I-C/toxicity , Protein Kinase Inhibitors/therapeutic use , Toll-Like Receptor 3/antagonists & inhibitors , Animals , Cell Line , ErbB Receptors/metabolism , Female , Humans , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Male , Mice , Mice, Inbred ICR , Pneumonia/chemically induced , Pneumonia/metabolism , Protein Kinase Inhibitors/pharmacology , Respiratory Mucosa/drug effects , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathology , Toll-Like Receptor 3/metabolism
8.
Neurol Res ; 43(4): 307-313, 2021 Apr.
Article in English | MEDLINE | ID: mdl-33274693

ABSTRACT

Background and purpose: To investigate the effect of Emodin on the inflammatory response of brain tissue and the expression of the TLR3 pathway in mice with herpes virus encephalitis.Method: Twenty male BALB/c mice were randomly divided into the NS group, HSV-1 group, HSV-1 + Emodin group and HSV-1 + ACV group. The histopathological features and the effect of TLR3 expression were observed by staining and immunohistochemistry (IHC) respectively. The gene expression of TLR3, trif, TRADD, TRAF6, traf3, p38, Nemo and IRF3 was detected by polymerase chain reaction (PCR). The protein production of TLR3 and its downstream molecules was detected by Western blot. The expression of IL-6, TNF-α and IFN-ß in the brain tissues was detected by ELISA.Result: Compared to the HSV-1 group, the pathological changes (inflammatory cell infiltration, necrotic temporal lobe and massive hemorrhage) were not as obvious as those in the HSV-1+emodin and HSV-1+ACV groups. The TLR3 staining increased significantly in the HSV-1 groups and decreased in the HSV-1 + emodin group. Compared with the NS group, the mRNA expression of TLR3, TRIF, TRADD, TRAF6, traf3, p38, NEMO and IRF3 decreased by 20%-60% in the HSV-1 + emodin group and 30% in the HSV-1 + ACV group, respectively. The expression of IL-6, TNF-α and IFN-ß decreased by 30%-50% in the HSV-1 + emodin group and showed no significant change in the HSV-1 + ACV group, respectively.Conclusion: Emodin could inhibit the inflammatory response in the brain of mice with herpes virus encephalitis. The inhibition of TLR3 expression may play an important role in this process.


Subject(s)
Brain/metabolism , Emodin/therapeutic use , Encephalitis, Herpes Simplex/metabolism , Herpes Simplex/metabolism , Herpesvirus 1, Human , Toll-Like Receptor 3/biosynthesis , Animals , Brain/drug effects , Brain/pathology , Central Nervous System/drug effects , Central Nervous System/metabolism , Central Nervous System/pathology , Emodin/pharmacology , Encephalitis, Herpes Simplex/drug therapy , Encephalitis, Herpes Simplex/pathology , Herpes Simplex/drug therapy , Herpes Simplex/pathology , Male , Mice , Mice, Inbred BALB C , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Signal Transduction/drug effects , Signal Transduction/physiology , Toll-Like Receptor 3/antagonists & inhibitors
9.
Phytomedicine ; 79: 153328, 2020 Dec.
Article in English | MEDLINE | ID: mdl-33007730

ABSTRACT

BACKGROUND: Chaiqin chengqi decoction (CQCQD) is a Chinese herbal formula derived from dachengqi decoction. CQCQD has been used for the management of acute pancreatitis (AP) in the West China Hospital for more than 30 years. Although CQCQD has a well-established clinical efficacy, little is known about its bioactive ingredients, how they interact with different therapeutic targets and the pathways to produce anti-inflammatory effects. PURPOSE: Toll-like receptor 4 (TLR4) and the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated pro-inflammatory signaling pathways, play a central role in AP in determining the extent of pancreatic injury and systemic inflammation. In this study, we screened the bioactive ingredients using a pharmacological sub-network analysis based on the TLR4/NLRP3 signaling pathways followed by experimental validation. METHODS: The main CQCQD bioactive compounds were identified by UPLC-QTOF/MS. The TLR4/NLRP3 targets in AP for CQCQD active ingredients were confirmed through a pharmacological sub-network analysis. Mice received 7 intraperitoneal injections of cerulein (50 µg/kg; hourly) to induce AP (CER-AP), while oral gavage of CQCQD (5, 10, 15 and 20 g/kg; 3 doses, 2 hourly) was commenced at the 3rd injection of cerulein. Histopathology and biochemical indices were used for assessing AP severity, while polymerase chain reaction, Western blot and immunohistochemistry analyses were used to study the mechanisms. Identified active CQCQD compounds were further validated in freshly isolated mouse pancreatic acinar cells and cultured RAW264.7 macrophages. RESULTS: The main compounds from CQCQD belonged to flavonoids, iridoids, phenols, lignans, anthraquinones and corresponding glycosides. The sub-network analysis revealed that emodin, rhein, baicalin and chrysin were the compounds most relevant for directly regulating the TLR4/NLRP3-related proteins TLR4, RelA, NF-κB and TNF-α. In vivo, CQCQD attenuated the pancreatic injury and systemic inflammation of CER-AP and was associated with reduced expression of TLR4/NLRP3-related mRNAs and proteins. Emodin, rhein, baicalin and chrysin significantly diminished pancreatic acinar cell necrosis with varied effects on suppressing the expression of TLR4/NLRP3-related mRNAs. Emodin, rhein and chrysin also decreased nitric oxide production in macrophages and their combination had synergistic effects on alleviating cell death as well as expression of TLR4/NLRP3-related proteins. CONCLUSIONS: CQCQD attenuated the severity of AP at least in part by inhibiting the TLR4/NLRP3 pro-inflammatory pathways. Its active ingredients, emodin, baicalin, rhein and chrysin contributed to these beneficial effects.


Subject(s)
Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/pharmacology , Inflammasomes/drug effects , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Pancreatitis/drug therapy , Acinar Cells/drug effects , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Ceruletide/toxicity , Emodin/pharmacology , Flavonoids/pharmacology , Inflammasomes/metabolism , Male , Mice , Mice, Inbred C57BL , Pancreatitis/chemically induced , Pancreatitis/metabolism , Pancreatitis/pathology , RAW 264.7 Cells , Toll-Like Receptor 3/antagonists & inhibitors
10.
J Clin Endocrinol Metab ; 104(10): 4607-4618, 2019 10 01.
Article in English | MEDLINE | ID: mdl-31199456

ABSTRACT

OBJECTIVE: This study explored whether circulating exosomes effectively participate in the inflammatory response in Hashimoto thyroiditis (HT). DESIGN: Exosomes were extracted from the serum of 30 patients with HT and 30 healthy control (HC) subjects. The expression of thyroperoxidase (TPO), thyroglobulin, high mobility group box 1 (HMGB1), heat shock protein 60 (HSP60), major histocompatibility complex class II (MHC-II), and intercellular adhesion molecule 1 (ICAM1) in exosomes was determined by Western blotting. Flow cytometry and immunofluorescence were performed to confirm that exosomes were taken up by healthy peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs). Then, either DCs or PBMCs were stimulated with HT exosomes (serum exosomes from patients with HT) or HC exosomes (serum exosomes from HC subjects) in the presence or absence of Toll-like receptor (TLR)2/3 inhibitors. RESULTS: TPO, HSP60, and MHC-II expression was higher in HT exosomes than in HC exosomes. Exosomes were mainly taken up by CD14+ monocytes and CD11c+ DCs. After DCs were stimulated by HT exosomes, significant elevations were observed in MyD88, TRIF, and p-P65 expression; median fluorescence intensity of CD40 and CD83; and IL-6 production. After stimulating PBMCs with HT exosomes, CD11c+TLR2+/TLR3+ and CD4+IFN-γ+Th1/IL-17A+Th17A cell percentages were significantly elevated, and CD4+CD25+Foxp3+ Treg cell percentage was significantly decreased. HT exosomes induced increased IL-17A and IFN-γ production, whereas IL-10 production was suppressed. However, addition of TLR2 or TLR3 inhibitor reversed most of the abovementioned results. CONCLUSIONS: Our study demonstrates that HT exosomes can present antigens to DCs and bind TLR2/3, causing DC activation via the nuclear factor κB signaling pathway, leading to an imbalance in CD4+ T lymphocyte differentiation, and potentially contributing to HT onset.


Subject(s)
Exosomes/immunology , Hashimoto Disease/immunology , T-Lymphocytes, Regulatory/immunology , Th1 Cells/immunology , Th17 Cells/immunology , Adaptor Proteins, Vesicular Transport/metabolism , Adult , Autoantigens/metabolism , CD4-Positive T-Lymphocytes , Case-Control Studies , Cell Differentiation , Chaperonin 60/metabolism , Dendritic Cells , Female , HMGB1 Protein/metabolism , Histocompatibility Antigens Class II/metabolism , Humans , Intercellular Adhesion Molecule-1/metabolism , Interleukin-10/metabolism , Interleukin-6/metabolism , Iodide Peroxidase/metabolism , Iron-Binding Proteins/metabolism , Male , Middle Aged , Mitochondrial Proteins/metabolism , Monocytes , Myeloid Differentiation Factor 88/metabolism , Thyroglobulin/metabolism , Toll-Like Receptor 2/antagonists & inhibitors , Toll-Like Receptor 3/antagonists & inhibitors
11.
Cells ; 8(1)2019 01 05.
Article in English | MEDLINE | ID: mdl-30621275

ABSTRACT

There is an active search for the ideal strategy to potentialize the effects of Mesenchymal Stem-Cells (MSCs) over the immune system. Also, part of the scientific community is seeking to elucidate the therapeutic potential of MSCs secretome and its extracellular vesicles (EVs), in order to avoid the complexity of a cellular therapy. Here, we investigate the effects of human adipose MSCs (AMSCs) licensing with INF-γ and TLR3 agonist over AMSCs proliferation, migration, as well as the immunomodulatory function. Furthermore, we evaluated how the licensing of AMSCs affected the immunomodulatory function of AMSC derived-secretome, including their EVs. INF-γ licensed-AMSCs presented an elevated expression of indoleamine 2,3-dioxygenase (IDO), accompanied by increased ICAM-1, as well as a higher immunosuppressive potential, compared to unlicensed AMSCs. Interestingly, the conditioned medium obtained from INF-γ licensed-AMSCs also revealed a slightly superior immunosuppressive potential, compared to other licensing strategies. Therefore, unlicensed and INF-γ licensed-AMSCs groups were used to isolate EVs. Interestingly, EVs isolated from both groups displayed similar capacity to inhibit T-cell proliferation. EVs isolated from both groups shared similar TGF-ß and Galectin-1 mRNA content but only EVs derived from INF-γ licensed-AMSCs expressed IDO mRNA. In summary, we demonstrated that INF-γ licensing of AMSCs provides an immunosuppressive advantage both from a cell-cell contact-dependent perspective, as well as in a cell-free context. Interestingly, EVs derived from unlicensed and INF-γ licensed-AMSCs have similar ability to control activated T-cell proliferation. These results contribute towards the development of new strategies to control the immune response based on AMSCs or their derived products.


Subject(s)
Extracellular Vesicles/immunology , Extracellular Vesicles/metabolism , Immune Tolerance , Interferon-gamma/immunology , Mesenchymal Stem Cells/immunology , Mesenchymal Stem Cells/metabolism , Adipose Tissue/cytology , Cell Movement , Cell Proliferation , Cell Survival , Cells, Cultured , Galectin 1/metabolism , Humans , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Intercellular Adhesion Molecule-1/metabolism , Mesenchymal Stem Cells/cytology , Toll-Like Receptor 3/antagonists & inhibitors , Transforming Growth Factor beta/metabolism
12.
Sci Rep ; 8(1): 15841, 2018 10 26.
Article in English | MEDLINE | ID: mdl-30367171

ABSTRACT

Recognition of nucleic acids by endosomal Toll-like receptors (TLR) is essential to combat pathogens, but requires strict control to limit inflammatory responses. The mechanisms governing this tight regulation are unclear. We found that single-stranded oligonucleotides (ssON) inhibit endocytic pathways used by cargo destined for TLR3/4/7 signaling endosomes. Both ssDNA and ssRNA conferred the endocytic inhibition, it was concentration dependent, and required a certain ssON length. The ssON-mediated inhibition modulated signaling downstream of TLRs that localized within the affected endosomal pathway. We further show that injection of ssON dampens dsRNA-mediated inflammatory responses in the skin of non-human primates. These studies reveal a regulatory role for extracellular ssON in the endocytic uptake of TLR ligands and provide a mechanistic explanation of their immunomodulation. The identified ssON-mediated interference of endocytosis (SOMIE) is a regulatory process that temporarily dampens TLR3/4/7 signaling, thereby averting excessive immune responses.


Subject(s)
Clathrin/metabolism , Endocytosis/drug effects , Oligonucleotides/pharmacology , Toll-Like Receptor 3/metabolism , Toll-Like Receptor 4/metabolism , Toll-Like Receptor 7/metabolism , Animals , Cells, Cultured , Chemokines/metabolism , Cytokines/metabolism , DNA, Single-Stranded/pharmacology , Endosomes/metabolism , Humans , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Macaca fascicularis , Poly I-C/pharmacology , Signal Transduction/drug effects , Skin/metabolism , Skin/pathology , Toll-Like Receptor 3/antagonists & inhibitors , Toll-Like Receptor 4/antagonists & inhibitors , Toll-Like Receptor 7/antagonists & inhibitors
13.
J Neuroimmunol ; 324: 16-21, 2018 11 15.
Article in English | MEDLINE | ID: mdl-30195920

ABSTRACT

Brain capillary endothelial cells are the component of blood brain barrier, and the first line of defense against viruses invading into brain. We demonstrate that treatment of hCMEC/D3 cells, a human brain capillary endothelial cell line, with a Toll-like receptor 3 (TLR3) agonist polyinosinic-polycytidylic acid (poly IC) induces the expression of interferon (IFN)-stimulated gene 60 (ISG60), and this reaction was mediated by IFN-ß. Knockdown of ISG60 increased the poly IC-induced expression of IFN-ß and an IFN-ß-inducible chemokine CXCL10. This indicates that ISG60 constitutes a negative feedback loop in the downstream of TLR3/IFN-ß. ISG60 in brain capillary endothelial cells may contribute to prevent excess immune reactions associated with viral infections.


Subject(s)
Endothelial Cells/metabolism , Feedback, Physiological/physiology , Intracellular Signaling Peptides and Proteins/biosynthesis , Signal Transduction/physiology , Toll-Like Receptor 3/biosynthesis , Capillaries/cytology , Capillaries/drug effects , Capillaries/metabolism , Cell Line , Endothelial Cells/drug effects , Feedback, Physiological/drug effects , Humans , Intracellular Signaling Peptides and Proteins/genetics , Poly I-C/pharmacology , Signal Transduction/drug effects , Toll-Like Receptor 3/antagonists & inhibitors , Toll-Like Receptor 3/genetics
14.
PLoS Pathog ; 14(5): e1007075, 2018 05.
Article in English | MEDLINE | ID: mdl-29782532

ABSTRACT

Toll-like receptor 3 (TLR3) senses dsRNA intermediates produced during RNA virus replication to activate innate immune signaling pathways through adaptor protein TRIF. Many viruses have evolved strategies to block TLR3-mediated interferon signaling via targeting TRIF. Here we studied how hepatitis C virus (HCV) antagonizes the TLR3-mediated interferon signaling. We found that HCV-encoded NS4B protein inhibited TLR3-mediated interferon signaling by down-regulating TRIF protein level. Mechanism studies indicated that the downregulation of TRIF by NS4B was dependent on caspase8. NS4B transfection or HCV infection can activate caspase8 to promote TRIF degradation, leading to suppression of TLR3-mediated interferon signaling. Knockout of caspase8 can prevent TRIF degradation triggered by NS4B, thereby enhancing the TLR3-mediated interferon signaling activation in response to HCV infection. In conclusion, our work revealed a new mechanism for HCV to evade innate immune response by blocking the TLR3-mediated interferon signaling via NS4B-induced TRIF degradation.


Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Hepacivirus/physiology , Interferons/metabolism , Signal Transduction/physiology , Toll-Like Receptor 3/antagonists & inhibitors , Viral Nonstructural Proteins/physiology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , CRISPR-Cas Systems , Caspase 8/genetics , Caspase 8/metabolism , Caspase 9/genetics , Caspase 9/metabolism , Cell Line, Tumor , Gene Knockout Techniques , HEK293 Cells , Humans , Immune Evasion/physiology , Immunity, Innate/immunology , Membrane Proteins/physiology , Toll-Like Receptor 3/metabolism , Transfection
15.
Mol Med Rep ; 18(2): 1682-1691, 2018 Aug.
Article in English | MEDLINE | ID: mdl-29845244

ABSTRACT

The aim of the present study was to explore the effect of Bushen recipe and its disassembled prescriptions on liver injury and chronic hepatitis B. Liver injury was induced in normal and hepatitis B virus (HBV)­transgenic mice through injection of Concanavalin A, followed by treatment with Bushen recipe and its disassembled prescriptions including the Bushen­yang, the Bushen­yin and the QingHua groups as well as the GanYanLing group (positive control). Subsequently, their liver function indexes were investigated by a microplate method and liver sections were blindly evaluated using an optical microscope by a pathologist. Subsequently, the activation state of Toll­like receptor (TLR)3/9 signaling pathway in liver tissues was analyzed by western blotting. Additionally, the inflammatory factors produced following liver injury in peripheral blood were detected via ELISA. Following intervention with the Bushen recipe and its disassembled prescriptions, the liver function indexe alanine aminotransferase had declined, whereas cholinesterase increased. The pathological alterations of liver tissue in HBV transgenic mice were reversed by Bushen recipe and its disassembled prescriptions. In addition, the TLR3/9 signaling pathway in liver tissues of HBV transgenic mice was inhibited and inflammatory factors such as interleukin (IL)­6, IL­1, tumor necrosis factor­α and interferon­Î³ were reduced significantly. In conclusion, the present study demonstrated that Bushen recipe and its disassembled prescriptions repaired liver injury induced by Concanavalin A through inhibition of TLR3/9 signaling pathway.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Chemical and Drug Induced Liver Injury/drug therapy , Drugs, Chinese Herbal/pharmacology , Genome , Toll-Like Receptor 3/genetics , Toll-Like Receptor 9/genetics , Animals , Chemical and Drug Induced Liver Injury/genetics , Chemical and Drug Induced Liver Injury/immunology , Chemical and Drug Induced Liver Injury/pathology , Concanavalin A , Gene Expression Regulation , Hepatitis B virus/genetics , Interferon-gamma/antagonists & inhibitors , Interferon-gamma/genetics , Interferon-gamma/immunology , Interleukin-1/antagonists & inhibitors , Interleukin-1/genetics , Interleukin-1/immunology , Interleukin-6/antagonists & inhibitors , Interleukin-6/genetics , Interleukin-6/immunology , Male , Mice , Mice, Transgenic , Signal Transduction , Toll-Like Receptor 3/antagonists & inhibitors , Toll-Like Receptor 3/immunology , Toll-Like Receptor 9/antagonists & inhibitors , Toll-Like Receptor 9/immunology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology , Virus Integration
16.
Article in English | MEDLINE | ID: mdl-29686974

ABSTRACT

Dengue virus (DENV) is a mosquito-borne single-stranded RNA virus causing human disease with variable severity. The production of massive inflammatory cytokines in dengue patients has been associated with dengue disease severity. However, the regulation of these inflammatory responses remains unclear. In this study, we report that SIRT6 is a negative regulator of innate immune responses during DENV infection. Silencing of Sirt6 enhances DENV-induced proinflammatory cytokine and chemokine production. Overexpression of SIRT6 inhibits RIG-I-like receptor (RLR) and Toll-like receptor 3 (TLR3) mediated NF-κB activation. The sirtuin core domain of SIRT6 is required for the inhibition of NF-κB p65 function. SIRT6 interacts with the DNA binding domain of p65 and competes with p65 to occupy the Il6 promoter during DENV infection. Collectively, our study demonstrates that SIRT6 negatively regulates DENV-induced inflammatory response via RLR and TLR3 signaling pathways.


Subject(s)
DEAD Box Protein 58/antagonists & inhibitors , Dengue Virus/immunology , Dengue/immunology , Dengue/pathology , Sirtuins/metabolism , Toll-Like Receptor 3/antagonists & inhibitors , Aedes , Animals , Cell Line , DNA-Binding Proteins , Dengue/virology , Enzyme Activation/physiology , HEK293 Cells , Humans , Immunity, Innate/immunology , Inflammation , Macrophages/immunology , Mice , RAW 264.7 Cells , RNA Interference , RNA, Small Interfering/genetics , Receptors, Immunologic , Sirtuins/genetics , Transcription Factor RelA/metabolism
17.
Stem Cells ; 36(8): 1198-1209, 2018 08.
Article in English | MEDLINE | ID: mdl-29676038

ABSTRACT

The process by which committed precursors mature into cardiomyocytes is poorly understood. We found that TLR3 inhibition blocked cardiomyocyte maturation; precursor cells committed to the cardiomyocyte lineage failed to express maturation genes and sarcomeres did not develop. Using various approaches, we found that the effects of TLR3 upon cardiomyocyte maturation were dependent upon the RelA subunit of nuclear factor kappa B (NFκB). Importantly, under conditions that promote the development of mature cardiomyocytes NFκB became significantly enriched at the promoters of cardiomyocyte maturation genes. Furthermore, activation of the TLR3-NFκB pathway enhanced cardiomyocyte maturation. This study, therefore, demonstrates that the TLR3-NFκB pathway is necessary for the maturation of committed precursors into mature cardiomyocytes. Stem Cells 2018;36:1198-1209.


Subject(s)
Cell Differentiation , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , NF-kappa B/metabolism , Toll-Like Receptor 3/metabolism , Animals , Animals, Newborn , Cellular Reprogramming , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression Regulation , Mice, Inbred C57BL , MicroRNAs/genetics , MicroRNAs/metabolism , Promoter Regions, Genetic/genetics , Protein Subunits/metabolism , Toll-Like Receptor 3/antagonists & inhibitors , Transcription Factor RelA/metabolism
18.
Eur J Med Chem ; 151: 450-461, 2018 May 10.
Article in English | MEDLINE | ID: mdl-29649741

ABSTRACT

A series of diverse small molecules have been designed and synthesized through structure-based drug design by taking advantage of fragment merging and elaboration approaches. Compounds ZL0420 (28) and ZL0454 (35) were identified as potent and selective BRD4 inhibitors with nanomolar binding affinities to bromodomains (BDs) of BRD4. Both of them can be well docked into the acetyl-lysine (KAc) binding pocket of BRD4, forming key interactions including the critical hydrogen bonds with Asn140 directly and Tyr97 indirectly via a H2O molecule. Both compounds 28 and 35 exhibited submicromolar potency of inhibiting the TLR3-dependent innate immune gene program, including ISG54, ISG56, IL-8, and Groß genes in cultured human small airway epithelial cells (hSAECs). More importantly, they also demonstrated potent efficacy reducing airway inflammation in a mouse model with low toxicity, indicating a proof of concept that BRD4 inhibitors may offer the therapeutic potential to block the viral-induced airway inflammation.


Subject(s)
Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Drug Design , Inflammation/drug therapy , Nuclear Proteins/antagonists & inhibitors , Respiratory Mucosa/drug effects , Toll-Like Receptor 3/antagonists & inhibitors , Transcription Factors/antagonists & inhibitors , Animals , Cell Cycle Proteins , Cell Line , Humans , Immunity, Innate , Inflammation/immunology , Male , Mice, Inbred C57BL , Molecular Docking Simulation , Nuclear Proteins/immunology , Respiratory Mucosa/immunology , Structure-Activity Relationship , Toll-Like Receptor 3/immunology , Transcription Factors/immunology
19.
Sci Rep ; 8(1): 5165, 2018 03 26.
Article in English | MEDLINE | ID: mdl-29581525

ABSTRACT

Deficiency of ribosomal proteins (RPs) leads to Diamond Blackfan Anemia (DBA) associated with anemia, congenital defects, and cancer. While p53 activation is responsible for many features of DBA, the role of immune system is less defined. The Innate immune system can be activated by endogenous nucleic acids from non-processed pre-rRNAs, DNA damage, and apoptosis that occurs in DBA. Recognition by toll like receptors (TLRs) and Mda5-like sensors induces interferons (IFNs) and inflammation. Dying cells can also activate complement system. Therefore we analyzed the status of these pathways in RP-deficient zebrafish and found upregulation of interferon, inflammatory cytokines and mediators, and complement. We also found upregulation of receptors signaling to IFNs including Mda5, Tlr3, and Tlr9. TGFb family member activin was also upregulated in RP-deficient zebrafish and in RPS19-deficient human cells, which include a lymphoid cell line from a DBA patient, and fetal liver cells and K562 cells transduced with RPS19 shRNA. Treatment of RP-deficient zebrafish with a TLR3 inhibitor decreased IFNs activation, acute phase response, and apoptosis and improved their hematopoiesis and morphology. Inhibitors of complement and activin also had beneficial effects. Our studies suggest that innate immune system contributes to the phenotype of RPS19-deficient zebrafish and human cells.


Subject(s)
Anemia, Diamond-Blackfan/immunology , Anemia, Diamond-Blackfan/metabolism , Immunity, Innate/physiology , Zebrafish/immunology , Zebrafish/metabolism , Activin Receptors/antagonists & inhibitors , Activins/metabolism , Animals , Arginine/analogs & derivatives , Arginine/pharmacology , Benzamides/pharmacology , Benzhydryl Compounds/pharmacology , Complement C3a/antagonists & inhibitors , Complement C3a/metabolism , Dioxoles/pharmacology , Disease Models, Animal , Humans , Interferons/metabolism , K562 Cells , RNA, Small Interfering/metabolism , Receptor, Transforming Growth Factor-beta Type I/antagonists & inhibitors , Ribosomal Proteins/metabolism , Toll-Like Receptor 3/antagonists & inhibitors , Toll-Like Receptor 3/metabolism , Transforming Growth Factor beta/metabolism , Tumor Suppressor Protein p53/metabolism , Up-Regulation , Zebrafish/embryology , Zebrafish Proteins/metabolism
20.
J Immunol ; 200(7): 2291-2303, 2018 04 01.
Article in English | MEDLINE | ID: mdl-29445009

ABSTRACT

Binge/moderate alcohol suppresses TLR4-MyD88 proinflammatory cytokines; however, alcohol's effects on TLR-TRIF signaling, especially after in vivo exposure in humans, are unclear. We performed a comparative analysis of the TLR4-MyD88, TLR4-TRIF, and TLR3-TRIF pathways in human monocytes following binge alcohol exposure. Mechanistic regulation of TLR-TRIF signaling by binge alcohol was evaluated by analyzing IRF3 and TBK1, upstream regulator protein phosphatase 1 (PP1), and immunoregulatory stress proteins HspA1A and XBP-1 in alcohol-treated human and mouse monocytes/macrophages. Two approaches for alcohol exposure were used: in vivo exposure of primary monocytes in binge alcohol-consuming human volunteers or in vitro exposure of human monocytes/murine macrophages to physiological alcohol concentrations (25-50 mM ethanol), followed by LPS (TLR4) or polyinosinic-polycytidylic acid (TLR3) stimulation ex vivo. In vivo and in vitro binge alcohol exposure significantly inhibited the TLR4-MyD88 cytokines TNF-α and IL-6, as well as the TLR4-TRIF cytokines/chemokines IFN-ß, IP-10, and RANTES, in human monocytes, but not TLR3-TRIF-induced cytokines/chemokines, as detected by quantitative PCR and ELISA. Mechanistic analyses revealed TBK-1-independent inhibition of the TLR4-TRIF effector IRF3 in alcohol-treated macrophages. Although stress protein XBP-1, which is known to regulate IRF3-mediated IFN-ß induction, was not affected by alcohol, HspA1A was induced by in vivo alcohol in human monocytes. Alcohol-induced HspA1A was required for inhibition of TLR4-MyD88 signaling but not TLR4-TRIF cytokines in macrophages. In contrast, inhibition of PP1 prevented alcohol-mediated TLR4-TRIF tolerance in macrophages. Collectively, our results demonstrate that in vivo and in vitro binge alcohol exposure in humans suppresses TLR4-MyD88 and TLR4-TRIF, but not TLR3-TRIF, responses. Whereas alcohol-mediated effects on the PP1-IRF3 axis inhibit the TLR4-TRIF pathway, HspA1A selectively suppresses the TLR4-MyD88 pathway in monocytes/macrophages.


Subject(s)
Adaptor Proteins, Vesicular Transport/antagonists & inhibitors , Binge Drinking/pathology , Ethanol/toxicity , Macrophages/immunology , Monocytes/immunology , Myeloid Differentiation Factor 88/antagonists & inhibitors , Toll-Like Receptor 3/antagonists & inhibitors , Toll-Like Receptor 4/antagonists & inhibitors , Adolescent , Adult , Animals , Cell Line , Chemokine CCL5/antagonists & inhibitors , Chemokine CXCL10/antagonists & inhibitors , Female , HSP70 Heat-Shock Proteins/metabolism , Humans , Inflammation/pathology , Interferon-beta/antagonists & inhibitors , Interleukin-6/antagonists & inhibitors , Lipopolysaccharides/immunology , Macrophages/drug effects , Male , Mice , Middle Aged , Monocytes/drug effects , Poly I-C/immunology , RAW 264.7 Cells , Receptors, Neuropeptide Y/metabolism , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/antagonists & inhibitors , X-Box Binding Protein 1/drug effects , Young Adult
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