scholarly journals Chicken DDX1 Acts as an RNA Sensor to Mediate IFN-β Signaling Pathway Activation in Antiviral Innate Immunity

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhenyu Lin ◽  
Jie Wang ◽  
Wenxian Zhu ◽  
Xiangyu Yu ◽  
Zhaofei Wang ◽  
...  
Keyword(s):  
Viral Infections ◽  
Rna Virus ◽  
Direct Interaction ◽  
Poly I:C ◽  
Polycytidylic Acid ◽  
Pulldown Assay ◽  
Pathway Activation ◽  
Viral Yield ◽  
Antiviral Innate Immunity

Chickens are the natural host of Newcastle disease virus (NDV) and avian influenza virus (AIV). The discovery that the RIG-I gene, the primary RNA virus pattern recognition receptor (PRR) in mammals, is naturally absent in chickens has directed attention to studies of chicken RNA PRRs and their functions in antiviral immune responses. Here, we identified Asp-Glu-Ala-Asp (DEAD)-box helicase 1 (DDX1) as an essential RNA virus PRR in chickens and investigated its functions in anti-RNA viral infections. The chDDX1 gene was cloned, and cross-species sequence alignment and phylogenetic tree analyses revealed high conservation of DDX1 among vertebrates. A quantitative RT-PCR showed that chDDX1 mRNA are widely expressed in different tissues in healthy chickens. In addition, chDDX1 was significantly upregulated after infection with AIV, NDV, or GFP-expressing vesicular stomatitis virus (VSV-GFP). Overexpression of chDDX1 in DF-1 cells induced the expression of IFN-β, IFN-stimulated genes (ISGs), and proinflammatory cytokines; it also inhibited NDV and VSV replications. The knockdown of chDDX1 increased the viral yield of NDV and VSV and decreased the production of IFN-β, which was induced by RNA analog polyinosinic-polycytidylic acid (poly[I:C]), by AIV, and by NDV. We used a chicken IRF7 (chIRF7) knockout DF-1 cell line in a series of experiments to demonstrate that chDDX1 activates IFN signaling via the chIRF7 pathway. Finally, an in-vitro pulldown assay showed a strong and direct interaction between poly(I:C) and the chDDX1 protein, indicating that chDDX1 may act as an RNA PRR during IFN activation. In brief, our results suggest that chDDX1 is an important mediator of IFN-β and is involved in RNA- and RNA virus-mediated chDDX1-IRF7-IFN-β signaling pathways.

scholarly journals TRIM25 Identification in the Chinese Goose: Gene Structure, Tissue Expression Profiles, and Antiviral Immune Responses In Vivo and In Vitro

2016 ◽  
Vol 2016 ◽  
pp. 1-14 ◽  
Author(s):  
Yunan Wei ◽  
Hao Zhou ◽  
Anqi Wang ◽  
Lipei Sun ◽  
Mingshu Wang ◽  
...  
Keyword(s):  
Rna Virus ◽  
Expression Profiles ◽  
Critical Role ◽  
Coiled Coil ◽  
Poly I:C ◽  
Positive Role ◽  
Antiviral Immune Response ◽  
Polycytidylic Acid

The retinoic acid-inducible gene I (RIG-I) and the RIG-I-like receptor (RLR) protein play a critical role in the interferon (IFN) response during RNA virus infection. The tripartite motif containing 25 proteins (TRIM25) was reported to modify caspase activation and RIG-I recruitment domains (CARDs) via ubiquitin. These modifications allow TRIM25 to interact with mitochondrial antiviral signaling molecules (MAVs) and form CARD-CARD tetramers. Goose TRIM25 was cloned from gosling lungs, which possess a 1662 bp open reading flame (ORF). This ORF encodes a predicted 554 amino acid protein consisting of a B-box domain, a coiled-coil domain, and a PRY/SPRY domain. The protein sequence has 89.25% sequence identity withAnas platyrhynchosTRIM25, 78.57% withGallus gallusTRIM25, and 46.92% withHomo sapiensTRIM25. TRIM25 is expressed in all gosling and adult goose tissues examined. QRT-PCR revealed that goose TRIM25 transcription could be induced by goose IFN-α, goose IFN-γ, and goose IFN-λ, as well as a35 s polyinosinic-polycytidylic acid (poly(I:C)), oligodeoxynucleotides 2006 (ODN 2006), and resiquimod (R848) in vitro; however, it is inhibited in H9N2 infected goslings for unknown reasons. These data suggest that goose TRIM25 might play a positive role in the regulation of the antiviral immune response.

scholarly journals Basolateral activation with TLR agonists induces polarized cytokine release and reduces barrier function in RPE in vitro

2020 ◽  
Author(s):  
Laura Terheyden ◽  
Johann Roider ◽  
Alexa Klettner
Keyword(s):  
Cell Viability ◽  
Barrier Function ◽  
Neuronal Cell ◽  
Poly I:C ◽  
Rpe Cells ◽  
Ussing Chambers ◽  
Tlr Agonists ◽  
Danger Signals ◽  
Polycytidylic Acid

Abstract Purpose Systemic inflammation may be of importance in the development of AMD. RPE cells can recognize danger signals with toll-like receptors (TLR) and may react in a pro-inflammatory manner. In this study, we evaluated the basal and apical secretions of TNFα, IL-6, and IL-1β in primary RPE cells and RPE/choroid explant cells under basolateral stimulation of TLR2, 3, and 4; the effects on barrier function; and their influence on neuronal cell viability. Methods RPE/choroid tissue explants were prepared from porcine eyes and cultivated in modified Ussing chambers; primary porcine RPE cells on transwell plates. Cells were basally stimulated with agonists Pam2CSK4 (Pam; TLR2), polyinosinic/polycytidylic acid (Poly I:C; TLR3), and lipopolysaccharide (LPS; TLR4) for 24 h. Supernatants were evaluated with ELISA for cytokines TNFα, IL-6, and IL-1β. Apical supernatants were applied to SHSY-5Y cells, and cell viability was evaluated in MTT assay. Barrier function was tested by measuring transepithelial electrical resistance (TER) and occludin immunostaining. Results None of the tested TLR agonists was toxic on RPE cells after 24 h of exposure. Unstimulated RPE cells secreted hardly any cytokines. Pam induced IL-6, IL-1ß, and TNFα on the basal and apical sides at all concentrations tested. Poly I:C induced IL-6 and TNFα primarily at the basal side at lower but on both sides at higher concentrations. LPS induced IL-6, IL-1ß, and TNFα apically and basally at all concentrations tested. In the RPE/choroid, a strong difference between apical and basal secretions could be found. IL-6 was constitutively secreted basally, but not apically, but was induced by all agonists on both sides. IL-1ß and TNFα alpha were strongly induced on the basal side by all agonists. TER was reduced by all agonists, with Pam and LPS being effective in all concentrations tested. Occludin expression was unaltered, but the distribution was influenced by the agonists, with a less distinct localization at the cell borders after treatment. None of the agonists or supernatants of treated RPE and RPE/choroid organ cultures exerted any effect on viability of SHSY-5Y cells. Conclusions Danger signals activating TLRs can induce polarized cytokine expression and contribute to the loss of barrier function in the RPE.

p38 MAPK activation controls the TLR3-mediated up-regulation of cytotoxicity and cytokine production in human NK cells

Blood ◽  
2004 ◽  
Vol 104 (13) ◽  
pp. 4157-4164 ◽  
Author(s):  
Simona Pisegna ◽  
Gianluca Pirozzi ◽  
Mario Piccoli ◽  
Luigi Frati ◽  
Angela Santoni ◽  
...  
Keyword(s):  
Nk Cells ◽  
P38 Mapk ◽  
Cytokine Production ◽  
Viral Infections ◽  
Nk Cell ◽  
Mitogen Activated Protein Kinase ◽  
Poly I:C ◽  
Central Component ◽  
Polycytidylic Acid

Abstract Natural killer (NK) cells are a component of the innate immunity against viral infections through their rapid cytotoxic activity and cytokine production. Although the synthetic double-stranded (ds) RNA polyinosinic-polycytidylic acid (poly I:C), a mimic of a common product of viral infections, is known to rapidly up-regulate their in vivo functions, NK cell ability to directly respond to dsRNA is still mostly unknown. Our results show that treatment with poly I:C significantly up-regulates both natural and CD16-mediated cytotoxicity of highly purified human NK cells. Poly I:C also induces the novel capability of producing CXCL10 chemokine in human NK cells and synergistically enhances interferon-γ (IFN-γ) production induced by either adaptive or innate cytokines. In accordance with the expression of Toll-like receptor-3 (TLR3) and of TRIF/TICAM-1 adaptor, poly I:C stimulation induces the activation of interferon regulatory factor-3 (IRF-3) transcription factor and of p38 mitogen-activated protein kinase (MAPK) in human NK cells. Finally, we demonstrate that p38 MAPK activity is required for the dsRNA-dependent enhancement of cytotoxicity and CXCL10 production. The occurrence of dsRNA-induced signaling and functional events closely correlates with the TLR3 mRNAprofile in different NK cell populations. Taken together, these data identify p38 as a central component of NK cell ability to directly respond to dsRNA pathogen-associated molecular pattern (PAMP).

scholarly journals HCFC2 is needed for IRF1- and IRF2-dependent Tlr3 transcription and for survival during viral infections

2017 ◽  
Vol 214 (11) ◽  
pp. 3263-3277 ◽  
Author(s):  
Lei Sun ◽  
Zhengfan Jiang ◽  
Victoria A. Acosta-Rodriguez ◽  
Michael Berger ◽  
Xin Du ◽  
...  
Keyword(s):  
Viral Infections ◽  
Poly I:C ◽  
Type I ◽  
Induced Mutations ◽  
Herpes Simplex Virus 1 ◽  
Double Stranded Rna ◽  
Polycytidylic Acid ◽  
Interferon Regulatory Factor 1 ◽  
Mouse Macrophages ◽  
Simplex Virus

Transcriptional regulation of numerous interferon-regulated genes, including Toll-like receptor 3 (Tlr3), which encodes an innate immune sensor of viral double-stranded RNA, depends on the interferon regulatory factor 1 (IRF1) and IRF2 transcription factors. We detected specific abrogation of macrophage responses to polyinosinic-polycytidylic acid (poly(I:C)) resulting from three independent N-ethyl-N-nitrosourea–induced mutations in host cell factor C2 (Hcfc2). Hcfc2 mutations compromised survival during influenza virus and herpes simplex virus 1 infections. HCFC2 promoted the binding of IRF1 and IRF2 to the Tlr3 promoter, without which inflammatory cytokine and type I IFN responses to the double-stranded RNA analogue poly(I:C) are reduced in mouse macrophages. HCFC2 was also necessary for the transcription of a large subset of other IRF2-dependent interferon-regulated genes. Deleterious mutations of Hcfc2 may therefore increase susceptibility to diverse infectious diseases.

scholarly journals Single-stranded DNA oligonucleotides inhibit TLR3-mediated responses in human monocyte-derived dendritic cells and in vivo in cynomolgus macaques

Blood ◽  
2012 ◽  
Vol 120 (4) ◽  
pp. 768-777 ◽  
Author(s):  
Annette E. Sköld ◽  
Maroof Hasan ◽  
Leonardo Vargas ◽  
Hela Saidi ◽  
Nathalie Bosquet ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Proinflammatory Cytokines ◽  
Viral Infections ◽  
Human Monocyte ◽  
Poly I:C ◽  
Single Stranded Dna ◽  
Dna Oligonucleotides ◽  
Polycytidylic Acid ◽  
Cynomolgus Macaques

Abstract TLR3 is a key receptor for recognition of double-stranded RNA and initiation of immune responses against viral infections. However, hyperactive responses can have adverse effects, such as virus-induced asthma. Strategies to prevent TLR3-mediated pathology are therefore desired. We investigated the effect of single-stranded DNA oligonucleotides (ssDNA-ODNs) on TLR3 activation. Human monocyte-derived dendritic cells up-regulate maturation markers and secrete proinflammatory cytokines on treatment with the synthetic TLR3 ligand polyinosine-polycytidylic acid (poly I:C). These events were inhibited in cultures with ssDNA-ODNs. Poly I:C activation of nonhematopoietic cells was also inhibited by ssDNA-ODNs. The uptake of poly I:C into cells was reduced in the presence of ssDNA-ODNs, preventing TLR3 engagement from occurring. To confirm this inhibition in vivo, we administered ssDNA-ODNs and poly I:C, alone or in combination, via the intranasal route in cynomolgus macaques. Proinflammatory cytokines were detected in nasal secretions in the poly I:C group, while the levels were reduced in the groups receiving ssDNA-ODNs or both substances. Our results demonstrate that TLR3-triggered immune activation can be modulated by ssDNA-ODNs and provide evidence of dampening proinflammatory cytokine release in the airways of cynomolgus macaques. These findings may open novel perspectives for clinical strategies to prevent or treat inflammatory conditions exacerbated by TLR3 signaling.

TLR3-induced activation of mast cells modulates CD8+ T-cell recruitment

Blood ◽  
2005 ◽  
Vol 106 (3) ◽  
pp. 978-987 ◽  
Author(s):  
Zane Orinska ◽  
Elena Bulanova ◽  
Vadim Budagian ◽  
Martin Metz ◽  
Marcus Maurer ◽  
...  
Keyword(s):  
Mast Cell ◽  
T Cell ◽  
Mast Cells ◽  
Cd8 T Cell ◽  
Poly I:C ◽  
Cell Recruitment ◽  
Polycytidylic Acid ◽  
Peritoneal Mast Cells

AbstractMast cells play an important role in host defense against various pathogens, but their role in viral infection has not been clarified in detail. dsRNA, synthesized by various types of viruses and mimicked by polyinosinic-polycytidylic acid (poly(I:C)) is recognized by Toll-like receptor 3 (TLR3). In this study, we demonstrate that poly(I:C) injection in vivo potently stimulates peritoneal mast cells to up-regulate a number of different costimulatory molecules. Therefore, we examined the expression and the functional significance of TLR3 activation in mast cells. Mast cells express TLR3 on the cell surface and intracellularly. After stimulation of mast cells with poly(I:C) and Newcastle disease virus (NDV), TLR3 is phosphorylated and the expression of key antiviral response cytokines (interferon β, ISG15) and chemokines (IP10, RANTES) is upregulated. Interestingly, mast cells activated via TLR3-poly(I:C) potently stimulate CD8+ T-cell recruitment. Indeed, mast-cell–deficient mice (KitW/KitW-v) given an intraperitoneal injection of poly(I:C) show a decreased CD8+ T-cell recruitment, whereas granulocytes normally migrate to the peritoneal cavity. Mast-cell reconstitution of KitW/KitW-v mice normalizes the CD8+ T-cell influx. Thus, mast cells stimulated through engagement of TLR3 are potent regulators of CD8+ T-cell activities in vitro and in vivo.

scholarly journals PSV-18 Program Chair Poster Pick: Poly(I:C) dose response and its effects on piglet sickness behaviors

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 218-218
Author(s):  
Haley E Rymut ◽  
Courtni R Bolt ◽  
Megan P Corbett ◽  
Laurie A Rund ◽  
Rodney W Johnson ◽  
...  
Keyword(s):  
Viral Infection ◽  
Viral Infections ◽  
Sickness Behavior ◽  
Activity Level ◽  
Poly I:C ◽  
Activity Levels ◽  
Post Injection ◽  
Mixed Effect ◽  
Polycytidylic Acid ◽  
Sickness Behaviors

Abstract The synthetic polyinosinic-polycytidylic acid Poly(I:C) is commonly used to mimic viral infection in rodents. A Poly(I:C) injection activates inflammatory receptors that recognize viral patterns. This immune-stimulating agent can be used to understand the effects of viral infection on pork production. The objective of this study was to compare the effects of multiple Poly(I:C) doses on sickness behaviors across time. Female and male pigs from the University of Illinois Swine herd, averaging 24.5 kg, were injected with 1 mg/kg or 0.5 mg/kg of Poly(I:C), or comparable volume of Saline at approximately 60 days of age. Post injection, sickness behaviors and activity levels were measured in 15-minute intervals by a trained experimenter for three hours. Observed sickness behaviors included vomiting, diarrhea, or shivering. The activity levels were scored 1 through 8 with lower levels indicating pigs running, medium levels indicating walking, and higher levels indicating laying behaviors. The binary sickness behavior and discrete activity levels from 130 observations from 10 pigs evenly distributed across sexes were analyzed using generalized mixed effect models. The model included the effects of Poly(I:C) dose, hour, sex, and interactions, and a repeated structure within pig. The administration of Poly(I:C) had a significant effect on activity level within one-hour post-injection, and Poly(I:C)-treated pigs had lower activity levels than the Saline group. The Poly(I:C) effect had a lower significance in males (P < 0.001) than females (P < 0.004). The Poly(I:C) treatment also had a significant effect on the probability of sickness behavior (P < 0.005). The probability of sickness behavior was 0.03, 0.18, and 0.23 for pigs treated with Saline, 0.5, and 1.0 mg/kg of Poly(I:C), respectively. Our results suggest that Poly(I:C) could be an effective agent in studying sickness response to viral infections in pigs. This study is supported by USDA NIFA AFRI, grant number 2018-67015-27413.

In vitro validation of camphene as a potential antiviral agent against betanodavirus causing viral nervous necrosis in barramundi

2021 ◽  
Vol 19 ◽  
Author(s):  
Ruby Singh ◽  
Prachi Srivastava ◽  
Deepika Anand ◽  
Pani K. Prasad
Keyword(s):  
Mtt Assay ◽  
Viral Infections ◽  
Rna Virus ◽  
Antiviral Agent ◽  
Sea Bass ◽  
Viral Nervous Necrosis ◽  
Cytotoxicity Studies ◽  
In Silico Studies ◽  
Juvenile Stages

Introduction:: Viral infections are major threat to aquaculture industry throughout the world. Betanodavirus is one of the most infectious virus that causes highest mortality in larval and juvenile stages of Lates calcarifer commonly known as Barramundi. Methods:: Methods: It is a single stranded positive sense RNA virus and causes viral nervous necrosis (VNN). VNN is caused by RNA virus which gets transmitted both horizontally and vertically so the most effective method against this virus is to vaccinate the fish, however vaccination becomes difficult since the disease is associated with the outbreaks in larval and juvenile stages which are not that much immunocompetent. In our previous in silico studies we proved the stability of camphene as a better phytochemical agent. Results:: In continuation to prove the authenticity of Camphene as potential antiviral agent against betanodavirus, its in vitro validation was performed. Sea bass kidney cell line (SISK) was selected for carrying out the in vitro studies and cytotoxicity studies of Camphene in the SISK was was done by MTT assay. Based on the analysis of MTT assay different dosage of camphene were selected viz.,(0.2, 0.5, 1, 1.5, 2, 2.5, 5, 10, 20, 30 μg/ml). The SISK cells were infected with virus inoculum (200μl). Further the antiviral activity of Camphene on infected SISK cells by Betanodavirus was elucidated with the help of quantitative Real time PCR(qPCR) on the 3rd and 5th day of infection. Conclusion:: Analysis of results depictecd that the dose of camphene 2 to 10 μg/ml is the safest dose against Betanodavirus. Hence this is aptly revealed that camphene can be used as potential antiviral agent against Betanodavirus.

scholarly journals Surfactant protein C-deficient mice are susceptible to respiratory syncytial virus infection

2009 ◽  
Vol 297 (1) ◽  
pp. L64-L72 ◽  
Author(s):  
Stephan W. Glasser ◽  
Teah L. Witt ◽  
Albert P. Senft ◽  
John E. Baatz ◽  
Dusti Folger ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Protein C ◽  
Viral Infections ◽  
Surfactant Protein ◽  
Poly I:C ◽  
Cell Hyperplasia ◽  
Surfactant Protein C ◽  
Syncytial Virus ◽  
Tlr3 Signaling

Patients with mutations in the pulmonary surfactant protein C (SP-C) gene develop interstitial lung disease and pulmonary exacerbations associated with viral infections including respiratory syncytial virus (RSV). Pulmonary infection with RSV caused more severe interstitial thickening, air space consolidation, and goblet cell hyperplasia in SP-C-deficient ( Sftpc−/−) mice compared with SP-C replete mice. The RSV-induced pathology resolved more slowly in Sftpc−/−mice with lung inflammation persistent up to 30 days postinfection. Polymorphonuclear leukocyte and macrophage counts were increased in the bronchoalveolar lavage (BAL) fluid of Sftpc−/−mice. Viral titers and viral F and G protein mRNA were significantly increased in both Sftpc−/−and heterozygous Sftpc+/−mice compared with controls. Expression of Toll-like receptor 3 (TLR3) mRNA was increased in the lungs of Sftpc−/−mice relative to Sftpc+/+mice before and after RSV infection. Consistent with the increased TLR3 expression, BAL inflammatory cells were increased in the Sftpc−/−mice after exposure to a TLR3-specific ligand, poly(I:C). Preparations of purified SP-C and synthetic phospholipids blocked poly(I:C)-induced TLR3 signaling in vitro. SP-C deficiency increases the severity of RSV-induced pulmonary inflammation through regulation of TLR3 signaling.

scholarly journals Interleukin-38 ameliorates poly(I:C) induced lung inflammation: therapeutic implications in respiratory viral infections

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xun Gao ◽  
Paul Kay Sheung Chan ◽  
Grace Chung Yan Lui ◽  
David Shu Cheong Hui ◽  
Ida Miu-Ting Chu ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Viral Infections ◽  
Inflammatory Responses ◽  
Inflammatory Diseases ◽  
Poly I:C ◽  
Therapeutic Implications ◽  
Cytokines And Chemokines ◽  
Respiratory Viral Infections ◽  
Pro Inflammatory Cytokines

AbstractInterleukin-38 has recently been shown to have anti-inflammatory properties in lung inflammatory diseases. However, the effects of IL-38 in viral pneumonia remains unknown. In the present study, we demonstrate that circulating IL-38 concentrations together with IL-36α increased significantly in influenza and COVID-19 patients, and the level of IL-38 and IL-36α correlated negatively and positively with disease severity and inflammation, respectively. In the co-cultured human respiratory epithelial cells with macrophages to mimic lung microenvironment in vitro, IL-38 was able to alleviate inflammatory responses by inhibiting poly(I:C)-induced overproduction of pro-inflammatory cytokines and chemokines through intracellular STAT1, STAT3, p38 MAPK, ERK1/2, MEK, and NF-κB signaling pathways. Intriguingly, transcriptomic profiling revealed that IL-38 targeted genes were associated with the host innate immune response to virus. We also found that IL-38 counteracts the biological processes induced by IL-36α in the co-culture. Furthermore, the administration of recombinant IL-38 could mitigate poly I:C-induced lung injury, with reduced early accumulation of neutrophils and macrophages in bronchoalveolar lavage fluid, activation of lymphocytes, production of pro-inflammatory cytokines and chemokines and permeability of the alveolar-epithelial barrier. Taken together, our study indicates that IL-38 plays a crucial role in protection from exaggerated pulmonary inflammation during poly(I:C)-induced pneumonia, thereby providing the basis of a novel therapeutic target for respiratory viral infections.

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