Knockdown Of Retinoic Acid-inducible gene-I Research Articles

Involvement of retinoic acid‑inducible gene‑I in radiation‑induced senescence of human umbilical vein endothelial cells.

In 2012, the threshold radiation dose (0.5 Gy) for cardiovascular and cerebrovascular diseases was revised, and this threshold dose may be exceeded during procedures involving radiation such as interventional radiology. Therefore, in addition to regulating radiation dose, it is necessary to develop strategies to prevent and mitigate the development of cardiovascular disease. Cellular senescence is irreversible arrest of cell proliferation. Although cellular senescence is one of the mechanisms for suppressing cancer, it also has adverse effects. For example, senescence of vascular endothelial cells is involved in development of vascular disorders. However, the mechanisms underlying induction of cellular senescence are not fully understood. Therefore, the present study explored the factors involved in the radiation-induced senescence in human umbilical vein endothelial cells (HUVECs). The present study reanalyzed the gene expression data of senescent normal human endothelial cells and fibroblast after irradiation (NCBI Gene Expression Omnibus accession no. GSE130727) and microarray data of HUVECs 24 h after irradiation (NCBI Gene Expression Omnibus accession no. GSE76484). Numerous genes related to viral infection and inflammation were upregulated in radiation-induced senescent cells. In addition, the gene group involved in the retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR) signaling pathway, which plays an important role to induce anti-viral response, was altered in irradiated HUVECs. Therefore, to investigate the involvement of RIG-I and melanoma differentiation-associated gene 5 (MDA5), which are RLRs, in radiation-induced senescence of HUVECs, the protein expression of RIG-I and MDA5 and the activity of senescence-associated β-galactosidase (SA-β-gal), a representative senescence marker, were analyzed. Of note, knockdown of RIG-I in HUVECs significantly decreased radiation-increased proportion of cells with high SA-β-gal activity (i.e., senescent cells), whereas this phenomenon was not observed in MDA5-knockdown cells. Taken together, the present results suggested that RIG-I, but not MDA5, was associated with radiation-induced senescence in HUVECs.

RIG-I promotes cell proliferation in esophageal squamous cell carcinoma by facilitating p21 degradation.

Retinoic acid-inducible gene-I (RIG-I) is considered a key sensor for host recognition of RNA virus infections. Recent studies have shown that RIG-I also regulates carcinogenesis. However, the role of RIG-I in esophageal squamous cell carcinoma (ESCC) remains unclear. We investigated the RIG-I expression in ESCC cells using a public database, immunohistochemistry, and Western blotting. We evaluated the proliferative activity of ESCC cells using CCK-8, colony formation, and EdU staining assays. Further, we determined the ESCC cell-cycle changes using flow cytometry and the ubiquitination of p21 in the cells using cycloheximide chase and ubiquitination assays. Finally, we verified the in vivo effects of RIG-I on ESCC cells by constructing xenograft models. RIG-I was highly expressed in ESCC cells and significantly promoted their proliferation and cell-cycle. Moreover, RIG-I knockdown inhibited xenograft growth in nude mice. Furthermore, RIG-I accelerated the cell-cycle by promoting the ubiquitination and degradation of p21. Overall, this study revealed that the increased expression of RIG-I due to ESCC accelerated the progression of esophageal cancer by promoting the ubiquitination and degradation of p21, which is related to the prognosis of ESCC. Thus, RIG-I may be a novel therapeutic target for ESCC treatment.

TRIM25 Suppresses Rabies Virus Fixed HEP-Flury Strain Production by Activating RIG-1-Mediated Type I Interferons.

Rabies remains a great threat to public health worldwide. So far, the mechanism of rabies virus (RABV) infection is not fully understood, and there is no effective treatment for rabies. Identifying more host restriction factors of RABV will spur the development of novel therapeutic interventions against rabies. Accumulating studies suggest that tripartite motif-containing (TRIM) proteins have great effects on virus replication. TRIMs control the antiviral responses through either direct interaction with viral proteins or indirect regulation of innate immune signaling molecules in the host. The role of TRIM25 in rabies virus (RABV) infection is poorly understood. Using next-generation sequencing, we found that TRIM25 is upregulated during HEP-Flury infection. Knockdown of TRIM25 enhances HEP-Flury production, while overexpression of TRIM25 suppresses HEP-Flury replication. Knockdown of interferon α and interferon β weakens the anti-RABV response induced by TRIM25 overexpression, and potentiates RABV production. Furthermore, we found that TRIM25 regulates type-I interferon response by targeting retinoic acid-inducible gene I (RIG-I) during HEP-Flury infection. Knockdown of RIG-I weakens the anti-HEP-Flury response induced by TRIM25 overexpression, indicating that TRIM25 regulates RABV production via the RIG-I-IFN axis. In addition, we observed that TRIM25 does not directly interact with HEP-Flury structural proteins, suggesting that TRIM25 regulates HEP-Flury production indirectly. Taken together, our work identifies TRIM25 as a new host factor involved in HEP-Flury infection, which may be a potential target for the development of antiviral drugs against RABV.

Replication of Porcine Astrovirus Type 1-Infected PK-15 Cells In Vitro Affected by RIG-I and MDA5 Signaling Pathways.

The interferon (IFN) system is an extremely powerful antiviral response in animal cells. The subsequent effects caused by porcine astrovirus type 1 (PAstV1) IFN activation are important for the host's response to viral infections. Here, we show that this virus, which causes mild diarrhea, growth retardation, and damage of the villi of the small intestinal mucosa in piglets, induces an IFN response upon infection of PK-15 cells. Although IFN-β mRNA was detected within infected cells, this response usually occurs during the middle stages of infection, after genome replication has taken place. Treatment of PAstV1-infected cells with the interferon regulatory factor 3 (IRF3) inhibitor BX795 decreased IFN-β expression, whereas the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) inhibitor BAY11-7082 did not. These findings indicate that PAstV induced the production of IFN-β via IRF3-mediated rather than NF-κB-mediated signaling pathways in PK-15 cells. Moreover, PAstV1 increased the protein expression levels of retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) in PK-15 cells. The knockdown of RIG-I and MDA5 decreased the expression levels of IFN-β and the viral loads and increased the infectivity of PAstV1. In conclusion, PAstV1 induced the production of IFN-β via the RIG-I and MDA5 signaling pathways, and the IFN-β produced during PAstV1 infection inhibited viral replication. These results will help provide new evidence that PAstV1-induced IFNs may protect against PAstV replication and pathogenesis. IMPORTANCE Astroviruses (AstVs) are widespread and can infect multiple species. Porcine astroviruses produce mainly gastroenteritis and neurological diseases in pigs. However, astrovirus-host interactions are less well studied, particularly with respect to their antagonism of IFN. Here, we report that PAstV1 acts via IRF3 transcription pathway activation of IFN-β. In addition, the knockdown of RIG-I and MDA5 attenuated the production of IFN-β induced by PAstV1 in PK-15 cells and increased efficient viral replication in vitro. We believe that these findings will help us to better understand the mechanism of how AstVs affect the host IFN response.

RIG-I Promotes Tumorigenesis and Confers Radioresistance of Esophageal Squamous Cell Carcinoma by Regulating DUSP6.

We investigated the expression and biological function of retinoic acid inducible gene I (RIG-I) in esophageal squamous cell carcinoma (ESCC). Materials and methods: An immunohistochemical analysis was performed on 86 pairs of tumor tissue and adjacent normal tissue samples of patients with ESCC. We generated RIG-I-overexpressing ESCC cell lines KYSE70 and KYSE450, and RIG-I- knockdown cell lines KYSE150 and KYSE510. Cell viability, migration and invasion, radioresistance, DNA damage, and cell cycle were evaluated using CCK-8, wound-healing and transwell assay, colony formation, immunofluorescence, and flow cytometry and Western blotting, respectively. RNA sequencing was performed to determine the differential gene expression between controls and RIG-I knockdown. Tumor growth and radioresistance were assessed in nude mice using xenograft models. RIG-I expression was higher in ESCC tissues compared with that in matched non-tumor tissues. RIG-I overexpressing cells had a higher proliferation rate than RIG-I knockdown cells. Moreover, the knockdown of RIG-I slowed migration and invasion rates, whereas the overexpression of RIG-I accelerated migration and invasion rates. RIG-I overexpression induced radioresistance and G2/M phase arrest and reduced DNA damage after exposure to ionizing radiations compared with controls; however, it silenced the RIG-I enhanced radiosensitivity and DNA damage, and reduced the G2/M phase arrest. RNA sequencing revealed that the downstream genes DUSP6 and RIG-I had the same biological function; silencing DUSP6 can reduce the radioresistance caused by the overexpression of RIG-I. RIG-I knockdown depleted tumor growth in vivo, and radiation exposure effectively delayed the growth of xenograft tumors compared with the control group. RIG-I enhances the progression and radioresistance of ESCC; therefore, it may be a new potential target for ESCC-targeted therapy.

RIG-I Promotes Cell Viability, Colony Formation, and Glucose Metabolism and Inhibits Cell Apoptosis in Colorectal Cancer by NF-κB Signaling Pathway.

Background Retinoic acid-inducible gene-I (RIG-I) has crucial effects on various cancers, while RIG-I's detailed roles and mechanism in colorectal cancer (CRC) are uncovered. Methods qRT-PCR was used to detect the expression of RIG-I in CRC, adjacent nontumor specimens, and five cell lines. CCK-8, colony formation, and flow cytometry assays were conducted to study CRC cell viabilities. Extracellular acidification rates, lactate analysis, and ATP analysis were conducted to study the cell viabilities and glucose metabolism of CRC cells. Western blot is used to determine the proteins of NF-κBp65 in the nucleus and cytoplasm. Results This study revealed the upregulation of RIG-I in CRC tissues and cells and that high RIG-I expression was correlated with poor prognosis of CRC patients. In addition, silencing RIG-I inhibited cell viability as well as colony formation and promoted cell apoptosis in CRC cells, while RIG-I knockdown suppressed transplanted tumor growth and facilitated apoptosis in nude mice. Moreover, silencing RIG-I inhibited glucose metabolism by decreasing extracellular acidification rate, lactate production, adenosine triphosphate, and content of hypoxia-inducible factor 1α and pyruvate kinase isoform. 2.2-Deoxy-d-glucose, a glycolysis inhibitor, reduced the growth of CRC cells and promoted apoptosis in vitro and in vivo. In addition, RIG-I knockdown decreased NF-κB nuclear translocation. Besides, inhibiting NF-κB effectively eliminated RIG-I overexpression roles in cell viability and glucose metabolism in CRC cells. Conclusion In summary, this study revealed that RIG-I mediated CRC cell proliferation, apoptosis, and glucose metabolism at least partly by NF-κB signaling pathway.

PolyIC Induces Retinoic Acid-inducible Gene-I and Melanoma Differentiation-associated Gene 5 and Modulates Inflammation in Podocytes.

Viral infection often exacerbates proteinuria, which has been suggested to be due to antiviral responses of podocytes. We examined the effect of polyinosinic-polycytidylic acid (polyIC) on the expression of retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) in differentiated human podocytes in culture. The podocytes were treated with 2 ng/ml to 500 μg/ml of polyIC for 3 to 36 h, and also transfected with siRNA against RIG-I and MDA5. F-actin staining was performed to assess actin reorganization. PolyIC induced the expression of RIG-I and MDA5 in dose- and time-dependent manner, accompanied with interferon-β (IFN-β) and interleukin-6 (IL-6) up-regulation and actin reorganization. Temporal knockdown of RIG-I by siRNA decreased IFN-β expression, while MDA5 siRNA inhibited IFN-β and IL-6 expression. Actin reorganization was attenuated by RIG-I and MDA5 knockdown. RIG-I and MDA5 may play a role in the antiviral responses of podocytes.

Interferon-alpha enhances the antitumour activity of EGFR-targeted therapies by upregulating RIG-I in head and neck squamous cell carcinoma

Background:The epidermal growth factor receptor (EGFR)-targeted therapies have been tested in the clinic as treatments for head and neck squamous cell carcinoma (HNSCC). Owing to intrinsic or acquired resistance, EGFR-targeted therapies often lead to a low response rate and treatment failure. Interferon-alpha (IFNα) is a chemosensitising agent and multi-functional cytokine with a tumour inhibitory effect. However, the synergic effect of IFNα and EGFR-targeted therapies (erlotinib and nimotuzumab) and their mechanisms in HNSCC remain unclear.Methods:The interactions between IFNα, erlotinib, and nimotuzumab were evaluated in vitro in HNSCC cells. The synergistic effect of IFNα (20 000 IU per day, s.c.), erlotinib (50 mg kg−1 per day, i.g.) and nimotuzumab (10 mg kg−1 per day, i.p.) was further confirmed in vivo using HNSCC xenografts in nude mice. The upregulation of retinoic-acid inducible gene I (RIG-I) induced by IFNα and EGFR-targeted therapies and its mechanism were detected in vitro and in vivo.Results:IFNα enhances the antitumour effects of erlotinib and nimotuzumab on HNSCC cells both in vitro and in vivo. Importantly, both IFNα and EGFR-targeted therapies promote the expression of RIG-I by activating signal transducers and activators of transcription 1 (STAT1) in HNSCC cells. RIG-I knockdown reduced the sensitivity of HN4 and HN30 cells to IFNα, erlotinib, and nimotuzumab. Moreover, IFNα transcriptionally induced RIG-I expression in HNSCC cells through STAT1.Conclusions:IFNα enhances the effect of EGFR-targeted therapies by upregulating RIG-I, and its expression may represent a predictor of the effectiveness of a combination treatment including IFNα in HNSCC.

Interferon (IFN)-induced protein 35 (IFI35) negatively regulates IFN-β-phosphorylated STAT1-RIG-I-CXCL10/CCL5 axis in U373MG astrocytoma cells treated with polyinosinic-polycytidylic acid

Interferon (IFN)-stimulated genes (ISGs) exert multiple functions in immune system. IFN-induced protein 35 (IFI35) is a member of ISGs, and has been suggested to regulate innate immune reaction. However, the physiological functions and pathological roles of IFI35 in the central nervous system are not characterized well. In the present study, we found that the expression of IFI35 was induced by a Toll-like receptor 3 (TLR3) ligand polyinosinic-polycytidylic acid (poly IC) in U373MG human astrocytoma cells. Knockdown of IFI35 using RNA interference resulted in increased expression of IFN-β, phosphorylated STAT1 (P-STAT1), retinoic acid-inducible gene-I (RIG-I), CXCL10 and CCL5 induced by poly IC. Poly IC-induced expression of CXCL10 and CCL5 was decreased by knockdown of RIG-I. These results suggest that IFI35 may negatively regulate the TLR3-IFN-β-P-STAT1-RIG-I-CXCL10/CCL5 axis in U373MG cells, and IFI35 may play a role at least partially in the regulation of innate immune reactions in astrocytes.

Mumps virus induces innate immune responses in mouse ovarian granulosa cells through the activation of Toll-like receptor 2 and retinoic acid-inducible gene I

Mumps virus (MuV) infection may lead to oophoritis and perturb ovarian function. However, the mechanisms underlying the activation of innate immune responses to MuV infection in the ovary have not been investigated. This study showed that Toll-like receptor 2 (TLR2) and retinoic acid-inducible gene I (RIG-I) cooperatively initiate innate immune responses to MuV infection in mouse ovarian granulosa cells. Ovarian granulosa cells infected with MuV significantly produced pro-inflammatory cytokines and chemokines, including interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), monocyte chemotactic protein 1 (MCP-1), and type 1 interferons (IFN-α and IFN-β). Knockdown of RIG-I significantly decreased MuV-induced cytokine expression. TLR2 deficiency reduced the expression of IL-1β, TNF-α, and MCP-1 but did not affect the expression of IFN-α and IFN-β in granulosa cells after infection with MuV. Intraperitoneal injection of MuV induced the ovarian innate immune responses in vivo, which suppressed estradiol synthesis and induced granulosa cell apoptosis. The results provide novel insights into the mechanisms underlying MuV-induced innate immune responses in the mouse ovary.

RIG-I specifically mediates group II type I IFN activation in nervous necrosis virus infected zebrafish cells

The type I interferon (IFN) response has been shown to be crucial for the survival of zebrafish larvae infected with nervous necrosis virus (NNV). Teleost type I IFNs can be divided into two groups, based on their cysteine content. While teleost group I IFNs have been extensively studied in terms of their regulation and anti-viral properties, the characteristics of teleost group II IFNs have been relatively unexplored. In this study, we describe the mechanism by which group II IFNs are activated in response to NNV infection in a zebrafish cell line, by focusing on the relationship between type I IFNs and pattern recognition receptors. Expression profile analysis of infected cells by microarray and qPCR revealed signaling activation of two pattern recognition receptors (PRRs): RIG-I like receptors (RLRs) and MyD88-dependent Toll-like receptors (TLRs). Knockdown of retinoic acid-inducible gene I (RIG-I) specifically repressed induction of group II IFNs (IFNϕ2, IFNϕ3) by NNV infection. Furthermore, Ingenuity Pathway Analysis (IPA) was used to demonstrate that RIG-I knockdown results in down-regulation of the inflammatory response in NNV-infected cells. Taken together, our results indicate that RIG-I plays an essential role in zebrafish group II type I IFN induction and the inflammatory response to NNV infection.

RIG-I detects HIV-1 infection and mediates type I interferon response in human macrophages from patients with HIV-1-associated neurocognitive disorders.

The aim of this study was to explore the precise role of retinoic acid-inducible gene-I (RIG-I) signaling in human immunodeficiency virus type 1 (HIV-1)-infected macrophages from patients with HIV-1-associated neurocognitive disorders (HAND). Postmortem brain tissues were collected from patients with HIV-1-associated dementia and were compared to samples collected from HIV serum-positive patients without dementia and HIV serum-negative patients. A human monocyte-derived macrophage (MDM) primary culture system was established to evaluate the expression of RIG-I in these samples. Knockdown of RIG-I pathways genes was employed and STAT1 expression and phosphorylation levels were examined to explore the molecular mechanisms of HAND. The expression of RIG-I in postmortem brain tissue from HAND patients was significantly higher than in patients who were HIV serum-positive without dementia or HIV serum-negative. Moreover, we demonstrated that HIV-1 infection could result in a significant increase in the level of RIG-I in human MDMs. Moreover, a correlation was found between the increase in RIG-I expression and STAT1 expression and phosphorylation. Accordingly, knockdown of RIG-I decreased the phosphorylation of STAT1 and downregulated interferon-related genes. These observations highlight the importance of RIG-I signaling in anti-HIV innate immunity in macrophages, which may be beneficial for the treatment of HIV and aid in the understanding of the neuropathogenesis of HAND.

A RING finger protein 114 (RNF114) homolog from Chinese sturgeon (Acipenser sinensis) possesses immune-regulation properties via modulating RIG-I signaling pathway-mediated interferon expression

Ubiquitin ligases play important roles in immune regulation. The human RNF114 (RING finger protein 114), an ubiquitin ligase, was recently reported to be involved in immune response to double-stranded RNA in disease pathogenesis. Here, we identified a RNF114 homolog in Chinese sturgeon (Acipenser sinensis) and investigated its potential role in immune response. The full-length cDNA of Chinese sturgeon RNF114 (csRNF114) contains an open reading frame (ORF) of 681 nucleotides coding a protein of 227 amino acids. csRNF114 shares the highest identity of 76% at amino acid level to other RNF114 homologs, clustering with bony fish RNF114s based on phylogenetic analysis. The main structural features of csRNF114, including a C3HC4 (Cys3-His-Cys4) RING domain, a C2HC (Cys2-His-Cys)-type zinc finger motif, a C2H2 (Cys2-His2)-type zinc finger motif, and a UIM (ubiquitin-interacting motif), take csRNF114 as an ubiquitin ligase. csRNF114 mRNA was widely expressed in various tissues and significantly up-regulated in poly(I:C)-treated Chinese sturgeon. Over-expression of csRNF114 in HEK293T cells significantly promoted both basal and poly(I:C)-induced activation of interferon regulatory transcription factor 3 (IRF3) and nuclear factor-κB (NF-κB) downstream retinoic acid inducible gene I (RIG-I) signaling pathway and expression of target genes type I interferon (IFN), which was nearly abolished by knockdown of RIG-I with specific human siRNA and by mutation of the C3HC4 RING domain (C28A/C31A) in csRNF114 as well. Furthermore, csRNF114 associated with ubiquitinated proteins in HEK293T cells, for which the C3HC4 RING domain was essential. These data suggested that an ubiquitin ligase RNF114 homolog with a potential role in antiviral response possibly through modulating RIG-I signaling pathway was cloned from Chinese sturgeon, which might contribute to our understanding of the immune biology of Chinese sturgeon.

RIG-I knockdown impedes neurogenesis in a murine model of Japanese encephalitis.

Retinoic acid inducible gene I (RIG-I) is a well established pattern recognition receptor (PRR) in neurons infected with Japanese encephalitis virus (JEV) as reported previously from our laboratory. Japanese encephalitis (JE) virus infection in brain has been shown to decrease the proliferation of neural stem/progenitor cells (NSPCs) which has its implications in neurological sequelae in JE survivors. We have found that ablation of RIG-I both in vivo and in vitro models results in significant decrease in NSPC proliferation post JEV infection. We hypothesize that knockdown of RIG-I diminishes the expression of antiviral molecules resulting in an increase in viral replication, which in turn results in enhancement of the expression of cell cycle inhibitors, hence affecting the proliferation of NSPCs.

Roles of TLR3 and RIG-I in mediating the inflammatory response in mouse microglia following Japanese encephalitis virus infection.

Japanese encephalitis virus (JEV) infection can cause central nervous system disease with irreversible neurological damage in humans and animals. Evidence suggests that overactivation of microglia leads to greatly increased neuronal damage during JEV infection. However, the mechanism by which JEV induces the activation of microglia remains unclear. Toll-like receptor 3 (TLR3) and retinoic acid-inducible gene I (RIG-I) can recognize double-stranded RNA, and their downstream signaling results in production of proinflammatory mediators. In this study, we investigated the roles of TLR3 and RIG-I in the inflammatory response caused by JEV infection in the mouse microglial cell line. JEV infection induced the expression of TLR3 and RIG-I and the activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK). Knockdown of TLR3 and RIG-I attenuated activation of ERK, p38MAPK, activator protein 1 (AP-1), and nuclear factor κB (NF-κB). Secretion of TNF-α, IL-6, and CCL-2, which was induced by JEV, was reduced by TLR3 and RIG-I knockdown and inhibitors of phosphorylated ERK and p38MAPK. Furthermore, viral proliferation was increased following knockdown of TLR3 and RIG-I. Our findings suggest that the signaling pathways of TLR3 and RIG-I play important roles in the JEV-induced inflammatory response of microglia.

Sustained Activation of Interferon Regulatory Factor 3 during Infection by Paramyxoviruses Requires MDA5

Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are the main cytosolic sensors of single-stranded RNA viruses, including paramyxoviruses, and are required to initiate a quick and robust innate antiviral response. Despite different ligand-binding properties, the consensus view is that RIG-I and MDA5 trigger common signal(s) to activate interferon regulatory factor 3 (IRF-3) and NF-κB, and downstream antiviral and proinflammatory cytokine expression. Here, we performed a thorough analysis of the temporal involvement of RIG-I and MDA5 in the regulation of IRF-3 during respiratory syncytial virus (RSV) infection. Based on specific RNA interference-mediated knockdown of RIG-I and MDA5 in A549 cells, we confirmed that RIG-I is critical for the initiation of IRF-3 phosphorylation, dimerization and downstream gene expression. On the other hand, our experiments yielded the first evidence that knockdown of MDA5 leads to early ubiquitination and proteasomal degradation of active IRF-3. Conversely, ectopic expression of MDA5 prolonged RIG-I-induced IRF-3 activation. Altogether, we provide novel mechanistic insight into the temporal involvement of RIG-I and MDA5 in the innate antiviral response. While RIG-I is essential for initial IRF-3 activation, engagement of induced MDA5 is essential to prevent early degradation of IRF-3, thereby sustaining IRF-3-dependent antiviral gene expression. MDA5 plays a similar role during Sendai virus infection suggesting that this model is not restricted to RSV amongst paramyxoviruses.

Classical swine fever virus triggers RIG-I and MDA5-dependent signaling pathway to IRF-3 and NF-κB activation to promote secretion of interferon and inflammatory cytokines in porcine alveolar macrophages.

BackgroundClassical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious disease of pigs. The RNA helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5) are differentially involved in the detection of various RNA viruses. In present study, we investigated the roles of RIG-I and MDA-5 in eliciting antiviral and inflammatory responses to CSFV shimen strain in Porcine alveolar macrophages (PAMs).MethodsCSFV Shimen strain was used as challenge virus in this study and PAMs were cultured in vitro. Interferon regulatory factor (IRF)-3 and nuclear factor-kappa B (NF-κB) translocation was detected using immunofluorescent staining; RIG-I, MDA5, interferon promoter-stimulating factor 1 (IPS-1), IRF-3 and NF-κB expression was measured by Western Blotting; Interferon beta (IFN-β), IFN-α, interleukin-1beta (IL-1β), IL-6 and tumor necrosis factor (TNF-α) expression was tested by Enzyme-linked immunosorbent assays (ELISA) and shRNA-mediated knockdown of MDA5 or RIG-I was performed.ResultsThe findings suggested that the initial response to CSFV infection resulted in the higher expression of RIG-I and MDA5 leading to the activation of IPS-1, IRF-3 and NF-κB in a dose-dependent manner. Evaluation of IFN-α, IFN-β, IL-1β, IL-6 or TNF-α expressed by PAMs showed significant differences between infected and uninfected cells. CSFV infected cells induced to express high levels of IFN-α, IFN-β, IL-1β, IL-6 and TNF-α in a dose-dependent way within 24 h post-infection (hpi). At the same time, CSFV improved the nuclear translocation of IRF-3 and NF-κB. We also directly compared and assessed the roles of RIG-I and MDA5 in triggering innate immune actions during CSFV infection through shRNA-mediated knockdown of MDA5 or RIG-I. We found that, compared to the control, the production of IFN-α, IFN-β, IL-1β, IL-6 and TNF-α in response to CSFV infection was heavily reduced in RIG-I knockdown cells while it was moderately decreased in MDA5 knockdown cells. PAMs derived from knockdown of both RIG-I and MDA5 almost failed to produce IFNs and inflammatory cytokines.ConclusionsIt indicates that CSFV can be recognized by both RIG-I and MDA5 to initiate the RIG-I signaling pathway to trigger innate defenses against infection.

Polyinosinic-Polycytidylic Acid Induces the Expression of Interferon-Stimulated Gene 20 in Mesangial Cells

Background/Aims: Interferon (IFN)-stimulated gene 20 (ISG20) is a 3′-to-5′ exonuclease specific for single-stranded RNA and involved in host defense reactions against RNA viruses. The expression and the role of ISG20 in mesangial cells have not been reported. Methods: Normal human mesangial cells were cultured and treated with polyinosinic-polycytidylic acid (poly (I:C)), an authentic double-stranded RNA which mimics viral infection to cells. The effect of RNA interference of Toll-like receptor 3 (TLR3) or IFN-β on the ISG20 expression was examined. The effect of a blocking antibody against the receptor for IFN-β or anti-inflammatory steroid dexamethasone was also examined. Results: Treatment of cells with poly (I:C) induced the expression of ISG20. The poly (I:C)-induced expression of ISG20 was inhibited by knockdown of TLR3, IFN regulatery factor 3 (IRF3) or IFN-β. Blocking of the receptor for IFN-β suppressed and overexpression of IFN-β enhanced ISG20 expression. The poly (I:C)-induced expressions of IFN-β and ISG20 were inhibited by dexamethasone. Transfection of mesangial cells with poly (I:C) or 5′-triphosphate single-stranded RNA as a complex with cationic lipid also induced the expression of ISG20, and this was inhibited by knockdown of retinoic acid-inducible gene-I (RIG-I). Conclusion: Poly (I:C) induces the expression of ISG20 in mesangial cells. ISG20 may be involved in anti-viral reactions in renal mesangial cells. TLR3, IRF3 and de novo synthesized IFN-β may mediate the poly (I:C)-induced expression of ISG20, and RIG-I may mediate ISG20 expression induced by poly (I:C)/cationic lipid complex.

RIG‐I mediates nonsegmented negative‐sense RNA virus‐induced inflammatory immune responses of primary human astrocytes

While astrocytes produce key inflammatory mediators following exposure to neurotropic nonsegmented negative-sense RNA viruses such as rabies virus and measles virus, the mechanisms by which resident central nervous system (CNS) cells perceive such viral challenges have not been defined. Recently, several cytosolic DExD/H box RNA helicases including retinoic acid-inducible gene I (RIG-I) have been described that function as intracellular sensors of replicative RNA viruses. Here, we demonstrate that primary human astrocytes constitutively express RIG-I and show that such expression is elevated following exposure to a model neurotropic RNA virus, vesicular stomatitis virus (VSV). Evidence for the functional nature of RIG-I expression in these cells comes from the observation that this molecule associates with its downstream effector molecule, interferon promoter stimulator-1, following VSV infection and from the finding that a specific ligand for RIG-I elicits astrocyte immune responses. Importantly, RIG-I knockdown significantly reduces inflammatory cytokine production by VSV-infected astrocytes and inhibits the production of soluble neurotoxic mediators by these cells. These findings directly implicate RIG-I in the initiation of inflammatory immune responses by human glial cells and provide a potential mechanism underlying the neuronal cell death associated with acute viral CNS infections.

RIG-I mediates rhabdovirus-induced inflammatory immune responses of primary human astrocytes (136.3)

Abstract Astrocytes are ideally positioned to respond to invading pathogens of the central nervous system (CNS) and produce key inflammatory mediators following exposure to neurotropic rhabdoviruses such as rabies virus and vesicular stomatitis virus (VSV). However, the mechanisms by which resident CNS cells perceive such viral challenges have not been defined. Recently, several cytosolic DExD/H box RNA helicases including retinoic acid-inducible gene I (RIG-I) have been described that function as intracellular sensors of replicative RNA viruses. Here, we demonstrate that primary human astrocytes constitutively express RIG-I and show that such expression is elevated following exposure to VSV. Evidence for the functional nature of RIG-I expression in these cells comes from the observation that this molecule associates with its downstream effector molecule, interferon promoter stimulator-1, following VSV infection and from the finding that a specific ligand for RIG-I elicits astrocyte immune responses. Importantly, RIG-I knockdown significantly reduces inflammatory cytokine production by VSV-infected astrocytes and inhibits the production of soluble neurotoxic mediator(s) by these cells. These findings directly implicate RIG-I in the initiation of inflammatory immune responses by human glial cells and provide a potential mechanism underlying the inflammation and neuronal cell damage associated with acute rhabdoviral infections of the CNS.