Induction Of IFN-β mRNA Research Articles

Multiple unfolded protein response pathways cooperate to link cytosolic dsDNA release to stimulator of interferon gene activation.

The double-stranded DNA (dsDNA) sensor STING has been increasingly implicated in responses to "sterile" endogenous threats and pathogens without nominal DNA or cyclic di-nucleotide stimuli. Previous work showed an endoplasmic reticulum (ER) stress response, known as the unfolded protein response (UPR), activates STING. Herein, we sought to determine if ER stress generated a STING ligand, and to identify the UPR pathways involved. Induction of IFN-β expression following stimulation with the UPR inducer thapsigargin (TPG) or oxygen glucose deprivation required both STING and the dsDNA-sensing cyclic GMP-AMP synthase (cGAS). Furthermore, TPG increased cytosolic mitochondrial DNA, and immunofluorescence visualized dsDNA punctae in murine and human cells, providing a cGAS stimulus. N-acetylcysteine decreased IFN-β induction by TPG, implicating reactive oxygen species (ROS). However, mitoTEMPO, a mitochondrial oxidative stress inhibitor did not impact TPG-induced IFN. On the other hand, inhibiting the inositol requiring enzyme 1 (IRE1) ER stress sensor and its target transcription factor XBP1 decreased the generation of cytosolic dsDNA. iNOS upregulation was XBP1-dependent, and an iNOS inhibitor decreased cytosolic dsDNA and IFN-β, implicating ROS downstream of the IRE1-XBP1 pathway. Inhibition of the PKR-like ER kinase (PERK) pathway also attenuated cytoplasmic dsDNA release. The PERK-regulated apoptotic factor Bim was required for both dsDNA release and IFN-β mRNA induction. Finally, XBP1 and PERK pathways contributed to cytosolic dsDNA release and IFN-induction by the RNA virus, Vesicular Stomatitis Virus (VSV). Together, our findings suggest that ER stressors, including viral pathogens without nominal STING or cGAS ligands such as RNA viruses, trigger multiple canonical UPR pathways that cooperate to activate STING and downstream IFN-β via mitochondrial dsDNA release.

Rubella Virus Triggers Type I Interferon Antiviral Response in Cultured Human Neural Cells: Involvement in the Control of Viral Gene Expression and Infectious Progeny Production

The type I interferon (IFN) response is one of the primary defense systems against various pathogens. Although rubella virus (RuV) infection is known to cause dysfunction of various organs and systems, including the central nervous system, little is known about how human neural cells evoke protective immunity against RuV infection, leading to controlling RuV replication. Using cultured human neural cells experimentally infected with RuV RA27/3 strain, we characterized the type I IFN immune response against the virus. RuV infected cultured human neural cell lines and induced IFN-β production, leading to the activation of signal transducer and activator of transcription 1 (STAT1) and the increased expression of IFN-stimulated genes (ISGs). Melanoma-differentiation-associated gene 5 (MDA5), one of the cytoplasmic retinoic acid-inducible gene I (RIG-I)-like receptors, is required for the RuV-triggered IFN-β mRNA induction in U373MG cells. We also showed that upregulation of RuV-triggered ISGs was attenuated by blocking IFN-α/β receptor subunit 2 (IFNAR2) using an IFNAR2-specific neutralizing antibody or by repressing mitochondrial antiviral signaling protein (MAVS) expression using MAVS-targeting short hairpin RNA (shRNA). Furthermore, treating RuV-infected cells with BX-795, a TANK-binding kinase 1 (TBK1)/I kappa B kinase ε (IKKε) inhibitor, robustly reduced STAT1 phosphorylation and expression of ISGs, enhancing viral gene expression and infectious virion production. Overall, our findings suggest that the RuV-triggered type I IFN-mediated antiviral response is essential in controlling RuV gene expression and viral replication in human neural cells.

Avian IRF1 and IRF7 Play Overlapping and Distinct Roles in Regulating IFN-Dependent and -Independent Antiviral Responses to Duck Tembusu Virus Infection

Avian interferon regulatory factors 1 and 7 (IRF1 and IRF7) play important roles in the host’s innate immunity against viral infection. Our previous study revealed that duck tembusu virus (DTMUV) infection of chicken fibroblasts (DF1) and duck embryo fibroblasts (DEFs) induced the expression of a variety of IFN-stimulated genes (ISGs), including VIPERIN, IFIT5, CMPK2, IRF1, and IRF7. IRF1 was further shown to play a significant role in regulating the up-expression of VIPERIN, IFIT5, and CMPK2 and inhibiting DTMUV replication. In this study, we confirm, through overexpression and knockout approaches, that both IRF1 and IRF7 inhibit DTMUV replication, mainly via regulation of type I IFN expression, as well as the induction of IRF1, VIPERIN, IFIT5, CMPK2, and MX1. In addition, IRF1 directly promoted the expression of VIPERIN and CMPK2 in an IFN-independent manner when IRF7 and type I IFN signaling were undermined. We also found that non-structural protein 2B (NS2B) of DTMUV was able to inhibit the induction of IFN-β mRNA triggered by Newcastle disease virus (NDV) infection or poly(I:C) treatment, revealing a strategy employed by DTMUV to evade host’s immunosurveillance. This study demonstrates that avian IRF7 and IRF1 play distinct roles in the regulation of type I IFN response during DTMUV infection.

Tryptanthrin attenuates TLR3-mediated STAT1 activation in THP-1 cells.

Upon viral infection, dysregulated immune responses are associated with the disease exacerbation and poor prognosis. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway are essential for the innate immune responses against invading viruses as well as for sustained activation of macrophages. Tryptanthrin, a natural alkaloid, exhibits various bioactivities, including anti-microbial and anti-inflammatory effects. The aim of this study was to elucidate the effects of tryptanthrin on toll-like receptor 3 (TLR3)–mediated STAT1 activation in macrophages in vitro. Using phorbol myristate acetate (PMA)–differentiated THP-1 cells, we analyzed the protein level of phosphorylated-STAT1 (p-STAT1) upon stimulation with polyinosinic-polycytidylic acid (poly IC), a well-known TLR3 ligand, with and without tryptanthrin. We found that tryptanthrin decreased the protein level of p-STAT1 in a concentration-dependent manner after poly IC stimulation. On the other hand, tryptanthrin did not affect the levels of p-STAT1 upon stimulation with lipopolysaccharide from Escherichia coli. Consistently, tryptanthrin suppressed poly IC–induced mRNA expression of interferon (IFN)–stimulated genes which are regulated by STAT1. Moreover, tryptanthrin decreased the protein level of phosphorylated-IFN regulatory factor 3 and the subsequent IFN-β mRNA induction after poly IC stimulation. Tryptanthrin is a promising therapeutic agent for the aberrant activation of macrophages caused by viral infection.

TNF-Mediated Inhibition of Classical Swine Fever Virus Replication Is IRF1-, NF-κB- and JAK/STAT Signaling-Dependent.

The sera from pigs infected with virulent classical swine fever virus (CSFV) contain substantial amounts of tumor necrosis factor (TNF), a prototype proinflammatory cytokine with pleiotropic activities. TNF limits the replication of CSFV in cell culture. In order to investigate the signaling involved in the antiviral activity of TNF, we employed small-molecule inhibitors to interfere specifically with JAK/STAT and NF-κB signaling pathways in near-to-primary endothelial PEDSV.15 cells. In addition, we knocked out selected factors of the interferon (IFN) induction and signaling pathways using CRISPR/Cas9. We found that the anti-CSFV effect of TNF was sensitive to JAK/STAT inhibitors, suggesting that TNF induces IFN signaling. Accordingly, we observed that the antiviral effect of TNF was dependent on intact type I IFN signaling as PEDSV.15 cells with the disrupted type I IFN receptor lost their capacity to limit the replication of CSFV after TNF treatment. Consequently, we examined whether TNF activates the type I IFN induction pathway. With genetically modified PEDSV.15 cells deficient in functional interferon regulatory factor 1 or 3 (IRF1 or IRF3), we observed that the anti-CSFV activity exhibited by TNF was dependent on IRF1, whereas IRF3 was dispensable. This was distinct from the lipopolysaccharide (LPS)-driven antiviral effect that relied on both IRF1 and IRF3. In agreement with the requirement of IRF1 to induce TNF- and LPS-mediated antiviral effects, intact IRF1 was also essential for TNF- and LPS-mediated induction of IFN-β mRNA, while the activation of NF-κB was not dependent on IRF1. Nevertheless, NF-κB activation was essential for the TNF-mediated antiviral effect. Finally, we observed that CSFV failed to counteract the TNF-mediated induction of the IFN-β mRNA in PEDSV.15 cells, suggesting that CSFV does not interfere with IRF1-dependent signaling. In summary, we report that the proinflammatory cytokine TNF limits the replication of CSFV in PEDSV.15 cells by specific induction of an IRF1-dependent antiviral type I IFN response.

Cytoplasmic RNA Sensor Pathways and Nitazoxanide Broadly Inhibit Intracellular Mycobacterium tuberculosis Growth.

SummaryTo establish stable infection, Mycobacterium tuberculosis (MTb) must overcome host innate immune mechanisms, including those that sense pathogen-derived nucleic acids. Here, we show that the host cytosolic RNA sensing molecules RIG-I-like receptor (RLR) signaling proteins RIG-I and MDA5, their common adaptor protein MAVS, and the RNA-dependent kinase PKR each independently inhibit MTb growth in human cells. Furthermore, we show that MTb broadly stimulates RIG-I, MDA5, MAVS, and PKR gene expression and their biological activities. We also show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits intracellular MTb growth and amplifies MTb-stimulated RNA sensor gene expression and activity. This study establishes prototypic cytoplasmic RNA sensors as innate restriction factors for MTb growth in human cells and it shows that targeting this pathway is a potential host-directed approach to treat tuberculosis disease.

In vitro and in vivo roles of sphingosine kinase 2 during dengue virus infection.

There is growing evidence of the influence of sphingosine kinase (SK) enzymes on viral infection. Here, the role of sphingosine kinase 2 (SK2), an isoform of SK prominent in the brain, was defined during dengue virus (DENV) infection. Chemical inhibition of SK2 activity using two different SK2 inhibitors, ABC294640 and K145, had no effect on DENV infection in human cells in vitro. In contrast, DENV infection was restricted in SK2-/- immortalized mouse embryonic fibroblasts (iMEFs) with reduced induction of IFN-β mRNA and protein, and mRNA for the IFN-stimulated genes (ISGs) viperin, IFIT1, IRF7 and CXCL10 in DENV-infected SK2-/- compared to WT iMEFs. Intracranial (ic) DENV injection in C57BL/6 SK2-/- mice induced body weight loss earlier than in WT mice but DENV RNA levels were comparable in the brain. Neither SK1 mRNA or sphingosine-1-phosphate (S1P) levels were altered following ic DENV infection in WT or SK2-/- mice but brain S1P levels were reduced in all SK2-/- mice, independent of DENV infection. CD8 mRNA was induced in the brains of both DENV-infected WT and SK2-/- mice, suggesting normal CD8+ T-cell infiltration into the DENV-infected brain independent of SK2 or S1P. Thus, although SK2 may be important for replication of some viruses SK2 activity does not affect DENV infection in vitro and SK2 or S1P levels do not influence DENV infection or T-cell infiltration in the context of infection in the brain.

Cyclin-dependent kinase activity is required for type I interferon production

Recognition of nucleic acids results in the production of type I IFNs, which activate the JAK/STAT pathway and promote the expression of IFN-stimulated genes. In a search for modulators of this pathway, we discovered an unexpected requirement for cyclin-dependent kinases (CDK) in the production of type I IFN following nucleic acid sensing and virus infection. Inhibition of CDK activity or knockdown of CDK levels leads to a striking block in STAT activation and IFN-stimulated gene expression. CDKs are not required for the initial nucleic acid sensing leading to IFN-β mRNA induction, nor for the response to exogenous IFN-α/β, but are critical for IFN-β release into culture supernatants, suggesting a posttranscriptional role for CDKs in type I IFN production. In the absence of CDK activity, we demonstrate a translational block specific for IFN-β, in which IFN-β mRNA is removed from the actively translating polysomes, while the distribution of other cellular mRNAs or global translation rates are unaffected. Our findings reveal a critical role for CDKs in the translation of IFN-β.

The vitamin D analogue ED71 but Not 1,25(OH)2D3 targets HIF1α protein in osteoclasts.

Although both an active form of the vitamin D metabolite, 1,25(OH)2D3, and the vitamin D analogue, ED71 have been used to treat osteoporosis, anti-bone resorbing activity is reportedly seen only in ED71- but not in 1,25(OH)2D3 -treated patients. In addition, how ED71 inhibits osteoclast activity in patients has not been fully characterized. Recently, HIF1α expression in osteoclasts was demonstrated to be required for development of post-menopausal osteoporosis. Here we show that ED71 but not 1,25(OH)2D3, suppress HIF1α protein expression in osteoclasts in vitro. We found that 1,25(OH)2D3 or ED71 function in osteoclasts requires the vitamin D receptor (VDR). ED71 was significantly less effective in inhibiting M-CSF and RANKL-stimulated osteoclastogenesis than was 1,25(OH)2D3 in vitro. Downregulation of c-Fos protein and induction of Ifnβ mRNA in osteoclasts, both of which reportedly block osteoclastogenesis induced by 1,25(OH)2D3 in vitro, were both significantly higher following treatment with 1,25(OH)2D3 than with ED71. Thus, suppression of HIF1α protein activity in osteoclasts in vitro, which is more efficiently achieved by ED71 rather than by 1,25(OH)2D3, could be a reliable read-out in either developing or screening reagents targeting osteoporosis.

Two Phases of Inflammatory Mediator Production Defined by the Study of IRAK2 and IRAK1 Knock-in Mice

The roles of IL-1R-associated kinase (IRAK)2 and IRAK1 in cytokine production were investigated using immune cells from knock-in mice expressing the TNFR-associated factor 6 (TRAF6) binding-defective mutant IRAK2[E525A] or the catalytically inactive IRAK1[D359A] mutant. In bone marrow-derived macrophages (BMDMs), the IRAK2-TRAF6 interaction was required for the late (2-8 h) but not the early phase (0-2 h) of il6 and tnfa mRNA production, and hence for IL-6 and TNF-α secretion by TLR agonists that signal via MyD88. Loss of the IRAK2-TRAF6 interaction had little effect on the MyD88-dependent production of anti-inflammatory molecules produced during the early phase, such as Dual Specificity Phosphatase 1, and a modest effect on IL-10 secretion. The LPS/TLR4-stimulated production of il6 and tnfa mRNA and IL-6 and TNF-α secretion was hardly affected, because the Toll/IL-1R domain-containing adapter-inducing IFN-β (TRIF) signaling pathway was used instead of the IRAK2-TRAF6 interaction to sustain late-phase mRNA production. IRAK1 catalytic activity was not rate limiting for il6, tnfa, or il10 mRNA production or the secretion of these cytokines by BMDMs, but IFN-β mRNA induction by TLR7 and TLR9 agonists was greatly delayed in plasmacytoid dendritic cells (pDCs) from IRAK1[D359A] mice. In contrast, IFN-β mRNA production was little affected in pDCs from IRAK2[E525A] mice, but subsequent IFN-α mRNA production and IFN-α secretion were reduced. IFN-β and IFN-α production were abolished in pDCs from IRAK1[D359A] × IRAK2[E525A] double knock-in mice. Our results establish that the IRAK2-TRAF6 interaction is rate limiting for the late, but not the early phase of cytokine production in BMDM and pDCs, and that the IRAK2-TRAF6 interaction is needed to sustain IκB-inducing kinase β activity during prolonged activation of the MyD88 signaling network. [corrected]

Autophagy in tumor cells and the host STING pathway are critical for innate immune sensing of tumors and bridging to an adaptive immune response (P2183)

Abstract Spontaneous T cell responses against tumors occur frequently. However, the mechanisms by which innate immune responses become induced in response to cancer, and how they can bridge to T cell priming against tumor antigens, have remained poorly defined. We recently reported that host type I IFN signals are required for a spontaneous T cell response against tumors. In the current work, we have mapped the sensing mechanism to one involving STING/TBK1/IRF3 which drives IFN-β transcription by host APCs. The tumor-derived ligand that triggers this pathway appears to be DNA. Using the DNA-specific dye DRAQ5 or EDU labeling, we demonstrated transfer of tumor DNA to host APCs via single cell ImageStream analysis; this was confirmed using PCR specific for tumor DNA. This was associated with IFN-β mRNA induction and nuclear translocation of IRF-3, which were dependent upon host STING. We hypothesized that autophagosome provide a mechanism for delivery of protected tumor DNA into APCs. By inhibition of tumor cell autophagy using the chemical 3-MA, or shRNA silencing of Becn or ATG5, we found marked reduction in the induction of host IFN-β production and spontaneous T cell responses in vivo. Our data suggest that tumor cell autophagy is required for optimal T cell priming against tumor antigens in vivo, that occurs through DNA-mediated activation of host IFN-β production via a STING-dependent pathway.

Abstract SY26-03: Innate immune sensing of tumors via the host STING pathway as a bridge to a spontaneous adaptive T cell response.

Abstract Recent evidence has suggested that clinical responses to immunotherapeutic interventions occur predominantly in patients having a pre-existing anti-tumor immune response and a T cell-inflamed tumor microenvironment. It has therefore become critical to understand the mechanism by which a subset of patients generates this phenotype, which should instruct on how to deliberately facilitate generation of appropriate tumor-based inflammation and thereby expand the fraction of patients deriving clinical benefit from immunotherapy. However, the mechanisms by which innate immune responses become activated in response to cancer, and how they can bridge to spontaneous T cell priming against tumor antigens, have remained poorly defined. We recently reported that host type I IFN signals are required for a spontaneous T cell response against tumors. In the current work, we have mapped the sensing mechanism to one involving STING/TBK1/IRF3 which drives IFN-β transcription by host APCs. Spontaneous T cell responses against tumors were eliminated in mice deficient in STING or IRF3, and rejection of immunogenic tumors was abrogated. The tumor-derived ligand that triggers this pathway appears to be DNA. Using the DNA-specific dye DRAQ5 or EDU labeling and single cell ImageStream analysis, we demonstrated that in vivo transfer of tumor DNA to host APCs occurs in vivo; this was confirmed using PCR specific for tumor DNA. APC uptake of tumor DNA was associated with IFN-β mRNA induction and nuclear translocation of IRF-3, which were dependent upon host STING. We hypothesized that autophagosome provide a mechanism for delivery of protected tumor DNA into APCs. Inhibition of tumor cell autophagy using the chemical 3-MA, or shRNA silencing of Becn or ATG5, led to a marked reduction in the induction of host IFN-β production and spontaneous T cell responses in vivo. Our data suggest that tumor cell autophagy is required for optimal T cell priming against tumor antigens in vivo, that occurs through DNA-mediated activation of host IFN-β production through a STING-dependent pathway. Analysis of these biologic steps in human melanoma metastases is ongoing. Strategies to mimic activation of the STING pathway should be considered as a mechanism to create an inflamed tumor microenvironment with the hope of expanding the clinical efficacy of T cell-based immunotherapies. Citation Format: Thomas Frank Gajewski. Innate immune sensing of tumors via the host STING pathway as a bridge to a spontaneous adaptive T cell response. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY26-03. doi:10.1158/1538-7445.AM2013-SY26-03

Priming of NK Cell Anti-Viral Effector Mechanisms by Direct Recognition of Human Cytomegalovirus

Natural killer (NK) cells play an important role in the defense against viral infections. Activation of resting NK cells is tightly controlled by the balance of surface inhibitory and activating receptors and aided by cytokines released by accessory cells along the anti-viral response. On the other hand, NK cells express functional pattern recognition receptors (PRRs) whose function has been mostly addressed by the use of synthetic agonists. The present study was undertaken to investigate whether NK cells could directly recognize a complex pathogen such as Human Cytomegalovirus (HCMV). Exposure of primary human NK cells to HCMV (TB40/E strain) induced the expression of CD69, promoted IFNγ secretion, and increased their cytotoxic activity against HCMV-infected autologous monocyte-derived dendritic cells. The divergent response induced by infective and UV-inactivated virions indicated the involvement of different NK cell sensors in the recognition of HCMV. The fact that NK cell activation could be partially prevented by blocking mAb specific for IFNAR and TLR2, together with the induction of IFNβ mRNA, supported the involvement of IFNβ and TLR2 in the response to HCMV. Thus, our data indicate that simultaneous activation of several PRRs leads to the autonomous priming of NK cell effector functions and could be a previously unappreciated mechanism presumably contributing to the control of HCMV infection.

Stress Granule Formation Induced by Measles Virus Is Protein Kinase PKR Dependent and Impaired by RNA Adenosine Deaminase ADAR1

ADAR1, an interferon (IFN)-inducible double-stranded (ds) RNA-specific adenosine deaminase, downregulates host innate responses, including activation of the dsRNA-dependent protein kinase (PKR) and induction of IFN-β mRNA. Conversely, PKR amplifies IFN-β induction by measles virus (MV) and inhibits virus protein synthesis. Formation of stress granules (SGs), cytoplasmic aggregates of stalled translation complexes and RNA-binding proteins, is a host response to virus infection mediated by translation initiation factor eIF2α phosphorylation. We examined the roles of PKR and ADAR1 in SG formation using HeLa cells stably deficient in either PKR (PKR(kd)) or ADAR1 (ADAR1(kd)) compared to control (CON(kd)) cells. Infection with either wild-type (WT) MV or an isogenic mutant lacking C protein expression (C(ko)) comparably induced formation of SG in ADAR1(kd) cells, whereas only the C(ko) mutant was an efficient inducer in control cells. Both ADAR1 and PKR colocalized with SG following infection. MV-induced; SG formation was PKR dependent but impaired by ADAR1. Complementation of ADAR1(kd) cells by expression of either p150 WT isoform or the p150 Zα (Y177A) Z-DNA-binding mutant of ADAR1 restored suppression of host responses, including SG formation and PKR activation. In contrast, neither the p110 WT isoform nor the p150 catalytic (H910A, E912A) mutant of ADAR1 complemented the ADAR1(kd) phenotype. These results further establish ADAR1 as a suppressor of host innate responses, including activation of PKR and the subsequent SG response.

IFN-β Expression Is Directly Activated in Human Neutrophils Transfected with Plasmid DNA and Is Further Increased via TLR-4–Mediated Signaling

Upon LPS binding, TLR4 activates a MyD88-dependent pathway leading to the transcriptional activation of proinflammatory genes, as well as a MyD88-independent/TRIF-dependent pathway, responsible for the transcriptional induction of IFN-β. Previous findings delineated that human neutrophils are unable to induce the transcription of IFN-β in response to TLR4 stimulation. Because neutrophils do not express protein kinase C ε, a molecule recently reported as essential for initiating the MyD88-independent/TRIF-dependent pathway, we optimized an electroporation method to transfect PKCε into neutrophils with very high efficiency. By doing so, a significant IFN-β mRNA expression was induced, in the absence of LPS stimulation, not only in PKCε-overexpressing neutrophils but also in cells transfected with a series of empty DNA plasmids; however, LPS further upregulated the IFN-β transcript levels in plasmid-transfected neutrophils, regardless of PKCε overexpression. Phosphoimmunoblotting studies, as well as chromatin immunoprecipitation assays targeting the IFN-β promoter, revealed that IFN-β mRNA induction occurred through the cooperative action of IRF3, activated by transfected DNA, and NF-κB, activated by LPS. Additional immunoblotting and coimmunoprecipitation studies revealed that neutrophils constitutively express various cytosolic DNA sensors, including IFN-inducible protein 16, leucine-rich repeat (in Flightless I) interacting protein-1, and DDX41, as well as that IFN-inducible protein 16 is the intracellular receptor recognizing transfected DNA. Consistently, infection of neutrophils with intracellular pathogens, such as Bartonella henselae, Listeria monocytogenes, Legionella pneumophila, or adenovirus type 5, promoted a marked induction of IFN-β mRNA expression. Taken together, these data raise questions about the role of PKCε in driving the MyD88-independent/TRIF-dependent response and indicate that human neutrophils are able to recognize and respond to microbial cytosolic DNA.

STING regulates IFN-β expression during endoplasmic reticulum stress (57.4)

Abstract STING (TMEM173, MPYS,MITA) located in the endoplasmic reticulum (ER), plays a critical role in type I IFN responses to intracellular DNA. STING activates TBK1, resulting in phosphorylated IRF3 that regulates IFN-β transcription. Our previous work showing markedly enhanced LPS-induced IFN-β during ER stress implicated increased IRF3 recruitment to the ifnb1 promoter. In this study, utilizing STING-/- mouse embryonic fibroblasts (MEFs) and RNAi (in MEFs and macrophages) we determined that STING was essential for synergistic induction of IFN-β mRNA in response to LPS and ER stress (thapsigargin/TPG treatment). TPG treatment alone induced re-localization and clustering of STING and TBK1 in MEFs. Although ER stress resulted in nuclear translocation of IRF3 in a STING-independent manner, TPG and TPG+LPS-induced IRF3 phosphorylation (as detected by immunofluorescence and western blot respectively) depended upon STING. In comparison, STING was not required for LPS or LPS+TPG induced NF-κB phosphorylation. These results indicate that ER stress alone results in IRF3 nuclear translocation and phosphorylation. In mice, treatment with the ER stress inducer tunicamycin was sufficient for induction of serum-detectable IFN-β, comparable with low dose LPS. Our results also suggest that the synergistic IFN-β responses during LPS and ER stress involve STING-dependent IRF3 phosphorylation.

Validation of efficient high-throughput plasmid and siRNA transfection of human monocyte-derived dendritic cells without cell maturation

Transfection of primary immune cells is difficult to achieve at high efficiency and without cell activation and maturation. Dendritic cells (DCs) represent a key link between the innate and adaptive immune systems. Delineating the signaling pathways involved in the activation of human primary DCs and reverse engineering cellular inflammatory pathways have been challenging tasks. We optimized and validated an effective high-throughput transfection protocol, allowing us to transiently express DNA in naïve primary DCs, as well as investigate the effect of gene silencing by RNA interference. Using a high-throughput nucleofection system, monocyte-derived DCs were nucleoporated with a plasmid expressing green fluorescent protein (GFP), and transfection efficiency was determined by flow cytometry, based on GFP expression. To evaluate the effect of nucleoporation on DC maturation, the expression of cell surface markers CD86 and MHCII in GFP-positive cells was analyzed by flow cytometry. We established optimal assay conditions with a cell viability reaching 70%, a transfection efficiency of over 50%, and unchanged CD86 and MHCII expression. We examined the impact of small interfering RNA (siRNA)-mediated knockdown of RIG-I, a key viral recognition receptor, on the induction of the interferon (IFN) response in DCs infected with Newcastle disease virus. RIG-I protein was undetectable by Western blot in siRNA-treated cells. RIG-I knockdown caused a 75% reduction in the induction of IFNβ mRNA compared with the negative control siRNA. This protocol should be a valuable tool for probing the immune response pathways activated in human DCs.

Foot-and-Mouth Disease Virus Replicates Only Transiently in Well-Differentiated Porcine Nasal Epithelial Cells

Three-dimensional (3D) porcine nasal mucosal and tracheal mucosal epithelial cell cultures were developed to analyze foot-and-mouth disease virus (FMDV) interactions with mucosal epithelial cells. The cells in these cultures differentiated and polarized until they closely resemble the epithelial layers seen in vivo. FMDV infected these cultures predominantly from the apical side, primarily by binding to integrin alphav beta6, in an Arg-Gly-Asp (RGD)-dependent manner. However, FMDV replicated only transiently without any visible cytopathic effect (CPE), and infectious progeny virus could be recovered only from the apical side. The infection induced the production of beta interferon (IFN-beta) and the IFN-inducible gene Mx1 mRNA, which coincided with the disappearance of viral RNA and progeny virus. The induction of IFN-beta mRNA correlated with the antiviral activity of the supernatants from both the apical and basolateral compartments. IFN-alpha mRNA was constitutively expressed in nasal mucosal epithelial cells in vitro and in vivo. In addition, FMDV infection induced interleukin 8 (IL-8) protein, granulocyte-macrophage colony-stimulating factor (GM-CSF), and RANTES mRNA in the infected epithelial cells, suggesting that it plays an important role in modulating the immune response.

Oncogenic Ras Promotes Reovirus Spread by Suppressing IFN-β Production through Negative Regulation of RIG-I Signaling

Reovirus is the first naturally occurring human virus reported to exploit activated Ras signaling in the host cell for infection, and is currently undergoing clinical trials as a cancer therapeutic. Recent evidence suggests that Ras transformation promotes three reoviral replication steps during the first round of infection: uncoating of the incoming virion, generation of progeny viruses with enhanced infectivity, and virus release through enhanced apoptosis. Whether oncogenic Ras also enhances reovirus spread in subsequent rounds of infection through other mechanisms has not been examined. Here, we show that compared with nontransformed cells, Ras-transformed cells are severely compromised not only in their response to IFN-beta, but also in the induction of IFN-beta mRNA following reovirus infection. Defects in both IFN-beta production and response allow for efficient virus spread in Ras-transformed cells. We show that the MEK/ERK pathway downstream of Ras is responsible for inhibiting IFN-beta expression by blocking signaling from the retinoic acid-inducible gene I (RIG-I) which recognizes viral RNAs. Overexpression of wild-type RIG-I restores INF-beta expression in reovirus-infected Ras-transformed cells. In vitro-synthesized viral mRNAs also invoke robust RIG-I-mediated IFN-beta production in transfected nontransformed cells, but not in Ras-transformed cells. Collectively, our data suggest that oncogenic Ras promotes virus spread by suppressing viral RNA-induced IFN-beta production through negative regulation of RIG-I signaling.

Z Proteins of New World Arenaviruses Bind RIG-I and Interfere with Type I Interferon Induction

The retinoic acid-inducible gene I product (RIG-I) is a cellular sensor of RNA virus infection that regulates the cellular beta interferon (IFN-beta) response. The nucleoproteins (NP) of arenaviruses are reported to antagonize the IFN response by inhibiting interferon regulatory factor 3 (IRF-3). Here, we demonstrate that the Z proteins of four New World (NW) arenaviruses, Guanarito virus (GTOV), Junin virus (JUNV), Machupo virus (MAVC), and Sabia virus (SABV), bind to RIG-I, resulting in downregulation of the IFN-beta response. We show that expression of the four NW arenavirus Z proteins inhibits IFN-beta mRNA induction in A549 cells in response to RNA bearing 5' phosphates (5'pppRNA). NW arenavirus Z proteins interact with RIG-I in coimmunoprecipitation studies and colocalize with RIG-I. Furthermore, expression of Z proteins interferes with the interaction between RIG-I and MAVS. Z expression also impedes the nuclear factor kappa light chain enhancer of activated B cells (NF-kappaB) and IRF-3 activation. Our results indicate that NW arenavirus Z proteins, but not Z protein of the Old World (OW) arenavirus lymphocytic choriomeningitis virus (LCMV) or Lassa virus, bind to RIG-I and inhibit downstream activation of the RIG-I signaling pathway, preventing the transcriptional induction of IFN-beta.