IRF3-dependent Gene Research Articles

The Cytomegalovirus M35 Protein Directly Binds to the Interferon-β Enhancer and Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes.

Induction of type I interferon (IFN) gene expression is among the first lines of cellular defense a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system, showing that M35 interferes with type I IFN induction downstream of pattern-recognition receptor (PRR) activation. Here, we report structural and mechanistic details of M35's function. Determination of M35's crystal structure combined with reverse genetics revealed that homodimerization is a key feature for M35's immunomodulatory activity. In electrophoretic mobility shift assays (EMSAs), purified M35 protein specifically bound to the regulatory DNA element that governs transcription of the first type I IFN gene induced in nonimmune cells, Ifnb1. DNA-binding sites of M35 overlapped with the recognition elements of interferon regulatory factor 3 (IRF3), a key transcription factor activated by PRR signaling. Chromatin immunoprecipitation (ChIP) showed reduced binding of IRF3 to the host Ifnb1 promoter in the presence of M35. We furthermore defined the IRF3-dependent and the type I IFN signaling-responsive genes in murine fibroblasts by RNA sequencing of metabolically labeled transcripts (SLAM-seq) and assessed M35's global effect on gene expression. Stable expression of M35 broadly influenced the transcriptome in untreated cells and specifically downregulated basal expression of IRF3-dependent genes. During MCMV infection, M35 impaired expression of IRF3-responsive genes aside of Ifnb1. Our results suggest that M35-DNA binding directly antagonizes gene induction mediated by IRF3 and impairs the antiviral response more broadly than formerly recognized. IMPORTANCE Replication of the ubiquitous human cytomegalovirus (HCMV) in healthy individuals mostly goes unnoticed but can impair fetal development or cause life-threatening symptoms in immunosuppressed or -deficient patients. Like other herpesviruses, CMV extensively manipulates its hosts and establishes lifelong latent infections. Murine CMV (MCMV) presents an important model system as it allows the study of CMV infection in the host organism. We previously showed that during entry into host cells, MCMV virions release the evolutionary conserved protein M35 protein to immediately dampen the antiviral type I interferon (IFN) response induced by pathogen detection. Here, we show that M35 dimers bind to regulatory DNA elements and interfere with recruitment of interferon regulatory factor 3 (IRF3), a key cellular factor for antiviral gene expression. Thereby, M35 interferes with expression of type I IFNs and other IRF3-dependent genes, reflecting the importance for herpesviruses to avoid IRF3-mediated gene induction.

STAT2 hinders STING intracellular trafficking and reshapes its activation in response to DNA damage

In cancer cells, endogenous or therapy-induced DNA damage leads to the abnormal presence of DNA in the cytoplasm, which triggers the activation of cGAS (cyclic GMP-AMP synthase) and STING (stimulator of interferon genes). STAT2 suppresses the cGAMP-induced expression of IRF3-dependent genes by binding to STING, blocking its intracellular trafficking, which is essential for the full response to STING activation. STAT2 reshapes STING signaling by inhibiting the induction of IRF3-dependent, but not NF-κB-dependent genes. This noncanonical activity of STAT2 is regulated independently of its tyrosine phosphorylation but does depend on the phosphorylation of threonine 404, which promotes the formation of a STAT2:STING complex that keeps STING bound to the endoplasmic reticulum (ER) and increases resistance to DNA damage. We conclude that STAT2 is a key negative intracellular regulator of STING, a function that is quite distinct from its function as a transcription factor.

Abstract C038: Repeat RNA dysregulation of cellular states in the pancreatic cancer microenvironment

Abstract Aberrant transcription of the repeat RNAs is a common feature in epithelial cancers including PDAC, but the function of these non-coding RNAs in cancer development is relatively unexplored. We have found that these repeat RNAs are sensed and replicate like retroviruses, and now have identified the ability of these viral-like elements to be transmitted from cancer cells through extracellular vesicles (EVs). PDAC-derived EVs applied to cancer-associated fibroblasts (CAFs) activates interferon-stimulated genes (ISGs) and is able to drive CAFs towards an inflammatory CAF (iCAF) phenotype with concordant loss of myofibroblast CAF (myCAF) marker genes. Using in-vitro transcription, we demonstrate that individual repeat RNAs (HSATII, HERVK (env), LINE-1 5’UTR and LINE-1 3’UTR) are sufficient to induce ISG response in CAFs with HSATII and HERVK (env) having the most potent ISG response. In contrast, PDAC cells were found to induce epithelial-mesenchymal transition (EMT) with loss of epithelial gene expression. To determine the potential sensor of HSATII repeat RNAs, we utilized CRISPR/Cas9 knockout of the viral RNA sensors RIG-I, MDA5, and MAVS in PDAC and CAF cells. Notably, these sensors were important for PDAC repeat RNA sensing and response, but not in CAF cells. Evaluating the innate immune pathway further downstream, we used genetic knockout of IRF3 with CRISPR/Cas9 knockout and find significant downregulation of key EMT genes that are shared with myCAF markers (ACTA2, FN1, SERPINE1). Interestingly, HSATII RNA activated IRF3 dependent EMT genes in PDAC cells, but induced IRF3 degradation in CAF cells that results in loss of myCAF gene expression. Furthermore, we found that conditioned media from HSATII transfected CAF activates EMT-related gene expression (ACTA2, FN1, SERPINE1) in PDAC cell lines, which indicates an cell extrinsic mechanism to augment EMT induction in PDAC cells. We utilized next generation spatial transcriptomic platforms NanoString GeoMx and CosMx to understand the spatial distribution of repeat RNAs in human PDAC tumors. We find that repeat RNAs can be found as a gradient from PDAC cells to the surround tumor microenvironment consistent with delivery of these RNA species. Analysis of over 300,000 individual cells in 3 PDAC tumor specimens, we find that high repeat PDAC cells have lost epithelial gene expression and high repeat CAFs have lost myCAF gene expression. Altogether, these findings support the “infection” of repeat RNAs disrupts cellular identity in both tumor cells and the CAF microenvironment as a mechanism for tumor progression. Citation Format: Eunae You, Luli Zou, Patrick Danaher, Ildiko E. Phillips, Michael J. Raabe, Bidish Patel, Amaya Pankaj, Khrystyna North, Sean Kim, Youngmi Kim, Martin Aryee, David T. Ting. Repeat RNA dysregulation of cellular states in the pancreatic cancer microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C038.

DDX50 Is a Viral Restriction Factor That Enhances IRF3 Activation.

The transcription factors IRF3 and NF-κB are crucial in innate immune signalling in response to many viral and bacterial pathogens. However, mechanisms leading to their activation remain incompletely understood. Viral RNA can be detected by RLR receptors, such as RIG-I and MDA5, and the dsRNA receptor TLR3. Alternatively, the DExD-Box RNA helicases DDX1-DDX21-DHX36 activate IRF3/NF-κB in a TRIF-dependent manner independent of RIG-I, MDA5, or TLR3. Here, we describe DDX50, which shares 55.6% amino acid identity with DDX21, as a non-redundant factor that promotes activation of the IRF3 signalling pathway following its stimulation with viral RNA or infection with RNA and DNA viruses. Deletion of DDX50 in mouse and human cells impaired IRF3 phosphorylation and IRF3-dependent endogenous gene expression and cytokine/chemokine production in response to cytoplasmic dsRNA (polyIC transfection), and infection by RNA and DNA viruses. Mechanistically, whilst DDX50 co-immunoprecipitated TRIF, it acted independently to the previously described TRIF-dependent RNA sensor DDX1. Indeed, shRNA-mediated depletion of DDX1 showed DDX1 was dispensable for signalling in response to RNA virus infection. Importantly, loss of DDX50 resulted in a significant increase in replication and dissemination of virus following infection with vaccinia virus, herpes simplex virus, or Zika virus, highlighting its important role as a broad-ranging viral restriction factor.

Abstract 1234: G4 binders as potential immunostimulatory compounds for cancer therapy

Abstract G-quadruplex (G4) is non-canonical nucleic acid structure involved in a plethora of fundamental biological processes. In past decades, it has been studied as a promising pharmaceutical target for anticancer therapy. However, no G4-targeting agent has shown significant clinical effects up to date. Pyridostatin (PDS), a well-known G4 binder, can induce double-stranded DNA breaks and genome instability. We have recently shown that PDS and other G4 binders can trigger micronuclei formation in cancer cells at non-cytotoxic concentrations [1]. As micronuclei can play a crucial role in linking genome instability to innate immunity, we have here wondered whether G4 binders can induce immune gene activation in human MCF-7 breast cancer cells. By using RNA-Seq technology, we show that non-cytotoxic doses of PDS can activate Interferon beta- and IRF3-dependent genes leading to an innate immune gene response in human cancer cells. In addition, immune gene activation follows the induction of micronuclei formation supporting cytoplasmic DNA as a trigger of immune gene activation. Immunofluorescence assays showed that cGAS (a cytoplasmic DNA sensor) can bind to and is activated by micronuclei. In addition, cytostatic PDS concentrations activate STING and IRF3 factors leading to activated immune gene expression. STING inhibition with siRNAs, CRISPR knockout or a chemical inhibitor resulted in a suppression of immune gene expression and Interferon-beta (IFN-B) production in human MCF-7 and murine B16 cell lines, showing that STING is required for PDS-induced immune gene activation in cancer cells. We have then extended the study to a structurally-unrelated G4 binder, PhenDC3, and the results show that PhenDC3 can induce micronuclei formation and IFN-B production in a STING-dependent manner. Then, using TCGA-BRCA dataset, we show that specific gene expression patterns mediated by PDS correlate with immunological hot features of human tumors, such as anti-tumor immune cell infiltration. We could also define a specific PDS gene signature that can predict a better survival of breast cancer patients, suggesting an impact of immunomodulation potential of PDS or other G4 binders in human patients. Thus, our results provide strong evidence that G4 binders can activate innate immune genes in human and murine cancer cells with therapeutic potentials, and that G4 binder-induced micronuclei accumulation can trigger the cGAS-STING signaling pathway. The findings may open to the development of novel immunotherapeutic anticancer strategies based on G4-targeting compounds.

Cerium dioxide nanoparticles exacerbate house dust mite induced type II airway inflammation

BackgroundNanomaterial inhalation represents a potential hazard for respiratory conditions such as asthma. Cerium dioxide nanoparticles (CeO2NPs) have the ability to modify disease outcome but have not been investigated for their effect on models of asthma and inflammatory lung disease. The aim of this study was to examine the impact of CeO2NPs in a house dust mite (HDM) induced murine model of asthma.ResultsRepeated intranasal instillation of CeO2NPs in the presence of HDM caused the induction of a type II inflammatory response, characterised by increased bronchoalveolar lavage eosinophils, mast cells, total plasma IgE and goblet cell metaplasia. This was accompanied by increases in IL-4, CCL11 and MCPT1 gene expression together with increases in the mucin and inflammatory regulators CLCA1 and SLC26A4. CLCA1 and SLC26A4 were also induced by CeO2NPs + HDM co-exposure in air liquid interface cultures of human primary bronchial epithelial cells. HDM induced airway hyperresponsiveness and airway remodelling in mice were not altered with CeO2NPs co-exposure. Repeated HMD instillations followed by a single exposure to CeO2NPs failed to produce changes in type II inflammatory endpoints but did result in alterations in the neutrophil marker CD177. Treatment of mice with CeO2NPs in the absence of HDM did not have any significant effects. RNA-SEQ was used to explore early effects 24 h after single treatment exposures. Changes in SAA3 expression paralleled increased neutrophil BAL levels, while no changes in eosinophil or lymphocyte levels were observed. HDM resulted in a strong induction of type I interferon and IRF3 dependent gene expression, which was inhibited with CeO2NPs co-exposure. Changes in the expression of genes including CCL20, CXCL10, NLRC5, IRF7 and CLEC10A suggest regulation of dendritic cells, macrophage functionality and IRF3 modulation as key early events in how CeO2NPs may guide pulmonary responses to HDM towards type II inflammation.ConclusionsCeO2NPs were observed to modulate the murine pulmonary response to house dust mite allergen exposure towards a type II inflammatory environment. As this type of response is present within asthmatic endotypes this finding may have implications for how occupational or incidental exposure to CeO2NPs should be considered for those susceptible to disease.

Hypothiocyanous Acid Suppresses PolyI:C-Induced Antiviral Responses by Modulating IRF3 Phosphorylation in Human Airway Epithelial Cells.

Pattern recognition receptors recognize RNA viruses and trigger type I and III interferon (IFN) production and apoptosis to limit viral replication and spread. Some innate immune cells produce oxidants in response to viral infection to protect against invasion. Recent studies have demonstrated the virucidal activity of hypothiocyanous acid (HOSCN), an oxidant generated by the peroxidase-catalyzed reaction of thiocyanate with hydrogen peroxide. However, the effects of HOSCN on host antiviral responses are still unknown. In this study, we aimed to clarify the role of HOSCN in host antiviral responses against RNA viruses in airway epithelial cells using polyinosinic-polycytidylic acid (polyI:C), a mimic of viral RNA. Our results show that HOSCN repressed antiviral responses in NCI-H292 human airway epithelial cells. HOSCN decreased polyI:C-induced apoptosis and the expression levels of IFNB1, IFNL1, IFNL2 and IFNL3 mRNAs. In addition, the induction of other interferon regulatory factor 3 (IRF3)-dependent genes was also suppressed by HOSCN. Further analyses focused on IRF3 revealed that HOSCN inhibited the phosphorylation of IRF3 at Ser386 and Ser396 as well as its dimerization and nuclear translocation by inhibiting the phosphorylation of TANK-binding kinase 1 (TBK1). Furthermore, HOSCN led to the phosphorylation of IRF3 at residues other than Ser386 and Ser396, implying that HOSCN may cause a conformational change in IRF3 to impair its function. Collectively, these results suggest that HOSCN plays a novel signaling role in the antiviral response, acting as a negative regulator of apoptotic and TBK1-IRF3 signaling pathways and limiting IRF3-dependent gene expression.

Lipopolysaccharide Adsorbed to the Bio-Corona of TiO2 Nanoparticles Powerfully Activates Selected Pro-inflammatory Transduction Pathways.

It is known that the adsorption of bioactive molecules provides engineered nanoparticles (NPs) with novel biological activities. However, the biological effects of the adsorbed molecules may also be modified by the interaction with NP. Bacterial lipopolysaccharide (LPS), a powerful pro-inflammatory compound, is a common environmental contaminant and is present in several body compartments such as the gut. We recently observed that the co-incubation of LPS with TiO2 NPs markedly potentiates its pro-inflammatory effects on murine macrophages, suggesting that, when included in a NP bio-corona, LPS activity is enhanced. To distinguish the effects of adsorbed LPS from those of the free endotoxin, a pellet fraction, denominated P25/LPS, was isolated by centrifugation from a mixture of P25 TiO2 NP (128 µg/ml) and LPS (10 ng/ml) in the presence of fetal bovine serum. Western blot analysis of the pellet eluate indicated that the P25/LPS fraction contained, besides proteins, also LPS, pointing to the presence of LPS-doped NP. The effects of adsorbed or free LPS were then compared in Raw264.7 murine macrophages. RT-PCR was used to evaluate the induction of cytokine genes, whereas active, phosphorylated isoforms of proteins involved in signaling pathways were assessed with western blot. At a nominal LPS concentration of 40 pg/ml, P25/LPS induced the expression of both NF-κB and IRF3-dependent cytokines at levels comparable with those observed with free LPS (10 ng/ml), although with different time courses. Moreover, compared to free LPS, P25/LPS caused a more sustained phosphorylation of p38 MAPK and a more prolonged induction of STAT1-dependent genes. Cytochalasin B partially inhibited the induction of Tnfa by P25/LPS, but not by free LPS, and suppressed the induction of IRF3-dependent genes by either P25/LPS or free LPS. These data suggest that, when included in the bio-corona of TiO2 NP, LPS exhibits enhanced and time-shifted pro-inflammatory effects. Thus, in assessing the hazard of NP in real life, the enhanced effects of adsorbed bioactive molecules should be taken into account.

Transcriptional and non-transcriptional functions of IRF3 in host defense

Abstract Innate immune response is the first line of host defense against microbial infection. Interferon Regulatory Factor 3 (IRF3), a critical transcription factor, is rapidly activated during virus infection to trigger numerous antiviral genes, including the interferons. Our studies have revealed that in addition to triggering these genes, IRF3 activates direct apoptosis of virus-infected cells by a newly discovered antiviral apoptotic pathway, RIPA (Chattopadhyay et al, Immunity 2016, EMBO J, 2010). In RIPA, IRF3 is differentially modified by linear polyubiquitination of two lysine residues. Moreover, a knock-in mouse strain, without the transcriptional activity of IRF3, can still mount antiviral response by its RIPA branch. Importantly, the use of pathway-specific mutants of IRF3 revealed that both transcriptional, and RIPA, branches contribute to the overall antiviral functions of IRF3. To investigate the contribution of the transcriptional branch of IRF3, we screened a shRNA library of the human ISGs. Our screen identified a small subset of novel IRF3-dependent genes, which exhibit antiviral functions in human and mouse cells. These newly-identified ISGs are protective against a wide range of clinically relevant human viruses, e.g. RSV, HPIV3, HSV-1, HCMV. In-depth investigation revealed that these ISGs regulate cellular autophagy pathway to control virus replication. The presentation will highlight how both pathways of IRF3 mount an optimum host response against viral as well as non-viral pathogenesis.

BRD4 Couples NF-κB/RelA with Airway Inflammation and the IRF-RIG-I Amplification Loop in Respiratory Syncytial Virus Infection.

The airway mucosa expresses protective interferon (IFN) and inflammatory cytokines in response to respiratory syncytial virus (RSV) infection. In this study, we examine the role of bromodomain containing 4 (BRD4) in mediating this innate immune response in human small airway epithelial cells. We observe that RSV induces BRD4 to complex with NF-κB/RelA. BRD4 is functionally required for expression of the NF-κB-dependent inflammatory gene regulatory network (GRN), including the IFN response factor 1 (IRF1) and IRF7, which mediate a cross talk pathway for RIG-I upregulation. Mechanistically, BRD4 is required for cyclin-dependent kinase 9 (CDK9) recruitment and phospho-Ser 2 carboxy-terminal domain (CTD) RNA polymerase (Pol) II formation on the promoters of IRF1, IRF7, and RIG-I, producing their enhanced expression by transcriptional elongation. We also find that BRD4 independently regulates CDK9/phospho-Ser 2 CTD RNA Pol II recruitment to the IRF3-dependent IFN-stimulated genes (ISGs). In vivo, poly(I·C)-induced neutrophilia and mucosal chemokine production are blocked by a small-molecule BRD4 bromodomain inhibitor. Similarly, BRD4 inhibition reduces RSV-induced neutrophilia, mucosal CXC chemokine expression, activation of the IRF7-RIG-I autoamplification loop, mucosal IFN expression, and airway obstruction. RSV infection activates BRD4 acetyltransferase activity on histone H3 Lys (K) 122, demonstrating that RSV infection activates BRD4 in vivo These data validate BRD4 as a major effector of RSV-induced inflammation and disease. BRD4 is required for coupling NF-κB to expression of inflammatory genes and the IRF-RIG-I autoamplification pathway and independently facilitates antiviral ISG expression. BRD4 inhibition may be a strategy to reduce exuberant virus-induced mucosal airway inflammation.IMPORTANCE In the United States, 2.1 million children annually require medical attention for RSV infections. A first line of defense is the expression of the innate gene network by infected epithelial cells. Expression of the innate response requires the recruitment of transcriptional elongation factors to rapidly induce innate response genes through an unknown mechanism. We discovered that RSV infection induces a complex of bromodomain containing 4 (BRD4) with NF-κB and cyclin-dependent kinase 9 (CDK9). BRD4 is required for stable CDK9 binding, phospho-Ser 2 RNA Pol II formation, and histone acetyltransferase activity. Inhibition of BRD4 blocks Toll-like receptor 3 (TLR3)-dependent neutrophilia and RSV-induced inflammation, demonstrating its importance in the mucosal innate response in vivo Our study shows that BRD4 plays a central role in inflammation and activation of the IRF7-RIG-I amplification loop vital for mucosal interferon expression. BRD4 inhibition may be a strategy for modulating exuberant mucosal airway inflammation.

HIV Vpu Interferes with NF-κB Activity but Not with Interferon Regulatory Factor 3.

The accessory HIV protein Vpu inhibits a number of cellular pathways that trigger host innate restriction mechanisms. HIV Vpu-mediated degradation of tetherin allows efficient particle release and hampers the activation of the NF-κB pathway thereby limiting the expression of proinflammatory genes. In addition, Vpu reduces cell surface expression of several cellular molecules such as newly synthesized CD4. However, the role of HIV Vpu in regulating the type 1 interferon response to viral infection by degradation of the interferon regulatory factor 3 (IRF3) has been subject of conflicting reports. We therefore systematically investigated the expression of IRF3 in primary CD4(+) T cells and macrophages infected with HIV at different time points. In addition, we also tested the ability of Vpu to interfere with innate immune signaling pathways such as the NF-κB and the IRF3 pathways. We report here that HIV Vpu failed to degrade IRF3 in infected primary cells. Moreover, we observed that HIV NL4.3 Vpu had no effect on IRF3-dependent gene expression in reporter assays. On the other hand, HIV NL4.3 Vpu downmodulated NF-κB-dependent transcription. Mutation of two serines (positions 52 and 56) involved in the binding of NL4.3 Vpu to the βTrCP ubiquitin ligase abolishes its ability to inhibit NF-κB activity. Taken together, these results suggest that HIV Vpu regulates antiviral innate response in primary human cells by acting specifically on the NF-κB pathway. HIV Vpu plays a pivotal role in enhancing HIV infection by counteraction of Tetherin. However, Vpu also regulates host response to HIV infection by hampering the type 1 interferon response. The molecular mechanism by which Vpu inhibits the interferon response is still controversial. Here we report that Vpu affects interferon expression by inhibiting NF-κB activity without affecting IRF3 levels or activity. These data suggest that Vpu facilitates HIV infection by regulating NF-κB transcription to levels sufficient for viral transcription while limiting cellular responses to infection.

A role for APPL1 in TLR3/4-dependent TBK1 and IKKε activation in macrophages.

Endosomes have important roles in intracellular signal transduction as a sorting platform. Signaling cascades from TLR engagement to IRF3-dependent gene transcription rely on endosomes, yet the proteins that specifically recruit IRF3-activating molecules to them are poorly defined. We show that adaptor protein containing a pleckstrin-homology domain, a phosphotyrosine-binding domain, and a leucine zipper motif (APPL)1, an early endosomal protein, is required for both TRIF- and retinoic acid-inducible gene 1-dependent signaling cascades to induce IRF3 activation. APPL1, but not early endosome Ag 1, deficiency impairs IRF3 target gene expression upon engagement of both TLR3 and TLR4 pathways, as well as in H1N1-infected macrophages. The IRF3-phosphorylating kinases TBK1 and IKKε are recruited to APPL1 endosomes in LPS-stimulated macrophages. Interestingly, APPL1 undergoes proteasome-mediated degradation through ERK1/2 to turn off signaling. APPL1 degradation is blocked when signaling through the endosome is inhibited by chloroquine or dynasore. Therefore, APPL1 endosomes are critical for IRF3-dependent gene expression in response to some viral and bacterial infections in macrophages. Those signaling pathways involve the signal-induced degradation of APPL1 to prevent aberrant IRF3-dependent gene expression linked to immune diseases.

The Small GTPase Arf6 Is Essential for the Tram/Trif Pathway in TLR4 Signaling

Recognition of lipopolysaccharides (LPS) by Toll-like receptor 4 (TLR4) at the plasma membrane triggers NF-κB activation through recruitment of the adaptor proteins Mal and MyD88. Endocytosis of the activated TLR4 allows recruitment of the adaptors Tram and Trif, leading to activation of the transcription factor IRF3 and interferon production. The small GTPase ADP-ribosylation factor 6 (Arf6) was shown to regulate the plasma membrane association of Mal. Here we demonstrate that inhibition of Arf6 also markedly reduced LPS-induced cytokine production in Mal(-/-) mouse macrophages. In this article, we focus on a novel role for Arf6 in the MyD88-independent TLR4 pathway. MyD88-independent IRF3 activation and IRF3-dependent gene transcription were strictly dependent on Arf6. Arf6 was involved in transport of Tram to the endocytic recycling compartment and internalization of LPS, possibly explaining its requirement for LPS-induced IRF3 activation. Together, these results show a critical role for Arf6 in regulating Tram/Trif-dependent TLR4 signaling.

Caspase-8 Blocks Kinase RIPK3-Mediated Activation of the NLRP3 Inflammasome

Caspase-8 deficiency in certain cells prompts chronic inflammation. One mechanism suggested to account for this inflammation is enhanced signaling for necrotic cell death, mediated by the protein kinases RIPK1 and RIPK3 that caspase-8 can cleave. We describe an activity of caspase-8 in dendritic cells that controls the initiation of inflammation in another way. Caspase-8 deficiency in thesecells facilitated lipopolysaccharide-induced assembly and function of the NLRP3 inflammasome. This effect depended on the functions of RIPK1 and RIPK3, as well as of MLKL and PGAM5, two signaling proteins recently shown to contribute to RIPK3-mediated induction of necrosis. However, although enhancement of inflammasome assembly in the caspase-8-deficient cells shares proximal signaling events with the induction of necrosis, it occurred independently of cell death. These findings provide new insight into potentially pathological inflammatory processes to which RIPK1- and RIPK3-mediated signaling contributes.

Hepatitis C Virus Reveals a Novel Early Control in Acute Immune Response

Recognition of viral RNA structures by the intracytosolic RNA helicase RIG-I triggers induction of innate immunity. Efficient induction requires RIG-I ubiquitination by the E3 ligase TRIM25, its interaction with the mitochondria-bound MAVS protein, recruitment of TRAF3, IRF3- and NF-κB-kinases and transcription of Interferon (IFN). In addition, IRF3 alone induces some of the Interferon-Stimulated Genes (ISGs), referred to as early ISGs. Infection of hepatocytes with Hepatitis C virus (HCV) results in poor production of IFN despite recognition of the viral RNA by RIG-I but can lead to induction of early ISGs. HCV was shown to inhibit IFN production by cleaving MAVS through its NS3/4A protease and by controlling cellular translation through activation of PKR, an eIF2α-kinase containing dsRNA-binding domains (DRBD). Here, we have identified a third mode of control of IFN induction by HCV. Using HCVcc and the Huh7.25.CD81 cells, we found that HCV controls RIG-I ubiquitination through the di-ubiquitine-like protein ISG15, one of the early ISGs. A transcriptome analysis performed on Huh7.25.CD81 cells silenced or not for PKR and infected with JFH1 revealed that HCV infection leads to induction of 49 PKR-dependent genes, including ISG15 and several early ISGs. Silencing experiments revealed that this novel PKR-dependent pathway involves MAVS, TRAF3 and IRF3 but not RIG-I, and that it does not induce IFN. Use of PKR inhibitors showed that this pathway requires the DRBD but not the kinase activity of PKR. We then demonstrated that PKR interacts with HCV RNA and MAVS prior to RIG-I. In conclusion, HCV recruits PKR early in infection as a sensor to trigger induction of several IRF3-dependent genes. Among those, ISG15 acts to negatively control the RIG-I/MAVS pathway, at the level of RIG-I ubiquitination.These data give novel insights in the machinery involved in the early events of innate immune response.

The role of TBK1 and IKKϵ in the expression and activation of Pellino 1

Mammalian Pellino isoforms are phosphorylated by IRAK (interleukin receptor associated kinase) 1/IRAK4 in vitro, converting them into active E3 ubiquitin ligases. In the present paper we report a striking enhancement in both transcription of the gene encoding Pellino 1 and Pellino 1 protein expression when murine BMDMs (bone-marrow-derived macrophages) are stimulated with LPS (lipopolysaccharide) or poly(I:C). This induction occurs via a TRIF [TIR (Toll/interleukin-1 receptor)-domain-containing adaptor-inducing interferon-β]-dependent IRAK-independent pathway and is prevented by inhibition of the IKK [IκB (inhibitor of nuclear factor κB) kinase]-related protein kinases, TBK1 {TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)-associated nuclear factor κB activator]-binding kinase 1} and IKKε. Pellino 1 is not induced in IRF3 (interferon regulatory factor 3)-/- BMDMs, and its induction is only reduced slightly in type 1 interferon receptor-/- BMDMs, identifying Pellino 1 as a new IRF3-dependent gene. We also identify Pellino 1 in a two-hybrid screen using IKKε as bait, and show that IKKε/TBK1 activate Pellino 1 in vitro by phosphorylating Ser76, Thr288 and Ser293. Moreover, we show that the E3 ligase activity of endogenous Pellino 1 is activated in LPS- or poly(I:C)-stimulated macrophages. This occurs more rapidly than the increase in Pellino 1 mRNA and protein expression, is prevented by the inhibition of IKKε/TBK1 and is reversed by phosphatase treatment. Thus IKKε/TBK1 mediate the activation of Pellino 1's E3 ligase activity, as well as inducing the transcription of its gene and protein expression in response to TLR3 and TLR4 agonists.

Chikungunya Virus Induces IPS-1-Dependent Innate Immune Activation and Protein Kinase R-Independent Translational Shutoff

Chikungunya virus (CHIKV) is an arthritogenic mosquito-transmitted alphavirus that is undergoing reemergence in areas around the Indian Ocean. Despite the current and potential danger posed by this virus, we know surprisingly little about the induction and evasion of CHIKV-associated antiviral immune responses. With this in mind we investigated innate immune reactions to CHIKV in human fibroblasts, a demonstrable in vivo target of virus replication and spread. We show that CHIKV infection leads to activation of the transcription factor interferon regulatory factor 3 (IRF3) and subsequent transcription of IRF3-dependent antiviral genes, including beta interferon (IFN-β). IRF3 activation occurs by way of a virus-induced innate immune signaling pathway that includes the adaptor molecule interferon promoter stimulator 1 (IPS-1). Despite strong transcriptional upregulation of these genes, however, translation of the corresponding proteins is not observed. We further demonstrate that translation of cellular (but not viral) genes is blocked during infection and that although CHIKV is found to trigger inactivation of the translational molecule eukaryotic initiation factor subunit 2α by way of the double-stranded RNA sensor protein kinase R, this response is not required for the block to protein synthesis. Furthermore, overall diminution of cellular RNA synthesis is also observed in the presence of CHIKV and transcription of IRF3-dependent antiviral genes appears specifically blocked late in infection. We hypothesize that the observed absence of IFN-β and antiviral proteins during infection results from an evasion mechanism exhibited by CHIKV that is dependent on widespread shutoff of cellular protein synthesis and a targeted block to late synthesis of antiviral mRNA transcripts.

Activation of the Interferon Response by Human Cytomegalovirus Occurs via Cytoplasmic Double-Stranded DNA but Not Glycoprotein B

In vitro infection of cells with the betaherpesvirus human cytomegalovirus (HCMV) stimulates an innate immune response characterized by phosphorylation of the transcription factor interferon regulatory factor 3 (IRF3) and subsequent expression of IRF3-dependent genes. While previous work suggests that HCMV envelope glycoprotein B is responsible for initiating this reaction, the signaling pathways stimulated by virus infection that lead to IRF3 phosphorylation have largely been uncharacterized. Recently, we identified Z DNA binding protein 1 (ZBP1), a sensor of cytoplasmic DNA, as an essential protein for this response. We now describe a human fibroblast cell line exhibiting a recessive defect that results in the absence of activation of IRF3 following treatment with HCMV but not Sendai virus or double-stranded RNA. In addition, we show that while exposure of these cells to soluble HCMV glycoprotein B is capable of triggering IRF3-dependent gene transcription, transfection of the cells with double-stranded DNA is not. Furthermore, we show that overexpression of ZBP1 in these cells reestablishes their ability to secrete interferon in response to HCMV and that multiple ZBP1 transcriptional variants exist in both wild-type and mutant cells. These results have two major implications for the understanding of innate immune stimulation by HCMV. First, they demonstrate that HCMV glycoprotein B is not the essential molecular pattern that induces an IRF3-dependent innate immune response. Second, IRF3-terminal signaling triggered by HCMV particles closely resembles that which is activated by cytoplasmic double-stranded DNA.

Human T Cell Lymphotropic Virus 1 Manipulates Interferon Regulatory Signals by Controlling the TAK1-IRF3 and IRF4 Pathways

We previously reported that human T cell lymphotropic virus 1 (HTLV-1) Tax oncoprotein constitutively activates transforming growth factor-beta-activated kinase 1 (TAK1). Here, we established Tax-positive HuT-102 cells stably transfected with a short hairpin RNA vector (HuT-shTAK1 cells) and investigated the physiological function of TAK1. Microarray analysis demonstrated that several interferon (IFN)-inducible genes, including chemokines such as CXCL10 and CCL5, were significantly down-regulated in HuT-shTAK1 cells. In contrast, Tax-mediated constitutive activation of nuclear factor-kappaB (NF-kappaB) was intact in HuT-shTAK1 cells. IFN-regulatory factor 3 (IRF3), a critical transcription factor in innate immunity to viral infection, was constitutively activated in a Tax-dependent manner. Activation of IRF3 and IRF3-dependent gene expressions was dependent on TAK1 and TANK-binding kinase 1 (TBK1). On the other hand, IRF4, another member in the IRF family of transcription factors overexpressed in a Tax-independent manner, negatively regulated TAK1-dependent IRF3 transcriptional activity. Together, HTLV-1 manipulates IFN signaling by regulating both positive and negative IRFs.

TAG, a splice variant of the adaptor TRAM, negatively regulates the adaptor MyD88–independent TLR4 pathway

Toll-like receptor 4 (TLR4) signals the induction of transcription factor IRF3-dependent genes from the early endosome via the adaptor TRAM. Here we report a splice variant of TRAM, TAG ('TRAM adaptor with GOLD domain'), which has a Golgi dynamics domain coupled to TRAM's Toll-interleukin 1 receptor domain. After stimulation with lipopolysaccharide, TRAM and TAG localized to late endosomes positive for the GTPase Rab7a. TAG inhibited activation of IRF3 by lipopolysaccharide. Knockdown of TAG with small interfering RNA enhanced induction of the chemokine CCL5 (RANTES), but not of interleukin 8, by lipopolysaccharide in human peripheral blood mononuclear cells. TAG displaced the adaptor TRIF from TRAM. TAG is therefore an example of a specific inhibitor of the adaptor MyD88-independent pathway activated by TLR4. Targeting TAG could be useful in the effort to boost the immunostimulatory effect of TLR4 without causing unwanted inflammation.