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Patients with chronic hepatitis B (CHB) undergoing interferon (IFN)-α-based therapies often exhibit a poor HBeAg serological response. Thus, there is an unmet need for new therapies aimed at CHB. This study comprised two clinical trials, including 130 CHB patients, who were treatment-naïve; in the first, 92 patients were systematically analyzed ex vivo for interleukin-2 receptor (IL-2R) expression and inhibitory molecules expression after receiving Peg-IFN-α-2b therapy. In our second clinical trial, 38 non-responder patients, in whom IFN-α therapy had failed, were treated with or without low-dose IL-2 for 24 weeks. We then examined the hepatitis B virus (HBV)-specific CD8+ T-cell response and the clinical outcome, in these patients. Although the majority of the participants undergoing Peg-IFN-α-2b therapy were non-responders, we observed a decrease in CD25 expression on their CD4+ T cells, suggesting that IFN-α therapy may provide a rationale for sequential IL-2 treatment without increasing regulatory T cells (Tregs). Following sequential therapy with IL-2, we demonstrated that the non-responders experienced a decrease in the numbers of Tregs and programmed cell death protein 1 (PD-1) expression. In addition, sequential IL-2 administration rescued effective immune function, involving signal transducer and activator of transcription 1 (STAT1) activation. Importantly, IL-2 therapy significantly increased the frequency and function of HBV-specific CD8+ T cells, which translated into improved clinical outcomes, including HBeAg seroconversion, among the non-responder CHB patients. Our findings suggest that sequential IL-2 therapy shows efficacy in rescuing immune function in non-responder patients with refractory CHB.

Acute exposure of cancer cells to high concentrations of type I interferon (IFN-I) drives growth arrest and apoptosis, whereas chronic exposure to low concentrations provides important prosurvival advantages. Tyrosine-phosphorylated IFN-stimulated gene (ISG) factor 3 (ISGF3) drives acute deleterious responses to IFN-I, whereas unphosphorylated (U-)ISGF3, lacking tyrosine phosphorylation, drives essential constitutive prosurvival mechanisms. Surprisingly, programmed cell death-ligand 1 (PD-L1), often expressed on the surfaces of tumor cells and well recognized for its importance in inactivating cytotoxic T cells, also has important cell-intrinsic protumor activities, including dampening acute responses to cytotoxic high levels of IFN-I and sustaining the expression of the low levels that benefit tumors. More thorough understanding of the newly recognized complex roles of IFN-I in cancer may lead to the identification of novel therapeutic strategies.

Background Dysregulation of transcription and cytokine expression has been implicated in the pathogenesis of a variety inflammatory diseases. The resulting imbalance between inflammatory and resolving transcriptional programs can cause an overabundance of pro-inflammatory, classically activated macrophage type 1 (M1) and/or helper T cell type 1 (Th1) products, such as IFNγ, TNFα, IL1-β, and IL12, that prevent immune switching to resolution and healing. The low molecular weight fraction of human serum albumin (LMWF5A) is a novel biologic drug that is currently under clinical investigation for the treatment of osteoarthritis and the hyper-inflammatory response associated with COVID-19. This study aims to elucidate transcriptional mechanisms of action involved with the ability of LMWF5A to reduce pro-inflammatory cytokine release. Methods ELISA arrays were used to identify cytokines and chemokines influenced by LMWF5A treatment of LPS-stimulated peripheral blood mononuclear cells (PBMC). The resulting profiles were analyzed by gene enrichment to gain mechanistic insight into the biologic processes and transcription factors (TFs) underlying the identified differentially expressed cytokines. DNA-binding ELISAs, luciferase reporter assays, and TNFα or IL-1β relative potency were then employed to confirm the involvement of enriched pathways and TFs. Results LMWF5A was found to significantly inhibit a distinct set of pro-inflammatory cytokines (TNFα, IL-1β, IL-12, CXCL9, CXCL10, and CXCL11) associated with pro-inflammatory M1/Th1 immune profiles. Gene enrichment analysis also suggests these cytokines are, in part, regulated by NF-κB and STAT transcription factors. Data from DNA-binding and reporter assays support this with LMWF5A inhibition of STAT1α DNA-binding activity as well as a reduction in overall NF-κB-driven luciferase expression. Experiments using antagonists specific for the immunomodulatory and NF-κB/STAT-repressing transcription factors, peroxisome proliferator-activated receptor (PPAR)γ and aryl hydrocarbon receptor (AhR), indicate these pathways are involved in the LMWF5A mechanisms of action by reducing LMWF5A drug potency as measured by TNFα and IL-1β release. Conclusion In this report, we provide evidence that LMWF5A reduces pro-inflammatory cytokine release by activating the immunoregulatory transcription factors PPARγ and AhR. In addition, our data indicate that LMWF5A suppresses NF-κB and STAT1α pro-inflammatory pathways. This suggests that LMWF5A acts through these mechanisms to decrease pro-inflammatory transcription factor activity and subsequent inflammatory cytokine production.

Suppressing cellular signal transducers of transcription 2 (STAT2) is a common strategy that viruses use to establish infections, yet the detailed mechanism remains elusive, owing to a lack of structural information about the viral-cellular complex involved. Here, we report the cryo-EM and crystal structures of human STAT2 (hSTAT2) in complex with the non-structural protein 5 (NS5) of Zika virus (ZIKV) and dengue virus (DENV), revealing two-pronged interactions between NS5 and hSTAT2. First, the NS5 methyltransferase and RNA-dependent RNA polymerase (RdRP) domains form a conserved interdomain cleft harboring the coiled-coil domain of hSTAT2, thus preventing association of hSTAT2 with interferon regulatory factor 9. Second, the NS5 RdRP domain also binds the amino-terminal domain of hSTAT2. Disruption of these ZIKV NS5-hSTAT2 interactions compromised NS5-mediated hSTAT2 degradation and interferon suppression, and viral infection under interferon-competent conditions. Taken together, these results clarify the mechanism underlying the functional antagonism of STAT2 by both ZIKV and DENV.

Significance Macrophages regulate many aspects of the innate immune response and the activation of adaptive immunity following exposure to microbial ligands. However, macrophages can also contribute to inflammation underlying diseases such as atherosclerosis and obesity. Epigenetic regulators control inflammatory gene regulation and, as such, are potential targets for modulation of the inflammatory response. Here, we show that inhibitors and degraders of the bromodomain protein BRD9, a subunit of the noncanonical BAF complex, limit inflammation by specifically blocking the induction of interferon-stimulated genes. This effect overlaps with the transcriptional responses with the BET inhibitor JQ1 but affects fewer genes and is more specific in scope. Our results suggest that BRD9 inhibitors/degraders may be therapeutically relevant agents to limit interferon-associated inflammation., Macrophages induce a number of inflammatory response genes in response to stimulation with microbial ligands. In response to endotoxin Lipid A, a gene-activation cascade of primary followed by secondary-response genes is induced. Epigenetic state is an important regulator of the kinetics, specificity, and mechanism of gene activation of these two classes. In particular, SWI/SNF chromatin-remodeling complexes are required for the induction of secondary-response genes, but not primary-response genes, which generally exhibit open chromatin. Here, we show that a recently discovered variant of the SWI/SNF complex, the noncanonical BAF complex (ncBAF), regulates secondary-response genes in the interferon (IFN) response pathway. Inhibition of bromodomain-containing protein 9 (BRD9), a subunit of the ncBAF complex, with BRD9 bromodomain inhibitors (BRD9i) or a degrader (dBRD9) led to reduction in a number of interferon-stimulated genes (ISGs) following stimulation with endotoxin lipid A. BRD9-dependent genes overlapped highly with a subset of genes differentially regulated by BET protein inhibition with JQ1 following endotoxin stimulation. We find that the BET protein BRD4 is cobound with BRD9 in unstimulated macrophages and corecruited upon stimulation to ISG promoters along with STAT1, STAT2, and IRF9, components of the ISGF3 complex activated downstream of IFN-alpha receptor stimulation. In the presence of BRD9i or dBRD9, STAT1-, STAT2-, and IRF9-binding is reduced, in some cases with reduced binding of BRD4. These results demonstrate a specific role for BRD9 and the ncBAF complex in ISG activation and identify an activity for BRD9 inhibitors and degraders in dampening endotoxin- and IFN-dependent gene expression.

Highlights • IFN stimulated binding of ISRE by ISGF3 is cell specific, particularly for ISRE in enhancer regions. • IFN stimulated binding of ISRE in enhancer regions associates with differential expression. • The homeostatic, chromatin state of an ISRE is predictive of IFN stimulated binding., The type I interferon (IFN) signaling pathway involves binding of the transcription factor ISGF3 to IFN-stimulated response elements, ISREs. Gene expression under IFN stimulation is known to vary across cell types, but variation in ISGF3 binding to ISRE across cell types has not been characterized. We examined ISRE binding patterns under IFN stimulation across six cell types using existing ChIPseq datasets. We find that ISRE binding is largely cell specific for ISREs distal to transcription start sites (TSS) and largely conserved across cell types for ISREs proximal to TSS. We show that bound ISRE distal to TSS associate with differential expression of ISGs, although at weaker levels than bound ISRE proximal to TSS. Using existing ATACseq and ChIPseq datasets, we show that the chromatin state of ISRE at homeostasis is cell type specific and is predictive of cell specific, ISRE binding under IFN stimulation. Our results support a model in which the chromatin state of ISRE in enhancer elements is modulated in a cell type specific manner at homeostasis, leading to cell type specific differences in ISRE binding patterns under IFN stimulation.

We previously identified a subset of interferon-stimulated genes (ISGs) upregulated by West Nile virus (WNV) infection in wild-type mouse embryo fibroblasts (MEFs) after viral proteins had inhibited type I interferon (IFN)-mediated JAK-STAT signaling and also in WNV-infected RIG-I-/-, MDA5-/-, STAT1-/-, STAT2-/-, IFNAR-/-, IRF3-/-, IRF7-/-, and IRF3/7-/- MEFs. In this study, ISG upregulation by WNV infection in IFNAR-/- MEFs was confirmed by transcriptome sequencing (RNA-seq). ISG upregulation by WNV infection was inhibited in RIG-I/MDA5-/- MEFs. ISGs were upregulated in IRF1-/- and IRF5-/- MEFs but only minimally upregulated in IRF3/5/7-/- MEFs, suggesting redundant IRF involvement. We previously showed that a single proximal interferon-stimulated response element (ISRE) in the Oas1a and Oas1b promoters bound the ISGF3 complex after type I IFN treatment. In this study, we used wild-type and mutant promoter luciferase reporter constructs to identify critical regions in the Oas1b and Ifit1 promoters for gene activation in infected IFNAR-/- MEFs. Two ISREs were required in both promoters. Mutation of these ISREs in an Ifit1 promoter DNA probe reduced in vitro complex formation with infected nuclear extracts. An NF-κB inhibitor decreased Ifit1 promoter activity in cells and in vitro complex formation. IRF3 and p50 promoter binding was detected by chromatin immunoprecipitation (ChIP) for upregulated ISGs with two proximal ISREs. The data indicate that ISREs function cooperatively to upregulate the expression of some ISGs when type I IFN signaling is absent, with the binding complex consisting of IRF3, IRF5, and/or IRF7 and an NF-κB component(s) as well as other, as-yet-unknown factors. IMPORTANCE Type I IFN signaling in mammalian cells induces formation of the ISGF3 transcription factor complex, which binds to interferon stimulated response elements (ISREs) in the promoters of interferon-stimulated genes (ISGs) in the cell nucleus. Flavivirus proteins counteract type I IFN signaling by preventing either the formation or nuclear localization of ISGF3. A subset of ISRE-regulated ISGs was still induced in West Nile virus (WNV)-infected mouse embryo fibroblasts (MEFs), indicating that cells have an alternative mechanism for activating these ISGs. In this study, cellular components involved in this ISG upregulation mechanism were identified using gene knockout MEFs and ChIP, and critical promoter regions for gene activation were mapped using reporter assays. The data indicate a cooperative function between two ISREs and required binding of IRF3, IRF5, and/or IRF7 and an NF-κB component(s). Moreover, type I IFN signaling-independent ISG activation requires different additional promoter activation regions than type I IFN-dependent activation.

Recently, 94 inborn errors of immunity (IEI) patients suffering from COVID-19 have been described, overall demonstrating a mild phenotype [1] although more severe disease manifestations have been suggested for patients with alterations in the interferon (IFN) signaling pathway, including auto-antibodies against type I IFN [2]. Patients with STAT1 GOF mutations show a complex and often severe phenotype, combining an increased susceptibility of fungal, (myco-) bacterial and viral infections as well as autoimmune and autoinflammatory manifestations [3]. Characteristically, in response to type I and type II IFN stimulation, these patients show STAT1 hyperphosphorylation [3, 4]. Whether in the context of SARS-CoV-2 infection, the hyperactivation of the IFN-JAK1/2-STAT1 pathway would be protective (antiviral effect) or deleterious (hyperinflammation) is unclear. Ruxolitinib (a selective JAK1/2 inhibitor) has been successfully used in STAT1 GOF patients controlling many disease manifestations [5] and also resulted in improved pulmonary function and faster recovery from lymphopenia in previously healthy individuals suffering from severe COVID-19 [6]., This work was supported by the Instituto de Salud Carlos III, Madrid (Spain) [Sara Borrell, CD20/00124 to P.B.L, Juan Rodés JR18/00042 to P.O, FIS PI19/01471 to O.N] and the Consejería de Salud, Junta Andalucía [SA0051/2020 to O.N]. A.G-V was supported by the Instituto de Salud Carlos III, cofinanced by the European Development Regional Fund (“A way to achieve Europe”), Subprograma Miguel Servet (CP19/00159).

Interferon β (IFN-β) signaling activates the transcription factor complex ISGF3 to induce gene expression programs critical for antiviral defense and host immune responses. It has also been observed that IFN-β activates a second transcription factor complex, γ-activated factor (GAF), but the significance of this coordinated activation is unclear. We report that in murine lung epithelial cells (MLE12) high doses of IFN-β indeed activate both ISGF3 and GAF, which bind to distinct genomic locations defined by their respective DNA sequence motifs. In contrast, low doses of IFN-β preferentially activate ISGF3 but not GAF. Surprisingly, in MLE12 cells GAF binding does not induce nearby gene expression even when strongly bound to the promoter. Yet expression of interferon stimulated genes is enhanced when GAF and ISGF3 are both active compared to ISGF3 alone. We propose that GAF may function as a dose-sensitive amplifier of ISG expression to enhance antiviral immunity and establish pro-inflammatory states.

Stimulation of cells by type I interferons (IFN) leads to the differential expression of 100s of genes known as interferon stimulated genes, ISGs. The collection of ISGs differentially expressed under IFN stimulation, referred to as the IFN signature, varies across cell types. Non-canonical IFN signaling has been clearly associated with variation in IFN signature across cell types, but the existence of variation in canonical signaling and its impact on IFN signatures is less clear. The canonical IFN signaling pathway involves binding of the transcription factor ISGF3 to IFN-stimulated response elements, ISREs. We examined ISRE binding patterns under IFN stimulation across six cell types using existing ChIPseq datasets available on the GEO and ENCODE databases. We find that ISRE binding is cell specific, particularly for ISREs distal to transcription start sites, potentially associated with enhancer elements, while ISRE binding in promoter regions is more conserved. Given variation of ISRE binding across cell types, we investigated associations between the cell type, homeostatic state and ISRE binding patterns. Taking a machine learning approach and using existing ATACseq and ChIPseq datasets available on GEO and ENCODE, we show that the epigenetic state of an ISRE locus at homeostasis and the DNA sequence of the ISRE locus are predictive of the ISRE’s binding under IFN stimulation in a cell type, specific manner, particularly for ISRE distal to transcription start sites.

Natural killer (NK) cells are important components of the innate immune defense against infections and cancers. Signal transducer and activator of transcription 1 (STAT1) is a transcription factor that is essential for NK cell maturation and NK cell-dependent tumor surveillance. Two alternatively spliced isoforms of STAT1 exist: a full-length STAT1α and a C-terminally truncated STAT1β isoform. Aberrant splicing is frequently observed in cancer cells and several anti-cancer drugs interfere with the cellular splicing machinery. To investigate whether NK cell-mediated tumor surveillance is affected by a switch in STAT1 splicing, we made use of knock-in mice expressing either only the STAT1α (Stat1α/α) or the STAT1β (Stat1β/β ) isoform. NK cells from Stat1α/α mice matured normally and controlled transplanted tumor cells as efficiently as NK cells from wild-type mice. In contrast, NK cells from Stat1β/β mice showed impaired maturation and effector functions, albeit less severe than NK cells from mice that completely lack STAT1 (Stat1-/- ). Mechanistically, we show that NK cell maturation requires the presence of STAT1α in the niche rather than in NK cells themselves and that NK cell maturation depends on IFNγ signaling under homeostatic conditions. The impaired NK cell maturation in Stat1β/β mice was paralleled by decreased IL-15 receptor alpha (IL-15Rα) surface levels on dendritic cells, macrophages and monocytes. Treatment of Stat1β/β mice with exogenous IL-15/IL-15Rα complexes rescued NK cell maturation but not their effector functions. Collectively, our findings provide evidence that STAT1 isoforms are not functionally redundant in regulating NK cell activity and that the absence of STAT1α severely impairs, but does not abolish, NK cell-dependent tumor surveillance.

The mammalian genome comprises nuclear DNA (nDNA) derived from both parents and mitochondrial DNA (mtDNA) that is maternally inherited and encodes essential proteins required for oxidative phosphorylation. Thousands of copies of the circular mtDNA are present in most cell types that are packaged by TFAM into higher-order structures called nucleoids1. Mitochondria are also platforms for antiviral signalling2 and, due to their bacterial origin, mtDNA and other mitochondrial components trigger innate immune responses and inflammatory pathology2,3. We showed previously that cytoplasmic release of mtDNA activates the cGAS–STING–TBK1 pathway resulting in interferon-stimulated gene (ISG) expression that promotes antiviral immunity4. Here, we find that persistent mtDNA stress is not associated with basally activated NF-κB signalling or interferon gene expression typical of an acute antiviral response. Instead, a specific subset of ISGs that includes Parp9 remains activated by the unphosphorylated form of ISGF3 that enhances nDNA damage and repair responses. In cultured primary fibroblasts and cancer cells, the chemotherapeutic drug doxorubicin causes mtDNA damage and release, which leads to cGAS–STING–dependent ISG activation. In addition, mtDNA stress in TFAM-deficient mouse melanoma cells produces tumours that are more resistant to doxorubicin in vivo. Finally, Tfam+/− mice exposed to ionizing radiation exhibit enhanced nDNA repair responses in spleen. Therefore, we propose that damage to and subsequent release of mtDNA elicits a protective signalling response that enhances nDNA repair in cells and tissues, suggesting that mtDNA is a genotoxic stress sentinel. Persistent mitochondrial DNA stress is shown to upregulate nuclear DNA damage and repair responses via activation of the cGAS–STING pathway and a subset of interferon-stimulated genes.

Platelet-derived growth factor receptor-β (PDGFR-β) has been reported to promote phenotypic transformation of vascular smooth muscle cells (VSMCs). The purpose of this study was to investigate the role of the PDGFR-β/IRF9/SIRT-1/NF-κB pathway in VSMC phenotypic transformation after subarachnoid hemorrhage (SAH). SAH was induced using the endovascular perforation model in Sprague-Dawley rats. PDGFR-β small interfering RNA (siRNA) and IRF9 siRNA were injected intracerebroventricularly 48 h before SAH. SIRT1 activator (resveratrol) and inhibitor (EX527) were administered intraperitoneally 1 h after SAH induction. Twenty-four hours after SAH, the VSMC contractile phenotype marker α-smooth muscle actin (α-SMA) decreased, whereas the VSMC synthetic phenotype marker embryonic smooth muscle myosin heavy chain (Smemb) increased. Both PDGFR-β siRNA and IRF9 siRNA attenuated the induction of nuclear factor-κB (NF-κB) and enhanced the expression of α-SMA. The SIRT1 activator (resveratrol) preserved VSMC contractile phenotype, significantly alleviated neurological dysfunction, and reduced brain edema. However, these beneficial effects of PDGFR-β siRNA, IRF9 siRNA and resveratrol were abolished by the SIRT1 inhibitor (EX527). This study shows that PDGFR-β/IRF9/SIRT-1/NF-κB signaling played a role in the VSMC phenotypic transformation after SAH. Inhibition of this signaling cascade preserved the contractile phenotype of VSMCs, thereby improving neurological outcomes following SAH.

The survival rate for pancreatic ductal adenocarcinoma (PDAC) remains low. More therapeutic options to treat this disease are needed, for the current standard of care is ineffective. Using an animal model of aggressive PDAC (Kras/p48TGFβRIIKO), we discovered an effect of TGFβ signaling in regulation of G-CSF secretion in pancreatic epithelium. Elevated concentrations of G-CSF in PDAC promoted differentiation of Ly6G+ cells from progenitors, stimulated IL10 secretion from myeloid cells, and decreased T-cell proliferation via upregulation of Arg, iNOS, VEGF, IL6, and IL1b from CD11b+ cells. Deletion of csf3 in PDAC cells or use of a G-CSF–blocking antibody decreased tumor growth. Anti–G-CSF treatment in combination with the DNA synthesis inhibitor gemcitabine reduced tumor size, increased the number of infiltrating T cells, and decreased the number of Ly6G+ cells more effectively than gemcitabine alone. Human analysis of human datasets from The Cancer Genome Atlas and tissue microarrays correlated with observations from our mouse model experiments, especially in patients with grade 1, stage II disease. We propose that in aggressive PDAC, elevated G-CSF contributes to tumor progression through promoting increases in infiltration of neutrophil-like cells with high immunosuppressive activity. Such a mechanism provides an avenue for a neoadjuvant therapeutic approach for this devastating disease. Cancer Immunol Res; 5(9); 718–29. ©2017 AACR.

Stringent regulation of antiviral signaling and cellular autophagy is critical for the host response to virus infection. However, little is known how these cellular processes are regulated in the absence of type I interferon signaling. Here, we show that ATF3 is induced following Japanese encephalitis virus (JEV) infection, and regulates cellular antiviral and autophagy pathways in the absence of type I interferons in mouse neuronal cells. We have identified new targets of ATF3 and show that it binds to the promoter regions of Stat1, Irf9, Isg15 and Atg5 thereby inhibiting cellular antiviral signaling and autophagy. Consistent with these observations, ATF3-depleted cells showed enhanced antiviral responses and induction of robust autophagy. Furthermore, we show that JEV replication was significantly reduced in ATF3-depleted cells. Our findings identify ATF3 as a negative regulator of antiviral signaling and cellular autophagy in mammalian cells, and demonstrate its important role in JEV life cycle.

Type I interferons (IFN-I) are critical in antimicrobial and antitumor defense. Although IFN-I signal via the interferon-stimulated gene factor 3 (ISGF3) complex consisting of STAT1, STAT2, and IRF9, IFN-I can mediate significant biological effects via ISGF3-independent pathways. For example, the absence of STAT1, STAT2, or IRF9 exacerbates neurological disease in transgenic mice with CNS production of IFN-I. Here we determined the role of IFN-I-driven, ISGF3-independent signaling in regulating global gene expression in STAT1-, STAT2-, or IRF9-deficient murine mixed glial cell cultures (MGCs). Compared with WT, the expression of IFN-α-stimulated genes (ISGs) was reduced in number and magnitude in MGCs that lacked STAT1, STAT2, or IRF9. There were significantly fewer ISGs in the absence of STAT1 or STAT2 versus in the absence of IRF9. The majority of ISGs regulated in the STAT1-, STAT2-, or IRF9-deficient MGCs individually were shared with WT. However, only a minor number of ISGs were common to WT and STAT1-, STAT2-, and IRF9-deficient MGCs. Whereas signal pathway activation in response to IFN-α was rapid and transient in WT MGCs, this was delayed and prolonged and correlated with increased numbers of ISGs expressed at 12 h versus 4 h of IFN-α exposure in all three IFN-I signaling-deficient MGCs. In conclusion, 1) IFN-I can mediate ISG expression in MGCs via ISGF3-independent signaling pathways but with reduced efficiency, with delayed and prolonged kinetics, and is more dependent on STAT1 and STAT2 than IRF9; and 2) signaling pathways not involving STAT1, STAT2, or IRF9 play a minor role only in mediating ISG expression in MGCs.

Cells maintain the balance between homeostasis and inflammation by adapting and integrating the activity of intracellular signaling cascades, including the JAK-STAT pathway. Our understanding of how a tailored switch from homeostasis to a strong receptor-dependent response is coordinated remains limited. Here, we use an integrated transcriptomic and proteomic approach to analyze transcription-factor binding, gene expression and in vivo proximity-dependent labelling of proteins in living cells under homeostatic and interferon (IFN)-induced conditions. We show that interferons (IFN) switch murine macrophages from resting-state to induced gene expression by alternating subunits of transcription factor ISGF3. Whereas preformed STAT2-IRF9 complexes control basal expression of IFN-induced genes (ISG), both type I IFN and IFN-γ cause promoter binding of a complete ISGF3 complex containing STAT1, STAT2 and IRF9. In contrast to the dogmatic view of ISGF3 formation in the cytoplasm, our results suggest a model wherein the assembly of the ISGF3 complex occurs on DNA., A rapid cellular response to interferons (IFNs) is critical for establishing antimicrobial immunity, but how cells switch from from homeostasis to IFN signaling is not fully understood. Here, the authors provide evidence that IFNs induce gene expression by alternating subunits of transcription factor ISGF3.

PKZ is a fish-specific protein kinase containing Zα domains. PKZ is known to induce apoptosis through phosphorylating eukaryotic initiation factor 2α kinase (eIF2α) in the same way as double-stranded RNA-dependent protein kinase (PKR), but its exact role in detecting pathogens remains to be fully elucidated. Herein, we have found that PKZ acts as a fish-specific DNA sensor by initiating IFN expression through IRF3- or ISGF3-like mediated pathways. The expression pattern of PKZ is similar to those of innate immunity mediators stimulated by poly (dA:dT) and poly (dG:dC). DNA-PKZ interaction can enhance PKZ phosphorylation and dimerization in vitro. These findings indicate that PKZ participates in cytoplasmic DNA-mediated signaling. Subcellular localization assays have also shown that PKZ is located in the cytoplasm, which suggests that PKZ acts as a cytoplasmic PRR. Meanwhile, co-IP assays have shown that PKZ can separately interact with IRF3, STING, ZDHHC1, eIF2α, IRF9, and STAT2. Further investigations have revealed that PKZ can activate IRF3 and STAT2; and that IRF3-dependent and ISGF3-like dependent mediators are critical for PKZ-induced IFN expression. These results demonstrate that PKZ acts as a special DNA pattern-recognition receptor, and that PKZ can trigger immune responses through IRF3-mediated or ISGF3-like mediated pathways in fish.

Type I interferon response plays a prominent role against viral infection, which is frequently disrupted by viruses. Here, we report Bcl-2 associated transcription factor 1 (Bclaf1) is degraded during the alphaherpesvirus Pseudorabies virus (PRV) and Herpes simplex virus type 1 (HSV-1) infections through the viral protein US3. We further reveal that Bclaf1 functions critically in type I interferon signaling. Knockdown or knockout of Bclaf1 in cells significantly impairs interferon-α (IFNα) -mediated gene transcription and viral inhibition against US3 deficient PRV and HSV-1. Mechanistically, Bclaf1 maintains a mechanism allowing STAT1 and STAT2 to be efficiently phosphorylated in response to IFNα, and more importantly, facilitates IFN-stimulated gene factor 3 (ISGF3) binding with IFN-stimulated response elements (ISRE) for efficient gene transcription by directly interacting with ISRE and STAT2. Our studies establish the importance of Bclaf1 in IFNα-induced antiviral immunity and in the control of viral infections., Author summary Alphaherpesvirus, such as Pseudorabies virus (PRV) and Herpes simplex virus type 1 (HSV-1), can establish persistent infection and cause various diseases in hosts. Interferon (IFN) response is hosts’ first defense system against viral infection. Here, we report alphaherpesvirus induces degradation of a host protein, Bclaf1, via its expressed viral protein US3 upon infection. We further show that Bclaf1 is a novel regulator of IFN pathway by enhancing the IFN induced transcriptions of anti-viral genes. In the absence of Bclaf1, IFN induced anti-viral activity is greatly reduced. Our study highlight the importance of Bclaf1 in IFN mediated antiviral function and reveal a strategy employed by alphaherpesvirus to counteract hosts’ defense.

The transcription factor ISGF3, comprised of IRF9 and tyrosine‐phosphorylated STATs 1 and 2, transmits the signal from the type I interferon receptor to the genome. We have discovered a novel phosphorylation of STAT2 on T387 that negatively regulates this response. In most untreated cell types, the majority of STAT2 is phosphorylated on T387 constitutively. In response to interferon‐β, the T387A mutant of STAT2 is much more effective than wild‐type STAT2 in mediating the expression of many interferon‐stimulated genes, in protecting cells against virus infection, and in inhibiting cell growth. Interferon‐β‐treated cells expressing wild‐type STAT2 contain much less ISGF3 capable of binding to an interferon‐stimulated response element than do cells expressing T387A STAT2. T387 lies in a cyclin‐dependent kinase (CDK) consensus sequence, and CDK inhibitors decrease T387 phosphorylation. Using CDK inhibitors to reverse the constitutive inhibitory phosphorylation of T387 of U‐STAT2 might enhance the efficacy of type I interferons in many different clinical settings.

After viral infection, type I and III interferons (IFNs) are coexpressed by respiratory epithelial cells (RECs) and activate the ISGF3 transcription factor (TF) complex to induce expression of a cell-specific set of interferon-stimulated genes (ISGs). Type I and III IFNs share a canonical signaling pathway, suggesting that they are redundant. Animal and in vitro models, however, have shown that they are not redundant. Because TFs dictate cellular phenotype and function, we hypothesized that focusing on TF-ISG will reveal critical combinatorial and nonredundant functions of type I or III IFN. We treated BEAS-2B human RECs with increasing doses of IFNβ or IFNλ1 and measured expression of TF-ISG. ISGs were expressed in a dose-dependent manner with a nonlinear jump at intermediate doses. At subsaturating combinations of IFNβ and IFNλ1, many ISGs were expressed in a pattern that we modeled with a cubic equation that mathematically defines this threshold effect. Uniquely, IFNβ alone induced early and transient IRF1 transcript and protein expression, while IFNλ1 alone induced IRF1 protein expression at low levels that were sustained through 24 h. In combination, saturating doses of these 2 IFNs together enhanced and sustained IRF1 expression. We conclude that the cubic model quantitates combinatorial effects of IFNβ and IFNλ1 and that IRF1 may mediate nonredundancy of type I or III IFN in RECs.

Interferon stimulated genes (ISGs), a collection of genes important in the early innate immune response, are upregulated in response to stimulation by extracellular type I interferons. The regulation of ISGs has been extensively studied in cells exposed to significant interferon stimulation, but less is known about ISG regulation in homeostatic regimes in which extracellular interferon levels are low. Using a collection of pre-existing, publicly available microarray datasets, we investigated ISG regulation at homeostasis in CD4, pulmonary epithelial, fibroblast and macrophage cells. We used a linear regression model to predict ISG expression levels from regulator expression levels. Our results suggest significant regulation of ISG expression at homeostasis, both through the ISGF3 molecule and through IRF7 and IRF8 associated pathways. We find that roughly 50% of ISGs have expression levels significantly correlated with ISGF3 expression levels at homeostasis, supporting previous results suggesting that homeostatic IFN levels have broad functional consequences. We find that ISG expression levels varied in their correlation with ISGF3, with epithelial and macrophage cells showing more correlation than CD4 and fibroblast cells. Our analysis provides a novel approach for decomposing and quantifying ISG regulation.

Equine herpesvirus type 4 (EHV-4) is mildly pathogenic but is a common cause of respiratory disease in horses worldwide. We previously demonstrated that unlike EHV-1, EHV-4 is not a potent inducer of type-I IFN and does not suppress that IFN response, especially during late infection, when compared to EHV-1 infection in equine endothelial cells (EECs). Here, we investigated the impact of EHV-4 infection in EECs on type-I IFN signaling molecules at 3, 6, and 12 hpi. Findings from our study revealed that EHV-4 did not induce nor suppress TLR3 and TLR4 expression in EECs at all the studied time points. EHV-4 was able to induce variable amounts of IRF7 and IRF9 in EECs with no evidence of suppressive effect on these important transcription factors of IFN-α/β induction. Intriguingly, EHV-4 did interfere with the phosphorylation of STAT1/STAT2 at 3 hpi and 6 hpi, less so at 12 hpi. An active EHV-4 viral gene expression was required for the suppressive effect of EHV-4 on STAT1/STAT2 phosphorylation during early infection. One or more early viral genes of EHV-4 are involved in the suppression of STAT1/STAT2 phosphorylation observed during early time points in EHV-4-infected EECs. The inability of EHV-4 to significantly down-regulate key molecules of type-I IFN signaling may be related to the lower severity of pathogenesis when compared with EHV-1. Harnessing this knowledge may prove useful in controlling future outbreaks of the disease.

WhereasVHLinactivation is a primary event in clear cell renal cell carcinoma (ccRCC), the precise mechanism(s) of how this interacts with the secondary mutations in tumor suppressor genes, includingPBRM1,KDM5C/JARID1C,SETD2, and/orBAP1, remains unclear. Gene expression analyses reveal that VHL, PBRM1, or KDM5C share a common regulation of interferon response expression signature. Loss of HIF2α, PBRM1, or KDM5C inVHL-/-cells reduces the expression of interferon stimulated gene factor 3 (ISGF3), a transcription factor that regulates the interferon signature. Moreover, loss of SETD2 or BAP1 also reduces the ISGF3 level. Finally, ISGF3 is strongly tumor-suppressive in a xenograft model as its loss significantly enhances tumor growth. Conversely, reactivation of ISGF3 retards tumor growth by PBRM1-deficient ccRCC cells. Thus afterVHLinactivation, HIF induces ISGF3, which is reversed by the loss of secondary tumor suppressors, suggesting that this is a key negative feedback loop in ccRCC.

Cells maintain the balance between homeostasis and inflammation by adapting and integrating the activity of intracellular signalling cascades, including the JAK-STAT pathway. Our understanding how a tailored switch from homeostasis to a strong receptor-dependent response is coordinated remains limited. We used an integrated transcriptomic and proteomic approach to analyze transcription-factor binding, gene expression and in vivo proximity-dependent labelling of proteins in living cells under homeostatic and interferon (IFN)-induced conditions. We show that interferons (IFN) switch murine macrophages from resting-state to induced gene expression by alternating subunits of transcription factor ISGF3. Whereas preformed STAT2-IRF9 complexes control basal expression of IFN-induced genes (ISG), both type I IFN and, unexpectedly, IFNγ cause promoter binding of a complete ISGF3 complex containing STAT1, STAT2 and IRF9. In contrast to the dogmatic view of ISGF3 formation in the cytoplasm, our results suggest a model wherein the assembly of the ISGF3 complex occurs on DNA.

Interferon (IFN)-I and IFN-II both induce IFN-stimulated gene (ISG) expression through Janus kinase (JAK)-dependent phosphorylation of signal transducer and activator of transcription (STAT) 1 and STAT2. STAT1 homodimers, known as γ-activated factor (GAF), activate transcription in response to all types of IFNs by direct binding to IFN-II activation site (γ-activated sequence)-containing genes. Association of interferon regulatory factor (IRF) 9 with STAT1–STAT2 heterodimers [known as interferon-stimulated gene factor 3 (ISGF3)] or with STAT2 homodimers (STAT2/IRF9) in response to IFN-I, redirects these complexes to a distinct group of target genes harboring the interferon-stimulated response element (ISRE). Similarly, IRF1 regulates expression of ISGs in response to IFN-I and IFN-II by directly binding the ISRE or IRF-responsive element. In addition, evidence is accumulating for an IFN-independent and -dependent role of unphosphorylated STAT1 and STAT2, with or without IRF9, and IRF1 in basal as well as long-term ISG expression. This review provides insight into the existence of an intracellular amplifier circuit regulating ISG expression and controlling long-term cellular responsiveness to IFN-I and IFN-II. The exact timely steps that take place during IFN-activated feedback regulation and the control of ISG transcription and long-term cellular responsiveness to IFN-I and IFN-II is currently not clear. Based on existing literature and our novel data, we predict the existence of a multifaceted intracellular amplifier circuit that depends on unphosphorylated and phosphorylated ISGF3 and GAF complexes and IRF1. In a combinatorial and timely fashion, these complexes mediate prolonged ISG expression and control cellular responsiveness to IFN-I and IFN-II. This proposed intracellular amplifier circuit also provides a molecular explanation for the existing overlap between IFN-I and IFN-II activated ISG expression.

IL-27 is a cytokine exerting pleiotropic immunomodulatory effects on a broad spectrum of immune cells. Optimal IL-27 production downstream of TLR3/4 ligand stimulation relies on autocrine type I IFN signaling, defining a first and second phase in IL-27 production. This work shows that IL-1 receptor-associated kinase 1 (IRAK1) limits TLR3/4- and IFNAR-induced IL-27 production. At the mechanistic level, we identified IRAK1 as a novel regulator of STAT1, IRF1, and IRF9. We found hyperactivation of STAT1 together with increased nuclear levels of IRF1 and IRF9 in IRAK1-deficient murine macrophages compared with control cells following stimulation with LPS and poly(I:C). IRAK1-deficient human microglial cells showed higher basal levels of STAT1 and STAT2 compared with control cells. Blocking the kinase activity of TBK1/IKKε in IRAK1 knockdown human microglial cells reduced the high basal levels of STAT1/2, uncovering a TBK1/IKKε kinase–dependent mechanism controlling basal levels of STAT1/2. Stimulating IRAK1 knockdown human microglial cells with IFN-β led to increased IL-27p28 expression compared with control cells. In IRAK1-deficient murine macrophages, increased IL-27 levels were detected by ELISA following IFN-β stimulation compared with control macrophages together with increased nuclear levels of p-STAT1, IRF1, and IRF9. Treatment of wild-type and IRAK1-deficient murine macrophages with fludarabine similarly reduced TLR3/4-induced IL-27 cytokine levels. To our knowledge, this work represents the first report placing IRAK1 in the IFNAR pathway and identifies IRAK1 as an important regulator of STAT1, controlling IL-27 production downstream of TLR3/4 and IFNAR signaling pathways.

To subvert host antiviral immune responses, many viruses have evolved countermeasures to inhibit IFN signaling pathway. Porcine bocavirus (PBoV), a newly identified porcine parvovirus, has received attention because it shows clinically high co-infection prevalence with other pathogens in post-weaning multisystemic wasting syndrome (PWMS) and diarrheic piglets. In this study, we screened the structural and non-structural proteins encoded by PBoV and found that the non-structural protein NP1 significantly suppressed IFN-stimulated response element (ISRE) activity and subsequent IFN-stimulated gene (ISG) expression. However, NP1 affected neither the activation and translocation of STAT1/STAT2, nor the formation of the heterotrimeric transcription factor complex ISGF3 (STAT1/STAT2/IRF9). Detailed analysis demonstrated that PBoV NP1 blocked the ISGF3 DNA-binding activity by combining with the DNA-binding domain (DBD) of IRF9. In summary, these results indicate that PBoV NP1 interferes with type I IFN signaling pathway by blocking DNA binding of ISGF3 to attenuate innate immune responses., Highlights • Porcine bocavirus (PBoV) NP1 interferes with the IFN α/β signaling pathway. • PBoV NP1 does not prevent STAT1/STAT2 phosphorylation and nuclear translocation. • PBoV NP1 inhibits the DNA-binding activity of ISGF3. • PBoV NP1 interacts with the DNA-binding domain of IRF9.

Background: In spite of modern therapies for non-small-cell lung cancer (NSCLC), prognosis for many patients is still poor and survival rates are low. Immunotherapy is the possibility to improve the lung immune response surrounding the tumour. However, this approach requires detailed understanding of the local immune-responses of NSCLC patients. Methods: We analysed samples from three different regions within the lungs of NSCLC patients, whereas we distinguished between patients suffering from adenocarcinoma and squamous cell carcinoma. Expression of type 1 T helper (Th1)/type 1 cytotoxic (Tc1) factors was assessed by quantitative real-time PCR, western blot analyses or immunohistochemistry. Cytotoxic cell activity of CD8+ T cells was determined via co-culture with autologous tumour cells and apoptosis assay. Results: We found decreased levels of the transcription factor T-box expressed in T cells (T-bet or Tbx21) and of the downstream activated IFN-γ-dependent pSTAT1α isoform in the lung tumour areas of patients with NSCLC as compared with tumour-free control regions. In these patients, reduced T-bet and pSTAT1α levels were found associated with increased immunosuppressive markers like cytotoxic T lymphocyte-associated protein 4, programmed cell death 1 and with a suppression of the Th1 cell cytokine production and Tc1 cell activity. Conclusions: These findings confirm a central role of T-bet in targeted immunotherapy for patients with NSCLC.

IFNs, which transduce pivotal signals through Stat1 and Stat2, effectively suppress the replication of Legionella pneumophila in primary murine macrophages. Although the ability of IFN-γ to impede L. pneumophila growth is fully dependent on Stat1, IFN-αβ unexpectedly suppresses L. pneumophila growth in both Stat1- and Stat2-deficient macrophages. New studies demonstrating that the robust response to IFN-αβ is lost in Stat1-Stat2 double-knockout macrophages suggest that Stat1 and Stat2 are functionally redundant in their ability to direct an innate response toward L. pneumophila. Because the ability of IFN-αβ to signal through Stat1-dependent complexes (i.e., Stat1-Stat1 and Stat1-Stat2 dimers) has been well characterized, the current studies focus on how Stat2 is able to direct a potent response to IFN-αβ in the absence of Stat1. These studies reveal that IFN-αβ is able to drive the formation of a Stat2 and IFN regulatory factor 9 complex that drives the expression of a subset of IFN-stimulated genes, but with substantially delayed kinetics. These observations raise the possibility that this pathway evolved in response to microbes that have devised strategies to subvert Stat1-dependent responses.

The progesterone receptor (PR) regulates transcriptional programs that drive proliferation, survival, and stem cell phenotypes. Although the role of native progesterone in the development of breast cancer remains controversial, PR clearly alters the transcriptome in breast tumors. This study identifies a class of genes, Interferon (IFN)-stimulated genes (ISGs), potently downregulated by ligand-activated PR which have not been previously shown to be regulated by PR. Progestin-dependent transcriptional repression of ISGs was observed in breast cancer cell line models and human breast tumors. Ligand-independent regulation of ISGs was also observed, as basal transcript levels were markedly higher in cells with PR knockdown. PR repressed ISG transcription in response to IFN treatment, the canonical mechanism through which these genes are activated. Liganded PR is robustly recruited to enhancer regions of ISGs, and ISG transcriptional repression is dependent upon PR's ability to bind DNA. In response to PR activation, key regulatory transcription factors that are required for IFN-activated ISG transcription, STAT2 and IRF9, exhibit impaired recruitment to ISG promoter regions, correlating with PR/ligand-dependent ISG transcriptional repression. IFN activation is a critical early step in nascent tumor recognition and destruction through immunosurveillance. As the large majority of breast tumors are PR positive at the time of diagnosis, PR-dependent downregulation of IFN signaling may be a mechanism through which early PR-positive breast tumors evade the immune system and develop into clinically relevant tumors. Implications: This study highlights a novel transcriptional mechanism through which PR drives breast cancer development and potentially evades the immune system. Mol Cancer Res; 15(10); 1331–40. ©2017 AACR.

STAT1, which carries tumor suppressor functions in several models, consists of two isoforms, namely STAT1α and STAT1β. The biological function and significance of STAT1β has never been examined in human cancer. We examined STAT1β function in esophageal squamous cell carcinoma (ESCC) by transfecting a STAT1β gene into various ESCC cell lines. The interaction between STAT1α and STAT1β was examined by using co-immunoprecipitation and confocal microscopy. The prognostic significance of STAT1β expression, detectable by immunohistochemistry and western blot, was evaluated in a large cohort of ESCC patients. Enforced expression of STAT1β induced and prolonged the expression and phosphorylation of STAT1α in ESCC cells, and these effects were amplified by gamma-interferon (IFN-γ). We also found that STAT1β interacts with STAT1α and decreases STAT1α degradation by the proteasome. Moreover, STAT1β substantially increased the DNA binding and transcription activity of STAT1. STAT1β also sensitized ESCC cells to chemotherapeutic agents, including cisplatin and 5-flurouracil. Using western blot and immunohistochemistry, we found that STAT1β was frequently decreased in esophageal cancer, as compared to their adjacent benign esophageal epithelial tissue. Loss of STAT1β significantly correlated with lymph node metastasis, invasion and shorter overall survival in ESCC patients. Therefore, STAT1β plays a key role in enhancing the tumor suppressor function of STAT1α, in ESCC, in a manner that can be amplified by IFN-γ.

Interferon (IFN)-stimulated genes (ISGs) are antiviral effectors that are induced by IFNs through the formation of a tripartite transcription factor ISGF3, which is composed of IRF9 and phosphorylated forms of STAT1 and STAT2. However, we found that IFN-independent ISG expression was detectable in immortalized cell lines, primary intestinal and liver organoids, and liver tissues. The constitutive expression of ISGs was mediated by the unphosphorylated ISGF3 (U-ISGF3) complex, consisting of IRF9 together with unphosphorylated STAT1 and STAT2. Under homeostatic conditions, STAT1, STAT2, and IRF9 were found in the nucleus. Analysis of a chromatin immunoprecipitation sequencing data set revealed that STAT1 specifically bound to the promoters of ISGs even in the absence of IFNs. Knockdown of STAT1, STAT2, or IRF9 by RNA interference led to the decreased expression of various ISGs in Huh7.5 human liver cells, which was confirmed in mouse embryonic fibroblasts (MEFs) from STAT1-/-, STAT2-/-, or IRF9-/- mice. Furthermore, decreased ISG expression was accompanied by increased replication of hepatitis C virus and hepatitis E virus. Conversely, simultaneous overexpression of all ISGF3 components, but not any single factor, induced the expression of ISGs and inhibited viral replication; however, no phosphorylated STAT1 and STAT2 were detected. A phosphorylation-deficient STAT1 mutant was comparable to the wild-type protein in mediating the IFN-independent expression of ISGs and antiviral activity, suggesting that ISGF3 works in a phosphorylation-independent manner. These data suggest that the U-ISGF3 complex is both necessary and sufficient for constitutive ISG expression and antiviral immunity under homeostatic conditions.

Glial activation and subsequent release of neurotoxic proinflammatory factors are believed to play an important role in the pathogenesis of several neurological disorders including Parkinson’s disease (PD). Inhibition of glial activation and inflammatory processes may represent a therapeutic target to alleviate neurodegeneration. Securinine, a major natural alkaloid product from the root of the plant Securinega suffruticosa, has been reported to have potent biological activity and is used in the treatment of neurological conditions such as amyotrophic lateral sclerosis, poliomyelitis, and multiple sclerosis. In this study, we explored the underlying mechanisms of neuroprotection elicited by securinine, particularly its anti-inflammatory effects in glial cells. Our results demonstrate that securinine significantly and dose-dependently suppressed the nitric oxide production in microglia and astrocytic cultures. In addition, securinine inhibited the activation of the inflammatory mediator NF-κB, as well as mitogen-activated protein kinases in lipopolysaccharide- (LPS-) stimulated BV2 cells. Additionally, securinine also inhibited interferon-γ- (IFN-γ-) induced nitric oxide levels and iNOS mRNA expression. Furthermore, conditioned media (CM) from securinine pretreated BV2 cells significantly reduced mesencephalic dopaminergic neurotoxicity compared with CM from LPS stimulated microglia. These findings suggest that securinine may be a potential candidate for the treatment of neurodegenerative diseases related to neuroinflammation.

□ A new formulation (NF) of subcutaneous (sc) interferon (IFN) β-1a was developed in an attempt to improve injection tolerability and immunogenicity. We compared antiviral and IFNβ-stimulated gene (ISG) activities of IFNβ-1a sc NF with IFNβ-1a sc original formulation and IFNβ-1b sc. When equivalent unit amounts were compared, the IFNβ formulations demonstrated similar antiviral activity and induced similar levels of ISG mRNA. However, on a weight basis (ng/mL), significantly more IFNβ-1b sc was needed to equal the antiviral activity of either IFNβ-1a sc formulation, and both IFNβ-1a sc formulations induced significantly higher levels of ISG mRNA than IFNβ-1b sc.

The use of pegylated interferon-α (pegIFN-α) has replaced unmodified recombinant IFN-α for the treatment of chronic viral hepatitis. While the superior antiviral efficacy of pegIFN-α is generally attributed to improved pharmacokinetic properties, the pharmacodynamic effects of pegIFN-α in the liver have not been studied. Here, we analyzed pegIFN-α-induced signaling and gene regulation in paired liver biopsies obtained prior to treatment and during the first week following pegIFN-α injection in 18 patients with chronic hepatitis C. Despite sustained high concentrations of pegIFN-α in serum, the Jak/STAT pathway was activated in hepatocytes only on the first day after pegIFN-α administration. Evaluation of liver biopsies revealed that pegIFN-α induces hundreds of genes that can be classified into four clusters based on different temporal expression profiles. In all clusters, gene transcription was mainly driven by IFN-stimulated gene factor 3 (ISGF3). Compared with conventional IFN-α therapy, pegIFN-α induced a broader spectrum of gene expression, including many genes involved in cellular immunity. IFN-induced secondary transcription factors did not result in additional waves of gene expression. Our data indicate that the superior antiviral efficacy of pegIFN-α is not the result of prolonged Jak/STAT pathway activation in hepatocytes, but rather is due to induction of additional genes that are involved in cellular immune responses. published

We previously showed that a fish interferon (IFN) regulatory factor 9 (IRF9) homologue, crucian carp Carassius auratus IRF9, displays constitutively nuclear localization and involvement in fish IFN-dependent JAK-STAT signaling; however, little is known about the expression regulation of fish IRF9. Here, we characterized the expression of zebrafish IRF9 by promoter analysis. Zebrafish IRF9 gene promoter contained several putative transcription factor binding sites, including one ISRE (IFN-stimulated response element), one GAS (IFN gamma activation sequence) and three GATEs (IFN gamma activated transcriptional element, GATE1/2/3). Further sequence analyses revealed that GAS and GATE motifs existed in all promoters of IRF9 from mammals and fishes. Luciferase assays confirmed that zebrafish IRF9 promoter could be activated by zebrafish IFN phi s and zebrafish IFN gamma 2, as well as transcription factors IRF3, IRF7, and combination of IRF9 and STAT2. Treatment of recombinant crucian carp IFN protein or overexpression of zebrafish IFN gamma 2 both led to significant increase in crucian carp IRF9 mRNA and protein in cultured fish cells. Comparison of IFN-stimulated promoter activity revealed much more significant induction of zebrafish IRF9 by zebrafish IFN gamma 2 than by zebrafish IFN phi s. Mutation analyses showed that the putative GAS and GATE3 contributed to zebrafish IFN gamma 2-triggered IRF9 expression, whereas the putative ISRE and the other two GATEs were not functional for induction of zebrafish IRF9. These results together indicated that the expression property of IRF9 might be conserved from fish to mammals and that some not yet identified mechanisms could exist in IRF9 gene transcription regulation in response to IFNs. (C) 2013 Elsevier Ltd. All rights reserved.

A single high dose of interferon-β (IFNβ) activates powerful cellular responses, in which many anti-viral, pro-apoptotic, and anti-proliferative proteins are highly expressed. Since some of these proteins are deleterious, cells downregulate this initial response rapidly. However, the expression of many anti-viral proteins that do no harm is sustained, prolonging a substantial part of the initial anti-viral response for days and also providing resistance to DNA damage. While the transcription factor ISGF3 (IRF9 and tyrosine-phosphorylated STATs 1 and 2) drives the first rapid response phase, the related factor un-phosphorylated ISGF3 (U-ISGF3), formed by IFNβ-induced high levels of IRF9 and STATs 1 and 2 without tyrosine phosphorylation, drives the second prolonged response. The U-ISGF3-induced anti-viral genes that show prolonged expression are driven by distinct IFN stimulated response elements (ISREs). Continuous exposure of cells to a low level of IFNβ, often seen in cancers, leads to steady-state increased expression of only the U-ISGF3-dependent proteins, with no sustained increase in other IFNβ-induced proteins, and to constitutive resistance to DNA damage., IFNβ-dependent increases in STAT1, STAT2, and IRF9 mediate resistance to viruses and DNA damage IFNβ induces the formation of a novel transcriptional complex, U-ISGF3, which contains un-phosphorylated STATs. U-ISGF3 regulates the expression of a subset of IFNβ-stimulated genes to promote resistance to virus infection and DNA damage.