Subset Of IFN-stimulated Genes Research Articles

Shared and Distinctive Transcriptomic and Proteomic Pathways in Adult and Juvenile Dermatomyositis.

Transcript and protein expression were interrogated to examine gene locus and pathway regulation in the peripheral blood of active adult dermatomyositis (DM) and juvenile DM patients receiving immunosuppressive therapies. Expression data from 14 DM and 12 juvenile DM patients were compared to matched healthy controls. Regulatory effects at the transcript and protein level were analyzed by multi-enrichment analysis for assessment of affected pathways within DM and juvenile DM. Expression of 1,124 gene loci were significantly altered at the transcript or protein levels across DM or juvenile DM, with 70 genes shared. A subset of interferon-stimulated genes was elevated, including CXCL10, ISG15, OAS1, CLEC4A, and STAT1. Innate immune markers specific to neutrophil granules and neutrophil extracellular traps were up-regulated in both DM and juvenile DM, including BPI, CTSG, ELANE, LTF, MPO, and MMP8. Pathway analysis revealed up-regulation of PI3K/AKT, ERK, and p38 MAPK signaling, whose central components were broadly up-regulated in DM, while peripheral upstream and downstream components were differentially regulated in both DM and juvenile DM. Up-regulated components shared by DM and juvenile DM included cytokine:receptor pairs LGALS9:HAVCR2, LTF/NAMPT/S100A8/HSPA1A:TLR4, CSF2:CSF2RA, EPO:EPOR, FGF2/FGF8:FGFR, several Bcl-2 components, and numerous glycolytic enzymes. Pathways unique to DM included sirtuin signaling, aryl hydrocarbon receptor signaling, protein ubiquitination, and granzyme B signaling. The combination of proteomics and transcript expression by multi-enrichment analysis broadened the identification of up- and down-regulated pathways among active DM and juvenile DM patients. These pathways, particularly those which feed into PI3K/AKT and MAPK signaling and neutrophil degranulation, may be potential therapeutic targets.

Abstract 673: CHROMR Governs Interferon Signaling And Cholesterol Homeostasis In Human Atherosclerotic Plaque

Long noncoding RNAs (lncRNA) have emerged as critical regulators of gene expression, yet their potential contribution to human disease remains poorly understood. We previously identified the primate-specific lncRNA CHROMR as a key regulator of cholesterol homeostasis. Here, we report a novel function for CHROMR in controlling interferon signaling, which is known to regulate innate and adaptive immune responses that drive atherosclerosis. Using single cell RNA-sequencing of human atheroma, we observed robust expression of CHROMR in plaque immune cell populations, which correlated strongly with the expression of two subsets of interferon-stimulated genes (ISG). Interrogation of these genesets using DisGeNET, a comprehensive catalogue of genes and variants associated to human diseases, revealed associations with atherosclerosis and coronary artery disease. Differential expression analysis of CHROMR -expressing versus non-expressing immune cells showed higher levels of ISGs particularly in the monocyte-macrophage cluster. To assess the impact of CHROMR depletion on inflammatory gene expression, we knocked down CHROMR in THP1 macrophages using GapmeR antisense oligonucleotides (or controls) and treated with the TLR3 agonist poly(I:C) to stimulate the interferon response. Transcriptome profiling and unsupervised hierarchical clustering of genes differentially expressed in CHROMR -sufficient and -depleted macrophages revealed that CHROMR silencing markedly repressed expression of key ISGs, including MX1 , IFI44L , STAT1 , and CXCL and CCL family chemokines ( CXCL10 , CXCL11, CCL2 ). Conversely, overexpression of CHROMR in THP1 macrophages increased expression of these ISGs. Furthermore, CHROMR depletion in macrophages reduced histone acetylation at regulatory regions of ISG loci. Mechanistically, we showed that CHROMR sequesters the interferon regulatory factor (IRF)-2-dependent transcriptional co-repressor IRF2BP2, thereby licensing IRF-dependent signaling and transcription of the ISG network. Taken together, our work unveils a critical role for CHROMR in coordinating interferon signaling responses in macrophages and suggests that targeting this lncRNA may reduce inflammation in atherosclerotic plaques.

Interferons reshape the 3D conformation and accessibility of macrophage chromatin

SummaryEngagement of macrophages in innate immune responses is directed by type I and type II interferons (IFN-I and IFN-γ, respectively). IFN triggers drastic changes in cellular transcriptomes, executed by JAK-STAT signal transduction and the transcriptional control of interferon-stimulated genes (ISG) by STAT transcription factors. Here, we study the immediate-early nuclear response to IFN-I and IFN-γ in murine macrophages. We show that the mechanism of gene control by both cytokines includes a rapid increase of DNA accessibility and rearrangement of the 3D chromatin contacts particularly between open chromatin of ISG loci. IFN-stimulated gene factor 3 (ISGF3), the major transcriptional regulator of ISG, controlled homeostatic and, most notably, induced-state DNA accessibility at a subset of ISG. Increases in DNA accessibility correlated with the appearance of activating histone marks at surrounding nucleosomes. Collectively our data emphasize changes in the three-dimensional nuclear space and epigenome as an important facet of transcriptional control by the IFN-induced JAK-STAT pathway.

ILF3 contributes to the establishment of the antiviral type I interferon program.

Upon detection of viral infections, cells activate the expression of type I interferons (IFNs) and pro-inflammatory cytokines to control viral dissemination. As part of their antiviral response, cells also trigger the translational shutoff response which prevents translation of viral mRNAs and cellular mRNAs in a non-selective manner. Intriguingly, mRNAs encoding for antiviral factors bypass this translational shutoff, suggesting the presence of additional regulatory mechanisms enabling expression of the self-defence genes. Here, we identified the dsRNA binding protein ILF3 as an essential host factor required for efficient translation of the central antiviral cytokine, IFNB1, and a subset of interferon-stimulated genes. By combining polysome profiling and next-generation sequencing, ILF3 was also found to be necessary to establish the dsRNA-induced transcriptional and translational programs. We propose a central role for the host factor ILF3 in enhancing expression of the antiviral defence mRNAs in cellular conditions where cap-dependent translation is compromised.

Early transcriptional responses to influenza virus infections in vivo

Abstract Influenza A virus (IAV) infects a broad range of cell types within the respiratory tract. The cells initially targeted by the virus in a naïve host are the site of primary replication and virus spread. These cells are difficult to detect using replication competent IAV as the virus rapidly spreads to secondary cells. To overcome this obstacle we utilize a fluorescence expressing single cycle IAV (scIAV) to identify the cells initially infected in the mouse lung. Using this tool, we observed two distinct populations of epithelial cells during the early stages of infection: cells with high virus replication and cells with low virus replication. This suggests that some cells within the lung are innately permissive to virus replication while others are able to blunt replication. We have determined that this phenotype is not due to coinfection of a cell with multiple virions, and it appears to be independent of type I IFN. The level of replication is also not dependent on epithelial cell type. There are distinct cellular pathways that are significantly impacted in each population of infected cells compared to uninfected cells. Additionally, there are subsets of interferon-stimulated genes that are specifically upregulated in each population. These data indicate that different levels of virus replication may activate distinct cellular responses to infection. Fluorescent reporter scIAVs could be used to further elucidate the mechanism of cellular permissibility and the early innate immune response to IAV infection.

44: Interferon-dependent increase in STAT1, STAT2, and IRF9 prolongs resistance to viruses and DNA damage

A single high dose of IFN β activates powerful cellular responses in which many antiviral, pro-apoptotic, and anti-proliferative proteins are highly expressed. The expression of IFN-stimulated genes (ISGs) is mediated by ISGF3, consisting of IRF9 and tyrosine phosphorylated STATs 1 and 2. This initial response to IFN β is rapid and transient, so that the expression of most ISGs returns to basal levels within a day. However, the expression of some ISGs is prolonged for many days. We found that high levels of STAT1, STAT2, and IRF9 proteins that lack tyrosine phosphorylation are critical for the prolonged second phase response to IFN β . The expression of the STAT1, STAT2, and IRF9 proteins is increased by IFN β at late times (>24 h), and these proteins form a complex resembling ISGF3 (named unphosphorylated ISGF3, U-ISGF3), and bind to ISREs of target genes independently of tyrosine phosphorylation of STATs 1 and 2. U-ISGF3 induces only a subset of ISGs (20–30% of the total), which are mostly known to be antiviral genes and are also highly expressed in therapy-resistant cancer cells, but not cytotoxic ISGs. We observed that, in the absence of interferon treatment, high levels of U-ISGF3 significantly reduced the replication of many RNA viruses. High U-ISGF3 levels are correlated with resistance of cancer cells to DNA damage induced by radiation, etoposide, or doxorubicin. Knocking STAT1 and IRF9 down to decrease U-ISGF3 levels sensitized cancer cells to doxorubicin-induced DNA damage. Continuous exposure of cells to a low level of IFN β , often seen in cancers, leads to steady-state increases in the expression of only the U-ISGF3-dependent genes, with no sustained increases of IFN β -induced cytotoxic genes. Our study has elucidated that the U-ISGF3-dependent response to IFN β prolongs the antiviral effects, but leads to DNA damage resistance, which inhibits responses to cancer therapy.

Identification of an intrahepatic transcriptional signature associated with self-limiting infection in the woodchuck model of hepatitis B

The woodchuck model of hepatitis B virus (HBV) infection displays many characteristics of human infection and has particular value for characterizing the host immune responses during the development of chronic infection. Using the newly developed custom woodchuck microarray platform, we compared the intrahepatic transcriptional profiles of neonatal woodchucks with self-limiting woodchuck hepatitis virus (WHV) infection to those woodchucks progressing to persistent WHV infection. This revealed that WHV does not induce significant intrahepatic gene expression changes during the early-acute stage of infection (8 weeks), suggesting it is a stealth virus. At the mid-acute phase of infection (14 weeks), resolution was associated with induction of a prominent cytotoxic T-cell signature. Strikingly, this was accompanied by high-level expression of PD-1 and various other inhibitory T-cell receptors, which likely act to minimize liver damage by cytotoxic T cells during viral clearance. In contrast to the expression of perforin and other cytotoxic effector genes, the interferon-γ (IFN-γ) signaling response in the mid-acute phase was comparable to that in chronically infected adult animals. The absence of a strong IFN-α/β transcriptional response indicated that type I IFN is not a critical mediator of self-limiting infection. Nevertheless, a number of antiviral genes, including viperin, were differentially expressed during resolving infection, suggesting that a subset of IFN-stimulated genes (ISG) may play a role in the control of WHV replication. We identified new immune pathways associated with the clearance of hepadnavirus infection revealing novel molecular targets with potential for the therapeutic treatment of chronic hepatitis B.

Essential Cell-Autonomous Role for Interferon (IFN) Regulatory Factor 1 in IFN-γ-Mediated Inhibition of Norovirus Replication in Macrophages

Noroviruses (NVs) cause the majority of cases of epidemic nonbacterial gastroenteritis worldwide and contribute to endemic enteric disease. However, the molecular mechanisms responsible for immune control of their replication are not completely understood. Here we report that the transcription factor interferon regulatory factor 1 (IRF-1) is required for control of murine NV (MNV) replication and pathogenesis in vivo. This led us to studies documenting a cell-autonomous role for IRF-1 in gamma interferon (IFN-γ)-mediated inhibition of MNV replication in primary macrophages. This role of IRF-1 in the inhibition of MNV replication by IFN-γ is independent of IFN-αβ signaling. While the signal transducer and activator of transcription STAT-1 was also required for IFN-γ-mediated inhibition of MNV replication in vitro, class II transactivator (CIITA), interferon regulatory factor 3 (IRF-3), and interferon regulatory factor 7 (IRF-7) were not required. We therefore hypothesized that there must be a subset of IFN-stimulated genes (ISGs) regulated by IFN-γ in a manner dependent only on STAT-1 and IRF-1. Analysis of transcriptional profiles of macrophages lacking various transcription factors confirmed this hypothesis. These studies identify a key role for IRF-1 in IFN-γ-dependent control of norovirus infection in mice and macrophages.

The Role of Nuclear Factor κB in the Interferon Response

The nuclear factor κB (NF-κB) transcription factor regulates the expression of genes involved in cell survival and immune responses. We have identified a novel interferon (IFN)-activated signaling pathway that leads to NF-κB activation and demonstrate that a subset of IFN-stimulated genes and microRNAs that play key roles in cellular response to IFN is regulated by NF-κB. This review focuses on the IFN-induced NF-κB activation pathway and the role of NF-κB in the expression of IFN-induced coding and noncoding genes, antiviral activity and apoptosis, and the therapeutic application of IFN in cancer and infectious disease.

Switching Roles

Signaling by type I interferon (IFN) switches PLZF from being a transcriptional repressor to an activator required for the expression of a subset of IFN-stimulated genes.

Non‐conventional signal transduction by type 1 interferons: The NF‐κB pathway

Type I interferons (IFNs) regulate diverse cellular functions by modulating the expression of IFN-stimulated genes (ISGs) through the activation of the well established signal transduction pathway of the Janus Kinase (JAK) and signal transducers and activators of transcription (STAT) proteins. Although the JAK-STAT signal transduction pathway is critical in mediating IFN's antiviral and antiproliferative activities, other signaling pathways are activated by IFNs and regulate cellular response to IFN. The NF-kappaB transcription factor regulates the expression of genes involved in cell survival and immune responses. We have identified a novel IFN mediated signal pathway that leads to NF-kappaB activation and demonstrate that a subset of ISGs that play key roles in cellular response to IFN is regulated by NF-kappaB. This review focuses on the IFN-induced NF-kappaB activation pathway and the role of NF-kappaB in ISG expression, antiviral activity and apoptosis, and the therapeutic application of IFN in cancer and infectious disease.

Cutting Edge: Apoptosis-Regulating Signal Kinase 1 Is Required for Reactive Oxygen Species-Mediated Activation of IFN Regulatory Factor 3 by Lipopolysaccharide

IFN regulatory factor (IRF) 3 participates in the transcriptional induction of IFN-alpha, IFN-beta, and a subset of IFN-stimulated genes (ISGs) as a result of viral infection. In addition, bacterial cell wall components such as LPS activate IRF3 in a p38-dependent manner. In this study we show that IRF3-mediated ISG induction by LPS requires the production of reactive oxygen species (ROS) by the NADPH-dependent oxidase NOX4. Furthermore, we present evidence that LPS-mediated ROS production leads to activation of apoptosis-regulating-signal kinase (ASK) 1, a MAPK kinase kinase family member capable of activating the MAP kinase 6/p38 axis. ASK1 kinase activity proved essential for IRF3-mediated ISG induction by LPS. Thus, our results presented here suggest a novel role for ROS and ASK1 in the innate immune response as signaling intermediates in the IRF3 activation pathway.

Promoter Organization of the Interferon-A Genes Differentially Affects Virus-induced Expression and Responsiveness to TBK1 and IKK∈

Virus-induced expression of interferon (IFN)-A genes is regulated by two members of the IFN regulatory factor (IRF) family, IRF-3 and IRF-7, which are activated by phosphorylation during viral infection by the IKK-related serine/threonine kinases TBK1 and IkappaB kinase epsilon (IKKepsilon). In this study, we demonstrate that three IRF-binding sites located in the virus-responsive element mediate the transcriptional activation of the IFN-A4 promoter by IRF-3. The precise arrangement of these IRF elements is required for synergistic activation of the IFN-A4 promoter following Newcastle disease virus infection or activation by TBK1 or IKKepsilon. The ordered assembly of IRF-3 multimers on the promoter also determines cooperative recruitment of IRF-3 and CREB-binding protein and differential virus-induced expression of IFN-A4 gene promoter compared with IFN-A11. Naturally occurring nucleotide substitutions disrupt two of the IRF elements in the IFN-A11 gene promoter, leading to a dramatic decrease in IRF-3 and CREB-binding protein recruitment and in IRF-3-dependent transcription. Transcription of the IFN-A4 promoter by IRF-7 is mediated by two IRF elements; promoter mutants that carry a reversed IRF element retain the ability to respond to IKKepsilon or TBK1 expression in the presence of IRF-7 but lose the capacity to respond to virus or kinase-induced IRF-3. Interestingly, IKKepsilon or TBK1 stimulates the IRF-7-mediated transcription of IFN-A11, although at a lesser extent compared with IFN-A4. Our data indicate that virus-induced expression of IFN-A genes is dictated by the organization of IRF elements within the IFN-A promoters and that the differential IFN-A gene expression, based on the IRF-3 responsiveness, is partially compensated in the presence of IRF-7 when both factors are activated by IKKepsilon or TBK1.

Interferonα Activates NF-κB in JAK1-deficient Cells through aTYK2-dependentPathway

In addition to activating members of the STAT transcription factor family, interferon alpha/beta (IFNalpha/beta) activates the NF-kappaB transcription factor. To determine the role of the Janus tyrosine kinase (JAK)-STAT pathway in NF-kappaB activation by IFN, we examined NF-kappaB activation in JAK1-deficient mutant human fibrosarcoma cells. In wild-type fibrosarcoma cells (2fTGH), IFN activates STAT1, STAT2, and STAT3, as well as NF-kappaB complexes comprised of p50 and p65. In contrast, in JAK1-deficient cells, IFN induces NF-kappaB activation and NF-kappaB dependent gene transcription but does not activate these STAT proteins and has no effect on STAT-dependent gene transcription. Expression of a catalytically inactive TYK2 tyrosine kinase in JAK1-deficient cells, as well as in the highly IFN-sensitive Daudi lymphoblastoid cell line, abrogates NF-kappaB activation by IFN. Moreover, IFN does not promote NF-kappaB activation in TYK2-deficient mutant fibrosarcoma cells. Our results demonstrate a dichotomy between the classical JAK-STAT pathway and the NF-kappaB signaling pathway. In the IFN signaling pathway leading to STAT activation, both JAK1 and TYK2 are essential, whereas NF-kappaB activation requires only TYK2.

Role of Nuclear Factor-κB in the Antiviral Action of Interferon and Interferon-regulated Gene Expression

Interferons (IFNs) play critical roles in host defense by modulating the expression of various genes via tyrosine phosphorylation of STAT transcription factors. IFN-alpha/beta activates another important transcription factor, nuclear factor-kappaB (NF-kappaB), but its role in IFN-mediated activity is poorly understood. Sensitivity to the antiviral and gene-inducing effects of IFN was examined in normal fibroblasts and in NF-kappaB knockout fibroblasts from p50- and p65-null mice. Antiviral assays demonstrated that NF-kappaB knockout fibroblasts were sensitized to the antiviral action of IFN. Moreover, analysis of IFN-stimulated gene expression by real-time PCR demonstrated selective effects of NF-kappaB on gene expression. Our results demonstrate that a subset of IFN-stimulated genes is regulated through an NF-kappaB-dependent pathway and that NF-kappaB may regulate the sensitivity of cells to IFN-mediated antiviral activity.