Interferon Regulatory Factor Family Research Articles

Irf8 Regulates Cytokine Signaling and Proliferation of Hematopoietic Stem Cells

Irf8 is a member of the Interferon Regulatory Factor family, playing diverse functions in the development of innate and adaptive immune cells and in defenses against intracellular pathogens. We found, for the first time, that the loss of Irf8 results in a severe decrease in number and the reconstitution capacity of long-term hematopoietic stem cells (LT-HSCs). The fact that Irf8deficient hematopoietic microenvironment did not affect the reconstitution ability of HSCs indicates that Irf8 regulates HSCs in a cell autonomous manner. The effect of Irf8 depletion on HSC's number and the reconstitution capacity is unlikely due to the influence of a myeloproliferative syndrome, as the disease is not transplantable. In addition, the number of LT-HSCs is also decreased in E14.5 fetal liver of Irf8deficient mice, when the myeloproliferative disorder has not been developed. A cell cycle analysis show that the number of cycling LT-HSCs is greatly reduced in Irf8deficient mice. In addition, LT-HSCs in Irf8deficient mice failed to enter cell cycle in response to the interferon treatment. Genome-wide transcription profiling and activity of cis-regulatory element analysis of LT-HSCs reveal that the expression of key regulators of cytokine/growth factor signaling and factors controlling HSC self-renewal are dysregulated in Irf8deficient mice. These results indicate that Irf8 plays a critical role in regulating cytokine signaling and cell cycle of HSCs and that Irf8 plays an orchestrated role in enhancing innate and adaptive immunity starting from the top of hematopoietic hierarchy. DisclosuresNo relevant conflicts of interest to declare.

Interferon regulatory factor transcript levels correlate with clinical outcomes in human glioma.

Members of the interferon regulatory factor (IRF) gene family are crucial regulators of type I interferon signaling, which may play a role in the resistance of glioma to immune checkpoint blockade. However, the expression profiles, potential functions, and clinical significance of IRF family members remain largely unknown. Here, we examined IRF transcript levels and clinicopathological data from glioma patients using several bioinformatic databases, including ONCOMINE, GEPIA, TCGA, and cBioPortal. We found that IRF1, IRF2, IRF5, IRF8 and IRF9 were significantly upregulated in glioma compared to normal brain tissue. Higher IRF1, IRF2, IRF3, IRF4, IRF5, IRF7, IRF8 and IRF9 mRNA levels correlated with more advanced tumor grades and poorer outcomes. Moreover, although IRFs mutation rates were low (ranging from 0.5% to 2.3%) in glioma patients, genetic alterations in IRFs were associated with more favorable patient survival. Functional analysis showed that IRFs participated in glioma pathology mainly through multiple inflammation- and immunity-related pathways. Additionally, correlations were identified between IRFs and infiltration of immune cells within glioma tissues. Collectively, these results indicate that IRF family members, including IRF1, IRF2, IRF5, IRF8 and IRF9, may serve as prognostic biomarkers and indicators of immune status in glioma patients.

Evolution of the IRF Family in Salmonids.

Interferon regulatory factors (IRFs) as a family, are major regulators of the innate antiviral response in vertebrates principally involved in regulating the expression of interferons (IFNs) and interferon-stimulated genes (ISGs). To date, nine IRFs have been identified in mammals with a 10th member also found in several avian and fish species. Through genome mining and phylogenetic analysis, we identified and characterised 23 irf genes in 6 salmonid species. This larger repertoire of IRF in salmonids results from two additional whole-genome duplications which occurred in early teleosts and salmonids, respectively. Synteny analysis was then used to identify and confirm which paralogues belonged to each subgroup and a new nomenclature was assigned to the salmonid IRFs. Furthermore, we present a full set of Real-Time PCR primers for all rainbow trout IRFs, confirmed by sequencing to ensure paralogue specificity. RT PCR was then used to examine the response of all trout irf genes in vivo, following Vibrio anguillarum and poly I:C stimulation, indicating potential functional divergence between paralogues. Overall, this study presents a comprehensive overview of the IRF family in salmonids and highlights some novel roles for the salmonid-specific IRFs in immunity.

Interferon regulatory factor 1 (IRF1) and anti-pathogen innate immune responses.

The eponymous member of the interferon regulatory factor (IRF) family, IRF1, was originally identified as a nuclear factor that binds and activates the promoters of type I interferon genes. However, subsequent studies using genetic knockouts or RNAi-mediated depletion of IRF1 provide a much broader view, linking IRF1 to a wide range of functions in protection against invading pathogens. Conserved throughout vertebrate evolution, IRF1 has been shown in recent years to mediate constitutive as well as inducible host defenses against a variety of viruses. Fine-tuning of these ancient IRF1-mediated host defenses, and countering strategies by pathogens to disarm IRF1, play crucial roles in pathogenesis and determining the outcome of infection.

Decreased interferon regulatory factor 6 expression due to DNA hypermethylation predicts an unfavorable prognosis in clear cell renal cell carcinoma

Background: Emerging evidences have indicated that IRF6, as a member of the Interferon regulatory factors (IRFs) family, plays important roles in a variety of tumors. However, the expression status of IRF6 and its prognostic value in clear cell renal cell carcinoma (ccRCC) remain unclear.Methods: In this study, we used TCGA-KIRC, GEO and TIP databases and immunohistochemistry staining to determine the expression profile, clinico-pathological features and prognostic value of IRF6 in ccRCC. MSP and demethylation analysis were utilized to verify the regulatory effect of DNA methylation on IRF6 expression.Results: Our results found that IRF6 expression was downregulated in ccRCC tissues and cell lines, and decreased IRF6 expression was associated with worse clinicopathological features and poorer prognosis. Besides, the results of multivariate Cox regression analysis also confirmed that decreased IRF6 expression was an independently risk factor predictor of shorter Overall Survival (OS) (HR: 0.8524, 95%CI: 0.7614-0.9543, P=0.0056) and Disease Free Survival (DFS) (HR: 0.7024, 95%CI: 0.6087-0.8104, P<0.0001) in ccRCC patients. Moreover, the results of MSP and demethylation analysis validated that decreased IRF6 expression was caused by DNA hypermethylation. Furthermore, our results showed that IRF6 expression was associated with the infiltration levels of multiple immune cells in ccRCC.Conclusions: These findings demonstrated that IRF6 expression was significantly reduced in ccRCC and DNA hypermethylation played an important role in decreased IRF6 expression. In addition, the decrease of IRF6 was related to the unfavorable prognosis of ccRCC patients and the alterations of tumor immune cells infiltration.

Identification of the Prognostic Value and Clinical Significance of Interferon Regulatory Factors (IRFs) in Colon Adenocarcinoma.

BackgroundColon adenocarcinoma (COAD) is one of the most common malignant tumors and has high incidence and mortality rates. The interferon regulatory factor (IRF) family is known as a key transcription factor in the IFN signaling pathway and cellular immunity. This research explored the relationship between the IRF family and COAD through use of bioinformatics technology.Material/MethodsUsing the UALCAN and GEPIA databases, we analyzed the transcription and prognostic value of IRFs in COAD, and GSCALite was used in cancer genomics analysis. TIMER, LinkedOmics, and Metascape were used to assess the potential function of IRFs in COAD.ResultsThe transcription levels of IRF3 were elevated in COAD tissues, while IRF2/4/6 were downregulated compared with normal patients in subgroup analyses of race, age, weight, sex, nodal metastasis, individual cancer stages, TP53 mutation status, and histological subtypes. IRF3 and IRF7 in COAD were significantly associated with a poor prognosis. Drug sensitivity analysis revealed that the expression level of IRF2/4/8 was negatively associated with drug resistance. A significant correlation was found between the IRF family and immune cell infiltration. Moreover, enrichment analysis revealed that the IRFs were associated with response to tumor necrosis factor, transcription misregulation in cancer, and JAK-STAT signaling pathway. We also identified several kinase and miRNA targets of the IRF family in COAD.ConclusionsWe identified IRF3 and IRF7 as prognostic biomarkers in COAD, and the IRF family was associated with immune cell infiltration and gene regulation networks, providing additional evidence showing the significant role of the IRF family in COAD.

Ubiquitination modification: critical regulation of IRF family stability and activity.

Interferon regulatory factors (IRFs) play pivotal and critical roles in innate and adaptive immune responses; thus, precise and stringent regulation of the stability and activation of IRFs in physiological processes is necessary. The stability and activities of IRFs are directly or indirectly targeted by endogenous and exogenous proteins in an ubiquitin-dependent manner. However, few reviews have summarized how host E3 ligases/DUBs or viral proteins regulate IRF stability and activity. Additionally, with recent technological developments, details about the ubiquitination of IRFs have been continuously revealed. As knowledge of how these proteins function and interact with IRFs may facilitate a better understanding of the regulation of IRFs in immune responses or other biological processes, we summarized current studies on the direct ubiquitination of IRFs, with an emphasis on how these proteins interact with IRFs and affect their activities, which may provide exciting targets for drug development by regulating the functions of specific E3 ligases.

Poxviral Targeting of Interferon Regulatory Factor Activation.

As viruses have a capacity to rapidly evolve and continually alter the coding of their protein repertoires, host cells have evolved pathways to sense viruses through the one invariable feature common to all these pathogens—their nucleic acids. These genomic and transcriptional pathogen-associated molecular patterns (PAMPs) trigger the activation of germline-encoded anti-viral pattern recognition receptors (PRRs) that can distinguish viral nucleic acids from host forms by their localization and subtle differences in their chemistry. A wide range of transmembrane and cytosolic PRRs continually probe the intracellular environment for these viral PAMPs, activating pathways leading to the activation of anti-viral gene expression. The activation of Nuclear Factor Kappa B (NFκB) and Interferon (IFN) Regulatory Factor (IRF) family transcription factors are of central importance in driving pro-inflammatory and type-I interferon (TI-IFN) gene expression required to effectively restrict spread and trigger adaptive responses leading to clearance. Poxviruses evolve complex arrays of inhibitors which target these pathways at a variety of levels. This review will focus on how poxviruses target and inhibit PRR pathways leading to the activation of IRF family transcription factors.

Molecular characterization of the interferon regulatory factor (IRF) family and functional analysis of IRF11 in the large yellow croaker (Larimichthys crocea)

Interferon regulatory factors (IRFs) are a family of transcription factors involved in regulating interferon (IFN) responses and immune cell development. A total of 11 IRFs have been identified in teleost fish. Here, a complete repertoire of 11 IRFs (LcIRFs) in the large yellow croaker (Larimichthys crocea) was characterized with the addition of five newly identified members, LcIRF2, LcIRF5, LcIRF6, LcIRF10, and LcIRF11. These five LcIRFs possess a DNA-binding domain (DBD) at the N-terminal that contains five to six conserved tryptophan residues and an IRF-association domain (IAD) or IAD2 at the C-terminal that is responsible for interaction with other IRFs or co-modulators. Phylogenetic analysis showed that the 11 LcIRFs were divided into four clades including the IRF1 subfamily, IRF3 subfamily, IRF4 subfamily, and IRF5 subfamily. These are evolutionarily related to their respective counterparts in other fish species. The 11 LcIRFs were constitutively expressed in all examined tissues, although at different expression levels. Upon polyinosinic: polycytidylic acid (poly (I:C)) stimulation, the expression of all 11 LcIRFs was significantly induced in the head kidney and reached the highest levels at 6 h post-stimulation (except LcIRF4). LcIRF1, LcIRF3, LcIRF7, LcIRF8, and LcIRF10 were more strongly induced by poly (I:C) than the other LcIRFs. Significant induction of all LcIRFs was observed in the spleen, with LcIRF2, LcIRF5, LcIRF6, LcIRF7, LcIRF9, and LcIRF11 reaching their highest levels at 48 h LcIRF3 and LcIRF11 showed a stronger response to poly (I:C) in the spleen than the other LcIRFs. In addition, LcIRF1, LcIRF3, LcIRF7, LcIRF9, LcIRF10, and LcIRF11 were significantly induced by Vibro alginolyticus in both the spleen and the head kidney, with LcIRF1 strongly induced. Thus, LcIRFs exhibited differential inducible expression patterns in response to different stimuli in different tissues, suggesting that LcIRFs have different functions in the regulation of immune responses. Furthermore, overexpression of LcIRF11 activated the promoters of LcIFNc, LcIFNd, and LcIFNh, and differentially induced the expression levels of LcIFNs and IFN-stimulated genes (ISGs). Overexpression of LcIRF11 in epithelioma papulosum cyprinid (EPC) cells inhibited the replication of viral genes after infection of spring viremia of carp virus (SVCV). These data suggested that LcIRF11 may function as a positive regulator in regulating the cellular antiviral response through induction of type I IFN expression. Taken together, the present study reported molecular characterization and expression analysis of 11 IRFs in the large yellow croaker, and investigated the role of LcIRF11 in the antiviral response, which laid a good foundation for further study on the evolution and functional characterization of fish IRFs.

2293-PUB: Epigenomic Analysis of AgRP Neurons Reveals That Leptin Induces Satiety via IRF3

AgRP neurons in the arcuate nucleus (ARC) of the hypothalamus play a dominant role in maintaining energy balance and are dysregulated in obesity, but prior studies examining their transcriptional regulation have been confounded by the rich cellular diversity of the ARC and the low abundance of these neurons. We have employed AgRP neuron-specific Translating Ribosomal Affinity Purification followed by RNA-sequencing (TRAP-seq) and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) to determine the transcriptomic and epigenomic landscape of these neurons during the fed, fasted and leptin-treated states. These rich datasets were computationally integrated to identify candidate transcription factors (TFs) potentially mediating the homeostatic control of food intake, of which the Interferon Regulatory Factor (IRF) family of TFs were enriched during the leptin-treated state. Cell-type specific functional studies have revealed that IRF3 plays a key role in mediating the satiety-inducing effects of leptin. Thus, we have identified a novel role for AgRP-neuron expressed IRF3 in regulating the satiety-evoking effects of leptin. Disclosure F.D. Heyward: None. R. Ivison: None. L.T.Y. Tsai: None. E. Rosen: None. Funding American Diabetes Association (1-19-PMF-008 to F.D.H.); Burroughs Wellcome Fund; American Heart Association

Expression pattern of the interferon regulatory factor family members in influenza virus induced local and systemic inflammatory responses

Type I interferon is considered to be a key cytokine in influenza virus-induced acute lung injury (ALI), in which IRF3 and IRF7 play particularly important roles. However, whether all nine members of IRF family are involved in influenza virus-induced immune response is currently unknown. In this study, we found that all members of IRF family responded to influenza virus. The IRF family expression profile seems to be related to the pathogenicity of the particular influenza virus strain. The influenza virus mainly relies on endosomal TLR signals and the positive feedback loop of IFN-I to cause either direct or indirect different expression of all IRF family members locally or systemically. Interestingly, IRF6 was somewhat different from other IRF family members during influenza virus infection. Overall, the expression profile of the IRF family may be a valuable reference for the prevention and treatment of influenza complications, which encourage further, more in-depth research.

IRF1 negatively regulates NF-κB signaling by targeting MyD88 for degradation in teleost fish

MyD88 is considered as one of the most crucial adaptors in TLR signaling pathway. MyD88 may be influential to interferon regulatory factors (IRFs), while the way that IRFs regulate MyD88 is not fully understood. In this study, we demonstrated that the member of IRF family named IRF1 in miiuy croaker played a role as a negative regulator of MyD88-mediated NF-κB signaling and promoted the degradation of MyD88. Firstly, we found the strong inhibitory effect of IRF1 on MyD88-mediated NF-κB signaling pathway. Secondly, we confirmed that IRF1 could enhance the degradation of MyD88, while the knockdown of IRF1 presented an opposite result. Furthermore, the DBD domain of IRF1 was necessary for the inhibition to MyD88. In addition, it could be found that IRF1 could promote MyD88 degradation through ubiquitin-proteasome pathway. Our findings suggest that miiuy croaker IRF1 negatively regulates the cellular response by targeting MyD88 for degradation, which provides new insights into the regulatory mechanism in teleost.

Identification and functional analysis of two interferon regulatory factor 3 genes and their involvement in antiviral immune responses in the Chinese giant salamander Andrias davidianus

Interferon regulatory factor 3 (IRF3), a crucial member of interferon regulatory factor (IRF) family, plays an important role in innate immunity in vertebrates. However, there are no reports on the characterization and especially their respective functional analysis of two IRF3 genes in some species. In this study, two IRF3 genes as well as their roles in the immune response were identified and investigated in Chinese giant salamander, Andrias davidianus. The complete open reading frames of AdIRF3A and AdIRF3B were 1, 113 bp and 1, 380 bp in length, encoding 370 and 459 amino acids, respectively. Both AdIRF3A and AdIRF3B protein contain an IRF and an IRF3 domain. Phylogenetic analysis indicated that AdIRF3s clustered together with other IRF3 proteins. Tissue distribution analysis showed that AdIRF3s were expressed in all tissues tested, with highest expression levels in blood. Both AdIRF3s actively responded to Chinese giant salamander iridovirus (GSIV) and poly (I:C) challenge in A. davidianus. AdIRF3A/B silencing significantly suppressed the DNA virus and viral RNA analog-induced expression of IFN-inducible genes. Luciferase reporter assay further confirmed the regulatory role of AdIRF3s in IFN signaling. These results provide new insights into the origin or evolution of IRF3 in amphibians and even in vertebrates.

Expression, subcellular localization, and potential antiviral function of three interferon regulatory factors in the big-belly seahorse (Hippocampus abdominalis)

Interferon regulatory factors (IRFs) are among the most important transcription mediators and have multiple biological functions, such as antiviral and antimicrobial defense, cell differentiation, immune modulation, and apoptosis. Three IRF family members (HaIRF4-like, HaIRF6, and HaIRF8) of the big belly seahorse (Hippocampus abdominalis) were molecularly and functionally characterized at the sequence and transcriptional level. The coding sequences of HaIRF4-like, HaIRF6, and HaIRF8 were 1214, 1485, and 1266 bp in length, encoding proteins of size 46.21, 55.32, and 47.56 kDa, respectively. Potential viral transcription and replication was detected against VHSV infection using qPCR in HaIRFs-transfected FHM cells. IRFs significantly reduced viral gene expression at 24 h and 48 h post infection and the expression of interferon-stimulated genes (ISGs) was modulated at transcriptional level upon HaIRF overexpression in FHM cells. Subcellular HaIRF localization was observed using GFP-tagged expression vectors in FHM cells. HaIRF4-like and HaIRF8 were localized to the nucleus, whereas HaIRF6 was observed in the cytoplasm. All three IRFs were ubiquitously expressed in all analyzed tissues of the big belly seahorse. The mRNA expression of IRF4-like, IRF6, and IRF8 increased significantly post injection in the blood and gills following LPS, poly (I:C), and Streptococcus iniae challenge. These findings demonstrate that seahorse IRFs are involved in host defense mechanisms against immune stimulants and HaIRFs induce interferon and ISGs which trigger antiviral activity against viral infections in the host.

Development of the First Transgenic Animal Model for IRF4-Induced Lymphoid Malignancy

Introduction The transcription factor IRF4, a member of Interferon Regulatory Factor (IRF) family, is a critical regulator for the production of functional B and T lymphocytes. The dysregulation of IRF4 expression has been frequently implicated in various mature B- and T-lymphoid malignancies such as multiple myeloma and peripheral T-cell lymphomas in which IRF4 acts as an oncogene. However, in vivo model systems to investigate the oncogenic property of IRF4 has not been established. One of common downstream targets of IRF4 across different cancers is the oncogene, MYC, which promotes cell proliferation. Other downstream targets or collaborating factors of IRF4 in these malignancies have not been fully elucidated. Here, we established the first transgenic animal model for IRF4-induced lymphoma, which displays multiple spectrum of tumors providing an in vivo platform to study IRF4 oncogenicity. Methods and Results To identify the specific lineages and stages of the hematopoietic cells that can be transformed by IRF4, we utilized the zebrafish lck promoter, which is active both in T- and B-lymphocytes. We overexpressed human IRF4 gene in zebrafish under this promoter along with a fluorescent marker gene. We confirmed successful expression of both IRF4 and marker genes in the lymphocyte population. Strikingly, multiple F0 transgenic zebrafish showed an expansion of fluorescent signals arising from the thymus by 5 months, while control fish showed thymic involution after 4 months. Approximately 20% of F1 animals developed lymphoma within 5 months and progressed to leukemia by 8 months. Histopathological examination revealed massive infiltration of abnormal lymphocytes into skin, spinal cord, muscles, and liver, which resembles clinical and pathological features observed in several types of human mature T-cell neoplasms. Interestingly, the tumor onset and progression were significantly accelerated when crossed with p53e7/e7-mutant fish, which possesses transactivation-dead p53 variant. This reveals the synergy between IRF4 expression and p53 loss of function, which have also been observed in several types of human lymphomas. Notably, analysis of gene expression profiles using the single-cell RNA-sequencing platform revealed a simultaneous development of both B- and T-cell tumors, which consist of multiple clones at the early stage of tumorigenesis. T-cell-derived tumors became dominant at the late stage of tumorigenesis. Importantly, the expressions of mycb, the zebrafish orthologue of human MYC, as well as of other lymphoid transcription factors were highly upregulated in those tumors in parallel with IRF4 expression. Conclusion Our study demonstrated that IRF4 serves as a driver oncogene in the development of T- and B-cell malignancies. IRF4 accelerates the tumor progression by taking advantage of the impaired function of p53, as demonstrated by massive infiltration into distal organs recapitulating human lymphomas. Taken together, our zebrafish IRF4 model provides a very powerful platform to investigate the plausible mechanisms and pathways through which IRF4 exert its oncogenic property in lymphomagenesis. Disclosures Iida: Teijin Pharma: Research Funding; Astellas: Research Funding; Gilead: Research Funding; Sanofi: Research Funding; MSD: Research Funding; Abbvie: Research Funding; Kyowa Kirin: Research Funding; Chugai: Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Daichi Sankyo: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

Mucosal immunoglobulin IgT plays a key role in the oral immunity of teleosts

Interferon regulatory factor (Irf) family represents one of the most important transcription factor families, with multiple biological roles. In this study, we characterized five Irf family members (irf4a, irf4b, irf6, irf8 and irf10) in Megalobrama amblycephala at the cDNA and (predicted) amino acid levels, analyzed them phylogenetically, and developed gene-specific primers for qPCR analysis. All five irfs were constitutively expressed in all examined tissues, but their transcription was significantly higher in lymphoid organs and tissues, such as kidney, spleen and intestine. Exceptions were irf8, which was expressed at a high level in heart and brain tissues, and irf6, expressed at low levels in most tissues. After a bacterial immune challenge with Aeromonas hydrophila, the expression of irfs in liver was up-regulated: mairf4a 8.12-fold, mairf4b 29.9-fold, mairf6 1.38-fold and mairf10 1.65-fold (mairf8 was an exception: 0.07-fold). In spleen, kidney, intestine and gills, transcript levels of studied irfs increased only at specific time-points. The results suggested that irfs are involved in the immune response to bacterial infection in M. amblycephala, which will help elucidate the biological functions of irfs in the immune system of teleost fish.

Involvement of Interferon Regulatory Factor 7 in Nicotine's Suppression of Antiviral Immune Responses.

Nicotine, the active ingredient in tobacco smoke, suppresses antiviral responses. Interferon regulatory factors (IRFs) regulate transcription of type I interferons (IFNs) and IFN-stimulated genes (ISGs) in this response. IRF7 is a key member of the IRF family. Expression of Irf7 is elevated in the brains of virus-infected animals, including human immunodeficiency virus-1 transgenic (HIV-1Tg) rats. We hypothesized that IRF7 affects nicotine's modulation of antiviral responses. Using CRISPR/Cas9 system, IRF7-mutant cell lines were created from human embryonic kidney 293FT cells in which 16 nicotinic acetylcholine receptors (nAChRs) were detected. Decreased expression of IRF7 was confirmed at both the mRNA and protein levels, as was IRF7-regulated cell growth in two IRF7-mutant cell lines, designated IRF7-Δ7 and IRF7-Δ11. In IRF7-Δ7 cells, expression of two nAChR genes, CHRNA3 and CHRNA9, changed modestly. After stimulation with polyinosinic-polycytidylic acid (poly I:C) (0.25μg/ml) for 4h to mimic viral infection, 293FT wild-type (WT) and IRF7-Δ7 cells were treated with 0, 1, or 100μM nicotine for 24h, which increased IFN-β expression in both types of cells but elevation was higher in WT cells (p < 0.001). Expression was significantly suppressed in WT cells (p < 0.001) but not in IRF7-Δ7 cells by 24-h nicotine exposure. Poly I:C stimulation increased mRNA expression of retinoic-acid-inducible protein I (RIG-I), melanoma-differentiation-associated gene 5 (MDA5), IFN-stimulated gene factor 3 (ISG3) complex, and IFN-stimulated genes (IRF7, ISG15, IFIT1, OAS1); nicotine attenuated mRNA expression only in WT cells. Overall, IRF7 is critical to nicotine's effect on the antiviral immune response. Graphical Abstract Involvement of IRF7 in nicotine's suppression of poly I:C-induced antiviral immune responses. PAMPs, such as a synthetic viral analogue of dsRNA poly I:C attack cells, will be recognized by PRRs, and the host innate immunity against viral infection will be activated. PRRs signaling trigger phosphorylation of IRF7 and IRF3 to induce their translocation to the nucleus and result in the production of type I IFNs. Then IFNs bind to IFNAR to activate the transcription factor ISGF3, a complex consisting of STAT1, STAT2, and IRF9. Further, it induces the expression of ISGs, including IFIT1, OAS1, IRF7, ISG15, etc. Nicotine suppresses the immune responses stimulated by poly I:C. In the IRF7-mutant cells, nicotine's suppressive effects on poly I:C-stimulated immune responses were restrained.

Direct Inhibition of IRF-Dependent Transcriptional Regulatory Mechanisms Associated With Disease.

Interferon regulatory factors (IRFs) are a family of homologous proteins that regulate the transcription of interferons (IFNs) and IFN-induced gene expression. As such they are important modulating proteins in the Toll-like receptor (TLR) and IFN signaling pathways, which are vital elements of the innate immune system. IRFs have a multi-domain structure, with the N-terminal part acting as a DNA binding domain (DBD) that recognizes a DNA-binding motif similar to the IFN-stimulated response element (ISRE). The C-terminal part contains the IRF-association domain (IAD), with which they can self-associate, bind to IRF family members or interact with other transcription factors. This complex formation is crucial for DNA binding and the commencing of target-gene expression. IRFs bind DNA and exert their activating potential as homo or heterodimers with other IRFs. Moreover, they can form complexes (e.g., with Signal transducers and activators of transcription, STATs) and collaborate with other co-acting transcription factors such as Nuclear factor-κB (NF-κB) and PU.1. In time, more of these IRF co-activating mechanisms have been discovered, which may play a key role in the pathogenesis of many diseases, such as acute and chronic inflammation, autoimmune diseases, and cancer. Detailed knowledge of IRFs structure and activating mechanisms predisposes IRFs as potential targets for inhibition in therapeutic strategies connected to numerous immune system-originated diseases. Until now only indirect IRF modulation has been studied in terms of antiviral response regulation and cancer treatment, using mainly antisense oligonucleotides and siRNA knockdown strategies. However, none of these approaches so far entered clinical trials. Moreover, no direct IRF-inhibitory strategies have been reported. In this review, we summarize current knowledge of the different IRF-mediated transcriptional regulatory mechanisms and how they reflect the diverse functions of IRFs in homeostasis and in TLR and IFN signaling. Moreover, we present IRFs as promising inhibitory targets and propose a novel direct IRF-modulating strategy employing a pipeline approach that combines comparative in silico docking to the IRF-DBD with in vitro validation of IRF inhibition. We hypothesize that our methodology will enable the efficient identification of IRF-specific and pan-IRF inhibitors that can be used for the treatment of IRF-dependent disorders and malignancies.

Regulating IRFs in IFN Driven Disease.

The Interferon regulatory factors (IRFs) are a family of transcription factors that play pivotal roles in many aspects of the immune response, including immune cell development and differentiation and regulating responses to pathogens. Three family members, IRF3, IRF5, and IRF7, are critical to production of type I interferons downstream of pathogen recognition receptors that detect viral RNA and DNA. A fourth family member, IRF9, regulates interferon-driven gene expression. In addition, IRF4, IRF8, and IRF5 regulate myeloid cell development and phenotype, thus playing important roles in regulating inflammatory responses. Thus, understanding how their levels and activity is regulated is of critical importance given that perturbations in either can result in dysregulated immune responses and potential autoimmune disease. This review will focus the role of IRF family members in regulating type I IFN production and responses and myeloid cell development or differentiation, with particular emphasis on how regulation of their levels and activity by ubiquitination and microRNAs may impact autoimmune disease.

Full characterization and transcript expression profiling of the interferon regulatory factor (IRF) gene family in Atlantic cod (Gadus morhua)

Atlantic cod (Gadus morhua) represents a unique immune system among teleost fish, making it a species of interest for immunological studies, and especially for investigating the evolutionary history of immune gene families. The interferon regulatory factor (IRF) gene family encodes transcription factors which function in the interferon pathway, but also in areas including leukocyte differentiation, cell growth, autoimmunity, and development. We previously characterized several IRF family members in Atlantic cod (Irf4a, Irf4b, Irf7, Irf8, and two Irf10 splice variants) at the cDNA and putative amino acid levels, and in the current study we took advantage of the new and improved Atlantic cod genome assembly in combination with rapid amplification of cDNA ends (RACE) to characterize the remaining family members (i.e. Irf3, Irf5, Irf6, Irf9, and two Irf2 splice variants). Real-time quantitative PCR (QPCR) was used to investigate constitutive expression of all IRF transcripts during embryonic development, suggesting several putative maternal transcripts, and potential stage-specific roles. QPCR studies also showed 11 of 13 transcripts were responsive to stimulation with poly(I:C), while 6 of 13 transcripts were responsive to lipopolysaccharide (LPS) in Atlantic cod head kidney macrophages, indicating roles for cod IRF family members in both antiviral and antibacterial responses. This study is the first to investigate expression of the complete IRF family in Atlantic cod, and suggests potential novel roles for several of these transcription factors within immunity as well as in early development of this species.