Role Of Interferon Regulatory Factor Research Articles

Interferon regulatory factor 7 (IRF7) is required for the optimal initial control but not subsequent clearance of lymphocytic choriomeningitis virus infection in mice

The role of IRF7 in the host response to lymphocytic choriomeningitis virus (LCMV) Armstrong 53b infection of mice was investigated. Intracranial infection of IRF7 KO mice was associated with delayed onset of LCM, increased survival and significantly reduced expression of the Ifng gene in the brain but not in the periphery. IRF7 KO mice showed impaired control of LCMV replication and delayed clearance of LCMV. Similar numbers of activated anti-LCMV-GP33–41 CD8+ T cells were present in the brain and spleens of infected WT and IRF7 KO mice. While plasma IFN-β was increased to similar levels, IFN-α was markedly reduced in IRF7 KO compared with WT mice. Compared with IFN-β, IFN-α was a less potent inhibitor of LCMV infection in vitro. In conclusion, IRF7 (1) is required for the early innate control of LCMV infection, likely through the regulation of the appropriate type I IFN response, and (2) is not required for the antiviral CD8+ T cell-dependent clearance of LCMV from infected tissues.

Identification of three new cis-regulatory IRF5 polymorphisms: in vitro studies

BackgroundPolymorphisms in the interferon regulatory factor 5 (IRF5) gene are associated with susceptibility to systemic lupus erythematosus, rheumatoid arthritis and other diseases through independent risk and protective haplotypes. Several functional polymorphisms are already known, but they do not account for the protective haplotypes that are tagged by the minor allele of rs729302.MethodsPolymorphisms in linkage disequilibrium (LD) with rs729302 or particularly associated with IRF5 expression were selected for functional screening, which involved electrophoretic mobility shift assays (EMSAs) and reporter gene assays.ResultsA total of 54 single-nucleotide polymorphisms in the 5' region of IRF5 were genotyped. Twenty-four of them were selected for functional screening because of their high LD with rs729302 or protective haplotypes. In addition, two polymorphisms were selected for their prominent association with IRF5 expression. Seven of these twenty-six polymorphisms showed reproducible allele differences in EMSA. The seven were subsequently analyzed in gene reporter assays, and three of them showed significant differences between their two alleles: rs729302, rs13245639 and rs11269962. Haplotypes including the cis-regulatory polymorphisms correlated very well with IRF5 mRNA expression in an analysis based on previous data.ConclusionWe have found that three polymorphisms in LD with the protective haplotypes of IRF5 have differential allele effects in EMSA and in reporter gene assays. Identification of these cis-regulatory polymorphisms will allow more accurate analysis of transcriptional regulation of IRF5 expression, more powerful genetic association studies and deeper insight into the role of IRF5 in disease susceptibility.

Abstract A79: Regulation of the breast tumor microenvironment by interferon regulatory factor 5 (IRF5).

Abstract Interferon Regulatory Factor 5 (IRF5) is a member of the IRF family of proteins which predominately serve as immune regulators in response to extracellular pathogens. IRF5 has also been characterized as a tumor suppressor gene where loss of its expression has been observed in a variety of tumor types including blood cancers, breast and colon cancer. In cell types that constitutively express IRF5, such as immune and epithelial cells, it generally resides in the cytoplasm. Once activated by a stimulus that induces post-translational modification(s) of IRF5, it undergoes dimerization and translocation to the nucleus. In the nucleus, IRF5 acts as a transcription factor binding to specific DNA sequences in the promoter regions of target genes known as interferon-sensitive response elements (ISRE) or interferon regulatory elements (IRE). Upon binding to these elements, IRF5 can positively regulate transcription of its target genes, such as pro-inflammatory cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-12, and interferon (IFN)-α, or negatively regulate targets such as IL-10. Most of our current understanding of IRF5 expression and function comes from examining its role in the immune system. Recent data from our lab has shown that IRF5 is a new tumor suppressor gene in breast cancer that controls metastasis in part by regulating expression of the chemokine receptor CXCR4. We detected a graded decrease in IRF5 expression that correlated with advanced disease stage in patients with ductal carcinoma in situ (DCIS), and almost complete loss of IRF5 expression in nearly all of the breast tissues examined from patients diagnosed with invasive ductal carcinoma (IDC). IRF5 expression was easily detectable in mammary epithelial cells from healthy donors and patients with atypical ductal hyperplasia (ADH). Interestingly, in these same tumor specimens from IDC patients, we noticed that a large percentage of infiltrating immune cells expressed high levels of IRF5. Together, these findings suggest at least two distinct functions for IRF5 in the breast tumor microenvironment: 1) in the tumor cell itself and 2) in the infiltrating immune cells of IDC patients. We hypothesize that it is the initial loss of IRF5 expression in mammary epithelial cells of patients with DCIS that reprograms the tumor microenvironment to allow for tumor progression and metastasis. To begin to understand why loss of IRF5 in breast tumor cells allows for disease progression, we worked primarily in an in vitro 3D culture model of a highly invasive human breast cancer cell line, MDA-MB-231, which does not express detectable levels of IRF5. A population of MDA-MB-231 cells were retrovirally infected with either an empty vector or IRF5 pBabe plasmid (EV-231 or IRF5-231) and were used to assess cell proliferation, migration, invasion, chemotaxis, and gene expression profiles. In particular, a focused tumor cytokine array was performed to measure differences in chemokine and chemokine receptor expression between EV-231 and IRF5-231 cells grown in 3D culture. Co-culture of these cells with immune cell types allowed for characterization of an immune response. This work demonstrates that loss of IRF5 in breast cancer cell lines allows for increased tumor cell migration/invasion and significantly alters the expression of several genes important in the regulation of immune cell trafficking and activation. The data supports an otherwise unknown role for IRF5 as an immune regulator in epithelial cells. The ability of IRF5 to slow mammary cell growth and metastasis, as well as shape the tumor-immune microenvironment, points to the importance of studying this new gene regulator in mammary epithelial cells. Citation Format: Erica M. Pimenta, Betsy J. Barnes. Regulation of the breast tumor microenvironment by interferon regulatory factor 5 (IRF5). [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A79.

Lenalidomide Efficacy in Activated B-Cell-Like Subtype Diffuse Large B-Cell Lymphoma Is Dependent Upon IRF4 and Cereblon Expression

AbstractAbstract 3287 Background:Durable responses with lenalidomide monotherapy have been reported in patients with non-Hodgkin lymphoma. In relapsed/refractory diffuse large B-cell lymphoma (DLBCL), higher responses were observed in the activated B-cell-like (ABC) subtype than in the germinal centre B-cell (GCB)-like subtype (Czuczman, et al. British Journal of Haematology, 2011, 154, 477–481). Herein, the molecular mechanisms involved in the differential efficacy of lenalidomide in DLBCL subtypes were investigated. Methods:A panel of DLBCL cell lines, with 5 of ABC-subtype and 11 of non-ABC subtype, was collected and cell of origin subtype was confirmed based on literature, molecular and genetic analysis. The direct antiproliferative effect of lenalidomide on DLBCL cells was assessed using the 3H-thymidine incorporation assay and apoptosis analysis. The molecular mechanisms involved in the antiproliferative efficacy of lenalidomide in DLBCL subtypes were investigated by western blot, immunohistochemistry (IHC) and qRT-PCR analysis of key signaling events during B-cell receptor (BCR)-dependent NF-κB activation. The critical roles of interferon regulatory factor 4 (IRF4), and cereblon (CRBN) in lenalidomide efficacy were established by knock-in or knock-down of these proteins in sensitive ABC cells. Finally, a mouse xenograft model was used to confirm the antitumor effect of lenalidomide and the relevance of the molecular mechanism involved. Results:Using DLBCL cell lines, lenalidomide treatment was found to preferentially suppress proliferation of ABC-DLBCL cells in vitro at a concentration range of 0.01–100 μM (the median plasma concentration at Cmax for patients receiving 25 mg lenalidomide is 2.2 μM) and delay tumor growth in a human tumor xenograft model of OCI-Ly10 cells (lenalidomide 3–30 mg/kg, p.o. qdX28), with minimal effect on non-ABC-DLBCL cells.This tumoricidal effect of lenalidomide was associated with downregulation of IRF4, a survival factor in ABC-DLBCL cells. Treatment with lenalidomide for 1–3 days, similar to the inhibitors of PKCb and MALT1 (LY-333,531 and z-VRPR-fmk, respectively), was found to significantly (p<0.05) downregulate IRF4 protein levels in sensitive cell lines such as OCI-Ly10 and U2932. IRF4 inhibition by lenalidomide reduced CARD11-BCL-10-MALT1 complex activity of ABC-DLBCL cells (as measured by BCL-10 cleavage) and resulted in downregulation of B-cell receptor (BCR)-dependent NF-κB activity. An NF-κB-driven luciferase assay revealed that lenalidomide (1 μM) inhibited transcriptional activity of NF-κB up to 56% in the sensitive ABC-DLBCL cell lines OCI-Ly10 (p <0.05) and U2932 (p <0.01) after 2-day drug treatment. Lenalidomide also significantly (p <0.05) inhibited DNA binding by Rel A/p65, p50 and c-rel/p70 in 4 lines of ABC cells. While IRF4-specific siRNA mimicked the effects of lenalidomide reducing NF-κB activation, IRF4 overexpression conferred cell resistance to lenalidomide, indicating the crucial role of IRF4 inhibition in lenalidomide efficacy in ABC DLBCL.Furthermore, knockdown of CRBN in OCI-Ly10 (p <0.05) and U2932 (p <0.01) conferred resistance to lenalidomide as demonstrated by the abrogation of the inhibitory effects of lenalidomide on IRF4 expression, BCL-10 cleavage, NF-κB activity, and proliferation of these cells, whereas the activity of inhibitors to PKC β and IKKα/β (LY-333,531 and CC-415501, respectively) remained unaffected. These data indicate that antitumor effects of lenalidomide on ABC-DLBCL cells require the presence of cereblon. Conclusions:These data may provide a mechanism for the preferential efficacy of lenalidomide in ABC-DLBCL observed in clinical studies. These findings suggest that lenalidomide has direct antitumor activity against DLBCL cells, preferentially ABC-DLBCL cells, by blocking IRF4 expression and the BCR-NF-κB signaling pathway in a cereblon-dependent manner (also see Figure below). [Display omitted] Disclosures:Zhang:Celgene Corp: Employment, Equity Ownership. Kosek:Celgene Corp: Employment, Equity Ownership. Wang:Celgene Corporation: Employment, Equity Ownership. Heise:Celgene Corporation: Employment, Equity Ownership. Schafer:Celgene: Employment, Equity Ownership. Chopra:Celgene Corporation: Employment, Equity Ownership.

A Role for Interferon Regulatory Factor 4 in the Development of CLL

AbstractAbstract 714Chronic Lymphocytic Leukemia (CLL) representing 30% of all the adult leukemia is a heterogeneous B cell malignancy with complex clinical behavior and pathophysiology. Even though an increasing body of literature has helped in understanding the biology of CLL the precise molecular events that underlie the pathogenesis of this disease are still not fully understood. Recent Whole Genome Sequencing and Genome Wide Association Studies have linked different genetic loci to the origin and evolution of the disease. A Genome Wide Association Study (GWAS) by Di Bernardo et al identified Single Nucleotide Polymorphisms in the 3'UTR of Interferon Regulatory Factor 4 (IRF4) as a familial risk allele for CLL. These SNPs are associated with lower mRNA levels of IRF4 in individuals carrying the risk allele. Another study by Havelange V. et al identified heterozygous mutations in the DNA binding domain of IRF4 in patients with CLL. These studies have linked lower levels of IRF4 to the pathogenesis of CLL. Even though studies have linked low levels of IRF4 to the pathogenesis of CLL, whether low levels of IRF4 indeed contribute to CLL development remains to be determined.In rodent, CLL cells are presumably derived from B1 cells. Like CLL cells, B1 cells are B220+CD5+ and possess self-reactive B cell receptors (BCR). B1 cells normally reside in peritoneal and pleural cavities and constitute only about 5% of total B lymphocytes in mice. However, B1 cell population is dramatically elevated in the VH11 knock-in mice. VH11 family of variable IgH chain is expressed exclusively on B1 cells where it pairs with Vk9 to form BCR that recognizes phosphatidylcholine on senescent RBCs. We did not observe an overt increase of CLL development in IRF4 deficient mice. However, when we crossed the VH11 knock-in mice to an IRF4 deficient mice it resulted in a CLL like lymphoproliferative disorder with marked expansion of CD5+ B220low/int population in blood, spleen and other lymphoid organs. To monitor pathogenesis of CLL, we collected blood from saphenous vein of IRF4−/−VH11 and IRF4+/+VH11 littermate control mice monthly. A total of ten mice from each group were used for this study. The appearance of a monoclonal B220lowCD5+ population and its percentage among peripheral blood mononuclear cells (PBMC) were used for the initial CLL diagnosis. The diagnostic criterion for CLL in VH11 mice is the appearance of monoclonal B220lowCD5+ cells constituting over 20% of PBMCs. If the percentage of the B220lowCD5+ clone is under 20% of the PBMC, the diagnosis would be monoclonal B cell lymphocytosis (MBL), a disease which can progress into CLL. Interestingly, 6 out of 10 IRF4−/−VH11 mice developed CLL after just 5 months while the four remaining IRF4−/−VH11 mice developed MBL. In contrast, none of the IRF4+/+VH11 control mice developed CLL or MBL. Two of the mice developed aggressive CLL where leukemic cells infiltrated liver and other non-lymphoid organs. BrdU incorporation assay identified spleen as the primary site of CLL cells expansion although proliferating CLL cells were also identified in lymph nodes and bone marrow. We further isolated CLL cells from IRF4−/−VH11 mice and transplanted them into immunodeficient mice (Rag2−/−gamma−/−). Our result shows that the disease was transplantable as all the transplanted mice succumbed to CLL within 1 month post-transplantation. Treatment of IRF4−/−VH11 CLL cells with specific Syk and Btk inhibitors targeting BCR signaling showed that like human CLL, the leukemic cells were depended on active BCR signaling for their survival and proliferation.In summary, we provide evidence for a causal relationship between low levels of IRF4 and development of CLL. Further studies are needed to provide mechanistic insights into how IRF4 is involved in the development and progression of this disease. The fine dissection of this relationship will allow for development of new therapeutic strategies and identification of new prognostic markers for diagnosis and treatment of CLL. Disclosures:No relevant conflicts of interest to declare. Disclosures:No relevant conflicts of interest to declare.

Critical Role for Interferon Regulatory Factor 3 (IRF-3) and IRF-7 in Type I Interferon-Mediated Control of Murine Norovirus Replication

Human noroviruses (HuNoV) are the major cause of epidemic, nonbacterial gastroenteritis in the world. The short course of HuNoV-induced symptoms has implicated innate immunity in control of norovirus (NoV) infection. Studies using murine norovirus (MNV) confirm the importance of innate immune responses during NoV infection. Type I alpha and beta interferons (IFN-α/β) limit HuNoV replicon function, restrict MNV replication in cultured cells, and control MNV replication in vivo. Therefore, the cell types and transcription factors involved in antiviral immune responses and IFN-α/β-mediated control of NoV infection are important to define. We used mice with floxed alleles of the IFNAR1 chain of the IFN-α/β receptor to identify cells expressing lysozyme M or CD11c as cells that respond to IFN-α/β to restrict MNV replication in vivo. Furthermore, we show that the transcription factors IRF-3 and IRF-7 work in concert to initiate unique and overlapping antiviral responses to restrict MNV replication in vivo. IRF-3 and IRF-7 restrict MNV replication in both cultured macrophages and dendritic cells, are required for induction of IFN-α/β in macrophages but not dendritic cells, and are dispensable for the antiviral effects of IFN-α/β that block MNV replication. These studies suggest that expression of the IFN-α/β receptor on macrophages/neutrophils and dendritic cells, as well as of IRF-3 and IRF-7, is critical for innate immune responses to NoV infection.

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.

Implications of post-translational modifications of IRF7 on pDC IFN-alpha response

Background We previously showed that plasmacytoid dendritic cells (pDCs) derived from females can produce significantly more IFN-alpha in response to HIV-1 and HIV-1-encoded TLR7/8 ligands than pDCs derived from males, resulting in stronger secondary activation of CD8+ T cells (Meier et al., Nat Med 2009). Given the crucial role of interferon regulatory factor 7 (IRF7) in the regulation of type I IFN production by pDCs, the goal of the current study was to investigate its impact on the observed differences.

Role of Interferon Regulatory Factor 7 in T Cell Responses during Acute Lymphocytic Choriomeningitis Virus Infection

Type I interferons (IFNs), predominantly IFN-α and -β, play critical roles in both innate and adaptive immune responses against viral infections. Interferon regulatory factor 7 (IRF7), a key innate immune molecule in the type I IFN signaling pathway, is essential for the type I IFN response to many viruses, including lymphocytic choriomeningitis virus (LCMV). Here, we show that although IRF7 knockout (KO) mice failed to control the replication of LCMV in the early stages of infection, they were capable of clearing LCMV infection. Despite the lack of type I IFN production, IRF7 KO mice generated normal CD4(+) T cell responses, and the expansion of naïve CD8(+) T cells into primary CD8(+) T cells specific for LCMV GP(33-41) was relatively normal. In contrast, the expansion of the LCMV NP(396)-specific CD8(+) T cells was severely impaired in IRF7 KO mice. We demonstrated that this defective CD8(+) T cell response is due neither to an impaired antigen-presenting system nor to any intrinsic role of IRF7 in CD8(+) T cells. The lack of a type I IFN response in IRF7 KO mice did not affect the formation of memory CD8(+) T cells. Thus, the present study provides new insight into the impact of the innate immune system on viral pathogenesis and demonstrates the critical contribution of innate immunity in controlling virus replication in the early stages of infection, which may shape the quality of CD8(+) T cell responses.

Splenocyte Apoptosis and Autophagy Is Mediated by Interferon Regulatory Factor 1 During Murine Endotoxemia

Sepsis-induced lymphocyte and dendritic cell apoptosis contributes to immunosuppression, which results in an inability to eradicate the primary infection as well as a propensity to acquire new, secondary infections. Another cellular process, autophagy, is also activated in immune cells and plays a protective role. In the present study, we demonstrate that interferon regulatory factor 1 (IRF-1) regulates both immune cell apoptosis and autophagy in a murine endotoxemia model. Interferon regulatory factor 1 is activated at an early phase through a Toll-like receptor 4-dependent, myeloid differentiation primary response gene 88-independent manner in splenocytes. Furthermore, IRF-1 knockout (KO) mice are protected from a lethal endotoxemia model. This protection is associated with decreased apoptosis and increased autophagy in splenocytes. Interferon regulatory factor 1 KO mice experience decreased apoptotic cell loss, especially in CD4⁺ T lymphocytes and myeloid antigen-presenting cells. Meanwhile, IRF-1 KO mice demonstrate increased autophagy and improved mitochondrial integrity. This increased autophagy in KO mice is attributable, at least in part, to deactivation of mammalian target of rapamycin/P70S6 signaling--a main negative regulator of autophagy. Therefore, we propose a novel role for IRF-1 in regulating both apoptosis and autophagy in splenocytes in the setting of endotoxemia with IRF-1 promoting apoptosis and inhibiting autophagy.

Inherent baseline levels of phosphorylated IRF7 impact pDC IFNα response to TLR7 stimulation (108.8)

Abstract Background: We previously showed that plasmacytoid dendritic cells (pDCs) derived from females can produce significantly more IFNα in response to HIV-1 and HIV-1-encoded TLR7/8 ligands than pDCs derived from males, resulting in stronger secondary activation of CD8+ T cells. Given the crucial role of interferon regulatory factor 7 (IRF7) in the regulation of type I IFN production by pDCs, we hypothesized that IRF7is involved in the differences observed in IFNα production. Methods: Fresh PBMC were isolated from HIV-1-negative subjects enrolled at Massachusetts General Hospital. Levels of IRF7 were measured in pDCs by phospho-flow cytometry, In-Cell Western blotting and qRT-PCR at baseline and at different time-points after TLR7 stimulation. IFNα levels were quantified by flow cytometry, ELISA and qRT-PCR Results: We observed a higher percentage of pDCs expressing phosphorylated IRF7 at baseline in females than in males using flow cytometry (p&amp;lt;0.05). This difference persisted after TLR7 stimulation and was associated with higher IFNα production by females. Conclusion: These data show that sex differences in the baseline level of phosphorylated IRF7 in pDCs might account for described higher IFNα production upon TLR7 stimulation, and might provide new insights into the mechanisms underlying faster HIV-1 disease progression in females compared to males after controlling for viral load.

Enhanced interferon regulatory factor 3 binding to the interleukin‐23p19 promoter correlates with enhanced interleukin‐23 expression in systemic lupus erythematosus

To examine the role of interferon regulatory factor 3 (IRF-3) in the regulation of interleukin-23 (IL-23) production in patients with systemic lupus erythematosus (SLE). Bone marrow-derived macrophages were isolated from both wild-type and IRF3(-/-) C57BL/6 mice. These cells were stimulated with the Toll-like receptor 3 (TLR-3) agonist poly(I-C), and IL-23p19 cytokine levels were analyzed by enzyme-linked immunosorbent assay. IRF-3 binding to the IL-23p19 gene promoter region in monocytes from patients with SLE and healthy control subjects was analyzed by chromatin immunoprecipitation (ChIP) assay. Luciferase reporter gene assays were performed to identify key drivers of IL-23p19 promoter activity. TANK-binding kinase 1 (TBK-1) protein levels were determined by Western blotting. ChIP assays demonstrated that IRF-3 was stably bound to the human IL-23p19 promoter in monocytes; this association increased following TLR-3 stimulation. Patients with SLE demonstrated increased levels of IRF-3 bound to the IL-23p19 promoter compared with control subjects, which correlated with enhanced IL-23p19 production in monocytes from patients with SLE. Investigations of the TLR-3-driven responses in monocytes from patients with SLE revealed that TBK-1, which is critical for regulating IRF-3 activity, was hyperactivated in both resting and TLR-3-stimulated cells. Our results demonstrate for the first time that patients with SLE display enhanced IL-23p19 expression as a result of hyperactivation of TBK-1, resulting in increased binding of IRF-3 to the promoter. These findings provide novel insights into the molecular pathogenesis of SLE and the potential role for TLR-3 in driving this response.

Role of interferon regulatory factor 7 in serum‐transfer arthritis: Regulation of interferon‐β production

Innate immune responses activate synoviocytes and recruit inflammatory cells into the rheumatoid joint. Type I interferons (IFNs) play a role in autoimmunity, and IFN gene transcription is activated by IFN-regulatory factors (IRFs) in response to innate sensor recognition. The purpose of this study was to examine the effect of genetic deficiency of IRF-7 in a passive K/BxN serum-transfer model of arthritis. Passive-transfer arthritis was induced in IRF-7(-/-) mice, and additional groups were treated with IFNβ or poly(I-C). Clinical arthritis scoring, histologic assessment, micro-computed tomography, and synovial tissue quantitative polymerase chain reaction analysis were performed. Mouse serum was analyzed by enzyme-linked immunosorbent assay (ELISA). In the passive K/BxN serum-transfer model, arthritis severity was significantly increased in IRF-7(-/-) mice compared with wild-type (WT) mice. In addition, expression of IFNβ in synovium and serum was decreased, potentially contributing to increased arthritis. IRF-7(-/-) mice injected with replacement IFNβ had a decrease in arthritis. Poly(I-C) treatment diminished arthritis in IRF-7(-/-) mice, restored synovial IFNβ gene expression, and increased serum levels of IFNβ. In vitro studies demonstrated that poly(I-C) stimulation of fibroblast-like synoviocytes (FLS) from IRF-7(-/-) mice resulted in increased induction of proinflammatory gene expression as compared with FLS from WT mice; however, IFNβ expression was not significantly different. In contrast, peritoneal macrophages from IRF-7(-/-) mice showed significantly less induction of IFNβ in response to poly(I-C) stimulation. IRF-7 deficiency exacerbates arthritis and replacement treatment with IFNβ or poly(I-C) decreases arthritis severity. Both macrophage- and synoviocyte-specific roles of IRF-7 likely contribute to the increased arthritis. IRF-7 might play an antiinflammatory role in passive-transfer arthritis through regulation of macrophage IFNβ production.

Interferon regulatory factor 1 regulation of oligodendrocyte injury and inflammatory demyelination

Oligodendrocyte injury and inflammatory demyelination are key pathological abnormalities of multiple sclerosis (MS), and its animal model, i.e., the experimental autoimmune encephalomyelitis (EAE). Traditionally, they are viewed as destructive processes secondary to a dysregulated autoimmune reaction. New evidence emerged over the last decade indicating that oligodendrocytes are not merely immune targets but rather active participants in the neuroimmune network and, in fact, can regulate the events leading to inflammatory demyelination. In this review, we are discussing the role of interferon regulatory factor 1 (IRF-1) as a master transcription factor orchestrating oligodendrocyte injury and inflammatory demyelination in MS and EAE. We are also discussing the significance of IRF-1 signaling in the induction of oligodendrocyte pyroptosis, a Caspase 1-dependent pro-inflammatory cell death, as a disease-enhancing mechanism. Finally, we are drawing attention to IRF-1 as a potential therapeutic target in MS and to the importance of investigating other oligodendrocyte-dependent disease mechanisms.

Interferon Regulatory Factor-1 Regulates the Autophagic Response in LPS-Stimulated Macrophages through Nitric Oxide

The pathogenesis of sepsis is complex and, unfortunately, poorly understood. The cellular process of autophagy is believed to play a protective role in sepsis; however, the mechanisms responsible for its regulation in this setting are ill defined. In the present study, interferon regulatory factor 1 (IRF-1) was found to regulate the autophagic response in lipopolysaccharide (LPS)-stimulated macrophages. In vivo, tissue macrophages obtained from LPS-stimulated IRF-1 knockout (KO) mice demonstrated increased autophagy and decreased apoptosis compared to those isolated from IRF-1 wild-type (WT) mice. In vitro, LPS-stimulated peritoneal macrophages obtained from IRF-1 KO mice experienced increased autophagy and decreased apoptosis. IRF-1 mediates the inhibition of autophagy by modulating the activation of the mammalian target of rapamycin (mTOR). LPS induced the activation of mTOR in WT peritoneal macrophages, but not in IRF-1 KO macrophages. In contrast, overexpression of IRF-1 alone increased the activation of mTOR and consequently decreased autophagic flux. Furthermore, the inhibitory effects of IRF-1 mTOR activity were mediated by nitric oxide (NO). Therefore, we propose a novel role for IRF-1 and NO in the regulation of macrophage autophagy during LPS stimulation in which IRF-1/NO inhibits autophagy through mTOR activation.

Blocking of interferon regulatory factor 1 reduces tumor necrosis factor α–induced interleukin‐18 bioactivity in rheumatoid arthritis synovial fibroblasts by induction of interleukin‐18 binding protein a: Role of the nuclear interferon regulatory factor 1–NF‐κB–c‐jun complex

To examine the role of interferon regulatory factor 1 (IRF-1) in tumor necrosis factor α (TNFα)-induced interleukin-18 binding protein a (IL-18BPa) expression in rheumatoid arthritis synovial fibroblasts (RASFs). TNFα-induced IRF-1 expression was assessed by real-time quantitative polymerase chain reaction and Western blotting. The effect of TNFα on IRF-1 was assessed using nuclear and cytoplasmic extracts, Western blots, and immunofluorescence. Chemical inhibitors of NF-κB or MAP kinases were used to analyze the signaling pathways of TNFα-induced IRF-1 expression and IRF-1 nuclear translocation. Control and IRF-1 small interfering RNA (siRNA) were used to analyze the effect of IRF-1 down-regulation on TNFα-induced IL-18BP expression. IL-18BPa expression was assessed by enzyme-linked immunosorbent assay, and IL-18 was assessed at the transcription and bioactivity levels using KG-1 cells. TNFα induced RASF IRF-1 expression at the messenger RNA and protein levels, with a maximal effect at 2 hours (P < 0.05; n ≥ 3). Furthermore, TNFα induced nuclear translocation of IRF-1, with maximal translocation at 2 hours (∼6 fold-induction) (P < 0.05; n = 4). Blocking of the NF-κB or JNK-2 pathways reduced TNFα-induced IRF-1 nuclear translocation by 35% and 50%, respectively (P < 0.05; n ≥ 4). Using siRNA to knock down IRF-1, we observed reduced IL-18BPa expression. Additionally, IL-18 bioactivity was higher when siRNA was used to knock down IRF-1 expression. These results show that IRF-1 is a key regulator of IL-18BPa expression and IL-18 bioactivity in RASFs. Regulation of IRF-1 will be a new therapeutic target in RA.

Abstract C45: Inhibition of antiapoptotic BCL2 members facilitates interferon regulatory factor-1 (IRF1) mediated prodeath signaling in breast cancer cells

Abstract Antiestrogens (AEs) have long been a standard treatment for estrogen receptor-positive (ER+) breast cancer patients. However, antiestrogen resistance, either de novo or acquired, is a major hurdle for effective therapy in breast cancer patients. To address this, the underlying mechanisms of antiestrogen resistance must be understood. Antiestrogens are known to induce apoptosis in sensitive breast cancer cells. The tumor suppressor gene interferon regulatory factor-1 (IRF1) plays a key role in signaling to proapoptotic BH3 only BCL2 (B-cell lymphoma 2) family members (i.e., BIK, PUMA, and BMF). In an attempt to understand IRF1-mediated signaling in AE sensitivity, AE-sensitive and -resistant breast cancer cells were made to overexpress IRF1 or a dominant negative IRF1 (dnIRF1; lacks the transactivation domain of IRF1 but retains the DNA-binding domain). We found that IRF1 regulates the transcription of proapoptotic BIK, BMF, and PUMA, which are necessary to induce apoptosis in AE-sensitive cells. Conversely, AE-resistant breast cancer cells have low IRF1 expression and increased levels of antiapoptotic BCL2 and BCLW. Previous work by our group showed that the inflammatory cytokine IFNγ can be used to induce IRF1 expression in AE-resistant cells, and restores the responsiveness of these cells to fulvestrant (Faslodex; ICI 182,780) through BH3-mediated apoptosis, suggesting a key role of IRF1 in endocrine-resistant breast cancer. We also discovered that the pan BCL2 inhibitor GX15-070 (obatoclax) significantly reduces cell proliferation and, in combination with fulvestrant or tamoxifen, results in a synergistic inhibition of cell growth in AE-resistant breast cancer cells. Treatment with obatoclax in AE-resistant cells results in a reduction in Beclin1 (BECN1) protein levels with a corresponding increase in cleaved caspase-7, indicating a possible role of autophagy and apoptosis in GX15-070-induced cell death. Moreover, overexpression of BECN1 in AE-resistant cells enhances obatoclax-mediated cell death. Collectively, our data demonstrate that a combination of obatoclax and an antiestrogen increases the levels of free BH3 domain proteins, thus facilitating mitochondrial dysfunction, and rendering breast cancer cells more susceptible to tumor cell death by IRF1-mediated signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C45.

PS1-116 Role of interferon regulatory factor 7 (IRF7) in the host response to lymphocytic choriomeningitis infection

PS1-116 Role of interferon regulatory factor 7 (IRF7) in the host response to lymphocytic choriomeningitis infection

New evidence for the role of interferon regulatory factor 5 in the development of systemic lupus erythematosus: Comment on the article by Tada et al

New evidence for the role of interferon regulatory factor 5 in the development of systemic lupus erythematosus: Comment on the article by Tada et al

Important role of interferon regulatory factor (IRF)-3 in the interferon response of mouse macrophages upon infection by Newcastle disease virus

Newcastle disease virus (NDV) is an interesting agent for activating innate immune activity in macrophages including secretion of TNF-α and IFN-α, upregulation of TRAIL and activation of NF-κB and iNOS. However, the molecular mechanism of such cellular activities remains largely unknown. Tumor selectivity of replication of NDV has been described to be linked to deviations in tumor cells of the type I interferon response. We therefore focused on the interferon response to NDV of macrophages as part of innate anti-viral and anti-tumor activity. In particular, we investigated the functional significance of the interferon regulatory factor genes (IRF)-3 and IRF-7. Deletion of the IRF-3 or IRF-7 gene was found to increase susceptibility of mouse macrophages to virus infection. Surprisingly, NDV replicated better in IRF-3 KO than in IRF-7 KO macrophages. Further analysis showed that IRF-3 KO macrophages have a lower basal and NDV-induced RIG-I expression in comparison to IRF-7 KO macrophages. This might explain why, in IRF-3 KO macrophages, the secretion of type I interferons after NDV infection is delayed, when compared to IRF-7 KO and wild-type macrophages. In addition, IRF-3 KO cells showed reduced NDV-induced levels of IRF-7. This effect could be prevented by priming the cells first by interferon-α. Further results indicated that an early production of type I interferon rather than high maximal levels at later time points are important for resistance to infection by NDV. In conclusion, these results demonstrate an important role of IRF-3 for the innate anti-viral response to NDV of mouse macrophages.