Interferon Regulatory Factor 5 Expression Research Articles

Nanoparticle-Delivered IRF5 siRNA Facilitates M1 to M2 Transition, Reduces Demyelination and Neurofilament Loss, and Promotes Functional Recovery After Spinal Cord Injury in Mice.

Macrophage activation and persistent inflammation contribute to the pathogenesis of spinal cord injury (SCI), and different phenotypes of macrophages play diverse roles in the pathological process of SCI. After SCI, there is an acute phase of alternatively activated (M2) macrophage infiltration, followed by a long-lasting phase of classically activated (M1) macrophage accumulation in the wound. The long-lasting predominance of M1 macrophages may derail healing and compromise organ functions. Based on the previous findings that the transcription factor interferon regulatory factor 5 (IRF5) up-regulates genes associated with M1 macrophages, we attempted to examine the effect of silencing IRF5 on SCI progression. IRF5 expression was assessed with Western blotting or immunohistochemistry. Macrophage phenotypes were measured with flow cytometry or immunohistochemistry. M1- or M2-related cytokines were measured with a Luminex assay kit. IRF5 siRNA was delivered into the macrophages infiltrated into the wound of SCI mice through lipidoid nanoparticle. Locomotor functions were measured with Basso Mouse Scale (BMS) scoring. Myelination was assessed with luxol fast blue staining. Myelin binding protein, neurofilaments, synaptic markers, and cytokines in the wound area were measured with Western blotting. The Mann-Whitney U test was used for statistical analyses. After SCI, significant elevation of IRF5 was evident on day 1, peaked on day 7, and gradually decreased thereafter. Similar dynamic change in the expression of CD86, a typical M1 marker, was observed. In contrast, there was a transient increase in the expression of CD206, a typical M2 marker, which peaked 6h after SCI, and returned to baseline within 1day. Macrophages isolated from the epicenter at day 3 after SCI were predominantly M1 phenotype, and a siRNA-mediated knockdown of IRF5 resulted in a reduced expression of M1 macrophage markers and increased expression of M2 macrophage markers. Nanoparticle-mediated delivery of IRF5 siRNA to SCI mouse model resulted in a dramatic decrease in the number of M1 macrophages and a significant increase in the number of M2 macrophages in the wound. This was associated with a robust inflammation resolution, attenuation of demyelination and neurofilament loss, and significant improvement of locomotor function (p < 0.05). IRF5 may serve as a therapeutic target to promote post-SCI recovery.

Abstract 3677: Loss of IRF5 induces spontaneous murine mammary tumorigenesis

Abstract Despite significant research efforts, breast cancer is still the most common malignancy in women and the most common cause of cancer death worldwide. A major challenge in breast cancer treatment is disease heterogeneity that is contributed by many factors including intrinsic cell factors, microenvironment, angiogenesis and tumor-specific immune responses. Interferon regulatory factor 5 (IRF5) is a transcription factor that controls inflammatory and immune responses. Analysis of over 3,000 human breast cancer tissues revealed that high expression of IRF5 correlates with increased survival and lower incidence of metastasis, whereas the lower quartile of IRF5 expression is a significant marker of poor prognosis for recurrence-free survival. These data support a tumor suppressor role for IRF5. To examine how loss of IRF5 expression may play a role in spontaneous mammary tumorigenesis, we generated Irf5 knockout (ko) mice in a BALB/C background. In mammary glands harvested from one-year-old female virgin ko mice (n = 15), we found an increased incidence (16%) of mammary carcinoma in situ as compared to their wild-type littermates (4%; n = 18). Of interest, the number of tumor bearing mice was significantly higher (38%) in retired ko female breeders (n = 8); however, there were no abnormalities detected in one-year-old male ko virgins or breeders (n = 6). Cellular and molecular analysis of female ko and wild-type littermates revealed that Irf5 deficiency enhanced mammary epithelial cell proliferation as indicated by an increased number of Ki67 expressing cells, and resulted in abnormal hyperbranching, which may contribute to mammary tumorigenesis. Irf5 ko breeders also showed impaired mammary gland involution after weaning, which has been shown to facilitate mammary tumor formation in humans. Taken together, these findings demonstrate that IRF5 is a gender-specific tumor suppressor in breast cancer. In this ongoing study, we are further investigating the intrinsic and extrinsic (microenvironment) role of IRF5 in mammary tumor initiation and progression. Citation Format: Dan Li, Xiaohui Bi, Corey Goyeneche, Amy Pitler, Betsy Barnes. Loss of IRF5 induces spontaneous murine mammary tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3677.

Mangiferin inhibits macrophage classical activation via downregulating interferon regulatory factor5 expression.

Mangiferin is a natural polyphenol and the predominant effective component of Mangifera indica Linn. leaves. For hundreds of years, Mangifera indica Linn. leaf has been used as an ingredient in numerous traditional Chinese medicine preparations for the treatment of bronchitis. However, the pharmacological mechanism of mangiferin in the treatment of bronchitis remains to be elucidated. Macrophage classical activation is important role in the process of bronchial airway inflammation, and interferon regulatory factor 5 (IRF5) has been identified as a key regulatory factor for macrophage classical activation. The present study used the THP-1 human monocyte cell line to investigate whether mangiferin inhibits macrophage classical activation via suppressing IRF5 expression in vitro. THP-1 cells were differentiated to macrophages by phorbol 12-myristate 13-acetate. Macrophages were polarized to M1 macrophages following stimulation with lipopolysaccharide (LPS)/interferon-γ (IFN-γ). Flow cytometric analysis was conducted to detect the M1 macrophages. Reverse transcription-quantitative polymerase chain reaction was used to investigate cellular IRF5 gene expression. Levels of proinflammatory cytokines and IRF5 were assessed following cell culture and cellular homogenization using enzyme-linked immunosorbent assay. IRF5 protein and nuclei co-localization was performed in macrophages with laser scanning confocal microscope immunofluorescence analysis. The results of the present study demonstrated that mangiferin significantly inhibits LPS/IFN-γ stimulation-induced classical activation of macrophages in vitro and markedly decreases proinflammatory cytokine release. In addition, cellular IRF5 expression was markedly downregulated. These results suggest that the inhibitory effect of mangiferin on classical activation of macrophages may be exerted via downregulation of cellular IRF5 expression levels.

An IRF5 Decoy Peptide Reduces Myocardial Inflammation and Fibrosis and Improves Endothelial Cell Function in Tight-Skin Mice

Interferon regulatory factor 5 (IRF5) has been called a “master switch” for its ability to determine whether cells mount proinflammatory or anti-inflammatory responses. Accordingly, IRF5 should be an attractive target for therapeutic drug development. Here we report on the development of a novel decoy peptide inhibitor of IRF5 that decreases myocardial inflammation and improves vascular endothelial cell (EC) function in tight-skin (Tsk/+) mice. Biolayer interferometry studies showed the Kd of IRF5D for recombinant IRF5 to be 3.72 ± 0.74x10-6M. Increasing concentrations of IRF5D (0–100 μg/mL, 24h) had no significant effect on EC proliferation or apoptosis. Treatment of Tsk/+ mice with IRF5D (1mg/kg/d subcutaneously, 21d) reduced IRF5 and ICAM-1 expression and monocyte/macrophage and neutrophil counts in Tsk/+ hearts compared to expression in hearts from PBS-treated Tsk/+ mice (p<0.05). EC-dependent vasodilatation of facialis arteries isolated from PBS-treated Tsk/+ mice was reduced (~15%). IRF5D treatments (1mg/kg/d, 21d) improved vasodilatation in arteries isolated from Tsk/+ mice nearly 3-fold (~45%, p<0.05), representing nearly 83% of the vasodilatation in arteries isolated from C57Bl/6J mice (~55%). IRF5D (50μg/mL, 24h) reduced nuclear translocation of IRF5 in myocytes cultured on both Tsk/+ cardiac matrix and C57Bl/6J cardiac matrix (p<0.05). These data suggest that IRF5 plays a causal role in inflammation, fibrosis and impaired vascular EC function in Tsk/+ mice and that treatment with IRF5D effectively counters IRF5-dependent mechanisms of inflammation and fibrosis in the myocardium in these mice.

A protease-activated receptor 2 agonist (AC-264613) suppresses interferon regulatory factor 5 and decreases interleukin-12p40 production by lipopolysaccharide-stimulated macrophages: Role of p53.

The transcription factor interferon regulatory factor 5 (IRF5) has a key role in the production of interleukin (IL)-12 by macrophages. IRF5 is also a central mediator of toll-like receptor signaling and is a direct target of p53. Activation of protease-activated receptor 2 (PAR-2) upregulates p53 and suppresses apoptosis. This study investigated the influence of human neutrophil elastase (HNE) and PAR-2 agonists on expression of IRF5 and IL-12p40 by macrophages stimulated with lipopolysaccharide. Granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent macrophages showed upregulation of IRF5 expression, while HNE reduced expression of p53 and IRF5 in a concentration-dependent manner. HNE also caused a concentration-dependent decrease of IRF5 in macrophages transfected with small interfering RNA to silence p53, while silencing of β-arrestin 2 blunted the reduction of p53 or IRF5 by HNE. Incubation of macrophages with a PAR-2 agonist, AC-264613, caused a decrease of IRF5 expression and also significantly reduced p53 protein expression. HNE upregulated the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) and caused transactivation of TLR4, while AC-264613 did not promote TLR4 transactivation. In conclusion, the PAR-2 agonist AC-264613 attenuated IRF5-associated IL-12p40 production by macrophages.

IRF5 regulates lung macrophages M2 polarization during severe acute pancreatitisin vitro

AIMTo investigate the role of interferon regulatory factor 5 (IRF5) in reversing polarization of lung macrophages during severe acute pancreatitis (SAP) in vitro.METHODSA mouse SAP model was established by intraperitoneal (ip) injections of 20 μg/kg body weight caerulein. Pathological changes in the lung were observed by hematoxylin and eosin staining. Lung macrophages were isolated from bronchoalveolar lavage fluid. The quantity and purity of lung macrophages were detected by fluorescence-activated cell sorting and evaluated by real-time polymerase chain reaction (RT-PCR). They were treated with IL-4/IRF5 specific siRNA (IRF5 siRNA) to reverse their polarization and were evaluated by detecting markers expression of M1/M2 using RT-PCR.RESULTSSAP associated acute lung injury (ALI) was induced successfully by ip injections of caerulein, which was confirmed by histopathology. Lung macrophages expressed high levels of IRF5 as M1 phenotype during the early acute pancreatitis stages. Reduction of IRF5 expression by IRF5 siRNA reversed the action of macrophages from M1 to M2 phenotype in vitro. The expressions of M1 markers, including IRF5 (S + IRF5 siRNA vs S + PBS, 0.013 ± 0.01 vs 0.054 ± 0.047, P < 0.01), TNF-α (S + IRF5 siRNA vs S + PBS, 0.0003 ± 0.0002 vs 0.019 ± 0.018, P < 0.001), iNOS (S + IRF5 siRNA vs S + PBS, 0.0003 ± 0.0002 vs 0.026 ± 0.018, P < 0.001) and IL-12 (S + IRF5 siRNA vs S + PBS, 0.000005 ± 0.00004 vs 0.024 ± 0.016, P < 0.001), were decreased. In contrast, the expressions of M2 markers, including IL-10 (S + IRF5 siRNA vs S + PBS, 0.060 ± 0.055 vs 0.0230 ± 0.018, P < 0.01) and Arg-1 (S + IRF5 siRNA vs S + PBS, 0.910 ± 0.788 vs 0.0036 ± 0.0025, P < 0.001), were increased. IRF5 siRNA could reverse the lung macrophage polarization more effectively than IL-4.CONCLUSIONTreatment with IRF5 siRNA can reverse the pancreatitis-induced activation of lung macrophages from M1 phenotype to M2 phenotype in SAP associated with ALI.

Onco-exaptation of an endogenous retroviral LTR drives IRF5 expression in Hodgkin lymphoma.

The transcription factor interferon regulatory factor 5 (IRF5) is upregulated in Hodgkin lymphoma (HL) and is a key regulator of the aberrant transcriptome characteristic of this disease. Here we show that IRF5 upregulation in HL is driven by transcriptional activation of a normally dormant endogenous retroviral LOR1a long terminal repeat (LTR) upstream of IRF5. Specifically, through screening of RNA-sequencing libraries, we detected LTR-IRF5 chimeric transcripts in multiple HL cell lines but not in normal B-cell controls. In HL, the LTR was in an open and hypomethylated epigenetic state, and we further show the LTR is the site of transcriptional initiation. Among HL cell lines, usage of the LTR promoter strongly correlates with overall levels of IRF5 mRNA and protein, indicating that LTR transcriptional awakening is a major contributor to IRF5 upregulation in HL. Taken together, oncogenic IRF5 overexpression in HL is the result of a specific LTR transcriptional activation. We propose that such LTR derepression is a distinct mechanism of oncogene activation ('onco-exaptation'), and that such a mechanism warrants further investigation in molecular and cancer research.

Interferon regulatory factor 5 in human autoimmunity and murine models of autoimmune disease.

Interferon regulatory factor 5 (IRF5) has been demonstrated as a key transcription factor of the immune system, playing important roles in modulating inflammatory immune responses in numerous cell types including dendritic cells, macrophages, and B cells. As well as driving the expression of type I interferon in antiviral responses, IRF5 is also crucial for driving macrophages toward a proinflammatory phenotype by regulating cytokine and chemokine expression and modulating B-cell maturity and antibody production. This review highlights the functional importance of IRF5 in a disease setting, by discussing polymorphic mutations at the human Irf5 locus that lead to susceptibility to systemic lupus erythematosus, rheumatoid arthritis, and inflammatory bowel disease. In concordance with this, we also discuss lessons in IRF5 functionality learned from murine in vivo models of autoimmune disease and inflammation and hypothesize that modulation of IRF5 activity and expression could provide potential therapeutic benefits in the clinic.

Irf5 deficiency in macrophages promotes beneficial adipose tissue expansion and insulin sensitivity during obesity

Accumulation of visceral adipose tissue correlates with elevated inflammation and increased risk of metabolic diseases. However, little is known about the molecular mechanisms that control its pathological expansion. Transcription factor interferon regulatory factor 5 (IRF5) has been implicated in polarizing macrophages towards an inflammatory phenotype. Here we demonstrate that mice lacking Irf5, when placed on a high-fat diet, show no difference in the growth of their epididymal white adipose tissue (epiWAT) but they show expansion of their subcutaneous white adipose tissue, as compared to wild-type (WT) mice on the same diet. EpiWAT from Irf5-deficient mice is marked by accumulation of alternatively activated macrophages, higher collagen deposition that restricts adipocyte size, and enhanced insulin sensitivity compared to epiWAT from WT mice. In obese individuals, IRF5 expression is negatively associated with insulin sensitivity and collagen deposition in visceral adipose tissue. Genome-wide analysis of gene expression in adipose tissue macrophages highlights the transforming growth factor β1 (TGFB1) gene itself as a direct target of IRF5-mediated inhibition. This study uncovers a new function for IRF5 in controlling the relative mass of different adipose tissue depots and thus insulin sensitivity in obesity, and it suggests that inhibition of IRF5 may promote a healthy metabolic state during this condition.

Abstract 20: Role of IRF5-IRF4 Regulatory Axis in Microglial Polarization After Neonatal Stroke

Background: Perinatal arterial ischemic stroke (PAIS) is an important cause of neonatal death and long-term disability. Once PAIS occurs, microglia are activated and innate immune responses are mounted in the brain. Microglial activation is characterized as M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotype depending on the progression of brain injury. Recent studies with adult models of peripheral inflammation have found that interferon regulatory factor 5 (IRF5) regulates expression of pro-inflammatory cytokines, while IRF4 controls M2 macrophage polarization. The balance of the IRF5-IRF4 regulatory axis is critical in establishing M1/M2 macrophage phenotypes; however, it is unknown if microglial activation is regulated by the same mechanism in neonatal brains after PAIS. We hypothesize that IRF5-IRF4 regulatory axis directs M1/M2 microglial polarization and manipulation of the axis confers neuroprotection against PAIS. Methods: IRF5/IRF4 inducible conditional knockout (ICKO) mice in which IRF5 or IRF4 was deleted specifically in microglia by Tamoxifen induction were subjected to 60-minute Rice-Vanucci Modeling (RVM) at post-natal day 10 (P10) to induce PAIS. Stroke outcomes were evaluated at 7d of RVM. C57BL6 P10 pups will also be used for examination of microglial activation and IRF5/IRF4 expression by flow cytometry (FC), RT-PCR, IHC, and western blotting. Results: Microglia exhibited high levels of M2 marker CD206 at 3d and M1 marker MHCII at 7d of PAIS by FC analysis (n= 6st, 3sh/gp). IRF4 mRNA in FC-sorted microglia was upregulated 24h after stroke and peaked at 3d, but declined to baseline at 7d. Conversely, IRF5 was not upregulated until 3d and peaked at 7d. Correspondingly, IRF4 and IRF5 expression in the nuclear fraction was increased at 3d and 7d respectively. Similar patterns of IRF5/IRF4 expression was seen in Iba-1 + cells in the brain by IHC (n= 8st, 4sh/gp). IRF4 ICKO mice had worsened stroke outcomes while IRF5 ICKO showed protective effects 7d after PAIS. Conclusions: PAIS led to a transient M2 phenotype of microglia that switched to M1 polarization at a later time. IRF5-IRF4 regulatory axis plays an important role in mediating M1/M2 polarization of microglia and is a promising target of interventional therapy for PAIS.

A conserved region within interferon regulatory factor 5 controls breast cancer cell migration through a cytoplasmic and transcription-independent mechanism.

BackgroundMigration of breast cancer cells out of a duct or lobule is a prerequisite for invasion and metastasis. However, the factors controlling breast cancer cell migration are not fully elucidated. We previously found that expression of the transcription factor interferon regulatory factor 5 (IRF5) is significantly decreased as a breast lesion progresses from a non-malignant stage to ductal carcinoma in situ and is eventually lost in ~80% of invasive ductal carcinomas examined. Human in vitro and murine in vivo models of invasive breast cancer confirmed an important role for IRF5 in regulating cell motility, invasion and/or metastasis; yet, the mechanism(s) by which this occurs is not known. Since IRF5 is primarily expressed in the cytoplasm of human mammary epithelial cells, we hypothesized that IRF5 may function in a transcription-independent manner to control intrinsic cell migration.ResultsA series of IRF5 deletion mutants were tested in cell motility, invasion and migration assays. A novel, conserved 10 amino acid domain was identified that regulates mammary epithelial cell migration. This region (∆115-125) is downstream of IRF5′s DNA binding domain and therefore when absent, retains IRF5 transcription activity but loses cell migration control. An IRF5 construct with a mutated nuclear localization signal further confirmed that IRF5 controls migration in a cytoplasmic and transcription-independent manner. Candidate cytoskeletal molecules were identified in MDA-MB-231 cells to interact with IRF5 by immunoprecipitation and mass spectrometry analysis. α6-tubulin was independently confirmed to interact with endogenous IRF5 in MCF-10A cells. Alterations in F-actin bundling after staining EV- and IRF5-231 cells with phalloidin suggests that IRF5 may control cell migration/motility through its interaction with cytoskeletal molecules that contribute to the formation of F-actin networks. Last and most notably, we found that IRF5′s control of cell migration is not restricted to mammary epithelial cells but functions in other epithelial cell types suggesting a more global role for this newly identified cell migratory function of IRF5.ConclusionsThese findings are significant as they identify a new regulator of epithelial cell migration and provide specific insight into the mechanism(s) by which loss of IRF5 expression in mammary epithelial cells contributes to breast cancer metastasis.

IRF5 is a novel regulator of CXCL13 expression in breast cancer that regulates CXCR5(+) B- and T-cell trafficking to tumor-conditioned media.

Clinical studies using prognostic and predictive signatures have shown that an immune signal emanating from whole tumors reflects the level of immune cell infiltration--a high immune signal linked to improved outcome. Factors regulating immune cell trafficking to the tumor, however, are not known. Previous work has shown that expression of interferon regulatory factor 5 (IRF5), a critical immune regulator, is lost in ~80% of invasive ductal carcinomas examined. We postulated that IRF5-positive and -negative breast tumors would differentially regulate immune cell trafficking to the tumor. Using a focused tumor inflammatory array, differences in cytokine and chemokine expression were examined between IRF5-positive and -negative MDA-MB-231 cells grown in three-dimensional culture. A number of cytokines/chemokines were found to be dysregulated between cultures. CXCL13 was identified as a direct target of IRF5 resulting in the enhanced recruitment of B and T cells to IRF5-positive tumor-conditioned media. The ability of IRF5 to regulate mediators of cell migration was confirmed by enzyme-linked immunosorbent assay, chromatin immunoprecipitation assay, small interfering RNA knockdown and immunofluorescence staining of human breast tumor tissues. Analysis of primary immune cell subsets revealed that IRF5 specifically recruits CXCR5(+) B and T cells to the tumor; CXCR5 is the receptor for CXCL13. Analysis of primary breast tumor tissues revealed a significant correlation between IRF5 and CXCL13 expression providing clinical relevance to the study. Together, these data support that IRF5 directly regulates a network of genes that shapes a tumor immune response and may, in combination with CXCL13, serve as a novel prognostic marker for antitumor immunity.

177 The Alpha-ketoglutarate Receptor GPR99 Regulates Pathological Cardiac Hypertrophy

IntroductionGPR99, a member of G-protein coupled receptor family, is expressed in the heart. Previous studies suggested that this receptor can bind to alpha-ketoglutarate, a metabolite that is elevated in the...

Transcription factor IRF5 drives P2X4R+-reactive microglia gating neuropathic pain.

In response to neuronal injury or disease, microglia adopt distinct reactive phenotypes via the expression of different sets of genes. Spinal microglia expressing the purinergic P2X4 receptor (P2X4R) after peripheral nerve injury (PNI) are implicated in neuropathic pain. Here we show that interferon regulatory factor-5 (IRF5), which is induced in spinal microglia after PNI, is responsible for direct transcriptional control of P2X4R. Upon stimulation of microglia by fibronectin, IRF5 induced de novo expression of P2X4R by directly binding to the promoter region of the P2rx4 gene. Mice lacking Irf5 did not upregulate spinal P2X4R after PNI, and also exhibited substantial resistance to pain hypersensitivity. Furthermore, we found that expression of IRF5 in microglia is regulated by IRF8. Thus, an IRF8-IRF5 transcriptional axis may contribute to shifting spinal microglia toward a P2X4R-expressing reactive state after PNI. These results may provide a new target for treating neuropathic pain.

IRF5 is a novel regulator of CXCL13 expression in breast cancer that increases CXCR5+ B and T cell trafficking to the tumor (TUM7P.955)

Abstract Clinical studies using predictive signatures have shown that a high immune signal from whole tumor expression profiles correlate with improved outcome in some cancers, including breast cancer. Factors regulating immune cell trafficking to the tumor, however, are not known. Utilizing MDA-MB-231 breast cancer cells grown in 3D culture, we examined whether intratumoral expression of interferon regulatory factor 5 (IRF5) regulates the profile of cytokines/chemokines expressed. Previous work has shown that IRF5 expression is lost in ~80% of all invasive ductal carcinomas examined. Using focused arrays, we found a number of cytokines/chemokines that were dysregulated between IRF5-positive and -negative MDA-MB-231 cells. CXCL13 was identified as a direct target of IRF5. Using tumor-conditioned media (TCM) from IRF5-positive and -negative cells, we found a significant increase in B and T cell trafficking to TCM from IRF5-positive cells. Subset analysis revealed that primary CXCR5+ B and T cells showed significantly reduced migration to IRF5-negative TCM or IRF5-positive TCM depleted of CXCL13. CXCR5 is the primary receptor for CXCL13. Analysis of human breast tumor tissues revealed a positive correlation between IRF5 and CXCL13 expression providing clinical relevance to this study. Data support that IRF5 directly regulates a network of genes that shapes a tumor immune response and may serve as a novel prognostic marker for chemotherapy and immunotherapy response.

Association of interferon regulatory factor 5 (IRF5) markers with an increased risk of lupus and overlapping autoimmunity in a Kuwaiti population

Background: Variations in IRF5 have been associated with an increased risk of developing lupus (SLE).Aim: To explore the association of IRF5 markers (rs2004640, rs10954213) with susceptibility to SLE and to the development of more than one autoimmune disease (OP) and to analyse the influence of rs2004640 on IRF5 isoform expression.Methods: A total of 300 age/gender/ethnicity-matched Kuwaitis were screened using the TaqMan®SNP Genotyping Assay. Data analysis was conducted in three groups: patients with high/low anti-dsDNA antibody titre, lupus nephritis/non-nephritis and disease onset age ≤28 years/>28 years. cDNA samples with defined rs2004640 genotypes were amplified using specific primers and transcripts associated with each of the exon1 variants were detected on polyacrylamide-gel.Results: A significant association was observed between the IRF5 markers (rs2004640, rs10954213) and susceptibility to SLE (p < 0.0001) and OP (p < 0.025). Stratified analysis showed a significant association of the rs2004640 T-allele with a high titre of anti-dsDNA antibody (p = 0.0035, OR = 1.92) and a low disease onset age ≤28 years (p = 0.0026, OR = 1.94). Additionally, the influential role of the rs2004640 T allele on IRF5 expression was observed.Conclusions: This study is the first to investigate the association of IRF5 markers with OP and the very first to explore the association of these markers with SLE in Arabs. The novelty of this study is the association of the rs2004640 T-allele with increased anti-dsDNA antibody production and SLE in a younger age group.

IRF5 is a target of BCR-ABL kinase activity and reduces CML cell proliferation

Interferon regulatory factor 5 (IRF5) modulates the expression of genes controlling cell growth and apoptosis. Previous findings have suggested a lack of IRF5 transcripts in both acute and chronic leukemias. However, to date, IRF5 expression and function have not been investigated in chronic myeloid leukemia (CML). We report that IRF5 is expressed in CML cells, where it interacts with the BCR-ABL kinase that modulates its expression and induces its tyrosine phosphorylation. Tyrosine-phosphorylated IRF5 displayed reduced transcriptional activity that was partially restored by imatinib mesylate (IM). Interestingly, a mutant devoid of a BCR-ABL consensus site (IRF5(Y104F)) still presented significant tyrosine phosphorylation. This finding suggests that the oncoprotein phosphorylates additional tyrosine residues or induces downstream signaling pathways leading to further IRF5 phosphorylation. We also found that ectopic expression of IRF5 decreases the proliferation of CML cell lines by slowing their S-G2 transition, increasing the inhibition of BCR-ABL signaling and enhancing the lethality effect observed after treatment with IM, α-2-interferon and a DNA-damaging agent. Furthermore, IRF5 overexpression successfully reduced the clonogenic ability of CML CD34-positive progenitors before and after exposure to the above-indicated cytotoxic stimuli. Our data identify IRF5 as a downstream target of the BCR-ABL kinase, suggesting that its biological inactivation contributes to leukemic transformation.

In Vivo Silencing of the Transcription Factor IRF5 Reprograms the Macrophage Phenotype and Improves Infarct Healing

The aim of this study was to test whether silencing of the transcription factor interferon regulatory factor 5 (IRF5) in cardiac macrophages improves infarct healing and attenuates post-myocardial infarction (MI) remodeling. In healing wounds, the M1 toward M2 macrophage phenotype transition supports resolution of inflammation and tissue repair. Persistence of inflammatory M1 macrophages may derail healing and compromise organ functions. The transcription factor IRF5 up-regulates genes associated with M1 macrophages. Here we used nanoparticle-delivered small interfering ribonucleic acid (siRNA) to silence IRF5 in macrophages residing in MIs and in surgically-induced skin wounds in mice. Infarct macrophages expressed high levels of IRF5 during the early inflammatory wound-healing stages (day 4 after coronary ligation), whereas expression of the transcription factor decreased during the resolution of inflammation (day 8). Following in vitro screening, we identified an siRNA sequence that, when delivered by nanoparticles to wound macrophages, efficiently suppressed expression of IRF5 in vivo. Reduction of IRF5 expression, a factor that regulates macrophage polarization, reduced expression of inflammatory M1 macrophage markers, supported resolution of inflammation, accelerated cutaneous and infarct healing, and attenuated development of post-MI heart failure after coronary ligation as measured by protease targeted fluorescence molecular tomography-computed tomography imaging and cardiac magnetic resonance imaging (p < 0.05). This work identified a new therapeutic avenue to augment resolution of inflammation in healing infarcts by macrophage phenotype manipulation. This therapeutic concept may be used to attenuate post-MI remodeling and heart failure.

Abstract P5-04-14: Loss of interferon regulatory factor 5 expression in breast cancer alters the tumor microenvironment to promote metastasis

Abstract Staining of over 300 human breast cancer tissue samples demonstrates that over 75% of invasive ductal carcinomas, regardless of subtype, have lost expression of interferon regulatory factor 5 (IRF5). Loss of IRF5 correlates with higher tumor grades, indicating that it may be an important biomarker for disease progression and metastasis. IRF5 is an important transcriptional regulator in immune cells, yet its role in the immune response and other cellular processes in ductal epithelial cells has never been examined. Levels of IRF5 are lowest in human breast cancer cell lines that are most invasive. Re-introduction of IRF5 expression in a xenograft mouse model of breast cancer decreased the number and size of primary tumors as well as completely abrogated tumor spread. Since breast cancer survival is dependent on hindering tumor spread, the focus of this study is to elucidate the mechanisms by which IRF5 inhibits metastasis. Through cell based assays we found that overexpression of IRF5 in MDA-MB-231 cells inhibits cell movement by 50%. We also characterized the protein domain necessary for this function, and found that it is not dependent on the DNA binding domain. This is the first evidence of a non-transcriptional role for IRF5 and may explain its cytoplasmic localization in normal ductal epithelial cells. A more obvious aspect of metastatic inhibition by IRF5 comes from its role as an immune regulator. IRF5 positively regulates the expression of many proteins that are considered potent activators of an inflammatory immune response. To gain insight into the cytokines and receptors that IRF5 regulates, we isolated MDA-MB-231 cells that stably express IRF5 from 3D acini and performed a focused microarray analysis of gene expression. We found that cells expressing IRF5 had significantly elevated levels of immune stimulating genes such as CXCL5, CXCL13, IL-33, and IFNg among many others. In transwell assays using IRF5 positive or negative tumor conditioned media, we found that IRF5 positive media caused increased trafficking of human lymphocytes. These data indicate that signaling from IRF5 positive and negative tumor cells is distinct and that IRF5 may play a role in shaping the tumor-immune microenvironment. We postulate that a loss of IRF5 in breast tumor hinders a favorable immune response and allows cell movement beyond the basement membrane. Genes upregulated by IRF5 in MDA-MB-231CXCL5128.3CXCR215.78CXCL1312.85CXCL812.01VegF R310.48IL-3310.31IFNg8.55CCR47.06Numerical values indicate the fold change in gene expression when compared to control. The ability of IRF5 to inhibit cell migration within the tumor cell itself and alter the surrounding microenvironment so drastically offers some explanation as to why tumor progression depends on its downregulation. With the advent of immunotherapy, the IRF5 status of a breast tumor may not only be important in determining metastatic potential, but also in determining the most appropriate immunotherapy since we expect that IRF5 positive and negative tumors will require altered treatment regimens. Further characterizing how IRF5 shapes the tumor microenvironment is essential for the development of clinically relevant immunotherapy strategies. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-04-14.

181 M1 MACROPHAGES AND IRF5 EXACERBATE ATHEROSCLEROTIC DISEASE

Introduction Atherosclerosis is an inflammatory disease that occurs in vascular sites subjected to complex blood flow, e.g. oscillatory (OSS) or low shear stress (LSS), the latter leading to the formation of a macrophage-rich plaque which is vulnerable to rupture and thrombotic events. Monocyte-derived macrophages can be conditioned towards a pro-inflammatory phenotype (M1) or a regulatory one (M2) and may determine atherosclerotic plaque outcome. Interferon Regulatory Factor 5 (IRF5) has been shown to be instrumental for M1 macrophage polarisation. We aimed to establish the occurrence of macrophage polarisation and the expression of IRF5 in murine atherosclerosis. Methods A perivascular shear stress modifying cast was tied around a carotid artery, mimicking LSS and OSS in ApoE−/− mice fed a high fat diet. Homeostatic Ly-6Clo monocyte recruitment to the carotid artery was observed after labelling them in vivo with green fluorescent microspheres. We examined the expression of the macrophage polarisation markers CD206 (M2), iNOS and IRF5 (M1) in the cast-treated carotid artery by Immunohistochemistry and Confocal Immunolocalisation. Results Labelled Ly-6Clo monocytes were more prevalent in the upstream half of LSS-modulated plaques (p=0.0006) in comparison to the downstream half and OSS-modulated lesions. These monocytes co-localised with the expression of iNOS not CD206. In addition, most macrophages in the LSS-induced carotid artery lesions (vulnerable phenotype) expressed iNOS (p=0.006) and IRF5 (p=0.03) compared to OSS-modulated lesions (stable phenotype) while CD206 expression showed no differences. Conclusions Ly-6Clo monocyte recruitment and M1 macrophages were significantly more prevalent in low shear stress-modulated plaques. These differences suggest low shear stress promotes monocyte recruitment and macrophage polarisation towards the pro-inflammatory M1 phenotype.