Interferon Regulatory Factor 5 mRNA Research Articles

The primate-specific presence of interferon regulatory factor-5 pseudogene 1.

Interferon regulatory factor 5 (IRF5) is a key transcription factor in inflammatory and immune responses, with its dysregulation linked to autoimmune diseases. Using bioinformatic approaches, including Basic Local Alignment Search Tool (BLAST) for sequence similarity searches, BLAST-Like Alignment Tool (BLAT) for genome-wide alignments, and several phylogenetics software, such as Multiple Alignment using Fast Fourier Transform (MAFFT), for phylogenetic analyses, we characterized the structure, origin, and evolutionary history of the human IRF5 pseudogene 1 (IRF5P1). Our analyses reveal that IRF5P1 is a chimeric processed pseudogene containing sequences derived from multiple sources, including IRF5-like sequences from disparate organisms. We find that IRF5P1 is specific to higher primates, likely originating through an ancient retroviral integration event approximately 60 million years ago. Interestingly, IRF5P1 resides within the triple QxxK/R motif-containing (TRIQK) gene, and its antisense strand is predominantly expressed as part of the TRIQK pre-messenger RNA (mRNA). Analysis of publicly available RNA-seq data suggests potential expression of antisense IRF5P1 RNA. We hypothesize that this antisense RNA may regulate IRF5 expression through complementary binding to IRF5 mRNA, with human genetic variants potentially modulating this interaction. The conservation of IRF5P1 in the primate lineage suggests its positive effects on primate evolution and innate immunity. This study highlights the importance of investigating pseudogenes and their potential regulatory roles in shaping lineage-specific immune adaptations.

The prognostic impact of transforming growth factor beta 2 (TGFB2) mRNA levels, in conjunction with interferon-gamma receptor activation of interferon regulatory factor 5 (IRF5) and expression of CD276/B7-H3 in low-grade gliomas.

e14564 Background: Low-grade glioma (LGG) tumors are characterized by a low infiltration of immune cells, requiring therapeutic interventions to boost the immune response. Methods: We analyzed mRNA expression datasets from the UCSC Xena web platform to compare normal brain tissue with LGG tumor samples to screen for upregulated genes. We also used cBioportal to determine the relationship between mRNA expression levels of 513 LGG patients and their overall survival (OS) outcomes. Results: Tumor-associated macrophage (TAM) markers, MSR1/CD204, CD86, and CD68, exhibited a 6-fold (P<0.0001), 8.9-fold (P<0.0001), and 15.6-fold increases in mRNA expression levels, respectively in LGG tumors. Both TGFB1 (4.1-fold increase, P<0.0001) and TGFB2 (2.2-fold increase, P<0.0001) were upregulated in brain tumors, suggesting that TGFB ligands are pivotal in establishing an immunosuppressive TME. In addition, mRNA upregulation of interferon-gamma receptors, IFNGR1 and IFNGR2, and signaling molecules STAT1, IRF1, and IRF5, pointed to an essential role for IFN-γ mediated remodeling of the TME. A tumor-associated antigen, CD276/B7-H3, mRNA expression showed a significant (P<0.0001) 4.03-fold increase in tumor tissue. Multivariate Cox proportional hazards models investigating the interaction of TGFB2 and activation of IFNGR2, STAT1, IRF1, or IRF5 showed that the prognostic impact of high mRNA levels (25 percentile cut-off) of TGFB2high was independent of IFNGR2, STAT1, IRF1, or IRF5 mRNA levels (TGFB2high HR (95% CI) = 4.07 (2.35-7.06), 6 (3.62-10.11), 4.38 (2.67-7.17), and 4.48 (2.82-7.12) for models with IFNGR2, STAT1, IRF1, or IRF5 respectively). Patients with high levels of TGFB2 were overrepresented by LGG patients with IDH wild-type mutation status. The prognostic impact of TGFB2high and IDH wild-type observed by the increases in hazard ratios for TGFB2 (HR (95% CI range) = 2.02 (1.05-3.89)) and IDH wild-type (HR (95% CI range) = 4.44 (1.9-10.4)) were independent predictors of survival requiring risk stratification of patients identify LGG patients with IDH wild-type and TGFB2high in the design of clinical trials. The ratio of TGFB1:TGFB2 expression levels was also a prognostic indicator for OS, whereby patients who exhibited a 2.5-fold greater level of TGFB1 compared to TGFB2 mRNA levels experienced improved survival outcomes. A 1.8-fold higher TGFB2 levels, the mRNA expression corresponded to worse OS. Conclusions: The specific abrogation of TGFB2 and not TGFB1 by OT101, a TGFB2-specific synthetic phosphorothioate antisense oligodeoxynucleotide, is a prerequisite to employing a strategy to target TGFB pathways. IRF5 and CD276/B7-H3 as prognostic markers are potential targets for combination therapies with TGFB2 inhibitors.

Transforming Growth Factor Beta 2 (TGFB2) mRNA Levels, in Conjunction with Interferon-Gamma Receptor Activation of Interferon Regulatory Factor 5 (IRF5) and Expression of CD276/B7-H3, Are Therapeutically Targetable Negative Prognostic Markers in Low-Grade Gliomas.

LGG tumors are characterized by a low infiltration of immune cells, requiring therapeutic interventions to boost the immune response. We conducted a study analyzing mRNA expression datasets from the UCSC Xena web platform. To screen for upregulated genes, we sought to compare normal brain tissue with LGG tumor samples. We also used cBioportal to determine the relationship between mRNA expression levels of 513 LGG patients and their overall survival (OS) outcomes. Three tumor-associated macrophage (TAM) markers, MSR1/CD204, CD86, and CD68, exhibited a 6-fold (p < 0.0001), 8.9-fold (p < 0.0001), and 15.6-fold increase in mRNA expression levels, respectively, in LGG tumors. In addition, both TGFB1 (4.1-fold increase, p < 0.0001) and TGFB2 (2.2-fold increase, p < 0.0001) ligands were also upregulated in these tumors compared to normal brain tissue, suggesting that TGFB ligands are pivotal in establishing an immunosuppressive, angiogenic, and pro-tumorigenic TME in gliomas mediated through TAMs. In addition, mRNA upregulation of interferon-gamma receptors, IFNGR1 and IFNGR2, and the downstream signaling molecules STAT1, IRF1, and IRF5, pointed to an essential role for IFN-γ mediated remodeling of the TME. Interestingly, the mRNA expression of a tumor-associated antigen, CD276/B7-H3, showed a significant (p < 0.0001) 4.03-fold increase in tumor tissue, giving further insights into the roles of macrophages and tumor cells in supporting the immunosuppressive TME. Multivariate Cox proportional hazards models investigating the interaction of TGFB2 and activation of IFNGR2, STAT1, IRF1, or IRF5 showed that the prognostic impact of high mRNA levels (25th percentile cut-off) of TGFB2 was independent of IFNGR2, STAT1, IRF1, or IRF5 mRNA levels (TGFB2high HR (95% CI) = 4.07 (2.35-7.06), 6 (3.62-10.11), 4.38 (2.67-7.17), and 4.48 (2.82-7.12) for models with IFNGR2, STAT1, IRF1, or IRF5, respectively) and age at diagnosis. Patients with high levels of TGFB2 and IFNGR2 were over-represented by LGG patients with isocitrate dehydrogenase wild-type (IDHwt) mutation status. The prognostic impact of high levels of TGFB2 and IDH wild-type observed by the increases in hazard ratios for TGFB2 (HR (95% CI range) = 2.02 (1.05-3.89)) and IDH wild-type (HR (95% CI range) = 4.44 (1.9-10.4)) were independent predictors of survival, suggesting that risk stratification of patients identifies LGG patients with IDH wild-type and high levels of TGFB2 in the design of clinical trials. Furthermore, we have additional IRF5 and CD276/B7-H3 as prognostic markers that can also be targeted for combination therapies with TGFB2 inhibitors. In support of these findings, we demonstrated that low levels of gene methylation in TGFB2, IFNGR2, IRF1, IRF5, STAT1, and CD276 were associated with significantly worse overall survival (OS) outcomes. This suggests that potential mechanisms to increase the expression of these prognostic markers occur via the action of demethylation enzymes.

Interferon Regulatory Factor 5 Gene Polymorphisms and mRNA Expression Levels Are Associated with Neuromyelitis Optica Spectrum Disorder.

Interferon regulatory factor 5 (IRF5) is a critical transcription factor in the toll-like receptor signaling pathway. It is associated with autoimmune disorders, such as rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease. However, the relationship between the functional single nucleotide polymorphisms (SNPs) of IRF5 and its mRNA expression level in patients with neuromyelitis optica spectrum disorder remains unclear. The present study aimed to investigate the relationship between polymorphisms and mRNA expression levels of the IRF5 gene with the incidence of neuromyelitis optica spectrum disorder (NMOSD) in northern Chinese Han people. Two loci of the IRF5 gene (rs2004640 and rs2280714) of 164 patients with NMOSD and 269 healthy subjects were genotyped using the multiple SNaPshot technique. The frequencies of alleles, genotypes, and haplotypes were compared. Stratified analysis was performed according to age, sex, AQP4 status, onset age, and Expanded Disability Status Scale (EDSS) score. The IRF5 mRNA levels in peripheral blood mononuclear cells (PBMCs) of 64 NMOSD patients (32 patients in the acute stage and 32 patients in the remission stage) and 35 healthy subjects were detected by real-time PCR. The association of SNP polymorphisms with the mRNA expression level was determined by nonparametric tests. Allele and genotype frequency distributions of rs2004640 showed significant differences between both groups. Compared to healthy controls, the frequency of rs2004640 T allele markedly increased in patients (OR = 1.51, 95% CI = 1.09-2.08, P = 0.005). Minor allele T and GT genotype of rs2004640 that significantly increases the risk of NMOSD were discovered using genetic inheritance models (codominant, dominant, and overdominant) and haplotype analyses. Subsequent haplotype analyses revealed that the major haplotype "T-A" containing the risk alleles (the SNP sequence of the alleles was rs2004640 and rs2280714) had adverse effects on NMOSD. Based on the stratification analysis according to the EDSS score, the GT genotype frequency in the EDSS ≥ 4 group (38.2%) was markedly lower than that in the EDSS < 4 group (61.8%) (OR = 0.32, 95% CI = 0.15-0.68, P = 0.0054), with a significant difference. The IRF5 mRNA expression level was increased in NMOSD patients compared to that in normal subjects. IRF5 gene polymorphisms may be tightly associated with the genesis and progression of NMOSD in northern Chinese Han people. IRF5 mRNA expression was increased in patients with NMOSD and significantly increased in patients with acute phase. Perhaps IRF5 expression levels can be used as a predictor of disease activity in the future.

Interferon Regulatory Factor 5 siRNA-Loaded Folate-Modified Cationic Liposomes for Acute Lung Injury Therapy.

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is an overwhelming pulmonary inflammation with limited clinical treatment strategies. Interferon regulatory factor 5 (IRF5) is a crucial regulator of inflammation factors, which can be upregulated under an inflammatory state and related to the efferocytosis of macrophages. Herein, IRF5 was knockdown by small interfering RNA (siIRF5) to promote the anti-inflammatory effect of macrophages. Macrophage-targeting cationic liposome modified by folate (FA-LP) was developed to deliver siIRF5 (FA-LP/siIRF5). Liposomes were characterized for their particle size, zeta potential, protein adsorption and hemolysis of red blood cells. The amount of IRF5 mRNA and the expression of IRF5 were measured using quantitative reverse transcription PCR (RT-qPCR) and western blot, respectively. The phenotype and efferocytosis of macrophages and the regulatory pathway of efferocytosis and biodistribution of liposomes in the ALI mice model were investigated. Data revealed that FA-LP/siIRF5 could obviously downregulate the expression of IRF5 in macrophages, skewing the polarization of macrophages to M2 phenotype (anti-inflammatory state) and thus improving their efferocytosis. Moreover, regulation of efferocytosis of macrophages by siIRF5 is related to the NF- B pathway. The in vivo biodistribution of FA-LP exhibited higher accumulation in the inflammatory lungs, suggesting that FA-LP could be considered as a promising gene delivery system and FA-LP/siIRF5 is an alternative strategy for the treatment of ALI/ARDS. To the best of our knowledge, this is the first study reporting that siIRF5 can be used for the treatment of ALI/ARDS.

IRF5 is increased in labouring myometrium and regulates pro-labour mediators.

Preterm birth continues to be the leading cause of neonatal mortality and morbidities that can extend into adult life. Few treatment options stem from our incomplete understanding of the mechanisms of human labour and delivery. Activation of the inflammatory response in gestational tissues by inflammation and/or infection leads to the production of pro-inflammatory and pro-labour mediators, thus preterm birth. Interferon regulatory factor 5 (IRF5) has recently emerged as an important pro-inflammatory transcription factor involved in acute and chronic inflammation. The aims of this study were to determine the expression of IRF5 in human myometrium from labouring and non-labouring women, and whether IRF5 is involved in the genesis of pro-inflammatory and pro-labour mediators induced by pro-inflammatory cytokines or toll-like receptor (TLR) ligands. IRF5 mRNA and protein expression was significantly higher in human myometrium after spontaneous term labour, compared to non-labouring tissues. IRF5 mRNA expression was also significantly higher in primary myometrial cells treated with the pro-inflammatory cytokines IL1B or TNF. In primary myometrial cells, IRF5 knockdown by siRNA (siIRF5) was associated with significantly decreased expression and or secretion of pro-inflammatory cytokines (IL1A, IL6), chemokines (CXCL8, CCL2), adhesion molecules (ICAM1, VCAM1) and contraction-associated proteins PTGS2, PGF2α and PTGFR when in the presence of IL1B, TNF, fsl-1 (TLR2/6 ligand) or flagellin (TLR5 ligand). siIRF5-transfected cells also displayed decreased NF-κB RELA transcriptional activity in the presence of these preterm birth mediators. Our study suggests a novel role for IRF5 in the regulation of the inflammatory response in human myometrium.

Interferon regulatory factor 4/5 signaling impacts on microglial activation after ischemic stroke in mice.

Microglial activation is a key element in initiating and perpetuating inflammatory responses to stroke. Interferon regulatory factor 5 (IRF5) and IRF4 signaling have been found critical in mediating macrophage pro‐inflammatory (M1) and anti‐inflammatory (M2) phenotypes, respectively, in peripheral inflammation. We hypothesize that the IRF5/4 regulatory axis also mediates microglial activation after stroke. C57BL6 mice of 8–12 weeks were subject to a 90‐min middle cerebral artery occlusion, and the brains evaluated at 24 h, 3, 10 and 30 days after reperfusion. Flow cytometry was utilized to examine microglial activation and cytokine expression. RT‐PCR was performed for mRNA levels of IRF5/4 in sorted microglia. Microglial expression of IRF5/4 was examined by immunohistochemistry, and brain cytokine levels were determined by ELISA. Our results revealed that the IRF5 mRNA level in sorted microglia increased at 3 days of stroke; whereas IRF4 mRNA level exhibited biphasic increases, with a transient rise at 24 h and a peak at 10 days. The same pattern was seen in IRF5/4 protein colocalization with Iba‐1+ cells by IHC. Intracellular levels of TNF‐α and IL‐1β in microglia peaked at 3 days of stroke, and IL‐4+ IL‐10+ double‐positive microglia significantly increased at day 10. Brain levels of these cytokines were consistent with microglial cytokine changes. Worse behavior test results were seen at 3 days vs. 10 days of stroke. We conclude that microglia phenotypes are dynamic to ischemic stroke, and IRF5/4 signaling may regulate microglial M1/M2 activation and impact on stroke outcomes.

Interferon regulatory factor 5 (IRF5) suppresses hepatitis C virus (HCV) replication and HCV-associated hepatocellular carcinoma

Hepatitis C virus (HCV) infection is a major risk factor for the development of chronic liver disease. The disease typically progresses from chronic HCV to fibrosis, cirrhosis, hepatocellular carcinoma (HCC), and death. Chronic inflammation associated with HCV infection is implicated in cirrhosis and HCC, but the molecular players and signaling pathways contributing to these processes remain largely unknown. Interferon regulatory factor 5 (IRF5) is a molecule of interest in HCV-associated HCC because it has critical roles in virus-, Toll-like receptor (TLR)-, and IFN-induced signaling pathways. IRF5 is also a tumor suppressor, and its expression is dysregulated in several human cancers. Here, we present first evidence that IRF5 expression and signaling are modulated during HCV infection. Using HCV infection of human hepatocytes and cells with autonomously replicating HCV RNA, we found that levels of IRF5 mRNA and protein expression were down-regulated. Of note, reporter assays indicated that IRF5 re-expression inhibited HCV protein translation and RNA replication. Gene expression analysis revealed significant differences in the expression of cancer pathway mediators and autophagy proteins rather than in cytokines between IRF5- and empty vector-transfected HCV replicon cells. IRF5 re-expression induced apoptosis via loss in mitochondrial membrane potential, down-regulated autophagy, and inhibited hepatocyte cell migration/invasion. Analysis of clinical HCC specimens supports a pathologic role for IRF5 in HCV-induced HCC, as IRF5 expression was down-regulated in livers from HCV-positive versus HCV-negative HCC patients or healthy donor livers. These results identify IRF5 as an important suppressor of HCV replication and HCC pathogenesis.

IRF5 is elevated in childhood-onset SLE and regulated by histone acetyltransferase and histone deacetylase inhibitors.

Interferon regulatory factor 5 (IRF5) plays a critical role in the induction of type I interferon, proinflammatory cytokines and chemokines, and participates in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). However, the relationship between IRF5 and childhood-onset SLE remains elusive. In the present study, we demonstrated that levels of mRNA expression of IRF5, IFN-α, and Sp1 were significantly increased in childhood-onset SLE, as seen on quantitative real-time PCR, and the expression of Sp1 and IFN-α was positively correlated with IRF5. In addition to being used as antitumor drugs, a number of histone deacetylase inhibitors (HDACi) display potent anti-inflammatory properties; however, their effects on IRF5 expression remain unclear. In this study, we identified that HDACi trichostatin A (TSA) and histone acetyltransferase (HAT)-p300 downregulated IRF5 promoter activity, mRNA expression, and protein level, whereas the HAT-p300/CBP-associated factor had no effect. Moreover, TSA inhibited the production of TNF-α and IL-6 in differentiated THP-1cells. Furthermore, chromatin immunoprecipitation assays revealed that TSA inhibited DNA binding of Sp1, RNA polymerase II, HDAC3, and p300 to the core promoter region of IRF5. Our results suggest that HDACi may have therapeutic potential in patients with autoimmune diseases such as SLE through repression of IRF5 expression.

Differences in IP‑10, TLR4 and IRF5/3 between SVR and non‑SVR HCV‑1 patients treated with PEG‑IFN and ribavirin.

The present study aimed to investigate alterations in Toll‑like receptor4 (TLR4), interferon regulatory factor5 (IRF5) and interferon‑γ‑inducible protein‑10 (IP‑10), and evaluate whether these factors may be associated with a sustained virological response (SVR) among patients with hepatitisC virus genotype‑1 (HCV‑1) who were treated with peginterferon plus ribavirin (PEG‑IFN‑RBV). A total of 31 Chinese patients infected with HCV‑1 were enrolled in the present study and 25patients obtained SVR. The expression levels of IP‑10 declined significantly during PEG‑IFN‑RBV therapy at the 24 and 48week time‑points, compared with the baseline (P<0.005, 0.001 and 0.001, respectively). In addition, it was observed that IRF5 mRNA expression and the number of TLR4+ peripheral blood mononuclear cells exhibited similar correlations with IP‑10 concentration (R2=0.0726, P=0.001, R2=0.1634, P<0.0001, respectively) in the SVR group patients; however, these correlations were not observed to be present in the non‑SVR group patients. In conclusion, the results of the present study suggest that marked alterations in IP‑10, TLR4 and IRF5 expression may serve as indicators for the development of SVR in patients with HCV‑1 treated with PEG‑IFN‑RBV.

Abstract 45: IRF5/IRF4 Regulatory Axis Mediated Microglial M1/M2 Polarization After Ischemic Stroke

Introduction: Microglia have a prominent role in initiating, sustaining and resolving post-stroke inflammation. The pathology of stroke largely depends on the spectrum of microglial activation with the classically (M1; pro-inflammatory) and/or alternatively activated (M2; anti-inflammatory) phenotype. It has been reported that interferon regulatory factor 5 (IRF5) and IRF4 regulate the M1 and M2 phenotype of macrophages respectively in systemic inflammation; however, it is unknown if microglial activation is regulated by the same mechanism after stroke. We hypothesize that IRF5/IRF4 directs M1/M2 microglial polarization respectively after stroke and the balance of the IRF5/IRF4 regulatory axis influences stroke outcomes. Methods: C57BL6 mice (8-12 weeks) were subjected to a 90-minute middle cerebral artery occlusion (MCAO). Stroke outcomes were evaluated at 12h, 3d, 10d of MCAO. Protein and mRNA levels of IRF4/5 at each time point were examined by IHC, Western blot and RT-PCR. Microglial phenotypes were evaluated by flow cytometry (FC) and determined by the ratio of MHC-II + (M1) or CD206 + (M2) microglia number over the total microglia. IRF4/IRF5 conditional knockout (CKO) mice will also be utilized to examine the effect of gene deletion on stroke outcomes. Results: IRF4 protein level increased at 10d in the ipsilateral brain lysates, but IRF5 level decreased at day 10 compared to that of 3d. M2 microglia number was higher at 10d vs. 3d by FC analysis; while M1 microglia ratio was lower at 10d vs. 3d. The Iba1 + IRF4 + (M2) and Iba1 + IRF5 + (M1) cell number by IHC showed the same pattern as that in FC data. In addition, IRF4 mRNA in FC-sorted microglia was up-regulated at 3d of stroke and peaked at 10d, but IRF5 mRNA significantly increased at 3d vs. sham and declined to baseline at 10d. IRF5-CKO mice had significantly smaller infarct volume and improved behavior deficits than IRF5 flox/flox mice while IRF4-CKO mice had worse outcomes than that of IRF4 flox/flox mice. Conclusion: IRF5-mediated pro-inflammatory phenotype (M1) is predominate in the acute stage of stroke that switches to IRF4-mediated anti-inflammatory phenotype (M2) in the chronic stage. IRF5/IRF4 regulatory axis regulates the microglial M1/M2 activation and impacts stroke outcomes.

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.

Molecular Effects of Autoimmune-Risk Promoter Polymorphisms on Expression, Exon Choice, and Translational Efficiency of Interferon Regulatory Factor 5

The rs2004640 single nucleotide polymorphism and the CGGGG copy-number variant (rs77571059) are promoter polymorphisms within interferon regulatory factor 5 (IRF5). They have been implicated as susceptibility factors for several autoimmune diseases. IRF5 uses alternative promoter splicing, where any of 4 first exons begin the mRNA. The CGGGG indel is in exon 1A's promoter; the rs2004640 allele creates a splicing recognition site, enabling usage of exon 1B. This study aimed at characterizing alterations in IRF5 mRNA due to these polymorphisms. Cells with risk polymorphisms exhibited ~2-fold higher levels of IRF5 mRNA and protein, but demonstrated no change in mRNA stability. Quantitative PCR demonstrated decreased usage of exons 1C and 1D in cell lines with the risk polymorphisms. RNA folding analysis revealed a hairpin in exon 1B; mutational analysis showed that the hairpin shape decreased translation 5-fold. Although translation of mRNA that uses exon 1B is low due to a hairpin, increased IRF5 mRNA levels in individuals with the rs2004640 risk allele lead to higher overall protein expression. In addition, several new splice variants of IRF5 were sequenced. IRF5's promoter polymorphisms alter first exon usage and increase transcription levels. High levels of IRF5 may bias the immune system toward autoimmunity.

IRF5 is a specific marker of inflammatory macrophages in vivo.

Macrophages are an integral part of the innate immune system and key players in pathogen clearance and tissue remodelling. Both functions are accomplished by a pivotal network of different macrophage subtypes, including proinflammatory M1 and anti-inflammatory M2 macrophages. Previously, our laboratory identified the transcription factor interferon regulatory factor 5 (IRF5) as the master regulator of the M1 macrophage polarisation. IRF5 was found to be highly expressed in human M1 compared to M2 macrophages. Furthermore, IRF5 dictates the expression of proinflammatory genes such as IL12b and IL23a whilst repressing anti-inflammatory genes like IL10. Here we show that murine bone marrow derived macrophages differentiated in vitro with GM-CSF are also characterised by high levels of IRF5 mRNA and protein and express proinflammatory cytokines upon LPS stimulation. These macrophages display characteristic expression of M1-marker MHC II but lack the M2-marker CD206. Significantly, we develop intracellular staining of IRF5- expressing macrophages and utilise it to recapitulate the in vitro results in an in vivo model of antigen-induced arthritis, emphasising their physiological relevance. Thus, we establish the species-invariant role of IRF5 in controlling the inflammatory macrophage phenotype both in vitro and in in vivo.

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.

Methylation profile of the promoter region of IRF5 in primary Sjögren's syndrome

The transcription factor interferon regulatory factor 5 (IRF5), in the type I interferon pathway is involved in the genetic susceptibility to various autoimmune diseases. A 5-bp insertion/deletion (CGGGG indel) polymorphism in the promoter region of IRF5 associated with primary Sjögren's syndrome (pSS) could be epigenetically deregulated in this condition. Therefore, we investigated DNA methylation patterns of the promoter region of IRF5 to determine whether its epigenetic deregulation could explain the increased expression of IRF5 mRNA in pSS patients, along with the risk of pSS induced by the genetic polymorphism. DNA extracted from total peripheral blood mononuclear cells, isolated CD4(+) T cells, B lymphocytes and monocytes from 19 pSS patients and 24 healthy controls underwent methylation analysis by pyrosequencing. Salivary gland epithelial cells (SGECs) were cultured from minor salivary glands. Regions of interest in the CGGGG repeat and ATG initiation codon region were amplified by PCR and analysed by pyrosequencing. The effect of the demethylating agent 5-AzaC on IRF5 mRNA expression in controls was quantified by RT-PCR. Among the healthy controls, the mean methylation of the nine CpG pairs of the CGGGG repeat region and the 18 CpG pairs of the ATG region was < 15% in CD4(+) T cells, B lymphocytes, monocytes and SGECs. Patients and controls did not differ in methylation profiles as regards CD4(+) T cells and B lymphocytes. IRF5 mRNA expression did not differ with or without 5-AzaC in controls. The absence of aberrant DNA methylation profiles for the putative regulatory regions of IRF5 in CD4(+) T cells, B lymphocytes, and monocytes from patients with pSS, does not support the hypothesis that epigenetic deregulation in combination with the genetic polymorphism explains the increase in IRF5 mRNA levels in pSS patients.

Irf5‐deficient mice are protected from pristane‐induced lupus via increased Th2 cytokines and altered IgG class switching

Polymorphisms in the transcription factor interferon (IFN) regulatory factor 5 (IRF5) have been identified that show a strong association with an increased risk of developing the autoimmune disease systemic lupus erythematosus (SLE). A potential pathological role for IRF5 in SLE development is supported by the fact that increased IRF5 mRNA and protein are observed in primary blood cells of SLE patients and this correlates with an increased risk of developing the disease. Here, we demonstrate that IRF5 is required for pristane-induced SLE via its ability to control multiple facets of autoimmunity. We show that IRF5 is required for pathological hypergammaglobulinemia and, in the absence of IRF5, IgG class switching is reduced. Examination of in vivo cytokine expression (and autoantibody production) identified an increase in Irf5(-/-) mice of Th2 cytokines. In addition, we provide clear evidence that loss of Irf5 significantly weakens the in vivo type I IFN signature critical for disease pathogenesis in this model of murine lupus. Together, these findings demonstrate the importance of IRF5 for autoimmunity and provide a significant new insight into how overexpression of IRF5 in blood cells of SLE patients may contribute to disease pathogenesis.

Protection of Irf5-deficient mice from pristane-induced lupus involves altered cytokine production and class switching (159.11)

Abstract Polymorphisms in the transcription factor interferon (IFN) regulatory factor 5 (IRF5) have been identified that show strong association with increased risk of developing the autoimmune disease systemic lupus erythematosus (SLE). A potential pathologic role for IRF5 in SLE development is supported by the fact that increased IRF5 mRNA and protein abundance are observed in primary blood cells of SLE patients that correlate with increased risk of developing the disease. Here, we demonstrate in clean littermates that IRF5 is required for pristane-induced lupus via its ability to control multiple facets of autoimmunity. We show that IRF5 has a distinct influence on pathological hypergammaglobulinemia and provide evidence for its role in regulating IgG1 class switching and antigen specificity. Examination of in vivo cytokine expression (and autoantibody production) identified an imbalance in Irf5-/- mice favoring Th2 polarization. In addition, we provide clear evidence that loss of Irf5 significantly weakens the in vivo type I IFN signature that is critical for disease pathogenesis in this model of murine lupus. Together, these findings demonstrate the global effect that IRF5 has on autoimmunity and provides significant new insight into how overexpression of IRF5 in blood cells of SLE patients may contribute to disease pathogenesis.

Altered IRF5 exon usage and increased expression of IRF7 targets in cells with an allele associated with risk for lupus (47.27)

Abstract Background. The single nucleotide polymorphism rs2004640 within interferon regulatory factor 5 (IRF5) is a well established genetic risk locus for systemic lupus erythematosus (SLE). The overall effects on the cell, as well as the mechanisms through which this polymorphism enhances the risk for lupus remain elusive. Since an alternate first exon is created by this polymorphism, we hypothesized that the risk haplotype causes changes in splicing patterns. We further hypothesized that the risk allele may alter the expression levels of the transcriptional targets of either IRF5 or IRF7, which is modulated by IRF5. Methods. Peripheral blood mononuclear cells were isolated from lupus unaffected individuals after informed consent. DNA was extracted and genotyped for the allele at rs2004640. IRF 5 splice variant usage in EBV-transformed cells from nine race, sex, and age-matched pairs differing in rw2004640 genotype were compared using quantitative RT-PCR. Expression of genes regulated by IRF5 and IRF7 was also compared using quantitative RT-PCR. Statistical differences were evaluated using the Wilcoxon signed rank sum test. Results. Cells with the risk haplotype had more IRF5 mRNA (1.5-fold, p=0.05), but less exon 1C (1.9-fold, p=0.017) and less exon 1D (3.5-fold, p=0.012) than those with the protective type. Additionally, transcription of three targets of IRF7 was increased in risk cells, (average 1.5-fold increase, p&amp;lt;0.05).

Association of IRF5 polymorphisms with activation of the interferon α pathway

ObjectiveThe genetic association of interferon regulatory factor 5 (IRF5) with systemic lupus erythematosus (SLE) susceptibility has been convincingly established. To gain understanding of the effect of IRF5 variation in individuals...