Polymorphisms in the interferon regulatory factor 5 (IRF5) gene have been consistently replicated and shown to confer risk for or protection from the development of systemic lupus erythematosus (SLE). IRF5 expression is significantly upregulated in SLE patients and upregulation associates with IRF5-SLE risk haplotypes. IRF5 alternative splicing has also been shown to be elevated in SLE patients. Given that human IRF5 exists as multiple alternatively spliced transcripts with distinct function(s), it is important to determine whether the IRF5 transcript profile expressed in healthy donor immune cells is different from that expressed in SLE patients. Moreover, it is not currently known whether an IRF5-SLE risk haplotype defines the profile of IRF5 transcripts expressed. Using standard molecular cloning techniques, we identified and isolated 14 new differentially spliced IRF5 transcript variants from purified monocytes of healthy donors and SLE patients to generate an IRF5 variant transcriptome. Next-generation sequencing was then used to perform in-depth and quantitative analysis of full-length IRF5 transcript expression in primary immune cells of SLE patients and healthy donors by next-generation sequencing. Evidence for additional alternatively spliced transcripts was obtained from de novo junction discovery. Data from these studies support the overall complexity of IRF5 alternative splicing in SLE. Results from next-generation sequencing correlated with cloning and gave similar abundance rankings in SLE patients thus supporting the use of this new technology for in-depth single gene transcript profiling. Results from this study provide the first proof that 1) SLE patients express an IRF5 transcript signature that is distinct from healthy donors, 2) an IRF5-SLE risk haplotype defines the top four most abundant IRF5 transcripts expressed in SLE patients, and 3) an IRF5 transcript signature enables clustering of SLE patients with the H2 risk haplotype.
Interferon regulatory factor 5 gene (IRF5) polymorphisms are strongly associated with several diseases, including systemic lupus erythematosus (SLE). The association includes risk and protective components. They could be due to combinations of functional polymorphisms and related to cis-regulation of IRF5 expression, but their mechanisms are still uncertain. We hypothesised that thorough testing of the relationships between IRF5 polymorphisms, expression data from multiple experiments and SLE-associated haplotypes might provide useful new information.
Expression data from four published microarray hybridisation experiments with lymphoblastoid cell lines (57 to 181 cell lines) were retrieved. Genotypes of 109 IRF5 polymorphisms, including four known functional polymorphisms, were considered. The best linear regression models accounting for the IRF5 expression data were selected by using a forward entry procedure. SLE-associated IRF5 haplotypes were correlated with the expression data and with the best cis-regulatory models.
A large fraction of variability in IRF5 expression was accounted for by linear regression models with IRF5 polymorphisms, but at a different level in each expression data set. Also, the best models from each expression data set were different, although there was overlap between them. The SNP introducing an early polyadenylation signal, rs10954213, was included in the best models for two of the expression data sets and in good models for the other two data sets. The SLE risk haplotype was associated with high IRF5 expression in the four expression data sets. However, there was also a trend towards high IRF5 expression with some protective and neutral haplotypes, and the protective haplotypes were not associated with IRF5 expression. As a consequence, correlation between the cis-regulatory best models and SLE-associated haplotypes, regarding either the risk or protective component, was poor.
Our analysis indicates that although the SLE risk haplotype of IRF5 is associated with high expression of the gene, cis-regulation of IRF5 expression is not enough to fully account for IRF5 association with SLE susceptibility, which indicates the need to identify additional functional changes in this gene.
systemic lupus erythematosus; IRF5; lymphoblastoid cell lines; cis-regulation; disease susceptibility; linear regression models
High serum interferon α (IFNα) activity is a heritable risk factor for systemic lupus erythematosus (SLE). Auto-antibodies found in SLE form immune complexes which can stimulate IFNα production by activating endosomal Toll-like receptors and interferon regulatory factors (IRFs), including IRF5. Genetic variation in IRF5 is associated with SLE susceptibility; however, it is unclear how IRF5 functional genetic elements contribute to human disease.
1034 patients with SLE and 989 controls of European ancestry, 555 patients with SLE and 679 controls of African–American ancestry, and 73 patients with SLE of South African ancestry were genotyped at IRF5 polymorphisms, which define major haplotypes. Serum IFNα activity was measured using a functional assay.
In European ancestry subjects, anti-double-stranded DNA (dsDNA) and anti-Ro antibodies were each associated with different haplotypes characterised by a different combination of functional genetic elements (OR > 2.56, p >003C; 1.9×10−14 for both). These IRF5 haplotype-auto-antibody associations strongly predicted higher serum IFNα in patients with SLE and explained > 70% of the genetic risk of SLE due to IRF5. In African–American patients with SLE a similar relationship between serology and IFNα was observed, although the previously described European ancestry-risk haplotype was present at admixture proportions in African–American subjects and absent in African patients with SLE.
The authors define a novel risk haplotype of IRF5 that is associated with anti-dsDNA antibodies and show that risk of SLE due to IRF5 genotype is largely dependent upon particular auto-antibodies. This suggests that auto-antibodies are directly pathogenic in human SLE, resulting in increased IFNα in cooperation with particular combinations of IRF5 functional genetic elements.
SLE is a systemic autoimmune disorder affecting multiple organ systems including the skin, musculoskeletal, renal and haematopoietic systems. Humoral autoimmunity is a hallmark of SLE, and patients frequently have circulating auto-antibodies directed against dsDNA, as well as RNA binding proteins (RBP). Anti-RBP autoantibodies include antibodies which recognize Ro, La, Smith (anti-Sm), and ribonucleoprotein (anti-nRNP), collectively referred to as anti-retinol-binding protein). Anti-retinol-binding protein and anti-dsDNA auto-antibodies are rare in the healthy population.1 These auto-antibodies can be present in sera for years preceding the onset of clinical SLE illness2 and are likely pathogenic in SLE.34
The 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 without SLE, a study was undertaken to examine whether such genetic variation predisposes to activation of the interferon α (IFNα) pathway.
Using a computer simulated approach, 14 single nucleotide polymorphisms (SNPs) and haplotypes of IRF5 were tested for association with mRNA expression levels of IRF5, IFNα and IFN-inducible genes and chemokines in lymphoblastoid cell lines (LCLs) from individuals of European (CEU), Han Chinese (CHB), Japanese (JPT) and Yoruba Nigerian (YRI) backgrounds. IFN-inducible gene expression was assessed in LCLs from children with SLE in the presence and absence of IFNα stimulation.
The major alleles of IRF5 rs13242262 and rs2280714 were associated with increased IRF5 mRNA expression levels in the CEU, CHB+JPT and YRI samples. The minor allele of IRF5 rs10488631 was associated with increased IRF5, IFNα and IFN-inducible chemokine expression in CEU (pc=0.0005, 0.01 and 0.04, respectively). A haplotype containing these risk alleles of rs13242262, rs10488631 and rs2280714 was associated with increased IRF5, IFNα and IFN-inducible chemokine expression in CEU LCLs. In vitro studies showed specific activation of IFN-inducible genes in LCLs by IFNα.
SNPs of IRF5 in healthy individuals of a number of ethnic groups were associated with increased mRNA expression of IRF5. In European-derived individuals, an IRF5 haplotype was associated with increased IRF5, IFNα and IFN-inducible chemokine expression. Identifying individuals genetically predisposed to increased IFN-inducible gene and chemokine expression may allow early detection of risk for SLE.
Systemic lupus erythematosus (SLE) is a severe multi-system autoimmune disease which results from both genetic predisposition and environmental factors. Many lines of investigation support interferon alpha (IFN-α) as a causal agent in human lupus, and high levels of serum IFN-α are a heritable risk factor for SLE. Interferon regulatory factors (IRFs) are a family of transcription factors involved in host defense, which can induce transcription of IFN-α and other immune response genes following activation. In SLE, circulating immune complexes which contain nucleic acid are prevalent. These complexes are recognized by endosomal Toll-like receptors, resulting in activation of downstream IRF proteins. Genetic variants in the IRF5 and IRF7 genes have been associated with SLE susceptibility, and these same variants are associated with increased serum IFN-α in SLE patients. The increase in serum IFN-α related to IRF5 and 7 genotypes is observed only in patients with particular antibody specificities. This suggests that chronic stimulation of the endosomal Toll-like receptors by autoantibody immune complexes is required for IRF SLE-risk variants to cause elevation of circulating IFN-α and subsequent risk of SLE. Recently, genetic variation in the IRF8 gene has been associated with SLE and multiple sclerosis, and studies support an impact of IRF8 genotype on the IFN-α pathway. In summary, the SLE-associated polymorphisms in the IRF family of proteins appear to be gain-of-function variants, and understanding the impact of these variants upon the IFN-α pathway in vivo may guide therapeutic strategies directed at the Toll-like receptor/IRF/IFN-α pathway in SLE.
Interferon Alpha; Genetics; Systemic Lupus Erythematosus; Interferon Regulatory Factor; Autoantibodies; Autoimmunity
Interferon-α (IFNα) is a heritable risk factor for systemic lupus erythematosus (SLE). Genetic variation near IRF7 is implicated in SLE susceptibility. SLE-associated autoantibodies can stimulate IFNα production through the Toll-like receptor/IRF7 pathway. This study was undertaken to determine whether variants of IRF7 act as risk factors for SLE by increasing IFNα production and whether autoantibodies are important to this phenomenon.
We studied 492 patients with SLE (236 African American, 162 European American, and 94 Hispanic American subjects). Serum levels of IFNα were measured using a reporter cell assay, and single-nucleotide polymorphisms (SNPs) in the IRF7/PHRF1 locus were genotyped.
In a joint analysis of European American and Hispanic American subjects, the rs702966 C allele was associated with the presence of anti–double-stranded DNA (anti-dsDNA) antibodies (odds ratio [OR] 1.83, P = 0.0069). The rs702966 CC genotype was only associated with higher serum levels of IFNα in European American and Hispanic American patients with anti-dsDNA antibodies (joint analysis P = 4.1 × 10−5 in anti-dsDNA–positive patients and P = 0.99 in anti-dsDNA–negative patients). In African American subjects, anti-Sm antibodies were associated with the rs4963128 SNP near IRF7 (OR 1.95, P = 0.0017). The rs4963128 CT and TT genotypes were associated with higher serum levels of IFNα only in African American patients with anti-Sm antibodies (P = 0.0012). In African American patients lacking anti-Sm antibodies, an effect of anti-dsDNA–rs702966 C allele interaction on serum levels of IFNα was observed, similar to the other patient groups (overall joint analysis P = 1.0 × 10−6). In European American and Hispanic American patients, the IRF5 SLE risk haplotype showed an additive effect with the rs702966 C allele on IFNα level in anti-dsDNA–positive patients.
Our findings indicate that IRF7/PHRF1 variants in combination with SLE-associated autoantibodies result in higher serum levels of IFNα, providing a biologic relevance for this locus at the protein level in human SLE in vivo.
Genetic variants of the interferon (IFN) regulatory factor 5 (IRF5) gene are associated with systemic lupus erythematosus (SLE) susceptibility. The contribution of these variants to IRF-5 expression in primary blood cells of SLE patients has not been addressed, nor has the role of type I IFN. The aim of this study was to determine the association between increased IRF-5 expression and the IRF5 risk haplotype in SLE patients.
IRF-5 transcript and protein levels in 44 Swedish patients with SLE and 16 healthy controls were measured by quantitative real-time PCR, minigene assay, and flow cytometry. The rs2004640, rs10954213, rs10488631 and the CGGGG indel were genotyped in these patients. Genotypes of these polymorphisms defined a common risk and protective haplotype.
IRF-5 expression and alternative splicing were significantly upregulated in SLE patients versus healthy donors. Enhanced transcript and protein levels were associated with the risk haplotype of IRF5; rs10488631 gave the only significant independent association that correlated with increased transcription from non-coding exon 1C. Minigene experiments demonstrated an important role for rs2004640 and the CGGGG indel, along with type I IFNs in regulating IRF-5 expression.
This study provides the first formal proof that IRF-5 expression and alternative splicing are significantly upregulated in primary blood cells of SLE patients. The risk haplotype is associated with enhanced IRF-5 transcript and protein expression in SLE patients.
Interferon regulatory factor 2 (IRF2) negatively regulates type I interferon (IFN) responses, while it plays a role in induction of Th1 differentiation. Previous linkage and association studies in European-American populations suggested genetic role of IRF2 in systemic lupus erythematosus (SLE); however, this observation has not yet been confirmed. No studies have been reported in the Asian populations. Here we investigated whether IRF2 polymorphisms contribute to susceptibility to SLE in a Japanese population. Association study of 46 IRF2 tag single nucleotide polymorphisms (SNPs) detected association of an intronic SNP, rs13146124, with SLE. When the association was analyzed in 834 Japanese patients with SLE and 817 healthy controls, rs13146124 T was significantly increased in SLE compared with healthy controls (dominant model, P = 5.4×10−4, Bonferroni-corrected P [Pc] = 0.026, odds ratio [OR] 1.48, 95% confidence interval [CI] 1.18–1.85). To find causal SNPs, resequencing was performed by next-generation sequencing. Twelve polymorphisms in linkage disequilibrium with rs13146124 (r2: 0.30–1.00) were identified, among which significant association was observed for rs66801661 (allele model, P = 7.7×10−4, Pc = 0.037, OR 1.53, 95%CI 1.19–1.96) and rs62339994 (dominant model, P = 9.0×10−4, Pc = 0.043, OR 1.46, 95%CI 1.17–1.82). The haplotype carrying both of the risk alleles (rs66801661A–rs62339994A) was significantly increased in SLE (P = 9.9×10−4), while the haplotype constituted by both of the non-risk alleles (rs66801661G–rs62339994G) was decreased (P = 0.0020). A reporter assay was carried out to examine the effect of the IRF2 haplotypes on the transcriptional activity, and association of the IRF2 risk haplotype with higher transcriptional activity was detected in Jurkat T cells under IFNγ stimulation (Tukey's test, P = 1.2×10−4). In conclusion, our observations supported the association of IRF2 with susceptibility to SLE, and the risk haplotype was suggested to be associated with transcriptional activation of IRF2.
Systemic lupus erythematosus (SLE) is a complex trait characterised by the production of a range of auto-antibodies and a diverse set of clinical phenotypes. Currently, ∼8% of the genetic contribution to SLE in Europeans is known, following publication of several moderate-sized genome-wide (GW) association studies, which identified loci with a strong effect (OR>1.3). In order to identify additional genes contributing to SLE susceptibility, we conducted a replication study in a UK dataset (870 cases, 5,551 controls) of 23 variants that showed moderate-risk for lupus in previous studies. Association analysis in the UK dataset and subsequent meta-analysis with the published data identified five SLE susceptibility genes reaching genome-wide levels of significance (Pcomb<5×10−8): NCF2 (Pcomb = 2.87×10−11), IKZF1 (Pcomb = 2.33×10−9), IRF8 (Pcomb = 1.24×10−8), IFIH1 (Pcomb = 1.63×10−8), and TYK2 (Pcomb = 3.88×10−8). Each of the five new loci identified here can be mapped into interferon signalling pathways, which are known to play a key role in the pathogenesis of SLE. These results increase the number of established susceptibility genes for lupus to ∼30 and validate the importance of using large datasets to confirm associations of loci which moderately increase the risk for disease.
Genome-wide association studies have revolutionised our ability to identify common susceptibility alleles for systemic lupus erythematosus (SLE). In complex diseases such as SLE, where many different genes make a modest contribution to disease susceptibility, it is necessary to perform large-scale association studies to combine results from several datasets, to have sufficient power to identify highly significant novel loci (P<5×10−8). Using a large SLE collection of 870 UK SLE cases and 5,551 UK unaffected individuals, we firstly replicated ten moderate-risk alleles (P<0.05) from a US–Swedish study of 3,273 SLE cases and 12,188 healthy controls. Combining our results with the US-Swedish data identified five new loci, which crossed the level for genome-wide significance: NCF2 (neutrophil cytosolic factor 2), IKZF1 (Ikaros family zinc-finger 1), IRF8 (interferon regulatory factor 8), IFIH1 (interferon-induced helicase C domain-containing protein 1), and TYK2 (tyrosine kinase 2). Each of these five genes regulates a different aspect of the immune response and contributes to the production of type-I and type-II interferons. Although further studies will be required to identify the causal alleles within these loci, the confirmation of five new susceptibility genes for lupus makes a significant step forward in our understanding of the genetic contribution to SLE.
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multiple genetic risk factors, high levels of interferon alpha (IFN-α), and the production of autoantibodies against components of the cell nucleus. Interferon regulatory factor 5 (IRF5) is a transcription factor which induces the transcription of IFN-α and other cytokines, and genetic variants of IRF5 have been strongly linked to SLE pathogenesis. IRF5 functions downstream of Toll-like receptors and other microbial pattern-recognition receptors, and immune complexes made up of SLE-associated autoantibodies seem to function as a chronic endogenous stimulus to this pathway. In this paper, we discuss the physiologic role of IRF5 in immune defense and the ways in which IRF5 variants may contribute to the pathogenesis of human SLE. Recent data regarding the role of IRF5 in both serologic autoimmunity and the overproduction of IFN-α in human SLE are summarized. These data support a model in which SLE-risk variants of IRF5 participate in a “feed-forward” mechanism, predisposing to SLE-associated autoantibody formation, and subsequently facilitating IFN-α production downstream of Toll-like receptors stimulated by immune complexes composed of these autoantibodies.
A haplotype of the interferon regulatory factor 5 (IRF5) gene has been associated with the risk of developing systemic lupus erythematosus (SLE), and our previous studies have demonstrated that high levels of serum interferon-α (IFNα) activity are a heritable risk factor for SLE. The aim of this study was to determine whether the IRF5 SLE risk haplotype mediates the risk of SLE by predisposing patients to the development of high levels of serum IFNα activity.
IFNα levels in 199 SLE patients of European and Hispanic ancestry were measured with a sensitive functional reporter cell assay. The rs2004640, rs3807306, rs10488631, and rs2280714 single-nucleotide polymorphisms (SNPs) in IRF5 were genotyped in these patients. Haplotypes were categorized as SLE risk, neutral, or protective based on published data.
SLE patients with risk/risk and risk/neutral IRF5 genotypes had higher serum IFNα activity than did those with protective/protective and neutral/protective genotypes (P = 0.025). This differential effect of IRF5 genotype on serum IFNα levels was driven largely by SLE patients who were positive for either anti–RNA binding protein (anti-RBP) or anti–double-stranded DNA (anti-dsDNA) autoantibodies (P = 0.012 for risk/risk or risk/neutral versus protective/protective or neutral/protective). The rs3807306 genotype was independently associated with high serum IFNα in this autoantibody group. We found no difference in IFNα activity according to IRF5 genotype in patients lacking either type of autoantibody or in patients positive for both classes of autoantibody.
The IRF5 SLE risk haplotype is associated with higher serum IFNα activity in SLE patients, and this effect is most prominent in patients positive for either anti-RBP or anti-dsDNA autoantibodies. This study demonstrates the biologic relevance of the SLE risk haplotype of IRF5 at the protein level.
Interferon response factor 5 (IRF5) regulates innate immune responses to viral infection. IRF5 genetic variants have been shown to be strongly associated with risk for systemic lupus erythematosus (SLE). Functional roles of IRF5 exon 6 structural variants that occur as part of a SLE risk-associated haplotype, including a 30-bp in/del (in/del-10) and a 48-bp splice-site variant (SV-16), have not been established. In this study, we used IRF5 deficient cells overexpressing human IRF5 variants to investigate the roles of exon 6 in/del-10 and SV-16 in regulation of the apoptosis response, nuclear translocation, and ability to transactivate IRF5 responsive cytokines. We found that expression of IRF5 isoforms including either SV-16 or in/del-10 confers ability of IRF5 to impair the apoptotic response and correlates with reduced capacity for IRF5 nuclear translocation in MEFs after a DNA-damaging stimulus treatment. Interestingly, the presence or absence of both SV-16 and in/del-10 results in abrogation of both the anti-apoptotic and enhanced nuclear translocation effects of IRF5 expression. Only cells expressing IRF5 bearing SV-16 show increased IL-6 production upon LPS stimulation. MEFs expressing hIRF5 variants containing in/del-10 showed no significant difference from the control; however, cells carrying hIRF5 lacking both SV-16 and in/del-10 showed reduced IL-6 production. Our overall findings suggest that exon 6 SV-16 is more potent than in/del-10 for IRF5-driven resistance to apoptosis and promotion of cytokine production; however, in/del-10 co-expression can neutralize these effects of SV-16.
SLE; IRF5 variants; exon 6; apoptosis; nuclear translocation
Although the transcription factors IRF-3 and IRF-7 are considered master regulators of type I interferon (IFN) induction and IFN stimulated gene (ISG) expression, Irf3−/−×Irf7−/− double knockout (DKO) myeloid dendritic cells (mDC) produce relatively normal levels of IFN-β after viral infection. We generated Irf3−/−×Irf5−/−×Irf7−/− triple knockout (TKO) mice to test whether IRF-5 was the source of the residual induction of IFN-β and ISGs in mDCs. In pathogenesis studies with two unrelated positive-sense RNA viruses (West Nile virus (WNV) and murine norovirus), TKO mice succumbed at rates greater than DKO mice and equal to or approaching those of mice lacking the type I IFN receptor (Ifnar−/−). In ex vivo studies, after WNV infection or exposure to Toll-like receptor agonists, TKO mDCs failed to produce IFN-β or express ISGs. In contrast, this response was sustained in TKO macrophages following WNV infection. To define IRF-regulated gene signatures, we performed microarray analysis on WNV-infected mDC from wild type (WT), DKO, TKO, or Ifnar−/− mice, as well as from mice lacking the RIG-I like receptor adaptor protein MAVS. Whereas the gene induction pattern in DKO mDC was similar to WT cells, remarkably, almost no ISG induction was detected in TKO or Mavs−/− mDC. The relative equivalence of TKO and Mavs−/− responses suggested that MAVS dominantly regulates ISG induction in mDC. Moreover, we showed that MAVS-dependent induction of ISGs can occur through an IRF-5-dependent yet IRF-3 and IRF-7-independent pathway. Our results establish IRF-3, -5, and -7 as the key transcription factors responsible for mediating the type I IFN and ISG response in mDC during WNV infection and suggest a novel signaling link between MAVS and IRF-5.
Host pathogen sensors, including those of the Toll-like receptor and RIG-I like receptor (RLR) families, detect viral infection in cells. Signaling through these receptors triggers expression of type I interferon (IFN) and IFN-stimulated genes (ISGs), in part through the IRF family of transcription factors. Previous studies with West Nile virus (WNV) showed that IRF-3 and IRF-7 control IFN expression in fibroblasts and neurons, whereas macrophages and myeloid dendritic cells (mDC) retained the ability to induce IFN-β without IRF-3 and IRF-7. In the current study, we generated Irf3−/−×Irf5−/−×Irf7−/− (TKO) mice to characterize the contributions of specific IRF transcription factors to IFN and ISG induction in response to WNV infection in cells and in mice. We found that induction of IFN and ISGs was largely abolished in TKO mDC, but sustained in TKO macrophages. Because IFN and ISG induction also was absent in mDC lacking MAVS, a key mediator of RLR signaling, our results suggest a novel signaling link between IRF-5 and MAVS. This study establishes the molecular pathways responsible for IFN induction in mDC and suggests a cross-talk between IRF-5 and RLR signaling pathways.
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 that IRF5 is required for pristane-induced SLE 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 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.
interferon regulatory factor 5 (IRF5, IRF-5); systemic lupus erythematosus (SLE); autoantibody; type I interferon; Th2
Systemic lupus erythematosus (SLE) is the prototype autoimmune disease where genes regulated by type I interferon (IFN) are over-expressed and contribute to the disease pathogenesis. Because signal transducer and activator of transcription 4 (STAT4) plays a key role in the type I IFN receptor signaling, we performed a candidate gene study of a comprehensive set of single nucleotide polymorphism (SNPs) in STAT4 in Swedish patients with SLE. We found that 10 out of 53 analyzed SNPs in STAT4 were associated with SLE, with the strongest signal of association (P = 7.1 × 10−8) for two perfectly linked SNPs rs10181656 and rs7582694. The risk alleles of these 10 SNPs form a common risk haplotype for SLE (P = 1.7 × 10−5). According to conditional logistic regression analysis the SNP rs10181656 or rs7582694 accounts for all of the observed association signal. By quantitative analysis of the allelic expression of STAT4 we found that the risk allele of STAT4 was over-expressed in primary human cells of mesenchymal origin, but not in B-cells, and that the risk allele of STAT4 was over-expressed (P = 8.4 × 10−5) in cells carrying the risk haplotype for SLE compared with cells with a non-risk haplotype. The risk allele of the SNP rs7582694 in STAT4 correlated to production of anti-dsDNA (double-stranded DNA) antibodies and displayed a multiplicatively increased, 1.82-fold risk of SLE with two independent risk alleles of the IRF5 (interferon regulatory factor 5) gene.
IRF5 is a transcription factor involved both in the type I interferon and the toll-like receptor signalling pathways. Previously, IRF5 has been found to be associated with systemic lupus erythematosus, rheumatoid arthritis and inflammatory bowel diseases. Here we investigated whether polymorphisms in the IRF5 gene would be associated with yet another disease with features of autoimmunity, multiple sclerosis (MS).
We genotyped nine single nucleotide polymorphisms and one insertion-deletion polymorphism in the IRF5 gene in a collection of 2337 patients with MS and 2813 controls from three populations: two case–control cohorts from Spain and Sweden, and a set of MS trio families from Finland.
Two single nucleotide polymorphism (SNPs) (rs4728142, rs3807306), and a 5 bp insertion-deletion polymorphism located in the promoter and first intron of the IRF5 gene, showed association signals with values of p<0.001 when the data from all cohorts were combined. The predisposing alleles were present on the same common haplotype in all populations. Using electrophoretic mobility shift assays we observed allele specific differences in protein binding for the SNP rs4728142 and the 5 bp indel, and by a proximity ligation assay we demonstrated increased binding of the transcription factor SP1 to the risk allele of the 5 bp indel.
These findings add IRF5 to the short list of genes shown to be associated with MS in more than one population. Our study adds to the evidence that there might be genes or pathways that are common in multiple autoimmune diseases, and that the type I interferon system is likely to be involved in the development of these diseases.
Genetic variation in interferon regulatory factor 5 (IRF5) has been associated with risk of developing systemic lupus erythematosus (SLE), and this association is largely dependent upon anti-Ro autoantibodies. We studied a unique cohort of anti-Ro positive individuals with diverse diagnoses to determine if IRF5 genotype associated with maternal diagnosis or progression of autoimmunity.
We genotyped haplotype-tagging polymorphisms in IRF5 in 93 European ancestry subjects recruited to the Research Registry for Neonatal Lupus who all had high titer anti-Ro autoantibodies and a child with neonatal lupus (NL), and allele frequencies were compared to non-autoimmune controls. The mothers diagnoses included SLE, Sjogren’s syndrome (SS), undifferentiated autoimmune syndrome (UAS), and asymptomatic.
The SLE-risk haplotype of IRF5 was enriched in all anti-Ro positive subjects except those with SS (OR = 2.55, p=8.8×10−4). Even asymptomatic individuals with anti-Ro antibodies were enriched for the SLE-risk haplotype (OR=2.69, p=0.019). The same haplotype was more prevalent in subjects who were initially asymptomatic, but developed symptomatic SLE during follow up (OR=5.83, p=0.0024). Interestingly, SS was associated with two minor IRF5 haplotypes, and these same haplotypes were decreased in frequency in those with SLE and UAS.
The IRF5 SLE-risk haplotype was associated with anti-Ro antibodies in asymptomatic individuals as well as progression to SLE in asymptomatic anti-Ro positive individuals. SS in NL mothers was associated with different IRF5 haplotypes. These data suggest that IRF5 polymorphisms play a role in serologic autoimmunity in humans and may promote the progression to clinical autoimmunity.
systemic lupus erythematosus; interferon; autoantibodies; neonatal lupus; Sjogren’s syndrome
Interferon regulatory factor 5 (IRF5) polymorphisms are strongly associated with an increased risk of developing the autoimmune disease systemic lupus erythematosus. In mouse lupus models, IRF5-deficiency was shown to reduce disease severity consistent with an important role for IRF5 in disease pathogenesis. However these mouse studies were confounded by the recent demonstration that the IRF5 knockout mouse line contained a loss-of-function mutation in the dedicator of cytokinesis 2 (DOCK2) gene. As DOCK2 regulates lymphocyte trafficking and Toll-like receptor signaling, this raised the possibility that some of the protective effects attributed to IRF5 deficiency in the mouse lupus models may instead have been due to DOCK2 deficiency. We have therefore here evaluated the effect of IRF5-deficiency in the MRL/lpr mouse lupus model in the absence of the DOCK2 mutation. We find that IRF5-deficient (IRF5−/−) MRL/lpr mice develop much less severe disease than their IRF5-sufficient (IRF5+/+) littermates. Despite markedly lower serum levels of anti-nuclear autoantibodies and reduced total splenocyte and CD4+ T cell numbers, IRF5−/− MRL/lpr mice have similar numbers of all splenic B cell subsets compared to IRF5+/+ MRL/lpr mice, suggesting that IRF5 is not involved in B cell development up to the mature B cell stage. However, IRF5−/− MRL/lpr mice have greatly reduced numbers of spleen plasmablasts and bone marrow plasma cells. Serum levels of B lymphocyte stimulator (BLyS) were markedly elevated in the MRL/lpr mice but no effect of IRF5 on serum BLyS levels was seen. Overall our data demonstrate that IRF5 contributes to disease pathogenesis in the MRL/lpr lupus model and that this is due, at least in part, to the role of IRF5 in plasma cell formation. Our data also suggest that combined therapy targeting both IRF5 and BLyS might be a particularly effective therapeutic approach in lupus.
Polymorphisms 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.
Polymorphisms 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.
A 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.
We 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.
The transcription factor interferon regulatory factor 5 (IRF5) has been identified as a human systemic lupus erythematosus (SLE) susceptibility gene by numerous joint linkage and genome-wide association studies. Although IRF5 expression is significantly elevated in primary blood cells of SLE patients, it is not yet known how IRF5 contributes to SLE pathogenesis. Recent data from mouse models of lupus indicate a critical role for IRF5 in the production of pathogenic autoantibodies and the expression of Th2 cytokines and type I IFN. In the current study, we examined the mechanism(s) by which loss of Irf5 protects mice from pristane-induced lupus at early time points of disease development. We demonstrate that Irf5 is required for Ly6C(hi) monocyte trafficking to the peritoneal cavity (PC), which is believed to be one of the initial key events leading to lupus pathogenesis in this model. Chemotaxis assays using peritoneal lavage from pristane-injected Irf5+/+ and Irf5−/− littermates support an intrinsic defect in Irf5−/− monocytes. We found the expression of chemokine receptors CXCR4 and CCR2 to be dysregulated on Irf5−/− monocytes and less responsive to their respective ligands, CXCL12 and CCL2. Bone marrow reconstitution experiments further supported an intrinsic defect in Irf5−/− monocytes since Irf5+/+ monocytes were preferentially recruited to the PC in response to pristane. Together, these findings demonstrate an intrinsic role for IRF5 in the response of monocytes to pristane, and their recruitment to the primary site of inflammation that is thought to trigger lupus onset in this experimental model of SLE.
To confirm and define the genetic association of STAT4 and systemic lupus erythematosus, investigate the possibility of correlations with differential splicing and/or expression levels, and genetic interaction with IRF5.
30 tag SNPs were genotyped in an independent set of Spanish cases and controls. SNPs surviving correction for multiple tests were genotyped in 5 new sets of cases and controls for replication. STAT4 cDNA was analyzed by 5’-RACE PCR and sequencing. Expression levels were measured by quantitative PCR.
In the fine-mapping, four SNPs were significant after correction for multiple testing, with rs3821236 and rs3024866 as the strongest signals, followed by the previously associated rs7574865, and by rs1467199. Association was replicated in all cohorts. After conditional regression analyses, two major independent signals represented by SNPs rs3821236 and rs7574865, remained significant across the sets. These SNPs belong to separate haplotype blocks. High levels of STAT4 expression correlated with SNPs rs3821236, rs3024866 (both in the same haplotype block) and rs7574865 but not with other SNPs. We also detected transcription of alternative tissue-specific exons 1, indicating presence of tissue-specific promoters of potential importance in the expression of STAT4. No interaction with associated SNPs of IRF5 was observed using regression analysis.
These data confirm STAT4 as a susceptibility gene for SLE and suggest the presence of at least two functional variants affecting levels of STAT4. Our results also indicate that both genes STAT4 and IRF5 act additively to increase risk for SLE.
Association studies; systemic lupus erythematosus; STAT4 transcription factor; Interferon regulatory factor; genetic predisposition to disease
Interferon Regulatory Factor 8 (IRF8) is required for development, maturation and expression of anti-microbial defenses of myeloid cells. BXH2 mice harbor a severely hypomorphic allele at Irf8 (Irf8R294C) that causes susceptibility to infection with intracellular pathogens including Mycobacterium tuberculosis. We report that BXH2 are completely resistant to the development of cerebral malaria (ECM) following Plasmodium berghei ANKA infection. Comparative transcriptional profiling of brain RNA as well as chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq) was used to identify IRF8-regulated genes whose expression is associated with pathological acute neuroinflammation. Genes increased by infection were strongly enriched for IRF8 binding sites, suggesting that IRF8 acts as a transcriptional activator in inflammatory programs. These lists were enriched for myeloid-specific pathways, including interferon responses, antigen presentation and Th1 polarizing cytokines. We show that inactivation of several of these downstream target genes (including the Irf8 transcription partner Irf1) confers protection against ECM. ECM-resistance in Irf8 and Irf1 mutants is associated with impaired myeloid and lymphoid cells function, including production of IL12p40 and IFNγ. We note strong overlap between genes bound and regulated by IRF8 during ECM and genes regulated in the lungs of M. tuberculosis infected mice. This IRF8-dependent network contains several genes recently identified as risk factors in acute and chronic human inflammatory conditions. We report a common core of IRF8-bound genes forming a critical inflammatory host-response network.
Cerebral malaria is a severe and often lethal complication from infection with Plasmodium falciparum which is driven in part by pathological host inflammatory response to parasitized red cells′ adherence in the brain microvasculature. However, the pathways that initiate and amplify this pathological neuroinflammation are not well understood. As susceptibility to cerebral malaria is variable and has been shown to be partially heritable, we have studied this from a genetic perspective using a mouse model of infection with P. berghei which induces experimental cerebral malaria (ECM). Here we show that mice bearing mutations in the myeloid transcription factor IRF8 and its heterodimerization partner IRF1 are completely resistant to ECM. We have identified the genes and associated networks that are activated by IRF8 during ECM. Loss-of-function mutations of several IRF8 targets are also shown to be protective. Parallel analysis of lungs infected with Mycobacterium tuberculosis show that IRF8-associated core pathways are also engaged during tuberculosis where they play a protective role. This contrast illustrates the balancing act required by the immune system to respond to pathogens and highlights a lynchpin role for IRF8 in both. Finally, several genes in these networks have been individually associated with chronic or acute inflammatory conditions in humans.
Interferon (IFN) regulatory factor 5 (IRF-5) is a transcription factor involved in the regulation of host defense. Previous reports have demonstrated a significant association of various IRF-5 polymorphisms with systemic lupus erythematosus (SLE), among Caucasians. This case-control study aimed to investigate whether IRF-5 polymorphisms were involved in the genetic predisposition to primary Sjögren Syndrome (pSS), an autoimmune disease closely related to SLE.
We analyzed IRF-5 rs2004640, rs2070197, rs10954213, and rs2280714 polymorphisms in a cohort of 212 pSS patients and 162 controls, all of Caucasian origin. The four studied polymorphisms were genotyped by competitive allele specific polymerase chain reaction (PCR) using FRET technology.
The IRF-5 rs2004640 GT or TT genotypes (T allele carriers) were found among 87% of pSS patients compared with 77% in controls (P=0.01; OR1.93, 95%IC [1.15–3.42]). Likewise, IRF-5 rs2004640 T allele was found on 59% of chromosomes in pSS patients compared with 52% in controls (P=0.04; OR 1.36, 95% CI [1.01–1.83]). No significant association was evidenced with rs2070197, rs10954213, and rs2280714 when analyzed independently. Nevertheless, haplotype reconstructions based on the four studied polymorphisms suggest that various allele combinations of rs2004640 and rs2070197 could define susceptibility or protective haplotypes.
We demonstrated for the first time a significant association of IRF-5 rs2004640 T allele with pSS. These results, which require further replication on larger sample sized populations suggest that, beside association with identical major histocompatibility complex (MHC) gene polymorphisms, pSS and SLE also share IRF-5 polymorphism as a common genetic susceptibility factor.
Alleles; Case-Control Studies; Genetic Predisposition to Disease; genetics; Genotype; Haplotypes; Humans; Interferon Regulatory Factors; genetics; Lupus Erythematosus, Systemic; genetics; Polymorphism, Single Nucleotide; genetics; Sjogren's Syndrome; genetics; IRF-5; Sjögren's syndrome; genetic polymorphism; haplotype
Genetic variants of interferon regulatory factor 5 (IRF5) are associated with susceptibility to systemic lupus erythematosus (SLE). IRF5 regulates the expression of proinflammatory cytokines and type I interferons (IFN) believed to be involved in SLE pathogenesis. The aim of this study was to determine the activation status of IRF5 by assessing its nuclear localization in immune cells of SLE patients and healthy donors, and to identify SLE triggers of IRF5 activation.
IRF5 nuclear localization in subpopulations of peripheral blood mononuclear cells (PBMC) from 14 genotyped SLE patients and 11 healthy controls was assessed using imaging flow cytometry. IRF5 activation and function were examined after ex vivo stimulation of healthy donor monocytes with SLE serum or components of SLE serum. Cellular localization was determined by ImageStream and cytokine expression by Q-PCR and ELISA.
IRF5 was activated in a cell type-specific manner; monocytes of SLE patients had constitutively elevated levels of nuclear IRF5 compared to NK and T cells. SLE serum was identified as a trigger for IRF5 nuclear accumulation; however, neither IFNα nor SLE immune complexes could induce nuclear localization. Instead, autoantigens comprised of apoptotic/necrotic material triggered IRF5 nuclear accumulation in monocytes. Production of cytokines IFNα, TNFα and IL6 in monocytes stimulated with SLE serum or autoantigens was distinct yet correlated with the kinetics of IRF5 nuclear localization.
This study provides the first formal proof that IRF5 activation is altered in monocytes of SLE patients that is in part contributed by the SLE blood environment.
Systemic lupus erythematosus (SLE) is a genetically complex disease with heterogeneous clinical manifestations. A polymorphism in the STAT4 gene has recently been established as a risk factor for SLE, but the relationship with specific SLE subphenotypes has not been studied. We studied 137 SNPs in the STAT4 region genotyped in 4 independent SLE case series (total n = 1398) and 2560 healthy controls, along with clinical data for the cases. Using conditional testing, we confirmed the most significant STAT4 haplotype for SLE risk. We then studied a SNP marking this haplotype for association with specific SLE subphenotypes, including autoantibody production, nephritis, arthritis, mucocutaneous manifestations, and age at diagnosis. To prevent possible type-I errors from population stratification, we reanalyzed the data using a subset of subjects determined to be most homogeneous based on principal components analysis of genome-wide data. We confirmed that four SNPs in very high LD (r2 = 0.94 to 0.99) were most strongly associated with SLE, and there was no compelling evidence for additional SLE risk loci in the STAT4 region. SNP rs7574865 marking this haplotype had a minor allele frequency (MAF) = 31.1% in SLE cases compared with 22.5% in controls (OR = 1.56, p = 10−16). This SNP was more strongly associated with SLE characterized by double-stranded DNA autoantibodies (MAF = 35.1%, OR = 1.86, p<10−19), nephritis (MAF = 34.3%, OR = 1.80, p<10−11), and age at diagnosis<30 years (MAF = 33.8%, OR = 1.77, p<10−13). An association with severe nephritis was even more striking (MAF = 39.2%, OR = 2.35, p<10−4 in the homogeneous subset of subjects). In contrast, STAT4 was less strongly associated with oral ulcers, a manifestation associated with milder disease. We conclude that this common polymorphism of STAT4 contributes to the phenotypic heterogeneity of SLE, predisposing specifically to more severe disease.
Systemic lupus erythematosus is a chronic disabling autoimmune disease, most commonly striking women in their thirties or forties. It can cause a wide variety of clinical manifestations, including kidney disease, arthritis, and skin disorders. Prognosis varies greatly depending on these clinical features, with kidney disease and related characteristics leading to greater morbidity and mortality. It is also complex genetically; while lupus runs in families, genes increase one’s risk for lupus but do not fully determine the outcome. It is thought that the interactions of multiple genes and/or interactions between genes and environmental factors may cause lupus, but the causes and disease pathways of this very heterogeneous disease are not well understood. By examining relationships between subtypes of lupus and specific genes, we hope to better understand how lupus is triggered and by what biological pathways it progresses. We show in this work that the STAT4 gene, very recently identified as a lupus risk gene, predisposes specifically to severe manifestations of lupus, including kidney disease.