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.
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 (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
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 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
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
Systemic sclerosis (SSc) is a fibrotic autoimmune disease in which the genetic component plays an important role. One of the strongest SSc association signals outside the human leukocyte antigen (HLA) region corresponds to interferon (IFN) regulatory factor 5 (IRF5), a major regulator of the type I IFN pathway. In this study we aimed to evaluate whether three different haplotypic blocks within this locus, which have been shown to alter the protein function influencing systemic lupus erythematosus (SLE) susceptibility, are involved in SSc susceptibility and clinical phenotypes. For that purpose, we genotyped one representative single-nucleotide polymorphism (SNP) of each block (rs10488631, rs2004640, and rs4728142) in a total of 3,361 SSc patients and 4,012 unaffected controls of Caucasian origin from Spain, Germany, The Netherlands, Italy and United Kingdom. A meta-analysis of the allele frequencies was performed to analyse the overall effect of these IRF5 genetic variants on SSc. Allelic combination and dependency tests were also carried out. The three SNPs showed strong associations with the global disease (rs4728142: P = 1.34×10−8, OR = 1.22, CI 95% = 1.14–1.30; rs2004640: P = 4.60×10−7, OR = 0.84, CI 95% = 0.78–0.90; rs10488631: P = 7.53×10−20, OR = 1.63, CI 95% = 1.47–1.81). However, the association of rs2004640 with SSc was not independent of rs4728142 (conditioned P = 0.598). The haplotype containing the risk alleles (rs4728142*A-rs2004640*T-rs10488631*C: P = 9.04×10−22, OR = 1.75, CI 95% = 1.56–1.97) better explained the observed association (likelihood P-value = 1.48×10−4), suggesting an additive effect of the three haplotypic blocks. No statistical significance was observed in the comparisons amongst SSc patients with and without the main clinical characteristics. Our data clearly indicate that the SLE risk haplotype also influences SSc predisposition, and that this association is not sub-phenotype-specific.
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.
Recently, a new genetic factor within the interferon regulatory factor 5 (IRF5) gene was demonstrated for systemic lupus erythematosus (SLE) through linkage and association: the rs2004640‐T allele. IRF5 is involved in the production of rheumatoid arthritis (RA) cytokines, and SLE already shares with RA one genetic factor within the tyrosine phosphatase PTPN22 gene.
To test the hypothesis that the SLE IRF5 genetic factor could also be shared with RA.
Patients and methods
100 French Caucasian trio families with RA were genotyped and analysed with the transmission disequilibrium test, the frequency comparison of the transmitted and untransmitted alleles, and the genotype relative risk. 97% power was available to detect at least a trend in favour of a factor similar to that reported for SLE.
The analysis showed the absence of linkage and association globally and in “autoimmune” RA subsets, with a weak non‐significant trend against the IRF5rs20046470‐T allele. Given the robustness of familial‐based analysis, this slight negative trend provided strong evidence against even a weaker factor than that reported for SLE.
Our results exclude the IRF5rs2004640‐T allele as a major genetic factor for RA in this French Caucasian population.
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.
Both genetic and environmental interactions affect systemic lupus erythematosus (SLE) development and pathogenesis. One known genetic factor associated with lupus is a haplotype of the interferon regulatory factor 5 (IRF5) gene. Analysis of global gene expression microarray data using gene set enrichment analysis identified multiple interferon- and inflammation-related gene sets significantly overrepresented in cells with the risk haplotype. Pathway analysis using expressed genes from the significant gene sets impacted by the IRF5 risk haplotype confirmed significant correlation with the interferon pathway, Toll-like receptor pathway, and the B-cell receptor pathway. SLE patients with the IRF5 risk haplotype have a heightened interferon signature, even in an unstimulated state (P = 0.011), while patients with the IRF5 protective haplotype have a B cell interferon signature similar to that of controls. These results identify multiple genes in functionally significant pathways which are affected by IRF5 genotype. They also establish the IRF5 risk haplotype as a key determinant of not only the interferon response, but also other B-cell pathways involved in 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 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
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.
Recently, two studies provided convincing evidence that IFN regulatory factor 5 (IRF5) gene polymorphisms are significantly associated with systemic lupus erythematosus (SLE) in several white populations. To replicate the association with SLE in an Asian population, we examined the genetic effects in our SLE cohort from a Korean population. A total of 1,565 subjects, composed of 593 cases and 972 controls, were genotyped using the TaqMan® (Applied Biosystems, Foster City, CA, USA) method. The genetic effects of polymorphisms on the risk of SLE were evaluated using χ2 tests and a Mantel–Haenszel meta-analysis. Statistical analysis revealed results in the Korean population were similar to the previous reports from white populations. The rs2004640 T allele had a higher frequency in SLE cases (0.385) than controls (0.321; odds ratio (OR) = 1.32, P = 0.0003). In combined analysis, including all seven independent cohorts from the three studies so far, robust and consistent associations of the rs2004640 T allele with SLE were observed. The estimate of risk was OR = 1.44 (range, 1.34–1.55), with an overall P = 1.85 × 10-23 for the rs2004640 T allele. The haplotype (rs2004640T–rs2280714T) involved in both the alternative splice donor site and the elevated expression of IRF5 also had a highly significant association with SLE (pooled, P = 2.11 × 10-16). Our results indicate that the genetic effect on the risk of SLE mediated by IRF5 variants can be generally accepted in both white and Asian populations.
The interferon regulatory factor 5 gene (IRF5) has been shown to play a crucial role in harmful immune responses by induction of proinflammatory cytokines. Functional genetic variants associated with increasd IRF5 expression of specific isoforms are associated with systemic lupus erythematosus (SLE) and it is possible that they may also predispose to other autoimmune disorders. We tested the association of two IRF5 SNPs, correlated with IRF5 expression and SLE risk, in 947 nuclear family trios type 1 diabetes (T1D) using the transmission disequilibrium test. Our results suggest that the functional IRF5 variations do not confer an obvious risk for T1D.
Systemic sclerosis (SSc) and systemic lupus erythematosus (SLE) are related chronic autoimmune diseases of complex aetiology in which the interferon (IFN) pathway plays a key role. Recent studies have reported an association between IRF7 and SLE which confers a risk to autoantibody production. A study was undertaken to investigate whether the IRF7 genomic region is also involved in susceptibility to SSc and the main clinical features.
Two case-control sets of Caucasian origin from the USA and Spain, comprising a total of 2316 cases of SSc and 2347 healthy controls, were included in the study. Five single nucleotide polymorphisms (SNPs) in the PHRF1-IRF7-CDHR5 locus were genotyped using TaqMan allelic discrimination technology. A meta-analysis was performed to test the overall effect of these genetic variants on SSc.
Four out of five analysed SNPs were Significantly associated with the presence of anticentromere autoantibodies (ACA) in the patients with SSc in the combined analysis (rs1131665: pFDR=6.14 × 10−4, OR=0.78; rs4963128: pFDR=6.14 × 10−4, OR=0.79; rs702966: pFDR=3.83 × 10−3, OR=0.82; and rs2246614: pFDR=3.83 × 10−3, OR=0.83). Significant p values were also obtained when the disease was tested globally; however, the statistical significance was lost when the ACA-positive patients were excluded from the study, suggesting that these associations rely on ACA positivity. Conditional logistic regression and allelic combination analyses suggested that the functional IRF7 SNP rs1131665 is the most likely causal variant.
The results show that variation in the IRF7 genomic region is associated with the presence of ACA in patients with SSc, supporting other evidence that this locus represents a common risk factor for autoantibody production in autoimmune diseases.
IgA nephropathy (IgAN) and nephritis in Systemic Lupus Erythematosus (SLE) are two common forms of glomerulonephritis in which genetic findings are of importance for disease development. We have recently reported an association of IgAN with variants of TGFB1. In several autoimmune diseases, particularly in SLE, IRF5, STAT4 genes and TRAF1-C5 locus have been shown to be important candidate genes. The aim of this study was to compare genetic variants from the TGFB1, IRF5, STAT4 genes and TRAF1-C5 locus with susceptibility to IgAN and lupus nephritis in two Swedish cohorts.
Patients and Methods
We genotyped 13 single nucleotide polymorphisms (SNPs) in four genetic loci in 1252 DNA samples from patients with biopsy proven IgAN or with SLE (with and without nephritis) and healthy age- and sex-matched controls from the same population in Sweden.
Genotype and allelic frequencies for SNPs from selected genes did not differ significantly between lupus nephritis patients and SLE patients without nephritis. In addition, haplotype analysis for seven selected SNPs did not reveal a difference for the SLE patient groups with and without nephritis. Moreover, none of these SPNs showed a significant difference between IgAN patients and healthy controls. IRF5 and STAT4 variants remained significantly different between SLE cases and healthy controls. In addition, the data did not show an association of TRAF1-C5 polymorphism with susceptibility to SLE in this Swedish population.
Our data do not support an overlap in genetic susceptibility between patients with IgAN or SLE and reveal no specific importance of SLE associated SNPs for the presence of lupus nephritis.
Polymorphisms in the transcription factor interferon regulatory factor 5 (IRF5) are strongly associated in human genetic studies with an increased risk of developing the autoimmune disease systemic lupus erythematosus. However, the biological role of IRF5 in lupus pathogenesis has not previously been tested in an animal model. In this study we show that IRF5 is absolutely required for disease development in the FcγRIIB−/−Yaa and FcγRIIB−/− lupus models. In contrast to IRF5-sufficient FcγRIIB−/−Yaa mice, IRF5-deficient FcγRIIB−/−Yaa mice do not develop lupus manifestations and have a phenotype comparable to wildtype mice. Strikingly, full expression of IRF5 is required for the development of autoimmunity as IRF5-heterozygotes had dramatically reduced disease. One effect of IRF5 is to induce the production of the type I interferon IFN-α, a cytokine implicated in lupus pathogenesis. To address the mechanism by which IRF5 promotes disease, we evaluated FcγRIIB−/−Yaa mice lacking the type I interferon receptor IFNAR1. Unlike the IRF5-deficient and IRF5-heterozygous FcγRIIB−/−Yaa mice, IFNAR1-deficient FcγRIIB−/−Yaa mice maintained a substantial level of residual disease. Furthermore, in FcγRIIB−/− mice lacking Yaa, IRF5-deficiency also markedly reduced disease manifestations indicating that the beneficial effects of IRF5 deficiency in FcγRIIB−/−Yaa mice are not due only to inhibition of the enhanced TLR7 signaling associated with the Yaa mutation. Overall, we demonstrate that IRF5 plays an essential role in lupus pathogenesis in murine models and that this is mediated through pathways beyond that of type I interferon production.
Systemic Lupus Erythematosus (SLE) is a systemic autoimmune disease in which the type I interferon pathway has a crucial role. We have previously shown that three genes in this pathway, IRF5, TYK2 and STAT4, are strongly associated with risk for SLE. Here, we investigated 78 genes involved in the type I interferon pathway to identify additional SLE susceptibility loci. First, we genotyped 896 single-nucleotide polymorphisms in these 78 genes and 14 other candidate genes in 482 Swedish SLE patients and 536 controls. Genes with P<0.01 in the initial screen were then followed up in 344 additional Swedish patients and 1299 controls. SNPs in the IKBKE, TANK, STAT1, IL8 and TRAF6 genes gave nominal signals of association with SLE in this extended Swedish cohort. To replicate these findings we extracted data from a genomewide association study on SLE performed in a US cohort. Combined analysis of the Swedish and US data, comprising a total of 2136 cases and 9694 controls, implicates IKBKE and IL8 as SLE susceptibility loci (Pmeta=0.00010 and Pmeta=0.00040, respectively). STAT1 was also associated with SLE in this cohort (Pmeta=3.3 × 10−5), but this association signal appears to be dependent of that previously reported for the neighbouring STAT4 gene. Our study suggests additional genes from the type I interferon system in SLE, and highlights genes in this pathway for further functional analysis.
systemic lupus erythematosus; type I interferon system; candidate gene study; single nucleotide polymorphism; IKBKE; IL8
Interferon regulatory factor (IRF)-5 is a transcription factor involved in type I interferon signaling whose germ line variants have been associated with autoimmune pathogenesis. Since relationships have been observed between development of autoimmunity and responsiveness of melanoma to several types of immunotherapy, we tested whether polymorphisms of IRF5 are associated with responsiveness of melanoma to adoptive therapy with tumor infiltrating lymphocytes (TILs).
140 TILs were genotyped for four single nucleotide polymorphisms (rs10954213, rs11770589, rs6953165, rs2004640) and one insertion-deletion in the IRF5 gene by sequencing. Gene-expression profile of the TILs, 112 parental melanoma metastases (MM) and 9 cell lines derived from some metastases were assessed by Affymetrix Human Gene ST 1.0 array.
Lack of A allele in rs10954213 (G > A) was associated with non-response (p < 0.005). Other polymorphisms in strong linkage disequilibrium with rs10954213 demonstrated similar trends. Genes differentially expressed in vitro between cell lines carrying or not the A allele could be applied to the transcriptional profile of 112 melanoma metastases to predict their responsiveness to therapy, suggesting that IRF5 genotype may influence immune responsiveness by affecting the intrinsic biology of melanoma.
This study is the first to analyze associations between melanoma immune responsiveness and IRF5 polymorphism. The results support a common genetic basis which may underline the development of autoimmunity and melanoma immune responsiveness.
Increased IFN-α signaling is a primary pathogenic factor in systemic lupus erythematosus (SLE). STAT4 is a transcription factor that is activated by IFN-α signaling, and genetic variation of STAT4 has been associated with risk of SLE and rheumatoid arthritis. We measured serum IFN-α activity and simultaneous IFN-α-induced gene expression in PBMC in a large SLE cohort. The risk variant of STAT4 (T allele; rs7574865) was simultaneously associated with both lower serum IFN-α activity and greater IFN-α-induced gene expression in PBMC in SLE patients in vivo. Regression analyses confirmed that the risk allele of STAT4 was associated with increased sensitivity to IFN-α signaling. The IFN regulatory factor 5 SLE risk genotype was associated with higher serum IFN-α activity; however, STAT4 showed dominant influence on the sensitivity of PBMC to serum IFN-α. These data provide biologic relevance for the risk variant of STAT4 in the IFN-α pathway in vivo.
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.
To analyze if genetically determined Amerindian ancestry predicts the increased presence of risk alleles of known susceptibility genes for systemic lupus erythematosus.
Single nucleotide polymorphisms within 16 confirmed genetic susceptibility loci for SLE were genotyped in a set of 804 Mestizo lupus patients and 667 Mestizo normal healthy controls. In addition, 347 admixture informative markers were genotyped. Individual ancestry proportions were determined using STRUCTURE. Association analysis was performed using PLINK, and correlation of the presence of risk alleles with ancestry was done using linear regression.
A meta-analysis of the genetic association of the 16 SNPs across populations showed that TNFSF4, STAT4, PDCD1, ITGAM, and IRF5 were associated with lupus in a Hispanic-Mestizo cohort enriched for European and Amerindian ancestry. In addition, two SNPs within the MHC region, previously associated in a genome-wide association study in Europeans, were also associated in Mestizos. Using linear regression we predict an average increase of 2.34 risk alleles when comparing a lupus patient with 100% Amerindian ancestry to an SLE patient with 0% American Indian Ancestry (p<0.0001). SLE patients with 43% more Amerindian ancestry are predicted to carry one additional risk allele.
Amerindian ancestry increased the number of risk alleles for lupus.
Ro52 is a member of the TRIM family of single-protein E3 ligases and is also a target for autoantibody production in systemic lupus erythematosus and Sjögren's syndrome. We previously demonstrated a novel function of Ro52 in the ubiquitination and proteasomal degradation of IRF3 following TLR3/4 stimulation. We now present evidence that Ro52 has a similar role in regulating the stability and activity of IRF7. Endogenous immunoprecipitation of Ro52-bound proteins revealed that IRF7 associates with Ro52, an effect which increases following TLR7 and TLR9 stimulation, suggesting that Ro52 interacts with IRF7 post-pathogen recognition. Furthermore, we show that Ro52 ubiquitinates IRF7 in a dose-dependent manner, resulting in a decrease in total IRF7 expression and a subsequent decrease in IFN-α production. IRF7 stability was increased in bone marrow-derived macrophages from Ro52-deficient mice stimulated with imiquimod or CpG-B, consistent with a role for Ro52 in the negative regulation of IRF7 signalling. Taken together, these results suggest that Ro52-mediated ubiquitination promotes the degradation of IRF7 following TLR7 and TLR9 stimulation. As Ro52 is known to be IFN-inducible, this system constitutes a negative-feedback loop that acts to protect the host from the prolonged activation of the immune response.