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
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.
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
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
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-α (IFNα) levels are elevated in many patients with systemic lupus erythematosus (SLE) and may play a primary role in its pathogenesis. The purpose of this study was to determine whether serum IFNα activity in SLE patients and their healthy first-degree relatives is highest in early adulthood, when the incidence of SLE is greatest.
Serum samples from 315 SLE patients, 359 healthy first-degree relatives, and 141 healthy unrelated donors were measured for IFNα activity using a functional reporter cell assay. IFNα activity was analyzed in relation to age, and subgroups with high levels of IFNα activity were identified within the large data sets using a Mann-Whitney sliding window segmentation algorithm. The significance of each subgrouping was ranked by Kruskal-Wallis testing.
Age was inversely correlated with IFNα activity in female SLE patients (r = −0.20, P = 0.001) as well as their healthy female first-degree relatives (r = −0.16, P = 0.02). In male patients and their healthy male first-degree relatives, there was no significant overall correlation between age and serum IFNα activity. The segmentation algorithm revealed significantly increased IFNα activity between the ages of 12 and 22 years in female SLE patients and between the ages of 16 and 29 years in male SLE patients. Both male and female healthy first-degree relatives had significantly decreased IFNα activity after the age of 50 years.
Serum IFNα activity is higher in younger individuals in the SLE family cohorts, and this tendency is accentuated in affected individuals. This age-related pattern of IFNα activity may contribute to the increased incidence of SLE in early adulthood, and interestingly, males and females had similar age-related patterns of IFNα activity.
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.
Results from two studies have implicated the interferon regulatory gene IRF5 as a susceptibility gene in systemic lupus erythematosus (SLE). In this study, we conducted a family-based association analysis in 380 UK SLE nuclear families. Using a higher density of markers than has hitherto been screened, we show that there is association with two SNPs in the first intron, rs2004640 (P = 3.4 × 10−4) and rs3807306 (P = 4.9 × 10−4), and the association extends into the 3′-untranslated region (UTR). There is a single haplotype block encompassing IRF5 and we show for the first time that the gene comprises two over-transmitted haplotypes and a single under-transmitted haplotype. The strongest association is with a TCTAACT haplotype (T:U = 1.92, P = 5.8 × 10−5), which carries all the over-transmitted alleles from this study. Haplotypes carrying the T alleles of rs2004640 and rs2280714 and the A allele of rs10954213 are over-transmitted in SLE families. The TAT haplotype shows a dose-dependent relationship with mRNA expression. A differential expression pattern was seen between two expression probes located each side of rs10954213 in the 3′-UTR. rs10954213 shows the strongest association with RNA expression levels (P = 1 × 10−14). The A allele of rs10954213 creates a functional polyadenylation site and the A genotype correlates with increased expression of a transcript variant containing a shorter 3′-UTR. Expression levels of transcript variants with the shorter or longer 3′-UTRs are inversely correlated. Our data support a new mechanism by which an IRF5 polymorphism controls the expression of alternate transcript variants which may have different effects on interferon signalling.
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 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.
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.
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.
Vitamin D deficiency is widespread and has been associated with many chronic diseases, including autoimmune disorders. A study was undertaken to explore the impact of low vitamin D levels on autoantibody production in healthy individuals, as well as B cell hyperactivity and interferon α (IFNα) activity in patients with systemic lupus erythematosus (SLE).
Serum samples from 32 European American female patients with SLE and 32 matched controls were tested for 25-hydroxyvitamin D (25(OH)D) levels, lupus-associated autoantibodies and serum IFNα activity. Isolated peripheral blood mononuclear cells were tested for intracellular phospho-ERK 1/2 as a measure of B cell activation status.
Vitamin D deficiency (25(OH)D <20 ng/ml) was significantly more frequent among patients with SLE (n=32, 69%) and antinuclear antibody (ANA)-positive controls (n=14, 71%) compared with ANA-negative controls (n=18, 22%) (OR 7.7, 95% CI 2.0 to 29.4, p=0.003 and OR 8.8, 95% CI 1.8 to 43.6, p=0.011, respectively). Patients with high B cell activation had lower mean (SD) 25(OH)D levels than patients with low B cell activation (17.2 (5.1) vs 24.2 (3.9) ng/ml; p=0.009). Patients with vitamin D deficiency also had higher mean (SD) serum IFNα activity than patients without vitamin D deficiency (3.5 (6.6) vs 0.3 (0.3); p=0.02).
The observation that ANA-positive healthy controls are significantly more likely to be deficient in vitamin D than ANA-negative healthy controls, together with the finding that vitamin D deficiency is associated with certain immune abnormalities in SLE, suggests that vitamin D plays an important role in autoantibody production and SLE pathogenesis.
Interferon-alpha (IFN-α) is a primary pathogenic factor in systemic lupus erythematosus (SLE), and high IFN-α levels may be associated with particular clinical manifestations. The prevalence of individual clinical and serologic features differs significantly by ancestry. We used multivariate and network analyses to detect associations between clinical and serologic disease manifestations and serum IFN-α activity in a large diverse SLE cohort.
1089 SLE patients were studied (387 African-American, 186 Hispanic-American, and 516 European-American). Presence or absence of ACR clinical criteria for SLE, autoantibodies, and serum IFN-α activity data were analyzed in univariate and multivariate models. Iterative multivariate logistic regression was performed in each background separately to establish the network of associations between variables that were independently significant following Bonferroni correction.
In all ancestral backgrounds, high IFN-α activity was associated with anti-Ro and anti-dsDNA antibodies (p-values 4.6×10−18 and 2.9 × 10−16 respectively). Younger age, non-European ancestry, and anti-RNP were also independently associated with increased serum IFN-α activity (p≤6.7×10−4). We found 14 unique associations between variables in network analysis, and only 7 of these associations were shared by more than one ancestral background. Associations between clinical criteria were different in different ancestral backgrounds, while autoantibody-IFN-α relationships were similar across backgrounds. IFN-α activity and autoantibodies were not associated with ACR clinical features in multivariate models.
Serum IFN-α activity was strongly and consistently associated with autoantibodies, and not independently associated with clinical features in SLE. IFN-α may be more relevant to humoral tolerance and initial pathogenesis than later clinical disease manifestations.
systemic lupus erythematosus; interferon alpha; autoantibodies; ancestry
Systemic lupus erythematosus (SLE) is a highly heterogeneous disorder, characterized by differences in autoantibody profile, serum cytokines, and clinical manifestations. SLE-associated autoantibodies and high serum interferon alpha (IFN-α) are important heritable phenotypes in SLE which are correlated with each other, and play a role in disease pathogenesis. These two heritable risk factors are shared between ancestral backgrounds. The aim of the study was to detect genetic factors associated with autoantibody profiles and serum IFN-α in SLE.
We undertook a case-case genome-wide association study of SLE patients stratified by ancestry and extremes of phenotype in serology and serum IFN-α. Single nucleotide polymorphisms (SNPs) in seven loci were selected for follow-up in a large independent cohort of 538 SLE patients and 522 controls using a multi-step screening approach based on novel metrics and expert database review. The seven loci were: leucine-rich repeat containing 20 (LRRC20); protein phosphatase 1 H (PPM1H); lysophosphatidic acid receptor 1 (LPAR1); ankyrin repeat and sterile alpha motif domain 1A (ANKS1A); protein tyrosine phosphatase, receptor type M (PTPRM); ephrin A5 (EFNA5); and V-set and immunoglobulin domain containing 2 (VSIG2).
SNPs in the LRRC20, PPM1H, LPAR1, ANKS1A, and VSIG2 loci each demonstrated strong association with a particular serologic profile (all odds ratios > 2.2 and P < 3.5 × 10-4). Each of these serologic profiles was associated with increased serum IFN-α. SNPs in both PTPRM and LRRC20 were associated with increased serum IFN-α independent of serologic profile (P = 2.2 × 10-6 and P = 2.6 × 10-3 respectively). None of the SNPs were strongly associated with SLE in case-control analysis, suggesting that the major impact of these variants will be upon subphenotypes in SLE.
This study demonstrates the power of using serologic and cytokine subphenotypes to elucidate genetic factors involved in complex autoimmune disease. The distinct associations observed emphasize the heterogeneity of molecular pathogenesis in SLE, and the need for stratification by subphenotypes in genetic studies. We hypothesize that these genetic variants play a role in disease manifestations and severity in SLE.
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
Excessive activity of dendritic cells (DCs) is postulated as a central disease mechanism in Systemic Lupus Erythematosus (SLE). Vitamin D is known to reduce responsiveness of healthy donor DCs to the stimulatory effects of Type I IFN. As vitamin D deficiency is reportedly common in SLE, we hypothesized that vitamin D might play a regulatory role in the IFNα amplification loop in SLE. Our goals were to investigate the relationship between vitamin D levels and disease activity in SLE patients and to investigate the effects of vitamin D on DC activation and expression of IFNα-regulated genes in vitro.
In this study, 25-OH vitamin D (25-D) levels were measured in 198 consecutively recruited SLE patients. Respectively, 29.3% and 11.8% of African American and Hispanic SLE patient had 25-D levels <10 ng/ml. The degree of vitamin D deficiency correlated inversely with disease activity; R = −.234, p = .002. In 19 SLE patients stratified by 25-D levels, there were no differences between circulating DC number and phenotype. Monocyte-derived DCs (MDDCs) of SLE patients were normally responsive to the regulatory effects of vitamin D in vitro as evidenced by decreased activation in response to LPS stimulation in the presence of 1,25-D. Additionally, vitamin D conditioning reduced expression of IFNα-regulated genes by healthy donor and SLE MDDCs in response to factors in activating SLE plasma.
We report on severe 25-D deficiency in a substantial percentage of SLE patients tested and demonstrate an inverse correlation with disease activity. Our results suggest that vitamin D supplementation will contribute to restoring immune homeostasis in SLE patients through its inhibitory effects on DC maturation and activation. We are encouraged to support the importance of adequate vitamin D supplementation and the need for a clinical trial to assess whether vitamin D supplementation affects IFNα activity in vivo and, most importantly, improves clinical outcome.
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
Current therapies for systemic lupus erythematosus (SLE), a debilitating, potentially lethal, multifactorial systemic autoimmune disease, are limited to suppressing disease activity and are associated with multiple adverse effects. Recent advances in basic and translational sciences have elucidated a crucial role for the interferon-alpha (IFNα) pathway in the pathogenesis of this enigmatic disease. The so-called “type I interferon signature” has emerged as a major risk factor for disease activity of SLE. Multiple genes encoding for molecules within the type I interferon pathway have been associated with SLE in genome wide association studies. In addition, innate immune receptors are thought to be triggered by either endogenous and/or exogenous stimuli that lead to hypersecretion of IFNα. We review the multiple emerging treatment strategies targeting IFNα-related pathways. These include monoclonal antibodies against IFNα, anti-IFNα antibody-inducing vaccines, and inhibitors of toll-like receptors. We also summarize the current status of these pharmaceutical agents in early clinical trials.
pDC; TLR; IRF; JAK/STAT; Pin1; interferonopathies; virome; proteasome
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 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.
The C1858T polymorphism in PTPN22 has been associated with risk of systemic lupus erythematosus (SLE), as well as multiple other autoimmune diseases. We have previously shown that high serum interferon alpha (IFN-α) activity is a heritable risk factor for SLE, and we hypothesized that the PTPN22 risk variant may shift serum cytokine profiles to higher IFN-α activity resulting in risk of disease.
IFN-α was measured in 143 SLE patients using a functional reporter cell assay, and TNF-α was measured with ELISA. The rs2476601 SNP in PTPN22 (C1858T) was genotyped in the same patients. Patients were grouped using a clustering algorithm into four cytokine groups (IFN-α predominant, IFN-α and TNF-α correlated, TNF-α predominant, and IFN-α and TNF-α both low).
SLE patients carrying the risk allele of PTPN22 had higher serum IFN-α activity than patients lacking the risk allele (p=0.027). TNF-α levels were lower in risk allele carriers (p=0.030), and the risk allele was more common in patients with an IFN-α predominant or IFN-α and TNF-α correlated cytokine profile as compared to patients with TNF-α predominance or both cytokines low (p=0.002). 25% of male patients carried the risk allele, compared to 10% of female patients (p=0.02), however cytokine skewing was similar in both sexes.
The autoimmune disease risk allele of PTPN22 is associated with skewing of serum cytokine profiles toward higher IFN-α activity and lower TNF-α in SLE patients in vivo. This serum cytokine pattern may be relevant in other autoimmune diseases associated with the PTPN22 risk allele.