The recent identification of IRF5
as an SLE susceptibility gene has provided a critical link between the type I IFN pathway and disease pathogenesis. Given the role of IRF-5 in host defense and in type I IFN
gene regulation (6
), it is reasonable to hypothesize that genetic variants associated with IRF-5 expression may contribute to increased IFNα levels in SLE patients. Here, we demonstrate a direct association of IRF-5
transcript levels with SLE. Total IRF-5
expression and expression from non-coding Ex1A and 1D were significantly upregulated independent of genotype in primary PBMC from SLE patients compared to healthy donors (). IRF-5
alternative splicing was also enhanced in SLE patients () providing the first evidence that IRF-5 isoforms specific to SLE may be identified. IRF-5 exists as multiple alternatively spliced isoforms, each with distinct cell type-specific expression, regulation, and function (29
). Previous studies have shown that the identical polypeptides V3 (Ex1C) and V4 (Ex1A) are the primary inducers of virus-mediated IFNα (29
). These isoforms are highly expressed in pDC of healthy donors. The mechanism(s) of enhanced IRF-5
alternative splicing in SLE is not known but data presented here provide initial evidence that U1 snRNP, SRp20 and SRp40 may be involved. snRNPs are components of the spliceosome and are essential for the removal of introns; SR proteins are critical splicing factors involved in regulating/selecting splice sites. snRNPs, specifically U1 snRNP, are the major SLE autoantigens in addition to dsDNA (35
). U1 snRNP was recently shown to induce robust type I IFN (36
). The ability of U1 to alter the utilization of Ex1A and splicing of Ex5-7 suggests that it may also be a critical factor in regulating IRF-5
alternative splicing. Equally important, SRp20 and p40 have been shown to be autoantigens in patients with SLE (32
) and overexpression in our minigene assays showed enhanced transcription and alternative splicing of IRF-5
. Additional experiments are necessary to confirm an association of autoantigen levels in serum of SLE patients with IRF-5
expression and/or alternative splicing.
Significant effort has gone into the replication and expansion of IRF5
genotype data in SLE (16
). Multiple risk and protective haplotypes have been predicted, yet information is significantly lacking regarding the functional consequence of these polymorphisms. We identified an IRF5
risk haplotype, containing the risk alleles of SNPs rs2004640, rs10954213, rs10488631, and the CGGGG indel (24
), that in part explains the association between SLE and elevated IRF-5 expression. Transcripts were increased 30–85% in patients having the risk (H2) versus protective haplotype (H3) in homozygous form. The most significant correlation with the H2 haplotype came from utilization of Ex1C. Contrary to published data by Graham et al.
), detection of Ex1B-associated transcripts was negligible in either healthy donors or SLE patients. Our data instead support findings by others indicating an overall low utilization of Ex1B (20
). Transcription from Ex1C is generally negligible in unstimulated healthy donor PBMC, yet stimulation with IFNα leads to enhanced expression (29
). We determined that the risk alleles of rs10488631, and not the other variants, were independently associated with increased IRF-5
expression from Ex1C (20
). SNPs rs2004640 and rs10954213 have been associated with IRF-5
transcription in HapMap samples; however, neither were independently associated (19
). It is difficult to determine the biological significance of single polymorphisms since patient samples contain multiple polymorphisms that may or may not be associated with the disease. Furthermore, SLE patients that are homozygous for a single risk variant are rare. Indeed, the CGGGG polymorphism accounts for the association signal observed from rs2004640 and rs10954213 (24
). Minigene reporter assays demonstrated that risk alleles of both the CGGGG indel and rs2004640 were associated with increased transcription from Ex1A and 1D (). The fact that neither of these variants gave independent association with IRF-5
expression may be due to their being masked by risk alleles of rs10954213 or rs10488631, or due to a genuine joint effect by these alleles.
By FACS and immunoblot analysis, we show a direct correlation between increased IRF-5
transcription and protein expression in blood cells of SLE patients (). Similar to Q-PCR data, the risk allele of rs10488631 was the best independent predictor of IRF-5 protein expression; the rs10954213 risk allele was also significantly associated. Observed differences between IRF-5 transcript and protein expression associated with rs10954213 may be due to its function in stabilizing IRF-5 proteins (22
While our data support an association of the H2 haplotype with enhanced IRF-5 expression in SLE, these may not be the only variants contributing to expression; other factors independent of genotype may also contribute. Stratification of IRF-5
expression by rs10488631 and the CGGGG indel in healthy donors and SLE patients support this idea (). Differences in expression between these groups could be a result of functional variants that have yet to be identified. The other possibility is that disease-associated factors contribute to IRF-5 expression. Niewold et al.
) reported that an IRF5
risk haplotype (rs2004640, rs3807306, rs10488631, and rs2280714) was associated with increased serum IFNα activity. High IFNα levels detected in SLE patients could be a factor contributing to enhanced IRF-5 expression (11
). Minigene experiments confirmed that both genotype and soluble IFNα contribute to IRF-5
expression (). Patients with SLE have long been known to display elevated type I IFN in their serum. We provide the first detailed evidence supporting a role for the IRF5
risk haplotype in directing expression of both IRF-5 and IFNα, whereby IRF-5 expression is linked to circulating IFNα levels in patients.