Abstract
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.
Highlights
Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease characterized by elevated type I interferon (IFN) production, a break of immune tolerance to self-antigens, persistent production of pathogenic autoantibodies, complement activation, and immune complex (IC) deposition resulting in inflammation and end organ damage [1]
The aim of this study was to use a combination of molecular cloning and next-generation sequencing technologies to obtain sufficient coverage depth in primary immune cells of SLE patients and healthy donors to clearly determine 1) whether SLE patients express an interferon regulatory factor 5 (IRF5) transcript signature that is distinct from healthy donors, and 2) whether an IRF5-SLE risk haplotype determines the IRF5 transcript signature, controlling and/or contributing to the global pathologic function(s) of IRF5 in SLE
We have demonstrated a significant association between elevated IRF5 expression in primary Mo of SLE patients with the IRF5-SLE H2 risk haplotype [15]; in addition, we recently showed that IRF5 is constitutively activated in Mo, and not T or natural killer (NK) cells, from SLE patients compared to healthy donors [37]
Summary
Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease characterized by elevated type I interferon (IFN) production, a break of immune tolerance to self-antigens, persistent production of pathogenic autoantibodies, complement activation, and immune complex (IC) deposition resulting in inflammation and end organ damage [1]. While the underlying etiology of SLE remains obscure, several lines of evidence document the importance of genetic factors [2,3] One such factor, the interferon regulatory factor 5 (IRF5) gene, was identified in the susceptibility to develop SLE [4]. The interferon regulatory factor 5 (IRF5) gene, was identified in the susceptibility to develop SLE [4] This initial finding, and the subsequent replication in numerous patient cohorts [4,5,6,7,8,9,10], marked an important break-through in our understanding of SLE pathogenesis given the critical role of IRF5 in regulating type I IFN expression and mediating Toll-like receptor (TLR) signaling [11,12,13,14]. Further studies have shown that IRF5-SLE risk haplotypes are associated with serum IFNa activity in SLE patients [17,18]
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