Interferon regulatory factor 5 in human autoimmunity and murine models of autoimmune disease

Accepted Manuscript Interferon Regulatory Factor 5 (IRF5) in human autoimmunity and murine models of autoimmune disease Hayley L. Eames, Alastair L. Corbin, Irina A. Udalova PII:

S1931-5244(15)00223-6

DOI:

10.1016/j.trsl.2015.06.018

Reference:

TRSL 935

To appear in:

Translational Research

Received Date: 15 April 2015 Revised Date:

29 June 2015

Accepted Date: 30 June 2015

Please cite this article as: Eames HL, Corbin AL, Udalova IA, Interferon Regulatory Factor 5 (IRF5) in human autoimmunity and murine models of autoimmune disease, Translational Research (2015), doi: 10.1016/j.trsl.2015.06.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Interferon Regulatory Factor 5 (IRF5) in human autoimmunity and murine models of autoimmune disease Hayley L. Eames*1, Alastair L. Corbin* and Irina A. Udalova*2 *

Correspondence: 1 Dr. Hayley L. Eames, [email protected] 2 Prof. Irina A. Udalova, [email protected]

Running title

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IRF5 in autoimmunity

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Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics Rheumatology and Musculoskeletal Sciences, University of Oxford, Roosevelt Drive, Headington, Oxford, OX3 7FY, United Kingdom, +44(0)1865 612600

Abstract

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Interferon Regulatory Factor 5 (IRF5) has been demonstrated as a key transcription factor of the immune system, playing important roles in modulating inflammatory immune responses in numerous cell types including dendritic cells, macrophages and B-cells. As well as driving the expression of Type I Interferon in anti-viral responses, IRF5 is also crucial for driving macrophages toward a pro-inflammatory phenotype by regulating cytokine and chemokine expression, and modulating B-cell

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maturity and antibody production. This review highlights the functional importance of IRF5 in a disease setting, by discussing polymorphic mutations at the human Irf5 locus that lead to susceptibility to Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA) and Inflammatory Bowel Disease (IBD). In concordance with this, we

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also discuss lessons in IRF5 functionality learned from murine in vivo models of autoimmune disease and inflammation, and hypothesise that modulation of IRF5

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activity and expression could provide potential therapeutic benefits in the clinic. Introduction

The Interferon Regulatory Factor (IRF) family of transcription factors consists of nine family members in mammals: IRF1, IRF2, IRF3, IRF4 (also known as PIP, LSIRF or ICSAT), IRF5, IRF6, IRF7, IRF8 (also known as ICSBP) and IRF9 (also known as ISGF3γ) (1). The IRFs were first characterised in the late 1980’s as transcriptional regulators of Type I interferon (IFN) production: driving expression of the single Ifnb1 gene, and 13 closely related Ifna genes. The process of IFNα and IFNβ production, and therefore IRF activity, is crucial to the first line of defence against viral pathogens, as Type I IFN drives the recognition and degradation of viral RNA/DNA,

ACCEPTED MANUSCRIPT inhibits protein synthesis to prevent viral replication and particle assembly, and finally drives apoptosis of infected cells to contain the viral infection. Additionally, Type I IFN functions in both innate and T-cell mediated immunity, by promoting differentiation of dendritic cells, protecting CD8+ cells from antigen-induced cell death, and polarising CD4+ T-cells to a TH1 phenotype (2, 3). Many of the IRFs are now described as direct transducers of viral-mediated signalling, and their

encoded IRF proteins (vIRFs) (4).

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importance in Type I IFN production is highlighted by the existence of multiple viralThese vIRFs are homologous to host IRF

proteins, but lack crucial features for true IRF activity, to enable viruses to circumvent host immune responses by mimicry to dampen Type I IFN production (5-7).

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In order to mediate gene expression, IRF proteins typically bind the DNA via their highly conserved N-terminal DNA binding domain, at a putative binding motif known

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as an Interferon Stimulated Response Element (ISRE), which consists of two GAAA half sites (8). Additionally, the IRFs can cooperate with other transcription factor family members to drive gene expression during an immune response. IRF1, IRF3 and IRF7 are integral to the IFNβ enhanceosome – a cooperative transcription factor complex containing two IRF homo- or hetero-dimers, one NFκB dimer (RelA/p50) and one AP1 complex (ATF2/c-Jun) that assembles at the Ifnb1 promoter, in a

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virally-inducible region of clustered binding sites, known as the four Positive Regulatory Domains (PRD-IV) of Ifnb1 (9-11). By interacting with one another whilst bound to the DNA, the enhanceosome complex is more stable and efficient at inducing transcription than any of the individual components bound independently to the promoter, and is therefore the optimal condition for IFNβ production in response

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to viral infection (12). Overexpression of IRF3 can activate Ifnb1 expression even in the absence of viral infection (13), but IRF3 alone is insufficient for induction of Ifna

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genes, which requires IRF7 or IRF5 (14, 15). Plasmacytoid dendritic cells (pDCs) are specialised cells that can produce large quantities of IFNα, as can monocytes and B-cells, in which both IRF5 and IRF7 are expressed constitutively and have key non-redundant roles in IFNα production (14). Activation of IRF5 by phosphorylation is virus-specific – IRF5 association with the Ifna promoter in vivo is only observed following infection with Newcastle Disease Virus (NDV), and not Sendai virus, leading to induction of the Ifna8 subtype, rather than the Ifna1 subtype induced by IRF7 following NDV infection (14). This highlights a distinct and important role for IRF5 in Type I IFN production, in isolation from IRF3 and IRF7 activity. Beyond Type I IFN production in response to virus, it is now becoming clear that the

ACCEPTED MANUSCRIPT IRFs can also play pivotal roles in both transcriptional regulation and development of other aspects of host defence. IRF5 in particular, due to its expression in multiple immune cell types, including macrophages, B-cells and dendritic cells (16), has been associated with susceptibility to numerous autoimmune diseases and cancer. In this review, regulation of Irf5 expression and consequences of single nucleotide polymorphisms (SNPs) at the Irf5 gene locus will be discussed in the context of Parallel to this, the phenotypic consequence of IRF5

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autoimmune disease.

deficiency in multiple murine models of autoimmune disease and infection will be discussed, to give an insight into the essential role of IRF5 in the immune system in a complex in vivo setting.

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IRF5 gene structure and splicing

In humans, the Irf5 gene is located on chromosome 7q32 and consists of 9 coding

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exons plus one non-coding exon in the 5’ UTR, of which there are 3 variants (Exon 1A, 1B, and 1C), each encoding an alternative promoter for the Irf5 gene (P-V1, PV2, P-V3), upstream of the ATG start codon in Exon 2 (17).

Splicing of Irf5

transcripts is a complex process, with transcription initiated at one of the three discrete transcription start sites (TSS), and multiple variants initiated at each promoter, leading to a total of nine IRF5 transcripts (see Figure 1), which have been

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demonstrated to have differential expression in different cell types. IRF5-v1, -v2, and -v3/v4 are expressed in human primary pDCs and macrophages, whereas IRF5-v5 and –v6 were identified in human primary peripheral blood mononuclear cells (PBMCs) from healthy donors, and immortalised T-cell/B-cell malignancies (17). The

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severely truncated IRF5-v7, -v8 and -v9 variants have only been detected on the PCR level in human cancer cell lines (17).

Additionally, 14 new Irf5 transcript

variants that derive from Exon 1A have also been recently identified by a

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combination of next-generation sequencing and molecular cloning, which exhibit differential expression in healthy donors and SLE patients (18). This highlights the complexity in expression and splicing of Irf5 and the potential for this gene to act as a biomarker for autoimmune disease. It is well described that phosphorylation events are required to activate IRF5 protein (19), but little is known about how Irf5 expression itself is regulated. The majority of Irf5 transcripts (-v1, -v4, -v5, -v6, -v7, - v8) derive from Exon 1A, which contains an IRF binding site that is stimulated in virus-infected cells, and these transcripts have the highest translation efficiency (17, 20). Exon 1C, the TSS for –v3, also contains an IRF binding site, which is responsive to IFN stimulation via the binding of the

ACCEPTED MANUSCRIPT ISGF3 complex (containing IRF9) (17), suggesting that the Irf5 locus can be regulated by other IRF family members. The fact that Irf5 expression is induced in human PBMC-derived or murine bone marrow-derived macrophages following differentiation with GM-CSF in vitro suggests transcriptional regulation by members of the STAT family of transcription factors (21-23).

This is further highlighted in a

study that used ChIP-seq datasets to identify transcription factor binding sites in the

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Irf5 locus, which identified a STAT2 binding site in the Exon 1C promoter, as well as numerous other binding sites across the 5’ UTR including those for PU.1, IRF4, PAX5, TCF12, p53, EBF, AP1, Myc and NFκB (24). These transcription factors therefore also have the potential for regulation of Irf5 expression, although further

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experimental studies in multiple cell types are required to confirm this.

Another characteristic of the Irf5 locus is a strong CpG island (CGI) that spans the

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promoter region containing Exons 1A and 1B and contains multiple binding sites for the transcription factor Sp1 (25). Active CGI promoters are often enriched for Sp1 binding sites, as DNA binding by Sp1 can in turn recruit RNA Polymerase II and TATA-binding protein (TBP) to the region, to promote transcription even in the absence of a TATA box (26). This is in line with the fact that CGI regions are GCrich, predominantly non-methylated and associated with transcription initiation (27).

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Similarly, approximately 70% of promoters across the genome also contain CGIs – mostly those associated with housekeeping genes, but also tissue-specific genes and those involved in developmental regulation (27-29). A study of the chromatin environment at LPS-inducible genes in macrophages showed that CGIs are relatively nucleosome-deficient in a manner that is independent of SWI/SNF chromatin

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remodelling activity (30), suggesting that CGI-associated loci are relatively ‘open’ for access by transcriptional machinery in macrophages. The presence of a CGI at the

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Irf5 locus suggests either basal levels of expression, or the opportunity for rapid Irf5 expression upon induction, which is in line with the known roles of IRF5 in innate immunity, as discussed later in this review. It has also been shown that CGIs in the Irf7 promoter can become methylated in the 2fTGH cell line, leading to stable Irf7 gene silencing by direct inhibition of transcription factor binding (31). At first glance, this could be considered an explanation for the low Irf5 expression in the T-cell compartment of the immune system (16), compared to myeloid cells. However, no difference in methylation profile of Irf5 locus in primary Sjögren’s syndrome (pSS) or inflammatory bowel disease (IBD) patients compared to healthy controls has been observed (32, 33); suggesting that the Irf5 locus is not epigenetically controlled, at least during inflammatory disease states.

ACCEPTED MANUSCRIPT As well as the use of alternative promoter exons, some of the known Irf5 transcript variants also exhibit distinctive insertion/deletion patterns in Exon 6 as a result of alternative splicing events (See Figure 1 and Figure 2). Alternative splicing is the processing of pre-mRNA to mature mRNA transcripts via the removal of intronic sequences, leaving only the coding exons. As part of the process, exons can also be excluded from the primary transcript by ‘exon skipping’, or the boundaries of exons

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can be altered by utilisation of alternative 3’ acceptor splice junctions (changes the 5’ boundary of downstream exon) or 5’ donor splice junctions (changes the 3’ boundary of upstream exon). In Exon 6 of the Irf5 gene, there are two constitutively active 3’ acceptor splice sites. As a result of this, the Irf5 transcripts –v1 and –v5 contain a

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48bp insertion in Exon 6 known as SV-16 (34). Additionally, -v1, -v3 and –v4 have another 30bp in-frame deletion known as indel-10 or rs60344245 in Exon 6, which is not present in the other Irf5 variants (34). Transcripts –v3 and –v4 are the only

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variants that possess both of these Exon 6 indels. It is thought that these indel repeat motifs are located in a putative PEST domain within the resulting IRF5 protein isoform – a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) – that has only recently expanded in evolution, and may represent an important protein-protein interaction interface (17). Alteration of sequence in this domain may therefore affect IRF5 activity via modulation of cofactor binding. PEST domains are

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also normally present in proteins characterised by rapid protein turnover, so it can also be hypothesised that indels in this region could influence the stability of different IRF5 isoforms (35). Functionally, although these Exon 6 indels in isolation do not seem to increase the risk of autoimmune disease (36), they have been shown to play

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a role differential IRF5 activity. Splice variant SV-16 is the more potent indel and promotes resistance to apoptosis and cytokine release, whereas coexpression of indel-10 appears capable of neutralising the effects of SV-16 (34). The presence of

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these indels on Irf5 risk haplotypes for autoimmune disease is therefore likely to modulate IRF5 activity in combination with other polymorphisms. Alternative splicing can also result in the severely truncated –v7, -v8 and –v9 transcripts of Irf5 (See Figure 1) (17). The transcript variant –v7 would lack the majority of the DBD when translated due to skipping of Exon 2, and thus may function as an endogenous dominant negative (DN) mutant of full-length IRF5 protein. The variant –v8 has a large single deletion that spans most of Exons 6 and 7, which would affect the suggested PEST sequence and the IRF Association Domain (IAD) upon translation, which is responsible for dimerisation of IRF family members and many other protein-protein interactions, including with the coactivator

ACCEPTED MANUSCRIPT p300 and NFκB RelA (37, 38). An early termination codon is inserted in –v9, so this variant would lack the entire C-terminus including the IAD and serine rich region (SRR) in the resulting protein isoform, where most of the protein-activating phosphorylation events occur (19). IRF5 Polymorphisms

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Genome-wide association studies (GWAS) that compare the genome sequence between healthy and diseased cohorts are becoming a useful tool to identify gene mutations that predispose to pathogenesis.

Single-nucleotide polymorphisms

(SNPs) – variation of a single nucleotide within a population that result in two

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different alleles (one associated with disease risk, and one with disease protection) at a particular genomic locus – have now been described for multiple diseases and ethnic groups, which has lead to the identification of many new susceptibility loci that

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can now be the focus of further study and therapeutic design. Multiple SNPs in the Irf5 gene have now been described, as shown in Figure 2, and in some cases the functional consequences have also been identified. Importantly, recent functional analyses of association of common genetic variants with variations in gene expression confirmed that IRF5 presents one of the most significant expression

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quantitative trait loci in myeloid cells stimulated with bacterial LPS (39, 40). The most commonly associated Irf5 SNP seems to be rs2004640, which is located 2bp downstream of Exon 1B in the promoter region of the Irf5 locus. The T risk allele creates a 5’ donor splice site, which causes Exon 1B to be spliced onto Exon 2 – the first coding exon (41). In contrast, when the protective G allele is present, the splice

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junction is not recognised and the unspliced transcript is targeted by non-sense mediated decay (41, 42). The rs2004640 SNP therefore causes an increase in Exon

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1B driven Irf5 transcripts, such as variants –v2 and –v9, whereas Exon 1A is usually the predominant promoter exon for expressed transcripts.

Autoimmune risk

haplotypes that include rs2004640 exhibit high levels of IRF5 and IFNα (43, 44). It has also been demonstrated that monocyte-derived macrophages and dendritic cells from rs2004640 risk-associated allele carriers produce increased inflammatory cytokines such as TNFα, IL-12p40, IL-8 and IL-1β following stimulation of TLR2 by MDP (45). Another common functional Irf5 SNP is the rs77571059 polymorphism, also described as the CGGGG indel, which is located 64bp upstream of Exon 1A. This 5bp copy-number variant results in a risk allele with 4 copies of the CGGGG repeat sequence, compared to the protective allele which has only 3 copies. This additional sequence forms an extra binding motif in the CGI region of Irf5 promoter

ACCEPTED MANUSCRIPT for the transcription factor Sp1, which causes increased expression of Irf5 (46). The functional rs10954213 Irf5 SNP is also often included in autoimmune disease risk haplotypes. The A risk allele of this SNP creates a functional polyadenylation signal, resulting in an Irf5 transcript with a shorter 3’ UTR.

Human Irf5 transcripts can

therefore contain one or two polyA sites corresponding to the risk or protective alleles of the rs10954213 SNP, resulting in short or long 3’ UTRs respectively (36, 47).

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Longer transcripts contain two AU-rich elements, leading to rapid mRNA turnover; whereas shorter transcripts have a longer half-life, and are more upregulated in cells stimulated with Type I IFN (48). Finally, the rs10488631 SNP, which seems to be commonly strongly associated with autoimmune diseases (see Tables 1 and 2), is The C risk allele of

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located approximately 4kb to the 3’ of the Irf5 locus (49).

rs10488631 is associated with increased expression of Irf5 transcripts utilising the Exon 1C promoter (43). Moreover, increased serum IFNα activity is observed when

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this SNP is included in a risk haplotype with rs3807306 (44).. However, the individual contribution of this SNP to autoimmune phenotypes currently requires further investigation. Interestingly, the C risk allele of rs10488631 is also independently associated with SLE as a polymorphism of the Tnp03 locus, which encodes the nuclear import receptor Transportin 3, and is found in the same region as the Irf5 locus in the genome (50). This review will now focus on the specific association of

IRF5 and SLE

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these and other less well-described Irf5 SNPs in a range of autoimmune diseases.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune inflammatory

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disease that can result in patients experiencing a wide range of clinical phenotypes. Human SLE can consist of a facial butterfly rash (malar rash) or life-threatening manifestations such as nephritis if the disease progresses to become systemic (51).

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Patients are predominantly women, and 95% possess anti-nuclear antibodies (ANA), which recognise ‘self’ chromatin components including double-stranded DNA (dsDNA), histones and nucleosomes, that break self-tolerance to become pathogenic, resulting in an autoimmune response. ANAs have been identified in multiple autoimmune diseases, including SLE, Sjögren’s syndrome, systemic sclerosis and rheumatoid arthritis (52). In SLE, immune complexes involving ANAs are often deposited around the body, including on the renal glomerular basement membrane, which ultimately results in glomerulonephritis due to the inflammatory response that ensues (53). Concomitant with ANA production, SLE patients also reproducibly exhibit an IFN-signature that correlates with disease severity – serum levels of IFNα increase according to flares of skin rash and fever; and the highest

ACCEPTED MANUSCRIPT levels of IFNα are observed during the peak of disease (54, 55). Microarray studies have also shown an upregulation of genes associated with the IFN pathway in PBMCs from approximately 50% of SLE patients (56). Although the etiology of SLE remains unclear, genetic factors have been established as a key line of enquiry, and several genetic associations with SLE susceptibility

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have been identified, including Ptpn22 (57), the HLA genes (58), Stat4 (59), and importantly in the context of this review – the Irf5 locus. The Irf5 polymorphism rs2004640 was first associated with SLE in a joint linkage and association analysis with the Tyk2 locus in Swedish and Finnish populations, with an adjusted p-value of 7.9x10-6 (60). Table 1 lists a wide range of GWAS studies that have since been

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documented to show robust associations between Irf5 polymorphisms and SLE, in multiple cohorts and a range of ethnicities. The strongest Irf5 association with SLE is

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the rs2004640 SNP, which is consistently associated in worldwide cohorts including those from Europe, America and Japan (41, 49, 61, 62), although only a weak association was observed in a case-control study from China (63).

Significant

enrichment of the rs2004640 T risk allele can be observed in SLE patients compared to matched healthy controls (43).

European and South American SLE cohorts have

also been strongly associated with the rs77571059 promoter indel upstream of Exon

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1A (46, 62), and the rs10488631 SNP located 4kb downstream of Irf5 is also strongly associated with European SLE patients (49). Elevated IRF5 protein expression in SLE patient monocytes correlates with the Irf5 risk haplotype of rs2004640, rs10954213, rs10488631 and rs77571059 (43), suggesting increased IRF5 activity in an SLE autoimmune setting, which is further confirmed by constitutively elevated

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nuclear IRF5 levels in SLE monocytes (64). In concordance with this, the Irf5 risk haplotype in SLE corresponds to high serum levels of IFNα (44) – a key

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characteristic of SLE pathology and known transcriptional gene target of IRF5. Stimulation of healthy monocytes in vitro with SLE serum (in particular SLE autoantigens composed of necrotic/apoptotic material) stimulates IFNα, TNFα and IL-6 production that coincides with the kinetics of IRF5 nuclear translocation (64). As well as increased expression of Irf5 in SLE, RNA-seq studies have also identified that SLE patients express an Irf5 transcript signature that is distinct from healthy donors (18), and parallel to this spliceosome component activity is increased in PBMCs from SLE patients, indicating an increase in Irf5 alternative splicing in autoimmune conditions (43).

As the IRF5 isoforms have been shown to have

differential abilities to regulate proinflammatory cytokine expression, in particular

ACCEPTED MANUSCRIPT those involved in SLE pathogenesis, this also represents an important factor driving inflammation in SLE patients (18). The critical role for IRF5 in SLE pathogenesis can also be highlighted using murine models of the disease and Irf5-/- mice. In the pristane model of murine SLE, Irf5-/mice experienced poor lymphocyte activation and T-cells were differentiated to a TH2

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rather than TH1 phenotype, corresponding with reduced IL-12, IL-23 and in particular IFNα levels, which are critical for SLE disease pathogenesis (65). A reduction in Ly6Chi monocytes migrating to the peritoneal cavity due to down-regulated expression of chemokine receptors CXCR4 and CCR2 has also been observed in the absence of IRF5, which is thought to be a key initial event in the pathogenesis of Concordant with this, the FcγRIIB-/-Yaa and

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the pristane-induced model (66).

FcγRIIB-/- murine lupus models showed that IRF5 is crucial for disease development,

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in particular IFNα production (67), as was also the case for Irf5-/--MLR/lpr mice (68). A reduction in IgG class-switching in B-cells in the Irf5-/- mice was also demonstrated upon pristane induction of SLE (69), which correlates with observations that IRF5 also plays a crucial role in B-cell development. Irf5-/- mice experience attenuated plasma cell maturation, resulting in splenomegaly due to accumulation of immature B-cells in the spleen. This is due a lack of expression of the IRF5-target gene Prdm1

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(BLIMP1), a plasma cell commitment factor (70). It has also been observed that IRF5 controls IgG2a antibody production – the characteristic IgG of antiviral and autoimmune responses – via direct binding to the γ2a locus and regulation of Ikaros expression (71, 72). A lack of mature B-cells and therefore antibodies means that

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fewer ANA immune complexes can form in Irf5-/- mice, which would normally drive SLE pathogenesis. These described B-cell differences were put into question when it was identified that some of the Irf5-/- colonies worldwide had acquired a

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spontaneous mutation in the Dock2 gene, which we have discussed in the Conclusion section of this review. Consistent with the importance of a Type I IFN signature in SLE, Irf5-/-mice also exhibit a marked reduction in serum Type I IFN levels upon Newcastle disease virus (NDV) infection (73) and poor survival of vesicular stomatitis virus (VSV) and Herpes simplex virus (HSV-1) infection associated with decreased IFNα production (74). In conclusion, SNPs in the Irf5 locus therefore seem to both increase IRF5 expression, and drive alternative splicing of Irf5 to generate alternative isoforms in SLE patients, which have differential activity. In vivo studies have also established key roles for IRF5 in both myeloid and B-cells in SLE.

ACCEPTED MANUSCRIPT IRF5 and RA Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease, with a prevalence of 1% in the population (75) and a disproportionately high incidence in older individuals and women compared to men (3:1 ratio) (76). The target tissue in RA is primarily the synovial joints – often those of the hands, feet and knees - and

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clinical features comprise a syndrome of joint pain, stiffness and swelling, as well as increased susceptibility to infections, cardiovascular disease and lymphoma (76). RA joints are characterised by a massive leukocytic infiltrate into the synovium (synovitis); which leads to chronic inflammation, pannus formation and subsequent irreversible joint damage, due to cartilage degradation and erosion of juxta-articular

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bone (75, 77).

There is strong evidence that multiple genetic factors confer predisposition to RA,

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although geographic location and ethnicity play a factor in the strength of such associations. Variation in the human leukocyte antigen (HLA) gene is a crucial determinant of RA susceptibility, in particular the Shared Epitope (SE) – a molecular structure consisting of a conserved amino acid sequence at aa70-74 of the third hypervariable region of the DRB1 chain, which is found in both the HLA-DRB1*01 and *04 alleles - is strongly associated with RA (78). The SE also determines greater

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disease severity with an increased incidence of bone erosion and earlier disease onset (79). A majority of RA patients are positive for autoantibodies against the Fc portion of the IgG molecule (Rheumatoid Factor – RF), and the presence of RF correlates with a severe form of RA, although it sometimes can be detected in normal

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healthy individuals or patients with conditions other than RA (80). RA patients also produce autoantibodies against cyclic citrullinated peptides (anti-CCP), and these are rarely detected in healthy individuals, therefore can be considered a more specific

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disease marker than RF (81). Anti-CCP antibodies can be detected years before the appearance of any RA clinical symptoms and are indicators of a greater degree of inflammation and a more destructive disease (81). Smoking is associated with antiCCP positive RA, and anti-CCP antibodies display a strong association with the HLADRB1 SE, whereas anti-CCP negative RA is associated with HLADRB1*03 allele, which suggests that there are at least two distinct diseases within the syndrome of RA (82). In line with the genetic component of RA, multiple polymorphisms in the Irf5 locus have been associated with RA, although only to a weak/moderate extent (see Table 2).

In particular the rs2004640 and rs3757385 SNPs have been identified in

ACCEPTED MANUSCRIPT independent studies including Norwegian, Spanish, Swedish and Dutch nationalities (46, 83, 84). Equally however, there are also studies that do not agree with Irf5 associations with RA, so the link is not as robust as is observed with SLE (85, 86). Of the Irf5 SNPs that have been associated with RA, those cohorts that were phenotyped for the type of RA they were suffering, also showed inconsistent results. Multiple studies show association of Irf5 with anti-CCP negative or RF negative RA,

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suggesting a less severe form of the disease, however in terms of the degree of erosion associated with Irf5 SNPs the results are unclear (83, 87). It is possible that IRF5 is involved in more acute inflammatory events, rather than the systemic inflammation characteristic of RA, and therefore increases the susceptibility of a

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patient to RA at the early stages of onset, rather than being a factor in prolonged disease status. The early events leading to the development of RA remain unclear, but formation of anti-CCP antibodies is believed to be a key pathogenic event.

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Neutrophils isolated from RA patients display enhanced neutrophil extracellular trap (NET) formation, characterized by the presence of citrullinated autoantigens, which in turn leads to significantly augmented inflammatory responses in synovial fibroblasts (88).

The inflamed RA synovium consists largely of activated macrophages (30-40%) and

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T-cells (~30%), although B-cells and dendritic cells are also present (75, 77). Resident tissue cells, including activated synovial fibroblasts, chondrocytes and osteoclasts, have also been shown to promote perpetuation of inflammation in the RA joint, as well as mediate the associated cartilage and bone damage (89). RA synovial fluid contains a large number of activated neutrophils and a wide range of

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effector molecules, including pro-inflammatory cytokines (IL-1β, TNF, IL-6), chemokines (IL-8, IP-10, MCP-1, RANTES), and matrix metalloproteases (MMP-1, -

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3, -9, -13); which interact with one another in a complex manner resulting in chronic and persistent inflammation (90). The recruitment, activation and function of each cell type present in the RA synovium is directed principally by this network of secreted effector molecules. TNF is considered the prime inflammatory mediator in the RA joint, due to its ability to induce degradation of cartilage (91) and bone (92) in vitro via chondrocyte and osteoclast activation; and also the observation that TNF is spontaneously produced by dissociated RA synovial mononuclear cell cultures to chronic levels (93), along with IL-1β (93), IL-6 (94), IL-8 (95) and GM-CSF (96). Importantly, if TNF activity is blocked in these cultures, spontaneous production of IL1β protein and IL1b mRNA is significantly reduced (97), suggesting a hierarchy of control within the effector molecules associated with the RA synovium. Consistent

ACCEPTED MANUSCRIPT with this idea, TNF blockade also inhibits expression of IL-6 and IL-8, and spontaneous production of GM-CSF (responsible for MHC class II expression on antigen-presenting cells) (96, 98). IRF5 has been shown to be extremely important for increased and prolonged TNF production in inflammatory (M1) macrophages, compared to anti-inflammatory (M2)

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macrophages in vitro (21, 22). ChIP seq studies of genome-wide IRF5 recruitment have extended this observation to many inflammatory genes, which IRF5 regulates in co-operation with NFκB (37).

In line with this, mice lacking IRF5 (Irf5-/-) exhibit

severely impaired serum cytokine production and are protected against lethal LPSand CpG-induced endotoxic shock (99). Moreover, in the acute Antigen-Induced

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Arthritis (AIA) murine model of arthritis, IRF5 deficiency limits the neutrophil influx into the inflamed joint in the early stages of arthritis leading to the reduction in the

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TH1/TH17 and γδT IL-17+ cells in the joint at the later stages (Weiss M et al submitted PNAS 2015), which suggests a further role for IRF5 in chemokine expression. Interestingly, no difference was observed between wild type and Irf5-/mice in the collagen-induced arthritis (CIA) murine model, based on clinical score of paws and Type II collagen autoantibody production (71). Both AIA and CIA are antigen-dependent and T-cell/B-cell driven models, however there are multiple

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reasons for this apparent difference in arthritic phenotype in the mouse. The AIA model is acute (the mouse is immunised with methylated BSA emulsified in complete Freund’s adjuvant intra-dermally at the base of the tail, and again intra-articularly leading to rapid localised inflammation in the knee joint (100)); whereas the CIA model is more systemic (the mouse is immunised intra-dermally at the base of the

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tail with Type II collagen emulsified in complete Freund’s adjuvant, which elicits arthritis approximately 21 days later (101)). This therefore may highlight a role for

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IRF5 in the early stages of autoimmunity rather than in more prolonged inflammation. Additionally, it is preferable in the CIA model to monitor each mouse individually over a timecourse, as paw swelling in each mouse can begin to occur on different days, and therefore harvesting all mice on the same day post-immunisation rather than post-onset may result in the mice being at different stages of disease and therefore not comparable, which may be the case for the CIA study in question. The CIA model on the C57BL/6 background is also known to be milder than the disease observed on the DBA/1 background (102), so backcrossing mice to a H-2q (DBA/1like) rather than H-2b haplotype would increase the severity of disease on the C57BL/6 background (103), which would allow for perhaps more subtle differences to be identified.

ACCEPTED MANUSCRIPT IRF5 was also shown to play a central role in defining an inflammatory macrophage phenotype: IRF5 expression is induced during differentiation of human monocytes and murine bone marrow into inflammatory macrophages to transcriptionally regulate characteristic pro-inflammatory markers (22, 23). In line with this, IRF5 was highly expressed in macrophages of inflamed knee in the AIA model (23). As a result, Irf5-/macrophages are polarised to an anti-inflammatory phenotype and Irf5-/- mice are

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unable to mount essential TH1 responses to chronic Leishmania donovani infection due to a shift toward increased TH2 cytokine expression (104). Additionally, in response to lipopolysaccharide Irf5-/- mice are unable to promote robust TH1/TH17 responses (22), which are especially prevalent in autoimmune conditions. In stark

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contrast to these observed Irf5-/- phenotypes in vivo, Irf4-/- mice demonstrate increased susceptibility to endotoxic shock due to increased levels of TNF, IL-12 and IL-6, and exhibit faulty TH2 responses to Nippostrongylus brasiliensis helminth

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infection (105, 106). Interestingly, when Irf4-/- mice are crossed with autoimmune C57BL/6-(Fas)lpr mice to study lupus, there is an increased activation of antigenpresenting cells resulting in a massive increase in plasma levels of TNF and IL12p40, despite overall protection from glomerulonephritis (107). Consistent with this, ectopic expression of IRF4 in Irf4-/- bone marrow derived macrophages can rescue the expression of characteristic M2 markers such as Arg1, Chi3l3, Fizz1 and Mrc1

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(108), and ectopic expression of IRF5 in M2 macrophages can drive expression of M1 markers such as IL-12p40, IL-12p35 and IL-23p19 (22). It can therefore be said that IRF5 and IRF4 have mirroring transcriptional roles in modulating M1-M2 macrophage plasticity. This opposition of activity is thought to be due to the fact that

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both IRF4 and IRF5 can interact with the same region of MyD88 downstream of TLR signalling (109).

As levels of IRF4 remain the same throughout macrophage

polarisation, whereas IRF5 expression levels change when macrophages are

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polarised to an M1 phenotype (22), this suggests that IRF5 has to overcome the presence of IRF4 and the ‘default’ setting of an M2 phenotype in order to mediate its proinflammatory activity.

Interestingly, recent ChIP-seq studies have shown that

IRF4 is recruited to the Irf5 promoter in murine bone marrow-derived dendritic cells (110), and has been shown to negatively regulate Irf5 promoter reporter activities (111); suggesting yet another level of direct competition between the two transcription factors in myeloid cells. The key role of macrophages and cytokines in the RA joint suggests that dampening of IRF5 activity in synovial macrophages could provide new avenues for development of RA targeted therapies. IRF5 and IBD

ACCEPTED MANUSCRIPT The inflammatory bowel diseases (IBDs) are a heterogeneous group of chronicremittent, progressive inflammatory disorders of the gut. Patients present with debilitating symptoms such as bloody diarrhoea, intestinal cramping, and severe weight loss. Crohn’s Disease (CD), which can affect the entire gastrointestinal tract, and Ulcerative Colitis (UC), in which inflammation is limited to the colonic and rectal mucosae, are the most common forms of IBD (112). As with many autoimmune

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conditions, the aetiology of the IBDs is currently unknown, with environmental, host genetic factors, and the host’s microbiota understood to contribute to disease pathogenesis (112). Mutations in Card15/Nod2 evidence this point, as impaired bacterial recognition leads to microbial outgrowth, decreased mucosal barrier

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function, and compensatory pathologic inflammation (113, 114).

Most IBD cases have multifactorial causes, however, intestinal pathology can result

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from monogenetic defects such as Wiskott-Aldrich syndrome, NEMO deficiency, XIAP deficiency, and IPEX syndrome (primary immunodeficiencies) (115-120). Similarly, IL-10 and IL-10 receptor (IL10R) pathway deficiencies predispose to early onset IBD, which is hard to control with current therapies: patients have a poor prognosis except after haematopoietic stem cell transplantation therapy (121-123). Mice and humans lacking T regulatory cells are unable to generate peripheral

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tolerance to the microbiota, a process in which IL-10 keeps pro-inflammatory activities of leukocytes in check, and are predisposed to IBD (124, 125). SNPs in IL10 are also associated with UC in adults (126, 127). The association of mutations in the IL-23 and IL-17 pathways with IBD highlighted the role of IL-17-producing TH17

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cells in mucosal homeostasis, adding a new cell class to the TH1/TH2 paradigm, and new therapeutic targets (128, 129). IL-23 was shown to drive development of TH17 cells that promote intestinal barrier function, whilst mutations in the IL-23 pathway

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are known to be key drivers of CD and are strongly linked to UC (130-133). Targeted blocking of IL-17A in isolation in the clinic proved unsuccessful, in fact it increased the number of adverse events compared to placebo, whereas the neutralisation of IL23 with monoclonal antibodies protected mice from experimental colitis and now shows promise in human clinical trials (134-137). Thus our understanding of the genetic predisposition to IBD can be described as pertaining to bacterial sensing, barrier function, autophagy and impaired tolerance (112).

Gain of function Irf5 mutations that were first linked to SLE have now also demonstrated association to IBD – in particular UC and CD – as shown in Table 3. The rs77571059 CGGGG insertion haplotype was linked with moderate strength to

ACCEPTED MANUSCRIPT Belgian UC patients, and this was confirmed in an independent study in a North American cohort (138, 139).

A separate report indicated that the effect of the

rs77571059 indel is not to alter the methylation status of the Irf5 promoter in IBD, therefore increased Irf5 expression associated with this SNP is not due to a more open chromatin state (33). Weaker associations with IBD have also been described for the rs3807306 and rs4728142 SNPs - predominantly explained by an association

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with UC, with very weak association to CD (140). Studies in Chinese patients also weakly associated the rs3807306 SNP with CD at low frequencies (141), however in contrast to the previous study rs4728412 was strongly associated with CD (141, 142). The haplotype GGATT, comprising SNPs rs4728142, rs2004640, rs3807306,

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rs7808907, and rs1874328, was strongly associated with CD in the Han Chinese UC cohort, but was only present in a minority of cases tested (141).

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The specific effects of Irf5 mutations in IBD patients have not been thoroughly investigated to date. Based on the effects of gain of function mutations in other autoimmune diseases reviewed here, and the effect of mutations in the IL-10, IL-23 and NOD2 pathways, it appears likely that increased Irf5 expression will affect the response to TLR and NOD-like receptor (NLR) sensing of bacterial antigens. Increased signalling via TLRs and NLRs leads to higher expression of

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proinflammatory cytokines such as TNF and IL-1β, and decreased expression of IL10, which could combine to promote a pro-inflammatory environment and contribute to intestinal pathology (143, 144). As IRF5 was also demonstrated to generate TH1/TH17 responses in vitro (22), another likely explanation for the increased risk of

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IBD could be through preferential differentiation and maintenance of pathogenic IFNγ+ TH17 cells in the intestine driven by increased IL-12 and IL-23 expression by antigen presenting cells (129, 144-146). Limited murine models demonstrating a role

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for IRF5 in gut pathology have currently been described; however in line with a role for IRF5 in cytokine production in the gut, protease-activated receptor 1 (PAR1) has been shown to inhibit IRF5-driven IL-12/IL-23 secretion by gut macrophages, leading to suppression of mucosal TH1/TH17 responses in chronic gastritis mediated by Helicobacter pylori infection (147). Further in vivo work to investigate the role of IRF5 in the gut would be beneficial.

Conclusion This review has aimed to summarise what is currently known about regulation of Irf5 gene expression in autoimmune and inflammatory settings.

The combination of

ACCEPTED MANUSCRIPT GWAS studies with in vitro experiments and in vivo murine models of autoimmune disease enables a thorough study of the role of IRF5 in the immune system. There are a few limitations that should be considered when designing in vivo experiments for IRF5, in order to obtain the best quality data on the topic. Firstly, it is important to bear in mind that it was problematically identified in 2012 that some Irf5-/- colonies worldwide had developed a spontaneous genomic duplication and frame-shift

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mutation in the Dock2 gene, which had been inadvertently been bred to homozygosity and led to reduced expression of DOCK2 in the Irf5-/- mice (148). This was discovered when Irf5-/- mice were further backcrossed to C57BL/6 and their Bcell phenotype (no splenic marginal zone B-cells and a marked reduction in the Of interest, the reported

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percentage of mature B-cells) was lost (148).

monocyte/macrophage phenotypes of Irf5-/- mice appeared to not be affected. DOCK2 (Dedicator of Cytokinesis 2) is a hematopoietic cell-specific guanine

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exchange factor that mediates immune responses via Rac activation, and Dock2-/mice are defective in neutrophil, B-cell and T-cell migration due to defective chemokine receptor signalling, and have excessive TH2 responses due to increased expression of IL-4RA on T-cells (149-151). This led to conflicting results within the field, including data regarding Type I IFN and antibody production (152), and led to the necessity for rederivation of many Irf5-/- colonies in order to gain confidence in Of note, the

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observed phenotypes – particularly those associated with B-cells.

observations described for IRF5 in the MRL/lpr model of SLE have been verified as independent of Dock2 mutations (153). Irf5-/- mouse colonies should therefore now be routinely screened for Dock2 mutations.

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Secondly, there is also data to show that different strains of mice express different steady-state levels of Irf5 mRNA in their spleens, and within each strain female mice

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express higher basal levels of Irf5 mRNA than their age-matched male counterparts (154). It is therefore important to design experiments using mice of the same age and gender to ensure an equal background level of expression. Finally, there are fundamental differences in the Irf5 gene locus between mice and humans – in contrast to the human Irf5 gene, murine Irf5 is located on chromosome 6 and is primarily expressed as a full-length transcript with only a single splice variant (73). This suggests that although in vitro murine IRF5 protein can be activated by both TBK1 and MyD88 to form homodimers, and regulate transcription of Type I IFN and cytokine production like its human counterpart (73), there may be limitations or simplifications related to IRF5 activity in the mouse that are not accurate with the human system.

ACCEPTED MANUSCRIPT How the Irf5 locus itself is regulated transcriptionally still remains relatively elusive information; however it is clear that dysregulation of Irf5 expression in the immune system due to single nucleotide polymorphisms leads to susceptibility of patients to autoimmune disorders – the most significant associations being with Systemic Lupus Erythematosus. Further to this, there are also GWAS studies associating IRF5 with additional autoimmune diseases that were beyond the scope of this review. For

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example the rs2004640 and rs2280714 SNPs are weakly associated with Systemic Sclerosis (155, 156), the rs77571059 CGGGG indel is weakly associated with Primary Sjögren’s Syndrome (157), and the rs3807306 and rs4728142 SNPs are weakly associated with Multiple Sclerosis (158). In particular, the role of IRF5 in

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Multiple Sclerosis could warrant further study, as IRF5 can be induced in spinal microglia following peripheral nerve injury (159); and Irf5 SNPs can result in poor pharmacological responses of MS patients to IFNβ therapy (160). Interestingly, the

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opposite alleles of rs2004640 and rs77571059 to those associated with the TH1/TH17 diseases mentioned in this review have also been associated with susceptibility to the TH2 disease of asthma (161), which again highlights the importance of IRF5 in the macrophage plasticity paradigm. The disease scope for IRF5-based therapeutics is therefore wide, and could be a beneficial strategy, as specifically targeting a defined role of a transcription factor could rule out problems of off-target effects of

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therapy. For example, although anti-TNF biologics can be beneficial in numerous autoimmune diseases, they are not curative and therapeutic effects can be only partial. Complete blockade of TNF activity is not always beneficial, as the body requires some TNF to enable other more balanced immune responses to occur.

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However, blocking IRF5 may not be easy, as transcription factors are often considered “non-druggable”, but perhaps other steps, such as the regulation of IRF5 might be more tractable. In conclusion, further studies are warranted and strategies

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are required to target IRF5 activity by therapeutic intervention. Acknowledgements

The authors declare no conflicts of interest associated with this review article. All authors have read the journal’s authorship agreement and policy on disclosure of potential conflicts of interest. HLE, ALC and IAU all contributed to the writing of the article, and have reviewed and approved the manuscript. This work was funded by The Kennedy Institute Trustees’ Research Fund.

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ACCEPTED MANUSCRIPT Tables and Table Legends SNP

Gene region

R/P alleles

Population

Cases/Controls

Odds Ratio (95% CI)

Probability

Strength

Reference

rs752637

Promoter

G/A

European

1383/1614

0.80 (0.71-0.90)

p= 2.4x10-4

Weak

(49)

Norwegian

154/756

1.95 (1.50-2.53)

p= 3.75x10-7

Moderate

(162)

North American/Spanish/ Swedish/Argentine

1661/2508

1.47 (1.36-1.60)

p= 4.2x10

Strong

(61)

North American

370/462

1.68 (1.20-2.34)

South American/Spanish/ German/Italian

1488/1466

1.54 (1.39-1.71)

European

1383/1614

0.73 (0.65-0.81)

Japanese/Korean

277/201

1.31 (1.14-1.49)

T/G

3' UTR

A/T

European

1383/1614

rs10488631

3' UTR

C/T

European

1383/1614

rs10954213

3' UTR

G/A

rs77571059 (CGGGG in/del)

Promoter

South American/Spanish/ German/Italian South American/Spanish/ German/Italian

4/3 repeats

Swedish

p= 8x10-3

Weak

(163)

Strong

(62)

p= 4.3x10-8

Moderate

(49)

p= 8.3x10-5

Weak

(41)

p= 2.38x10

-16

0.87 (0.77-0.99)

p= 2.9x10-2

Weak

(49)

2.02 (1.72-2.38)

p= 7.8x10-18

Strong

(49)

M AN U

rs2280714

RI PT

Promoter

SC

rs2004640

-21

1488/1466

1.35 (1.21-1.51)

p= 3.49x10-7

Moderate

(62)

1488/1466

1.60 (1.44-1.80)

p= 1.58x10-19

Strong

(62)

485/563

1.69 (1.42-2.02)

p= 4.6x10-9

Strong

(46)

Table 1 - Association of SNPs in the Irf5 locus with SLE

TE D

Summary of GWAS studies shown to demonstrate association of the Irf5 locus with Systemic Lupus Erythematosus (SLE), including the population dynamics, number of cases and controls, odds ratio and P-values determined. alleles, CI = Confidence Interval.

AC C

EP

Moderate (^-6-8), Strong (^-9+)

R/P = Risk/Protective

P-value strength classification: Weak (^-3-5),

ACCEPTED MANUSCRIPT SNP

Gene region

R/P alleles

Population

Cases/Controls

Odds Ratio (95% CI)

Probability

Strength

Reference

rs729302

Promoter

A/C

Japanese (anti-CCP negative)

1942/1598

1.22 (1.09-1.35)

p= 4x10-3

Weak

(164)

Norwegian (non-erosive)

1140 (380 families)

N/A

p= 5.1x10-3

Weak

(83)

Spanish (anti-CCP negative, SE positive)

4620/3741

0.88 (0.83-0.94)

p= 6.5x10

-5

Weak

(84)

Swedish/Dutch (anti-CCP negative)

1530/881

N/A

p= 3.6x10-3

Weak

(165)

-3

Weak

Promoter

A/C

rs10488631

3' UTR

C/T

rs77571059 (CGGGG in/del)

3' UTR

Promoter

1140 (380 families)

N/A

Swedish/Dutch (anti-CCP negative)

1530/881

N/A

Swedish/Dutch (anti-CCP negative)

1530/881

N/A

European (anti-CCP positive)

6768/8806

1.25 (1.14-1.37)

Swedish (anti-CCP negative)

2300/1836

A/C

rs3807306

rs10954213

p= 4.2x10

Norwegian (RF negative/nonerosive)

G/A

4/3 repeats

Norwegian (RF negative/nonerosive)

1140 (380 families)

Swedish (RF negative/antiCCP negative)

2300/1836

RI PT

Promoter

T/G

1.27 (1.08-1.50)

Weak

p= 2.2x10

-3

Weak

(165)

p= 9.1x10-5

Weak

(165)

p= 2.8x10-6

Moderate

(166)

p= 4.1x10-3

Weak

(167)

-3

Weak

p= 1.4x10

(83)

N/A

1.29 (1.14-1.16) 1.27 (1.13-1.43)

(83)

p= 1.7x10

-3

SC

rs3757385

Promoter

M AN U

rs2004640

p= 8.04x10

-4

Weak

p= 7.9x10-5

Weak

-5

Weak

(167) p= 7.3x10

Table 2 - Association of SNPs in the Irf5 locus with RA

Summary of GWAS studies shown to demonstrate association of the Irf5 locus with Rheumatoid Arthritis (RA), including the population dynamics, number of cases and

TE D

controls, odds ratio and P-values determined. R/P = Risk/Protective alleles, CI = Confidence Interval. P-value strength classification: Weak (^-3-5), Moderate (^-6-8),

EP

Strong (^-9+)

Gene region

R/P alleles

Population

Cases/Controls

Odds Ratio (95% CI)

Probability

Strength

Reference

rs3807306

Promoter

T/G

Belgian (UC patients)

427/534

1.36 (1.12-1.64)

p=1.8x10-3

Weak

(138)

Belgian (UC patients)

427/534

1.50 (1.24-1.83)

p= 4.2x10-5

Weak

(138)

European (UC patients)

6687/19718

1.07 (1.03-1.11)

p= 1.74x10-8

Moderate

(168)

Belgian (UC patients)

427/534

2.42 (1.76-3.34)

p= 5.3x10-8

Moderate

(138)

AC C

SNP

rs4728142

rs77571059 (CGGGG in/del)

Promoter

Promoter

A/G

4/3 repeats

Table 3 - Association of SNPs in the Irf5 locus with IBD Summary of GWAS studies shown to demonstrate association of the Irf5 locus with Inflammatory Bowel Disorder (IBD), including the population dynamics, number of cases and controls, odds ratio and P-values determined. alleles, CI = Confidence Interval. Moderate (^-6-8), Strong (^-9+)

R/P = Risk/Protective

P-value strength classification: Weak (^-3-5),

ACCEPTED MANUSCRIPT Figure Legends Figure 1 - Alternatively spliced isoforms of IRF5 Irf5 gene structure showing intron and exon boundaries (not to scale), including the alternative promoter exons – Ex1A, Ex1B and Ex1C – at the 5’ UTR of the locus. Alternative splicing results in 9 different transcript variants of IRF5 (v1-v9), each with

missing regions of RNA following splicing events.

RI PT

deletions in different domains of the IRF5 protein structure – dashed lines indicate

Figure 2 – Autoimmune disease associated polymorphisms at the human Irf5 locus

Irf5 gene structure showing intron and exon boundaries (not to scale), including the

SC

alternative promoter exons – Ex1A, Ex1B and Ex1C – at the 5’ UTR of the locus. Approximate location of each single nucleotide polymorphism (SNP) in the Irf5 locus is indicated along the Irf5 gene structure, with the SNP number highlighted in bold,

AC C

EP

TE D

brackets (Risk>Protective).

M AN U

and the exact SNP location in (italics). Risk and protective alleles are indicated in

ACCEPTED MANUSCRIPT

Figure 1 – Alternatively spliced isoforms of IRF5

Irf5 gene Ex1A Ex1B

Ex1C

Ex2

Ex3

V3 V4

IRF5 protein

Ex8 Ex9

EP AC C

V9

Ex7

TE D

V5

V8

Ex6

M AN U

V2

V7

Ex4 Ex5

SC

V1

V6

3’ UTR

Coding region

RI PT

5’ UTR

DBD

IAD

SRR

DNA-Binding Domain

IRF Association Domain

SerineRich Region

AC C EP TE D

SC

Ex8

(7:g.128954671)

Ex7

rs2280714 (C>T)

Ex6

(7:g.128954129)

Irf5 Coding region

rs10488631 (T>C)

Ex4 Ex5

(7:g.128949373)

Ex3

rs10954213 (G>A)

Ex1C Ex2

RI PT

5’ UTR

(7:g.128947320_128947349)

SV-16 (48bp splice insertion) rs60344245 (30bp indel)

M AN U

(7:g.128940626)

rs3807306 (G>T)

(7:g.128939366)

Ex1A Ex1B

rs752637 (T>C)

(7:g.128938247)

rs2004640 (T>G)

(7:g.128937873_128937877)

Chromosome 7 Forward strand

rs77571059 (CGGGG indel 4>3)

(7:g.128937250)

rs3757385 (T>G)

(7:g.128933913)

rs4728142 (G>A)

(7:g.128928906)

rs729302 (A>C)

ACCEPTED MANUSCRIPT

Figure 2 – Autoimmune disease associated polymorphisms at the human Irf5 locus 3’ UTR

Ex9