Objective

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.

Methods

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.

Results

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.

Conclusion

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.

Recent evidence indicates a new role for histone deacetylases (HDACs) in the activation of genes governing the host immune response. Virus, along with other pathogenic stimuli, triggers an antiviral defense mechanism through the induction of IFN, IFN-stimulated genes, and other proinflammatory cytokines. Many of these genes have been shown to be regulated by transcription factors of the IFN regulatory factor (IRF) family. Recent studies from IRF5 knockout mice have confirmed a critical role for IRF5 in virus-induced type I IFN expression and proinflammatory cytokines IL-6, IL-12, and TNF-α; yet, little is known of the molecular mechanism of IRF5-mediated proinflammatory cytokine expression. In this study, we show that both HDACs and histone acetyltransferases (HATs) associate with IRF5, leading to alterations in its transactivation ability. Using the HDAC inhibitor trichostatin A, we demonstrate that ISRE, IFNA, and IL6 promoters require HDAC activity for transactivation and transcription, whereas TNFα does not. Mapping the interaction of corepressor proteins (HDAC1, silencing mediator of retinoid and thyroid receptor/nuclear corepressor of retinoid receptor, and Sin3a) and HATs to IRF5 revealed distinct differences, including the dependence of IRF5 phosphorylation on HAT association resulting in IRF5 acetylation. Data presented in this study support a mechanism whereby virus triggers the dynamic conversion of an IRF5-mediated silencing complex to that of an activating complex on promoters of target genes. These data provide the first evidence, to our knowledge, of a tightly controlled transcriptional mechanism whereby IRF5 regulates proinflammatory cytokine expression in conjunction with HATs and HDACs.

Introduction

New signaling pathways of the interleukin (IL) family, interferons (IFN) and interferon regulatory factors (IRF) have recently been found within tumor microenvironments and in metastatic sites. Some of these cytokines stimulate while others inhibit breast cancer proliferation and/or invasion. IRFs, a family of nine mammalian transcription factors, have multiple biologic functions that when dysregulated may contribute to tumorigenesis; most well-known are their roles in regulating/initiating host immunity. Some IRF family members have been implicated in tumorigenesis yet little is still known of their expression in primary human tumors or their role(s) in disease development/progression. IRF5 is one of the newer family members to be studied and has been shown to be a critical mediator of host immunity and the cellular response to DNA damage. Here, we examined the expression of IRF5 in primary breast tissue and determined how loss of expression may contribute to breast cancer development and/or progression.

Methods

Formalin-fixed paraffin-embedded archival breast tissue specimens from patients with atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) were examined for their expression of IRF1 and IRF5. Knockdown or overexpression of IRF5 in MCF-10A, MCF-7 and MDA-MB-231 mammary epithelial cell lines was used to examine the role of IRF5 in growth inhibition, invasion and tumorigenesis.

Results

Analysis of IRF expression in human breast tissues revealed the unique down-regulation of IRF5 in patients with different grades of DCIS and IDC as compared to IRF1; loss of IRF5 preceded that of IRF1 and correlated with increased invasiveness. Overexpression of IRF5 in breast cancer cells inhibited in vitro and in vivo cell growth and sensitized them to DNA damage. Complementary experiments with IRF5 siRNAs made normal mammary epithelial cells resistant to DNA damage. By 3-D culture, IRF5 overexpression reverted MDA-MB-231 to normal acini-like structures; cells overexpressing IRF5 had decreased CXCR4 expression and were insensitive to SDF-1/CXCL12-induced migration. These findings were confirmed by CXCR4 promoter reporter assays.

Conclusions

IRF5 is an important tumor suppressor that regulates multiple cellular processes involved in the conversion of normal mammary epithelial cells to tumor epithelial cells with metastatic potential.

Toll–IL-1–resistance (TIR) domain–containing adaptor-inducing IFN-β (TRIF)–related adaptor molecule (TRAM) is the fourth TIR domain–containing adaptor protein to be described that participates in Toll receptor signaling. Like TRIF, TRAM activates interferon regulatory factor (IRF)-3, IRF-7, and NF-κB-dependent signaling pathways. Toll-like receptor (TLR)3 and 4 activate these pathways to induce IFN-α/β, regulated on activation, normal T cell expressed and secreted (RANTES), and γ interferon–inducible protein 10 (IP-10) expression independently of the adaptor protein myeloid differentiation factor 88 (MyD88). Dominant negative and siRNA studies performed here demonstrate that TRIF functions downstream of both the TLR3 (dsRNA) and TLR4 (LPS) signaling pathways, whereas the function of TRAM is restricted to the TLR4 pathway. TRAM interacts with TRIF, MyD88 adaptor–like protein (Mal)/TIRAP, and TLR4 but not with TLR3. These studies suggest that TRIF and TRAM both function in LPS-TLR4 signaling to regulate the MyD88-independent pathway during the innate immune response to LPS.

Transcription factors of the interferon regulatory factor (IRF) family have been identified as critical mediators of early inflammatory gene transcription in infected cells. We recently determined that, besides IRF-3 and IRF-7, IRF-5 serves as a direct transducer of virus-mediated signaling. In contrast to that mediated by the other two IRFs, IRF-5-mediated activation is virus specific. We show that, in addition to Newcastle disease virus (NDV) infection, vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1) infection activates IRF-5, leading to the induction of IFNA gene subtypes that are distinct from subtypes induced by NDV. The IRF-5-mediated stimulation of inflammatory genes is not limited to IFNA since in BJAB/IRF-5-expressing cells IRF-5 stimulates transcription of RANTES, macrophage inflammatory protein 1β, monocyte chemotactic protein 1, interleukin-8, and I-309 genes in a virus-specific manner. By transient- transfection assay, we identified constitutive-activation (amino acids [aa] 410 to 489) and autoinhibitory (aa 490 to 539) domains in the IRF-5 polypeptide. We identified functional nuclear localization signals (NLS) in the amino and carboxyl termini of IRF-5 and showed that both of these NLS are sufficient for nuclear translocation and retention in infected cells. Furthermore, we demonstrated that serine residues 477 and 480 play critical roles in the response to NDV infection. Mutation of these residues from serine to alanine dramatically decreased phosphorylation and resulted in a substantial loss of IRF-5 transactivation in infected cells. Thus, this study defines the regulatory phosphorylation sites that control the activity of IRF-5 in NDV-infected cells and provides further insight into the structure and function of IRF-5. It also shows that the range of IRF-5 immunoregulatory target genes includes members of the cytokine and chemokine superfamilies.

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.

Objective

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.

Methods

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.

Results

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.

Conclusions

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.