Betsy J. Barnes

LPS-TLR4 Signaling to IRF-3/7 and NF-κB Involves the Toll Adapters TRAM and TRIF

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.

Virus-specific Activation of a Novel Interferon Regulatory Factor, IRF-5, Results in the Induction of Distinct Interferon α Genes

Interferon regulatory factor (IRF) genes encode DNA-binding proteins that are involved in the innate immune response to infection. Two of these proteins, IRF-3 and IRF-7, serve as direct transducers of virus-mediated signaling and play critical roles in the induction of type I interferon genes. We have now shown that another factor, IRF-5, participates in the induction of interferon A (IFNA) and IFNB genes and can replace the requirement for IRF-7 in the induction of IFNA genes. We demonstrate that, despite the functional similarity, IRF-5 possesses unique characteristics and does not have a redundant role. Thus, 1) activation of IRF-5 by phosphorylation is virus-specific, and its in vivo association with the IFNA promoter can be detected only in cells infected with NDV, not Sendai virus, while both viruses activate IRF-3 and IRF-7, and 2) NDV infection of IRF-5-overexpressing cells preferentially induced the IFNA8 subtype, while IFNA1 was primarily induced in IRF-7 expressing cells. These data indicate that multiple signaling pathways induced by infection may be differentially recognized by members of the IRF family and modulate transcription of individual IFNA genes in a virus and cell type-specific manner.

The Interferon Regulatory Factor, IRF5, Is a Central Mediator of Toll-like Receptor 7 Signaling

Interferon regulatory factors (IRFs) are critical components of virus-induced immune activation and type I interferon regulation. IRF3 and IRF7 are activated in response to a variety of viruses or after engagement of Toll-like receptor (TLR) 3 and TLR4 by double-stranded RNA and lipopolysaccharide, respectively. The activation of IRF5, is much more restricted. Here we show that in contrast to IRF3 and IRF7, IRF5 is not a target of the TLR3 signaling pathway but is activated by TLR7 or TLR8 signaling. We also demonstrate that MyD88, interleukin 1 receptor-associated kinase 1, and tumor necrosis factor receptor-associated factor 6 are required for the activation of IRF5 and IRF7 in the TLR7 signaling pathway. Moreover, ectopic expression of IRF5 enabled type I interferon production in response to TLR7 signaling, whereas knockdown of IRF5 by small interfering RNA reduced type I interferon induction in response to the TLR7 ligand, R-848. IRF5 and IRF7, therefore, emerge from these studies as critical mediators of TLR7 signaling.

Comparative analysis of IRF and IFN-alpha expression in human plasmacytoid and monocyte-derived dendritic cells

Plasmacytoid dendritic cells (PDC) produce high levels of type I IFN upon stimulation with viruses, while monocytes and monocyte‐derived dendritic cells (MDDC) produce significantly lower levels. To find what determines the high production of type I IFN in PDC, we examined the relative levels of IRF transcription factors, some of which play critical roles in the induction of IFN. Furthermore, to determine whether the differences could result from expression of distinct IFNA subtypes, the profile of IFNA genes expressed was examined. PDC responded equally well to stimulation with HSV‐1 and Sendai virus (SV) by producing high levels of type I IFN, whereas the MDDC and monocyte response to SV were lower, and neither responded well to HSV‐1. All three populations constitutively expressed most of the IRF genes. However, real‐time RT‐PCR demonstrated increased levels of IRF‐7 transcripts in PDC compared with monocytes. As determined by intracellular flow cytometry, the PDC constitutively expressed significantly higher levels of IRF‐7 protein than the other populations while IRF‐3 levels were similar among populations. Analysis of the profile of IFNA genes expressed in virus‐stimulated PDC, monocytes and MDDC demonstrated that each population expressed IFNA1 as the major subtype but that the range of the subtypes expressed in PDC was broader, with some donor and stimulus‐dependent variability. We conclude that PDC but not MDDC are uniquely preprogrammed to respond rapidly and effectively to a range of viral pathogens with high levels of IFN‐α production due to the high levels of constitutively expressed IRF‐7.

Review: On the Role of IRF in Host Defense

Transcription factors of the interferon (IFN) regulatory factor (IRF) family have been shown to play an essential role in the regulated expression of type I IFN genes, IFN-stimulated genes (ISG), and other cytokines and chemokines. Three members of the IRF family, IRF-3, IRF-5, and IRF-7, have been identified as acting as direct transducers of virus-mediated signaling. In infected cells, these factors are activated by phosphorylation on the serine residues, transported to the nucleus, where they bind to the promoters of IFNA and IFNB genes and tether histone transacetylases to the transcription complex enhanceosome. IFNB and IFNA subtypes are expressed at different levels in infected cells. The ratio between the relative levels of IRF-3 and IRF-7 was shown to play an essential role in the inducible expression of type I IFN genes, whereas IRF-3 alone is sufficient for expression of the IFNB gene. IRF-5 was identified recently as another inducer of IFNA genes, which has two unique properties: (1) its activation is virus specific, and (2) the profile of IFNA genes induced by IRF-5 is distinct from that induced by IRF-7. Several viruses target functions of IRF to eliminate the early inflammatory response. Kaposis sarcoma herpesvirus (KSHV) encodes a cluster of four genes with homology to cellular IRF. Three of these vIRF were shown to inhibit induction of IFN genes and ISG in infected cells and function as dominant negative mutants of cellular IRF. The unique properties of previously uncharacterized vIRF-2 and vIRF-3 are discussed.

Multiple Regulatory Domains of IRF-5 Control Activation, Cellular Localization, and Induction of Chemokines That Mediate Recruitment of T Lymphocytes

ABSTRACT 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.

Two Discrete Promoters Regulate the Alternatively Spliced Human Interferon Regulatory Factor-5 Isoforms MULTIPLE ISOFORMS WITH DISTINCT CELL TYPE-SPECIFIC EXPRESSION, LOCALIZATION, REGULATION, AND FUNCTION

Interferon regulatory factor-5 (IRF-5) is a mediator of virus-induced immune activation and type I interferon (IFN) gene regulation. In human primary plasmacytoid dendritic cells (PDC), IRF-5 is transcribed into four distinct alternatively spliced isoforms (V1, V2, V3, and V4), whereas in human primary peripheral blood mononuclear cells two additional new isoforms (V5 and V6) were identified. The IRF-5 V1, V2, and V3 transcripts have different noncoding first exons and distinct insertion/deletion patterns in exon 6. Here we showed that V1 and V3 have distinct transcription start sites and are regulated by two discrete promoters. The V1 promoter (P-V1) is constitutively active, contains an IRF-E consensusbinding site, and is further stimulated in virus-infected cells by IRF family members. In contrast, endogenous V3 transcripts were up-regulated by type I IFNs, and the V3 promoter (P-V3) contains an IFN-stimulated responsive element-binding site that confers responsiveness to IFN through binding of the ISGF3 complex. In addition to V5 and V6, we have identified three more alternatively spliced IRF-5 isoforms (V7, V8, and V9); V5 and V6 were expressed in peripheral blood mononuclear cells from healthy donors and in immortalized B and T cell malignancies, whereas expression of V7, V8, and V9 transcripts were detected only in human cancers. The results of this study demonstrated the existence of multiple IRF-5 spliced isoforms with distinct cell type-specific expression, cellular localization, differential regulation, and dissimilar functions in virus-mediated type I IFN gene induction.

Signaling through IFN Regulatory Factor-5 Sensitizes p53-Deficient Tumors to DNA Damage–Induced Apoptosis and Cell Death

Human IFN regulatory factor-5 (IRF-5) is a candidate tumor suppressor gene that mediates cell arrest, apoptosis, and immune activation. Here we show that ectopic IRF-5 sensitizes p53-proficient and p53-deficient colon cancer cells to DNA damage-induced apoptosis. The combination IFN-beta and irinotecan (CPT-11) cooperatively inhibits cell growth and IRF-5 synergizes with it to further promote apoptosis. The synergism is due to IRF-5 signaling since a striking defect in apoptosis and cell death was observed in IRF-5-deficient cells, which correlated well with a reduction in DNA damage-induced cellular events. Components of this IRF-5 signaling pathway are investigated including a mechanism for DNA damage-induced IRF-5 activation. Thus, IRF-5-regulated pathways may serve as a target for cancer therapeutics.

Genetic variants and disease‐associated factors contribute to enhanced interferon regulatory factor 5 expression in blood cells of patients with systemic lupus erythematosus

OBJECTIVE Genetic variants of the interferon (IFN) regulatory factor 5 gene (IRF5) are associated with susceptibility to systemic lupus erythematosus (SLE). 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 IFNs. 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 polymerase chain reaction, minigene assay, and flow cytometry. Single-nucleotide polymorphisms rs2004640, rs10954213, and rs10488631 and the CGGGG insertion/deletion were genotyped in these patients. Genotypes of these polymorphisms defined both a common risk haplotype and a common protective haplotype. RESULTS IRF-5 expression and alternative splicing were significantly up-regulated in SLE patients compared with healthy donors. Enhanced transcript and protein levels were associated with the risk haplotype of IRF5; rs10488631 displayed the only significant independent association that correlated with increased transcription from the noncoding first exon 1C. Minigene experiments demonstrated an important role for rs2004640 and the CGGGG insertion/deletion, along with type I IFNs, in regulating IRF5 expression. CONCLUSION This study provides the first formal proof that IRF-5 expression and alternative splicing are significantly up-regulated in primary blood cells of patients with SLE. Furthermore, the risk haplotype is associated with enhanced IRF-5 transcript and protein expression in patients with SLE.

IRF-5 Is a Mediator of the Death Receptor-induced Apoptotic Signaling Pathway

The efficient and regulated response to cellular stress is coordinated by a genetic regulatory network in which a given transcription factor controls the expression of diverse target genes depending on the cell type and/or nature of the stimuli. The tumor suppressor p53 is thought to preferentially regulate the balance between cell survival and death. The interferon regulatory factor 5 (IRF-5), known to be involved in the innate immune response to pathogens, is also a critical regulator of DNA damage-induced apoptosis. Here, we provide direct evidence that IRF-5 promotes apoptosis upon signaling through tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptors (DR). We report that IRF-5 sensitizes tumor cells to TRAIL-induced apoptosis and cell death that is further enhanced by type I interferons. Cells deficient of IRF-5 gave a significantly diminished response to these agents. IRF-5 is involved in DR signaling upstream of caspase 8, in part because of an IRF-5-dependent increase in caspase 8 activation. We provide evidence that TRAIL induces a signaling cascade that leads to the phosphorylation and nuclear localization of IRF-5, resulting in transactivation of key DR signaling components. The results presented here identify IRF-5 as a new mediator of DR signaling and provides molecular insight into the mechanism of TRAIL-induced IRF-5 signaling.