Interferon Regulatory Factors in Immune Cell Development and Host Response to Infections

No part of this digital document may be reproduced, stored in a retrieval system or transmitted commercially in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

Chapter I

Angela Battistini Molecular Pathogenesis Unit, Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy

Abstract

The interferon regulatory factors (IRFs) constitute a family of nine cellular transcriptional regulators whose first two members, IRF-1 and -2, were originally identified in the context of the regulation of the virus-inducible enhancer-like elements of the human Interferon (IFN)- gene. Subsequent studies have revealed seven additional cellular members that are all critically involved in multiple aspects of cell physiology including regulation of cell growth and differentiation of haematopoietic cells, inflammation and immunity in response to pathogen- and danger-derived signals and regulation of oncogenesis. The distinct and not overlapping roles of each family member are thought to be the result of slightly different DNA binding specificity, pattern of expression and/or association with their partners in transcription. In this chapter, the present knowledge of the roles of these transcription factors in regulating immune cell development and function and in activating antiviral signaling pathways, are summarized. A brief overview of the mechanisms of viral evasion and subversion of IRF activity is also provided.

Introduction

IFNs constitute a heterogeneous family of proteins identified more than 50 years ago based on their ability to interfere with viral replication within host cells in a cell type-specific, but virus-non specific manner. Since their identification, cloning of genes, purification of the 2 Angela Battistini proteins, definition of IFN receptors and of signal transduction pathways triggered by ligand- receptors interactions, while aided the understanding of the molecular mechanisms of IFN action also revealed that many different stimuli in addition to viruses, can trigger IFN production. About ten distinct IFNs have been identified in mammals; seven of these have been described for humans. They are typically divided in three classes: Type I IFN, Type II IFN, and Type III IFN based on the specific cell surface receptor they bind. Type I IFN, that comprises IFN- constituted by several partially homologous genes, and IFN- represented by a single gene, are the best characterized and the most broadly expressed and represent the first line of defense against virus infections. They restrict virus replication and pave the way to activate the adaptive arm of the immune response. Upon binding to their specific receptors, they activate a cascade of signaling pathways leading to activation of transcription factors that translocate in the nucleus, bind to upstream sequence elements named IFN-stimulated response elements (ISRE), and activate the transcription of a large number of cellular target genes called Interferon-stimulated genes (ISGs) (Platanias LC et al. 2005; Decker T et al. 2005; Theofilopoulos AN, et al. 2005; Samarajiwa SA, et al. 2009). While some of these genes encode proteins that modulate viral replication, the effect of others, extend beyond the infected cell. Thus, in addition to their antiviral and antibacterial activity, IFNs have essential functions in disparate biological processes including immune cell development and function, regulation of cell growth and oncogenesis, inflammation and autoimmunity. Beside ISGs, are components of a family of transcription factors named Interferon Regulatory Factors (IRFs). This family is presently composed of nine mammalian members coded by distinct, but related genes. All IRF share a homologous DNA-binding domain and a less conserved C-terminus, that determines their transcriptional activity that can result in activation, repression or dual activity on target genes. Although IRFs were first characterized as transcriptional inducers of type I IFN and ISGs, gene-targeting studies performed for all IRFs have revealed the different roles played by each member of the family. The distinct and not overlapping functions of each family member are thought to be the result of slightly different DNA binding specificity within the broad IRF consensus sequence, patterns of expression and/or association with other regulators (Taniguchi T et al. 2001; Takaoka A et al. 2008). The first two members, IRF-1 and IRF-2, were initially identified as regulators of virus-inducible enhancer-like elements of the human IFN- gene. Subsequent studies, however, showed that IRF-1 is not essential for IFN gene expression, while IRF-3, IRF-7 and IRF-9 are more specifically involved in IFN induction and antiviral response. Other members, including IRF-1, IRF-2, IRF-4 and IRF-8, profoundly affect the development and function of various immune cells, while a distinct role of IRF-5 in inflammatory cytokines expression and of IRF-6 in epitelial differentiation has been defined (Takaoka A et al. 2008; Battistini A 2009; Savitsky D et al. 2010). As intracellular mediators of the induction and biological activities of IFNs, their central role in innate immunity to pathogens has recently been stressed by the elucidation of their crucial involvement in the regulation of gene expression in responses triggered by pattern recognition receptors (PRRs), also through IFN-independent mechanisms (Honda K and Taniguchi T 2006a). The recognition that some IRFs have also a crucial role in the development of various immune cells put these transcription factors at the interface between innate and adaptive immunity. Interferon Regulatory Factors in Immune Cell Development … 3

Here, I review present findings on the characteristics of these transcription factors and on their role in regulating immune cell development and function and activation of antiviral signaling pathways. Finally, I provide a brief overview of the principal mechanisms of viral evasion and subversion of IRF activity. Although not touched here, IRFs, specifically IRF-1, IRF-2, IRF-3, IRF-5 and IRF-8 also profoundly affect cell growth, survival, apoptosis and oncogenesis, as extensively reviewed elsewhere (Taniguchi T et al. 2001; Takaoka A et al. 2008; Savitsky D et al. 2010)

IRFs: Characteristics and Post-Translational Modifications

To date nine human cellular IRF genes (IRF2, IRF3, IRF4/PIP/LSIRF/ ICSAT, IRF5, IRF6, IRF7, IRF8/ICSBP, and IRF9/p48/ISGF3), and some virus-encoded analogues of cellular IRFs (v-IRF-1-4), have been identified (Taniguchi T et al. 2001; Takaoka A et al. 2008; Paun A and Pitha PM 2007). Their origin is phylogenetically linked with the appearance of multicellularity in animals (Nehyba J et al. 2009) and, interestingly, they coevolved with the rel/NF-B family with which they can cooperate in regulating gene expression, representing the major signaling system employed in innate immunity (Hiscott J 2007a; Ghosh S and Hayden MS 2008; Takaoka A et al. 2008). Based on phylogenetical analysis, the nine mammalian members of the family can be subdivided in four groups IRF-1- G (IRF-1, IRF-2), IRF-3-G (IRF-3, IRF-7), IRF-4-G (IRF-4, IRF-8, IRF-9), and IRF-5-G (IRF-5, IRF-6) (Nehyba J et al. 2009). IRFs share structural features consisting in a highly conserved N-terminal DNA binding domain (DBD) of around 115 aminoacids, that is characterized by a wing-type helix-loop- helix motif containing five tryptofan residues shared with the Myb DBD (Nguyen H et al. 1997). This domain is responsible for binding to DNA sequences termed the IFN-regulatory factor element (IRF-E) present in the IFN-β promoter whose consensus sequence G (A) AAAG/C T/C GAAA G/C T/C (Tanaka N et al. 1993) is almost indistinguishable from the interferon-stimulated response element (ISRE, A/GNGAAANNGAAACT) activated by IFN signaling (Darnell JE Jr et al. 1994). A subset of ISREs designated ETS/IRF response element (EIRE, GGAAANNGAAA) (Eisenbeis CF et al. 1995; Meraro D et al. 2002), or the IRF-Ets composite element (EICE, GGAANNGAAA) (Meraro D et al. 2002) and the IRF/ETS composite sequence with the polarity opposite of that in the EICE and EIRE (IECS, GAAANN[N]GGAA) Tamura T et al. 2005) are bound by selected IRFs, IRF-8 and IRF-4, in complexes with other cell type-specific trancription factors. The C-terminal portion varies among the members and acts as regulatory domain that classifies IRFs into two groups: those (IRF-3, IRF-5, IRF-7, IRF-9) that activate and those (IRF-1, IRF-2, IRF-4, IRF-8) that can either activate or repress gene transcription, depending on the target gene and on their partners in regulation of gene transcription (Taniguchi T et al. 2001). These interactions are mediated by two motifs named IRF-associated domain (IAD): IAD1, which was initially found in IRF-8 and is conserved in all IRFs apart IRF-1 and IRF-2, and IAD2 that is, instead, present only in IRF-1 and IRF-2. Through these domains IRFs can associate with other IRFs or other transcription factors and coactivators of transcription. The formation of these complexes modulates the ability of IRFs to bind DNA target sequences and activate gene transcription (Sharf R et al. 1997; Meraro D et al. 1999). IRF-8 can form multiple protein complexes with both IRF-1 and IRF-2 resulting in increased binding activity 4 Angela Battistini to ISRE (Sharf R et al. 1997) and with IRF-4 and PU.1, a member of the ETS family, regulating gene expression in a cell-type-specific manner (Brass AL et al. 1996). IRF-9 acts as a DNA-binding subunit that associates with signal and activator of transcription (Stat) 1 and Stat2 to form the ISGF3 heterodimeric factor in response to type I IFN signaling (Veals SA et al. 1992). IRF-1, IRF-3 and IRF-7 form part of large protein complex that include CREB-binding protein (CBP/p300), NF-B, AP1, participating in the formation of the IFN- enhanceosome/DRAF1 (Wathelet MG et al. 1998; Harada H et al. Cell 1990). Similarly, IRF- 1 and IRF-2 form a complex with multiple histone acetylases, PCAF, CBP, and p300/CREB to bind to ISRE (Masumi A et al. 1999). Depending on the promoter and on the nature of protein-protein interaction these complexes can serve as activators or repressors (Eisenbeis CF et al. 1995; Harada H et al. Cell 1990; Nelson N et al. 1993; Zhang L et al. 1997; Vaughan PS et al. 1995). Most of these interactions are regulated by different and multiple post-translational modifications that have a big impact on IRF activities and that include acetylation, phosphorylation, sumoylation and ubiquitination . Both IRF-1 and IRF-2 are acetylated in vitro by p300 and to a lesser degree by p300/CBP-associated factor (PCAF) (Masumi A et al. 2001). Two lysine residues in the DBD of IRF-2, Lys-75 and Lys-78, are the major acetylation sites. Acetylation of IRF-2, leads to inhibition of histone acetylation by p300, suggesting a possible mechanism for transcriptional repression mediated by IRF-2. Mutation of Lys-75, indeed, diminished the IRF-2-dependent activation of histone H4 promoter activity, although acetylation of IRF-2 did not alter DNA binding activity in vitro. The fact that IRF-2 was shown acetylated only in growing NIH 3T3 cells, but not in growth-arrested cells, suggests that this modification plays a role in the transforming activity of IRF-2 (Masumi A et al. 2003). Lys-78 is a residue located within a region important for ISRE binding activity and is conserved throughout the IRF family. Interestingly, the equivalent residue in IRF-7 at Lys- 92 was shown to be acetylated in vivo and in vitro by the histone acetyltransferases PCAF and GCN5. However, intriguingly acetylation of Lys- 92 seems to negatively modulate IRF-7 DNA binding (Caillaud A et al. 2002). IRF-3 is acetylated when incorporated into the large holocomplex that includes p300 and a p300 lacking histone acetylase activity failed to confer IRF-3 DNA binding activity, indicating that acetylation of IRF-3 is important for the DNA binding activity of the IRF-3 holocomplex (Suhara W et al. 2002). Acetylation within DBD of both IRF-9 and STAT2 is also critical for the ISGF3 complex activation and antiviral gene regulation (Tang X et al.2007). Phosphorylation, at both serine/threonine and tyrosine residues, also deeply affects IRF activity and is a major mechanism governing IRF-3-, IRF-5- , IRF-7-mediated induction of type I IFN and ISGs as well as IRF-1-, IRF-2- and IRF-8-protein-protein interactions and formation of heterodimers. Post-translational modifications of IRF-1 have been initially suggested in studies on viral-induced human IFN- gene transcription (Watanabe N et al. 1991). Subsequently, it has been reported that the serine/threonine, casein kinase II, directly interacted with IRF-1 and IRF-1 was phosphorylated at two clustered sites, one located in the DBD between amino acids 140-150, the other in the C-terminal acidic activation domain between amino acids 219- Interferon Regulatory Factors in Immune Cell Development … 5

230. Mutations of the four phosphoaceptor residues in the C-terminal transactivation domain, significantly decreased transactivation by IRF-1 (Lin R and Hiscott J 1999a) IRF-2 is phosphorylated in vivo and several potential phosphorylation sites for a variety of serine/threonine protein kinases have been identified (Birnbaum MJ et al. 1997). Recombinant casein kinase II also phosphorylates IRF-2 at the C-terminal end (Lin R and Hiscott J 1999a). Serine phosphorylation of IRF-8 at a unique serine residue within its IAD has also been reported, upon the association with Trip15 a component of the COP9/signalosome complex. The phosphorylated residue is essential for efficient association with IRF-1 and thus for the repressor activity exerted by IRF-8 on IRF-1 (Cohen H et al. 2000). Tyrosine phosphorylation of IRF-1, IRF-2 and IRF-8 has also crucial importance in regulating the activity of these IRFs. IRF-8 and IRF-2 are constitutively phosphorylated at tyrosine residues in vivo in the DBD, whereas Tyr-phosphorylated IRF-1 is induced by type II IFN. This modification negatively regulates IRF-8’own DNA binding activity but it is essential for the formation of heterodimers with IRF-1 and IRF-2. Dephosphorylated IRF-1 and IRF-2 bind DNA less effectively, whereas dephosphorylated IRF-8 becomes an active repressor that binds directly to the DNA and down-regulates IFN-stimulated gene expression. Tyrosine residues 110 in the DBD and 211 in the IAD are important for the formation of IRF- 8 heterodimers with IRF-1 and IRF-2, while mainly tyrosine residue 48, which is shared with IRF-4, affects the ability of IRF-8 to to synergize and form an efficient heterocomplex with PU1 (Meraro D et al. 2002; Sharf R et al. 1997). Phosphorylation of tyrosines 220 and 262 within the IRF-2 IAD also facilitates its interaction with IRF-8 (Meraro D et al. 1999). Thus, phosphorylation events in both the DBD and the IAD may be responsible for the modulation of IRF activities either by promoting interaction with other transcription factors or by enhancing or preventing binding to target DNA sequences. Moreover, regulation of IRF-8 transcriptional activity may vary depending on the cell type in which is expressed and the different heterocomplexes that can form with tissue- restricted factors. In this respect, the impact of tyrosine phosphorylation of IRF-1, IRF-2 and IRF-8 in allowing the formation of transcriptionally active heterocomplexes has also been well defined in the context of hematopoietic cytokines-induced myeloid differentiation. Phosphorylation of IRF-8 tyrosines 92 and 95 have been defined as necessary for interaction of IRF-8 with PU.1 and IRF-1 at composite ETS/IRF sequences in the CYBB and NCF2 genes that mediate stage-specific transcriptional activation in differentiating myeloid cells (Eklund E et al. 1998; Kautz B et al. 2001). A conserved tyrosine residue Tyr-109, in the IRF domain of IRF-2 is also required for IRF-8 interaction with the NF1 gene-cis elements-bound PU.1-IRF-2 heterodimer and activationn of transcription (Huang W et al. 2007; Huang W et al. 2006 Zhu C et al. 2004). Consistently, tyrosine phosphorylation may also mediate the leukemia-suppressing effects of IRF-8 as it has been shown that activation of SHP1/SHP2 protein tyrosine phosphatases inhibited expression of myeloid specific genes and accelerated progression to leukemia (Kautz B et al. 2001; Konieczna I et al. 2008). Serine/threonine phosphorylation of IRF-3, IRF-5 and IRF-7 is a crucial event in their activation and is mediated by two related kinases, the inhibitor of B kinase (IKK)and the TANK-binding kinase 1 (TBK1), whose role in signaling pathways arising from activation of pathogen sensors has been extensively reported (see a latter section). These kinases phosphorylate different Ser/Thr residues at the C-terminal portion of the proteins (Fitzgerald KA et al. 2003a; Sharma S et al. 2003). IRF-3 has two phosphorylation sites earlier 6 Angela Battistini recognized, the first includes Ser385 and 386, and the second a cluster located between amino acids 396 and 405 (Yoneyama M et al. 1998; Lin R et al. 1998b; Hiscott J 2007b). Phosphorylation at Ser339 has also recently been reported as important in generating a hyperactivated IRF-3 (Clement JF et al. 2008). Crystal studies for IRF-3 have supported a model of activation that foresees that C-terminal phosphorylation induces a conformational change in the autoinhibitory domain of the protein, that allows homo- or heterodimeration, nuclear localization, association with the co-activator CBP/p300 and binding to target sequences to stimulate promoter activation (Lin R et al. 1999b; Kumar KP et al. 2000; Qin BY et al. 2003). Like IRF-3, IRF-7 transcriptional activity depends on phosphorylation of Serine residues in the C-terminal region (Lin R et al. 2000a; Caillaud A et al. 2005; tenOever BR et al. 2004). Pathogenic infection triggers IRF-7 phosphorylation and formation of homo or heterodimers with IRF-3 that translocate into the nucleus, where, upon binding to coactivators, activate transcription of target genes (Yang H et al. 2003; Whatelet MG et al.1998). In line with the model suggested for IRF-3 activation, a general model where, in the absence of a virus signal, through intramolecular interactions the inhibitory domain (ID) maintains IRF-7 as a “closed” structure efficiently masking the N-terminal DBD, the transactivation domains and the C-terminal signal response domains, thereby preventing the downstream activation, has been suggested (Lin R et al. 2000a). Accordingly, deletion of an autoinhibitory domain of IRF-7 (human IRF-7 aa 247-467) generates a constitutively active form of IRF-7. Similarly, Serine 471/472 and 477/479 appear to be important for activation since substitution of the serine 477/479 with the phosphomimetic Asp also leads to a consitutively active form of IRF-7 (tenOever BR et al. 2004). The role of phosphorylation in IRF-5 activity is still not well understood. A serine cluster is present at the C-terminal of IRF-5, potential target of kinases; however, the exact definition of kinases and specific residues interested has proven difficult also due to the many isoforms described for this protein (Mancl ME et al.2005). Studies on IRF-5 activation during viral infection have provided varied results. Activation reported for Newcastle disease virus (NDV) infection, vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1) (Barnes BJ et al. 2002b) was not confirmed. It has been reported that expression of TBK1 or IKKε kinases increases IRF-5 phosphorylation and promotes its dimerization, nuclear accumulation, binding to CBP/p300, and binding to DNA, as is the case of IRF-3 and IRF-7. The exact sites on IRF-5 that are phosphorylated by TBK1 or IKKε remain, however, to be determined (Cheng TF et al. 2006). Recently, crystal structure studies indicated that phosphorylation is likely to activate IRF-5 by triggering conformational rearrangements that switch the C-terminal segment from an autoinihibitory to a dimerization role allowing nuclear translocation and binding to DNA as, already suggested for IRF-3 and IRF-7 (Chen W et al. 2008)). A similar function of an inhibitory domain of IRF-4 has been reported (Brass AL et al. 1996) thus supporting this model as a general mechanism for activation of IRFs. Recently, IKKα has been indicated as the kinase that phosphorylates IRF-5 in the TLR7/9-MyD88-dependent signaling (see a latter section). However, this modification results in negative regulation of IRF-5 activation. Phosphorylation of IRF-5 by IKKα, indeed, inhibits TRAF6 mediated K63 ubiquitination that is required for its nuclear translocation and is essential for IRF-5 activity (Balkhi MY et al. 2008; Balkhi MY et al. 2010). The functional significance of these observations in a physiological setting remains yet to be determined and still requires investigations. Interestingly, upon triggering of the same pathway during Interferon Regulatory Factors in Immune Cell Development … 7

TLR7/9-mediated dendritic cell activation, IKKα is instead required for activation of IRF-7 (Hoshino K et al. 2006). IRF-6 phosphorylation is induced by cell proliferation. Kinase(s) involved in IRF-6 modification has not been identified and phosphorylated IRF6 is not readily observed in the nucleus suggesting that IRF-6 may not follow stereotypical IRF activation pathways. IRF-6 phosphorylation, instead, has revealed to be a signal for its proteasome-dependent degradation. These observations in mammary epithelial cells support a model in which IRF-6, in collaboration with maspin, promotes mammary epitelial cell differentiation by regulating exit from the cell cycle and entry into the G(0) phase of cellular quiescence (see below) (Bailey CM et al. 2008a,b). Phosphorylation of some IRFs thus not only renders them active but may also serve as a signal for proteasomal degradation to extinguish IRF-mediated responses by regulating their expression (Higgs R and Jeffrey CA 2008). In this respect, it has become clear that ubiquitin-mediated degradation of IRF-3 and IRF-7 is an effective mechanism to limit type I IFN production. Several endogenous proteins have been described that, after viral infection, contribute to polyubiquitination and subsequent proteosomal degradation of these IRFs (Lin, R., et al. 1998; Nakagawa K et al. 2006; Xiong H et al. 2005; Higgs R and Jefferies CA 2008). These include Ro52, Pin1, RBCK1, and Cul- 1. In particular, peptide-prolyl isomerase Pin1 was shown to interact with phosphorylated IRF-3 at Ser339 following double-stranded (ds)RNA stimulation. This interaction leads to polyubiquitination and then proteasome-dependent degradation of IRF-3 (Saitoh T et al. 2006). In Sendai virus-infected cells polyubiquitination of IRF-3 is mediated in part by a Cullin-based ubiquitin ligase and is dependent on TBK-1-mediated phosphoryaltion (Bibeau- Poirier A et al. 2006). The E3 ubiquitin ligase Ro52 a member of the TRIM family of single- protein E3 ligases (Meroni G and Diez-Roux G 2005) is similarly directly responsible for ubiquitintaing and targeting of IRF-3 and IRF-7 promoting their degradation (Higgs R et al. 2010). Interestingly, this ligase also targets IRF-8 but on the contrary, enhancing its activity (Kong HJ et al. 2007). Ubiquitination indeed, does not always mediate degradation, the specific lysine targeted to form polyubiquitin chains appearing critical in determining the functional effects of this modification: Lys48-linked polyubiquitination generally results in proteosomal degradation whereas Lys 63-linked polyubiquitination have a regulatory role that does not involve proteolysis (Malynn BA and Ma A 2010). Recently, the ubiquitin E3 ligase RAUL was added to the list of ligases that can directly catalize lysine 48-linked polyubiquitination of both IRF-3 and IRF-7 inducing their proteosomal-mediated degradation (Yu Yet al. 2010). Ubiquitin-mediated activation of IRF-7 has also been reported. IRF-7 activity is increased following Lys63-linked ubiquitination mediated by TRAF6, a E3 ligase in the TLR pathway, and by Rip1 a E3 ligase stimulated by the Epstein-Barr virus viral oncoprotein LMP1 (Kawai T et al. 2004; Huye LE et al. 2007). Like IRF-3 and IRF-7, IRF-1 and IRF-8 are regulated by ubiquitin-mediated proteosomal degradation and the carboxyl-terminal domain of the proteins controls their ubiquitination (Nakagawa K and Yokosawa H 2000; Xiong H et al. 2005). A role for discrete motifs in the enhancer domain of IRF-1 in directing polyubiquitination and degradation has also been precisely defined. This region is not subject to modification by ubiquitin, but rather it contains both an ubiquitination signal and a distinct degradation signal. Removal of the C- terminal 70 amino acids from IRF-1 inhibits both its degradation and polyubiquitination, whereas removal of the C-terminal 25 amino acids inhibits degradation of the protein but does 8 Angela Battistini not prevent its ubiquitination (Pion E et al. 2009). Very recently, it has been identified the E3 ubiquitin ligase CHIP as able to form a complex with IRF-1 and mediate its proteosomal degradation under stress conditions (Narayan V et al. 2011). The E3 ligase Cbl induces ubiquitination-mediated degradation of IRF-8 (Xiong H et al. 2005) in contrast with the Ro52-mediated ubiquitination that, instead, as mentioned above, increases IRF-8 activity (Kong HJ et al. 2007). Recently, IRF-2 has been recognized as a substrate of the murine double minute 2 (MDM2) a member of the RING finger family of ubiquitin-protein ligase E3. Interestingly, this ligase is best known as a specific and crucial negative regulator of p53 (Pettersson S et al. 2009). Thus, even if the physiological significance of the interaction between MDM2 and IRF-2 has not yet been defined, it is conceivable that it impacts on IRF-2 functions in growth control and immunity. Recent evidences in our laboratory suggest that also IRF-1 can serve as specific substrate of MDM2 E3 ligase and the HIV-1 transactivator Tat protein may favour the IRF-1-MDM2 interaction leading to accellerate IRF-1 proteosomal-mediated degradation in infected cells (Remoli AL and Battistini A, unpublished work). In addition to cellular proteins, also viral proteins can act as E3 ubiquitin ligases and target IRFs for degradation, thus limiting their antiviral activity or instead activating them and these effects are described in more detail in a latter section. Regulation of IRF activity is also dependent upon SUMOylation. SUMOylation is the covalent attachment of SUMO, the small ubiquitin-like peptide, to lysine residues of targeted substrates and represents another important post-translational control of function of several protein including transcription factors (Gill G 2003; Johnson ES 2004; Hay RT 2005). In mammals, four different isoforms, termed SUMO-1, -2, -3 and -4, have been identified so far. SUMO-2/3 are 95% identical to each other and 50% identical with SUMO-1. Like ubiquitination, SUMOylation requires three-step enzymatic reactions that involve an activating enzyme E1 (SAE1/SAE2), a conjugating enzyme E2 (Ubc9), and different E3 SUMO ligases (Bettermann K et al. 2011; Praefcke GJ et al. 2011). SUMOylation, in general, has been found to inhibit transcription factor activities by several means including changes in protein stability, in localization, or promotion of interactions with transcriptional repressors (Hay RT 2005; Gill G 2005; Garcia-Dominguez M and Reyes JC 2009). Among the IRFs, IRF-1, -2, -3 and -7 have been reported as modified by sumoylation. While SUMOylation of IRF-1 inhibits its activity by sequestering IRF-1 into nuclear bodies, SUMOylation of IRF-2 does not modify its nuclear localization but, instead, increases its ability to inhibit IRF-1 transcriptional activity (Nakagawa K et al. 2002; Park SM et al. 2010; Kim EJ et al. 2008; Han KJ et al. 2008). SUMOylation of IRF-7 and IRF-3 is dependent on the activation of Toll-like receptor and RIG-I pathways but not on the IFN-stimulated pathways. However, SUMOylation of IRF-3 and IRF-7 was not dependent on their phosphorylation. This modification suppresses their transactivation activities and IFN induction and thus is an integral part of IRF-3 and IRF-7 activity that contributes to post- activation attenuation of IFN production through inhibition of IFN transcription (Kubota T et al. 2008). Up to now, considerably fewer SUMO E3 ligases than ubiquitin E3 ligases have been discovered. Recently, it has been identified TRIM28 as the SUMOE3 ligase specific for IRF- 7 that targets lysine 444 and lysine 446 of the protein and has little effect on the closely related IRF-3 (Liang Q et al. 2011). Conversely, the general SUMO E3 ligase PIAS1 has been shown to display no preference between IRF-3 and IRF-7 (Chang TH et al. 2009). Interferon Regulatory Factors in Immune Cell Development … 9

Interestingly, it has recently been reported that the SUMOylated Lys residues in IRF-7 are the same residues involved in TRAF6-mediated K63-linked ubiquitination that instead, as mentioned above, leads to IRF-7 activation. Since both ubiquitination and SUMOylation are reversible they could act as competitive mechanisms of IRF-7 modulation depending on cells and stimuli (Liang Q et al. 2011).

IRF-1: The Multifaceted IRF

Among IRFs, IRF-1 has revealed to be the most multifunctional as it is implicated in the regulation of a broad spectrum of biological functions including hemaetopoietic differentiation, development and activation of immune cells, antiviral and antibacterial responses and regulation of cell cycle and apoptosis in response to a variety of genotoxic stresses (Taniguchi T et al. 2001; Takaoka A et al. 2008; Savitsky D et al. 2010). IRF-1 was initially isolated by its affinity to specific target sequences in the virus- inducible enhancer-like elements of the human IFN- gene promoter (Fujita T et al. 1988). Subsequent studies, in IRF-1 null mice, showed that IRF-1, even if able (Fujita T et al. 1989a; Pine R et al. 1990) was not essential for type I IFN gene expression (Matsuyama T et al.1993; Ruffner H, et al. 1993). IRF-1 instead exerts a variety of non-redundant functions in several aspect of cell physiology depending on the cell type and state of differentiation, the nature of the stimulus and its partners in transcription. IRF-1 is expressed at low basal levels in all cell types examined, with the exception of early embryonal cells, and a variety of stimuli can induce IRF-1 mRNA accumulation including type I and type II IFNs, ds RNA, cytokines and hormones (Fujita T et al. 1989b). The remarkable IRF-1 functional diversity in regulating cellular responses reflects the differential modulation of distinct sets of genes implicated in antiviral and antibacterial as well as antiproliferative, antiapoptotic, antioncogenic and immunomodulatory responses (Kröger A et al. 2002). IRF-1 is manly a weak transcriptional activator and so far only two gene promoters have been shown to be repressed by IRF-1, the IL-4 promoter in response to type II IFN and the Foxp3 promoter during T regulatory cell differentiation (Elser B et al. 2002; Fragale A et al. 2008). IRF-1 activity is primarily regulated at the transcriptional level that dictates the abundance of the protein that is very instable with a very short half-life of around 30 minutes. However, posttranscriptional modifications including phosphorylation and proteasome- mediated degradation as well as interactions with other transcriptional factors or cofactors, as described above, also dictate specific activities (Taniguchi T et al. 2001; Kröger A et al. 2002). IRF-1 contributes to the host defense by different mechanisms: by affecting immune cell development, by modulating innate and adaptive immune responses and by direct induction of antiviral and antibacterial genes.

IRF-1 in Regulation of Immune Cell Development Several studies have implicated IRF-1 in the development and function of multiple cell populations of the immune system. It affects NK and Dendritic cell development and activities, T cell selection and maturation, as well as leukomogenic development (Taniguchi T et al. 2001; Takaoka A et al. 2008; Savitsky D et al. 2010; Battistini A 2009). Earlier studies in mice deficient in IRF-1 showed a marked impairment of CD8+ T and NK cell maturation, impaired IL-12 production in multiple cell types, and exclusive T helper type 10 Angela Battistini

II (Th2) differentiation associated with defects in T helper type I (Th1) responses (Matsuyama T et al.1993). In developing tymocytes, IRF-1 regulates the expression of genes important for positive selection of CD8+ T cells and for their functionality as TAP1 and LMP2 (Penninger JM et al. 1997; White LC et al. 1996). IRF-1 has also a highly complex role in the differentiation of Th cells. Although naive CD4+ T cells develop in IRF-1-/- mice, IRF-1 is indispensable for the differentiation of Th1 cells (Lohoff M et al. 1997; Taki S et al. 1997). The expression of a number of genes pivotal in Th1 differentiation is, indeed, affected by IRF-1, which has therefore been called a “super” Th1 cell transcription factor (Lohoff M and Mak TW 2005). These genes include p40 and p35 subunits of Interleukin (IL)-12, a cytokine essential for Th1 differentiation and direct target of IRF-1 transcriptional activity (Maruyama S et al. 2003; Kollet JI and Petro TM. 2006). Impaired production by macrophages and DCs of IL-12, may, thus, account for the lack of Th1 differentiation in IRF- 1 deficient mice (Lohoff M et al. 1997; Taki S et al. 1997). Interestingly, in T and NK cells, IRF-1 itself is a target of IL-12 (Coccia EM et al. 1999a; Galon J et al. 1999) and in turn it controls and is indispensable for the expression of the gene encoding the IL-12 receptor 1 (Kano S et al. 2008). Moreover, IRF-1 has also been shown to repress IL-4 gene transcription and production of IL-4 (Elser B et al. 2002), the key cytokine in Th2 differentiation, thereby directly inhibiting differentiation of Th2 cells. IRF-1 is also substantially induced by IFN- (Pine R et al. 1994; Coccia EM et al. 2000) that, in antigen presenting cells, leads to increased production of IL-12 by NK cells, a source of IFN- during infection, and in turn stimulates IL-12 production by macrophages. Thus, IRF-1 participates with IFN- and IL-12 in an autocrine positive amplification loop that both induces and perpetuates the Th1 response, while inhibits Th2 differentiation. The effect of IRF-1 on T cell differentiation may, however, be elicited in part also through its ability to support the differentiation and function of specific dendritic cell (DC) subsets that direct Th differentiation. Recent data from our laboratory showed that IRF-1 is involved in the development and function of specific subsets of DC whose cytokine profile preferentially drives a Th1 polarization (Gabriele L et al. 2006). The existence of distinct subsets of Dc that can be distinguished on the basis of phenotypic and functional features, localization and differentiation status (Shortman K and Liu YI 2002; Ardavin, C. 2003; Belz GT et al. 2012) accounts for their functional diversity in evoking immune responses. In this respect, the role of DCs is dichotomous in that they may both present antigens and the appropriate stimulatory molecules to initiate an adaptive immune response, or they may induce tolerance and release anti-inflammatory signals.. IRF-1-/- mice have reduced numbers of CD8+ DC subset that fails to undergo full maturation in response to viral or bacterial stimuli and are unable to stimulate the proliferation of allogeneic T cells. IRF-1-/- DCs present, instead, a tolerogenic cytokine profile (Gabriele L et al. 2006). This may account for the impaired responses of these mice to infectious agents and for their reduced susceptibility to induction of autoimmune disorders. Consistently, IRF-1 has been also implicated in the generation of T regulatory (Treg) cells, a subset of CD4+ T cells critical in inducing and maintaining tolerance. Mice devoid of IRF-1 show a selective and marked increase in the thymus and in all peripheral lymphoid organs of highly activated and differentiated CD4+CD25+Foxp3+ Treg cells. The molecular basis of these effects relies on the binding of IRF-1 to a highly conserved IRF-E sequence in the promoter of Foxp3 gene the unique marker of Treg cells and on negative regulation of promoter transcriptional activity (Fragale Interferon Regulatory Factors in Immune Cell Development … 11

A et al. 2008). Thus, IRF-1 may also be considered a critical element in the intimate crosstalk between Treg cells and tolerogenic DCs. Several studies have also evidentiated the role of IRF-1 in the differentiation of myeloid lineages. IRF-1 is expressed in immature myeloid bone marrow cells (Liebermann DA and Hoffman B 2002) and its expression significantly increases during granulocytic differentiation of both human and murine myeloid progenitors (Abdollahi A et al.1991; Coccia EM et al. 2001). Consistently, IRF-1-deficient mice show an increased number of immature granulocytic precursors associated with a decreased number of mature granulocytic elements in response to both G-CSF and M-CSF (Testa U et al. 2004).

IRF-1 in Regulation of Immune Responses The role of IRF-1 in host defense from pathogens is double: indirect through effects on differentiation of immune cells as described above, and on the stimulation of IFN expression and direct through transcriptional stimulation of antiviral genes, even in an IFN-independent manner. Despite the earlier proposed importance of IRF-1 in type I IFN gene regulation in response to viral infection was not supported by experiments on genetically modified mice (Matsuyama T et al. 1993; Ruffner H, et al. 1993), as mentioned above, the observation that IRF-1, together with IRF-3 and IRF-7, can be found in both the IFN-A and IFN-B enhanceosomes (Thanos D and Maniatis T 1995; Au WC et al. 1995) suggests that IRF-1 can play a role in IFN-induction. This apparent discrepancy has recently been solved in the context of the studies of the signaling pathways triggered by pathogen sensors that have showed that IRF-1 indeed induces IFN- expression, but in a cell type- and ligand-specific manner (Negishi H et al. 2006). In this respect, IRFs, in particular IRF-3, IRF-5 and IRF-7 gained, in the last years, much attention as essential regulators downstream of pathogen sensors named pattern recognition receptors (PRRs). These receptors are utilized by the innate immune system to sense pathogens or dangers through recognition of pathogen- associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). PRRs are present on the cell membrane or in the cytoplasm of both professional innate immune cells including macrophages and dendritic cells as well as in T cells and also in non professional cells such as epitelial and endothelial cells and fibroblasts that also contribute to innate immunity (Honda K and Taniguchi T 2006; Akira et al. 2006; Takeuchi O and Akira S 2010, 2011) Differences in the signaling programs triggered by distinct PRRs are associated with the different pathogens in order to elicit the appropriate immune response and disarm the specific pathogen. Several classes of PRRs have been described that comprise retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs), Toll-like receptors (TLRs), NOD-like receptors (NLRs), C-type lectins receptors and the IFI200 family member absent in melanoma 2 (AIM2). Whereas TLRs sense PAMPs in the extracellular space and endosomes, NLRs, RLRs and AIM2 function as pathogen sensors in intracellular compartments (Akira S et al. 2006; Robinson MJ et al. 2006; Sansonetti PJ 2006; Kawai T and Akira S 2010, 2011; Wilkins C and Gale M Jr. 2010; Barber GN 2011). Downstream of these molecules three major pathways are activated: the NF-B pathway, the mitogen-activated protein kinases (MAPKs) and the IRF pathway (Honda K and Taniguchi T 2006; Akira S et al. 2006; O’Neill LA and Bowie AG 2007). Type I IFN production in response to PRR signaling essentially 12 Angela Battistini depends on the presence and the activation of IRFs and the role of individual IRFs varies depending on the cellular source and the PRR used (Honda K and Taniguchi T 2006). TLRs, together with the cytosolic RIG-I-like receptors are the best-characterized class of PRR. Activation mediated by TLRs relies on two different pathways: one dependent on the adaptor protein MYD88 and the other related to the adaptor TRIF. MyD88 is recruited by all TLRs with the exception of TLR3, which instead, signals only through TRIF (Honda K and Taniguchi T 2006; O’Neill LA and Bowie AG 2007; Kawai T and Akira S 2010). IRF-1 has been shown to play a role in IFN induction in the MyD88–dependent signaling in myeloid DCs upon CpG-B-TLR9 engagement. As IRF-7 and IRF-5 (see latter section), IRF-1 interacts directly with the adaptor MyD88 and in IFN--treated DCs, TLR9 engagement results in IRF- 1 increased expression and post-translational modifications that allow optimal induction of a specific set of genes specifically up-regulated by IRF-1 including IFN-, iNOS and IL-12 p35 (Negishi H et al. 2006; Schmitz F et al. 2007). Thus, a ‘licensing’ process whereby interaction of IRF-1 with TLR9-activated MyD88 increases the rate of nuclear translocation of IRF-1 and transcriptional activation of target genes, relative to that noted with IFN--induced IRF-1 that has not been ‘licensed’ by MyD88, it has been proposed (Negishi H et al. 2006). Interestingly, a role of IRF-1 in IFN- expression has been described also in primary macrophages, in response to tumor necrosis factor (TNF) (Yarilina A et al. 2008). In these cells, TNF induces an autocrine loop characterized by low and sustained production of IFN- that acted in synergy with canonical TNF signals to induce sustained expression of genes encoding inflammatory molecules, together with classic interferon-response molecules, including those known to mediate antiviral response. This TNF-mediated IFN- production is essentially dependent on IRF-1. It is, however, worth noting that in contrast to PRRs that use IRF-3 or IRF-7 (see below), to induce massive but rapid and transient IFN- production in virus-infected fibroblasts and pDCs (Honda K et al. 2005), the IRF1-dependent type I IFN induction, in myeloid DCs on CpG-B-TLR9 engagement and in TNF-stimulated macrophages, is markedly weaker and delayed. Thus, even if it has to be established whether and how the small amounts of interferon induced contribute to overall interferon production in the setting of an infection, it can be postulated that IRF-1-MyD88-dependent activation of IFN- production may explain the antimicrobial synergism between IFN-γ and TLR, while the local production of TNF-stimulated IFN- in macrophages, may be important in regulating inflammation in noninfectious settings. IRF-1-mediated IFN- induction thus seems a tailored response in specific settings. IRF-1 may contribute to the innate immune response to infections mediated by PRRs also by regulating PRRs gene expression in response to type I IFN. TLR3, TLR4, TLR6, TLR9 and RIG-I gene promoters are all bound and transcriptionally regulated by IRF-1 (Guo Z et al. 2005; Nhu QM et al. 2006; Fragale A et al. 2011; Su ZZ et al. 2007). As for a direct role of IRF-1 in host defence, early studies already indicated that IRF-1 was able to induce typical IFN functions including antiviral state against several RNA viruses (Kimura T et al. 1994; Pine R. 1992), also consistent with the finding that enforced expression of IRF-1 prevented VSV, EMCV, and HCV replication in an IFN-independent way (Pine R. 1992; Kanazawa N, et al. 2004). This phenotype was not a consequence of IFN production but was dependent on direct induction of a set of ISGs affecting viral replication (Chang CH, et al. 1992; Coccia E.M., et al. 1999b; Kirchhoff S et al. 1995). Several primary IRF-1 target genes have, since then, been recognized (Kroeger A et al. JICR 2002) including Interferon Regulatory Factors in Immune Cell Development … 13 the more recently identified viperin, an highly conserved antiviral gene, that interferes with the propagation of a broad spectrum of DNA and RNA viruses. In particular, it has been reported that IRF-1 is essential, whereas the type I IFN system is dispensable, for VSV- induced viperin gene transcription (Stirnweiss A et al. 2010). The direct role of IRF-1 in inducing an antiviral state and in controlling IFN-independent signaling events leading to ISGs expression and antiviral immunity has been recently confirmed by elegant data showing that IRF-1 plays a unique role in regulating MAVS- dependent signaling from perixosomes (Dixit E et al. 2010). The RIG-I-like receptor (RLR) adaptor protein MAVS (Belgnaoui SM et al. 2011) is located in both mithocondria and perixosomes. Whereas mithocondrial MAVS activates an IFN-dependent signaling though IRF-3 with delayed kinetics which amplifies and stabilizes the antiviral response, perixosomal MAVS induces, exclusively through IRF-1, a rapid IFN-independent expression of defense factors that include a number of ISGs, providing a rapid and short-term protection upstream the mithondrial-activated IFN. As in the case of direct viperin induction, this IRF-1-mediated type I IFN-independent mechanism of enhanced ISGs expression, while may provide a redundant mechanism to protect cells from viral infections, becomes particularly important for pathogens that evade innate immunity by disrupting the induction and function of IFN (see a latter section). Consistently, in a in vitro screening of more than 380 human ISGs tested for their ability to inhibit the replication of several human and animal viruses, including Hepatitis C virus, Yellow fever virus, West Nile virus, Chikungunya virus, Venezuelan equine encephalitis virus and Human immunodeficiency virus type-1, IRF-1 has recently been identified as an effector with the broader antiviral properties (Schoggins JW et al. 2011). Similarly, in the context of HIV infection in DCs, it was demonstrated that a distinct subset of ISGs is directly stimulated by IRF-1 without detectable production of type I or type II IFN. These genes include MX1, STAT1, OASE, ISG15, IFI35, IFIT5, some of which such as MxA, OAS1-3 and ISG15, have intrinsic unique antiviral actions and could substitute for absent type I IFNs (Harman AN et al. 2011). However, how these genes affect HIV replication, whether ISGs benefit the host, the virus, or both still remains to be determined. The observation that different patterns of ISG are stimulated by other viruses and by HIV in other cell types (Coberley CR, et al. 2004; van 't Wout AB et al. 2003) may suggest direct viral modulation in DC to enable successful HIV-1 transfer to T cells. In T cells, that are primary target of HIV replication, IRF-1, on the other hand, is similarly induced, but promptly hijacked by the virus on the viral promoter to induce viral replication when the viral transactivator Tat is still absent or at sub-threshold amounts (Sgarbanti M et al. 2002, 2008). Later on in infection IRF-1 is also sequestered by Tat and this results in a substantial quenching of IRF-1 transcriptional activity (Remoli AL et al. 2006). Thus, even if mechanisms involved are completely different, in T cells as in DC, HIV-1-induced IRF-1 expression may, instead to protect, constitute one of the viral strategies developed to evade the host immune response as described in more details in a latter section. Overall IRF-1, as initially highlighted by knockout mice studies and fully demonstrated by subsequent observations, exerts profound and multifarious effects in the regulation of both innate and adaptive immunity orchestrating host responses that largely extend beyond its role in IFN signaling. In addition, IRF-1 has key roles in cell cycle regulation and apoptosis in response to a variety of genotoxic stress and thus in oncogenesis, all issues not covered here but extensively reviewed elsewhere. (Taniguchi T et al. 2001 ; Tamura T et al. 2008 ; Savitsky D et al. 2010). 14 Angela Battistini

IRF-2: The Neglected IRF

IRF-2 has been discovered by cross-hybridization with IRF-1 cDNA during the study of transcriptional regulation of mammalian IFN- gene upon viral infection (Harada H et al. 1989). In contrast to IRF-1, IRF-2 expression is constitutive in most cell types and due to higher stability, more represented in the cell, but as IRF-1 it is also inducible by Type I IFN and viral infection and also by IRF-1 (Harada H et al. 1989; Coccia EM et al. 1999b). Similar to IRF-1, IRF-2 is expressed in many immune cells including B and T cells, macrophages and DCs (Taniguchi T et al. 2001; Nelson N et al. 1996). IRF-2 was originally described mainly as a transcriptional repressor by competing for binding of especially IRF-9 and IRF-1 to conserved ISRE/IRF-E sequences (Harada et al. 1989; Tanaka et al. 1993; Yamamoto et al. 1994; Lin R et al. 1994; Hida S et al. 2000). Biologically, IRF-2 plays an important role in cell growth regulation, and has been shown to be a potential oncogene, as overexpression of IRF-2 causes anchorage-independent growth in NIH 3T3 cells and tumor formation in mice, which can be reverted by the concomitant expression of IRF-1. The IRF-2-mediated inhibition of the proapoptotic and growth regulatory functions of IRF-1 is considered mainly responsible for the oncogenic transformation of NIH/3T3 cells overexpressing IRF-2 (Harada H et al. 1993). IRF-2 was also shown to reverse N-Ras-induced growth inhibition in a myeloid cell line (Passioura T et al. 2005). In line with its recognized oncogenic potential, IRF-2 has recently been reported to serve as a baseline transactivator of the human papilloma virus (HPV) type-16 major early promoter, that drives the expression of the E7 oncogenic protein (Lace MJ et al. 2010). IRF-2 is also associated with oncogenic progression in human esophageal carcinomas (Wang Y et al. 2007) and by modulating its sub cellular localization, regulates the activity of NF-B that is known to be persistently active during cancer progression (Karin M 2006) (Chae M et al. 2008). In addition to its oncogenic potential, IRF-2 has emerged as a complex and distinct mediator of gene expression and cell cycle control. In addition to a C-terminal repressor domain that may contribute to post-induction repression of IRF-1-dependent-gene activation, IRF-2 presents indeed, in its central region, a latent activation domain (Yamamoto H et al. 1994) and under certain conditions may activate gene transcription. In particular, IRF-2 can actively induce the expression of genes such as the cell cycle-regulated histone H4 (Vaughan PS et al. 1995, 1998), the gp91 (phox) (Luo W and Skalnik DG 1996) the vascular cell adhesion molecule (VCAM) 1 (Jesse TL et al. 1998) and transporter associated with antigen processing (TAP)-1 (Rouyez MC et al. 2005). As an activator IRF-2 can also cooperate with IRF-1 in transcriptional stimulation of several immunomodulatory genes including MHC class II (Xi H et al. 1999), Il-12p40 (Salkowski CA, et al. 1999) and IL-7 protein (Oshima S et al. 2004). Moreover, IRF-2, probably in association with IRF-1, down-regulates the expression of IL-4 (Elser B et al. 2002), thus contributing in the induction of a Th1 response.

IRF-2 in Regulation of Immune Cell Development As for other IRFs, studies in IRF-2 knockout mice have revealed its physiological roles. In particular, IRF-2 null mice exhibit immunological abnormalities as bone marrow suppression of hematopoiesis and B lymphopoiesis and mortality following lymphocytic choriomeningitis virus infection (Matsuyama T et al. 1993). These mice also spontaneously Interferon Regulatory Factors in Immune Cell Development … 15 develop inflammatory skin disease as they age, involving CD8+ T cells, that exhibit a hyper- responsiveness to antigen stimulation in vitro and up-regulated ISGs expression (Hida S et al. 2000). Subsequent analyses of these mice have revealed that IRF-2 is also important for the development of NK and Dendritic cells. IRF-2-/- mice, indeed, selectively lack mature NK cells probably due to an accelleration of apoptosis (Lohoff M et al. 2000; Taki S et al. 2005). The defective NK cell differentiation and the lack of IL-12 in macrophages in IRF-2-/- mice (Salkowski CA et al. 1999) then contribute to the defective Th1 response, and indeed, these mice as IRF-1-/- mice, are susceptible to infections that require a Th1 response as Leishmania major (Lohoff M et al. 2000). IRF-2 is also required for limiting the generation of basophils. The expansion of basophils in naive IRF2-/- mice results in an increase in IL4 production and Th2 polarization (Hida S et al. 2005). Thus, in addition to the positive role exerted in concert with IRF-1 in Th1 polarization, the negative regulatory role on the basophil population size is critically important for preventing excess Th2 polarization and to a proper Th1/Th2 balance in naive animals. In myeloid cell development IRF-2 has an opposite role as compared with that of IRF-1: it inhibits granulopoiesis by suppressing the IRF-1 stimulatory effect on the late differentiation marker lactoferrin (Coccia EM et al. 2001), while it stimulates the unrelated megakaryocytic lineage by up-regulating transcription factors that are important in megakaryocyte development in both the early and later stages of the differentiation process (Stellacci E et al. 2004). IRF-2 has recently also been implicated in differentiation of the erythroid lineage. IRF-2-deficient mice have been shown to have impaired erythropoiesis characterized by decreased numbers of late erythroblasts accompanied by an increased number of early erythroid progenitors (Mizutani T et al. 2008). IRF-2 has important roles also in the development of myeloid DC. IRF2-/- mice exhibit a selective loss of splenic and epidermal CD4+CD8- DCs and this defect has been ascribed to an abnormally increased type I IFN signaling since the deficiency was rescued by introduction in IRF2-/- mice a null mutation for the IFN receptor complex (Honda K, et al. 2004; Ichikawa E et al. 2004). By inhibiting type I IFN, IRF-2 has been shown also to preserves self-renewal and multi-lineage differentiation capacity of hematopoietic stem cells (Sato T et al. 2009).

IRF-2 in Regulation of Immune Responses A specific role of IRF-2 in activation of immune cells has not been reported. In the context of innate immunity IRF-2 is, instead, considered a unique negative regulator, attenuating type I IFN and type I IFN-induced gene transcription, in order to balance the beneficial and harmful effects of IFN-alpha/beta signaling. Surprisingly, however, as mentioned above mice deficient for IRF-2 exhibit several immunological abnormalities and cannot survive lymphocytic choriomeningitis virus infection thus suggesting a role for IRF-2 in antiviral defense (Matsuyama T et al. 1993; Hida S et al. 2000). The molecular bases of these aspects have not been elucidated and await extensive investigations. As mentioned above, IRF-2, in cooperation with IRF-1, also contributes in the induction of a Th1 response. Even if an involvement of IRF-2 in PRRs signaling pathway has not been reported, IRF- 2, as IRF-1, participates in the regulation of TLR gene expression. A role for IRF-2 in constitutive expression of murine TLR3 and TLR9 genes has been, in fact, reported (Heinz S et al. 2003; Guo Z et al. 2005). Whether IRF-2 can form heterodimers and also cooperate with 16 Angela Battistini

IRF-1 in IFN-mediated up-regulation of these genes remains to be exactly defined, however, it has been shown that a synergistic stimulation of the TLR3 promoter occurs upon IRF-1 and IRF-2 co-expression (Fragale A et al. 2011).

IRF-3, IRF-7 and IRF-9: The Interferonic and Antiviral IRF

IRF-3 and IRF-7 the two IRFs with the greatest sequence homology to one another are key regulators of type I IFN gene expression upon infections. The cellular events that control their activity have been elucidated recently and constitute the response of the immune system to invading pathogens (Honda K and Taniguchi T 2006). IRF-3 is constitutively expressed in most cell types (Au WC et al. 1995) while IRF-7 is expressed at discrete levels only in specialized immune cells, such as B cells and DCs and in most cell types is inducible by IFN-mediated signaling (Sato M et al. 1998, Au WC et al. 1998, Marie I et al. 1998). Early studies, mainly carried out in virus-infected fibroblasts, identified IRF-3 and IRF-7 as the two IRFs mainly involved in regulating the early and late phases of IFN expression, respectively (Marie I et al. 1998; Lin R et al. 2000a). Their essential and distinct roles in regulating type I IFN expresssion was then demonstrated by studies in IRF-3 and IRF-7 knockout mice (Sato M et al. 2000). In the initial hypothesis, IRF- 3 being considered mainly responsible for the initial induction of the IFN- gene whereas IRF-7, whose up-regulation is mediated by type I IFNs themselves, involved in the late phase of type I IFN gene induction (Sato M et al. 1998a, 2000). A more detailed analysis in IRF-7 knockout mice, however, subsequently indicated that small basal amounts of IRF-7 are crucial even in the initial phase of type I IFN gene induction by viruses-activated cytosolic receptor pathways. The formation of the heterodimer IRF-3/IRF-7, or the homodimer of IRF- 7, rather than the homodimer of IRF-3 seems thus more important for the production of type I IFN (Honda K et al. 2005a). The homo and heterodimeric complexes also act differently on the type I IFN gene family members. IRF-3 potently activates the IFN- gene rather than the IFN- genes (except IFN-a4), whereas IRF-7 preferentially stimulates the IFN-a and IFN-a5 genes (Sato M et al. 2000). Both factors are, expressed in the cytoplasm in a latent form and, upon viral infection undergo phosphorylation, and dimerization. Modified IRFs then translocate in the nucleus and homo or heterodimers of IRF-3 and IRF-7 in a complex with coactivators such as CBP/p300 bind to target DNA sequences (Lin R et al. 1998; Yoneyama M, et al. 1998; Weaver BK et al. 1998.; Sato M, et al. 1998a,b; Marié I et al. 1998; Wathelet MG, et al.1998). As mentioned in the previous section, their transcriptional activity is regulated by virus and dsRNA-induced C-terminal phosphorylation at several serine/threonine residues (Mori M, et al.2004; Panne D et al. 2007) by the TBK1 and IKK kinases (Fitzgerald and others 2003a; Sharma and others 2003). These kinases are activated upon engagement of a number of PRRs and depending on the nature the pathogen and host cell type, activate a cascade of events leading to the transcriptional activation of powerful antiviral genes such as the type I IFNs and/or the triggering of inflammatory responses through interleukin-1 (IL-1) and IL-18 production (Honda K and Taniguchi T 2006; Akira S et al. 2006; Kawai T and Akira S 2010; 2011). Interferon Regulatory Factors in Immune Cell Development … 17

The IRF-3-mediated induction pathway of type I IFN has been recognized to be activated by cytosolic recognition systems mediated by RIG-I-like receptors i.e. RIG-I and MDA5 two RNA helicase enzymes, present in all cell types that produce IFN. They recognize dsRNA of many replicating viruses in a TLR-independent manner (Meylan E and Tschopp J 2006; Kawai T and Akira S 2010) and are essential for IRF activation and type I IFN gene induction in response to viral infections in many cell types except pDCs where IFN induction is, instead, primarily regulated by IRF-7 (Yoneyama M, et al. 2004; Kato H et al. 2005; Yoneyama M et al. 2005; Kato H, et al. 2006). They activate the downstream pathway trough an adaptor molecule identified by several groups and named VISA, Cardiff, MAVS or IPS-1 (Meylan E et al. 2005; Xu LG et al. 2005; Kawai T et al. 2005; Seth RB et al. 2005) to transmit signals to IKKs, including NEMO/IKK, TANK and TBK1/IKK (Zhao T et al. 2007; Xu LG et al. 2005; Kawai T et al. 2005) that trigger activation of NF-B and IRF3/7. Studies in IRF-7-/- mice (Honda K et al. 2005a,b; Honda K and Taniguchi T 2006) have demonstrated that IRF-7 is essential in TLR-dependent signaling pathway. The specific ligands recognized by IFN-inducing TLRs include dsRNA derived from dsRNA viruses or from ssRNA viruses as replication intermediates for TLR3 (Alexopoulou L et al. 2001; Akira S et al 2006), LPS for TLR4 (Poltorak A et al 1998), ssRNA as replication intermediates for TLR7 and 8 (Hemmi H et al. 2002; Heil F et al. 2004), and unmethylated CpG-DNA for TLR9 (Hemmi H et al. 2000). In addition, TLR3 recognizes some DNA viruses or parasites indicating that TLR3 can recognize structures other than dsRNA (Flandin JF et al.2006; Tabeta K et al. 2004; Zhang SY et al. 2007). Upon engagement of TLR3 and TLR4, both of which use the TRIF-dependent pathway, IRF-3 and IRF-7 are both activated (Kawai T et al. 2001; Doyle S et al. 2002; Fitzgerald KA et al. 2003b; Sakaguchi S et al. 2003). The induction of IFN- by LPS-engaged TLR4 is mainly mediated by IRF-3 homodimers being absent in IRF-3-/- DCs, but nearly normal in IRF-7-/- cells (Honda K et al. 2005a). However, in the absence of IRF-3 and in particular conditions of IRF-7 overexpression, (e.g. in conventional DC and monocytes after IFN- priming), IRF-7 may participate in IFN- induction on exposure to LPS (Richez C et al. 2009; Sakaguchi S et al. 2003). As described for TLR4, IRF-3 is involved in TLR3-mediated induction of type I IFN expression however, IFN mRNA is still expressed in IRF-3 knock-out DCs upon dsRNA stimulation even if at lower extent, but is completely abolished in double IRF-3/7 double knock-out cells. This indicates that, in DC activated by TLR3 signaling pathways, IRF-7 is required for maximal induction (Savitsky D et al. 2010). IRF-7 is specifically activated downstream of pathways induced by TLR7 (expressed in mouse cDCs and human plamacytoid DCs), TLR8 (expressed in human myeloid DC) and TLR9 (expressed in plamacytoid DCs). These receptors, in contrast to TLR3- and TLR4- mediated type I IFN induction which depends on TRIF, exclusively utilize MyD88 as signaling adaptor (Akira S et al. 2006) and IRF-7 (but not IRF-3), physically interacts with MyD88 in the endosomal compartment where these TLRs are expressed (Honda K et al. 2004b; Kawai T et al. 2004). Regulation of type I IFN in pDCs has been extensively studied. pDCs are professional IFN-producing cells that in response to TLR7 and TLR9 engagement release the larger amounts of type I IFN in the body (Colonna M et al. 2004). pDCs express high levels of TLR7 and TLR9 in endosomes and, in these cells, IRF-7 is the main regulator essential for the robust MyD88-dependent IFN gene induction (Honda K et al. 2005a). Consistently, higher constitutive expression of IRF-7 in pDCs as compared with other cell 18 Angela Battistini types has been reported (Izaguirre B et al. 2003). Notably, a spatiotemporal regulation of MyD88-IRF-7 signalling, operating in these cells, is critical for the specific ability of pDCs to produce high-levels of type I IFNs (Honda K et al. 2005b). In these TLR signaling pathways, IRF-7 can also be activated by the IRAK1/4-IKK protein kinase cascade, which is known to be involved in NF-B activation (Uematsu S et al. 2005; Hoshino K et al. 2006). More recently, it has been reported that phosphatidylinositol-3 kinase (PI3K) is also required for optimal IRF-7 nuclear translocation and IFN production in pDCs on TLR triggering (Guiducci C et al. 2008). In line with the observation that type I IFNs are also produced in response to bacterial and even parasitic infections (Bogdan C et al. 2004, Uematsu S and Akira S 2007), it has recently also been reported IFN induction by the MyD88-IRF1 and -IRF7 pathways in response to ligands for TLR2/1 and TLR2/6 heterodimers, that instead has long been associated with pro-inflammatory cytokines but not type I interferon responses (Dietrich N et al. 2010). In addition to RNA-sensing receptors, DNA recognizing receptors that utilize the IRF-3/7 to induce IFN expression in response to DNA from pathogens or synthetic dsDNA in a TLR- independent manner, have been identified (Stockinger S et al. 2004; Hochrein H, et al. 2004; Stetson DB and Medzhitov R 2006; Ishii KJ et al. 2006). Different cytosolic DNA sensors have been, so far, reported and characterized including DAI (Takaoka A et al. 2007; Wang Z et al. 2008) the 3' repair exonuclease 1 (Trex1) (Stetson DB, et al. 2008) and the adaptor protein named STING/MITA that localizes to the outer membrane of mitochondria and following DNA-mediated stimuli, links IPS-1 adaptor to TBK1 and IRF-3 (Ishikawa H and Barber GN 2008; Zhong B et al. 2008). In these pathways, while IRF-3 seems sufficient for induction of IFN-, for IFN- induction IRF-7 is also required (Takaoka A et al. 2007). Very recently, a novel innate sensor, the DNA sensor IFI-16/p204, responsible for HSV containment at the site of acute infection that functions in a IRF-3-dependent, but IRF-7- and TLR-independent manner, has been reported (Conrady CD et al. 2012). Interestingly, IRF-3 has been recently also involved in the signaling pathway of NOD2 a member of the nucleotide-binding domain (NBD)- and leucine-rich-region (LRR)-containing family of cytoplasmic proteins known as NLRs nucleotide-binding domain (NOD)-like receptors (NLR) protein family, that have rapidly emerged as central regulators of immunity and inflammation with demonstrated relevance to human diseases (Schroder K and Tschopp J 2010; Joao G et al. 2011; Kanneganti TD 2010). Much attention has focused on the ability of several NLRs to activate the inflammasome complex and drive proteolytic processing of inflammatory cytokines, however, NLRs also regulate important inflammasome-independent functions in the immune system (Jenny PY et al. 2010; Kanneganti TD 2010). Indeed, viral infection can activate NOD2, leading to its translocation to the mitochondria, where it associates with the mitochondrial MAVS adaptor in the RIG-I-like receptor signaling pathway and forms a newly defined multimeric complex, that can act as central platforms for innate antiviral responses, that together regulate the production of antiviral type I IFN and inflammatory cytokines (Sabbah A et al. 2009). Interestingly, in response to Helicobacter pylori infection, IRF-7, as IRF-3, has been shown to be activated by a NLR receptor, NOD1, a sister molecule of NOD2 mainly expressed in APCs and epithelial cells (Watanabe T et al. 2010). Besides regulating type I IFN expression, IRF-3 and IRF-7 have been also implicated in the induction of another class of IFN family members, type III IFN (IFN lamda) genes that Interferon Regulatory Factors in Immune Cell Development … 19 have similar antiviral activities as type I IFN. IFN-lambda1 gene is regulated by virus- activated IRF-3 and IRF-7, thus resembling that of the IFN- gene, whereas IFN-lambda2/3 gene expression is mainly controlled by IRF-7, thus resembling those of IFN- genes (Osterlund PI et al. 2007). In addition to their essential roles in the transcriptional induction of immediate-early IFN genes, IRF-3 and IRF-7 also exert host defense activities through direct and distinct induction of target genes in an IFN-independent manner. Several biochemical and gene profiling studies have delineated dsRNA and virus-induced genes and attempts to dissect IRF-3 and IRF-7 target genes by using cells lacking all type I interferon genes or cells expressing the constitutively activated form of the proteins have been performed (Geiss G et al. 2001; Elco C Pet al. 2005; Grandvaux N et al. 2002; Barnes BJ et al. 2004 Andersen J et al. 2008; Goubau D et al. 2009; Honda K, et al. 2006). In particular, activated IRF-3 leads to selective regulation of IFN-responsive genes involved in the establishment of an antiviral state including ISG54, ISG56, and ISG60, guanylate binding protein 1 (GBP1), 2-5 oligoadenylate synthase (2-5 OAS), ISG15, IP-10 and the CC chemokine RANTES (Grandvaux N et al. 2002; Nakaya T et al. 2001; Lin R et al. 1999c). More recently, transcriptional profiles of active IRF-3- or IRF-7-transduced primary human macrophages confirmed the previously reported IRF-3 and IRF-7 target genes, and also identified additional differentially modulated genes in both a IFN-dependent and IFN-independent manner. These results demonstrated that IRF-3 or IRF-7 differentially regulates apoptotic and anti-tumor gene programs in primary macrophages. In particular, the pro-apoptotic BH3-only protein Noxa was identified, in macrophages as a primary IRF-3-target gene and essential regulator of IRF-3-, dsRNA- and vesicular stomatitis virus-induced cell death, while IRF-7 increases the expression of a broad range of ISGs including immunomodulatory cytokines as well as genes involved in antigen processing and presentation, probably in an IFN-mediated manner (Romieu-Mourez R et al. 2006; Goubau D et al. 2009). Overexpression of IRF-7 has been, on the other hand, reported to directly stimulate transcription of the immunoproteosoma subunit LMP2 (Sgarbanti M et al. 2007) involving IRF-7 also in adaptive immunity. IRF-9 also known as ISGF3 or p48, like IRF-1 and IRF-2, is constitutively expressed in a variety of cell types and is inducible by type II IFN and viral infection (Levy DE et al. 1990). As IRF-3 and IRF-7, it plays a major role in antiviral defense as part of a ternary complex termed the IFN-stimulated gene factor 3 (ISGF3) that is an essential mediator of type I IFN signaling (Schindler C et al. 1992; Bluyssen AR et al. 1996). Following detection of PAMPS by PRRs, the newly secreted IFNs, indeed, bind to cell surface receptors of neighboring cells and in an autocrine and paracrine manner induce, via the JAK/STAT signaling pathway (Darnell JE Jr et al. 1994), the formation of the heterocomplex ISGF3 that is composed by IRF-9 and activated tyrosine-phosphorylated signal transducer and activator (STAT)-1 and STAT2 (Fu X Y et al. 1990; Levy DE et al. 1989; Veals SA et al. 1992). This complex is formed within minutes of IFN treatment and participates in the transcriptional activation of a large number of IFN-inducible antiviral genes by binding specifically to the ISRE present on target promoters. These genes include all bona fide ISGs including IRF-7 that in a positive loop further stimulates type I IFN expression as described above. Interestingly, early studies identified the binding of IRF-9 to the IFN- promoter in response to viral infection, suggesting a direct role also in IFN stimulation (Harada H et al. 1996; Kawakami T, et al. 1995; Yoneyama M, et al. 1996). IRF-9 is the major DNA binding 20 Angela Battistini component of the ISGF3 complex (Veals SA et al. 1993), it can also form a complex with the STAT1 homodimer and with STAT2 alone and these complexes bind to DNA with the same specificity as ISGF3 (Kraus TA et al. 2003). Accordingly, MEF from IRF-9 knockout mice do not respond to type I and type II IFNs (Kimura T et al. 1996; Harada H et al. 1996). A role in IFN-γ-mediated antiviral activity in humans mediated by an ISGF3 complex containing unphosphorylated STAT2 has also been recently reported (Morrow AN, et al. 2011). The importance of IRF-9 in the ISGF3 complex activity has been further confirmed by recent data, obtained by combining mathematical modeling with experimental analysis, that have shown that IRF-9 is a crucial factor for accelerating IFNα-induced early antiviral signaling. Increasing the initial IRF-9 concentration by over-expression, results in higher levels of phosphorylated STAT proteins in the nucleus, and consequently augmented expression of IFNα-target genes (Maiwald T et al. 2010). Furthermore, through a combination of both in vitro biochemistry and in vivo transcriptional profiling it has been demonstrated that in the absence of IFN signaling, an IFN-like transcriptome is still induced. This transcriptional activity is mediated from ISRE sequences that bind to both ISGF3 as well as to IRF7. A significant overlap between the genes induced by IRF-7, IRF-3 and ISGF3 was found and slight differences in the ISRE encoded upstream of target genes defined the specificity of the single transcription factors. These results while indicate a redundancy in the induction of an antiviral state by IRF-3, -7, -9 further support their IFN-independent transcriptional activity on ISGs stimulation (Schmid S et al. 2010).

IRF-4 and IRF-8: The Hematopoietic IRF

Of all members of the IRF family, IRF-4 and IRF-8 are those predominantly expressed in hematopoietic cells, with IRF-4 largely restricted to lymphocytes and expressed in all stages of B-cell development and in mature T cells and macrophages (Eisenbeis CF et al. 1995; Marecki S et al. 1999) and IRF-8 primarily detected in cells of myeloid lineages but also expressed in preB-cells and activated T cells (Nelson N et al. 1996; Cattoretti G et al. 2006). These factors share a high degree of sequence homology, predominantly in the DBD, and can serve as either transciptional activator or repressor depending on protein interaction partners and/or context of DNA binding. They both have a weak DNA binding affinity that is increased by association with other interacting factors including IRF-1 and IRF-2, IRF-9 and tissue-restricted factors as PU1 and E47 (Marecki S et al. 2002; Levi BZ et al. 2002; Ozato K et al. 2007).

IRF-4 and IRF-8 in Regulation of Immune Cell Development The roles of IRF-4 and -8 in immune cell development and function have been well documented and both factors exert cell-intrinsic and non-redundant roles as well as some shared functions. The role of IRF-4 seems predominant in lymphoid differentiation while that of IRF-8 in myeloid lineage selection. As for other IRFs, much of what is known about their functions has come from studies of mice bearing a null mutation of the gene. IRF-4 null mice develop progressive generalized lymphadenopathy due to an increase in the number of CD4+ and CD8+ T and B cells. In addition, IRF-4 null mice exhibit a marked reduction in serum immunoglobulin concentrations and do not mount detectable antibody responses. Due to impaired T lymphocyte functions such as cytokine production and activity, cytotoxic activity Interferon Regulatory Factors in Immune Cell Development … 21 and proliferation, these mice could not generate cytotoxic or antitumor responses. IRF-4 is thus essential for the function and homeostasis of mature B and T lymphocytes that is more evident after antigen stimulation (Mittrücker HW et al. 1997; Lu R et al. 2003). Consistently, IRF-4 expression is largely restricted to lymphocytes and IRF-4 is expressed in all stages of B cell development as well as in mature T cells. Interestingly, in both T and B cells, IRF-4 expression is essentially regulated by pathways known to induce lymphocyte activation and not by type I or type II IFN like as instead occurs for other members of the IRF family (Gupta S et al. 1999; Matsuyama T et al. 1995; Yamagata T. et al. 1996; Eiseinbeis CF et al. 1995). IRF-8-/- mice, instead, exhibit a marked expansion of granulocytes and, to a lesser extent, macrophages and are markedly immunodeficient because of deficiencies in type I IFN and IL-12 production (Holtschke T et al. 1996; Giese NA et al. 1997). Interestingly, BXH2 mice that bear a point mutation affecting a single amino acid change in the IAD domain of IRF-8 exhibit an almost identical phenotype (Turcotte K et al. 2005). Since the IAD domain is responsible for the ability of IRF-8 to heterodimerize, this finding indicates that almost all activities of IRF-8 are dependent on its interactions with other proteins. IRF-8 regulates the development and function of macrophages, dendritic cells and B cells (Lu R et al. 2008; Tamura T et al. 2005a; Lee CH et al. 2006). IRF-8 has a key intrinsic role in myeloid cell lineage selection driving differentiation toward macrophages and inhibiting granulocytic (neutrophilic) differentiation and accordingly, IRF-8 over-expression in null cells drives the differentiation of myeloid progenitors into macrophages (Tamura T et al. 2000). IRF-8 also inhibits myeloid cell growth and promotes apoptosis and regulate macrophage functions (Tamura T et al. 2000; Tsujimura H et al. 2002; Gabriele L et al. 1999) by controlling many genes that are involved in cell growth regulation and in apoptosis of myeloid cells, as well as genes important for macrophage activities including those encoding Cdkn2b, Bcl-Xl, Blimp-1, Cathepsin C, Cystatin C, lysozyme M, and prosaposin (Tamura T et al. 2005b; Takaoka A et al. 2008). By complexing with IRF-1 and PU.1, IRF-8 also regulates genes stimulated by IFN- and by Toll-like receptor engagement. These genes include IL-12, gp91phox, iNOs, TLR4, Fc receptor I, IRF-8 itself, IL-18, Nramp1 and Rantes (Xiong H et al. 2003; Kim YM et al. 2000; Marecki S et al. 2001; Liu J and Ma X. 2006; Alter-Koltunoff M et al. 2003). Furthermore, mice lacking IRF-8 exhibit an increased expansion of neutrophils at the expense of monocytes/macrophages that develops in a syndrome that resembles the human a chronic myeloid leukemia (CML) disease (Holtschke T et al. 1996; Tsujimura H et al. 2002). Particularly worth noting is that the IRF-8 expression levels are significantly decreased in CML and acute myelogenous leukemia cells from patients (Schmidt M et al. 1998). Very recently, a loss-of function mutation in the human IRF-8 gene that also results in a very high neutrophil count and an absence of circulating monocytes and dendritic cells has been reported (Hambleton S et al. 2011). Thus, these observations support a conservation of IRF8’s function between mice and humans also in tumor suppression as reviewed elsewhere (Takaoka A et al. 2008; Savitsky D et al. 2010). The role of IRF-4 in myeloid differentiation is less understood. IRF-4, like IRF-8, is expressed in macrophages (Marecki S et al. 1999) and even if IRF-4-/- mice do not display abnormalities in myeloid differentiation, recently, based on studies in double KO mice and gene introduction experiments, it has been demonstrated that IRF-4 has activities similar to IRF-8 in regulating myeloid cell development. IRF-4 indeed promotes macrophages, while 22 Angela Battistini hindering granulocytic cell differentiation. Of note, IRF- 4 as IRF-8, targets the IECS sequence in the promoter of macrophage-related genes as Mmp12 and Mrc1 that are however specifically induced by IRF-4 but not IRF-8, indicating also for IRF-4 an intrinsic role in myeloid differentiation. The regulation of other macrophage-related genes as those encoding as Blimp-1, Cathepsin C, Cystatin C and CSF1/M-CSF receptor is instead shared with IRF-8. Interestingly both IRFs also induce the expression of IRF-5 another IRF preferentially expressed in immune cells, including macrophages, and that can also in part mediate the positive effects of IRF-4/8 on the innate immune responses in macrophages (Yamamoto M et al. 2011). Interestingly, while IRF-8 seems specifically required for M1 macrophage polarization, via induction of Il-12, IRF-4 has been shown as essential for M1 polarization (Satoh T et al. 2010). Furthermore, IRF-8-/-IRF-4-/- double knockout mice exhibit a more severe CML-like disease than IRF8-/-mice (Yamamoto M, et al. 2011). Regarding lymphoid cell development, IRF-4 and IRF-8 each exerts both a redundant and a cell-intrinsic role. The initial identification of IRF-4 and -8 as transcription factors that bind to an EICE motif located in the immunoglobulin Ek30 and l gene enhancer regions provided the first evidence that IRF-4 and -8 might play an overlapping role in pre-B-cell development (Brass AL et al. 1996; Brass AL et al. 1999). As in the myeloid lineage, they, indeed, cooperate during B cell differentiation (Lu R 2008). Both are expressed in immature stages of B cell development including pre-B cells in the bone marrow (Cattoretti G et al. 2006) and are involved in the transcriptional network controlling B cell lineage specification, commitment and differentiation in bone marrow (Lu R 2008), the regulation of the pre-B to B cell development depending on their heterodimerization (Lu R et al. 2003). The two factors exert a redundant role in promoting the transition from pre-B to IgM+B cells. In double knockout mice, but not single null mice, B cells are arrested at the large pre-B stage (Lu R et al. 2003) and either IRF-4 or IRF-8 are able to rescue the defect (Ma S et al. 2006). IRF-4 and -8 orchestrate the transition from large pre-B to small pre-B by inducing the expression of the Ikaros family transcription factors Ikaros and Aiolos (Ma S et al. 2008) that down regulate the pre-B-cell receptor (BCR) by suppressing the expression of the surrogate light chain (SLC) (Thompson EC et al. 2007). In addition, IRF-4 inhibits IL-7 signaling, a proliferative signal for pre-B cell that must be down-regulated for light chain rearrangement (Johnson K et al. 2008), and up-regulates the expression of the chemokine receptor CXCR4 that in response to its ligand CXCL12 promotes migration of pre-B cells away from Il-7-producing stromal cells, thus contributing to limit proliferative signals (Tokoyoda K et al. 2004). Although IRF-4 and -8 function redundantly to control pre- B-cell development, IRF-4, but not IRF-8, is uniquely required for receptor editing. Expression of IRF-4 but not IRF-8 is, indeed rapidly induced by self-antigen in immature B (Pathak S. et al. 2008). They also play a critical and non-redundant role in Germinal Center (GC) reaction and plasma cell generation. In the dark zone while IRF-8 expression increases, IRF-4 is suppressed and in the light zone as centrocytes differentiate in plasma cells, the expression of IRF-8 declines and that of IRF-4 increases (Cattoretti G et al. 2006; Falini B, et al. 2000; Sciammas R et al. 2006; Lee CH et al. 2006; Klein U et al. 2006). Studies in the recently developed knock-out-conditional mice allowed to define more precisely the role of IRF-8, revealing that IRF-8 indeed controls the size of both the splenic marginal zone and follicular B cell population (Feng J et al. 2011). The molecular bases of these effects have been defined by the elucidation of the direct target genes of IRF-4 and IRF-8 in these cells. So IRF-8 controls expression of B cell-specific genes, as activation-induced cytidine deaminase, BCL6 and MDM2 in GC B cells (Lee CH et Interferon Regulatory Factors in Immune Cell Development … 23 al. 2006; Cattoretti G et al. 2006; Zhou JX et al. 2009), while IRF-4 has identified as transcriptional repressor that directly down-regulates BCL6 expression in GC B cells (Saito M et al. 2007). IRF-4 has been also shown as a transcriptional regulator of Fas apoptosis inhibitory molecule (FAIM), an inhibitor of Fas-mediated apoptosis in B cells. FAIM is preferentially expressed in germinal center B cells and thus IRF-4-mediated regulation of FAIM most likely occurs as part of germinal center formation. Interestingly, FAIM also enhances CD40-induced IRF-4 expression in B cells indicating a positive feedback regulatory loop mediated by IRF-4 in B cell differentiation (Kaku H and Rothstein TL 2009). IRF-4, together with the transcriptional repressor Blimp-1 that is directly induced by IRF-4, (Sciammas R et al. 2006) is also required for the development of antibody-producing plasma cells and their differentiation (Klein U et al. 2006). Interestingly, in plasma cells, also IRF-4 is induced by Blimp-1 (Sciammas R and Davis MM 2004) indicating a positive loop initiated by IRF-4. Of note, Blimp-1 and IRF-4 have recently been predicted as targets of MicroRNAs 125a and 125b. Over-expression of miR-125b inhibits the induction of Blimp-1 expression and IgM secretion in an LPS-responsive B cell line. Furthermore, the differentiation of primary B cells and the survival of cultured myeloma cells were inhibited by miR- 125b(Gururajan M et al. 2010). These findings thus suggest that miR-125b promotes B lymphocyte diversification in GC by inhibiting premature utilization of IRF-4 as an essential transcription factors for plasma cell differentiation. In B cell differentiation, IRF-4 and IRF-8 have, thus, not only critical functions for light chain rearrangement, as initially suggested, but also act throughout B cell development at multiple stages, from B cell lineage specification to commitment and differentiation including pre-B cell development, receptor editing, germinal- center reaction and plasma cell differentiation with overlapping as well as specific effects. The specific role of IRF-4 and IRF-8 in regulating Th1/Th2 differentiation is, instead, quite different, IRF-4 promoting Th2 while IRF-8 promoting Th1 differentiation, respectively. IRF-4-/- T cells do not undergo Th2 differentiation in vitro and do not produce IL-4 and other Th2 cytokines. Moreover, when treated with IL-4 in vitro, they do not express GATA3, a hallmark of Th2 differentiation (Giese NA et al. 1997; Lohoff M et al. 2002; Tominaga N et al. 2003). The fact that IRF-4 in association with NFAT1 and/or NFAT2 activates the IL-4 promoter (Rengarajan J et al. 2002; Hu CM et al. 2002) and also influences the expression of GFI-1, a transcriptional repressor required for Th2 proliferation, indicates that IRF-4 exerts a cell-intrinsic role (Lohoff M et al. 2002; Tominaga N et al. 2003; Zhu J et al. 2002). Interestingly, it has recently been reported that IRF-4 plays a dual role in the regulation of Th2 cytokines: it inhibits Th2 cytokine production in naïve CD4+ T cells while it stimulates Th2 cytokines production in effector/memory CD4+ T cells (Honma K et al. 2008). Furthermore, IRF-4 contributes to Th2 heterogeneity promoting the expression of a specific subset of Th2 cytokines. When Th2 cells are separated based on levels of IL-10 secretion, IRF-4 expression segregates into the subset of Th2 cells expressing high levels of IL-10. Interestingly, in PU.1-deficient T cells there was an increase in IRF-4 binding to the IL-10 gene, and in the ability of IRF-4 to induce IL-10 production indicating a specific IRF-4- induced positive transcriptional regulation of this cytokine that is impaired by the presence of PU1 and by the formation of the heterodimer IRF-4/PU1 (Ahyi AN et al. 2009). The role of IRF-8 is instead to stimulate Th1 differentiation. Like IRF-1-/- mice, IRF-8 null mice show a defective Th1 response and a Th2 prone phenotype (Giese NA et al. 1997). 24 Angela Battistini

This may be due to the inability of DCs and macrophages to produce IL-12 as indicated above (Masumi A et al. 2002). IRF-4 and IRF-8 also exert opposite functions in the differentiation of Th17 a recently characterized, unique subset of T helper cells that has critical roles in the pathogenesis of autoimmunity and tissue inflammation (Weaver CT et al. 2006; Peters A et al. 2011; Ghoreschi K et al. 2011). Specifically, IRF-4 is a key regulator of development, expansion, survival and activity (Brüstle A et al. 2007), while IRF-8 is an intrinsic transcriptional inhibitor of Th17 cell differentiation (Ouyang X et al. 2011). The lack of Th17 differentiation in IRF-4-/- mice seems to be partially due to a lack of IL-6-mediated down-regulation of the transcription factor Foxp3, the hallmark of Treg cells. The expression of the orphan nuclear receptor RORT the lineage-specific transcription factor essential in Th17 differentiation (Ivanov II et al. 2006) is, also, markedly decreased in IRF-4-/- T cells in Th17-polarizing conditions. The importance of IRF-4 in Th17 cell activity has been further stressed by the observation that that mice deficient in IBP (IRF-4-binding protein), the protein that sequesters IRF-4 and prevents it from targeting IL-17 and IL-21 genes, rapidly developed rheumatoid arthritis-like joint disease and large-vessel vasculitis, a pathology associated with inappropriate synthesis of IL-17 and IL-21 (Chen Q et al. 2008). As opposite, mice with a conventional knockout, as well as a T cell-specific deletion, of the IRF-8 gene exhibited more efficient Th17 cells, in part due to IRF-8 physical interaction with RORγt (Ouyang X et al. 2011). As in the case of other IRFs, the extrinsic effect of IRF-4 and IRF-8 on T cell differentiation may be elicited in part through their ability to support the differentiation and function of specific DC subsets that direct Th differentiation. IRF-4 and IRF-8 have been the first IRFs implicated in DC differentiation. Their expression pattern is DC subset-specific and mirrors requirement of these IRFs for DC subset development as well documented by studies in null mice. Expression of IRF-4 is high in in double-negative (DN) and CD4+ DC and IRF-4 is either not expressed or expressed at low levels in CD8+ DCs and pDc (Suzuki S et al. 2004). Conversely, IRF-8 is highly expressed in CD8 + and pDCs and at low levels in CD8 - DCs (Schiavoni G et al. 2002; Tsujmura H et al. 2003). So, IRF-4 is necessary for the generation of CD4+ DC and IRF-8 for that of CD8a+ DC and pDCs while both support the development of CD4-CD8 - DN DC development as evidentiated by the analysis of single and double knock-out mice (Aliberti J et al. 2003; Suzuki S et al. 2004; Tamura T et al. 2005a). More recently, a mutation in IRF-8 that abolishes the development of CD8 + DC without impairing the pDC development has been reported indicating that there is a mechanistic separation that underlies the development of different DC subsets by IRF-8. As previously mentioned, the mutation is within the IAD important for the interaction of IRF-8 with partner proteins and abrogates the binding to PU.1, SpiB and IRF-2 indicating that these functional differences rely on the IRF-8-partner interactions (Tailor P et al. 2008). Recently, it has been reported that the basic helix-loop-helix transcription factor (E protein) E2-2/Tcf4, an essential transcriptional regulator of the pDC lineage in mice, directly activates IRF-8 and IRF-7 genes (Cisse B et al. 2008). IRF-8 is also required for full differentiation and function of epidermal Langherans cells (LCs) and dermal DCs (Schiavoni G et al. 2002). The requirement of IRF-4 and IRF-8 for DC in vitro differentiation also diverges, in that fms- like tyrosine kinase 3 ligand (Flt3L)-mediated DC differentiation depends mainly on IRF-8, Interferon Regulatory Factors in Immune Cell Development … 25 whereas granulocyte macrophage colony-stimulating factor (GMCSF)-mediated differentiation depends on IRF-4 (Suzuki S et al. 2004; Tamura T et al. 2005a).

IRF-4 and IRF-8 in Regulation of Immune Responses IRF-4 and IRF-8 also play distinct roles in innate immune responses elicited by PRR engagement and in particular, they affect the TLR-Myd88 signaling in opposite manner negatively and positively, respectively (Negishi H et al. 2005; Honma K et al. 2005; Tsujimura H et al. 2004; Zhao J et al. 2006). Upon TLR activation, IRF-4 expression is induced and part of the synthesized protein localizes in the cytoplasm where it forms a complex with MyD88. IRF-4 binds to the same region as does IRF-5, thus can compete with the IRF-5 for binding. This results in an attenuation of the MyD88-dependent activation of IRF-5 and induction of pro-inflammatory cytokines following TLR ligation. Consistently, IRF-4 knockout mice produce enhanced levels of pro-inflammatory cytokines, upon TLR ligation, and are highly sensitive to endotoxic shoch induced by TLR9 stimulation (Honma K et al. 2005; Negishi H et al. 2005). In contrast with this negative role, it has recently been reported that in macrophages, upon stimulation with the TLR4 ligand LPS or the TLR9 ligand CpG DNA, IRF-4 may also exert a positive role on IL6 expression. This could, however, be ascribed to the stimulatory effect exert by both IRF-4 and IRF-8, in macrophages, on IRF-5 expression, as mentioned above (Yamamoto M et al. 2011). As a central member in the IFN signaling cascade and in the second wave of IFN synthesis in DCs, IRF-8 plays, instead, a key role in antiviral innate immunity. IRF-8 as IRF-4 is involved in the TLR-9-MyD88 pathway and interacts with TRAF6 (Zhao J et al. 2006) but not with MyD88 (Negishi H et al. 2005). Interestingly, IRF-8-/- DCs fail to produce pro-inflammatory cytokines in response to CpG but not in response to LPS that signals through TLR4 (Tsujimura H et al. 2004). This indicates that among TLRs, differential signals are required to activate NF-B, while TLR9 signaling in DCs uniquely depends on IRF-8. In response to various PAMPs, both in pDCs and cDCs, IRF-8, but not IRF-4 is required for the expression of pro-inflammatory cytokines including IL12p40 and type I IFN. In particular, IRF-8 participates in the second amplifying wave of IFN production, as demonstrated by the observation that in IRF-8-/- DCs the early IRF-3/7-dependent phase of type I IFN production following virus infection occurs, but the second, which enables high IFN production, is significantly reduced (Masumi A et al. 2002; Tailor P et al. 2007). The notion that IRF-8 acts on a late phase of transcription is also consistent with the observation that IRF-8 augments expression of some IFN-responsive genes with a delayed time course in macrophages (Kanno Y et al. 2005). Complementary to these data, a different and so far never described mechanism of IFN- rapid induction that involves cooperation between IRF-8 and IRF-3 in monocytes after pathogenic stimulation, has very recently been described (Li P et al. 2011). IRF-3 is a previously unidentified interaction partner of IRF-8 and IRF-3/8 interaction seems not dependent on DBD and IAD of the proteins. Interestingly, in a complex with PU1, IRF-8 binds constitutively to the IFN- promoter in monocytes, before induction, and facilitates the recruitment of IRF-3. These data while further stress the role of IRF-8 in IFN transcription, also suggest that, in specialized cells, IRF-8 can serve as a transcription factor that stably binding, poises the IFN promoter to assume an open chromatin configuration for rapid induction a cell-type-restricted manner. 26 Angela Battistini

Finally, as IRF-1 and IRF-2, IRF-8 has been involved in the regulation of the expression of some TLR genes including TLR4 (Rehli M et al. 2000), TLR9 (Schroder K et al. 2007), TLR11 (Cai Z et al. 2009) and TLR3 (Fragale A et al. 2011). In a complex with member of the Ets family such as PU1, IRF-4 and IRF-8, can also bind and directly stimulate the expression of specific antiviral genes in an IFN- independent manner. This is the case of ISG15 that contains a unique ISRE element named EIRE that also recruits non-IRFs and can be bound by hetero-complexes formed between PU.1 and IRF-8 or IRF-4 (Meraro D JI 2002) allowing for ISGS15 specific regulation in a cell-type-restricted manner. In this respect, in immune cells where the expression of IRF-4 and IRF-8 is restricted, they can also be stimulated by antigenic stimulation and by type II IFN, respectively. Consistently, although ISG15 is induced by type I IFN in many cell types, its expression in monocytes and lymphocytes is very high, and it is even secreted as a soluble immunomodulatory protein, stressing the existence of cell specific mechanisms of expression regulation mediated by IRFs (D’Cunha J et al. 1996). Very recently, a direct role of IRF- 4 in a complex with NFATc1 and NFATc2, in cell type specific transcriptional regulation of the anti-HIV gene, APOBEC3G has been reported, evidentiating a positive role of IRF-4 in innate immune responses. Interestingly, IRF-8 is also able to bind and stimulate the APOBEC3G promoter and thus since, IRF-4 expression is restricted to lymphocytes, whereas IRF-8 is primarily detected in cells of myeloid lineage and A3G expression is detected in both cell types, it has been postulated that IRF-4 may critically participate in the regulation of A3G promoter activity in T cells, whereas IRF-8 likely exerts that role in macrophages (Farrow MA, et al. 2011) Hence, the similar but distinct transcription factors IRF-4 and IRF-8 make a critical contribution to diverse immune responses by acting in professional APCs as well as in lymphoid cells at several levels, affecting not only differentiation but also activities of these immune cell populations, with both overlapping as well as distinct roles.

IRF-5: The Inflammatory Cytokines Regulator

IRF-5 is a recently characterized member of the IRF family, identified as a regulator of type I IFN gene expression (Barnes BJ et al. 2001;Barnes B et al. 2002a). Human IRF-5 exists as multiple alternatively spliced isoforms, some of these being transcriptionally inactive and functioning as dominant negative mutants. Each isoform displays distinct cell type-specific expression, regulation, cellular localization, and different functions in virus-mediated type I IFN gene induction (Mancl ME et al. 2005; Cheng TF et al. 2006; Lin R et al. 2005). Unlike HuIRF-5, MuIRF-5 is expressed as a single major transcript that is expressed at very low levels and activates the murine IFNA4 and IFNB promoters but not other murine IFNA promoters (Barnes BJ et al. 2001,2003; Paun A et al. 2008). IRF-5 expression is constitutive in B cells and DCs but it is also inducible by type I IFN and TLR signaling (Yanai H et al. 2007; Takaoka A et al. 2005; Mancl ME et al. 2005). It is mainly expressed in the cytoplasm and upon phosphorylation it translocates in the nucleus. Phosphorylation occurs upon many stimuli including viral infection, TLR signaling and DNA damage. (Barnes BJ et al. 2001; Lin R et al. 2005). Initial in vitro studies indicated that IRF-5 serves as a direct transducer of virus-mediated signaling and its activation is virus specific. Newcastle disease virus (NDV), Vescicular stomatitis virus (VSV), and herpes simplex virus type (HSV)-1, have been shown Interferon Regulatory Factors in Immune Cell Development … 27 to activate IRF-5, whereas Sendai virus (SeV) and dsRNA poly(I) poly(C) (pI:C), which activate IRF-3 and IRF-7, do not activate IRF-5 (Barnes B, et al. 2002; Barnes BJ et al. 2001,2002). In infected cells, IRF-5, as IRF-7, activates expression of IFN- , however, the subtypes of IFN- induced by IRF-5 and IRF-7 are different, the IFNA1 being the mayor subtype induced by IRF-7 and the IFNA8 the subtype preferentially induced by IRF-5 in NDV-infected cells (Barnes B et al. 2002; Barnes BJ et al. 2001). Gene-disruption studies have confirmed that IRF-5 plays an intrinsic part in innate immunity but have revealed a unique role in TLR-mediated induction of inflammatory responses (Takaoka A et al. 2005). Thus, the distinct signature of IRF-5 is the induction of early inflammatory cytokines and chemokines rather than type I IFN. IRF-5 seems involved selectively in the induction of pro- inflammatory cytokines by virtually all TLRs and consistently, IRF-5 binding sites are present in the promoter of several gene encoding key pro-inflammatory cytokines such as IL- 12, TNF- and IL-6. Accordingly, IRF-5 null mice show resistance to lethal shock induced by either unmethylated DNA, the ligand of TLR9 or lipopolysaccharide, the ligand of TLR4, which correlates with a marked decrease in the serum levels of pro-inflammatory cytokines (Takaoka A et al. 2005; Honda K and Taniguchi T 2006; Paun A et al. 2008; Barnes BJ et al. 2004). A fine, cell type-specific regulation of TNF secretion by IRF-5 has recently been reported. DCs, but not macrophages, have high levels of IRF-5 protein that is responsible for the late- phase expression of TNF. Interestingly, while IRF-5 can directly bind to TNF promoter in an upstream region of the promoter, its recruitment to a downstream region depends on protein- protein interactions with NF-B RelA, and is essential for maintaining TNF gene transcription over a prolonged period of time (Krausgruber T et al. 2010). IRF-5, like IRF-7, is activated by TLR7 and TLR9 and interacts with the adaptors MyD88 and TRAF6. The domain of MyD88 interested in the binding is, however, different: IRF-7 binds to the dead domain, whereas IRF-5 interacts with the central region of the protein (Kawai T et al. 2004; Takaoka A et al. 2005; Schoenemeyer A et al. 2005). As mentioned above, IRF-4 binds to the same region of MyD88 as does IRF-5 and competes with IRF-5 for binding (Negishi H et al. 2005) thus, a selective attenuation of pro-inflammatory cytokines induction following the MyD88-dependent activation of IRF-5 can occur in those cells, such as pDCs or B cells, that also express IRF-4. Even if the exact mechanisms of IRF-5 stimulation/activation are not fully understood, recently, the importance of phosphorylation at serine/threonine residues in a C-terminal autoinhibitory domain of the protein, for IRF-5 dimer formation and association with CBP/p300 in the nucleus, has been reported (Chen W et al. 2008). In addition, TRAF6-mediated K63- linked ubiquitination is important for IRF-5 nuclear localization upon TLR7/9 signaling (Balkhi MY, et al. 2008). Several initial studies, employing over-expression or RNA interference assays using cultured cell lines, had suggested that IRF5 plays a role in inducing antiviral responses (Barnes BJ et al. 2003). These findings have been then confirmed in IRF-5-/- mice that are highly susceptible to viral infection accompanied by a decrease in type I IFN induction in the sera (Yanai H et al. 2007; Yasuda K et al. 2007). These studies have also indicated a cell- type-specific role of IRF-5 in cytokine gene induction, mainly affecting DCs and pDCs, rather than macrophages (Schoenemeyer A et al. 2005; Paun A et al. 2008). The exact mechanisms of activation and function of IRF-5, including its relationship with NF-κB and 28 Angela Battistini

IRF-7, remain, however, to be exactly defined (Negishi H et al. 2006; Yasuda K et al. 2007; Barnes BJ et al. 2003; Paun A et al. 2008). Studies In IRF-5-/- mice have also demonstrated that the TLR3-mediated induction of proinflammatory cytokine genes is, similarly dependent on IRF-5 (Takaoka A et al. 2005), indicating that IRF-5 is involved in TRIF-mediated signaling pathways. In this respect, IRF-5 has been proposed also as an important mediator in TLR synergy, mediating the crosstalk between the MyD88 and TRIF pathways. Under physiological conditions, it is likely that immune cells simultaneously detect several PAMPs via more than one of TLRs and produce a response more complex than that mediated by a single TLR. IRF-5 seems an essential transcription factor for this synergism in the induction of specific target genes as IL-6, IL- 12p40, and IL-23p19 gene expression, downstream of both adaptors (Ouyang X et al. 2007). Recently, the role of IRF-5 in host defense mediated by TLR engagement has been extended to parasitic infection. It has indeed been reported that TLR7-mediated activation of IRF-5 is essential for the development of host-protective Th1 responses to L. Donovani at late stages of infection (Paun A et al. 2011). IRF-5 is also involved in the RIG-I signaling pathway (Yanai H et al. 2007; Paun A et al. 2008). IRF5-/- mice are highly vulnerable to infection with VSV or NDV in a cell-type specific manner, in that only macrophages but not MEF from these mice are defective in type I IFN production upon viral challenge. Phoshphorylation and nuclear translocation of IRF- 5 upon infection with these viruses was also demonstrated (Yanai H et al. 2007). The mechanism of IRF-5 activation by RIG-I and its specific contribution to transcriptional regulation of Type I IFN and pro-inflammatory cytokines are however, still poorly defined. Indeed, although IRF-5 like IRF-3 and IRF-7 is phosphorylated by IKK and TBK1 in co- transfected cells, the phosphorylation of IRF-5 does not lead to IRF-5 nuclear localization or activation (Lin R et al. 2005). Moreover, NDV and the RIG-I pathway seem weaker activators of IRF-5 than TLR7/9-activated MyD88 pathway suggesting that IRF-5 may have a key role in the TLR but not in the RIG-I-mediated antiviral and inflammatory response. More recently, IRF-5 was also shown to cooperate with NOD2 and TBK1 in triggering expression of Type I IFN in response to Mycobacterium tuberculosis (Pandey AK et al. 2009). A specific role of IRF-5 in immune cell development is so far unknown. Recently, it has been, however, reported that IRF-5 is involved in B-cell maturation and in the commitment to terminal B-cell differentiation by stimulation of Blimp-1 expression, a master regulator of plasma cell differentiation. IRF5-/- mice mice develop an age-related splenomegaly, associated with a disruption of normal splenic architecture and a dramatic accumulation of CD19+B220- B cells with a defect in TLR7 and TLR9-induced IL-6 production. Splenic B cells from IRF5-/- mice mice also exhibit a decreased level of plasma cells (Lien C et al. 2010). Furthermore, the involvement of IRF-5 in autoimmune diseases as systemic Lupus erythematosus (SLE) and the significance of IRF-5 polymorphisms in these pathologies have been suggested but this issue still remains to be fully addressed (Graham RR et al. 2006; Graham RR et al. 2007). The current conventional wisdom is that IRF-5 may drive SLE development by causing the aberrant production of type I IFN through TLR9 and/or TLR7 signaling activated by immune-complexes (Kyogoku C and Tsuchiya N 2007). Recently, in mice has been demonstrated a requirement for IRF-5 in the production of auto-antibodies of the IgG2a subtype, the most prominent isotype in inducing autoimmunity and IRF-5 is also Interferon Regulatory Factors in Immune Cell Development … 29 required for the secretion of pathogenic antibodies through its direct control of class switch recombination of the gamma2a locus (Savitsky DA et al. 2010). Finally, along with numerous studies on its role on the innate immune responses, compelling evidences also indicate that like IRF-1, IRF-5 is involved in DNA-damage responses and tumor suppression as extensively reviewed elsewhere (Tamura T et al. 2008; Takaoka A et al. 2008; Savitsky D et al. 2010).

IRF-6: The Morphogenic IRF

IRF-6 is a novel and unique member of the family that has not been linked to functions or regulatory pathways described for other members. IRF-6 is structurally related to IRF-5 but seems to have completely different functions that, together with regulation of its expression, remain to be exactly defined (Bailey CM and Hendrix MJ 2008). Early studies in humans regarding mutations in IRF-6 indicated that IRF-6 is involved in two related syndromes, the Van der Woude syndrome, a rare autosomal dominant developmental malformation usually associated with bilateral lower lip pits and the popliteal pterygium syndrome (VWS) a disorder with a similar orofacial phenotype that also includes skin and genital anomalies. The gene encoding IRF-6 is located in the critical region for the Van der Woude syndrome locus at chromosome 1q32–q41. Expression analyses showed that high levels of IRF-6 mRNA are associated with the medial edge of the fusing palate, tooth buds, hair follicles, genitalia and skin. The haploinsufficiency of IRF-6 disrupts orofacial development and is consistent with dominant-negative mutations disturbing development of the skin and genitalia (Kondo S et al. 2002; Dissemond J et al. 2004). Since these initial observations, multiple reports have been published demonstrating novel mutations in the IRF- 6 gene locus that cause VWS or its more severe counterpart Popliteal Pterygium Syndrome (Bailey CM and Hendrix MJ 2008). Studies in mice that fail to express functional IRF-6 confirmed its morphogenetic role. IRF-6-/- mice die shortly after birth due to complications from a severe skin defect. These have abnormal skin, limb and craniofaccial development along with a defect in keratinocytes differentiation. The major histological feature is the absence of a normal stratified epidermis and keratinocytes do not stop to proliferate and fail to differentiate (Ingraham CR et al. 2006; Richardson RJ et al. 2006). Recent attempts to identify molecular mechanisms responsible for these IRF-6 activities indicated that the proliferation-differentiation balance of keratinocytes essential for palate fusion and skin differentiation could be, at least in part, determined by reciprocal regulation of IRF-6 and the DeltaNp63 isoform of p63 in which p63 stimulates IRF-6 expression that in turn induces proteasome-mediated DeltaNp63 degradation allowing keratinocytes to exit the cell cycle, thereby limiting their ability to proliferate (Moretti F et al. 2010; Thomason HA et al. 2010). Moreover, IRF-6 has been reported as a primary Notch target in keratinocytes and keratinocyte-derived SCC cells which contributes to the role of this pathway in differentiation and tumor suppression (Restivo G et al. 2011). A role of IRF-6 in tumor suppression related to its role in cell cycle arrest has, thus, been suggested and extended to mammary carcinoma cells. IRF-6 has been shown to act in a coordinated manner with maspin, a known tumor suppressor in normal breast tissue, in promoting mammary epithelial cell differentiation by acting on the cell cycle. This interaction occurs via the conserved IAD of IRF-6 and is 30 Angela Battistini regulated by IRF-6 phosphorylation. The loss of the maspin-IRF-6 interaction may promote neoplastic transformation (Bailey CM and Hendrix MJ 2008). Consistently, a function for IRF-6 in suppression of tumorigenesis in stratified epithelia has been reported. Genome-wide analysis in primary human keratinocytes, indeed, identified a subset of direct IRF-6 target genes. Most of these genes are involved in cell cycle, cell adhesion and motility and control of epidermal precursor proliferation/differentiation switch (Botti E et al. 2011). To date, IRF-6 expression has only been reported in epithelial-derived tissues and major focus of IRF-6 research defined its essential role in normal epidermal development and differentiation, while much less is known regarding its role, if any, in immune cell development and immune responses. Nevertheless, the role of IRF-6 in keratinocytes, indirectly supports a possible role in innate immune responses mediated by epitelial cells that have functional TLRs and that are able to produce antimicrobial substances to kill pathogenic bacteria and fungi (Mempel M et al. 2003; Pivarcsi A et al. 2003; Furrie E et al. 2005). In this respect, IRF-6 translocation into the nucleus in response to TLR3-ligand poly I:C in epitelial cells has recently been reported (Bailey CM and Hendrix MJ 2008). The direct role of IRF-6 in TLR-mediated host immune responses against pathogens and whether or not nuclear IRF-6 promotes quiescence and differentiation in these cells through IFN and IFN-associated genes expression, remain to be established.

Viral Evasion and Subversion of IRF Activity

As the host has evolved factors and strategies for detecting and responding to viral infections, viruses have developed countermeasures to inhibit these processes. Viral strategies can involve both evasion/inhibition and subversion of host components to viral own advantage. Multiple strategies may be utilized by the same virus as well as common strategies are used by different viruses in order to evade the immune system and to establish long-term latency in the host (Garcia-Sastre A. 2002; Katze MG et al. 2002; Levy DE and Garcia-Sastre A 2001; Unterholzner L and Bowie AG 2008). Due to genomic constraints, many pathogenic viruses focus their efforts on a small number of host targets that are key players in the antiviral response. Thus, common host molecules that connect virus recognition to downstream effectors of type I IFN production and inflammatory cytokine expression are usually targeted by viral immune evasion proteins. In this respect, transcription factors are particularly good targets for viruses that need to prevent the activation of the innate immune system with a limited number of viral protein products. Given the important and unique functions exerted by IRFs on the immune system, it is expected that several viruses have evolved strategies aimed at the attenuation, destruction or hijacking of IRFs activities. IRF-3 and IRF-7 as crucial factors for the initial induction of type I IFN during viral infection, as well as direct effectors of some IFN activities, are prime candidates for viral inhibition. They are indeed directly or indirectly inactivated by viral factors. As indirect mean to disarm them, the phosphoproteins of many non-segmented negative strand RNA viruses, such as Rabies virus, Borna disease virus and Ebola virus prevent the activation of IRF-3 by inhibiting the kinase TBK1 and thus IRF-3 phosphorylation and nuclear translocation (Hartman AL et al. 2008; Unterstab G et al. 2005; Brzózka K et al. 2005). West Nile virus (WNV) a highly pathogenic recently re-emergent flavivirus, instead, delays activation of IRF- Interferon Regulatory Factors in Immune Cell Development … 31

3 (Fredericksen BL and Gale M Jr 2006). The WNV nonstructural (NS)-1 protein, indeed, inhibits nuclear translocation of IRF-3 and NF-B upon TLR3 ligation by poly(I:C) (Wilson JR et al. 2008). To inactivate antiviral innate immunity some viruses also exploits the host SUMOylation system as also an integral part of IRF-3 and IRF-7 activity regulation. VSV or EMCV- induced SUMOylation of IRF-3 and IRF-7 as consequence of TLR and RIG-I activation but not of IFN signaling was, recently, reported (Kubota T, et al 2008). The VP35, dsRNA binding protein, a component of filoviruses Ebola and Marburg virus nucleocapsid complex has been shown to block recruitment of IRF-7 to the IFN- and IFN- genes, in murine DCs, by interacting with the SUMO E2 enzyme Ubc9, and the E3 ligase PIAS1 and promoting SUMOylation of IRF-7 thus inhibiting IFN transcription. A similar effect of VP35 was observed for IRF-3 (Chang TH et al. 2009). The VP35 protein, on the other hand, has been shown to inhibit the activation of IRF-3 also by binding to dsRNA and inhibiting RIG-I signaling (Basler CF et al. 2000). VP35, also acts as a pseudo-substrate for IKK and TBK1 kinases thus inhibiting both IRF-3/7 activation (Prins KC et al. 2009; Basler CF and Amarasinghe GK 2009). TBK1 and DEAD box protein 3 (Ddx3) kinases are also inhibited by vaccinia virus-encoded N1L and K7 proteins resulting in IRF-3/-7 lack of activation (DiPerna G et al. 2004; Schroder M et al. 2008). More recently, another vaccinia virus protein, the virulence factor C6 has been shown to inhibit IRF-3 and IRF-7 activation at the level of the TBK1/IKKε complex (Unterholzner L et al. 2011). As Ebola VP35 protein, the V protein from several paramyxoviruses, including mumps and parainfluenza 5, inhibits dsRNA- mediated signaling, acting as IRF-3 mimics that compete with cellular IRF-3 for phosphorylation by IKKε /TBK1 (Lu LL, et al. 2008). The Measles Virus V protein abolishes TLR7/9/MyD88-dependent IFN induction in human pDC by competing with IRF-7 for the binding to IKKα, the other kinase in the pathway that phosphorylates IRF-7. Moreover, it also binds to IRF-7 directly quenching IRF- 7 transcriptional activity (Pfaller C. K and Conzelmann KK 2008). The human parainfluenza virus type 2 V protein prevents TLR7- and TLR9-dependent IFN-α induction in pDCs by binding to IRF-7 through the Trp-rich motif in the C-terminal domain unique to V protein, but instead to directly block IRF-7 transcriptional activity, specifically affects TLR7/9- dependent IRF-7 activation processes (Kitagawa Y et al. 2011). Enterovirus 71(EV71), a member of the Picornaviridae family, that causes hand-foot-and- mouth disease and neurological complications in young children, inactivates IRF-3 and drastically suppresses interferon-stimulated gene expression. This involves the EV71 3C protein, which upon association with RIG-I disrupts the formation of a functional RIG-I complex. This precludes the recruitment of the adaptor IPS-1 by RIG-I and subsequent nuclear translocation of IRF-3 (Lei X et al. 2010). V71 3C also interacts with the TRIF adaptor in TLR signaling and induces its cleavage (Lei X et al. 2011). Even if not directly checked, it is conceivable that these mechanisms also result in impairment of IRF-7 activation, and thus despite viral engagement of different PRRs, the expression of IFN- is not stimulated. The viral gene products derived from ORF3b, ORF-6 and N proteins of severe acute respiratory syndrome coronavirus (SARS-Co) inhibit both interferon synthesis and signaling through inhibition of IRF-3 phosphorylation and nuclear translocation (Kopecky-Bromberg SA et al. 2007). Activation of IFN- IRF-3-dependent promoters, as well as nuclear 32 Angela Battistini translocation of IRF-3 was similarly blocked by the nucleoprotein (NP) of Lymphocytic Choriomeningitis virus, a prototypic arenavirus (Martínez-Sobrido L et al. 2007). Activation of IRF-3 and/or IRF-7 is also prevented by Non-structural proteins 3 and 4 (NS3/4A) of Hepatitis C virus (HCV) that cleave the TLR adaptors MAVS/IPS-1/VISA/CARDIF and TRIF (Li K et al. 2005; Seth RB et al. 2006; Meylan E, et al. 2005; Lin R et al. 2006). The HCV NS3 protein also interacts directly with TBK1 inhibiting association between TBK1 and IRF-3, and thus IRF-3 activation (Otsuka M et al. 2005). IRF-3 is also inhibited by Varicella- zoster virus (VZV), an alphaherpesvirus that is restricted to humans. The viral immediate- early protein 62 (IE62), even in the absence of viral replication, was sufficient to block TBK1-mediated IFN- secretion and IRF-3 function. IE62-mediated inhibition was mapped to blocking phosphorylation of at least three serine residues on IRF-3. However, the exact mechanism has not been elucidated since IE62 binding to TBK1 or IRF-3 was not detected and it was shown that IE62 does not perturb TBK1-IRF-3 complex formation (Sen N et al. 2010). The NS1 protein from Influenza virus, in addition to sequestering dsRNA, the viral mediator of RIG-I activation, directly interacts with RIG-I/MAVS blocking IRF-3 binding and activation (Mibayashi M, et al. 2007). The adaptor MAVS is also targeted by the PICORNA Hepatitis A virus through its 3ABC protein that degrades MAVS in the mithocondria (Yang Y, et al. 2007). The existence of an evasion mechanism based on the inhibition of the RIG-I sensor through the action of the HIV-1 protease (PR) has been also described. The ectopic expression of PR resulted in the inhibition of IRF-3 phosphorylation and in the decreased expression of IFN and interferon-stimulated genes (Solis M et al. 2011). The other cytosolic RNA helicase, MDA5, is targeted by the V protein from several viruses including Mumps virus, Sendai virus, Paramyxovirususes Simian virus 5, Human parainfluenza virus 1, and Hendra virsu that directly interact with MDA5 but with not the sister cytosolic RNA sensor, RIG-I (Andrejeva J et al. 2004). Instead of inhibiting IRF-3 activation, some viruses eliminate IRF-3 altogether. In the early steps of HIV-1 infection the relative levels of IRF-3 protein are decreased due to the ubiquitin-associated proteasome degradation induced by HIV-1 proteins Vpr and Vif (Okumura A et al. 2008; Doehle BP et al. 2009). Similarly, ICP0 protein from bovine herpes virus as well as the N (pro) protein of pestivirus classical swine fever virus (CSFV) interact with IRF-3 and mediate its proteasome-dependent degradation (Saira K et al. 2007; Bauhofer O et al. 2007). In addition, the same virus CSFV causes an inhibition of transcription of the IRF-3 gene in infected cells. (La Rocca SA et al. 2005). The ICP0 of Human Herpes Virus 1 also interacts with IRF-3 but instead of degrading it, it sequesters IRF-3 and its coactivator CBP/p300 in nuclear bodies preventing the binding to target DNA (Melroe GT et al. 2007) thus indicating that the mechanism of viral evasion mediated by ICP0 appears to be only partially conserved between herpes viruses. Competition with IRF-3 for the CBP/p300 binding is another mechanism to impair IRF-3 activity. It is exerted by the adenovirus E1A gene product (Juang YT et al. 1998) and by the E6 protein from the oncogenic human papillomavirus type 16 (HPV-16) that bind both CBP and p300 and inhibit induction of IFN- mRNA following Sendai virus infection (Ronco LV, et al. 1998; Patel D et al. 1999). Interaction between IRF-3 and the cotranscriptional activator CBP is also targeted by a conserved herpesviral kinase, designated ORF36 in murine - herpesvirus 68 (MHV-68), that specifically binds to the activated form of IRF-3 in the Interferon Regulatory Factors in Immune Cell Development … 33 nucleus, inhibiting IRF-3/CBP interaction and thereby suppressing the recruitment of RNA polymerase II to the IFN- promoter (Hwang S et al. 2009). Rotavirus NSP1 antagonizes IFN signaling by inducing degradation of not only IRF-3 but also IRF-5 and IRF-7. This multiple attack could allow the virus to replicate in specialized trafficking cells (e.g. macrophages and dendritic cells) and to cross the gut barrier (Barro M and Patton JT 2005; GraV JW et al. 2002; Barro M and Patton JT 2007). Accelerated proteasome-dependent degradation of IRF-7 in most cell types, except lymphoid tissues infected with NDV, has also been reported. (Prakash A and Levy DE 2006). Several viruses also target IRF-1. The core structural protein of HCV, at variance with HCV nonstructural proteins that target IRF-3, exerts a transcriptional repression of several interferon-stimulated genes including IL-15, IL-12, and LMP2, by suppressing IRF-1 synthesis (Ciccaglione AR et al. 2007). In T cells, IRF-1 is targeted by HIV-1 and hijacked on LTR to stimulate viral transcription in the early phases of infection (Sgarbanti M et al. 2002,2008). Interestingly, recently it has been reported that also in monocyte-derived DCs, HIV-1 exploits IRF-1 to subvert the IFN induction pathway and to increase its own replication early after infection (Harman AN et al. 2011). IRF-1 is also specifically targeted by the HIV-1 trans-activator Tat that sequesters it quenching its transcriptional activity on target genes (Remoli AL et al. 2006). On the other hand, interactions between Tat and IRF-1 could also be exploited to host’s own advantage since IRF-1 has been shown to act as a genetic adjuvant in Tat-based vaccination strategies, increasing Th1 and CTL responses induced by Tat used as an antigen (Castaldello A et al. 2010). All these described anti-IFN activity being critical for evasion of host immunity in the context of some infections may, however, also contribute to persistence of viral infection and to virus-induced carcinogenesis as in the case of Epstein Barr virus (EBV), Kaposi sarcoma- associated herpes virus (KSHV)/HHV8 (Human Herpes virus 8) and Human Papilloma virus (HPV). EBV can hijack IRF-7 to regulate its replication and induce transformation. Interestingly, IRF-7 was initially identified as a factor able to bind the Qp promoter in EBV type III latency (Zhang L and Pagano JS 1997). EBV-encoded latent membrane protein (LMP)-1, the major EBV oncoprotein, and one of the key viral proteins required for the transformation of primary B cells (Zhang L et al. 2004) is able to induce the expression of IRF-7 and activate it through receptor interactin protein (RIP)-1 and TRAF6. This LMP-1-mediated activation of IRF-7 is thought to increase the EBV transformation process (Song YJ et al. 2008; Zhang L, et al. 2004). The induction of IRF-7 may, indeed, cause the silencing of Qp promoter in EBV type III latency and it has been suggested that along with LMP1 could mediate EBV transformation. At the same time, the EBV immediate early protein BZLF-1 binds IRF-7 directly preventing it from activating type I IFN promoters (Hahn AM, et al. 2005; Ning S, et al. 2011). Another oncogenic DNA virus, the KSHV/HHV8, encodes multiple regulatory proteins, including four IRFs viral homologues, expressed during the lytic replication cycle or in the latent phase capable of modulating the antiviral response of the host. The K-bZIP, that is turned on by the immediate-early replication and transcription factor activator (RTA) competes with IRF-3 for binding sites in the IFN- promoter, blocking promoter activation in response to multiple stimuli and also prevents the activation of RANTES and CXCL11 primary IRF-3 target genes (Lefort S et al. 2007). Similarly, KSHV latency-associated 34 Angela Battistini nuclear antigen (LANA-1) inhibits IFN- transcription and synthesis by competing with the binding of IRF-3 to the IFN- promoter (Cloutier N and Flamand L 2010). Recently, it has been reported that RTA also blocks innate immunity by targeting cellular TRIF and specifically degrading TRIF by shortening its half-life without, however, direct interaction (Ahmad H et al. 2011). IRF-7 is targeted by at least two proteins from KSHV/HHV8. The RTA was first documented as an E3 ligase that promoted IRF-7 ubiquitination and proteasome-mediated degradation (Yu Y et al. 2005). The viral immediate-early protein, namely ORF45, instead, interacts with IRF-7 and in consequence, IRF-7 phosphorylation is inhibited and the accumulation of IRF-7 in the nucleus in response to viral infection is blocked (Zhu FX, et al. 2002). HHV8 also encodes four viral interferon regulatory factors (vIRF-1-4) analogues, three of which have been cloned and characterized as dominant negative factors that directly interfere with host IRFs to inhibit virus-mediated induction of type I IFN genes (Cunningham C et al. 2003). In particular, vIRF-1 and v-IRF-2 act as dominant-negative inhibitors of IRF-1 and IRF-3 by preventing their association with coactivators CREB-binding protein (CBP)/p300) (Burysek L et al. 1999; Lin R et al. 2001; Burysek L et al. 1999). Moreover, vIRF-2 targets STAT1 and IRF-9 components of ISGF3 complex (Mutocheluh M et al. 2011). v-IRF-3 also known as LANA-2, which is expressed in human B cell lymphoma-Primary effusion lymphoma (PEL) lines and is required for their continuous proliferation, interacts with and inhibits the DNA binding activity of IRF-5 and IRF-7 suppressing IFN- and ISGs production (Wies E et al. 2009; Joo CH et al. 2007). The function of vIRF-3/IRF-5 interaction is, however, not limited to the IFN system as IRF5- mediated cell growth regulation was significantly altered by over-expression of v-IRF-3 in B cells and vIRF3 was able to prevent IRF5-mediated growth inhibition and G2/M cell cycle arrest (Bi X et al. 2011). Thus, the role of KSHV/HHV8-encoded IRFs, as the IRF-7 piracy by EBV, is not limited to inhibition of the IFN response, but can contribute to the persistence of viral infections and to virus-induced carcinogenesis. This is the case also of HPV. The HPV16 encoded E7 proteins binds to and inactivates IRF-1 inhibiting IRF1-mediated antioncogenic activity (Park JS et al. 2000; Perea SE et al. 2000) and thereby overcoming host immunity against cervical tumor development. Intriguingly, another protein of HPV-16, the E5 protein that is expressed before virus integration and plays an important role in the early phases of tumorigenesis, instead of repressing, stimulates IFN-β and ISGs expression through the induction of IRF-1 (Muto V et al. 2011). However, this IFN signature, instead of protecting cells from infection, has been associated with loss of HPV16 episomes and cervical tumor progression (Pett MR et al. 2006), thus confirming that HPV-targeting of IRF-1 at several levels may be considered a mechanism of progression to transformation. IRF-9, component of the ISGF-3 complex in the type I IFN signaling, is dismantled by the HPV E7 oncoprotein by direct protein-protein interaction and inhibition of nuclear translocation that results in loss of the ISGF3 complex formation and activity (Barnard P and McMillan NA 1999). Human cytomegalovirus instead disrupts multiple levels of the IFN- signal transduction pathway by decreasing the expression of Jak-1 and IRF-9 (Miller DM et al. 1999). Finally, IRFs can be targeted also by viral-encoded miRNA an emerging intense area of research. Very recently, miR-K12-11, a viral miRNA encoded by Kaposi's sarcoma- associated herpesvirus (KSHV) has been shown as critical for the modulation of IFN signaling that acts through targeting IKK and by decreasing IKK-mediated IRF-3/IRF-7 Interferon Regulatory Factors in Immune Cell Development … 35 phosphorylation. This resulted in suppression of antiviral immunity and together with other viral strategies, indicated above, may contribute to maintenance of latency (Liang D et al. 2011). It is predictable that this field of research will soon expand and increasing number of viral encoded miRNA, affecting IRF expression and activity, will be identified.

Conclusion

A prompt and efficacious cellular response is key to host defense against pathogens and dangers. In the present chapter I have summarized our current knowledge of the characteristics of the IRF family members, of how, directly or indirectly, these transcription factors crucially impact a number of aspects of host defense and of how their activities are hijacked or subverted by viruses. Since their discovery more than 20 years ago, members of this family of transcriptional regulators have grown remarkably in the number and diversity of functions. Although the first two members, IRF-1 and IRF-2, were originally identified as intracellular mediators of the induction and biological activities of IFN, studies in the past several years have provided strong evidences that the IRF proteins play key roles in a number of aspects of host defense beyond their functions in the IFN system. These range from activation or attenuation of immune responses by essentially all IRFs, to regulation of the development of hematopietic and epitelial cells by specifically IRF-1, -2, -4, -6 and -8, with overlapping as well as unique functions. In recent years, these factors gained a renewed attention with the recognition of their involvement in signaling pathways arising from pattern recognition receptors and danger signals. Multiple IRFs share signaling molecules in these pathways and are activated upon different as well as common stimuli and function as a platform to induce an overlapping but distinct set of genes to evocate the appropriate response. Each member of the family, indeed, exerts various distinct regulatory effects and may regulate positively or negatively target genes depending on cells in which is expressed, promoter context or association with other transcription factors. Through both their common and specific features IRFs thus contribute to the establishment of the necessary diversity in the host defense system. Given their crucial and unique functions on the immune system it is thus not surprising that pathogens put forward a multitude of successful strategies aimed at the attenuation, destruction or hijacking of IRF activities. Furthermore, IRF functions are not limited to the host immune response against pathogens. Although only briefly sketched or not touched at all in this chapter, there are a number of studies that have demonstrated the role of IRFs in regulation of cell growth restriction and apoptosis and thus in oncogenesis and antitumor immunity as well as in allergy, autoimmunity and inflammatory diseases. Further studies aimed at the elucidation of the full range of target genes triggered by IRFs often in a cell type- and expression level- specific fashion, as well as further insights into the cross-talk with other transcriptional regulator, functional deregulation of IRF activation and significance of IRF polymorphisms in diseases, will undoubtedly have important therapeutic implications for many human diseases. Translation of the basic knowledge on the multifarious activities of IRFs into novel therapeutic strategies for the treatment not only of infectious and inflammatory diseases but also of autoimmunity and cancer thus represents a future challenging goal.

36 Angela Battistini

Acknowledgments

I apologize to all of the Authors whose outstanding papers, significant to the subject, could not be cited or discussed here due to space constraints. I thank all members of my laboratory in particular A. Fragale, G. Marsili, R. Orsatti, E. Perrotti, A.L. Remoli and M. Sgarbanti for their dedication, stimulating discussions and support and Sabrina Tocchio for invaluable editorial assistance. This work was in part supported by grants from ACC-ISS program 3 and from the AIDS project -Ministry of Health, Italy

References

Abdollahi, A; Lord, KA; Hoffman-Liebermann, B; Liebermann, DA. Interferon regulatory factor 1 is a myeloid differentiation primary response gene induced by interleukin 6 and leukemia inhibitory factor: role in growth inhibition. Cell Growth Differ. 1991, 2: 401- 407. Ahmad, H; Gubbels, R; Ehlers, E; Meyer, F; Waterbury, T; Lin, R; and Zhang, L. Kaposi Sarcoma-associated Herpes virus Degrades Cellular Toll-Interleukin-1 Receptor Domain- containing Adaptor-inducing -Interferon (TRIF). J. Biol. Chem. 2011, 286:7865-7872. Ahyi, AN; Chang, HC; Dent, AL; Nutt, SL; Kaplan, MH. IFN regulatory factor 4 regulates the expression of a subset of Th2 cytokines. J. Immunol. 2009, 183:1598-606. Akira, S; Uematsu, S; Takeuchi, O. Pathogen recognition and innate immunity. Cell 2006, 124:783-801. Alexopoulou, L; Holt AC; Medzhitov, R; Flavel, RA. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 2001, 413:732–738. Aliberti, J; Schulz, O; Pennington, DJ; Tsujimura, H; Reis e Sousa, C; Ozato, K; Sher, A. Essential role for ICSBP in the in vivo development of murine CD8+ dendritic cells. Blood 2003, 101:305-310. Alter-Koltunoff, M; Ehrlich, S; Dror, N; Azriel, A; Eilers, M; Hauser, H; Bowen, H; Barton, CH; Tamura, T; Ozato, K; Levi, BZ. Nramp1-mediated innate resistance to intraphagosomal pathogens is regulated by IRF-8, PU.1, and Miz-1. J. Biol. Chem. 2003, 278:44025-44032. Andersen, J; VanScoy, S; Cheng, TF; Gomez, D; Reich, NC. IRF-3-dependent and augmented target genes during viral infection. Genes Immunol. 2008, 9:168-175. Andrejeva, J; Childs, KS; Young, DF; Carlos, TS; Stock, N; Goodbourn, S; Randall, RE. The V proteins of paramyxoviruses bind the IFN-inducible RNA helicase, mda-5, and inhibit its activation of the IFN-beta promoter. Proc. Natl. Acad. Sci. USA 2004, 101:17264- 17269. Ardavin, C. Origin, precursors and differentiation of mouse dendritic cells. Nat. Rev. Immunol. 2003, 3:582-590. Au, WC; Moore, PA; Lowther, W; Juang, YT; Pitha, PM. Identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes. Proc. Natl. Acad. Sci. USA 1995, 92:11657-11661. Interferon Regulatory Factors in Immune Cell Development … 37

Au, WC; Moore, PA; LaFleur, DW; Tombal, B; Pitha, PM. Characterization of the interferon J. Biol. Chem. 1998, 273:29210-29217. Au, WC; Yeow, WS; Pitha, PM. Analysis of functional domains of interferon Virology 2001, 280:273-282. Bailey, CM; Abbott, DE; Margaryan, NV; Khalkhali-Ellis, Z; Hendrix, MJ. Interferon regulatory factor 6 promotes cell cycle arrest and is regulated by the proteasome in a cell cycle dependent manner. Mol. Cell. Biol. 2008a, 28:2235-2243. Bailey, CM; Hendrix, MJ. IRF6 in development and disease: a mediator of quiescence and differentiation. Cell Cycle 2008b, 7:1925-1930. Balkhi, MY; Fitzgerald, KA; Pitha, PM. Functional regulation of MyD88-activated interferon regulatory factor 5 by K63- linked polyubiquitination. Mol. Cell Biol, 2008, 28:7296– 7308. Balkhi, MY; Fitzgerald, KA; Pitha, PM. IKKalpha negatively regulates IRF-5 function in a MyD88-TRAF6 pathway. Cell Signal. 2010, 22:117-127. Barber, GN. Cytoplasmic DNA innate immune pathways. Immunol. Rev. 2011, 243:99-108. Barnes, BJ; Moore, PA; Pitha, PM. Virus-specific activation of a novel interferon regulatory factor, IRF-5, results in the induction of distinct interferon alpha genes. J. Biol. Chem. 2001, 276: 23382-23390. Barnes, B; Lubyova, B; Pitha, PM. On the role of IRF in host Barnes, BJ; Kellum, MJ; Field, AE; Pitha, PM. Multiple regulatory domains of IRF-5 control activation, cellular localization, and induction of chemokines that mediate recruitment of T lymphocytes. Mol. Cell Biol. 2002, 22:5721-5740. Barnes, BJ; Field, AE; Pitha-Rowe, PM. Virus-induced heterodimer formation between IRF-5 and IRF-7 modulates assembly of the IFNA enhanceosome in vivo and transcriptional activity of IFNA genes. J. Biol. Chem. 2003, 278:16630-16641. Barnes, BJ; Richards, J; Mancl, M; Hanash, S; Beretta, L; Pitha, PM. Global and distinct targets of IRF-5 and IRF-7 during innate response to viral infection. J. Biol. Chem. 2004, 279: 45194-45207. Barro, M; Patton, JT. Rotavirus nonstructural protein 1 subverts innate immune response by inducing degradation of IFN regulatory factor 3. Proc. Natl. Acad. Sci. USA, 2005; 102: 4114-4119. Barro, M; Patton, JT. Rotavirus NSP1 inhibits expression of type I interferon by antagonizing the function of interferon regulatory factors IRF3, IRF5, and IRF7. J. Virol., 2007; 81: 4473–4481. Basler, CF; Amarasinghe, GK. Evasion of interferon J. Interferon Cytokine. Res. 2009, 29: 511-520. Battistini, A. Interferon regulatory factors in hematopoietic cell differentiation and immune regulation. J. Interferon Cytokine Res. 2009, 29:765-780. Bauhofer, O; Summerfield, A; Sakoda, Y; Tratschin, JD; Hofmann, MA; Ruggli, N. Classical swine fever virus Npro interacts with interferon regulatory factor 3 and induces its proteasomaldegradation. J. Virol. 2007, 81: 3087-3096. Belgnaoui, SM; Paz, S; Hiscott, J. Orchestrating the interferon Curr. Opin. Immunol, 2011; 23: 564-572. Belz, GT; Nutt, SL.Transcriptional programming of the dendritic cell network. Nat. Rev. Immunol. 2012, 12:101-113. doi: 10.1038/nri3149. 38 Angela Battistini

Bettermann, K; Benesch, M; Weis, S; Haybaeck, J. SUMOylation in carcinogenesis. Cancer Lett. 2012, 316: 113-125. Bi, X; Yang, L; Mancl, ME; Barnes, BJ. Modulation of interferon regulatory factor 5 activities by the Kaposi sarcoma-associated herpesvirus-encoded viral interferon regulatory factor 3 contributes to immune evasion and lytic induction. J. Interferon Cytokine Res. 2011, 31:373-382. Bibeau-Poirier, A; Gravel, SP; Clement, JF; Rolland, S; Rodier, G; Coulombe, P; Hiscott, J; Grandvaux, N; Meloche, S; Servant, MJ. Involvement of the IκB kinase (IKK)-related kinasesTank-binding kinase 1/IKKi and cullin-based ubiquitin ligases in IFN regulatory factor-3 degradation. J. Immunol. 2006, 177: 5059-5067. Birnbaum, MJ; van Zundert, B; Vaughan, PS; Whitmarsh AJ; van Wijnen, AJ; Davis, RJ; Stein, GS; Stein, JL. Phosphorylation of the oncogenic transcription factor interferon regulatory factor 2 (IRF2) in vitro and in vivo. J. Cell Biochem. 1997, 66: 175-183. Bluyssen, AR; Durbin, JE; Levy, DE. ISGF3 gamma p48, a specificity switch for interferon activated transcription factors. Cytokine Growth Factor Rev. 1996, 7:11-17. Botti, E; Spallone, G; Moretti, F; Marinari, B; Pinetti, V; Galanti, S; De Meo, PD; De Nicola, F; Ganci, F; Castrignanò, T; Pesole, G; Chimenti, S; Guerrini, L; Fanciulli, M; Blandino, G; Karin, M; Costanzo, A. Developmental factor IRF6 exhibits tumor suppressor activity in squamous cell carcinomas. Proc. Natl. Acad. Sci. USA2011, 16:108:13710-13715. Brass, AL; Kehrli, E; Eisenbeis, CF; Storb, U; Singh, H. Pip, a lymphoid-restricted IRF, contains a regulatory domain that is important for autoinhibition and ternary complex formation with the Ets factor PU.1. Genes Dev. 1996, 10: 2335-2347. Brass, AL; Zhu, AQ; Singh, H. Assembly requirements of PU.1-Pip (IRF-4) activator complexes: inhibiting function in vivo using fused dimers. EMBO J. 1999, 18: 977-991. Brüstle, A; Heink, S; Huber, M; Rosenplanter, C; Stadelmann, C; Yu, P; Arpaia, E; Mak, TW; Kamradt, T; Lohoff, M. The development of inflammatory T(H)-17 cells requires interferon-regulatory factor 4. Nat. Immunol. 2007, 8:958-966. Brzózka, K; Finke, S; Conzelmann, KK. Identification of the rabies virus alpha/beta interferon antagonist: phosphoprotein P interferes with phosphorylation of interferon regulatory factor 3. J. Virol. 2005, 79:7673-7681. Burysek, L; Yeow, WS; Lubyova, B; Kellum, M; Schafer, SL; Huang, YQ; Pitha, PM. Functional analysis of human herpesvirus 8-encoded viral interferon regulatory factor 1 and its association with cellular interferon regulatory factors and p300. J. Virol. 1999, 73:7334-7342. Cai, Z; Shi, Z; Sanchez, A; Zhang, T; Liu, M; Yang, J; Wang, F; Zhang, D. Transcriptional regulation of Tlr11 gene expression in epithelial cells. J. Biol. Chem. 2009, 284:33088- 33096. Caillaud, A; Prakash, A; Smith, E; Masumi, A; Hovanessian, AG; Levy, DE; and Marie, IJ. Acetylation of interferon regulatory factor-7 by p300/CREB-binding protein (CBP)- associated factor (PCAF) impairs its DNA binding. J. Biol. Chem. 2002, 277: 49417- 49421. Caillaud, A; Hovanessian, AG; Levy, DE; Marie, IJ. Regulatory serine residues mediate phosphorylation-dependent and phosphorylation-independent activation of interferon regulatory factor 7. J. Biol. Chem. 2005, 280: 17671-17677. Interferon Regulatory Factors in Immune Cell Development … 39

Castaldello, A; Sgarbanti, M; Marsili, G; Brocca-Cofano, E; Remoli, AL; Caputo, A; Battistini, A. Interferon Regulatory Factor-1 Acts as a Powerful Adjuvant in tat DNA Based Vaccination. J. Cell. Physiol. 2010, 224: 702-709. Cattoretti, G; Shaknovich, R; Smith, PM; Jäck, HM; Murty, VV; Alobeid, B. Stages of germinal center transit are defined by B cell transcription factor coexpression and relative abundance. J. Immunol. 2006, 177:6930-6939. Chae, M; Kim, K; Park, MS; Jang, IS; Seo, T; Kim, DM; Kim, IC; Lee, H; Park, J. IRF-2 regulates NF-kappaB activity by modulating the subcellular localization of NF-kappaB. Biochem. Biophys. Res. Commun. 2008, 370: 519-524. Chang, CH; Hammer, J; Loh, JE; Fodor, WL; Flavell, RA. The activation of major histocompatibility complex class I genes by interferon regulatory factor-1 (IRF-1). Immunogenetics 1992, 35:378-384. Chang, TH; Kubota, T; Matsuoka, M; Jones, S; Bradfute, SB; Bray, M; Ozato, K. Ebola Zaire virus blocks type I interferon production by exploiting the host SUMO modification machinery. PLoS Pathog. 2009, 5: e1000493. Chen, Q; Yang, W; Gupta, S; Biswas, P; Smith, P; Bhagat, G; Pernis, AB. IRF-4-binding protein inhibits interleukin-17 and interleukin-21 production by controlling the activity of IRF-4 transcription factor. Immunity 2008, 29:899-911. Chen, W; Lam, SS; Srinath, H; Jiang, Z; Correia, JJ; SchiVer, CA; Fitzgerald, KA; Lin, K; Royer, WE Jr. Insights into interferon regulatory factor activation from the crystal structure of dimeric IRF5. Nat. Struct. Mol. Biol. 2008, 15:1213-1220. Chen, W; Royer WE Jr. Structural insights into interferon regulatory factor activation. Cell Signal. 2010, 22:883-887. Cheng, TF; Brzostek, S; Ando, O; Van Scoy, S; Kumar, KP; Reich, NC. Differential activation of IFN regulatory factor (IRF)-3 and IRF-5 transcription factors during viral infection. J. Immunol. 2006, 176: 7462-7470. Childs, KS; Goodbourn, S. Identification of novel co-repressor molecules for Interferon Regulatory Factor-2. Nucleic Acids Res. 2003, 31: 3016-3026. Ciccaglione, AR; Stellacci, E; Marcantonio, C; Muto, V; Equestre, M; Marsili, G; Rapicetta, M; Battistini, A. Repression of interferon regulatory factor 1 by hepatitis C virus core protein results in inhibition of antiviral and immunomodulatory genes. J. Virol. 2007, 81:202-214. Cisse, B; Caton, ML; Lehner, M; Maeda, T; Scheu, S; Locksley, R; Holmberg, D; Zweier, C; den Hollander, NS; Kant, SG; Holter, W; Rauch, A; Zhuang, Y; Reizis, B. Transcription factor E2-2 is an essential and specific regulator of plasmacytoid dendritic cell development. Cell 2008, 135:37-48. Clement, JF; Bibeau-Poirier, A; Gravel, SP; Grandvaux, N; Bonneil, E; Thibault, P; Meloche, S; Servant, M.J. Phosphorylation of IRF-3 on Ser 339 generates a hyperactive form of IRF-3 through regulation of dimerization and CBP association. J. Virol. 2008, 82: 3984. Cloutier, N; Flamand, L. Kaposi sarcoma-associated herpesvirus latency-associated nuclear antigen inhibits interferon (IFN) beta expression by competing with IFN regulatory factor-3 for binding to IFNB promoter. J. Biol. Chem. 2010, 285:7208-7221. Coberley, CR; Kohler, JJ; Brown, JN; Oshier, JT; Baker, HV; Popp, MP; Sleasman, JW; Goodenow, MM. Impact on genetic networks in human macrophages by a CCR5 strain of human immunodeficiency virus type 1. J. Virol. 2004, 78:11477-11486. 40 Angela Battistini

Coccia, EM; Passini, N; Battistini, A; Sinigaglia, F; Rogge, L. Interleukin-12 induces expression of interferon regulatory factor-1 via signal transducer and activator of transcription-4 in human T helper type 1 cells. J. Biol. Chem. 1999a, 274:6698-6703. Coccia, EM; Del Russo, N; Stellacci, E; Orsatti, R; Benedetti, E; Marziali, G; Hiscott, J; Battistini, A. Activation and Repression of the 2-5A Synthetase and p21 Gene Promoters by IRF-1 and IRF-2. Oncogene 1999b, 18: 2129-2137. Coccia, EM; Stellacci, E; Marziali, G; Weiss, G; Battistini A. IFN-gamma and IL-4 differently regulate inducible NO synthase gene expression through IRF-1 modulation. Int. Immunol. 2000,12:977-985. Coccia, EM; Stellacci, E; Valtieri, M; Masella, B; Feccia, T; Marziali, G; Hiscott, J; Testa, U; Peschle, C, Battistini, A. Ectopic expression of interferon regulatory factor-1 potentiates granulocytic differentiation. Biochem J. 2001, 360:285-294. Cohen, H; Azriel, A; Cohen, T; Meraro, D; Hashmueli, S; Bech-Otschir, D; Kraft, R; Dubiel, W; Levi, BZ. Interaction between interferon consensus sequence-binding protein and COP9/signalosome subunit CSN2 (Trip15). A possible link between interferon regulatory factor signaling and the COP9/signalosome. J. Biol. Chem. 2000, 275:39081-39089. Colonna, M; Trinchieri, G; Liu YJ. Plasmacytoid dendritic cells in immunity. Nat. Immunol. 2004, 5: 1219-1226. Conrady, CD; Zheng, M; Fitzgerald, KA; Liu, C; Carr, DJ. Resistance to HSV-1 infection in the epithelium resides with the novel innate sensor, IFI-16. Mucosal. Immunol. 2012, doi: 10.1038/mi.2011.63. Cunningham, C; Barnard, S; Blackbourn, DJ; Davison, AJ. Transcription mapping of human herpesvirus 8 genes encoding viral interferon regulatory factors. J. Gen. Virol. 2003, 84:1471-1483. D’Cunha, J; Knight, E Jr; Haas, AL; Truitt, RL; Borden, EC. Immunoregulatory properties of ISG15, an interferon-induced cytokine. Proc. Natl. Acad. Sci. USA 1996, 93:211-215. Darnell, JE Jr; Kerr, IM; Stark, GR. Jak.-STAT pathways Science 1994, 264:1415-1421. Decker, T; Müller, M; Stockinger, S. The yin and yang of type I interferon activity in bacterial infection. Nat. Rev. Immunol. 2005, 5:675-687. Desterro, JM; Rodriguez MS; Hay RT. SUMO-1 modification of IkappaBalpha inhibits NF- kappaB activation. Mol. Cell. 1998, 2: 233-239. Dietrich, N; Lienenklaus, S; Weiss, S; Gekara, NO. Murine toll-like receptor 2 activation induces type I interferon responses from endolysosomal compartments. PLoS One. 2010, 5:e10250. DiPerna, G; Stack, J; Bowie, AG; Boyd ,A; Kotwal, G; Zhang, Z; Arvikar, S; Latz, E; Fitzgerald, KA; Marshall, WL. Poxvirus protein N1L targets the I-kappaB kinase complex, inhibits signaling to NF-kappaB by the tumor necrosis factor superfamily of receptors, and inhibits NF-kappaB and IRF3 signaling by toll-like receptors. J. Biol. Chem. 2004, 279: 36570-36578. Dissemond, J; Haberer, D; Franckson, T; Hillen, U. The Van der Woude syndrome: a case report and review of the literature. J. Eur. Acad. Dermatol. Venereol. 2004, 18:611-613. Dixit, E; Boulant, S; Zhang, Y; Lee, AS; Odendall, C; Shum, B; Hacohen, N; Chen, ZJ; Whelan, SP; Fransen, M; Nibert, ML; Superti-Furga, G; Kagan, JC. Peroxisomes are signaling platforms for antiviral innate immunity Cell 2010, 141: 668-681. Interferon Regulatory Factors in Immune Cell Development … 41

Doehle, BP; Hladik, F; McNevin, JP; McElrath, MJ; Gale, M Jr. Human immunodeficiency virus type 1 mediates global disruption of innate antiviral signaling and immune defenses within infected cells. J. Virol. 2009, 83:10395-10405. Doyle, S; Vaidya, S; O'Connell, R; Dadgostar, H; Dempsey, P; Wu, T; Rao, G; Sun, R; Haberland, M; Modlin, R; Cheng, G. IRF3 mediates a TLR3/TLR4-specific antiviral gene program. Immunity 2002, 17:251-263. Drew, PD; Franzoso, G; Carlson, LM; Biddison, WE; Siebenlist, U; Ozato, K. Interferon regulatory factor-2 physically interacts with NF-kappa B in vitro and inhibits NF-kappa B induction of major histocompatibility class I and beta 2-microglobulin gene expression in transfected human neuroblastoma cells. J. Neuroimmunol. 1995, 63:157-162. Eisenbeis, CF; Singh, H; Storb, U. Pip, a novel IRF family member, is a lymphoid-specific, PU.1-dependent transcriptional activator. Genes Dev. 1995, 9:1377-1387. Eklund, EA; Jalava A; Kakar R. PU.1, interferon regulatory factor 1, and interferon consensus sequence-binding protein cooperate to increase gp91phox expression. J. Biol. Chem. 1998, 273:13957-13965. Elco, CP; Guenther, JM; Williams, BRG; Sen, GC. Analysis of genes induced by Sendai virus infection of mutant cell lines reveals essential roles of interferon regulatory factor 3, NF-kappaB, and interferon but not toll-like receptor 3. J. Virol. 2005, 79:3920-3929. Elser, B; Lohoff, M; Kock, S; Giaisi, M; Kirchhoff, S; Krammer, PH; Li-Weber, M. IFN Immunity 2002, 17:703-712. Falini, B; Fizzotti, M; Pucciarini, A; Bigerna, B; MaraWoti, T; Gambacorta, M; Pacini, R; Alunni, C; Natali-Tanci, L; Ugolini, B; Sebastiani, C; Cattoretti, G; Pileri, S; Dalla- Favera, R; Stein, H. A monoclonal antibody (MUM1p) detects expression ofthe MUM1/IRF4 protein in a subset of germinal center B cells, plasma cells, and activated T cells. Blood 2000, 95:2084-2092. Farrow, MA; Kim, EY; Wolinsky, SM; Sheehy, AM. NFAT and IRF proteins regulate transcription of the anti-HIV gene, APOBEC3G. J. Biol. Chem. 2011, 286:2567-2577. Feng, J; Wang, H; Shin, D-M; Masiuk, M; Qi, C-F; Morse, HC. IFN regulatory factor 8 restricts the size of the marginal zone and follicular B cell pools. J. Immunol. 2011, 186: 1458–1466. Fitzgerald, KA; McWhirter, SM; Faia, KL; Rowe, DC; Latz, E; Golenbock, DT; Coyle, AJ; Liao, SM; Maniatis, T. IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 2003a, 4:491-496. Fitzgerald, KA; Rowe, DC; Barnes, BJ; Caffrey, DR; Visintin, A; Latz, E; Monks, B; Pitha, PM; Golenbock, DT. LPS-TLR4 signaling to IRF-3/7 and NF-kappaB involves the toll adapters TRAM and TRIF. J. Exp. Med. 2003b, 198:1043-1055. Flandin, JF; Chano, F; Descoteaux, A. RNA interference reveals a role for TLR2 and TLR3 in the recognition of Leishmania donovani promastigotes by interferon-gamma-primed macrophages. Eur. J. Immunol. 2006, 36:411-420. Fragale, A; Gabriele, L: Stellacci, E; Borghi, P; Perrotti, E; Ilari, R; Lanciotti, A; Remoli, AL; Venditti, M; Belardelli, F; Battistini, A. IFN regulatory factor-1 negatively regulates CD4+ CD25+ regulatory T cell differentiation by repressing Foxp3 expression. J. Immunol. 2008, 181:1673-1682. Fragale, A; Stellacci, E; Ilari, R; Remoli, AL; Lanciotti, A; Perrotti, E; Shytaj, I; Orsatti, R; Lawrence, HR; Lawrence, NJ; Wu, J; Rehli, M; Ozato, K; Battistini, A. Critical role of IRF-8 in negative regulation of TLR3 J. Immunol. 2011, 186:1951-1962. 42 Angela Battistini

Fredericksen, BL; Gale, M; Jr. West Nile virus evades activation of interferon regulatory factor 3 through RIG-I-dependent and -independent pathways without antagonizing host defense signaling. J. Virol. 2006, 80:2913-2923. Fu, XY; Kessler DS; Veals SA; Levy DE; Darnell JE; Jr. ISGF3, the transcriptional activator induced by interferon alpha, consists of multiple interacting polypeptide chains. Proc. Natl. Acad. Sci. USA 1990, 87: 8555-8559. Fujita, T; Sakakibara, J; Sudo, Y; Miyamoto, M; Kimura, Y; Taniguchi, T. Evidence for a nuclear factor(s), IRF-1, mediating induction EMBO J. 1988, 7:3397-3405. Fujita, T; Kimura, Y; Miyamoto, M; Barsoumian, EL; Taniguchi, T. Induction of endogenous IFN Nature. 1989a, 337:270-272. Fujita, T; Reis, LF; Watanabe, N; Kimura, Y; Taniguchi, T; Vilcek, J. Induction of the transcription Proc. Natl. Acad. Sci. USA. 1989b, 86:9936-9940. Furrie, E; Macfarlane, S; Thomson, G; Macfarlane, GT. Toll-like receptors-2, -3 and -4 expression patterns on human colon and their regulation by mucosal-associated bacteria. Immunology 2005, 115:565-574. Gabriele, L; Fragale, A; Borghi, P; Sestili, P; Stellacci, E; Venditti, M; Schiavoni, G; Sanchez, M; Belardelli, F; Battistini, A. IRF-1 deficiency skews the differentiation of dendritic cells toward plasmacytoid and tolerogenic features. J. Leukoc. Biol. 2006, 80:1500-1511. Galon, J; Sudarshan, C; Ito, S; Finbloom, D; O’Shea, JJ. IL-12 induces IFN regulating factor- 1 (IRF-1) gene expression in human NK and T cells. J. Immunol. 1999, 162:7256-7262. Garcia-Dominguez, M; Reyes, JC. SUMO association with repressor complexes, emerging routes for transcriptional control. Biochim. Biophys. Acta 2009, 1789:451-459. Garcia-Sastre, A. Mechanisms of inhibition of the host interferon α/β-mediated antiviral responses by viruses. Microbes Infect. 2002; 4: 647-655. Geiss, G; Jin, G; Guo, J; Bumgarner, R; Katze, MG; Sen, GC. A comprehensive view of regulation of gene expression by double-stranded RNA-mediated cell signaling. J. Biol. Chem. (2001) 276, 30178-30182. Ghoreschi, K; Laurence, A; Yang, XP; Hirahara, K; O'Shea, JJ. T helper 17 cell heterogeneity and pathogenicity in autoimmune disease. Curr. Opin. Immunol. 2011, 23:702-706. Ghosh, S; Hayden, MS. New regulators of NF-jB in inflammation. Nat. Rev. Immunol. 2008, 8: 837-848. Giese, NA; Gabriele, L; Doherty, TM; Klinman, DM; Tadesse-Heath, L; Contursi, C; Epstein, SL; Morse, HC 3rd. Interferon (IFN) consensus sequence-binding protein, a transcription factor of the IFN regulatory factor family, regulates immune responses in vivo through control of interleukin 12 expression. J. Exp. Med. 1997, 186:1535-1546. Gill, G. Post-translational modification by the small ubiquitin-related modifier SUMO has big effects on transcription factor activity. Curr. Opin. Genet. Dev. 2003, 13:108-113. Gill, G. Something about SUMO inhibits transcription. Curr. Opin. Genet. Dev. 2005, 15:536-541. Goubau, D; Romieu-Mourez, R; Solis, M; Hernandez, E; Mesplède, T; Lin, R; Leaman, D; Hiscott, J. Transcriptional re-programming of primary macrophages reveals distinct apoptotic and anti-tumoral functions of IRF-3 and IRF-7. Eur. J. Immunol. 2009, 39:527- 540. Graff, JW; Mitzel, DN; Weisend, CM; Flenniken, ML; Hardy, ME. Interferon regulatory factor 3 is a cellular partner of rotavirus NSP1. J. Virol 2.002, 76:9545–9550. Interferon Regulatory Factors in Immune Cell Development … 43

Graham, RR; Kozyrev, SV; Baechler, EC; Reddy, MV; Plenge, RM; Bauer, JW; Ortmann, WA; Koeuth, T; Gonzalez Escribano, MF; Pons-Estel, B; Petri, M; Daly, M; Gregersen, PK; Martin, J; Altshuler, D; Behrens, TW; Alarcon-Riquelme, ME. A common haplotype of interferon regulatory factor 5 (IRF5) regulates splicing and expression and is associated with increased risk of systemic lupus erythematosus. Nat. Genet. 2006, 38:550-555. Graham, RR; Kyogoku, C; Sigurdsson, S; Vlasova, IA; Davies, LR; Baechler, EC; Plenge, RM; Koeuth, T; Ortmann, WA; Hom, G; Bauer, JW; Gillett, C; Burtt, N; Cunninghame Graham, DS; Onofrio, R; Petri, M; Gunnarsson, I; Svenungsson, E; Rönnblom, L; Nordmark, G; Gregersen, PK; Moser, K; Gaffney, PM; Criswell, LA; Vyse, TJ; Syvänen, AC; Bohjanen, PR; Daly, MJ; Behrens, TW; Altshuler, D. Three functional variants of IFN Proc. Natl. Acad. Sci. USA 2007, 104:6758-6763. Grandvaux, N; Servant, MJ; tenOever, B; Sen, GC; Balachandran, S; Barber, GN; Lin, R, Hiscott, J. Transcriptional profiling of interferon J. Virol. 2002, 76:5532-5539. Guo, Z; Garg, S; Hill, KM; Jayashankar, L; Mooney, MR; Hoelscher, M; Katz, JM; Boss, JM; Sambhara, S. A distal regulatory region is required for constitutive and IFN-beta- induced expression of murine TLR9 gene. J. Immunol. 2005, 175:7407-7418. Gupta, S; Jiang, M; Anthony, A; Pernis, AB. Lineage-specific modulation of interleukin 4 signaling by interferon regulatory factor 4. J. Exp. Med. 1999, 190:1837-1848. Gururajan, M; Haga, CL; Das, S; Leu, CM; Hodson, D; Josson, S; Turner, M; Cooper, MD. MicroRNA 125b inhibition of B cell differentiation in germinal centers. Int Immunol. 2010, 22:583-592. Hahn, AM; Huye, LE; Ning, S; Webster-Cyriaque, J: Pagano, JS. Interferon regulatory factor 7 is negatively regulated by the Epstein-Barr virus immediate-early gene, BZLF-1. J. Virol. 2005, 79:10040-10052. Hambleton, S; Salem, S; Bustamante, J; Bigley, V; Boisson-Dupuis, S; Azevedo, J; Fortin, A; Haniffa, M; Ceron-Gutierrez, L; Bacon, CM; Menon, G; Trouillet, C; McDonald, D; Carey, P; Ginhoux, F; Alsina, L; Zumwalt, TJ; Kong, XF; Kumararatne, D; Butler, K; Hubeau, M; Feinberg, J; Cant, A; Abel, L; Chaussabel, D; Doffinger, R; Talesnik, E; Grumach, A; Duarte, A; Abarca, K; Moraes-Vasconcelos, D; Burk, D; Berghuis, A; Geissmann, F; Collin, M; Casanova, JL; Gros P. IRF8 mutations and human dendritic- cell immunodeficiency. N. Engl. J. Med. 2011, 365:127-138. Han, KJ; Jiang, L; Shu, HB. Regulation of IRF2 transcriptional activity by its sumoylation. Biochem. Biophys. Res. Commun. 2008, 372:772-778. Harada, H; Fujita, T; Miyamoto, M; Kimura, Y; Maruyama, M; Furia, A; Miyata, T; Taniguchi, T. Structurally similar but functionally distinct factors, IRF-1 and IRF-2, bind to the same regulatory elements of IFN Cell 1989, 58:729-739. Harada, H; Kitagawa, M; Tanaka, N; Yamamoto, H; Harada, K; Ishihara, M; Taniguchi, T. Anti-oncogenic and oncogenic potentials of interferon regulatory factors-1 and -2. Science 1993, 259:971-974. Harada, H; Matsumoto, M; Sato, M; Kashiwazaki, Y; Kimura, T; Kitagawa, M; Yokochi, T; Tan, RS; Takasugi, T; Kadokawa, Y; Schindler, C; Schreiber, RD; Noguchi, S; Taniguchi, T. Regulation of IFN Genes. Cells. 1996, 1:995-1005. Harada, H; Willison, K; Sakakibara, J; Miyamoto, M; Fujita, T; Taniguchi, T. Absence of the type I IFN Cell 1990, 3:303-312. 44 Angela Battistini

Harman, AN; Lai, J; Turville, S; Samarajiwa, S; Gray, L; Marsden, V; Mercier, S; Jones, K; Nasr, N; Rustagi, A; Cumming, H; Donaghy, H; Mak, J; Gale, M; jr, Churchill, M; Hertzog, P; Cunningham, AL. HIV infection of dendritic cells subverts the IFN induction pathway via IRF-1 and inhibits type 1 IFN production. Blood 2011, 118: 298-308. Hartman, AL; Bird, BH; Towner, JS; Antoniadou, ZA; Zaki, SR; Nichol, ST. Inhibition of IRF-3 activation by VP35 is critical for the high level of virulence of ebola virus. J. Virol. 2008, 82:2699-2704. Hay, RT. SUMO: a history of modification. Mol. Cell. 2005, 18:1-12. Heil, F; Hemmi, H., Hochrein, H; Ampenberger, F; Kirschning, C; Akira, S; Lipford, G; Wagner, H; Bauer S. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 2004, 303:1526-1529. Heinz, S., Haehnel, V; Karaghiosoff, M; Schwarzfischer, L; Muller, M; Krause, SW; Rehli, M. Species-specific regulation of Toll-like receptor 3 genes in men and mice. J. Biol. Chem. 2003, 278:21502-21509. Hemmi, H., Kaisho, T; Takeuchi, O; Sato, S; Sanjo, H; Hoshino, K; Horiuchi, T; Tomizawa, H; Takeda, K; Akira S. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat. Immunol. 2002, 3:196-200. Hemmi, H; Takeuchi, O; Kawai, T; Kaisho, T; Sato, S; Sanjo, H; Matsumoto, M; Hoshino, K; Wagner, H; Takeda K; Akira S. A Toll-like receptor recognizes bacterial DNA. Nature 2000, 408:740-745. Hida, S; Ogasawara, K; Sato, K; Abe, M; Takayanagi, H; Yokochi, T; Sato, T; Hirose, S; Shirai, T; Taki, S; Taniguchi, T. CD8(+) T cell-mediated skin disease in mice lacking IRF-2, the transcriptional attenuator of interferon-alpha/beta signaling. Immunity 2000, 13:643–655. Hida, S; Tadachi, M; Saito, T; Taki, S. Negative control of basophil expansion by IRF-2 critical for the regulation of Th1/Th2 balance. Blood 2005, 106:2011–2017. Higgs, R; Jefferies, CA. Targeting IRFs by ubiquitination: regulating antiviral responses. Biochem. Soc. Trans. 2008, 36:453-458. Higgs, R; Lazzari, E; Wynne, C; Ní Gabhann, J; Espinosa, A; Wahren-Herlenius, M; Jefferies, CA. Self protection from anti-viral responses Ro52 promotes degradation of the transcription factor IRF7 downstream of the viral Toll-Like receptors. PLoS One 2010, 5: e11776. Hilton, L; Moganeradj, K; Zhang, G; Chen, YH; Randall, RE; McCauley, JW; Goodbourn, S. The NPro product of bovine viral diarrhea virus inhibits DNA binding by interferon regulatory factor 3 and targets it for proteasomal degradation. J. Virol. 2006, 80:11723- 11732. Hiscott, J. Convergence of the NF-B and IRF pathways in the regulation of the innate antiviral response. Cytokine Growth Factor Rev. 2007a, 18:483-490. Hiscott, J. Triggering the innate antiviral response through IRF-3 activation. J. Biol. Chem. 2007b, 282:15325-15329. Hochrein, H; Schlatter, B; O’Keeffe, M; Wagner, C; Schmitz, F; Schiemann, M; Bauer, S; Suter, M; Wagner, H. Herpes simplex virus type-1 induces IFN-alpha production via Toll-like receptor 9-dependent and -independent pathways. Proc. Natl. Acad. Sci. USA 2004, 101:11416-11421. Holtschke, T; Löhler, J; Kanno, Y; Fehr, T; Giese, N; Rosenbauer, F; Lou, J; Knobeloch, KP; Gabriele, L; Waring, JF; Bachmann, MF; Zinkernagel, RM; Morse, HC 3rd; Ozato, K; Interferon Regulatory Factors in Immune Cell Development … 45

Horak, I.. Immunodeficiency and chronic myelogenous leukemia-like syndrome in mice with a targeted mutation of the ICSBP gene. Cell 1996, 87:307-317. Honda, K; Mizutani, T; Taniguchi, T. Negative regulation of IFN-alpha/beta signaling by IFN regulatory factor 2 for homeostatic development of dendritic cells. Proc. Natl. Acad. Sci. USA 2004, 101:2416-2421. Honda, K; Yanai, H; Mizutani, T; Negishi, H; Shimada, N; Suzuki, N; Ohba, Y; Takaoka, A; Yeh, WC; Taniguchi, T. Role of a transductional-transcriptional processor complex involving MyD88 and IRF-7 in Toll-like receptor signaling. Proc. Natl. Acad. Sci. USA 2004, 101:15416–15421. Honda, K; Yanai, H; Negishi, H; Asagiri, M; Sato, M; Mizutani, T; Shimada, N; Ohba, Y; Takaoka, A; Yoshida, N; Taniguchi, T. IRF-7 is the master regulator of type-I interferon- dependent immune responses. Nature 2005a, 434:772-777. Honda, K; Ohba, Y; Yanai, H; Negishi, H; Mizutani, T; Takaoka, A; Taya, C; Taniguchi, T. Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-I interferon induction. Nature.2005b, 434:1035-1040. Honda, K; Taniguchi, T. IRFs: master regulators of signaling by Toll-like receptors and cytosolic pattern-recognition receptors. Nat. Rev. Immunol. 2006a, 6:644-658. Honda, K; Takaoka, A; Taniguchi, T. Type I interferon Immunity. 2006b, 25:349-360. Honma, K; Kimura, D; Tominaga, N; Miyakoda, M; Matsuyama, T; Yui, K. Interferon regulatory factor 4 differentially regulates the production of Th2 cytokines Proc. Natl. Acad. Sci. USA. 2008, 105:15890-15895. Honma, K; Udono, H; Kohno, T; Yamamoto, K; Ogawa, A; Takemori, T; Kumatori, A; Suzuki, S; Matsuyama, T; Yui, K. Interferon regulatory factor 4 negatively regulates the production of proinflammatory cytokines by macrophages in response to LPS. Proc. Natl. Acad. Sci. USA 2005, 102:16001-16006. Hornung, V; Ellegast, J; Kim, S; Brzozka, K; Jung, A; Kato, H; Poeck, H; Akira, S; Conzelmann, KK; Schlee, M; Endres, S; Hartmann, G. 50-Triphosphate RNA is the ligand for RIG-I. Science 2006, 314:994-997. Hoshino, K; Sugiyama, T; Matsumoto, M; Tanaka, T; Saito, M; Hemmi, H; Ohara, O; Akira, S; Kaisho, T. IkappaB kinase-alpha is critical for interferon-alpha production induced by Toll-like receptors 7 and 9. Nature. 2006, 440:949–953. Hoshino, T; Sugiyama, M; Matsumoto, T; Tanaka, M; Saito, H; Hemmi, O; Ohara, S; Akira, T; Kaisho, I. kappaB kinase-alpha is critical for interferon-alpha production induced by Toll-like receptors 7 and 9. Nature 2006, 440: 949-953. Hu, CM; Jang, SY; Fanzo, JC; Pernis, AB. Modulation of T cell cytokine production by interferon regulatory factor-4. J. Biol. Chem. 2002, 277:49238-49246. Huang, W; Horvath, E; Eklund, EA. PU.1, interferon regulatory factor (IRF) 2, and the interferon consensus sequence-binding protein (ICSBP/IRF8) cooperate to activate NF1 transcription in differentiating myeloid cells. J. Biol. Chem. 2007, 282:6629-6643. Huang, W; Saberwal, G; Horvath, E; Zhu, C; Lindsey, S; Eklund, EA. Leukemia-associated, constitutively active mutants of SHP2 protein tyrosine Mol. Cell. Biol. 2006, 26:6311- 6332. Huye, LE; Ning, S; Kelliher, M; Pagano, JS. Interferon regulatory factor 7 is activated by a viral oncoprotein through RIP-dependent ubiquitination. Mol. Cell. Biol. 2007, 27:2910- 2918. 46 Angela Battistini

Hwang, S; Kim, KS; Flano, E; Wu, TT; Tong, LM; Park, AN; Song, MJ; Sanchez, DJ; O'Connell, RM; Cheng, G; Sun. Conserved herpesviral kinase promotes viral persistence by inhibiting the IRF-3-mediated type I interferon response. Cell Host Microbe 2009, 5:166-178. Ichikawa, E; Hida, S; Omatsu, Y; Shimoyama, S; Takahara, K; Miyagawa, S; Inaba, K; Taki, S. Defective development of splenic and epidermal CD4 + dendritic cells in mice deWcient for IFN regulatory factor-2. Proc. Natl. Acad. Sci. USA 2004, 101:3909-3914. Ingraham, CR; Kinoshita, A; Kondo, S; Yang, B; Sajan, S; Trout, KJ; Malik, MI; Dunnwald, M; Goudy, SL; Lovett, M; Murray, JC; Schutte, BC. Abnormal skin Nat. Genet. 2006 38:1335-1340. Inohara, N; Nunez, G. NODs: intracellular proteins involved in inflammation and apoptosis. Nat. Rev. Immunol. 2003, 3:371–382. Ishii, KJ; Coban, C; Kato, H; Takahashi, K; Torii, Y; Takeshita, F; Ludwig, H; Sutter, G; Suzuki, K; Hemmi, H; Sato, S; Yamamoto, M; Uematsu, S; Kawai, T; Takeuchi, O; Akira, S. A Toll-like receptor-independent antiviral response induced by double-stranded B-form DNA. Nat. Immunol. 2006, 7:40-48. Ishikawa, H; Barber, GN. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 2008, 455:674-678. Itoh, S; Harada, H; Fujita, T; Mimura, T; Taniguchi, T. Sequence of a cDNA Nucleic Acids Res. 1989, 17:8372. Ivanov, II; McKenzie, BS; Zhou, L; Tadokoro, CE; Lepelley, A; Lafaille, JJ; Cua, DJ;Littman, DR. The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 2006, 126:1121-1133. Izaguirre, A; Barnes, BJ; Amrute, S; Yeow, WS; Megjugorac, N; Dai, J; Feng, D; Chung, E; Pitha, PM; Fitzgerald-Bocarsly, P. Comparative analysis of IRF and IFN-alpha expression in human plasmacytoid and monocyte-derived dendritic cells. J. Leukoc. Biol. 2003, 74:1125-1138. Jesse, TL; LaChance, R; Iademarco, MF; Dean, DC. Interferon regulatory factor-2 is a transcriptional activator in muscle where it regulates expression of vascular cell adhesion molecule-1. J. Cell Biol. 1998, 140:1265-1276. Johnson, ES. Protein modification by SUMO, Annu. Rev. Biochem. 2004, 73:355-382. Johnson, K; Hashimshony, T; Sawai, CM; Pongubala, JM; Skok, JA; Aifantis, I; Singh, H. Regulation of immunoglobulin light-chain recombination by the transcription factor IRF- 4 and the attenuation of interleukin-7 signaling. Immunity 2008, 28:335–345. Joo, CH; Shin, YC; Gack, M; Wu, L; Levy, D; Jung, JU. Inhibition of interferon regulatory factor 7 (IRF7)-mediated interferon signal transduction by the Kaposi’s sarcoma- associated herpesvirus viral IRF homolog vIRF3. J. Virol. 2007, 81:8282-8292. Juang, YT; Lowther, W; Kellum, M; Au, WC; Lin, R; Hiscott, J; Pitha, PM. Primary activation of interferon A and interferon B gene transcription by interferon regulatory factor 3. Proc. Natl. Acad. Sci. USA 1998, 95:9837-9842. Kaku, H; Rothstein, TL. Fas apoptosis inhibitory molecule expression in B cells is regulated through IRF4 in a feed-forward mechanism. J. Immunol. 2009, 183:5575-5581. Kanazawa, N; Kurosaki, M; Sakamoto, N; Enomoto, N; Itsui, Y; Yamashiro, T; Tanabe, Y; Maekawa, S; Nakagawa, M; Chen, CH; Kakinuma, S; Oshima, S; Nakamura T; Kato T; Wakita T; Watanabe M. Regulation of hepatitis C virus replication by interferon regulatory factor 1. J. Virol. 2004, 78:9713-9720. Interferon Regulatory Factors in Immune Cell Development … 47

Kanneganti, TD; Lamkanfi, M; Nunez, G. Intracellular NOD-like receptors in host defense and disease. Immunity. 2007, 27:549-559. Kanneganti, TD. Central roles of NLRs and inflammasomes in viral infection Nat. Rev. Immunol. 2010, 10:688-698. Kanno, Y; Levi, BZ; Tamura, T; Ozato, K. Immune cell specific amplification of interferon signaling by the IRF-4/8-PU.1 complex. J. Interferon Cytokine. Res. 2005, 25:770-779. Kano, S; Sato, K; Morishita, Y; Vollstedt, S; Kim, S; Bishop, K; Honda, K; Kubo, M; Taniguchi, T. The contribution of transcription factor IRF-1 to the interferon-gamma- interleukin 12 signaling axis and Th1 versus TH-17 differentiation of CD4+ T cells. Nat. Immunol. 2008, 9:9-10. Karin, M. Nuclear factor-kappaB in cancer development and progression. Nature. 2006, 441:431-436. Kato, H; Sato, S; Yoneyama, M; Yamamoto, M; Uematsu, S; Matsui, K; Tsujimura, T; Takeda, K; Fujita, T; Takeuchi, O; Akira, S. Cell type-specific involvement of RIG-I in antiviral response. Immunity. 2005, 23:19-28. Kato, H; Takeuchi, O; Sato, S; Yoneyama, M; Yamamoto, M; Matsui, K; Uematsu, S; Jung, A; Kawai, T; Ishii, KJ; Yamaguchi, O;, Otsu, K; Tsujimura, T;, Koh, CS; Reis e Sousa, C;, Matsuura, Y; Fujita, T; Akira, S. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature. 2006, 441:101-105. Katze, MG; He, Y; Gale, M Jr. Viruses and interferon: a fight for supremacy. Nat. Rev. Immunol. 2002; 2: 675-687. Kautz, B; Kakar, R; David, E; Eklund EA. SHP1 protein-tyrosine phosphatase inhibits gp91PHOX and p67PHOX expression by inhibiting interaction of PU.1, IRF1, interferon consensus sequence-binding protein, and CREB-binding protein with homologous cis elements in the CYBB and NCF2 genes. J. Biol. Chem. 2001, 276:37868-37878. Kawai, T; Takeuchi, O; Fujita, T; Inoue, J; Mühlradt, PF; Sato, S; Hoshino, K; Akira, S. Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J. Immunol. 2001, 167:5887-5894. Kawai, T; Sato, S; Ishii, KJ; Coban, C; Hemmi, H; Yamamoto, M; Terai, K; Matsuda, M; Inoue, J; Uematsu, S; Takeuchi, O; Akira, S. Interferon-alpha induction through Toll-like receptors involves a direct interaction of IRF7 with MyD88 and TRAF6. Nat. Immunol. 2004, 5:1061-1068. Kawai, T; Takahashi, K; Sato, S; Coban, C; Kumar, H; Kato, H; Ishii, KJ; Takeuchi, O; Akira S. IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction. Nat Immunol. 2005, 6:981-988. Kawai, T; Akira, S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. 2010, 11:373-384. Kawai, T; Akira, S. Toll- like receptors and their cross-talk with other innate receptors in infection and immunity. Immunity. 2011, 34:637-650. Kawakami, T; Matsumoto, M; Sato, M; Harada, H; Taniguchi, T; Kitagawa, M. Possible involvement of the transcription FEBS Lett. 1995, 358:225-229. Kim, EJ; Park, JS; Um, SJ. Ubc9-mediated sumoylation leads to transcriptional repression of IRF-1. Biochem. Biophys. Res. Commun. 2008, 377:952-956. 48 Angela Battistini

Kim, YM; Im, JY; Han, SH; Kang, HS; Choi, I. IFN-gamma up-regulates IL-18 gene expression via IFN consensus sequence-binding protein and activator protein-1 elements in macrophages. J. Immunol. 2000, 165, 3198-3205. Kimura, T; Nakayama, K; Penninger, J; Kitagawa, M; Harada, H; Matsuyama, T; Tanaka, N; Kamijo, R; Vilcek, J; Mak, TW; et al. Involvement of the IRF-1 transcription factor in antiviral responses to interferons. Science. 1994, 264:1921-1924. Kimura, T; Kadokawa, Y; Harada, H; Matsumoto, M; Sato, M; Kashiwazaki, Y; Tarutani, M; Tan, RS; Takasugi, T; Matsuyama, T; Mak, TW; Noguchi, S; Taniguchi, T. Essential and non-redundant roles of p48 (ISGF3 gamma) and IRF-1 in both type I and type II interferon Genes Cells. 1996, 1:115-124. Kitagawa, Y; Yamaguchi, M; Zhou, M; Komatsu, T; Nishio, M; Sugiyama, T; Takeuchi, K; Itoh, M; Gotoh, B. A tryptophan-rich motif in the human parainfluenza virus type 2 V protein is critical for the blockade of toll-like receptor 7 (TLR7)- and TLR9-dependent signaling. J. Virol. 2011, 85: 4606-4611. Klein, U; Casola, S; Cattoretti, G; Shen, Q; Lia, M; Mo, T; Ludwig, T; Rajewsky, K; Dalla- Favera, R. Transcription factor IRF4 controls plasma cell differentiation and class-switch recombination. Nat. Immunol. 2006, 7:773-782. Kollet, JI; Petro, TM. IRF-1 and NF-kappaB p50/cRel bind to distinct regions of the proximal murine IL-12 p35 promoter during costimulation with IFN-gamma and LPS. Mol. Immunol. 2006, 43:623-633. Kondo, S; Schutte, BC; Richardson, RJ; Bjork, BC; Knight, AS; Watanabe, Y; Howard, E; de Lima, RL; Daack-Hirsch, S; Sander, A; McDonald-McGinn, DM; Zackai, EH; Lammer, EJ; Aylsworth, AS; Ardinger, HH; Lidral, AC; Pober, BR; Moreno, L; Arcos-Burgos, M; Valencia; Houdayer, C; Bahuau, M; Moretti-Ferreira, D; Richieri-Costa, A; Dixon, MJ; Murray, JC. Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes. Nat. Genet. 2002 32:285-289. Kondo, M; Wagers, AJ; Manz, MG; Prohaska, SS; Scherer, DC; Beilhack, GF; Shizuru, JA; Weissman, IL. Biology of hematopoietic stem cells and progenitors: implications for clinical application. Annu. Rev. Immunol. 2003, 21: 759-806. Kong, HJ; Anderson, DE; Lee, CH; Jang, MK; Tamura, T; Tailor, P; Cho, HK; Cheong, J; Xiong, H; Morse, III HC; Ozato, K. Cutting edge: autoantigen ro52 is an interferon inducible E3 ligase that ubiquitinates IRF-8 and enhances cytokine expression inmacrophages. J. Immunol. 2007, 179: 26–30. Konieczna, I; Horvath, E; Wang, H; Lindsey, S; Saberwal, G; Bei, L; Huang, W; Platanias, L; Eklund, EA. Constitutive activation of SHP2 in mice cooperates with ICSBP deficiency to accelerate progression to acute myeloid leukemia. J. Clin. Invest. 2008, 118:853-867. Kopecky-Bromberg, SA; Martínez-Sobrido, L; Frieman, M; Baric, RA; Palese, P. Severe acute respiratory syndrome coronavirus open reading frame (ORF) 3b, ORF 6, and nucleocapsid proteins function as interferon antagonists. J. Virol. 2007 81:548-557. Kozyrev, SV; Alarcon-Riquelme, ME. The genetics and biology of Irf5-mediated signaling in lupus. Autoimmunity. 2007, 40:591–601. Kraus, TA; Lau, JF; Parisien, JP; Horvath, CM. A hybrid IRF9-STAT2 protein recapitulates interferon-stimulated gene expression and antiviral response. J. Biol. Chem. 2003 278:13033-13038. Interferon Regulatory Factors in Immune Cell Development … 49

Krausgruber, T; Saliba, D; Ryzhakov, G; Lanfrancotti, A; Blazek, K; Udalova, IA. IRF5 is required for late-phase TNF secretion by human dendritic cells. Blood. 2010, 115:4421- 4430. Kröger, A; Köster, M; Schroeder, K; Hauser, H; Mueller, PP. Activities of IRF-1. J. Interferon Cytokine. Res. 2002, 22:5-14. Kubota, T; Matsuoka, M; Chang, TH; Tailor, P; Sasaki, T; Tashiro, M; Kato, A; Ozato K. Virus infection triggers SUMOylation of IRF3 and IRF7, leading to the negative regulation of type I interferon gene expression. J. Biol. Chem. 2008, 283: 25660-25670. Kumar, KP; McBride, KM; Weaver, BK; Dingwall, C; Reich, NC. Regulated nuclear- cytoplasmic localization Kyogoku, C; Tsuchiya, N. A compass that points to lupus regulatory factor-7 and its potential role in the transcription activation of interferon A genes. regulatory factor 7 and its association with IRF-3. defense. J. Interferon Cytokine Res. 2002, 22:59-71. responses by Ebola and Marburg viruses. antiviral response through the mitochondrial antiviral signaling (MAVS) adapter.. and transcriptional activation in response to IFNs and other extracellular signaling proteins..-gamma represses IL-4 expression via IRF-1 and IRF-2. expression by Src homology 2 domain-containing protein tyrosine phosphatase-2 activity in human myeloid dendritic cells. and silencing properties to human IFN-beta gene regulatory elements.-alpha and IFN-beta genes by a regulatory transcription factor, IRF- 1. factor IRF-1 and interferon-beta mRNAs by cytokines and activators of second- messenger pathways. regulatory factor 5 (IRF5) define risk and protective haplotypes for human lupus. regulatory factor 3 target genes: direct involvement in the regulation of interferon-stimulated genes. and IFN-inducible genes.-alpha/beta genes: evidence for a dual function of the transcription factor complex ISGF3 in the production and action of IFN-alpha/beta. system in EC cells: transcriptional activator (IRF-1) and repressor (IRF- 2) genes are developmentally regulated. [corrected] gene induction by the interferon regulatory factor family of transcription factors.in naive vs. effector/memory CD4+ T cells. phosphatase inhibit NF1 transcriptional activation by the interferon consensus sequence binding protein., R, limb and craniofacial morphogenesis in mice deficient for interferon regulatory factor 6 (Irf6). coding for human IRF-2.. factor ISGF3 gamma in virus-induced expression of the IFN-beta gene. responses, as revealed by gene targeting studies., C of interferon regulatory factor 3, a subunit of double-stranded RNA-activated factor 1. Mol. Cell Biol.: Genetic studies on type I interferon pathway. Genes. Immun. 2007, 8:445-455. La Rocca, SA; Herbert, RJ; Crooke, H; Drew, TW; Wileman, TE; Powell, PP. Loss of interferon regulatory factor 3 in cells infected with classical swine fever virus involves the N-terminal protease, Npro. J. Virol. 2005 79:7239-7247. Lace, MJ; Anson, JR; Haugen, TH; Turek, LP. Interferon regulatory factor (IRF)-2 activates the HPV Virology. 2010, 399:270-279. Lee, CH; Melchers, M; Wang, H; Torrey, TA; Slota, R; Qi, CF; Kim, JY; Lugar, P; Kong, HJ; Farrington, L; van der Zouwen, B; Zhou, JX; Lougaris, V; Lipsky, PE; Grammer, AC; Morse, HC 3rd. Regulation of the germinal center gene program by interferon (IFN) regulatory factor 8/IFN consensus sequence-binding protein. J. Exp. Med. 2006, 203: 63- 72. Lefort, S; Soucy-Faulkner, A; Grandvaux, N; Flamand, L. Binding of Kaposi's sarcoma- associated herpesvirus K-bZIP to interferon J. Virol. 2007, 81:10950-10960. 50 Angela Battistini

Lei, X; Liu, X; Ma, Y; Sun; Yang, Y; Jin, Q; He, B; Wang, J. The 3C protein of enterovirus 71 inhibits retinoid acid-inducible gene I-mediated interferon regulatory factor 3 activation and type I interferon responses. J. Virol. 2010 84:8051-8061. Lei, X; Sun; Liu, X; Jin, Q; He, B; Wang, J. Cleavage of the adaptor protein TRIF by enterovirus 71 3C inhibits antiviral responses mediated by Toll-like receptor 3. J. Virol. 2011, 85:8811-8818. Levi, BZ; Hashmueli, S; Gleit-Kielmanowicz, M; Azriel, A; Meraro, D. ICSBP/IRF-8 transactivation: a tale of protein-protein interaction. J. Interferon Cytokine. Res. 2002, 22:153-160. Levy, DE; Kessler, DS; Pine, R; Darnell, JE, Jr. Cytoplasmic activation of ISGF3, the positive regulator of interferon-alpha-stimulated transcription, reconstituted in vitro. Genes Dev. 1989, 3:1362–1371. Levy, DE; Lew, DJ; Decker, T; Kessler, DS; Darnell, JE Jr. Synergistic interaction between interferon EMBO J. 1990 9:1105-1111. Levy, DE; Garcia-Sastre, A. The virus battles: IFN induction of the antiviral state and mechanisms of viral evasion. Cytokine. Growth Factor Rev. 2001, 12: 143-156. Li, K; Foy, E; Ferreon, JC;Nakamura, M; Ferreon, AC; Ikeda, M; Ray, SC; Gale M Jr; Lemon SM. Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-likereceptor 3 adaptor protein TRIF. Proc. Natl. Acad. Sci. USA. 2005, 102:2992-2997. Li, P; Wong, JJ; Sum, C; Sin, WX; Ng, KQ; Koh, MB; Chin, KC. IRF8 and IRF3 cooperatively regulate rapid interferon-β induction in human blood monocytes. Blood. 2011, 117:2847-2854. Liang, D; Gao, Y; Lin, X; He, Z; Zhao, Q; Deng, Q; Lan, K. A human herpesvirus mRNA attenuates interferon signaling and contributes to maintenance of viral latency by targeting IKKε. Cell Res. 2011, 21:793-806. Liang, Q; Deng, H; Li, X; Wu, X; Tang, Q; Chang, TH; Peng, H; Rauscher, FJ 3rd; Ozato, K; Zhu, F. Tripartite motif-containing protein 28 is a small ubiquitin-related modifier e3 ligase and negative regulator of IFN regulatory factor 7. J. Immunol. 2011, 187:4754- 4763. Liebermann, DA; Hoffman, B. Myeloid differentiation (MyD) primary response genes in hematopoiesis. Oncogene 2002, 21:3391-3402. Lien, C; Fang, CM; Huso, D; Livak, F; Lu, R; Pitha, PM. Critical role of IRF-5 in regulation of B-cell differentiation. Proc. Natl. Acad. Sci. USA 2010, 107:4664-4668. Lin, R; Mustafa, A; Nguyen, H; Gewert, D; Hiscott, J. Mutational analysis of interferon (IFN) regulatory factors 1 and 2;Effects on the induction of IFN-beta gene expression. J. Biol. Chem. 1994, 269:17542-17549. Lin, R; Heylbroeck, C; Pitha, PM; Hiscott, J. Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential and proteasome-mediated degradation. Mol. Cell Biol. 1998, 18:2986-2996. Lin, R; Hiscott, J. A role for casein kinase II phosphorylation in the regulation of IRF-1 transcriptional activity. Mol. Cell Biochem. 1999a, 191169-180. Lin, R; Mamane, Y; Hiscott, J. Structural and functional analysis of interferon regulatory factor 3: localization of the transactivation and autoinhibitory domains. Mol. Cell Biol. 1999b, 19: 2465-2474. Interferon Regulatory Factors in Immune Cell Development … 51

Lin, R; Heylbroeck, C; Genin, P; Pitha, PM; Hiscott, J. Essential role of interferon regulatory factor 3 in direct activation of RANTES chemokine transcription. Mol. Cell. Biol. 1999c,19:959-966. Lin, R; Genin, P; Mamane, Y; Hiscott, J. Selective DNA binding and association with the CREB binding protein coactivator contribute to differential activation of alpha/beta interferon genes by interferon regulatory factors 3 and 7. Mol. Cel.l Biol. 2000a, 20:6342–6353. Lin, R., Mamane, Y., and Hiscott, J. Multiple regulatory domains control IRF-7 activity in response to virus infection. J. Biol. Chem. 2000b, 275:34320-34327. Lin, R; Genin, P; Mamane, Y; Sgarbanti, M; Battistini, A; Harrington, Jr WJ; Barber, GN; Hiscott, J. HHV-8 encoded vIRF-1 represses the interferon antiviral response by blocking IRF-3 recruitment of the CBP/p300 coactivators. Oncogene. 2001, 20:800-811. Lin, R; Yang, L; Arguello, M; Penafuerte, C; Hiscott, J. A CRM1-dependent nuclear export pathway is involved in the regulation of IRF-5 subcellular localization J. Biol. Chem. 2005, 280:3088-3095. Lin, R; Lacoste, J; Nakhaei, P; Sun, Q; Yang, L; Paz, S; Wilkinson, P; Julkunen, I; Vitour, D; Meurs, E; Hiscott, J. Dissociation of a MAVS/IPS-1/VISA/Cardif-IKKepsilon molecular complex from the mitochondrial outer membrane by hepatitis J. Virol. 2006, 80:6072- 6083. Liu, J; Ma X. Interferon regulatory factor 8 regulates RANTES gene transcription in cooperation with interferon regulatory factor-1, NF-kappaB, and PU.1. J. Biol. Chem. 2006, 281:19188-19195. Lohoff, M; Ferrick, D; Mittrucker, HW; Ducan, GS; Bischof, S; Rollinghoff, M; Mak, TW. Interferon regulatory factor-1 is required for a T helper 1 immune response in vivo. Immunity 1997, 6:681-689. Lohoff, M; Duncan, GS; Ferrick, D; Mittrücker, HW; Bischof, S; Prechtl, S; Röllinghoff, M; Schmitt, E; Pahl, A; Mak, TW. Deficiency in the transcription J. Exp. Med. 2000, 192:325-336. Lohoff, M; Mittrucker, HW; Prechtl, S; Bischof, S; Sommer, F; Kock, S; Ferrick, DA; Duncan, GS; Gessner, A; Mak, TW. Dysregulated T helper cell differentiation in the absence of interferon regulatory factor 4. Proc. Natl. Acad. Sci. USA. 2002, 99:11808- 11812. Lohoff, M; Mak, TW. Roles of interferon-regulatory factors in T-helper-cell differentiation. Nat. Rev. 2005, 5:125-133. Lu, G; Reinert, JT; Pitha-Rowe, I; Okumura, A; Kellum, M; Knobeloch, KP; Hassel, B; Pitha, PM. ISG15 enhances the innate antiviral response Cell Mol. Biol. 2006, 52:29-34. Lu, LL; Puri, M; Horvath, CM; Sen, GC. Select paramyxoviral V proteins inhibit IRF3 activation by acting as alternative substrates for inhibitor of kappaB kinase epsilon (IKKe)/TBK1. J. Biol. Chem. 2008 283:14269-14276. Lu, R. Interferon regulatory factor 4 and 8 in B-cell development. Trends Immunol. 2008, 29:487-492. Lu, R; Pitha, PM. Monocyte differentiation to macrophage requires interferon regulatory factor 7. J. Biol .Chem. 2001, 276:45491-45496. Lu, R; Medina, KL; Lancki, DW; Singh, H IRF-4,8 orchestrate the pre-B-to-B transition in lymphocyte development. Genes Dev. 2003, 17:1703-1708. 52 Angela Battistini

Luo, W; Skalnik, DG. Interferon regulatory factor-2 directs transcription from the gp91(phox) promoter. J. Biol. Chem. 1996, 271:23445-23451. Ma, S; Turetsky, A; Trinh, L; Lu, R.. IFN regulatory factor 4 and 8 promote Ig light chain kappa locus activation in pre-B cell development. J. Immunol. 2006, 177:7898-7904. Ma, S; Pathak, S; Trinh, L; Lu, R. Interferon regulatory factors 4 and 8 induce the expression of Ikaros and Aiolos to down-regulate pre-B-cell receptor and promote cell-cycle withdrawal in pre-B-cell development. Blood. 2008, 111, 1396-1403. Magalhaes, JG; Sorbara, MT; Girardin, SE; Philpott, DJ. What is new with Nods? Current Opin. Immunol. 2011, 23:29–34. Maiwald, T; Schneider, A; Busch, H; Sahle, S; Gretz, N; Weiss, TS; Kummer, U; Klingmüller, U. Combining theoretical analysis and experimental data generation reveals IRF9 as a crucial factor for accelerating interferon α-induced early antiviral signalling. FEBS J. 2010 277:4741-4754. Malynn, BA; Ma, A. Ubiquitin makes its mark on immune regulation Immunity. 2010, 33:843-852. Mancl, ME; Hu, G; Sangster-Guity, N; Olshalsky, SL; Hoops, K; Fitzgerald-Bocarsly, P; Pitha, PM; Pinder, K; Barnes, BJ. 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. J. Biol. Chem. 2005, 280: 21078-21090. Marecki, S; Atchison, ML; Fenton, MJ. Differential expression and distinct functions of IFN regulatory factor 4 and IFN consensus sequence binding protein in macrophages. J. Immunol. 1999, 163:2713-2722. Marecki, S; Fenton, MJ. The role of IRF-4 in transcriptional regulation. J. Interferon Cytokine. Res. 2002, 22:121-133. Marecki, S; Riendeau, CJ; Liang, MD; Fenton, MJ. PU.1 and multiple IFN regulatory factor proteins synergize to mediate transcriptional activation of the human IL-1 beta gene. J. Immunol. 2001, 166:6829-6838. Marié, I; Durbin, JE; Levy, DE. Differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor-7. EMBO J. 1998, 17:6660- 6669. Martínez-Sobrido, L; Giannakas, P; Cubitt, B; García-Sastre, A. Differential inhibition of type I interferon induction by arenavirus nucleoproteins. J. Virol. 2007, 81:12696-12703. Maruyama, S; Sumita, K; Shen, H; Kanoh, M; Xu, X; Sato, M; Matsumoto, M; Shinomiya, H; Asano, Y. Identification of IFN regulatory factor-1 binding site in IL-12 p40 gene promoter. J. Immunol. 2003, 170:997-1001. Masumi, A; Wang, IM; Lefebvre, B; Yang, XJ; Nakatani, Y; Ozato K. The histone acetylase PCAF is a phorbol-ester-inducible coactivator of the IRF family that confers enhanced interferon responsiveness. Mol. Cell. Biol. 1999, 19:1810–1820. Masumi, A; Ozato, K. Coactivator p300 acetylates the interferon regulatory factor-2 in U937 cells following phorbol ester treatment. J. Biol. Chem. 2001, 276:20973-20980. Masumi, A; Tamaoki, S; Wang, IM; Ozato, K; Komuro, K. IRF-8/ICSBP and IRF-1 cooperatively stimulate mouse IL-12 promoter activity in macrophages. FEBS Lett. 2002, 531:348–353. Masumi, A; Yamakawa, Y; Fukazawa, H; Ozato, K; Komuro, K. Interferon regulatory factor- 2 regulates cell growth J. Biol. Chem. 2003 278:25401-25407. Interferon Regulatory Factors in Immune Cell Development … 53

Matsuyama, T; Kimura, T; Kitagawa, M; Pfeffer, K; Kawakami, T; Watanabe, N; Kündig, TM; Amakawa, R; Kishihara, K; Wakeham, A; et al. Targeted disruption of IRF-1 or IRF-2 results in abnormal type I IFN gene induction and aberrant lymphocyte development. Cell. 1993 75:83-97. Melroe, GT; Silva, L; Schaffer, PA; Knipe, DM. Recruitment of activated IRF-3 and CBP/p300 to herpes simplex virus ICP0 nuclear foci: potential role in blocking IFN-β induction. Virology 2007, 360:305-321. Mempel, M; Voelcker, V; Köllisch, G; Plank, C; Rad, R; Gerhard, M; Schnopp, C; Fraunberger, P; Walli, AK; Ring, J; Abeck, D; Ollert, M. Toll-like receptor expression in human keratinocytes: nuclear factor kappaB controlled gene activation by Staphylococcus aureus is toll-like receptor 2 but not toll-like receptor 4 or platelet activating factor receptor dependent. J. Invest. Dermatol. 2003, 121:1389-1396. Meraro, D; Hashmueli, S; Koren, B; Azriel, A; Oumard, A; Kirchhoff, S; Hauser, H; Nagulapalli, S; Atchison, ML; Levi, BZ. Protein-protein and DNA-protein interactions affect the activity of lymphoid-specific IFN regulatory factors. J. Immunol. 1999, 163:6468-6478. Meraro, D; Gleit-Kielmanowicz, M; Hauser, H; Levi, BZ. IFN J. Immunol. 2002, 168:6224- 6231. Meroni, G; Diez-Roux, G. TRIM/RBCC, a novel class of ‘single protein RING finger’ E3 ubiquitin ligases. BioEssays 2005, 27:1147-1157. Meylan, E; Curran, J; Hofmann, K; Moradpour, D; Binder, M; Bartenschlager, R; Tschopp, J. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis. Nature. 2005, 437:1167-1172. Meylan, E; Tschopp, J. Toll-like receptors and RNA helicases: two parallel ways to trigger antiviral responses. Mol. Cell.. 2006, 22:561-569. Mibayashi, M; Martínez-Sobrido, L; Loo, YM; Cárdenas, WB; Gale, M Jr; García-Sastre, A. Inhibition of retinoic acid-inducible gene I-mediated induction of beta interferon by the NS1 protein of influenza A virus. J. Virol. 2007, 81:514-524. Miller, DM; Zhang, Y; Rahill, BM; Waldman, WJ; Sedmak, DD. Human cytomegalovirus inhibits IFN-alpha-stimulated antiviral and immunoregulatory responses by blocking multiple levels of IFN-alpha signal transduction. J. Immunol. 1999, 162: 6107-6113. Mittrücker, HW; Matsuyama, T; Grossman, A; Kündig, TM; Potter, J; Shahinian, A; Wakeham, A; Patterson, B; Ohashi, PS; Mak, TW. Requirement for the transcription factor LSIRF/IRF4 for mature B and T lymphocyte function. Science. 1997, 275:540- 543. Miyamoto, M; Fujita, T; Kimura, Y; Maruyama, M; Harada, H; Sudo, Y; Miyata, T; Taniguchi, T. Regulated expression of a gene encoding Cell. 1988, 54:903-913. Mizutani, T; Tsuji, K; Ebihara, Y; Taki, S; Ohba, Y; Taniguchi, T; Honda, K. Homeostatic erythropoiesis by the transcription factor IRF2 through attenuation of type I interferon signaling. Exp. Hematol. 2008, 36:255–264. Moretti, F; Marinari, B; Lo Iacono, N; Botti, E; Giunta, A; Spallone, G; Garaffo, G; Vernersson-Lindahl, E; Merlo, G; Mills, AA; Ballarò, C; Alemà, S; Chimenti, S; Guerrini, L; Costanzo, A. A regulatory feedback loop involving p63 and IRF6 links the pathogenesis of 2 genetically different human ectodermal dysplasias. J. Clin. Invest. 2010, 120:1570-1577. 54 Angela Battistini

Mori, M; Yoneyama, M; Ito, T; Takahashi, K; Inagaki, F; Fujita, T. Identification of Ser-386 of interferon regulatory factor 3 as critical target for inducible phosphorylation that determines activation. J. Biol. Chem. 2004, 279:9698-9702. Morrow, AN; Schmeisser, H; Tsuno, T; Zoon, KC. A novel role for IFN-stimulated gene factor 3II in IFN-γ signaling and induction of antiviral activity in human cells. J. Immunol. 2011, 186:1685-1693. Muto, V; Stellacci, E; Lamberti, AG; Perrotti, E; Carrabba, A; Matera, G; Sgarbanti, M; Battistini, A; Liberto, MC; Focà, A. Human papillomavirus type 16 E5 protein induces expression of beta interferon J. Virol. 2011, 85:5070-5080. Mutocheluh, M; Hindle, L; Areste, C; Chanas, SA; Butler, LM; Lowry, K; Shah, K; Evans, DJ; Blackbourn, DJ. Kaposi's sarcoma-associated herpesvirus viral interferon J. Gen. Virol. 2011, 92:2394-2398. Nakagawa, K; Yokosawa, H. Degradation of transcription factor IRF-1 by the ubiquitinproteasome pathway. The C-terminal region governs the protein stability. Eur J. Biochem. 2000, 267:1680-1686. Nakagawa, K.; Yokosawa, H. PIAS3 induces SUMO-1 modification and transcriptional repression of IRF1, FEBS Lett. 2002, 530:204-208. Nakaya, T; Sato, M; Hata, N; Asagiri, M; Suemori, H; Noguchi, S; Tanaka, N; Taniguchi. T. Gene induction pathways mediated by distinct IRFs during viral infection. Biochem. Biophys. Res. Commun. 2001, 283: 1150-1156. Narayan, V; Pion, E; Landré, V; Müller, P; Ball, KL. Docking-dependent ubiquitination of the interferonJ. Biol. Chem. 2011, 286:607-611. Negishi, H; Ohba, Y; Yanai, H; Takaoka, A; Honma, K; Yui, K; Matsuyama, T; Taniguchi, T; Honda, K. Negative regulation of Toll-like-receptor signaling by IRF-4. Proc. Natl. Acad. Sci. USA 2005, 102:15989-15994. Negishi, H; Fujita, Y; Yanai, H; Sakaguchi, S; Ouyang, X; Shinohara, M; Takayanagi, H; Ohba, Y; Taniguchi, T; Honda, K. Evidence for licensing of IFN-gamma-induced IFN regulatory factor 1 transcription factor by MyD88 in Toll-like receptor-dependent gene induction program. Proc. Natl. Acad. Sci. USA 2006, 103:15136-15141. Nehyba, J; Hrdlicková, R; Bose, HR. Dynamic evolution of immune system regulators: the history of the interferon regulatory factor family. Mol. Biol. Evol. 2009, 26:2539-2550. Nelson, N; Kanno, Y; Hong, C; Contursi, C; Fujita, T; Fowlkes, BJ; O'Connell, E; Hu-Li, J; Paul, WE; Jankovic, D; Sher, AF; Coligan, JE; Thornton, A; Appella, E; Yang, Y; Ozato, K. Expression of IFN regulatory factor family proteins in lymphocytes. Induction of Stat- 1 and IFN consensus sequence binding protein expression by T cell activation. J. Immunol. 1996, 156:3711-3720. Nelson, N; Marks, MS; Driggers, PH; Ozato, K. Interferon consensus sequence-binding protein, a member of the interferon regulatory factor family, suppresses interferon- induced gene transcription. Mol. Cell Biol. 1993, 13:588-599. Ng, SL; Friedman, BA; Schmid, S; Gertz, J; Myers, RM; Tenoever, BR; Maniatis, T. IκB kinase (IKK) regulates the balance between type I and type II interferon responses. Proc. Natl. Acad. Sci. USA 2011, 108:21170-21175. Nguyen, H; Hiscott, J; Pitha, PM. The growing family of interferon Cytokine. Growth Factor Rev. 1997, 8:293-312. Nhu, QM; Cuesta, N; Vogel, SN. Transcriptional regulation of lipopolysaccharide (LPS)- induced Toll-like receptor (TLR J. Endotoxin. Res. 2006, 12:285-295. Interferon Regulatory Factors in Immune Cell Development … 55

Ning, S; Pagano, JS; Barber, GN. IRF7: activation, regulation, modification and function. Genes. Immun. 2011, 12:399-414. doi: 10.1038/gene.2011.21 O’Neill, LA; Bowie, AG. The family of five: TIR-domain-containing adaptors in Toll-like receptor signaling. Nat. Rev. Immunol. 2007, 7:353-364. Okumura, A; Alce, T; Lubyova, B; Ezelle, H; Strebel, K; Pitha, PM. HIV-1 accessory proteins VPR and Vif modulate antiviral response by targeting IRF-3 for degradation. Virology 2008, 373:85-97. Oshima, S; Nakamura, T; Namiki, S; Okada, E; Tsuchiya, K; Okamoto, R; Yamazaki, M; Yokota, T; Aida, M; Yamaguchi, Y; Kanai, T; Handa, H; Watanabe, M. Interferon regulatory factor 1 (IRF-1) and IRF-2 distinctively up-regulate gene expression Mol Cell Biol. 2004, 24:6298-6310. Osterlund, PI; Pietilä, TE; Veckman, V; Kotenko, SV; Julkunen, I. IFN regulatory factor family members differentially regulate the expression of type III IFN (IFN-lambda) genes. J. Immunol. 2007, 179:3434-3442. Otsuka, M; Kato, N; Moriyama, M; Taniguchi, H; Wang, Y; Dharel, N; Kawabe, T; Omata, M. Interaction between the HCV NS3 protein and the host Hepatology. 2005, 41:1004- 1012. Ouyang, X; Negishi, H; Takeda, R; Fujita, Y; Taniguchi, T; Honda, K. Cooperation between MyD88 and TRIF pathways in TLR synergy via IRF5 activation. Biochem. Biophys. Res. Commun. 2007, 354:1045-1051. Ouyang, X; Zhang, R; Yang, J; Li, Q; Qin, L; Zhu, C; Liu, J; Ning, H; Shin, MS; Gupta, M; Qi, CF; He, JC; Lira, SA; Morse, HC 3rd; Ozato, K; Mayer, L; Xiong, H. Transcription factor IRF8 directs a silencing programme for TH17 cell differentiation. Nat. Commun. 2011, 2:314. Ozato, K; Tailor, P; Kubota, T. The interferon regulatory factor family in host defense: mechanism of action. J. Biol. Chem. 2007, 282:20065-20069. Pandey, AK; Yang, Y; Jiang, Z; Fortune, SM; Coulombe, F; Fitzgerald, KA; Sassetti, CM; Kelliher, MA. NOD2, RIP2 and IRF5 play a critical role in the type I interferon response to Mycobacterium tuberculosis. PLoS Pathog. 2009, 5:e1000500. Panne, D; McWhirter, SM; Maniatis, T; Harrison, SC. Interferon response factor 3 is regulated by a dual phosphorylation dependent switch. J. Biol. Chem. 2007, 282:22816- 22822. Park, JS; Kim, EJ; Kwon, HJ; Hwang, ES; Namkoong, SE; Um, SJ. Inactivation of interferon regulatory factor-1 tumor suppressor protein by HPV E7 oncoprotein. Implication for the E7-mediated immune evasion mechanism in cervical carcinogenesis. J. Biol. Chem. 2000, 275:6764–6769. Park, SM; Chae, M; Kim, BK; Seo, T; Jang, IS; Choi, JS; Kim, IC; Lee, JH; Park, J. SUMOylated IRF-1 shows oncogenic potential by mimicking IRF-2. Biochem. Biophys. Res. Commun. 2010, 391:926-930. Passioura, T; Shen, S; Symonds, G; Dolnikov, A. A retroviral library genetic screen identifies IRF-2 as an inhibitor Oncogene. 2005, 24:7327-7336. Pathak, S; Ma, S; Trinh, L; Lu, R. A role for interferon regulatory factor 4 in in receptor editing. Mol. Cell Biol. 2008, 28:2815–2824. Paun, A; Pitha, PM. The IRF family, revisited. Biochimie 2007, 89:744-753. 56 Angela Battistini

Paun A, Reinert JT, Jiang Z, Medin C, Balkhi MY, Fitzgerald KA, Pitha PM (2008) Functional characterization of murine interferon regulatory factor 5 (IRF-5) and its role in the innate antiviral response. J. Biol. Chem. 2008, 283:14295-14308. Paun, A; Reinert, JT; Jiang, Z; Medin; Balkhi, M; Fitzgerald, KA; Pitha, PM. Functional characterization of murine interferon regulatory factor 5 (IRF-5) and its role in the innate antiviral response. J. Biol. Chem. 2008, 283: 14295-14308. Paun, A; Bankoti, R; Joshi, T; Pitha, PM; Stäger, S. Critical role of IRF-5 in the development of T helper 1 responses to Leishmania donovani infection. PLoS Pathog. 2011, 7:e1001246. Penninger, JM; Sirard, C; Mittrücker, HW; Chidgey, A; Kozieradzki, I; Nghiem, M; Hakem, A; Kimura, T; Timms, E; Boyd, R; Taniguchi, T; Matsuyama, T; Mak, TW. The interferon regulatory transcription factor IRF-1 controls positive and negative selection of CD8+ thymocytes. Immunity. 1997, 7:243-254. Peters, A, Lee, Y, Kuchroo, VK. The many faces of Th17 cells. Trends Immunol. 2011, 32:395-401. Pett, MR; Herdman, MT; Palmer, RD; Yeo, GS; Shivji, MK; Stanley, MA; Coleman, N. Selection of cervical keratinocytes containing integrated HPV16 associates with episome loss and an endogenous antiviral response. Proc. Natl. Acad. Sci. U S A 2006, 103:3822- 3827. Pietilä, TE; Veckman, V; Lehtonen, A; Lin, R; Hiscott, J; Julkunen, I. Multiple NF-kappaB and IFN regulatory factor family transcription factors regulate CCL19 gene expression in human monocyte-derived dendritic cells. J. Immunol. 2007, 178:253-261. Pine, R; Decker, T; Kessler, DS; Levy, DE; Darnel, JE Jr. Purification and cloning of interferon-stimulated gene factor 2 (ISGF2): ISGF2 (IRF-1) can bind to the promoters of both beta interferon- and interferon-stimulated genes but is not a primary transcriptional activator of either. Mol. Cell. Biol. 1990, 10:2448-2457. Pine, R. Constitutive expression of an ISGF2/IRF1 transgene J. Virol. 1992, 66:4470-4478. Pion, E; Narayan, V; Eckert, M; Ball, KL. Role of the IRF-1 enhancer domain in signalling polyubiquitination and degradation. Cell Signal. 2009, 21:1479-1487. Pivarcsi, A; Bodai, L; Réthi, B; Kenderessy-Szabó, A; Koreck, A; Széll, M; Beer, Z; Bata- Csörgoo, Z; Magócsi, M; Rajnavölgyi, E; Dobozy, A; Kemény, L. Expression and function of Toll-like receptors Int. Immunol. 2003, 15:721-730. Platanias, LC. Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat. Rev. Immunol 2005, 5:375-386. Poltorak, A; He, X; Smirnova, I; Liu, M.Y; Van Huffel, C; Du, X; Birdwell, D; Alejos. E; Silva. M; Galanos, C; Freudenberg, M; Ricciardi-Castagnoli, P; Layton, B; Beutler, B. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science. 1998, 282:2085-2088. Praefcke, GJ; Hofmann, K; Dohmen, RJ. SUMO playing tag with ubiquitin. Trends Biochem. Sci. 2012, 37:23-31. Epub 2011 Oct 20. Prakash, A; Levy, DE. Regulation of IRF7 through cell typespecific protein stability. Biochem Biophys. Res. Commun. 2006, 342:50-56. Prins, KC; Cardenas, WB; Basler, CF. Ebola virus protein VP35 impairs the function of interferon regulatory factor-activating kinases IKKepsilon and TBK-1. J. Virol. 2009, 83: 3069-3077. Interferon Regulatory Factors in Immune Cell Development … 57

Qin, BY; Liu, C; Lam, SS; Srinath, H; Delston, R; Correia, JJ; Derynck, R; Lin, K. Crystal structure of IRF-3 reveals mechanism of autoinhibition and virus-induced phosphoactivation. Nat. Struct. Biol.. 2003, 10:913-921. Ramos, HJ; Gale, M Jr. RIG-I Like receptors Curr. Opin. Virol. 2011, 1:167-176. Rehli, M; Poltorak, A; Schwarzfischer, L; Krause, SW; Andreesen, R; Beutler, B. PU.1 and interferon consensus sequence-binding protein regulate the myeloid expression of the human Toll-like receptor 4 gene. J. Biol. Chem. 2000, 275:9773-9781. Remoli, AL; Marsili, G; Perrotti, E; Gallerani, E; Ilari, R; Nappi, F; Cafaro, A; Ensoli, B; Gavioli, R; Battistini, A. Intracellular HIV-1 Tat protein represses constitutive LMP2 transcription increasing proteasome activity by interfering with the binding of IRF-1 to STAT1. Biochem. J. 2006, 396:371-380. Rengarajan, J; Mowen, KA; McBride, KD; Smith, ED; Singh, ED; Glimcher, LH. Interferon regulatory factor 4 (IRF4) interacts with NFATc2 to modulate interleukin 4 gene expression. J Exp Med. 2002, 95:1003-1012 Restivo, G; Nguyen, BC; Dziunycz, P; Ristorcelli, E; Ryan, RJ; Özuysal, ÖY; Di Piazza, M; Radtke, F; Dixon, MJ; Hofbauer, GF; Lefort, K; Dotto, GP. IRF6 is a mediator of Notch pro-differentiation and tumour suppressive function in keratinocytes. EMBO J. 2011, 30:4571-4585. Richardson, RJ; Dixon, J; Malhotra, S; Hardman, MJ; Knowles, L; Boot-Handford, RP; Shore, P; Whitmarsh, A; Dixon, MJ. Irf6 is a key determinant of the keratinocyte proliferation-differentiation switch. Nat. Genet. 2006, 38:1329-1334. Richez, C; Yasuda, K; Watkins, AA; Akira, S; Lafyatis, R; van Seventer, JM; Rifkin, IR. TLR4 J. Immunol. 2009, 182:820-828. Robinson, MJ; Sancho, D; Slack, EC; LeibundGut-Landmann, S; Reis e Sousa, C; Rouyez, MC; Lestingi, M; Charon, M; Fichelson, S; Buzyn, A; Dusanter-Fourt, I. IFN J. Immunol. 2005, 174:3948-3958. Robinson, MJ. Myeloid C-type lectins in innate immunity. Nat. Immunol. 2006, 7:1258-1265. Romieu-Mourez, R; Solis, M; Nardin, A; Goubau, D; Baron-Bodo, V; Lin, R; Massie, B; Salcedo, M; Hiscott, J. Distinct roles for IFN regulatory factor (IRF)-3 and IRF-7 in the activation of antitumor properties of human macrophages. Cancer Res. 2006, 66:10576- 10585. Ronco, LV; Karpova, AY; Vidal, M; Howley, PM. Human papillomavirus 16 E6 oncoprotein binds to interferon regulatory factor-3 and inhibits its transcriptional activity. Genes. Dev. 1998, 12:2061-2072. Ruffner, H; Reis, LF; Näf, D; Weissmann, C. Induction of type I interferon genes and interferon-inducible genes in embryonal stem cells devoid of interferon regulatory factor 1. Proc. Natl. Acad. Sci. U S A. 1993, 90:11503-11507. Sabbah, A; Chang, TH; Harnack, R; Frohlich, V; Tominaga, K; Dube, PH; Xiang, Y; Bose, S. Activation of innate immune antiviral responses by Nod2. Nat. Immunol. 2009, 10:1073- 1080. Saira, K; Zhou, Y; Jones, C. The infected cell protein 0 encoded by bovine herpesvirus 1 (bICP0) induces degradation of interferon response factor 3 and, consequently, inhibits β interferon promoter activity. J. Virol. 2007, 81:3077-3086. Saito, M; Gao, J; Basso, K; Kitagawa, Y; Smith, PM; Bhagat, G; Pernis, A; Pasqualucci, L; Dalla-Favera, R. A signaling pathway mediating downregulation of BCL6 in germinal 58 Angela Battistini

center B cells is blocked by BCL6 gene alterationsin B cell lymphoma. Cancer Cell. 2007, 12, 280-292. Saitoh, T; Tun-Kyi, A; Ryo, A; Yamamoto, M; Finn, G; Fujita, T; Akira, S; Yamamoto, N; Lu, K.P; and Yamaoka, S. Negative regulation of interferon-regulatory factor 3- dependent innate antiviral response by the prolyl isomerase Pin1. Nat. Immunol. 2006, 7:598-605. Sakaguchi, S; Negishi, H; Asagiri, M; Nakajima, C; Mizutani, T; Takaoka, A; Honda, K; Taniguchi, T. Essential role of IRF-3 in lipopolysaccharide-induced interferon-beta gene expression and endotoxin shock. Biochem. Biophys. Res. Commun. 2003, 306:860-866. Salkowski, CA, Kopydlowski, K, Blanco, J, Cody, MJ, McNally, R, Vogel, SN. IL-12 is dysregulated in macrophages from IRF-1 and IRF-2 knockout mice. J. Immunol. 1999, 163:1529-1536. Samarajiwa, SA; Forster, S; Auchettl, K; Hertzog, PJ. INTERFEROME: the database of interferon regulated genes. Nucleic Acids Res. 2009, 37(Database issue):D852-7. Sansonetti, PJ. The innate signaling of dangers and the dangers of innate signaling. Nat. Immunol. 2006 Dec;7(12):1237-42. Sato, M; Hata, N; Asagiri, M; Nakaya, T; Taniguchi, T; Tanaka, N. Positive feedback regulation of type I IFN FEBS Lett. 1998a; 441:106-109. Sato, M; Tanaka, N; Hata, N; Oda, E; Taniguchi, T. Involvement of the IRF family transcription FEBS Lett. 1998b, 425:112-116. Sato, M; Suemori, H; Hata, N; Asagiri, M; Ogasawara, K; Nakao, K; Nakaya, T; Katsuki, M; Noguchi, S; Tanaka, N; Taniguchi, T. Distinct and essential roles of transcription Immunity. 2000, 13:539-548. Sato, T; Onai, N; Yoshihara, H; Arai, F; Suda, T; Ohteki, T. Interferon regulatory factor-2 protects quiescent hematopoietic stem cells from type I interferon-dependent exhaustion. Nat. Med. 2009, 15:696–700. Satoh, T; Takeuchi, O; Vandenbon, A; Yasuda, K; Tanaka, Y; Kumagai, Y; Miyake, T; Matsushita, K; Okazaki, T; Saitoh, T; Honma, K; Matsuyama, T; Yui, K; Tsujimura, T; Standley, DM; Nakanishi, K; Nakai, K; Akira, S. The Jmjd3-Irf4 axis regulates M2 macrophage polarization and host responses against helminth infection. Nat. Immunol. 2010, 11: 936-944. Savitsky, D; Tamura, T; Yanai, H; Taniguchi, T. Regulation of immunity and oncogenesis by the IRF transcription factor family. Cancer Immunol. Immunother. 2010, 59:489-510. Savitsky, DA; Yanai, H; Tamura, T; Taniguchi, T; Honda, K. Contribution of IRF5 in B cells to the development of murine SLE-like disease through its transcriptional control of the IgG2a locus. Proc. Natl. Acad. Sci. U S A. 2010, 107:10154-10159. Schiavoni, G; Mattei, F; Sestili, P; Borghi, P; Venditti, M; Morse, III HC; Belardelli, F; Gabriele, L. ICSBP is essential for the development of mouse type I interferon-producing cells and for the generation and activation of CD8+ dendritic cells. J .Exp. Med. 2002, 196:1415-1425. Schindler, C; Fu, XY; Improta, T; Aebersold, R; Darnell, JE Jr. Proc. Natl. Acad. Sci. USA. 1992, 89:7836-7839. Schmid, S; Mordstein, M; Kochs, G; García-Sastre, A; Tenoever, BR. Transcription factor redundancy ensures induction of the antiviral state. J. Biol. Chem. 2010, 285:42013- 42022. Interferon Regulatory Factors in Immune Cell Development … 59

Schmidt, M; Nagel, S; Proba, J; Thiede, C; Ritter, M; Waring, JF; Rosenbauer, F; Huhn, D; Wittig, B; Horak, I; Neubauer, A. Lack of interferon consensus sequence binding protein (ICSBP) transcripts in human myeloid leukemias. Blood. 1998; 91: 22-29. Schmitz, F; Heit, A; Guggemoos, S; Krug, A; Mages, J; Schiemann, M; Adler, H; Drexler, I, Haas, T, Lang, R, Wagner, H. Interferon-regulatory-factor 1 controls Toll-like receptor 9- mediated IFN Eur. J. Immunol. 2007, 37:315-327. Schoenemeyer, A; Barnes, BJ; Mancl, ME; Latz, E; Goutagny, N; Pitha, PM; Fitzgerald, KA; Golenbock, DT. The interferon regulatory factor, IRF5, is a central mediator of toll-like receptor 7 signaling. J. Biol. Chem. 2005, 280:17005-17012. Schoggins, JW; Wilson, SJ; Panis, M; Murphy, MY; Jones, CT; Bieniasz, P; Rice, CM. A diverse range of gene products are effectors of the type I interferon antiviral response. Nature. 2011, 472:481-485. Schroder, K; Lichtinger, M; Irvine, KM; Brion, K; Trieu, A; Ross, IL; Ravasi, T; Stacey, KJ; Rehli, M; Hume, DA; Sweet, MJ. PU.1 and ICSBP control constitutive and IFN-gamma- regulated Tlr9 gene expression in mouse macrophages. J. Leukoc. Biol. 2007, 81:1577- 1590. Schroder, K; Tschopp, J. The inflammasomes. Cell 2010, 140:821- 832. Schroder, M; Baran, M; Bowie, AG. Viral targeting of DEAD box protein 3 reveals its role in TBK1/IKKepsilon-mediated IRF activation. EMBO J. 2008, 27:2147-2157. Sciammas, R; Davis, MM. Modular nature of Blimp-1 in the regulation of gene expression during B cell maturation. J. Immunol. 2004, 172, 5427-5440. Sciammas, R; Shaffer, AL; Schatz, JH; Zhao, H; Staudt, LM; Singh, H. Graded expression of interferon regulatory factor-4 coordinates isotype switching with plasma cell differentiation. Immunity. 2006, 25, 225-236. Sen, N; Sommer, M; Che, X; White, K; Ruyechan, WT; Arvin, AM. Varicella-zoster virus immediate-early protein 62 blocks interferon regulatory factor 3 (IRF3) phosphorylation at key serine residues: a novel mechanism of IRF3 inhibition among herpesviruses. J. Virol. 2010, 84:9240-9253. Seth, RB; Sun, L; Ea, CK; Chen, ZJ. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3. Cell. 2005, 122:669-682. Seth, RB; Sun, L; Chen, ZJ. Antiviral innate immunity pathways. Cell Res. 2006, 16:141– 147. Sgarbanti, M; Borsetti, A; Moscufo, N; Bellocchi, MC; Ridolfi, B; Nappi, F; Marsili, G; Marziali, G; Coccia, EM; Ensoli, B; Battistini, A. Modulation of human immunodeficiency virus 1 replication by interferon regulatory factors. J. Exp. Med. 2002, 195:1359-1370. Sgarbanti, M; Marsili, G; Remoli, AL; Orsatti, R; Battistini A. IRF-7: new role in the regulation of genes involved in adaptive immunity. Ann. N Y Acad. Sci. 2007, 1095:325- 333. Sgarbanti, M; Remoli, AL; Marsili, G; Ridolfi, B; Borsetti, A; Perrotti, E; Orsatti, R; Ilari, R; Sernicola, L; Stellacci, E; Ensoli, B; Battistini, A. IRF-1 is required for full NF-B transcriptional activity at the human immunodeficiency virus type 1 long terminal repeat enhancer. J. Virol. 2008, 82: 3632-3641. Sharf, R; Meraro, D; Azriel, A; Thornton, AM; Ozato, K; Petricoin, EF; Larner, AC; Schaper, F; Hauser, H; Levi, BZ. Phosphorylation events modulate the ability of interferon 60 Angela Battistini

consensus sequence binding protein to interact with interferon regulatory factors and to bind DNA. J. Biol. Chem. 1997, Apr 11;272(15):9785-9792. Sharma, S; tenOever, BR; Grandvaux, N; Zhou, GP; Lin, R; Hiscott, J. Triggering the interferon antiviral response through an IKK-related pathway. Science. 2003, 300:1148- 1151. Shortman, K; Liu, YJ. Mouse and human dendritic cell subtypes. Nat. Rev. Immunol. 2002, 2:151-161. Solis, M; Nakhaei, P; Jalalirad, M. RIG-I-mediated antiviral signaling is inhibited in HIV J. Virol. 2011, 85:1224-1236. Song, YJ; Izumi, KM; Shinners, NP; Gewurz, BE; Kie, VE. IRF7 activation by Epstein-Barr virus latent membrane protein 1 requires localization at activation sites and TRAF6, but not TRAF2 or TRAF3. Proc. Natl. Acad. Sci. USA 2008, 105:18448–18453. Staal, A; Enserink, JM; Stein, JL; Stein, GS; van Wijnen, A. J. Molecular characterization of celtix-1, a bromo domain protein interacting with the transcription factor interferon regulatory factor 2. J. Cell Physiol. 2000, 185: 269-279. Stellacci, E; Testa, U; Petrucci, E; Benedetti, E; Orsatti, R; Feccia, T; Stafsnes, M; Coccia, EM.; Marziali, G; Battistini, A. Interferon regulatory factor-2 drives megakaryocytic differentiation. Biochem J. 2004, 377:367-378. Stetson, DB; Medzhitov, R. Recognition of cytosolic DNA activates an IRF3-dependent innate immune response. Immunity 2006, 24:93-103. Stetson, DB; Ko, JS; Heidmann, T; Medzhitov, R. Trex1 prevents cell-intrinsic initiation of autoimmunity. Cell 2008, 134:587-598. Stirnweiss, A; Ksienzyk, A; Klages, K; Rand, U; Grashoff, M; Hauser, H; Kröger, A. IFN regulatory factor-1 bypasses IFN-mediated antiviral effects through Viperin gene induction. J. Immunol. 2010, 184:5179-5185. Stockinger, S; Reutterer, B; Schal o, B; Schellack, C; Brunner, S; Materna, T; amamoto, M; Akira, S; Taniguchi, T; Murray, PJ; Mu ller, M; Decker, T. IFN regulatory factor 3- dependent induction of type I IFNs by intracellular bacteria is mediated by a TLR- and Nod2-independent mechanism. J. Immunol. 2004, 173:7416-7425. Su, ZZ; Sarkar, D; Emdad, L; Barral, PM; Fisher, PB. Central role of interferon regulatory factor-1 (IRF-1) in controlling retinoic acid inducible gene-I (RIG-I) expression. J. Cell Physiol. 2007, 213:502-510. Suhara, W; Yoneyama, M; Fujita, T. Direct Involvement of CREB-binding Protein/p300 in Sequence-specific DNA Binding of Virus-activated Interferon Regulatory Factor-3 Holocomplex. J. Biol. Chem. 2002, 277:22304-22313. Suzuki, S; Honma, K; Matsuyama, T; Suzuki, K; Toriyama, K; Akitoyo, I; Yamamoto, K; Suematsu, T; Nakamura, M; Yui, K; Kumatori A. Critical roles of interferon regulatory factor 4 in CD11bhighCD8alpha-dendritic cell development. Proc. Natl. Acad. Sci. USA 2004, 101:8981-8986. Tabeta, K; Georgel, P; Janssen, E; Du, X; Hoebe, K; Crozat, K; Mudd, S; Shamel, L; Sovath, S; Goode, J; Alexopoulou, L; Flavell, RA; Beutler, B. Toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection. Proc. Natl. Acad. Sci. USA 2004, 101:3516-3521. Tailor, P; Tamura, T; Kong, HJ; Kubota, T; Kubota, M; Borghi, P; Gabriele, L; Ozato K. The feedback phase of type I interferon induction in dendritic cells requires interferon regulatory factor 8. Immunity 2007, 27:228-239. Interferon Regulatory Factors in Immune Cell Development … 61

Tailor, P; Tamura, T; Morse, HC 3rd; Ozato, K. The BXH2 mutation in IRF8 differentially impairs dendritic cell subset development in the mouse. Blood 2008, 111:1942-1945. Takaoka, A; Yanai, H; Kondo, S; Duncan, G; Negishi, H; Mizutani, T; Kano, S; Honda, K; Ohba, Y; Mak, TW; Taniguchi T. Integral role of IRF-5 in the gene induction Nature. 2005, 434:243-249. Takaoka, A; Wang, Z; Choi, MK; Yanai, H; Negishi, H; Ban, T; Lu, Y; Miyagishi, M; Kodama, T; Honda, K; Ohba, Y; Taniguchi T. DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. Nature. 2007, 448:501-505. Takaoka, A; Tamura, T; Taniguchi, T. Interferon regulatory factor family of transcription factors and regulation of oncogenesis. Cancer Sci. 2008, 99:467-478. Takeuchi, O; Akira, S. Pattern recognition receptors and inflammation. Cell. 2010, 140:805- 820. Taki, S; Sato, T; Ogasawara, K; Fukuda, T; Sato, M; Hida, S; Suzuki, G; Mitsuyama, M; Shin, EH; Kojima, S; Taniguchi, T; Asano, Y. Multistage regulation of Th1-type immune responses by the transcription factor IRF-1. Immunity. 1997, 6:673-679. Taki, S; Nakajima, S; Ichikawa, E; Saito, T; Hida, S. IFN J. Immunol. 2005, 174:6005-6012. Tamura, T; Nagamura-Inoue, T; Shmeltzer, Z; Kuwata, T; Ozato, K. ICSBP directs bipotential myeloid progenitor cells to differentiate into mature macrophages. Immunity 2000, 13:155-165. Tamura, T; Tailor, P; Yamaoka, K; Kong, HJ; Tsujimura, H; O'Shea, JJ; Singh, H; Ozato, K. IFN regulatory factor-4 and -8 govern dendritic cell subset development and their functional diversity. J. Immunol. 2005a, 174:2573-2581. Tamura, T; Thotakura, P; Tanaka, TS; Ko, MS; Ozato, K. Identification of target genes and a unique cis element regulated by IRF-8 in developing macrophages. Blood. 2005b, 106:1938-1947. Tanaka, N; Kawakami, T; Taniguchi, T. Recognition DNA sequences of interferon regulatory factor 1 (IRF-1) and IRF-2, regulators of cell growth and the interferon system. Mol. Cell Biol. 1993, 13:4531-4538. Tang, X; Gao, JS; Guan, YJ; McLane, KE; Yuan, ZL; Ramratnam, B; Chin, YE. Acetylation- dependent signal transduction for type I interferon receptor. Cell. 2007, 131:93-105. Taniguchi, T; Ogasawara K; Takaoka A; Tanaka N. IRF family of transcription factors as regulators of host defense. Annu. Rev. Immunol. 2001, 19:623-655. tenOever, BR; Sharma, S; Zou, W; Sun, Q; Grandvaux, N; Julkunen, I;Hemmi, H; Yamamoto, M; Akira, S; Yeh, WC; Lin, R; and Hiscott, J. Activation of TBK1 and IKKvarepsilon kinases by vesicular stomatitis virus infection and the role of viral ribonucleoprotein in the development of interferon antiviral immunity. J. Virol. 2004, 78:10636-10649. Testa, U; Stellacci, E; Pelosi, E; Sestili, P; Venditti, M; Orsatti, R; Fragale, A; Petrucci, E; Pasquini, L; Belardelli, F; Gabriele, L; Battistini, A. Impaired myelopoiesis in mice devoid of interferon regulatory factor 1. Leukemia. 2004, 18:1864-1871. Thanos, D; Maniatis, T. Virus induction Cell 1995, 83:1091-100. Thefilopoulos, AN; Baccala, R; Bautler, B; Kono, DH. Type I interferons (alpha/beta) in immunity and autoimmunity. Annu. Rev. Immunol. 2005, 23:307-336. Thomason, HA; Zhou, H; Kouwenhoven, EN; Dotto, GP; Restivo, G; Nguyen, BC; Little, H; Dixon, MJ; van Bokhoven, H; Dixon, J. Cooperation between the transcription factors 62 Angela Battistini

p63 and IRF6 is essential to prevent cleft palate in mice. J. Clin. Invest. 2010, 120:1561- 1569. doi: 10.1172/JCI40266. Thompson, EC; Cobb, BS; Sabbattini, P; Meixlsperger, S; Parelho, V; Liberg, D; Taylor, B; Dillon, N; Georgopoulos, K; Jumaa, H; Smale, ST; Fisher, AG; Merkenschlager, M. Ikaros DNA-binding proteins as integral components of B cell developmental-stage- specific regulatory circuits. Immunity. 2007, 26:335-344. Ting, JP; Duncan, JA; Lei, Y. How the noninflammasome NLRs function in the innate immune system. Science. 2010, 327:286-290. Tokoyoda, K; Egawa, T; Sugiyama, T; Choi, BI; Nagasawa, T. Cellular niches controlling B lymphocyte behavior within bone marrow during development. Immunity. 2004, 20:707- 718. Tominaga, N; Ohkusu-Tsukada, K; Udono, H; Abe, R; Matsuyama, T; Yui, K. Development of Th1 and not Th2 immune responses in mice lacking IFN-regulatory factor-4. Int. Immunol. 2003, 15:1-10. Tsujimura, H; Nagamura-Inoue, T; Tamura, T; Ozato, K. IFN consensus sequence binding protein/IFN regulatory factor-8 guides bone marrow progenitor cells toward the macrophage lineage. J. Immunol. 2002, 169: 1261-1269. Tsujimura, H; Tamura, T; Kong, HJ; Nishiyama, A;Ishii, KJ; Klinman, DM; Ozato, K Toll- like receptor 9 signaling activates NfkappaB through IFN regulatory factor-8/IFN consensus sequence binding protein in dendritic cells. J. Immunol. 2004, 172:6820-6827. Tsujmura, H; Tamura, T; Ozato, K; Cuttin, E. IFN consensus sequence binding protein/IFN regulatory factor 8 drives the development of type 1 IFN-producing plasmacytoid dendritic cells. J. Immunol. 2003, 170:1131-1135. Turcotte, K; Gauthier, S; Tuite, A; Mullick, A; Malo, D; Gross, P. A mutation in the Icsbp1 gene causes susceptibility to infection and a chronic myeloidleukemia–like syndrome in BXH-2 mice. J. Exp. Med. 2005, 201:881-890. Uematsu, S; Akira, S. Toll-like receptors and Type I interferons. J Biol Chem 282: 15319– 15323; Bogdan C, Mattner J, Schleicher U (2004) The role of type I interferons in non- viral infections. Immunol. Rev. 2007, 202: 33-48. Unterholzner, L; Bowie, AG. The interplay between viruses Biochem. Pharmacol. 2008, 75:589-602. Unterholzner, L; Sumner, RP; Baran, M; Ren, H; Mansur, DS; Bourke, NM; Randow, F; Smith, GL; Bowie, AG. Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7. PLoS Pathog. 2011, 7:e1002247. Unterstab, G; Ludwig, S; Anton, A; Planz, O; Dauber, B; Krappmann, D; Heins, G; Ehrhardt, C; Wolff, T. Viral targeting of the interferon-β-inducing Traf family member-associated NFkB activator (TANK)-binding kinase-1. Proc. Natl. Acad. Sci. USA 2005, 102:13640- 13645. van 't Wout AB, Lehrman GK, Mikheeva SA, O'Keeffe GC, Katze MG, Bumgarner RE, Geiss GK, Mullins JI. Cellular gene expression upon human immunodeficiency virus type 1 infection of CD4(+)-T-cell lines. J. Virol. 2003, 77:1392-1402. Vaughan, PS; Aziz, F; van Wijnen, AJ; Wu, S; Harada, H; Taniguchi, T; Soprano, KJ; Stein, JL; Stein, GS. Activation of a cell cycle-regulated histone gene by the oncogenic transcription factor IRF-2. Nature. 1995, 377:362-365. Interferon Regulatory Factors in Immune Cell Development … 63

Vaughan, PS; van der Meijden, CM; Aziz, F; Harada, H; Taniguchi, T; van Wijnen, AJ; Stein, JL; Stein, GS Cell cycle regulation of histone H4 gene transcription requires the oncogenic factor IRF-2. J. Biol. Chem. 1998, 273:194-199. Veals, SA; Schindler, C; Leonard, D; Fu, XY; Aebersold, R; Darnell, JE; Jr., and Levy, DE. Subunit of an alpha-interferon-responsive transcription factor is related to interferon regulatory factor and Myb families of DNA-binding proteins. Mol. Cell Biol. 1992, 12:3315-3324. Veals, SA; Santa Maria, T; Levy, DE. Two domains of ISGF3 gamma that mediate protein- DNA Mol. Cell Biol. 1993, 13:196-206. Wang, H; Lee, CH; Qi, C; Tailor, P; Feng, J; Abbasi, S; Atsumi, T; Morse, HC 3rd. IRF8 regulates B-cell lineage specification, commitment, and differentiation. Blood. 2008, 112: 4028-4038. Wang, Y; Liu, DP; Chen, PP; Koeffler, HP; Tong, XJ; Xie, D. Involvement of IFN regulatory factor (IRF)-1 and IRF-2 in the formation and progression of human esophageal cancers. Cancer Res. 2007. 67: 2535-2543. Wang, Z; Choi, MK; Ban, T; Yanai, H; Negishi, H; Lu, Y; Tamura, T; Takaoka, A; Nishikura, K; Taniguchi, T. Regulation of innate immune responses by DAI (DLM- 1/ZBP1) and other DNAsensing molecules. Proc. Natl. Acad. Sci. USA 2008, 105:5477- 5482. Watanabe, N; Sakakibara, J; Hovanessian, AG; Taniguchi, T; Fujita, T. Activation of IFN- beta element by IRF-1 requires a posttranslational event in addition to IRF-1 synthesis. Nucleic Acids Res. 1991, 19:4421-4428. Watanabe, T; Asano, N; Fichtner-Feigl, S; Gorelick, PL; Tsuji, Y; Matsumoto, Y; Chiba, T; Fuss, IJ; Kitani, A; Strober W: NOD1 contributes to mouse host defense against Helicobacter pylori via induction of type I IFN and activation of the ISGF3 signaling pathway. J. Clin. Invest. 2010, 120:1645-1662. Wathelet, MG; Lin, CH; Parekh, BS; Ronco, LV; Howley, PM; Maniatis, T. Virus infection Mol. Cell. 1998, 1:507-518. Weaver, BK; Kumar, KP; Reich, NC. Interferon regulatory factor 3 and CREB-binding protein/p300 are subunits of double-stranded RNA-activated transcription factor DRAF1. Mol. Cell Biol. 1998, 18:1359-1368. Weaver, CT; Harrington, LE;, Mangan, PR; Gavrieli, M; Murphy KM. Th17: An effector CD4 T cell lineage with regulatory T cell ties. Immunity. 2006, 24:677-688. White, LC; Wright, KL; Felix, NJ; Ruffner, H; Reis, LF; Pine, R; Ting, JP. Regulation of LMP2 and TAP1 genes Immunity 1996, 5:365-376. Wies, E; Hahn, AS; Schmidt, K; Viebahn, C; Rohland, N; Lux, A; Schellhorn, T; Holzer, A; Jung, JU; Neipel, F. The Kaposi’s sarcoma-associated herpesvirus-encoded vIRF-3 inhibits cellular IRF-5. J. Biol. Chem. 2009, 284:8525-8538. Wilkins, C; Gale, M Jr. Recognition of viruses by cytoplasmic sensors. Curr. Opin. Immunol. 2010, 22:41-47. Wilson, JR; de Sessions, PF; Leon, MA; Scholle, F. West Nile virus nonstructural protein 1 inhibits TLR3 signal transduction. J. Virol. 2008, 82:8262-8271. Xi, H; Eason, DD; Ghosh, D; Dovhey, S; Wright, KL; Blanck, G. Co-occupancy of the interferon regulatory element of the class II transactivator (CIITA) type IV promoter by interferon regulatory factors 1 and 2. Oncogene. 1999, 43:5889-5903. 64 Angela Battistini

Xiong, H; Li, H; Kong, HJ; Chen, Y; Zhao, J; Xiong, S; Huang, B; Gu, H; Mayer, L; Ozato, K; Unkeless, JC. Ubiquitin-dependentdegradation of interferon regulatory factor-8 mediated by Cbl down-regulates interleukin-12 expression. J. Biol. Chem. 2005, 280: 23531-23539. Xiong, H; Zhu, C; Li, H; Chen, F; Mayer, L; Ozato, K; Unkeless, JC; Plevy, SE. Complex formation of the interferon (IFN) consensus sequence-binding protein with IRF-1 is essential for murine macrophage IFN-gamma-induced iNOS gene expression. J. Biol. Chem. 2003, 278, 2271–2277. Xu, LG; Wang, YY; Han, KJ; Li, LY; Zhai, Z; Shu, HB. VISA is an adapter protein required for virus-triggered IFN-beta signaling. Mol. Cell. 2005, 19:727-740. Yamagata, T; Nishida, J; Tanaka, S; Sakai, R; Mitani, K; Yoshida, M; Taniguchi, T; Yazaki, Y; Hirai, H. A novel interferon regulatory factor family transcription factor, ICSAT/Pip/LSIRF, that negatively regulates the activity of interferon-regulated genes. Mol. Cell Biol. 1996, 16:1283-1294. Yamamoto, H; Lamphier, MS; Fujita, T; Taniguchi, T; Harada, H. The oncogenic transcription factor IRF-2 possesses a transcriptional repression and a latent activation domain. Oncogene 1994, 9:1423-1428. Yamamoto, M; Kato, T; Hotta, C; Nishiyama, A; Kurotaki, D; Yoshinari, M; Takami, M; Ichino, M; Nakazawa, M; Matsuyama, T; Kamijo, R; Kitagawa, S; Ozato, K; Tamura, T. Shared and distinct functions of the transcription factors IRF4 and IRF8 in myeloid cell development. PLoS One. 2011, 6:e25812. Yanai, H; Chen, HM; Inuzuka, T; Kondo, S; Mak, TW; Takaoka, A; Honda, K; Taniguchi, T. Role of IFN regulatory factor 5 transcription factor in antiviral immunity and tumor suppression. Proc. Natl. Acad. Sci. USA 2007, 104:3402-3407. Yang, H; Lin, CH; Ma, G; Baffi, MO; Wathelet, MG. Interferon regulatory factor-7 synergizes with other transcription J, Biol, Chem. 2003, 278:15495-15504. Yang, Y; Liang, Y; Qu, L; Chen, Z; Yi, M; Li, K; Lemon, SM. Disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor. Proc, Natl, Acad ,Sci, USA 2007, 104:7253-7258. Yarilina A, Park-Min KH, Antoniv T, Hu X, Ivashkiv LB. TNF activates an IRF1-dependent autocrine loop leading to sustained expression of chemokines and STAT-1 dependent type I interferon-response genes. Nature Immunol. 2008, 9:378-387. Yasuda, K; Richez, C; Maciaszek, JW; Agrawal, N; Akira, S; Marshak-Rothstein, A; Rifkin, IR.. Murine dendritic cell type I IFN production induced by human IgG-RNA immune complexes is IFN regulatory factor (IRF)5 and IRF7 dependent and is required for IL-6 production. J, Immunol. 2007, 178:6876-6885. Yoneyama, M; Suhara, W; Fukuhara, Y; Sato, M; Ozato, K; Fujita, T. Autocrine amplification of type I interferon gene expression mediated by interferon stimulated gene factor 3 (ISGF3). J, Biochem. 1996, 120:160-169. Yoneyama, M; Suhara, W; Fukuhara, Y; Fukada, M; Nishida, E; Fujita, T. Direct triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF-3 andCBP/p300. Embo, J. 1998, 17:1087-1095. Yoneyama, M; Kikuchi, M; Natsukawa, T; Shinobu, N; Imaizumi, T; Miyagishi, M; Taira, K; Akira, S; Fujita, T. The RNA Nat. Immunol. 2004, 5:730-737. Yoneyama, M; Kikuchi, M; Matsumoto, K; Imaizumi, T; Miyagishi, M; Taira, K; Foy, E; Loo, YM; Gale, M Jr;; Akira, S; Yonehara, S; Kato, A; Fujita, T. Shared and unique Interferon Regulatory Factors in Immune Cell Development … 65

functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity J. Immunol. 2005, 175:2851-2858 Yu, Y; Hayward, GS. The ubiquitin Immunity. 2010, 33:863-877. Yu, Y; Wang, SE; Hayward, GS. The KSHV immediate-early transcription factor RTA encodes ubiquitin E3 ligase activity that targets IRF7 for proteosome-mediated degradation Immunity 2005, 22:59.-70. Zhang, L; Pagano, JS. IRF-7, a new interferon regulatory factor associated with Epstein-Barr Virus latency. Mol. Cell Biol. 1997, 17: 5748-5757. Zhang, L; Zhang, J; Lambert, Q; Der, CJ; Del Valle, L; Miklossy, J; Khalili, K; Zhou, Y; Pagano, JS. Interferon regulatory factor 7 is associated with Epstein-Barr virus- transformed central nervous system lymphoma and has oncogenic properties. J. Virol, 2004, 78:12987-12995. Zhang, SY; Jouanguy, E; Ugolini, S; Smahi, A; Elain, G; Romero, P; Segal, D; Sancho- Shimizu, V; Lorenzo, L; Puel, A; Picard, C; Chapgier, A; Plancoulaine, S; Titeux, M; Cognet, C; von Bernuth, H; Ku, CL; Casrouge, A; Zhang, XX; Barreiro, L; Leonard, J; Hamilton, C; Lebon, P; Heron, B; Vallee, L; Quintana-Murci, L; Hovnanian, A; Rozenberg, F; Vivier, E; Geissmann, F; Tardieu, M; Abel, L; Casanova, JL. TLR3 deficiency in patients with herpes simplex encephalitis. Science. 2007, 317:1522-1527. Zhao, J; Kong, HJ; Li, H; Huang, B; Yang, M; Zhu, C; Bogunovic, M; Zheng, F; Mayer, L; Ozato, K; Unkeless, J; Xiong, H. IRF-8/interferon (IFN) consensus sequence-binding protein is involved in Toll-like receptor (TLR) signaling and contributes to the cross-talk between TLR and IFN-gamma signaling pathways. J, Biol, Chem. 2006, 281:10073- 10080. Zhao, T; Yang, L; Sun, Q; Arguello, M; Ballard, DW; Hiscott, J; Lin, R. The NEMO adaptor bridges the nuclear factor-kappaB and interferon regulatory factor signaling pathways. Nat, Immunol. 2007, 8:592-600. Zhong, B; Yang, Y; Li, S; Wang, YY; Li, Y; Diao, F; Lei, C; He, X; Zhang, L; Tien, P; Shu, HB. The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation. Immunity. 2008, 29:538-550. Zhou, JX; Lee, CH; Qi, CF; Wang, H; Naghashfar, Z; Abbasi S; Morse HC 3rd. IFN regulatory factor 8 regulates MDM2 in germinal center B cells. J. Immunol. 2009, 183:3188-3194. Zhu, C; Saberwal, G; Ly, Y; Platanias, F; L. C. and Eklund E. A. The interferon consensus sequence-binding protein activates transcription of the gene encoding neurofibromin. J, Biol, Chem. 2004, 279:50874-50885. Zhu, FX; King, SM; Smith, EJ; Levy, DE; Yuan, YA. Kaposi's sarcoma-associated herpesviral protein inhibits virus-mediated induction of type I interferon by blocking IRF-7 phosphorylation and nuclear accumulation. Proc. Natl, Acad. Sci. USA 2002, 99:5573-5578.

Author Disclosure Statement

The author declares that no competing financial interests exist. Correspondence to: Dr. Angela Battistini; Molecular Pathogenesis Unit 66 Angela Battistini

Department of Infectious, Parasitic and Immune-Mediated Diseases Istituto Superiore di Sanità Viale Regina Elena, 299 00161 Rome, Italy

Tel: (+39 06) 49903266 Fax: (+39 06) 49902082 E-mail: [email protected]