Role Of IRF-1 Research Articles

Abstract 118: Effect of Interferon Regulatory Factor-1 on Vascular Dementia Using Mouse Chronic Cerebral Hypoperfusion Model

Objective: Accumulation of inflammatory cells around the damaged blood vessels is an important feature of the pathophysiology of Binswanger disease due to hypoxic conditions. Recent reports indicate that expression of interferon regulatory factor (IRF)-1-dependent genes in neurons plays a role in ischemic neuronal death. However, the roles of IRF-1 in vascular dementia are not well investigated. Therefore, we assessed the effect of IRF-1 on cognitive function using vascular dementia mouse model by chronic cerebral hypoperfusion. Methods: Male 10-week-old C57BL/6 (wild-type; WT) and IRF-1-knockout (IRF-1KO) mice were used in this study. Chronic cerebral hypoperfusion mouse model was generated by bilateral common carotid artery stenosis (BCAS). Six weeks after BCAS, the mice were subjected to the Morris water maze test 5 times a day for 5 days. After the Morris water maze task, cerebral blood flow (CBF) was evaluated by laser speckle flowmetry. Expression of NADPH oxidase subunits and inflammatory cytokines in cortex and hippocampus were determined by real-time RT-PCR. Results: Body weight before and 6 weeks after BCAS operation did not differ in all groups. In the Morris water maze task, escape latency was significantly prolonged in sham-operated IRF-1KO mice compared with sham-operated WT mice. Moreover, BCAS-treated IRF-1KO mice exhibited more prolonged escape latency compared with sham-operated IRF-1KO and BCAS-treated WT mice. Although BCAS treatment reduced CBF in WT mice, there was no significant difference in CBF between sham-operated WT and IRF-1KO mice. On the other hand, after BCAS treatment, CBF was more markedly decreased in IRF-1KO mice than WT mice. In sham-operated groups, mRNA expression of NDADPH oxidase subunits and inflammatory cytokines in the brain did not differ between WT and IRF-1KO mice. In contrast, BCAS-treated IRF-1KO mice showed the tendency to increase mRNA expression of p22phox, p47phox, TNF-α and MCP-1 than BCAS-treated WT mice. Conclusion: These results suggest that IRF-1 has a protective effect on cognitive decline due to the prevention of CBF reduction, and the increases in NADPH oxidase and inflammatory cytokines in mouse chronic cerebral hypoperfusion model.

IFN Regulatory Factor-1 Modulates the Function of Dendritic Cells in Patients with Acute Coronary Syndrome

Background: Atherosclerosis is widely recognized as a complex inflammatory disease involving pathogenic immune response of T cells and antigen-presenting cells (APCs) such as dendritic cells (DCs) and macrophages. Accumulating evidence has revealed that mature DCs play critical roles in the differentiation of effector T cells into CD4+ T cells, which effectively participate in the onset of acute coronary syndrome (ACS). IFN regulatory factor (IRF)-1 has been shown to be involved in various immune processes. The role of IRF-1 in DCs in the pathogenesis of ACS has not been investigated. Methods and Results: We examined the relative mRNA and protein expression of IRF-1 in human monocyte-derived DCs in patients with coronary artery disease (CAD). The overexpression or silencing of IRF-1 expression in DCs in patients with ACS was performed to explore the possible role of IRF-1 in the maturation and function of DCs involved in ACS. The results showed that the relative expression of IRF-1 in DCs is obviously increased in patients with ACS. The overexpression or silencing of IRF-1 expression could effectively promote or attenuate the maturation and function of DCs. In addition, we revealed that the MAPK pathway (phosphorylation of JNK, p38 and ERK1/2) might be downstream of IRF-1 signalling pathway in activation of circulating DCs in ACS patients. Conclusion: The present data demonstrate that IRF-1 could effectively promote the immune maturation and function of DCs in ACS patients.

K63-linked polyubiquitination of IRF1: an essential step in the IL-1 signaling cascade.

K63-linked polyubiquitination of IRF1: an essential step in the IL-1 signaling cascade.

Abstract 370: Interferon Regulatory Factors 3 and 7 Regulate Vein Graft Remodeling and Vascular Inflammation

Introduction: Type I interferons (IFN) are implicated in the development of vascular proliferative diseases. Pathway analysis by gene set enrichment analysis of hypercholesterolemic ApoE3*Leiden murine vein grafts revealed that the type I IFN pathway belonged to the top 15 of significant regulated pathways. The transcriptional regulators of type I IFN and type I IFN responsive genes are the interferon regulatory factors (IRF). Activation of TLR3 results in activation of type I IFN in a IRF3 and IRF7 dependent manner. TLR3 has been shown to be protective in vascular remodeling. The aim of this study was to investigate the role of IRF3 and IRF7 on vein graft remodeling. Methods and Results: The importance of IRFs in vein graft remodeling is illustrated by the increase in vein graft thickening in Irf3 -/- and Irf7 -/- mice in comparison to control mice (n=9/group, Irf3 -/- ; 39%, p=0.185, Irf7 -/- ; 68% p=0.003). Also an increase in outward remodeling ( Irf3 -/- ; 26%, p=0.081, Irf7 -/- ; 42%, p=0.049) was observed comparable to that of Tlr3 -/- (n=8, 52%, p<0.001) Immunohistochemical analysis revealed that both Irf3 -/- and Irf7 -/- mice showed a significant higher influx of macrophages in the vessel wall than the control mice whereas the Irf7 -/- mice also showed a significant decrease in collagen content. RNA levels of typical type I IFN responsive genes such as Mx1, Ifit1-3 and Oas2 were down regulated in the knockout vein grafts in comparison to control vein grafts. Activation of both Irf3 -/- and Irf7 -/- bone marrow derived macrophages with LPS and poly:(IC) resulted in a significant increase in TNFα production. Conclusions: IRFs regulates vein graft remodeling since Irf3 -/- and especially Irf7 -/- vein grafts show increased vessel wall thickening and outward remodeling. This increased remodeling is the result of a pro-inflammatory response as reflected by the increase in macrophages in the vein graft wall.

A Role for IRF8 in B Cell Anergy

B cell central tolerance is a process through which self-reactive B cells are removed from the B cell repertoire. Self-reactive B cells are generally removed by receptor editing in the bone marrow and by anergy induction in the periphery. IRF8 is a critical transcriptional regulator of immune system development and function. A recent study showed that marginal zone B cell and B1 B cell populations are dramatically increased in IRF8-deficient mice, indicating that there are B cell-developmental defects in the absence of IRF8. In this article, we report that mice deficient for IRF8 produced anti-dsDNA Abs. Using a hen egg lysozyme double-transgenic model, we further demonstrate that B cell anergy was breached in IRF8-deficient mice. Although anergic B cells in the IRF8-proficient background were blocked at the transitional stage of development, anergic B cells in the IRF8-deficient background were able to mature further, which allowed them to regain responses to Ag stimulation. Interestingly, our results show that IRF8-deficient B cells were more sensitive to Ag stimulation and were resistant to Ag-induced cell death. Moreover, our results show that IRF8 was expressed at a high level in the anergic B cells, and an elevated level of IRF8 promoted apoptosis in the transitional B cells. Thus, our findings reveal a previously unrecognized function of IRF8 in B cell anergy induction.

A role for IRF4 in the development of CLL

AbstractInterferon regulatory factor 4 (IRF4) is a critical transcriptional regulator of B-cell development and function. A recent genome-wide single-nucleotide polymorphism (SNP) association study identified IRF4 as a major susceptibility gene in chronic lymphocytic leukemia (CLL). Although the SNPs located in the IRF4 gene were linked to a downregulation of IRF4 in CLL patients, whether a low level of IRF4 is critical for CLL development remains unclear. In rodents, CLL cells are derived from B1 cells whose population is dramatically expanded in immunoglobulin heavy chain Vh11 knock-in mice. We bred a Vh11 knock-in allele into IRF4-deficient mice (IRF4−/−Vh11). Here, we report that IRF4−/−Vh11 mice develop spontaneous early-onset CLL with 100% penetrance. Further analysis shows that IRF4−/−Vh11 CLL cells proliferate predominantly in spleen and express high levels of Mcl-1. IRF4−/−Vh11 CLL cells are resistant to apoptosis but reconstitution of IRF4 expression in the IRF4−/−Vh11 CLL cells inhibits their survival. Thus, our study demonstrates for the first time a causal relationship between low levels of IRF4 and the development of CLL. Moreover, our findings establish IRF4−/−Vh11 mice as a novel mouse model of CLL that not only is valuable for dissecting molecular pathogenesis of CLL but could also be used for therapeutic purposes.

The Roles of IRF-3 and IRF-7 in Innate Antiviral Immunity against Dengue Virus

We investigated the roles of IFN regulatory factor (IRF)-3 and IRF-7 in innate antiviral immunity against dengue virus (DENV). Double-deficient Irf-3(-/-)7(-/-) mice infected with the DENV2 strain S221 possessed 1,000-150,000 fold higher levels of viral RNA than wild-type and single-deficient mice 24 h postinfection (hpi); however, they remained resistant to lethal infection. IFN-α/β was induced similarly in wild-type and Irf-3(-/-) mice post-DENV infection, whereas in the Irf-7(-/-) and Irf-3(-/-)7(-/-) mice, significantly low levels of IFN-α/β expression was observed within 24 hpi. IFN-stimulated gene induction was also delayed in Irf-3(-/-)7(-/-) mice relative to wild-type and single-deficient mice. In particular, Cxcl10 and Ifnα2 were rapidly induced independently of both IRF-3 and IRF-7 in the Irf-3(-/-)7(-/-) mice with DENV infection. Higher levels of serum IFN-γ, IL-6, CXCL10, IL-8, IL-12 p70, and TNF were also observed in Irf-3(-/-)7(-/-) mice 24 hpi, at which time point viral titers peaked and started to be cleared. Ab-mediated blockade experiments revealed that IFN-γ, CXCL10, and CXCR3 function to restrict DENV replication in Irf-3(-/-)7(-/-) mice. Additionally, the IFN-stimulated genes Cxcl10, Ifit1, Ifit3, and Mx2 can be induced via an IRF-3- and IRF-7-independent pathway that does not involve IFN-γ signaling for protection against DENV. Collectively, these results demonstrate that IRF-3 and IRF-7 are redundant, albeit IRF-7 plays a more important role than IRF-3 in inducing the initial IFN-α/β response; only the combined actions of IRF-3 and IRF-7 are necessary for efficient control of early DENV infection; and the late, IRF-3- and IRF-7-independent pathway contributes to anti-DENV immunity.

Deletion of Irf5 protects hematopoietic stem cells from DNA damage-induced apoptosis and suppresses γ-irradiation-induced thymic lymphomagenesis

Repeated low-dose γ-irradiation (IR) induces thymic lymphoma in mice because of oncogenic mutations propagating from a primitive hematopoietic stem/progenitor cell (HSC) in the bone marrow. It is well known that IR-induced thymic lymphomagenesis is markedly enhanced by p53 deficiency, yet data also indicate that p53-dependent apoptosis can actively drive tumor formation in this model. The latter was recently expounded on by findings from Puma-deficient mice, indicating that loss of this proapoptotic p53 target gene results in protection from IR-induced lymphomagenesis rather than enhanced susceptibility to. Similar to Puma, the transcription factor interferon regulatory factor 5 (Irf5) has been reported as a p53 target gene and is required for DNA damage-induced apoptosis. To date, no studies have been performed to elucidate the in vivo role of IRF5 in tumorigenesis. Given its essential role in DNA damage-induced apoptosis, we explored the tumor suppressor function of IRF5 in IR-induced thymic lymphomagenesis. Somewhat surprisingly, we found that thymic lymphoma development was significantly suppressed in Irf5(-/-) mice as compared with wild-type littermates. Suppression was due, in part, to reduced thymocyte and HSC apoptosis, resulting in reduced compensatory proliferation, and reduced replication stress-associated DNA damage. The observed effects were independent of p53 or Puma as these proteins were upregulated in Irf5(-/-) mice in response to IR. This study demonstrates an important new role for IRF5 in maintaining HSC homeostasis after IR and supports the non-redundant functions of IRF5, p53 and PUMA in DNA damage-induced lymphomagenesis. We propose that IRF5 may be an attractive target for developing therapeutic agents to ameliorate radiation-induced bone marrow injury.

Role of IRF4 in IFN-Stimulated Gene Induction and Maintenance of Kaposi Sarcoma–Associated Herpesvirus Latency in Primary Effusion Lymphoma Cells

IFN regulatory factor (IRF) 4 is a hematopoietic cell-specific transcription factor that regulates the maturation and differentiation of immune cells. Using an inducible expression system, we found that IRF4 directly induced a specific subset of IFN-stimulated genes (ISGs) in a type I IFN-independent manner in both epithelial and B cell lines. Moreover, Kaposi sarcoma-associated herpesvirus (KSHV)-encoded viral FLICE inhibitory protein (vFLIP) enhances IRF4-mediated gene induction. Coexpression of IRF4 with vFLIP significantly increased ISG60 (IFIT3) and Cig5 (RSAD2) transcription that was dependent on the ability of vFLIP to activate NF-κB. A vFLIP mutant (A57L) defective in NF-κB activation failed to enhance IRF4-mediated ISG induction. Thus, we provide a physiologically relevant mechanism by which viral protein-mediated NF-κB activation modulates specific ISG induction by IRF4. In contrast, IRF4 also acted as a negative regulator of KSHV replication and transcription activator expression after induction of KSHV lytic reactivation in KSHV-positive primary effusion lymphoma cells. Taken together, these results suggest a dual role for IRF4 in regulating ISG induction and KSHV lytic reactivation in primary effusion lymphoma cells.

Dysregulation of interferon regulatory factors impairs the expression of immunostimulatory molecules in hepatitis C virus genotype 1-infected hepatocytes

BackgroundIL-7 and IL-15 are produced by hepatocytes and are critical for the expansion and function of CD8 T cells. IL-15 needs to be presented by IL-15Rα for efficient stimulation of...

RNAi Induces Innate Immunity through Multiple Cellular Signaling Pathways

Background & AimsOur previous results showed that the knockdown of woodchuck hepatitis virus (WHV) by RNA interference (RNAi) led to upregulation of interferon stimulated genes (ISGs) in primary hepatocytes. In the present study, we tested the hypothesis that the cellular signaling pathways recognizing RNA molecules may be involved the ISG stimulation by RNAi.MethodsPrimary murine hepatocytes (PMHs) from wild type mice and WHV transgenic (Tg) mice were prepared and treated with defined siRNAs. The mRNA levels of target genes and ISGs were detected by real-time RT-PCR. The involvement of the signaling pathways including RIG-I/MDA5, PKR, and TLR3/7/8/9 was examined by specific inhibition and the analysis of their activation by Western blotting.ResultsIn PMHs from WHV Tg mice, specific siRNAs targeting WHV, mouse β-actin, and GAPDH reduced the levels of targeted mRNAs and increased the mRNA expression of IFN-β, MxA, and IP-10. The enhanced ISG expression by siRNA transfection were abolished by siRNA-specific 2′-O-methyl antisense RNA and the inhibitors 2-AP and chloroquine blocking PKR and other TLR-mediated signaling pathways. Furthermore, Western blotting revealed that RNAi results in an increase in PKR phosphorylation and nuclear translocation of IRF3 and NF-êB, indicating the possible role of IRF3 in the RNAi-directed induction of ISGs. In contrast, silencing of RIG-I and MDA5 failed to block RNAi-mediated MxA induction.ConclusionsRNAi is capable of enhancing innate immune responses through the PKR- and TLR-dependent signaling pathways in primary hepatocytes. The immune stimulation by RNAi may contribute to the antiviral activity of siRNAs in vivo.

P182 Role of IRF8 as a molecular integrator that orchestrates CD8 T effector differentiation

Compared to CD4 T cells, the differentiation pathway for CD8 T cells is still underexplored. We found that the CD8 activation/differentiation is accomplished by two ways, by high dose antigen-MHC/co-stimulation or by low-dose antigen-MHC/co-stimulation plus simultaneous gc-cytokine signaling. We asked if these two modes of activation are qualitatively different. Naïve OT-I CD8T cells were stimulated by an OVA257–264-peptide in the context of Class I MHC (on ex vivo maturated dendritic cells) in the presence or absence of exogenously added recombinant IL-15 (5 nM). Another set of cells were also stimulated by IL-15 alone. RNA extraction from these cells was followed by cDNA synthesis/labeling and hybridization to Affymetrix cDNA array was performed. Results from triplicate independent array analyses were statistically evaluated to identify induced genes that are specific for each type of stimulation. To validate the array results, qPCR was performed on the expression of selected genes. To further assess the relevance of the chosen one, IRF8, CD8 T cells were isolated from OT-ITg-IRF8 knockout mice and their effector differentiation was determined by intracellular cytokine staining (IFNg, TNFa), by qPCR (granzyme/perforin) and by cytotoxicity assay. Gene induction pattern was compared at 6 h and 12 h following stimulation. At 6hr, 3 modes of stimulation (cytokine alone, antigen alone, combination of both) showed rather overlapping gene expression pattern. However the pattern showed dramatic change as early as 12 h point. Antigen-stimulated gene pool has shrunk to <500 genes and that of IL-15 < 1200. In contrast the combinatorial stimulation induced >1600 genes and 769 of them were only seen with the combination of two stimulations. Thus the high-dose antigen/MHC/costimulation and low-dose antigen/MHC/co-stimulation + cytokine are qualitatively different stimuli. The list of unique genes that were induced upon the combinatorial stimulation contains a lot of known genes that are involved in the CD8 T cell activation, including IL-2, CD25, and IL-12Rbeta. We then focused our attention on transcription factors (TFs) in this gene pool because we were interested in delineating the transcription network during CD8 T cell differentiation. We identified c-jun and IRF8. We then chose IRF8 for subsequent studies because the role of IRF8 in CD8 T cell activation has been uncharacterized. So we tested if CD8 T cell differentiation is compromised in the absence of IRF8. Expression of the two known T-box TFs, T-bet and eomesdermin, was not affected in the absence or presence of IRF8, excluding the possibility that IRF8 operates upstream of the T-box TFs. In contrast, the depletion of IRF8 in OT-I T cells (generated by crossing OT-I Tg mice and IRF8 knockout mice) has functionally crippled the CD8 T cells because they failed to produce IRFg or exert cytotoxic activity againt OVA-expressing target cells upon stimulation. qPCR also validated that the induction of IRF8 mRNA requires the cooperation of gc-cytokine signal and antigen-MHC/co-stimulation stimulation. These results define a new role for IRF8 as an integrator of two distinct activation signal pathways (TCR and gc-cytokine), and uncover a potential novel intracellular pathways originated from IRF8 in the differentiation of CD8 T cells responding to suboptimal dose of antigen.

O007 IRF-5; Mediates cross talk between innate and adaptive immunity

Introduction IRF-5 is a transcription factors activated by TLR7 and TLR9 during innate immune responses. IRF-5 activates not only Type I IFN, but also inflammatory cytokines and chemokines. Most importantly a distinct genetic variations in the IRF-5 gene show a strong association with autoimmune diseases such as Lupus (SLE). Here we analyzed the role of IRF-5 in the induction of inflammatory cytokines and B cells activation, two hallmarks of SLE. Methods The two strains of IRF-5 knockout mice were evaluated for their rsponse to antigens in vivo. The IgG switching was anlayzed in isolated B cells in vitro. The molecular mechanism involved in the alteration of the antigenic response was determined on the molecular levels. Results Using two independently generated strains of IRF5-deficient mice Irf 5−/− mice we showed that these mice exhibit high susceptibility to VSV and HSV-1 infection, reduction in serum levels of type I IFN as well as inflammatory cytokines,including interferon in response to viral infection, high sussceptibilty to virus (VSV) and parasite (Laishmania donovai) infection and a defect in generation of the TH-1 response. IRF-5, but not IRF-7 is also critical for B cells differention to plasma cells, IgG2a/c responses to antigens and to polyoma virus infection. This defect is due to the intrinsic deletion of IRF-5 in B cells, as Rag1 knockout mice reconstituted with Irf5 −/− B cells show a decrease in IgG2a/c expression after viral infection. Irf 5−/−B cells in vitro have diminished TLR and cytokine-induced class switching to IgG2a/c. Addressing the molecular mechanism of these defects, we show that IRF-5 regulates Blimp-1 and Ikaros expression, factors essential for plasma cells differentiation and IgG2a/c expression respectively. Reconstitution of IRF-5 in Irf 5−/− B cells rescue Blimp-1 expression, down-regulates Ikaros levels and increases switching to IgG2a/c. The IRF site in Blimp-1 and ikzf1 promoters binds IRF-5. Conclusion Collectively these results identify the role of IRF-5 both in the innate and adaptive immune respose and indicate its a critical role in the cross talk between the innate and adaptive immune responses.

P119 Type I and III interferon are attenuated in a human in vitro model of alternatively activated macrophages and is mediated by IRF4

Introduction Human monocytes may be polarized in vitro into classically or alternatively activated macrophages (CAM and AAM) after treatment with IFN-g or IL-4, respectively [1] . In vivo, AAM are closely associated with amplifying Th2 responses, and are implicated in the pathogenesis of allergic diseases and asthma. A current impediment to investigating the biology of human AAM is wide donor-to-donor variability and the quantity of primary macrophages that survive in vitro polarization. To overcome this impediment, we have successfully established a THP-1 derived -CAM and -AAM populations demonstrating typical macrophage-oriented morphological characteristics, gene and protein expression profile in vitro. The THP1-AAM model preferentially expressed interferon alpha/beta pathway genes, type I and III interferon genes, in addition to IRF4 compared to THP1-CAM. Methods THP-1 cells were activated with PMA for 48 h and polarized for 96 h with either IFN-g (500 IU/mL) or IL-4 (100 IU/mL). After polarization, the THP1-CAM and THP1-AAM were treated with LPS (100 ng/mL) and expression of IFN and inflammatory genes were measured by qRT-PCR or ELISA. Expression of IRF4 in THP1-AAM and -CAM was determined by qRT-PCR. To determine the role of IRF4 in THP1-AAM, the gene for IRF4 was silenced with siRNA prior to polarization of the THP-1 cells, and expression of interferon and inflammatory genes were measured in the IRF-silenced vs. control cells. Results THP1-AAM preferentially expressed mannose receptor and select chemokines including CCL18 and CCL22 while THP1-CAM expressed CXCL10 and TNF-alpha among other phenotype dependent markers. Upon LPS treatment, THP1-AAM preferentially expressed IL-10 in the supernatant while THP1-CAM expressed IFN-g, and IL-1Beta. Using THP1-AAM to understand interferon subtype expression in response to TLR4 challenge, we demonstrated that THP1-AAM express significantly attenuated level of interferon alpha/beta pathway, IFN-Beta, and IFN-Lambda1 genes compared to THP1-CAM upon LPS treatment. Expression of phenotype-oriented genes and IFN subtype expression profile were reproducible in primary human monocyte derived macrophages. To further investigate the mechanism for IFN attenuation in THP1-AAM, we demonstrate that IRF4 is up-regulated only in THP1-AAM. Silencing of IRF4 resulted in attenuation of expression of multiple AAM-oriented genes. Moreover, IRF4 silencing led to an enhanced expression of IFN-Beta and IFN-Lambda1 in response to LPS treatment pointing towards its critical role in mediating the AAM phenotype and its response to infection. Conclusion THP-1 derived -CAM and -AAM populations are a reliable and reproducible in vitro models to investigate a spectrum of molecular mechanisms governing classic, and alternative phenotypes in macrophage populations and their role in pathologic processes. THP1-AAM express a unique pattern of anti-inflammatory and type I and III interferon genes that may provide a signature response to TLR4 insult. Furthermore, IRF4 is a critical factor in mediating human monocytes during the alternatively activated polarization process and may be a potential therapeutic target for treatment of allergic inflammation of the upper airways.

The role of IRF4 in regulating effector CD8+ T cell development via repression of EOMES expression. (111.51)

Abstract CD8+ T cell development in the thymus generates a predominant population of conventional naïve cells, along with minor populations of ‘innate’ T cells that resemble memory cells. Recent studies analyzing a variety of knockout or knock-in mice have indicated that impairments in the TCR signaling pathway produce increased numbers of innate CD8+ T cells, characterized by their high expression of CD44, CD122, CXCR3, and the transcription factor Eomesodermin. One component of this altered development is a non-CD8+ T cell-intrinsic role for IL-4. To determine whether reduced TCR signaling within the CD8+ T cells might also contribute to this pathway, we investigated the role of the transcription factor IRF4. IRF4 is upregulated following TCR stimulation in wild-type T cells; however, this upregulation is impaired in Itk-/- T cells, which have reduced responses to TCR signaling. Further, analysis of IRF4-deficient CD8+ thymocytes showed normal development of thymic CD8+ T cells. Interestingly, IRF4-deficient peripheral CD8+ T cells acquired a memory phenotype and expressed the transcription factor Eomesodermin. We also show that activation of naïve IRF4-deficient CD8+ T cells leads to rapid and robust expression of Eomesodermin. Together, these data indicate that IRF4 upregulation following CD8+ T cell activation normally suppresses Eomesodermin expression, thereby regulating the differentiation pathway of CD8+ effector T cells.

Shared and Distinct Functions of the Transcription Factors IRF4 and IRF8 in Myeloid Cell Development

Interferon regulatory factor (IRF) 8 and IRF4 are structurally-related, hematopoietic cell-specific transcription factors that cooperatively regulate the differentiation of dendritic cells and B cells. Whilst in myeloid cells IRF8 is known to modulate growth and differentiation, the role of IRF4 is poorly understood. In this study, we show that IRF4 has activities similar to IRF8 in regulating myeloid cell development. The ectopic expression of IRF4 in myeloid progenitor cells in vitro inhibits cell growth, promotes macrophages, but hinders granulocytic cell differentiation. We also show that IRF4 binds to and activates transcription through the IRF-Ets composite sequence (IECS). Furthermore, we demonstrate that Irf8 -/- Irf4 -/- mice exhibit a more severe chronic myeloid leukemia (CML)-like disease than Irf8 -/- mice, involving a disproportionate expansion of granulocytes at the expense of monocytes/macrophages. Irf4 -/- mice, however, display no obvious abnormality in myeloid cell development, presumably because IRF4 is expressed at a much lower level than IRF8 in granulocyte-macrophage progenitors. Our results also suggest that IRF8 and IRF4 have not only common but also specific activities in myeloid cells. Since the expression of both the IRF8 and IRF4 genes is downregulated in CML patients, these results may add to our understanding of CML pathogenesis.

PS2-062. Novel role of IRF5 in transcriptional inhibition of human IL-10 gene expression

PS2-062. Novel role of IRF5 in transcriptional inhibition of human IL-10 gene expression

The Varicella-Zoster Virus ORF47 Kinase Interferes with Host Innate Immune Response by Inhibiting the Activation of IRF3

The innate immune response constitutes the first line of host defence that limits viral spread and plays an important role in the activation of adaptive immune response. Viral components are recognized by specific host pathogen recognition receptors triggering the activation of IRF3. IRF3, along with NF-κB, is a key regulator of IFN-β expression. Until now, the role of IRF3 in the activation of the innate immune response during Varicella-Zoster Virus (VZV) infection has been poorly studied. In this work, we demonstrated for the first time that VZV rapidly induces an atypical phosphorylation of IRF3 that is inhibitory since it prevents subsequent IRF3 homodimerization and induction of target genes. Using a mutant virus unable to express the viral kinase ORF47p, we demonstrated that (i) IRF3 slower-migrating form disappears; (ii) IRF3 is phosphorylated on serine 396 again and recovers the ability to form homodimers; (iii) amounts of IRF3 target genes such as IFN-β and ISG15 mRNA are greater than in cells infected with the wild-type virus; and (iv) IRF3 physically interacts with ORF47p. These data led us to hypothesize that the viral kinase ORF47p is involved in the atypical phosphorylation of IRF3 during VZV infection, which prevents its homodimerization and subsequent induction of target genes such as IFN-β and ISG15.

STAT1 Is a Master Regulator of Pancreatic β-Cell Apoptosis and Islet Inflammation

Cytokines produced by islet-infiltrating immune cells induce β-cell apoptosis in type 1 diabetes. The IFN-γ-regulated transcription factors STAT1/IRF-1 have apparently divergent effects on β-cells. Thus, STAT1 promotes apoptosis and inflammation, whereas IRF-1 down-regulates inflammatory mediators. To understand the molecular basis for these differential outcomes within a single signal transduction pathway, we presently characterized the gene networks regulated by STAT1 and IRF-1 in β-cells. This was done by using siRNA approaches coupled to microarray analysis of insulin-producing cells exposed or not to IL-1β and IFN-γ. Relevant microarray findings were further studied in INS-1E cells and primary rat β-cells. STAT1, but not IRF-1, mediates the cytokine-induced loss of the differentiated β-cell phenotype, as indicated by decreased insulin, Pdx1, MafA, and Glut2. Furthermore, STAT1 regulates cytokine-induced apoptosis via up-regulation of the proapoptotic protein DP5. STAT1 and IRF-1 have opposite effects on cytokine-induced chemokine production, with IRF-1 exerting negative feedback inhibition on STAT1 and downstream chemokine expression. The present study elucidates the transcriptional networks through which the IFN-γ/STAT1/IRF-1 axis controls β-cell function/differentiation, demise, and islet inflammation.

TLR3 and TLR4 are innate antiviral immune receptors in human microglia: Role of IRF3 in modulating antiviral and inflammatory response in the CNS

In the CNS, microglia are the primary targets of HIV infection. In this study, we investigated the effect of activation of the innate antiviral receptors TLR3 and TLR4 on HIV infection of primary human microglia, as well as microglial cell signaling and gene expression. Ligands for both TLR3 and TLR4 potently inhibited HIV replication in microglia through a pathway requiring IRF3. Surprisingly, a remarkably similar pattern of cell signaling and gene expression was observed in TLR3- and TLR4-activated microglia, suggesting a relatively minor role for MyD88 following TLR4 activation in these cells. HIV did not activate IRF3 but rather decreased IRF3 protein, indicating that HIV does not activate TLR3 or RIG-like helicases in microglia. Taken together, these results indicate that activation of TLR3 or TLR4 will elicit antiviral immunity, in addition to inducing proinflammatory responses. We suggest that a balanced expression between inflammatory and innate immune genes might be achieved by IRF3 over-expression.