Characterization of an interferon-stimulated response element (ISRE) in the Il23a promoter.
ABSTRACT: We have demonstrated previously that IFN-? plays a protective role in the initiation of chronic intestinal inflammation through attenuation of Toll-like receptor-mediated IL-23 induction in macrophages. Here, an interferon-stimulated response element (ISRE) is identified in a region of conserved nucleotide sequences in the Il23a promoter. This ISRE mediated, in part, Il23a promoter induction by LPS and inhibition of LPS-induced activity by IFN-?. LPS and IFN-? recruit interferon regulatory factors (IRFs) to the Il23a ISRE in murine bone marrow-derived macrophages (BMMs). Functionally, IRF-1 is a negative regulator of Il23a in LPS-stimulated BMMs. IRF-1(-/-) BMMs demonstrated enhanced LPS-induced Il23a expression compared with WT BMMs. Moreover, IRF-1 deficiency resulted in prolonged occupancy of RelA on the Il23a promoter. Consequently, IRF-1(-/-) mice were more susceptible to colonic injury by trinitrobenzenesulfonic acid, and IL-10/IRF-1 double-deficient (IL-10/IRF-1(-/-)) mice demonstrated more severe colonic inflammation compared with IL-10(-/-) mice. The severity of colitis in both models correlated with increased colonic IL-23. CD11b(+) lamina propria mononuclear cells, comprising predominantly macrophages, were identified as the major source of IL-23 in colitis-prone mice. Basal and heat-killed Escherichia coli-stimulated levels of Il23a were increased in IL-10/IRF-1(-/-) compared with WT and IL-10(-/-) colonic CD11b(+) lamina propria mononuclear cells. In conclusion, these experiments characterize IRF-ISRE interactions on the Il23a promoter, which have in vivo relevance as a homeostatic checkpoint in chronic intestinal inflammation.
Project description:IL-23 regulation is a central event in the pathogenesis of the inflammatory bowel diseases. We demonstrate that IFN-gamma has anti-inflammatory properties in the initiation phase of IL-23-mediated experimental colitis. IFN-gamma attenuates LPS-mediated IL-23 expression in murine macrophages. Mechanistically, IFN-gamma inhibits Il23a promoter activation through altering NF-kappaB binding and histone modification. Moreover, intestinal inflammation is inhibited by IFN-gamma signaling through attenuation of Il23a gene expression. In germ-free wild-type mice colonized with enteric microbiota, inhibition of colonic Il23a temporally correlates with induction of IFN-gamma. IFN-gammaR1/IL-10 double-deficient mice demonstrate markedly increased colonic inflammation and IL23a expression compared with those of IL-10(-/-) mice. Colonic CD11b(+) cells are the primary source of IL-23 and a target for IFN-gamma. This study describes an important anti-inflammatory role for IFN-gamma through inhibition of IL-23. Converging genetic and functional findings suggest that IL-23 and IFN-gamma are important pathogenic molecules in human inflammatory bowel disease.
Project description:A family of interferon (IFN) regulatory factors (IRFs) have been shown to play a role in transcription of IFN genes as well as IFN-stimulated genes. We report the identification of a member of the IRF family which we have named IRF-3. The IRF-3 gene is present in a single copy in human genomic DNA. It is expressed constitutively in a variety of tissues and no increase in the relative steady-state levels of IRF-3 mRNA was observed in virus-infected or IFN-treated cells. The IRF-3 gene encodes a 50-kDa protein that binds specifically to the IFN-stimulated response element (ISRE) but not to the IRF-1 binding site PRD-I. Overexpression of IRF-3 stimulates expression of the IFN-stimulated gene 15 (ISG15) promoter, an ISRE-containing promoter. The murine IFNA4 promoter, which can be induced by IRF-1 or viral infection, is not induced by IRF-3. Expression of IRF-3 as a Gal4 fusion protein does not activate expression of a chloramphenicol acetyltransferase reporter gene containing repeats of the Gal4 binding sites, indicating that this protein does not contain the transcription transactivation domain. The high amino acid homology between IRF-3 and ISG factor 3 gamma polypeptide (ISGF3 gamma) and their similar binding properties indicate that, like ISGF3 gamma, IRF-3 may activate transcription by complex formation with other transcriptional factors, possibly members of the Stat family. Identification of this ISRE-binding protein may help us to understand the specificity in the various Stat pathways.
Project description:<h4>Background</h4>Interferon-induced 35-kDa protein (IFP35) plays important roles in antiviral defense and the progression of some skin cancer diseases. It can be induced by interferon-? (IFN-?) in multiple human cells. However, the mechanisms by which IFN-? contributes to IFP35 induction remain to be elucidated.<h4>Methods/principal findings</h4>We identified the transcription start sites of IFP35 by 5' rapid amplification of cDNA ends (RACE) and cloned the promoter of IFP35. Sequence analysis and luciferase assays revealed two GC boxes and an IFN-stimulated response element (ISRE) in the 5' upstream region of the transcription start sites, which were important for the basal transcription of IFP35 gene. Furthermore, we found that interferon regulatory factor 1 (IRF-1) and IRF-2 could bind to IFP35 promoter and upregulate endogenous IFP35 protein level. Depletion of endogenous IRF-1 by interfering RNA reduced the constitutive and IFN-?-dependent expression of IFP35, whereas depletion of IRF-2 had little effect on IFN-?-inducible IFP35 expression. Moreover, IRF-1 was recruited to the ISRE site in IFP35 promoter in IFN-? treated HeLa cells, as demonstrated by electrophoretic mobility shift and chromatin immunoprecipitation assays.<h4>Conclusions/significance</h4>These findings provide the first evidence that IRF-1 and IRF-2 are involved in constitutive IFP35 expression in HeLa cells, while IRF-1 also activates IFP35 expression in an IFN-?-inducible manner. Our data therefore identified a new IRF-1 and IRF-2 target gene, which may expand our current understanding of the versatile functions of IRF-1 and IRF-2.
Project description:ICSBP (IRF-8) is a transcription factor of the IRF family expressed only in the immune system. It is induced in macrophages by gamma interferon (IFN-gamma) and contributes to macrophage functions. By interacting with Ets family protein PU.1, ICSBP binds to the IRF/Ets composite element and stimulates transcription. ICSBP binds to another DNA element, the IFN-stimulated response element (ISRE), a common target of the IRF family. Limited knowledge as to how ICSBP and other IRF proteins regulate ISRE-dependent transcription in IFN-gamma-activated macrophages is available. By mass-spectrometric analysis of ISRE-bound proteins in macrophages, we identified TEL, another Ets member, as a factor recruited to the element in an IFN-gamma-dependent manner. In vitro analysis with recombinant proteins indicated that this recruitment is due to a direct interaction between ICSBP and TEL, which is enhanced by the presence of ISRE. Significantly, the interaction with TEL in turn resulted in the recruitment of the histone deacetytase HDAC3 to the ISRE, causing increased repression of IFN-gamma-mediated reporter activity through the ISRE. This repression may provide a negative-feedback mechanism operating after the initial transcriptional activation by IFN-gamma. By associating with two different Ets family proteins, ICSBP exerts a dual function in IFN-gamma-dependent gene regulation in an immune system-specific manner.
Project description:IRF-7 mediates robust production of type I IFN via MyD88 of the TLR9 pathway in plasmacytoid dendritic cells (pDCs). Previous in vitro studies using bone marrow-derived dendritic cells lacking either Irf7 or Irf3 have demonstrated that only IRF-3 is required for IFN-? production in the TLR4 pathway. Here, we show that IRF-7 is essential for both type I IFN induction and IL-1? responses via TLR4 in mice. Mice lacking Irf7 were defective in production of both IFN-? and IL-1?, an IFN-?-induced pro-inflammatory cytokine, after LPS challenge. IFN-? production in response to LPS was impaired in IRF-7-deficient macrophages, but not dendritic cells. Unlike pDCs, IRF-7 is activated by the TRIF-, but not MyD88-, dependent pathway via TBK-1 in macrophages after LPS stimulation. Like pDCs, resting macrophages constitutively expressed IRF-7 protein. This basal IRF-7 protein was completely abolished in either Ifnar1 -/- or Stat1 -/- macrophages, which corresponded with the loss of LPS-stimulated IFN-? induction in these macrophages. These findings demonstrate that macrophage IRF-7 is critical for LPS-induced type I IFN responses, which in turn facilitate IL-1? production in mice.
Project description:Ubiquitously expressed interferon regulatory factor 3 (IRF-3) is directly activated after virus infection and functions as a key activator of the immediate-early alpha/beta interferon (IFN) genes, as well as the RANTES chemokine gene. In the present study, a tetracycline-inducible expression system expressing a constitutively active form of IRF-3 (IRF-3 5D) was combined with DNA microarray analysis to identify target genes regulated by IRF-3. Changes in mRNA expression profiles of 8,556 genes were monitored after Tet-inducible expression of IRF-3 5D. Among the genes upregulated by IRF-3 were transcripts for several known IFN-stimulated genes (ISGs). Subsequent analysis revealed that IRF-3 directly induced the expression of ISG56 in an IFN-independent manner through the IFN-stimulated responsive elements (ISREs) of the ISG56 promoter. These results demonstrate that, in addition to its role in the formation of a functional immediate-early IFN-beta enhanceosome, IRF-3 is able to discriminate among ISRE-containing genes involved in the establishment of the antiviral state as a direct response to virus infection.
Project description:Hepatitis C virus (HCV) infection induces the alpha-chemokine interleukin-8 (CXCL-8), which is regulated at the levels of transcription and mRNA stability. In the current study, CXCL-8 regulation by double-stranded (ds)RNA pathways was analyzed in the context of HCV infection. A constitutively active mutant of the retinoic acid-inducible gene I (RIG-I), RIG-N, activated CXCL-8 transcription. Promoter mutagenesis experiments indicated that NF-kappaB and interferon (IFN)-stimulated response element (ISRE) binding sites were required for the RIG-N induction of CXCL-8 transcription. IFN-beta promoter stimulator 1 (IPS-1) expression also activated CXCL-8 transcription, and mutations of the ISRE and NF-kappaB binding sites reduced and abrogated CXCL-8 transcription, respectively. In the presence of wild-type RIG-I, transfection of JFH-1 RNA or JFH-1 virus infection of Huh7.5.1 cells activated the CXCL-8 promoter. Expression of IFN regulatory factor 3 (IRF-3) stimulated transcription from both full-length and ISRE-driven CXCL-8 promoters. Chromatin immunoprecipitation assays demonstrated that IRF-3 and NF-kappaB bound directly to the CXCL-8 promoter in response to virus infection and dsRNA transfection. RIG-N stabilized CXCL-8 mRNA via the AU-rich element in the 3' untranslated region of CXCL-8 mRNA, leading to an increase in its half-life following tumor necrosis factor alpha induction. The data indicate that HCV infection triggers dsRNA signaling pathways that induce CXCL-8 via transcriptional activation and mRNA stabilization and define a regulatory link between innate antiviral and inflammatory cellular responses to virus infection.
Project description:The fungal analog zymosan induces IL-23 and low amounts of IL-12 p70. This study addresses the molecular mechanisms underlying this cytokine pattern in human monocyte-derived dendritic cells. The transcriptional regulation of il23a, one of the chains of IL-23, depended on the activation of c-Rel and histone H3 phosphorylation, as judged from the association of c-Rel with the il23a promoter and the correlation between IL-23 production and Ser-10-histone H3 phosphorylation. Consistent with its reduced ability to produce IL-12 p70, zymosan induced a transient occupancy of the il12a promoter by c-Rel, blocked the production of IL-12 p70 and the transcription of il12a induced by other stimuli, and triggered the expression and nuclear translocation of the transcriptional repressors of the Notch family hairy and enhancer of split (Hes)-1, Hes5, hairy/enhancer-of-split related with YRPW motif protein (Hey)-1, and transducin-like enhancer of split (TLE). Zymosan also induced the interaction of Hes1 and TLE with histone H3 phosphorylated on Ser-10 and deacetylated on Lys-14. Inhibition of class III histone deacetylases increased the production of IL-12 p70 and partially blunted the inhibitory effect of zymosan on the production of IL-12 p70 elicited by LPS and IFN-?. These results indicate that the selective induction of IL-23 by ?-glucans is explained by the activation of c-Rel associated with Ser-10-histone H3 phosphorylation in the il23a promoter mediated by mitogen- and stress-activated kinase and/or protein kinase A and inhibition of il12a transcription by a mechanism involving activation of several corepressors with the ability to bind TLE and to promote histone deacetylation.
Project description:Tyrosine kinase 2 (Tyk2), a central component of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling, has major effects on innate immunity and inflammation. Mice lacking Tyk2 are resistant to endotoxin shock induced by lipopolysaccharide (LPS), and Tyk2 deficient macrophages fail to efficiently induce interferon alpha/beta after LPS treatment. However, how Tyk2 globally regulates transcription of downstream target genes remains unknown. Here we examine the regulatory role of Tyk2 in basal and inflammatory transcription by comparing gene expression profiles of peritoneal macrophages from Tyk2 mutant and wildtype control mice that were either kept untreated or exposed to LPS for six hours.Untreated Tyk2-deficient macrophages exhibited reduced expression of immune response genes relative to wildtype, in particular those that contain interferon response elements (IRF/ISRE), whereas metabolic genes showed higher expression. Upon LPS challenge, IFN-inducible genes (including those with an IRF/ISRE transcription factor binding-site) were strongly upregulated in both Tyk2 mutant and wildtype cells and reached similar expression levels. In contrast, metabolic gene expression was strongly decreased in wildtype cells upon LPS treatment, while in Tyk2 mutant cells the expression of these genes remained relatively unchanged, which exaggerated differences already present at the basal level. We also identified several 5'UR transcription factor binding-sites and 3'UTR regulatory elements that were differentially induced between Tyk2 deficient and wildtype macrophages and that have not previously been implicated in immunity.Although Tyk2 is essential for the full LPS response, its function is mainly required for baseline expression but not LPS-induced upregulation of IFN-inducible genes. Moreover, Tyk2 function is critical for the downregulation of metabolic genes upon immune challenge, in particular genes involved in lipid metabolism. Together, our findings suggest an important regulatory role for Tyk2 in modulating the relationship between immunity and metabolism.
Project description:There is a debate on whether invertebrates possess an antiviral immunity similar to the interferon (IFN) system of vertebrates. The Vago gene from arthropods encodes a viral-activated secreted peptide that restricts virus infection through activating the JAK-STAT pathway and is considered to be a cytokine functionally similar to IFN. In this study, the first crustacean IFN regulatory factor (IRF)-like gene was identified in Pacific white shrimp, Litopenaeus vannamei. The L. vannamei IRF showed similar protein nature to mammalian IRFs and could be activated during virus infection. As a transcriptional regulatory factor, L. vannamei IRF could activate the IFN-stimulated response element (ISRE)-containing promoter to regulate the expression of mammalian type I IFNs and initiate an antiviral state in mammalian cells. More importantly, IRF could bind the 5'-untranslated region of L. vannamei Vago4 gene and activate its transcription, suggesting that shrimp Vago may be induced in a similar manner to that of IFNs and supporting the opinion that Vago might function as an IFN-like molecule in invertebrates. These suggested that shrimp might possess an IRF-Vago-JAK/STAT regulatory axis, which is similar to the IRF-IFN-JAK/STAT axis of vertebrates, indicating that invertebrates might possess an IFN system-like antiviral mechanism.