Interferon-stimulated Response Element Research Articles

Chromium(VI) Stimulates Fyn to Initiate Innate Immune Gene Induction in Human Airway Epithelial Cells

Mechanisms for pathogenic metal signaling in airway injury or disease promotion are poorly understood. It is widely believed that one mechanism for pathogenic and possible carcinogenic effects of inhaled chromium (Cr(VI)) is inhibition of inducible gene transactivation. However, we recently reported that Cr(VI) inhibition of Sp1-dependent transactivation required signal transducer and activator of transcription 1 (STAT1)-dependent expression of an inhibitory protein in airway epithelium. Thus, Cr(VI) exposures can induce genes, and we hypothesized that this induction resulted from Cr(VI) signaling through an innate immune-like STAT1-dependent pathway initiated by Fyn. Exposure of human airway epithelial (BEAS-2B) cells to Cr(VI) selectively transactivated the STAT-responsive interferon-stimulated response element (ISRE) and induced ISRE-driven transactivation of interferon regulatory factor 7 (IRF7), without affecting the gamma interferon-activated site (GAS)-driven IRF1 expression. Cr(VI)-induced IRF7 was absent or greatly reduced in cells that lacked STAT1, were treated with the Src family kinase inhibitor, PP2, or lacked Fyn. Expressing Fyn, but not Src, in mouse embryonic fibroblasts cells null for Src, Yes, and Fyn restored Cr(VI)-stimulated STAT1 tyrosine phosphorylation and IRF7 expression. Finally, shRNA knockdown of Fyn in BEAS-2B cells prevented Cr(VI)-activated STAT1 transactivation of IRF7. These data support a novel mechanism through which Cr(VI) stimulates Fyn to initiate interferon-like signaling for STAT1-dependent gene transactivation.

Distinct Interaction Networks of the MUM1/IRF4 in B- and T-Cell Lymphomas Identified by Tandem Mass Spectrometry.

Abstract 3925Poster Board III-861 IntroductionMultiple myeloma oncogene 1/interferon regulatory factor 4 (MUM1/IRF4) is a 52-kDa transcriptional activator protein which plays an important role in interferon-stimulated response element (ISRE)-regulated signal transduction mechanisms important in lymphoid cell development and differentiation. MUM1/IRF4 is also critical for the generation of immunoglobulin-secreting plasma cells. MUM1/IRF4 is expressed in the activated B-cell (ABC)-like subset of diffuse large B-cell lymphomas (DLBCL) and is targeted by chromosomal translocations in a subset of multiple myelomas and peripheral T-cell lymphomas. Despite its importance in lymphocyte and lymphoma biology, and its addiction demonstrated in multiple myelomas, the proteomic network of MUM1/IRF4 interacting proteins has not been determined. MethodsWe determined the proteomic interaction networks of MUM1/IRF4 in B-cell and T-cell lymphoma contexts, by using a functional proteomic approach. Total cell lysates were prepared from MUM1/IRF4 expressing cell lines including OCI-LY10 ((ABC)-like DLBCL) and HH (mycosis fungoides derived-T-cell) and, as a negative control, from non-MUM1/IRF4 expressing SUDHL4 cell line. Immunoprecipitates of the MUM1/IRF4 expressing B- and T-cell line were compared to that of hyperimmune mouse immunoglobulin by 1-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis. Coomassie-stained protein bands from both immunocomplexes were excised and analyzed by electrospray liquid chromatography tandem mass spectrometry (ESI-LC/MS/MS). Peptide sequences were identified by searching the MS/MS data against human IPI protein using database X!Tandem with k-score plug-in. Proteins found in the two control sets (SUDHL4 and IgG isotype) were manually subtracted from the proteins found in the experimental set. MS/MS results were validated using immunoprecipitation and Western blotting and some of the interacting proteins were further confirmed by reciprocal immunoprecipitation. ResultsA total of 163 and 94 proteins were identified from 12 Coomassie-stained bands which were unique to the MUM1/IRF4 immunocomplex in OCI-Ly10 and HH cell lines, respectively. Previously reported proteins in the MUM1/IRF4 signal pathway were identified including FK506 and TRAF family protein. More importantly, many proteins previously not associated with MUM1/IRF4 were identified. In common to both subsets were proteins such as ARHGDIA-involved in RhoA-mediated signaling. In the ABC-like B-cell (OCI-LY10) specific context, these included proteins known to be critical regulators of lymphocyte proliferation (RAB2A), motility, trafficking and cell adhesion. In the T-cell specific context, proteins known to play important roles in endocytosis (Reggies) and T-cell activation (GDI2, Guanine nucleotide exchange factors) were identified in the MUM1/IRF4 interactome. A subset of the proteins identified by MS were confirmed by western blotting and reciprocal immunoprecipitation. ConclusionsOur studies reveal that although a minor subset of MUM1/IRF4 interacting proteins are common in B- and T-cells, MUM1/IRF4 exhibits distinct interaction partners dependent of specific cellular contexts. The diverse interaction networks implicate MUM1/IRF4 in previously undescribed functional roles in lymphocyte-specific subsets. Comprehensive elucidation of the protein-protein interaction networks in the specific cellular contexts will provide opportunities for exploitation of the knowledge for design of rational interventions targeting the critical nodes and modules in MUM1/IRF4-deregulation in B- and T-cell malignancies. Disclosures:No relevant conflicts of interest to declare.

Molecular structural and functional characterization of STAT1 gene regulatory region in teleost Channa argus

The transcription factor STAT1 is involved in signal transduction of type I and II interferons (IFNs). However, the molecular characteristics of the STAT1 regulatory region still remain to be elucidated in teleosts. In the present study, the complete cDNA and the regulatory region of the STAT1 gene were isolated from snakehead (Channa argus). More than 2.4kb 5′-flanking region of STAT1 shares the regulatory elements of IFN-stimulated response element (ISRE) and IFN-γ activation site (GAS). Consensus ISRE and GAS were located from −373 to −361 and −716 to −724 in the promoter region, respectively. Moreover, it is noticeable that the crucial elements of ISRE (+698 to +710) and GAS (+294 and +301) are present in the first intron of snakehead STAT1. Comparisons of six vertebrate STAT1 5′-flanking regions all present the common sequence characteristics of IFN-induced gene promoter, which include ISRE, GAS and Sp1 sites. In order to further characterize the snakehead STAT1 regulatory region, six reporter constructs of snakehead STAT1 promoter and first intron were generated to examine the specificity to human interferon-gamma (hIFN-γ). Only those constructs containing the ISRE element showed notable reporter activity after stimulation of Hela cells with hIFN-γ. However, sequential deletions of putative transcription factor binding sites indicated that GAS elements have little effect on the promoter and intronic activity in response to hIFN-γ. Taken together, these results suggest that the regulatory mechanisms of IFN-signalling appear to be mediated in a similar manner in fish and mammals.

Effect of Type-I Interferon on Retroviruses

Type-I interferons (IFN-I) play an important role in the innate immune response to several retroviruses. They seem to be effective in controlling the in vivo infection, though many of the clinical signs of retroviral infection may be due to their continual presence which over-stimulates the immune system and activates apoptosis. IFN-I not only affect the immune system, but also operate directly on virus replication. Most data suggest that the in vitro treatment with IFN-I of retrovirus infected cells inhibits the final stages of virogenesis, avoiding the correct assembly of viral particles and their budding, even though the mechanism is not well understood. However, in some retroviruses IFN-I may also act at a previous stage as some retroviral LTRs posses sequences homologous to the IFN-stimulated response element (ISRE). When stimulated, ISREs control viral transcription. HIV-1 displays several mechanisms for evading IFN-I, such as through Tat and Nef. Besides IFN-α and IFN-β, some other type I IFN, such as IFN-τ and IFN-ω, have potent antiviral activity and are promising treatment drugs.

Palmitoylation of Interferon-α (IFN-α) Receptor Subunit IFNAR1 Is Required for the Activation of Stat1 and Stat2 by IFN-α

Type I interferons (IFNs) bind IFNAR receptors and activate Jak kinases and Stat transcription factors to stimulate the transcription of genes downstream from IFN-stimulated response elements. In this study, we analyze the role of protein palmitoylation, a reversible post-translational lipid modification, in the functional properties of IFNAR. We report that pharmacological inhibition of protein palmitoylation results in severe defects of IFN receptor endocytosis and signaling. We generated mutants of the IFNAR1 subunit of the type I IFN receptor, in which each or both of the two cysteines present in the cytoplasmic domain are replaced by alanines. We show that cysteine 463 of IFNAR1, the more proximal of the two cytoplasmic cysteines, is palmitoylated. A thorough microscopic and biochemical analysis of the palmitoylation-deficient IFNAR1 mutant revealed that IFNAR1 palmitoylation is not required for receptor endocytosis, intracellular distribution, or stability at the cell surface. However, the lack of IFNAR1 palmitoylation affects selectively the activation of Stat2, which results in a lack of efficient Stat1 activation and nuclear translocation and IFN-alpha-activated gene transcription. Thus, receptor palmitoylation is a previously undescribed mechanism of regulating signaling activity by type I IFNs in the Jak/Stat pathway.

The EBV-encoded latent membrane proteins, LMP2A and LMP2B, limit the actions of interferon by targeting interferon receptors for degradation

Although frequently expressed in EBV-positive malignancies, the role that Latent membrane protein 2A and 2B (LMP2A, LMP2B) play in the oncogenic process remains obscure. Here we demonstrate a novel function for these proteins in epithelial cells, namely, their ability to modulate signalling from type I/II interferon receptors (IFNRs). We show that LMP2A and LMP2B-expressing epithelial cells display decreased responsiveness to interferon (IFN)α and IFNγ, as assessed by STAT1 phosphorylation, ISGF3 and GAF-mediated binding to ISRE and GAS elements and luciferase reporter activation. Transcriptional profiling highlighted the extent of this modulation, with both viral proteins impacting “globally” on interferon-stimulated gene expression. Whilst not affecting the levels of cell surface IFNRs, LMP2A and LMP2B accelerated the turnover of IFNRs through processes requiring endosome acidification. This function may form part of EBVs strategy to limit anti-viral responses and defines a novel function for LMP2A and LMP2B in modulating signalling from receptors that participate in innate immune responses.

Type I interferon drives tumor necrosis factor–induced lethal shock

Tumor necrosis factor (TNF) is reputed to have very powerful antitumor effects, but it is also a strong proinflammatory cytokine. Injection of TNF in humans and mice leads to a systemic inflammatory response syndrome with major effects on liver and bowels. TNF is also a central mediator in several inflammatory diseases. We report that type I interferons (IFNs) are essential mediators of the lethal response to TNF. Mice deficient in the IFN-α receptor 1 (IFNAR-1) or in IFN-β are remarkably resistant to TNF-induced hypothermia and death. After TNF injection, IFNAR-1−/− mice produced less IL-6, had less bowel damage, and had less apoptosis of enterocytes and hepatocytes compared with wild-type (WT) mice. Extensive gene expression analysis in livers of WT and IFNAR-1−/− mice revealed a large deficiency in the response to TNF in the knockout mice, especially of IFN-stimulated response element–dependent genes, many of which encode chemokines. In livers of IFNAR-1−/− mice, fewer infiltrating white blood cells (WBCs) were detected by immunohistochemistry. Deficiency of type I IFN signaling provided sufficient protection for potentially safer therapeutic use of TNF in tumor-bearing mice. Our data illustrate that type I IFNs act as essential mediators in TNF-induced lethal inflammatory shock, possibly by enhancing cell death and inducing chemokines and WBC infiltration in tissues.

Interferon-mediated enhancement of in vitro replication of porcine circovirus type 2 is influenced by an interferon-stimulated response element in the PCV2 genome

Porcine circovirus type 2 (PCV2) is an economically important swine pathogen. It has been shown that treatment of PCV2-infected cells with interferon gamma (INF-γ) or alpha (IFN-α) increases PCV2 replication in vitro, and that an interferon-stimulated response element (ISRE)-like sequence was identified in the PCV2 genome. To determine if the ISRE is involved in viral response to IFNs, several ISRE mutants of PCV2 were created by serial mutations of the ISRE sequence. Treatment of ISRE mutants-infected cells with IFN-γ or IFN-α showed a progressive diminishment in the enhanced viral replication in correlation with increasing alterations to the ISRE sequence. To determine if the ISRE was necessary and sufficient for IFN-mediated enhancement of PCV2 replication, DNA fragments spanning the ISRE-containing promoter region of the rep gene from wildtype and ISRE-mutant PCV2 were tested in a luciferase reporter gene system. 3D4/31 cells transfected with reporter gene constructs were treated with IFN-γ or IFN-α, respectively. The results showed that the untreated controls for both ISRE-mutant and wildtype PCV2 had higher levels of luciferase reporter activity than IFN-treated samples, indicating that, when removed from the context of whole viral genome, the ISRE-like activity of the sequence was lost. Furthermore, treatment with IFNs diminished the promoter activity regardless of the mutation, suggesting that other cis elements in the viral genome may be required for regulating the ISRE-mediated gene transcription. In conclusion, the PCV2 ISRE, when present in the context of intact virus but not in isolation, influences the interferon-mediated enhancement of PCV2 replication in vitro.

Expression regulation and functional characterization of a novel interferon inducible gene Gig2 and its promoter

Grass carp hemorrhagic virus (GCHV)-induced gene 2 (Gig2) is a novel gene previously identified from UV-inactivated GCHV-treated Carassius auratus blastulae embryonic (CAB) cells, suggesting that it should play a pivotal role in the interferon (IFN) antiviral response. In this study, a polyclonal anti-Gig2 antiserum was generated and used to study the inductive expression pattern by Western blot analysis, showing no basal expression in normal CAB cells but a significant up-regulation upon UV-inactivated GCHV, polyinosinic:polycytidylic acid (Poly I:C) and recombinant IFN (rIFN). However, constitutive expression of Gig2 is observed in all tested tissues from grass carp (Ctenopharyngodon idellus), and Poly I:C injection increases the relative amount of Gig2 protein in skin, spleen, trunk kidney, gill, hindgut and thymus. Moreover, the genomic sequence covering the whole Gig2 ORF and the upstream promoter region were amplified by genomic walking. Significantly, the Gig2 promoter contains three IFN-stimulated response elements (ISREs), nine GAAA/TTTC motifs and five γ-IFN activating sites (GAS), which are the characteristics of genes responsive to both type I IFN and type II IFN. Subsequently, the complete Gig2 promoter sequence was cloned into pGL3-Basic vector, and its activity was measured by luciferase assays in the transfected CAB cells. The Gig2 promoter-driven construct is highly induced in CAB cells after treatment with Poly I:C or rIFN, and the functional capability is dependent on IFN regulatory factor 7 (IRF7), because its activity can be stimulated by IRF7. Collectively, the data provide strong evidence that Gig2 is indeed a novel IFN inducible gene and its expression is likely dependent on IRF7 upon Poly I:C or IFN.

Cholera toxin inhibits IL-12 production and CD8α+ dendritic cell differentiation by cAMP-mediated inhibition of IRF8 function

Prior studies have demonstrated that cholera toxin (CT) and other cAMP-inducing factors inhibit interleukin (IL)-12 production from monocytes and dendritic cells (DCs). We show that CT inhibits Th1 responses in vivo in mice infected with Toxoplasma gondii. This correlated with low serum IL-12 levels and a selective reduction in the numbers of CD8α+ conventional DCs (cDCs) in lymphoid organs. CT inhibited the function of interferon (IFN) regulatory factor (IRF) 8, a transcription factor known to positively regulate IL-12p35 and p40 gene expression, and the differentiation of CD8α+ and plasmacytoid DCs (pDCs). Fluorescence recovery after photobleaching analysis showed that exposure to CT, forskolin, or dibutyryl (db) cAMP blocked LPS and IFN-γ–induced IRF8 binding to chromatin. Moreover, CT and dbcAMP inhibited the binding of IRF8 to the IFN-stimulated response element (ISRE)–like element in the mouse IL-12p40 promoter, likely by blocking the formation of ISRE-binding IRF1–IRF8 heterocomplexes. Furthermore, CT inhibited the differentiation of pDCs from fms-like tyrosine kinase 3 ligand–treated bone marrow cells in vitro. Therefore, because IRF8 is essential for IL-12 production and the differentiation of CD8α+ cDCs and pDCs, these data suggest that CT and other Gs-protein agonists can affect IL-12 production and DC differentiation via a common mechanism involving IRF8.

Activation of interferon signaling pathway in primary effusion lymphoma cells by inhibition of viral interferon regulatory factor 1 of Kaposi's sarcoma-associated herpesvirus

Kaposi's Sarcoma-Associated Herpesvirus (KSHV), also known as human Herpesvirus 8, is associated with several malignant disorders, including Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease. KSHV ORF-K9 encodes viral interferon regulatory factor 1 (vIRF1), an early lytic gene product homologous to cellular interferon regulatory factors. The IRF family members are transcription regulatory factors that bind to interferon-stimulated response elements (ISRE) to regulate interferon-responsive genes involved in pathogen response, cytokine signaling, cell growth regulation, and hematopoietic development. As an oncogenic protein, vIRF1 potentially contributes to KSHV-driven malignancies. In this study, KSHV vIRF1 was suppressed with peptide-conjugated antisense phosphorodiamidate morpholino oligonucleotides (PPMO) and the effect was examined. PPMO are single-stranded DNA analogues that have a modified backbone and which penetrate cells readily. Treatment of PEL cells with anti-K9 PPMO led to reduction of vIRF1 expression. The anti-K9 PPMO also reduced expression of vIRF1 in 293 cells transiently transfected with a K9 plasmid. PEL cells that were treated with anti-K9 PPMO exhibited reduced RTA and vIL-6 expression and reduced level of KSHV DNA copies. The inhibition of vIRF1 in PEL cells led to elevation of protein levels of important genes in IFN signaling, including IRF3 and STAT1, indicating the activation of the pathway. The results demonstrate that targeting of vIRF1 mRNA with PPMO can potently reduce vIRF1 protein translation, and indicate that further exploration of these compounds in an animal model is warranted.

PLZF Outreach: A Finger in Interferon's Pie

In this issue of Immunity, Xu et al. (2009) find that the transcription factor PLZF activates interferon-stimulated genes and facilitates natural killer cell functions. Interferon-induced PLZF phosphorylation and histone deacetylase 1 recruitment probably mediates the repressor-to-activator conversion.

PKR , a p53 target gene, plays a crucial role in the tumor-suppressor function of p53

Type I IFN-induced expression of dsRNA-activated protein kinase (PKR) during viral infection is a well-established antiviral mechanism. However, little is known about the expression of PKR in the context of p53 and about PKR involvement in p53-mediated tumor suppression. Here, we report that PKR is a p53 target gene and plays an important role in the tumor-suppressor function of p53. Activation of p53 by genotoxic stress induces a significant level of PKR expression by acting on the newly identified cis-acting element (ISRE), which is separated from the IFN-stimulated responsive element on the PKR promoter, resulting in translational inhibition and cell apoptosis. The genotoxin-mediated inhibition of translation is associated with the p53/PKR/elF2a (eukaryotic initiation factor-2alpha) pathway. To some extent, p53 activation induced by DNA damage facilitates cell apoptosis by activating PKR. PKR-knockdown human colon cancer cells grew rapidly in nude mice and proved resistant to anti-cancer drugs. These data indicate that p53-mediated tumor suppression can be attributed at least in part to the biological functions of PKR induced by p53 in genotoxic conditions.

IRF-1 and p65 mediate upregulation of constitutive HLA-A antigen expression by hepatocellular carcinoma cells

Malignant transformation of hepatocytes is frequently associated with upregulation of HLA-A expression. Currently there is no information available regarding the mechanisms underlying this phenotypic change. We investigated HLA-A expression in 165 paraffin embedded tissues and 21 fresh tissues from liver cancer patients. Utilizing truncated HLA-A promoter–reporter constructs and gel-shift assay we had identified the regulatory elements and transcription factors required for HLA-A upregulation. 54% of the paraffin embedded tissues showed increased HLA-A expression in their cancerous part. 43% of the fresh liver cancer tissues had increased HLA-A complex expression with the HLA-A heavy chain gene demonstrating the highest level of upregulation (62%). Enhanced HLA-A expression in the liver cell lines QGY7701 and BEL7402 was found to be mediated by binding of interferon regulatory factor 1 (IRF-1) to interferon stimulated response element, and of nuclear transcription factor p65 binding to enhancer A element in the HLA-A promoter of these cell lines. The in vivo relevance of these findings was indicated by the association of the enhanced expression of IRF-1 and accumulation of nuclear p65 with HLA-A upregulation in 8 of the 21 liver cancer lesions investigated. Our results indicated that HLA-A upregulation in liver cancer was mediated by both increased nuclear aggregation of transcription factor p65 and upregulation of transcription factor IRF-1.

Severe acute respiratory syndrome coronavirus nucleocapsid protein does not modulate transcription of the human FGL2 gene

Among the structural and nonstructural proteins of severe acute respiratory syndrome coronavirus (SARS-CoV), the nucleocapsid (N) protein plays pivotal roles in the biology and pathogenesis of viral infection. N protein is thought to dysregulate cell signalling and the transcription of cellular genes, including FGL2, which encodes a prothrombinase implicated in vascular thrombosis, fibrin deposition and pneumocyte necrosis. Here, we showed that N protein expressed in cultured human cells was predominantly found in the cytoplasm and was competent in repressing the transcriptional activity driven by interferon-stimulated response elements. However, the expression of N protein did not influence the transcription from the FGL2 promoter. More importantly, N protein did not modulate the expression of FGL2 mRNA or protein in transfected or SARS-CoV-infected cells. Taken together, our findings did not support the model in which SARS-CoV N protein specifically modulates transcription of the FGL2 gene to cause fibrosis and vascular thrombosis.

369 Neutrophil Recruitment Is Deficient in Bacteria-Depleted Mice and Is Necessary for Recovery from DSS-Induced Colitis via TLR4-Dependent MIP2 Expression

Background: Interleukin (IL)-12 and 23 are heterodimeric cytokines composed of a common p40 subunit and p35 and p19 subunits, respectively. IL-23 is strongly implicated in the pathogenesis of inflammatory bowel disease. Aim: The aim of this study is to characterize the molecular regulation of Il23a gene expression and promoter activation in murine macrophages and experimental colitis. Results: Interferon-γ (IFN-γ) inhibits LPS-induced Il23a mRNA and IL-23 protein expression in murine bone marrow-derived macrophages (BMMs). A conserved nucleotide sequence across multiple species was identified in the promoter of the Il23a gene that contains an interferon stimulated response element (ISRE). In LPS and IFN-γ activated BMMs, chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrate that interferon regulatory factors (IRFs) interact with this ISRE. Using a 1.8 kb murine Il23a luciferase reporter plasmid, LPS induces and IFN-γ inhibits LPSinduced p19 promoter activity in BMMs. Mutations in the ISRE abrogate IFN-γ inhibition of LPS-induced promoter activity. To understand the contribution of specific IRFs, IRF-1 was inhibited in BMMs by siRNA and experiments were performed in IRF-1 deficient (-/-) macrophages. IRF-1 knockdown and IRF-1-/BMMs demonstrate increased LPS-induced IL-23 expression. Primary response genes have promoters that either exist in an open chromatin structure or undergo rapid nucleosomal remodeling. In contrast, secondary response genes with delayed induction kinetics require new protein synthesis prior to transcription initiation. Il23a expression in LPS-activated BMMs demonstrates rapid induction kinetics and unlike Il12b (established secondary response gene) is not dependent on new protein synthesis. Interestingly, LPS plus IFN-γ activated BMMs demonstrate loss of the IFN-γ inhibitory effect, suggesting IFN-γ mediated regulation of Il23a requires new protein synthesis. Il23a regulation was then studied in murine colitis models. Following intrarectal administration of TNBS, IRF-1-/mice exhibit increased inflammation and increased colonic IL-23 expression compared to wild type (WT) mice. Additionally, we demonstrate mucosal regulation of IL-23 by the enteric microbiota using germ-free and conventionalized WT and colitis-prone IL-10-/mice. Enteric bacteria induce significantly higher intestinal IL-23 expression in IL-10-/-mice compared withWTmice. Conclusions: IFN-γ and IRF-1 negatively regulate Il23a in murine macrophages and experimental colitis. Regulation of intestinal Il23a by the enteric microflora is a significant event in the initiation of chronic intestinal inflammation.

367 Molecular Regulation of Il23a Gene Expression in Murine Macrophages and Experimental Colitis

Background: Interleukin (IL)-12 and 23 are heterodimeric cytokines composed of a common p40 subunit and p35 and p19 subunits, respectively. IL-23 is strongly implicated in the pathogenesis of inflammatory bowel disease. Aim: The aim of this study is to characterize the molecular regulation of Il23a gene expression and promoter activation in murine macrophages and experimental colitis. Results: Interferon-γ (IFN-γ) inhibits LPS-induced Il23a mRNA and IL-23 protein expression in murine bone marrow-derived macrophages (BMMs). A conserved nucleotide sequence across multiple species was identified in the promoter of the Il23a gene that contains an interferon stimulated response element (ISRE). In LPS and IFN-γ activated BMMs, chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrate that interferon regulatory factors (IRFs) interact with this ISRE. Using a 1.8 kb murine Il23a luciferase reporter plasmid, LPS induces and IFN-γ inhibits LPSinduced p19 promoter activity in BMMs. Mutations in the ISRE abrogate IFN-γ inhibition of LPS-induced promoter activity. To understand the contribution of specific IRFs, IRF-1 was inhibited in BMMs by siRNA and experiments were performed in IRF-1 deficient (-/-) macrophages. IRF-1 knockdown and IRF-1-/BMMs demonstrate increased LPS-induced IL-23 expression. Primary response genes have promoters that either exist in an open chromatin structure or undergo rapid nucleosomal remodeling. In contrast, secondary response genes with delayed induction kinetics require new protein synthesis prior to transcription initiation. Il23a expression in LPS-activated BMMs demonstrates rapid induction kinetics and unlike Il12b (established secondary response gene) is not dependent on new protein synthesis. Interestingly, LPS plus IFN-γ activated BMMs demonstrate loss of the IFN-γ inhibitory effect, suggesting IFN-γ mediated regulation of Il23a requires new protein synthesis. Il23a regulation was then studied in murine colitis models. Following intrarectal administration of TNBS, IRF-1-/mice exhibit increased inflammation and increased colonic IL-23 expression compared to wild type (WT) mice. Additionally, we demonstrate mucosal regulation of IL-23 by the enteric microbiota using germ-free and conventionalized WT and colitis-prone IL-10-/mice. Enteric bacteria induce significantly higher intestinal IL-23 expression in IL-10-/-mice compared withWTmice. Conclusions: IFN-γ and IRF-1 negatively regulate Il23a in murine macrophages and experimental colitis. Regulation of intestinal Il23a by the enteric microflora is a significant event in the initiation of chronic intestinal inflammation.

368 Recombinant Tumor Necrosis Factor Alpha (TNFα) Peptide-Based Vaccine Ameliorates Acute and Chronic Murine Colitis

Background: Interleukin (IL)-12 and 23 are heterodimeric cytokines composed of a common p40 subunit and p35 and p19 subunits, respectively. IL-23 is strongly implicated in the pathogenesis of inflammatory bowel disease. Aim: The aim of this study is to characterize the molecular regulation of Il23a gene expression and promoter activation in murine macrophages and experimental colitis. Results: Interferon-γ (IFN-γ) inhibits LPS-induced Il23a mRNA and IL-23 protein expression in murine bone marrow-derived macrophages (BMMs). A conserved nucleotide sequence across multiple species was identified in the promoter of the Il23a gene that contains an interferon stimulated response element (ISRE). In LPS and IFN-γ activated BMMs, chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrate that interferon regulatory factors (IRFs) interact with this ISRE. Using a 1.8 kb murine Il23a luciferase reporter plasmid, LPS induces and IFN-γ inhibits LPSinduced p19 promoter activity in BMMs. Mutations in the ISRE abrogate IFN-γ inhibition of LPS-induced promoter activity. To understand the contribution of specific IRFs, IRF-1 was inhibited in BMMs by siRNA and experiments were performed in IRF-1 deficient (-/-) macrophages. IRF-1 knockdown and IRF-1-/BMMs demonstrate increased LPS-induced IL-23 expression. Primary response genes have promoters that either exist in an open chromatin structure or undergo rapid nucleosomal remodeling. In contrast, secondary response genes with delayed induction kinetics require new protein synthesis prior to transcription initiation. Il23a expression in LPS-activated BMMs demonstrates rapid induction kinetics and unlike Il12b (established secondary response gene) is not dependent on new protein synthesis. Interestingly, LPS plus IFN-γ activated BMMs demonstrate loss of the IFN-γ inhibitory effect, suggesting IFN-γ mediated regulation of Il23a requires new protein synthesis. Il23a regulation was then studied in murine colitis models. Following intrarectal administration of TNBS, IRF-1-/mice exhibit increased inflammation and increased colonic IL-23 expression compared to wild type (WT) mice. Additionally, we demonstrate mucosal regulation of IL-23 by the enteric microbiota using germ-free and conventionalized WT and colitis-prone IL-10-/mice. Enteric bacteria induce significantly higher intestinal IL-23 expression in IL-10-/-mice compared withWTmice. Conclusions: IFN-γ and IRF-1 negatively regulate Il23a in murine macrophages and experimental colitis. Regulation of intestinal Il23a by the enteric microflora is a significant event in the initiation of chronic intestinal inflammation.

Role of a distal enhancer in the transcriptional responsiveness of the human CD200 gene to interferon-γ and tumor necrosis factor-α

CD200 plays an important role in prevention of graft rejection, autoimmune diseases and spontaneous abortion by delivering an immunoregulatory signal through interaction with its receptor. It also plays a role in regulating tumor immunity. We previously documented evidence for C/EBPβ as being important in the regulation of constitutive expression of CD200. However, the molecular mechanism(s) controlling inducible expression of CD200 are yet to be explored. Here we address the regulated expression of human CD200 by T cells in response to Con A, IFN-γ or/and TNF-α. A prominent DNase I hypersensitivity site (DHS) was localized ∼5.4kb upstream of the major transcriptional start site. Four cis-elements were identified within this genomic region: one nuclear factor-κB (NF-κB) site, one IFN-γ activation site (GAS) element and two IFN-stimulated response element (ISRE) for binding of interferon-regulatory factors (IRFs), respectively. Mutation of the NF-κB site, GAS or one but not the other of ISREs dramatically reduced the luciferase activity. These findings were further confirmed by chromatin immunoprecipitation (ChIP) assays using antibodies against NF-κB p65, STAT1α, and IRF-1. All the above findings suggest that IFN-γ and TNF-α induce CD200 expression through a 5′ upstream enhancer and that NF-κB, STAT1 and IRF-1 play pivotal roles in this process.

Selective Transcriptional Down-Regulation of Human Rhinovirus-Induced Production of CXCL10 from Airway Epithelial Cells via the MEK1 Pathway

Human rhinovirus (HRV) infections can trigger exacerbations of lower airway diseases. Infection of airway epithelial cells induces production of a number of proinflammatory chemokines that may exacerbate airway inflammation, including CXCL10, a chemoattractant for type 1 lymphocytes and NK cells. Primary human bronchial epithelial cells and the BEAS-2B human bronchial epithelial cell line were used to examine the role of MAPK pathways in HRV-16-induced production of CXCL10. Surprisingly, PD98059 and U0126, two inhibitors of the MEK1/2-ERK MAPK pathway, significantly enhanced HRV-16-induced CXCL10 mRNA and protein. This enhancement was not seen with IFN-beta-induced production of CXCL10. Studies using small interfering RNA revealed that knockdown of MEK1, but not MEK2, was associated with enhanced HRV-induced CXCL10 production. Promoter construct studies revealed that PD98059 and U0126 enhanced HRV-16-induced transcriptional activation of CXCL10. HRV-16-induced promoter activation was regulated by two NF-kappaB binding sites, kappaB1 and kappaB2, and by an IFN-stimulated response element. Inhibitors of the MEK1/2-ERK pathway did not alter HRV-16-induced activation of tandem repeat kappaB1 or kappaB2 constructs, nor did they alter HRV-16-induced nuclear translocation/binding of NF-kappaB to either kappaB1 or kappaB2 recognition sequences. Furthermore, PD98059 and U0126 did not alter phosphorylation or degradation of IkappaBalpha. In contrast, inhibitors of the MEK1/2-ERK pathway, and small interfering RNA knockdown of MEK1, enhanced nuclear translocation/binding of IFN regulatory factor (IRF)-1 to the IFN-stimulated response element recognition sequence in HRV-16 infected cells. We conclude that activation of MEK1 selectively down-regulates HRV-16-induced expression of CXCL10 via modulation of IRF-1 interactions with the gene promoter in human airway epithelial cells.