IRF-1 Gene Expression Research Articles

Type I IFN Signature in plasmacytoid dendritic cells from young Sle 1,2,3 lupus prone mice. (171.1)

Abstract The importance of the Type I Interferon (IFN) Signature in systemic lupus erythmatosus is well documented in patients and mouse models. We have earlier observed a distinct IFN gene Signature in the myeloid dendritic cells (mDC) of young Sle 1,2,3 lupus prone mice. In this study, we grew bone-marrow precursors in the presence of Flt3L to generate plasmacytoid dendritic cells (pDC) and analyzed the IFN Signature. The percentage of pDCs generated in culture did not differ between the Sle and the control C57BL/6 (B6) mice. We sorted pDCs (B220+CD11c+) and mDCs (CD11c+CD11b+) from the culture and analyzed the constitutive expression of IFN responsive genes (IFNb, IRF7, ISG15, Mx-1 and CXCL10) by real-time RT-PCR as sign of IFN Signature. There was minimal IFNb expression in both B6 and Sle pDCs; while the IFNb expression in mDCs was higher and much higher in Sle vs B6 mDCs like we observe in cultures supplemented with GMCSF. IRF7 gene expression was higher in pDCs than mDCs and much higher in Sle pDCs. The expression of the other genes in pDCs was lower than in mDCs in both strains, although in Sle pDCs their expression was higher than in B6 pDCs. Our results indicate that plasmacytoid DCs from young pre-diseased Sle1,2,3 lupus prone mice express an IFN Signature similarly to lupus myeloid DCs. Moreover, these results highlight the important role for IRF7 in the activation of lupus pDCs and suggest a probable mechanism for the hyper-activation of the IFN Signature in lupus.

Synoviocyte innate immune responses: TANK-binding kinase-1 as a potential therapeutic target in rheumatoid arthritis

Innate immune responses in the rheumatoid synovium contribute to inflammation and joint destruction in RA. Two IκB kinase (IKK)-related kinases, TNF receptor associated factor (TRAF) family member-associated nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) activator (TANK)-binding kinase 1 (TBK1) and IKKε, potentially regulate synovitis by activating IFN response genes. These kinases induce the expression of inflammatory mediators such as C-X-C motif ligand 10 (CXCL10)/IFN-γ-induced protein 10 kDa (IP-10) in fibroblast-like synoviocytes (FLS). Since IP-10 is a promising therapeutic target in RA, we evaluated whether blocking TBK1 might be an effective way to modulate IP-10 expression. Wild-type (WT) and IKKε(-/-) FLS were transfected with TBK1 or control small interfering RNA (siRNA) and stimulated with polyinosinic acid : polycytidylic acid [poly(I:C)]. Gene expression was assayed using quantitative PCR. Cytokine production in culture supernatants was measured by Luminex multiplex analysis. IFN-regulatory factor (IRF3) dimerization was determined by native PAGE. IFN-β and IP-10 promoter activity was measured using luciferase reporter constructs. Initial studies showed that siRNA markedly decreased TBK1 expression in cultured FLS. Poly(I:C)-induced IRF7 gene expression was inhibited in the absence of TBK1, but not IKKε. IRF3 gene expression was similar to WT cells in TBK1 or IKKε-deficient FLS. IRF3 dimerization required both TBK1 and IKKε. Surprisingly, IRF3-mediated gene and protein expression of IFN-β and IP-10 was dependent on TBK1, not IKKε. Promoter constructs showed that TBK1 decreased IP-10 gene transcription and IP-10 mRNA stability was unaffected by TBK1 deficiency. Based on the selective regulation of IP-10 in FLS, TBK1 appears to be the optimal IKK-related kinase to target in RA.

Counter‐regulation mechanism of IL‐4 and IFN‐α signal transduction through cytosolic retention of the pY‐STAT6:pY‐STAT2:p48 complex

IFN-α and IL-4 induce Th1 and Th2 responses, respectively, and often display antagonistic actions against each other. To elucidate the molecular mechanism of counter-regulation, we have investigated the signal interception by IFN-α and IL-4, employing a human B-cell line Ramos, sensitive to both cytokines. In these cells, IFN-α effectively inhibited IL-4-induced Fc epsilon receptor II (CD23) expression, whereas IL-4 suppressed IFN-α-mediated IRF7 expression. The counter-regulatory action by IL-4 and IFN-α proceeded with a delayed kinetics requiring 4 h. Notably, IFN-α did not affect the IL-4-induced tyrosine phosphorylation of STAT6, but induced a time-dependent cytoplasmic accumulation of phosphotyrosine(pY)-STAT6 and a corresponding decrease in nuclear pY-STAT6. By confocal analysis and co-immunoprecipitation assays, we demonstrated the colocalization and molecular interaction of IL-4-induced pY-STAT6 with IFN-α-induced pY-STAT2:p48 in the cytosol. In addition, the over-expression of STAT2 or STAT6 induced the concomitant cytosolic accumulation of pY-STAT6 or pY-STAT2, leading to the suppression of IL-4-induced CD23 or IFN-α-induced IRF7 gene expression, respectively. Our data suggest that the signals ensued by IFN-α and IL-4 induce cytoplasmic sequestration of IL-4-activated STAT6 and IFN-α-activated STAT2:p48 in B cells through the formation of pY-STAT6:pY-STAT2:p48 complex, which provides a novel mechanism by which IFN-α and IL-4 cross-regulate their signaling into the nucleus.

Activated IL-1R1 Signaling Pathway Induces Th17 Cell Differentiation in Patients with Relapsing Remitting Multiple Sclerosis (83.18)

Abstract BACKGROUND: IL-1β plays a crucial role in the development of human Th17 cells. However, its mechanisms of action are still not completely elucidated. Recent has study discovered that Th17 cells are characterized by IL-1R1 expression. RESULTS: Our study has revealed that IL-1R1 and IL-1R2 gene expression is significantly increased in both naïve and memory CD4+ T-cells derived from patients with relapsing-remitting multiple sclerosis (RRMS) in comparison to matched healthy controls. IL-1R1 expression is up-regulated in the in vitro differentiated Th17 cells in comparison to the Th1 and Th2 cell subsets. IL-17A production was induced by IL-1β. This induction was blocked by IL-1R antagonist in the naïve CD4+cells cultured in the presence of Th17 polarizing cytokines. When IL-1R1 gene expression was silenced using siRNA, human naïve CD4+ T-cells cultured in the presence of Th17 polarizing cytokines had a significantly decreased expression of IL-21, IL-22, IL-23R, IRF4, RORC, IL-17A and IL-17F genes, confirming that IL-1R1 signaling induces Th17 cell differentiation. IRF4 gene expression silencing, similarly inhibited Th17 cell differentiation in the presence of IL-1b, indicating that IL-1R1 signals upstream of IRF4. CONCLUSION: Our study has identified that IL-1R1-mediated signaling pathway may be constitutively activated, leading to an increased Th17 cell expansion and autoimmune response in patients with RR MS.

Type I Interferons and Interferon Regulatory Factors Regulate TNF-Related Apoptosis-Inducing Ligand (TRAIL) in HIV-1-Infected Macrophages

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family that participates in HIV-1 pathogenesis through the depletion of CD4+ T cells. TRAIL is expressed on the cell membrane of peripheral immune cells and can be cleaved into a soluble, secreted form. The regulation of TRAIL in macrophages during HIV-1 infection is not completely understood. In this study, we investigated the mechanism(s) of TRAIL expression in HIV-1-infected macrophages, an important cell type in HIV-1 pathogenesis. A human monocyte-derived macrophage (MDM) culture system was infected with macrophage-tropic HIV-1ADA, HIV-1JR-FL, or HIV-1BAL strains. TRAIL, predominantly the membrane-bound form, increased following HIV-1 infection. We found that HIV-1 infection also induced interferon regulatory factor (IRF)-1, IRF-7 gene expression and signal transducers and activators of transcription 1 (STAT1) activation. Small interfering RNA knockdown of IRF-1 or IRF-7, but not IRF-3, reduced STAT1 activation and TRAIL expression. Furthermore, the upregulation of IRF-1, IRF-7, TRAIL, and the activation of STAT1 by HIV-1 infection was reduced by the treatment of type I interferon (IFN)-neutralizing antibodies. In addition, inhibition of STAT1 by fludarabine abolished IRF-1, IRF-7, and TRAIL upregulation. We conclude that IRF-1, IRF-7, type I IFNs, and STAT1 form a signaling feedback loop that is critical in regulating TRAIL expression in HIV-1-infected macrophages.

Control of ER stress by a chemical chaperone counteracts apoptotic signals in IFN-γ-treated murine hepatocytes

Apoptosis of hepatocytes plays a key role in the pathogenesis of immune-mediated hepatitis. However, the detailed mechanisms of apoptotic signaling remain unclear. In this study, we investigated the involvement of ER stress in a model of IFN-gamma-induced apoptosis of hepatocytes in vitro, using a chemical chaperone reagent, glycerol. IFN-gamma-induced apoptotic events (mitochondrial release of cytochrome c, enzymatic activation of caspase-3 and -9) were markedly inhibited by glycerol. Glycerol induced partial inhibition of cytotoxicity indicated by lactate dehydrogenase release from the cytosol but had no inhibitory effect on the induction of IRF-1 gene expression and reactive oxygen species, required for hepatocyte apoptosis by IFN-gamma. Induction of caspase-4 and -12 gene expression, positively correlated with ER stress, was attenuated by glycerol. Gene analysis revealed that induction of ER stress-related genes, C/EBP homologue protein (CHOP/GADD153) and TRB3, was suppressed completely by glycerol treatment. These results suggest that ER stress plays a crucial role in mediating apoptosis of hepatocytes induced by IFN-gamma, and a chemical chaperone is an effective inhibitor of the ER stress.

Irf4 is a positional and functional candidate gene for the control of serum IgM levels in the mouse

Natural IgM are involved in numerous immunological functions but the genetic factors that control the homeostasis of its secretion and upholding remain unknown. Prompted by the finding that C57BL/6 mice had significantly lower serum levels of IgM when compared with BALB/c mice, we performed a genome-wide screen and found that the level of serum IgM was controlled by a QTL on chromosome 13 reaching the highest level of association at marker D13Mit266 (LOD score=3.54). This locus was named IgMSC1 and covered a region encompassing the interferon-regulatory factor 4 gene (Irf4). The number of splenic mature B cells in C57BL/6 did not differ from BALB/c mice but we found that low serum levels of IgM in C57BL/6 mice correlated with lower frequency of IgM-secreting cells in the spleen and in the peritoneal cavity. These results suggested that C57BL/6 mice have lower efficiency in late B-cell maturation, a process that is highly impaired in Irf4 knockout mice. In fact, we also found reduced Irf4 gene expression in B cells of C57BL/6 mice. Thus, we propose Irf4 as a candidate for the IgMSC1 locus, which controls IgM homeostatic levels at the level of B-cell terminal differentiation.

KAP1 regulates type I interferon/STAT1-mediated IRF-1 gene expression

Signal transducers and activators of transcription (STATs) mediate cell proliferation, differentiation, and survival in immune responses, hematopoiesis, neurogenesis, and other biological processes. Recently, we showed that KAP1 is a novel STAT-binding partner that regulates STAT3-mediated transactivation. KAP1 is a universal co-repressor protein for the KRAB zinc finger protein superfamily of transcriptional repressors. In this study, we found KAP1-dependent repression of interferon (IFN)/STAT1-mediated signaling. We also demonstrated that endogenous KAP1 associates with endogenous STAT1 in vivo. Importantly, a small-interfering RNA-mediated reduction in KAP1 expression enhanced IFN-induced STAT1-dependent IRF-1 gene expression. These results indicate that KAP1 may act as an endogenous regulator of the IFN/STAT1 signaling pathway.

IFN-β Provides Immuno-Protection in the Retina by Inhibiting ICAM-1 and CXCL9 in Retinal Pigment Epithelial Cells

The retinal pigment epithelial (RPE) cell is a potent regulatory cell that facilitates normal physiologic processes and plays a critical role in a variety of retinal diseases. We evaluated IFN-beta production in human RPE cells through TLR signaling and investigated the effects of IFN-beta on RPE cells. RPE cells treated with poly(I:C) or infected with an RNA virus produce IFN-beta. Kinetic studies revealed that IFN-beta levels continue to increase over a 48-h period and this was associated with the up-regulation of IRF-7 gene expression, a known positive feedback molecule for IFN-beta production. Microarray analysis revealed that in IFN-beta treated cells, 480 genes of 22,283 genes were up or down-regulated by >2-fold. We hypothesize that IFN-beta induction during TLR signaling in the retina is an immunosuppressive factor produced to limit immunopathologic damage. Cytokine activation of RPE cells results in the production of the chemokines, CXCL9 and CXCL10, and the adhesion molecule, ICAM-1. Pretreatment of RPE cells with IFN-beta resulted in inhibition of ICAM-1 production and elimination of CXCL9 production. This treatment did not alter CXCL10 production. Anti-IFN-beta Ab blocked the inhibitory action of IFN-beta. Real time PCR analysis revealed that IFN-beta treatment inhibited gene expression of sICAM-1 and CXCL9. The results indicate a critical role for RPE cell derived IFN-beta in the down-regulation of CXCL9 and ICAM-1 expression in the retina and suggest that the inhibition of CXCL9 is an immuno-suppressive mechanism that protects the retina from excessive inflammation.

786 PEGINTERFERON PLUS RIBAVIRIN AND SUSTAINED VIROLOGICAL RESPONSE ON HCV CIRRHOSIS: EFFECT ON LIVER-RELATED EVENT FREE SURVIVAL AND FACTORS PREDICTING RESPONSE: A RETROSPECTIVE MULTICENTER ANALYSIS

IRF-1 gene expression in liver biopsies prior was 2.37±1.7 and was downregulated in liver biopsies after to 1.66±1.43 (p = 0.022). In patients with NR the basal expression was 1.72±3.6 and interestingly, was up-regulated in liver biopsies after of the treatment 2.37±1.7 (p = 0.035). Conclusions: Our study has demonstrated that IRF-1 gene have different expression patterns in HCV patients with SVR and NR. IRF-1 gene expression signature will probably used in the future for optimised treatment.

IRF-1Gene Variations Influence IgE Regulation and Atopy

The development of atopic diseases is characterized by skewed immune responses to common allergens. Only recently, interferons have been identified to play a crucial role in these mechanisms. Because interferon regulatory factor (IRF)-1 is critical for interferon expression, we tested the hypotheses that genetic changes in this essential transcription factor may have consequences for the development of atopy. The IRF-1 gene locus was resequenced in 80 human chromosomes. Association and haplotype analyses were performed in a cross-sectional study population of German children from Dresden (n = 1,940), and results were replicated in a second population sample from Munich (n = 1,159), both part of the ISAAC (International Study of Asthma and Allergy in Childhood) phase II. Promoter polymorphism effects were studied using electrophoretic mobility shift assay and colorimetric binding assays. Allele-specific IRF-1 gene expression was studied in vitro using luciferase reporter assays, whereas we assessed ex vivo expression of IRF-1 by real-time polymerase chain reaction and IFN-gamma protein by Luminex technology (Bio-Rad, Hercules, CA). Statistical analyses were performed using SAS/Genetics (SAS 9.1.3; SAS Institute, Cary, NC). By resequencing, 49 polymorphisms were identified within the IRF-1 gene. Four blocks containing 11 polymorphisms were significantly associated with atopy, total IgE levels, or specific IgE levels in both populations (P < 0.05). Two polymorphisms changed transcription factor binding of nuclear factor (NF)-kappaB and EGR1 (early growth response 1) to the IRF-1 promoter, altered gene expression in vitro (P = 0.0004), and altered IRF-1 mRNA and IFN-gamma protein expression ex vivo. Our results suggest that functionally relevant IRF-1 polymorphisms influence atopy risk, potentially by altering transcription factor binding, IRF-1 gene expression, and IFN-gamma regulation.

Role of IFN regulatory factor 5 transcription factor in antiviral immunity and tumor suppression

Host defense consists of two main aspects, namely, immune response to invading pathogens and suppression of tumor development. A family of transcription factors, IFN regulatory factors (IRFs), has recently gained much attention in terms of its critical role in linking these two aspects of host defense, wherein IRF5 was previously shown to play a critical role in the induction of proinflammatory cytokines by activation of Toll-like receptors. In the present study, using IRF5 gene-targeted mice (Irf5(-/-) mice), we demonstrate another facet of the IRF5 function in the regulation of immune response and tumor suppression. We show that IRF5 is critical for antiviral immunity by showing that Irf5(-/-) mice are highly vulnerable to viral infections, accompanied by a decrease in type I IFN induction in the sera. Furthermore, we show that Irf5(-/-) fibroblasts are resistant to apoptosis upon viral infection, resulting in an enhanced viral propagation. Finally, we provide evidence that IRF5 is critical for the induction of apoptosis, but not in cell cycle arrest, in response to DNA damage and that IRF5 functions as a tumor suppressor by acting on a pathway that may be distinct from that for p53. These results, together with the dual regulation of IRF5 gene expression by IFN signaling and p53, may provide a new link in the transcriptional network underlying antiviral immunity and tumor suppression.

Involvement of TBK1 and IKKϵ in lipopolysaccharide‐induced activation of the interferon response in primary human macrophages

Interferon (IFN) is an important effector of the innate immune response, induced by different viral or bacterial components through Toll-like receptor-dependent and -independent mechanisms. In human macrophages and macrophage-activated killer cells, we demonstrate that (i) the type I IFN response to lipopolysaccharide (LPS) is weak compared to the host response to virus infection; (ii) there is a temporal difference in the induction of tank-binding kinase-1 (TBK1) and IkappaB kinase (IKK)-related kinase epsilon (IKKepsilon) kinase activities in response to LPS, with TBK1 activated early and IKKepsilon induced in the late phase of IFN induction; and (iii) interferon regulatory factor (IRF)-7 is induced following LPS treatment, but there is no evidence that IRF-7 becomes activated by phosphorylation in vivo. Specifically, TBK1 kinase activity is rapidly increased after LPS stimulation (15 min) whereas IKKepsilon activation occurs at 8 h. RNA interference-mediated inhibition of TBK1 and IKKepsilon expression in macrophages interfere with IFNB and IRF7 gene expression following LPS activation. Macrophage priming with rIFN-alpha increased IRF-7 expression, led to a sharp up-regulation of the IFNB gene and to a rapid induction of IFNA2 upon LPS stimulation. These data support a differential role of TBK1 and IKKepsilon in the downstream response mediated by IRF-3 and IRF-7 to LPS in primary human macrophages.

The genetics and biology of Irf5-mediated signaling in lupus

Recently much attention was attracted to the importance of the type I interferon pathway in the initiation and development of the autoimmune disease systemic lupus erythematosus (SLE). Many SLE patients have increased serum levels of IFN-α and display an IFN gene expression “signature” characterized by strong overexpression of IFN-responsive genes in leukocytes and target tissues. Moreover, about 20% of cancer patients treated with IFN-α therapy manifest symptoms resembling SLE and some later develop the disease. One of the key genes of the IFN-α pathway, IRF5, was found to be strongly associated with SLE. Two functional SNPs lead to alternative splicing and altered steady-state level of IRF5 gene expression. Besides, the gene has a polymorphic inserion/deletion in exon 6, which contributes to the diversity in the isoform pattern of IRF5. Interestingly, recent studies have not found association of IRF5 with the other autoimmune diseases, such as rheumatoid arthritis or psoriasis, suggesting the unique role for IRF5 in the development of lupus. Here, we present the current knowledge on IRF5 genetics and its biological function and discuss the possible ways in which IRF5 contributes to susceptibility to SLE.

Transdermal delivery of interferon‐γ (IFN‐γ) mediated by penetratin, a cell‐permeable peptide

IFN-gamma (interferon-gamma) has several applications in the treatment of IFN-gamma-related skin disorders. While systemic delivery - the major route used to administer IFN-gamma - results in significant side effects and toxicity, including fever, fatigue, nausea, vomiting and neurotoxicity, transdermal delivery has a very low transduction efficiency. In order to improve the efficiency of transdermal IFN-gamma delivery, we introduced a Pen (penetratin) peptide, a 16-amino-acid-long polypeptide corresponding to the third helix of the DNA-binding domain (homoeodomain) of Antennapedia (a Drosophila transcription factor). The human IFN-gamma gene was then fused with a gene fragment that encodes the Pen of Antennapedia in a bacterial expression vector, producing a genetic in-frame Pen-IFN-gamma. The expressed and purified Pen-IFN-gamma was then found to have a much more efficient transduction profile than native IFN-gamma. In addition, compared with native IFN-gamma, Pen-IFN-gamma exhibited similar activities when added exogenously to a culture medium: (i) induction of IRF-1 gene expression, and (ii) NF-kappaB (nuclear factor kappaB) luciferase reporter activation. These results indicate that the transdermal delivery system using Pen may be an excellent way to replenish IFN-gamma in the various disorders related to this cytokine.

Differential Regulation of Interferon Regulatory Factor (IRF)-7 and IRF-9 Gene Expression in the Central Nervous System during Viral Infection

Interferon regulatory factors (IRFs) are a family of transcription factors involved in the regulation of the interferons (IFNs) and other genes that may have an essential role in antiviral defense in the central nervous system, although this is currently not well defined. Therefore, we examined the regulation of IRF gene expression in the brain during viral infection. Several IRF genes (IRF-2, -3, -5, -7, and -9) were expressed at low levels in the brain of uninfected mice. Following intracranial infection with lymphocytic choriomeningitis virus (LCMV), expression of the IRF-7 and IRF-9 genes increased significantly by day 2. IRF-7 and IRF-9 gene expression in the brain was widespread at sites of LCMV infection, with the highest levels in infiltrating mononuclear cells, microglia/macrophages, and neurons. IRF-7 and IRF-9 gene expression was increased in LCMV-infected brain from IFN-gamma knockout (KO) but not IFN-alpha/betaR KO animals. In the brain, spleen, and liver or cultured glial and spleen cells, IRF-7 but not IRF-9 gene expression increased with delayed kinetics in the absence of STAT1 but not STAT2 following LCMV infection or IFN-alpha treatment, respectively. The stimulation of IRF-7 gene expression by IFN-alpha in glial cell culture was prevented by cycloheximide. Thus, (i) many of the IRF genes were expressed constitutively in the mouse brain; (ii) the IRF-7 and IRF-9 genes were upregulated during viral infection, a process dependent on IFN-alpha/beta but not IFN-gamma; and (iii) IRF-7 but not IRF-9 gene expression can be stimulated in a STAT1-independent but STAT2-dependent fashion via unidentified indirect pathways coupled to the activation of the IFN-alpha/beta receptor.

IFN-α and IL-12 activate IFN regulatory factor 1 (IRF-1), IRF-4, and IRF-8 gene expression in human NK and T cells

IFN-alpha and IL-12 are macrophage-derived cytokines that enhance innate and Th1 immune responses. However, there is little information regarding IFN-alpha and IL-12 target genes that would be involved in mediating the immunostimulatory effects of these cytokines. The interferon regulatory factor (IRF) family of transcription factors is known to be involved in controlling lymphocyte differentiation and functions. In this work we have studied the effect of IFN-alpha and IL-12 on the expression of IRF transcription factors in human NK and T cells. Both IFN-alpha and IL-12 strongly up-regulated IRF-1, IRF-4, and IRF-8 mRNA and protein expression. The binding of IRF-4 and IRF-8 to the lambdaB gene enhancer sequence was also increased following IFN-alpha- and IL-12-treatment of NK and T cells. A GAS element from the promoter region of the IRF-4 gene was identified. Following stimulation of cells with IFN-alpha or IL-12, Stat4 was found to bind to this IRF-4 GAS element, as detected by EMSA and DNA affinity binding, implying that the IRF-4 gene is directly activated by both cytokines. Our results suggest that IFN-alpha and IL-12 may enhance innate and Th1 immune responses by inducing IRF-1, IRF-4, and IRF-8 gene expression.

Interferon regulatory factor-1 down-regulates cytokine-induced IP-10 expression in pancreatic islets

Background. Interferon (IFN)-γ acts synergistically with interleukin (IL)-1β and tumor necrosis factor (TNF)-α to activate isoform of nitric oxide synthetase (iNOS) gene expression, induce apoptosis, and impair glucose-stimulated insulin release (GSIR) in pancreatic islets. This effect is an important mechanism of islet dysfunction in models of pancreatitis, type I diabetes, and islet allograft rejection. We tested the hypothesis that transcription factor interferon regulatory factor (IRF)-1 plays a regulatory role in both this cytokine-induced islet injury and cytokine-induced gene expression for chemotactic chemokines. Methods. Isolated islets from wild-type (WT) or IRF-1−/− C57BL/6 mice were cultured in a mixture of IL-1β, TNF-α, and IFN-γ ± the iNOS inhibitor L-NMMA. The following end points were assessed: i) GSIR; ii) rates of apoptosis; and iii) gene expression for iNOS, IRF-1 and inducible protein (IP)-10, monocyte chemoattractive protein (MCP)-1, macrophage inflammatory protein-1α, and macrophage inflammatory protein-1β. Results. Cytokine-treated WT islets demonstrated an increase in IRF-1 and iNOS gene expression, inhibition of GSIR, increased rates of apoptosis, and increased gene transcription and protein release for IP-10 and MCP-1. Cytokine-treated IRF-1−/− islets demonstrated relatively less iNOS gene expression, preserved GSIR, reduced rates of apoptosis, and a more marked increase in transcription for IP-10 and MCP-1 and in IP-10 protein release. L-NMMA-cotreated WT islets were completely resistant to cytokine-induced dysfunction and apoptosis but demonstrated the same degree of cytokine-induced chemokine gene expression as cytokine-treated WT without L-NMMA. Conclusion. IFN-γ, IL-1β, and TNF-α in combination induce chemokine gene expression in pancreatic islets. Transcription factor IRF-1 mediates cytokine-induced islet dysfunction, apoptosis, and iNOS gene expression but down-regulates IP-10 gene expression. (Surgery 2003;134:134-41.)

Impairment of interferon-induced IRF-7 gene expression due to inhibition of ISGF3 formation by trichostatin A.

Two members of the signal transducer and activator of transcription family, STAT1 and STAT2, form, together with interferon regulatory factor 9 (IRF-9), the ISGF3 complex that activates the expression of the interferon-stimulated genes (ISG). The ISGF3 complex also participates in the virus-induced alpha/beta interferon (IFN-alpha/beta) gene amplification cascade by up-regulating IRF-7 gene expression. Here, we show that treatment of cells with trichostatin A (TSA), a deacetylase inhibitor, inhibits the virus-induced activation of IFN-alpha/beta promoters and dramatically reduces the ability of different ISG promoters to respond to IFN stimulation. Impairment of IFN-alpha/beta and ISG expression by TSA in infected cells is due to the blockage of interferon-stimulated ISGF3 complex formation, which leads to the abolition of IRF-7 gene expression. We also show that the TSA-dependent inhibition of ISGF3 is related to impaired nuclear accumulation of STAT2. Our data suggest that an acetylation/deacetylation mechanism participates in the regulation of cellular distribution and function of STAT2 in IFN-alpha/beta signaling.

Synergistic stimulation of MHC class I and IRF-1 gene expression by IFN-gamma and TNF-alpha in oligodendrocytes

Synergistic stimulation of MHC class I and IRF-1 gene expression by IFN-gamma and TNF-alpha in oligodendrocytes