Interferon regulatory factor 1(IRF-1) activates anti-tumor immunity via CXCL10/CXCR3 axis in hepatocellular carcinoma (HCC).

Interferon regulatory factor 1 (IRF-1) is a tumor suppressor gene in cancer biology with anti-proliferative and pro-apoptotic effect on cancer cells, however mechanisms of IRF-1 regulating tumor microenvironment (TME) in hepatocellular carcinoma (HCC) remain only partially characterized. Here, we investigated that IRF-1 regulates C-X-C motif chemokine 10 (CXCL10) and chemokine receptor 3 (CXCR3) to activate anti-tumor immunity in HCC. We found that IRF-1 mRNA expression was positively correlated with CXCL10 and CXCR3 through qRT-PCR assay in HCC tumors and in analysis of the TCGA database. IRF-1 response elements were identified in the CXCL10 promoter region, and ChIP-qPCR confirmed IRF-1 binding to promote CXCL10 transcription. IRF-2 is a competitive antagonist for IRF-1 mediated transcriptional effects, and overexpression of IRF-2 decreased basal and IFN-γ induced CXCL10 expression. Although IRF-1 upregulated CXCR3 expression in HCC cells, it inhibited proliferation and exerted pro-apoptotic effects, which overcome proliferation partly mediated by activating the CXCL10/CXCR3 autocrine axis. In vitro and in vivo studies showed that IRF-1 increased CD8+ T cells, NK and NKT cells migration, and activated IFN-γ secretion in NK and NKT cells to induce tumor apoptosis through the CXCL10/CXCR3 paracrine axis. Conversely, this effect was markedly abrogated in HCC tumor bearing mice deficient in CXCR3. Therefore, the IRF-1/CXCL10/CXCR3 axis contributes to the anti-tumor microenvironment in HCC.

Mechanisms of concanavalin A-induced cytokine synthesis by hepatic stellate cells: Distinct roles of interferon regulatory factor-1 in liver injury.

Mice depleted of hepatic stellate cells (HSCs) are protected from concanavalin A (ConA)-induced liver injury that is mediated by the activation of interferon regulatory factor 1 (IRF1). The aim of this study was to determine the mechanisms of ConA-mediated signaling and synthesis/release of mediators by HSCs that damage hepatocytes. Primary cultures of wildtype (WT) and IRF1-knockout (KO) HSCs and hepatocytes were used, and ConA-induced liver damage in interferon (IFN)αß receptor-deficient (IFNαßR-KO) mice was determined. Specific binding of ConA to HSCs induced rapid activation of JAK2 and STAT1. ConA-induced expression of IRF1, IFNß, tumor necrosis factor α, and CXCL1 was abrogated by selective inhibition of JAK2 and STAT1. Despite activating JAK2/STAT1, ConA failed to stimulate expression of inflammatory cytokines in HSCs from IRF1-KO mice. ConA-conditioned WT-HSC medium caused activation of JNK and caspase 3, and apoptosis of hepatocytes from WT but not from IRF1-KO or IFNαßR-KO mice. Conversely, ConA-conditioned medium of IRF1-KO HSCs failed to cause apoptosis of WT or IRF1-KO hepatocytes. IFNαßR-KO mice were protected from ConA-induced liver damage, and ConA-induced hepatic expression of IRF1 and pro-inflammatory cytokines and chemokines, and infiltration of neutrophils were significantly lower in IFNαßR-KO than in WT mice. These results demonstrate distinct roles of IRF1 in hepatic inflammation (HSCs) and injury (hepatocytes) and can be an important target for intervention in acute liver injury.

IRF1 up-regulates isg15 gene expression in dsRNA stimulation or CSFV infection by targeting nucleotides -487 to -325 in the 5' flanking region.

Interferon (IFN)-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that is heavily involved in immune response elicitation. As an important member of interferon regulatory factor (IRF) family, IRF1 can activate the expression of multiple genes, including the human optineurin gene (Sudhakar et al., 2013). In this study, a sequence in the promoter region of the optineurin gene was compared to the 5' flanking region of the porcine isg15 gene. Porcine IRF1 also possesses antiviral activity against several swine viruses (Li et al., 2015), but the mechanism is not well understood. Herein, we report that porcine IRF1 and ISG15 were up-regulated in porcine kidney (PK-15) cells following stimulation with double-stranded RNA (dsRNA) or classical swine fever virus (CSFV) infection. We also found that siRNA-mediated knockdown of IRF1 expression resulted in lower ISG15 expression in response to polyinosinic:polycytidylic acid [poly(I:C)] or CSFV infection. The overexpression of IRF1 resulted in ISG15 up-regulation. IRF1 was shown to translocate to the nucleus in response to dsRNA stimulation. To further identify the functional domain of the isg15 gene that promotes IRF1 transcriptional activity, firefly luciferase and ISG15 reporter systems were constructed. The results of the firefly luciferase and ISG15 reporter assay suggested that IRF1 mediates the up-regulation of ISG15. Nucleotides -487 to -325, located in the 5' flanking region of the isg15 gene, constituted the promoter region of IRF1. ChIP assay indicated that IRF1 protein was able to interact with the DNA in the 5'fr of isg15 gene in cells. As an innate immune response protein with broad-spectrum antiviral activity, the up-regulation of ISG15 mediated by IRF1 in porcine cells is reported for the first time. These results warrant further investigation into the antiviral activity of porcine IRF1 against reported swine viruses.

Role of interferon regulatory factor 1 in governing Treg depletion, Th1 polarization, inflammasome activation and antitumor efficacy of cyclophosphamide.

The antitumor effectiveness of cyclophosphamide (CTX) and other chemotherapeutics was shown to rely not only on direct cytotoxicity but also on immunogenic tumor cell death and systemic immunomodulatory mechanisms, including regulatory T cell (Treg) depletion, Th1 cell polarization, type I interferon (IFN) and proinflammatory cytokine production. IFN regulatory factor (IRF)-1 is a transcriptional regulator of IFNs and IFN-inducible genes, involved in the control of Th1 and Treg differentiation and in sterile inflammation. Aim of this study was to explore the role of IRF-1 in CTX-induced antitumor effects and related immune activities. This study shows for the first time that IRF-1 is important for the antitumor efficacy of CTX in mice. Moreover, experiments in tumor-bearing C57BL/6 mice showed that Irf1 gene expression in the spleen was transiently increased following CTX administration and correlated with the induction of Th1 cell expansion and of Il12p40 gene expression, which is the main Th1-driving cytokine. At the same time, CTX administration reduced both Foxp3 expression and Treg cell percentages. These effects were abrogated in Irf1-/- mice. Further experiments showed that the gene and/or protein expression of caspase-1, iNOS, IL-1ß, IL-6 and CXCL10 and the levels of nitric oxide were modulated following CTX in an IRF-1-direct- or -indirect-dependent manner, and highlighted the importance of caspase-1 in driving the sterile inflammatory response to CTX. Our data identify IRF-1 as important for the antitumor efficacy of CTX and for the regulation of many immunomodulatory activities of CTX, such as Th1 polarization, Treg depletion and inflammation.

Enhanced Inflammasome Activity in Systemic Lupus Erythematosus Is Mediated via Type I Interferon-Induced Up-Regulation of Interferon Regulatory Factor 1.

OBJECTIVE: The inflammasome complex is a driver of organ damage in patients with systemic lupus erythematosus (SLE). Although type I interferons (IFNs) are well established as mediators of SLE pathogenesis, their role in inflammasome activation in SLE has not been assessed. The aim of this study was to examine type I IFNs as regulators of the inflammasome. METHODS: SLE patients fulfilled ≥4 American College of Rheumatology criteria and were recruited from the University of Michigan Lupus Cohort. Primary monocytes were isolated from SLE patients or healthy controls by negative selection, treated with inflammasome activators in the presence or absence of IFNα, and IL-1ß secretion was measured by enzyme-linked immunosorbent assay. Expression levels of IFN and inflammasome-related molecules were assessed by real-time polymerase chain reaction and Western blotting. IFN regulatory factor 1 (IRF-1) expression was specifically down-regulated by small interfering RNA (siRNA) transfection and a chemical inhibitor. RESULTS: Monocytes from patients with SLE exhibited increased expression and enhanced activation of the inflammasome by ATP when compared with control monocytes. Expression of inflammasome and IFN-regulated genes was significantly correlated in monocytes from SLE patients but not in control monocytes. Inflammasome activity was increased after prolonged exposure to IFNα. Reduction of IRF-1 expression via siRNA blocked caspase 1 up-regulation after treatment with IFNα. Importantly, hyperactivity of the inflammasome in the monocytes of SLE patients was significantly reduced after knockdown or inhibition of IRF-1. CONCLUSION: Prolonged type I IFN exposure, as seen in SLE patients, primes monocytes for robust inflammasome activation in an IRF-1-dependent manner. IRF-1 inhibition may serve as a novel target for treatment of SLE-associated inflammation and organ damage.

Increase of IRF-1 gene expression and impairment of T regulatory cells suppression activity on patients with myelodysplastic syndrome: A longitudinal one-year study.

Studies have demonstrated that abnormalities in interferon regulatory factor-1 (IRF-1) expression might develop myelodysplastic syndromes (MDS). IRF-1 was described as modulator of T regulatory (Treg) cells by suppressing Foxp3 on mice. We aimed to determine the role of Treg and IRF-1 in MDS. Thirty-eight MDS patients fulfilling WHO criteria and classified according to risk scores were evaluated at time 0 (T0) and after 12 months (T12) for: Treg suppression activity in coculture with T effector (Teff) cells; IRF-1 and Foxp3 genetic expression by qRT-PCR; IL-2, -4, -6, -10, -17, TNFα and IFNγ production by Cytometric Bead Array. No differences in Foxp3 expression (T0=0.06±0.06 vs T12=0.06±0.12, p=0.5), Treg number (T0=5.62±2.84×105 vs T12=4.87±2.62×105; p=0.3) and Teff percentage (T0=16.8±9.56% vs T12=13.1±6.3%; p=0.06) were observed on T12. Low risk MDS patients showed a higher number of Treg (5.2±2.6×105) versus high risk group (2.6±1.2×105, p=0.03). Treg suppression activity was impaired on T0 and T12.Cytokine production and IRF-1 expression were increased on T12. The correlation between IRF-1 and FoxP3 was negative (r2=0.317, p=0.045) on T0. These results suggest a hyper activity of the immune system, probably secondary to Treg suppression activity impairment. This state may induce the loss of tolerance culminating in the proliferation of MDS clones.

Sublytic C5b-9 Induces Glomerular Mesangial Cell Apoptosis through the Cascade Pathway of MEKK2-p38 MAPK-IRF-1-TRADD-Caspase 8 in Rat Thy-1 Nephritis.

The apoptosis of glomerular mesangial cells (GMCs) in the early phase of rat Thy-1 nephritis (Thy-1N), a model of human mesangioproliferative glomerulonephritis (MsPGN), is primarily triggered by sublytic C5b-9. However, the mechanism of GMC apoptosis induced by sublytic C5b-9 remains unclear. In this study, we demonstrate that expressions of TNFR1-associated death domain-containing protein (TRADD) and IFN regulatory factor-1 (IRF-1) were simultaneously upregulated in the renal tissue of Thy-1N rats (in vivo) and in GMCs under sublytic C5b-9 stimulation (in vitro). In vitro, TRADD was confirmed to be a downstream gene of IRF-1, because IRF-1 could bind to TRADD gene promoter to promote its transcription, leading to caspase 8 activation and GMC apoptosis. Increased phosphorylation of p38 MAPK was verified to contribute to IRF-1 and TRADD production and caspase 8 activation, as well as to GMC apoptosis induced by sublytic C5b-9. Furthermore, phosphorylation of MEK kinase 2 (MEKK2) mediated p38 MAPK activation. More importantly, three sites (Ser153/164/239) of MEKK2 phosphorylation were identified and demonstrated to be necessary for p38 MAPK activation. In addition, silencing of renal MEKK2, IRF-1, and TRADD genes or inhibition of p38 MAPK activation in vivo had obvious inhibitory effects on GMC apoptosis, secondary proliferation, and urinary protein secretion in rats with Thy-1N. Collectively, these findings indicate that the cascade axis of MEKK2-p38 MAPK-IRF-1-TRADD-caspase 8 may play an important role in GMC apoptosis following exposure to sublytic C5b-9 in rat Thy-1N.

Interferon regulatory factor 1 attenuates vascular remodeling; roles of angiotensin II type 2 receptor.

We previously reported interferon regulatory factor (IRF) 1 plays physiological roles in "growth"-regulated angiotensin II type 2 (AT2) receptor expression in fibroblasts. Here, we investigated whether IRF-1 is involved in attenuation of vascular remodeling in association with AT2 receptor upregulation. Neointimal area in injured artery after 14 days of cuff placement was significantly increased in IRF-1 knockout mice (IRF-1KO) and AT2 receptor knockout mice (AT2KO) compared with wild-type mice (WT: C57BL/6J). Treatment with compound 21 attenuated neointima formation in both WT and IRF-1KO. AT2 receptor mRNA expression after 7 days of cuff placement was significantly decreased in IRF-1KO compared with WT; however, IRF-1 expression did not differ between AT2KO and WT. Apoptotic changes in injured artery after 14 days of cuff placement were significantly attenuated in IRF-1KO, with a decrease in interleukin-1ß-converting enzyme and inducible nitric oxide synthase mRNA levels. These results indicate IRF-1 is one of the key transcriptional factors for the prevention of neointimal formation involving AT2 receptors.

Gene Regulatory Network Inference of Immunoresponsive Gene 1 (IRG1) Identifies Interferon Regulatory Factor 1 (IRF1) as Its Transcriptional Regulator in Mammalian Macrophages.

Immunoresponsive gene 1 (IRG1) is one of the highest induced genes in macrophages under pro-inflammatory conditions. Its function has been recently described: it codes for immune-responsive gene 1 protein/cis-aconitic acid decarboxylase (IRG1/CAD), an enzyme catalysing the production of itaconic acid from cis-aconitic acid, a tricarboxylic acid (TCA) cycle intermediate. Itaconic acid possesses specific antimicrobial properties inhibiting isocitrate lyase, the first enzyme of the glyoxylate shunt, an anaplerotic pathway that bypasses the TCA cycle and enables bacteria to survive on limited carbon conditions. To elucidate the mechanisms underlying itaconic acid production through IRG1 induction in macrophages, we examined the transcriptional regulation of IRG1. To this end, we studied IRG1 expression in human immune cells under different inflammatory stimuli, such as TNFα and IFNγ, in addition to lipopolysaccharides. Under these conditions, as previously shown in mouse macrophages, IRG1/CAD accumulates in mitochondria. Furthermore, using literature information and transcription factor prediction models, we re-constructed raw gene regulatory networks (GRNs) for IRG1 in mouse and human macrophages. We further implemented a contextualization algorithm that relies on genome-wide gene expression data to infer putative cell type-specific gene regulatory interactions in mouse and human macrophages, which allowed us to predict potential transcriptional regulators of IRG1. Among the computationally identified regulators, siRNA-mediated gene silencing of interferon regulatory factor 1 (IRF1) in macrophages significantly decreased the expression of IRG1/CAD at the gene and protein level, which correlated with a reduced production of itaconic acid. Using a synergistic approach of both computational and experimental methods, we here shed more light on the transcriptional machinery of IRG1 expression and could pave the way to therapeutic approaches targeting itaconic acid levels.

Transcription factor IRF1 is responsible for IRF8-mediated IL-1ß expression in reactive microglia.

Interferon regulatory factor-8 (IRF8) plays a crucial role in the transformation of microglia to a reactive state by regulating the expression of various genes. In the present study, we show that IRF1 is required for IRF8-induced gene expression in microglia. Peripheral nerve injury induced IRF1 gene upregulation in the spinal microglia in an IRF8-dependent manner. IRF8 transduction in cultured microglia induced de novo gene expression of IRF1. Importantly, knockdown of the IRF1 gene in IRF8-transduced microglia prevented upregulation of interleukin-1ß (IL-1ß). Therefore, our findings suggest that expression of IL-1ß is dependent on IRF1 in IRF8-expressing reactive microglia.

Interferon regulatory factor-1 signaling regulates the switch between autophagy and apoptosis to determine breast cancer cell fate.

Interferon regulatory factor-1 (IRF1) is a tumor suppressor that regulates cell fate in several cell types. Here, we report an inverse correlation in expression of nuclear IRF1 and the autophagy regulator ATG7 in human breast cancer cells that directly affects their cell fate. In mice harboring mutant Atg7, nuclear IRF1 was increased in mammary tumors, spleen, and kidney. Mechanistic investigations identified ATG7 and the cell death modulator beclin-1 (BECN1) as negative regulators of IRF1. Silencing ATG7 or BECN1 caused estrogen receptor-α to exit the nucleus at the time when IRF1 nuclear localization occurred. Conversely, silencing IRF1 promoted autophagy by increasing BECN1 and blunting IGF1 receptor and mTOR survival signaling. Loss of IRF1 promoted resistance to antiestrogens, whereas combined silencing of ATG7 and IRF1 restored sensitivity to these agents. Using a mathematical model to prompt signaling hypotheses, we developed evidence that ATG7 silencing could resensitize IRF1-attenuated cells to apoptosis through mechanisms that involve other estrogen-regulated genes. Overall, our work shows how inhibiting the autophagy proteins ATG7 and BECN1 can regulate IRF1-dependent and -independent signaling pathways in ways that engender a new therapeutic strategy to attack breast cancer.

Oncogenic Ras inhibits IRF1 to promote viral oncolysis.

Oncolytic viruses exploit common molecular changes in cancer cells, which are not present in normal cells, to target and kill cancer cells. Ras transformation and defects in type I interferon (IFN)-mediated antiviral responses are known to be the major mechanisms underlying viral oncolysis. Previously, we demonstrated that oncogenic RAS/Mitogen-activated protein kinase kinase (Ras/MEK) activation suppresses the transcription of many IFN-inducible genes in human cancer cells, suggesting that Ras transformation underlies type I IFN defects in cancer cells. Here, we investigated how Ras/MEK downregulates IFN-induced transcription. By conducting promoter deletion analysis of IFN-inducible genes, namely guanylate-binding protein 2 and IFN gamma inducible protein 47 (Ifi47), we identified the IFN regulatory factor 1 (IRF1) binding site as the promoter region responsible for the regulation of transcription by MEK. MEK inhibition promoted transcription of the IFN-inducible genes in wild type mouse embryonic fibroblasts (MEFs), but not in IRF1(-/-) MEFs, showing that IRF1 is involved in MEK-mediated downregulation of IFN-inducible genes. Furthermore, IRF1 protein expression was lower in RasV12 cells compared with vector control NIH3T3 cells, but was restored to equivalent levels by inhibition of MEK. Similarly, the restoration of IRF1 expression by MEK inhibition was observed in human cancer cells. IRF1 re-expression in human cancer cells caused cells to become resistant to infection by the oncolytic vesicular stomatitis virus strain. Together, this work demonstrates that Ras/MEK activation in cancer cells downregulates transcription of IFN-inducible genes by targeting IRF1 expression, resulting in increased susceptibility to viral oncolysis.

MiR-23a facilitates the replication of HSV-1 through the suppression of interferon regulatory factor 1.

MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate gene expression. It has been reported that miRNAs are involved in host-virus interaction, but evidence that cellular miRNAs promote virus replication has been limited. Here, we found that miR-23a promoted the replication of human herpes simplex virus type 1 (HSV-1) in HeLa cells, as demonstrated by a plaque-formation assay and quantitative real-time PCR. Furthermore, interferon regulatory factor 1 (IRF1), an innate antiviral molecule, is targeted by miR-23a to facilitate viral replication. MiR-23a binds to the 3'UTR of IRF1 and down-regulates its expression. Suppression of IRF1 expression reduced RSAD2 gene expression, augmenting HSV-1 replication. Ectopic expression of IRF1 abrogated the promotion of HSV-1 replication induced by miR-23a. Notably, IRF1 contributes to innate antiviral immunity by binding to IRF-response elements to regulate the expression of interferon-stimulated genes (ISGs) and apoptosis, revealing a complex interaction between miR-23a and HSV-1. MiR-23a thus contributes to HSV-1 replication through the regulation of the IRF1-mediated antiviral signal pathway, which suggests that miR-23a may represent a promising target for antiviral treatments.

Interferon regulatory factor 1 is an independent predictor of platinum resistance and survival in high-grade serous ovarian carcinoma.

OBJECTIVE: High-grade serous ovarian cancer (HGSOC) that is resistant to platinum-based chemotherapy has a particularly poor prognosis. Response to platinum has both prognostic survival value and dictates secondary treatment strategies. Using transcriptome analysis, we sought to identify differentially expressed genes/pathways based on a tumor's platinum response for discovering novel predictive biomarkers. METHODS: Seven primary HGSOC tumor samples, representing two extremes of platinum sensitivity/timing of disease recurrence, were analyzed by RNA-Seq, Ingenuity Pathways Analysis (IPA) and Upstream Regulator Analysis (URA), and used to explore differentially expressed genes and prevalent molecular and cellular processes. Progression-free and overall survival (PFS, OS) was estimated using the Kaplan-Meier method in two different sample sets including GEO and TCGA data sets. RESULTS: IPA and URA highlighted an IRF1-driven transcriptional program (P=0.0017; z-score of 3.091) in the platinum sensitive improved PFS group. QRT-PCR analysis of 31 HGSOC samples demonstrated a significant difference in PFS between low and high IRF1 expression groups (P=0.048) and between groups that were platinum sensitive versus not (P=0.016). In a larger validation data set, increased levels of IRF1 were associated with both increased PFS (P=0.043) and OS (P=0.019) and the effect on OS was independent of debulking status (optimal debulking, P=0.025; suboptimal, P=0.041). CONCLUSION: Transcriptome analysis identifies IRF1, a transcription factor that functions both in immune regulation and as a tumor suppressor, as being associated with platinum sensitivity and an independent predictor of both PFS and OS in HGSOC.

Osteopontin enhances the expression of HOTAIR in cancer cells via IRF1.

Osteopontin (OPN), a secreted phosphoglycoprotein, plays important roles in tumor growth, invasion, and metastasis for many types of cancers. The long, noncoding RNA HOTAIR has been strongly associated with the invasion and metastasis of cancer cells. In this study, we found that recombinant human OPN could induce HOTAIR expression in a time- and dose-dependent manner, and our data also showed that OPN transcriptionally activated the expression of HOTAIR in cancer cells. Furthermore, through chromatin immunoprecipitation and luciferase activity assays, we found that IRF1 could bind to the HOTAIR promoter region and decrease its transcriptional activity, and cellular overexpression of IRF1 downregulated the level of HOTAIR. The receptor CD44 has also been verified as a regulator of OPN-induced HOTAIR expression. Interestingly, our data demonstrated that OPN could regulate PI3K/AKT and IRF1 expression and signaling, thereby influencing the expression of HOTAIR. In hepatocellular carcinoma samples, levels of HOTAIR correlated with the expression of OPN and IRF1. We therefore conclude that OPN, as an extracellular matrix protein, can stimulate the expression of HOTAIR by attenuating the inhibitory effect of IRF1, and this results in promotion of the invasion and metastasis of cancer cells.

A novel role for interferon regulatory factor 1 (IRF1) in regulation of bone metabolism.

Increased risk of bone fractures is observed in patients with chronic inflammatory conditions, such as inflammatory bowel disease and rheumatoid arthritis. Members of the Interferon Response Factor family of transcriptional regulators, IRF1 and IRF8, have been identified as genetic risk factors for several chronic inflammatory and autoimmune diseases. We have investigated a potential role for the Irf1 gene in bone metabolism. Here, we report that Irf1(-/-) mutant mice show altered bone morphology in association with altered trabecular bone architecture and increased cortical thickness and cellularity. Ex vivo studies on cells derived from bone marrow stimulated with Rank ligand revealed an increase in size and resorptive activity of tartrate-resistant acid-positive cells from Irf1(-/-) mutant mice compared with wild-type control mice. Irf1 deficiency was also associated with decreased proliferation of bone marrow-derived osteoblast precursors ex vivo, concomitant with increased mineralization activity compared with control cells. We show that Irf1 plays a role in bone metabolism and suggest that Irf1 regulates the maturation and activity of osteoclasts and osteoblasts. The altered bone phenotype of Irf1(-/-) mutants is strikingly similar to that of Stat1(-/-) mice, suggesting that the two interacting proteins play a critical enabling role in the common regulation of these two cell lineages.

EGFR activation suppresses respiratory virus-induced IRF1-dependent CXCL10 production.

Airway epithelial cells are the primary cell type involved in respiratory viral infection. Upon infection, airway epithelium plays a critical role in host defense against viral infection by contributing to innate and adaptive immune responses. Influenza A virus, rhinovirus, and respiratory syncytial virus (RSV) represent a broad range of human viral pathogens that cause viral pneumonia and induce exacerbations of asthma and chronic obstructive pulmonary disease. These respiratory viruses induce airway epithelial production of IL-8, which involves epidermal growth factor receptor (EGFR) activation. EGFR activation involves an integrated signaling pathway that includes NADPH oxidase activation of metalloproteinase, and EGFR proligand release that activates EGFR. Because respiratory viruses have been shown to activate EGFR via this signaling pathway in airway epithelium, we investigated the effect of virus-induced EGFR activation on airway epithelial antiviral responses. CXCL10, a chemokine produced by airway epithelial cells in response to respiratory viral infection, contributes to the recruitment of lymphocytes to target and kill virus-infected cells. While respiratory viruses activate EGFR, the interaction between CXCL10 and EGFR signaling pathways is unclear, and the potential for EGFR signaling to suppress CXCL10 has not been explored. Here, we report that respiratory virus-induced EGFR activation suppresses CXCL10 production. We found that influenza virus-, rhinovirus-, and RSV-induced EGFR activation suppressed IFN regulatory factor (IRF) 1-dependent CXCL10 production. In addition, inhibition of EGFR during viral infection augmented IRF1 and CXCL10. These findings describe a novel mechanism that viruses use to suppress endogenous antiviral defenses, and provide potential targets for future therapies.

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

BACKGROUND: IL-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 CD8 T cells. METHODS: We analysed the hepatic levels of IL-7, IL-15, IL-15Rα and interferon regulatory factors (IRF) in patients with chronic hepatitis C (CHC) (78% genotype 1) and the role of IRF1 and IRF2 on IL-7 and IL-15Rα expression in Huh7 cells with or without hepatitis C virus (HCV) replicon. RESULTS: Hepatic expression of both IL-7 and IL-15Rα, but not of IL-15, was reduced in CHC. These patients exhibited decreased hepatic IRF2 messenger RNA levels and diminished IRF2 staining in hepatocyte nuclei. We found that IRF2 controls basal expression of both IL-7 and IL-15Rα in Huh7 cells. IRF2, but not IRF1, is downregulated in cells with HCV genotype 1b replicon and this was accompanied by decreased expression of IL-7 and IL-15Rα, a defect reversed by overexpressing IRF2. Treating Huh7 cells with IFNα plus oncostatin M increased IL-7 and IL-15Rα mRNA more intensely than either cytokine alone. This effect was mediated by strong upregulation of IRF1 triggered by the combined treatment. Induction of IRF1, IL-7 and IL-15Rα by IFNα plus oncostatin M was dampened in replicon cells but the combination was more effective than either cytokine alone. CONCLUSIONS: HCV genotype 1 infection downregulates IRF2 in hepatocytes attenuating hepatocellular expression of IL-7 and IL-15Rα. Our data reveal a new mechanism by which HCV abrogates specific T-cell responses and point to a novel therapeutic approach to stimulate anti-HCV immunity.

JUNB/AP-1 controls IFN-γ during inflammatory liver disease.

Understanding the molecular pathogenesis of inflammatory liver disease is essential to design efficient therapeutic approaches. In hepatocytes, the dimeric transcription factor c-JUN/AP-1 is a major mediator of cell survival during hepatitis, although functions for other JUN proteins in liver disease are less defined. Here, we found that JUNB was specifically expressed in human and murine immune cells during acute liver injury. We analyzed the molecular function of JUNB in experimental models of hepatitis, including administration of concanavalin A (ConA) or α-galactosyl-ceramide, which induce liver inflammation and injury. Mice specifically lacking JUNB in hepatocytes displayed a mild increase in ConA-induced liver damage. However, targeted deletion of Junb in immune cells and hepatocytes protected against hepatitis in experimental models that involved NK/NKT cells. The absence of JUNB in immune cells decreased IFN-γ expression and secretion from NK and NKT cells, leading to reduced STAT1 pathway activation. Systemic IFN-γ treatment or adenovirus-based IRF1 delivery to Junb-deficient mice restored hepatotoxicity, and we demonstrate that Ifng is a direct transcriptional target of JUNB. These findings demonstrate that JUNB/AP-1 promotes cell death during acute hepatitis by regulating IFN-γ production in NK and NKT cells and thus functionally antagonizes the hepatoprotective function of c-JUN/AP-1 in hepatocytes.

Interferon regulatory factor-1 in flagellin-induced reprogramming: potential protective role of CXCL10 in cornea innate defense against Pseudomonas aeruginosa infection.

PURPOSE: We previously showed that pre-exposure of the cornea to Toll-like receptor (TLR)5 ligand flagellin induces strong protective innate defense against microbial pathogens and hypothesized that flagellin modulates gene expression at the transcriptional levels. Thus, we sought to determine the role of one transcription factor, interferon regulatory factor (IRF1), and its target gene CXCL10 therein. METHODS: Superarray was used to identify transcription factors differentially expressed in Pseudomonas aeruginosa-challenged human corneal epithelial cells (CECs) with or without flagellin pretreatment. The expression of CXCL10, IRF1, LI-8(CXCL2), and IFNγ was determined by PCR, immunohistochemistry, Western/dot blotting, and/or ELISA. IRF1 knockout mice, CXCL10 and IFNγ neutralization, and NK cell depletion were used to define in vivo regulation and function of CXCL10. The severity of P. aeruginosa was assessed using clinical scoring, slit-lamp microscopy, bacterial counting, polymorphonuclear leukocytes (PMN) infiltration, and macrophage inflammatory protein 2/Chemokine (C-X-C motif) ligand 2 (MIP-2/CXCL2) expression. RESULTS: Flagellin pretreatment drastically affected P. aeruginosa-induced IRF1 expression in human CECs. However, flagellin pretreatment augmented the P. aeruginosa-induced expression of Irf1 and its target gene Cxcl10 in B6 mouse corneas. Irf1 deficiency reduced infection-triggered CXCL10 expression, increased keratitis severity, and attenuated flagellin-elicited protection compared to values in wild-type (WT) controls. CXCL10 neutralization in the cornea of WT mice displayed pathogenesis similar to that of IRF1⁻/⁻ mice. IFNγ receptor neutralization and NK cell depletion prevented flagellin-augmented IRF1 and CXCL10 expression and increased the susceptibility to P. aeruginosa infection in mouse corneas. CONCLUSIONS: IRF1 plays a role in the corneal innate immune response by regulating CXCL10 expression. IFNγ-producing NK cells augment the epithelial expression of IRF1 and CXCL10 and thus contribute to the innate defense of the cornea against P. aeruginosa infection.