Myeloid neddylation targets IRF7 and promotes host innate immunity against RNA viruses.

Neddylation, an important type of post-translational modification, has been implicated in innate and adapted immunity. But the role of neddylation in innate immune response against RNA viruses remains elusive. Here we report that neddylation promotes RNA virus-induced type I IFN production, especially IFN-α. More importantly, myeloid deficiency of UBA3 or NEDD8 renders mice less resistant to RNA virus infection. Neddylation is essential for RNA virus-triggered activation of Ifna gene promoters. Further exploration has revealed that mammalian IRF7undergoes neddylation, which is enhanced after RNA virus infection. Even though neddylation blockade does not hinder RNA virus-triggered IRF7 expression, IRF7 mutant defective in neddylation exhibits reduced ability to activate Ifna gene promoters. Neddylation blockade impedes RNA virus-induced IRF7 nuclear translocation without hindering its phosphorylation and dimerization with IRF3. By contrast, IRF7 mutant defective in neddylation shows enhanced dimerization with IRF5, an Ifna repressor when interacting with IRF7. In conclusion, our data demonstrate that myeloid neddylation contributes to host anti-viral innate immunity through targeting IRF7 and promoting its transcriptional activity.

Involvement of Interferon Regulatory Factor 7 in Nicotine's Suppression of Antiviral Immune Responses.

Nicotine, the active ingredient in tobacco smoke, suppresses antiviral responses. Interferon regulatory factors (IRFs) regulate transcription of type I interferons (IFNs) and IFN-stimulated genes (ISGs) in this response. IRF7 is a key member of the IRF family. Expression of Irf7 is elevated in the brains of virus-infected animals, including human immunodeficiency virus-1 transgenic (HIV-1Tg) rats. We hypothesized that IRF7 affects nicotine's modulation of antiviral responses. Using CRISPR/Cas9 system, IRF7-mutant cell lines were created from human embryonic kidney 293FT cells in which 16 nicotinic acetylcholine receptors (nAChRs) were detected. Decreased expression of IRF7 was confirmed at both the mRNA and protein levels, as was IRF7-regulated cell growth in two IRF7-mutant cell lines, designated IRF7-Δ7 and IRF7-Δ11. In IRF7-Δ7 cells, expression of two nAChR genes, CHRNA3 and CHRNA9, changed modestly. After stimulation with polyinosinic-polycytidylic acid (poly I:C) (0.25 µg/ml) for 4 h to mimic viral infection, 293FT wild-type (WT) and IRF7-Δ7 cells were treated with 0, 1, or 100 µM nicotine for 24 h, which increased IFN-ß expression in both types of cells but elevation was higher in WT cells (p < 0.001). Expression was significantly suppressed in WT cells (p < 0.001) but not in IRF7-Δ7 cells by 24-h nicotine exposure. Poly I:C stimulation increased mRNA expression of retinoic-acid-inducible protein I (RIG-I), melanoma-differentiation-associated gene 5 (MDA5), IFN-stimulated gene factor 3 (ISG3) complex, and IFN-stimulated genes (IRF7, ISG15, IFIT1, OAS1); nicotine attenuated mRNA expression only in WT cells. Overall, IRF7 is critical to nicotine's effect on the antiviral immune response. Graphical Abstract Involvement of IRF7 in nicotine's suppression of poly I:C-induced antiviral immune responses. PAMPs, such as a synthetic viral analogue of dsRNA poly I:C attack cells, will be recognized by PRRs, and the host innate immunity against viral infection will be activated. PRRs signaling trigger phosphorylation of IRF7 and IRF3 to induce their translocation to the nucleus and result in the production of type I IFNs. Then IFNs bind to IFNAR to activate the transcription factor ISGF3, a complex consisting of STAT1, STAT2, and IRF9. Further, it induces the expression of ISGs, including IFIT1, OAS1, IRF7, ISG15, etc. Nicotine suppresses the immune responses stimulated by poly I:C. In the IRF7-mutant cells, nicotine's suppressive effects on poly I:C-stimulated immune responses were restrained.

Central Role of the NF-κB Pathway in the Scgb1a1-Expressing Epithelium in Mediating Respiratory Syncytial Virus-Induced Airway Inflammation.

Lower respiratory tract infection with respiratory syncytial virus (RSV) produces profound inflammation. Despite an understanding of the role of adaptive immunity in RSV infection, the identity of the major sentinel cells initially triggering inflammation is controversial. Here we evaluate the role of nonciliated secretoglobin (Scgb1a1)-expressing bronchiolar epithelial cells in RSV infection. Mice expressing a tamoxifen (TMX)-inducible Cre recombinase-estrogen receptor fusion protein (CreERTM) knocked into the Scgb1a1 locus were crossed with mice that harbor a RelA conditional allele (RelAfl ), with loxP sites flanking exons 5 to 8 of the Rel homology domain. The Scgb1a1CreERTM/+ × RelAfl/fl mouse is a RelA conditional knockout (RelACKO) of a nonciliated epithelial cell population enriched in the small bronchioles. TMX-treated RelACKO mice have reduced pulmonary neutrophilic infiltration and impaired expression and secretion of NF-κB-dependent cytokines in response to RSV. In addition, RelACKO mice had reduced expression levels of interferon (IFN) regulatory factor 1/7 (IRF1/7) and retinoic acid-inducible gene I (RIG-I), components of the mucosal IFN positive-feedback loop. We demonstrate that RSV replication induces RelA to complex with bromodomain-containing protein 4 (BRD4), a cofactor required for RNA polymerase II (Pol II) phosphorylation, activating the atypical histone acetyltransferase (HAT) activity of BRD4 required for phospho-Ser2 Pol II formation, histone H3K122 acetylation, and cytokine secretion in vitro and in vivo TMX-treated RelACKO mice have less weight loss and reduced airway obstruction/hyperreactivity yet similar levels of IFN-γ production despite higher levels of virus production. These data indicate that the nonciliated Scgb1a1-expressing epithelium is a major innate sensor for restricting RSV infection by mediating neutrophilic inflammation and chemokine and mucosal IFN production via the RelA-BRD4 pathway.IMPORTANCE RSV infection is the most common cause of infant hospitalizations in the United States, resulting in 2.1 million children annually requiring medical attention. RSV primarily infects nasal epithelial cells, spreading distally to produce severe lower respiratory tract infections. Our study examines the role of a nonciliated respiratory epithelial cell population in RSV infection. We genetically engineered a mouse that can be selectively depleted of the NF-κB/RelA transcription factor in this subset of epithelial cells. These mice show an impaired activation of the bromodomain-containing protein 4 (BRD4) coactivator, resulting in reduced cytokine expression and neutrophilic inflammation. During the course of RSV infection, epithelial RelA-depleted mice have reduced disease scores and airway hyperreactivity yet increased levels of virus replication. We conclude that RelA-BRD4 signaling in nonciliated bronchiolar epithelial cells mediates neutrophilic airway inflammation and disease severity. This complex is an attractive target to reduce the severity of infection.

Expression of interferon regulatory factor 7 correlates with the expression of Epstein-Barr Virus latent membrane protein 1 and cervical lymph node metastasis in nasopharyngeal cancer.

Interferon regulatory factor 7 (IRF7) has oncogenic properties in several malignancies such as Epstein-Barr virus (EBV)-associated lymphoma. However, there is no evidence whether IRF7 is associated with the oncogenesis of nasopharyngeal cancer (NPC), the pathogenesis of which is closely associated with EBV. Herein, we report that expression of IRF7 was increased in normal nasopharyngeal cells that expressed the EBV principal oncoprotein, latent membrane protein 1 (LMP1). In addition, IRF7 was mainly expressed in the nucleus in both normal nasopharyngeal cells and nasopharyngeal cancer cells that expresses LMP1. On immunohistochemical analysis, IRF7 was predominantly localized in the nucleus in biopsy samples of NPC tissues. In total, IRF7 expression was detected with 36 of 49 specimens of these tissues. Furthermore, the expression score of IRF7 correlated with the expression score of LMP1. Moreover, the expression score of IRF7 is associated with cervical lymph-node metastasis, which reflects the highly metastatic nature of this cancer. Taken together, our results suggest that expression of IRF7 is one of the metastatic effectors of LMP1 signalling in EBV-associated NPC.

Restoring PU.1 induces apoptosis and modulates viral transactivation via interferon-stimulated genes in primary effusion lymphoma.

Primary effusion lymphoma (PEL), which is an aggressive subgroup of B-cell lymphoma associated with Kaposi sarcoma-associated herpes virus/human herpes virus-8, is refractory to the standard treatment, and exhibits a poor survival. Although PU.1 is downregulated in PEL, the potential role of its reduction remains to be elucidated. In this investigation, we analyzed the DNA methylation of PU.1 cis-regulatory elements in PEL and the effect of restoring PU.1 on PEL cells. The mRNA level of PU.1 was downregulated in PEL cells. The methylated promoter and enhancer regions of the PU.1 gene were detected in PEL cells. Suppression of cell growth and apoptosis were caused by the restoration of PU.1 in PEL cells. A microarray analysis revealed that interferon-stimulated genes (ISGs) including pro-apoptotic ISGs were strongly increased in BCBL-1 cells after the induction of PU.1. Reporter assays showed that PU.1 transactivated pro-apoptotic ISG promoters, such as the XAF1, OAS1 and TRAIL promoters. Mutations at the PU.1 binding sequences suppressed its transactivation. We confirmed the binding of PU.1 to the XAF1, OAS1 and TRAIL promoters in a chromatin immunoprecipitation assay. PU.1 suppressed ORF57 activation by inducing IRF7. The reinduction of PU.1 reduced formation of ascites and lymphoma cell infiltration of distant organs in PEL xenograft model mice. Collectively, PU.1 has a role in tumor suppression in PEL and its down-regulation is associated with PEL development. Restoring PU.1 with demethylation agents may be a novel therapeutic approach for PEL.

Prevention of TLR9 Pathway in Warm Ischemia in Porcine Donor Liver after Cardiac Death.

OBJECTIVE: To investigate effect of warm ischemia after cardiac death on activation of TLR9 pathway in porcine liver. METHODS: Donor of cardiac death (DCD) model was established with Duroc, Landrace, Large White crossbred pigs. Liver tissues from the animals were harvested at 0, 5, 10, 15, 25 and 30 minutes after warm ischemia for analysis of expression of TLR9, IRF7, IFN-ß, and TNF-α at mRNA and protein levels by real-time PCR and western blot, respectively, and for assessment of NF-κB/DNA binding activity by western blot detection of p65 protein. RESULTS: Ischemia induced TLR9, IRF7, IFN-ß, and TNF-α expression at both mRNA and protein levels in an ischemic time dependent manner. Among them, expression of TNF-α and IFN- ß was induced later than TLR9 and IRF7 did. Ischemia also enhanced NF-κB binding to DNA in the DCD liver tissue. Pretreatment with iCpG specifically blocked activation of TLR9 pathway triggered by ischemia in liver and protected the animals from ischemia-caused liver tissue damage. CONCLUSION: Warm ischemia activates TLR9 pathways in the porcine liver tissue. Blocking TLR9 pathway could offer protection from ischemia-caused liver tissue in DCD liver transplantation.

Transcriptional Repression of IFN Regulatory Factor 7 by MYC Is Critical for Type I IFN Production in Human Plasmacytoid Dendritic Cells.

Type I IFNs are crucial mediators of human innate and adaptive immunity and are massively produced from plasmacytoid dendritic cells (pDCs). IFN regulatory factor (IRF)7 is a critical regulator of type I IFN production when pathogens are detected by TLR 7/9 in pDC. However, hyperactivation of pDC can cause life-threatening autoimmune diseases. To avoid the deleterious effects of aberrant pDC activation, tight regulation of IRF7 is required. Nonetheless, the detailed mechanisms of how IRF7 transcription is regulated in pDC are still elusive. MYC is a well-known highly pleiotropic transcription factor; however, the role of MYC in pDC function is not well defined yet. To identify the role of transcription factor MYC in human pDC, we employed a knockdown technique using human pDC cell line, GEN2.2. When we knocked down MYC in the pDC cell line, production of IFN-stimulated genes was dramatically increased and was further enhanced by the TLR9 agonist CpGB. Interestingly, MYC is shown to be recruited to the IRF7 promoter region through interaction with nuclear receptor corepressor 2/histone deacetylase 3 for its repression. In addition, activation of TLR9-mediated NF-κB and MAPK and nuclear translocation of IRF7 were greatly enhanced by MYC depletion. Pharmaceutical inhibition of MYC recovered IRF7 expression, further confirming the negative role of MYC in the antiviral response by pDC. Therefore, our results identify the novel immunomodulatory role of MYC in human pDC and may add to our understanding of aberrant pDC function in cancer and autoimmune disease.

microRNA-762 promotes breast cancer cell proliferation and invasion by targeting IRF7 expression.

OBJECTIVES: miRNAs play crucial roles in human tumourigenesis. This study was performed to measure expression and function of miR-762 in breast cancer. MATERIALS AND METHODS: Expression of miR-762 in breast tissues and cell lines (SK-BR-3, DA-MB-435s, MCF-7 and MDA-MB-231, HBL-100) was measured by using real-time RT-PCR. We restored expression of miR-762 in MCF-7 cells to measure its functional roles. Luciferase assays were performed to reveal the target gene of miR-762. RESULTS: Expression of miR-762 was high in both breast cancer cell lines and specimens, and its overexpression increased breast cancer cell proliferation and invasion. Interferon regulatory factor 7 (IRF7) is a direct target of miR-762 and overexpression of miR-762 reduced expression of IRF7. Moreover, IRF7 was repressed, its levels inversely correlated to miR-762 expression. IRF7 rescued miR-762-induced cell invasion and proliferation. CONCLUSIONS: These results demonstrate that miR-762 tumour effect was achieved by targeting IRF7 in human breast cancer specimens.

Role of STAT1 in Chlamydia-Induced Type-1 Interferon Production in Oviduct Epithelial Cells.

We previously reported that Chlamydia muridarum-infected murine oviduct epithelial cells (OE cells) secrete interferon ß (IFN-ß) in a mostly TLR3-dependent manner. However, C. muridarum-infected TLR3-deficient OE cells were still able to secrete detectable levels of IFN-ß into the supernatants, suggesting that other signaling pathways contribute to Chlamydia-induced IFN-ß synthesis in these cells. We investigated the role of STAT1 as a possible contributor in the Chlamydia-induced type-1 IFN production in wild-type (WT) and TLR3-deficient OE cells to ascertain its putative role at early- and late-times during Chlamydia infection. Our data show that C. muridarum infection significantly increased STAT1 gene expression and protein activation in WT OE cells; however, TLR3-deficient OE cells showed diminished STAT1 protein activation and gene expression. There was significantly less IFN-ß detected in the supernatants of C. muridarum-infected OE cells derived from mice deficient in STAT1 when compared with WT OE cells, which suggest that STAT1 is required for the optimal synthesis of IFN-ß during infection. Real-time quantitative polymerase chain reaction analyses of signaling components of the type-1 IFN signaling pathway demonstrated equal upregulation in the expression of STAT2 and IRF7 genes in the WT and TLR3-deficient OE cells, but no upregulation in these genes in the STAT1-deficient OE cells. Finally, experiments in which INFAR1 was blocked with neutralizing antibody revealed that IFNAR1-mediated signaling was critical to the Chlamydia-induced upregulation in IFN-α gene transcription, but had no role in the Chlamydia-induced upregulation in IFN-ß gene transcription.

IRF7 promotes glioma cell invasion by inhibiting AGO2 expression.

Interferon regulatory factor 7 (IRF7) is the master transcription factor that plays a pivotal role in the transcriptional activation of type I interferon genes in the inflammatory response. Our previous study revealed that IRF7 is an important regulator of tumor progression via the expression of inflammatory cytokines in glioma. Here, we report that IRF7 promotes glioma invasion and confers resistance to both chemotherapy and radiotherapy by inhibiting expression of argonaute 2 (AGO2), a regulator of microRNA biogenesis. We found that IRF7 and AGO2 expression levels were negatively correlated in patients with glioblastoma multiforme. Ectopic IRF7 expression led to a reduction in AGO2 expression, while depletion of IRF7 resulted in increased AGO2 expression in the LN-229 glioma cell line. In an in vitro invasion assay, IRF7 overexpression enhanced glioma cell invasion. Furthermore, reconstitution of AGO2 expression in IRF7-overexpressing cells led to decreased cell invasion, whereas the reduced invasion due to IRF7 depletion was rescued by AGO2 depletion. In addition, IRF7 induced chemoresistance and radioresistance of glioma cells by diminishing AGO2 expression. Finally, AGO2 depletion alone was sufficient to accelerate glioma cell invasion in vitro and in vivo, indicating that AGO2 regulates cancer cell invasion. Taken together, our results indicate that IRF7 promotes glioma cell invasion and both chemoresistance and radioresistance through AGO2 inhibition.

Differentiation of neurons restricts Arbovirus replication and increases expression of the alpha isoform of IRF-7.

UNLABELLED: Susceptibility to alphavirus infection is age dependent, and host maturation is associated with decreased virus replication and less severe encephalitis. To identify factors associated with maturation-dependent restriction of virus replication, we studied AP-7 rat olfactory bulb neuronal cells, which can differentiate in vitro. Differentiation was associated with a 150- to 1,000-fold decrease in replication of the alphaviruses Sindbis virus and Venezuelan equine encephalitis virus, as well as La Crosse bunyavirus. Differentiation delayed synthesis of SINV RNA and protein but did not alter the susceptibility of neurons to infection or virion maturation. Additionally, differentiation slowed virus-induced translation arrest and death of infected cells. Differentiation of uninfected AP-7 neurons was associated with changes in expression of antiviral genes. Expression of key transcription factors was increased, including interferon regulatory factor 3 and 7 (IRF-3 and IRF-7) and STAT-1, suggesting that neuronal maturation may enhance the capacity for antiviral signaling upon infection. IRF-7 produced by undifferentiated AP-7 neurons was exclusively the short dominant negative γ-isoform, while that produced by differentiated neurons was the full-length α-isoform. A similar switch in IRF-7 isoforms also occurred in the brains of maturing C57BL/6J mice. Silencing of IRF expression did not improve virus multiplication in differentiated neurons. Therefore, neuronal differentiation is associated with upregulation of transcription factors that activate antiviral signaling, but this alone does not account for maturation-dependent restriction of virus replication. IMPORTANCE: Viral encephalomyelitis is an important cause of age-dependent morbidity and mortality. Because mature neurons are not readily regenerated, recovery from encephalitis suggests that mature neurons utilize unique antiviral mechanisms to block infection and/or clear virus. To identify maturational changes in neurons that may improve outcome, we compared immature and mature cultured neurons for susceptibility to three encephalitic arboviruses and found that replication of Old World and New World alphaviruses and a bunyavirus was reduced in mature compared to immature neurons. Neuronal maturation was associated with increased baseline expression of interferon regulatory factor 3 and 7 mRNAs and production of distinct isoforms of interferon regulatory factor 7 protein. Overall, our studies identified maturational changes in neurons that likely contribute to assembly of immunoregulatory factors prior to infection, a more rapid antiviral response, increased resistance to virus infection, and improved survival.

IRF7 in the Australian black flying fox, Pteropus alecto: evidence for a unique expression pattern and functional conservation.

As the only flying mammal, bats harbor a number of emerging and re-emerging viruses, many of which cause severe diseases in humans and other mammals yet result in no clinical symptoms in bats. As the master regulator of the interferon (IFN)-dependent immune response, IFN regulatory factor 7 (IRF7) plays a central role in innate antiviral immunity. To explore the role of bat IRF7 in the regulation of the IFN response, we performed sequence and functional analysis of IRF7 from the pteropid bat, Pteropus alecto. Our results demonstrate that bat IRF7 retains the ability to bind to MyD88 and activate the IFN response despite unique changes in the MyD88 binding domain. We also demonstrate that bat IRF7 has a unique expression pattern across both immune and non-immune related tissues and is inducible by double-strand RNA. The broad tissue distribution of IRF7 may provide bats with an enhanced ability to rapidly activate the IFN response in a wider range of tissues compared to other mammals. The importance of IRF7 in antiviral activity against the bat reovirus, Pulau virus was confirmed by siRNA knockdown of IRF7 in bat cells resulting in enhanced viral replication. Our results highlight the importance of IRF7 in innate antiviral immunity in bats.

Negative regulation of type I IFN expression by OASL1 permits chronic viral infection and CD8⁺ T-cell exhaustion.

The type I interferons (IFN-Is) are critical not only in early viral control but also in prolonged T-cell immune responses. However, chronic viral infections such as those of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in humans and lymphocytic choriomeningitis virus (LCMV) in mice overcome this early IFN-I barrier and induce viral persistence and exhaustion of T-cell function. Although various T-cell-intrinsic and -extrinsic factors are known to contribute to induction of chronic conditions, the roles of IFN-I negative regulators in chronic viral infections have been largely unexplored. Herein, we explored whether 2'-5' oligoadenylate synthetase-like 1 (OASL1), a recently defined IFN-I negative regulator, plays a key role in the virus-specific T-cell response and viral defense against chronic LCMV. To this end, we infected Oasl1 knockout and wild-type mice with LCMV CL-13 (a chronic virus) and monitored T-cell responses, serum cytokine levels, and viral titers. LCMV CL-13-infected Oasl1 KO mice displayed a sustained level of serum IFN-I, which was primarily produced by splenic plasmacytoid dendritic cells, during the very early phase of infection (2-3 days post-infection). Oasl1 deficiency also led to the accelerated elimination of viremia and induction of a functional antiviral CD8 T-cell response, which critically depended on IFN-I receptor signaling. Together, these results demonstrate that OASL1-mediated negative regulation of IFN-I production at an early phase of infection permits viral persistence and suppresses T-cell function, suggesting that IFN-I negative regulators, including OASL1, could be exciting new targets for preventing chronic viral infection.

Interferon regulatory factor 7 deficiency prevents diet-induced obesity and insulin resistance.

Obesity-related inflammation has been implicated in the pathogenesis of insulin resistance and type 2 diabetes. In this study, we addressed the potential role of interferon regulatory factor 7 (IRF7), a master regulator of type I interferon-dependent immune responses, in the regulation of energy metabolism. The expression levels of IRF7 were increased in white adipose tissue, liver tissue, and gastrocnemius muscle of both diet-induced obese mice and ob/ob mice compared with their lean counterparts. After feeding a high-fat diet (HFD) for 24 wk, IRF7 knockout (KO) mice showed less weight gain and adiposity than wild-type controls. KO of IRF7 improved glucose and lipid homeostasis and insulin sensitivity. Additionally, KO of IRF7 ameliorated diet-induced hepatic steatosis. Next, we assessed the inflammatory state of the IRF7 KO mice on the HFD. These mice showed less macrophage infiltration into multiple organs and were protected from local and systemic inflammation. This study demonstrates a role for IRF7 in diet-induced alterations in energy metabolism and insulin sensitivity. Our results also suggest that IRF7 is involved in the etiology of metabolic abnormalities, which suggests a new strategy for treating obesity and type 2 diabetes.

Breast cancer-derived transforming growth factor-ß and tumor necrosis factor-α compromise interferon-α production by tumor-associated plasmacytoid dendritic cells.

We previously reported that plasmacytoid dendritic cells (pDCs) infiltrating breast tumors are impaired for their interferon-α (IFN-α) production, resulting in local regulatory T cells amplification. We designed our study to decipher molecular mechanisms of such functional defect of tumor-associated pDC (TApDC) in breast cancer. We demonstrate that besides IFN-α, the production by Toll-like receptor (TLR)-activated healthy pDC of IFN-ß and TNF-α but not IP-10/CXCL10 nor MIP1-α/CCL3 is impaired by the breast tumor environment. Importantly, we identified TGF-ß and TNF-α as major soluble factors involved in TApDC functional alteration. Indeed, recombinant TGF-ß1 and TNF-α synergistically blocked IFN-α production of TLR-activated pDC, and neutralization of TGF-ß and TNF-α in tumor-derived supernatants restored pDCs' IFN-α production. The involvment of tumor-derived TGF-ß was further confirmed in situ by the detection of phosphorylated Smad2 in the nuclei of TApDC in breast tumor tissues. Mechanisms of type I IFN inhibition did not involve TLR downregulation but the inhibition of IRF-7 expression and nuclear translocation in pDC after their exposure to tumor-derived supernatants or recombinant TGF-ß1 and TNF-α. Our findings indicate that targeting TApDC to restore their IFN-α production might be an achievable strategy to induce antitumor immunity in breast cancer by combining TLR7/9-based immunotherapy with TGF-ß and TNF-α antagonists.

Silencing of Irf7 pathways in breast cancer cells promotes bone metastasis through immune escape.

Breast cancer metastasis is a key determinant of long-term patient survival. By comparing the transcriptomes of primary and metastatic tumor cells in a mouse model of spontaneous bone metastasis, we found that a substantial number of genes suppressed in bone metastases are targets of the interferon regulatory factor Irf7. Restoration of Irf7 in tumor cells or administration of interferon led to reduced bone metastases and prolonged survival time. In mice deficient in the interferon (IFN) receptor or in natural killer (NK) and CD8(+) T cell responses, metastasis was accelerated, indicating that Irf7-driven suppression of metastasis was reliant on IFN signaling to host immune cells. We confirmed the clinical relevance of these findings in over 800 patients in which high expression of Irf7-regulated genes in primary tumors was associated with prolonged bone metastasis-free survival. This gene signature may identify patients that could benefit from IFN-based therapies. Thus, we have identified an innate immune pathway intrinsic to breast cancer cells, the suppression of which restricts immunosurveillance to enable metastasis.

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

OBJECTIVES: 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. METHODS: 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-ß and IP-10 promoter activity was measured using luciferase reporter constructs. RESULTS: 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-ß 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. CONCLUSION: Based on the selective regulation of IP-10 in FLS, TBK1 appears to be the optimal IKK-related kinase to target in RA.

Beta interferon-mediated activation of signal transducer and activator of transcription protein 1 interferes with Rickettsia conorii replication in human endothelial cells.

Infection of the endothelial cell lining of blood vessels with Rickettsia conorii, the causative agent of Mediterranean spotted fever, results in endothelial activation. We investigated the effects of R. conorii infection on the status of the Janus kinase (JAK)-signal transducer and activator of transcription protein (STAT) signaling pathway in human microvascular endothelial cells (HMECs), the most relevant host cell type, in light of rickettsial tropism for microvascular endothelium in vivo. R. conorii infection induced phosphorylation of STAT1 on tyrosine 701 and serine 727 at 24, 48, and 72 h postinfection in HMECs. Employing transcription profile analysis and neutralizing antibodies, we further determined that beta interferon (IFN-ß) production and secretion are critical for STAT1 activation. Secreted IFN-ß further amplified its own expression via a positive-feedback mechanism, while expression of transcription factors interferon regulatory factor 7 (IRF7) and IRF9, implicated in the IFN-ß-STAT1 feedback loop, was also induced. Metabolic activity of rickettsiae was essential for the IFN-ß-mediated response(s) because tetracycline treatment inhibited R. conorii replication, IFN-ß expression, and STAT1 phosphorylation. Inclusion of IFN-ß-neutralizing antibody during infection resulted in significantly enhanced R. conorii replication, whereas addition of exogenous IFN-ß had the opposite inhibitory effect. Finally, small interfering RNA-mediated knockdown further confirmed a protective role for STAT1 against intracellular R. conorii replication. In concert, these findings implicate an important role for IFN-ß-mediated STAT1 activation in innate immune responses of vascular endothelium to R. conorii infection.

IL-29/IL-28A suppress HSV-1 infection of human NT2-N neurons.

The newly identified cytokines, IL-28/IL-29 (also termed type III IFNs), are able to inhibit a number of viruses. Here, we examined the antiviral effects of IL-29/IL-28A against herpes simplex virus type 1 (HSV-1) in human NT2-N neurons and CHP212 neuronal cells. Both IL-29 and IL-28A could efficiently inhibit HSV-1 replication in neuronal cells, as evidenced by the reduced expression of HSV-1 DNA and proteins. This inhibitory effect of IL-29 and IL-28A against HSV-1 could be partially blocked by antibody to IL-10Rß, one of the key receptors for IL-29 and IL-28A. To explore the underlying antiviral mechanisms employed by IL-29/IL-28A, we showed that IL-29/IL-28A could selectively induce the expression of several Toll-like receptors (TLRs) as well as activate TLR-mediated antiviral pathway, including IFN regulatory factor 7, IFN-α, and the key IFN-α stimulated antiviral genes.

Expression profiles of carp IRF-3/-7 correlate with the up-regulation of RIG-I/MAVS/TRAF3/TBK1, four pivotal molecules in RIG-I signaling pathway.

The cytoplasmic helicase protein RIG-I (retinoic acid-inducible gene I) and downstream signaling molecules, MAVS (mitochondrial antiviral signaling protein), TRAF3 (TNF-receptor-associated factor 3) and TBK1 (TANK-binding kinase 1), have significant roles in the recognition of cytoplasmic 5'-triphosphate ssRNA and short dsRNA, and phosphorylation of IRF-3 (interferon regulatory factor 3) and IRF-7 which is responsible for the induction of type I interferons (IFN). In the present study, the full-length cDNAs of RIG-I, MAVS, TRAF3 and TBK1 were cloned and identified in common carp (Cyprinus carpio L.). The deduced protein of carp RIG-I is of 946 aa (amino acids), consisting of two CARDs (caspase-recruitment domain), a DEXDc (DExD/H box-containing domain), a HELICc (helicase superfamily c-terminal domain) and a RD (regulatory domain). Carp MAVS is of 585 aa, containing a CARD, a proline-rich region and a TM (transmembrane domain). Carp TRAF3 encodes a protein of 573 aa, including a RING (really interesting new gene), two TRAF-type zinc fingers, a coiled coil and a MATH-TRAF3 (meprin and TRAF homology) domain. Carp TBK1 is of 727 aa and contains a S_TKc domain (Serine/Threonine protein kinases, catalytic domain). Carp RIG-I, MAVS, TRAF3 and TBK1 mRNAs are ubiquitously expressed in all tissues examined. In response to SVCV infection, carp RIG-I and MAVS mRNAs were up-regulated at different levels in spleen, head kidney and intestine tissues at different time points. Similarly, both carp IRF-3 and IRF-7 mRNAs were significantly up-regulated in the detected tissues. Especially in intestine, the IRF-3 and IRF-7 mRNAs of carp increased and reached 25.3-fold (at 3 dpi) and 224.7-fold (at 5 dpi). Noteworthily, a significant growth of carp TRAF3 and TBK1 mRNA was also mainly found in intestine (7.0-fold and 11.3-fold at 5 dpi, respectively). These data implied that the expression profiles of IRF-3/-7 mRNAs in carp correlate with the up-regulation of RIG-I/MAVS/TRAF3/TBK, and carp RIG-I and MAVS may be involved in antiviral responses through the RIG-I viral recognition signaling pathway in a TRAF3/TBK1-dependent manner.