Interferon regulatory factor (IRF) genes encode DNA-binding proteins that are involved in the innate immune response to infection. Two of these proteins, IRF-3 and IRF-7, serve as direct transducers of virus-mediated signaling and play critical roles in the induction of type I interferon genes. We have now shown that another factor, IRF-5, participates in the induction of interferon A (IFNA) and IFNB genes and can replace the requirement for IRF-7 in the induction of IFNA genes. We demonstrate that, despite the functional similarity, IRF-5 possesses unique characteristics and does not have a redundant role. Thus, 1) activation of IRF-5 by phosphorylation is virus-specific, and its in vivo association with the IFNA promoter can be detected only in cells infected with NDV, not Sendai virus, while both viruses activate IRF-3 and IRF-7, and 2) NDV infection of IRF-5-overexpressing cells preferentially induced the IFNA8 subtype, while IFNA1 was primarily induced in IRF-7 expressing cells. These data indicate that multiple signaling pathways induced by infection may be differentially recognized by members of the IRF family and modulate transcription of individual IFNA genes in a virus and cell type-specific manner.

Plasmacytoid dendritic cells (PDC) produce high levels of type I IFN upon stimulation with viruses, while monocytes and monocyte-derived dendritic cells (MDDC) produce significantly lower levels. To find what determines the high production of type I IFN in PDC, we examined the relative levels of IRF transcription factors, some of which play critical roles in the induction of IFN. Furthermore, to determine whether the differences could result from expression of distinct IFNA subtypes, the profile of IFNA genes expressed was examined. PDC responded equally well to stimulation with HSV-1 and Sendai virus (SV) by producing high levels of type I IFN, whereas the MDDC and monocyte response to SV were lower, and neither responded well to HSV-1. All three populations constitutively expressed most of the IRF genes. However, real-time RT-PCR demonstrated increased levels of IRF-7 transcripts in PDC compared with monocytes. As determined by intracellular flow cytometry, the PDC constitutively expressed significantly higher levels of IRF-7 protein than the other populations while IRF-3 levels were similar among populations. Analysis of the profile of IFNA genes expressed in virus-stimulated PDC, monocytes and MDDC demonstrated that each population expressed IFNA1 as the major subtype but that the range of the subtypes expressed in PDC was broader, with some donor and stimulus-dependent variability. We conclude that PDC but not MDDC are uniquely preprogrammed to respond rapidly and effectively to a range of viral pathogens with high levels of IFN-alpha production due to the high levels of constitutively expressed IRF-7.

Recent studies implicate the interferon (IFN) regulatory factors (IRF) IRF-3 and IRF-7 as key activators of the alpha/beta IFN (IFN-alpha/beta) genes as well as the RANTES chemokine gene. Using coexpression analysis, the human IFNB, <em>IFNA1</em>, and RANTES promoters were stimulated by IRF-3 coexpression, whereas the IFNA4, IFNA7, and <em>IFNA1</em>4 promoters were preferentially induced by IRF-7 only. Chimeric proteins containing combinations of different IRF-7 and IRF-3 domains were also tested, and the results provided evidence of distinct DNA binding properties of IRF-3 and IRF-7, as well as a preferential association of IRF-3 with the CREB binding protein (CBP) coactivator. Interestingly, some of these fusion proteins led to supraphysiological levels of IFN promoter activation. DNA binding site selection studies demonstrated that IRF-3 and IRF-7 bound to the 5'-GAAANNGAAANN-3' consensus motif found in many virus-inducible genes; however, a single nucleotide substitution in either of the GAAA half-site motifs eliminated IRF-3 binding and transactivation activity but did not affect IRF-7 interaction or transactivation activity. These studies demonstrate that IRF-3 possesses a restricted DNA binding site specificity and interacts with CBP, whereas IRF-7 has a broader DNA binding specificity that contributes to its capacity to stimulate delayed-type IFN gene expression. These results provide an explanation for the differential regulation of IFN-alpha/beta gene expression by IRF-3 and IRF-7 and suggest that these factors have complementary rather than redundant roles in the activation of the IFN-alpha/beta genes.

The use of type I interferons (IFNs) in cancer therapy has been limited by ineffective dosing and significant toxicity. Here, we exploited the tumor-homing ability of proangiogenic Tie2-expressing monocytes (TEMs) to deliver IFN-alpha to tumors. By transplanting hematopoietic progenitors transduced with a Tie2 promoter/enhancer-driven Ifna1 gene, we turned TEMs into IFN-alpha cell vehicles that efficiently targeted the IFN response to orthotopic human gliomas and spontaneous mouse mammary carcinomas and obtained significant antitumor responses and near complete abrogation of metastasis. TEM-mediated IFN-alpha delivery inhibited tumor angiogenesis and activated innate and adaptive immune cells but did not impair myelopoiesis and wound healing detectably. These results illustrate the therapeutic potential of gene- and cell-based IFN-alpha delivery and should allow the development of IFN treatments that more effectively treat cancer.

IRF-7 plays an essential role in virus-activated transcription of IFNA genes. To analyze functional domains of IRF-7 we have constructed an amino-terminal deletion mutant of IRF-7 (237-514) which exerted a dominant negative (DN) effect on virus-induced expression of the endogenous Type I IFN genes. Focusing on the molecular mechanism underlying the dominant negative effect of IRF-7 DN, we found that virus-activated transcription of endogenous IFNA genes requires full-length IRF-7 and that Serine 483 and 484 play an essential role. While IRF-7 DN had no effect on virus-stimulated nuclear translocation of IRF-3 and IRF-7, the binding of IRF-7 DN to IRF-3 and IRF-7 was detected by GST pull-down assay as well as by immunoprecipitation in infected cells, indicating that IRF-7 DN targets both IRF-7 and IRF-3. The region by which IRF-7 interacts with IRF-3 was mapped between amino acid 418 and 473. Overexpression of IRF-7 DN in virus-infected 2FTGH cells resulted in an inhibition of IFN synthesis and in a significant reduction of binding of both IRF-3 and IRF-7 to the IFNA1 promoter. Interestingly, the IRF-7 DN-mediated suppression of IFNA gene expression can be negated by overexpression of IRF-3. Altogether these results suggest that the IRF-3/IRF-7 complexes are biologically active and are involved in virus-activated transcription of endogenous IFNA genes.

Type I interferons constitute an important part of the innate immune response against viral infection. Unlike the expression of interferon (IFN) B gene, the expression of IFNA genes is restricted to the lymphoid cells. Both IFN regulatory factor 3 and 7 (IRF-3 and IRF-7) were suggested to play positive roles in these genes expression. However, their role in the differential expression of individual subtypes of human IFNA genes is unknown. Using various IFNA reporter constructs in transient transfection assay we found that overexpression of IRF-3 in virus infected 2FTGH cells selectively activated IFNA1 VRE, whereas IRF-7 was able to activate IFNA1, A2, and A4. The binding of recombinant IRF-7 and IRF-3 to these VREs correlated with their transcriptional activation. Nuclear proteins from infected and uninfected IRF-7 expressing 2FTGH cells formed multiple DNA-protein complexes with IFNA1 VRE, in which two unique DNA-protein complexes containing IRF-7 were detected. In 2FTGH cells, virus stimulated expression of IFNB gene but none of the IFNA genes. Reconstitution of IRF-7 synthesis in these cells resulted, upon virus infection, in the activation of seven endogenous IFNA genes in which IFNA1 predominated. These studies suggest that IRF-7 is a critical determinant for the induction of IFNA genes in infected cells.

A cluster of 11 interferon (IFN) genes were identified in the Atlantic salmon genome linked to the growth hormone 1 gene. The genes encode three different IFN subtypes; IFNa (two genes), IFNb (four genes) and IFNc (five genes), which show 22-32% amino acid sequence identity. Expression of the fish IFNs were studied in head kidney, leukocytes or TO cells after stimulation with the dsRNA poly I:C or the imidazoquinoline S-27609. In mammals, poly I:C induces IFN-beta through the RIG-I/MDA5 or the TLR3 pathway, both of which are dependent on NF-kB. In contrast, S-27609 induces mammalian IFN-alpha in plasmacytoid dendritic cells through the TLR7 pathway independent of NF-kappaB. The presence of an NF-kappaB site in their promoters and their strong up-regulation by poly I:C, suggest that salmon IFNa1/IFNa2 are induced through similar pathways as IFN-beta. In contrast, the apparent lack of NF-kappaB motif in the promoter and the strong upregulation by S-27609 in head kidney and leukocytes, suggest that IFNb genes are induced through a pathway similar to mammalian IFN-alpha. IFNc genes showed expression patterns different from both IFNa and IFNb. Taken together, salmon IFNa and IFNb are not orthologs of mammalian IFN-beta and IFN-alpha, respectively, but appear to utilize similar induction pathways.

The imidazoquinolineamine derivative 1-(2-methyl propyl)-1H-imidazole [4,5-c]quinoline-4-amine (imiquimod) has been shown to induce alpha interferon (IFN-alpha) synthesis both in vivo and in peripheral blood mononuclear cells in vitro. In this study, we show that, in these cells, imiquimod induces expression of several IFNA genes (IFNA1, IFNA2, IFNA5, IFNA6, and IFNA8) as well as the IFNB gene. Imiquimod also induced the expression of interleukin (IL)-6, IL-8, and tumor necrosis factor alpha genes. Expression of all these genes was transient, independent of cellular protein synthesis, and inhibited in the presence of tyrosine kinase and protein kinase C inhibitors. Infection with Sendai virus led to expression of a similar set of cytokine genes and several of the IFNA genes. Imiquimod stimulates binding of several induction-specific nuclear complexes: (i) the NF-kappa B-specific complexes binding to the kappa B enhancer present in the promoters of all cytokine genes, but not in IFNA genes, and (ii) the complex(es) binding to the A4F1 site, 5'-GTAAAGAAAGT-3', conserved in the inducible element of IFNA genes. These results indicate that imiquimod, similar to viral infection, stimulates expression of a large number of cytokine genes, including IFN-alpha/beta, and that the signal transduction pathway induced by both of these stimuli requires tyrosine kinase and protein kinase activity.

Epidemiological studies suggest that chronic exposure to air pollution increases susceptibility to respiratory infections, including tuberculosis in humans. A possible link between particulate air pollutant exposure and antimycobacterial immunity has not been explored in human primary immune cells. We hypothesized that exposure to diesel exhaust particles (DEP), a major component of urban fine particulate matter, suppresses antimycobacterial human immune effector cell functions by modulating TLR-signaling pathways and NF-κB activation. We show that DEP and H37Ra, an avirulent laboratory strain of Mycobacterium tuberculosis, were both taken up by the same peripheral human blood monocytes. To examine the effects of DEP on M. tuberculosis-induced production of cytokines, PBMC were stimulated with DEP and M. tuberculosis or purified protein derivative. The production of M. tuberculosis and purified protein derivative-induced IFN-γ, TNF-α, IL-1β, and IL-6 was reduced in a DEP dose-dependent manner. In contrast, the production of anti-inflammatory IL-10 remained unchanged. Furthermore, DEP stimulation prior to M. tuberculosis infection altered the expression of TLR3, -4, -7, and -10 mRNAs and of a subset of M. tuberculosis-induced host genes including inhibition of expression of many NF-κB (e.g., CSF3, IFNG, IFNA, IFNB, IL1A, IL6, and NFKBIA) and IFN regulatory factor (e.g., IFNG, IFNA1, IFNB1, and CXCL10) pathway target genes. We propose that DEP downregulate M. tuberculosis-induced host gene expression via MyD88-dependent (IL6, IL1A, and PTGS2) as well as MyD88-independent (IFNA, IFNB) pathways. Prestimulation of PBMC with DEP suppressed the expression of proinflammatory mediators upon M. tuberculosis infection, inducing a hyporesponsive cellular state. Therefore, DEP alters crucial components of antimycobacterial host immune responses, providing a possible mechanism by which air pollutants alter antimicrobial immunity.

We investigated the antiviral activity and gene induction properties of interferon gamma (IFN-γ) compared to type I IFN (IFNa1) in Atlantic salmon. IFN-γ protected salmon cells against infectious pancreatic necrosis virus (IPNV)-induced cytopathic effect (CPE), reduced virus titers, and inhibited the synthesis of the viral structural protein VP3. Moreover, IFN-γ showed potent antiviral activity against salmonid alphavirus 3 (SAV3) measured as a reduction in virus nsP1 transcripts. IFN-γ (a type II IFN) had less specific antiviral activity against IPNV than IFNa1, showing a half-maximal effective concentration of 1.6 ng/ml versus 31 pg/ml determined in the CPE reduction assay. Compared to IFNa1, IFN-γ was a more effective inducer of the antiviral protein GBP, several interferon regulatory transcription factors (IRFs), and the chemokine IP-10. The antiviral activity of IFN-γ may also in part be ascribed to upregulation of Mx, ISG15, and viperin. These are typical type I IFN-induced genes in mammals and were also more strongly induced by IFNa1 than by IFN-γ in salmon cells. Fish and mammalian IFN-γ thus show strikingly similar gene induction properties. Interestingly, the antiviral activity of IFN-γ against IPNV and SAV3 and its ability to induce Mx and ISG15 markedly decreased in the presence of neutralizing antiserum against IFNa1. In contrast, antiIFNa1 had no effect on the induction of IRF-1 and IP-10 by IFN-γ. This suggests that the antiviral activity of IFN-γ is partially dependent on IFNa induction. However, because antiIFNa1 could not abolish the IFN-γ-mediated induction of Mx and ISG15 completely, IFN-γ may possibly also induce such genes directly.

Pancreatic carcinoma cells lines are known to have a high incidence of homozygous deletion of the candidate tumor suppressor gene p16 (MTS1/CDKN2), which resides in the chromosome 9p21 region. Here we: (a)examined a series of these cell lines for the incidence of codeletion of genes located near p16, in particular, the gene for the enzyme 5'-deoxy-5'-methylthioadenosine phosphorylase (MTAP) and the genes of the IFN-alpha and -beta cluster (IFNs); and (b) investigated whether therapeutic strategies could be developed that target malignant cells that have undergone the codeletion of such genes. Five of the eight pancreatic carcinoma cell lines were p16(-), MTAP was codeleted in all five cases. Because MTAP phosphorolyzes 5'-deoxy-5'-methylthioadenosine (MTA), generated as a byproduct of polyamine synthesis, to the salvageable purine base adenine, loss of this pathway in p16(-), MTAP(-) cells might sensitize these cells to methotrexate (MTX), the mechanism of action of which involves, in part, an inhibition of purine de novo synthesis. MTAP(+) normal keratinocytes and pancreatic carcinoma lines had relatively poor sensitivity, in terms of efficacy, to the purine nucleotide-starving actions of MTX. This may be in part due to the MTAP-dependent salvage of adenine moieties from endogenously generated MTA, because the MTAP inhibitor 5'-chloro-5'-de- oxyformycin A potentiates the antipurine actions of MTX in some of these MTAP(+) lines. Also, exogenous MTA (10 microM) reverses the growth-inhibitory actions of MTX in these lines. In contrast, MTAP(-) cell lines, which cannot recycle purines from endogenous MTA, have a relatively high sensitivity to the antipurine actions of MTX, which is not modulated by 5'-chloro-5'-deoxyformycin A or exogenous MTA. Thus the MTAP loss in malignant cells may be an example of gene deletion chemoselectivity, in which genetic deletions that occur as part of the oncogenic process render these cells more sensitive to particular anticancer agents than normal cells, which have not undergone such deletions. We also examined whether the loss of IFN genes sensitize cells to the growth-inhibitory actions of these cytokines. Three of the five p16(-) cell lines bore homozygous deletions of IFNA1 and IFNB1 genes, representing each end of the IFN-alpha,-beta gene cluster; one cell line bore a codeletion of the IFNA1 gene but retained the IFNB1 locus. Whereas the cell lines that were most sensitive to the growth-inhibitory effects of IFN-beta or IFN-alpha(2b), tended to be those with IFN deletions, there were enough exceptions to this pattern to indicate that the IFN genotype does not reliably predict IFN responsiveness.

HIV-1 is transmitted primarily across mucosal surfaces and rapidly spreads within the intestinal mucosa during acute infection. The type I interferons (IFNs) likely serve as a first line of defense, but the relative expression and antiviral properties of the 12 IFNα subtypes against HIV-1 infection of mucosal tissues remain unknown. Here, we evaluated the expression of all IFNα subtypes in HIV-1-exposed plasmacytoid dendritic cells by next-generation sequencing. We then determined the relative antiviral potency of each IFNα subtype ex vivo using the human intestinal Lamina Propria Aggregate Culture model. IFNα subtype transcripts from the centromeric half of the IFNA gene complex were highly expressed in pDCs following HIV-1 exposure. There was an inverse relationship between IFNA subtype expression and potency. IFNα8, IFNα6 and IFNα1IFNα1 were both highly expressed but exhibited relatively weak antiviral activity. The relative potencies correlated with binding affinity to the type I IFN receptor and the induction levels of HIV-1 restriction factors Mx2 and Tetherin/BST-2 but not APOBEC3G, F and D. However, despite the lack of APOBEC3 transcriptional induction, the higher relative potency of IFNα8 and IFNα1IFNα1) subtypes significantly induced HIV-1 GG-to-AG hypermutation. The results unravel non-redundant functions of the IFNα subtypes against HIV-1 infection, with strong implications for HIV-1 mucosal immunity, viral evolution and IFNα-based functional cure strategies.

Type I interferon (IFN) plays a critical role in the innate immunity against viral infection. Expression of IFNA genes in infected cells is cell type-dependent and is regulated at the transcriptional level. The present study is focused on the molecular mechanism underlying the differential expression of human IFNA1 and A2 genes. Two nucleotides, at positions -98 and -81 of IFNA1 and A2 promoter, were pivotal to the differential expression. The DNA pull-down and chromatin precipitation assays have shown that nuclear interferon regulatory factor (IRF)-3 and IRF-7 as well as IRF-1 bind to IFNA1 virus-responsive element (VRE). Interestingly, overexpression of IRF-7 increased the otherwise weak binding of both IRF-3 and IRF-7 to IFNA2 VRE. These data together with the results of two-step chromatin immunoprecipitation strongly suggest that the IRF-3 and IRF-7 bind to IFNA1 promoter as a dimer. Furthermore, binding of IRF-3 and IRF-7 to IFNA VRE is associated with the presence of acetylated histone H3, suggesting that histone acetyltransferase(s) is tethered together with virus-activated IRF-3 and IRF-7 to the IFNA1 promoter. In addition, the constitutively active IRF-3 (5D) and IRF-7 (2D) mutants activate the endogenous IFNA genes in uninfected cells; however, the expression profile of IFNA is not identical to that induced by viral infection.

Pancreatic cancer tends to be highly resistant to current therapy and remains one of the great challenges in biomedicine with very low 5-year survival rates. Here, we report that zalcitabine, an antiviral drug for human immunodeficiency virus infection, can suppress the growth of primary and immortalized human pancreatic cancer cells through the induction of ferroptosis, an iron-dependent form of regulated cell death. Mechanically, this effect relies on zalcitabine-induced mitochondrial DNA stress, which activates the STING1/TMEM173-mediated DNA sensing pathway, leading to macroautophagy/autophagy-dependent ferroptotic cell death via lipid peroxidation, but not a type I interferon response. Consequently, the genetic and pharmacological inactivation of the autophagy-dependent ferroptosis pathway diminishes the anticancer effects of zalcitabine in cell culture and animal models. Together, these findings not only provide a new approach for pancreatic cancer therapy but also increase our understanding of the interplay between autophagy and DNA damage response in shaping cell death.Abbreviations: ALOX: arachidonate lipoxygenase; ARNTL/BMAL1: aryl hydrocarbon receptor nuclear translocator-like; ATM: ATM serine/threonine kinase; ATG: autophagy-related; cGAMP: cyclic GMP-AMP; CGAS: cyclic GMP-AMP synthase; ER: endoplasmic reticulum; FANCD2: FA complementat