Presence Of IFN-β Research Articles

Aromatic amino acid metabolites alter interferon signaling and influenza pathogenesis.

The ability of gut microbial metabolites to influence the host is increasingly recognized. The microbiota extensively metabolizes the three aromatic amino acids, tryptophan, tyrosine, and phenylalanine. Previously we have found that a metabolite of tyrosine, 4-OH-phenylpropionic acid, can enhance type I interferon (IFN) signaling and protect from influenza pathogenesis in a murine model. Herein we screened 17 related aromatic amino acid metabolites for effects on IFN signaling in human lung epithelial cells and monocytes alone and in the presence of IFN-β, influenza, and LPS. While the tryptophan family metabolites reduced IFN signaling in both cell types, the tyrosine and phenylalanine metabolites had varied effects, which were cell-type dependent. Pooled treatment of all these metabolites reduced IFN signaling in both cell types and suggested a tryptophan metabolite effect dominance. Strikingly, when all the metabolites were pooled together, we found reduced influenza recovery in both cell types. RNA sequencing further validated reduced viral loads and decreased IFN signaling. Single gene silencing of significantly upregulated genes identified by RNA sequencing (EGR2, ATP6VD02, SPOCK1, and IL31RA) did not completely abrogate the metabolite induced decrease in IFN signaling. However, these upregulated targets suggested a mechanistic link to TGF-beta signaling. Treatment with a TGF-beta inhibitor and combined targeted gene silencing led to a significant reversal of metabolite induced IFN signaling suppression. Finally, we demonstrated that intranasal administration of these metabolites prior to influenza infection led to reduced animal morbidity, viral titers, and inflammation. Our work implies that microbial metabolites can alter IFN signaling mechanistically through TGF-beta and promote beneficial outcomes during influenza infection.

Interferon-β Decreases the Hypermetabolic State of Red Blood Cells from Patients with Multiple Sclerosis.

Multiple sclerosis (MS) is an inflammatory disease characterized by damage to the myelin sheath surrounding axons in the central nervous system. While the exact mechanism of this destruction is unknown, excess nitric oxide (NO) and adenosine triphosphate (ATP) have been measured in tissues and fluids obtained from people with MS. Here, incubation of interferon-beta (IFN-β), an MS drug with an unknown mechanism of action, with red blood cells (RBCs) obtained from people with MS provide evidence of a potential hypermetabolic state in the MS RBC that is decreased with IFN-β intervention. Specifically, binding of all three components of an albumin/C-peptide/Zn2+ complex to MS RBCs was significantly increased in comparison to control RBCs. For example, the binding of C-peptide to MS RBCs was significantly increased (3.4 ± 0.1 nM) compared to control RBCs (1.6 ± 0.2 nM). However, C-peptide binding to MS RBCs was reduced to a value (1.6 ± 0.3 nM) statistically equal to that of control RBCs in the presence of 2 nM IFN-β. Similar trends were measured for albumin and Zn2+ binding to RBCs when in the presence of IFN-β. RBC function was also affected by incubation of cells with IFN-β. Specifically, RBC-derived ATP and measurable membrane GLUT1 were both significantly decreased (56 and 24%, respectively) in the presence of IFN-β. Collectively, our results suggest that IFN-β inhibits albumin binding to the RBC, thereby reducing its ability to deliver ligands such as C-peptide and Zn2+ to the cell and normalizing the basal hypermetabolic state.

A 77 Amino Acid Region in the N-Terminal Half of the HSV-1 E3 Ubiquitin Ligase ICP0 Contributes to Counteracting an Established Type 1 Interferon Response.

ABSTRACTHerpes simplex virus 1 (HSV-1) is a human pathogen capable of establishing lifelong latent infections that can reactivate under stress conditions. A viral immediate early protein that plays important roles in the HSV-1 lytic and latent infections is the viral E3 ubiquitin ligase, ICP0. ICP0 transactivates all temporal classes of HSV-1 genes and facilitates viral gene expression. ICP0 also impairs the antiviral effects of interferon (IFN)-β, a component of host innate defenses known to limit viral replication. To begin to understand how ICP0 allows HSV-1 to disarm the IFN-β response, we performed genetic analyses using a series of ICP0 truncation mutants in the absence and presence of IFN-β in cell culture. We observed that IFN-β pretreatment of cells significantly impaired the replication of the ICP0 truncation mutants, n212 and n312, which code for the first 211 and 311 amino acids of ICP0, respectively; this effect of IFN-β correlated with decreased HSV-1 early and late gene expression. This increased sensitivity to IFN-β was not as apparent with the ICP0 mutant, n389. Our mapping studies indicate that loss of 77 amino acids from residues 312 to 388 in the N-terminal half of ICP0 resulted in a virus that was significantly more sensitive to cells pre-exposed to IFN-β. This 77 amino acid region contains a phospho-SUMO-interacting motif or -SIM, which we propose participates in ICP0’s ability to counteract the antiviral response established by IFN-β.IMPORTANCE Interferons (IFNs) are secreted cellular factors that are induced by viral infection and limit replication. HSV-1 is largely refractory to the antiviral effects of type 1 IFNs, which are synthesized shortly after viral infection, in part through the activities of the viral regulatory protein, ICP0. To understand how ICP0 impedes the antiviral effects of type 1 IFNs, we used a series of HSV-1 ICP0 mutants and examined their viral replication and gene expression levels in cells stimulated with IFN-β (a type 1 IFN). Our mapping data identifies a discrete 77 amino acid region in the N-terminal half of ICP0 that facilitates HSV-1 resistance to IFN-β. This region of ICP0 is modified by phosphorylation and binds to the posttranslational modification SUMO, suggesting that HSV, and potentially other viruses, may counteract type 1 IFN signaling by altering SUMO and/or SUMO modified cellular proteins.

Transcription from the proximal promoter of ELAC1, a gene for tRNA repair, is upregulated by interferons

tRNase ZS (ELAC1) and TRNT1 function in tRNA recycling. Recently, we have shown that these genes are upregulated in the cells infected with Theiler's mouse encephalitis virus (TMEV), implying that tRNA recycling functions in response to viral infection. To address the molecular mechanism underlying the ELAC1 upregulation in the cells infected with TMEV, we performed luciferase assays using various plasmid constructs harboring the ELAC1 promoter region. The luciferase expression from a construct containing the full-length ELAC1 promoter was augmented by TMEV, poly IC, IFN-β, or IFN-γ. We identified four IFN-stimulated responsible elements (ISREs) in the proximal promoter region. The luciferase expression from the constructs that lack all the ISREs was strongly reduced compared with that from the constructs with the four ISREs in the presence of IFN-β or IFN-γ. The observation that the ISREs from the ELAC1 promoter are essential for the gene upregulation by IFN-β or IFN-γ suggests that the ELAC1 gene is upregulated by IFNs.

Mathematical model of STAT signalling pathways in cancer development and optimal control approaches.

In various diseases, the STAT family display various cellular controls over various challenges faced by the immune system and cell death programs. In this study, we investigate how an intracellular signalling network (STAT1, STAT3, Bcl-2 and BAX) regulates important cellular states, either anti-apoptosis or apoptosis of cancer cells. We adapt a mathematical framework to illustrate how the signalling network can generate a bi-stability condition so that it will induce either apoptosis or anti-apoptosis status of tumour cells. Then, we use this model to develop several anti-tumour strategies including IFN-β infusion. The roles of JAK-STATs signalling in regulation of the cell death program in cancer cells and tumour growth are poorly understood. The mathematical model unveils the structure and functions of the intracellular signalling and cellular outcomes of the anti-tumour drugs in the presence of IFN-β and JAK stimuli. We identify the best injection order of IFN-β and DDP among many possible combinations, which may suggest better infusion strategies of multiple anti-cancer agents at clinics. We finally use an optimal control theory in order to maximize anti-tumour efficacy and minimize administrative costs. In particular, we minimize tumour volume and maximize the apoptotic potential by minimizing the Bcl-2 concentration and maximizing the BAX level while minimizing total injection amount of both IFN-β and JAK2 inhibitors (DDP).

Cathepsin H deficiency decreases hypoxia-ischemia-induced hippocampal atrophy in neonatal mice through attenuated TLR3/IFN-\u03b2 signaling

BackgroundCathepsin H (CatH) is a lysosomal cysteine protease with a unique aminopeptidase activity. Its expression level is increased in activated immune cells including dendritic cells, macrophages, and microglia. We have previously reported that CatH deficiency impairs toll-like receptor 3 (TLR3)-mediated activation of interferon regulatory factor 3 (IRF3), and the subsequent secretion of interferon (IFN)-β from dendritic cells. Furthermore, there is increasing evidence that IFN-β secreted from microglia/macrophages has neuroprotective effects. These observations prompted further investigation into the effects of CatH deficiency on neuropathological changes.MethodsIn this study, neuropathological changes were examined using histochemical staining (both hematoxylin-eosin (H&E) and Nissl) of the hippocampus of wild-type (WT) and CatH-deficient (CatH−/−) mice after hypoxia-ischemia (HI). The density and the localization of CatH and TLR3 were examined by immunofluorescent staining. CatH processing in microglia was assayed by pulse-chase experiments, while immunoblotting was used to examine TLR3 expression and IRF3 activation in microglia/macrophages in the presence of poly(I:C). Microglial cell death was examined by fluorescence-activated cell sorting (FACS), and primary astrocyte proliferation in the presence of IFN-β was examined using scratch wound assay.ResultsWT mice displayed severe atrophy in association with neuronal death and moderate astrogliosis in the hippocampus following neonatal HI. Somewhat surprisingly, CatH−/− mice showed marked neuronal death without severe atrophy in the hippocampus following HI. Furthermore, there was notable microglia/macrophages cell death and strong astrogliosis in the hippocampus. The TLR3 and phosphorylated IRF3 expression level in the hippocampus or splenocytes (mainly splenic macrophages); from CatH−/− mice was lower than in WT mice. In vitro experiments demonstrated that recombinant IFN-β suppressed HI-induced microglial cell death and astrocyte proliferation.ConclusionThese observations suggest that CatH plays a critical role in the proteolytic maturation and stabilization of TLR3, which is necessary for IFN-β production. Therefore, impaired TLR3/IFN-β signaling resulting from CatH deficiency may induce microglial cell death after activation and astrogliosis/glial scar formation in the hippocampus following HI injury, leading to suppression of hippocampal atrophy.

Human Norovirus NTPase Antagonizes Interferon-β Production by Interacting With IkB Kinase ε.

Human norovirus (HuNoV) is the leading cause of epidemic acute gastroenteritis worldwide. Type I interferons (IFN)-α/β are highly potent cytokines that are initially identified for their essential roles in antiviral defense. It was reported that HuNoV infection did not induce IFN-β expression but was controlled in the presence of IFN-β in human intestinal enteroids and a gnotobiotic pig model, suggesting that HuNoV has likely developed evasion countermeasures. In this study, we found that a cDNA clone of GII.4 HuNoV, the predominantly circulating genotype worldwide, inhibits the production of IFN-β and identified the viral NTPase as a key component responsible for such inhibition. HuNoV NTPase not only inhibits the activity of IFN-β promoter but also the mRNA and protein production of IFN-β. Additional studies indicate that NTPase inhibits the phosphorylation and nuclear translocation of interferon-regulatory factor-3 (IRF-3), leading to the suppression of IFN-β promoter activation. Mechanistically, NTPase interacts with IkB kinase ε (IKKε), an important factor for IRF-3 phosphorylation, and such interaction blocks the association of IKKε with unanchored K48-linked polyubiquitin chains, resulting in the inhibition of IKKε phosphorylation. Further studies demonstrated that the 1-179 aa domain of NTPase which interacts with IKKε is critical for the suppression of IFN-β production. Our findings highlight the role of HuNoV NTPase in the inhibition of IFN-β production, providing insights into a novel mechanism underlying how HuNoV evades the host innate immunity.

Type I interferon/IRF7 axis instigates chemotherapy-induced immunological dormancy in breast cancer

Neoadjuvant and adjuvant chemotherapies provide survival benefits to breast cancer patients, in particular in estrogen receptor negative (ER−) cancers, by reducing rates of recurrences. It is assumed that the benefits of (neo)adjuvant chemotherapy are due to the killing of disseminated, residual cancer cells, however, there is no formal evidence for it. Here, we provide experimental evidence that ER− breast cancer cells that survived high-dose Doxorubicin and Methotrexate based chemotherapies elicit a state of immunological dormancy. Hallmark of this dormant phenotype is the sustained activation of the IRF7/IFN-β/IFNAR axis subsisting beyond chemotherapy treatment. Upregulation of IRF7 in treated cancer cells promoted resistance to chemotherapy, reduced cell growth and induced switching of the response from a myeloid derived suppressor cell-dominated immune response to a CD4+/CD8+ T cell-dependent anti-tumor response. IRF7 silencing in tumor cells or systemic blocking of IFNAR reversed the state of dormancy, while spontaneous escape from dormancy was associated with loss of IFN-β production. Presence of IFN-β in the circulation of ER− breast cancer patients treated with neoadjuvant Epirubicin chemotherapy correlated with a significantly longer distant metastasis-free survival. These findings establish chemotherapy-induced immunological dormancy in ER− breast cancer as a novel concept for (neo)adjuvant chemotherapy activity, and implicate sustained activation of the IRF7/IFN-β/IFNAR pathway in this effect. Further, IFN-β emerges as a potential predictive biomarker and therapeutic molecule to improve outcome of ER− breast cancer patients treated with (neo)adjuvant chemotherapy.

IFN-β affects the angiogenic potential of circulating angiogenic cells by activating calpain 1.

Circulating angiogenic cells (CACs) are monocyte-derived cells with endothelial characteristics, which contribute to both angiogenesis and arteriogenesis in a paracrine way. Interferon-β (IFN-β) is known to inhibit these divergent processes in animals and patients. We hypothesized that IFN-β might act by affecting the differentiation and function of CACs. CACs were cultured from peripheral blood mononuclear cells and phenotypically characterized by surface expression of monocytic and endothelial markers. IFN-β significantly reduced the number of CACs by 18-64%. Apoptosis was not induced by IFN-β, neither in mononuclear cells during differentiation, nor after maturation to CACs. Rather, IFN-β impaired adhesion to, and spreading on, fibronectin, which was dependent on α5β1 (VLA-5)-integrin. IFN-β affected the function of VLA-5 in mature CACs, leading to rounding and detachment of cells, by induction of calpain 1 activity. Cell rounding and detachment was completely reversed by inhibition of calpain 1 activity in mature CACs. During in vitro capillary formation, CAC addition and calpain 1 inhibition enhanced sprouting of endothelial cells to a comparable extent, but were not sufficient to rescue tube formation in the presence of IFN-β. We show that the IFN-β-induced reduction of the numbers of in vitro differentiated CACs is based on activation of calpain 1, resulting in an attenuated adhesion to extracellular matrix proteins via VLA-5. In vivo, this could lead to inhibition of vessel formation due to reduction of the locally recruited CAC numbers and their paracrine angiogenic factors.

IFIT1 Differentially Interferes with Translation and Replication of Alphavirus Genomes and Promotes Induction of Type I Interferon.

Alphaviruses are a group of widely distributed human and animal pathogens. It is well established that their replication is sensitive to type I IFN treatment, but the mechanism of IFN inhibitory function remains poorly understood. Using a new experimental system, we demonstrate that in the presence of IFN-β, activation of interferon-stimulated genes (ISGs) does not interfere with either attachment of alphavirus virions to the cells, or their entry and nucleocapsid disassembly. However, it strongly affects translation of the virion-delivered virus-specific RNAs. One of the ISG products, IFIT1 protein, plays a major role in this translation block, although an IFIT1-independent mechanism is also involved. The 5’UTRs of the alphavirus genomes were found to differ significantly in their ability to drive translation in the presence of increased concentration of IFIT1. Prior studies have shown that adaptation of naturally circulating alphaviruses to replication in tissue culture results in accumulation of mutations in the 5’UTR, which increase the efficiency of the promoter located in the 5’end of the genome. Here, we show that these mutations also decrease resistance of viral RNA to IFIT1-induced translation inhibition. In the presence of higher levels of IFIT1, alphaviruses with wt 5’UTRs became potent inducers of type I IFN, suggesting a new mechanism of type I IFN induction. We applied this knowledge of IFIT1 interaction with alphaviruses to develop new attenuated variants of Venezuelan equine encephalitis and chikungunya viruses that are more sensitive to the antiviral effects of IFIT1, and thus could serve as novel vaccine candidates.

Reversible Silencing of Cytomegalovirus Genomes by Type I Interferon Governs Virus Latency

Herpesviruses establish a lifelong latent infection posing the risk for virus reactivation and disease. In cytomegalovirus infection, expression of the major immediate early (IE) genes is a critical checkpoint, driving the lytic replication cycle upon primary infection or reactivation from latency. While it is known that type I interferon (IFN) limits lytic CMV replication, its role in latency and reactivation has not been explored. In the model of mouse CMV infection, we show here that IFNβ blocks mouse CMV replication at the level of IE transcription in IFN-responding endothelial cells and fibroblasts. The IFN-mediated inhibition of IE genes was entirely reversible, arguing that the IFN-effect may be consistent with viral latency. Importantly, the response to IFNβ is stochastic, and MCMV IE transcription and replication were repressed only in IFN-responsive cells, while the IFN-unresponsive cells remained permissive for lytic MCMV infection. IFN blocked the viral lytic replication cycle by upregulating the nuclear domain 10 (ND10) components, PML, Sp100 and Daxx, and their knockdown by shRNA rescued viral replication in the presence of IFNβ. Finally, IFNβ prevented MCMV reactivation from endothelial cells derived from latently infected mice, validating our results in a biologically relevant setting. Therefore, our data do not only define for the first time the molecular mechanism of IFN-mediated control of CMV infection, but also indicate that the reversible inhibition of the virus lytic cycle by IFNβ is consistent with the establishment of CMV latency.

Sema4A inhibits the therapeutic effect of IFN-β in EAE

Approximately one-third of patients with multiple sclerosis (MS) respond poorly to interferon-beta (IFN-β) therapy. Serum Sema4A is increased in MS patients, and those who have high Sema4A do not respond to IFN-β therapy. In this study, we investigated whether recombinant Sema4A abrogates the efficacy of IFN-β in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Administration of Sema4A concurrently with IFN-β diminished the efficacy of IFN-β in EAE. These effects of Sema4A were attributed to promote Th1 and Th17 differentiation and to increase adhesive activation of T cells to endothelial cells, even in the presence of IFN-β.

Sema4A as a biomarker predicting responsiveness to IFN β treatment

Approximately one-third of patients with multiple sclerosis (MS) exhibit markedly high-level-expression of Sema4A. The expression of Sema4A is increased on DCs in MS patients and shed from these cells in a metalloproteinase-dependent manner. DC-derived Sema4A is critical for Th17 cell differentiation, and MS patients with high Sema4A levels exhibit Th17 skewing. Furthermore, patients with high Sema4A levels have more severe disabilities and are unresponsive to IFN-β treatment. We investigated whether recombinant Sema4A abrogates the efficacy of IFN-β in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Administration of Sema4A concurrently with IFN-β abrogated the efficacy of IFN-β. These effects of Sema4A were attributed to promote Th1 and Th17 differentiation and to increase adhesive activation of T cells to endothelial cells, even in the presence of IFN-β.Thus unresponsiveness to IFN-β treatment of MS patients with high Sema4A was also confirmed by model mice EAE. We recommend assaying Sema4A first, and then selecting DMD other than IFN-β for patients with high Sema4A.

Immunoregulatory Effects of Interferon-β in Suppression of Th17 cells

To investigate the immunoregulatory effects of interferon (IFN)-β on CD4+ T cells, we examined the response of CD4+ T cells from IFN-β(+/+) and IFN-β(-/-) mice to CD3/CD28 activation and to differentiation to Th17 lineage, analyzing the expression of signaling effectors, cell surface receptors, production of IL-17, and gene expression profiles. We provide evidence of increased phosphorylation of the membrane proximal kinase associated with TCR activation, ZAP-70, in IFN-β(-/-) T cells compared with IFN-β(+/+) T cells. Anti-CD3/anti-CD28 antibody stimulation of whole splenocytes or CD4+ T cells from IFN-β(-/-) mice results in secretion of IL-17A, in contrast to identical stimulation of cells from IFN-β(+/+) mice, which fails to increase IL-17A production. After CD3/CD28 activation, IFN-β(-/-) CD4+ T cells express higher levels of IRF-4, required for Th17 differentiation, and increased expression of CCR6, IL-23R, IL-6R, and CXCR4, compared with activated IFN-β(+/+) T cells. Notably, cell surface expression of IL-6R and IL-23R is significantly higher in the IFN-β(-/-) CD4+ T cells, with an increased number of double-positive CCR6+IL-23R+ and IL-6R+IL-23R+ CD4+ T cells. On polarization to Th17 lineage, CD4+ T cells from IFN-β(-/-) mice exhibit a more Th17-primed transcriptome compared with CD4+ T cells from IFN-β(+/+) mice. Indeed, when CD4+ T cells from IFN-β(+/+) mice are polarized to Th17 lineage in the presence of IFN-β, many Th17-associated genes are down-regulated. Employing a MOG-peptide-induced experimental autoimmune encephalomyelitis model of multiple sclerosis, we identify a greater proportion of Th17 cells in the lymph nodes of IFN-β(-/-) mice compared with IFN-β(+/+) mice, and increased numbers of CD4+ T cells in the central nervous system of IFN-β(-/-) mice, regardless of the stage of disease. Taken together, our data indicate an immunoregulatory role for IFN-β in the suppression of Th17 cells.

IL7Rα Expression and Upregulation by IFNβ in Dendritic Cell Subsets Is Haplotype-Dependent

The IL7Rα gene is unequivocally associated with susceptibility to multiple sclerosis (MS). Haplotype 2 (Hap 2) confers protection from MS, and T cells and dendritic cells (DCs) of Hap 2 exhibit reduced splicing of exon 6, resulting in production of relatively less soluble receptor, and potentially more response to ligand. We have previously shown in CD4 T cells that IL7Rα haplotypes 1 and 2, but not 4, respond to interferon beta (IFNβ), the most commonly used immunomodulatory drug in MS, and that haplotype 4 (Hap 4) homozygotes have the highest risk of developing MS. We now show that IL7R expression increases in myeloid cells in response to IFNβ, but that the response is haplotype-dependent, with cells from homozygotes for Hap 4 again showing no response. This was shown using freshly derived monocytes, in vitro cultured immature and mature monocyte-derived dendritic cells, and by comparing homozygotes for the common haplotypes, and relative expression of alleles in heterozygotes (Hap 4 vs not Hap 4). As for T cells, in all myeloid cell subsets examined, Hap 2 homozygotes showed a trend for reduced splicing of exon 6 compared to the other haplotypes, significantly so in most conditions. These data are consistent with increased signaling being protective from MS, constitutively and in response to IFNβ. We also demonstrate significant regulation of immune response, chemokine activity and cytokine biosynthesis pathways by IL7Rα signaling in IFNβ -treated myeloid subsets. IFNβ-responsive genes are over-represented amongst genes associated with MS susceptibility. IL7Rα haplotype may contribute to MS susceptibility through reduced capacity for IL7Rα signalling in myeloid cells, especially in the presence of IFNβ, and is currently under investigation as a predictor of therapeutic response.

Constitutive Phosphorylation of Interferon Receptor A-Associated Signaling Proteins in Systemic Lupus Erythematosus

BackgroundOverexpression of type I interferon (IFN-I)-induced genes is a common feature of systemic lupus erythematosus (SLE) and its experimental models, but the participation of endogenous overproduction of IFN-I on it is not clear. To explore the possibility that abnormally increased IFN-I receptor (IFNAR) signaling could participate in IFN-I-induced gene overexpression of SLE, we examined the phosphorylation status of the IFNAR-associated signaling partners Jak1 and STAT2, and its relation with expression of its physiologic inhibitor SOCS1 and with plasma levels of IFNα and IFN-like activity.Methodology/Principal FindingsPeripheral blood mononuclear cells (PBMC) from SLE patients with or without disease activity and healthy controls cultured in the presence or in the absence of IFNβ were examined by immunoprecipitation and/or western blotting for expression of the two IFNAR chains, Jak1, Tyk2, and STAT2 and their phosphorylated forms. In SLE but not in healthy control PBMC, Jak1 and STAT2 were constitutively phosphorylated, even in the absence of disease activity (basal pJak1: controls vs. active SLE p<0.0001 and controls vs. inactive SLE p = 0.0006; basal pSTAT2: controls vs. active and inactive SLE p<0.0001). Although SOCS1 protein was slightly but significantly decreased in SLE in the absence or in the presence of IFNβ (p = 0.0096 to p<0.0001), in SOCS1 mRNA levels were markedly decreased (p = 0.036 to p<0.0001). IFNβ induced higher levels of the IFN-I-dependent MxA protein mRNA in SLE than in healthy controls, whereas the opposite was observed for SOCS1. Although there was no relation to increased serum IFNα, active SLE plasma could induce expression of IFN-dependent genes by normal PBMC.Conclusions/SignificanceThese findings suggest that in some SLE patients IFN-I dependent gene expression could be the result of a low IFNAR signaling threshold.

TRIM79α, an interferon-stimulated gene product, restricts tick-borne encephalitis virus replication by degrading the viral RNA polymerase.

In response to virus infection, type I interferons (IFNs) induce several genes, most of whose functions are largely unknown. Here, we show that the tripartite motif (TRIM) protein, TRIM79α, is an IFN-stimulated gene (ISG) product that specifically targets tick-borne encephalitis virus (TBEV), a Flavivirus that causes encephalitides in humans. TRIM79α restricts TBEV replication by mediating lysosome-dependent degradation of the flavivirus NS5 protein, an RNA-dependent RNA polymerase essential for virus replication. NS5 degradation was specific to tick-borne flaviviruses, as TRIM79α did not recognize NS5 from West Nile virus (WNV) or inhibit WNV replication. In the absence of TRIM79α, IFN-β was less effective in inhibiting tick-borne flavivirus infection of mouse macrophages, highlighting the importance of a single virus-specific ISG in establishing an antiviral state. The specificity of TRIM79α for TBEV revealsa remarkable ability of the innate IFN response to discriminate between closely related flaviviruses.

Abstract 1499: Gene therapy in a mouse model for malignant melanoma: Combining the activities of the p53/Arf and interferon pathways

Abstract Background: Malignant melanoma cells typically maintain wild-type p53 in a non-functional state, due in part to the loss of ARF (p14ARF in humans, p19ARF in mice). Data have shown that interferon-β (IFNβ) can activate the transcription of p53 and interact with the ARF pathway as well. By using a p53-driven adenovirus, we sought to combine the action and regulation of p53, p19ARF and IFNβ for the treatment of malignant melanoma in an immunocompetent mouse model. Methods: p53-responsive AdPG adenoviral vectors (Bajgelman and Strauss, 2008) containing p53, p19ARF and IFNβ were produced and expression verified by immunofluorescence and ELISA. In vitro evaluation of the antiproliferative effect of single or co-transduction of B16F10 cells (B16, p53 wild-type) was done by FACS after annexin/PI staining. C57/Bl6 mice were injected subcutaneously with B16 cells and tumors were allowed to develop (∼0.5 cm2). At this point, three consecutive intra-tumor injections (3 day interval between injections) of individual or combined adenoviral vectors were performed. Animals were then sacrificed and tumors collected and analyzed. Results: The combination of IFNβ and p19ARF proved to be more effective in inhibiting proliferation of B16 cells than either treatment alone, both in vitro and in vivo. In vitro we observed that neither AdPGp19 nor AdPGIFNβ alone altered the cell cycle, though co-transduction with these vectors resulted in extensive cell death. Annexin-V staining was also increased by the combined, but not individual, treatments. Cell death was observed only after 90-120h of incubation, yet prior to this event, indicators of senescence (positive SA-βGal staining, loss of histone H3 acetylatation at Lys9) were revealed in both single and combined vector treatments. In vivo, we observed that the combined gene transfer strategy led to reduced tumor volume, reduced incorporation of BrdU and increased staining by TUNEL in the primary tumor. Assays are in progress to determine if the combined treatment is effective in reducing metastases and prolonging survival. Conclusion: The use of p53-responsive vectors for the restoration of ARF expression in the presence of IFNβ represents a potential strategy for melanoma tumor suppression. While mechanistic studies are underway, we propose that the complementation of the p53/ARF and interferon pathways in the primary tumor may lead to reduction of metastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1499.

Effects of type I interferons on IGF-mediated autocrine/paracrine growth of human neuroendocrine tumor cells

We recently demonstrated that interferon (IFN)-beta has a more potent antitumor activity than IFN-alpha in BON cells, a neuroendocrine tumor (NET) cell line. The present study showed the role of type I IFNs in the modulation of the insulin-like growth factor (IGF) system in NETs. BON cells expressed IGF-I, IGF-II, IGF-I receptor, and insulin receptor mRNA. In addition, IGF-I and IGF-II stimulated the proliferation of BON cells and induced an inhibition of DNA fragmentation (apoptosis). As evaluated by quantitative RT-PCR, treatment with IFN-alpha (100 IU/ml) or IFN-beta (100 IU/ml) inhibited the expression of IGF-II mRNA (-42% and -65%, respectively, both P < 0.001), whereas IGF-I receptor mRNA was significantly upregulated by IFN-alpha (+28%, P < 0.001) and downregulated by IFN-beta (-47%, P < 0.001). Immunoreactive IGF-II concentration decreased in the conditioned medium during IFN-alpha (-16%, P < 0.05) and IFN-beta (-69%, P < 0.001) treatment. Additionally, IGF-I receptor bioactivity was reduced (-54%) after IFN-beta treatment. Scatchard analysis of (125)I-labeled IGF-I binding to cell membrane of BON cells revealed a dramatic suppression of maximum binding capacity only in the presence of IFN-beta. Finally, the proapoptotic activity of IFN-beta was partially counteracted by the coadministration of IGF-I and IGF-II (both at 50 nM). In conclusion, these data demonstrate that the IGF system has an important role in autocrine/paracrine growth of BON cells. The more potent antitumor activity of IFN-beta compared with IFN-alpha could be explained by several effects on this system: 1) both IFNs inhibit the transcription of IGF-II, but the suppression is significantly higher after IFN-beta than IFN-alpha and 2) only IFN-beta inhibits the expression of IGF-I receptor.

Interferon-β expressed by a rabies virus-based HIV-1 vaccine vector serves as a molecular adjuvant and decreases pathogenicity

Type I interferon is important in anti-viral responses and in coordinating the innate immune response. Here we explore the use of interferon-β to adjuvant the response to a rabies virus (RV) vaccine vector expressing both HIV-1 Gag and IFN-β. Viral load and immune responses of immunized mice were analyzed over time. Our results indicate that the RV expressing IFN-β (IFN(+)) is highly attenuated when compared to control RV and demonstrate that the expression of IFN-β reduces viral replication approximately 100-fold. Despite the decrease in replication, those mice immunized with the IFN(+) RV had a significantly greater number of activated CD8+ T cells. The increased activation of CD8+ T cells was dependent on IFN-β signaling, as we saw no difference following infection of IFNAR−/− mice. Although mice immunized with IFN(+) have a greater primary immune response than controls, immunized mice that were challenged with vaccinia-expressing Gag had no significant difference in the number or functionality of CD8+ T cells. The increased CD8+ T cell activation in the presence of IFN-β, even with greatly reduced viral replication, indicates the beneficial effect of IFN-β for the host.