Exogenous IFN-α Research Articles

Antiviral activity and host gene induction by tamarin and marmoset interferon-α and interferon-γ in the GBV-B primary hepatocyte culture model

GBV-B induces hepatitis in tamarins and marmosets and is a surrogate model for HCV infections. Here, we cloned and characterized the antiviral activity of tamarin and marmoset interferon (IFN)alpha and IFN gamma. Potent antiviral activity was observed for tamarin and marmoset IFN alpha in primary hepatocyte cultures infected with GBV-B. The antiviral activity was greater in cultures exposed to IFN alpha prior to GBV-B infection, suggesting that either GBV-B was capable of inhibition of the antiviral activity of exogenous IFN alpha or that the preexisting endogenous IFN response to the virus reduced efficacy to exogenous IFN alpha. IFN gamma also exhibited antiviral activity in GBV-B infected hepatocytes. The transcriptional response to IFN alpha in marmoset hepatocytes was characterized using human genome microarrays. Since the GBV-B hepatocyte culture model possesses a functional innate immune response, it will provide opportunities to explore the nature of the antiviral response to a virus closely related to HCV.

IFN-α Enhances Peptide Vaccine-Induced CD8+ T Cell Numbers, Effector Function, and Antitumor Activity

Type I IFNs, including IFN-alpha, enhance Ag presentation and promote the expansion, survival, and effector function of CD8(+) CTL during viral infection. Because these are ideal characteristics for a vaccine adjuvant, we examined the efficacy and mechanism of exogenous IFN-alpha as an adjuvant for antimelanoma peptide vaccination. We studied the expansion of pmel-1 transgenic CD8(+) T cells specific for the gp100 melanocyte differentiation Ag after vaccination of mice with gp100(25-33) peptide in IFA. IFN-alpha synergized with peptide vaccination in a dose-dependent manner by boosting relative and absolute numbers of gp100-specific T cells that suppressed B16 melanoma growth. IFN-alpha dramatically increased the accumulation of gp100-specific, IFN-gamma-secreting, CD8(+) T cells in the tumor through reduced apoptosis and enhanced proliferation of Ag-specific CD8(+) T cells. IFN-alpha treatment also greatly increased the long-term maintenance of pmel-1 CD8(+) T cells with an effector memory phenotype, a process that required expression of IFN-alpha receptor on the T cells and IL-15 in the host. These results demonstrate the efficacy of IFN-alpha as an adjuvant for peptide vaccination, give insight into its mechanism of action, and provide a rationale for clinical trials in which vaccination is combined with standard-of-care IFN-alpha therapy for melanoma.

Intranasal Administration of Alpha Interferon Reduces Seasonal Influenza A Virus Morbidity in Ferrets

The type I interferon (IFN) response represents one of the first lines of defense against influenza virus infections. In this study, we assessed the protective potential of exogenous IFN-alpha against seasonal and highly pathogenic influenza viruses in ferrets. Intranasal treatment with IFN-alpha several hours before infection with the H1N1 influenza A virus strain A/USSR/90/77 reduced viral titers in nasal washes at least 100-fold compared to mock-treated controls. IFN-treated animals developed only mild and transient respiratory symptoms, and the characteristic fever peak seen in mock-treated ferrets 2 days after infection was not observed. Repeated application of IFN-alpha substantially increased the protective effect of the cytokine treatment. IFN-alpha did not increase survival after infection with the highly pathogenic H5N1 avian influenza A virus strain A/Vietnam/1203/2004. However, viral titers in nasal washes were significantly reduced at days 1 and 3 postinfection. Our study shows that intranasal application of IFN-alpha can protect ferrets from seasonal influenza viruses, which replicate mainly in the upper respiratory tract, but not from highly pathogenic influenza viruses, which also disseminate to the lung. Based on these results, a more intensive evaluation of IFN-alpha as an emergency drug against pandemic influenza A is warranted.

내독소내성 마우스에서 Nitric Oxide 생성에 미치는 림프구 부전

본 연구에서는 내독소내성 상태에 있는 마우스 세포의 싸이토카인 생성능을 측정하기 위하여 대식세포-림프구 공동배양계를 이용한 nitric oxide (NO) 생성을 조사하였다. 마우스 복강 대식세포에 lipopolysaccharide (LPS)와 interferon-g (IFN-g)를 처리시 NO 생성이 증가되었으며, tumor necrosis factor-a (TNF-a) 또한 LPS처럼 NO 합성을 자극하는 것을 관찰할 수 있었다. 한편, 대식세포를 비장세포와 공동배양시, LPS 단독처리만으로도 NO 합성이 증가되었다. 반면, 2.5 mg/kg LPS로 전처리하고 치사량의 LPS를 2차 투여한 마우스의 경우, 마우스의 치사 및 혈중 TNF-a와 IFN-g가 증가되지 않았다. 또한 LPS-내성 마우스로부터 분리한 대식세포를 정상 비장세포와 공동배양시 LPS에 의한 NO생성이 일어나지 않았으며, 외래 TNF-a에 의한 NO 생성도 일어나지 않았다. 이와 아울러 정상 대식세포와 LPS 내성 마우스로부터 분리한 비장세포를 공동배양하였을 때, LPS 자극으로 인한 NO 생성이 일어나지 않았으며, 이러한 억제현상은 외래 IFN-g 또는 IFN-g 생성을 촉진시키는 concanavalin A (ConA)에 의해서 다시 역전되었다. 이러한 결과는 대식세포 뿐만 아니라 림프구도 LPS 내성에 관여하는 것을 보여준다고 사료된다. INF-g는 TNF-a 발현을 증가시키기 때문에, 림프구의 INF-g 합성 감소는 LPS에 내성을 보이는 대식세포의 TNF-a 합성 저하와 상호작용으로 내독소내성 상태를 유도하며 과도한 염증반응을 억제하는 것으로 사료된다. 따라서 LPS 내독소내성은 중환자의 심각한 패혈증에 대한 예방법으로 활용될 수 있을 것으로 기대된다. In this study, nitric oxide (NO) production in a macrophage-lymphocyte co-culture system was used to assess the cytokine producing capability of cells during endotoxin tolerance in mice. Incubation of peritoneal macrophages with interferon-<TEX>$\tau$</TEX> (IFN-<TEX>$\tau$</TEX>) in the presence of lipopolysaccharide (LPS) augmented NO synthesis. Exogenous tumor necrosis factor-<TEX>$\alpha$</TEX>(TNF-<TEX>$\alpha$</TEX>) could also replace LPS for the stimulation of NO production. Macrophages co-cultured with splenic lymphocytes showed augmented NO synthesis by LPS alone. However, pretreatment of mice with 2.5 mg/kg LPS completely prevented the lethality and the increase of blood TNF-<TEX>$\alpha$</TEX> and IFN-<TEX>$\tau$</TEX> after the second challenge with a lethal dose of LPS. In addition, when macrophages prepared from LPS-tolerant mice were co-cultured with normal splenocytes, LPS also could not induce the production of NO, even in the presence of exogenous TNF-<TEX>$\alpha$</TEX>. Moreover, when normal macrophages were co-cultured with splenocytes obtained from LPS-tolerant mice, stimulation with LPS could not evoke the NO production enhancement. However, this down-regulation was able to reverse by exogenous IFN-<TEX>$\tau$</TEX> or concanavalin A (ConA), a stimulator of IFN-<TEX>$\tau$</TEX> production. Our results indicate that not only macrophages but also lymphocytes contribute to LPS tolerance. As INF-<TEX>$\tau$</TEX> can enhance the expression of TNF-<TEX>$\alpha$</TEX>, the decrease of INF-<TEX>$\tau$</TEX>synthesis from lymphocytes may orchestrate with the decrease of TNF-<TEX>$\alpha$</TEX> synthesis from LPS-tolerant macrophages for the production of tolerant state and the prevention of excessive inflammation. Therefore, LPS tolerance may be exploited for prophylaxis of severe sepsis in patients at risk.

Interferons modulate FcɛRI-dependent production of autoregulatory IL-10 by circulating human monocytoid dendritic cells

Immature human blood monocytoid dendritic cells (mDCs) express high-affinity receptors for IgE (Fc epsilon RI), yet their exact function and regulation remain poorly understood. We sought to characterize Fc epsilon RI-dependent cytokine responses and their regulation in circulating human blood mDCs. Fc epsilon RI-dependent cytokine responses of circulating mDCs were studied by using anti-Fc epsilon RI alpha stimulation. Plasmacytoid dendritic cell (pDC) cross-regulation through Toll-like receptor 9 on these responses was investigated by examining the effects of exogenous IFN-alpha pretreatment and by coculturing pDCs and mDCs stimulated with CpG. Culture supernatants were analyzed by means of ELISA to determine cytokine levels. Cell markers were determined by means of flow cytometry. mDCs express marked levels of Fc epsilon RI (net mean fluorescence intensity, 196 +/- 49; n = 4). After Fc epsilon RI-dependent activation in mDCs, TNF-alpha (2189 +/- 864 pg/10(6) mDCs, n = 3) levels were upregulated within 4 hours, whereas IL-10 (112 +/- 47 pg/10(6) mDCs, n = 3) levels were detectable only after 24 hours of incubation. After adding IL-10-neutralizing antibody, TNF-alpha Fc epsilon RI-dependent responses were significantly augmented (3903 +/- 197 pg/10(6) mDCs, P < .01, n = 3). Conversely, recombinant IL-10 dose-dependently inhibited Fc epsilon RI-mediated TNF-alpha responses up to 86% +/- 3% (n = 3, P < .001). Pretreatment of mDCs with IFN-alpha (100 U/mL) enhanced Fc epsilon RI-dependent secretion of IL-10 by 3.2-fold (183 +/- 11 pg/10(6) mDCs, n = 4) compared with that seen in untreated cells (57 +/- 33 pg/10(6) mDCs, P < .001, n = 4). In pDC/mDC cocultures pretreated with CpG, Fc epsilon RI-dependent IL-10 secretion by mDCs was similarly augmented by 3-fold. Autocrine secretion of IL-10, a critical autoregulator of Fc epsilon RI-dependent proinflammatory responses in mDCs, is cross-regulated by IFN-alpha, a major product of Toll-like receptor 9 responses in pDCs that normally promotes T(H)1 immunity.

Gene expression analysis of macrophages derived from ankylosing spondylitis patients reveals interferon‐γ dysregulation

To determine whether macrophages, a type of cell implicated in the pathogenesis of ankylosing spondylitis (AS), exhibit a characteristic gene expression pattern. Macrophages were derived from the peripheral blood of 8 AS patients (median disease duration 13 years [range <1-43 years]) and 9 healthy control subjects over 7 days with the use of granulocyte-macrophage colony-stimulating factor. Cells were stimulated for 24 hours with interferon-gamma (IFN gamma; 100 units/ml), were left untreated for 24 hours, or were treated for 3 hours with lipopolysaccharide (LPS; 10 ng/ml). RNA was isolated and examined by microarray and real-time quantitative reverse transcription-polymerase chain reaction analysis. Microarray analysis revealed 198 probe sets detecting the differential expression of 141 unique genes in untreated macrophages from AS patients compared with healthy controls. Clustering and principal components analysis clearly distinguished AS patients and controls. Of the differentially expressed genes, 78 (55%) were IFN-regulated, and their relative expression indicated a "reverse" IFN signature in AS patient macrophages, where IFN gamma-up-regulated genes were underexpressed and down-regulated genes were overexpressed. Treatment of macrophages with exogenous IFN gamma normalized the expression of these genes between patients and controls. In addition, the messenger RNA encoded by the IFN gamma gene was approximately 2-fold lower in AS patient macrophages at baseline (P = 0.004) and was poorly responsive to LPS (P = 0.018), as compared with healthy controls. Our findings reveal consistent differences in gene expression in macrophages from AS patients, with evidence of a striking "reverse" IFN signature. Together with poor expression and responsiveness of the IFN gamma gene, these results suggest that there may be a relative defect in IFN gamma gene regulation, with autocrine consequences and implications for disease pathogenesis.

IFN-α-Induced Signal Transduction, Gene Expression, and Antitumor Activity of Immune Effector Cells Are Negatively Regulated by Suppressor of Cytokine Signaling Proteins

Proteins belonging to the suppressors of cytokine signaling (SOCS) family have been shown to regulate cytokine signal transduction in various cell types but their role in modulating the response of immune cells to IFN-alpha has not been fully explored. We hypothesized that SOCS proteins would inhibit the antitumor activity of IFN-alpha-stimulated immune cells. Transcripts for SOCS1, SOCS2, SOCS3, and cytokine-inducible Src homology 2-containing protein were identified in total human PBMC (PBMCs, NK cells, and T cells) within 1-2 h of stimulation with IFN-alpha (10(3)-10(5) U/ml). Immunoblot analysis confirmed the expression of these factors at the protein level. Transcripts for SOCS proteins were rapidly but variably induced in PBMCs from patients with metastatic melanoma following the i.v. administration of IFN-alpha-2b (20 million units/m(2)). Overexpression of SOCS1 and SOCS3, but not SOCS2, in the Jurkat T cell line inhibited IFN-alpha-induced phosphorylated STAT1 and the transcription of IFN-stimulated genes. Conversely, small inhibitory RNA-mediated down-regulation of SOCS1 and SOCS3 in Jurkat cells and normal T cells enhanced the transcriptional response to IFN-alpha. Loss of SOCS1 or SOCS3 in murine immune effectors was associated with enhanced IFN-induced phosphorylated STAT1, transcription of IFN-stimulated genes, and antitumor activity. Of note, IFN-alpha treatment eliminated melanoma tumors in 70% of SOCS1-deficient mice, whereas IFN-treated SOCS-competent mice all died. The antitumor effects of IFN-alpha in tumor-bearing SOCS1-deficient mice were markedly inhibited following depletion of CD8(+) T cells. These results indicate that the antitumor response of immune effector cells to exogenous IFN-alpha is regulated by SOCS proteins.

Innate Cytokine Responses in Porcine Macrophage Populations: Evidence for Differential Recognition of Double-Stranded RNA

Pulmonary airways are vulnerable to infection because of exposure to Ag during respiration. The innate, antiviral response must be activated rapidly after pathogen recognition, and alveolar macrophages (AMphi) play a role in this response. TLR3 and protein kinase R (PKR) recognize dsRNA, a replication intermediate of RNA viruses, and initiate transcription of IFN-alphabeta. In this study, synthetic dsRNA poly(I:C) was used to investigate innate responses of porcine AMphi compared with responses of peritoneal macrophages (PMphi). Poly(I:C) triggered IFN-alphabeta in AMphi and PMphi, but levels in AMphi were higher. In contrast, mRNA levels of IFN-stimulated genes, Mx and PKR, were greater in PMphi than AMphi. Low levels of Mx and PKR transcription in AMphi were not due to deficient type I IFN receptor signaling, as exogenous IFN-alpha induced nuclear translocation of phosphorylated STAT1. To investigate the differential mechanism by which IFN-alphabeta transcription is activated in AMphi and PMphi, 2-aminopurine (2-AP) was used to block dsRNA-mediated activation of PKR. IFN-alphabeta, Mx, and PKR mRNA levels in AMphi after poly(I:C) treatment were unaffected by 2-AP; conversely, transcription of IFN-alphabeta, Mx, or PKR remained at baseline levels in PMphi. Phosphorylated PKR was detected in PMphi, but not AMphi, after poly(I:C) treatment. In addition to IFN-alphabeta gene induction, mRNA levels of TNF-alpha and RANTES were higher in AMphi than PMphi after poly(I:C) stimulation. In summary, differential dsRNA-induced cytokine expression patterns between AMphi and PMphi provide evidence that dsRNA recognition and subsequent signaling is likely mediated via TLR3 in AMphi and PKR in PMphi.

Evaluation of Immunomodulators, Interferons and Known in Vitro SARS-CoV Inhibitors for Inhibition of SARS-Cov Replication in BALB/c Mice

Compounds approved for therapeutic use and in vitro inhibitors of severe acute respiratory syndrome coronavirus (SARS-CoV) were evaluated for inhibition in the mouse SARS-CoV replication model. A hybrid interferon, interferon alpha (IFN-alpha) B/D, and a mismatched double-stranded (ds) RNA interferon (IFN) inducer, Ampligen (poly I:poly C124), were the only compounds that potently inhibited virus titres in the lungs of infected mice as assessed by CPE titration assays. When mice were dosed intraperitoneally (i.p.) with IFN-alpha B/D once daily for 3 days beginning 4 h after virus exposure, SARS-CoV replication in the lungs of infected mice was reduced by 1 log10 at 10,000 and 32,000 IU; at the highest dose of 100,000 IU, virus lung titres were below detectable limits. Ampligen used i.p. at 10 mg/kg 4 h prior to virus exposure also reduced virus lung titres to below detectable limits. Nelfinavir, beta-D-N4-hydroxycytidine, calpain inhibitor VI, 3-deazaneplanocin A and Alferon (human leukocyte IFN-alpha-n3) did not significantly reduce lung virus titres in mice. Anti-inflammatory agents, chloroquine, amodiaquin and pentoxifylline, were also inactive in vivo, suggesting that although they may be useful in ameliorating the hyperinflammatory response induced by the virus infection, they will not significantly reduce the replication of the virus, the inducer of inflammatory response. Thus, anti-inflammatory agents may only be useful in treating virus lung infections if used in combination with agents that inhibit virus replication. In summary, the data suggest that induction of IFN by mismatched dsRNA or actual treatment with exogenous IFN-alpha can inhibit SARS-CoV replication in the lungs of mice.

Interferon-α: A Key Factor in Autoimmune Disease?

Interferon (IFN)-alpha is an effective drug for treatment of uveitis in Behçet's disease. This study was undertaken to investigate the mechanism of action of IFN-alpha in the treatment of various types of noninfectious sight-threatening uveitis. Eleven patients with refractory uveitis, and 13 healthy individuals were enrolled. The number of circulating plasmacytoid dendritic cells (pDCs) and their capacity to produce IFN-alpha in culture on stimulation with synthetic oligodinucleotides containing the CpG-motif were studied. Peripheral blood CD4+ T-cell phenotype and activation status were evaluated by flow cytometry at 0, 2, and 8 weeks after treatment for expression of CD69, CD62L, chemokine receptors (CCR4, CXCR3, and CCR5), and intracellular cytokines (TNF-alpha, IFN-gamma, and IL-10). All patients experienced a positive clinical response to IFN-alpha treatment. There was no significant difference between patients and control subjects in the number of circulating pDCs, but there was a significant decrease in the capability of patients' pDCs to produce IFN-alpha in response to CpG (P < 0.001). Peripheral blood CD4+ T cells expressed reduced levels of surface CD62L (P < 0.005) as a measure of activation and higher levels of chemokine receptors CXCR3, CCR4, and CCR5 (P < 0.005, P < 0.05, and P < 0.05, respectively); in addition, intracellular T-cell IL-10 levels were increased once the treatment was initiated (P < 0.01). The data suggest that IFN-alpha may control uveitis by promoting induction of IL-10-producing T-cells, possibly T-regulatory cells. Dysregulation of the T-cell population in patients with uveitis may be associated with a defect in the pDCs' ability to produce IFN-alpha, which can be circumvented with administration of exogenous IFN-alpha.

Immunocorrection of altered cytokine production in neurological patients

Well established psycho-neuro-endocrine-immune interactions play a significant role in the pathogenesis of neurological diseases. The major objective for this study was to investigate cytokine production and new approaches for immunocorrection in patients with Parkinson's (PD) and Wilson's diseases (WD). Cytokine profile was studied in 16 patients 5 times with 3-month intervals (I, II, III, IV, V). Mitogen-induced and plasma level of 8 cytokines, their gene expression in the peripheral immunocompetent cells, and response of immunocytes to recombinant interferons, interferon-inducing chemicals and the nootropic drug Semax were assessed. Plasma level of IL-6 significantly decreased 36% at the interval V in WD patients. Significant difference in plasma IL-12 was observed between PD and WD patients at the interval V. Significant decrease of interferon (IFN)-γ secretion in vitro was observed at interval III. In vitro secretion of IFN-α was significantly lower at II and III intervals vs. I interval (8.0±2.1U/ml, and 10.8±2.4U/ml vs. 24.0±3.6U/ml) in PD patients Pretreatment of cells with exogenous IFN-α did not affect secretion of endogenous IFN in cell cultures at the interval I, but recovered its diminished secretion in vitro at the interval III. Similar pattern in IFN secretion was observed in the Semax-treated cultures. Data revealed alterations in the interferon system which may increase risk of neurological patients for the viral infections.

Hepatology highlights

Hepatology highlights

Differential regulation of the protein tyrosine kinase activity followingiInterleukin-2 (IL-2), Interferron gamma (IFN?) and SRBC administration in brain tumor induced conditions: SRBC acting as a dual potentiator in regulating the cytokine profile

Protein tyrosine kinases (PTKs) act as an important class of signal transducer in cytokine mediated signaling. Defects in phosphorylation of tyrosine residues of intracellular substrates of the immunocytes are a noted phenomenon in glioma induced immune suppression. Administration of BRMs like Interleukin2 (IL2), Interferon ? (IFN ?) and SRBC in glioma induced experimental models, improved their survival status by immune potentiation. It was shown that SRBC exerts the maximum anti-tumor immune boosting by augmenting the functional status of the two immunocytes-microglia and lymphocytes when compared with IL2 and IFN ?. The present study focuses on the differential modulation of the protein tyrosine kinase activity in lymphocytes and microglia following the administration of the 3 BRMs. Our findings indicate that PTKs actively transduce signals on administration of exogenous IL-2. But exogenous IFN ? administration fails to elicit the enzyme activity. With SRBC administration, a differential PTK activity modulation was observed in the two immunocytes. SRBC not only shifted the cytokine profile to Th1 subset of lymphocytes but also simultaneously upregulated the expression of the activation marker IL2R?/CD25 thereby resulting in auto-activation of the hosts immunocytes.

Specific Engagement of TLR4 or TLR3 Does Not Lead to IFN-β-Mediated Innate Signal Amplification and STAT1 Phosphorylation in Resident Murine Alveolar Macrophages

The innate immune response must be mobilized promptly yet judiciously via TLRs to protect the lungs against pathogens. Stimulation of murine peritoneal macrophage (PMphi) TLR4 or TLR3 by pathogen-associated molecular patterns (PAMPs) typically induces type I IFN-beta, leading to autocrine activation of the transcription factor STAT1. Because it is unknown whether STAT1 plays a similar role in the lungs, we studied the response of resident alveolar macrophages (AMphi) or control PMphi from normal C57BL/6 mice to stimulation by PAMPs derived from viruses (polyriboinosinic:polyribocytidylic acid, specific for TLR3) or bacteria (Pam(3)Cys, specific for TLR2, and repurified LPS, specific for TLR4). AMphi did not activate STAT1 by tyrosine phosphorylation on Y701 following stimulation of any of these three TLRs, but readily did so in response to exogenous IFN-beta. This unique AMphi response was not due to altered TLR expression, or defective immediate-early gene response, as measured by expression of TNF-alpha and three beta chemokines. Instead, AMphi differed from PMphi in not producing bioactive IFN-beta, as confirmed by ELISA and by the failure of supernatants from TLR-stimulated AMphi to induce STAT1 phosphorylation in PMphi. Consequently, AMphi did not produce the microbicidal effector molecule NO following TLR4 or TLR3 stimulation unless exogenous IFN-beta was also added. Thus, murine AMphi respond to bacterial or viral PAMPs by producing inflammatory cytokines and chemokines, but because they lack the feed-forward amplification typically mediated by autocrine IFN-beta secretion and STAT1 activation, require exogenous IFN to mount a second phase of host defense.

Pkr Gene Expression and Response to Pegylated Interferon plus Ribavirin Therapy in Chronic Hepatitis C

Pegylated interferon (PEG-IFN) alpha combined with ribavirin is the current standard treatment for hepatitis C, but around 50% of patients do not respond for reasons that are not fully understood. To explore the regulation of IFN-inducible protein kinase (PKR), we have measured PKR mRNA levels in peripheral blood mononuclear cells (PBMCs) and in liver biopsies from patients with chronic hepatitis C. PBMCs were also analysed after in vitro incubation with IFN and during antiviral therapy. Non-responders to PEG-IFN plus ribavirin had pre-treatment PKR mRNA levels in PBMCs (0.1+/-0.0074) and in liver (0.102+/-0.051) that were significantly higher than those of responders (PBMCs: 0.023+/-0.014, P=0.0005; liver: 0.034+/-0.020; P=0.0002). On the other hand, PKR mRNA levels in PBMCs were similar in non-responders and in responders after in vitro exposure to IFN (0.434+/-0.301 vs 0.403+/-0.222; P=NS) and during therapy (0.31+/-0.10 vs 0.30+/-0.12; P=NS). These results indicate that in hepatitis C, non-responsiveness to IFN-alpha is associated with pre-treatment up-regulation of the PKR gene, evidence that the infecting hepatitis C virus is able to stimulate endogenous IFN production, being resistant to its antiviral effect. On the other hand, the PKR gene response to exogenous IFN was similar in responders and non-responders, at least in PBMCs, suggesting that variations in its activation are not major determinants of the outcome of antiviral treatment.

Changes in gene expression induced by phosphorothioate oligodeoxynucleotides (including G3139) in PC3 prostate carcinoma cells are recapitulated at least in part by treatment with interferon-beta and -gamma.

G3139 is an antisense bcl-2 phosphorothioate oligodeoxyribonucleotide that is currently being evaluated in Phase III clinical trials in several human cancers. The aim of the present work was to further identify the apparent non-bcl-2-dependent mechanism of this action of this compound in PC3 prostate cancer cells. We performed Affymetrix U95A oligonucleotide microarray studies on mRNA isolated from cells treated with G3139 and related oligonucleotides. Hierarchical clustering revealed the presence of a set of genes of which the expression was elevated on both 1 and 3 days after oligonucleotide treatment. Significantly, the persistence of expression of the up-regulation of these genes, many of which are members of the IFN cascade, was greater for G3139 than for any other oligomer evaluated. Furthermore, many of the genes with the greatest up-regulation of expression are also those of which the expression is up-regulated after treatment of cells with IFNs. Treatment of PC3 cells with either IFN-beta or -gamma recapitulated some of the aspects of the molecular and phenotypic changes observed after treatment with a G3139/Lipofectin complex. These include down-regulation of bcl-2 protein expression itself, down-regulation of protein kinase C alpha protein expression (but not that of other protein kinase C isoforms), alteration in p21/Waf1/Cip1 protein expression, up-regulation of MHC-I cell surface expression, and profound suppression of cell growth in the absence of a notable increase in cellular apoptosis. However, G3139 (when complexed with Lipofectin) did not induce the up-regulation of expression of either type I or type II IFNs, nor could IFNs be found in conditioned media from treated cells. Oligonucleotide microarray experiments demonstrated that G3139 could induce elements of the IFN cascade in PC3 cells in vitro. In addition, the cellular phenotype obtained after treatment with exogenous IFN could, at least in part, recapitulate that obtained after G3139 treatment. Nevertheless, the oligonucleotide microarray experiments we performed also demonstrated that there are extremely large qualitative and quantitative differences between the two treatments.

Human cytomegalovirus UL83-coded pp65 virion protein inhibits antiviral gene expression in infected cells.

The initial interaction of human cytomegalovirus with fibroblasts triggers, and then partially blocks, an innate immune response pathway that leads to the induction of IFN-responsive genes and proinflammatory chemokines. Infection of fibroblasts with human cytomegalovirus inhibited their ability to respond to exogenous IFN. Consistent with the observation that the block did not depend on de novo viral protein synthesis, ectopic expression of the viral UL83-coded pp65, an abundant virion protein, inhibited IFN signaling. Furthermore, DNA array analysis showed that infection with a pp65-deficient mutant virus caused a much stronger induction of many IFN-response and proinflammatory chemokine RNAs than infection with wild-type virus. The nuclear DNA-binding activities of transcription factors NF-kappaB and IRF1 were induced to a much greater extent after infection with the pp65-deficient mutant than with wild-type virus. IFN-stimulated gene factor 3 DNA-binding was modestly enhanced, whereas IRF3 activity was not affected by mutation of pp65. Together, these results imply that pp65, which is delivered to newly infected cells in the virion, antagonizes a pathway that affects NF-kappaB and IRF1 and prevents the accumulation of mRNAs encoded by numerous cellular antiviral genes.

Respiratory syncytial virus (RSV) nonstructural (NS) proteins as host range determinants: a chimeric bovine RSV with NS genes from human RSV is attenuated in interferon-competent bovine cells.

Bovine respiratory syncytial virus (BRSV) escapes from cellular responses to alpha/beta interferon (IFN-alpha/beta) by a concerted action of the two viral nonstructural proteins, NS1 and NS2. Here we show that the NS proteins of human RSV (HRSV) are also able to counteract IFN responses and that they have the capacity to protect replication of an unrelated rhabdovirus. Even combinations of BRSV and HRSV NS proteins showed a protective activity, suggesting common mechanisms and cellular targets of HRSV and BRSV NS proteins. Although able to cooperate, NS proteins from BRSV and HRSV showed differential protection capacity in cells from different hosts. A chimeric BRSV with HRSV NS genes (BRSV h1/2) was severely attenuated in bovine IFN competent MDBK and Klu cells, whereas it replicated like BRSV in IFN-incompetent Vero cells or in IFN-competent human HEp-2 cells. After challenge with exogenous IFN-alpha, BRSV h1/2 was better protected than wild-type BRSV in human HEp-2 cells. In contrast, in cells of bovine origin, BRSV h1/2 was much less resistant to exogenous IFN than wild-type BRSV. These data demonstrate that RSV NS1 and NS2 proteins are major determinants of host range. The differential IFN escape capacity of RSV NS proteins in cells from different hosts provides a basis for rational development of attenuated live RSV vaccines.

Degradation of STAT1 and STAT2 by the V proteins of simian virus 5 and human parainfluenza virus type 2, respectively: consequences for virus replication in the presence of alpha/beta and gamma interferons.

Human cell lines were isolated that express the V protein of either simian virus 5 (SV5) or human parainfluenza virus type 2 (hPIV2); the cell lines were termed 2f/SV5-V and 2f/PIV2-V, respectively. STAT1 was not detectable in 2f/SV5-V cells, and the cells failed to signal in response to either alpha/beta interferons (IFN-alpha and IFN-beta, or IFN-alpha/beta) or gamma interferon (IFN-gamma). In contrast, STAT2 was absent from 2f/PIV2-V cells, and IFN-alpha/beta but not IFN-gamma signaling was blocked in these cells. Treatment of both 2f/SV5-V and 2f/PIV2-V cells with a proteasome inhibitor allowed the respective STAT levels to accumulate at rates similar to those seen in 2fTGH cells, indicating that the V proteins target the STATs for proteasomal degradation. Infection with SV5 can lead to a complete loss of both phosphorylated and nonphosphorylated forms of STAT1 by 6 h postinfection. Since the turnover of STAT1 in uninfected cells is longer than 24 h, we conclude that degradation of STAT1 is the main mechanism by which SV5 blocks interferon (IFN) signaling. Pretreatment of 2fTGH cells with IFN-alpha severely inhibited both SV5 and hPIV2 protein synthesis. However, and in marked contrast, pretreatment of 2fTGH cells with IFN-gamma had little obvious effect on SV5 protein synthesis but did significantly reduce the replication of hPIV2. Pretreament with IFN-alpha or IFN-gamma did not induce an antiviral state in 2f/SV5-V cells, indicating either that the induction of an antiviral state is completely dependent on STAT signaling or that the V protein interferes with other, STAT-independent cell signaling pathways that may be induced by IFNs. Even though SV5 blocked IFN signaling, the addition of exogenous IFN-alpha to the culture medium of 2fTGH cells 12 h after a low-multiplicity infection with SV5 significantly reduced the subsequent cell-to-cell spread of virus. The significance of the results in terms of the strategy that these viruses have evolved to circumvent the IFN response is discussed.

Inhibition of the Constitutive and Induced IFN-β Production by IL-4 and IL-10 in Murine Peritoneal Macrophages

We had previously reported that freshly harvested peritoneal macrophages (PM) are in a type I IFN-mediated antiviral state, which is lost during in vitro culture of PM, concomitantly with a progressive decline in the expression of IFN-β. We report herein that in vitro culture of PM in the presence of IL-4 or IL-10 results in an enhanced decay of the IFN-β-mediated antiviral state to vesicular stomatitis virus (VSV). Moreover, IL-4 and IL-10 inhibited the production of type I IFN induced by LPS or NDV infection, as assessed by IFN production and induction of IFN-mediated antiviral state. The accumulation and physiological turnover of IFN-β mRNA was not affected by IL-4 or IL-10. Finally, neither IL-10 nor IL-4 exerted any inhibitory effect on the antiviral activity induced by exogenous type-I IFN. These results suggest that Th2 cytokines, such as IL-4 and IL-10, act as negative regulators of the type I IFN-mediated antiviral response in PM and may represent stop signals for the constitutive or induced type I IFN expression in PM.