Early expression of IFN-alpha/beta and iNOS in the brains of Venezuelan equine encephalitis virus-infected mice.

To investigate the roles of type I interferon (IFN-alpha/beta) and other mediators of innate immune responses (e.g., inducible nitric oxide synthase [iNOS]) in early dissemination of Venezuelan equine encephalitis virus (VEE) infection, we used mice with targeted deletions in either their IFN-alpha/beta-receptor (IFNAR-1-/-) or interferon regulatory factor 2 (IRF-2-/-) genes. Following footpad infection, both IFNAR-1-/- and IRF-2-/- mice were more susceptible than control mice to VEE. The IFNAR-1-/- mice also exhibit accelerated VEE dissemination to serum, spleen, and brain, and compared with control mice, they evidenced faster kinetics in the upregulation of proinflammatory genes. In contrast, in IRF-2-/- mice, iNOS gene induction was completely absent following peripheral virulent VEE infection. In evaluating the role of cells involved in iNOS production, primary microglial cell cultures were found to be highly permissive to VEE infection. Moreover, VEE infection increased levels of nitric oxide (NO) in resting microglial cultures but decreased NO production in IFN-gamma-stimulated microglia. Thus, these findings suggest that reactive nitrogen species play an important contributory role in VEE dissemination and survival of the host. Our results further suggest the necessity for a carefully balanced host response that follows a middle course between immunopathology and insufficient inflammatory response to VEE infection.     

Paramyxovirus Sendai virus V protein counteracts innate virus clearance through IRF-3 activation, but not via interferon, in mice

The present study was undertaken to clarify the role of Sendai virus (SeV) V protein, which has been shown to downregulate IFN-beta induction through inhibition of IRF-3 activation, in viral pathogenesis. Mice infected with rSeV mutants, deficient in V expression or expressing V lacking the C-terminus, had several-fold higher IFN activity levels in the lungs than those in wild-type virus-infected mice, and the mutant viruses were rapidly excluded from the lung from the early phase of infection before induction of acquired immunity. In addition, the unique early clearance of the mutants did not occur in IRF-3 knockout (KO) mice. However, high titers of IFN were detected even in the infected KO mice. Furthermore, early clearance of the mutant viruses was also observed in IFN signaling-deficient mice, IFN-alpha/beta receptor KO mice and STAT1 KO mice. These results indicate that SeV V protein counteracts IRF-3-mediated innate antiviral immunity for efficient virus replication and pathogenesis in mice, but it is not IFN.     

Antiviral activity of bacteria-derived human alpha interferons against encephalomyocarditis virus infection of mice.

Bacteria-derived human leukocyte interferon (IFN) subtypes, IFN-alpha A, -alpha B, and -alpha D, and two hybrid IFNs, IFN-alpha AD and -alpha DA, were examined for both in vitro and in vivo antiviral activity. Two of these materials in highly purified form (IFN-alpha D and -alpha D) protect mice against lethal doses of encephalomyocarditis virus infection. A single dose of 1 microgram of protein of IFN-alpha D 3 h before infection conferred protection in both BDF1 and CD-1 mice against encephalomyocarditis virus infection, and multiple treatments with IFN-alpha D or IFN-alpha AD extend the mean survival time of infected mice. On a weight basis, IFN-alpha AD was approximately 100-fold more effective than IFN-alpha D. There is a direct correlation between the antiviral activity of the various human IFN species in L-929 cells and in mice for both single and multiple treatments before infection, but none of the cloned human IFN subtypes were effective when administered 24 h after infection. Mixtures of the two parental materials, IFN-alpha A and -alpha D, were not as protective as the hybrid molecule IFN-alpha AD, suggesting that IFNs with unique and altered species specificity can be produced by recombinant DNA methods.     

Multiple sclerosis and hepatitis C virus infection are associated with single nucleotide polymorphisms in interferon pathway genes.

We have studied 35 single nucleotide polymorphisms (SNPs) in the interferon (IFN) pathway to determine their contribution to multiple sclerosis (MS) and hepatitis C virus (HCV) infection. A total of 182 patients with MS, 103 patients with chronic hepatitis C, and 118 control subjects were enrolled in the study. Of the 35 SNPs studied, 3 were in IFN-alpha receptor (IFNAR-1), 10 in IFN-alpha/beta receptor (IFNAR-2), 9 in Stat1, 5 in Stat2, and 8 in IFN regulatory factor-1 (IRF-1). Compared to controls, Stat1 gene polymorphisms were significantly more frequent in MS patients (rs# 2066802 OR = 7.46, 95% CI = 2.22-25.10; rs# 1547550 OR = 1.69, 95% CI = 1.01-2.81) and in HCV patients (rs# 2066802 OR = 5.95, 95% CI = 1.55-22.81; rs# 1547550 OR = 2.30, 95% CI = 1.24-4.24). Also one IRF-1 gene SNP was associated with MS (rs# 2070721 OR = 2.05, 95% CI = 1.03-4.09), and four IRF-1 gene SNPs were associated with HCV infection (rs# 2070721 OR = 2.59, 95% CI = 1.23-5.43; rs# 2070723 OR = 4.8, 95% CI = 1.26-18.20; rs# 2070728 OR = 9.81, 95% CI = 1.21-79.4; rs# 2070729 OR = 3.6, 95% CI = 1.23-10.48; rs# 839 OR = 4.67, 95%CI = 1.29-16.87). Characteristic nucleotide combinations on single chromosomes (haplotype) generated block structures, including SNPs, that differed between patients and controls. Using a permutation test to detect differences in haplotype distribution between groups, the CCATTGA and the CCGAA haplotypes in the IRF-1 gene were more frequent in MS (p = 0.03) and in HCV patients (p = 0.001) than in controls. In conclusion, our data show that genetic variants in the IRF-1 and Stat1 genes of the IFN pathway are associated with MS and HCV infection.     

Effect of deficiency of the double-stranded RNA-dependent protein kinase, PKR, on antiviral resistance in the presence or absence of ribonuclease L: HSV-1 replication is particularly sensitive to deficiency of the major IFN-mediated enzymes.

Control of viral replication by interferon (IFN) is thought to be principally mediated by the 2',5'-oligoadenylate synthetase (OAS)/RNAse L, double-stranded dependent protein kinase (PKR), and myxovirus resistance protein (Mx) pathways. In this study, we monitored the constitutive and IFN-induced antiviral activity in mouse embryo fibroblasts lines derived from mice with targeted disruption of either PKR or PKR/RNAse L genes. At high multiplicity of infection (moi = 10), the absence of PKR had no effect on replication of vesicular stomatitis virus (VSV) but moderately enhanced encephalomyocarditis virus (EMCV) growth and greatly increased replication of herpes simplex virus-1 (HSV-1). Replication of EMCV, HSV-1, and VSV was modestly higher in PKR-/- RNAse L-/- fibroblasts when compared with control cells. Although the antiviral action of IFN-alpha was unaffected by the absence of PKR, IFN action was significantly impaired in the double knockout cells but was dependent on the stage of the virus cycle. At early stages, it appeared that anti-EMCV and anti-HSV-1 action of IFN-alpha was significantly compromised, although weak residual antiviral activity was seen. The action of IFN-alpha against VSV was specifically compromised at a late stage of virus replication. The results showed that PKR is an important mediator in constitutive resistance against HSV-1 and that RNAse L is also necessary for the full antiviral activity of IFN against a variety of viruses. These results supported the existence of novel pathways aimed toward specific stages of the virus life cycle.     

Tissue selectivity of interferon-stimulated gene expression in mice infected with Dam(+) versus Dam(-) Salmonella enterica serovar Typhimurium strains

The host interferon (IFN) system plays an important role in protection against microbial infections. Salmonella enterica serovar Typhimurium is highly virulent in the mouse model, whereas mutants that lack DNA adenine methylase (Dam(-)) are highly attenuated and elicit fully protective immune responses against murine typhoid fever. We examined the expression of IFN-responsive genes in several mouse tissues following infection with Dam(+) or Dam(-) Salmonella. Infection of mice with Dam(+) Salmonella resulted in the induction of host genes known to be indicators of IFN bioactivity and regulated by either IFN-alpha/beta (Mx1) or IFN-gamma (class II transactivator protein [CIITA] and inducible nitric oxide synthase [iNOS]) or by both IFN-alpha/beta and IFN-gamma (RNA-specific adenosine deaminase [ADAR1] and RNA-dependent protein kinase [PKR]) in a tissue-specific manner compared to uninfected animals. Since the Mx1 promoter is IFN-alpha/beta specific and the Mx1 gene is not inducible directly by IFN-gamma, these data suggest a role of IFN-alpha/beta in the host response to Salmonella infection. Mice infected with Dam(-) Salmonella showed reduced expression of the same set of IFN-stimulated genes (ISGs) as that observed after infection with wild-type Salmonella. The reduced capacity to induce ISGs persisted in Dam(-)-vaccinated mice after challenge with the virulent (Dam(+)) strain. Finally, although no Dam(-) organisms were recovered from the liver or spleen after oral infection of mice, ADAR, PKR, Mx, and CIITA expression levels were elevated in these tissues relative to those in uninfected mice, suggestive of the distant action of a signaling molecule(s) in the activation of ISG expression.     

Comparative study of various immunomodulators for macrophage and natural killer cell activation and antiviral efficacy against exotic RNA viruses

Several immunomodulators were compared for immunomodulatory and antiviral activity in B6C3F1 female mice. Our results demonstrate that murine recombinant gamma interferon (rIFN-G), human recombinant alpha A/D interferon (rIFN-A), ampligen (a polyribonucleotide) and CL246,738 modulate nonspecific immunity and are effective antiviral agents in vivo. Administration of each of these agents 1 day before cell harvest induced high levels of splenic natural killer (NK) cell activity against YAC-1 target cells. rIFN-G was also a potent activator of peritoneal macrophages (M phi), as evidenced by high levels of antitumor activity and changes in ectoenzyme phenotype that is characteristic of tumoricidal M phi. rIFN-A, ampligen and CL246,738 induced moderate to low levels of M phi activation by these criteria. In vivo protection experiments showed that repeated therapeutic treatment with rIFN-A protected mice against i.p. infection with Venezuelan equine encephalitis (an alpha togavirus, VEE), Banzi (a flavivirus) and herpes simplex virus type 2 (HSV-2). Similar treatment with rIFN-G was effective against VEE and HSV-2, but ineffective against Banzi virus. A single prophylactic i.p. dose of ampligen 1 day before virus challenge was very effective against Banzi virus, moderately effective against HSV-2, and ineffective against VEE and Caraparu (a bunyavirus) infection. A single prophylactic oral dose of CL246,738 provided almost complete protection of mice against VEE, Banzi, and HSV-2, and also increased the mean survival time for Caraparu infected mice. Collectively, these results indicate that rIFN-A, r-IFN-G, ampligen and CL246,738 may be useful in prophylactic or early therapeutic treatment of several serious virus infections. Since these agents stimulate NK cells and M phi, their antiviral activity may result, in part, from the alterations they induce in the natural immune system.     

Virulence criteria for Brucella abortus strains as determined by interferon regulatory factor 1-deficient mice

Interferon regulatory factor 1-deficient (IRF-1(-/-)) mice infected with virulent Brucella abortus 2308 at 5 x 10(5) CFU developed acute hepatitis similar to many natural hosts but, unlike natural hosts, IRF-1(-/-) mice were unable to resolve infection and died. In contrast, IRF-1(-/-) mice survived when infected at 5 x 10(5) CFU with several attenuated Brucella strains (S19, RB51, cbp, and cyd). The survival of infected IRF-1(-/-) mice is likely a function of the level of virulence of each Brucella strain and the extent of retained immunity. Further, these findings suggest that adaptive immunity may be important to the survival of IRF-1(-/-) mice since attenuated Brucella strains can protect IRF-1(-/-) mice against lethal challenge with virulent Brucella: Using the IRF-1(-/-) mouse model, the following set of criteria were identified to define Brucella virulence: (i) the day of death for 50% of mice infected with 5 x 10(5)CFU of Brucella, (ii) the extent of liver toxicity, and (iii) the minimum immunizing dose of Brucella to protect against challenge with virulent S2308. Thus, IRF-1(-/-) mice are important to determining the level of Brucella virulence, to evaluating Brucella mutants for attenuation, and to investigating adaptive immunity in brucellosis.     

Role of interferon in lethality and lymphoid atrophy induced by Coxsackievirus B3 infection in mice.

To assess the importance of interferon (IFN) in the pathology of coxsackievirus B3 (CVB-3) infection, we evaluated both mortality rate and lymphoid involution in young adult BALB/C mice infected with lethal doses of the virus and treated either with anti-IFN antibody or with murine IFN-alpha/beta. Administration of antibody to IFN caused a profound worsening of the pathology and an increase in the mortality rate in infected animals. Treatment with murine IFN exerted a significant ameliorative effect on lethality when administered concomitantly with or soon after virus infection. The extent of this protection was correlated with the plasma levels of exogenous or endogenous IFN at 6 h postinfection, whereas no correlation with IFN titers was found later. The effects of IFN apparently were not directly mediated by antiviral effects, because at the times studied, no relation was found between IFN levels and virus titers, at least in the plasma of the infected animals. Lymphoid atrophy, assessed by measuring spleen weight, was only partially reversed by early IFN treatment. These data suggest that IFN production is critical during the early phases of infection, whereas it does not seem to play a significant protective role at later stages.     

Production of type I (alpha/beta) interferon after virus infection of cloned, alloantigen-sensitized mouse T lymphocytes

Mouse T lymphocytes sensitized to alloantigens were cloned by limiting dilution in the presence of interleukin 2. Clones were tested for surface markers Thy-1, Lyt-1 and Lyt-2, and for cytotoxic function. Production of interferon (IFN) by clones either (a) stimulated with allogeneic cells; (b) activated with concanavalin A (Con A); or (c) infected with Semliki Forest virus or Newcastle disease virus were assayed. All clones produced IFN upon Con A stimulation and most after virus infection. Analysis of the IFN produced by a single clone, using anti-IFN antisera, showed that while Con A stimulation induced production of type II IFN (IFN-gamma), the IFN produced after virus infection was type I IFN (IFN-alpha/beta).     

Suppression of innate immune cytokines and interferon regulatory factor-1 by endogenous interferon-alpha in response to respiratory syncytial virus in neonate mononuclear cells

Respiratory syncytial virus (RSV) infections are extremely common in early childhood but are most severe in infants in the first few months of life. Unresponsive adaptive immunity and hyporesponsive innate immunity were previously found to be the typical responses of neonate mononuclear cells (MCs) to live RSV. Investigating the mechanism of innate immune hyporesponsiveness in neonate MCs to live RSV revealed that in contrast to the previously reported low expression of interferon (IFN)-gamma, IFN-alpha expression in response to live RSV was significantly greater than that observed in adult MCs. Inhibition of live RSV-induced IFN-alpha with anti-IFN-alpha antibodies in neonate MCs led to significant increases in innate cytokine [IFN-gamma, interleukin (IL)-12, IL-18 and tumor necrosis factor (TNF)-alpha] but not adaptive immune cytokine [IL-2] production. Although MCs from adults responded to live RSV with upregulation of interferon regulatory factor-1 (IRF-1) mRNA, IRF-1 mRNA in RSV-treated neonate MCs was not detectable. However, in the presence of anti-IFN-alpha antibodies, live RSV induced detectable IRF-1 mRNA expression in neonate MCs. These data support the possibility that the severity of early life RSV-induced illnesses may occur via a mechanism in which live RSV induces IFN-alpha that in turn leads to innate immune suppression in neonate MCs.     

Oromucosal interferon therapy: marked antiviral and antitumor activity.

Oromucosal administration of murine interferon-alpha/beta (IFN-alpha/beta) or individual recombinant species of murine IFN-alpha, IFN-beta, or IFN-gamma or recombinant human IFN-alpha1-8, which is active in the mouse, exerted a marked antiviral activity in mice challenged systemically with a lethal dose of encephalomyocarditis virus (EMCV), vesicular stomatitis virus (VSV), or varicella zoster virus (VZV). The effects observed were dose dependent and similar in magnitude to those observed following parenteral administration of the same dose of IFN. No antiviral activity was observed after oromucosal administration of murine IFN-alpha/beta in animals in which the IFN receptor had been inactivated by homologous recombination. In contrast to parenteral treatment, oromucosal IFN therapy was found to be ineffective when IFNs were administered before virus infection. Oromucosal administration of IFN-alpha also exerted a marked antitumor activity in mice injected i.v. with highly malignant Friend erythroleukemia cells or other transplantable tumors, such as L1210 leukemia, which has no known viral etiology, the EL4 tumor, or the highly metastatic B16 melanoma. These results show that high doses of IFN can be administered by the oromucosal route apparently without ill effect, raising the possibility that the oromucosal route will prove to be an effective means of administering high doses of IFN that are clinically effective but poorly tolerated.     

Role of IFN-alpha/beta and IL-12 in the activation of natural killer cells and interferon-gamma production during experimental infection with Trypanosoma cruzi

Control of Trypanosoma cruzi infection depends largely upon the production of interferon (IFN)-gamma. During experimental infection this cytokine is produced early, mainly by natural killer (NK) cells and later by T cells. As NK cells have been reported to participate in defence against T. cruzi, it is of importance to study the regulation of NK cell functions during infection with the parasite. Several innate cytokines regulate NK cell activity, among them being interferon (IFN)-alpha and IFN-beta (type 1 IFNs) and interleukin (IL)-12, which have all been reported to be involved in protection against T. cruzi. The role of these cytokines in regulation of NK cell functions and disease outcome were studied by infection of mutant mice lacking the IFN-alpha/beta receptor (IFNalpha/betaR-/-) or IL-12 (IL-12-/-) with T. cruzi. IFNalpha/betaR-/- mice were unable to activate the cytotoxic response but produced IFN-gamma, and were not more susceptible than controls. IL-12-/- mice were extremely susceptible and failed to produce T cell-derived IFN-gamma and nitric oxide (NO), although NK cytotoxicity was induced. The results indicate that IL-12 protects against T. cruzi by initiating T cell-mediated production of IFN-gamma, but that endogenous IFN-alpha/beta and NK cell cytotoxicity are not of major importance in defence.     

Induction of the human protein P56 by interferon, double-stranded RNA, or virus infection

P56 is the most abundant protein induced by interferon (IFN) treatment of human cells. To facilitate studies on its induction pattern and cellular functions, we expressed recombinant P56 as a hexahistidine-tagged protein in Escherichia coli and purified it to apparent homogeneity using affinity chromatography. A polyclonal antibody raised against this recombinant protein was used to show that P56 is primarily a cytoplasmic protein. Cellular expression of P56 by transfection did not inhibit the replication of vesicular stomatitis virus and encephalomyocarditis virus. P56 synthesis was rapidly induced by IFN-beta, and the protein had a half-life of 6 h. IFN-gamma or poly(A)(+) could not induce the protein, but poly(I)-poly(C) or an 85-bp synthetic double-stranded RNA efficiently induced it. Similarly, infection of GRE cells, which are devoid of type I IFN genes, by vesicular stomatitis virus, encephalomyocarditis virus, or Sendai virus caused P56 induction. Surprisingly, Sendai virus could also induce P56 in the mutant cell line P2.1, which cannot respond to either IFN-alpha/beta or double-stranded RNA. Induction of P56 in the P2.1 cells and the parental U4C cells by virus infection was preceded by activation of IRF-3 as judged by its translocation to the nucleus from the cytoplasm.     

Type I interferon is a powerful inhibitor of in vivo HIV-1 infection and preserves human CD4(+) T cells from virus-induced depletion in SCID mice transplanted with human cells.

Although several studies are available on the in vitro inhibitory activities of type I interferon (IFN) on HIV-1 replication, the role of these cytokines in the pathogenesis of AIDS is still matter of conjecture. Both beneficial and adverse effects have been envisaged and considered as a possible rationale for the development of either IFN or anti-IFN therapies in HIV-1-infected patients. In the present study, we have evaluated the efficacy of human type I IFN on HIV-1 infection and virus-induced depletion of human CD4 T cells in two models established in SCID mice. In SCID mice transplanted with human U937 cells (U937-SCID mouse model), continuous treatment with type I consensus IFN (CIFN) resulted in a total suppression of HIV-1 infection. This inhibitory effect was superior to that obtained after AZT treatments. Results from an ensemble of experiments in SCID mice transplanted with either control or genetically modified human U937 cells transduced with a Tat-inducible IFN-alpha gene (LTR-IFN-A2 U937) indicated that low levels of IFN-alpha, produced locally as a result of virus infection, were extremely effective in inhibiting acute HIV infection and virus replication. Of interest, LTR-IFN-A2 U937 cells conferred a strong anti-HIV-1 protection to coinjected bystander U937 cells. Notably, experiments with SCID mice reconstituted with human PBL (hu-PBL-SCID mouse model) showed that treatment with CIFN inhibited HIV-1 replication more effectively than AZT treatment. Remarkably, treatment with CIFN resulted in a clear-cut protection from the virus-induced depletion of human CD4 T cells, which was also associated with the generation of an antibody response toward HIV-1 antigens in 50% of the virus-injected xenografts. These results suggest that type I IFN efficiently preserves human CD4(+) cells from virus-induced damage in hu-PBL-SCID mice, not only by inducing an antiviral state in target cells but also by stimulating anti-HIV-1 human immune responses in vivo.