Allan Randrup Thomsen

Lambda Interferon (IFN-λ), a Type III IFN, Is Induced by Viruses and IFNs and Displays Potent Antiviral Activity against Select Virus Infections In Vivo

ABSTRACT Type III interferons (IFNs) (interleukin-28/29 or lambda interferon [IFN-λ]) are cytokines with IFN-like activities. Here we show that several classes of viruses induce expression of IFN-λ1 and -λ2/3 in similar patterns. The IFN-λs were—unlike alpha/beta interferon (IFN-α/β)—induced directly by stimulation with IFN-α or -λ, thus identifying type III IFNs as IFN-stimulated genes. In vitro assays revealed that IFN-λs have appreciable antiviral activity against encephalomyocarditis virus (EMCV) but limited activity against herpes simplex virus type 2 (HSV-2), whereas IFN-α potently restricted both viruses. Using three murine models for generalized virus infections, we found that while recombinant IFN-α reduced the viral load after infection with EMCV, lymphocytic choriomeningitis virus (LCMV), and HSV-2, treatment with recombinant IFN-λ in vivo did not affect viral load after infection with EMCV or LCMV but did reduce the hepatic viral titer of HSV-2. In a model for a localized HSV-2 infection, we further found that IFN-λ completely blocked virus replication in the vaginal mucosa and totally prevented development of disease, in contrast to IFN-α, which had a more modest antiviral activity. Finally, pretreatment with IFN-λ enhanced the levels of IFN-γ in serum after HSV-2 infection. Thus, type III IFNs are expressed in response to most viruses and display potent antiviral activity in vivo against select viruses. The discrepancy between the observed antiviral activity in vitro and in vivo may suggest that IFN-λ exerts a significant portion of its antiviral activity in vivo via stimulation of the immune system rather than through induction of the antiviral state.

An important role for type III interferon (IFN-lambda/IL-28) in TLR-induced antiviral activity.

Type III IFNs (IFN-λ/IL-28/29) are cytokines with type I IFN-like antiviral activities, which remain poorly characterized. We herein show that most cell types expressed both types I and III IFNs after TLR stimulation or virus infection, whereas the ability of cells to respond to IFN-λ was restricted to a narrow subset of cells, including plasmacytoid dendritic cells and epithelial cells. To examine the role of type III IFN in antiviral defense, we generated IL-28Rα-deficient mice. These mice were indistinguishable from wild-type mice with respect to clearance of a panel of different viruses, whereas mice lacking the type I IFN receptor (IFNAR−/−) were significantly impaired. However, the strong antiviral activity evoked by treatment of mice with TLR3 or TLR9 agonists was significantly reduced in both IL-28RA−/− and IFNAR−/− mice. The type I IFN receptor system has been shown to mediate positive feedback on IFN-αβ expression, and we found that the type I IFN receptor system also mediates positive feedback on IFN-λ expression, whereas IL-28Rα signaling does not provide feedback on either type I or type III IFN expression in vivo. Finally, using bone-marrow chimeric mice we showed that TLR-activated antiviral defense requires expression of IL-28Rα only on nonhemopoietic cells. In this compartment, epithelial cells responded to IFN-λ and directly restricted virus replication. Our data suggest type III IFN to target a specific subset of cells and to contribute to the antiviral response evoked by TLRs.

Sensing of RNA Viruses: a Review of Innate Immune Receptors Involved in Recognizing RNA Virus Invasion

ABSTRACT Our knowledge regarding the contribution of the innate immune system in recognizing and subsequently initiating a host response to an invasion of RNA virus has been rapidly growing over the last decade. Descriptions of the receptors involved and the molecular mechanisms they employ to sense viral pathogen-associated molecular patterns have emerged in great detail. This review presents an overview of our current knowledge regarding the receptors used to detect RNA virus invasion, the molecular structures these receptors sense, and the involved downstream signaling pathways.

Functional epistasis on a common MHC haplotype associated with multiple sclerosis

Genes in the major histocompatibility complex (MHC) encode proteins important in activating antigen-specific immune responses. Alleles at adjacent MHC loci are often in strong linkage disequilibrium; however, little is known about the mechanisms responsible for this linkage disequilibrium. Here we report that the human MHC HLA-DR2 haplotype, which predisposes to multiple sclerosis, shows more extensive linkage disequilibrium than other common caucasian HLA haplotypes in the DR region and thus seems likely to have been maintained through positive selection. Characterization of two multiple-sclerosis-associated HLA-DR alleles at separate loci by a functional assay in humanized mice indicates that the linkage disequilibrium between the two alleles may be due to a functional epistatic interaction, whereby one allele modifies the T-cell response activated by the second allele through activation-induced cell death. This functional epistasis is associated with a milder form of multiple-sclerosis-like disease. Such epistatic interaction might prove to be an important general mechanism for modifying exuberant immune responses that are deleterious to the host and could also help to explain the strong linkage disequilibrium in this and perhaps other HLA haplotypes.

Role of CD40 ligand and CD28 in induction and maintenance of antiviral CD8+ effector T cell responses.

The primary aim of this report was to evaluate the immune responses of CD40 ligand-deficient (CD40L−/−) mice infected with two viruses known to differ markedly in their capacity to replicate in the host. Lymphocytic choriomeningitis virus (LCMV) is a natural mouse pathogen that replicates widely and extensively, whereas vesicular stomatitis virus (VSV) spreads poorly. We found that the primary response of CD40L−/− mice toward VSV is significantly impaired; proliferation of both CD4+ and CD8+ cells is reduced 2- to 3-fold, few CD8+ cells acquire an activated phenotype, and little functional activity is induced. Very similar results were obtained in VSV-infected, CD28-deficient mice. In contrast, neither CD40L nor CD28 was required for induction of a primary CD8+ response toward LCMV. Surprisingly, lack of CD4+ T cells had no impact on the primary immune response toward any of the viruses, even though the CD40 ligand dependence demonstrated for VSV would be expected to be associated with CD4 dependence. Upon coinfection of VSV-infected mice with LCMV, the requirement for CD40 ligand (but not CD28) could be partially bypassed, as evidenced by a 3-fold increase in the frequency of VSV-specific CD8+ T cells on day 6 postinfection. Finally, despite the fact that the primary LCMV-specific CD8+ response is virtually unimpaired in CD40L−/− mice, their capacity to maintain CD8+ effector activity and to permanently control the infection is significantly reduced. Thus, our results demonstrate that the importance of CD40/CD40L interaction for activation of CD8+ T cells varies between viruses and over time.

TLR2 and TLR9 Synergistically Control Herpes Simplex Virus Infection in the Brain

Viruses are recognized by the innate immune system through pattern recognition receptors (PRRs). For instance, HSV virions and genomic DNA are recognized by TLR2 and TLR9, respectively. Although several viruses and viral components have been shown to stimulate cells through TLRs, only very few studies have defined essential roles for single TLRs in innate immune defense in vivo. This could suggest that PRRs act in concert to mount the first line of defense against virus infections. To test this hypothesis we have examined the host response of C57BL/6, TLR2−/−, TLR9−/−, and TLR2/9−/− mice toward HSV-2 infection. After a systemic infection, the cytokine serum response was markedly reduced in the double knockout mice, but only partly affected in either strain of the single knockout mice. This was supported by in vitro data showing that HSV-induced cytokine expression relayed on TLR2 and TLR9 in a cytokine- and cell type-dependent manner. With respect to the cellular response to infection, we found that recruitment but not activation of NK cells was impaired in TLR2/9−/− mice. Importantly, the viral load in the brain, but not liver, was significantly higher in the brain of TLR2/9−/− mice whereas the viral loads in organs of single knockout mice were statistically indistinguishable from C57BL/6 mice. In the brain we found that TNF-α and the IFN-stimulated gene CXCL9 were expressed during infection and were dependent on either TLR2 or TLR9. Thus, TLR2 and TLR9 synergistically stimulate innate antiviral activities, thereby protecting against HSV infection in the brain.

CXCL10 Is the Key Ligand for CXCR3 on CD8+ Effector T Cells Involved in Immune Surveillance of the Lymphocytic Choriomeningitis Virus-Infected Central Nervous System

IFN-γ-inducible protein 10/CXCL10 is a chemokine associated with type 1 T cell responses, regulating the migration of activated T cells through binding to the CXCR3 receptor. Expression of both CXCL10 and CXCR3 are observed during immunopathological diseases of the CNS, and this receptor/ligand pair is thought to play a central role in regulating T cell-mediated inflammation in this organ site. In this report, we investigated the role of CXCL10 in regulating CD8+ T cell-mediated inflammation in the virus-infected brain. This was done through analysis of CXCL10-deficient mice infected intracerebrally with lymphocytic choriomeningitis virus, which in normal immunocompetent mice induces a fatal CD8+ T cell-mediated meningoencephalitis. We found that a normal antiviral CD8+ T cell response was generated in CXCL10-deficient mice, and that lack of CXCL10 had no influence on the accumulation of mononuclear cells in the cerebrospinal fluid. However, analysis of the susceptibility of CXCL10-deficient mice to lymphocytic choriomeningitis virus-induced meningitis revealed that these mice just like CXCR3-deficient mice were partially resistant to this disease, whereas wild-type mice invariably died. Furthermore, despite marked up-regulation of the two remaining CXCR3 ligands: CXCL9 and 11, we found a reduced accumulation of CD8+ T cells in the brain parenchyma around the time point when wild-type mice succumb as a result of CD8+ T cell-mediated inflammation. Thus, taken together these results indicate a central role for CXCL10 in regulating the accumulation of effector T cells at sites of CNS inflammation, with no apparent compensatory effect of other CXCR3 ligands.

Persistent Virus Infection despite Chronic Cytotoxic T-Lymphocyte Activation in Gamma Interferon-Deficient Mice Infected with Lymphocytic Choriomeningitis Virus

ABSTRACT The role of gamma interferon (IFN-γ) in the permanent control of infection with a noncytopathic virus was studied by comparing immune responses in wild-type and IFN-γ-deficient (IFN-γ −/−) mice infected with a slowly invasive strain of lymphocytic choriomeningitis virus (LCMV Armstrong). While wild-type mice rapidly cleared the infection, IFN-γ −/− mice became chronically infected. Virus persistence in the latter mice did not reflect failure to generate cytotoxic T-lymphocyte (CTL) effectors, as an unimpaired primary CTL response was observed. Furthermore, while ex vivo CTL activity gradually declined in wild-type mice, long-standing cytolytic activity was demonstrated in IFN-γ −/− mice. The prolonged effector phase in infected IFN-γ −/− mice was associated with elevated numbers of CD8+ T cells. Moreover, a higher proportion of these cells retained an activated phenotype and was actively cycling. However, despite the increased CD8+ T-cell turnover, which might have resulted in depletion of the memory CTL precursor pool, no evidence for exhaustion was observed. In fact, at 3 months postinfection we detected higher numbers of LCMV-specific CTL precursors in IFN-γ −/− mice than in wild-type mice. These findings indicate that in the absence of IFN-γ, CTLs cannot clear the infection and are kept permanently activated by the continuous presence of live virus, resulting in a delicate new balance between viral load and immunity. This interpretation of our findings is supported by mathematical modeling describing the effect of eliminating IFN-γ-mediated antiviral activity on the dynamics between virus replication and CTL activity.

TLR3 deficiency renders astrocytes permissive to herpes simplex virus infection and facilitates establishment of CNS infection in mice

Herpes simplex viruses (HSVs) are highly prevalent neurotropic viruses. While they can replicate lytically in cells of the epithelial lineage, causing lesions on mucocutaneous surfaces, HSVs also establish latent infections in neurons, which act as reservoirs of virus for subsequent reactivation events. Immunological control of HSV involves activation of innate immune pattern-recognition receptors such as TLR3, which detects double-stranded RNA and induces type I IFN expression. Humans with defects in the TLR3/IFN pathway have an elevated susceptibility to HSV infections of the CNS. However, it is not known what cell type mediates the role of TLR3 in the immunological control of HSV, and it is not known whether TLR3 sensing occurs prior to or after CNS entry. Here, we show that in mice TLR3 provides early control of HSV-2 infection immediately after entry into the CNS by mediating type I IFN responses in astrocytes. Tlr3-/- mice were hypersusceptible to HSV-2 infection in the CNS after vaginal inoculation. HSV-2 exhibited broader neurotropism in Tlr3-/- mice than it did in WT mice, with astrocytes being most abundantly infected. Tlr3-/- mice did not exhibit a global defect in innate immune responses to HSV, but astrocytes were defective in HSV-induced type I IFN production. Thus, TLR3 acts in astrocytes to sense HSV-2 infection immediately after entry into the CNS, possibly preventing HSV from spreading beyond the neurons mediating entry into the CNS.

The importance of lytic and nonlytic immune responses in viral infections

Antiviral immune effector mechanisms can be divided broadly into lytic and nonlytic components. We use mathematical models to investigate the fundamental question of which type of response is required to combat different types of viral infection. According to our model, the relative roles of the two types of component depend on the cytopathicity of the virus relative to its rate of replication. If the viral cytopathicity is low relative to the rate of viral replication, the model predicts that a combination of lytic and nonlytic effector mechanisms is likely to be required to resolve the disease, particularly if the virus replicates at a fast rate. By contrast, if viral cytopathicity is high relative to the replication rate of the virus, then lytic and nonlytic mechanisms can, in principle, resolve the infection independently. We discuss our findings in the context of specific viral infections and use our model to interpret empirical data.