Interferon-lambda contributes to innate immunity of mice against influenza A virus but not against hepatotropic viruses

Virus-infected cells secrete a broad range of interferon (IFN) subtypes which in turn trigger the synthesis of antiviral factors that confer host resistance. IFN-alpha, IFN-beta and other type I IFNs signal through a common universally expressed cell surface receptor, whereas IFN-lambda uses a distinct receptor complex for signaling that is not present on all cell types. Since type I IFN receptor-deficient mice (IFNAR1(0/0)) exhibit greatly increased susceptibility to various viral diseases, it remained unclear to which degree IFN-lambda might contribute to innate immunity. To address this issue we performed influenza A virus infections of mice which carry functional alleles of the influenza virus resistance gene Mx1 and which, therefore, develop a more complete innate immune response to influenza viruses than standard laboratory mice. We demonstrate that intranasal administration of IFN-lambda readily induced the antiviral factor Mx1 in mouse lungs and efficiently protected IFNAR1(0/0) mice from lethal influenza virus infection. By contrast, intraperitoneal application of IFN-lambda failed to induce Mx1 in the liver of IFNAR1(0/0) mice and did not protect against hepatotropic virus infections. Mice lacking functional IFN-lambda receptors were only slightly more susceptible to influenza virus than wild-type mice. However, mice lacking functional receptors for both IFN-alpha/beta and IFN-lambda were hypersensitive and even failed to restrict usually non-pathogenic influenza virus mutants lacking the IFN-antagonistic factor NS1. Interestingly, the double-knockout mice were not more susceptible against hepatotropic viruses than IFNAR1(0/0) mice. From these results we conclude that IFN-lambda contributes to inborn resistance against viral pathogens infecting the lung but not the liver.     

Effects of type I interferons on Friend retrovirus infection

The type I interferon (IFN) response plays an important role in the control of many viral infections. However, since there is no rodent animal model for human immunodeficiency virus, the antiviral effect of IFN-alpha and IFN-beta in retroviral infections is not well characterized. In the current study we have used the Friend virus (FV) model to determine the activity of type I interferons against a murine retrovirus. After FV infection of mice, IFN-alpha and IFN-beta could be measured between 12 and 48 h in the serum. The important role of type I IFN in the early immune defense against FV became evident when mice deficient in IFN type I receptor (IFNAR(-/-)) or IFN-beta (IFN-beta(-/-)) were infected. The levels of FV infection in plasma and in spleen were higher in both strains of knockout mice than in C57BL/6 wild-type mice. This difference was induced by an antiviral effect of IFN-alpha and IFN-beta and was most likely mediated by antiviral enzymes as well as by an effect of these IFNs on T-cell responses. Interestingly, the lack of IFNAR and IFN-beta enhanced viral loads during acute and chronic FV infection. Exogenous IFN-alpha could be used therapeutically to reduce FV replication during acute but not chronic infection. These findings indicate that type I IFN plays an important role in the immediate antiviral defense against Friend retrovirus infection.     

A family of interferon-induced Mx-related mRNAs encodes cytoplasmic and nuclear proteins in rat cells.

Mouse Mx protein, an interferon (IFN)-induced nuclear protein, confers selective resistance to influenza virus. We show here that, as with influenza virus-resistant Mx+ mouse embryo cells, influenza virus mRNA accumulation and protein synthesis are strongly inhibited in rat embryo cells treated with IFN-alpha/beta. IFN-alpha/beta induced in rat cells the synthesis of Mx-related mRNAs migrating on Northern (RNA) gels as two bands of about 3.5 and 2.5 kilobases which directed the synthesis of three electrophoretically distinct proteins called rat Mx proteins 1, 2, and 3. The three rat proteins were antigenically related to the mouse Mx protein but differed in molecular weight and intracellular location. Rat Mx protein 1 was found predominantly in the nucleus and, on the basis of several criteria, resembled the nuclear mouse Mx protein. It was induced by IFN-alpha/beta in all 28 inbred rat strains tested. Rat Mx proteins 2 and 3 differed from protein 1 at the carboxy terminus and were predominantly cytoplasmic like the human Mx homolog. Sequence data of partial cDNA clones indicate that three Mx-related genes, rather than one, exist in the rat.     

Interferon-λ Contributes to Innate Immunity of Mice against Influenza A Virus but Not against Hepatotropic Viruses

Virus-infected cells secrete a broad range of interferon (IFN) subtypes which in turn trigger the synthesis of antiviral factors that confer host resistance. IFN-α, IFN-β and other type I IFNs signal through a common universally expressed cell surface receptor, whereas IFN-λ uses a distinct receptor complex for signaling that is not present on all cell types. Since type I IFN receptor-deficient mice (IFNAR1^0/0) exhibit greatly increased susceptibility to various viral diseases, it remained unclear to which degree IFN-λ might contribute to innate immunity. To address this issue we performed influenza A virus infections of mice which carry functional alleles of the influenza virus resistance gene Mx1 and which, therefore, develop a more complete innate immune response to influenza viruses than standard laboratory mice. We demonstrate that intranasal administration of IFN-λ readily induced the antiviral factor Mx1 in mouse lungs and efficiently protected IFNAR1^0/0 mice from lethal influenza virus infection. By contrast, intraperitoneal application of IFN-λ failed to induce Mx1 in the liver of IFNAR1^0/0 mice and did not protect against hepatotropic virus infections. Mice lacking functional IFN-λ receptors were only slightly more susceptible to influenza virus than wild-type mice. However, mice lacking functional receptors for both IFN-α/β and IFN-λ were hypersensitive and even failed to restrict usually non-pathogenic influenza virus mutants lacking the IFN-antagonistic factor NS1. Interestingly, the double-knockout mice were not more susceptible against hepatotropic viruses than IFNAR1^0/0 mice. From these results we conclude that IFN-λ contributes to inborn resistance against viral pathogens infecting the lung but not the liver.     

The interferon system of teleost fish

Interferons (IFNs) are secreted proteins, which induce vertebrate cells into an antiviral state. In mammals, three families of IFNs (type I IFN, type II IFN and IFN-lambda) can be distinguished on the basis of gene structure, protein structure and functional properties. Type I IFNs, which include IFN-alpha and IFN-beta, are encoded by intron lacking genes and have a major role in the first line of defense against viruses. The human IFN-lambdas have similar biological properties as type I IFNs, but are encoded by intron containing genes. Type II IFN is identical to IFN-gamma, which is produced by T helper 1 cells in response to mitogens and antigens and has a key role in adaptive cell mediated immunity. IFNs, which show structural and functional properties similar to mammalian type I IFNs, have recently been cloned from Atlantic salmon, channel catfish, pufferfish, and zebrafish. Teleost fish appear to have at least two type I IFN genes. Phylogenetic sequence analysis shows that the fish type I IFNs form a group separated from the avian type I IFNs and the mammalian IFN-alpha, -beta and -lambda groups. Interestingly, the fish IFNs possess the same exon/intron structure as the IFN-lambdas, but show most sequence similarity to IFN-alpha. Recently, IFN-gamma genes have also been cloned from several fish species and shown to have the same exon/intron structure as mammalian IFN-gamma genes. The antiviral effect of mammalian type I IFN is exerted through binding to the IFN-alpha/beta-receptor, which triggers signal transduction through the JAK-STAT signal transduction pathway resulting in expression of Mx and other antiviral proteins. Putative IFN receptor genes have been identified in pufferfish. Several interferon regulatory factors and members of the JAK-STAT pathway have also been identified in various fish species. Moreover, Mx and several other interferon stimulated genes have been cloned and studied in fish. Furthermore, antiviral activity of Mx protein from Atlantic salmon and Japanese flounder has recently been demonstrated.     

Mx is dispensable for interferon-mediated resistance of chicken cells against influenza A virus

The type I interferon (IFN) system plays an important role in antiviral defense against influenza A viruses (FLUAV), which are natural chicken pathogens. Studies of mice identified the Mx1 protein as a key effector molecule of the IFN-induced antiviral state against FLUAV. Chicken Mx genes are highly polymorphic, and recent studies suggested that an Asn/Ser polymorphism at amino acid position 631 determines the antiviral activity of the chicken Mx protein. By employing chicken embryo fibroblasts with defined Mx-631 polymorphisms and retroviral vectors for the expression of Mx isoforms in chicken cells and embryonated eggs, we show here that neither the 631Asn nor the 631Ser variant of chicken Mx was able to confer antiviral protection against several lowly and highly pathogenic FLUAV strains. Using a short interfering RNA (siRNA)-mediated knockdown approach, we noted that the antiviral effect of type I IFN in chicken cells was not dependent on Mx, suggesting that some other IFN-induced factors must contribute to the inhibition of FLUAV in chicken cells. Finally, we found that both isoforms of chicken Mx protein appear to lack GTPase activity, which might explain the observed lack of antiviral activity.     

Type I IFN negatively regulates CD8+ T cell responses through IL-10-producing CD4+ T regulatory 1 cells

Pleiotropic, immunomodulatory effects of type I IFN on T cell responses are emerging. We used vaccine-induced, antiviral CD8(+) T cell responses in IFN-beta (IFN-beta(-/-))- or type I IFN receptor (IFNAR(-/-))-deficient mice to study immunomodulating effects of type I IFN that are not complicated by the interference of a concomitant virus infection. Compared with normal B6 mice, IFNAR(-/-) or IFN-beta(-/-) mice have normal numbers of CD4(+) and CD8(+) T cells, and CD25(+)FoxP3(+) T regulatory (T(R)) cells in liver and spleen. Twice as many CD8(+) T cells specific for different class I-restricted epitopes develop in IFNAR(-/-) or IFN-beta(-/-) mice than in normal animals after peptide- or DNA-based vaccination. IFN-gamma and TNF-alpha production and clonal expansion of specific CD8(+) T cells from normal and knockout mice are similar. CD25(+)FoxP3(+) T(R) cells down-modulate vaccine-primed CD8(+) T cell responses in normal, IFNAR(-/-), or IFN-beta(-/-) mice to a comparable extent. Low IFN-alpha or IFN-beta doses (500-10(3) U/mouse) down-modulate CD8(+) T cells priming in vivo. IFNAR- and IFN-beta-deficient mice generate 2- to 3-fold lower numbers of IL-10-producing CD4(+) T cells after polyclonal or specific stimulation in vitro or in vivo. CD8(+) T cell responses are thus subjected to negative control by both CD25(+)FoxP3(+) T(R) cells and CD4(+)IL-10(+) T(R1) cells, but only development of the latter T(R) cells depends on type I IFN.     

TRAF6 and MEKK1 play a pivotal role in the RIG-I-like helicase antiviral pathway

Type I interferons (IFN-alpha/beta) are essential for immune defense against viruses and induced through the actions of the cytoplasmic helicases, RIG-I and MDA5, and their downstream adaptor molecule IPS-1. TRAF6 and the downstream kinase TAK1 have been shown to be essential for the production of proinflammatory cytokines through the TLR/MyD88/TRIF pathway. Although binding of TRAF6 with IPS-1 has been demonstrated, the role of the TRAF6 pathway in IFN-alpha/beta production has not been fully understood. Here, we demonstrate that TRAF6 is critical for IFN-alpha/beta induction in response to viral infection and intracellular double-stranded RNA, poly(I:C). Activation of NF-kappaB, JNK, and p38, but not IRF3, was impaired in TRAF6-deficient mouse embryo fibroblasts in response to vesicular stomatitis virus and poly(I:C). However, TAK1 was not required for IFN-beta induction in this process, since normal IFN-alpha/beta production was observed in TAK1-deficient mouse embryo fibroblasts. Instead, another MAP3K, MEKK1, was important for the activation of the IFN-beta promoter in response to poly(I:C). Forced expression of MEKK1 in combination with IRF3 was sufficient for the induction of IFN-beta, whereas suppression of MEKK1 expression by small interfering RNA inhibited the induction of IFN-beta by poly(I:C). These data suggest that IPS-1 requires TRAF6 and MEKK1 to activate NF-kappaB and mitogen-activated protein kinases that are critical for the optimal induction of type I interferons.     

Mx1 causes resistance against influenza A viruses in the Mus spretus-derived inbred mouse strain SPRET/Ei

Inbred SPRET/Ei mice, derived from Mus spretus, were found to be extremely resistant to infection with a mouse adapted influenza A virus. The resistance was strongly linked to distal chromosome 16, where the interferon-inducible Mx1 gene is located. This gene encodes for the Mx1 protein which stimulates innate immunity to Orthomyxoviruses. The Mx1 gene is defective in most inbred mouse strains, but PCR revealed that SPRET/Ei carries a functional allele. The Mx1 proteins of M. spretus and A2G, the other major resistant strain derived from Mus musculus, share 95.7% identity. We were interested whether the sequence variations between the two Mx1 alleles have functional significance. To address this, we used congenic mouse strains containing the Mx1 gene from M. spretus or A2G in a C57BL/6 background. Using a highly pathogenic influenza virus strain, we found that the B6.spretus-Mx1 congenic mice were better protected against infection than the B6.A2G-Mx1 mice. This effect may be due to different Mx1 induction levels, as was shown by RT-PCR and Western blot. We conclude that SPRET/Ei is a novel Mx1-positive inbred strain useful to study the biology of Mx1.     

Interferon-regulated Mx genes are not responsive to interleukin-1, tumor necrosis factor, and other cytokines.

Accumulation of Mx gene products in cells of patients and experimental animals has been recognized as a useful marker for detecting minute quantities of biologically active interferon (IFN). Goetschy et al. (J. Goetschy, H. Zeller, J. Content, and M. A. Horisberger, J. Virol. 63:2616-2622, 1989) reported that not only IFNs but also interleukin-1 (IL-1) and tumor necrosis factor (TNF) were potent inducers of the human Mx genes. However, we observed no Mx induction in cultured human fibroblasts or in human peripheral blood mononuclear cells treated with various concentrations of IL-1 alpha or TNF-alpha. Mx induction was found in the spleens of mice treated with TNF-alpha or IL-1 alpha, but this effect could be neutralized with antibodies to murine IFN-alpha/beta. Of the other cytokines that we tested (IL-2, IL-6, and granulocyte-macrophage colony-stimulating factor), only IL-2 induced the Mx genes in peripheral blood mononuclear cells, but antibodies to human IFN-beta efficiently neutralized this effect. Our results thus indicate that IFNs are the only cytokines with intrinsic Mx-inducing activity.     

Critical role for the oligoadenylate synthetase/RNase L pathway in response to IFN-beta during acute ocular herpes simplex virus type 1 infection

We previously demonstrated that IFN-beta transgene treatment protects mouse trigeminal ganglia (TG) cells from acute HSV-1 infection in vitro. However, IFN-alpha6 transgene treatment does not provide protection against acute HSV-1 infection in vitro, even though equivalent levels of IFN are expressed with both transgene treatments. In the present study we show that IFN-beta transgene treatment before acute ocular HSV-1 infection protects mice from HSV-1-mediated mortality, whereas IFN-alpha6 transgene treatment does not reduce mortality. Treatment with the IFN-beta and IFN-alpha6 transgenes was associated with increased expression of oligoadenylate synthetase (OAS)1a mRNA in the eye. However, protein kinase R mRNA was not up-regulated in the eye. In TG, only IFN-beta transgene treatment reduced infectious virus levels. Furthermore, in the absence of a functional OAS pathway, corneal HSV-1 Ag expression was more widespread, and the ability of IFN-beta transgene treatment to reduce infectious HSV-1 in eyes and TG was lost. Along with selective up-regulation of OAS1a mRNA expression in TG from IFN-beta transgene-treated mice, we found increased levels of phospho-STAT1. Likewise, p38 MAPK phosphorylation was increased in TG from IFN-beta transgene-treated mice, compared with both IFN-alpha6 and vector-treated mice. We also observed a time-dependent increase in JNK phosphorylation in TG from IFN-beta transgene-treated vs IFN-alpha6 and vector-treated mice. Our results demonstrate that IFN-beta is a potent antiviral cytokine that exerts protection against ocular HSV-1 infection via selective up-regulation of OAS1a mRNA in TG and by altering the phosphorylation of proteins in antiviral signaling cascades.     

Mx protein: constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus

Mx+ mice are much more resistant to influenza virus than Mx- strains. The resistance is mediated by interferon (IFN) alpha/beta. After IFN treatment, Mx+ but not Mx- cells accumulate Mx protein and become specifically resistant to orthomyxoviruses. cDNA encoding Mx protein was cloned and sequenced. Southern analyses indicate that Mx- alleles derive from their Mx+ counterpart by deletions. IFN-treated Mx+ cells contained a 3.5 kb Mx mRNA, while Mx- cells showed only traces of shorter Mx RNA. Mx- cells transformed with Mx cDNA expressed Mx protein constitutively to varying extents; resistance of individual cells to influenza virus correlated with Mx protein expression. Thus, specific resistance to influenza virus in vivo may be attributed to Mx protein expression and is independent of other IFN-mediated effects.     

cDNA structures and regulation of two interferon-induced human Mx proteins.

Human cells treated with interferon synthesize two proteins that exhibit high homology to murine Mx1 protein, which has previously been identified as the mediator of interferon-induced cellular resistance of mouse cells against influenza viruses. Using murine Mx1 cDNA as a hybridization probe, we have isolated cDNA clones originating from two distinct human Mx genes, designated MxA and MxB. In human fibroblasts, expression of MxA and MxB is strongly induced by alpha interferon (IFN-alpha), IFN-beta, Newcastle disease virus, and, to a much lesser extent, IFN-gamma, MxA and MxB proteins have molecular masses of 76 and 73 kilodaltons, respectively, and their sequences are 63% identical. A comparison of human and mouse Mx proteins revealed that human MxA and mouse Mx2 are the most closely related proteins, showing 77% sequence identity. Near their amino termini, human and mouse Mx proteins contain a block of 53 identical amino acids and additional regions of very high sequence similarity. These conserved sequences are also present in a double-stranded RNA-inducible fish gene, which suggests that they may constitute a functionally important domain of Mx proteins. In contrast to mouse Mx1 protein, which accumulates in the nuclei of IFN-treated mouse cells, the two human Mx proteins both accumulate in the cytoplasm of IFN-treated cells.