A Study On The Expression, Regulation And Antiviral Activity Of Interferon-λ In Human Neuronal Cells

Interferon lambda(IFN-λ), the newly discovered typeⅢfamily of IFNs, comprises threestructurally related cytokines, IFN-λ1, IFN-λ2 and IFN-λ3. IFN-λgene expression hasbeen identified mainly in antigen-presenting cells after Toll-like receptor(TLR) triggeringor a panel of different virus infection. Once binding to its heterodimeric receptor complexcomposed of interleukin-28 receptorαsubunit(IL-28Rα) and IL-10Rβ, IFN-λactivatesboth janus kinases-signal transducers and activators of transcription(Jak-STAT) pathwayand the mitogen-activated protein(MAP) kinases pathway, through which it exerts antiviral,antitumor and immunoregulatory activities as typeⅠIFNs.Until now, most of the studies on IFN-λhave focused on immune system. Little is knownabout whether central nerve system(CNS) expresses IFN-λas well as its function in CNS.Thus we examined the gene expression and regulation of IFN-λin human neuronal cells. Inaddition, we investigated the antiviral ability of IFN-λand the mechanism(s) involved inthe IFN-λaction. This study has provided not only direct experimental evidence tosupport the notion that IFN-λplays a role in the innate immunity against viral infection inhuman neurons, but also therapitic potential for HSV-1 or HIV induced neurological andpsychiatric disorders.1. Expression and regulation of IFN-λand IFN-λreceptor in human neuronal cellsIn order to determine whether human neuronal cells express IFN-λand IFN-λreceptor, wefirst examined endogenous IFN-λmRNA expression in human neuronal cells. Humanbrain tissue and a set of human neuronal cells, including primary human neurons, NT2-Nneurons and neuroblastoma cell lines(CHP212, and SH-SY5Y) expressed IFN-λ1 mRNA.However, IFN-λ2/3 mRNA was not detectable in these cells. There is a gradual and steady increase in the levels of IFN-λ1 during the course of NT2-N differentiation.Human neuronal cells also express all 10 TLRs. The activation of TLR-3 bypolyriboinosinic polyribocytidylic acid (PolyⅠ: C) induces IFN-λ, while other TLR ligands(TLR-4, 5, 7, 8, 9) had little impact on IFN-λexpression in human neuronal cells. ThisTLR-3-mediated induction of IFN-λwas dose-and time-dependent. Human brain tissue,primary human neurons and the neuronal cell lines also expressed mRNA for both IL-28Rαand IL-10Rβ, the heterodimeric IFN-λreceptor complex. Moreover, we observed agradual and steady increase in the levels of IL-28Rαand IL-10Rβduring the course ofneuronal differentiation.2. IFN-λinhibits HSV-1 infection of human neuronal cellsTo confirm the antiviral ability of IFN-λin human neuronal cells, we further examinedwhether IFN-λcould inhibit HSV-1 infection. Human NT2-N neuron and humanneuroblastoma cell line CHP212 could effectively support HSV-1 replication. Infectedcells became rounding and detached and HSV-1 DNA gradually increased over the timecourse of the infection. IFN-λsuppressed HSV-1 infection at different level of virusreplication. By using real-time PCR, we observed both IFN-λ1 and IFN-λ2 significantlyinhibited HSV-1 DNA synthesis in dose-and time-dependent manner. The degree ofIFN-λ-mediated inhibition of HSV-1 was similar to that of typeⅠIFN. The anti-HSV-1effect of IFN-λ1 and IFN-λ2 was also supported by the reduced expression of HSV-1gDprotein detected by immunohistochemistry method. Antibody to IL-10Rβpartiallyneutralized the inhibition of HSV-1 by both IFN-λ1 and IFN-λ2, suggesting the anti-HSV-1effect of IFN-λis mediated through its Specific receptor.3. IFN-λinhibits HIV-1 replication in human neuronal cellsTo further demonstrate antiviral the function of IFN-λin human neuronal cells, we examinewhether IFN-λhas the ability to supress HIV-1. Because HIV-1 does not infect humanneurons that have no CD4 receptor for HIV, we used pseudotyped HIV-1 for the experiment.HIV-1 virions pseudotyped with the envelope from amphotropic murine leukemia virus(MLV) (HIV-1 entry receptor independent) were used to study the impact of IFN-λon HIVreplication. IFN-λinhibited pseudotyped HIV-1 infection of the neuronal cells andinduced the expression of apolipoprotein B mRNA editing enzyme 3G(APOBEC3G) andAPOBEC3F, the anti-HIV intracellular factors, suggesting induction of APOBEC3G/F may be one of the mechanisms for the inhibitory effect of IFN-λon HIV-1.We also determined whether endogenous IFN-λin human neurons has the ability to inhibitHIV-1. We determined that activation of TLR-3 could inhibit HIV-1 replication. Andantibody to IL-10Rβsignificantly neutralized the antiviral activity of PolyⅠ: C, while controlIgG had little effect. These findings suggest the role of endogenous IFN-λinTLR-3-mediated anti-HIV-1 effect.4. IFN-λactivates IFN-αpathwayTypeⅠIFNs play a vital role in host resistance to viral infection. After recognizing themolecular pattern of virus, TLR in host cell will be activated and sequentially induce IFNregulatory factors(IRFs). IRFs is phosphalated and translocated into neuclei, then bindsto TypeⅠIFN promoter, resulting in IFN-α/βproduction. After secreting from the cells,TypeⅠIFN binds to their receptors and activates Jak-stat signaling, resulting in inducing aspectrum of IFN stimulated genes(ISGs). To investigation of the mechanisms involved inthe IFN-λaction, we examine whether IFN-λactivates typeⅠIFN pathway. Both IFN-λ1and IFN-λ2 induce the expression of TLR-3, 9 and IRF-7, resulting in the activation ofIFN-α. IFN-λalso induces all ISGs we detect, such as myxovirus resistance-A(MxA),oligoadenylate synthetase-1(OAS-1), RNA-activated protein kinase R(PKR) and ISG56.These findings suggest the activation of IFN-αpathway may be involved in the antiviraleffect of IFN-λin human neuronal cells.Conclusion: Human neuronal cells expressed endogenous IFN-λ1, but not IFN-λ2/3.Human neuronal cells also expressed IFN-λreceptor complex, IL-28Rαand IL-10Rβ.Activation of TLR-3 by PolyⅠ:C induced the expression of both IFN-λ1 and IFN-λ2/3 in theneuronal cells. IFN-λwas able to inhibit pseudotyped HIV replication and HSV-1 in theneuronal cells. These data provide direct and compelling evidence that there isintracellular expression and regulation of IFN-λin the neuronal cells, which may have animportant role in human innate neuronal protection against viral infections in the CNS.Future studies are needed in order to determine the in vivo antiviral role of IFN-λin theCNS.