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IL-27 is a novel member of the IL-12 cytokine family. IL-27 has pro- and anti-inflammatory properties, and it controls the responses of adaptive immunity. It promotes the differentiation of naïve Th cells and suppresses the effector functions of Th17 cells. Biologically active IL-27 is a heterodimer composed of EBV-induced gene 3 (EBI3) and p28 proteins. We report that TLR-dependent expression of IL-27 in human macrophages is mediated by IFN-alpha. Stimulation of macrophages with agonists for TLR3 {polyinosinic:polycytidylic acid [poly(I:C)]}, TLR4 (LPS), or TLR7/8 (R848) results in concurrent expression of EBI3 and p28. The p28 expression is inhibited with neutralizing anti-IFN-alpha antibodies. Unlike poly(I:C), LPS, and R848, TLR2 agonist (S)-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys4-OH trihydrochloride does not stimulate macrophages to produce IFN-alpha, and therefore, it is not able to turn on the expression of p28. There is an IFN-stimulated response element (ISRE) in the p28 gene promoter. IFN-alpha enhances the expression of IFN regulatory factor 1 (IRF-1) in macrophages and induces binding of IRF-1 to the p28 ISRE site. The data provide a mechanistic basis for the IFN-alpha-mediated activation of IL-27. The data emphasize a role of IFN-alpha in immune responses, which rely on the recognition of pathogens by TLRs.

Epithelial cells of the lung are the primary targets for respiratory viruses. Virus-carried single-stranded RNA (ssRNA) can activate Toll-like receptors (TLRs) 7 and 8, whereas dsRNA is bound by TLR3 and a cytoplasmic RNA helicase, retinoic acid-inducible protein I (RIG-I). This recognition leads to the activation of host cell cytokine gene expression. Here we have studied the regulation of influenza A and Sendai virus-induced alpha interferon (IFN-alpha), IFN-beta, interleukin-28 (IL-28), and IL-29 gene expression in human lung A549 epithelial cells. Sendai virus infection readily activated the expression of the IFN-alpha, IFN-beta, IL-28, and IL-29 genes, whereas influenza A virus-induced activation of these genes was mainly dependent on pretreatment of A549 cells with IFN-alpha or tumor necrosis factor alpha (TNF-alpha). IFN-alpha and TNF-alpha induced the expression of the RIG-I, TLR3, MyD88, TRIF, and IRF7 genes, whereas no detectable TLR7 and TLR8 was seen in A549 cells. TNF-alpha also strongly enhanced IKK epsilon mRNA and protein expression. Ectopic expression of a constitutively active form of RIG-I (deltaRIG-I) or IKK epsilon, but not that of TLR3, enhanced the expression of the IFN-beta, IL-28, and IL-29 genes. Furthermore, a dominant-negative form of RIG-I inhibited influenza A virus-induced IFN-beta promoter activity in TNF-alpha-pretreated cells. In conclusion, IFN-alpha and TNF-alpha enhanced the expression of the components of TLR and RIG-I signaling pathways, but RIG-I was identified as the central regulator of influenza A virus-induced expression of antiviral cytokines in human lung epithelial cells.

Dendritic cells (DCs) respond to microbial infections by undergoing phenotypic maturation and by producing multiple cytokines. In the present study, we analyzed the ability of influenza A and Sendai viruses to induce DC maturation and activate tumor necrosis factor alpha (TNF-alpha), alpha/beta interferon (IFN-alpha/beta), and IFN-like interleukin-28A/B (IFN-lambda2/3) and IL-29 (IFN-lambda1) gene expression in human monocyte-derived myeloid DCs (mDC). The ability of influenza A virus to induce mDC maturation or enhance the expression of TNF-alpha, IFN-alpha/beta, interleukin-28 (IL-28), and IL-29 genes was limited, whereas Sendai virus efficiently induced mDC maturation and enhanced cytokine gene expression. Influenza A virus-induced expression of TNF-alpha, IFN-alpha, IFN-beta, IL-28, and IL-29 genes was, however, dramatically enhanced when cells were pretreated with IFN-alpha. IFN-alpha priming led to increased expression of Toll-like receptor 3 (TLR3), TLR7, TLR8, MyD88, TRIF, and IFN regulatory factor 7 (IRF7) genes and enhanced influenza-induced phosphorylation and DNA binding of IRF3. Influenza A virus also enhanced the binding of NF-kappaB to the respective NF-kappaB elements of the promoters of IFN-beta and IL-29 genes. In mDC IL-29 induced MxA protein expression and possessed antiviral activity against influenza A virus, although this activity was lower than that of IFN-alpha or IFN-beta. Our results show that in human mDCs viruses can readily induce the expression of IL-28 and IL-29 genes whose gene products are likely to contribute to the host antiviral response.

Toll-like receptors (TLRs) mediate host cell activation by various microbial components. TLR2, TLR3, TLR4, TLR7, TLR8, and TLR9 are the receptors that have been associated with virus-induced immune response. We have previously reported that all these TLRs, except TLR9, are expressed at mRNA levels in human monocyte-derived macrophages. Here we have studied TLR2, TLR3, TLR4, and TLR7/8 ligand-induced IFN-alpha, IFN-beta, IL-28, and IL-29 expression in human macrophages. IFN-alpha pretreatment of macrophages was required for efficient TLR3 and TLR4 agonist-induced activation of IFN-alpha, IFN-beta, IL-28, and IL-29 genes. TLR7/8 agonist weakly activated IFN-alpha, IFN-beta, IL-28, and IL-29 genes, whereas TLR2 agonist was not able to activate these genes. IFN-alpha enhanced TLR responsiveness in macrophages by up-regulating the expression of TLR3, TLR4, and TLR7. IFN-alpha also enhanced the expression of TLR signaling molecules MyD88, TIR domain-containing adaptor inducing IFN-beta, IkappaB kinase-epsilon, receptor interacting protein 1, and IFN regulatory factor 7. Furthermore, the activation of transcription factor IFN regulatory factor 3 by TLR3 and TLR4 agonists was dependent on IFN-alpha pretreatment. In conclusion, our results suggest that IFN-alpha sensitizes cells to microbial recognition by up-regulating the expression of several TLRs as well as adapter molecules and kinases involved in TLR signaling.

NK and T cell-derived IFN-gamma is a key cytokine that stimulates innate immune responses and directs adaptive T cell response toward Th1 type. IL-15, IL-18, and IL-21 have significant roles as activators of NK and T cell functions. We have previously shown that IL-15 and IL-21 induce the expression of IFN-gamma, T-bet, IL-12R beta 2, and IL-18R genes both in NK and T cells. Now we have studied the effect of IL-15, IL-18, and IL-21 on IFN-gamma gene expression in more detail in human NK and T cells. IL-15 clearly activated IFN-gamma mRNA expression and protein production in both cell types. IL-18 and IL-21 enhanced IL-15-induced IFN-gamma gene expression. IL-18 or IL-21 alone induced a modest expression of the IFN-gamma gene but a combination of IL-21 and IL-18 efficiently up-regulated IFN-gamma production. We also show that IL-15 activated the binding of STAT1, STAT3, STAT4, and STAT5 to the regulatory sites of the IFN-gamma gene. Similarly, IL-21 induced the binding of STAT1, STAT3, and STAT4 to these elements. IL-15- and IL-21-induced STAT1 and STAT4 activation was verified by immunoprecipitation with anti-phosphotyrosine Abs followed by Western blotting with anti-STAT1 and anti-STAT4 Abs. IL-18 was not able to induce the binding of STATs to IFN-gamma gene regulatory sites. IL-18, however, activated the binding of NF-kappa B to the IFN-gamma promoter NF-kappa B site. Our results suggest that both IL-15 and IL-21 have an important role in activating the NK cell-associated innate immune response.