CpG and poly(I:C) stimulation of dendritic cells and fibroblasts limits herpes simplex virus type 1 infection in an IFNβ-dependent and -independent way

Viral activation of toll-like receptors (TLRs) on dendritic cells (DCs) leads to production of various cytokines, including antiviral type I interferons (IFNs). Synthetic ligands specific for TLRs are also able to induce the production of type I IFNs (IFNα/β) by DCs, suggesting that these ligands have potential as antiviral drugs. In this in vitro study we extensively investigated the antiviral activity of various TLR ligands. Mouse bone marrow (BM) cells were differentiated into plasmacytoid and conventional DCs (pDCs and cDCs), stimulated with various TLR ligands and tested the antiviral abilities of collected supernatants in an in vitro herpes simplex virus type 1 (HSV-1) infection model. We observed a significant IFNβ-, (but not IFNα-) dependent reduction in HSV-1 infection when a mixed pDC/cDC population was stimulated with the TLR9 ligand CpG. In the absence of pDCs, TLR stimulation resulted in less pronounced antiviral effects. The most pronounced antiviral effect was observed when both DC subsets were stimulated with poly(I:C). A similar noticeable antiviral effect was observed when fibroblasts (L929 cells) were stimulated directly with poly(I:C). These poly(I:C)-mediated antiviral effects were only partially IFNβ-mediated and probably TLR independent. These data demonstrate that TLR ligands are not only able to produce type I IFN but can indeed act as antiviral drugs. In particular poly(I:C), which exerts its antiviral effects even in the absence of DCs, may become a promising drug e.g. to prevent respiratory infections by topical intranasal application.     

Selenium suppresses the activation of transcription factor NF-kappa B and IRF3 induced by TLR3 or TLR4 agonists

Toll-like receptors (TLRs) play an important role in recognition of microbial components and induce innate immune responses by recognizing invading microbial pathogens leading to the activation of the adaptive immune responses. The microbial components trigger the activation of two downstream signaling pathways of TLRs; MyD88- and TRIF-dependent pathways leading to the expression of pro-inflammatory cytokines and type I interferons (IFNs). The MyD88- and TRIF-dependent pathways lead to the activation of NF-kappa B and IRF3 through the activation of IKK-beta and TBK1, respectively. Selenium is an essential trace element nutrient possessing anticarcinogenic properties. Here, we attempted to identify the molecular targets of selenium in TLR signaling pathways. Selenium inhibited NF-kappaB activation induced by poly[I:C] (TLR3 agonist), LPS (TLR4 agonist) or overexpression of MyD88 or IKK-beta which is the key kinase of MyD88-dependent signaling pathway. Selenium inhibited IRF3 activation induced by poly[I:C], LPS or the overexpression of TRIF or TBK1. Selenium also suppressed the expression of COX-2 and iNOS and the endogenous IFN beta mRNA induced by poly[I:C] or LPS. Therefore, our results suggest that selenium can modulate both MyD88- and TRIF-dependent signaling pathways of TLRs leading to decreased inflammatory gene expression.     

Interferons and their role In inflammation.

Cytokines are pleiotropic molecules showing a wide variety of biologic functions on various cells and tissues, and several different cytokines exert similar and overlapping functions on certain cells. Interferons (IFNs), among the first cytokines identified, play a crucial role in human disease. The IFN cytokine family consists of type I IFNs (IFN-a and IFN-b) and type II IFN (IFN-g). In the first decades of IFN research, type I IFNs were considered primarily as viral inhibitors, whereas type II IFN, also termed "immune IFN", was generally considered to be uniquely involved in immune reactions. This view has changed considerably in the last years. The importance of type I IFNs in inflammation, immunoregulation and T-cell responses has been identified and has changed dramatically our interpretation of the biological relevance of type I and II IFNs. Recent data suggest that IFN-a is a multifunctional immunomodulatory cytokine with profound effects on the cytokine cascade including several anti-inflammatory properties, whereas IFN-g remains a classical proinflammatory cytokine. These different effects on critical mediators of inflammation may also explain why type I and II IFNs are clinically successful in different diseases. These newly identified immunoregulatory and anti-inflammatory functions of type I IFNs may be of importance in the treatment of diseases such as chronic viral hepatitis or multiple sclerosis and help to explain some of the mechanisms of IFNs.     

Novel plant-derived recombinant human interferons with broad spectrum antiviral activity

Type I interferons (IFNs) are potent mediators of the innate immune response to viral infection. IFNs released from infected cells bind to a receptor (IFNAR) on neighboring cells, triggering signaling cascades that limit further infection. Subtle variations in amino acids can alter IFNAR binding and signaling outcomes. We used a new gene crossbreeding method to generate hybrid, type I human IFNs with enhanced antiviral activity against four dissimilar, highly pathogenic viruses. Approximately 1400 novel IFN genes were expressed in plants, and the resultant IFN proteins were screened for antiviral activity. Comparing the gene sequences of a final set of 12 potent IFNs to those of parent genes revealed strong selection pressures at numerous amino acids. Using three-dimensional models based on a recently solved experimental structure of IFN bound to IFNAR, we show that many but not all of the amino acids that were highly selected for are predicted to improve receptor binding.     

Novel plant-derived recombinant human interferons with broad spectrum antiviral activity

Type I interferons (IFNs) are potent mediators of the innate immune response to viral infection. IFNs released from infected cells bind to a receptor (IFNAR) on neighboring cells, triggering signaling cascades that limit further infection. Subtle variations in amino acids can alter IFNAR binding and signaling outcomes. We used a new gene crossbreeding method to generate hybrid, type I human IFNs with enhanced antiviral activity against four dissimilar, highly pathogenic viruses. Approximately 1400 novel IFN genes were expressed in plants, and the resultant IFN proteins were screened for antiviral activity. Comparing the gene sequences of a final set of 12 potent IFNs to those of parent genes revealed strong selection pressures at numerous amino acids. Using three-dimensional models based on a recently solved experimental structure of IFN bound to IFNAR, we show that many but not all of the amino acids that were highly selected for are predicted to improve receptor binding.     

Distinct functions of IRF-3 and IRF-7 in IFN-alpha gene regulation and control of anti-tumor activity in primary macrophages

Type I IFN (IFN-alpha/beta) have important biological functions ranging from immune cell development and activation, to tumor cell killing and most importantly inhibition of virus replication. Following viral infection or activation of Toll-like receptors (TLRs) via distinct ligands, IFN-alpha/beta are produced. Two members of the interferon regulatory factor (IRF) family - IRF-3 and IRF-7 - are the major modulators of IFN gene expression. Activation of IRF-3 and IRF-7 by TBK1/IKKvarepsilon mediated phosphorylation promotes IFN gene expression and potentiates the production of IFN responsive genes important to the development of an effective antiviral immune response. IFN treatment can augment anti-tumor properties and they are potentially key players in cancer therapy. For example, adoptive transfer of IFN-gamma-activated macrophages can mediate tumor cell killing via direct cell-cell contact, as well as release of soluble cytotoxic pro-inflammatory molecules. A recent study investigated whether IRF-3 and IRF-7 could mediate the acquisition of new anti-tumor effector functions in macrophages. Adenovirus mediated transduction of the active form of IRF-7 into primary macrophages resulted in the production of type I IFN, upregulation of target genes including TRAIL and increased tumoricidal activity of macrophages; in contrast, the active form of IRF-3 led to induction of cell death. These studies indicate that IRF-7 transduced macrophages may be an attractive candidate for in vivo adoptive therapy of cancer.     

Antiviral activity of type I interferons and interleukins 29 and 28a (type III interferons) against Apeu virus

Interferons (IFNs) are cytokines with important immunomodulatory activity in vertebrates. Although type I IFNs and interleukins (IL) 29 and 28a (type III IFNs) bind to different cellular receptors and have distinct structures, most of their biological activities are redundant. Apeu virus (APEUV) is a member of the Bunyaviridae family isolated from the Brazilian rain forest. In this paper we evaluated the antiviral activity of type I and type III IFNs against APEUV. All tested IFNs were able to induce an antiviral state against the virus in a dose-dependent way. The activity of type III IFNs did not need the presence of type I IFNs. Mixing both types of IFNs did not improve the biological activity of each type alone. The tested IFNs were also able to protect human peripheral blood mononuclear cells from infection. IFN alpha2, IFN beta, IL-29 and IL-28a induced the expression of 2′,5′-oligoadenylate synthetase (2′5′OAS) and 6–16 genes. Although MxA gene was related to antiviral activity against Bunyaviruses, there was no induction of MxA in our model. We were able to show activity of type I and type III IFNs against a RNA virus, and that this activity is not dependent on MxA gene.     

Antiviral activity of type I interferons and interleukins 29 and 28a (type III interferons) against Apeu virus

Interferons (IFNs) are cytokines with important immunomodulatory activity in vertebrates. Although type I IFNs and interleukins (IL) 29 and 28a (type III IFNs) bind to different cellular receptors and have distinct structures, most of their biological activities are redundant. Apeu virus (APEUV) is a member of the Bunyaviridae family isolated from the Brazilian rain forest. In this paper we evaluated the antiviral activity of type I and type III IFNs against APEUV. All tested IFNs were able to induce an antiviral state against the virus in a dose-dependent way. The activity of type III IFNs did not need the presence of type I IFNs. Mixing both types of IFNs did not improve the biological activity of each type alone. The tested IFNs were also able to protect human peripheral blood mononuclear cells from infection. IFN alpha2, IFN beta, IL-29 and IL-28a induced the expression of 2′,5′-oligoadenylate synthetase (2′5′OAS) and 6–16 genes. Although MxA gene was related to antiviral activity against Bunyaviruses, there was no induction of MxA in our model. We were able to show activity of type I and type III IFNs against a RNA virus, and that this activity is not dependent on MxA gene.     

The important roles of type I interferon and interferon-inducible genes in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a severe autoimmune disease that causes multiple-organ dysfunction mainly affecting women in their childbearing years. Type I IFN synthesis is usually triggered by viruses, and its production is tightly regulated and limited in time in health individuals. However, many patients with systemic autoimmune diseases including SLE have signs of aberrant production of type I interferon (IFN) and display an increased expression of IFN-inducible genes. Continuous type I IFNs derived from activated plasmacytoid dendritic cells (pDCs) by interferogenic immune complexes (ICs) and migration of these cells to tissues both break immune tolerance and promote an on-going autoimmune reaction in human body. By the means of detecting type I IFNs and IFN-inducible genes, it can help with diagnosis and evaluation of SLE in early stage and more efficiently. Anti-IFN-α monoclonal antibodies in SLE patients were recently reported and is now being investigated in phase II clinical trails. In this review, we focus on recent research progress in type I IFN and IFN-inducible genes. Possible mechanisms behind the dysregulated type I IFN system in SLE and how they contribute to the development of an autoimmune process, and act as a biomarker and therapeutic target will be reviewed.     

TBK1-targeted suppression of TRIF-dependent signaling pathway of toll-like receptor 3 by auranofin

Toll-like receptors (TLRs) play an important role in induction of innate immune responses. The stimulation of TLRs by microbial components triggers two branches of downstream signaling pathways: myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent signaling pathways. Auranofin, a sulfur-containing gold compound (Au[I]), has been widely used for the treatment of rheumatoid arthritis. Since dysregulation of TLRs can lead to severe systemic inflammatory and joint destructive process in rheumatoid arthritis, auranofin-mediated modulation of TLR activation may have therapeutic potential against such diseases. Previously, we demonstrated that auranofin suppressed TLR4 signaling pathway by inhibiting TLR4 dimerization induced by LPS. Here, we examined the effect of auranofin on signal transduction via the TRIF-dependent pathway induced by a TLR3 agonist. Auranofin inhibited nuclear factor-κB and interferon (IFN) regulatory factor 3 (IRF3) activation induced by polyinosinic-polycytidylic acid (poly[I:C]). Auranofin inhibited poly[I:C]-induced phosphorylation of IRF3 as well as IFN-inducible genes such as IFN inducible protein-10. Furthermore, auranofin inhibited TBK1 kinase activity in vitro. All the results suggest that auranofin suppress TLR signaling at multiple steps.     

TANK-binding kinase 1 as a novel therapeutic target for viral diseases

Introduction: TANK-binding kinase 1 (TBK1) is vital for the induction of antiviral innate immune responses. Both RNA and DNA viral infection induces TBK1 activation, triggers phosphorylation of interferon regulatory factor (IRF) 3 and subsequent expression of type I interferons (IFNs; IFN-α/β). Type I IFNs can induce the expression of numerous antiviral genes called interferon-stimulated genes (ISGs) to build a remarkable antiviral state and limit viral replication. Thus, optimal TBK1 activity is crucial for IRF3-induced type I IFNs expression and ISGs-mediated viral elimination.

Areas covered: This review provides an overview of the diverse roles of TBK1 in antiviral innate immune responses, the regulatory mechanisms of TBK1 activity and the implication in antiviral development.

Expert opinion: TBK1 is a key kinase against antiviral infection via inducing type I IFNs expression. Multiple types of post-translational modifications of TBK1 tightly regulate TBK1 activity and subsequent TBK1-dependent antiviral responses. The identified regulators of TBK1 unveil regulatory mechanisms of host antiviral innate immunity and immuno-escape mechanism of virus provide strategies to control viral diseases by modulating TBK1 activity.     

The role of myeloid receptors on murine plasmacytoid dendritic cells in induction of type I interferon

This study tested the hypothesis that a set of predominantly myeloid restricted receptors (F4/80, CD36, Dectin-1, CD200 receptor and mannan binding lectins) and the broadly expressed CD200 played a role in a key function of plasmacytoid DC (pDC), virally induced type I interferon (IFN) production. The Dectin-1 ligands zymosan, glucan phosphate and the anti-Dectin-1 monoclonal antibody (mAb) 2A11 had no effect on influenza virus induced IFNα/β production by murine splenic pDC. However, mannan, a broad blocking reagent against mannose specific receptors, inhibited IFNα/β production by pDC in response to inactivated influenza virus. Moreover, viral glycoproteins (influenza virus haemagglutinin and HIV-1 gp120) stimulated IFNα/β production by splenocytes in a mannan-inhibitable manner, implicating the function of a lectin in glycoprotein induced IFN production. Lastly, the effect of CD200 on IFN induction was investigated. CD200 knock-out macrophages produced more IFNα than wild-type macrophages in response to polyI:C, a MyD88-independent stimulus, consistent with CD200's known inhibitory effect on myeloid cells. In contrast, blocking CD200 with an anti-CD200 mAb resulted in reduced IFNα production by pDC-containing splenocytes in response to CpG and influenza virus (MyD88-dependent stimuli). This suggests there could be a differential effect of CD200 on MyD88 dependent and independent IFN induction pathways in pDC and macrophages. This study supports the hypothesis that a mannan-inhibitable lectin and CD200 are involved in virally induced type I IFN induction.     

Suppression of MyD88- and TRIF-dependent signaling pathways of Toll-like receptor by (-)-epigallocatechin-3-gallate, a polyphenol component of green tea

Toll-like receptors (TLRs) play an important role in recognition of microbial components and induction of innate immunity. The microbial components trigger the activation of two downstream signaling pathways of TLRs; MyD88- and/or TRIF-dependent pathways leading to activation of NF-kappaB. (-)-Epigallocatechin-3-gallate (EGCG), a flavonoid found in green tea, is known to inhibit NF-kappaB activation induced by many pro-inflammatory stimuli. EGCG was shown to inhibit the activity of IKKbeta which is the key kinase in the canonical pathway for NF-kappaB activation in MyD88-dependent pathway of TLRs. However, it is not known whether EGCG inhibits TRIF-dependent pathway through which more than 70% of lipopolysaccharide (LPS)-induced genes are regulated. Therefore, we attempted to identify the molecular target of EGCG in TRIF-dependent pathways of TLR3 and TLR4. EGCG inhibited the activation of IFN regulatory factor 3 (IRF3) induced by LPS, poly[I:C], or the overexpression of TRIF. The inhibition of IRF3 activation by EGCG was mediated through the suppression of the kinase activity of TBK1. However, EGCG did not inhibit activation of IRF3 induced by overexpression of constitutively active IRF3. These results suggest that the molecular target of EGCG is TBK1 in TRIF-dependent signaling pathways of TLR3 and TLR4. Therefore, our results suggest that green tea flavonoids can modulate both MyD88- and TRIF-dependent signaling pathways of TLRs and subsequent inflammatory target gene expression.     

Toll-like receptor 7-mediated enhancement of contextual fear memory in mice

Toll-like receptor (TLR) 7 recognizes viral single-stranded RNA and triggers production of the type I interferons (IFNs) IFN-α and IFN-β. Imiquimod, a synthetic TLR7 ligand, induces production of type I IFNs and is used clinically as an antiviral and antitumor drug. In the present study, we examined the effect of imiquimod on conditioned and innate fear behaviors in mice. Imiquimod was administered 2, 4, or 15 h before contextual fear conditioning. Imiquimod treatment 4 or 15 h before fear conditioning significantly enhanced context-dependent freezing behavior. This imiquimod-induced enhancement of fear-related behaviors was observed 120 h after fear conditioning. In contrast, imiquimod failed to enhance context-dependent freezing behavior in TLR7 knockout mice. Imiquimod had no significant effect on pain threshold or on innate fear-related behavior, as measured by the elevated plus-maze. The levels of type I IFN mRNA in the brain were significantly increased at 2 h after imiquimod treatment. Imiquimod also increased interleukin (IL)-1β mRNA expression in the brain at 4 h following administration, while mRNA expression of F4/80, a macrophage marker, was unaffected by imiquimod treatment. Our findings suggest that TLR7-mediated signaling enhances contextual fear memory in mice, possibly by inducing the expression of type I IFNs and IL-1β in the brain.     

Type I Interferons as Regulators of Human Antigen Presenting Cell Functions

Type I interferons (IFNs) are pleiotropic cytokines, initially described for their antiviral activity. These cytokines exhibit a long record of clinical use in patients with some types of cancer, viral infections and chronic inflammatory diseases. It is now well established that IFN action mostly relies on their ability to modulate host innate and adaptive immune responses. Work in recent years has begun to elucidate the mechanisms by which type I IFNs modify the immune response, and this is now recognized to be due to effects on multiple cell types, including monocytes, dendritic cells (DCs), NK cells, T and B lymphocytes. An ensemble of results from both animal models and in vitro studies emphasized the key role of type I IFNs in the development and function of DCs, suggesting the existence of a natural alliance between these cytokines and DCs in linking innate to adaptive immunity. The identification of IFN signatures in DCs and their dysregulation under pathological conditions will therefore be pivotal to decipher the complexity of this DC-IFN interaction and to better exploit the therapeutic potential of these cells.