Interferons induce an antiviral state in human pancreatic islet cells

Enterovirus infections, in particular those with Coxsackieviruses, have been linked to the development of type 1 diabetes (T1D). Although animal models have demonstrated that interferons (IFNs) regulate virus-induced T1D by acting directly on the beta cell, little is known on the human pancreatic islet response to IFNs. Here we show that human islet cells respond to IFNs by expressing signature genes of antiviral defense. We also demonstrate that they express three intracellular sensors for viral RNA, the toll like receptor 3 (TLR3) gene, the retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated gene-5 (MDA-5), which induce type I IFN production in infected cells. Finally, we show for the first time that the IFN-induced antiviral state provides human islets with a powerful protection from the replication of Coxsackievirus. This may be critical for beta cell survival and protection from virus-induced T1D in humans.     

Sec13 is a positive regulator of VISA-mediated antiviral signaling

Viral infection triggers the innate antiviral immune response that rapidly produces type I interferons in most cell types to combat viruses invading. Upon viral infection, the cytoplasmic RNA sensors RIG-I/MDA5 recognize viral RNA, and then RIG-I/MDA5 is transported to mitochondria interacting with VISA through the CARD domain. From there, VISA recruits downstream antiviral signaling pathways molecules, such as TRAFs and TBK1. Eventually, IRF3 is phosphorylated and type I IFNs are induced to fight as the first line of defense against viruses. However, it remains unclear how VISA acts as a scaffold to assemble the signalosome in RIG-I-mediated antiviral signaling. Here, we demonstrated Sec13 as a novel component that was involved in VISA-mediated antiviral signaling pathway. The co-immunoprecipitation assays showed that Sec13 specifically interacts with VISA. Overexpression of Sec13 increases VISA’s aggregation and ubiquitination and significantly enhances the phosphorylation and dimerization of IRF3, facilitating the IFN-β production. Conversely, the knockdown of Sec13 attenuates Sendai virus-induced and VISA-mediated IRF3 activation and the production of IFNβ, thus weakens antiviral immune activity.     

Recognition of viruses by innate immunity

Summary: The innate immune system plays critical roles in recognizing viral infections and evoking initial anti-viral responses. Nucleotides from RNA viruses are recognized by retinoic acid-inducible gene I (RIG-I)-like helicases (RLHs) and Toll-like receptors (TLRs), and the recognition triggers signaling cascades that induce anti-viral mediators such as type I interferons (IFNs) and pro-inflammatory cytokines. The RLH signaling pathways play essential roles in the recognition of RNA viruses in various cells, with the exception of plasmacytoid dendritic cells (pDCs). However, TLRs are important for the production of type I IFNs in pDCs but not in other cell types. The contributions of RLHs and TLRs to the production of type I IFNs in response to RNA viruses vary depending on the route of infection. Specifically, local infections induce IFNs through RLHs but not TLRs, whereas systemic infections strongly stimulate TLRs in pDCs. In this review, we discuss recent advances toward clarifying the signaling pathways activated by RLHs and TLRs.     

Identification of the role of RIG-I, MDA-5 and TLR3 in sensing RNA viruses in porcine epithelial cells using lentivirus-driven RNA interference

Pathogen recognition receptors are essential for antiviral host immune responses. These specialized receptors detect conserved viral compounds and induce type I interferons (IFN) and pro-inflammatory cytokines. Here we evaluated the contribution of RIG-I, MDA-5 and TLR3 to the recognition of classical swine fever (CSFV), foot-and-mouth disease virus (FMDV), vesicular stomatitis virus (VSV) and influenza A virus (IAV) to IFN-β responses in the porcine epithelial cell line PK-15. To this end, we identified porcine gene specific small interfering RNA sequences and employed a lentivirus (LV)-based system to deliver the corresponding short hairpin RNA. With this, gene knockdown cell lines were created and tested with regard to the knockdown levels over time and following IFN-β stimulation. During several passages of the transduced cells, the expression of both the reporter gene eGFP and the reduced RNA levels of the targeted gene were stable, although the latter was relatively variable. IFN-β induced IFN-responsive genes such as RIG-I, but the levels of the silenced cell line remained reduced compared to the control cells. Based on virus-induced IFN-β mRNA responses, our results indicate that in PK-15 cells FMDV-detection is solely mediated by MDA-5, whereas VSV and IAV are mainly detected by RIG-I with a minor contribution of MDA-5, and CSFV is sensed by MDA-5, RIG-I and TLR3.     

Viral RNA Induces Type I Interferon-Dependent Cytokine Release and Cell Death in Mesangial Cells via Melanoma-Differentiation-Associated Gene-5 : Implications for Viral Infection-Associated Glomerulonephritis

Viral RNA can trigger interferon signaling in dendritic cells via the innate recognition receptors melanoma-differentiation-associated gene (MDA)-5 and retinod-inducible gene (RIG)-I in the cytosol or via Toll-like receptors (TLRs) in intracellular endosomes. We hypothesized that viral RNA would also activate glomerular mesangial cells to produce type I interferon (IFN) via TLR-dependent and TLR-independent pathways. To test this hypothesis, we examined Toll/Interleukin-1 receptor domain-containing adaptor-inducing interferon-β (TRIF)-deficient mice, which lack a key adaptor for TLR3 signaling. In primary mesangial cells, poly I:C RNA-mediated IFN-β induction was partially TRIF dependent; however, when poly I:C RNA was complexed with cationic lipids to enhance cytosolic uptake, mesangial cells produced large amounts of IFN-α and IFN-β independent of TRIF. Mesangial cells expressed RIG-I and MDA-5 and their mitochondrial adaptor IFN-β promoter stimulator-1 as well, and small interfering RNA studies revealed that MDA5 but not RIG-I was required for cytosolic poly I:C RNA signaling. In addition, mesangial cells produced Il-6 on stimulation with IFN-α and IFN-β, suggesting an autocrine proinflammatory effect. Indeed, blockade of IFN-αβ or lack of the IFNA receptor reduced viral RNA-induced Il-6 production and apoptotic cell death in mesangial cells. Furthermore, viral RNA/cationic lipid complexes increased focal necrosis in murine nephrotoxic serum nephritis in association with increased renal mRNA expression of IFN-related genes. Thus, TLR-independent recognition of viral RNA is a potent inducer of type I interferon in mesangial cells, which can be an important mediator of virally induced glomerulonephritis.Chronic viral infections can trigger de novo immune complex glomerulonephritis, eg, hepatitis C virus-associated glomerulonephritis, but more frequently, acute viral infections trigger disease activity of pre-existing glomerulonephritis, like in IgA nephropathy, lupus nephritis, or renal vasculitis.¹ Viral infections activate systemic antiviral immunity, which may contribute to disease flares of glomerulonephritis by enhancing autoantibody production, immune complex formation, or by systemic interferon (IFN) release.² In fact, rapid production of type I IFN is a central element of antiviral immunity because type I IFNs inhibit viral replication in the infected cells and have pleiotrophic immunomodulatory effects on macrophages, T cells, and natural killer cells.^2,3The main source of type I IFNs is plasmacytoid dendritic cells (pDCs) in the intravascular compartment.⁴ Several viral components can induce type I IFNs in pDCs. For example, viral proteins activate Toll-like receptor (TLR) 2 and 4 signaling at the cell surface.⁵ By contrast, different shapes of viral nucleic acids can activate TLRs in intracellular endosomes, ie, TLR3 (double-stranded (dsRNA)), TLR7/8 (single-stranded (ssRNA)), and TLR9 (CpG-DNA).⁵ Furthermore, viral RNA can also be detected in the intracellular cytosol, eg, via melanoma-differentiation-associated gene (MDA)-5 (dsRNA) and retinoic-acid-inducible protein (RIG) (dsRNA and 5′-triphosphate RNA).^5,6,7,8 Ligation of any of these innate RNA recognition receptors rapidly triggers the production of type I IFN in pDCs.It is thought that most cells can produce type I IFNs when they are infected with a DNA or RNA virus. For example, TLR3-mediated recognition of viral dsRNA in pancreatic islet cells can trigger autoimmune pancreatic islet destruction via local production of IFN-α.⁹ But whether locally produced type I IFNs contribute to viral infection-induced glomerulonephritis is not known.¹⁰ In fact, to our best knowledge, IFN release by glomerular cells, including mesangial cells, has not been reported. The only nucleic acid-specific TLR expressed by mesangial cells is TLR3, and mesangial cells produce Il-6 and CCL2 on exposure to viral dsRNA.¹¹ However, whether this effect is mediated via endosomal TLR3 or by cytosolic dsRNA receptors is not known. We hypothesized that viral RNA will trigger an innate antiviral response program in glomerular mesangial cells, including the release of type I IFN, and that this effect is mediated by TLR-dependent as well as TLR-independent RNA recognition.     

Polyinosinic-polycytidylic acid-mediated stimulation of human gammadelta T cells via CD11c dendritic cell-derived type I interferons

The recognition of pathogen-associated molecular patterns (PAMPs) by the innate immune system is a crucial step in inducing effective immune responses. Double-stranded RNA [mimicked by polyinosinic-polycytidylic acid (poly(I:C)], synthesized by various types of viruses, represents one important member of these immunostimulatory microbial components. Here we report that poly(I:C) has potent gammadelta T-cell costimulatory capacity. Within peripheral blood mononuclear cells, poly(I:C)-stimulated gammadelta T cells expressed increased levels of CD69 and exhibited significantly enhanced antigen-mediated proliferation in response to isopentenylpyrophosphate (IPP). Among several recombinant cytokines tested, type I interferons (IFN-alpha, IFN-beta) and interleukin-15 (IL-15) showed a similar activation pattern of gammadelta T cells. gammadelta T-cell clones and purified gammadelta T cells did not respond to poly(I:C), indicating indirect effects of this compound. Depletion of CD11c(+) dendritic cells (DC), which express Toll-like receptor 3 (TLR3), known to recognize poly(I:C), abrogated poly(I:C)-mediated stimulation of gammadelta T cells. In addition, the supernatant of poly(I:C)-treated CD11c(+) DC was able to mimic the stimulatory effects of poly(I:C) on gammadelta T cells. Experiments with neutralizing antibodies indicated that type I IFNs, but not IL-15, contributed to the poly(I:C)-mediated activation of gammadelta T cells. In conclusion, gammadelta T-cell activation by immunostimulatory double-stranded RNA, such as poly(I:C), is indirectly mediated via type I IFNs derived from TLR3-expressing CD11c(+) DCs. These results suggest that upon confrontation with certain viruses, gammadelta T cells can be rapidly activated by type I interferons and may contribute to effective antiviral responses.     

Recognition of cytosolic DNA activates an IRF3-dependent innate immune response

Nucleic acid recognition upon viral infection triggers type I interferon production. Viral RNA is detected by both endosomal, TLR-dependent and cytosolic, RIG-I/MDA5-dependent pathways. TLR9 is the only known sensor of foreign DNA; it is unknown whether innate immune recognition of DNA exists in the cytosol. Here we present evidence that cytosolic DNA activates a potent type I interferon response to the invasive bacterium Listeria monocytogenes. The noninvasive Legionella pneumophila triggers an identical response through its type IV secretion system. Activation of type I interferons by cytosolic DNA is TLR independent and requires IRF3 but occurs without detectable activation of NF-kappaB and MAP kinases. Microarray analyses reveal a unique but overlapping gene-expression program activated by cytosolic DNA compared to TLR9- and RIG-I/MDA5-dependent responses. These findings define an innate immune response to DNA linked to type I interferon production.     

Plasmacytoid dendritic cell precursors/type I interferon-producing cells sense viral infection by Toll-like receptor (TLR) 7 and TLR9

Plasmacytoid dendritic cell (pDC) precursors, also called type I IFN (//)-producing cells (IPCs), are the key effectors in the innate immune system because of their extraordinary capacity to produce type I IFNs against microbial infection, particularly viral infection. In contrast to myeloid DCs, human pDC/IPCs selectively express Toll-like receptor (TLR) 7 and TLR9 within the endosomal compartment. These receptors are specifically designed to recognize the nucleoside-based products derived from RNA viruses and DNA viruses. Therefore, this expression profile potentially enables pDC/IPCs to sense a variety of viruses. Stimulation of TLR7 or TLR9 leads to type I IFN responses through the MyD88 pathway. Thus, pDC/IPCs may play a central role in host defense against viral infection through the TLR7 and TLR9 system.     

Soluble G protein of respiratory syncytial virus inhibits Toll-like receptor 3/4-mediated IFN-beta induction

Monocyte-derived dendritic cells (mDCs) recognize viral RNA extrinsically by Toll-like receptor (TLR) 3 on the membrane and intrinsically retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5) in the cytoplasm to induce type I IFNs and mDC maturation. When mDCs were treated with live or UV-irradiated respiratory syncytial virus (RSV), early ( approximately 4 h) induction of IFN-beta usually occurs in other virus infections was barely observed. Live RSV subsequently replicated to activate the cytoplasmic IFN-inducing pathway leading to robust type I IFN induction. We found that RSV initial attachment to cells blocked polyI:C-mediated IFN-beta induction, and this early IFN-beta-modulating event was abrogated by antibodies against envelope proteins of RSV, demonstrating the presence of a IFN-regulatory mode by early RSV attachment to host cells. By IFN-stimulated response element (ISRE) reporter analysis in HEK293 cells, polyI:C- or LPS-mediated ISRE activation was dose dependently inhibited by live and inactive RSV to a similar extent. Of the RSV envelope proteins, simultaneously expressed or exogenously added RSV G or soluble G (sG) proteins inhibited TLR3/4-mediated ISRE activation in HEK293 cells. sG proteins expressed in cells did not affect the RIG-I/MDA5 pathway but inhibited the TLR adaptor TRIF/TICAM-1 pathway for ISRE activation. Finally, extrinsically added sG protein suppressed the production of IFN-beta in mDCs. Although the molecular mechanism of this extrinsic functional mode of the RSV G glycoprotein (G protein) remains undetermined, G proteins may neutralize the fusion glycoprotein function that promotes IFN-mediated mDC modulation via TLR4 and may cause insufficient raising cell-mediated immunity against RSV.     

The type I interferon response during viral infections: a "SWOT" analysis

The type I interferon (IFN) response is a strong and crucial moderator for the control of viral infections. The strength of this system is illustrated by the fact that, despite some temporary discomfort like a common cold or diarrhea, most viral infections will not cause major harm to the healthy immunocompetent host. To achieve this, the immune system is equipped with a wide array of pattern recognition receptors and the subsequent coordinated type I IFN response orchestrated by plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (cDCs). The production of type I IFN subtypes by dendritic cells (DCs), but also other cells is crucial for the execution of many antiviral processes. Despite this coordinated response, morbidity and mortality are still common in viral disease due to the ability of viruses to exploit the weaknesses of the immune system. Viruses successfully evade immunity and infection can result in aberrant immune responses. However, these weaknesses also open opportunities for improvement via clinical interventions as can be seen in current vaccination and antiviral treatment programs. The application of IFNs, Toll-like receptor ligands, DCs, and antiviral proteins is now being investigated to further limit viral infections. Unfortunately, a common threat during stimulation of immunity is the possible initiation or aggravation of autoimmunity. Also the translation from animal models to the human situation remains difficult. With a Strengths-Weaknesses-Opportunities-Threats ("SWOT") analysis, we discuss the interaction between host and virus as well as (future) therapeutic options, related to the type I IFN system.     

MDA5/RIG-I and virus recognition

The innate immune system initially recognizes RNA virus infection and evokes antiviral responses by producing type I interferons (IFNs). Toll-like receptors (TLRs) and cytoplasmic retinoic acid-inducible gene I (RIG-I)-like helicases (RLHs) are the two major receptor systems for detecting RNA viruses. The RLH signaling pathways play essential roles in the recognition of RNA viruses in various cells, with the exception of plasmacytoid dendritic cells, which utilize TLRs for virus recognition. The route of infection determines the cell types responsible for type I IFN production. Recent studies have suggested that TLRs are critical for activation of adaptive immune responses against several virus infections, although it may be premature to draw such a conclusion for virus infections in general. In this review, we will discuss recent advances toward clarifying the signaling pathways activated by RLHs and TLRs.     

Activation of the TLR3 pathway regulates IFNbeta production in chickens

Toll-like receptors (TLRs) play key roles in the response to pathogens and in mammals the host response to virus critically relies on TLR3 to detect viral-derived dsRNA. However, in chickens there is a paucity of information about this pathway, and in view of the recent concerns with regard to highly pathogenic avian influenza, there is a clear need for understanding these antiviral pathways. Furthermore, TLR3 engagement is important to the outcome of viral infection because of its role in the induction of interferons (IFNs) and the diverse antiviral effects that these molecules induce. With this in mind, we have investigated the role of TLR3 and its impact on the production of IFNs. We show that in the chicken, poly(I:C), a dsRNA analogue, rapidly induces type 1 IFN similar to that seen in mammals. Furthermore, IFN can activate the upregulation of TLR3, which in some cell types induces them to become responsive to dsRNA. These data highlight the similar function that TLR3 plays in chickens and mammals. To determine the role of chicken TLR3 in response to poly(I:C), we used RNAi-mediated gene silencing to show that poly(I:C)-stimulated IFNbeta expression involves TLR3 signalling. The interrelationship between TLR3 and interferon as well as the observed increase in TLR3 and IFNbeta expression during H5N1 avian influenza infection indicates the importance of these molecules in viral infections in chickens.     

TLR ligands induce synergistic interferon-β and interferon-λ1 gene expression in human monocyte-derived dendritic cells

Toll-like receptors (TLRs) are pattern-recognition receptors of the innate immune system that recognize various pathogen-associated molecules. TLR ligands are potent activators of immune cells and certain TLR ligands have a synergistic ability to induce the production of pro-inflammatory cytokines. In the present study we have analyzed the potential synergy between TLR3, TLR4 and TLR7/8 ligands in type I and type III interferon (IFN) gene expression in human monocyte-derived dendritic cells (moDCs). We show that stimulation of moDCs with TLR7/8 ligand R848 together with TLR3 or TLR4 ligands, polyI:C or LPS, respectively, leads to a synergistic expression of IFN-β and IFN-λ1 mRNAs. Neutralization of type I IFNs as well as IFN priming prior to stimulation suggest that IFN-dependent positive feedback loop is at least partly responsible for the mechanism of synergy. Enhanced expression of TLR3 and especially TLR7, which are both under the regulation of type I IFNs, correlated to synergistic TLR ligand-dependent induction of IFN-β and IFN-λ1 genes. NF-κB, PI3 kinase and MAP kinase pathways were involved in TLR ligand-induced IFN gene expression as evidenced by pharmacological signaling inhibitors. The data indicates that IFNs contribute to TLR-dependent gene activation in human DCs stimulated with multiple TLR ligands.     

TLR3 in antiviral immunity: key player or bystander?

Toll-like receptor 3 (TLR3), which recognizes double-stranded (ds)RNA, was the first identified antiviral TLR and, because dsRNA is a universal viral molecular pattern, TLR3 has been assumed to have a central role in the host response to viruses. However, this role has recently been questioned by in vivo studies and the discovery of several other antiviral pattern-recognition receptors. In this review, the function of TLR3 in the context of these other receptors, namely TLR7, 8 and 9 and the newly identified dsRNA-receptor retinoic-acid inducible gene-I (RIG-I) is discussed. Also, recent research concerning the expression profile of TLR3, its evasion by viruses and a potential role in crosspriming is addressed, which reveals a clearer appreciation of the contribution of TLR3 to antiviral immunity.     

CHID1 positively regulates RLR antiviral signaling by targeting the RIG-I/VISA signalosome

Retinoic acid-inducible gene-I (RIG-I) belongs to the RIGI-like receptors (RLRs), a class of primary pattern recognition receptors. It senses viral double-strand RNA in the cytoplasm and delivers the activated signal to its adaptor virus-induced signaling adapter (VISA), which then recruits the downstream TNF receptor-associated factors and kinases, triggering a downstream signal cascade that leads to the production of proinflammatory cytokines and antiviral interferons (IFNs). However, the mechanism of RIG-I-mediated antiviral signaling is not fully understood. Here, we demonstrate that chitinase domain-containing 1 (CHID1), a member of the chitinase family, positively regulates the RLR antiviral signaling pathway by targeting the RIG-I/VISA signalosome. CHID1 overexpression enhances the activation of nuclear factor κB (NF-кB) and interferon regulatory factor 3 (IRF3) triggered by Sendai virus (SeV) by promoting the polyubiquitination of RIG-I and VISA, thereby potentiating IFN-β production. CHID1 knockdown in human 239T cells inhibits SeV-induced activation of IRF3 and NF-κB and the induction of IFN-β. These results indicate that CHID1 positively regulates RLR antiviral signal, revealing the novel mechanism of the RIG-I antiviral signaling pathway.     

The role of viral nucleic acid recognition in dendritic cells for innate and adaptive antiviral immunity

Dendritic cells which are located at the interface of innate and adaptive immunity are targets for infection by many different DNA and RNA viruses. Dendritic cell subpopulations express specific nucleic acid recognition receptors belonging to the Toll-like receptor family (TLR3, 7, 8, 9) and the cytosolic RNA helicase family (RIG-I, MDA5, LGP2). Activation of dendritic cells by viral DNA and RNA via these receptors is essential for triggering the innate antiviral immune response and shaping the ensuing adaptive antiviral immunity. This review will summarize our current knowledge of viral nucleic acid recognition and signaling by Toll-like receptors and RNA helicases focusing on recent evidence for their specific functions in antiviral defense in vivo.     

The role of viral nucleic acid recognition in dendritic cells for innate and adaptive antiviral immunity

Dendritic cells which are located at the interface of innate and adaptive immunity are targets for infection by many different DNA and RNA viruses. Dendritic cell subpopulations express specific nucleic acid recognition receptors belonging to the Toll-like receptor family (TLR3, 7, 8, 9) and the cytosolic RNA helicase family (RIG-I, MDA5, LGP2). Activation of dendritic cells by viral DNA and RNA via these receptors is essential for triggering the innate antiviral immune response and shaping the ensuing adaptive antiviral immunity. This review will summarize our current knowledge of viral nucleic acid recognition and signaling by Toll-like receptors and RNA helicases focusing on recent evidence for their specific functions in antiviral defense in vivo.