Hepatitis C Virus Pathogen Associated Molecular Pattern (PAMP) Triggers Production of Lambda-Interferons by Human Plasmacytoid Dendritic Cells

Plasmacytoid Dendritic Cells (pDCs) represent a key immune cell in the defense against viruses. Through pattern recognition receptors (PRRs), these cells detect viral pathogen associated molecular patterns (PAMPs) and initiate an Interferon (IFN) response. pDCs produce the antiviral IFNs including the well-studied Type I and the more recently described Type III. Recent genome wide association studies (GWAS) have implicated Type III IFNs in HCV clearance. We examined the IFN response induced in a pDC cell line and ex vivo human pDCs by a region of the HCV genome referred to as the HCV PAMP. This RNA has been shown previously to be immunogenic in hepatocytes, whereas the conserved X-region RNA is not. We show that in response to the HCV PAMP, pDC-GEN2.2 cells upregulate and secrete Type III (in addition to Type I) IFNs and upregulate PRR genes and proteins. We also demonstrate that the recognition of this RNA is dependent on RIG-I-like Receptors (RLRs) and Toll-like Receptors (TLRs), challenging the dogma that RLRs are dispensable in pDCs. The IFNs produced by these cells in response to the HCV PAMP also control HCV replication in vitro. These data are recapitulated in ex vivo pDCs isolated from healthy donors. Together, our data shows that pDCs respond robustly to HCV RNA to make Type III Interferons that control viral replication. This may represent a novel therapeutic strategy for the treatment of HCV.     

Hepatitis C Virus Core Protein Inhibits Interferon Production by a Human Plasmacytoid Dendritic Cell Line and Dysregulates Interferon Regulatory Factor-7 and Signal Transducer and Activator of Transcription (STAT) 1 Protein Expression

Plasmacytoid Dendritic Cells (pDCs) represent a key immune cell population in the defense against viruses. pDCs detect viral pathogen associated molecular patterns (PAMPs) through pattern recognition receptors (PRR). PRR/PAMP interactions trigger signaling events that induce interferon (IFN) production to initiate local and systemic responses. pDCs produce Type I and Type III (IFNL) IFNs in response to HCV RNA. Extracellular HCV core protein (Core) is found in the circulation in chronic infection. This study defined how Core modulates PRR signaling in pDCs. Type I and III IFN expression and production following exposure to recombinant Core or β-galactosiade was assessed in human GEN2.2 cells, a pDC cell line. Core suppressed type I and III IFN production in response to TLR agonists and the HCV PAMP agonist of RIG-I. Core suppression of IFN induction was linked with decreased IRF-7 protein levels and increased non-phosphorylated STAT1 protein. Circulating Core protein interferes with PRR signaling by pDCs to suppress IFN production. Strategies to define and target Core effects on pDCs may serve to enhance IFN production and antiviral actions against HCV.     

HIV and HCV Activate the Inflammasome in Monocytes and Macrophages via Endosomal Toll-Like Receptors without Induction of Type 1 Interferon

Innate immune sensing of viral infection results in type I interferon (IFN) production and inflammasome activation. Type I IFNs, primarily IFN-α and IFN-β, are produced by all cell types upon virus infection and promote an antiviral state in surrounding cells by inducing the expression of IFN-stimulated genes. Type I IFN production is mediated by Toll-like receptor (TLR) 3 in HCV infected hepatocytes. Type I IFNs are also produced by plasmacytoid dendritic cells (pDC) after sensing of HIV and HCV through TLR7 in the absence of productive pDC infection. Inflammasomes are multi-protein cytosolic complexes that integrate several pathogen-triggered signaling cascades ultimately leading to caspase-1 activation and generation pro-inflammatory cytokines including interleukin (IL)-18 and IL-1β. Here, we demonstrate that HIV and HCV activate the inflammasome, but not Type I IFN production, in monocytes and macrophages in an infection-independent process that requires clathrin-mediated endocytosis and recognition of the virus by distinct endosomal TLRs. Knockdown of each endosomal TLR in primary monocytes by RNA interference reveals that inflammasome activation in these cells results from HIV sensing by TLR8 and HCV recognition by TLR7. Despite its critical role in type I IFN production by pDCs stimulated with HIV, TLR7 is not required for inflammasome activation by HIV. Similarly, HCV activation of the inflammasome in monocytes does not require TLR3 or its downstream signaling adaptor TICAM-1, while this pathway leads to type I IFN in infected hepatocytes. Monocytes and macrophages do not produce type I IFN upon TLR8 or TLR7 sensing of HIV or HCV, respectively. These findings reveal a novel infection-independent mechanism for chronic viral induction of key anti-viral programs and demonstrate distinct TLR utilization by different cell types for activation of the type I IFN vs. inflammasome pathways of inflammation.     

Type III IFNs in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity

Bats are known to harbor a number of emerging and re-emerging zoonotic viruses, many of which are highly pathogenic in other mammals but result in no clinical symptoms in bats. The ability of bats to coexist with viruses may be the result of rapid control of viral replication early in the immune response. IFNs provide the first line of defense against viral infection in vertebrates. Type III IFNs (IFN-λs) are a recently identified IFN family that share similar antiviral activities with type I IFNs. To our knowledge, we demonstrate the first functional analysis of type III IFNs from any species of bat, with the investigation of two IFN-λ genes from the pteropid bat, Pteropus alecto. Our results demonstrate that bat type III IFN has similar antiviral activity to type I and III IFNs from other mammals. In addition, the two bat type III IFNs are differentially induced relative to each other and to type I IFNs after treatment or transfection with synthetic dsRNA. Infection with the bat paramyxovirus, Tioman virus, resulted in no upregulation of type I IFN production in bat splenocytes but was capable of inducing a type III IFN response in three of the four bats tested. To our knowledge, this is the first report to describe the simultaneous suppression of type I IFN and induction of type III IFN after virus infection. These results may have important implications for the role of type III IFNs in the ability of bats to coexist with viruses.     

Herpesviruses and the Type III Interferon System

Type III interferons (IFNs) represent the most recently discovered group of IFNs. Together with type I IFNs (e.g. IFN-α/β), type III IFNs (IFN-λ) are produced as part of the innate immune response to virus infection, and elicit an anti-viral state by inducing expression of interferon stimulated genes (ISGs). It was initially thought that type I IFNs and type III IFNs perform largely redundant functions. However, it has become evident that type III IFNs particularly play a major role in antiviral protection of mucosal epithelial barriers, thereby serving an important role in the first-line defense against virus infection and invasion at contact areas with the outside world, versus the generally more broad, potent and systemic antiviral effects of type I IFNs. Herpesviruseses are large DNA viruses, which enter their host via mucosal surfaces and establish lifelong, latent infections. Despite the importance of mucosal epithelial cells in the pathogenesis of herpesviruses, our current knowledge on the interaction of herpesviruses with type III IFN is limited and largely restricted to studies on the alphaherpesvirus herpes simplex virus (HSV). This review summarizes the current understanding about the role of IFN-λ in the immune response against herpesvirus infections.     

Induction of an Antiviral State and Attenuated Coxsackievirus Replication in Type III Interferon-Treated Primary Human Pancreatic Islets

Type III interferons (IFNs), also called lambda interferons (IFN-λ), comprise three isoforms, IFN-λ1 (interleukin-29 [IL-29]), IFN-λ2 (IL-28A), and IFN-λ3 (IL-28B). Only limited information is available on their expression and biological functions in humans. Type I and type II IFNs protect human pancreatic islets against coxsackievirus infection, and this is important since such viruses have been proposed to play a role in the development of human type 1 diabetes. Here we investigated whether type III IFN is expressed during infection of human islet cells with coxsackievirus and if type III IFN regulates permissiveness to such infections. We show that human islets respond to a coxsackievirus serotype B3 (CVB3) infection by inducing the expression of type III IFNs. We also demonstrate that islet endocrine cells from nondiabetic individuals express the type III IFN receptor subunits IFN-λR1 and IL-10R2. Pancreatic alpha cells express both receptor subunits, while pancreatic beta cells express only IL-10R2. Type III IFN stimulation elicited a biological response in human islets as indicated by the upregulated expression of antiviral genes as well as pattern recognition receptors. We also show that type III IFN significantly reduces CVB3 replication. Our studies reveal that type III IFNs are expressed during CVB3 infection and that the expression of the type III IFN receptor by the human pancreatic islet allows this group of IFNs to regulate the islets'' permissiveness to infection. Our novel observations suggest that type III IFNs may regulate viral replication and thereby contribute to reduced tissue damage and promote islet cell survival during coxsackievirus infection.     

Herpesviruses and the Type Ⅲ Interferon System

Type Ⅲ interferons (IFNs) represent the most recently discovered group of IFNs.Together with type Ⅰ IFNs (e.g.IFN-α/β),type Ⅲ IFNs (IFN-λ) are produced as part of the innate immune response to virus infection,and elicit an anti-viral state by inducing expression of interferon stimulated genes (ISGs).It was initially thought that type Ⅰ IFNs and type Ⅲ IFNs perform largely redundant functions.However,it has become evident that type Ⅲ IFNs particularly play a major role in antiviral protection of mucosal epithelial barriers,thereby serving an important role in the first-line defense against virus infection and invasion at contact areas with the outside world,versus the generally more broad,potent and systemic antiviral effects of type Ⅰ IFNs.Herpesviruseses are large DNA viruses,which enter their host via mucosal surfaces and establish lifelong,latent infections.Despite the importance of mucosal epithelial cells in the pathogenesis of herpesviruses,our current knowledge on the interaction of herpesviruses with type Ⅲ IFN is limited and largely restricted to studies on the alphaherpesvirus herpes simplex virus (HSV).This review summarizes the current understanding about the role of IFN-λ in the immune response against herpesvirus infections.     

An important role for type III interferon (IFN-lambda/IL-28) in TLR-induced antiviral activity

Type III IFNs (IFN-lambda/IL-28/29) are cytokines with type I IFN-like antiviral activities, which remain poorly characterized. We herein show that most cell types expressed both types I and III IFNs after TLR stimulation or virus infection, whereas the ability of cells to respond to IFN-lambda was restricted to a narrow subset of cells, including plasmacytoid dendritic cells and epithelial cells. To examine the role of type III IFN in antiviral defense, we generated IL-28Ralpha-deficient mice. These mice were indistinguishable from wild-type mice with respect to clearance of a panel of different viruses, whereas mice lacking the type I IFN receptor (IFNAR(-/-)) were significantly impaired. However, the strong antiviral activity evoked by treatment of mice with TLR3 or TLR9 agonists was significantly reduced in both IL-28RA(-/-) and IFNAR(-/-) mice. The type I IFN receptor system has been shown to mediate positive feedback on IFN-alphabeta expression, and we found that the type I IFN receptor system also mediates positive feedback on IFN-lambda expression, whereas IL-28Ralpha signaling does not provide feedback on either type I or type III IFN expression in vivo. Finally, using bone-marrow chimeric mice we showed that TLR-activated antiviral defense requires expression of IL-28Ralpha only on nonhemopoietic cells. In this compartment, epithelial cells responded to IFN-lambda and directly restricted virus replication. Our data suggest type III IFN to target a specific subset of cells and to contribute to the antiviral response evoked by TLRs.     

Microbial pathogenesis and type III interferons

The innate immune system possesses a multitude of pathways to sense and respond to microbial pathogens. One such family are the interferons (IFNs), a family of cytokines that are involved in several cellular functions. Type I IFNs are appreciated to be important in several viral and bacterial diseases, while the recently identified type III IFNs (IFNL1, IFNL2, IFNL3, IFNL4) have been studied primarily in the context of viral infection. Viral and bacterial infections however are not mutually exclusive, and often the presence of a viral pathogen increases the pathogenesis of bacterial infection. The role of type III IFN in bacterial and viral-bacterial co-infections has just begun to be explored. In this mini review we discuss type III IFN signaling and its role in microbial pathogenesis with an emphasis on the work that has been conducted with bacterial pathogens.     

USP18-based negative feedback control is induced by type I and type III interferons and specifically inactivates interferon α response

Type I interferons (IFN) are cytokines that are rapidly secreted upon microbial infections and regulate all aspects of the immune response. In humans 15 type I IFN subtypes exist, of which IFN α2 and IFN β are used in the clinic for treatment of different pathologies. IFN α2 and IFN β are non redundant in their expression and in their potency to exert specific bioactivities. The more recently identified type III IFNs (3 IFN λ or IL-28/IL-29) bind an unrelated cell-type restricted receptor. Downstream of these two receptor complexes is a shared Jak/Stat pathway. Several mechanisms that contribute to the shut down of the IFN-induced signaling have been described at the molecular level. In particular, it has long been known that type I IFN induces the establishment of a desensitized state. In this work we asked how the IFN-induced desensitization integrates into the network built by the multiple type I IFN subtypes and type III IFNs. We show that priming of cells with either type I IFN or type III IFN interferes with the cell's ability to further respond to all IFN α subtypes. Importantly, primed cells are differentially desensitized in that they retain sensitivity to IFN β. We show that USP18 is necessary and sufficient to induce differential desensitization, by impairing the formation of functional binding sites for IFN α2. Our data highlight a new type of differential between IFNs α and IFN β and underline a cross-talk between type I and type III IFN. This cross-talk could shed light on the reported genetic variation in the IFN λ loci, which has been associated with persistence of hepatitis C virus and patient's response to IFN α2 therapy.     

Understanding Interferon Subtype Therapy for Viral Infections: Harnessing the Power of the Innate Immune System.

Type I and III interferons (IFNs) of the innate immune system belong to a polygenic family, however the individual subtype mediators of the antiviral response in viral infections have been hindered by a lack of reagents. Evaluation studies using different IFN subtypes have distinguished distinct protein properties with different efficacies towards different viruses, opening promising avenues for immunotherapy. This review largely focuses on the application of IFN-α/β and IFN-λ therapies for viral infections, influenza, herpes, HIV and hepatitis. Such IFN subtype therapies may help to cure patients with virus infections where no vaccine exists. The ability of cell types to secrete a number of IFN subtypes from a multi-gene family may be an intuitive counterattack on viruses that evade IFN subtype responses. Hence, clinical use of virus-targeted IFN subtypes may restore antiviral immunity in viral infections. Accumulating evidence suggests that individual IFN subtypes have differential efficacies in selectively activating immune cell subsets to enhance antiviral immune responses leading to production of sustained B and T cell memory. Cytokine therapy can augment innate immunity leading to clearance of acute virus infections but such treatments may have limited effects on chronic virus infections that establish lifelong latency. Therefore, exploiting individual IFN subtypes to select those with the ability to sculpt protective responses as well as reinstating those targeted by viral evasion mechanisms may inform development of improved antiviral therapy.     

Amino acid differences in interferon-tau (IFN-τ) of Bos taurus Coreanae and Holstein

Interferons (IFNs) are commonly grouped into type I and type II IFN. Type I IFNs are known as antiviral IFNs including IFN-α, IFN-β, and IFN-ω whereas type II IFN is referred to immune IFN and IFN-γ is only member of the type II IFN. Type I IFNs are induced by virus invading however type II IFN is produced by mitogenic or antigenic stimuli. IFN-τ was first identified in ruminant ungulates as a pregnancy recognition hormone, trophoblastin. IFN-τ constitutes a new class of type I IFN, which possesses the common features of type I IFN, such as the ability to prevent viral infection and to limit cell proliferation. In addition, IFN-τ is unique in that it is induced by pregnancy unlike other type I IFNs. We cloned Bos taurus (B. T.) Coreanae IFN-τ from peripheral blood mononuclear cells. The amino acid sequence of B. T. Coreanae IFN-τ shares only 90.3% identity with that of Holstein dairy cow. Recombinant B. T. Coreanae and Holstein IFN-τ proteins were expressed in Escherichia coli and the antiviral activity of IFN-τ proteins were examined. Both recombinant proteins were active and protected human WISH and bovine MDBK cells from the cytopathic effect of vesicular stomatitis virus. The recombinant IFN-τ protein of B. T. Coreanae and Holstein properly induced the expression of antiviral genes including 2',5'-oligoadenylate synthetase (OAS) and Mx GTPase 1 (Mx-1).     

Activation of Type I and III Interferon Response by Mitochondrial and Peroxisomal MAVS and Inhibition by Hepatitis C Virus

Sensing viruses by pattern recognition receptors (PRR) triggers the innate immune system of the host cell and activates immune signaling cascades such as the RIG-I/IRF3 pathway. Mitochondrial antiviral-signaling protein (MAVS, also known as IPS-1, Cardif, and VISA) is the crucial adaptor protein of this pathway localized on mitochondria, peroxisomes and mitochondria-associated membranes of the endoplasmic reticulum. Activation of MAVS leads to the production of type I and type III interferons (IFN) as well as IFN stimulated genes (ISGs). To refine the role of MAVS subcellular localization for the induction of type I and III IFN responses in hepatocytes and its counteraction by the hepatitis C virus (HCV), we generated various functional and genetic knock-out cell systems that were reconstituted to express mitochondrial (mito) or peroxisomal (pex) MAVS, exclusively. Upon infection with diverse RNA viruses we found that cells exclusively expressing pexMAVS mounted sustained expression of type I and III IFNs to levels comparable to cells exclusively expressing mitoMAVS. To determine whether viral counteraction of MAVS is affected by its subcellular localization we employed infection of cells with HCV, a major causative agent of chronic liver disease with a high propensity to establish persistence. This virus efficiently cleaves MAVS via a viral protease residing in its nonstructural protein 3 (NS3) and this strategy is thought to contribute to the high persistence of this virus. We found that both mito- and pexMAVS were efficiently cleaved by NS3 and this cleavage was required to suppress activation of the IFN response. Taken together, our findings indicate comparable activation of the IFN response by pex- and mitoMAVS in hepatocytes and efficient counteraction of both MAVS species by the HCV NS3 protease.     

A vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type I interferon

Type I interferons (IFN) comprise a family of cytokines that signal through a common cellular receptor to induce a plethora of genes with antiviral and other activities. Recombinant IFNs are used for the treatment of hepatitis C virus infection, multiple sclerosis, and certain malignancies. The capability of type I IFN to suppress virus replication and resultant cytopathic effects is frequently used to measure their bioactivity. However, these assays are time-consuming and require appropriate biosafety containment. In this study, an improved IFN assay is presented which is based on a recombinant vesicular stomatitis virus (VSV) replicon encoding two reporter proteins, firefly luciferase and green fluorescent protein. The vector lacks the essential envelope glycoprotein (G) gene of VSV and is propagated on a G protein-expressing transgenic cell line. Several mammalian and avian cells turned out to be susceptible to infection with the complemented replicon particles. Infected cells readily expressed the reporter proteins at high levels five hours post infection. When human fibroblasts were treated with serial dilutions of human IFN-β prior to infection, reporter expression was accordingly suppressed. This method was more sensitive and faster than a classical IFN bioassay based on VSV cytopathic effects. In addition, the antiviral activity of human IFN-λ (interleukin-29), a type III IFN, was determined on Calu-3 cells. Both IFN-β and IFN-λ were acid-stable, but only IFN-β was resistant to alkaline treatment. The antiviral activities of canine, porcine, and avian type I IFN were analysed with cell lines derived from the corresponding species. This safe bioassay will be useful for the rapid and sensitive quantification of multi-species type I IFN and potentially other antiviral cytokines.     

Mechanisms of Antiviral Immune Evasion of SARS-CoV-2

Coronavirus disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is characterized by a delayed interferon (IFN) response and high levels of proinflammatory cytokine expression. Type I and III IFNs serve as a first line of defense during acute viral infections and are readily antagonized by viruses to establish productive infection. A rapidly growing body of work has interrogated the mechanisms by which SARS-CoV-2 antagonizes both IFN induction and IFN signaling to establish productive infection. Here, we summarize these findings and discuss the molecular interactions that prevent viral RNA recognition, inhibit the induction of IFN gene expression, and block the response to IFN treatment. We also describe the mechanisms by which SARS-CoV-2 viral proteins promote host shutoff. A detailed understanding of the host-pathogen interactions that unbalance the IFN response is critical for the design and deployment of host-targeted therapeutics to manage COVID-19.     

Role of type I interferons in the activation of autoreactive B cells

Type I interferons (IFNs) are a family of cytokines involved in the defense against viral infections that play a key role in the activation of both the innate and adaptive immune system. IFNs both directly and indirectly enhance the capacity of B lymphocytes to respond to viral challenge and produce cytotoxic and neutralizing antibodies. However, prolonged type I IFN exposure is not always beneficial to the host. If not regulated properly IFN can drive autoantibody production as well as other parameters of systemic autoimmune disease. Type I IFNs impact B-cell function through a variety of mechanisms, including effects on receptor engagement, Toll-like receptor expression, cell migration, antigen presentation, cytokine responsiveness, cytokine production, survival, differentiation and class-switch recombination. Type I IFNs are also cytotoxic for a variety of cell types and thereby contribute to the accumulation of cell debris that serves as a potential source for autoantigens. Type I IFN engagement of a variety of accessory cells further promotes B-cell survival and activation, as exemplified by the capacity of type I IFNs to increase the level of B-cell survival factors, such as B lymphocyte stimulator, produced by dendritic cells. Therefore, it is not surprising that the loss of expression of the type I IFN receptor can have dramatic effects on the production of autoantibodies and on the clinical features of systemic autoimmune diseases such as systemic lupus erythematosus.     

Ⅲ型干扰素——新型抗病毒细胞因子

干扰素（IFN）是抗病毒感染的第一道防线,Ⅰ型和Ⅱ型干扰素不仅可抑制病毒,而且还能参与天然免疫反应和获得性免疫反应。最近干扰素家族增添一位新成员：Ⅲ型干扰素,即IFN-λ,因其具有类似干扰素的抗病毒活性且能诱导干扰素相关基因的表达而命名。IFN-λ受体与Ⅰ型干扰素的受体不同,但具有与Ⅰ型干扰素类似的诱导表达方式和信号转导通路,并能激活一系列相似的干扰素刺激基因。就IFN-λ家族及其受体、基因表达和信号转导机制、抗病毒作用等进行综述。     

Sendai virus infection induces efficient adaptive immunity independently of type I interferons

Adaptive immunity in response to virus infection involves the generation of Th1 cells, cytotoxic T cells, and antibodies. This type of immune response is crucial for the clearance of virus infection and for long-term protection against reinfection. Type I interferons (IFNs), the primary innate cytokines that control virus growth and spreading, can influence various aspects of adaptive immunity. The development of antiviral immunity depends on many viral and cellular factors, and the extent to which type I IFNs contribute to the generation of adaptive immunity in response to a viral infection is controversial. Using two strains (Cantell and 52) of the murine respiratory Sendai virus (SeV) with differential abilities to induce type I IFN production from infected cells, together with type I IFN receptor-deficient mice, we examined the role of type I IFNs in the generation of adaptive immunity. Our results show that type I IFNs facilitate virus clearance and enhance the migration and maturation of dendritic cells after SeV infection in vivo; however, soon after infection, mice clear the virus from their lungs and efficiently generate cytotoxic T cells independently of type I IFN signaling. Furthermore, animals that are unresponsive to type I IFN develop long-term anti-SeV immunity, including CD8+ T cells and antibodies. Significantly, this memory response is able to protect mice against challenge with a lethal dose of virus. In conclusion, our results show that primary and secondary anti-SeV adaptive immunities are developed normally in the absence of type I IFN responsiveness.