Efficacy of IFN-λ1 to Protect Human Airway Epithelial Cells against Human Rhinovirus 1B Infection

Impaired interferon (IFN) production has been observed in various obstructive respiratory diseases. This contributes to enhanced sensitivity towards viral infections triggering acute exacerbations. To compensate for this impaired host IFN response, there is need to explore new therapeutic strategies, like exogenous administration of IFNs as prophylactic treatment. In the present study, we examined the protective potential of IFN-λ1 and compared it with the previously established protecting effect of IFN-β. A549 cells and human primary bronchial epithelial cells were first treated with either IFN-β (500 IU/ml) or IFN-λ1 (500 ng/ml) for 18 h. For infection, two approaches were adopted: i) Continuous scenario: after pre-treatment, cells were infected immediately for 24 h with human rhinovirus 1B (HRV1B) in IFN-containing medium, or were cultured for another 72 h in IFN-containing medium, and then infected for 24 h with HRV1B, ii) Pre-treatment scenario: IFN-containing medium was replaced after 18 h and cells were infected for 4 h either immediately after pre-treatment or after additional culturing for 72 h in IFN-free medium. The protective effect was evaluated in terms of reduction in the number of viral copies/infectious progeny, and enhanced expression of IFN-stimulated genes (ISGs). In both cell types and in both approaches, IFN-λ1 and IFN-β treatment resulted in pronounced and long-lasting antiviral effects exemplified by significantly reduced viral copy numbers and diminished infectious progeny. This was associated with strong up-regulation of multiple ISGs. However, in contrast to the IFN-β induced expression of ISGs, which decreased over time, expression of ISGs induced by IFN-λ1 was sustained or even increased over time. Here we demonstrate that the protective potential of IFN-λ1 is comparable to IFN-β. Yet, the long-lasting induction of ISGs by IFN-λ1 and most likely less incitement of side effects due to more localized expression of its receptors could make it an even more promising candidate for prophylactic treatment than IFN-β.     

Rhinovirus and dsRNA Induce RIG-I-Like Receptors and Expression of Interferon β and λ1 in Human Bronchial Smooth Muscle Cells

Rhinovirus (RV) infections cause exacerbations and development of severe asthma highlighting the importance of antiviral interferon (IFN) defence by airway cells. Little is known about bronchial smooth muscle cell (BSMC) production of IFNs and whether BSMCs have dsRNA-sensing receptors besides TLR3. dsRNA is a rhinoviral replication intermediate and necrotic cell effect mimic that mediates innate immune responses in bronchial epithelial cells. We have explored dsRNA-evoked IFN-β and IFN-λ₁ production in human BSMCs and potential involvement of TLR3 and RIG-I-like receptors (RLRs). Primary BSMCs were stimulated with 0.1–10 µg/ml dsRNA, 0.1–1 µg/ml dsRNA in complex with the transfection agent LyoVec (dsRNA/LyoVec; selectively activating cytosolic RLRs) or infected with 0.05–0.5 MOI RV1B. Both dsRNA stimuli evoked early (3 h), concentration-dependent IFN-β and IFN-λ₁ mRNA expression, which with dsRNA/LyoVec was much greater, and with dsRNA was much less, after 24 h. The effects were inhibited by dexamethasone. Further, dsRNA and dsRNA/LyoVec concentration-dependently upregulated RIG-I and MDA5 mRNA and protein. dsRNA and particularly dsRNA/LyoVec caused IFN-β and IFN-λ₁ protein production (24 h). dsRNA- but not dsRNA/LyoVec-induced IFN expression was partly inhibited by chloroquine that suppresses endosomal TLR3 activation. RV1B dose-dependently increased BSMC expression of RIG-I, MDA5, IFN-β, and IFN-λ₁ mRNA. We suggest that BSMCs express functional RLRs and that both RLRs and TLR3 are involved in viral stimulus-induced BSMC expression of IFN-β and IFN-λ₁.     

Leukocyte-Derived IFN-α/β and Epithelial IFN-λ Constitute a Compartmentalized Mucosal Defense System that Restricts Enteric Virus Infections

Epithelial cells are a major port of entry for many viruses, but the molecular networks which protect barrier surfaces against viral infections are incompletely understood. Viral infections induce simultaneous production of type I (IFN-α/β) and type III (IFN-λ) interferons. All nucleated cells are believed to respond to IFN-α/β, whereas IFN-λ responses are largely confined to epithelial cells. We observed that intestinal epithelial cells, unlike hematopoietic cells of this organ, express only very low levels of functional IFN-α/β receptors. Accordingly, after oral infection of IFN-α/β receptor-deficient mice, human reovirus type 3 specifically infected cells in the lamina propria but, strikingly, did not productively replicate in gut epithelial cells. By contrast, reovirus replicated almost exclusively in gut epithelial cells of IFN-λ receptor-deficient mice, suggesting that the gut mucosa is equipped with a compartmentalized IFN system in which epithelial cells mainly respond to IFN-λ that they produce after viral infection, whereas other cells of the gut mostly rely on IFN-α/β for antiviral defense. In suckling mice with IFN-λ receptor deficiency, reovirus replicated in the gut epithelium and additionally infected epithelial cells lining the bile ducts, indicating that infants may use IFN-λ for the control of virus infections in various epithelia-rich tissues. Thus, IFN-λ should be regarded as an autonomous virus defense system of the gut mucosa and other epithelial barriers that may have evolved to avoid unnecessarily frequent triggering of the IFN-α/β system which would induce exacerbated inflammation.     

Lambda Interferon Renders Epithelial Cells of the Respiratory and Gastrointestinal Tracts Resistant to Viral Infections

Virus-infected cells secrete a broad range of interferons (IFN) which confer resistance to yet uninfected cells by triggering the synthesis of antiviral factors. The relative contributions of the various IFN subtypes to innate immunity against virus infections remain elusive. IFN-α, IFN-β, and other type I IFN molecules signal through a common, universally expressed cell surface receptor, whereas type III IFN (IFN-λ) uses a distinct cell-type-specific receptor complex for signaling. Using mice lacking functional receptors for type I IFN, type III IFN, or both, we found that IFN-λ plays an important role in the defense against several human pathogens that infect the respiratory tract, such as influenza A virus, influenza B virus, respiratory syncytial virus, human metapneumovirus, and severe acute respiratory syndrome (SARS) coronavirus. These viruses were more pathogenic and replicated to higher titers in the lungs of mice lacking both IFN receptors than in mice with single IFN receptor defects. In contrast, Lassa fever virus, which infects via the respiratory tract but primarily replicates in the liver, was not influenced by the IFN-λ receptor defect. Careful analysis revealed that expression of functional IFN-λ receptor complexes in the lung and intestinal tract is restricted to epithelial cells and a few other, undefined cell types. Interestingly, we found that SARS coronavirus was present in feces from infected mice lacking receptors for both type I and type III IFN but not in those from mice lacking single receptors, supporting the view that IFN-λ contributes to the control of viral infections in epithelial cells of both respiratory and gastrointestinal tracts.The interferon (IFN) system represents a major element of the innate immune response against viral infections (10, 13, 14). Virus-induced IFN is a complex mixture of biologically active molecules, which includes type I and type III IFN. Type I IFN consists of 14 different IFN-α subtypes in the mouse as well as IFN-β, IFN-κ, IFN-ɛ, and limitin, which all signal through the same universally expressed cell surface receptor complex (IFNAR) (30). Type III IFN includes IFN-λ1, IFN-λ2, and IFN-λ3 (21, 28), of which only the latter two are encoded by genes that are expressed in the mouse (22). Type III IFN uses a distinct receptor complex (IL28R) for signaling (21, 28), which appears to be expressed on only a few cell types, including epithelial cells (29). Binding of type I IFN and type III IFN to their cognate receptor complexes triggers signaling cascades that result in the activation of a large number of genes, many of which encode antiviral proteins (10, 32). Type I IFN and type III IFN trigger highly similar gene expression profiles in responsive cells, suggesting that both IFN types might serve similar functions. However, it has to date been largely unclear to which extent IFN-λ might contribute to innate immunity.Using knockout mouse strains that lack receptors for type I IFN (IFNAR1^0/0), type III IFN (IL28Rα^0/0), or both (IFNAR1^0/0IL28Rα^0/0), we have recently shown that IFN-λ contributes to resistance against influenza A virus (FLUAV) (26). Here, we used the same mouse strains to investigate the relative contribution of IFN-λ in resistance against additional viral pathogens that infect the respiratory and gastrointestinal tract and to visualize IFN-λ-responsive cells. We found that the double-knockout mice showed enhanced susceptibility to various viruses that primarily replicate in lung epithelial cells. Our analysis further revealed that epithelial cells of both lung and gastrointestinal tracts can strongly respond to IFN-λ and that IFN-λ inhibited the replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in both lung and gastrointestinal tracts.     

TLR3, TLR4 and TLRs7–9 Induced Interferons Are Not Impaired in Airway and Blood Cells in Well Controlled Asthma

Defective Rhinovirus induced interferon-β and interferon-λ production has been reported in bronchial epithelial cells from asthmatics but the mechanisms of defective interferon induction in asthma are unknown. Virus infection can induce interferon through Toll like Receptors (TLR)3, TLR7 and TLR8. The role of these TLRs in interferon induction in asthma is unclear. This objective of this study was to measure the type I and III interferon response to TLR in bronchial epithelial cells and peripheral blood cells from atopic asthmatics and non-atopic non-asthmatics. Bronchial epithelial cells and peripheral blood mononuclear cells from atopic asthmatic and non-atopic non-asthmatic subjects were stimulated with agonists to TLR3, TLR4 & TLRs7–9 and type I and III interferon and pro-inflammatory cytokine, interleukin(IL)-6 and IL-8, responses assessed. mRNA expression was analysed by qPCR. Interferon proteins were analysed by ELISA. Pro-inflammatory cytokines were induced by each TLR ligand in both cell types. Ligands to TLR3 and TLR7/8, but not other TLRs, induced interferon-β and interferon-λ in bronchial epithelial cells. The ligand to TLR7/8, but not those to other TLRs, induced only type I interferons in peripheral blood mononuclear cells. No difference was observed in TLR induced interferon or pro-inflammatory cytokine production between asthmatic and non-asthmatic subjects from either cell type. TLR3 and TLR7/8,, stimulation induced interferon in bronchial epithelial cells and peripheral blood mononuclear cells. Interferon induction to TLR agonists was not observed to be different in asthmatics and non-asthmatics.     

Interferon alfacon 1 inhibits SARS-CoV infection in human bronchial epithelial Calu-3 cells

The primary targets for SARS-CoV infection are the epithelial cells in the respiratory and intestinal tract. The angiotensin-converting enzyme 2 (ACE-2) has been identified as a functional receptor for SARS-CoV. ACE-2 has been shown to be expressed at the apical domain of polarized Calu-3 cells. In this report, interferon alfacon 1 was examined for inhibitory activities against SARS-CoV on human lung carcinoma epithelial Calu-3 cell line and the other three African green monkey kidney epithelial cell lines. Interferon alfacon 1 demonstrated significant antiviral activity in neutral red uptake assay and virus yield reduction assay. The data might provide an important insight into the mechanism of pathogenesis of SARS-CoV allowing further development of antiviral therapies for treating SARS infections.     

Toll-Like Receptor Expression and Induction of Type I and Type III Interferons in Primary Airway Epithelial Cells

Interferons (IFNs) are a critical component of the first line of antiviral defense. The activation of Toll-like receptors (TLRs) expressed by dendritic cells triggers different signaling cascades that result in the production of large amounts of IFNs. However, the functional consequences of TLR activation and differential IFN production in specific cell populations other than antigen-presenting cells have not yet been fully elucidated. In this study, we investigated TLR expression and polarization in airway epithelial cells (AECs) and the consequences of TLR agonist stimulation for the production of type I (IFN-α/β) and type III (IFN-λ) IFNs. Our results show that the pattern of expression and polarization of all TLRs in primary AEC cultures mirrors that of the human airways ex vivo and is receptor specific. The antiviral TLRs (TLR3, TLR7, and TLR9) are mostly expressed on the apical cell surfaces of epithelial cells in the human trachea and in primary polarized AECs. Type III IFN is the predominant IFN produced by the airway epithelium, and TLR3 is the only TLR that mediates IFN production by AECs, while all TLR agonists tested are capable of inducing AEC activation and interleukin-8 production. In response to influenza virus infection, AECs can produce IFN-λ in an IFNAR- and STAT1-independent manner. Our results emphasize the importance of using primary well-differentiated AECs to study TLR and antiviral responses and provide further insight into the regulation of IFN production during the antiviral response of the lung epithelium.     

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.     

Human but Not Mouse Hepatocytes Respond to Interferon-Lambda In Vivo

The type III interferon (IFN) receptor is preferentially expressed by epithelial cells. It is made of two subunits: IFNLR1, which is specific to IFN-lambda (IFN-λ) and IL10RB, which is shared by other cytokine receptors. Human hepatocytes express IFNLR1 and respond to IFN-λ. In contrast, the IFN-λ response of the mouse liver is very weak and IFNLR1 expression is hardly detectable in this organ. Here we investigated the IFN-λ response at the cellular level in the mouse liver and we tested whether human and mouse hepatocytes truly differ in responsiveness to IFN-λ. When monitoring expression of the IFN-responsive Mx genes by immunohistofluorescence, we observed that the IFN-λ response in mouse livers was restricted to cholangiocytes, which form the bile ducts, and that mouse hepatocytes were indeed not responsive to IFN-λ. The lack of mouse hepatocyte response to IFN-λ was observed in different experimental settings, including the infection with a hepatotropic strain of influenza A virus which triggered a strong local production of IFN-λ. With the help of chimeric mice containing transplanted human hepatocytes, we show that hepatocytes of human origin readily responded to IFN-λ in a murine environment. Thus, our data suggest that human but not mouse hepatocytes are responsive to IFN-λ in vivo. The non-responsiveness is an intrinsic property of mouse hepatocytes and is not due to the mouse liver micro-environment.     

Inhibition of IFN-γ-dependent antiviral airway epithelial defense by cigarette smoke

Background

Although individuals exposed to cigarette smoke are more susceptible to respiratory infection, the effects of cigarette smoke on lung defense are incompletely understood. Because airway epithelial cell responses to type II interferon (IFN) are critical in regulation of defense against many respiratory viral infections, we hypothesized that cigarette smoke has inhibitory effects on IFN-γ-dependent antiviral mechanisms in epithelial cells in the airway.

Methods

Primary human tracheobronchial epithelial cells were first treated with cigarette smoke extract (CSE) followed by exposure to both CSE and IFN-γ. Epithelial cell cytotoxicity and IFN-γ-induced signaling, gene expression, and antiviral effects against respiratory syncytial virus (RSV) were tested without and with CSE exposure.

Results

CSE inhibited IFN-γ-dependent gene expression in airway epithelial cells, and these effects were not due to cell loss or cytotoxicity. CSE markedly inhibited IFN-γ-induced Stat1 phosphorylation, indicating that CSE altered type II interferon signal transduction and providing a mechanism for CSE effects. A period of CSE exposure combined with an interval of epithelial cell exposure to both CSE and IFN-γ was required to inhibit IFN-γ-induced cell signaling. CSE also decreased the inhibitory effect of IFN-γ on RSV mRNA and protein expression, confirming effects on viral infection. CSE effects on IFN-γ-induced Stat1 activation, antiviral protein expression, and inhibition of RSV infection were decreased by glutathione augmentation of epithelial cells using N-acetylcysteine or glutathione monoethyl ester, providing one strategy to alter cigarette smoke effects.

Conclusions

The results indicate that CSE inhibits the antiviral effects of IFN-γ, thereby presenting one explanation for increased susceptibility to respiratory viral infection in individuals exposed to cigarette smoke.     

Interferon and Ribavirin Combination Treatment Synergistically Inhibit HCV Internal Ribosome Entry Site Mediated Translation at the Level of Polyribosome Formation

Purpose

Although chronic hepatitis C virus (HCV) infection has been treated with the combination of interferon alpha (IFN-α) and ribavirin (RBV) for over a decade, the mechanism of antiviral synergy is not well understood. We aimed to determine the synergistic antiviral mechanisms of IFN-α and RBV combination treatment using HCV cell culture.

Methods

The antiviral efficacy of IFN-α, RBV alone and in combination was quantitatively measured using HCV infected and replicon cell culture. Direct antiviral activity of these two drugs at the level of HCV internal ribosome entry site (IRES) mediated translation in Huh-7 cell culture was investigated. The synergistic antiviral effect of IFN-α and RBV combination treatment was verified using both the CalcuSyn Software and MacSynergy Software.

Results

RBV combination with IFN-α efficiently inhibits HCV replication cell culture. Our results demonstrate that IFN-α, interferon lambda (IFN-λ) and RBV each inhibit the expression of HCV IRES-GFP and that they have a minimal effect on the expression of GFP in which the translation is not IRES dependent. The combination treatments of RBV along with IFN-α or IFN-λ were highly synergistic with combination indexes <1. We show that IFN-α treatment induce levels of PKR and eIF2α phosphorylation that prevented ribosome loading of the HCV IRES-GFP mRNA. Silencing of PKR expression in Huh-7 cells prevented the inhibitory effect of IFN-α on HCV IRES-GFP expression. RBV also blocked polyribosome loading of HCV-IRES mRNA through the inhibition of cellular IMPDH activity, and induced PKR and eIF2α phosphorylation. Knockdown of PKR or IMPDH prevented RBV induced HCV IRES-GFP translation.

Conclusions

We demonstrated both IFN-α and RBV inhibit HCV IRES through prevention of polyribosome formation. The combination of IFN-α and RBV treatment synergistically inhibits HCV IRES translation via using two different mechanisms involving PKR activation and depletion of intracellular guanosine pool through inhibition of IMPDH.     

Intranasal Treatment with Poly(I·C) Protects Aged Mice from Lethal Respiratory Virus Infections

In the 2002-2003 severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, no patients under 24 years of age died, while mortality was greater than 50% in those over 65 years. Greater than 90% of all deaths from influenza A virus (IAV) occur in the elderly (>65 years of age). To address this age-related susceptibility to SARS-CoV and IAV, we infected C57BL/6 (B6) mice with mouse-adapted SARS-CoV (MA15) or IAV (PR8), both of which cause severe disease in aged mice. Intranasal pretreatment of aged mice with poly(I·C) (a TLR3 agonist) and, to a lesser extent, CpG, R848, or lipopolysaccharide (TLR9, TLR7/8, or TLR4 agonists), provided a high level of protection [90% to 100% survival rate after poly(I·C) treatment] against lethal MA15 or IAV challenge and reduced pathological changes and virus loads in the lungs at early times after infection. Poly(I·C) pretreatment upregulated beta interferon (IFN-β), IFN-γ, IL-1β, and tumor necrosis factor (TNF) gene expression in the lungs. Intranasal pretreatment with IFN-β or IFN-γ but not IL-1β or TNF also protected aged mice, consistent with the notion that poly(I·C) pretreatment functioned, at least in part, by inducing IFN-β and IFN-γ. We also identified a potential cellular target for poly(I·C) by showing that treatment inhibited virus replication in primary human airway epithelial cells. These results suggest that intranasal poly(I·C) should be evaluated as a prophylactic agent in aged individuals at high risk for contracting SARS-CoV or IAV infections.     

Interferon-λ Contributes to Innate Immunity of Mice against Influenza A Virus but Not against Hepatotropic Viruses

Virus-infected cells secrete a broad range of interferon (IFN) subtypes which in turn trigger the synthesis of antiviral factors that confer host resistance. IFN-α, IFN-β and other type I IFNs signal through a common universally expressed cell surface receptor, whereas IFN-λ uses a distinct receptor complex for signaling that is not present on all cell types. Since type I IFN receptor-deficient mice (IFNAR1^0/0) exhibit greatly increased susceptibility to various viral diseases, it remained unclear to which degree IFN-λ might contribute to innate immunity. To address this issue we performed influenza A virus infections of mice which carry functional alleles of the influenza virus resistance gene Mx1 and which, therefore, develop a more complete innate immune response to influenza viruses than standard laboratory mice. We demonstrate that intranasal administration of IFN-λ readily induced the antiviral factor Mx1 in mouse lungs and efficiently protected IFNAR1^0/0 mice from lethal influenza virus infection. By contrast, intraperitoneal application of IFN-λ failed to induce Mx1 in the liver of IFNAR1^0/0 mice and did not protect against hepatotropic virus infections. Mice lacking functional IFN-λ receptors were only slightly more susceptible to influenza virus than wild-type mice. However, mice lacking functional receptors for both IFN-α/β and IFN-λ were hypersensitive and even failed to restrict usually non-pathogenic influenza virus mutants lacking the IFN-antagonistic factor NS1. Interestingly, the double-knockout mice were not more susceptible against hepatotropic viruses than IFNAR1^0/0 mice. From these results we conclude that IFN-λ contributes to inborn resistance against viral pathogens infecting the lung but not the liver.