IFN-λ Response Research Articles

Apoptotic Caspases Suppress Expression of Endogenous Retroviruses in HPV31+ Cells That Are Associated with Activation of an Innate Immune Response

Avoidance of an immune response is critical to completion of the human papillomavirus (HPV) life cycle, which occurs in the stratified epithelium and is linked to epithelial differentiation. We previously demonstrated that high-risk HPVs use apoptotic caspases to suppress an antiviral innate immune response during the productive phase of the life cycle. We found that caspase-8 and caspase-3 suppress a type I IFN-β and type III IFN-λ response by disabling the MDA5/MAVS double-stranded RNA (dsRNA) sensing pathway, indicating that immunogenic RNAs increase upon differentiation in HPV+ cells. In this study, we demonstrate that caspase inhibition results in aberrant transcription of a subset of endogenous retroviruses (ERVs) that have been shown to activate an IFN response through dsRNA-sensing pathways. We show that the increase in ERV transcription is accompanied by an enrichment in dsRNA formation. Additionally, we demonstrate that the robust increase in ERV expression requires activation of the JAK/STAT-signaling pathway, indicating that this subset of ERVs is IFN-inducible. Overall, these results suggest a model by which caspase activity blocks the reactivation of ERVs through the JAK/STAT pathway, protecting HPV+ cells from an increase in immunogenic dsRNAs that otherwise would trigger an IFN response that inhibits productive viral replication.

Swine acute diarrhea syndrome coronavirus Nsp1 suppresses IFN-λ1 production by degrading IRF1 via ubiquitin–proteasome pathway

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel porcine enteric coronavirus that causes acute watery diarrhea, vomiting, and dehydration in newborn piglets. The type III interferon (IFN-λ) response serves as the primary defense against viruses that replicate in intestinal epithelial cells. However, there is currently no information available on how SADS-CoV modulates the production of IFN-λ. In this study, we utilized IPI-FX cells (a cell line of porcine ileum epithelium) as an in vitro model to investigate the potential immune evasion strategies employed by SADS-CoV against the IFN-λ response. Our results showed that SADS-CoV infection suppressed the production of IFN-λ1 induced by poly(I:C). Through screening SADS-CoV-encoded proteins, nsp1, nsp5, nsp10, nsp12, nsp16, E, S1, and S2 were identified as antagonists of IFN-λ1 production. Specifically, SADS-CoV nsp1 impeded the activation of the IFN-λ1 promoter mediated by MAVS, TBK1, IKKε, and IRF1. Both SADS-CoV and nsp1 obstructed poly(I:C)-induced nuclear translocation of IRF1. Moreover, SADS-CoV nsp1 degraded IRF1 via the ubiquitin-mediated proteasome pathway without interacting with it. Overall, our study provides the first evidence that SADS-CoV inhibits the type III IFN response, shedding light on the molecular mechanisms employed by SADS-CoV to evade the host immune response.

A251 GUT-SPECIFIC DOWNREGULATION OF INTERFERON-LAMBDA RESPONSES OCCURS IN INFLAMMATORY BOWEL DISEASES

Abstract Background Inflammatory bowel diseases (IBD) affect 1:140 Canadians, resulting in significant inflammation and gut damage. Unlike type I and II interferons (IFNs), the most recently discovered IFNs, interferon-lambdas (IFN-λs), uniquely downregulate gut inflammation and promote gut healing in mouse models. However, it remains unknown if the important anti-inflammatory IFN-λ signaling pathways induced downstream of the IFN-λR1/IL-10RB heterodimeric receptor are altered in IBD. Aims To compare intestinal and peripheral IFN-λR levels and responses in people with active or remission IBD and those without IBD. We hypothesized that intestinal IFN-λR1 levels and downstream activities are decreased in people living with IBD, which could contribute to IBD pathology (gut inflammation and mucosal damage). Methods During routine colonoscopy, uninflamed biopsy samples were obtained from the terminal ileum and sigmoid colon. IFN-λR1 levels were quantified in intestinal biopsy and blood samples from persons with or without IBD (n=12 adults and n=12 pediatric) by immunohistochemistry and flow cytometry. Fresh intestinal biopsies were also cultured ex vivo for 24 hr in media +/- IFN-λ3 and gene expression was quantified by RT-qPCR. Results In IBD gut tissues, a significant decrease in IFN-λR1+ cells was more prominent in IBD patients with active disease (pampersand:003C0.01, 30-50% reduction). The decrease was more prominent in IBD patients with active disease. Consequently, there was a significantly lower IFN-λ-driven upregulation of genes such as IFIT1 and MX1 in IBD as compared to non-IBD tissues (pampersand:003C0.05, 4-7-fold reduction). Interestingly, IFN-λR1 levels were not decreased in peripheral blood immune cells, indicating the dysregulation of IFN-λR responses is likely gut-specific. Conclusions Our findings demonstrate that people living with IBD have dysregulated IFN-λ responses in the gut which could lead to the lower induction of key anti-inflammatory pathways. This work supports the further study of mechanisms regulating the IFN-λ system to develop strategies to restore and promote IFN-λ responses as a novel potential future IBD therapy. Funding Agencies University of Manitoba, Weston Family Foundation, The Children's Hospital Research Institute of Manitoba.

Current Landscape of IFN-λ: Induction, Inhibition, and Potential Clinical Applications to Treat Respiratory Viral Infections.

IFN-λ or type III IFN is an important mediator of antiviral response. Several respiratory viruses induce the production of IFN-λ during their course of infection. However, they have also developed intricate mechanisms to inhibit its expression and activity. Despite a considerable amount of research on the regulatory mechanisms of respiratory viruses on the IFN-λ response, little is still known about the effect of this cytokine on immune cells and the antiviral effects of all IFN-λ isoforms, and a better understanding of the detrimental effects of IFN-λ treatment is required. Here we highlight the relevance of IFN-λ as an antiviral cytokine in the respiratory tract. Data from studies invitro, ex vivo, experimental animal models, and ongoing clinical trials emphasize the therapeutic opportunity that IFN-λ represents to treat and prevent different types of respiratory viral infections.

Role of Innate Interferon Responses at the Ocular Surface in Herpes Simplex Virus-1-Induced Herpetic Stromal Keratitis.

Herpes simplex virus type 1 (HSV-1) is a highly successful pathogen that primarily infects epithelial cells of the orofacial mucosa. After initial lytic replication, HSV-1 enters sensory neurons and undergoes lifelong latency in the trigeminal ganglion (TG). Reactivation from latency occurs throughout the host's life and is more common in people with a compromised immune system. HSV-1 causes various diseases depending on the site of lytic HSV-1 replication. These include herpes labialis, herpetic stromal keratitis (HSK), meningitis, and herpes simplex encephalitis (HSE). HSK is an immunopathological condition and is usually the consequence of HSV-1 reactivation, anterograde transport to the corneal surface, lytic replication in the epithelial cells, and activation of the host's innate and adaptive immune responses in the cornea. HSV-1 is recognized by cell surface, endosomal, and cytoplasmic pattern recognition receptors (PRRs) and activates innate immune responses that include interferons (IFNs), chemokine and cytokine production, as well as the recruitment of inflammatory cells to the site of replication. In the cornea, HSV-1 replication promotes type I (IFN-α/β) and type III (IFN-λ) IFN production. This review summarizes our current understanding of HSV-1 recognition by PRRs and innate IFN-mediated antiviral immunity during HSV-1 infection of the cornea. We also discuss the immunopathogenesis of HSK, current HSK therapeutics and challenges, proposed experimental approaches, and benefits of promoting local IFN-λ responses.

A171 INTESTINAL INTERFERON-LAMBDA RECEPTOR 1 EXPRESSION AND RESPONSES ARE SIGNIFICANTLY DECREASED IN PEDIATRIC INFLAMMATORY BOWEL DISEASE PATIENTS

Abstract Background While interferon-lambdas (IFN-λs) were initially discovered for their role in antiviral immunity at mucosal barriers such as the lung and gut, there are many unanswered questions for how IFN-λs uniquely dampen inflammatory immune responses. In mouse colitis models, IFN-λs were shown to play a significant role in promoting epithelial barrier integrity and mucosal healing. Microbes present in the gut naturally induce IFN-λs, but how chronic inflammation, such as in patients with inflammatory bowel diseases (IBD), affects IFN-λ biology has not been well studied, especially in humans. Purpose Here, we tested the hypothesis that children with active, more severe IBD would present with lower intestinal IFN-λR levels and IFN-λ responses which could contribute to IBD pathology through exacerbated inflammation, decreased mucosal healing and impaired barrier function. Method We screened 14 novel antibodies to find the optimal clone that accurately stains the unique IFN-λ receptor subunit (IFN-λR1) protein in human intestinal tissue and IFN-λR1 levels were quantified by immunohistochemistry and flow cytometry in biopsy samples from children without IBD, Crohn’s disease, or ulcerative colitis (n=35 total). Fresh patient biopsies (ascending colon or terminal ileum) were also treated in media +/- IFN-λ for 24hrs using our novel ex vivo biopsy assay and changes in gene expression were quantified by RT-qPCR. Intestinal washes were also collected and microbes profiled by shotgun metagenomics. Result(s) We identified 2 new antibodies that accurately stained human cell lines and immune cells known to express IFN-λR1 protein (gut epithelial cells and B cells). We found that IFN-λ receptor (IFN-λR) levels are significantly reduced in gut epithelial and immune cells within pediatric IBD intestinal tissue, even at non-inflamed sites (p<0.01, 30-50% reduction) and this was even more pronounced when comparing moderate/severe disease compared to children with no disease activity. This led to lower IFN-λ responsiveness in IBD compared to non-IBD biopsies when investigating IFN-stimulated gene expression (p<0.05, up to 7-fold reduction). Paired patient gut microbe analyses identified specific species that correlated with changes in IFN-λ receptor expression. Conclusion(s) Together, our findings demonstrate pediatric IBD patients may be less able to induce critical IFN-λ-mediated antimicrobial responses and protective anti-inflammatory pathways. This work supports the goal to restore and promote IFN-λ responses as a novel therapeutic strategy for pediatric IBD. Please acknowledge all funding agencies by checking the applicable boxes below Other Please indicate your source of funding; Children's Hospital Research Institute of Manitoba Disclosure of Interest None Declared

The autophagy gene Epg5 promotes susceptibility to enteric viral infection

Abstract Mutations in the autophagy gene EPG5 are responsible for Vici syndrome, a multisystem human genetic disease with a combined immunodeficiency component. However, the mechanism(s) by which EPG5 regulates immune signaling are incompletely understood. Previously, we found that Epg5−/− mice are resistant to influenza and exhibit hyperinflammation in the lungs including elevated IL1B/IL-1β and TNF/TNFa. Here, we show that the disruption of Epg5 results in protection against multiple enteric viruses including norovirus and rotavirus. RNA sequencing revealed that IFN-λ responses are highly upregulated in the intestines of Epg5−/− mice. Further, mice lacking Epg5 exhibit substantial alterations of the intestinal microbiota. Surprisingly, germ-free Epg5−/− mice showed persistent inflammation of both the intestine and lung, suggesting a microbiota independent mechanism. Epg5−/−Ifnlr1−/− mice, which lack the receptor for IFN-λ, regained susceptibility to viral infection but maintained microbial dysbiosis, indicating that IFN-λ signaling is the primary mediator of resistance to enteric viruses but is dispensable for microbial dysbiosis in Epg5−/− mice. Intriguingly, epg-5-mutant Caenorhabditis elegans animals are also resistant to Orsay virus, which is an intestinal cell-tropic virus and the only known virus that naturally infects C. elegans. This study unveils an important role for autophagy gene Epg5 in protection of mammalian hosts by modulating intestinal IFN-λ responses, which is unexpectedly independent of the microbiota, and raises the possibility of a conserved viral resistance mechanism that is shared between C. elegans and mammals.

An endogenously activated antiviral state restricts SARS-CoV-2 infection in differentiated primary airway epithelial cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the coronavirus disease-19 (COVID-19) pandemic, was identified in late 2019 and caused >5 million deaths by February 2022. To date, targeted antiviral interventions against COVID-19 are limited. The spectrum of SARS-CoV-2 infection ranges from asymptomatic to fatal disease. However, the reasons for varying outcomes to SARS-CoV-2 infection are yet to be elucidated. Here we show that an endogenously activated interferon lambda (IFNλ1) pathway leads to resistance against SARS-CoV-2 infection. Using a well-differentiated primary nasal epithelial cell (WD-PNEC) culture model derived from multiple adult donors, we discovered that susceptibility to SARS-CoV-2 infection, but not respiratory syncytial virus (RSV) infection, varied. One of four donors was resistant to SARS-CoV-2 infection. High baseline IFNλ1 expression levels and associated interferon stimulated genes correlated with resistance to SARS-CoV-2 infection. Inhibition of the JAK/STAT pathway in WD-PNECs with high endogenous IFNλ1 secretion resulted in higher SARS-CoV-2 titres. Conversely, prophylactic IFNλ treatment of WD-PNECs susceptible to infection resulted in reduced viral titres. An endogenously activated IFNλ response, possibly due to genetic differences, may be one explanation for the differences in susceptibility to SARS-CoV-2 infection in humans. Importantly, our work supports the continued exploration of IFNλ as a potential pharmaceutical against SARS-CoV-2 infection.

Murine Type III interferons are functionally redundant and correlate with bacterial burden during influenza/bacterial super-infection.

BackgroundType III interferon, or interferon lambda (IFNλ) is a crucial antiviral cytokine induced by influenza infection. While IFNλ is important for anti-viral host defense, published data demonstrate that IFNλ is pathogenic during influenza/bacterial super-infection. It is known that polymorphisms in specific IFNλ genes affect influenza responses, but the effect of IFNλ subtypes on bacterial super-infection is unknown.MethodsUsing an established model of influenza, Staphylococcus aureus super-infection, we studied IFNλ3-/- and control mice to model a physiologically relevant reduction in IFNλ and to address its role in super-infection.ResultsSurprisingly, IFNλ3-/- mice did not have significantly lower total IFNλ than co-housed controls, and displayed no change in viral or bacterial clearance. Importantly, both control and IFNλ3-/- mice displayed a positive correlation between viral burden and total IFNλ in the bronchoalveolar lavage during influenza/bacterial super-infection, suggesting that higher influenza viral burden drives a similar total IFNλ response regardless of IFNλ3 gene integrity. Interestingly, total IFNλ levels positively correlated with bacterial burden, while viral burden and bronchoalveolar lavage cellularity did not.ConclusionsThese data suggest IFNλ2 can compensate for IFNλ3 to mount an effective antiviral and defense, revealing a functional redundancy in these highly similar IFNλ subtypes. Further, the IFNλ response to influenza, as opposed to changes in cellular inflammation or viral load, significantly correlates with susceptibility to bacterial super-infection. Moreover, the IFNλ response is regulated and involves redundant subtypes, suggesting it is of high importance to pulmonary pathogen defense.

IFN-λ Regulates Neutrophil Biology to Suppress Inflammation in Herpes Simplex Virus-1-Induced Corneal Immunopathology.

HSV-1 infection of the cornea causes a severe immunoinflammatory and vision-impairing condition called herpetic stromal keratitis (SK). The virus replication in corneal epithelium followed by neutrophil- and CD4+ T cell-mediated inflammation plays a dominant role in SK. Although previous studies demonstrate critical functions of type I IFNs (IFN-α/β) in HSV-1 infection, the role of recently discovered IFN-λ (type III IFN), specifically at the corneal mucosa, is poorly defined. Our study using a mouse model of SK pathogenesis shows that HSV-1 infection induces a robust IFN-λ response compared with type I IFN production at the corneal mucosal surface. However, the normal progression of SK indicates that the endogenous IFN responses are insufficient to suppress HSV-1-induced corneal pathology. Therefore, we examined the therapeutic efficacy of exogenous rIFN-λ during SK progression. Our results show that rIFN-λ therapy suppressed inflammatory cell infiltration in the cornea and significantly reduced the SK pathologic condition. Early rIFN-λ treatment significantly reduced neutrophil and macrophage infiltration, and IL-6, IL-1β, and CXCL-1 production in the cornea. Notably, the virucidal capacity of neutrophils and macrophages measured by reactive oxygen species generation was not affected. Similarly, ex vivo rIFN-λ treatment of HSV-1-stimulated bone marrow-derived neutrophils significantly promoted IFN-stimulated genes without affecting reactive oxygen species production. Collectively, our data demonstrate that exogenous topical rIFN-λ treatment during the development and progression of SK could represent a novel therapeutic approach to control HSV-1-induced inflammation and associated vision impairment.

Murine astrovirus tropism for goblet cells and enterocytes facilitates an IFN-λ response in vivo and in enteroid cultures.

Although they globally cause viral gastroenteritis in children, astroviruses are understudied due to the lack of well-defined animal models. While murine astroviruses (muAstVs) chronically infect immunodeficient mice, a culture system and understanding of their pathogenesis is lacking. Here, we describe a platform to cultivate muAstV using air-liquid interface (ALI) cultures derived from mouse enteroids, which support apical infection and release. Chronic muAstV infection occurs predominantly in the small intestine and correlates with higher interferon-lambda (IFN-λ) expression. MuAstV stimulates IFN-λ production in ALI, recapitulating our in vivo findings. We demonstrate that goblet cells and enterocytes are targets for chronic muAstV infection in vivo, and that infection is enhanced by parasite coinfection or type 2 cytokine signaling. Depletion of goblet cells from ALI limits muAstV infection in vitro. During chronic infection, muAstV stimulates IFN-λ production in infected cells and induces ISGs throughout the intestinal epithelium in an IFN-λ-receptor dependent manner. Collectively, our study provides insights into the cellular tropism and innate immune responses to muAstV and establishes an enteroid-based culture system to propagate muAstV in vitro.

Articles of Significant Interest in This Issue

Viral infection of intestinal epithelial cells (IECs) is inhibited by type III interferons (IFN-λs), rather than the more broadly active type I IFNs (IFN-αs and IFN-β [IFN-α/β]). Van Winkle et al. (e00603-20) found that the intestinal epithelial IFN-λ response provides antiviral protection while minimizing epithelial cytotoxicity compared to IFN-β. These findings offer new insight into the benefits of an IFN-λ response at the intestinal barrier and suggest how dysregulation of this response could promote inflammatory disease.

The Coronavirus PEDV Evades Type III Interferon Response Through the miR-30c-5p/SOCS1 Axis.

Porcine epidemic diarrhea virus (PEDV) is an economically important pathogen that has evolved several mechanisms to evade type I IFN responses. Type III interferon (IFN-λ), an innate cytokine that primarily targets the mucosal epithelia, is critical in fighting mucosal infection in the host and has been reported to potently inhibit PEDV infection in vitro. However, how PEDV escapes IFN-λ antiviral response remains unclear. In this study, we found that PEDV infection induced significant IFN-λ expression in type I IFN-defective Vero E6 cells, but virus-induced endogenous IFN-λ did not reduce PEDV titers. Moreover, we demonstrated that PEDV escaped IFN-λ responses by substantially upregulating the suppressor of cytokine signaling protein 1 (SOCS1) expression, which impaired the induction of IFN-stimulated genes (ISGs) and dampened the IFN-λ antiviral response and facilitated PEDV replication in Vero E6 cells. We further showed that PEDV infection increased SOCS1 expression by decreasing host miR-30c-5p expression. MiR-30c-5p suppressed SOCS1 expression through targeting the 3′ untranslated region (UTR) of SOCS1. The inhibition of IFN-λ elicited ISGs expression by SOCS1 was specifically rescued by overexpression of miR-30c-5p. Collectively, our findings identify a new strategy by PEDV to escape IFN-λ-mediated antiviral immune responses by engaging the SOCS1/miR-30c axis, thus improving our understanding of its pathogenesis.

A Secreted Viral Nonstructural Protein Determines Intestinal Norovirus Pathogenesis

Murine norovirus (MNoV) infects a low percentage ofenteric tuft cells and can persist in these cells for months following acute infection. Both tuft-cell tropism and resistance to interferon-λ (IFN-λ)-mediated clearance during persistent infection requires the viral nonstructural protein 1/2 (NS1/2). We showthat processing of NS1/2 yields NS1, an unconventionally secreted viral protein that is central for IFN-λ resistance. MNoV infection globally suppresses intestinal IFN-λ responses, which is attributable to secreted NS1. MNoV NS1 secretion is triggered by caspase-3 cleavage of NS1/2, and a secreted form of human NoV NS1 is also observed. NS1 secretion is essential for intestinal infection and resistance to IFN-λ invivo. NS1 vaccination alone protects against MNoV challenge, despite the lack of induction of neutralizing anti-capsid antibodies previously shown to confer protection. Thus, despite infecting a low number of tuft cells, NS1 secretion allows MNoV to globally suppress IFN responses and promote persistence.

HIV-1 and HIV-2 induce Interferon-lambda production by plasmacytoid Dendritic Cells

Abstract Type III Interferons (IFN-λ) were barely studied during HIV infection, despite their antiviral properties similar to those of type I IFN and their important role during HCV infection, where IFN-λ3 gene polymorphisms correlate with viral clearance. We tested IFN-λ responses to HIV-1, and also to HIV-2, which is less pathogenic. We first stimulated PBMC (EFS-Inserm convention) enriched for dendritic cells (DC), the main IFN producers. Purified HIV-1 virions or H9 T cells chronically infected with HIV-1 induced transcription of IFN-λ1 and 2/3 mRNA (RT-qPCR), as well as the secretion of IFN-λ (ELISA). Similar results were obtained with HIV-2-infected cells or virions. Intracellular labeling for IFN-λ1 was detected by flow cytometry in BDCA-4+ plasmacytoid DC (pDC), but not in BDCA-1+ or -3+ conventional DC (cDC). This was confirmed in > 95% purified cell populations. Recognition of HIV-1 or HIV-2 by pDC induced their maturation (surface CD83 and CCR7 overexpression), and bystander maturation of cDC. Therefore, IFN-λ production by human blood pDC was induced at comparable levels by HIV-1 or HIV-2, either as cell-free viruses or as infected cells, indicating that it is not a clue for the difference in host-pathogen relationship between the two viruses, and that IFN-λ may contribute to the Interferon-Stimulated-Gene response during HIV infection. Knowledge of IFN-λ responses to HIV will help modulating IFN responses toward viral control and against pathogenic immune hyperactivation.

IFN-lambda regulates dendritic cell function to mediate protective immunity against influenza A virus infection

Abstract Influenza A virus (IAV) is a public health threat with constant potential for pandemic outbreak in addition to causing annual, seasonal epidemics. While an IAV vaccine is available, it is rapidly subverted through viral genetic drift, necessitating strategies to limit IAV infection and drive robust, protective immune responses. Type III interferon (IFN), IFN-lambda (IFNλ), is the most highly produced IFN in the lung during IAV infection and restricts IAV in vitro and in vivo. Further, IFNλ influences CD4 T cell skewing following IAV vaccination, but the contribution of IFNλ to the critically protective CD8 T cell response against IAV has not been defined. Here we evaluated the role of IFNλ signaling in the development of CD8 T cell responses against IAV infection in a murine model. Our studies reveal that IFNλ signaling is essential to support cytokine-producing IAV-specific effector CD8 T cells as the CD8 T cell response against IAV is significantly reduced in IFNλ receptor knock out (IFNλR KO) mice. Importantly, IFNλR KO mice exhibited reduced immune protection upon heterologous IAV re-challenge that is dependent upon T cell responses. This immune defect was not T cell intrinsic but instead mapped to altered function and migration of dendritic cells (DCs) in IFNλR KOs, thus linking IFNλ signaling in DCs with modulation of the T cell response. Transcriptomic analysis of DCs revealed the dysregulation of a set of genes implicated in antigen uptake, processing and/or presentation, downstream of IFNλ, but not type I IFN. The function of these genes in the IFNλ response for programming CD8 T cells against IAV will be presented. Our findings reveal a unique role for IFNλ in the governance of DC function and subsequent downstream T cell function.

Immune Cell Profiling of IFN-λ Response Shows pDCs Express Highest Level of IFN-λR1 and Are Directly Responsive via the JAK-STAT Pathway.

The interferon lambda (IFN-λ) cytokines have well-known antiviral properties, yet their contribution to immune regulation is not well understood. Epithelial cells represent the major target cell of IFN-λ; peripheral blood mononuclear cells are generally considered nonresponsive, with the exception of plasmacytoid dendritic cells (pDCs). In this study we aimed to define the potential for discrete subpopulations of cells to directly respond to IFN-λ. Analysis of peripheral blood leukocytes reveals that, while pDCs uniformly express the highest levels of IFN-λ receptor, a small proportion of B cells and monocytes also express the receptor. Nevertheless, B cells and monocytes respond poorly to IFN-λ stimulation in vitro, with minimal STAT phosphorylation and interferon-stimulated gene (ISG) induction observed. We confirm that pDCs respond to IFN-λ in vitro, upregulating their expression of pSTAT1, pSTAT3, and pSTAT5. However, we found that pDCs do not upregulate pSTAT6 in response to IFN-λ treatment. Our results highlight unique aspects of the response to IFN-λ and confirm that while the IFN-λ receptor is expressed by a small proportion of several different circulating immune cell lineages, under normal conditions only pDCs respond to IFN-λ stimulation with robust STAT phosphorylation and ISG induction. The difference in STAT6 responsiveness of pDCs to type I and type III interferons may help explain the divergence in their biological activities.

Interferon lambda inhibits dengue virus replication in epithelial cells.

BackgroundIn viral disease, infection is controlled at the cellular level by type I interferon (IFN-I), but dengue virus (DENV) has the ability to inhibit this response. Type III interferon, also known as lambda IFN (IFN-III or IFN-λ), is a complementary pathway to the antiviral response by IFN-I. This work analyzed the IFN-λ (IFN-III) mediated antiviral response against DENV serotype 2 (DENV-2) infection.MethodsDengue fever patients were sampled to determine their IFN-λ levels by ELISA. To study the IFN-λ response during DENV infection we selected the epithelial cell line C33-A, and we demonstrated that it is permissive to DENV-2 infection. The effect of IFN-λ on virus replication was determined in these cells, in parallel to the expression of IFN-stimulated genes (ISGs), and Suppressor of Cytokine Signaling (SOCS), genes measured by RT-qPCR.ResultsWe found increased (~1.8 times) serological IFN-λ in dengue fever patients compared to healthy blood donors. IFN-λ inhibited DENV-2 replication in a dose-dependent manner in vitro. The reduction of viral titer corresponded with increased ISG mRNA levels (MX1 and OAS1), with the highest inhibition occurring at ISG’s peak expression. Presence of IFN-negative regulators, SOCS1 and SOCS3, during DENV-2 infection was associated with reduced IFN-λ1 expression.ConclusionsEvidence described here suggests that IFN-λ is a good candidate inhibitor of viral replication in dengue infection. Mechanisms for the cellular and organismal interplay between DENV and IFN- λ need to be further studied as they could provide insights into strategies to treat this disease. Furthermore, we report a novel epithelial model to study dengue infection in vitro.

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.

Prominent amphibian (Xenopus laevis) tadpole type III interferon response to the frog virus 3 ranavirus.

Ranaviruses (Iridoviridae) are posing an increasing threat to amphibian populations, with anuran tadpoles being particularly susceptible to these viral infections. Moreover, amphibians are the most basal phylogenetic class of vertebrates known to possess both type I and type III interferon (IFN)-mediated immunity. Moreover, little is known regarding the respective roles of the IFN mediators in amphibian antiviral defenses. Accordingly, we transcriptionally and functionally compared the amphibian Xenopus laevis type I (IFN) and III (IFN-λ) IFNs in the context of infections by the ranavirus frog virus 3 (FV3). X. laevis IFN and IFN-λ displayed distinct tissue expression profiles. In contrast to our previous findings that X. laevis tadpoles exhibit delayed and modest type I IFN responses to FV3 infections compared to the responses of adults, here we report that tadpoles mount timely and robust type III IFN gene responses. Recombinant forms of these cytokines (recombinant X. laevis IFN [rXlIFN] and rXlIFN-λ) elicited antiviral gene expression in the kidney-derived A6 cell line as well as in tadpole leukocytes and tissues. However, rXlIFN-λ was less effective than rXlIFN in preventing FV3 replication in A6 cells and tadpoles and inferior at promoting tadpole survival. Intriguingly, FV3 impaired A6 cell and tadpole kidney type III IFN receptor gene expression. Furthermore, in A6 cultures rXlIFN-λ conferred equal or greater protection than rXlIFN against recombinant viruses deficient for the putative immune evasion genes, the viral caspase activation and recruitment domain (vCARD) or a truncated vIF-2α gene. Thus, in contrast to previous assumptions, tadpoles possess intact antiviral defenses reliant on type III IFNs, which are overcome by FV3 pathogens. Anuran tadpoles, including those of Xenopus laevis, are particularly susceptible to infection by ranavirus such as FV3. We investigated the respective roles of X. laevis type I and type III interferons (IFN and IFN-λ, respectively) during FV3 infections. Notably, tadpoles mounted timely and more robust IFN-λ gene expression responses to FV3 than adults, contrasting with the poorer tadpole type I IFN responses. However, a recombinant X. laevis IFN-λ (rXlIFN-λ) conferred less protection to tadpoles and the A6 cell line than rXlIFN, which may be explained by the FV3 impairment of IFN-λ receptor gene expression. The importance of IFN-λ in tadpole anti-FV3 defenses is underlined by the critical involvement of two putative immune evasion genes in FV3 resistance to IFN- and IFN-λ-mediated responses. These findings challenge the view that tadpoles have defective antiviral immunity and suggest, rather, that their antiviral responses are predominated by IFN-λ responses, which are overcome by FV3.