Visualizing the Selectivity and Dynamics of Interferon Signaling In Vivo.

Interferons (IFN) are pleiotropic cytokines essential for defense against infection, but the identity and tissue distribution of IFN-responsive cells in vivo are poorly defined. In this study, we generate a mouse strain capable of reporting IFN-signaling activated by all three types of IFNs and investigate the spatio-temporal dynamics and identity of IFN-responding cells following IFN injection and influenza virus infection. Despite ubiquitous expression of IFN receptors, cellular responses to IFNs are highly heterogenous in vivo and are determined by anatomical site, cell type, cellular preference to individual IFNs, and activation status. Unexpectedly, type I and II pneumocytes, the primary target of influenza infection, exhibit striking differences in the strength and temporal dynamics of IFN signaling associated with differential susceptibility to the viral infection. Our findings suggest that time- and cell-type-dependent integration of distinct IFN signals govern the specificity and magnitude of IFN responses in vivo.

Interferon-λ Mediates Non-redundant Front-Line Antiviral Protection against Influenza Virus Infection without Compromising Host Fitness.

Lambda interferons (IFNλs) or type III IFNs share homology, expression patterns, signaling cascades, and antiviral functions with type I IFNs. This has complicated the unwinding of their unique non-redundant roles. Through the systematic study of influenza virus infection in mice, we herein show that IFNλs are the first IFNs produced that act at the epithelial barrier to suppress initial viral spread without activating inflammation. If infection progresses, type I IFNs come into play to enhance viral resistance and induce pro-inflammatory responses essential for confronting infection but causing immunopathology. Central to this are neutrophils which respond to both cytokines to upregulate antimicrobial functions but exhibit pro-inflammatory activation only to type I IFNs. Accordingly, Ifnlr1-/- mice display enhanced type I IFN production, neutrophilia, lung injury, and lethality, while therapeutic administration of PEG-IFNλ potently suppresses these effects. IFNλs therefore constitute the front line of antiviral defense in the lung without compromising host fitness.

Activation of Epithelial Signal Transducer and Activator of Transcription 1 by Interleukin 28 Controls Mucosal Healing in Mice With Colitis and Is Increased in Mucosa of Patients With Inflammatory Bowel Disease.

BACKGROUND & AIMS: We investigated the roles of interleukin 28A (also called IL28A or interferon λ2) in intestinal epithelial cell (IEC) activation, studying its effects in mouse models of inflammatory bowel diseases (IBD) and intestinal mucosal healing. METHODS: Colitis was induced in C57BL/6JCrl mice (controls), mice with IEC-specific disruption of Stat1 (Stat1IEC-KO), mice with disruption of the interferon λ receptor 1 gene (Il28ra-/-), and mice with disruption of the interferon regulatory factor 3 gene (Irf3-/-), with or without disruption of Irf7 (Irf7-/-). We used high-resolution mini-endoscopy and in vivo imaging methods to assess colitis progression. We used 3-dimensional small intestine and colon organoids, along with RNA-Seq and gene ontology methods, to characterize the effects of IL28 on primary IECs. We studied the effects of IL28 on the human intestinal cancer cell line Caco-2 in a wound-healing assay, and in mice colon wounds. Colonic biopsies and resected tissue from patients with IBD (n = 62) and patients without colon inflammation (controls, n = 23) were analyzed by quantitative polymerase chain rection to measure expression of IL28A, IL28RA, and other related cytokines; biopsy samples were also analyzed by immunofluorescence to identify sources of IL28 production. IECs were isolated from patient tissues and incubated with IL28; signal transducer and activator of transcription 1 (STAT1) phosphorylation was measured by immunoblots and confocal imaging. RESULTS: Lamina propria cells in colon tissues of patients with IBD, and mice with colitis, had increased expression of IL28 compared with controls; levels of IL28R were increased in the colonic epithelium of patients with IBD and mice with colitis. Administration of IL28 induced phosphorylation of STAT1 in primary human and mouse IECs, increasing with dose. Il28ra-/-, Irf3-/-, Irf3-/-Irf7-/-, as well as Stat1IEC-KO mice, developed more severe colitis after administration of dextran sulfate sodium than control mice, with reduced epithelial restitution. Il28ra-/- and Stat1IEC-KO mice also developed more severe colitis in response to oxazolone than control mice. We found IL28 to induce phosphorylation (activation) of STAT1 in epithelial cells, leading to their proliferation in organoid culture. Administration of IL28 to mice with induced colonic wounds promoted mucosal healing. CONCLUSIONS: IL28 controls proliferation of IECs in mice with colitis and accelerates mucosal healing by activating STAT1. IL28 might be developed as a therapeutic agent for patients with IBD.

Interferon-λ restricts West Nile virus neuroinvasion by tightening the blood-brain barrier.

Although interferon-λ [also known as type III interferon or interleukin-28 (IL-28)/IL-29] restricts infection by several viruses, its inhibitory mechanism has remained uncertain. We used recombinant interferon-λ and mice lacking the interferon-λ receptor (IFNLR1) to evaluate the effect of interferon-λ on infection with West Nile virus, an encephalitic flavivirus. Cell culture studies in mouse keratinocytes and dendritic cells showed no direct antiviral effect of exogenous interferon-λ, even though expression of interferon-stimulated genes was induced. We observed no differences in West Nile virus burden between wild-type and Ifnlr1(-/-) mice in the draining lymph nodes, spleen, or blood. We detected increased West Nile virus infection in the brain and spinal cord of Ifnlr1(-/-) mice, yet this was not associated with a direct antiviral effect in mouse neurons. Instead, we observed an increase in blood-brain barrier permeability in Ifnlr1(-/-) mice. Treatment of mice with pegylated interferon-λ2 resulted in decreased blood-brain barrier permeability, reduced West Nile virus infection in the brain without affecting viremia, and improved survival against lethal virus challenge. An in vitro model of the blood-brain barrier showed that interferon-λ signaling in mouse brain microvascular endothelial cells increased transendothelial electrical resistance, decreased virus movement across the barrier, and modulated tight junction protein localization in a protein synthesis- and signal transducer and activator of transcription 1 (STAT1)-independent manner. Our data establish an indirect antiviral function of interferon-λ in which noncanonical signaling through IFNLR1 tightens the blood-brain barrier and restricts viral neuroinvasion and pathogenesis.

Peginterferon Lambda-1a, a New Therapeutic for Hepatitis C Infection, from Bench to Clinic.

Chronic infection with hepatitis C virus (HCV) is estimated to affect approximately 3% of the world's population and cause 350,000 deaths each year. For a number of years, the standard of care has been combination therapy with recombinant alfa interferons-originally as native proteins but more recently as polyethyleneglycol-modified derivatives-and ribavirin, with the recent addition of an NS3 protease inhibitor for HCV genotype 1. However, therapeutic alfa interferons are associated with a significant burden of treatment-limiting adverse events, including musculoskeletal and influenza-like symptoms, hematologic cytopenias, autoimmune disease, fatigue, and other neurologic events. In 2003, a team at ZymoGenetics (now a fully owned subsidiary of Bristol-Myers Squibb) and a second, independent group simultaneously identified a new class of interferons-the type III lambda interferons-with near-identical activity to the type I alfa interferons in hepatocytes but with an unrelated and less ubiquitous receptor. Subsequent evaluation of the type III interferon system demonstrated antiviral activity against HCV in vitro with limited activity in peripheral blood mononuclear cells and other nonhepatocyte cell types, supporting its development as a potentially better-tolerated therapy for viral hepatitis. Peginterferon lambda-1a (Lambda) is an investigational type III therapeutic agent originally developed at ZymoGenetics that is currently in Phase 3 studies for the treatment of HCV. In this review, we describe the selection of the Lambda molecule and its preclinical and early clinical development, and how the resulting data have helped to establish the differentiated safety profile for Lambda-with fewer influenza-like and musculoskeletal symptoms and less hematologic toxicity than the alfa interferons-that was seen in later studies.

IL-28A (IFN-λ2) modulates lung DC function to promote Th1 immune skewing and suppress allergic airway disease.

IL-28 (IFN-λ) cytokines exhibit potent antiviral and antitumor function but their full spectrum of activities remains largely unknown. Recently, IL-28 cytokine family members were found to be profoundly down-regulated in allergic asthma. We now reveal a novel role of IL-28 cytokines in inducing type 1 immunity and protection from allergic airway disease. Treatment of wild-type mice with recombinant or adenovirally expressed IL-28A ameliorated allergic airway disease, suppressed Th2 and Th17 responses and induced IFN-γ. Moreover, abrogation of endogenous IL-28 cytokine function in IL-28Rα(-/-) mice exacerbated allergic airway inflammation by augmenting Th2 and Th17 responses, and IgE levels. Central to IL-28A immunoregulatory activity was its capacity to modulate lung CD11c(+) dendritic cell (DC) function to down-regulate OX40L, up-regulate IL-12p70 and promote Th1 differentiation. Consistently, IL-28A-mediated protection was absent in IFN-γ(-/-) mice or after IL-12 neutralization and could be adoptively transferred by IL-28A-treated CD11c(+) cells. These data demonstrate a critical role of IL-28 cytokines in controlling T cell responses in vivo through the modulation of lung CD11c(+) DC function in experimental allergic asthma.

Cutting edge: involvement of the type I IFN production and signaling pathway in lipopolysaccharide-induced IL-10 production.

Macrophages respond to LPS by the rapid activation of proinflammatory cytokines that serve to initiate host defense against microbial invasion. To prevent injury to the host from excess production of these cytokines, IL-10 is up-regulated to feedback inhibit the proinflammatory response. However, the molecular events responsible for LPS-induced up-regulation of IL-10 remain to be elucidated. In this study, we provide evidence that production of and signaling by type I IFN is required for LPS-induced IL-10 up-regulation. In addition, we demonstrate that defect in type I IFN production and signaling results in a trend toward LPS-mediated superinduction of proinflammatory genes and cytokines in bone marrow-derived macrophages. Our findings suggest a novel anti-inflammatory role for the type I IFN production and signaling pathway in regulating LPS response in bone marrow-derived macrophages.

Rescue of TRAF3-null mice by p100 NF-kappa B deficiency.

Proper activation of nuclear factor (NF)-kappaB transcription factors is critical in regulating fundamental biological processes such as cell survival and proliferation, as well as in inflammatory and immune responses. Recently, the NF-kappaB signaling pathways have been categorized into the canonical pathway, which results in the nuclear translocation of NF-kappaB complexes containing p50, and the noncanonical pathway, which involves the induced processing of p100 to p52 and the formation of NF-kappaB complexes containing p52 (Bonizzi, G., and M. Karin. 2004. Trends Immunol. 25:280-288). We demonstrate that loss of tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) results in constitutive noncanonical NF-kappaB activity. Importantly, TRAF3-/- B cells show ligand-independent up-regulation of intracellular adhesion molecule 1 and protection from spontaneous apoptosis during in vitro culture. In addition, we demonstrate that loss of TRAF3 results in profound accumulation of NF-kappaB-inducing kinase in TRAF3-/- cells. Finally, we show that the early postnatal lethality observed in TRAF3-deficient mice is rescued by compound loss of the noncanonical NF-kappaB p100 gene. Thus, these genetic data clearly demonstrate that TRAF3 is a critical negative modulator of the noncanonical NF-kappaB pathway and that constitutive activation of the noncanonical NF-kappaB pathway causes the lethal phenotype of TRAF3-deficient mice.

Interleukin-29 uses a type 1 interferon-like program to promote antiviral responses in human hepatocytes.

Interleukin-28A (IL-28A), IL-28B and IL-29 are a family of class II cytokines that stimulate antiviral responses through a heterodimeric receptor that is distinct from the type I interferon (IFN) receptor. To better understand how this newly described family of cytokines regulates the antiviral state, we compared various cellular responses elicited by IL-29 and IFN-alpha. Here we show that these cytokines stimulate similar patterns of signal transducer and activator of transcription 1 (STAT-1), -2, -3, and -5 phosphorylation and nearly identical patterns of gene expression when analyzed in two distinct cell types by microarray analysis. Interestingly, the IL-29 receptor is preferentially expressed on primary hepatocytes within normal liver and pegylated forms of IL-29 and IFN-alpha induced equivalent 2'5' oligoadenylate synthetase (OAS) and MX1 gene expression in this cell type. Pegylated IL-29 also produced a significant reduction in human hepatitis B and hepatitis C viral load in vitro and reduced the cytopathic effect caused by the fully replicating flavivirus, West Nile virus. In conclusion, IL-29 and IFN-alpha stimulate identical antiviral responses despite their utilization of different receptors. This fact, combined with significant receptor expression in hepatitis virus-infected livers, suggests that IL-29 may have therapeutic value against chronic viral hepatitis in human patients.

Toll-like receptors induce a phagocytic gene program through p38.

Toll-like receptor (TLR) signaling and phagocytosis are hallmarks of macrophage-mediated innate immune responses to bacterial infection. However, the relationship between these two processes is not well established. Our data indicate that TLR ligands specifically promote bacterial phagocytosis, in both murine and human cells, through induction of a phagocytic gene program. Importantly, TLR-induced phagocytosis of bacteria was found to be reliant on myeloid differentiation factor 88-dependent signaling through interleukin-1 receptor-associated kinase-4 and p38 leading to the up-regulation of scavenger receptors. Interestingly, individual TLRs promote phagocytosis to varying degrees with TLR9 being the strongest and TLR3 being the weakest inducer of this process. We also demonstrate that TLR ligands not only amplify the percentage of phagocytes uptaking Escherichia coli, but also increase the number of bacteria phagocytosed by individual macrophages. Taken together, our data describe an evolutionarily conserved mechanism by which TLRs can specifically promote phagocytic clearance of bacteria during infection.

T3JAM, a novel protein that specifically interacts with TRAF3 and promotes the activation of JNK(1).

Previous studies suggest that localization of tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family members is important for regulating their signal transduction. During a screen for TRAF3-associated proteins that potentially alter TRAF3 subcellular localization and enable signal transduction, we identified a novel protein, T3JAM (TRAF3-interacting Jun N-terminal kinase (JNK)-activating modulator). This protein associates specifically with TRAF3 but not other TRAF family members. Coexpression of T3JAM with TRAF3 recruits TRAF3 to the detergent-insoluble fraction. More importantly, T3JAM and TRAF3 synergistically activate JNK but not nuclear factor (NF)-kappaB. Our studies indicate that T3JAM may function as an adapter molecule that specifically regulates TRAF3-mediated JNK activation.

The signaling adaptors and pathways activated by TNF superfamily.

Members of the TNF receptor superfamily play pivotal roles in numerous biological events in metazoan organisms. Ligand-mediated trimerization by corresponding homo- or heterotrimeric ligands, the TNF family ligands, causes recruitment of several intracellular adaptors, which activate multiple signal transduction pathways. While recruitment of death domain (DD) containing adaptors such as Fas associated death domain (FADD) and TNFR associated DD (TRADD) can lead to the activation of a signal transduction pathway that induces apoptosis, recruitment of TRAF family proteins can lead to the activation of transcription factors such as, NF-kappaB and JNK thereby promoting cell survival and differentiation as well as immune and inflammatory responses. Individual TNF receptors are expressed in different cell types and have a range of affinities for various intracellular adaptors, which provide tremendous signaling and biological specificities. In addition, numerous signaling modulators are involved in regulating activities of signal transduction pathways downstream of receptors in this superfamily. Most of the TNF receptor superfamily members as well as many of their signaling mediators, have been uncovered in the last two decades. However, much remains unknown about how individual signal transduction pathways are regulated upon activation by any particular TNF receptor, under physiological conditions.

Toll-like receptor 3 mediates a more potent antiviral response than Toll-like receptor 4.

We have recently described an IFN regulatory factor 3-mediated antiviral gene program that is induced by both Toll-like receptor (TLR)3 and TLR4 ligands. In our current study, we show that activation of IFN/viral response gene expression in primary macrophage cells is stronger and prolonged with TLR3 stimulation compared with that of TLR4. Our data also reveal that the cytoplasmic tails of both TLR3 and TLR4 can directly interact with myeloid differentiation factor 88 (MyD88). However, although Toll/IL-1 receptor homology domain-containing adaptor protein/MyD88 adaptor-like is able to associate with TLR4, we were unable to detect any interaction between Toll/IL-1 receptor homology domain-containing adaptor protein/MyD88 adaptor-like and TLR3. By using quantitative real-time PCR assays, we found that TLR3 expression is inducible by both TLR3 and TLR4 ligands, while TLR4 expression is not inducible by these same stimuli. Furthermore, using cells derived from mice deficient in the IFN-alphabetaR, we show that both TLR3 and TLR4 require IFN-beta autocrine/paracrine feedback to induce TLR3 expression and activate/enhance genes required for antiviral activity. More specifically, a subset of antiviral genes is initially induced independent of IFN-beta, yet the cytokine further enhances expression at later time points. This was in contrast to a second set of genes (including TLR3) that is induced only after IFN-beta production. Taken together, our data argue that, despite both TLR3 and TLR4 being able to use IFN-beta to activate/enhance antiviral gene expression, TLR3 uses multiple mechanisms to enhance and sustain the antiviral response more strongly than TLR4.