Structure-function of type I and III interferons.

Type I and type III interferons (IFNs) are major components in activating the innate immune response. Common to both are two distinct receptor chains (IFNAR1/IFNAR2 and IFNLR1/IL10R2), which form ternary complexes upon binding their respective ligands. This results in close proximity of the intracellularly associated kinases JAK1 and TYK2, which cross phosphorylate each other, the associated receptor chains, and signal transducer and activator of transcriptions, with the latter activating IFN-stimulated genes. While there are clear similarities in the biological responses toward type I and type III IFNs, differences have been found in their tropism, tuning of activity, and induction of the immune response. Here, we focus on how these differences are embedded in the structure/function relations of these two systems in light of the recent progress that provides in-depth information on the structural assembly of these receptors and their functional implications and how these differ between the mouse and human systems.

Interferon-epsilon is a novel regulator of NK cell responses in the uterus.

The uterus is a unique mucosal site where immune responses are balanced to be permissive of a fetus, yet protective against infections. Regulation of natural killer (NK) cell responses in the uterus during infection is critical, yet no studies have identified uterine-specific factors that control NK cell responses in this immune-privileged site. We show that the constitutive expression of IFNε in the uterus plays a crucial role in promoting the accumulation, activation, and IFNγ production of NK cells in uterine tissue during Chlamydia infection. Uterine epithelial IFNε primes NK cell responses indirectly by increasing IL-15 production by local immune cells and directly by promoting the accumulation of a pre-pro-like NK cell progenitor population and activation of NK cells in the uterus. These findings demonstrate the unique features of this uterine-specific type I IFN and the mechanisms that underpin its major role in orchestrating innate immune cell protection against uterine infection.

Epithelially Restricted Interferon Epsilon Protects Against Colitis.

BACKGROUND & AIMS: Type I interferon (T1IFN) signalling is crucial for maintaining intestinal homeostasis. We previously found that the novel T1IFN, IFNε, is highly expressed by epithelial cells of the female reproductive tract, where it protects against pathogens. Its function has not been studied in the intestine. We hypothesize that IFNε is important in maintaining intestinal homeostasis. METHODS: We characterized IFNε expression in mouse and human intestine by immunostaining and studied its function in the dextran sulfate sodium (DSS) colitis model using both genetic knockouts and neutralizing antibody. RESULTS: We demonstrate that IFNε is expressed in human and mouse intestinal epithelium, and expression is lost in inflammation. Furthermore, we show that IFNε limits intestinal inflammation in mouse models. Regulatory T cell (Treg) frequencies were paradoxically decreased in DSS-treated IFNε-/- mice, suggesting a role for IFNε in maintaining the intestinal Treg compartment. Colitis was ameliorated by transfer of wild-type Tregs into IFNε-/- mice. This demonstrates that IFNε supports intestinal Treg function. CONCLUSIONS: Overall, we have shown IFNε expression in intestinal epithelium and its critical role in gut homeostasis. Given its known role in the female reproductive tract, we now show IFNε has a protective role across multiple mucosal surfaces.

Interferon-ε is a tumour suppressor and restricts ovarian cancer.

High-grade serous ovarian cancers have low survival rates because of their late presentation with extensive peritoneal metastases and frequent chemoresistance1, and require new treatments guided by novel insights into pathogenesis. Here we describe the intrinsic tumour-suppressive activities of interferon-ε (IFNε). IFNε is constitutively expressed in epithelial cells of the fallopian tube, the cell of origin of high-grade serous ovarian cancers, and is then lost during development of these tumours. We characterize its anti-tumour activity in several preclinical models: ovarian cancer patient-derived xenografts, orthotopic and disseminated syngeneic models, and tumour cell lines with or without mutations in Trp53 and Brca genes. We use manipulation of the IFNε receptor IFNAR1 in different cell compartments, differential exposure status to IFNε and global measures of IFN signalling to show that the mechanism of the anti-tumour activity of IFNε involves direct action on tumour cells and, crucially, activation of anti-tumour immunity. IFNε activated anti-tumour T and natural killer cells and prevented the accumulation and activation of myeloid-derived suppressor cells and regulatory T cells. Thus, we demonstrate that IFNε is an intrinsic tumour suppressor in the female reproductive tract whose activities in models of established and advanced ovarian cancer, distinct from other type I IFNs, are compelling indications of potential new therapeutic approaches for ovarian cancer.

Characterization of Monoclonal Antibodies to Measure Cell Surface Protein Levels of Human Interferon-Lambda Receptor 1.

Type III interferons (IFN-lambdas, IFN-λs) are important antiviral cytokines that can also modulate immune responses by acting through a heterodimeric receptor composed of the specific and limited expressed IFN-λR1 chain and the ubiquitous IL-10R2 chain, which is shared with IL-10 family cytokines. Conflicting data have been reported regarding which cells express the IFN-λR1 subunit and directly respond to IFN-λs. This is, in part, owing to transcript levels of the IFN-λR1 gene, IFNLR1, not always correlating with cell surface protein levels. In this study, we tested a panel of novel monoclonal antibodies (mAbs) that specifically recognize human IFN-λR1. Initially, antigen specificity was confirmed by enzyme-linked immunosorbent assay (ELISA), from which a subset of antibodies was selected for additional flow cytometry and neutralization assays. We further characterized two antibodies based on their strong ELISA binding activity (HLR1 and HLR14) and found only HLR14 could reliably detect cell surface IFN-λR1 protein on a variety of cell lines by flow cytometry. HLR14 could also detect IFN-λR1 protein on certain primary human blood cells, including plasmacytoid dendritic cells and B cells from peripheral blood. Availability of the HLR14 mAb will enable the quantification of IFN-λR1 protein levels on cells and better characterization of the cell specificity of the IFN-λ response.

Type I interferon antagonism of the JMJD3-IRF4 pathway modulates macrophage activation and polarization.

Metabolic adaptations can directly influence the scope and scale of macrophage activation and polarization. Here we explore the impact of type I interferon (IFNß) on macrophage metabolism and its broader impact on cytokine signaling pathways. We find that IFNß simultaneously increased the expression of immune-responsive gene 1 and itaconate production while inhibiting isocitrate dehydrogenase activity and restricting α-ketoglutarate accumulation. IFNß also increased the flux of glutamine-derived carbon into the tricarboxylic acid cycle to boost succinate levels. Combined, we identify that IFNß controls the cellular α-ketoglutarate/succinate ratio. We show that by lowering the α-ketoglutarate/succinate ratio, IFNß potently blocks the JMJD3-IRF4-dependent pathway in GM-CSF and IL-4 activated macrophages. The suppressive effects of IFNß on JMJD3-IRF4-dependent responses, including M2 polarization and GM-CSF-induced inflammatory pain, were reversed by supplementation with α-ketoglutarate. These results reveal that IFNß modulates macrophage activation and polarization through control of the cellular α-ketoglutarate/succinate ratio.

Epigenetic Activation of Plasmacytoid DCs Drives IFNAR-Dependent Therapeutic Differentiation of AML.

Pharmacologic inhibition of epigenetic enzymes can have therapeutic benefit against hematologic malignancies. In addition to affecting tumor cell growth and proliferation, these epigenetic agents may induce antitumor immunity. Here, we discovered a novel immunoregulatory mechanism through inhibition of histone deacetylases (HDAC). In models of acute myeloid leukemia (AML), leukemia cell differentiation and therapeutic benefit mediated by the HDAC inhibitor (HDACi) panobinostat required activation of the type I interferon (IFN) pathway. Plasmacytoid dendritic cells (pDC) produced type I IFN after panobinostat treatment, through transcriptional activation of IFN genes concomitant with increased H3K27 acetylation at these loci. Depletion of pDCs abrogated panobinostat-mediated induction of type I IFN signaling in leukemia cells and impaired therapeutic efficacy, whereas combined treatment with panobinostat and IFNα improved outcomes in preclinical models. These discoveries offer a new therapeutic approach for AML and demonstrate that epigenetic rewiring of pDCs enhances antitumor immunity, opening the possibility of exploiting this approach for immunotherapies. SIGNIFICANCE: We demonstrate that HDACis induce terminal differentiation of AML through epigenetic remodeling of pDCs, resulting in production of type I IFN that is important for the therapeutic effects of HDACis. The study demonstrates the important functional interplay between the immune system and leukemias in response to HDAC inhibition. This article is highlighted in the In This Issue feature, p. 1397.

Structural integrity with functional plasticity: what type I IFN receptor polymorphisms reveal.

The type I IFNs activate an array of signaling pathways, which are initiated after IFNs bind their cognate receptors, IFNα/ß receptor (IFNAR)1 and IFNAR2. These signals contribute to many aspects of human health including defense against pathogens, cancer immunosurveillance, and regulation of inflammation. How these cytokines interact with their receptors influences the quality of these signals. As such, the integrity of receptor structure is pivotal to maintaining human health and the response to immune stimuli. This review brings together genome wide association studies and clinical reports describing the association of nonsynonymous IFNAR1 and IFNAR2 polymorphisms with clinical disease, including altered susceptibility to viral and bacterial pathogens, autoimmune diseases, cancer, and adverse reactions to live-attenuated vaccines. We describe the amino acid substitutions or truncations induced by these polymorphisms and, using the knowledge of IFNAR conformational changes, IFNAR-IFN interfaces and overall structure-function relationship of the signaling complexes, we hypothesize the effect of these polymorphisms on receptor structure. That these predicted changes to IFNAR structure are associated with clinical manifestations of human disease, highlights the importance of IFNAR structural integrity to maintaining functional quality of these receptor-mediated responses. Type I IFNs are pivotal to innate immune responses and ultimately, to human health. Understanding the consequences of altered structure on the actions of these clinically significant cell receptors provides important information on the roles of IFNARs in health and disease.

A hot spot on interferon α/ß receptor subunit 1 (IFNAR1) underpins its interaction with interferon-ß and dictates signaling.

The interaction of IFN-ß with its receptor IFNAR1 (interferon α/ß receptor subunit 1) is vital for host-protective anti-viral and anti-proliferative responses, but signaling via this interaction can be detrimental if dysregulated. Whereas it is established that IFNAR1 is an essential component of the IFNAR signaling complex, the key residues underpinning the IFN-ß-IFNAR1 interaction are unknown. Guided by the crystal structure of the IFN-ß-IFNAR1 complex, we used truncation variants and site-directed mutagenesis to investigate domains and residues enabling complexation of IFN-ß to IFNAR1. We have identified an interface on IFNAR1-subdomain-3 that is differentially utilized by IFN-ß and IFN-α for signal transduction. We used surface plasmon resonance and cell-based assays to investigate this important IFN-ß binding interface that is centered on IFNAR1 residues Tyr240 and Tyr274 binding the C and N termini of the B and C helices of IFN-ß, respectively. Using IFNAR1 and IFN-ß variants, we show that this interface contributes significantly to the affinity of IFN-ß for IFNAR1, its ability to activate STAT1, the expression of interferon stimulated genes, and ultimately to the anti-viral and anti-proliferative properties of IFN-ß. These results identify a key interface created by IFNAR1 residues Tyr240 and Tyr274 interacting with IFN-ß residues Phe63, Leu64, Glu77, Thr78, Val81, and Arg82 that underlie IFN-ß-IFNAR1-mediated signaling and biological processes.

Auto-phosphorylation Represses Protein Kinase R Activity.

The central role of protein kinases in controlling disease processes has spurred efforts to develop pharmaceutical regulators of their activity. A rational strategy to achieve this end is to determine intrinsic auto-regulatory processes, then selectively target these different states of kinases to repress their activation. Here we investigate auto-regulation of the innate immune effector protein kinase R, which phosphorylates the eukaryotic initiation factor 2α to inhibit global protein translation. We demonstrate that protein kinase R activity is controlled by auto-inhibition via an intra-molecular interaction. Part of this mechanism of control had previously been reported, but was then controverted. We account for the discrepancy and extend our understanding of the auto-inhibitory mechanism by identifying that auto-inhibition is paradoxically instigated by incipient auto-phosphorylation. Phosphor-residues at the amino-terminus instigate an intra-molecular interaction that enlists both of the N-terminal RNA-binding motifs of the protein with separate surfaces of the C-terminal kinase domain, to co-operatively inhibit kinase activation. These findings identify an innovative mechanism to control kinase activity, providing insight for strategies to better regulate kinase activity.

Interferon epsilon promotes HIV restriction at multiple steps of viral replication.

Interferon epsilon (IFNɛ) is a type I IFN that is expressed constitutively in the female reproductive tract (FRT), and contributes to protection in models of sexually transmitted infections. Using multiple cell systems, including reporter cell lines and activated peripheral blood lymphocytes (PBLs), we show that recombinant IFNɛ impairs HIV infection at stage(s) post HIV entry and up to the translation of viral proteins. Consistent with this, IFNɛ upregulated a number of host cell restriction factors that block HIV at these stages of the replication cycle. The potency of IFNɛ induction of these HIV restriction factors was comparable to conventional type I IFNs, namely IFNα and IFNß. IFNɛ also significantly reduced the infectivity of progeny virion particles likely by inducing expression of HIV restriction factors, such as IFITM3, which act at that stage of infection. Thus, our data demonstrate that human IFNɛ suppresses HIV replication at multiple stages of infection.

Purification and biological characterization of soluble, recombinant mouse IFNß expressed in insect cells.

Interferon ß (IFNß) is a member of the type I interferon family of cytokines widely recognised for their anti-viral, anti-proliferative and immunomodulatory properties. Recombinant, biologically active forms of this cytokine are used clinically for the treatment of multiple sclerosis and in laboratories to study the role of this cytokine in health and disease. Established methods for expression of IFNß utilise either bacterial systems from which the insoluble recombinant proteins must be refolded, or mammalian expression systems in which large volumes of cell culture are required for recovery of acceptable yields. Utilising the baculovirus expression system and Trichoplusia ni (Cabbage Looper) BTI-TN-5B1-4 cell line, we report a reproducible method for production and purification of milligram/litre quantities of biologically active murine IFNß. Due to the design of our construct and the eukaryotic nature of insect cells, the resulting soluble protein is secreted allowing purification of the Histidine-tagged natively-folded protein from the culture supernatant. The IFNß purification method described is a two-step process employing immobilised metal-ion affinity chromatography (IMAC) and reverse-phase high performance liquid chromatography (RP-HPLC) that results in production of significantly more purified IFNß than any other reported eukaryotic-based expression system. Recombinant murine IFNß produced by this method was natively folded and demonstrated hallmark type I interferon biological effects including antiviral and anti-proliferative activities, and induced genes characteristic of IFNß activity in vivo. Recombinant IFNß also had specific activity levels exceeding that of the commercially available equivalent. Together, our findings provide a method for production of highly pure, biologically active murine IFNß.

Structural basis of a unique interferon-ß signaling axis mediated via the receptor IFNAR1.

Type I interferons are important in regulating immune responses to pathogens and tumors. All interferons are considered to signal via the heterodimeric IFNAR1-IFNAR2 complex, yet some subtypes such as interferon-ß (IFN-ß) can exhibit distinct functional properties, although the molecular basis of this is unclear. Here we demonstrate IFN-ß can uniquely and specifically ligate to IFNAR1 in an IFNAR2-independent manner, and we provide the structural basis of the IFNAR1-IFN-ß interaction. The IFNAR1-IFN-ß complex transduced signals that modulated expression of a distinct set of genes independently of Jak-STAT pathways. Lipopolysaccharide-induced sepsis was ameliorated in Ifnar1(-/-) mice but not Ifnar2(-/-) mice, suggesting that IFNAR1-IFN-ß signaling is pathologically relevant. Thus, we provide a molecular basis for understanding specific functions of IFN-ß.

Silencing of Irf7 pathways in breast cancer cells promotes bone metastasis through immune escape.

Breast cancer metastasis is a key determinant of long-term patient survival. By comparing the transcriptomes of primary and metastatic tumor cells in a mouse model of spontaneous bone metastasis, we found that a substantial number of genes suppressed in bone metastases are targets of the interferon regulatory factor Irf7. Restoration of Irf7 in tumor cells or administration of interferon led to reduced bone metastases and prolonged survival time. In mice deficient in the interferon (IFN) receptor or in natural killer (NK) and CD8(+) T cell responses, metastasis was accelerated, indicating that Irf7-driven suppression of metastasis was reliant on IFN signaling to host immune cells. We confirmed the clinical relevance of these findings in over 800 patients in which high expression of Irf7-regulated genes in primary tumors was associated with prolonged bone metastasis-free survival. This gene signature may identify patients that could benefit from IFN-based therapies. Thus, we have identified an innate immune pathway intrinsic to breast cancer cells, the suppression of which restricts immunosurveillance to enable metastasis.

The interferons and their receptors--distribution and regulation.

The interferons (IFNs) were originally described over 50 years ago, identified by their ability to confer viral resistance to cells. We now know that they are much more than just anti-viral cytokines collectively having roles in both innate and adaptive immune responses, in tumor surveillance and defense, and modulation of immune cell function. Three types of IFN have now been described, simply referred to as type I, II and III. Distinguishable by the unique receptors that they rely on for signal transduction, the three types of IFN have specific and varied roles in the maintenance of human health and defense against pathogens. In mounting an IFN-mediated immune response, the human body has developed the ability to regulate IFN-mediated signal transduction. Like all cytokines, the ability of a cell to respond to IFN is completely dependent on the presence of its cognate receptor on the surface of the target cell. Thus, one of the major mechanisms used by the human body to regulate the strength and duration of the IFN response is through regulation of receptor levels, thereby altering the cytokine-specific responsiveness of the target cell. This review will discuss the receptor system utilized by the type I IFNs and compare it with that of the type II and III IFNs, which also regulate immune responses through controlling receptor level on the cell surface.

Type I-IFNs control GVHD and GVL responses after transplantation.

Although the effects of type II-IFN (IFN-γ) on GVHD and leukemia relapse are well studied, the effects of type I-interferon (type I-IFN, IFN-α/ß) remain unclear. We investigated this using type I-IFN receptor-deficient mice and exogenous IFN-α administration in established models of GVHD and GVL. Type I-IFN signaling in host tissue prevented severe colon-targeted GVHD in CD4-dependent models of GVHD directed toward either major histocompatibility antigens or multiple minor histocompatibility antigens. This protection was the result of suppression of donor CD4(+) T-cell proliferation and differentiation. Studies in chimeric recipients demonstrated this was due to type I-IFN signaling in hematopoietic tissue. Consistent with this finding, administration of IFN-α during conditioning inhibited donor CD4(+) proliferation and differentiation. In contrast, CD8-dependent GVHD and GVL effects were enhanced when type I-IFN signaling was intact in the host or donor, respectively. This finding reflected the ability of type I-IFN to both sensitize host target tissue/leukemia to cell-mediated cytotoxicity and augment donor CTL function. These data confirm that type I-IFN plays an important role in defining the balance of GVHD and GVL responses and suggests that administration of the cytokine after BM transplantation could be studied prospectively in patients at high risk of relapse.

Suppressor of cytokine signaling (SOCS) 1 inhibits type I interferon (IFN) signaling via the interferon alpha receptor (IFNAR1)-associated tyrosine kinase Tyk2.

Type I IFNs are critical players in host innate and adaptive immunity. IFN signaling is tightly controlled to ensure appropriate immune responses as imbalance could result in uncontrolled inflammation or inadequate responses to infection. It is therefore important to understand how type I IFN signaling is regulated. Here we have investigated the mechanism by which suppressor of cytokine signaling 1 (SOCS1) inhibits type I IFN signaling. We have found that SOCS1 inhibits type I IFN signaling not via a direct interaction with the IFN α receptor 1 (IFNAR1) receptor component but through an interaction with the IFNAR1-associated kinase Tyk2. We have characterized the residues/regions involved in the interaction between SOCS1 and Tyk2 and found that SOCS1 associates via its SH2 domain with conserved phosphotyrosines 1054 and 1055 of Tyk2. The kinase inhibitory region of SOCS1 is also essential for its interaction with Tyk2 and inhibition of IFN signaling. We also found that Tyk2 is preferentially Lys-63 polyubiquitinated and that this activation reaction is inhibited by SOCS1. The consequent effect of SOCS1 inhibition of Tyk2 not only results in a reduced IFN response because of inhibition of Tyk2 kinase-mediated STAT signaling but also negatively impacts IFNAR1 surface expression, which is stabilized by Tyk2.