The interferons: 50 years after their discovery, there is much more to learn

The interferons (IFNs) and their receptors represent a subset of the class 2 alpha-helical cytokines that have been in chordates for millions of years. This brief review focuses on the discovery and purification of interferons, cloning of human IFN-alpha and IFN-beta, interferon receptors, activities and therapeutic uses of interferons, and the side effects of interferons.     

Innate immune response to viral infection

In viral infections the host innate immune system is meant to act as a first line defense to prevent viral invasion or replication before more specific protection by the adaptive immune system is generated. In the innate immune response, pattern recognition receptors (PRRs) are engaged to detect specific viral components such as viral RNA or DNA or viral intermediate products and to induce type I interferons (IFNs) and other pro-inflammatory cytokines in the infected cells and other immune cells. Recently these innate immune receptors and their unique downstream pathways have been identified. Here, we summarize their roles in the innate immune response to virus infection, discrimination between self and viral nucleic acids and inhibition by virulent factors and provide some recent advances in the coordination between innate and adaptive immune activation.     

Regulation of receptor activator of NF-kappa B ligand-induced osteoclastogenesis by endogenous interferon-beta (INF-beta ) and suppressors of cytokine signaling (SOCS). The possible counteracting role of SOCSs- in IFN-beta-inhibited osteoclast formation

Bone resorption and the immune system are correlated with each other, and both are controlled by a variety of common cytokines produced in the bone microenvironments. Among these immune mediators, the involvement of type I interferons (IFNs) in osteoclastic bone resorption remains unknown. In this study, we investigated the participation of IFN-beta and suppressors of cytokine signaling (SOCS)-1 and -3 in osteoclastogenesis. Addition of exogenous IFN-beta to osteoclast progenitors (bone-derived monocytes/macrophages) inhibited their differentiation toward osteoclasts induced by the receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor with/without transforming growth factor-beta, where inhibition was associated with down-regulation of the gene expressions of molecules related to osteoclast differentiation. In addition, RANKL induced the expression of IFN-beta; furthermore, neutralizing antibody against type I IFNs accelerated the osteoclast formation, indicating type I IFNs as potential intrinsic inhibitors. On the other hand, RANKL also induced the expression of SOCS-1 and -3, suppressors of the IFN signaling. Pretreatment with RANKL for a sufficient time for the induction of SOCSs attenuated phosphorylation of STAT-1 in response to IFN-beta in osteoclast progenitors, causing a decrease in the binding activity of nuclear extracts toward the interferon-stimulated response element. mRNA levels of STAT-1, STAT-2, and IFN-stimulated gene factor-3gamma, comprising IFN-stimulated gene factor-3, were not altered by RANKL. Thus, although the inhibitory cytokine such as IFN-beta is produced in response to RANKL, the inhibition of osteoclastogenesis may be rescued by the induction of signaling suppressors such as SOCSs.     

RNase L: its biological roles and regulation

2'-5'oligoadenylate-dependent ribonuclease L (RNase L) is one of the key enzymes involved in the function of interferons (IFNs), a family of cytokines participating in innate immunity against viruses and other microbial pathogens. Upon binding with its activator, 5'-phosphorylated, 2'-5' linked oligoadenylates (2-5A), RNase L degrades single-stranded viral and cellular RNAs and thus plays an important role in the antiviral and antiproliferative functions of IFNs. In recent years, evidence has revealed that RNase L displays a broad range of biological roles which are summarized in this review.     

Class II cytokine receptors and their ligands: key antiviral and inflammatory modulators

Class II cytokine receptors were originally defined on the basis of sequence homologies in the extracellular domains of receptors for interferons (IFNs) and interleukin-10 (IL-10), and the ligands, known as class II cytokines, also have a common structure. More recently, a series of new receptors and cytokines that belong to this family have been discovered. The therapeutic potential of the 'old' members of this family, IFNs and IL-1, is recognized in the clinic, and the existence of structurally related molecules is raising expectations for additional clinical applications. In this review, I discuss both structural and biological data that are emerging about this family of receptors and ligands, to highlight the potential applications of modulating the activity of these cytokines.     

Induction of type I interferons by bacteria

Type I interferons (IFNs) are secreted cytokines that orchestrate diverse immune responses to infection. Although typically considered to be most important in the response to viruses, type I IFNs are also induced by most, if not all, bacterial pathogens. Although diverse mechanisms have been described, bacterial induction of type I IFNs occurs upon stimulation of two main pathways: (i) Toll‐like receptor (TLR) recognition of bacterial molecules such as lipopolysaccharide (LPS); (ii) TLR‐independent recognition of molecules delivered to the host cell cytosol. Cytosolic responses can be activated by two general mechanisms. First, viable bacteria can secrete stimulatory ligands into the cytosol via specialized bacterial secretion systems. Second, ligands can be released from bacteria that lyse or are degraded. The bacterial ligands that induce the cytosolic pathways remain uncertain in many cases, but appear to include various nucleic acids. In this review, we discuss recent advances in our understanding of how bacteria induce type I interferons and the roles type I IFNs play in host immunity.     

Interferons: Success in anti-viral immunotherapy

The interferons (IFNs) are glycoproteins with strong antiviral activities that represent one of the first lines of host defense against invading pathogens. These proteins are classified into three groups, Type I, II and III IFNs, based on the structure of their receptors on the cell surface. Due to their ability to modulate immune responses, they have become attractive therapeutic options to control chronic virus infections. In combination with other drugs, Type I IFNs are considered as “standard of care” in suppressing Hepatitis C (HCV) and Hepatitis B (HBV) infections, while Type III IFN has generated encouraging results as a treatment for HCV infection in phase III clinical trials. However, though effective, using IFNs as a treatment is not without the need for caution. IFNs are such powerful cytokines that affect a wide array of cell types; as a result, patients usually experience unpleasant symptoms, with a percentage of patients suffering system wide effects. Thus, constant monitoring is required for patients treated with IFN in order to reach the treatment goals of suppressing virus infection and maintaining quality of life.     

Biological activity of interleukins-28 and -29: comparison with type I interferons

Despite binding to receptors distinct from those of type I interferons (IFNs), human interleukins-28A, -28B and -29 (IL-28A, IL-28B and IL-29; alternatively named IFN lambda-2 {IFN-lambda2}, IFN-lambda3 and IFN-lambda1, respectively, or collectively, type III IFNs), a small family of three structurally-related cytokines, are, like IFNs, known to induce antiviral activity. To further biologically characterize IL-28A and IL-29, we compared their activities with those of IFNs in a range of human cell lines. We found that they induced antiviral activity in fewer cell lines and more weakly than IFNs; also IL-28A was less active than IL-29. Additionally, we showed IL-28A and IL-29 induced reporter genes--protein MxA promoter linked to luciferase, or interferon stimulated response element (ISRE) linked to secreted alkaline phosphatase (SEAP)--more weakly than IFN. Antiproliferative activity was induced by IFNs in most cell lines, but only in one human glioblastoma cell line, LN319, was dose-dependent IL-29-growth inhibition demonstrable. Polymerase chain reaction (PCR) quantification of messenger (m) RNA of IL-28/29 receptor subunits, IL-28Ralpha and IL-10Rbeta, indicated variable expression levels; although their expression was highest in the responsive LN319 cell line, lower but significant expression of both mRNAs was found in relatively unresponsive cell lines. In conclusion, we found IL-28A and IL-29 act similarly to IFNs, but are less effective generally and have activity in a more limited range of cell lines.     

Cytokine Activation by Antibody Fragments Targeted to Cytokine-Receptor Signaling Complexes

Exogenous cytokine therapy can induce systemic toxicity, which might be prevented by activating endogenously produced cytokines in local cell niches. Here we developed antibody-based activators of cytokine signaling (AcCS), which recognize cytokines only when they are bound to their cell surface receptors. AcCS were developed for type I interferons (IFNs), which induce cellular activities by binding to cell surface receptors IFNAR1 and IFNAR2. As a potential alternative to exogenous IFN therapy, AcCS were shown to potentiate the biological activities of natural IFNs by ∼100-fold. Biochemical and structural characterization demonstrates that the AcCS stabilize the IFN-IFNAR2 binary complex by recognizing an IFN-induced conformational change in IFNAR2. Using IFN mutants that disrupt IFNAR1 binding, AcCS were able to enhance IFN antiviral potency without activating antiproliferative responses. This suggests AcCS can be used to manipulate cytokine signaling for basic science and possibly for therapeutic applications.     

Splenic Red Pulp Macrophages Produce Type I Interferons as Early Sentinels of Malaria Infection but Are Dispensable for Control

Type I interferons (T1IFNs) are among the earliest cytokines produced during infections due to their direct regulation by innate immune signaling pathways. Reports have suggested that T1IFNs are produced during malaria infection, but little is known about the in vivo cellular origins of T1IFNs or their role in protection. We have found that in addition to plasmacytoid dendritic cells, splenic red pulp macrophages (RPMs) can generate significant quantities of T1IFNs in response to P. chabaudi infection in a TLR9-, MYD88-, and IRF7-dependent manner. Furthermore, T1IFNs regulate expression of interferon-stimulated genes redundantly with Interferon-gamma (IFNG), resulting in redundancy for resistance to experimental malaria infection. Despite their role in sensing and promoting immune responses to infection, we observe that RPMs are dispensable for control of parasitemia. Our results reveal that RPMs are early sentinels of malaria infection, but that effector mechanisms previously attributed to RPMs are not essential for control.     

Corticosteroids and interferons inhibit cytokine-induced production of IL-8 by human endothelial cells

IL-8, secreted by endothelial cells at the site of inflammation, participates in recruitment and transmigration of leukocytes. IL-8 may also have pathophysiological consequences in inflammatory and immunological disorders. We have investigated the effect of interferons (IFNs) and glucocorticosteroids (GCs) on cytokine induced secretion and production of IL-8 by human umbilical endothelial cells (HUVEC). There was a low spontaneous secretion of IL-8 by unstimulated HUVEC which increased after 6 or 24 h of stimulation with the pro-inflammatory cytokines TNF-alpha or IL-1beta. IFN-gamma as well as the GCs, Dexamethasone and Budesonide, inhibited TNF-alpha induced IL-8 secretion in a dose-dependent manner. IFNs may have a general modulating effect, since IFN-alpha also inhibited the TNF-alpha-induced IL-8 secretion. There was a slight, but significant, increase in the content of intracellular IL-8 in stimulated HUVEC. However, there was no difference between stimulation with IL-1beta or TNF-alpha alone or in combination with IFNs or GCs, whereas inhibition of IL-8 secretion with monensin increased IL-8 content suggesting that IFNs and GCs inhibit synthesis rather than secretion of IL-8. In conclusion, IFNs or GCs may be useful for inhibiting IL-8 production by endothelial cells and could thus be used for therapeutic modulation of the inflammatory response.     

Inhibitory effects of human immunodeficiency virus gp120 and Tat on CpG-A-induced inflammatory cytokines in plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs), not only inhibit viral replication, but also play an essential role in linking the innate and adaptive immune system. In this study, we explored the effects of human immunodeficiency virus (HIV) gp120 and tat on CpG-A-induced inflammatory cytokines in pDCs. The results provided fundamental insights into HIV pathogenesis that may hold promise for preventative and even curative strategies. pDCs were isolated using blood DC antigen 4 (BDCA-4) DC isolation kit, and the purity was analyzed using BDCA-2 antibody by flow cytometry. pDCs and peripheral blood mononuclear cells (PBMCs) were stimulated by either CpG-A (5 μg/ml), gp120 (0.5 μg/ml), tat (0.5 μg/ml), or CpG-A treatment combined with gp120 or tat. The production of type I interferons (IFNs) and other inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interlukine-6 (IL-6), and interferon-gamma-inducible protein-10 (IP-10) in the culture supernatant, was determined by enzyme-linked immunosorbent assay. The results showed that CpG-A induced high levels of type I IFNs and other inflammatory cytokines, including TNF-α, IL-6, and IP-10, in pDCs. Concomitant treatment with gp120 reduced the levels of IFN-α, IFN-β, TNF-α, IL-6, and IP-10 induced by CpG-A in pDCs by 79%, 53%, 60%, 50%, and 34%, respectively, while tat suppressed them by 88%, 66%, 71%, 64%, and 53%, respectively. Similar results were demonstrated in CpG-A-treated PBMCs. In conclusion, gp120 and tat are effective inhibitors of the CpG-A-mediated induction of type I IFNs and other inflammatory cytokines from pDCs and PBMCs.     

Viral transformation for production of personalized type I interferons

Type I interferons (IFN) are cytokines with many functions and have been widely used to treat many human diseases such as hepatitis C virus infection. Using the viral transformation and priming properties of Epstein-Barr virus, we have developed a system that can produce high levels of “personalized” IFNs, which are produced from the cells of the patient to whom the IFNs are to be administrated. We demonstrate the feasibility of the system. This seems to be the first report for the establishment of a personalized IFN-production system. The personalized IFNs could have a longer circulation time, fewer side effects but higher efficacy. We anticipate that the system can provide an improved form of IFN for medical uses.     

TLR-dependent and TLR-independent pathways of type I interferon induction in systemic autoimmunity

We formulate a two-phase paradigm of autoimmunity associated with systemic lupus erythematosus, the archetypal autoimmune disease. The initial Toll-like receptor (TLR)-independent phase is mediated by dendritic cell uptake of apoptotic cell debris and associated nucleic acids, whereas the subsequent TLR-dependent phase serves an amplification function and is mediated by uptake of TLR ligands derived from self-antigens (principally nucleic acids) complexed with autoantibodies. Both phases depend on elaboration of type I interferons (IFNs), and therapeutic interruption of induction or activity of these cytokines in predisposed individuals might have a substantial mitigating effect in lupus and other autoimmune diseases.     

Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA

In vitro-transcribed mRNA has great therapeutic potential to transiently express the encoded protein without the adverse effects of viral and DNA-based constructs. Mammalian cells, however, contain RNA sensors of the innate immune system that must be considered in the generation of therapeutic RNA. Incorporation of modified nucleosides both reduces innate immune activation and increases translation of mRNA, but residual induction of type I interferons (IFNs) and proinflammatory cytokines remains. We identify that contaminants, including double-stranded RNA, in nucleoside-modified in vitro-transcribed RNA are responsible for innate immune activation and their removal by high performance liquid chromatography (HPLC) results in mRNA that does not induce IFNs and inflammatory cytokines and is translated at 10- to 1000-fold greater levels in primary cells. Although unmodified mRNAs were translated significantly better following purification, they still induced high levels of cytokine secretion. HPLC purified nucleoside-modified mRNA is a powerful vector for applications ranging from ex vivo stem cell generation to in vivo gene therapy.     

Microbial pathogenesis and type III interferons

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