Interferon Alpha and Beta

Interferon alpha and beta

Discovery and structure

Type I interferons (IFN-I) comprise a wide class of structurally related cytokines, mostly recognized by their pivotal role in antiviral responses.1The most extensively studied members of IFN-I are interferon-α (IFN-α) and interferon-β (IFN-β). IFN-I regulate lymphocyte development, immune responses and the maintenance of immunological memory of cytotoxic T cells. In addition, they have a protective role in various pathophysiologic processes, but also detrimental effects on several autoimmune diseases.2

At the end of 1950s interferon (IFN) was first described as a substance inducing the antiviral state in cells.3 Later, interferons were grouped as type I IFNs (acid-stable at pH 2 and heat-stable) and type II IFNs, which are acid-labile, but so far there is only one member in this group – IFN-. (Reviewed in4). More recently, type III IFNs were described, IFNs-ʎ (lambda).

Type I IFNs are structurally related proteins that act on a common cell-surface IFN-α receptor (IFNAR). Members of this family include: IFN-α (alpha), IFN-β (beta), IFN-κ (kappa), IFN-δ (delta), IFN-ε (epsilon), IFN-τ (tau), IFN-ω (omega), and IFN-ζ (zeta, also known as limitin).

All human type I IFN genes are clustered in the same locus on the short arm of chromosome 9. Homology between human IFN- and IFN- is about 30% and 45% at the amino acid and nucleotide level, respectively.IFN-, and  (IFN-αβ) genes lack introns; this feature allows a more rapid transcription which is particularly convenientin defense against viral infections.4

Thirteen genes code for structurally different forms of IFN- but only a single gene codes for human IFN-. IFN- subtypes are - IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21. All eutherians produce IFN-I, but IFNA2, the first discovered and prototypical member of the family, is restricted to humans and closely related hominids.5

Human IFN- subtypes are composed of 165 or 166 amino acids and murine IFN- subtypes consist of 166 or 167 amino acids. Human and murine IFN- have 166 and 161 amino acids, respectively. 6

IFN- and IFN-β are 15–21 kD and 22 kD in size respectively.IFN- and IFN-β have a globular structure composed of five a-helices. Their receptors, IFNAR1 and IFNAR2, belong to the class II cytokine receptor family for a-helical cytokines.6

Receptors and signaling

IFN-α and β bind both to a specific cell surface receptor complex – IFNAR- on both,the virus infected cell and nearby uninfected cells. IFNAR is present in low numbers (100–5000molecules/cell) on the surface of all vertebrate cells. The receptor complex consists of two known subunits, IFNAR-1 and IFNAR-2.

Mature human IFNAR-1, resulting from removal of the peptide leader sequence, is a 530 amino acid residue integral membrane protein. It is composed of an extracellular domain of 409 amino acid residues, a transmembrane domain of 21 residues and an intracellular domain of 100 residues. Mature human IFNAR-2 has been isolated as three forms. The full length receptor chain comprised of 487 amino acids, is referred to as IFNAR-2c and is 115kDa in size.7

Antiviral activity mediated by the IFNAR requires induction of an enzyme 2’–5’- oligoadenylate synthetase (2’, 5’-AS), a double-strand RNA dependent protein kinase (PKR), as well as a myxovirus (influenza) resistance (MxA) protein. These molecules inhibit viral replication and degrade viral components. Induction of 2’–5’-AS activates ribonuclease (RNase) L, a latent cellular endoribonuclease that mediates antiviral activity8.

Type I IFNssignaling pathwaysare initiated by Janus kinase (Jak) phosphorylation of downstream proteins. The canonical route involves STAT1 and STAT2 activation by Jak (JAK-STAT pathway). Other alternative networks have been discovered, such asthe activation of the pleiotropic mTOR pathway through PI3K, or MAPK p38 activation by vav or another GTP exchange factor. In addition to operate through transcriptional control, type I IFN can directly influence translation through the mTOR pathway.9, 10

IFN-αβ strongly activates STAT1 and STAT2 and induces the formation heterotrimeric transcription factor complex interferon-stimulated gene factor 3 (ISGF3).11 ISGF3 translocates to the nucleus and induces the transcription of hundreds of IFN-stimulated genes involved in the generation of the antiviral state.

Negative regulation of type I IFN signaling is accomplished by various mechanisms, including receptor internalization and degradation, dephosphorylation of JAKs and STATs by several phosphatases, induction of suppressors of cytokine signaling (SOCS) and repression of STAT-mediated gene activation by protein inhibitors of activated STATs (PIAS).12

In summary, the overall IFNαβ signalinginvolves five major steps: (a) IFN-driven dimerization of the receptor outside the cell which leads to (b) initiation of a tyrosine phosphorylation cascade inside the cell, resulting in (c) dimerization of the phosphorylated STATs, activating them for (d) transport into the nucleus, where they (e) bind to specific DNA sequences and stimulate transcription.13

Cellular sources and targets

Small amounts of IFN-I are produced under healthy conditions (IFNα by leukocytes and IFNβ by fibroblasts) but their production increases enormously by viral infections or exposure to double-stranded nucleic acids.14

Basically, all nucleated cells can produce IFN-αβin response to viral infection, but plasmacytoid dendritic cells (pDCs), or “natural IFN-producing cells” (NIPCs) produce up to 1000-fold more IFN-αβ (and IFN-λ) than other cell types.15, 16

Even though pDCs express constitutivelyall protein machinery for rapid IFN-I production, they are mostly dispensable for drivinglocal anti-viral responses.17Initially, local infected cells are the main source of IFN-I, but after systemic viral spread, pDCs on the spleen becomes the most important source.Mast cell can also secrete IFN-I. Due to their tissue localization, their importance as local sentinels of viral infections should be further evaluated.

The relative biologic activities of the different IFN- subtypes vary markedly, and, for example, on a molar basis, IFN-8 and IFN- are more efficient antiviral agents than are many other IFN- subtypes.18

Different stimuli trigger IFN-I expression. Viruses and double-stranded RNA are the most efficient natural inducers of type I IFNs, but other infectious agents, such as protozoan parasites, may induce their production. 19,20 Besides, pathogen-associated molecular patterns, danger signals and cellular stress induced by viral infection can initiate synergistic pathways that promote IFN-I production21.

Probably, all cells in the organism can produce type I IFNs, but in the absence of viral infection IFN synthesis is shut off, and most cells do not release measurable amounts of it.4

Beside pDCs it seems that relevant levels of type I IFNs are also produced by conventional myeloid DCs (CD11chiB220−Ly6C−)22,23especially when infected with certain types of DC-tropic dsRNA viruses.24

It is remarkable that the induction of most IFN-α genes is dependent on IFN-β signaling and IFN-β-induced interferon regulatory factor 7 (IRF7).

The type I IFN gene induction is initiated by the recognition of double-stranded (ds) RNA that is produced by many viruses during their replication cycle. These patterns can be roughly divided as being “cytosolic” or “endosomic”.

“Cytosolic” receptors, such as RIG-I, MDA5 and LGP-2 , are expressed ubiquitously and are localized to the cell’s cytosol where they detect viral nucleic acids produced upon infection.25, 26

The first identified cytosolic sensor is dsRNA dependent protein kinase (PKR), whose catalytic activity is stimulated by its binding to dsRNA. However, although PKR contributes to type I IFN production in response to the synthetic dsRNA analog poly(I:C), gene targeting in mice has shown that it is superfluous for IFN responses to viral infection.27,28 MDA5 recognizes mainly dsRNA,RIG-1 can detect single stranded RNA (ssRNA) of viral origin, but also short dsRNA fragments.29 Host ssRNA cannot be recognized through this receptor because of the presence of the methyl guanosine cap or a monophosphate at the 5’ end.

“Endosomic” receptors consist of members of the Toll-like receptor [TLR] family, which detect viral nucleic acids in endosomes and only in specialized cell types.

Nine TLRs have been identified in humans, of which three (TLR3, TLR7 and TLR9) recognize nucleic acid components and stimulate type I IFN production. Interestingly, TLR expression is also regulated by IFNs, further highlighting the amplifying principle of the IFN response.30 The TLR “endosomic” mode of recognition does not require that the IFN-producing cells are infected themselves and hence does not need to be ubiquitous. 31

Four IRF family members (IRF1, IRF3, IRF5 and IRF7) are positive regulators of type I IFN production. These molecules regulate transcription at distinct type I IFN loci, thereby determining which type I IFN subtypes are expressed in the initiation and propagation of the IFN response.32

Type I IFN induction is mediated by an initial complex composed minimally of TRAF3, NEMO (IKKγ) and TANK; this complex controls the activity of noncanonical kinases TBK1 and IKKɛ that specifically phosphorylate transcription factors IRF3 and IRF7, leading to their dimerization and nuclear translocation, and transcriptional activation of type I IFN genes.

IRF3, but not IRF7, is produced constitutively in nucleated cells; IFN-B activates IRF7 transcription promoting a positive feedback loop that ends up with stimulation of all types of IFN-alpha and also IFN Beta. Plasmocytoid DCs express IRF7 constituvely, explaining why they are a potent source of IFN-I.

Both IRFs and NF-κB bind to the IFNβ promoter in a temporally coordinated fashion to drive its transcription. Secreted IFNβ then binds to and activates the type I IFN receptor (a heterodimer of IFNAR1 and IFNAR2) in an autocrine or paracrine manner.

Role in immune regulation and cellular networks

Type I IFNs are the main players in defense against viral infection. Their effects go beyond directly killingviral-infected cells; they also orchestrate also adaptive immune responses. IFN-inducible genes (about 1000) participate in different biological processes, such as cell metabolism, cell survival, proliferation and tissue repair. 33Particularly, production of IFNβ is very important because it induces other cells (infected or non-infected) to make IFNα, thus amplifying and maintaining the IFN response.

The regulatory effects of type I IFNs on innate immune cells occur at several stages of differentiation, including the pluripotent HSC, immature and mature DC. Type I IFNs do not only regulate innate but adaptive immune responses too. IFN-I can directly influence immune cells through IFNAR or, indirectly,by inducing chemokines for recruitment of immune cells to the site of infection. IFN-I induce secretion of a second wave of cytokines like IL-15 that regulate NK and memory CD8+ T cell numbers and activities. NK cells show low responsiveness to IFN-I, instead, they are stimulated by IL-15-trans presented by IFN-I activated DCs.34, 35 Also, type I IFN - dependent release of IL-15 leads to rapid and efficient memoryCD8+ T cell response in a TORc1 dependent manner.34

IFNs are critical for the stimulation DCs, enhancing its capability to present antigens, and for the activation of naïve T cells.22Beside immune cells, type I IFNs can regulate the lifespan of various other cell types. For example, IFN-I have been reported to trigger apoptosis of tumor cells as well as virus-infected cells.

On the one hand, cytotoxic T cells are induced to proliferate by type I IFNs. Additionally, IFNs also stimulate production of the chemokines CXCL9, CXCL10 and CXCL11, which attract CTL (cytotoxic lymphocytes) to the sites of infection. The expression of MHC class I molecules is increased on all types of cells due the type I IFNs – so making the recognition of infected cells more easy.

In contrast, IFN-αβ seem to have a potent antiproliferative and proapoptotic effects on T cells36, which is contradictary to the clonal expansion of effector T cells during infection when large amounts of IFNs are produced. It can be that early in the antiviral response, T cells are under the control of regulatory processes that downregulate the transcriptional response to IFNs, thereby facilitating proliferation of effector cells.37

Type I IFNs regulate CD4+ T helper cell development. IFN-αβ contributes to various functions of T helper type 1 (Th1) cells, particularly the secretion of interleukin-2 (IL-2) by memory cells. Conversely, IFN-αβ restricts the development of alternative populations such as Th2 and Th17. 38

The antiproliferative and proapoptotic effects of IFN-αβ are associated with a variety of molecular changes, including increases in both cyclin kinase inhibitors and several proapoptotic molecules (Fas/FasL, p53, Bax, Bak) as well as with activation of procaspases 8 and 3.36

Type I IFNs also exert a variety of effects on the development and function of B cells. Thus, IFN-αβ enhance BCR-dependent mature B2 cell responses and increase survival and resistance to Fas-mediated apoptosis.39

Signaling by IFNAR, acting directly or indirectly through other cytokines/chemokines, is also required for normal development and proliferation of the B1 subset40, which is thought to be a major producer of autoantibodies. Moreover, type I IFNs, acting indirectly through DC activation, exert strong adjuvant effects by markedly enhancing antibody responses and promoting Ig isotype switching.41

IFNs-I also affect monocyte and/or macrophage function and differentiation. Thus, IFNs-I markedly support the differentiation of monocytes into DC with high capacity for antigen presentation, stimulate macrophage antibody-dependent cytotoxicity, and positively or negatively regulate the production of various cytokines (e.g., TNF, IL-1, IL-6, IL-8, IL-12, and IL-18) by macrophages 42In addition, autocrine IFN-I is required for the enhancement of macrophage phagocytosis by macrophage colony-stimulating factor and IL-4 43and for the lipopolysaccharide-, virus-, and IFN-γ–induced oxidative burst through the generation of nitric oxide synthase 2.

Role in host defense and autoimmunity

Type I IFNs are critical for the host innate and adaptive immune responses against several pathogens, mainly viruses. Its relevance as a defense mechanism varies according to host (genetics) and pathogen factors. IFN-Iarenecessaryfor eradication of most viral infections,but dispensable in some cases44. A negative effect on elimination of some intracellular bacteria, fungi and, surprisingly, in some chronic viral infections have been described (Reviewed in45). Novel pathways of type I IFN-mediated immunoregulation are being discovered. Deficient mice of the IFN-stimulated gene cholesterol 25-hydroxylase overproduce inflammatory interleukin-1 (IL-1) family cytokines and are more resistant to infection by the intracellular bacteriaListeria monocytogenes.46

Several lines of evidence strongly suggest that these cytokines are directly activating cells and effector pathways of pathogenic significance in systemic autoimmune disease.Type I IFNs promote DC activation and, then, enhance its capability of antigen presentation to autorreactive T cell clones. Thus, viral infections may boost autoimmune responsesunder aninflammationcontext.

Compelling evidence supports raised levels of type I IFN in systemic autoimmune diseases, but also in organ-specific conditions.Elevated type I IFN levels in the serum of patients with systemic autoimmunity were described in 1970th 47 but were largely ignored. The involvement of INF-α in autoimmunity wasfirst suggested by Rönnblom and colleagues. They demonstrated that elevated serum IFN-α levels could be driven by immune complexes.48,49IFN was produced, when immunoglobulin from patients with systemic lupus erythematosus (SLE) was combined with plasmid DNA or apoptotic cells and added to peripheral blood mononuclear cells (PBMCs),

Subsequently, Blanco and colleagues50 demonstrated that serum from patients with SLE was capable of inducing the maturation of monocytes into DCs in an IFN-α-dependent manner. Chronic DC maturation in the presence of increased IFN levels might have a central role in autoimmunity by activating autoreactive T cells to drive the autoimmune destruction of target tissues. The capacity of self-antigen-containing immune complexes to stimulate IFN production further contributes to a self-propagating loop of tissue damage.

Recently, novel immune mechanisms that lead to IFN-I release in response to host nucleic acids have been discovered, wherein, these cytokines set up an appropriate scenario for antigen presentation and proliferation of auto-rreactive lymphocytes. In psoriatic patients, it has been demonstrated that antimicrobial peptide secretion, secondary to skin infection, promotes autoimmune responses. Coupling of LL-37 with host nucleic acids enhance pDC stimulation and IFNrelease.51 As demonstrated in this first study, other types of antimicrobial peptides linked to self-RNA or -DNA can enhance autoimmune responses in systemic diseases.52, 53 Structural features of antimicrobial peptides, which share cationic properties, permit to activate TLR954

In some patients treatment of chronic viral disease or malignancy with high-dose IFN has been associated with the generation of autoimmunity. The autoimmune phenomena that manifest are quite diverse, ranging from the induction of autoantibodies to the development of autoimmune diseases including SLE, polymyositis, and rheumatoid arthritis (RA). 55

Interestingly, the autoimmune symptoms that manifest can resolve after the cessation of treatment,56 suggesting that, although type I IFNs can initiate symptoms of autoimmunity, additional factors are required for the initiation of a self-propagating loop. Additionally, low IFN levels are found in most patients in multiple sclerosis. 57

It should be noted, that IFN-α and IFN-β are widely used in the clinical practice for treatment of hairy cell leukemia, malignant melanoma, AIDS-related Kaposi`s sarcoma, hepatitis C infections,multiple sclerosis, genital warts, hepatitis C with HIV coinfection, hepatitis B, general viral infections, myelogenous leukemia, cutaneous T-cell lymphoma, follicular non-Hodgkin's lymphoma, renal cell. 58, 59Several drugs are registered, namely: Infergen (IFN--α-con-1), Alferon-N (IFN--α-n3 leukocyte derived), Roferon-A (Recombinant IFN-α-2a), Intron A (Recombinant IFN--α-2b), PEG Intron (PEG recombinant IFN--α-2b) and Avonex (IFN-β-1a). Additionally, they are tested in clinical trials for safety and potential treatment of lymphomas60 and hepatocellular carcinoma, 61, 62

Role in allergic disease

The importance of IFN-αβ-mediated suppression of allergic T cell subsets is underscored by studies demonstrating that pDCs from asthmatic patients secrete less IFN-αβ than healthy donor pDCs in response to viral infections and toll-like receptor (TLR) ligands.63, 64Similarly,impaired IFN-β levels, in response to rhinovirus infection, were found in epithelial cells from asthmatic children.65 The cause of this deficiency is partially understood. Recently, Gielen et al.observed an upregulation of supressor of cytokine signalling (SOCS), an inhibitor of IFN-I production, in bronchial epithelial cells of asthmatic patients.66

Likewise, Gill et al.67 compared the induction of IFN-a by influenza virus in pDCs isolated from patients with asthma or healthy subjects and found that influenza virus infection promoted significantly less IFN-α secretion by pDCs from patients with asthma patients.