Interferons at age 50: past, current and future impact on biomedicine

Abstract

The family of interferon (IFN) proteins has now more than reached the potential envisioned by early discovering virologists: IFNs are not only antivirals with a spectrum of clinical effectiveness against both RNA and DNA viruses, but are also the prototypic biological response modifiers for oncology, and show effectiveness in suppressing manifestations of multiple sclerosis. Studies of IFNs have resulted in fundamental insights into cellular signalling mechanisms, gene transcription and innate and acquired immunity. Further elucidation of the multitude of IFN-induced genes, as well as drug development strategies targeting IFN production via the activation of the Toll-like receptors (TLRs), will almost certainly lead to newer and more efficacious therapeutics. Our goal is to offer a molecular and clinical perspective that will enable IFNs or their TLR agonist inducers to reach their full clinical potential.

Major milestones and discoveries in 50 years of interferon research

Figure 1. Mammalian Toll-like receptors (TLRs) and their ligands.

TLR1, TLR2, TLR4, TLR5 and TLR6 are located on the cell surface. Their extracellular domains (depicted as rods) bind specific microbial products that act as ligands and the intracellular domains (depicted as spheres) signal via specific cytoplasmic signalling proteins. TLRs function as homodimers or heterodimers.The ligands specific for several such dimers are listed at the top of the figure. Several other TLRs, such as TLR3, TLR7/8 and TLR9, recognize specific nucleic acids that are often produced by viruses. They span the endosomal membrane with the ligand-binding domains inside the lumen and the signalling domains in the cytoplasm. They also function as dimers and recognize double-stranded (ds) RNA, single-stranded (ss) RNA or dsDNA containing CpG sequences. GPI, glycosylphosphatidylinisotol; LPS, lipopolysaccharide.

Figure 2. Different interferon (IFN) signalling pathways activated by dsRNA and viruses.

Extracellular double-stranded (ds) RNA or intracellular dsRNA produced during viral replication can activate different signalling pathways triggered by either membrane-bound Toll-like receptor 3 (TLR3) or cytoplasmic retinoic acid-inducible gene I (RIG-I; also known as DDX58) or melanoma differentiation associated protein 5 (MDA5; also known as IFIH1). a | TLR3 recognizes dsRNA in the lumen of the endosome, which causes phosphorylation of specific tyrosine residues in TLR3 by an unidentified protein tyrosine kinase (PTK). TLR3 dimerizes, binds to CD14 and activates the signalling complex assembled by TLR adaptor molecule 1 (TRIF). Two major pathways bifurcate from TRIF. One, composed of tumour necrosis factor (TNF) receptor-associated factor 3 (TRAF3) and TANK-binding kinase (TBK1/IKKE), leads to phosphorylation of the transcription factor IFN regulatory factor 3 (IRF3). IRF3 requires further phosphorylation by the phosphatidylinositol 3-kinase (P13K)/AKT pathway for its full activation, which is initiated by binding PI3K to phosphorylated TLR3. The other branch acts through TRAF6 and transforming growth factor-β-activated kinase 1 (TAK1; also known as MAP3K7) leading to the activation of nuclear factor-κB (NFκB), JUN and activating transcription factor 2 (ATF2) transcription factors. The activated transcription factors translocate from the cytoplasm to the nucleus, bind to the cognate sites in the promoters of the target genes and singly or in combinations induce their transcription. b | The cytoplasmic RNA helicases RIG-I and MDA5 recognize dsRNA or 5′ triphosphorylated single-stranded (ss) RNA and use the mitochondrial membrane-bound protein IFN-β-promoter stimulator 1 (IPS1; also known as VISA) as the specific adaptor. IPS1 functions like TRIF and activates the same transcription factors leading to the induction of similar genes. In addition, they cause apoptosis by activating caspases 8 and 10 through the interaction of FADD with IPS1. Solid arrows denote steps that have been fully delineated, stippled arrows show steps that contain as yet unknown intermediaries. AIP3, atrophin-1 interacting protein 3; CCL5, chemokine (C-C motif) ligand 5; CXCL10, chemokine (C-X-C motif) ligand 10; IFIT1/2, interferon-induced protein with tetratricopeptide repeats 1/2; IKK, inhibitor of NFκB kinase; SELE, selectin E (endothelial adhesion molecule 1).

Figure 3. Receptor activation or ligand–receptor complex assembled by type I, type II or type III interferons.

Type I interferons (IFNs) (α, β ω, κ, ɛ, δ (pigs), τ (ruminants)) interact with IFN (α, β and ω) receptor 1 (IFNAR1) and IFNAR2; type II IFN-γ with IFN-γ receptor 1 (IFNGR1) and IFNGR2; and type III IFN-λs with IFN-λ receptor 1 (IFNLR1; also known as IL28RA) and interleukin 10 receptor 2 (IL10R2; also known as IL10RB). Type II IFN-γ is an antiparallel homodimer exhibiting a two-fold axis of symmetry. It binds two IFNGR1 receptor chains, assembling a complex that is stabilized by two IFNGR2 chains. These receptors are associated with two kinases from the JAK family: JAK1 and TYK2 for type I and III IFNs; JAK1 and JAK2 for type II IFN. All IFN receptor chains belong to the class 2 helical cytokine receptor family, which is defined by the structure of the extracellular domains of their members: approximately 200 amino acids structured in two subdomains of 100 amino acids (fibronectin type III modules), themselves structured by seven β-strands arranged in a β-sandwich. The 200 amino-acids domain usually contain the ligand binding site. IFNAR2, IFNLR1, IL10R2, IFNGR1 and IFNGR2 are classical representatives of this family, while IFNAR1 is atypical as its extracellular domain is duplicated. GAS, IFN-γ-activated site; IRF9, IFN regulatory factor 9; ISGF3, IFN-stimulated gene factor 3, refers to the STAT1–STAT2–IRF9 complex; ISRE, IFN-stimulated response element; P, phosphate; STAT1/2, signal transducers and activators of transcription 1/2.

Figure 4. Complexity of the signalling response.

Different types of cells respond differentially to a single type of interferon (IFN) by varying the activation of specific signal transducers and activators of transcription (STATs), additional transcription factors (TFs) and kinases in addition to the Janus kinases (JAKs). Priming of cells by pre-treatment with another cytokine modulates the response further by increasing the amounts of negative regulators and by modulating other processes. Most genes require STATs, with or without additional TFs, and several genes respond only to activated TFs and not to STATs. The STATs bind to IFN-γ-activated site (GAS) elements or, together with IFN regulatory factor (IRF) proteins, to IFN-stimulated response elements (ISREs), and the TFs bind to specific binding elements (TFBE). CIS, cytokine inducible SH2-containing protein; PTP, protein tyrosine phosphatase; SOCS1, suppressor of cytokine signalling 1.

Figure 5. Potential drug targets in the interferon (IFN) system.

Examples of potential or developmental drugs targeted at different steps in the pathways are presented. IPS1, IFN-β promoter stimulator 1 (also known as VISA); ISG, IFN-stimulated gene; JAK, Janus kinase; RNASEL, ribonulcease L; SOCS1, suppressor of cytokine signalling 1; TRAIL, tumour necrosis factor-related apoptosis-inducing ligand (also known as APO2L); TRIF, TLR adapter molecule 1.