Interferons Horizon Therapeutics

Abstract

Interferons (IFNs) are a family of multi-functional proteins, called cytokines, that are produced by immune cells such as leukocytes, natural killer (NK) cells, macrophages, fibroblasts, and epithelial cells. The minute amount of these α-helical glycoproteins, produced by mammalian cells, are firm components of the innate arm of the immune system providing rapid and broad protection against numerous types of invading pathogens. Interferons, from their discovery in the 19th century, have always held out a promise of important clinical utility first as an antiviral agent and more recently holding anti-inflammatory and regenerative effects for treating various neurological diseases such as multiple sclerosis, encephalopathies, Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), etc. IFNs elicit anti-viral and anti-inflammatory properties by inducing transcription of multiple IFN stimulated genes (ISG), a response that is partly mediated by Interferon regulatory factors (IRFs). This chapter provides a brief introduction of the interferon system as well as an in-depth assessment of the interferon signature and the various assay procedures for synthesizing non-natural interferon analogs for structural analysis, which may be helpful in designing improved products and act as a diagnostic tool for neurodegenerative disorders.

1. Introduction

The name of the interferons comes from their capability to intrude with the product of new contagion patches. When the vulnerable system is attacked, they get actuated due to viral infection or other unknown substances and the white blood cells in the body produces interferons, which are a group of proteins called cytokines. Interferons (IFNs) are a group of soluble α-helical glycoproteins [1] that are produced and released by the innate arm of immune cells such as leukocytes, natural killer (NK) cells, fibroblasts, and epithelial cells in response to virus infection (or any other stimuli). They bind to specific IFN receptors on cells to trigger multiple signaling pathways that result in the expression of IFN-stimulated genes (ISGs). The ISG products then render the cell resistant to subsequent virus infection. Interferons do not directly kill the virus or cancerous cells rather they boost the vulnerable system response and reduce the growth of cancer cells by regulating the exertion of several genes that control the stashing of multitudinous cellular proteins that affect their growth. In extreme cases, an indecorous prosecution of these pathways or their inordinate activation can result in cell death. IFNs can play beneficial roles in the nervous system because of their tremendous capacity for upregulating immune responses. The three major types of interferons, that is, IFN-α, β and γ act as implicit curatives for a number of diseases. IFN-α has been used for the treatment of hepatitis B and C, and in several types of cancers, including hairy cell leukemia, chronic myeloid leukemia, Kaposi's sarcoma, and Erdheim–Chester disease (or polyostotic sclerotic histiocytosis), a rare complaint of bone marrow inflammation that can also affect the cerebellum [2]. IFN-β, an immunosuppressive cytokine, is the first medicine shown to promote clinical improvement in multiple sclerosis by inhibiting IL-12 production and inducing IL-10 [3]. Genetic research reveals the role of IFN-β in regulating mitochondrial dynamics to prevent neurodegeneration. IFN-β rescues mitochondrial abnormalities and neuronal survivance in Parkinson’s disease in vivo [4]. Finally, IFN-γ has been used in the treatment of chronic granulomatous disease, a rare hereditable complaint in which the phagocytic cells have disabled capacity to kill ingested microbes, resulting in recurring bacterial and fungal infections [5]. IFNs, presumably in confluence with other cytokines, hold a prominent role in various therapies against diseases due to their incredible wide-ranging and pronounced immunological properties.

2. Origin and classification of interferons

Among the major discoveries in science, the discovery of interferon was a fortuitous one. The 60-year history of exploration on IFN abounds with big and small breakthroughs and are been recorded in the literature. However, information on the succession in lines of thought that led from one discovery to the next is dispersed, and many of those linkages may only be recorded in the memory of ‘veteran’ interferon workers. New generations of interferon workers tend to rely on handbooks or laboratory manuals, whereas background about sophisticated pathways of discovery is usually omitted. Therefore, this historical section related to molecular structure, production, and action of IFN will be considered from the viewpoint of how our insights have grown within the environment of evolving tools and general knowledge in cellular and molecular biology.

The basic phenomenon of interference was first described in the year 1935 with the capability of one contagion to interfere with the replication of another (challenge) contagion [6]. Thus, the hunt was underway for the mediator of viral interference for about 20 years until Alick Isaacs and Jean Lindenmann coined the term interferon (IFN) to it, in 1957 [7]. When heat- or UV-inactivated influenza virus was injected into the 10-day-old fragmented chorioallantoic membrane of chick embryos, a substance was released that inhibited viral multiplication. Hemagglutination, or the virus's ability to interact with and agglutinate red blood cells, was used to quantify influenza viral production (or inhibition). The interfering chemical was given the name "interferon." The titration ended when a well (on a plate of tiny wells) was identified with partial agglutination; the reciprocal of the influenza dilution thus measured was used as the interferon titer (concentration). Interferon molecules produced by infected cells function via autocrine and paracrine signaling to transform host cells into antiviral cells [8]. They have profound immunomodulatory as well as antiviral properties. They were initially classified as leukocyte, fibroblast, or immune IFNs, based on their cellular origin. Type I IFN (leukocyte and fibroblast IFN) and type II IFN (immune IFN) are two types of IFNs that are now known to be made up of over 20 distinct proteins [9].

3. IFNs family background

Even though IFNs were initially classified as antiviral agents, Isaac and Lindenmann could not have anticipated the enormous impact their discovery would have, and the extent to which they would be pertinent far beyond the discipline of Virology. From the discovery in the 1960s that IFNs also played a role in the control of cell growth and animal tumors up to the recent findings that they are pivotal regulators of both innate and adaptive immune responses, the result is that vertebrate life would be permanently threatened without IFNs.

Multiple criteria, including sequence identity, genetic loci, cell of origin, receptor distribution, and downstream reactions, have been used to classify IFNs. Although IFNs are expressed at low levels in the body at rest, they are activated to varying degrees depending on the stimuli, as a result, they play a dynamic and pathogen-specific function in the immune response. IFNs modulate the immune system’s ability by promoting transcription of interferon signaling genes (ISGs) after they are generated and released by immune cells.

IFNs were classified as type-I (pH stable) or type-II (pH unstable) based on their pH sensitivity. The designation of IFN-α/β and IFN-γ as type-I and type-II IFNs, respectively, was further verified by analysis of their unique amino acid sequences and crystal structures [10, 11, 12, 13]. The type-I family has been expanded to 16 members which include 12 IFN-αs that are encoded by 13 genes (IFN-α1/13 encode the same protein) [14, 15, 16, 17, 18], IFN-β (the well-known IFNs and the first to be cloned, purified, and sequenced) [17, 19, 20], IFN-ϵ [21], IFN-κ [22], and IFN-ω [23]. Type-II family includes only one member, that is, interferon- γ, produced by NK cells and T-cells (in response to cytokines IL-12 and IL-18). Both types of IFN promote an “antiviral state” by snooping with cell proliferation and viral replication mechanisms. Moreover, IFNs render infected cells to become more susceptible to apoptosis (procaspase activity) and recognition by CD8+ cytotoxic T-cells by upregulating the expression of class I-major histocompatibility complex (MHC-I) on infected cells [24]. In 2003, the genome analysis discovered a novel type-III family of IFNs (IFN-λ), which were shown to be comparable to the IL-10 family of cytokines [16, 25, 26, 27], particularly IL-22 [28] based on sequence and subsequent structural studies. In humans, there are four different subtypes of Type III IFN, namely, IFN-λ1 (IL-29), IFN-λ2 (IL-28A), IFN-λ3 (IL-28B), and IFN-λ4 [29]. These IFN present similar biological effects to type-I IFNs, playing an important role in host defense against viral infections.

After a brief introduction of some of the cardinal features of the three types of interferons, we will now discuss the type of receptors involved in signal transduction pathways and biological activities elicited by them and then focus on the regulation of these IFN responses using transcription regulatory factors.

4. IFN induction and signaling mechanism

IFNs are incredibly effective at limiting virus replication and transmission, but because they are not normally expressed, IFN synthesis must be triggered promptly and strongly upon host contact with the virus. Because all viruses proliferate inside host cells, identifying bacterial or viral nucleic acids (e.g., RNA or DNA genome) upon microbial challenge, is an efficient technique for eliciting innate immune responses. These foreign substances are firstly identified by a specialized group of proteins known as Toll-like receptors (TLRs) which are, further, a type of pattern-recognition receptors (PRRs), that are expressed on sentinel cells. These receptors are either cytosolic or endosomal membrane proteins [30]. The binding of dsRNA/dsDNA to the helicase domain of RIG-I (Retinoic acid-inducible gene-I) and MDA5 (melanoma differentiation-associated gene-5), respectively, induces caspase activation following activation of tumor necrosis factor (TNF) receptor-associated factor (TRAF)-associated NF-κB activator, TANK binding kinase 1 (TBK1) and inhibitor of NF-κB kinase IKKε Figure 1 [31, 32, 33]. IRF-3 and IRF-7 are expressed ubiquitously as inactive monomers in the cytosol but when cells are stimulated with poly (I:C) or virus infection, they get phosphorylated by the serine/threonine kinases, homodimerized, and are then translocated from cytosol to nucleus and binds to responsive elements for IFN-β gene transcription. After this, the secreted IFN binds to their specific cognate cell surface receptors, the heterodimeric IFNAR1/IFNAR2 complex for type I IFNs, dimers of the heterodimeric IFNGR1/IFNGR2 complex for type II IFN and the heterodimeric IFNLR1/IL10R2 complex for type III IFNs as represented in Table 1 [32], present on the infected cell's surface, causing an autocrine signaling cascade that mobilizes other interferon response components and changes the gene expression patterns, resulting in an interferon response. IFNs can also bind to the interferon receptor produced by nearby non-virus infected cells, operating in a paracrine manner to enhance interferon response and aid these cells in combating viral infection [33].

Figure 1.

The Mechanism of production of IFNs [30, 31, 32].

Interferons	Discovery	Gene	Family members	Receptors	IFN- producing cells	Signalling	References
Type-I IFN	1957	Chr 9	IFN-α (13 subtypes), IFN-β, IFN-ε, IFN-κ, IFN-ω	IFNAR1/IFNAR2 subunits	All nucleated cells	TYK2, JAK1, STAT1/2 forms ternary complex ISGF3, bind to ISRE/GAS	[17, 34, 35, 36, 37, 38]
Type-II IFN	1965	Chr 12	IFN-γ	IFNGR1/IFNGR2 subunits	B and T-lymphocytes, NK cells, APCs	JAK1/2, STAT1, bind to GAS/ISRE	[34, 35, 39, 40, 41, 42]
Type-III IFN	2003	Chr 19	IFN-λ1, IFN-λ2, IFN-λ3, IFN-λ4	IFNLR1/IL10R2 complex	All nucleated cells, dendritic cells, and epithelial cells	TYK2. JAK1, ISGF3, bind to ISRE	[29, 34, 35, 43, 44, 45]

Table 1.

Classification of interferons in humans.

The IFN signal transduction pathway has been appropriately described in multiple comprehensive reviews Figure 2 [17, 29, 38, 40, 41, 42, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57]. The type I IFNs bind to their related heterodimeric cell surface receptors, IFNAR1 and IFNAR2, which signals through the activation of Janus activated kinases (JAKs), specifically TYK2 (Tyrosine kinase 2) and JAK1, respectively, causing tyrosine phosphorylation of the receptors’ intracellular domains and recruitment of signal transducers and activator of transcription (STAT), STAT1 and STAT2 proteins, which in turn forms a trimeric complex, called ISGF3 (IFN stimulated gene factor 3) that consists of STAT1, STAT2, and IRF9 [17, 38]. The ISGF3 then translocate to the nucleus and binds to the IFN stimulated response element (ISRE) in the promoter region of IFN-stimulated genes (ISGs) and initiates transcription of antiviral genes. IRF2 acts as a transcriptional attenuator of ISGF3-mediated transcriptional activation within the nucleus, hence, the absence of IRF2 would result in increased Type I IFN signaling [46]. IFNAR activation also activates STAT1, STAT3, STAT4, STAT5, and STAT6 homodimers, as well as STAT1–STAT2, STAT1–STAT3, STAT1–STAT4, STAT1–STAT5, STAT2–STAT3 and STAT5–STAT6 heterodimers which bind and activates GAS (IFN-γ activated sequence) motifs, found in the promoter region of ISGs resulting in their gene expression [47, 48, 49, 50, 51]. Type I IFN signaling may also activate other signaling pathways that do not rely on the so-called JAK/STAT pathway. They are the non-canonical modifiers of Type I signaling called the mitogen-activated protein kinase (MAPK)/c-Jun amino-terminal kinase (JNK) pathways and the phosphoinositide 3-kinase (PI3K) pathway, which leads to diverse effects on the cell [52]. Furthermore, there is sufficient evidence that the function of distinct STATs may be modulated to account for individual responses. For example, a recent study found that STAT1 inhibits the IFN-α dependent induction of IFN-γ expression, whereas surprisingly, IFN-α or IFN-β mediated activation of STAT4 is essential for the IFN-γ synthesis during viral infection [53]. As a result, the functional diversity of type I IFN-regulated pathways allows for the transcriptional activation of a plethora of genes that facilitate the induction of physiologic responses.

Figure 2.

Signal transduction mechanism by Type I, Type II and Type III IFN receptors and production of ISGs [17, 29, 33, 38, 39, 40, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55].

Type II IFNs or IFN-γ is biologically active in its noncovalently coupled homodimer form. The extracellular domain of the two IFNGR1/CD119 subunit attaches to this homodimer, which then interacts with IFNGR2 to form a functional IFN- γ receptor complex. The receptor complex's IFNGR1 subunits are linked to JAK1, while the IFNGR2 subunits are linked to JAK2 [40]. When JAK1 and JAK2 are activated, the receptor is phosphorylated, and STAT1 is recruited and phosphorylated. The phosphorylation of STAT1 causes it to homodimerize and translocate to the nucleus. STAT1 homodimers attach to Gamma activated sequence (GAS) sites in the promoters of target genes once they reach the nucleus, regulating their transcription [41, 42]. IFN-γ signaling is dependent on weak type I IFN signaling, which is mediated by low type I IFN constitutive production [54]. Many of the IFN-gamma/STAT1 signaling-induced target genes are transcription factors that cause the expression of secondary response genes. IFN-gamma signaling can also activate the MAPK, PI3K/AKT/mTOR, and NF-kappa B signaling pathways, which control the expression of a variety of additional genes [55]. IFN-gamma signaling plays an important role in host defense by promoting macrophage activation, upregulating the expression of antigen processing and presentation molecules, driving the development and activation of Th1 cells, enhancing natural killer cell activity, regulating B cell functions, and inducing the production of chemokines that promote effector cell trafficking to sites of inflammation.

Type III interferons (IFN-λs) communicate with the body via a unique heterodimeric receptor complex, comprising of the IFN-λR1 subunit and interleukin-10R (IL-10R), shared by a number of cytokines in the IL-10 superfamily [29]. Despite the fact that IFN-λ and type I IFNs are structurally disparate and are engaged in different types of receptors, they both share the same JAK/STAT signal transduction pathway to trigger interferon-stimulated genes (ISGs), which have antiviral and immunoregulatory functions. So, these findings initially surmised that the Type I and Type III IFNs were functionally redundant. They were further distinguished by the kinetics of Type III interferons, which had a lower amplitude than Type I interferons while having long-lasting ISG expression [44, 45].

However, dysregulation of the IFN production and function would lead to immunological pathogenesis, such as inflammatory diseases, autoimmune and neurodegenerative disorders, via inappropriately stimulating inflammatory responses or dampening microbial controls. Thus, IFN response must be tightly regulated in order to develop protective immunity against microbial infections, curing autoimmune disorders and neurodegenerative diseases while avoiding detrimental toxicity induced by improper or prolonged gene expression.

5. IRF-mediated regulation of IFNs

Interferon regulatory factors (IRFs) are a group of transcription factors that are involved in a range of aspects of the innate and adaptive immune responses, including immune cell proliferation and differentiation, as well as modulating pathogenic responses [58]. Were first discovered and identified in the promoter region of the human interferon-β gene (IFN β1) during 1988, when a mouse cDNA clone encodes a protein that has specificity towards the IFN-β gene containing virus-inducible enhancer element, was identified [59]. During that period, there was no other homology present in accordance with this gene or other proteins. So, it was recognized and named as the IFN-regulatory factor 1 (IRF1). Further, cDNA clone that was identified later, subsequent cross-hybridization with IRF1 cDNA was named as IRF2. This signified the formal acknowledgment and birth of the massive IRF family [60]. IRFs specifically recognize the ISRE (Interferon-Stimulated Response Element), a conserved DNA consensus sequence, and become functionally active in the form of homodimers or heterodimers. The IRF family of transcription factors comprises of several members, namely, IRF1, IRF2, IRF3, IRF4 (also known as PIP, LSIRF or ICSAT), IRF5, IRF6, IRF7, IRF8 (also known as ICSBP), and IRF9 (also known as ISGF3γ/p48) were identified in Mus musculus and Homosapiens [61, 62]; IRF10 is observed in birds [63] and fishes [64], IRF11 is found in lower vertebrates, such as teleost fishes and zebrafish [65]. IRF1 and IRF2 have been extensively studied at the molecular level due to their unique properties of regulating gene expression despite having structural similarities. Although the former functions as a transcriptional activator and the latter repress IRF1 function by competing for the same cis-elements within type-I IFN (IFN-α/β) and IFN-inducible genes, they possess a high degree of structural similarity [66]. IRF-3, IRF-5, and IRF-7 are the three members of the IRF family which are induced by Type I IFNs, downstream of PRRs that detect viral DNA/RNA, resulting in a feedforward loop that maximally drives IFN expression [67]. Other members such as IRF-4, IRF-5, and IRF-8 are some of the key regulators of myeloid cell proliferation and phenotypic differentiation, which aids in modulating the inflammatory responses [68]. The fourth member of the family, IRF-9, controls the expression of a wide range of IFN stimulating genes. Researchers have discovered that, like the IFN-β gene, the IFN-λ1 gene is controlled by both IRF3 and IRF7, but IRF7 is the primary regulator of the IFN-λ2/3 genes [69, 70]. Understanding how their levels and activity are controlled is crucial since changes in either can lead to dysregulated immune responses and the development of autoimmune and neurodegenerative diseases.

6. Interferon system dysfunction and related disorders

Interferons are used to treat a number of diseases such as those that are caused by viruses (such as hepatitis B and C virus) or due to inflammation (like multiple sclerosis and systemic sclerosis) as depicted in Table 1 [34, 71, 72]. They also act as antineoplastic agents to treat malignancies (such as breast carcinoma, nodular lymphoma, chronic myelogenous leukemia (CML), Kaposi’s sarcoma, and renal adenocarcinoma) [73]. The expression levels of IFNs, as well as their actions, are superbly controlled in order to protect host cells from potential toxicity resulting from excessive responses. However, persistent and dysregulated IFN expression causes many diseases such as Type I interferonopathy, a type of inherited CNS disease. They have also been associated with the development or worsening of autoimmune diseases such as psoriasis, systemic lupus erythematosus (SLE), and, in rare cases, rheumatoid arthritis (RA) [74]. This was observed in their mRNA expression patterns that contain the interferon signature. Moreover, a lot of murine Alzheimer's disease (AD) models, as well as wild-type mouse brains challenged with generic nucleic acid-containing amyloid fibrils, showed an increased IFN-stimulated gene (ISG) signature [75]. These findings all point to IFNs that have a negative impact on the brain. Interferon overactivation is also associated with low levels of apoptotic particle clearance, resulting in an accumulation of apoptotic products (such as DNA-CpG motifs and U-RNAs). Similar abnormalities are seen in patients with primary Sjogren's syndrome, systemic scleroderma, and polymyositis, as well as a few cases of rheumatoid arthritis. Immunomodulation treatments aiming at lowering interferon overactivity are being tried in people with such diseases [76].

Shreds of evidence have shown that the development of autoimmune illnesses in certain people who were given IFN-α suggests that this cytokine plays a key role in breaking tolerance and triggering autoimmune responses in such patients [77]. Similarly, IFN-γ may also contribute to autoimmune disorders in addition to its host defense actions. Although IFN-γ production has been reported to be disease-limiting in experimental allergic encephalomyelitis (EAE), it may have a role in autoimmune nephritis [78]. Moreover, increased vulnerability to infection with certain viruses and intracellular bacteria appears to be linked to the loss of functioning IFNGR1 that is involved in Type II IFN signaling [79].

Recent research suggests that even mild-to-moderate acute COVID-19 infection results in a continuing, prolonged inflammatory response, which is not seen with common coronavirus infection [80]. After surviving acute coronavirus disease 2019 (COVID-19) infection, some individuals develop post-acute COVID syndrome (long COVID (LC)) that lasts longer than 12 weeks. The mechanisms behind this activation are still being investigated, but they might include antigen persistence, autoimmunity triggered by antigenic cross-reactivity, or a reflection of damage repair. These findings show that individuals with COVID-19 have an aberrant immunological profile at long intervals after infection, indicating the presence of an LC syndrome [80]. In this aspect, learning more about the immunological components of diverse pathologies has yielded common themes. Because of these unifying concepts, immune-based therapeutics for viral respiratory diseases, autoimmune and neurodegenerative disorders must be identified.

7. Closing remarks and outlook

There is a worldwide interest in repurposing existing drugs and understanding mechanisms against viral, autoimmune, and neurodegenerative diseases. Structural determination, interaction with different co-solutes, and binding studies can facilitate the process of vaccine development, help in understanding the mechanism of anti-inflammation, and design a potent inhibitor for drug discovery. The interaction studies with different proteins will stabilize and/or destabilize, allowing deeper insight into various interactions (attractive and repulsive forces) to maintain a high functional protein population as this can probably be helpful for pre-clinical toxicological studies. Furthermore, beyond the therapeutic benefit to the individual patient, IFN therapy may aid public health measures aimed at delaying the spread of pandemic diseases and also minimizing the deterioration of symptoms in cases of autoimmune diseases and neurodegenerative disorders by reducing the time it takes for their symptoms to deteriorate. However, the most difficult element of creating therapy options for immune modulation against such illnesses is disentangling beneficial from harmful signals. So, for that purpose, targeted immune regulation can temper maladaptive factors enabling beneficial immune response against disorders which might help reduce its severity in the future.