**2.2. Pathways of induction and major roles of interferons**

In this chapter, we describe and review different cytokines. They will be categorized according to their type, followed by presentation of their function and a brief scope: IFN (IFN-α, β and γ), IL (IL-1, IL-2 and others), TNF (TNF-α and TNF-β) and others. A brief explanation of different cytokines activities also will be done, comprising pro- and anti-inflammatory action, cellular immune responses and performance in hematopoiesis. Methods to reach these objectives include a literature search in the most relevant sources of information, including

As key results, this chapter will provide a better understanding on cytokines types and functions, with organized concepts about this subject. As we aim to provide a comprehensive review of the available data regarding cytokines, this chapter will be a valuable source of

Interferon family represents a widely expressed group of cytokines. It includes three main classes, designated as type I IFNs, type II IFN and type III IFNs. The two main type I IFNs includes IFN-α (further classified into 13 different subtypes such as IFN-α1, -α2, -α4, -α5, -α6, -α7, -α8, -α10, -α13, -α14, -α16, -α17 and -α21), and IFN-β. The term *interferon* derives from the ability of these cytokines to interfere with viral replication. Type I IFNs present a potent antiviral effect by inhibiting viral replication, increasing the lysis potential of natural killer (NK) cells and the expression of MHC class I molecules on virus-infected cells, and stimulating the development of Th1 cells. During an infectious process, this type of interferon becomes abundant and is easily detectable in the blood. On the other hand, type II IFN has only one representative, IFN-γ. This cytokine plays a major role is macrophage activation both in innate and adaptive immune responses. Type III IFNs, also denoted IL-28/29, present similar biological effects to type I IFN, playing an important role in host defense against viral infections [5–8].

Interferon was the first described member of the class of protein molecules now known as cytokines. Nowadays, interferons are well known to participate in innate immune system, mediating responses against viral infections. This role of the IFNs was first described in the 1930s, when a research conducted by Hoskins demonstrated that rabbits previously infected by the herpes simplex virus were protected against subsequent infections by the same type of virus. In 1937, a few years after Hoskins' experiment, Findlay and MacCallum showed that the virus-infected animals were also resistant to infections caused by antigenically different viruses, corroborating and complementing the existing evidence regarding IFNs functions at that time. Their findings, however, were only confirmed in 1957, when Isaacs and Lindenmann, through cell cultures research, demonstrated that cells infected by a virus had the ability to produce a protein that could make other cells resistant to other viruses. Glasgow, in 1966, theorized that the interferon production was not limited to primary infection by viruses, and that this cytokine might play a role following re-infection. Therefore, the concept of "immune

information for readers who seek a thorough and structured synthesis on this topic.

PubMed/Medline, Scopus and Web of Science databases.

**2. Interferons**

66 Autoantibodies and Cytokines

**2.1. History**

There are several isotypes of type I IFNs. In humans, there are multiple forms of IFN-α, only one type of IFN-β and additional isotypes, as IFN-δ, IFN-ε, IFN-κ, IFN-τ and IFN-ω (IFN-δ and IFN-τ have been only described in pigs and cattle). This sort of cytokines presents similar structure, binding to the same cell surface receptor, and they are coded by a family of linked genes located on the human chromosome 9 [7, 12].

Type I IFN synthesis is induced by microbial challenge (i.e., viral and bacterial infections or microbial nucleic acids exposure) when the pattern recognition receptors (PRRs) sense these microorganisms. These receptors can be found in the cytosol or in the endosome. Once a virus infects a cell, the cell activates signals that lead to phosphorylation, dimerization and passage to the nucleus of the interferon response factor 3 (IRF3). Along with IRF3, other transcription factors, such as nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1), activate the transcription of IFN-β gene. After this process, secreted IFN-β binds to the interferon receptor (IFNAR) on the surface of the infected cell, producing an autocrine signaling to mobilize other interferon response factors and alter gene expression patterns to provide interferon response. Besides autocrine signaling, IFN-β also binds to the interferon receptor expressed by neighboring non-virus infected cells, acting in a paracrine manner to promote interferon response in order to help these cells to resist viral infection [5, 13, 14].

Interferon response comprises a series of reactions that alter the expression of a variety of human genes. These reactions are mediated by the binding with type I interferon receptors, which consists of the IFNAR1 and IFNAR2 transmembrane proteins, and two associated cytoplasmic tyrosine kinases, the Janus kinase 1 (Jak1) and tyrosine kinase 2 (TyK2). In addition to IRF3, another transcription factor induced by interferon response is interferon response factor 7 (IRF7), which is responsible to initiate IFN-α transcription without the need of NF-κB and AP-1. The canonical pathway that mediates the biological effects of IFNs corresponds to the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Both the antiviral and inflammatory effects of IFN-α/IFN-β are specifically mediated by STAT1 and STAT2. This pathway, however, does not work in isolated manner. It extensively communicates with other signal transduction pathways, therefore recruiting several effector molecules to promote a potent effect against viral infections, antiproliferative and antitumor activities, in addition to the immunomodulatory effects. In healthy individuals, these type I IFN genes are strictly regulated, with almost no constitutive IFN-α production [7, 15, 16].

ligands to NK cell receptors expression in virus-infected cells. Thirdly, they lead to NK cells

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NK cells are lymphocytes of innate immune system, which provide defense against viral infections by secreting cytokines (mainly IFN-γ) and killing infected cells. When IFN-α or IFN-β bind to interferon receptors on circulating NK cells, these are activated and directed to infected tissues, where they attack virus-infected cells. It is possible to say that NK cells play, in innate immune response, similar functions than cytotoxic T cells in adaptive immune

Type II and type III IFNs do not share homogeneity with type I IFN in terms of induction, and the signaling pathways are, therefore, through their own receptors. Nevertheless, the signal pathways involved with type I IFN and type II IFN, as well as the target genes used by these cytokines, somewhat overlap. IFN-γ receptor (IFNGR) is composed by two structurally homologous polypeptides that belong to the type II cytokine receptor family, named IFN-γR1 and IFN-γR2. IFN-γ (originally designated as macrophage-activating factor) binds and induces dimerization of the two receptor chains. This process leads to the activation of JAK1 and JAK2 kinases and, subsequently, to the phosphorylation and dimerization of STAT1, which stimulates the transcription of several genes. The genes induced by this cytokine encode

several different molecules that mediate the biological activities of IFN-γ [5, 14, 25].

Unlike IFN-α or IFN-β, the gene that encodes IFN-γ is located on the human chromosome 12. This unique specimen of type II IFN is the primary cytokine involved in macrophage activation (named as classical activation) and plays a critical role in immunity against intracellular microorganisms. In innate immune system, IFN-γ is the main cytokine produced by NK cells, acting as a mediator of innate immunity. Despite belonging to the *interferon* family, IFN-γ does not produce a potent antiviral effect, running primarily as an activator of effector cells of the immune system. In adaptive immunity, IFN-γ is produced by T cells in response to antigen recognition, and its secretion is increased by IL-12 and IL-18. In addition, B cells and professional antigen-presenting cells (e.g., monocyte/macrophage and dendritic cells) are also involved in this cytokine production. While IL-12 and IL-18 control the production of IFN-γ by promoting its synthesis, IL-4 and IL-10 correspond to the negative regulators of type II IFN

Regarding biological activities, both type I and type II IFN are essential in the immediate cellular response to viral infections. IFN-γ acts on immune cell activation and induction of the major histocompatibility complex (MHC) molecules, which is important at a later stage of the response. Thus, this cytokine establishes an antiviral state for long-term control, coordinating the transition from innate to adaptive immunity. IFN-γ plays a role in macrophage activation, triggering microbicide effector functions in these cells. Macrophages activated by IFN-γ promote more intensive pinocytosis and phagocytosis, in addition to an improved microbial killing ability. Furthermore, IFN-γ acts as a cell growth inhibitor and presents the ability of

In summary, in the early stages of infection, NK cells are the main producers of IFN-γ, whose major role is macrophage activation. Once activated, macrophages release cytokines

to eradicate virus-infected cells [8, 21, 22].

response [23, 24].

production [5, 8, 25].

triggering apoptosis [25, 26].

A high number of cells produce IFN-α and IFN-β, including macrophages, fibroblasts, and endothelial cells, specialized leukocytes, called interferon-producing cells (IPCs), or natural interferon-producing cells, secrete up to 1000 times more interferon than the others after microbial challenge. These cells, also known as plasmacytoid dendritic cell (pDCs), are present in the blood, comprising less than 1% of the total peripheral blood mononuclear cells. In terms of morphology, they are similar to plasmocytes, another type of cell responsible for the massive production of this cytokine. IPCs express toll-like receptors (TLRs) 6, 7, 9 and 10, which are critical components of innate immunity, acting as pathogen sensors. Toll-like receptors act on innate immunity cells by detecting conserved patterns of pathogenic microorganisms. These cells, when activated by these receptors, lead to maturation of antigen-presenting cells and production of inflammatory cytokines. Hence, IPCs become responsive to a variety of viral infections through quick secretion of massive amounts of type I IFN. In other words, these cells can produce substantial amounts of type I IFN in response to stimulation with a wide range of DNA and RNA viruses, which signal through TLR9 and TLR7, respectively. During an antiviral immune response, therefore, IPCs are able to promote the function of NK cells, B and T cells, and myeloid dendritic cells through type I IFN. IPCs still differentiate into a unique type of mature dendritic cell, which allows the direct regulation of the function of T cells and links innate and adaptive immune responses. This process occurs at a later stage of viral infection [11, 17–20].

The whole process mentioned above can be summarized through the following explanation. On the first day after stimulation by viral infection (microbial challenge), IPCs produce massive amounts of type I IFN. On the following 2 days, IPCs differentiate into a type of dendritic cell called a plasmacytoid dendritic cell, which maintains the ability to produce interferon. During the infection process, these cells cluster into the T cell areas of the draining lymph nodes. Although there is some similarity between plasmacytoid dendritic cells and myeloid dendritic cells (known as conventional dendritic cells), it is believed that plasmacytoid dendritic cells do not have a substantial involvement in T cell activation in adaptive immunity, which is the main function of conventional dendritic cells. Therefore, in the context of innate immunity, conventional dendritic cells produce relatively small amounts of type I IFN, but produce large amounts of IL-12, a cytokine that interacts with type I IFN to activate the NK cell response to viral infection [7, 11].

IFNs, besides being first line of defense against viral infections, play important roles in immunosurveillance for malignant cells. More specifically, type I interferons present a potent antiviral activity, which is associated with several physiological changes. For ease of understanding, the role of type I interferons, in which IFN-α and IFN-β are the major actors, can be divided in three main functions. Firstly, these cytokines stimulate resistance to viral replication in all cells through cellular genes activation, with the consequent destruction of the viral mRNA and inhibition of the viral proteins translation. Secondly, they promote an increase in ligands to NK cell receptors expression in virus-infected cells. Thirdly, they lead to NK cells to eradicate virus-infected cells [8, 21, 22].

other signal transduction pathways, therefore recruiting several effector molecules to promote a potent effect against viral infections, antiproliferative and antitumor activities, in addition to the immunomodulatory effects. In healthy individuals, these type I IFN genes are strictly regu-

A high number of cells produce IFN-α and IFN-β, including macrophages, fibroblasts, and endothelial cells, specialized leukocytes, called interferon-producing cells (IPCs), or natural interferon-producing cells, secrete up to 1000 times more interferon than the others after microbial challenge. These cells, also known as plasmacytoid dendritic cell (pDCs), are present in the blood, comprising less than 1% of the total peripheral blood mononuclear cells. In terms of morphology, they are similar to plasmocytes, another type of cell responsible for the massive production of this cytokine. IPCs express toll-like receptors (TLRs) 6, 7, 9 and 10, which are critical components of innate immunity, acting as pathogen sensors. Toll-like receptors act on innate immunity cells by detecting conserved patterns of pathogenic microorganisms. These cells, when activated by these receptors, lead to maturation of antigen-presenting cells and production of inflammatory cytokines. Hence, IPCs become responsive to a variety of viral infections through quick secretion of massive amounts of type I IFN. In other words, these cells can produce substantial amounts of type I IFN in response to stimulation with a wide range of DNA and RNA viruses, which signal through TLR9 and TLR7, respectively. During an antiviral immune response, therefore, IPCs are able to promote the function of NK cells, B and T cells, and myeloid dendritic cells through type I IFN. IPCs still differentiate into a unique type of mature dendritic cell, which allows the direct regulation of the function of T cells and links innate and adaptive immune responses. This process occurs at a later stage of

The whole process mentioned above can be summarized through the following explanation. On the first day after stimulation by viral infection (microbial challenge), IPCs produce massive amounts of type I IFN. On the following 2 days, IPCs differentiate into a type of dendritic cell called a plasmacytoid dendritic cell, which maintains the ability to produce interferon. During the infection process, these cells cluster into the T cell areas of the draining lymph nodes. Although there is some similarity between plasmacytoid dendritic cells and myeloid dendritic cells (known as conventional dendritic cells), it is believed that plasmacytoid dendritic cells do not have a substantial involvement in T cell activation in adaptive immunity, which is the main function of conventional dendritic cells. Therefore, in the context of innate immunity, conventional dendritic cells produce relatively small amounts of type I IFN, but produce large amounts of IL-12, a cytokine that interacts with type I IFN to activate the NK

IFNs, besides being first line of defense against viral infections, play important roles in immunosurveillance for malignant cells. More specifically, type I interferons present a potent antiviral activity, which is associated with several physiological changes. For ease of understanding, the role of type I interferons, in which IFN-α and IFN-β are the major actors, can be divided in three main functions. Firstly, these cytokines stimulate resistance to viral replication in all cells through cellular genes activation, with the consequent destruction of the viral mRNA and inhibition of the viral proteins translation. Secondly, they promote an increase in

lated, with almost no constitutive IFN-α production [7, 15, 16].

viral infection [11, 17–20].

68 Autoantibodies and Cytokines

cell response to viral infection [7, 11].

NK cells are lymphocytes of innate immune system, which provide defense against viral infections by secreting cytokines (mainly IFN-γ) and killing infected cells. When IFN-α or IFN-β bind to interferon receptors on circulating NK cells, these are activated and directed to infected tissues, where they attack virus-infected cells. It is possible to say that NK cells play, in innate immune response, similar functions than cytotoxic T cells in adaptive immune response [23, 24].

Type II and type III IFNs do not share homogeneity with type I IFN in terms of induction, and the signaling pathways are, therefore, through their own receptors. Nevertheless, the signal pathways involved with type I IFN and type II IFN, as well as the target genes used by these cytokines, somewhat overlap. IFN-γ receptor (IFNGR) is composed by two structurally homologous polypeptides that belong to the type II cytokine receptor family, named IFN-γR1 and IFN-γR2. IFN-γ (originally designated as macrophage-activating factor) binds and induces dimerization of the two receptor chains. This process leads to the activation of JAK1 and JAK2 kinases and, subsequently, to the phosphorylation and dimerization of STAT1, which stimulates the transcription of several genes. The genes induced by this cytokine encode several different molecules that mediate the biological activities of IFN-γ [5, 14, 25].

Unlike IFN-α or IFN-β, the gene that encodes IFN-γ is located on the human chromosome 12. This unique specimen of type II IFN is the primary cytokine involved in macrophage activation (named as classical activation) and plays a critical role in immunity against intracellular microorganisms. In innate immune system, IFN-γ is the main cytokine produced by NK cells, acting as a mediator of innate immunity. Despite belonging to the *interferon* family, IFN-γ does not produce a potent antiviral effect, running primarily as an activator of effector cells of the immune system. In adaptive immunity, IFN-γ is produced by T cells in response to antigen recognition, and its secretion is increased by IL-12 and IL-18. In addition, B cells and professional antigen-presenting cells (e.g., monocyte/macrophage and dendritic cells) are also involved in this cytokine production. While IL-12 and IL-18 control the production of IFN-γ by promoting its synthesis, IL-4 and IL-10 correspond to the negative regulators of type II IFN production [5, 8, 25].

Regarding biological activities, both type I and type II IFN are essential in the immediate cellular response to viral infections. IFN-γ acts on immune cell activation and induction of the major histocompatibility complex (MHC) molecules, which is important at a later stage of the response. Thus, this cytokine establishes an antiviral state for long-term control, coordinating the transition from innate to adaptive immunity. IFN-γ plays a role in macrophage activation, triggering microbicide effector functions in these cells. Macrophages activated by IFN-γ promote more intensive pinocytosis and phagocytosis, in addition to an improved microbial killing ability. Furthermore, IFN-γ acts as a cell growth inhibitor and presents the ability of triggering apoptosis [25, 26].

In summary, in the early stages of infection, NK cells are the main producers of IFN-γ, whose major role is macrophage activation. Once activated, macrophages release cytokines that participate in T cells activation, therefore initiating the adaptive immune response. After being produced and entering the infected site, the effector T cells become, in turn, the main source of IFN-γ and cell-mediated cytotoxicity. Besides the effects on host defense, IFN-γ is also involved in the protection against tumor development [5, 26].

Due to the ability to increase immune response, type I and type II IFN have been explored in clinical trials as treatments for several conditions. It has been found that these cytokines are involved with the improvement of several conditions, such as hepatitis B and C virus infections, autoimmune diseases and certain types of leukemia and lymphomas. Hence, this class of cytokines, which play a paramount role in the immune system, consist of valuable treatment strategy. Still, in order to obtain full advantage of the therapeutic potential of interferons, further researches are needed to elucidate the core mechanisms of their effects [31, 32].

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Tumor necrosis factor (TNF) is a cytokine that had the name derived from it discovery in 1975 as a molecule that caused in vitro necrosis of tumors. Shortly thereafter, it was observed that TNF expression was promoted by immune system cells. These discoveries were important to a posterior characterizing of the TNF superfamily and the TNF receptor superfamily, which has more than 40 members, being the most outstanding TNF-α (commonly named as TNF) and TNF-β (also named Iymphotoxin), but also including cytokines and membrane proteins that have similar sequence homologies and a homotrimeric pyramidal structure (e.g. CD40 ligand, FAS ligand, OX40 ligand, GITR ligand and other several proteins). The binding of this family of cytokines with their respective receptors triggers especially inflam-

TNF-β, a type II transmembrane protein, is an important key in the development of lymph nodes and Peyer's patches, and also for the maintenance of secondary lymphoid organs. The expression of TNF-β is mainly stimulated by lymphocytes. TNF-α will be better described in

Although it were discovered many receptors along the decades, two are best known: TNFR1 (55 kD) and TNFR2 (75 kD). Both receptors are plasma membrane trimmers, while TNFR1 is expressed by most human cells and TNFR2 is mainly produced by immune system cells. It is important to mention that TNFR2 have a higher affinity to TNF. They are related to inflammatory reactions, so that a cytokine bind to the receptor, it induces the recruitment of proteins

The production of this cytokine is performed by different cells from the immune system, which includes T cells, NK cells, macrophages and monocytes. The stimulus for TNF expression includes different factors, such as bind to pathogen lipopolysaccharide (LPS) and other parts with toll-like receptors (TLRs), and also by other cytokines, highlighting IFN-y [33, 35]. It is primary secreted as a nonglycosylated type II membrane protein arranged as homotrimer. TNF membrane releases a trimeric soluble cytokine (a polypeptide that weighs around 17-kDa with triangular pyramid shape) through proteolytic cleavage by metalloprotease TNF-converting enzyme, and this is the circulating form that is found in blood plasma, and that allows a potent capacity to displacement in the body, thus it endocrine function. It is not

**3. Tumor necrosis factor**

matory reactions [33–37] .

the following topics [38, 39].

that are important for the process [35, 40] .

**3.1. Expression and structure of tumor necrosis factor alpha**

Type III IFN (IL-28/29 or IFN-λ), likewise type I IFN, present antiviral activity. Type III IFN is subdivided in IFN-λ1 and IFN-λ2/3, which are expressed in identical patterns. The signaling pathway related to IFN-λ is similar to IFN-α/IFN-β, involving mechanisms relying on IRFs and NF-κB actions, with the last one playing an essential role in regulating type III IFN expression. Nevertheless, the expression of IFN-λ is more flexible when compared to type I IFN, once it also involves independent actions of NF-kB and IRFs, allowing the production of this cytokine in response to a wider range of stimuli. Most classes of virus and some bacterial products induce IFN-λ expression, and almost all cell types, mostly pDCs, produce type III IFN after virus infection. However, different from the other types of IFN, macrophages are not involved in IFN-λ expression. Regarding biological activities, IFN-λ acts as the first line in host defense against viral infections, besides regulating innate and adaptive immune responses. Recently, a new member of the Interferon Lambda family was identified, the IFN-λ4. This cytokine presents strong antiviral activity and has been recently described to be related to hepatitis C treatment failure. Several *in vivo* studies have shown that IFN-λ can be developed as a potent antiviral agent, covering a wide spectrum of viral infections, with the additional benefit of not promoting the unwanted pro-inflammatory effects of IFN-α [6, 27–29].

### **2.3. Interferons and related diseases**

The first sign that type I IFN was somehow involved with human autoimmune diseases came from the observation of an increased incidence of autoantibodies and autoimmune diseases after type I IFN treatment. Hence, when considering the indication of IFN-α therapy for some conditions (e.g., hepatitis C virus infection), it is important to scrutinize the presence of autoantibodies in the patient, since they may increase the risk for autoimmune disease development with this kind of treatment [14]. As previously mentioned, pDCs are responsible for producing high levels of type I IFN in response to nucleic acid-containing immune complexes through the activation of TLRs 7 and 9 [11]. These immune complexes are prevalent in autoimmune conditions, such as systemic lupus erythematosus (SLE), which makes this process highly relevant for the development of autoimmunity. It has been described that, in autoimmune diseases, several key immune effector cells, such as B cells, T effector cells and regulatory T cells are modulated by IFN-α. Hence, type I IFN plays a substantial role in this kind of condition [16].

Regarding type II IFN, IFN-γ may contribute to the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus, multiple sclerosis and type I diabetes mellitus. The role of this cytokine in autoimmune diseases (both in promoting and suppressing the condition) has been shown in several mouse models. The administration of IFN-γ at very early stages of experimental autoimmune encephalomyelitis exacerbates the disease, while its administration at a later stage reduces disease severity. Hence, the absence of biomarkers that could indicate the best stage of the disease to initiate IFN-γ treatment consists in a limiting factor for its therapeutic use [25, 26, 30]. This subject will be reported in the topic "Cytokines and autoantibodies".

Due to the ability to increase immune response, type I and type II IFN have been explored in clinical trials as treatments for several conditions. It has been found that these cytokines are involved with the improvement of several conditions, such as hepatitis B and C virus infections, autoimmune diseases and certain types of leukemia and lymphomas. Hence, this class of cytokines, which play a paramount role in the immune system, consist of valuable treatment strategy. Still, in order to obtain full advantage of the therapeutic potential of interferons, further researches are needed to elucidate the core mechanisms of their effects [31, 32].
