**2.1. Classification of IFNs**

IFNs were first introduced in 1957 as antiviral molecules. Based on their receptor types on the cell membrane surface, IFNs are classified into type I and type II. IFN type I mainly consists of IFN-α/β, while IFN type II consists of IFN-γ. IFN type I is a family of cytokines in which their amino acid sequence similarity reaches 30–80%. They are produced by a wide variety of cells, including fibroblasts, epithelial cells, and hepatocytes [12, 13]. However, in most viral infections, plasmacytoid dendritic cells (pDCs) are probably the major source of these cytokines. In contrast, IFN type II (IFN-γ) is a single gene cytokine unrelated in structure to IFN-α/β, which is produced largely by macrophages, natural killer (NK) cells, and T lymphocytes [12].

### **2.2. Immune response for HCV infection and IFN induction**

a 20–25-year period, and these individuals are at an increased risk for developing end-stage hepatic diseases or hepatocellular carcinoma [4]. Therefore, aggressive antiviral treatments to successfully induce viral remission constitute a major strategy for reducing the morbidity and

Immunotherapy with interferon-alpha (IFN-α) is commonly used to treat CHC and several types of malignancies owing to its antiviral, antiproliferative, and immunoregulatory effects [5]. In clinical trials, more than 50% of CHC patients treated with combination therapy using IFN-α and ribavirin achieved a sustained viral response, defined as undetectable HCV in the blood 6 months following the end of treatment [4]. Despite the efficacy of IFN-α in CHC treatment, IFN-α therapy causes serious side effects; early signs include somatic symptoms (anorexia, pain, insomnia, fever, and fatigue). Prolonged therapy causes neuropsychiatric symptoms including depressive states, anhedonia, anxiety, and cognitive impairment. In particular, depression is a serious and frequently occurring side effect of IFN-α therapy, and this leads to discontinuation of the therapy in up to 45% of patients [6, 7]. Therefore, in order to avoid the discontinuation of IFN-α therapy owing to depressive symptoms induced by the cytokine, it is

important to identify the risk factor(s) leading to the associated depressive symptoms.

(IDO1) activity, is increased in patients receiving IFN-α therapy [8].

A number of findings suggest that the neuropsychiatric side effects observed during IFN-α therapy may be linked to aberrations in the tryptophan (TRP)-kynurenine (KYN) pathway [8, 9]. Clinical studies have found that IFN-α therapy reduces plasma TRP and serotonin (5-hydroxythrptamine; 5-HT) levels [8] and increases KYN levels in plasma and cerebrospinal fluid (CSF). In addition, the KYN/TRP ratio, an index of indoleamine 2,3-dioxygenase 1

IDO1 is an extrahepatic enzyme that catalyzes the conversion of TRP to KYN, which can produce many neuroactive metabolites such as 3-hydroxykynurenine (3-HK), kynurenic acid (KA), and quinolinic acid (QUIN). Intriguingly, QUIN levels in CSF have been found to correlate with the severity of depressive pathology [10], and post-mortem studies have shown increased microglia QUIN levels in the frontal cortex of severely depressed patients [11].

In the current chapter, we present the findings of our latest study, which demonstrates the association between IFN treatment and changes in the TRP-KYN pathway in the blood of HCV patients. To do so, we investigated the effect of chronic *Ifn* gene expression on depression-like behavior and levels of brain TRP-KYN metabolites in mice. Our results suggest the possibility for the prediction of onset risk of depression as a side effect in HCV patients.

**2. Molecular characteristics and antiviral mechanisms of interferons** 

IFNs were first introduced in 1957 as antiviral molecules. Based on their receptor types on the cell membrane surface, IFNs are classified into type I and type II. IFN type I mainly consists of IFN-α/β, while IFN type II consists of IFN-γ. IFN type I is a family of cytokines in which their

mortality associated with CHC.

38 Pharmacokinetics and Adverse Effects of Drugs - Mechanisms and Risks Factors

**(IFNs)**

**2.1. Classification of IFNs**

The host response against HCV infection is first triggered when a pathogen-associated molecular pattern (PAMP), presented by an infecting virus, is recognized and engaged by specific PAMP receptors expressed on the host cells. This leads to the activation of signals that ultimately induce the expression of antiviral effector genes [14, 15] (**Figure 1**). For RNA viruses, protein, and nucleic acid products of infection or replication have been identified as viral PAMPs. These are engaged by specific toll-like receptors (TLRs) or nucleic acid-binding proteins that serve as PAMP receptors [15–17]. The viral RNA of HCV contains each of these PAMP signatures, and is adequate to trigger the host response when introduced into naïve cells [18, 19]. In hepatocytes, which is the target cell of HCV infection, independent pathways

**Figure 1.** The host innate response to HCV infection. Adapted from Ref. [14]. (1) HCV RNA binding to RIG-I or TLR3 results in the activation IRF-3. The dimer of phospho-IRF-3 translocates to the nucleus, interacts with transcription partners and binds to the cognate-DNA PRD in the promoter region of IRF-3 target genes. (2) IRF-3 activation leads to the induction of IFN-β production. (3) Secreted IFN-β from the infected cells binds to the IFN-α/β receptor, and results in activation of the JAK-STAT pathway. The ISGF3 complex translocates to the nucleus, where it binds to the ISRE on target genes to direct ISG expression. IRF-7 is one of the ISGs and it is activated after expression through viral PAMP signaling. (4) The IRF-7 dimer and heterodimer with IRF-3 binds to VRE in the promotor region of IFN-α genes resulting in the production of various IFN-α subtypes and establishing a positive-feedback loop for IFN amplification. It is the IFN-α component of the host response that is exploited by the current IFN-based therapy for HCV infection [14].

of retinoic acid-inducible gene I (RIG-I) and TLR3 signaling construct two major pathways of host defense triggered by double-stranded (ds) RNA [19–21]. Viral PAMP binding to RIG-I or TLR3 results in the phosphorylation and activation of interferon regulatory factor 3 (IRF-3) by TANK-binding kinase 1 (TBK-1) and I kappa B kinase ε (IKK-ε) [14, 22]. The dimer of phospho-IRF-3 translocates to the cell nucleus, interacts with its transcription partners, including CREB-binding protein (CBP)/p300, and binds to the cognate-DNA positive regulatory domain (PRD) in the promoter region of IRF-3 target genes, such as IFN-β [14, 23]. The engagement of PAMP receptors also leads to the synthesis of IFN-α/β, tumor necrosis factor (TNF), and a variety of other cytokines, which are largely produced by mainly pDCs that express TLRs in abundance. IFN-α/β produced by pDCs activates NK cells, thereby enhancing their cytotoxic potential and stimulating their production of IFN-γ. IFN-α/β produced by pDCs also modulates the activation of CD8<sup>+</sup> T cells, which produce additional IFN-γ and represent the central players in the pathogen-specific adaptive immune response [12].

### **2.3. The antiviral effect of IFNs on HCV**

IFN-α mediates a wide range of biological activities including antiproliferation, immunomodulation, and antiviral responses. IFN-α/β acts to induce the antiviral response in cells. These cells can be far from IFN-α/β production site and IFN-α/β interacts with specific cell surface receptors, type I IFN receptors (interferon-alpha receptor 1 (IFNΑR1) and IFNΑR2; **Figure 1**). IFNARs signal to the nucleus via Janus kinase-1 (Jak1) and tyrosine kinase 2 (Tyk2) phosphorylation of the signal transducers and activators of transcription (STATs) [24]. The classic IFN-α/β signaling pathways activate STAT1/STAT2 heterodimers and the trimeric IFN-stimulated gene factor (ISGF) complex containing IRF-9, which activate the expression of specific subsets of genes controlled by promoters containing interferon-stimulated response elements (ISRE; **Figure 1**) [15]. Interferon-stimulated genes (ISGs) are the genetic effectors of the host response, although the details of the signaling mechanisms by which IFN-α/β and IFN-γ induce the transcription of ISGs are still being defined [25]. IRF-7 is a transcription factor and an ISG. It is expressed in many tissue types, including complex liver tissue, in response to IFN. IRF-7 is activated after expression via viral PAMP signaling pathways that overlie with the IRF-3 activation pathway. IRF-7 phosphorylation, dimerization, and heterodimerization with IRF-3 lead to bind its cognate virus-responsive element (VRE) in the promotor region of IFN-α genes. Then, this binding results in the production of various IFN-α subtypes. The transcription effector action of IRF-7 also promotes diversification of the ISG response, establishing a positive-feedback loop that amplifies IFN production, and antiviral action [14]. This increases the plenty of RIG-I and viral PAMP signaling modules whose continued signaling acts to amplify IFN production and the host response. The medicinal administration of IFN-α promotes an antiviral reaction against HCV infection by stimulating ISG expression via the IFN-α/β receptor and the JAK-STAT pathway. In addition to stimulating ISG expression, IFN-α induces or promotes the maturation of immune effector cells, and enhances the production of other cytokines by resident hepatic cells to indirectly modulate the cell-mediated defenses and adaptive immunity to HCV [15]. Viral trigger and control of the host response may elucidate cellular tolerance for HCV RNA replication and influence the outcome of infection.

**Figure 2.** Schematic overview of the TRP-KYN pathway. IDO1 catabolizes L-TRP to N-formyl-L-kynurenine, which is converted to L-KYN by formamidase. L-KYN is further metabolized to AA by kynureninase (KYNU), to KA by kynurenine aminotransferases (KATs), and to 3-HK by kynurenine 3-monooxygenase (KMO). KMO is then metabolized

A Critical Risk Factor for a Major Side Effect of Interferon-Alpha Therapy: Activated Indoleamine…

http://dx.doi.org/10.5772/intechopen.71013

41

to 3-HAA by 3-hydroxyanhranilate 3,4-dioxygenase (3-HAAO). 3-HAA is further metabolized to QUIN.

A Critical Risk Factor for a Major Side Effect of Interferon-Alpha Therapy: Activated Indoleamine… http://dx.doi.org/10.5772/intechopen.71013 41

of retinoic acid-inducible gene I (RIG-I) and TLR3 signaling construct two major pathways of host defense triggered by double-stranded (ds) RNA [19–21]. Viral PAMP binding to RIG-I or TLR3 results in the phosphorylation and activation of interferon regulatory factor 3 (IRF-3) by TANK-binding kinase 1 (TBK-1) and I kappa B kinase ε (IKK-ε) [14, 22]. The dimer of phospho-IRF-3 translocates to the cell nucleus, interacts with its transcription partners, including CREB-binding protein (CBP)/p300, and binds to the cognate-DNA positive regulatory domain (PRD) in the promoter region of IRF-3 target genes, such as IFN-β [14, 23]. The engagement of PAMP receptors also leads to the synthesis of IFN-α/β, tumor necrosis factor (TNF), and a variety of other cytokines, which are largely produced by mainly pDCs that express TLRs in abundance. IFN-α/β produced by pDCs activates NK cells, thereby enhancing their cytotoxic potential and stimulating their production of IFN-γ. IFN-α/β produced by pDCs also modu-

IFN-α mediates a wide range of biological activities including antiproliferation, immunomodulation, and antiviral responses. IFN-α/β acts to induce the antiviral response in cells. These cells can be far from IFN-α/β production site and IFN-α/β interacts with specific cell surface receptors, type I IFN receptors (interferon-alpha receptor 1 (IFNΑR1) and IFNΑR2; **Figure 1**). IFNARs signal to the nucleus via Janus kinase-1 (Jak1) and tyrosine kinase 2 (Tyk2) phosphorylation of the signal transducers and activators of transcription (STATs) [24]. The classic IFN-α/β signaling pathways activate STAT1/STAT2 heterodimers and the trimeric IFN-stimulated gene factor (ISGF) complex containing IRF-9, which activate the expression of specific subsets of genes controlled by promoters containing interferon-stimulated response elements (ISRE; **Figure 1**) [15]. Interferon-stimulated genes (ISGs) are the genetic effectors of the host response, although the details of the signaling mechanisms by which IFN-α/β and IFN-γ induce the transcription of ISGs are still being defined [25]. IRF-7 is a transcription factor and an ISG. It is expressed in many tissue types, including complex liver tissue, in response to IFN. IRF-7 is activated after expression via viral PAMP signaling pathways that overlie with the IRF-3 activation pathway. IRF-7 phosphorylation, dimerization, and heterodimerization with IRF-3 lead to bind its cognate virus-responsive element (VRE) in the promotor region of IFN-α genes. Then, this binding results in the production of various IFN-α subtypes. The transcription effector action of IRF-7 also promotes diversification of the ISG response, establishing a positive-feedback loop that amplifies IFN production, and antiviral action [14]. This increases the plenty of RIG-I and viral PAMP signaling modules whose continued signaling acts to amplify IFN production and the host response. The medicinal administration of IFN-α promotes an antiviral reaction against HCV infection by stimulating ISG expression via the IFN-α/β receptor and the JAK-STAT pathway. In addition to stimulating ISG expression, IFN-α induces or promotes the maturation of immune effector cells, and enhances the production of other cytokines by resident hepatic cells to indirectly modulate the cell-mediated defenses and adaptive immunity to HCV [15]. Viral trigger and control of the host response may elucidate cellular tolerance for HCV RNA replication and influence the

T cells, which produce additional IFN-γ and represent the central

lates the activation of CD8<sup>+</sup>

outcome of infection.

**2.3. The antiviral effect of IFNs on HCV**

players in the pathogen-specific adaptive immune response [12].

40 Pharmacokinetics and Adverse Effects of Drugs - Mechanisms and Risks Factors

**Figure 2.** Schematic overview of the TRP-KYN pathway. IDO1 catabolizes L-TRP to N-formyl-L-kynurenine, which is converted to L-KYN by formamidase. L-KYN is further metabolized to AA by kynureninase (KYNU), to KA by kynurenine aminotransferases (KATs), and to 3-HK by kynurenine 3-monooxygenase (KMO). KMO is then metabolized to 3-HAA by 3-hydroxyanhranilate 3,4-dioxygenase (3-HAAO). 3-HAA is further metabolized to QUIN.
