**4.4. The effects of Ido1 gene-deficiency on depression-like behavior, changes in TRP metabolism, 5-HT, and its turnover in the frontal cortex of mice following chronic Ifn-γ gene expression**

Additionally, we evaluated the role of IDO1 in the development of depression-like behavior after *Ifn-γ* gene transfer using *Ido1* gene knockout (KO) mice, and determined the levels of TRP metabolites in the frontal cortex.

**Figure 5.** The effect of chronic *Ifn-γ* gene expression on the TRP-KYN pathway and depression-like behavior in mice. Original data from Ref. [47]. (a) Behavioral changes in mice 28 days after *Ifn-γ* gene transfer. Open field test shows locomotor activity of mice in a novel environment. Y-maze test shows short-term memory. Forced swim test shows depression-like behavior. Immobility time was significantly increased in IFN-γ-transfected (+) mice, compared to IFNγ-transfected (−) mice. The open bar shows IFN-γ-transfected (−) mice, and the closed bar shows IFN-γ-transfected (+) mice. Each column represents the mean ± SEM (n = 9–16). \**p* <0.05 *versus* IFN-γ-transfected (−) mice. (b) (c) Changes in the levels of TRP and its metabolites in the serum and frontal cortex of mice after *Ifn-γ* gene transfer. TRP-KYN metabolite concentrations were determined in the serum (b) and the frontal cortex (c) of mice 35 days after *Ifn-γ*-gene transfer. The open bar shows IFN-γ-transfected (−) mice, and the closed bar shows IFN-γ-transfected (+) mice. Each column

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represents the mean ± SEM (n = 15–20). \*\**p*<0.01, \*\*\**p*<0.001 *versus* IFN-γ-transfected (−) mice [47].

The increase in time spent in an immobile posture in the *Ifn-γ*-transfected (+)/wild type mice was significantly improved in *Ido1* KO mice (**Figure 6a**). In wild type mice, *Ifn-γ* gene transfer significantly increased the concentrations of KYN and 3-HK in the frontal cortex by 4.7- and 2.5-fold, respectively. In contrast, *Ido1* KO mice withdrew these changes in *Ifn-γ* gene transfer A Critical Risk Factor for a Major Side Effect of Interferon-Alpha Therapy: Activated Indoleamine… http://dx.doi.org/10.5772/intechopen.71013 49

In order to clarify whether the activation of IDO1 by IFN-γ-affected behaviors, three tests, open-field test (OFT), the Y-maze test, and forced swimming test (FST), were performed in mice. Mice were transfected with either a pCpG-mcs plasmid (control vector) that did not contain the *Ifn-γ* gene [IFN-γ-transfected (−) mice] or a pCpG-Muγ plasmid that long-lasting expressed *Ifn-γ* [IFN-γ transfected (+) mice]. No significant differences in locomotor activity of the OFT was observed between IFN-γ transfected (−) and (+) mice. Similarly, in the Y-maze test, no significant differences in the alternation behavior were detected between the two groups of mice. However, in the FST, immobility time was significantly longer in IFN-γtransfected (+) mice (**Figure 5a**). Our findings strongly suggest that IDO1 induction by IFN-γ is a critical factor in depression-like behaviors but not in short-term memory or locomotor

**4.3. Changes in the levels of TRP and its metabolites in the serum and frontal cortex of** 

mid, we measured TRP metabolites in the serum and frontal cortex of these mice.

In order to further elucidate the relationship between the IDO1-induced KYN pathway and the development of depression-like behavior in mice transfected with the pCpG-Muγ plas-

The serum and the frontal cortex were corrected from mice immediately following behavioral testing to determine the levels of TRP, KYN, KA, 3-HK, 3-HAA, and AA (**Figure 5b** and **c**). The concentration of serum TRP was significantly decreased in IFN-γ transfected (+) mice compared to IFN-γ-transfected (−) mice. In contrast, the levels of serum KYN and 3-HK were significantly increased in the IFN-γ-transfected (+) mice (**Figure 5b**). In the frontal cortex, IFN-γ transfected (+) mice had significantly higher KYN and 3-HK levels than the IFN-γ-transfected (−) mice. The TRP and KA levels in the frontal cortex tended to be lower in the IFN-γ-transfected (+) mice (**Figure 5c**). The activation of IDO1 by *Ifn-γ* gene transfer significantly modified the levels of TRP and its metabolites not only in the serum, but also in the frontal cortex of mice. These results suggest that an alternative explanation for the participation of IDO1 in IFNγ-induced depression-like behavior is the generation of neuroactive TRP metabolites. This interpretation is consistent with our clinical data and previous studies by O'Connor et al.

**4.4. The effects of Ido1 gene-deficiency on depression-like behavior, changes in TRP metabolism, 5-HT, and its turnover in the frontal cortex of mice following chronic Ifn-γ** 

Additionally, we evaluated the role of IDO1 in the development of depression-like behavior after *Ifn-γ* gene transfer using *Ido1* gene knockout (KO) mice, and determined the levels of

The increase in time spent in an immobile posture in the *Ifn-γ*-transfected (+)/wild type mice was significantly improved in *Ido1* KO mice (**Figure 6a**). In wild type mice, *Ifn-γ* gene transfer significantly increased the concentrations of KYN and 3-HK in the frontal cortex by 4.7- and 2.5-fold, respectively. In contrast, *Ido1* KO mice withdrew these changes in *Ifn-γ* gene transfer

activity in mice.

and Wichers et al. [32, 35].

TRP metabolites in the frontal cortex.

**gene expression**

**mice following chronic Ifn-γ gene expression**

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

**Figure 5.** The effect of chronic *Ifn-γ* gene expression on the TRP-KYN pathway and depression-like behavior in mice. Original data from Ref. [47]. (a) Behavioral changes in mice 28 days after *Ifn-γ* gene transfer. Open field test shows locomotor activity of mice in a novel environment. Y-maze test shows short-term memory. Forced swim test shows depression-like behavior. Immobility time was significantly increased in IFN-γ-transfected (+) mice, compared to IFNγ-transfected (−) mice. The open bar shows IFN-γ-transfected (−) mice, and the closed bar shows IFN-γ-transfected (+) mice. Each column represents the mean ± SEM (n = 9–16). \**p* <0.05 *versus* IFN-γ-transfected (−) mice. (b) (c) Changes in the levels of TRP and its metabolites in the serum and frontal cortex of mice after *Ifn-γ* gene transfer. TRP-KYN metabolite concentrations were determined in the serum (b) and the frontal cortex (c) of mice 35 days after *Ifn-γ*-gene transfer. The open bar shows IFN-γ-transfected (−) mice, and the closed bar shows IFN-γ-transfected (+) mice. Each column represents the mean ± SEM (n = 15–20). \*\**p*<0.01, \*\*\**p*<0.001 *versus* IFN-γ-transfected (−) mice [47].

mice (**Figure 6b**). The levels of KYN and 3-HK in the frontal cortex after *Ifn-γ* gene transfer were considerably lower in *Ido1* KO mice than in wild type mice. Even though we cannot exclude the possibility that genetic deficient in *Ido1* and the resulting modifications in TRP metabolites could influence other behavioral tests, our results clearly demonstrate that *Ido1* KO mice do not show depression-like behavior and do not intensify TRP metabolites after *Ifn-γ* gene transfer.

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Other studies have emphasized that the 5-HT pathway is also relevant to depression. In a clinical study, it has been shown that levels of TRP and 5-hydroxytryptophan, a precursor of 5-HT, were significantly decreased from their baseline levels in the serum of HCV patients during IFN-α therapy [44]. Thus, we speculate that biological mechanisms underlying the IFN-α treatment induced-depressive symptoms are linked not only to the activated IDO1 and KYN pathway but also to a dysfunction of the 5-HT system. To clarify on the basis of the neurotransmitter changes in depression-like behavior after *Ifn-γ* gene transfer, we measured the concentrations of 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the frontal cortex of wild type and *Ido1* KO mice (**Figure 6c**). We showed that *Ifn-γ* gene transfer produced a trend toward increased 5-HIAA levels in wild type mice but not in *Ido1* KO mice. These results indicated that *Ifn-γ* gene transfer induced a potential increase in IDO1-induced 5-HT turnover. A raised 5-HT turnover suggests a process by which the availability of 5-HT to be released by neurons is decreased to compensate for neuronal dysfunction associated with depression-like behavior promoted by *Ifn-γ* gene transfer. Correspondingly, previous clinical studies have shown that brain 5-HT turnover is significantly increased in MDD patients without medication and decreased following selective serotonin reuptake inhibitors (SSRI) therapy [45, 46].

Taken together, an alternative interpretation for the involvement of IDO1 in IFN-γ-induced depression-like behavior may be that depression is related to not only the generation of neuroactive TRP metabolites but also to the alteration of serotoninergic neurotransmission.

The levels of TRP metabolites in the serum of HCV patients changed significantly. In particular, the increase in serum 3-HK concentration in depressive HCV patients was much larger than that in HCV patients without depressive symptoms. The ratios of serum KYN/ TRP, reflecting IDO1 activity, and 3-HK/KA were increased in depressive and non-depressed HCV patients with therapy. However, the increase in serum KYN/TRP and 3-HK/KA ratios in depressive patients was much higher than that of non-depressive HCV patients. When the *Ifn-γ* gene was transfected into normal mice, depression-like behavior significantly increased. Additionally, *Ifn-γ* gene transfer to mice induced dramatic changes in TRP metabolite concentrations in the serum and the prefrontal cortex. On the other hand, genetic deletion of *Ido1* abrogated the enhanced depression-like behavior after *Ifn-γ* gene transfer. In conclusion, our results clearly show that IDO1 is a critical molecular regulator of the depressive pathology induced as a side effect of interferon therapy. Moreover, the depressive symptoms are induced via increases in degradation of TRP and neuroactive metabolites along the KYN

**5. Conclusion**

**Figure 6.** The effects of *Ido1* gene-deficiency on depression-like behavior, changes in TRP metabolism, 5-HT, and its turnover in the frontal cortex of mice following chronic *Ifn-γ* gene expression. Original data from Ref. [47]. (a) Abnormal behavior in a forced swimming test after *Ifn-γ* gene transfer in mice was improved in *Ido1* gene deficient mice. The Y axis shows the percent value of immobility time in IFN-γ-transfected (+) mice, compared to the time (100%) in IFN-γ-transfected (−) mice (n = 8–15). (b) The level of TRP metabolites in the frontal cortex of mice 35 days after *Ifn-γ*-gene transfer (n = 6–15). (c) The amount of 5-HT, 5-HIAA, and 5-HIAA/5-HT ratio as an index of serotonin turnover in the frontal cortex of mice 35 days after *Ifn-γ*-gene transfer (n = 6–15). The open bar represents wild type and the closed bar, *Ido1* gene deficient mice. IFNγ-transfected (−) mice were injected with the control plasmid (pCpG-mcs), and IFN-γ-transfected (+) mice were injected with the IFN-γ-expressing pCpG-Muγ plasmid. Each column represents the mean ± SEM. \**p*<0.05, \*\*\**p*<0.001 *versus* IFN-γtransfected (−) wild type mice, # *p*<0.05, ##*p*<0.01, ###*p*<0.001 *versus* IFN-γ-transfected (+) wild type mice [47].

mice (**Figure 6b**). The levels of KYN and 3-HK in the frontal cortex after *Ifn-γ* gene transfer were considerably lower in *Ido1* KO mice than in wild type mice. Even though we cannot exclude the possibility that genetic deficient in *Ido1* and the resulting modifications in TRP metabolites could influence other behavioral tests, our results clearly demonstrate that *Ido1* KO mice do not show depression-like behavior and do not intensify TRP metabolites after *Ifn-γ* gene transfer.

Other studies have emphasized that the 5-HT pathway is also relevant to depression. In a clinical study, it has been shown that levels of TRP and 5-hydroxytryptophan, a precursor of 5-HT, were significantly decreased from their baseline levels in the serum of HCV patients during IFN-α therapy [44]. Thus, we speculate that biological mechanisms underlying the IFN-α treatment induced-depressive symptoms are linked not only to the activated IDO1 and KYN pathway but also to a dysfunction of the 5-HT system. To clarify on the basis of the neurotransmitter changes in depression-like behavior after *Ifn-γ* gene transfer, we measured the concentrations of 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the frontal cortex of wild type and *Ido1* KO mice (**Figure 6c**). We showed that *Ifn-γ* gene transfer produced a trend toward increased 5-HIAA levels in wild type mice but not in *Ido1* KO mice. These results indicated that *Ifn-γ* gene transfer induced a potential increase in IDO1-induced 5-HT turnover. A raised 5-HT turnover suggests a process by which the availability of 5-HT to be released by neurons is decreased to compensate for neuronal dysfunction associated with depression-like behavior promoted by *Ifn-γ* gene transfer. Correspondingly, previous clinical studies have shown that brain 5-HT turnover is significantly increased in MDD patients without medication and decreased following selective serotonin reuptake inhibitors (SSRI) therapy [45, 46].

Taken together, an alternative interpretation for the involvement of IDO1 in IFN-γ-induced depression-like behavior may be that depression is related to not only the generation of neuroactive TRP metabolites but also to the alteration of serotoninergic neurotransmission.
