| |
Interferon-α-induced modulation of glucocorticoid and serotonin receptors as a mechanism of depression
|
| |
| |
Journal of Hepatology June 2005
Article in Press
Wei Caia, Vladimir I. Khaoustova, Qing Xiead, Tianhong Panb, Weidong Leb, Boris Yoffeac
a Department of Medicine, Veterans Affairs Medical Center (151B), 2002 Holcombe Blvd., Houston, TX 77030, USA
b Department of Neurology; Baylor College of Medicine, Houston, TX, USA
c Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA
d Department of Infectious Diseases, Ruijin Hospital, Shanghai Second Medical
University, China
ABSTRACT
Background/Aims
The mechanism of interferon (IFN)-α-induced depression remains poorly understood. Recently, modulation of glucocorticoid receptor (GR) and serotonin receptor 1A (5-HTR1A) were implicated in mechanism(s) leading to depression. To gain insight into this mechanism, we assessed the effect of IFN-α on the modulation of GR and 5-HTR1A expression.
Methods
Hepatoblastoma, myelocyte-derived and T cell leukemia-derived cell lines were treated with titrated doses of IFN-α for different incubation times and analyzed by Western blot, RT-PCR, and microarrays. Dose- and time-dependent decreases of proteins and mRNA levels of GR and 5-HTR1A were observed.
Results
The expression of GR and 5-HTR1A in cells treated for 6 days decreased by 74 and 72%, respectively. Recovery was observed following IFN-α withdrawal. Co-incubation with tricyclic antidepressants (desipramine) or serotonin reuptake inhibitors (fluoxetine) attenuated the effect of IFN-α on GR or 5-HTR1A. GR and 5-HTR1A were unaffected by treatment with either IFN-γ or TUDCA. However, the effect of IFN-α on GR was abolished when used in combination with TUDCA.
Conclusions
In conclusion, IFN-α downregulated GR and 5-HTR1A levels in cell lines. These levels of GR and 5-HTR1A, following IFN-α-induced downregulation, recovered after withdrawal of IFN-α or addition of desipramine or fluoxetine. These data provide insights regarding pathogenesis of IFN-α-induced depression.
1. Introduction
Hepatitis C virus (HCV) infection is a major health problem estimated to affect 350 million individuals worldwide and a leading cause of liver cirrhosis and hepatocellular carcinoma [1]. IFN-α-based therapy is the only effective treatment for HCV infection [2]. However, this treatment may result in depression, and adversely affect outcome because of its negative impact on patients’ quality of life, its interference with treatment adherence, and its serious complications, including suicide [3,4]. In a recent report, 33% HCV patients between the 2nd and 5th months of IFN-α therapy developed major depressive disorder (MDD) [5]. It is unknown whether subjects with preexisting psychiatric disorders, including HCV patients, are more likely to develop the neuropsychiatric complications of IFN-α therapy. Importantly, depression is also seen in other conditions following treatment with IFN-α, such as hepatitis B, hairy cell leukemia, AIDS-related Kaposi's sarcoma, chronic myelogenous leukemia, and melanoma [6].
The mechanism of IFN-α-induced depression remains unclear. Recent observations reported that MDD has been linked to the activation and dysregulation of the negative feedback system of the hypothalamic-pituitary-adrenal (HPA) axis. Glucocorticoid receptors (GR), located in the hippocampal area, are key elements in this negative feedback system. Both mineralocorticoid (type 1) and glucocorticoid (type 2) receptors are involved in the regulation of the HPA axis, but GR is primarily involved in termination of the stress response and in the pathogenesis of MDD [7,8]. In several studies the levels of GR were found to be significantly lower in depressed patients than in the healthy controls [9,10] and GR mRNA levels were decreased in the dentate gyrus and hippocampal subfields in animals that had been subjected to chronic stress [11,12]. Furthermore, treatment with tricyclic antidepressants (TAD) demonstrated upregulation of GR in the hippocampus and hypothalamus [7,13]. These data were confirmed by experiments in vitro that demonstrated desipramine-induced increase in GR gene promoter activity, GR mRNA levels, and binding activity [14]. However, other researchers have found that treatment with desipramine had no effect on GR expression [15,16]. Importantly and pertinent to our study, it was reported that GR located on lymphocytes could be used as a marker in predicting the response to anti-depressive treatment [17]. These observations were confirmed by a recent study that examined desipramine, imipramine, and serotonergic antidepressant mirtazapine, for their effects on GR expression in primary human leukocytes and in monocytic U-937 cells [18].
Recently, the impact of IFN-α on the serotonin (5-HT) system was implicated as one of the mechanisms of depression. Studies in IFN-α-treated patients showed correlation between the severity of depression and plasma tryptophan (a serotonin precursor) levels and serum 5-HT concentrations and that in animals IFN-α alters 5-HT receptors in the brain [19,20]. The serotonin 1A receptor (5-HTR1A) is the most abundant among other receptors in the hippocampus and has been implicated in affective disorders [21–26] including report of reduced 5-HTR1A mRNA levels in subjects who died by suicide [15]. Selective serotonin reuptake inhibitors (SSRI) acting primarily through the inhibition of serotonin transporters have been used for the treatment of IFN-α-induced depression [2]. Whereas in some studies SSRI have been shown to increase 5-HTR1A mRNA with depression, no changes in 5-HTR1A mRNA expression have been noted in others [25,27].
To gain insight into the mechanism(s) of IFN-α induced MDD, we assessed the in vitro effects of IFN-α and the combination of IFN-α with antidepressants on modulation of GR and 5-HTR1A expressions.
4. Discussion
Clinical observations indicate that a significant number of HCV patients undergoing treatment with IFN-α therapy develop severe depression [5]. Depression is a relative contraindication for the treatment with IFN-α therapy because of the risk of exacerbation of depressive disorders and increased susceptibility for suicide [6]. IFN-α-induced depression is preventable by pretreatment with antidepressants [2]. These findings suggest a direct relationship between IFN-α treatment and depression. However, the pathophysiology of IFN-α-induced depression remains unclear. Several potential mechanisms of development depression were proposed including the impact of IFN-α on the HPA axis and serotonin pathway. Recent studies suggest that IFN-α stimulates the HPA axis, and this may contribute to the induction of depression. In clinical studies, IFN-α stimulated plasma ACTH and cortisol secretion in patients and controls [33,34]. Glucocorticoids, the final product of HPA axis, and their receptors are crucial mediators in the endocrine-immune interaction. Dysregulation and dysfunction of corticosteroids and their receptors have been implicated in the pathogenesis of MDD [8]. Several studies have observed a reduction in the levels of GR in depressed subjects [9,10]. GR, as a member of the nuclear receptor family, can alter the expression of specific target genes that involved in brain function and adaptation to stress [35]. Exposure to chronic stress exacerbates several neuropsychiatric disorders including depression, and differentially regulates the GR levels [36].
The complex physiological actions of 5-HT are mediated by a considerable number of 5-HT receptors [37]. Molecular and functional evidence exists for 16 different subtypes of 5-HT receptors [25]. Several studies show that IFN-α-induced depression is associated with the modulation of levels of 5-HT receptors in the brain [19,20]. A significant decrease in 5-HTR1A mRNA in the hippocampus of subjects with history of MDD has been reported [24]. Furthermore, a decrease in the 5-HTR1A binding potential, as determined by positron emission tomography, has been demonstrated in forebrain areas of depressed patients [38]. Furthermore, a recent repot demonstrated the modulation of 5HTR1A on circulating lymphocytes of depressed subjects [39].
Our experiments demonstrated the dose- and time-dependent decreases of mRNA levels and protein expression of GR and 5-HTR1A in IFN-α treated cells. The expression of GR and 5-HTR1A in cells treated continuously for 6 days with IFN-α was decreased by 75 and 72%, respectively. Interestingly, GR and 5-HTR1A nearly completely recovered within 3 days of culture without IFN-α, suggesting reversibility of IFN-α effects. Microarrays confirmed that serotonin and glucocorticoid related genes were downregulated, supporting our data and previous studies [9–11,15,25,40].
Both TAD and SSRI effectively ameliorate IFN-α-induced depression in patients [2,3]. Our results demonstrate that an IFN-α-induced downregulation of GR and 5HTR1A was almost completely restored by treatment with desipramine and fluoxetine. The majority of studies using desipramine indicated an upregulation of GR in the brain [7,13,14], while few studies showed no effect [15,16]. A recent study has demonstrated that antidepressant treatment directly induces GR upregulation, which in turn leads to enhanced susceptibility of the HPA axis to negative feedback by glucocorticoids and, therefore, to normalization of HPA hyperactivity in depressed patients [7]. The mechanism of the effects of antidepressants on 5-HTR1A could be different and independent from the effect of antidepressants on GR; it selectively and potently inhibits 5-HT re-uptake. Furthermore, the prophylactic use of fluoxetine lowered the incidence of IFN-α induced depression [41]. These data suggest that the prophylactic use of antidepressants, such as desipramine or fluoxetine, might prevent the development of IFN-α-induced depression.
In control experiments, we also investigated the effects of IFN-γ. It plays an important role as an antiviral agent and as an immunomodulator. Furthermore, it was suggested that it might affect the 5-HT metabolism and levels of GR in the macrophages [42,43]. However, no differences in levels of GR and 5-HTR1A were observed in our experiments.
TUDCA is a hydrophilic bile acid that protects liver cells by inhibiting apoptosis [28]. Importantly, TUDCA modulates GR activation by inducing nuclear translocation of GR [32]. In our studies, TUDCA caused only a moderate increase in the GR levels. However, when used in combination with IFN-α, TUDCA abolished the effect of IFN-α on GR. It is conceivable that IFN-α-induced downregulation of GR by affecting GR translocation, and these effects were restored by treatment with TUDCA. Interestingly, no changes were observed in levels of 5-HTR1A following treatment with TUDCA.
In summary, our results demonstrate that a) IFN-α downregulates the expression of GR and 5HTR1A in lymphoid and hepatic cell lines; b) IFN-α may play a critical role in regulating expression of GR and 5-HTR1A; and c) desipramine and fluoxetine decreases the effect of IFN-α on GR and 5-HTR1A. These in vitro results are consistent with clinical and animals studies regarding the modulation of levels of GR and 5-HT receptors in depression [9,10,19,20]. Recent reports suggest that lymphocytes reflect the metabolic processes of brain cells and that GR and 5HT1A receptors located on lymphocytes could be used as a marker in predicting the response to anti-depressive treatment [17,39,44]. It is conceivable that assessment of GR and 5HTR1A could reflect the stage and the severity of depression in patients undergoing IFN-α based therapy. Thus, if our observations will be confirmed in patients, then measurement of GR and 5HTR1A could lead to simple data-driven tests for predicting the development of depression and offer an opportunity to tailor specific therapy for individuals undergoing IFN-α treatment for HCV as well as cancer. Thus, a large number of patients will benefit from IFN-α therapy.
2. Materials and methods
2.1. Cell line and culture
In our study we used human hepatoblastoma cell line (Huh7), myelocyte-derived cell line (P815) and T cell leukemia-derived cell line (Jurkat).
2.2. Drugs
IFN-α and IFN-γ were gifts from the Schering Corporation (Kenilworth, NJ) and InterMune, Inc. (Brisbane, CA), respectively. Fluoxetine, desipramine and tauroursodeoxycholic acid (TUDCA) were purchased from Sigma (St. Louis, MO).
2.3. Drug treatment protocols
A) To establish experimental conditions cells were treated with titrated doses of IFN-α (3 to 300ng/ml) for different incubation times. B) To assess the recovery time of GR-α and 5-HTR1A following treatment with IFN-α, cells were treated with a high dose of IFN-α (300ng/ml) for 3 days, and then analyzed following incubation for additional 24, 48, and 72h with and without IFN-α. C) To evaluate the effect(s) of antidepressants, cells were cultured with IFN-α in combination with titrated doses of desipramine or fluoxetine. D) In control experiments, cells were cultured with titrated doses of IFN-γ and TUDCA and the effect of TUDCA was assessed by co-incubation with optimal concentrations of drugs.
2.4. Western blot analysis
GR, 5-HTR1A and PCNA proteins from cell lysates were separated on polyacrylamide-SDS gels and transferred to membrane as described [28] and after blocking and washing were incubated with primary antibodies from (Santa Cruz Biotechnology, CA): anti-GR, anti-5-HTR1A, or anti-PCNA. Immunoreactivity was detected with secondary antibodies and the ECL system (Amersham Inc., IL). β-actin was assessed for adjustment of protein loading. The relative intensity of bands was measured using National Institutes of Health's image analysis software (ImageJ1.22d, NIH, USA). All values were normalized to their respective lane of loading controls. Statistical analysis was preformed using Student's t-test. P values of 0.05 were considered statistically significant.
2.5. Reverse transcription-polymerase chain reaction (RT-PCR).
The amplification of 5-HTR1A gene with the specific primers (Forward: 5′-CTCATGGTGTCGGTGTTGG-3′, Reverse: 5′-GTGTAGCCATGATCCTTGC-3′) was obtained with 1 cycle at 95°C for 15min, followed by 40 cycles at 95°C for 45s, 61°C for 45s, 72°C for 45s and 1 cycle at 72°C for 10min. The amplification of GR-α gene with the specific primers (Forward: 5′-GCAGTTTCACTCTCAATGGG-3′, Reverse: 5′-CTGGCCCTTCAAATGTTGCT-3′) was obtained with 1 cycle at 95°C for 15min, followed by 35 cycles at 94°C for 30s, 59°C for 45s, 72°C for 45s and 1 cycle at 72°C for 10min. Aliquots of the reaction products were analyzed by electrophoresis.
2.6. Affymetrix genechip arrays
For microarray analysis, the GeneChip System (Affymetrix, CA) was used. The targets for GeneChip analyses were prepared according to the current expression analysis technical manual
Microarrays were processed in a human genome GeneChip Array (HG-U133A). The Affymetrix software (Microarray Suite 5.0) extracted fluorescence intensities from each element on the microarrays as detected by confocal laser scanning according to the manufacturer's recommendations.
3. Results
3.1. The effect of IFN-α on GR and 5-HTR1A in different cell lines
Human GR are comprised of two isoforms, α and β. GR-α is the predominant isoform of the receptor [29,30]. In contrast to GR-β, GR-α is detected in all cells and tissues. Among 16 different subtypes of 5-HT receptors [23], we assessed the expression of 5-HTR1A because of evidence that dysregulation of 5-HTR1A has been implicated in depressive disorders [21–23].
In order to establish the optimal IFN-α concentration and time for inducing GR and 5-HTR1A modulation, Huh7, P815, and Jurkat cells were treated with different doses of IFN-α for various incubation times. The dose- and time-dependent decreases of mRNA levels and protein expression of GR and 5-HTR1A were observed with different concentrations of IFN-α and varying incubation periods: 1, 2 or 3 days. Dose- and time-dependent decreases in the expression of GR and 5-HTR1A were observed (Fig. 1). The expression of GR was decreased by 10, 11 and 11%, and 5-HTR1A by 6, 7 and 11%, respectively, after 1, 2 and 3 days of exposure to low dose of IFN-α (3ng/ml). At the highest dose of IFN-α (300ng/ml) the GR levels decreased by 12, 18 and 52% and 5-HTR1A levels decreased by 5, 15 and 63%, respectively, in the same time frame. Based on these results, treatment with 300ng/ml concentration of IFN-α for 3 to 6 days was chosen as the optimal concentration and time for assessment of GR and 5-HTR1A modulations.
3.2. The recovery time of GR and 5-HTR1A following IFN-α-induced downregulation
Cells were treated for 3, 4, 5, and 6 days with IFN-α and/or for 3 days with IFN-α and then 1, 2, and 3 additional days without IFN-α. The expression of GR and 5-HTR1A was analyzed by Western blot. The expression of GR and 5-HTR1A in cells treated continuously with IFN-α for 6 days was decreased by 74 and 72%, respectively. The levels of GR and 5-HTR1A in cells treated for 3 days with IFN-α and then additionally for 3 days without IFN-α, recovered more than twofold from the levels following 3 days of IFN treatment.
These results were confirmed by RT-PCR. The mRNA levels of GR and 5-HTR1A decreased in cells treated with IFN-α for 6 days by 70 and 67%, respectively. When cells were treated for 3 days with IFN-α and then additionally for 3 days without IFN-α, the levels of GR and 5-HTR1A recovered by at least twofold. Thus, these data demonstrated the partial recovery of the levels of GR and 5-HTR1A to the base line of the controls occurred within 3 days after withdrawal of IFN-α following IFN-α induced downregulation. Similar results were observed with Huh7 and P815 cell lines (data not shown).
3.3. The effect of antidepressants on GR and 5-HTR1A following IFN-α-induced downregulation
Since previous studies showed that TAD and SSRI modulate the expression of GR and 5-HTR1A [15] we examined the effect of desipramine and fluoxetine on cells cultured with and without IFN-α. The titration with different doses of desipramine and fluoxetine from 0.1 to 10μM was performed. Both drugs were non-toxic and not effective on GR and 5-HTR1A expression at 0.1–1.0μM, whereas they were both toxic at 10μM and induced cell death after 3 days of treatment. However, 1.0μM concentrations desipramine and fluoxetine to IFN-α-treated Jurkat cells significantly decreased the effect of IFN-α on levels of GR and 5-HTR1A. The levels of GR and 5-HTR1A were elevated more than twofold when compared with the levels of expression in cells treated with IFN-α alone. These findings were confirmed by RT-PCR analysis and demonstrated that addition of desipramine to IFN treatment significantly reduced the effect of IFN-α on the mRNA levels of GR and 5-HTR1A (up to twofold).
3.4. The effect of IFN-α on cell cycle
PCNA, the proliferating cell nuclear antigen, is one of several essential factors for proper chromatin assembly and its expression is closely associated with cell cycle progression [31]. To assess whether observed modulation of GR and 5-HTR1A by IFN-α is cell cycle related, we evaluated the levels of PCNA. No difference in PCNA levels was observed in cells treated with different doses of IFN-α (Fig. 1). These data demonstrate that modulation of GR and 5-HTR1A is not cell cycle dependent and was not related to potential antiproliferative effect of IFN-α.
3.5. The effect of IFN-γ on GR and 5-HTR1A
IFN-γ is one of the pro-inflammatory cytokines and plays an important role as an antiviral agent and in modulating nearly all phases of immune and inflammatory responses. To assess the specificity of IFN-α, Jurkat and Huh7 cells were treated with different concentrations (20 to 200ng/ml) of IFN-γ for 3 days. No difference in expression of both GR and 5-HTR1A were observed between treated and non-treated cells (data not shown). These results suggest specificity of IFN-α effects on GR and 5-HTR1A and may explain absence of neuropsychiatric complications of IFN-γ in clinical settings.
3.6. The effect of TUDCA on GR following IFN-α-induced downregulation
TUDCA, an endogenous bile acid, is a relatively hydrophilic molecule compared with other nuclear receptor ligands. TUDCA might interact with the cell membrane and then modulate cytoplasmic events, such as GR activation [32]. Our results demonstrate that the expression of GR increased by 15% when Huh7 cells were treated with 200μM TUDCA alone for 3 days. Interestingly, combination treatment of TUDCA with IFN-α decreased the downregulation of the expression of GR by twofold. TUDCA did not have any effects on expression levels of 5-HTR1A.
3.7. Affymetrix genechip arrays
To confirm our RT-PCR results we performed selected experiments with the use of the Affymetrix GeneChip Array. Following IFN-α treatment most of the genes were upregulated and expression of at least 400 genes increased from two to hundredfold (data not shown). However, several serotonin and glucocorticoid related genes were downregulated: 5-HTR1A by 2.5, serotonin receptor 7 by 6.4, glucocorticoid regulated kinase by 3.5, GR DNA binding factor 1 by 2.7 and glucocorticoid regulated kinase-like by twofold, respectively. Only one serotonin related gene, acetylserotonin o-methyltransferase, was upregulated twofold and no single glucocorticoid related genes were upregulated. Interestingly, downregulation of 5-HTR1A and GR DNA binding factor 1 induced with IFN-α was reduced with desipramine.
|
|
| |
| |
|
|
|
