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Antidepressant effects of TNF-α blockade in an animal model of depression
Journal of Psychiatric Research 47 (2013) 611e616
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Antidepressant effects of TNF-a blockade in an animal model of depression Ute Krügel a, *,1, Johannes Fischer a,1, Susanne Radicke a, Ulrich Sack c, Hubertus Himmerich b a
Rudolf Boehm Institute of Pharmacology and Toxicology, Medical Faculty, University of Leipzig, Härtelstraße 16-18, D-04107 Leipzig, Germany Department of Psychiatry and Psychotherapy, Medical Faculty, University of Leipzig, Semmelweisstraße 10, 04103 Leipzig, Germany c Department of Clinical Immunology, Medical Faculty, University of Leipzig, Johannisallee 30, 04103 Leipzig, Germany b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 October 2012 Received in revised form 26 November 2012 Accepted 11 January 2013
Pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-a) have repeatedly been shown to play a pivotal role in the pathophysiology of depression. Therefore, we tested the possible antidepressant-like effect of the anti-TNF-a drug etanercept in an animal model of chronic mild stress. Male Wistar rats were assigned to a non-restrained and a restrained protocol for 5 weeks. From beginning of the third week the animals were treated either with Ringer solution daily or with etanercept twice a week (0.3 mg/kg, i.p.) instead of Ringer solution (n ¼ 12 each). As reference, imipramine (10 mg/kg, i.p.) was administered in a third restraint group daily. Naïve non-treated non-restrained rats served as healthy controls (n ¼ 12). In the forced swim test (FST) depression-like behaviour induced by restraint was recorded as enhanced immobile time and reduced climbing activity of the vehicle-treated group in comparison to the naïve and the non-restrained vehicle treated group. The treatment with etanercept significantly reduced the depression-like effects resulting in reduced immobile time in the FST and intensified climbing behaviour (p < 0.01, p < 0.05), both similar to the antidepressive-like effect of imipramine (p < 0.01 both). The repeated restraint induced a loss of body weight gain in the Ringertreated group which was not reversed, neither by imipramine nor by etanercept. The antidepressant effects of blocking TNF-a using etanercept may be caused by enhancement of serotonergic or noradrenergic neurotransmission or normalization of stress hormone secretion which has to be substantiated in further studies. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Body weight Depression Etanercept Forced swim test Imipramine Rat
1. Introduction Changes regarding the immune system and specifically the cytokine system e in which tumour necrosis factor-alpha (TNF-a) is a pro-inflammatory key signalling molecule e have been shown to be involved in the development of psychiatric disorders (Himmerich et al., 2009). Especially, TNF-a might contribute to the pathogenesis of depression, because plasma levels of TNF-a and its soluble receptors have been found to be elevated in acutely depressed patients (Himmerich et al., 2008), and experimental stimulation of TNF-a production leads to depression-like emotional and cognitive disturbances in humans (Reichenberg et al., 2001). It has been postulated that the activation of the cytokine system might play a causative role in the depression-related activation of the hypothalamicepituitaryeadrenal (HPA) system (Maes et al., * Corresponding author. Tel.: þ49 341 9713007; fax: þ49 341 9724609. E-mail address: [email protected] (U. Krügel). 1 Both authors equally contributed to this work. 0022-3956/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jpsychires.2013.01.007
1993). Experimental studies applying immune stimulation in humans (Reichenberg et al., 2001, 2002) as well as in rodents (Linthorst and Reul, 1999; Pollak and Yirmiya, 2002; Engler et al., 2011) support the view that inflammatory cytokines are causally involved in the neuroendocrinological and behavioural alterations of patients with depressive disorders. As pro-inflammatory cytokines and serotonergic homeostasis have both been implicated in the pathophysiology of depression, Zhu et al. hypothesized that cytokines might activate neuronal serotonin transporters. This idea underlines the theory of a serotonin deficiency during depression and the pharmacodynamic mechanism of selective serotonin reuptake inhibitors (SSRI) in the treatment of depression, because SSRIs lead to recovery from depression via deactivation of serotonin transporters. Indeed, Zhu et al. found TNF-a stimulated serotonin uptake in both a rat embryonic raphe cell line and in mouse midbrain and striatal synaptosomes. These results provide evidence that pro-inflammatory cytokines can acutely regulate neuronal serotonin transporter activity. A mitogen-activated protein kinase may be involved in
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this mechanism (Zhu et al., 2006; Himmerich and Sheldrick, 2010). Moreover, pro-inflammatory cytokines such as TNFa affect the tryptophan metabolism directly or indirectly by stimulating the enzyme indoleamine 2,3-dioxygenase, which leads to a peripheral depletion of tryptophan (Wichers and Maes, 2002). The aromatic amino acid tryptophan functions as a precursor for the monoamine neurotransmitter serotonin in the brain. In other psychiatric disorders, for example narcolepsy, it has been hypothesized that TNF-a might also contribute to the destruction of neurons (Himmerich et al., 2006, 2009; Lotrich, 2012; Raison and Miller, 2011; Müller and Schwarz, 2007). Cytokines may additionally influence glutamatergic signalling. For example, interleukin (IL)-18 impairs long term potentiation and glutamatergic neurotransmission (Curran and O’Connor, 2001); however, it is not yet clear, whether TNF-a might also act on glutamatergic signalling. It has been postulated on the basis of in-vitro as well as in-vivo studies that the therapeutic action of antidepressants may be partially caused by their influence on cytokine production in addition to their direct effect on monoaminergic neurotransmission. Antidepressants have been, for example, shown to decrease IL-1b (Himmerich et al., 2010b), interferon (IFN)-g (Himmerich et al., 2010a) and TNF-a production (Ignatowski et al., 1996; Nickola et al., 2001). Moreover, TNF-a has been shown to modulate norepinephrine (NE) signalling. For instance, it inhibits the release of NE in hypothalamic and hippocampal slices from rats (Reynolds et al., 2005; Elenkov et al., 1992). However, after chronic administration of the antidepressant drug desipramine, a NE reuptake blocker and active metabolite of imipramine, to rats, TNF-a no longer inhibited but rather facilitated NE release from electrically depolarized hippocampal slices (Reynolds et al., 2005). Therefore, we have to assume that modulation of TNF-a signalling may be one of the mechanisms how antidepressants work. Taken together, pro-inflammatory cytokines like TNF-a might exert their depressogenic effects by an activation of the HPA axis, an activation of neuronal serotonin transporters, the stimulation of the indoleamine 2,3-dioxygenase, by immunologically mediated destruction of neurons, and the release of glutamate. Antidepressants, in turn, may unfold their therapeutic action in part by modulating the cytokine system. Hence, reducing the proinflammatory cytokine production could be a possible mechanism of antidepressants (Himmerich et al., 2010a, 2010b). Moreover, anti-TNF-a-agents such as etanercept, a human TNFa receptor p75-Fc fusion protein, have been shown to exert antidepressant effects in patients with moderate to severe psoriasis (Tyring et al., 2006). Therefore, blocking TNF-a might be a novel strategy against depression which should be tested in an animal paradigm. Within the immune system, TNF-a is a cytokine that is involved in the development and maintenance of the immune response. It is a pivotal cytokine in inflammation, and its critical role has been demonstrated in a number of diseases such as rheumatoid arthritis, ankylosing spondylitis, Crohn’s disease and psoriasis. Etanercept binds to TNF-a, thereby blocking its interaction with cell surface receptors and attenuating its pro-inflammatory effects (Zhou, 2005). Etanercept is approved and generally well-tolerated for the treatment of immune-mediated inflammatory conditions including rheumatoid arthritis, juvenile idiopathic arthritis, psoriasis, psoriatic arthritis and ankylosing spondylitis. In humans, etanercept is absorbed slowly from the site of subcutaneous injection, with time to peak concentration at approximately 48e60 h, and is cleared slowly from the body with a halflife period of 70e100 h. The absolute bioavailability of etanercept is about 58% in healthy subjects following subcutaneous administration (Zhou, 2005).
In the reported experiment we tested the effects of etanercept and imipramine on stress-induced depression-like behaviour in rats. Imipramine, in contrast to etanercept, has a much shorter halflife period compared to etanercept. The half-life of imipramine is between 9 and 20 h (Ciraulo et al., 1988). Therefore, imipramine is taken about one to three times daily for antidepressant treatment in humans, whereas etanercept is injected subcutaneously twice a week. Accordingly, we applied imipramine daily and etanercept twice a week in the current investigation. However, to our knowledge, pharmacokinetic data of etanercept are not available in rats. In rats or mice, repeated restraint stress induces repeated transient elevations of plasma corticosterone (Strausbaugh et al., 1999) and subsequent reduction of glucocorticoid receptor expression (Chiba et al., 2012). This form of chronic mild stress also evokes depression-like behavioural changes accompanied by working memory and learning deficits (Albonetti and Farabollini, 1993; Beck and Luine, 2002; Regenthal et al., 2009) and appears therefore to be an appropriate model for depression. In order to test antidepressant effects, the forced swim test (FST) has a high predictive value for the efficacy of drugs in antidepressant therapies. Originally, the reduction of immobile time in an inescapable water basin in favour of climbing (escape) behaviour and active swimming after acute antidepressant administration was measured. Furthermore, the FST serves as a marker for the behavioural state associated with depression-like symptoms (Kitada et al., 1981; Cryan et al., 2005; Porsolt et al., 1979). We hypothesized, that etanercept given in a subchronic treatment regime could be beneficial for the behavioural outcome in an animal experiment using a restraint stress paradigm which induces depression-like behavioural changes. A dose of etanercept of 0.3 mg/kg body weight was chosen on the base of various studies at rodents in which this compound was effective between 0.15 and 0.8 mg/kg (Inglis et al., 2005; Venegas-Pont et al., 2010; Haugen et al., 2008). 2. Experimental procedures 2.1. Animals Adult male Wistar rats (outbreed, 12e14 weeks old, n ¼ 72, Janvier, Le Genest Saint Isle, France) were housed in standard laboratory cages in groups of four animals for two weeks for acclimatisation. The animals were allowed free access to food and water under a 12-h lightedark schedule (lights on 7:00 a.m.e 7:00 p.m.). The experiments were approved by the Animal Welfare Office (Leipzig, Germany; TVV10/11) according to the German guidelines for the use of animals in biomedical research. All efforts were made to minimize the number of animals used and their suffering. 2.2. Drug application Animals of a body weight of 371 4 g were randomly assigned to six experimental groups (n ¼ 12 each). The first group (1) was group housed and served as healthy “naïve” control without restraint or treatment. The animals of the other groups were single housed: (2) no restraint, treatment with Ringer solution (2 ml/kg i.p.) daily, (3) no restraint, treatment with Etanercept (0.3 mg/kg i.p.) instead of Ringer twice a week, (4) restraint, treatment with Ringer solution (2 ml/kg i.p.) daily, (5) restraint, treatment with imipramine (10 mg/kg i.p.) daily, (6) restraint, treatment with Etanercept (0.3 mg/kg i.p.) instead of Ringer twice a week. Etanercept (EnbrelÒ) (Pfizer Pharma GmbH, Berlin, Germany) was suspended according to the manufactures instructions and further diluted in Ringer solution. Etanercept and imipramine
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hydrochloride (SigmaeAldrich, Taufkirchen, Germany) were given in a dosage and volume adjusted to the body weight. 2.3. Restrained stress protocol The animals exposed to chronic mild stress by repeated restraint were placed into perforated plexi glass tubes (6.5 cm inner diameter 20 cm length) for four hours per day for 35 subsequent days (modified from Regenthal et al., 2009) (Fig. 1). The restraint allowed normal breathing and limited movements of head and limbs. The animals were restrained unforeseeable at various day times and places out of their home cages daily. After about three days all animals stepped into the tubes voluntarily. Beginning from the day 14 for 21 subsequent days about one hour after each restraint, the animals were treated as indicated above with vehicle, imipramine or etanercept. At day 35, 24 h after the last drug administration, the FST was performed with all animals including the naïve “healthy” control group. During the experimental period, the body weight of all animals was recorded twice a week. 2.4. Forced swim test The behavioural testing was performed as previously described (Regenthal et al., 2009; Porsolt et al., 1977). Briefly, in a preceding trial the rats were placed in a cylindrical basin (0.5 m height 0.25 m diameter) filled with water (25 1 C) up to a height of 0.35 m for 15 min. After 24 h, the experimental trial with an observation period of seven minutes followed. The time of clearly defined episodes of immobility (floating and movements to keep the head out of the water only) and of climbing at the basin wall were registered by an experimenter blind to the treatments. After the swim sessions the animals were taken out and gently dried with towels before they were placed back to their home cages. 2.5. Data analysis Statistical analysis was performed using SigmaStat (Version 4.0; integrated with SigmaPlot 11; Systat Software Inc., San Jose Cal. U.S.A.). Data were analysed as appropriate using two way ANOVA (handling treatment as factors) or one way ANOVA followed by Student-NewmaneKeuls pair wise multiple comparison. The normal body weight gain was tested by one way repeated measures ANOVA (time as factor) with post hoc Student-NewmaneKeuls pair wise multiple comparison. A value of p < 0.05 was considered to be significant. 3. Results 3.1. Forced swim test Immobility of rodents in the FST is taken as a measure of depressive-like behaviour which can be abolished by treatment
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with antidepressants (Porsolt et al., 1977). One way ANOVA revealed a main effect of the repeated restraint on immobility [F(2, 32) ¼ 6.30, p ¼ 0.005] (Fig. 2A) and climbing activity [F(2, 32) ¼ 12.78, p < 0.001] (Fig. 2B), indicating the induction of depressive-like behavioural responses. As expected, post hoc analysis also indicates that single housed and daily injected animals tend towards higher immobility (p ¼ 0.16) and showed lower climbing activity (p < 0.001) than naïve animals. As the main result of this study a significant overall effect on immobility by treatment with etanercept compared to that with Ringer solution was found [two way ANOVA: for handling F(1,46) ¼ 0.17, p ¼ 0.72; for treatment F(1,46) ¼ 6.37, p ¼ 0.015 and interaction of both F(1,46) ¼ 16.32, p ¼ 0.001]. Similarly, a significant overall effect on climbing by treatment with etanercept was found [two way ANOVA: for handling F(1,46) ¼ 0.13, p ¼ 0.72; for treatment F(1,46) ¼ 4.37, p ¼ 0.04; and interaction of both F(1,46) ¼ 1.89, p ¼ 0.18]. The post hoc analysis shows that etanercept significantly reduced the immobility of the restrained but not that of the non-restrained animals (p ¼ 0.004). Etanercept also evoked a considerable enhancement of climbing activity in the repeatedly stressed animals (p ¼ 0.02). The effects of etanercept on the restraint-induced behaviour were compared with those of imipramine [F(2,33) ¼ 15.06, p < 0.001 for immobility and F(2,33) ¼ 5.64, p ¼ 0.008 for climbing]. One way ANOVA analysis of antidepressive-like treatment revealed that imipramine reduced the restraint-induced effects on immobility and escape attempts (p < 0.001 and p ¼ 0.006). Furthermore, the effects of imipramine and etanercept on immobility and climbing did not differ. 3.2. Body weight At the beginning of the experiment the mean body weight of the experimental groups did not differ. Over the period of the experimental protocol naïve rats showed a normal weight gain [one way repeated measure ANOVA: F(2,35) ¼ 55.08, p < 0.001]. The introduction of the restraint protocol suppressed it within 2 and 5 weeks (p < 0.001 both). No significant influence on the body weight by single housing and daily injections was detectable albeit the climbing behaviour was affected. As expected, the two way analysis of body weight for the factors handling and treatment with etanercept revealed a significant overall effect of restraint [F(1,46) ¼ 43.55, p < 0.001] and a post hoc determined reduction of body weight in the restrained Ringer-treated group compared to the non-restrained one. No overall effects were found for treatment with etanercept [F(1,46) ¼ 2.16, p ¼ 0.149] and for the interaction of restraint with treatment [F(1,46) ¼ 0.28, p ¼ 0.598]. The main observation by post hoc analysis was that etanercept did not abolish the restraint-induced reduction of body weight (p ¼ 0.51). Further one way analysis revealed that, compared to naïve rats, the body weight of non-restrained etanercept-treated animals was reduced by trend only (p ¼ 0.106). The reference imipramine even caused a decline of body weight compared to both, the Ringer- and the etanercept-treated restrained groups (p ¼ 0.002; p ¼ 0.004).
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4. Discussion
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4.1. TNF-a blockers as a possibly novel class of antidepressants 5 weeks
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Fig. 1. Schematic presentation of the time course of the experimental protocol. Note that a non-restrained untreated “naïve” group as healthy control runs in parallel.
Using a rat model of depression induced by repeated restraint stress we could show that the treatment with etanercept reduces immobile time in the FST and induces climbing behaviour compared to vehicle-treated animals. These effects were similar to the observations after imipramine treatment. Further, etanercept did
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Fig. 2. Restraint-induced effects on behaviour in the forced swim test (FST) are influenced by etanercept and imipramine. Effects on immobility (A) and climbing (B) after three weeks of additional treatment with either Ringer solution (Ri), etanercept (Eta; 0.3 mg/kg every third day, i.p.) or imipramine (Imi; 10 mg/kg daily, i.p.) following two weeks of restraint alone are shown. Data are given as the mean S.E.M. (n ¼ 12 each), *p < 0.05, **p < 0.01, ***p < 0.001.
not scale down the restraint-induced weight changes, similar to imipramine which potentiated body weight loss. Our results demonstrate that blocking of TNF-a by etanercept has antidepressant-like effects, supporting the repeatedly reported involvement of pro-inflammatory cytokines in the pathophysiology of depression (Himmerich et al., 2008; Müller and Schwarz, 2007; Reichenberg et al., 2001; Lotrich, 2012; Raison and Miller, 2011). That treatment with etanercept diminishes depression-like behaviour of rats also confirms observations in patients with psoriasis who showed improvements in symptoms of depression which were only weekly correlated with objective measures of skin clearance or joint pain (Tyring et al., 2006). The effects of etanercept in the present study were similar to that of intracerebroventricularly administered anti-TNF-a antibody shown to reverse depression-like behaviour induced by TNFa in mice (Kaster et al., 2012). Furthermore, these authors found that TNF-a-induced depression-like behaviour was prevented by the classic antidepressant imipramine. This result is in line with
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Fig. 3. Restraint- and treatment-induced effects on the body weight of rats. Body weights are shown before, after two weeks of repeated restraint (untreated) and after three subsequent weeks with additional treatment either with Ringer solution (Ri), etanercept (Eta; 0.3 mg/kg every third day, i.p.) or imipramine (Imi; 10 mg/kg daily, i.p.). Data are given as the mean S.E.M. (n ¼ 12 each), *p < 0.05, **p < 0.01, ***p < 0.001.
the suggested reduced production of pro-inflammatory cytokines, e.g. of TNF-a, by current antidepressants in rats and humans (Reynolds et al., 2005; Himmerich et al., 2010a, 2010b). Specifically the relatively strong effects of etanercept seen in the FST which were comparable to imipramine may be another brick in the cytokine hypothesis of depression and give a hint that TNFa antagonism may become a future antidepressant treatment strategy. However, from this animal study, one can not conclude that TNF-a antagonism would reverse the emotional and cognitive disturbances seen in humans after induction of the TNF-a system (Reichenberg et al., 2001) which are typical symptoms of depressed patients. Therefore, these results should be interpreted with caution regarding their relevance for the treatment of depression in humans. Notably, etanercept induced antidepressive-like behaviour in favour to augmented escape attempts in the FST. A similar response was found for imipramine-treated restrained rats. Increased climbing behaviour is a feature of antidepressive drugs preferentially modulating noradrenergic neurotransmission, e.g. reboxetine, but not of such enhancing serotonergic activity, e.g. citalopram or sertraline (Page et al., 2003; Mikail et al., 2012; own observations). The observed climbing behaviour during the treatment with the TNF-a blocker etanercept and imipramine is in agreement with data of Reynolds et al. (2005) demonstrating that TNF-a itself reduces noradrenergic neurotransmission in hippocampal slices of rats. This might be at least one possible pathway for the antidepressant-like action of etanercept in addition to other pathways derived from the physiological functions of TNF-a (for review see Santello and Volterra, 2012). As etanercept has beneficial effects in stress-induced depression, TNF-a blockers or similar cytokine-blocking agents could be alternative antidepressive agents with new mechanisms of action. Until now only antidepressants targeting on monoamines like imipramine are commonly used in the psychopharmacological antidepressant therapy (Covington et al., 2010), even though fewer than 35 percent of patients achieve remission with the first applied antidepressant drug (Trivedi et al., 2008). Thus the development of new antidepressant drug targets and new antidepressant drugs is an important demand in psychopharmacological research and development. In this regard, our study might contribute to the development of novel antidepressants on the basis of cytokine antagonism.
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4.2. Limitations of current TNF-a blockers as antidepressants Although stressful life events only partly contribute to the incidence of depression in humans, and a multidimensional causality of depression is widely accepted, chronic mild stress is a model with a high validity to induce antidepressant-reversible depressive-like effects in rodents (Willner, 2005). This is supported by the behavioural outcome of animals induced by repeated stressors (isolated housing, injections, restraint) and by the effects of the accompanied antidepressant treatment found in the present study as well as by activation of the hypothalamicepituitarye adrenal axis and of inflammatory mechanisms (Strausbaugh et al., 1999). However, an animal experiment can only be a first step for the development of a new antidepressant drug, because data derived from rodents can not easily transferred for human therapies. For example, imipramine and anti-TNF-a drugs are known to induce a weight gain in humans (Zimmermann et al., 2003; Brown et al., 2012), but to provoke weight loss in rats caused by reduction of food intake as it was also the case in our study (Hughes and Pither, 1987; Mogensen et al., 1994). As to be seen in Fig. 3, non-restraint rats gained weight over the whole period of the investigation, but rats undergoing the stress paradigm stopped gaining weight after the restraint started. As a side effect, noradrenergic mechanisms also may contribute to the lack of body weight gain in restrained imipramine-treated rats since the selective NE reuptake inhibitor, LY368975, reduced food intake in rats (Gehlert et al., 1998). Though the underlying mechanism of weight stagnancy in the restrained etanercept-treated animals remains unclear, obviously the health status has an important impact on the weight development as etanercept-treated non-restrained animals showed only a slight tendency of weight reduction. In this regard it has to be acknowledged that TNF-a is able to inhibit the release of NE in brain slices from rats (Elenkov et al., 1992; Reynolds et al., 2005) and anti-TNF-a medication might therefore lead to an increase in NE signalling and in less weight gain compared to placebo-treated animals. Despite of the limitations of animal models to measure “mental” disturbances as well as to transfer doseeresponse data, it seems to be promising to test anti-TNF-a agents against depression. An advantage of TNF-a blockers over totally new medicinal products is that they are already in use for rheumatoid arthritis, ankylosing spondylitis, psoriasis, psoriatic arthritis, uveitis and inflammatory bowel disease (Sfikakis and Tsokos, 2011). However, when treating depression with anti-TNF-a drugs, safety issues have also to be addressed such as reactivation of tuberculosis and exacerbation of pre-existing multiple sclerosis (Gregory et al., 2012). The most common adverse effect of etanercept treatment is injection site reaction, which is generally self-limiting and often does not require treatment. Therapy with etanercept may be associated with an increased risk for infection, the development of malignancy, demyelinating disease and congestive heart failure (Kerensky et al., 2012). Therefore, contraindications are a medical history of serious infections in the last 6 months, tuberculosis, pneumocystis carinii pneumonia, congestive heart failure, a malignancy or demyelinating disease (Miyasaka et al., 2006). In this respect, the next generation of non-antibody anti-TNFa drugs including progranulin or the soluble preligand-binding assembly domain (Sfikakis and Tsokos, 2011) might be potential antidepressant anti-TNF-a drugs without compromising the protective role of TNF-a in host defence and autoimmunity. After the results of this preliminary investigation have been promising, further animal studies including behavioural experiments, e.g. on drive or anxiety and the analysis of hormones of the HPA axis, e.g. glucocorticoids, have to be done to underline the
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validity of this stress paradigm and to investigate the effect of etanercept on stress hormones. 4.3. Conclusion In summary, etanercept given in a subchronic treatment regime was beneficial for the behavioural outcome in an animal model of restraint stress which induced depression-like behavioural changes. This result implies that TNF-a blockers might become a possibly novel class of antidepressants. Role of funding source This work was supported by the Claussen-Simon-Foundation. The authors were fully responsible for the idea of the study as well as the experiments, analysis, interpretation of data and writing the manuscript. Contributors All contributors to the aforementioned text are cited above. Conflict of interest Dr. Himmerich received speaker honoraria from AstraZeneca, Lilly and Servier, consulting fees from Bristol-Myers Squibb, and chemical substances for study support from AstraZeneca, Novartis and Wyeth. The other authors have no conflict of interest to report. Acknowledgement The authors would like to acknowledge the excellent technical assistance of Mrs. A.-K. Krause, Mrs. K. Bauer and Mr. L. Feige. References Albonetti ME, Farabollini F. Effects of single and repeated restraint on the social behavior of male rats. Physiology & Behavior 1993;53:937e42. Beck KD, Luine VN. Sex differences in behavioral and neurochemical profiles after chronic stress: role of housing conditions. Physiology & Behavior 2002;75: 661e73. Brown RA, Spina D, Butt S, Summers GD. Long-term effects of anti-tumour necrosis factor therapy on weight in patients with rheumatoid arthritis. Clinical Rheumatology 2012;31:455e61. Chiba S, Numakawa T, Ninomiya M, Richards MC, Wakabayashi C, Kunugi H. Chronic restraint stress causes anxiety- and depression-like behaviors, downregulates glucocorticoid receptor expression, and attenuates glutamate release induced by brain-derived neurotrophic factor in the prefrontal cortex. Progress in Neuro-Psychopharmacology & Biological Psychiatry 2012;39:112e9. Ciraulo DA, Barnhill JG, Jaffe JH. Clinical pharmacokinetics of imipramine and desipramine in alcoholics and normal volunteers. Clinical Pharmacology & Therapeutics 1988;43:509e18. Covington HEI, Vialou V, Nestler EJ. From synapse to nucleus: novel targets for treating depression. Neuropharmacology 2010;58:683e93. Cryan JF, Valentino RJ, Lucki I. Assessing substrates underlying the behavioral effects of antidepressants using the modified rat forced swimming test. Neuroscience and Biobehavioural Reviews 2005;29:547e69. Curran B, O’Connor JJ. The pro-inflammatory cytokine interleukin-18 impairs longterm potentiation and NMDA receptor-mediated transmission in the rat hippocampus in vitro. Neuroscience 2001;108:83e90. Elenkov IJ, Kovacs K, Duda E, Stark E, Vizi ES. Presynaptic inhibitory effect of TNF-alpha on the release of noradrenaline in isolated median eminence. Journal of Neuroimmunology 1992;41:117e20. Engler H, Doenlen R, Engler A, Riether C, Prager G, Niemi MB, et al. Acute amygdaloid response to systemic inflammation. Brain, Behavior, and Immunity 2011; 25:1384e92. Gehlert DR, Dreshfield L, Tinsley F, Benvenga MJ, Gleason S, Fuller RW, et al. The selective norepinephrine reuptake inhibitor, LY368975, reduces food consumption in animal models of feeding. Journal of Pharmacology and Experimental Therapeutics 1998;287:122e7. Gregory AP, Dendrou CA, Attfield KE, Haghikia A, Xifara DK, Butter F, et al. TNF receptor 1 genetic risk mirrors outcome of anti-TNF therapy in multiple sclerosis. Nature 2012;488:508e11.
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Haugen E, Tang MS, Isic A, Andersson B, Fu M. TNFalpha antagonist upregulates interleukin-6 in rats with hypertensive heart failure. International Journal of Cardiology 2008;130:64e8. Himmerich H, Beitinger PA, Fulda S, Wehrle R, Linseisen J, Wolfram G, et al. Plasma levels of tumor necrosis factor alpha and soluble tumor necrosis factor receptors in patients with narcolepsy. Archives of Internal Medicine 2006;166:1739e43. Himmerich H, Berthold-Losleben M, Pollmacher T. The relevance of the TNF-alpha system in psychiatric disorders. Fortschritte der Neurologie e Psychiatrie 2009; 77:334e45. Himmerich H, Fulda S, Linseisen J, Seiler H, Wolfram G, Himmerich S, et al. Depression, comorbidities and the TNF-alpha system. European Psychiatry 2008;23:421e9. Himmerich H, Fulda S, Sheldrick AJ, Plumakers B, Rink L. IFN-gamma reduction by tricyclic antidepressants. The International Journal of Psychiatry in Medicine 2010a;40:413e24. Himmerich H, Milenovic S, Fulda S, Plumakers B, Sheldrick AJ, Michel TM, et al. Regulatory T cells increased while IL-1beta decreased during antidepressant therapy. Journal of Psychiatric Research 2010b;44:1052e7. Himmerich H, Sheldrick AJ. TNF-alpha and ghrelin: opposite effects on immune system, metabolism and mental health. Protein and Peptides Letters 2010;17: 186e96. Hughes RN, Pither JM. Chronic imipramine effects on exploratory behavior in rats. Pharmacology and Biochemical Behaviour 1987;27:359e62. Ignatowski TA, Chou RC, Spengler RN. Changes in noradrenergic sensitivity to tumor necrosis factor-alpha in brains of rats administered clonidine. Journal of Neuroimmunology 1996;70:55e63. Inglis JJ, Nissim A, Lees DM, Hunt SP, Chernajovsky Y, Kidd BL. The differential contribution of tumour necrosis factor to thermal and mechanical hyperalgesia during chronic inflammation. Arthritis Research and Therapy 2005;7:R807e16. Kaster MP, Gadotti VM, Calixto JB, Santos AR, Rodrigues AL. Depressive-like behavior induced by tumor necrosis factor-a in mice. Neuropharmacology 2012;62:419e26. Kerensky TA, Gottlieb AB, Yaniv S, Au SC. Etanercept: efficacy and safety for approved indications. Expert Opinion on Drug Safety 2012;11:121e39. Kitada Y, Miyauchi T, Satoh A, Satoh S. Effects of antidepressants in the rat forced swimming test. European Journal of Pharmacology 1981;72:145e52. Linthorst AC, Reul JM. Inflammation and brain function under basal conditions and during long-term elevation of brain corticotropin-releasing hormone levels. Advances in Experimental Medicine and Biology 1999;461:129e52. Lotrich F. Inflammatory cytokines, growth factors, and depression. Current Pharmaceutical Design 2012;18:5920e35. Maes M, Scharpe S, Meltzer HY, Bosmans E, Suy E, Calabrese J, et al. Relationships between interleukin-6 activity, acute phase proteins, and function of the hypothalamicepituitaryeadrenal axis in severe depression. Psychiatry Research 1993;49:11e27. Mikail HG, Dalla C, Kokras N, Kafetzopoulos V, Papadopoulou-Daifoti Z. Sertraline behavioral response associates closer and dose-dependently with cortical rather than hippocampal serotonergic activity in the rat forced swim stress. Physiology & Behavior 2012;107:201e6. Miyasaka N, Takeuchi T, Eguchi K. Guidelines for the proper use of etanercept in Japan. Modern Rheumatology 2006;16:63e7. Mogensen J, Pedersen TK, Holm S. Effects of chronic imipramine on exploration, locomotion, and food/water intake in rats. Pharmacology and Biochemical Behaviour 1994;47:427e35. Müller N, Schwarz MJ. Immunological aspects of depressive disorders. Nervenarzt 2007;78:1261e73. Nickola TJ, Ignatowski TA, Reynolds JL, Spengler RN. Antidepressant drug-induced alterations in neuron-localized tumor necrosis factor-alpha mRNA and a2adrenergic receptor sensitivity. Journal of Pharmacology and Experimental Therapeutics 2001;297:680e7.
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Journal of Psychiatric Research journal homepage: www.elsevier.com/locate/psychires
Antidepressant effects of TNF-a blockade in an animal model of depression Ute Krügel a, *,1, Johannes Fischer a,1, Susanne Radicke a, Ulrich Sack c, Hubertus Himmerich b a
Rudolf Boehm Institute of Pharmacology and Toxicology, Medical Faculty, University of Leipzig, Härtelstraße 16-18, D-04107 Leipzig, Germany Department of Psychiatry and Psychotherapy, Medical Faculty, University of Leipzig, Semmelweisstraße 10, 04103 Leipzig, Germany c Department of Clinical Immunology, Medical Faculty, University of Leipzig, Johannisallee 30, 04103 Leipzig, Germany b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 October 2012 Received in revised form 26 November 2012 Accepted 11 January 2013
Pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-a) have repeatedly been shown to play a pivotal role in the pathophysiology of depression. Therefore, we tested the possible antidepressant-like effect of the anti-TNF-a drug etanercept in an animal model of chronic mild stress. Male Wistar rats were assigned to a non-restrained and a restrained protocol for 5 weeks. From beginning of the third week the animals were treated either with Ringer solution daily or with etanercept twice a week (0.3 mg/kg, i.p.) instead of Ringer solution (n ¼ 12 each). As reference, imipramine (10 mg/kg, i.p.) was administered in a third restraint group daily. Naïve non-treated non-restrained rats served as healthy controls (n ¼ 12). In the forced swim test (FST) depression-like behaviour induced by restraint was recorded as enhanced immobile time and reduced climbing activity of the vehicle-treated group in comparison to the naïve and the non-restrained vehicle treated group. The treatment with etanercept significantly reduced the depression-like effects resulting in reduced immobile time in the FST and intensified climbing behaviour (p < 0.01, p < 0.05), both similar to the antidepressive-like effect of imipramine (p < 0.01 both). The repeated restraint induced a loss of body weight gain in the Ringertreated group which was not reversed, neither by imipramine nor by etanercept. The antidepressant effects of blocking TNF-a using etanercept may be caused by enhancement of serotonergic or noradrenergic neurotransmission or normalization of stress hormone secretion which has to be substantiated in further studies. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Body weight Depression Etanercept Forced swim test Imipramine Rat
1. Introduction Changes regarding the immune system and specifically the cytokine system e in which tumour necrosis factor-alpha (TNF-a) is a pro-inflammatory key signalling molecule e have been shown to be involved in the development of psychiatric disorders (Himmerich et al., 2009). Especially, TNF-a might contribute to the pathogenesis of depression, because plasma levels of TNF-a and its soluble receptors have been found to be elevated in acutely depressed patients (Himmerich et al., 2008), and experimental stimulation of TNF-a production leads to depression-like emotional and cognitive disturbances in humans (Reichenberg et al., 2001). It has been postulated that the activation of the cytokine system might play a causative role in the depression-related activation of the hypothalamicepituitaryeadrenal (HPA) system (Maes et al., * Corresponding author. Tel.: þ49 341 9713007; fax: þ49 341 9724609. E-mail address: [email protected] (U. Krügel). 1 Both authors equally contributed to this work. 0022-3956/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jpsychires.2013.01.007
1993). Experimental studies applying immune stimulation in humans (Reichenberg et al., 2001, 2002) as well as in rodents (Linthorst and Reul, 1999; Pollak and Yirmiya, 2002; Engler et al., 2011) support the view that inflammatory cytokines are causally involved in the neuroendocrinological and behavioural alterations of patients with depressive disorders. As pro-inflammatory cytokines and serotonergic homeostasis have both been implicated in the pathophysiology of depression, Zhu et al. hypothesized that cytokines might activate neuronal serotonin transporters. This idea underlines the theory of a serotonin deficiency during depression and the pharmacodynamic mechanism of selective serotonin reuptake inhibitors (SSRI) in the treatment of depression, because SSRIs lead to recovery from depression via deactivation of serotonin transporters. Indeed, Zhu et al. found TNF-a stimulated serotonin uptake in both a rat embryonic raphe cell line and in mouse midbrain and striatal synaptosomes. These results provide evidence that pro-inflammatory cytokines can acutely regulate neuronal serotonin transporter activity. A mitogen-activated protein kinase may be involved in
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this mechanism (Zhu et al., 2006; Himmerich and Sheldrick, 2010). Moreover, pro-inflammatory cytokines such as TNFa affect the tryptophan metabolism directly or indirectly by stimulating the enzyme indoleamine 2,3-dioxygenase, which leads to a peripheral depletion of tryptophan (Wichers and Maes, 2002). The aromatic amino acid tryptophan functions as a precursor for the monoamine neurotransmitter serotonin in the brain. In other psychiatric disorders, for example narcolepsy, it has been hypothesized that TNF-a might also contribute to the destruction of neurons (Himmerich et al., 2006, 2009; Lotrich, 2012; Raison and Miller, 2011; Müller and Schwarz, 2007). Cytokines may additionally influence glutamatergic signalling. For example, interleukin (IL)-18 impairs long term potentiation and glutamatergic neurotransmission (Curran and O’Connor, 2001); however, it is not yet clear, whether TNF-a might also act on glutamatergic signalling. It has been postulated on the basis of in-vitro as well as in-vivo studies that the therapeutic action of antidepressants may be partially caused by their influence on cytokine production in addition to their direct effect on monoaminergic neurotransmission. Antidepressants have been, for example, shown to decrease IL-1b (Himmerich et al., 2010b), interferon (IFN)-g (Himmerich et al., 2010a) and TNF-a production (Ignatowski et al., 1996; Nickola et al., 2001). Moreover, TNF-a has been shown to modulate norepinephrine (NE) signalling. For instance, it inhibits the release of NE in hypothalamic and hippocampal slices from rats (Reynolds et al., 2005; Elenkov et al., 1992). However, after chronic administration of the antidepressant drug desipramine, a NE reuptake blocker and active metabolite of imipramine, to rats, TNF-a no longer inhibited but rather facilitated NE release from electrically depolarized hippocampal slices (Reynolds et al., 2005). Therefore, we have to assume that modulation of TNF-a signalling may be one of the mechanisms how antidepressants work. Taken together, pro-inflammatory cytokines like TNF-a might exert their depressogenic effects by an activation of the HPA axis, an activation of neuronal serotonin transporters, the stimulation of the indoleamine 2,3-dioxygenase, by immunologically mediated destruction of neurons, and the release of glutamate. Antidepressants, in turn, may unfold their therapeutic action in part by modulating the cytokine system. Hence, reducing the proinflammatory cytokine production could be a possible mechanism of antidepressants (Himmerich et al., 2010a, 2010b). Moreover, anti-TNF-a-agents such as etanercept, a human TNFa receptor p75-Fc fusion protein, have been shown to exert antidepressant effects in patients with moderate to severe psoriasis (Tyring et al., 2006). Therefore, blocking TNF-a might be a novel strategy against depression which should be tested in an animal paradigm. Within the immune system, TNF-a is a cytokine that is involved in the development and maintenance of the immune response. It is a pivotal cytokine in inflammation, and its critical role has been demonstrated in a number of diseases such as rheumatoid arthritis, ankylosing spondylitis, Crohn’s disease and psoriasis. Etanercept binds to TNF-a, thereby blocking its interaction with cell surface receptors and attenuating its pro-inflammatory effects (Zhou, 2005). Etanercept is approved and generally well-tolerated for the treatment of immune-mediated inflammatory conditions including rheumatoid arthritis, juvenile idiopathic arthritis, psoriasis, psoriatic arthritis and ankylosing spondylitis. In humans, etanercept is absorbed slowly from the site of subcutaneous injection, with time to peak concentration at approximately 48e60 h, and is cleared slowly from the body with a halflife period of 70e100 h. The absolute bioavailability of etanercept is about 58% in healthy subjects following subcutaneous administration (Zhou, 2005).
In the reported experiment we tested the effects of etanercept and imipramine on stress-induced depression-like behaviour in rats. Imipramine, in contrast to etanercept, has a much shorter halflife period compared to etanercept. The half-life of imipramine is between 9 and 20 h (Ciraulo et al., 1988). Therefore, imipramine is taken about one to three times daily for antidepressant treatment in humans, whereas etanercept is injected subcutaneously twice a week. Accordingly, we applied imipramine daily and etanercept twice a week in the current investigation. However, to our knowledge, pharmacokinetic data of etanercept are not available in rats. In rats or mice, repeated restraint stress induces repeated transient elevations of plasma corticosterone (Strausbaugh et al., 1999) and subsequent reduction of glucocorticoid receptor expression (Chiba et al., 2012). This form of chronic mild stress also evokes depression-like behavioural changes accompanied by working memory and learning deficits (Albonetti and Farabollini, 1993; Beck and Luine, 2002; Regenthal et al., 2009) and appears therefore to be an appropriate model for depression. In order to test antidepressant effects, the forced swim test (FST) has a high predictive value for the efficacy of drugs in antidepressant therapies. Originally, the reduction of immobile time in an inescapable water basin in favour of climbing (escape) behaviour and active swimming after acute antidepressant administration was measured. Furthermore, the FST serves as a marker for the behavioural state associated with depression-like symptoms (Kitada et al., 1981; Cryan et al., 2005; Porsolt et al., 1979). We hypothesized, that etanercept given in a subchronic treatment regime could be beneficial for the behavioural outcome in an animal experiment using a restraint stress paradigm which induces depression-like behavioural changes. A dose of etanercept of 0.3 mg/kg body weight was chosen on the base of various studies at rodents in which this compound was effective between 0.15 and 0.8 mg/kg (Inglis et al., 2005; Venegas-Pont et al., 2010; Haugen et al., 2008). 2. Experimental procedures 2.1. Animals Adult male Wistar rats (outbreed, 12e14 weeks old, n ¼ 72, Janvier, Le Genest Saint Isle, France) were housed in standard laboratory cages in groups of four animals for two weeks for acclimatisation. The animals were allowed free access to food and water under a 12-h lightedark schedule (lights on 7:00 a.m.e 7:00 p.m.). The experiments were approved by the Animal Welfare Office (Leipzig, Germany; TVV10/11) according to the German guidelines for the use of animals in biomedical research. All efforts were made to minimize the number of animals used and their suffering. 2.2. Drug application Animals of a body weight of 371 4 g were randomly assigned to six experimental groups (n ¼ 12 each). The first group (1) was group housed and served as healthy “naïve” control without restraint or treatment. The animals of the other groups were single housed: (2) no restraint, treatment with Ringer solution (2 ml/kg i.p.) daily, (3) no restraint, treatment with Etanercept (0.3 mg/kg i.p.) instead of Ringer twice a week, (4) restraint, treatment with Ringer solution (2 ml/kg i.p.) daily, (5) restraint, treatment with imipramine (10 mg/kg i.p.) daily, (6) restraint, treatment with Etanercept (0.3 mg/kg i.p.) instead of Ringer twice a week. Etanercept (EnbrelÒ) (Pfizer Pharma GmbH, Berlin, Germany) was suspended according to the manufactures instructions and further diluted in Ringer solution. Etanercept and imipramine
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hydrochloride (SigmaeAldrich, Taufkirchen, Germany) were given in a dosage and volume adjusted to the body weight. 2.3. Restrained stress protocol The animals exposed to chronic mild stress by repeated restraint were placed into perforated plexi glass tubes (6.5 cm inner diameter 20 cm length) for four hours per day for 35 subsequent days (modified from Regenthal et al., 2009) (Fig. 1). The restraint allowed normal breathing and limited movements of head and limbs. The animals were restrained unforeseeable at various day times and places out of their home cages daily. After about three days all animals stepped into the tubes voluntarily. Beginning from the day 14 for 21 subsequent days about one hour after each restraint, the animals were treated as indicated above with vehicle, imipramine or etanercept. At day 35, 24 h after the last drug administration, the FST was performed with all animals including the naïve “healthy” control group. During the experimental period, the body weight of all animals was recorded twice a week. 2.4. Forced swim test The behavioural testing was performed as previously described (Regenthal et al., 2009; Porsolt et al., 1977). Briefly, in a preceding trial the rats were placed in a cylindrical basin (0.5 m height 0.25 m diameter) filled with water (25 1 C) up to a height of 0.35 m for 15 min. After 24 h, the experimental trial with an observation period of seven minutes followed. The time of clearly defined episodes of immobility (floating and movements to keep the head out of the water only) and of climbing at the basin wall were registered by an experimenter blind to the treatments. After the swim sessions the animals were taken out and gently dried with towels before they were placed back to their home cages. 2.5. Data analysis Statistical analysis was performed using SigmaStat (Version 4.0; integrated with SigmaPlot 11; Systat Software Inc., San Jose Cal. U.S.A.). Data were analysed as appropriate using two way ANOVA (handling treatment as factors) or one way ANOVA followed by Student-NewmaneKeuls pair wise multiple comparison. The normal body weight gain was tested by one way repeated measures ANOVA (time as factor) with post hoc Student-NewmaneKeuls pair wise multiple comparison. A value of p < 0.05 was considered to be significant. 3. Results 3.1. Forced swim test Immobility of rodents in the FST is taken as a measure of depressive-like behaviour which can be abolished by treatment
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with antidepressants (Porsolt et al., 1977). One way ANOVA revealed a main effect of the repeated restraint on immobility [F(2, 32) ¼ 6.30, p ¼ 0.005] (Fig. 2A) and climbing activity [F(2, 32) ¼ 12.78, p < 0.001] (Fig. 2B), indicating the induction of depressive-like behavioural responses. As expected, post hoc analysis also indicates that single housed and daily injected animals tend towards higher immobility (p ¼ 0.16) and showed lower climbing activity (p < 0.001) than naïve animals. As the main result of this study a significant overall effect on immobility by treatment with etanercept compared to that with Ringer solution was found [two way ANOVA: for handling F(1,46) ¼ 0.17, p ¼ 0.72; for treatment F(1,46) ¼ 6.37, p ¼ 0.015 and interaction of both F(1,46) ¼ 16.32, p ¼ 0.001]. Similarly, a significant overall effect on climbing by treatment with etanercept was found [two way ANOVA: for handling F(1,46) ¼ 0.13, p ¼ 0.72; for treatment F(1,46) ¼ 4.37, p ¼ 0.04; and interaction of both F(1,46) ¼ 1.89, p ¼ 0.18]. The post hoc analysis shows that etanercept significantly reduced the immobility of the restrained but not that of the non-restrained animals (p ¼ 0.004). Etanercept also evoked a considerable enhancement of climbing activity in the repeatedly stressed animals (p ¼ 0.02). The effects of etanercept on the restraint-induced behaviour were compared with those of imipramine [F(2,33) ¼ 15.06, p < 0.001 for immobility and F(2,33) ¼ 5.64, p ¼ 0.008 for climbing]. One way ANOVA analysis of antidepressive-like treatment revealed that imipramine reduced the restraint-induced effects on immobility and escape attempts (p < 0.001 and p ¼ 0.006). Furthermore, the effects of imipramine and etanercept on immobility and climbing did not differ. 3.2. Body weight At the beginning of the experiment the mean body weight of the experimental groups did not differ. Over the period of the experimental protocol naïve rats showed a normal weight gain [one way repeated measure ANOVA: F(2,35) ¼ 55.08, p < 0.001]. The introduction of the restraint protocol suppressed it within 2 and 5 weeks (p < 0.001 both). No significant influence on the body weight by single housing and daily injections was detectable albeit the climbing behaviour was affected. As expected, the two way analysis of body weight for the factors handling and treatment with etanercept revealed a significant overall effect of restraint [F(1,46) ¼ 43.55, p < 0.001] and a post hoc determined reduction of body weight in the restrained Ringer-treated group compared to the non-restrained one. No overall effects were found for treatment with etanercept [F(1,46) ¼ 2.16, p ¼ 0.149] and for the interaction of restraint with treatment [F(1,46) ¼ 0.28, p ¼ 0.598]. The main observation by post hoc analysis was that etanercept did not abolish the restraint-induced reduction of body weight (p ¼ 0.51). Further one way analysis revealed that, compared to naïve rats, the body weight of non-restrained etanercept-treated animals was reduced by trend only (p ¼ 0.106). The reference imipramine even caused a decline of body weight compared to both, the Ringer- and the etanercept-treated restrained groups (p ¼ 0.002; p ¼ 0.004).
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4. Discussion
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Fig. 1. Schematic presentation of the time course of the experimental protocol. Note that a non-restrained untreated “naïve” group as healthy control runs in parallel.
Using a rat model of depression induced by repeated restraint stress we could show that the treatment with etanercept reduces immobile time in the FST and induces climbing behaviour compared to vehicle-treated animals. These effects were similar to the observations after imipramine treatment. Further, etanercept did
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Fig. 2. Restraint-induced effects on behaviour in the forced swim test (FST) are influenced by etanercept and imipramine. Effects on immobility (A) and climbing (B) after three weeks of additional treatment with either Ringer solution (Ri), etanercept (Eta; 0.3 mg/kg every third day, i.p.) or imipramine (Imi; 10 mg/kg daily, i.p.) following two weeks of restraint alone are shown. Data are given as the mean S.E.M. (n ¼ 12 each), *p < 0.05, **p < 0.01, ***p < 0.001.
not scale down the restraint-induced weight changes, similar to imipramine which potentiated body weight loss. Our results demonstrate that blocking of TNF-a by etanercept has antidepressant-like effects, supporting the repeatedly reported involvement of pro-inflammatory cytokines in the pathophysiology of depression (Himmerich et al., 2008; Müller and Schwarz, 2007; Reichenberg et al., 2001; Lotrich, 2012; Raison and Miller, 2011). That treatment with etanercept diminishes depression-like behaviour of rats also confirms observations in patients with psoriasis who showed improvements in symptoms of depression which were only weekly correlated with objective measures of skin clearance or joint pain (Tyring et al., 2006). The effects of etanercept in the present study were similar to that of intracerebroventricularly administered anti-TNF-a antibody shown to reverse depression-like behaviour induced by TNFa in mice (Kaster et al., 2012). Furthermore, these authors found that TNF-a-induced depression-like behaviour was prevented by the classic antidepressant imipramine. This result is in line with
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Fig. 3. Restraint- and treatment-induced effects on the body weight of rats. Body weights are shown before, after two weeks of repeated restraint (untreated) and after three subsequent weeks with additional treatment either with Ringer solution (Ri), etanercept (Eta; 0.3 mg/kg every third day, i.p.) or imipramine (Imi; 10 mg/kg daily, i.p.). Data are given as the mean S.E.M. (n ¼ 12 each), *p < 0.05, **p < 0.01, ***p < 0.001.
the suggested reduced production of pro-inflammatory cytokines, e.g. of TNF-a, by current antidepressants in rats and humans (Reynolds et al., 2005; Himmerich et al., 2010a, 2010b). Specifically the relatively strong effects of etanercept seen in the FST which were comparable to imipramine may be another brick in the cytokine hypothesis of depression and give a hint that TNFa antagonism may become a future antidepressant treatment strategy. However, from this animal study, one can not conclude that TNF-a antagonism would reverse the emotional and cognitive disturbances seen in humans after induction of the TNF-a system (Reichenberg et al., 2001) which are typical symptoms of depressed patients. Therefore, these results should be interpreted with caution regarding their relevance for the treatment of depression in humans. Notably, etanercept induced antidepressive-like behaviour in favour to augmented escape attempts in the FST. A similar response was found for imipramine-treated restrained rats. Increased climbing behaviour is a feature of antidepressive drugs preferentially modulating noradrenergic neurotransmission, e.g. reboxetine, but not of such enhancing serotonergic activity, e.g. citalopram or sertraline (Page et al., 2003; Mikail et al., 2012; own observations). The observed climbing behaviour during the treatment with the TNF-a blocker etanercept and imipramine is in agreement with data of Reynolds et al. (2005) demonstrating that TNF-a itself reduces noradrenergic neurotransmission in hippocampal slices of rats. This might be at least one possible pathway for the antidepressant-like action of etanercept in addition to other pathways derived from the physiological functions of TNF-a (for review see Santello and Volterra, 2012). As etanercept has beneficial effects in stress-induced depression, TNF-a blockers or similar cytokine-blocking agents could be alternative antidepressive agents with new mechanisms of action. Until now only antidepressants targeting on monoamines like imipramine are commonly used in the psychopharmacological antidepressant therapy (Covington et al., 2010), even though fewer than 35 percent of patients achieve remission with the first applied antidepressant drug (Trivedi et al., 2008). Thus the development of new antidepressant drug targets and new antidepressant drugs is an important demand in psychopharmacological research and development. In this regard, our study might contribute to the development of novel antidepressants on the basis of cytokine antagonism.
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4.2. Limitations of current TNF-a blockers as antidepressants Although stressful life events only partly contribute to the incidence of depression in humans, and a multidimensional causality of depression is widely accepted, chronic mild stress is a model with a high validity to induce antidepressant-reversible depressive-like effects in rodents (Willner, 2005). This is supported by the behavioural outcome of animals induced by repeated stressors (isolated housing, injections, restraint) and by the effects of the accompanied antidepressant treatment found in the present study as well as by activation of the hypothalamicepituitarye adrenal axis and of inflammatory mechanisms (Strausbaugh et al., 1999). However, an animal experiment can only be a first step for the development of a new antidepressant drug, because data derived from rodents can not easily transferred for human therapies. For example, imipramine and anti-TNF-a drugs are known to induce a weight gain in humans (Zimmermann et al., 2003; Brown et al., 2012), but to provoke weight loss in rats caused by reduction of food intake as it was also the case in our study (Hughes and Pither, 1987; Mogensen et al., 1994). As to be seen in Fig. 3, non-restraint rats gained weight over the whole period of the investigation, but rats undergoing the stress paradigm stopped gaining weight after the restraint started. As a side effect, noradrenergic mechanisms also may contribute to the lack of body weight gain in restrained imipramine-treated rats since the selective NE reuptake inhibitor, LY368975, reduced food intake in rats (Gehlert et al., 1998). Though the underlying mechanism of weight stagnancy in the restrained etanercept-treated animals remains unclear, obviously the health status has an important impact on the weight development as etanercept-treated non-restrained animals showed only a slight tendency of weight reduction. In this regard it has to be acknowledged that TNF-a is able to inhibit the release of NE in brain slices from rats (Elenkov et al., 1992; Reynolds et al., 2005) and anti-TNF-a medication might therefore lead to an increase in NE signalling and in less weight gain compared to placebo-treated animals. Despite of the limitations of animal models to measure “mental” disturbances as well as to transfer doseeresponse data, it seems to be promising to test anti-TNF-a agents against depression. An advantage of TNF-a blockers over totally new medicinal products is that they are already in use for rheumatoid arthritis, ankylosing spondylitis, psoriasis, psoriatic arthritis, uveitis and inflammatory bowel disease (Sfikakis and Tsokos, 2011). However, when treating depression with anti-TNF-a drugs, safety issues have also to be addressed such as reactivation of tuberculosis and exacerbation of pre-existing multiple sclerosis (Gregory et al., 2012). The most common adverse effect of etanercept treatment is injection site reaction, which is generally self-limiting and often does not require treatment. Therapy with etanercept may be associated with an increased risk for infection, the development of malignancy, demyelinating disease and congestive heart failure (Kerensky et al., 2012). Therefore, contraindications are a medical history of serious infections in the last 6 months, tuberculosis, pneumocystis carinii pneumonia, congestive heart failure, a malignancy or demyelinating disease (Miyasaka et al., 2006). In this respect, the next generation of non-antibody anti-TNFa drugs including progranulin or the soluble preligand-binding assembly domain (Sfikakis and Tsokos, 2011) might be potential antidepressant anti-TNF-a drugs without compromising the protective role of TNF-a in host defence and autoimmunity. After the results of this preliminary investigation have been promising, further animal studies including behavioural experiments, e.g. on drive or anxiety and the analysis of hormones of the HPA axis, e.g. glucocorticoids, have to be done to underline the
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validity of this stress paradigm and to investigate the effect of etanercept on stress hormones. 4.3. Conclusion In summary, etanercept given in a subchronic treatment regime was beneficial for the behavioural outcome in an animal model of restraint stress which induced depression-like behavioural changes. This result implies that TNF-a blockers might become a possibly novel class of antidepressants. Role of funding source This work was supported by the Claussen-Simon-Foundation. The authors were fully responsible for the idea of the study as well as the experiments, analysis, interpretation of data and writing the manuscript. Contributors All contributors to the aforementioned text are cited above. Conflict of interest Dr. Himmerich received speaker honoraria from AstraZeneca, Lilly and Servier, consulting fees from Bristol-Myers Squibb, and chemical substances for study support from AstraZeneca, Novartis and Wyeth. The other authors have no conflict of interest to report. Acknowledgement The authors would like to acknowledge the excellent technical assistance of Mrs. A.-K. Krause, Mrs. K. Bauer and Mr. L. Feige. References Albonetti ME, Farabollini F. Effects of single and repeated restraint on the social behavior of male rats. Physiology & Behavior 1993;53:937e42. Beck KD, Luine VN. Sex differences in behavioral and neurochemical profiles after chronic stress: role of housing conditions. Physiology & Behavior 2002;75: 661e73. Brown RA, Spina D, Butt S, Summers GD. Long-term effects of anti-tumour necrosis factor therapy on weight in patients with rheumatoid arthritis. Clinical Rheumatology 2012;31:455e61. Chiba S, Numakawa T, Ninomiya M, Richards MC, Wakabayashi C, Kunugi H. Chronic restraint stress causes anxiety- and depression-like behaviors, downregulates glucocorticoid receptor expression, and attenuates glutamate release induced by brain-derived neurotrophic factor in the prefrontal cortex. Progress in Neuro-Psychopharmacology & Biological Psychiatry 2012;39:112e9. Ciraulo DA, Barnhill JG, Jaffe JH. Clinical pharmacokinetics of imipramine and desipramine in alcoholics and normal volunteers. Clinical Pharmacology & Therapeutics 1988;43:509e18. Covington HEI, Vialou V, Nestler EJ. From synapse to nucleus: novel targets for treating depression. Neuropharmacology 2010;58:683e93. Cryan JF, Valentino RJ, Lucki I. Assessing substrates underlying the behavioral effects of antidepressants using the modified rat forced swimming test. Neuroscience and Biobehavioural Reviews 2005;29:547e69. Curran B, O’Connor JJ. The pro-inflammatory cytokine interleukin-18 impairs longterm potentiation and NMDA receptor-mediated transmission in the rat hippocampus in vitro. Neuroscience 2001;108:83e90. Elenkov IJ, Kovacs K, Duda E, Stark E, Vizi ES. Presynaptic inhibitory effect of TNF-alpha on the release of noradrenaline in isolated median eminence. Journal of Neuroimmunology 1992;41:117e20. Engler H, Doenlen R, Engler A, Riether C, Prager G, Niemi MB, et al. Acute amygdaloid response to systemic inflammation. Brain, Behavior, and Immunity 2011; 25:1384e92. Gehlert DR, Dreshfield L, Tinsley F, Benvenga MJ, Gleason S, Fuller RW, et al. The selective norepinephrine reuptake inhibitor, LY368975, reduces food consumption in animal models of feeding. Journal of Pharmacology and Experimental Therapeutics 1998;287:122e7. Gregory AP, Dendrou CA, Attfield KE, Haghikia A, Xifara DK, Butter F, et al. TNF receptor 1 genetic risk mirrors outcome of anti-TNF therapy in multiple sclerosis. Nature 2012;488:508e11.
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