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Immunomodulatory activity of ketamine in human astroglial A172 cells: Possible relevance to its rapid antidepressant activity
Journal of Neuroimmunology 282 (2015) 33–38
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Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Immunomodulatory activity of ketamine in human astroglial A172 cells: Possible relevance to its rapid antidepressant activity Yael Yuhas a,d,⁎, Shai Ashkenazi a,c,d, Eva Berent a, Abraham Weizman b,d,e a
Laboratory of Pediatric Infectious Diseases, Felsenstein Medical Research Center, Petach Tikva, Israel Laboratory of Biological Psychiatry, Felsenstein Medical Research Center, Petach Tikva, Israel Department of Pediatrics A, Schneider Children's Medical Center of Israel, Petach Tikva, Israel d Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel e Research Unit, Geha Mental Health Center, Petach Tikva, Israel b c
a r t i c l e
i n f o
Article history: Received 19 June 2014 Received in revised form 9 March 2015 Accepted 12 March 2015 Keywords: Ketamine Astroglial cells Cytokines Immunomodulation Antidepressant
a b s t r a c t To determine if the immunomodulatory effect of ketamine is relevant to its rapid antidepressant activity, cultured human astroglial cells were incubated with ketamine, cytokine mix, or both. At 24 h, ketamine dosedependently (100–500 μM) decreased IL-6 and TNFα production and gene expression and, at clinically relevant concentration (100 μM), augmented IL-β release and gene expression in both unstimulated and cytokinestimulated cells. In unstimulated cells, ketamine also increased IL-8 production and mRNA expression. The reduction in IL-6 mRNA was significant within 1 h in unstimulated cells and at 4 h after stimulation. Ketamine suppressed the production of the only established depression-relevant proinflammatory cytokines, IL-6 and TNFα. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Ketamine, a widely used anesthetic drug, has recently been found to be a potent and fast-acting antidepressive agent. In sub-anesthetic doses, it exerts a marked, rapid (within 2 h) and long-term effect, and is efficient in patients with treatment-resistant depression (Duman et al., 2012; Matthews and Zarate, 2013). Several molecular mechanisms have been suggested to explain the antidepressive activity of ketamine based on its properties as a nonselective, noncompetitive high-affinity, N-methyl-D-aspartate (NMDA) receptor antagonist and agonist of the serine/threonine kinase signaling pathway, the mammalian target of rapamycin (mTOR). These include an increase in neuroplasticity and synaptogenesis via modulation of glutamatergic signaling, stimulation of mTOR, an essential protein-regulating factor in synaptic plasticity (Li et al., 2010; Duman et al., 2012), and NMDA receptor blockade, as well as immunomodulatory activity and inhibition of glycogen synthase kinase-3β (GSK-3β) (Zunszain et al., 2013). However, the cellular mechanisms underlying ketamine's unique rapid antidepressant activity are still unclear. It has become evident that neuroinflammation plays an important role in major depression. An elevated inflammatory response was found in patients with major depression (Dantzer et al., 2008; Miller ⁎ Corresponding author at: Felsenstein Medical Research Center, Petach Tikva 49101, Israel. E-mail address: [email protected] (Y. Yuhas).
http://dx.doi.org/10.1016/j.jneuroim.2015.03.012 0165-5728/© 2015 Elsevier B.V. All rights reserved.
et al., 2009; Dowlati et al., 2010; Dean, 2011; Sukoff Rizzo et al., 2012; Miller, 2013) and in the brains of suicide victims (Erhardt et al., 2013), and there are reports linking increased inflammation with treatment resistance (Miller et al., 2009, 2013; Raison et al., 2013). Additionally, clinical observations and studies in both animal models and human volunteers have shown that depressive syndromes very often occur during the course of infective diseases, which are accompanied by elevated levels of inflammatory mediators (Miller et al., 2009). Recent experimental findings suggest that ketamine inhibits inflammatory responses and reduces proinflammatory cytokine production (Loix et al., 2011), raising the possibility that these activities account for ketamine's rapid antidepressant effect (Yang, 2011). Specifically, ketamine inhibited lipopolysaccharide (LPS)- or lipoteichoic acid-induced production of tumor necrosis factor (TNF)α, interleukin (IL)-1β, and IL-6 in mouse macrophage Raw264.7 cells (Chang et al., 2005; Wu et al., 2008; Chang et al., 2010), LPS-induced production of TNFα, IL1β, and IL-6 in rat astrocytes (Wu et al., 2012); the production of nitric oxide and IL-1β- but not TNFα production in primary cultured rat microglial cells (Chang et al., 2009), and LPS-induced production of TNFα in rat mixed glial cells (Shibakawa et al., 2005). Ketamine also attenuated the production of IL-1α, IL-1β, IL-6, IL-10, TNFα, and interferon (IFN)γ in serum of rats injected with LPS but had only a minimal effect on inflammation caused by traumatic brain injury (Ward et al., 2011). In human cells, ketamine inhibited TNFα, IL-6, and IL-8 production induced in vitro by LPS in whole blood (Kawasaki et al., 1999). In addition, an in vivo study found that continuous administration
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of ketamine (as anesthesia) during elective coronary artery bypass grafting attenuated the response of proinflammatory cytokines (IL-6 and IL-8) during and after surgery (Welters et al., 2011). However, according to a recent meta-analysis, intraoperative ketamine significantly inhibits only the early postoperative IL-6 inflammatory response (Dale et al., 2012). Previous studies of ketamine and inflammatory mediators have been performed in animal models or in vitro, in cells induced by bacterial components. Little is known about the effect of ketamine on human brain cells, and it is not clear whether the changes that occur in peripheral blood cells in response to ketamine are associated with a corresponding immune response in the central nervous system. The aim of the present study was to investigate the in vitro effect of ketamine on inflammatory processes in human astroglial cells. This design was chosen because astroglial cells are the equivalent in the brain of peripheral macrophages in the immune system and serve as the main source of brain inflammatory mediators. The immunomodulatory effects of ketamine were investigated in both unstimulated cells and cells simulated by a mix of cytokines known to be elevated in human astroglial cells during both infective and sterile inflammation. 2. Methods 2.1. Reagents The cell culture medium and its supplement were obtained from Biological Industries (Beit HaEmek, Israel). Recombinant human IL-1β, IFNγ, and TNFα were obtained from ProSpec-Tany TechnoGene Ltd. (Rehovot, Israel). Ketamine was a gift from Dr. Udi Lebel (Felsenstein Medical Research Center, Petach Tikva, Israel). 2.2. Cell culture Human-derived glioblastoma (astroglial cells) A-172 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in Dulbecco modified Eagle medium (DMEM) at 37 °C in a humidified incubator in 5% CO2 atmosphere. The medium was supplemented with 10% heat-inactivated fetal bovine serum and L-glutamine (2 mM) and with penicillin (100 U/ml), streptomycin (100 mg/l), and nystatin (12.5 U/ml). The cells were incubated in serum-free medium for 24 h and then exposed to a cytokine mix of TNFα, IL-1β, and IFNγ (100 ng/ml) with or without ketamine or to ketamine alone. Evaluation of neutral red uptake yielded no differences in viability among the differently treated cells.
were performed in a Step One Plus RT-PCR system (Applied Biosystems). The mRNA results for all cytokines were normalized to human RPLPO ribosomal protein mRNA.
2.6. Statistical analysis Results are presented as mean ± standard errors of the mean. Unpaired t test was used to compare results among treatments. Significance was defined a priori as a p value of b0.05.
3. Results 3.1. Effect of ketamine on inflammatory mediator production: cytokine array analysis To determine the effect of ketamine on inflammatory processes, we screened 35 inflammatory mediators using a cytokine array assay. Cells were incubated with medium alone, ketamine alone (500 μM), cytokine mix, or cytokine mix with ketamine. The supernatants were collected after 24 h and analyzed for cytokine levels. The untreated A172 cells spontaneously secreted high levels of monocyte chemotactic protein 1 (MCP1), migration inhibitory factor (MIF), and Serpine E1, and low levels of granulocyte macrophage colony stimulating factor (GM-CSF), IL-8, and IL-23. Incubation with ketamine was associated with a strong increase in levels of IL-1β (undetected in untreated cells) and IL-8 and a slight increase in the level of GM-CSF (Fig. 1). Treatment with cytokine mix led to an increase in the following mediators: complement component 5a, GM-CSF, chemokine (C-X-C motif) ligand 1 (CXCL1), IL-1 receptor antagonist (IL-1ra), IL-6, IL-8, CXCL10 (also known as Interferon gamma-induced protein 10, IP-10), CXCL11 (also known as interferoninducible T-cell alpha chemoattractant, I-TAC, and Interferon-gammainducible protein 9, IP-9) and RANTES (regulated on activation, normal T-cell expressed and secreted; also known as chemokine C–C motif ligand 5 CCL5). The addition of ketamine to the array was not associated with a change in the level of any of the mediators (data not shown).
Ketamine GM-CSF
PC
PC IL-1β
2.3. Cytokine array One milliliter of culture medium was centrifuged to remove cell debris, and the supernatants were analyzed using a Proteome Profiler Array Human Cytokine Panel A kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. 2.4. Cytokine determination The concentrations of IL-6, TNFα, IL-1β and IL-8 were determined using an enzyme-linked immunoassay (ELISA) kit from R&D Systems, according to the manufacturer's instructions.
PC
IL-8 IL-23 MCP-1 MIF
Control GM-CSF PC
PC IL-23
2.5. Real-time polymerase chain reaction (RT-PCR) RNA was isolated with TRIzol Reagent (Invitrogen, Life Technologies, Carlsbad, CA) and transcribed to cDNA using the High Capacity cDNA RT kit (Applied Biosystems, Foster City, CA). The cDNA samples were examined for IL-6, TNFα, IL-1β, and IL-8 using TaqMan Assay-onDemand and a TaqMan Gene Expression mix. All reagents, primers, and probes were obtained from Applied Biosystems. RT-PCR reactions
SerpinE1
PC
MCP-1MIF
IL-8
Serpin E1
Fig. 1. Effect of ketamine on the production of inflammatory mediators. A172 cells were treated with ketamine (500 μM) for 24 h. The culture medium was collected and analyzed using a human cytokine array. The upregulated genes are highlighted by rectangles. PC, positive control. A blot representative of one of two similar experiments is shown.
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3.2. Stimulating effect of ketamine on IL-1β production To further explore the inductive effect of ketamine on IL-1β found in our array analysis, cells were incubated with medium, ketamine, cytokine mix, or cytokine mix with ketamine, and IL-1β levels in the supernatant were measured after 24 h by quantitative ELISA. The results confirmed the array screening data. The addition of ketamine (500 μM) alone increased IL-1β concentration from 22 ± 0.5 pg/ml to 62 ± 7 pg/ml (p = 0.02, n = 3). The addition of ketamine (500 μM) to cells incubated with the cytokine mix (which contains IL-1β) increased IL-β concentration by 21%, from 70 ± 2 ng/ml to 89 ± 3 ng/ml (p = 0.001, n = 3). RT-PCR analysis of RNA extracted from the cells at 24 h after stimulation showed that ketamine increased the expression of IL-1β mRNA in the unstimulated cells in a concentration-dependent manner and augmented the expression of IL-β mRNA in the cytokine-stimulated cells (Fig. 2A, B). The ketamine-induced increase in IL-β mRNA expression was detected within 4 h in the unstimulated and cytokine-stimulated cells (Fig. 2C, D).
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(Fig. 3), cytokine mix alone led to high IL-8 mRNA levels. However, there was no further increase in IL-8 protein or mRNA in cells coincubated with cytokine mix and ketamine. 3.4. Inhibitory effect of ketamine on IL-6 production To evaluate the effect of ketamine on IL-6 levels, the level of IL-6 was measured in the supernatant of cells exposed to ketamine, cytokine mix, or cytokine mix with different concentrations of ketamine. In the cytokine-stimulated cells, ketamine decreased IL-6 production after 24 h in a concentration-dependent manner: by 89% at a ketamine concentration of 500 μM (p = 0.001); by 60% at a concentration of 250 μM (p b 0.01); and by 33% at a concentration of 100 μM (p b 0.05, n = 5 in each group) (Fig. 4A). On RNA analysis, ketamine significantly reduced the expression of IL-6 mRNA in stimulated cells at 4 h (p b 0.001) and 24 h (p b 0.001) after the addition of cytokine mix. IL-6 mRNA expression was also significantly reduced at 1 h and 4 h in cells incubated with ketamine alone compared to untreated cells (Fig. 4B, C).
3.3. Stimulating effect of ketamine on IL-8 production 3.5. Inhibitory effect of ketamine on TNFα production To further explore the inductive effect of ketamine on IL-8 expression in the array analysis, cells were incubated with ketamine, cytokine mix, or cytokine mix combined with ketamine. RT-PCR analysis of IL-8 mRNA extracted from cells at 24 h showed that ketamine alone increased IL-8 mRNA levels in a concentration-dependent manner
* 10 8 6 *
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After 24 h of stimulation, ketamine reduced the expression of TNFα mRNA in response to cytokine mix in a concentration-dependent manner (Fig. 5A). In addition, ketamine (500 μM) significantly decreased TNFα gene expression in unstimulated cells (Fig. 5D). Ketamine
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Fig. 2. Effect of ketamine on IL-1β gene expression. A. RT-PCR analysis of IL-1β mRNA expression in A172 cells treated with different concentrations of ketamine for 24 h (p b 0.001 for ketamine 50 μM and 500 μM and p b 0.05 for ketamine 250 μM, vs untreated cells, n = 4 in each group). B. RT-PCR analysis of cells treated with cytokine mix and different concentrations of ketamine for 24 h (p b 0.001 for ketamine 500 μM, and p = 0.01 for ketamine 250 μM vs mix, n = 4). C. RT-PCR analysis of cells treated with cytokine mix and ketamine (500 μM) for 1 and 4 h (p b 0.05 for mix vs mix with ketamine for 4 h, n = 4 in each group). D. RT-PCR analysis of cells treated with ketamine alone (500 μM) for 1 h and 4 h (p b 0.01 for ketamine vs control at 4 h, n = 4). Data are presented as mean ± SE.
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*
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Fig. 3. Effect of ketamine on IL-8 gene expression. RT-PCR analysis of IL-8 mRNA in A172 cells treated with different concentrations of ketamine for 24 h (p b 0.05 for ketamine 50 μM and 250 μM, and p b 0.01 for ketamine 500 μM, vs untreated cells, n = 4 in each group). Data are presented as mean ± SE of one of 3 similar experiments.
also attenuated the release of TNFα; incubation of the cells with IL-1β together with IFNγ induced the production of TNFα, and the addition of ketamine significantly reduced TNFα levels in the supernatant 24 h later (Fig. 5B). After 1 h of incubation with the cytokine mix, cells exhibited a strong expression of TNFα regardless of the presence or absence of ketamine. However, in contrast to the ketamine-induced decrease in TNFα expression at 24 h, TNFα mRNA expression increased after 4 h of incubation in both cytokine-stimulated and unstimulated cells (Fig. 5C, D).
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4. Discussion
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The major finding of our study is that ketamine exhibits immunomodulatory activity in both unstimulated and cytokine-stimulated human astroglial cells. Ketamine augmented IL-1β and IL-8 production and suppressed IL-6 and TNFα production by the astroglial cells. It is noteworthy that in the unstimulated cells, IL-6 gene expression significantly decreased as early as 1 h after the addition of ketamine. Previous in vitro studies have reported immunomodulatory effects of ketamine on peripheral immune cells, such as mouse macrophages (Chang et al., 2005; Wu et al., 2008; Chang et al., 2010) and human whole blood cells (Kawasaki et al., 1999), in addition to rat astrocytes (Wu et al., 2012), rat microglial cells (Chang et al., 2009), and rat mixed glial cells (Shibakawa et al., 2005). Ketamine also inhibited endotoxin-induced expression of TNFα, IL-6, and IL-8 in human blood in vivo (Taniguchi and Yamamoto, 2005). In general, these studies indicate that ketamine attenuates the production of proinflammatory cytokines stimulated by bacterial components (for review see Loix et al., 2011), suggesting that this factor may account for the fast-acting antidepressant effect of ketamine (Yang, 2011). Accordingly, in the present in vitro experiments, ketamine decreased IL-6 production and gene expression within 4 h in cytokinestimulated astroglial cells and within 1 h in unstimulated cells. Furthermore, ketamine decreased the expression of TNFα mRNA in both stimulated and unstimulated astroglial cells within 24 h and significantly reduced TNFα release in response to IFNγ and IL-1β at 24 h after induction. Interestingly, there was a rapid elevation in TNFα gene expression following exposure of 4 h to ketamine in unstimulated and stimulated cells, but both the gene and protein levels dropped significantly at 24 h. The clinical significance of this observation is as yet unclear. Several meta-analyses support the notion that depression is accompanied by activation of the immune response. A 2010 meta-analysis of 24 studies reported that the only cytokines found to be increased in patients with major depression compared to controls were TNFα and
10000 mix 9000 mix+ 8000 Ket (500µM) 7000 6000 5000 4000 3000 2000 1000 * 0 Time 4h
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Fig. 4. Effect of ketamine on IL-6 production and gene expression. A. IL-6 levels in cell supernatants 24 h after stimulation with cytokine mix in the presence and absence of ketamine (p b 0.05 for mix with ketamine 100 μM, p = 0.005 for ketamine 250 μM and p = 001 for ketamine 500 μM, vs mix, n = 5 for each treatment). Data represent the results of one of three similar experiments (means ± SE). B. IL-6 mRNA expression at 4 and 24 h after the addition of cytokine mix with or without ketamine (500 μM) (p b 0.001 for mix vs mix with ketamine at 4 h and 24 h, n = 4 in each group). C. IL-6 mRNA expression in cells treated for 1 h or 4 h with ketamine (500 μM) alone (p b 0.001 and p = 0.01 for ketamine vs untreated cells at 1 h and 4 h, respectively, n = 4 in each group).
IL-6 (Dowlati et al., 2010). Two years later, a larger meta-regression study found significantly above-normal blood levels of IL-6 and TNFα, as well as the soluble IL-2 receptor, in European patients with major depression (Liu et al., 2012). The association between major depression
Y. Yuhas et al. / Journal of Neuroimmunology 282 (2015) 33–38
B 5000
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Fig. 5. Effect of ketamine on TNFα production and gene expression. A. TNFα mRNA expression in cells treated with cytokine mix (mix) or with mix together with different concentrations of ketamine 24 h after stimulation (p b 0.05 and p b 0.001 for mix vs mix with ketamine 250 μM and 500 μM, respectively, n = 4 in each group). B. TNFα concentration in cell's supernatants 24 h after stimulation with IFNγ and IL-1β in the presence or absence of ketamine (p = 0.02, n = 5). C. TNFα mRNA expression in cells incubated with mix or mix with ketamine for 1 h and 4 h (p b 0.04 for 4 h, n = 4 in each group). D. TNFα mRNA expression in cells treated with ketamine alone for 1 h, 4 h, and 24 h (p b 0.05 and p b 0.01 for ketamine vs untreated cells at 4 h and 24 h, respectively, n = 4 in each group).
and elevated circulatory levels of IL-6, but not other cytokines, was noted in a third meta-analysis (Hiles et al., 2012a). IL-6 elevation was higher in patients with a diagnosis of depressive disorder than in patients with only depressive symptoms; in patients recruited from inpatient or outpatient settings than in patients from the general community; and in patients who were not selected for a particular comorbidity than in patients selected for cardiovascular disease (Hiles et al., 2012a,b). Moreover, IL-6 knockout mice exhibit resistance to stressinduced development of depression-like behaviors (Chourbaji et al., 2006). Together, these findings point to the specificity of IL-6 involvement in major depression rather than a global elevation in cytokines. A pharmacological meta-analysis showed that treatment with selective serotonin reuptake inhibitors was associated with a reduction in circulatory IL-6 and TNFα levels, consistent with the possibility that they contribute to depressive symptoms and that antidepressants inhibit the effects of proinflammatory cytokines in the brain (Hannestad et al., 2011). Accordingly, another pharmacological meta-analysis reported that overactive IL-6 is significantly attenuated following antidepressant treatment (Hiles et al., 2012b). Activation of the mTOR signaling pathway leads to significant attenuation of overactive stimulated microglial cells by repressing the expression levels of neurotoxic proinflammatory mediators and cytokines, while enhancing the release of the antiinflammatory cytokine IL-10 (Dello Russo et al., 2009; Powell et al., 2012; Dello Russo et al.,
2013). It is possible that ketamine-induced stimulation of mTOR signaling (Hashimoto, 2011; Duman et al., 2012) was involved in the suppression of TNFα and IL-6 production in our human glial cells. It is noteworthy that the effective anti-inflammatory concentrations of ketamine (100 μM) have been found to be clinically relevant (Chang et al., 2005; Wu et al., 2008). We observed that levels of IL-1β and IL-8 released were augmented by ketamine in human astroglial cells. This finding differs from studies in rat macrophage and astroglial cells which showed a ketamineinduced reduction of these cytokines (Shibakawa et al., 2005; Chang et al., 2009; Wu et al., 2012). This difference most probably stems from differences between human astroglial cells and rat immune and glial cells. The relevance of our finding to the rapid antidepressant activity of ketamine is unclear, as the various meta-analyses did not identify any role of IL-1β or IL-8 in the pathophysiology of depression (Dowlati et al., 2010; Hiles et al., 2012b) or in the response to antidepressant treatment (Hannestad et al., 2011; Hiles et al., 2012a). The cause for the inconsistency between the in vitro up-regulation of IL-β expression and down-regulation of both IL-6 and TNFα following exposure of A172 human astroglial cells to ketamine is as yet unclear. Further molecular study is needed to clarify this discrepancy. In conclusion, the present study demonstrates for the first time that ketamine suppresses the in vitro production of the only established depression-relevant proinflammatory cytokines, IL-6 (within
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1 h) and TNFα (within 24 h), in human astroglial cells. This antineuroinflammatory activity may be involved in the rapid antidepressant effect of ketamine. Further in vivo studies with ketamine, mTOR agonists/antagonists, and IL-6/TNFα antagonists are needed to elucidate the complex relationship among ketamine, specific proinflammatory cytokines, neuroinflammation biomarkers, and the rapid antidepressant activity of ketamine. Nevertheless, it appears that it is important to carry out the ketamine studies in relevant human-derived cells as it has been previously studied only in animal models. References Chang, Y., Chen, T.L., Sheu, J.R., Chen, R.M., 2005. Suppressive effects of ketamine on macrophage functions. Toxicol. Appl. Pharmacol. 204, 27–35. Chang, Y., Lee, J.-J., Hsieh, C.-Y., Hsiao, G., Chou, D.-S., Sheu, J.-R., 2009. Inhibitory effects of ketamine on lipopolysaccharide-induced microglial activation. Mediat. Inflamm. http://dx.doi.org/10.1155/2009/705379 (March 30 (Epub)). Chang, H.C., Lin, K.H., Tai, Y.T., Chen, J.T., Chen, R.M., 2010. Lipoteichoic acid-induced TNF-α and IL-6 gene expressions oxidative stress production in macrophages are suppressed by ketamine through downregulating Toll-like receptor 2-mediated activation of ERK1/2 and NFκB. Shock 33, 485–492. Chourbaji, S., Urani, A., Inta, I., Sanchis-Segura, C., Brandwein, C., Zink, M., Schwaninger, M., Gass, P., 2006. IL-6 knockout mice exhibit resistance to stress-induced development of depression-like behaviors. Neurobiol. Dis. 23, 587–594. Dale, O., Somogyi, A.A., Li, Y., Sullivan, T., Shavit, Y., 2012. Does intraoperative ketamine attenuate inflammatory reactivity following surgery? A systematic review and meta-analysis. Anesth. Analg. 115, 934–943. Dantzer, R., O-Connor, J.C., Freund, G.G., Johnson, R.W., Kelley, K.W., 2008. From inflammation to sickness and depression: when the immune system subjugates the brain. Nat. Rev. Neurosci. 9, 46–56. Dean, B., 2011. Understanding the role of inflammatory-related pathways in the pathophysiology and treatment of psychiatric disorders: evidence from human peripheral studies and CNS studies. Int. J. Neuropsychopharmacol. 14, 997–1012. Dello Russo, C., Lisi, L., Tringali, G., Navarra, P., 2009. Involvement of mTOR kinase in cytokine-dependent microglial activation and cell proliferation. Biochem. Pharmacol. 78, 1242–1251. Dello Russo, C., Lisi, L., Feinstein, D.L., Navarra, P., 2013. mTOR kinase, a key player in the regulation of glial functions: relevance for the therapy of multiple sclerosis. Glia 61, 301–311. Dowlati, Y., Hermann, N., Swardfager, W., Liu, H., Sham, L., Reim, E.K., Lanctot, K.L., 2010. A meta-analysis of cytokines in major depression. Biol. Psychiatry 67, 446–457. Duman, R.S., Li, N., Liu, R.-J., Durin, V., Aghajanian, G., 2012. Signaling pathways underlying the rapid antidepressant actions of ketamine. Neuropharmacology 62, 35–41. Erhardt, S., Lim, C.K., Linderholm, K.R., Janelidze, S., Lindqvist, D., Samuelsson, M., Lundberg, K., Postolache, T.T., Träskman-Bendz, L., Guillemin, G.J., Brundin, L., 2013. Connecting inflammation with glutamate agonism in suicidality. Neuropsychopharmacology 38, 743–752. Hannestad, J., DellaGioia, N., Block, M., 2011. The effects of antidepressant medication treatment on serum levels of inflammatory cytokines: a meta-analysis. Neuropsychopharmacology 36, 2452–2459. Hashimoto, K., 2011. Role of the mTOR signaling pathway in the rapid antidepressant action of ketamine. Expert. Rev. Neurother. 11, 33–36. Hiles, S.A., Baker, A.L., de Malmanche, T., Attia, J., 2012a. A meta-analysis of differences in IL-6 and IL-10 between people with and without depression: exploring the causes of heterogeneity. Brain Behav. Immun. 26, 1180–1188.
Hiles, S.A., Baker, A.L., de Malmanche, T., Attia, J., 2012b. Interleukin-6, C-reactive protein and interleukin-10 after antidepressant treatment in people with depression: a metaanalysis. Psychol. Med. 42, 2015–2026. Kawasaki, T., Ogata, M., Kawasaki, C., Ogata, J.-I., Inoue, Y., Shigematsu, A., 1999. Ketamine suppresses proinflammatory cytokine production in human whole blood in vitro. Anesth. Analg. 89, 665–669. Li, N., Lee, B., Liu, R.-J., Banasr, M., Dwyer, J.M., Iwata, M., Li, X.-Y., Aghajanian, G., Duman, R.S., 2010. mTOR-dependent synapse formation underlies the rapid antidepressant effects of NMDA antagonists. Science 329, 959–964. Liu, Y., Ho, R.C.-H., Mak, A., 2012. Interleukin (IL)-6, tumour necrosis factor alpha (TNF-α) and soluble interleukin-2 receptors (sIL-2R) are elevated in patients with major depressive disorder: a meta-analysis and meta-regression. J. Affect. Disord. 139, 230–239. Loix, S., De Kock, M., Henin, P., 2011. The anti-inflammatory effects of ketamine: state of the art. Acta Anaesthesiol. Belg. 62, 47–58. Matthews, D.C., Zarate Jr., A.A., 2013. Current status of ketamine and related compounds for depression. J. Clin. Psychiatry 74, 516–517. Miller, A.H., 2013. Conceptual confluence: the kynurenine pathway as a common target for ketamine and the convergence of the inflammation and glutamate hypotheses of depression. Neuropsychopharmacology 38, 1607–1608. Miller, A.H., Maletic, V., Raison, C.L., 2009. Inflammation and its discontents: the role of cytokines in the pathophysiology of major depression. Biol. Psychiatry 65, 732–741. Miller, A.H., Haroon, E., Raison, C.L., Felger, J.C., 2013. Cytokine targets in the brain: impact on neurotransmitters and neurocircuits. Depress. Anxiety 30, 297–306. Powell, J.D., Pollizzi, K.N., Heikamp, E.B., Horton, M.R., 2012. Regulation of immune responses by mTOR. Annu. Rev. Immunol. 30, 39–68. Raison, C.L., Rutherford, R.E., Woolwine, B.J., Shuo, C., Schettler, P., Drake, D.F., Haroon, E., Miller, A.H., 2013. A randomized controlled trial of the tumor necrosis factor antagonist infliximab for treatment-resistant depression: the role of baseline inflammatory biomarkers. JAMA Psychiatry 70, 31–41. Shibakawa, Y.S., Sasaki, Y., Goshima, Y., Echigo, N., Kamiya, Y., Kurahashi, K., Yamada, Y., Andoh, T., 2005. Effects of ketamine and propofol on inflammatory responses of primary glial cell cultures stimulated with lipopolysaccharide. Br. J. Anaesth. 95, 803–810. Sukoff Rizzo, S.J., Neal, S.J., Hughes, Z.A., Beyna, M., Rosenzweig-Lipson, S., Moss, S.J., Brandon, N.J., 2012. Evidence for sustained election of IL-6 in the CNS as a key contributor of depressive-like phenotypes. Transl. Psychiatry 2, e199. Taniguchi, T., Yamamoto, K., 2005. Anti-inflammatory effects of intravenous anesthetics on endotoxemia. Mini Rev. Med. Chem. 5, 241–245. Ward, J.L., Harting, M.T., Cox Jr., C.S., Mercer, D.W., 2011. Effects of ketamine on endotoxin and traumatic brain injury-induced cytokine production in the rat. J. Trauma 70, 1471–1479. Welters, I.D., Feurer, M.K., Preiss, V., Muller, M., Scholz, S., Kwapisz, M., Mogk, M., Neuhauser, C., 2011. Continuous S-(+)-ketamine administration during elective coronary artery bypass graft surgery attenuates pro-inflammatory cytokine response during and after cardiopulmonary bypass. Br. J. Anaesth. 106, 172–179. Wu, G.-J., Chen, T.-L., Ueng, Y.-F., Chen, R.-M., 2008. Ketamine inhibits tumor necrosis factor-α and interleukin-6 gene expression in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation. Toxicol. Appl. Pharmacol. 228, 105–113. Wu, Y., Li, W., Zhou, C., Lu, F., Gao, T., Liu, Y., Cao, J., Zhang, Y., Zhang, Y., Zhou, C., 2012. Ketamine inhibits lipopolysaccharide-induced astrocyte activation by suppressing TLR4/NF-kB pathway. Cell. Physiol. Biochem. 30, 609–617. Yang, C., 2011. Does ketamine exert a fast-acting antidepressant effect via inhibition of pro-inflammatory cytokines? Psychol. Med. 41, 1787–1789. Zunszain, P.A., Horowitz, M.A., Cattaneo, A., Lupi, M.M., Pariante, C.M., 2013. Ketamine: synaptogenesis, immunomodulation and glycogen synthase kinase-3 as underlying mechanisms of its antidepressant properties. Mol. Psychiatry 1–6.
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Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Immunomodulatory activity of ketamine in human astroglial A172 cells: Possible relevance to its rapid antidepressant activity Yael Yuhas a,d,⁎, Shai Ashkenazi a,c,d, Eva Berent a, Abraham Weizman b,d,e a
Laboratory of Pediatric Infectious Diseases, Felsenstein Medical Research Center, Petach Tikva, Israel Laboratory of Biological Psychiatry, Felsenstein Medical Research Center, Petach Tikva, Israel Department of Pediatrics A, Schneider Children's Medical Center of Israel, Petach Tikva, Israel d Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel e Research Unit, Geha Mental Health Center, Petach Tikva, Israel b c
a r t i c l e
i n f o
Article history: Received 19 June 2014 Received in revised form 9 March 2015 Accepted 12 March 2015 Keywords: Ketamine Astroglial cells Cytokines Immunomodulation Antidepressant
a b s t r a c t To determine if the immunomodulatory effect of ketamine is relevant to its rapid antidepressant activity, cultured human astroglial cells were incubated with ketamine, cytokine mix, or both. At 24 h, ketamine dosedependently (100–500 μM) decreased IL-6 and TNFα production and gene expression and, at clinically relevant concentration (100 μM), augmented IL-β release and gene expression in both unstimulated and cytokinestimulated cells. In unstimulated cells, ketamine also increased IL-8 production and mRNA expression. The reduction in IL-6 mRNA was significant within 1 h in unstimulated cells and at 4 h after stimulation. Ketamine suppressed the production of the only established depression-relevant proinflammatory cytokines, IL-6 and TNFα. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Ketamine, a widely used anesthetic drug, has recently been found to be a potent and fast-acting antidepressive agent. In sub-anesthetic doses, it exerts a marked, rapid (within 2 h) and long-term effect, and is efficient in patients with treatment-resistant depression (Duman et al., 2012; Matthews and Zarate, 2013). Several molecular mechanisms have been suggested to explain the antidepressive activity of ketamine based on its properties as a nonselective, noncompetitive high-affinity, N-methyl-D-aspartate (NMDA) receptor antagonist and agonist of the serine/threonine kinase signaling pathway, the mammalian target of rapamycin (mTOR). These include an increase in neuroplasticity and synaptogenesis via modulation of glutamatergic signaling, stimulation of mTOR, an essential protein-regulating factor in synaptic plasticity (Li et al., 2010; Duman et al., 2012), and NMDA receptor blockade, as well as immunomodulatory activity and inhibition of glycogen synthase kinase-3β (GSK-3β) (Zunszain et al., 2013). However, the cellular mechanisms underlying ketamine's unique rapid antidepressant activity are still unclear. It has become evident that neuroinflammation plays an important role in major depression. An elevated inflammatory response was found in patients with major depression (Dantzer et al., 2008; Miller ⁎ Corresponding author at: Felsenstein Medical Research Center, Petach Tikva 49101, Israel. E-mail address: [email protected] (Y. Yuhas).
http://dx.doi.org/10.1016/j.jneuroim.2015.03.012 0165-5728/© 2015 Elsevier B.V. All rights reserved.
et al., 2009; Dowlati et al., 2010; Dean, 2011; Sukoff Rizzo et al., 2012; Miller, 2013) and in the brains of suicide victims (Erhardt et al., 2013), and there are reports linking increased inflammation with treatment resistance (Miller et al., 2009, 2013; Raison et al., 2013). Additionally, clinical observations and studies in both animal models and human volunteers have shown that depressive syndromes very often occur during the course of infective diseases, which are accompanied by elevated levels of inflammatory mediators (Miller et al., 2009). Recent experimental findings suggest that ketamine inhibits inflammatory responses and reduces proinflammatory cytokine production (Loix et al., 2011), raising the possibility that these activities account for ketamine's rapid antidepressant effect (Yang, 2011). Specifically, ketamine inhibited lipopolysaccharide (LPS)- or lipoteichoic acid-induced production of tumor necrosis factor (TNF)α, interleukin (IL)-1β, and IL-6 in mouse macrophage Raw264.7 cells (Chang et al., 2005; Wu et al., 2008; Chang et al., 2010), LPS-induced production of TNFα, IL1β, and IL-6 in rat astrocytes (Wu et al., 2012); the production of nitric oxide and IL-1β- but not TNFα production in primary cultured rat microglial cells (Chang et al., 2009), and LPS-induced production of TNFα in rat mixed glial cells (Shibakawa et al., 2005). Ketamine also attenuated the production of IL-1α, IL-1β, IL-6, IL-10, TNFα, and interferon (IFN)γ in serum of rats injected with LPS but had only a minimal effect on inflammation caused by traumatic brain injury (Ward et al., 2011). In human cells, ketamine inhibited TNFα, IL-6, and IL-8 production induced in vitro by LPS in whole blood (Kawasaki et al., 1999). In addition, an in vivo study found that continuous administration
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of ketamine (as anesthesia) during elective coronary artery bypass grafting attenuated the response of proinflammatory cytokines (IL-6 and IL-8) during and after surgery (Welters et al., 2011). However, according to a recent meta-analysis, intraoperative ketamine significantly inhibits only the early postoperative IL-6 inflammatory response (Dale et al., 2012). Previous studies of ketamine and inflammatory mediators have been performed in animal models or in vitro, in cells induced by bacterial components. Little is known about the effect of ketamine on human brain cells, and it is not clear whether the changes that occur in peripheral blood cells in response to ketamine are associated with a corresponding immune response in the central nervous system. The aim of the present study was to investigate the in vitro effect of ketamine on inflammatory processes in human astroglial cells. This design was chosen because astroglial cells are the equivalent in the brain of peripheral macrophages in the immune system and serve as the main source of brain inflammatory mediators. The immunomodulatory effects of ketamine were investigated in both unstimulated cells and cells simulated by a mix of cytokines known to be elevated in human astroglial cells during both infective and sterile inflammation. 2. Methods 2.1. Reagents The cell culture medium and its supplement were obtained from Biological Industries (Beit HaEmek, Israel). Recombinant human IL-1β, IFNγ, and TNFα were obtained from ProSpec-Tany TechnoGene Ltd. (Rehovot, Israel). Ketamine was a gift from Dr. Udi Lebel (Felsenstein Medical Research Center, Petach Tikva, Israel). 2.2. Cell culture Human-derived glioblastoma (astroglial cells) A-172 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in Dulbecco modified Eagle medium (DMEM) at 37 °C in a humidified incubator in 5% CO2 atmosphere. The medium was supplemented with 10% heat-inactivated fetal bovine serum and L-glutamine (2 mM) and with penicillin (100 U/ml), streptomycin (100 mg/l), and nystatin (12.5 U/ml). The cells were incubated in serum-free medium for 24 h and then exposed to a cytokine mix of TNFα, IL-1β, and IFNγ (100 ng/ml) with or without ketamine or to ketamine alone. Evaluation of neutral red uptake yielded no differences in viability among the differently treated cells.
were performed in a Step One Plus RT-PCR system (Applied Biosystems). The mRNA results for all cytokines were normalized to human RPLPO ribosomal protein mRNA.
2.6. Statistical analysis Results are presented as mean ± standard errors of the mean. Unpaired t test was used to compare results among treatments. Significance was defined a priori as a p value of b0.05.
3. Results 3.1. Effect of ketamine on inflammatory mediator production: cytokine array analysis To determine the effect of ketamine on inflammatory processes, we screened 35 inflammatory mediators using a cytokine array assay. Cells were incubated with medium alone, ketamine alone (500 μM), cytokine mix, or cytokine mix with ketamine. The supernatants were collected after 24 h and analyzed for cytokine levels. The untreated A172 cells spontaneously secreted high levels of monocyte chemotactic protein 1 (MCP1), migration inhibitory factor (MIF), and Serpine E1, and low levels of granulocyte macrophage colony stimulating factor (GM-CSF), IL-8, and IL-23. Incubation with ketamine was associated with a strong increase in levels of IL-1β (undetected in untreated cells) and IL-8 and a slight increase in the level of GM-CSF (Fig. 1). Treatment with cytokine mix led to an increase in the following mediators: complement component 5a, GM-CSF, chemokine (C-X-C motif) ligand 1 (CXCL1), IL-1 receptor antagonist (IL-1ra), IL-6, IL-8, CXCL10 (also known as Interferon gamma-induced protein 10, IP-10), CXCL11 (also known as interferoninducible T-cell alpha chemoattractant, I-TAC, and Interferon-gammainducible protein 9, IP-9) and RANTES (regulated on activation, normal T-cell expressed and secreted; also known as chemokine C–C motif ligand 5 CCL5). The addition of ketamine to the array was not associated with a change in the level of any of the mediators (data not shown).
Ketamine GM-CSF
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PC IL-1β
2.3. Cytokine array One milliliter of culture medium was centrifuged to remove cell debris, and the supernatants were analyzed using a Proteome Profiler Array Human Cytokine Panel A kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. 2.4. Cytokine determination The concentrations of IL-6, TNFα, IL-1β and IL-8 were determined using an enzyme-linked immunoassay (ELISA) kit from R&D Systems, according to the manufacturer's instructions.
PC
IL-8 IL-23 MCP-1 MIF
Control GM-CSF PC
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2.5. Real-time polymerase chain reaction (RT-PCR) RNA was isolated with TRIzol Reagent (Invitrogen, Life Technologies, Carlsbad, CA) and transcribed to cDNA using the High Capacity cDNA RT kit (Applied Biosystems, Foster City, CA). The cDNA samples were examined for IL-6, TNFα, IL-1β, and IL-8 using TaqMan Assay-onDemand and a TaqMan Gene Expression mix. All reagents, primers, and probes were obtained from Applied Biosystems. RT-PCR reactions
SerpinE1
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Serpin E1
Fig. 1. Effect of ketamine on the production of inflammatory mediators. A172 cells were treated with ketamine (500 μM) for 24 h. The culture medium was collected and analyzed using a human cytokine array. The upregulated genes are highlighted by rectangles. PC, positive control. A blot representative of one of two similar experiments is shown.
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3.2. Stimulating effect of ketamine on IL-1β production To further explore the inductive effect of ketamine on IL-1β found in our array analysis, cells were incubated with medium, ketamine, cytokine mix, or cytokine mix with ketamine, and IL-1β levels in the supernatant were measured after 24 h by quantitative ELISA. The results confirmed the array screening data. The addition of ketamine (500 μM) alone increased IL-1β concentration from 22 ± 0.5 pg/ml to 62 ± 7 pg/ml (p = 0.02, n = 3). The addition of ketamine (500 μM) to cells incubated with the cytokine mix (which contains IL-1β) increased IL-β concentration by 21%, from 70 ± 2 ng/ml to 89 ± 3 ng/ml (p = 0.001, n = 3). RT-PCR analysis of RNA extracted from the cells at 24 h after stimulation showed that ketamine increased the expression of IL-1β mRNA in the unstimulated cells in a concentration-dependent manner and augmented the expression of IL-β mRNA in the cytokine-stimulated cells (Fig. 2A, B). The ketamine-induced increase in IL-β mRNA expression was detected within 4 h in the unstimulated and cytokine-stimulated cells (Fig. 2C, D).
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(Fig. 3), cytokine mix alone led to high IL-8 mRNA levels. However, there was no further increase in IL-8 protein or mRNA in cells coincubated with cytokine mix and ketamine. 3.4. Inhibitory effect of ketamine on IL-6 production To evaluate the effect of ketamine on IL-6 levels, the level of IL-6 was measured in the supernatant of cells exposed to ketamine, cytokine mix, or cytokine mix with different concentrations of ketamine. In the cytokine-stimulated cells, ketamine decreased IL-6 production after 24 h in a concentration-dependent manner: by 89% at a ketamine concentration of 500 μM (p = 0.001); by 60% at a concentration of 250 μM (p b 0.01); and by 33% at a concentration of 100 μM (p b 0.05, n = 5 in each group) (Fig. 4A). On RNA analysis, ketamine significantly reduced the expression of IL-6 mRNA in stimulated cells at 4 h (p b 0.001) and 24 h (p b 0.001) after the addition of cytokine mix. IL-6 mRNA expression was also significantly reduced at 1 h and 4 h in cells incubated with ketamine alone compared to untreated cells (Fig. 4B, C).
3.3. Stimulating effect of ketamine on IL-8 production 3.5. Inhibitory effect of ketamine on TNFα production To further explore the inductive effect of ketamine on IL-8 expression in the array analysis, cells were incubated with ketamine, cytokine mix, or cytokine mix combined with ketamine. RT-PCR analysis of IL-8 mRNA extracted from cells at 24 h showed that ketamine alone increased IL-8 mRNA levels in a concentration-dependent manner
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After 24 h of stimulation, ketamine reduced the expression of TNFα mRNA in response to cytokine mix in a concentration-dependent manner (Fig. 5A). In addition, ketamine (500 μM) significantly decreased TNFα gene expression in unstimulated cells (Fig. 5D). Ketamine
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Fig. 2. Effect of ketamine on IL-1β gene expression. A. RT-PCR analysis of IL-1β mRNA expression in A172 cells treated with different concentrations of ketamine for 24 h (p b 0.001 for ketamine 50 μM and 500 μM and p b 0.05 for ketamine 250 μM, vs untreated cells, n = 4 in each group). B. RT-PCR analysis of cells treated with cytokine mix and different concentrations of ketamine for 24 h (p b 0.001 for ketamine 500 μM, and p = 0.01 for ketamine 250 μM vs mix, n = 4). C. RT-PCR analysis of cells treated with cytokine mix and ketamine (500 μM) for 1 and 4 h (p b 0.05 for mix vs mix with ketamine for 4 h, n = 4 in each group). D. RT-PCR analysis of cells treated with ketamine alone (500 μM) for 1 h and 4 h (p b 0.01 for ketamine vs control at 4 h, n = 4). Data are presented as mean ± SE.
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*
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Fig. 3. Effect of ketamine on IL-8 gene expression. RT-PCR analysis of IL-8 mRNA in A172 cells treated with different concentrations of ketamine for 24 h (p b 0.05 for ketamine 50 μM and 250 μM, and p b 0.01 for ketamine 500 μM, vs untreated cells, n = 4 in each group). Data are presented as mean ± SE of one of 3 similar experiments.
also attenuated the release of TNFα; incubation of the cells with IL-1β together with IFNγ induced the production of TNFα, and the addition of ketamine significantly reduced TNFα levels in the supernatant 24 h later (Fig. 5B). After 1 h of incubation with the cytokine mix, cells exhibited a strong expression of TNFα regardless of the presence or absence of ketamine. However, in contrast to the ketamine-induced decrease in TNFα expression at 24 h, TNFα mRNA expression increased after 4 h of incubation in both cytokine-stimulated and unstimulated cells (Fig. 5C, D).
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4. Discussion
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The major finding of our study is that ketamine exhibits immunomodulatory activity in both unstimulated and cytokine-stimulated human astroglial cells. Ketamine augmented IL-1β and IL-8 production and suppressed IL-6 and TNFα production by the astroglial cells. It is noteworthy that in the unstimulated cells, IL-6 gene expression significantly decreased as early as 1 h after the addition of ketamine. Previous in vitro studies have reported immunomodulatory effects of ketamine on peripheral immune cells, such as mouse macrophages (Chang et al., 2005; Wu et al., 2008; Chang et al., 2010) and human whole blood cells (Kawasaki et al., 1999), in addition to rat astrocytes (Wu et al., 2012), rat microglial cells (Chang et al., 2009), and rat mixed glial cells (Shibakawa et al., 2005). Ketamine also inhibited endotoxin-induced expression of TNFα, IL-6, and IL-8 in human blood in vivo (Taniguchi and Yamamoto, 2005). In general, these studies indicate that ketamine attenuates the production of proinflammatory cytokines stimulated by bacterial components (for review see Loix et al., 2011), suggesting that this factor may account for the fast-acting antidepressant effect of ketamine (Yang, 2011). Accordingly, in the present in vitro experiments, ketamine decreased IL-6 production and gene expression within 4 h in cytokinestimulated astroglial cells and within 1 h in unstimulated cells. Furthermore, ketamine decreased the expression of TNFα mRNA in both stimulated and unstimulated astroglial cells within 24 h and significantly reduced TNFα release in response to IFNγ and IL-1β at 24 h after induction. Interestingly, there was a rapid elevation in TNFα gene expression following exposure of 4 h to ketamine in unstimulated and stimulated cells, but both the gene and protein levels dropped significantly at 24 h. The clinical significance of this observation is as yet unclear. Several meta-analyses support the notion that depression is accompanied by activation of the immune response. A 2010 meta-analysis of 24 studies reported that the only cytokines found to be increased in patients with major depression compared to controls were TNFα and
10000 mix 9000 mix+ 8000 Ket (500µM) 7000 6000 5000 4000 3000 2000 1000 * 0 Time 4h
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Fig. 4. Effect of ketamine on IL-6 production and gene expression. A. IL-6 levels in cell supernatants 24 h after stimulation with cytokine mix in the presence and absence of ketamine (p b 0.05 for mix with ketamine 100 μM, p = 0.005 for ketamine 250 μM and p = 001 for ketamine 500 μM, vs mix, n = 5 for each treatment). Data represent the results of one of three similar experiments (means ± SE). B. IL-6 mRNA expression at 4 and 24 h after the addition of cytokine mix with or without ketamine (500 μM) (p b 0.001 for mix vs mix with ketamine at 4 h and 24 h, n = 4 in each group). C. IL-6 mRNA expression in cells treated for 1 h or 4 h with ketamine (500 μM) alone (p b 0.001 and p = 0.01 for ketamine vs untreated cells at 1 h and 4 h, respectively, n = 4 in each group).
IL-6 (Dowlati et al., 2010). Two years later, a larger meta-regression study found significantly above-normal blood levels of IL-6 and TNFα, as well as the soluble IL-2 receptor, in European patients with major depression (Liu et al., 2012). The association between major depression
Y. Yuhas et al. / Journal of Neuroimmunology 282 (2015) 33–38
B 5000
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24 h
Fig. 5. Effect of ketamine on TNFα production and gene expression. A. TNFα mRNA expression in cells treated with cytokine mix (mix) or with mix together with different concentrations of ketamine 24 h after stimulation (p b 0.05 and p b 0.001 for mix vs mix with ketamine 250 μM and 500 μM, respectively, n = 4 in each group). B. TNFα concentration in cell's supernatants 24 h after stimulation with IFNγ and IL-1β in the presence or absence of ketamine (p = 0.02, n = 5). C. TNFα mRNA expression in cells incubated with mix or mix with ketamine for 1 h and 4 h (p b 0.04 for 4 h, n = 4 in each group). D. TNFα mRNA expression in cells treated with ketamine alone for 1 h, 4 h, and 24 h (p b 0.05 and p b 0.01 for ketamine vs untreated cells at 4 h and 24 h, respectively, n = 4 in each group).
and elevated circulatory levels of IL-6, but not other cytokines, was noted in a third meta-analysis (Hiles et al., 2012a). IL-6 elevation was higher in patients with a diagnosis of depressive disorder than in patients with only depressive symptoms; in patients recruited from inpatient or outpatient settings than in patients from the general community; and in patients who were not selected for a particular comorbidity than in patients selected for cardiovascular disease (Hiles et al., 2012a,b). Moreover, IL-6 knockout mice exhibit resistance to stressinduced development of depression-like behaviors (Chourbaji et al., 2006). Together, these findings point to the specificity of IL-6 involvement in major depression rather than a global elevation in cytokines. A pharmacological meta-analysis showed that treatment with selective serotonin reuptake inhibitors was associated with a reduction in circulatory IL-6 and TNFα levels, consistent with the possibility that they contribute to depressive symptoms and that antidepressants inhibit the effects of proinflammatory cytokines in the brain (Hannestad et al., 2011). Accordingly, another pharmacological meta-analysis reported that overactive IL-6 is significantly attenuated following antidepressant treatment (Hiles et al., 2012b). Activation of the mTOR signaling pathway leads to significant attenuation of overactive stimulated microglial cells by repressing the expression levels of neurotoxic proinflammatory mediators and cytokines, while enhancing the release of the antiinflammatory cytokine IL-10 (Dello Russo et al., 2009; Powell et al., 2012; Dello Russo et al.,
2013). It is possible that ketamine-induced stimulation of mTOR signaling (Hashimoto, 2011; Duman et al., 2012) was involved in the suppression of TNFα and IL-6 production in our human glial cells. It is noteworthy that the effective anti-inflammatory concentrations of ketamine (100 μM) have been found to be clinically relevant (Chang et al., 2005; Wu et al., 2008). We observed that levels of IL-1β and IL-8 released were augmented by ketamine in human astroglial cells. This finding differs from studies in rat macrophage and astroglial cells which showed a ketamineinduced reduction of these cytokines (Shibakawa et al., 2005; Chang et al., 2009; Wu et al., 2012). This difference most probably stems from differences between human astroglial cells and rat immune and glial cells. The relevance of our finding to the rapid antidepressant activity of ketamine is unclear, as the various meta-analyses did not identify any role of IL-1β or IL-8 in the pathophysiology of depression (Dowlati et al., 2010; Hiles et al., 2012b) or in the response to antidepressant treatment (Hannestad et al., 2011; Hiles et al., 2012a). The cause for the inconsistency between the in vitro up-regulation of IL-β expression and down-regulation of both IL-6 and TNFα following exposure of A172 human astroglial cells to ketamine is as yet unclear. Further molecular study is needed to clarify this discrepancy. In conclusion, the present study demonstrates for the first time that ketamine suppresses the in vitro production of the only established depression-relevant proinflammatory cytokines, IL-6 (within
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1 h) and TNFα (within 24 h), in human astroglial cells. This antineuroinflammatory activity may be involved in the rapid antidepressant effect of ketamine. Further in vivo studies with ketamine, mTOR agonists/antagonists, and IL-6/TNFα antagonists are needed to elucidate the complex relationship among ketamine, specific proinflammatory cytokines, neuroinflammation biomarkers, and the rapid antidepressant activity of ketamine. Nevertheless, it appears that it is important to carry out the ketamine studies in relevant human-derived cells as it has been previously studied only in animal models. References Chang, Y., Chen, T.L., Sheu, J.R., Chen, R.M., 2005. Suppressive effects of ketamine on macrophage functions. Toxicol. Appl. Pharmacol. 204, 27–35. Chang, Y., Lee, J.-J., Hsieh, C.-Y., Hsiao, G., Chou, D.-S., Sheu, J.-R., 2009. Inhibitory effects of ketamine on lipopolysaccharide-induced microglial activation. Mediat. Inflamm. http://dx.doi.org/10.1155/2009/705379 (March 30 (Epub)). Chang, H.C., Lin, K.H., Tai, Y.T., Chen, J.T., Chen, R.M., 2010. Lipoteichoic acid-induced TNF-α and IL-6 gene expressions oxidative stress production in macrophages are suppressed by ketamine through downregulating Toll-like receptor 2-mediated activation of ERK1/2 and NFκB. Shock 33, 485–492. Chourbaji, S., Urani, A., Inta, I., Sanchis-Segura, C., Brandwein, C., Zink, M., Schwaninger, M., Gass, P., 2006. IL-6 knockout mice exhibit resistance to stress-induced development of depression-like behaviors. Neurobiol. Dis. 23, 587–594. Dale, O., Somogyi, A.A., Li, Y., Sullivan, T., Shavit, Y., 2012. Does intraoperative ketamine attenuate inflammatory reactivity following surgery? A systematic review and meta-analysis. Anesth. Analg. 115, 934–943. Dantzer, R., O-Connor, J.C., Freund, G.G., Johnson, R.W., Kelley, K.W., 2008. From inflammation to sickness and depression: when the immune system subjugates the brain. Nat. Rev. Neurosci. 9, 46–56. Dean, B., 2011. Understanding the role of inflammatory-related pathways in the pathophysiology and treatment of psychiatric disorders: evidence from human peripheral studies and CNS studies. Int. J. Neuropsychopharmacol. 14, 997–1012. Dello Russo, C., Lisi, L., Tringali, G., Navarra, P., 2009. Involvement of mTOR kinase in cytokine-dependent microglial activation and cell proliferation. Biochem. Pharmacol. 78, 1242–1251. Dello Russo, C., Lisi, L., Feinstein, D.L., Navarra, P., 2013. mTOR kinase, a key player in the regulation of glial functions: relevance for the therapy of multiple sclerosis. Glia 61, 301–311. Dowlati, Y., Hermann, N., Swardfager, W., Liu, H., Sham, L., Reim, E.K., Lanctot, K.L., 2010. A meta-analysis of cytokines in major depression. Biol. Psychiatry 67, 446–457. Duman, R.S., Li, N., Liu, R.-J., Durin, V., Aghajanian, G., 2012. Signaling pathways underlying the rapid antidepressant actions of ketamine. Neuropharmacology 62, 35–41. Erhardt, S., Lim, C.K., Linderholm, K.R., Janelidze, S., Lindqvist, D., Samuelsson, M., Lundberg, K., Postolache, T.T., Träskman-Bendz, L., Guillemin, G.J., Brundin, L., 2013. Connecting inflammation with glutamate agonism in suicidality. Neuropsychopharmacology 38, 743–752. Hannestad, J., DellaGioia, N., Block, M., 2011. The effects of antidepressant medication treatment on serum levels of inflammatory cytokines: a meta-analysis. Neuropsychopharmacology 36, 2452–2459. Hashimoto, K., 2011. Role of the mTOR signaling pathway in the rapid antidepressant action of ketamine. Expert. Rev. Neurother. 11, 33–36. Hiles, S.A., Baker, A.L., de Malmanche, T., Attia, J., 2012a. A meta-analysis of differences in IL-6 and IL-10 between people with and without depression: exploring the causes of heterogeneity. Brain Behav. Immun. 26, 1180–1188.
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