Amelioration of experimental autoimmune encephalomyelitis by clozapine is not associated with defective CD4 T cell responses
© The Author(s). 2017
Received: 12 January 2017
Accepted: 16 March 2017
Published: 29 March 2017
Atypical antipsychotic agents, such as clozapine, are used for treating psychosis and depression and have recently been found to modulate neuroinflammation. We have shown previously that treatment of mice with the atypical antipsychotic agents, clozapine or risperidone, attenuates disease severity in experimental autoimmune encephalomyelitis (EAE); however, the mechanism by which they are protective is unknown. In this study, we investigated the effects of clozapine on CD4+ T cell responses and found that clozapine did not significantly affect the expansion of myelin-specific T cells, their differentiation into pathogenic subsets, or their encephalitogenic capacity to induce EAE. Interestingly, although clozapine enhanced differentiation of regulatory T (Treg) cells, in vivo neutralization of Tregs indicated that Tregs were not responsible for the protective effects of clozapine during the induction and effector phase of EAE. Taken together, our studies indicate that clozapine does not mediate its protective effects by directly altering CD4 T cells.
KeywordsEAE MS Atypical antipsychotics Neuroinflammation
Multiple sclerosis (MS) is a debilitating disease of the central nervous system (CNS) that is mediated by inflammatory demyelination of the myelin sheaths that surround neuronal axons . Immune-mediated demyelination impairs nerve conduction and causes the clinical symptoms of MS, which are diverse and include loss of coordination, visual disturbances, fatigue, and paralysis; the degree of which is assessed using the Expanded Disability Status Scale . Although there are now a number of therapeutic options for relapsing-remitting MS patients, they are not similarly effective in all patients and only one has shown potential in the primary progressive forms of MS. This is not surprising given that blood-brain barrier (BBB) disruption is minimal in the progressive forms  and current therapeutic strategies are protein - based such as glatiramer acetate, interferon-β, and natalizumab, which have limited capacity to cross the intact BBB . In addition, while ocrelizumab has shown limited potential in the treatment of primary progressive MS , its efficacy in patients may partly be determined by the severity of BBB disruption. Therefore, effective therapeutic options are urgently needed.
MS is considered a neuroinflammatory disease and shares some pathophysiological similarities with several neuropsychiatric disorders. For example, it is becoming increasingly evident that neuropsychiatric disorders such as schizophrenia and major depressive disorders are associated with inflammation in the CNS and characterized by chronic microglial activation . These diseases are associated with elevated inflammatory markers and, in particular, the expression of inflammatory cytokines interleukin (IL-) 1β, IL-6, and tumor necrosis factor-α, which were found in post-mortem samples from suicide victims with major depression  and schizophrenia . Clinical treatments for psychiatric disease like fluoxetine, risperidone, quetiapine, and clozapine have recently been acknowledged for their immunomodulatory effects in various models of inflammation [9–12] and show promise as immune-modulating agents. Abnormal serum cytokine levels in schizophrenic patients are normalized by treatment with atypical antipsychotics in some studies , suggesting an immune - altering effect.
Recently, we have shown that the atypical antipsychotic agents risperidone and clozapine are effective at reducing disease in experimental autoimmune encephalomyelitis (EAE) , demonstrating that atypical antipsychotic agents are potential treatments for MS. While these agents show promise as therapeutics in MS and other neuroinflammatory disorders, the mechanism by which they reduce disease and alter inflammation is not completely understood. Given that EAE is a disease that is driven by autoreactive Th1 and Th17 cells and that defective development of these subsets greatly alters the susceptibility of mice to EAE, this study aimed to investigate if clozapine were able to reduce disease by impairing CD4 T cell-mediated induction of EAE.
Materials and methods
All mice used were female and aged between 8 and 12 weeks. C57BL6/J were purchased from the Biomedical Research Unit of the Malaghan Institute of Medical Research (Wellington, NZ) and housed in the Victoria University of Wellington animal facility. 2D2 TCRMOG35-55 (CD45.2) mice expressing the T cell receptor (TCR) specific for myelin oligodendrocyte glycoprotein (MOG) 35-55 and B6-SJptprca (CD45.1) mice were bred at the Victoria University of Wellington animal facility. Food and water were available ad libitum.
Induction of EAE and clozapine treatment
EAE was induced by subcutaneous (s.c) immunization in the rear flanks of mice with MOG35-55 (50 μg/mouse; Genscript, Piscataway, NJ, USA) emulsified in complete Freund’s adjuvant (500 μg/mouse Mycobacterium tuberculosis) followed by intraperitoneal (i.p) injection of 200 ng/mouse pertussis toxin on days 0 and 2. Mice were weighed and scored daily for signs of disease by a non-blinded investigator, using the following disease rating scale: 0, normal; 1, partial tail paralysis; 2, full tail paralysis; 3, full paralysis of one hind limb; 4, full paralysis of both hind limbs; and 5, moribund. Clozapine was kindly supplied by Douglas Pharmaceuticals Ltd. (Auckland, New Zealand) and added to drinking water 1 day prior to EAE induction at a concentration calculated to achieve a daily dose of 60 mg/kg. For regulatory T (Treg) neutralization, mice were injected with 200 μg of anti-CD25 monoclonal antibody (PC61; BioXCell, West Lebanon, NH, USA) or rat IgG (Sigma-Aldrich, St. Louis, MO, USA) i.p 3 days prior to EAE induction and maintained with repeat injections at 7 and 14 days post immunization (d.p.i).
Adoptively transferred EAE
Donor C57BL6/J mice were immunized for EAE as described above but without the pertussis toxin. Spleens and draining lymph nodes were harvested at 12 d.p.i, and single-cell suspensions were made by passage through a 70-μm cell strainer (BD Biosciences, Franklin Lakes, NJ). Red blood cells were lysed using Red Blood Cell Lysis Buffer Hybri-Max (Sigma-Aldrich, St. Louis, MO). Splenocytes were washed and re-suspended in culture medium containing Dulbecco’s modified Eagle medium (DMEM), 100 U/mL penicillin, 100 μg/mL streptomycin, 10 mM HEPES, 2 mM l-glutamine, 50 μM 2-mercaptoethanol, non-essential amino acids and 10% fetal calf serum (all from Life Technologies, Carlsbad, CA, USA). Donor cells were cultured in tissue culture flasks at 1 × 107 cells/mL with IL-12p70 (20 ng/mL; Peprotech, Rocky Hill, NJ, USA), XMG1.2 (10 μg/mL; BioXCell, USA), MOG35-55 (50 μg/mL), and either vehicle or clozapine (20 μM) at 37 °C and 5% CO2. After 96 h of stimulation, non-adherent donor cells were harvested and injected into recipient naïve C57BL/6J mice (2 × 107 cells/mouse) followed by pertussis toxin (200 ng/mouse) on days 0 and 2. Mice were monitored daily for disease as described above.
In vivo proliferation assay
Splenocytes and lymph node cells were isolated from 2D2 TCRMOG35-55 mice as described and labelled with CellTrace CFSE Cell Proliferation Kit (Life Technologies) according to the manufacturer’s protocol. CFSE-labelled 2D2 TCRMOG35-55 cells (2 × 107) were injected i.p into B6-SJptprca 1 day prior to EAE induction. At day 0, mice were immunized to induce EAE and euthanized 5 days post immunization. Proliferation of CD45.2+CD4+ cells were assessed in peripheral blood (cardiac puncture), draining lymph nodes (inguinal and mesenteric), and spleen by flow cytometry.
In vitro assays
Splenocytes, isolated as described above, were seeded in flat bottom 96-well plates (BD Biosciences) at 1 × 106 cells/well. Splenocytes were stimulated with MOG35-55 (50 μg/mL) (Genscript) for 72 h before supernatant was collected for cytokine detection.
T cell differentiation
Splenocytes were plated at 1 × 106 cells/well in flat bottom 96-well plates (BD Biosciences). Th1 cells were induced by addition of IL-12p70 (20 ng/mL) and 11B11 (10 μg/mL; a gift from the Malaghan Institute of Medical Research). Th17 cells were induced by addition of transforming growth factor (TGF-) β1 (5 ng/mL; eBioscience, San Diego, CA), IL-6 (20 ng/mL; BD Biosciences), and XMG1.2 (10 μg/mL; BioXCell). Tregs were induced by addition of TGF-β1 (5 ng/mL), MP5-20F3 (10 μg/mL), XMG1.2 (10 μg/mL), and 11B11 (10 μg/mL). Clozapine or vehicle (AcOH) was added at indicated final concentrations. Cells were incubated for 72 h at 37 °C and 5% CO2 after which supernatant was collected and T cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA), 500 ng/mL ionomycin (Sigma-Aldrich) and Golgi Stop (BD Biosciences) used according to the manufacturer’s recommendations for a further 5 h before analysis of T cell subsets by intracellular flow cytometry.
For the detection of immune cells, cytokines, and transcription factors, the following antibodies were used: CD4 (GK1.5; Biolegend, San Diego, CA, USA), CD45 (30-F11; Biolegend), CD25 (PC61; Biolegend), CD8 (53-6.7; Biolegend), CD11b (M1/70; Biolegend), CD3 (17A2; Biolegend), IFN-γ (XMG1.2; BD Biosciences), T-bet (04-46; BD Biosciences), RORγT (Q31-378; BD Biosciences), IL-17A (TC11-18H10; BD Biosciences), Foxp3 (150D; Biolegend), CD45.1 (A20; BD Biosciences), CD45.2 (104; BD Biosciences), goat antirabbit FITC (BD Biosciences), and rabbit antimouse dopamine D1 and D2 receptor (both from Merck, Darmstadt, Germany). For cell surface staining, cells were incubated with Fc Block (2.4G2; BD Biosciences) for 15 min prior to staining with fluorescently labelled antibodies for 20 min on ice. For intracellular staining, cells were fixed, permeabilized, and stained using Transcription Factor Buffer Set (BD Biosciences) according to the manufacturer’s protocol. Flow cytometry was performed on a BD FACS Canto II (BD Biosciences) and analyzed using Flowjo software (Treestar Inc., Ashland, OR, USA).
Cytokines in the supernatant were measured by LEGENDplex Mouse Inflammation Panel multi-analyte flow assay kit (Biolegend) or sandwich ELISA (BD Biosciences) according to the manufacturer’s recommendations.
Data were graphed and analyzed by one-way ANOVA, two-way ANOVA, Student’s t test, and Mann-Whitney test as indicated in each figure using GraphPad Prism software (GraphPad, La Jolla, CA, USA). Dunnett’s and Sidak’s post hoc multiple comparison test was performed to determine significant differences between test groups. P values of <0.05 were considered significant.
Clozapine treatment delayed onset and reduced severity of EAE
Treatment with clozapine did not alter MOG-specific T cell expansion upon EAE induction
Clozapine did not alter pathogenic T cell subsets but promoted Tregs
EAE is a disease that is driven by pathogenic Th1 cells expressing T-bet and IFN-γ as well as Th17 cells expressing RORγT and IL-17A. This dependence was demonstrated when it was shown that mice which have defective Th1 and Th17 development (i.e., IL-12p40-deficient mice) were resistant to EAE . In addition, severity of EAE is regulated by Foxp3-expressing regulatory T cells (Tregs) that have immunosuppressive capacity . Clozapine has previously been shown to decrease production of IFN-γ and enhance IL-4 and IL-10 in poly (I:C)-stimulated PBMC cultures from schizophrenia patients, which suggests an effect on CD4+ T cell differentiation . To assess development of Th1, Th17, and Treg cell subsets during EAE, we measured RORγT, T-bet, and Foxp3 expression in peripheral CD4+ T cells during EAE as these transcription factors are master regulators of Th1 , Th17 , and Treg  cells, respectively.
Protection from EAE by clozapine was not dependent on regulatory CD4+ T cells
Clozapine did not alter encephalitogenicity of MOG35-55-specific CD4+ T cells
Atypical antipsychotics like clozapine readily cross the BBB and are used for treating psychiatric diseases like schizophrenia. Recently, these medicines have been recognized as immune-modulating agents as they are able to suppress inflammation associated with schizophrenia . Few studies are available describing the effects of clozapine treatment on the immune response, but these have shown that clozapine can inhibit the activation of immune cells like microglia and T cells in response to inflammatory stimuli [12, 30, 31]. Given that both schizophrenia and multiple sclerosis are inflammatory diseases of the central nervous system and that clozapine is able to reduce inflammation in the CNS, we wished to investigate whether clozapine could be re-purposed to treat progressive forms of multiple sclerosis for which there is only one effective therapeutic option available . Indeed, we have shown that clozapine is able to reduce disease in EAE, indicating that it is a great candidate for multiple sclerosis; however, the precise mechanism is not yet known. In this study, we show that clozapine treatment does not significantly alter the development of robust antigen-specific T cell responses despite effective suppression of EAE disease.
The effect of clozapine on T cell activation has previously been investigated in human PBMCs and is consistent with our findings, showing that clozapine inhibits secretion of IFN-γ after stimulation of CD3 and CD28 . In our study, we showed that clozapine inhibits IFN-γ secretion in response to an antigen; however, in contrast to the previous study, we did not observe significant alterations in T cell differentiation to Th1 or Th17 in vitro or during EAE.
One of the most striking findings in this study is that clozapine promotes in vitro differentiation of Tregs and expression of Foxp3, a key transcription factor for the development and function of Tregs. Although clozapine had the potential to augment Treg function, it did not appear to be important for disease protection during EAE as neutralization of CD25+ Tregs had no effect on disease protection. Interestingly, while clozapine promoted Treg differentiation, little effect was observed during differentiation to Th1 and Th17 cells. This observation could be explained by clozapine activating the Akt pathway as has reported in various tissues [32–34], and since this pathway is important for CD3, CD28 and cytokine signalling . Akt is upstream of the mammalian target of rapamycin (mTOR) and regulates T cell differentiation during activation. For example, suppression of mTOR activity promotes the generation of Foxp3+ Tregs , and the deletion of mTOR in CD4 T cells enhances differentiation of Tregs but not Th1, Th2, or Th17 cells , indicating a potential role for mTOR in the mechanism of action of clozapine.
Despite the effects of clozapine in an in vitro differentiation model, we did not find that these alterations were important for disease protection during EAE as no difference in Th1, Th17, or Treg differentiation was detected with treatment during the induction and effector phase of disease even though mice were protected from disease. This suggests that protection may not be mediated through alteration of the T cell response directly. It is possible that clozapine alters the ability of macrophages and microglia to become activated and initiate disease in the CNS independent of CD4 T cells, although this is yet to be shown. However, clozapine has recently been shown to alter the production of inflammatory cytokines from in vitro cultured microglia , and we have shown previously that clozapine suppresses the production of IL-12 in bone marrow-derived macrophages stimulated with lipopolysaccharide indicating that clozapine is able to alter macrophage activation . This effect may be important given that once autoreactive T cells initiate the inflammatory response in the CNS, inflammatory monocytes are recruited and their numbers correlate to severity of disease . The ability of clozapine to alter macrophage activation to be less inflammatory may contribute to attenuated disease during treatment as these cells are predominant in the demyelinating regions of the brain in EAE and MS .
In conclusion, while clozapine is effective at reducing EAE in mice, we did not find defective antigen-specific T cell responses. Given that we have recently shown clozapine to inhibit the activation of microglia in the CNS during EAE  and that clozapine alters the activation of LPS-stimulated macrophages directly , instead, we believe the mechanism of protection may involve other immune pathways including the alteration of myeloid activation in the CNS specifically. We have recently shown that clozapine effectively reduces established EAE disease when given therapeutically at 12 or 20 d.p.i, indicating that clozapine has potential as a therapy for MS . Finally, it has been suggested that prospective therapies for progressive MS have immune-modulating properties that target myeloid cell activation in the CNS and readily cross an intact BBB , further supporting the feasibility of using clozapine as a potential therapy for progressive MS.
Central nervous system
Days post immunization
Experimental autoimmune encephalomyelitis
Myelin oligodendrocyte glycoprotein
Phorbol 12-myristate 13-acetate
T cell receptor
Transforming growth factor
Regulatory T cell
The authors would like to thank Katharina Robichon for her help and technical advice.
This study was funded by the Neurological Foundation of New Zealand (1433-PG) and the Ministry of Business, Innovation, and Employment (RTVU1503).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
PZ performed all of the experiments, analyzed the data, and wrote the manuscript. BC contributed to the experimental design. ACL contributed to the experimental design and data analysis and edited the final manuscript. All authors read and approved the final manuscript.
ACL and BC hold a patent for the use of clozapine during MS. The authors declare that they have no competing interests.
Consent for publication
The Victoria University of Wellington Animal Ethics Committee approved all experimental procedures used in this study (2014-R23).
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