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ACTIVATION OF THE ADENOSINE A2A RECEPTOR ATTENUATES EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS AND IS ASSOCIATED WITH INCREASED INTRACELLULAR CALCIUM LEVELS
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YUMEI LIU, a HAIFENG ZOU, a PING ZHAO, a BO SUN, a JINGHUA WANG, a QINGFEI KONG, a LILI MU, a SIHAN ZHAO, a GUANGYOU WANG, a DANDAN WANG, a YAO ZHANG, a JIAYING ZHAO, a PENGQI YIN, a LEI LIU, b XIULI ZHAO c* AND HULUN LI a,d*
may offer a therapeutic option for this disease. Ó 2016 Published by Elsevier Ltd on behalf of IBRO.
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a Department of Neurobiology, Neurobiology Key Laboratory, Harbin Medical University, Education Department of Heilongjiang Province, Harbin, Heilongjiang 150086, China
Key words: multiple sclerosis, adenosine A2A receptor, experimental autoimmune encephalomyelitis, intracellular calcium.
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b Department of Psychiatry, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, China
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c Department of Neurology, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, China
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d Key Laboratories of Education Ministry for Myocardial Ischemia Mechanism and Treatment, Harbin, Heilongjiang 150086, China
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Abstract—Multiple sclerosis (MS) is a common autoimmune disease that inevitably causes inflammatory nerve demyelination. However, an effective approach to prevent its course is still lacking and urgently needed. Recently, the adenosine A2A receptor (A2AR) has emerged as a novel inflammation regulator. Manipulation of A2AR activity may suppress the MS process and protect against nerve damage. To test this hypothesis, we treated murine experimental autoimmune encephalomyelitis (EAE), a model for MS, with the selective A2AR agonist, CGS21680 (CGS). We evaluated the effects of CGS on the pathological features of EAE progression, including CNS cellular infiltration, inflammatory cytokine expression, lymphocyte proliferation, and cell surface markers. Treatment with CGS significantly suppressed specific lymphocyte proliferation, reduced infiltration of CD4+ T lymphocytes, and attenuated the expression of inflammatory cytokines, which in turn inhibited the EAE progression. For the first time, we demonstrate that CGS can increase the intracellular calcium concentration ([Ca2+]i) in murine lymphocytes, which may be the mechanism underlying the suppressive effects of CGS-induced A2AR activation on EAE progression. Our findings strongly suggest that A2AR is a potential therapeutic target for MS and provide insight into the mechanism of action of A2AR agonists, which
*Corresponding authors. Address: Department of Neurology, The First Affiliated Hospital of Harbin Medical University, 23 Youzheng Street, Nangang District, Harbin, Heilongjiang 150001, China. (X. Zhao). E-mail addresses:
[email protected] (X. Zhao),
[email protected] (H. Li). Abbreviations: A2AR, adenosine A2A receptor; CFA, complete Freund’s adjuvant; CGS, CGS21680; CRAC, Ca2+ release-activated calcium; EAE, experimental autoimmune encephalomyelitis; FITC, fluorescein isothiocyanate; MS, multiple sclerosis; PBS, phosphatebuffered saline; PE, phycoerythrin. http://dx.doi.org/10.1016/j.neuroscience.2016.05.028 0306-4522/Ó 2016 Published by Elsevier Ltd on behalf of IBRO. 1
INTRODUCTION
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Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease that is considered to have CD4+-mediated autoimmune pathogenesis. Murine experimental autoimmune encephalomyelitis (EAE), an animal model of MS, is a T cell-mediated autoimmune disease that can be induced by immunization with myelin protein peptides and used to assess the efficacy of putative therapeutic agents (Paterson, 1986; Dittel, 2008). The CD4+ T helper (Th) cell plays a key role in autoimmune disease (Bonnie, 2008). Studies show that the balance of Th1, Th2, and regulatory T (Treg) cells plays a role in EAE pathogenesis (Chen et al., 2006). When the Th1:Th2 cell ratio shifts to a predominantly Th2 profile, the effects of proinflammatory Th1 cytokines, such as IFN-c and TNF-a, are countered, and the severity of autoimmune disease is alleviated (Yoles et al., 2001; Weaver et al., 2005). Recent reports show that IL-17, a proinflammatory cytokine released from Th17 cells, also plays an important role in the pathogenesis of allergic and autoimmune diseases (Nakae et al., 2003a,b; Mu et al., 2009), whereas Treg and Th2 cytokines play an antiinflammatory role and maintain tolerance to self-antigens. Therefore, a shift from a Treg/Th2 profile to a Th1/Th17 profile may be responsible for the development and/or progression of EAE. Adenosine released from metabolically active cells and generated extracellularly by degradation of released adenosine triphosphate (ATP) regulates the function of immune cells and other cell types (Erdmann et al., 2005; Hasko´ and Pacher, 2008). Through its binding to the adenosine A2A receptor (A2AR), adenosine can modulate numerous cellular functions and protect cells and tissues from inflammation. Therefore, adenosine is said to ‘‘put the brake on inflammation” (Kirkpatrick, 2002). The importance of A2AR in mediating this negative feedback loop has been demonstrated by the finding that A2AR knockout mice lack the ability to control inflammation,
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which leads to extensive and fatal tissue destruction (Ohta and Sitkovsky, 2001). The expression of A2AR is the highest in immune cells, including monocytes and macrophages (Khoa et al., 2001), lymphocytes (Koshiba et al., 1999; Apasov et al., 2000; Armstrong et al., 2001), dendritic cells, neutrophils, natural killer cells (Williams et al., 1997; Harish et al., 2003; Raskovalova et al., 2005), and natural killer T cells (Lappas et al., 2006). Numerous studies have shown that A2ARs are the primary adenosine receptors that mediate lymphocyte responses (Day et al., 2004; Odashima et al., 2005a; Blackburn et al., 2009; Lau et al., 2009; Alam et al., 2009). When activated, adenosine receptors can downregulate inflammation and protect against tissue damage (Link et al., 2000; Ohta and Sitkovsky, 2001). Many studies have suggested that adenosine can inhibit cytokine production in vitro and in vivo via stimulating A2ARs. Schnurr et al. (2004) have found that adenosine significantly inhibits the production of IFN-a, IL-6, and IL-12 by activated plasmacytoid dendritic cells via A2ARs. Lappas et al. (2005) have reported that A2AR activation in CD4+ T cells inhibits IFN-c release. Another study has demonstrated that A2AR activation exerts strong inhibitory actions on both Th1 and Th2 cells during early and late stages of lymphocyte activation (Cso´ka et al., 2008). The A2AR agonist, ATL313, can inhibit lymphocyte proliferation through its actions on both T lymphocytes and APCs and can significantly reduce IFN-c release in a dose-dependent manner (Sevigny et al., 2007). A2AR stimulation in human Th cells isolated from blood or gastric biopsy specimens efficiently suppresses IL-2, IFN-c, and TNF-a production (Alam et al., 2009). Lukashev et al. (2003) have shown that A2ARs are variably expressed in T cell subsets and may regulate cytokine production in activated T lymphocytes; however, they focused only on Th1 and Th2 cells. A2AR expression in Th17 and Treg subsets was not measured. In MS and EAE, T cells are predominately of the Th1 or Th17 phenotype, as indicated by their ability to generate IFN-c and IL17, but less IL-4 (a Th2 cytokine). Recent reports have indicated that loss of the A2AR exacerbated EAE pathology in mice (Yao et al., 2012; Wang et al., 2014). In addition, Mills et al. (2008, 2012) reported that A2AR lymphocyte expression is essential for limiting the severity of EAE, and they also demonstrated that A2AR (/) lymphocytes are more proliferative and produce more IFN-c than their wild-type counterparts. These results indicate that the A2AR plays a key role in regulating the lymphocyte inflammatory responses. Calcium signals are essential for diverse cellular functions, including differentiation, effector function, and gene transcription. In the brain, adenosine A1, A2B, and A3 receptors can promote inositol triphosphate receptorregulated calcium release from intracellular stores (Doengi et al., 2008). In lymphocytes and other cell types, store-operated Ca2+ entry through Ca2+ releaseactivated calcium (CRAC) channels is the main mechanism for entry of extracellular Ca2+ across the plasma membrane (Lewis, 2001). Opening of CRAC channels leads directly to sustained increase of [Ca2+]i, which, in
turn, affects T cell functions that have long-term consequences, including lymphocyte proliferation and the differentiation of naı¨ ve T cells into various effector or memory T cells. The aims of our study were to determine whether a relationship exists between A2AR expression and the severity of EAE, to evaluate the effects of an A2AR agonist on the development and progression of EAE, and to elucidate a plausible mechanism that would explain the effects of the A2AR agonist on EAE. We hypothesized that stimulation of A2AR activity in mice would result in inhibition of EAE development and/or progression and alleviation of disease signs.
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EXPERIMENTAL PROCEDURES
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Induction and clinical assessment of EAE
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Female C57BL/6 mice, 6–8- week-old, were purchased from the Peking Vital River Laboratory Animal Ltd. (Peking, China). All animals were bred and maintained in accordance with the guidelines for the Care and Use of Laboratory Animals published by the China National Institute of Health. Mice in the EAE group were immunized subcutaneously with 200 lg of myelin oligodendrocyte glycoprotein (MOG35–55, MEVGWYRSPFSRVVHLYRNGK, AC Scientific, China) peptide emulsified in incomplete Freund’s adjuvant (IFA, St. Louis, MO, USA) that contained 250 lg of Mycobacterium tuberculosis H37RA extract (Difco, Detroit, MI, USA). Each mouse received 200 ng of pertussis toxin (Campbell, CA, USA) in 200 ll of phosphate-buffered saline (PBS) intravenously on day 0 and day 2 of immunization. The control group was immunized with complete Freund’s adjuvant (CFA) alone without MOG35–55 peptide and no follow-up intravenous PT administration. Animals in all groups were weighed at the beginning of the experiment and every day thereafter until the end of the study period (28 days postimmunization). Mice were monitored daily after immunization. The clinical signs of EAE were assigned using the following scoring system: 0, normal mouse without signs of disease; 1, limp tail; 2, limp tail and hind limb weakness; 3, partial hind limb paralysis; 4, complete hind limb paralysis and/or forelimb paralysis; and 5, moribund state or death due to EAE. Mean clinical scores for each group were calculated daily by adding the scores of individual mice (including mice not developing signs of EAE) and dividing by the number of mice in the group. Average day of onset was calculated by adding the first days of clinical signs of individual mice and dividing by the number of mice in the group.
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Treatment with the CGS21680
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2(4-((2-Carboxymethyl)phenyl)ethylamino)-5 -Nethylcarboxamidoadenosine (CGS-21680) was obtained from Tocris, UK. CGS-21680 was dissolved in 10% DMSO and diluted in 0.9% NaCl for i.p. On the day of immunization, the mice were arbitrarily assigned into
Please cite this article in press as: Liu Y et al. Activation of the adenosine A2A receptor attenuates experimental autoimmune encephalomyelitis and is associated with increased intracellular calcium levels. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.05.028
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four groups: CFA (control), EAE, CGS-1, and CGS-5. Mice in the latter two groups were immunized with MOG35–55 peptide, as were the EAE mice, and treated with 0.01 mg/kg CGS21680 (CGS-1) and 0.05 mg/kg CGS21680 (CGS-5) (Tocris, UK) (Fozard et al., 2002) intraperitoneally every days for 28 days postimmunization.
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Lymphocyte isolation and culture
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Lymphocytes were isolated from the lymph nodes of EAE and CFA mice on 7, 14, 21 and 28 days postimmunization with MOG35–55. Cells were 95% viable, as determined by Trypan blue exclusion. After washing 3 times with Hank’s balanced salt solution, 2 106 cells/mL cells were cultured in EAE lymphocyte culture medium containing RPMI 1640 medium plus 10% fetal bovine serum at 37 °C with 5% CO2.
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Histology
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Lumbar spinal cord sections of were removed from mice at the peak of clinical disease and fixed in 10% formalin. Paraffin-embedded transverse sections were stained with H&E or Luxol fast blue for visualization of inflammatory infiltrates and demyelination, respectively, by light microscopy.
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Lymphocyte isolation from nerve tissue
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Lymphocyte in the brain and spinal was examined using Percoll gradient method as described previously (Gelderblom et al., 2009; Zhang et al., 2014). Mice were euthanized and perfused with PBS at the peak of clinical disease. Brains were dissected with cerebella removed, and spinal cords were isolated from mice. Cerebrum and spinal cords were pressed through 150-lm and 75lm filters. Next, cells were separated from myelin and debris using 70% and 30% Percoll (GE Healthcare, Pittsburgh, USA) gradients, respectively, followed by centrifugation. Cells from the interface of 70%/30% Percoll gradients were then incubated with fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (eBioscience, San Diego, CA, USA) for 30 min and were then analyzed on FacsCalibur (BD Biosciences, Franklin Lakes, USA) by a technician without previous knowledge of grouping using and acquired data were processed with FlowJo software (TreeStar Inc., Ashland, USA).
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Measurement of A2AR expression by immunohistochemistry and flow cytometry Mice were euthanized 14 days postimmunization. Spleens were isolated and snap frozen in liquid nitrogen. A cryostat was used to generate 10-lm-thick sections, which were mounted on glass slides. Sections were incubated with goat anti-mouse A2AR (1:200, Santa Cruz Biological, CA, USA), followed by incubation with horseradish peroxidase-conjugated anti-goat IgG (1:1000). 3,30 -Diaminobenzidine was used as a chromogen to visualize labeled antigens. Nuclei were later stained with hematoxylin, and tissue sections were digitally imaged using Image Pro Plus software (Media
Cybernetics, Silver Springs, MD, USA). The results were averaged and expressed as positive cells/ocular field. For flow cytometry analysis, lymphocytes were isolated from lymph nodes of EAE and CFA mice at 7, 14, 21, or 28 days postimmunization with MOG35–55 and analyzed for A2AR expression by fluorescent staining. The fluorochrome-conjugated antibodies used for the direct staining of lymphocytes were FITC-conjugated anti-CD4 and PerCP-Cy5.5-conjugated anti-A2AR (Santa Cruz Biological, CA, USA). Two-color immunofluorescent analysis was performed on the BD FACS Calibur flow cytometer. Isotype-matched, PerCPCy5.5- and FITC–conjugated monoclonal antibodies (mAbs) of irrelevant specificity were used as negative controls.
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T lymphocyte proliferation assay
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Lymphocytes were resuspended at a final concentration of 5 105 cells/well and cultured in flat-bottomed, 96well microplates. After exposure to PBS (control), AChR97–116 (10 lg/ml, Sangon Ltd, China), concanavalin A (ConA, 5 lg/ml, Sigma–Aldrich, Shanghai, China), MOG35–55 (20 lg/ml), CGS ( 10 nM) or CGS ( 10 nM) + SCH58261 (10 nM) for 72 h, cell viability was measured by the Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) with water-soluble tetrazolium salt-8 (WST-8) reagent, as previously described (Chang et al., 2006; Liu et al., 2010). Briefly, 10 lL of CCK-8 solution were added to each well, followed by incubation for 3–4 h at 37 °C and measurement of the optical density at 490 nm.
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Detection of Th cell subtypes
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Intracellular and extracellular cytokine detection was carried out by flow cytometry as described previously (Lohr et al., 2006). Briefly, all cells were incubated for 5 h with Brefeldin A (1:1000 dilution, eBioscience, Inc.), an inhibitor of intracellular protein transport, and then washed and resuspended in FACS medium with 1% bovine serum albumin. The cells were first incubated with FITC-conjugated anti-mouse-CD4 (eBioscience, Inc.) for 30 min at 4 °C. After fixation and permeabilization, intracytoplasmic staining was carried out using one of the following fluorescent-labeled antibodies: phycoerythrin (PE)-conjugated anti-mouse-IFN-c, IL-4, Foxp3, or IL-17 (all from BD Biosciences). Samples were analyzed within 24 h on the BD FACScan System equipped with Cell Quest software (BD Biosciences). Isotype-matched PEand FITC-conjugated mAbs of irrelevant specificity were used as negative controls.
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Cytokine assays
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Lymphocytes from either EAE or CFA mice (2 10 cells/ mL) were cultured in the presence of MOG35–55 (20 lg/ mL) with or without CGS (0.01 lM). Cell culture supernatants were collected at 72 h and frozen at 80 °C until needed for the cytokine assays. IFN-c, IL4, TGF-b, and IL-17 levels were measured using ELISA 6
Please cite this article in press as: Liu Y et al. Activation of the adenosine A2A receptor attenuates experimental autoimmune encephalomyelitis and is associated with increased intracellular calcium levels. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.05.028
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kits (BD Biosciences). A standard curve for each assay was generated using recombinant mouse cytokines, and the concentration of each cytokine in the cell supernatants was determined by interpolation from the respective standard curve.
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Measurement of [Ca2+]i
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Relative changes in lymphocyte [Ca2+]i were derived from changes in the intensity of Fluo-3-loaded cells (Sei et al., 1999; Fujigaki et al., 2006). Lymphocytes (2 106 cells/mL) from EAE mice were cultured in the presence of MOG35–55 (20 lg/mL) alone, with CGS (0.01 lM) or with CGS21680 (0.01 lM) and SCH51280 (0.01 lM). These cells were loaded with 1 lM Fluo-3AM (ABCAM, UK) by incubation in subdued light for 30 min at 25 °C. Fluo-3loaded cells were stained with Percp-Cy5.5-conjugated anti-CD4 mAb to label T cells selectively. Finally, cells were washed 3 times with Mg2+- and Ca2+-free PBS, resuspended in 1 ml PBS, and analyzed on the BD FACScan System.
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Statistical analysis
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Data are expressed as mean ± standard deviation (SD) or standard error (SE). Differences between groups were analyzed by the two-tailed Student’s t-test for paired and unpaired data in the GraphPad software package (GraphPad Software, Inc., CA, USA). A pvalue less than 0.05 was considered statistically significant.
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RESULTS
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A2AR expression is increased in spleen and lymphocytes of EAE mice To measure lymphocyte expression of A2AR in mice with EAE, spleen sections were examined by immunohistochemistry, and semiquantitative analysis was performed blindly by investigators. A2AR expression was detected in the spleens of mice with EAE and mice treated only with CFA. However, the number of A2AR+ spleen cells in EAE mice was higher (p < 0.001) compared to that in CFA controls (Fig. 1). Examination of A2AR+/CD4+ double-labeled T cells by flow cytometry was used to evaluate A2AR expression over the time course of EAE progression. Compared to that in CFA controls, the number of A2AR+/CD4+ T cells in EAE mice was higher at 7 (p < 0.01), 14 (p < 0.05), and 21 (p < 0.05) days postimmunization. No significant difference was observed in the number of double-positive cells between the EAE and CFA groups at 28 days postimmunization, which was the end of the study period (Fig. 2). Effect of CGS21680 (CGS) on T lymphocyte proliferation Proliferation of T cells from EAE mice was markedly greater when exposed to myelin oligodendrocyte glycoprotein (MOG)35–55 peptide than when exposed to the negative control, acetylcholine receptor (AChR)97–116
Fig. 1. Expression of A2AR in CFA and EAE mice. (A) Spleen section harvested from either EAE or CFA control mice at the peak of clinical disease were screened for A2AR+ cells. Arrow indicates A2AR positive cells. Scale bar = 50 lm. (B) Graphic representation of the data shown in (A). Each column represents the mean ± SE of six sections per group. ***p < 0.001 vs. CFA group, two-tailed Student’s t-test for unpaired data.
peptide, suggesting that EAE lymphocyte proliferation was specific to MOG35–55 (Fig. 3A). MOG35–55 had a greater stimulatory effect on the proliferation of T cells from EAE mice compared to CFA mice (p < 0.01). Con A, used as a positive control, exerted similar effects on the proliferation of T cells from EAE and CFA mice. Treatment with 10 nM CGS for 72 h significantly suppressed the MOG35–55-induced proliferation of T cells derived from EAE mice. This inhibitory effect of CGS was abrogated by the addition of the A2AR antagonists SCH58261. The effect of A2AR stimulation on T cell proliferation was assessed by treating lymphocyte cultures with different concentrations of CGS21680, 109, 108, 107, 106, 105 and 104 M.
Please cite this article in press as: Liu Y et al. Activation of the adenosine A2A receptor attenuates experimental autoimmune encephalomyelitis and is associated with increased intracellular calcium levels. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.05.028
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Fig. 2. A2AR expression in CD4+ T cells in EAE mice. (A) Representative density plot showing A2AR expression in CD4+ T cells isolated from mice at 7, 14, 21, and 28 days postimmunization. (B) Graphic representation of the data shown in (A). Data are expressed as the mean ± SD of nine mice/group from three independent experiments. *p < 0.05, **p < 0.01 vs. CFA group.
Fig. 3. Effect of CGS on lymphocyte proliferation. (A) Lymphocytes were stimulated with no antigen (0 Ag, control), AChR (10 lg/ml), ConA (5 lg/ ml), MOG (20 lg/ml), MOG + CGS (10 nM) or MOG + CGS (10 nM) + SCH58261 (10 nM) for 72 h. CGS21680 treatment decreased MOGspecific lymphocyte proliferation compared with cells stimulated with MOG35–55 only (**p < 0.01). AChR and ConA were used as negative and positive controls, respectively. Each column represents the mean ± SE of n = 6/group from three independent experiments. *p < 0.01, #p < 0.05, p < 0.05. (B) Lymphocyte proliferation was decreased following CGS21680 treatment in a dose-dependent manner. *p < 0.05, **p < 0.01, *** p < 0.001 vs. 0 M CGS group.
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We also observed that proliferation of T cells from EAE mice exhibited a significant, negative dose-dependent response to increasing CGS concentrations from 108 to 104 M (Fig. 3B).
CGS alters the distribution of Th subsets in vitro The effect of CGS (10 nM) on the distribution of CD4+ Th subsets derived from EAE and CFA mice was examined by flow cytometry. This analysis revealed a decrease in the Th1 (CD4+/IFN-c+; p < 0.05), Th2 (CD4+/IL-4+; p < 0.05), and Th17 (CD4+/IL-17+; p < 0.01) subsets and an increase in the Treg subset (CD4+/CD25+/ Foxp3+; p < 0.001) in CGS-treated lymphocyte cultures of EAE mice compared to those not treated with CGS (Fig. 4A, B). To confirm the observed Th profile, a cytokine profile analysis was performed. Supernatants of CGS-treated cells exhibited lower mean concentrations of IFN-c (p < 0.01), IL-4 (p < 0.05), and IL-17 (p < 0.05) compared to supernatants of untreated cells (Fig. 4C). In contrast, the mean concentration of transforming growth factor beta (TGF-b), produced by Treg cells, was elevated (p < 0.05). These results show
that A2AR activation downregulated Th1, Th2, and Th17 responses and upregulated Treg responses in EAE mice.
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CGS reduces the clinical severity of EAE
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C57BL/6 mice with EAE were treated with CGS to determine whether A2AR activation could protect against disease progression (Fig. 5). Treatment with CGS at 0.01 mg/kg or 0.05 mg/kg significantly decreased the mean clinical severity score around the peak of clinical disease. Body weights of mice treated with the higher CGS dose were significantly higher than those of untreated mice around the peak of clinical disease, and their weights were similar to those of the CFA controls at the end of the study period. No significant differences in cumulative and peak disease scores were observed between the two CGS doses.
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CGS alters the distribution of Th subsets in vivo Treatment of EAE mice with CGS (0.01 mg/kg) resulted in reductions in Th1 (p < 0.05), Th2 (p < 0.001), and Th17 (p < 0.001) cells and an increase in Treg cells (p < 0.001), compared to untreated mice (Fig. 6A and B).
Please cite this article in press as: Liu Y et al. Activation of the adenosine A2A receptor attenuates experimental autoimmune encephalomyelitis and is associated with increased intracellular calcium levels. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.05.028
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Fig. 5. Clinical scores and body weights. Clinical scores (A) and body weights (B) were measured in CFA mice (d), EAE mice (j), and EAE mice treated with 0.01 mg/kg CGS (CGS-1, ) and 0.05 mg/kg CGS (CGS-5, .). n = 10 for EAE, CGS-1, and CGS-5 groups; n = 7 for CFA group. *p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01 vs. EAE group.
Fig. 4. Effect of CGS on distribution of Th subsets in vitro. (A) Representative density plot showing expression of IFN-c, IL-4, IL-17, and Foxp3 in CD4+ T cells from CFA and EAE mice cultured with or without 10 nM CGS for 72 h. (B) Graphic representation of the data shown in (A). Each column represents the mean ± SD of eight mice/group from three independent experiments. *p < 0.05, ** p < 0.01, ***p < 0.001 vs. EAE group. (C) Cytokine concentrations in supernatants of CFA and EAE lymphocytes with or without exposure to 10 nM CGS for 72 h. Each column represents the mean ± SE of seven mice/group from three independent experiments. *p < 0.05, **p < 0.01 vs. EAE group.
Effect of CGS on nerve tissue in EAE mice
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To evaluate the effect of CGS on EAE at the tissue level, spinal cords from treated and untreated EAE mice were examined histologically for cellular infiltration and nerve demyelination at 14 days postimmunization. As detected by H&E and Luxol fast blue staining, spinal cord sections from CGS-treated EAE mice exhibited markedly fewer inflammatory infiltrates and demyelinated foci compared to untreated EAE mice (Figs. 7 and 8). The infiltration of lymphocytes in nerve tissues was examined in EAE and CGS-1 groups. Based on the FACS data obtained CGS-1 and CGS-5 treatment significantly reduced the number of CD4+ lymphocyte compared to EAE controls. (p < 0.05, p < 0.001, Fig. 7).
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Similarly, the mean concentrations of IFN-c, IL-4, and IL-17 (all p < 0.05) were decreased in the CGS-treated EAE mice (Fig. 6C). However, the mean TGF-b concentration was elevated (p < 0.05) in response to CGS treatment. These in vivo results support the in vitro findings that A2AR activation results in downregulation of Th1, Th2, and Th17 responses and upregulation of Treg responses in EAE mice.
Using flow cytometry with Fluo-3 AM, we examined the effects of CGS on intracellular calcium concentration ([Ca2+]i) in T lymphocytes from EAE mice. Changes in the percentage of Fluo-3+ cells reflect changes in the [Ca2+]i of CD4+ cells. This analysis demonstrated that the mean [Ca2+]i of CGS-treated cells was elevated (p < 0.001) compared to untreated cells (Fig. 9). The increased effect of CGS21680 was abrogated following the addition of SCH58261.
Please cite this article in press as: Liu Y et al. Activation of the adenosine A2A receptor attenuates experimental autoimmune encephalomyelitis and is associated with increased intracellular calcium levels. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.05.028
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Fig. 6. Effect of CGS on distribution of Th subsets in vivo. (A) Representative density plot showing expression of IFN-c, IL-4, IL-17, and Foxp3 in CD4+ T cells from CFA, EAE, and CGS-treated EAE mice given 0.01 mg/kg CGS. Lymphocytes from mice in all groups were isolated at the clinical peak of disease. (B) Graphic representation of the data shown in (A). Each column represents the mean ± SD of seven mice/group from 3 independent experiments. * p < 0.05, ***p < 0.001 vs. EAE group. (C) Cytokine concentrations in supernatants of stimulated T cells from CFA, EAE, and CGStreated EAE mice. Each column represents the mean ± SE of five to six mice/group from three independent experiments. *p < 0.05 vs. EAE group.
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DISCUSSION
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A2AR plays an important role in immune modulation and clinical immunotherapy. In a previous study, we demonstrated that an A2AR agonist protected rats from experimental autoimmune myasthenia gravis (EAMG) by decreasing lymphocyte proliferation and anti-AChR IgG levels, as well as by partially correcting the imbalance between Th1/Th2/Th17 and Treg subsets (Li et al., 2012). In this study, we characterized the expression of A2AR in T cells and the effect of an A2AR agonist on the pathology and clinical severity of murine EAE, which, to our knowledge, has not been done before. We demonstrated that A2AR expression was increased in mice presenting with EAE. High levels of
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Fig. 7. Effect of CGS on nerve tissue inflammation in EAE mice. Representative H&E photomicrographs of transverse lumbar spinal cord sections from (A) CFA mice, (B) EAE mice, (C) EAE mice treated with 0.01 mg/kg CGS, and (D) EAE mice treated with 0.05 mg/kg CGS. Tissue from mice in all groups was harvested at the peak of clinical disease. Arrow indicates inflammation infiltration typically seen in EAE and CGS-1 groups. Scale bar = 100 lm. (E) Representative density plot showing the distribution of CD4+ cells in nerves tissue. (F) Graphic representation of the data shown in (E). Each column represents mean ± SE. *p < 0.05, ###p < 0.001 vs. EAE group.
A2AR expression were detected in spleen sections, and the A2AR density on CD4+ T cells of EAE mice was increased compared to CFA controls. We hypothesized
Please cite this article in press as: Liu Y et al. Activation of the adenosine A2A receptor attenuates experimental autoimmune encephalomyelitis and is associated with increased intracellular calcium levels. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.05.028
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Fig. 8. Effect of CGS on nerve tissue demyelination in EAE mice. Representative Luxol fast blue photomicrographs of transverse lumbar spinal cord sections from (A) CFA mice, (B) EAE mice, (C) EAE mice treated with 0.01 mg/kg CGS, and (D) EAE mice treated with 0.05 mg/kg CGS. Tissue from mice in all groups was harvested at the peak of clinical disease. Arrow indicates demyelinated fragment typically seen in EAE and CGS-1 groups. Scale bar = 100 lm.
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that there were low levels of adenosine and/or A2AR receptor activity in EAE; therefore, an increase in adenosine production or administration of an adenosinelike compound would lead to increased activation of the A2AR receptor and subsequent protection of nerve tissue from disease. In support of our hypothesis, Mills et al. (2012) reported that A2AR lymphocyte expression is essential for limiting the severity of EAE. Koshiba et al. (1999) observed that activation of human peripheral T cells with phorbol myristate acetate and ionomycin
resulted in increased A2AR expression, and our results are consistent with those previously found that high levels of A2A receptor were expressed on the choroid plexus of EAE mice (Mills et al., 2012) and in lymphocytes from MS patients (Vincenzi et al., 2013). Other research has suggested that the activation of mouse CD4+ Th cells significantly upregulates A2AR mRNA expression (Lappas et al., 2005; Murphree et al., 2005). It has been shown that A2AR expression in human Th cells is induced 2–4 h after activation, and this expression level returns
Please cite this article in press as: Liu Y et al. Activation of the adenosine A2A receptor attenuates experimental autoimmune encephalomyelitis and is associated with increased intracellular calcium levels. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.05.028
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Fig. 9. Effect of CGS on [Ca2+]i in lymphocytes of EAE mice. Data shown are the results of Ca-Fluo-3 AM analysis. (A) Histograms show the distribution of [Ca2+]i measured 12 h after the addition of 0.01 lM CGS or 0.01 lM CGS + 0.01 lM SCH to lymphocyte cultures. (B) Representative density plot showing the distribution of [Ca2+]i in CD4+ cells after exposure to 0.01 lM CGS or 0.01 lM CGS + 0.01 lM SCH. (C) Graphic representation of the data shown in (B). Changes in percentage of Fluo-3+ cells reflect changes in [Ca2+]i. Each column represents the mean ± SE of n = 6/group from three independent experiments. ***p < 0.001 vs. EAE group, ###p < 0.001 vs. EAE group.
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to baseline by 24 h postactivation (Alam et al., 2009). More recently, Arosio et al. (2000) have shown that patients with multiple cognitive domain-mild cognitive impairment have lower A2AR mRNA levels and receptor density compared to Alzheimer’s dementia patients and normal subjects. Based on their findings, A2AR expression may be useful in differentiating the type of cognitive impairment. Examination of clinical EAE scores revealed that CGS treatment can alleviate EAE induced by immunization with the encephalitogenic peptide, MOG35–55. Although the body weights of CGS-treated EAE mice did decrease around the peak of clinical disease, the weights of mice treated with the higher CGS dose remained significantly higher than those of their untreated counterparts, and their weights did approach the weights of the CFA controls at the end of the study period. This observed weight loss may be partly explained by the fact that treatment with A2AR agonists increases energy expenditure in mice, resulting in leaner body mass (Gnad et al., 2014). It has been found that very late antigen 4 (VLA-4) was upregulated in MS patients and the A2AR stimulation was able to reduce VLA-4 expression in lymphocytes from MS patients(Vincenzi et al., 2013). This information revealed that A2AR agonists could limit lymphocyte migration across the blood–brain barrier, which is consistent with our findings that A2AR activation decreased inflammatory cell infiltration and demyelination in the spinal cord following EAE induction in mice. Recently, Loram et al. (2015) have shown that a single
intrathecal administration of an A2AR agonist was able to attenuate motor symptoms induced EAE in rats. Our study utilized intraperitoneal approach different from the intrathecal approach in Loram et al’s study (Loram et al., 2015), providing evidence for translational application of A2AR agonists through less invasive i.p. route. Mills et al. (2008) confirmed that SCH58261, a selective antagonist of A2A receptors, can protect mice against MOG-induced brain damage by specifically inhibiting A2AR signaling. Mice with genetic inactivation of the A2A receptor developed serious EAE (Yao et al., 2012; Wang et al., 2014), and these A2AR knockout mice displayed increased inflammatory cell infiltration in CNS. Obviously, the effects of A2ARs on neuroinflammation and neuronal damage in the brain are complex and paradoxical. Recently, Wang et al. (2013) confirmed that chronic treatment with caffeine, a non-selective antagonist of adenosine receptors, protected mice against EAE development. The protective effect of chronic caffeine treatment was associated with up-regulation of brain A1R, but not A2AR. And researchers speculate that SCH58261 can influence adhesion molecules ICAM-1at the choroid plexus with EAE (Mills et al., 2008; Yao et al., 2012). In the peripheral tissues, we consider A2AR activation can inhibit both Th1 and Th2 cell development and effector functions(Cso´ka et al., 2008), and the production of proinflammatory cytokines such as TNF-a, IL-6, and IL-12 (Ohta and Sitkovsky, 2001; Day et al., 2003, 2004; Sitkovsky et al., 2004). In keeping with these, A2AR agonists can promote the generation of
Please cite this article in press as: Liu Y et al. Activation of the adenosine A2A receptor attenuates experimental autoimmune encephalomyelitis and is associated with increased intracellular calcium levels. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.05.028
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Foxp3+ regulatory T cells and the production of transforming growth factor-b (Zarek et al., 2008). Our Th cell profile data showed that CGS corrected the imbalance between Th1/Th2/Th17 and Treg subsets, as well as abrogated EAE-associated lymphocyte proliferation in vitro. These results are consistent with those from our previous research (Li et al., 2012), which indicated that A2AR agonists can inhibit effector T cells and induce proliferation of Tregs. We found that CGS can shift the balance between Th1/Th2/Th17 and Treg subsets in favor of Tregs both in vitro and in vivo. Consistent with its effects on the Th subsets, CGS suppressed the release of key Th1, Th2, and Th17 cytokines (IFN-c, IL-4, and IL-17, respectively). In the study by Sevigny et al. (2007), ATL313 suppressed IFN-c but not IL-4. This finding suggests the likelihood of variable specificities among these A2AR agonists. Based on examination of the spinal cord sections, we found that CGS can reduce the severity of inflammation and tissue damage in murine EAE. These results are consistent with those of studies using other disease models, which have reported reduced inflammation and tissue injury in response to A2AR activation. For instance, administration of an A2AR agonist attenuated colitis by changing the balance of proinflammatory and anti-inflammatory cytokines expressed by Th cells during their activation (Naganuma et al., 2006). ATL146e, another A2AR agonist, can attenuate gastric mucosal inflammation in rats (Odashima et al., 2005b). Other reports have suggested that Tregs exert their suppressive action through the production of adenosine (Kobie et al., 2006; Deaglio et al., 2007). Zarek et al. (2008) have demonstrated that adenosine can divert CD4+ T cells away from Th17 differentiation and toward Foxp3+ differentiation and can increase TGF-b production with a concomitant decrease of the proinflammatory cytokine, IL-6. These investigators have also showed that A2AR stimulation can prevent T cell apoptosis and Th1/Th2 development. Other investigators have shown that adenosine promotes the generation of LAG-3+ Tregs (Himer et al., 2010). In our study, these histological findings further support the role of A2AR activation in regulating the inflammatory response and subsequent tissue injury. We also found that CGS can alter the [Ca2+]i, which may at least partly explain the mechanism of action of this A2AR agonist in alleviating EAE. In cells of the immune system, including T cells, B cells, mast cells and many other cell types, Ca2+ signals control differentiation, proliferation, effector function and gene transcription (Hogan et al., 2003; Lewis, 2007). Few studies have discussed the effect of adenosine on the calcium in lymphocytes. Recently, Chimote et al. (2013) identified adenosine or the A2A receptor agonist CGS21680 inhibits KCa3.1, a Ca2+-activated K+ channel expressed in lymphocytes via A2A receptor, resulting in decreased T cell motility. This mechanism is likely to contribute to decrease infiltration of lymphocytes in nerve tissues following A2AR activation. Lymphocyte proliferation and differentiation require sustained Ca2+ influx to keep cytoplasmic Ca2+ concentrations at higher than basal
levels for several hours. In lymphocyte cultures, the [Ca2+]i was increased for at least 12 h after the addition of CGS. This result indicates that CGS is capable of keeping cytoplasmic Ca2+ concentrations at high levels for long periods of time. Based on our findings, we can conclude that murine EAE is associated with increased A2AR expression in splenic T cells. A2AR stimulation with CGS suppresses EAE progression, as evidenced by reduced clinical severity and nerve tissue damage. CGS can alter the profile of autoreactive Th1, Th2, Th17, and Treg subsets, resulting in suppression of IFN-c, IL-4, and IL17 release and induction of TGF-b release. A2AR activation also increases the [Ca2+]i in lymphocytes. The A2AR may be a useful therapeutic target for alleviating MS and perhaps other T cell-mediated autoimmune diseases. Our data provide additional evidence that supports the role of A2ARs in adenosinemediated regulation of the inflammatory response.
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Acknowledgments—This work was supported by National Nature Science Foundation of China (81100883, 81371323, 8117112, 81100853), China Postdoctoral Science Foundation (2013M540303) Heilongjiang Provincial Nature Science Foundation (H201408), the Science and Technology Study Project of the Education Department of Heilongjiang Province (11541114) and the Harbin Medical University Innovation Fund for Postgraduates (HCXB2010001).
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(Accepted 13 May 2016) (Available online xxxx)
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