Activation of the adenosine A2A receptor exacerbates experimental autoimmune neuritis in Lewis rats in association with enhanced humoral immunity

Journal of Neuroimmunology 293 (2016) 129–136

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Activation of the adenosine A2A receptor exacerbates experimental autoimmune neuritis in Lewis rats in association with enhanced humoral immunity Min Zhang a, Xiao-Li Li a, Heng Li a, Shan Wang a, Cong-Cong Wang a, Long-Tao Yue b, Hua Xu c, Peng Zhang a, Hui Chen a, Bing Yang a, Rui-Sheng Duan a,⁎ a b c

Department of Neurology, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014, PR China Central Laboratory, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014, PR China Department of Neurology, Taian City Central Hospital, Taian 271000, PR China

a r t i c l e

i n f o

Article history: Received 20 September 2015 Received in revised form 6 January 2016 Accepted 3 March 2016

Keywords: A2A receptor Experimental autoimmune neuritis CGS21680

a b s t r a c t Accumulated evidence demonstrated that Adenosine A2A receptor (A2AR) is involved in the inflammatory diseases. In the present study, we showed that a selective A2AR agonist, CGS21680, exacerbated experimental autoimmune neuritis in Lewis rats induced with bovine peripheral myelin. The exacerbation was accompanied with reduced CD4+ Foxp3+ T cells, increased CD4+ CXCR5+ T cells, B cells, dendritic cells and antigen-specific autoantibodies, which is possibly due to the inhibition of IL-2 induced by CGS21680. Combined with previous studies, our data indicate that the effects of A2AR stimulation in vivo are variable in different diseases. Caution should be taken in the use of A2AR agonists. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Guillain–Barré syndrome (GBS), characterized by symmetrical limb weakness, is a group of acute disorders affecting the peripheral nervous system (PNS). Acute inflammatory demyelinating polyneuropathy (AIDP) and acute motor axonal neuropathy (AMAN) are the most common subtypes of GBS. AIDP is the predominant form in North America and Europe (van den Berg et al., 2014). Experimental autoimmune neuritis (EAN) can be induced in susceptible animal species by immunization with peripheral myelin, myelin protein P2 or P0 or by transferred T lymphocytes sensitized to these proteins (Mäurer and Gold, 2002). Because EAN resembles AIDP in clinical and histological features, EAN animal serves as a valuable animal model to study the pathogenesis of GBS and screen potential therapies. Adenosine is a naturally occurring nucleoside. It participates in a range of physiological and pathophysiological processes through binding

Abbreviations: A2AR, A2A receptor; EAN, experimental autoimmune neuritis; Treg, regulatory T cell; Tfh, T follicular helper cell; Teff, effector T cell; GBS, Guillain–Barré syndrome; PNS, peripheral nervous system; CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; BPM, bovine peripheral myelin; MNCs, mononuclear cells; AIDP, acute inflammatory demyelinating polyneuropathy; AMAN, acute motor axonal neuropathy; i.p., intraperitoneally; p.i., post-immunization; CFSE, carboxy-fluorescein diacetate, succinimidyl ester; FBS, fetal bovine serum; ConA, Concanavalin A; HE, hematoxylin and eosin; LFB, Luxol fast blue. ⁎ Corresponding author. E-mail address: [email protected] (R.-S. Duan).

http://dx.doi.org/10.1016/j.jneuroim.2016.03.002 0165-5728/© 2016 Elsevier B.V. All rights reserved.

its receptors on cell membrane. Four subtypes of its receptors have been identified: A1, A2A, A2B and A3, all of which are 7-transmembrane proteins belonging to the G-protein-coupled receptor family. A2A receptors (A2ARs) are widely expressed in spleen, thymus, leukocytes, blood platelets, heart, blood vessels, and brain. Previous reports showed that A2AR stimulation inhibited inflammatory responses by modulating the functions of macrophages, neutrophils, NK cells, NKT cells and lymphocytes. Its anti-inflammatory functions have been proved in a number of animal models, like ischemia–reperfusion injury, inflammatory bowel disease and pulmonary inflammation. These indicate that A2AR may be a therapeutic target in diseases due to overactive inflammatory response, such as autoimmunity and graft rejection (Milne and Palmer, 2011; Haskó et al., 2008). Reports on the effects of A2AR stimulation in nervous system inflammation are discordant. A2AR knockout mice, compared with wild type, develop more severe experimental autoimmune encephalomyelitis (EAE) and show more inflammatory cell infiltration in the central nervous system (Yao et al., 2012). However, CD73−/− (the cell surface enzyme that catalyzes the formation of extracellular adenosine) mice are resistant to EAE induction and A2AR antagonist protects mice from EAE development (Mills et al., 2008). A2AR deficiency exacerbates white matter lesions caused by chronic cerebral hypo-perfusion in mice, while its deficiency attenuates brain injury induced by transient ischemia (Chen et al., 1999; Yu et al., 2004; Duan et al., 2009). Distinct effects of A2AR stimulation on bone marrow-derived cells versus nonbone marrow-derived cells, different pathological processes, and

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different glutamate concentrations in local environment contribute to these discordant results (Milne and Palmer, 2011). Yet, the role of A2AR on GBS/EAN has not been reported. In the present study, we evaluated the effects of A2AR stimulation with CGS21680, a selective A2AR agonist, on EAN in Lewis rats induced with bovine peripheral myelin (BPM). Clinical symptoms and body weights were recorded throughout the experiments. To investigate the mechanisms of action, we further analyzed the immune cell populations, cytokine profiles of mononuclear cells (MNCs) from draining lymph nodes, and autoantibody levels in serum. Our data showed that although lymphocyte proliferation and pro-inflammatory cytokine production were inhibited, CGS21680 intervention promoted the development of EAN. The aggravation was accompanied with reduced proportion of CD4+ Foxp3+ regulatory T (Treg) cells, increased CD4+ CXCR5+ T follicular helper (Tfh) cells, B cells, dendritic cells and antigen-specific autoantibodies, which was possibly due to the inhibition of IL-2 production induced by CGS21680. 2. Materials and methods 2.1. Animals and reagents Female Lewis rats aged 6–8 weeks (body weight, 140–160 g) were purchased from Vital River Corporation (Beijing, China). All rats were housed under specific pathogen-free conditions in the local animal facility with free access to water and food. All experiment protocols have been approved by the institutional ethics committee. BPM was prepared from fresh adult bovine cauda equina according to the method previously reported (Norton and Poduslo, 1973). P0 peptide 180–199 (SSKRGRQTPVLYAMLDHSRS) was purchased from AC Scientific, Inc. (Xian, China). CGS21680 was purchased from Tocris Bioscience (Bristol, UK). 2.2. Induction of EAN and assessment of clinical symptoms Rats were immunized subcutaneously in both hind footpads with a total 200 μl inoculum containing 5 mg BPM and 1 mg Mycobacterium tuberculosis (strain H37RA; Difco, Detroit, MI, USA) emulsified in 100 μl saline and 100 μl incomplete Freund adjuvant (Sigma-Aldrich, USA). Clinical scores and body weights were monitored daily from day 0 until day 17 post-immunization (p.i.). Clinical symptoms were graded as follows: 0 = no illness; 1 = flaccid tail; 2 = moderate paraparesis; 3 = severe paraparesis; 4 = tetraparesis; 5 = death; intermediate scores of 0.5 increments were given to rats with intermediate signs. 2.3. Intervention with CGS21680 Administration of CGS21680 (at a dose of 1 mg/kg in PBS) started on day 5 p.i. Rats in experimental group were injected with CGS21680 intraperitoneally (i.p.) every two days until the end of the experiments. Rats in control group were given equal volume of PBS in the same way. The doses (1 mg/kg/i.p.) and the treatment regimen (every two days, start on day 5 p.i.) were determined according to previous reports (Li et al., 2011, 2012; Tang et al., 2010).

were washed twice and re-suspended at a concentration of 2 × 106 cells/ml in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, grand land, NY, USA) and 1% (v/v) penicillin– streptomycin (containing 10,000 IU/ml penicillin and 10,000 μg/ml streptomycin; Hyclone, Logan, UT, USA) for the following experiments. 2.6. Proliferation assay with CFSE 10 × 106 MNCs from each group were re-suspended in 2 ml RPMI 1640. Cell suspensions were added with carboxy-fluorescein diacetate, succinimidyl ester (CFSE, final concentration 2.5 μM, Invitrogen, UK) and thoroughly mixed. After incubation in the dark for 15 min at 37 °C, the staining process was quenched by adding 10 ml ice-cold complete RPMI 1640 (containing 10% FBS) and incubated on ice for 5 min. Then cells were washed twice with RPMI 1640. Cell pellets were resuspended in complete RPMI 1640 (containing 10% FBS). The stained MNCs (1 × 106 cells/ml, 1 ml/well) were cultured in triplicates in 24well culture plates in the dark at 37 °C. Each well was supplied with 50 μl of Concanavalin A (ConA, final concentration 5 μg/ml, SigmaAldrich, USA) or 50 μl of P0 peptide (final concentration 10 μg/ml). 72 h later, cells were collected and stained with PE-labeled anti-rat CD4 antibody (Biolegend, San Diego, USA) for 30 min at 4 °C. Finally, cells were analyzed with a flow cytometer. 2.7. Flow cytometry analysis of immune cell populations, cell surface molecules and intracellular cytokines For cell surface molecule detection, 1 × 106 MNCs from each rat were stained with FITC anti-CD3 (Biolegend, USA), FITC anti-CD4 (Biolegend, USA), FITC anti-CD8 (Biolegend, USA), FITC anti-CD161a (BD Pharmingen, USA), FITC anti-MHC classII(Biolegend, USA), PE anti-CD80 (Biolegend, USA), FITC anti-CD86 (Biolegend, USA), Alexa Fluor® 647 anti-CD103 (Biolegend, USA), FITC anti-CD19 (Abcam, USA), PE anti-γδ TCR (Biolegend, USA) or anti-CXCR5 (Abcam, USA) antibody respectively. For CXCR5 detection, cells were incubated further with Alexa Fluor® 488-conjugated goat anti-rabbit IgG antibody (Abcam, USA) after primary antibody staining. For Foxp3 detection, cells were first stained with FITC anti-CD4 antibody, then fixed and permeabilized with Foxp3 staining buffer set (eBioscience, USA) according to the instruction and finally stained with PE-Cyanine5 anti-Foxp3 antibody (eBioscience, USA). After staining, cells were analyzed with a flow cytometer. For intracellular cytokine detection, MNCs were incubated in the presence of Cell Stimulation Cocktail Plus Protein Transport Inhibitors (eBioscience, USA) according to the instruction for 4 h at 37 °C. Then cells were collected and fixed with 2% paraformaldehyde for 20 min at 4 °C. After washes with permeabilization wash buffer (Biolegend, USA) according to the instruction, cells were incubated with PE anti IL-10 (BD Pharmingen, USA), PE anti-IL-4 (Biolegend, USA), PE anti-IL17 A (eBioscience, USA), PE anti-TNF-α (Biolegend, USA) or PE antiIFN-γ (Biolegend, USA) antibody respectively for 30 min at 4 °C. Cells were analyzed with a flow cytometer. 2.8. ELISA analysis of IL-2 concentration in MNC culture supernatants

Animals were sacrificed on day 17 p.i. just after the symptoms of two groups peaked. Serum, inguinal and popliteal lymph nodes, spleen, and sciatic nerves of each rat were collected for the following experiments.

MNCs (1 × 106 cells/ml) were cultured in triplicates in the presence of P0 peptide (final concentration 10 μg/ml) for 72 h at 37 °C in the incubator. Then the supernatants were collected and stored at −80 °C for further IL-2 detection. IL-2 concentrations were measured with rat IL-2 ELISA kit (Dakewe Bioengineering, Beijing) according to the instruction. The results were expressed as pg/ml.

2.5. Preparation of lymph node mononuclear cells

2.9. ELISA analysis of IgG and IgG isotype levels in serum

Lymph nodes were grinded through cell strainers in RPMI 1640 medium (HyClone, Beijing, China). The mononuclear cells (MNCs)

Flat-bottomed high binding 96-well microplates were coated with 100 μl of P0 peptide (10 μg/ml) overnight at 4 °C. The uncoated sites

2.4. Collection of samples

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were blocked with 1% bovine serum albumin (BSA). Diluted serum samples (1: 100) were added in triplicates and incubated for 2 h at 37 °C. After three washes, biotinylated anti-rat IgG, anti-rat IgG1, anti-rat IgG2a, or anti-rat IgG2b (1:500, Biolegend, USA) were added and incubated for 1 h at 37 °C. Then streptavidin-HRP (1: 1000, Biosynthesis Biotechnology, China) were added and incubated for 30 min at 37 °C. Color was developed with tetramethylbenzidine (TMB) solution (Tiangen Biotechnology, Beijing, China) and stopped with 2 M H2SO4. Color absorbance at 450 nm was analyzed with a microplate reader. Between each step, plates were washed thoroughly with washing buffer. 2.10. Histological analysis Sciatic nerves were fixed in 4% paraformaldehyde (PFA) in PBS and then embedded in paraffin. Paraffin-embedded tissue sections (3 μm) were dewaxed and stained with hematoxylin and eosin (HE). The numbers of inflammatory cells were counted under a microscope. The results were expressed as the number of infiltrating cells per mm2 tissue sections. 2.11. Immunohistochemistry Paraffin tissue sections of sciatic nerves (3 μm) were deparaffinized and rehydrated. The sections were exposed to citrate in a pressure cooker for antigen retrieval. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 15 min. After three washes with PBS, the sections were incubated overnight at 4 °C with mouse anti-rat CD68 antibody (1:200, Abcam, USA). Antibody binding to tissue sections was detected with HRP-conjugated goat anti-mouse secondary antibodies (Zhongshan Goldenbridge Biotechnology, Beijing, China), followed by development of color with diaminobenzidine (DAB) substrate (Zhongshan Goldenbridge Biotechnology, Beijing, China). Slides were viewed under a microscope. The results were expressed as cells per mm2. 2.12. Luxol fast blue staining Paraffin embedded tissue sections of sciatic nerves were dewaxed and hydrated to 95% ethyl alcohol. The sections were stained with 0.1% Luxol fast blue (LFB, Sigma-Aldrich, USA) solution at 56 °C overnight and then differentiated with 0.05% lithium carbonate solution. After counterstained with hematoxylin, the sections were observed under a microscope. The severity of demyelination was evaluated semi-quantitatively according to previous reports: 0 = normal, 1 = less than 25% demyelination fibers, 2 = 25%–50% demyelination, 3 = 50% –75% demyelination, 4 = more than 75% demyelination (Zhao et al., 2010). 2.13. Statistical analysis Data are representative of two independent experiments. Statistical data analysis was performed using GraphPad Prism 6. Clinical scores and demyelination scores were analyzed using Mann–Whitney test. The other results were analyzed using student's t-test. Data were expressed as mean ± SD. Significance level was set at p b 0.05. 3. Results 3.1. CGS21680 intervention promotes the development of EAN To evaluate the effects of A2AR stimulation on EAN, CGS21680 was injected i.p. at a dose of 1 mg/kg every two days since day 5 p.i. until the end of the experiment. The doses of CGS21680 (1 mg/kg/i.p.) were determined based on previous reports (Tang et al., 2010; Li et al., 2012). The treatment started on day 5 p.i. Although, at this time, rats have not shown clinical symptoms, the in vivo immune response has

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been established (Li et al., 2011). Rats in control group received the same volume of vehicle. All immunized rats developed EAN. Rats in control group showed clinical symptoms on day 12 p.i, while rats injected with CGS21680 showed neurological deficits on day 11 p.i. The symptoms progressed rapidly and peaked around day 15 p.i. Rats in control group started to recover on day 16 p.i., while rats in CGS21680 treatment group on day 17 p.i. Clinical scores were higher in the CGS21680 intervention group than those in control group on days 11, 13 and 14 p.i. (p b 0.05) (Fig. 1A). Body weights in the CGS21680 intervention group were much less than those in control group on days 10, 12, 13 and 14 p.i. (p b 0.05, 0.01, 0.01 and 0.05 respectively) (Fig. 1B). These data indicate that CGS21680 intervention exacerbates the illness of the EAN.

3.2. CGS21680 intervention promotes inflammatory cell infiltration and demyelination of sciatic nerves To examine the histological changes of target tissues, sections of sciatic nerves were stained with HE, anti-CD68 antibody and LFB. Compared with control group, sciatic nerves from CGS21680 intervention group showed more macrophage infiltration and demyelination (Fig. 2).

3.3. CGS21680 intervention elevates the levels of P0 peptide-specific antibodies in serum Previous study showed a time-dependent increase of anti-P0 peptide 180–199 antibodies in serum of animal models induced with BPM (Zhu et al., 2007). Here we detected the P0 peptide-specific antibodies in serum, which served as indicators of antigen specific humoral immune response in EAN. Our data showed that the levels of anti-P0 peptide IgG antibody in serum from the CGS21680 intervention group were higher than those from the control group. IgG subtypes analysis showed that the levels of IgG1 and IgG2a were preferably elevated in CGS21680 intervention group (Fig. 3).

3.4. CGS21680 intervention suppresses Th1 and Th17 cytokines, and powerfully inhibits lymphocyte proliferation and IL-2 secretion Clinical scores, body weights, peripheral nerves histological changes, and autoantibody titers indicated that CGS21680 intervention promoted the development of EAN in Lewis rats induced with BPM. To examine the effects of CGS21680 on immune response in EAN, draining lymph nodes were harvested on day 17 p.i. Cytokine profiles of lymph node MNCs were analyzed by flow cytometry. Our data showed that, compared with the control group, the percentages of TNF-α, IFN-γ, and IL-17 positive cells among MNCs were decreased (p b 0.01, 0.01 and 0.05 respectively) after CGS21680 intervention, while IL-4 or IL-10 producing cells were less affected (Fig. 4). Lymph node MNCs were stained with CFSE to assess the effects of CGS21680 on lymphocyte proliferation. CFSE passively diffuses into cells and becomes florescent after it is cleaved by intracellular esterase. CFSE forms covalent bonds with cytoplasmic components, then it is divided equally between daughter cells. Up to eight successive generations of cell division could be determined when analyzed by flow cytometry. Compared with control group, both P0 peptide activated antigen-specific (p b 0.001) and ConA activated antigen-nonspecific (p b 0.001) MNCs and CD4+ T cell proliferations were significantly suppressed after CGS21680 treatment (Fig. 5). The levels of IL-2 in MNC culture supernatants in the presence of P0 peptide were measured by ELISA method. Compared with control group, IL-2 production was inhibited after CGS21680 intervention (p b 0.01). And the production of IL-2 by MNCs was reduced to a quite low level (Fig. 5C).

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Fig. 1. CGS21680 intervention exaggerated EAN. EAN was induced in Lewis rats by immunization with BPM (5 mg/animal) and Mycobacterium tuberculosis (1 mg/animal) in incomplete Freund adjuvant. From day 5 p.i., animals from each group were injected i.p. every two days with CGS21680 (1 mg/kg) or vehicle respectively (n = 5 per group). (A) Clinical scores and (B) body weights were assessed daily and shown as mean ± SD (*p b 0.05, **p b 0.01).

3.5. CGS21680 intervention reduces the proportions CD4+ Foxp3+ Treg cells while increases CD4+ CXCR5+ Tfh cells, B cells and dendritic cells in draining lymph nodes Inhibitions of pro-inflammatory cytokine production and lymphocyte proliferation could not explain EAN exacerbation induced by CGS21680 treatment. To illuminate how CGS21680 intervention promoted the development of EAN in Lewis rat, other lymphocyte populations were analyzed, including CD4+ Foxp3+ Treg cells, CD4+ CXCR5+ Tfh cells, CD19+ B cells, CD103+ dendritic cells (DCs), γδ T cells, NK cells and NKT cells. Compared with control group, Treg cells were decreased (p b 0.01), while the percentages of Tfh cell, B cells and DCs in the draining lymph nodes were increased after CGS21680 treatment (Fig. 6). The proportions of γδ T cells, NK cells and NKT cells showed no statistical differences between two groups (data not shown).

3.6. CGS21680 intervention increases the expressions of MHC classII and CD86 MHC classII, CD86 and CD80 are cell surface molecules important for immune response initiation and modulation, and play important roles in antibody affinity maturation. The expressions of theses molecules on MNCs were determined by flow cytometry. Compared with control group, both the percentages of positive cells and the mean fluorescence intensities of MHC classII and CD86 were increased after CGS21680 treatment (Fig. 7). 4. Discussion In present study, we found that CGS21680 intervention aggravated EAN in Lewis rats induced with BPM. Although CGS21680 inhibited pro-inflammatory cytokine production and lymphocyte proliferation,

Fig. 2. CGS21680 intervention promoted macrophage infiltration and demyelination of sciatic nerves. Sciatic nerves were collected after the animals were sacrificed on day 17 p.i. The sciatic nerve sections were stained with HE, anti-CD68 antibody and Luxol Fast blue. (A) Representative micrographs showed the histological changes of sciatic nerves between two groups. (B) Numbers of inflammatory cells, macrophages (CD68+ cells) per mm2 sciatic nerve section and the scores of demyelination. The results are expressed as mean ± SD (*p b 0.05).

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Fig. 3. CGS21680 intervention enhanced the production of P0 peptide-specific antibodies. After the animals were sacrificed on day 17 p.i., the sera were collected and the levels of P0 peptide-specific antibodies were measured by ELISA method. The results show the OD values and are expressed as mean ± SD (*p b 0.05, **p b 0.01).

it decreased the proportions of Treg cells, increased Tfh as well as B cells, and promoted the formation of autoantibodies. Our data showed that A2AR stimulation by CGS21680 promoted the development of EAN in Lewis rats in association with enhanced humoral immunity. Treg cells are important for maintenance of immune homeostasis and prevention of autoimmunity (Campbell and Koch, 2011). In EAN animals, early Treg cell depletion exacerbated neurological deficits (Meyer Zu Hörste et al., 2014). Accumulated Treg cells were found in the sciatic nerves during the recovery phase of EAN (Zhang et al., 2009). A number of therapeutic chemicals exerted their effects partially through up-regulation of Treg cells in EAN (Li et al., 2011; Xu et al., 2014). In present study, we found CGS21680 intervention decreased the proportion of Treg cells and inhibited IL-2 production. Treg cells

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express high level of CD25, the alpha chain of IL-2 receptor. IL-2 singling is critical for the survival and/or function of Treg cells in peripheral (Thornton et al., 2004). It has been proved that IL-2 deficient mice developed fatal autoimmune diseases and lymphoproliferation (Horak et al., 1995). IL-2 neutralization by specific antibody for a limited period could reduce the number of Treg cells and elicit autoimmune diseases as well (Malek and Castro, 2010). A decrease of Treg cell: effector T (Teff) cell ratio was found in the islet in nonobese diabetic mouse due to defective IL-2 production (Tang et al., 2008). Single nucleotide polymorphisms in the IL-2Rα genes were reported to be associated with autoimmune disease susceptibility in human (Ballesteros-Tato, 2014). Supplement of low dose of exogenous IL-2 has been proved effective in prevention or treatment of EAE and experimental autoimmune myasthenia gravis (EAMG) (Webster et al., 2009; Liu et al., 2010). Besides, Previous studies showed that A2AR signaling inhibited IL-2 production (Erdmann et al., 2005; Naganuma et al., 2006; Alam et al., 2009). Consistent with these reports, in the present study, the ex vivo lymphocyte proliferation assay showed that lymphocyte proliferation and IL-2 production were significantly inhibited by CGS21680, and such effect was persistent even without the presence of CGS21680. It's likely that CGS21680 inhibited the IL-2 production by lymphocytes, which led to the local deprivation of IL-2 for Treg cells and caused the reduced numbers and functions of Treg cells. The protective role of Treg cells in EAN was undermined. Besides decreased Treg cells, we also found increased Tfh cells after CGS21680 intervention. Tfh cells are critical for the effective development of humoral immunity, and function primarily for the formation of germinal center, where they interact with B cells and facilitate

Fig. 4. CGS21680 intervention reduced the numbers of IFN-γ, TNF-α and IL-17 positive cells, while had less effect on IL-4 and IL-10. Lymph node MNCs were obtained on day 17 p.i. The IFN-γ+, TNF-α+, IL-17+, IL-4+ and IL-10+ cells of lymph node MNCs were analyzed by flow cytometry. (A) Representative scatter plots of cytokine positive cells among lymph node MNCs. (B) Percentages of positive cells among lymph node MNCs. The results are expressed as mean ± SD (*p b 0.05, **p b 0.01).

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Fig. 5. CGS21680 intervention significantly suppressed the proliferation of lymphocytes and the secretion of IL-2 by lymphocytes. Lymph node MNCs were obtained on day 17 p.i. MNCs were stained with CFSE and cultured in the presence of P0 peptide or ConA for 72 h in vitro. Then the cells were collected and counterstained with PE-labeled anti-CD4 antibodies and analyzed with a flow cytometer. The concentrations of IL-2 in the MNC culture supernatants in the presence of P0 peptide were detected by ELISA method. (A) Representative flow cytometry analysis of MNC proliferation. Percentages of CFSElow cells among MNCs and percentages of CFSElowCD4+ cells among CD4+ cells are shown in (B). (C) The levels of IL-2 in MNC culture supernatants. The results are expressed as mean ± SD (***p b 0.001).

antibody affinity maturation and isotype switch (Crotty, 2014). Transcription factor Bcl-6 is essential for the differentiation of Tfh cells. Bcl6 expression could be repressed by STAT5 pathway, which is primarily

induced by IL-2 singling (Johnston et al., 2012). Supplement of exogenous IL-2 has been proved to limit the development of humoral immunity by suppressing Tfh cell differentiation (Ballesteros-Tato et al.,

Fig. 6. CGS21680 intervention down-regulated Treg cells, while enhanced the formation of Tfh cells, B cells and DCs. Lymph node MNCs were obtained on day 17 p.i. Treg (CD4+ Foxp3+) cells, Tfh (CD4+ CXCR5+) cells, B (CD19+) cells and DC (CD103+) cells were analyzed by flow cytometry. (A) Representative contour plots of FACS analysis of CD4+ Foxp3+ cells, CD4+ CXCR5+ cells, CD19+ cells and CD103+ cells. (B) Percentages of CD4+ Foxp3+ cells CD4+ CXCR5+ cells among lymph node CD4+ cells. (C) Percentages of CD19+ cells and CD103+ cells among MNCs. The results are expressed as mean ± SD (*p b 0.05, **p b 0.01).

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Fig. 7. CGS21680 intervention increased the expression CD86 and MHC classII. Lymph node MNCs were obtained on day 17 p.i. Expressions of CD80, CD86 and MHC classII were analyzed by flow cytometry. (A) Representative contour plots of positive cells among lymph node MNCs. Percentages of positive cells among MNCs and the mean fluorescence intensity of CD80, CD86 and MHC classII are shown in (B) and (C) respectively. The results are expressed as mean ± SD (*p b 0.05, **p b 0.01).

2012). In the germinal center, B cells and Tfh cells form a symbiotic relationship. B cell-specific expression of CD86 was essential for the Tfh cell formation (Salek-Ardakani et al., 2011). Treg cells could downregulate the expression of CD86 on DCs and B cells. Treg cell depletion promoted the formation of Tfh cells, germinal centers and plasma and memory B cells (Wing et al., 2014). Consistent with previous reports, our study showed that along with the decreased of IL-2 production and reduced Treg cells, the proportions of Tfh cells, B cells and DCs in the draining lymph nodes were increased and the expression of CD86 and MHC classII molecules were enhanced after CGS21680 intervention. It's reasonable that both the attenuated IL-2 signaling and downregulated Treg cells promoted the formation of Tfh cells and contributed to the enhanced humoral immunity in EAN. As myelin protein P0 accounts for more than half of total proteins in BPM (Xia et al., 2012) and increased P0 peptide-specific antibodies correlated with EAN development in Lewis rats immunized with BPM (Zhu et al., 1994, 2007), we analyzed the levels of P0 peptide 180– 199-specific IgG and IgG subtypes in serum from EAN rats. Data showed that CGS21680 intervention increased P0 peptide-specific IgG, IgG1 and IgG2a production. Autoantibodies participated in the pathogenesis of EAN by inducing the formation of immune complex and promoting infiltration of macrophages in peripheral nervous system (Tran et al., 2010). In line with increased P0 peptide-specific antibodies, histological analysis of sciatic nerves from CGS21680 treatment group showed more macrophage infiltration and more demyelination sites compared with those from control group. Previous study also showed that autoantibodies against neurofascin and gliomedin took part in the development

of EAN induced with BPM by disrupting the organization of nodes of Ranvier, which led to the conduction slowing or block (Lonigro and Devaux, 2009). It's reasonable to speculate that such antibodies also increased along with the enhanced humoral immunity elicited by CGS21680 intervention and contributed to the aggravation of EAN. In EAE, stimulation of A2ARs on hematopoietic cells and nonhematopoietic cells had opposite effects. Lack of A2AR expression on hematopoietic cells gave rise to more severe EAE, while lack of A2AR signaling in nonhematopoietic cells protected mice from EAE development (Mills et al., 2012). Our present study showed that A2AR stimulation promoted the development of EAN in association with down-regulated Treg cells and up-regulated Tfh cells, which has not been reported. Whether A2AR stimulation on bone marrow-derived cells or non-bone marrow-derived cells exerts different effects in EAN needs further studies. As for the effects of A2AR stimulation on lymphocytes, discordant results have been reported. There are reports showed that repetitive CGS21680 treatment in early stage or CGS21680 treatment in long period with long intervals inhibited IL-17 production and promoted Foxp3 expression possibly by enhance TGF-β production (Zarek et al., 2008; Li et al., 2012). However, there is also report showed that A2AR stimulation did not affect TGF-β production (Naganuma et al., 2006). In experimental autoimmune uveitis (EAU), a single injection with CGS21680 or adenosine receptor non-selective agonist at early stage inhibited Th17 response, while treatment at a late stage enhanced Th17 response via γδ T cells. A2AR stimulation inhibited immune response during the quiescent phase of disease, but A2AR stimulation promoted immune

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response when inflammation had already occurred. The number of Foxp3+ cells was not significantly affected in this study (Liang et al., 2014). In our study, CGS21680 was applied when immune response had already occurred and the injections were repeated every other day until the end of experiments. Besides decreased IFN-γ, TNF-α and IL-17, we found decreased Treg cells and increased Tfh cells, in accompany with more vigorous inhibition of lymphocyte proliferation and IL-2 production. IL-2 is essential for the maintenance and function of Treg cells in peripheral (Malek and Castro, 2010). Previous studies have proved that CGS21680-mediated A2AR signaling preferentially inhibited IL-2 production in a dose-dependent manner (Erdmann et al., 2005; Alam et al., 2009). It's plausible that IL-2 deprivation in microenvironment lead to down-regulation of Treg cells as documented in other reports (Shevach, 2012). Combined with previous reports, our data indicate that the effects of A2AR stimulation in vivo are inflammatory environment dependent and affected by multiple factors. In summary, our study shows that A2AR activation by its selective agonist CGS21680 exacerbates EAN in Lewis rats induced with BPM. The exacerbation is associated with reduced proportion of Treg cells, increased formation of Tfh cells and B cells, and elevated levels of autoantibodies. A2AR is a valuable therapeutic target. A number of its agonists and antagonists have been developed and some of them have entered into clinical trials. However, caution should be taken in use of A2AR agonists treating nervous system autoimmune diseases. Conflict of interest All authors declare no conflict of interest. Acknowledgment This work was funded by Taishan Scholars Construction Engineering of Shandong Province (ts20130914) and partially supported by grants from the National Natural Science Foundation of China (81171128 and 81471222). References Alam, M.S., et al., 2009. A2A adenosine receptor (AR) activation inhibits pro-inflammatory cytokine production by human CD4+ helper T cells and regulates Helicobacterinduced gastritis and bacterial persistence. Mucosal Immunol. 2 (3), 232–242. Ballesteros-Tato, A., 2014. Beyond regulatory T cells: the potential role for IL-2 to deplete T-follicular helper cells and treat autoimmune diseases. Immunotherapy 6 (11), 1207–1220. Ballesteros-Tato, A., et al., 2012. Interleukin-2 inhibits germinal center formation by limiting T follicular helper cell differentiation. Immunity 36 (5), 847–856. Campbell, D.J., Koch, M. a, 2011. Phenotypical and functional specialization of FOXP3+ regulatory T cells. Nat. Rev. Immunol. 11 (2), 119–130. Chen, J.F., et al., 1999. A(2A) adenosine receptor deficiency attenuates brain injury induced by transient focal ischemia in mice. J. Neurosci. 19 (21), 9192–9200. Crotty, S., 2014. Review T follicular helper cell differentiation, function, and roles in disease. Immunity 41 (4), 529–542. Duan, W., et al., 2009. Adenosine A2A receptor deficiency exacerbates white matter lesions and cognitive deficits induced by chronic cerebral hypoperfusion in mice. J. Neurol. Sci. 285 (1–2), 39–45. Erdmann, A.A., et al., 2005. Activation of Th1 and Tc1 cell adenosine A2A receptors directly inhibits IL-2 secretion in vitro and IL-2-driven expansion in vivo. Blood 105 (12), 4707–4714. Haskó, G., et al., 2008. Adenosine receptors: therapeutic aspects for inflammatory and immune diseases. Nat. Rev. Drug Discov. 7 (9), 759–770. Horak, I., et al., 1995. Interleukin-2 deficient mice: a new model to study autoimmunity and self-tolerance. Immunol. Rev. 148, 35–44. Johnston, R.J., et al., 2012. STAT5 is a potent negative regulator of TFH cell differentiation. J. Exp. Med. 209 (2), 243–250.

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