Animals
Eight-week-old C57BL/6 mice (female) were purchased from Charles River Laboratories (CRL, Beijing, China). The transgenic line B6. Cg-Gt (ROSA) 26Sortm9 (CAG-tdTomato) Hze/J (Ai9) was purchased from Jackson Laboratory (Stock No: 007909). A2ARflox/flox mice (female) have been described previously [20]. All mice were bred and housed under pathogen-free conditions at Wenzhou Medical University.
EAE induction and behavioral scoring
EAE model was produced following the procedure described previously [16]. Briefly, 1:1 emulsion of MOG35-55 peptide (1 mg/ml in PBS) (Anaspec) and complete Freund’s adjuvant (CFA, Sigma) was injected subcutaneously (50 μl) into each flank. In addition, pertussis toxin (PTX, 20 ng) (Biological Laboratories Inc.) was given intravenously (200 μl in PBS) at the time of immunization and again two days later. The EAE mice were daily scored based on the disease symptom severity scale (from 0 to 15), as we described previously [21] .
In vivo assay of CP permeability
Briefly, at day 8, 10, or 12 post-immunization, the EAE mice were anesthetized with 4.5% isoflurane and then treated with intravenous injection (retro-orbitally) of 100 µl of 4 kDa Dextran Alexa Fluor 488 (final concentration 2 mg/100 µl). Fifteen minutes later, the mice were deeply anesthetized with isoflurane and then perfused with cold PBS and 4% formaldehyde (PFA). The brains were dissected out and embedded with O.C.T. matrix. The frozen sections (20 µm thick) were used to evaluate the fluorescence intensity with a confocal microscope (LSM880, Zeiss).
A2AR antagonist treatment
For the treatment of the A2AR antagonist KW6002 in vivo, mice were intraperitoneally injected with vehicle (DMSO/PBS) or KW6002 (5 mg/kg) every day. To isolate the effective time window of KW6002, mice were intraperitoneally injected with vehicle (DMSO/PBS) or KW6002 (5 mg/kg) every day in the phase of day 8-14 or 8-12 post-immunization.
Over-expression of A2AR in the CP cell line ZM310
The A2AR gene Adora2a (mouse, NM_009630.3) was cloned into the vector FV115 (CMV-MCS-3Flag-Ubi-ZsGreen-IRES-Puromycin) with the seamless cloning method and was used for the lentivirus package (1.1 × 10 8). The empty vector was used as the control group.
The Z310 cells (2 × 10 4) were cultured in a DMEM medium (DMEM/F12 and 10% fetal bovine serum) at 37 °C for 24 h. The cells were then transfected with the constructed lentivirus (MOI (multiplicity of infection) = 20). Forty-eight hours following the transfection, the A2AR positive cells were screened with various concentrations of puromycin (1μg/ml, 2.5μg/ml, 5μg/ml, 10μg/ml). The ZsGreen positive cells were then used for TEER and cell migration assays.
Primary culture of the CP epithelium
The CP cell cultures were produced as described previously [22]. Briefly, following perfusion with PBS, the CP tissues were dissected and digested with 0.25% trypsin. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 medium (Invitrogen) containing 10% fetal bovine serum (FBS), 1 mM L-glutamine, 1 mM sodium pyruvate, penicillin/streptomycin (100 U/ml), insulin (5 ng/ml), 20 μM arabinosylcytosine (Ara-C), sodium selenite (5 ng/ml), and epidermal growth factor (EGF) (10 ng/ml) (Sigma-Aldrich). Then, the cells were cultured in the L-polylysine-coated 24-well plates or polycarbonate filters according to the experimental goal.
Effects of CGS21680 and NFkB/STAT3 inhibitor
Following cultivation of the abovementioned primary CP cells for three days, the cells were treated with CGS21680 (100 nM), or vehicle (DMSO). Then, 30 min later, the cells were collected for qPCR or Western Blot. In addition, the 3-days-cultured primary CP cells were pretreated with JSH-23 (10 μM, a NFκB transcriptional activity inhibitor, 749886-87-1, Sigma-Aldrich), Stattic (100 μM, a specific STAT3 inhibitor, 573099, Millipore) or vehicle for 1 h. Then, CGS21680 (100 nM, C141, Sigma-Aldrich) or vehicle were added. Finally, 30 min later, the cells were collected for qPCR.
TEER measurement
The primary CP cells were isolated as described above and cultured on polycarbonate filters in a transwell chamber (pore size: 5 μm, diameter: 6.5 mm). Six days later, the A2AR agonist CGS21680 (100 nM) was added into the upper chamber of the transwell. Then, TEER was monitored within 2 h (MERS00002, Millipore). For the rat CP epithelium cell line (ZM310), the A2AR-labelled, empty vector-labelled or blank ZM310 cells were cultured on the same filters for 4 days, then TEER was measured.
T cell migration assay
The method was used as described previously [23]. Briefly, the A2AR-labelled, empty vector-labelled or blank ZM310 cells were grown on filters as mentioned above for 3 days. The CD4+ T cells were isolated from the spleen with Anti-Mouse CD4 Magnetic Particles (BD, #551539). Then, the medium of the upper chamber was changed to 100 μl 1640 medium containing CD4+ T cells (1 × 106 cells/ml), and the insert was plated into a new well filled with RPMI 1640 (0.5% FBS). Twenty-four hours later, the migrating cells in the lower chamber were subjected to the crystal violet staining and counted under a microscope (DM750, Leica).
Quantitative real-time PCR (qPCR)
Following perfusion with PBS, the CP was dissected from the ventricles for RNA isolation using Trizol (Invitrogen) and cDNA synthesis with PrimeScript™ 1st Strand cDNA synthesis Kit (Takara, 6110A). Quantitative PCR was performed with SYBR-Green premix Extaq (Takara) and detected with a Real-Time PCR System (CFX96; Bio-Rad). The following primers were used: ppia (forward 5’-AGCATACAGGTCCTGGCATCTTGT-3’ and reverse 5’ -CAAAGACCACATGCTTGCCATCCA-3’),
icam1 (forward 5’-AGATCACATTCACGGTGCTGGCTA-3’ and reverse 5’-AGCTTTGGGATGGTAGCTGGAAGA-3’),
ccl2 (forward 5’-CATCCACGTGTTGGCTCA-3’ and reverse 5’-GATCATCTTGCTGGTGAATGAGT-3’),
cxcl10 (forward 5’-AACTGCATCCATATCGATGAC-3’ and reverse 5’-GTGGCAATGATCTCAACAC-3’), ccl5 (forward 5’-GCTGCTTTGCCTACCTCTCC-3’ and reverse 5’-TCGAGTGACAAACACGACTGC-3’), ccl20 (forward 5’- GCCTCTCGTACATACAGACGC-3’ and reverse 5’- CCAGTTCTGCTTTGGATCAGC-3’), and adora2a (forward, 5’-CCGAATTCCACTCCGGTACA-3’ and, reverse 5’-CAGTTGTTCCAGCCCAGCAT-3’;). The Ct values were converted to relative quantification data using a 2−ΔΔCt method.
Immunofluorescent and hematoxylin-eosin (HE) staining
Anesthetized mice were fully perfused with PBS and 4% PFA (paraformaldehyde) and the brains were isolated and fixed with PFA. Before staining, the brain slices (30 μm thick) were washed with PBS three times and then incubated for 30 min in PBS containing 0.3% Triton X-100 and 3% serum. Later, the slices were incubated with primary antibodies for 12 hours: rabbit anti-ICAM1 (1:100, Abcam) or mouse anti-A2AR (1:300, Wako). The sections were then washed with PBS and incubated for 2 h at room temperature with Alexa Fluor 488 antibodies (Abcam; 1:400). The slices were then washed and mounted on slides with VECTASHIELD mounting media (containing DAPI). For CD3 staining, the isolated CP tissues were fixed with 2.5% PFA for 30 min and then transferred to PBS. The following protocol was similar to the above procedure with rat anti-CD3 (1:100, CD3-FITC, Abcam). Images were acquired with a confocal microscope (LSM880, Zeiss).
As described earlier [16], tissue slices were stained with hematoxylin-eosin.
CP-specific knockdown of A2ARs
The focal knockdown of A2ARs in the CP was achieved using the method described previously [24]. In brief, 2.11 μl of CRE-TAT recombinase (20 μg for each ventricle, Millipore) or sterile PBS were injected into each of the lateral ventricles (AP: 0.98, ML: −1.3, DV: 2.6). The injection rate was 1 μl/min, and the needle was kept inside the brain for other 5 min before a withdrawal. Mice were allowed to recover for two weeks before the MOG immunization.
Flow cytometry analysis of immune cells
Following perfusion with PBS, the spleen, CSF (5 μl per mouse) and spinal cord were isolated from PBS or CRE-TAT treated mice at day 14 post immunization. Cell suspensions of the spleen and spinal cord were prepared and incubated with ACK buffer (C3702, Beyotime) to lyse red blood cells. With a centrifugation at 1500 rpm for 5 min, the cells were resuspended with 100 μl of staining buffer (70-S1001, Multisciences). Next, 10 μl of Clear Back (FcR blocking, MTG-001, MBL) were mixed with 50 μl of cell suspension (≤1 × 106) and incubated for 5 min at room temperature. After that, 10 μl of T-Select MHC Tetramer (I-Ab MOG35-55 Tetramer-PE, TS-M704-1, MBL) were added and incubated for 45 min at 4°C. Then, the samples were stained with fluorochrome-conjugated monoclonal antibodies against FVS510 (564406, BD Pharmingen), CD3-FITC (11-0032-82, Invitrogen), CD4-PerCP-Cy5.5 (550954, BD Pharmingen) and CD196-AF647 (561753, BD Pharmingen) for 30 min at 4°C. Next, the staining buffer (3 ml per sample) was added into the sample solution and mixed well. Following a centrifugation at 1500 rpm for 5 min, the cells were resuspended with 200 μl of staining buffer. At last, the stained cells were acquired with a flow cytometer (NovoCyte Quanteon, Agilent) and analyzed with NovoExpress software (Agilent).
Western Blot analysis of signaling molecules
The primary CP cells were isolated as started above and cultured in L-polylysine-coated plates for 2 days. Then, the cells were respectively treated with vehicle, 100 nM CGS21680 (HY-13201, MCE), 30 μM JSH-23 (an inhibitor of nuclear translocation of p65) (HY-13982, MCE), 20 μM Stattic (an inhibitor of STAT3 phosphorylation (Y705 and S727)) (HY-13818, MCE), a combination of JSH-23 (30 μM) and CGS21680 (100 nM), or a combination of Stattic (20 μM) and CGS21680 (100 nM). The cells were collected and prepared for RNA or protein isolation. The proteins were transferred from polyacrylamide gels to PVDF membranes (Invitrogen) at 100 V for 120 min. Membranes were blocked using bovine serum albumin (5% w/v). Membranes were incubated with primary antibodies overnight at 4 °C and secondary antibodies at room temperature for 90 min. Primary antibodies were anti-Stat3 (12640S, CST; 1:1000, rabbit), anti-Phospho-Stat3 (Tyr 705) (9145S, CST; 1:1000, rabbit), anti-NFκB-p65 (8242S, CST; 1:1000, rabbit) and anti-Phospho-NFκB-p65 (Ser 536) (3033S, CST; 1:1000, rabbit) (Ser 536) (9145S, CST; 1:1000, rabbit). The secondary antibody was IRDyer 800CW (LICOR; 1:5000). Signal intensities were calculated using ImageJ software and normalized to values of Stat3 or p65.
Statistical analyses
Statistical analysis of behavioral deficit scores in different groups during the EAE disease courses were determined utilizing Two-way ANOVA for repeated-measurements (RM). One-way ANOVA was used to estimate leukocyte trafficking determinant expression during EAE disease course. The Student’s t-test was used to compare two groups of drug treatment, over-expression or CRE-TAT injection. All statistical analyses were performed with GraphPad Prism 6.0.