Mice
Female C57BL/6 mice (6-8 weeks old, 20 ± 2 g) were purchased from Vitallihua Experimental Animal Co., Ltd. (Beijing, China). All the mice were housed in a specific pathogen-free facility. All animals and experiments used in this study were approved and performed in accordance with the guidelines of the Animal Ethics Committee of Xiamen University.
EAE induction and ATO treatment
To induce EAE, mice were immunized with 200 μg of MOG35-55 peptide (BAM Biotech Co., Ltd., Xiamen, China) in complete freund's adjuvant (Sigma, MO, USA) supplemented with 2.5 mg/mL H37RA (Cohesion Biosciences, CA, England). 500 ng of pertussis toxin (List Biological Laboratories Inc., CA, USA) was administered intraperitoneally on the day of immunization and 48 h later. The clinical symptoms were scored as follows: 0, normal; 1, tail paralysis; 2, partial hindlimbs paralysis; 3, complete hindlimbs paralysis; and 4, complete hindlimbs paralysis and partial forelimbs weakness. Animals (n = 30) were randomly divided into three groups: control, no treatment; EAE, MOG treatment; and EAE + ATO (MOG combined ATO treatment). Ten days post-immunization, mice in the EAE + ATO group was intraperitoneally injected with ATO (0.5 mg/kg/day) for 8 days. On day 22 post-immunization, mice were sacrificed and peripheral blood, spinal cord, and spleen were collected and used for further experiments.
Histopathology, Immunohistochemistry and Immunofluorescence
The spinal cord was dissected and fixed with ice-cold 4% paraformaldehyde overnight at 4 ℃, embedded in paraffin, cut into 5 μm slices, and stained with luxol fast blue (LFB) and hematoxylin and eosin (HE). LFB-stained sections were scored for demyelination as follows: 0, none; 1, rare foci; 2, a few areas of demyelination; 3, one to two large areas of demyelination; and 4, extensive demyelination. HE-stained sections were also scored for inflammation as follows: 0, none; 1, a few scattered inflammatory cells; 2, perivascular infiltrates; 3, extensive perivascular cuffing with extension into adjacent parenchyma; and 4, extensive cell infiltration in the white matter [19]. Additionally, the sections were subjected to indirect immunostaining. The primary antibodies were used as follows: anti-CD4 (1:70, Servicebio, Wuhan, China), anti-MBP (1:50, Boster, Wuhan, China), and anti-Iba-1 (1:200, Servicebio). Staining was quantified using the HALO™ image analysis software (Indica Labs, NM, USA).
Proinflammatory cytokine detection
Blood was obtained from the eyeball and kept at room temperature for 30 min. After centrifugation at 4000 rpm for 20 min, the serum was transferred into a new tube and stored at -80 ℃. The concentration of IFN-γ in serum was measured using a commercial V-PLEX proinflammatory panel 1 kit (MSD, NJ, USA).
Flow cytometry
Red blood cells were removed to obtain peripheral and spleen lymphocytes. The cells were incubated in anti-CD4-FITC (Biolegend, CA, USA) at 4 °C for 30 min. The stained CD4+ T cells were examined by flow cytometry with Beckman Cytoflex S (CA, USA). All data from flow cytometry were processed using FlowJo software V.10.
Apoptosis
Apoptosis of CD4+ T cells was detected with the Annexin V-FITC/PI Detection Kit (Meilunbio, Dalian, China). Briefly, after washing with PBS, cells were stained with Annexin V-FITC and PI for 15 min at room temperature. Subsequently, cells were detected by flow cytometry with Beckman Cytoflex S. Apoptosis signals of spinal cords were examined with the Apoptosis Detection Kit (Servicebio).
JC-1 staining
CD4+ T cells were sorted from the spleen with the Mouse CD4+ T cell Isolation Kit (Miltenyi, Bergisch Gladbach, Germany), seeded in 12-well plates, and treated with 5 μg/mL ConA or ATO (1, and 2 μM) for 24 h. JC-1 staining was performed to monitor the change of mitochondrial membrane potential. Briefly, the CD4+ T cells were incubated with 1640 RPMI medium contained 10 mg/mL JC-1 probe (Sigma) for 30 min at 37 °C. After washing three times with PBS, the stained cells were examined by flow cytometry with Beckman Cytoflex S.
Transmission electron microscope (TEM)
CD4+ T cells were cultured and treated as described above. After washing with PBS, the cells were fixed with 2.5% glutaraldehyde overnight at 4 °C. The following day they were fixed again with 1% osmiun tetroxide for 2.5 h at room temperature. Subsequently, the cells were embeded after dehydration. Ultra-thin sections were counterstained with uranyl acetate for 30 min and lead citrate for 30 s and observed by a TEM (HT7800, Hitachi).
Caspase 3 Activity Assays
Total protein was extracted from CD4+ T cells to measure Caspase 3 activity with the Caspase 3 Activity Assay kit (Applygen, Beijing, China). Briefly, BCA assay was used to determine the protein concentrations; then, 10 μL of Caspase 3 substrate was incubated with 10 μL of the total protein (30 μg) in a final volume of 100 μL for 3 h at 37 °C. The absorbance of p-nitroanilide was measured using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 405 nm in turn to calculate the Caspase 3 activity.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from spinal cords or spleen with Trizol (TansGen, Beijing, China). Reverse transcription was performed using the cDNA Synthesis SuperMix for qPCR kit (TansGen). The mRNA expression was quantified using qPCR SuperMix kit (TansGen) and normalized to β-actin. The sequences of primers are as follows: IL-1β, forward 5′-TCGCAGCAGCACATCAACAAGAG-3′, reverse 5′-TGCTCATGTCCTCATCCTGGAAGG-3′; IL-2, forward 5′-GGAGCAGCTGTTGATGGACCTAC-3, reverse 5′-AATCCAGAACATGCCGCAGAG-3′; IL-6, forward 5′-TGGGACTGATGCTGGTGACA-3′, reverse 5′-ACAGGTCTGTTGGGAGTGGT-3′; IFN-γ, forward 5′-CGGCACAGTCATTGAAAGCCTA-3′, reverse 5′-GTTGCTGATGGCCTGATTGTC-3′; TNF-α, forward 5′-GCCTCTTCTCATTCCTGCTTGTGG-3′, reverse 5′-GTGGTTTGTGAGTGTGAGGGTCTG-3′; MBP, forward 5′-GCTCTGGCAAGGACTCACACAC-3′, reverse 5′-TGGAGGTGGTGTTCGAGGTGTC-3′; β-actin, forward 5′-CATCCGTAAAGACCTCTATGCCAAC-3′, reverse 5′-ATGGAGCCACCGATCCACA-3′.
Western blot
Spinal cords were homogenized and CD4+ T cells were lysed with cold RIPA buffer (Proteintech, IL, USA) supplemented with a protease inhibitor cocktail (Sigma). After centrifugation at 4 °C for 15 min, the supernatants were collected and used for western blot analysis. The following antibodies were used: anti-Bax (1:1000, Proteintech, IL, USA), anti-MBP (1:1000, Proteintech), anti-cleaved Caspase 3 (1:1000, Affinity, OH, USA), anti-cleaved Caspase 9 (1:1000, Affinity), anti-Bcl-2 (1:1000, Proteintech), anti-cleaved PARP (1:1000, Affinity), anti-β-actin (1:5000, Bioworld, MN, USA).
Statistical analysis
Data are analyzed with GraphPad Prism 6 software (La Jolla, CA, USA) and represented as means ± SD of three separate experiments. All analysis of multiple comparisons were performed using a one-way ANOVA. A p value<0.05 was considered to be statistically significant.