Fingolimod Modulates Th1 And Treg Cell Differentiation And Function In Experimental Autoimmune Encephalomyelitis Through The Akt-m TOR Axis

Part one The effect of fingolimod on prevention and treatment inexperimental autoimmune encephalomyelitis miceObjective: This study chose different doses of fingolimod to manage EAE mice and studied their effect of prevention and treatment on EAE to select a safe and effective dose preparing for the next expreiment.Methods:1 EAE induction. C57BL/6 mice at 8–10 weeks of age was induced by immunization with 250-μg myelin oligodendrocyte glycoprotein(MOG) p35–55 dissolved in complete Freund’s adjuvant(CFA), which contained 4-mg/ml heat-killed Mycobacterium tuberculosis H37 Ra. After emusfication, as per 0.1 ml is administered subcutaneously into mice by dividing each side of the spine into four points. At time 0 and at 48 h post-immunization, mice were injected intraperitoneally with 500-ng pertussis toxin.2 After being induced, EAE mice were dealt with different doses of fingolimod(2.0 mg/kg,1.0 mg/kg,0.3 mg/kg,0.1 mg/kg). Mice were examined daily after induction for clinical signs of EAE for 30 days and scored according to the following criteria: 0, no paralysis; 1, tail paralysis; 2, hindlimb weakness or partial paralysis; 3, hindlimb paralysis; 4, forelimb and hindlimb paralysis; and 5, moribund and death. When clinical signs were inter-mediate between two grades of the disease, 0.5 was added to the lower score. The effect of different doses of fingolimod was evaluated and the effective dose was chose for the following experiment.3 Daily treatment with fingolimod(1.0 mg/kg) began when a clinical score of ≥1 was reached and clinical score of EAE mice were evaluated every day. The therapeutic effect of fingolimod was assessed according the above criterion.4 From the first day after EAE mice model set up, fingolimod(1.0 mg/kg) were given. On post-immunization day 21(at the peak time of EAE), mice were induced to death by deep anesthesia and cardiac perfusion with 4% paraformaldehyde(PFA), embedded in paraffin, using standard methodology. Spinal cord sections were stained with hematoxylin & eosin(H&E) for evaluating overall histology and inflammation. And spinal cord sections were stained with Luxol Fast Blue(LFB) for evaluating demyelination. The degrees of inflammation and demyelination were assessed semi-quantitatively on three non-serial sections of each mouse in a blinded manner, as described in previous study. The level of inflammation was quantified as the following standardas: 0, no inflammation; 1, cellular infiltrates only around blood vessel and meninges; 2, mild cellular infiltrates in parenchyma(1–10/section); 3, moderate cellular infiltrates in parenchyma(11–100/section); 4, serious cellular infiltrates in parenchyma(> 100/section). The level of demyelination was quantified as the following standardas: 0, normal white matter; 1, rare foci; 2, a few areas of demyelination; 3, confluent perivascular or subpial demyelination; 4, massive demyelination involving one half of the spinal cord; 5, extensive demyelination involving the whole cord.After perfusion with 4% glutaraldehyde for 0.5 h, the spinal cords(n = 1) from each group were dissected out rapidly and made into small blocks, pre-fixed with 4%(v/v) glutaraldehyde, and immersed in PBS. After washing in PBS, the blocks were post-fixed in 1%(w/v) buffered osmium tetroxide for 1 h. After dehydration with graded acetone, blocks were embedded in epoxy resin Epon 812. Sections were cut and stained with uranyl acetate and lead citrate. Demyelination was measured with a JEM-1230 electron microscope.5 Using SPSS18.0 software for statistical analysis, data are presented as the mean ± SD. Significant differences in clinical scores between pairs of groups were examined using Mann Whitney U test. The comparison of the sample rate used χ2 test and other statistical comparisons between two groups were analyzed by ANOVA analysis. A P value of <0.05 was considered statistically significant.Results:1 Using four different doses(2.0 mg/kg, 1.0 mg/kg, 0.3 mg/ml, 0.1 mg/kg) of fingolimod preventing EAE mice, the effect of 2.0 mg/kg, 1.0 mg/kg and 0.3 mg/kg were obvious. Through the comparison between groups, the effect of 1.0 mg/kg group and 2.0 mg/kg group was equal, and the effect of 1.0 mg/kg group is better than 0.3 mg/kg and 0.1 mg/kg group. Subsequently, we chose the dose of 1.0 mg/kg for further study.2 When a clinical score of ≥1 was reached, EAE mice were divided into two groups randomly(EAE group and EAE plus fingolimod treatment group). We found that fingolimod could revese the clinical score of EAE significantly.3 Through the spinal cord slice for HE staining, LFB staining and electron microscope, there was almost no inflammatory infiltration or demyelination in the dorsal lateral funiculus of lumbar spinal cord in healthy control mice, and there were more inflammatory infiltration or demyelination in EAE group than in the health control group. Moreover, there were less inflammatory cells infiltration and myelin depigmentation in EAE mice treating with fingolimod than in EAE group. The results suggested that fingolimod treatment reduced inflammatory infiltration or demyelination obviously in EAE mice. Part Two Fingolimod regulates Th1/Treg cell differentiation and functionin experimental autoimmune encephalomyelitis miceObjective: This study chose fingolimod to manage EAE mice and analyzed its influence to the differentiation and function of Th1 and Treg cells to explore the immunomodulatory effects.Methods:1 EAE induction. C57BL/6 mice at 8–10 weeks of age was induced by immunization with 250-μg myelin oligodendrocyte glycoprotein(MOG) p35–55 dissolved in complete Freund’s adjuvant(CFA), which contained 4-mg/ml heat-killed Mycobacterium tuberculosis H37 Ra. After emusfication, as per 0.1 ml is administered subcutaneously into mice by dividing each side of the spine into four points. At time 0 and at 48 h post-immunization, mice were injected intraperitoneally with 500-ng pertussis toxin.2 At the peak time of EAE, in accordance with the requirement for preparation of single cell suspension, flow cytometry was used to test the proportion of Th1 and Treg cells in the spleens of three groups(health control group, EAE group, EAE plus fingolimod treatment group).Splenocytes(n = 6) were harvested and washed with RPMI 1640 medium. Samples were stimulated in triplicate with MOG35-55 in 24-well plates for 24 h. After that, cells were stimulated with 50-ng/ml phorbol 12-myristate 13-acetate(PMA), 1-ug/ml ionomycin and 5-μg/ml of brefeldin A, and culture medium was collected after 13 h. Treg cells were detected directly without stimulation. Cell-surface proteins were labeled with FITC-anti-CD4 or FITC-anti-CD4/APC-anti-CD25 antibodies. Intracellular cytokines were labeled with PE-anti-IFN-γ and APC-anti-IL4 antibodies(for Th1/Th2 cells) or PE-anti-Foxp3 antibody. A minimum of 10,000 cells were analyzed by flow cytometry on a FACSCalibur flow cytometer.3 Spleens were dissected at the peak time of disease, the concentrations of IFN-γ and TGF-β in the splenocyte supernatants of C57/BL6 mice were determined using ELISA and the levels of IFN-γ and TGF-β mRNA gene in splenocytes were tested by real-time PCR. Cell supernatants were collected to measure IFN-γ and TGF-β concentrations by quantitative ELISA and the test was carried out in accordance with the manufacturer’s recommendations.Total RNA was extracted and RNA reverse transcribed into cDNA. qRT–PCR parameters: Initial incubations were performed at 94°C for 30 s, followed by 40 cycles of 94°C for 15 s and 60°C for 15 s and 72°C for 10 s.4 Spinal cords were dissected at the peak time of EAE. The levels of IFN-γ and TGF-β mRNA were determined by real-time PCR and the expression of TGF-β was detected with immunohistochemical assay in the three groups(health control group, EAE group, EAE plus fingolimod treatment group).Mice(n=6) from each group mice were induced to death by deep anesthesia and cardiac perfusion with 4% paraformaldehyde(PFA), and spinal cords were embedded in paraffin. The spinal cord tissue sections(5 μm/each) were incubated with anti-TGF-β at 4 °C overnight. After being washed, the bound antibodies were detected with biotinylated secondary antibodies, and visualized using diaminobenzidine, followed by examination under a light microscope.5 Using SPSS18.0 software for statistical analysis, data are presented as the mean±SD. Statistical comparisons between two groups were analyzed by ANOVA analysis. A P value of <0.05 was considered statistically significant.Results:1 There were different changes in the frequencies of Th1 and Treg cells in the spleens of EAE mice. Compared with health control group, the proportion of Th1 cells increased, while Treg cells decreased significantly in the EAE group. The proportion of Th1 cells decreased, while Treg cells increased significantly in EAE plus fingolimod treatment group compared with EAE group.2 Compared with health control group, IFN-γ concentration increased and TGF-β concentration decreased significantly in the spleens of EAE mice. In the contraty, IFN-γ concentration decreased and TGF-β concentration increased significantly in EAE plus fingolimod treatment group compared with EAE group. Furthermore, the same changes also happened in gene expression.3 Using immunohistohistochemical method to detect the expression of TGF-β in the spinal cord of mice, the study showed that the expression of TGF-β weakened in EAE group compared with control group, and the expression of TGF-β strengthened in fingolimod group compared with EAE group.4 Using real-time PCR method, the study showed that IFN-γ mRNA upregulated and TGF-β mRNA downregulated obviously in EAE group compared with the health control group. In the contrary, IFN-γ mRNA downregulated and TGF-β mRNA upregulated obviously in fingolimod treatment group compared with the EAE group. Part three Fingolimod ameliorates experimental autoimmuneencephalomyelitis mice through Akt-mTOR signalingpathwayObjective: This study chose fingolimod to manage EAE mice and discussed the immune regulation of the Akt-mTOR signaling pathways acted on EAE mice for further, expecting to provide new ideas for treating MS and find a new direction for the application of fingolimod in other nervous system diseases.Methods:1 EAE induction. C57BL/6 mice at 8–10 weeks of age was induced by immunization with 250-μg myelin oligodendrocyte glycoprotein(MOG) p35–55 dissolved in complete Freund’s adjuvant(CFA), which contained 4-mg/ml heat-killed Mycobacterium tuberculosis H37 Ra. After emusfication, as per 0.1 ml is administered subcutaneously into mice by dividing each side of the spine into four points. At time 0 and at 48 h post-immunization, mice were injected intraperitoneally with 500-ng pertussis toxin.2 Spleens were dissected at the peak time of disease. Real-time PCR method was used to test the expression of T-bet and Foxp3 mRNA in the three groups of mice.3 Spleens were dissected at the peak time of disease, and total proteins were extracted from spleens for Western blot analysis. Akt, pAkt, s6 k and ps6 k expression in the spleens of mice were examined in healthy control group, EAE group and EAE plus fingolimod treatment group.The proteins were prepared according to the manufacturer’s instruction. The concentrations of proteins were measured using a BCA protein assay reagent kit. Equal amounts of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electro-phoresis(SDS-PAGE) and transferred onto polyvinylidene fluoride membranes. Nonspecific reactivity was blocked by 5% non-fat dry milk in TBST buffer at room temperature for 1 h, followed by incubation with primary antibodies at 4°C for 12 h. After being washed with PBST for three times, the bound antibodies were detected with corresponding secondary antibodies. The relative levels of target protein to control expression were quantified by densitometric scanning.4 Using SPSS18.0 software for statistical analysis, data are presented as the mean ± SD. Statistical comparisons between two groups were analyzed by ANOVA analysis. A P value of <0.05 was considered statistically significant.Results:1 Compared with health control group, the T-bet mRNA upregulated and Foxp3 mRNA downregulated notably in the spleens of EAE mice. After treatment with fingolimod, the T-bet mRNA downregulated and Foxp3 mRNA upregulated notably compared with EAE mice.2 At the peak time of disease, the expression of pAkt/Akt and ps6k/s6 k enhanced significantly in EAE group compared with health control group, while after fingolimod treatment, the expression of pAkt/Akt and ps6k/s6 k diminished notably compared with EAE group.Conclusion:1 Fingolimod treatment could prevent the EAE onset and reduced inflammatory infiltration or demyelination obviously in EAE mice. And fingolimod treatment also reverses the progression of EAE.2 The onset of EAE mice affected the differentiation and function of Th1 and Treg cells, and fingolimod treatment modulated the differentiation and function of Th1 and Treg cells.3 The onset of EAE mice activated Akt-mTOR signaling pathways. Fingolimod regulated the differentiation and function of Th1 and Treg cells by inhibiting this signal pathway. These findings suggested us Akt-mTOR pathway inhibitor may become a new target for the treatment of MS.