The Expression Of S1P1 And Inflammtory Chemokine Receptors In ??T17 Cells Of EAMG Lewis Rat

Background and objectiveMyasthynia gravis?MG?is a typical autoimmune disease.Most patients have autoantibodies against the acetylcholine receptor?AchR?and block the binding of acetylcholine to the receptor at the neuromuscular junction?NMJ?,which would block the transmission of the signal of nerve-muscle and leads to muscle weakness.IL-17 is a typical inflammatory-regulatory cytokine,closely involved in the occurrence and development of autoimmune diseases.In vivo,??T17 is not only an important source of IL-17,but also is believed to have the effect of inducing Th17 to produce IL-17.Therefore,in recent years,the role of??T17 in autoimmune diseases has attracted more and more attention.However,the relationship between??T17 and MG is not clear.We detected the increase in thymic??T cells in MG patients by flow cytometry in the earlier period;and the results of immunofluorescence showed that S1P1 positive??T cells were mostly distributed at the corticomedullary junction?CMJ?,which suggests that there is an increase in the output of??T cells in the thymus of MG patients.To further analyze the characteristics of migrating-out from thymus and inflammatory chemotaxis of??T17 cells under MG pathological conditions.In this experiment,Lewis rats were immunized with 97-118 peptides of human recombinant AChR?subunit,and experimental autoimmune myasthynia gravis?EAMG?model was established to detect the expression of S1P1 and the inflammatory chemokine receptors such CCR9 on the surface of??T cells and analyse of the effect of S1P1 signal on the activation and function of??T.Methods1.Induction and evaluation of Lewis rat EAMG:The EAMG model was established by immunizing Lewis rats with human recombinant AChR?subunit 97-118 peptide.42 rats were randomly divided into two groups,of which 22 were EAMG-induced group and 20 were control group.Intradermal injections of the hindpaw foot pads and shoulders were performed every 28 days according to 200?g of peptide dose per mouse.Induction was performed a total of three times.Complete Freund's adjuvant?CFA?was used for the first induction,and incomplete Freund's adjuvant?IFA?was then used.After the induction began,the body weights of the two groups were measured every 7 days,and muscle function was measured by a cage-grabbed experiment.Before induction and 15 days after the second and third induction,peripheral blood from the tail veins of the two groups of rats was collected and the concentration of anti-human recombinant AChR?subunit 97-118 peptide antibody in serum was determined by ELISA.If the serum OD value of the EAMG-induced group/the control group serum OD value was 2.1,the antibody was considered as positive.After 15 days of the third induction,electromyograms were performed on all rats.Action potentials of the EAMG induction group and the control group were recorded,and the ratio of the 4th wave to the 1st wave amplitude was calculated.A decrease of 10%or more in the average amplitude of the stimulation at 3Hz and 5Hz was considered as an abnormal decrease in amplitude of the low-frequency repetitive nerve electrical stimulation,that is positive.Finally,the rats were scored according to the EAMG rat scoring rules.2.The axillary and in guinal lymph nodes and thymus tissues of the EAMG group and the control group were isolated by aseptic surgery,ground and filtered in an ultra-clean bench,and centrifuged to prepare a single cell suspension.Cells were labeled with anti-CD3-FITC,anti-??TCR-PE,and anti-IL-17-APC flow-fluorescent antibodies.The proportion of??T and??T17 cells was detected by FCM;anti-S1P1-Alexa Fluor 680,anti-CCR2-Alexa Fluor 700,and anti-CCR6-Alexa Fluor405 and anti-CCR9-Alexa Fluor 700 antibody was used to label and detect expression of S1P1 and corresponding chemokine receptors on the surface of??T and??T17cells.3.Immunohistochemical staining of 3?m paraffin sections of lymph nodes and thymus tissues of experimental rats was performed to observe the expression and distribution of??T cells.Anti-??TCR and S1P1 immunofluorescent antibodies were used to double-label frozen rat lymph nodes and thymus tissue to observe the distribution and expression of S1P1⁺??T cells.4.The spleen of Lewis rats was aseptically isolated,ground,filtered,and centrifuged to prepare a single cell suspension.The cells were incubated at 37?,5%CO₂ for 1h and 40 min,washed and adherent stromal cells were obtained.??T cells were isolated from rat lymph nodes by immunomagnetic beads,and the positive rate of flow identification was>90%.A mixed culture system was established with a ratio of stromal cells to??T cells of 1:1,and IL-2?2000 UI/mL?and anti-CD3 antibody?50?g/10⁵ cells?were added.The mixed cultured cells were divided into four groups:EAMG Group Blank,EAMG-S1P stimulation group,control group Blank and control-S1P stimulation group,after 36 hours,the flow antibody labeled CCR2 and CCR6 in??T of EAMG group,and the total protein of each group was extracted.Biolegend LEGENDplex^TM Multi-Analyte Flow kit was used to detect the concentrations of IL-2,IL-4,IL-5,IL-6,IL-9,IL-10,IL-13,IL-17A,IL-17F,IL-22,IFN-?,GM-CSF and TNF-?in the total proteins extracted from each group.5.Statistical analysis:SPSS 20.0 statistical software was used to test the normality.If the data was in accordance with the normal distribution,the t-test was used for analysis.If the data were not consistent,ANOVA was used for analysis.The results were descripted as mean±standard deviation,P<0.05 indicates that the difference was statistically significant.Results1.The results of evaluation of EAMG Lewis rat:The weight of the control group increased steadily,but the body weight of the EAMG-induced group gradually decreased after the first immunization.The powerful grabbing time of rats in the experimental-inducing EAMG group were siginificant reduction.The results of ELISA showed that on 15th day after the second and third induction,the ratio of serum OD values in the EAMG-induced group to the serum OD value in the control group was 3.94±0.85 and 7.72±1.65,respectively.That is,the anti-human recombinant AChR?subunit 97-118 peptide antibody in the peripheral blood of EAMG-induced group was positive;in addition,according to the electromyogram results,except for the four negative rats,the amplitude attenuation rates of the 3Hz and 5Hz amplitudes of the EAMG induction group were 15.90±4.30%and15.60±3.65%,respectively,which was statistically significant compared with the control group?6.60±3.20%,8.70±2.30%,P<0.05?.At the end of the experiment,the scores of rats in the experimental-inducing EAMG group are followed,4 rats were 0points;6 rats were1 point;10 rats were 2 points and 2 rats were 3 points.2.Expression and distribution of??T in lymph nodes and thymus of EAMG rats:FCM results showed that in the EAMG group,the lymph node?1.22±0.12%?and the thymus?0.43±0.11%?of the CD3⁺??T cells were higher than the control group?0.84±0.16%?and the thymus?0.22±0.07%,P<0.05?.Thymus??T cells were mostly located in the subcapsular region,CMJ and medulla region.EAMG group S1P1⁺??T increased significantly in thymic vascular rich CMJ,and the proportion of S1P1^high??T cells?54.33±18.41%?was significantly higher than the control group?5.72±1.45%,P<0.05?.Lymph node??T cells were mostly distributed in the medulla,and the medulla area S1P1⁺??T in the EAMG group was significantly increased compared with the control group.The proportion of S1P1^high??T in the EAMG group lymph node cells was 38.99±10.09%,which was significantly higher than the control group?13.68±3.39%,P<0.05?.The above data suggest that both thymic output and lymphatic metastasis of??T cells in the EAMG group increased.3.Expression of S1P1 and inflammatory chemokine receptors in??T17 cells of EAMG rat thymus:FCM results showed that the proportion of??T17 in??T cells in EAMG group was 12.25±2.79%,which was significantly higher than that in control group?5.81±1.52%,P<0.05?;in??T17 cells,the positive rate of S1P1 in EAMG group was 97.34±1.83%compared with the control group?95.87±2.07%?,there was no significant difference?P>0.05?,but the mean fluorescence intensity of S1P1 in??T17 cells?377.5±89.90?was significantly higher than that in the control group?250.17±21.62,P<0.05?.It suggests that S1P1 expression is up-regulated in EAMG thymic??T17 cells.In addition,in??T17 cells,the proportion of S1P1/CCR9 double positive cells in the EAMG group was 85.08±4.19%,which was significantly higher than that in the control group?64.97±6.12%,P<0.05?.The proportion of CCR6-positive??T17 cells?29.69±6.31%?was also significantly higher than that of the control group?15.33±3.60%,P<0.05?,suggesting that EAMG mice had increased??T17 cells homing from the thymus gland.4.Expression of S1P1 and chemokine receptors on??T17 cells in EAMG rat lymph nodes:FCM results showed that the proportion of??T17 in??T cells in EAMG group was 14.58±2.92%,which was significantly higher than that in control group?6.13±1.92%,P<0.05?;the proportion of S1P1⁺cells in??T17 cells?97.44±2.61%?was significantly higher than that in control group?82.66±6.55%,P<0.05?;the proportions of CCR2⁺and S1P1/CCR9 double positive cells in??T17 were70.18±7.49%and 84.68±9.59%,respectively,which were significantly higher than those in the control group?40.42±5.18%,54.21±7.83%,P<0.05?;and the proportion of CCR6⁺cells?32.93±4.96%?was significantly lower than the control group?47.54±6.42%,P<0.05?.5.Effect of S1P1 signal on??T cell function:Under the stimulation of S1P,the concentrations of IL-2,IL-6,TNF-?,GM-CSF,IL-17A and IL-17F in EAMG group and control group were significantly higher than those in the blank group?P<0.05?;but there was no significant difference in the expression of IFN-?,IL-22,IL-13,IL-10,IL-9,IL-5 and IL-4?P>0.05?.In addition,FCM detection found that CCR2⁺??T cells?47.90±7.84%?in the EAMG-S1P stimulation group were significantly higher than those in the blank group?2.38±1.84%,P<0.05?.ConclusionsThe percentage of??T17 cells in the thymus and lymph nodes of EAMG Lewis rats increased,and the expression of S1P1,CCR9 and CCR6 on the surface of thymic??T17 cells were up-regulated,which was beneficial to the thymus output.The positive rate of S1P1,CCR9,and CCR2 in??T17 cells of lymph nodes increased,and the positive rate of CCR6 decreased,which is favorable to the chemotaxis of inflammation.In addition,S1P1 receptor signaling can also promote??T cells to upregulate CCR2,and promote the secretion of IL-17 and other cytokines,affecting the function of??T.