The Character And Clinical Significance Of T Follicular Helper On Experimental Allergic Encephalomyelitis In Mouse

Objective: Observe the change of T follicular helper(Tfh) cells inexperimental autoimmune encephalomyelitis(EAE), to explore the relationshipof Tfh cells and disease. Methods:60female C57BL/6J mice were randomlydivided into three groups: namely, normal group, the EAE control group andmethylprednisolone treatment group (n=20). The myelin oligodendrocyteglycoprotein35-55(MOG35-55) and complete Freund’s adjuvant (CFA) immunizingantigen, hypodermic injection to build EAE model.The next day after build themodel,the prednisolone treatment group were given a intraperitoneal injection ofprednisolone25mg/kg dose, dosing1times a day, for14consecutive days,meanwhile each of normal group and the EAE control group was givenisopyknic saline. Record the morbidity of C57BL/6J mice,and the delitescence,the progression and the maximal neurological dysfunction score. Killed the miceduring the peak period if attacked, or30days after build the model. Collectbrain,spinal cord and arterial blood from mice. Observed the pathologicalchanges of the brain by HE staining through light microscope.The level ofinterleukin-4(IL-4)、 interleukin-10(IL-10) in spinal cord was analyzed byradioimmunoassay. The proportion of Tfh in CD4+T cell were checked by FCM;the level of interleukin-21(IL-21)、 chemokine CXC ligand13(CXCL13)secreted by Tfh was detected by ELISA.Results:(1)There was no incidence of EAE in the normal group，but completely incidence occurred in the EAE controlgroup and the methylprednisolone treatment group. We counted the days duringthe delitescence and the progression in the EAE control group and themethylprednisolone treatment group.As results,the delitescence of the EAEcontrol group were12.95±1.28days and those of the methylprednisolonetreatment group were16.10±1.29days. Through statistics analysis, we found thedelitescence of methylprednisolone treatment groupwas longer than that of theEAE control group (P<0.01). The progression also differed between groups.Theprogression of the EAE control group were4.70±0.92days and those of themethylprednisolone treatment group were3.00±0.97days,which showed that themethylprednisolone treatment group had much shorter progression than those ofthe EAE contral group (P<0.01). The neurological dysfunction score of the EAEcontrol group were3.60±1.27marks and those of the methylprednisolonetreatment group were2.35±0.88marks.The difference between two groups hadstatistics meaning (P<0.01).(2) Through light microscope, we observed that theslice of brain and spinal cord in the normal group kept still. But changes of theEAE group and the methylprednisolone treatment group in the peak period ofdisease showed perivascular inflammatory cell infiltration which is givenpriority to with lymphocyte infiltration and accompanied by a small number ofmononuclear cells, and demyelination in white matter typically withinflammatory cuff around small blood vessels. The methylprednisolonetreatment group showed less infiltration than the EAE control group.(3)The subsets of CD4+CXCR5+cells, CD4+ICOS+cells and Tfh cells in CD4+cellsmeasured with flow cytometry in the normal control group,the EAE controlgroup and the methylprednisolone treatment group: The subsets ofCD4+CXCR5+cells in CD4+cells in the normal control group were5.00±1.82%，and those of the EAE control group and the methylprednisolone treatmentgroup were25.97±3.14%and15.15±1.67%.The subsets of CD4+ICOS+cellsin CD4+cells in the normal control group were17.31±1.96%，and those of theEAE control group and the methylprednisolone treatment group were34.91±2.74%and23.09±2.39%.The subsets of Tfh cells in CD4+cells in thenormal control group were2.81±0.62%，and those of the EAE control group andthe methylprednisolone treatment group were8.58±1.27%and5.70±1.02%.Thesubsets of CD4+CXCR5+cells, CD4+ICOS+cells and Tfh cells in CD4+cells inthe EAE control group were higher than those of the normal group (P＜0.01).The subsets of CD4+CXCR5+cells, CD4+ICOS+cells and Tfh cells inCD4+cells in the methylprednisolone treatment group were lower than those inthe EAE control group (P＜0.01) but higher than those in the normal group (P＜0.01).(4)Levels of IL-21molecules and CXCL13molecules on the surface ofTfh cells measured with flow cytometry in the normal control group,the EAEcontrol group and the methylprednisolone treatment group: Levels of IL-21molecules on the surface of Tfh cells in the normal control group were1.62±0.29pg/ml，nd those of the EAE control group and the methylprednisolonetreatment group were3.47±0.35pg/ml and2.45±0.27pg/ml.Levels of CXCL13 molecules on the surface of Tfh cells in the normal control group were1.73±0.43pg/ml， and those of the EAE control group and themethylprednisolone treatment group were3.75±0.52pg/ml and2.64±0.27pg/ml.Levels of IL-21molecules and CXCL13molecules on the surface of Tfhcells in the EAE control group were higher than those in the normal group (P＜0.01). Levels of IL-21molecules and CXCL13molecules on the surface of Tfhcells in the methylprednisolone treatment group were lower than those in theEAE control group (P＜0.01) but higher than those in the normal group (P＜0.01).(4)IL-4and IL-10levels in medulla oblongata of mice in the normalcontrol group,the EAE control group and the methylprednisolone group:IL-4levels in medulla oblongata of the normal group were24.98±2.23pg/ml,whilethose of the EAE control group were11.17±2.07pg/ml and themethylprednisolone treatment group were18.48±2.23pg/ml.IL-4levels inmedulla oblongata of the EAE control group lower than those of the normalgroup significantly（P<0.01). And the IL-4levels in medulla oblongata of themethylprednisolone treatment group surmounted significantly than the EAEcontrol group but lower than those of the normal group（P <0.01). IL-10levelsin medulla oblongata of the normal group were2.60±0.17pg/ml,while those ofthe EAE control group were1.14±0.13pg/ml and the methylprednisolonetreatment group were2.05±0.15pg/ml.IL-10levels in medulla oblongata of theEAE control group significantly lower than those of the normal group（P<0.01).And the IL-10levels in medulla oblongata of the methylprednisolone treatment group surmounted significantly than the EAE control group，but significantlylower than those of the normal group（P <0.01).(5)Correlative analysis: Levelsof IL-4and IL-10of the normal control group, the EAE control group and themethylprednisolone treatment group were significantly negatively correlatedwith the subsets of Tfh cells in CD4+cells(P<0.01).And levels of IL-21andCXCL13in medulla oblongata of the normal control group, the EAE controlgroup and the methylprednisolone treatment group were positively correlatedwith the subsets of Tfh cells in CD4+cells(P<0.01). Levels of Tfh cells andIL-21molecules and CXCL13molecules on the surface of Tfh cells of the EAEcontrol group and the methylprednisolone treatment group were negativelycorrelated with the delitescence significantly (P<0.01),but positively correlatedwith the progression and the neurological dysfunction scoret in the peak period(P<0.01).In the meanwhile,IL-10levels in medulla oblongata of the EAEcontrol group and the methylprednisolone treatment group were positivelycorrelated with the delitescence(P<0.01),and were negatively correlated with theprogression and the neurological dysfunction score in the peak period (P<0.01).Conclusion:1.MOG35-55can induce C57BL/6mice to produce EAE modelsuccessfully because of the neurological disturbances of EAE mice wereremarkable,also because of the vessels in brain and spinal cord weredemylinated and infiltrated with inflammatory cell infiltration.2.IL-4levels andIL-10levels in medulla oblongata of the EAE control group significantly lowerthan those of the normal group,levels of IL-4and IL-10of the normal control group were significantly negatively correlated with the subsets of Tfh cells inCD4+cells,but positively correlated with the progression, the peak period andthe neurological dysfunction score.It shows the fact that IL-4and IL-10caninhibiting the inflammatory response.3.Levels of Tfh cells and IL-21moleculesand CXCL13molecules on the surface of Tfh cells in the EAE control groupwere higher than those in the normal group,and significantly negativelycorrelated with the process of delitescence,and were significantly positivelycorrelated with the progression, the peak period and the neurologicaldysfunction score.All of these shows that Tfh cells may take part in nosogenesisof EAE.4.Methylprednisolone may treat EAE by inhibiting the abnormallyincteaseing of Tfh cells,IL-21and CXCL13,it may also functioning bypromoting the levels of IL-4and IL-10.