The Therapeutic Effect And Mechanism Of Breg, PD1, PDL1 In The Experimental Autoimmune Encephalomyelitis By Tolerance Dendritic Cell

Background and objective :Multiple sclerosis(MS)is a chronic inflammatory autoimmune disease of central nervous system(CNS)which is characterized by the axonal destruction and demyelination of brain and spinal cord.MS patients express different clinical symptoms,severe cases even can cause physical disability and then greatly reduce the quality of life.Experimental autoimmune encephalomyelitis(EAE)is a common animal model of MS.As professional antigen presenting cell,dendritic cell(DC)plays an important role in the pathogenesis of MS.Tolerogenic dendritic cell(t DC)is a special type of DC,which can induce the formation of regulatory T cell(Treg)and promote the secretion of inflammatory suppression factors,which have potential therapeutic for MS.Programmed death 1(PD1)and programmed death ligand 1(PDL1)belong to the B7 superfamily,play a role in the pathogenesis of MS via negatively regulate function.Regulatory B cell(Breg)is a kind of B cells,which can inhibit the development of the disease by secreting inflammatory suppression factors MS.1,25(OH)2D3 is an immunomodulator,which can effectively induce the formation of t DC(VD3-DC)that plays therapeutic effect in a certain degree after being returned to the onset of EAE mice.In this experiment,mononuclear cells,derived from bone marrow,are induced to be DC in vitro and then are induced to be VD3-DC with 1,25(OH)2D3 which has the immunomodulatory effect.Next,we detect the expression of PDL1 on the surface of DC and VD3-DC and the both will be back to the diseased EAE mice.We observe: the clinical symptoms of EAE mice after reinfusion therapy;the expression of PD1 on the surface of T cells in spleen and lymph nodes;the proportion of Breg in spleen and lymph nodes;the inflammatory cell infiltration and demyelination in the spinal cord of mice through hematoxylin eosin(HE)staining and Luxol fast blue(LFB)staining.Our research purpose is to explore the therapeutic effect and mechanism of Breg,PD1,PDL1 in the experimental autoimmune encephalomyelitis by VD3-DC.Methods:1.C57BL/6 mice were injected subcutaneously(s.c.)with MOG33–55 in complete freund’s adjuvant(CFA)containing H37RA(Mycobacterium tuberculosis).Mice were injected intraperitoneally(i.p.)with 500 ng pertussis toxin(PTX)on day 0and day 2.2.Mononuclear cells derived from bone marrow in cultured medium added to interleukin-4(IL-4)and granulocyte-macrophage colony-stimulating factor(GM-CSF)(10ng/ml)to induce DC and then added 1,25(OH)2D3(10-8M)into the medium to generate VD3-DC.Next,in order to promote DC and VD3-DC maturity with adding in lipopolysaccharide(LPS)for 24 h to stimulate,detect the expression of PDL1 on the surface of DC and VD3-DC using flow cytometry.3.EAE mice were divided into three groups,at day 8,DC/VD3-DC were harvested,then incubated by MOG antigen 4 hours and washed MOG antigen,EAE mice were injected respectively PBS,DC and VD3-DC via tail wein(EAE mice induced by 10,13,16 days),observed the clinical symptoms.4.On the peak of treatment,separated the spleen and lymph node cells from EAE mice,detect the expression of PD1 and the proportion Breg using flow cytometry.5.On the peak of treatment,separated the spinal cord,observe the spinal cord inflammatory cell infiltration and demyelination by HE and LFB staining.Results:1.According to our previous experiments,we can successfully induce EAE mice animal model and VD3-DC by the same experiment methods and obtain the number of PDL1 on the surface of VD3-DC more than DC,and the difference was statistically significant;2.Compared with PBS control group,the clinical symptoms of EAE mice can relieve with DC/VD3-DC treatment and the latter effect is more significant.3.Compared with PBS control group,the expression of PD1 on the surface of T lymphocytes in spleen and lymph nodes increased both DC and VD3-DC groups.4.Compared with PBS control group,the proportion of Breg(CD19+CD5+CD1d+)increased markedly among the spleen-B cells in VD3-DC group,however,the proportion of Breg reduced in DC group,and the difference had no statistical significance.In addition,the proportion of Breg augmented on the lymph nodes-B cells both DC and VD3-DC groups,but the difference had no statistical significance.5.Compared with PBS control group,we can know from the results of HE and LFB staining,the treatment with DC/VD3-DC can decrease inflammatory cell infiltration and demyelination in spinal cord and the effect of VD3-DC group is more notable.Conclusions:1.The expression of PDL1 on the surface of VD3-DC increase.2.VD3-DC treatment can alleviate the clinical symptoms of EAE mice.3.The expression of PD1 and the proportion increase on the surface of B/T cells in the spleen and lymph nodes with VD3-DC.4.VD3-DC treatment can reduce the inflammatory cell infiltration and demyelination in spinal cord.