The Regulation Of B7-H4 Mediated Mesenchymal Stem Cells On Immune Microenvironment In EAE

Objective:To investigate the effects of B7-H4 molecule-mediated mesenchymal stem cell transplantation on immune microenvironment in mouse experimental autoimmune encephalomyelitis(EAE)model and its possible mechanism.Methods:1)The mouse models of experimental autoimmune encephalomyelitis(EAE)induced by peptide myelin oligodendrocyte glycoprotein(MOG35-55),and green fluorescent protein(GFP)-labeled mouse mesenchymal stem cell line C3H10 T1/2(C3H10)were used to treatment.Mice were randomly divided into normal group,EAE group(1 w after immunization,injection 100?l PBS),C3H10 group(1 w after immunization,transplantation C3H10 Cells 1×10~6cell/mice),C3H10-B7H4 group(1 w after immunization,transplantation C3H10-B7H4 cells(B7-H4 targeting shRNA transfected C3H10 cells)1×10~6cell/mice),C3H10-NC group(1 w after immunization,transplantation C3H10-NC cells(transfer the empty plasmid to C3H10cells)1×10~6cell/mice).Observe indicators:1)The neurological function scores of each group were observed every day.Additionally,the migration and survival of stem cells in the microenvironment of spinal cord lesions and the myelination,axonal damage and glial cell response were observed by histopathology at the peak of the onset(20 days after immunization).The inflammatory cell infiltration,inflammatory cell composition and the expression of negative co-stimulatory molecules PD-L1 and B7-H4 were analyzed.2)Anatomical observation of optic nerve in each group was performed,and through pathological examination of optic nerve inflammatory infiltration and myelination,while the retinal morphological observation and thickness detection were performed to study the apoptosis and necrosis of ganglion cells.3)The B cells and subgroups of peripheral blood and spleen for each group were detected at the peak of the onset.In addition,the morphology of spleen lymphocyte germinal center and the distribution of spleen T and B cells were observed.Result:1)Neurological deficit score showed that the transplanted with mouse mesenchymal stem C3H10 cells could alleviate the symptoms of EAE mice,delay the onset time and decrease the neurological deficit score.However,the therapeutic effect was significantly attenuated when down-regulate the expression of B7-H4 on C3H10 cells.The results showed that stem cells could migrate into the spinal cord and survived.Pathological studies showed that inflammatory cell infiltration,demyelination and glial cell proliferation were observed in the spinal cord tissue except the normal group.However,the degree of inflammatory cell infiltration,myelination and glial cell proliferation in C3H10transplantation group was significantly lighter than that in C3H10-B7H4 cell transplantation group and the number of PD-L1 and B7-H4 positive cells was significantly higher than that of EAE model group and C3H10-B7H4 group.2)The anatomical observation of each group optic nerve showed that there was mild edema in the optic nerve,especially in the capsule layer in EAE group and parallel to the C3H10-B7H4 group,but there was no significant difference between C3H10 group,C3H10-NC group and normal group.The Pathological staining showed that inflammatory cell infiltration,demyelination,axonal damage and ganglion cell apoptosis were observed in the optic nerve tissues after MOG induction for EAE group and were similar with C3H10-B7H4 group,while C3H10 group or C3H10-NC group was lighter than model group.The thickness of retina and the bipolar cells were found to be significantly thinner than those in the normal group and was not different from that of the C3H10-B7H4 group.Conversely,C3H10 and C3H10-NC group could protect neurons and significantly inhibit retinal thinning.3)The ratio of B lymphocytes in the peripheral blood of each group of mice was similar in the peak time,while the proportion of B cells in spleen cells in EAE model group was similar to that in C3H10-B7H4 group,but lower than the normal group.Interestingly,the ratio of spleen B cells in C3H10 transplantation was similar to that in the normal group.The difference of memory and plasma cells distribution between peripheral blood and spleen showed that the memory cells and plasma cells were mainly distributed in the spleen for EAE group.After C3H10 stem cell transplantation,the memory cell distribution was statistically different,while the plasma cells distribution were not statistically differences.The difference of memory and plasma cells distribution between peripheral blood and spleen,there was no significant difference in C3H10-B7H4transplantation group after down-regulation of B7-H4 molecule.Histopathological findings showed that the spleen of EAE group was significantly bigger than normal group,and the spleen volume of the other groups were slightly increased,but were smaller than the model group.Pathological findings showed that the spleen germinal center of each group was significantly increased after MOG induction,and the spleen germinal center of EAE model group was significantly increased compared with other treatment groups,and a large amount of T cell infiltration was observed in center,and mixed with B cells.The spleen germinal center for C3H10 group and C3H10-NC group also can be seen a large number of B cell aggregation and the presence of T lymphocyte infiltration.However,compared with the EAE group,the number of T cells was significantly reduced,but it is surprising that the C3H10-B7H4 group is similar to the model group,showing a large number of T,B cell infiltration.Conclusions:1)C3H10 transplantation can reduce the EAE disease,delay the onset,andits protective effect mediated by B7-H4.Stem cells can migrate and survive in the spinal cord lesions,B7-H4-mediated mesenchymal stem cells C3H10 involved in the inhibition of proinflammatory cell infiltration,myelin sheath loss and glial cell proliferation,and the regulation of spinal cord lesions microenvironment.Moreover,B7-H4-mediated mesenchymal stem cells can affect the expression of PD-L1 and B7-H4 in the tissue,and provide the appropriate microenvironment for the protection of nerve cells and promoting myelination and axon repair and regeneration.2)Stem cell transplantation can inhibit inflammatory cell infiltration,demyelination,axonal damage and ganglion cell apoptosis,prevent retinal atrophy,and thereby protecting the multiple sclerosis related optic nerve injury.B7-H4 molecules involved in the protection of myelination and axonal injury,inhibition of ganglion cells and bipolar cell apoptosis,necrosis,protection of retinal neurons to prevent retinal thinning.3)B cells as antigen presenting cells(APC)can induce T cell activation,and then causing demyelination and induced the occurrence of EAE.B7-H4 mediates the therapeutic effect of mesenchymal stem cells on EAE and inhibits B cells as APC cells presenting antigens,activating T cells,inhibiting plasma cell differentiation and producing related antibodies.