Effects Of CD4~+T Cells O-GlcNAc Level In Central Nervous System Diseases

Background:Activation of the immune system after central nervous system(CNS)diseases can be divided into classic pathway,and alternative pathway.Immune activation in multiple sclerosis(MS)belongs to classic pathway,and immune activation during stroke belongs to alternative pathway.MS is a chronic immune-mediated demyelinating disease,mainly caused by immune cells infiltration into the CNS.CD4~+T cell is believed to be one of the most important pathogenic immune cell type.Intracerebral hemorrhage(ICH)accounts for about 20%of all strokes with high morbidity and mortality,and lacks effective treatment.CD4~+T cells are important peripheral-derived lymphocytes involved in secondary brain injury following ICH.O-GlcNAc is a special type of glycosylation.It is a reversible modification transferring the monosaccharide group to the serine/threonine residue of the intracellular protein.It is widely involved in many processes such as cell signal transduction,immune cell functions and tumor cell migration.We aimed to determine how the level of O-GlcNAcylatin in CD4~+T cells changes after MS and ICH,and further to clarify whether these changes affect the outcomes of MS and ICH.Methods:The O-GlcNAcylation in peripheral CD4~+T cells from mice model of MS and ICH was explored by flow cytometry and western blot.To clarify how the CD4~+T cell O-GlcNAcylation affects MS and ICH,we constructed CD4~+T cells specific Ogt downregulated mice(Ogt-KD)by gene strategy.The gene profile was compared between KD mice and the controls using isolated peripheral CD4~+T cells,and the relevant pathways were further indentified after a Gene Ontology analysis of the differentially expressed genes.The relationship between CD4~+T cell migration ability and its O-GlcNAc level was explored by Transwell experiments.The O-GlcNAc modification of the key protein Dedicator of cytokinesis(DOCK8)was confirmed by immunoprecipitation experiments.After overexpressing DOCK8,mass spectrometry was applied to find its O-GlcNAc modification sites.The O-GlcNAc site point mutation plasmid of DOCK8 was constructed and transfected into 293T and Jurkat cells to identify the key modification sites.Results:In the mouse models of MS and ICH,the level of splenic CD4~+T cells O-GlcNAcylation was higher than the controls.By constructing CD4~+T cell specific Ogt downregulated mice,we found that decrease the level of O-GlcNAcylation in CD4~+T cells significantly improved the severity of ICH and experimental autoimmune encephalomyelitis(EAE),which is a classic mouse model of MS.Less CD4~+T cells infiltration into CNS could be seen in both EAE and ICH mice.Furthermore,splenic CD4~+T cells in KD and control EAE mice were verified to have different gene profile,and then Gene Ontology analysis of the differentially expressed genes revealed that the significant changed pathway involved in cell adhesion and migration.Through Transwell experiments,we confirmed that the level of O-GlcNAcylation was up-regulated after migration of CD4~+T cells;up-regulating or down-regulating the level of O-GlcNAcylation,and the migration ability of CD4~+T cells increased or decreased accordingly.DOCK8 can be O-GlcNAcylated.After overexpressing DOCK8,we performed mass spectrometry analysis to find that DOCK8 has multiple O-GlcNAcylation sites.The O-GlcNAcylation site point mutation plasmid was constructed and transfected into 293T and Jurkat cells.It was found that the Thr1920 mutation site significantly affected the O-GlcNAcylation level of DOCK8 and involved in cell migration.Conclusion:The level of O-GlcNAcylation in splenic CD4~+T cells was elevated in EAE and ICH mice.After reducing the level of O-GlcNAcylation in CD4~+T cells,the symptoms of the EAE and ICH mice were alleviated.The protective effects can be explained by DOCK8 mediated less CD4~+T cell infiltration into CNS.Manipulating the level of O-GlcNAc modification in CD4~+T cells may be an important therapeutic target for MS and ICH.