Roles Of FasL In Mediating The Subsets Of CD4~+T Cells After Experimental Stroke And The Underlying Mechanisms

Background and Objective:The peripheral CD4+T lymphocytes infiltrated into the brain parenchyma and acted on the microglia after ischemic stroke.It is closely related to the occurrence,development and prognosis of brain immune inflammation.Different subsets of CD4+T lymphocytes mediate different inflammatory effects.Among them,Th1/Th17 mediated pro-inflammatory effect,while the Th2/Treg cells mediated inflammatory effect.Fas/FasL is expressed in a variety of immune cells and involved in the transmission of inflammatory signals.This study aims to investigate the effect of FasL on modulating the subtypes of CD4+T cells and microglial phenotypes.We also show the possible molecular mechanism involved in neuronal apoptosis.Methods:Both FasL-mutant(gld)and wild-type(B6)mice were experienced middle cerebral artery occlusion(MCAO).The CD4+T cells subpopulations Th17/Treg were assayed by flow cytometry(FACS)and the expression levels of Th17 and Treg cell phenotype and function markers were measured by Real-time PCR(qPCR).CD4+T cells and LPS-stimulated microglia were cocultured 24h,The mRNA of classical activated microglia(M1)markers(iNOS,TNF-a,CD 16,CD32,CD86)and alternatively activated microglia(M2)markers(Ym-1/2,CD206,Arg-1)in microglia cells were measured by Q-PCR,the protein expression of NF-?B pathway was determined by Western blot.Primary cortical neuron from B6/gld mice was subjected to oxygen glucose deprivation(OGD),co-culture supernatant was added to and the cell viability and apoptosis were measured by MTT.Meanwhile,the protein expression of Bax/Bcl-2 was determined by Western blot.Results:The results of flow cytometry and Q-PCR showed that the Th1 7/Treg balance was turned to Treg both in the brain and peripheral blood in gld mice after MCAO.The mRNA level of Ym-1 was significantly increased with the decrease of P65 nuclear transfer after the co-culture of CD4+T cells and LPS-stimulated microglia in gld mice,while the mRNA level of CD86 was obviously down-regulated.When the NF-?B pathway was inhibited,the protein expression of iNOS and COX-2 were markedly reduced in both mice.Besides,compared with wild-type mice,coculture-conditioned medium prepared from gld mice protected neuron against OGD injury.Meanwhile,the expression of Bax/Bcl-2 was decreased,which indicated that the degree of apoptosis was decreased.Conclusions:After ischemic stroke,FasL-mutant decreases Th17/Treg ratio and reduces inflammation,then attenuates the M1-microglial polarization through the inhibition of NF-?B pathway.Finally,it abates the damage of ischemic neuron.