Roles Of FasL In Modulating Functions Of CD8~+T Cells After Ischemic Stroke And The Underlying Mechanisms

Background:Immuno-inflammatory responses,which occur throughout the whole process of ischemic stroke,are considered to be important modulating factors of disease progression,cerebral injury and repair after brain ischemia.Killing target cells via cytotoxicity,CD8+T cells play a crucial role in antigen specific immune responses.After ischemic stroke,peripheric CD8+T cells pass through the broken blood-brain barrier and infiltrate into the brain parenchyma.CD8+T cells have been proved to aggravate cerebral ischemia injury,however,little is known about the underlying mechanisms.In the periphery,CD8+T cells are able to promote apoptosis of target cells through FasL expressed on their membrane.Nevertheless,emerging evidences suggest that FasL plays important roles in pro-inflammation.Blocking FasL is capable of attenuating T cells-related immuno-inflammatory responses after ischemic stroke.In this study,we explored whether FasL was able to modulate CD8+T cells functions after cerebral ischemia.Meanwhile,the roles of FasL in CD8+T cells induced neuronal death and microglial polarization were also demonstrated in this study.Furthermore,we elucidated the underlying molecular mechanism of how FasL regulated CD8+T cells functions.Methods:In vivo,middle cerebral artery occlusion(MCAO)was performed in both wide-type(WT)and FasL mutant(gld)mice.We assessed the infarct volumes and functional outcomes different time points after reperfusion,the numbers and functions of infiltrated CD8+T cells in ischemic hemisphere were evaluated by flow cytometry at the same time.Magnetic-activated cell sorting was used to isolate CD8+T cells from spleens of WT and gld mice.Then CD8+T cells from WT and gld mice were injected intravenously into mature T cells deficient Rag1-/-recipients respectively and then MCAO were induced.After 72 h,infarct volume,functional outcomes were measured.Meanwhile,q-PCR was used to detect the expression of pro-inflammatory factors in ischemic hemisphere and immunofluorescence was applied to assess neuronal survival and glial activation in ipsilateral brain.In vitro,CD8+T cells from WT and gld mice were co-cultured with post oxygen glucose deprivation(OGD)neurons,and the cell viability was measured by MTT and lactate dehydrogenase(LDH)assay.Microglia were co-cultured with CD8+T cells from different mice and the microglial polarization were evaluated by q-PCR.In some experiments,FasL and Fas neutralizing antibodies were used to block FasL on CD8+T cells and Fas on microglia,respectively.After co-culturing,q-PCR and flow cytometry were sed to measure the functions of CD8+T cells and polarization of microglia.In addition,flow cytometry was conducted to detect the activation of MAPK signaling pathway of CD 8+T cells after FasL stimulation.At last,PD98059,SB203580 and SP600125 were used to inhibit the activation of MAPK pathway and flow cytometry,q-PCR and cytometric beads array were applied to assess the functions of CD8+T cells.Results:1)Compared with WT mice,the infarct volumes and neurologic impairments were significantly attenuated in gld mice after MCAO.2)In the ischemic hemisphere,the numbers of CD8+T cells in gld mice were invariable compared with WT mice.However,the expression of activation markers and cytotoxicity cytokines in CD8+T cells were significantly decreased in the ischemic hemisphere of gld mice.3)gld CD8+T cells-reconstituted Ragl-/-mice showed less infarct volume and lighter neurologic impairment compared with WT group.The expressions of pro-inflammatory cytokines in the ischemic hemisphere were significantly downregulated in g1d CD8+ T cells-reconstituted Rag1-/-mice.More surviving neurons and less activated microglia were seen in the ischemic penumbra of post stroke Ragr1-/-mice inj ected with gld CD8+T cells in contrast with WT CD8+T cells.4)CD8+T cells from gld mice alleviated post-OGD neuronal death compared with WT control.5)CD8+T cells from gld mice significantly decreased M1 microglial polarization compared with WT control.After blocking FasL on CD8+T cells or Fas on microglia,the cytotoxicity of CD8+T cells after co-culturing was markedly restrained.6)Expression of p-ERK1/2,p-JNK,p-p3 8 and the cytotoxic markers in CD8+T cells were enhanced after FasL stimulation.Inhibiting MAPK signaling pathway was able to block FasL-induced activation of CD8+T cells.Conclusions:1)FasL mutation attenuated infarct volumes,neurologic impairments and CD8+T cells cytotoxicity in experimental stroke.Meanwhile,FasL mutation alleviated CD8+T cell-induced neuronal death and microglia activation in ischemic penumbra,thus leading to the inhibition of brain immuno-inflammatory responses.2)After ischemic stroke,CD8+T cells killed neurons through FasL.Besides,FasL deficiency weakens CD8+T cells induced M1 microglia polarization and microglia induced CD8+T cells cytotoxicity enhancement.3)FasL promoted cytotoxicity of CD8+T cells through activating MAPK signaling pathway.