Double Negative T Lymphocytes Mice Up-regulate M2 Microglia In Ischemic Stroke Through FasL

Background and objectives:Ischemic stroke leads to significant morbidity and mortality all around the world.Inflammation is involved in almost whole process of stroke pathology.The crosstalk between T lymphocyte and microglia plays an important role in ischemia reperfusion injury.Early reperfusion strategies remain the treatment of choice but can initiate and augment an inflammatory response causing secondary brain damage.The understanding of post-ischemic inflammation is very limited.The FasL mutation gld mice demonstrated attenuated ischemic brain damage after experimental stroke,with double negative(DN)T lymphocytes accumulating in peripheral blood and spleen.However the biological actions of DN T lymphocytes are no clear.This study aims to explore the effects of DN T lymphocytes in experimental stroke.Methods:In vivo,the subtypes of T lymphocytes including CD4+ T lymphocytes,CD8 + T lymphocytes,DN T lymphocytes,Th17 and Treg cell in peripheral blood and spleen were measured by flow cytometry after cerebral ischemia.Meanwhile,infiltrating T lymphocytes in infarct hemisphere and activated microglia in brain after ischemia stroke were detected by flow cytometry and q-PCR.In vitro,different subtypes T lymphocytes isolated from C57BL/6 or gld mice were co-cultured with primary microglia,which were subjected to different treatments.Flow cytometry and Real-time PCR were used to measure the phenotype of primary microglia.CBA assay were used to quantify the levels of cytokines and chemokines.The apoptosis of OGD-neurons treated with the supernatant co-cultured with primary microglia were meansured with Annexin-V and PI.Results:The results of flow cytometry showed that both in peripheral blood and spleen,DN T lymphocytes were accumulated in gld mice.DN T lymphocytes in peripheral blood were decreased after ischemic stroke,while the regulatory Treg cell in peripheral blood were increased after ischemic stroke.In vivo,infiltrating T lymphocytes were proved to be recuited to ischemic brain after MCAO both in B6 and gld mice by flow cytometry.While DN T lymphocytes were significantly accumulated in ischemic cerebral hemisphere in gld mice 3 days after MCAO.gld mice up-regulated the mRNA expression of Foxp3 and down-regulated RoRc in ischemic hemisphere after MCAO.It's revealed that expression of M1(CD86)and M2(CD206)markers kept rising within 72 h after MCAO.While gld mice was more inclined to promoting M2 microglia polarizationIn vitro primary microglia underwent a shift to alternative-activated microglia when co-cultured with different subtypes of T lymphocytes isolated from spleen in gld mice,whereas emerged a shift to classical-activated microglia when co-cultured with T splenic lymphocytes isolated from C57BL/6 mice.Subtype of T lymphocytes co-cultured with microglia exerted DN T isolated from gld mice up-regulated some anti-inflammatory cytokines,like IL-4 and IL-10,which reduced the apoptosis of OGD-neurons.Conclusions:The neuroprotective effects in gld mice may be related to the immune regulation of DN T lymphocytes on microglial polarization via FasL in stroke.