Roles Of Neuronal Soluble Fas Ligand In Mediating M1-microglia Polarization After Experimental Stroke And The Underlying Mechanisms

Objectives:Ischemic stroke is a leading cause of morbidity and mortality worldwide, which is a heavy burden for family and society. Unfortunately, recombinant tissue plasminogen activator (rt-PA) is the only approved treatment. Inflammation occurs throughout the progression of cerebral ischemia/reperfusion and appears to play important roles in its prognosis. Microglia constitute the first line of immune defense in the brain and can be activated soon after stroke onset. Microglia exert dual roles, including classical activation phenotypes (M1) typically releasing pro-inflammatory mediators, andalternative activation phenotypes (M2) possessing anti-inflammatory properties, which makes it a promising target for therapeutic strategies in ischemic stroke. Neuron can control microglial activation and function by both contact-dependent and contact-independent mechanisms, contributing to maintenance of CNShomeostasis, however, the involved molecular mechanisms are barely known. Previous studies showed that FasL triggered inflammation, activated microglia and exacerbated brain injury after ischemic stroke. In this study, we explored the polarization of microglia in FasL mutant (Gld) mice and the involvement of neuronal sFasL in modulating microglial polarization after ischemia. Meanwhile, the potential mechanisms of sFasL in triggering JAK2/STAT3 and NF-κB signal transduction pathways were demonstrated in this study.In addition, the effect of Ml/M2-conditioned medium from WT/Gld mice on the fate of ischemic neurons was also elucidated.Methods:In vivo, middle cerebral artery occlusion (MCAO) was inducedin both FasL mutant C57BL/6J (WT) and B6 Smn.C3H-FasLgld (Gld) mice. At 24h and 72h after reperfusion, Real-time PCR and immunostaining were employed to assess microglial activation and polarization. Meanwhile, behavior tests were used to test neurologic defilicts of ischemic ice. In vitro, primary cortical neuron or microglia or neuron-microglia coculture from WT/Gldmice was subjected to oxygen glucose deprivation (OGD). sFasL level in the neuronal supernatant was evaluated by ELISA. Neuronal-conditioned medium or exogenous sFasL was applied to primary microglia with or without FasL neutralizing antibody.Protein expression of JAK2/STAT3 and NF-κB pathways were determined by Western blotting. In some experiment, AG490 and JSH-23 were employed to inhibit tha activation of JAK2/STAT3 and NF-κB pathways, respectively. In addition, M1 or M2 microglial-conditioned medium from both wild-type and gld were prepared and applied to the post-OGD neuronal cultures for 6 hours The effect of microglia phenotype from WT/Gld mice on the fate of ischemic neurons was further elucidated. Neurotoxicity and cell viability were assessed by MTT assay, lactate dehydrogenase assayand calcein AM and propidium iodideand staining.Results:(1)In vivo, neuronal apoptosis was attenuated in Gldmice after MCAO, accompanied by reduced brain damage and improved neurological performance.(2)In vivo, compared with WT mice, the numbers of M1 microglia were decreased whilethenumbersofM2microgliawereincreasedinperi-infarctareaofipisilateralhemisp hereinGld mice after MCAO.(3)In vitro,6 h OGD neuronal-conditioned medium induce M1-microglial polarization.(4) A significant increase of sFasL expression was observed in the post-OGD neuronal medium at 3h and 6h.Meanwhile, this M1 phenotype shift induced by post-OGD neuronal medium was partially blocked by utilization of FasL neutralizing antibody or Gldneuronal-conditioned medium treatment.(5) Exogenous sFasL could trigger M1-microglial polarization, accompanied by the upregulation of p-JAK2, p-STAT3 and enhanced phosphorylation of IκBa and nuclear translocation of p50/p65. Consistently, the M1 markers were down-regulated after JAK2/STAT3 or NF-KBsignaling pathway was inhibited.(6) M1-conditioned media exacerbated oxygen glucose deprivation induced neuronal death. In contrast, maintaining the M2 phenotype of microglia protected neurons against oxygen glucose deprivation.(7) Compared with wild-type primary microglia, microglia with FasL mutant protected the survival and increased the cell viability of post-OGD neuron under M1 microglial-conditioned medium treatment.Conclusions:(1) FasL mutation attenuated neuronal apotosis and M1-microglial polarizationand improved neurological performance in experimental stroke.(2) The ischemic neuron triggered microglial pro-inflammation in a contact-independent way and sFasL released by ischemic neuron took a significant role in M1-phenotype shift, which might involve the activation of the JAK2/STAT3 and NF-kB signal pathways.(3) Inhibition of sFasL protected neuron against M1 microglial-induced injury after ischemia.