TREM2,A New Target For Regulating Microglia Activation State, Involved In Neuroprotective Effect By Relieving Neuroinflammation

Cardiovascular and cerebrovascular diseases have been regarded as the first killer to human health. As a big aging population country, the incidence of stroke is higher in China, which seriously impairs national health. Since 1994, stroke has been the first couse of death in the big city of China. Demograghic data suggested that 429 ~ 620 out of100,000 people suffer from this disease, 116~142 deaths out of 100,000 people each year.The brain is prone to be injured after ischemia-reperfusion, because it is the most sensitive organ to ischemia and hypoxia. To some extent, the brain dysfunction negatively affects our spirit, emotion, behavior, consciousness and other organs. Neuroinflammation often plays a special role in the entire pathological process of ischemia-reperfusion injury. To date, the secondary brain injury caused by severe neuroinflammation has been the tough problem in clinical practice. Therefore, inhibiting the neuroinflammation will shed some light on the treatment of stroke. Given to that microglia is a representative cell in brain immune system, more and more people draw their attention to the function of microglia during neuroinflammation. In different periods of ischemia-reperfusion injury, the function of microglia changes over time. Classical activated microglia(M1 phenotype) releases a large amount of inflammatory cytokines, oxygen free radicals and other harmful substances in the early inflammation, which destroies the neuronal structure and function;However, selective activated microglia(M2 phenotype) secretes neurotrophic substances,removes necrotic or apoptotic neuronal debris and promotes the formation of glial scar tissue in the late inflammation. Thus regulating microglia activation state to alleviate neuroinflammation may be the new target for the treatment of stroke. As an immunoglobulin-like receptor, TREM2(Triggering Receptor II Expressed by Myeloid cells) plays a "negative regulator" beneficial role in autoimmune and inflammatory processes, highly expressed in microglia. So is TREM2 one of the key targets for regulating microglia activation state? Is it a possible that ameliorating neurological damage after cerebral ischemia by regulating the expression of TREM2? In response to these scientific questions, we designed the vitro and vivo experiments and confirmed that up-regulation of TREM2 could relieve neuroinflammation and improve neurological function after ischemia by switching microglia to M2 from M1. Our research initially stated that TREM2 is the important signaling molecule regulating microglia activation state, which may provide a new target for developing new drugs to treat ischemic brain injury.Experiment 1 The Expression of TREM2 in Microglia of Different Functional Activation States[Objective] To observe the expression of TREM2 in M1 and M2 activation state and verify the relationship between TREM2 and the different activation state of microglia.[Methods] Directionally transform primary microglia into the M1 state by LPS / IFN-γ and transform primary microglia into the M2 state by IL-4 / IL-13. Use the method of Western Blot and Immunofluorescence staining to measure the expression of TREM2, M1 marker i NOS and M2 marker Arg-1.[Results] In LPS / IFN-γ group, the expression of i NOS is significantly higher than control group(P <0.05), and in IL-4 / IL-13 group, the expression of Arg-1 is also significantly higher compared with that of control group(P <0.05). We separated microglia into different activation state successfully and then found the expression of TREM2 in IL-4 / IL-13 group was increased, compared with the control group(P <0.05).The results of Double immunofluorescence staining and Western Blot are almost unanimous.[Conclusion] The expression of TREM2 was increased in the M2 microglia and decreased in the M1 microglia.Experiment II Variation of microglia activation state and the expression of TREM2 after ischemic brain injury[Objective] To measure the expression of TREM2, M1 and M2 markers after ischemic brain injury at different time points.[Methods] SPF male C57 BL / 6 mice were divided into Sham group and MCAO group,and the later were randomly divided into four groups again. Focal cerebral ischemia was induced by MCAO using an intraluminal filament technique as described in our previous studies. Obtain ischemic penumbra of brain tissue(n = 6) at 6h, 24 h, 3d and 7d after reperfusion respectively. Immunohistochemistry staining and Western Blot were used to detect the expression of i NOS, IL-6, Arg-1, BDNF and TREM2.[Results] 6 hours after reperfusion, i NOS and IL-6 were significantly increased compared with the Sham group(P <0.01, P <0.001, respectively). M1 markers i NOS and IL-6showed a higher level than the Sham group at 6h, 24 h. M2 markers Arg-1 and BDNF were highest at 7d after reperfusion(P <0.001). The expression of TREM2 was gradually increased at 3d after reperfusion(P <0.001).[Conclusion] M1 microglia dominated in early cerebral ischemic injury, while M2 microglia dominated in the late ischemia injury. TREM2 was increased in M1 / M2 transition period after ischemia reperfusion.Experiment Ⅲ Suppression of TREM2 Enhanced M1 Microglia Polarization and aggravated cerebral ischemia-reperfusion injury[Objective] To investigate the effect of down-regulation TREM2 on neurological behavior,infarct volumes, apoptosis after cerebral ischemia-reperfusion injury.[Methods] Male C57 BL / 6 mice were divided into the following three groups: MCAO group, si RNA group, scr RNA group. TREM2-si RNA and control si RNA were injected intracerebroventricularly 72 hours before the onset of MCAO for a duration of 60 min. 3d after ischemia-reperfusion injury, TTC staining, neurobehavioral scores(Longa score) and TUNEL staining were conducted to assess brain function.[Results] TUNEL staining showed that the number of apoptotic neuron was significantly increased in TREM2-si RNA group compared with MCAO group(P <0.05), while infarct volume and neurological behavior score in TREM2-si RNA group revealed no significant difference.[Conclusion] Suppression of TREM2 aggravated neuronal apoptosis after stroke.Experiment IV Up-regulation of TREM2 Enhanced M2 Microglia Polarization and played neuroprotective role after cerebral ischemia- reperfusion injury[Objective] To verify the neuroprotective effects of TREM2 after cerebral ischemiareperfusion injury when TREM2 was up-regulated.[Methods] 32 male C57 BL / 6 mice were divided into 4 groups: MCAO group, Hsp602.5μg group, Hsp60 3.75μg group, Hsp60 5μg group. We administrated Hsp60 1 hour by intraperitoneal injection before making MCAO model.Also 24 male C57 BL / 6 mice were divided into Sham group, MCAO group, LV+MCAO group. TREM2-vector was injected intracerebroventricularly 10 days before the onset of MCAO for a duration of 60 min. We assessed brain function by TTC staining, neurobehavioral scores(Longa score) and TUNEL staining.[Results] We found that the infarct size was reduced and the neurological outcome was improved in Hsp60 5μg group as compared with MCAO group(P <0.001). In Hsp603.75μg group and Hsp60 5μg group, TUNEL staining showed that the number of apoptotic cells was significantly lower than MCAO group(P <0.01).Compared with MCAO group,the infarct size was decreased(P<0.05) and the expression of i NOS was reduced in LV+MCAO group(P<0.01), while the expression of Arg-1 was increased(P<0.001).[Conclusion] Hsp60 activated TREM2 and improved neurological function after ischemia-reperfusion injury.ExperimentⅤ Regulating TREM2 by Lentiviral transfection altered the microglia activation state after OGD model[Objective] To further verify the role of TREM2 in altering the microglia activation state in vitro.[Methods] N9 microglias were divided into three groups: control-vector group,TREM2-vector group and TREM2-si RNA group. Microglias were transfected with a lentivirus for 3 days and microglias were treated by OGD model for 4h, then regained the oxygen and glucose for 24 h. i NOS, IL-6, BDNF, Arg-1 were measured by western blot.[Results] Compared with the control-vector group, the expression of M2 markers BDNF and Arg-1 were increased in TREM2-vector group(P <0.05); Meanwhile, the level of i NOS and IL-6 in TREM2-si RNA group are higher than the control-vector group(P<0.001).[Conclusion] up-regulation of TREM2 promoted microglia switching to M2 from M1.