Roles And Mechanisms Of Tim-3 In Regulating Microglia And Traumatic Brain Injury

BackgroundMacrophages play a key role in the regulation of immune homeostasis and their dysfunction is closely related to varieties of acute and chronic,uncontrollable inflammatory diseases and thus become targets for intervention and treatment.As macrophages in central nervous system,microglia play an important role in maintaining neuroimmune homeostasis through phagocytic defense,immune response,neurotrophic,and synaptic remodeling.Once microglia are over-activated,the immune response would be out of control and large amounts of inflammatory cytokines are secreted,which may cause immune damage.The function of microglia is closely related to its polarized states.Microglia exhibit two phenotypes,"classic activation"?M1?and"alternative activation"?M2?,depending on the environment in which they are located and the surrounding stimuli.M1 type microglia induce the activation of iNOS and NF-?B pathways to produce various pro-inflammatory cytokines such as TNF-?,IL-1?and IL-6 as well as superoxide,reactive oxygen species and nitric oxide.M2 type microglia promote tissue repair,secrete neurotrophic factors and anti-inflammatory cytokines such as TGF-?.Microglia play a role in enhancing the inflammatory response?M1?or promoting inflammatory repair?M2?by polarizing in different directions of M1/M2.For example,in the case of brain trauma,over-activation of microglia and enhanced polarization of M1 are important factors in triggering"secondary injury"and post-traumatic stress disorder?PTSD?.In addition,as in Alzheimer's disease,premature polarization of microglia to M2 is not conducive to the degeneration of amyloid?-protein?A??.Thus timely regulation of the polarization of microglia is important for prevention and treatment of brain damage and dysfunction.Traumatic brain injury?TBI?lead to high healthcare cost and death tolls around the world due to high morbidity and mortality during wartime and peacetime.Furthermore,uncontrollably amplified cascades of inflammatory response is one of the pathogenesis of TBI and the out of control of microglia activation is a key factor in the cascades of amplified inflammatory response.Due to the complexity of the pathogenesis of TBI,effective interventions have been insufficient so far.Therefore,an deeper exploration of the mechanism of TBI and the search for potential intervention targets are of great significance for reducing combat downsizing and facilitating social burden.T cell immunoglobulin and mucin-domain containing molecule-3?Tim-3?is widely expressed in activated T cells,macrophages,dendritic cells and other immune cells.It is a newly discovered immune checkpoint molecule.In recent years,Tim-3 has attracted widespread attention in the immunology community due to its important immunemodulatory function.Currently,research on Tim-3 has focused on its regulation of T cell function.Our laboratory has long been concerned with the regulation of macrophage function and was the first to discover that Tim-3 regulated macrophage function through various mechanisms in vitro and Tim-3 played an important role in maintaining natural immune homeostasis.Moreover,further reseearches suggested that intervention with Tim-3 signaling influenced disease progression by remodeling macrophage function.Therefore,whether the function of microglia can be regulated by interfering with the signal of Tim-3,and then affecting the occurrence and deterioration of TBI,has become an important scientific problem in this study.ObjectiveTo explore the roles and mechanisms of Tim-3 in regulating microglia polarization;to establish traumatic brain injury model in mice;to construct and multiply the Tim-3?HAVCR2?knockout mice;to explore the roles of Tim-3 in regulating traumatic brain injury.Methods and ResultsThis thesis is composed of four parts,the roles and mechanisms of Tim-3 in regulating microglia polarization,establishment and evaluation of a mouse model of traumatic brain injury,construction and identification of Tim-3 knockout mice,and primary exploration of the roles of Tim-3 in regulating traumatic brain injury.1 The roles and mechanisms of Tim-3 in regulating microglia polarizationTim-3 and its ligand expressions in BV-2 were analyzed using flow cytometry assay.Cytokines secreted by BV-2 treated with Tim-3 blocking fusion protein were analyzed using ELISA and realtime fluorescence quantitative PCR to observe the polarization of BV-2.The mechanisms were analyzed using western blot.RESULT:T cell immunoglobulin and mucin-domain containing molecule-3?Tim-3?and its ligand Galectin-9?Gal-9?were expressed in BV-2.M1 type associated pro-inflammation cytokines secreted by BV-2 including IL-6,IL-1?,TNF-?and IL-12 were significantly promoted while M2 type associated anti-inflammation cytokines secreted by BV-2 such as TGF-?and IL-10 were inhibited when treated with Tim-3 blocking fusion protein.Tim-3significantly regulated the polarization of BV-2 via inhibition both of RIG-I and NF-?B signaling pathway.2 Establishment and evaluation of mice model of traumatic brain injurymale C57BL/6 mice were randomly divided into Sham group?control group?and CCI group?controlled cortical impact?each with 20.After abdominal anesthesia,the dura mater was exposed.The mouse cortex was impacted by e-controlled-CCI instrument;the NSS score is recorded according to the neurological severity scale?NSS?before and after the impact and then the brain tissue is taken for HE staining.NSS score and HE staining results were used to evaluate the model establishment.RESULT:Mice of both groups survived.Mice model of CCI group was moderate TBI model based on NSS scores and HE staining results.3 Construction and identification of Tim-3 knockout miceFlox^+/+homozygous mice were screened by commercialized HAVCR2-Floxed recombinant transgenic mice;Flox^+/+Cre+mice was screened by Flox^+/+homozygous mice and commercial EIIa-Cre recombinant transgenic mice(Cre^+/+or Cre^+/-mice express Cre enzyme).Mice genomic DNA,brain tissue RNA and protein were extracted,and the knockout effect of Tim-3 was examined by PCR,realtime fluorescence quantitative PCR and Western blotting.RESULTS:Mice genomic DNA identification showed that Flox^+/+homozygous mice and Flox^+/+Cre+mice were obtained;realtime fluorescence quantitative PCR and Western blotting showed that the level of Tim-3 mRNA and protein in brain tissue was significantly decreased.4 Primary exploration of the roles of Tim-3 in regulating traumatic brain injuryTBI?CCI?models were established using Wild type?WT?mice and Tim-3 knockout?Knock out,KO?model mice,20 mice per group.NSS scores were performed according to the neurological neverity scale?NSS?at 12 hours and 24 hours after injury to assess the establishment of the model and the difference in neurological function damage;and RNA were extracted from the peri-contusional cerebral cortex.realtime fluorescence quantitative PCR was used to detect the difference of inflammatory factor expression in WT mice and KO mice after injury,and thus to explore the roles of Tim-3 in regulating traumatic brain injury.RESULTS:The moderate TBI?CCI?model was successfully replicated in WT and KO mice;Tim-3 reduced the degree of neurological damage and inhibited the expression of type I interferon 12 hours after injury;the NSS scores and the expression levels of inflammatory factors such as TNF-?and IL-1?were not significantly different 24 hours after injury.Conclusion1 Tim-3 played a role in regulating the polarization of BV-2 via inhibiting its M1polarization and promoting its M2 polarization.2 Moderate TBI mice models were established successfully using wild-type C57BL/6 mice.3 Tim-3 knockout mice were constructed and multiplied,which laid a foundation for studying the role of Tim-3 in regulating brain dysfunction diseases such as traumatic brain injury.4 The TBI?CCI?models were constructed using wild C57BL/6 mice and Tim-3knockout mice which were constructed and expanded by our laboratory.The role of Tim-3in regulating traumatic brain injury was initially explored.Tim-3 had a protective effect on neurological damage and inhibited the expression of type I interferons 12 hours after injury.InnovationIn this study,the immune checkpoint molecule Tim-3 was used as the research object,and its roles and molecular mechanisms in regulating the polarization of microglia BV-2were deeply explored at the cellular level.Mice controlled cortical impact models were established and constructed.Tim-3 knockout mice were initially used to simulate traumatic brain injury models.In the research,it was found that Tim-3 had an protective effect on neurological function and inhibited the expression of type I interferon 12 hours after injury.But its effect on inflammatory factors such as TNF-?and IL-1?has not yet obtained clear results.Relevant experiments will be adjusted and optimiozed to get further study.