Biological Roles Of CKLF1 In Neuron-microglia Interactions After Stroke

Stroke is a sudden cerebrovascular disease with high mortality and disability rates.87%of stroke patients are ischemic,caused by the interruption of blood flow in a brain-supplying artery from a thrombus.Neuronal degeneration and necrosis caused by ischemia in the ischemic core leading to irreversible damage.The ischemic penumbra which is adjected to ischemic core develops to the final infarction window under ischemia.Neurons located in the penumbra are silent in terms of electrophysiological function,but still,maintain metabolic activity.Therefore,improving the survival rate of neurons located in the penumbra is of vital importance to the recovery of neurological function and prognosis.As the resident immune cells in brain tissue,microglia are rapidly recruited to the injured site and further activated to perform their function.Activated microglia have dual functions at different stages of ischemic stroke.On the one hand,microglia secrete pro-inflammatory and cytotoxic substances to exacerbate the damage.On the other hand,microglia phagocytose cell debris and secrete anti-inflammatory cytokine to promote the recovery of the penumbra.This dual-action relates to the different phenotypes of microglia under pathological stages after cerebral ischemia.Activated microglia are classified into M1 type producing deleterious effect and M2 type exerting neuroprotective function.The microglia are first polarized into the M2 type in the penumbra area and play a protective role by swallowing damaged neurons,clearing cell debris,and releasing pro-repair factors.With the prolonged period of ischemia and hypoxia,the core area of the infarction spreads to the penumbra area,and the microglia transform into pro-inflammatory M1 type.Regulating the different polarization states of microglia is an effective new treatment strategy for cerebral ischemia.Therefore,understanding how microglial polarization is controlled after cerebral ischemia will help the development of neuro-protective agents.Early theories believed that neurons are the victims of microglia after cerebral ischemia.However,studies have found that neurons may control microglia under ischemic conditions.Biker et al.proposed that neurons regulate microglia through "on"and "off" signals in 2007.Subsequently study found that neuron regulate the activation and polarization of microglia by releasing chemokines,glutamate,ATP.Chemokines have a small molecular weight and a variety of chemokine receptors are expressed on the surface of microglia cell membranes.Therefore,chemokines seem an ideal messenger of neuron-microglia interactions.Chemokine-like factor-1(CKLF1)is the first chemokine reported by Chinese scientists.Our previous research found that CKLF1 is highly expressed in brain tissue only after cerebral ischemia.Inhibiting or knocking out CKLF1 can effectively improve the neurobehavioral deficits,reduce cerebral infarction edema in animal stroke model.Our previous studies found that CKLF1 only co-localized with neurons detected by immunofluorescence in a model of hypertension combined with cerebral ischemia.Exogenous administration of C27(an activity peptide of CKLF1)to microglia can promote M1 polarization.However,the regulation of CKLF1 and the mechanism of its effect on the phenotypic changes of microglia have not yet been elucidated,which severely hinders its research as a new therapeutic target for stroke.To detect the cellular source of CKLF1,we established the primary culture of various types of cells in the brain,including neurons,microglia,astrocytes,and oligodendrocytes.The results showed that only neurons CKLF1 mRNA and protein expression increased significantly after OGD/R.Immunofluorescence detection also found that the neurons were co-located with CKLF1 in rat MCAO model.After confirming that neurons are the main cellular source of CKLF1 after stroke,we explored the transcriptional regulation of CKLF1 in neurons.We predicted the transcription factor of CKLF1 through the database.Combined with the previous research,we believe that NF-?B is the transcription factor of CKLF1.To verify such prediction,we performed a glucose and oxygen deprivation model on primary neuronal cells and administered NF-?B inhibitors.The experimental results found that inhibiting NF-?B can significantly reduce the expression of CKLF1 mRNA and protein.To further study whether NF-?B directly regulates the transcription of CKLF1,Chromatin Immunoprecipitation(ChIP)experiments were performed.Through chromatin co-precipitation experiments,we found that NF-?B directly binds to the CKLF1 promoter,which was increased after stroke.We next performed the dual-luciferase reporter assay,it is found that NF-?B can directly promote the transcription of CKLF1 with the binding site at-249 to-238 bp.Based on the above experiment,we explored whether CKLF1 mediates the neuron-microglia interaction in vitro and in vivo.Neuronal medium transformed experiments were performed in vitro.Blocking the activity of CKLF1 and inhibiting the activity of NF-?B can both significantly reduce the influence of conditioned medium on the polarization of microglia.Such results were tested in vivo.We construct neuronal specially CKLF1 depletion of penumbra rat.On this basis,a model of rat MCAO was established.Then,we found that the polarization of microglia to M1 type was significantly reduced,while the polarization to M2 type was significantly increased,after specifically knocking out CKLF1 in neurons.At the same time,the depletion of CKLF1 in the neuron of penumbra reduced the number of activated microglia.After confirming that CKLF1 mediates the interaction between neurons and microglia,we have made a preliminary exploration of the mechanism by which CKLF1 promotes the polarization of microglia to M1 type.Based on the results of previous studies,we observed the effect of CKLF1 on the MAPK signal pathway in microglia.Exogenous administration C27(activity peptide of CKLF1)to BV2 microglia can promote the phosphorylation of p38 and JNK with no difference on ERK.We also verified such results in the above-mentioned rats that specifically knocked out CKLF1 in the ischemic brain region.TREM-2 is a membrane protein that maintains the homeostasis of microglia,and its lack of expression on the cell membrane can promote the polarization of microglia to M1 type.In this experiment,after exogenous administration of C27 to BV2 microglia cells,the expression of TREM-2 was detected.The results showed that the expression of TREM-2 decreased under the action of C27 in a dose-dependent manner.It was also found that neuron-specific knock-out CKLF1 can promote the expression of TREM-2 in microglia.It is also found that C27 may be the TREM-2 protein that degrades cell membranes through the autophagy-lysosome pathway.This study reported a molecule that mediates neuron-microglia interactions after stroke,CKLF1,which provides a further explanation for the cellular functions of neurons after stroke.Our group has reported that inhibiting CKLF1 can effectively improve stroke.In this experiment,we further studied the mechanism of CKLF1 aggravating stroke damage,which provides a relevant basis for the development of drugs targeting CKLF1,In this paper,the cell source of CKLF1 was determined by relevant technical means,and on this basis,the transcriptional regulation of CKLF1 was analyzed.This will be beneficial to future basic research on CKLF 1.