Astrocyte KDM4A Aggravates Cerebral Ischemia And Reperfusion Injury

AimFor ischemic stroke,more serious injuries such as infarct site expansion and aggravated neuron cell damage will be happen in the process of blood flow reperfusion,this is so called cerebral ischemia reperfusion injury（CIRI）,and there are no effective treatment strategies at present.Exploration of the pathogenesis of CIRI and search for effective therapeutic targets are hot and difficult issues in the field of cerebrovascular diseases.After CIRI,peripheral immune cells such as neutrophils invade the brain and trigger immune response,which is an important cause of aggravating brain tissue injury.Effective strategies to reduce neutrophils infiltration are of great significance for alleviating brain tissue injury caused by CIRI.However,our knowledge about the immune response is still very limited,and the complex mechanism of neutrophil infiltration after CIRI remains need to be further studied.Astrocytes are involved in regulating the immune response after brain injury.Astrocytes regulate the infiltration of peripheral immune cells into the injured brain tissue by releasing many inflammatory factors.Studies have shown that astrocytes are the main source of CXC chemokines ligand（CXCL）1 and other chemokines that regulate neutrophil infiltration.These chemokines are regulated by the transcription factor nuclear factor kappa-B（NFκB）.In addition to transcription factors,gene expression is also affected by histone modification.However,the histone modification enzymes that play a key role in the regulation of inflammatory factors after CIRI remain unclear.Lysine-specific demethylase 4 A（KDM4A）is an important member of the Jumonji domain 2 family histone demethylases.KDM4A is high sensitive to hypoxia and its expression is upregulated upon hypoxia condition.KDM4A participates in the regulation of gene expression by interacting with NFκB and other transcription factors,and then regulates a variety of physiological and pathological processes.However,the role and mechanism of astrocyte KDM4A in the pathological processes of CIRI is completely unknown.To prove this,in the present project we plan to employ astrocyte Kdm4a conditional knockout mice（Kdm4a KO）and establish the CIRI models in vivo and in vitro.By using methods of pharmocology,molecular biology,cell biology and other multidisciplinary research methods,we aim to determine the deleterious role of astrocyte KDM4A post-CIRI via inducing neutrophil infiltration and aggravating inflammation injury,explore the underlying mechanism（s）,and provide a promising therapeutic target for the prevention and treatment of CIRI.Methods1.The role of astrocyte KDM4A in the process of CIRIMiddle cerebral artery occlusion/reperfusion（MCAO/R）surgery was performed in C57BL/6J mice,penumbra area was separated 24 h after reperfusion,protein expression of KDM family was detected by Western Blot assay,KDM4A expression and localization was determined by immunofluorescence.Astrocytes,microglia,and neurons were primary cultured,KDM4A gene and protein level were determined by real-time polymerase chain reaction（RT-PCR）and Western Blot assay upon oxygen glucose deprivation/reoxygenation（OGD/R）condition.Kdm4a KO mice were generated and underwent MCAO/R surgery.Beheavior deficits were determined by beheavior test,infarct volume was calculated by 2,3,5-Triphenyltetrazolium chloride（TTC）staining,blood brain barrier injury was assayed by Evans Blue staining,astrogliosis was evaluated by immunofluorescence,neuronal apoptosis was observed by terminal deoxynucleotidyl transferase mediated nick end labeling（TUNEL）staining.2.Mechanism of KDM4A aggravates CIRITo further investigate the mechanism astrocyte KDM4A affects stroke outcome.Astrocytes were isolated by magnet-activated cell sorting in penumbra area after MCAO/R in Kdm4a KO and Control mice.RNA-Sequnce was performed to detect the gene expression difference between two groups.The penumbra area was separated,cell suspension was prepared,and the infiltrated peripheral immune cells were detected by flow cytometry.Immunofluorescence staining was used to evaluate neutrophil infiltration.Antibodies were injected into Kdm4a KO and Control mice through tail vein to neutralize neutrophils,and MCAO/R injury were performed,behavioral test and TTC staining were performed to evaluate the injury degree.To study the mechanism of astrocyte KDM4A regulating neutrophil infiltration.Astrocytes were isolated from penumbra area in Kdm4a KO and Control mice after MCAO/R by fluorescence-activated cell sorting,inflammatory factor of two groups were determined by proteome profiler array.The differences of inflammatory factors were further confirmed by enzyme-linked immunosorbent assay（ELISA）and flow cytometry.Astrocytes and neutrophils were co-cultured by Transwell method,and anti-CXCL1antibody was added.The effect of astrocyte Kdm4a knockout on the number of infiltrated neutrophils was detected under OGD/R injury.To explore the mechanism of KDM4A upregulating CXCL1.Chromatin immunoprecipitation assay was performed to detect the recruitment of transcription factors at the promoter of Cxcl1.Immunoprecipitation assay was used to confirm the interaction between KDM4A and NFκB in astrocytes.Promoter luciferase reporter assay was performed to determine whether NFκB binding sequence is involved in the activation of Cxcl1 gene promoter upon OGD/R condition.NFκB was silenced in astrocytes by small interfering RNA,and CXCL1 levels were detected by ELISA to evaluate whether NFκB interacts with KDM4A to regulate Cxcl1 transcription.Results1.The KDM4A in astrocytes is upregulated after CIRI and aggravates the brain injuryWestern Blot assay showed that KDM4A was upregulated in penumbra area after MCAO/R.RT-PCR and Western blot analysis showed that KDM4A expression in astrocytes was remarkably elevated after OGD/R injury.Immunofluorescence results showed that KDM4A was upregulated and primarily co-localized with astrocyte marker glial fibrillary acidic protein in stroke mice.Kdm4a KO mice were successfully generated,using Kdm4a^flox/floxmice as Control,stroke outcome was determined in Kdm4a KO and Control mice.Compared with Control group,Kdm4a KO mice exhibited less impairment in the components of the neurological deficit and foot-fault test,while displayed longer period in rotator test.TTC staining results showed that Kdm4a KO had significantly smaller infarcts than Control mice.Examination of blood brain barrier permeability revealed that the Kdm4a KO mice had significantly smaller Evans blue staining area and less Evans blue extravasation compared to Control mice.Astrogliosis was determined by immunofluorescence staining,results showed that Kdm4a KO mice had smaller number of GFAP⁺reactive astrocytes in penumbra area compared with Control mice.TUNEL staining showed that the number of apoptotic neurons was significantly lower in Kdm4a KO mice when compared to Control mice after MCAO/R.These results suggest that selective knockout of astrocyte Kdm4a plays a protective role after MCAO/R.2.Astrocyte KDM4A contributes to neutrophil infiltration following CIRI through stimulation of CXCL1 expression via interacting with NFκBRNA-Sequence results showed that Kdm4a deficiency affected thousands of genes in astrocytes in penumbra area.Flow cytometry and immunofluorescence assay showed that Kdm4a knockout in astrocytes significantly reduced the neutrophils infiltration in penumbra area after MCAO/R injury.When depleting neutrophils in Control and Kdm4a KO mice by injection of antibodies,the behavior deficiency and infarct volumes were similar between two groups after MCAO/R.These collective findings suggest that astrocyte KDM4A may lead to an accumulation of neutrophils in the ischemic brain,potentially aggravating brain injury caused by CIRI.The proteome profiler array and ELISA results showed that Kdm4a KO astrocytes displayed significant lower CXCL1 expression compared with Control mice after MCAO/R.Flow cytometry analysis showed that less CXCL1 positive astrocytes were observed in penumbra area of Kdm4a KO mice after MCAO/R.The results of Transwell experiment showed that after OGD/R injury,a large number of neutrophils infiltrated into the astrocyte layer,while the number of infiltrated neutrophils was significantly reduced in Kdm4a KO group compared with the Control group.Blocking CXCL1 by antibody in conditional medium,the proportions of neutrophils in the Kdm4a KO and Control groups become equal.These results indicated that astrocyte KDM4A induced neutrophil infiltration through upregulation of CXCL1.Chromatin immunoprecipitation assay showed that NFκB and KDM4A were recruited at the Cxcl1 promoter in astrocytes after OGD/R injury.Co-immunoprecipitation assay showed that NFκB and KDM4A interacted with each other in astrocytes after OGD/R injury.Promotor luciferase reporter assay showed that NFκB binding sequence was involved in the activation of Cxcl1 gene promoter after OGD/R injury.ELISA results showed that silencing NFκB reduced CXCL1 levels in astrocytes upon OGD/R injury.These results indicate KDM4A upregulates CXCL1 in astrocytes via interacting with NFκB.Conclusion1.The KDM4A in astrocytes was upregulated after CIRI and aggravates the brain injury.2.KDM4A interacts with NFκB and regulates astrocytes releasing CXCL1,induces neutrophils infiltration and aggravates brain injury caused by CIRI.