The Role Of Zhx2 In EAE By Activating Nlrp3 Inflammasome And Inducing Pyroptosis In Macrophages

BackgroundThe experimental autoimmune encephalomyelitis model(EAE)is identified as the ideal model of human classic model of multiple sclerosis(MS).MS is an autoimmune disease mainly mediated by T cells and simultaneously mediated by multiple immune cells.As a chronic demyelinating disease,MS is mainly characterized by local inflammatory infiltration,demyelination and axonal injury in the central nervous system(CNS).Recently,MS has a gradual increase in incidence,most of which occurs in young adults.The recurrence rate and disability rate are extremely high,which seriously affect the normal work and life quality of patients.Therefore,uncovering the mechanism of MS development is of vital importance for the diagnosis and treatment of MS.MS is a complex nervous system disease,and its pathogenesis remains unclear so far.In EAE,pyroptosis occurs in peripheral myeloid cells to induce inflammation and promotes differentiation of T cells in the spleen and lymph nodes into Th1 and Th17 cells,thereby aggravating neuroinflammation and demyelination of EAE.It is reported that Gsdmd(Gasdermin D)mediated pyroptosis plays a key role in the development of EAE.In response to external stimulation,the GSDMD is cleaved and the activated form N-GSDMD moves to the cell membrane to induce pyroptosis.In addition,pyroptosis occurs in microglia and oligodendrocytes during inflammatory stimulation,and caspase-3/7 is involved in the process of pyroptosis in microglia.The occurrence of pyroptosis depends on inflammasome activation.Inhibition of caspase-1 activation by VX-765 can further inhibit inflammasome activation,thus reducing the severity of EAE.Among NLRP3,NLRC4,AIM2 inflammasomes,NLRP3 inflammasome is the most widely studied.When stimulated by DAMPs or PAMPs,sensor molecule NLRP3 responds to dangerous signals and rapidly recruits ASC and pro-caspase-1 to form inflammasome complex.The abnormal activation of NLRP3 inflammasome is involved in many diseases,such as infection,metabolic disorder,inflammation and tumor.The activation of NLRP3 inflammasome also occurs in MS patients.The activation of NLRP3 inflammasome and GSDMD mediated pyroptosis in macrophages are involved in the occurrence and development of infectious diseases and autoimmune diseases.Furthermore,in EAE,a large number of monocyte-derived macrophages infiltrate into the central nervous system and distribute in the demyelinating lesion area,promoting the loss of axons.On the other hand,activated macrophages secrete inflammatory cytokines,which promotes the migration of Thl and Th17 cells and aggravates the progress of EAE.References indicate the key role of macrophages in EAE pathogenensis.To explore the exact role of macrophages in the development of EAE would provide effective strategies for the MS intervention.ZHX2 is first identified as a negative regulator of AFP.Literature has shown that ZHX2 plays a pro-inflammatory role in different inflammatory diseases.In atherosclerosis,Zhx2 alleviates atherosclerosis by promoting apoptosis of macrophages.In sepsis,Zhx2 enhances the glycolysis of macrophages,leading to increased lactic acid production and aggravating sepsis.However,the role of ZHX2 in MS remains unclear.RNA-seq showed that the transcription level of Nlrp3、Il-1β、Gsdmd were decreased in BMDM from the myeloid cell specific knockout Zhx2,suggesting that ZHX2 may be involved in the activation of NLRP3 inflammasome and pyroptosis regulation.Since pyroptosis and macrophages play key roles in the development of EAE,we proposed a hypothesis that Zhx2 may regulate the pyroptosis of macrophages to participate in EAE pathogenesis.Objectives:This study takes EAE as the model to explore the key role of Zhx2 in the development of EAE and related molecular mechanism.The objectives are as follows:1.To identify the Zhx2 expression in EAE.2.To explore the role of Zhx2 in the development of EAE.3.To clarify the molecular mechanisms of Zhx2 in EAE.(1)To investigate the effect of myloid cell specific knockout Zhx2 on EAE development.(2)To reveal the effect of ZHX2 on NLRP3 inflammasome and pyroptosis.(3)To elucidate the mechanism of ZHX2 regulating NLRP3 inflammasome.Methods and resultsⅠ.Zhx2 promotes the development of EAE1.Establishment and identification of EAE model in miceEAE model was established with C57BL/6 mice aged 6～8 weeks.Compared with the control group,EAE mice showed significant weight loss and clinical score increase at 8 days after immunization.H&E staining showed that the mice with EAE showed obvious inflammatory cell infiltration,which indicated that the EAE model was successfully established.2.Enhanced expression of Zhx2 in mice with EAEEAE model was induced in WT mice.The expression of Zhx2 in the CNS of EAE mice was significantly higher than that in control mice detected by qPCR,WB and IHC.Moreover,microglia and monocyte-derived macrophages in the CNS of EAE mice were sorted.The expression of Zhx2 in microglia and macrophage from EAE mice was higher than that of control mice detected by qPCR.The above results indicate that Zhx2 participates in the development of EAE.3.Zhx2 participates in EAE pathogenesis through promoting pyroptosisIn order to address the role of Zhx2 in the development of EAE,tamoxifen-induced Zhx2 systemic knockout(KO-Zhx2)mice were used as the model for subsequent studies.It has been reported that Gsdmd mediated pyroptosis is involved in the progression of EAE.EAE model was induced in WT and KO-Zhx2 mice.After MOG35-55 immunization,Disulfiram was injected intraperitoneally daily.The results showed that compared with the WT mice,the weight of the KO-Zhx2 mice were not significantly decreased,and the clinical score was significantly reduced.And after Disulfiram administration,WT group had no significant weight loss,and the clinical score was significantly reduced,which were similar to KO-Zhx2 mice.Besides,flow cytometry was used to detect the proportion of immune cells.The results showed that the proportion of Th17 cells in the LN,the proportion of Th17 cells and macrophages in CNS decreased.The proportion of the above immune cells in the WT mice given Disulfiram also decreased.These results suggest that Zhx2 knockout alleviates EAE progression,and Zhx2 participates in the development of EAE through pyroptosis.Ⅱ.The Role of Zhx2 in macrophages in EAE1.Myloid cell specific knockout Zhx2 attenuates EAE severityTo address the role of Zhx2 in macrophages in the development of EAE,myeloid knockout Zhx2(MKO-Zhx2)mice were used as the model in the subsequent studies.EAE model were induced in WT and MKO-Zhx2 mice respectively.We found that there was no significant decrease in body weight and clinical score decreased significantly in the MKO-Zhx2 mice.H&E staining also showed that inflammation was significantly reduced in MKO-Zhx2 mice.The expression of microglia specific marker Iba1 was detected using IHC staining.The result showed that the microglia in the CNS of WT mice were circular and activated,while the microglia of MKO-Zhx2 mice were quiescent and branched.The above results indicate that myloid specific knockout Zhx2 can significanty alleviate EAE.2.ZHX2 promotes the activation of NLRP3 inflammasome in macrophagesTo further clarify the mechanisms of ZHX2 regulating macrophages in the development of EAE,BMDM of WT and MKO-Zhx2 mice were cultured respectively.RNA-sequence showed that the transcription levels of Nlrp3,Il-1β and Gsdmd were decreased in BMDM of MKO-Zhx2 mice,suggesting that ZHX2 may be involved in the activation of NLRP3 inflammasomes and GSDMD mediated pyroptosis.PEM and BMDM were extracted from WT and MKO-Zhx2 mice.LPS and ATP/Nig were used to stimulate NLRP3 inflammasome activation.The results showed that the secretion of IL-1β and ASC specks were decreased in PEM of MKO-Zhx2 mice.Consistently,the secretion of IL-1β was significantly reduced in THP1 cells after ZHX2 knockdown.On the contrary,the secretion of IL-1β was increased in THP1 with ZHX2 overexpression.In EAE model of WT and MKO-Zhx2 mice,ASC specks in CNS and the levels of IL-1β and IL-18 in serum were significantly decreased in MKO-Zhx2 mice.These results suggest that ZHX2 promotes the activation of NLRP3 inflammasomes in macrophages.3.ZHX2 promotes GSDMD-mediated pyroptosis of macrophages3.1 ZHX2 promotes pyroptosis of macrophagesPEM and BMDM of WT and MKO-Zhx2 mice were isolated and cultured.The PEM of WT mice showed obvious pyroptosis morphology after LPS+ATP/Nig stimulation,and the cells were swollen and rounded.However,the pyroptosis of PEM from MKO-Zhx2 mice was significantly reduced,and the cell death ratios,the expression of N-Gsdmd and the secretion of LDH were significantly decreased.Consistently,interfering ZHX2 in THP1 cells resulted in significantly decreased cell death rates and LDH secretion.On the contrary,the proportion of cell death was increased and the secretion of LDH was also enhanced in THP1 cells overexpressing ZHX2.These results suggest that ZHX2 can promote pyroptosis of macrophages.3.2 ZHX2 promotes pyroptosis by regulating GSDMDGSDMD siRNA and Disulfiram were used to treat PEM from WT and MKO-Zhx2 mice.After stimulation with LPS+ATP/Nig,the percentage of cell death was significantly reduced in macrophages from WT and MKO-Zhx2 mice after inhibiting GSDMD,as well as the secretion of LDH.These results suggest that ZHX2 promotes GSDMD-mediated pyroptosis in macrophages.4.ZHX2 regulates NLRP3 inflammasome4.1 ZHX2 promotes the expression of NLRP3In order to further clarify the mechanism of ZHX2 regulating NLRP3 inflammasome,PEM and BMDM of WT and MKO-Zhx2 mice were isolated and cultured.The expression of Nlrp3 of PEM and BMDM in MKO-Zhx2 mice was decreased detected by qPCR and WB.Similarly,after interfering ZHX2 in THP1 cells,the expression of NLRP3 was also decreased.In addition,the expression of NLRP3 was increased in THP1 cells with ZHX2 overexpression.These results suggest that ZHX2 can promote the expression of NLRP3.4.2 ZHX2 regulates the transcription of NLRP3As ZHX2 is a transcription factor,ZHX2 could promote the activity of NLRP3 promoter detected by luciferase report experiment in HEK 293 cells.In addition,the ChIP showed that ZHX2 could combine with NLRP3 promoter.Moreover,the activity of NLRP3 promoter was still promoted by ZHX2 in HEK 293 cells treated with NF-κB inhibitor.In addition,the expression of Nlrp3 in BMDM of WT mice was still higher than that of MKO-Zhx2 mice after treating with the NF-κB inhibitor.Consistently,ZHX2 still promoted the expression of NLRP3 in THP1 cells with ZHX2 overexpression when treated with NF-κB inhibitors.These results suggest that ZHX2 probably regulates the transcription of NLRP3 partially dependent of the NF-κB pathway.4.3 ZHX2 promotes the assembly of NLRP3 inflammasomeTo further identify the role of ZHX2 in NLRP3 inflammasome activation,NLRP3 inflammasome system was reconstituted in HEK293 cells together with ZHX2 co-transfection.The results showed that ZHX2 overexpression promoted the assembly of NLRP3 inflammasomes.PEM and BMDM were extracted from WT and MKO-Zhx2 mice and stimulated with LPS for 30min or 12h respectively,followed by stimulation with ATP for 30min.The activation of caspase-1 and the secretion of IL-1β were decreased in PEM and BMDM from MKO-Zhx2 mice.These results suggest that ZHX2 not only regulates the transcription of NLRP3,but also promotes the assembly of NLRP3 inflammasome.In conclusion,this study was performed to clarify the role of Zhx2 in the development of EAE for the first time.We found that ZHX2 activated the NLRP3 inflammasome and induced GSDMD mediated pyrotosis to aggravate EAE,which would provide a novel potential target for the intervention of EAE.In addition,we found that ZHX2 regulated NLRP3 transcrition and NLRP3 inflammasome assembly,however the mechanisms require further investigation.