The Function And Molecular Mechanism Of Histone Demethylase KDM4B On NLRP3 Inflammasome Activation

ObjectiveInnate immunity is regarded as the first line of defense to distinguish "self" from"non-self".If the innate immunity is inappropriately regulated,it will cause severe immune damage,inflammation and even death.Among them,the inflammatory response depends on the release of inflammatory cytokines.The inflammatory response includes:priming and activating processes,and then the priming process is induced by LPS(and others)and activatited the NF-κB signaling pathway,causing the release of cytokines IL-6,TNF-α.And also induces the up-regulation of mRNA levels of pro-IL-1B and NLRP3.The activation process means that DAMP and PAMP activate NLRP3 inflammasome,thereby promoting the secretion and release of IL-1β.Therefore,NLRP3 plays a key role in the inflammasome activation.NLRP3 inflammasome is involved in the development of various diseases.Therefore,the regulation of NLRP3 is crucial to the homeostasis.Various factors regulate NLRP3 inflammasome including:transcription regulation,post-transcriptional regulation,translation level regulation and post-translation regulation.In recent years,more and more epigenetic regulation has been found to be involved in regulating inflammation.Among them,histone modification is one of the important ways of epigenetic regulation,and histone methylation modification is one of the most widely studied modification.Histone methylation mainly occurs on the lysine and arginine of histone H3 or H4,to regulate gene expression.Histone H3K9 methylation and H3K27 methylation are two important inhibition modification.Recent studies have found that inhibitors of histone demethylase JMJD3 inhibit the activation of inflammatory genes in human primary macrophages by demethyltransferasing H3K27me3,and inhibit the secretion of the inflammatory cytokine TNF-α.Therefore,the dynamic regulation of histone methylation modification on inflammatory response may be a potential new treatment method for inflammasome-related diseases.However,the H3K9 methylation modification on the regulation of inflammatory cytokines has not been reported.Therefore,we focused on the regulation of H3K9me3 demethylation on the inflammation response.We used H3K9me3 demethylase KDM4 family members(especially KDM4B)to deeply study the regulatory effects and molecular mechanisms of H3K9me3 on NLRP3 inflammasome.In the present study,we show that KDM4B enhances NLRP3 expression via promoting NLRP3 transcription.Furthermore,KDM4family members broad-spectrum inhibitor ML324 inhibits NLRP3 expression,the cleavage of Caspase-1and subsequent IL-1β secretion in peritoneal macrophages.Therefore,we identified ML324 as a negative regulator of NLRP3 inflammasome.Besides,our data suggested that KDM4B is a potential target for the therapies of inflammatory diseases.Materials and Method1.ML324 influences NLRP3 inflammasome and NLRP3 expression1.1 Mouse peritoneal primary macrophages were pretreated with dimethylsuphoxide(DMSO)or ML 324(20mM)for 2 hours then stimulated with LPS for 6 hours,followed by ATP(5mM))、Nig(50μM)or poly(dA:dT)(200ng/ml)treatment for 45-60min.The level of IL-1β,IL-6 and TNF-α in the culture supernatants were determined by ELISA.1.2 Mouse peritoneal primary macrophages were treated with dimethylsuphoxide(DMSO)or different doses of ML324(5mM,10mM,20mM,50mM)for 2h,then stimulated with LPS(200 ng/ml)for 6h,and next stimulated with ATP for 45～60min.The level of IL-1β,IL-6 and TNF-α in the culture supernatants were determined by ELISA.1.3 Mouse peritoneal primary macrophages were pretreated with dimethylsuphoxide(DMSO)or ML324(20mM)for 2 hours,then stimulated with LPS(200 ng/ml)for different time points(0h,4h,8h),and then stimulated with ATP for 45～60min.The cell supernatant was concentrated with an ultrafiltration tube.The Caspase-1 cleavage(secretion of p10 and p20)in thesupernatants(SN)and the expression of Caspase-1 in the cell lysate(CL)were determined through Western blot.1.4 Mouse peritoneal primary macrophages were treated with dimethylsuphoxide(DMSO)or different doses of ML324(5mM,10mM,20mM,50mM)for 2h,followed by stimulation with LPS(200 ng/ml)for 6h,and then stimulated with ATP for 45min～60min.The Caspase-1 cleavage(secretion of p10 and p20)in thesupernatants(SN)and the expression of Caspase-1 in the cell lysate(CL)were determined through Western blot.1.5 Mouse peritoneal primary macrophages were pretreated with dimethylsuphoxide(DMSO)or ML324(20mM)for 2 hours,then stimulated with LPS(200 ng/ml)for different time points(0,4h,8h),and detectd the protein expression level about inflammasome components by Western blot.1.6 Mouse peritoneal primary macrophages were pretreated with dimethylsuphoxide(DMSO)or ML324(20mM)for 2 hours,then stimulated with LPS(200 ng/ml)for 2 hours,the mRNA level of IL-1β,IL-6,TNF-α and NLRP3 were determined by RT-PCR.1.7 Mouse peritoneal primary macrophages were pretreated with dimethylsuphoxide(DMSO)or different doses of ML324(5mM,10mM,20mM,50mM)for 2h,and then stimulated with LPS(200 ng/ml)for 2h,the mRNA level of IL-1β,IL-6,TNF-α and NLRP3 were determined by RT-PCR.2.The effect of KDM4B on NLRP3 expression2.1 HEK293T cells were transfected with KDM4A-Flag,KDM4B-Flag,KDM4C-Flag,KDM4D-Flag pcmv-Flag plasmid.The effect of KDM4 family members on the protein level of NLRP3 was detected by Western bolt.2.2 Designed and synthesized the KDM4B specific siRNA,and transfected KDM4B-siRNA and Ctrl-siRNA by transfection reagentsINTERFERIN(?)in mouse primary peritoneal macrophages and further cultured for 48h.Then detected expression of KDM4B by Western blot to select the best efficient interference siRNA.2.3 Transfected the best KDM4B-siRNA and Ctrl-siRNA into the mouse peritoneal macrophages,and continued to culture for 48 hours,stimulated the transfected cells with LPS(200 ng/ml)at different time points(0,4h,8h),and detectd the protein expression level of NLRP3 inflammasome components by Western blot.2.4 Transfected the best KDM4B-siRNA and Ctrl-siRNA into the mouse peritoneal macrophages,and continued to culture for 48 hours,stimulated the transfected cells with LPS(200 ng/ml)2 hours,then the mRNA level of IL-1β,IL-6,TNF-α and NLRP3 were determined by RT-PCR.2.5KDM4B+/+ and KDM4B-/-mice peritoneal primary macrophages were stimulated with LPS(200 ng/ml)at different time points(0,4h,8h),and detected the protein expression level of NLRP3 inflammasome components by Western blot.2.6KDM4B+/+ and KDM4B-/-miceperitoneal primary macrophages were stimulated with LPS(200 ng/ml)for 2 hours,then the mRNA level of IL-1β,IL-6,TNF-α and NLRP3 were determined by RT-PCR.2.7Two groups of experiments were designed:KDM4B+/+ and KDM4B-/-miceperitoneal primary macrophages are stimulated with LPS(200 ng/ml)at different time points(0min,30min,60min);Transfected the best KDM4B-siRNA and Ctrl-siRNA into the mouse peritoneal macrophages,and continued to culture for 48 hours,stimulated the transfected cells with LPS(200 ng/ml)at different time points(0min,30min,60min).The expressions of protein associated with priming process of NLRP3 inflammasome activation were detected by Western blot.3.KDM4B regulates NLRP3 expression3.1 HEK293T cells were transfected with KDM4B-Flag,KDM4B(H189A)-Flag and PCMV-Flag plasmid,and then collected RNA,the effects of the two plasmids on NLRP3 mRNA levels were detected by RT-PCR.3.2KDM4B+/+ and KDM4B-/-miceperitoneal primary macrophages stimulated with LPS(200 ng/ml)at different time points(0h,1h,4h),and then detected whether RNA polymerase II-specific antibody binds to the TSS region of NLRP3 promoter by CHIP-qPCR experiment to detect whether KDM4B affects NLRP3 transcription.3.3KDM4B+/+ and KDM4B-/-mice peritoneal primary macrophages were stimulated with LPS(200 ng/ml)at different time points(0h,1h,4h),and then detected whether KDM4B-specific antibody binds to the TSS region of NLRP3 promoter by CHIP-qPCR experiment.4.KDM4B influences NLRP3 inflammasome activity4.1 Transfected the best KDM4B-siRNA and Ctrl-siRNA into the mouse peritoneal macrophages,and continued to culture for 48 hours,stimulated the transfected cells with LPS(200 ng/ml)for 6 hours,and then stimulated with ATP(5mM))、Nig(50μM)or poly(dA:dT)(200ng/ml)for 45～60min.The level of IL-1β,IL-6 and TNF-α in the culture supernatants were determined by ELISA.4.2 Transfected the best KDM4B-siRNA and Ctrl-siRNA into the mouse peritoneal macrophages,and continued to culture for 48 hours,stimulated the transfected cells with LPS(200 ng/ml)for different time points(0h,4h,8h),and then stimulated with ATP for 45～60min.The cell supernatant was concentrated with an ultrafiltration tube.The Caspase-1 cleavage(secretion of p10 and p20)in thesupernatants(SN)and NLRP3 expression in the cell lysate(CL)were determined through Western blot.4.3 KDM4B+/+ and KDM4B-/-mice peritoneal primary macrophages were stimulated with LPS(200 ng/ml)for 6 hours,and then useingATP(5mM))、Nig(50μM)or poly(dA:dT)(200ng/ml)stimulated for 45～60min.The level of IL-1β,IL-6 and TNF-α in the culture supernatants were determined by ELISA.4.4 KDM4B+/+and KDM4B-/-mice peritoneal primary macrophages were stimulated with LPS(200 ng/ml)for different time(0,4h,8h),and then stimulated with ATP for 45-60min.The cell supernatant was concentrated with ultrafiltration tubes.The Caspase-1 cleavage(secretion of p10 and p20)in thesupernatants(SN)and NLRP3 expression in the cell lysate(CL)were determined through Western blot.5.ML324 influences NLRP3 inflammasome activity in vivo.5.1 ML-324 influences LPS-induced endotoxernia in vivo.C57BL/6J mice(female,5-7weeks)were intraperitoneally injected with dimethylsuphoxide(DMSO),or ML-324(40mg/kg)for 3 hours,followed by LPS(10mg/kg)for 2hours.Secretions of IL-1β,IL-6 and TNF-α in peripheral blood serum were determined by ELISA.5.2 ML324 influences Alum-induced peritonitis in vivo.C5 7BL/6mice(female,5-7weeks)were intraperitoneally injected with dimethylsuphoxide(DMSO),or ML324(40mg/kg)for 3 hours,and then were treated with Alum for 16h to induce the peritonitis model.Absolute number of PECs,neutrophils or Ly6C+monocytes recruited to the peritoneum were analysed by flow cytometry.Results1.KDM4 inhibitor ML324 inhibits NLRP3 inflammasome activation and NLRP3 expression.1.1ML324 inhibits NLRP3 inflammasome activationMouse peritoneal primary macrophages were pretreated with dimethylsuphoxide(DMSO)or ML324(20mM)for 2 hours then stimulated with LPS for 6 hours,followed by ATP(5mM)),Nig(50μM)or poly(dA:dT)(200ng/ml)treatment for 45～60min.ELISA date suggested that ML324 significantly promoted the secretion of IL-1β induced by ATP and Nig,the level of IL-6 and TNF-α had no significantly changed,while did not affect the IL-1β induced by poly(dA:dT).Mouse peritoneal primary macrophages were treated with dimethylsuphoxide(DMSO)or different doses of ML324(5mM,10mM,20mM,50mM)for 2h then stimulated with LPS(200 ng/ml)for 6h,and next stimulated with ATP for 45～60min.ELISA date suggested that IL-1β secretion gradually decreased with the increase of ML324 dose,while the level of IL-6 and TNF-α had no significantly changed.1.2 ML324 inhibits cleavage of Caspase-1Mouse peritoneal primary macrophages were pretreated with dimethylsuphoxide(DMSO)or ML324(20mM)for 2 hours,then stimulated with LPS(200 ng/ml)for different time points(0h,4h,8h),and then stimulated with ATP for 45～60min.Western blot date suggested that the Caspase-1 cleavage(secretion of p10 and p20)reduced in thesupernatants(SN)and Caspase-1 expression decrease in the cell lysate(CL)after ML324 treatment.Mouse peritoneal primary macrophages were treated with dimethylsuphoxide(DMSO)or different doses of ML324(5mM,10mM,20mM,50mM)for 2h,followed by stimulation with LPS(200 ng/ml)for 6h,and then stimulated with ATP for 45min～60min.Western blot date suggested that the Caspase-1 cleavage(secretion of p10 and p20)gradually decreased with the increase of ML324 dose in thesupernatants(SN)and Caspase-1 expression have no significantly changes in the cell lysate(CL)after ML324 treatment.1.3 ML324 inhibits NLRP3 expressionMouse peritoneal primary macrophages were pretreated with dimethylsuphoxide(DMSO)or ML324(20mM)for 2 hours,then stimulate with LPS(200 ng/ml)for different time points(0,4h,8h),Western blot date suggested that ML324 inhibits NLRP3 expression,while there is no obvious change of the level of ASC and Caspase-1.Mouse peritoneal primary macrophages were pretreated with dimethylsuphoxide(DMSO)or ML324(20mM)for 2 hours,then stimulated with LPS(200 ng/ml)for 2 hours,RT-PCR date suggested that ML324 inhibits NLRP3 expression,while there is no obvious change of IL-1β,IL-6 and TNF-α.Mouse peritoneal primary macrophages were pretreated with dimethylsuphoxide(DMSO)or different doses of ML324(5mM,10mM,20mM,50mM)for 2h,and then stimulated with LPS(200 ng/ml)for 2h,RT-PCR date suggested that ML324 inhibits NLRP3 expression in a dose-dependent manner.2.KDM4B enhances NLRP3 expression2.1 KDM4B,differing from KDM4A/C/D,enhances NLRP3 expressionHEK293T cells were transfected with KDM4A-Flag,KDM4B-Flag,KDM4C-Flag,KDM4D-Flag and pcmv-Flag plasmid.Western blot date suggested that KDM4B,differing from KDM4A/C/D,enhance the expression of NLRP3 at the protein level.2.2 KDM4B knockdowninhibitsNLRP3 expressionDesigned and synthesized the KDM4B specific siRNA,and transfected KDM4B-siRNA and Ctrl-siRNA by transfection reagents interferin in mice primary peritoneal macrophages and further cultured for 48h.Then Western bolt dates choose the best one which is most interference efficient.Transfected the best KDM4B-siRNA and Ctrl-siRNA into the mouse peritoneal macrophages,and continued to culture for 48 hours,stimulated the transfected cells with LPS(200 ng/ml)at different time points(0,4h,8h),then the cells were lysed after 24 hours;Transfected the best KDM4B-siRNA and Ctrl-siRNA into the mice peritoneal macrophages,and continued to culture for 48 hours,stimulated the transfected cells with LPS(200 ng/ml)2 hours.Then Western bolt and RT-PCR date suggested that KDM4B knockdown could inhibit the NLRP3 expression at both of the protein and mRNA level,whereas the expression of ASC and Caspase-1did not significantly changed at the protein level,IL-1β,IL-6 and TNF-α had not changed at the mRNA level either.2.4 KDM4B deficiency inhibitsNLRP3 expression.KDM4B+/+ and KDM4B-/-mice peritoneal primary macrophages were stimulated with LPS(200 ng/ml)at different time points(0,4h,8h),then the cells were lysed;Took KDM4B+/+and KDM4B+/+ miceperitoneal primary macrophages were stimulated with LPS(200 ng/ml)for 2 hours,collected RNA;And then Western bolt and RT-PCR dates suggested that KDM4B knockdown could inhibit the NLRP3 expression at both of the protein and mRNA level,whereas the expression of ASC and Caspase-1did not significantly changed at the protein level,IL-1β,IL-6 and TNF-α did not changed at the mRNA level either..2.5 KDM4B had no effects on the priming process of NLRP3 inflammasome activationTwo groups of experiments were designed:Took KDM4B+/+ and KDM4B-/-mice peritoneal primary macrophages were stimulated with LPS(200 ng/ml)at different time points(0min,30min,60min);Transfected the best KDM4B-siRNA and Ctrl-siRNA into the mice peritoneal macrophages,and continued to culture for 48 hours,stimulated the transfected cells with LPS(200 ng/ml)at different time points(0min,3 0min,60min).The expressions of protein associated with priming process of NLRP3 inflammasome activation,such as p-I κBα,were did not changed.3.KDM4B enhances NLRP3 transcription3.1 KDM4B enhances NLRP3 expression through demethylase activity.HEK293T cells were transfected with KDM4B-Flag,KDM4B(H189A)-Flag and pcmv-Flag plasmid,collected RNA,RT-PCR date suggested that KDM4B enhances NLRP3 expression at the mRNA level,andafter transfected with KDM4B(H189A)-Flag,the promoting effect disappeared3.2 KDM4B enhances the transcription initiation of NLRP3 promoter region to enhance the transcription of NLRP3.Took KDM4B+/+ and KDM4B-/-mice peritoneal primary macrophages were stimulated with LPS(200 ng/ml)at different time points(0h,1h,4h),CHIP-qPCR experiment date suggested that RNA polymerase Ⅱ-specific antibody binds to the TSS region of NLRP3 promoter,further suggested that KDM4B affects NLRP3 transcription.3.3 KDM4B binds to the NLRP3 promoter regionTook KDM4B+/+ and KDM4B-/-mice peritoneal primary macrophages were stimulated with LPS(200 ng/ml)at different time points(0h,1h,4h),CHIP-qPCR experiment date suggested that KDM4B-specific antibody binds to the TSS region of NLRP3 promoter.4.KDM4B enhances NLRP3 inflammasome activation4.1 KDM4B knockdown inhibits NLRP3 inflammasome-induced IL-1β secretion and Caspase-1 cleavage.Transfected the best KDM4B-siRNA and Ctrl-siRNA into the mouse peritoneal macrophages,and continued to culture for 48 hours,stimulated the transfected cells with LPS(200 ng/ml)for 6 hours,and then stimulated with ATP(5mM)),Nig(50pM)or poly(dA:dT)(200ng/ml)for 45-60min.ELISA date suggested that after KDM4B-siRNA treatment,ATP and Nig-induced the IL-1β secretion decreased,IL-6 and TNF-α did not change significantly,while poly(dA:dT)induced IL-1β secretion was basically unchangedTransfected the best KDM4B-siRNA and Ctrl-siRNA into the mouse peritoneal macrophages,and continued to culture for 48 hours,stimulated the transfected cells with LPS(200 ng/ml)for different time points(0h,4h,8h),and then stimulated with ATP for 45～60min.The cell supernatant was concentrated with an ultrafiltration tube.Western blot date suggested that the Caspase-1 cleavage(secretion of p10 and p20)reduced in thesupernatants(SN)and Caspase-1 expression decrease in the cell lysate(CL)after transfected the KDM4B-siRNA.4.2 KDM4B deficiency inhibits NLRP3 inflammasome-induced IL-1β secretion and Caspase-1 cleavage.KDM4B+/+and KDM4B-/-mice peritoneal primary macrophages were stimulated with LPS(200 ng/ml)for 6 hours,and then usingATP(5mM)),Nig(50μM)or poly(dA:dT)(200ng/ml)stimulated for 45～60min.ELISA date suggested that in KDM4B-/-mice peritoneal primary macrophages,ATP and Nig-induced the IL-1βsecretion decreased,IL-6 and TNF-α did not change significantly,while poly(dA:dT)induced IL-1β secretion was basically unchanged either.KDM4B+/+ and KDM4B-/-mice peritoneal primary macrophages were stimulated with LPS(200 ng/ml)for different time(0,4h,8h),and then stimulated with ATP for 45～60min.The cell supernatant was concentrated with ultrafiltration tubes.Western blot date suggested that the Caspase-1 cleavage(secretion of p10 and p20)reduced in thesupernatants(SN)and Caspase-1 expression decrease in the cell lysate(CL)in KDM4B-/-mice peritoneal primary macrophages.5.ML324 attenuatesNLRP3 inflammasome activition in vivo5.1 ML-324 suppressesLPS-inducedsecretions of IL-1β and TNF-α.C57BL/6J mice(female,5-7 weeks)were intraperitoneally injected with dimethylsuphoxide(DMSO),or ML-324(40mg/kg)for 3 hours,followed by LPS(10mg/kg)for 2hours.Obtain blood by removing eyeballs,ELISA dates suggested that in ML324 treatment group,secretions of IL-10 in peripheral blood serum was lower than that in control group,with no difference in secretion of IL-6 and TNF-α.5.2 ML324 ameiliates Alum-inudced peritonitis in vivo.C57BL/6 mice(female,5-7weeks)were intraperitoneally injected with dimethylsuphoxide(DMSO),or ML324(40mg/kg)for 3 hours,and then were treated with Alum for 16h to induce the peritonitis model.Absolute number of PECs,neutrophils or Ly6C+monocytes recruited to the peritoneum were analysed by flow cytometry.The date suggested that the total number of PECs,neutrophils,and Ly6C+monocytes in the ML324 treated experimental group were less than those of wild type.This shows that ML324 can inhibit the activation of NLRP3 inflammasome and the enrichment of immune cells.Conclusion1.Histone demethylase KDM4B selectively promotes NLRP3 transcription and NLRP3 inflammasome activation by binding to the NLRP3 gene promoter region.2.KDM4 inhibitor ML324 inhibits NLRP3 expression and NLRP3 inflammasome activation,and alleviates the progress of NLRP3 inflammasome-associated disease model in mice.Innovation and significance1.Revealed the role of H3K9me3 demethylase KDM4B in NLRP3 transcription and NLRP3 inflammasome activation,suggesting that H3K9me3 demethylation may participate in the regulation of inflammatory response by regulating NLRP3 expression.2.Revealed the role of KDM4B and KDM4 family inhibitor ML324 inNLRP3 inflammasome activation,and provided a new target for the treatment of diseases related to imbalance of NLRP3 inflammasome activation.