The Regulatory Effect Of HDAC10 On NLRP3 Inflammasome Activation And Its Molecular Mechanism

ObjectiveNLRP3 inflammasome is one of the most widely studied inflammasomes at present,and plays a critical regulatory role in innate immunity.The NLRP3 inflammasome is mainly composed of NLRP3,ASC and pro-caspase-1.It is reported that the NLRP3 inflammasome is involved in the development of a variety of inflammatory diseases,including autoinflammatory syndrome,cardiovascular disease,neurodegenerative diseases and tumors.Therefore,precise regulation of NLRP3 inflammasome activation is essential for adequate immune protection and may be a potential target for the treatment of a variety of inflammatory diseases.Recent studies have indicated that post-translational modifications provide a new perspective for us to study the regulatory mechanism of NLRP3 inflammasome assembly and activation.Acetylation is an important post-translational modification for NLRP3 inflammasome activation.Up to now,reports about the acetylation modification of NLRP3 inflammasome activation remains to be clarified.Histone deacetylase 10(HDAC10)is a member of HDAC family.However,the regulatory mechanism of HDAC10 in NLRP3 inflammasome activation is still unclear.In this study,we investigated the regulatory effect of HDAC10 on NLRP3 inflammasome activation and explored its functional mechanism.Materials and Methods1.Detection of NLRP3 acetylation1.1 Detection of the acetylation effect of histone acetyltransferases(CBP,P300,Tip60)on NLRP3 The acetylation effect of endogenous CBP,P300 and Tip60 on NLRP3 was detected by Western Blot.The acetylation effect of exogenous CBP,P300 and Tip60 on NLRP3 was detected by Co-immunoprecipitation(Co-IP)and Western Blot.1.2 Detection of the regulatory effect of the histone deacetylase inhibitor on the acetylation of NLRP3The regulatory effect of histone deacetylase inhibitor TSA on the acetylation of NLRP3 was detected by Co-IP and Western Blot.2.Detection of the effect of HDAC10 on NLRP32.1 Screening of HDAC family members interacted with NLRP3 proteinsHDAC family members molecules that could interact with NLRP3 proteins were screened by Co-IP and Western Blot.2.2 Construction of truncated mutants of HDAC10 and NLRP3 proteins2.3 Detection of the NLRP3 truncated mutant responsible for the interaction with HDAC10 proteinThe interaction between the NLRP3 truncated mutants(?PYD,?NACHT,?LRR)and HDAC10 proteins was defined by Co-IP and Western Blot.The interaction between the NLRP3 domains(1-93aa,220-536aa,742-99 1aa)and HDAC 10 proteins was detected by Co-IP and Western Blot.2.4 Detection of the HDAC10 truncated mutant responsible for the interaction with NLRP3 proteinThe interaction between the HDAC10 truncated mutants(?DAC,?LRD)and NLRP3 proteins was detected by Co-IP and Western Blot.2.5 To explore the physiological significance of HDAC10 in disease modelsLPS was used to stimulate mouse peritoneal macrophages for 6h,and ATP was added to stimulate cells for 30 minutes before collecting cellular proteins.The protein expression levels of NLRP3,HDAC 10,cleaved caspase-1,cleaved IL-1? were detected by Western Blot.Animal models related to NLRP3 inflammasome activation was searched by GEO database.Oligo package was used to evaluate the quality of microarrays.RMA was used to preprocess the original data and annotate the probe with the latest official annotation file.GSEA analysis was performed to analyze the data.T he expression of HDAC10 in animal models of NLRP3 inflammasome activation wasdetected.2.6 Regulation effect of HDAC10 on NLRP3 protein2.6.1 To investigate the regulatory effect of HDAC10 on NLRP3 proteinThe effect of HDAC10 of different concentrations on the protein expression level of NLRP3 was detected by Western Blot.2.6.2 To study the regulatory effect of domain of HDAC10 protein on NLRP3 The effect of the domain of HD AC 10 protein on the protein expression level of NLRP3 was detected by Western Blot.2.6.3 Cycloheximide(CHX)experiment was used to detect the regulatory effect of HDAC10 on NLRP3Treatment with CHX for different time points(Oh,4h,8h,12h),NLRP3 protein expression levels at different time were detected by Western Blot.2.6.4 Detection of the regulatory effect of HDAC10 on NLRP3 by proteasome inhibitor or autophagy inhibitorProteasome inhibitor MG132 or autophagy inhibitor chloroquine(CQ)were added to inhibit proteasome and lysosomal activity respectively.NLRP3 protein expression was detected by Western Blot.3.To investigate the regulatory effect of HDAC10 on NLRP3 inflammasome activation3.1 The regulatory effect of HDAC10 on NLRP3 inflammasome activation in mouse peritoneal-derived macrophagesMouse peritoneal macrophages were transfected with siRNA specifically targeting HDAC10(Si-HDAC10)and Si-NC as the control group.LPS was used to stimulate cells for 6h,and ATP was added to stimulate cells for 30 minutes before collecting cellular proteins.The protein expression levels of NLRP3,ASC,pro-caspase-1,cleaved caspase-1,pro-IL-1? and cleaved IL-1? were detected by Western Blot.The level of IL-1? secretion in the supernatant was detected by ELISA.LPS was used to stimulate cells for 2h.The expression changes of NLRP3,ASC,CASP1 and IL-1? at the mRNA level were detected by qRT-PCR.And the non-specific inflammatory factors TNF-?and IL-6 were detected at the mRNA level by qRT-PCR.3.2 The regulatory effect of HDAC10 on NLRP3 inflammasome activation in mouse bone marrow-derived macrophagesBone marrow-derived macrophages were transfected with Si-HDAC10 and Si-NC as the control group.LPS was used to stimulate cells for 6h,and ATP was added to stimulate cells for 30 minutes before collecting cellular proteins.The protein expression levels of NLRP3,ASC,pro-caspase-1,cleaved caspase-1,pro-IL-1? and cleaved IL-1?were detected by Western Blot.The level of IL-1? secretion in the supernatant was detected by ELISA.LPS was used to stimulate cells for 2h.The expression changes of NLRP3,ASC,CASP1 and IL-1? at the mRNA level were detected by qRT-PCR.And the non-specific inflammatory factors TNF-? and IL-6 were detected at the mRNA level was detected by qRT-PCR3.3 The regulatory effect of TSA on NLRP3 inflammasome activation in mouse peritoneal-derived macrophagesMacrophages were treated with TSA.The protein expression levels of NLRP3,ASC,pro-caspase-1,cleaved caspase-1,pro-IL-1? and cleaved IL-1? were detected by Western Blot.3.4 The regulatory effect of TSA on NLRP3 inflammasome activation in mouse bone marrow-derived macrophagesMacrophages were treated with TSA.The protein expression levels of NLRP3,ASC,pro-caspase-1,cleaved caspase-1,pro-IL-1? and cleaved IL-1? were detected by Western Blot.4.To study the regulatory mechanism of HDAC10 on NLRP3 inflammasome activation4.1 Detection of the deacetylation of HDAC10 on NLRP3HEK293T cells were transfected with HDAC10 plasmids of different concentrations,Tip60 plasmid and the blank vector as the control group.The acetylation level of NLRP3 was detected by Western Blot.4.2 To verify the effect of HDAC10 on the deacetylation level of NLRP3 in mouse peritoneal-derived macrophagesMouse peritoneal macrophages were transfected with Si-HDAC10 and Si-NC as the control group.LPS was used to stimulate cells for 6h,and ATP was added to stimulate cells for 30 minutes before collecting cellular proteins.The protein expression levels of NLRP3,ASC,pro-caspase-1,cleaved caspase-1,pro-IL-1? and cleaved IL-1? as well as the acetylation level of NLRP3 were detected by Western Blot.4.3 To study the effect of HDAC10 on the ubiquitination level of NLRP3HEK293T cells were co-transfected with NLRP3,ubiquitin(Ub)and HDAC10 plasmids,and empty vector as control group.The effect of HDAC10 on the ubiquitination level of NLRP3 was detected by ubiquitination experiments.4.4 To verify the effect of the deacetylation on the ubiquitination level of NLRP3HEK293T cells were co-transfected with NLRP3 and Ub plasmids before treated with TSA.The ubiquitination and acetylation effects of NLRP3 were detected by Co-IP and Western Blot.4.5 To verify the effect of HDAC10 on the ubiquitination level of NLRP3 in mouse peritoneal-derived macrophagesMouse peritoneal macrophages were transfected with Si-HDAC10 and Si-NC as the control group.LPS and ATP were used to stimulate cells.The ubiquitination level of NLRP3 was detected by ubiquitination experiments..Results1.NLRP3 could be acetylated.1.1 Histone acetyltransferases induced acetylation modification of NLRP3Our data showed that the acetylation level of endogenous NLRP3 was significantly enhanced by overexpression of CBP,P300,and Tip60.Co-IP data showed that the acetylation level of exogenous NLRP3 was significantly enhanced by overexpression of CBP,P300,and Tip60.These results indicated that histone acetyltransferases induced acetylation modification of NLRP3,suggesting NLRP3 could be acetylated.1.2 TSA enhanced acetylation modification of NLRP3Our data showed that the acetylation level of endogenous NLRP3 was significantly enhanced in TSA-treated cells.Co-IP data showed that the acetylation level of exogenous NLRP3 was significantly enhanced in TSA-treated cells.These results indicated that TSA enhanced acetylation modification of NLRP3,suggesting NLRP3 could be acetylated.Accordingly,we speculated that acetylation of NLRP3 might be negatively regulated through the deacetylation effect of histone deacetylases.2.HDAC10 negatively regulated NLRP32.1 The interaction between HDAC10 and NLRP3The members of the HDAC family that might bind to NLRP3 were screened by Co-IP and Western Blot.The results showed that HDAC10 could interact with NLRP3 in HEK293T cells2.2 The NACHT domain of NLRP3 mediated the binding of NLRP3 to HDAC10 The results showed that NLRP3 ?NACHT lost the ability to interact with HDAC 10,indicating that the NACHT domain of NLRP3 was required for the interaction between NLRP3 and HDAC 10.2.3 The DAC domain of HDAC10 mediated the binding of HDAC10 to NLRP3 The results showed that HDAC10 ?DAC lost the ability to interact with NLRP3,indicating that the DAC domain of HDAC 10 was required for the interaction between HDAC10 and NLRP32.4 HDAC10 was significantly down-regulated during NLRP3 inflammasome activationThe results showed that the protein level of HDAC 10 was significantly down-regulated during NLRP3 inflammasome activation.GESA analysis results showed that HDAC 10 expression was significantly decreased during NLRP3 inflammasome activation of the acute kidney injury(AKI)models,and HDAC10 was related to immune response and inflammatory response.HDAC 10 could play a role in regulating the immune response of lysine metabolism and regulation,and the regulation of acetyltransferase complex The above results suggested that HDAC 10 could negatively regulate the activation of NLRP3 inflammasome2.5 HDAC10 inhibited NLRP3 expression2.5.1 HDAC10 inhibited NLRP3 expression in a dose-dependent mannerThe results showed that HDAC10 overexpression significantly reduced the expression level of NLRP3 protein in a dose-dependent manner,suggesting that HDAC10 could inhibit NLRP3 protein expression in a dose-dependent manner.2.5.2 The LRD domain of HDAC10 mediated the negative regulatory effect of HDAC10 on NLRP3The results showed that HDAC10 overexpression significantly reduced the expression level of NLRP3 protein.HDAC10 ?LRD lost the ability to reduce the expression of NLRP3,which suggested that HDAC10 negatively regulated the expression of NLRP3 by its LRD domain2.5.3 HDAC10 significantly decreased the protein stability of NLRP3The results showed that HDAC10 could greatly decrease the protein stability of NLRP3,suggesting that HDAC10 could promote the degradation of NLRP3 protein.2.5.4 HDAC10 promoted proteasomal degradation of NLRP3The experimental results showed that HDAC10-induced NLRP3 protein degradation could be reversed by MG 132,but not by chloroquine.These results indicated that HDAC10 negatively regulated NLRP3 by proteasome mediated pathway.3.HDAC10 negatively regulated NLRP3 inflammasome activation3.1.1 Knockdown of HDAC10 significantly increased the activation of NLRP3inflammasome in mouse peritoneal-derived macrophages The results of Western Blot showed that the protein levels of NLRP3,cleaved caspase-1 and cleaved IL-1? were significantly increased in mouse peritoneal macrophages And the level of IL-1? secretion increased significantly in the supernatant.The results of qRT-PCR showed that knockdown of HDAC 10 had no regulatory effects on mRNA expression of NLRP3 and IL-1?.Similarly,non-specific inflammatory factors(TNF-?and IL-6)mRNA expression were not influenced by HDAC10 knockdown.The above results indicated that HDAC 10 inhibited NLRP3 inflammasome activation by regulating protein level rather than mRNA level of NLRP33.1.2 Knockdown of HDAC10 significantly increased the activation of NLRP3 inflammasome in mouse bone marrow-derived macrophagesThe results showed that the protein levels of NLRP3,cleaved IL-1?,and cleaved caspase-1 were significantly increased in mouse bone marrow-derived macrophages.The above results indicated that HDAC10 negatively regulated NLRP3 inflammasome activation by inhibiting the protein level of NLRP3.3.2.1 NLRP3 inflammasome activation was significantly enhanced in TSA-treated mouse peritoneal-derived macrophagesThe results showed that the protein levels of NLRP3,cleaved caspase-1 and cleaved IL-1? were significantly increased in TSA-treated mouse macrophages,which further indicated that HDAC10 could negatively regulate NLRP3 inflammasome activation.3.2.2 NLRP3 inflammasome activation was significantly enhanced in TSA-treated mouse bone marrow-derived macrophagesThe results showed that the protein levels of NLRP3,cleaved caspase-1 and cleaved IL-1? were significantly increased in TSA-treated mouse macrophages,which further indicated that HDAC10 could negatively regulate NLRP3 inflammasome activation.4.HDAC10 significantly reduced the acetylation of NLRP3The results showed that overexpression of HDAC10 significantly reduced the acetylation level of NLRP3,suggesting that HDAC10 could inhibit the acetylation effect of NLRP3 and exert a deacetylation effect on NLRP3.5.HDAC10 inhibited the activation of NLRP3 inflammasome through its deacetylation effectWestern Blot results showed the protein levels of NLRP3,cleaved caspase-1 and cleaved IL-1? were significantly increased.And the acetylation level of NLRP3 was also up-regulated.These data indicated that HDAC10 inhibited NLRP3 inflammasome activation through its deacetyiation effect.6.HDAC10 promoted the ubiquitination modification of NLRP3The results showed that HDAC10 upregulated the ubiquitination level of NLRP3,which indicated that HDAC10 could promote the ubiquitination modification of NLRP3.7.Acetylation could suppress ubiquitination of NLRP3The results showed that after TSA treatment,the acetylation level of NLRP3 was significantly enhanced,and its protein ubiquitination level was significantly reduced,which suggested that acetylation could inhibit the ubiquitination effect of NLRP3.8.HDAC10 could promote the ubiquitination of NLRP3 by its deacetylationThe results of ubiquitination analysis experiments showed that HDAC10 knockdown greatly reduced the ubiquitination level of NLRP3.This result indicated that HDAC10 positively regulated the ubiquitination effect of NLRP3 by its deacetylation.HDAC10 negatively regulated NLRP3 by promoting ubiquitination of NLRP3.Conclusions1.Histone acetyltransferases(CBP,p300,Tip60)and histone deacetylase inhibitor(TSA)could enhance acetylation modification of NLRP3,suggesting NLRP3 could be acetylated.2.We demonstrated for the first time that class ? histone deacetylase HDAC10 could directly bind to NLRP3,and have the deacetylation effect on NLRP3.3.HDAC10 negatively regulated the activation of NLRP3 inflammasome by inhibiting the protein expression of NLRP3.Innovations and significances1.In our study,it was discovered for the first time that NLRP3 could be acetylated under the action of histone acetyltransferases(CBP,p300,Tip60)or histone deacetylase inhibitor(TSA).HDAC10 inhibited the protein expression of NLRP3 by directly interacting with NLRP3 and its deacetylation effect.2.We revealed that HDAC10 could negatively regulate NLRP3 protein expression and further inhibit NLRP3 inflammasome activation,which clarified a novel regulatory mechanism of NLRP3 inflammasome activation and provided a potential therapeutic target for NLRP3 inflammasome-related disease.