The Role Of RIPK3/MLKL-mediated Necroptosis In The Pathogenesis Of Pulpitis

Background and objectivePulpitis is a typical inflammatory disease of dental pulp caused primarily by bacterial infection,leading to toothache,tissue necrosis,and abscess formation.Immune cells that infiltrate inflamed dental pulp exert their antimicrobial activity to defend against pathogenic bacteria;during this process,excessive inflammation inevitably causes significant damage to pulp cells and collateral tissue.A low level of inflammation is critical,at least initially,to promote reparative processes,but uncontrolled pulpitis will inhibit reparative events.How to control the excessive inflammatory response and maintain the balance between inflammation and repair is one of the key issues of vital pulp therapy.Necroptosis is a caspase-independent mode of regulated necrosis defined by activation of receptor-interacting protein kinase 3(RIPK3)and its downstream substrate,the pseudokinase mixed lineage kinase domain-like(MLKL).Upon phosphorylation by active RIPK3,MLKL oligomerizes and translocates to cellular membranes,leading to the rupture of plasma membrane and cell death.It is widely accepted that necroptosis is a highly proinflammatory type of cell death because of the passive release of intracellular contents following plasma membrane rupture.Recent studies have reported that necroptotic signaling can activate the NLRP3 inflammasome and promote the secretion of proinflammatory cytokines in a cell-intrinsic manner.Indeed,the role of necroptosis in the pathogenesis of bacterial infection is increasingly appreciated,such as pneumonia and periodontitis.However,whether necroptosis is involved in the pathogenesis of pulpitis has not yet been reported.Thus,the current study was designed to investigate whether necroptosis is implicated in human dental pulp inflammation and mouse experimental pulpitis,as well as to determine the impact of necroptosis on the inflammatory response in human dental pulp fibroblasts(HDPFs)and the underlying mechanism in vitro.This study will contribute to unveiling the key regulators of pulpitis and providing targets for the vital pulp therapy.Methods1.The expression of RIPK3/MLKL signaling pathway in inflamed human dental pulpTo examine whether necroptosis is implicated in the pathogenesis of dental pulp inflammation,western blotting and immunohistochemistry were utilized to detected the expression levels of RIPK3,p-RIPK3,MLKL,and p-MLKL in inflamed and healthy pulp tissues.Then the expression of p-MLKL in different cell types within human dental pulp were determined by immunofluorescence double staining.Specific cell markers CD68,fibroblast surface protein(FSP)and CD31 were used to identified macrophages,fibroblasts and vascular endothelial cells,respectively.2.The role of necroptosis in the pathogenesis of mouse experimental pulpitisAn experimental pulpitis model was established by exposing the pulp chamber in the maxillary first molars of mice,and the expressions of IL-6 and p-MLKL were detected by immunohistochemistry.To explore the potential role of necroptosis in the development of dental pulp inflammation in vivo,GSK872 or vehicle was administered to mice in the experimental pulpitis model.The histological characteristics of the pulps were examined using HE staining,and the expression of the IL-6 mRNA and IL-6,pMLKL protein were detected by qPCR and immunohistochemistry,respectively.The effect of N-cadherin knockdown on the proliferation and migration of human dental pulp stem cells3.RIPK3/MLKL signaling pathway mediates necroptosis in human dental pulp fibroblastsTo investigate whether they undergo necroptosis in the context of inflammation,HDPFs were treated in vitro with LPS in the presence or absence of the Smac mimetic AZD5582 and the pan-caspase inhibitor zVAD-fmk.Cell viability,necrosis,ultrastructural characteristics and hallmarks of necroptosis activation(including RIPK3,p-RIPK3,MLKL and p-MLKL)were detected by CCK-8 assay,Hoechst33342/PI staining,transmission electron microscopy and western blotting,respectively.Furthermore,shRNA-mediated knockdown of MLKL or specific inhibitors of RIPK3(GSK872)or MLKL(NSA)were also used to confirm the involvement of RIPK3 and MLKL in LAZ-induced cell death of HDPFs.4.Effect of necroptosis on the inflammatory response of human dental pulp fibroblastsThe levels of 21 cytokines/chemokines in the culture medium of HDPFs were measured using MILLIPLEX? MAP assays.qPCR and western blotting were used to verify the expression of mRNA and protein levels of proinflammatory cytokines that were significantly up-regulated.Western blotting and immunofluorescence staining were used to examined the underlying mechanism.Furthermore,shRNA-mediated knockdown of MLKL was also used to confirm the effect of necroptosis on the expression of proinflammatory cytokines and the related molecular mechanism in HDPFs.5.A preliminary study on the molecular mechanism regulating necroptosis in human dental pulp fibroblasts based on proteomicsTMT quantitative proteomics was used to compare the differentially expressed proteins in HDPFs of necroptosis group and normal control group.Combined with bioinformatics analysis,candidate proteins of interest that may be involved in the regulation of necroptosis in HDPFs were screened out.Western blotting and immunofluorescence staining were further used to verify and preliminarily explore the molecular mechanism regulating necroptosis in HDPFs.Results1.The expression of RIPK3/MLKL signaling pathway in inflamed human dental pulpCompared with healthy pulp tissues,the levels of key molecules of necroptotic signaling pathway RIPK3,p-RIPK3,MLKL and p-MLKL were higher in inflamed pulp tissues,which were consistent with the increased expressions of NF-κB p65 and proinflammatory cytokine IL-6.The expression of RIPK3 was co-localized with its downstream p-MLKL in the same tissue specimen.p-MLKL,the marker of necroptosis,was expressed in CD68+ macrophages,FSP+ pulp fibroblasts and CD31+ vascular endothelial cells.2.The role of necroptosis in the pathogenesis of mouse experimental pulpitisp-MLKL is highly expressed in inflamed pulp tissues of experimental pulpitis model in mice.GSK872,the specific RIPK3 inhibitor,alleviated the inflammatory response and tissue damage of dental pulp,as well as the expression of p-MLKL.3.RIPK3/MLKL signaling pathway mediates necroptosis in human dental pulp fibroblastsLPS/AZD5582/zVAD-fmk(LAZ)treatment decreased cell survival and induced cell necrosis in HDPFs after LAZ exposure.The expressions of p-RIPK3 and p-MLKL,and the ratios of p-RIPK3/RIPK3 and p-MLKL/MLKL were upregulated in a timedependent manner following LAZ priming of HDPFs.Pretreatment with GSK872 or NS A,as well as shRNA-mediated knockdown of MLKL,protected HDPFs against LAZ-induced cytotoxicity and reduced the ratio of necrotic cells in response to LAZ priming.4.Effect of necroptosis on the inflammatory response of human dental pulp fibroblastsThe levels of the cytokine IL-6 and the chemokine IL-8 were significantly higher in the culture medium of HDPFs treated with LAZ than in that of HDPFs treated with LPS,and the addition of GSK872 or NSA suppressed these levels dramatically.LAZ stimulation of HDPFs promoted phosphorylation of the NF-κB p65 subunit,thus increasing the ratio of p-p65 to p65.In addition,immunofluorescent staining revealed that the nuclear translocation of p65 was much stronger in LAZ-treated HDPCs than in cells stimulated by LPS alone.Silencing of MLKL decreased the expression of p-p65 and the p-p65/p65 ratio upon LAZ challenge and retarded the translocation of p65 to the nucleus triggered by LAZ priming.Immunofluorescent double staining revealed that LAZ treatment induced the expression of p-MLKL as well as the nuclear translocation of p65.5.A preliminary study on the molecular mechanism regulating necroptosis in human dental pulp fibroblasts based on proteomicsCompared with the control group,a large number of interferon-stimulated genes were up-regulated in HDPFs after necroptosis induction,including OAS1,IFIT3,MX1,IFIT1,IFIT2,MX2,etc.The JAK-STAT signaling pathway was identified as one of the significantly enriched signaling pathways of differentially expressed proteins.LAZ stimulation of HDPFs significantly up-regulated the levels of IFIT3,STAT1 and pSTAT1 in a time-dependent manner.The expression of IFIT3 was significantly increased and co-localized with pMLKL in inflamed pulp tissues and necroptotic HDPFs.Conclusions1.The RIPK3/MLKL-mediated necroptosis is activated in inflamed human dental pulp.Necroptosis occurred not only in the immune cells(e.g.,macrophages),but also in the nonimmune cells(e.g.,pulp fibroblasts and vascular endothelial cells),which may play an important role in the pathogenesis of pulpitis.2.Necroptosis participates in the pathogenesis of experimental pulpitis in mice.GSK872 ameliorates dental pulp inflammation in the mouse experimental pulpitis model,probably by inhibiting the RIPK3/MLKL signaling pathway.3.LPS/AZD5582/zVAD-fmk treatment induces RIPK3/MLKL-dependent necroptosis in HDPFs.4.RIPK3/MLKL-mediated necroptosis promotes the production of proinflammatory cytokines in HDPFs in a cell-intrinsic manner via the NF-κB pathway.5.IFIT3 may promote the formation of RIPK3-MLKL necrosome,the subsequent necroptosis and inflammatory response in HDPFs by regulating the JAK-STAT signalling pathway,thus participating in the pathogenesis of pulpitis.