The Role Of Osteoclast Extracellular Traps In The Pathogenesis Of Rheumatoid Arthritis

Rheumatoid arthritis（RA）is a common autoimmune disease in humans and animals that is susceptible to genetic and environmental factors and manifests itself primarily as chronic synovitis with irreversible joint damage,ultimately leading to physical disability or even life-threatening injury.However,there is no specific drug to treat RA.SIRT1 has been shown to be an important determinant of RA disease course changes and is the most studied member of the acetylase（Sirtuins）family and has been shown to be closely associated with immunology and endocrinology,however,its detailed relationship with RA pathogenesis is unclear.Osteoclasts（OCs）are differentiated from bone marrow macrophages and are the only cells in the body capable of degrading bone.OCs can secrete extracellular vesicles to exchange information with osteoblasts,which play a key role in maintaining bone homeostasis and bone growth processes and are key cells in the pathogenesis of RA.Current studies on the effects of OCs on RA are limited to the enhanced bone resorption function of OCs leading to joint damage,however,the specific pathogenic mechanism remains unclear.In this study,we will investigate the reasons for the enhanced bone resorption function of OCs.The release of Extracellular traps（ETs）,initially thought to be a physiological process used to help protect the host from pathogen invasion,has since been found to be associated with many other important physiological and pathological processes,particularly playing a key role in a variety of autoimmune diseases.ETs consist of extracellular DNA（eDNA）bound to components such as elastase（Ela）,myeloperoxidase（MPO）,and high mobility histone B1（HMGB1）.It is generally believed that ETs are produced through two pathways,NADPH oxidase（NOX）-dependent manner and NOX-independent manner,and their generation is closely linked to p-AKT and p-ERK regulation.A variety of cells（such as neutrophils,macrophages,etc.）can secrete ETs,but there have been no reported studies on the production of ETs by OCs.In the course of our preliminary RA study,we discovered for the first time in China and abroad that OCs can produce ETs substances,namely osteoclast extracellular traps（OETs）.It was also found that OETs play a key role in the pathogenesis of RA,and the SIRT1/p-AKT/p-ERK pathway was also found to be closely associated with the formation of OETs.Therefore,in this study,in order to more comprehensively explore the pathogenic mechanism of OETs on RA,we will systematically investigate the mechanism of SIRT1/p-AKT/p-ERK pathway mediating the onset of RA caused by OETs,which provides new ideas for the cure of RA.First,to clarify the ability of OCs to release ETs and the effect of ETs formation on the osteoclastic ability of OCs,we induced mouse bone marrow macrophages into OCs in vitro using RANKL and M-CSF,and detected the production of eDNA by OCs induced by different concentrations of collagen type II（CII）or foponol 12-tetradecanoate 13-acetate（PMA）at different time points using a fluorescent enzyme marker.We found that OCs released peak eDNA at 8 μM CII concentration for 6 h of treatment（P < 0.001）;OCs released peak eDNA at 4 μM PMA concentration for 6 h of treatment（P < 0.001）.We identified the morphology and structure of OETs with the aid of scanning electron microscopy and used immunofluorescence microscopy to determine that OETs contain key components such as eDNA,Ela,MPO and HMGB1.Next,to investigate the reason for the enhanced bone resorption function of OCs,we used scanning electron microscopy to observe the bone resorption traps in bone fragments,and the results showed that the production of OETs significantly increased the number of bone resorption traps and thus enhanced the bone destruction ability of OCs,while HMGB1 antibody,DNase I（eDNA inhibitor）and Cl-amidine（histone guanylation inhibitor）effectively inhibited the number of bone resorption traps caused by OCs and reduced the bone destruction ability of OCs.Then,to explore the expression of pro-inflammatory factors induced by the release of OETs from OCs,we used ELISA to detect the expression levels of TIL-6,IL-1β and TNF-α in the culture medium.The results showed that CII significantly induced high expression of TIL-6,IL-1β and TNF-α（P < 0.001）,while HMGB1 antibody,DNase I（eDNA inhibitor）and Cl-amidine（histone guanylation inhibitor）significantly inhibited the expression of inflammatory cytokines IL-6,IL-1β and TNF-α（P < 0.001）.In summary,in vitro,both PMA and CⅡ induced the release of OETs from OCs;and increased the release of proinflammatory factors such as TIL-6,IL-1β and TNF-α;the production of OETs could enhance the ability of OCs to damage bone tissue,and the inhibition of OETs could effectively reduce the ability of OCs to damage bone.Second,to further investigate the mechanism by which the SIRT1/p-AKT/p-ERK pathway mediates the formation of CII-induced OETs,we first induced mouse bone marrow macrophage-derived OCs by pretreatment with various inhibitors of RANKL and M-CSF,including Staurosporine（PKC inhibitor）,U0126（ERK and MEK inhibitor）,PD98059（ERK inhibition）,DPI（NADPH oxidase inhibitor）,LY294002（PI3 kinase inhibitor and AKT inhibitor）,GW5074（c-Raf inhibitor）,and OETs formation were analyzed by laser confocal microscopy and luminescence zymography.The results showed that the CII-induced group had significant OETs production（P <0.001）,and ERK inhibitor and AKT inhibitor could inhibit CII-induced OETs production（P < 0.001）,which indicated that ERK and AKT had regulatory effects on the formation of OETs;further,we used Western Blot technique to analyze the correlation between ERK and AKT phosphorylation and The correlation of OETs generation showed that CII could significantly induce high expression of p-ERK and p-AKT（P < 0.001）,AKT inhibitor significantly inhibited p-ERK expression（P <0.001）,while ERK inhibitor had no significant inhibitory effect on p-AKT expression（P > 0.05）,indicating that AKT is upstream of ERK expression.Then,we examined the interactions of CⅡ with human and mouse SIRT1 proteins by ZDOCK software,respectively,and the results showed that CⅡ formed hydrogen bonds with HIS 363,ARG 274,GLY 415 of human SIRT1 protein and ARG 438,ASP 290,GLN 286,TYR272 of mouse SIRT1 protein,respectively,indicating that CⅡ could bind to human and mouse SIRT1 proteins;then,we constructed SIRT1 knockdown RAW264.7 cells with the help of CRISPR/Cas9 technology and examined the relationship between SIRT1,AKT and ERK in the formation of CII-induced OETs by Western Blot technique,and the results showed that CII induction could stimulate high expression of SIRT1（P <0.001）,which is consistent with the computer analysis of CⅡ strong effect on SIRT1 results;Western Blot also showed that knockdown of SIRT1 significantly inhibited CⅡinduction of p-ERK and p-AKT expression（P < 0.001）,while ERK and AKT inhibitors had no significant effect on SIRT1 expression（P > 0.001）,this result indicates that during CⅡ induction of SIRT1 is located upstream of ERK and AKT during the formation of OETs and has a regulatory role on p-ERK and p-AKT expression.In addition,we investigated the relationship between ROS and OETs production using a fluorescent zymography combined with the NADPH oxidase（NOX）inhibitor DPI,and we found that the mitochondrial ROS content was significantly enhanced in the CIIinduced group compared with the control group（P < 0.001）,and the cytoplasmic ROS content was not significantly different（P > 0.05）,and the release of OETs was not significantly inhibited by DPI pretreatment（P > 0.05）,indicating that CII-induced OETs production was not associated with NOX.In summary,in vitro findings suggest that CII-induced OCs mediate the production of OETs via the SIRT1/p-AKT/p-ERK pathway and that the production pathway is NOX non-dependent.Finally,we used CII as an inducer to construct a mouse model of RA,combined with three candidate inhibitors of OETs（HMGB1 antibody,DNase I and Cl-amidine）to validate the mechanism of OETs production in vivo and to examine the possibility of HMGB1,DNase I and histone guanylation as therapeutic targets for RA.We used ELISA to detect the expression levels of TNF-α and IL-6 in the serum and synovial fluid of RA mice;co-localized OCs,eDNA,Ela,RAGE,MPO,Histone and HMGB1 in the synovial fluid of RA mice with the help of immunofluorescence;observed the pathological changes and inflammatory cell infiltration in the joints of RA mice by HE staining;analyzed the expression of SIRT1/p-AKT/p-ERK pathway proteins in the synovial fluid of RA mice by Western Blot.The results showed that OETs in the synovial fluid of the RA mouse model constructed by CII induction were continuously produced and accompanied by IL-6,IL-1β,and TNF-α production,while the expression levels of SIRT1,p-AKT,and p-ERK were significantly enhanced（P < 0.001）.Continuous use of three inhibitors of OETs（HMGB1 antibody,DNase I and Cl-amidine）significantly improved inflammatory pathological changes in the joints and significantly reduced the expression levels of inflammatory cytokines IL-6,IL-1β and TNF-α（p < 0.001）.In summary,the in vivo model results suggest that OETs are key factors in the development of RA,;their mechanism of development is mediated by the SIRT1/p-AKT/p-ERK pathway;HMGB1,eDNA and histone guanylation are potential targets for the treatment of RA,consistent with the results of in vitro experiments.In conclusion,in this study,we discovered for the first time in China and abroad that CⅡ can induce the production of OETs and clarified the structure of OETs and their components;we initially elucidated the SIRT1/p-AKT/p-ERK pathway as the molecular mechanism mediating the production of OETs through in vitro experiments;we completed the construction of a mouse RA model using CⅡ induction and verified in vivo that the SIRT1/p-AKT/p-ERK pathway-mediated induction of OETs by CII is the pathogenesis of RA,especially the results of treatment with antibodies,enzyme inhibitors and small molecule inhibitors identified HMGB1,eDNA and histone guanylation as potential targets for the treatment of RA..In conclusion,this study provides an important theoretical basis for the in-depth exploration of the pathogenesis of RA,and also provides new targets for the prevention and treatment of RA.