The Role And Mechanism Of Extracellular ADP And Its Receptors In Rheumatoid Arthritis

Inflammation plays an important role in maintaining the homeostasis of the whole body.The inflammatory response is also the key strategy for host against the infectious agents or tissue damages,which has the potential in inflammatory diseases.Rheumatoid arthritis(RA)is a typical chronic autoimmune disease caused by excessive activation of the host immune system.The main clinical manifestations are persistent joint synovial inflammation and a large number of immune cells infiltration,including neutrophils,which destroy the joint cartilage and bone.At present,the pathogenic mechanism,pathological processes and treatments of rheumatoid arthritis are not fully understood.The process of activation and regulation of local inflammatory response during RA has been a hot topic in this field.Meanwhile,a series of "danger signals" including HMGB1,HSP,IL-33,and extracellular nucleotides have been found play an important role in the development and progression of RA,and their excessive and chronic release leads to abnormal joint inflammation and cartilage destruction.These endogenous molecules from different joint cells consist a feedback loops outside the cell,amplifying joint damage and local inflammatory responses.When released into the extracellular environment,these mediators may cross-link different receptors on the same cell,thereby simultaneously activate many signaling pathways within immune cells leading to a destructive inflammatory response.Therefore,the development of prevention and treatment strategies for RA in the future may depend on the early detection and suppression of these "danger signals" and related signaling pathways.Thus,the study of the function and regulation mechanism of extracellular "danger signal" in local inflammation of RA has great potential in research and application.Adenosine diphosphate(ADP)is one of the important members among the extracellular nucleotide family.It is abundance in vivo and is also the secondary metabolites of ATP,which is involved in platelet activation and aggregation.Furthermore,it also participates in platelet-mediated inflammatory responses.At the same time,our previous study found that extracellular ADP acts as "danger signal" that can be released in large quantities during inflammation and tissue damage,as well as in the joint tissues of RA mice,and ADP receptors are highly expressed,consisting with expression data in the human RA disease database.So,does ADP released from RA joints regulate the occurrence of local inflammation?What are the important functions in the development of RA disease?Our study illustrates a feedback regulation of extracellular ADP during RA formation,providing theoretical basis and experimental support for the developing and applications of the drugs that target "danger signals".First,we explored the induction process of extracellular ADP by local inflammation in RA.We have found that extracellular ADP,as an intercellular signaling molecule,can be released in large quantities not only in cell model but also in the joint tissues of RA model mice,and it rapidly response to the injury with high concentration,consisting with high expression of ADP receptor P2Y1 in RA patients.Then,we constructed collagen-induced arthritis mice model to further investigate the function of extracellular ADP in the pathological process of RA.We found that ADP treated mice are significantly higher than the control mice in terms of clinical scores,joint thickness,and degree of swelling.As well,ADP also affects the pathological morphology of joints,promotes the infiltration of inflammatory neutrophils and increases joint inflammation,affecting the pathological process.In vivo,using the P2Y1 knockout mice to establish a RA model,the knockout mice were significantly reduced in clinical scores and joint swelling.After treating with the P2Y1 specific antagonist,the mice of joint swelling can be slightly inhibited;The ADP-promoting infiltration of neutrophils into inflammatory joints is also obviously blocked.This suggests that extracellular ADP may act as a "danger signal" to promote inflammatory neutrophil infiltration and increase the local inflammatory response in RA joints.We then focused on the biological function of extracellular ADP-induced neutrophil infiltration in RA.We firstly used microarray to screen for differential genes and biological process,suggesting the role of extracellular ADP in influencing the chemotactic migration of neutrophils.On this basis,the use of in vivo cell migration model confirmed that extracellular ADP can cause the ability of neutrophils to migrate to the site of inflammation.Meanwhile,it was found in vitro Transwell experiments that ADP-induced macrophages supernatant had a strong ability to attract neutrophils.And the chemokine CXCL-2 secreted by ADP-induced macrophages plays a key role in this process.In in vitro and in vivo models,the specific CXCL-2 antibody was used to block the function of CXCL-2,which can significantly inhibit the migration of the cells.At the same time,we found that macrophages were co-localized with CXCL-2 in the joint tissue of RA mice,revealing the pivotal role of CXCL-2 secreted by macrophages in ADP-intensified RA local inflammation.Finally,the mechanism of ADP-induced transcriptional regulation of CXCL-2 was studied.We used common bioinformatics software to predict transcription factor binding sites that may be involved in the regulation of ADP.Followed by comparisons and screenings of methods such as dual luciferase reporter genes and specific site mutations,the results showed that ADP can induce macrophages transcription factor NF-?B to enter the nucleus and bind to the CXCL-2 promoter region,promoting the transcription and expression of its gene.Furthermore,we found that ADP activates P2Y,receptor,coupling G?q to increase intracellular Ca2+ signal by calcium flux assay experiment.And,further functional experiments confirmed that ADP-induced macrophage CXCL-2 secretion was decreased after inhibiting Ca2+ signaling.In conclusion,these results basically illustrate the molecular mechanism of extracellular ADP-induced CXCL-2 expression in macrophage-mediated inflammatory response.In summary,our study demonstrated extracellular ADP as an important"danger signal" molecule in the development of RA.Extracellular ADP induces CXCL-2-mediated neutrophil migration through activating P2Y1 receptor on macrophages,and participates in the occurrence and development of inflammatory responses,aggravate the pathological process of RA.Our study also illustrated that extracellular ADP regulated CXCL-2 expression mainly through Ca2+-NF-?B signal pathway.These results lay the foundation for further development of specific therapeutic strategies and drugs based on extracellular danger signals and their receptors for inflammatory diseases.