Study Of Extracellular ATP Activating P2X7R-NF-?B Pathway To Promote The Maturation Of Bone Marrow-derived Dendritic Cells Of Mice

Background:The maturation state of dendritic cells(DCs)could play an important role in immune activities.Generally,mature DC promotes immune rejection,while immature DC induces immune tolerance.Thus intervening the maturation of DC has become a useful method to induce immune tolerance.Our research group had previously found that TLR2/MyD88/NF-?B(p65)pathway could promote DC maturation,T lymphocytes proliferation and the release of inflammatory cytokines during immune rejection;and the inhibition of TLR2/MyD88/NF-?B(p65)pathway significantly improved corneal allograft tolerance and prolonged the survival duration of grafts in rats.However,the upstream mechanism of NF-?B pathway to promote DC maturation is still unclear and needs further exploration.The activation of T lymphocytes requires three signals.Interestingly,in addition to these three traditional pathways,recent studies have recognized the fourth signal,which is extracellular adenosine triphosphate(ATP)acting as autocrine stimuli by activating its receptor P2X7R.At present,there is no research regarding the relationship between P2X7R and NF-?B pathway in DC.Here we aimed to find out the connection between P2X7R and NF-?B(p65)pathway in DC maturation.Method:Bone marrow derived dendritic cells were collected and induced in mice,which were divided into immature DCs(6d group),mature DCs(8d group),enhanced mature DCs(LPS group),P2X7R agonist BzATP stimulation group(Bz group),and P2X7R inhibitor OxATP stimulation group(Ox group).Relative mRNA expression of P2X7R and NF-?B(p65)was detected by RT-qPCR.Protein expression of P2X7R and NF-?B(p65)was detected by Western-blot.Supernatant inflammatory cytokine concentration of IFN-? and IL-12 was detected by Enzyme-Linked Immuno Sorbent Assay(ELISA).Protein interaction between P2X7R and NF-?B(p65)was detected by co-immunoprecipitation assay.And intracellular ATP concentration was detected by ATP assay.Results:Results showed that according to the order of 6d group,8d group and LPS group,DCs experienced morphological changes into more mature appearances,with elevated levels of inflammatory cytokines IFN-,y and IL-12,which demonstrated the maturation process of DC.Along with DC maturation,the mRNA and protein expression of P2X7R and NF-?B(p65)were both increased,with elevated intracellular ATP levels.P2X7R agonist stimulated the morphological changes of DCs into the appearance of mature DCs,and promoted the release of IFN-y and IL-12,as well as the expression of NF-?B(p65).Whereas,P2X7R inhibitor had the opposite influences.Co-immunoprecipitation assay confirmed the binding of P2X7R and NF-?B(p65).Main Conclusion:Our study suggested that extracellular ATP could promote DC maturation and release of inflammatory cytokines through the binding of P2X7R and NF-?B(p65).This is the first study to show the P2X7R-NF-?B(p65)pathway in DC.Interference with this pathway may be a new target for regulating immune responses in areas like infectious diseases,inflammation,transplantation,tumor and autoimmune diseases,which could have significant importance for studying the regulation of corneal transplantation immunity.In addition,intracellular ATP level could be a new indicator of the maturity state of DC.