The Function Of ADP In Macrophages Antibacterial Immune Regulation

Recent studies found that damaged cells and immune cells released extracellular nucleotides(ATP,ADP,UTP and UDP)in a short time,when tissue inflammation,ischemic or hypoxic injury.Extracellular nucleotides,as a danger signal,can directly combine with purine receptors on cell membrane and active receptors,leading to an immune response and inflammation.ATP or UDP in extracellular space activates immune cells to release inflammatory cytokines like IL-1? and IL-18,thereby promote inflammation response and kill pathogens in monocytes/macrophages.ADP changes the shape of platelets,activates fibrinogen-receptor and initiates platelet aggregation by simultaneous activation of P2Y1 and P2Y12.In P2Y12 knocked-out mice the LPS-induced systemic inflammation is more severe.Moreover,ADP can enhance microglial migration function by activating of P2Y12 and P2Y13.Our previous studies show that,during bacterial infections,the released UDP and ATP activates macrophages,and clear exogenous pathogens by phagocytosis and secreting inflammatory cytokines,ultimately resisting bacteria.During previous studies,we find that bacterial infections also lead to ADP release,and ADP can influence immune functions of macrophages.However,as ATP and UDP are all explored in depth,there are few discussions about effects of ADP during bacterial infections.As a result,we will deeply explore the mechanism of ADP-mediated immune cells' functions in bacterial infections.This paper can be divided into three parts:a)We construct bacteria-infected cell model by using LPS and PAM3CSK4 to imitate gram-negative/positive bacterium and stimulating mice macrophage cell line RAW264.7 and bone marrow macrophages.From this cell model,we detect the cell supernatants and find that ADP is released from macrophages in short time when infected by bacteria.Peritoneal inject E.coli to establish mouse acute peritonitis model.The results demonstrate that,in the ADP-treated group there are less bacteria in the peritoneal fluid,and the survival rate is also higher than control group.b)As we all know that organism firstly starts the innate immune system,in which macrophages play an important role of recognizing and cleaning pathogens.Then we explore the relation between ADP and phagocytic function or chemotactic migration function in macrophages.The transwell migration experiment result suggests that extracellular ADP not only promote the migration function in macrophages,but also stimulate the transfection and expression of cytokines IL-1?,IL-1?,IL-6,CCL20,MCP-1,GM-CSF.The release of chemotactic factor MCP-1 encourages the recruitment of monocytes/macrophages.c)For further explore the mechanism of ADP-mediated immune cells'functions,we use P2Y1 selected inhibitor MRS2179 and P2Y12/13 inhibitor forskolin to block ADP-induced signal path.In vitro research result,forskolin can inhibit ADP-induced monocytes/macrophages recruitment and MCP-1 release.However,MRS2179 is not noticeable in inhibiting ADP-induced effects.In vivo experiment,we construct bacteria-infected mice model,E.coli infected peritonitis model.In this model we detect the numbers of macrophages and residual bacteria in mice ascites by using flow cytometry,and MCP-1 concentration in mice serum.The results show that ADP-induced macrophages recruiting to bacterial infection area,and MCP-1 releasing,bacteria clearance and antibacterial ability are all weakened by foskolin.Foskolin also decreased the survival rate of peritonitis mouse.In summary,we prove that ADP is released as a danger signal during bacterial infections.Moreover we also support that ADP can collect macrophages to bacterial infections area and induce macrophages to release chemokine by activating P2Y12/13 receptors,and then recruit more monocytes/macrophages.As a result ADP promote the efficiency of bacterial clearance and achieve the target of anti-bacteria and protective.Therefore,these results above supply the theoretical foundation for further ADP regulation mechanism exploration during bacterial infections.Furthermore,it provides new thinking of preventing bacterial infections and researching antibacterials.