Mechanism Of Interaction Between TLR9 And Caveolin-1 In Inflammatory Preconditioning

Sepsis is a systemic inflammatory response caused by an infection.It not only occurs systemic inflammatory response and immune dysfunction,but also accumulates changes in the function of multiple organs in the body,which can be life-threatening in severe cases.The development of sepsis is related to the balance between anti-inflammatory and pro-inflammatory in the body.When pathogens such as bacteria,fungi,and parasites invade the host body,Polymorphonuclear neutrophil(PMN),as the first line of defense of innate immunity,It will engulfs pathogens and releases a series of cytokines that activate the innate immune system.PMNs are key cells involved in the inflammatory response and play an important role in the immune response to sepsis.In the previous work,we established the intraperitoneal injection of Escherichia coli(E.coli)in mice to induce septic peritonitis,and found that pre-injection of low doses of E.coli into mice could make mice resistant to lethal doses of E.coli.It can be obtained within 2-4 hours after the injection of low-dose E.coli,and we call this phenomenon E.coli-induced"Inflammation preconditioning"(InP).Through the adoptive transfer experiment of peritoneal lavage fluid,it was found that membrane surface TLR9 positive neutrophils(Membrane Toll like receptor 9 positive PMN,mTLR9~+PMN)play a protective role.In the previous work,we previously proved through various experimental methods that in the inflammation pre-conditioning(InP)model,the PMN cell membrane protein Caveolin-1(Cav-1)co-localizes with mTLR9 in the same region,and bound at the cell membrane.The research group put forward the hypothesis that Cav-1 may be the molecule responsible for the transport and anchoring of mTLR9 on the PMN cell membrane,and it is also the protein that plays a synergistic role with it.And the correlation study between Cav-1 and mTLR9 can distinguish the functional difference between membrane TLR9 and plasma TLR9 in PMN,and determine the functional mechanismof mTLR9~+PMN.This topic uses clinical sepsis models,mice and HL60 in vitro inflammatory preconditioning models to explore the interaction mechanismof mTLR9 and Cav-1,and provide new ideas for the prevention and treatment of subsequent sepsis.Methods:(1)Flow cytometry analysis of mTLR9,Cav-1,TLR9 downstreamsignaling molecules My D88,TRAF3 in neutrophils of Normal group,sepsis Recovery group and sepsis Dead group,IRF3 expression level.(2)Co-immunoprecipitation was used to analyze the interaction between TLR9and Cav-1 in peritoneal lavage fluid cells of mouse InP model and HL60 in vitro inflammatory preconditioning model,and to determine its binding site.(3)The Tol2 vector was linearized by PCR,and it was connected with the target gene gh1 with a homologous sequence corresponding to the end of the vector of about20 bp by homologous recombination to construct the recombinant plasmid Tol2-ef1α-EGFP-ef1α-gh1-PA,microinjected into WT zebrafish,screened zebrafish expressing green fluorescence throughout the body,reared to sexual maturity,and mated with WT zebrafish,screened for fluorescence,and raised to sexual maturity,sniped the tail to extract mRNA,and identified by Real Time-PCR gh1 expression at the mRNA level.Results:(1)Flow cytometry results showed that mTLR9 and Cav-1 were highly expressed in sepsis patients,and the expression levels of TLR9 downstreamsignaling molecules My D88,TRAF3,and IRF3 in neutrophils in Recovery group were higher than those in other groups.(2)Co-immunoprecipitation was used to determine the interaction between TLR9and Cav-1 in the peritoneal lavage fluid cells of the mouse InP model and the HL60 in vitro inflammatory preconditioning model,and to determine the binding of TLR9 to the CSD of Cav-1 through its Caveolin-1 binding motif(CBM)sequence.(3)It was confirmed that the transgenic zebrafish Tg(ef1α:EGFP;ef1α:gh1)was successfully constructed by using Tol2 transposon.Conclusions:(1)The high expression of mTLR9 and Cav-1 in peripheral blood neutrophils has a protective effect on recovery fromsepsis.(2)Determine the interaction between TLR9 and Cav-1 in the InP model and determine its binding site.(3)Green fluorescently labeled transgenic zebrafish Tg(ef1α:EGFP;ef1α:gh1)was successfully constructed using Tol2 transposon,which provided experimental animals for subsequent experiments.