| Bacterial peritonitis accompanied by excessive inflammatory response is essentially a cytokine storm happened in the body,and large number of anti-inflammatory cytokines and pro-inflammatory cytokines rapidly produced in immune cells.In this process,polymorphonuclear neutrophil(PMN)serves as the immune system's first line of host defense and exerts anti-inflammatory effect.As the disease progresses,PMN releases a series of pro-inflammatory mediators,triggering cytokine storms and aggravating the inflammatory response.Therefore,neutrophils have a double-sided function in hyperinflammatory diseases,with both anti-inflammatory and pro-inflammatory properties.In the previous studies,we found that TLR9 recognized pathogen-associated molecular patterns(PAMPs)or risk-associated molecular patterns(DAMPs)in PMN during MPLA-mediated inflammation preconditioning.Then TLR9 transferred from endosome to the cell membrane for binding to caveolin-1,which activated the neutrophil subtype with anti-inflammatory properties,named mTLR9+PMN which play a key role in this protection and protect the body from excessive inflammation.This prompts us to study the signaling pathways and mechanisms of mTLR9+PMN,and we shoud further study the anti-inflammatory factors.In the study of the polarization of macrophages in inflammatory response,it was found that interferon regulatory factors(IRFs)play an essential role.In particular,IRF4 and IRF5 play important roles in regulating the polarization of anti-inflammatory phenotype of macrophages(M2)and pro-inflammatory phenotype of macrophages(M1).The impact of IRF4 and IRF5 binding to MyD88 competitively for macrophage differentiation and polarization into M1 and M2 is the decisive factor.M1 highly express IRF5 which bind to MyD88 to induce a lot of pro-inflammatory cytokines,such as TNF-?,M2 overexpress IRF4 which bind to MyD88 to produce a large number of anti-inflammatory cytokines,such as IL-10,IL-4,IL-1 for the host anti-inflammatory responses to avoid excessive inflammation.At present,there are few studies on the function and gene transcription regulation of IRFS in neutrophil.Based on the above research,we suggest that we should study the anti-inflammatory mechanism of IRFs in mTLR9+PMN.Therefore,we further speculate that in bacterial peritonitis,a hyperinflammatory disease,mTLR9+PMN induces anti-inflammatory pathway of IRF4/MyD88/IL-10,which plays a role in suppressing inflammation,and pro-inflammatory pathway of IRF5/MyD88/TNF-?,which promotes the occurrence and development of inflammation.The contest between these two signaling pathways determines the fate of the host.Therefore,we believe that in MPLA-mediated inflammation preconditioning,mTLR9+PMN activates IRF4/MyD88/IL-10 pathway and inhibits the release of pro-inflammatory factors,which has a certain protective effect in bacterial peritonitis mice.In view of this,we established bacterial peritonitis model in mice with E.coli,applied MPLA to mediate inflammation preconditioning,and used TLR9 and Cav-1knockout mice.Then we observed the survival time and histopathological changes in mice,the expression of proteins and inflammatory factors.By exploring the signaling pathways of IRF4/IRF5 and MyD88 induced by MPLA to study the protective mechanism of mTLR9+PMN in inflammatory preconditioning.At the same time,we constructed transgenic zebrafish Tg(Tol2-EF1?: MyD88-EGFP-PA)to achieve a zebrafish model of MyD88 overexpression,so that we can further study the signaling pathways in gene function.This will provide a new scientific evidence and scientific research ideas for the prevention and treatment of bacterial peritonitis.The experimental results are as follows.(1)The effect of TLR9 in MPLA mediated inflammation preconditioning(InP).InP group with tlr9-/-mice(n=8).First,we found that InP had no effect on the survival of tlr9-/-mice,with only 1 mouse alive at 72 h;InP had serious damage of intestine in the tlr9-/-mice as same as the model group;Reintroduction of neutrophils from wild type mice(WT mice)lavage fluid into tlr9-/-mice could partially restore its InP effect,about 5 mice alive;Expression of TLR9,MyD88 proteins and cytokines of TNF-?,IL-6 were markedly decreased in tlr9-/-mice of InP.(2)The protective effect of mTLR9 in InP is Cav-1 dependent.InP group with cav-1-/-mice(n=8).First,we found that InP had no effect in cav-1-/-mice,about 5 mice died at 72 h;Severe damage of intestinal was also observed in cav-1-/-mice of InP;The expression of TLR9 on the memberane was not detected in cav-1-/-mice of InP;And the expression of TNF-? and IL-6 were markedly decreased.(3)The effect of IRF4/IRF5 in MPLA mediated inflammation preconditioning(InP).InP group with WT mice(n=8).The expression of IRF4 gradually increased,but the expression of IFR5 gradually decreased,and the ratio of IRF4 to IRF5 increased gradually with the time of MPLA effected;The co-expression of IRF4 and IRF5 with MyD88 by in InP Co-immunoprecipitation;We also detected the expression of IRF4 increased significantly,while the expression of IRF5 decreased in nucleus of InP;And InP group increased the secretion of the IL-10,while reduced the secretion of TNF-?.(4)Construction of transgenic zebrafish Tg(Tol2-EF1?: MyD88-EGFP-PA).The Tol2 transposon was used to successfully construct the MyD88 over-expressed recombinant plasmid,which microinjected into single-cell stage of the zebrafish with TP m RNA;Then screened out the embryos of green fluorescent protein expressing in the whole-body,and cultured to F0 generation.It is convenient for future screening and identification of stable inherited F1 and homozygous F2 generation transgenic zebrafish. |
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