Study On The Molecular Mechanism Of CD11b Regulating The Production Of IL-12 In Macrophages Stimulated By LPS

Background:Endotoxic shock(ES)is caused by various pathogenic microbes or toxin infection,as a systemic response syndrome that characterized by tissue perfusion,hypotension and organ dysfunction,it can severely contribute to the severe critical clinical death.Although technology of modern treatment is progressing rapidly,ES remains difficultly to be solved.The imbalance of inflammatory mediators is a key factor during the development of ES.IL-12 is closely relevant to ES,which is a heterodimeric cytokine that is involved in immune regulation and anti-infection processes.In an endotoxic shock mouse model,we found that CDl1b inhibitor effectively elevated IL-12 level in the peripheral blood.However,the relationship between CD11b and IL-12 remains elusive.In this study,we investigated whether CD11b is involved in regulating the production of IL-12 stimulated with LPS and its mechanism.Method:Firstly,we established a mouse model of endotoxic shock and tested the effect of CD11b inhibitors to survival rate.To determine the expression of IL-12 in peripheral blood of endotoxic shock mice,we used flow cytometry microarray assay.Secondly,RAW264.7 macrophages were treated with shRNA interference or inhibitors respectively.The effects of CD11b on IL-12p35 and p40 subunits for both transcription and translation levels were investigated by real-time quantitative PCR and western blot.Finally,RAW264.7 macrophages were treated with transiently transfected shRNA or inhibitors,and then the effect of CD11b on the activation of MAPK and NF-κB signaling pathway was investigated by Western blotting.Results:Compared to the control group,CDllb inhibitor Gu-4 significantly increased 50%of survival rate and elevated IL-12 level in the peripheral blood.Knocking down the expression of CD11b or inhibiting the activation of CD11b increased IL-12p35 and IL-12p40 subunits at both transcriptional and protein levels in macrophages stimulated with LPS.In addition,Western blotting experiments indicated that knocking down the expression of CD11b or inhibiting the activation of CD11b significantly inhibited the phosphorylation of JNK and IKKα/β in macrophages stimulated with LPS.And activation of NF-κB was significantly attenuated.There was no significant effect of phosphorylation levels of ERK.The results suggested that CDllb inhibited LPS-stimulated IL-12 production is involved in JNK and NF-κB signaling.Conclusion:This study suggested that CD11b inhibited IL-12 production in LPSstimulated macrophages mainly via JNK and NF-κB signaling pathways,and inhibiting the function of CD11b may be of help for attenuating excessive inflammatory responses induced by LPS.Understanding the key links and regulatory mechanisms of inflammatory mediators provides a new theoretical basis for improving the treatment of ES.