The Formation Of Endosomal Akt/IKKα/IKKβ Signalosome Facilitate Dendritic Cell Cross-presentation By Mediating Endosomal Recruitment Of Sec61

Dendritic cells(DCs)are the major antigen presenting cells that mediate cross-presentation.Their special intracellular microenvironment makes cross-presentation much more efficient than other antigen presenting cells such as macrophages cell.The antigens and endotoxin components of pathogenic microorganisms can be recognized by different pattern recognition receptors and activate NF-κB,Akt and other signal pathways.But until now,the specific mechanism of Akt and canonical NF-κB signaling pathways,which can be activated by antigens and inflammatory stimuli,in the pathogenic microorganisms induced-cross-presentation is remains unclear Therefore,Firstly,Bone marrow-derived dendritic cells were treated with commericial model antigen OVA(contains low-doses endotoxin)and model antigen OVAET(endotoxin-free)respectively to detect the activity of Akt and NF-κB signaling pathway by immunoblotting.Then,co-immunoprecipitation was performed to detect the translocation of Akt,IKKα,and IKKβ kinases on Rab5+ early endosomes.Moreover,downregulate the corresponding kinase activity to detect the effect of these kinases on cross-presentation by means of immunofluorescence and flow cytometry,respectively.Finally,by use of co-immunoprecipitation and immunofluorescence to explore the effect of the corresponding kinase on endoplasmic reticulum associated protein Sec61 endosomal recruitment.The results showed that loading OVA for 15-60 min,the phosphorylation levels of Akt and IKKα/β increased significantly,and the phosphorylation level reached the maximum at 30min.At the same time,the degradation of p100 and IκBα also increased significantly;while loading OVAET neither increases IKKβ phosphorylation nor causes IκBα degradation,suggesting that while canonical NF-κB signaling efficiently achieved by inflammatory stimuli,noncanonical NF=κB signaling and Akt kinase activation obviously acquired by the antigen stimulation.And then the inhibition of IKKa/Akt and IKKβ also obviously impaired DC abilities of cross-presentation.Interestingly,antigens and inflammatory stimuli were revealed to augment the endosomal recruitments of IKKa,IKKβ and phosphorylated Akt.Finally,IKKa and Akt were demonstrated to contribute to IKKβ-mediated the endosomal translocation of Sec61and facilitate DC cross-presentation.All these data indicate that antigen-induced noncanonical NF-κB signaling promote the endosomal recruitments of canonical NF-κB signaling,which in turn augment Sec61 translocation and DC cross-presentation.These findings improve our understanding of the cell-biological mechanisms of pathogenic microorganisms affecting the endosomal translocation of Sec61 and the effective initiation of adaptive immune responses under inflammatory conditions.