The Roles Of K48 Ubiquitination Of Mannose Receptor In Nicotine-enhanced Cross-presentation Of Dendritic Cells

Dendritic cells(DCs)are known as initiators of adaptive immunity.In addition to classical MHC I-restricted endogenous antigen presentation,Surface molecules such as lectin,CD40,langerin,heat shock protein mediated cross-presentation allows DCs presenting intracellular antigen and induces protective immunity against intracellular microbes infection or against tumors.Cross-presentation by dendritic cells(DCs)requires ubiquitination-derived antigen degradation,the endosomal translocation of endoplamic reticulum(ER)associated protein p97 and Sec61.Our previous studies showed that nicotine treatment facilitated to endosomal translocation of endociytosis antigen mediated by mannose receptor(MR)and endosomal recruitment of p97/Sec61 through up-regulation of MR expression.Although the ubiquitination of mannose receptor augment antigenic translocation toward to endosomes,increase the endosomal recruitment of p97,and enhance cross-presentation,the effect of nicotine on MR ubiquitination,MR ubiquitinated manner on antigenic endosomal translocation and the mechanism of ubiquitinated MR-increased endosomal recruitment of p97 and Sec61 are still poorly understood.To address this object,bone marrow-derived dendtritic cells(BMDCs)and the model antigen ovalbumin(OVA)were used for this recearch.Firstly,mechanism of nicotine-increased MR expression was determined by western blot.Secondly,co-immunoprecipitation was applied to determine the effect of nicotine on the level and manner of MR ubiquitination.Thirdly,in ubiquitin silencing condition,the K48C/R peptide of ubiquitin mutant chains was applied to modify nicotine-increased ubiquination manner of MR,and the effect of nicotine-increased ubiquitination manner of MR on endosomal orientation of exogenous antigen,the distribution of p97/Sec61,cross presentation were determined by confocal microscope observation.Finally,IFN-?-Enzyme linked immunospot assay(ELISPOT)was used to confirm the cross-presentation dependent antigenic specific cross priming.The results showed that:Firstly,the treatment with nicotine efficiently induced phosphorylation of phosphoinositide 3-kinase(PI3K),protein kinase B(Akt)and the downstream mTOR-p70S6 from 5 to 30 min.The pretreatment with kinase inhibitors wortmannin,LY294002,Rapamycin and LY2584702 prior to nicotine stimulation obviously abolished the effect of nicotine on MR expression,indicating that nicotine treatment upregulated MR expression via PI3K-Akt-mTOR-p70S6 pathway.Secondly,co-immunoprecipitation analyses with MR,ubiquitin and K48 ubiquitin monoclonal antibodies revealed that the treatment with nicotine evidently increased the level of MR K48 ubiquination.Thirdly,confocal microscopy observation demonstrated that the inhibition of MR ubiquitination and modification of MR K48 ubiquitination not only abolished nicotine-increased the translocation of exogenous antigen,the co-localizations of cross-presented ovalbumin with the marker of early endosome(EEA1/Rab5),but also obviously reversed the effect of nicotine on the co-localizations of p97 and Sec61 with EEA1/Rab5 and with endoplasmic reticulum(ER)marker calnexin.Fourthly,the effect of nicotine-enhanced cross-priming was also abrogated by either the ubiquitination inhibition or the manner modification of K48 ubiquination.In conclusion,the research discovered the effect of nicotine on MR ubiquitination,and the roles of MR K48 ubiquitination in nicotine-increased endosomal translocation of exogenous antigen and the endosomal recruitments of p97/Sec61,which provide evidences for further exploration.