The Roles Of Mannose Receptor And TLR4Mediated Pathway In Nicotine-enhanced Cross-Presentation Of Dendritic Cells

Dendritic cells (DCs) is an important type of antigen presenting cells (APC), which has nicotinic acetylcholine receptor (nAchR) expression. Our previous studies showed that nicotine treatment could not only up-regulate surface molecules ex pression in murine bone marrow-derived DCs but also augment DCs-mediated CTL priming and reveals anti-tumor effects, indicating that nicotine treatment increases DCs’cross-presentation abilities. Mannose receptor (MR), an important pathgon recognition receptor in DCs, has been found play important role in the process of cross-presentation,which include endosomal recruitment of TAP and cytosolic antigen translocation. But, until to now, little is known about the roles of MR-mediated endocytosis and TAP endosomal recruitment in nicotine-increases cross-presentation. Moreover, the effect of nicotine on human PBMC-derived DCs’cross presentation is still to clarify. To address this object, murine bone marrow derived DCs was treated with nicotine and the expression of MR, TLR4was firstly determined by flow cytometry and Western blot respectively. With inhibitors to inhibit related kinases activities, the mechanism of nicotine-increase MR and TLR4expression was then investigated. With SiRNA transfection to knock down MR and MyD88expression, the roles of MR and TLR4signal pathway in nicotine-increase DCs’ cross-presentation was explored by confocal microscope observation and flow cytometry respectively. Lastly and importantly, the effect of nicotine on human PBMC-derived DCs’ cross-presentation was determined by flow cytometry and confocal microscope. he key point of our project is to explore the mechanisms of nAchR regulating cross-presentation, which could provide fundamental theory support for DCs clinical use to deal with tumor and viral infection.The results showed that:firstly, nicotine treatment could not only increase MR and TLR4expression but also augment the expression of CD80and4-1BBL in DCs, which result in160%endocytosis increase of treated DCs. The pretreatment of nAchR specific antagonist alpha-bungarotoxin and non-specific anatagonist tubocurarine obviously decreased nicotine’s effect on the upregulation of CD80and4-1BBL, indicating that nicotine upregulates MR, CD80,4-1BBL expression through nAchR.Secondly, the phosphorylation of ERK and PI3K was efficiently induced by nicotine treatment, which reach the peak at5-15min and5-30min respectively. The usages of LY294002and Wortmannin obviously inhibited the effect of nicotine on TLR4and MR expression, indicating that nicotine upregulates TLR4and MR expression by activating PI3K-Akt pathway. Thirdly, confocal microscope observation showed that nicotine treatment not only incerases OVA translocation to early endosome but also promote MR endosomal recruitment, whereas MR knock down abolished endosomal translocation of OVA and MR, indicating that nicotine promotes OVA endosomal translocation by up-regulating MR expression. Fourthly, the SIINFEKL-H2Kb formation and colocalization of SIINFEKL with EEA1, Rab7, MHC class Ⅰ but not class Ⅱ molecules, which were abolished by MR knock down, were efficiently increased by the nicotine treatment, indicating that nicotine-increased cross-presentation was MR-mediated OVA endosomal translocation dependent. Fifthly, the formation of SIINFEKL-H2Kb complex, endosomal recruitment of TAP and colocalization of SIINFEKL with EEA1, MHC class Ⅰ but class Ⅱ molecules, which could be abolished by MyD88SiRNA transfection, were augmented by LPS treatment, indicating that MyD88-dependent endosomal recruitment of TAP play important role in nicotine increased murine DCs’ cross presentation. Most importantly, not only nicotine upregulated DCs surface molecules nAchR, MR, TLR4, CD80, CD86and4-1BBL by nAchR-PI3K-Akt pathway but also the role of MyD88-dependent TAP endosomal recruitment could also be achieved in human PBMC derived DCs. Also these results indicated that nicotine increased DCs’ cross presentation by regulating endosomal recruitment of TAP and cytosolic antigen translocation, which could provide fundamental theory support for DCs clinical use to deal with tumor and viral infection.