Ex VivoNicotine Stimulation Augments the Efficacy of Human Peripheral Blood Mononuclear Cell-Derived Dendritic Cell Vaccination via Activating Akt-S6 Pathway

Abstract

Our previous studies showed that α7 nicotinic acetylcholine receptor (nAchR) agonist nicotine has stimulatory effects on murine bone marrow-derived semimature DCs, but the effect of nicotine on peripheral blood mononuclear cell- (PBMC-) derived human semimature dendritic cells (hu-imDCs) is still to be clarified. In the present study, hu-imDCs (cultured 4 days) were conferred with ex vivo lower dose nicotine stimulation and the effect of nicotine on surface molecules expression, the ability of cross-presentation, DCs-mediated PBMC priming, and activated signaling pathways were determined. We could demonstrate that the treatment with nicotine resulted in increased surface molecules expression, enhanced hu-imDCs-mediated PBMC proliferation, upregulated release of IL-12 in the supernatant of cocultured DCs-PBMC, and augmented phosphorylation of Akt and ribosomal protein S6. Nicotine associated with traces of LPS efficiently enhanced endosomal translocation of internalized ovalbumin (OVA) and increased TAP-OVA colocalization. Importantly, the upregulation of nicotine-increased surface molecules upregulation was significantly abrogated by the inhibition of Akt kinase. These findings demonstrate that ex vivo nicotine stimulation augments hu-imDCs surface molecules expression via Akt-S6 pathway, combined with increased Ag-presentation result in augmented efficacy of DCs-mediated PBMC proliferation and Th1 polarization.

Increased translocation of antigens to endosomes and TLR4 mediated endosomal recruitment of TAP contribute to nicotine augmented cross-presentation

“…Our previous studies showed that nicotine treatment promotes semi-mature DCs cross-priming [7–11]. The mechanism of antigen uptake determined the entrance of antigens into a specific intracellular pathway, which is required for efficient cross-presentation [24].…”

“…MR internalized OVA target into early-endosomes, inhibit endosomes’ mature into lysosomes [15]. The treatment with nicotine not only induced the activation of Erk and p38 (Supplementary Figure S1) but also promoted the phosphorylation of PI3K and increased co-stimulatory molecules expression [10–11]. Interestingly, the downstream kinases of PI3K, such as Akt, mTOR and p70S6, were efficiently activated with nicotine (Supplementary Figure S2).…”

“…Bone marrow-derived DCs were generated by culturing progenitors for four days [11] in RPMI 1640 medium supplemented with 10 ng/ml GM-CSF, 1 ng/ml IL-4, and 10% FBS. Non-adherent cells were gently washed out with PBS on day 4 of culture; the remaining loosely adherent clusters were used as semi-matured DCs.…”

“…Nonneuronal cells such as DCs, epithelial cells and endothelial cells express nicotinic acetylcholine receptor (nAChR) [4]. Despite that nAChR activation promoted tumor metastasis and increased overall mortality [5–6], nicotine up-regulate surface molecule expressiones, augment DCs-dependent T cell activation on murine and human semi-mature DCs [7–11]. As model antigen ovalbumin (OVA) or tumor lysates do not originate from DCs, nicotine increasing DCs-dependent CTL priming indicates that α7 nAChR activation has positive effect on DCs cross-presentation, which occurs via vacuolar or endosome-to-cytosol pathway [12].…”

“…In the endosome-to-cytosol pathway, receptors such as mannose receptor (MR) mediate antigen uptake, recruit internalized antigens toward endosomes [14–16]. Despite that the activation of α7 nAChR increases antigen internalization and promotes cross-presentation [7–11], the exact effect of α7 nAChR activation on MR expression and the mechanism of nicotine-increased cross-presentation are still uncertain.…”