Nicotine induces TIPE2 upregulation and Stat3 phosphorylation contributes to cholinergic anti-inflammatory effect.

Cholinergic anti-inflammatory pathway has therapeutic effect on inflammation-associated diseases. However, the exact mechanism of nicotine-mediated anti-inflammatory effect is still unclear. TIPE2, a new member of tumor necrosis factor-α-induced protein-8 family, is a negative regulator of immune homeostasis. However, the roles of TIPE2 in cholinergic anti-inflammatory effect are still uncertain. Here, we demonstrated that nicotine exerts its anti-inflammatory effect by TIPE2 upregulation and phosphorylated stat3 mediated the inhibition of NF-κB activation, which was supported by the following evidence: firstly, both nicotine and TIPE2 inhibit pro-inflammatory cytokine release via NF-κB inactivation. Secondly, nicotine upregulates TIPE2 expression via α7 nicotinic acetylcholine receptor. Moreover, the enhancement of stat3 phosphorylation and decrease of LPS-induced p65 translocation were achieved by nicotine treatment. Importantly, nicotine treatment augments the interaction of phosphorylated stat3 and p65, indicating that the inhibitory effect of nicotine on NF-κB activation was mediated with protein-protein interactions. Hence, this study revealed that TIPE2 upregulation and stat3 phosphorylation contribute to nicotine-mediated anti-inflammation effect, indicating that TIPE2 and stat3 might be potential molecules for dealing with inflammation-associated diseases.

Nicotine up-regulated 4-1BBL expression by activating Mek-PI3K pathway augments the efficacy of bone marrow-derived dendritic cell vaccination.

PURPOSE: To explore the role of 4-1BBL in nicotine-treated immature dendritic cells (imDCs) mediated anti-tumor effects. METHODS: Bone marrow-derived imDCs were stimulated with nicotine and 4-1BBL expression was determinated by flow cytometry, Western blot and RT-PCR respectively. Then, the roles of 4-1BBL in nicotine-augmented DCs-dependent T cell proliferation, CTL priming and anti-tumor effects were investigated by BrdU cell proliferation assay, enzyme-linked immunospot assay and in vivo preventive effect on tumor development, respectively. Finally, using relative kinase inhibitors, the mechanism of 4-1BBL up-regulation by nicotine stimulation and the roles of Mek-PI3K signal pathways in nicotine-augmented DCs-dependent T cell proliferation were explored by Western blot and BrdU cell proliferation assay, respectively. RESULTS: Firstly, nicotine could up-regulate 4-1BBL expression in both protein and mRNA levels. Secondly, the effects of nicotine-augmented DCs-dependent T-cell proliferation, CTL priming and anti-tumor effects could be significantly abolished by blocking CD80, CD86 and 4-1BBL activity, respectively. Thirdly, the combined blockages of CD80/CD86, CD80/4-1BBL, CD86/4-1BBL or CD80/CD86/4-1BBL signals could decrease 53.2 %, 29.6 %, 27.9 % and 54.5 % nicotine-enhanced T cell proliferation, respectively. Importantly, nicotine-induced 4-1BBL up-regulation could be decreased by the usage of Mek-PI3K pathway kinase inhibitors. The pre-treatment of Mek-p38-PI3K kinase inhibitors could obviously abolish nicotine-augmented DCs-dependent T cell proliferation. CONCLUSIONS: CD80/CD86 and 4-1BBL are critical for nicotine augmented DCs-mediated anti-tumor effects. 4-1BBL and CD80/CD86 could be considered as potential candidates for preventive and therapeutic tumor vaccination.

TGF-ß of lung cancer microenvironment upregulates B7H1 and GITRL expression in dendritic cells and is associated with regulatory T cell generation.

The effects of TGF-ß on dendritic cells (DCs) on the tumor microenvironment are not well understood. We report, here, the establishment of an in vitro lung cancer microenvironment by co-incubation of seminaphtharhodafluor (SNARF) labeled Lewis lung cancer (LLC) cells, carboxyfluorescein succinimidyl ester (CFSE) labeled fibroblasts and 4-chloromethyl-7-hydroxycoumarin (CMHC) labeled DCs. Raw 264.7, EL4 and NCI-H446 cells were able to synthesize TGF-ß which was determined by flow cyto-metry and western blotting, respectively. Furthermore, TGF-ß efficiently increased regulatory T-cell (Treg) expansion and upregulated DC B7H1 and GITRL expression. TGF-ß and the co-incubation of LLC cells, fibroblasts with DCs could augment the expression of B7H1 and GITRL molecules of DCs. The data presented here indicate that the B7H1 and GITRL molecules may play an important role in TGF-ß-induced Treg expansion of lung cancer microenvironment.

Nicotine stimulated bone marrow-derived dendritic cells could augment HBV specific CTL priming by activating PI3K-Akt pathway.

Our previous studies have revealed that nicotine-treated immature dendritic cells (imDCs) have anti-tumor effects in murine lymphoma models. The present study is to explore HBV-specific CTL priming and its cytolytic activities of nicotine-treated murine DCs, the mechanism of α7 nicotinic acetylcholine receptor (nAChR) up-regulation by nicotine and the efficiency of nicotine with other cytokines. To address these hypotheses, bone marrow-derived imDCs were stimulated by nicotine and expression of α7 nAChR was firstly determined by flow cytometry and Western blot. Then, DCs-dependent HBV-specific T cell proliferation and IL-12 secretion were secondly determined by BrdU cell proliferation assay and ELISA, respectively. The HBV-specific CTL priming and its activities were further explored by intraperitoneal transfer of nicotine treated imDCs. The mechanism of nicotine up-regulating α7 nAChR was finally explored by Western blot. The results showed that: first, the maximal activation of PI3K and Akt was reached at 30 and 60-120 min respectively after nicotine stimulation. Nicotine up-regulated the expression of α7 nAChR by activating PI3K-Akt pathway in murine DCs; secondly, nicotine stimulation could enhance DCs' ability of HBV-specific T cell proliferation and IL-12 secretion; thirdly, adoptive transfer of nicotine stimulated DCs could induce HBV specific CTL priming in vivo and those CTL had cytolytic activities; fourthly, nicotine had equal efficiencies to 2 ng/ml IFN-γ in DCs-mediated T cell proliferation. All these data presented here indicated that nicotine treated imDCs might be considered as a potential candidate for HBV immunotherapy.

Lipopolysaccharide and dose of nicotine determine the effects of nicotine on murine bone marrow-derived dendritic cells.

The reported effects of nicotine on dendritic cells (DCs) are controversial. To investigate the factors which determine the effects of nicotine on DCs, immature dendritic cells (imDCs) induced from murine bone marrow were treated with different doses of nicotine with or without lipopolysaccharides (LPS). The morphology and expression of the co-stimulatory molecules CD80, CD86, CD40 and CD54 were observed and determined by microscopy and flow cytometry, respectively. The results showed that, firstly, nicotine treatment promoted the development of DC precursors into imDCs with a semi-mature phenotype revealed by a higher expression of CD11c and more branched projections. Secondly, lower doses of nicotine (16.5 ng/ml), but not higher (200 µg/ml), up-regulated the expression of the co-stimulatory molecules CD80, CD40 and CD54 on imDCs. Co-administration of LPS and nicotine revealed differential effects on co-stimulatory molecule expression on imDCs. Thirdly and importantly, treatment with lower doses of nicotine (16.5 ng/ml) did not augment expression of the CD80, CD86, CD40 and CD54 molecules in mature DCs. Fourthly and interestingly, high doses of nicotine (more than 165 µg/ml) revealed pro-apoptotic activity but lower doses of nicotine (16.5-0.165 ng/ml) achieved an anti-apoptotic effect on imDCs. All data presented here indicate that the controversial effects of nicotine on DCs may be due to the LPS of the nicotinic environment and the dose of nicotine used.