Effect of cigarette smoke on mucosal vaccine response with activation of plasmacytoid dendritic cells: The outcomes of in vivo and in vitro experiments.

Mucosal vaccines offer several advantages over transdermal vaccines, including the ability to acquire systemic and mucosal immunities. Smoking is a huge public health threat and major risk factor for various diseases that exacerbate or prolong respiratory symptoms and conditions. However, its impact on the efficacy of mucosal vaccines remains partially explored. Thus, this study investigates the effects of smoking on mucosal vaccine reactivity by assessing the induction of Th1 immunity, a vital response in infection defense. Cigarette smoke condensate was prepared as a substitute for mainstream smoke. We intranasally administered diphtheria toxoid as an antigen and natural CpG oligonucleotide G9.1, which enhances the Th1-type antibody (Ab) response in a plasmacytoid dendritic cells (pDCs) dependent manner, as an adjuvant to mice to assess the effect of cigarette smoke condensate on Ab responses. The mechanism of its effect was evaluated using human peripheral blood mononuclear cells and their pDC-rich fraction cultured with or without G9.1. In mice, cigarette smoke condensate tended to decrease diphtheria toxoid-specific Ab response, with a higher reduction in Th1-type IgG2 Ab response than in Th2-type IgG1 Ab response. In human peripheral blood mononuclear cells, cigarette smoke condensate significantly reduced the induction of IFN-α production by G9.1. Moreover, G9.1-induced increases in the CD83 expression in pDCs and the CD80 expression in DCs were suppressed via treatment with cigarette smoke condensate. Among the mechanisms suggested were decreased expression of toll-like receptor 9 mRNA, decreased expression of mRNA for IFN regulatory factor 7, and increased CpG methylation of its promoter region. The analysis of Tbet and GATA3 expressions revealed that cigarette smoke condensate exhibits Th1-directed immunostimulatory activity at a steady state but becomes more Th2-directed under G9.1 stimulation. In conclusion, smoking could reduce mucosal vaccine responses by decreasing pDC activation and, consequently, Th1-dominant immunity.

A palindromic CpG-containing phosphodiester oligodeoxynucleotide as a mucosal adjuvant stimulates plasmacytoid dendritic cell-mediated T(H)1 immunity.

BACKGROUND: CpG oligodeoxynucleotides (ODNs), resembling bacterial DNA, are currently tested in clinical trials as vaccine adjuvants. They have the nuclease-resistant phosphorothioate bond; the immune responses elicited differ according to the CpG ODN sequence and vaccination method. To develop a CpG ODN that can induce plasmacytoid dendritic cell (pDC)-mediated T(H)1 immunity through the mucosa, we constructed phosphodiester G9.1 comprising one palindromic CpG motif with unique polyguanosine-runs that allows degradation similar to naturally occurring bacterial DNA. METHODS: T(H)1 and T(H)2 immunity activation was evaluated by cytokine production pattern and T-bet/GATA-3 ratio in human peripheral blood mononuclear cells and mouse bone marrow cells. Adjuvanticity was evaluated in mice administered G9.1 with diphtheria toxoid (DT) through nasal vaccination. RESULTS: G9.1 exhibited stronger IFN-α-inducing activity than A-class CpG ODN2216 and increased T-bet/GATA-3 ratio by enhancing T-bet expression. Nasally administered G9.1 plus DT induced DT-specific mucosal IgA and serum IgG, but not IgE, responses with antitoxin activity in C57BL/6 and BALB/c mice, possibly due to IFN/BAFF production. Induction of T(H)1, but not T(H)2-type Abs depended completely on pDCs, the first in vivo demonstration by CpG ODNs. CONCLUSIONS: G9.1 is a promising mucosal adjuvant for induction of pDC-mediated T(H)1 immunity.

Glutathione S-transferase M1 inhibits dexamethasone-induced apoptosis in association with the suppression of Bim through dual mechanisms in a lymphoblastic leukemia cell line.

Glutathione S-transferase mu (GSTM1) is mainly known as a detoxification enzyme but it has also been shown to be a negative regulator of apoptosis-related signaling cascades. Recently GSTM1 has been reported to be a significant risk factor for hematological relapse in childhood acute lymphoblastic leukemia, although the underlying mechanism remains largely unknown. Glucocorticoids play a crucial role in the treatment of childhood acute lymphoblastic leukemia, therefore we hypothesized that GSTM1 plays important roles in glucocorticoid-induced apoptotic pathways. To clarify the relationship between GSTM1 and drug resistance, GSTM1 was transfected into a T-acute lymphoblastic leukemia cell line, CCRF-CEM (CEM), and we established the GSTM1-expressing cell lines CEM/M1-4 and CEM/M1-9. Transduction of GSTM1 into CEM selectively decreased cellular sensitivity to dexamethasone in a manner that was independent of glutathione conjugation, but was due to apoptosis inhibition. Dexamethasone-induced p38-MAPK and Bim activation were concomitantly suppressed. Interestingly, nuclear factor kappa b (NF-kappaB) p50 activity was upregulated in GSTM1-expressing CEM. Inhibition of NF-kappaB by the pharmacological agent BAY11-7082 greatly enhanced the sensitivity of the GSTM1-expressing CEM to dexamethasone and was accompanied by an increase in Bim expression. Thus, we propose that GSTM1, a novel regulator of dexamethasone-induced apoptosis, causes dexamethasone resistance by suppression of Bim through dual mechanisms of both downregulation of p38-MAPK and upregulation of NF-kappaB p50.

Nicotine induces human neutrophils to produce IL-8 through the generation of peroxynitrite and subsequent activation of NF-kappaB.

Leukocytosis in tobacco smokers has been well recognized; however, the exact cause has not been elucidated. To test the hypothesis that tobacco nicotine stimulates neutrophils in the respiratory tract to produce IL-8, which causes neutrophilia in vivo, we examined whether nicotine induces neutrophil-IL-8 production in vitro; the causative role of NF-kappaB in its production, in association with the possible production of reactive oxygen intermediates that activate NF-kappaB; and the nicotinic acetylcholine receptors (nAChRs) involved in IL-8 production. Nicotine stimulated neutrophils to produce IL-8 in both time- and concentration-dependent manners with a 50% effective concentration of 1.89 mM. A degradation of IkappaB-alpha/beta proteins and an activity of NF-kappaB p65 and p50 were enhanced following nicotine treatment. The synthesis of superoxide and the oxidation of dihydrorhodamine 123 (DHR) were also enhanced. The NOS inhibitor, nomega-Nitro-l-arginine methyl ester, prevented nicotine-induced IL-8 production, with an entire abrogation of DHR oxidation, IkappaB degradation, and NF-kappaB activity. Neutrophils spontaneously produced NO whose production was not increased, but rather decreased by nicotine stimulation, suggesting that superoxide, produced by nicotine, generates peroxynitrite by reacting with preformed NO, which enhances the NF-kappaB activity, thereby producing IL-8. The nAChRs seemed to be involved in IL-8 production. In smokers, blood IL-8 levels were significantly higher than those in nonsmokers. In conclusion, nicotine stimulates neutrophil-IL-8 production via nAChR by generating peroxynitrite and subsequent NF-kappaB activation, and the IL-8 appears to contribute to leukocytosis in tobacco smokers.

Discovery of immunostimulatory CpG-DNA and its application to tuberculosis vaccine development.

DNA containing an unmethylated CpG motif has a potent immunostimulatory effect on the vertebrate immune system. Because such CpG motifs are relatively common in bacterial DNA, but rare in mammalian animal and plant DNA, they may be an evolutionary adaptation augmenting innate immunity, most likely in response to pathogens that replicate within the host cells, such as viruses and intracellular bacteria. Microbial infection induces innate immunity by triggering pattern-recognition systems. The infected cells produce proinflammatory cytokines that directly combat microbial invaders and express costimulating surface molecules, which develop adaptive immunity by inducing distinct T cell differentiation. Bacterial DNA with unmethylated CpG-DNA stimulates vertebrate immature immune cells to induce maturation and to produce TNF-alpha as well as Th1-type cytokines, IL-12 and IFN-gamma. Therefore, CpG-DNA functions as an adjuvant for regulating the initiation of Th1 differentiation. The roles of immunostimulatory CpG motifs in DNA vaccine developments and in therapeutic applications have been discussed.