Lycopene Inhibits NF-kB-Mediated IL-8 Expression and Changes Redox and PPARγ Signalling in Cigarette Smoke–Stimulated Macrophages

Increasing evidence suggests that lycopene, the major carotenoid present in tomato, may be preventive against smoke-induced cell damage. However, the mechanisms of such a prevention are still unclear. The aim of this study was to investigate the role of lycopene on the production of the pro-inflammatory cytokine IL-8 induced by cigarette smoke and the possible mechanisms implicated. Therefore, human THP-1 macrophages were exposed to cigarette smoke extract (CSE), alone and following a 6-h pre-treatment with lycopene (0.5–2 µM). CSE enhanced IL-8 production in a time- and a dose-dependent manner. Lycopene pre-treatment resulted in a significant inhibition of CSE-induced IL-8 expression at both mRNA and protein levels. NF-kB controlled the transcription of IL-8 induced by CSE, since PDTC prevented such a production. Lycopene suppressed CSE-induced NF-kB DNA binding, NF-kB/p65 nuclear translocation and phosphorylation of IKKα and IkBα. Such an inhibition was accompanied by a decrease in CSE-induced ROS production and NOX-4 expression. Lycopene further inhibited CSE-induced phosphorylation of the redox-sensitive ERK1/2, JNK and p38 MAPKs. Moreover, the carotenoid increased PPARγ levels which, in turn, enhanced PTEN expression and decreased pAKT levels in CSE-exposed cells. Such effects were abolished by the PPARγ inhibitor GW9662. Taken together, our data indicate that lycopene prevented CSE-induced IL-8 production through a mechanism involving an inactivation of NF-kB. NF-kB inactivation was accompanied by an inhibition of redox signalling and an activation of PPARγ signalling. The ability of lycopene in inhibiting IL-8 production, NF-kB/p65 nuclear translocation, and redox signalling and in increasing PPARγ expression was also found in isolated rat alveolar macrophages exposed to CSE. These findings provide novel data on new molecular mechanisms by which lycopene regulates cigarette smoke-driven inflammation in human macrophages

Correction: Lycopene Inhibits NF-kB-Mediated IL-8 Expression and Changes Redox and PPARγ Signalling in Cigarette Smoke-Stimulated Macrophages.

[This corrects the article DOI: 10.1371/journal.pone.0019652.]

Effects of lycopene, alone and in combination with cigarette smoke extract (CSE), on intracellular IL-8 mRNA levels, NF-kB/p65 nuclear translocation, ROS production and PPARγ expression in isolated rat alveolar macrophages (AMs).

<p>Panel A: IL-8 levels by reverse transcription polymerase chain reaction in cells treated for 6 h with lycopene (2 µM) followed by a 24-h CSE (0.5%) exposure. Panel B: representative western blot analysis of nuclear NF-kB (p65)-associated proteins in cells pre-treated for 6 h with lycopene (2 µM) and, then exposed to CSE (0.5%) for 3 h. Panel C: ROS levels in cells pre-treated for 6 h with lycopene (2 µM) and, then exposed to CSE (0.5%) for 3 h. Panel D: PPARγ: representative western blot analysis of PPARγ protein in cells pre-treated for 6 h with lycopene (2 µM) and, then exposed to CSE (0.5%) for 3 h. In panels A and C, values were the means ± SEM of three experiments. Values not sharing the same letter were significantly different (P<0.05, Fisher's test).</p

Effects of lycopene on CSE-stimulated NF-kB activation in human THP-1 cells.

<p>Panel A: IL-8 production in culture medium after addition of the NF-kB inhibitor pyrrolidine dithiocarbamate (PDTC). THP-1 cells were pre-incubated with PDTC (10 µM) for 30 min and, then treated with CSE was added for 3 h and 24 h. Panel B: NF-kB DNA binding activity in cells pre-treated for 6 h with lycopene (2 µM) and, then exposed to CSE (0.5%) for 3 h; the specifity was demonstrated by using excess unlabeled NF-kB oligonucleotides ( = cold ssNF-kB) which competed away binding. Panel C: representative western blot analysis of nuclear NF-kB (p65)-associated proteins in cells pre-treated for 6 h with lycopene (2 µM) and, then exposed to CSE (0.5%) for 3 h; panel D: representative western blot analyses of cytosolic p-IKKα and p-IkBα in cells pre-treated for 6 h with lycopene (2 µM) and, then exposed to CSE (0.5%) for 3 h; the values indicated represented the ratio protein/actin. In panel A, values were the means ± SEM of three experiments. Values not sharing the same letter were significantly different (P<0.05, Fisher's test).</p

Effects of lycopene on CSE-induced reactive oxygen species (ROS) production in human THP-1 cells.

<p>Panel A: ROS levels after addition of the NAD(P)H oxidase-4 (NOX-4) inhibitor diphenyleneiodonium (DPI) in cells exposed to CSE for 3 h and 24 h. Cells were pre-incubated with DPI at the concentration of 10 µM for 1 h and then washed prior to the addition of CSE. Panel B: ROS levels in cells pre-treated for 6 h with lycopene (2 µM) and, then exposed to CSE (0.5%) for 3 h and 24 h. Panel C: representative western blot analysis of NOX-4 in cells pre-treated for 6 h with lycopene (2 µM) and, then exposed to CSE (0.5%) for 3 h and 24 h; the values indicated represented the ratio NOX-4/actin. In panels A and B, values were the means ± SEM of five experiments. Values not sharing the same letter were significantly different (P<0.005, Tukey's test).</p

Effects of lycopene on CSE-induced MAPK phosphorylation in human THP-1 cells.

<p>Representative western blot analyses of JNK, ERK1/2 and p38 in cells pre-treated for 6 h with lycopene (2 µM) and, then exposed to CSE (0.5%) for 3 h. The values indicated represented the ratio phosphorylated protein/total protein.</p

Effects of lycopene on CSE-inhibited PPARγ signalling in human THP-1 cells.

<p>Panel A: PPARγ mRNA and panel B: representative western blot analysis of PPARγ protein cells pre-treated for 6 h with lycopene (2 µM) and, then exposed to CSE (0.5%) for 3 h. Panel C: representative western blot analysis of PTEN in cells pre-treated for 6 h with lycopene (2 µM) and/or with the PPARγ inhibitor GW9662 and, then exposed to CSE (0.5%) for 24 h. Panel D: representative western blot analysis of AKT in cells pre-treated for 6 h with lycopene (2 µM) and/or with the PPARγ inhibitor GW9662 and, then exposed to CSE (0.5%) for 24 h. GW9662 was pre-incubated at the concentration of 10 µM. The values indicated represented the ratio protein/actin (PTEN) and phosphorylated protein/total protein (AKT). In panel A, values were the means ± SEM of three experiments. Values not sharing the same letter were significantly different (P<0.05, Fisher's test).</p