Pharmacological Evaluation of Sirtuin 1 Inhibition and Activation in U937 Cells and in Primary Human Monocyte Derived Macrophages

A Hodzic, P Bahra, E Hardaker. Novartis, Horsham, West Sussex, UK

Macrophages are one of the most predominant inflammatory cell types involved in chronic inflammatory diseases such as COPD. Activation of lung macrophages leads to increased expression of pro-inflammatory cytokines (e.g. IL-8) and matrix metalloproteinases (e.g. MMP-9). Sirtuin1 (Sirt-1) is a NAD+-dependent protein deacetylase involved in negatively regulating transcription factors such as NF-ϰB. It has been shown that oxidative stress can decrease levels of Sirt-1 in macrophages resulting in increased pro-inflammatory cytokine release (Yang et.al., 2007).The aim of this study was to pharmacologically evaluate and compare the effect of a Sirt-1 activator (SRT2172; Nakamaru et.al.,2009) and a Sirt-1 inhibitor (EX527; Solomon et.al., 2006) on phorbol ester (PMA) stimulated IL-8 release and MMP-9 expression in a human macrophage cell line (U937) and PMA-stimulated IL-8 release from human monocyte derived macrophages (MDMs).

Human blood monocytes were isolated using monocyte isolation kit II (Miltenyi Biotech). MDMs were generated over 12 day culture of monocytes in RPMI 1640, 10% FBS, 2mM L-glutamine + 2ng/ml granulocyte macrophage colony stimulation factor (GMCSF). U937 cells were cultured in RPMI 1640, 10% FBS, 2mM L-glutamine. MDMs were pre-treated for 30 min in 96 well plates (1x105 cells/well) with the Sirt-1 activator and inhibitor followed by stimulation with PMA (50ng/ml, 24h). U937 were pre-treated in 48 well plates (2x105cells/well) followed by stimulation with PMA (50ng/ml, 48h). Cell-free supernatants were collected and assayed for IL-8 release (ELISA, R&D Systems) and MMP-9 protein levels (Amersham Biotrak). Total RNA (RNasey kit; Qiagen) was prepared for real-time MMP-9 gene analysis (Applied Biosystems). Data are expressed as mean ±standard error of the mean (sem). Statistical significance was assumed when P<0.05 using a one –way ANOVA with Dunnet’s multiple comparisons test.

Treatment of U937 cells with the Sirt-1 activator (SRT2172) attenuated PMA induced IL-8 release (Max inhibition 99.1±1.1% at 30µM, P<0.05; n=3), whereas the Sirt-1 inhibitor (EX527) concentration dependently augmented PMA-induced IL-8 release (max increase of 270±42.9% above PMA control at 30µM, P<0.05; n=3). PMA-induced MMP-9 gene expression in U937 cells was reduced by SRT2172 (≥3µM) treatment whilst total PMA stimulated MMP-9 protein levels were reduced back to baseline (P<0.05; n=3). In contrast, the Sirt-1 inhibitor had no effect on MMP-9 gene expression. In MDMs, PMA-induced IL-8 release was concentration dependently reduced by the Sirt-1 activator (84.9±4.6% max inhibition at 30µM, P<0.05; n=4) whereas the Sirt-1 inhibitor augmented IL-8 release from ≥10µM to a max of 370±11.0% (30uM) above PMA control (P<0.05; n=3). These data demonstrate the role of Sirt-1 in modulating pro-inflammatory mediator release from PMA-stimulated U937 cells and MDMs. Activators of Sirt-1 may have therapeutic potential in chronic inflammatory diseases such as COPD where macrophages play a pivotal role.

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