Tania O. Crișan1, Maartje C.P. Cleophas1, Kathrin Buffen1, Tim L. Jansen2, Charles Dinarello1,3, Mihai G. Netea1, Leo A.B. Joosten1
1Department of Internal Medicine, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands 2Department of Rheumatology, VieCuri Med Centre, Venlo, The Netherlands 3Division of Infectious Diseases, University of Colorado School of Medicine, Aurora, Colorado, USA.
Background: In gout, it has become apparent that metabolic triggers are important players in the degree of inflammation in gout. The high serum urate levels observed in patients with gout, not only predispose to MSU crystallization but also can prime the inflammatory responses in relation to gout-related stimuli. In this study we investigate the mechanisms of soluble urate-induced priming of monocytes and the shift in cytokine production consisting in high IL-1beta but low IL-1 receptor antagonist (Ra) specific to uric acid.
Methods: PBMCs or monocytes from healthy volunteers were pretreated with uric acid for 24h and then subjected to 24h stimulation with TLR2 or TLR4 ligands in the presence or absence of MSU. Cytokine production was assessed using specific sandwich ELISA kits. Samples were lysed and protein profiler (phosphokinase) was used or western blot for AMPK and Akt was performed. Autophagic activity was assessed via western blot for LC3 in human macrophages and fluorescence microscopy of LC3-GFP expressing HeLa cells.
Results: Increased phosphorylation of Akt and PRAS 40 (proline rich Akt substrate 40 kDa) was observed upon uric acid treatment while AMPK phosphorylation was not affected. Western blot for LC3-I and LC3-II fractions as well as fluorescence microscopy revealed lower autophagic activity in cells exposed to uric acid compared to control conditions.
Conclusions: In this study we investigate the mechanism through which high uric acid concentrations modify inflammatory responses by enhancing IL-1 beta transcription and decreasing IL-1Ra. We show that the Akt-PRAS40-mTOR pathway is activated in response to uric acid. As a consequence, autophagy activity is diminished and this can, at least in part, explain the induction of IL-beta. Other processes downstream Akt and PRAS40 phosphorylation are under investigation to demonstrate the link with the specific cytokine profile determined by uric acid in human cells. Key words Uric acid, IL-1beta, Akt, autophagy.