OP0303 MONOSODIUM URATE AND CALCIUM PYROPHOSPHATE CRYSTAL-INDUCED INTERLEUKIN 1 PRODUCTION DEPENDS ON GLUCOSE UPTAKE THROUGH GLUT1 TRANSPORTER
Authors: Félix Renaudin, Laure Campillo-Gimenez, Florence Castelli, François Fenaille, Aurélie Prignon, Christèle Combes, Martine Cohen Solal, Frederic Lioté, Hang Korng Ea
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background</h3> Monosodium urate (MSU) and monoclinic calcium pyrophosphate dihydrated (mCPPD)crystals are responsible for relapsing acute arthritis which is driven by interleukin 1β (IL-1β).IL-1β production relies on NLRP3 inflammasome activation leading to ASC and caspase-1 recruitment. In tumor cells and in LPS-stimulated macrophages a switch of cell metabolisms toward glycolysis favors IL-1β production. <h3>Objectives</h3> The aims of this study were to assess 1/whether crystal-induced NLRP3 activation and IL-1β production involved glucose metabolism and 2/how MSU and mCPPD crystals induced glucose uptake focusing on the role of glucose transporter Glut1. <h3>Methods</h3> Synthetic and pyrogen-free MSU and mCPPD crystals were used to stimulate THP-1 cells and mouse bone marrow-derived macrophages (BMDM). Cells were stimulated in presence or absence of glucose (2g/L) and pyruvate (10mM). Glycolysis was inhibited by 2-deoxy-glucose (2DG) and the role of glucose transporter 1 (Glut1) was assessed by pharmacological inhibitor (STF-31) and siRNA. IL-1β production was quantified by ELISA.Metabolomic analysis was performed by mass spectrometry. Glucose uptake was determined using <sup>18</sup>F-DG (Fluor18 labeled-2DG) and positron emission tomography (PET). Glut1 membrane localization was assessed by flux cytometry and confocal microscopy, ASC speck formation by confocal microscopy. <i>In vivo</i>, we used murine air pouch model to assess the effects of 2DG and Glut1 inhibition in crystal-mediated inflammation.Glut1 membrane localization of circulating neutrophils was compared to synovial fluid neutrophils harvested during gout flare. <h3>Results</h3> <i>In vitro</i>, both MSU and mCPPD crystal-induced IL-1β secretion and ASC speck formation were inhibited when cells were cultured in glucose-free medium or in presence of 2DG.Similarly,MSU and mCPPD crystal-induced inflammation was abrogated in mice treated with 2DG.In THP-1 cells stimulated by MSU and mCPPD crystals, metabolomic analysis displayed alteration of glycolysis pathway and Krebs cycle and decrease of intracellular ATP production. Interestingly, MSU and mCPPD crystals increased glucose uptake<i>in vitro, ex vivo and in vivo</i>. Crystal-induced IL-β secretion was decreased by STF-31 and in THP-1 cells transfected with Glut1 siRNA. Next, we observed that MSU and mCPPD crystals increased membrane localization of Glut1 in THP-1, BMDM and neutrophils infiltrated in air pouch lavages and membranes. Glut1 membrane localization was positively correlated with IL-1β production. Moreover, during gout flare, the proportion of Glut1 positive neutrophils was higher in synovial fluid neutrophils than in circulating neutrophils. Finally, STF-31 treatment decreased, <i>in vitro,</i> glucose uptake induced by MSU and mCPPD crystals, and <i>in vivo</i> MSU and mCPPD crystal-induced inflammation. <h3>Conclusion</h3> Glucose uptake through Glut1 transporter enhanced IL-1β production induced by MSU and mCPPD crystals. Studies to decipher how these crystals induced Glut1 membrane localization are ongoing. Similarly, we investigate how glycolysis regulates NLRP3 and ASC activation. Decreasing Glut1 membrane localization might be a potential target to dampen crystal-induced inflammation. <h3>References</h3> <b>Disclosure of Interests:</b> Félix Renaudin: None declared, Laure Campillo-Gimenez: None declared, Florence Castelli: None declared, François Fenaille: None declared, Aurélie Prignon: None declared, Christèle Combes: None declared, martine Cohen Solal Speakers bureau: Amgen and Lilly, Frederic Lioté Grant/research support from: institutional grants from Grunenthal, Ipsen Pharma/Menarini, Novartis, SOBI for the European Crystal Network Workshops, Consultant for: Grunenthal, Novartis, Hang Korng Ea: None declared