ObjectiveMacrophage activation by monosodium urate (MSU) and calcium pyrophosphate (CPP) crystals mediates an interleukin (IL)-1β-dependent inflammation during gout and pseudo-gout flare, respectively. Since metabolic reprogramming of macrophages goes along with inflammatory responses dependently on stimuli and tissue environment, we aimed to decipher the role of glycolysis and oxidative phosphorylation in the IL-1β-induced microcrystal response.MethodsBriefly, an in vitro study (metabolomics and real-time extracellular flux analysis) on MSU and CPP crystal-stimulated macrophages was performed to demonstrate the metabolic phenotype of macrophages. Then, the role of aerobic glycolysis in IL-1β production was evaluated, as well in vitro as in vivo using 18F-fluorodeoxyglucose positron emission tomography imaging and glucose uptake assay, and molecular approach of glucose transporter 1 (GLUT1) inhibition.ResultsWe observed that MSU and CPP crystals led to a metabolic rewiring toward the aerobic glycolysis pathway explained by an increase in GLUT1 plasma membrane expression and glucose uptake on macrophages. Also, neutrophils isolated from human synovial fluid during gout flare expressed GLUT1 at their plasma membrane more frequently than neutrophils isolated from bloodstream. Both glucose deprivation and treatment with either 2-deoxyglucose or GLUT1 inhibitor suppressed crystal-induced NLRP3 activation and IL-1β production, and microcrystal inflammation in vivo.ConclusionIn conclusion, we demonstrated that GLUT1-mediated glucose uptake is instrumental during the inflammatory IL-1β response induced by MSU and CPP crystals. These findings open new therapeutic paths to modulate crystal-related inflammation.

Background: Calcium pyrophosphate (CPP) microcrystal deposition is associated with wide clinical phenotypes, including acute and chronic arthritis, that are interleukin 1β (IL-1β)-driven. Two CPP microcrystals, namely monoclinic and triclinic CPP dihydrates (m- and t-CPPD), have been identified in human tissues in different proportions according to clinical features. m-CPP tetrahydrate beta (m-CPPTβ) and amorphous CPP (a-CPP) phases are considered as m- and t-CPPD crystal precursors in vitro.Objectives: We aimed to decipher the inflammatory properties of the three crystalline phases and one amorphous CPP phase and the intracellular pathways involved.Methods: The four synthesized CPP phases and monosodium urate crystals (MSU, as a control) were used in vitro to stimulate the human monocytic leukemia THP-1 cell line or bone marrow-derived macrophages (BMDM) isolated from WT or NLRP3 KO mice. The gene expression of pro- and anti-inflammatory cytokines was evaluated by quantitative PCR; IL-1β, IL-6 and IL-8 production by ELISA; and mitogen-activated protein kinase (MAPK) activation by immunoblot analysis. NF-κB activation was determined in THP-1 cells containing a reporter plasmid. In vivo, the inflammatory potential of CPP phases was assessed with the murine air pouch model via cell analysis and production of IL-1β and CXCL1 in the exudate. The role of NF-κB was determined by a pharmacological approach, both in vivo and in vitro.Results: In vitro, IL-1β production induced by m- and t-CPPD and m-CPPTβ crystals was NLRP3 inflammasome dependent. m-CPPD crystals were the most inflammatory by inducing a faster and higher production and gene expression of IL-1β, IL-6, and IL-8 than t-CPPD, m-CPPTβ and MSU crystals. The a-CPP phase did not show an inflammatory property. Accordingly, m-CPPD crystals led to stronger activation of NF-κB, p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) MAPKs. Inhibition of NF-κB completely abrogated IL-1β and IL-8 synthesis and secretion induced by all CPP crystals. Also, inhibition of JNK and ERK1/2 MAPKs decreased both IL-1β secretion and NF-κB activation induced by CPP crystals. In vivo, IL-1β and CXCL1 production and neutrophil infiltration induced by m-CPPD crystals were greatly decreased by NF-κB inhibitor treatment.Conclusion: Our results suggest that the inflammatory potential of different CPP crystals relies on their ability to activate the MAPK-dependent NF-κB pathway. Studies are ongoing to investigate the underlying mechanisms.

The biological effects and cellular activations triggered by monosodium urate (MSU) and calcium pyrophosphate dihydrate (monoclinic: m-CPPD) crystals might be modulated by protein coating on the crystal surface. This study is aimed at: (i) Identifying proteins adsorbed on m-CPPD crystals, and the underlying mechanisms of protein adsorption, and (ii) to understand how protein coating did modulate the inflammatory properties of m-CPPD crystals. The effects of protein coating were assessed in vitro using primary macrophages and THP1 monocytes. Physico-chemical studies on the adsorption of bovine serum albumin (BSA) upon m-CPPD crystals were performed. Adsorption of serum proteins, and BSA on MSU, as well as upon m-CPPD crystals, inhibited their capacity to induce interleukin-1-β secretions, along with a decreased ATP secretion, and a disturbance of mitochondrial membrane depolarization, suggesting an alteration of NLRP3 inflammasome activation. Proteomic analysis identified numerous m-CPPD-associated proteins including hemoglobin, complement, albumin, apolipoproteins and coagulation factors. BSA adsorption on m-CPPD crystals followed a Langmuir-Freundlich isotherm, suggesting that it could modulate m-CPPD crystal-induced cell responses through crystal/cell-membrane interaction. BSA is adsorbed on m-CPPD crystals with weak interactions, confirmed by the preliminary AFM study, but strong interactions of BSA molecules with each other occurred favoring crystal agglomeration, which might contribute to a decrease in the inflammatory properties of m-CPPD crystals. These findings give new insights into the pathogenesis of crystal-related rheumatic diseases and subsequently may open the way for new therapeutic approaches.

Mechanical overload and aging are the main risk factors of osteoarthritis (OA). Galectin 3 (GAL3) is important in the formation of primary cilia, organelles that are able to sense mechanical stress. The objectives were to evaluate the role of GAL3 in chondrocyte primary cilium formation and in OA in mice. Chondrocyte primary cilium was detected in vitro by confocal microscopy. OA was induced by aging and partial meniscectomy of wild-type (WT) and Gal3-null 129SvEV mice (Gal3−/−). Primary chondrocytes were isolated from joints of new-born mice. Chondrocyte apoptosis was assessed by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), caspase 3 activity and cytochrome c release. Gene expression was assessed by qRT-PCR. GAL3 was localized at the basal body of the chondrocyte primary cilium. Primary cilia of Gal3−/− chondrocytes were frequently abnormal and misshapen. Deletion of Gal3 triggered premature OA during aging and exacerbated joint instability-induced OA. In both aging and surgery-induced OA cartilage, levels of chondrocyte catabolism and hypertrophy markers and apoptosis were more severe in Gal3−/− than WT samples. In vitro, Gal3 knockout favored chondrocyte apoptosis via the mitochondrial pathway. GAL3 is a key regulator of cartilage homeostasis and chondrocyte primary cilium formation in mice. Gal3 deletion promotes OA development.

Adenine phosphoribosyltransferase (APRT) deficiency is a genetic disease characterized by an increased production of 2,8 dihydroxyadenine (2,8-DHA) precipitating in urine, leading to a crystalline nephropathy and end-stage renal disease. Here, we describe the high prevalence of granuloma (88%) in biopsies from patients with APRT deficiency. A murine model of 2,8-DHA nephropathy was generated, showing that anakinra or dexamethasone, combined with allopurinol, improved renal function to a larger extent than allopurinol alone, the standard therapy. Inflammation plays a critical role in the development of 2,8-DHA nephropathy, and therapy based upon drugs targeting innate immunity could improve renal function recovery.

BackgroundOsteoarthritis (OA) is the most common adult rheumatic disease characterized by cartilage degradation, synovial inflammation and subchondral bone remodeling. Risk factors associated with OA are age, genetics, mechanical overload and presence of calcium-containing microcrystals. Calcium pyrophosphate crystals including monoclinic and triclinic dihydrate phases (m- and t-CPPD) are found in 40% of end-stage OA patients. Frequently asymptomatic, it can give rise to synovitis contributing to OA lesion worsening. CPP crystal-induced inflammation is orchestrated mainly by interleukin (IL)-1β. Secretion of IL-1β required a cytokine maturation process which depends on the NLRP3 inflammasome activation. This intracellular multiprotein complex can be stimulated by ATP-dependent potassium (K+) efflux, reactive oxygen species (ROS) generation, lysosomal or mitochondrial alterations.ObjectivesThe objectives were to identify intracellular pathways induced by CPPD crystals and leading to NLRP3 activation and IL-1β production.MethodsThe effects of CPPD crystals were assessed in human THP-1 cell line and bone marrow-derived macrophages (BMDM) from wild type (wt), NLRP3 (nlrp3–/–) or P2X7 receptor (p2x7–/–) knock out mice. Cells were primed before stimulation with synthetic m- and t-CPPD crystals in presence or absence of K+-enriched media (KCl 50mM – to block K+ efflux), N-acetyl-L-cystein (NAC 50mM – an intracellular ROS scavenger) or oxidized ATP (oxATP 200μM – an antagonist of ATP). NLRP3 expression was determined by western blotting, IL-1β and extracellular ATP (ATPe) concentrations were measured in cell culture supernatants whereas ROS production and mitochondrial membrane potential were evaluated using fluorescent probes (DFDA and JC-1, respectively).ResultsFirst, IL-1β production induced by CPPD crystals was lacking in nlrp3–/– BMDM but important in wt BMDM. Second, we showed that m-CPPD crystals induced a higher NLRP3 expression and IL-1β production than t-CPPD suggesting a differential modulation of NLRP3 activation. We observed that CPPD-induced IL-1β secretion was completely abrogated when K+ efflux or intracellular ROS were inhibited. Moreover, we demonstrated a stronger decrease in mitochondrial membrane potential following m-CPPD than t-CPPD crystal stimulation, combined with a de novo ROS generation. These two latter effects were inhibited when K+ efflux was blocked. Finally, we found that m- and t-CPPD crystals differentially brought on an ATP release and that IL-1β production was partially inhibited by oxATP. However, although ATPe can trigger K+ efflux through P2X7 receptor opening, crystal-mediated IL-1β production was similar between wt and p2x7r–/– BMDM.ConclusionsIL-1β production triggered by m- and t-CPPD crystals occurred through a modulation of ROS production and mitochondrial disruption. Interestingly, K+ efflux, associated with ATP release, could be the initial signal of CPPD-induced IL-1β maturation, independently of P2X7 receptor involvement. These results highlight a new molecular pathway of ...

ObjectiveLow-grade inflammation plays a pivotal role in osteoarthritis (OA) through exposure to reactive oxygen species (ROS). In chondrocytes, NADPH oxidase 4 (NOX4) is one of the major ROS producers. In this study, we evaluated the role of NOX4 on joint homoeostasis after destabilisation of the medial meniscus (DMM) in mice.MethodsExperimental OA was simulated on cartilage explants using interleukin-1β (IL-1β) and induced by DMM in wild-type (WT) and NOX4 knockout (NOX4-/-) mice. We evaluated NOX4 expression, inflammation, cartilage metabolism and oxidative stress by immunohistochemistry. Bone phenotype was also determined by micro-CT and histomorphometry.ResultsWhole body NOX4 deletion attenuated experimental OA in mice, with a significant reduction of the OARSI score at 8 weeks. DMM increased total subchondral bone plate (SB.Th), epiphysial trabecular thicknesses (Tb.Th) and bone volume fraction (BV/TV) in both NOX4-/-and wild-type (WT) mice. Interestingly, DDM decreased total connectivity density (Conn.Dens) and increased medial BV/TV and Tb.Th only in WT mice. Ex vivo, NOX4 deficiency increased aggrecan (AGG) expression and decreased matrix metalloproteinase 13 (MMP13) and collagen type I (COL1) expression. IL-1β increased NOX4 and 8-hydroxy-2'-deoxyguanosine (8-OHdG) expression in WT cartilage explants but not in NOX4-/-. In vivo, absence of NOX4 increased anabolism and decreased catabolism after DMM. Finally, NOX4 deletion decreased synovitis score, 8-OHdG and F4/80 staining following DMM.ConclusionNOX4 deficiency restores cartilage homoeostasis, inhibits oxidative stress, inflammation and delays OA progression after DMM in mice. These findings suggest that NOX4 represent a potential target to counteract for OA treatment.

BackgroundMonosodium 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.ObjectivesThe 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.MethodsSynthetic 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 18F-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. In vivo, 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.Results In vitro, 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 uptakein vitro, ex vivo and in vivo. 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, in vitro, glucose uptake induced by MSU and mCPPD crystals, and in vivo MSU and mCPPD crystal-induced inflammation.ConclusionGlucose 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 a...