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International audience; Nutrition appears to be an important environmental factor involved in the onset of inflammatory bowel diseases (IBD) through yet poorly understood biological mechanisms. Most studies focused on fat content in high caloric diets, while refined sugars represent up to 40% of caloric intake within industrialized countries and contribute to the growing epidemics of inflammatory diseases. Herein we aim to better understand the impact of a high-fat-high-sucrose diet on intestinal homeostasis in healthy conditions and the subsequent colitis risk. We investigated the early events and the potential reversibility of high caloric diet-induced damage in mice before experimental colitis. C57BL/6 mice were fed with a high-fat or high-fat high-sucrose or control diet before experimental colitis. In healthy mice, a high-fat high-sucrose diet induces a pre-IBD state characterized by gut microbiota dysbiosis with a total depletion of bacteria belonging to Barnesiella that is associated with subclinical endoscopic lesions. An overall down-regulation of the colonic transcriptome converged with broadly decreased immune cell populations in the mesenteric lymph nodes leading to the inability to respond to tissue injury. Such in-vivo effects on microbiome and transcriptome were partially restored when returning to normal chow. Long-term consumption of diet enriched in sucrose and fat predisposes mice to colitis.This enhanced risk is preceded by gut microbiota dysbiosis and transcriptionalreprogramming of colonic genes related to IBD. Importantly, diet-induced transcriptome and microbiome disturbances are partially reversible after switching back to normal chow with persistent sequelae that may contribute to IBD predisposition in the general population.

International audience

International audience

International audience; Objective. Decreased expression of collagen type II in favour of collagen type I or X is one hallmark of chondrocyte phenotype changes in osteoarthritic (OA) cartilage. MicroRNA-(miR-) 29b was previously shown to target collagens in several tissues. We studied whether it could contribute to collagen imbalance in chondrocytes with an impaired phenotype. Methods. After preliminary microarrays screening, miR-29b levels were measured by RT-quantitative PCR in in vitro models of chondrocyte phenotype changes (IL-1í µí»½ challenge or serial subculturing) and in chondrocytes from OA and non-OA patients. Potential miR-29b targets identified in silico in 3 í® í°-UTRs of collagens mRNAs were tested with luciferase reporter assays. The impact of premiR-29b overexpression in ATDC5 cells was studied on collagen mRNA levels and synthesis (Sirius red staining) during chondrogenesis. Results. MiR-29b level increased significantly in IL-1í µí»½-stimulated and weakly in subcultured chondrocytes. A 5.8-fold increase was observed in chondrocytes from OA versus non-OA patients. Reporter assays showed that miR-29b targeted COL2A1 and COL1A2 3 í® í°-UTRs although with a variable recovery upon mutation. In ATDC5 cells overexpressing premiR-29b, collagen production was reduced while mRNA levels increased. Conclusions. By acting probably as a posttranscriptional regulator with a different efficacy on COL2A1 and COL1A2 expression, miR-29b can contribute to the collagens imbalance associated with an abnormal chondrocyte phenotype.

International audience

International audience

Microsomal prostaglandin E synthase (mPGES)-1 is a newly identified inducible enzyme of the arachidonic acid cascade with a key function in prostaglandin (PG)E2 synthesis. We investigated the kinetics of inducible cyclo-oxygenase (COX)-2 and mPGES-1 expression with respect to the production of 6-keto-PGF1alpha and PGE2 in rat chondrocytes stimulated with 10 ng/ml IL-1beta, and compared their modulation by peroxisome-proliferator-activated receptor (PPAR)gamma agonists. Real-time PCR analysis showed that IL-1beta induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1 expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF1alpha and PGE2 peaked 24 hours after stimulation with IL-1beta; the induction of PGE2 was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-Delta12,14prostaglandin J2 (15d-PGJ2) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 microM), with more potency on PGE2 level than on 6-keto-PGF1alpha level (-90% versus -66% at 10 microM). A high dose of 15d-PGJ2 partly decreased COX-2 expression but decreased mPGES-1 expression almost completely at both the mRNA and protein levels. Rosiglitazone was poorly effective on these parameters even at 10 microM. Inhibitory effects of 10 microM 15d-PGJ2 were neither reduced by PPARgamma blockade with GW-9662 nor enhanced by PPARgamma overexpression, supporting a PPARgamma-independent mechanism. EMSA and TransAM analyses demonstrated that mutated IkappaBalpha almost completely suppressed the stimulating effect of IL-1beta on mPGES-1 expression and PGE2 production, whereas 15d-PGJ2 inhibited NF-kappaB transactivation. These data demonstrate the following in IL-1-stimulated rat chondrocytes: first, mPGES-1 is rate limiting for PGE2 synthesis; second, activation of the prostaglandin cascade requires NF-kappaB activation; third, 15d-PGJ2 strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ2 occurs independently of PPARgamma through inhibition of the NF-kappaB pathway; fifth, mPGES-1 is the main target of 15d-PGJ2.

International audience; ObjectiveTo investigate the effect of pioglitazone on ă inflammation-induced bone loss and changes in bone microarchitecture in ă rats with adjuvant-induced arthritis (AIA), focusing on the contribution ă of interleukin-17 (IL-17) and the balance of RANKL and osteoprotegerin ă (OPG). ă MethodsMale Lewis rats sensitized with Freund's complete adjuvant were ă treated orally for 21 days with 30 mg/kg/day of pioglitazone or vehicle. ă Arthritis severity was evaluated by clinical and histologic examination. ă Bone mineral density (BMD) was assessed by dual x-ray absorptiometry. ă The therapeutic effect of pioglitazone on changes of the bone ă architecture was determined by micro-computed tomography (micro-CT). ă Levels of RANKL, OPG, and IL-17 were determined by serum immunoassay and ă by synovial tissue immunohistochemistry. Messenger RNA for IL-17 and ă retinoic acid receptor-related orphan nuclear receptor t (RORt) was ă evaluated by quantitative reverse transcription-polymerase chain ă reaction and IL-17 promoter activity by gene-reporter assay. ă ResultsMicro-CT analysis revealed that pioglitazone treatment reduced ă arthritis severity and bone erosion scores and increased BMD in ă comparison to vehicle treatment. Cortical bone thickness was preserved, ă although the major beneficial effect of pioglitazone was on indices of ă the trabeculae, especially trabecular separation. Pioglitazone reduced ă the ratio of RANKL to OPG, in both the serum and the inflamed synovium. ă Circulating levels of IL-17 were significantly reduced by pioglitazone ă treatment, as were the percentages of IL-17-positive cells, mainly ă polymorphonuclear cells, in the inflamed synovium. Induction of IL-17 ă was strictly dependent on the binding of RORt to IL-17 promoter, and ă lentiviral overexpression of peroxisome proliferator-activated receptor ă (PPAR) reduced the expression of RORt. ă ConclusionPioglitazone decreased the level of inflammatory bone ă destruction and protected the bone microarchitecture in rats with AIA by ă controlling the circulating and local expression of IL-17, with a ă subsequent decrease in the RANKL-to-OPG ratio. Along with the inhibition ă of RORt expression after PPAR overexpression, these findings provide ă evidence of the major contribution of reduced IL-17/RANKL-dependent ă osteoclastogenesis.

International audience; Peroxisome proliferators activated receptors (PPAR) are ligand-inducible nuclear transacting factors comprising three subtypes, PPARα, PPARβ/δ and PPARγ, which play a key role in lipids and glucose homeostasis. All PPAR subtypes have been identified in joint or inflammatory cells and their activation resulted in a transcriptional repression of pro-inflammatory cytokines (IL-1, TNFα), early inflammatory genes (NOS2, COX-2, mPGES-1) or matrix metalloproteases (MMP-1, MMP-13), at least for the γ subtype. PPAR full agonists were also shown to stimulate IL-1 receptor antagonist (IL-1Ra) production by cytokine-stimulated articular cells in a subtype-dependent manner. These anti-inflammatory and anti-catabolic properties were confirmed in animal models of joint diseases where PPAR agonists reduced synovial inflammation while preventing cartilage destruction or inflammatory bone loss, although many effects required much higher doses than needed to restore insulin sensitivity or to lower circulating lipid levels. However, these promising effects of PPAR full agonists were hampered by their ability to reduce the growth factor-dependent synthesis of extracellular matrix components or to induce chondrocyte apoptosis, by the possible contribution of immunosuppressive properties to their anti-arthritic effects, by the increased adipocyte differentiation secondary to prolonged stimulation of PPARγ, and by a variable contribution of PPAR subtypes depending on the system. Clinical data are scarce in rheumatoid arthritis (RA) patients whereas thousands of patients worldwilde, treated with PPAR agonists for type 2 diabetes or dyslipidemia, are paradoxically prone to suffer from osteoarthritis (OA). Whereas high dosage of full agonists may expose RA patients to cardiovascular adverse effects, the proof of concept that PPAR agonists have therapeutical relevance to OA may benefit from an epidemiological follow-up of joint lesions in diabetic or hyperlipidemic patients treated for long periods of time with glitazones or fibrates. Additionally, cellular and animal studies are required to assess whether partial agonists of PPAR (SPPARMs) may preserve therapeutical properties with potentially less safety concern.; Les récepteurs activés par les proliférateurs de peroxysomes (PPAR) sont des facteurs de transcription inductibles par des ligands dont il existe trois sous-types, PPARα, PPARβ/δ et PPARγ jouant un rôle primordial dans le métabolisme des lipides et l'homéostasie du glucose. Ces trois sous-types sont également exprimés dans les cellules résidentes de l'articulation et celles de l'infiltrat inflammatoire, et leur activation réprime l'expression de cytokines pro-inflammatoires (IL-1, TNFα), de gènes précoces de l'inflammation (iNOS, COX-2, mPGES-1) ou de métalloprotéases matricielles (MMP-1, MMP-13), tout du moins pour le sous-type γ. Les agonistes de PPAR sont également capables de stimuler la production de l'antagoniste du récepteur à l'IL-1 (IL-1Ra) par les cellules articulaires soumises à un stimulus cytokinique. Ces potentialités anti-inflammatoires et anti-cataboliques ont été confirmées dans des modèles animaux d'arthropathie, dans lesquels les agonistes de PPAR diminuent l'inflammation synoviale et préviennent la destruction du cartilage ou la perte osseuse inflammatoire, même si les posologies requises sont très supérieures à celles permettant de normaliser les taux circulants de lipides ou de rétablir la sensibilité des tissus périphériques à l'insuline. Ces effets prometteurs des agonistes sont cependant pondérés par leur capacité à diminuer l'effet trophique des facteurs de croissance sur la biosynthèse des composants matriciels ou à provoquer une apoptose chondrocytaire, par leur effet immunosuppresseur responsable d'une surestimation des propriétés anti-inflammatoires dans les polyarthrites expérimentales, par le renforcement de la différenciation adipocytaire lors d'une stimulation prolongée de PPARγ, et par la contribution variable des sous-types selon le système expérimental. Les données cliniques restent anecdotiques dans la polyarthrite rhumatoïde alors que, paradoxalement, des milliers de patients qui sont traités quotidiennement par des agonistes de PPAR pour un diabète de type 2 ou une dyslipidémie, souffrent conjointement d'arthrose. Alors que les fortes posologies d'agonistes exposeraient les patients polyarthritiques à un risque important d'effets indésirables, notamment cardiovasculaires, la démonstration de l'intérêt des agonistes de PPAR dans l'arthrose pourrait être apportée par une étude épidémiologique de l'incidence ou de l'évolution de la maladie arthrosique chez les patients diabétiques et/ou dyslipidémiques traités par des glitazones ou des fibrates. Il sera également opportun d'étudier si des agonistes partiels de PPAR permettront de conserver certaines potentialités thérapeutiques tout en diminuant le risque d'effets indésirables.

Coatings, and particularly Chemical Vapor Deposited (CVD) diamond coatings, are promising regarding hard to machine materials and some applications are already ready industrially. The excellent thermo-mechanical properties of diamond impose this solution to the machining of non-ferrous materials. However, the coated tools are difficult to understand. As a substrate-coating system, the tool is itself a composite material, bringing some difficulties. Edge preparation technologies are various: it is difficult to identify the optimal process. It has also to be noticed that the tool-piece interaction in machining operations is not completely understood yet, and that an intense research work is done to understand these phenomena. The thermo-mechanical loading on the coating is difficult to determine, and the developments on the coatings needs experimental data which are cost and time consuming.In this work, we will show the interest and improvement brought by such a coating in comparison to other coatings used in machining. Then, the stress state of the system carbide – coating is study by the mean of numerical and analytical models, as well as by the mean of experimental measurements.The influences of the different configurations of the geometry and of the deposition process on the residual stresses into the layer are studied. These high stresses have a direct impact on the adhesion of the layer, which will be discussed and measured experimentally into the second part of our work. To study the adhesion phenomena deeply, an experimental device has been designed. The obtained results are detailed and discussed. With these data, the Energy release rate of the interface is calculated and proposed as an adhesion criterion at the end of this work, Les revêtements diamant CVD (Chemical Vapour Deposition), sont très prometteurs pour l’usinage des matériaux modernes dit difficile à usiner et quelques applications sont déjà opérationnelles, comme l'usinage du graphite. Les propriétés thermomécaniques du diamant l'imposent comme la solution pour l'usinage des matériaux non-ferreux. Toutefois, la maîtrise de la technologie des outils revêtus est difficile. En tant que système substrat-couche fine, l'outil devient un composite, apportant son lot de difficultés. Le chargement thermomécanique sur le revêtement est difficile à déterminer et les développements sur les revêtements nécessitent des données expérimentales qui sont longues et coûteuses à obtenir. En particulier, l'adhésion de la couche sur l'outil est un point crucial qu'il est difficile de quantifier aujourd'hui.Dans ce travail, nous montrons l'intérêt et le gain réalisé par l'emploi d'un tel revêtement en comparaison avec d'autres revêtements. Puis, l'état de contrainte du système carbure-diamant est étudié en détail au moyen de modélisations ainsi qu'à travers des mesures expérimentales. L'influence des différentes configurations de la géométrie et du procédé de dépôt de la couche sur les contraintes résiduelles thermiques est étudiée. Ces fortes contraintes résiduelles ont un impact direct sur l'adhésion, impact discuté et mesuré expérimentalement dans la seconde partie de notre travail. Afin d'étudier ces phénomènes, un montage a été conçu et réalisé. Les résultats obtenus sont détaillés et discutés. Le taux de restitution d'énergie critique de l'interface sera calculé et proposé en tant que critère d’adhésion dans la fin de ce travail

International audience; Microsomal prostaglandin E synthase (mPGES)-1 is a newly identified inducible enzyme of the arachidonic acid cascade with a key function in prostaglandin (PG)E 2 synthesis. We investigated the kinetics of inducible cyclo-oxygenase (COX)-2 and mPGES-1 expression with respect to the production of 6-keto-PGF 1α and PGE 2 in rat chondrocytes stimulated with 10 ng/ml IL-1β, and compared their modulation by peroxisome-proliferator-activated receptor (PPAR)γ agonists. Real-time PCR analysis showed that IL-1β induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1 expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF 1α and PGE 2 peaked 24 hours after stimulation with IL-1β; the induction of PGE 2 was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-∆ 12,14 prostaglandin J 2 (15d-PGJ 2) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 µM), with more potency on PGE 2 level than on 6-keto-PGF 1α level (-90% versus-66% at 10 µM). A high dose of 15d-PGJ 2 partly decreased COX-2 expression but decreased mPGES-1 expression almost completely at both the mRNA and protein levels. Rosiglitazone was poorly effective on these parameters even at 10 µM. Inhibitory effects of 10 µM 15d-PGJ 2 were neither reduced by PPARγ blockade with GW-9662 nor enhanced by PPARγ overexpression, supporting a PPARγ-independent mechanism. EMSA and TransAM ® analyses demonstrated that mutated IκBα almost completely suppressed the stimulating effect of IL-1β on mPGES-1 expression and PGE 2 production, whereas 15d-PGJ 2 inhibited NF-κB transactivation. These data demonstrate the following in IL-1-stimulated rat chondrocytes: first, mPGES-1 is rate limiting for PGE 2 synthesis; second, activation of the prostaglandin cascade requires NF-κB activation; third, 15d-PGJ 2 strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ 2 occurs independently of PPARγ through inhibition of the NF-κB pathway; fifth, mPGES-1 is the main target of 15d-PGJ 2 .