Glycolytic reprogramming in macrophages and MSCs during inflammation

BackgroundDysregulated inflammation is associated with many skeletal diseases and disorders, such as osteolysis, non-union of fractures, osteonecrosis, osteoarthritis and orthopaedic infections. We previously showed that continuous infusion of lipopolysaccharide (LPS) contaminated polyethylene particles (cPE) caused prolonged inflammation and impaired bone formation. However, the metabolic and bioenergetic processes associated with inflammation of bone are unknown. Mitochondria are highly dynamic organelles that modulate cell metabolism and orchestrate the inflammatory responses that involve both resident and recruited cells. Glycolytic reprogramming, the shift from oxidative phosphorylation (OXPHOS) to glycolysis causes inappropriate cell activation and function, resulting in dysfunctional cellular metabolism. We hypothesized that impaired immunoregulation and bone regeneration from inflammatory states are associated with glycolytic reprogramming and mitochondrial dysfunction in macrophages (Mφ) and mesenchymal stromal cells (MSCs).MethodsWe used the Seahorse XF96 analyzer and real-time qPCR to study the bioenergetics of Mφ and MSCs exposed to cPE. To understand the oxygen consumption rate (OCR), we used Seahorse XF Cell Mito Stress Test Kit with Seahorse XF96 analyzer. Similarly, Seahorse XF Glycolytic Rate Assay Kit was used to detect the extracellular acidification rate (ECAR) and Seahorse XF Real-Time ATP Rate Assay kit was used to detect the real-time ATP production rates from OXPHOS and glycolysis. Real-time qPCR was performed to analyze the gene expression of key enzymes in glycolysis and mitochondrial biogenesis. We further detected the gene expression of proinflammatory cytokines in Mφ and genes related to cell differentiation in MSC during the challenge of cPE.ResultsOur results demonstrated that the oxidative phosphorylation of Mφ exposed to cPE was significantly decreased when compared with the control group. We found reduced basal, maximal and ATP-production coupled respiration rates, and decreased proton leak in Mφ during challenge with cPE. Meanwhile, Mφ showed increased basal glycolysis and proton efflux rates (PER) when exposed to cPE. The percentage (%) of PER from glycolysis was higher in Mφ exposed to cPE, indicating that the contribution of the glycolytic pathway to total extracellular acidification was elevated during the challenge of cPE. In line with the results of OCR and ECAR, we found Mφ during cPE challenge showed higher glycolytic ATP (glycoATP) production rates and lower mitochondrial ATP (mitoATP) production rates which is mainly from OXPHOS. Interestingly, MSCs showed enhanced glycolysis during challenge with cPE, but no significant changes in oxygen consumption rates (OCR). In accordance, seahorse assay of real-time ATP revealed glycoATP rates were elevated while mitoATP rates showed no significant differences in MSC during challenge with cPE. Furthermore, Mφ and MSCs exposed to cPE showed upregulated gene expression levels of glycolytic regulators and Mφ exposed to cPE expressed higher levels of pro-inflammatory cytokines.ConclusionThis study demonstrated the dysfunctional bioenergetic activity of bone marrow-derived Mφ and MSCs exposed to cPE, which could impair the immunoregulatory properties of cells in the bone niche. The underlying molecular defect related to disordered mitochondrial function could represent a potential therapeutic target during the resolution of inflammation

Étude prospective comparant un ancillaire à usage unique contre un ancillaire traditionnel et des guides de coupes sur mesure contre des guides de coupes standard pour la pose d'une prothèse totale de genou

Introduction. Rising budgetary constraints on health facilities require technical innovations. With an emphasis on cost reduction, operating room (OR) efficiency and enhanced patient outcomes, patient specific cutting guides and single-use disposable instrumentation have emerged as potential beneficial innovations. The aim of this study was to evaluate the impact of patient specific cutting guides and single use disposable instrumentation for TKA on OR and sterilization times.Material and methods: it was a monocentric prospective interventional full factorial design study, including 136 patients who underwent TKA. We compared patient-specific (PSG, n=68) to conventional cutting guides (CVG, n=68), and single use (SUI, n=68) to conventional instrumentation (CVI, n=68). Preoperative alignment showed 58% varus and 23% valgus knees. The following data were recorded in the OR and sterilization department: number and type of instrument trays, operating time, scrub nurse time, room occupancy time, decontamination, assembly and packaging, sterilization processes, quality assurance. The primary outcome was time savings between instrumentation and cutting blocks. Secondary outcomes were difference in the number of instrument trays, Oxford Knee Score (OKS), mechanical axis using the HKA angle.Results: median operating time was 80 min (Q1-Q3: 73-90). Operating time was significantly increased for SUI compared to CVI group (median SUI: 83 min versus median CVI: 78min, +5min, p=0.0072). There was no significant difference between PSG and CVG for operating time (median: 80 min for both groups, p=0.83). Median sterilization duration was 1261 min (Q1-Q3: 934-1603). It was significantly in favor of SUI (median: 936 min) over CVI (median: 1565 min) (+629min, pConclusion: SUI lowers the number of instrument trays and sterilization duration, in primary TKA. PSG is not associated with significant OR or sterilization time reduction. The use of SUI will also reduce the risk for noncompliance of sterilized instrument trays.Introduction : Les contraintes budgétaires croissantes sur les établissements de soins font mettre en place des mesures innovantes pour diminuer les coûts. L'objectif de notre étude est de mesurer l'effet des guides de coupe sur mesure et des ancillaires jetables lors des arthroplasties totales de genou sur les gains de temps au bloc opératoire et en stérilisation.Matériel et méthodes : Il s'agit d'une étude monocentrique prospective interventionnelle, en plan factoriel, incluant 136 patients qui ont eu une arthroplastie totale de genou et comparant les guides de coupes sur mesure (GCM, n=68) aux guides de coupes standard (GCS, n=68), et les ancillaires usage unique (AUU, n=68) aux ancillaires conventionnels (ACV, n=68). En préopératoire, 58 % des genoux étaient en varus et 23% en valgus. Les données suivantes ont été recueillies au bloc opératoire et en stérilisation: nombre et type de boite utilisées, temps de chirurgie, d'instrumentiste, d'utilisation de salle, et de nettoyage manuel au bloc, réception des boites, utilisation laveur, conditionnement, autoclave, validation, et temps total.Résultats : Un total de 404 boites ont été stérilisées pour les 136 patients: 252 dans le groupe ACV et 152 dans le groupe AUU; 215 dans le groupe GCV et 189 dans le groupe GSM. Les temps de chirurgie, d'instrumentiste, et d'utilisation de salle n'étaient pas significativement différents entre les groupes GCV et GSM; le temps de chirurgie était significativement allongé (+4mn, P=0.0084) dans le groupe AUU comparé au groupe ACV. Les temps de traitement des boites n'étaient pas significativement différents entre les groupes GCV et GSM. En revanche ces mêmes temps étaient nettement en faveur du groupe AUU (médiane temps total de stérilisation par patient: 936 min; Q1 - Q3: 718 - 1096) par rapport au groupe ACV (médiane: 1556 min; Q1 - Q3: 1350 - 1799) (PDiscussion : Il est reconnu que les guides de coupes sur mesure sont équivalents en terme de précision aux ancillaires métalliques standards. En revanche, l'utilisation d'ancillaire à usage unique permet un gain de temps IBODE important (mise à disposition des ancillaires) et en stérilisation et cette information est nouvelle.Conclusion : L'utilisation des ancillaires à usage unique lors de la réalisation des arthroplasties par prothèses totales de genou permet un gain de temps IBODE et en stérilisation

Importance of Early Diagnosis and Care in Knee Dislocations Associated with Vascular Injuries

International audienceBackground: Arterial injury secondary to acute knee dislocation (KD) is a rare but devastative complication. The aim of this study is to evaluate functional sequelae and factors of poor prognosis.Methods: A retrospective monocentric series of consecutive KD with acute ischemia by popliteal artery injury was analyzed between 2005 and 2017. The main outcome was the amputation rate.Results: Sixteen dislocations were included. Nine (56%) were due to public road accidents, 5 (31%) were due to falls from height, and 2 (13%) were due to sports injuries. Dislocation had occurred in the posterior location in 8 (50%) cases. Regarding arterial injury, there were 7 (44%) ruptures, 7 (44%) dissections, and 2 (13%) isolated thromboses. Eleven (69%) KDs with vascular trauma were associated with signs of acute ischemia. Revascularization was achieved by anatomical venous bypass in 14 (88%), resection and direct anastomosis in one (6%), and isolated thrombectomy in one (6%). Median time to surgery (time between trauma and vascular repair) was 7 hours (3.25-60.92 hours). Primary revascularization was performed in 12 (75%) cases. In three cases (19%), orthopedic reduction and stabilization were performed first. In one case, (6%) three-step management with vascular shunt at first, then with knee stabilization, and finally vascular bypass was carried out. Stabilization was achieved by using an external fixator in 13 (82%) cases, by open reduction and internal fixation in one case (6%), by ligamentoplasty in one (6%), and by using a long leg cast in one (6%). Fasciotomy was required in 12 (75%) cases. Two patients had early vascular complications, and 2 had early systemic complications. Three secondary transfemoral amputations were performed. Median follow-up duration was 23 months. No secondary amputation was recorded. At the end of follow-up, functional outcomes were evaluated using the Oxford Knee Score (OKS). The median OKS was 30 versus the pretrauma median OKS of 47 (P < 0.00028). No risk factor associated with limb amputation has been highlighted.Conclusions: Analysis of these results provided indications for therapeutic management of this condition. This study shows poor functional outcomes because of severity of vascular lesion in patients with orthopedic trauma but with healthy arteries

Metabolic profile of mesenchymal stromal cells and macrophages in the presence of polyethylene particles in a 3D model

Abstract Background Continuous cross talk between MSCs and macrophages is integral to acute and chronic inflammation resulting from contaminated polyethylene particles (cPE); however, the effect of this inflammatory microenvironment on mitochondrial metabolism has not been fully elucidated. We hypothesized that (a) exposure to cPE leads to impaired mitochondrial metabolism and glycolytic reprogramming and (b) macrophages play a key role in this pathway. Methods We cultured MSCs with/without uncommitted M0 macrophages, with/without cPE in 3-dimensional gelatin methacrylate (3D GelMA) constructs/scaffolds. We evaluated mitochondrial function (membrane potential and reactive oxygen species—ROS production), metabolic pathways for adenosine triphosphate (ATP) production (glycolysis or oxidative phosphorylation) and response to stress mechanisms. We also studied macrophage polarization toward the pro-inflammatory M1 or the anti-inflammatory M2 phenotype and the osteogenic differentiation of MSCs. Results Exposure to cPE impaired mitochondrial metabolism of MSCs; addition of M0 macrophages restored healthy mitochondrial function. Macrophages exposed to cPE-induced glycolytic reprogramming, but also initiated a response to this stress to restore mitochondrial biogenesis and homeostatic oxidative phosphorylation. Uncommitted M0 macrophages in coculture with MSC polarized to both M1 and M2 phenotypes. Osteogenesis was comparable among groups after 21 days. Conclusion This work confirmed that cPE exposure triggers impaired mitochondrial metabolism and glycolytic reprogramming in a 3D coculture model of MSCs and macrophages and demonstrated that macrophages cocultured with MSCs undergo metabolic changes to maintain energy production and restore homeostatic metabolism

Impact du stress neonatal sur l'oligodendrogénèse du cortex préfrontal et du comportement adulte. Identification de mécanismes activité dépendants

International audienceExposure to stress during early life (infancy/childhood) has long-term effects on the structure and function of the prefrontal cortex (PFC), and increases the risk for adult depression and anxiety disorders. However, little is known about the molecular and cellular mechanisms of these effects. Here, we focused on changes induced by chronic maternal separation during the first 2 weeks of postnatal life. Unbiased mRNA expression profiling in the medial PFC (mPFC) of maternally separated (MS) pups identified an increased expression of myelin-related genes and a decreased expression of immediate early genes. Oligodendrocyte lineage markers and birthdating experiments indicated a precocious oligodendrocyte differentiation in the mPFC at P15, leading to a depletion of the oligodendrocyte progenitor pool in MS adults. We tested the role of neuronal activity in oligodendrogenesis, using designed receptors exclusively activated by designed drugs (DREADDs) techniques. hM4Di or hM3Dq constructs were transfected into mPFC neurons using fast-acting AAV8 viruses. Reduction of mPFC neuron excitability during the first 2 postnatal weeks caused a premature differentiation of oligodendrocytes similar to the MS pups, while chemogenetic activation normalised it in the MS animals. Bidirectional manipulation of neuron excitability in the mPFC during the P2-P14 period had long lasting effects on adult emotional behaviours and on temporal object recognition: hM4Di mimicked MS effects, while hM3Dq prevented the pro-depressive effects and short-term memory impairment of MS. Thus, our results identify neuronal activity as a critical target of early-life stress and demonstrate its function in controlling both postnatal oligodendrogenesis and adult mPFC-related behaviours