RESUMEN
OBJECTIVE: A number of studies have demonstrated that molecules called 'alarmins' or danger-associated molecular patterns (DAMPs), contribute to inflammatory processes in the OA joint. Metabolic reprogramming of immune cells, including macrophages, is emerging as a prominent player in determining immune cell phenotype and function. The aim of this study was to investigate if basic calcium phosphate (BCP) crystals which are OA-associated DAMPs, impact on macrophage phenotype and metabolism. METHODS: Human monocyte derived macrophages were treated with BCP crystals and expression of M1 (CXCL9, CXCL10) and M2 (MRC1, CCL13)-associated markers was assessed by real-time PCR while surface maturation marker (CD40, CD80 & CD86) expression was assessed by flow cytometry. BCP induced metabolic changes were assessed by Seahorse analysis and glycolytic marker expression (hexokinase 2(HK2), Glut1 and HIF1α) was examined using real-time PCR and immunoblotting. RESULTS: Treatment with BCP crystals upregulated mRNA levels of CXCL9 and CXCL10 while concomitantly downregulating expression of CCL13 and MRC1. Furthermore, BCP-treated macrophages enhanced surface expression of the maturation makers, CD40, CD80 and CD86. BCP-treated cells also exhibited a shift towards glycolysis as evidenced by an increased ECAR/OCR ratio and enhanced expression of the glycolytic markers, HK2, Glut1 and HIF1α. Finally, BCP-induced macrophage activation and alarmin expression was reduced in the presence of the glycolytic inhibitor, 2-DG. CONCLUSIONS: This study not only provides further insight into how OA-associated DAMPs impact on immune cell function, but also highlights metabolic reprogramming as a potential therapeutic target for calcium crystal-related arthropathies.
Asunto(s)
Fosfatos de Calcio/farmacología , Citocinas/efectos de los fármacos , Glucólisis/efectos de los fármacos , Macrófagos/efectos de los fármacos , Osteoartritis/inmunología , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Antígenos CD40/metabolismo , Quimiocina CXCL10/efectos de los fármacos , Quimiocina CXCL10/genética , Quimiocina CXCL10/inmunología , Quimiocina CXCL9/efectos de los fármacos , Quimiocina CXCL9/genética , Quimiocina CXCL9/inmunología , Citocinas/genética , Regulación hacia Abajo , Transportador de Glucosa de Tipo 1/efectos de los fármacos , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Glucólisis/genética , Hexoquinasa/efectos de los fármacos , Hexoquinasa/genética , Hexoquinasa/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Activación de Macrófagos , Macrófagos/inmunología , Macrófagos/metabolismo , Glicoproteínas de Membrana/efectos de los fármacos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Proteínas Quimioatrayentes de Monocitos/efectos de los fármacos , Proteínas Quimioatrayentes de Monocitos/genética , Proteínas Quimioatrayentes de Monocitos/inmunología , Osteoartritis/genética , Fenotipo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Inmunológicos/efectos de los fármacos , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Regulación hacia ArribaRESUMEN
Limitations associated with demineralised bone matrix and other grafting materials have motivated the development of alternative strategies to enhance the repair of large bone defects. The growth plate (GP) of developing limbs contain a plethora of growth factors and matrix cues which contribute to long bone growth, suggesting that biomaterials derived from its extracellular matrix (ECM) may be uniquely suited to promoting bone regeneration. The goal of this study was to generate porous scaffolds from decellularised GP ECM and to evaluate their ability to enhance host mediated bone regeneration following their implantation into critically-sized rat cranial defects. The scaffolds were first assessed by culturing with primary human macrophages, which demonstrated that decellularisation resulted in reduced IL-1ß and IL-8 production. In vitro, GP derived scaffolds were found capable of supporting osteogenesis of mesenchymal stem cells via either an intramembranous or an endochondral pathway, demonstrating the intrinsic osteoinductivity of the biomaterial. Furthermore, upon implantation into cranial defects, GP derived scaffolds were observed to accelerate vessel in-growth, mineralisation and de novo bone formation. These results support the use of decellularised GP ECM as a scaffold for large bone defect regeneration.