Osteoarthritis-associated basic calcium phosphate crystals alter immune cell metabolism and promote M1 macrophage polarization

Abstract

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 reprog-ramming of immune cells, including macrophages, is emerging as a prominent player in determiningimmune 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 surfacematuration marker (CD40, CD80&CD86) expression was assessed byflow cytometry. BCP inducedmetabolic changes were assessed by Seahorse analysis and glycolytic marker expression (hexokinase2(HK2), Glut1 and HIF1a) was examined using real-time PCR and immunoblotting.Results:Treatment with BCP crystals upregulated mRNA levels of CXCL9 and CXCL10 while concomi-tantly downregulating expression of CCL13 and MRC1. Furthermore, BCP-treated macrophages enhancedsurface expression of the maturation makers, CD40, CD80 and CD86. BCP-treated cells also exhibited ashift towards glycolysis as evidenced by an increased ECAR/OCR ratio and enhanced expression of theglycolytic markers, HK2, Glut1 and HIF1a. Finally, BCP-induced macrophage activation and alarminexpression 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 onimmune cell function, but also highlights metabolic reprogramming as a potential therapeutic target forcalcium crystal-related arthropathies