Rheumatoid arthritis peripheral CD14+ monocytes are hyper-inflammatory, hyper-glycolytic and retain a memory bias toward M1 macrophages
Trudy McGarry1, Megan Hanlon2, Clare Cunningham2, Douglas J Veale1 and Ursula Fearon2.
1. Centre for Arthritis and Rheumatic Diseases, St. Vincent's University Hospital and University College Dublin. 2. Molecular Rheumatology, Trinity Biomedical Sciences Institute, Trinity College Dublin
Myeloid cells with a monocyte/macrophages phenotype are present in large numbers in the rheumatoid arthritis (RA) joint, significantly contributing to disease.
This study aimed to assess whether peripheral monocytes in RA are pre-programmed to become M1 pro-inflammatory macrophages.
Blood was collected from healthy donors, at-risk individuals (Those with arthralgia, ACPA+/RF+, normal CRP and no evidence of synovitis) and established RA patients. CD14+ monocytes were isolated from peripheral blood mononuclear cells using a CD14 magnetic bead separation kit. Cells were stimulated with LPS (100ng/ml) for 3-24 hours and to assess the effects of STAT3 inhibition, cells were pre-treated with STATTIC (10µM) for 30mins. A Human Cytokine and Chemokine PCR array was carried out and those genes most differentially expression were further validated in a larger cohort of patients using RT-PCR. The metabolic profile of cells was analysed using Seahorse XFE Technology, which concomitantly analysis glycolysis and mitochondrial respiration in real-time. Gene and protein expression of key inflammatory and glycolytic markers was also carried out by RT-PCR, western blotting and ELISA.
CD14+ RA monocytes are hyper-inflammatory upon stimulation, with significantly higher expression of IL-1β, TNFα, IL-6, IL-27, CXCL10 and CXCL11 compared to healthy controls, which is indicative of a M1-like pro-inflammatory phenotype. These hyper-inflammatory cells are highly glycolytic, with increased expression of HIF1α and PFKFB3, a key glycolytic enzyme. Both baseline glycolysis and the maximal glycolytic capacity are increased in RA CD14+ monocytes, with no changes observed in mitochondrial respiration. This hyper-inflammatory, hyper-glycolytic phenotype is mediated by STAT3, as selective STAT3 inhibition can significantly decrease M1-like cytokines and PFKFB3 expression. Finally, this pro-inflammatory phenotype in evident in CD14+ monocytes from arthralgia ACPA+/RF+ people at risk of developing disease, demonstrating that these processes may precede clinical manifestations in RA.
This study demonstrates the unique inflammatory and metabolic phenotype of RA monocytes, suggesting that peripheral CD14+ monocytes may be pre-programmed to become M1-like pro-inflammatory macrophages. In addition, the observation of this phenotype in at-risk individuals indicates that these features may precede clinical manifestations of RA and therefore could be useful as a biomarker for early diagnosis.