Background/Purpose: Synovial macrophages play a key role in RA disease progression, yet the diversity of macrophage subsets within the joint remains unknown. The concept of macrophage polarization into M1 inflammatory macrophages and M2 tissue-resolving macrophages, parallelled by changes in the bioenergetic cell profile, has received much attention. However the diversity and plasticity of macrophage subsets and their metabolic profile within the joint has yet to be elucidated.

Methods: Synovial tissue biopsies from Rheumatoid Arthritis (RA), Psoriatic Arthritis (PsA) and Osteoarthritis (OA) were obtained through key-hole arthroscopy. To phenotype distinct macrophage subtypes in the RA joint, synovial tissue biopsies (~10-15) were enzymatically and physically digested using an enzymatic cocktail and the GentleMACs dissociation system to yield a synovial single cell suspension. RA, PsA and OA synovial cell suspensions along with synovial fluid mononuclear cells were stained using a specific antibody panel (CD40, CD45, CD64, CD68, CD163, CD206, CD253) and analysed by advanced-multicolour flow cytometry analysis. Blood was also obtained from healthy and RA donors, CD14+ cells sorted and differentiated into macrophages for 8 days and polarised to either M1 (LPS/IFNγ) or M2 (IL-4) macrophages. Inflammatory and metabolic genes were measured by RT-PCR. The two major energy-using pathways, glycolysis (ECAR) and oxidative phosphorylation (OCR) were measured by Seahorse XFE technology.

Results: M1 cells displayed higher expression of pro-inflammatory genes and demonstrated a pro-glycolytic phenotype with significant increases in HIF1α, HK2, LDHA and PFKFB3, compared to M2, this effect was then further exacerbated in RA macrophages compared to healthy control. Using seahorse technology we demonstrate that in RA, both M1 and M2 macrophages display higher ECAR and OCR profiles compared to HC indicating that RA macrophages are more metabolically active compared to HC. Furthermore we note increased ECAR:OCR profiles in RA vs HC, thus RA macrophages utilise glycolysis more readily than HC. Flow cytometry analysis of RA synovial tissue and fluid revealed that CD68+ macrophages display markers typical of both M1 (CD40+CD253+) and M2 (CD206+CD163+). A significant increase in the frequency of CD68+ and CD64+ macrophages in the synovial tissue compared to fluid was observed, along with significant increases in marker expression of CD40, CD163 and CD206. Further analysis revealed a spectrum of macrophage subtypes exists within the inflamed joint, with a dominant macrophage subtype consisting of double positive CD206+ CD163+ identified, which is enriched in the synovial tissue compared to synovial fluid. Furthermore, we demonstrate that the frequency of this enriched population is significantly increased in RA synovial tissue compared to that of PsA and OA synovial tissue.

Conclusion: This study demonstrates distinct metabolic profiles in M1/M2 RA macrophages; associated with differences in key inflammatory mediators. Furthermore, we have identified, for the first time, a dominant subtype of RA tissue-specific macrophages, suggesting that these cells remain plastic and their function dependent on their microenvironment.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/distinct-macrophage-phenotype-and-bioenergetic-profiles-in-rheumatoid-arthritis/