Distinct macrophage phenotype and bioenergetic profiles in Rheumatoid Arthritis
Megan M. Hanlon, Mary Canavan, Trudy McGarry, Candice Low, Douglas J. Veale, Ursula Fearon.
Molecular Rheumatology, Trinity Biomedical Sciences Institute, Trinity College Dublin
Synovial macrophages play a key role in RA disease progression, however, the diversity and plasticity of macrophage subsets and their metabolic profile within the joint has yet to be elucidated.
To phenotype distinct macrophage subsets within the RA joint, and determine the metabolic, inflammatory and phagocytosis function of RA macrophages compared to healthy controls (HC).
Synovial-tissue biopsies from RA, PsA and OA, obtained through arthroscopy, were digested to yield a single cell suspension. Biopsy suspensions and synovial fluid mononuclear cells were analysed using advanced flow-cytometry with the following antibody panel (CD40,-CD45,-CD64,-CD68,-CD163,-CD206,-CD253). CD14+ monocytes were sorted from RA and HC bloods and differentiated/polarized into M1/M2 macrophages. Inflammatory (IL-8,-MCP-1,-IL-1b,-CCR5,-IRAK1,-OSM) and metabolic (HIF1a,-PFKFB3,-PKM2,-LDHA,-HK2) markers were measured by RT-PCR, and phagocytosis by OVA luciferase-yellow assays. Glycolysis (ECAR) and oxidative phosphorylation (OCR) were quantified by Seahorse -XFE- technology.
RA synovial-tissue and fluid CD68+ macrophages displayed markers typical of both M1(CD40+CD253+) and M2(CD206+CD163+). A significant increase in frequency of CD68+ and CD64+ macrophages in synovial-tissue compared to fluid was observed (p<0.05), with significant increases in marker expression of CD40,CD163,CD206 (p<0.07). A spectrum of macrophage-subtypes within the inflamed joint was observed, with significant enrichment of a dominant double positive CD206+CD163+ macrophage-subtype in the synovial-tissue versus synovial fluid demonstrated (p<0.05). Increased frequency of CD206+CD163+ macrophages and higher expression of activation marker CD40 were demonstrated in RA synovial-tissue compared to PsA and OA. M1 macrophages demonstrate a pro-glycolytic phenotype with significant increases in HIF1a,-HK2,-PKM2,-and PFKB3, compared to M2, effects exacerbated in RA macrophages compared to HC. In parallel, using seahorse-technology RA M1 and M2 macrophages displayed higher ECAR and OCR profiles, in addition to an increased ECAR:OCR ratio compared to HC (p<0.05), evidence that RA macrophages switch to a glycolytic profile. This was paralleled by increased intracellular-cytokine expression of IL-1b,-IL-6 and TNFa and gene expression of IL-8,-IL-1b, OSM and MCP-1 (p<0.05). Finally, phagocytic ability of RA M1 was impaired compared to HC.
We have identified, for the first time, a dominant macrophages subtype enriched in RA synovial-tissue. Furthermore, RA M1/M2 have distinct metabolic profiles associated with differences in key inflammatory mediators and phagocytic function.