• Background/Purpose: Synovial macrophages are key effector cells in rheumatoid arthritis (RA), where they are a major source of pro-inflammatory cytokines and contribute to the cartilage and bone destruction. Macrophages are phagocytic cells present in all tissues and show a remarkable plasticity in response to environmental signals. However, the polarization state of macrophages in RA has not been fully uncovered. To dissect the molecular basis for the tissue-damaging effects of macrophages in RA joints, we have characterized the phenotype and transcriptome of RA synovial macrophages. Moreover, we have studied the macrophage polarizing ability of the synovial fluid of RA (RA-SF) patients.
• Methods: Human monocytes (obtained from buffy coats from normal donors) and macrophages from RA-SF (RA-SF MØ) were isolated by Ficoll gradient and subsequent magnetic cell sorting using anti-CD14 microbeads. Monocytes were cultured for 7 days in RPMI containing GM-CSF or M-CSF to generate GM-CSF-polarized macrophages (GM-MØ) or M-CSF-polarized macrophages (M-MØ). The phenotypic and transcriptomic characterization of ex-vivo isolated CD14+ RA-SF macrophages was accomplished by flow cytometry and quantitative real time PCR. In normal and synovial tissues, the expression of macrophage-polarization markers was analyzed by immunofluorescence labeling. To assess the RA-SF polarizing ability, RA-SF  was added onto monocytes or M-MØ (ratio 1:1 in culture medium) in the presence or absence of a blocking anti-activin A antibody for 72 hours and the expression of macrophage-polarization markers was analyzed by qRT-PCR and Western Blot.
• Results: Flow cytometry and gene profiling indicated that RA-SF macrophages express pro-inflammatory polarization markers (MMP12, EGLN3, CCR2), lack expression of markers associated to homeostatic and anti-inflammatory polarization (IGF1, HTR2B), and exhibit a transcriptomic profile that resembles the activin A-dependent gene signature of pro-inflammatory in vitro generated macrophages. In vitro experiments on monocytes and macrophages indicated that RA-SF promote the acquisition of pro-inflammatory markers (INHBA, MMP12, EGLN3, CCR2), but led to a significant reduction in the expression genes associated to homeostasis and inflammation resolution (FOLR2, SERPINB2, IGF1, CD36), thus confirming pro-inflammatory polarization ability of RA-SF. Importantly, the macrophage polarizing ability of RA-SF was inhibited by an anti-activin A neutralizing antibody, thus demonstrating that activin A mediates the pro-inflammatory macrophage polarizing ability of RA-SF. Moreover, and in line with these findings, multicolor immunofluorescence evidenced that macrophages within RA synovial membranes (RA-SM) express pro-inflammatory polarization markers whose expression is activin A-dependent.
• Conclusion: Altogether, our results demonstrate that macrophages from RA synovial fluid and membrane exhibit an MMP12+ EGLN3+ CCR2+ pro-inflammatory polarization state whose acquisition is partly dependent on activin A from the synovial fluid.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/macrophages-from-the-synovium-of-active-rheumatoid-arthritis-exhibit-an-activin-a-dependent-pro-inflammatory-profile/