Rheumatoid arthritis (RA) is a chronic inflammatory disease whose pathogenesis 
and severity correlates with the presence of macrophage-derived pro-inflammatory 
cytokines within the inflamed synovium. Macrophage-derived cytokines fuel the 
pathological processes in RA and are targets of clinically successful therapies. 
However, although macrophage polarization determines cytokine production, the 
polarization state of macrophages in RA joints remains poorly defined. To 
dissect the molecular basis for the tissue-damaging effects of macrophages in RA 
joints, we undertook the phenotypic and transcriptomic characterization of ex 
vivo isolated CD14(+) RA synovial fluid (RA-SF) macrophages. Flow cytometry and 
gene profiling indicated that RA-SF macrophages express pro-inflammatory 
polarization markers (MMP12, EGLN3, CCR2), lack expression of markers associated 
with 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 fact, high levels of 
Smad-activating activin A were found in RA-SF and, accordingly, the Smad 
signalling pathway was activated in ex vivo-isolated RA-SF macrophages. In vitro 
experiments on monocytes and macrophages indicated that RA-SF promoted the 
acquisition of pro-inflammatory markers (INHBA, MMP12, EGLN3, CCR2) but led to a 
significant reduction in the expression of genes associated with homeostasis and 
inflammation resolution (FOLR2, SERPINB2, IGF1, CD36), thus confirming the 
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, multicolour immunofluorescence evidenced that macrophages 
within RA synovial membranes (RA-SM) also express pro-inflammatory polarization 
markers whose expression is activin A-dependent. Altogether, our results 
demonstrate that macrophages from RA synovial fluids and membranes exhibit an 
MMP12(+) EGLN3(+) CCR2(+) pro-inflammatory polarization state whose acquisition 
is partly dependent on activin A from the synovial fluid.