Role of the Epigenetic Regulator EZH2 in Proinflammatory Macrophage Polarisation and Signalling in Rheumatoid Arthritis
Michelle Trenkmann, Eimear Linehan, Mary Canavan, Douglas Veale, Ursula Fearon
Centre for Arthritis and Rheumatic Diseases, Dublin Academic Medical Centre University College Dublin, Ireland
Macrophages (M??) polarise along a spectrum of proinflammatory (M1) to regulatory/wound-healing (M2) phenotypes and are key pathogenic cells in rheumatoid arthritis (RA). Epigenetic mechanisms determine cell fate and have been found aberrantly regulated in RA.
To examine a role of the histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2) in RA macrophage polarisation and activation.
Monocyte-derived M?? were differentiated from peripheral blood monocytes using M-CSF, transfected with EZH2 siRNA, stimulated with LPS+IFN?? (M1) or IL-4 (M2), and analysed by multicolour flow cytometry, quantitative real-time PCR and Western blot.
Polarisation was confirmed by induction of CD40, CD64 and CD80 (M1) or CD206 (M2) (RA n=8, HC n=9). CD64 expression was higher in RA than HC M?? (64±16% vs. 35±23% CD64+ cells; p<0.01) indicating skewing towards a proinflammatory M1 phenotype in RA. EZH2 was upregulated in M1-polarised M? (HC: 9.25±3.1-fold, RA: 13.22±7.13-fold; p<0.01). Silencing of EZH2 inhibited M1 polarisation (i.e. less CD80+, CD64+ and CD40+ cells), whereas unpolarised M0 M? showed increased expression of CD40 and CD80 (p<0.05 all)(n=5). EZH2 silencing increased STAT1 mRNA and protein expression in M0 and M1 M? (p<0.05) without affecting STAT1 phosphorylation. In contrast, we demonstrated increased phospho-STAT3 in EZH2-silenced M1 M? with levels of total STAT3 remaining unchanged.
RA M?? show an intrinsic shift towards a proinflammatory M1-like phenotype and M1-polarised M?? induce EZH2. EZH2 silencing skewed M0 M?? towards a more inflammatory/activated phenotype but attenuated the inflammatory phenotype of M1 M??, possibly mediated through differential regulation of JAK/STAT signalling.