15A138

Role of the Epigenetic Regulator EZH2 in Proinflammatory Macrophage Polarisation and Signalling in Rheumatoid Arthritis

Author(s)

Michelle Trenkmann, Eimear Linehan, Mary Canavan, Douglas Veale, Ursula Fearon

Department(s)/Institutions

Centre for Arthritis and Rheumatic Diseases, Dublin Academic Medical Centre University College Dublin, Ireland

Introduction

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.

Aims/Background

To examine a role of the histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2) in RA macrophage polarisation and activation.

Method

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.

Results

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.

Conclusions

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.

Click to access the login or register cheese