AB0048 Proinflammatory Macrophage Polarisation in Rheumatoid Arthritis and its Regulation by the Histone Methyltransferase EZH2

BackgroundMacrophages (MΦ) are tissue-resident innate immune cells central to the pathogenesis of rheumatoid arthritis (RA) that produce inflammatory cytokines and degradative enzymes in great abundance. MΦ polarise along a spectrum of classical (M1) to alternative (M2) activation gearing their function towards proinflammatory to regulatory/wound-healing/proangiogenic activities. Differentiation and polarisation are epigenetically regulated and an associated imbalance might contribute to chronic inflammation in RA.ObjectivesTo examine MΦ polarisation and epigenetic regulation in RA.MethodsMonocyte-derived MΦ (MDM) were obtained by in-vitro differentiation from RA and healthy control (HC) peripheral blood monocytes using M-CSF. THP-1 monocytes were differentiated into MΦ using PMA. M0 MΦ were polarised with LPS+IFNγ (M1) or IL-4 (M2) or cultured with RA synovial fluid (SF). Additionally, MΦ were transfected with siRNA for the histone methyltransferase Enhancer of Zeste homologue 2 (EZH2). MΦ polarisation and activation were analysed by flow cytometry and gene expression by quantitative real-time PCR and Western blot.ResultsAll MDM analysed were >90% CD68+ confirming efficient in-vitro MΦ differentiation. M1-polarised MDM showed induction of M1 markers (CD40, CD64) and the activation marker CD80 while M2-polarised MDM upregulated CD206. Similar results were obtained for THP-1 MΦ (increase in CD40+ and CD80+ cells by LPS+IFNγ, increase in M2 marker CD209 under IL-4). 71±16% of RA M0 MDM were positive for CD64 as opposed to 28±14% in HC (p