AB0048 Proinflammatory Macrophage Polarisation in Rheumatoid Arthritis and its Regulation by the Histone Methyltransferase EZH2
Authors: M. Trenkmann, E. Linehan, M. Canavan, D. Veale, U. Fearon
Journal: Annals of the Rheumatic Diseases (2015)

<h3>Background</h3> Macrophages (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. <h3>Objectives</h3> To examine MΦ polarisation and epigenetic regulation in RA. <h3>Methods</h3> Monocyte-derived MΦ (MDM) were obtained by <i>in-vitro</i> 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. <h3>Results</h3> All MDM analysed were &gt;90% CD68<sup>+</sup> confirming efficient <i>in-vitro</i> 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<sup>+</sup> and CD80<sup>+</sup> 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&lt;0.005); this difference was maintained under M1 polarisation (87±6% vs. 59±22% CD64<sup>+</sup>, p&lt;0.05) indicating an intrinsic shift towards a proinflammatory M1 phenotype in RA. In support of this, HC M0 MDM expressed higher levels of proangiogenic VEGF than RA M0 MDM (2.5-fold, p&lt;0.01). The histone 3 lysine 27 (H3K27) demethylase JMJD3 was significantly induced in both M1- and M2-polarised MDM (3.4±0.9- and 2.6±0.5-fold, p&lt;0.05) whereas the H3K27 methyltransferase EZH2 was upregulated only in M1-polarised MDM (11.4±1.7-fold, p&lt;0.005) which was confirmed at the protein level by Western blot. Silencing of EZH2 reduced the numbers of CD64<sup>+</sup> and CD40<sup>+</sup> cells among M1-polarised MDM as well as CD206<sup>+</sup> cells among M2 MDM (RA and HC). Similarly, among THP-1 M1 MΦ, reduced numbers of CD40<sup>+</sup> and CD80<sup>+</sup> cells were observed upon siEZH2 transfection. Furthermore, EZH2 siRNA significantly inhibited the induction of IL6 and IL1B in M1 MDM but increased the expression of IL1B in M0 MDM (p&lt;0.05). Finally, culture of M0 MDM with RA SF induced an M1-M2-intermediate state with an increase in the numbers of CD40<sup>+</sup>, CD64<sup>+</sup> and CD206<sup>+</sup> cells (p&lt;0.01). <h3>Conclusions</h3> We demonstrate skewing of RA MDM towards a proinflammatory M1-like phenotype, differential induction of EZH2 and JMJD3 in M1- and M2-polarised MDM and a hybrid M1-M2 phenotype of MDM exposed to an RA synovial environment. Our data underline the complexity of macrophage polarisation in RA and the importance of epigenetic regulation in this process. <h3>Disclosure of Interest</h3> None declared