Background/Purpose:

In rheumatoid arthritis (RA), synovial tissue macrophages (MΦ) are inherently involved in disease pathogenesis by producing inflammatory mediators and matrix-destructive enzymes. Depending on their polarization, MΦ show proinflammatory (classically activated M1 MΦ) or regulatory/wound-healing (alternatively activated M2 MΦ) behavior. Epigenetic mechanisms play a crucial role in the plasticity of cells and aberrant epigenetic regulation of pathogenetically relevant processes is a hallmark of many human diseases such as RA. In this study we examined if the epigenetic regulator Enhancer of Zeste Homolog 2 (EZH2) mediates differentiation and polarization of RA macrophages.

Methods:

Peripheral blood monocytes from healthy controls (HC) or RA patients were treated with M-CSF to obtain monocyte-derived MΦ (MDM). Efficient in-vitro MΦ differentiation was confirmed by CD68 staining (>95%). M0 MΦ were transfected with siRNA for histone 3 lysine 27 (H3K27) methyltransferase EZH2 or the H3K27 demethylase JMJD3 and/or stimulated with LPS+IFNγ or IL-4 to drive M1 or M2 polarization, respectively. Multicolor flow cytometry, quantitative real-time PCR (qPCR) and Western blot were performed to analyze MΦ polarization and gene expression.

Results: M1 MDM induced M1 markers CD40 and CD64 and the activation marker CD80 whereas M2 MDM upregulated CD206 (RA n=8 and HC n=9), thus confirming polarization. RA M0 MDM showed much higher expression of CD64 than HC MΦ (64±16% vs. 35±23% CD64+ cells; p<0.01) indicating skewing towards a proinflammatory M1 phenotype in RA. JMJD3 was significantly induced in both M1- and M2-polarized MDM (3.45±1.3- and 2.1±0.66-fold, p<0.005) whereas EZH2 was upregulated only in M1-polarized MDM (9.25±3.1-fold, p<0.0001)(n=9 HC) which was confirmed at the protein level by Western blot. Silencing of EZH2 (n=5 each) significantly inhibited M1 polarization (i.e. reduced numbers of CD80⁺, CD64⁺ and CD40⁺cells), whereas, interestingly, unpolarized M0 MDM showed increased expression of CD40 and CD80 (p<0.05 all). Consistent with this, EZH2 silencing resulted in increased expression of VEGF (2.2±0.6-fold) and IL1B (3.4±2-fold) in M0 MDM, but inhibition of IL1B induction in M1 MDM (-75±15%) (all p<0.05). Looking for potential targets of EZH2 that might mediate the observed effects on MΦ polarization and cytokine expression, we analyzed genes involved in MΦ plasticity and interferon signaling (KLF4, STAT1, STAT3, STAT6, IRF4 and IRF5). Silencing of EZH2 increased STAT1 mRNA and STAT1 protein in M0 and M1 MDM (p<0.05), with no change observed for STAT1 phosphorylation. In contrast, we found reduced phospho-STAT3 in EZH2-silenced M1 MDM with levels of total STAT3 remaining unchanged. 

Conclusion: Our data show an intrinsic shift of RA MDM towards a proinflammatory M1-like phenotype and induction of EZH2 in M1-polarized MΦ. We found EZH2 silencing to skew MΦ plasticity towards an inflammatory/activated phenotype under neutral conditions but attenuation of the inflammatory phenotype in a proinflammatory milieu. Mechanistically, different arms of the JAK/STAT pathway appear to be involved in these processes underpinning the importance of EZH2 in inflammatory MΦ signaling.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/role-of-the-epigenetic-regulator-ezh2-in-proinflammatory-macrophage-polarization-and-signaling-in-rheumatoid-arthritis/