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EZH2 deficiency attenuates Treg differentiation in rheumatoid arthritis
Journal of Autoimmunity xxx (xxxx) xxxx
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EZH2 deficiency attenuates Treg differentiation in rheumatoid arthritis Xin-yue Xiaoa,1, Yue-ting Lia,1, Xu Jianga, Xin Jia, Xin Lub, Bo Yangb, Li-jun Wud, Xiao-han Wange, Jing-bo Guof, Li-dan Zhaoa, Yun-yun Feia, Hua-xia Yanga, Wen Zhanga, Feng-chun Zhanga, Fu-lin Tanga, Jian-min Zhangc, Wei Hec, Hua Chena,∗∗, Xuan Zhanga,∗ a
Department of Rheumatology and Clinical Immunology, Peking Union Medical College Hospital, Clinical Immunology Center, Chinese Academy of Medical Sciences and Peking Union Medical College, The Ministry of Education Key Laboratory, Beijing, 100730, China b Department of Orthopedics, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100730, China c Department of Immunology & National Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS) & Peking Union Medical College, Beijing, 100005, China d Department of Rheumatology and Clinical Immunology, People's Hospital of Xinjiang Uygur Autonomous Region, Urumchi, 830001, China e Department of Rheumatology, AnYang District Hospital, AnYang, HeNan Province, 455000, China f Department of Traditional Chinese Medicine, 256th Clinical Department of Bethune International Peace Hospital of PLA, Shijiazhuang, 050800, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Rheumatoid arthritis EZH2 CD4+ T cells Differentiation Treg
The chromatin modifier enhancer of zeste homolog 2 (EZH2) methylates lysine 27 of histone H3 (H3K27) and regulates T cell differentiation. However, the potential role of EZH2 in the pathogenesis of rheumatoid arthritis (RA) remains elusive. We analyzed EZH2 expression in PBMC, CD4+ T cells, CD19+ B cell, and CD14+ monocytes from active treatment-naïve RA patients and healthy controls (HC). We also suppressed EZH2 expression using EZH2 inhibitor GSK126 and measured CD4+ T cell differentiation, proliferation and apoptosis. We further examined TGFβ-SMAD and RUNX1 signaling pathways in EZH2-suppressed CD4+ T cells. Finally, we explored the regulation mechanism of EZH2 by RA synovial fluid and fibroblast-like synoviocyte (FLS) by neutralizing key proinflammatory cytokines. EZH2 expression is lower in PBMC and CD4+ T cells from RA patients than those from HC. EZH2 inhibition suppressed regulatory T cells (Tregs) differentiation and FOXP3 transcription, and downregulated RUNX1 and upregulated SMAD7 expression in CD4+ T cells. RA synovial fluid and fibroblast-like synoviocytes suppressed EZH2 expression in CD4+ T cells, which was partially neutralized by anti-IL17 antibody. Taken together, EZH2 in CD4+ T cells from RA patients was attenuated, which suppressed FOXP3 transcription through downregulating RUNX1 and upregulating SMAD7 in CD4+ T cells, and ultimately suppressed Tregs differentiation. IL17 in RA synovial fluid might promote downregulation of EZH2 in CD4+ T cells. Defective EZH2 in CD4+ T cells might contribute to Treg deficiency in RA.
1. Introduction
regulation of cytokine-induced transcriptional factors, which is highly environment-dependent as well as epigenetically regulated [6]. Epigenetic processes such as DNA methylation, histone modification, nucleosome positioning, miRNAs and DNA supercoiling stresses are essential for the regulation of gene expression [7,8] and bridges the genes and the diseases [9]. The evidence of epigenetic dysregulation in autoimmune diseases is accumulating [10]. For instance, epigenetic modifications by DNMT1 and TET2 regulate T cell differentiation and are implicated in autoimmune diseases including systemic lupus erythematosus (SLE) and Sjögren syndrome [11,12]. The concordance of RA between identical twins is 12–35%, implicating other etiology
Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by progressive joint destruction and subsequent disability [1]. Synovium inflammation is a hallmark of RA, which consists of massive activated CD4+ T cells [2], suggesting disturbed homeostasis of CD4+ T cells plays a critical role in the development of RA [3,4]. Furthermore, CD4+ T cell depletion using anti-CD4 antibody prevented experimental arthritis [5]. Naïve CD4+ T cells differentiate into T cell subsets including T helper 1 (Th1), T helper 17 (Th17), and regulatory T cells (Treg) by the
∗
Corresponding author. Corresponding author. E-mail addresses: [email protected] (H. Chen), [email protected] (X. Zhang). 1 Xin-yue Xiao and Yue-ting Li contributed equally to this manuscript. ∗∗
https://doi.org/10.1016/j.jaut.2020.102404 Received 4 November 2019; Received in revised form 2 January 2020; Accepted 5 January 2020 0896-8411/ © 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: Xin-yue Xiao, et al., Journal of Autoimmunity, https://doi.org/10.1016/j.jaut.2020.102404
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days, then were stained with Ki67 and measured by a BD FACSAria II flow cytometry. Apoptosis of cells were measured by Annexin V and 7AAD (BD Pharmingen). For T cell differentiation, naïve CD4+ T cells (1 × 10 6 cells/ml) were stimulated with anti-CD3 (5 μg/ml)/anti-CD28 (5 μg/ml) for 5 days. For Th17 differentiation, IL-1β (10 ng/ml), IL-6(10 ng/ml), TGF-β (10 ng/ml) and IL-23(10 ng/ml) were supplemented; for Th1 differentiation, IL-2 (10 ng/ml) and IL-12 (10 ng/ml) were supplemented. PMA, ionomycin and Golgistop (BD Pharmingen) were added in the last 4 h. Cells were harvested, surface stained, permeabilized, stained with intracellular IL-17A and IFN-gamma and detected by flow cytometry. For Treg differentiation, IL-2 (10 ng/ml) and TGF-β (10 ng/ml) were supplemented. After surface staining of CD4 and CD25, cells were permeabilized, stained with FOXP3 and detected by flow cytometry. Jurkat T cells were obtained from National Infrastructure of Cell Line Resource (Beijing, China) and were maintained in RPMI-1640. Jurkat T cells were transfected with control siRNA (si-NC), or EZH2 siRNA (siEZH2-1, siEZH2-2) (100 nM) using a Gene Pulser Xcell Electroporation Systems (Biorad) according to the manufacturer’ s instructions. Naïve CD4+ T cells (1 × 106) were stimulated with anti-CD3 mAb (5 μg/ml), anti-CD28 (5 μg/ml) mAb supplemented with 10% RA synovial fluid and anti-IL1β(5 μg/ml), anti-IFNγ(5 μg/ml), anti-IL6(5 μg/ ml), anti-IL17A(5 μg/ml), or anti-TNFα(5 μg/ml) neutralized antibody. Cells were harvested after 4 days and analyzed by flow cytometry. FLS (1 × 105 cells/well) were seeded at in DMEM supplemented with 10% FCS and incubated overnight. Then naïve T cells (1 × 106) were added and were stimulated with anti-CD3 mAb (5 μg/ml), antiCD28 (5 μg/ml) mAb, TGF-β (20 ng/mL) and IL-2 (20 ng/ml) for 4 days. Cells were harvested and analyzed by flow cytometry.
beyond genetic background is involved [13,14]. Recent studies suggested epigenetic modifications contribute to the tumor-like activation of fibroblast-like synoviocyte (FLS) from RA [15–17]. Furthermore, the DNA methylation landscapes of peripheral blood mononuclear cells (PBMC) and FLS are distinctly different among RA patients, healthy control and osteoarthritis [18,19]. However, whether the aberrant epigenetic changes regulate CD4+ T cell differentiation in RA patients remains unknown. The chromatin modifier enhancer of zeste homolog 2 (EZH2), a catalytic subunit of Polycomb repressive complex 2 (PRC2), methylates lysine 27 of histone H3 (H3K27) to silence gene transcription in cell cycle and differentiation [20]. Accumulating evidence has highlighted the links between EZH2 and T cell differentiation [21]. EZH2 silences the expression of Tbx1 and Gata3 in T helper (Th) cells and EZH2 can also regulate T cell polarization [22,23]. Additionally, overexpression of EZH2 in FLS from RA patients represses Wnt inhibitor SFRP1 and subsequently activates the pathogenic FLS [24], which is potentially regulated by Lnc-IL7R [25]. Taken together, these studies indicate that EZH2 might participate the abnormal T cell polarization and contribute to the development of RA. In this study, we reported that EZH2 expression was lower in CD4+ T cells from RA patients. EZH2 inhibition attenuated the FOXP3 expression and Treg differentiation, by downregulating RUNX1 and upregulating SMAD7 expression. Furthermore, synovial fluid and FLS from RA patients downregulated EZH2 expression in CD4+ T cells. 2. Materials and methods 2.1. Patients and healthy controls A total of 52 treatment-naïve active RA patients fulfilled the 2010 ACR revised criteria for RA were enrolled. The disease activity was measured by the 28-joint DAS with CRP (DAS28-CRP) (Supplementary Table 1). The study was approved by the Institutional Review Board of Peking Union Medical College Hospital and written informed consent was obtained from all participants.
2.4. Real-time quantitative PCR Fresh cells were harvested and resuspended in TRIzol (Invitrogen Life Technologies) and stored at − 70 °C. Total RNA was extracted using the RNeasy Mini Kit (Qiagen). Reverse transcription reactions were prepared using the SYBR Premix Ex Taq System (Takara). Realtime PCR was performed using a 7900HT Fast Real-Time PCR System (Applied Biosystems). Relative expression of EZH2, FOXP3, IL17A and IFNγ against GAPDH were calculated using the comparative ΔΔCT method.
2.2. Antibodies and reagents The following fluorochrome-conjugated or unlabeled monoclonal antibodies were used: CD4 (RPA-T4), CD14 (61D3), IFN-γ (B27), FOXP3 (PCH101), CD25 (BC96), IL-17 (64DEC17) (eBioscience, San Diego, CA, USA), CD19(HIB19), Ki67(KI671-A), CD90(5E10) (Biolegend, USA). The corresponding IgG isotype antibodies were used as control. Annexin V Apoptosis Detection Kit was obtained from BD Bioscience (559763). Recombinant IL-2, IL-4, IL-6, IL-1β, IL-23, and TGF-β were purchased from PeproTech (Rocky Hill, NJ, USA). Phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma-Aldrich (St Louis, MO, USA). Antibodies against β-actin (4970) were purchased from Cell Signaling Technology (Danvers, MA). Antibody against EZH2 was purchased from Affinity (AF5150) for WB and BD bioscience (562478) for flowcytometry. Antibody against SMAD7 (ab216428) and RUNX1 (EPR3099) were purchased from Abcam (USA). siRNA was purchased from Ruibo biotech (Guangzhou, China)
2.5. Western blotting Cells were lysed by incubation for 1 h in a RIPA lysis buffer 1 mM PMSF and a proteinase inhibitor cocktail (BD Biosciences, Franklin Lakes, NJ). The lysates were kept on ice and vortexed every 10 min for 1 h before centrifugation at 12 000 g at 4 °C. Equal amounts of protein were separated by SDS-PAGE (Invitrogen), transferred to PVDF membranes (Millipore, Billerica, MA, USA), blocked with 5% dried milk in PBS containing 0.5% Tween 20, incubated with indicated antibodies, then incubated with HRP-conjugated secondary antibody and finally developed using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Boston, MA, USA).
2.3. Cell separation, culture, stimulation and electrotransfection PBMC were prepared with Ficoll-Hypaque density-gradient centrifugation and synovial cell suspension was prepared by enzyme digestion. Naïve CD4+ T cells, CD4+ T cells, CD19+ B cells and CD14+ monocytes were isolated from PBMC and synovium using magnetic isolation kits accordingly (Miltenyi Biotec, Germany), respectively. Naïve CD4+ T cells were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) in 5% CO2 at 37 °C. For proliferation assays, naïve CD4+ T cells (1 × 10 7 cells/ml) were stimulated with anti-CD3 (5 μg/ml) and anti-CD28 (5 μg/ml) mAbs for 3
2.6. Statistical analysis Data were summarized with mean ± SD. Normal distributed data was analyzed by Kolmogorov–Smirnov and Shapiro–Wilk tests (P > 0.05). The Student's t-test for continuous variables with a normal distribution. For the data with a normal distribution and homogeneity of variance, one-way analysis of variance. All data were analyzed using SPSS 22.0 software (SPSS, Chicago, IL, USA). 2
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Fig. 1. EZH2 expression in PBMC and CD4+ T cells from RA and HC. (A) EZH2 mRNA expression in PBMC, CD4+ T cells, CD14+ monocytes and CD19+ B cells from HCs and RA patients (n = 22). (B) EZH2 protein expression in peripheral CD4+cells from HCs and RA patients (n = 5). (C) EZH2 protein expression in peripheral CD4+ CD45RA+ naïve T cells from HCs and RA patients (n = 20). (**: p < 0.01, ****: p < 0.0001).
3. Results
which plays a role in the pathogenesis of RA.
3.1. Lower EZH2 expression in CD4+ T cells from RA patients
3.3. Attenuated EZH2 promotes SMAD7 and suppresses RUNX1 in CD4+ T cells
We first screened expression profile of epigenetic modification-related genes including PRMT5, TRIM22, TET1, TET2, TET3 and GADD45B, RFX1, EZH2 and SIRT1, in PBMC, CD4+ T cells, CD19+B cells and CD14+monocytes from treatment-naïve RA patients and healthy controls (HC) using quantitative PCR (Fig. 1A and S1). We found that expression of EZH2, TET3 and GADD45B were lower and expression of PRMT5 and TET1 were higher in PBMCs from RA. We confirmed that expression of EZH2 and TET3 were lower in CD4+ T cells from RA. As the expression of EZH2 was higher than TET2 in PBMC and T cells, we chose the EZH2 for further research. Given EZH2 expression was significantly lower in PBMC and CD4+ T cells from RA than those from HCs (Fig. 1A), we decided to focus on potential regulation of CD4+ T cells by EZH2. We further confirmed that protein expression of EZH2 in CD4+ T cells and CD4+CD45RA+ naïve T cells from RA patients was also significantly lower than those from HC (Fig. 1B and C). Together, our data indicated that EZH2 expression was lower in CD4+ T cells from RA patients.
We further explored the EZH2-mediated Treg differentiation regulation mechanism. Given that Treg differentiation is primarily driven by TGF-β signaling [30–35], we further investigated whether EZH2 modulated TGF-β-SMAD signaling. We found that EZH2 inhibition upregulated mRNA expression of SMAD7 but not SMAD2, SMAD3 or SMAD4 (Fig. 4A), which was confirmed by Western blot (Fig. 4B). To reveal whether EZH2 directly bind to the SMAD7, we further evaluated EZH2 binding sites in three lymphoma cell lines using chromatin immunoprecipitation followed by sequencing (ChIP-seq) [36]. As expected, EZH2 binding in SMAD7 locus was two-fold higher than isotype control (Fig. 4C). Besides, we found that EZH2-inhibition downregulated the expression of RUNX1 (Fig. 4D), which is a key transcription factor in the induction and suppressive function of regulatory T cells [33,34,37,38]. Together, these data indicated that SMAD7 and RUNX1 might participate in EZH2-mediated Treg differentiation.
3.2. Attenuated EZH2 suppressed Treg differentiation
3.4. Synovial fluid and FLS from RA patients suppress EZH2 expression in CD4+ T cells
Next, we examined whether EZH2 regulated CD4+ T cell differentiation. Given EZH2 depletion induced a lower frequency of murine Treg and a controversial higher population of Th1 [26,27], we examined Th1, Th17, Treg differentiation in naïve CD4+ T cells treated with GSK126, an EZH2 and H3K27me3 inhibitor [28]. Notably, we found that induction of CD4+CD25highFOXP3+ Treg was attenuated by GSK126 (Fig. 2A, 44.6 ± 16.9% vs. 22.8 ± 19.2%). However, no significant change of Th1 and Th17 differentiation, T cell proliferation or apoptosis was observed (Fig. 2B–E). Consistently, we found that GSK126 downregulated mRNA expression of FOXP3 but not IL17A or IFNγ in Jurkat T cells (Fig. 3A). We also transfected two EZH2 siRNAs into Jurkat T cells (Fig. S3), and found that EZH2-silencing reduced FOXP3 but not IL17A or IFNγ expression (Fig. 3B). Accordingly, we also confirmed Treg population was smaller in RA patients compared with those in HCs (Fig. S2) [29]. Together, lower level of EZH2 in CD4+ T cells from RA potential contributed to defective Treg differentiation,
Beyond lower peripheral Treg population, synovial-resident Treg population is also lower in RA patients [39]. Therefore, we treated CD4+ T cells with synovial fluid from RA and OA patients and examined the dynamics of EZH2. Consistently, we found a significantly lower EZH2 expression in CD4+ T cells treated with SF from RA patients compare to those from OA patients (Fig. 5A), suggesting that the RA synovial milieu suppressed the EZH2 expression. In addition, we also conducted the inhibition experiment of individual cytokines where CD4+ T cells were exposed to RA synovial fluid. We stimulated T cells in 10% RA synovial fluid supplemented with anti-IL1β, anti-IFNγ, antiIL6, anti-IL17, or anti-TNFα neutralized antibody. We found that IL17A neutralization reduced RA synovial fluid inhibition of EZH2 in T cells (Fig. S4) . We further examined whether FLS regulated the FOXP3 expression in CD4+ T cells. As expected, FLS from RA patients downregulated the ratio of Tregs (Fig. 5B) and the expression of EZH2 in T cells (Fig. 5C). Together, the synovial microenvironment of RA patients 3
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potential therapeutic target of EZH2/SMAD7 interaction in RA. Treg plays a pivotal role in maintaining immune homeostasis and Treg deficiency is implicated in the pathogenesis of autoimmune diseases [40], which is orchestrated by genetic and environmental factors [41]. We and others have shown a lower frequency of Treg in RA patients [29]. In line with it, Treg depletion exacerbates arthritis and adoptive transfer of Treg is curative in collagen-induced arthritis model [42,43]. Epigenetic reprogramming is essential for stabilizing Treg lineage [44] during Treg development. Brooks and Guo et al. reveals that Treg has a distinct H3K27me3 landscape compared to naive or other CD4+ Th subsets [8,45]. Treg-specific deletion of Ezh2 results in spontaneous autoimmunity in the mouse model [46]. Pharmacological inhibition of EZH2 destabilizes FOXP3 expression [47]. We also observed that inhibition of EZH2 reduced Treg differentiation. However, there is no H3K27me3 occupancy in the FOXP3 locus in both naïve and Treg, suggesting that EZH2 might regulate FOXP3 indirectly [45]. We sought to explore the underlying mechanism for EZH2 to regulate FOXP3. FOXP3 is the master regulator of Treg development and function [48,49]. Disruption of FOXP3 abrogates thymic Treg development, causing T cells hyperactivation with self-antigens and autoimmune polyendocrinopathy, IBD, and allergy [50]. FOXP3 is mainly regulated by TGFβ signaling, and SMAD2/3/4 are important transcriptional factors in promoting TGFβ signaling while SMAD7 plays an inhibitory role [30,51–53]. EZH2 inhibition reduces p‐SMAD2/3 nuclear translocation and in fibroblasts [54], suppresses the TGF-β-Smad-ASCL1 pathway in small cell lung cancer [55], and attenuates renal fibrosis by promoting Smad7 expression [56]. Besides, Runx1 and AML1 interact with FOXP3 to regulate Treg function [33]. Runx1‐CBFβ complex also facilitates FOXP3 promoter activity [34,35]. Given EZH2 positively regulated Treg differentiation, we investigated whether EZH2 regulates TGFβSMAD signaling and RUNX1 in CD4+ T cells. We demonstrated that the inhibition of EZH2 upregulated the SMAD7 and downregulated RUNX1, which are both essential for the differentiation of Treg. Notably, EZH2 usually methylates lysine 27 of histone H3 (H3K27) to silence gene transcription in classical way, but it can also promote target genes expression in independent of catalytic activity [57]. How EZH2 inhibition downregulated the expression of RUNX1 in T cells still need further study. Decreased frequencies of Treg in the peripheral blood have been reported in autoimmune diseases including RA [58,59]. Meanwhile, Treg was also reduced in inflammatory synovial tissue [39]. So, it would be interesting to explore the effect of synovial fluid and fibroblast-like synoviocytes on EZH2 expression. Consistently, we found a significantly lower expression level of EZH2 in RA SF cocultured T cells compared with osteoarthritis SF, suggesting that inflammatory cytokines in the synovium inflammatory compartment may participate in the regulation of EZH2 expression. For example, MMPs are known to be involved in the conversion of TGF-β from the latent to the active form [60]. Moreover, we detected a decreased trend of EZH2 in T cells after cocultured with RA FLS. Combining the phenomenon that a large amount of T cell infiltration in RA synovium, we speculate that EZH2 participates in the inhibition of Treg differentiation by RA FLS.
Fig. 2. EZH2 Inhibition attenuates Treg differentiation Naïve CD4+ T cells pretreated with EZH2 inhibitor GSK126 or DMSO, then were stimulated with anti-CD3 and anti-CD28 mAbs with Th1, Th17 and Treg-polarizing condition, respectively. The cytokine production, proliferation and apoptosis were measured by flow cytometry on day 5, 3 and 3 accordingly. (A) Treg differentiation, (B) Th1 differentiation, (C) Th17 differentiation, (D) T cell apoptosis, and (E) proliferation of naïve CD4+ T cells treated with GSK126 or DMSO (n = 7). (*: p < 0.05).
contributed to the attenuated EZH2 expression.
5. Conclusions
4. Discussion
In summary, we demonstrated that epigenetic regulator EZH2 was significantly lower in CD4+ T cells from RA patients, which was partially driven by synovial fluid and FLS from RA. Lower level of EZH2 promoted SMAD7 and attenuated RUNX1, which suppressed FOXP3 transcription and subsequently inhibited Treg polarization and ultimately played a role in the pathogenesis of RA.
To elucidate the potential epigenetic regulators participating in the pathogenesis of RA, we screened a serial of epigenetic modificationrelated regulators and identified EZH2, an H3K27 methyltransferase, was significantly lower in CD4+T cells from RA patients. We demonstrated that deficiency of EZH2 suppressed FOXP3 expression through upregulating SMAD7 and downregulating RUNX1 expression in CD4+T cells, which contributed to Treg differentiation. Our study identified a new epigenetic modifier of Treg development in RA and indicates the
Author contributions X.X., Y.L., X.J., X.Z. and H.C. participated in the design of the study. 4
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Fig. 3. The mRNA expressions of FOXP3, IFNγ, IL17A and IL17F in EZH2-suppressed Jurkat T cells. (A) The mRNA expressions of FOXP3, IL17A, IL17F and IFNγ in Jurkat T cells treated with GSK126 for 48 h. (B) The mRNA expressions of FOXP3, IL17A, IL17F and IFNγ in Jurkat T cells after EZH2-silencing for 48 h. Data were representative of three independent experiments.
analysis, Investigation, Data curation, Writing - original draft, Visualization. Yue-ting Li: Conceptualization, Methodology, Software, Formal analysis, Investigation, Data curation, Writing - original draft, Visualization. Xu Jiang: Conceptualization. Xin Ji: Resources. Xin Lu: Resources. Bo Yang: Resources. Li-jun Wu: Resources. Xiao-han Wang: Resources. Jing-bo Guo: Resources. Li-dan Zhao: Resources. Yun-yun Fei: Resources. Hua-xia Yang: Resources. Wen Zhang: Resources. Feng-chun Zhang: Resources. Fu-lin Tang: Resources. Jian-min Zhang: Resources. Wei He: Resources. Hua Chen: Conceptualization, Writing - review & editing. Xuan Zhang: Conceptualization, Project administration, Funding acquisition.
X.L., B.Y., L.W., X.W., J.G., L.Z., Y.F., H.Y., W.Z., F.Z., F.T., J.Z., W.H., H.C., X.Z. contributed to sample collection. X.Z. supervised the study. X.X. and Y.L. performed all the experiments and statistical analyses. X.X. and Y.L. prepared the manuscript. X.Z. and H.C. revised the manuscript. All authors read, provided feedback and approved the final manuscript. Funding This study was supported by grants from the National Natural Science Foundation of China (81788101, 81630044, 81601432, 81550023, 81325019, 81771763, 81273312, 91542000, 81801633), Chinese Academy of Medical Science Innovation Fund for Medical Sciences(CIFMS2016-12M-1–003, 2017–12M-1–008, 2017-I2M-3–011, 2016–12M-1–008), Grant from Medical Epigenetics Research Center, Chinese Academy of Medical Sciences (2017PT31035).
Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
CRediT authorship contribution statement Xin-yue Xiao: Conceptualization, Methodology, Software, Formal
Fig. 4. Attenuated EZH2 upregulates SMAD7 expression and downregulates RUNX1 expression. (A) mRNA and (B) protein expression of SMAD7 in CD4+ T cells treated with GSK126 or DMSO. (C) EZH2 ChIP-seq enrichment of SMAD7 gene in WSUDLCL, Karpass and Pfeiferr T cell lines. (D) GSK126 downregulated RUNX1 expression in CD4+ T cells. (*: p < 0.05). 5
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Fig. 5. Synovial fluid and FLS from RA patients downregulate EZH2 expression in T cells. (A) EZH2 mRNA expression in CD4+ T cells treated with synovial fluid (SF) from RA patients (n = 3) or osteoarthritis (n = 3). (B) Treg differentiation in the presence of FLS from RA patients (n = 4). (C) EZH2 expression in CD4+ T cells in the presence of FLS from RA patients (n = 4). (*: p < 0.05, ***: p < 0.001).
Acknowledgments [14]
We are sincerely grateful to all patients participating our research. Appendix A. Supplementary data
[15]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jaut.2020.102404.
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EZH2 deficiency attenuates Treg differentiation in rheumatoid arthritis Xin-yue Xiaoa,1, Yue-ting Lia,1, Xu Jianga, Xin Jia, Xin Lub, Bo Yangb, Li-jun Wud, Xiao-han Wange, Jing-bo Guof, Li-dan Zhaoa, Yun-yun Feia, Hua-xia Yanga, Wen Zhanga, Feng-chun Zhanga, Fu-lin Tanga, Jian-min Zhangc, Wei Hec, Hua Chena,∗∗, Xuan Zhanga,∗ a
Department of Rheumatology and Clinical Immunology, Peking Union Medical College Hospital, Clinical Immunology Center, Chinese Academy of Medical Sciences and Peking Union Medical College, The Ministry of Education Key Laboratory, Beijing, 100730, China b Department of Orthopedics, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100730, China c Department of Immunology & National Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS) & Peking Union Medical College, Beijing, 100005, China d Department of Rheumatology and Clinical Immunology, People's Hospital of Xinjiang Uygur Autonomous Region, Urumchi, 830001, China e Department of Rheumatology, AnYang District Hospital, AnYang, HeNan Province, 455000, China f Department of Traditional Chinese Medicine, 256th Clinical Department of Bethune International Peace Hospital of PLA, Shijiazhuang, 050800, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Rheumatoid arthritis EZH2 CD4+ T cells Differentiation Treg
The chromatin modifier enhancer of zeste homolog 2 (EZH2) methylates lysine 27 of histone H3 (H3K27) and regulates T cell differentiation. However, the potential role of EZH2 in the pathogenesis of rheumatoid arthritis (RA) remains elusive. We analyzed EZH2 expression in PBMC, CD4+ T cells, CD19+ B cell, and CD14+ monocytes from active treatment-naïve RA patients and healthy controls (HC). We also suppressed EZH2 expression using EZH2 inhibitor GSK126 and measured CD4+ T cell differentiation, proliferation and apoptosis. We further examined TGFβ-SMAD and RUNX1 signaling pathways in EZH2-suppressed CD4+ T cells. Finally, we explored the regulation mechanism of EZH2 by RA synovial fluid and fibroblast-like synoviocyte (FLS) by neutralizing key proinflammatory cytokines. EZH2 expression is lower in PBMC and CD4+ T cells from RA patients than those from HC. EZH2 inhibition suppressed regulatory T cells (Tregs) differentiation and FOXP3 transcription, and downregulated RUNX1 and upregulated SMAD7 expression in CD4+ T cells. RA synovial fluid and fibroblast-like synoviocytes suppressed EZH2 expression in CD4+ T cells, which was partially neutralized by anti-IL17 antibody. Taken together, EZH2 in CD4+ T cells from RA patients was attenuated, which suppressed FOXP3 transcription through downregulating RUNX1 and upregulating SMAD7 in CD4+ T cells, and ultimately suppressed Tregs differentiation. IL17 in RA synovial fluid might promote downregulation of EZH2 in CD4+ T cells. Defective EZH2 in CD4+ T cells might contribute to Treg deficiency in RA.
1. Introduction
regulation of cytokine-induced transcriptional factors, which is highly environment-dependent as well as epigenetically regulated [6]. Epigenetic processes such as DNA methylation, histone modification, nucleosome positioning, miRNAs and DNA supercoiling stresses are essential for the regulation of gene expression [7,8] and bridges the genes and the diseases [9]. The evidence of epigenetic dysregulation in autoimmune diseases is accumulating [10]. For instance, epigenetic modifications by DNMT1 and TET2 regulate T cell differentiation and are implicated in autoimmune diseases including systemic lupus erythematosus (SLE) and Sjögren syndrome [11,12]. The concordance of RA between identical twins is 12–35%, implicating other etiology
Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by progressive joint destruction and subsequent disability [1]. Synovium inflammation is a hallmark of RA, which consists of massive activated CD4+ T cells [2], suggesting disturbed homeostasis of CD4+ T cells plays a critical role in the development of RA [3,4]. Furthermore, CD4+ T cell depletion using anti-CD4 antibody prevented experimental arthritis [5]. Naïve CD4+ T cells differentiate into T cell subsets including T helper 1 (Th1), T helper 17 (Th17), and regulatory T cells (Treg) by the
∗
Corresponding author. Corresponding author. E-mail addresses: [email protected] (H. Chen), [email protected] (X. Zhang). 1 Xin-yue Xiao and Yue-ting Li contributed equally to this manuscript. ∗∗
https://doi.org/10.1016/j.jaut.2020.102404 Received 4 November 2019; Received in revised form 2 January 2020; Accepted 5 January 2020 0896-8411/ © 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: Xin-yue Xiao, et al., Journal of Autoimmunity, https://doi.org/10.1016/j.jaut.2020.102404
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days, then were stained with Ki67 and measured by a BD FACSAria II flow cytometry. Apoptosis of cells were measured by Annexin V and 7AAD (BD Pharmingen). For T cell differentiation, naïve CD4+ T cells (1 × 10 6 cells/ml) were stimulated with anti-CD3 (5 μg/ml)/anti-CD28 (5 μg/ml) for 5 days. For Th17 differentiation, IL-1β (10 ng/ml), IL-6(10 ng/ml), TGF-β (10 ng/ml) and IL-23(10 ng/ml) were supplemented; for Th1 differentiation, IL-2 (10 ng/ml) and IL-12 (10 ng/ml) were supplemented. PMA, ionomycin and Golgistop (BD Pharmingen) were added in the last 4 h. Cells were harvested, surface stained, permeabilized, stained with intracellular IL-17A and IFN-gamma and detected by flow cytometry. For Treg differentiation, IL-2 (10 ng/ml) and TGF-β (10 ng/ml) were supplemented. After surface staining of CD4 and CD25, cells were permeabilized, stained with FOXP3 and detected by flow cytometry. Jurkat T cells were obtained from National Infrastructure of Cell Line Resource (Beijing, China) and were maintained in RPMI-1640. Jurkat T cells were transfected with control siRNA (si-NC), or EZH2 siRNA (siEZH2-1, siEZH2-2) (100 nM) using a Gene Pulser Xcell Electroporation Systems (Biorad) according to the manufacturer’ s instructions. Naïve CD4+ T cells (1 × 106) were stimulated with anti-CD3 mAb (5 μg/ml), anti-CD28 (5 μg/ml) mAb supplemented with 10% RA synovial fluid and anti-IL1β(5 μg/ml), anti-IFNγ(5 μg/ml), anti-IL6(5 μg/ ml), anti-IL17A(5 μg/ml), or anti-TNFα(5 μg/ml) neutralized antibody. Cells were harvested after 4 days and analyzed by flow cytometry. FLS (1 × 105 cells/well) were seeded at in DMEM supplemented with 10% FCS and incubated overnight. Then naïve T cells (1 × 106) were added and were stimulated with anti-CD3 mAb (5 μg/ml), antiCD28 (5 μg/ml) mAb, TGF-β (20 ng/mL) and IL-2 (20 ng/ml) for 4 days. Cells were harvested and analyzed by flow cytometry.
beyond genetic background is involved [13,14]. Recent studies suggested epigenetic modifications contribute to the tumor-like activation of fibroblast-like synoviocyte (FLS) from RA [15–17]. Furthermore, the DNA methylation landscapes of peripheral blood mononuclear cells (PBMC) and FLS are distinctly different among RA patients, healthy control and osteoarthritis [18,19]. However, whether the aberrant epigenetic changes regulate CD4+ T cell differentiation in RA patients remains unknown. The chromatin modifier enhancer of zeste homolog 2 (EZH2), a catalytic subunit of Polycomb repressive complex 2 (PRC2), methylates lysine 27 of histone H3 (H3K27) to silence gene transcription in cell cycle and differentiation [20]. Accumulating evidence has highlighted the links between EZH2 and T cell differentiation [21]. EZH2 silences the expression of Tbx1 and Gata3 in T helper (Th) cells and EZH2 can also regulate T cell polarization [22,23]. Additionally, overexpression of EZH2 in FLS from RA patients represses Wnt inhibitor SFRP1 and subsequently activates the pathogenic FLS [24], which is potentially regulated by Lnc-IL7R [25]. Taken together, these studies indicate that EZH2 might participate the abnormal T cell polarization and contribute to the development of RA. In this study, we reported that EZH2 expression was lower in CD4+ T cells from RA patients. EZH2 inhibition attenuated the FOXP3 expression and Treg differentiation, by downregulating RUNX1 and upregulating SMAD7 expression. Furthermore, synovial fluid and FLS from RA patients downregulated EZH2 expression in CD4+ T cells. 2. Materials and methods 2.1. Patients and healthy controls A total of 52 treatment-naïve active RA patients fulfilled the 2010 ACR revised criteria for RA were enrolled. The disease activity was measured by the 28-joint DAS with CRP (DAS28-CRP) (Supplementary Table 1). The study was approved by the Institutional Review Board of Peking Union Medical College Hospital and written informed consent was obtained from all participants.
2.4. Real-time quantitative PCR Fresh cells were harvested and resuspended in TRIzol (Invitrogen Life Technologies) and stored at − 70 °C. Total RNA was extracted using the RNeasy Mini Kit (Qiagen). Reverse transcription reactions were prepared using the SYBR Premix Ex Taq System (Takara). Realtime PCR was performed using a 7900HT Fast Real-Time PCR System (Applied Biosystems). Relative expression of EZH2, FOXP3, IL17A and IFNγ against GAPDH were calculated using the comparative ΔΔCT method.
2.2. Antibodies and reagents The following fluorochrome-conjugated or unlabeled monoclonal antibodies were used: CD4 (RPA-T4), CD14 (61D3), IFN-γ (B27), FOXP3 (PCH101), CD25 (BC96), IL-17 (64DEC17) (eBioscience, San Diego, CA, USA), CD19(HIB19), Ki67(KI671-A), CD90(5E10) (Biolegend, USA). The corresponding IgG isotype antibodies were used as control. Annexin V Apoptosis Detection Kit was obtained from BD Bioscience (559763). Recombinant IL-2, IL-4, IL-6, IL-1β, IL-23, and TGF-β were purchased from PeproTech (Rocky Hill, NJ, USA). Phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma-Aldrich (St Louis, MO, USA). Antibodies against β-actin (4970) were purchased from Cell Signaling Technology (Danvers, MA). Antibody against EZH2 was purchased from Affinity (AF5150) for WB and BD bioscience (562478) for flowcytometry. Antibody against SMAD7 (ab216428) and RUNX1 (EPR3099) were purchased from Abcam (USA). siRNA was purchased from Ruibo biotech (Guangzhou, China)
2.5. Western blotting Cells were lysed by incubation for 1 h in a RIPA lysis buffer 1 mM PMSF and a proteinase inhibitor cocktail (BD Biosciences, Franklin Lakes, NJ). The lysates were kept on ice and vortexed every 10 min for 1 h before centrifugation at 12 000 g at 4 °C. Equal amounts of protein were separated by SDS-PAGE (Invitrogen), transferred to PVDF membranes (Millipore, Billerica, MA, USA), blocked with 5% dried milk in PBS containing 0.5% Tween 20, incubated with indicated antibodies, then incubated with HRP-conjugated secondary antibody and finally developed using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Boston, MA, USA).
2.3. Cell separation, culture, stimulation and electrotransfection PBMC were prepared with Ficoll-Hypaque density-gradient centrifugation and synovial cell suspension was prepared by enzyme digestion. Naïve CD4+ T cells, CD4+ T cells, CD19+ B cells and CD14+ monocytes were isolated from PBMC and synovium using magnetic isolation kits accordingly (Miltenyi Biotec, Germany), respectively. Naïve CD4+ T cells were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) in 5% CO2 at 37 °C. For proliferation assays, naïve CD4+ T cells (1 × 10 7 cells/ml) were stimulated with anti-CD3 (5 μg/ml) and anti-CD28 (5 μg/ml) mAbs for 3
2.6. Statistical analysis Data were summarized with mean ± SD. Normal distributed data was analyzed by Kolmogorov–Smirnov and Shapiro–Wilk tests (P > 0.05). The Student's t-test for continuous variables with a normal distribution. For the data with a normal distribution and homogeneity of variance, one-way analysis of variance. All data were analyzed using SPSS 22.0 software (SPSS, Chicago, IL, USA). 2
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Fig. 1. EZH2 expression in PBMC and CD4+ T cells from RA and HC. (A) EZH2 mRNA expression in PBMC, CD4+ T cells, CD14+ monocytes and CD19+ B cells from HCs and RA patients (n = 22). (B) EZH2 protein expression in peripheral CD4+cells from HCs and RA patients (n = 5). (C) EZH2 protein expression in peripheral CD4+ CD45RA+ naïve T cells from HCs and RA patients (n = 20). (**: p < 0.01, ****: p < 0.0001).
3. Results
which plays a role in the pathogenesis of RA.
3.1. Lower EZH2 expression in CD4+ T cells from RA patients
3.3. Attenuated EZH2 promotes SMAD7 and suppresses RUNX1 in CD4+ T cells
We first screened expression profile of epigenetic modification-related genes including PRMT5, TRIM22, TET1, TET2, TET3 and GADD45B, RFX1, EZH2 and SIRT1, in PBMC, CD4+ T cells, CD19+B cells and CD14+monocytes from treatment-naïve RA patients and healthy controls (HC) using quantitative PCR (Fig. 1A and S1). We found that expression of EZH2, TET3 and GADD45B were lower and expression of PRMT5 and TET1 were higher in PBMCs from RA. We confirmed that expression of EZH2 and TET3 were lower in CD4+ T cells from RA. As the expression of EZH2 was higher than TET2 in PBMC and T cells, we chose the EZH2 for further research. Given EZH2 expression was significantly lower in PBMC and CD4+ T cells from RA than those from HCs (Fig. 1A), we decided to focus on potential regulation of CD4+ T cells by EZH2. We further confirmed that protein expression of EZH2 in CD4+ T cells and CD4+CD45RA+ naïve T cells from RA patients was also significantly lower than those from HC (Fig. 1B and C). Together, our data indicated that EZH2 expression was lower in CD4+ T cells from RA patients.
We further explored the EZH2-mediated Treg differentiation regulation mechanism. Given that Treg differentiation is primarily driven by TGF-β signaling [30–35], we further investigated whether EZH2 modulated TGF-β-SMAD signaling. We found that EZH2 inhibition upregulated mRNA expression of SMAD7 but not SMAD2, SMAD3 or SMAD4 (Fig. 4A), which was confirmed by Western blot (Fig. 4B). To reveal whether EZH2 directly bind to the SMAD7, we further evaluated EZH2 binding sites in three lymphoma cell lines using chromatin immunoprecipitation followed by sequencing (ChIP-seq) [36]. As expected, EZH2 binding in SMAD7 locus was two-fold higher than isotype control (Fig. 4C). Besides, we found that EZH2-inhibition downregulated the expression of RUNX1 (Fig. 4D), which is a key transcription factor in the induction and suppressive function of regulatory T cells [33,34,37,38]. Together, these data indicated that SMAD7 and RUNX1 might participate in EZH2-mediated Treg differentiation.
3.2. Attenuated EZH2 suppressed Treg differentiation
3.4. Synovial fluid and FLS from RA patients suppress EZH2 expression in CD4+ T cells
Next, we examined whether EZH2 regulated CD4+ T cell differentiation. Given EZH2 depletion induced a lower frequency of murine Treg and a controversial higher population of Th1 [26,27], we examined Th1, Th17, Treg differentiation in naïve CD4+ T cells treated with GSK126, an EZH2 and H3K27me3 inhibitor [28]. Notably, we found that induction of CD4+CD25highFOXP3+ Treg was attenuated by GSK126 (Fig. 2A, 44.6 ± 16.9% vs. 22.8 ± 19.2%). However, no significant change of Th1 and Th17 differentiation, T cell proliferation or apoptosis was observed (Fig. 2B–E). Consistently, we found that GSK126 downregulated mRNA expression of FOXP3 but not IL17A or IFNγ in Jurkat T cells (Fig. 3A). We also transfected two EZH2 siRNAs into Jurkat T cells (Fig. S3), and found that EZH2-silencing reduced FOXP3 but not IL17A or IFNγ expression (Fig. 3B). Accordingly, we also confirmed Treg population was smaller in RA patients compared with those in HCs (Fig. S2) [29]. Together, lower level of EZH2 in CD4+ T cells from RA potential contributed to defective Treg differentiation,
Beyond lower peripheral Treg population, synovial-resident Treg population is also lower in RA patients [39]. Therefore, we treated CD4+ T cells with synovial fluid from RA and OA patients and examined the dynamics of EZH2. Consistently, we found a significantly lower EZH2 expression in CD4+ T cells treated with SF from RA patients compare to those from OA patients (Fig. 5A), suggesting that the RA synovial milieu suppressed the EZH2 expression. In addition, we also conducted the inhibition experiment of individual cytokines where CD4+ T cells were exposed to RA synovial fluid. We stimulated T cells in 10% RA synovial fluid supplemented with anti-IL1β, anti-IFNγ, antiIL6, anti-IL17, or anti-TNFα neutralized antibody. We found that IL17A neutralization reduced RA synovial fluid inhibition of EZH2 in T cells (Fig. S4) . We further examined whether FLS regulated the FOXP3 expression in CD4+ T cells. As expected, FLS from RA patients downregulated the ratio of Tregs (Fig. 5B) and the expression of EZH2 in T cells (Fig. 5C). Together, the synovial microenvironment of RA patients 3
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potential therapeutic target of EZH2/SMAD7 interaction in RA. Treg plays a pivotal role in maintaining immune homeostasis and Treg deficiency is implicated in the pathogenesis of autoimmune diseases [40], which is orchestrated by genetic and environmental factors [41]. We and others have shown a lower frequency of Treg in RA patients [29]. In line with it, Treg depletion exacerbates arthritis and adoptive transfer of Treg is curative in collagen-induced arthritis model [42,43]. Epigenetic reprogramming is essential for stabilizing Treg lineage [44] during Treg development. Brooks and Guo et al. reveals that Treg has a distinct H3K27me3 landscape compared to naive or other CD4+ Th subsets [8,45]. Treg-specific deletion of Ezh2 results in spontaneous autoimmunity in the mouse model [46]. Pharmacological inhibition of EZH2 destabilizes FOXP3 expression [47]. We also observed that inhibition of EZH2 reduced Treg differentiation. However, there is no H3K27me3 occupancy in the FOXP3 locus in both naïve and Treg, suggesting that EZH2 might regulate FOXP3 indirectly [45]. We sought to explore the underlying mechanism for EZH2 to regulate FOXP3. FOXP3 is the master regulator of Treg development and function [48,49]. Disruption of FOXP3 abrogates thymic Treg development, causing T cells hyperactivation with self-antigens and autoimmune polyendocrinopathy, IBD, and allergy [50]. FOXP3 is mainly regulated by TGFβ signaling, and SMAD2/3/4 are important transcriptional factors in promoting TGFβ signaling while SMAD7 plays an inhibitory role [30,51–53]. EZH2 inhibition reduces p‐SMAD2/3 nuclear translocation and in fibroblasts [54], suppresses the TGF-β-Smad-ASCL1 pathway in small cell lung cancer [55], and attenuates renal fibrosis by promoting Smad7 expression [56]. Besides, Runx1 and AML1 interact with FOXP3 to regulate Treg function [33]. Runx1‐CBFβ complex also facilitates FOXP3 promoter activity [34,35]. Given EZH2 positively regulated Treg differentiation, we investigated whether EZH2 regulates TGFβSMAD signaling and RUNX1 in CD4+ T cells. We demonstrated that the inhibition of EZH2 upregulated the SMAD7 and downregulated RUNX1, which are both essential for the differentiation of Treg. Notably, EZH2 usually methylates lysine 27 of histone H3 (H3K27) to silence gene transcription in classical way, but it can also promote target genes expression in independent of catalytic activity [57]. How EZH2 inhibition downregulated the expression of RUNX1 in T cells still need further study. Decreased frequencies of Treg in the peripheral blood have been reported in autoimmune diseases including RA [58,59]. Meanwhile, Treg was also reduced in inflammatory synovial tissue [39]. So, it would be interesting to explore the effect of synovial fluid and fibroblast-like synoviocytes on EZH2 expression. Consistently, we found a significantly lower expression level of EZH2 in RA SF cocultured T cells compared with osteoarthritis SF, suggesting that inflammatory cytokines in the synovium inflammatory compartment may participate in the regulation of EZH2 expression. For example, MMPs are known to be involved in the conversion of TGF-β from the latent to the active form [60]. Moreover, we detected a decreased trend of EZH2 in T cells after cocultured with RA FLS. Combining the phenomenon that a large amount of T cell infiltration in RA synovium, we speculate that EZH2 participates in the inhibition of Treg differentiation by RA FLS.
Fig. 2. EZH2 Inhibition attenuates Treg differentiation Naïve CD4+ T cells pretreated with EZH2 inhibitor GSK126 or DMSO, then were stimulated with anti-CD3 and anti-CD28 mAbs with Th1, Th17 and Treg-polarizing condition, respectively. The cytokine production, proliferation and apoptosis were measured by flow cytometry on day 5, 3 and 3 accordingly. (A) Treg differentiation, (B) Th1 differentiation, (C) Th17 differentiation, (D) T cell apoptosis, and (E) proliferation of naïve CD4+ T cells treated with GSK126 or DMSO (n = 7). (*: p < 0.05).
contributed to the attenuated EZH2 expression.
5. Conclusions
4. Discussion
In summary, we demonstrated that epigenetic regulator EZH2 was significantly lower in CD4+ T cells from RA patients, which was partially driven by synovial fluid and FLS from RA. Lower level of EZH2 promoted SMAD7 and attenuated RUNX1, which suppressed FOXP3 transcription and subsequently inhibited Treg polarization and ultimately played a role in the pathogenesis of RA.
To elucidate the potential epigenetic regulators participating in the pathogenesis of RA, we screened a serial of epigenetic modificationrelated regulators and identified EZH2, an H3K27 methyltransferase, was significantly lower in CD4+T cells from RA patients. We demonstrated that deficiency of EZH2 suppressed FOXP3 expression through upregulating SMAD7 and downregulating RUNX1 expression in CD4+T cells, which contributed to Treg differentiation. Our study identified a new epigenetic modifier of Treg development in RA and indicates the
Author contributions X.X., Y.L., X.J., X.Z. and H.C. participated in the design of the study. 4
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Fig. 3. The mRNA expressions of FOXP3, IFNγ, IL17A and IL17F in EZH2-suppressed Jurkat T cells. (A) The mRNA expressions of FOXP3, IL17A, IL17F and IFNγ in Jurkat T cells treated with GSK126 for 48 h. (B) The mRNA expressions of FOXP3, IL17A, IL17F and IFNγ in Jurkat T cells after EZH2-silencing for 48 h. Data were representative of three independent experiments.
analysis, Investigation, Data curation, Writing - original draft, Visualization. Yue-ting Li: Conceptualization, Methodology, Software, Formal analysis, Investigation, Data curation, Writing - original draft, Visualization. Xu Jiang: Conceptualization. Xin Ji: Resources. Xin Lu: Resources. Bo Yang: Resources. Li-jun Wu: Resources. Xiao-han Wang: Resources. Jing-bo Guo: Resources. Li-dan Zhao: Resources. Yun-yun Fei: Resources. Hua-xia Yang: Resources. Wen Zhang: Resources. Feng-chun Zhang: Resources. Fu-lin Tang: Resources. Jian-min Zhang: Resources. Wei He: Resources. Hua Chen: Conceptualization, Writing - review & editing. Xuan Zhang: Conceptualization, Project administration, Funding acquisition.
X.L., B.Y., L.W., X.W., J.G., L.Z., Y.F., H.Y., W.Z., F.Z., F.T., J.Z., W.H., H.C., X.Z. contributed to sample collection. X.Z. supervised the study. X.X. and Y.L. performed all the experiments and statistical analyses. X.X. and Y.L. prepared the manuscript. X.Z. and H.C. revised the manuscript. All authors read, provided feedback and approved the final manuscript. Funding This study was supported by grants from the National Natural Science Foundation of China (81788101, 81630044, 81601432, 81550023, 81325019, 81771763, 81273312, 91542000, 81801633), Chinese Academy of Medical Science Innovation Fund for Medical Sciences(CIFMS2016-12M-1–003, 2017–12M-1–008, 2017-I2M-3–011, 2016–12M-1–008), Grant from Medical Epigenetics Research Center, Chinese Academy of Medical Sciences (2017PT31035).
Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
CRediT authorship contribution statement Xin-yue Xiao: Conceptualization, Methodology, Software, Formal
Fig. 4. Attenuated EZH2 upregulates SMAD7 expression and downregulates RUNX1 expression. (A) mRNA and (B) protein expression of SMAD7 in CD4+ T cells treated with GSK126 or DMSO. (C) EZH2 ChIP-seq enrichment of SMAD7 gene in WSUDLCL, Karpass and Pfeiferr T cell lines. (D) GSK126 downregulated RUNX1 expression in CD4+ T cells. (*: p < 0.05). 5
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Fig. 5. Synovial fluid and FLS from RA patients downregulate EZH2 expression in T cells. (A) EZH2 mRNA expression in CD4+ T cells treated with synovial fluid (SF) from RA patients (n = 3) or osteoarthritis (n = 3). (B) Treg differentiation in the presence of FLS from RA patients (n = 4). (C) EZH2 expression in CD4+ T cells in the presence of FLS from RA patients (n = 4). (*: p < 0.05, ***: p < 0.001).
Acknowledgments [14]
We are sincerely grateful to all patients participating our research. Appendix A. Supplementary data
[15]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jaut.2020.102404.
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