Epigenetic modification of mesenchymal stromal cells enhances their suppressive effects on the Th17 responses of cells from rheumatoid arthritis patients

Background The aim of this study was to investigate if epigenetically modified human mesenchymal stromal cells (hMSCs) can regulate the Th17-related immune responses. Methods We tested epigenetic drug combinations at various doses and selected the four combinations that resulted in maximal interleukin (IL)-10 and indoleamine 2,3-dioxygenase gene expression in hMSCs. We examined the effects of epigenetically modified hMSCs (epi-hMSCs) on CD4+ T-cell proliferation and inflammatory cytokine secretion under Th0- and Th17-polarizing conditions using mixed lymphocyte reactions and enzyme-linked immunosorbent assays (ELISAs). We determined Th17 cytokine levels and the percentage of Th17 cells among synovial fluid mononuclear cells (SFMCs) from rheumatoid arthritis (RA) patients by ELISA and flow cytometry. Results Epi-hMSCs inhibited the development of IL-17-producing cells in culture. The percentages of IL-17+ and interferon (IFN)-γ+ cells among peripheral blood mononuclear cells from healthy donors were lower under both the Th0 and Th17 conditions in the presence of epi-hMSCs than in the presence of no or untreated hMSCs. Epi-hMSC-treated RA patient SFMCs secreted lower levels of IL-17 and IFN-γ than RA patient SFMCs cultured without hMSCs or with untreated hMSCs. Conclusions An optimal combination of hypomethylating agents and histone deacetylase inhibitors can enhance the immunomodulatory potential of hMSCs, which may be useful for RA treatment.


Background
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by uncontrolled synovitis and subsequent destruction of the cartilage and bone. In RA, interleukin (IL)-17 induces the production of proinflammatory mediators, such as IL-1 and tumor necrosis factor (TNF)-α, from synovial fibroblasts, macrophages, and chondrocytes. The levels of IL-17 produced by CD4 + T cells in the synovium are significantly higher in patients with RA than in patients with osteoarthritis [1,2]. T-helper (Th)17 cells, which are defined by their selective IL-17 secretion, are a distinct lineage of CD4 + helper T cells. Their development and activity are regulated by Th1 and Th2 cytokines [3][4][5].
Mesenchymal stromal cells (MSCs) are present in various tissues, including the bone marrow (BM), umbilical cord blood, and adipose tissues. They possess intrinsic immunoregulatory properties that modulate innate and adaptive immunity [6][7][8][9]. As a result, MSCs have been widely studied as a promising platform for a cell-based therapy to prevent or treat RA. The proof of principle has been established in preclinical studies [10][11][12][13][14]. BM-MSCs can suppress T-cell proliferation via contact-dependent mechanisms and produce soluble immunoregulatory factors, such as IL-10 [15]. The immune-altering abilities of MSCs have been exploited for therapeutic purposes, as in the ongoing clinical trials of the transfer of MSCs for the treatment of rheumatic diseases [12].
DNA methyltransferases and histone deacetylases (HDAC) are epigenetic modulators that regulate DNA methylation and the histone acetylation status of genomic DNA, respectively, thereby regulating the expression of target genes. Different cell types possess different methylation and histone status profiles. Responses to hypomethylating agents (HMAs) or HDAC inhibitors (HDACi) vary according to individual cellular characteristics. HMAs and HDACi increase the efficiency of induced pluripotent stem cell generation and somatic cell reprogramming through epigenetic modifications [16][17][18][19]. Recent studies showed that an HMA mitigated graft-versus-host disease and experimental colitis by converting effector T cells to regulatory T cells [20][21][22][23]. In our previous study, epigenetic modification of human MSCs (hMSCs) with HMAs plus HDACi resulted in higher expression of Runx-2, BDNF, and Sox-9 than the control treatment. HMAs and HDACi enhance the in-vitro differentiation of MSCs, suggesting that epigenetic modification could alter the function of hMSCs [22].
In this study, we aimed to investigate if epigenetic modification could enhance the immunoregulatory properties of hMSCs, enabling them to inhibit the production of proinflammatory cytokines by T cells and suppress Th17 responses in cells from patients with RA.

Patient populations and study design
We obtained synovial fluid from six RA patients, all of whom fulfilled the 1987 revised criteria of the American College of Rheumatology (formerly the American Rheumatism Association) [24]. We also obtained peripheral blood from six healthy volunteers.

Culture and assessment of MSCs
We acquired hMSCs, which were isolated and expanded from healthy donor BM, from the Catholic Institute of Cell Therapy (Catholic Medical Center, Seoul, Republic of Korea). We expanded hMSCs up to passage 3. After the third passage, we assessed the cells for the MSC phenotype CD73 + CD90 + CD105 + CD11 − CD19 − CD34 − CD44 − HLA-DR − using APC-conjugated anti-CD73, FITC-conjugated anti-CD90, PerCP 5.5-conjugated anti-CD105, APC-conjugated anti-CD11, PE-conjugated anti-CD19, FITC-conjugated anti-CD34, PerCP 5.5-conjugated anti-CD45, and FITCconjugated anti-HLADR. For the phenotypic analysis of MSCs, each sample was divided into three aliquots to prevent the occurrence of interference waves. All antibodies were purchased from BD Biosciences (San Diego, CA, USA). We determined the level of nonantigen-specific fluorescence by incubating cell aliquots with isotype-matched monoclonal control antibodies. We analyzed the samples with a FACSCalibur™ flow cytometer (BD Biosciences, San Diego, CA, USA) using FACSDiva™ software. For each analysis, we assayed a minimum of 10,000 cells.

Isolation of peripheral blood mononuclear cells and synovial fluid mononuclear cells
We prepared peripheral blood mononuclear cells (PBMCs) from heparinized blood and synovial fluid mononuclear cells (SFMCs) from heparinized synovial fluids by Ficoll-Paque (GE Healthcare Life Sciences, Little Chalfont, UK) density-gradient centrifugation. Cells were cultured as described previously [25]. Briefly, we prepared cell suspensions (10 6 cells/ml) in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. We dispensed 1-ml aliquots of the suspensions into 24-well plates (Nunc, Roskilde, Denmark) for incubation. For cytokine detection at the single-cell level by flow cytometric analysis, we stimulated PBMCs with 50 ng/ml phorbol 12-myristate 13-acetate and 1 μg/ml ionomycin in the presence of GolgiStop™ (BD Biosciences) for 4 h.

CD4 + T-cell isolation by magnetic-activated cell sorting (MACS)
Anti-CD4 microbeads were used according to the recommendations of the manufacturer (Miltenyi Biotec, Sunnyvale, CA, USA). PBMCs were resuspended in 80 μl FCS staining buffer. Anti-CD4 microbeads (20 μl) were added and incubated for 15 min at 6-12°C. Saturating amounts of fluorochrome-conjugated antibodies were added, and cells were incubated for an additional 10 min. Cells were diluted in 2.5 ml 2% FCS staining buffer, pelleted, resuspended in 500 μl FCS, and magnetically separated, usually on an Auto-MACS magnet (Miltenyi Biotec, Bergisch Gladbach, Germany) fitted with a MACS mass spectrophotometry column. Flow-through and two 1-ml washes were collected as the negative fraction. After removal from the magnet, enriched cells were collected in two 0.5-ml aliquots from the column. Alternatively, cells stained with PE-conjugated anti-CD4 were washed, magnetically labeled with anti-PE microbeads (20 μl added to an 80 μl cell suspension for 15 min at 6-12°C), and magnetically separated as described above. The purity of cells was assessed by flow cytometric analysis of stained cells on a FACS Vantage sorter (BD Biosciences). Most of the isolated cells (> 97%) exhibited the CD4 T-cell marker.

Suppression assay
PBMCs were collected from healthy donors (n = 3). We stimulated CD4 + T cells with anti-CD3 (1 μg/ml) and T cell-depleted, irradiated (3000 rad) antigen-presenting cells (non-CD4 + T cells) in the presence or absence of epi-hMSCs. We used effector T and antigen-presenting cells (non-CD4 + T cells) from the same donor. The purities of the T-cell subsets were > 95% as determined by flow cytometry (data not shown). We examined the proliferation of the CD4 + T cells by adding [ 3 H]-thymidine (1 mCi/well; GE Healthcare) to the cultures for the final 8 h of incubation. We assessed [ 3 H]-thymidine incorporation using a liquid β-scintillation counter (Beckman Coulter, Brea, CA, USA). We also confirmed the immunomodulatory effect of MSCs by analyzing the epigenetic reprogramming effects on the T-cell activation/proliferation during Th17 differentiation using carboxyfluorescein succinimidyl ester (CFSE; Sigma Aldrich) labelling.

Enzyme-linked immunosorbent assays
We measured the levels of cytokines, such as IL-17, IFN-γ, IL-10, and IL-2, in the CD4 + T-cell culture supernatants using sandwich enzyme-linked immunosorbent assays (ELISAs; R&D Systems) according to the manufacturer's instructions. We measured the absorbance at 405 nm using an ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Statistical analysis
We performed statistical analyses with SPSS software (version 16.0; SPSS, Chicago, IL, USA). Continuous variables are summarized as mean ± standard deviation (SD). Categorical variables are summarized as a percentage of the group total. We performed independent t tests for continuous variables. We used the nonparametric Wilcoxon signed-rank test to compare T-cell proliferation, cytokine production, and gene expression among the control and treatment groups. We performed chi-squared/Fisher's exact tests for categorical variables. A P value < 0.05 was considered statistically significant.

The expression of IDO and IL-10 by epi-hMSCs
We selected four of the 36 combinations of HMAs and HDACi based on their ability to significantly upregulate the expression of IL-10 and IDO over those in untreated hMSCs: 2 μM 5-AZA + 5 mM VPA (A2V5), 2 μM 5-AZA + 10 mM VPA (A2V10), 100 nM DEC + 100 nM TSA (D100T100), and 100 nM DEC + 500 nM TSA (D100T500). We found that the A2V10 combination had an additive effect, whereas the A2V5, D100T100, and D100T500 combinations had synergistic effects (Fig. 1a). An appreciable increase in protein expression was confirmed upon use of the four combinations selected on the basis of the gene expression results (Fig. 1b). We did not observe a higher rate of apoptosis in the drug treatment groups than in the untreated control (data not shown). Thus, the selected dosing combinations effectively increased immune regulatory molecule expression without inducing toxicity.

Discussion
In the present study, we observed that epi-hMSCs induced by treatment with an HMA plus an HDACi had greater immunoregulatory properties than untreated hMSCs. These findings demonstrate the potential of epi-hMSCs as a treatment option for RA. Previous studies have reported that the immunomodulatory effects of hMSCs are associated with their abilities to inhibit Th1 and Th17 cell differentiation [26,27]. The modulation of key markers on MSCs will be critical for developing high-performance cells for clinical application in Th17-dependent immunological diseases, such as RA. IDO is a powerful immunomodulatory factor that is secreted by hMSCs. Moreover, IL-10 secretion by MSCs is critical for their immunomodulatory functions in immune disease models, including those for arthritis [28].
In this study, we investigated if treatment with an HMA in combination with an HDACi could increase the gene expression of IL-10 and IDO by MSCs, based on the success of our previous approach to upregulate key differentiation factors in MSCs [22]. The enhanced IL-10 and IDO expression in epi-hMSCs after combination treatment confirmed that epigenetic modification can modulate gene expression levels. The epi-hMSCs also had greater immunosuppressive effects on T-cell proliferation and cytokine expression, and Th17 cell differentiation, than the untreated hMSCs.
Th17 cells protect the host against extracellular pathogens that are encountered at mucosal surfaces. They also play a detrimental role in experimental murine models of inflammatory diseases, such as multiple sclerosis and RA, as well as in human inflammatory bowel disease and psoriasis [29][30][31][32]. A previous study demonstrated that MSCs inhibit human Th17 cell differentiation and function [33]. IL-2 supports the proliferation [34][35][36][37] and survival [38] of T cells, as well as the differentiation of naive T cells into effector and memory cells, including Th17 cells [39][40][41][42]. In our study, coculture with epi-hMSCs suppressed the production of IL-2 compared with its expression in the cultures under Th17 conditions alone or with untreated hMSCs. Effector T cells, including Th17 cells, may differ in patients with RA and healthy individuals due to the continuous stimulation and attempts at immunosuppression in the setting of autoimmunity [43]. Importantly, coculture with epi-hMSCs, as opposed to no or untreated hMSCs, resulted in lower Th17 cytokine secretion and proliferation by cells from patients with RA. These findings support the potential of epi-hMSCs for the treatment of RA.
Although the results of this study on epi-hMSCs are promising, they are limited by the fact that we did not demonstrate such effects in in-vivo models. However, as effective regulation of Th17 immune responses was observed during proliferation and differentiation of Th17 cells and cytokine secretion, the results suggest that epigenetic modification of MSCs deserves further study.

Conclusions
We found that treatment with the combination of an HMA and an HDACi increased the immunomodulatory properties of hMSCs. Our results support the approach of enhancing the function of hMSCs via epigenetic modification. Further studies on the safety of epi-hMSCs are required prior to their use as therapeutics in RA and related diseases. In addition, future research should focus on the development of novel epigenetic markers to select optimal hMSCs and methodologies to increase the therapeutic effects of epi-hMSCs.