The influence of bone marrow-and synovium-derived mesenchymal stromal cells from osteoarthritis patients on regulatory T cells in co-culture

Summary

There is increasing evidence that inflammation in the synovium plays a major role in the progression of osteoarthritis (OA). However, the immunogenic properties of mesenchymal stromal cells (MSCs), which are considered to regulate immunity in various diseases, remain largely unknown in OA. The purpose of this study was to determine the influence of MSCs from OA patients on regulatory T cells (T_regs) in an allogeneic co-culture model. Bone marrow (BM) and synovial membrane (SM) were harvested from hip joints of OA patients and co-cultured with lymphocytes enriched in CD4⁺CD25⁺CD127^– regulatory T cells (T_reg⁺LC) from healthy donors. T_reg proportions and MSC markers were assessed by flow cytometry. Cytokine levels were assessed after 2 and 5 days of co-cultivation. Additionally, T_reg⁺LC cultures were analysed in the presence of interleukin (IL)-6 and MSC-supernatant complemented medium. B-MSCs and S-MSCs were able to retain the T_reg proportion compared to lymphocyte monocultures. T cell–MSC co-cultures showed a significant increase of IL-6 compared to MSC cultures. S-MSCs produced higher amounts of IL-6 compared to B-MSCs, both in single and T cell co-cultures. The effect of retaining the T_reg percentage could be reproduced partially by IL-6 addition to the medium, but could only be observed fully when using MSC culture supernatants. Our data demonstrate that retaining the T_reg phenotype in MSC–T cell co-cultures can be mediated by MSC derived from OA patients. IL-6 plays an important role in mediating these processes. To our knowledge, this study is the first describing the interaction of MSCs from OA patients and T_regs in an allogeneic co-culture model.

Introduction

While, in the past, osteoarthritis (OA) has generally been considered a ‘non-inflammatory’ disease, numerous recent studies have suggested that inflammation may play an important role in its progression [1–3]. These inflammatory processes take place in the synovial membrane [4], and are characterized by lymphocyte and macrophage invasion [5,6] and elevated proinflammatory cytokines [7].

Because there are currently no therapeutic approaches to halt OA progression, much hope has been expressed regarding the development of new therapeutic strategies, including cell-based approaches. In this context, mesenchymal stem or stromal cells (MSCs) have been investigated extensively throughout the past two decades mainly for their regenerative potential [8–10]. Their immunosuppressive competence has, however, become another important field of research (overview in [11] and [12]). Therefore, MSCs have been investigated in animal models of multiple sclerosis [13], pulmonary fibrosis [14], renal failure [15] and myocardial infarction [16]. In a clinical setting, MSCs have been used successfully as an immunosuppressive treatment in patients with severe graft-versus-host disease [17].

MSCs were also identified to play a crucial role in modulating the inflammatory processes in rheumatoid arthritis [18]. In an animal model of collagen-induced arthritis, MSCs reduced inflammation significantly in the joints by reducing proliferation and modulating cytokine expression [19].

The mechanisms of MSC-mediated immunosuppression are unclear and still controversial [20,21], while representing a promising target of cell-based therapies in diseases with important inflammatory processes. MSCs have been proved to suppress T cell proliferation successfully both in vitro and in vivo [22,23]. Recent studies have also shown that MSCs regulate and recruit regulatory T cells (T_regs) in a co-culture approach [24–26]. T_regs themselves have been identified as key players in numerous diseases, among them rheumatoid arthritis [2,27]; however, until recently they have not been associated with OA pathogenesis [28,29].

Although an important number of Phase I/II studies using MSCs in OA have been started (overview on [30]) and these cells have already been used in small patient series [31], the underlying processes of both the regenerative properties and, more importantly, the immunosuppressive capacities of MSCs in OA, are only poorly understood.

The aim of this study, therefore, was to analyse the effect of human MSCs from OA patients on T_regs in an allogeneic lymphocyte co-culture model. We compared MSCs derived from the bone marrow of a joint-adjacent bone and from the synovium of the affected joint to investigate whether the synovial MSCs located within the tissue affected by inflammation exerted different immunomodulatory properties.

Methods

Patients

MSCs were isolated from bone marrow and synovial membrane of 34 patients (age 68 ± 12 years, 19 female and 15 male) that had been collected during total hip arthroplasty for primary OA Kellgren grades III and IV. Exclusion criteria for these patients were a history of cancer, rheumatic diseases and acute or chronic infections.

Lymphocytes were extracted from whole blood samples of 16 young healthy donors (28 ± 7 years, five female and 11 male). Exclusion criteria for these donors were a history of cancer, rheumatic diseases, acute and chronic infections, cartilage injury and OA.

The study protocol was approved by the ethics committee of the University of Heidelberg, Germany. Both patients and blood donors provided informed consent. The procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 2000.

MSCs

Mononuclear cells (MNCs) were isolated from bone marrow samples by Ficoll Paque plus (GE Healthcare, Uppsala, Sweden) gradient centrifugation. MNCs were then resuspended in culture medium at a density of 5 × 10⁵ cells/cm² (= 2·5 × 10⁶ cells/ml). Culture medium contained Dulbecco's modified Eagle's medium low glucose (DMEM-LG; Invitrogen, Karlsruhe, Germany), supplemented with 10% fetal calf serum (FCS; Biochrom, Berlin, Germany) and 1% penicillin/streptomycin (Invitrogen). The cells were cultured in 175 cm² cell culture flasks (Nunc, Roskilde, Denmark) at 37°C with 6% CO₂ in a humidified atmosphere. After 24 h, with the first media exchange, non-adherent cells were discarded; afterwards, medium replacement was carried out every 72 h until the cells reached an 80% confluent layer. The cells were then detached with trypsin (Biochrom), washed with complete medium and counted (trypan blue 0·4%; Sigma-Aldrich, Steinheim, Germany). Afterwards, MSCs were replated and cultured under the conditions described above until reaching confluence at passage 2. The ability of MSC to differentiate into chondrogenic, adipogenic and osteogenic lineages was demonstrated according to protocols described previously [32].

MSCs were allogeneic to the lymphocytes in all co-culture experiments.

Lymphocytes

Peripheral blood mononuclear cells (PBMC) were collected from whole blood samples using Ficoll Paque plus (GE Healthcare, Uppsala, Sweden) gradient centrifugation. PBMC were then separated into a mixture of CD4⁺CD25^– and CD4⁺CD25⁺CD127^– cells using magnetic separation (CD4⁺CD25⁺CD127^dim/– regulatory T cell Isolation Kit II, LS and LD columns, MidiMACS™ separator, all from Miltenyi Biotec, Bergisch Gladbach, Germany). The isolated cells were then analysed for CD4, CD25, CD127 and forkhead box protein 3 (FoxP3) (see below).

Co-culture assays

MSCs derived from bone marrow (B-MSCs) and synovium (S-MSCs) from 18 patients were co-cultured with CD4⁺ T cells enriched in T_regs for 5 days in DMEM-LG (Invitrogen) supplemented with 10% FCS (Biochrom) and 1% penicillin/streptomycin (Invitrogen). The cells were resuspended in 48-well plates, each well containing 1 ml of medium and cells in various concentrations: T cells/MSCs 4:3 (37 500 T cells/cm² and 28 125 MSCs/cm²), 2:1 (37 500 T cells/cm² and 18 750 MSCs/cm²) and 4:1 (37 500 T cells/cm² and 9375 MSCs/cm²). Single-cell cultures of T cells (37 500 cells/cm²) and MSCs (28 125, 18 750, 9375 MSCs/cm²) were used as controls. At day 2, the well plates were centrifuged at 488 g for 10 min. Supernatants were collected for cytokine analysis (see below). For all cultures, the whole medium was then replaced. After 5 days of co-culture, supernatants were again collected as described above and analysed for cytokines (see below). The cells were then resuspended in phosphate-buffered saline (PBS; Invitrogen) with 0·5% FCS (Biochrom) and 2 mM ethylenediamine tetraacetic acid (EDTA) (Sigma-Aldrich). The lymphocytes were thus separated from the MSCs, washed and prepared for flow cytometry (see below). MSCs were detached with trypsin as described above, washed in whole medium and resuspended in PBS with 0·5% FCS and 2 mM EDTA. MSCs were then prepared for flow cytometry (see below).

Interleukin (IL)-6 and MSC supernatant supplemented lymphocyte cultures

CD4⁺ T cells enriched in T_regs were generated as described above by magnetic bead separation. The cells were resuspended in 48-well plates, each well containing 1 ml of medium (see above) and 50 000 T cells. In one group, the medium was supplemented with 5 ng/ml IL-6 (Miltenyi Biotec); in another, 10 ng/ml IL-6 was added to the medium. A third group was supplemented with supernatants from passage 2 bone marrow-derived MSCs cultured in DMEM-LG with 10% FCS and 1% penicillin/streptomycin. Cell cultures without supplementation to the media were used as controls. At day 2, the 48-well plates were centrifuged at 488 g for 10 min. Supernatants were collected and analysed for cytokines (see below). For all cultures, the whole medium was then replaced. After 5 days of culture, supernatants were collected as described above and analysed for cytokines (see below). The cells were then resuspended in PBS (Invitrogen) with 0·5% FCS and 2 mM EDTA (Sigma-Aldrich) and prepared for flow cytometry.

Flow cytometry analysis

One-colour cytometry (MSCs) and three-and four-colour cytometry (T cells) was performed using a MACS Quant™ analyser and MACS Quantify version 2.1 software (Miltenyi Biotec). Positive fluorescence was defined as any event above the background fluorescence, which was defined by a line where 99·5% of the events in isotype antibody-labelled cells were considered negative.

The following anti-human antibodies were used in the experiments: for T cell analysis, CD4 fluorescein isothiocyanate (FITC) mouse immunoglobulin (Ig)G1, CD25 phycoerythin (PE) or allophycocyanin (APC) mouse IgG2b (Miltenyi Biotec), CD127 APC or PE-Cy5 mouse IgG2a (BD Biosciences, Heidelberg, Germany). FoxP3 intracellular staining was performed with the FoxP3 staining buffer set and FoxP3-PE mouse IgG1 antibodies (BD Biosciences), according to the manufacturer's protocol. For MSC analysis, CD105 PE mouse IgG1 (Miltenyi Biotec), CD10 FITC mouse IgG1, CD13 PE mouse IgG1, CD14 FITC mouse IgG1, CD34 PE mouse IgG1, CD44 FITC mouse IgG2b, CD45 FITC mouse IgG1, CD49a PE mouse IgG1, CD90 FITC mouse IgG1, CD140b PE mouse IgG2a CD146 PE mouse IgG1, CD166 PE mouse IgG1 (BD Biosciences) and human leucocyte antigen (HLA)-ABC PE (Dako, Glostrup, Denmark) were used. Isotype-matched control antibodies were used for assessment of background fluorescence.

Cytokine analysis

Multiple simultaneous cytokine detection for IL-2, IL-4, IL-6, IL-10, IL-17a, tumour necrosis factor (TNF)-α and interferon (IFN)-γ was performed using the human T helper type 1 (Th1)/Th2/Th17 cytokine kit (BD Biosciences) on a MACS Quant™ analyser and MACS Quantify version 2.1 software (Miltenyi Biotec) as well as the FCAP Array software, version 1.0.1 (BD Biosciences). The assays were performed with undiluted supernatants and with supernatants diluted to 1:10 with PBS (Invitrogen). In addition, enzyme-linked immunosorbent assays (ELISA, n = 5 per group) were performed with commercial kits for detection of IL-1ra (BioSource Europe SA, Nivelles, Belgium) and IL-1β and IL-8 (Invitrogen Corporation, Camarillo, CA, USA), according to the manufacturers' protocols.

Statistical analysis

Statistical analysis was performed using spss software (SPSS Inc., released 2009; PASW Statistics for Windows, version 18.0; SPSS Inc., Chicago, IL, USA). One-way analyses of variance (anova) followed by Bonferroni adjustment were performed to compare the different groups of lymphocyte cultures after creating interindividual differences for each patient. Differences were considered statistically significant for P-values smaller 0·05. Results are shown as means ± standard deviation (s.d.).

Results

B-MSC and S-MSC retain CD127 down-regulation and up-regulate FoxP3 in an allogeneic lymphocyte co-culture

An important variation could be observed of T_reg percentages after magnetic separation (Fig. 1a). Because of the donor-associated varying baseline T_reg percentages before co-culture, intraindividual differences between the T_reg percentages after single-and co-cultures at day 5 and the initial T_reg percentage (day 0) were calculated in each group. There were no significant differences in CD4 expression (P = 0·522 between the groups) and in the percentages of CD4⁺CD25⁺ cells (P = 0·258) between the groups.

Figure 1.

(a) CD4⁺CD25⁺CD127⁻ Treg percentages of total CD4⁺ T cells at d0. Magnetic separation of CD4⁺CD25⁺CD127⁻ cells was performed with each circle representing a single culture. Percentages varied from less than 20% to over 70%. (b) Gating strategy for regulatory T-cells (CD127⁻/FoxP3⁺).

T_regs were defined as CD4⁺CD25⁺CD127^– or CD4⁺CD25⁺FoxP3 cells, respectively. The gating strategy is demonstrated in Fig. 1b. There was a negative correlation between CD127 and FoxP3 expression, the mean intraindividual difference between CD127^– and FoxP3⁺ cells being 4·62 ± 6·31%.

Both B-MSC– and S-MSC–lymphocyte co-cultures showed no significant changes in the T_reg proportion, while we observed a significant decrease in the proportion of T_reg in T cell monoculture (Fig. 2). This was the case for CD4⁺CD25⁺CD127^– cells (Fig. 2a, P < 0·001 for both T cell single-culture versus B-MSC/T cell co-culture and S-MSC/T cell co-culture) and CD4⁺FoxP3⁺ cells (Fig. 2b, P = 0·006 for T cell single-culture versus B-MSC/T cell and P = 0·005 versus S-MSC/T cell co-cultures). There were no statistical differences between S-MSC/T cell and B-MSC/T cell co-cultures regarding CD127 and FoxP3 expression. The MSC effect on T_reg-enriched CD4⁺ lymphocyte culture was independent of the T cell : MSC ratio (Fig. 2c).

Figure 2.

(a) Intraindividual differences between the baseline CD4⁺CD25⁺CD127⁻ Treg percentage in Treg-enriched CD4⁺ lymphocyte cultures and the Treg percentages at d5. Treg⁺LC: CD4⁺ lymphocytes enriched in Tregs cultured alone. Treg⁺LC/B-MSC: CD4⁺ lymphocytes enriched in Tregs cultured with bone-marrow-derived MSC. Treg⁺LC/S-MSC: CD4⁺ lymphocytes enriched in Tregs cultured with synovium-derived MSC. Results are displayed as mean-SEM. ***P < 0.001. (b) Intraindividual differences between the baseline Treg (CD4⁺CD25⁺FoxP3⁺) percentage in Treg-enriched, CD4⁺ lymphocyte cultures and the Treg percentages at d5. Treg⁺LC: CD4⁺ lymphocytes enriched in Tregs cultured alone. Treg⁺LC/B-MSC: CD4⁺ lymphocytes enriched in Tregs cultured with bone-marrow-derived MSC. Treg⁺LC/S-MSC: CD4⁺ lymphocytes enriched in Tregs cultured with synovium-derived MSC. Results are displayed as mean-SD. **P < 0.01. (c) MSC effects on Treg percentages in cocultures with Treg enriched CD4⁺ lymhocytes are independent of MSC/lymphocyte ratio (4:3, 2:1, 4:1) in both B-MSC and S-MSC.

S-MSC and B-MSC express typical stem cell markers before and after allogeneic lymphocyte co-culture

Both S-MSCs and B-MSCs matched all criteria for MSCs according to the International Society for Cellular Therapy [33]; they were plastic-adherent and could be differentiated successfully into chondrogenic, osteogenic and adipogenic lineage (data not shown). Flow cytometry revealed the typical expression of mesenchymal stromal cell markers, MSCs being positive for CD90, CD105, CD73 and negative for CD45, CD34, CD14, among others. The surface marker profile of MSCs used in our experiments is shown in Table 1. There were no significant differences in surface profiles between B-MSC and S-MSC before co-culture, except for CD146, which showed very low expression levels on S-MSCs and was highly donor-dependent in B-MSCs.

Table 1.

Surface markers on bone marrow-and synovium-derived mesenchymal stromal cells before co-culture

	B-MSC	S-MSC
CD90	+	+
CD105	+	+
CD73	+	+
CD34	−	−
CD45	−	−
CD14	−	−
CD10	−	−
CD13	+	+
CD44	+	+
CD49a	+	+
CD140b	+	+
CD166	+	+
HLA-ABC	+	+
CD146	+/−	−

B-MSC: bone marrow mesenchymal stromal cells; S-MSC: synovium mesenchymal stromal cells.

IL-6 release is increased in MSC–lymphocyte co-cultures

Cytometric bead array for several cytokines (n = 10 for day 2 and n = 5 for day 5) revealed high levels of IL-6 in cultures with MSCs, while IL-2, 4, TNF-α and IFN-γ were not detectable both in diluted and undiluted supernatants; IL-10 and IL-17a could be detected only sporadically in some supernatants without differences among the groups (data not shown). Neither IL-1ra, IL-1β nor IL-8 were detectable in the supernatants.

CD4⁺ T cells enriched in T_regs showed no significant IL-6 production when compared to co-cultures of S-MSCs and T cells and S-MSC single cultures (P < 0·001 for comparison with S-MSC single-cell and T cell co-cultures at day 2, P < 0·05 for comparison of S-MSC single-cell cultures and P < 0·001 for comparison of S-MSC/T cell co-cultures at day 5, Fig. 3a,b). IL-6 production in S-MSCs was significantly higher than in B-MSC cultures at day 2 (P < 0·001, Fig. 3a) and significantly higher in S-MSC/T cell co-cultures than in S-MSCs cultured alone (P = 0·01). At day 5, we observed an important decrease of IL-6 production in all groups, while the IL-6 quantity remained significantly higher in S-MSC/T cell co-cultures when compared to B-MSC/T cell co-cultures (P = 0·006; Fig. 3b).

Figure 3.

(a) IL-6 levels in the different groups at d2 of culture. T-cells in single culture showed no significant IL-6 production. S-MSC cultures showed significantly higher IL-6 levels compared to B-MSC at d2 and d5. *P < 0.05, ***P < 0.001. (b) IL-6 levels in the different groups at d5 of culture. IL-6 production was drastically reduced at d5 compared to d2. **P < 0.01.

MSC supernatants retain the T_reg proportions in T_reg⁺ lymphocyte cultures

In order to determine whether or not the effects of MSCs on T_regs in co-culture could be reproduced by IL-6, CD4⁺ lymphocyte cultures enriched in T_regs were supplemented either with 5 ng/ml IL-6, 10 ng/ml IL-6 or supernatants from B-MSC cultures in passage 2. To assess the effective IL-6 concentrations in our supplemented media, IL-6 concentrations were analysed by cytometric bead array at days 2 and 5 of lymphocyte culture. The effective concentrations at both time-points were reduced to approximately a third of the initially administered concentrations (Table 2). However, in both the 5 ng/ml and the 10 ng/ml supplemented groups, the natural IL-6 level found in the B-MSC supernatants had been surmounted effectively.

Table 2.

Mean interleukin (IL)-6 concentrations after days 2 and 5 of regulatory T cell (T_reg)+ lymphocyte culture

	T cells	T cells+ 5 ng/ml IL-6	T cells+ 10 ng/ml IL-6	T cells+ B-MSC supernatants
Mean IL-6 concentration (pg/ml) day 2	31·21 ± 26·49	1851·5 ± 312·44	3117·34 ± 717·3	895·09 ± 118·9
Mean IL-6 concentration (pg/ml) day 5	9·95 ± 15·81	1812·52 ± 416·45	3748·6 ± 923·12	694·32 ± 254·71

B-MSC: bone marrow mesenchymal stromal cells. The effective concentrations at these time-points were approximately three times lower than the originally administered concentration. Results are displayed as means ± standard deviation.

Figure 4a,b shows the effects of IL-6 and B-MSC supernatant supplementation on the CD4⁺ cultures. We could detect a significant decrease of the T_reg proportion in non-supplemented T cell cultures compared to both the initial T_reg percentage (P < 0·001, Fig. 4a) and T cell cultures supplemented with MSC supernatant (P = 0·003; Fig. 4a). There was no change in the CD4⁺ percentages between the groups (Fig. 4b).

Figure 4.

(a) Effects of IL-6 and B-MSC supernatants on CD4⁺ lymphocytes enriched in Tregs (CD4⁺CD25⁺CD127⁻). d0: baseline Treg percentage in CD4⁺ cultures before experiments. d5: Treg percentage in lymphocyte cultures without media supplementation. d5 + 5 ng/ml/d5 + 10 ng/ml: Treg percentage in lymphocyte cultures with 5 or 10 ng/ml IL-6 supplementation. d5 + MSC supernatant: Treg percentage in lymphocyte cultures with supplementation of B-MSC cultures derived supernatants. **P < 0.01, ***P < 0.001. (b) Effects of IL-6 and B-MSC supernatants on CD4⁺ lymphocytes enriched in Tregs. No significant differences in the percentage of CD4⁺ were observed between the groups.

This effect seemed to be partially reproducible by IL-6 at concentrations of 5 and 10 ng/ml; however, the full effect of retaining the T_reg phenotype was observed only with MSC supernatants.

Discussion

Our findings show that mesenchymal stromal cells from OA patients modulate T cells effectively, maintaining a regulatory phenotype in an allogeneic co-culture approach with T cells from young and healthy donors. We chose to use this approach in order to attribute findings in the co-culture to MSCs connected to the disease rather than using T_regs from OA patients who also may have been preconditioned. Because of the unique ability of MSCs to escape allorecognition [34], allogeneic co-cultures are an adequate model for the investigation of MSC–lymphocyte interactions [24].

To our knowledge, this is the first study to report effective immunomodulatory capacities of MSCs from OA patients, and more specifically from OA synovium. MSC–T_reg interactions have been reported in other contexts than OA, most importantly in transplantation immunology [35]; however, correlating these findings to OA remains a challenge in this early phase of research.

Interactions between MSCs and T cells

MSCs from healthy donors have been shown to recruit regulatory subsets from CD3⁺/CD45RA⁺ and CD3⁺/CD45RO⁺ fractions [24]. In these experiments, MSCs maintained FoxP3 expression and promoted CD127 down-regulation in purified T_reg subsets. It is known that the suppressive effects of T_regs are lost when cultured ex vivo, and recent findings suggest that, with time, a shift of these cells will occur towards effector memory-like cells that produce IL-6, IL-17 and IFN-γ [36]. This effect can be prevented by co-culture with MSCs [25]. MSCs seem to not only promote CD4⁺ T_reg generation, but also generation of CD8⁺ regulatory subsets [26].

In our experiments, we found that both FoxP3 expression and absence of CD127 expression was maintained in CD4⁺ T cells enriched in T_regs when co-cultured with MSCs. Our data thus support previous findings that FoxP3 is correlated inversely with CD127 expression [24,37]. The synovial MSCs were able to effectively maintain the T_reg proportions comparable to B-MSCs. These findings suggest that MSCs from OA patients effectively retain the T_reg subpopulation, but do not recruit T_regs from the CD4⁺ fraction, as in the study by di Ianni et al. [24]. Whether this is related to the disease remains to be identified in future experiments; however, the differences observed may also be due to variations in the experimental setting.

OA, synovium and MSC regeneration and immunomodulation potential

There is discussion as to whether OA affects MSC ability to differentiate into various tissues. The chondrogenic potential of MSCs has been reported to be reduced in advanced OA [38]; however, other studies suggest that the chondrogenic potential of MSCs from OA or rheumatoid arthritis patients is equal to MSC from healthy donors [39,40]. To this day, whether or not OA affects MSC immunomodulatory potential is unknown. We believe that we are the first to report that synovial MSCs from an OA-affected joint effectively maintain the T_reg proportions in a lymphocyte co-culture, thus suggesting that the inflammatory milieu has no deteriorating effects on MSC immunoregulatory potential.

There has been growing evidence of distinct properties of synovial membrane-derived human mesenchymal stromal cells [41–43]. Some studies suggest that S-MSC may be discriminated from B-MSC by their transcriptional profiles [44]. Apart from a negative CD146 expression in S-MSCs, there were no differences regarding the surface markers between B-MSC and S-MSC in our study. Also, no differences in plastic adherence or differentiation potential between these cells were observed in our experiments. While the synovium is located in the centre of joint inflammation associated with OA [5,7], this did not seem to have an influence on the immunomodulatory properties of MSCs in our experiments. The elevated IL-6 production, however, suggests that S-MSC from OA patients exert distinct properties in this particular setting. The question of whether or not the higher IL-6 secretion by S-MSCs is caused by the inflammatory conditions in the joint cannot be answered from our data, but it must be an aim of future experiments to link the degree of synovial inflammation to IL-6 secretion and MSC immunomodulatory potential.

We have used an in-vitro model in our experiments; thus, it is difficult to draw any conclusions regarding the in-vivo situation. However, these are only initial findings stating that the interaction of MSCs and regulatory T cells may play a role in the osteoarthritic joint in vivo, as has been suggested for numerous other diseases, including rheumatoid arthritis [2,27]. Future experiments will need to determine whether these findings will allow the application of some of the therapeutic strategies for rheumatoid arthritis locally to the OA-affected joint.

Influence of IL-6 in MSC-T_reg interaction

IL-6 was the predominant cytokine in the co-cultures, which is why we chose to supplement T_reg-enriched lymphocyte cultures with this cytokine. Our data suggest that IL-6 plays a role in S-MSC-and B-MSC-mediated immunomodulation, as supplementation of IL-6 to the culture media was shown to partially reproduce the MSC-mediated T_reg maintenance. To our knowledge, this is the first study to report that MSC from OA patients may exert some of their effects via IL-6 and thus may play an important role in shifting the balance of regulatory and effector T cells in OA.

The full effect of T_reg maintenance by MSCs, as seen in the MSC–lymphocyte co-cultures, was observed in the group supplemented with MSC supernatants. In our opinion, MSC–T_reg interaction therefore seems to be based on paracrine effects rather than on cell–cell interaction. However, other soluble factors, that remain to be detected, appear to be involved in these processes, although none of the other cytokines analysed in our experiments seem to be of major importance in this particular in-vitro setting.

The role of IL-6 in immunomodulation is discussed most controversially. IL-6, along with IL-8, has been shown to promote T_reg migration to certain tumours [45] and may therefore play a crucial role in T_reg signalling. A recent study in type II diabetes has also shown that the percentage of CD4⁽⁺⁾CD25^(hi)CD127^(–) T_regs are correlated positively with plasma IL-6 [46].

Mesenchymal stem cells from mice were shown to promote the differentiation of uncommitted naive T cells to FoxP3⁺ regulatory T cells [47]. This study has shown that the major cytokines involved in these processes were transforming growth factor (TGF)-β and IL-6 and that immunomodulation could be blocked by administration of anti-TGF-β or anti-IL-6, suggesting paracrine mechanisms. In another study in mice, excessive IL-6 production caused an increase in natural T_regs, which maintained their immunosuppressive capacities both in vitro and in vivo [48]. This suggests that IL-6 may be a key mediator in effector/regulatory T cell balance.

However, there are also data that are in contrast to our findings. In another study, using T_regs derived from the rheumatic synovium, blocking IL-6 or TNF increased the suppressive immunomodulatory capacities of these cells [49]. This study suggests that T_reg function varies importantly with the inflammatory milieu in the joint.

These findings are not in accordance with our experiments, in which IL-6 maintained the percentage of T_regs. This discrepancy may be due to differences in the underlying pathologies, but also reflect the dual role of IL-6. However, the functional consequences remain to be identified in upcoming experiments. We have chosen to carry out our study on T_regs taken from healthy donors in order to provide information on the immunomodulatory capacities of MSCs and ruling out possible influences of OA on T_regs and thus on T_reg–MSC interaction. An important question is whether or not T_regs taken from OA patients differ in their interaction with MSCs. Our findings therefore raise several questions that need to be verified by future research. Understanding the effects of MSCs in local joint inflammation in OA may pave the way for future cell-based approaches, as MSCs can be supposed to not only exert their regenerative potential when administered, but also intervene in local immunity.

Limitations

This study has several limitations. First, a comparison to MSCs taken from healthy donors would be useful to detect whether the inability to recruit T_regs from a CD4⁺ population, as has been shown by other groups, should be interpreted as a reduced capacity of immunomodulation in OA. This question has not yet been addressed due to ethical questions, as synovium from healthy individuals is not easily available. Taking synovium from a young population undergoing hip or knee arthroscopy might falsify the results, as it is known that cartilage injuries are found in up to 65% of the patients during arthroscopy, irrespective of the underlying pathology [50]. In our opinion, comparing healthy tissue to tissue derived from OA patients might reveal whether or not OA can be considered to be causing immunodepression in the joint synovium.

For this study, T_regs from healthy individuals were chosen in order to examine the effect of MSCs from OA patients on functional T lymphocytes. However, as stated above, it is necessary to conduct further research on how MSCs and T_regs taken from the same patients interact. In our experiments, blood volumes up to 150 ml were necessary to isolate a sufficient number of T_regs to conduct the co-culture experiments. While this is unproblematic for healthy individuals, in the context of a perioperative setting of a total hip arthroplasty with its high blood loss these volumes were considered too important to be taken from the OA patients. We are currently working on optimizing the isolation procedures as well as on methods that can provide T_regs from OA patients without taking important blood samples, such as collecting cells during the intraoperative autotransfusion procedure.

This study addressed only changes in phenotypical T_reg properties and its important activation marker FoxP3. Our experiments cannot provide information on functional changes in T_reg suppression potency. These experiments will need to be carried out in future to determine whether MSC immunomodulation has an effect on the functional properties of T_regs.

Joint inflammation may have differed among the patients recruited in this study; whether this has an effect on MSC immunomodulatory processes in vitro will need to be determined in future experiments. Therefore, it may be necessary to correlate inflammation in the synovium with the in-vitro immunomodulatory properties of MSCs.

We were able to detect IL-6 as an important factor in MSC–T_reg interaction; however, future studies should focus upon other possible cytokines involved.

Disclosure

The authors declare that they have no competing interests.