Adipose-Derived Mesenchymal Stem Cells from Arthritis Patients: Differential Modulation of CD4 T Cell Activation and Cytokine Production

Introduction

Mesenchymal stem cells (MSCs) are multipotent stem cells that can be isolated from various sources, including umbilical cord/placental tissues, bone marrow, adipose tissue, dental tissue, and other tissues in the body [1–13]. MSCs can differentiate into a variety of mesenchymal tissues, such as bone, cartilage, tendon, fat, bone marrow stroma, muscle, and periodontal tissue [2,5,6]. They are also characterized by self-renewal capacity and immunomodulatory properties [7,14]. MSCs were shown to suppress tumor growth, inhibit angiogenesis, regulate cellular signaling, and induce apoptosis in cancer cells [15]. MSCs also exert immunomodulatory properties on the immune cell subsets and local and systemic innate and adaptive immune responses, and can create inflammatory microenvironments [14]. These properties make MSCs a promising source for cell therapy in inflammation, immune diseases, and organ transplantation [14].

Due to the low invasiveness and repeatability of the isolation procedure, adipose tissue is one of the most popular sources of MSCs. Those MSCs are called adipose-derived stem cells (ASCs). ASCs have similar characteristics to other MSCs, including the ability to differentiate into multiple cell types and their immunomodulatory properties [3,4]. They have been investigated for their potential use in regenerative dentistry and the treatment of degenerative and inflammatory diseases [4]. ASCs have been extensively studied for their immunomodulatory effects in different contexts, including sepsis, transplant medicine, autoimmune diseases, and tissue repair [16,17]. ASCs are present in damaged and inflamed tissues, where factors secreted by ASCs, such as cytokines, chemokines, and extracellular matrix protein modulate the immune response and contribute to tissue repair and regeneration [17]. ASCs can also suppress the immune response of the host and have the potential to be expanded in culture, which makes them suitable for stem cell-based strategies [17]. The use of ASCs is also being considered in the treatment of rheumatoid arthritis (RA). Rheumatoid arthritis and osteoarthritis (OA) are 2 distinct forms of arthritis with different pathogeneses.

The RA pathogenesis involves a combination of genetic and environmental factors that trigger an autoimmune response. The immune system mistakenly attacks the host tissues, leading to chronic inflammation and the formation of a hyperplastic synovium called the pannus [18]. The pannus invades and destroys the adjacent cartilage and bone, resulting in joint deformity and functional impairment [19]. The inflamed synovium in RA is a major source of cytokines and proteinases that mediate cartilage destruction [19]. The disease also involves systemic inflammation, affecting multiple organs outside the joints and leading to complications such as cardiovascular diseases [18].

The pathogenesis of OA is primarily driven by mechanical factors and the aging process. OA is characterized by the progressive degeneration of articular cartilage, accompanied by changes in the subchondral bone and synovium [19]. Mechanical stress on the joint, such as repetitive use or joint instability, can lead to cartilage damage and initiate the disease process [20]. Chondrocytes, the cells responsible for maintaining cartilage homeostasis, respond to mechanical signals and molecular factors by producing inflammatory mediators and matrix-degrading enzymes [19]. The imbalance between cartilage degradation and repair processes eventually leads to loss of cartilage integrity and development of OA [21].

While both RA and OA cause joint inflammation, the underlying mechanisms and immune responses differ. In RA, the inflammatory process is the main component of disease pathogenesis, with CD4+ T cells and pro-inflammatory mediators (including cytokines) perpetuating chronic inflammation and joint destruction [22]. In OA, the inflammation is milder and primarily driven by mechanical factors, although synovial inflammation can also occur [23]. The inflammatory mediators involved in OA are different from those in RA, and the extent of systemic inflammation is generally lower in OA [24]. Therefore, OA is used as a comparative control in studies on rheumatoid arthritis. Mesenchymal stem cells may be an attractive therapeutic option for patients with RA or OA due to the regenerative and immunomodulatory abilities of these cells. The infrapatellar fat pad (IFP) is an alternative, rich and easily available source of mesenchymal stem cells (adipose-derived stem cells, ASCs). In a previous study we found that ASCs from RA and OA patients maintained the ability to differentiate into adipocytes, chondrocytes, and osteoblasts [25]. In addition, RA- and OA-derived ASCs showed a similar phenotype and comparable abilities to inhibit CD4+ T cell proliferation, which was dependent on the induction of soluble factors [25]. These results were the basis for the present research on the therapeutic potential of ASCs in the treatment of patients with RA. We also demonstrated that ASCs collected from 2 tissue locations from RA patients – subcutaneous adipose tissue and intra-articular adipose tissue – have similar immunomodulatory properties [26], but information regarding other effects on CD4+ T cell activities is lacking.

The current study aimed to evaluate the impact of ASCs, isolated from intra-articular adipose tissue (infrapatellar fat pad of the knee joint) of patients with RA, on CD4+ T cells activation and development of this cell subset towards Th17 (IL-17 secreting cells) and T regulatory (Treg) cells. We hypothesized that ASCs located in the RA joint promote the development of the pro-inflammatory Th17 subset while failing to induce Tregs.

Material and Methods

PATIENTS:

Enrolled patients met the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) criteria for RA or OA [27,28]. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the National Institute of Geriatrics, Rheumatology, and Rehabilitation in Warsaw, Poland (approval number DL/14.01.2016). All patients gave their voluntary, written consent to the use of their tissues in the study. Demographic and clinical data of the participants are presented in Table 1.

ISOLATION OF ASCS AND PERIPHERAL BLOOD IMMUNE CELLS:

ASCs were isolated from the infrapatellar fat pad (IPFP) of RA and OA patients who underwent total knee replacement surgery. The adipose tissue was dissected and digested using a 0.25% trypsin solution (Sigma-Aldrich, St Louis, MO, USA) for 20 minutes at 37°C with agitation. The digested tissue was then filtered, washed with phosphate-buffered saline (PBS), and centrifuged for 3 minutes at 1200×g. The stromal vascular fraction (SVF) was collected from the pellet, followed by 2 additional centrifugations in PBS. If needed, red blood cells were lysed. The resulting cells were seeded into 25-cm2 culture flasks in a medium designed for ASC culture, obtained from Lonza (Lonza Group, Lonza Walkersville, Inc., Walkersville, MD, USA) [29]. ASCs between passages 3 and 5 were used in all experiments (Walkersville, MD, USA). The differentiation capacity towards adipocytes, osteoblasts, and chondrocytes and the specific phenotype of the obtained ASCs were assessed following the “Minimal criteria for defining multipotent mesenchymal stromal cells” according to The International Society for Cellular Therapy position statement, as in our previous study [25]. The experiments performed are shown in Figure 1.

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Knowing that the activity of ASCs may result in different effects on CD4⁺ lymphocytes, depending on whether ASCs are cultured solely in the presence of an isolated CD4⁺ cell population (CD4/ASC culture variant) or in the presence of peripheral blood mononuclear cells (PBMC; PBMC/ASC culture variant), 2 types of co-culture were performed in this study. PBMCs were isolated from buffy coats obtained from healthy donors by density gradient centrifugation with Ficoll-Paque (GE Healthcare, Uppsala, Sweden). CD4⁺ cells were isolated from PBMCs using the EasySep Human CD4⁺ T Cell Isolation Kit (Stemcell Technologies, Vancouver, Canada). For each well of a 24-well plate, 6×10⁴ ASCs were seeded. We used untreated ASCs and ASCs pre-stimulated with cytokine. For pre-stimulation, ASCs were treated with recombinant human tumor necrosis factor (TNF) and interferon (IFN-γ) at a concentration of 10 ng/mL each (ASCs_TI). TNF and IFN-γ were obtained from R&D Systems (BioTeche Minneapolis, MN, USA).

PBMCs were stimulated with 2.5 μg/mL of phytohemagglutinin (PHA, Sigma-Aldrich), and CD4⁺ cells were activated using Dynabeads™ Human T-Activator CD3/CD28 (Thermo Fisher Scientific, Waltham, MA, USA). Activated cells were co-cultured with ASCs at a ratio of 1.2×10⁶ PBMCs per well in 2 mL of RPMI (PAN Biotech UK Ltd., Wimborne, UK) supplemented with 10% fetal calf serum (Biochrom, Berlin, Germany), 200 U/mL penicillin, 200 μg/mL streptomycin (Polfa Tarchomin S.A., Warsaw, Poland), and 5 μg/mL plasmocin (InvivoGen, San Diego, CA, USA).

At the end of the 96-hour co-culture, the PBMCs or CD4⁺ were harvested for cytometric and RNA analyses of CD4⁺ cells. Where PBMCs were harvested, CD4⁺ cells were purified using the EasySep Human CD4⁺ T Cell Isolation Kit (Stemcell Technologies, Vancouver, Canada). Co-culture supernatants were collected to measure the concentrations of soluble factors. Supernatants from separately cultured ASCs, PBMCs, and/or CD4⁺ cells served as controls for co-culture.

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To analyze the phenotype of CD4⁺ T lymphocytes, PBMC or CD4⁺ T cells harvested from the co-culture with ASCs were incubated for 30 minutes on ice with specific monoclonal antibodies targeting human antigens CD3-BV510, CD4-FITC, CD25-PE, CD127-PE-Cy7, and HLA-DR-PerCP (all from Becton Dickinson, San Diego, CA, USA). In the subsequent step, the cells were fixed and permeabilized using the FoxP3/transcription factor Staining Buffer Set (Thermo Fisher Scientific, Waltham, MA, USA). Intracellular staining was then performed using an anti-FoxP3-AF 647 antibody (Becton Dickinson, San Diego, CA, USA). Following a washing step, the cells were acquired and analyzed using a FACS Canto cell sorter/cytometer and Diva software. Isotype controls were appropriately included in all experiments to ensure an accurate interpretation of the results.

QUANTIFICATION OF SOLUBLE FACTORS IN CULTURE SUPERNATANTS:

The concentrations of soluble factors were determined using specific commercial enzyme-linked immunosorbent assays (ELISAs) in duplicate, following manufacturers’ instructions. The optical density readings were obtained using a Multiskan GO spectrophotometer (Thermo Fisher Scientific) and analyzed using Skanit Software 3.2. Details of the kits used for cytokines detection are depicted in Table 2.

BLOCKING EXPERIMENTS:

To counteract the effects of TGFβ, the neutralizing antibody (from Thermo Fisher Scientific) was used at a concentration of 50 mg/mL. Indomethacin (from Sigma-Aldrich) was used at a concentration of 1 mM to inhibit the synthesis of prostaglandin 2 (PGE2). To hinder kynurenine production, 1 mM 1-methyltryptophan (1-MT) (from Sigma-Aldrich, Germany) was employed. The concentrations of blocking agents used in this study were optimized in previous experiments [25,29]. ASCs were cultured with blocking agents for 48-hour, before establishing the co-culture with PBMC or CD4+ T cells. PBMCs were used as non-stimulated or pre-activated cells using PHA. Just before establishing the co-culture, ASCs and PBMCs were washed with PBS and then cultured together for 5 days. At the end of the co-culture, PBMCs were harvested for cytometric analysis, and the culture supernatants were collected for soluble factors quantification.

STATISTICAL ANALYSES:

Statistical analyses and visualization of the data were performed using GraphPad Prism software version 7 (GraphPad Software, Boston, MA, USA). To assess the distribution of the data, the Shapiro-Wilk test was used. To compare the effects of ASCS obtained from patients with RA and OA, unpaired sample tests, specifically the Mann-Whitney or Kruskal-Wallis tests, were employed. To evaluate the impact of blocking agents, the Friedman test and Dunn’s multiple comparison test were used. Statistical significance was determined by considering probability values below 0.05.

Results

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Flow cytometry analysis was used to investigate the expression of surface proteins (CD69, CD25, and HLA-DR), and the transcription factor FoxP3 produced by CD4+ T cells in the CD4/ASCs and PBMCs/ASCs co-cultures. As a result of co-cultures, the expression of CD69 molecule on CD4+ T cells was increased, except in the co-culture with-stimulated OA/ASCsTI. Distinct effects were observed regarding the expression of CD25. In the CD4/ASCs co-culture, CD25 expression was diminished after co-culturing with OA-derived ASCs (P<0.05) but increased following co-culture with RA-derived ASCs (P<0.05). In PBMCs/ASCs variant, the decrease in CD25 expression was observed when OA-derived ASCsTI (P<0.01) and RA-derived ASCs (P<0.05) were present in co-culture. Furthermore, variations in the expression of HLA-DR were observed. In the case of CD4/ASCs co-cultures, independently of the source of origin of the ASCs, there was a decrease in HLA-DR expression on CD4+ T cells (P<0.01). In ASC/PBMCs co-cultures, such an effect was observed only when OA-derived ASCs (P<0.001) were used, indicating impaired immunoregulatory properties of ASCs from RA patients. This was confirmed by further analysis of CD4+ lymphocytes expressing the transcription factor FoxP3, which is characteristic for Treg [30]. Only ASCs from OA patients were able to increase the percentage of Treg in PBMCs/ASCs culture (P<0.01 for CD4/ASC and P<0.001 for PBMC/ASCs) (Figure 2).

EFFECTS OF PBMCS/ASCS CO-CULTURE ON PRO-INFLAMMATORY CYTOKINES PRODUCTION:

Investigated cytokines contribute to the complex network of immune responses and play important roles in both physiological and pathological processes, including inflammation and autoimmune diseases. TNF production in co-cultures was downregulated irrespective of the co-culture variant (P<0.0001 for all variants). Unexpectedly, IFNγ concentration was reduced in the PBMCs/ASCs co-cultures (P<0.05 for OA and P<0.001 for RA), but it was elevated in the CD4/ASCs variant (P<0.0001 for OA and P<0.001 for RA). There was no significant effect of co-cultures on IL-4 secretion. Nevertheless, it should be noted that IL-4 secretion was low in our culture settings. Separately cultured ASCs did not produce TNF, IFNγ, or IL-4 (Figure 3).

We observed significant increases in the concentrations of IL-17AF (CD4p<0.0001 for OA and P<0.01 for RA; PBMC P<0.0001 for OA and P<0.0001 for RA), IL-17A (CD4, P<0.0001 for OA and P<0.0001 for RA; PBMC P<0.0001 for OA and P<0.0001 for RA), and IL-17F (CD4, P<0.01 for OA and P<0.01 for RA; PBMC P<0.01 for OA and P<0.01 for RA) in co-culture with RA- and OA-derived ASCs (Figure 4). Additionally, we observed a slight reduction in IL-21 secretion in CD4/ASCs variant from RA patients (P<0.01), while IL-21 secretion remained low in all PBMCs/ASCs variants, regardless of the disease origin. Furthermore, IL-22 concentrations were unaffected in CD4/ASCs variant but showed an increase in the PBMCs/ASCs variant (P<0.01 for OA and P<0.001 for RA). Notably, IL-26 production was increased in both co-culture variants except for PBMCs/ASCs from OA patients’ CD4 (P<0.0001 for OA and P<0.0001 for RA) and for PBMC (P<0.05 for RA). Importantly, ASCs cultured separately did not secrete any of the examined soluble factors (Figure 5). Interestingly, except for IL-26, ASCs obtained from RA patients or OA patients similarly modulated the production of pro-inflammatory cytokines.

EFFECTS OF ASC CO-CULTURE ON ANTI-INFLAMMATORY CYTOKINES PRODUCTION:

The following substances related to immunosuppressive ASCs properties were measured in cell culture supernatants: IL-35, IL-1Rα, Galectin-3, and leukemia inhibitory factor (LIF). In the CD4/ASCs co-culture, we noticed an increase in the concentration of IL-35 (P<0.0001 for OA and P<0.0001 for RA). However, in PBMCs/ASCs variant this increase in IL-35 occurred only in cultures containing OA-derived ASCs (P<0.01). Interestingly, ASCs themselves also produced small amounts of IL-35, and this secretion was enhanced by stimulation with TI. Notably, RA-derived ASCs produced significantly less IL-35 compared to those obtained from OA patients (P<0.01). CD4/ASCs co-culture resulted in a marked increase in the production of IL-1Ra (P<0.0001 for OA and P<0.001 for RA). In PBMCs/ASCs co-culture, this increase was less prominent and statistically significant only for ASCs derived from RA patients (P<0.01). ASCs cultured separately secreted small amounts of IL-1Ra. IL-1Ra levels did not differ significantly between ASCs lines derived from RA patients and OA patients. Only in the CD4/ASCs co-culture variant was there a significant increase in the levels of galectin-3 (P<0.05 for OA and P<0.05 for RA). ASCs cultured separately also produced galectin-3, but no differences were found between the patients’ groups. TI stimulation did not seem to affect the production of this cytokine (Figure 6).

KYNURENINE PATHWAY AND PGE2 WERE INVOLVED IN TREG GENERATION IN ASCS/PBMCS CO-CULTURES:

Produced by MSC prostaglandin E2 (PGE2), transforming growth factor beta (TGF-β) and indoleamine 2,3-digoxygenase (IDO) mediate the immunoregulatory effects of these cells [31]. To confirm the participation of the above-mentioned factors in ASC-induced Treg propagation, we performed blocking experiments.

The presence of ASCs or ASCsTI, significantly increased the percentage of Treg in PBMCs culture. However, the addition of indomethacin to those co-cultures led to a decrease in Treg percentages (P<0.001) Addition of 1-MT to the PBMCs/ASCsTI co-cultures the resulted in a significant reduction of Treg percentages (P<0.01) (Figure 7).

KYNURENINE PATHWAY AND PGE2 WERE INVOLVED IN THE MODULATION OF CYTOKINE PRODUCTION IN ASCS/PBMCS CO-CULTURES:

The addition of indomethacin and 1-MT to the co-cultures of ASCs and PBMCs resulted in a significant reduction of Treg percentages in different co-culture variants. Similar results were obtained with ASCs derived from RA patients or OA patients.

Regarding soluble factors, in PBMCs/ASCs and PBMCs/ASCsTI co-cultures, we observed a decrease in the concentrations of TNF. Furthermore, addition of indomethacin to the PBMCs/ASCs or PBMCs/ASCsTI co-cultures, led to a significant increase in TNF concentrations compared to the ASCs/PBMCs co-culture (P<0.0001 for OA and P<0.0001 for RA). Intriguingly, when 1-MT was added to the PBMCs/ASCsTI variants, we observed a significant increase in TNF concentrations (P<0.0001 for OA and P<0.01 for RA). These observations were consistent, regardless of whether cells were derived from patients with RA or OA. In PBMCs/ASCs and PBMCs/ASCsTI co-cultures, a decrease in IFNγ concentrations was observed. On the other hand, in the PBMCs/ASCs variants, the addition of indomethacin led to an increase in IFNγ levels compared to the non-stimulated PBMCs/ASCs variants (P<0.0001 for OA and P<0.0001 for RA). Furthermore, blocking TGFβ using a neutralizing antibody resulted in increased IFNγ production, particularly in co-culture of OA-derived ASCs (P<0.001). In PBMCs/ASCsTI cultures, the addition of 1-MT led to significant increase in IFNγ levels, but this effect was observed only in OA-derived ASCs variants (P<0.01) (Figure 8). Co-culturing PBMCs with ASCs led to a significant increase in IL-17 concentration. Blocking experiments with soluble factors did not significantly alter this effect (Figure 9).

Discussion

The use of MSCs is one of the most promising therapeutic methods in RA. The therapeutic potential of MSCs has been extensively studied in vitro and in vivo. MSCs have been shown to improve the course of RA in several animal models [32–37]. However, the results of use of MSCs in RA patients have not been consistently positive [38–40], contrary to MSCs application in OA, where these cells showed beneficial effects [41]. Thus, there is a need to better understand the properties of MSCs in RA treatment. It is widely accepted that the immunomodulatory effects of MSCs are activated only in response to the surrounding inflammatory environment [42]. To mimic the in vivo inflammatory environment and enhance the immune regulatory function (and thus the therapeutic potential) of MSCs, we pre-stimulated ASCs in some experiments before their culture with peripheral blood cells. Interferon gamma, a leading pro-inflammatory cytokine against viral and bacterial infections, and TNF, secreted mainly by macrophages, are the main inflammatory cytokines used to condition MSCs to enhance their therapeutic potential [43]. In previous studies, we obtained data confirming the differentiation potency, characteristic phenotype, and immunoregulatory activity (reflected by the reduction of CD4+ T cell proliferation) of ASCs derived from IPFP of patients with RA and OA [25]. Here, we assessed the immunosuppressive functions of ASCs based on their ability to promote the Treg population and to create an anti-inflammatory environment. The results of our co-culture experiments extended knowledge of the immunomodulatory effects of ASCs on phenotype and secretory activities of isolated CD4+ T cells and PBMCs.

Cytometric analyses confirmed that ASCs can modulate the expression of surface activation markers CD69, CD25, and HLA-DR in the CD4+ T cells (in co-cultures of ASCs with purified CD4+ T cell population as well as with PBMCs). Similar results were obtained previously [26,44–47]. Interestingly, RA-derived ASCs did not modulate HLA-DR expression, which may reflect impaired function of these cells. In addition to the expression of extracellular activation markers, we performed cytometric analysis of the expression of the transcription factor FoxP3, a marker of Tregs [30]. Tregs possess immunosuppressive and immunoregulatory properties. Their activity leads to natural attenuation of immune response and maintains autotolerance [48]. Some regulatory lymphocyte subsets secrete anti-inflammatory cytokines such as IL-10, TGFβ, and IL-35 [49]. One of the therapeutic approaches in RA and other autoimmune diseases is to enhance the suppressive activity of Tregs. It has been demonstrated that ASCs derived from healthy donors promote Treg FoxP3+ cells expansion but inhibit Th17 differentiation [50–53]. Therefore, ASCs activity could be beneficial in controlling RA disease. On the other hand, RA-derived ASCs did not modulate FoxP3 expression, nor did they increase IL-35 production in ASCs co-culture with PBMC, suggesting functional impairment of ASCs in RA. However, we observed an increase of FoxP3 expression and IL-35 concentrations in co-cultures with OA-derived ASCs. It is worth noting that in our experience, increased Treg differentiation occurred only in the case of ASC co-culture with PBMC. This is consistent with the observations of human bone marrow mesenchymal stem cells BM-MSCs, which can stimulate the formation of classical Treg only in the presence of monocytes/PBMCs, but fail to exert such an effect when co-cultured with purified CD4+ T cells [54]. Th17 lymphocytes express RORc transcription factor and secrete IL-17A as well as other pro-inflammatory cytokines participating in the pathogenesis of RA (eg, IL-17F, IL-17AF, IL-21, IL-22, IL-26, TNF, and IL-6) [55]. Although ASCs were able to effectively reduce TNF production by CD4+ T cells, they also stimulated the production of cytokines associated with the function of Th17 cells, indicating the reduced anti-inflammatory potential of ASCs. Another research group reported that MSCs derived from the BM of RA patients displayed impaired abilities in inhibiting differentiation of Th17 cells [56]. Our data also indicate that ASCs obtained from patients with other rheumatic diseases can increase secretion of Th17-related cytokines [29].

Inconsistent therapeutic effects of MSCs application in RA may result from the loss of suppressive function of MSCs in the inflammatory microenvironment of rheumatoid joint [57–59]. This is supported by the results obtained in animal studies on adjuvant-induced arthritis (AIA) in rats. BM-MSCs from healthy rats exerted immunosuppressive properties in vitro but lost them after intraperitoneal administration to AIA rats [60]. Nevertheless, other authors demonstrated that MSCs administration in AIA limited inflammation, cartilage destruction, joint edema, bone resorption, and pro-inflammatory cytokines secretion, as well as up-regulation of anti-inflammatory IL-10 [35–37,57]. Moreover, studies on collagen-induced arthritis (another animal model of RA) and preliminary clinical trials produced inconsistent results concerning MSCs administration [61–64]. The net effect of the activity of MSCs from healthy donors is dependent on stimuli provided by the local environment. Pro-inflammatory cytokines such as TNF, IFNγ, and interleukin IL-1α/β have been demonstrated to induce or enhance immunosuppressive functions of MSCs [65,66]. In our setting, the addition of TNF or IFN to the cultured cells did not enhance the immunosuppressive properties of ASCs, perhaps due to the reduced sensitivity of ASCs to inflammatory factors present at high concentrations in the inflamed joint. However, other pro-inflammatory factors/pathways may be involved. Published reports indicated that stimulation of inflammatory pathways by Toll-like receptors can promote pro-inflammatory activities of MSCs [67,68].

The immunomodulatory effects on T lymphocytes are more pronounced in ASC/PBMC co-cultures, mirroring the complex cellular environment of the joint. This suggests that the influence of ASCs is mediated through interactions with various cell types within the PBMC population. However, even in ASC/PBMC co-cultures, cells derived from RA patients appear to be functionally impaired, particularly in their capacity to generate regulatory T cells (Tregs).

A limitation of our study is that we did not compare RA-derived ASCs with those from healthy donors, such as individuals who had undergone surgery after knee injuries. However, we focused primarily on comparisons of ASCs from RA and OA patients because autologous transplantation of the latter has been shown to benefit patients. To this end, we ensured that tissues from RA and OA patients underwent identical procedures, both surgically and in the laboratory. Time was of the essence, because tissues were processed within half an hour of collection, minimizing the risk of tissue degradation (which might be difficult to ensure if trauma tissues were collected from another hospital). Although this approach limited our ability to compare with healthy donors, it allowed us to maintain a high level of consistency and reliability within the constraints of our study. The ultimate goal of the study was to assess the suitability of ASCs for autologous cell transplantation in individuals with RA.

A limitation of this work is that we were not able to investigate all the mechanisms by which ASCs can exert their immunomodulatory effects. For instance, we did not examine the influence of miRNA-containing vesicles produced/secreted by ASC as well as immunomodulatory effects dependent on cell-to-cell contact. However, the results of our blocking experiments indicated that the metabolic processes involving kynurenine pathway and soluble PGE2 are associated with the immunomodulatory activities of ASC, in particular TNF and IFN production and Tregs development. Furthermore, obesity has been shown to alter the properties and phenotype of mesenchymal stem cells derived from human subcutaneous adipose tissue or visceral fat [70–72]. However, there is a lack of data on the properties of ASCs from IPFP in obese individuals. Unfortunately, our study had a relatively small number of obese patients, and we did not observe significant differences in the effects of activity between ASC lines from nonobese and obese patients, which prevented us from exploring this topic.

Conclusions

The results of this study indicate that ASCs can influence the phenotype of CD4+ T cells and the production of pro-inflammatory as well as anti-inflammatory cytokines. However, the immunomodulatory abilities of ASCs from RA patients are impaired, which calls into question their therapeutic usefulness. Further research is necessary to better understand the biology of ASCs and their therapeutic potential.