Serum IFN-γ levels predict the therapeutic effect of mesenchymal stem cell transplantation in active rheumatoid arthritis
To explore the mechanism of the different clinical efficacies of mesenchymal stem cell transplantation (MSCT) and identify a possible serum biomarker for predicting the therapeutic effect of MSCT in rheumatoid arthritis (RA) patients.
A total of 105 patients with persistently active RA and poor responses to traditional medication were randomly divided into MSCT and control groups. Outcomes were evaluated according to the 28-joint Disease Activity Score and Health Assessment Questionnaire, serological indicators, regulatory T cell (Treg) to T helper 17 (Th17) cell ratio, and inflammatory cytokine levels. Twelve weeks after MSCT, the outcomes of the MSCT group were evaluated according to the European League against Rheumatism response criteria. Patients with a good or moderate response were added to the response group, and those with no response were added to the no-response group.
No serious adverse events were reported for either MSCT subgroup (28 in the response group and 24 in the no-response group). The therapeutic effects lasted for 48 weeks without continuous administration. Notably, a transient increase in serum IFN-γ (>2 pg/ml) levels was observed in the response group, but not in the no-response group. Furthermore, an increase in IL-10 levels and the Treg/Th17 ratio and a reduction in IL-6 levels appeared 2–3 weeks after the transient IFN-γ increase.
Allogeneic MSCT is safe and feasible, and we propose high serum IFN-γ levels as a potent biomarker for predicting MSCT response.
Trial registration chictr.org, ChiCTR-ONC-16008770. Registered 3 July 2016, http://www.chictr.org.cn/showproj.aspx?proj=14820
KeywordsMesenchymal stem cell Rheumatoid arthritis IFN-γ Clinical trial
mesenchymal stem cell transplantation
28-joint Disease Activity Score
Health Assessment Questionnaire
regulatory T cell
T helper 17
European League against Rheumatism
disease-modifying antirheumatic drugs
mesenchymal stem cells
peripheral blood mononuclear cells
systemic lupus erythematosus
non-steroidal anti-inflammatory drugs
umbilical cord-derived human MSCs
anti-cyclic citrullinated peptide antibody
one-way analysis of variance
erythrocyte sedimentation rate
tumor necrosis factor-alpha
IL 2 receptor alpha
Rheumatoid arthritis (RA) is chronic synovitis characterized by articular inflammation, synovial joint damage and deteriorating disability over time. The basic pathogenic mechanisms that initiate and promote RA progression are likely to be systemic and synovial inflammation due to unbalanced immune homeostasis, possibly involving genetic and environmental factors.
In the BeSt study, 508 RA patients were treated with hormones, disease-modifying antirheumatic drugs (DMARDs), or biological agents and were followed for up to 5 years; according to the results, more than 50% of the RA patients did not achieve clinical remission after treatment . Therefore, despite great medical advances, the following problems remain: (i) some patients cannot tolerate DMARDs or biologics; and (ii) biologic agents are expensive and weaken the immune system, thus potentially increasing the risk of infections. Consequently, novel treatment options for RA patients are urgently needed.
Mesenchymal stem cells (MSCs) are multipotent cells derived from bone marrow or other connective tissues, such as the umbilical cord; MSCs have the capacity to self-renew and differentiate into mesenchymal tissues . In addition to their differentiation ability, MSCs have immunomodulatory effects on many diseases [3, 4]. MSCs can inhibit the proliferation of activated peripheral blood mononuclear cells (PBMCs) and T lymphocytes  to induce regulatory T cell (Treg) differentiation and inhibit T helper 17 (Th17) cell function ; it is through these mechanisms that MSCs exert their immunomodulatory effects on RA. The immunomodulatory abilities of MSCs are thought to depend on interferon-γ (IFN-γ)-induced indoleamine 2,3-dioxygenase (IDO). The unique immunomodulatory properties of MSCs make them a promising candidate cell therapy for tissue repair, potentially for use in treating immune disorders, such as graft-versus-host disease (GVHD) , and autoimmune diseases, such as systemic lupus erythematosus (SLE) ; however, contradictory results have been reported for using MSC transplantation (MSCT) to treat RA [9, 10]. There are huge individual variations in the clinical efficacy of MSCT for the same disease, but the reasons for this are unclear. For the widespread clinical application of MSCT, a better understanding of the biological properties of MSCs is required. This information would help clarify the mechanisms of MSC-based transplantation for immunomodulation and improve clinical efficacy.
In this study, we aimed to determine the different clinical efficacy of MSCT in RA patients and to identify a possible serum biomarker for predicting the therapeutic effects of MSCT.
All patients met the American College of Rheumatology 1987 (ACR1987) criteria for RA classification and had no other autoimmune or systemic diseases. Written informed consent was provided in accordance with the Declaration of Helsinki. The study was registered at Chictr.org (identifier: ChiCTR-ONC-16008770) and approved by the Ethics Committee of Daping Hospital, Third Military Medical University of the Chinese People’s Liberation Army (YIYANLUNSHEN (2016) NO. 007). The enrolled RA patients responded poorly to regular clinical strategies, including DMARDs, non-steroidal anti-inflammatory drugs (NSAIDs), steroids and biologics, or could not tolerate their serious side effects; thus, the patients maintained active disease conditions (Additional file 1: Table S1). Overall, 105 RA patients were enrolled from July 2016 to March 2017.
Patients were randomly divided into MSCT and control groups. Source and preparation of umbilical cord-derived human MSCs (UCMSCs) were detailed in Additional file 2. The patients received either 1 × 106 cells/kg body weight in 50 mL of 1% albumin in physiological saline as the treatment or 50 mL of 1% albumin in physiological saline without UCMSCs as the control via intravenous infusion. If the status of the patient continued to improve, a withdrawal schedule was used to taper off conventional drug treatment in the following order: prednisone acetate, then NSAIDs and DMARDs. All treatment modifications were agreed upon by the rheumatologist in charge.
Assessment of disease status
Serum levels of anti-cyclic citrullinated peptide antibody (anti-CCP) and rheumatoid factor (RF) were determined by indirect immunofluorescence using kits purchased from Euroimmun (Lübeck, Germany). The percentages of peripheral blood Tregs and Th17 cells were analyzed by flow cytometry (NovoCyte™, ACEA Biosciences, San Diego, CA USA). Monoclonal antibodies against CD4, CD25, Foxp3 and IL-17A were purchased from BD. Flow cytometry data were analyzed with NovoExpress software (ACEA Biosciences, San Diego, CA USA). A bead-based multiplex cytokine assay was custom-designed to quantify the following cytokines: TNF-a, IFN-γ, IL-1β, IL-2R, IL-6, IL-8, and IL-10. The assays were performed according to their instructions, and measurements were made with a Luminex 200 system (Millipore Corporation, Bedford, Massachusetts USA).
Statistical analyses were conducted using the t test for parametric data and the Mann–Whitney U test for non-parametric data. One-way analysis of variance (ANOVA), followed by the Bonferroni test, was used when there were more than two groups. All statistical tests were two-sided, and the significance level was set at P < 0.05. All analyses were conducted with SPSS 17.0 (SPSS, Inc). The data are shown as the mean ± standard error of the mean.
No serious acute adverse events occurred during or after MSCT. Three patients had chills or fever (≤ 39 °C) after MSC infusion but recovered within 3 h without any intervention. No patients developed graft-versus-host disease (GVHD), and no serious infections occurred. No significant abnormalities were found according to routine blood tests, liver and kidney function analysis, chest radiography, urine analysis, or electrocardiography.
Safety evaluation on patients between response and no-response group
Measures (normal value range)
Total protein (65–85 g/L)
73.24 ± 4.32
72.52 ± 3.46
72.63 ± 5.21
74.64 ± 3.68
Albumin (40–55 g/L)
33.23 ± 5.31
32.57 ± 3.54
32.54 ± 4.63
40.63 ± 3.25*
Globulin (20–40 g/L)
30.62 ± 3.57
31.39 ± 4.36
31.58 ± 4.23
32.13 ± 3.86
Cholesterol (3.1–5.72 mmol/L)
4.03 ± 1.34
4.23 ± 1.53
4.15 ± 1.26
4.28 ± 1.93
Triglyceride (0.30–1.7 mmol/L)
1.46 ± 0.69
1.49 ± 0.83
1.51 ± 0.38
1.43 ± 0.58
Creatinine (41–73 μmol/L)
46.53 ± 13.52
45.36 ± 12.38
43.36 ± 12.78
45.25 ± 14.21
Urea (2.6–7.5 mmol/L)
4.23 ± 1.35
4.34 ± 1.28
4.12 ± 1.24
4.15 ± 1.87
Fasting blood glucose (3.9–6.1 mmol/L)
4.74 ± 0.83
4.84 ± 0.96
4.67 ± 0.73
4.75 ± 0.86
White blood cell (3.5–9.5) × 109
5.65 ± 1.35
5.78 ± 1.26
5.84 ± 1.39
5.59 ± 1.47
Hemoglobin (115–150 g/L)
102.35 ± 18.79
103.24 ± 19.21
101.15 ± 19.82
113.46 ± 16.62*
Platelet (94–268) × 109
253.23 ± 68.31
262.31 ± 72.78
263.12 ± 70.24
223.19 ± 65.87*
Assessment of disease activity
Serological autoantibody profile findings
RA is a severe, progressive, systemic inflammatory disease of unknown etiology. RA development is closely related to immune system disorders, which are mostly categorized by unbalanced immune homeostasis. Further tests showed that the percentage of CD4+CD25+Foxp3+ Tregs was increased and that the percentage of CD4+IL-17A+ Th17 cells was decreased in the response group compared to those in the no-response and control group (Fig. 3c). These findings support that MSCs play important roles in regulating immune homeostasis. In particular, a low ratio of CD4+CD25+Foxp3+ Treg to CD4+IL-17A+ Th17 cells (Treg/Th17) indicates an unbalanced immune status and excessive inflammation microenvironment. This low ratio was observed in all patients before MSCT, but it was gradually reversed in the response group after MSCT (Fig. 3d).
IFN-γ predicts clinical response to MSCT in RA patients
A decrease in the number of bone marrow MSCs can be found in RA-like autoimmune diseases, and this decrease grows more severe with disease progression . Recent studies have shown that MSCs can ameliorate arthritis in RA patients and animal models and that the immunosuppressive effect of MSCs may be turned off or even switched to a stimulatory effect . However, many aspects of the role of MSCs in treating RA remain unclear. Our present study has substantiated the clinical safety and efficacy of MSCT for treating RA patients. However, consistent with the study design, the clinical efficacy of MSCT varied greatly. Twelve weeks after MSCT, 54% of the patients in the MSCT group achieved a good or moderate response, whereas the other 46% of patients in the MSCT group had no clinical response.
The intravenous infusion of UCMSCs is a safe and effective practice that can improve immune function and serologic indices. In addition to a significant decrease in disease activity as assessed by the DAS28 value and HAQ score, MSC infusion reversed the inflammatory microenvironment and rebalanced the immune system. In the MSCT response group, evidence of the clinical benefits was obtained, and the clinical manifestation improvements were likely related to the increased Treg/Th17 ratio, which resulted in decreased levels of inflammatory cytokine expression, ESR and CRP. These results suggest that anti-inflammation along with improved immune-modulation and induced immune-tolerance are likely to be the major mechanisms of MSCT. However, the effects of MSCs in vivo are not permanent. In the present study, 8% of patients had disease relapse at 24 weeks after a prior good or moderate response. Disease activity indices, such as ESR and CRP levels, reverted slightly toward baseline levels, concomitant with relapsed joint swelling and pain. Because of the safety profile of MSC infusion in clinical applications, another MSC infusion after 6 months may be necessary for RA patients with partial relapse.
Considering there are no significant differences in age, gender and disease status between response and non-response groups, as mentioned in Additional file 1: Table S1, endogenous factors which may affect the immunoregulatory ability of MSCs come into our sight. To further investigate the cause of such varying responses, we focused on MSC responsiveness to the host microenvironment and MSC participation in immune homeostasis. Several reports have indicated that MSCs are not constitutively immunosuppressive; instead, they need to be activated by the inflammatory environment of the host to develop their immunoregulatory ability . This finding was based on the observation that anti-IFN-γ receptor antibodies blocked the immunosuppressive effect of MSCs. In addition, the presence of other inflammatory cytokines can influence the phenotype and immunosuppressive effect of MSCs. Inflammatory stimuli induced-MSCs can secrete high levels of soluble molecules that can regulate immune homeostasis; these molecules include IDO, nitric oxide (NO), prostaglandin E2 (PGE2), TNF-α-stimulated gene 6 protein (TSG-6), heme oxygenase-1 (HO-1), chemokine (C–C motif) ligand 2 (CCL2), IL-10 and galectin . In our in vitro study, we also confirmed that stimulated with IFN-γ could significantly increase the expression of IDO. According to data from animal models of RA, MSCs are most effective when transplanted after the onset of an inflammatory response. In a mouse GVHD model, MSC transplantation on the same day as bone marrow transplantation (BMT) showed no protective effects , whereas MSC transplantation 3, 8, or 20 days after BMT significantly ameliorated GVHD progression . In line with this observation, we also observed increased IFN-γ levels prior to decreases in the DAS28 value and increases in the Treg/Th17 ratio and IL-10 levels. These data suggest that inflammation occurred before the MSCs exerted their immunosuppressive effects.
Since MSCT efficacy varies greatly among patients, biomarkers that predict MSCT response are urgently needed. Dander et al.  identified two biomarkers for acute GVHD, TNF receptor (TNFR) I and IL 2 receptor alpha (IL-2Rα), that may explain the patient response. The levels of these two factors were consistently decreased in the responder patients, but they were increased before the MSC infusion. This observation agrees with other studies indicating that MSCs need an inflammatory environment to be activated before exerting their therapeutic effects . In the present study, we provide evidence that high serum IFN-γ levels, before or after MSCT, are positively associated with a reduced DAS28 value in RA patients and may be used as a biomarker to predict the clinical efficacy of or select RA patients for allogeneic MSCT.
Further investigation of IFN-γ-primed MSCs in animal models of RA will be crucial for developing novel MSC-based therapies for RA patients who respond poorly to MSCT. The current patients are still being followed, and more patients are being recruited to further confirm the safety and efficacy of MSCT as a new treatment modality for RA.
In the present study, we demonstrate that (1) allogenic MSC transplantation is safe, as no serious adverse events were observed during or after MSC transplantation; (2) MSCT was effective in some RA patients, as suggested by the reductions in the DAS28 value, HAQ score, CRP level, and ESR; (3) MSCT improved the autoantibody profile of patients who responded to the therapy; (4) the potential mechanisms of MSCT are likely to be the anti-inflammatory or immunomodulatory effects of MSCs, as well as restored immune balance and tolerance; (5) serum IFN-γ levels may be a key factor that predicts the therapeutic effects of MSCs on RA patients.
CL and XX conceived the project and designed the experiments. YY, XH, RZ, WG, MZ and WX conducted the experiments. YY and XH wrote the manuscript. XX, CL, and DJ revised the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Consent for publication
Ethics approval and consent to participate
The study was approved by the Ethics Committee of Daping Hospital, Third Military Medical University of the Chinese People’s Liberation Army. Written informed consent was provided in accordance with the Declaration of Helsinki.
This work was supported by the National Natural Science Foundation of China (NSFC, Nos. 81571912 and 81372027) and the Natural Scientific Foundation of Chongqing (Nos. cstc2014jcyjys10002 and cstc2015shmszx120108).
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