Introduction
The allo-transplantation of vascularized composite tissue (VCA) has been increasingly used in recent years to reconstruct tissues following amputation or severe injury (1,2). However, the immunosuppressive agents that are administered post-transplantation may cause life-threatening complications, including nephrotoxicity, opportunistic infections and tumorigenesis (3). The cumulative side effects of chronic immunosuppression outweigh the benefits of VCA. In the last decade, cellular therapy has attracted increased attention in the field of allo-transplantation. Mesenchymal stem cells (MSCs), particularly adipose-derived stem cells (ADSCs), can regulate the host immune response to allografts and, in addition to immunosuppressants, improve graft survival (4,5).
The combination therapy of MSCs and immunosuppres-sants induces immune tolerance to VCA, indicating that a cellular component can reduce the dependency on immuno-suppressants in maintaining allografts. However, cell-based therapy also has safety concerns, including the increased risk of infection during ex vivo expansion and cell aggregation during systematic infusion (6,7). Therefore, it is essential to understand the interactions between ADSCs and host immune cells in order to improve the outcomes of cellular therapy in allo-transplantation.
ADSCs secrete immunomodulatory cytokines, including prostaglandin E2 (PGE-2), which inhibit the proliferation of peripheral blood mononuclear cells (PBMCs) in a mixed lymphocyte reaction (8), and express higher levels of cyclooxygenase-2 (COX-2) and indoleamine-2,3- dioxygenase when co-cultured with lymphocytes or pro-inflammatory cytokines (9). In addition, ADSCs and other MSCs regulate the function of T cells, the major driver of allo-rejection, and dendritic cells and macrophages during allo- transplantation (10,11).
The studies performed so far on the mechanisms of ADSC-mediated immunosuppression have not analyzed the molecular changes induced by ADSCs in lymphocytes. The aim of the present study was to determine the effect of ADSCs on T cells; to this end, ADSCs were isolated from adipose tissues and their interaction with the human Jurkat T cell line was investigated.
Materials and methods
Isolation and expansion of ADSCs, and co-culture with Jurkat cells
The human ADSCs were cultured as described previously (12). Briefly, adipose tissue was obtained by liposuction of the abdominal wall from three different donors (samples 1, 2 and 3; females aged 36, 54 and 56 years; Shanghai 9th People's Hospital, Shanghai, China), who had provided informed consent. The tissues were digested in 0.01% collagenase IV (Roche Diagnostics GmbH, Mannheim, Germany) for 1 h, washed twice with PBS, and seeded in 10-cm culture dishes at the density of 1x105 cells/ml with low-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; ScienCell Research Laboratories, Inc., San Diego, CA, USA), 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cells were cultured at 37˚C under 5% CO2 until they reached 80-90% confluence, following which they were dissociated with 0.05% Trypsin-EDTA and passaged. The cells of passages 2-5 were combined, and used for further characterization and in vitro differentiation. The ADSCs were identified by immune- detection of surface CD29 (1:100, cat. no. B195249), CD44 (1:100, cat. no. B162932), CD90 (1:100, cat. no. B205317), CD34 (1:100, cat. no. B203565) and CD45 (1:100, cat. no. B215193) (all BioLegend, Inc., San Diego, CA, USA). The cells were stained with the labeled antibodies for 15 min in the dark at 4˚C and analyzed using the BD FACSCalibur flow cytom-eter (BD Biosciences, San Jose, CA, USA). Adipogenesis, osteogenesis and chondrogenesis were induced by suitable differentiation media (human adipose-derived stem cell adipogenic differentiation medium, HUXMD-90031; human adipose-derived stem cell osteogenic differentiation medium, HUXMD-90021; human adipose-derived stem cell chondro-genic differentiation medium, HUXMD-9004; all Cyagen Bioscience, Inc., Guangzhou, China) at 37˚C under 5% CO2 for >28 days, and the ensuing differentiated cells were identified by staining with oil red, alizarin red and alcian blue, respectively. Images were captured using an inverted microscope (Leica Microsystems GmbH, Wetzlar, Germany).
The Jurkat cells (purchased from GENE, Inc., Shanghai, China) were suspended in RPMI 1640 medium (HyClone; GE Healthcare, Logan, UT, USA) with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin, and seeded in 100-mm dishes at the density of 1x106 cells each. The culture medium was replaced every second day. The ADSCs and Jurkat cells were co-cultured for subsequent experiments in the same media in a 0.4-µm Transwell system (Corning Incorporated, Corning, NY, USA), wherein the ADSCs were seeded in the upper chamber and Jurkat cells in the lower chamber at the ratio of 1:5. The Jurkat cells were treated with 40 µM of the JNK inhibitor SP600125 (Selleck Chemicals, Houston, TX, USA) or DMSO (1 µl/ml cell suspension) for 30 min at 37°C per the requirements of the experiment.
Proliferation, cell cycle and apoptosis assays
The effect of the ADSCs on Jurkat cell proliferation was measured using a CCK-8 (Doijndo Molecular Technologies, Inc., Kumamoto, Japan) assay according to the manufacturer's protocol. The Jurkat cells were seeded into the lower chamber of a 24-well Transwell plate at a density of 1×105 cells/ml per well in 600 µl medium. The upper chambers were filled with either ADSC suspension or sterile culture medium (control). The cells were cultured for 1, 3, or 5 days, and then incubated with 60 µl CCK-8 per well at 37°C for 3 h. The supernatants were collected and the absorbance was measured at 450 nm with a microplate reader (Molecular Devices LLC, Sunnyvale, CA, USA). Each test sample was assayed in triplicate and the experiment was repeated three times.
For cell cycle profiling, the Jurkat cells were harvested following 24 h of mono- or co-culture, and fixed overnight with 70% ethanol. The cells were stained with the reagents provided in a Cell Cycle kit (Qihai Biotechnology, Shanghai, China) as per the manufacturer's protocol. To detect apoptosis, the mono- and co-cultured Jurkat cells were harvested and washed twice with phosphate-buffered saline (PBS), and stained using the FITC-Annexin V/PI Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA) as per the manufacturer's protocol. All stained samples were analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA), and the ModFit LT v2.0 program (BD Biosciences).
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis
Total RNA was extracted from the Jurkat cells harvested following 48 h of mono- or co-culture using an RNA isolation kit (Takara Bio, Inc., Otsu, Japan). The purity of the RNA was evaluated by calculating the A260/A280 ratio, and samples with ratios between 1.8 and 2.0 were used for further analysis. The mRNA was reverse transcribed into cDNA using the PrimeScript™ II First Strand cDNA Synthesis kit (Takara Bio, Inc.). The RT-qPCR analysis was accomplished with a SYBR-Green PCR Master mix kit (Applied Biosystems; Thermo Fisher Scientific, Inc., MA, USA) containing 2 µl cDNA and 0.5 µl of each primer (10 µM), according to the manufacturer's protocol. The following primer pairs were used for gene amplification: TGF-β1, forward 5'-ACA CCA ACT ATT GCT TCA G-3' and reverse 5'-TGT CCA GGC TCC AAA TG-3'; TNF-a, forward 5'-CTC GAA CCC CGA GTG ACA AG-3' and reverse 5'-TGA GGT ACA GGC CCT CT G AT-3'; β-actin, forward 5'-AAG CAG GAG TAT GAC GAG TCC G-3' and reverse 5'-GCC TTC ATA CAT CTC AAG TTG G-3', with the following thermal cycling conditions for 40 cycles in total: 10 min at 95°C; 15 sec at 95°C; and 30 sec at 60°C. The relative gene expression levels were calculated using the 2-ΔΔCq method (13), and normalized against those of β-actin. Three independent experiments were performed.
ELISA
After 72 h, the supernatants were collected from the mono- and co-cultured Jurkat cells and stored at -80°C until use. The levels of TGF-β1 were measured using an ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's protocol.
Western blotting
Total protein was extracted from the Jurkat cells harvested following 24, 48 or 72 h of co-culture with RIPA lysis buffer as described previously (14). The nuclear proteins were extracted with NE-PER nuclear and cytoplasmic extraction reagents (Pierce; Thermo Fisher Scientific, Inc.) from the 48 and 72 h co-cultured cells. Equal quantities of protein (40 µg) per sample were loaded on an 12% SDS-polyacrylamide gel, electrophoresed and transferred onto a PVDF membrane and blocked with TBS with Tween-20 containing 5% non-fat dry milk for 1 h at room temperature. The membranes were probed with primary antibodies against phosphorylated (p)-P65 (1:2,000, cat. no. AF2006, Affinity Biosciences, Cambridge, UK), P65 (1:2,000, cat. no. 10745-1-AP, ProteinTech Group, Inc., Wuhan, China), inhibitor of NF-κB (IκB) kinase (IKK)β (1:1,000, cat. no. Ab124957, Abcam, Cambridge, MA, USA), IκBα (1:1,000, cat. no. 4812, Cell Signaling Technology, Inc., Danvers, MA, USA), B-cell lymphoma 2 (Bcl-2; 1:2,000, cat. no. 12789-1-AP, ProteinTech Group, Inc.), Bcl-2-associated X protein (Bax; 1:5,000, cat. no. 50599-2-IG, Protein Tech Group, Inc), JNK1/2 (1:1,000, cat. no. 9252, Cell Signaling Technology, Inc.), p-JNK1/2 (1:1,000, cat. no. 4668, Cell Signaling Technology, Inc.), extracellular signal-regulated kinase (ERK)1/2 (1:1,000, cat. no. 9102, Cell Signaling Technology, Inc.), p-ERK1/2 (1:2,000, cat. no. 4370, Cell Signaling Technology, Inc.), P38 (1:1,000, cat. no. 9212, Cell Signaling, Technology, Inc.), p-P38 (1:1,000, cat. no. 9211, Cell Signaling Technology, Inc.), mothers against decapentaplegic (Smad)2/3 (1:1,000, cat. no. AF6367, Affinity Biosciences), p-Smad2/3 (1:200, cat. no. MAB8935, R&D Systems, Inc.) and the loading control β-actin (1:200, cat. no. BM0627, Boster Biological Technology, Wuhan, China). This step was followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1:50,000, goat anti-mouse, cat no. BA1051, Boster Biological Technology) for 2 h at room temperature. The specific protein bands were visualized using an enhanced chemiluminescence detection kit (GE Healthcare Life Sciences, Chalfont, UK), and the ratio of the grayscale values of the target protein were calculated.
Statistical analysis
All data are presented as the mean ± standard deviation. Statistical significance was calculated with Student's t-test and one-way analysis of variance; Tukey's multiple comparison test was used for post hoc analysis. Statistical analyses were performed using Prism 6.0 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.
Results
Effect of ADSCs on Jurkat cell proliferation, cell cycle and apoptosis
The ADSCs isolated from human adipose tissue were characterized using MSC surface markers CD90, CD44, CD29, CD34 and CD45 (12). The multipotency of the ADSCs was determined by the adipogenic, osteogenic and chondrogenic differentiation assays (Fig. 1A-H).
The effect of ADSCs on Jurkat cell behavior was assessed using established proliferation, cell cycle and apoptosis assays. Although no significant differences were observed in the proliferation rates of the mono- and co-cultured Jurkat cells on days 1 and 3, the ADSC-co-cultured cells exhibited a significantly lower proliferation rate on day 5 compared with the control cells (P<0.05; Fig. 2A). Following 24 h of co-culture with ADSCs, the proportion of Jurkat cells in the G0/G1 and G2/M phases increased significantly and there was a concomitant decrease in the proportion of cells in the S phase compared with the control cells (Fig. 2B). Finally, the Jurkat cells co-cultured with ADSCs for 48 h exhibited significantly higher apoptotic rates than the control cells (Fig. 3A-D). The Jurkat cells co-cultured with ADSCs for 48 h also showed significantly higher levels of pro-apoptotic Bax and lower levels of anti-apoptotic Bcl-2 compared with the control cells (Fig. 3E). Taken together, the ADSCs suppressed Jurkat cell proliferation by arresting them at the G0/G1 and G2/M phases, and inducing apoptosis.
Effect of ADSCs on Jurkat cell cytokine secretion
The mRNA and protein levels of TNF-α and TGF-β1 were measured in the Jurkat cells co-cultured with ADSCs by RT-qPCR and ELISA analyses, respectively. Compared with the control cells, the mRNA levels of TNF-α and TGF-β1 were signifi-cantly decreased in the co-cultured cells at 48 h, whereas the secreted levels of TGF-β1 were significantly increased in the supernatants of the co-cultured cells at 72 h. (Fig. 4).
Regulation of Jurkat cell signaling pathways by ADSCs
In addition to the cells co-cultured with ADSCs for 48 h showing higher levels of pro-apoptotic Bax and lower levels of anti-apoptotic Bcl-2, the NF-κB p-P65 subunit levels in the cellular and nuclear fractions were marginally lower in the co-cultured cells compared with those in the control cells after 72 h. No apparent differences were observed in the levels of P65, IκBα or IKKβ between the two groups, as well as the level of Erk, P38 and Smad2/3 (Figs. 5A-D and 6). By contrast, the level of p-JNK1/2 was higher in the co-cultured cells after 48 h (Fig. 6A-C). These findings indicated that the ADSCs induced apoptosis of the Jurkat cells by suppressing NF-κB P65 subunit phosphorylation and activating the JNK signaling pathway.
ADSC induces apoptosis in Jurkat cells via the JNK pathway
In order to evaluate the role of the activation of JNK in ADSC-induced Jurkat cell apoptosis, the latter were treated with 40 µM of the specific JNK inhibitor SP600125 prior to the co-culture, and the percentage of apoptotic cells was assessed. As shown in Fig. 7A-E, the inhibition of JNK in Jurkat cells significantly inhibited ADSC-induced apoptosis.
Discussion
As the prognosis of allo-transplantation is dependent on the host immune response to the allografts, reducing T cell-mediated acute rejection is vital in order to protect the allograft. The immunomodulatory effects of ADSCs observed in pre-clinical studies make it a suitable addition to routine immunosuppressants for preventing allograft rejection (4,5). Therefore, it is important to determine the specific effects of ADSCs on immune cells, particularly CD4+ and CD8+ T cells, which are the major elicitors of acute and chronic rejection (15,16).
Gonzalez-Rey et al (17) reported suppressive effects of human ADSCs on the proliferation of activated PBMCs and production of TNF-α. A study on the miniature swine hind-limb model showed that the infusion of ADSCs prolonged allograft survival, increased regulatory T cells in allografts and blood, and decreased the levels of circulating pro-inflammatory cytokine TNF-α (4). In addition, Matula et al (18) reported a similar inhibitory effect of ADSCs on Jurkat T cells following 3 and 4 days of co-culture. However, Mousavi et al (19) did not report any differences in the proliferation rates of Jurkat cells cultured with or without ADSC for 2 and 3 days. In the present study, ADSC co-culture reduced Jurkat cell viability on day 5, with no significant effects observed until day 3, indicating a time-dependent effect of the ADSCs on Jurkat cells.
The NFAT and NF-κB signaling pathways synergistically activate T cells (20). NF-κB is a transcription factor that regulates the expression of inflammatory cytokines and genes essential for the proliferation and survival of T cells (21). MSCs have been shown to inhibit NF-κB signaling in responder cells (22-24). Consistent with this, the present study observed a reduction in the levels of p-p65 and its nuclear translocation in Jurkat cells co-cultured with ADSCs, thereby confirming the suppressive effect of ADSCs on T cells.
Consistent with the pro-apoptotic effects of MSCs on T cells reported in previous studies (25,26), the present study observed increased apoptosis in the Jurkat cells co-cultured with ADSCs. Previous studies have also reported an inhibitory effect of MSCs on T cell apoptosis (27-29). This ambiguity may be the result of differences in responder cells (28), different tissue sources of MSCs (e.g. bone marrow, placenta or adipose tissue) (27,29), and different T-cell stimulators (e.g., PHA or anti-Fas antibody) (27,29) used in these studies. Therefore, MSCs from different origins may affect T cell function via different mechanisms.
Apoptosis can be induced by either inhibiting survival signals, for example, BCL-2 as an inner mitochondrial membrane anti-apoptotic protein, or by increasing death signals, for example BAX as a pro-apoptotic effector of mitochondrial outer membrane permeabilization (30). Furthermore, Bcl-2-associated apoptosis in Jurkat cells is associated with the activation of JNK (31). The present study observed increased levels of p-JNK1/2 in the co-cultured Jurkat cells, indicating the ADSC-mediated activation of JNK. The involvement of the JNK pathway in ADSC-induced apoptosis was further verified by the JNK-specific inhibitor SP600125 inhibiting apoptosis in the co-cultured Jurkat cells. However, the inhibitory effect was not complete, indicating the involvement of other pathways in ADSC-induced apoptosis. Higher levels of secreted TGF-β1, an activator of JNK, were present in the superna-tants of the co-cultured Jurkat cells. However, the mRNA levels of TGF-β1 were significantly lower in the co-cultured Jurkat cells; it is likely that TGF-β1 was secreted by the ADSCs and not Jurkat cells. TGF-β1 is an important component of the ADSC secretome, and has immunosuppressive and apoptotic effects on T cells (32,33). Taken together, the ADSCs induced apoptosis in Jurkat cells by activating the JNK pathway.
MSCs have shown potential in improving transplantation outcomes (34-36). A previous study showed that the inclusion of bone marrow-derived MSCs (BMSCs) during renal transplantation improved renal function at the one-year follow-up (34). In addition, the BMSCs also reduced tubulitis, interstitial fibrosis and tubule atrophy post-transplantation (35). The immunomodulatory effects ADSCs, although evident in pre-clinical and experimental studies, have not been verified in clinical trials. In the present study, the mechanism underlying the effect of ADSC on T cells was elucidated, to further underscore the clinical potential of ADSCs in immunological diseases and transplantation, and to improve current understanding of stem cell-based therapy.
In conclusion, the present study showed that ADSCs suppressed Jurkat T cell viability by inducing apoptosis and cell cycle arrest by activating the JNK signaling and mitochondrial apoptotic pathways. These findings provide novel insights into the molecular mechanisms underlying the immunomodulatory effects of ADSCs, and provide an experimental basis for the clinical application of ADSCs.