A potent immunomodulatory role of exosomes derived from mesenchymal stromal cells in preventing cGVHD

Human MSCs were isolated from bone marrow (BM) samples and identified as previously described [1, 4]. Briefly, the bone marrow aspirates (at least 20 mL) were diluted with cultured medium in 1:1 and MSC were isolated with Ficoll-Paque solution (1.077 g/mL; Amersham Biosciences, Uppsala, Sweden) after centrifugation at 800 g for 20 min. The isolated MSC cells were resuspended and cultured at a density of 5000 cells/cm². The medium contains low glucose Dulbecco’s modified Eagle’s medium (L-DMEM; Hyclone, Logan, UT, USA) and 10% fetal bovine serum (FBS; Hyclone). The adherent cells were cultured with medium changes every 3 days. When they were 70–80% confluent, cells were detached by trypsin-EDTA and passaged at a ratio of 1:3 and the third passage MSCs were used for exosome preparations. All these MSCs have been tested for their ability to differentiate into osteoblasts, adipocytes, and chondrocytes. Flow cytometry was performed using a FACSort and analyzed with CellQuest software (Becton Dickinson, San Jose, CA, USA). Only the cells exhibited surface expression of mesenchymal markers (CD73, CD105, and CD166) and cell adhesion molecules (CD29, CD44, and CD90) but negative for hematopoietic markers (CD14, CD19, CD31, CD34, CD45, and HLA-DR) were identified as human MSCs, which fulfill the minimal definition criteria proposed by the International Society for Cellular Therapy. Human dermal fibroblasts purchased from ScienCell were used as control cells. Human dermal fibroblasts were cultured in DMEM/high glucose medium (Invitrogen) that contained 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C.

To manufacture exosomes, cells were cultured with exosome-free FBS for 48 h, which was prepared by a sequential centrifugation procedure as 200×g for 10 min, 2000×g for 20 min, 10,000×g for 30 min, and 110,000×g for 7 h at 4 °C, followed by filtration using a 0.22-μm filter [22]. The culture supernatant was collected and performed ultracentrifugation with the same sequential centrifugation procedure as above. The pellet was washed twice with PBS and then filtered through the 0.22-μm filter. The prepared exosomes were stored at − 20 °C until use. The electronic microscopy was utilized for characterization of isolated exosomes. After fixation with 2% paraformaldehyde, the exosomes were negatively stained with phosphotungstic acid for 1 min and examined with a transmission electron microscopy (hitachi H-7650). Markers of exosomes, including CD63, CD9, and CD81, were analyzed by western blot as previously described [23]. The primary antibodies included antibodies against CD63, CD9, and CD81 (Abcam, Cambridge, MA, USA).

The mouse cGVHD model was established as previously described [24]. Briefly, 10- to 12-week-old BALB/cJ^H-2d female mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., China) as recipients received irradiation followed by a tail vein injection of 8 × 10⁶ bone marrow cells and 8 × 10⁶ spleen cells from B10.D2 male mice, the donors purchased from Jackson Laboratories, Bar Harbor, USA. The animal experimental design and procedures were reviewed and approved by the animal experimental ethics committee of Guangdong General Hospital. Recipient mice were monitored every 3 days with respect to the clinical score, body weight loss, and activities beginning at day 14 after bone marrow transplantation (BMT). Mice assigned a clinical score above 0.6 were regarded as established cGVHD. The sry gene on Y chromosome was detected in blood DNA from the female recipient mice on day 20 after BMT. The genotype result showed that all the representative recipient mice presented with sry gene expression, indicating that these mice were indeed transplanted successfully (Additional file 1: Figure S1). On day 22 after BMT, cGVHD mice received a tail vein injection of MSCs-exo or Fib-exo in a 100-μl volume at a dose of 1 μg/μl. The exosome injections were administered once per week for 6 weeks. Blank control mice received equal amounts of a PBS injection. The disease score and skin score were determined as previously described [24], and survival was checked daily for 60 days. The criteria of skin score were briefly determined as follows: healthy appearance = 0, skin lesions with alopecia less than 1 cm² in area = 1, skin lesions with alopecia 1 to 2 cm² in area = 2, and skin lesions with alopecia more than 2 cm² in area = 3. Additionally, animals were assigned 0.3 point each for skin disease (lesions or scaling) on the ears, tail, and paws with minimum score as 0 and maximum score as 3.9. The clinical disease score was based on the clinical manifestation of the skin, body weight, and hunch. When the body weight was loss between 2 and 8%, the mouse had score a point and when more than 8%, the mouse would gain 2 points. For hunch position, the mouse would get a score when hunch quiescent and 2 points when hunch affect action. So the minimum clinical disease score was 0, and the maximum was 7.9.

Tissues were fixed with 4% formalin overnight, embedded in paraffin and cut into 6 μm slices. H&E and Masson staining (Masson’s trichrome staining kit, Sigma) were performed separately on consecutive tissue sections, and images were obtained using a microscope (Leica DM4000, Wetzlar, Germany). Quantification of fibrosis was conducted using ImageJ (NIH) as the percentage of blue collagen-stained area relative to the total tissue in one field.

PBMCs were isolated from healthy donors and patients with active clinical manifestations of cGVHD, who provided written informed consent in accordance with the Declaration of Helsinki. This experiment was approved by the Ethics Committees of Guangdong General Hospital. Healthy PBMCs were initially stimulated with 2.5 μg/ml PHA (Sigma, USA) and treated with Fib-exo or MSCs-exo (10 μg/ml or 50 μg/ml) for 5 days to detect Treg cells. In addition, other healthy PBMCs were cultured under the inductive condition of Th17 cells (25 μL/well Human T-Activator CD3/CD28 Dynabeads in 24-well plates (Life Technologies, Thermo Fisher Scientific, USA), Il-6100 ng/ml, and TGF-β 20 ng/ml) and treated with individual exosomes to detect the percentage of Th17 cells. Finally, patients’ PBMCs were stimulated with Human T-Activator CD3/CD28 Dynabeads and treated with PBS, Fib-exo, or MSCs-exo (50 μg/ml) for 5 days. The CD4 T subsets were determined by flow cytometry and qPCR.

The cells were isolated from the spleen, lymph nodes (LNs), or lung, and cell surface protein expression was detected and quantified by flow cytometry. A single-cell suspension of lung tissue was prepared to evaluate the infiltration of CD4 T cells. Briefly, the lungs were removed, dissociated, and digested in collagenase D (2 mg/mL) at 37 °C for 30 min. The digested lungs were filtered through 40 mm cell straining to remove the debris. For intracellular cytokine detection, the cells were re-stimulated for 5 h with PMA (20 ng/ml)/ionomycin (1 μM) (Sigma, USA). Golgi-stop was added in the last hour, and intracellular cytokine staining was performed using a BD Biosciences Cytofix/Cytoperm kit as recommended (BD Pharmingen, San Diego, CA, USA). Flow cytometry analysis was performed on a Bectone-Dickinson FACSCalibur (BD Biosciences) using protein-specific monoclonal antibodies as previously described [25]. The data were analyzed using FlowJo software (TreeStar).

The relevant cytokines, including IL-17, IL-21, IL-22, IL-2, IL-12, and IL-10, in mouse serum were analyzed using a Luminex MAGPIX system (Luminex Corp., Austin, TX) according to the manufacturer’s instructions [26]. The secretion of IL-17 and IL-10 cytokines from cultured PBMCs obtained from active cGVHD patients was detected by ELISA. The assays were performed using human IL-17A and IL-10 ELISA kits (eBioscience, USA) according to the manufacturer’s instructions. Samples were detected in triplicate relative to standards supplied by the manufacturer and analyzed for significant differences among different groups.

Real-time PCR analysis was performed to detect the mRNA levels of relevant transcription factors as previously described [23]. Briefly, total RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA, USA) and converted into first-strand cDNA using random hexamer primers and the Reverse Transcriptase Superscript II Kit (Invitrogen, Carlsbad, CA, USA). PCR was then performed in a total volume of 20 μL that contained 2 μL of cDNA, 10 μL of 2 × SYBR Premix Ex Taq, 0.8 μL of 50 × ROX Reference Dye (TaKaRa Biotechnology Co., Ltd., Dalian, China), and 10 μmol/L of the primer pairs, which are listed in Additional file 2: Table S1. Gapdh was used as a reference gene. The PCR amplification protocol consisted of 95 °C for 30 s and up to 40 cycles of 95 °C for 5 s and 60 °C for 34 s according to the manufacturer’s instructions.

Statistical analysis was performed with SPSS software version 19.0 (IBM, Ehningen, Germany). The data are presented as the mean value ± standard error of the mean (SEM) and were statistically analyzed using a one-factor analysis of variance (ANOVA). Survival curves were plotted as Kaplan-Meier curves and analyzed with log-rank tests. P values < 0.05 were considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001).

Exosomes derived from MSCs were obtained through sequential ultracentrifugation in this study. A transmission electron image presented the typical rounded shape of an exosome, approximately 100 nm (Fig. 1a). The western blot results showed that Fib-exo and MSCs-exo were positive for CD9, CD81, and CD63, markers of exosomes (Fig. 1b), thereby confirming the presence of exosomes.

To assess the efficacy of MSCs-exo as a therapeutic intervention for cGVHD, we used a typical B10.D2 in the BALB/c mouse of the cGVHD model. Receipt mice were administered tail vein injections of PBS, Fib-exo, or MSCs-exo once per week from day 22 of BMT, when these mice had developed the clinical features of cGVHD. Thirty-five days after BMT, PBS-treated cGVHD mice served as a blank control, which showed clear weight loss and dermal lesions characterized by hair loss, redness, flaking, scabbing, or a hunched posture (Fig. 1c). In contrast to Fib-exo-treated cGVHD mice, which presented similar clinical signs to the PBS-treated mice, mice that received a MSCs-exo injection displayed little clinical features (Fig. 1c). In addition, these MSCs-exo-treated mice had a significant improvement of survival (Fig. 1d) and had the lowest disease scores and skin scores in the cGVHD pathology among all animals (Fig. 1e, f).

The observed trends in cGVHD disease were also verified by histopathology. Both cGVHD mice with PBS or Fib-exo application presented typical histopathological features compatible with sclerodermic fibrotic cGVHD (Fig. 2a, b). A thickened reticular dermis and an increase in the extracellular matrix constituents were observed in PBS and Fib-exo-treated mice (Fig. 2a, b). By contrast, MSCs-exo-treated mice exhibited less epidermal fibrosis with a decreased thickness of the dermis and less loss of hair follicles (Fig. 2a, b). The statistics of Masson-positive fibrosis area percentage showed the minimal fibrosis in the MSCs-exo group (Fig. 2b). In addition, these cGVHD mice with PBS or Fib-exo application developed pulmonary cGVHD (Fig. 2c, d). These mice displayed narrower small airways with a significant increase in the peribronchiolar and perivascular collagen deposition (Fig. 2d), indicating the presence of fibroproliferative disease. By contrast, mice treated with MSCs-exo manifested an organized, regular structure of the small airway in the lung, with little Masson-positive fibrosis (Fig. 2c, d). When the liver tissue was analyzed, similar results were observed. cGVHD mice with PBS or Fib-exo treatment presented with severe portal fibrosis and inflammatory cell infiltration, which were significantly suppressed by MSCs-exo treatment (Fig. 2e, f). Overall, these results showed that MSCs-exo had an inhibitory effect on cGVHD.

It is of note that alloreactive T cells were required for the induction of cGVHD. To understand the therapeutic mechanism of exosomes from MSCs, the activation of effector CD4+ T cells in the lymphocytes of cGVHD mice was first evaluated by CD44 expression using flow cytometry. As shown in Fig. 3a, the percentages of CD4 + CD44+ in lymphocytes from the cGVHD with PBS and Fib-exo treatment were similar, whereas a reduced percentage was observed in MSCs-exo treated mice, which indicates that MSCs-exo inhibited the activation of CD4+ T cells. Furthermore, the expression of CCR6, which facilitates Th17 cell recruitment, was significantly reduced in the MSCs-exo group (Fig. 3b). Considering the involvement of the lung in T cell-targeted organs in cGVHD, single-cell suspensions of lung tissues were prepared, and the infiltration of CD4 T cells was detected. We found a substantial reduction of CD4+ T cells in the lungs of the MSCs-exo treated mice (Fig. 3c), which was consistent with less infiltration of leukocytes, as shown in Fig. 2c. These findings indicate that the MSCs-exo treatment suppressed the migration and infiltration of CD4+ T cells into the target organ during cGVHD.

Th17 cells are expanded in the spleen and draining lymph nodes of cGVHD mice, and IL-17 has been implicated to be a central mediator of cGVHD pathology. To determine whether the protective effect of exosomes from MSCs was dependent on the Th17-pathway, we collected splenocytes and lymphocytes from LNs and detected Th17 and Treg cells by flow cytometry. As expected, cGVHD is typically accompanied by the presence of IL-17-expressing CD4+ T (Th17) cells (Fig. 4a, b). Strikingly, a significant reduction of Th17 cells was observed in the splenocytes (Fig. 4a) and lymph node cells (Fig. 4b) from MSCs-exo mice compared with the controls. It is interesting to note that the effect was more prominent in splenocytes.

Given the centrality of IL-10 in the suppression of the inflammatory response in transplantation rejection and several autoimmune diseases, we investigated IL-10-producing regulatory T cells (Treg) by flow cytometry. As shown in Fig. 4c, MSCs-exo upregulated the frequency of IL-10-expressing cells. Foxp3, a critical transcriptional factor for Treg differentiation, was also increased in MSCs-exo-treated cGVHD mice (Fig. 4d). In addition, these relevant transcription factors were measured by qPCR. mRNA expression of the detected genes, including RORγt, Stat3, Foxp3, and T-bet, in PBMCs from PBS-treated cGVHD mice was similar to that in the Fib-exo group (Fig. 5a–d), excluding the possible effect of exosomes from fibroblast cells. By contrast, the MSCs-exo treatment induced a significant reduction of RORγt, a transcription factor that is selectively expressed in Th17 cells, and STAT3, which is required for RORγt expression and IL-21 production (Fig. 5a, b). However, T-bet, which is important for Th1 cell development, was almost equivalent between these groups (Fig. 5d), indicating that MSCs-exo exerted no obvious effect on Th1 generation. Taken together, these observations suggested that MSCs-exo inhibited cGVHD by promoting the expansion of Treg cells while inhibiting the pro-inflammatory Th17 cells that mediate cGVHD.

Th17-relevant cytokine production, also a hallmark of GVHD, was detected in the serum obtained from individual group mice by Luminex. On day 39 after BMT, the expression of IL-17A, IL-21, IL-22, IL-2, IL-10, and IL-12 was similar in serum obtained from the PBS and Fib-exo groups (Fig. 5e–j). By contrast, the MSCs-exo-treated group presented significantly lower levels of IL-17A (Fig. 5e), the typical cytokine produced by Th17 cells; IL-22 (Fig. 5g); and IL-21 (Fig. 5f), which has an essential role in all phases of GVHD pathophysiology to mediate tissue damage. IL-2, a T cell growth factor for amplifying lymphocyte responses, was also reduced in the MSCs-exo-treated group compared with the PBS and Fib-exo groups (Fig. 5i). It is interesting to note that there was no significant difference in IL-12, a cytokine involved in the differentiation of naive T cells into Th1 cells, between the different groups (Fig. 5j). As for the important regulatory cytokine IL-10, we found that MSCs-exo induced an approximately two-fold elevation of IL-10 (Fig. 5h). Thus, MSCs-exo may exert marked immunosuppressive effects on cytokine production.

Our data confirm that Th17 cells are critical to the development of cGVHD and that MSCs-exo alleviate cGVHD in murine models, in part, by inhibiting Th17 cells and promoting Treg cells. To confirm that this effect is not restricted to mouse model, we tested the effects of MSCs-exo on human PBMCs in vitro. We initially isolated and cultured CD3+ cells and investigated their uptake of PKH26-labeled exosomes. The actin filaments of CD3+ cells were labeled with phalloidin. As shown in Fig. 6a, PKH26-labeled exosomes were present in the cytoplasm of cultured CD3 cells, which indicates exosome uptake by CD3 cells. Furthermore, MSCs-exo upregulated the percentage of CD25+Foxp3+CD4+ Treg in PBMCs from healthy donors, with a more significant effect in the higher dose MSCs-exo group (Fig. 6b). When PBMCs from healthy donors were cultured under Th17 culture conditions, the differentiation of Th17 cells was markedly suppressed by 50 μg/ml MSCs-exo (Fig. 6c). In addition, PBMCs from patients with active cGVHD were also stimulated with T-activator CD3/CD28 and treated with PBS, Fib-exo, and MSCs-exo. Consistently, IL-17-expressing CD4+ T cells were reduced in the MSCs-exo group (Fig. 7a), whereas the production of IL-10, the anti-inflammatory cytokine that antagonizes pro-inflammatory T cell subsets, such as Th1 and Th17 cells, was significantly upregulated (Fig. 7b). The mRNA expression of relevant transcription factors was detected by qPCR. Similarly, MSCs-exo suppressed the expression of both RORγt and Stat3 and promoted the upregulation of Foxp3 (Fig. 8a–c), further indicating the involvement of the Th17/Treg balance in the mitigated effect of MSCs-exo in cGVHD. Moreover, the typical cytokines IL-17 and IL-10 were evaluated in the supernatants using ELISA. As expected, MSCs-exo inhibited the production of IL-17 while promoted IL-10 generation in PBMCs from human cGVHD (Fig. 8e, f). These data confirmed that MSCs-exo could modulate the development of Th17 and Treg cells in the setting of active cGVHD.