Osthole enhances the immunosuppressive effects of bone marrow‐derived mesenchymal stem cells by promoting the Fas/FasL system

Abstract Thanks to the advantages of easy harvesting and escape from immune rejection, autologous bone marrow‐derived mesenchymal stem cells (BMSCs) are promising candidates for immunosuppressive therapy against inflammation and autoimmune diseases. However, the therapy is still challenging because the immunomodulatory properties of BMSCs are always impaired by immunopathogenesis in patients. Because of its reliable and extensive biological activities, osthole has received increased clinical attention. In this study, we found that BMSCs derived from osteoporosis donors were ineffective in cell therapy for experimental inflammatory colitis and osteoporosis. In vivo and in vitro tests showed that because of the down‐regulation of Fas and FasL expression, the ability of osteoporotic BMSCs to induce T‐cell apoptosis decreased. Through the application of osthole, we successfully restored the immunosuppressive ability of osteoporotic BMSCs and improved their treatment efficacy in experimental inflammatory colitis and osteoporosis. In addition, we found the immunomodulatory properties of BMSCs were enhanced after osthole pre‐treatment. In this study, our data highlight a new approach of pharmacological modification (ie osthole) to improve the immune regulatory performance of BMSCs from a healthy or inflammatory microenvironment. The development of targeted strategies to enhance immunosuppressive therapy using BMSCs may be significantly improved by these findings.

a key issue in BMSC cytotherapy. Emerging evidence suggests that BMSCs respond positively to the microenvironment because they are plastic stem cells, and the microenvironment can affect the immunosuppressive properties of exogenous BMSCs. 4,5 As shown in recent studies, BMSCs from different donors have different immunoregulatory characteristics. For instance, BMSCs isolated from healthy candidates are more effective than those derived from patients with lupus for the treatment of autoimmune diseases and the migration capacity of BMSCs harvested from patients with systemic lupus erythematosus is also impaired. 6 In accordance with previous studies, we found that the treatment effect of BMSCs from osteoporosis mice on immune inflammatory diseases such as colitis is worse than that of BMSCs from normal mice. We also found that the immunoregulation of BMSCs was affected by the Fas / FasL pathway in osteoporosis. These findings suggest that the pathological condition of the donor may affect the immunosuppressive effects of BMSCs. 1,7 Although recent studies have shown that the recognition of small molecular compounds can counteract inflammatory insults in BMSCs, 8 a pharmacological solution that promotes the immunosuppressive effects of BMSCs derived from inflammatory microenvironment has not yet been established.
Osthole is a natural pyroxanthin originally extracted from the Cnidium plant, which is commonly used in the clinical practice of traditional Chinese medicine. 9 Osthole has been proven to have various functions, such as osteogenesis, hepatoprotection, cardiovascular protection, anti-inflammation, anti-tumour and anti-microbial activities. 10 Its immunoregulatory capability has been further demonstrated in bronchial epithelial cells, peripheral cone cells and peripheral blood mononuclear cells. 11,12 Osthole has been shown to induce apoptosis and cell cycle arrest in breast cancer cells by inhibiting signal transducer and activator of transcription 3 (STAT 3) phosphorylation and nuclear migration. 13 However, the effects of osthole on the immunomodulatory properties of BMSCs derived from inflammatory and healthy microenvironments have not yet been evaluated. As an extension of our previous investigations, 1,7 this study attempts to assess the immunomodulatory ability potential of BMSCs from osteoporosis mice for the treatment of colitis and osteoporosis. In the present study, we used osthole-modified BMSCs to improve the immunoregulatory function in vitro, to treat inflammatory colitis and oestrogen deficiency-induced osteoporosis in a mice model, and to investigate the underlying mechanisms.

| Animals
All animal experiments were conducted under the experimental animal guidelines of the National Institutes of Health and approved by the Animal Care and Use Committee of Chongqing Medical University. Female C57BL/6 mice (21-25 g) aged 8 weeks were purchased from the animal centre of Chongqing Medical University. All mice were bred in specific pathogen-free conditions and had free access to food and water. C57BL/6 mice were used as the sources for the osteoporosis model, experimental colitis model, BMSC culture and other cell experiments. Dextran sulphate sodium salt (DSS; 3%; MP Biomedical, Irving, California, USA) was fed to the mice in water for 10 days to induce experimental colitis. To establish the oestrogen deficiency-induced osteoporosis model, the mice were anaesthetized and received bilateral ovariectomy or sham operation.

| BMSC and T-cell isolation and coculture
BMSCs isolated from the femurs and tibias and T cells isolated from the spleen of C57BL/6 mouse were cultured as described previously. 1 BMSCs were passaged for three generations, and the purity and specificity were verified by conventional characterization, surface marker identification, and self-renewal and multi energy differentiation assays. BMSCs from healthy sham and osteoporosis donors were transplanted and designated as S/BMSCs and O/BMSCs, respectively.
The spleen single cell suspension was harvested by crushing the spleen of the mice. After red blood cells were removed using a red blood cell lysis buffer (Sigma-Aldrich, St. Louis, USA), spleen cells were cultured with RPMI 1640, supplemented with 10% foetal bovine serum, 50 mm 2-ME, 2 mm L-glutamine (Sigma-Aldrich) and 1% penicillin streptomycin. Anti-CD3 and anti-CD28 (1 mg/mL; Santa Cruz Biotechnology) were used to activate T-cell proliferation for 48 hours. A total of 2 × 10 6 activated T cells were then added to sixwell plates pre-seeded with 2 × 10 5 BMSCs for 48 hours of coculture and further analysis.

| BMSC pre-treatment with osthole
Osthole with purity above 98% was purchased from Medchem Express (Shanghai, China). In accordance with previous studies, 14 confluent BMSCs were incubated with basal medium containing 10 -5 mol/L osthole for 3 days and the resulting osthole-modified BMSCs were used for subsequent studies. After 48 hours of coculture, the T cells that migrated to the lower chamber were counted under a fluorescence microscope (Olympus Optical, Tokyo, Japan).

| Gene expression assay
The level of monocyte chemotactic protein 1 (MCP-1) in the supernatants of BMSCs and tumour necrosis factor (TNF)α and interferon (IFN)γ levels in the serum were measured using commercialized inflammatory cytokine ELISA kits (Beyotime, Haimen, China). For

Western blot analysis, primary antibodies (Santa Cruz Biotechnology)
were used to detect the protein expression of Fas, FasL and β-actin.

Enhanced chemiluminescence (ECL) kits (Amersham Biosciences)
were used to explore the blots on the membranes. ImageJ software was used to quantify the grey values of blots as described previously. 18 Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis using the primers listed in Table 1 was performed as previously described to measure the mRNA expression of Fas, FasL and β-actin. 18

| Micro-computed tomography (CT) examination
Micro-CT (GE, Germany) was used to scan the femurs. The X-ray source was set to 80 kV and 500 μ for a micro focus. Areas of interest were selected manually in the marrow cavity. Bone morphology parameters were evaluated, including bone mineral density (BMD), bone volume/tissue volume (BV/TV), trabecula number (Tb.N), trabecula thickness (Tb. Th) and trabecular thickness (Tb. Sp).

| Statistical analysis
The data were presented as the mean ± SD. Comparisons were conducted using Student's t test or one-way ANOVA. Survival was compared with the Kaplan-Meier log-rank test, whereas bodyweight changes were analysed with the matched-pair signed-rank test. Each experiment was performed with at least three replicates.
Differences were considered significant at P values < .05.

| Osthole restores the O/BMSC-mediated T-cell migration and apoptosis in vitro
The BMSCs used in our study highly expressed mesenchymal stem cell markers (Sca-1 and CD29) were negative for haematopoietic cell mark- migration triggered by O/BMSCs ( Figure 1D). As an explanation for the T-cell migration modulation, we found that pre-treatment with osthole significantly increased MCP-1 secretion by O/BMSCs ( Figure 1E), which is consistent with the early findings indicating that MCP-1 is a key chemokine secreted by BMSCs to promote T-cell migration. 1 Figure 1F). These findings illustrated that osthole can rescue O/BMSC-induced T-cell migration and apoptosis.

| Osthole rescues the O/BMSC-mediated immunosuppression in colitis model
We further explored whether osthole pre-treatment can restore the therapeutic effect of O/BMSCs in vivo (Figure 2A). Ten days after

| Osthole rescues the O/BMSC-mediated immunosuppression in osteoporosis model
It has been reported that the ovariectomy model simulates bone pathogenesis caused by microenvironmental changes in oestrogen deficiency and secondary inflammation. Therefore, we next investigated whether our method was also suitable for the treatment of os-

teoporosis induced by ovariectomy through intravenous injections of O/BMSC, S/BMSCs and osthole-modified O/BMSCs in ovariectomy-
induced osteoporosis mice ( Figure 3A). As shown in Figure 3B and

| Osthole increases S/BMSC-induced T-cell migration and apoptosis
We also investigated whether osthole pre-treatment can improve the expression of Fas and FasL in BMSCs from healthy donors.
Western blotting showed that osthole pre-treatment increased the accumulation of Fas and FasL proteins in S/BMSCs ( Figure 4A-C). However, after pre-treatment, we did not observe substantial changes in Fas and FasL at the mRNA level ( Figure 4D-E). We confirmed that S/BMSCs pre-treated with osthole can promote T-cell migration compared to the control ( Figure 4F). We also found that pre-treatment with osthole increased the amount of S/BMSC-secreted MCP-1, a key chemokine for T-cell recruitment ( Figure 4G). Next, by using the direct coculture model in vitro, we explored whether BMSCs pre-treated with osthole could promote the apoptosis of adjacent T cells. Apoptosis assays showed that compared to naïve S/BMSCs, osthole-modified S/BMSCs increased the apoptosis rate of cocultured T cells ( Figure 4H).
These findings indicated that the immunomodulatory effects of BMSCs from healthy individuals can also be improved by osthole pre-conditioning through increased T-cell migration and apoptosis in vitro.

| Osthole enhanced S/BMSC cytotherapy effects in osteoporosis model
Next  Inflammatory diseases caused by cell dysfunction include inflammatory colitis, osteoporosis and graft versus host disease (GVHD). 1,15 At present, there is no ideal treatment with an ideal therapeutic rate for these diseases. We and others have reported that the key driving forces of pro-inflammatory cytokines, such as TNFα, IL-1β and IFNγ, accumulate in inflammatory colitis. 2,5 We also demonstrated that the BMSCs derived from healthy donors induced T-cell apoptosis and further reduced pro-inflammatory cytokine expression after systematic injection into mice. 20  found that osthole up-regulated Fas and FasL at the protein level but not at the mRNA level. As important modulators of cell function, mi-croRNAs (miRNAs) can inhibit gene expression post-transcriptionally by binding to the 3'UTR of mRNA. 1 We speculated that the reason osthole up-regulated Fas and FasL at the protein but not mRNA level may be due to the fact that osthole inhibits the expression of some miRNAs. [37][38][39] It is also important to explore whether existing miR-NAs can be regulated by osthole.

| D ISCUSS I ON
Previous studies have shown that BMSCs genetically modified with viral vectors harbour potential risks in clinical applications 40 ; therefore, natural small molecules would be more practical and safer for the improvement of cell therapy. Our findings, combined with other previously published results, 41,42 indicate that osthole-based cell therapy is an ideal optional solution to improve cytotherapy mediated by BMSCs derived from either healthy or inflammatory conditions. Our results further demonstrate that the use of allogeneic BMSCs without gene manipulation for the clinical treatment of immune and inflammatory diseases is feasible.
In conclusion, our data revealed a new approach of pharmaceutical modification as a functional and convenient solution to improve the immune regulation of BMSCs derived from healthy and inflammatory conditions, and provide a method to improve the clinical application of autologous BMSC-based immunotherapy.

ACK N OWLED G EM ENT
This work was supported by the National Natural Science Foundation of China (81800979 to Yang Yu, 31970783 to Deqin Yang, 81700958 to Liang Chen, 32000577 to Bingyi Shao, and 81800958 to Dou Lei).

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available on request from the authors.