Mol Neurobiol DOI 10.1007/s12035-016-9840-3
Rapamycin Augments Immunomodulatory Properties of Bone Marrow-Derived Mesenchymal Stem Cells in Experimental Autoimmune Encephalomyelitis Mansoureh Togha 1 & Mehrdad Jahanshahi 2 & Leila Alizadeh 3 & Soodeh Razeghi Jahromi 4,3 & Gelareh Vakilzadeh 5 & Bahram Alipour 6 & Ali Gorji 3,7 & Amir Ghaemi 8,9
Received: 26 October 2015 / Accepted: 4 March 2016 # Springer Science+Business Media New York 2016
Abstract The immunomodulatory and anti-inflammatory properties of bone marrow-derived mesenchymal stem cells (BM-MSCs) have been considered as an appropriate candidate for treatment of autoimmune diseases. Previous studies have revealed that treatment with BM-MSCs may modulate immune responses and alleviate the symptoms in experimental autoimmune encephalomyelitis (EAE) mice, an animal model of multiple sclerosis. Therefore, the present study was designed to examine immunomodulatory effects of BMMSCs in the treatment of myelin oligodendrocyte glycoprotein (MOG) 35-55-induced EAE in C57BL/6 mice. MSCs were obtained from the bone marrow of C57BL mice, cultured with DMEM/F12, and characterized with flow cytometry for the presence of cell surface markers for BM-MSCs. Following three passages, BM-MSCs were injected intraperitoneally into EAE mice alone or in combination with rapamycin. Immunological and histopathological effects of BM-MSCs and addition of rapamycin to BM-MSCs were evaluated. The results demonstrated that adding rapamycin to BM-
MSCs transplantation in EAE mice significantly reduced inflammation infiltration and demyelination, enhanced the immunomodulatory functions, and inhibited progress of neurological impairments compared to BM-MSC transplantation and control groups. The immunological effects of rapamycin and BM-MSC treatments were associated with the inhibition of the Ag-specific lymphocyte proliferation, CD8+ cytolytic activity, and the Th1-type cytokine (gamma-interferon (IFN-γ)) and the increase of Th-2 cytokine (interleukin-4 (IL-4) and IL-10) production. Addition of rapamycin to BMMSCs was able to ameliorate neurological deficits and provide neuroprotective effects in EAE. This suggests the potential of rapamycin and BM-MSC combined therapy to play neuroprotective roles in the treatment of neuroinflammatory disorders.
Keywords Multiple sclerosis . Stem cells . Neuropharmacology . Cytokine . Neuroprotection
Mansoureh Togha and Mehrdad Jahanshahi contributed equally to this work. * Amir Ghaemi
[email protected];
[email protected]
1
Iranian Center of Neurological Research, Neuroscience Research Institute, Tehran University of Medical Sciences, Tehran, Iran
2
Neuroscience Research Center, Department of Anatomy, Faculty of Medicine, Golestan University of Medical Sciences, Gorgan, Iran
3
Shefa Neuroscience Research Center, Tehran, Iran
4
Multiple Sclerosis Research Center-Neuroscience Institute, Sina Hospital, Tehran University of Medical Sciences, Tehran, Iran
5
School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
6
Iranian Blood Transfusion Organization Research Center, Tehran, Iran
7
Epilepsy Research Center, Klinik und Poliklinik für Neurochirurgie, Department of Neurology, Westfälische Wilhelms-Universität Münster, Münster, Germany
8
Infectious Diseases Research Center, Department of Microbiology, Golestan University of Medical Sciences, P.O. Box 49175-1141, Gorgan, Iran
9
Department of Virology, Institute Pasteur of Iran, Tehran, Iran
Mol Neurobiol
Introduction Multiple sclerosis (MS) can be considered as an inflammatory neurodegenerative disorder. Neuronal demyelination and axonal injury play major roles in pathophysiology of MS [1, 2]. Experimental autoimmune encephalomyelitis (EAE) is an organ-specific cell-mediated autoimmune demyelinating disease, characterized by neurological deficits and lymphocytic and mononuclear cell infiltration of the central nervous system (CNS) [3, 4]. A number of therapeutic strategies have been studied in an attempt to minimize or ameliorate tissue damage in EAE model. However, due to the complexity of EAE pathogenesis and the heterogeneity among patients, combination therapy strategies that target a range of various disease mechanisms might be more efficient than monotherapy [5, 6]. Transplantation of bone marrow-derived mesenchymal stem cells has been extensively studied in EAE experimental animal model [7–9] based on their self-renewal capacity, the ability to migrate into inflammation sites, potential of differentiation into multiple lineages, and immunomodulatory and neuroprotective properties [10–13]. Different immunomodulatory and neuroprotective mechanisms are involved in the ameliorative effects of bone marrow-derived mesenchymal stem cells (BM-MSCs) in the treatment of EAE [14]. It has been emphasized that BM-MSCs require being primed by inflammatory signals to induce their maximal immunosuppressive activities [15]. Previous studies have also demonstrated that BM-MSC immunosuppressive efficacy may be improved through stimulation by toll-like receptor (TLR) agonists [16] and gamma-interferon (IFN-γ) [17]. Rapamycin is a potent immunomodulator with fewer side effects compared to other immunosuppressants, e.g., cyclosporine [18]. Rapamycin has been used for a decade in transplant patients to prevent graft rejection [19]. Rapamycin has been reported to increase the percentage of regulatory T (Treg) cells in vivo and promote immunomodulation [20–22]. Rapamycin selectively promotes expansion of functional human Tregs and depletes human CD4+ CD25− T effector cells [23]. In EAE animal model, rapamycin was highly effective in preventing the onset and severity of disease [21]. More recently, it has been shown that rapamycin can increase the immunosuppressive activity of MSCs in vitro [24]. Furthermore, mammalian target of rapamycin (mTOR) inhibition can extend the life span of MSCs [25]. Besides their role in cell culture, it has been demonstrated that rapamycin pretreatment can optimize the immunomodulatory potential of MSCs in a murine model of acute graft-versus-host disease [26]. All together, these observations suggest that rapamycin has the potential to improve therapeutic effects of BM-MSCs in the treatment of EAE. In this study, we investigated whether adding rapamycin to BM-MSC transplantation could augment their therapeutic and immunomodulatory effects in EAE mice. As a result, we
demonstrate that the combination treatment additively ameliorated EAE in mice by reducing disease severity, CNS inflammation, and demyelination compared to BM-MSC treatment alone. Moreover, this combination therapy enhanced immunomodulatory properties, suggesting the potential of combination therapy to improve the recovery of patients with MS.
Materials and Methods Reagents Roswell Park Memorial Institute (RPMI) cell culture medium, penicillin/streptomycin, and fetal bovine serum (FBS) were purchased from Gibco (Invitrogen, Germany). Hooke kit for EAE induction [myelin oligodendrocyte glycoprotein (MOG) 35–55/complete Freund’s adjuvant (CFA) emulsion and pertussis toxin (PTX):5×] and MOG 35–55 for in vitro stimulation of cells were purchased from Hooke Laboratories (Hooke Laboratories, EK-0115, Lawrence, MA, USA). Rapamycin (Sigma-Aldrich, St. Louis, MO) was dissolved in a vehicle solution of phosphate buffer containing DMSO 1/500. Reagents required for histopathological analysis, including paraffin, xylol, alcohol, phosphate buffer saline (PBS), hematoxylin/eosin (H&E), and luxol fast blue (LFB) solution, were obtained from Sigma (Sigma-Aldrich, Germany). Cytokine ELISA kits were obtained from eBioscience (eBioscience, San Diego, CA). Animals C57BL/6 female mice (n = 10, 8–10 weeks old) were purchased from Pasture Institute, Tehran, Iran. The animals were housed in a pathogen-free animal condition with standard humidity and 22–23 °C temperature and 12/12 (7–19) dark/light cycle. Four animals were housed in each cage and maintained 1 week for acclimatization and then weighted once a week during the experiment. All the experiments were approved by the Ethical Committee of Golestan University of Medical Sciences, Gorgan, Iran. Murine Bone Marrow Mesenchymal Cell Extraction and Culture Expansion BM-MSCs were isolated from C57BL/6 mice (6–8 weeks old) and collected by marrow aspiration of the shafts of femurs and tibias into DMEM/F12 (Gibco, BRL, Eggenstein, Germany) supplemented with 10 % FBS using a 21-G needle. The cells were then washed in PBS and centrifuged at 400×g for 5 min. The cells were seeded onto 25-cm2 flasks containing DMEM/F12 medium supplemented with 10 % FBS, 100 U/ml penicillin/streptomycin (Gibco, BRL, Eggenstein, Germany), and 2.5 μg/ml
Mol Neurobiol
fungizone (Invitrogen, Paisley, Scotland). Culture medium was changed at day 2 to remove non-adherent cells. Whole medium was subsequently replaced weekly. The cells were grown for 2–3 weeks until almost confluent. Adherent cells were then detached by 0.25 % trypsin-EDTA and replated using a 1:3 dilution until third passage.
upper and lower back of MOG 35–55 (200 μg) peptide emulsified in CFA containing 400 μg Mycobacterium tuberculosis. Two hours later, and again 24 h thereafter, the mice received an intraperitoneal injection of PTX (0.1 ml/animal/day).
BM-MSC Surface Marker Analysis
One week after the injection, the mice were examined daily for development of disabilities. Most of the EAE mice demonstrated clinical symptoms after approximately 10 days postinjection. Mice that developed no clinical symptoms were excluded. Clinical scores were blindly registered according to standard protocol: 0, no disease; 1, floppy tail; 2, hind limb weakness; 3, partial hind limb paralysis; 4, complete hind limb paralysis; and 5, moribund stage [28]. Following the appearance of EAE symptoms, mice were scored for disease severity and categorized into separate test groups to obtain equally weighted average disease scores before experimental interventions.
The expressions of surface antigens CD29, CD166, CD90, CD44, CD73, and CD105 (as positive markers) and CD45, CD34, and CD14 (as negative markers) on BM-MSCs were evaluated by flow cytometry using the following monoclonal antibodies: fluorescein isothiocyanate (FITC)-labeled antiCD45 (50 μg/ml), anti-CD44 (50 μg/ml), anti-CD90 (50 μg/ ml), and anti-CD105 (50 μg/ml) and phycoerythrin (PE)-labeled anti-CD14 (25 μg/ml), anti-CD34 (25 μg/ml), antiCD29 (20 μg/ml), anti-CD73 (20 μg/ml), and anti-CD166 (50 μg/ml; all from BD Biosciences, USA). The passage 3 of BM-MSCs was used for analysis of cell determinant markers. Briefly, cells were incubated with the specific antibodies or isotype control antibodies with 3 % bovine serum albumin (Sigma, USA) in PBS for 30 min at 4 °C, followed by fixation with paraformaldehyde 1 % (Sigma, USA). Final analysis was performed using flow cytometry (Partec PAS III, Germany). CellQuest software was used for data analysis (FCS EXPRESS). Osteogenic and Adipogenic Differentiation Assays The potency of BM-MSCs for differentiation to adipogenic and osteogenic lineages was assessed as previously described [27]. Briefly, adipogenic differentiation was induced by adding 1-methyl-3-isobutylxanthine (0.5 mmol/l), dexamethasone (1 mol/l), insulin (10 g/ml), and indomethacin (0.2 mol/ l; all from Sigma-Aldrich) to subconfluent cultures of BMMSCs. Differentiation medium was changed each 3 days till 21 days. Adipogenesis was confirmed after 21 days and by Oil Red O staining and the accumulation of neutral lipids in fat vacuoles. For osteogenic differentiation, cells were plated in 6-well culture plates at 3 × 103 cells/cm2 in BM-MSC medium supplemented with dexamethasone (0.1 mol/l), ascorbic acid (0.05 mmol/l), and glycerophosphate (10 mmol/l). Matrix mineralization was confirmed by calcium phosphate deposits after 0.2 % Alizarin Red S (2 % aqueous solution, pH 4.1–4.3, adjusted with ammonium hydroxide) staining for 20 min. EAE Induction C57BL/6 female mice were immunized with Hooke kits (Hooke Laboratories, EK-0115, Lawrence, MA, USA) according to the manufacturer’s instructions. Briefly, after a mild anesthesia, EAE mice received a subcutaneous injection in the
Clinical Scoring of Disease and Disability
BM-MSC and Rapamycin Treatments The ability of combined treatment BM-MSCs/rapamycin to reduce the symptoms of EAE was examined by administration of these treatments into EAE mice via intraperitoneal route. Ten days post-EAE induction, animals were blindly divided into three groups (n = 10), including (1) PBS (EAE-PBS) group, (2) BM-MSC (EAE-BM-MSC) group (twice with 2 × 106 BM-MSC injections at days 10 and 17 after EAE induction), and (3) combination of BM-MSC-rapamycin (EAE-BM-MSC/rapamycin) group. For combination group, rapamycin (0.3 mg/kg) was administered in 0.1 ml distilled water once daily for 7 days beginning on day 10 post-EAE induction in combination with 2 × 106 BM-MSCs two times at 10 and 17 after EAE induction. We evaluated the neurological functions of EAE mice treated daily until 50-day post-MOG injection. Histopathological Studies At the end of the follow-up period (50-day post-EAE induction), five mice per group were anesthetized with ketamine (IP, 50 mg/kg) and xylezine (4 mg/kg). Thereafter, mice were perfused with PBS containing 4 % paraformaldehyde (PFA). Spinal cord sections (L1 to L3) were dissected and immersed in 4 % PFA overnight and then embedded in paraffin. Finally, coronal sections were obtained in 5-μm thickness. The quantification of neurological damage in EAE mice was performed via histological analysis of 5-μm paraffin CNS sections of EAE-PBS-, EAE-BMMSC-, and EAE-BM-MSC/rapamycin-treated groups. Paraffin sections were stained with LFB to assess myelin damage and with H&E to assess infiltrating cells. The
Mol Neurobiol
sections were observed with an Olympus light microscope (BX51, Olympus, Japan) and photographed with an Olympus digital camera (Olympus, Japan) linked to a microscope. ImageJ software was used to calculate the extent of demyelination in the spinal cord as the percentage of volume fraction in damage tissue/total area for 16 sections in each animal (n = 5). Furthermore, H&E staining was performed to study infiltration of hematogenous cells into all the parts of CNS [29]. The numbers of perivascular inflammatory infiltrates were calculated and expressed as the numbers of inflammatory infiltrates per square millimeter.
MOG-Specific Lymphocyte Proliferation Assay Single-cell suspension of mononuclear cells was isolated individually from the spleen of each mouse (n = 3) at 1 week after the last treatment, suspended to 5 × 105/ml with RPMI complete medium (RPMI 1640 containing 10 % FCS, 2 mM L-glutamine, 1 % HEPES, 50 μM βmercaptoethanol, 200 U/ml penicillin, 200 μg/ml streptomycin, and 100 U/ml of mycostatin) and seeded in 96well flat-bottom plates. Each cell sample was plated in triplicate for each mouse. The culture was stimulated with 10 μM MOG 35–55 peptide, 2 μg/ml phytohemagglutinin (PHA; Sigma Chemicals) as a positive control, bovine serum albumin (BSA) as an irrelevant control, and medium as a negative control. After incubation for 3 days at 37 °C with 5 % CO2, the proliferation responses were detected by a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 5 mg/ml, Sigma) method. Dimethyl sulfoxide (DMSO; 100 μl) was added to dissolve produced formazan crystals. Plates were read at 540 nm, and the results were expressed as stimulation index (SI). The SI was determined as follows: OD values of stimulated cells (Cs) minus relative cell numbers of unstimulated cells (Cu) by relative OD values of unstimulated cells [30]. SI = (Cs _ Cu)/Cu % Cytotoxity ¼ ðexperimental value − effector cell controlÞ−
Spleen Cytokine Assay Mononuclear cells from spleens of treated mice (n = 3) were prepared and incubated with RPMI-1640 at a concentration of 5 × 105 cells/well in 96-well plates (Nunk/Denmark) for 2 days. The RPMI was supplemented with 10 % FCS, 1 % L-glutamine,
Fig. 1 Characterization of bone marrow stem cells (BM-MSCs). BM- MSC differentiation to adipogenic and osteogenic. a Adipogenic differentiation was determined by Oil Red O staining for lipid vacuoles. b Differentiation into osteocytes after induction culture was assessed by Alizarin Red staining for calcium mineralization. c BM-MSC phenotype identification by flow cytometry. Dot plots represent the surface markers of BM-MSCs. The samples were analyzed using flow cytometry during the third passage, evaluating the cell surface markers CD90, CD44, CD105, CD73, CD29, CD166, CD34, CD45, and CD14. Gates were set using isotype controls
Cell Cytotoxicity Assay One week after the final treatment, mice (n = 3) were sacrificed and their splenocytes were isolated. For each sample obtained from individual mouse, single-cell suspension of mononuclear cells (used as the effector cells) was cocultured in RPMI 1640 medium with washed target cells EL4 at 50:1 effector-to-target cell (E/T) ratios, in which maximal release of LDH was observed. For preparation of the target cells, EL4 cells were stimulated with 10-μM MOG 35–55 peptide and then incubated for 4 h. After centrifugation, the supernatants (50 μl/well) were transferred to the 96-well flat-bottom plates, and cytotoxic T lymphocyte (CTL) activity was determined by measuring LDH release using Cytotoxicity Detection Kit (LDH) according to the procedures stated by the manufacturer (TaKaRa Company). Several controls were used for the cytotoxicity assay. BHigh control^ was the total LDH released from the target cells, and all EL4 cells were lysed by medium containing 1 % Triton X-100. BLow control^ was the natural release of LDH from the target cells, which was obtained by adding EL4 cells only in the assay medium. BT cell control^ that was used to measure the natural release of LDH from T cells was obtained by adding the different ratios of T cells only in the assay medium. The assay for all samples, including the controls, was performed in triplicates. The LDH-mediated conversion of the tetrazolium salt into red formazan product was measured at 490 nm after incubation at room temperature for 30 min [31]. The percentage of specific cytolysis was determined by the following formula: low control 100 high control−low control
1 % HEPES, 0.1 % 2ME, and 0.1 % penicillin/streptomycin and pulsed with 2 μg/ml PHA or 10 μM MOG 35–55 peptide after 3 days. Cell-free supernatants were harvested and assayed for interleukin-4 (IL-4), interleukin-10 (IL-10), and IFN-γ at 48 h using a sandwich-based ELISA kit (eBioscience, USA) and performed according to the manufacturer’s protocol. All studies were performed in triplicate for each mouse.
Mol Neurobiol
Mol Neurobiol
Statistical Analysis Statistical analysis was performed using the SPSS 15 software. Statistical analysis for significant differences on clinical scores was performed with the non-parametric Mann– Whitney U test for the clinical course of EAE. Lymphocyte proliferation, CTL, and cytokine assay were analyzed by a one-way ANOVA. Data are presented as mean ± SD. Differences were considered statistically significant when the P value was less than 0.05.
Results
(n = 10 mice/group). The mice were monitored to day 50 (50-day post-EAE induction). As presented in Fig. 2, the cumulative scores and severity of EAE were ameliorated in EAE-BM-MSCs (1.75 ± 0.11, 0 % mortality, P ≤ 0.001, Mann–Whitney U test) and EAE-BM-MSC/rapamycin groups (1.09 ± 0.16, 0 % mortality, P ≤ 0.001, Mann– Whitney U test) compared to EAE-PBS mice (2.93 ± 0.23, 40 % mortality). Additionally, treatment with the combination of BM-MSCs-rapamycin caused a significant reduction in the mean clinical scores compared to BM-MSCs alone (P ≤ 0.01, Mann–Whitney U test). Rapamycin Improved BM-MSC Effects on Demyelination and Inflammation
BM-MSC Preparation and Characterization BM-MSCs were successfully isolated from C57BL/6 mice bone marrow. A homogeneous BM-MSC population was obtained from C57BL/6 mice after three passages ex vivo. To validate the multipotentiality of the BM-MSC cultures, in vitro differentiation was induced into adipogenic and osteogenic lineages under culture conditions. The results showed that BM-MSCs were able to differentiate into adipocytes and osteocytes, confirming that the expanded cells are of BMMSC origin. Adipogenesis was evident by formation of lipid droplets and transformation of BM-MSCs into round cells following Oil Red O staining; approximately 55–60 % of the cells appeared to be involved in adipoblast differentiation (Fig. 1a). Osteogenesis was indicated by the formation of Alizarin Red-positive calcified deposits, so about 70 % of the cells showed differentiation to osteoblast (Fig. 1b). Based on the adherent properties of BM-MSCs, the cells were extracted from the femurs and tibias and characterized by flow cytometry by the expression of common stem cell surface markers. After the third passage, the majority of BMMSCs expressed the mesenchymal cell surface markers, lymphocyte differentiation marker CD73 (96.08 %; integral unit), CD29 (95.46 %; Thy-1), and CD90 (95.06 %); activated leukocyte cell adhesion molecule CD166 (81.08 %); and early adhesion and hyaluronan markers CD44 (83.26 %) and CD105 (49.41 %). The majority of BM-MSCs were negative for CD45, a hematopoietic cell surface marker; CD34 (0.05 %); and CD14 (0.32 %, Fig. 1c). The data reported are representative of three independent experiments, each performed in duplicates. Rapamycin Enhanced BM-MSC Improvement of the Clinical Score In order to assess the additive effect of rapamycin to BMMSCs on EAE progression, PBS, BM-MSCs, or a combination of BM-MSCs/rapamycin were administered intraperitoneally in C57BL/6 mice 10 days after MOG immunization
The clinical scores of the disease were recorded for 50 days, and then, the mice were sacrificed and their spinal cords and brains were harvested. Tissue samples were processed and stained with H&E (Fig. 3a) and LFB (Fig. 3b) to assess for the presence of inflammatory foci and demyelination. Cell infiltration analysis of brains obtained from EAE-BM-MSC mice on day 50 post-EAE induction revealed a significant reduction in the total number of the inflammatory infiltrates of mononuclear cells (44.1 ± 2.31) compared to EAE-PBS mice (73.1 ± 6.3 cells per field, P ≤ 0.01, post hoc). Combined administration of BM-MSC and rapamycin significantly decreased the number of infiltrating cells present in the brain compared to the EAE-PBS and the EAE-BM-MSC mice (25.4 ± 2.6 cells per field, P ≤ 0.001, post hoc; Fig. 3c). Quantification of demyelination by density analysis of LFB-stained spinal cord sections obtained from EAE-PBS mice demonstrated a significant decrease in the myelinated area compared to EAE-BM-MSC mice. EAE-BM-MSC/ rapamycin mice had a significant reduction in the demyelination compared to the EAE-PBS and the EAE-BM-MSC mice (P ≤ 0.05, post hoc). Rapamycin Decreased BM-MSC Response on T Cell Proliferation The immunomodulatory effects of BM-MSCs represent an important mechanism by which these cells can induce protection in EAE model. To determine whether the combined treatment with BM-MSCs/rapamycin influenced the cell-mediated response, we performed MOG-specific lymphocyte proliferation in vitro. Splenocytes were isolated from treated mice and divided into four parts stimulated with MOG antigen, PHA as a positive control, BSA as an irrelevant control, and medium as a negative control. As shown in Fig. 4, the lymphocyte proliferation rate was significantly lower in EAE-BM-MSCs/ rapamycin and EAE-BM-MSC mice compared to EAE-PBS mice (P ≤ 0.001). Furthermore, the combined group had a less significant lymphocyte proliferation response than EAE-BM-
Mol Neurobiol
Fig. 2 The combination of BM-MSC/rapamycin attenuates clinical EAE severity in mice. a During 50-day clinical follow-up, the disease severity of C57BL/6-EAE in mice transplanted with combination of BM-MSC/ rapamycin was significantly reduced compared to EAE-BM-MSC and EAE-PBS mice. Data are expressed as the mean clinical score of all the mice in the group versus the time post-MOG injection. b Cumulative scores (CSs) were calculated as the sum of all EAE clinical scores
(mean ± SD, n = 7) from disease onset to day 50 post-challenge, divided by the number of mice in each group. Development of EAE was significantly reduced in combined treated mice (P < 0.001). Graphs represent the combination of all independent experiments. Clinical severity of EAE was graded by the widely used scale ranging from 0 to 5. Data are expressed as the mean ± SD. ** and *** indicate significance at P ≤ 0.01 and P ≤ 0.001, respectively
MSC group (P ≤ 0.01). These results suggest that CD4 lymphocytes from EAE-BM-MSC/rapamycin mice exhibited a lower proliferation response.
produced significant higher level of IL-10 compared to EAE-BM-MSC mice (P ≤ 0.001). However, there was no significant difference in the levels of IL-4 between EAE-BMMSC/rapamycin and EAE-BM-MSC groups. These results suggest that splenocytes of EAE-BM-MSC/rapamycin mice had a lower ratio of Th1/Th2 compared to BM-MSC and PBS mice.
Rapamycin Augmented BM-MSC Anti-Inflammatory Effect Cellular immune response was evaluated by cytokine secretion assay of the in vitro stimulated splenocytes. The Th1/Th2 cytokine ratio is a key regulator of clinical EAE disease. IFN-γ secretion is a marker of Th1-type antigen-specific cellular immune responses, while IL-4 and IL-10 are recognized as Th2-type humoral-mediated immune response. The cytokine profiles induced by combined treatment were investigated following stimulation with MOG antigen. As shown in Fig. 5a, EAE-BM-MSCs/rapamycin and EAE-BM-MSCs mice significantly generated less IFN-γ than EAE-PBS mice (P ≤ 0.001). IL-4 and IL-10 levels were significantly increased when the combination of BM-MSCs/rapamycin or BM-MSCs alone was administered compared to PBS group (P ≤ 0.001; Fig. 5b, c). Furthermore, stimulated IFN-γ production was higher in the BM-MSC group compared to BM-MSCs/ rapamycin (P ≤ 0.001). BM-MSC/rapamycin treatment
Rapamycin Reduced BM-MSC Effects on the CTL Response Several evidences have emphasized the contribution of CD8 T cells in animal models of CNS autoimmunity. In our study, the CTL response in immunized mice was examined using the LDH release assay. The effector:target cell ratio up to the maximum ratio of 100:1 was used in the present study. As shown in Fig. 6, the cytolytic activity was significantly higher in mice treated with the PBS than in those treated with the combined treatment of BM-MSCs/rapamycin or BM-MSCs alone (P < 0.001). Furthermore, BM-MSC group had a significantly higher antigen-specific CTL response than the group treated with the combined treatment (P ≤ 0.01).
Mol Neurobiol
Fig. 3 The combination of BM-MSC/rapamycin decreases demyelination and inflammation outcomes of EAE mice. H&E (a) and LFB (b) staining were performed to assess the extent of demyelination and mononuclear cell infiltration in the CNS of EAE mice. In H&E panel, arrows show the inflammatory cells. In LFB panel, the blue areas show intact region (in normal group), whereas the pink areas show demyelinating region. c Graphs representing the number of infiltrates (n = 3 mice, from two independent experiments). Comparisons of the
H&E images show a decrease in the number of inflammatory cells in the brain after administration of combined treatment compared with BM-MSC and PBS groups (in EAE group, ***P ≤ 0.001). d Graphs representing the quantification of demyelination by density analysis of LFB-stained spinal cord sections obtained from combined EAE-BMMSC/rapamycin treatment demonstrated a significant increase in the myelinated area as compared to EAE-PBS mice (*P ≤ 0.05)
Discussion
the compounds with potent immunosuppressive activity aimed at inducing immunomodulation and redirecting immune responses are mesenchymal stem cells and rapamycin. Rapamycin inhibits the mammalian target of rapamycin pathway, which is downstream of costimulatory molecules (such as CD28) and cytokine receptors [34]. Rapamycin, also known as sirolimus, is a macrocyclic triene anti-biotic displaying immunomodulatory activity. It has been widely used in preventing allogenic transplant rejection and in treating some autoimmune diseases [22]. A therapeutic role of rapamycin has already been suggested in several animal models of autoimmune diseases, such as EAE [22], autoimmune arthritis [35], and the non-obese diabetic [36] mice. The animal studies suggest that the beneficial effects of rapamycin
MS, an inflammatory disease of the CNS, is characterized by an autoimmune response against myelin proteins, which results in progressive neurological dysfunction [32]. The available immunomodulatory treatments, such as beta-interferon, are only partially effective, and more effective treatment strategies to reduce the chronic and progressive aspects of the disease are needed [6]. Considering the complex pathophysiology of MS, combination therapy offers an advantage over monotherapy. Combined therapy is considered to be most effective when the two immunomodulatory agents affect different mechanisms of action and alleviate adverse events by using lower doses of individual medications [5, 33]. Among
Mol Neurobiol
Fig. 4 The combination of BM-MSC/rapamycin diminishes T cell proliferation. Splenocyte proliferation levels after in vitro stimulation with MOG35–55 peptide.C57BL/6 mice were immunized by MOG 35–55. After 10 days, the mice were treated IP with BM-MSC/ rapamycin and BM-MSCs (test groups) and PBS (negative control groups). One week after final treatment, spleens of individual mice (n = 3) were removed and lymphocyte proliferation was evaluated with MTT method. Formazan crystal formation after incubation of MTT was determined by solving the crystals in DMSO, and optical density (OD) was read at 540 nm. All tests were performed in triplicate for each mouse. Values are the mean ± SD of the mean for the experiments. Lymphocyte proliferation of the EAE-PBS group was significantly higher than those in EAE-BM-MSC and EAE-BM-MSC/rapamycin groups (P < 0.001). *** indicates statistically significant difference between the marked groups as determined by one-way ANOVA
are associated with its regulatory properties on T cell activation and proliferation [22]. Despite its significant advantages, the previous studies suggested that high-dose rapamycin can potently induce cell death [32]. Systemic administration of rapamycin showed specific adverse events including reversible cytopenia that seems to be dose dependent [26]. Therefore, a suboptimal dose of rapamycin was chosen for use in our study. It has been shown that administration of suboptimal doses of rapamycin is not sufficient for the suppression of immune responses [37]. Thus, the additive effect of 0.3 mg/kg rapamycin to BM-MSCs was used to suppress EAE in this study, and it was investigated whether the combination of a two BM-MSCs and repeated administration of rapamycin at suboptimal doses were capable of modulating immune responses and ameliorating EAE severity. The present study shows that although BM-MSC transplantation alone had significant effects on EAE mice compared to the control animals, the additive effect of rapamycin further protected EAE mice by reduction of clinical severity, inflammatory infiltration, and demyelination as well as by enhancement of the immunomodulatory responses. These findings indicate that the therapeutic effect of combined treatment on the clinical and histopathological features of EAE is likely immune mediated. Improvement of the clinical score was accompanied with a reduction in demyelinating
and inflammation areas in the CNS. This was established by decreased IFN-γ secretion, splenocyte proliferation, and CD8 cytolytic activity stimulated by MOG 35–55 peptide in the combined treatment group. Our study also revealed that the additive effect of rapamycin on BM-MSCs significantly increased the production of IL-4 and IL-10 and polarized the cytokine profile toward Th2. In keeping with our results, previous investigations revealed that the activation of myelin-specific CD4 T cells is critical to the pathogenesis of EAE. Those cells expressing Th1-type cytokines, including IFN-γ and TNF-α, are required for clinical severity of EAE, whereas Th2-type cytokines, including IL-4, IL-10, and TGF-β, are protective [38, 39]. IFN-γ responses to myelin basic protein-derived peptides and shifting the cytokine pattern of myelin-autoreactive T cells to a Th1 profile correlate with clinical disease progression [40]. In our study, combined application of rapamycin and BMMSCs was associated with the inhibition of the CD8+ cytolytic activity. While much emphasis had been focused on the role of CD4+ T cells in MS, recent evidence have proposed that CD8+ T lymphocytes might also have a role in this disease. Huseby et al. have shown that CD8 T cell clones, specific for the class I-restricted fragment of myelin basic protein, induce severe EAE with paralysis and other neurological deficits [41]. Furthermore, the increased number of CD8+ T cells within the inflammatory brain lesions and oligoclonal expansions of CD8+ T cells in the brain and cerebrospinal fluid of MS patients support the pathogenic role of CD8+ T in MS [42]. It has been shown that significant increases of CD8+ T cells are generated upon immunization with MOG 35–55 in C57BL/6 mice [43]. Several studies have confirmed the immunosuppressive characteristics of BM-MSCs. It has been shown that BMMSCs can modulate the functions of both T and B lymphocytes. BM-MSCs can inhibit the production of IFN-γ by CD4+ T and CD8+ T cells, whereas they can restore the secretion of IL-4 by CD4+ and CD8+ T cells [44]. Therefore, the immunomodulatory features of BM-MSCs, together with their capacity to differentiate into neuronal lineages, support the neuroprotective effects of BM-MSCs in EAE and suggest the potential therapeutic role in neuroinflammatory disorders [45]. The present study complements these findings by demonstrating that BM-MSCs generate a more efficient effect on clinical severity of EAE, antiinflammatory, and neuroprotective activities when combined with suboptimal doses of rapamycin. This may be partially due to the enhanced additive clinical and immunological effects. The findings are in accordance with previous results showing that a low concentration of rapamycin can attain enhanced immunoregulatory potential of MSCs [24]. In this regard, MSCs and rapamycin have both been reported to increase the proportion of Tregs through direct [46].
Mol Neurobiol
Mol Neurobiol
Fig.
5 The combination of BM-MSC/rapamycin induces antiinflammatory effect. Determination of the ratios of TH1/TH2 cytokines by enzyme-linked immunosorbent assay (ELISA).Collected supernatants (n = 3) from stimulated splenocytes were screened for the presence of IFN-γ (a), IL-4 (b), and IL-10 (c) and to determine the phenotype of the immune responses. The data is represented as the mean ± SD. Each sample was examined in triplicate. *** (P < 0.001) indicates statistically significant difference between the marked groups as determined by oneway ANOVA. BM-MSC/rapamycin and BM-MSC treatment groups show significant differences with PBS control group (P < 0.001)
In agreement with our synergistic effect findings, it has been reported that MSCs combined with low-dose rapamycin exerted immunosuppressive effects via cell-cell contact and the regulation of cytokine secretion and significantly prolonged allograft survival in the diabetic mice. The study presented that MSCs combined with rapamycin downregulated IL-2 and IFN-γ expressions and modulate T cell phenotypes toward Th1 suppression compared with the rapamycin group [47]. Kim et al. findings represented that rapamycin pretreatment of MSCs in a murine model of acute graftversus-host disease (aGVHD) were associated with a reduction of Th17 cells and an increase in regulatory T cells [26]. Synergism has also been stated with systemic administration of rapamycin together with MSCs in a heart transplant mode [48]. The suppressive effects of MSCs and rapamycin were also seen to be additive in vitro. Kim et al. have shown that rapamycin can induce autophagy of MSCs while preserving the expression of surface markers and multilineage differentiation potentials of MSCs under in vitro conditions. That may
underlie the effective immunomodulatory effect of MSCs by rapamycin treatment [26]. B7-H1, also known as CD274 or programmed cell death 1 ligand (PD-L1), has been reported for a synergistic interaction of MSCs and RAPA in the suppression of immune responses [49]. Several studies have verified that MSCs combined with other immunosuppressive drugs induce allograft immune tolerance through immunosuppression in vivo. Hou et al. has exhibited that a treatment with combined treatment with a low dose of minocycline and mesenchymal stem cells exceeded the effects of either treatment alone and exerted synergistic therapeutic effects on EAE progression [50]. Consequently, the additive effect of rapamycin on BM-MSC protective function meets potent immunosuppressive and protective effects in current investigation and characteristically protecting and inhibiting EAE development, thus rationale for testing these medications in combination therapy. Acknowledgments This study was supported by Research Deputy at Golestan University of Medical Sciences, Gorgan, Iran and Tehran University of Medical Sciences, Tehran, Iran. Compliance with Ethical Standards All the experiments were approved by the Ethical Committee of Golestan University of Medical Sciences, Gorgan, Iran. Disclosure interests.
References 1.
2.
3.
4.
5.
6. Fig. 6 The combination of BM-MSC/rapamycin decreases CTL response. Analysis of the cytotoxic activity of CD8 induced by combined group of BM-MSC/rapamycin. CTL activity of the lymphocytes from immunized mice (three mice per group) was measured at 100:1 E/T ratio by LDH release assay kit (TaKaRa) as described in BMaterials and Methods^ section. Specific lyses of target cells are shown with non-specific background lysis subtracted. ** and *** indicate significance at P ≤ 0.01 and P ≤ 0.001 as determined by oneway ANOVA, respectively
All the authors declare that they have no conflict of
7.
8.
Graber JJ, McGraw CA, Kimbrough D, Dhib-Jalbut S (2010) Overlapping and distinct mechanisms of action of multiple sclerosis therapies. Clin Neurol Neurosurg 112(7):583–591. doi:10.1016/j. clineuro.2010.05.002 Murray TJ (2009) The history of multiple sclerosis: the changing frame of the disease over the centuries. J Neurol Sci 277(Suppl 1): S3–8. doi:10.1016/s0022-510x(09)70003-6 Breij EC, Brink BP, Veerhuis R, van den Berg C, Vloet R, Yan R, Dijkstra CD, van der Valk P, Bo L (2008) Homogeneity of active demyelinating lesions in established multiple sclerosis. Ann Neurol 63(1):16–25. doi:10.1002/ana.21311 Ransohoff RM (2012) Animal models of multiple sclerosis: the good, the bad and the bottom line. Nat Neurosci 15(8):1074– 1077. doi:10.1038/nn.3168 Conway D, Cohen JA (2010) Combination therapy in multiple sclerosis. Lancet Neurol 9(3):299–308. doi:10.1016/s14744422(10)70007-7 Stuart WH (2007) Combination therapy for the treatment of multiple sclerosis: challenges and opportunities. Curr Med Res Opin 23(6):1199–1208. doi:10.1185/030079907x187838 Fransson M, Piras E, Wang H, Burman J, Duprez I, Harris RA, LeBlanc K, Magnusson PU, Brittebo E, Loskog AS (2014) Intranasal delivery of central nervous system-retargeted human mesenchymal stromal cells prolongs treatment efficacy of experimental autoimmune encephalomyelitis. Immunology 142(3):431– 441. doi:10.1111/imm.12275 Guo Y, Chan KH, Lai WH, Siu CW, Kwan SC, Tse HF, Wing-Lok Ho P, Wing-Man Ho J (2013) Human mesenchymal stem cells
Mol Neurobiol
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
upregulate CD1dCD5(+) regulatory B cells in experimental autoimmune encephalomyelitis. Neuroimmunomodulation 20(5):294– 303. doi:10.1159/000351450 Wang X, Kimbrel EA, Ijichi K, Paul D, Lazorchak AS, Chu J, Kouris NA, Yavanian GJ, Lu SJ, Pachter JS, Crocker SJ, Lanza R, Xu RH (2014) Human ESC-derived MSCs outperform bone marrow MSCs in the treatment of an EAE model of multiple sclerosis. Stem Cell Rep 3(1):115–130. doi:10.1016/j.stemcr.2014.04. 020 Bonab MM, Sahraian MA, Aghsaie A, Karvigh SA, Hosseinian SM, Nikbin B, Lotfi J, Khorramnia S, Motamed MR, Togha M, Harirchian MH, Moghadam NB, Alikhani K, Yadegari S, Jafarian S, Gheini MR (2012) Autologous mesenchymal stem cell therapy in progressive multiple sclerosis: an open label study. Curr Stem Cell Res Ther 7(6):407–414 Mirmosayyeb O, Meamar R, Tanhaie AP, Eskandari N, Shaygannejad V (2014) Mesenchymal stem cell therapy in multiple sclerosis: an updated review of the current clinical trials. Multiple Scler Relat Disord 3(6):750. doi:10.1016/j.msard.2014. 09.180 Rivera FJ, Aigner L (2012) Adult mesenchymal stem cell therapy for myelin repair in multiple sclerosis. Biol Res 45(3):257–268. doi: 10.4067/s0716-97602012000300007 Shirian S, Ebrahimi-Barough S, Saberi H, Norouzi-Javidan A, Mousavi SM, Derakhshan MA, Arjmand B, Ai J (2015) Comparison of capability of human bone marrow mesenchymal stem cells and endometrial stem cells to differentiate into motor neurons on electrospun poly(epsilon-caprolactone) scaffold. Molecular neurobiology. doi:10.1007/s12035-015-9442-5 Bai L, Lennon DP, Eaton V, Maier K, Caplan AI, Miller SD, Miller RH (2009) Human bone marrow-derived mesenchymal stem cells induce Th2-polarized immune response and promote endogenous repair in animal models of multiple sclerosis. Glia 57(11):1192– 1203. doi:10.1002/glia.20841 Krampera M, Cosmi L, Angeli R, Pasini A, Liotta F, Andreini A, Santarlasci V, Mazzinghi B, Pizzolo G, Vinante F, Romagnani P, Maggi E, Romagnani S, Annunziato F (2006) Role for interferongamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells. Stem Cells (Dayton, Ohio) 24(2):386– 398. doi:10.1634/stemcells.2005-0008 Opitz CA, Litzenburger UM, Lutz C, Lanz TV, Tritschler I, Koppel A, Tolosa E, Hoberg M, Anderl J, Aicher WK, Weller M, Wick W, Platten M (2009) Toll-like receptor engagement enhances the immunosuppressive properties of human bone marrow-derived mesenchymal stem cells by inducing indoleamine-2,3-dioxygenase-1 via interferon-beta and protein kinase R. Stem Cells (Dayton, Ohio) 27(4):909–919. doi:10.1002/stem.7 Ryan JM, Barry F, Murphy JM, Mahon BP (2007) Interferongamma does not break, but promotes the immunosuppressive capacity of adult human mesenchymal stem cells. Clin Exp Immunol 149(2):353–363. doi:10.1111/j.1365-2249.2007.03422.x Daoud D, Scheld HH, Speckmann EJ, Gorji A (2007) Rapamycin: brain excitability studied in vitro. Epilepsia 48(4):834–836. doi:10. 1111/j.1528-1167.2006.00976.x Saunders RN, Metcalfe MS, Nicholson ML (2001) Rapamycin in transplantation: a review of the evidence. Kidney Int 59(1):3–16. doi:10.1046/j.1523-1755.2001.00460.x Donia M, Mangano K, Amoroso A, Mazzarino MC, Imbesi R, Castrogiovanni P, Coco M, Meroni P, Nicoletti F (2009) Treatment with rapamycin ameliorates clinical and histological signs of protracted relapsing experimental allergic encephalomyelitis in Dark Agouti rats and induces expansion of peripheral CD4 + CD25 + Foxp3+ regulatory T cells. J Autoimmun 33(2):135–140. doi:10.1016/j.jaut.2009.06.003 Esposito M, Ruffini F, Bellone M, Gagliani N, Battaglia M, Martino G, Furlan R (2010) Rapamycin inhibits relapsing experimental
autoimmune encephalomyelitis by both effector and regulatory T cells modulation. J Neuroimmunol 220(1-2):52–63. doi:10.1016/j. jneuroim.2010.01.001 22. Lisi L, Navarra P, Cirocchi R, Sharp A, Stigliano E, Feinstein DL, Dello Russo C (2012) Rapamycin reduces clinical signs and neuropathic pain in a chronic model of experimental autoimmune encephalomyelitis. J Neuroimmunol 243(1-2):43–51. doi:10.1016/j. jneuroim.2011.12.018 23. Strauss L, Whiteside TL, Knights A, Bergmann C, Knuth A, Zippelius A (2007) Selective survival of naturally occurring human CD4 + CD25 + Foxp3+ regulatory T cells cultured with rapamycin. J Immunol 178(1):320–329 24. Girdlestone J, Pido-Lopez J, Srivastava S, Chai J, Leaver N, Galleu A, Lombardi G, Navarrete CV (2015) Enhancement of the immunoregulatory potency of mesenchymal stromal cells by treatment with immunosuppressive drugs. Cytotherapy 17(9):1188–1199. doi:10.1016/j.jcyt.2015.05.009 25. Gharibi B, Farzadi S, Ghuman M, Hughes FJ (2014) Inhibition of Akt/mTOR attenuates age-related changes in mesenchymal stem cells. Stem Cells (Dayton, Ohio) 32(8):2256–2266. doi:10.1002/ stem.1709 26. Kim KW, Moon SJ, Park MJ, Kim BM, Kim EK, Lee SH, Lee EJ, Chung BH, Yang CW, Cho ML (2015) Optimization of adipose tissue-derived mesenchymal stem cells by rapamycin in a murine model of acute graft-versus-host disease. Stem Cell Res Ther 6:202. doi:10.1186/s13287-015-0197-8 27. Bianco P, Kuznetsov SA, Riminucci M, Gehron Robey P (2006) Postnatal skeletal stem cells. Methods Enzymol 419:117–148. doi: 10.1016/s0076-6879(06)19006-0 28. Jahromi SR, Arrefhosseini SR, Ghaemi A, Alizadeh A, Sabetghadam F, Togha M (2014) Effect of oral genistein administration in early and late phases of allergic encephalomyelitis. Iran J Basic Med Sci 17(7):509–515 29. Vakilzadeh G, Khodagholi F, Ghadiri T, Darvishi M, Ghaemi A, Noorbakhsh F, Gorji A, Sharifzadeh M (2014) Protective effect of a cAMP analogue on behavioral deficits and neuropathological changes in cuprizone model of demyelination. Molecular neurobiology. doi:10.1007/s12035-014-8857-8 30. Tahamtan A, Ghaemi A, Gorji A, Kalhor HR, Sajadian A, Tabarraei A, Moradi A, Atyabi F, Kelishadi M (2014) Antitumor effect of therapeutic HPV DNA vaccines with chitosan-based nanodelivery systems. J Biomed Sci 21:69. doi:10.1186/s12929-014-0069-z 31. Sajadian A, Tabarraei A, Soleimanjahi H, Fotouhi F, Gorji A, Ghaemi A (2014) Comparing the effect of toll-like receptor agonist adjuvants on the efficiency of a DNA vaccine. Arch Virol 159(8): 1951–1960. doi:10.1007/s00705-014-2024-4 32. Khoury S (2014) Immunology of MS. Multiple Scler Relat Disord 3(6):766. doi:10.1016/j.msard.2014.09.011 33. Paintlia AS, Paintlia MK, Singh I, Skoff RB, Singh AK (2009) Combination therapy of lovastatin and rolipram provides neuroprotection and promotes neurorepair in inflammatory demyelination model of multiple sclerosis. Glia 57(2):182–193. doi:10.1002/glia. 20745 34. Cruz-Guilloty F, Saeed AM, Duffort S, Cano M, Ebrahimi KB, Ballmick A, Tan Y, Wang H, Laird JM, Salomon RG, Handa JT, Perez VL (2014) T cells and macrophages responding to oxidative damage cooperate in pathogenesis of a mouse model of age-related macular degeneration. PLoS One 9(2):e88201. doi:10.1371/ journal.pone.0088201 35. Cejka D, Hayer S, Niederreiter B, Sieghart W, Fuereder T, Zwerina J, Schett G (2010) Mammalian target of rapamycin signaling is crucial for joint destruction in experimental arthritis and is activated in osteoclasts from patients with rheumatoid arthritis. Arthritis Rheum 62(8):2294–2302. doi:10.1002/art.27504 36. Battaglia M, Stabilini A, Draghici E, Migliavacca B, Gregori S, Bonifacio E, Roncarolo MG (2006) Induction of tolerance in type
Mol Neurobiol
37.
38.
39.
40.
41.
42.
43.
1 diabetes via both CD4 + CD25+ T regulatory cells and T regulatory type 1 cells. Diabetes 55(6):1571–1580. doi:10.2337/db051576 Iwata H, Nagano T, Toyo-oka K, Hirose H, Hamaoka T, Fujiwara H (1994) Suppression of allograft responses by combining alloantigen-specific i.v. pre-sensitization with suboptimal doses of rapamycin. Int Immunol 6(1):93–99 Sonobe Y, Jin S, Wang J, Kawanokuchi J, Takeuchi H, Mizuno T, Suzumura A (2007) Chronological changes of CD4(+) and CD8(+) T cell subsets in the experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Tohoku J Exp Med 213(4):329–339 Montero E, Nussbaum G, Kaye JF, Perez R, Lage A, Ben-Nun A, Cohen IR (2004) Regulation of experimental autoimmune encephalomyelitis by CD4+, CD25+ and CD8+ T cells: analysis using depleting antibodies. J Autoimmun 23(1):1–7. doi:10.1016/j.jaut. 2004.05.001 Hedegaard CJ, Krakauer M, Bendtzen K, Lund H, Sellebjerg F, Nielsen CH (2008) T helper cell type 1 (Th1), Th2 and Th17 responses to myelin basic protein and disease activity in multiple sclerosis. Immunology 125(2):161–169. doi:10.1111/j.1365-2567. 2008.02837.x Huseby ES, Liggitt D, Brabb T, Schnabel B, Ohlen C, Goverman J (2001) A pathogenic role for myelin-specific CD8(+) T cells in a model for multiple sclerosis. J Exp Med 194(5):669–676 Fletcher JM, Lalor SJ, Sweeney CM, Tubridy N, Mills KH (2010) T cells in multiple sclerosis and experimental autoimmune encephalomyelitis. Clin Exp Immunol 162(1):1–11. doi:10.1111/j.13652249.2010.04143.x Leuenberger T, Paterka M, Reuter E, Herz J, Niesner RA, Radbruch H, Bopp T, Zipp F, Siffrin V (2013) The role of CD8+ T cells and their local interaction with CD4+ T cells in myelin oligodendrocyte glycoprotein35-55-induced experimental autoimmune encephalomyelitis. J Immunol 191(10):4960–4968. doi:10.4049/jimmunol. 1300822
44.
45.
46.
47.
48.
49.
50.
Gerdoni E, Gallo B, Casazza S, Musio S, Bonanni I, Pedemonte E, Mantegazza R, Frassoni F, Mancardi G, Pedotti R, Uccelli A (2007) Mesenchymal stem cells effectively modulate pathogenic immune response in experimental autoimmune encephalomyelitis. Ann Neurol 61(3):219–227. doi:10.1002/ana.21076 Al Jumah MA, Abumaree MH (2012) The immunomodulatory and neuroprotective effects of mesenchymal stem cells (MSCs) in experimental autoimmune encephalomyelitis (EAE): a model of multiple sclerosis (MS). Int J Mol Sci 13(7):9298–9331. doi:10.3390/ ijms13079298 Akiyama K, Chen C, Wang D, Xu X, Qu C, Yamaza T, Cai T, Chen W, Sun L, Shi S (2012) Mesenchymal-stem-cell-induced immunoregulation involves FAS-ligand-/FAS-mediated T cell apoptosis. Cell Stem Cell 10(5):544–555. doi:10.1016/j.stem.2012.03.007 Cheng PP, Liu XC, Ma PF, Gao C, Li JL, Lin YY, Shao W, Han S, Zhao B, Wang LM, Fu JZ, Meng LX, Li Q, Lian QZ, Xia JJ, Qi ZQ (2015) iPSC-MSCs combined with low-dose rapamycin induced islet allograft tolerance through suppressing Th1 and enhancing regulatory T-cell differentiation. Stem Cells Dev 24(15):1793– 1804. doi:10.1089/scd.2014.0488 Ge W, Jiang J, Baroja ML, Arp J, Zassoko R, Liu W, Bartholomew A, Garcia B, Wang H (2009) Infusion of mesenchymal stem cells and rapamycin synergize to attenuate alloimmune responses and promote cardiac allograft tolerance. Am J Transplant 9(8):1760– 1772. doi:10.1111/j.1600-6143.2009.02721.x Wang H, Qi F, Dai X, Tian W, Liu T, Han H, Zhang B, Li H, Zhang Z, Du C (2014) Requirement of B7-H1 in mesenchymal stem cells for immune tolerance to cardiac allografts in combination therapy with rapamycin. Transpl Immunol 31(2):65–74. doi:10.1016/j.trim. 2014.06.005 Hou Y, Ryu CH, Park KY, Kim SM, Jeong CH, Jeun SS (2013) Effective combination of human bone marrow mesenchymal stem cells and minocycline in experimental autoimmune encephalomyelitis mice. Stem Cell Res Ther 4(4):77. doi:10.1186/scrt228
