If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Correspondence to: C.H. Ryu, Department of Clinical Pathology Laboratory Science, Daejeon Health Institute of Technology, 21 Chungjeong St., Dong-gu, Daejeon 34504, Korea.
Correspondence to: S-S. Jeun, Department of Neurosurgery, Seoul St. Mary's Hospital, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 137-701, Korea.
Department of Biomedicine & Health Science, College of Medicine, The Catholic University of Korea, Seoul, Republic of KoreaDepartment of Neurosurgery, Seoul St. Mary's Hospital, The Catholic University of Korea, Seoul, Republic of Korea
The combined treatment improves the clinical score of EAE mice.
•
The combined treatment regulates inflammatory cytokines in EAE mice.
•
The combined treatment modulates the levels of Th1 and Th2 cytokines in EAE mice.
•
The combined treatment reduced the blood-brain barrier disruption in EAE mice.
Abstract
Methylprednisolone (MP) has been recommended as a standard drug in MS therapies. We previously demonstrated that IFNβ-secreting human bone marrow-derived mesenchymal stem cells (MSCs-IFNβ) exert immunomodulatory effects in experimental autoimmune encephalomyelitic (EAE) mice. In this study, we evaluated whether a combined treatment of MP and MSCs-IFNβ had enhanced therapeutic effects on EAE mice. The combination treatment resulted in enhanced immunomodulatory effects, including reduced production of pro-inflammatory cytokines and increased production of anti-inflammatory cytokines. Thus, our results provide a framework for designing novel experimental protocols to enhance the therapeutic effects of existing MS treatments.
Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS) that is caused by specific adaptive immune responses to self-antigens. It is characterized by multi-focal demyelination, axonal damage, disruption of the blood-brain barrier (BBB), and lymphocyte infiltration by autoreactive B and T cells (
). In a commonly used animal model of CNS autoimmune disease, experimental allergic encephalomyelitis (EAE) is induced in mice by immunization with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55), resulting in an ascending spastic/flaccid paralysis caused by autoreactive immune cells in the CNS (
). A number of experimental treatments have been developed to improve the survival of patients with MS. Approved therapies include glatiramer acetate, interferon-beta (IFN-β), and mitoxantrone, all of which mainly target the immunological aspects of MS (
Copolymer 1 reduces relapse rate and improves disability in relapsing-remitting multiple sclerosis: results of a phase III multicenter, double-blind placebo-controlled trial. The Copolymer 1 Multiple Sclerosis Study Group.
Interferon beta-1b is effective in relapsing-remitting multiple sclerosis. I. Clinical results of a multicenter, randomized, double-blind, placebo-controlled trial. The IFNB Multiple Sclerosis Study Group.
). However, many patients do not respond optimally to these drugs. Therefore, there is an unmet need to develop more effective therapeutic protocols for MS.
Methylprednisolone (MP) is a synthetic glucocorticoid (GC) whose structure is similar to that of a natural hormone produced by the adrenal glands. It is commonly used for the treatment of respiratory, inflammatory, allergic, and autoimmune diseases such as MS, lupus erythematosus, and arthritis (
). However, long-term use of high-dose MP has been associated with many side effects such as hyperglycemia, decreased resistance to infection, facial swelling, weight gain, congestive cardiac insufficiency, fluid and sodium retention, edema, hypertension, glaucoma, and psychosis (
). Recently, low- or suboptimal-dose MP was found to attenuate clinical severity in EAE when combined with minocycline, atovastatin, ulinastatin, and erythoropoietin (
). Thus, synergistic combinations of low-dose MP and other drugs could result in improved clinical efficacy while avoiding the undesirable side effects and toxicity of high-dose MP.
Interferon-beta (IFN-β), an approved drug for treating MS, has potent anti-inflammatory and immunomodulatory activities (
). However, IFN-β therapy has been limited by the short half-life of IFN-β and its difficulty in crossing the BBB. Mesenchymal stem cells (MSCs) have attracted much attention in tissue engineering as vehicles for gene therapy and as support cells for engraftment (
). We previously used hBM-MSCs as IFN-β gene delivery vehicles and showed that these cells exhibited targeted migration to the CNS and enhanced degradation capabilities. We also showed that IFN-β-secreting hBM-MSCs (MSCs-IFNβ) reduced the clinical severity of EAE, reduced the extent of inflammatory cell infiltration, alleviated demyelination, and promoted a shift in the cytokine response from Th1 to Th2 in EAE treatment (
). Despite its impressive therapeutic effects in the EAE model, patients with MS who receive IFN-β therapy generally have poor clinical outcomes. Adding to these difficulties, the disease symptoms and pathologic aspects of MS are complex. Therefore, therapeutic strategies for MS could be designed to target multiple levels of MS pathology. One such approach is combination therapy with conventional immunomodulatory drugs. Here we report for the first time that a combined treatment consisting of MP and MSCs-IFNβ has enhanced therapeutic efficacy compared with each treatment individually. These results provide invaluable insight into the potential of combined treatments with standard MS drugs and may offer new therapeutic options for treating patients with MS.
2. Materials and methods
2.1 Cell culture and IFN-β expression
Human bone marrow-derived MSCs (hBM-MSCs) were purchased from the Catholic Institute of Cell Therapy (CIC, Seoul, Korea). All work with human-derived MSCs was approved by the Institutional Review Board of Seoul St. Mary's Hospital. MSCs were cultured at a concentration of 5 × 104 cells/cm2 in low glucose Dulbecco's Modified Eagle's Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 20% fetal bovine serum and used for experiments during passages 5 to 8. The recombinant adenoviral vector encoding the gene for EGFP (Ad-GFP) and mouse IFN-β (Ad-IFN-β) was constructed using the Ad-Easy vector system by following the manufacturer's instructions (Quantum Biotechnologies, Carlsbad, CA, USA). MSCs were transduced with adenoviral vectors driving the expression of EGFP (Ad-GFP) and IFN-β (Ad-IFN-β) as previously described. Briefly, hBM-MSCs were incubated with premixed virus-Fe3+ complexes (multiplicity of infection = 50) for 4 h at 37 °C, washed twice with PBS, and incubated in maintenance medium.
2.2 Assessment of IFNβ expression and viability in MSCs-GFP and MSCs-IFNβ
BM-MSCs seeded at 24-well plates (2 × 104 cells) or 96-well plates (5 × 103 cells). To identify the IFN-β expression, BM-MSCs were infected with Ad-GFP and Ad-IFN-β, as described previously. After 24 h, the medium was removed, and MSCs-GFP cells were treated with MP (10 mM). Expression of IFN-β was determined using the ELISA kit (R&D Systems, Madison, WI, USA) in culture supernatant. The viability of cells was determined by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay (Sigma-Aldrich). The optical density (OD) of each well at 570 nm was determined using Spectramax Plus 384 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).
2.3 EAE induction and treatments
The mice had free access to gamma lay sterilized diet (TD 2018S, Harlan Laboratories, Inct/America), autoclaved R/O water, and lived on a 12-h light/dark cycle at a specific pathogen-free animal facility. Breeding conditions of the animal room were 22 ± 5 °C in temperature and 50 ± 10% in humidity. All animal protocols were approved by the Institutional Animal Care and Use Committee of the Medical College of Catholic University. EAE was induced in female C57BL/6 mice (9 weeks old, weighing 18 to 20 g) (Orient Bio Inc., Seongnam, Korea) using the Hooke Labs EAE induction kit (Hooke Labs, Lawrence, MA, USA) according to the manufacturer's instructions. In brief, mice were subcutaneously injected with an emulsion of MOG35-55, complete Freund's adjuvant (CFA), and Mycobacterium tuberculosis H37Ra. At 24 h post immunization, the mice received an intraperitoneal pertussis toxin injection. Mice were scored daily according to the following criteria: 0, no clinical signs; 0.5, partial tail paralysis; 1, complete tail paralysis; 1.5, hind limb weakness; 2, strong hind limb weakness; 2.5, partial hind limb paralysis; 3, complete hind limb paralysis; 3.5, partial front limb paralysis; 4, complete hind limb paralysis and front limb paralysis; and 5, moribund or dead. Mice were randomly divided into four groups and injected intravenously (IV) or intraperitoneally on day 14 post immunization with the following treatments: PBS (saline 100 μl/each mouse, intravenous injection, n = 9); MSCs-GFP (1.0 × 106 cells/each mouse, intravenous injection, n = 9); MSCs-IFNβ (1.0 × 106 cells/each mouse, intravenous injection, n = 9); MP (10 mg/kg, intraperitoneal injection, n = 9); and a combination of MP and MSCs-IFNβ (n = 9); Combination (MP: 10 mg/kg, intraperitoneal injection; MSCs-IFNβ: 1.0 × 106 cells/each mouse, intravenous injection, n = 9). Methylprednisolone (MP) was purchased from Sigma-Aldrich (St. Louis, MO, USA), dissolved in PBS and filter-sterilized.
2.4 Histological evaluation of demyelination and inflammatory infiltration
Histological analysis was performed on 4% paraformaldehyde-fixed, OCT-embedded sections of lumbar spinal cords of EAE mice harvested at day 30 post immunization. Frozen sections were stained with luxol fast blue (LFB) and hematoxylin-eosin (H&E) to evaluate the extents of inflammatory infiltration and demyelination, respectively. For immunofluorescence staining, the sections were incubated at 4 °C overnight with the following antibodies: polyclonal rabbit anti-ionized calcium-binding adaptor molecule 1 (Iba1; Wako Pure Chemical Industries, Osaka, Japan) or polyclonal rabbit anti-mouse myelin basic protein (MBP; Millipore, Billerica, MA, USA). Fluorescence images were acquired using an LSM700 confocal microscope (Carl Zeiss, Oberkochen, Germany). All images were evaluated using MetaMorph software (Molecular Devices).
2.5 Determination of cytokines by ELISA
Splenocytes (1 × 106 cells) plated on 60 mm plates and cultured with MOG35–55 (10 μg/ml) (Sigma Aldrich) for 7 days in RPMI1640 medium (Gibco, Carlsbad, CA, USA). The culture supernatants were harvested by centrifugation at 1200g at room temperature for 5 min. The concentrations of IFN-γ, TNF-α, IL-12, IL-4, and IL-10 in the culture supernatants were quantified by the Quantikine immunoassay kit (R&D Systems), according to the manufacturer's instructions. The optical density of each well at 450 nm was determined using Spectramax Plus 384 Microplate Reader (Molecular Devices).
2.6 Lymphocyte characterization by flow cytometry
Mice were sacrificed at day 30 post immunization and splenocytes were obtained from spleens for flow cytometry (FACS) analysis. Fluorescence-activated cell sorting was performed to evaluate lymphocyte surface markers. For intracellular cytokine staining, splenocytes were re-stimulated for 48 h with MOG35-55 (10 μg/ml). The splenocytes (1 × 105 cells) were stained with phycoerythrin or fluorescein isothiocyanate-conjugated rat anti-mouse CD4, CD45, CD8, CD45R, CD25 and Foxp3 antibodies (all from BD Bioscience, Franklin Lakes, NJ, USA). Samples were analyzed by FACS with MoFlo XDP and Summit software (Beckman Coulter, Inc., Fullerton, CA, USA).
2.7 Determination of caspase3/7 activity
The caspase-3/7 activity in CD4+ T cells was determined using a caspase-Glo 3/7 Assay (Promega, Madison, WI, USA). Briefly, purified CD4+ T cells were isolated by FACS and washed twice with cold PBS following MOG re-stimulation. An equal volume of caspase-3/7 detection reagent was added to the CD4+ T cell extracts, and the mixtures were incubated at 37 °C for 4 h. The luminescence emitted by each sample was measured with a SpectraMax L luminometer (Molecular Devices, Sunnyvale, CA, USA).
2.8 Fluorescent detection of Evans blue dye
The uptake of Evans blue dye (EB, Sigma-Aldrich), a tracer marker of circulation into the brain and spinal cord tissues, was measured using a spectrophotometer. Briefly, 4% EB was infused by IV injection via the tail vein into each mouse in the control groups, as well as into individual mice from the experimental EAE groups identified to be at the peak of clinical disease. After 4 h, the mice were sacrificed by transcardiac perfusion with PBS, and the spinal cords were removed and homogenized in 0.5% Triton X-100. The supernatants were plated in a flat-bottom 96 well plate (100 μl/well), and the resultant fluorescence was quantified using a microplate fluorescence reader (Perkin Elmer, Wellesley, MA, USA) (excitation: 620 nm, emission: 680 nm). Values are presented as fluorescence intensities and represent the average values of at least three mice per group.
2.9 Statistical analysis
Quantification was performed by an examiner blinded to the treatment status of each animal. All data reported using SPSS 13.0 software (SPSS lnc, Chicago, IL, USA) and shown as means ± SEM (Standard error of mean). The statistical significance of clinical score was analyzed by the Kruskal-Waills test with post-hoc Bonferroni corrections. The others statistical comparisons between the groups were examined by using one-way analysis of variance (ANOVA) with post-hoc Bonferroni corrections. The probability values <0.05 were considered significant.
3. Results
3.1 Combined treatment ameliorated clinical severity and histological outcomes of EAE mice
We identified whether MP affected the viability and IFN-β expression of MSCs-IFNβ. MTT and ELISA analysis showed that MSCs-IFNβ were not affected by MP treatment (Supplementary Fig. S1 A and B). To evaluate the therapeutic efficacy of combination therapy with MP and MSCs-IFNβ on EAE mice, treatments were initiated on day 14 post immunization, a day at which all mice showed similar symptoms. PBS-treated mice developed disease symptoms by 7–14 days post immunization. Mice treated with MSCs-GFP, MSCs-IFNβ, or MP treatment alone exhibited only slightly altered disease symptoms; however, mice treated with the combination exhibited reduced disease severity. We also evaluated the clinical score achieved by each animal from day 0 to day 46 after immunization. Mean/max clinical score of EAE was significantly reduced by combination treatment, compared with MSCs-GFP, MSCs-IFN β, or MP treatment alone (Maximum clinical score, PBS: 4.1 ± 0.5; MSCs-GFP: 3.6 ± 0.3; MSCs-IFNβ: 2.9 ± 0.2; MP: 3.1 ± 0.2; Combination: 2.1 ± 0.1, P < 0.01) (Mean clinical score, PBS: 3.4 ± 0.1; MSCs-GFP: 2.9 ± 0.4; MSCs-IFNβ: 2.5 ± 0.2; MP: 2.7 ± 0.4; Combination: 1.5 ± 0.3, P < 0.05 or P < 0.01) (Fig. 1A). The area under the curve (AUC) of clinical score in each group was also significantly reduced by combination treatment, compared with MSCs-GFP, MSCs-IFN β, or MP treatment alone (Maximum clinical score, PBS: 139.4 ± 4.6; MSCs-GFP: 120 ± 5.1; MSCs-IFNβ: 112.2 ± 4.8; MP: 117.6 ± 3.2; Combination: 98.6 ± 4.8, P < 0.01) (Mean clinical score, PBS: 118.3 ± 10.0; MSCs-GFP: 100.9 ± 6.0; MSCs-IFNβ: 95.6 ± 10.9; MP: 101.9 ± 8.1; Combination: 74.3 ± 8.7, P < 0.05 or P < 0.01) (Supplementary Fig. S2). To evaluate the extent of inflammatory infiltration and remyelination in the spinal cord of EAE mice, we performed H&E, Iba-1, LFB, and MBP staining. Infiltrated mononuclear and activated microglial cells were particularly apparent in the white matter of the lumbar spinal cord. The extent of inflammatory cell infiltration was decreased by the combined treatment compared with MSCs-GFP, MSCs-IFNβ, or MP treatments alone (Fig. 1B). Similarly, combined treatment increased remyelination and MBP expression in the white matter of the lumbar spinal cord of EAE mice (Fig. 1C). These results suggest that the reduced clinical severity achieved with the combination therapy was due to less inflammatory infiltration and increased remyelination in the EAE mice.
3.2 The combined treatment modulated the levels of Th1 and Th2 cytokines in EAE mice
Inflammatory cytokines are known to modulate the immune response during EAE (
EAE was induced in female C57 BL/6 mice by immunization with the MOG 35-55. After 14 days of EAE induction, mice were treated with MSCs-GFP (1 × 106 cells, i.v.), MSCs-IFNβ (1 × 106 cells, i.v.), MP (10 mg/kg, i.p.), or combined conditions. (A) The mean daily clinical score of each group was assessed from day 1 until day 46 post immunization. The mean/max clinical score of EAE was quantified. Columns, mean; bars, SEM. *P < 0.05; **P < 0.01, Kruskal-Wallis test with post-hoc Bonferroni corrections. (B) H&E (upper panel) and Iba-1 (bottom panel) staining were performed to assess the extent of inflammatory cell infiltration in the lumbar spinal cords of EAE mice. (C) The representative sections were analyzed for LFB (upper panel) and MBP (bottom panel) staining to detect remyelination in the lumbar spinal cords of EAE mice. Scale bar, 50 μm. The results are representative of three independent experiments.
Splenocytes (5 × 105/well) isolated from the five groups of EAE mice 16 days after PBS, MSCs-GFP, MSCs-IFNβ, MP, or Combination treatment, and then the cells were stimulated in vitro with MOG (10 μg/ml) for 7 days. (A) pro-inflammatory (IFN-γ, TNF-α, IL-17, IL-12) and (B) anti-inflammatory (IL-4, IL-10) cytokines were detected by ELISA in the supernatants of splenocytes culture. Columns, mean; bars, SEM. *P < 0.05; **P < 0.01, One-way ANOVA with post-hoc Bonferroni corrections. The results are representative of three independent experiments.
). We investigated whether the effects of the combination treatment could be attributed to changes in the MOG-reactivated T cell populations in the spleens of EAE mice. The populations of MOG-reactivated CD4+CD45+ and CD8+ T cells in the MSCs-GFP, MSCs-IFNβ, and MP treatment groups were decreased compared with that of the PBS treatment group. Moreover, the combination treatment group exhibited significantly decreased CD4+CD45+ and CD8+ T cell populations compared with MSCs-GFP, MSCs-IFNβ, and MP treatment group (CD4+CD45+, PBS: 52.9 ± 5.62; MSCs-GFP: 49.5 ± 1.95; MSCs-IFNβ: 43.6 ± 1.74; MP: 48.7 ± 1.53; Combination: 35 ± 3.68, P < 0.05 or P < 0.01) (CD8+, PBS: 42.5 ± 2.12; MSCs-GFP: 39 ± 1.41; MSCs-IFNβ: 32.5 ± 2.12; MP: 31.5 ± 0.7; Combination: 24 ± 1.41, P < 0.05 or P < 0.01) (Fig. 3A ). CD4+CD25+ T regulatory cells were recently reported to play pivotal roles in controlling immune balance by regulating CD4+ and CD8+ T cells (
). We found that the populations of MOG-reactivated CD4+CD25+Foxp3+ cells (Tregs) in the MSCs-GFP, MSCs-IFNβ, and MP treatment groups were increased compared with that in the PBS treatment group. Moreover, the combination treatment group exhibited a significantly increased population of MOG-reactivated Treg cells compared with MSCs-GFP, MSCs-IFNβ, and MP treatment group (CD4+CD25+, PBS: 1.7 ± 0.74; MSCs-GFP: 2.6 ± 0.23; MSCs-IFNβ: 3.8 ± 0.07; MP: 3.2 ± 0.23; Combination: 7.2 ± 0.48, P < 0.05 or P < 0.01) (Fig. 3B). Thus, the combination treatment appears to inhibit MOG-reactivated T cells by increasing the splenic Treg population in EAE mice. Furthermore, the combination treatment group exhibited increased caspase3/7 activity in MOG-reactivated CD4+ T cells compared with MSCs-GFP, MSCs-IFNβ, and MP treatment groups (Caspase 3/7 activity, PBS: 114,158.5 ± 3947.7; MSCs-GFP: 147,500 ± 3535.5; MSCs-IFNβ: 164,904.5 ± 1679.3; MP: 131,862.5 ± 9700.7; Combination: 183,310.5 ± 6095.9) (Fig. 3C). These data suggest that the combination treatment of MP and MSCs-IFNβ could reduce the population of MOG-reactivated T cells by increasing the Treg population, as well as by initiating apoptotic signaling.
Fig. 3MOG-reactivated T cells population of EAE spleen were regulated by combination treatment.
Single cell suspensions from the pooled spleen were reactivated with MOG (10 μg/ml) for 48 h. The frequency of CD4+CD45+, CD8+, CD45R+ and CD4+CD25+Foxp3+ Treg cells was analyzed by FACS. (A) Representative FACS dots showed that the frequency of CD4+CD45+, CD8+ and CD45R+ T cells of each group. The percentage of CD4+CD45+ and CD8+ T cell in each group was quantified. (B) Representative FACS dots showed that the frequency of CD25+Foxp3+ T cells of each group in the CD4+ gate. The percentage of CD4+CD25+Foxp3+ T cell in each group was quantified. (C) CD4+ T cells were analyzed for apoptosis using the caspase-Glo 3/7 kit. Columns, mean; bars, SE. *P < 0.05; **P < 0.01, One-way ANOVA with post-hoc Bonferroni corrections. The results are representative of three independent experiments.
3.4 The combined treatment regulates BBB permeability in the spinal cords of EAE mice
Disruption of the BBB in CNS is known to be related to the pathogenesis of inflammation and demyelination after MS. To determine whether the combination treatment attenuates BBB disruption, we injected EB dye intravenously into EAE mice at day 7 post treatment. Compared to the PBS treatment group, EB-stained brain and spinal cord tissue in the other four treatment groups exhibited a smaller amount of EB extravasation. Moreover, the combination treatment group showed significantly decreased EB contents in the brain and spinal cord compared with MSCs-GFP, MSCs-IFNβ, and MP treatment group (Brain, PBS: 0.4 ± 0.05; MSCs-GFP: 0.3 ± 0.04; MSCs-IFNβ: 0.3 ± 0.03; MP: 0.35 ± 0.05; Combination: 1.3 ± 0.03, P < 0.01) (Spinal cord, PBS: 0.7 ± 0.04; MSCs-GFP: 0.5 ± 0.05; MSCs-IFNβ: 0.5 ± 0.04; MP: 0.4 ± 0.05; Combination: 0.3 ± 0.03, P < 0.05 or P < 0.01) (Fig. 4A ). Occludin is a major component of BBB tight junctions and is also a well-established marker of tight junctions (
). We analyzed occludin expression by immunofluorescence staining of spinal cord sections from EAE mice. Occludin expression levels were elevated in mice treated with MSCs-GFP, MSCs-IFNβ, and MP compared with mice treated with PBS alone. The fluorescence intensity in the spinal cords from MSCs-GFP, MSCs-IFNβ-, and MP-treated mice was slightly increased compared with those in the spinal cords from PBS-treated mice. However, the combined treatment resulted in significantly increased fluorescence intensity compared with MSCs-GFP, MSCs-IFNβ, and MP treatment alone (Occludin, PBS: 2800 ± 300; MSCs-GFP: 3350 ± 186; MSCs-IFNβ: 3720 ± 365; MP: 3150 ± 253; Combination: 9600 ± 751, P < 0.01) (Fig. 4B). These results suggest that combined treatment reduced BBB disruption in CNS of EAE mice via restoration of tight junctions.
Fig. 4The combined treatment attenuates BBB disruption in EAE mice.
The integrity of BBB in each group was detected by quantitative measurement for EB content at day 7 after treatment (n = 3/each). (A) The mice were injected intravenously with 4% Evans blue (EB) dye, and extravasation of EB dye into the brain (left) and spinal cord (right) was measured at 5 h later. The BBB disruption indicated the extravasation of EB. The intensity of EB in the brain and spinal cord tissue was measured at 620 nm using a fluorometer. Columns, mean; bars, SE. *P < 0.05; **P < 0.01, One-way ANOVA with post-hoc Bonferroni corrections. (B) immunofluorescence staining of occludin in the lumbar spinal cords (upper panel). Scale bar, 50 μm. The fluorescence intensity of occludin for each group was statistically analyzed (bottom panel). Columns, mean; bars, SE. **P < 0.01, One-way ANOVA with post-hoc Bonferroni corrections. The results are representative of three independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
In this study, we identified that combined treatment with low dose MP (10 mg/kg) and MSCs-IFNβ effectively attenuated clinical severity and suppressed histopathological events in EAE. IFN-β approved by the FDA for MS treatment: IFN-β1b 250 μg subcutaneous (s.c.) every other days, IFN-β1a 22 μg s.c. three times per week, and IFN-β1a 30 μg intramuscular (i.m.) once a week (
Human biologic response modification by interferon in the absence of measurable serum concentrations: a comparitive trial of subcutaneous and intravenous interferon-beta serine.
). However, administration of a recombinant protein is typically associated with peak and trough kinetics governed by the biologic half-life of the protein. IFN-β has a very short half-life in vivo (
), thus necessitating a regimen of frequent injections to compensate for rapid systemic clearance and degradation. However, long-term repeated injections of IFNβ are associated with several clinically relevant side effects, including depression, inflammation, and liver toxicity (
). Thus, effective drug treatment requires high and multiple doses, which is costly, inconvenient and limited by CNS side effects. Combination therapy can provide several advantages because of the enhanced efficacy compared with higher dose monotherapy, low risk of adverse reactions relative to higher dose monotherapy, lower costs and improved medication concordance (
). Moreover, MP did not affect on viability and secretion capacity of MSCs-IFNβ (Supplementary Fig. S1 B and C). In this regard, MP and MSCs-IFNβ meet these criteria thus rationale for testing these treatments in combination therapy, which may play a major role in improving efficacy and safety in MS patients.
The balance between the pro- and anti-inflammatory cytokines in organ-specific autoimmunity is pivotal in the determination of resistance or susceptibility. In EAE, disease susceptibility is thought to correlate with the expression of pro-inflammatory Th1 cytokines, while anti-inflammatory Th2 have been associated with remission and recovery (
). Here, combination treatment enhanced the immunomodulatory effects, which suppressed pro-inflammatory cytokines (IFN-γ and TNF-α) and conversely increased anti-inflammatory cytokines (IL-4 and IL-10) in EAE spleen. Consistent with these findings, previously prednison and IFN-β1a are reported to attenuate EAE development by promoting Th1/Th2-biased immunity in EAE model (
). Thus, combination treatment may affect the resistance via modulation of the Th1/Th2 cytokine balance, which plays an important role in the pathogenesis and prognosis of MS. The Tregs are play a crucial role in the control of immune responses by down-regulating the function of effector CD4+ or CD8+ T cells and abrogate autoimmune disease such as MS (
). Similarly we observed that combined treatment decrease the population of reactivated CD4 and CD8 T cells, and increase the population of activated CD4+CD25+Foxp3+ Treg cells. Tregs suppress T cell function by several mechanisms including binding to effector T cell subsets and preventing secretion of their cytokines. Recently, Pandiyan et al. show that Treg cells induced apoptosis of effector CD4+ T cells in vitro and in vivo in a mouse model of inflammatory disease (
). Furthermore, combination treatment increased apoptosis of MOG-reactivated CD4+ T cell in EAE. Therefore, combination treatment of MP and MSCs-IFNβ could increase the apoptosis of reactivated T cells by regulating Treg suppressive functions.
The possible therapeutic effect of the combined treatment might involve modulation of BBB dysfunction. An altered BBB permeability is thought to be a key initiating factor in the pathogenesis of MS (
). The integrity of the BBB is critically dependent on tight junctions between cerebral endothelial cells. Tight junctions are specialized cell-cell adhesion structures and critical components of the BBB that have previously been shown to be abnormally expressed in MS (
). In this study, we found that the combined treatment effectively reduced BBB disruption via restoration of tight junctions in the spinal cords from EAE mice. Thus, reduced BBB permeability may suggest decreased immune cell infiltration into CNS then consequently reduced CNS inflammation and demyelination, which was in coincidence with the results of our histological evaluation in combination-treated EAE mice.
5. Conclusion
This study demonstrated that the combination of MP and MSCs-IFNβ exhibits enhanced therapeutic effects by decreasing inflammatory cell infiltration, suppressing demyelination, immunomodulatory effects by promoting a shift from the Th1 to the Th2 cytokine balance, Treg cell-mediated suppression of activated T cells, stabilizing the BBB, and preventing the progression of disease in EAE mice. Thus, combination treatments may provide a promising therapeutic protocol for clinical therapies of MS.
The following are the supplementary data related to this article.
Supplementary Fig. S1The effect of MP on viability and secretory capacity of MSCs-IFNβ
(A) Viability of MSCs-GFP and MSCs-IFNβ was analyzed with the MTT assay 24 h after MP (0 to 160 μM) treatment. (B) For the quantification of secreted IFN-β in MSCs-IFNβ, supernatants were isolated on different days (n = 3/each group) with or without MP (10 mM) treatment and then assessed by ELISA. (C) To compare the longevity of IFN-β expression between recombinant IFN-β (rIFN-β) (1 × 103 units/ml) treated MSCs-GFP and MSCs-IFNβ, we collected the supernatants on different days and then performed the ELISA assay. MSCs-GFP was used as a control. Columns, mean; bars, SEM. The results are representative of three independent experiments.
Supplementary Fig. S2The area under the curve (AUC) of each clinical score curve.
Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, One-way ANOVA with post-hoc Bonferroni corrections.
Author contribution
MJK wrote the draft manuscript, performed most of the experiments, collected data, and analyzed statistical data analysis. SSJ and CHR contributed to the conception and design of the study, interpretation of data, and editing of the manuscript. JYL, SAP and WSK prepared the BM-MSCs and tested the cell in vitro, and editing of the manuscript. All authors read and approved the final version of the manuscript.
Competing interests
The authors declare that they have no conflict of interest.
Acknowledgments
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2014 R1A1A2054772, 2016 R1D1A1B03931146) and Ministry of Health & Welfare, Republic of Korea (HI16C0812).
References
Amedei A.
Prisco D.
D'elios M.M.
Multiple sclerosis: the role of cytokines in pathogenesis and in therapies.
Human biologic response modification by interferon in the absence of measurable serum concentrations: a comparitive trial of subcutaneous and intravenous interferon-beta serine.
Copolymer 1 reduces relapse rate and improves disability in relapsing-remitting multiple sclerosis: results of a phase III multicenter, double-blind placebo-controlled trial. The Copolymer 1 Multiple Sclerosis Study Group.
Interferon beta-1b is effective in relapsing-remitting multiple sclerosis. I. Clinical results of a multicenter, randomized, double-blind, placebo-controlled trial. The IFNB Multiple Sclerosis Study Group.
30134-0&id=fx1.jpg){kind=link}
30134-0&id=gr1.jpg){kind=link}
30134-0&id=gr2.jpg){kind=link}
30134-0&id=gr3.jpg){kind=link}
30134-0&id=gr4.jpg){kind=link}
30134-0&id=gr5.jpg){kind=link}
30134-0&id=gr6.jpg){kind=link}