Synergistic potential of bone marrow mesenchymal stem cells and miR181-a combinational therapy against multiple sclerosis

Abstract

Background

Multiple sclerosis (MS) is a progressive autoimmune disease characterized by massive inflammatory infiltration, demyelination, and subsequent axonal injury and neuronal damage in the central nervous system (CNS). The etiology of MS remains unclear and there is not yet a definitive therapeutic schedule for the disease. Bone marrow mesenchymal stem cells (BMSCs), exhibiting neuroimmune-modulatory functions to alleviate various autoimmune diseases, show great potential in the treatment of MS. However, the instability of BMSCs-mediated immunosuppression in vivo has limited their application. MiR181-a, a positive regulator of immune balance, which has a preference for T cells and B cells differentiation, but degrade rapidly upon entering systemic circulation due to their unstable molecular structure.

Methods

We propose a synergistic therapy approach that combines the penetrative targeting capability of BMSCs with the immuno-modulatory effects of miR181-a by overexpressing miR181-a to BMSCs through lentivirus packaging system. With this strategy, on the basis of the establishment of the experimental autoimmune encephalomyelitis (EAE) model, miR181-a overexpressing BMSCs (miR181a-BMSCs) would have a stronger immuno-modulatory treatment benefit, in terms of attenuating MS development.

Results

Indicate that this method prolongs the modulatory effects of BMSCs and resulted in significantly enhancements of the proliferation of regulatory B cells (Bregs), regulatory T cells (Tregs) and the inhibition of Th17 cells compared to the traditional BMSCs group. Moreover, 10-fold miRNA’s concentration in the exosome of miR181a-BMSCs, leading to an increased duration of miRNAs to exert their biological effects. By immunotherapy and synergistic treatment, the effectiveness of the treatment is significantly enhanced, showing consistent results in different groups of the animal model.

Conclusions

This strategy takes advantage of BMSCs and miRNA and thus presents an effective synergistic strategy for the treatment of autoimmune diseases.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-025-04401-7.

Introduction

Multiple sclerosis (MS), a progressive autoimmune disease, is characterized by inflammation, demyelination, and neurodegeneration of the central nervous system (CNS) [1, 2]. Notably, the incidence and prevalence of MS are currently still on the rise, particularly among individuals aged 20 to 40 years [3]. At present, the treatment of MS is mainly symptomatic, which is often accompanied by a series of side effects, such as nausea, vomiting, and immune dysfunction [4, 5]. Therefore, new therapeutic methods are continually being explored to treat MS. The experimental autoimmune encephalomyelitis (EAE) animal model is one of the most widely utilized animal models in MS research, as both the clinical and pathological characteristics were akin to those observed in MS [6]. B cells drive autoimmunity by serving as a source of pathogenic autoantibodies and cytokines [7]. Numerous studies have shown an inverse correlation between the quantity and functionality of the regulatory B cells (Bregs) and the severity of MS [8]. Currently, the specific role and mechanism of Bregs in MS remain inadequately understood. Bregs assist in activating of CD4⁺ T cells [7], directly inhibit the activity of pro-inflammatory T cells (Th1 and Th17 cells) through the secretion of the anti-inflammatory cytokine such as IL-10 [9], and facilitate the conversion of T cells into regulatory T cells (Tregs) [10]. In the investigation of MS treatment, growing evidence indicates that the dysregulation of B cells and T cells, particularly their regulatory roles in inflammation and immune response [11–13]. Consequently, current studies focus on exploring the induction, function, and regulation of B cells and T cells in MS [14], aiming to identify more effective therapeutic strategies that modulate their interaction and alleviate MS. Despite considerable efforts, current therapeutic strategies remain limited. Thus, modulating or restoring the immunomodulatory function of Bregs to influence the balance of inflammatory factors and the interaction between Th17 cells and Tregs may offer a promising therapeutic approach.

Bone marrow mesenchymal stem cells (BMSCs) are adult stem cells distinguished by their immunomodulatory properties and multi-lineage differentiation potential, which contribute to the regulation of immune responses [15]. BMSCs ameliorate neurological deficits and facilitate functional recovery in MS [16, 17]. Research indicates that BMSCs are capable of modulating B and T lymphocytes, enhance the proliferation of Tregs through the secretion of TGF-β, which also facilitates the differentiation of T cells into Tregs and ameliorates the progression of autoimmune diseases [18, 19]. Owing to their migratory abilities, homing capacity, immunomodulatory functions, and anti-inflammatory effects, BMSCs have emerged as a promising therapeutic approach for the treatment of autoimmune diseases [20, 21]. Despite the clinical data demonstrating the positive efficacy of BMSCs, several challenges remain regarding their therapeutic application, including limited therapeutic effects, difficulties in long-term consolidation [22, 23]. These challenges underscore the necessity for additional research and exploration. Researchers are investigating strategies to enhance the success rate of BMSCs transplantation, including co-treatment with neurotrophic factors, drugs, and electroacupuncture [24, 25]. The combined treatment involving BMSCs is still in the preliminary stage, it provides a novel therapeutic approach for autoimmune diseases.

MicroRNAs (miRNAs) possess the capacity to modulate various cellular processes, including development, proliferation, differentiation, and plasticity [26]. Several studies have demonstrated that miRNAs play a crucial role in controlling oligodendrocyte differentiation and myelin formation, and their dysregulation has significant implications in demyelinating diseases [27], particularly MS. Research indicates that in patients with MS, miR181-a is significantly decreased during the acute phase of the disease [28, 29] (Supplementary Fig. 1). Previous studies have highlighted the role of miR181 family members in immune cell development, particularly in B cell and T cell differentiation and activity [30, 31]. MiR181-a reduces T cell polarization to the Th1 phenotype and promotes Tregs differentiation [32]. Abnormal miR181-a expression may influence the differentiation and activity of B cells and T cells, thereby affecting the progression of EAE [32, 33]. However, due to their unstable structure, miRNAs degrade rapidly in systemic circulation. Thus, a stable miR181-a vector is necessary to effectively exert its biological effects [34, 35].

Currently, extracellular vesicles, including microvesicles and exosomes, are recognized as carriers of miRNA released by various living cells [36]. Due to the immunomodulatory functions of BMSCs, their capacity for multidirectional differentiation, and their ease of acquisition and transplantation, they are considered the optimal carriers for miRNA [37, 38]. In this study, we employed the lentiviral packaging system, known for its high gene introduction efficiency [39], to combine the immunosuppressive properties of miR181-a with the cell-targeting capability of BMSCs (miR181a-BMSCs) and investigate whether miR181a-BMSCs could exert a synergistic effect and maintain long-term, stable expression of miR181-a. We predict that the combined treatment of miR181-a and BMSCs will exhibit a more significant immunomodulatory therapeutic effect in mitigating the progression of EAE compared to conventional stem cell therapies. Through in vitro and in vivo experiments, we observed that co-culture of lymphocytes with miR181a-BMSCs enhanced the secretion of the anti-inflammatory cytokine IL-10, reduced the secretion of pro-inflammatory cytokines IFN-γ, IL-17, and TGF-β, and promoted the differentiation of peripheral blood B cells and CD4⁺ T cells toward an anti-inflammatory phenotype. Reinfusion of miR181a-BMSCs delayed the progression of EAE, promoted IL-10 secretion by B cells, and induced the differentiation of CD4⁺ T cells towards an anti-inflammatory phenotype. During this process, the reinfusion of miR181a-BMSCs indirectly modulated the function of CD4⁺ T cells by regulating the IL-10 secretion of Bregs. Given that the infiltration of peripheral immune cells into the white matter of the spinal cord correlates with the degree of paralysis, inflammatory infiltration and demyelination lesions were analyzed using hematoxylin-eosin (H&E) staining and immunofluorescence staining. The results showed that miR181a-BMSCs reduced the severity inflammation and demyelination, demonstrating a stronger immunomodulatory therapeutic effect in alleviating the progression of MS. The long-term objective of this study is to propose a more effective approach for a range of autoimmune diseases, including MS, by utilizing a synergistic strategy to achieve sustained and stable outcomes.

Materials and methods

Animals

C57BL/6 female mice (14–16 g, 6–8 weeks), obtained from Beijing Huafu Kang Co, were used in EAE models. All animals were housed under standard conditions, including a 12-hour light/dark cycle, humidity maintained between 50% and 54%, and a temperature of 21–25 °C, with ad libitum access to food and water. In this study, mice were randomly assigned to three groups: EAE group (received immune emulsion and PBS injection, PBS + EAE), BMSCs treatment group for EAE (BMSCs + EAE), and miR181a-BMSCs treatment group for EAE (miR181a-BMSCs + EAE). At the peak phase (16 days post-immunization), experimental animals showed obvious clinical symptoms, including listlessness, decreased appetite, weight loss, instability in standing, abnormal gait, motor coordination disorder, and severe onset mice reached bilateral hind limb paralysis, and the mice were sacrificed at this stage for subsequent experiments. All experimental procedures were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals issued by the China National Health Research Institute, and the experimental protocols received approval from the Ethics Committee of Harbin Medical University. The work has been reported in line with the ARRIVE guidelines 2.0. No specific inclusion or exclusion criteria were established, and no mice were excluded from the study. All animals included in the study completed the entire experimental process. Five animals per group were used for clinical score, weight measurement and onset incidence analysis. Ten animals per group were used to analyze the day of onset, cumulative scores, and peak clinical scores. Four animals per group were used for in vitro analysis, while five animals per group were used for in vivo experimental analysis. Three animals per group were subjected to H&E staining and fluorescent myelin staining analysis. All outcome assessments were performed by experimentalists blinded to the group assignments.

EAE induction

On day 0, mice were subcutaneously injected with 100 µg of recombinant mouse myeloid oligodendrocyte glycoprotein (rmMOG) emulsified in complete Freund’s adjuvant (CFA, Sigma-Aldrich, USA), which contains Mycobacterium tuberculosis (DIFCO). Mice were subsequently injected with pertussis toxin (PT, 200ng/mouse, List Biological Laboratories, USA) via the tail vein on days 0 and 2. Clinical scoring criteria for EAE ranged from 0 to 5 scale, with daily assessments according to the following scale: 0, no clinical signs of EAE; 0.5, tail weakness; 1, limp tail; 2, hind limb weakness or impaired gait; 2.5, unilateral hind limb paralysis; 3, complete hind limb paralysis; 4, forelimb and hind limb paralysis; 4.5, moribund; 5, death [5]. When clinical symptoms fell between two grades, an additional 0.5 points were added to the lower score.

BMSCs isolation and culture

Healthy, female wild-type C57BL/6 mice (11–12 g, 6–8 weeks) were anesthetized with isoflurane (RWD, China) and subsequently euthanized by cervical dislocation, in strict accordance with ethical guidelines, to ensure a humane and painless death. Bilateral femora and tibia of mice were aseptically excised, with surrounding muscle and connective tissue carefully dissected, exposing the bone marrow cavity. The cavity was then washed with sterile PBS using a 27-gauge needle until the bone appeared white. Bone marrow was collected, followed by lysis of erythrocytes using ACK Lysis Buffer, centrifugation, and resuspension. BMSCs were cultured in complete medium (10% fetal bovine serum, 1% penicillin/streptomycin in high-glucose DMEM) at 37 °C, 5% CO₂, and saturated humidity. After 24 to 48 h, the upper medium was removed, and non-adherent suspended cells were washed away with phosphate-buffered saline (PBS). The medium was then changed every 2 to 3 days. When the cell density reached 80%, the cells were passaged with 0.05% trypsin/EDTA. BMSCs from passages 3 to 5 were used in our experiments.

Lentiviral packaging system and establishment of miR181a-BMSCs

The lentivirus packaging cell line used in this study was the human epithelial cell line, 293T, obtained from GeneCopoeia (USA). Amplification was conducted in six-well plates using DMEM high-glucose medium (Thermo Fisher Scientific, USA), containing 1% penicillin/streptomycin and 10% fetal bovine serum. The entire vector system (pEZX-MR04, pRSV Rev, pMDLg/pRRE, pLP/VSVG, mixed at a ratio of 2:1:1:1; Invitrogen, USA) and Lipofectamine 3000 (Thermo Fisher Scientific, USA) were added to HEK293T cells (2 × 10⁶ cells/well) for co-transfection. After 72 h, the cell supernatant containing the virus was collected and centrifuged at 4000 rpm for 10 min at 4 °C. BMSCs were directly infected with 2 mL of viral supernatant (5 × 10⁶ TU/ml) at a density of 0.5 × 10⁵ cells per well and subsequently incubated at 37 °C in 5% CO₂. After 24 h, 1 mL of supernatant was removed, and 1 mL of viral supernatant was added to enhance the infection. After 72 h, the successfully transfected cells were examined under a fluorescence microscope. The transfected BMSCs were treated with 3.5 µg/mL Puromycin (puro, Yeasen, China) for cell selection. The selection medium was changed every 2 to 3 days until all green fluorescent protein-negative cells were removed and miR181a-BMSCs were obtained. Exosomes were extracted from the cells using ultracentrifugation.

RNA extraction and qPCR

Total RNA was extracted from BMSCs and miR181a-BMSCs using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions. RNA concentration and purity were determined using NanoDrop software. Subsequently, cDNA was synthesized from total RNA using the All-in-One miR First-Strand cDNA Synthesis Kit (GeneCopoeia, USA). Real-time PCR was performed utilizing Hieff qPCR SYBR Green Master Mix (High ROX Plus) (Yeasen, China) and All-in-One™ miRNA Universal Adaptor PCR Primer (GeneCopoeia, USA). The primer sequences included: miR181a-F, 5′-CCG ACA ACC ACT ACC TCA-3′ and 3′-CGT GAA GAA TGT GCG AGA − 5′; U6, 5′-CTC GCT TCG GCA GCA CA-3′ and 3′-AAC GCT TCA CGA ATT TGC GT-5′. The data were analyzed using a relative quantitative method, specifically the 2-^ΔΔCt method, to calculate the relative expression of target genes. Subsequently, differences between groups were statistically evaluated using a t-test with a P value < 0.05 considered statistically significant.

BMSCs and miR181a-BMSCs treatment

Cells were prepared into a single-cell suspension dissolved in 200 µl PBS, 5 × 10⁶ cells/ml. On day 8 and 10, mice from both the BMSCs and miR181a-BMSCs groups received tail vein transplantation of stem cells (1 × 10⁶ cells per injection). The untreated EAE (control) group received an equivalent volume of PBS. From day 0 to day 28, mouse weights were recorded daily. Disease progression curves were plotted based on clinical scores, following the EAE standardized scoring scheme.

Co-culture of peripheral lymphocyte with BMSCs or miR181a-BMSCs

Spleens and lymph nodes were collected from 6 to 8 week-old EAE wild-type female C57BL/6 mice using sterile techniques, and T and B lymphocytes were subsequently isolated. A co-culture system of lymphocytes, BMSCs, and miR181a-BMSCs was established. BMSCs or miR181a-BMSCs were cultured separately in six-well plates for 24 to 48 h in complete stem cell medium. After ensuring the cells were in good condition (95–100% confluence, spindle-shaped monolayer, uniform adherent growth with a swirl pattern, and no signs of contamination), the complete medium was discarded, and peripheral blood T and B lymphocytes were added. The ratio of lymphocytes to BMSCs was set at 10:1 in 10% RPMI-1640 medium (supplemented with 5 µL/mL of rmMOG, 10% heat-inactivated FCS, 1% penicillin/streptomycin, 1% L-glutamine, 5 mM 2-mercaptoethanol, and non-essential amino acids). The groups included: the lymphocyte-only (Control - LN group), the co-culture group with BMSCs (BMSCs + LN group), and the co-culture group with miR181a-BMSCs (miR181a-BMSCs + LN group). Then placed in an incubator at 37 °C with 5% CO₂ for 48 h.

ELISA

Quantitative analysis of IFN-γ, TNF-β, IL-6, IL-17, IL-10, and IL-4 levels was performed using ELISA with commercially available kits (Absin, China). Measurements were conducted on serum samples from three groups of mice, as well as on the supernatants derived from a 48-hour culture of BMSCs, miR181a-BMSCs, and lymphocytes stimulated with rmMOG (2 mg/mL).

Flow cytometry

For cell surface staining, mononuclear cell suspensions were prepared as described above and subsequently stained with the following antibodies: PerCP/Cyanine5.5 anti-mouse CD4 antibody (Biolegend, USA), PE anti-mouse CD25 antibody (eBioscience, USA), PE anti-mouse CD19 antibody (Biolegend, USA), PE/Cyanine7 anti-mouse CD1d antibody (Biolegend, USA), and PerCP/Cyanine5.5 anti-mouse CD5 antibody (Biolegend, USA), APC anti-mouse/rat CD29 antibody (Biolegend, USA), APC anti-mouse/human CD44 antibody (Biolegend, USA), APC anti-mouse Ly-6 A/E (Sca-1) antibody (Biolegend, USA), APC anti-mouse CD 31 antibody (Biolegend, USA), APC anti-mouse CD117(c-kit) antibody (Biolegend, USA).

For the staining of intracellular cytokines (secreted by CD4⁺ T cells), including IL-17 (APC anti-mouse IL-17 antibody, Biolegend, USA) and Foxp3 (APC anti-mouse Foxp3 antibody, eBioscience, USA), cells were stimulated with ionomycin (1 µg/mL, Enzo Life Sciences, USA), PMA (50 ng/mL, Abcam, UK), and Brefeldin A (3 µg/mL, Biolegend, USA) for 4 h. For the staining of intracellular cytokine (secreted by Bregs), IL-10 (APC anti-mouse IL-10 antibody, Biolegend, USA) was assessed following stimulation with ionomycin, Brefeldin A, PMA for 4 h.

Single-cell suspensions were prepared, as described above. After 4 h of stimulation, incubated with antibodies specific for cell surface markers diluted in PBS buffer for 30 min at 4 °C. Following washing twice with staining buffer, the cells were fixed and permeabilized with Fixation/Permeabilization Solution (BD Biosciences, USA) at 4 °C for 30 min. Intracellular marker antibodies were diluted in perm/wash buffer (BD Biosciences, USA) and incubated in the dark. Flow cytometry data were acquired using a flow cytometer (BD FACS Verse, BD Biosciences, USA) and analyzed with FlowJo software (Tree Star, USA).

Histopathological assessment

After euthanasia, the murine spinal cord was promptly collected and embedded in OCT for freezing. The frozen spinal cord tissue was sectioned into 8 μm thick slices. H&E staining and immunofluorescence staining were employed to assess the infiltration of inflammatory cells and demyelination. For H&E staining, tissue sections were fixed with cold acetone at 4 °C for 15 min and washed three times with PBS (5 min each). The tissue sections were stained with hematoxylin and eosin for 3 min, washed with running water for 7 min, and dehydrated through increasing concentrations of alcohol (80%, 95%, 100%; 10 s each), followed by treatment with a series of three xylene solutions (5 min each). Finally, the spinal cord tissue was sealed with neutral resin. For demyelination staining, the sections were washed with PBS for 20 min. Then the sections were incubated with FluoroMyelin Green stains (Molecular Probes, USA) for 20 min at room temperature (RT), followed by incubation with DAPI (Abcam, UK) for 3 min at RT. Finally, the sections were washed three times with PBS (10 min each) and subsequently sealed with Fluoromount-G to prevent fluorescence quenching.

Differentiation induction of miR181a-BMSCs

For the chondrogenic differentiation experiment involving miR181a-BMSCs, 4 × 10⁵ miR181a-BMSCs were collected. The complete culture medium for chondrogenic differentiation was added to a 15 mL centrifuge tube for induction and incubated with 5% CO₂ at 37 °C. When the cells clumped together, gently flick the tube to resuspend the chondrosphere. Replace the complete medium every 2–3 days. When the chondrosphere reaches a diameter of 1.5 to 2 mm after 21 days, prepare frozen sections and stain them with Alcian Blue 8GX solution.

For the osteogenic and adipogenic differentiation experiments, miR181a-BMSCs were coated with gelatin and seeded in a 6-well plate at a density of 4 × 10⁴ cells/cm². Then, 2 mL of 10% DMEM medium was added and cultured in an incubator with 5% CO₂ at 37 °C. When the cell confluence reached 70%, the medium was replaced with 2 mL of osteogenic differentiation medium, which was refreshed every 3 days. After 2–4 weeks of induction, the formation of calcium nodules was observed and stained with Alizarin Red S. When the cell confluence reached 100%, the medium was replaced with 2 mL of adipogenic differentiation medium (A) After 3 days of induction, the cells were switched to adipogenic differentiation medium (B) Medium A and B were used alternately. When the induction period resulted in lipid droplets of appropriate size, Oil Red O staining was performed. All differentiation reagents were obtained from Oricell, USA.

Statistical analysis

All statistical analyses were conducted using GraphPad Prism 10.1.2 (GraphPad, USA). Data are presented as mean ± standard deviation (SD). The Shapiro-Wilk test was employed to evaluate the data for normal distribution before the application of parametric tests. Comparisons between groups were performed using independent sample t-tests or one-way analysis of variance (ANOVA). P value less than 0.05 was considered indicative of statistical significance.

Results

Preparation and characterization of miR181a-BMSCs

BMSCs extracted from healthy adult C57BL/6 exhibited adherent growth in radial or spiral patterns (Fig. 1A). Adherent 293T cells (Fig. 1B) were used as lentivirus packaging cells. The basic characteristics of the miR181-a plasmid are shown in Fig. 1C. Using CMV as a promoter, eGFP (enhanced green fluorescent protein) was incorporated into the plasmid, which also contains ampicillin resistance. The GFP signal was visualized using fluorescence microscopy, with a transfection efficiency exceeding 95% (Fig. 1D). The viral supernatant was collected and subsequently transfected into BMSCs in an optimal growth state. After 72 h, green fluorescence was observed in successfully transfected cells (Fig. 1E). After puromycin (Yeasen, China) positive selection, miR181a-BMSCs with successful expression exhibited similar morphological differences compared to normal BMSCs, both displaying a spindle-shaped morphology (Fig. 1F). A stable cell line expressing miR181-a was successfully established (Fig. 1G). According to qPCR analysis, the expression of miR181-a in the miR181a-BMSCs group was significantly elevated by approximately 100-fold compared to untreated BMSCs (Fig. 1H). The level of miR181-a in exosomes from miR181a-BMSCs was significantly elevated (Fig. 1I). Exosomes secreted by BMSCs may exert enhanced therapeutic effects through sustained release [40, 41].

Fig. 1.

Preparation and characteristics of miR181a-BMSCs. (A, B) Morphology of BMSCs (A) and HEK293T cells (B). (C) Basic information of the plasmid miR181-a. (D) HEK293T cells, 72 h post-transfection. (E) Green fluorescence was detected in BMSCs 72 h post-viral transfection. (F) BMSCs after transfection for 72 h; growth status of miR181a-BMSCs. (G) MiR181a-BMSCs exhibited eGFP expression after puromycin selection. (H, I) Expression of miR181-a in BMSCs, miR181a-BMSCs (H) and exosomes (I) was detected by qPCR. Mean ± SD, n = 4/group, *P < 0.05, **P < 0.01, ***P < 0.001, t-test

Flow cytometry was used to assess the surface markers of both BMSCs and miR181a-BMSCs. The results indicated that CD29, CD44, CD31, CD117, and Ly6A/E in miR181a-BMSCs were consistent with the expression of BMSCs (Fig. 2A). Over 95% of the miR181a-BMSCs surface antigens were positive for CD29, CD44, and Ly6A/E, and negative for CD31 and CD117 (Fig. 2B). Chondrogenic (Fig. 2C), osteogenic (Fig. 2D), and adipogenic (Fig. 2E) differentiation experiments revealed significant morphological changes, consistent with the fundamental characteristics of BMSCs and their multidirectional differentiation potential.

Fig. 2.

Surface marker expression and differentiation potential of miR181a-BMSCs. (A, B) The surface markers of stem cells were characterized through flow cytometry. (C) Identification of chondrogenic differentiation. (D) Identification of osteogenic differentiation. (E) Identification of adipogenic differentiation. These experiments were repeated three times independently

Enhanced immunomodulatory function of miR181a-BMSCs co-culture with lymphocytes

CD4⁺ T cells and B cells are known to be the main pathogenic cells in MS. To assess the regulatory effect of miR181a-BMSCs treatment on peripheral B and T cells, lymphocytes were separated at the peak phase of disease (16 days post-immunization), were co-cultured with BMSCs or miR181a-BMSCs for 48 h, and B and T cell subsets were analyzed by flow cytometry. Compared with the Control-LN and BMSCs + LN groups, the proportion of anti-inflammatory Bregs (CD5⁺CD1d⁺CD19⁺IL10⁺) (Fig. 3A) and Tregs (CD4⁺CD25⁺Foxp3⁺) (Fig. 3B) was significantly increased following co-culture with miR181a-BMSCs. Simultaneously, the proportion of pro-inflammatory Th17 cells (CD4⁺IL17⁺) (Fig. 3C) was significantly reduced. Based on these results, miR181a-BMSCs exhibited more pronounced regulatory effects on B and T cells compared to BMSCs, primarily through the regulation of IL-10 secretion in Bregs and the reduction of Th17 differentiation. Additionally, an increase in Tregs differentiation was observed.

Fig. 3.

Enhanced synergistic anti-inflammatory effects of miR-181a and BMSCs. (A, B, C) Percentage of Bregs (CD5⁺CD1d⁺CD19⁺IL10⁺) (A) Tregs (CD4⁺CD25⁺Foxp3⁺) (B) and Th17 cells (CD4⁺IL17⁺) (C) in lymphocytes after co-cultures with miR181a-BMSCs. (D-I) ELISA detection of cytokines in supernatants from lymphocytes co-cultured with miR181a-BMSCs. Mean ± SD, n = 4/group, *P < 0.05, **P < 0.01, ***P < 0.001, One-way ANOVA. These experiments were repeated three times independently

To investigate the influence of pro-inflammatory and anti-inflammatory factors on the immune response, ELISA was conducted to characterize the cytokine profiles of the co-culture supernatants. The results indicated that, compared to the Control-LN group, BMSCs promoted the secretion of IFN-γ (Fig. 3D), IL-6 (Fig. 3E), TNF-β (Fig. 3F), IL-4 (Fig. 3G), and IL-10 (Fig. 3H). In contrast, IFN-γ significantly decreased following miR181a-BMSCs treatment, while IL-4 showed no significant difference. Compared to the BMSCs + LN group, miR181a-BMSCs further promoted the secretion of TNF-β and IL-10, while increased the ability to inhibit the secretion of IFN-γ, IL-6, TNF-β, and IL-4. Additionally, compared to the Control-LN group, miR181a-BMSCs promoted a greater secretion of the anti-inflammatory cytokine IL-10 and exhibited a stronger inhibitory effect on the pro-inflammatory factors IFN-γ and IL-17 (Fig. 3I). Based on alterations in inflammatory cytokines, we hypothesize that miR181a-BMSCs regulate the immune response by modulating B and T cells subsets responsible for secreting inflammatory factors.

MiR181a-BMSCs ameliorated EAE clinical course

We established B cell-dominated (rmMOG-induced) EAE models (Fig. 4A). To evaluate whether miR181a-BMSCs have a superior effect compared to BMSCs, tail vein reinfusions of BMSCs or miR181a-BMSCs were performed on the 8th and 10th days, and clinical scores and body weights were recorded from the 0th to 28th days (Fig. 4B&C). Compared to the PBS + EAE and the BMSCs + EAE groups, mice in miR181a-BMSCs + EAE group showed a lower incidence of disease (Fig. 4D), and a significantly delayed onset (Fig. 4E). The clinical scores at the peak phase and the cumulative scores were significantly lower in the miR181a-BMSCs + EAE group compared to the PBS + EAE and the BMSCs + EAE groups (Fig. 4F&G). Overall, miR181a-BMSCs significantly delayed the onset time, reduced the severity of the disease, and provided more effective treatment than BMSCs in the EAE model.

Fig. 4.

MiR181a-BMSCs ameliorated EAE clinical course. (A) EAE was induced in C57BL/6 mice, which were subsequently administered injections via the tail vein on the 8th and 10th days following immunization. Clinical symptoms were monitored and recorded throughout the study. (B) Clinical score evolution, n = 5/group. (C) Weight measurement, n = 5/group. (D) Onset incidence, n = 5/group. (E) Day of onset, n = 10/group. (F) Clinical scores at peak phase, n = 10/group. (G) Cumulative score (sum of clinical scores from 0 to 28 days after immunization for each mouse), n = 10/group. Data are represented as Mean ± SD and analyzed B-C, E-G with One-way ANOVA, D Log-rank (Mantel-Cox) test, *p < 0.05, **p < 0.01, ***p < 0.001

From the in vitro results, we observed that the infusion of miR181a-BMSCs could regulate the secretion of cytokines, primarily secreted by CD4⁺ T cells and B cells. Therefore, to evaluate the impact of miR181a-BMSCs reinfusion on the differentiation of peripheral blood B cells and CD4⁺ T cells, the proportions of CD4⁺ T cell and B cell subsets in peripheral blood during the peak phase of the disease were assessed by flow cytometry. The percentages of Bregs (Fig. 5A) and Tregs (Fig. 5B) were significantly higher in the miR181a-BMSCs + EAE groups compared to the BMSCs + EAE group. In the CD4⁺ T cell subset, the percentage of Th17 cells (Fig. 5C) secreting pro-inflammatory cytokines was significantly lower in the miR181a-BMSCs + EAE group compared to the BMSCs + EAE group (Fig. 5B). Infusion of miR181a-BMSCs promoted the proliferation of anti-inflammatory Bregs and Tregs and suppressed the differentiation of pro-inflammatory Th17 cells.

Fig. 5.

Flow Cytometry and ELISA analysis of Bregs, Tregs, Th17 cells and cytokines by Group. (A)Percentage of Bregs in lymphocytes at the peak of EAE in each group, n = 5. (B) Percentage of Tregs, n = 5. (C) Percentage of Th17 cells, n = 5. (D-I) The expression of cytokines in the peripheral blood of mice at the peak of EAE, n = 4. Mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, One-way ANOVA. These experiments were repeated three times independently

To assess inflammatory factors in vivo after treatment with miR181a-BMSCs, ELISA was used to quantify cytokines in the serum of mice at the peak phase. Compared with the PBS + EAE group, IFN-γ (Fig. 5D), IL-6 (Fig. 5E), and IL-17 (Fig. 5F) decreased in both the BMSCs + EAE and miR181a-BMSCs + EAE groups, while TGF-β (Fig. 5G) and IL-10 (Fig. 5H) increased. There was no significant difference in IL-4 (Fig. 5I) among the groups. Except for IL-6, the alterations in other inflammatory factors were more pronounced in the miR181a-BMSCs group than in the BMSCs group.

Impact of miR181a-BMSCs treatment on neuroinflammation and spinal cord demyelination

The spinal cord is considered the primary site of disease initiation and progression in the EAE model. Spinal cord injuries typically present with prominent demyelinating lesions, which are localized and can be readily detected via histological analysis [42, 43]. Consequently, to analyze the pathological changes more effectively, we focused on the spinal cord. At the peak phase, spinal cord samples from each group were analyzed using H&E staining (Fig. 6A&C) and fluorescent myelin staining (Fig. 6B&D) to assess inflammatory infiltration and demyelination. Lesion areas are marked with boxes, black arrows indicate sites of inflammatory infiltration, and white arrows denote demyelination. Both H&E and green fluorescent myelin staining revealed pronounced inflammatory infiltration and demyelination in the EAE group at the peak phase. In comparison to the EAE group, both BMSCs and miR181a-BMSCs groups exhibited significantly reduced inflammatory cell infiltration and improved myelin integrity. Notably, the miR181a-BMSCs group showed a more substantial reduction in inflammatory cell infiltration and demyelination compared to the BMSCs group, indicating a more pronounced therapeutic effect of miR181a-BMSCs in EAE group.

Fig. 6.

MiR181a-BMSCs treatment alleviates neuroinflammation and spinal cord demyelination in EAE. (A) H&E staining revealed neuroinflammatory infiltration. (B) Fluorescent myelin staining revealed spinal cord demyelination. (C)Percentage of inflammatory infiltration area. (D)Percentage of demyelination area. Mean ± SD, n = 3/group, *P < 0.05, **P < 0.01, ***P < 0.001, One-way ANOVA. These experiments were repeated three times independently

Discussion

Bone marrow mesenchymal stem cells hold significant promise in the fields of immune regulation and tissue regeneration. Recent evidence strongly suggests that exosomes are vital for stem cell therapy through a paracrine mechanism [44]. Due to their differentiation and regenerative capabilities, as well as their lower immunogenicity and higher survival rate post-transplantation compared to stem cells, exosomes have garnered substantial attention from both clinicians and researchers [45]. Studies have demonstrated that exosomes derived from BMSCs exhibit immunomodulatory effects on autoimmune diseases, as well as protective and reparative effects on damaged brain and CNS tissues through various mechanisms, thereby influencing disease progression [46].

In our study, miR181a-BMSCs exhibited notable therapeutic effects, evidenced by a reduced degree of demyelination in the CNS, decreased inflammatory cell infiltration, and lower secretion of inflammatory cytokines in peripheral blood. We observed increased expression of miR181-a in exosomes secreted by miR181a-BMSCs, with the anti-inflammatory effects potentially mediated through BMSCs therapy or BMSCs exosome therapy. Currently, the extraction of miR181a-BMSCs exosomes poses several challenges, such as complications in the extraction process, low concentration levels, and a suboptimal utilization rate. Given that BMSCs can serve as carriers to substantially secrete exosomes, which could stabilize and prolong the therapeutic effect of miR181-a, thereby enhancing the potential synergistic therapeutic impact of BMSCs and miR181-a. As a result, in this study, miR181a-BMSCs have been selected for infusion therapy.

In MS, pathogenic T cell responses are recognized as key drivers of autoimmune inflammation [9]. However, accumulating evidence indicates that B cells can modulate T cell functions through various mechanisms, including antigen presentation, cytokine secretion, and immune regulation [7, 8]. Therefore, the alleviation of EAE pathogenesis may involve both B cells and T cells, we investigated the therapeutic effect of miR181a-BMSCs on EAE with a focus on B cell and T cell interactions.

In vitro co-culture experiments demonstrated that the IL-4, IFN-γ, and IL-17 in the supernatant of the miR181a-BMSCs + LN group were significantly lower compared to the BMSCs + LN group, while the anti-inflammatory cytokine IL-10 was significantly increased. Additionally, the proportion of anti-inflammatory Bregs and Tregs increased, and the proportion of pro-inflammatory Th17 cells decreased, indicating that overexpression of miR181-a enhances the immunosuppressive function of BMSCs by regulating anti-inflammatory cytokines. In the in vivo experiments, the injection of miR181a-BMSCs demonstrated a significant therapeutic effect. Compared to the PBS + EAE group, the INF-γ, IL-6, and IL-17 in peripheral blood decreased at the peak phase, while the anti-inflammatory cytokine IL-10 increased significantly. The expression of INF-γ, IL-17, and IL-10 in the miR181a-BMSCs + EAE group exhibited more significant changes compared to the BMSCs + EAE group. In the context of changes in B cells and CD4⁺ T cell subsets, administration of miR181a-BMSCs significantly promoted the differentiation of anti-inflammatory Bregs and Tregs, while inhibiting the differentiation of pro-inflammatory Th17 cells.

It is noteworthy that an opposing trend in IL-6 was observed in vitro and in vivo, with IL-6 increased in vitro and decreased in vivo. IL-6 and TGF-β play a crucial role in Th17 differentiation, while IL-6 suppresses TGF-β induced Treg differentiation. This leads to dysregulation of the Th17/Treg balance, and excessive IL-6 secretion which contributes to the development of various pathological conditions [47]. IL-6 is not only produced by autoimmune-mediated cells. It is reported that mesenchymal cells, fibroblasts, and other cell types have also been found to produce IL-6 under both physiological and pathological conditions [48]. The potential influence of T cell adhesion cytokines and T cell soluble cytokines on IL-6 should also be considered [49]. Therefore, IL-6 is affected by a variety of cytokines in vivo, the relationship between the changes in IL-6 and BMSCs requires further exploration for clarification.

The consistent results from in vitro and in vivo studies demonstrated that miR181a-BMSCs significantly enhanced the immunomodulatory capacity of BMSCs, delayed disease onset, and more effectively alleviated symptoms. IL-10 is a cytokine that downregulates the production of pro-inflammatory cytokines, including IL-2, IL-3, IFN-γ, and tumor necrosis TNF-α, while also inhibiting the immune response of Th1 cells [8]. It has been established that Bregs are a key component of the immunosuppressive cell family and play a crucial role in immune homeostasis and various immune conditions. Similar to Tregs, Bregs have the ability of mitigating autoimmune diseases, suppressing hyperactive effector cells, and preventing allograft rejection [50]. Bregs play an essential role in mitigating inflammation in autoimmune and infectious diseases [8]. IL-10⁺ Bregs exert their effects by stimulating Tregs and promoting the conversion of CD4⁺ effector T cells into Tregs [51]. Our results showed that overexpression of miR181-a enhanced the ability of BMSCs to promote the differentiation of IL-10⁺ Bregs and Tregs, while inhibiting the secretion of pro-inflammatory cytokines such as IL-17. Briefly, by increasing the secretion of the anti-inflammatory cytokine IL-10 in B cells, the activation of Bregs were enhanced, thereby regulating the inflammatory response and T cell activation, which ameliorated the pathogenesis of EAE (Fig. 7).

Fig. 7.

MiR181a-BMSCs ameliorate EAE by regulating IL-10⁺Bregs and modulating CD4⁺ T cells

In addition to the aforementioned mechanisms, miR181-a regulates oxidative stress in various cell types, particularly in inflammation and immune functions [52]. In BMSCs, the upregulation of miR181-a expression alleviates oxidative stress-induced damage while promoting repair and regeneration. BMSCs reduce reactive oxygen species (ROS) and inflammation through antioxidative mechanisms, thus contributing to neuroprotection, tissue repair, and mitigating demyelination [53, 54]. Therefore, miR181-a modification of BMSCs enhances their immunomodulatory function and may promote neuroprotection by regulating oxidative stress, thus opening new research avenues for neurodegenerative and autoimmune diseases. In this study, BMSCs were administered via tail vein injection, typically resulting in their initial capture in the pulmonary capillaries, where they are temporarily retained during pulmonary circulation [55]. The lung, as a key immune organ, may enhance the immunomodulatory and anti-inflammatory effects of BMSCs within the pulmonary capillaries, thereby supporting the systemic immune system [56]. Furthermore, after filtration through the lung, BMSCs migrate to other target organs via the bloodstream, thereby contributing to systemic repair and exerting broad therapeutic potential [57, 58]. While miR181a-BMSCs show promising therapeutic potential in the EAE model, the safety assessment of their clinical application remains crucial. For instance, it is important to evaluate whether miR181a-BMSCs, when introduced into the systemic circulation, may influence immune tolerance, trigger an excessive immune response, or promote tumorigenesis in certain cases [59, 60]. Therefore, further studies that comprehensively assess immune responses, tumor risk, genetic stability, and cellular distribution are necessary to ensure the safety and efficacy of MSC-based therapeutic strategies in clinical settings, establishing a solid foundation for future clinical trials.

Conclusion

This study demonstrated a pronounced therapeutic effect of miR181a-BMSCs in the treatment of EAE. MiR181a-BMSCs reduced peripheral lesions in the CNS, decreased the secretion of pro-inflammatory cytokines, and improved clinical symptoms. Additionally, miR181a-BMSCs promoted the differentiation of Bregs and Tregs, while decreasing IL-17 secretion. BMSCs exhibit significant capabilities in differentiation, immunomodulation, and nerve regeneration. Their self-secreted exosomes possess immunomodulatory properties that prolong immune response regulation, and offering long-term therapeutic benefits in vivo. MiR181-a plays a crucial role in activating Bregs and T cells, while simultaneously reducing differentiation into the pathogenic phenotype. MiR181-a also promotes the production of Tregs and exerts anti-inflammatory effects in the treatment of neuroinflammatory diseases. In addition, the present data suggest that treatment with miR181a-BMSCs may offer a long-term synergistic therapeutic effect on the progression of EAE. The incorporation of miR181-a into the transplantation of BMSCs exhibits potent immunosuppressive and protective effects, with the synergistic interaction between BMSCs and miR181-a enhancing therapeutic outcomes. The detailed therapeutic mechanism will be explored in subsequent experiments, and further exploration as a potential combination therapy is warranted.

Electronic supplementary material

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Abbreviations

MS

Multiple sclerosis

CNS

Central nervous system

BMSCs

Bone marrow mesenchymal stem cells

EAE

Experimental autoimmune encephalomyelitis

Bregs

Regulator B cells

Tregs

Regulatory T cells

MiRNAs

MicroRNAs

H&E

Hematoxylin-eosin

rmMOG

Recombinant mouse myeloid oligodendrocyte glycoprotein

CFA

Complete Freund’s adjuvant

PT

Pertussis toxin

PBS

Phosphate-buffered saline

Puro

Puromycin

RT

Room temperature

eGFP

Enhanced green fluorescent protein

ROS

Reactive oxygen species

Author contributions

XX and XJ Liu conceived and designed the experiment. XX Yang, YW¹, XC Peng and YW² conducted a literature search. XX and SJ Chen conducted the experiments. SF Zhang, YP Wang and JF Wu analyzed the data. YM Liu, BS and HL Li helped with the analysis though constructive discussions. YZ, LL Mu and QF Kong contributed reagents, materials, and analysis tools. XX drafted the manuscript. SJ Chen revised the manuscript. The final draft was read and approved by all authors.

Funding

This work was supported by the Natural Science Foundation of Heilongjiang Province of China (LH2022H004), the National Natural Science Foundation of China (82371821, 82271845), and the Special Project of Traditional Chinese Medicine in Heilongjiang Province of China (ZYW2023-132).

Data availability

All data and materials are available in the manuscript.

Declarations

Ethics approval and consent to participate

This study does not involve clinical experiments. All animal handling and experimental procedures were performed in accordance with the guidelines of the Care and Use of Laboratory Animals published by the China National Institute of Health. Animal ethics review was approved by the Medical Ethics Committee of Harbin Medical University. Approved Project Title: The role and mechanism of BMSCs combined with miRNA in the treatment of multiple sclerosis. Approval number: HMUIRB20180010. Date of approval: 29 May 2018. 293T used in this study were purchased from GeneCopoeia, the original source (GeneCopoeia) has confirmed that there was initial ethical approval for collection of human cells, and that the donors had signed informed consent.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.