Previous studies have demonstrated the effectiveness of MSC-EV in treating various autoimmune diseases using animal models. Reduction in clinical scores, demyelination, and neuroinflammation has been observed with MSC-EV therapy in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis, with an associated upregulation of Treg cells [22]. Additionally, MSC-EV treatment has been shown to decrease uveitis in experimental autoimmune uveitis rats [23]. Furthermore, MSC-EV therapy delayed the inflammatory response in rheumatoid arthritis by reducing cartilage damage and decreasing pro-inflammatory cytokines [12].

In the present study, we examined the therapeutic effects of CM-EV in SLE. CM-EV treatment significantly increased survival rates, decreased anti-dsDNA antibody levels, ameliorated renal histopathology and IgG and C3 deposition in the kidneys, and significantly decreased BUN levels at 24 weeks of age. Although ASC-EV significantly reduced the incidence of severe proteinuria, it did not improve survival rate.

Compared with those in ASC-EV, TGF-β1 production and expression of miR-155-5p and miR-142-3p in CM-EV significantly increased. NTA showed that CM-EV output from iMSC culture supernatant primed with conditioned media obtained from disease-conditioned immune cells was significantly higher than ASC-EV output from unprimed media.

In autoimmune diseases, miR-10-5p, miR-142-3p, miR-146a-5p, miR-155-5p, and miR-216a-5p contribute to the immunoregulatory effects of MSCs and EV derived from MSCs [24,25,26]. In this study, the expression of miR-155-5p and miR-142-3p was significantly higher in CM-EV than in ASC-EV. EV-derived miR-155-5p reduces B cell proliferation and PI3K/AKT-induced activation [24]. miR-142-3p expression in CD4 + T lymphocytes from patients with SLE is significantly lower than that of those from the healthy control group, resulting in T cell activation and B cell hyperstimulation [25].

An imbalance in the M1:M2 ratio and abnormal activation of macrophages contribute to the pathogenesis of SLE. MSCs can induce macrophage polarization toward the M2 phenotype through the TGF-β/Akt/FoxO1 pathway [27]. miRNA-155-5p in MSC-derived EV contributes to this mechanism [24]. Based on flow cytometry analysis of splenocytes, CM-EV treatment significantly increased the expression of anti-inflammatory M2 macrophages compared with that in the control group. Moreover, TGF-β is a major immunomodulating cytokines produced by MSCs. The activation of the TGF-β/NF-kB signaling pathway decreases the proportion of Th17 and Th1 and increases the proportion of Treg and Th2 [28]. In this study, the proportions of Treg, Th17, Th1, and Th2 cells were not significantly affected by the treatments.

After disease onset, the expression of lupus-specific miRNAs, including miR-31, miR-96, miR-127, miR-155, miR-182, miR-183, and miR-379, was significantly upregulated in the splenocytes of NZB/W F1 mice [29]. Consistent with previous finding, lupus-specific miRNAs showed the highest expression in the splenocytes of NZB/W F1 mice in the C group but the lowest expression in the CM group. Particularly, the expression levels of miR-31-5p, miR-182-5p, and miR-183-5p were significantly different between the groups, with miR-182-5p and miR-183-5p being significantly downregulated in the CM group compared with that in the C group.

miR-31 targets forkhead-box p3 (Foxp3); therefore, miR-31 upregulation is associated with Treg dysfunction in lupus [30]. In SLE, miR-182–96-183 upregulation induces a decrease in Foxo1/3a and/or microphthalmia-associated transcription factor (MITF), which plays a role in regulating T cell homeostasis and tolerance, resulting in T and B cell activation, autoantibody production, immune tolerance breakdown, and autoimmunity development [31]. Therefore, decreased expression of the SLE disease-specific miRNAs (miR-31, miR-182, and miR-183) in the spleen may contribute to the alleviation of SLE.

Patients with active SLE have significantly higher serum levels of IL-1β and TNF-α than those with inactive SLE disease and healthy individuals [32]. In the present study, serum levels of IL-1β were significantly lower in the C group than in the E and CM groups, while serum levels of TNF-α were significantly lower in the E group than in the C group.

Furthermore, cell apoptosis was significantly higher in the renal tubular epithelium of mice in the C group than in that of those in the E and CM groups. Renal tubular epithelial cells affect the pathological process of acute and chronic kidney disease and play a major role in the incidence of lupus nephritis [33]. Therefore, suppressing apoptosis in the renal tubular epithelium may contribute to alleviating SLE.

In this study, there was no positive-treatment control group using current treatment drugs; however, the prednisolone treatment group (methylprednisolone 5 mg/kg/day) in our previous SLE study had a survival rate of 93.3% at 42 weeks of age [18]. Therefore, the survival rates of the CM-EV treatment group in this study and those of the prednisolone treatment group in the previous study were similar. CM-EV might be an alternate treatment if prednisolone is not working or if there are severe adverse effects.

There are obvious limitations in evaluating treatment effect in SLE mouse models [2, 19]; human SLE exhibits various clinical symptoms, such as nephritis, arthritis, and dermatitis. However, the NZB/W F1 mouse model of SLE is mainly characterized by the development of autoantibodies and lupus nephritis [2]. Therefore, although there are various evaluation criteria for human clinical symptoms, only the survival rate and the incidence of severe proteinuria were considered significant clinical markers in this experiment.

The amount of ASC-EV used in this experiment improved several indicators of SLE (decrease in the incidence of severe proteinuria and anti-dsDNA antibody concentration, reduction of proinflammatory macrophages, improvement of kidney histopathology, and reduction of apoptosis in renal tubular epithelial cells). However, this did not significantly increase survival rate. Since the therapeutic efficacy of EV is dose-dependent [34], it was expected that increasing the amount or frequency of administration of ASC-EV would show better effects in treating SLE.

Cytokine and histopathological examinations were performed on surviving individuals, revealing that the degree of kidney pathology was similar between the ASC-EV group and CM-EV group. The mice that died in the ASC-EV group showed severe proteinuria, indicating that they likely had a poor score in kidney pathology. Further research is needed to explore the mechanisms underlying the differences in survival rates between the ASC-EV and CM-EV groups. Further, herein, CM-EV showed a significant increase in TGF-β1 production and miR-155-5p and miR-142-3p expression compared to those in ASC-EV. The expression of TGF-β1, miR-155-5p, and miR-142-3p can significantly facilitate the treatment of autoimmune diseases [24,25,26]. Additionally, CM-EV treatment significantly reduced the expression of lupus-specific miRNAs (miR-182-5p and miR-183-5p) in the spleen, indicating that it was more effective than ASC-EV treatment in controlling SLE.

MSCs are a key source of various immunosuppressive soluble factors. One of the negative effects of MSC therapy or MSC-EV therapy is that the inclusion of many different materials makes it difficult to determine which specific components play the most significant role in disease suppression. In this study, we focused on TGF-β1, IL-1Ra, and PGE2, which are known to play the most important roles among the available factors known to contribute to immune regulation in autoimmune diseases [35], and measured their production. The results indicated that the production of TGF-β1 in CM-EV was significantly higher than that in ASC-EV. Therefore, it is thought that CM-EV exerted a greater influence on M2 polarization, resulting in a more effective therapeutic effect.

Latini et al. reported that miR-142 was significantly downregulated in SLE patients, while miR-155 also showed a trend of decreased expression [36]. MDM2, a protein involved in cell cycle regulation and immune modulation, is a common target of both miR-142 and miR-155 [36]. MDM2 was observed to be overexpressed in SLE patients, contributing to disease progression [36]. Their findings suggested that miR-142 and miR-155 play a crucial role in SLE by influencing MDM2 expression. Moreover, the therapeutic effect of mycophenolic acid was shown to be mediated by the upregulation of miR-142-3p/5p and miR-146a through histone modification at the promoter region, revealing a potential mechanism of action [37]. CM-EV showed a significantly higher expression of miR-142 and miR-155 than ASC-EV in this study. Therefore, it is believed that CM-EV exhibited better therapeutic effects (as observed in lupus-specific miRNA expression and survival rates) through the regulation of MDM2 and histone modification.