Mesenchymal Stem Cell Exosome-derived MiR-223 Alleviates Acute Graft-versus-host Disease Via Reducing the Migration of Donor T Cells

Background: Mesenchymal stem cells (MSCs) have been utilized in treating acute graft-versus-host disease (aGvHD) as they show strong immunosuppressive capacity through the release of various mediators, including immunosuppressive molecules, growth factors, chemokines, and exosomes. MicroRNAs (miRNAs) derived from MSC exosomes (MSCs-Exo) play a critical role in the regulation of immune responses. However, the function of miRNAs in treating aGvHD remains unknown. Here, we performed expression profiling of exosome-miRNAs from human umbilical cord MSCs (huc-MSCs) and murine compact bone MSCs (mb-MSCs) to investigate their immunoregulation effects in aGvHD.

Methods: Huc-MSCs-Exo and mb-MSCs-Exo were isolated and constructed MSCs-Exo-derived miRNA expression profiling using high-throughput sequencing. High expression of miR-223 was identified in both kinds of MSCs-Exo by bioinformatics analysis and quantitative real-time PCR (qPCR). In vitro cell crawling assay, transmigration assay and adhesion assay were subsequently applied to investigate the regulation of miR-223 on T cells. MiR-223 target gene was analyzed by western blot, luciferase analysis, and qPCR. Moreover, murine aGvHD model was established by infusing splenocytes and bone marrow nuclear cells from C57BL/6j mice (H-2Kb) into BALB/c recipient mice (H-2Kd). For therapeutic effect, MSCs or miR-223 Agomir were injected via tail vein. The general conditions of the mice in each group were monitored. Hematoxylin-eosin (H&E) staining was used to detect pathological changes of mice spleen, liver, and intestine. Mechanistically, immunofluorescence and flow cytometry were used to evaluate donor T cell migration, and enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of serum inflammatory cytokines IFN-γ, TNF-α, and IL-17.

Results: High-throughput sequencing revealed high expression of miR-223 in huc-MSCs-Exo and mb-MSCs-Exo. MiR-223 could restrain adhesion and migration of T cells by inhibiting ICAM-1 expression in mouse lymphatic endothelial cells. MiR-223Agomir infusion attenuated aGvHD clinical symptoms, reduced the donor T cell infiltration into the spleen, liver, and intestine, and decreased inflammatory cytokines IFN-γ, TNF-α, and IL-17.

Conclusion: MSCs-Exo-derived miR-223 could attenuate aGvHD in mice through decreasing donor T cell migration. Our results unveil a new role of MSCs-Exo containing miR-223 in the treatment of aGvHD.