BACKGROUND: Acute rejection (AR) is a key adverse factor affecting the prognosis of organ transplantation. Considering the many complex mechanisms underlying AR, new treatment methods remain in the stage of continuous exploration. Mesenchymal stem cells (MSCs) have been widely used in various refractory immune-related diseases, but the role of MSC-derived exosomes (MSC-Exos) in alleviating graft rejection has not been extensively studied. Soluble fibronectin-like protein 2 (sFgl2) is thought to induce an anti-inflammatory phenotype in immune cells. In this study, we constructed MSC-Exos carrying sFgl2 (sFgl2-MSC-Exos) and explored the main mechanism underlying the reduction in acute rejection after mouse heart transplantation.
METHODS: The mouse heart transplantation model established using microsurgical techniques was randomly divided into an untreated group, a MSC-Exo treatment group and a sFgl2-MSC-Exo treatment group. Enrichment of DIR-labeled MSC-Exos and sFgl2-MSC-Exos in cardiac grafts after intravenous administration was evaluated by in vivo imaging. In addition to evaluating the survival time of the cardiac grafts, the remaining mice in each group were sacrificed 7 days after surgery to obtain cardiac grafts and blood for experiments. Hematoxylin-eosin (HE) staining was used to evaluate the severity of acute rejection of the cardiac grafts. Moreover, infiltration of macrophages, CD4T cells and regulatory T cells (Tregs) in cardiac grafts was analyzed by immunohistochemistry (IHC). Macrophage subsets and Treg groups in peripheral blood were quantitatively detected by flow cytometry. The polarization and function of bone marrow-derived macrophages (BMDMs) in response to different exosome treatments were evaluated using flow cytometry, ELISA, and a coculture system. In addition, the phosphorylation levels of key proteins in the SHP2-STAT3 signaling pathway were detected by Western blotting to elucidate the mechanism of sFgl2-MSC-Exos. +
RESULTS: In vivo, compared with the untreated group, MSC-Exo treatment significantly prolonged the survival time of mouse cardiac grafts and effectively reduced myocardial necrosis in the grafts and inflammatory cell infiltration. Notably, the protective effect of MSC-Exos was further enhanced because of the presence of sFgl2. MSC-Exos carrying sFgl2 further reduced the proportion of CD4T cells and increased the proportion of Tregs. In addition, sFgl2-MSC-Exos reduced the infiltration of M1 macrophages and increased the infiltration of M2 macrophages in cardiac tissue. In vitro experiments revealed that macrophages could bind to MSC-Exos and that the presence of sFgl2 could activate the SHP2/STAT3 signaling pathway and enhance MSC-Exo-induced macrophage polarization to the M2 phenotype. Blocking the CD32b receptor and inhibiting the activation of SHP2 reversed this effect. +
CONCLUSION: The sFgl2-MSC-Exos activated the SHP2-STAT3 pathway through the CD32b receptor on macrophages, thereby promoting the polarization of M2 macrophages while inhibiting the polarization of M1 macrophages and reducing the acute rejection of mouse heart transplantation.
