hUC-EVs-ATO reduce the severity of acute GVHD by resetting inflammatory macrophages toward the M2 phenotype

Abstract

Background: Both extracellular vesicles from mesenchymal stromal cell-derived human umbilical cords (hUC-EVs) and arsenic trioxides (ATOs) have been demonstrated to treat acute graft-versus-host disease (aGVHD) via immunomodulation. Apart from immunomodulation, hUC-EVs have a unique function of drug delivery, which has been proposed to enhance their efficacy. In this study, we first prepared ATO-loaded hUC-EVs (hUC-EVs-ATO) to investigate the therapeutic effect and potential mechanisms of hUC-EVs-ATO in a mouse model of aGVHD after allogeneic hematopoietic stem cell transplantation (HSCT).

Methods: An aGVHD model was established to observe the therapeutic effects of hUC-EVs-ATO on aGVHD. Target organs were harvested for histopathological analysis on day 14 after transplantation. The effects of hUC-EVs-ATO on alloreactive CD4⁺ were evaluated by flow cytometry in vivo and in vitro. Flow cytometry, RT-PCR, immunofluorescence colocalization analysis and Western blot (Wb) analysis were performed to examine macrophage polarization after hUC-EV-ATO treatment. The cytokines in serum were measured by a cytometric bead array (CBA). TEM, confocal microscopy and Wb were performed to observe the level of autophagy in macrophages. A graft-versus-lymphoma (GVL) mouse model was established to observe the role of hUC-EVs-ATO in the GVL effect.

Results: The clinical manifestations and histological scores of aGVHD in the hUC-EVs-ATO group were significantly reduced compared with those in the ATO and hUC-EVs groups. The mice receiving hUC-EVs-ATO lived longer than the control mice. Notably, hUC-EVs-ATO interfering with alloreactive CD4⁺ T cells differentiation were observed in aGVHD mice but not in an in vitro culture system. Additional studies showed that depletion of macrophages blocked the therapeutic effects of hUC-EVs-ATO on aGVHD. Mechanistically, hUC-EVs-ATO induced autophagic flux by inhibiting mammalian target of rapamycin (mTOR) activity to repolarize M1 to M2 macrophages. Additionally, using a murine model of GVL effects, hUC-EVs-ATO were found not only to reduce the severity of aGVHD but also to preserve the GVL effects. Taken together, hUC-EVs-ATO may be promising candidates for aGVHD treatment.

Conclusions: hUC-EVs-ATO enhanced the alleviation of aGVHD severity in mice compared with ATO and hUC-EVs without weakening GVL activity. hUC-EVs-ATO promoted M1 to M2 polarization via the mTOR-autophagy pathway. hUC-EVs-ATO could be a potential therapeutic approach in aGVHD after allo-HSCT.

Keywords: Acute graft-versus-host disease; Autophagy; Macrophage; hUC-EVs-ATO.