Contributions
Abstract: EP1295
Type: E-Poster Presentation
Session title: Stem cell transplantation - Experimental
Background
Acute graft-versus-host disease (aGVHD), a serious complication, frequently occurs following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are considered promising candidates for treating GVHD, primarily due to their immunomodulation ability and safety. Recently, EVs have been exploited for delivery of exogenous therapeutic agents and internalized by macrophages, leading to a switch from the M1 to M2 phenotype (Mol Ther. 2010; Stem Cells Transl Med. 2017). Arsenic trioxide (As2O3), a promising regent for dysregulated immune diseases. In our previous study, we found that As2O3 prolonged the survival of aGVHD mice by polarizing macrophages toward the M2 status (Sci China Life Sci. 2020). Autophagy is a conserved cellular degradation pathway that maintains intracellular homeostasis. It has been elucidated that downregulation of autophagy levels limits repolarization of M2 to M1 (Nat Rev Immunol.2019). Based on this knowledge, herein, we hypothesized that EVs loaded with As2O3 could reset the M1 to M2 phenotype through autophagy for a greater therapeutic response against aGVHD.
Aims
To investigate the therapeutic effect of hUC-MSC-EVs-ATO in aGVHD mouse model and explore the immune mechanism of hUC-MSC-EVs-ATO in vivo and vitro.
Methods
hUC-MSC-EVs-ATO was injected intravenouslly at the onset of the murine aGVHD model. Moreover, we administered clodronate liposomes by intraperitoneal injection to deplete mouse macrophages before using hUC-MSC-EVs-ATO. We also established a murine GVL model with supplementation of A20 leukemia cells. At the experimental endpoint, mice were sacrificed, and target organs were extracted to analyses the phenotypic alterations in macrophages by enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemistry. Finally, mTOR, p-mTOR, p62, LC3-I and LC3-II in BMDM-M1 and RAW264.7-M1 cells with or without hUC-MSC-EVs-ATO treatment were analyzed by western blot, and autophagic vacuoles were observed by electron microscopy.
Results
The severity and histological score of aGVHD were significantly reduced in response to hUC-MSC-EVs-ATO treatment. Mice receiving hUC-MSC-EVs-ATO exhibited longer lives compared to control mice. hUC-MSC-EVs-ATO reduced the number of Th1 and Th17 cells, while anti-inflammatory M2 and Treg cells were increased in aGVHD target organs liver and spleen. Notably, depletion of macrophages blocked these therapeutic effects and the effects on T-cells by hUC-MSC-EVs-ATO. In vitro, hUC-MSC-EVs-ATO repolarized inflammatory M1 macrophages to the anti-inflammatory M2 phenotype in both LPS/IFN-γ-conditioned bone marrow-derived and RAW264.7 macrophages. Furthermore, we demonstrated that hUC-MSC-EVs-ATO repolarized M2 to M1 macrophages partly through induction of autophagic flux by suppressing mammalian target of rapamycin (mTOR) activity. The mTOR signaling pathway activator MHY1485 reduced autophagy and repolarized M1 to M2 macrophages. Specifically, using murine model graft-versus-leukemia effects (GVL), we show here that treatment not only reduces aGVHD but also preserves GVL.
Conclusion
hUC-MSC-EVs-ATO alleviated aGVHD by switching the M1 phenotype to the M2 phenotype through inducing autophagy. Our findings demonstrate that hUC-MSC-EVs-ATO represents a potential therapeutic agent against GVHD, while preserving GVL activity.
Keyword(s): Acute graft-versus-host disease, As2O3, Macrophage, Mesenchymal stem cell
Abstract: EP1295
Type: E-Poster Presentation
Session title: Stem cell transplantation - Experimental
Background
Acute graft-versus-host disease (aGVHD), a serious complication, frequently occurs following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are considered promising candidates for treating GVHD, primarily due to their immunomodulation ability and safety. Recently, EVs have been exploited for delivery of exogenous therapeutic agents and internalized by macrophages, leading to a switch from the M1 to M2 phenotype (Mol Ther. 2010; Stem Cells Transl Med. 2017). Arsenic trioxide (As2O3), a promising regent for dysregulated immune diseases. In our previous study, we found that As2O3 prolonged the survival of aGVHD mice by polarizing macrophages toward the M2 status (Sci China Life Sci. 2020). Autophagy is a conserved cellular degradation pathway that maintains intracellular homeostasis. It has been elucidated that downregulation of autophagy levels limits repolarization of M2 to M1 (Nat Rev Immunol.2019). Based on this knowledge, herein, we hypothesized that EVs loaded with As2O3 could reset the M1 to M2 phenotype through autophagy for a greater therapeutic response against aGVHD.
Aims
To investigate the therapeutic effect of hUC-MSC-EVs-ATO in aGVHD mouse model and explore the immune mechanism of hUC-MSC-EVs-ATO in vivo and vitro.
Methods
hUC-MSC-EVs-ATO was injected intravenouslly at the onset of the murine aGVHD model. Moreover, we administered clodronate liposomes by intraperitoneal injection to deplete mouse macrophages before using hUC-MSC-EVs-ATO. We also established a murine GVL model with supplementation of A20 leukemia cells. At the experimental endpoint, mice were sacrificed, and target organs were extracted to analyses the phenotypic alterations in macrophages by enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemistry. Finally, mTOR, p-mTOR, p62, LC3-I and LC3-II in BMDM-M1 and RAW264.7-M1 cells with or without hUC-MSC-EVs-ATO treatment were analyzed by western blot, and autophagic vacuoles were observed by electron microscopy.
Results
The severity and histological score of aGVHD were significantly reduced in response to hUC-MSC-EVs-ATO treatment. Mice receiving hUC-MSC-EVs-ATO exhibited longer lives compared to control mice. hUC-MSC-EVs-ATO reduced the number of Th1 and Th17 cells, while anti-inflammatory M2 and Treg cells were increased in aGVHD target organs liver and spleen. Notably, depletion of macrophages blocked these therapeutic effects and the effects on T-cells by hUC-MSC-EVs-ATO. In vitro, hUC-MSC-EVs-ATO repolarized inflammatory M1 macrophages to the anti-inflammatory M2 phenotype in both LPS/IFN-γ-conditioned bone marrow-derived and RAW264.7 macrophages. Furthermore, we demonstrated that hUC-MSC-EVs-ATO repolarized M2 to M1 macrophages partly through induction of autophagic flux by suppressing mammalian target of rapamycin (mTOR) activity. The mTOR signaling pathway activator MHY1485 reduced autophagy and repolarized M1 to M2 macrophages. Specifically, using murine model graft-versus-leukemia effects (GVL), we show here that treatment not only reduces aGVHD but also preserves GVL.
Conclusion
hUC-MSC-EVs-ATO alleviated aGVHD by switching the M1 phenotype to the M2 phenotype through inducing autophagy. Our findings demonstrate that hUC-MSC-EVs-ATO represents a potential therapeutic agent against GVHD, while preserving GVL activity.
Keyword(s): Acute graft-versus-host disease, As2O3, Macrophage, Mesenchymal stem cell