PGE2 Dependent Inhibition of Macrophage Pyroptosis By MSCs Contributes to Alleviating aGVHD

Introduction Mesenchymal stem cells (MSCs) are being recognized as one of the treatment options for acute graft versus host disease (aGVHD), but their therapeutic mechanisms have not been fully elucidated. Pyroptosis, a novel form of inflammation related programmed cell death, often occurs in myeloid cells. Many studies have found that macrophage pyroptosis plays an important role in multiple inflammatory and autoimmune diseases (Journal of Autoimmunity, 2018). As an immune disease with involvement of various inflammatory factors, aGVHD exhibits macrophage dysfunction according to our previous study (Sci China Life Sci, 2020). However, whether macrophages undergo pyroptosis and their role in aGVHD remain unknown. MSCs have been reported to inhibit pyroptosis, and some cytokines that suppress pyroptosis can also be secreted by MSCs (Nature Immunology, 2016). Whether inhibition of macrophage pyroptosis represents a therapeutic mechanism for MSCs to alleviate aGVHD needs further exploration. Methods Twenty patients with aGVHD and 20 patients without aGVHD after hematopoietic stem cell transplantation were enrolled in our study. Macrophages were derived from CD14+ monocytes of patients and the THP-1 cell line. CD4+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy volunteers. MSCs were obtained from fresh umbilical cord of healthy puerpera. Morphological analysis of macrophages was performed by scanning electron microscopy. Expression of GSDMD and NLRP3 inflammasome associated components was assessed by real-time transcription-polymerase chain reaction (RT-PCR), western blot and immunofluorescent staining. The subgroup of CD4+T cells was analyzed by flow cytometry. RT-PCR, ELISA and RNA interference were used to evaluate relevant immunomodulatory factors which were involved in the inhibitory effect of MSCs on macrophage pyroptosis. Additionally, an aGVHD mouse model was established to observe the therapeutic effect and mechanism of MSCs on macrophage pyroptosis. Results Scanning electron microscopy images showed the formation of membrane pores in macrophages of aGVHD patients. Meanwhile, expression of the pyroptosis executioner GSDMD, NLRP3 inflammasome associated components, IL-1β, IL-18, and LDH release were elevated in macrophages from aGVHD patients, indicating that macrophages in aGVHD underwent NLRP3 inflammasome activation and pyroptosis. Furthermore, NLRP3 inhibition reduced macrophages pyroptosis, suggesting that macroph