Contributions
Abstract: S231
Type: Oral Presentation
Session title: Stem cell transplantation - Preclinical/experimental
Background
Pyroptosis, a novel form of inflammation-related programmed cell death, often occurs in myeloid cells. Many studies have found that macrophage pyroptosis plays an essential role in multiple inflammatory and autoimmune diseases (Journal of Autoimmunity, 2018). Acute GVHD, an immune disease involving various inflammatory factors, is characterized by macrophage dysfunction, as reported in our previous study (Sci China Life Sci, 2020). However, whether macrophages undergo pyroptosis in aGVHD remains unknown. Disulfiram, an inhibitor of pore formation by GSDMD, could block pyroptosis and cytokine release. Recently, disulfiram treatment was found to be safe to attenuate some immune and inflammatory diseases (Journal of Experimental Medicine, 2019; Nature Immunology, 2020). However, the therapeutic effects of disulfiram on aGVHD remain unknown.
Aims
This study aimed to explore macrophage pyroptosis in aGVHD and investigate the therapeutic effects of pyroptosis inhibitors on aGVHD.
Methods
Macrophages and CD4+ T cells were isolated from the bone marrow and spleens of C57 mice. Morphological analysis was performed by scanning electron microscopy. The expression of GSDMD was assessed by RT-PCR and WB. Cell death was measured by flow cytometry, Hoechst 33342/PI staining and lactate dehydrogenase (LDH) release assays. Inflammatory factors were tested by ELISA and immunohistochemistry. The subgroup of CD4+ T cells was analysed by flow cytometry. The pyroptosis inhibitor disulfiram was administered to the GVHD mouse model to observe its therapeutic effect.
Results
Scanning electron microscopy images showed the formation of membrane pores in the peritoneal macrophages of aGVHD mice. Additionally, more PI positivity and more LDH release were observed in peritoneal macrophages in aGVHD mice. The expression of GSDMD was elevated in the macrophages of aGVHD mice. IL-1β and IL-18 were increased in both serum and target tissues in aGVHD mice. Additionally, elevated levels of IL-1β and IL-18 were observed in the serum of aGVHD patients. These results indicated that macrophages in aGVHD underwent pyroptosis.
Because CD4+ T cells play a critical role in the pathogenesis of aGVHD, we investigated the effect of macrophage pyroptosis on CD4+ T cells. In vitro, macrophage pyroptosis increased the proportion of CD69+, Th1 and Th17 cells in CD4+ T cells, suggesting that macrophage pyroptosis might be involved in the development of aGVHD.
Administration of the pyroptosis inhibitor disulfiram into aGVHD model mice markedly improved the survival of the mice and attenuated the clinical and histopathological scores. Peritoneal macrophages that were PI positive and GSDMD in macrophages of target tissues were decreased in aGVHD mice. IL-1β and IL-18 levels were also decreased in both serum and target tissues in aGVHD mice. Furthermore, disulfiram reduced the proportions of CD69+, Th1 and Th17 cells among CD4+ cells in the spleen of aGVHD mice. These results revealed that macrophage pyroptosis could be a potential therapeutic target in aGVHD and that the pyroptosis inhibitor disulfiram could alleviate aGVHD by inhibiting macrophage pyroptosis.
Conclusion
Our results firstly demonstrated that macrophage pyroptosis plays a crucial role in the pathogenesis of aGVHD by promoting the activation and differentiation of CD4+ T cells. The pyroptosis inhibitor disulfiram markedly improved the survival and reduced the severity of aGVHD in mice. Macrophage pyroptosis may be a novel supplement to the pathological mechanism in aGVHD and provides a potential therapeutic target for aGVHD.
Keyword(s): Acute graft-versus-host disease
Abstract: S231
Type: Oral Presentation
Session title: Stem cell transplantation - Preclinical/experimental
Background
Pyroptosis, a novel form of inflammation-related programmed cell death, often occurs in myeloid cells. Many studies have found that macrophage pyroptosis plays an essential role in multiple inflammatory and autoimmune diseases (Journal of Autoimmunity, 2018). Acute GVHD, an immune disease involving various inflammatory factors, is characterized by macrophage dysfunction, as reported in our previous study (Sci China Life Sci, 2020). However, whether macrophages undergo pyroptosis in aGVHD remains unknown. Disulfiram, an inhibitor of pore formation by GSDMD, could block pyroptosis and cytokine release. Recently, disulfiram treatment was found to be safe to attenuate some immune and inflammatory diseases (Journal of Experimental Medicine, 2019; Nature Immunology, 2020). However, the therapeutic effects of disulfiram on aGVHD remain unknown.
Aims
This study aimed to explore macrophage pyroptosis in aGVHD and investigate the therapeutic effects of pyroptosis inhibitors on aGVHD.
Methods
Macrophages and CD4+ T cells were isolated from the bone marrow and spleens of C57 mice. Morphological analysis was performed by scanning electron microscopy. The expression of GSDMD was assessed by RT-PCR and WB. Cell death was measured by flow cytometry, Hoechst 33342/PI staining and lactate dehydrogenase (LDH) release assays. Inflammatory factors were tested by ELISA and immunohistochemistry. The subgroup of CD4+ T cells was analysed by flow cytometry. The pyroptosis inhibitor disulfiram was administered to the GVHD mouse model to observe its therapeutic effect.
Results
Scanning electron microscopy images showed the formation of membrane pores in the peritoneal macrophages of aGVHD mice. Additionally, more PI positivity and more LDH release were observed in peritoneal macrophages in aGVHD mice. The expression of GSDMD was elevated in the macrophages of aGVHD mice. IL-1β and IL-18 were increased in both serum and target tissues in aGVHD mice. Additionally, elevated levels of IL-1β and IL-18 were observed in the serum of aGVHD patients. These results indicated that macrophages in aGVHD underwent pyroptosis.
Because CD4+ T cells play a critical role in the pathogenesis of aGVHD, we investigated the effect of macrophage pyroptosis on CD4+ T cells. In vitro, macrophage pyroptosis increased the proportion of CD69+, Th1 and Th17 cells in CD4+ T cells, suggesting that macrophage pyroptosis might be involved in the development of aGVHD.
Administration of the pyroptosis inhibitor disulfiram into aGVHD model mice markedly improved the survival of the mice and attenuated the clinical and histopathological scores. Peritoneal macrophages that were PI positive and GSDMD in macrophages of target tissues were decreased in aGVHD mice. IL-1β and IL-18 levels were also decreased in both serum and target tissues in aGVHD mice. Furthermore, disulfiram reduced the proportions of CD69+, Th1 and Th17 cells among CD4+ cells in the spleen of aGVHD mice. These results revealed that macrophage pyroptosis could be a potential therapeutic target in aGVHD and that the pyroptosis inhibitor disulfiram could alleviate aGVHD by inhibiting macrophage pyroptosis.
Conclusion
Our results firstly demonstrated that macrophage pyroptosis plays a crucial role in the pathogenesis of aGVHD by promoting the activation and differentiation of CD4+ T cells. The pyroptosis inhibitor disulfiram markedly improved the survival and reduced the severity of aGVHD in mice. Macrophage pyroptosis may be a novel supplement to the pathological mechanism in aGVHD and provides a potential therapeutic target for aGVHD.
Keyword(s): Acute graft-versus-host disease