Donor Macrophage Pyroptosis Contributed to the Development of aGVHD

Introduction

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 diseases, such as sepsis, liver injury, and autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis (Journal of Autoimmunity, 2018). Acute GVHD (graft versus host disease) is an immune disease involving various inflammatory factors, and macrophage dysfunction was observed in our previous study (Sci China Life Sci, 2020). During the initiation of aGVHD, large amounts of DAMPs and PAMPs are also released by radiotherapy and infection, which may trigger pyroptosis. However, whether macrophages undergo pyroptosis in aGVHD remains unknown and needs further exploration.

Methods

The peritoneal macrophages from aGVHD mice were collected after transplantation. Scanning electron microscopy was used to observe cell morphology. Flow cytometry, LDH release and PI staining were used to evaluate cell death. Target tissues, including skin, liver, colon and ileum, were obtained after transplantation for histopathologic analysis, immunofluorescence, immunohistochemical staining and Wb tests. Pyroptosis-related inflammatory factors, including IL-1β and IL-18, were evaluated by a BCA assay and immunohistochemistry. Macrophages and CD4⁺ T cells were isolated from the bone marrow and spleens of C57 mice. Macrophage-specific GSDMD knockdown mice using adeno-associated serotype 9 virus (AAV9) viral vectors and macrophage conditional GSDMD KO mice (GSDMD^rlysm) were used to demonstrate the role of macrophage pyroptosis in the development of aGVHD. Intestinal tissues and peripheral blood from patients with aGVHD and without aGVHD were collected to further validate the occurrence of macrophage pyroptosis in aGVHD.

Results

Scanning electron microscopy images showed the formation of membrane pores in the peritoneal macrophages of aGVHD mice. Moreover, cell death and LDH release were significantly increased in the peritoneal macrophages of aGVHD mice. The number of GSDMD⁺ macrophages and expression levels of IL-1β and IL-18 were significantly increased in the target tissues of aGVHD mice. The expression of GSDMD-N was also significantly increased in the liver tissue of aGVHD mice. These results indicated that aGVHD was accompanied by macrophage pyroptosis. In addition, elevated levels of IL-1β and IL-18 were observed in the serum and colons of aGVHD patients, which further demonstrated the occurrence of macrophage pyroptosis in aGVHD.

To determine whether macrophage pyroptosis contributed to the development of aGVHD, we used macrophage-specific GSDMD knockdown mice with AAV9 viral vectors and WT mice as recipients or donors to compare their disease severity and overall survival. Less pathological damage to tumor tissues, lower clinical scores and longer survival were observed in recipients with macrophage-specific GSDMD knockdown in their donors, indicating that macrophage pyroptosis in donor mice promoted the development of aGVHD. Furthermore, similar results were observed in recipients when macrophage-conditioned GSDMD KO mice (GSDMD^rlysm) were their donors.

Given the important role of CD4⁺T cells in the pathogenesis of aGVHD, the effects of macrophage pyroptosis on CD4⁺ T cells in vitro were further investigated. In vitro, macrophage pyroptosis promoted the proliferation of CD4⁺ T cells and increased the proportion of CD69⁺ cells among CD4⁺T cells. The proportions of Th1 and Th17 cells were increased, and the proportion of Tregs was decreased after coculturing with pyroptosed macrophages. These results suggested that macrophage pyroptosis might be involved in the development of aGVHD by affecting CD4⁺T cells. In addition, a reduced proportion of activated CD4⁺T cells, Th1, Th17 cells and an increased proportion of Tregs were observed in recipients when their donors were macrophage-specific GSDMD knockdown or GSDMD^rlysm, further demonstrating that macrophage pyroptosis from donors promoted the development of aGVHD by affecting the function of CD4⁺ T cells.

Conclusions

In conclusion, macrophage pyroptosis derived from donors were first found in aGVHD, and they might participate in the development of aGVHD by affecting the activation, proliferation and differentiation of CD4⁺ T cells.