Peritoneal Cavity Cells Ameliorate Graft-Versus-Host Disease of Mice after MHC Mismatched Bone Marrow Transplantation
Authors: Liu Zhigang , Zhang Wanhua , Chen Xinchuan , He Chuan , Ji Jie , Liu Ting 
Published: October 2019

Background: Peritoneal cavity cells are a group of isolated cells with unique functions, they have great anti-inflammatory, immune modulation and tissue recovering abilities. Several studies have independently reported the therapeutic function of peritoneal cavity cells on mice models with ulcerative colitis or other inflammatory bowel diseases. In the current study, we further investigated the effect of peritoneal cells on treatment of GVHD on mice models.

Methods: We selected a dose of 7.0Gy of TBI for BABL/c mice. And 4 hours after TBI, BABL/c mice were infused with 5*106BMCs cells or 1*106spleen cells of C57BL/6 mice through tail veins to construct MHC mismatched myeloablative allogeneic hematopoietic stem cell transplantation model. The mice were randomly separated into two groups, which were infused with normal saline and 5* 106peritoneal cavity cells on day 0, 3, and 5. Survival time, weight changes, GVHD score were evaluated. Peritoneal cavity cells of GFP C57BL/6 transgenic mice were injected into mice recipients at day 5 after transplantation to observe the distribution of GFP positive cells in mice recipients. Flow cytometry was used to test proportion of CD4/CD8 cells and proportions of effector T cells and Naïve T cells in CD4 and CD8 cells of spleen and bone marrow in BMT group and peritoneal cavity cells group at different time points after transplantation. TNF-α, IFN-γ, IL-2, IL-17, IL-15, IL-4, IL-10 and TGF-β in plasma were studied with the method of Luminex at different time points in two groups. Expression of IL-10 and TGF-β in colons, intestines, and livers were assessed with immunofluorescence staining.

Results: Mice in peritoneal cavity cells group had significant longer survival time and rapidly weight loss recover. Mice in peritoneal group had better performance in activity, unhairing, and skin changes as well. Counts of blood cells and chimeric status at day 7, 14, 21, 28 after transplantation showed that blood count recovered and stable chimerism in both groups. Small living animal imaging technology found that peritoneal cavity cells concentrate in colons and intestines after injection of GFP C57BL/6 transgenic mice peritoneal cavity cells. Fluorescence microscope showed that large amounts of green fluorocyte distributed mostly in colons and intestines, with few in liver. Flow cytometry proved that many GFP positive cells in intestines and colons (30%, and 15%, respectively), and a few in livers and lungs (approximately 5%), while negative in control group. We analyzed the lymphocyte subsets of spleen and bone marrow in two groups with flow cytometry and found that peritoneal cells treatment could increase the proportion of CD4 cells and decrease CD8 cells. In CD4 subsets, proportion of effective T cell decreased apparently 3 weeks after transplantation, and count of naïve T cells increased, which is not found in BMT group. Flow cytometry also showed that proportion of Treg cells, Th2 cells and NK cells were significantly higher in peritoneal cavity cells group, while proportion of Th1 cells were lower. TGF-β, IL-10 and IL-4 were significantly higher in peritoneal cavity cell treatment group, while TNF-α, IFN-γ, IL-15, IL-2 were lower. Immunofluorescence staining also showed that TGF-β and IL-10 were strongly expressed in colons and intestines, but not in BMT group.

Conclusion: These results demonstrate that peritoneal cavity cells could ameliorate graft-versus-host disease of mice after MHC mismatched bone marrow transplantation. Survival time was prolonged, and weight loss, GVHD score, and pathologic injuries to tissues improved after infusion of 5* 106peritoneal cavity cells into BMT mice. Peritoneal cavity cells injected to BMT mice concentrate mainly in colons and intestines, which functioned as anti-inflammation and tissue repairing cells. These cells could modulate differentiation of T lymphocytes of mice recipients, decrease proportion of CD8 cells and increase CD4 cells, increase proportion of Tn cells and decrease that of Te cells in CD4 subsets, and increase proportion of Tregs, Th2 and NK cells and decrease that of Th1 cells. Peritoneal cavity cells could influence levels of cytokines by increasing anti-inflammatory factors including TGF-β, IL-10, IL-4, and significantly decreasing inflammatory factors like IFN-γ, TNF-α, and IL-15.

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Disclosures

Liu: West China Hospital of Sichuan University: Employment.