Mechanism Study Of The Augmentation Of Mouse Chronic Graft-Versus-Host-Disease By Zymosan

Objective:The aim is to investigate the machenism of macrophages and regulatory T cells(Treg) in zymoan augment mice chronic GVHD in a MHC-matched mouse GVHD model(H2d to H2d), based on busulfan and fludarabine as a conditioning regimen.Methods:BALB/C mice were conditioned with busulfan and fludarabine and transplanted with splenocytes and bone marrow from DBA/2 mice,similar as the MHC-matched mouse c GVHD model(H2d to H2d) we've published. BALB/C mice were assigned into two groups(n=8 for each group): zymosan group(100*106 splenocytes+10*106 bone marrow cells+zymosan); control group(100*106 splenocytes+10*106 bone marrow cells+pbs). GVHD was monitored by the lost of body weight, GVHD clinical scores and the survival of each group. Histology of the GVHD target organs(skin, small intestine, large intestine, liver and lung) were used to analyze the severity of GVHD. Different types of macrophage cells in GVHD target organs in recipients were detected by flow cytometry: the proportion of classically activated macrophage and alternatively activated macrophages respectively. Cytokines from macrophage cells in GVHD target organs in recipients, including IL-4,IL-10,IL12 and IL23,and the percentage regulatory T cells(Treg) were measured. For in vitro experiments, macrophage in GVHD target organs from BALB/C mice which were conditioned with busulfan and fludarabine were enriched by anti-CD11 b beads. Macrophage were assigned into two groups: zymosan group(culture with zymosan in 24h); control group(culture with PBS in 24h). The proportion of types of macrophage cells were checked by flow cytometry.Results:(1)Establishment of c GVHD mouse model: Busulfan 15ug/g and fludarabine 30ug/g twice a day for four days as a conditioning regimen followed by transplantation of 100*106 SPL and 10 *106+BM cells, lethal GVHD can be induced, showing death, lost of body weight, hunch back, diarrhea and hair lost. Histology of the GVHD target organs looked like scleroderma, a DBA/2?Balb/c mouse GVHD model(H2d to H2d). The survival in zymosan group was shorter than control group. Furthermore, we observed that zymosan group markedly increased lymphocyte infiltration and tissue damage in GVHD target organs.(2) The machenism of macrophage and treg in zymoan augment mice chronic GVHD: In vivo, proportion of alternatively activated macrophages(M2) in liver and lung of the mice in the experimental group was significantly lower than that in the control group(P < 0.05), and experimental group and the control group in the classical activated macrophages(M1) were without significant difference. Coincidently, the concentration of IL-4 and IL-10 secreted by M2 from the zymosan group weresignificantly decreased(p<0.001). The concentration of IL-12 and IL-23 had no difference in the two groups. Compared with the control group, the CD80 and CD86 of mouse liver macrophages in the experimental group were significantly increased(p<0.05).In vitro, the proportion of M1 in the experimental group was significantly increased(p<0.05), and the proportion of M2 was significantly decreased(p<0.05). Mice in the experimental group were significantly decreased in Treg cells.Conclusion:(1) Zymosan can augment mouse chronic graft-versus-host-disease.(2) The machenism of macrophage and Treg in zymoan augment mice chronic GVHD could be that zymosan make the donor alternatively activated macrophages reduced, and then concentration of IL-4 and IL-10 are lower. Meanwhile, zymosan led to tregs reduction. Zymosan could reduce suppressive immunoreactions, and by this way augment target tissue damage and augment GVHD.