FK228 Reduced Donor-Specific Antibody Production In Pre-Sensitized Skin Transplant Mice Though IRE1α-XBP1 Pathway

Chapter 1 Effects of FK228 on the production of donor-specific antibodies in sensitized skin transplant mice and the underlying mechanismObjective:To observe the survival of allogeneic presensitized skin transplant in mice with or without HDACI-FK228 treatment,and further explore the role and mechanisms of FK228 on the production of donor-specific antibodies.Methods:Allogeneic mouse tail-back ectopic skin transplantation was used to establish the model.The first presensitized skin transplant from BALB/C to C57BL/6 mice were performed at-14 days.The secondary skin graft was performed on the same recipient mice on day 0.The recipient mice were divided into three groups randomly:syngeneic group（C57BL/6→C57BL/6+vehicle）,allogeneic group（BALB/c→C57BL/6+vehicle）,FK228 treatment group（BALB/c→C57BL/6+FK228）.FK228 was injected intraperitoneally with the dose of 0.5 mg/（kg*d）from day 0 to day the observation ended,n=12 in each group.The survival of the skin graft（n=6）was monitored daily after surgery and the survival time was recorded.The loss of skin graft was defined as necrotic area of the grafted skin graft>90%.The peripheral blood samples of recipient mice were obtained on day 0,7 and 14,and the antibody binding assay was used to detect the level of donor-specific antibody（DSA）-IgG and IgM via flow cytometry.In addition,the spleen and transplanted skin samples of recipient mice（n=6）collected on the 10th day.The transplanted skin tissue was stained with HE to evaluate the pathological changes.The level of cytokines in serum were detected by ELISA.The proportion of CD4⁺PD-1⁺CXCR5⁺Tfh,and B220^lowCD138⁺ plasma cells were detected by Flow cytometry.The totalRNA of the spleen lymphocytes was extracted,and RT-PCR technology was used to detect the mRNA levels of Hdac1 and Hdac2,the levels of B cell-related Pax5,Bcl-6,Bach2,and plasma cell generation-related Blimp-1,Irf4,Xbp1.Total spleen lymphocytes protein was extracted,and Western blot was used to detect the levels of HDAC1,HDAC2 and histone H2A,H3 acetylation,the expression level of IRE1α-XBP1 pathway protein,BLIMP-1,AID.Results:The median survival time of grafts after first and secondary skin transplantation in the allogeneic group was 12 and 11 days,respectively.After the second allograft skin graft,the survival of the skin graft was significantly prolonged due to the use of FK228（Median survival time,MST=25 days,P<0.0001）.The results of HE staining of the skin grafts revealed that compared with the syngeneic group,there were obvious epidermal damage,hair follicle destruction,severe mononuclear cell infiltration without keratinization in the allogeneic group and FK228 group.Compared with the allogeneic group,the skin lesion in the FK228 group alleviated significantly.The results of flow cytometry data showed that compared with the syngeneic group,the levels of DSA-IgG and IgM in the circulation of the recipient mice in allogeneic group and FK228 group were upregulated distinctively on the day of the secondary skin transplantation.Furthermore,the DSA-IgM and IgG levels of the allogeneic mice on the 7th and 14th days increased significantly compared to the syngeneic mice.Those in the FK228 group on were obviously lower than the allogeneic group（P<0.01）.Compared with the allogeneic group,the proportion of CD4⁺PD-1⁺CXCR5⁺Tfh and B220^lowCD138⁺plasma cells in the FK228group significantly decreased（P<0.05）.ELISA results showed that the level of IL-21 in FK228 group was significantly lower than that in the allogeneic group（P<0.05）.RT-PCR results showed that compared with the syngeneic group,Hdac1 and Hdac2 highly expressed in the allogeneic group（P<0.05）;however,comparing with the allogeneic group,the transcriptional levels of Hdac1 and Hdac2 significantly decreased in the FK228 group.There was no significant difference in the transcription levels of B cell-related key transcription factors Pax5,Bcl-6,and Bach2 in the spleen of the allogeneic group and the FK228 group（P>0.05）.However,FK228 significantly down-regulated the mRNA transcription levels of Prdm-1 and Xbp1（P<0.05）.Western blot results showed that compared with the syngeneic group,the expression of HDAC1 and HDAC2 in the spleen of the allogeneic group increased,and so did the levels of AID,BLIMP-1,p-IRE1α and XBP1（P<0.05）;compared with the allogeneic group,the expressions of HDAC1 and HDAC2 in FK228 group were inhibited,and the levels of acetylated histones H2A and H3 significantly increased（P<0.05）.At the same time,the levels of AID,BLIMP-1,p-IRE1α and XBP1 significantly decreased（P<0.05）.Conclusions:FK228 significantly prolonged the skin graft survival in presensitized mice and decreased the DSA levels in vivo.FK228 down-regulated the transcription of Pridm-1 and Xbp1,thereby inhibiting the generation of plasma cells.In addition,FK228 down-regulated Tfh ratio as well as IL-21 level.During endoplasmic reticulum stress UPR,FK228 inhibited the activity of IRE1α-XBP1 pathway,thereby affecting the modification and synthesis of the antibody.In addition,FK228 also interfered with antibody secretion by inhibiting the expression of BLIMP-1,and further interfered with the CSR process by down-regulating the level of AID.Chapter 2 FK228 inhibits B cells in vitro and its mechanismObjective: To observe the role of FK228 on B cells in vitro,and reveal the underlyding mechanisms.Methods: Primary CD19+ B cells were isolated from the spleen of C57BL/6 mice by magnetic bead sorting,and stimulated with anti-CD40 antibody（1 ug/ml）+ IL4（10 ng/ml）.The experiment was divided into control and FK228 groups.The cells were cultured for 2 days,and the effects of FK228 on B cell proliferation and apoptosis were evaluated by CCK8 and Annexin V-PI.Cultured for 6 days,the culture supernatant was collected,and IgG1 and IgM in the supernatant were detected by ELISA.The cell pellets in each group were collected,followed byRNA and protein extraction.The mRNA levels of Prdm-1,Irf4 and Xbp1 were detected by RT-PCR.Western blot technology was used to detect the levels of HDAC1,HDAC2,acetylated histones H2A and H3 in B cells.To detect the effect of FK228 on antibody synthesis and modification-related phosphorylation the level of IRE1α and the expression level of XBP1 in the IRE1α-XBP1 pathway of endoplasmic reticulum stress,as well as levels of BLIMP-1 and AID.Flow cytometry was used to detect the proportion of B220^lowCD138⁺ plasma cells in the cell pellet,as well as the expression of cell surface signaling molecules CD80 and MHC-Ⅱ.Results: When the reagent concentration reached 5 and 8 n M,the effects of FK228 on B cell proliferation and apoptosis were significantly different.When the reagent concentration increased to a 5,8 n M,it could promote the apoptosis of B cells.ELISA showed that compared with the control group,FK228 significantly decreased the levels of IgM and IgG1（P < 0.05）.Western results showed that FK228 also inhibited the expression of HDAC1 and HDAC2,and up-regulated the levels of acetylated histones H2A and H3 in vitro.At theRNA level,FK228 significantly inhibited the relative expression levels of Prdm-1 and Xbp1（P < 0.05）,but had no significant effect on the transcription of Irf4.The results of flow detection showed that FK228 significantly reduced the proportion of B220^lowCD138⁺ plasma cells,and the expression of CD80 and MHC-Ⅱ on the cell surface also decreased.During endoplasmic reticulum stress UPR,FK228 significantly decreased the expression levels of AID,BLIMP-1,XBP1,and p-IRE1α（P < 0.05）.Conclusions: FK228 inhibits the proliferation and promotes the apoptosis of B cells.FK228 inhibited B cell differentiation and the secretion of IgG1 and IgM in vitro.FK228 suppressed the expression of HDAC1 and HDAC2,promoted the acetylation of histones H2A and H3,downregulated the transcription levels of Pridm-1 and Xbp1 and inhibited the differentiation of B cells to Plasma cells.In addition,FK228 inhibited antibody synthesis and modification related the activity of the IRE1α-XBP1 pathway during the endoplasmic reticulum stress UPR.Furthermore,FK228 interfered with BLIMP-1,AID.