| Objective To establish murine model of actue graft-versus-host disease(aGVHD)after allogeneic hematopoietic stem cell transplantation(allo-HSCT)with total body irradiation(TBI).To determine the role of CHI3L1 in aGVHD.To investigate the effect of CHI3L1 on the differentiation and function of Tregs.Methods(1)C57BL/6(H-26,?)mice were donor and BALB/C(H-2d,?)mice were recipient.Thirty SPF BALB/C mice were randomly divided into six groups and then received 6.5Gy,7.0Gy,7.5Gy,8.0Gy,8.5Gy and 9.0Gy X ray TBI.After TBI,twenty BALB/C mice were randomly divided into four groups and then received 1 ×106,3× 106,5×106,,10×106 C57BL/6 bone marrow cells(BMCs)via tail vein injection,respectively.aGVHD models were established by coinfusion of 1×106,3×106,5×106,,10×106 splenocytes via tail vein,respectively.We observed survival rate each goup.Histological analysis including lung,liver,gut and skin were obtained from recipients after transplantation and were detected by HE staining.The relative percentages of donor chimerism in the recipients were determined by flowcytometry.(2)After TBI,thirty SPF grade BALB/C mice were randomly divided into three groups:the recipient BALB/C mice were received only 5×106 C57BL/6 BMCs via tail vein injection;the recipient BALB/C mice were received 5 × 106 C57BL/6 BMCs and 3×106 C57BL/6 splenocytes via tail vein injection;the recipient BALB/C mice were received 5×106 C57BL/6 BMCs and 3 × 106 CHI3L1-konckout(CHI3L1-KO)C57BL/6 splenocytes via tail vein injection.We observed clinical manifestations of aGVHD and survival after allo-HSCT.Histopathology and the donor chimera were detected in recipient mice after 20 days post allo-HSCT.The survival rate was compared by Log-rank test.(3)Plasma concentrations of cytokines were detected using a Cytometric Bead Array(CBA)kit in recipient mice after 20 days post allo-HSCT.Plasma concentrations of interleukin(IL)-21 were detected using an ELISA kit in recipient mice after 20 days post allo-HSCT.Plasma concentrations of chemokines were detected using a chemokines chip in recipient mice after 20 days post allo-HSCT.Absolute number of splenocytes,the proportion of CD3+,CD4+and CD8+in spleen were detected using flow analysis in recipient mice after 20 days post allo-HSCT.Intracellular cytokine IL-6?IL-10?IFN-??TNF-? on CD4+T cell and CD8+T cell were analyzed through flow analysis in recipient mice after 20 days post allo-HSCT.The proportion of Tfh cells in spleen was detected using flow analysis in recipient mice after 20 days post allo-HSCT.(4)CFSE labeled CD3+ T cells from B6 or CHI3L1-KO mice were cultured with irradiated non-T cells(2600cGy)(1:1),while anti-CD3 mAb(145-2C11;Bio Express)were added at the dose of 0.5?g/ml.After 5 days of culture,the proliferation of effective T cells was evaluated by CFSE dilution by flow cytometry.(5)The expression of IFN-?,TNF-?,IL-6,IL-10,IL-21,CXCL9,CXCL11 and CXCL13 mRNA from liver,lung,small intestine and skin was measured with Real-time PCR.(6)The expression of ICOS,AKT/P-AKT and ERK/P-ERK from liver,lung,small intestine and skin was measured with Western blot.(7)CD4+T cells derived from WT and CHI3L1-KO C57BL/6 mice spleen were selected by Mini MACS,and cultured in 24 wells plate for 3 weeks,which coated with 2ug/ml anti-CD3 monoclonal antibody,the medium system included lug/ml anti-CD28 monoclonal antibody,IL-2 100U/ml with RPMI 1640 medium and 15%FBS.After 3 weeks,CD4+CD25+T cells were detected by flow cytometry.(8)CD4+CD25+Tregs were isolated from the spleen of B6 or CHI3L1-KO mice through magnetic selection with CD4+CD25+T cell isolation kit and washed with culture medium.Then Tregs were added to CFSE-labeled CD4+CD25-T cells(2×105;1:4)from B6 mice that were activated by anti-CD3 mAb coated at the dose of 0.5 ?g/ml in 96-well flat-bottom plates.The ability of Tregs to inhibit the activation and proliferation of CD4+CD25-T cells was determined by measuring the proliferation of CFSE-labeled CD4+T cells via flow cytometry.(9)After TBI,thirty SPF grade BALB/C mice were randomly divided into three groups:the recipient BALB/C mice were received only 5× 106 C57BL/6 BMCs via tail vein injection;the recipient BALB/C mice were received 5×106 C57BL/6 BMCs and 3×106 C57BL/6 splenocytes via tail vein injection;the recipient BALB/C mice were received 5×106 C57BL/6 BMCs and 3×106 CHI3L1-konckout(CHI3L1-KO)C57BL/6 splenocytes via tail vein injection.The proportion of CD3+CD4+CD25+Foxp3+Treg cells in spleen was detected using flow analysis in recipient mice after 20 days post allo-HSCT.(10)The expression of Sirt-1 mRNA from spleen,liver,lung,small intestine and skin was measured with Real-time PCR.Results(1)All mice receiving 6.5Gy irradiation achieved long-term survival.The median survival time of mice conditioned with 7.0Gy,7.5Gy TBI was 32d,25d respectively and in above groups 35%or 15%mice survived 40 d after TBI.The mice conditioned with 8.0Gy,8.5Gy and 9.0Gy all died within 20 days.The survival time was compared by Log-rank test,with ? 2-25.83,P<0.0001.All mice survived 100d post-HSCT when reconstituted with 5x106 BMCs.No overt aGVHD was observed when mice were reconstituted with BMCs alone.Moderate aGVHD was induced when mice were infused with 1×106splenocytes.Overt aGVHD was induced when mice were infused with 3×106splenocytes.Severe aGVHD was induced when mice were infused with5×106 or10×106 splenocytes.Complete donor chimerism was achieved on 14d after allo-HSCT.(2)Compared with WT group,CHG3L1-KO group induced more serious aGVHD,pathological changes of lung,liver,small intestine and skin are more severe from CHI3L1-KO group.There were significant differences in survival rate and body weight between the two experimental groups(P<0.0001).The donor chimeric rate of the two experimental groups achieved complete chimerism.(3)Compared to WT group,the levels of IFN-?,TNF-?,IL-6 and IL-21 in the serum were significantly higher from CHI3L1-KO group(P<0.0001).The absolute number of spleen cells in CHI3L1-KO group was significantly higher than that in WT group(P<0.0001),and the levels of CXCL9,CXCL10 and CXCL13 in the serum were also significantly higher(P<0.0001).The percentage of CD3+,CD4+and CD8+cells in spleen from CHI3L1-KO group was significantly higher than that from WT group(P<0.0001),and the difference was statistically significant(P<0.05);CD3+CD4+IFN-?+T cells,CD3+CD4+TNF-?+T cells,CD3+CD4+ IL-6+T cells,CD8+IFN-?+T cells,CD3+CD8+TNF-?+T cells and CD3 CD8 IL-6 T cells in the spleen from CHI3L1-KO group increased significantly(P<0.0001);CD3+CD4+IL10+T cells and CD3+CD8+IL10+T cells in the spleen from CHI3L1-KO group decreased significantly(P<0.0001).The proportion of Tfh cells in spleen from CHI3L1-KO group was significantly increased compared with that from WT group(P<0.0001),and the difference was statistically significant(P<0.0001).(4)proliferative capacity of CD3+ T cells was increased in CHI3L1-KO mice,compared with WT group(P<0.0001).(5)Compared with WT group,the expression levels of IFN-?,TNF-?,IL-6,IL-21,CXCL9,CXCL10 and CXCL13 mRNA in lung,liver,small intestine and skin from CHI3L1-KO group were significantly higher,while the level of IL-10 in the lung,liver and small intestine and skin was significantly reduced,the gap was statistically significant.(6)Compared with WT group,the protein expression of ICOS,P-AKT and P-ERK in lung,liver,small intestine and skin from CHI3L1-KO group was significantly enhanced.(7)Compared with CD4+T cells in WT mice,the ability of CD4+T cells to differentiate into CD3+CD4+CD25+Foxp3+Treg cells in CHI3L1-KO mice was significantly attenuated,the difference was statistically significan(P<0.0001).(8)Compared with WT mice,CD3+CD4+CD25+Foxp3+Treg cells in CHI3L1-KO mice inhibited the proliferation of CD4+CD25-T cells,but the difference was not statistically significant.(9)Compared with WT group,the proportion of CD3+CD4+CD25+Foxp3+Treg in CHI3L1-KO group was significantly decreased,and the difference was statistically significant(P<0.0001).(10)Compared with WT group,the expression level of Sirt-1mRNA in the spleen,lung,liver,small intestine and skin from CHI3L1-KO group was significantly higher,the difference was statistically significant(P<0.0001).Conclusions 8.0Gy was myeloablative conditioning in BALB/c mice.Hematopoietic recovery could be achieved when BALB/c mice were reconstituted with 5×106 BMCs.aGVHD could be induced when mice were injected with 3×106 splenocytes.CHI3L1 deficient donor splenocytes accentuated the pathogenesis of acute graft-versus-host diseases and inhibit the differentiation of Treg cells in spleen. |
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