The Role And Mechanism Of IL-Y In The Progression Of Chronic Graft Versus Host Disease In Mice

Objective:To investigate the role and mechanism of IL-Y in the progression of murine chronic graft-versus-host disease(cGVHD).Methods:(1)The cDNA encoding mouse IL-27p28 and IL-14p40 were amplified by PCR from the total RNA extracted from spleen cells of C57BL/6 mice stimulated with LPS.IL-27p28 and IL-14p40 gene were fused via a hydrophobic polypeptide linker(Gly4Ser)3 by overlap extension PCR to obtain mscIL-Y fusion gene.The mscIL-Y fusion gene was cloned into eukaryotic expression vector mini-circle after added Igk signal sequence and restriction enzyme cutting site of Nhel I in the 5' end and Sal I and label of Flag in the 3'end,then the positive recombinant clone was analyzed by digestion of restriction endonuclease and DNA sequencing.(2)To established two models of chronic graft-versus-host disease in mice.Fourteen SPF grade BALB/c mice were randomly assigned to two groups:1)Model of C57BL/6?BALB/c:Fourteen 6-8 weeks of BALB/c(H2Kd,?)mice were randomly assigned to two groups:the concentration of the experimental group was 120ug/2ml IL-Y plasmid by hydrodynamic gene transfer(mini-IL-Y),and the control group was 120ug/2ml empty plasmid(mini-circle).The recipient BALB/c mice were conditioned with 650cGy total body irradiation(TBI)after 24h receiving an HGT injection.Irradiation was followed by the infusion of 5×106C57BL/6(H2Kb,?)bone marrow cells and whole splenocytes(1x106).2)Model of DBA/2->BALB/c:Fourteen 6-8 weeks of BALB/c mice were randomly assigned to two groups:the concentration of the experimental group was 120ug/2ml IL-Y plasmid by hydrodynamic gene transfer(mini-IL-Y),and the control group was 120ug/2ml empty plasmid(mini-circle).The recipient BALB/c mice were conditioned with 650cGy TBI after 24h receiving an HGT injection.Irradiation was followed by the infusion of 7.5×106 DBA/2(H2Kd,?)bone marrow cells and 4×107 CD25-splenocytes.(3)Clinical manifestations of a GVHD,survival and weight change were observed after allogeneic bone marrow transplantation.Urine protein level detected weekly and serum anti-ds-DNA antibody level were detected at the eighth week after transplantation.(4)The kidney,skin,liver,lung,salivary glands and small intestine of mice were collected at the eighth week after transplantation for histopathological examination.(5)The percentage of CD4,CD8,CD44 and CD62L of lymphocytes in mice spleen cells were measured by flow analysis after 8-weeks post BMT,whereas the proportion of regulatory T cells(Treg),follicular helper T cells(Tfh)and B cells,follicular B cells,germinal center B cell,marginal zone B cell and plasma cells were also detected.At the same time,the expression of co-stimulatory molecules on the surface of B cells was also detected.In addition,intracellular cytokine IL-4,IL-17A,IFN-y,TNF-a on CD4+T cell and CD8+T cell were analyzed through flow analysis.(6)The levels of IL-2,IL-4,IL-17A,IFN-y,TNF-a and IL-21 in serum were assessed by Cytometric Beads Array after 8-week cells transfer.Results:(1)IL-Y eukaryotic expression vector was successfully constructed,and positive recombinant vector was identified by digestion of restriction endonuclease and DNA sequencing.In addition,the overexpression of IL-Y in mouse liver was proved by immunohistochemistry and western blot.(2)Two mouse cGVHD models were successfully constructed.(3)Urine protein and the serum of anti-dsDNA antibody(IgG)were significantly up-regulated by IL-Y administration.Weight loss was more significant in the IL-Y administration.(4)In both models,IL-Y administrated recipients were induced severe clinical GVHD with significant pathology damage compared with the control,including inflammatory cell infiltration,cellular swelling and hepatic small sinus space in the liver;glomerular structure destruction and crescent formation in the kidney;dermal fibrosis,deposition of collagen fiber and consumption of subcutaneous fat in the skin;destroyed bronchi and alveoli structure in the lung;the damage of hair follicle and small intestinal villus;the inflammatory cell infiltration and glands atrophy in the salivary glands,at 8 weeks after transplantation.(5)Further study indicated that the percentages and number of effector(CD44+CD62L-)CD4+T cells and effector(CD44+CD62L-)CD8+T cells were increased in IL-Y group whereas naive(CD44-CD62L+)CD4+Tcells and naive(CD44-CD62L+)CD8+T cells were significantly decreased.At the same time,IL-Y administration had significantly lower frequency Treg.Although the percentage and number of Tfh cells in the IL-Y group were not significantly increased compared with the control,the proportion of ICOS+Tfh cells was significantly increased.In addition,it was found that the proportion and number of total B cells,germinal center B cells and plasma cells in spleen lymphocytes of the IL-Y group were significantly increased,with higher levels of MHC-II and CD86 co-stimulatory molecular.At the same time,germinal center B cells,marginal zone B cell and plasma cells in lung lymphocytes of the IL-Y group were also significantly enhanced.Moreover,intracellular staining showed that IL-Y increased the percentage and number of TNF-a secreted CD4+T cells and TNF-a secreted CD8+T cells in the spleen and liver lymphocyte.(6)CBA detection of 8-week serum after transplantation found that the levels of TNF-?were significantly increased in IL-Y group.Conclusion:Our study demonstrates that IL-Y can aggravate the development of cGVHD.The main mechanism is to promote the proliferation of effector T cells,promote the secretion of TNF-a by T cells,promote the proliferation of ICOS+Tfh cells,inhibit Tregs augmentation,and enhance the differentiation of B cells into GC B-cells and plasma cells as well as the ability to increase the production of high-affinity autoantibodies by B cells,and so on work together.