Bronchus Epithelium Cells Production Of Galectin-9Induced By IFN-γ Contributes To Immune Protection In Lung After Hematopoietic Stem Cell Transplantation

Object: The immune tolerance of lung in Acute Graft-versus-host Disease was knownwithout certainty. Establish relative immune tolerance for lung in a murine model ofaGVHD induced by allogeneic bone marrow transplantation (allo-BMT) for providingexperimental model and study of immune tolerance in the lung after allo-BMT.Methods: Establish C57BL/6J→B6D2F1, C57BL/6J→BALB/C (spleen cells: bonemarrow cells=2:1) and C57BL/6J→BALB/C (spleen cells: bone marrow cells=4:1) modelsof aGVHD, and C57BL/6J→C57BL/6J model was used as control. The clinical scores,survival, the numbers of peripheral white blood cells, and chimeras were observed;pathological injuries were compared between the lung and traditional target organs (liver,small intestines, skin); the expression of IFN-γ in lung after transplantation wasinvestigated by ELISA. Result: Allogeneic HSCT mice had enhanced survival in42post-transplantation(surivival:20%) and recipients of allogeneic grafts showed classical symptoms andhistological injury, and pathological changes of lung was not as serious as liver, smallintestine, skin at day28after transplantation; syngeneic mice all survived at day42aftertransplantation, without GVHD symptoms and pathological changes. In MHC-disparatemice (2:1and4:1groups) died significantly faster at a median of12days aftertransplantation, with severe changes of clinical symptoms and pathology of classical organs,and MHC-disparate mice (4:1groups) had developed severe interstitial pneumonitis; Thenumbers of peripheral white blood cells in dead and moribund mice was (1.69±0.12)×109/L, and donor–derived Y chromosome was detected at day30and day60aftertransplantation. the mean IFN-γ concentration in the lung of allogeneic HSCT mice wasobviously increased in the first and second week after transplantation, and the peakconcentration was (188.6±12.7) pg/ml; the IFN-γ concentration of MHC-disparate mice(2:1and4:1groups) were slightly increased in the first and second week aftertransplantation, and there were statistically significant difference with allogeneic group (P<0.05).Conclusion: there was relative immune tolerance for lung in a murine model of aGVHDinduced by hematopoietic stem cell transplantation, and it was associated with the infusionof lymphocytes, the degree of their MHC-similarity, IFN-gamma concentration of lungsafter transplantation. Object: the aim of this study was to explore the expression and function of Gal-9in anIFN-gamma-associated model of acute GVHD.Methods: Establish C57BL/6J→B6D2F1, IFN-γ-/-→B6D2F1and C57BL/6J→IFN-γR-/-models of aGVHD, and C57BL/6J→C57BL/6J model was used as control. The clinicalscores, survival, pathological changes, total cell counts and protein BALF, and lung wet/dryratio were observed. The cell apoptosis in the lung after transplantation at day7wasinvestigated by TUNEL assay, and T cell subset and cytokines in serum were analyzed byFlow cytometry and ELISA. The expression of Gal-9in lung and traditional target organs(liver, small intestine, and skin) were compared by immunohistochemistry,immunofluorescence, ELISA assay and Western blot. That whether Gal-9play specificeffect on IPS induced by IFN-γ-/-signaling was further determined. Targeted sequentiallyGal-9recombinant protein to the airways in recipients of IFN-γ-/-grafts or IFN-γR-/-recipients of wt grafts, and survival and lung pathology were observed, and cytokines ofIFN-gamma, TNF-a, IL-4and IL-17were detected.Result: Syngeneic mice all survived at day42after transplantation, without GVHDsymptoms and pathological changes; Allogeneic wild type mice had enhanced survival in24post-transplantation (survival＞50%) and pathological changes of lung was not asserious as liver, small intestine, skin at day28after transplantation; the recipients ofIFN-γ-/-grafts and IFN-γR-/-recipients of allogeneic grafts all died within day14aftertransplantation, with severe pathological changes of lung; recipients of IFN-γ-/-grafts or IFN-γR-/-recipients of wt grafts resulted in a significant increasing of BALF total cells,total protein and lung wet/dry ratio at weeks1after SCT compared to wild typecontrols;The number of apoptotic TUNEL-positive cells increased in recipient of wt graftsin the early time, and diminished in recipients of IFN-γ-/-grafts or IFN-γR-/-recipients of wtgrafts; The numbers of donor CD4+and CD8+T cells in recipients of IFN-γ-/-donor orIFN-γR-/-recipients of wt grafts were significantly increased on day7after transplantation.Cytokine analysis of sera also revealed significantly greater IL-4levels in recipients ofIFN-γ-/-donor, and systemic IFN-γ and IL-17production were increased in IFN-γ R-/-recipient; the high expression of Gal-9in the lung of allogeneic mice was observed byImmunohistochemistry, immunofluorescence, ELISA assay and Western blot.Gal-9recombinant protein significantly enhanced survival and lung pathology in recipients ofIFN-γ-/-grafts or IFN-γR-/-recipients of wt grafts; recipients of IFN-γ-/-grafts with Gal-9recombinant protein showed less concentration of IL-4and IL-17, and concentration ofIFN-γ and IL-17were decreased in IFN-γR-/-recipients of wt grafts.Conclusion: IFN-γ-associated Gal-9in the lung after transplantation promoted T-cellapoptosis and decreased cytokine secretion after hematopoietic stem cell transplantation,providing potential evidence in preventing lung injury after transplantation. Object: To explore the expression of Gal-9induced by IFN-γ in bronchus epithelium cellsand the protective effect of Gal-9promoted T-cell apoptosis.Methods: Primary human bronchial epithelial cells (NHBECs) were acquired frombronchial brushing from the normal trachea and central airways, and immunofluorescentstaining of CK-18, CK-19and vimentin was performed on formalin-fixed cells to assesspurity of NHBECs. When the cells were grown to80%confluence, NHBECs werestimulated with IFN-γ from0.08to50ng/ml for24h, or stimulated with10ng/ml for0,3,6,12,24, and48h, and the expression of Gal-9in HBEs was analyzed by Real-time PCRand Western blot. Purified T cells by CD3magnetic bead separation and autoMACS systemwere cultured in RPMI1640containing10%FCS and were stimulated for12h with ConAand recombinant growth factor IFN-γ, or IL-4, or IL-17. The number of T cells subsets andCD69and Tim-3was detected by Flow cytometry. T cells were co-cultured with HBE afterstimulation with10ng/ml IFN-γ for24h. Neutralizing monoclonal antibody against humanTim-3and Transwell system were used, and T cells apoptosis was determined by flowcytometry. The activation of Caspase family induced by T-cell apoptosis was detected byspectral photomer, and a caspase3inhibitor (z-DEVD-fmk) or a caspase4inhibitor(Ac-LEVD-cho) was added into the culture.Result: CK18and CK19were all expressed in cytoplasm, and the positive expressionswere present more than90%, vimentin was no expression in those NHBECs. The Gal-9expression stimulated by IFN-γ was enhanced in a concentration-and time-dependent manner, and Gal-9mRNA and protein reached a maximal level at24h after the stimulationwith10ng/ml IFN-γ. The number of CD3+cells by CD3magnetic bead separation was86.2±5.21%and the number of CD69+positive cells with ConA-treatment was(72.11±3.140)%by flow cytometry. The number of Th1, Th2, and Th17cells afterstimulation with recombinant growth factor IFN-γ, or IL-4, or IL-17was (71.3±2.09)%,(62.3±3.51)%and (42.3±6.12)%; Activated-T cells were cocultured with HBE afterstimulation with10ng/ml IFN-γ for24h and the level of apoptosis increased significantly(18.5±1.46%) compared with1.9±0.12%, and the inhibition of T cells apoptosis is partlyindependently of anti-Tim-3pathway (8.1±0.33%). Apoptosis analysis was performed in24-well transmembrane culture, and the percentage of apoptosis was (12.6±2.31)%. Theactivation of Caspase-3and Caspase-4was enhanced in T cells were cocultured with HBEafter stimulation with10ng/ml IFN-γ for24h. Caspase3inhibitor exhibited significantinhibitory effect on HBE-derived Gal-9-induced T cells apoptosis with (13.4±2.13)%, butCaspase-4inhibitor exhibited no or only weak inhibitory activity on T cells apoptosis.Conclusion: Up-regulation of Gal-9in bronchus epithelium cells stimulated by IFN-γcontributes to apoptosis of Th1/Th17cells in both Tim-3-dependent and-independentpathways, via activation of caspase-3.