The Role Of Tim-3/Galectin-9Pathway In Acute Allograft Rejection After Lung Transplantation

Part IIncreased expression of Tim-3and its ligand Galectin-9in rat allografts during acute rejection episodesBackground:Lung transplantation has recently become the ultimate and effective therapeutic option for several end-stage lung diseases. Despite improvements in operative techniques and the advances in immunosuppressants, the success of clinical lung transplantation is still poor in comparison to other solid organ transplants. Acute rejection (AR) episodes are a significant cause of morbidity for the transplant patient. Therefore, monitoring and diagnosis of AR at the early stage and performing effective treatment, it is crucial for improving the prognosis of patients suffering from lung transplant. Recently, a large number of papers suggested that the expression of Tim-3and its ligand Galectin-9in human renal allograft rejection was significantly increased, and indicated that they may be used as possible biomarks for renal allograft rejection.Objective: To detect the expression of Tim-3and its ligand Galectin-9in rat allografts during AR episodes after lung transplantation, and to explore the impact on AR occurrence after lung transplantation.Methods:Using rat orthotopic left lung transplantation model, and lung transplantation was performed between rat Lewis to Lewis for syngrafts, and rat Fisher344to Lewis for allografts. All recipient rats did not receive any immunosuppressive agents. Recipients were sacrificed and grafts were harvested at day3,7, or10after transplantation. The grade of acute rejection was histopathologically evaluated. T lymphocyte infiltrating in lung grafts were detected by immunohistochemistry. Surface expression of Tim-3on T lymphocytes infiltrating in rat lung grafts was examined by double staining Immunofluorescence. The intragraft gene expression levels of Tim-3, Galectin-9and several related cytokines were quantitatively measured by real-time polymerase chain reaction (RT-PCR). Meanwhile, correlation analysis was performed.Results: The severity of acute rejection in allografts was gradually aggravated with time. At the same time, no rejection or even minimal rejection was observed in syngrafts. A decreased CD4/CD8ratio was associated with the severity of acute rejection occurring. Then, Tim-3and its ligand Galectin-9were markedly up-regulated in allografts by immunohistochemistry (P<0.05), and Tim-3expression on CD4+and CD8+T cells in allografts was increased (P<0.05). All molecules studied were markedly up-regulated in allografts compared with syngrafts by RT-PCR (P<0.05). A positive correlation was found between Tim-3and IFN-y mRNA levels, and IL-17mRNA levels. Also, there was a correlation between Tim-3and Galectin-9. In addition, there was a correlation of Galectin-9mRNA with IFN-y mRNA, even with the severity of rejection grade.Conclusion:Our study firstly showed that the expression of Tim-3and its ligand Galectin-9in allografts was significantly enhanced during AR episodes, and might play an important role in the pathogenesis of a rat lung transplant rejection, implying a new possible strategy for detecting acute pulmonary allograft rejection. Part IISynthesis of recombinant human Galectin-9in vitro and detection of the impact on T lymphocytes from lung transplant miceBackground: Galactoside binding lectin-9(Galectin-9) is a sugar-binding protein and a member of galectin family, which has a wide distribution in tissues and plays an important role in mediating cell differentiation, apoptosis, adhesion, the inter-cell aggregation, tumor metastasis regulation and inflammatory responses. In recent years, researchers performed experimental relevant research by synthesis of recombinant Galectin-9protein in vitro and had a deeper understanding of its biological properties.Objective:Recombinant human Galectin-9protein in vitro was synthesized by the whole gene synthesis and the impact on T lymphocytes from lung transplant recipients was detected in this study, which was to lay the foundation for further study of its biological function.Methods:In this study, human Galectin-9gene was optimized by codon optimization software, and optimized Galectin-9gene sequences were synthesized by the whole gene synthesis. Then the recombinant expression plasmid pET28a-Galectin-9was constructed by using genetic engineering techniques, following by DNA sequence analysis and identification. The recombinant plasmid transfected competent E. coli BL21(DE3), and protein expression vector was constructed. Expression, purification and identification of human recombinant Galectin-9protein antigen activity were carried out. Mouse model of orthotopic left lung transplantation was performed between BALB/c to C57BL/6for allografs, and spleen cells were harvested at day3after transplantation, and T lymphocytes were isolated and cultured for72hours in vitro within different concentrations of human recombinant Galectin-9protein. Proliferation of T lymphocytes was detected by MTT assay, and the levels of inflammatory cytokines in the cell supernatant were measured by ELISA.Results:Human Galectin-9gene sequences were synthesized by the whole gene synthesis, and recombinant plasmid pET28a-Galectin-9was successfully constructed, which was correct by restriction analysis and DNA sequencing analysis. The recombinant plasmid was transformed into the expression host, and the product was induced and purified. SDS-PAGE and Western Blotting analysis showed that the expression of protein was recombinant human Galectin-9protein and of antigenic activity. Proliferation of T lymphocytes was significantly inhibited by recombinant human Galectin-9protein and dose-dependent inhibition was found in vitro. The inflammatory cytokines, IFN-y and IL-17secreted from T lymphocytes were significantly reduced (P<0.05) and dose-dependent inhibition was also found, while the secretion of IL-4was not obvious (P>0.05).Conclusion:The prokaryotic expression vector pET28a-Galectin-9containing human Galectin-9sequences was successfully constructed, induced and purified, and the purity of the recombinant human Galectin-9protein was>90%in this article. The significant inhibition of sensitized T lymphocyte proliferation and the reduced release of inflammatory cytokines were found in vitro, which laid a foundation for further relevant studies in vivo. Part IIIActivation of Tim-3/Galectin-9pathway attenuates acute allograft rejection after lung transplantation in miceBackground:Current studies showed that Tim-3preferentially expressed on the surface of Thl and Th17cells and played an important role in autoimmune and alloimmune diseases. Galectin-9was identified as the Tim-3ligand and thereby established Tim-3/Galectin-9pathway as an important regulator of Thl immunity as well as Thl7. In recent years, domestic and foreign researchers employed the recombinant Galectin-9protein to activate Tim-3/Galectin-9pathway in animal models of autoimmune diseases and alloimmune diseases, and protective effects were detected, but whether the similar protective effect for lung allograft has not yet been reported.Objective: To investigate the effect of recombinant human Galectin-9protein on acute rejection grade of recipient mice suffering from allogeneic lung transplantation and explore its immunomodulating role in a mouse allogeneic lung transplantation model.Methods:Murine orthotopic left lung allograft transplantation model from BALB/c to C57BL/6was established. The study was divided into the experimental group and control group. Recipients in experimental group were administered with recombinant human Galectin-9protein for7days since day1post-transplant while recipients in control group were treated with PBS. Allografts were obtained at day7from two groups after transplantation, respectively. Pathological evaluation of allograft rejection was performed to estimate the severity of rejection on day7post-transplant. Tim-3+, CD4+and CD8+T cells infiltrating in allografts were analyzed by immunohistochemistry. Real-time quantitative PCR was utilized to detect related gene mRNA expression in allograft lungs. The number of apoptotic cells in allografts was detected by TUNNEL assay.Results:Compared with the control group, histological examination showed that the severity of rejection in treated group with recombinant human Galectin-9protein was significantly reduced (P<0.05). Immunohistochemistrical findings suggested that the infiltration of CD4+and CD8+T cells and Tim-3+cells in allografts were significantly decreased (P<0.05). Administration of recombinant human Galectin-9decreased the expression of Tim-3, IFN-y and IL-17mRNA in allografts markedly, while the levels of Foxp3and Galectin-9were significantly elevated (P<0.05). Furthermore, the number of apoptotic cells in the experimental group by apoptosis detection was significantly increased (P<0.05).Conclusion: Activation of Tim-3/Galectin-9pathway by administration of recombinant human Galectin-9protein significantly reduced acute allograft rejection in mice with allogeneic lung transplant. The mechanism was to decrease the infiltration of T lymphocytes, to induce Tim-3+cells apoptosis, to reduce the release of inflammatory cytokines and to down-regulate Thl and Th17immune responses. It might be a new therapeutic strategy for further study on anti-rejection after lung transplantation.