Bone Marrow Mesenchymal Stem Cells Can Protect Murine Lung Allograft From Immune Rejection

PartⅠEstablish Mice Orthotopic Left Lung Transplantation Model (First Report in China)Objective To investigate how to establish stable mice orthotopic left lung transplant model.Methods Male FVB mice were randomly divided into recipient and donor. Three-cuff technique which was established by our group was used to establish mice orthotopic left lung transplant model.Results More than 90% of the recipient animals survived after surgery. Cold ischemia time is 35.6±5.9min, warm ischemia time is 25.3±7.2min, donor lung back up time is 21±5.6min, and the whole surgery time is 85±15min. The long term survival time achieved more than 30 days with functional lung graft. Micro CT shows clear left lung field one month after surgery, left bronchus cuff is still open. The artery blood analysis shows no difference before and after clipping right lung helium. Histologically, the lung graft appeared very similar to the right lung one month after surgery. The pulmonary alveolar is aerated well.Conclusion Based on our experience of the rats orthotopic left lung transplantation model, through practicing, we independently established successful mice orthotopic left lung transplantation model which is the first time report in China. Compared to the method established by the other country, our method is easier to perform and more stable. This will benefit the basic research related to lung transplant a lot. PartⅡNon-invasive evaluation of lung allograft acute rejection and monitoring the effect of cyclosporine A treatment by using bioluminescence imagingObjectives we investigated the possibility of using bioluminescence imaging(BLI) to non-invasively detect the murine lung allograft rejection and evaluate the immunosuppressive effect of cyclosporine.Background Non-invasive surveillance for allograft rejection has great potential in clinical application. Currently, the standard diagnosing and staging rejection after organ transplantation is biopsy, which is not only invasive but also prone to sampling errors. The motivation of this study is to investigate the possibility of using bioluminescence imaging for non-invasively detecting the lung allograft rejection and evaluating Cyclosporine A immune suppressive effect.Methods and Results Mice orthotopic left lung transplantation model was established by using transgeneic mice L2G85(H-2q) which have Luciferase-eGFP reporter gene as donor and either Balb/c(H-2d) or FVB(H-2q) as recipients. Bioluminescence imaging signal emitting from the transplanted lung graft was measured at regular time point until graft lost was present. At different time point, the animals were sacrificed; lung grafts were explanted for HE staining. All the lung grafts were rejected within 7 days after transplant. The BLI signal strength reduced to 1.35±0.1% of day 1 baseline value at day 7. At 2 weeks after surgery, the bioluminescence signal strength decreased to the background level. However, in syngeneic transplant group, the BLI signal was 89.6±4.5% of day 1 value at two week time point. We use cyclosporine A to treat allogeneic lung transplant recipients at different dose, 10mg/kg/d,20mg/kg/d, and 30mg/kg/d. We found that at day 28 after transplant, the BLI signal in the 20mg/kg/d group is. much stronger than 10mg/kg/d compared to the baseline day one value (41.05±4% VS 14.87±3%, P<0.05), but there is no significant difference of the BLI signal strength compared to the baseline day one value between 20mg/kg/d and 30mg/kg/d group (41.05±4% VS 44.49±4%,P>0.05).Conclusion Bioluminescence imaging technique is a reliable method for non-invasively longitudinal evaluation of lung allograft rejection and monitoring cyclosporine immune suppressive effect. This technique may have great promise in the clinic for establishing individual immune-suppressive strategy in the future. Part III Bone marrow mesenchymal stem cells can protect murine lung allograft from immune rejectionObjectives Bone marrow mesenchymal stem cells(BMSC) has immune modulatory effect. After systematic administration, the cells will be trapped in the lung. Here we investigated if the cells can protect lung allograft from immune rejection after being trapped in the lung and the possible mechanism.Method The whole experiment was divided into two parts. First part, to monitor bone marrow mesenchymal stem cell engraftment into the lung; Severe Combined Immunodeficiency mice were divided into normal group, sham group which has been performed open-close chest surgery and lung transplant group which has been performed mice orthotopic left lung transplantation surgery.1X106 BMSC from L2G85 source were injected through tail vein into these mice The cells survival was monitored by bioluminescence imaging(n=5/group). Second part, to monitor the effect of BMSC on the immune rejection against the lung allograft; Mice orthotopic left lung transplant model was established by using L2G85 as donor and Balb/c as recipient. All the recipient animals were randomly divided into three groups:control, bone marrow mesenchymal stem cells treatment, and cyclosporine treatment group(n=20/group). The control group received PBS 100ul tail vein injection every day for total 5 days; BMSC treatment group received BMSC which were from FVB source,1X106/d, total 5 days; The cyclosporine treatment group received cyclosporine 20mg/kg/d intraperitoneal injection until the experiment end time point. Some of the recipient animals were sacrificed at day 7 after surgery. The lung grafts were removed and graft infiltration lymphocytes were isolated. The percentages of CD4+FOXP3+Treg and CD8+IFN-y+Tc were assessed by flow cytometry assay. The histological changes were assessed by HE staining. The splenocytes were obtained for enzyme-linked immunosorbent spot assay to test recipient immune response type. The remaining animals were monitored bioluminescence signal emitted from the donor lung allograft at regular time point until day 21.Results The first experiment, in normal group, sham group, and lung transplant group, bioluminescence imaging(BLI) shows that bone marrow mesenchymal stem cells were trapped in the lung earlier time after being injected into the animal through tail vein. The BLI signal strength is comparative between left lung and right lung. After 24h, the BLI signal strength reduced tremendously among all the group, in which, normal group reduced the most, lung transplantation group right lung and sham group reduced the second, and lung transplantation group left lung reduced the least. At day 3, BLI signal cannot be detected in all the animals except the lung transplant group left lung; The second part experiment, at day 7 after transplant, compared to the baseline BLI signal strength, there is no significant difference between bone marrow mesenchymal stem cells treatment group and cyclosporine treatment group (66.7±8% vs 63.9±4.5%) P>0.05. The BLI singal in MSC group is stronger than in PBS group compared to the baseline. The difference is significant (66.7±8% vs 1.62±0.4%) P<0.05. At day 14 after transplant, the BLI signal decreased to the background level in the PBS group, however, the relative signal strength compared to the baseline in MSC treatment group is 42.3±3.5%, and the cyclosporine treatment group is 46.6±5.4%. There is no significant difference between MSC treatment group and the cyclosporine treatment group, P>0.05. When compare these two groups to PBS group, the difference is significant, P<0.05. At day 21 after surgery, the BLI signal in MSC treatment group decreased to background level which is 0.21±.5% compared to the baseline strength. However, the relativity compared to baseline in cyclosporine treatment group is 41.64±3.5%. The difference between these two groups is significant, P<0.05. The histological observations of lung graft at day 7 after surgery showed that in the MSC treatment group and the cyclosporine treatment group, the lung tissue appears normal and the pulmonary alveolar were aerated well, but the PBS group showed alveolar collapse, alveolar septal thickening, interstitial lymphocyte infiltration in the lung. In the graft infiltrating lymphocytes, the percentage of CD4+FOXP3+Treg in MSC treatment group is much higher than in PBS group (32.04±2.5% VS 15.5±1.7%) P<0.05. The percentage of CD8+IFN-γ+ in MSC treatment group is lower than in PBS group (27.6±1.4% VS 55.2±6.7%) P<0.05. ELISPOT results showed that IFN-γspots are 404±35/106cell in PBS group,200±23/106 cell in MSC group, and 196±26/106 cell in cyclosporine group. The IL-4 spots are 38.7±5.4/106cell in PBS group,75±10/106cell in MSC group, and 18±2.9/106cell in cyclosporine group.Conclusion In this experiment, we have shown that at earlier time point, bone marrow mesenchymal stem cells were trapped in the lung after systematically administration. After 24h, the bioluminescence signal emitted from the cells decreased tremendously; however, mechanical ventilation and lung transplantation can improve the cells engraftment into the lung. Multiple dose administration of bone marrow mesenchymal stem cells can protect the lung allograft from immune rejection. The mechanism is related to T regulatory cells induction and Thl to Th2 immune response deviation. These research results have great application potential in clinic organ transplantation.