Combined Donor Specific Transfusion (DST) And Anti-CD154 Therapy Achieves Airway Allograft Tolerance: Microarray-based Gene Expression Study

PartⅠThe heterotopic tracheal allograft as an animal model ofobliterative bronchiolitis in mouseBackground and Objective: Obliterative bronchiolitis(OB)is the most important cause ofgraft dysfunction following lung transplantation. Time cost and technique requirement limitthe application of animal models of in situ lung transplant in the research of OB. Clinicalspecimen especially special stage is usually beyond our reach. We established a new mousemodel of chronic allograft rejection using heterotopically transplanted trachea to provide a novel modeling method for OB research.Methods: In experimental group, 9 BALB / c mi ce served as donors, and C57BL / 6 asrecipients, while in control group, 9 BALB / c mice served as donor and recipientsrespectively. The trachea and bronchus of BALB / c mice were harvested as donor andthen were heterotopically transplanted into a subcutaneous pouch in the dorsal surface ofrecipient mouse. All grafts were harvested 5,15,25 days after transplantation, paraffinimbedding, and stained with hematoxylin and eosin (HE) to observe the histopathology ofthe grafts. OB incidence in two groups was compared.Results: 18 mice all survived with no infection. In HE staining, the experimental groupshowed the histopathology of OB; the transplanted tracheas were obliterative, inflammatorycell infiltrated, tracheal epithelium completely shed, and fibroblasts predominantlyproliferated. While in the control group, there was no apparent histological change innormal trachea. The incidence of OB in the experimental group was significantly higherthanthe control group p=0.000).Conclusions: Heterotopic tracheal allografting in mouse as a model of chronic allograftrejection after lung transplantation displays many advantages. It provides all alternation forstudying chronic allograft rejection after lung transplantation.PartⅡGene expression profile of obliterative airway in a mouseheterotopic tracheal transplantation modelBackground and Objective Obliterative bronchiolitis is the leading cause of morbidityand late mortality after lung transplantation. However, different immunosuppressionstrategies are of unproven value till now. The molecular mechanisms of obliterativebronchiolitis following lung transplantation remains unclear. The genome-wide screening capability of microarray analysis suggests the potential to identify new surrogate markersfor OB and to better understand its underlying mechanisms. We analyzed the geneexpression profile in obliterative bronchiolitis following lung transplantation in a mouseheterotopic tracheal transplantation model to provide basis for prevention.Methods Heterotopic tracheal transplantation was performed using BALB/c as donors andBALB/c or C57BL/6 as recipients. Tracheal grafts were harvested on day 5, 15, 25 andpathological changes were analyzed in one half over time, and the other half were subjectedto microarray analyses to examine changes in gene expression patterns at day 15 (preOBperiod). Differential expression of selected genes was confirmed by Real-time quantitativeor semi quantitative RT-PCR.Results Compared with the isograft control, expression levels of 422 genes wereup-regulated in the allograft. A large proportion of the up-regulated genes in thistransplant-associated profile were functionally linked with cell survival and differentiationincluding ubiquitin-proteasome pathway, transforming growth factorβand other concernedcell cycles and cell differentiatation.Conclusions OB presented as a multistage, ongoing and nonequilibrium process. Theanalyses with much more genes associated with cell differentiation and less withimmunological cells suggest the development of new therapeutic strategies perhaps requiresa paradigm shift away from alloimmunity and inflammation toward one of tissueremodeling and myofibroblast transdifferentiation.PartⅢCombined donor specific transfusion and anti-CD154 therapy achievesairway allograft tolerance: microarray-based gene expression studyBackground and Objective The induction of specific tolerance would be the ultimateachievement and solution to chronic rejection in transplant immunology. Although numerous studies have demonstrated donor specific transfusion and anti-CD154 inducedtolerance after lung, heart, liver, islet transplantation, the mechanisms underlying thistolerance protocol esp. intragraft changes that occur during the early stages of toleranceinduction have not been characterized. This study aimed to define the molecular mechanismduring induction of obliterative bronchiolitis following heterotopic tracheal transplantationtolerance using microarrays.Methods Tolerance induction was achieved in tracheal allografts from BALB/c toC57BL/6 mice by donor specific transfusion of splenocytes intravenously andintraperitoneal injection of anti-CD154 mAbs, We investigated gene expression analysis intolerant murine heterotopic tracheal allografts by means of gene microarrays. Differentialexpression of selected genes was confirmed by Real-time quantitative RT-PCR.Results Comparative analysis between DST/anti-CD154 protocol induced tolerant andrejected grafts revealed many genes were specifically downregulated in the tolerantallografts including RAP1 pathway, CD40/CD40L concerned T cell surface molecules andmany profibrotic genes which were upregulated in the rejecting allografts. In the tolerantallografts there were also marked expressions of a number of genes for proinflammatoryfactors.Conclusions These results suggest that a vital role for RAP1 pathway, CD40/CD40Lconcerned T cell surface molecules and many profibrotic genes in inducing airwaytransplant tolerance in this model. Determining the mechanisms whereby these and manyother genes remains to be elucidated contributes to transplant tolerance may help in thedevelopment of new strategies to improve long-term graft outcome. Also, these resultsprovide evidence immunological tolerance can be induced in the presence of prominentproinflammatory gene expression.