Evaluation Of Innate Immune Molecules MIF And TLR5 As Monitoring Targets For Allograft Rejection

Background and ObjectivesOrgan transplantation is an important means of managing end-organ failure. However, acute allograft rejection remains a major complication after transplantation. Thus how to diagnose the acute rejection timely is still one of the hot topics of in this field. Traditional techniques to predict rejection include clinical symptoms, hematological indexes, immunopathology, genomics and imaging techniques et al. Biopsies were considered to be the gold standard for monitoring allograft well-being after transplantation. However, biopsies are invasive, associated with morbidity if performed on a routine basis and can potentially miss focal rejection. Because of the widely-influence of the transplantation rejection and the complex immunological mechanism, now it is hard to find an accepted, no invasive immunological indexe with high sensitivity and specificity. Therefore, how to find a new noninvasive index that can monitor the transplant rejection effectively and make the timely diagnosis and differential diagnosis before the transplant organ dysfunction has become the hot topic in transplantation immunology.Molecular imaging is a newly emerging science; it integrates the principles of cell and molecular biology, immunology, nuclear medicine and diagnostic imaging. The application of molecular imaging technology in studying the transplantation immunological phenomenon will become one of the important methods to monitor the transplant rejection. In addition, the molecular imaging informations are important for the individualized application, efficacy and the risk factors evaluation of immunosuppressant after transplantation.Transplantation rejection is a complicated process.Numerous immune molecules participate in it. The innate immune system is an important part of the immune system. The innate immune system can not only play the nonspecific devouring and scavenging effect, but also can regulate the adaptive immune system. At different stages of organ transplantation, there is a complex and close connection between the innate immune system and the adaptive immune system.Recently, cytokines infiltrated in the grafts and their specific antibodies are becoming the targets of molecular imaging for transplantation rejection, and nuclear medicine imaging with radioisotope-labeled cytokine or a related antibody for detection of transplantation rejection has been reported, such as 123I or 111In labeled major MHC classⅡmolecules and 99mTc labeled MCP-1. However, non-specificity, a side effect or slow clearance affected these agents' application in clinic.Macrophage migration inhibitory factor (MIF) was originally identified as a kind of lymphokine that recruits the macrophages to inflamed sites and was involved in cell-mediated immunity and delayed type hypersensitivity (DTH). Recently, MIF was found to be one of the major cytokines involved in the allograft rejection. In addition, the important role of MIF in the development of acute GVHD in a mouse model of allogeneic stem cell transplantation has also been reported. In "Part one" of this dissertation, we choose MIF as a new target of molecular imaging in allograft rejection.The aim of this study was to better understand the relationship between MIF and acute skin allograft rejection, then clarify whether 131I-anti-MIF mAb may enable non-invasive evaluation of acute rejection specifically.Besides of this, accurately evaluation of the immunosuppressive curative effect and monitoring the recipients' immune status is another key objective for transplant monitoring. Under the guidance of transplant monitoring, we can realize the individualized application of the immunosuppressant. However, since the immunosuppressant could inhibit the proliferation of T cells, B cell and the secretion of cytokines, so if we still use the cytokine as the molecular imaging agent, it can not evaluate the receptor's immunity state accurately. Thus finding a new immune molecule will be useful for monitoring the immunosuppressive curative effect after transplantation in clinic.TLRs can specifically identify the pathogen-associated molecular patterns (PAMPs), and play important roles not only in the activation of innate immunity, but also in the induction and regulation of adaptive immunity. So the TLRs can also be regard as an important bridge between innate immunity and adaptive immunity. TLRs can be activated by autologous antigens, allogenic antigens and heterologous antigens. The latest study in 2008 showed that existence of natural Tregs was necessary for the long-term survival of heart and skin allograft in mice, the CpG-specific activator of TLR9 can inhibit the function of Tregs and promote the differentiation of Thl effective T cells to inhibit the survival of the allograft. Activation of TLR7 can promote the allograft rejection; activation of TLR4 can inhibit the long-term graft of heart survival, prevented the accumulation of the CD4+/Foxp3+Treg within grafts. So we propose that the activation of TLRs may be critical in the allograft rejection.It was found recently that TLR5 is the only negative regulatory TLRs which may be directly or indirectly involved in the process of autoimmune diseases. TLR5 can be activated by bacterial Flagellin, a specific ligand to TLR5, and mediate the immune response by stimulating the production of TNF-a, IL-Iβ, IL-8 and other pro-inflammatory cytokine through the activation of NF-κB (nuclear transcription factor-KB). Previous studies showed that TLR5 was found selectively high-expressed on Tregs, and Flagellin can enhance the inhibition function of Treg's on effective T cells. Recently a paper in 2008 reported that Flagellin can protect the body from damage of chemicals, bacteria, viruses and radiation through binding to TLR5.In view of the important role of TLR5 in induction immune tolerance and its regulatory effect on the Treg, we propose that TLR5 may be another important immune molecule in allograft rejection. In "Part two" of this dissertation, we studied the expression of TLR5 dynamically during the skin allograft rejection progress; detected the effect of Flagellin on the survival time and the expression of TLR5, and then revealed the regulatory role of the immunosuppressant Rapamycin (Rapa) on the expression of TLR5. The aim of this study was to clarify the relationship between TLR5 and allograft rejection process, then evaluated whether TLR5 can be a new noninvasive index for monitoring the allograft rejection. Methods1. The expression of MIF in allograft model:The skin allotransplantation and isotransplantation mice were respectively sacrificed at 1,7 and 14 days after transplantation. The expression of MIF mRNA in grafts was quantified by RT-PCR. Meaningwhile the protein expression was analyzed by immunohistochemical staining after transplantation.2. The preparation and the biology active identification of anti-MIF McAb:anti-MIF McAb was made with hybridoma technology whose specificity and biological activities were identified with Western blotting, ELISA and MMI.3. The molecular imaging of 131I-anti-MIF McAb in skin allotransplantation mice: Radioiodination of the anti-MIF McAb and control IgG was performed by the Iodogen method, The skin allotrantinsplantation and isotransplantation mice were injected intravenously via the tail vein with a PBS solution (100μL) of I31I-anti-MIF McAb (185 kBq) respectively. At each point of time (1,7 and 14 days after transplantation), five mice of each group were sacrificed at 24 h after injection, blood, grafts, the recipient normal skins of opposite side and the organs were weighed and counted in the gamma counter. Then the%ID/g and the T/NT (target/nontarget) ratios of each group were calculated. The skin allotransplantation mice were injected intravenously via the tail vein with a PBS solution (100μL) of 131I-anti-MIF mAb or 131I-IgG (3.7 MBq) respectively. At each point of time (1,7 and 14 days after transplantation), five mice of each group were imaged by whole-body autoradiography. Images were performed at 24 h after injection.4. The expression of TLR5 in allograft model:The skin allotransplantation and isotransplantation mice were respectively sacrificed at 1,7,14and 21 days after transplantation. The expression of TLR5 mRNA in grafts was quantified by RT-PCR. Then the protein expression was analyzed by immunohistochemical staining.5. The regulatory role of Rapa on the expression of TLR5:The skin allotransplantation mice were intraperitoneally injected with Rapa (2.5mg/kg-d) 1 time/day (Rapa Group) or NS (Control Group) respectively. The grafts from two groups were collected at 1,7,14 and 21 days after transplantation. The expression of TLR5 mRNA in grafts was quantified by RT-PCR. The T cells collected on day 14 after transplantation were treated with different concentrations of Rapa in different times to study the effect of Rapa on the secretion of IL-10 by ELISA.6. The extraction and purification of Flagellin:Flagellin was extracted and purified from the Salmonella Paratyphoid A.Then the purity and the specificity of purified flagellin was identified by Western Blotting.7. The regulatory role of Flagellin on the expression of TLR5:The recipient BALB/c mice were injected with NS or Flagellin respectively to observe the function of Flagellin on the expression of TLR5 mRNA and the survival time of the grafts.Then the correlation between them was clarified. The T cells collected on day 14 after transplantation were treated with different concentrations of Flagellin in different times to study the effect of Flagellin on the expression of TLR5.8. Radioiodination of the Flagellin was performed by the Iodogen method.Then we identified its labeling efficiency, radiochemical purity and stability in vitro.Results1. The expression of MIF in allograft model:The distribution of MIF mRNA expression within grafts was assessed by RT-PCR. There was no change in MIF gene expression in day 1 isografts or allografts compared with the normal skin of the opposite side. However, there was a marked increase of MIF mRNA expression in day 7 allografts. With the degree of the allograft rejection intensified, the expression of MIF mRNA kept on increasing, at day 14, it reached its peak. In contrast, MIF mRNA levels in isotransplantation mice barely changed through the experiment. Consistent with the result of RT-PCR, immunohistochemistry staining showed the isografts showing little change in MIF expression compared with the normal skin. In contrast, the allografts showed a marked up-regulation in MIF immunostaining. The expression of MIF was closely relevant to the degree of the rejection.2. Three stable cell lines were obtained and named as 5G2D7,5G2C7 and 5G2 E3 with high specificity and biological activity to MIF.3. The biodistribution of 131I-anti-MIF mAb was studied at each point of time (day 1, 7, and day 14 after transplantation) in the skin allotransplantation and isotransplantation mice.131I-anti-MIF mAb showed a considerably higher uptake and a lower decrease in the allografts compared with the normal skin of the opposite side. There was no statistically significant difference in the radioactivity of 131I-anti-MIF mAb between the isografts and the normal skin. The biodistribution of 131I-anti-MIF mAb in organs indicated this imaging agents were excreted from the kidney or swallowed by reticuloendothelium of liver. There was no difference between the 131I-anti-MIF mAb group and the 131I-IgG group in the early stage of allograft rejection. With the development of the allograft rejection, the T/NT ratio of the 131I-anti-MIF mAb group increased continually, at day 14 it was 17.13, which is the highest. The T/NT ratio of the 131I-IgG group decreased during the progression of rejection. Both of the imaging agents showed a few uptakes in the grafts at day 1. Nevertheless, with the development of the allograft rejection, the 'i-anti-MIF mAb group showed much more clear images than the 131I-IgG, which is in accordance with its high T/NT ratio.4. The expression of TLR5 in allograft model increased gradually after transplantation, and on the day 14 reached its peak. In contrast, TLR5 mRNA levels in isotransplantation mice barely changed through the experiment.5. At 10,14 and 21 days after transpantation, there was a markedly increase of TLR5 mRNA expression within grafts after treated with Rapa. The secretion of IL-10 was increased gradually when treated with Rapa (100 nmol/L) and reached its peak after 72h incubarition.6. Flagellin was successfully extracted and purified from Salmonella Paratyphoid A. It can provide properties of high specificity and high purity.7. Compared with normal saline treatment group, the expression of TLR5 mRNA in Flagellin treatment group increased obviously (p< 0.05) and the survival time of the grafts was extended to 21.57±1.4 days. There was positive relationship between the expression of TLR5 and the survival time of the grafts. In vitro experiment, the expression of TLR5 mRNA markedly increased when treated with Flagellin (100ng/ml) and reached its peak after 6h incubarition.8. The preparation 131I-Flagellin was successfully. The labeling efficiency of 131I-Flagellin was 91.6%. The specific activity of 131I-Flagellin was 35.27 GBq/μmol.When placed in room temperature after 72 hours, it still has good stability and immunological activity.Conclusions and Potential Impact1. MIF expression correlated closely with the gradually increasing allograft rejection.2. We successfully construct the anti-MIF McAb with high specificity and biological activity to MIF.3. The higher T/NT ratio of 131I-anti-MIF mAb and its specificity targeting the grafts provide a possible method for molecular imaging of allograft rejection.4. There was positive relationship between the expression of TLR5 and the degree of the allograft rejection.5. The immunosuppressant Rapa could increase the expression of TLR5 mRNA and up-regulate the secretion of IL-10.6. Successfully extracted and purified Flagellin.7. TLR5's specific ligands Flagellin could increase the expression of TLR5 mRNA and extend the survival time of the grafts.8. The preparation 131I-Flagellin was successfully and it can provide propertie of good stability in vitro. Points of Innovation1. For the first time, MIF was choosed to be a new molecular imaging agent of the allograft rejection monitoring. It can provide properties of high sensitivity and high specificity.2. For the first time the phosphor screen autoradiography technique was used to dynamically monitor the whole processes of allograft rejection with clear and visualized imaging data.3. For the first time we claryfied the expression of TLR5 was closely relevant to the degree of the rejection and prepared the radioiodinated Flagellin (the specific ligand of TLR5). These results will provide basises for taking TLR5 as a new index for accurately evaluation of the immunosuppressive curative effect after transplantation.