Effects Of Autoimmune Regulator(Aire) On Promiscuous Gene Expression Of MTEC

Self-tolerance is the non-response of immune system to self components in physiological conditions. Two kinds of mechanisms are involved in maintaining self-tolerance: central tolerance and peripheral tolerance. Thymus plays important roles in central tolerance. Various immature T clones that express particular TCR could be produced by random rearrangement of TCRαand TCRβgene segments during the maturation of thymus. The immature T clones that have high affinity with tissue restricted antigens (TRA) are induced to apoptosis and deleted. The deletion of self-reactive T clones in thymus is called negative selection. Several thousands of TRAs could be expressed by mTECs and DCs in cortico-medullary junction, which is called promiscuous gene expression. Promiscuous gene expression of thymus might play an important role in negative selection of self-reactive T clones. However, the mechanisms that could be involved in regulation of promiscuous gene expression are not well understood.Autoimmune regulator (Aire), whose mutation is associated with the disease of autoimmune polygland syndrom 1 (APS I) in human, is mainly expressed in mTEC. The expression of many TRAs in thymus decrease in Aire knockout mice, So Aire might play the function in negative selection by regulating the promiscuous gene expression in physiological conditions.In this research, we have studied the regulatory functions of Aire on promiscuous gene expression from two parts: Part I. Effects of Aire on TRA expression in mTEC of Balb/c mice1. Purity and identification of primarily cultured TECThe primarily cultured TECs were identified by immunochemistry methods. The purity of Balb/c-TEC increases with time and at d8 the purity could be about 85%.2. Aire and TRAs expression in primarily cultured mTECThe expression level of Aire and TRAs in mTEC was analyzed By RT-PCR. The result showed that the expression level of Aire and TRAs in Balb/c-mTEC attenuated gradually as culture time, was higher at d3, weaker at d8 and disappeared at d11.3. Effects of Aire on TRAs expression of Balb/c-mTECAire was transfected transiently to mTEC at different culture days-before and after the disappearance of promiscuous gene expression-and expression of TRAs was detected by RT-PCR. The results showed that Aire could promote TRAs expression only at being transfected before the disappearance of TRA expression, and different TRAs were regulated at different levels by Aire. Aire could extend the expression time of TRAs in mTEC. Thymocytes were important resource of upstream activation signals of Aire.4. Effects of Aire on inducing thymocytes to apoptosis by mTECIncreased TRAs expression in mTEC by transfected with Aire could promote apoptosis of T cells.Conclusions:a. The expression level of Aire and TRAs in Balb/c-mTEC attenuated gradually with culture time.b. Aire can promote the expression level and extend the expression time of TRAs in mTEC at the early phase of culture. Thymocytes are important resource of activation signals of Aire protein.c. The ability of Aire-mTEC in inducing apoptosis of thymocytes improved obviously.Part II. Effects of Aire on TRAs expression in mTEC of T1D models1. TRAs expression and Treg production in thymus of STZ-induced models There were obvious"Three highs and one low"signs of diabetes mellitus, the urine glucose were all strong positive, serum IAA levels increased obviously with apparent islitis in experimental group, which showed that T1D models were induced by STZ successfully. The thymi of model mice diminished much and lost normal physiological structure of cortico-medullary junction. In the mean time, the whole CD4+CD25+Treg populations decreased which might be an important reason of T1D.Expression of TRAs in thymus of experimental group decreased obviously, which might be important for T1D because of abnormal central tolerance.2. TRAs expression and Treg production in thymus of NOD miceThe body weights of NOD mice we bought(10-12 week old,female) were similar with Balb/c mice essentially, the urine glucose were all negative, the blood glucose levels were all normal, simultaneously with the volumes of thymi. On the other hand, serum IAA levels of NOD mice were higher than Balb/c mice and many pathobiologic changes took place in several glands (e.g.,infiltration of inflammatory cells in islets,salivary glands,thyroids and mixing of cortico-medullary junction in thymus). Production of CD4+CD25+Treg decreased and the decreased part might be the antigen-specific Treg, not the whole Treg library. MHCII molecules expression on NOD-mTECs was lower than Balb/c-mTEC obviously, which might be an important reason of disorder in negative selection of NOD mice.The expression of Aire and TRAs in NOD-mTEC decreased obviously, which showed manifest defects in promiscuous gene expression of NOD-mTEC. The expression of Aire and TRAs in NOD-mTEC attenuated gradully as culture went on, similar with Balb/c-mTEC.3. Effects of Aire on TRA expression in NOD-mTECAire could recover the defected expression of TRAs in NOD-mTEC and extend the expression time. The thymocytes can promote the recovery of Aire to defected TRAs expression in NOD-mTEC.Conclusions:a. There are several defects in thymus of T1D models, which might be important reasons of T1D.b. Aire can recover the defected TRAs expression of NOD-mTEC, and thymocytes can promote the recovery of Aire to defected TRAs expression in NOD-mTEC.Innovation of this thesisa. Transfering-cultured mTEC cell lines lost TRA expression during the long-time culture procedure and can't be used in research of promiscuous gene expression. We established an Aire transiently-transfecting model of primarily cultured Balb/c-mTEC initiately,and depending on this model we researched the effects of Aire on promiscuous gene expression of thymus and found direct proofs of Aire to promote the TRA expression in mTEC.b. On the basis of the discovery that Aire can promote promiscuous gene expression of thymus, we researched the cross-talk between thymocytes and stromal cells in thymic microenviroment. We found that thymocytes might promote the regulation of Aire to TRAs expression in mTEC, Aire can promote apoptosis of thymocytes by promoting TRA expression in mTEC. c. After discovering the defects of TRAs expression in thymi of T1D models, we then established another model of Aire transiently-transfecting NOD-mTEC. We found that Aire can recover the TRA expression in pathological mTEC.Position and value of the resultsThe research methods in this thesis have never been found in or out of our country, and the results might bring fresh methods and outlets to the research of promiscuous gene expression in mTEC. Maybe they can play important roles in the investigation of acting mechanisms of Aire, TRA expression in mTEC and the etiological factors of autoimmune diseases, meanwhile bring significant evidences to the gene therapies of multiple autoimmune diseases.