The Effect Of Autoimmune Regulator (AIRE) On The Functions Of APC In Peripheral Immune Tolerance

Self-tolerance is the condition of non-responsiveness to self antigens in immune system under the physiological state. If the self-tolerance is broken down, it will lead to autoimmune diseases. The maintance of self-tolerance are carried out through central and peripheral tolerance mechanisms. And the thymus is the main site in inducing T lymphocytes central tolerance. In the thymus, T lymphoctyes undergo a series of selections. Thymocytes with low to middium affinity for self-peptide-MHC complexes expressed on cortical epithelial cells are positively selection, and differenciate CD4 or CD8 single positive (SP) cells (positive selection). The SP cells then migrate to the medulla. Thymocytes recognizing the self-peptide-MHC complexes too strongly, thus being self reactivity is deleted (negative selection). More and more evidence suggests a functional role for the thymic expression of tissue specific genes, termed promiscuous gene expression, in the induction of T cell tolerance. Medullary epithelial cells (mTECs) were identified as the specialized cell type which express of a broad range of tissue-specific genes. Self-antigens expressed by mTECs represent almost all parenchymal organs. The self antigens expressed by mTECs are present by themselves or cross-present by dendritic cells, to induce the negtive selection of self reactive T lymphocytes. Although most autoreactive T lymphocytes are deleted by central tolerance mechanism, there are also a few cells can escape from thymus and migrate to peripheral circulation. But only 3–5% of the general population suffers from autoimmune diseases. It must be several peripheral tolerance mechanisms can prevent the diseases. Generally the peripheral tolerance mechanisms include clonal ignorance, anergy, and deletion. Since CD4~+CD25~+ regulatory T cells are identified and characterized with immune suppresion ability by Sakaguchi and his collegors in 1995, more and more researchers pay close attention to the key role of this subsets in peripheral immunotolerance. But so far there are many questions about its generation and function.Autoimmune regulator (AIRE) is a kind of transcript factor, specially expressed on medulla thymic epithelial cells and macrophages and dendritic cells in peripheral spleen, lymph nodes. The single mutation of AIRE will lead autoimmune polyglandular syndrome typ1(APS1), also named Autoimmune Polyendocrinopathy-Candidiasis- Ectodermal Dystrophy Phenotyp(eAPECED), which mediated by autoimmune response and characterized with multi-organs damage. The clinical features is autoimmune diseases affected multi-glanders. Its manifestions include addison's disease, hypoparathyroidism, thyroid disorder, diabetes, autoimmune hepatitis and so on. Recently reports suggest AIRE expressed in thymus can regulate the expression of tissue specific antigens on mTECs, thus play a critical role in negetive seletion and deletion of autoreactive T cells of central tolerance. And it leads to the autoimmunity in APS1 patients. However, the role of AIRE in peripheral immunotolerance is unclear. At the same time besides the autoimmunity, APS1 patients often suffer from the immune deficiency with candidiasis. But this deficiency can not explained by central tolerance. Therefore we suppose that the peripheral role of AIRE may be the reason of candidiasis.Type 1diabetes mellitus is a common autoimmune disease. Now its morbility increased year by year in the world. Central and peripheral tolerance dysfunctions are considered the important reasons of T1DM. But still have others mechanisms unknown. People established the animal models by various methods for deeply studying. Non obese diabetes mice are considered the ideal model of T1DM since they can imitate the progress of human diabetes better. And it has been used broadly in many fields including mechanisms exploration and drugs exploitation.In this study, we study the T1DM and NOD mice to know the peripheral effects of AIRE. And through use the monocyte-macrophage cell linee RAW264.7 to study the effects of AIRE on APC functions, we explore the mechanisms of AIRE in peripheral immune system.Our rearch includes three parts:1. The distributions of AIRE in tissuses and cells In order to understand the distribution information of AIRE, and furtherly study its function, we use RT-PCR to dectect the AIRE mRNA expression in various tissues and cells in Balb/c mice. The results showed AIRE expressed on thymus and bone marrow these two kinds of central organs, its also expressed on peripheral organs such as spleen and lymph nodes. In adition, AIRE also expressed in genital system including ovary and testical. On the cell level AIRE mainly expressed on antigen presenting cells, including bone marrow induced DCs, spleenic DCs and PBMCs. These results indicated AIRE expressed on peripheral tissues and APCs may play a critical role in peripheral immune system.2. The changes of AIRE expression in peripheral immunotolerance disorder Furtherly in order to know the role AIRE in pheriphral tolerance, we studyed on the T1DM patients and NOD mice. First in order to establish the model of autoimmune disease, we dectected the state of peripheral immune. The results showed there have several autoimmunity and dysfunction of peripheral tolerance: the infiltration of lymphocytes in pancrea, thyroid and salivary gland; Th1/Th2 increased; the expression of TRA in spleenic DCs in earlier incubation. However the increase numbers of CD4~+CD25~+Treg may supress the immune response and maintain the normal levels of blood/urine glucose and insulin autoantibody, at the same time impose the decrease of proliferation of spleenocytes. In order to furtherly study the effect of AIRE in peripheral tolerance, we detected the expression of AIRE in peripheral APCs in NOD mice and PBMCs in T1DM patients. It showed AIRE mRNA decreased in two models. It indicated the abnormal of AIRE expression may be the important reason for peripheral autoimmunity and abnormity of peripheral tolerance. By transfected AIRE plasmid into spleenic DCs, and detected the changes of TRA, we found AIRE is positive related with some TRA, such as insulin. It suggested the role of AIRE in peripheral may partly attributed to the regualtion on TRA in APCs.3. The effects of AIRE on APCs functions In order to identify the mechanisms of AIRE's function in peripheral immune system, we use AIRE transfected RAW264.7 cell to study the effects of AIRE on APCs funtions. Results showed after AIRE transfected the levels of MHC-II molecule and CD86 were decreased, thus which may affect the ability of antigen presentation and stimulation to T cells of APCs. After transfection, the expressions of TLR1-9 showed differenct changes. It suggested AIRE can affect the recognization ability both to self and foreign antigens (such as candida albicans) of APCs. We cocultured the spleenocytes with AIRE transfected RAW264.7 cells directly or with its supernatant, we found AIRE can through regulate the TLR9 directly or by producing TGF-β1 indirectly to induce the CD4~+CD25~+Treg and its function gene Foxp3 expression increased.From above studies, we made the following conclusions:1. AIRE can not only express in central immune organs and cells, but also express in peripheral immune organs and APCs.2. AIRE expressed on peripheral immune tissues and cells pacipatiate in maintaince of peripheral tolerance. And the effect of AIRE on the exprssion of TRA(insulin) on dendritic cells may be one of the peripheral tolerance mechanisms.3. AIRE regulates many molecules expressions on APCs, which affects the ability of antigen recognization, presentation and stimulation T cells of APCs. In addition, through the regulation to TLR9 on APCs, AIRE may directly or indirectly affect the generation and function of CD4+CD25+Tregs. These ensured the normal immune defense and maintance of peripheral immunotolerance.This research provides the theory evidences for complete understanding the role of AIRE in immunotolerance. And it furtherly explained the reason about concomitance of autoimmunity and immune deficiency in APS1. Based on the effects of AIRE on APC, through the effect of TLRs to Tregs, we can effective induce tolerance(prevent autoimmune disease, anti-infection and graft rejection and so on) or break tolerance(anti-tumor). It guides a new way to effectively treat diseases.