| Autoimmune regulator (Aire) is a transcriptional regulator, which is considered as an important factor in maintenance of self-tolerance because its mutations could cause autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Studies have revealed that Aire is primarily expressed in lymphoid organs, especially in thymus. Within the thymus, it is expressed predominantly in medullary thymic epithelia (mTEC). Aire expressed in mTEC could play the function in central tolerance by regulating the expression of peripheral tissue-specific antigens (PTAs) and deleting the self-reactive T cells. But, the function of above perhaps is not the only role of Aire in maintenance of central tolerance. Other studies have demonstrated that mTEC can express many chemokines regulated by Aire, including CCL22, and these chemokines can attract thymocytes towards mTEC to finish negative selection.Studies have revealed that Aire is not only expressed in thymus, but also in peripheral lymphoid organs, such as lymph nodes and spleen. Recent data have also shown that Aire is expressed in appendix and embryo in low level. However, the function of Aire expressed in peripheral lymphoid organs is not clear so far. The facts that Aire in thymus can regulate the expression of CCL22 to induce central tolerance and some APCs in peripheral can secrete CCL22 to attract CD4+CD25+Treg make the possibility that in peripheral the expression of CCL22 can be regulated by Aire to maintain immune tolerance. To test this hypothesis, we investigated whether Aire could regulate the expression of CCL22 in APCs.In this study, RAW264.7 cells which belong to macrophage cell line were used to establish two kinds of cell models: transiently and stable transfected cells with Aire. 1 Expression changes of CCL22 in RAW264.7 cells transfected with Aire transientlyIn this research, RAW264.7 cell models transiently transfected with pEGFP-C1 or pEGFP-C1-Aire were established to study whether CCL22 could be regulated by Aire. Firstly, to identify whether the plasmids were the right ones, the plasmids were digested by enzymes: pEGFP-C1 was digested by one enzyme and pEGFP-C1-Aire was digested by one and two kinds of enzymes respectively. Results showed that the products of digestion were the same size as expected, indicting the plasmids were the ones needed. Secondly, RAW264.7 cells were transfected with pEGFP-C1 or pEGFP-C1-Aire transiently respectively, and then transfection effects were identified by fluorescence microscope and analyzed expression level of Aire mRNA. The results revealed that the transfection was successful. Thirdly, mRNA expression level of CCL22 (48h after transfection) was analyzed by RT-PCR. The results showed that compared with vector group, CCL22 of Aire group was up-regulated and statistical significance (P<0.05), making the possibility that expression level of CCL22 in RAW264.7 cells could be up-regulated by Aire.2 Expression changes of CCL22 in RAW264.7 cells transfected with Aire stablyIn order to better understand the role that Aire plays on the expression level of CCL22 and avoid stochastic factors, stable transfected cell lines expressing GFP and GFP-Aire were established. The stable transfected cell lines were generated using selection marker G418.The positive cell clones were identified by RT-PCR, fluorescence microscope and immunohistochemistry. The results showed that the stable transfected cell lines were achieved successfully. After that, mRNA expression level of CCL22 was analyzed by RT-PCR. The results revealed that CCL22 expression level in Aire group was significantly up-regulated and statistically significant (P<0.05), showing that the expression level of CCL22 could be up-regulated in RAW264.7 stable transfected cell lines by Aire.This study demonstrated that CCL22 expression level can be up-regulated by Aire in RAW264.7 cells and brought possibility that CCL22 could be up-regulated by Aire in peripheral APCs. This study provided experimental basis for studying relationship between Aire and CD4+CD25+Treg, and so offered a new point of view to understand function and significance of Aire in peripheral immune tolerance.
|
PDF Full Text Request