DNA methylation signatures of the AIRE promoter in thymic epithelial cells, thymomas and normal tissues.

Mutations in the AIRE gene cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), which is associated with autoimmunity towards several peripheral organs. The AIRE protein is almost exclusively expressed in medullary thymic epithelial cells (mTEC) and CpG methylation in the promoter of the AIRE gene has been suggested to control its tissue-specific expression pattern. We found that in human AIRE-positive medullary and AIRE-negative cortical epithelium, the AIRE promoter is hypomethylated, whereas in thymocytes, the promoter had high level of CpG methylation. Likewise, in mouse mTECs the AIRE promoter was uniformly hypomethylated. In the same vein, the AIRE promoter was hypomethylated in AIRE-negative thymic epithelial tumors (thymomas) and in several peripheral tissues. Our data are compatible with the notion that promoter hypomethylation is necessary but not sufficient for tissue-specific regulation of the AIRE gene. In contrast, a positive correlation between AIRE expression and histone H3 lysine 4 trimethylation, an active chromatin mark, was found in the AIRE promoter in human and mouse TECs.

Chagasic thymic atrophy does not affect negative selection but results in the export of activated CD4+CD8+ T cells in severe forms of human disease.

Extrathymic CD4+CD8+ double-positive (DP) T cells are increased in some pathophysiological conditions, including infectious diseases. In the murine model of Chagas disease, it has been shown that the protozoan parasite Trypanosoma cruzi is able to target the thymus and induce alterations of the thymic microenvironment and the lymphoid compartment. In the acute phase, this results in a severe atrophy of the organ and early release of DP cells into the periphery. To date, the effect of the changes promoted by the parasite infection on thymic central tolerance has remained elusive. Herein we show that the intrathymic key elements that are necessary to promote the negative selection of thymocytes undergoing maturation during the thymopoiesis remains functional during the acute chagasic thymic atrophy. Intrathymic expression of the autoimmune regulator factor (Aire) and tissue-restricted antigen (TRA) genes is normal. In addition, the expression of the proapoptotic Bim protein in thymocytes was not changed, revealing that the parasite infection-induced thymus atrophy has no effect on these marker genes necessary to promote clonal deletion of T cells. In a chicken egg ovalbumin (OVA)-specific T-cell receptor (TCR) transgenic system, the administration of OVA peptide into infected mice with thymic atrophy promoted OVA-specific thymocyte apoptosis, further indicating normal negative selection process during the infection. Yet, although the intrathymic checkpoints necessary for thymic negative selection are present in the acute phase of Chagas disease, we found that the DP cells released into the periphery acquire an activated phenotype similar to what is described for activated effector or memory single-positive T cells. Most interestingly, we also demonstrate that increased percentages of peripheral blood subset of DP cells exhibiting an activated HLA-DR+ phenotype are associated with severe cardiac forms of human chronic Chagas disease. These cells may contribute to the immunopathological events seen in the Chagas disease.

Autoimmune regulator deficiency results in decreased expression of CCR4 and CCR7 ligands and in delayed migration of CD4+ thymocytes.

Autoimmune regulator (Aire) has been viewed as a central player in the induction of tolerance. This study examines whether Aire can modulate the production of the thymic chemokines involved in corticomedullary migration and thus play a role in intrathymic thymocyte migration and maturation. Aire deficiency resulted in reduced gene expression and protein levels of the CCR4 and CCR7 ligands in whole thymi of mice, as determined by quantitative PCR analysis and ELISA. The expression of the CCR4 ligands coincided with Aire expression in the CD80(high) medullary thymic epithelial cells, whereas the expression of the CCR7 ligands was detected in other cell populations. Also, the expression pattern of the CCR4 and CCR7 ligands follows that of Aire during postnatal but not during embryonic development. In vitro, overexpression of Aire resulted in an up-regulation of selected CCR4 and CCR7 ligands, which induced selective migration of double-positive and single-positive CD4(+) cells. In vivo, Aire deficiency resulted in a diminished emigration of mature CD4(+) T cells from the thymi of 5-day-old mice. In conclusion, Aire regulates the production of CCR4 and CCR7 ligands in medullary thymic epithelial cells and alters the coordinated maturation and migration of thymocytes. These results suggest a novel mechanism behind the Aire-dependent induction of central tolerance.

Modulation of Aire regulates the expression of tissue-restricted antigens.

Intrathymic expression of tissue-restricted antigens (TRAs) has been viewed as the key element in the induction of central tolerance and recently, a central role for the autoimmune regulator (Aire) has been suggested in this process. The aim of this study was to establish whether down or up-regulation of Aire leads to alterations in TRA expression and whether this is limited to thymic epithelial cells. This study also characterized whether TRAs follow Aire expression during normal development, and whether thymic microenvironment plays a role in the expression of Aire and TRAs. We did several in vivo and in vitro experiments to manipulate Aire expression and measured expression of four TRAs (Trefoil factor-3, Insulin-2, Major urinary protein-1 and Salivary protein-1) by real-time RT-PCR. Aire had an allele dose-dependent effect on TRA expression in the thymuses of mice from two strains, C57BL/6J and Balb/c, but had no effect on TRA expression in the lymph nodes. In the thymus, Aire and TRAs were both localized in the medulla and were co-expressed during normal development and involution. In the primary stromal cells as well as thymic epithelial cell line, the adenoviral over-expression of Aire resulted in an increase in TRA expression. By manipulating in vitro organ-cultures we showed that thymic microenvironment plays a dominant role in Aire expression whereas TRAs follow the same pattern. The data underline a direct role for Aire in TRA expression and suggest that modulation of Aire has a potential to control central tolerance and autoimmunity.