Modulation of Aire regulates the expression of tissue-restricted antigens

Abstract

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.

Fig. 1

Dose-dependent effect of Aire on TRA expression in C57Bl/6 and Balb/c mice. Four to six weeks old C57Bl/6 or Balb/c mice were genotyped by PCR (A) and whole thymuses from WT, Aire HET and Aire KO mice were analyzed for TRA gene expression by real-time PCR. TRA expression followed the expression of Aire in a dose-dependent manner in C57Bl/6 (B) as well as Balb/c (C) mice. Data are mean with S.E.M. of triplicate measurements of one out of two representative experiments.

Fig. 2

Expression of Ins2 in the lymph nodes of WT vs. Aire KO. Thymuses or inguinal lymph nodes were collected from 4- to 6-week-old mice and analyzed for Aire or Ins2 gene expression by real-time PCR. Aire expression relative to the epithelial marker, K8, was higher in the lymph nodes compared to the whole thymus of WT mice (A). The expression of Ins2 was unaffected in the Aire KO mice compared to the WT mice (B). Data are mean with S.E.M. of triplicate measurements of one out of two representative experiments.

Fig. 3

TRA expression in mTEC vs. cTEC populations of WT and Aire KO mice. Thymuses were stained with anti-EpCAM and anti-Aire antibodies and analyzed by immunofluorescent microscopy (A) or were enzyme digested and FACS-sorted according to the expression of EpCAM and analyzed for the expression level of TRAs by real-time PCR (B). Medullary compartment of thymus was distinctly characterized by high-EpCAM expression and by the presence of Aire-positive cells. TRAs were highly expressed in the thymic medulla but not in cortex of the WT mice. Aire KO mice showed virtually no expression of TRAs either in medulla or cortex. Data in (B) are mean with S.E.M. of triplicate measurements of one out of two representative experiments.

Fig. 4

Aire and TRA expression during development. Thymuses were collected from normal WT mice at indicated time-points and the gene expression level analyzed by real-time PCR. Aire expression, as well as the expression of most of the ectopic genes reached their peak at D11 after birth followed by a gradual decrease. Data are mean with S.E.M. of triplicate measurements of one out of two representative experiments. Dotted line corresponds to polynomial estimation.

Fig. 5

Over-expression of Aire induces TRA expression in primary thymic stromal cells and in thymic epithelial cell line. Infection with AdAire-GFP (as described in the methods) caused an induction of Aire protein (A). Monolayers of primary thymic stromal cells (B) or thymic epithelial cell-line TEC 1C6 (C) were infected with equal amounts of Ad-GFP or AdAire-GFP and, 48 h later, the cells were harvested for RNA purification and real-time PCR. Aire over-expression resulted in increased TRA expression in both cell types. Data are mean with S.E.M. of triplicate measurements of one out of two representative experiment.

Fig. 6

Requirement of thymic microenivironment for Aire and TRA expression. Thymuses from E17.5 old embryos of C57BL/6 mice were collected and either left intact (3D lobes), disaggregated (3D > 2D) or disaggregated and re-aggregated (RTOC) as described in the methods. Disaggregation resulted in down-regulation of Aire and TRA expression. This effect was restored by reaggregation. Data are mean with S.E.M. of triplicate measurements of one out of two representative experiment.