The absence of the autoimmune regulator gene (AIRE) impairs the three-dimensional structure of medullary thymic epithelial cell spheroids.

BACKGROUND: Besides controlling the expression of peripheral tissue antigens, the autoimmune regulator (AIRE) gene also regulates the expression of adhesion genes in medullary thymic epithelial cells (mTECs), an essential process for mTEC-thymocyte interaction for triggering the negative selection in the thymus. For these processes to occur, it is necessary that the medulla compartment forms an adequate three-dimensional (3D) architecture, preserving the thymic medulla. Previous studies have shown that AIRE knockout (KO) mice have a small and disorganized thymic medulla; however, whether AIRE influences the mTEC-mTEC interaction in the maintenance of the 3D structure has been little explored. Considering that AIRE controls cell adhesion genes, we hypothesized that this gene affects 3D mTEC-mTEC interaction. To test this, we constructed an in vitro model system for mTEC spheroid formation, in which cells adhere to each other, establishing a 3D structure. RESULTS: The comparisons between AIRE wild type (AIREWT) and AIRE KO (AIRE-/-) 3D mTEC spheroid formation showed that the absence of AIRE: i) disorganizes the 3D structure of mTEC spheroids, ii) increases the proportion of cells at the G0/G1 phase of the cell cycle, iii) increases the rate of mTEC apoptosis, iv) decreases the strength of mTEC-mTEC adhesion, v) promotes a differential regulation of mTEC classical surface markers, and vi) modulates genes encoding adhesion and other molecules. CONCLUSIONS: Overall, the results show that AIRE influences the 3D structuring of mTECs when these cells begin the spheroid formation through controlling cell adhesion genes.

Autoimmune regulator act in synergism with thymocyte adhesion in the control of lncRNAs in medullary thymic epithelial cells.

The autoimmune regulator (Aire) gene in medullary thymic epithelial cells (mTECs) encodes the AIRE protein, which interacts with its partners within the nucleus. This "Aire complex" induces stalled RNA Pol II on chromatin to proceed with transcription elongation of a large set of messenger RNAs and microRNAs. Considering that RNA Pol II also transcribes long noncoding RNAs (lncRNAs), we hypothesized that Aire might be implicated in the upstream control of this RNA species. To test this, we employed a loss-of-function approach in which Aire knockout mTECs were compared to Aire wild-type mTECs for lncRNA transcriptional profiling both in vitro and in vivo model systems. RNA sequencing enables the differential expression profiling of lncRNAs when these cells adhere in vitro to thymocytes or do not adhere to them as a way to test the effect of cell adhesion. Sets of lncRNAs that are unique and that are shared in vitro and in vivo were identified. Among these, we found the Aire-dependent lncRNAs as for example, Platr28, Ifi30, Morrbid, Malat1, and Xist. This finding represents the first evidence that Aire mediates the transcription of lncRNAs in mTECs. Microarray hybridizations enabled us to observe that temporal thymocyte adhesion modulates the expression levels of such lncRNAs as Morrbid, Xist, and Fbxl12o after 36 h of adhesion. This finding shows the existence of a synergistic mechanism involving a link between thymocyte adhesion, Aire, and lncRNAs in mTECs that might be important for immune self-representation.