Aire knockdown in medullary thymic epithelial cells affects Aire protein, deregulates cell adhesion genes and decreases thymocyte interaction

• Published in: Molecular immunology Vol. 77; pp. 157 - 173
• Main Authors: Pezzi, Nicole, Assis, Amanda Freire, Cotrim-Sousa, Larissa Cotrim, Lopes, Gabriel Sarti, Mosella, Maritza Salas, Lima, Djalma Sousa, Bombonato-Prado, Karina F., Passos, Geraldo Aleixo
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01-09-2016
• Subjects: Aire gene; AIRE Protein; Animals; Blotting, Western; Cell Adhesion - immunology; Cell Differentiation - immunology; Epithelial Cells - cytology; Epithelial Cells - immunology; Epithelial Cells - metabolism; Female; Gene Expression Profiling; Gene Expression Regulation - immunology; Gene Knockdown Techniques; In Situ Hybridization, Fluorescence; Mice; Mice, Inbred C57BL; mTEC; mTEC-thymocyte adhesion; Polymerase Chain Reaction; Self Tolerance - immunology; siRNA; Thymocyte; Thymocytes - cytology; Thymocytes - immunology; Thymocytes - metabolism; Thymus; Thymus Gland - cytology; Thymus Gland - immunology; Thymus Gland - metabolism; Transcription Factors - immunology; Transcription Factors - metabolism; Transcriptome - immunology; mTEC-thymocyte adhesion; siRNA; Aire gene; Thymocyte; mTEC; Thymus
• ISSN: 0161-5890; 1872-9142
• DOI: 10.1016/j.molimm.2016.08.003

•Aire mRNA levels are correlated to mTEC-thymocyte interaction.•Slight variations in Aire mRNA levels in mTEC cells are sufficient to:•Decrease Aire protein levels.•Disturb the expression levels of genes involved in cell adhesion.•Inhibit Aire protein nuclear localization.
We demonstrate that even a partial reduction of Aire mRNA levels by siRNA-induced Aire knockdown (Aire KD) has important consequences to medullary thymic epithelial cells (mTECs). Aire knockdown is sufficient to reduce Aire protein levels, impair its nuclear location, and cause an imbalance in large-scale gene expression, including genes that encode cell adhesion molecules. These genes drew our attention because adhesion molecules are implicated in the process of mTEC-thymocyte adhesion, which is critical for T cell development and the establishment of central self-tolerance. Accordingly, we consider the following: 1) mTECs contribute to the elimination of self-reactive thymocytes through adhesion; 2) Adhesion molecules play a crucial role during physical contact between these cells; and 3) Aire is an important transcriptional regulator in mTECs. However, its role in controlling mTEC-thymocyte adhesion remains unclear. Because Aire controls adhesion molecule genes, we hypothesized that the disruption of its expression could influence mTEC-thymocyte interaction. To test this hypothesis, we used a murine Aire+ mTEC cell line as a model system to reproduce mTEC-thymocyte adhesion in vitro. Transcriptome analysis of the mTEC cell line revealed that Aire KD led to the down-modulation of more than 800 genes, including those encoding for proteins involved in cell adhesion, i.e., the extracellular matrix constituent Lama1, the CAM family adhesion molecules Vcam1 and Icam4, and those that encode peripheral tissue antigens. Thymocytes co-cultured with Aire KD mTECs had a significantly reduced capacity to adhere to these cells. This finding is the first direct evidence that Aire also plays a role in controlling mTEC-thymocyte adhesion.