Meta-Analysis of Autoimmune Regulator-Regulated Genes in Human and Murine Models: A Novel Human Model Provides Insights on the Role of Autoimmune Regulator in Regulating STAT1 and STAT1-Regulated Genes

Abstract

Autoimmune regulator (AIRE) regulates promiscuous expression of tissue-restricted antigens in medullary epithelial cells (mTEC) of the thymus. To understand the diverse effects of AIRE, it is crucial to elucidate the molecular mechanisms underlying the process of AIRE-regulated gene expression. In this study, we generated a recombinant AIRE expression variant of the TEC 1A3 human cell line, TEC 1A3 AIREhi, to determine genes targeted by AIRE, and using microarray analysis, we identified 482 genes showing significant differential expression (P < 0.05; false discovery rate <5%), with 353 upregulated and 129 downregulated by AIRE expression. Microarray data were validated by quantitative PCR, confirming the differential expression of 12 known AIRE-regulated genes. Comparison of AIRE-dependent differential expression in our cell line model with murine datasets identified 447 conserved genes with a number of transcription regulatory interactions, forming several key nodes, including STAT1, which had over 30 interactions with other AIRE-regulated genes. As STAT1 mutations cause dominant chronic mucocutaneous candidiasis and decreased STAT1 levels in monocytes of autoimmune polyglandular syndrome 1 (APS-1) patients, it was important to further characterize AIRE–STAT1 interactions. TEC 1A3AIREhi were treated with the STAT1 phosphorylation inhibitors fludarabine and LLL3 showed that phosphorylated STAT1 (p-STAT1) was not responsible for any of the observed differential expression. Moreover, treatment of TEC 1A3 AIREhi with STAT1 shRNA did not induce any significant variation in the expression of unphosphorylated STAT1 (U-STAT1) downstream genes, suggesting that these genes were directly regulated by AIRE but not via U-STAT1. The novel model system we have developed provides potential opportunities for further analysis of the pathogenesis of (APS-1) and the wider roles of the AIRE gene.

Characterisation of APS‐1 Experimental Models Is Crucial for Development of Novel Therapies

“…In 4-week-old mice, no observable lymphocytic infiltration was detected in either the retina, lung, liver, and stomach ( Figure 4(a) ) or ovary and testis ( Figure 4(b) ). Thus, consistent with the previous studies, there was not any difference between Aire −/− mice and their Aire +/+ littermates at 4 weeks old [ 12 , 13 ].…”

“…In contrast, no such changes were observed in 12-week-old Aire +/+ mice. In agreement with the previous studies, the infiltrations showed a clear age dependence pattern, as tissue infiltration in 4-week-old Aire −/− mice was undetectable [12,13].…”

“…The Benoist and Mathis group engineered this APS-1 mouse model to carry a defective Aire gene that represents the most common APS-1 mutation, R257X, by introducing lox/Cre-mediated recombination in embryonic stem (ES) cells. These mice carry, in the homozygous state, a mutant allele bearing a deletion of exon 2 and portions of the upstream and downstream introns [ 13 ]. To confirm the expression pattern of Aire in the thymus of Aire +/+ and Aire −/− mice, thymi were collected from 4-week-old and 12-week-old mice for histological analysis.…”

“…Phenotypically, the Aire −/− mice did not differ from their Aire +/+ littermates. Therefore, tissue histology was investigated in 4-week-old and 12-week-old Aire −/− and Aire +/+ mice with special attention given to the retina, lung, liver, stomach, and reproductive organs based on previous studies [ 12 , 13 ].…”

“…AIRE is a transcriptional regulator that is highly expressed in medullary thymic epithelial cells (mTECs) [ 11 , 12 ]. One of the pivotal findings is that AIRE/Aire promotes self-tolerance in the thymus by regulating the promiscuous expression of a wide array of expressions of tissue-specific antigens (TSAs) that have the commonality of being tissue-restricted in their expression pattern in the periphery [ 13 , 14 ]. Autoreactive thymocytes (T cell precursors) that recognise these TSAs with high affinity undergo negative selection via apoptosis, or, alternatively, forkhead box P3 (FOXP3) + regulatory T cells (T reg ) are generated in order to prevent autoimmunity [ 15 ].…”