title: Expression profile of peripheral tissue antigen genes in medullary thymic epithelial cells (mTECs) is dependent on mRNA levels of autoimmune regulator (Aire).
abstract: In the thymus of non-obese diabetic (NOD) mice, the expression of the autoimmune regulator (Aire) gene varies with age, and its down-regulation in young mice precedes the later emergence of type 1 diabetes mellitus (T1D). In addition, the insulin (Ins2) peripheral tissue antigen (PTA) gene, which is Aire-dependent, is also deregulated in these mice. Based in these findings, we hypothesized that the imbalance in PTA gene expression in the thymus can be associated with slight variations in Aire transcript levels. To test this, we used siRNA to knockdown Aire by in vivo electro-transfection of the thymus of BALB/c mice. The efficiency of the electro-transfection was monitored by assessing the presence of irrelevant Cy3-labeled siRNA in the thymic stroma. Importantly, Aire-siRNA reached medullary thymic epithelial cells (mTECs) down-regulating Aire. As expected, the in vivo Aire knockdown was partial and transient; the maximum 59% inhibition occurred in 48 h. The Aire knockdown was sufficient to down-regulate PTA genes; however, surprisingly, several others, including Ins2, were up-regulated. The modulation of these genes after in vivo Aire knockdown was comparable to that observed in NOD mice before the emergence of T1D. The in vitro transfections of 3.10 mTEC cells with Aire siRNA resulted in samples featuring partial (69%) and complete (100%) Aire knockdown. In these Aire siRNA-transfected 3.10 mTECs, the expression of PTA genes, including Ins2, was down-regulated. This suggests that the expression profile of PTA genes in mTECs is affected by fine changes in the transcription level of Aire.
authors.affiliation: Molecular Immunogenetics Group, Department of Genetics, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), 14040-900 Ribeirão Preto, SP, Brazil.
authors.forename: Ernna H
authors.initials: EH
authors.lastname: Oliveira
authors.forename: Claudia
authors.initials: C
authors.lastname: Macedo
authors.forename: Paula B
authors.initials: PB
authors.lastname: Donate
authors.forename: Renata S
authors.initials: RS
authors.lastname: Almeida
authors.forename: Nicole
authors.initials: N
authors.lastname: Pezzi
authors.forename: Catherine
authors.initials: C
authors.lastname: Nguyen
authors.forename: Marcos A
authors.initials: MA
authors.lastname: Rossi
authors.forename: Elza T
authors.initials: ET
authors.lastname: Sakamoto-Hojo
authors.forename: Eduardo A
authors.initials: EA
authors.lastname: Donadi
authors.forename: Geraldo A
authors.initials: GA
authors.lastname: Passos
mesh_terms: D000818:Animals; D002460:Cell Line; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000378:metabolism; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics / Q000276:immunology*; D004847:Epithelial Cells / Q000276:immunology*; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D007328:Insulin / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D016688:Mice, Inbred NOD; D012333:RNA, Messenger / Q000096:biosynthesis; D034741:RNA, Small Interfering / Q000235:genetics; D013950:Thymus Gland / Q000276:immunology / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D059467:Transcriptome; D000096927:AIRE Protein
chemical_list: D024043:Collagen Type II; D019388:Cytochrome P-450 CYP1A2; D007328:Insulin; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014157:Transcription Factors; D000096927:AIRE Protein; C447413:Col2a1 protein, mouse; C561967:Ins2 protein, mouse