DNA methylation impairs TLR9 induced Foxp3 expression by attenuating IRF-7 binding activity in fulminant type 1 diabetes

• Published in: Journal of autoimmunity 2013-03, Vol.41, p.50-59
• Main Authors: Wang, Zhen, Zheng, Ying, Hou, Can, Yang, Lin, Li, Xia, Lin, Jian, Huang, Gan, Lu, Qianjin, Wang, Cong-Yi, Zhou, Zhiguang
• Format: Article
• Language: English
• Subjects: Allergy and Immunology; Base Sequence; Blotting, Western; Cells, Cultured; CTLA-4 Antigen - genetics; CTLA-4 Antigen - metabolism; Diabetes Mellitus, Type 1 - classification; Diabetes Mellitus, Type 1 - genetics; Diabetes Mellitus, Type 1 - metabolism; DNA Methylation; Forkhead Transcription Factors - genetics; Forkhead Transcription Factors - metabolism; Foxp3; Fulminant type 1 diabetes; Gene Expression; HEK293 Cells; Humans; Interferon Regulatory Factor-7 - genetics; Interferon Regulatory Factor-7 - metabolism; IRF-7; Leukocytes, Mononuclear - drug effects; Leukocytes, Mononuclear - metabolism; Molecular Sequence Data; Oligodeoxyribonucleotides - pharmacology; Promoter Regions, Genetic - genetics; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction - drug effects; TLR9; Toll-Like Receptor 9 - agonists; Toll-Like Receptor 9 - metabolism
• ISSN: 0896-8411; 1095-9157
• DOI: 10.1016/j.jaut.2013.01.009

Abstract Fulminant type 1 diabetes (FT1D) is an extremely aggressive disease characterized by the abrupt onset of insulin-deficient hyperglycemia. However, the precise mechanisms underlying disease etiology almost remain unclear. As mice deficient in regulatory T cells (Tregs) are prone to the development of an FT1D-like phenotype, we thus investigated whether FT1D patients manifest Treg deficiency and explored the related mechanisms. We first noted a significant reduction for Foxp3 and CTLA4 expression levels in PBMCs of FT1D patients. IRF-7 was found to selectively bind to the Foxp3 promoter, and by which it promotes Foxp3 transcription. Therefore, ectopic IRF-7 expression significantly promoted Foxp3 and CTLA4 expression in PBMCs, while knockdown of IRF-7 manifested opposite effect. Importantly, stimulation of PBMCs with CpG ODN, a ligand for TLR9, significantly induced Foxp3 expression, demonstrating that TLR9 signaling positively regulates Treg development. However, knockdown of IRF-7 expression almost completely diminished the enhancing effect of TLR9 signaling on Foxp3 expression, suggesting that IRF-7 is a downstream molecule of TLR9 signaling and is essential for TLR9 induced Treg generation. Of interestingly note, the Foxp3 promoter in FT1D patients was hypermethylated, indicating that DNA methylation could be a causative factor responsible for the reduced Foxp3 expression in FT1D patients. Indeed, our mechanistic studies revealed that DNA methylation blocked IRF-7 binding to the Foxp3 promoter. Together, our data support the notion that environmental insults in genetic predisposed subjects trigger Foxp3 promoter hypermethylation, which then prevents IRF-7 binding to the Foxp3 promoter and impairs Treg development/functionality contributing to the pathogenesis of FT1D.