title: DNA methylation impairs TLR9 induced Foxp3 expression by attenuating IRF-7 binding activity in fulminant type 1 diabetes.
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.
authors.affiliation: Diabetes Center, 2nd Xiangya Hospital, and Institute of Metabolism and Endocrinology, Key Laboratory of Diabetes Immunology, Ministry of Education, Central South University, 139 Renmin Middle Rd, Changsha, Hunan 410011, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Wang
authors.forename: Ying
authors.initials: Y
authors.lastname: Zheng
authors.forename: Can
authors.initials: C
authors.lastname: Hou
authors.forename: Lin
authors.initials: L
authors.lastname: Yang
authors.forename: Xia
authors.initials: X
authors.lastname: Li
authors.forename: Jian
authors.initials: J
authors.lastname: Lin
authors.forename: Gan
authors.initials: G
authors.lastname: Huang
authors.forename: Qianjin
authors.initials: Q
authors.lastname: Lu
authors.forename: Cong-Yi
authors.initials: CY
authors.lastname: Wang
authors.forename: Zhiguang
authors.initials: Z
authors.lastname: Zhou
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D060908:CTLA-4 Antigen / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D019175:DNA Methylation; D003922:Diabetes Mellitus, Type 1 / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D050839:Interferon Regulatory Factor-7 / Q000235:genetics / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D051217:Toll-Like Receptor 9 / Q000819:agonists / Q000378:metabolism*
chemical_list: D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; C526902:CpG ODN 2216; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; C494234:IRF7 protein, human; D050839:Interferon Regulatory Factor-7; D009838:Oligodeoxyribonucleotides; C495371:TLR9 protein, human; D051217:Toll-Like Receptor 9