Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.
Center of Excellence in Systems Biology, Research affairs, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
Front Endocrinol (Lausanne). 2021 Feb 15;11:619989. doi: 10.3389/fendo.2020.619989. eCollection 2020.
Graves' ophthalmopathy (GO) is a frequent extrathyroidal complication of Graves' hyperthyroidism. Orbital fibroblasts contribute to both orbital tissue inflammation and remodeling in GO, and as such are crucial cellular elements in active GO and inactive GO. However, so far it is largely unknown whether GO disease progression is associated with functional reprogramming of the orbital fibroblast effector function. Therefore, the aim of this study was to compare both the proteome and global DNA methylation patterns between orbital fibroblasts isolated from active GO, inactive GO and healthy controls.
Orbital fibroblasts from inactive GO (n=5), active GO (n=4) and controls (n=5) were cultured and total protein and DNA was isolated. Labelled and fractionated proteins were analyzed with a liquid chromatography tandem-mass spectrometer (LC-MS/MS). Data are available ProteomeXchange with identifier PXD022257. Furthermore, bisulphite-treated DNA was analyzed for methylation pattern with the Illumina Infinium Human Methylation 450K beadchip. In addition, RNA was isolated from the orbital fibroblasts for real-time quantitative (RQ)-PCR. Network and pathway analyses were performed.
Orbital fibroblasts from active GO displayed overexpression of proteins that are typically involved in inflammation, cellular proliferation, hyaluronan synthesis and adipogenesis, while various proteins associated with extracellular matrix (ECM) biology and fibrotic disease, were typically overexpressed in orbital fibroblasts from inactive GO. Moreover, orbital fibroblasts from active GO displayed hypermethylation of genes that linked to inflammation and hypomethylated genes that linked to adipogenesis and autoimmunity. Further analysis revealed networks that contained molecules to which both hypermethylated and hypomethylated genes were linked, including NF-κB, ERK1/2, Alp, RNA polymerase II, Akt and IFNα. In addition, NF-κB, Akt and IFNα were also identified in networks that were derived from the differentially expressed proteins. Generally, poor correlation between protein expression, DNA methylation and mRNA expression was observed.
Both the proteomics and DNA methylation data support that orbital fibroblasts from active GO are involved in inflammation, adipogenesis, and glycosaminoglycan production, while orbital fibroblasts from inactive disease are more skewed towards an active role in extracellular matrix remodeling. This switch in orbital fibroblast effector function may have therapeutic implications and further studies into the underlying mechanism are thus warranted.
甲状腺眼病（GO）是 Graves 甲亢的一种常见甲状腺外并发症。眼眶成纤维细胞有助于 GO 中的眼眶组织炎症和重塑，因此是活动型 GO 和非活动型 GO 中的重要细胞成分。然而，到目前为止，GO 疾病进展是否与眼眶成纤维细胞效应功能的功能重编程有关还在很大程度上是未知的。因此，本研究的目的是比较活动型 GO、非活动型 GO 和健康对照组中分离的眼眶成纤维细胞的蛋白质组和全基因组 DNA 甲基化模式。
培养非活动型 GO（n=5）、活动型 GO（n=4）和对照组（n=5）的眼眶成纤维细胞，分离总蛋白和 DNA。使用液相色谱串联质谱仪（LC-MS/MS）分析标记和分离的蛋白质。数据可在 ProteomeXchange 中使用标识符 PXD022257 获得。此外，用亚硫酸氢盐处理的 DNA 用 Illumina Infinium Human Methylation 450K 珠芯片分析甲基化模式。此外，从眼眶成纤维细胞中分离 RNA 进行实时定量（RQ）-PCR。进行网络和途径分析。
活动型 GO 的眼眶成纤维细胞显示出通常参与炎症、细胞增殖、透明质酸合成和脂肪生成的蛋白质表达上调，而各种与细胞外基质（ECM）生物学和纤维化疾病相关的蛋白质在非活动型 GO 的眼眶成纤维细胞中通常表达上调。此外，活动型 GO 的眼眶成纤维细胞表现出与炎症相关的基因高甲基化和与脂肪生成和自身免疫相关的基因低甲基化。进一步分析揭示了包含与高甲基化和低甲基化基因相关的分子的网络，包括 NF-κB、ERK1/2、Alp、RNA 聚合酶 II、Akt 和 IFNα。此外，NF-κB、Akt 和 IFNα也存在于由差异表达蛋白衍生的网络中。通常，蛋白质表达、DNA 甲基化和 mRNA 表达之间的相关性较差。
蛋白质组学和 DNA 甲基化数据均支持活动型 GO 的眼眶成纤维细胞参与炎症、脂肪生成和糖胺聚糖产生，而非活动型疾病的眼眶成纤维细胞则更倾向于在细胞外基质重塑中发挥积极作用。这种眼眶成纤维细胞效应功能的转变可能具有治疗意义，因此有必要进一步研究其潜在机制。
