a Department of Anthropology and Human Genetics, Faculty of Science , Charles University , Prague , Czech Republic.
b Department of Biology and Medical Genetics, First Faculty of Medicine , Charles University and General University Hospital in Prague , Prague , Czech Republic.
Autoimmunity. 2019 Mar;52(2):88-94. doi: 10.1080/08916934.2019.1608965. Epub 2019 May 6.
The elevated plasma cell-free DNA (cfDNA) concentrations were repeatedly reported in association with the process of inflammation. The qualitative and quantitative characteristics of plasma cfDNA in active (newly diagnosed) celiac disease patients (CD) have not yet been studied despite the fact that cfDNA of healthy individuals is able to regulate immune response. We determined the total cfDNA concentration and relative content of telomeric sequences in plasma cfDNA in CD (n = 10) and healthy age- and sex-matched controls (HC, n = 10) by quantitative PCR. To obtain the evidence that the observed biological effects are caused solely by cfDNA molecules, we applied the treatment of paired plasma samples with DNase. Using paired samples of plasma (non-treated/native and treated by DNase), we analyzed the contribution of cfDNA to the activation of TLR9 and TNF-α mRNA expression in THP1 monocytic cell line. There were no significant differences in the quantities of plasma cfDNA and relative contents of telomeric sequences in their pools. When we compared the levels of TNF-α mRNA expression in THP1 cells achieved after stimulation with native CD and HC plasma samples, we found significantly (p = .031) higher expression after stimulation with CD samples. We documented also the ability of cfDNA contained in CD plasma samples to stimulate the production of TLR9 mRNA. The TLR9 mRNA expression levels were significantly (p = .014) lowered after cfDNA removal from CD plasma samples. The design of our experiments allowed us to study the effects of cfDNA without its isolation from plasma. cfDNA contained in CD plasma samples differs significantly in its immunoregulatory capacity from cfDNA in HC plasma. The differences are caused neither by different concentrations of cfDNA in plasma samples nor by different relative abundance of telomeric sequences. Further studies are needed to elucidate the role of plasma cfDNA in celiac disease pathogenesis.
血浆无细胞游离 DNA（cfDNA）浓度升高与炎症过程反复相关。尽管健康个体的 cfDNA 能够调节免疫反应，但活动性（新诊断）乳糜泻患者（CD）血浆 cfDNA 的定性和定量特征尚未得到研究。我们通过定量 PCR 测定 CD（n=10）和年龄、性别匹配的健康对照（HC，n=10）血浆 cfDNA 的总 cfDNA 浓度和端粒序列的相对含量。为了获得观察到的生物学效应仅由 cfDNA 分子引起的证据，我们用 DNase 处理配对的血浆样本。我们使用未经处理/天然和用 DNase 处理的配对血浆样本，分析 cfDNA 对 TLR9 和 TNF-αmRNA 表达在 THP1 单核细胞系中的激活的贡献。它们的池中的血浆 cfDNA 数量和端粒序列的相对含量没有显着差异。当我们比较用天然 CD 和 HC 血浆样本刺激后 THP1 细胞中 TNF-αmRNA 表达的水平时，我们发现用 CD 样本刺激后表达明显更高（p=0.031）。我们还记录了 CD 血浆样本中包含的 cfDNA 刺激 TLR9mRNA 产生的能力。从 CD 血浆样本中去除 cfDNA 后，TLR9mRNA 表达水平显着降低（p=0.014）。我们的实验设计允许我们在不分离血浆的情况下研究 cfDNA 的作用。与 HC 血浆 cfDNA 相比，CD 血浆样本中的 cfDNA 在其免疫调节能力上存在显着差异。差异既不是由血浆样本中 cfDNA 的浓度不同引起的，也不是由端粒序列的相对丰度不同引起的。需要进一步研究阐明血浆 cfDNA 在乳糜泻发病机制中的作用。
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