Unterberg Matthias, Kreuzer Maxmiliane Julia, Schäfer Simon Thomas, Bazzi Zainab, Adamzik Michael, Rump Katharina
Klinik für Anästhesiologie, Intensivmedizin und Schmerztherapie, Universitätsklinikum Knappschaftskrankenhaus Bochum-Langendreer der Ruhr-Universität Bochum, In der Schornau 23-25, 44892 Bochum, Germany.
Klinik für Anaesthesiologie, Klinikum der Universität Ludwig-Maximilians Universität München, München, Germany.
PLoS One. 2016 Jun 1;11(6):e0156702. doi: 10.1371/journal.pone.0156702. eCollection 2016.
Sepsis, with a persistently high 90-day mortality of about 46%, is the third most frequent cause of death in intensive care units worldwide. Further understanding of the inflammatory signaling pathways occurring in sepsis is important for new efficient treatment options. Key regulator of the inflammatory response is the transcription factor NFκB. As we have recently shown, the -94 Ins/Del NFKB1 promoter polymorphism influences sepsis mortality. However, a molecular explanation is still missing. Thus, promoter activity might be varying depending on the NFKB1 genotype, explaining the genotype dependent mortality from sepsis, and one likely mechanism is the degree of promoter methylation. Therefore, we tested the hypothesis that NFκB mRNA expression is regulated by promoter methylation in human cell lines and primary immune cell cultures. First, we examined the methylation of the NFKB1 promoter in U937, REH and HL-60 cells. In the promoter region of nt+99/+229 methylation in all analyzed cell lines was below 1%. Following incubation with bacterial cell wall components, no significant changes in the frequency of promoter methylation in U937 and REH cells were measured and the methylation frequency was under 1%. However, NFκB1 mRNA expression was two-fold increased in U937 cells after 24 h incubation with LPS. By contrast, demethylation by 5-Aza-2'-deoxycytidine incubation enhanced NFκB1 expression significantly. In addition, we analyzed NFKB1 promoter methylation in primary cells from healthy volunteers depending on the NFKB1-94 Ins/Del genotype. Methylation in the promoter region from nt+402 to nt+99 was below 1%. Genotype dependent differences occurred in neutrophil cells, where DD-genotype was significantly more methylated compared to II genotype at nt+284/+402. Besides in the promoter region from nt-227/-8 in ID-genotypes methylation of neutrophils was significantly decreased compared to lymphocytes and in II-genotypes methylation in neutrophils was significantly decreased compared to lymphocytes and monocytes. In addition, CHART-PCR showed that the hypomethylated promoter regions are highly accessible. Therefore we assume that the demethylated regions are very important for NFKB1 promoter activity.
脓毒症是全球重症监护病房中第三大常见死因，其90天死亡率持续居高不下，约为46%。进一步了解脓毒症中发生的炎症信号通路对于开发新的有效治疗方案至关重要。炎症反应的关键调节因子是转录因子NFκB。正如我们最近所表明的，-94 Ins/Del NFKB1启动子多态性会影响脓毒症死亡率。然而，分子层面的解释仍然缺失。因此，启动子活性可能因NFKB1基因型而异，这就解释了脓毒症死亡率的基因型依赖性，一种可能的机制是启动子甲基化程度。因此，我们检验了一个假设，即NFκB mRNA表达在人类细胞系和原代免疫细胞培养物中受启动子甲基化调控。首先，我们检测了U937、REH和HL-60细胞中NFKB1启动子的甲基化情况。在所有分析的细胞系中，nt+99/+229区域的启动子甲基化率均低于1%。在用细菌细胞壁成分孵育后，未检测到U937和REH细胞启动子甲基化频率有显著变化，甲基化频率仍低于1%。然而，用脂多糖（LPS）孵育24小时后，U937细胞中NFκB1 mRNA表达增加了两倍。相比之下，用5-氮杂-2'-脱氧胞苷孵育进行去甲基化显著增强了NFκB1的表达。此外，我们根据NFKB1 -94 Ins/Del基因型分析了健康志愿者原代细胞中NFKB1启动子的甲基化情况。nt+402至nt+99区域的启动子甲基化率低于1%。在中性粒细胞中出现了基因型依赖性差异，在nt+284/+402处，DD基因型的甲基化程度明显高于II基因型。此外，在ID基因型中，与淋巴细胞相比，中性粒细胞在nt-227/-8区域的启动子甲基化显著降低；在II基因型中，与淋巴细胞和单核细胞相比，中性粒细胞的甲基化也显著降低。此外，CHART-PCR显示低甲基化的启动子区域具有高度可及性。因此我们认为去甲基化区域对NFKB1启动子活性非常重要。
