BACKGROUND Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb). Granuloma is a pathological feature of tuberculosis and is a tight immune cell aggregation caused by Mtb. The main constituent cells are macrophages and their derivative cells including epithelioid macrophages. However, the molecular mechanism of the transition has not been reported. The purpose of this study was to investigate whether early secreted antigenic target of 6-kDa (ESAT6) can induce the transition of bone marrow-derived macrophages (BMDMs) into epithelioid macrophages and its possible molecular mechanism. METHODS The recombinant ESAT6 protein was obtained from E.coli carrying esat6 gene after isopropyl β-d-thiogalactopyranoside (IPTG) induction. BMDMs were isolated from bone marrow of mice hind legs. Cells viability was detected by Cell Counting Kit 8 (CCK8) assays. The expression levels of mRNA and proteins were detected by qPCR and Western blot, or evaluated by flow cytometry. The expression level of nitric oxide (NO) was measured with a nitric oxide indicator. RESULTS ESAT6 could significantly induce mRNA and protein expression levels of a group of epithelioid macrophages marker molecules (EMMMs), including E-cadherin, junction plakoglobin, ZO1, desmoplakin, desmoglein3 and catenin porteins, in BMDMs. These events could be abrogated in macrophage from TLR2 deficiency mice. ESAT6 could also markedly induce iNOS/NO production that could significantly inhibit trimethylation of H3K27 in the cells. ESAT6-induced expressions of epithelioid macrophages marker molecules were significantly inhibited in the presence of H3K27 histone demethylase inhibitor GSK J1. Furthermore, ROS scavenging agent N,N'-Dimethylthiourea (DMTU) could markedly inhibit the transition induced by ESAT6 in macrophages. CONCLUSION This study demonstrates that ESAT6 bound with TLR2 can activate iNOS/NO and ROS signalings to reduce the trimethylation of H3K27 resulting in the increment of EMMMs expression that is beneficial to the transition of macrophages into epithelioid macrophages. However, hypoxia can inhibit this transition event. This study has provided new evidence of pathogenesis of granuloma caused by Mtb and also proposed new ideas for the treatment of TB.

中文翻译：

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结核分枝杆菌6-kDa的早期分泌抗原靶标通过下调巨噬细胞中的iNOS / NO介导的H3K27三甲基化来诱导巨噬细胞向上皮样巨噬细胞的转变。

背景技术结核病（TB）是由结核分枝杆菌（Mtb）引起的慢性感染性疾病。肉芽肿是结核病的病理特征，是由Mtb引起的紧密的免疫细胞聚集。主要组成细胞是巨噬细胞，其衍生细胞包括上皮样巨噬细胞。但是，尚未报道过渡的分子机理。这项研究的目的是调查早期分泌的6-kDa抗原靶标（ESAT6）是否可以诱导骨髓来源的巨噬细胞（BMDMs）转变为上皮样巨噬细胞及其可能的分子机制。方法通过异丙基β-d-硫代半乳糖吡喃糖苷（IPTG）诱导，从携带esat6基因的大肠杆菌中获得重组ESAT6蛋白。从小鼠后腿的骨髓中分离出BMDM。通过细胞计数试剂盒8（CCK8）测定法检测细胞活力。通过qPCR和Western印迹检测mRNA和蛋白质的表达水平，或通过流式细胞术评估mRNA和蛋白质的表达水平。用一氧化氮指示剂测量一氧化氮（NO）的表达水平。结果ESAT6可以在BMDMs中显着诱导一组上皮样巨噬细胞标记分子（EMMM）的mRNA和蛋白质表达水平，包括E-钙黏着蛋白，结斑珠蛋白，ZO1，桥粒白蛋白，桥粒蛋白3和连环蛋白门蛋白。这些事件可以在TLR2缺乏小鼠的巨噬细胞中消除。ESAT6还可以显着诱导iNOS / NO产生，从而显着抑制细胞中H3K27的三甲基化。在H3K27组蛋白脱甲基酶抑制剂GSK J1的存在下，ESAT6诱导的上皮样巨噬细胞标记分子的表达被显着抑制。此外，ROS清除剂N，N'-二甲基硫脲（DMTU）可以显着抑制ESAT6在巨噬细胞中诱导的过渡。结论这项研究表明，与TLR2结合的ESAT6可以激活iNOS / NO和ROS信号以减少H3K27的三甲基化，从而导致EMMMs表达的增加，这有利于巨噬细胞向上皮样巨噬细胞的转变。但是，缺氧可以抑制这种过渡事件。这项研究为由Mtb引起的肉芽肿的发病机理提供了新的证据，也为结核病的治疗提出了新的思路。-二甲基硫脲（DMTU）可以显着抑制ESAT6在巨噬细胞中诱导的过渡。结论这项研究表明，与TLR2结合的ESAT6可以激活iNOS / NO和ROS信号以减少H3K27的三甲基化，从而导致EMMMs表达的增加，这有利于巨噬细胞向上皮样巨噬细胞的转变。但是，缺氧可以抑制这种过渡事件。这项研究为由Mtb引起的肉芽肿的发病机理提供了新的证据，也为结核病的治疗提出了新的思路。-二甲基硫脲（DMTU）可以显着抑制ESAT6在巨噬细胞中诱导的过渡。结论这项研究表明，与TLR2结合的ESAT6可以激活iNOS / NO和ROS信号以减少H3K27的三甲基化，从而导致EMMMs表达的增加，这有利于巨噬细胞向上皮样巨噬细胞的转变。但是，缺氧可以抑制这种过渡事件。这项研究为由Mtb引起的肉芽肿的发病机理提供了新的证据，也为结核病的治疗提出了新的思路。结论这项研究表明，与TLR2结合的ESAT6可以激活iNOS / NO和ROS信号以减少H3K27的三甲基化，从而导致EMMMs表达的增加，这有利于巨噬细胞向上皮样巨噬细胞的转变。但是，缺氧可以抑制这种过渡事件。这项研究为由Mtb引起的肉芽肿的发病机理提供了新的证据，也为结核病的治疗提出了新的思路。结论这项研究表明，与TLR2结合的ESAT6可以激活iNOS / NO和ROS信号以减少H3K27的三甲基化，从而导致EMMMs表达的增加，这有利于巨噬细胞向上皮样巨噬细胞的转变。但是，缺氧可以抑制这种过渡事件。这项研究为由Mtb引起的肉芽肿的发病机理提供了新的证据，也为结核病的治疗提出了新的思路。