Table1_RETRACTED: Transforming Growth Factor-β1 Promotes M1 Alveolar Macrophage Polarization in Acute Lung Injury by Up-Regulating DNMT1 to Mediate the microRNA-124/PELI1/IRF5 Axis.docx

ObjectiveMacrophages function as key orchestrators in the pathogenesis of acute lung injury (ALI). The current study sets out to investigate the molecular mechanism of transforming growth factor-β (TGFβ1) in the regulation of M1 alveolar macrophage polarization in ALI by modulating DNA methyltransferase 1 (DNMT1), along with the microRNA (miR)-124/Pellino 1 (PELI1)/interferon regulatory factor 5 (IRF5) axis.
MethodsFirst, ALI mouse models were established, and the proportion of M1 and M2 macrophages in mouse lung tissues was detected using flow cytometry. The targeting relationship between miR-124 and PELI1 was verified with the help of a dual luciferase gene reporter assay. Following TGFβ1 knockdown, RT-qPCR and Western blot assay were performed to analyze the expression patterns of TGFβ1, DNMT1, miR-124, and PELI1 and M1/M2 polarization markers in the lung tissues of ALI mice. Immunofluorescence was further employed to detect nuclear translocation of IRF5 in macrophages.
ResultsThe polarization of M1 macrophages was found to be positively correlated with the severity of lung injury. TGFβ1, DNMT1, PELI1 were highly expressed, while miR-124 was down-regulated in ALI mice, and IRF5 was primarily distributed in the nucleus. TGFβ1 promoted the polarization of M1 alveolar macrophages by up-regulating DNMT1. Furthermore, DNMT1 down-regulated the expression of miR-124, which led to enhancement of M1 alveolar macrophage polarization. Meanwhile, over-expression of miR-124 inhibited the nuclear translocation of IRF5 and suppressed M1 alveolar macrophage polarization. On the other hand, over-expression of PELI1 reversed the above trends.
ConclusionCollectively, our findings indicated that TGFβ1 can promote the expression of DNMT1, which down-regulates miR-124 to activate PELI1 and nuclear translocation of IRF5, thereby aggravating ALI in mice.

研究目的：巨噬细胞（macrophages）作为急性肺损伤（acute lung injury, ALI）发病进程中的关键调控枢纽。本研究旨在探讨转化生长因子-β（transforming growth factor-β, TGFβ1）通过调控DNA甲基转移酶1（DNA methyltransferase 1, DNMT1）及微小RNA（microRNA, miR）-124/Pellino 1（PELI1）/干扰素调节因子5（interferon regulatory factor 5, IRF5）轴，在ALI中调控M1型肺泡巨噬细胞极化的分子机制。
实验方法：首先构建ALI小鼠模型，采用流式细胞术检测小鼠肺组织中M1、M2型巨噬细胞的比例；借助双荧光素酶基因报告实验验证miR-124与PELI1的靶向结合关系。在敲低TGFβ1后，通过逆转录实时定量聚合酶链反应（RT-qPCR）与蛋白质印迹（Western blot）实验，分析ALI小鼠肺组织中TGFβ1、DNMT1、miR-124、PELI1的表达模式，以及M1/M2型巨噬细胞极化标志物的表达水平；进一步采用免疫荧光实验检测巨噬细胞中IRF5的核转位情况。
实验结果：研究发现，M1型巨噬细胞极化程度与肺损伤严重程度呈正相关；ALI小鼠肺组织中TGFβ1、DNMT1、PELI1呈高表达状态，miR-124表达下调，IRF5主要分布于细胞核内。TGFβ1可通过上调DNMT1的表达，促进M1型肺泡巨噬细胞极化。进一步机制研究显示，DNMT1可下调miR-124的表达，进而增强M1型肺泡巨噬细胞极化；同时，过表达miR-124可抑制IRF5的核转位，并阻抑M1型肺泡巨噬细胞极化；反之，过表达PELI1可逆转上述实验趋势。
结论：综上，本研究结果表明，TGFβ1可上调DNMT1的表达，后者通过下调miR-124以激活PELI1并促进IRF5的核转位，最终加重小鼠ALI。