MicroRNA-mediated Krüppel-like factor 4 upregulation induces alternatively activated macrophage-associated marker and chemokine transcription in 4,4’-methylene diphenyl diisocyanate exposed macrophages

1. Occupational exposure to 4,4’-methylene diphenyl diisocyanate (MDI) is associated with occupational asthma (OA) development. Alveolar macrophage-induced recruitment of immune cells to the lung microenvironment plays an important role during asthma pathogenesis. Previous studies identified that MDI/MDI-glutathione (GSH)-exposure downregulates endogenous <i>hsa-miR-206-3p</i>/<i>hsa-miR-381-3p</i>. Our prior report shows that alternatively activated (M2) macrophage-associated markers/chemokines are induced by MDI/MDI-GSH-mediated Krüppel-Like Factor 4 (KLF4) upregulation in macrophages and stimulates immune cell chemotaxis. However, the underlying molecular mechanism(s) by which MDI/MDI-GSH upregulates KLF4 remain unclear. 2. Following MDI-GSH exposure, microRNA(miR)-inhibitors/mimics or plasmid transfection, endogenous <i>hsa-miR-206-3p</i>/<i>hsa-miR-381-3p,</i> KLF4, or M2 macrophage-associated markers (<i>CD206</i>, <i>TGM2</i>), and chemokines (<i>CCL17, CCL22, CCL24</i>) were measured by either RT-qPCR, western blot, or luciferase assay. 3. MDI-GSH exposure downregulates <i>hsa-miR-206-3p</i>/<i>hsa-miR-381-3p</i> by 1.46- to 9.75-fold whereas upregulates <i>KLF4</i> by 1.68- to 1.99-fold, respectively. <i>In silico</i> analysis predicts binding between <i>hsa-miR-206-3p/hsa-miR-381-3p</i> and <i>KLF4.</i> Gain- and loss-of-function, luciferase reporter assays and RNA-induced silencing complex-immunoprecipitation (RISC-IP) studies confirm the posttranscriptional regulatory roles of <i>hsa-miR-206-3p/hsa-miR-381-3p</i> and <i>KLF4</i> in macrophages. Furthermore, <i>hsa-miR-206-3p</i>/<i>hsa-miR-381-3p</i> regulate the expression of M2 macrophage-associated markers and chemokines <i>via</i> KLF4. 4. In conclusion, <i>hsa-miR-206-3p/hsa-miR-381-3p</i> play a major role in regulation of MDI/MDI-GSH-induced M2 macrophage-associated markers and chemokines by targeting the <i>KLF4</i> transcript, and KLF4-mediated regulation in macrophages.

1. 职业暴露于4,4’-亚甲基二苯基二异氰酸酯（4,4’-methylene diphenyl diisocyanate, MDI）与职业性哮喘（occupational asthma, OA）的发生密切相关。哮喘发病进程中，肺泡巨噬细胞介导的免疫细胞向肺微环境募集发挥着关键作用。既往研究证实，MDI/MDI-谷胱甘肽（glutathione, GSH）暴露会下调内源性hsa-miR-206-3p与hsa-miR-381-3p的表达。本团队前期研究显示，MDI/MDI-GSH可通过促进巨噬细胞中Krüppel样因子4（Krüppel-Like Factor 4, KLF4）的上调，诱导交替活化（M2型）巨噬细胞的标志性分子与趋化因子表达，进而刺激免疫细胞趋化。然而，MDI/MDI-GSH上调KLF4的具体分子机制仍未阐明。2. 经MDI-GSH暴露处理后，通过转染微小RNA（microRNA, miR）抑制剂/模拟物或质粒，分别采用实时定量聚合酶链式反应（RT-qPCR）、蛋白质免疫印迹（western blot）或荧光素酶报告基因实验，检测内源性hsa-miR-206-3p、hsa-miR-381-3p、KLF4、M2型巨噬细胞标志性分子（CD206、TGM2）以及趋化因子（CCL17、CCL22、CCL24）的表达水平。3. MDI-GSH暴露可使hsa-miR-206-3p与hsa-miR-381-3p的表达下调1.46至9.75倍，同时使KLF4的表达分别上调1.68至1.99倍。计算机预测（in silico）分析显示，hsa-miR-206-3p/hsa-miR-381-3p与KLF4之间存在结合位点。功能获得与功能缺失实验、荧光素酶报告基因实验以及RNA诱导沉默复合物免疫沉淀（RNA-induced silencing complex-immunoprecipitation, RISC-IP）验证了hsa-miR-206-3p/hsa-miR-381-3p对巨噬细胞中KLF4的转录后调控作用。进一步研究发现，hsa-miR-206-3p/hsa-miR-381-3p可通过KLF4调控M2型巨噬细胞标志性分子与趋化因子的表达。4. 综上，hsa-miR-206-3p/hsa-miR-381-3p可通过靶向KLF4转录本，调控MDI/MDI-GSH诱导的M2型巨噬细胞标志性分子与趋化因子表达，进而介导巨噬细胞中KLF4的调控效应。