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.