MicroRNA-146a and RBM4 form a negative feed-forward loop that disrupts cytokine mRNA translation following TLR4 responses in human THP-1 monocytes

Within hours after its initiation, the severe systemic inflammatory response of sepsis shifts to an adaptive anti-inflammatory state with coincident immunosuppression. This anti-inflammatory phenotype is characterized by diminished proinflammatory cytokine gene expression in response to toll-like receptor (TLR) stimulation with bacterial endotoxin/ LPS, also known as endotoxin tolerance/adaptation. Our and other studies have established that gene-specific reprogramming following TLR4 responses independently represses transcription and translation of proinflammatory genes such as TNFα. We also previously demonstrated that TNFα and IL-6 mRNA translation is repressed in endotoxin adapted THP-1 human monocytes by a miRNA-based mechanism involving the argonaute family protein Ago2. Here, we further define the molecular nature of reprogramming translation by showing that TLR4-induced microRNA-146 promotes a feed-forward loop that modifies the subcellular localization of the RNA-binding protein RBM4 and promotes its interaction with Ago2. This interaction results in assembly of a translation repressor complex that disrupts TNFα and IL-6 cytokine synthesis in endotoxin adapted THP-1 monocytes. This novel molecular path prevents phosphorylation of RBM4 on serine-309 by p38 MAPK, which leads to RBM4 accumulation in the cytosol and interaction with Ago2. We further find that microRNA-146a knockdown by antagomirs or inhibiting protein phosphatases by okadaic acid, increases p38 MAPK phosphorylation and results in RBM4 serine-309 phosphorylation and nuclear re-localization, which disrupts RBM4 and Ago2 interactions and restores TLR4-dependent synthesis of TNFα and IL-6. We conclude that miR-146a plays a diverse and critical role in limiting an excessive acute inflammatory reaction.