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/lipopolysaccharide (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 tumor necrosis factor alpha (TNFα). We also previously demonstrated that TNFα and interleukin (IL)-6 mRNA translation is repressed in endotoxin-adapted THP-1 human monocytes by an miRNA-based mechanism involving the argonaute family protein argonaute 2 (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 (RNA-binding motif protein 4) and promotes its interaction with Ago2. This interaction results in the 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 the phosphorylation of RBM4 on serine-309 by p38 MAPK (mitogen-activated protein kinase), which leads to RBM4 accumulation in the cytosol and interaction with Ago2. We further find that microRNA-146a knockdown by antagomirs or protein phosphatase inhibition by okadaic acid increases p38 MAPK phosphorylation and results in RBM4 serine-309 phosphorylation and nuclear relocalization, which disrupts RBM4 and Ago2 interactions and restores TLR4-dependent synthesis of TNFα and IL-6. We conclude that miR-146a has a diverse and critical role in limiting an excessive acute inflammatory reaction.

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/lipopolysaccharide (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 tumor necrosis factor alpha (TNFα). We also previously demonstrated that TNFα and interleukin (IL)-6 mRNA translation is repressed in endotoxin-adapted THP-1 human monocytes by an miRNA-based mechanism involving the argonaute family protein argonaute 2 (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 (RNA-binding motif protein 4) and promotes its interaction with Ago2. This interaction results in the 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 the phosphorylation of RBM4 on serine-309 by p38 MAPK (mitogen-activated protein kinase), which leads to RBM4 accumulation in the cytosol and interaction with Ago2. We further find that microRNA-146a knockdown by antagomirs or protein phosphatase inhibition by okadaic acid increases p38 MAPK phosphorylation and results in RBM4 serine-309 phosphorylation and nuclear relocalization, which disrupts RBM4 and Ago2 interactions and restores TLR4-dependent synthesis of TNFα and IL-6. We conclude that miR-146a has a diverse and critical role in limiting an excessive acute inflammatory reaction.

An anti-inflammatory phenotype with pronounced immunosuppression develops during sepsis, during which time neutrophils and monocytes/macrophages limit their Toll-like receptor 4 responses to bacterial lipopolysaccharide (LPS/endotoxin). We previously reported that during this endotoxin-tolerant state, distinct signaling pathways differentially repress transcription and translation of proinflammatory cytokines such as TNFα and IL-6. Sustained endotoxin tolerance contributes to sepsis mortality. While transcription repression requires chromatin modifications, a translational repressor complex of Argonaute 2 (Ago2) and RNA-binding motif protein 4 (RBM4), which bind the 3′-UTR of TNFα and IL-6 mRNA, limits protein synthesis. Here, we show that Dcp1 supports the assembly of the Ago2 and RBM4 repressor complex into cytoplasmic processing bodies (p-bodies) in endotoxin-tolerant THP-1 human monocytes following stimulation with LPS, resulting in translational repression and limiting protein synthesis. Importantly, this translocation process is reversed by Dcp1 knockdown, which restores TNFα and IL-6 protein levels. We also find this translational repression mechanism in primary macrophages of septic mice. Because p-body formation is a critical step in mRNA translation repression, we conclude that Dcp1 is a major component of the translational repression machinery of endotoxin tolerance and may contribute to sepsis outcome.