Identification of miR-181a target mRNAs by RIP-CHIP during terminal differentiation of DC-SIGN+ mo-DCs

DC-SIGN+ monocyte-derived dendritic cells (mo-DCs) play important roles in bacterial infections and inflammatory diseases, but the factors regulating their differentiation and proinflammatory status remain poorly defined. Here, we identify a micro-RNA, miR-181a, and a molecular mechanism that simultaneously regulate the acquisition of DC-SIGN+ expression and the activation state of DC-SIGN+ mo-DCs. Specifically, we show that miR-181a promotes DC-SIGN expression during terminal mo-DC differentiation and limits its sensitivity and responsiveness to TLR triggering and CD40 ligation. Mechanistically, miR-181a sustains ERK-MAPK signaling in mo-DCs, thereby enabling the maintenance of high levels of DC-SIGN and a high activation threshold. Low miR-181a levels during mo-DC differentiation, induced by inflammatory signals, do not support the high phospho-ERK signal transduction required for DC-SIGNhi mo-DCs and lead to development of proinflammatory DC-SIGNlo/- mo-DCs. Collectively, our study demonstrates that high DC-SIGN expression levels and a high activation threshold in mo-DCs are linked and simultaneously maintained by miR-181a. RIP-CHIP (RNA-Binding Protein Immunoprecipitation-Microarray (Chip) Profiling) was performed to identify miR-181a specific target genes and delineate the Ago-2 dependent miRNA-targetome during terminal differentiation of DC-SIGN+ mo-DCs. Here, CD14+ monocyte intermediates (designated progenitor cell-derived monocytes, or moPs) arising in cultures of cord blood CD34+ progenitor cells were transduced with miR-181a KD or scramble control vectors, and supplemented with GM-CSF and IL-4 for 24 hours; then Ago-2-dependent miRNAs and their associated target mRNAs were pulled down, isolated, and analyzed on Affymetrix microarray chips.