Regulation of IL-1β, TAB2, Pellino-1 and SMAD6 in LPS-activated moDCs. Immature human moDCs were stimulated with LPS and harvested at the indicated time points. (A) qRT-PCR kinetics analysis for the indicated genes, normalizing on GAPDH RNA levels. Data indicate the mean (± SD) of a triplicate qPCR, representing at least 3 independent experiments, each derived from a different DC preparation. (B) Immunoblot kinetics analysis for Pellino-1, SMAD6 and TAB2. An actin immunoblot is shown for equal loading control. Data are representative of at least 3 independent experiments.

Induction of IL-1β and caspase-1 in moDCs after miR-155 knockdown. Immature human moDCs were transfected either with anti-miR-155 or with scramble LNA oligonucleotides and stimulated or not with LPS for 24 h. Cells and supernatants were collected and after harvesting both total RNA and cells extracts were enriched. (A) Total RNA was used to quantify expression of the indicated genes by relative qRT-PCR, normalizing on GAPDH RNA levels. The graph shows a log₂-scale fold induction calculated by normalizing the anti-miR-155 LNA expression values on the scramble LNA values. Data indicate the mean (± SD) of a triplicate qPCR. (B) Cells extracts were used to perform immunoblots to assay the immature form of IL-1β (pro-IL-1β) and caspase-1. An actin immunoblot is shown for equal loading control. (C) Supernatants were used to perform quantitative sandwich enzyme immunoassays to measure secreted IL-1β. In all panels, data are representative of at least 3 independent experiments, each derived from a different DC preparation. Symbols: scr, scramble LNA; 155, anti-miR-155 LNA.

TAB2 is a direct target of miR-155 in moDCs. In A and B moDCs were treated, harvested and analyzed as described in Fig. 3. (A) qRT-PCR analysis for the indicated genes, normalizing on GAPDH RNA levels. (B) Immunoblots to assay Pellino-1, TAB2 and CEBPB. An actin immunoblot is shown for equal loading control. Symbols: scr, scramble LNA; 155, anti-miR-155 LNA. (C) miR-155 directly repress TAB2 mRNAs through 3′ UTR interactions. The full-length 3′ UTRs of the human genes BACH1 (positive control), Pellino-1, TAB-2 were cloned into a reporter vector, downstream of luciferase. These vectors were then cotransfected with the indicated amount of miR-155 or miR control precursor in 293T cells, and luciferase activity was quantified. The graph shows the percentage of remaining luciferase activity calculated by normalizing the miR-155 expression values on the miR-control values. Data are representative of at least 3 independent experiments. (D) Predicted interaction between the miR-155 seed and the seed matches on human Pellino-1 and TAB2 3′UTR mRNAs, determined with the software TargetScan. The type of seed match and the relative position on the 3′UTR mRNA are indicated. A-T and G-C base pairs are indicated with a line, whereas G:U bounds are indicated with a dot.

miR-155 expression analysis in LPS-stimulated moDCs and gene expression analysis of moDCs after miR-155 knockdown. (A) Microarray analysis of miRNA expression in moDCs after stimulation with LPS. The scatter plot shows averaged (n = 16, quadruplicate hybridizations of 2 independent technical replicates, performed on 2 different DC preparations) background-subtracted raw intensities for each probe on both channels for Cy3-labeled control (immature DCs) and Cy5-labeled 4 h or 16 h LPS-treated cells (mature DCs) and their respective dye-swaps. Each dot represents one miRNA probe [(1) hsa_miR-155, (2) ambi_miR-7084, (3) hsa_miR-340, (4) hsa_miR-368, (5) mmu_miR-350]. (B) miR-155 expression was determined by relative qRT-PCR and normalized on U6 RNA levels. The data indicate the mean (± SD) of a triplicate qPCR, representing at least 3 independent experiments, each derived from a different DC preparation. (C) Total RNA was used for gene expression analysis using Affymetrix microarrays. We tested 12,190 probe sets that passed the control criteria. The scatter plot shows the anti-miR-155 LNA expression values normalized to scramble LNA values, applying a cut-off of 1.5. Each dot represents one probe set. A list of the 1324 probe sets identified in presence of LPS is available in Table S1. UNT, untreated cells.