IL-10 inhibits miR-155 in primary BMDM and hPBMC. a, primary BMDM and hPBMC were stimulated with LPS (100 ng/ml) or LPS + IL-10 (20 ng/ml) and measured for miR-155 expression by RT-PCR. b, WT and IL-10-deficient BMDM (IL-10 KO) were untreated or pretreated with monoclonal (mAb) IL-10 antibody (5 μg/ml) for 1 h prior to stimulation with LPS (100 ng/ml) for 24 h. Cells were analyzed for IL-10 expression by enzyme-linked immunosorbent assay and for miR-155 expression by RT-PCR. In all cases, graphs are representative of at least three separate experiments. Error bars indicate S.D. Unstim, unstimulated.

IL-10 increases the expression of the miR-155 target, SHIP1. a, pMir-SHIP1 and pMir-SHIP1 mutant 3′-UTR luciferase plasmids were co-transfected with increasing amounts of pre-155 (1, 10, and 100 nm) in Raw264.7 cells. Luciferase activity was measured, and results were normalized for TK-Renilla activity. b, IL-10-deficient BMDM were stimulated with LPS (100 ng/ml, white bars) or LPS + IL-10 (20 ng/ml, gray bars) for the times indicated. mRNA expression for SHIP1 was measured by RT-PCR. c, IL-10-deficient BMDM were stimulated with IL-10 (20 ng/ml), LPS (100 ng/ml), or LPS + IL-10 for 24 h. SHIP1 and β-actin protein expression were measured. d, pMir-SHIP1 3′-UTR luciferase activity was measured in Raw264.7 cells after stimulation with LPS (100 ng/ml) or LPS + IL-10 (20 ng/ml) for 8 h. Luciferase activity was measured, and results were normalized for TK-Renilla activity. e, WT and Tg miR-155 splenocytes were stimulated with IL-10 (50 ng/ml) for 16 h. miR-155 and SHIP1 expression was measured by RT-PCR. In all cases, results were represented as -fold stimulation over non-stimulated control and expressed as mean ± S.D. for triplicate determinations where each experiment is representative of three separate experiments. Unstim, unstimulated.

IL-10 inhibits miR-155 expression in response to TLR4 stimulation. a, I-BMDM were stimulated with LPS (100 ng/ml), with LPS + IL-10 (20 ng/ml), or with IL-10 alone for the times indicated. Expression of miR-155, miR-21 and miR-146a was measured by RT-PCR. b, I-BMDM were pretreated for 5 min with increasing doses of IL-10 or pretreated for various times with IL-10 (20 ng/ml) prior to the addition of LPS (100 ng/ml) for 24 h. miR-155 expression was measured by RT-PCR. In both cases, results were normalized and represented as -fold stimulation over the non-stimulated control and are representative of at least three separate experiments.

IL-10 inhibits the transcription of miR-155. a, IL-10-deficient BMDM were stimulated with LPS (100 ng/ml) or LPS + IL-10 (20 ng/ml). pri-miR-155, pre-miR-155, and mature miR-155 were measured by RT-PCR. b, Raw264.7 cells were co-transfected with wild-type BIC promoter luciferase plasmid and either si-control or si-STAT3. Cells were stimulated with LPS (100 ng/ml) or with LPS + IL-10 (25 ng/ml) for 18 h. Unstim, unstimulated. c, Raw264.7 cells transfected with wild-type BIC, NF-κB mutant (kB mut), AP1 mutant (AP1 mut), or Ets1 mutant (Ets1 mut) promoter luciferase plasmids were stimulated with LPS (100 ng/ml) or with LPS + IL-10 at two different doses (25 and 50 ng/ml). Luciferase activity was measured where results were normalized for TK-Renilla activity and represented as -fold stimulation over the non-stimulated control. In all cases, results are expressed as mean ± S.D. for triplicate determinations and are representative of three separate experiments.

Schematic illustrating a possible role for miR-155 in TLR4 and SHIP1 signaling. In response to LPS, miR-155 expression is induced, resulting in a decrease in SHIP1 expression, thus allowing PI3K activation of NF-κB and MAPK to proceed and promote the pro-inflammatory response. However, in the presence of IL-10, miR-155 expression is inhibited, allowing SHIP1 expression to recover and promote the conversion of PIP3 back to its inactive PIP2 state, switching off the pro-inflammatory response. Mal, MyD88-like adapter protein.