Tacrolimus inhibits IL-12 p40 protein and mRNA expression.

<p>(<b>A</b>) Murine bone marrow-derived macrophages (BMDMs) were pretreated with the indicated concentrations of tacrolimus for 1 h. Cells were then either left untreated (NS) or stimulated with LPS (100 ng/ml) or LPS and IFN-γ (10 ng/ml) for 24 h. IL-12 p40 protein secretion was assayed from supernatants by ELISA. (<b>B</b>) BMDMs were incubated with the indicated concentrations of tacrolimus for 1 h followed by 1 h treatment with IFN-γ (10 ng/ml, where indicated) prior to LPS (100 ng/ml) stimulation for 4 h or left untreated (NS). Cells were harvested and total RNA was assayed for <i>Il12b</i> mRNA levels by real-time RT-PCR. Each result represents the mean ± standard error (SD) for duplicate assays from three independent experiments. * p<0.05.</p

11R-VIVIT inhibits pro-inflammatory cytokine expression in murine macrophages.

<p>BMDMs from WT mice were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or 11R-VEET for 1 h followed by LPS (100 ng/ml) and IFN-γ (10 ng/ml) for 24 h. IL-12 p70 (<b>A</b>), IL-23 (<b>B</b>) and TNF (<b>C</b>) protein secretion were assayed from supernatants by ELISA. Each result represents mean ± SE of duplicate assays from three independent experiments. * p<0.05.</p

VIVIT treatment reduces spontaneous secretion of colonic inflammatory cytokines.

<p>Explants from colons of VIVIT (n = 12 for <b>A</b>, <b>B</b>; n = 7 for <b>C</b>) and VEET (n = 9 for <b>A</b>, <b>B</b>; n = 6 for <b>C</b>) treated mice were incubated in RPMI medium for 24 h, released cytokines were then determined by ELISA. We noted a marked difference in the gut spontaneous secretion of TNF-α (<b>B</b>) in gut supernatants from VIVIT treated mice, with a statistically significant change in IL-12 p40 (<b>A</b>) and in interferon-γ (<b>C</b>) production compared to VEET treated mice. Values are normalized to weight of intestinal explants, and data are presented as mean ± SEM.</p

Inhibition of IL-12 p40 by 11R-VIVIT is independent of IL-10.

<p>(<b>A</b>) Murine BMDMs were either untreated or pretreated with the indicated concentrations of 11R-VIVIT (white bars) or 11R-VEET (black bars) for 1 h followed by LPS (100 ng/ml) alone or together with IFN-γ (10 ng/ml) for 24 h. IL-10 protein secretion was assayed from supernatants by ELISA. (<b>B</b>) BMDMs from <i>Il10</i>^−/− mice were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or 11R-VEET for 1 h followed by LPS (100 ng/ml) and LPS plus IFN-γ (10 ng/ml) for 24 h. IL-12 p40 protein secretion was assayed from supernatants by ELISA. Results represent the mean ± SD for duplicate assays from three independent experiments. * p<0.05, ** p<0.01.</p

11R-VIVIT reduces DNA binding to the NFAT/IRF8 site in the murine IL-12 p40 promoter.

<p>BMDMs were either untreated (lane 1) or pretreated with the indicated concentrations of 11R-VEET (lanes 2–4) or 11R-VIVIT (lanes 5–7) for 1 h. Cells were then treated for 1 h with IFN-γ (10 ng/ml) prior to LPS (100 ng/ml) stimulation for 4 h. Cells were harvested and nuclear extracts were prepared for EMSA. ³²P labeled oligonucleotide probe spanning the NFAT/IRF8 site of the IL-12 p40 promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034172#pone.0034172-Zhu1" target="_blank">[10]</a> was incubated with 10 mg of nuclear extracts on ice for 30 min prior to electrophoresis. For supershift assays (lanes 3,4 and 6,7), 10 mg nuclear extract were incubated with 3 mg of anti-NFAT c1 (lanes 3 and 6) or anti-IRF8 (lanes 4 and 7) antibodies for 30 min on ice prior to addition of the ³²P labeled probe and 30 min incubation on ice followed by electrophoresis. The arrows represent DNA-protein complexes formed before and after incubation with the indicated antibodies. The above result is representative of five independent experiments.</p

NFAT binds to <i>Nos2</i> promoter and its selective inhibition abrogates nitric oxide secretion in macrophages.

<p>(<b>A</b>) Nuclear extracts were prepared from BMDMs stimulated with LPS (lane 1) or LPS and IFN-γ (lanes 2–7). Extracts were either incubated with a labeled probe NFAT/ISRE (element from region II of the promoter scheme), a competitor ISRE oligonucleotide, or with the indicated antibodies. DNA-protein complexes (indicated by arrow) were separated by electrophoresis. EMSA revealed binding of both IRF8 and NFATc1 to the same <i>Nos2</i> promoter element. (<b>B</b>) BMDMs from WT mice were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or the control peptide for 1 h followed by LPS (100 ng/ml) and IFN-γ (10 ng/ml) for 24 h. Nitric oxide secretion was assayed from supernatants by Greiss reaction. Experiments were performed in duplicate and repeated three times (mean ± SEM). (<b>C</b>) BMDMs were either untreated or pretreated with the indicated concentrations of 11R-VIVIT for 1 h followed by 1 h treatment with LPS alone or IFN-γ (10 ng/ml) prior to LPS (100 ng/ml) treatment for 4 h. Cells were harvested and total RNA was assayed for <i>Nos2</i> mRNA levels by real-time RT PCR. Data is representative of three independent experiments. * p<0.05.</p

11R-VIVIT inhibits IL-12 p40 protein and mRNA expression.

<p>(<b>A</b>) Murine bone marrow-derived macrophages (BMDMs) were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or the control peptide 11R-VEET for 1 h followed by LPS (100 ng/ml) alone or together with IFN-γ (10 ng/ml) for 24 h. IL-12 p40 protein secretion was assayed from supernatants by ELISA. (<b>B</b>) BMDMs were either untreated or pretreated with the indicated concentrations of 11R-VIVIT for 1 h followed by 1 h treatment with IFN-γ (10 ng/ml) prior to LPS (100 ng/ml) treatment for 4 h. Cells were harvested and total RNA was assayed for <i>Il12b</i> mRNA levels by real-time RT-PCR. Results were normalized with the respect to the levels of <i>β-actin</i> mRNA, and represent the mean ± SE for duplicate assays from three independent experiments. * p<0.05, ** p<0.01.</p