TNF-α impairs DC-mediated MSC recruitment.

<p>Differentiated DC were stimulated with TNF-α in presence or absence of resveratrol, as indicated. <b>A.</b> Matrigel-coated transwell inserts were introduced on top of these wells and MSC were seeded in the upper compartment, at a ratio of 1MSC:10DC. MSC recruitment was allowed to take place for 24 h. MSC on the bottom of the filter were stained with DAPI and all migrated cells counted under a fluorescence microscope. The Box&Whiskers plot represents all values (min-to-max), and the median (dash) for each condition, across 6 different DC donors. Cnt – control conditions without DC in the bottom compartment. Comparisons were performed using Friedman test, followed by Dunns multiple comparisons test (* p<0.05, *** p<0.001). <b>B.</b> Culture supernatants from DC alone or from <b>C</b>. Top and Bottom compartments of transwell experiments described above, were collected, their protein content quantified and then resolved by gelatin zymography. Proteolytic white bands were revealed on a Coomassie Blue-stained background.</p

Resveratrol inhibits TNF-α-stimulation on DC.

<p>Differentiated DC were stimulated as indicated, with LPS or TNF-α, in presence or absence of resveratrol for 72 h, before surface staining for CD1a and: CD83 (top row), or CD86 (middle row) or HLA-DR (bottom row). Samples were analyzed by flow cytometry. <b>A.</b> Plots of a representative phenotypic profile, with values in each quadrant representing percentage of cells in that quadrant. <b>B.</b> Graphs represent isotype control-subtracted mean fluorescent intensity (MFI) of CD83 (top graph), CD86 (middle graph) or HLA-DR (bottom graph), over 6 independent experiments. <b>C.</b> Cell culture supernatants were collected and analyzed for the indicated cytokines by ELISA. Each donor is represented as a different symbol and the mean value, over 6 independent experiments, is represented as a dash. Statistical significance was calculated using the Wilcoxon signed rank test (*p<0.05).</p

Presence of resveratrol rescues MSC motility in co-cultures with DC.

<p>DC were differentiated and stimulated as indicated, before being added to MSC cultures (1MSC:10DC). The cultures were followed by time-lapse video-microscopy for 12 h. Image analysis was performed using ImageJ software. <b>A</b>. Images with representative pathways from cell tracking using ImageJ. Examples of MSC (red) and DC (yellow) tracks are highlighted. Scale bar: 100 µm. <b>B</b>. Quantification of MSC and DC travelling distance when cultured together, <i>versus</i> separately. Box&Whiskers plots represent median (dash) and range of values. Statistical significance was calculated using Mann-Whitney test (*p<0.05, **p<0.01, ***p<0.001).</p

Resveratrol inhibits DC stimulation without significant cell death.

<p>Differentiated DC were treated with 10, 25 or 50 µM resveratrol prior to stimulation with TNF-α or LPS. Cells were surface stained for lineage marker CD11c and activation marker CD83 (left panel), or AnnexinV and PI (right panel). Samples were analyzed by flow cytometry. Values on each quadrant represent percentage of cells in that quadrant (Cnt: control, rsv: resveratrol). Data is representative of three experiments.</p

Resveratrol inhibits T cell proliferation in response to TNF-α stimulated DC.

<p>Differentiated DC were stimulated with TNF-α, in presence or absence of resveratrol as indicated, for 24 h, before adding CFSE-labeled lymphocytes from a different individual. Lymphocytes cultured with DC differentiating cytokines (GM-CSF and IL-4), PHA, TNF-α alone or in presence of resveratrol were used as controls. After 7 days cells were stained for CD3 and analyzed by flow cytometry. <b>A.</b> Representative plots of lymphocytes cultured: alone with DC differentiating cytokines (left), with TNF-α-stimulated DC (middle), and alone stimulated with PHA (right). The CD3 positive population was gated for further analysis. <b>B.</b> Representative plot of the percentage of T lymphocytes with different numbers of cell divisions (generations - G), with ^a representing samples where TNF-α and resveratrol were removed before addition of lymphocytes. <b>C.</b> Quantification of the percentage of proliferating T cells for 5 independent experiments. Each experiment is represented as a different symbol and a dash represents the mean. Comparisons were performed using the Kruskal-Wallis followed by Dunns multiple comparisons test (* p<0.05).</p

Reversibility of MMP inhibition.

<p>(A–C) Examples of recordings showing the reversibility of the galardin effect on (A) standard, (B) stiff and (C) compliant CDLs. (D) Normalized results. Tissues in compliant, standard and stiff states were stimulated with 50 µM galardin, washed and treated again with galardin. Data are expressed as means ± SD.</p

Resveratrol inhibits NF-κB signaling in DC exposed to TNF-α.

<p>DC were stimulated with TNF-α, in presence or absence of resveratrol, for the indicated times. <b>A.</b> Cells were stained for NF-κB (p65 subunit, green staining) and nuclei were counterstained with DRAQ5 (red staining), before acquisition by ImageStream^X. Representative plot of the similarity coefficient between nucleus and NF-κB stainings of unstimulated DC (grey), DC stimulated with TNF-α alone (red), or in presence of resveratrol (Rsv, green). The yellow line corresponds to the gate for which nuclear translocation of NF-κB was considered. Three representative images of cells found in the indicated areas of the graph are shown. For the control condition, cells with a 0 similarity coefficient (no p65 translocation, grey line), and for the TNF-α -stimulated condition, cells with a similarity coefficient of 1 (NF-κB nuclear translocation, red line) are shown. BF: brightfield. <b>B.</b> Quantification of the percentage of cells with nuclear NF-κB along time (within the translocated gate) for different donors. Each symbol represents a different individual (the mean value is represented as a dash), and TNF-α stimulation in absence or presence of resveratrol is represented by closed or open symbols, respectively. Comparisons were performed using Friedman test, followed by Dunns multiple comparisons test (* p<0.05). <b>C.</b> Phosphorylation of p65 in response to TNF-α, in presence or absence of resveratrol or BAY 11-7082 (BAY) and <b>D.</b> MAPK signaling in response to TNF-α and/or resveratrol was evaluated by Western blot. DC were stimulated with TNF-α, in presence or absence of resveratrol, for indicated times. Cells were lysed and samples resolved in reducing gels, which were blotted onto nitrocellulose membranes, and probed with the indicated antibodies. The images are representative of three independent experiments. p: phosphorylated.</p

Mean values ± S.D. of unnormalized complex modulus (n = 7).

<p>Mean values ± S.D. of unnormalized complex modulus (n = 7).</p

MMPs of CDLs in compliant, standard and stiff conditions visualized by gelatin zymography.

<p>(A) Zymogram showing pro-enzyme and active MMP band profile of CDLs in the three mechanical states. (B) Zymogram comparing the band profiles of CDLs, compared with human cell line showing MMP-2 and MMP-9 activity; this zymogram was included because of its very good band separation. (C) Optical density of MMP activity in CDLs in the three mechanical states. (D) Comparative densitometric analysis of scanned gels of CDLs in the different mechanical states with and without 50 µM galardin. Data are expressed as means ± SD. The asterisk (*) represents statistically significant difference P<0.05. MMPs were detected in more than six animals for each of the three mechanical states. (E) Zymogram comparing standard CDLs with and without galardin treatment.</p

Schematic representation of the CDL dissection.

<p>Schematic representation of the CDL dissection.</p