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. 2013:2013:574029.
doi: 10.1155/2013/574029. Epub 2013 Jul 9.

Resveratrol prevents dendritic cell maturation in response to advanced glycation end products

Brigitta Buttari  1 , Elisabetta Profumo, Francesco Facchiano, Elif Inci Ozturk, Luca Segoni, Luciano Saso, Rachele Riganò
Affiliations

Resveratrol prevents dendritic cell maturation in response to advanced glycation end products

Brigitta Buttari et al. Oxid Med Cell Longev. 2013.

Abstract

Advanced glycation end products (AGEs), generated through nonenzymatic glycosylation of proteins, lipids, and nucleic acids, accumulate in the body by age thus being considered as biomarkers of senescence. Senescence is characterized by a breakdown of immunological self-tolerance, resulting in increased reactivity to self-antigens. Previous findings suggest that AGE and its receptor RAGE may be involved in the pathogenesis of autoimmune reactions through dendritic cell (DC) activation. The aim of this study was to investigate whether resveratrol, a polyphenolic antioxidant compound with tolerogenic effects on DCs, was able to counteract the mechanisms triggered by AGE/RAGE interaction on DCs. By immunochemical and cytofluorimetric assays, we demonstrated that in vitro pretreatment of human monocyte-derived DCs with resveratrol prevents DC activation in response to glucose-treated albumin (AGE-albumin). We found that resveratrol exerts an inhibitory effect on DC surface maturation marker and RAGE up-regulation in response to AGE-albumin. It also inhibited proinflammatory cytokine expression, allostimulatory ability upregulation, mitogen-activated protein (MAP) kinases, and NF-κB activation in AGE-albumin-stimulated DCs. We suggest that resveratrol, by dismantling AGE/RAGE signaling on DCs may prevent or reduce increased reactivity to self-molecules in aging.

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Figures

Figure 1
Figure 1
Molecular characterization of glycated albumin. (a) Structural analysis of the amino acid sequence of albumin. The analysis indicates that 22 (in bold) out of 60 lysine residues (K) are potential glycation sites. (b) Fluorescent AGE formation in an albumin solution incubated in the presence of D-glucose or D-mannitol (250 mM) after 10, 30, or 60 days. Data are expressed as means of arbitrary unit/mg of proteins ± SD (n = 3). *P < 0.05; †‡ P < 0.001. The black star indicates the moderately modified AGE-albumin used in the subsequent cellular studies. (c) SDS-PAGE analysis of albumin preparations (50 μg per lane) incubated or not with 250 mM glucose for the reported times, followed by Coomassie staining, according to standard protocol. (d) Size exclusion chromatography of albumin and AGE-albumin. Albumin was incubated for 30 days with 250 mM D-mannitol (albumin) or D-glucose (AGE-albumin). One representative experiment out of three is reported.
Figure 2
Figure 2
Flow cytometric analysis of phenotypic dendritic cell (DC) maturation. Five-day human DCs pretreated or not with resveratrol (Resv, 50 μM) were cultured for 18 hours with or without AGE-albumin (AGE, 30 μg/mL). DCs treated with LPS (0.2 μg/mL), albumin (Alb; 30 μg/mL), and resveratrol (Resv; 50 μM) were used as controls. Expression of surface molecules was analyzed by flow cytometry as described in Section 2. Phenotypic maturation of DCs was detected by the appearance of CD83 (a) and by the expression of surface molecules (b). After 18 hours of incubation LPS and AGE-albumin induced almost similar DC maturation. Pretreatment of iDCs with resveratrol prevented the phenotypic maturation of DCs induced by AGE-albumin (CD83 and HLA-DR: ∗†‡ P < 0.001; CD40 and CD80: ∗† P < 0.001, ‡ P < 0.01; CD86: *P < 0.001, †‡ P < 0.01). Results are expressed as positive cell percentages (a) and mean fluorescence intensity (MFI, B) (means ± SD, n = 4). Samples were analyzed on a FACSCanto cytofluorimeter using CellDIVA (BD-Biosciences). P values by one-way ANOVA with a Bonferroni post hoc test.
Figure 3
Figure 3
Analysis of RAGE expression on dendritic cells (DCs). Five-day human DCs pretreated or not with resveratrol (Resv, 50 μM) were cultured for 18 hours with or without AGE-albumin (AGE, 30 μg/mL). DCs treated with LPS (0.2 μg/mL), albumin (Alb; 30 μg/mL) and resveratrol (Resv; 50 μM) were used as controls. (a) Flow cytometric analysis of RAGE expression. AGE-albumin (AGE) induced a statistically significant upregulation of RAGE (panel (i)) whose expression remained elevated until 60 hours (panel (ii)). Pretreatment of iDCs with resveratrol prevented RAGE upregulation in response to AGE-albumin at all-time points investigated (panel (ii)). Results are expressed as mean fluorescence intensity (MFI) (means ± SD, n = 3). *P < 0.001. † P < 0.05. (b) Western blotting analysis of RAGE expression on DCs. Western blotting followed by densitometric analysis confirmed RAGE downregulation on AGE-albumin-stimulated DCs by resveratrol (means ± SD, n = 3). ∗† P < 0.001.
Figure 4
Figure 4
Cytokine production in dendritic cell (DC) culture supernatants. Five-day human DCs pretreated or not with resveratrol (Resv, 50 μM) were cultured for 18 hours with or without AGE-albumin (AGE, 30 μg/mL). DCs treated with LPS (0.2 μg/mL), albumin (Alb; 30 μg/mL), and resveratrol (Resv; 50 μM) were used as controls. Supernatants were collected after 18 hours to measure IL-12p70, TNF-α, IL-10, and IL-1β by specific ELISA experiments. LPS and AGE-albumin (AGE) triggered a statistically significant upregulation of all cytokine secretions. Pretreatment of iDC with resveratrol prevented the upregulation of all proinflammatory cytokines in response to AGE-albumin. Results are expressed as means ± SD of four independent experiments (IL-12p70: *P < 0.01, †‡ P < 0.001; TNF-α and IL-1β: ∗†‡ P < 0.001, § P < 0.05; IL-10: ∗† P < 0.001).
Figure 5
Figure 5
Allostimulatory ability of dendritic cells (DCs). Five-day human DCs were stimulated for 18 hours with LPS (0.2 μg/mL) (■), and AGE-albumin (AGE; 30 μg/mL) (⚫), AGE-albumin plus resveratrol (Resv, 50 μM) (◯) or left unstimulated (Ctrl) (□). After 18 hours, DCs were extensively washed and cultured with allogeneic T lymphocytes (1 × 105 cells/well) for 3 days at various stimulator-responder ratios (1 : 4 to 1 : 64 DC/T). (a) Proliferation of allogeneic T cells was measured by [3H]-methyl-thymidine incorporation. Data are presented as mean cpm ± SD of four independent experiments (at 1/16 DC/T cells ratio: AGE-albumin and LPS versus unstimulated: ∗† P < 0.001; AGE-albumin + resveratrol versus AGE-albumin: ‡ P < 0.001). P values by the one-way ANOVA with a Bonferroni post hoc test. (b) Proliferation of IFN-γ-producing CD4+ T cells was determined by staining allogeneic CD4+ T cells with CFDA-SE (2.5 μM) and culturing them with irradiated AGE-albumin-stimulated or unstimulated DCs at 1 : 32 DC : T cell ratio. CD4+ T-cell proliferative activity (CFDA-SE content), as well as their ability to produce IFN-γ was measured on day 3 by flow cytometry as described in Section 2. Figure shows a representative experiment out of 3 with similar results. The numbers show the percentage of proliferating IFN-γ-producing CD4+ T cells. Samples were analyzed on a FACSCanto cytofluorimeter using CellDiva software (BD-Biosciences).
Figure 6
Figure 6
MAPK and NF-κB activation in dendritic cells (DCs). Five-day human DCs pretreated or not with resveratrol (Resv, 50 μM) were cultured for 45 minutes with or without AGE-albumin (AGE, 30 μg/mL). DCs treated with albumin (Alb; 30 μg/mL), resveratrol (Resv; 50 μM), LPS (0.2 μg/mL), or PMA (0.2 μg/mL) were used as controls. Cells were then analyzed by cell-based ELISA MAPK assay to monitor p38 and ERK activation and by NF-κB (p65 and p50) transcription factor assay to monitor NF-κB activation. (a) AGE-albumin stimulation induced the activation of both MAPK p38 and ERK pathways in DCs. Pretreatment of DCs with resveratrol prevented the upregulation of both MAPKs in response to AGE-albumin (n = 4; p-p38/p38: ∗‡ P < 0.001, † P < 0.01; pERK/ERK: ∗‡ P < 0.001, † P < 0.05). (b) AGE-albumin stimulation significantly increased active p65 and p50 levels in DCs. Pretreatment of DCs with resveratrol prevented the upregulation of both active p50 and p65 in response to AGE-albumin. The results are expressed as arbitrary units (n = 4, p50 and p65: *P < 0.05; † P < 0.001).
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