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. 2014:2014:257543.
doi: 10.1155/2014/257543. Epub 2014 May 8.

Resveratrol counteracts inflammation in human M1 and M2 macrophages upon challenge with 7-oxo-cholesterol: potential therapeutic implications in atherosclerosis

Brigitta Buttari  1 , Elisabetta Profumo  1 , Luca Segoni  1 , Daniela D'Arcangelo  2 , Stefania Rossi  3 , Francesco Facchiano  3 , Luciano Saso  4 , Rita Businaro  5 , Luigi Iuliano  5 , Rachele Riganò  1
Affiliations

Resveratrol counteracts inflammation in human M1 and M2 macrophages upon challenge with 7-oxo-cholesterol: potential therapeutic implications in atherosclerosis

Brigitta Buttari et al. Oxid Med Cell Longev. 2014.

Abstract

the proinflammatory M1 subset and the anti-inflammatory M2 one. 7-oxo-cholesterol, the most abundant cholesterol autoxidation product within atherosclerotic plaque, is able to skew the M1/M2 balance towards a proinflammatory profile. In the present study, we explored the ability of the polyphenolic compound resveratrol to counteract the 7-oxo-cholesterol-triggered proinflammatory signaling in macrophages. Resveratrol-pretreated human monocyte-derived M1 and M2 macrophages were challenged with 7-oxo-cholesterol and analyzed for phenotype and endocytic ability by flow cytometry, for metalloproteinase- (MMP-) 2 and MMP-9 by gelatin zymography, and for cytokine, chemokine, and growth factor secretome by a multiplex immunoassay. We also investigated the NF-κB signaling pathway. In the M1 subset, resveratrol prevented the downregulation of CD16 and the upregulation of MMP-2 in response to 7-oxo-cholesterol, whereas in M2 macrophages it prevented the upregulation of CD14, MMP-2, and MMP-9 and the downregulation of endocytosis. Resveratrol prevented the upregulation of several proinflammatory and proangiogenic molecules in both subsets. We identified modulation of NF-κB as a potential mechanism implicated in 7-oxo-cholesterol and resveratrol effects. Our results strengthen previous findings on the immunomodulatory ability of resveratrol and highlight its role as potential therapeutic or preventive compound, to counteract the proatherogenic oxysterol signaling within atherosclerotic plaque.

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Figures

Figure 1
Figure 1
Flow cytometric analysis of differentiation and activation surface markers on M1 and M2 macrophage subsets. Resveratrol prevented 7-oxo-cholesterol (7oxo-C) induced CD16 and CD14 changes in M1 (a) and M2 (b) macrophage subsets. Polarized M1 and M2 macrophages pretreated or not with resveratrol for 1 hour were stimulated with 7oxo-C (15 μM) for 20 hours and then analyzed for surface molecule expression by flow cytometry. Macrophages stimulated with LPS (100 ng/mL) were used as positive control. The results of one representative experiment of three are shown. The number in the histograms shows the mean fluorescence intensity.
Figure 2
Figure 2
Analysis of macrophage endocytosis. Resveratrol prevented the impairment of endocytosis in M2 macrophages in response to 7-oxo-cholesterol (7oxo-C). M1 (a) and M2 (b) macrophages—pretreated or not with resveratrol (30 μM) for 1 hour and then incubated with 7oxo-C (15 μM) for 20 h or left unstimulated—were added with FITC-dextran (1 mg/mL) and incubated for 30 minutes at 37°C at 5% CO2. The cellular uptake was analyzed by flow cytometry. The results of one representative experiment of three are shown. The number in the histograms shows the mean fluorescence intensity.
Figure 3
Figure 3
Gel zymography for MMP-2 and MMP-9 detection. Pretreatment of cells with resveratrol prevented upregulation of MMP-2 (a) in M1 and M2 subsets and of MMP-9 (b) in M2 macrophages in response to 7-oxo-cholesterol (7oxo-C). Culture supernatants of polarized M1 (□) and M2 (■) macrophages treated or not with resveratrol (Resv; 30 μM) for 1 hour and then stimulated with 15 μM 7oxo-C for 20 hours or left unstimulated were subjected to acrylamide gel electrophoresis and the gelatinolytic activity was determined by classical zymography as described in Section 2. Results are expressed as means ± SD of four independent experiments (*P < 0.001; † P < 0.05). Representative Western blotting and zymograms are reported on the top of the bar plot.
Figure 4
Figure 4
Secretome profile of cytokines, chemokines, and growth factors in M1 and M2 macrophages pretreated or not with resveratrol before stimulation with 7-oxo-cholesterol. Polarized M1 (□) and M2 (■) macrophages were stimulated with 15 μM 7-oxo-cholesterol (7oxo-C) for 20 hours after pretreatment or not with resveratrol (30 μM) for 1 hour at 37°C, 5% CO2. At the end of incubation time, supernatants were analyzed for cytokines (a), chemokines (b), and growth factors (c) release using a commercially available multiplex bead-based sandwich immunoassay kit, as described in Section 2. Results are expressed as means ± SD of three independent experiments (*P < 0.01; † P < 0.001; ‡ P < 0.05).
Figure 5
Figure 5
NF-κB activation in M1 and M2 macrophages. 7-oxo-cholesterol (7oxo-C) stimulation significantly increased active NF-κB p50 (a) and p65 (b) levels in M2 subset. Pretreatment of M2 with resveratrol prevented the upregulation of both active p50 and p65 in response to 7-oxo-cholesterol. M1 (□) and M2 (■) macrophages pretreated or not with resveratrol (30 μM) for 30 minutes were cultured for 1 hour with or without 7oxo-C (15 μM). Cells were then analyzed by NF-κB (p50 and p65) transcription factor assay to monitor NF-κB activation. The results are expressed as arbitrary units (n = 3, p50: *P < 0.01; † P < 0.001; p65: *P < 0.05; † P < 0.01).
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