Abstract
Aims/hypothesis
We recently found that activation of the type III histone deacetylase sirtuin 1 suppresses T cell immune responses. Here we sought to determine the therapeutic potential of the sirtuin 1 activator resveratrol in the treatment of diabetes in the NOD mouse model of type 1 diabetes and the mechanisms underlying such potential.
Methods
NOD mice were fed or subcutaneously injected with resveratrol and evaluated for development of diabetes. Splenocytes from resveratrol-treated and control mice were analysed by gene array. The altered expression of inflammatory genes induced by resveratrol was validated and the role of changed gene expression in prevention of diabetes was determined.
Results
Resveratrol administration potently prevented and treated type 1 diabetes in NOD mice. Gene array analysis indicated a dramatic decrease in expression of Ccr6, which encodes chemokine (C-C motif) receptor (CCR) 6, in the splenocytes from resveratrol-treated mice. CCR6 abundance on IL-17-producing cells and CD11b+F4/80hi macrophages was inhibited by resveratrol treatment. Interestingly, CCR6+ IL-17-producing cells and CD11b+F4/80hi macrophages accumulated in the spleens and pancreatic lymph nodes, but their presence in the pancreas was reduced, suggesting that resveratrol blocks their migration from peripheral lymphoid organs to the pancreas. Indeed, the migration of splenocytes toward media containing chemokine (C-C motif) ligand 20 (CCL20) was impaired by resveratrol treatment. CCL20 peptides, which block CCR6 binding to CCL20, inhibited development of type 1 diabetes.
Conclusions/interpretation
Inhibition of CCR6-mediated migration of inflammatory cells by resveratrol may provide a powerful approach for treatment of type 1 diabetes and possibly of other inflammatory diseases.
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Introduction
Resveratrol (trans-3,4′,5-trihydroxystilbene), a compound of the polyphenol group that is found in various fruits and vegetables, and is abundant in grapes [1], has recently emerged as a potential therapeutic reagent for many diseases, such as type 2 diabetes [2], cardiovascular heart diseases [3], cancers [4] and disease associated with ageing [5]. It has been suggested that resveratrol has immunosuppressive activities and thus therapeutic potential in the treatment/prevention of autoimmune diseases [6]. Feeding with resveratrol has recently been found to protect mice from experimental autoimmune encephalomyelitis (EAE) [7, 8]. The root extract of the weed Polygonum cuspidatum, an important constituent of Chinese and Japanese folk medicines, is the richest known source of resveratrol and has been used for the treatment of lupus and rheumatoid arthritis for many years. In addition, recent studies have demonstrated that resveratrol has a protective effect on allograft rejection in mice [9, 10].
The possible mechanisms for the pharmacological effects of resveratrol, although extensively analysed, remain largely unknown. Administration of resveratrol to mice inhibits body weight and liver weight gain induced by high-energy diet, prevents pancreatic damage and extends the animals’ lifespan [11]. These therapeutic activities of resveratrol are associated with: (1) increased insulin sensitivity; (2) reduced IGF-1 levels; (3) increases in AMP-activated protein kinase and peroxisome proliferator-activated receptor-c coactivator 1a activity; (4) increased mitochondrial number; and (5) improved motor function [12, 13]. By suppressing inhibitor of nuclear factor-κΒ (NF-κB) (IκB) kinase (IKK)-mediated phosphorylation of IκB, resveratrol inhibits NF-κB and prevents overproduction of 12-O-tetradecanoylphorbol-13-acetate-induced cyclooxygenase-2, which is considered a major mechanism of its anti-inflammatory activities [14]. Resveratrol has also been found to have inhibitory effects on many other protein and lipid kinases, such as mitogen-activated protein kinase, plasma creatine kinase, Src family tyrosine kinases and phosphoinositide 3-kinase (PI3K). Resveratrol is well-known as an activator of sirtuin 1, an NAD-dependent sirtuin family deacetylase [15–17]. We recently demonstrated that sirtuin 1 suppresses T cell immunity in mice by deacetylating c-Jun, an activating protein-1 family transcription factor, and that resveratrol inhibits c-Jun acetylation in connection with its suppressive effect on T cell activation [18]. Adenovirus-mediated overproduction of sirtuin 1 protects pancreatic beta cells against cytokine toxicity by suppressing the NF-κB pathway [19].
Resveratrol has been found to prevent inflammation in mice by suppressing NF-κB-mediated production of inflammatory cytokines and by inducing apoptosis [7, 8]. In this report, we found that resveratrol inhibits chemokine (C-C motif) receptor (CCR) 6 production on immune cells, leading to minimal pancreatic islet infiltration and suppression of diabetes in the NOD mouse model of type 1 diabetes.
Methods
Mice
NOD (H-2g7), NOD.BDC2.5 and NOD/SCID mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and used according to the guidelines of the Northwestern University Animal Care and Use Committee.
Diabetes assessment
Assessment of blood glucose levels was by test strips and a monitoring system (Accu-Chek Advantage; Pompano Beach, FL, USA). A mouse was considered hyperglycaemic or diabetic when the blood glucose level reached 9–14 or 16 mmol/l, respectively, for two consecutive weeks.
Resveratrol treatment
For feeding, female NOD mice were given 250 mg/kg resveratrol or Mega resveratrol (Sigma, St Louis, MO, USA) of 99% purity as confirmed by HPLC in 100 μl PBS. Administration was daily by gavage from 8 weeks of age. For injections, NOD mice were injected every other day with 25 mg/kg (in 100 μl PBS) subcutaneously. The mice were monitored for blood glucose levels once a week up to 32 weeks of age.
Histology
Pancreas histology was assessed as described [20]. Pancreases were removed from NOD females, fixed in 10% formalin and embedded in paraffin. Sections of 7 μm thickness were cut at 100 μm intervals to prevent double-counting of the same islet. Two sections per pancreas were stained with haematoxylin and eosin and analysed by light microscopy. Insulitis scoring was performed according to the following criteria: severe insulitis, 50% or more of the islet area infiltrated; mild insulitis, <50% of the islet area infiltrated; peri-insulitis, infiltration restricted to the periphery of islets; no insulitis, absence of cell infiltration.
For immunochemistry analysis, paraffin-fixed pancreatic tissue sections were incubated overnight at 4°C with anti-insulin (Abcam, Cambridge, MA, USA) and anti-F4/80 antibodies, and with the Alexa 594 goat anti-rabbit and Alexa 488 goat anti-rat IgG antibodies (Invitrogen, Carlsbad, CA, USA) at room temperature for 1 h. After five extensive washings with 0.1% Triton X100-containing PBS, sections were stained for 5 min with 0.5 mg/ml DAPI followed by three additional washings. Tissue sections were visualised with fluorescent microscope.
Flow cytometry
For cell surface protein staining, cells were removed from spleens and pancreatic lymph nodes, and incubated with FITC, phycoerythrin (PE)-, Cy5- or antigen-presenting cell (APC)-conjugated antibodies against mouse CD4, CCR4, CCR6, CCR7, CD11b, CD80, CD86, MHCI and MHCII in combinations as described. Subsequently, the cells were washed and fixed with 1% formaldehyde for 20 min at room temperature and then analysed. Cell apoptosis was analysed by annexin V staining as reported [21]. All antibodies were purchased from Biolegend (San Diego, CA, USA).
For intracellular cytokine analysis of IFN-γ and IL-17, the splenic cells (2 × 106 cell/ml) were stimulated for 4 h with 10 ng/ml phorbol myristate acetate and ionomycin (1 μmol/l) in the presence of brefeldin A (10 μg/ml) to block cytokine secretion and facilitate intracellular accumulation. The antibodies used were peridinin chlorophyll protein-anti-CD4, phycoerythrin-anti-IFN-γ and APC-anti-IL-17 (BD Biosciences, San Diego, CA, USA). Isotype-matched controls were included in all experiments. Events were collected on FACScan, FACSCanto II (San Jose, CA, USA) or CyanADP (Daco, Carpinteria, CA, USA) flow cytometers and analysed with CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA) or Summit (Beckman-Coulter, Fullerton, CA, USA).
Adoptive transfer experiments
For disease transfer, 1 × 107 freshly isolated splenocytes from resveratrol-treated or PBS-treated mice were injected into 6–8-week-old NOD/SCID mice. Blood glucose levels were monitored weekly in the first 3 weeks after transfer and then every other day.
Splenocyte migration assay
Freshly isolated splenocytes were incubated on 24-well collagen-coated, polyester membrane Transwell inserts of 6.5 mm diameter and 3.0 μm pore size (Corning, Lowell, MA, USA). The confluent inserts were transferred to fresh wells containing 600 μl of TEM buffer with 10 ng/ml recombinant mouse C-C chemokine ligand 20 (CCL20) (R&D Systems, Minneapolis, MN, USA). Cells were allowed to transmigrate for 3 h at 37°C. These experiments were performed in triplicate, with migration across the basal in vitro blood–brain barrier without added chemokine serving as spontaneous migration.
Real-time PCR analysis
RNA was isolated from spleens, pancreatic lymph nodes or pancreases using TRIzol reagent (Invitrogen) and was reverse-transcribed using Moloney murine leukaemia virus reverse transcriptase (Invitrogen). Quantitative real-time RT-PCR was performed on a sequence detection system (Prism 7000; Applied Biosystems, Foster City, CA, USA). β-Actin gene was used as a reference for sample normalisation. Primers and probes for the following genes including Ifn-γ (also known as Ifng), Il10, Il17 (also known as Il17a), Cd11c (also known as Itgax), Cd11b (also known as Itgam), F4/80 (also known as Emr1), Ccr4 and Ccr6 were purchased as assays-on-demand (Applied Biosystems, Bedford, MA, USA). Standard amplification protocol was used following the manufacturer’s description.
Statistical analysis
The χ2 test was used for incidence of diabetes analysis among experimental and control groups. For the rest of the experiments, p values were calculated with the two-tailed Student’s unpaired t test.
Results
Resveratrol protects NOD mice from diabetes
To investigate the effects of resveratrol on type 1 diabetes mellitus, NOD mice were fed a daily dose of 250 mg resveratrol per kilogram of body weight, beginning at week 8 of age as recently reported [7]. Disease onset was significantly delayed and 30% of the mice remained free of type 1 diabetes mellitus for the duration of the experiment (Electronic supplementary material [ESM] Fig. 1). A regimen using subcutaneous injection of 25 mg resveratrol/kg every other day was even more effective, as more than 80% of the mice were protected from type 1 diabetes mellitus (Fig. 1a, ESM Fig. 1c), a finding that correlates well with the suggestion that the oral route is less efficient due to low absorption of the drug [15–17]. Analysis of the serum levels of the liver enzymes alanine aminotranferease (ALT) and aspartate aminotransferase (AST) did not reveal any liver damage in treated mice. The average ALT level in control mice and AST level in all groups were slightly higher than normal, possibly because some mice were diabetic [22]. Serum creatinine levels were all in the normal ranges in mice of all three groups, indicating that resveratrol treatment did not harm kidney functions (ESM Table 1).
To further investigate the preventive roles of resveratrol against type 1 diabetes mellitus, we performed histological analysis of islet infiltration upon treatment with resveratrol at a time point when 50% of the control mice manifested diabetes. In the PBS-treated mice, most of the islets exhibited intra-insulitis, while the mice receiving subcutaneous resveratrol had mild insulitis with most islets remaining healthy. Similarly, recipients of oral resveratrol also had mild insulitis in comparison with control mice (Fig. 1b–e). Enumeration of the islets indicated that resveratrol-treated animals had a significantly greater number of total islets than PBS-treated control mice (Fig. 1d). Indeed, resveratrol-treated groups had a higher percentage of insulitis-free islets (69% for injection and 31% for feeding delivery vs 16%) and a significantly reduced number of islets with mild (10% for injection and 25% for feeding vs 34%) or severe insulitis (1% for subcutaneous injection and 14% for oral feeding vs 21%) relative to PBS-treated mice (Fig. 1e). Further analysis of the effects of resveratrol indicated that lymphocytes from resveratrol-treated mice were less pathogenic. When total splenocytes from resveratrol-treated and control mice were adoptively transferred into NOD/SCID mice, only 20% (one of five) became diabetic in the resveratrol-treated group, while 60% (three of five) of the control mice became diabetic (ESM Fig. 2). Furthermore, all five control mice developed diabetes at 5 weeks after transfer, while two of five resveratrol-treated mice remained non-diabetic at this time-point (ESM Fig. 2). These results indicate that resveratrol prevents spontaneous diabetes mellitus in the NOD mouse model of type 1 diabetes and may reverse type 1 diabetes if given at advanced stages of the disease.
To investigate whether resveratrol can reverse diabetes, we initiated resveratrol treatment at week 13 of age, when essentially all NOD mice have insulitis and about 10–20% had hyperglycaemia. While about 50% of the control mice had diabetes at week 19 of age, all resveratrol-treated mice remained healthy at that time-point. Morover, two of six resveratrol-treated mice were still protected from diabetes at 29 weeks of age, whereas all control animals had diabetes, suggesting that resveratrol has protective effects even after insulitis development in NOD mice (Fig. 1f).
The effect of resveratrol on T cell activation and cytokine production in treated NOD mice
Intracellular staining analysis revealed a decrease in IFN-γ-producing CD4+ and CD8+ T cells in the spleens of resveratrol-treated mice, suggesting that resveratrol may prevent diabetes by suppressing IFN-γ production (Fig. 2a–c). Unexpectedly, the percentage of IL-17-producing CD4+ T cells was significantly increased in the spleens of resveratrol-treated mice (Figs 2c, 3c, ESM Fig. 3). Levels of IFN-γ- and IL-17-producing cells increased with age and diabetes development, as we observed significant higher percentages in diabetic mice than in young healthy NOD mice (ESM Fig. 3a). In addition, we observed significantly increased IL-10-producing CD4+ T cell population in treated mice when compared with control mice (Fig. 2c). Similarly, real-time RT-PCR analysis revealed increased Il17 and Il10 mRNA levels, but decreased Ifn-γ mRNA expression in pancreatic lymph nodes from resveratrol-treated mice (ESM Fig. 3c). However, levels of Foxp3 and Tgf-β (also known as Tgfb1) mRNA were indistinguishable between PBS- and resveratrol-treated mice (Fig. 2d). Since IFN-γ has suppressive functions in T helper (Th)17 polarisation [23, 24], the increase in IL-17-producing T cells may have been partially due to the decreased number of IFN-γ in spleen and pancreatic lymph nodes from resveratrol-treated mice. In fact, a recent elegant study demonstrated that absence of IFN-γ in CD4+ T cells resulted in augmented Th2 and Th17 differentiation and exacerbated tissue damage in lung and skin [25].
CD4+ regulatory T cells (Tregs) have been considered one of the key players in suppression of autoimmune diseases such as type 1 diabetes mellitus. We thus asked whether resveratrol prevents type 1 diabetes mellitus in NOD mice by upregulating Tregs. However, the percentages of forkhead box (FOX)P3+ Tregs in spleen, pancreatic lymph nodes and pancreas were indistinguishable between resveratrol-treated and control mice (ESM Fig. 4a). The cell surface expression of suppressive molecules, including cytotoxic T lymphocyte antigen 4, glucocorticoid-induced TNF receptor family-related gene and TGF-β, on Tregs was not affected by resveratrol treatment (ESM Fig. 4c). FOXP3+ CD8+ T cells have recently been characterised as a new subset of Tregs, which has been shown to protect rats from diabetes [26, 27]. However, resveratrol appears not to affect CD8+ Tregs in NOD mice, as the percentages of CD4−FOXP3+ cells, which presumably includes CD8+FOXP3+Tregs, were indistinguishable between resveratrol-treated and control mice (ESM Fig. 4b). In addition, treatment of mice with resveratrol did not affect in vivo T cell activation as analysed by cell surface expression of CD44, CD62L and CD69 (ESM Fig. 5a, b), nor did it affect the Fas ligand levels and apoptosis of T and B cells in the spleen (ESM Fig. 5c, d). Therefore, resveratrol appears not to affect Treg development or function, or to suppress T cell activation in mice.
Resveratrol downregulates Ccr6 expression
To investigate how resveratrol protects NOD mice from diabetes, we performed inflammatory gene array chip analysis of the expression profile of genes associated with inflammatory autoimmune diseases in resveratrol- and PBS-treated mice. The findings clearly demonstrate that Ccr6 expression was significantly downregulated by resveratrol, while the expression of other chemokine receptor genes remained at similar levels in both animal groups (Fig. 3a). To validate the array data, we analysed CCR6 protein expression on the surface of T cells from resveratrol-treated mice and indeed confirmed a significant reduction on T cells from the spleen and pancreatic lymph nodes of resveratrol-treated mice compared with PBS-treated controls (Fig. 3b). This in vivo Ccr6 downregulation in T cells by resveratrol was further confirmed by real-time PCR analysis and western blotting (Fig. 3c, d). The controls, CCR4 and CCR7, were comparable between resveratrol- and PBS-treated mice (ESM Fig. 6). However, analysis of chemokine gene expression showed no measurable effect of resveratrol treatment on any of the chemokines tested. Given that CCR6 is involved in trafficking of autoimmune lymphocytes to inflammatory tissues and organs [28, 29], downregulation of Ccr6 expression in this case may be one of the mechanisms by which resveratrol prevents type 1 diabetes in mice.
Reduced CCR6 expression in Th17 and CD11b+F4/80hi macrophages in resveratrol-treated mice
It is well established that chemokine receptors are differentially expressed on subsets of effector and memory T cells, and provide specific trafficking in the steady state and in inflammation [30]. Given that CCR6 was reduced on splenic cells and that the number of IFN-γ-producing cells was diminished but that of Th17 cells increased, we determined whether CCR6 downregulation occurred in Th17 cells. This could explain disease prevention by resveratrol, especially because CCR6 has been found to play a critical role in the trafficking of effector Th17 cells to the central nervous system in humans with multiple sclerosis and in mice with EAE [29]. The findings revealed a significant reduction of in CCR6 expression on IL-17-producing CD4+ and CD4− T cells from resveratrol-treated mice (Fig. 3e, f). Moreover, IL-17-producing cells accumulated in the spleen of resveratrol-treated mice (Fig. 2), while their presence in the pancreas was dramatically reduced as measured by Il17 mRNA expression (ESM Fig. 3c). These results suggest that reduced CCR6 expression on Th17 cells leads to reduced trafficking of the cells to the pancreas and their accumulation in the spleen.
During flow cytometry analysis, we noticed a reduction in the side scatter (SSC)hi and forward scatter (FSC)low population of total splenocytes of diabetic mice and found that this population was protected by resveratrol treatment (ESM Fig. 7a). Further characterisation demonstrated that this population consisted of CD11b+ macrophages (ESM Fig. 7a). Splenocyte cellularity analysis indicated that the total CD11b+ macrophage numbers in diabetic mice were reduced and that resveratrol treatment prevented this macrophage loss in the spleen (ESM Fig. 8). Interestingly, the levels of CCR6 on SSChi and SSClowCD11b+ macrophages were reduced in NOD mice treated with resveratrol (ESM Fig. 7b). As a control, CCR4 expression on macrophages was not affected by resveratrol treatment (ESM Fig. 7c).
CD11b+F4/80low and CD11b+F4/80hi cells were increased in spleens from resveratrol-treated mice (Fig. 4a). Resveratrol inhibited CCR6 expression on CD11b+F4/80hi, but not on CD11b+F4/80low cells in the spleen (Fig. 4a) and pancreatic lymph nodes (Fig. 4b). In addition, we observed significant reductions in CD80 and CD86, but not MHCI and MHCII expression on macrophages from resveratrol-treated mice (Fig. 4c, ESM Fig. 9). Collectively, our data suggest that resveratrol may protect mice from diabetes by suppressing migration and pathogenicities of CD11b+F4/80hi macrophages.
Resveratrol impairs splenocyte trafficking toward CCL20
CCR6 is critical in the regulation of T effector cell trafficking toward sites where the CCR6 ligand, CCL20, is produced [31–36]. Haematoxylin and eosin staining analysis of pancreatic tissue sections revealed that resveratrol suppressed insulitis (Fig. 1b, c). Together with our finding of reduced CCR6 expression on T cells and macrophages, it is possible that resveratrol may inhibit islet inflammation by blocking migration of inflammatory cells to pancreas. Indeed, mRNA levels of Ccr6, but not Ccr4 and Ccl20 in the pancreas were reduced by 71% in resveratrol-treated compared with PBS-treated mice (Fig. 5a). In correlation with this reduction of CCR6 levels in pancreatic tissues of resveratrol-treated mice, mRNA levels of Cd11b (81% reduction), Ifn-γ (63% reduction) and Il17 (92% reduction) were significantly reduced (Fig. 5b, ESM Fig. 3c), suggesting that resveratrol inhibits the migration of pathogenic CD11b+F4/80hi macrophages and inflammatory T lymphocytes into the pancreas. We then performed an immunochemistry analysis to compare F4/80+ macrophages in the pancreases from resveratrol-treated and control mice. Similar to our results in haematoxylin and eosin staining, insulin staining further confirmed a significantly higher number of islets in the pancreases of resveratrol-treated mice. Interestingly, both in the islet and the interstitial area, F4/80+ cell numbers in the pancreases from resveratrol-treated mice were dramatically reduced (Fig. 5d, e). Together with the increased F4/80+ macrophages in spleen and pancreatic lymph nodes (Fig. 4a, b), our results suggest that resveratrol may block pathogenic macrophage trafficking into the pancreases.
We therefore used an in vitro culture system and tested the migration ability of splenocytes from resveratrol-treated mice in a CCR6- and CCL20-dependent manner. Fewer than 3% of splenocytes from resveratrol-treated mice migrated into the compartment containing CCL20-rich medium. In contrast, the migration rate for splenocytes isolated from diabetic mice was 12% (Fig. 5c). Collectively, our data indicate that resveratrol inhibits inflammatory cell trafficking from peripheral lymphoid organs to the pancreas by suppressing CCR6 production.
Our finding that resveratrol prevented type 1 diabetes by suppressing production of CCR6, but not of its ligand CCL20, suggests an important role of CCR6–CCL20 crosstalk during diabetes development. We therefore asked whether blocking of CCR6+ cell trafficking using CCL20 peptide might suppress diabetes in NOD/SCID mice that had been adoptively transferred with splenocytes from diabetic NOD mice. Indeed, administration of CCL20 peptide every other day significantly delayed disease onset and protected more than 70% mice from diabetes even at 40 days after transfer (Fig. 5f). Therefore, in the mouse model of type 1 diabetes used by us, blocking of CCR6-mediated migration of inflammatory cells prevents diabetes mellitus and resveratrol treats the condition by inhibiting CCR6 production.
Discussion
Type 1 diabetes is an autoimmune disease in which a self-destructive immune process against the pancreatic beta cells leads to insulin deficiency. In this study, we found that administration of resveratrol, particularly by subcutaneous injection, in the NOD mouse model of type 1 diabetes delayed disease in more than 80% of the animals. Resveratrol downregulates CCR6 expression on multiple inflammatory cell types. This reduction of CCR6 expression appears to block the migration of pathogenic cells to the pancreas, since insulitis was dramatically inhibited by resveratrol treatment and splenocytes from resveratrol-treated mice have impaired ability to migrate towards CCL20, a CCR6 ligand [34]. Moreover, we found that CCL20 peptides, which have previously been shown to suppress EAE development in mice [24], displayed great efficacy in the treatment of diabetes in NOD/SCID mice that had received pathogenic splenocytes from diabetic mice.
CCR6 downregulation is an important mechanism of resveratrol in diabetes treatment and prevention. CCR6 has been found to be a critical mediator in pathogenic lymphocyte migration to sites of inflammation in different types of disease setting [37]. Rebodi et al. recently reported that CCR6 production is required for pathogenic Th17 cell migration to the central nervous system during EAE development in mice [29]. The involvement of Th17 cells in type 1 diabetes is still under debate [38–41], but recent studies have indicated that Th17 cell accumulation is involved in peripheral blood T cells from children with type 1 diabetes mellitus [42, 43]. Indeed, we found that CCR6 expression on CD4+ and CD4− IL-17-producing cells was inhibited by resveratrol treatment. This CCR6 reduction seems likely to inhibit IL-17-producing cell migration to the pancreas, because Il17 mRNA expression was decreased in pancreatic cells, but increased in the spleens of resveratrol-treated mice. It has been reported that CD11b+F4/80hi macrophages retain lymphocytes in insulitis in NOD mice and that macrophage depletion inhibits diabetes development accompanied by pancreatic lymphocyte infiltration [37]. Resveratrol appears to suppress insulitis by blocking CD11b+F4/80hi macrophage migration to the pancreas, because our data indicate that a reduction of Cd11b mRNA in pancreas is accompanied by an increase in percentage of CD11b+ macrophage in the spleens of resveratrol-treated mice. In addition, macrophages from resveratrol-treated mice had impaired ability to migrate towards CCL20 and administration of CCL20 peptides, which block CCR6+ cell trafficking in mice, suppressed diabetes in the NOD/SCID mouse model of type 1 diabetes when mice had been adoptively transferred with splenocytes from diabetic mice. Therefore, our studies reveal a novel mechanism in resveratrol treatment of type 1 diabetes and possibly of other types of autoimmune diseases
Resveratrol increases the level and activity of sirtuin 1, a mammalian NAD+-dependent histone deacetylase [15, 16, 44]. The deacetylase activity of sirtuin 1 is necessary for its suppressive activity in gene transcription. We have recently reported that sirtuin 1 inhibits T cell activation and that its upregulation is required for peripheral T cell tolerance [18]. Accumulating evidence indicates a function of resveratrol in cells and in mice that is independent of sirtuin 1 [45]. It has been reported that activation of many other proteins can be inhibited by resveratrol in cells. Many of these proteins are involved in regulation of immune response, such as NF-κB, PI3K, FOXO3A and Janus kinase 1. Therefore, sirtuin 1 may merely contribute to a small portion of the in vivo activity of resveratrol.
Moreover, numerous previous studies have revealed that resveratrol affects many cell biological responses such as insulin sensitivity, gluco/lipotoxicity, endoplasmic reticulum stress, oxidative stress etc., all of which contribute to diabetes, in particular type 2 diabetes [46]. Our findings here define one of the immune suppressive functions of resveratrol in protecting mice from diabetes. We believe, however, that other mechanisms also contribute to the glucose-lowering functions of resveratrol in our model.
Our study suggests that resveratrol is a potential therapeutic and/or preventive reagent for type 1 diabetes in humans. Interestingly, the Food and Drug Administration of the USA has approved resveratrol for human use as a food supplement (GRN000224). The current ‘mega doses’ of resveratrol for humans is 500–1,000 mg (about 7–15 mg/kg body weight) per day, which is still much lower than the doses used in this study. Results from our study, using a much lower dose by injection (25 mg/kg), showed a greater efficacy of resveratrol in protecting mice from type 1 diabetes, reflecting the poor intestinal absorption of resveratrol. Therefore, more efforts are needed in the pharmaceutical studies to improve resveratrol absorption. Interestingly, more potent sirtuin 1 activators (about 1,000-fold more potent than resveratrol) that are structurally unrelated to resveratrol have been recently identified [2]. It will be interesting to characterise these small molecule sirtuin 1 inhibitors in immune regulation and type 1 diabetes mellitus treatment in mouse models of diabetes and in humans.
Abbreviations
- APC:
-
Antigen-presenting cell
- ALT:
-
Alanine aminotranferease
- AST:
-
Aspartate aminotransferase
- CCL20:
-
C-C Chemokine ligand 20
- CCR:
-
Chemokine (C-C motif) receptor
- EAE:
-
Experimental autoimmune encephalomyelitis
- FOX:
-
Forkhead box
- FSC:
-
Forward scatter
- NF-κB:
-
Nuclear factor κB
- PI3K:
-
Phosphoinositide 3-kinase
- SSC:
-
Side scatter
- Th:
-
T helper
- Treg:
-
Regulatory T cell
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Acknowledgements
We thank J. Borensztajn (Northwestern University) for technical support. This study was supported by NIH R01AI079056, R21AG028493 and a ‘The Type I Diabetes Pathfinder Award’ (DK083050) to D. Fang, and by grant R21AI68746 from National Institutes of Health (to H. Zaghouani). D. M. Tartar was supported by a life science fellowship from the University of Missouri, Columbia. This paper was supported by research funds for 2010 from Chonbuk National University (to S.-M. Lee). This work was also supported by the Northwestern University Interdepartmental ImmunoBiology Flow Cytometry Core Facility.
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ESM Table 1
Effects of resveratrol on the liver and kidney functions of NOD mice (PDF 6 kb)
ESM Fig. 1
Resveratrol prevents NOD mice against autoimmune diabetes mellitus. a NOD mice were fed daily with resveratrol by gavage at 250 mg/kg per day. Blood glucose levels were monitored weekly; percentages of diabetic mice are shown. b The blood glucose levels of each mouse in the resveratrol-feeding (RES) (b) and injection (c) groups, as well as the respective control groups are shown (PDF 345 kb)
ESM Fig. 2
Effect of resveratrol on disease transfer. Splenocyte from PBS-treated (a) or from resveratrol-treated mice (b) were isolated and adoptively transferred into NOD/SCID mice at 1 × 107 cells per mouse. Blood glucose levels of recipients were monitored; percentages of diabetic mice are shown (n = 5) (PDF 17 kb)
ESM Fig. 3
The effects of resveratrol on cytokine production by CD4+ T cells in NOD mice. a Splenocytes from PBS- or resveratrol-treated (RES) mice, and (b) from 6-week-old young and 20-week-old diabetic mice were cultivated for 4 h with phorbol myristate acetate plus ionomycin in the presence of 10 μg/ml brefedlin A. Intracellular staining of IL-17 and IFN-γ was performed. FL, forward light scatter (FL1s show the fluorescence height of IL-17-FITC; FL2s show the fluorescence height of IFN-γ-PE). c Total RNA was isolated from spleens and pancreas tissues of resveratrol and PBS-treated mice. The expression of each indicated gene was analysed by real-time RT-PCR and ratios of resveratrol-/PBS-treated groups are shown. White bars, Il-17; black bars, Ifn-γ. Values are presented as mean ± SD and represent data from five pairs of mice in each group (PDF 126 kb)
ESM Fig. 4
Effects of resveratrol on Treg development in NOD mice. a Lymphocytes from spleens, pancreases and pancreatic lymph nodes (PLN) of PBS- or resveratrol-treated (RES) mice were stained with Cy5-conjugated anti-CD4 and phycoerythrin (PE)-anti-CD25 followed by an intracellular staining with FITC-anti-FOXP3 and analysed by flow cytometry. b FOXP3+ cells in CD4+ and CD4− populations of splenocytes are shown. c CD4+FOXP3+ Tregs in splenocytes were gated and their levels of cytotoxic T lymphocyte antigen 4 (CTLA-4), glucocorticoid-induced TNF receptor family-related gene (GITR) and membrane bound TGF-β were analysed. Pink, specific staining; blue, isotype control. FL2s show the fluorescence heights of CTLA4-PE (top panels), GITR-PE (middle panels) and TGF-β-PE (bottom panels) (PDF 63 kb)
ESM Fig. 5
T cell activation in resveratrol-treated NOD mice. a Cell surface expression of CD44 and CD62L,(FL1s show the fluorescence height of CD44-FITC.; FL4s show the fluorescence height CD62L-APC.) and (b) CD4 and CD69 on CD4+ T cells (FL1s show the fluorescence height of CD69-FITC.; FL3s show the fluorescence height of CD4-Cy5 were analysed by flow cytometry. (c) Fas ligand (FasL) expression on CD4+ T cells in PBS- and resveratrol-treated mice were analysed. The percentage of FasL-positive cells and their mean fluorescence incidence (MFI) are shown. (d) Cell apoptosis of T and B lymphocytes in PBS and RES-treated mice was analyzed by flow cytometry. The percentage of annexin V-positive cells and their mean fluorescence incidence (MFI) are shown (PDF 38 kb)
ESM Fig. 6
CCR4 and CCR7 expression on CD4+ cells in the spleen of resveratrol-treated and control mice. Splenocytes from PBS- or resveratrol-treated mice were stained with antibodies against CD4, CCR4 or CCR7 and analysed by flow cytometry. Left panels, PBS-treated control mice; right panels, resveratrol-treated mice. FL2s show the fluorescence height of CD4-PE; FL4s show the fluorescence height of CCR7-APC (top panels) and CCR4-APC (bottom panels) (PDF 86 kb)
ESM Fig. 7
CCR6 levels in macrophages from resveratrol-treated NOD mice. a Splenocytes from 6-week-old mice, and PBS- or resveratrol-treated mice were analysed by flow cytometry. FSC and SSC, and CD11b+ cells analysed against SSC are presented as indicated. b Expression of CCR6 or (c) CCR4 on the surfaces of each indicated population FL2s show the fluorescence height of CCR6-PE.; FL8s show the fluorescence height of CCR4-APC (PDF 84 kb)
ESM Fig. 8
Characterisation of splenocytes in resveratrol-treated mice. The percentages of CD4+ and CD8+ T cells, B220+ B cells, CD11b+ macrophages and CD11c+ dendritic cells in the spleen of resveratrol-treated (black bars) and PBS-treated diabetic (grey bars) or non-treated young mice (white bars) are shown (PDF 15 kb)
ESM Fig. 9
Characterisation of macrophages in resveratrol-treated NOD mice. CD11b+ macrophages were gated. Levels of (a) MHC class I (FL4 shows the fluorescence height of MHC I-APC) and (b) MHC II (FL1 shows the fluorescence height of MHC II-FITC), as well as those of co-stimulatory molecules (c) CD80 and (d) CD86 were analysed. FL2s show the fluorescence heights of CD80-PE (c) or CD86-PE (d) (PDF 20 kb)
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Lee, SM., Yang, H., Tartar, D.M. et al. Prevention and treatment of diabetes with resveratrol in a non-obese mouse model of type 1 diabetes. Diabetologia 54, 1136–1146 (2011). https://doi.org/10.1007/s00125-011-2064-1
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DOI: https://doi.org/10.1007/s00125-011-2064-1