Resveratrol differentially modulates inflammatory responses of microglia and astrocytes
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Inflammatory responses in the CNS mediated by activated glial cells play an important role in host-defense but are also involved in the development of neurodegenerative diseases. Resveratrol is a natural polyphenolic compound that has cardioprotective, anticancer and anti-inflammatory properties. We investigated the capacity of resveratrol to protect microglia and astrocyte from inflammatory insults and explored mechanisms underlying different inhibitory effects of resveratrol on microglia and astrocytes.
A murine microglia cell line (N9), primary microglia, or astrocytes were stimulated by LPS with or without different concentrations of resveratrol. The expression and release of proinflammatory cytokines (TNF-α, IL-1β, IL-6, MCP-1) and iNOS/NO by the cells were measured by PCR/real-time PCR and ELISA, respectively. The phosphorylation of the MAP kinase superfamily was analyzed by western blotting, and activation of NF-κB and AP-1 was measured by luciferase reporter assay and/or electrophoretic mobility shift assay.
We found that LPS stimulated the expression of TNF-α, IL-1β, IL-6, MCP-1 and iNOS in murine microglia and astrocytes in which MAP kinases, NF-κB and AP-1 were differentially involved. Resveratrol inhibited LPS-induced expression and release of TNF-α, IL-6, MCP-1, and iNOS/NO in both cell types with more potency in microglia, and inhibited LPS-induced expression of IL-1β in microglia but not astrocytes. Resveratrol had no effect on LPS-stimulated phosphorylation of ERK1/2 and p38 in microglia and astrocytes, but slightly inhibited LPS-stimulated phosphorylation of JNK in astrocytes. Resveratrol inhibited LPS-induced NF-κB activation in both cell types, but inhibited AP-1 activation only in microglia.
These results suggest that murine microglia and astrocytes produce proinflammatory cytokines and NO in response to LPS in a similar pattern with some differences in signaling molecules involved, and further suggest that resveratrol exerts anti-inflammatory effects in microglia and astrocytes by inhibiting different proinflammatory cytokines and key signaling molecules.
KeywordsNitric Oxide Curcumin Proinflammatory Cytokine Resveratrol Microglial Cell
Microglia, the resident macrophage-like cells in the brain, play an important role in host defense and tissue repair in CNS [1, 2]. Activated microglia produce a variety of pro-inflammatory mediators, including tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), IL-6, monocyte chemotactic protein 1 (MCP-1, CCL2), nitric oxide (NO), and reactive oxygen species (ROS). Activated microglia serve immune surveillance functions by removing foreign microorganisms, but may also result in excessive inflammatory responses in the CNS [1, 2]. Astrocytes are the main glial cell type in the brain involved in maintaining CNS homeostasis. They also respond promptly to injury and regulate neuroinflammatory events [2, 3, 4]. Both in vitro and in vivo studies have documented the ability of astrocytes to produce a variety of cytokines, including IL-1, IL-6, IL-10, interferon-α (INF-α), IFN-β, TNF-α, TNF-β; and chemokines, including RANTES (CCL5), IL-8 (CXCL8) and MCP-1 . Over-activation of glial cells and release of proinflammatory cytokines may lead to neuronal death [5, 6, 7], causing neuropathological changes in CNS diseases such as multiple sclerosis [8, 9], Parkinson's disease [10, 11], Alzheimer's disease  and AIDS dementia . Therefore, limiting inflammatory cytokine production by activated microglia and astrocytes should be beneficial for prevention of neuroinflammation and neurodegeneration.
Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a polyphenolic compound found in a large number of plant species that are components of human diet, including mulberries, peanuts, grapes and red wine. Accumulating evidence suggests that resveratrol may exert a protective effect in the CNS under pathological conditions, and that resveratrol is associated with reduced risks of cardiovascular disease, cancer, diabetes and AD [14, 15, 16, 17]. Resveratrol has also been proposed to be an anti-inflammatory molecule . In glial cells, resveratrol has been reported to inhibit LPS-induced production of NO and TNF-α by the murine microglia cell line N9 [19, 20]; to inhibit prostaglandin E2 (PGE2) and free radical production by rat primary microglia , and to inhibit NO and PGE2 by the rat astroglial cell line C6 . Microglia and astrocytes are two cell types with different biological characteristics and functions in the CNS, it is not clear if there are differences between these cells in response to LPS or if resveratrol inhibits the inflammatory responses of these cells to LPS through similar mechanisms.
In the present study, we first examined the expression of various proinflammatory cytokines (TNF-α, IL-1β, IL-6, MCP-1) and of iNOS by murine microglia and astrocytes in response to LPS, and the signaling molecules involved. We then determined the effects of resveratrol on microglial cell and astrocyte activation by LPS, and explored the underlying key signaling molecules.
Resveratrol, LPS and MTT were obtained from Sigma (St. Louis, MO). PD98059, SP600125, SB203580, sulfasalazine and curcumin were from Calbiochem (Darmstadt, Germany). Antibodies against both phosphorylated and unphosphorylated extracellular signal-regulated kinases (ERK1/2), p38, c-jun N-terminal kinase (JNK) were obtained from Cell Signaling Technology (New England Biolabs, Beverly, MA). Dual-Luciferase Reporter Assay System was from Promega Corporation (Woods Hollow Road, Madison, USA). LightShift Chemiluminescent EMSA kit was from Pierce (Pierce, Rockford, IL, USA). DMEM was purchased from Gibco BRL (Burlington, Ontario, Canada). Fetal bovine serum (FBS) was from Hyclone (Logan, UT). All other reagents were obtained from Sigma-Aldrich unless otherwise described.
Glial cell cultures
Murine primary microglia or astrocytes were cultured on slides in 24-well plates. The cells were fixed with 4% paraformaldehyde for 10 min at room temperature, washed with PBS, and then incubated with 5% BSA/PBS, 0.01% Tween-20 at room temperature for an additional 1 h. Rat anti-CD11b (1:25, BD Bioscience) or anti-GFAP (1:100, Zymed) antibody was applied to the slides and incubated overnight at 4°C. Rat IgG (Santa Cruz) was used as negative control. The slides were washed and incubated with FITC-conjugated goat anti-rat IgG (1:200) for 60 min, washed with PBS, stained with Hoechst 33342, and mounted. Immunofluorescence labeling was observed under a fluorescent microscope.
RT-PCR and real-time PCR
Cells were cultured in medium without FBS for 24 h, then treated with 0.5 μg/mL LPS with or without various concentrations of resveratrol for another 8 h. Total RNA was extracted from cells with Trizol reagent (Invitrogen) and depleted of contaminating DNA with RNase-free DNase. cDNA was synthesized from 2 μg RNA with M-MuLV reverse transcriptase and random hexamer according to manufacturer's instructions (Fermentas, Burlington, Ontario, Canada). A total of 2 μL reverse transcription products was used for PCR. PCR products were visualized by ethedium bromide staining in 1.5% agarose gel and quantified using Gel-Pro Analyzer software (Media Cybernetics Inc., Silver Spring, Maryland, USA). Amplification of the target cDNA was normalized to β-actin expression. All experiments were replicated at least three times.
Real-time PCR primers
Real-time PCR primers
antisense: 5'- TGAGATAGCAAATCGGCTGACGGT
Cells cultured in 96-well cell culture plates were treated with various concentrations of resveratrol with or without 0.5 μg/ml LPS for 24 hours. Then the culture medium was removed and the cells were incubated with MTT (0.25 mg/ml) for 5 h at 37°C. The formazan crystals in the cells were solubilized with DMSO. The absorbance at 550 nm was determined by a microplate reader Multiskan JX (Themo LabSystems). Cell viability was expressed as a percentage of control.
Cells were treated with different concentrations of resveratrol with or without 0.5 μg/ml LPS for 8 h. The supernatant was collected and LDH release was detected using a cytotox 96® nonradioactive cytotoxicity assay kit (Promega) according to manufacture's instructions. Cell viability was expressed as a percentage of control.
Production of NO was determined by measuring the accumulated level of nitrite (an indicator of NO) in the supernatant after 24 h of LPS treatment with or without different concentrations of resveratrol using a colorimetric reaction with Griess reagent . Briefly, 100 μL of supernatant were mixed with 100 μL Griess reagent [0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, 1% sulfanilamide, and 2.5% H3PO4]. After incubation at room temperature in the dark for 10 min, total nitrites were measured spectrophotometrically at 540 nm. The concentration of nitrite in the sample was determined from a NaNO2 standard curve.
Proinflammatory cytokine measurement by ELISA
Microglial cells (2 × 104/well) or astrocytes (1 × 104/well) were seeded into 48-well plates and cultured for 24 h. Cells were then washed twice with DPBS, and incubated in serum-free DMEM with or without different concentrations of resveratrol for 30 min followed by 0.5 μg/mL LPS for an additional 24 h. The supernatants were collected for measurement of TNFα, IL-6, and MCP-1; and cell lysate were made for detecting IL-1β; using ELISA as described by the manufacturer (Biosourse International).
N9 cells or murine primary astrocytes were grown in 60-mm dishes until subconfluency and then were cultured overnight in medium in the absence of FBS. The cells were pretreated with different concentrations of resveratrol for 1 h followed by LPS for different times (N9 cells: 30 min, astrocytes: 20 min), then were lysed with cold lysis buffer as described previously . Cell lysate proteins were electrophoresed on a 10% SDS-PAGE gel, and transferred onto polyvinylidene difluoride membranes (Millipore Corporation, Bedford, MA). The membranes were blocked with 5% nonfat milk, and then were incubated with anti-phosphorylated ERK1/2, p38 or JNK antibody overnight at 4°C. After incubation with an HRP-conjugated secondary antibody, the protein bands were detected with a Supersignal West Pico chemiluminescenct substrate (Pierce, Rockford, IL) and X-Omat BT film (Eastman Kodak Company, Rochester, New York). For detection of total ERK1/2, p38, or JNK, the membranes were stripped with Restore Western Blot Stripping Buffer (Pierce, Rockford, IL), followed by incubation with specific antibodies. Immunoblot results were quantified using Gel-Pro Analyzer software (Media Cybernetics Inc., Silver Spring, Maryland, USA).
Transient transfection and NF-κB luciferase reporter assay
One day before transfection, murine primary microglial cells (1 × 105/well) or astrocytes (0.5 × 105/well) were seeded into 24-well plates. Transient transfection of pNF-κB-Luc plasmid and control vector (a generous gift from Dr. J. Lu, Second Military Medical University, China) was performed using Lipofectamin 2000 according to the manufacture's recommendations. A pRL-TK-Renilla vector was used as an internal control for normalization of transfection and harvesting efficiency. The cells were cultured with transfection mixture for 5 h, and were then cultured in DMEM containing 10% FBS, 0.5 μg/mL LPS with or without different concentrations of resveratrol for 16 h. Luciferase activity of pNF-κB-Luc and pRL-TK constructs was measured sequentially using the Dual-Luciferase Reporter Assay System (Promega). Variation in transfection efficiency was normalized by dividing the promoter construct activity by the respective co-transfected pRL-TK luciferase activity. Promoter activity of the NF-κB was expressed in units relative to values measured in cells cultured with control medium.
Nuclear extract preparation and electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared as previously described . Protein concentration was determined using a Bio-Rad protein assay kit with bovine serum albumin standards. Activation of AP-1 was assayed by EMSA using a LightShift Chemiluminescent EMSA kit (Pierce, Rockford, IL) according to the manufacture's instruction. Briefly, 6 μg of nuclear extract proteins were pre-incubated with binding buffer (50% Glycerol, 100 mmol/L MgCl2, 1% NP-40 and 1 μg/μL Poly (dI•dC)) for 5 min and then incubated with double-stranded biotin-labeled oligonucleotide containing consensus AP-1 binding site (5'-CGCTTGATGATGAGTCAGCCGGAA-3') for 15 min at room temperature. For competition experiments, unlabelled oligonucleotides were added to the nuclear extracts at a 200-fold molar excess before the addition of the biotin-labeled probe. DNA-protein complexes were analyzed by electrophoresis in 4% polyacrylamide gels. Complexes were transferred to a nylon membrane and crosslinked to the membrane using a hand-held UV lamp equipped with 312 nm bulbs. Migration of the biotinylated oligonucleotides and their complexes was detected by chemiluminescence followed by exposure of the membrane to X-ray films.
Data are presented as mean ± SD. All experiments were performed at least three times. Data were analyzed by a 1-way or 2-way ANOVA with a post hoc Bonferroni test. Differences were considered significant at p < 0.05.
LPS induces proinflammatory cytokine and iNOS expression in microglia and astrocytes in which different signaling molecules are involved
Effects of resveratrol on cell viability and LPS-induced morphological changes in glial cells
Effects of resveratrol on pro-inflammatory cytokine gene expression and release
Effects of resveratrol on iNOS expression and NO production
Effect of resveratrol on MAP kinase activation by LPS
Effects of resveratrol on NF-κB and AP-1 activation by LPS
The present study demonstrates that murine microglia and astrocytes produce proinflammatory cytokines and NO in response to LPS in a similar pattern with some differences in the signaling molecules involved. Resveratrol limited LPS-stimulated microglia and astrocyte activation with different potencies and through inhibiting different signaling molecules. To our knowledge, this is the first report of a difference between microglial cell and astrocyte in response to LPS, and of a difference in the capacity of resveratrol to protect microglia and astrocytes from inflammatory insults.
Microglia are the main resident immuno-competent and phagocytic cells in CNS. Astrocytes also play an important role in regulating inflammation in the CNS. LPS is capable of inducing production of pro-inflammatory cytokines and NO in both microglia and astrocytes [23, 33, 34]. Our study shows that LPS significantly induces the expression and production of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and MCP-1), and enhances the expression of iNOS and production of NO, by primary microglia and primary astrocytes. N9 cells expressed pro-inflammatory cytokines and iNOs at the mRNA level in response to LPS in a pattern similar to that of primary microglia. These results indicate that the inflammatory responses of microglial cell and astrocyte to LPS are similar.
By binding to TLR4, LPS activates NF-κB through TAK1, and activates AP-1 through the TAK1-MAP kinase (ERK1/2, p38, JNK) pathway. NF-κB and AP-1 control inflammatory responses through the induction of inflammatory cytokines [28, 32]. It has been reported that, in microglia, LPS stimulates TNF-α expression through activation of ERK1/2, p38, JNK/AP-1 and NF-κB [34, 35, 36]; stimulates IL-6 and MCP-1 expression through JNK2 and AP-1; stimulates IL-6 expression through p38 [34, 37]; and stimulates iNOS expression through ERK1/2, p38 and NF-κB [35, 38]. Our data demonstrate that, in addition to these mechanisms, LPS stimulates IL-1β expression through activation of ERK1/2, JNK, p38 and AP-1; stimulates IL-6 and MCP-1 expression through ERK1/2; and stimulates iNOS expression through JNK and AP-1. Taken together, these data suggest that MAP kinases, NF-κB and AP-1 are differentially involved in the production of proinflammatory cytokines and iNOS in microglia in response to LPS.
There are only a few reports regarding the involvement of MAP kinases and transcription factors in LPS-induced expression of inflammatory mediators in astrocytes. Treatment of astrocytes with LPS alone induces iNOS expression through ERK1/2- and NF-κB-related signaling pathways . A combination of LPS and IFN-γ results in TNF-α and iNOS expression through activation of ERK1/2, p38 and JNK [39, 40]. Our present study shows that LPS significantly induces ERK1/2, p38, and JNK phosphorylation and NF-κB activation but only slightly activates AP-1 in astrocytes, and that LPS induces proinflammatory cytokine (TNF-α, IL-1β, IL-6 and MCP-1) and iNOS expression in astrocytes through activation of ERK1/2, p38, JNK and AP-1. NF-κB is only involved in LPS-induced TNF-α and iNOS expression in astrocytes. Comparison of the results for microglia with those for astrocytes shows that similar signaling molecules (MAP kinases, NF-κB and AP-1) are involved in LPS-induced TNF-α, IL-1β and IL-6 expression, except that p38 is involved in MCP-1 and iNOS expression only in astrocytes and not in microglia. This may be due to differences in the biological characteristics of these two cell types.
Resveratrol has been reported to inhibit LPS-induced NO and PGE2 production by rat astroglioma cells , and to inhibit TNF-α, iNOS expression and NO production by a mouse microglial cell line [19, 20]. Our results show that, in addition to inhibiting LPS-stimulated TNF-α and NO production, resveratrol also inhibits LPS-induced expression and production of IL-1β, IL-6, and MCP-1 in primary microglia and in the microglial cell line N9 (Fig. 4 and 5). At the tested concentrations (5-50 μM), resveratrol significantly inhibited LPS-induced proinflammatory cytokine production by primary microglia (TNF-α, IL-1β and IL-6) and astrocytes (IL-6), including significant inhibition at the lowest concentration of 5 μM. Furthermore, our results suggest that resveratrol differentially regulates the production of pro-inflammatory molecules and NO by microglia relative to astrocytes. Resveratrol dose-dependently inhibited the production of NO, TNF-α, IL-6 and MCP-1 by primary microglia in response to LPS, but only inhibited NO, TNF-α and MCP-1 production at a high concentration (50 μmol/L) and had no effect on IL-1β production in astrocytes (Fig. 5). Therefore, resveratrol has a more potent suppressive effect on the production of pro-inflammatory molecules by LPS-activated microglia.
Existing observations from both in vitro and in vivo studies have demonstrated that resveratrol has differential effects on MAP kinases and can inhibit the activation of NF-κB and/or AP-1 in a cell or tissue-specific manner [41, 42]. Bi et al.  reported that resveratrol treatment (48 h) of LPS-stimulated (1 μg/mL) N9 cells inhibited LPS-induced p38 phosphorylation. Our study shows that LPS activates p38 in microglia and astrocytes, but we found that resveratrol has no effect on p38 phosphorylation in these cells (Fig. 7). The discrepancy may be due to differences in cell origin and experimental conditions. As our studies show that resveratrol has no effect on LPS-induced phosphorylation of ERK1/2 in either microglia or astrocytes, and only slightly inhibits LPS-induced JNK phosphorylation in astrocytes, we then examined the effect of resveratrol on signaling molecules downstream of MAPKs. NF-κB is a common regulatory element in the promoter region of many pro-inflammatory cytokines. Our studies show that resveratrol attenuates LPS-stimulated NF-κB activation in murine primary microglia and astrocytes. Consistently, other researchers have reported that resveratrol can suppress LPS-induced degradation of IκBα in the microglial cell line N9 , and can suppress nuclear translocation and activation of NF-κB in rat C6 astroglioma cells . Resveratrol is an activator of SIRT1, which has been reported to inhibit NF-κB activity through deacetylation of the RelA/p65subunit of NF-κB . The inhibition of SIRT1 signalingby LPS is partially responsible for the activation of NF-κB pathways and subsequent generation of TNF-α in Kupffer cells and macrophages . Therefore, it should be quite interesting to investigate whether activation of SIRT1 signaling also contributes to the inhibitory effect of resveratrol on NF-κB activation by LPS in glial cells. Recent studies have shown that resveratrol inhibits LPS-induced NF-κB activation by targeting TANK-binding kinase 1 and RIP1 in the TRIF complex in a murine macrophage cell line . Whether the inhibitory effect of resveratrol on LPS-induced NF-κB activation in microglia and astrocytes is mediated by a similar mechanism as that in macrophages will require further investigation. In addition to NF-κB, AP-1 has also beenshown to be involved in inflammatory responses in responseto LPS. Our results show that AP-1 is involved in LPS-induced IL-1β expression and release by microglia and astrocytes. Resveratrol inhibits LPS-induced AP-1 activation in microglia but not astrocytes, which may explain why resveratrol inhibits IL-1β expression and release by microglia but not by astrocytes. AP-1 is formed by dimerization of Jun proteins (c-Jun, JunB and JunD) or heterodimerization of a Jun protein with a Fos protein (c-Fos, FosB, Fra-1 and Fra-2) , and resveratrol has been reported to inhibit c-fos mRNA expression and AP-1 DNA binding in mouse skin . We found that resveratrol has no effect on LPS-induced JNK phosphorylation but inhibits LPS-induced AP-1 activation in microglia. Contrarily, resveratrol slightly inhibits LPS-induced phosphorylation of JNK but has no effect on AP-1 activation by LPS in astrocytes. The contrary effect of resveratrol on JNK phosphorylation and AP-1 activation in microglia and astrocytes may be due to involvement of AP-1 components other than c-Jun in LPS-induced AP-1 activation in microglia, and LPS may activate different AP-1 components in microglia and astrocytes, but these possibilities need further investigation. Collectively, the different effects of resveratrol on proinflammatory cytokine and iNOS expression in response to LPS in microglia and astrocytes may be due to different effects of resveratrol on NF-κB and AP-1 activation in these two cell types. In addition, differences in biological characteristics of microglia and astrocytes may also contribute to their unique response to LPS and resveratrol.
Microglia and astrocytes play important roles in host defense during brain infection and inflammation. These cells produce pro-inflammatory mediators in response to pathologic stimuli such as LPS. As a potent source of proinflammatory cytokines and chemokines, microglia and astrocytes are pivotal in the progression of CNS diseases including Alzheimer's disease , multiple sclerosis [8, 9], Parkinson's disease [10, 11], and brain injury. Therefore, a therapeutic approach aimed at suppressing activation of microglia and astrocytes may alleviate inflammation in the CNS and thus retard the progression of these diseases. Our results suggest that the extent of inflammatory responses induced by LPS in microglia and astrocytes could be limited by resveratrol, with different potencies. Therefore, resveratrol is a natural product with therapeutic potential against CNS diseases involving overproduction of pro-inflammatory cytokines and NO.
This study was supported by grants from the National Basic Research Program of China (973 program) (2010CB529701), the National Natural Science Foundation of China (30970917), the Knowledge Innovation Project of the Chinese Academy of Sciences (KSCX2-YW-N-034), and the Science & Technology Commission of Shanghai Municipality (07DJ14005, 09ZR1436700).
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