The NAD-dependent deacetylase sirtuin 2 is a suppressor of microglial activation and brain inflammation
2013; Springer Nature; Volume: 32; Issue: 19 Linguagem: Inglês
10.1038/emboj.2013.200
ISSN1460-2075
AutoresTeresa F. Pais, Éva M. Szegő, Oldriska Marques, Leonor Miller‐Fleming, Pedro Antas, Patrícia Guerreiro, Rita Machado de Oliveira, Burcu Kasapoğlu, Tiago F. Outeiro,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoArticle6 September 2013free access Source Data The NAD-dependent deacetylase sirtuin 2 is a suppressor of microglial activation and brain inflammation Teresa Faria Pais Corresponding Author Teresa Faria Pais Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal Search for more papers by this author Éva M Szegő Éva M Szegő Department of Neurodegeneration and Restorative Research, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Oldriska Marques Oldriska Marques Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal Search for more papers by this author Leonor Miller-Fleming Leonor Miller-Fleming Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal Search for more papers by this author Pedro Antas Pedro Antas Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal Search for more papers by this author Patrícia Guerreiro Patrícia Guerreiro Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal Search for more papers by this author Rita Machado de Oliveira Rita Machado de Oliveira Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal Search for more papers by this author Burcu Kasapoglu Burcu Kasapoglu Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Göttingen, Germany Search for more papers by this author Tiago Fleming Outeiro Corresponding Author Tiago Fleming Outeiro Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal Department of Neurodegeneration and Restorative Research, University Medical Center Göttingen, Göttingen, Germany Instituto de Fisiologia, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal Search for more papers by this author Teresa Faria Pais Corresponding Author Teresa Faria Pais Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal Search for more papers by this author Éva M Szegő Éva M Szegő Department of Neurodegeneration and Restorative Research, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Oldriska Marques Oldriska Marques Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal Search for more papers by this author Leonor Miller-Fleming Leonor Miller-Fleming Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal Search for more papers by this author Pedro Antas Pedro Antas Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal Search for more papers by this author Patrícia Guerreiro Patrícia Guerreiro Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal Search for more papers by this author Rita Machado de Oliveira Rita Machado de Oliveira Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal Search for more papers by this author Burcu Kasapoglu Burcu Kasapoglu Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Göttingen, Germany Search for more papers by this author Tiago Fleming Outeiro Corresponding Author Tiago Fleming Outeiro Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal Department of Neurodegeneration and Restorative Research, University Medical Center Göttingen, Göttingen, Germany Instituto de Fisiologia, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal Search for more papers by this author Author Information Teresa Faria Pais 1, Éva M Szegő2, Oldriska Marques1, Leonor Miller-Fleming1, Pedro Antas1, Patrícia Guerreiro1, Rita Machado de Oliveira1, Burcu Kasapoglu3 and Tiago Fleming Outeiro 1,2,4 1Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal 2Department of Neurodegeneration and Restorative Research, University Medical Center Göttingen, Göttingen, Germany 3Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Göttingen, Germany 4Instituto de Fisiologia, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal *Corresponding authors. Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Av. Prof. Egas Moniz, Lisboa 1649-028, Portugal. Tel.:+351 217999496; Fax:+351 217999412; E-mail: [email protected] of Neurodegeneration and Restorative Research, University Medical Center Göttingen, Göttingen, 37073 Germany. Tel.:+49 5513913544; Fax:+49 5513922693; E-mail: [email protected] The EMBO Journal (2013)32:2603-2616https://doi.org/10.1038/emboj.2013.200 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Deleterious sustained inflammation mediated by activated microglia is common to most of neurologic disorders. Here, we identified sirtuin 2 (SIRT2), an abundant deacetylase in the brain, as a major inhibitor of microglia-mediated inflammation and neurotoxicity. SIRT2-deficient mice (SIRT2−/−) showed morphological changes in microglia and an increase in pro-inflammatory cytokines upon intracortical injection of lipopolysaccharide (LPS). This response was associated with increased nitrotyrosination and neuronal cell death. Interestingly, manipulation of SIRT2 levels in microglia determined the response to Toll-like receptor (TLR) activation. SIRT2 overexpression inhibited microglia activation in a process dependent on serine 331 (S331) phosphorylation. Conversely, reduction of SIRT2 in microglia dramatically increased the expression of inflammatory markers, the production of free radicals, and neurotoxicity. Consistent with increased NF-κB-dependent transcription of inflammatory genes, NF-κB was found hyperacetylated in the absence of SIRT2, and became hypoacetylated in the presence of S331A mutant SIRT2. This finding indicates that SIRT2 functions as a 'gatekeeper', preventing excessive microglial activation through NF-κB deacetylation. Our data uncover a novel role for SIRT2 opening new perspectives for therapeutic intervention in neuroinflammatory disorders. Introduction Microglia, the innate immune cells in the CNS, are adapted to sense and immediately react to pathogens, misfolded proteins, or molecules released by damaged cells. It is now known that microglia may adopt different activated phenotypes in response to external stimuli. Whether activated microglia is beneficial or noxious to the CNS is determined by several variables, including the balance between cytotoxic and neurotrophic molecules generated by activated microglia, and the intensity and timing of microglial activation (reviewed in Lucin and Wyss-Coray, 2009; Perry et al, 2010; and Saijo and Glass, 2011). A deleterious sustained inflammatory response mediated by microglia has been associated to several neurologic conditions, including CNS infections (Garden, 2002; Dellacasa-Lindberg et al, 2011), ischemic stroke (Yrjanheikki et al, 1998), and neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's disease (McGeer et al, 1988; Sulzer, 2007; Tai et al, 2007). In these diseases, secretion of inflammatory mediators by microglia, such as pro-inflammatory cytokines (i.e., tumour necrosis factor (TNF), IL-6, and IL-1β), metalloproteases, reactive oxygen species (ROS), nitric oxide (NO), and glutamate, are thought to contribute to neuronal cell death (Block et al, 2007). Therefore, dissecting the mechanisms that selectively shut off deleterious activation pathways might play an important role in controlling neurologic diseases. In fact, deregulation of microglial receptors CD200R and TREM2 signalling-mediated pathways, which were shown to prevent toxicity of microglia, is associated to increased inflammation and neurodegeneration in humans (Piccio et al, 2008; Walker et al, 2009; Guerreiro et al, 2012). SIRT2 is one of the seven known mammalian SIRTs, a family of NAD+-dependent deacetylases. SIRT1 and SIRT6 emerged recently as regulators of several age-related pathologies, including neurodegenerative diseases and peripheral inflammation (Galli et al, 2011). SIRT2 is expressed as two isoforms, resulting from alternative splicing. The shorter isoform lacks the first 37 amino acids (North et al, 2003), is preferentially expressed in the adult nervous system, and is 20 times more expressed in the hippocampus than other sirtuins (Pandithage et al, 2008). SIRT2 is predominantly cytosolic but the shuttling between the cytoplasm and nucleus explains that both α-tubulin (North et al, 2003) and histones (Vaquero et al, 2006) are deacetylated by this sirtuin. Several studies indicate that SIRT2 plays an important role in brain function. SIRT2 is required for myelination through deacetylation of the polarity protein Par-3, as recently demonstrated by Schwann cell-specific ablation of SIRT2 in mice (Beirowski et al, 2011). SIRT2 activity is inhibited by cyclin-dependent kinase-2 (Cdk2) and Cdk5-dependent phosphorylation at residue S331. Accordingly, the S331A SIRT2 mutant, which cannot be phosphorylated at this specific residue, reduces neurite length and tubulin acetylation to a higher extent than the wild-type (wt) protein (Pandithage et al, 2008). Also, SIRT2 has been implicated in neuronal degeneration in cellular and animal models of Parkinson's disease (PD). Chemical inhibition of SIRT2 rescued neuronal cells from alpha-synuclein-induced toxicity (Outeiro et al, 2007). Other SIRT2 substrates were recently identified, including the p65 subunit of NF-κB, a major transcription regulator of inflammation. Fibroblasts from SIRT2−/− mice show hyperacetylation of p65 concomitantly with increased expression of NF-κB-dependent genes induced by TNF (Rothgiesser et al, 2010a). The receptor-interacting protein 1 (RIP-1), which is also involved in inflammatory signalling pathways, was also found to be a SIRT2 substrate. The deacetylation of RIP-1 by SIRT2 stabilizes the RIP-1–RIP-3 protein complex required for TNF-induced cell death in fibroblasts (Narayan et al, 2012). Altogether, these results provide evidence that SIRT2 deacetylates important regulators of inflammation such as NF-κB and RIP-1. Stimulation of Toll-like receptors (TLRs) in microglia triggers CNS inflammation mainly through NF-κB-dependent transcriptional activation. TLRs recognize pathogen-associated molecular patterns (PAMPs) during infection whereas in non-infectious-mediated CNS injury, TLRs bind to endogeneous damage-associated molecular patterns (DAMPs) released by dead cells (Hanamsagar et al, 2012). TLR4 is a receptor for the Gram-negative bacterial cell wall component, lipopolysaccharide (LPS) (Poltorak et al, 1998). Microglial activation by LPS occurs in bacterial meningitis caused by Escherichia coli (Kim, 2003) and Citrobacter koseri (Liu and Kielian, 2009) or during Staphylococcus aureus-induced brain abscess (Stenzel et al, 2008). Moreover, peripheral infection by Gram-negative bacteria is also known to activate the innate immune response in the CNS through TLR4 (Chakravarty and Herkenham, 2005), which may also contribute to the pathology of chronic neurodegenerative diseases (Cunningham, 2013). Also, several endogeneous TLR4 ligands have been identified such as High mobility group box 1 protein (HMGB1) (Yang et al, 2010), heme (Figueiredo et al, 2007), fibrinogen (Smiley et al, 2001), beta-amyloid (Reed-Geaghan et al, 2009), and alpha-synuclein (Fellner et al, 2013). Therefore, microglial activation by TLR4 and by other TLRs may have implications in CNS infection, stroke, and neurodegenerative diseases, which makes TLR signalling pivotal in neuroinflammatory conditions. Here, we addressed the biological significance of SIRT2 in the inflammatory response in the brain, where it is highly expressed, and in a context of TLR activation. We investigated SIRT2 as a potential regulator of brain inflammatory responses mediated by microglia. Our findings provide the first direct evidence for a key inhibitory role of SIRT2 in microglia-mediated inflammation and neurotoxicity. This indicates that SIRT2 might be explored as a target to prevent deleterious inflammatory responses, which may ultimately bear major implications in CNS inflammatory diseases. Results SIRT2 regulates the inflammatory response to LPS in vivo First, we examined whether SIRT2 was expressed by microglia since, to our knowledge, this had not been previously reported. Immunohistochemical analysis of the mouse brain showed SIRT2 expression in cells co-stained for Iba-1, a specific microglial cell marker that is upregulated in activated cells (Figure 1A) (Ito et al, 1998; Harting and Knoll, 2010). We also found that both full-length (LSIRT2) and the shorter truncated (sSIRT2) alternative splice isoforms (Dryden et al, 2003) were expressed in primary cultures of microglia, in the N9 microglial cell line, as well as in other brain cells as previously described (Harting and Knoll, 2010). Interestingly, the adult brain is particularly enriched in the shorter isoform in comparison with other organs (Supplementary Figure S1A). Figure 1.SIRT2 regulates the brain response to LPS. (A) SIRT2 expression by microglial cells. A representative image of co-localization of SIRT2 with Iba-1 was obtained from brain sections stained for Iba-1 (red) and SIRT2 (green). (B) SIRT2 protein levels in mice injected intracortically with PBS or LPS (0.2 μg). Cortical extracts from mice injected with PBS or LPS were analysed by western blotting and stained for SIRT2. Quantification was done by densitometry analysis of the protein bands and normalized to β-actin levels (mean±s.d.; n=3, PBS; n=5, LPS; t-test, *P<0.05). (C) Microglial activation induced by intracortical injection of LPS. Representative images of brain sections of mice injected with PBS and LPS at day 2 and 7 post injection and stained for Iba-1. (D) Microglial activation in wild-type versus SIRT2−/− mice. SIRT2−/−mice show increased microglial activation induced by LPS injection on bright-field and confocal (below) microscopy images of mice brain sections stained for Iba-1. Confocal images reveal thicker microglial processes in SIRT2−/−mice. (E) mRNA levels of IL-1, IL-6, Mpa2l, TNF, and Ip-10 inflammatory genes were determined by qPCR, normalized to β-actin expression levels. IL-6, Mpa2I, TNF, and lp-10 expression is increased after 6 h in LPS-injected SIRT2−/−mice compared to LPS-injected WT mice (mean±s.d., n=4; two-way ANOVA, ***P<0.001). (F) Flow cytometry analysis of cortical cells isolated from injected mice and stained for CD11b, CD45 and IL-6 (mean±s.d.; n=3, PBS; n=4, LPS; two-way ANOVA, test, ***P<0.001). (G) Induction of free radicals by LPS. Representative images of wt and SIRT2−/− mouse brain sections stained for nitro tyrosine 5 days post injection with PBS or LPS (5 μg). (H) Neuronal cell loss induced by LPS. Mice brain sections were stained for NeuN and the number of positive cells counted and normalized to the numbers of cells in the cortice of wt mice 5 days after PBS or LPS (5 μg) injection (mean±s.d., n=3; two-way ANOVA, *P<0.05). Download figure Download PowerPoint To investigate whether SIRT2 plays a direct role in microglia activation, mice were injected with 0.2 μg of LPS intracortically. LPS is a general pro-inflammatory stimulus that binds to TLR4 exclusively expressed by microglia in the mouse brain (Lehnardt et al, 2002), and very relevant in the context of bacterial meningitis (Kim, 2003). This is also a well-established model of direct activation of innate immunity in the brain, which activates NF-κB-dependent pathways in microglia (Milatovic et al, 2003). Surprisingly, the levels of SIRT2, but not SIRT1 (Supplementary Figure S1B), were significantly reduced in the cortex of mice 2 days after injection of LPS (Figure 1B). At this time point, there was a clear increase in microglial Iba-1 immunoreactivity that was still observed at day 7 post injection (Figure 1C). To elucidate the role of SIRT2 in microglia activation in vivo, we used SIRT2−/− mice. The number of Iba-1-positive cells in the cortex of SIRT2−/− adult mice is not significantly different from wt mice, suggesting that SIRT2 is not crucial for microglia differentiation (Supplementary Figure S2A). Notably, 2 days after LPS injection, microglial cells displayed enhanced Iba-1 immunoreactivity and thicker processes around the injection site in SIRT2−/− mice, a morphological change associated with microglial activation (Figure 1D). Furthermore, the mRNA levels of pro-inflammatory factors such as interleukin-6 (IL-6), macrophage activating 2 like (Mpa2l), interferon gamma-induced protein 10 (Ip 10) and TNF were significantly upregulated (∼2-fold) in the cortex of SIRT2−/−mice (Figure 1E). Flow cytometry analysis of cortical cells stained intracellularly for IL-6 supported the induction of this cytokine (∼2-fold) within the CD11b-positive (microglia/macrophages) population isolated from SIRT2−/− mice (Figure 1F; Supplementary Figure S2B). There was no significant difference if cells isolated from LPS-injected mice were intracellular stained with an isotype control antibody (wt, 2.9±0.5 versus 3.9±0.7 in SIRT2−/− mice). The expression of CD45, a marker of microglia/macrophage activation (Kettenmann et al, 2011), was increased 48 h after LPS injection and was not significantly different between wt and SIRT2−/− mice (Figure 1F; Supplementary Figure S2B). These results clearly show, for the first time, that in the absence of SIRT2 there is an increase in pro-inflammatory cytokine response by microglia in vivo. Moreover, the response to LPS in SIRT2−/− mice was associated with a clear induction of free radicals, namely peroxynitrite, as measured by the increased staining for nitrotyrosine in cortical cells 5 days after injection of 5 μg of LPS (Figure 1G). We used a higher dose of LPS in these experiments based on the previous studies reporting LPS-induced neurodegeneration (de Pablos et al, 2006). Thus, to further test whether increased inflammation and oxidative stress could lead to neuronal cell loss, we stained the brain sections for NeuN, a neuron-specific protein (Mullen et al, 1992). We found a small but a significant decrease in NeuN-positive cells in SIRT2/− mice injected with LPS compared with wt mice 5 days after LPS injection (Figure 1H; Supplementary Figure S2C). Altogether, our results clearly indicate that loss of SIRT2 renders microglia more prone to activation by pro-inflammatory stimuli in vivo, accompanied by higher levels of oxidative stress and neuronal cell loss. SIRT2 inhibits microglial activation in a phosphorylation-dependent manner To clarify the molecular mechanisms involved in SIRT2-mediated microglia activation, we next used the well-characterized N9 microglial cell line (Stansley et al, 2012) (Figure 2A). The sSIRT2 form appeared as a doublet in non-activated N9 cells. The slower migrating band disappeared after N9 cell extracts were treated with λPP1 phosphatase (Supplementary Figure S3A), indicating that this band corresponds to a phosphorylated form of SIRT2 (psSIRT2). Interestingly, microglial stimulation with LPS and TNF (LPS+TNF) significantly decreased sSIRT2 phosphorylation compared with non-stimulated cells (CTR) (Figure 2B). Figure 2.SIRT2 inhibition of microglial activation depends on S331 phosphorylation. (A) Western blot analysis of microglia activated with LPS and TNF (LPS+TNF) shows clear reduction in a slower migrating protein band (higher MW) corresponding to psSIRT2. Arrows on the right indicate the full (LSIRT2) and shorter (sSIRT2) SIRT2 splicing variants. (B) Microglial activation with LPS+TNF leads to a significant decrease in the level of phosphorylated sSIRT2 (mean±s.d. of three independent experiments, t-test, **P<0.01). (C) Viral-transduced microglial cells were untreated or activated with LPS+TNF and supernatants analysed for nitrites and IL-6, and cells immunostained against CD40 for flow cytometry. The quantification of iNOS activity, IL-6 and CD40 expression results is presented as a relative percentage of the levels quantified in cells viral-transduced with empty vector (CTR) and stimulated with LPS+TNF (mean±s.d., n=4 independent experiments; one-way ANOVA, Tukey's multiple comparison test, P<0.01). ** and *** are defined by the 95% confidence interval (CI) of difference between means. (D) Western blot analysis showing reduction of SIRT2 protein levels in non-activated (−) or activated with LPS+TNF (+) N9 cells viral-transduced with shRNA specific for SIRT2 (sh2.1) versus control shRNA (shCtr). (E) IL-6 secretion in SIRT2 KD cells. KD or shCtr-transduced cells were left untreated (CTR, white squares) or activated with LPS+TNF (black squares). IL-6 levels were determined by ELISA. (F) Expression of membrane activation markers in SIRT2 KD cells. Quantification of CD40, CD80, and CD45 expression was done by flow cytometry analysis and represented by the Geo Mean of the histograms obtained for the fluorescence intensity of stained cells. In (E) and (F), data are presented as mean±s.d. and representative of three independent experiments done in triplicate, two-way ANOVA, ***P<0.001. ND, not detectable.Source data for this figure is available on the online supplementary information page. Source Data for Figure 2A [embj2013200-sup-0001-SourceData-S1.pdf] Source Data for Figure 2D [embj2013200-sup-0002-SourceData-S2.pdf] Download figure Download PowerPoint We next hypothesized that SIRT2 may regulate microglial responses in a phosphorylation-dependent manner since SIRT2 phosphorylation at S331 is known to inhibit its deacetylase activity (Pandithage et al, 2008). To address this, we tested whether major pathways associated with microglial responses were affected by overexpression of wt sSIRT2 or SIRT2 phospho-mutants (S331A, phospho-resistant; and S331D, phospho-mimetic). The S331D mutant form migrated at a slight higher MW, as expected from the negative charge of the aspartate (Supplementary Figure S3B). N9 microglial cells were stimulated with LPS+TNF 48 h after viral transduction with the different sSIRT2 forms. Wild-type sSIRT2 reduced significantly the NO (assayed as nitrite) generated by the inducible isoform of nitric oxide synthase (iNOS) (∼50%), secretion of the pro-inflammatory IL-6 cytokine (∼40%), and expression of the membrane protein and activation marker CD40 (Kettenmann et al, 2011) (∼20%) in N9 cells stimulated with LPS+TNF (Figure 2C). The phospho-resistant mutant (sSIRT2_S331A) was significantly more effective than the phospho-mimetic mutant (sSIRT2_S331D) in reducing iNOS activity (72 versus 32%), IL-6 secretion (68 versus 44%), and CD40 expression (21 versus 8%). In addition, while the sSIRT2_S331A mutant induced a significant decrease in iNOS activity and IL-6 secretion, the S331D mutant was less effective in reducing CD40 expression in comparison with wt sSIRT2. In order to examine the effect of decreased levels of SIRT2 in microglia, N9 cells were transduced with lentiviral particles producing different short hairpin RNAs (shRNAs) specific for SIRT2 (sh2.1) or a non-coding sequence (scrambled) as a control (shCtr). shRNA transduction significantly reduced the expression levels of SIRT2 (∼90%) (Figure 2D). Microglia activation phenotype was then studied in SIRT2 knock-down (KD) N9 cells. Contrary to SIRT2 overexpression, decrease in SIRT2 protein levels significantly (P<0.05) enhanced the secretion of IL-6 (Figure 2E) and CD40 expression (Figure 2F) induced by LPS+TNF. CD80, another microglia/macrophage activation marker (Kettenmann et al, 2011), was also increased in SIRT2 KD cells while the levels of CD45, a protein tyrosine phosphatase, did not differ between control and KD activated cells (Figure 2E). Additionally, we did not detect differences in MAPK signalling pathways, assessed by ERK1/2 and p38 phosphorylation levels, phagocytic activity, glutamate production or induction of IL-1β mRNA levels (Supplementary Figure S4). Taken together, the overexpression and KD of SIRT2 in microglia confirmed that SIRT2 is an inhibitor of microglial inflammatory responses, and support our observations in SIRT2−/−mice. On the basis of our results, SIRT2 is probably acting downstream of activation signalling cascades triggered by inflammatory stimuli, and its effect on microglia is modulated by phosphorylation at S331. SIRT2 modulates microglia sensing by TLR2, 3 and 4 We then tested whether SIRT2 regulates the microglial response to other TLRs using specific ligands for TLR2 (Pam3CSK4), TLR3 (Poly I:C), TLR4 (LPS), and TLR9 (CpG). We analysed several established activation markers, including IL-10 production, which is induced following TLR ligation in microglia (Jack et al, 2005). IL-10 is also part of a negative-feedback mechanism to restrain inflammatory response in LPS-activated macrophages (Chang et al, 2007). SIRT2 KD in N9 cells increased CD40 expression, IL-6 and IL-10 secretion, and iNOS activity induced by TLR4 and TLR2 activation. We did not observe major alterations in response to TLR9 activation (Figure 3). Interestingly, SIRT2 KD in microglia only affected TNF induction significantly upon TLR3 activation. Figure 3.SIRT2 regulates microglial response through TLR2, 3 and 4. (A) Representative histograms of the fluorescence intensity for CD40 showing the overlays of shCtr (filled) and sh2.1 (solid line) viral-transduced N9 cells that were stimulated with different TLR ligands. (B) Induction of IL-10, IL-6, TNF, and nitrites (NO2−) by TLR ligands in control (white bars) and SIRT2 KD (black bars) N9 cells. LPS (50 ng/ml), Pam2CSK4 (0.05 μg/ml), Poly I:C (10 μg/ml), and CpG (10 μg/ml) are ligands for TLR4, 2, 3, and 9, respectively. Data shown are presented as mean±s.d. and representative of three independent experiments done in triplicate, two-way ANOVA, ***P<0.001. Download figure Download PowerPoint Together, these results show that SIRT2 is able to regulate microglial activation induced by several stimuli and therefore widens the spectrum of conditions where SIRT2 modulation could be relevant to CNS injury. SIRT2 suppresses ROS/RNS production, activation-induced cell death in microglia, and microglial-induced neurotoxicity Microglial cells contribute to the oxidative damage in neurodegenerative diseases through the generation of ROS and reactive nitrogen species (RNS) (Reynolds et al, 2007). Moreover, the simultaneous activation of NADPH oxidase and iNOS produces peroxynitrite, a highly toxic oxidant that crosses cellular membranes and causes neuronal and oligodendroglial cell death (Mander and Brown, 2005). Interestingly, reduced levels of SIRT2 enhanced both intracellular ROS formation (Figure 4A) and iNOS protein levels both in activated N9 cells and in primary microglia (Figure 4B). The upregulation of iNOS protein levels was accompanied by a significant increase in its activity, measured by the levels of nitrites in the supernatants of activated cells (Figure 4B). The induction of iNOS in activated SIRT2 KD N9 cells was also observed at the mRNA level (Supplementary Figure S5D). These data demonstrate that the loss of SIRT2 increases microglial pro-inflammatory responses and production of ROS and RNS during microglial activation, which is in agreement with our observation in SIRT2−/− mice (Figure 1). Figure 4.SIRT2 suppresses the neurotoxic response of microglia. (A) Quantification of intracellular ROS by flow cytometry analysis is represented by the Geo Mean of the histograms obtained for the fluorescence intensity of the probe. Data shown are presented as mean±s.d. and representative of three independent experiments done in triplicate, two-way ANOVA, ***P<0.001. (B) Western blot analysis of iNOS expression in activated shCtr-transduced and SIRT2 KD cells and in primary microglial cultures prepared from wt and SIRT2−/− newborn mice. Quantification of RNS by assessing iNOS activity through measurement of nitrites accumulation in the culture supernatants. Results are presented as fold induction values compared to control activated microglia. (C) Effect of SIRT2 KD on microglial cell death. Cell viability was measured by reduction of MTT to formazan in activated control or SIRT2-KD N9 cells. t-test ***P<0.005. (D) The caspase-3 activity was analysed by detection of cleaved caspase-3 levels by western blotting in SIRT2 KD N9 cells and SIRT2−/− primary microglia. (E) Effect of microglial conditioned media (MCM) on neuronal survival. HT22 neurons were incubated with medium obtained from viral-transduced microglia non-treated or treated with LPS+TNF and cell viability measured by MTT assay. Data are presented as mean±s.d. of three independent experiments or representative of three independent experiments. t-test *P<0.05; **P<0.01.Source data for this figure is available on the online supplementary information page. Source Data for Figure 4B [embj2013200-sup-0003-SourceData-S3.pdf] Source Data for Figure 4D [embj2013200-sup-0004-SourceData-S4.pdf] Download figure Download PowerPoint We also investigated microglial activation in microglial cells knock-down for SIRT1, a sirtuin previously linked to inflammation (Schug et al, 2010). Although IL-6 was also increased in activated microglia upon a 50% decrease in SIRT1 protein
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