Resolvin D1 and E1 promote resolution of inflammation in microglial cells in vitro

Brain, Behavior, and Immunity xxx (2015) xxx–xxx

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Resolvin D1 and E1 promote resolution of inflammation in microglial cells in vitro C. Rey a,b,c, A. Nadjar a,b, B. Buaud c, C. Vaysse c, A. Aubert a,b, V. Pallet a,b, S. Layé a,b, C. Joffre a,b,⇑ a

INRA, Nutrition et Neurobiologie Intégrée, UMR 1286, 33076 Bordeaux, France Univ. Bordeaux, Nutrition et Neurobiologie Intégrée, UMR 1286, 33076 Bordeaux, France c ITERG, Institut des corps gras, 33600 Pessac, France b

a r t i c l e

i n f o

Article history: Received 17 September 2015 Received in revised form 17 December 2015 Accepted 19 December 2015 Available online xxxx Keywords: Microglial cells n-3 PUFA DHA EPA RvD1 RvE1 BV2 Neuroinflammation miRNA

a b s t r a c t Sustained inflammation in the brain together with microglia activation can lead to neuronal damage. Hence limiting brain inflammation and activation of microglia is a real therapeutic strategy for inflammatory disease. Resolvin D1 (RvD1) and resolvin E1 (RvE1) derived from n-3 long chain polyunsaturated fatty acids are promising therapeutic compounds since they actively turn off the systemic inflammatory response. We thus evaluated the anti-inflammatory activities of RvD1 and RvE1 in microglia cells in vitro. BV2 cells were pre-incubated with RvD1 or RvE1 before lipopolysaccharide (LPS) treatment. RvD1 and RvE1 both decreased LPS-induced proinflammatory cytokines (TNF-a, IL-6 and IL-1b) gene expression, suggesting their proresolutive activity in microglia. However, the mechanisms involved are distinct as RvE1 regulates NFjB signaling pathway and RvD1 regulates miRNAs expression. Overall, our findings support that pro-resolving lipids are involved in the resolution of brain inflammation and can be considered as promising therapeutic agents for brain inflammation. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Microglia respond to brain injury and inflammation with production of inflammatory factors, such as pro-inflammatory cytokines (Gehrmann et al., 1995). Functional consequences of cytokine production in the brain include alterations in cognition and emotional behavior (recently reviewed in Dantzer et al., 2008). If sustained, inflammation can also worsen the related injury, leading to neuronal damage that is the basis of a large variety of brain pathologies (Woodroofe and Cuzner, 1993; Woodroofe, 1995; Blais and Rivest, 2003; Laye, 2010; Solito and Sastre, 2012). Hence, the identification of mediators limiting the inflammation and/or involved in the resolution of inflammation is of growing interest as it may provide novel targets in brain damage prevention and treatment. Recent data emphasize the importance of specialized proresolving mediators (SPM) such as resolvins in the resolution of inflammation (Fredman and Serhan, 2011). Resolvins are

⇑ Corresponding author at: UFR Pharmacie 2eme tranche 2eme étage, Université Bordeaux Segalen – CC34, 146 rue Léo Saignat, 33076 Bordeaux cedex, France. E-mail address: [email protected] (C. Joffre).

endogenous lipid mediators derived from n-3 polyunsaturated fatty acids (PUFAs) with both anti-inflammatory and pro-resolutive activities without immune-suppression (Serhan et al., 2002, 2008; Serhan, 2014). Among the resolvins, resolvin D1 (RvD1, 7S,8R,17S-tri hydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid) and resolvin E1 (RvE1, 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapen taenoic acid) are of particular interest in the resolution of inflammation because they actively turn off the inflammatory response (Fredman and Serhan, 2011). They underlie most of the beneficial effects attributed to their precursors (Calder, 2013; Serhan and Chiang, 2013; Bazinet and Laye, 2014; Headland and Norling, 2015). RvD1 is synthesized by 15- and 5-lipoxygenases (LOX) from docosahexaenoic acid (DHA) and acts through the binding to its receptors, orphan receptor G protein coupling receptor 32 (GPR32) and lipoxin A4 receptor/formyl peptide receptor 2 (ALX/fpr2) (Krishnamoorthy et al., 2010). RvE1 is derived from eicosapentaenoic acid (EPA) by cytochrome P450 enzymes and 5-LOX (Serhan et al., 2000; Arita et al., 2005) and binds to its receptor ChemR23 (Chemokine-like receptor 1), a G protein-coupled receptor (Samson et al., 1998). The anti-inflammatory and pro-resolving activities of RvD1 and E1 have been reported both in vitro and in vivo, mostly on peripheral cells. In vitro studies report that RvD1 and E1 potently decrease pro-inflammatory cytokine expression

http://dx.doi.org/10.1016/j.bbi.2015.12.013 0889-1591/Ó 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: Rey, C., et al. Resolvin D1 and E1 promote resolution of inflammation in microglial cells in vitro. Brain Behav. Immun. (2015), http://dx.doi.org/10.1016/j.bbi.2015.12.013

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C. Rey et al. / Brain, Behavior, and Immunity xxx (2015) xxx–xxx

(Schwab et al., 2007; Tian et al., 2009; Recchiuti et al., 2011; Titos et al., 2011) and enhance macrophage phagocytic activity (Krishnamoorthy et al., 2010; Ohira et al., 2010). RvD1 associated with 1,25-vitamin D3 reverse most of the effect of Ab on macrophages extracted from Alzheimer’s disease patients (Mizwicki et al., 2013). In vivo, RvD1 administration decreases proinflammatory cytokine production in acute models of lung (Wang et al., 2011, 2014a; Zhou et al., 2013; Yaxin et al., 2014), or kidney injury (Chen et al., 2014) and in a model of allergic airways (Rogerio et al., 2012). RvE1 also exerts potent anti-inflammatory actions via the regulation of cytokine production in experimental models of colitis (Arita et al., 2005) and peritonitis (Schwab et al., 2007). Studies from Serhan and coworkers identified the possible pathways and led to a hypothetical scheme for RvD1/ALX- and RvE1/ChemR23-dependent signaling in human macrophages (Ohira et al., 2010; Fredman and Serhan, 2011; Recchiuti, 2013). The regulation of microRNAs (small non coding RNA of approximately 23 nucleotides that target specific mRNA) that are fined tuners of immune responses seems to be one of the key components of RvD1/ALX signaling pathways whereas RvE1 is likely to regulate the phosphorylation of signaling proteins such as Akt and MAP kinases. Contrary to macrophages, anti-inflammatory activities of RvD1 and RvE1 have been poorly reported in microglia, the brain innate immune cells. In vitro, RvE1 blocks lipopolysaccharide (LPS)-induced microgliosis and tumor necrosis factor (TNF)-a release in primary microglial cell culture (Xu et al., 2013). In vivo, RvE1 significantly alters the inflammatory profile and activation of microglia in mice (Harrison et al., 2015) and inhibits neuropathic pain and spinal cord microglial activation following peripheral

2. Materials and methods 2.1. Cell culture BV2 microglial cells (a murine microglial cell line kindly provided by Dr. Watterson, Northwestern University, Chicago, IL, USA) were grown in RPMI (Fisher Scientific, Illkirch, France) supplemented with 10% heat-inactivated fetal calf serum (Eurobio, Courtaboeuf, France), streptomycin sulfate (50 lg/mL), phenoxypenicillinic acid (65 lg/mL) and glutamine (2 mmol/L) in 5% CO2 at 37 °C as previously described (De Smedt-Peyrusse et al., 2008). Cells were seeded at a density of 1  106/well in 6-well culture plates. When cells reached 90% of confluency, they were serum-starved for 24 h. RvD1 and RvE1 concentrations were chosen on the basis of dose response experiments (from pilot studies and the literature) (Krishnamoorthy et al., 2010; Titos et al., 2011; Wang et al., 2011; Wu et al., 2013; Li et al., 2014) (Fig. S1).

ChemR23

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interaction p<0.0001

interaction p<0.0001

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nerve injury (Xu et al., 2013). However, the mechanisms underlying these effects are still unknown. Here, we studied the effect of RvD1 and RvE1 on microglia activity in vitro. To do so, BV2 microglial cells were pre-incubated with RvD1 or RvE1 and then incubated with LPS to activate microglial cells. We first assessed the time course of proinflammatory and anti-inflammatory microglial markers expression post-LPS treatment. Then, we evaluated the capacity of RvD1 and RvE1 to modulate the inflammatory response. We finally deciphered some of the signaling pathways potentially involved in the anti-inflammatory and pro-resolving effect of resolvins.

Fig. 1. Gene expression of the enzymes and receptors involved in RvE1 (ChemR23) and RvD1 (ALX/fpr2) pathways in response to LPS (1 lg/mL), measured by RT-qPCR. Values are means ± SEM (n = 6). Statistics were applied using a 2-way ANOVA with Fisher’s PLSD post hoc test. *p < 0.05, **p < 0.01, ***p < 0.0001.

Please cite this article in press as: Rey, C., et al. Resolvin D1 and E1 promote resolution of inflammation in microglial cells in vitro. Brain Behav. Immun. (2015), http://dx.doi.org/10.1016/j.bbi.2015.12.013

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BV2 cells were incubated with 10 nM of 17(S)-RvD1 and RvE1 (Bertin Pharma, Montigny le Bretonneux, France) or vehicle (RPMI) for 30 min before addition of 1 lg/mL of LPS (Escherichia coli, 0127: B8; Sigma–Aldrich, Lyon, France) under serum-free conditions. Three independent experiments were performed: first the timecourse of LPS-induced pro-inflammatory markers (IL-1b, TNF-a, IL-6, CD86), anti-inflammatory markers (IL-10, SOCS3, Ym1, Arg1, CD206, 15-LOX and 5-LOX) and resolvin receptors (ChemR23 and ALX/fpr2) between 2 h and 24 h (Figs. 1 and 2), second the effect of RvD1 and RvE1 on the inflammatory marker gene expression after 6 h and 24 h LPS treatment (Figs. 3 and 4) and third the signaling pathways activated by the resolvins (Figs. 5–8).

To measure cytokine and receptor expression, quantitative PCR was performed using the Applied Biosystems (Foster, CA) assay-on demand gene expression protocol as previously described (Mingam et al., 2008; Madore et al., 2013). We focused on the expression levels of IL-1b, IL-6, TNF-a, IL-10 and CD86 mRNA for pro-inflammatory markers, on the expression levels of CD206, SOCS3, Ym1, Arg1, 15-LOX and 5-LOX mRNA for antiinflammatory markers and on the expression of the resolvin receptors ChemR23 and ALX/fpr2. Data were analyzed using the comparative threshold cycle (Ct) method, results are expressed as relative fold change (Mingam et al., 2008; Madore et al., 2013, 2014; Delpech et al., 2015) to control target mRNA expression.

2.2. mRNA isolation and RT-qPCR

2.3. miRNA isolation and RT-qPCR

Total RNA was extracted from BV2 cells using TRIzol (Invitrogen, Life Technologies). One microgram of RNA was reverse transcribed to synthesize cDNA using L-MLV Reverse Transcriptase Kit (Invitrogen, Life Technologies, Saint Aubin, France).

Quantitative real-time PCR was performed with a miScript System (Qiagen). All procedures were performed according to the instructions provided by the manufacturer (Qiagen, Courtaboeuf, France). Total miRNA was isolated using the miRNeasy Mini kit

IL-6

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IL-10 Relative foldchange

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Fig. 2. Gene expression of microglial inflammatory markers over time in response to LPS, measured by RT-qPCR. BV2 cells were incubated with LPS (1 lg/mL). Data are represented as the fold change calculated relative to the control group (baseline = 1). Values are means ± SEM (n = 3). Statistics were applied using a 2-way ANOVA with Fisher’s PLSD post hoc test. *p < 0.05, **p < 0.01, ***p < 0.0001.

Please cite this article in press as: Rey, C., et al. Resolvin D1 and E1 promote resolution of inflammation in microglial cells in vitro. Brain Behav. Immun. (2015), http://dx.doi.org/10.1016/j.bbi.2015.12.013

C. Rey et al. / Brain, Behavior, and Immunity xxx (2015) xxx–xxx

50 mM, SDS 2%, Urea 5 M, Antiprotease/APPTase inhibitor), sonicated (puissance 80) and centrifuged (5 min at 3600 rpm). Protein concentration was assessed by bicinchoninic acid protein assay (Uptima, Montlucon, France), according to the manufacturer’s instructions as previously cited in De Smedt-Peyrusse et al. (2008). Equal amount of proteins (25 lg/well) were loaded onto SDS– PAGE gels (10%) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). Membranes were saturated by incubation with 5% milk in Tris-Buffered Saline (TBS) and Tween 0.1%v (Tris/HCl pH 7.5, NaCl 100 nM, Tween-20 0.1%) for 1 h and incubated overnight (4 °C) with antibodies to P-IkB-a (1:500e), IkB-a (1:500e), P-ERK (1/500e), ERK (1:1000e), P-P38 (1:500e), P38 (1:1000e), GAPDH (1:5000e) (Cell Signaling, Ozyme, Saint Quentin en Yvelines, France). After washing in TBS–Tween, membranes were incubated 1 h with peroxidase-conjugated secondary antibody to rabbit (1:5000e, Jackson ImmunoResearch) diluted in TBS–Tween supplemented with 5% milk. Membranes were washed and the complex was detected with an ECL kit. Between each revelation, membranes were incubated (10 min) in stripping Re-Blot Plus Strong solution (Merk Millipore, Molsheim, France) to remove

according to Ceppi et al. (2009). Two microgrammes of RNA were used for reverse transcriptions using miScript II RT kit that contained 2 ll miScript Reverse Transcriptase Mix, 2 ll 10 miScript Nucleics Mix and 4 ll 5 miScript HiSpec Buffer. The RT program was 37 °C for 60 min and 95 °C for 5 min. Real-time PCR reactions were performed with a Roche Light Cycler 480 system using miScript SYBR Green PCR Master Mix that contained 2.5 ll 10 miScript primer assay, 2.5 ll 10 miScript universal primer, 12.5 ll 2 Quantitect SYBR Green PCR Master Mix. The PCR program consisted of 45 cycles of 95 °C 15 min, 94 °C 15 s, 55 °C 30 s, 70 °C 30 s. Each reaction was run in duplicate and negative control reaction was performed. Data were analyzed using the comparative threshold cycle (Ct) method as described elsewhere (Gabriely et al., 2008; Ponomarev et al., 2011), and normalized to uniformly expressed snoRD6 (Qiagen) (Zhou et al., 2013). 2.4. Western blot analysis After treatment, cells were washed with PBS, scraped off and centrifuged. Cell pellets were crushed in 50 ll of lysis buffer (Tris

Anti-inflammatory markers

CD206 SOCS3 Arg1

TNFCD86 CD206 SOCS3 Arg1 Ym1

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Fig. 3. Gene expression of microglial inflammatory markers in response to RvD1. BV2 cells were pre-incubated with RvD1 (10 nM) for 30 min and then incubated with LPS (1 lg/mL) for 6 h (A) or 24 h (B). Data are represented as the fold change calculated relative to the LPS group (baseline = 1). Values are means ± SEM (n = 3). Statistics were applied using a non-parametric Mann–Whitney test. *p < 0.05, **p < 0.01, ***p < 0.0001.

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Fig. 4. Gene expression of microglial inflammatory markers in response to RvE1. BV2 cells were pre-incubated with RvE1 (10 nM) for 30 min and then incubated with LPS (1 lg/mL) for 6 h (A) or 24 h (B). Data are represented as the fold change calculated relative to the LPS group (baseline = 1). Values are means ± SEM (n = 3–4). Statistics were applied using a non-parametric Mann–Whitney test. *p < 0.05, **p < 0.01, ***p < 0.0001.

Please cite this article in press as: Rey, C., et al. Resolvin D1 and E1 promote resolution of inflammation in microglial cells in vitro. Brain Behav. Immun. (2015), http://dx.doi.org/10.1016/j.bbi.2015.12.013

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2.5. Enzyme-linked immunosorbent assay (ELISA) Culture medium was harvested 6 h post-LPS treatment for RvD1 and 24 h post-LPS treatment for RvE1. It was stored at 20 °C. Concentration of tumor necrosis factor alpha (TNF-a) and interleukin (IL)-6 in cell-free supernatant was measured by using enzymelinked immunosorbent assay (ELISA) according to the manufacturer’s specifications (R&D Systems, Minneapolis, Minnesota) (detection limit for TNF-a: 0.36–7.21 pg/mL and IL-6: 1.3–1.8 pg/mL). 2.6. Statistical analysis All data are expressed as means ± standard error of the mean (SEM). Statistical significance between multiple groups was analyzed by two-way ANOVA (time  LPS treatment) followed by Fisher LSD post hoc test when appropriate (receptor and inflammatory marker expression in response to LPS). For the analysis of inflammatory marker expression, a non-parametric Mann–Whitney test was used to compare the Rv + LPS group to the LPS group. For the other analyses, statistical significance was determined by one way ANOVA analysis followed by Newman–Keuls post hoc test. Statistical significance was defined for p values <0.05. 3. Results 3.1. Microglia express RvD1 and RvE1 receptor RvD1 and RvE1 exert their effects in airway epithelial cells, monocytes/macrophages via specific G-protein coupled seven transmembrane receptors ALX/fpr2 and ChemR23, respectively (Krishnamoorthy et al., 2010; Ohira et al., 2010; Oh et al., 2011; Hsiao et al., 2014; Herova et al., 2015). Hence we investigated the temporal profile of expression of these receptors in microglial

B

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IL-6

18 h

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We then assessed by RT-qPCR the time course of cytokine mRNA production as well as expression of microglia phenotype markers in response to LPS (Fig. 2). TNF-a, IL-1b and IL-6 mRNA expression was significantly increased by LPS (treatment effect IL-6: F(3,1) = 40,88, p < 0.0001; TNF-a: F(3,1) = 49.79, p < 0.0001; IL-1b: F(3,1) = 46.28, p < 0.0001). Although mRNA expression of IL-6 increased steadily from 6 h to 24 h, TNF-a and IL-1b mRNA expression peaked at 6 h and 24 h (time effect: IL-6 F(3,3) = 4.88, p < 0.05; TNF-a F(3,3) = 8.67, p < 0.01; IL-1b F(3,3) = 4.67, p < 0.05; interaction time  LPS treatment: IL-6 F(3,3) = 4.9, p < 0.05, TNF-a F(3,3) = 6.62, p < 0.01, IL-1b F(3,3) = 4.6, p < 0.05). No effect of LPS

6h

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3.2. LPS induces pro- and anti-inflammatory marker mRNA expression in microglial cells

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IL-6

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cells by RT-qPCR (Fig. 1). Our data showed that both ALX/fpr2 and ChemR23 mRNAs are expressed in BV2 cell culture. ChemR23 expression decreased 6 h and 18 h post-LPS and increased with a peak of expression at 24 h post-LPS (time effect F(6,3) = 59.25, p < 0.0001; interaction time  LPS treatment F(6,3) = 57.08, p < 0.0001). ALX/fpr2 expression also varied with LPS treatment (treatment effect F(6,1) = 93.25, p < 0.0001), increasing at 6 h and 18 h and decreasing at 24 h (interaction time  LPS treatment F (6,3) = 56.88, p < 0.0001). 6 h after LPS application, ALX/fpr2 mRNA levels reached a maximum. As RvD1 and RvE1 are biosynthesized through lipoxygenase pathways (Krishnamoorthy et al., 2010; Serhan et al., 2000; Arita et al., 2005), we investigated the possibility for microglial cells to express these enzymes. The mRNA expression of 5-LOX and 15LOX was then evaluated in microglia cells by RT-qPCR. BV2 cells expressed 5-LOX and 15-LOX over time (time effect 5-LOX F(6,3) = 109.121, p < 0.0001, 15-LOX F(6,3) = 381.67, p < 0.0001) when submitted to LPS. Their mRNA expression decreased between 2 h and 18 h and drastically peaked at 24 h (interaction time  LPS treatment: 5-LOX F(6,3) = 110.3, p < 0.0001, 15-LOX F(6,3) = 382.6, p < 0.0001).

Ratio of RvE1+LPS/LPS protein concentration

the previous antibody. Chemiluminescence was captured and quantified by Gene Tools software (Syngene, Cambridge, United Kingdom).

Fig. 5. Time dependent effect of RvD1 and RvE1 on LPS-induced IL-6 and TNF-a release. BV2 cells were pre-incubated with LPS 2, 6, 18 and 24 h (A) and preincubated with RvD1 (B) or RvE1 (C). IL-6 and TNF-a were measured in the cell supernatant with specific ELISA. (A) Effect of LPS treatment on IL-6 and TNF-a release over the 24 h of treatment. (B and C) Effect of RvD1 and RvE1 on IL-6 and TNF-a release. Data are represented as means ± SEM (n = 4–6). Statistics were applied using a two-way ANOVA (time  treatment) followed by post hoc Fisher LSD test when appropriate. *p < 0.05, **p < 0.01, ***p < 0.0001 control vs LPS.

Please cite this article in press as: Rey, C., et al. Resolvin D1 and E1 promote resolution of inflammation in microglial cells in vitro. Brain Behav. Immun. (2015), http://dx.doi.org/10.1016/j.bbi.2015.12.013

C. Rey et al. / Brain, Behavior, and Immunity xxx (2015) xxx–xxx

vE 1 R

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To investigate the potential anti-inflammatory effect of RvD1 and RvE1, BV2 cells were pretreated with 10 nM RvD1 or RvE1 for 30 min prior to LPS stimulation (1 lg/mL). A dose response for RvD1 and RvE1 activity was performed on cytokine expression (IL-6). Based on our data, all experiments were then performed on the 10 nM concentration (Fig. S1). No effect of RvD1 and RvE1 (1 nM, 10 nM and 100 nM) on cell viability was revealed (data not shown). In the presence of RvD1, the mRNA expression of IL6, TNF-a, IL-1b and CD86 significantly decreased 6 h post-LPS treatment (IL-6: p < 0.05; TNF-a: p < 0.05; IL-1b: p < 0.05; CD86: p < 0.05) (Fig. 3A). No effect of RvD1 was measured 24 h post-LPS exposure (Fig. 3B). In the presence of RvE1, only IL-6 mRNA expression was significantly diminished 6 h post-LPS (IL-6: p < 0.05) (Fig. 4A). Gene expression of IL-1b and IL-6 was significantly decreased after 24 h LPS (IL-6: p < 0.05; IL-1b: p < 0.05) (Fig. 4B). No effect of RvE1 was observed for TNF-a, CD86, CD206, SOCS3, Arg1, Ym1 at 6 h and 24 h. Moreover, we evaluated in a complementary experiment the effect of RvD1 or RvE1 added 30 min after the LPS treatment on pro-inflammatory cytokine mRNA expression after 6 h and 24 h LPS treatment. No significant effect of RvD1 was measured on cytokine mRNA expression, despite a tendency to a decrease at 6 h. RvE1 also tended to decrease mRNA expression

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3.3. RvD1 and RvE1 alters LPS-induced inflammatory marker mRNA expression in microglia

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was observed on the expression of the anti-inflammatory cytokine IL-10. To further describe microglia activation, we also measured the expression level of some phenotypic markers previously linked to inflammation (CD86) and/or phagocytic activity (SOCS3, Ym1, Arg1, CD206) of microglia. Our data showed that CD86 mRNA expression was modulated by LPS treatment (F(3,3) = 3.53, p < 0.05; interaction time  LPS treatment: F(3,3) = 2.96, p < 0.05). Expression of SOCS3 was significantly induced by LPS as well (treatment effect F(3,1) = 47.18, p < 0.0001), increasing between 2 h and 24 h LPS-treatment (time effect F(3,3) = 3.92, p < 0.05). Ym1 and Arg1 mRNA expression decreased between 2 h and 18 h post-LPS to increase back after 24 h LPS (treatment effect: Ym1 F (3,1) = 26.083, p < 0.01, Arg1 F(3,1) = 126,012, p < 0.0001; time effect: Ym1 F(3,3) = 36.39, p < 0.0001, Arg1 F(3,3) = 124,912, p < 0.0001, interaction time  LPS treatment: Ym1 F(3,3) = 36.22, p < 0.0001, Arg1 F(3,3) = 124,912, p < 0.0001). CD206 expression was significantly reduced by LPS (treatment effect F(3,1) = 49.38, p < 0.0001), after 18 h and 24 h (time effect F(3,3) = 5.99, p < 0.01; interaction time  LPS treatment: F(3,3) = 5.05, p < 0.01). Overall, most of the inflammatory markers and the receptors of RvD1 and RvE1 were regulated between 2 h and 24 h after LPS exposure. In the next set of experiments, we thus tested the impact of resolvins on microglial activity 6 h and 24 h post-LPS treatment, when the expression of resolvin receptors and cytokine production are the highest.

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Fig. 6. Total and phosphorylation of IjB-a and MAP kinase protein relative levels in response to LPS and RvE1 + LPS. BV2 cells were pre-incubated with RvE1 (10 nM) for 30 min and then incubated with LPS (1 lg/mL) for 15 min. Data are represented as means ± SEM of GAPDH-normalized amounts of protein expression ((A): n = 8; (B–D): n = 4). Statistics were applied using one way ANOVA analysis. *p < 0.05, **p < 0.01, ***p < 0.0001 control vs LPS; #p < 0.05, ##p < 0.01, ###p < 0.0001 LPS vs RvE1 + LPS.

Please cite this article in press as: Rey, C., et al. Resolvin D1 and E1 promote resolution of inflammation in microglial cells in vitro. Brain Behav. Immun. (2015), http://dx.doi.org/10.1016/j.bbi.2015.12.013

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C. Rey et al. / Brain, Behavior, and Immunity xxx (2015) xxx–xxx

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NFjB is a critical transcription factor for controlling the expression of pro-inflammatory cytokines in many cell types including microglia (Doyle and O’Neill, 2006). When LPS binds to its receptor it induces the phosphorylation of IjB-a that allows NFjB nuclear translocation and leads to the degradation of IjB-a. De Smedt-Peyrusse previously showed that DHA, the n-3 PUFA precursor of RvD1, inhibits LPS-induced NFjB, consequently inhibiting pro-inflammatory cytokine synthesis (De Smedt-Peyrusse et al., 2008). We thus tested whether RvD1 and RvE1, metabolites of n-3 PUFA, could act through this NFjB pathway. The increase IjB-a phosphorylation (p < 0.01) observed 15 min post-LPS was significantly inhibited by pretreatment with RvE1 (p < 0.05) (Fig. 6A). Moreover, LPS-induced IjB-a-decrease (p < 0.0001) was attenuated by pretreatment with RvE1 (p < 0.05) (Fig. 6A). While no significant effect of RvD1 on IjB-a phosphorylation was ever revealed, our data showed that RvD1 attenuated the decrease of IjB-a (p < 0.0001) (Fig. 7A).

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3.4. Mechanism of action of RvE1 and RvD1

Along with NFjB, the mitogen-activated protein kinase (MAPK) pathway is known to be involved in LPS-induced microglial inflammatory responses (Koistinaho and Koistinaho, 2002; Park et al., 2015). We next investigated the effect of resolvins on LPSinduced MAPK activation. The MAPK family includes p38 MAPK and extracellular signal-regulated kinases (ERK1/2, also known as p44/42 MAPK subfamilies in mammalian cells). Neither LPS affected p38 phosphorylation and degradation nor significant effect of RvD1 and RvE1 was observed on p38 pathway (Figs. 6B and 7B). Regarding p44 MAPK, no effect of LPS was observed on the phosphorylation but LPS decreased p44 expression (p < 0.05) (Figs. 6C and 7C). No significant effect of RvD1 or RvE1 was observed (Figs. 6C and 7C). No significant effect was revealed for p42 MAPK at all (LPS or resolvins) (Figs. 6D and 7D). To evaluate the role of ALX/fpr2 in the effects of RvD1, an ALX antagonist Boc-2 (100 lM) (Wang et al., 2014b) was administered 30 min prior to LPS treatment. IL-6 gene expression was evaluated 6 h post-LPS exposure. The inhibitory effect of resolvin on LPSinduced IL-6 mRNA expression was abolished by Boc-2 (100 lM) (NaCl vs LPS: p < 0.0001; RvD1 + LPS vs LPS: p < 0.05; LPS + RvD1 vs LPS + Boc2 (100 lM) + RvD1: p < 0.05) (Fig. 8). RvD1 is known to act via its receptor ALX/fpr2 to regulate specific miRNAs that are key regulators for resolution of inflammation (Bartel, 2009; Recchiuti, 2013). We thus assessed gene expression of miRNAs involved in the inflammatory response after LPS and

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at 6 h and increased it at 24 h (data not shown). We also measured IL-6 and TNF-a protein expression by ELISA (Fig. 5). LPS treatment significantly induced IL-6 and TNF-a over the 24 h of treatment (Fig. 5A). However, no significant effect of RVD1 or RVE1 on IL-6 and TNF-a release were measured (Fig. 5B and C).

Fig. 7. Total and phosphorylation of IjB-a and MAP kinase protein relative levels in response to LPS and RvD1 + LPS. BV2 cells were pre-incubated with RvD1 (10 nM) for 30 min and then incubated with LPS (1 lg/mL) for 15 min. Data are represented as means ± SEM of GAPDH-normalized amounts of protein expression ((A): n = 8; (B–D): n = 3). Statistics were applied using one way ANOVA analysis. *p < 0.05, **p < 0.01, ***p < 0.0001 control vs LPS; #p < 0.05, ##p < 0.01, ###p < 0.0001 LPS vs RvD1 + LPS.

Please cite this article in press as: Rey, C., et al. Resolvin D1 and E1 promote resolution of inflammation in microglial cells in vitro. Brain Behav. Immun. (2015), http://dx.doi.org/10.1016/j.bbi.2015.12.013

C. Rey et al. / Brain, Behavior, and Immunity xxx (2015) xxx–xxx

Fig. 8. IL-6 expression in response to Boc-2 inhibitor of ALX/fpr2. BV2 cells were pre-incubated with Boc-2 (100 lM) for 30 min and then incubated with LPS (1 lg/ mL) for 6 h. Data are represented as means ± SEM of the fold change calculated relative to the control group (n = 4–6). Statistics were applied using one way ANOVA analysis. *p < 0.05, **p < 0.01, ***p < 0.0001 control vs LPS, #p < 0.05, ## p < 0.01, ###p < 0.0001 LPS vs LPS + RvD1.

RvD1 treatment (Sheedy and O’Neill, 2008; Quinn and O’Neill, 2011; Thounaojam et al., 2013). We focused on four miRNAs: miR-155, miR-146, mi-219 and miR-21 known to be key actors in many inflammatory processes (Quinn and O’Neill, 2011) and that have been shown to be regulated by LPS in macrophages (O’Neill et al., 2011; Thounaojam et al., 2013). In response to LPS, miR-155, miR-146 and miR-21 gene expression was significantly up-regulated (mir-155: p < 0.0001; miR-146: p < 0.0001, mir-21: p < 0.0001) whereas miR-219 was significantly decreased (p < 0.01) (Fig. 9). Pretreatment with RvD1 potentiated miR-155, miR-21 and miR-146 expression (miR-155: p < 0.0001; miR-21: p < 0.01; miR-146: p < 0.01) and induced a decrease in miR-219 gene expression (p < 0.0001) (Fig. 9). 4. Discussion In this study, we report for the first time that RvD1 and RvE1 are potent inhibitors of LPS-induced TNF-a, IL-6 and IL-1b gene

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expression and modulators of microglial phenotype in vitro. Our data deepen our comprehension of the beneficial effects of n-3 PUFAs via novel pro-resolving lipid derivates that stimulates the return to tissue homeostasis in the central nervous system. Moreover, RvD1 differs from RvE1 not only by its molecular origin and structure but also by its mechanism of action on BV2 microglia cells. Here we show that RvD1 and RvE1 decreased the expression of the pro-inflammatory cytokines IL-1b, TNF-a and IL-6 induced after 6 h and/or 24 h LPS exposure in microglial cells. If antiinflammatory activity of RvD1 and RvE1 has been described in peripheral immune cells such as macrophages (Haworth et al., 2008; Jin et al., 2009; Tang et al., 2013), this effect has been poorly described in microglia. Indeed, DHA derived resolvins inhibited TNF-a-induced IL-1b mRNA expression in human glioma cell line and RvE1 blocks LPS-induced TNF-a release in primary microglial cell culture (Serhan et al., 2002; Hong et al., 2003; Xu et al., 2013). Harrison et al. show that intraperitoneally daily administration of 100 ng RvE1 injection induces a reduction of activated microglia during traumatic brain injury (Harrison et al., 2015). Hence, it is highly likely that protective effect of resolvins on the brain is supported by its effect on microglia. This idea is reinforced by our data showing that microglial cells expressed RvD1 and RvE1 receptors. The presence of ALX/fpr2 and ChemR23 on microglial cells suggests that RvD1 and RvE1 could bind to these receptors and induce resolution of inflammation. ALX/fpr2 expression level was significantly increased 6 h after LPS application. This is consistent with the results of Mou et al. showing in endothelial cells that the expression of this receptor can be up-regulated by proinflammatory factors IL-1b and LPS (Mou et al., 2012). The expression of ChemR23 showed a huge increase after 24 h LPS treatment. This could be explained by an increase of M1 microglia since Herova et al. (2015) showed an increased expression of ChemR23 in M1 macrophages but not M2 (Herova et al., 2015). An interesting finding in this respect is the concordance between the bioactive time of resolvin actions on inflammation and the time of resolvin receptor overexpression after LPS stimulation. ChemR23 was over-expressed 24 h post-LPS at the same time as the RvE1 was the most active on inflammatory marker expression, and the ALX/fpr2 expression was highly increased 6 h post-LPS at the same time as RvD1 was the most active. Interestingly, the mRNA expression did not necessarily match the protein expression. It has been proposed that three potential reasons for the lack of a strong correlation between mRNA and protein expression levels are: (i) translational regulation, (ii) differences in protein in vivo half-

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Fig. 9. miRNA expression in response to LPS and RvD1 + LPS treatment. BV2 cells were pre-incubated with RvD1 (10 nM) for 30 min and then incubated with LPS (1 lg/mL) for 24 h. Data are represented as means ± SEM of the fold change calculated relative to the control group (n = 10). Statistics were applied using one way ANOVA analysis. * p < 0.05, **p < 0.01, ***p < 0.0001 control group vs LPS; #p < 0.05, ##p < 0.01, ###p < 0.0001 LPS vs RvD1 + LPS.

Please cite this article in press as: Rey, C., et al. Resolvin D1 and E1 promote resolution of inflammation in microglial cells in vitro. Brain Behav. Immun. (2015), http://dx.doi.org/10.1016/j.bbi.2015.12.013

C. Rey et al. / Brain, Behavior, and Immunity xxx (2015) xxx–xxx

lives, and (iii) to the lack of surrounding cells leading to the accumulation of cytokines thus hiding resolvin effects on protein synthesis levels (Lee et al., 1993; Greenbaum et al., 2003; Beyer et al., 2004). We showed that RvD1 modified microglia phenotype and response to LPS. Phenotype modifications have been associated to function diversity including motility, phagocytosis, antigen presentation, synapse remodeling (Hanisch and Kettenmann, 2007; Eggen et al., 2013; Hanisch, 2013). Hence our findings support the hypothesis that dietary manipulation of n-3 PUFAs is likely to change microglial function and activity (Delpech et al., 2015) through modulation of RvD1. For RvD1, one of the mechanisms involved may be modulation of miRNA. This was already described in macrophages (Krishnamoorthy et al., 2010; Recchiuti et al., 2011). miRNAs have recently emerged as a major class of gene expression regulators linked to most biological functions including immune regulation (Ceppi et al., 2009; O’Neill et al., 2011; Recchiuti et al., 2011; Recchiuti and Serhan, 2012). miRNAs in macrophages downregulate the mRNA translation of key inflammatory cytokines (Fredman and Serhan, 2011). We found upregulation of miR-155, -146, and -21 expressions upon LPS treatment. These data are in agreement with the work of Ceppi et al. (2009) who reported that both miR-155 and miR-146 are upregulated upon LPS stimulation in human primary dendritic cells (Ceppi et al., 2009). miR-155 targets the proteins involved in the activation of NFjB, thus controlling tissue damage due to inflammation (Faraoni et al., 2009). miR-146 is involved as a negative regulator fine tuning the immune response (Quinn and O’Neill, 2011). These miRNAs play a key role in modulating the IL-1 and IL-6 pathways. miR-21 is involved as a central player in the inflammatory response (Quinn and O’Neill, 2011). miR-21 plays a key role in the resolution of inflammation and in negatively regulating the pro-inflammatory response in particular in macrophages (Sheedy

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and O’Neill, 2008). In our study, RvD1 up-regulated the expression of these 3 miRNAs. It is consistent with the findings of Recchiuti et al. (2011) that demonstrate that miR-21 and miR-146 were induced by RvD1 in a murine model of peritonitis (Recchiuti et al., 2011). In addition, our results further confirm that RvE1 mechanism of action may be associated to NFjB signaling pathway as previously hypothesized by Arita et al. (2005). However we cannot exclude that RvE1 acted through miRNAs despite it has never been reported in the literature. Fig. 10 summarized our findings on RvD1 and RvE1 mechanisms of action in microglia. As an experimental model we used BV2 cells that constitute a powerful tool for the screening of compounds. It also confers the significant advantage to generate sufficient cells to conduct biochemical studies and then to screen easily and rapidly the immunomodulatory potential of a molecule (Orihuela et al., 2015; Franco and Fernandez-Suarez, 2015). However Butovsky et al. (2014) showed that microglial cell lines do not express the microglia signature of the adult mice, likely because BV2 cells are not exposed to their natural microenvironment; they lack interactions with neurons and astrocytes. Signaling from surrounding other cell type plays a critical role in microglial phenotype determination (Olah et al., 2011). Thus the absence of neurons and astrocytes may also contribute to changes in microglial properties in vitro (Crain et al., 2013). Nevertheless they still behave similarly to primary microglial cells for their metabolic functions under LPS conditions (Orihuela et al., 2015). The BV2 cell line is widely used and characterized and is a valid substitute in many experimental settings, including cell–cell interaction studies (Henn et al., 2009; Gresa-Arribas et al., 2012). 90% of the genes induced in BV2 cells by LPS were also found in primary microglia, and around 50% were even found in hippocampal microglia after in vivo stimulation of mice by icv injection of LPS (Henn et al., 2009). In our study, we provide further information on the presence and activities of

Fig. 10 RvD1 and RvE1 signaling in microglia.

Please cite this article in press as: Rey, C., et al. Resolvin D1 and E1 promote resolution of inflammation in microglial cells in vitro. Brain Behav. Immun. (2015), http://dx.doi.org/10.1016/j.bbi.2015.12.013

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C. Rey et al. / Brain, Behavior, and Immunity xxx (2015) xxx–xxx

RvD1 and RvE1 receptors. Further studies are warranted to confirm in vivo the results we obtained in vitro. RvD1 and RvE1 are promising compounds in the control of brain inflammation. Indeed, these mediators are of natural origin and are active at very low concentrations (nM) as compared to their precursors EPA and DHA (lM) (Ariel and Serhan, 2007; Bannenberg and Serhan, 2010). Here, we used a low concentration of resolvins (10 nM) according to previous studies conducted in peripheral cells (Dartt and Schneider, 2010; Fredman et al., 2010; Keyes et al., 2010; Oh et al., 2011) and a dose response study (Fig. S1). Interventional studies in patients with mild cognitive impairment or in patients with Alzheimer’s disease showed beneficial effects of EPA and DHA, when exists, at doses higher than 1 g/day (Thomas et al., 2015). Hence, RvD1 and RvE1 may be beneficial at concentrations closed to 1 mg/day. Peripheral administration of RvD1 prevents cognitive decline, astrocytes atrophy and synaptic impairment induced by a peripheral surgery (Terrando et al., 2013). These results suggest that resolvins may cross the blood– brain barrier and exert their effect in the brain. Indeed, RvD1 and RvE1 are small lipophilic molecules that are derived from DHA and EPA, are produced by oxygenation and contained 3 hydroxyl groups. It was recently shown that non esterified fatty acids diffuse into the brain (Bazinet and Laye, 2014; Chen et al., 2015). Resolvins open novel strategies for the treatment of inflammatory diseases. A recent study demonstrated the immunoresolving action of RvD1 administered orally (Recchiuti et al., 2014). Oral delivery to mice elevated RvD1 in plasma, reduces acute inflammation and accelerates or initiates resolution. These results highlight the possibility to exploit the beneficial effect of RvD1 in human. The present study showed that stimulation of the resolution of inflammation would become a new strategy for brain inflammatory disease therapy. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbi.2015.12.013. References Ariel, A., Serhan, C.N., 2007. Resolvins and protectins in the termination program of acute inflammation. Trends Immunol. 28, 176–183. Arita, M., Bianchini, F., Aliberti, J., Sher, A., Chiang, N., Hong, S., Yang, R., Petasis, N.A., Serhan, C.N., 2005. Stereochemical assignment, antiinflammatory properties, and receptor for the omega-3 lipid mediator resolvin E1. J. Exp. Med. 201, 713– 722. Bannenberg, G., Serhan, C.N., 2010. Specialized pro-resolving lipid mediators in the inflammatory response: an update. Biochim. Biophys. Acta 1801, 1260–1273. Bartel, D.P., 2009. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233. Bazinet, R.P., Laye, S., 2014. Polyunsaturated fatty acids and their metabolites in brain function and disease. Nat. Rev. Neurosci. 15, 771–785. Beyer, A., Hollunder, J., Nasheuer, H.P., Wilhelm, T., 2004. Post-transcriptional expression regulation in the yeast Saccharomyces cerevisiae on a genomic scale. Mol. Cell. Proteomics 3, 1083–1092. Blais, V., Rivest, S., 2003. Role of the innate immune response in the brain. Med. Sci. (Paris) 19, 981–987. Butovsky, O., Jedrychowski, M.P., Moore, C.S., Cialic, R., Lanser, A.J., Gabriely, G., Koeglsperger, T., Dake, B., Wu, P.M., Doykan, C.E., Fanek, Z., Liu, L., Chen, Z., Rothstein, J.D., Ransohoff, R.M., Gygi, S.P., Antel, J.P., Weiner, H.L., 2014. Identification of a unique TGF-beta-dependent molecular and functional signature in microglia. Nat. Neurosci. 17, 131–143. Calder, P.C., 2013. n-3 fatty acids, inflammation and immunity: new mechanisms to explain old actions. Proc. Nutr. Soc. 72, 326–336. Ceppi, M., Pereira, P.M., Dunand-Sauthier, I., Barras, E., Reith, W., Santos, M.A., Pierre, P., 2009. MicroRNA-155 modulates the interleukin-1 signaling pathway in activated human monocyte-derived dendritic cells. Proc. Natl. Acad. Sci. U.S. A. 106, 2735–2740. Chen, J., Shetty, S., Zhang, P., Gao, R., Hu, Y., Wang, S., Li, Z., Fu, J., 2014. Aspirintriggered resolvin D1 down-regulates inflammatory responses and protects against endotoxin-induced acute kidney injury. Toxicol. Appl. Pharmacol. 277, 118–123. Chen, C.T., Kitson, A.P., Hopperton, K.E., Domenichiello, A.F., Trepanier, M.O., Lin, L. E., Ermini, L., Post, M., Thies, F., Bazinet, R.P., 2015. Plasma non-esterified docosahexaenoic acid is the major pool supplying the brain. Sci. Rep. 5, 15791.

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Please cite this article in press as: Rey, C., et al. Resolvin D1 and E1 promote resolution of inflammation in microglial cells in vitro. Brain Behav. Immun. (2015), http://dx.doi.org/10.1016/j.bbi.2015.12.013