microRNA-200b modulates microglia-mediated neuroinflammation via the cJun/MAPK pathway

Authors

  • Shweta P. Jadhav,

    1. Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
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  • Sandhya P. Kamath,

    1. Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
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  • Mahesh Choolani,

    1. Department of Obstetrics and Gynecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
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  • Jia Lu,

    1. Defence Medical and Environmental Research Institute, DSO National Laboratories, Singapore
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  • S. Thameem Dheen

    Corresponding author
    1. Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
    • Address correspondence and reprint requests to S. T Dheen, Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Blk MD10, 4 Medical Drive, Singapore 117597, Singapore. E-mail: antstd@nus.edu.sg

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Abstract

Chronic activation of microglia, the macrophages of the CNS, has been shown to enhance neuronal damage because of excessive release of proinflammatory cytokines and neurotoxic molecules in a number of neurodegenerative diseases. Recent reports showed altered microRNA (miRNA) expression in immune-mediated pathologies, thus suggesting that miRNAs modulate expression of genes involving immune responses. This study demonstrates that miRNA-200b is expressed in microglia and modulates inflammatory response of microglia by regulating mitogen-activated protein kinase pathway. miRNA-200b expression was found to be down-regulated in activated microglia in vivo (traumatic brain injury rat model) and in vitro. A luciferase assay and loss- and gain-of-function studies revealed c-Jun, the transcription factor of cJun-N terminal kinase (JNK) mitogen-activated protein kinase pathway to be the target of miR-200b. Knockdown of miR-200b in microglia increased JNK activity along with an increase in pro-inflammatory cytokines, inducible nitric oxide synthase expression and nitric oxide (NO) production. Conversely, over-expression of miRNA-200b in microglia resulted in a decrease in JNK activity, inducible nitric oxide synthase expression, NO production and migratory potential of activated microglia. Furthermore, miR-200b inhibition resulted in increased neuronal apoptosis after treatment of neuronal cells with conditioned medium obtained from microglial culture. Taken together, these results indicate that miRNA-200b modulates microglial inflammatory process including cytokine secretion, NO production, migration and neuronal survival.

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Chronic microglial activation is implicated in the pathogenesis of several neurodegenerative diseases. Recently, miRNAs, the novel epigenetic factors, have been shown to be involved in several diseases including neurological disorders. In this study, we demonstrate that miR-200b is expressed in microglia and modulates inflammatory response of activated microglia by altering the expression of proinflammatory cytokines, NO production and neuronal survival via targeting the cJun MAPK pathway. Thus, miRNA-200b may prove to be a useful therapeutic target in the context of chronic neuroinflammation.

Abbreviations used
ISH

in situ hybridization

JNK

cJun-N terminal kinase

LNA

locked nucleic acid

MAPK

mitogen-activated protein kinase

miRNA

microRNA

Microglia, the resident immune cells of the CNS, have been shown to mediate neuroinflammation in brain injury and neurodegenerative diseases (Dheen et al. 2007). In the healthy CNS, microglia actively monitor the surrounding tissue and maintain the internal milieu. Microglia are activated in response to detrimental signals such as neuronal injury or infection and express a range of molecular mediators which help in tissue repair, neurotrophic support and inflammatory response (Chao et al. 1992; Banati et al. 1993; Fetler and Amigorena 2005). However, chronic activation of microglia leads to excessive production of proinflammatory cytokines, chemokines as well as other cytotoxic molecules such as nitric oxide (NO) and reactive oxygen species (Vilhardt 2005). Indeed, activated microglia have been implicated in amplifying the neuronal cell death in a number of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis and traumatic brain injury (TBI) (Tuppo and Arias 2005; Phani et al. 2012; Rubio-Perez and Morillas-Ruiz 2012).

MicroRNAs (miRNAs), a family of small non-coding RNAs, have emerged as novel post-transcriptional regulators of gene expression. miRNAs bind to the 3′ UTR of their target mRNAs resulting in gene silencing either by mRNA translational inhibition or degradation (Eulalio et al. 2008; Bartel 2009; Fabian and Sonenberg 2012). Recent reports show miRNAs such as miR-155 and miR-21 influencing microglia-mediated immune response by enhancing proinflammatory cytokine production (Cardoso et al. 2012; Zhang et al. 2012). On the other hand, some miRNAs, such as miR-210, function as negative regulator for expression of proinflammatory cytokines in microglia (Qi et al. 2012).

Furthermore, miR-200b and miR-200c, members of miR-200 family have been shown to regulate the cellular processes of tissue remodelling (Katoh & Katoh 2008; Mongroo and Rustgi 2010) and proliferation (Burk et al. 2008; Gregory et al. 2008) and modulate innate immune response by suppressing the signalling pathways leading to nuclear factor κ-light-chain-enhancer of activated B cells activation (Wendlandt et al. 2012). miRNA-200b has also been shown to target several proteins including c-Jun, the substrate of cJun-N terminal kinase (JNK) mitogen-activated protein kinase (MAPK) in the brain, indicating that miR-200b may alter immune responses of microglia by targeting the JNK/MAPK pathway (Juhila et al. 2011).

MAPK signalling pathways including JNK/stress-activated protein kinase (SAPK), p38 and extracellular signal-regulated kinase pathway have been shown to regulate diverse biological functions such as proliferation, differentiation and apoptosis in various cell types (Sabapathy et al. 1999; Graves et al. 2000; Chang and Karin 2001; Yang et al. 2003). Activation of MAPKs in microglia leads to the phosphorylation of the transcription factor, cJun, which contributes to the activation of microglia via the transcription of its target genes: Tumor necrosis factor alpha (TNF α), IL-1β, cox2 and MCP-1 (Kyriakis and Avruch 2001; Babcock et al. 2003; Waetzig et al. 2005). Furthermore, JNKs have been implicated in the pathogenesis of various neurodegenerative diseases such as Parkinson's disease (Vila et al. 2004; Brecht et al. 2005) and Alzheimer's disease (Hashimoto et al. 2003; Colombo et al. 2007).

In this study, we hypothesize that miRNA-200b modulates the inflammatory response of microglia by targeting cJun, the JNK substrate. We demonstrate that miRNA-200b targets cJun and its expression is down-regulated in activated microglial cells in vitro and in vivo. Furthermore , miR-200b functions as the negative regulator of activation of microglia, involving microglial migration, NO production and secretion of proinflammatory cytokines. To our knowledge, this is the first study demonstrating the role of miRNA-200b in regulating the inflammatory response via the MAPK pathway in microglia.

Materials and methods

Ethics statement

Male Wistar rats (2 months old) were purchased from Laboratory Animals Centre, National University of Singapore. This study was approved by the National University of Singapore Institutional Animal Care and Use Committee (IACUC) and DSO National Laboratories Institutional Animal Care and Use Committee. All procedures were in accordance with IACUC guidelines. All efforts were made to minimize pain and the number of animals used.

BV2 cell culture and activation

Murine BV-2 microglial cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM, Cat No. D1152; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% foetal bovine serum (FBS, Cat No. SV30160.03, HyClone, Logan, UT, USA) and cultured at 37°C with 5% CO2. The cells were treated with lipopolysaccharide (LPS), a bacterial endotoxin (Cat No. L6529; Sigma-Aldrich) at 1 μg/mL for 1–6 h to activate microglia.

Animal model of traumatic brain injury

Experimental TBI was produced using the rodent fluid percussion (FP) model of brain injury (Dixon et al. 1987). Male Wistar rats (age 2 months) were deeply anesthetized with 5% isofluorane and a 1 : 1 N2O : O2 mixture. Craniotomy was performed midway between bregma and lambda and a fluid pressure pulse was applied to produce brief displacement and deformation of neural tissue. Sham-operated rats received only anaesthesia and a midline incision. After injury or sham operation, the scalp was sutured, and the animals were allowed to recover from anaesthesia and returned to their home cages. Twenty-four hours after surgery fresh brain tissue was collected and stored at −80°C for laser capture microdissection (LCM) analysis.

Laser capture microdissection

Whole fresh brains (three sham operated and three TBI) were isolated as described above and frozen in a cryostat (Model No. CM 3050 S; Leica Microsystems, GmbH, Wetzlar, Germany). The forebrain was sectioned coronally through the hippocampus at 10 μm thickness and mounted on membrane slides (Prod. No. 50103; Molecular Machines & Industries, Glattbrugg, Switzerland). The sections were stained with peroxidase-conjugated isolectin (1 : 50, Cat. No. L5391; Sigma-Aldrich) and slides placed on the microscope stage of MMI CellCut (Molecular Machines & Industries). A collection tube (Prod. No. 50210; Molecular Machines & Industries) adjusted onto the section was used to collect lectin-stained microglia cells cut by laser. A minimum of 600 cells were isolated per sample.

RNA isolation

Total RNA including miRNAs and small RNAs was extracted from BV2 and laser capture dissected microglial cells using the miRNeasy Mini kit (Cat No 217004; Qiagen, Valencia, CA, USA) according to manufacturer's instructions.

miRNA real-time PCR

cDNA conversion for miRNA quantification was performed using the Universal cDNA Synthesis Kit (Prod No. 203301; Exiqon, Vedbaek, Denmark) according to manufacturer's instructions. For miRNA quantification, the miRCURY LNA Universal RT microRNA PCR system (Prod No. 203400; Exiqon) was used in combination with pre-designed primers (Prod No. 204144, Prod No. 203907; Exiqon) for mmu-miR-200b and snRNA U6 (reference gene). The miRNA expression was quantified using real-time PCR system (Model No.7900HT; Applied Biosystems, Life technologies, Carlsbad, CA, USA).

mRNA real-time PCR

For mRNA analysis, cDNA conversion was carried using 2 μg of RNA, 2 μL of Oligo (dT) 15 primer (Cat No. C1101; Promega, Madison, WI, USA) 1 μL of M-MLV reverse transcriptase (Cat No. M1701; Promega), 5 μL of M-MLV RT 5X buffer (Cat No. M531A; Promega), 0.7 μL of RNasin (Cat No. N2515; Promega), 0.5 μL of dNTP mix (Cat No. U1240; Promega) and nuclease free water in a 25 μL reaction volume. PCR analysis was carried out using 1 μL of 1 : 10 diluted cDNA in a reaction mixture containing 5 μL of Fast SYBR green 2X Master mix (Cat No. 4385612; Applied Biosystems, Life technologies), 0.5 μL each of forward and reverse primer (Table 1 and 2) and the total volume adjusted to 10 μL using RNase-free water. The reaction was carried out in Applied Biosystems 7900HT Fast Real-Time PCR machine (Life technologies).

Table 1. Primers used for qRT-PCR analysis (mouse)
GeneForward primerReverse primer
cJun5′-AAAACCTTGAAAGCGCAAAA-3′5′-CGCAACCAGTCAAGTTCTCA-3′
IL65′- AGTTGCCTTCTTGGGACTGA-3′5′- TCCACGATTTCCCAGAGAAC-3′
IL-1β5′- GCC CAT CCT CTG TGA CTCAT-3′5′-AGG CCA CAG GTA TTT TGT CG-3′
TNF-α5′-CATCACAACCACTCCCACTG-3′5′- GTTCTGCCAGTTCCTTCTGC-3′
Β actin5′-GAAGAGCTATGAGCTGCCTGA-3′5′-GGATTCCATACCCAAGAAGGA-3′
Table 2. Primers used for qRT-PCR and semiquantitative PCR analysis (rat)
GeneForward primerReverse primer
cJun5′-CCACCGAGACCGTAAAGAAA-3′5′-GTCGTCACGGAATTCTTGGT-3′
CD11b5′-AGGCAGCTGAATGGAAGGAC-3′5′CGTAGCGAATGATCCCTGCT-3′
CNPase5′-GGTACTGGTCTGCCATTTCAA-3′5′-AAGATGGTGTCTGCTGATGCT-3′
GFAP5′-AGA AAA CCGCAT CAC CATTC-3′5′-GCACACCTCACA TCACATCC-3′
MAP25′-TGT TGCTGCCAAGAAAGATG-3′5′-ACGTGGCTGGACTCAATACC-3′
PECAM25′-CGA AATCTAGGCCTCAGCAC-3′5′-CTTTTTGTCCAC GGTCACCT-3′
GAPDH5′-ACATGCCGCCTGGAGAAACCTGCC-3′5′-TGCCAGCCCCAGCATCAAAGGGGA-3′

miRNA target prediction

The miRWalk database (http://www.ma.uni-heidelberg.de/appa/zmf/mirwalk/) was used to predict miRNA–mRNA interactions in the 3′UTR of cJun.

In situ hybridization

To check the localization of miR-200b in microglia, in situ hybridization was carried out using Exiqon's miRCURY LNA microRNA in situ hybridization Buffer Set (Prod No. 90000; Exiqon) as per manufacturer's instructions. Furthermore, the cells were counter-stained with 4',6-Diamidino-2-phenylindole, Dihydrochloride (DAPI), a nuclear dye (1 μg/mL, Cat. No. D1306; Molecular Probes, Invitrogen, Life Technologies) and coverslips were mounted on glass slides with fluorescent mounting medium (Prod No. 5302380; DAKO, Agilent Technologies, Santa Clara, CA, USA). Images were captured with Olympus FV1000 confocal microscope (Olympus Corporation, Tokyo, Japan).

miRNA knockdown and over-expression

For functional analysis of miR-200b in microglia, BV2 cells were seeded in six-well plates at a density of 2 × 105. Transfection was carried out using X-tremeGENE siRNA transfection reagent (Cat. No. 04476093001; Roche Applied Sciences, Basel, Switzerland) following manufacturer's instructions. 5′fluorescent labelled miRCURY LNA microRNA inhibitors: mmu-miR-200b (Prod No. 410124-04) and scrambled miRNA (Prod No. 199004-04) were purchased from Exiqon (Figure S1). Similarly, non-labelled mirVANA miRNA mimic for mmu-miR-200b (Cat. # 4464084) and negative control (Cat. # 4464076) were purchased from Ambion (Life Technologies). Transfection complexes were prepared in opti-MEM medium (Cat # 31905070, Invitrogen, Life technologies) and added to the cells at final concentration of 40 nM (inhibitors) or 20 nM (mimics).

Cell viability assay

Cell viability post-transfection was measured using the colorimetric MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), assay (Cat # G5421, CellTiter 96R AQueous Non-Radioactive Cell Proliferation Assay; Promega) as per manufacturer's instructions. Briefly 5000 cells were seeded in 96-well plates and transfected as mentioned above. Twenty-four hours post transfection, 20 μL of MTS/phenazine methosulfate solution was added to each well. Next, the plate was incubated at 37°C with 5% CO2 for 3 h and the absorbance was measured using a microplate spectrophotometer at 490 nm. % cell viability was plotted as a function of the absorbance (Figure S2).

Luciferase assay

pMiRTarget luciferase reporter vector containing the 3′UTR of cJun cloned into the multiple cloning site was purchased from OriGene Technologies (custom made). BV2 cells were cotransfected with 80 ng of the luciferase reporter plasmid along with miRNA mimics (final concentration 20 nM). Forty-eight hours post transfection, luciferase activity was evaluated using the ONE-Glo Luciferase assay system from Promega (Cat # E6110). pMiRTarget luciferase reporter vector also encodes for red fluorescent protein which was used as a reporter for transfection monitoring and normalization (Figure S3a–d). Each experiment was repeated twice in triplicate.

Protein isolation and western blotting

Total protein extraction

Total protein was extracted from BV2 cells using the M-PER reagent (Prod No. 78501, M-PER; Thermo scientific, Rockford, IL, USA) following manufacturer's protocol. The extracted protein was quantified using Bradford method (Prod No. 500-0006, Bio-Rad Laboratories, Hercules, CA, USA).

Nuclear protein extraction

Nuclear protein was extracted using the nuclear protein extraction kit (Cat No. 2900; Merck Millipore, Billerica, MA, USA) according to manufacturer's instructions.

Western blotting

Total protein of 40 μg and 20 μg of nuclear protein from each sample was denatured at 95°C for 5 min and separated on a 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene (PVDF) transfer membranes blocked with 3% bovine serum albumin and incubated with the primary antibodies, anti-SAPK/JNK (Cat # 9258, 1 : 1000; Cell Signaling Technology Inc., Danvers, MA, USA) or anti-phospho SAPK/JNK (Thr183/Tyr185, Cat # 9251, 1 : 1000; Cell Signaling Technology Inc.) or anti-cJun (Cat # 9165, 1 : 1000; Cell Signaling Technology Inc.), or anti-phospho cJun (Ser63, Cat # 9261, 1 : 1000; Cell Signaling Technology Inc.) overnight at 4°C. Following washing the blots were incubated with secondary horseradish peroxidase antibody (Part No 0031430/Part no 0031460; Thermo Scientific). All blots were developed with enhanced chemiluminescence reagent (Part No. 34077; Thermo Scientific) and quantified on densitometer (Bio-rad) using Quantity One software (Bio-rad). To normalize for the protein content of each lane the blots were stripped (Part No. 21059; Thermo Scientific) and reprobed with anti-beta actin (Cat # A2228, 1 : 5000; Sigma-Aldrich) or anti-lamin A antibody (Cat # MAB 3540, 1 : 500; Merck Millipore) for total or nuclear protein respectively.

Nitrite quantification

Nitric oxide production was quantified using the Nitric Oxide Colorimetric BioAssay kit (Cat # N2577-01 US Biologicals) by measuring the levels of the stable metabolite nitrite in the culture medium as described previously (Nayak et al. 2010). Nitrate standard curve was used to calculate the nitric oxide production in the samples.

Immunocytochemistry

About 2 × 104 cells were seeded on poly-lysine-coated coverslips in 24-well culture plates. Following transfection and LPS treatment the cells were fixed with 4% PF washed and blocked with 5% goat serum followed by incubation with the following antibodies – total cJun (Cat # 9165, 1 : 1000; Cell Signaling Technology Inc.), anti-phospho cJun (Ser63, Cat # 9261, 1 : 1000; Cell Signaling Technology Inc.), TNF α (Prod No. Ab2148P, 1 : 500; Merck Millipore) inducible nitric oxide synthase (iNOS) (Cat.SC-649, 1 : 200, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4°C. The cells were then incubated with secondary Cy3 antibody (1 : 200, Cat. No. AP132C; Merck Millipore) followed by counter-staining with DAPI and coverslips were mounted as described previously.

Neuronal cell culture and apoptosis assay

MN9D dopaminergic neuronal cell line was grown in DMEM supplemented with 10% FBS (Cat No. SV30160.03, HyClone) and cultured in 37°C with 5% CO2 and differentiated for 6 days with 1 mM sodium butyrate (Cat # B5887; Sigma-Aldrich) (Figure S5). Apoptosis assay was carried out using the Annexin V-Cy3 apoptosis Detection kit (ab14142, Abcam, Cambridge, UK) as per manufacturer's instructions. Briefly, 1 × 105 cells seeded on six-well plates followed by incubation with a 1 : 1 mixture of complete medium (500 μL) and conditioned medium (500 μL) for 24 h. The conditioned medium was obtained from untreated BV2 cells and BV2 cells transfected with anti-miR-200b (inhibitor) or miR-200b mimics or control probes. Following incubation the cells were gently trypsinized and the staining protocol was followed as per manufacturer's instructions. Flow-cytometry was performed on a BD LSRFortessa X-20 (BD Biosciences, San Jose, CA, USA) analyser.

ELISA assay for TNF α release

Following transfection and activation, media supernatants from BV2 cell culture were collected, and TNF α protein level was determined using commercially available ELISA kit (Cat No: 88-7324-22; EBiosciences, San Diego, CA, USA) according to the manufacturer's procedure. TNF α standard curve was used to calculate the TNF α level in the samples.

F-actin assay

Following transfection and LPS treatment, the BV2 cells were fixed with 4% PF as described above and incubated with a rhodamine-phalloidin dye (cat # PHDR1, Cytoskeleton) to stain the f-actin for 30 min at room temperature of 25°C. Furthermore, the cells were counter-stained with DAPI and mounted with coverslips (Figure S5).

In vitro cell migration assay

Forty-eight hours post transfection BV2 cells were trypsinized and seeded into transwell inserts (Prod No. 3422; Corning Life Sciences, Tewksbury, MA, USA) containing free DMEM (no serum). For the control groups complete medium (10% FBS, DMEM) was added to the wells while for the activated groups, 1 μg/mL LPS was added to the wells along with complete medium. The cells were incubated in transwell inserts and cultured overnight in an incubator. The next day the cells that had not migrated to the lower chamber were removed from the upper surface of the transwell membrane with a cotton swab. Migrating cells on the lower membrane surface were fixed with 100% methanol followed by washing and air drying. The cells were stained with crystal violet (0.5%)/methanol (25%) solution. Images were captured on Nikon microscope (Chiyoda, Tokyo, Japan) at × 100. Experiments were assayed in triplicate, and ≥ 5 fields were counted in each experiment.

Statistical analysis

Data are represented as mean ± SD from at least three independent experiments. Statistical significance was evaluated by either the Student's t-test or one-way anova analysis of variance followed by post hoc Tukey test. p values of *p < 0.05 were considered significant.

Results

miR-200b is localized in microglia and its expression is down-regulated upon LPS activation of BV2 microglia

In situ hybridization study revealed the expression of miR-200b in BV2 microglial cells (Fig. 1c). A time-dependent down-regulation of miR-200b expression was observed in activated BV2 microglia (Fig. 1d). Bioinformatic analysis using the mirWALK database predicted the transcription factor, cJun as a putative mRNA target of miR-200b (Fig. 1e). cJun expression was greatly enhanced in BV2 microglia at 1 h after LPS exposure and remained up-regulated at up to 6 h of LPS treatment (Fig. 1f) indicating an inverse relationship between cJun and miR-200b expression in LPS-activated microglia in vitro.

Figure 1.

miR-200b is localized in microglial cells and its expression is down-regulated in activated microglia: (a–c). In situ hybridization analysis using 5′ fluorescently labelled scrambled microRNA (miRNA) probe (a), U6 snRNA (used as positive control) (b) and miRNA probe mmu-miR-200b (c) revealed the expression of this miRNA in BV2 microglia (green). The nucleus is counter-stained with DAPI (DAPI – blue). Scale bar: 20 μm. (d) miR-200b expression was quantified by Real-Time PCR analysis using locked nucleic acid (LNA) primers specific for mmu-miR-200b. Results are represented as miRNA fold change with respect to untreated controls. Statistical analysis was carried out using anova with post hoc Tukey test. Data represent the mean ± SD (n = 5) **< 0.01; *< 0.05 (e) Alignment of cJun 3′UTR with seed region of miR-200b obtained from Targetscan website. (f) cJun mRNA expression was quantified using Real-Time PCR analysis after 1–6 h of LPS activation. Results are represented as mRNA fold change with respect to untreated controls. Statistical analysis was carried out using anova with post hoc Tukey test. Data represent the mean ± SD (n = 5) ***< 0.001;**< 0.01; *< 0.05 (g). miR-200b and cJun mRNA expression from LCM extracted microglial cells was quantified using Real-Time PCR analysis. Statistical analysis was carried out using Student's t-test. Results are represented as miRNA/mRNA fold change with respect to untreated controls. Data represent the mean ± SD (n = 4) **< 0.01; *< 0.05.

miR-200b expression is down-regulated in microglia in traumatic brain injury rat model

In this study, rat TBI model has been used to study microglia-mediated neuroinflammation in vivo. Since TBI is focal to the hippocampus, lectin-stained microglia cells were isolated from the hippocampus of brain sections of sham-operated and TBI rat brain using LCM. The cells isolated by LCM were confirmed to be microglia since the mRNA expression of oligodendrocyte (CNPase), astrocyte (Glial fibrillary acidic protein, GFAP), neuronal (microtubule-associated protein 2, MAP2) and endothelial (platelet-endothelial cell adhesion molecule-1, PECAM-2) specific genes was undetectable (Figure S4). qRT-PCR analysis revealed a down-regulation of miR-200b expression while cJun exhibited increased mRNA expression in activated microglia following TBI (Fig. 1g). These results confirm the inverse correlation between the expression of miR-200b and cJun in activated microglia in vivo.

miR-200b targets cJun by directly interacting with its 3′UTR in microglial cells

Cotransfection of BV2 cells with miR-200b mimics and the reporter plasmid containing cJun 3′ UTR resulted in a significant decrease in luciferase activity as compared to control mimics suggesting that miR-200b directly interacts with the cJun 3′UTR (Fig. 2a). Furthermore, inhibition of miR-200b using miRCURY LNA miRNA inhibitors resulted in a significant increase in expression levels of cJun mRNA and total protein (Fig. 2b–d). Conversely over-expression of miRNA-200b using mirVana miRNA mimics resulted in a significant decrease in cJun expression at the protein level (Fig. 2c–d). Taken together, these results suggest that cJun is a target of miR-200b in microglial cells.

Figure 2.

cJun is a target of miR-200b in microglial cells. (a) Luciferase activity assay using reporter pMirTarget with mouse cJun 3′ UTR was performed after cotransfection with miR-200b mimic or control mimic in BV-2 cells. The luciferase activity of the control mimic transfection was set to 1. Mean ± SD (n = 5), *p 0.05. (b) cJun mRNA expression in BV2 cells was quantified following knockdown of microRNA (miRNA)- 200b. Results are represented as mRNA fold change with respect to untreated controls. Mean ± SD (n = 5), *p 0.05. (c) cJun protein expression after miR-200b knockdown and over-expression was quantified using densitometry analysis following western blot. Results are represented as protein fold change with respect to untreated controls. Mean ± SD (n = 6) *< 0.05 Statistical analysis for (a–c) was carried out using Student's t-test. (d) Western blot of cJun after miR-200b knockdown and over-expression.

Knockdown of miR-200b increases cJun phosphorylation in LPS-activated BV2 microglia

Immunocytochemical analysis revealed an increase in cJun phosphorylation (Fig. 3a, b) and total protein expression in activated BV2 microglia (Fig. 3c, d). Western blot analysis confirmed an increase in cJun phosphorylation in BV2 microglia after LPS treatment (Fig. 3e–h). There was a further increase in phospho cJun expression in activated microglia after knockdown of miR-200b (Fig. 3e–f). Conversely, over-expression of miR-200b did not increase the phosphorylation of cJun significantly in activated microglia (Fig. 3g, h).

Figure 3.

Knockdown of miR-200b increases cJun phosphorylation in activated BV2 microglia. (a–d) Immunocytochemistry revealing the increase in phospho cJun (a, b, red) and total cJun (c, d, red) expression after activation of BV2 microglia. The cells are marked with lectin (green) and the nucleus is counter-stained with DAPI (DAPI – blue). Scale bars (a–d): 50 μm. (e, g) Activation of cJun in nuclear extracts of activated BV2 microglia was quantified using densitometry analysis following western blot. Statistical analysis was carried out using anova with post hoc Tukey test. Data represent the mean ± SD (n = 5), *< 0.05 (f, h). Western blot of phospho and total cJun after miR-200b knockdown and over-expression in nuclear extract of activated BV2 microglia.

miR-200b suppresses JNK activity by down-regulating cJun expression

Since JNKs, members of the MAPK signalling molecules, bind and phosphorylate c-Jun (Hibi et al. 1993) we examined if suppression of cJun expression by miR-200b alters JNK protein activity. Total- and phospho-JNK proteins were examined by western blot after miR-200b knockdown and over-expression. Phospho-JNK protein expression increased in activated microglia transfected with scrambled miRNA and control mimic (Fig. 4a–d). miR-200b knockdown further increased the JNK phosphorylation in activated cells as compared to scrambled miRNA (Fig. 4a, b). Conversely, over-expression of miR-200b decreased JNK phosphorylation in activated microglia, as compared to those from control group confirming the alteration of JNK activity concomitant with cJun activity by miR-200b (Fig. 4c–d).

Figure 4.

miR-200b suppresses cJun-N terminal kinase (JNK) activity by down-regulating cJun expression. (a) Activation of JNK in activated BV2 microglia measured by western blotting using antisera against phosphorylated (p) and total JNK. (b) Western blot of p- and total JNK in activated microglia after miR-200b knockdown. (c) Activation of JNK in activated BV2 microglia measured by western blotting using anti-sera against phosphorylated (p) and total JNK shows a reduction in JNK phosphorylation after miR-200b over-expression. (d) Western blot of p- and total JNK in activated microglia after miR-200b over-expression. Statistical analysis in (a, c) was carried out using anova with post hoc Tukey test. Data represent the mean ± SD (n = 6) *< 0.05.

miR-200b negatively regulates inflammatory cytokine response of microglia

Knockdown of miR-200b in activated BV2 microglia significantly increased the mRNA expression of TNF α and IL-6 while a statistically significant change in IL-1β expression was not observed (Fig. 5a–c). ELISA analysis revealed increased TNF α secretion in the medium by activated BV2 cells following miR-200b knockdown (Fig. 5d). In contrast, miR-200b over-expression significantly decreased TNF α protein secretion in activated BV2 microglia (Fig. 5e). Immunocytochemical analysis also revealed an increase in TNF α expression after miR-200b knockdown in activated microglial cells as compared to scrambled miR (Fig. 6ai–aiv) while the converse was observed after miR-200b over-expression (Fig. 6bi–biv). Thus, both gain- and loss-of-function studies indicate a suppressive role of miR-200b in microglial inflammatory response.

Figure 5.

miR-200b negatively regulates inflammatory cytokine response qRT-PCR analysis of (a). IL6 (b) IL-1 β and (c) TNF α shows an up-regulation in the mRNA expression of these cytokines after miR-200b knockdown in activated microglia as compared to control. (d, e) TNF α protein release in media measured by ELISA after miR-200b knockdown (d) and over-expression (e). Statistical analysis was carried out using anova with post hoc Tukey test. Data represent the mean ± SD (n = 5), ***< 0.001, **< 0.01, *< 0.05.

Figure 6.

miR-200b decreases TNF α expression in activated microglia ai–biv: Confocal images showing the expression of TNF α (red) in BV2 cells transfected with scrambled probe (green) (ai, aii), miR-200b antagomir (green) (aiii, aiv), control mimic (bi, bii), miR-200b mimic (biii, biv) (DAPI – blue). Cells in panels aii, aiv, bii and biv were activated with LPS respectively. Scale bars ai–biv: 50 μm.

miR-200b modulates iNOS expression and NO production in activated microglia

Immunofluorescence evaluation revealed that inhibition of miR-200b increased iNOS expression in activated microglia (Fig. 7ai–aiv), whereas down-regulation of iNOS expression was observed in activated microglia over-expressing miR-200b (Fig. 7bi–biv). Moreover, inhibition of miR-200b in activated microglia led to increased release of NO as compared to scrambled miR (Fig. 7c). Conversely, over-expression of miR-200b in activated microglia significantly decreased the NO release as compared to control (Fig. 7d).

Figure 7.

Effect of miR-200b modulation on iNOS expression and Nitrite production. (ai–biv): Confocal images showing the expression of iNOS (red) in BV2 cells transfected with scrambled probe (green) (ai, aii), miR-200b antagomir (green) (aiii, aiv), control mimic (bi, bii) and miR-200b mimic (biii, biv). Cells in panels aii, aiv, biii, biv were activated with LPS (DAPI – blue). Scale bars ai–biv: 50 μm. (c–d). Quantitative analysis of nitric oxide (NO) production using the Greiss assay shows increase in NO production after miR-200b knockdown (c) while a decrease in NO production is observed after miR-200b over-expression (d) in activated BV2 cells. Statistical analysis was carried out using anova with post hoc Tukey test. Data represent the mean ± SD (n = 4), ***< 0.001, **< 0.01, *< 0.05.

miR-200b inhibition in activated microglia increases neuronal apoptosis

To determine the effect of modulation of miR-200b-mediated microglial immune response on neuronal survival, fully differentiated MN9D neurons were used. More than 2-fold change in apoptosis was observed in neuronal cells treated with conditioned medium (CM) obtained from LPS-activated BV2 microglia transfected with scrambled miR (Fig. 8a–c). There was a further increase in number of apoptotic cells exposed to CM obtained from miR-200b knockdown BV2 microglia as compared to scrambled + LPS-treated BV2 microglia (Fig. 8a, c). In contrast, a decrease in apoptotic neuronal cells was observed in culture containing CM obtained from LPS-activated microglia over-expressing miR-200b as compared to miR-200b knockdown BV2 microglia, indicating enhanced neuronal survival (Fig. 8b–c).

Figure 8.

miR-200b inhibition in activated microglia increases neuronal apoptosis (a–b): Histogram overlay representing the flow cytometric analysis of Annexin V fluorescence intensity indicates increased neuronal apoptosis after miR-200b knockdown (a) as compared to miR-200b over-expression (b). (c) Quantitative analysis of apoptotic MN9D cells assessed in the presence of microglial conditioned medium (CM). Results expressed as fold change in apoptotic cells with respect to control cells. Statistical analysis was carried out using anova with post hoc Tukey test. Data represent the mean ± SD (n = 3) *< 0.05.

Over-expression of miRNA-200b reduces the migratory ability of activated microglia

As a part of the inflammatory process, microglia migrate towards the site of injury/infection. This is brought about by changes accompanying their actin cytoskeleton. An increase in f-actin microspike projections was observed in activated microglial cells after miR-200b knockdown (Figure S6aiv, c). In contrast, microspike projections were hardly detectable in activated microglia over-expressing miR-200b (Figure S6biv, d). MiR-200b inhibition did not show any significant change in microglial migratory ability (Fig. 9ai–aiv, c). However miR-200b over-expression significantly impaired microglial migration after activation (Fig. 9bi–biv, d).

Figure 9.

Over-expression of miRNA-200b reduces the migratory ability of activated microglia (ai–biv) Phase contrast images showing migrated BV2 cells (purple) after miR-200b inhibition (ai–aiv) and miR-200b over-expression (bi–biv) in activated microglia. Scale bars (a–d):100 μm (c–d). Quantitative analysis of BV2 cell migration following activation after miR-200b knockdown (c) and miR-200b over-expression (d). Statistical analysis was carried out using anova with post hoc Tukey test. Data represent the mean ± SD (n = 3), **< 0.01, *< 0.05.

Discussion

Microglial activation is generally associated with neuroprotection. However, chronic activation of microglia is considered to be the pathological hallmark of neuroinflammation and has been implicated in neurodegenerative diseases (Tuppo and Arias 2005; Phani et al. 2012; Rubio-Perez and Morillas-Ruiz 2012). Hence, understanding the molecular mechanism of microglial activation might help develop therapeutic strategies for neurological disorders. Several transcription factors and signalling molecules including those involved in MAPK pathways have been shown to regulate microglial activation (Dheen et al. 2007). This study demonstrates that miR-200b, a novel non-coding RNA, regulates microglia-mediated immune response by targeting cJun, a substrate of the JNK/MAPK pathway.

miRNAs are novel epigenetic factors controlling gene expression at the post-transcriptional level through imperfect base pairing with the 3′UTRs of their target mRNAs (Kim et al. 2009). Altered miRNA expression patterns have been associated with many types of human diseases, including neuronal disorders (Fiore et al. 2008). The members of miRNA-200 family such as miR-200b and miR-200c have been shown to regulate antibacterial innate immune response by targeting the TLR4 signalling pathway (Wendlandt et al. 2012). Recently, miR-200c was found to be expressed in microglia (Jovičić et al. 2013). Since miR-200c and miR-200b share overlapping targets owing to the same seed sequence, the above evidence suggests a role for this family of miRNAs in regulating microglial functions in the CNS. This study provides evidence, for the first time, that miR-200b is expressed in microglia cells and its expression is significantly down-regulated in activated microglial cells both in vitro and in vivo model of TBI.

This study establishes that miR-200b in microglia directly inhibits cJun, which is a transcription factor regulated by the JNK MAPKs (Hibi et al. 1993). In activated microglia, JNK expression was shown to be up-regulated in response to various stimuli such as LPS, hypoxia or stress, leading to the release of pro-inflammatory cytokines via cJun, thereby resulting in neuronal cell death (Kyriakis and Avruch 2001; Brecht et al. 2005; Waetzig et al. 2005; Mehan et al. 2011; Deng et al. 2012). In this study, suppression of JNK activity in activated microglia by miR-200b via down-regulation of cJun expression and function led to a reduction in the expression of proinflammatory cytokines such as TNF α and IL6 and the production of neurotoxic molecules such as NO.

It is well established that chronic activation of microglia in neuropathological conditions has deleterious consequences on the surrounding healthy neurons (Tuppo and Arias 2005; Phani et al. 2012; Rubio-Perez and Morillas-Ruiz 2012). Our findings suggest that loss of miR-200b increases microglia-mediated neurotoxic effect. This observed increase in neuronal apoptosis in the culture exposed to conditioned medium derived from microglia treated with miR-200b inhibitor, is most probably because of the result of an excessive release of inflammatory cytokines and NO in the conditioned medium mediated via increased cJun activity. However, further experiments are required to ascertain the possible neuroprotective role of microglial miR-200b since the over-expression of miR-200b failed to overcome the LPS-mediated neuronal apoptosis.

Impaired microglial migration has been shown to contribute to the pathogenesis of several brain diseases such as Prion disease (Ciesielski-Treska et al. 2004), Parkinson's disease (Park et al. 2008), Alzheimer's disease (Mizuno 2012) and inhibit axonal regeneration during acute CNS injuries (Vargas and Barres 2007). This study demonstrates that over-expression of miR-200b in microglia affects f-actin reorganization which may result in impaired migration (Lauffenburger and Horwitz 1996; Mitchison and Cramer 1996). While it remains unclear whether these observations are a result of a direct inhibition of cJun activity and its subsequent downstream targets or via targeting of other mRNA factors, this study provides a link between miR-200b-mediated regulation of various microglial functions.

Post-transcriptional reduction of protein expression of selected genes linked to a particular disease by a specific miRNA represents new therapeutic options. It has been recently shown that administration of miR-124 in vivo suppresses experimental autoimmune encephalitis by affecting macrophages, (Ponomarev et al. 2011) and that knockdown of brain enriched miR-181 provides protection against ischaemia-induced neuronal death (Ouyang et al. 2012). Collectively, all of these studies have indicated that manipulating miRNA levels could be a potential treatment strategy for reducing microglial immune response in CNS pathologies.

To conclude our results demonstrate that miR-200b reduces inflammatory response of activated microglia via targeting of cJun/MAPK signalling pathway. This results in reduction of microglial cytokine expression, NO production and migration and may therefore represent a valuable therapeutic strategy in the context of chronic neuroinflammation.

Acknowledgements and conflict of interest disclosure

This research was supported by the joint ASTAR-MINDEF-NUS grant (09/1/50/19/621 and R-181-000-141-305) and NMRC Exploratory/Developmental Grant (NMRC/EDG/1039/2011; R-181-000-139-275), Singapore. The authors declare no conflict of interest.

All experiments were conducted in compliance with the ARRIVE guidelines.

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