The Modulates Microglia Behavior through MicroRNA-Dependent Regulation of c-Maf

This information is current as Wei Su, Stephanie Hopkins, Nicole K. Nesser, Bryce of October 1, 2021. Sopher, Aurelio Silvestroni, Simon Ammanuel, Suman Jayadev, Thomas Möller, Jonathan Weinstein and Gwenn A. Garden J Immunol 2014; 192:358-366; Prepublished online 6 December 2013; doi: 10.4049/jimmunol.1301397 Downloaded from http://www.jimmunol.org/content/192/1/358

Supplementary http://www.jimmunol.org/content/suppl/2013/12/06/jimmunol.130139 http://www.jimmunol.org/ Material 7.DC1 References This article cites 78 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/192/1/358.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The p53 Transcription Factor Modulates Microglia Behavior through MicroRNA-Dependent Regulation of c-Maf

Wei Su,* Stephanie Hopkins,* Nicole K. Nesser,*,1 Bryce Sopher,* Aurelio Silvestroni,* Simon Ammanuel,*,2 Suman Jayadev,*,† Thomas Mo¨ller,*,3 Jonathan Weinstein,*,† and Gwenn A. Garden*,†

Neuroinflammation occurs in acute and chronic CNS injury, including stroke, traumatic brain injury, and neurodegenerative dis- eases. Microglia are specialized resident myeloid cells that mediate CNS innate immune responses. Disease-relevant stimuli, such as reactive oxygen species (ROS), can influence microglia activation. Previously, we observed that p53, a ROS-responsive transcription factor, modulates microglia behaviors in vitro and in vivo, promoting proinflammatory functions and suppressing downregulation of the inflammatory response and tissue repair. In this article we describe a novel mechanism by which p53 modulates the functional

differentiation of microglia both in vitro and in vivo. Adult microglia from p53-deficient mice have increased expression of the anti- Downloaded from inflammatory transcription factor c-Maf. To determine how p53 negatively regulates c-Maf, we examined the impact of p53 on known c-Maf regulators. MiR-155 is a microRNA that targets c-Maf. We observed that cytokine-induced expression of miR- 155 was suppressed in p53-deficient microglia. Furthermore, Twist2, a transcriptional activator of c-Maf, is increased in p53- deficient microglia. We identified recognition sites in the 39 untranslated region of Twist2 mRNA that are predicted to interact with two p53-dependent microRNAs: miR-34a and miR-145. In this article, we demonstrate that miR-34a and -145 are regulated

by p53 and negatively regulate Twist2 and c-Maf expression in microglia and the RAW macrophage cell line. Taken together, these http://www.jimmunol.org/ findings support the hypothesis that p53 activation induced by local ROS or accumulated DNA damage influences microglia functions and that one specific molecular target of p53 in microglia is c-Maf. The Journal of Immunology, 2014, 192: 358–366.

cute and chronic injury of the CNS is associated with Microglia, like macrophages, are capable of a variety of in- neuroinflammation (1). Microglia, the resident immu- flammatory responses to altered environmental stimuli (5). In A nocompetent cells of the CNS, participate in a neuro- macrophages, classical activation develops in response to proin- inflammatory response to ischemic or traumatic injury. In the flammatory cytokines such as IFN-g and TLR ligands. Classically uninjured CNS, microglia express little or no mRNA for activated macrophages perform functions important for pathogen associated with active inflammation, such as CD45, MHC class II, suppression, such as generation of reactive oxygen species (ROS) by guest on October 1, 2021 costimulatory molecules (CD80 and CD86), and integrins (2–4). and secretion of proinflammatory mediators, activities that may During the response to injury, microglia become activated, dem- cause injury to bystander cells.Incontrast,anti-inflammatory onstrating increased expression of activation markers (CD45, signals such as IL-4, -10, or -13, immune complexes, TGF-b,and MHC class II, CD86, and so on). They also change in morphology glucocorticoids lead to type II inflammation, in which proin- from cells with a small soma and fine ramifications to larger flammatory activities are suppressed and inflammatory cells pro- ameboid cells capable of migrating toward a site of injury or in- mote tissue remodeling. Microglia expression of activation markers fection. differs quantitatively, but not qualitatively, from that observed in peripheral macrophages (4). The classical activation state of mye- loid cells has been linked with promoting inflammation (6), whereas the alternative phenotype is anti-inflammatory and promotes tissue *Department of Neurology, University of Washington, Seattle, WA 98195; and †Center on Human Development and Disability, University of Washington, Seattle, repair (7). WA 98195 Although the roles microglia play in the injured CNS are still 1Current address: Oregon Health Sciences University, Portland, OR. under investigation, an emerging theme is that glia contribute to 2Current address: The Johns Hopkins University, Baltimore, MD. the pathogenesis of neurologic diseases via a non–cell-autonomous 3Current address: Lundbeck Research, Paramus, NJ. mechanism (8). Microglia behavior during inflammation can lead Received for publication May 28, 2013. Accepted for publication October 29, 2013. to 1) loss of normal function in other neuroglia that subsequently This work was supported by National Institutes of Health Grants R01NS073848, trigger downstream damage to vulnerable neurons; and 2) direct R03NS70141, and R21NS062269 (to G.A.G.), with facilities support from Grant neuronal toxicity (9). The response to CNS injury may trigger both P30-HD02274 to the University of Washington Center on Human Development processes if neural injury leads to promotion of neurotoxic, tissue- and Disability. destructive inflammatory responses while concurrently suppressing Address correspondence and reprint requests to Dr. Gwenn A. Garden, University of Washington, 1959 North East Pacific Street, Seattle, WA 98195. E-mail address: a potentially neuroprotective inflammatory response. [email protected] The transcription factor p53 is best known as a regulator of cell- The online version of this article contains supplemental material. cycle control and apoptosis in response to discrete cellular insults Abbreviations used in this article: FM, FACS medium; KO, knockout; MCAO, mid- (8). Under basal conditions, p53 is ubiquitinated and rapidly dle cerebral artery occlusion; miRNA, microRNA; q, quantitative; ROS, reactive degraded. However, p53 activity can be rapidly increased in re- oxygen species; UTR, untranslated region; WT, wild-type. sponse to a wide range of stresses, including DNA damage, hyp- Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 oxia, and oxidative damage (10). Previously, we identified p53 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301397 The Journal of Immunology 359 as a novel candidate modulator of microglia behavior and dem- mice (C57/BL6 from The Jackson Laboratory), were maintained in a specific onstrated ongoing p53 transcriptional activity in microglia cul- pathogen–free facility under the guidance of an Institutional Animal Care tured under basal conditions (11–13). Genetic knockout (KO) or and Use Committee–approved protocol. pharmacological inhibition of p53 results in microglia that are less Middle cerebral artery occlusion neurotoxic in response to proinflammatory stimuli (11, 14). We Middle cerebral artery occlusion (MCAO) was performed on anesthetized also observed that p53-deficient microglia express genes associ- 12- to 16-wk-old male C57/BL6 mice obtained from The Jackson Labo- ated with the anti-inflammatory/tissue repair phenotype of alter- ratory, using the transient intraluminal filament method (37) with laser natively activated macrophages (13). In addition, p53 is activated Doppler cerebral blood flow monitoring. The filament was inserted to in a subpopulation of microglia in the inflamed human brain (12), obtain a $ 70% occlusion for 15 min, followed by filament removal and documentation of reperfusion, a paradigm developed to result in ischemic but p53 activation is excluded from the population of microglia preconditioning (38). Mice with cerebral blood flow profiles outside labeled by CD163 (13), a marker of macrophages that have a predetermined expected range were excluded. Mice were monitored by adopted anti-inflammatory or tissue repair phenotypes (15). neurobehavioral assessments using a published scoring system (39) for 72 h MicroRNAs (miRNAs) are small noncoding RNAs (∼22 nu- following creation of the lesion. Animals with a detectable behavioral cleotides long) involved in many physiological and pathological deficit were excluded from further study. Using this paradigm, we could find no detectable region of infarction in the cortex by 2,3,5-triphenylte- processes (16–18). A single miRNA can regulate hundreds of trazolium chloride staining. genes, and # 90% of human genes are potentially regulated by miRNAs (19, 20). MiRNAs downregulate the expression of Ex vivo flow cytometry mRNA targets by binding to complementary sequences in the 39 Adult mice (12–16 wk old, n = 4 mice per genotype) were intracardially untranslated region (UTR). Several reports suggest that miRNAs perfused with cold saline solution. Cortical tissue was separated from play an important role in regulating the responses of innate immune whole brain and dissociated using the MACS Neural Dissociation Kit Downloaded from (Miltenyi Biotec) and a modified version of the Miltenyi gentleMACS cells (21), including microglia (18). For example, recent reports protocol. Preparation of single-cell suspensions from mouse neural tissue suggest that miR-124 is a key regulator of microglia quiescence in was done according to the manufacturer’s instructions. Depletion of my- the CNS, thereby helping to prevent CNS inflammation (18, 22). elin and positive selection of myeloid cells were performed in sequential Because a single miRNA can modulate expression of many genes myelin and CD11b magnetic selection steps (Miltenyi Biotec). The re- 3 during inflammation, miRNAs may serve as epigenetic regulators of maining cells were centrifuged at 300 g for 10 min at 4˚C; then the pellet was resuspended in FACS medium (FM) [10% FCS (Atlanta Bio- http://www.jimmunol.org/ microglia behavior (23). logicals), 10 mM HEPES pH7.3 (Life Technologies), in HBSS buffer As a transcriptional activator, p53 exerts its function through without calcium or magnesium (Life Technologies)] and incubated with promoting expression of target genes to initiate cellular responses. FC block (BD Pharmingen). Cells were next stained with fluorochrome- Recent work has revealed that miRNAs are important components conjugated Abs according to the manufacturer’s protocol: FITC-CD45 (BD Pharmingen, Transduction Labs), APC-F4/80 (Affymetrix eBio- in the p53 transcriptional network (24). Maturation of miRNAs is science), or isotype control and DAPI (Sigma-Aldrich). Cells with Abs promoted by p53-mediated transcription (25), and p53 also induces were incubated on ice for 45 min, washed three times with FM, and expression of specific miRNAs. For example, several groups resuspended in FM. All cells were sorted on a BD ARIA II cell sorter directly into TRIzol LS reagent (Invitrogen). Microglia were defined as the reported that p53 directly regulates the expression of the miR-34 positive intermediate family (26–30). The miR-34 family consists of miR-34a, miR- population of cells that were F4/80 , and CD45 , using a previously published approach (40). by guest on October 1, 2021 34b, and miR-34c, which are encoded by two different genes. In transformed cells, ectopic expression of miR-34a promotes p53- Cell culture mediated apoptosis, cell-cycle arrest, and senescence, whereas in- The RAW cell line was grown in high-glucose DMEM supplemented with activation of endogenous miR-34a strongly inhibits p53-dependent 10% FBS (Life Technologies), 25 U/ml penicillin, and 25 mg/ml strep- apoptosis (24). In addition to the miR-34 family, p53 also di- tomycin and incubated in a 37˚C cell incubator supplemented with 5% 3 4 rectly regulates expression of several additional miRNAs, in- CO2. RAW cells were plated onto a 24-well dish at a density of 5 10 , cluding miR-145 (24, 25). Overexpression of miR-145 reduces transfectedwith5pmolofMISSIONmiRNA mimics (Sigma-Aldrich), using jetPEI (Polyplus Transfection) reagents according to the manufacturer’s pro- c- expression, whereas suppression of endogenous miR-145 tocol. For IL-4 treatment, cells were treated with 10 ng/ml IL-4 for 24 h before enhances c-Myc expression (24), which partially accounts for harvest. For nutlin treatment, cells were treated with DMSO, 10 mM, 40 mM, miR-145–mediated inhibition of tumor cell growth both in vitro or 80 mM nutlin, for 6 h before harvest. Cells were lysed for total RNA 24 h and in vivo (31, 32). post transfection and for total protein 72 h post transfection. Mixed glia cul- tures were generated, using previously published methods, from cortical tissue We previously reported that microglia from p53-deficient mice dissected on postnatal day 3 or 4 (13). Cells were cultured in high-glucose had altered expression and inflammatory behaviors suggesting DMEM (Hyclone, Thermo Scientific) supplemented with 10% heat- that p53 transcriptional activity is a critical activator of proin- inactivated horse serum (Life Technologies), 10% nutrient mixture F-12 flammatory responses in microglia (13). In this article, we identify ham (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), 10 mM HEPES c-Maf, a transcription factor known to promote functional differ- (Sigma-Aldrich), and 20% L929-conditioned medium (41). Microglia were isolated from the cultures 7–10 d post dissection by collecting floating entiation and anti-inflammatory responses in both lymphocytes cells. Primary microglia were plated on poly-d-lysine–coated plates at and myeloid cells (33, 34), as a downstream p53 target in micro- a density of 5 3 105 per 35-mm plate or 1 3 106 per 60-mm plate in D10C glia. We determined that p53 negatively regulates c-Maf in micro- media. Cells were treated with IFN-g (10 U/ml) for 24 h, nutlin-3 (40 mM) glia by enabling the induction of miR-155, a miRNA that for 6 h, or vehicle control. suppresses c-Maf. In addition, Twist2, a transcriptional activator of Real-time quantitative RT-PCR c-Maf, is suppressed by p53-regulated miR-34a and miR-145. Taken together, we propose that p53 activation may influence microglia Total RNA was extracted using the High Pure RNA Isolation Kit (Roche) or miRNeasy kit (QIAGEN) according to the manufacturers’ instructions. responses to injury by suppressing c-Maf expression and the sub- cDNA was generated using the High Capacity cDNA Reverse Transcrip- sequent protective inflammatory response. tase Kit (Applied Biosciences). cDNA from adult mRNA transcripts was amplified using the Ovation quantitative (q)PCR system (NuGEN) at the Materials and Methods University of Washington Center on Human Development and Disability Animals Microarray and Bioinformatics Core Laboratory. cDNA from adult mouse miRNA was generated using miRNA-specific primers. qPCR was per- Mice deficient in p53 (p532/2) (35) and miR-155 (miR-155 KO) (36), and formed using the StepOnePlus Real Time PCR Instrument (Applied Bio- strain-matched p53-expressing mice (p53+/+), as well as miR-155–expressing sciences) with the TaqMan Assay, TaqMan MicroRNA 360 p53 TARGETS c-Maf IN MICROGLIA

2 2 Assay, or Roche Universal Probe Library. TaqMan probe and primers used 2.5-fold in p53 / microglia (Fig. 1), compared with p53+/+ cells, are as follows: cMaf: Mm02581355_s1; Twist2: Mm00492147_m1; miR- suggesting that basal p53 activity in normal microglia is sufficient to 34a: 000426; miR-145: 002278; and miR-155: 002571. PCR products were suppress c-Maf expression in vivo. normalized to TBP (Mm01277042_m1) or 18S (Mm03928990_g1) for TaqMan gene expression assays, and Sno202 (001232) and Sno234 MiR-155, a suppressor of c-Maf, is regulated by p53 (001234) for miRNA assays. Roche UPL primer probe sets are listed in Table I. Because c-Maf is a potential downstream target of p53 in microglia, next we asked how p53 influences the expression of c-Maf. The Western blot transcriptional repertoire of p53 has been intensely studied, but RAW cells were lysed with RIPA buffer (Thermo Scientific) containing direct binding of p53 to the c-Maf gene had not been identified (47). protease inhibitor mixture (Sigma-Aldrich). A total of 10 mg total proteins To assess whether p53 directly suppresses expression of c-Maf were run on NuPage 4–12% Bis Tris gel (Invitrogen) and transferred according to the manufacturer’s protocol (Invitrogen). Membranes were mRNA, we evaluated the c-Maf genomic sequence and observed probed for Twist2 (Ab66031, Rabbit polyclonal; Abcam), c-Maf (Ab76817, several p53 consensus binding sites 2 Kb upstream of the trans- mouse monoclonal; Abcam), and b-actin (SC-47778, mouse monoclonal; lational start site. One recent study reported that p53 promoted Santa Cruz Biotechnology) as primary Abs (1:1000 dilution), and anti- transcriptional activation of c-Maf during the process of lens de- Rabbit (SC2301; Santa Cruz Biotechnology) or anti-mouse (SC2302; velopment (48). Because this report suggested that a direct inter- Santa Cruz Biotechnology) as a secondary Ab (1:5000 dilution). Twist2, c-Maf protein levels were normalized to b-actin. All films were scanned action between p53 and the c-Maf gene led to regulation in the and analyzed using ImageJ 1.47 software. opposite direction of what was observed in microglia, we hypoth- esized that p53 represses c-Maf expression in microglia through an Results alternate mechanism. To determine how c-Maf expression levels are c-Maf expression is suppressed by p53 in microglia Downloaded from We previously reported that p53 supports the proinflammatory response in microglia (13). We observed that p53 KO (p532/2) microglia in neuron/microglia cocultures are functionally distinct from wild-type (WT) primary microglia in response to HIV gp120- coated protein (11). We further found that p532/2 microglia dem- http://www.jimmunol.org/ onstrated a blunted response to IFN-g, failing to increase expression of genes associated with classical macrophage activation or secrete proinflammatory cytokines, and tend to have gene expression pat- terns and functions similar to those of alternatively activated mac- rophages (13). The c-Maf transcription factor has been reported to promote expression of anti-inflammatory cytokines and suppress expres- sion of proinflammatory cytokines in macrophages (33, 34, 42).

We hypothesized that p53 promotes microglia proinflammatory by guest on October 1, 2021 behaviors by suppressing c-Maf–mediated gene transcription. We therefore measured c-Maf mRNA in microglia cultured from day 3–4 postnatal p53+/+ or p532/2 mice, and found that c-Maf was elevated by 2-fold in p532/2 cells compared with p53+/+ cells (data not shown), indicating a regulatory connection between p53 and c-Maf. However, cultured microglia are exposed to cytokines that promote cell division, and c-Maf expression may be regulated by these cytokines (43). To determine whether the regulation of c-Maf expression by p53 in cultured microglia was unique to the in vitro environment, we asked whether c-Maf expression was spe- cifically regulated by p53 in microglia from the adult brain. We obtained microglia from 12- to 16-wk-old adult mice by ex vivo flow cytometry, based on previously published criteria (44–46), and measured c-Maf mRNA by qRT-PCR. c-Maf mRNA was elevated by

FIGURE 2. miR-155, a negative suppressor of c-Maf, requires p53 for induction in microglia. (A) miR-155 expression in microglia cultured from p53+/+ and p532/2 neonatal mice was determined by miRNA qRT-PCR assay. Primary cultured microglia were treated with 10 U/ml IFN-g or vehicle control for 24 h before total RNAs were extracted (n = 3 from separate culture experiments and qPCR runs). **p , 0.001 for miR-155 level in p53+/+ versus p532/2 cells upon IFN-g treatment by two-way ANOVA post hoc comparison. (B) c-Maf mRNA levels in microglia cul- tured from JAX WT and miR-155 KO neonatal mice were detected by FIGURE 1. c-Maf is negatively regulated by p53 in adult mice micro- qRT-PCR (n $ 3 from separate culture experiments and qPCR runs). *p , glia. Expression of c-Maf mRNA is elevated in p532/2 mice. qRT-PCR for 0.05 by unpaired t test with Welch’s correction. (C) c-Maf mRNA levels c-Maf mRNA was performed on total RNA obtained from p53+/+ and were determined by qRT-PCR in microglia collected from adult JAX WT p532/2 adult mouse brain microglia extracted by ex vivo flow cytometry and miR-155 KO mice by ex vivo flow cytometry (n = 3, adult mice brains (n = 3, adult mice brains were used in ex vivo flow cytometry, statistics were used in ex vivo flow cytometry, statistics were determined from were determined from separate qPCR runs). *p , 0.005 by unpaired t test. separate qPCR runs). *p , 0.05 by unpaired t test. The Journal of Immunology 361

FIGURE 3. Classical activation of microglia is suppressed in miR-155 KO cells. qRT-PCR was performed on WT and miR-155 KO microglia treated with IFN-g or control for IL-1a (A), IL-1b (B), and the murine scavenger MARCO (C). n = 3 separate cultures and qPCR runs. *p , 0.01, **p , 0.0001 by two-way ANOVA comparison. negatively regulated by p53, we assessed whether other known We previously reported that three genes associated with a type I regulators of c-Maf expression are influenced by p53 in microglia. inflammatory response—IL-1a,IL-1b,andMARCO(49)—arenot 2/2

Because p53 generally acts as a transcriptional activator, we effectively induced by IFN-g in p53 microglia (13). Similar Downloaded from hypothesized that suppression of c-Maf was mediated by a p53- results were observed in miR-155 KO microglia for IL-1a (Fig. 3A) induced miRNA. The proinflammatory miRNA miR-155 nega- and MARCO (Fig. 3C), but not for IL-1b (Fig. 3B). These results tively regulates c-Maf expression in T cells (36). We therefore suggest that regulation of IL-1a and MARCO by p53 in microglia evaluated miR-155 expression in cultured primary microglia by is mediated through miR-155; however, an alternative pathway qRT-PCR. Under resting conditions, very little miR-155 is ex- might influence microglia IL-1b expression in response to IFN-g. pressed in microglia (Fig. 2A). Treatment with classical activation Twist2, a positive regulator of c-Maf, is regulated by p53- http://www.jimmunol.org/ cytokine IFN-g leads to a robust induction of miR-155 in p53+/+ inducible miRNAs cells. In contrast, induction of miR-155 in p532/2 cells was sig- nificantly repressed. Comparing IFN-g–induced miR-155 expres- c-Maf expression is also transcriptionally regulated by Twist2, a ba- sion between p53+/+ and p532/2 microglia cultures revealed that sic helix-loop-helix transcription factor. Twist2 promotes myeloid p53 significantly augments the induction of miR-155 (Fig. 2A). differentiation and interacts with the 59 regulatory region of the We next measured c-Maf mRNA expression in microglia cultures cMaf gene to increase c-Maf expression (50). We therefore inves- from miR-155 KO mice and found that c-Maf mRNA was elevated tigated the relationship among p53, Twist2, and c-Maf in microglia. by 4.5-fold over WT microglia (Fig. 2B). We also observed a 1.5- We observed that Twist2 was upregulated by 2.5-fold in cultured 2/2 +/+ fold increase of c-Maf mRNA in microglia collected by ex vivo p53 microglia compared with p53 cells on the microarray by guest on October 1, 2021 flow cytometry from miR-155 KO adult mice compared with analysis previously reported (13). An even larger increase in Twist2 WT mice (Fig. 2C), demonstrating that even under basal con- expression was confirmed by qPCR (Table I) on mRNA isolated 2/2 +/+ ditions c-Maf expression is suppressed by this pathway in vivo. from p53 and p53 microglia (Fig. 4). This finding suggests an Taken together, these data suggest that p53 negatively regulates alternative mechanism by which c-Maf expression is regulated by c-Mafexpressionbytranscriptional activation of miR-155. p53 via p53-dependent suppression of Twist2 expression. Because p53 is known as a transcriptional activator, we hy- miR-155 KO recapitulates a portion of the p53 KO phenotype pothesized that p53 also induces the expression of miRNAs that in microglia negatively regulate Twist2. The 39-UTR of Twist2 was analyzed in Previously, we reported that p532/2 microglia did not promote silico for the identification of miRNA targets. Using two inde- HIV-gp120 neurotoxicity (11) and have a muted transcriptional pendent software platforms, we identified binding regions for two response to a classical activation signal (13). If miR-155 is the specific miRNAs, miR-34a and miR-145, which are known p53 key mediator of the p53-dependent microglia proinflammatory transcriptional targets (26, 27, 31). We performed RT-PCR for response, we hypothesized that mediators of classical activation these two miRNAs in both cultured microglia (Fig. 5A, 5B) and influenced by p53 would be similarly regulated by miR-155. To adult microglia extracted by ex vivo flow cytometry (Fig. 5C, 5D) test this hypothesis, primary microglia cultured from WT and and observed p53-dependent expression of these two miRNAs in miR-155 KO mice were treated with 10 U/ml of IFN-g for 24 h, both systems. Of interest, in vitro these two p53-dependent miRNAs and mRNAs associated with classical activation were evaluated. are differentially regulated by p53, with miR-34a being turned on

Table I. Roche primer and probe sets used for qPCR

Gene Name Forward Primer (59–39) Reverse Primer (59–39) Roche Probe No. GAPDH AGCTTGTCATCAACGGGAAG TTTGATGTTAGTGGGGTCTCG 9 TBP TGTGCACAGGAGCCAAGA CCCCACCATGTTCTGGAT 33 IL1a TTGGTTAAATGACCTGCAACA GAGCGCTCACGAACAGTTG 52 IL1b AGTTGACGGACCCCAAAAG AGCTGGATGCTCTCATCAGG 38 MARCO ATGCTCGGTTACTCCAGAGG ATTGTCCAGCCAGATGTTCC 10 c-Maf CCTTCCTCTCCCGAATTTTT CCACGGAGCATTTAACAAGG 33 Twist2 CATGTCCGCCTCCCACTA GATGTGCAGGTGGGTCCT 10 The qPCR primer sequences and corresponding probes were designed using the ProbeFinder Software available at http:// www.roche-applied-science.com 362 p53 TARGETS c-Maf IN MICROGLIA

FIGURE 4. Expression of Twist2, a positive regulator of c-Maf, is influenced by p53 genotype. Expression of Twist2 mRNA is elevated in p532/2 microglia. qRT-PCR for Twist2 mRNA on total RNA extracted from microglia cultured from p53+/+ and p532/2 neonatal pups. n =3 separate cultures and qPCR runs. *p , 0.0001 by unpaired t test. by basal p53 activity and miR-145 being induced by higher level p53 activity following treatment with the MDM2 inhibitor nutlin. However, both miR-34a and miR-145 were significantly reduced in adult p532/2 microglia isolated by ex vivo sorting. Taken together, Downloaded from these findings support our hypothesis that p53 can suppress c-Maf expression through a pathway independent of miR-155: inducing miRNAs that suppress the c-Maf activator Twist2. To further examine the hypothesis that p53 regulates Twist2 via FIGURE 5. miRNAs that target Twist2 for suppression are targeted by induction of miR-145 and miR-34a, we employed the mouse p53 in microglia both in vivo and in vitro. (A and B) qRT-PCR was per- leukemic monocyte macrophage cell line (RAW cells). To deter- formed to detect miR-34a and miR-145 on total RNA extracted from

2 2 http://www.jimmunol.org/ mine if activation of p53 lessened Twist2 protein in a myeloid cell cultured microglia that were p53 / , p53+/+, or p53+/+ following p53 in- line, we exposed RAW cells to increasing concentrations of nutlin. duction with nutlin for 6 h (n = 3 separate cultures, statistics were deter- , , We observed greater p53 stabilization (Supplemental Fig. 1) and mined from separate qPCR runs). *p 0.01, **p 0.0001 by one-way ANOVA. (C and D) qRT-PCR was performed for miR-34a and miR-145 on decreasing Twist2 protein detected by Western blot with increasing 2 2 adult microglia extracted from p53 / and p53+/+ mice. n = 3 from sep- nutlin concentration (Fig. 6A, 6B). Next, miR-145 and miR-34a arate qPCR runs. *p , 0.02, **p , 0.002 by unpaired t test. mimics (Sigma-Aldrich) were transfected into RAW cells, and Twist2 mRNA levels were determined by qRT-PCR. Twist2 mRNA levels were significantly reduced when either miR-145 or miR-34a the anti-inflammatory phenotype (13). We performed a Pscan

was overexpressed in RAW cells. As shown in Fig. 6, 24 h by guest on October 1, 2021 promoter analysis (51) on microarray data obtained from forebrain post transfection, Twist2 mRNA was knocked down by 66% and microglia ex vivo extracted 3 d following 15-min MCAO [a less 64%, when miR-145 and miR-34a were expressed, respectively severe ischemic pulse that does not induce cortical infarction but (Fig. 6C). The levels of miR-145 and miR-34a before and after does result in ischemic preconditioning (38)] or sham surgery. We transfection were determined by the manufacturer’s recommended observed that 15-min ischemia leads to a significant (p , 0.02) miRNA RT-PCR assay from the same extracted RNA samples induction of p53 transcriptional targets in ex vivo sorted cortical (Supplemental Fig. 2). In addition, we checked the c-Maf mRNA microglia 3 d after the ischemic insult. These findings demonstrate level in RAW cells transfected with either miR-145 or miR-34a that the proinflammatory response supported by p53 could be mimics, and observed . 60% reduction in c-Maf mRNA levels in detected 3 d following this injury paradigm. To determine if this both conditions (Fig. 6D). To ensure that the observed level of model of neuroinflammation involves activation of miR-155, reduction in mRNA led to a physiologically relevant change in miR-145, and miR-34a in microglia, we obtained forebrain microglia Twist2 or c-Maf protein, we performed Western blot for Twist2 byexvivoflowcytometry3dafter15-minMCAO.Thepresence and c-Maf on lysates from RAW cells treated with miR-34a and of an inflammatory response to ischemia was confirmed by a 30% miR-145 mimics and compared the observed change in protein increase in microglia and a 10-fold increase in macrophages ob- relative to a physiological inducer of c-Maf, IL-4 (Fig. 6E–G). tained from cortex ipsilateral to the MCAO, compared with the The reduction in c-Maf protein by miR-34a and miR-145 in RAW contralateral side. We observed an increase in the level of miR-155 cells was significant, though less profound. Taken together, our (Fig. 7A). MiR-34a and MiR-145 were not induced. In addition, the data strongly suggest that miR-145 and miR-34a, the p53-induced expression of c-Maf mRNA was suppressed in microglia from the miRNAs, target Twist2 mRNA for suppression, resulting in de- ischemic side of the brain at both time points (Fig. 7B). Twist2 creased c-Maf expression. mRNA was not detected. In summary, these data suggest that neu- The in vivo response to CNS ischemia involves p53 activation, roinflammation in vivo is associated with induction of at least one induction of p53-dependent miRNAs, and suppression of c-Maf p53-dependent miRNA that suppresses c-Maf expression in micro- glia (Fig. 8). The finding also suggests that miR-155 is the dominant The data presented demonstrate that p53 influences c-Maf ex- mediator of c-Maf suppression during the response to CNS ischemia. pression in several paradigms, including cultured microglia, RAW cells, and microglia extracted from normal adult mouse brain. The question remains whether these miRNAs modulate inflammatory Discussion gene expression in vivo during neuroinflammation. To address this, Many studies suggest that inflammation influences tissue injury we induced neuroinflammation by transient MCAO, a model of and degeneration in the CNS. It is now believed that neuro- CNS ischemia in which we have previously demonstrated that loss inflammation is an important contributor to neurodegeneration. Neu- of p53 leads to increased microglia expressing CD206, a marker of rons are susceptible to oxidative damage from ROS induced by The Journal of Immunology 363

FIGURE 6. p53 activation suppresses Twist2 and c-Maf expression and transfection of miR-145 or miR-34a downregulates Twist2 and c-Maf in RAW cells. (A) p53 activation by nutlin causes a dose- dependent reduction of Twist2 protein in RAW cells. (B) Densitometry of Twist2-labeled bands relative to actin-labeled bands on Western blot of RAW cell lysates after treatment with DMSO or 10 mM, 40 mM, or 80 mM of nutlin. Data were plotted from three separate experiments. *p , 0.001, one- way ANOVA. (C) Transfection of miRNA mimics miR-145, and miR-34a significantly decreases Twist2 mRNA level in RAW cells at 24 h post transfection. n = 3 from separate transfections and qPCR runs. ***p , 0.001, one-way ANOVA. (D) c-Maf mRNA levels were determined by qRT-PCR in samples Downloaded from when cells were transfected with or without miRNA mimics. n = 3 from separate transfections and qPCR runs. **p , 0.01, one-way ANOVA. (E) Transfection of miR-145 and miR-34a mimics significantly de- creased Twist2 and c-Maf protein in RAW cells, whereas IL-4 treatment increased both Twist2 and c-Maf protein. (F) Densitometry of Twist2 protein http://www.jimmunol.org/ level in RAW cells transfected with miR-145 and miR-34a mimics, or treated with IL-4. Data were plotted from four separate experiments. *p , 0.05, **p , 0.001, one-way ANOVA. (G) c-Maf protein level in RAW cells when cells were transfected with miR-145 and miR-34a mimics, or treated with IL-4. Data were plotted from four separate experi- ments. *p , 0.05, one-way ANOVA. by guest on October 1, 2021

inflammation, and inflammatory mediators can be directly neurotoxic thus the modulation of microglia behavior may have important or can attract leukocytes with cytotoxic properties. However, influences on long-term functional outcome in CNS disease or injury microglia are also capable of promoting tissue repair in the CNS; states. Several studies have shown that microglia exhibit patterns of functional differentiation similar to those described for macrophages (52). Microglia express marker proteins associated with specific differentiation states in a number of CNS diseases and disease models (53, 54), demonstrating that microglia are capable of heterogeneous functional differentiation responses to disease-relevant stimuli. Inflammation is a complex response that evolved to restore homeostasis post infection or injury, and microglia are key regu- lators of the inflammatory responses in CNS. However, little is known regarding the endogenous molecular signals that determine which components of the microglia functional repertoire will be expressed in response to a changing neural environment. Identi- fying the molecular signals that control whether microglia exhibit destructive or protective functions is likely to suggest novel thera- peutic targets that may improve functional outcome in diseases FIGURE 7. In vivo CNS inflammation induces activation of miR-155 associated with neuroinflammation, such as Alzheimer’s disease and suppression of c-Maf in microglia. (A) qPCR for miR-155, miR-145, (55, 56), Parkinson’s disease (57), amyotrophic lateral sclerosis and miR-34a expression in microglia extracted by ex vivo flow cyometery from mice that were 3 d (A) after 15-min transient MCAO. miRNA levels (58), multiple sclerosis (1), traumatic brain injury (59), and stroke were normalized to both Sno202 and Sno234 RNAs. *p , 0.001, two-way (60). We previously identified p53 as a transcriptional regulator of ANOVA. (B) qPCR for c-Maf mRNA relative to housekeeping gene ex- microglia behavior (13) and now report that p53 acts through pression in microglia extracted 3 d following 15 min. transient MCAO. miRNA dependent regulation of c-Maf, a known transcriptional *p , 0.001, unpaired t test. regulator of the inflammatory response, as diagramed in Fig. 8. 364 p53 TARGETS c-Maf IN MICROGLIA

lating cellular differentiation (66). c-Maf is a long Maf family member that promotes the differentiation of Th2 lymphocytes (67) as well as expression of F4/80 (mouse homolog of EMR1: epidermal growth factor–like module-containing mucin-like hor- mone receptor–like 1) (68) and an anti-inflammatory cytokine pattern in macrophages (33, 34, 42). It was also demonstrated that c-Maf plays a role in regulating cell fate decision (69). Thus c-Maf transcriptional activity is a critical molecular determinant of func- tional differentiation in two different classes of leukocytes. As a FIGURE 8. Proposed model for p53 modulation of c-Maf expression. critical transcriptional activator of anti-inflammatory cytokine genes, p53-mediated transcriptional activity is required for the induction of p53-dependent miRNAs (miR-145 and miR-34a). These miRNAs target c-Maf must be suppressed during proinflammation and activated a transcriptional regulator, Twist2, for suppression, thereby inhibiting the during anti-inflammation. Oxidative stress has been shown to expression of the anti-inflammatory transcription factor c-Maf. In the absence negatively regulate c-Maf expression, but the precise mechanism by of p53, microglia also fail to induce expression of miR-155, a miRNA that which this occurs has not been determined (69). Our findings sug- directly targets c-Maf. When p53 is activated in microglia by ROS, sponta- gest that repression of c-Maf is one means by which p53 influences neous DNA damage, or cellular stress associated with CNS disease and in- the functional differentiation of microglia. jury, the effectors of such activation work together to suppress the expression of c-Maf, thereby suppressing the anti-inflammation and tissue repair p53 promotes proinflammatory behavior in microglia through behaviors of microglia and making it more likely for microglia to adopt miR-155 proinflammatory behaviors that can be injurious to surrounding neural cells. Downloaded from Initially, miR-155 was identified as a proinflammatory miRNA that contributes to macrophage activation by targeting anti-inflammatory The p53-mediated transcriptional activity is required for the in- genes (4, 22). Recent studies suggest that miR-155 promotes duction of p53-dependent miRNAs (miR-145 and miR-34a). skewing toward proinflammatory behaviors by targeting anti- These miRNAs target a transcriptional regulator, Twist2, for sup- inflammatory genes like the IL-13R (IL13Ra1) for degradation (4). pression, thereby inhibiting the expression of the anti-inflammatory MiR-155alsotargetsSMAD2,whichisinvolvedintheTGF-b http://www.jimmunol.org/ transcription factor c-Maf. In the absence of p53, microglia also fail anti-inflammatory signaling pathway (70, 71). Finally, miR-155 to induce expression of miR-155, a miRNA that directly targets inhibits CEBPb, which was recently shown to be important for the c-Maf. Thus, when p53 is activated in microglia by ROS, sponta- expression of a number of genes associated with anti-inflammatory neous DNA damage, or cellular stress associated with CNS disease or “alternative” activation states, such as Arg1, IL-10, IL13Ra1, and injury, the effectors of such activation work together to suppress and CD206 (72). In microglia, miR-155 has been shown to the expression of c-Maf, thereby suppressing the anti-inflammation downregulate suppressor of cytokine stimulus 1 expression (73), and tissue repair behaviors of microglia and making it more likely which also serves to promote proinflammatory behaviors. In this for microglia to adopt proinflammatory behaviors that can be article, we show that in microglia, miR-155 is induced in a p53- dependent manner by proinflammatory cytokines. We also ob- injurious to surrounding neural cells. by guest on October 1, 2021 served that in the absence of miR-155, c-Maf expression is in- A new role for p53 in promoting a proinflammatory response creased, confirming that the previously reported role for miR-155 P53 is a key modulator of stress responses becoming activated by in lymphocytes is recapitulated in microglia. Thus, in the setting physiological stressors, including but not limited to, DNA damage of oxidative stress (as occurs in chronic ischemia and neurode- (induced by UV, ionizing radiation, or chemical agents such as generative diseases), chronic p53 activation in microglia may hydrogen peroxide), oxidative stress, osmotic shock, ribonucleo- suppress the anti-inflammatory response by promoting expression tide depletion, and deregulated oncogene expression (61, 62). Such of miR-155. In this article, we demonstrate that neuroinflamma- activation is marked by two major events: a dramatic increase in tion caused by brief CNS ischemia leads to activation of p53- the half-life of the p53 protein, and a conformational change in mediated gene expression, induction of miR-155, and sup- the structure that activates p53 transcriptional activity through pression of c-Maf mRNA in microglia. These findings support phosphorylation of its N-terminal domain (63, 64). In microglia, the hypothesis that p53 has a functional impact on an in vivo however, it appears that p53 influences gene transcription even in inflammatory response. the absence of cellular stress. We previously reported that cultured How p53 may regulate miR-155 microglia have demonstrable p53 transcriptional activity under basal conditions (13), and in this article we show that microglia MiRNAs have an established role in regulating inflammatory re- extracted from uninjured adult brain demonstrate differential ex- sponses (23). For example, bic/miR155 plays an important role pression of c-Maf and its regulatory miRNAs. We and others have in the differentiation of B, T, and dendritic cells (36). In this ar- reported that p53 also influences microglia responses to inflam- ticle, we observed that induction of miR-155 expression by IFN-g +/+ matory stimuli, including the regulation of cytokine expression treatment was significantly higher in p53 microglia than in 2/2 (13), inducible NO synthase expression, and TNF-a secretion p53 microglia. This finding suggests that induction of miR-155 (14). Inhibiting p53-mediated transcription in microglia prevented is highly dependent on p53. The mechanism by which p53 reg- neurotoxicity, suggesting that targeting of p53-mediated pathways ulates miR-155 expression has not been determined. It has been in microglia may have therapeutic benefit in CNS injury and reported that p53 can directly regulate the transcription of certain disease exacerbated by an overexuberant inflammatory response. miRNAs, but there is no clear consensus sequence for p53 binding to regulatory elements in proximity to the Bic/miR155 gene. c-Maf transcription factor and its role in anti-inflammatory An alternative mechanism by which p53 may modulate miR-155 responses in CNS expression is that p53 promotes posttranscriptional maturation The Maf family of transcription factors consists of long and short of miRNAs (25). Drosha is a protein that mediates the processing members, as well as a viral oncogene, v-Maf (65). This family of of pri-miRNA transcripts into premiRNAs. 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