The Kat in the HAT: The Histone Acetyl Transferase Kat6b (MYST4) Is Downregulated in Murine Macrophages in Response to LPS
Epigenetic modulators, including histone methylases, demethylases, and deacetylases, have been implicated previously in theregulation of classical and alternative macrophage activation pathways. In this study, we show that the histone acetyl transferase(HAT) Kat6B (MYST4) is strongly suppressed (>80%) in macrophages by lipopolysaccharide (LPS) (M1 activation), whileKat6A, its partner in the MOZ/MORF complex, is reciprocally upregulated. This pattern of expression is not altered by LPStogether with the adenosine receptor agonist NECA (M2d activation). This is despite the observation that miR-487b, a putativeregulator of Kat6B expression, is mildly stimulated by LPS, but strongly suppressed by LPS/NECA. Other members of theMYST family of HATs (Kat5, Kat7, and Kat8) are unaffected by LPS treatment. Using the pLightswitch 3′UTR reporterplasmid, the miR-487b binding site in the Kat6b 3′UTR was found to play a role in the LPS-mediated suppression of Kat6Bexpression, but other as-yet unidentified factors are also involved. As Kat6B is a HAT that has the potential to modulate geneexpression by its effects on chromatin accessibility, we are continuing our studies into the potential roles of this epigeneticmodulator in macrophage activation pathways.
1. Introduction
Macrophages are tissue resident phagocytic cells that playkey roles in immune responses, tissue debridement, angiogenesis, and wound repair following injury. Macrophagestake up residence in developing tissues during embryogenesis, and their presence is maintained throughout the life ofthe animal in the uninjured state by a slow rate of turnover[1–6]. In response to injury or infection, monocytes arerecruited from the circulation by chemoattractants producedat the sites of injury, and these monocytes differentiate intomacrophages [7–9]. Both resident and recruited macrophages respond to local environmental stimuli, which modifythe gene expression profile of these cells to produce cytokines, growth, and angiogenic factors that mediate inflammatory and anti-inflammatory responses, angiogenesis, andtissue repair.The particular phenotype adopted by macrophages inresponse to environmental stimuli depends upon a varietyof factors. Macrophages can be “classically activated” toassume an inflammatory phenotype, characterized by theexpression of cytokines such as TNFα and IL-12, nitric oxide(NO) (via the inducible NO synthase), and proteolyticenzymes. This phenotype has been dubbed “M1” and isinduced, at least in vitro, by interferon-γ (IFN-γ) alone orin combination with endotoxin (lipopolysaccharide (LPS))HindawiMediators of InflammationVolume 2018, Article ID 7852742, 11 pageshttps://doi.org/10.1155/2018/7852742or other Toll-like receptor (TLR) agonists. An “alternativelyactivated” phenotype, canonically termed “M2,” induced byIL-4 and IL-13 exhibits an anti-inflammatory phenotype,characterized by low expression of inflammatory cytokinesand elevated expression of anti-inflammatory cytokinesincluding IL-10, IL-1Rα, and TGFβ, as well as arginase-1(in mice), CD206, and CD163 [10, 11].
Recently, recommendations for the description of macrophage activation havebeen proposed. These proposals clearly recognize theextreme flexibility of macrophage gene expression inresponse to external cues and recognize that the simple useof the term “M2” is misleading. A set of standards encompassing macrophage source, definition of activators, and aconsensus collection of markers to describe macrophage activation was proposed to provide a common framework for thebroad diversity of macrophage phenotypic modulation inresponse to exogenous stimuli [12]. In addition, these proposals recognize that additional modes of activation resultingin modifications of macrophage gene expression profileshave been described that do not conform to the simplifiedM1/M2 paradigm. In our studies, we have defined a pathwayof activation that switches macrophages from an M1 phenotype to an M2-like phenotype that we have previously termed“M2d”, which requires stimulation of TLRs 2, 4, 7, or 9,together with stimulation of adenosine A2A and A2B receptors (A2AR and A2BRs) [13–20]. This M2d phenotype ischaracterized by low expression of inflammatory cytokines,elevated expression of anti-inflammatory cytokines includingIL-10, upregulated expression of A2ARs and A2BRs, andstrongly upregulated expression of VEGF. The M2d pathwayof activation is independent of IL-4 and IL-13 and does notdepend upon either IL-10 or IL-6 stimulation [16].In our studies of the signaling pathways involved in macrophage activation, we carried out a detailed profiling ofmicroRNA (miRNA) expression by murine macrophagesactivated by LPS (M1 activation) and LPS together with theadenosine A2R agonist 5′-N-ethylcarboxamidoadenosine(NECA) (“M2d” activation). MiRNAs are single-strandedRNAs composed of 21–23 nucleotides. These RNAs mayfunction as posttranscriptional regulators of gene expressionby binding to the 3′ untranslated region (3′UTR) of mRNAs.Each miRNA contains a seeding region that plays a key rolein target binding and repression of gene expression [21].Repression of expression may occur at the translationallevel or by promoting mRNA degradation [22, 23].
Priorstudies have examined the effects of LPS on miRNAexpression in macrophages [24–33]. MiRNAs includingmiR-155, miR-146a, and miR-210 have previously beenshown to be involved in regulating the expression of cytokines such as TNFα, IL-6, and IL-10 [34–38]. In the studypresented here, we identified a limited subgroup of miRNAsthat were found to be regulated in response to LPS/NECA incomparison to LPS alone (Table 1). We initially chose toexamine the role of miR-487b, which we found to beupregulated by LPS, but strongly downregulated by the combination of LPS and NECA (M2d activation). Bioinformaticanalysis indicated a limited group of genes with conserved3′UTR binding sites for miR-487b, including the histone acetyl transferase (HAT) Kat6b (MYST4), which is the focus ofthis manuscript. Studies of other miRNAs differentially modulated by LPS/NECA are currently underway.Given the potential importance of epigenetic modifications in the regulation of macrophage differentiation andactivation, we examined the expression in macrophages ofKat6b and other members of the MYST family of genes inresponse to LPS (M1 activation) and LPS/NECA (M2d activation). Strong and sustained suppression of Kat6b wasobserved in response to LPS. In addition, MYST3 was reciprocally upregulated by LPS, while MYST1, MYST2, andMYST5 were only marginally affected. We also examinedthe role of miR-487b in regulating the expression of Kat6b(MYST4). Our results suggest that the miR-487b site in the3′UTR of the Kat6b gene may play a role in regulating theexpression of the Kat6b gene, but that other, as-yet unidentified pathways regulated by LPS also contribute to the LPSmediated suppression of Kat6b gene expression.
2. Materials and Methods
Thioglycolateinduced peritoneal macrophages were prepared as previously described. Briefly, C57BL/6J male mice (8 weeks ofage, Jackson Laboratories, Bar Harbor, Maine) were injectedintraperitoneally (ip) with thioglycolate broth (3.5 ml). Fourdays later, mice were sacrificed by cervical dislocation,injected ip with 3.5 ml sterile PBS, and the peritoneal cavityexudate was then harvested. The cells were pelleted, washed(3x) with PBS, resuspended in RPMI 1640 medium (Sigma,Table 1: MiRNAs differentially regulated in murine peritonealmacrophage by LPS (M1 activation) versus LPS/NECA (M2dactivation).2 Mediators of InflammationSt. Louis, MO) containing 10% heat-inactivated fetal bovineserum (FBS), (Atlanta Biologicals, Lawrenceville, GA), 1%Pen-Strep 100x Solution, and 2% L-glutamine 200 mM Solution (Sigma), and plated at a density of 8 × 106 cells per100 mm dish. The cells were incubated at 37°C in a 5%CO2 tissue culture incubator overnight. Nonadherent cellswere removed by washing, and the medium was replacedwith RPMI 1640-1% FBS. The adherent cells, consisting of>95% macrophages, were then treated as follows: (a) stimulated with 100 ng/ml E. coli LPS (TLR4 agonist, purified tobe free of TLR2 agonists, gift of Dr. Stefanie Vogel, University of Maryland), (b) stimulated with LPS together withNECA (1 μM), (c) stimulated with NECA alone (1 μM),and (d) left unstimulated as a control group. Macrophagesin each group were treated for 3, 6, 12, and 24 hours, andthen total RNA was isolated and harvested.Isolation of RNA. The cells from each plate were scrapedin TRIzol Reagent (Invitrogen), and the RNA was isolatedusing the Zymo Research Corporation’s isolation procedure.Ethanol (100%) was first added in a 1 : 1 volume ratio to thehomogenate samples in TRIzol and vortexed. The samplewas then loaded onto Zymo-Spin™ IIC Columns and treatedwith DNase I cocktail to remove DNA from the column.
The columns were washed (2x) with Direct-zol™ followed byRNA Wash Buffer and the flow-through discarded. TheZymo-Spin IIC Column was then transferred to an RNasefree tube, and 50 μl of DNase/RNase free water was addedto elute the RNA, which was then stored at −80°C.The RNA concentration for each sample wasdetermined using a NanoDrop 2000c spectrophotometer(Thermo Fisher Scientific, Waltham, MA). For cDNA preparation, reverse transcription was performed using TaqManReverse Transcription Reagents (Applied Biosystems/LifeTechnologies, Grand Island, NY), and all incubations wereperformed in a C1000 Thermo Cycler (Bio-Rad, Hercules,CA). Q-RT-PCR reactions were set up to determine theexpression of MYST 1, 2, 3, 4, and 5 RNA at each time pointin the differently treated cells. A 96-well plate obtained fromApplied Biosystems/Life Technologies (Grand Island, NY)was prepared for each MYST gene and for cyclophilin-D asan endogenous control. Each experiment was performed 3times, and the reactions were in duplicate for each sample.For the gene of interest, the TaqMan probe and primer mixture were diluted to a 1 : 20 ratio. The TaqMan probes andprimers used in these experiments were purchased fromApplied Biosystems and are shown in Table 2. To each reaction, the following components were added: 1 μl of 20x TaqMan probe and primer assay mixture, 10 μl of 2x TaqManUniversal PCR Master Mix (both from Applied Biosystems),cDNA template (5 μl), and water for a final volume of 20 μl.Additionally, a control with no template was included inthe experiment. Q-RT-PCR reactions were performed usingan ABI 7000 Real Time PCR Thermocycler. Fold expressionwas normalized to that of unstimulated macrophages usingthe ΔΔCt method.
All results were also normalized to theexpression of cyclophilin-D, shown in previous studies tobe constitutively expressed and minimally regulated by LPS.Western Blot Analysis. Macrophages were lysed byadding the radioimmunoprecipitation assay (RIPA) buffercontaining complete protease inhibitor cocktail (539134,Calbiochem, Billerica, MA). Samples were centrifuged at10,000g for 10 minutes, and an aliquot was used for aBradford-based protein determination. Cell lysates wereboiled for 5 minutes with SDS-Laemmli buffer, and aliquots containing 50 μg of protein were loaded onto 7.5%SDS-polyacrylamide gels for electrophoresis. Following electrophoresis, proteins were transferred to nitrocellulosemembranes (Protran, Whatman, Dassel, Germany) using aBio-Rad wet transfer system, according to the manufacturer’sinstructions. The membranes were then blocked with 5%low-fat milk in Tris-buffered saline with 0.1% Tween 20 for1 hour, washed, and then incubated overnight at 4°C withan anti-Kat6b primary antibody (Novus Biologicals, Littleton, CO), or with an anti-nucleophosmin (NPM) primaryantibody (Abcam, Cambridge, MA). The blots were thenwashed with Tris-buffered saline containing 0.1% Tween 20and incubated for 1 hour with HRP-conjugated secondaryIgG. Immunoreactive bands were developed using a chemiluminescent substrate, ECL Plus (GE Healthcare, Piscataway,NJ), and protein bands were detected by using a FluorChemanalyzer (San Jose, CA).Analysis of the Role of miRNA-487b in the Regulationof Kat6b Expression. To determine the role of miR-487b inthe regulation of Kat6b expression, two approaches wereused. The first used the 3′UTR of the Kat6b gene cloneddownstream of luciferase in a reporter plasmid, transfectedinto the RAW264.7 macrophage cell line. This 3′UTR contains a putative miR-487b binding site (Figure 1). A mutatedTable 2MYST family members(with alternative names)TaqMan primer/probeserial numberMYST1 (Kat8, MOF) Mm00458911_m1MYST2 (Kat7, ORC1, and HBO1) Mm00624391_m1MYST3 (Kat6A, MOZ, ZNF220,and RUNXBP2 Mm01211941_m1MYST4 (Kat6b, MORF, and Querkopf) Mm00450564_m1MYST5 (Kat5, TIP60, and HTATIP) Mm01231512_m1CCTTTGAAGAGTACGATTTCAAAACCAGFigure 1: Region of the Kat6b 3′UTR sequence containing theputative miR-487b conserved binding site, indicated in bold. Thecomplete 3′UTR (1995 bases) was inserted into the pLightswitch 3′UTR luciferase reporter plasmid, using the NHE1/XHO1restriction sites, generating the pKat6bLuc-3′UTR plasmid(SwitchGear Genomics, Carlsbad, CA, Product ID S810637).
Fivebases (TACGA) were deleted from the miR-487b binding site togenerate the mutant clone (pKat6bLucΔ3′UTR).Mediators of Inflammation 33′UTR lacking this miR-487b binding site was also tested(Figure 1). The second approach studied the effects of synthetic miR-487b mimics cotransfected into RAW264.7 cellswith 3′UTR reporter plasmids, to determine the effects ofmiR-487b overexpression.To determine whether miR-487b targets the expressionof Kat6b through an effect on the putative miR-487b targetsite in the 3′UTR of the Kat6b gene, RAW264.7 cells weretransiently transfected with one of the following pLightswitch 3′UTR luciferase reporter plasmid clones (SwitchGearGenomics, Carlsbad, CA): (a) pKat6bLuc-3′UTR, which contains the 3′UTR of the Kat6b gene cloned downstream of theluciferase gene in the pLightswitch-3′UTR plasmid, and alsocontains the RPL10 promoter 5′ of the luciferase insert. TheRPL10 promoter is a constitutive promoter that is only minimally affected by LPS; (b) pKat6bLucΔ3′UTR, which contains the Kat6b 3′UTR with a specific deletion of theputative miR-487b binding site (Figure 1); (c) pEmptyLuc,which is the pLightswitch plasmid without a 3′UTR insert;and (d) pGAPDHLuc-3′UTR, which is a control constructwith the GAPDH wild-type 3′UTR cloned downstream ofluciferase. Expression of luciferase from this plasmid is unaffected by LPS treatment.To determine whether the 3′UTRs were affected by thevarious treatments, RAW264.7 cells were transfected with5 μg of each plasmid using LipoD (SignaGen Laboratories,MD) according to the manufacturer’s protocol for 18 hours.The cells were then replated in six-well plates (0.5 × 106 cellsin 1.5 ml of RPMI-10% FBS) and incubated at 37°C in the 5%CO2 tissue culture incubator for 24 hours.
The medium waschanged to RPMI 1640-1% FBS, and the cells were then stimulated with LPS (100 ng/ml), NECA (1 mM), and LPS/NECAor were left untreated. The plates were then incubated at 37°Cfor 6 hours, and the cells were then washed with PBS, lysedusing Passive Lysis Buffer (Promega, Madison, WI), andassayed for firefly luciferase activity for each transfection.Changes in luciferase activity were determined by comparingthe normalized luciferase activities of each of the test luciferase reporter constructs that are treated versus untreatedtransfection. Each treatment group was performed in triplicate, and each experiment was performed in duplicate.The effects of a synthetic mimic (Qiagen, syn-mmu-miR487b-3p miScript miRNA mimic, cat. number MSY0003184)on the expression of luciferase from the Kat6b 3′UTR plasmids alone and in response to LPS, NECA, and LPS/NECAwere examined. A miR-433 mimic was used as a nonspecificcontrol. The miR-487b and 433 mimics were transfected intoRAW264.7 cells for 6 hours at a final concentration of 50 nMusing HiPerFect (Qiagen), as described by the manufacturer.Following mimic delivery, 5 μg of either pKat6bLuc-3′UTR,pKat6bMYST4Δ 3′UTR, or pEmptyLuc vectors was transfected into the RAW264.7 cells using LipoD (SignaGen Laboratories, MD), and the cells were processed and treated withLPS, NECA, and LPS/NECA as described above.Statistical analysis was performedwith the unpaired Student t-test or analysis of variancefollowed by Tukey multiple comparison test. A p value <0.05 was used to indicate statistical significance. 3. Results miR-487b Expression Is Regulated in MurineMacrophages by LPS and LPS/NECA. In prior studies of theresponse to LPS (M1 activation) and LPSA/NECA (M2d activation), mi-RNA profiling analysis demonstrated a subgroupof miRs that were differentially regulated in response to LPSand LPS/NECA. These miRs included miR-877, miR-377-5p,miR-546, and miR-494, which were upregulated by LPS/NECA in comparison to LPS or NECA alone, and miR487b, miR-212, miR-220, and miR-712, which were downregulated by LPS/NECA in comparison to LPS or NECAalone (Table 1).Q-RT-PCR analysis was used to confirm the regulationof miR-487b expression by LPS and LPS/NECA (Figure 2).LPS (100 ng/ml) upregulated miR-487b expression by ~2-fold. NECA alone had no effect, but NECA together withLPS strongly suppressed miR-487b expression in macrophages by ~80% in comparison to unstimulated macrophages. As miR-487b was upregulated by LPS (~2 fold)and strongly suppressed by LPS/NECA, we speculated thatthis miR might play a role in the switch of macrophagesfrom an M1 to an M2d phenotype. Bioinformatic analysisof potential targets of miR-487b using TargetScan® andhttp://www.mirdb.org identified several genes with putativemiR-487b target sites that are conserved across mammalian species. Kat6b was identified in these analyses as oneof a small group of genes that are likely potential targetsfor miR-487b (Table 3).4 Mediators of InflammationKat6b Expression Is Suppressed in Murine Macrophagesby LPS. Kat6b is a histone acetyl transferase (HAT) that playsan important role in modifying histones and transcriptionfactors and thus is involved in regulating gene expression.We initially analyzed the effect of LPS and LPS/NECA onthe expression of Kat6b by macrophages in response toLPS using Q-RT-PCR. The results of this analysis areshown in Figure 3(a). Kat6b mRNA was strongly and rapidly suppressed (>80%) by LPS treatment of murine macrophages within 3 hours of treatment. This suppression wasmaintained through 12 hours following LPS treatment, withKat6b mRNA levels still showing ~50% suppression by 24hours. As shown in Figure 3(a), treatment with NECA, anadenosine A2A and A2B receptor agonist, did not significantly change this expression pattern. In addition to suppressing Kat6b mRNA, LPS also suppressed Kat6b proteinexpression. Figure 4 shows a Western blot of Kat6b proteinexpression in macrophages after 8 and 20 hours of treatment with or without LPS. Nucleophosmin (NPM) wasused as a housekeeping gene whose expression is notaltered in response to LPS, and Kat6b levels were normalized to NPM expression. Kat6b protein expression wasclearly suppressed in the LPS-treated macrophages. Epigenetic modulation of chromatin structure through regulationof histone demethylases and histone acetyl deacetylases(HDACs) has been implicated previously in classical (M1)and alternative (M2) pathways of macrophage activation.The potent suppression of the HAT Kat6b by LPS observedhere suggests the potential importance of this gene in the regulation of macrophage phenotype.Differential Regulation of MYST Family Genes inMurine Macrophages in Response to LPS. As expressionof the HAT Kat6b was strongly suppressed in responseto LPS, we investigated the expression of additional members of the MYST family of genes (MYSTs 1–5) to determine the specificity of this LPS-mediated suppression ofKat6b. All members of this family contain a MYST regionof about 240 amino acids with a canonical acetyl-CoAbinding site and a C2HC-type zinc finger motif. Table 2 summarizes the nomenclature of the MYST family genes and theQ-RT-PCR primer/probes used to analyze their expression.The results of this analysis are shown in Figures 3(b)–3(e).MYST1 mRNA was not significantly regulated by LPStreatment (Figure 3(b)). Similarly, the levels of MYST2(Figure 3(c)) and MYST5 (Figure 3(d)) mRNAs remainedfairly constant through 24 hours following LPS treatment.
In contrast, MYST3 mRNA levels were significantly elevated at 3, 6, and 12 hours following LPS treatment, witha 3-4-fold increase in expression, returning to baseline by24 hours following LPS stimulation (Figure 3(e)). Treatmentof macrophages with NECA alone did not affect the expression of any MYST gene. Also, treatment of macrophages withLPS together with NECA (LPS/NECA) did not alter the LPSinduced modulation of MYST gene expression. It thus appears that Kat6b and MYST3 are reciprocally regulatedin response to LPS, with Kat6b being strongly suppressed,while MYST3 is induced. Role of the Putative miR-487b Binding Site in theKat6b 3′UTR in the Regulation of Kat6b Expression. Todetermine the role of the putative miR-487b binding site inthe Kat6b 3′UTR in regulating Kat6b expression in responseto LPS, we cloned the Kat6b 3′UTR into the pLightswitch3′UTR plasmid downstream of the luciferase open readingframe, to generate the pKat6bLuc-3′UTR reporter plasmid.A second plasmid with the miR-487b binding site deleted, designated as pKat6bLucΔ3′UTR, was also engineered.These plasmids were transfected into RAW264.7 macrophages, which were then treated with LPS and examined for luciferase activity. As shown in Figure 5, LPS stimulation of RAW264.7 cells transfected with pKat6bLuc-3′UTRinduced a ~35% decrease in luciferase activity, while in cells transfected with pKat6bLucΔ3′UTR, LPS induced a ~24%decrease in luciferase activity (p < 0 05). This suggests that the 3′UTR containing the miR-487b binding site plays aminor role in the suppression of the LPS-induced Table 3: Transcripts with conserved 3′UTR sites for miR-487b.Gene name Gene symbol Conserved sites 8-mer 7-merChemokine Z (C-X-C motif) ligand 12 CXC 12 1 1NOTCH-regulated ankyrin repeat protein NRARP 1 1Astrotactin-1 ASTN1 1 1Nasal embryonic LHRH factor NELF 1 1K(Lysine) acetyl transferase 6B KAT6B 1 1Protein kinase C, alpha PRKCA 1 1EF-hand domain family, member D2 EFHD2 1 1Mitogen-activated protein kinase kinase 4 MAP2K4 1 1Zinc finger protein 219 ZNF219 1 1Ring finger protein 165 RNF165 1 1Protocadherin 7 PCDH7 1 1EPH receptor A3 EPHA3 1 1Insulin receptor substrate 1 IRS1 1 1POU class 2 homeobox 1 POU2F1 1 1Mediators of Inflammation 5expression of luciferase, as the loss of the miR-487b binding site results in only a small increase in luciferaseexpression in response to LPS. This indicates that otherfactors in addition to miR-487b must be involved, asLPS still induces significant suppression in the absence ofthe miR-487b binding site.To determine the effect of overexpression of miR-487bon the expression of luciferase from the wild-type andmutant reporter plasmids in the presence or absence ofLPS, RAW264.7 macrophages were cotransfected withpKat6bLuc-3′UTR or pKat6bLucΔ3′UTR together with asynthetic miR-487b mimic or a nonspecific miR mimic(miR-433). As shown in Figure 5, the synthetic miR-487bmimic markedly suppressed luciferase activity from thewild-type vector (~35% suppression), while the nonspecificmimic had little effect. The effect of the miR-487b mimicwas lost in the mutant vector. These results support thepotential role of miR-487b to regulate Kat6b expressionthrough binding to its binding site in the Kat6b 3′UTR. However, as is also shown in Figure 5, LPS still induced suppression of luciferase from the mutant vector, and this was notsignificantly affected by the miR-487b mimic. This findingsuggests that while miR-487b can play a role in regulatingexpression, factors other than miR-487b must also play a rolein the LPS-induced suppression. 4. Discussion Macrophages play key roles in inflammation, wound healing,angiogenesis, and immune responses. Resting macrophagesregulate the maintenance of tissue integrity but, in responseto inflammatory stimuli, change their gene expression profileto produce inflammatory or anti-inflammatory cytokines,growth, and angiogenic factors. The rapid and profoundchanges in the expression of genes in macrophages are mediated at several levels, including transcriptional control, aswell as posttranscriptional regulation of translation andmRNA and protein stability.Classical (“M1”) activation of macrophages is induced byIFN-γ and/or TLR agonists such as LPS (TLR4 agonist) andis characterized by the rapid and transient induction ofinflammatory cytokines such as TNFα and IL-12 andNOS-3 (iNOS). In contrast, alternative activation pathwaysthat induce an anti-inflammatory phenotype have beendescribed. These have generally been termed “M2” macrophages [11, 39]. Activation by IL-4, for example, induces ananti-inflammatory phenotype termed M2a, characterized bylow expression of inflammatory cytokines and elevatedexpression of the anti-inflammatory cytokine IL-10, IL-1Rα,as well as markers such as CD206 (MR), CD163, MHCII,Ym1, FIZZ-1, and arginase-1 [39–41]. We have previouslydescribed an “alternatively activated” macrophage phenotypethat we have termed “M2d” [15, 16, 42]. This phenotype isinduced by TLR2, 4, 7, and 9 agonists in a MyD88-dependent manner, in synergy with agonists of adenosineA2A and A2B receptors [15, 18–20]. M2d macrophagesexpress low levels of inflammatory cytokines and high levelsof IL-10 and the angiogenic growth factor VEGF. Inductionof this phenotype involves transcriptional upregulation ofHIF1α and posttranscriptional suppression of phospholipase-Cβ2 (PLCβ2) [42, 43].To determine the mechanism of PLCβ2 suppression inresponse to M2a activation, we performed a global screeningof miRNAs expressed in response to LPS, to NECA (an ARagonist), and to LPS together with NECA (M2d activationC8 L8 C20 L20Kat6b (196 kDa)NPM (39 kDa)3′UTR of the Kat6b mRNA. The Kat6b 3′UTR was cloneddownstream of the luciferase open reading frame in thepLightswitch-3′UTR reporter plasmid (pKat6bLuc-3′UTR). Asecond plasmid was prepared with the miR-487b binding sitedeleted (pKat6bLucΔ3′UTR). RAW264.7 macrophages weretransfected with either pKat6bLuc-3′UTR or pKat6bLucΔ3′UTR.Transfected cells were then treated with LPS for 6 hr (100 ng/ml)and analyzed for luciferase expression (n = 3). RAW264.7 cellswere also cotransfected with either pKat6bLuc-3′UTR orpKat6bLucΔ3′UTR together with either a synthetic miR-487bmimic or a nonspecific miR mimic (miR-433). The cells were thentreated with LPS (100 ng/ml for 6 hr and analyzed for luciferaseexpression (n = 3). Data represent the mean ± SE. ∗ indicatessamples with luciferase expression significantly different fromcontrol luciferase expression (p < 0 05).Mediators of Inflammation 7conditions). MiRNAs that are regulated by LPS have beenpublished in prior studies [24, 33, 36, 44–47]. In the currentstudy, miRNAs specifically modulated by LPS with NECAversus LPS alone were identified. As shown in Table 3, a subgroup of miRNAs was either up- or downregulated inresponse to LPS/NECA versus LPS alone. We confirmedthe changes in expression of miR-487b, which was found tobe mildly induced by LPS, but strongly suppressed by LPSwith NECA (Table 3 and Figure 2). Bioinformatic analysisof potential targets of miR-487b using TargetScan andhttp://www.mirdb.org identified a group of genes with putative miR-487b target sites conserved across mammalian species (Table 3). The HAT Kat6b was identified in theseanalyses as one of a small group of genes that are potentialtargets for miR-487b.There has been much interest recently in the role ofepigenetic modulators in the regulation of macrophageactivation pathways [48–53]. In particular, chromatinremodeling induced by targeted epigenetic modificationssuch as histone methylation or demethylation, as wellas acetylation or deacetylation, may lead to gene activation or repression [40, 54]. Histone deacetylases(HDACs) have been shown to play an important rolein macrophage M1 and M2 activation; however, the roleof histone acetyl transferases (HATs) in regulating macrophage activation has received little attention. HATsand HDACs are families of enzymes that modulate chromatin structure, thus affecting inflammatory gene expression[55, 56]. Mice lacking HDAC3 display a polarizationphenotype similar to IL-4 induced alternative activationand are hyperresponsive to IL-4 stimulation, suggesting thatHDAC3 is an epigenomic brake in macrophage M2a activation [53, 57–59]. By extension, this would suggest that HATsmight provide a stimulus to M2 activation, in contrast to theeffects of HDACs. However, the role of HATs in regulatingmacrophage M1/M2 polarization remains to be determined.HAT complexes of the MYST family are named after thefour founding family members, MOZ, Ybf2 (Sas3), Sas3, andTip60 [60, 61]. Other members of this family include Esa1,MOF, MORF, MSL, and HBO1 (Table 2). MYST familyHATs are typically characterized by the presence of zincfingers and chromodomains and are involved in acetylationof lysine residues on histones H2A, H3, and H4 [62–64].As the HAT Kat6b was identified in this study as a potentialtarget of miR-487b, we examined the effects of LPS on theexpression of Kat6b and also of the other members of theKat family of HATs (Kat6a, Kat5, Kat7, and Kat8). Kat6aand Kat6B form stable multisubunit complexes, MOZ andMORF, respectively [65]. The MOZ/MORF complex isresponsible for acetylation of a substantial portion of histoneH3, and possibly of other histones. The HAT activity of theMOZ/MORF complex is required for normal development,including hematopoiesis and skeletogenesis. Mutations ofKat6B have been identified in patients with Say-BarberBiesecker syndrome and with genitopatellar syndrome[66–68]. In a form of acute myeloid leukemia, there is atranslocation of the N-terminal portion of Kat6b in framewith CBP [62]. A translocation resulting in fusion to TAFIIalso leads to acute myeloid leukemia [63]. Disruption ofKat6b also leads to a Noonan syndrome-like phenotypeand hyperactivated MAPK signaling in both humans andmice [69]. Mutant mice deficient in Kat6b are reported todevelop poorly, exhibiting growth retardation, facial dysmorphism, skeletal abnormalities, and developmental brainanomalies, leading to their designation as “Querkopf”(“Strange head”) mice [69]. No studies on the inflammatoryand immunological responses of these mice have beenreported to date.Since Kat6B (MYST4, MORF) was identified as apotential target of miR-487b (Figure 1), we studied theexpression of Kat6b, as well as the other members ofthe MYST family of HATs, in the response of macrophages to LPS (M1) and LPS/NECA (M2d) activation.As shown in Figure 3, LPS induced a rapid, strong, andsustained suppression of Kat6b mRNA expression. Strongsuppression (>80%) was observed by 3 hours followingLPS treatment and sustained through at least 12 hours.After 24 hours, 50% suppression was still apparent. Incontrast, Kat6A (MYST5, MOZ) expression was stimulated by LPS and showed a reciprocal pattern of expression to that of Kat6A. The other members of the MYSTfamily (Kats 5, 7, and 8) were not affected by LPS treatment. Surprisingly, the expression patterns of Kat6A andKat6B in response to LPS/NECA were the same as thosewith LPS alone, despite the fact that miR-487b expressionis strongly suppressed by LPS. We propose in the light ofpublished literature implicating HDACs in M2 activation[53, 57–59] that the strong downregulation of the HATKat6b induced by LPS may play a reciprocal role withHDACs in regulating macrophage polarization. We arecurrently testing this hypothesis.To determine the role of miR-487b in the regulation ofKat6B suppression by LPS in macrophages, we cloned theintact Kat6B 3′UTR and a mutated 3′UTR lacking themiR-487b core binding sequence into a luciferase reporterplasmid. These plasmids were transfected into the macrophage cell line RAW264.7 either alone or together with amiR-487b mimic. LPS suppressed luciferase expression inthe intact plasmid, and this suppression was only mildlyabrogated by loss of the miR-487b binding site; however,the synthetic miR-487b mimic markedly suppressed luciferase activity from the wild-type vector, while the nonspecific mimic had little effect (Figure 5). The suppressiveeffect of the miR-487b mimic was lost in the mutant vector. Together, these results suggest that while the miR487b site in the Kat6B 3′UTR plays a role in the LPSmediated suppression of Kat6B, other factors in additionto miR-487b must also contribute. As LPS/NECA stronglysuppresses miR-487b expression in comparison to LPS alone,the lack of effect of LPS/NECA on the expression of Kat6Balso clearly suggests that miR-487b is not the sole factorinvolved in the suppression of Kat6B by LPS. In this context,it is of interest to note that miR-487b was recently implicatedas a negative regulator of bone marrow-derived macrophageactivation by targeting IL-33 production. miR-487b suppressed IL-33 production during the differentiation of bonemarrow-derived macrophages by binding to the 3′UTR of8 Mediators of InflammationIL-13 mRNA and suppressing its translation [70]. Nevertheless, the role of miR-487b modulation of macrophage M1/M2polarization remains unclear.
In summary, we have found that the HAT Kat6B is strongly suppressed in macrophages by LPS (M1 activation), while Kat6A is reciprocally upregulated. This pattern is not altered by LPS/NECA (M2d activation), despite the observation that miR-487b, a putative regulator of Kat6B expression, is strongly suppressed by LPS/NECA. As Kat6B is a HAT WM-1119 that has the potential to modulate gene expression by its effects on chromatin accessibility, we are continuing our studies into the potential roles of this epigenetic modulator in macrophage activation pathways.