of March 17, 2018.This information is current as

Distinct Lineages of Lung Dendritic Cells Expression inCcr7Epigenetic Control of

Kondilis-Mangum, Paul A. Wade and Donald N. CookTimothy P. Moran, Hideki Nakano, Hrisavgi D.

ol.1401104http://www.jimmunol.org/content/early/2014/10/07/jimmun

published online 8 October 2014J Immunol 

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The Journal of Immunology

Epigenetic Control of Ccr7 Expression in Distinct Lineages ofLung Dendritic Cells

Timothy P. Moran,*,1 Hideki Nakano,† Hrisavgi D. Kondilis-Mangum,‡ Paul A. Wade,‡ and

Donald N. Cook†

Adaptive immune responses to inhaled allergens are induced following CCR7-dependent migration of precursor of dendritic cell

(pre-DC)–derived conventional DCs (cDCs) from the lung to regional lymph nodes. However, monocyte-derived (moDCs) in the

lung express very low levels of Ccr7 and consequently do not migrate efficiently to LN. To investigate the molecular mechanisms

that underlie this dichotomy, we studied epigenetic modifications at the Ccr7 locus of murine cDCs and moDCs. When expanded

from bone marrow precursors, moDCs were enriched at the Ccr7 locus for trimethylation of histone 3 lysine 27 (H3K27me3),

a modification associated with transcriptional repression. Similarly, moDCs prepared from the lung also displayed increased levels

of H3K27me3 at the Ccr7 promoter compared with migratory cDCs from that organ. Analysis of DC progenitors revealed that

epigenetic modification of Ccr7 does not occur early during DC lineage commitment because monocytes and pre-DCs both had low

levels of Ccr7-associated H3K27me3. Rather, Ccr7 is gradually silenced during the differentiation of monocytes to moDCs. Thus,

epigenetic modifications of the Ccr7 locus control the migration and therefore the function of DCs in vivo. These findings suggest

that manipulating epigenetic mechanisms might be a novel approach to control DC migration and thereby improve DC-based

vaccines and treat inflammatory diseases of the lung. The Journal of Immunology, 2014, 193: 000–000.

Themigration of lung dendritic cells (DCs) to lymph nodes(LN) is critical for orchestrating adaptive immune re-sponses against inhaled Ags or microbes (1–4). Mobili-

zation of lung DCs to LN is dependent on the chemokinereceptor CCR7 and its ligands, CCL19 and CCL21, which areproduced in afferent lymphatic vessels and the T cell zones of LN(5). Under homeostatic conditions, CCR7-dependent migration oflung DCs to draining LN is important for the induction of regu-latory T cells, which help maintain peripheral tolerance to in-nocuous environmental Ags (6). During infection or otherinflammatory insults, migratory DCs become licensed to inducecytotoxic T cells or effector Th cell differentiation (7). Theseeffector T cells not only assist with pathogen clearance but canalso mediate maladaptive immune responses leading to inflam-matory lung diseases such as asthma (3, 4, 8). Because migratory

DCs play a central role in shaping immune responses in the lungs,manipulating Ccr7 expression is a potential strategy for eitheraugmenting or suppressing adaptive immunity. However, sucha strategy requires an improved understanding of the molecularmechanisms responsible for regulating Ccr7 expression.As in other nonlymphoid tissues, DCs in the lungs are composed

of two major lineages: conventional DCs (cDCs) and monocyte-derived DCs (moDCs) (9, 10). Lung cDCs arise from circulatingprogenitor cells referred to as precursor of DCs (pre-DC), whichdescend from the common DC progenitor in the bone marrow(BM) via a pathway dependent on Flt3 ligand (FL) (11). In con-trast, lung-resident moDCs develop from emigrating peripheralblood monocytes independently of FL (10). There is growingevidence that DC functional specialization is dependent on theirdevelopmental lineage (12). In agreement with this, we recentlyreported that lung cDCs and moDCs differ significantly in theirability to express Ccr7 and migrate to regional LN (10). Duringsteady-state conditions, Ccr7 is expressed at low levels in lungcDCs and is markedly upregulated by microbial products, such asLPS and polyinosinic:polycytidylic acid [poly (I:C)]. By contrast,moDCs in the lung fail to express Ccr7, even after treatment withthese microbial products. Consequently, cDCs, but not moDCs canefficiently ferry Ag from the lung to regional LN. These findingsare in agreement with studies showing that moDCs from otherperipheral tissues, such as the skin and gut, also express low levelsof Ccr7 and migrate poorly to LN (13, 14). Taken together, thesestudies suggest that the capacity for peripheral DCs to expressCcr7 and migrate to LN is regulated in a lineage-specific manner.The unique transcriptional profiles of DC subsets are likely

important for dictating their functional and migratory properties.Although the transcriptomes of DC subsets are clearly dependenton lineage-specific transcription factors (15), epigenetic mecha-nisms are also essential for regulating gene expression duringDC development (16). Epigenetic mechanisms, which includeDNA methylation and posttranslational modifications of histonetails, regulate gene expression by governing chromatin accessi-bility to the transcriptional machinery (17). Although epigenetic

*Department of Pediatrics, Duke University Medical Center, Durham, NC 27710;†Laboratory of Respiratory Biology, National Institute of Environmental HealthSciences, National Institutes of Health, Research Triangle Park, NC 27709; and‡Laboratory of Molecular Carcinogenesis, National Institute of Environmental HealthSciences, National Institutes of Health, Research Triangle Park, NC 27709

1Current address: Department of Pediatrics, Division of Allergy, Immunology andRheumatology, University of North Carolina, Chapel Hill, NC

Received for publication June 5, 2014. Accepted for publication September 8, 2014.

This work was supported by National Institutes of Health Grant 5 T32 AI 007062-34and by the Intramural Research Program of the National Institutes of Health and theNational Institute on Environmental Health Sciences.

Address correspondence and reprint requests to Dr. Donald N. Cook, National Insti-tute of Environmental Health Sciences, National Institutes of Health, 111 T.W.Alexander Drive, Building 101, Room E244, Research Triangle Park, NC 27709.E-mail address: cookd@niehs.nih.gov

The online version of this article contains supplemental material.

Abbreviations used in this article: BM, bone marrow; cDC, conventional DC; ChIP,chromatin immunoprecipitation; DC, dendritic cell; FL, Flt3 ligand; H3K27me3,histone 3 lysine 27 trimethylation; LN, lymph node; moDC, monocyte-derivedDC; NIEHS, National Institute of Environmental Health Sciences; poly (I:C), poly-inosinic:polycytidylic acid; PRC2, polycomb repressive complex 2; pre-DC,precursor of DC; QPCR, quantitative PCR.

Copyright� 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401104

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modifications of multiple genes have been well studied in thecontext of Th cell differentiation (18–20), remarkably little isknown of epigenetic mechanisms that might be expected to in-fluence DC development or function (16). In the current study, weinvestigated whether epigenetic modifications at the Ccr7 locusinfluence the migratory properties of different DC subsets. Wefound that moDCs have increased repressive histone modificationsat the Ccr7 locus, which was associated with decreased Ccr7expression and a nonmigratory phenotype. Surprisingly, theserepressive epigenetic marks are not established during lineagecommitment within the BM but rather during monocyte differ-entiation into moDCs in peripheral tissues. Our findings suggestthat epigenetic modifications at the Ccr7 locus closely correlatewith the migratory properties, and hence the functional roles, ofnonlymphoid DC subsets. The transcriptional silencing of Ccr7during moDC differentiation might also have implications for thedevelopment of effective DC-based vaccines for immunotherapy.

Materials and MethodsMice

C57BL/6J and OT-II (C57BL/6-Tg(TcraTcrb)425Cbn/J) mice were pur-chased from The Jackson Laboratory (Bar Harbor, ME). Ccr7gfp reportermice were generated in our laboratory and have been described previously(10). Mice were housed in specific pathogen-free conditions at the NationalInstitute of Environmental Health Sciences (NIEHS) and used between 6and 12 wk of age in accordance with guidelines provided by the Institu-tional Animal Care and Use Committees.

Flow cytometric analysis

Cells were incubated with a nonspecific-binding blocking reagent mixtureof anti-mouse CD16/CD32 (2.4G2) and normal mouse and rat serum(Jackson ImmunoResearch Laboratories, West Grove, PA) for 5 min. Forstaining of surface Ags, cells were incubated with fluorochrome (allo-phycocyanin, allophycocyanin-Cy7, Alexa Fluor 488, Alexa Fluor 647,eFluor 450, eFluor 605 NC, FITC, PerCP-Cy5.5, or PE) or biotin-conjugated Abs against mouse CD3ε (145-2C11), CD11b (M1/70),CD11c (N418), CD14 (Sa2-8), CD19 (6D5), CD40 (1C10), CD49b(DX5), CD86 (GL1), CD88 (20/70), CD103 (M290), CD115 (AFS98),CD135 (A2F10), F4/80 (BM8), Ly-6C (AL-21), Ly-6G (1A8), MHC classII I-Ab (AFb.120), Sca-1 (D7), and TER-119 (BD Biosciences [San Jose,CA], BioLegend [San Diego, CA], and eBioscience [San Diego, CA]).Staining with biotinylated Abs was followed by fluorochrome-conjugatedstreptavidin. Stained cells were analyzed on a five-laser LSRII (BD Bio-sciences) or sorted on a five-laser ARIA-II flow cytometer (BD Bio-sciences), and the data were analyzed using FACS Diva (BD Bioscience)and FlowJo (Treestar, Ashland, OR) software. Dead cells were excludedbased on their forward and side scatter or uptake of 7-aminoactinomycinD. Only single cells were analyzed.

Generation and analysis of BM-elicited DCs

Marrow was collected from pulverized bones, and RBCs were lysed with0.15 M ammonium chloride and 1 mM potassium bicarbonate. BM cellswere then cultured in complete RPMI 1640 medium (RPMI 1640, 10% FBS[Gemini, West Sacramento, CA], penicillin/streptomycin, and 50 ng ml21

2-ME) supplemented with either 100 ng ml21 recombinant human FL togenerate FL-DCs or 5 ng ml21 GM-CSF and IL-4 (R&D Systems, Min-neapolis, MN) to generate GM-DCs. Recombinant human FL was pro-duced by the NIEHS Protein Expression Core Facility using previouslydescribed methods (21). Media was replaced every 3 d, at which time freshcytokines were also added. In some experiments 100 ng ml21 LPS (Sigma-Aldrich, St. Louis, MO) or 10 mg ml21 poly(I:C) (InvivoGen, San Diego,CA) was added during the last 24 h of culture to induce DC maturation.Cells were harvested on day 8 and stained with biotinylated anti-CD11cAb (eBioscience), followed by incubation with streptavidin Microbeads(Miltenyi, Auburn, CA), and finally isolated with an automated magnet-activated cell sorter to a purity of .90% CD11c+ cells. To assay T cellstimulating activity, BM-derived DCs were purified using MACS and thencultured with naive CD4+ OT-II cells (DC:T cell ratio = 1:2) in the pres-ence of 10 nM OVA323–339 peptide (New England Peptide, Gardner,MA) as described previously (8). Proliferation was inferred from recoveryof viable (trypan blue negative) T cells after 5 d of coculture. In someexperiments, DCs were centrifuged onto glass slides and photographed

using an Olympus BX51 microscope, DP70 digital camera, and DP soft-ware (Olympus, Center Valley, PA).

Preparation and analysis of lung DCs subsets

LungDC subsets were isolated and purified by flow cytometry-based sortingas described previously (22). To induce activation of lung DCs in vivo,mice were anesthetized by isoflurane inhalation and given 0.1 mg LPS byoropharyngeal aspiration 16 h prior to lung DC isolation. In someexperiments, purified DC subsets were cultured in complete RPMI 1640medium containing 100 ng ml21 LPS.

Purification of monocytes and pre-DCs from BM

Ly-6Chi monocytes (CD32CD11b+CD11c2CD192CD49b2CD115+F4/80hiI-A2Ly-6ChiLy-6G2Sca-12TER1192) or pre-DCs (CD32CD11b2

CD11c+CD192CD49b2I-A2CD135+Ly-6G2Sca-12TER1192) from BMwere enriched by MACS and then further purified by flow cytometry–based sorting. In some experiments, Ly-6Chi monocytes were cultured incomplete RPMI 1640 medium supplemented with 5 ng ml21 GM-CSF andIL-4 to generate moDCs. Culture medium was changed every 3 d withfresh cytokines added at that time.

Chemotaxis assays

LPS-matured, CD11c+ BM-elicited DCs (23 105) were added to the upperchamber of a Transwell insert (Corning, Corning, NY) with a 5 mm poly-carbonate membrane. Various concentrations of CCL19 (R&D) wereadded to the bottom chamber. After a 2 h incubation at 37˚C, migratedcells were collected from the bottom chamber and enumerated by flowcytometry using Accucount Beads (Spherotech, Lake Forest, IL) per themanufacturer’s instructions.

cDNA amplification and analysis

Total RNAwas isolated from cells using an RNeasy kit (Qiagen, Valencia, CA)or TRIzol reagent (Life Technologies) and converted to cDNAwith oligo dTandrandom hexamer primers using murine leukemia virus reverse transcriptase(Life Technologies, Grand Island, NY). PCR amplificationwas performed usingprimers listed in Supplemental Table I with Power SYBR Green PCR MasterMix (Life Technologies) and an Mx3000P quantitative PCR (QPCR) system(Agilent Technologies, Santa Clara, CA). The efficiency-corrected DCt foreach gene was determined and normalized to Gapdh expression.

Chromatin immunoprecipitation

Native chromatin was prepared from cells by small-scale micrococcal nu-clease digestion as previously described with modification (23). Briefly,CD11c+ BM-elicited DCs (4 3 106) or sorted lung DCs (0.2–1 3 106) werewashed and incubated in lysis buffer containing 0.4% Nonidet P-40 for10 min on ice. Nuclei were then washed and resuspended in digestion buffercontaining 25 U/ml micrococcal nuclease (Worthington Biochemical,Lakewood, NJ) and incubated at 37˚C for 5 min to generate mono- anddinucleosomes. The reaction was stopped by addition of 10 mM Tris-HCl(pH 8), 5 mM EDTA, and 10 mM sodium butyrate, and samples were in-cubated overnight at 4˚C. After centrifugation, the supernatant was harvestedand Triton X-100 was added to a concentration of 1% (v/v). For BM-elicitedDC samples, chromatin was precleared with protein A–Sepharose/salmonsperm DNA (EMDMillipore, Billerica, MA) and then incubated overnight at4˚C with 2.5 mg of either anti-trimethylated H3 lysine 27 (anti-H3K27me3;Active Motif, Carlsbad, CA), anti-acetylated H3 lysine 27 (anti-H3K27ac;Active Motif), or nonspecific anti-rabbit IgG Ab (Santa Cruz Biotechnology,Dallas, TX). After incubation with protein A–Sepharose/salmon sperm DNAfor 1 h, immunoprecipitates were washed vigorously, and DNAwas purified.For lung DC samples, chromatin was immunoprecipitated with the aboveAbs using the MAGnify ChIP System (Life Technologies) per the manu-facturer’s instructions. Bound and input fractions were quantified by QPCRusing an MxP3000p QPCR system (Agilent Technologies) with PowerSYBR Green PCR Master Mix (Life Technologies) and PCR primers listedin Supplemental Table I. PCR conditions were as follows: 10 min at 95˚C;15 s at 95˚C and 60 s at 62˚C for 40 cycles; and dissociation curve analysis toverify specific PCR product labeling. Analysis was performed using theefficiency-corrected DCt method, with amplification efficiency determinedfrom a standard curve that was generated from genomic DNA. Data arepresented as the signal from bound fractions normalized to the input fraction.

Statistics

Data are expressed as mean 6 SD or SEM as indicated. Statistical dif-ferences between groups were calculated using a two-tailed Student t test,unless indicated otherwise. A p value , 0.05 was considered significant.

2 EPIGENETIC CONTROL OF Ccr7 EXPRESSION IN LUNG DCs

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ResultsBM-elicited conventional DCs, but not moDCs, expressfunctional CCR7

Lung DCs are rare compared with many other cell types in thatorgan. Accordingly, we first used different culture conditions togenerate cDCs and moDCs from BM progenitors ex vivo (Fig. 1A)(24). Because FL is required for cDC development, we culturedBM progenitors in the presence of this cytokine to generate cDCs(FL-DCs). To generate moDCs, we cultured progenitors with GM-CSF and IL-4 (GM-DCs). Light microscopic and flow cytometricanalyses revealed that both cultures contained CD11chiI-Abhi DCshaving similar morphologies, including the presence of dendrites(Fig. 1B, 1C). FL-DCs and GM-DCs were similarly responsiveto maturation stimuli, as both upregulated expression of MHCclass II and costimulatory molecules following LPS treatment(Fig. 1D). Furthermore, LPS treatment induced similar expressionof Il6 and Il12b mRNA in FL-DCs and GM-DCs (data not shown).Importantly, LPS-matured FL-DCs and GM-DCs were equivalentin their ability to stimulate naive T cells (Fig. 1E). Thus, by thesestandard assays, FL-DCs and GM-DCs each displayed the char-acteristics of bona fide DCs. However, FL-DCs and GM-DCsdiffered considerably with regard to lineage-associated gene ex-pression. Compared with GM-DCs, FL-DCs expressed signifi-

cantly higher levels of mRNA for the cDC-specific transcriptionfactor Zbtb46 (Fig. 1F) (25, 26). In contrast, activated GM-DCs

highly expressed Nos2, consistent with a moDC phenotype

(Fig. 1F) (27). On the basis of these observations, we concluded

that FL-DCs and GM-DCs corresponded to cDCs and moDCs,

respectively.Ccr7gfp reporter mice are a convenient resource for assaying

Ccr7 expression in DCs (10). Using these mice as a source of BM,

we compared Ccr7gfp expression in FL-DCs and GM-DCs. Fol-

lowing LPS stimulation, FL-DCs rapidly upregulated Ccr7gfp

(Fig. 2A, 2B). In contrast, GM-DCs expressed very low levels of

Ccr7gfp following LPS treatment (Fig. 2A, 2B). Similar results

were observed when FL-DCs and GM-DCs were stimulated with

the TLR3 ligand poly(I:C) (Fig. 2B). To confirm that the GFP

fluorescence correlated with functional CCR7 protein, we studied

the abilities of FL-DCs and GM-DCs to undergo chemotaxis in

response to CCL19. FL-DCs migrated very efficiently to various

concentrations of CCL19, confirming that they expressed func-

tional CCR7 (Fig. 2C). Conversely, GM-DCs migrated poorly to

CCL19 (Fig. 2C), which was consistent with their low expression

of Ccr7gfp. Thus, although FL-DCs and GM-DCs were similar in

many measures of DC function, only FL-DCs expressed func-

tional CCR7 at high levels.

FIGURE 1. Culturing BM precursors with FL or GM-CSF/IL-4 yields DCs that resemble conventional or moDCs, respectively. (A) Schematic protocol

for the generation and maturation of BM-elicited DCs using FL (FL-DCs) or GM-CSF/IL-4 (GM-DCs). (B) Representative light micrographs of cytospin

preparations of FL- or GM-DCs. (C) Representative cytograms depicting surface I-Ab and CD11c expression on FL- and GM-DCs after maturation with

LPS. (D) Histograms depicting I-Ab, CD40, and CD86 expression on unstimulated (shaded histograms) or LPS-matured (bold lines) DCs. Data are from

a single experiment, representative of two. (E) Stimulation of naive CD4+ OT-II cells by LPS-matured FL- or GM-DCs in the presence or absence of

OVA323–339 peptide. Bar graphs represent the mean6 SD of viable T cells (duplicate samples). Data are from a single experiment, representative of two. (F)

Zbtb46 and Nos2 mRNA expression by MACS-purified CD11c+ FL- and GM-DCs. Bar graphs represent the mean 6 SD of mRNA amounts in arbitrary

units (AU) after normalization to Gapdh mRNA. Data are from a single experiment, representative of two.

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The Ccr7 locus is enriched for repressive histone modificationsin BM-elicited moDCs

The inability of GM-DCs to express Ccr7 despite having oth-erwise normal responsiveness to LPS (Fig. 1D) suggested thatthe Ccr7 locus in these cells might be inaccessible to transcrip-tional activators. It is well established that histone modificationshelp govern chromatin accessibility to the transcriptional ma-chinery, thereby regulating gene expression (17). In particular,trimethylation of lysine 27 on histone 3 (H3K27me3), which isprimarily mediated by the polycomb repressive complex 2(PRC2), is associated with transcriptional repression in variouscells including DCs and macrophages (28, 29). Analysis ofavailable chromatin immunoprecipitation (ChIP)-sequencingdata obtained from the Encyclopedia of DNA Elements (EN-CODE) project (30) revealed that the Ccr7 promoter is enrichedfor H3K27me3 in tissues that generally lack CCR7 expression(Fig. 3A), suggesting that H3K27me3 might also be associatedwith Ccr7 repression in moDCs. Therefore, using ChIP-QPCRassays, we investigated H3K27me3 modifications at the Ccr7

promoter in LPS-stimulated FL-DCs and GM-DCs. We foundthat the Ccr7 promoter is significantly enriched for H3K27me3in GM-DCs but not FL-DCs (Fig. 3B). GM-DCs also had similarenrichment of H3K27me3 at the first intron of Ccr7. To excludethe possibility that our findings at the Ccr7 locus were due toa widespread nonspecific increase in H3K27me3 in GM-DCs, westudied a distant locus containing the Magea2 gene. No signifi-cant differences were seen in H3K27me3 at that locus, indicatingthat repressive histone marks were preferentially increased at theCcr7 locus (Fig. 3B). The level of H3K27me3 at the Ccr7 pro-moter was inversely related to Ccr7 mRNA expression, consis-tent with transcriptional repression of Ccr7 (Fig. 3C). Similardifferences in Ccr7-associated H3K27me3 were observed inunstimulated DCs, indicating that the repressive histone markswere established prior to DC activation (Supplemental Fig. 1A).Because histone acetylation has been associated with transcrip-tional activation in human DCs (28), we also analyzed H3K27acetylation at the Ccr7 locus. We did not find significant dif-ferences in Ccr7-associated H3K27 acetylation between FL- andGM-DCs (Supplemental Fig. 1B), suggesting that Ccr7 expres-sion was predominantly regulated by repressive histone marks.Taken together, these findings suggest that lineage-specific re-pressive histone modifications help govern Ccr7 expression inBM-elicited DCs.

Nonmigratory lung-resident moDCs have increased levels ofCcr7-associated H3K27me3

Having established that the Ccr7 locus is associated with re-pressive histone marks in moDCs expanded from BM progeni-tors, we next investigated whether similar repressive histonemarks are associated with Ccr7 in lung-resident moDCs. Lung-resident DCs can be phenotypically divided into CD103+ orCD11bhi DCs. CD103+ DCs arise from pre-DC precursors andthus represent a pure population of cDCs (10). By contrast,CD11bhi DCs contain a mixture of FL-dependent cDCs and FL-independent moDCs, with the latter cells having a higher displaylevel of CD14 (10). However, because some CD11bhi DCs ex-press intermediate levels of CD14, this molecule cannot reliablydistinguish cDCs from moDCs. CD64 expression is reported todiscriminate between cDCs and moDCs (3, 31), but we recentlyfound that display levels of a different marker, CD88, is a morereliable marker to distinguish CD11bhi moDCs from CD11bhi

cDCs (H. Nakano, T.P. Moran, K. Nakano, C.D. Bortner, andD.N. Cook, submitted for publication). To confirm that CD88can also distinguish nonmigratory moDCs from migratory cDCs,we isolated lung-resident DCs from Ccr7gfp reporter mice byflow cytometric cell sorting (Fig. 4A). As expected, all lung-resident DC subsets expressed low levels of Ccr7gfp immedi-ately after isolation from naive mice (Fig. 4B). Stimulation withLPS resulted in significant induction of Ccr7gfp in CD103+ andCD11bhiCD88lo DCs (Fig. 4B). In contrast, CD11bhiCD88hi DCsexpressed very low levels of Ccr7gfp after activation, which isconsistent with their monocytic lineage (Fig. 4B). All lung DCsubsets expressed high levels of CD86 after LPS stimulation(data not shown), indicating normal responsiveness to TLR4ligands. Analysis of Ccr7 mRNA in wild-type mice confirmedthat this gene is more highly expressed in activated CD103+

cDCs and CD11bhiCD88lo cDCs compared with CD11bhiCD88hi

moDCs (Fig. 4C). Thus, CD88 display can identify nonmigra-tory CD11bhi moDCs in the lung.Using CD88 as a marker for moDCs, we purified migratory

CD103+ and CD11bhiCD88lo cDCs, as well as nonmigratoryCD11bhiCD88hi moDCs, from the lungs of LPS-treated mice.Chromatin was prepared from each DC subset and analyzed for

FIGURE 2. BM-elicited cDCs, but not moDCs, express functional

CCR7. (A) Analysis of Ccr7gfp fluorescence in CD11c+ FL- or GM-DCs

generated from wild-type (gray histograms) or Ccr7gfp reporter mice (bold

lines) after LPS maturation. The percentage of cells expressing Ccr7gfp is

indicated on each plot. (B) Comparison of Ccr7gfp expression by CD11c+

FL- or GM-DCs after stimulation with either media alone, 100 ng ml21

LPS, or 10 mg ml21 poly(I:C) for 24 h. Bar graphs represent the mean 6SD of duplicate samples. Data are from a single experiment, representative

of two. (C) Chemotaxis of LPS-matured FL- or GM-DCs in response to the

indicated concentrations of the CCR7 ligand, CCL19. Symbols represent

the mean 6 SD of duplicate samples. Data are from a single experiment,

representative of three.

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Ccr7-associated H3K27me3. Compared with CD103+ DCs andCD11bhiCD88lo cDCs, CD11bhiCD88hi moDCs had significantlyelevated levels of H3K27me3 at the Ccr7 promoter (Fig. 4D). Theamount of Ccr7-associated H3K27me3 was inversely related to theexpression of this gene, consistent with transcriptional silencing ofCcr7 in CD11bhiCD88hi moDCs (Fig. 4C). Levels of H3K27me3 ata distant locus (Hoxc10) were similar among all DC subsets, con-firming that the epigenetic differences seen at the Ccr7 promoter arenot due to a nonspecific genomewide increase of H3K27me3 inlung moDCs (Fig. 4D). Overall, these findings indicate that thenonmigratory status of lung-resident moDCs is associated with re-pressive histone modifications at the Ccr7 promoter.

BM monocytes have low levels of Ccr7-associated H3K27me3

Our observation that cDCs and moDCs have disparate levels ofCcr7-associated H3K27me3 is consistent with lineage-specificepigenetic repression of Ccr7 expression. This prompted us toinvestigate whether the divergence in H3K27me3 at the Ccr7 lo-cus in cDCs and moDCs occurs during lineage commitment.Monocyte-DC progenitors can give rise to both monocytes andpre-DCs (32). We therefore compared histone modifications in thelatter two types of progenitor cells following their purificationfrom BM. Unexpectedly, monocytes and pre-DCs had similaramounts of H3K27me3 at the Ccr7 promoter (Fig. 5A), and theseamounts were much lower than that seen for either BM-elicited or

lung moDCs (Figs. 3B, 4D). Thus, differentiation of monocyte-DC progenitors to monocytes in the BM is not immediately ac-companied by epigenetic repression of Ccr7.

Epigenetic repression of the Ccr7 promoter is graduallyestablished during monocyte differentiation into DCs

The similar amounts of H3K27me3 at the Ccr7 locus of monocytesand pre-DCs suggested that this epigenetic modification is ac-quired after monocytes enter peripheral tissues and differentiateinto moDCs. We first investigated whether monocyte emigrationinto pulmonary tissues triggers epigenetic repression of Ccr7.Airway exposure to LPS results in the recruitment of Ly-6Chi

monocytes to the lungs, which rapidly differentiate into Ly-6Chi

CD11bhi inflammatory DCs (33). Inflammatory DCs also expressCD88 but can be distinguished from lung-resident moDCs by theirhigh-level expression of Ly-6C (Fig. 5B). ChIP-QPCR analysis ofinflammatory DCs revealed that they had significantly lower levelsof Ccr7-associated H3K27me3 compared with lung-residentmoDCs (Fig. 5C). Indeed, inflammatory DCs and BM mono-cytes had similar low levels of H3K27me3 at the Ccr7 locus(Fig. 5A, 5C). Thus, extravasation of monocytes into the lungparenchyma does not immediately result in epigenetic repressionof Ccr7.We next investigated whether epigenetic repression of Ccr7 is

established during the later stages of monocyte differentiation into

FIGURE 3. The Ccr7 locus is enriched for repressive histone modifications in BM-elicited moDCs. (A) Structure of the Ccr7 locus, including relative

amounts of H3K27me3 in chromatin from the indicated organs of adult C57BL/6 mice (available through the Encyclopedia of DNA Elements [ENCODE]

project and analyzed using the University of California Santa Cruz Genome Browser web site). The location of primers designed to amplify the Ccr7

promoter and first intron are indicated by rectangles. (B) ChIP-QPCR analysis of the Ccr7 promoter, Ccr7 first intron, orMagea2 locus in LPS-matured FL-

or GM-DCs following immunoprecipitation with either anti-H3K27me3 (n) or nonspecific IgG (N) Abs. Bar graphs represent the mean 6 SD of triplicate

samples after normalization to input DNA. Data are from a single experiment, representative of two. (C) Ccr7 mRNA in mature FL- or GM-DCs. Bar

graphs represent the mean 6 SD of duplicate samples after normalization to Gapdh mRNA. Data are from a single experiment, representative of two.

***p , 0.001. AU, arbitrary unit.

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moDCs. We have previously shown that only a very small per-centage of adoptively transferred monocytes enter the lungs anddifferentiate into resident moDCs (10). Therefore, it was notpossible to accurately track the fate of large numbers of mono-cytes in the lung for an extended period. As an alternative ap-proach, we purified Ly-6Chi monocytes from BM and followedepigenetic changes at the Ccr7 promoter during ex vivo differ-entiation of these cells into moDCs (34). As expected, during 6 dof culture in the presence of GM-CSF and IL-4, monocytes dif-ferentiated into CD11chiI-Abhi moDCs (Fig. 6A). As we had seenpreviously with BM progenitors cultured under these same con-ditions, monocyte-derived cells failed to express Ccr7, even aftertheir stimulation with LPS (Fig. 6B). During ex vivo moDC dif-ferentiation, we observed a significant increase in Ccr7-associatedH3K27me3 (Fig. 6C). In contrast, the level of H3K27me3 at theHoxc10 locus remained stable during the culture period (Fig. 6C),indicating the observed changes at the Ccr7 locus were not due toa genomewide increase in H3K27me3. Consistent with this, wedid not detect increased expression of the PRC2 core subunitsEZH2, EED, SUZ12, and RbAp48 during moDC differentiation

(Supplemental Fig. 2). Overall, these findings suggest that epi-genetic silencing of Ccr7 occurs in a specific and gradual mannerduring the differentiation of monocytes into lung-resident moDCs.

DiscussionThe migration of DCs to lung-draining LN is critical for shapingnascent immune responses to inhaled Ags (1, 3, 10). Given that theDC network is composed of populations with distinct functionalroles, it is not surprising that only certain subsets possess theability to express Ccr7 and migrate to regional LN. We havepreviously demonstrated that the developmental lineage of DCs isimportant for determining their migratory potential (10). In thecurrent study, we investigated whether epigenetic mechanismsmight contribute to lineage-specific Ccr7 expression. We foundthat in BM-elicited or lung moDCs, the Ccr7 promoter is enrichedfor the repressive histone mark H3K27me3. Consequently, Ccr7induction is transcriptionally repressed in moDCs but not cDCs.The repressive histone modifications are not established duringlineage commitment in the BM but are formed during monocytedifferentiation into moDCs in the periphery. Our results suggest

FIGURE 4. Nonmigratory lung-resident moDCs have increased levels of Ccr7-associated H3K27me3. (A) Gating strategy for flow cytometry–based

sorting of lung-resident DC subsets from wild-type or Ccr7gfp reporter mice. (B) Analysis of Ccr7gfp expression on sorted lung-resident DCs before (shaded

histograms) or after (bold lines) ex vivo stimulation with 100 ng ml21 LPS for 24 h. Data are from a single experiment, representative of two. (C) Ccr7

mRNA expression in lung-resident DCs isolated from mice 16 h post-LPS inhalation. Bar graphs represent the mean 6 SEM (after normalization to Gapdh

mRNA) of combined data from two independent experiments. (D) ChIP-QPCR analysis of the Ccr7 promoter or Hoxc10 locus (reference gene) in lung-

resident DCs after immunoprecipitation with either anti-H3K27me3 (n) or nonspecific IgG (N) Abs. Bar graphs represent the mean 6 SEM (after nor-

malization to input DNA) of combined data from three independent experiments. ***p , 0.001. Auto, autofluorescence; AU, arbitrary unit.

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that a combination of lineage-specific epigenetic mechanisms andcues within the tissue microenvironment help determine the mi-gratory capacity, and consequently the functional role, of lung DCsubsets. The nonmigratory status of moDCs suggests that they donot function during Ag sensitization, which is thought to requireDC migration, but rather during the effector phase of immuneresponses within the lung. Indeed, a recent report found thatmoDCs were largely responsible for Ag presentation and proin-flammatory cytokine secretion in the lungs during allergen chal-lenge (3). Whether lung moDCs are more efficient than cDCs atstimulating effector or memory T cells is an important questionfor future studies.Although there has been recent progress in identifying the

transcriptional profiles of different DC subtypes (9, 12, 35), little isknown regarding the epigenetic mechanisms regulating gene ex-pression in DCs. This is in contrast to Th cell differentiation,where epigenetic changes at lineage-specific genes have been welldocumented (36). Epigenetic mechanisms are likely responsiblefor regulating several aspects of DC immunobiology. For example,DCs treated with histone deacetylase inhibitors have altered ex-pression of proinflammatory cytokines and costimulatory mole-cules, arguing that dynamic changes in histone modificationsare important during DC maturation (37). Furthermore, heritableepigenetic changes have been shown to influence DC functionduring adaptive immune responses. Splenic DCs from offspring ofallergen-sensitized mothers had altered genomic DNA methyla-tion patterns, which was associated with a Th2-skewing phenotypewhen compared with DCs from pups of naive mothers (38). Thecurrent study provides further insight into how epigenetic changescan influence DC function, specifically through regulation of theirmigration. By epigenetically silencing the Ccr7 gene, moDCsassume a nonmigratory phenotype within the lung.Unexpectedly, we found that epigenetic repression of Ccr7 is not

established early during DC lineage commitment, but rather dur-

ing monocyte differentiation into moDCs. Using an ex vivo modelfor generating moDCs from BM monocytes, we observed a grad-ual but specific increase in H3K27me3 at the Ccr7 promoter. It ispossible that ex vivo generation of moDCs from monocytes doesnot accurately reflect in vivo differentiation of moDCs in the lung.However, ex vivo–generated and lung-resident moDCs had com-parable surface marker phenotypes and histone modifications atthe Ccr7 locus, suggesting that they undergo similar differentia-tion pathways. Improvements in cellular fate-mapping and ChIPanalyses using small cell numbers will help facilitate future epi-genetic studies of lung-resident moDCs and other rare cell types.Interestingly, we observed low levels of H3K27me3 at the Ccr7

locus in monocytes, implying that Ccr7 retains the potential to beexpressed in these cells or their progeny. This might explaincontradicting reports that monocyte-derived APCs in the coloncan express Ccr7 and migrate to LN (39). Furthermore, epidermalLangerhans cells—which are derived from monocytes (40)—arereported to express Ccr7 and traffic to skin-draining LN (41). It isconceivable that tissue-specific factors can regulate epigeneticsilencing of the Ccr7 locus in monocyte-derived APCs. Althoughthe identity of these factors is unknown, several cytokines canmodulate Ccr7 expression in activated DCs, including thymicstromal lymphopoietin (42), IL-33 (43), and type I IFNs (44). Inaddition, our studies with BM-elicited moDCs and ex vivo–cul-tured monocytes suggest a role of GM-CSF and FL in determiningepigenetic modifications at the Ccr7 locus. The observation thatGM-CSF may lead to epigenetic repression of Ccr7 in monocyteshas significant implications for DC-based immunotherapies be-cause this cytokine is frequently used to generate DCs from pe-ripheral blood monocytes in humans. Because LN homing by DCsis considered necessary for optimal stimulation of Ag-specificT cells (45), understanding how GM-CSF and FL influence themigratory potential of DCs will be essential for designing effec-tive DC vaccines.

FIGURE 5. Monocytes and their immediate progeny have low levels of H3K27me3 at the Ccr7 promoter. (A) ChIP-QPCR analysis of the Ccr7 promoter

or Hoxc10 locus (reference gene) in monocytes (mono) or pre-DCs following immunoprecipitation with either anti-H3K27me3 (n) or nonspecific IgG (N)

Abs. Bar graphs represent the mean 6 SEM (after normalization to input DNA) of combined data from three independent experiments. (B) Gating strategy

for purifying lung-resident CD11bhiCD88hi moDCs and Ly-6ChiCD11bhi inflammatory DCs (infDCs) from LPS-treated lungs. The cytogram depicts CD88

and Ly-6C expression after gating on CD11chiAutofluorescence2I-AbhiCD11bhi lung cells. (C) ChIP-QPCR analysis of the Ccr7 promoter or Hoxc10 locus

(reference gene) in Ly-6Chi infDCs or CD88hi moDCs. Bar graphs represent the mean 6 SEM (after normalization to input DNA) of combined data from

three independent experiments. *p , 0.05.

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Although our studies demonstrate that Ccr7-associatedH3K27me3 increases during monocyte differentiation intomoDCs, the mechanisms by which occurs are currently unclear.Because trimethylation of H3K27 is mediated by the chromatinmodification complex PRC2, we studied expression of genesencoding its core subunits (EZH2, EED, SUZ12, and RbAp48)

during monocyte differentiation to moDCs (46). We found thatexpression of these genes generally decreased during moDC de-velopment (Supplemental Fig. 2), which is consistent with thepreviously reported decrease in Ezh2 expression during cellulardifferentiation (47). Therefore, it is likely that the increased Ccr7-associated H3K27me3 results from a selective recruitment ofPRC2 to the Ccr7 locus by specific DNA-binding proteins ortranscription factors (48). Identification of such factors shouldfurther our understanding of how the Ccr7 locus is regulated inmoDCs.Although our observations suggest that H3K27me3 leads to

silencing of Ccr7 in moDCs, demonstrating direct causality isdifficult. It is possible to inhibit H3K27me3 by targeting thehistone methyltransferase EZH2, but this can affect multipleaspects of cell differentiation and survival (49). Not surprisingly,we found that the EZH2 inhibitor 3-deazaneplanocin A inter-fered with DC development from BM progenitors and mono-cytes, thus precluding any specific studies on Ccr7 expression(T.P. Moran, unpublished observations). Strategies that specifi-cally inhibit EZH2 activity at the Ccr7 locus will be importantfor addressing this issue. It is also likely that H3K27me3 is butone of many mechanisms involved with regulating Ccr7 ex-pression. Monocytes and monocyte-derived Ly-6Chi inflamma-tory DCs have low levels of Ccr7-associated H3K27me3, yetthey do not express Ccr7 after stimulation with LPS (Fig. 6A)(10). This implies that in addition to a receptive chromatinconfiguration at the Ccr7 promoter, lineage-specific transcrip-tion factors are also necessary for driving gene expression. Sev-eral transcription factors have been reported to regulate Ccr7expression, including NF-kB (50), peroxisome proliferator-activated receptor g (51), Runx3 (52), liver X receptor (53), andIFN regulatory factor 4 (54). The migratory properties of cDCsand moDCs may be dependent on differential expression of thesetranscription factors. Additional epigenetic mechanisms, such asDNA methylation and noncoding RNAs, might also regulate Ccr7expression. The Ccr7 promoter does not contain any CpG islands,but it is possible that DNA methylation in upstream enhancers orintronic regions may influence Ccr7 expression. Noncoding RNAsthat inhibit CCR7 production have been described in humanT lymphocytes and breast cancer cells (55, 56) but not in APCs.Future studies will help clarify the relative contribution of lineage-specific transcription factors and other epigenetic marks to Ccr7regulation.Overall, our findings suggest that by controlling Ccr7 ex-

pression, lineage-specific epigenetic mechanisms can influencethe function of specific DC subsets. Identifying the endogenousand cell-intrinsic factors that direct Ccr7-specific epigeneticchanges will likely uncover pathways that can be targeted tomodulate DC migration. In turn, the ability to manipulate themigratory properties of DCs represents a novel strategy for im-proving DC-based immunotherapies and for treating immune-mediated diseases.

AcknowledgmentsWe thank Carl Bortner and Maria Sifre of the NIEHS Flow Cytometry Cen-

ter for help with cell sorting, the NIEHS Protein Expression Core Facility

for production of recombinant human FL, Ligon Perrow for mouse

colony management, Komal Patel for technical assistance, Thomas Randall

for bioinformatics consultation, and Michael Fessler and Harriet Kinyamu

for critical review of the manuscript.

DisclosuresThe authors have no financial conflicts of interest.

FIGURE 6. Epigenetic repression of the Ccr7 promoter is gradually

established during monocyte differentiation to moDCs. (A–C) Ly-6Chi

monocytes sorted from the BM of wild-type (A and C) or Ccr7gfp re-

porter mice (B) were cultured in the presence of GM-CSF and IL-4 for

6 d. Cells were analyzed by flow cytometry (A and B) or ChIP-QPCR (C)

at the indicated time points. (A) Cytograms showing CD11c and I-Ab

expression on monocytes at the indicated time points during culture.

Data are from a single experiment, representative of two. (B) Cytograms

depict Ccr7gfp expression by monocytes at the indicated time points

during culture. Cells were treated with 100 ng ml21 LPS for 24 h prior

to flow cytometric analysis. Data are from a single experiment, repre-

sentative of two. (C) Quantification of H3K27me3 by ChIP-QPCR

analysis of the Ccr7 promoter or Hoxc10 locus (reference gene) during

monocyte differentiation into moDCs. Bar graphs represent the mean 6SEM (after normalization to input DNA) of combined data from two

independent experiments. The line graph depicts the mean ratio (6SEM) of Ccr7-associated H3K27me3 to Hoxc10-associated H3K27me3.

**p , 0.01.

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