Cutting Edge: An Inactive Chromatin Configuration at the IL-10 Locus in Human Neutrophils

of February 20, 2013.This information is current as

NeutrophilsConfiguration at the IL-10 Locus in Human Cutting Edge: An Inactive Chromatin

CassatellaBrandau, Flavia Bazzoni, Gioacchino Natoli and Marco A.Renato Ostuni, Kirsten Bruderek, Bastian Schilling, Sven Nicola Tamassia, Maili Zimmermann, Monica Castellucci,

http://www.jimmunol.org/content/190/5/1921doi: 10.4049/jimmunol.1203022January 2013;

2013; 190:1921-1925; Prepublished online 25J Immunol 

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Cutting Edge: An Inactive Chromatin Configuration atthe IL-10 Locus in Human NeutrophilsNicola Tamassia,* Maili Zimmermann,* Monica Castellucci,* Renato Ostuni,†

Kirsten Bruderek,‡ Bastian Schilling,x Sven Brandau,‡ Flavia Bazzoni,*Gioacchino Natoli,† and Marco A. Cassatella*

To identify the molecular basis of IL-10 expression in hu-man phagocytes, we evaluated the chromatin modifica-tion status at their IL-10 genomic locus. We analyzedposttranslational modifications of histones associatedwith genes that are active, repressed, or poised for tran-scriptional activation, including H3K4me3, H4Ac,H3K27Ac, and H3K4me1 marks. Differently from au-tologous IL-10–producing monocytes, none of the marksunder evaluation was detected at the IL-10 locus of rest-ing or activated neutrophils from healthy subjects ormelanoma patients. By contrast, increased H3K4me3,H4Ac, H3K4me1, and H3K27Ac levels were detectedat syntenic regions of the IL-10 locus in mouse neutro-phils. Altogether, data demonstrate that human neutro-phils, differently from either monocytes or mouseneutrophils, cannot switch on the IL-10 gene becauseits locus is in an inactive state, likely reflecting a neutro-phil-specific developmental outcome. Implicitly, dataalso definitively settle a currently unsolved issue on thecapacity of human neutrophils to produce IL-10. TheJournal of Immunology, 2013, 190: 1921–1925.

Recent data have contributed to expand the view ofpolymorphonuclear neutrophils as versatile cells fortheir capacity to cross talk with cellular components

of the innate and adaptive immune systems, as well as for theirability to condition the evolution of various processes via therelease of newly synthesized cytokines (1). Accordingly, thecytokine repertoire that neutrophils can potentially express isquite vast and includes proinflammatory/anti-inflammatoryand immunoregulatory cytokines, chemokines, TNF super-family members, colony-stimulating, and angiogenic factors(2). Interestingly, although differences in the capacity to ex-press cytokines have been reported to occur between humanand mouse neutrophils, there is not a general consensus in the

literature whether human neutrophils produce IFN-g, IL-6, IL-17, or IL-10 (1). With regard to IL-10, our groups and othershave recently demonstrated that highly purified neutrophilpopulations, isolated by a variety of procedures from healthydonors, do not express or secrete IL-10, either spontaneously orupon treatment with a panel of stimuli (including serum am-yloid protein A [SAA], LPS, Pam3CSK4, polyinosinic:poly-cytidylic acid, R-848, curdlan, neutrophil-activating proteinderived from Helicobacter pylori, the chemoattractant fMLF,insoluble immunocomplexes, IFN-g, TNF-a, GM-CSF, or G-CSF) used singly and in combination (3). In contrast, weconfirmed (4) that autologous monocytes, stimulated under thesame experimental conditions as neutrophils, promptly pro-duce detectable amounts IL-10 (3). Our data were in line withanalogous observations previously made in several laboratories(5–9), but in contradiction with findings reported by other re-searchers, who had instead observed a neutrophil-derived IL-10production under resting or stimulatory conditions (10–14).In particular, the study by De Santo et al. (14), showing thatSAA-1 induces the differentiation of IL-10–secreting neu-trophils with potential immunosuppressive activities in mela-noma patients, has instigated great interest for its implicationsin the cancer setting. In contrast, there is no doubt that mouseneutrophils do produce IL-10, as reproducibly observed eitherin in vitro experiments (3) or under a variety of experimentalmodels (1), including during methicillin-resistant Staphylococcusaureus infection (15), pneumonia (16), and polymicrobial sepsis(17). Because of the importance of IL-10 either in controllingdegree and duration of inflammatory reactions (18), or inpromoting immunosuppression in tumors (18), a molecularunderstanding of how the expression of this cytokine is dif-ferentially regulated in different cell types has obvious impli-cations not only for a better comprehension of inflammatorydisease pathogenesis, but also for immunotherapeutic strate-gies in cancer patients.Changes in chromatin organization and posttranslational

modifications control the transcriptional outputs in response to

*Section of General Pathology, Department of Pathology and Diagnostics, University ofVerona, 37134 Verona, Italy; †Department of Experimental Oncology, European Insti-tute of Oncology, I-20139 Milan, Italy; ‡Department of Otorhinolaryngology, UniversityHospital Essen, 45147 Essen, Germany; and xDepartment of Dermatology, UniversityHospital Essen, 45147 Essen, Germany

Received for publication November 1, 2012. Accepted for publication December 28,2012.

This work was supported by the Ministero dell’Istruzione, dell’Universita e della Ricerca(Grant 2009MFXE7L_001 to M.A.C.), Associazione Italiana per la Ricerca sul Cancro(Grant IG-11782 to M.A.C.), the Deutsche Forschungsgemeinschaft (Grant BR 2278/2-1 to S.B.), and a Fondazione Italiana per la Ricerca sul Cancro Fellowship (to N.T.).

Address correspondence and reprint requests to Dr. Marco A. Cassatella, Section ofGeneral Pathology, Department of Pathology and Diagnostics, University of Verona,Strada Le Grazie 4, 37134 Verona, Italy. E-mail address: [email protected]

The online version of this article contains supplemental material.

Abbreviations used in this article: ChIP, chromatin immunoprecipitation; PT, primarytranscript; SAA, serum amyloid protein A; TF, transcription factor.

Copyright� 2013 by TheAmerican Association of Immunologists, Inc. 0022-1767/13/$16.00

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specific environmental conditions or developmental states (19,20). Covalent modifications of histones are considered to beinvolved in regulating chromatin organization and in positive ornegative control of gene expression occurring during normal em-bryonic development (21), cancer (22), and the evolution ofspecific acquired immune responses (23). Among a wide rangeof posttranscriptional modifications, methylation and acetyla-tion of specific histone residues are pivotal in the maintenanceof the expression of a given gene in an active or suppressed state(24, 25). Accordingly, methylation of histone H3 at K9 andK27, and histone H4 at K20 (H3K9, H3K27, and H4K20)are correlated with repression of transcription, whereas meth-ylation of histone H3 at K4, K36, and K79 (H3K4, H3K36,and H3K79) is implicated in activation of transcription. His-tone posttranslational modifications can be detected by chro-matin immunoprecipitation (ChIP) using specific Abs formodified histones. ChIP has been instrumental not only toclarify that histone modifications are often dynamic, as wellas reversible, but also to establish combinations of marks as-sociated to different states of gene activity (26). Active pro-moters are associated with trimethylated histone H3 at K4(H3K4me3), which usually co-occurs with different levels ofacetylated histone H3 or H4 (AcH3 and AcH4), dependingon the state of gene activity. Enhancers are marked byH3K4me1 and can be divided into poised or active based onthe absence or presence of acetylated histone, respectively(19, 20). Conversely, trimethylation of K27 of histone 3(H3K27me3) represents a mark for silent genomic regions (27).In this study, we asked whether the different ability of

human neutrophils and monocytes to activate IL-10 expres-sion might reflect a different basal chromatin organization ofthe human IL-10 locus, which would, in turn, depend on theaction of highly specific developmental inputs, selectivelyacting in either cell type (28).

Materials and MethodsCell purification and culture

Granulocytes were isolated from buffy coats of healthy volunteers by Ficoll-Paquegradient followed by dextran sedimentation and hypotonic lysis of erythrocytes,under endotoxin-free conditions (29). Neutrophils were then enriched to reacha purity of .99% by positively removing any contaminating cells from gran-ulocytes, using the EasySep neutrophil enrichment kit (Stem Cell Technologies)(3). Human monocytes were purified from PBMCs with anti-CD14 microbeads(.98%;Miltenyi) (3). In selected experiment, neutrophils were isolated from theperipheral blood of melanoma patients (all stage IV M1c) or healthy volunteers.In the latter case, diluted blood was subjected to density gradient centrifugation(LSM 1077-Lymphocyte Separation medium; PAA Laboratories GmbH), andneutrophils (purity . 98%) were then isolated by sedimentation over 1%polyvinyl alcohol followed by hypotonic lysis (0.2% NaCl) of erythrocytes (3).Mouse neutrophils were isolated from bone marrow of C57BL/6 mice, usinga discontinuous Percoll gradient according to standard procedures (30), andfurther purified by positive selection using anti–Ly-6G Ab conjugated withallophycocyanin (Miltenyi) and anti-allophycocyanin MicroBeads (Miltenyi).Immediately after purification, human or mouse cells were either subjected tolysis for ChIP assays or suspended in RPMI 1640 medium supplemented with10% low-endotoxin FBS (,0.5 endotoxin unit/ml; BioWhittaker), treated ornot with 100 ng/ml ultrapure Escherichia coli LPS (0111:B4; Alexis), 5 mg/mlApoSAA (#300-13; PeproTech), or 1mg/ml Pam3CSK4 (Invivogen), and thencultured at 37˚C, 5% CO2 atmosphere for up to 6 h. Human samples wereobtained following healthy donors’ and patients’ informed written consent.The study has been cleared by the Ethic Committees of both the AziendaOspedaliera Universitaria Integrata di Verona (Italy) and the UniversityHospital Essen (Germany).

Quantitative real-time RT-PCR

Primary transcript (PT) quantitative RT-PCR analyses were performed aspreviously described previously (31), using gene-specific primer pairs (Invi-

trogen) available in the public database RTPrimerDB (http://medgen.ugent.be/rtprimerdb), under the following entry codes: PT–IL-10 (8612), PT–TNF-a (8613), and GAPDH (3539). Data were calculated by Q-Genesoftware (http://www.gene-quantification.de/download.html) and expressedas mean normalized expression units after GAPDH normalization.

ChIP assays

ChIP experiments were performed as described elsewhere (31), with minormodifications. In brief, nuclear extracts were prepared from 1–8 3 106 cells(depending on the type of ChIP assay) and then sonicated and immuno-precipitated with mAbs recognizing H3K4me3 (Millipore), or polyclonal Absrecognizing H4Ac, H3K27me3, H3K9me3, and SP1 (Millipore), H3K4me1and H3K27Ac (Abcam), and C/EBPb, c-FOS, and NF-kB p50 (Santa Cruz).The coimmunoprecipitated material was then subjected to quantitative PCRanalysis using specific primers (purchased from Invitrogen), either spanningthe human IL-10 locus or the murine IL-10 locus. Sequences of the ChIPprimers used are available on request. The various histone modifications werequantified also at the promoter of prolactin because it is completely silent inmyeloid cells to establish the background levels of ChIP experiments.

Results and DiscussionChromatin organization of the IL-10 locus in human neutrophils andmonocytes

Toelucidatewhether cell-specifichistonemodification signaturesmight provide indicative elements for the differential expressionof IL-10 in human phagocytes, we initially performed a com-parative analysis of the chromatin status at the IL-10 locus inhuman neutrophils and autologous monocytes freshly isolatedfrom the peripheral blood. Neither induction of IL-10 PTs (datanot shown) nor RNA polymerase II recruitment at the IL-10promoter (data not shown) were detected in neutrophils stim-ulated with 100 ng/ml LPS or 5 mg/ml SAA. We thus scanned20 kb of the IL-10 genomic locus (spanning from215.0 to +7.5kb relative to the transcription start site; Fig. 1A) for the presenceof chromatin modifications associated with transcriptionallyactive genes (20), including: 1) histone H3 trimethylated at K4(H3K4me3), which is localized at a few nucleosomes sur-rounding the transcription start site and strongly correlateswith active transcription (32); 2) tetraacetylated histone H4(H4Ac) and histone H3 acetylated at K27 (H3K27Ac), whichare enriched in active euchromatin (33); and 3) histone H3monomethylated at K4 (H3K4me1), which is detected at bothpromoters (in association with H3K4me3) and enhancers (34).In addition, we tested the levels of marks that are negatively cor-related with gene expression, includingH3K9me3,H3K27me3,and DNA methylation (19).Neither H3K4me3 (Fig. 1B) nor acetylated histones (Fig. 1C)

were detectable at the IL-10 locus of freshly isolated neutrophils,whereas they were both present in autologous monocytes (Fig.1B, 1C). Moreover, no association of H3K27Ac was found atthe IL-10 genomic locus of neutrophils (Fig. 1D), whereasH3K4me1 was present, even though at much lower levels thanin monocytes (Fig. 1E). Interestingly, H3K9me3, H3K27me3(data not shown), and DNAmethylation (data not shown) wereall undetectable at the IL-10 genomic locus of either neutrophilsor monocytes, thus ruling out transcriptional repression medi-ated by these marks (20). Taken together, data are in keepingwith the notion that the IL-10 genomic locus is in an inactivestate in human neutrophils, whereas it is poised for activation inmonocytes (20).

Chromatin organization of the IL-10 locus in activated neutrophilsand monocytes

Because induction of inflammatory gene expression is asso-ciated with chromatin remodeling and changes in histone

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modifications (35), we examined chromatin marks in neu-trophils and monocytes stimulated with LPS, SAA, orPam3CSK4 (Supplemental Fig. 1). Although activationmarks (especially H3K4me3 and H3K27Ac) were all upreg-ulated in activated monocytes, no substantial changes wereobserved in activated neutrophils (Supplemental Fig. 1).Similar results for IL-10 promoter and intragenic regionsamplified by primer sets 5 and 8 (as depicted in Fig. 1A) wereobserved in neutrophils and monocytes, either cultured asearlier for up to 5 h (data not shown) or stimulated with 500mg/ml zymosan, 100 mg/ml b-glucan or b-glucan plus LPS(data not shown). Conversely, H3K4me3, H4Ac, H3K4me1,and H3K27Ac were all detected at the CCL4 gene promoterin neutrophils, being (with the exception of H3K4me1) fur-ther increased upon LPS, SAA, or Pam3CSK4 stimulation,even at relatively higher levels than in monocytes (Supple-mental Fig. 1). Furthermore, ChIP of a number of tran-scription factors (TFs), previously proposed to bind to and/ortransactivate the IL-10 gene in various cells of human ormouse origin (36, 37), revealed no binding of C/EBPb (Fig.2A, left graph), c-FOS (Fig. 2B, left graph), SP1 (Fig. 2C, leftgraph), and NF-kBp50 (Fig. 2D, left graph) to the IL-10promoter of neutrophils, either constitutively or upon acti-

vation with LPS. The same TFs, however, were found to bindto neutrophil promoters of other known target genes (1) (Fig.2, right graphs). Altogether, data are all consistent with aninactive state of the IL-10 genomic locus in human neu-trophils.

Chromatin organization of the IL-10 locus in neutrophils isolated frommelanoma patients

Subsequently, we examined chromatin marks also in mela-noma neutrophils, because they were reported to producedramatically high amounts of IL-10 (14), a finding that wefailed to reproduce (3). We observed that, similar to neutro-phils of healthy subjects, neutrophils isolated from stage IVmelanoma patients, and cultured without or with SAA orLPS, did not exhibit any H3K4me3 and H4Ac marks (Fig. 3)at regions of the IL-10 locus that showed very high signals inmonocytes. The latter observations suggest that no chromatin

FIGURE 1. Profiles of H3K4me3, H4Ac, H3K27Ac, and H3K4me1 levels

at the IL-10 genomic locus of human neutrophils and monocytes. (A) The

schemes illustrate the position, within the human IL-10 genomic locus, of

the 18 primer sets that were designed and used for our ChIP analysis. The

numbers above the representation of the IL-10 genomic locus (upper scheme)

indicate the distance from the IL-10 transcription start site, as expressed in

kilobytes. (B–E) Neutrophils (upper graphs) and monocytes (lower graphs),freshly isolated from the peripheral blood of healthy donors, were processed

for ChIP analysis using Abs toward H3K4me3 (B), H4Ac (C), H3K27Ac (D),

and H3K4me1 (E). Precipitated DNA samples were amplified using primer

pairs specific for the regions of the human IL-10 locus, as indicated in (A).

Results are expressed as percentages over input DNA. (B–E) One represen-

tative experiment out of three with similar results is demonstrated.

FIGURE 2. TF binding at the IL-10 genomic locus of human neutrophils.

Neutrophils were cultured in the presence or absence of 100 ng/ml LPS for

3 h and then were processed for ChIP analysis using Abs toward C/EBPb (A),

c-FOS (B), SP1 (C), and NF-kB p50 (D). Precipitated DNA samples were

amplified using primer pairs positioned near the TF binding regions in the

IL-10 (left graphs), CXCL8, or TNF-a promoters (right graphs). TF binding to

the IL-10 promoter was detected by using primer set 8 (see lower scheme inFig. 1A) for c-FOS, SP1, and NF-kB p50, and primer set 9 (see lower schemein Fig. 1A) for C/EBPb. Results are expressed as percentages over input

DNA. One representative experiment out of three with similar results is

depicted.

FIGURE 3. Profiles of H3K4me3 and H4Ac levels at the IL-10 locus of

neutrophils isolated from melanoma patients. Neutrophils isolated from

melanoma patients were cultured for 2 h in the presence or absence of 100

ng/ml LPS and 5 mg/ml SAA. Four regions of IL-10 locus that showed the

highest signals in monocytes (numbers relate to the scheme of Fig. 1A) were

analyzed by ChIP for H3K4me3 (A) and H4Ac (B) levels. Changes in

H3K4me3 (A) and H4Ac (B) levels were analyzed at the CCL4 and prolactin

promoters as well (left graphs). Data from quantitative PCR are expressed as

percentages over input DNA and are displayed as means 6 SE of three in-

dependent experiments.

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reorganization occurs at the IL-10 genomic locus of mela-noma neutrophils.

Chromatin organization of the IL-10 locus in mouse neutrophils

Finally, we measured chromatin marks and TF binding also inmouse neutrophils (Supplemental Fig. 2). As a result, we de-tected increased levels of H3K4me3, H4Ac, H3K4me1, andH3K27Ac at syntenic regions of the IL-10 locus of unsti-mulated cells (Supplemental Fig. 2A–E), as well as a detect-able C/EBPb, c-FOS, SP1, and NF-kBp50, binding to theIL-10 locus upon cell activation with LPS (Supplemental Fig.2F–I), thus in full agreement with the well-known potentialcapacity of mouse neutrophils to express and release IL-10 (3,15, 16, 38). All in all, our experiments clearly suggest thatdifferent regulatory circuits and lineage-specific TFs controlthe state of the IL-10 locus in human and mouse neutrophils.In conclusion, overall, data shown in this article are consistent

with the notion that human neutrophils, either from blood ofhealthy subjects or frommelanoma samples, cannot produce IL-10 upon activation. Because the contrasting results related to IL-10 expression by human neutrophils (5–14) were not explainedby different experimental settings, peculiar purification proto-cols, or cell desensitization (3), the results shown in this articleindicate that lack of IL-10 induction in human neutrophilscorrelates with an inactive and neutrophil-specific basal chro-matin organization. Accordingly, our data indicate that chro-matin at the IL-10 genomic locus is differentially organized inhuman neutrophils and monocytes, and only in the latter itappears to be poised for transcription in response to stimula-tion. Therefore, basal differences in the chromatin state of theIL-10 locus, rather than a different responsiveness to activatingsignals, likely account for the differential ability of neutrophilsand monocytes to switch on transcription of this specific gene.Although we cannot exclude that yet-undefined situationsmight provoke major reorganization of the IL-10 genomic locusof human neutrophils, thus making it permissive to activation,our current results exclude that human neutrophils can expressIL-10 under standard conditions. Consequently, because it hasbeen reported that interaction of neutrophils with invariant NKT cells would result in less neutrophil-derived IL-10, a phe-nomenon supposed to have major influences on the diseaseoutcome in melanoma patients (14), our current data promptfor a reevaluation of planning to target presumed immuno-suppressive, IL-10–producing neutrophils (14).

AcknowledgmentsWe are grateful to Federica Calzetti and Elena Caveggion for expert technical

assistance.

DisclosuresThe authors have no financial conflicts of interest.

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The Journal of Immunology 1925

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Tamassia N et al.�Supplemental Figure 1

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Tamassia N et al.�Supplemental Figure 2

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Tamassia N et al.: page 1�Supplemental Figure legends

Supplemental Figure 1. Dynamic changes of histone marks at the IL-10 locus in activated

neutrophils and monocytes. Neutrophils (upper graphs) and monocytes (lower graphs) were

cultured in the presence or absence of 100 ng/ml LPS, 5 μg/ml SAA and 1 μg/ml Pam3CSK4. After

2 h, cells were processed for ChIP analysis using antibodies toward H3K4me3 (A), H4Ac (B),

H3K27Ac (C) and H3K4me1 (D). The presence of histone modifications within the different

regions at the IL-10 locus (A-D, left graphs, numbered as depicted in Fig. 1A), or at the CCL4 and

PRL promoters (A-D, right graphs), was analyzed by qPCR. Results are expressed as percentages

over input DNA. Panels show one experiment representative of 3 performed with similar results.

Supplemental Figure 2. Profiles of H3K4me3, H4Ac, H3K27Ac and H3K4me1 levels and TF

binding at the IL-10 locus of mouse neutrophils. (A) The schemes illustrate the position, within

the murine IL-10 genomic locus, of the six primer sets that were designed and utilized for ChIP

analysis. Numbers above the representation of the IL-10 genomic locus (upper scheme) indicate the

distance from the IL-10 TSS, as expressed in kb. (B-E) Neutrophils were isolated by positive

selection with anti-Ly-6G beads from bone marrow of C57BL/6 mice and cultured without or with

100 ng/ml LPS for 2 h. Cells were then collected and processed for ChIP analysis using antibodies

toward H3K4me3 (B), -H4Ac (C), -H3K27Ac (D) and -H3K4me1 (E). Precipitated DNA samples

were amplified using primer pairs specific for the regions of the murine IL-10 locus, as indicated in

panel A, as well as for the PRL and SOD2 promoters. (F-I) Neutrophils were cultured in the

presence or absence of 100 ng/ml LPS for 3 h, and then processed for ChIP analysis using

antibodies toward C/EBP�, c-FOS, SP1 and NF-�B p50. Precipitated DNA samples were amplified

using primer pairs specific for the regions of the murine IL-10 locus, as indicated in panel A, as

well as for the PRL and CXCL2 promoters. TF binding to the IL-10 promoter was detected by

using primer set 4 (as depicted in the lower scheme of panel A) for C/EBP�, c-FOS and SP1, and

primer set 6 (as depicted in the lower scheme of panel A) for NF-�B p50. Results are expressed as

percentages over input DNA. Panel groups B-E and F-I depict representative experiments (n= 3 for

each group).

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