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Immunobiology 218 (2013) 76– 89

Contents lists available at SciVerse ScienceDirect

Immunobiology

jo u rn al homepage: www.elsev ier .de / imbio

he hypoxic environment reprograms the cytokine/chemokine expression profilef human mature dendritic cells

abiola Blengioa,1, Federica Raggia,1, Daniele Pierobonb,c, Paola Cappellob,c, Alessandra Evaa,irella Giovarelli b,c, Luigi Varesioa, Maria Carla Boscoa,∗

Laboratory of Molecular Biology, G. Gaslini Institute, Genova, ItalyCenter for Experimental Research and Medical Studies (CERMS), San Giovanni Battista Hospital, Torino, ItalyDepartment of Medicine and Experimental Oncology, University of Turin, Torino, Italy

r t i c l e i n f o

rticle history:eceived 6 December 2011eceived in revised form 31 January 2012ccepted 4 February 2012

eywords:hemokines/cytokinesendritic cellsene regulationypoxia

nflammation

a b s t r a c t

Myeloid dendritic cells (DCs) are professional antigen-presenting cells critical for the orchestration ofimmunity and maintenance of self-tolerance. DC development and functions are tightly regulated by acomplex network of inhibitory and activating signals present in the tissue microenvironment, and dys-regulated DC responses may result in amplification of inflammation, loss of tolerance, or establishmentof immune escape mechanisms. Generation of mature (m)DCs from monocytic precursors recruited atpathological sites occurs under condition of low partial oxygen pressure (pO2). However, the way in whichthe hypoxic microenvironment modulates the functions of these cells is still not clear. We demonstratethat chronic hypoxia (4 days, 1% O2) promotes the onset of a highly proinflammatory gene expressionprofile in mDCs generated from primary human monocytes, characterized by the modulation of a signif-icant cluster of genes coding for proinflammatory chemokines/cytokines and/or their receptors. Withinthe chemokine system, strong upregulation of genes encoding proteins chemotactic for neutrophils, suchas CXCL2, CXCL3, CXCL5, CXCL6, and CXCL8, and for activated/memory T lymphocytes, monocytes, andimmature (i) DCs, e.g. CCL20, CCL3 and CCL5, was observed, concomitant with decreased expression ofgenes coding for naive/resting T cells chemoattractants, CCL18 and CCL23. Other hypoxia-inducible genes

coded for cytokines with a primary role in inflammation and angiogenesis, including osteopontin, vas-cular endothelial growth factor, and IL-1�. mRNA modulation was paralleled by protein secretion. Theseresults suggest that conditions of reduced O2 availability reprograms mDCs toward a proinflammatorydirection by tuning the cytokine/chemokine repertoire, thus affecting their ability to regulate leukocytetrafficking and activation at pathological sites, with potential implications for the pathogenesis of chronic inflammatory diseases.

ntroduction

Myeloid dendritic cells (DCs) play a key role in the initia-

ion and amplification of innate and adaptive immunity and inhe maintenance of self-tolerance (Banchereau et al. 2000; Rossind Young 2005). They originate from bone marrow progenitors

Abbreviations: mDCs, mature dendritic cells; iDCs, immature DCs; H-mDCs,ypoxic mDCs; pO2, partial oxygen pressure; OPN, osteopontin; VEGF, vas-ular endothelial growth factor; IL-1, interleukin-1; MDMs, monocyte-derivedacrophages; PBMCs, peripheral blood mononuclear cells; qRT-PCR, real time PCR;

M, conditioned medium; CA12, carbonic anhydrase 12; HIFs, hypoxia-inducibleranscription factors; HRE, hypoxia responsive element.∗ Corresponding author at: Laboratorio di Biologia Molecolare, Istituto Gianninaaslini, Padiglione 2, L.go G.Gaslini 5, 16147 Genova Quarto, Italy.el.: +39 010 5636633; fax: +39 010 3733346.

E-mail address: mariacarlabosco@ospedale-gaslini.ge.it (M.C. Bosco).1 Both authors contributed equally to this work.

171-2985/$ – see front matter © 2012 Elsevier GmbH. All rights reserved.oi:10.1016/j.imbio.2012.02.002

© 2012 Elsevier GmbH. All rights reserved.

which circulate in peripheral blood as primary monocytes and arerecruited from the circulation by environmental factors to non-lymphoid peripheral tissues, where they differentiate and residein an “immature” stage (iDCs) at sites of potential pathogen entryor of chronic inflammation (Banchereau et al. 2000; Cavanaghand Von Andrian 2002). iDCs are specialized for antigen captureand processing, functioning as sentinels of the immune system(Mellman and Steinman 2001). Upon antigen uptake and activationby endogenous factors, such as proinflammatory cytokines and tis-sue damage-associated molecular patterns (DAMPs), or exogenousfactors, such as pathogen-associated molecular patterns (PAMPs),iDCs undergo phenotypic and functional changes that culminatein their maturation into (m)DCs, which express high surface lev-els of MHC/peptide complexes and costimulatory molecules and

have a higher capacity for antigen presentation and T-cell stimula-tion (Banchereau et al. 2000; Rossi and Young 2005). mDCs switchtheir chemokine receptor repertoire down-regulating inflamma-tory receptors, such as CCR1, CCR2, CCR5, CCR6, and upregulating

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hose required for homing to secondary lymphoid organs, namelyCR7, where they prime naive T cells triggering specific immuneesponses (Rossi and Young 2005; Cavanagh and Von Andrian 2002;llavena et al. 2000). DC maturation is also associated with pro-

ound changes in the expression profile of both homeostatic (CCL17,CL22, CCL19, and CCL18) and pro-inflammatory (CCL2, CCL3, CCL5,CL20, CXCL8, CX3CL1, CXCL10, and CXCL16) chemokines (Visserst al. 2001; Vulcano et al. 2003; Adema et al. 1997).

The local microenvironment contributes to the regulation ofC development and functional behavior (Lin et al. 2010; Laouit al. 2011; Allavena et al. 2000). A common feature of injured andnflamed tissues is represented by hypoxia, a condition of low par-ial oxygen pressure (pO2, 0–20 mm Hg) which arises as a result ofisorganized or dysfunctional vascular network and diminished O2upply and creates a unique microenvironment affecting cell phe-otype and functional behavior (Semenza 2001; Muz et al. 2009;osco et al. 2008b). Hence, DC development from monocytic pre-ursors recruited at pathological sites occurs under the setting ofeduced pO2, and recent evidence indicates that low O2 environ-ent can impact on DC differentiation, maturation, and functional

roperties promoting the acquisition of a “hypoxic” phenotype sub-tantially different from that of the normoxic counterpart (Ricciardit al. 2008; Elia et al. 2008; Mancino et al. 2008; Rama et al. 2008;osco et al. 2008b, 2011; Yang et al. 2009; Ogino et al. 2011; Jantscht al. 2008; Melillo 2011). The aim of this study was to charac-erize the molecular bases underlying the functional behavior of

DCs generated from primary human monocytes within a hypoxicnvironment similar to that present in diseased tissues.

We present data showing that mDC development under long-erm hypoxic conditions (1% O2, 4 days) is associated with profoundhanges in their cytokine/chemokine gene expression profile, lead-ng to the onset of a highly proinflammatory phenotype.

aterials and methods

eneration of human monocyte-derived mDCs

mDCs were generated as described previously (Bosco et al.011). Briefly, human peripheral blood mononuclear cells (PBMCs)ere collected from venous blood of healthy volunteers by den-

ity gradient separation over a Ficoll cushion (Histopaque, Sigma,ilano, Italy), and monocytes were isolated using paramagneticicroBeads (Human Monocyte Isolation Kit-II, Miltenyi Biotec,

ologna, Italy) at a purity of >93% CD14+. To generate mDCs, mono-ytes were plated into six-well culture plates (1.5 × 106 cells/ml)BD Falcon, Sacco, Milano) in RPMI 1640 (Euroclone, Celbio, Milano)upplemented with 10% heat-inactivated FCS (HyClone, Celbio) andncubated for 4 days under normoxic (20% O2) or hypoxic (1% O2)onditions in the presence of human recombinant GM-CSF and IL-4both 100 ng/ml), as detailed (Elia et al. 2008; Ricciardi et al. 2008). Aocktail of proinflammatory mediators containing TNF� (50 ng/ml),L-1� (50 ng/ml), IL-6 (10 ng/ml), and PGE2 (1 mM) was added forhe last 48 h to induce DC maturation. All cytokines were fromeproTech Inc. (DBA, Milano), GM-CSF was from Miltenyi, PGE2rom Sigma. Hypoxic conditions were obtained by culturing cellsn an anaerobic work-station incubator (BUGBOX, CARLI Biotec,oma, Italy) flushed with a mixture of 1% O2, 5% CO2, and 94% N2.edium was allowed to equilibrate in a loosely capped flask in the

ypoxic incubator for 2 h before use, and pO2 was monitored using portable oxygen analyzer (Oxi 315i/set, WTW, Germany).

low cytometry

Flow cytometry was performed as described (Bosco et al. 2011).ells resuspended with FACS buffer (PBS supplemented with 0.2%

ogy 218 (2013) 76– 89 77

BSA, 0.01% NaN3) were incubated with fluorochrome-conjugatedmAbs for 30 min at 4 ◦C, after blocking nonspecific sites with rab-bit IgG (Sigma). Fluorescence was quantitated on a FACSCaliburflow cytometer equipped with CellQuest software (BD-Biosciences,Milano). Cells were gated according to their light-scatter propertiesto exclude cell debris. The following mAbs were used: anti-CD83-PE, anti-CXCR4-PE, and anti-CCR7-APC (Biolegend, Campoverde,Milano), anti-CD86-PE (BD-Pharmingen, Milano), anti-CD1a-FITC(Serotec, SPACE, Milano). Proper isotype-matched control Abs(Biolegend) were used.

RNA isolation, GeneChip hybridization and array analysis

Gene expression profiling was performed as described previ-ously (Bosco et al. 2006, 2011). Briefly, total RNA was purifiedfrom different donor-derived mDCs using the RNeasy MiniKit fromQiagen (Milano) and controlled for integrity with an Agilent Bioan-alyzer 2100 (Agilent Technologies Europe, Waldbroon, Germany).RNA was quantified by NanoDrop (NanoDrop Technologies,Wilmington, USA) and reverse-transcribed into double-strandedcDNA on a GeneAmp PCR System 2700 thermal cycler (AppliedBiosystems, Milano) using the one-cycle cDNA synthesis kit(Affymetrix, Milano). cDNA derived from three donors was puri-fied and biotin labeled with the IVT-expressed kit (Affymetrix).Labeled cRNA was fragmented according to Affymetrix’s instruc-tions and used for hybridization to Affymetrix HG-U133 plus2.0 arrays (Genopolis Corporation, Milano) containing 54,000probe sets coding for 38,500 genes. Chips were stained withstreptavidin–phycoerythrin (Invitrogen Life Technologies, Milano)and scanned using an Affymetrix GeneChip Scanner 3000. Datacapturing was conducted with standard Affymetrix analysis soft-ware algorithms (Microarray Suite 5.0). Comparative analysis ofhypoxic relative to normoxic expression profiles was carried outon GeneSpring Expression Analysis Software Gx9.0 (Silicon Genet-ics, Redwood City, CA). Gene expression data were normalizedusing “per chip normalization” and “per gene normalization”algorithms implemented in the GeneSpring program. Gene expres-sion levels were averaged, and fold-change was calculated asthe ratio between the average expression level under hypoxiaand normoxia. We selected a modulated gene list of ≥two foldinduction/inhibition. The significance of gene expression differ-ences between the two experimental conditions was calculatedusing the Mann–Whitney U-test. Only genes that passed the testat a confidence level of 95% (P < 0.05) were considered signif-icant. Complete data set for each microarray experiment waslodged in the Gene Expression Omnibus public repository at NCBI(www.ncbi.nlm.nih.gov/geo/) [accession Nr. GSE22282].

HRE consensus elements consisting of a 4nt core (CGTG)flanked by degenerated sequences ((T|G|C)(A|G)(CGTG)(C|G|A)(G|C|T)(G|T|C)(C|T|G)) were mapped in the promoter regions ofgenes represented in the chip, as detailed (Ricciardi et al. 2008).

Real-time RT-PCR

Real time PCR (qRT-PCR) was performed on a 7500 Real TimePCR System (Applied), using SYBR Green PCR Master Mix andsense/antisense oligonucleotide primers from TIBMolbiol (Gen-ova, Italy) (listed in Table 1) or from Quiagen (Milano) (CCL18,CCR2, RSP18), as detailed (Bosco et al. 2006). Expression data werenormalized on the values obtained in parallel for three reference

genes (indicated in Table 1) selected among those not affectedby hypoxia in the Affymetrix analysis, using the Bestkeeper soft-ware, and relative expression values were calculated using Q-genesoftware.

78 F. Blengio et al. / Immunobiology 218 (2013) 76– 89

Table 1Primer pairs used for real-time quantitative RT-PCR.

Gene Primers

Actin related protein 2/3 complex, subunit 1B (ARPC1B)a For 5′-aacgagaacaagtttgctgtg-3′

Rev 5′-gatgggcttcttgatgtgc-3′

Arginase, type II (ARG2) For 5′-attggtctgagagacgtggac-3′

Rev 5′-gccagtgtagggtcaaatgc-3′

BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) For 5′-ttccatctctgctgctctc-3′

Rev 5′-tggtggaggttgtcagac-3′

Carbonic anhydrase XII (CA12) For 5′-cttggcatctgtattgtggtg-3′

Rev 5′-tgggcctcagtctccatc-3′

Chemokine (C-C motif) ligand 3 (CCL3) For 5′-tttcagacttcagaaggacacg-3′

Rev 5′-tgatgcagagaactggttgc-3′

Chemokine (C-C motif) ligand 5 (CCL5) For 5′-acagcctctcccacaggtac-3′

Rev 5′-caatgtaggcaaagcagcag-3′

Chemokine (C-C motif) ligand 20 (CCL20) For 5′-aatttattgtgggcttcacacg-3′

Rev 5′-acccaagtctgttttggatttg-3′

Chemokine (C-C motif) receptor 6 (CCR6) For 5′-accgattgcctactccttga-3′

Rev 5′-caatggccatgttcaagagat-3′

Chemokine (C-C motif) ligand 23 (CCL23) For 5′-ctttgaaacgaacagcgagtg-3′

Rev 5′-cttgtgtcccttcaccttg-3′

Chemokine (C-X-C motif) ligand 2 (CXCL2) For 5′-cggcagggaaatgtatgtgt-3′

Rev 5′-catgagaaatgttgaccaca-3′

Chemokine (C-X-C motif) ligand 3 (CXCL3) For 5′-tcatcaaacatagctcagtcctg-3′

Rev 5′-ggctgacacattatggtctcc-3′

Chemokine (C-X-C motif) ligand 5 (CXCL5) For 5′-tgtttgccgcttaagctttc-3′

Rev 5′-tggctcacactatagtcaattgc-3′

Chemokine (C-X-C motif) ligand 6 (CXCL6) For 5′-tttacgcgttacgctgagag-3′

Rev 5′-gacaaacttgcttcccgttc-3′

Chemokine (C-X-C motif) ligand 8 (CXCL8) For 5′-gtgtgaaggtgcagttttgc-3′

Rev 5′-tctgcacccagttttccttg-3′

Enolase 2 (ENO2) For 5′-cctgtatcgccacattgctc-3′

Rev 5′-catgagagccaccattgatc-3′

Interleukin 1, alpha (IL1a) For 5′-aagaccaaccagtgctgctg-3′

Rev 5′-aacaagtttggatgggcaac-3′

Interleukin 1, beta (IL1b) For 5′-tgatgtctggtccatatgaactg-3′

Rev 5′-tgtacaaaggacatggagaacac-3′

Interleukin 23, alpha subunit p19 (IL23A) For 5′-gccagcagctttcacagaag-3′

Rev 5′-ccctcttctcttagatccatgtg-3′

Interleukin 17 receptor B (IL17BR) For 5′-ggcagggatctatctaatgtgg-3′

Rev 5′-agatgggtaaaccacaagaacc-3′

Interleukin 1 receptor antagonist (IL1RN) For 5′-tcatgctctgttcttgggaat-3′

Rev 5′-gcttgtcctgctttctgttc-3′

Macrophage migration inhibitory factor (MIF) For 5′-gtccttctgccatcatgc-3′

Rev 5′-gaaggccatgagctggt-3′

Secreted phosphoprotein 1 (OPN) For 5′-tgacccatctcagaagcag-3′

Rev 5′-atggctttcgttggacttac-3′

Ribosomal protein S19 (RPS19)a For 5′-aaagacgtgaaccagcagg-3′

Rev 5′-ttctcatcgtagggagcaag-3′

Thrombospondin 1 (THBS1) For 5′-cagcattctccatcaggaac-3′

Rev 5′-gaggaatggactgttgatagc-3′

Vascular endothelial growth factor A (VEGFA) For 5′-gcagcttgagttaaacgaacg-3′

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a Indicates the reference genes used for data normalization. Primers for RSP18 (Q

LISA

Conditioned medium (CM) from monocyte-derived mDCs waseplaced on day 3 of generation with fresh medium supplementedith cytokines for 24 h, both under normoxic and hypoxic condi-

ions. On day 4, the following cytokines were measured in cell-freeupernatants by specific ELISA according to the manufacturer’snstructions: CCL20, CXCL5, OPN, VEGF CCL3, CCL5, and CCL18 (R&Dystems), CCL23 (RayBiotech, Tebubio, Magenta, Milano), CXCL2Promokine, PBI, Milano), CXCL8 and IL-1� (Biolegend, Campov-rde, Mlano). The optical density of the plates was determinedsing a Spectrafluor Plus plate reader from TECAN (Milano, Italy).ll assays were done in duplicate and repeated three times.

tatistical analysis

Analyses were performed using unpaired Student’s t-testGraph-Pad Prism version 5.00, GraphPad Software Inc., San Diego,SA). Data are the mean ± SE of six independent experiments,

ev 5′-gcagcgtggtttctgtatc-3′

n) were also used for data normalization.

unless differently specified. Results were considered significant ifP ≤ 0.05 (*P ≤ 0.05, **P ≤ 0.01, and ***P < 0.001).

Results

Characterization of the cytokine/chemokine gene expressionprofile of hypoxic mDCs

We have recently investigated the gene expresson profile ofmDCs generated from human monocytes under chronic hypoxicconditions (H-mDCs) and demonstrated profound changes in theexpression of a large number of genes clustered into various cate-gories according to GO data mining for biological processes, respectto mDCs generated under normal O2 levels (Bosco et al. 2011).A statistically significant portion of genes displaying at least 2-

fold differential expression levels in three different donors fellinto pathways implicated in immune regulation, inflammatoryresponses, angiogenesis, and cell migration, indicating a func-tional distinction between normoxic and hypoxic mDCs (Bosco

F. Blengio et al. / Immunobiology 218 (2013) 76– 89 79

Table 2Relative expression of genes encoding chemokines/cytokines and their receptors in H-mDCs vs mDCs.a

Gene bank accessionno.

Gene symbol Full name Main function(s) of geneproduct

Fold changeb HREc Modulated ind

Monocytes Macrophages iDCs

Chemokine/ReceptorsUp-regulatedNM 004591 CCL20 Chemokine (C-C motif)

ligand 20 (MIP-3�)Chemotactic factor foriDCs, effector/memory Tcells, and naive B cells;expressed at sites ofinflammation andimplicated in thepathogenesis of chronicinflammatory diseases,tumor progression, andmetastatic spread

9.35 + Up Up Up

BC005276 CXCL2 Chemokine (C-X-Cmotif) ligand 2 (Gro-�)

Chemotactic factor forneutrophils andhematopoietic stem cells;monocyte-arrestchemokine; expressed atsites of inflammation

5.63 + Up − Up

BG166705 CXCL5 Chemokine (C-X-Cmotif) ligand 5 (ENA78)

Chemotactic factor forneutrophils; plays a role ininflammation

5.26 + Up − −

AF043341 CCL5 Chemokine (C-C motif)ligand 5 (RANTES)

Chemotactic factor formonocytes, memory Thcells, basophils,eosinophils, iDCs, and NKcells; activating stimulusfor basophils andeosinophils; recruitsleukocytes at inflammatorysites

3.61 + − − Up

NM 002993 CXCL6 Chemokine (C-X-Cmotif) ligand 6 (GCP-2)

Chemotactic factor forneutrophils

3.58 + Down − −

NM 002090 CXCL3 Chemokine (C-X-Cmotif) ligand 3 (Gro-�)

Chemotactic factor forneutrophils;monocyte-arrestchemokine; plays a role ininflammation

3.49 − Up − −

NM 000584 CXCL8 Chemokine (C-X-Cmotif) ligand 6 (IL-8)

Neutrophil chemotacticand activating factor;proangiogenic chemokine

3.05 − − Up Up

NM 002983 CCL3 Chemokine (C-C motif)ligand 3 (MIP-1�)

Chemoattractant formonocytes, activated Tcells, NK cells, eosinophils,IDC s and hematopoieticprogenitors; plays anautocrine role in DCaccumulation at sites ofinflammation

2.96 + − − Up

NM 002995 XCL1 Chemokine (C motif)ligand 1

Chemotactic factor forlymphocytes

1.85 + − − −

NM 000647 CCR2 Chemokine (C-C motif)receptor 2

Receptor for CCL2, 6, 7, 8,12, and 13 chemokines;plays a role in DCrecruitment toinflammatory sites

1.68 + Down Up −

NM 016951 CKLF Chemokine-like factor Potent chemoattractant forneutrophils, monocytes,and lymphocytes; canstimulate the proliferationof murine skeletal musclecells

1.20 + − − −

Down-regulatedNM 002988 CCL18 Chemokine (C-C motif)

ligand 18Chemotactic factor fornaive CD4+ and CD8+ Tcells and nonactivated Tlymphocytes

4.05 − Down Down −

NM 005064 CCL23 Chemokine (C-C motif)ligand 23

Chemotactic for resting Tlymphocytes; strongsuppressor of colonyformation bymultipotentialhematopoietic progenitorcells

4.02 + Down Down −

80 F. Blengio et al. / Immunobiology 218 (2013) 76– 89

Table 2 (Continued )

Gene bank accessionno.

Gene symbol Full name Main function(s) of geneproduct

Fold changeb HREc Modulated ind

Monocytes Macrophages iDCs

NM 006419 CXCL13 Chemokine (C-X-Cmotif) ligand 13

B lymphocytechemoattractant

3.62 + − − −

NM 004367 CCR6 Chemokine (C-C motif)receptor 6

Receptor of CCL20,preferentially expressed byiDCs and memory T cells;regulates their migrationduring inflammatory andimmune responses

1.38 + − − −

Cytokine/ReceptorsUp-regulatedAB019562 OPN Osteopontin (SPP1) Pleiotropic

cytokine/extracellularmatrix phosphoprotein;plays a critical role in theactivation of type Iimmunity and tumorgrowth, progression andspread; endowed withendothelial cell (EC)adhesive capacity andchemotactic activity for EC,monocytes, and Tlymphocytes; potentialbiomarker andproangiogenic mediator incardiovascular disease andrheumatic arthritis

6.28 + Up Up −

M15329 IL-1a Interleukin 1, alpha Multifunctional,proinflammatory,proangiogenic cytokine;stimulates thymocyteproliferation, B-cellmaturation andproliferation; fibroblastgrowth factor; endogenouspyrogen; involved in tjhepathogenesis ofautoinflammatory diseases

4.52 − Up Up Up

U65590 IL-1RN Interleukin 1 receptorantagonist

Receptor for IL-1,antagonizes IL-1 activity

3.83 + Up Up −

NM 002183 IL-3RA Interleukin 3 receptor,alpha (low affinity)

Interleukin-3 receptor,mediates IL-3 pleiotropicactivities on hematopoieticcell proliferation,diferentiation, apoptosis,migration, and effectoractivities

3.12 + − Up −

NM 000576 IL-1b Interleukin 1, beta Multifunctionalproinflammatory,proangiogenic cytokine;stimulates thymocyteproliferation, B-cellmaturation andproliferation; endogenouspyrogens; fibroblastgrowth factor; involved intjhe pathogenesis ofautoinflammatory diseases

3.04 + − − Up

NM 001657 AREG Amphiregulin(schwannoma-derivedgrowth factor)

EGF family growth factor,interacts with theEGF/TGF-alpha receptor topromote epithelial cellgrowth and inhibitscarcinoma cellproliferation

2.72 + Up − −

NM 002415 MIF Macrophage migrationinhibitory factor

Pleiotropic cytokine withmultuple effects inimmunoregulation andinflammation, includinginhibition of macrophagemotility

2.66 + Up Up Up

F. Blengio et al. / Immunobiology 218 (2013) 76– 89 81

Table 2 (Continued )

Gene bank accessionno.

Gene symbol Full name Main function(s) of geneproduct

Fold changeb HREc Modulated ind

Monocytes Macrophages iDCs

NM 016584 IL-23A Interleukin 23, alphasubunit p19

Subunit of IL-23;proinflammatory,proangiogenic andprocarcinogenic cytokine;regulates memory T cellfunctions; involved in Th17cell differentiation andTh17-dependentautoimmune inflammatorydiseases

2.61 + Up − Up

U64094 IL-1RB Interleukin 1 receptor,type II

Receptor for IL-1a, IL-1b,and interleukin-1 receptorantagonist (IL-1ra)

2.51 − − − −

AI738556 TRAILR4 Tumor necrosis factorreceptor superfamily,member 10

Receptor for TRAIL,contains a truncatedcytoplasmic death domainand plays an inhibitory rolein TRAIL-induced cellapoptosis

2.49 − − Up −

AF117297 TNFRSF18 Tumor necrosis factorreceptor superfamily,member 18

Member of theTNF-receptor superfamily,binds TNFSF18; maymodulate T-lymphocytesurvival in peripheraltissues, important forinteractions betweenactivated T-lymphocytesand endothelial cells

2.41 + − − −

BC010943 OSMR Oncostatin M receptor Member of type 1 cytokinereceptor family;heterodimerizes with IL-6signal transducer to formthe type II oncostatin Mreceptor and with IL-31receptor A to form theIL-31 receptor; regulatescytokine production

2.38 + − − −

NM 000759 CSF3 Colony stimulatingfactor 3

Growth factor, stimulateshematopoiesis in the bonemarow, inducing theproduction, differentiation,and function ofgranulocytes

2.20 + Up Up −

NM 016629 TNFRSF21 Tumor necrosis factorreceptor superfamily,member 21

Member of theTNF-receptor superfamily;activates NF-kB andMAPK8/JNK and inducescell apoptosis

2.20 − Up − −

NM 002192 INHBA Inhibin, beta A(activin A)

Involved in: the regulationof hypothalamic, pituitary,and gonadal hormonesecretion, germ celldevelopment andmaturation, erythroiddifferentiation, nerve cellsurvival, embryonic axialdevelopment, and bonegrowth

2.17 + Up − −

NM 019618 IL-1F9 Interleukin 1 family,member 9

Member of the IL-1 family;functions as an agonist ofNF-kB activation throughthe orphanIL-1-receptor-relatedprotein 2; is part of anindependent signalingsystem analogous to IL-1a,IL-1b receptor agonist andIL-1R

2.10 − Up − −

NM 002182 IL-1RAP Interleukin 1 receptoraccessory protein

Is part of themembrane-bound form ofthe IL-1 receptor; mediatesinterleukin-1-dependentactivation of NF-kB

2.00 − Up Up −

82 F. Blengio et al. / Immunobiology 218 (2013) 76– 89

Table 2 (Continued )

Gene bank accessionno.

Gene symbol Full name Main function(s) of geneproduct

Fold changeb HREc Modulated ind

Monocytes Macrophages iDCs

NM 001718 BMP6 Bone morphogeneticprotein 6

Secreted protein memberof TGF-� superfamily;plays pleiotropic effects ondifferent cell types;induces cell death inactivated memory B cells

1.93 + − − −

NM 003855 IL-18R1 Interleukin 18receptor 1

Receptor for theproinflammatory cytokineIL-18; leads to NF-kBactivation

1.85 − Down Up −

AF022375 VEGFA Vascular endothelialgrowth factor

EC-specific mitogen; playsa central role in drivingangiogenesis andvasculogenesis viastimulation of EC survival,proliferation andchemotaxis; promotesmonocytic cellrecruitment/activation

1.63 + Up Up Up

NM 145259 ACVRLK7 Activin A receptor,type IC

Plays a role indifferentiation, growtharrest and apoptosis ofhuman monocyte-derivedDC and CD1c(+) andCD123(+) peripheral bloodDCs

1.58 − − − −

NM 005211 CSF1R Colony stimulatingfactor 1 receptor

Receptor for CSF-1;mediates biological effectsof this cytokine on celldifferentiation andfunction

1.32 + Down Up −

Down-regulatedNM 018725 IL17BR Interleukin 17

receptor BReceptor for IL-17;mediates NF-kB activationand CXCL8 production;plays a role in controllingthe growth and/ordifferentiation ofhematopoietic cells;immunoregulatory activity

3.05 − − Down −

NM 004619 TRAF5 TNFreceptor-associatedfactor 5

TNFR superfamily member;binds to TNFR cytoplasmicdomains and mediate TNFpro-apoptotic effects byactivating NF-kB and JNK

1.20 + − Down −

a mDCs and H-mDCs were generated by culturing human monocytes under normoxic (20% O2) and hypoxic (1% O2) conditions in the presence of GM-CSF/IL-4 for 4 daysand a cocktail of pro-inflammatory stimuli for the last 48 h. Gene expression profiling was then carried out independently by microarray analysis on the RNA purified fromthree different mDC and H-mDC preparations, and comparative analysis of gene expression differences between the two experimental conditions was conducted as describedin the Materials and Methods. A GeneBank accession number, a common gene symbol, a full name, a brief description of the gene product main functions, and the fold changevalue are specified for each gene. Genes in each group are ordered by fold change. Those validated by qRT-PCR are underlined.

b Results are expressed as log2 ratio of fold differences between hypoxic and normoxic samples (mean of expression level of three experiments). Genes up/downmodulatedby ≥2 fold are shown.

c The + sign indicates genes whose promoter contain members of the HRE family. and o

F

ettimr(CCggamc

d Comparison of microarray results with those previously obtained in monocytesang et al. (2009), Ricciardi et al. (2008), Elia et al. (2008) and Yang et al. (2009).

t al. 2011). To identify genes not previously characterized inerms of responsiveness to hypoxia in mDCs, further analysis ofhese pathways was carried out. We found profound differencesn the expression of a subset of 39 genes coding for proinflam-

atory cytokines/chemokines and/or their receptors in H-mDCselative to mDCs, the majority of which (85%) was upregulatedTable 2). Within the chemokine system, the CC chemokines,CL20, CCL3, and CCL5, and the CXC chemokines, CXCL2, CXCL3,XCL5, CXCL6, and CXCL8, were identified as hypoxia-inducibleenes, whereas expression of CCL18-, CCL23-, and CXCL13-coding

enes was highly inhibited by hypoxia. The genes coding for CCR2nd CCR6 were also selectively up- and down-regulated in H-DCs respect to mDCs. Other hypoxia-inducible genes coded for

ytokines with a primary role in angiogenesis and inflammation,

ther monocyte-derived cells exposed to hypoxia, as reported by Bosco et al. (2006),

such osteopontin (OPN), macrophage migration inhibiting factor(MIF), interleukin (IL)-23A, colony stimulating factor 3 (CSF3), andvascular endothelial growth factor (VEGF). Of interest is also theupregulation by hypoxia of genes encoding various components ofthe IL-1 family of ligands and receptors, such as IL-1�, IL-1�, IL-1 receptor antagonist (IL-1RN), IL-1 receptor type 2 (IL-1RB), IL-1family member 9 (IL1F9), IL-1 receptor accessory protein (IL1RAP),IL-18R1, which are critically implicated in acute and chronic inflam-mation. Finally, mDCs hypoxic transcriptome was characterizedby the differential modulation of genes encoding members of

the tumor necrosis factor receptor superfamily (TNFRSF), thatwere selectively up-regulated (TRAILR4; TNFRSF18; TNFRSF21) ordown-regulated (TNFR-associated factor 5, TRAF5). These resultsdemonstrate that mDC generation under conditions of reduced O2

nobiol

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obpiMi2rpfifsWli

FwroCD

F. Blengio et al. / Immu

vailability strongly influences their cytokine/chemokine reper-oire, suggesting that prolongued hypoxia can exert a profound

odulatory effect on mDC immune response.As shown in Table 2, some of the observed hypoxia-induced

hanges in gene expression were shared with other monocytic-ineage cells either exposed to acute hypoxia, such as primary

onocytes (Bosco et al. 2006) and monocyte-derived macrophagesMDMs) (Fang et al. 2009), or generated under chronic hypoxia,uch as monocyte-derived iDCs (Ricciardi et al. 2008). However,pecific differences existed among the four cell types (Table 2).nterestingly, CCL20, IL-1�, MIF, and VEGF were the only genesound commonly upregulated by hypoxia in all the myeloid lineageells examined, whereas other genes induced in H-mDCs were notffected (e.g. OSMR, IL-1RB, and BMP6) or even downregulated (e.g.XCL6, IL-18R1, and CSF1R) in the other mononuclear phagocyteopulations. We conclude that hypoxia can selectively modulatehe cytokine/chemokine network in cells belonging to the mono-ytic lineage depending on their differentiation/maturation stage.

Response to hypoxia is primarily under the molecular controlf a family of hypoxia-inducible transcription factors (HIFs), whichind to and transactivate the hypoxia responsive element (HRE)resent in the promoter of many hypoxia-inducible genes, activat-

ng gene transcription (Semenza 2001; Cummins and Taylor 2005;uz et al. 2009). Accumulation of the O2-sensitive HIF-1� subunit

n H-mDCs was recently reported (Elia et al. 2008; Mancino et al.008). The possible relationship between cytokine/chemokine-eceptor gene inducibility by hypoxia and HRE presence in theromoter was investigated by mapping the HRE sequences in therst 2000 bases upstream the transcription initiation site. The

requency of HRE+ genes spotted on the chip was about 60% repre-

enting the background of HRE-containing genes in our population.

e found that almost 70% of both upregulated and downregu-ated genes contained at least one member of the HRE familyn the promoter, whereas the other 30% were HRE- (Table 2),

Chemokines/ receptors

Cytokines/receptors

-4 -2 20

IL17BRIL23A

IL-1RNVEGFA

IL-1MIFIL-1OPN

CCL18CCL23CCR6CCL3CXCL8CXCL6CXCL2CCR2CCL5CXCL3CXCL5CCL20

ARG2THBS1BNIP3ENO2CA12

Controlgenes

L

ig. 1. qRT-PCR analysis of genes selected from the microarray profile. Equal amounts oere pooled and subjected to qRT-PCR, as detailed in the Materials and Methods. Expres

elation to the values obtained for three reference genes. Known hypoxia gene targets

f fold-changes (1% O2 relative to 20% O2) and are the mean of triplicate determinationhemokine/cytokine genes not previously reported to be modulated by hypoxia in mDCs

Cs are highlighted in boldface.

ogy 218 (2013) 76– 89 83

indicating the involvement of hypoxia-responsive factors otherthan HIFs in the transactivation of a substantial number of genes inH-mDCs, similarly to what previously shown in H-iDCs (Ricciardiet al. 2008).

Confirmation of microarray data by qRT-PCR analysis of selectedhypoxia-modulated genes

To validate the microarray results, mRNA levels of a subset of20 genes selected among those listed in Table 2 were quantified byqRT-PCR on total RNAs from the mDCs and H-mDCs preparationsanalyzed by microarray, using the primer pairs indicated in Table 1.The expression of known hypoxia target genes, such as arginase2 (ARG2), BCL2/adenovirus E1B interacting protein 3 (BNIP3), car-bonic anhydrase XII (CA12), enolase 2 (ENO2), and thrombospondin1 (THBS1) (Semenza 2001; Muz et al. 2009; Bosco et al. 2006, 2008b,2011), whose upregulation in H-mDCs was confirmed by microar-ray analysis (www.ncbi.nlm.nih.gov/geo/, accession Nr. GSE22282),was assessed in parallel as positive controls. Three reference genes(actin related protein 2/3 complex subunit 1B, ARCP1B; ribosomalproteins 18, RSP18; and RSP19) were used for data normalization(Table 1). Relative mRNA expression levels are shown in Fig. 1.We found full concordance between qRT-PCR and Affymetrix datawith respect to the direction of the expression changes. For eight ofvalidated genes, such as CCL20, CCL5, CCR6, CXCL6, CXCL8, IL-1�,IL-1�, and OPN, the extent of modulation was also of compara-ble magnitude, whereas fold-differences were higher according to

qRT-PCR for CCR2, CXCL3, CXCL5, MIF, and VEGF, and to microarrayfor CCL18, CCL23, CCL3, CXCL2, IL-1RN, IL-23A, and IL-17BR. Theseresults confirm hypoxia responsiveness of chemokine/cytokine-receptor genes identified by microarray.

64 108

og2 ratio

f total RNA from the three donor-derived mDCs and H-mDCs tested by microarraysion changes of 20 genes selected from those listed in Table 2 were evaluated inwere analyzed in parallel as positive controls. Results are expressed as log2 ratios for each target transcript. Genes are ordered by fold change within each group.are underlined, whereas those identified for the first time as hypoxia-responsive in

84 F. Blengio et al. / Immunobiol

Table 3Phenotype of monocyte-derived mDCs.a

Surface marker mDCs H-mDCs

CD1a 86 ± 3b 82 ± 4CD83 89 ± 2 86 ± 3CD86 97 ± 2 98 ± 1CCR7 77 ± 4 78 ± 2CXCR4 75 ± 3 77 ± 3

a Human monocytes were cultured with IL-4/GM-CSF for 48 h and with the proin-flammatory mediators, TNF�, IL-1�, IL-6, PGE2 for additional 48 h under 20%O2

(mDCs) or 1%O2 (H-mDCs). Surface expression of the indicated markers was deter-m

p

Cp

gindeam

FmfCmeomre

ined by flow cytometry, as detailed in Materials and Methods.b Data are expressed as the mean percentage of positive cells ± SE from 6 inde-

endent donors.

hronic hypoxia promotes the onset of a highly proinflammatoryhenotype in mDCs

We selected a few hypoxia-modulated chemokine and cytokineenes for further analysis, based on their relevance for mDCmmunoregulatory/inflammatory functions. To quantify the mag-itude of hypoxia-induced changes and address the issue ofonor-to-donor variability, we compared mRNA transcript lev-

ls by qRT-PCR in monocyte-derived mDCs generated from

panel of 6 independent donors under hypoxic and nor-oxic conditions. As confirmed by flow cytometry, both cell

ig. 2. Hypoxia-induced changes in chemokine/cytokine mRNA expression levels.DCs and H-mDCs were generated from six different donors, total RNA was isolated

rom 4 day-old cultures, reverse-transcribed, and tested by qRT-PCR for: (A) CCL18,CL20, CCL23, CCL3, CCL5, CXCL2, CXCL5, and CXCL8; (B) IL-1b, OPN, and VEGF. CA12RNA levels were assayed in parallel as a positive control. Expression changes were

valuated as detailed in the Materials and Methods. Data are expressed as log2 ratiof fold-changes (H-mDCs relative to mDCs), calculated on the basis of triplicateeasurements for each target gene/donor, relative to the values obtained for the

eference genes. Linear fold changes are indicated by the number associated withach bar.

ogy 218 (2013) 76– 89

populations displayed the typical mature DC immunophenotype,characterized by high surface expression of CD1a and CD83 differ-entiation/maturation markers, CD86 costimulatory molecule, CCR7and CXCR4 chemokine receptors (Table 3), in agreement with pre-vious observations (Elia et al. 2008; Bosco et al. 2011).

Fig. 2 shows the relative mRNA expression levels of selectedgenes in H-mDCs respect to mDCs. A strong and consistentupregulation of CCL20, CCL3, CCL5, CXCL2, CXCL5 and CXCL8 anddownregulation of CCL18 and CCL23 transcripts was triggered byhypoxia in H-mDCs vs mDCs from every donor tested, indepen-dently of the baseline levels, although significant variations in theextent of modulation was observed among the samples examined(Panel A). Significant upregulation of IL-1�, OPN, and VEGF mRNAwas also demonstrated in all H-mDC preparations, paralleling thatof CA12 assessed as an index of response to hypoxia (Panel B).

To determine whether the changes in mRNA levels were associ-ated with similar effects on protein secretion, chemokine/cytokinerelease by mDCs and H-mDCs was measured by ELISA after24 h culture, as detailed in the Materials and Methods. mDCsconstitutively secreted high amounts of OPN (36.5 ± 3.2 ng/ml),CXCL8 (17.1 ± 3.9 ng/ml), CCL18 (944.7 ± 131 pg/ml), and CCL23(468.1 ± 76.3 pg/ml), and lower levels of CXCL5 (139.7 ± 6.2 pg/ml),CCL3 (67.8 ± 15.7 pg/ml), VEGF (47.5 ± 37.5 pg/ml), CXCL2(37.5±5 pg/ml), IL-1� (28 ± 3 pg/ml), CCL5 (17.1 ± 1.8 pg/ml),and CCL20 (16.5 ± 5.9 pg/ml) (Fig. 3). Consistent with mRNA data,protein secretion was significantly and differentially modulatedin response to hypoxia. An increase of ≈8-fold in the amounts ofsecreted CCL20 (123.6 ± 34.3 pg/ml), CXCL5 (1221 ± 76.3 pg/ml),IL-1� (233.3 ± 59.1 pg/ml), OPN (282 ± 71 ng/ml), and VEGF(404.83 ± 61 pg/ml) was detectable in H-mDCs compared to mDCs.CCL5 (56.5 ± 2.9 pg/ml), CXCL2 (151.7 ± 8.6 pg/ml), and CXCL8(63.7 ± 7.2 ng/ml) secretion increased by about 3–4-fold, whereasCCL3 levels were enhanced by ≈1.5-fold (105 ± 22.8 pg/ml) fol-lowing generation under hypoxia. A concomitant 80 and 50%reduction in the amounts of secreted CCL18 (162.3 ± 64.6 pg/ml)and CCL23 (251.7 ± 51.7 pg/ml), respectively, was measured in thesupernatants of H-mDCs relative to mDCs. These data demonstratethat the chemokine gene expression profile triggered by hypoxia inmDCs was also reflected in terms of protein secretion, suggestingthat mDC generation under reduced pO2 results in the activationof a highly proinflammatory state.

Discussion

DCs have the dual ability to stimulate protective immunityto pathogens and maintain self-tolerance by orchestrating thecoordinated recruitment and activation of innate and adaptiveimmune cells in diseased tissues through the secretion of spe-cific cytokines/chemokines (Mellman and Steinman 2001; Rossiand Young 2005; Lebre and Tak 2009; Adema et al. 1997), anddysregulated chemotactic network may result in amplification ofinflammation, loss of tolerance, or establishment of immune escapemechanisms (Bosco et al. 2008b; Lin et al. 2010; Ben Baruch 2006;Laoui et al. 2011; Mantovani et al. 2010; Rot and Von Andrian2004). Understanding how the local pathologic environment mod-ulates DC immunogenic or tolerogenic properties may be critical fordesigning more effective DC-based immunotherapeutic strategiesfor tumors, chronic inflammatory diseases, and autoimmune disor-ders. Results shown in this study demonstrate that chronic hypoxicconditions, similar to those present in diseased tissues, can inducea highly proinflammatory phenotype in human monocyte-derivedmDCs by strictly regulating their cytokine/chemokine transcrip-

tional profile.

The influence of low pO2 on the development and biologicalfunctions of several immune cells is well recognized, and thecritical contribution of reduced oxygenation to the amplification

F. Blengio et al. / Immunobiology 218 (2013) 76– 89 85

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CC

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0

50

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0

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(pg

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*

100

200

300

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0

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Fig. 3. Hypoxic modulation of chemokine/cytokine secretion by mDCs. mDCs (open bars) and H-mDC (black bars) were generated from the same donors analyzed in Fig. 2,CM was replaced on day 3 of generation with fresh medium, and cell free supernatants were harvested after 24 h and assayed for chemokine and cytokine content by specificELISA. Results are expressed as pg or ng/1 × 106 cells/ml and are the mean of six independent experiments. Vertical bars represent the standard deviations. The numberassociated with each bar indicates the protein fold changes in hypoxic relative to normoxic cells (arbitrarily defined as equal to 1). P-value of H-mDCs relative to mDCsm .

os2oigcopaTtIst

easured by the Student’s t-test is indicated (*P ≤ 0.05, **P ≤ 0.01, and ***P < 0.001)

f innate inflammatory reactions has been documented in severaltudies (Sica et al. 2011; Cramer et al. 2003; Bosco and Varesio010; Nizet and Johnson 2009; Bosco et al. 2008b). Inducibilityf a significant cluster of genes coding for immunoregulatory,nflammatory, and proangiogenic cytokines was demonstrated byene expression profiling in different types of mononuclear phago-ytes either exposed to short-term hypoxia (typically 8–24 h)r generated under conditions of long-term hypoxia, includingrimary monocytes (Bosco et al. 2006), MDMs (Fang et al. 2009),nd monocyte-derived iDCs (Ricciardi et al. 2008; Yang et al. 2009).he results reported here extend to mDCs this trend of response

o hypoxia, demonstrating strong upregulation of OPN, MIF, VEGF,L-23A, CSF3, and of various components of the IL-1 receptor/liganduperfamily in mDCs generated under chronic hypoxia comparedo cells generated under normoxia. Given the stimulatory effect

of these cytokines on endothelial cell survival, proliferation,adhesion, and chemotaxis (Senger et al. 1996; Bosco and Varesio2010) and on monocyte and T lymphocyte recruitment/activation(O’Regan et al. 2000; Murakami et al. 2006; Murdoch et al. 2004;Ben Baruch 2006), it is conceivable that mDCs infiltrating hypoxictissues have an increased ability to induce neo-vascularizationand inflammation compared to their normoxic counterpart, withimplication for the pathogenesis of tumors, inflammatory andautoimmune diseases (Bosco and Varesio 2010; Xu et al. 2005;Roccaro et al. 2005; Dinarello 2011; Santos and Morand 2009).

Interestingly, several differences existed between the cytokine

gene expression profile of H-mDCs and that of the other hypoxicmononuclear phagocytes. Specifically, a number of genes upreg-ulated in H-mDCs, such as OSMR, IL-1RB, and BMP6, were notinduced in any of the other monocytic lineage populations, whereas

8 nobiol

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6 F. Blengio et al. / Immu

ther inducible genes such as IL-18R1 and CSF1R were downregu-ated in primary monocytes following exposure to hypoxia (Boscot al. 2006). On the other hand, the expression of genes previouslyhown to be modulated in hypoxic monocytes and iDCs, includingSF1, CSF3R, CSF2RA, and IL6ST (Bosco et al. 2006; Ricciardi et al.008), and/or in differentiated macrophages, such as IL18, IL12Bnd CSF2 (Fang et al. 2009), was not affected in H-mDCs. Collec-ively, these observations indicate that modulation of genes codingor proteins involved in the development and persistance of anngiogenic and inflammatory environment is a common feature ofhe hypoxic transcriptome of cells belonging to the monocytic lin-age, although different combinations of cytokines are selectivelyxpressed in mDCs, iDC, and their monocytic precursors in responseo low pO2, probably representing a regulatory mechanism of themplitude and duration of inflammatory responses.

High levels of chemokines are present in areas of inflamma-ion and in various chronic inflammatory conditions (Koch 2005;aggiolini and Loetscher 2000; Ben Baruch 2006; Mantovani et al.010; Pharoah et al. 2006; Bizzarri et al. 2006), and the chemotac-ic network is known to be highly sensitive to microenvironmentalhanges including hypoxia (Schioppa et al. 2003; Ricciardi et al.008; Elia et al. 2008; Fang et al. 2009; Bosco et al. 2004a,b, 2006,008a,b; Zampetaki et al. 2004; Murdoch et al. 2004). The findingseported here together with previous observations indicate thatow pO2 can significantly and differentially influence the migratoryehavior and the chemokine expression profile of cells belongingo the monocytic lineage, as depicted in Fig. 4. Specifically, hypoxiaan impair the chemotaxis of primary monocytes after recruitmento diseased tissues and promote their retainment/concentrationt hypoxic sites by concomitantly inducing downregulation ofC-chemokine receptors and upregulation of the monocyte-arresthemokines, CXCL2 and CXCL3 (Bosco et al. 2006, 2008b). In con-rast, prolongued exposure to hypoxia exerts a positive regulatoryontrol on the chemotactic response of monocyte-derived iDCs byriggering the onset of a migratory phenotype characterized bypregulation of chemokine receptors with consequent increasedesponsiveness to specific ligands, while inhibiting inflammatoryhemokine production by these cells (Ricciardi et al. 2008; Yangt al. 2009). In the present study, we show that DC maturationithin a chronic hypoxic microenvironment promotes a highlyroinflammatory state as compared to the normoxic counterpart.pecifically, our results provide the first evidence that genes codingor various members of the neutrophil-attracting CXC chemokineamily, such as CXCL2, CXCL3, CXCL5, CXCL6, and CXCL8 (Baggiolinind Loetscher 2000; Bizzarri et al. 2006; Rot and Von Andrian 2004),nd for the CCL20, CCL3, and CCL5 chemokines, predominantlyhemotactic for activated/memory T lymphocytes, monocytes, andDCs (Pharoah et al. 2006; Baggiolini and Loetscher 2000; Schutysert al. 2003; Ben Baruch 2006), are strongly induced in H-mDCs,hereas CCL18 and CCL23, that are specific chemoattractants

or naive/resting T cell constitutively expressed at high levelsn normoxic mDCs (Forssmann et al. 1997; Adema et al. 1997),re strongly downregulated under hypoxia. This response wasonsistently demonstrated in all H-mDCs preparations analyzed,lthough the level of expression differed among individual donors.ene expression was associated with parallel modulation of proteinecretion, suggesting that a dynamic change in chemokine produc-ion by mDCs may occur in vivo depending on the degree of localxygenation.

O2 levels in the body are quite heterogeneous. PhysiologicO2 in healthy tissues typically spans between 3.3% and 7.9%Sitkovsky and Lukashev 2008; Semenza 2001; Staples et al. 2011),

lthough O2 concentrations below 2.5% were reported in somereas of normal tissues, e.g. lymphoid organs, reflecting the extentf vascularization (Caldwell et al. 2001). pO2 in damaged and dis-ased tissues decreases to levels lower than those present in the

ogy 218 (2013) 76– 89

correspondent healthy tissue, ranging from ≈5% to almost anoxicconditions of 0.1% in different pathologic situations (Bosco andVaresio 2010; Distler et al. 2004; Beyer et al. 2009; Vaupel et al.1991; Semenza 2001; Evans et al. 2001; Ogino et al. 2011; Stapleset al. 2011). The 1% O2 concentration used in this study representsa good approximation of the hypoxic environment that character-izes many chronic inflammatory diseases, e.g. rheumatic arthritis,artherosclerosis, inflammatory skin disorders, and primary solidtumors, with a recognized pathogenetic relevance in these condi-tions (Distler et al. 2004; Muz et al. 2009; Bosco and Varesio 2010;Bjornheden et al. 1999; Semenza 2001; Beyer et al. 2009; Akhavaniet al. 2009), and is commonly used as prototypic experimental con-dition of hypoxia in most in vitro studies (Jögi et al. 2002; Schioppaet al. 2003; Bosco et al. 2004a, 2006, 2008a; Battaglia et al. 2008;Akhavani et al. 2009), including those on dendritic cells (Mancinoet al. 2008; Ricciardi et al. 2008; Elia et al. 2008; Jantsch et al. 2008;Bosco et al. 2011; Yang et al. 2009; Ogino et al. 2011). The find-ing that CCL20 was the only chemokine inducible by this level ofhypoxia in all types of mononuclear phagocytes, both in vitro andin vivo (Bosco et al. 2006, 2008a; Battaglia et al. 2008; Elia et al.2008; Fang et al. 2009), is noteworthy and points to a unique rolefor this chemokine in the control of the kinetics and compositionof the leukocyte infiltrate in diseased tissues. Interestingly, CCL20upregulation in H-mDCs was associated with downregulation ofits specific receptor, CCR6, which is suggestive of a negative feed-back mechanism regulating the autocrine activation of producingcells. Conversely, CCR2 was upregulated by hypoxia in mDCs, asopposed to what shown in their monocytic precursors (Bosco et al.2006) but similarly to what found in iDCs (Ricciardi et al. 2008; Yanget al. 2009), suggesting the existance of a common regulatory mech-anism of the chemotactic response of iDCs and mDCs generatedat hypoxic sites. Future studies will be aimed at comparing mDCsresponses to moderate (e.g. 2.5%) and severe (e.g. 0.5%, 0.1%) O2concentrations to recapitulate the various levels of hypoxia presentin vivo in specific pathologic situations for a better understandingof the patogenetic mechanisms of diseases.

The fine tuning of the chemokine repertoire in H-mDC is likelyto represent a critical set point for the control of their Th-polarizingactivity at site of inflammation, which is crucial for the outcome ofadaptive immunity (Sallusto et al. 1998; Baggiolini and Loetscher2000; Banchereau et al. 2000; Moser and Murphy 2000; Lebreand Tak 2009; Lanzavecchia and Sallusto 2001). Increased produc-tion of CCL3, CCL5, and CCL20 chemokines, which attract CCR5-and CCR6-expressing Th1 and Th17 activated/memory lympho-cytes (Wedderburn et al. 2000; Nistala et al. 2008; Brand 2009),is compatible with a Th1/Th17 shift of mDC responses underchronic hypoxia. Inhibition of CCL18 and CCL23 production alsosupports the notion of H-mDCs driving immune responses toward aTh1/Th17-polarized proinflammatory direction, given the reportedrole of these chemokines in the generation of regulatory T cellsand the maintenance of tolerance (Forssmann et al. 1997; Ademaet al. 1997; Vulcano et al. 2003). The observation that CCL18 andCCL23 expression is negatively affected by hypoxia not only inmDCs but also in primary monocytes and iDCs (Bosco et al. 2006;Ricciardi et al. 2008) raises the possibility that inhibition of toler-ance is a common feature of monocytic lineage cell adaptation tothe hypoxic environment. Increased expression of OPN and IL-23,which are involved in the pathway leading to Th1 immunity andTh17 differentiation (Brand 2009; Di Cesare et al. 2009), may rep-resent an additional evidence that DC maturation under chronichypoxia is associated with a Th1/Th17-biased transcriptional pro-file. These findings support and extend our recent observation that

mDCs generated under chronic hypoxia are induced to express theTREM-1 immunoregulatory signaling receptor, whose engagementtriggers secretion of several proinflammatory and Th1-primingcytokines, suggesting that the hypoxic environment stimulates the

F. Blengio et al. / Immunobiology 218 (2013) 76– 89 87

Fig. 4. Hypoxia differentially affect the chemotactic activity and migratory behavior of mDCs and their precursors. (a) Circulating monocytes are recruited to hypoxic areasof diseased tissues where they are retained through downregulation of CC-chemokine receptors and upregulation of monocyte-arrest CXC-chemokines (Bosco et al., 2006,2008b). (b) Monocyte to iDC differentiation under chronic hypoxia promotes the development of a migratory phenotype, characterized by increased expression of chemokinereceptors and responsiveness to specific ligands, while decreasing inflammatory chemokine poduction (Ricciardi et al. 2008; Bosco et al. 2008b). (c) DC maturation withint henod C-chema

Tmebbhntwa

hpodtoilcppmsb2rr

he hypoxic microenvironment promotes the development of a proinflammatory pirection triggering the production of neutrophil-attracting CXC-chemokines and Cnd the concomitant inhibition of naïve/resting T cell specific chemoattractants.

h1 polarizing activity of these cells (Bosco et al. 2011). Further-ore, these results are in line with a previous report by Mancino

t al. (2008), showing enhanced Th1-polarized inflammatory capa-ility of hypoxic myeloid-derived mDCs, while contrasting datay Yang et al. (2009), who proposed that mDCs generated underypoxic conditions are redirected towards a Th2 stimulating phe-otype. A possible explanation for these divergent results could behe different experimental protocols used for DC maturation, whichere recently shown to strongly influence the immunostimulatory

nd Th-priming activity of these cells (Landi et al. 2011).In conclusion, this study documents the critical role of chronic

ypoxic conditions mimicking those present in vivo in severalathologic situations in mediating the functional reprogrammingf monocyte-derived mDCs toward a Th1/Th17 proinflammatoryirection by tightly regulating their cytokine/chemokine secre-ion, confirming and expanding earlier evidence that the degree ofxygenation to which DCs are exposed during development is anmportant variable in the control of their functional behavior. Pro-ongued or excessive activation of Th1/Th17-mediated responsesould be detrimental to the host, representing a mechanism ofersistance and amplification of the inflammatory process withotential implications for the pathogenesis of chronic inflam-atory and autoimmune diseases, such as rheumatoid arthritis,

ystemic lupus erythematosus, inflammatory skin disorders and

owel diseases (Schutyser et al. 2003; Brand 2009; Nistala et al.008; Gudjonsson et al. 2004; Di Cesare et al. 2009). Ouresults provide novel mechanistic clues on the contribution ofeduced O2 availability to chronic inflammation by triggering mDC

type, driving mDC immune responses toward a Th1/Th17-polarized inflammatoryokines active on Th1/Th17 activated/memory lymphocytes, monocytes, and iDCs,

proinflammatory responses, and the challenge of future studies willbe to validate these data in vivo.

Acknowledgements

This work was supported by grants from the: Italian Health Min-istry, Regione Piemonte “Progetti di Ricerca Sanitaria Finalizzata eApplicata, Progetti strategici su tematiche di interesse regionale osovraregionale (IMMONC)”, Italian Association for Cancer Research(AIRC), and Compagnia San Paolo, Special Project Oncology.FR wassupported by a fellowship from the Italian Association for CancerResearch (AIRC).

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