Chronic hypoxia reprograms human immature dendritic cells by inducing a proinflammatory phenotype and TREM-1 expression

Authors

  • Daniele Pierobon,

    1. Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy
    2. Center for Experimental Research and Medical Studies (CERMS), Azienda Sanitaria Città della Salute e della Scienza, Torino, Italy
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    • These authors equally contributed to this work.

  • Maria Carla Bosco,

    1. Laboratory of Molecular Biology, G. Gaslini Institute, Genova, Italy
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    • These authors equally contributed to this work.

  • Fabiola Blengio,

    1. Laboratory of Molecular Biology, G. Gaslini Institute, Genova, Italy
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  • Federica Raggi,

    1. Laboratory of Molecular Biology, G. Gaslini Institute, Genova, Italy
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  • Alessandra Eva,

    1. Laboratory of Molecular Biology, G. Gaslini Institute, Genova, Italy
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  • Miriam Filippi,

    1. Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy
    2. Center for Experimental Research and Medical Studies (CERMS), Azienda Sanitaria Città della Salute e della Scienza, Torino, Italy
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  • Tiziana Musso,

    1. Department of Public Health and Pediatric Sciences, University of Torino, Torino, Italy
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  • Francesco Novelli,

    1. Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy
    2. Center for Experimental Research and Medical Studies (CERMS), Azienda Sanitaria Città della Salute e della Scienza, Torino, Italy
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  • Paola Cappello,

    1. Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy
    2. Center for Experimental Research and Medical Studies (CERMS), Azienda Sanitaria Città della Salute e della Scienza, Torino, Italy
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  • Luigi Varesio,

    1. Laboratory of Molecular Biology, G. Gaslini Institute, Genova, Italy
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    • These authors share senior authorship.

  • Mirella Giovarelli

    Corresponding author
    1. Center for Experimental Research and Medical Studies (CERMS), Azienda Sanitaria Città della Salute e della Scienza, Torino, Italy
    • Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy
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    • These authors share senior authorship.


Full correspondence: Prof. Mirella Giovarelli, Department of Molecular Biotecnology and Health Sciences, University of Torino, Via Nizza 52 -10126 Torino, Italy

Fax: +39 0116336887

e-mail: mirella.giovarelli@unito.it

Abstract

DCs are powerful antigen-presenting cells central in the orchestration of innate and acquired immunity. DC development, migration, and activities are intrinsically linked to the microenvironment. DCs migrate through pathologic tissues before reaching their final destination in the lymph nodes. Hypoxia, a condition of low partial oxygen pressure, is a common feature of many pathologic situations, capable of modifying DC phenotype and functional behavior. We studied human monocyte-derived immature DCs generated under chronic hypoxic conditions (H-iDCs). We demonstrate by gene expression profiling the upregulation of a cluster of genes coding for antigen-presentation, immunoregulatory, and pattern recognition receptors, suggesting a stimulatory role for hypoxia on iDC immunoregulatory functions. In particular, we show that H-iDCs express triggering receptor expressed on myeloid cells(TREM-1), a member of the Ig superfamily of immunoreceptors and an amplifier of inflammation. This effect is reversible because H-iDC reoxygenation results in TREM-1 down-modulation. TREM-1 engagement promotes upregulation of T-cell costimulatory molecules and homing chemokine receptors, typical of mature DCs, and increases the production of proinflammatory, Th1/Th17-priming cytokines/chemokines, resulting in increased T-cell responses. These results suggest that TREM-1 induction by the hypoxic microenvironment represents a mechanism of regulation of Th1-cell trafficking and activation by iDCs differentiated at pathologic sites.

Introduction

Myeloid DCs are central in the orchestration of innate and acquired immune responses and in the maintenance of self-tolerance [1]. DC development involves three functionally and phenotypically distinct stages for which the terms “precursors,” “immature,” and “mature” are commonly used [2-5]. DCs precursors originate in the bone marrow, circulate via the bloodstream to reach target tissues, and take up residence at sites of potential pathogen entry, where they differentiate into immature DCs (iDCs) specialized for antigen capture [2, 4, 6]. Peripheral blood monocytes recruited from the circulation to inflammatory sites can also serve as iDC precursors [7, 8]. iDC redistribution in the tissues is determined by the local microenvironment through the production of chemotactic mediators, activation of inflammatory chemokine receptors, and regulation of adhesion molecules [7, 8]. Tissue injury, inflammation, and transformation cause dramatic changes of the microenvironment, modulating iDC phenotype and function and promoting maturation into (m)DCs [7-14].

A common denominator of injured and inflamed tissues is the presence of low partial oxygen pressure (pO2), which creates a unique microenvironment affecting cell phenotype, gene expression profile, and functional behavior [10, 11, 15, 16]. Response to hypoxia is primarily under the molecular control of a family of hypoxia-inducible transcription factors, composed of the constitutive HIF-1β subunit and an O2-sensitive α subunit (HIF-1α/-2α), which binds and transactivates the hypoxia responsive element (HRE) present in the promoter of many hypoxia-inducible genes [11, 15-17]. DC development from monocytic precursors recruited at pathological sites occurs under the setting of reduced pO2. Recent studies have reported that HIF-1α accumulates in hypoxic DCs and that O2 levels similar to those present in diseased tissues can impact on DC differentiation, maturation, and activation [10, 11, 18-24]. Hypoxia promotes the onset of a migratory phenotype in iDCs through the upregulation of inflammatory chemokine receptors and motility-related genes with consequent increased responsiveness to specific chemoattractants [18-20] and a proinflammatory state in mDCs by increasing the expression of genes coding for proinflammatory and Th1-priming chemokines/cytokines [24].

DCs integrate stimulatory and inhibitory signals present in the microenvironment through a defined repertoire of cell surface receptors, and deregulated expression of these molecules may result in aberrant responses characterized by amplification of inflammation and loss of tolerance [5, 7-9, 25-27]. Elucidation of the effects of hypoxia on the pattern of receptor expression by iDCs and mDCs is important for our understanding of the regulatory control exerted by the pathologic microenvironment on their tolerogenic or immunogenic properties. We recently demonstrated that DCs maturation under chronic hypoxia (H-mDCs) induces profound changes in the expression of genes encoding various immune-related receptor family members [23], including the triggering receptor expressed on myeloid cells (TREM-1). The latter is a new hypoxia-inducible gene in H-mDCs, member of the Ig receptor superfamily, and strong amplifier of the inflammatory responses [28-30]. We also demonstrated the presence of mDCs expressing TREM-1 in vivo in the hypoxic synovial fluid of patients affected by juvenile idiopathic arthritis [23]. However, the impact of chronic hypoxia on the receptor expression profile of iDCs is largely unknown.

In this study, we show that iDCs, generated from human monocytes under chronic hypoxia, hereafter called hypoxia (H-iDCs), are functionally reprogrammed through the differential expression of genes coding for antigen processing and presentation molecules, immunoregulatory, and pattern recognition receptors (PRR). Interestingly, TREM-1 is one of the hypoxia-inducible gene targets in iDCs. TREM-1 engagement on H-iDCs triggers pheno-typic and functional properties typical of mature cells. These include enhanced expression of T-cell costimulatory molecules and chemokine homing receptors and increased production of several proinflammatory and Th1/Th17-priming cytokines/chemokines, resulting in Th1/Th17-cell priming. These findings highlight the potential of TREM-1 in shaping H-iDC maturation and T-cell stimulatory activity at pathologic sites.

Results

Regulation of iDC immune-related receptor gene expression profile by hypoxia

We reported that H-iDCs generated under chronic hypoxia redefine their transcriptome respect to iDCs generated under normoxia, displaying the expression of a statistically significant portion of genes related to immune regulation, inflammatory responses, angiogenesis, and migration [19]. To identify new genes responding to hypoxia in iDCs, further analysis was carried out.

We found profound differences in the expression of a prominent cluster of cell surface receptor-encoding genes (52), the majority of which (83%) was upregulated (Table 1). H-iDCs expressed higher levels of genes coding for both classical and nonclassical antigen-presenting receptors, including MHC class I and II molecules and tetraspanin family members (CD37, CD53, CD9) that associate with and are implicated in MHC-peptide assembly [31, 32]. We also observed hypoxia-dependent expression of genes coding for immunoregulatory signaling receptors implicated in the regulation of DC maturation/polarization, inflammatory and immune functions [26, 33]. The most relevant are: SLAM family member-9 (SLAMF9), low-affinity IgE receptor, FcεRII (CD23A), and IgG receptors, FcγRIIA/B (CD32), CD69, CD58, natural cytotoxicity triggering receptor 3 (LST1), TREM-1, leukocyte Ig-like receptor 9 (LIR9), and leukocyte membrane Ag (CMRF-35H), whereas expression of CD33 antigen-like 3 (SIGLEC15) and SLAMF1, among others, was downregulated. The hypoxic transcriptome was also characterized by the differential modulation of genes encoding PRR critical to host defense [34] and scavenger receptors implicated in the regulation of fatty acid and/or cholesterol uptake/transport [35], such as CD180, G-protein-coupled receptor (GPCR) 132 (G2A), TLR 8, CD14, MD-1, and CD36, that were upregulated, and CD163 Ag, TLR5, and MD-2, that were downregulated. These results demonstrate that iDCs generation under hypoxia strongly affects the resulting surface receptor repertoire. Interestingly, only a few of the observed hypoxia-induced changes in gene expression were shared with those detected in H-mDCs [18, 23] or monocytic precursors exposed to acute hypoxia [36], whereas most of the genes upregulated in H-iDCs were not affected or even downregulated in the other mono-nuclear phagocyte (MP) populations examined (Table 1). We conclude that hypoxia can selectively modulate the gene expression pattern of immune-related receptors in monocytic lineage cells depending on their differentiation/maturation stage.

Table 1. Relative expression of genes encoding immune-related cell surface receptors in H-iDCs versus iDCsa
      Modulated ind
Gene bank accession no.Gene symbolFull nameMain function(s) of gene productFold changebHREcmDCsMonocytes
  1. a

    iDCs and H-iDCs 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 days. Gene expression profiling was then carried out independently by microarray analysis on the RNA purified from three independent iDC and H-iDC preparations, and comparative analysis of gene expression differences between the two experimental conditions was conducted as described in 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 change value are specified for each gene. Genes in each group are ordered by fold changes. Randomly selected genes validated for qRT-PCR are underlined.

  2. b

    Results are expressed as the ratio between hypoxic and normoxic values (mean of expression level of three experiments). Genes up/downmodulated by ≥twofold are shown.

  3. c

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

  4. d

    Comparison of microarray results with those previously obtained in mDCs generated under long-term hypoxia and monocytes a exposed to short-term hypoxia, as reported by Bosco et al., [26,36] and Yang et al. [18].

Receptors for antigen presentation
 Upregulated
NM_001774CD37CD37 antigenMember of the tetraspanin family; associates with both MHC classes I and II and regulates peptide/MHC presentation and cell adhesion20.756+
AI022073CD53CD53 antigenMember of the tetraspanin family; associates with MHC molecules and regulates peptide/MHC presentation; complexes with integrins and plays a role in the regulation of leukocyte activation, growth, and motility; contributes to the transduction of CD2-generated signals in T and NK cells6.555Up
M28590CD74CD74 antigenAntigen associated with major histocompatibility complex, class II; involved in the presentation of MHC-II-restricted antigenic peptides5.801
NM_001769CD9CD9 antigen (p24)Member of the tetraspanin family; interacts with and promotes MHC II clustering and peptide/MHC presentation; facilitates the organization of multimolecular membrane complexes, including integrins; plays a role in leukocyte activation, differentiation4.849+Up
M90684HLA-GHistocompatibility antigen, class I, GNonclassical inhibitory HLA class I molecule; forms heterodimers with beta-2-microglobulin; involved in the presentation of MHC-I-restricted antigenic peptides4.252Down
U62824HLA-CMajor histocompatibility complex, class I, CHLA class I heavy chain; forms heterodimers with beta-2-microglobulin; involved in the presentation of MHC-I-restricted antigenic peptides3.801
NM_030893CD1ECD1E antigenA member of the CD1 family of transmembrane glycoproteins structurally related to the MHC proteins; forms heterodimers with beta-2-microglobulin. Mediates the presentation of lipid and glycolipid antigens of self or microbial origin to T cells3.494
AF005487HLA-DRB6Major histocompatibility complex, class II, DR beta 3HLA class II beta chain molecule; Involved in the presentation of MHC-II-restricted antigenic peptides3.448Up
LO7950HLA-BMajor histocompatibility complex, class I, BHLA class I heavy chain; forms heterodimers with beta-2-microglobulin; plays a central role in the immune system by presenting peptides derived from the endoplasmic reticulum lumen2.885
BG397856HLA-DQA1Major histocompatibility complex, class II, DQ alpha 1HLA class II alpha chain molecule; Involved in the presentation of antigenic peptides derived from extracellular proteins2.883+
NM_018950HLA-FMajor histocompatibility complex, class I, FIt encodes a nonclassical HLA class I heavy chain; forms heterodimers with beta-2-microglobulin; binds a restricted subset of peptides for immune presentation2.713+
NM_002119HLA-DOAMajor histocompatibility complex, class II, DO alphaHLA class II alpha chain molecule; regulates HLA-DM-mediated peptide loading on MHC class II molecules2.674
M17955HLA-DQB1Major histocompatibility complex, class II, DQ beta 1HLA class II beta chain molecule; involved in the presentation of antigenic peptides derived from extracellular proteins2.236Up
Immunoregulatory receptors
 Upregulated
NM_033438SLAMF9SLAM family member 9Member of the signaling leukocyte activation Ig subfamily; involved in innate and acquired immune-responses; promotes DC maturation10.684+Up
NM_002002FCER2Fc fragment of IgE, low affinity II, receptor for (CD23A)Low affinity IgE receptor alpha-isoform expressed on APCs; enhances IgE-mediated Ag presentation and regulate IgE homeostasis; plays important roles in the pathogenesis of airway allergic inflammation10.590+
L07555CD69CD69 antigen (p60, early T-cell activation antigen)Member of the calcium dependent lectin superfamily of type II transmembrane receptors; inducer of inflammatory mediator production and cytotoxic activity; plays a role in defense response and heterophilic cell adhesion7.812Up
NM_170699GPBAR1G protein-coupled bile acid receptor 1Member of the G-protein-coupled receptor (GPCR) superfamily; functions as a cell surface receptor for bile acids; mediates immunosuppressive effect of bile acids; is implicated in the suppression of inflammatory responses5.459
NM_000610CD44CD44 antigen (homing function and Indian blood group system)Receptor for hyaluronic acid, osteopontin, collagens, and matrix metalloproteinases; involved in cell–cell interaction, leukocyte adhesion, recirculation and homing, hematopoiesis, and tumor spreading; plays a role in DC-mediated T cell activation4.919Up
AA700015CD58CD58 antigen, (lymphocyte function-associated antigen 3, LFA-3))Member of the immunoglobulin superfamily; binds the T lymphocyte CD2 protein, and functions in adhesion and activation of T lymphocytes4.537+
AI735692LST1Leukocyte specific transcript 1Gene encoded within the TNF region of the human MHC; plays immunosuppressive functions4.367+
NM_018643TREM-1Triggering receptor expressed on myeloid cells 1Ig superfamily immunoregulatory receptor expressed on monocytes/macrophages and neutrophils; stimulates their proinflammatory functions and acts as an inflammatory amplifier via functional interaction with TLRs; involved in inflammatory diseases and septic shock4.280+UpUp
U90940FCGR2BFc fragment of IgG, low affinity IIb, receptor for (CD32)ITIM-containing subunit of the low affinity IgG receptor; inhibits cell activation; involved in myeloid cell effector and regulatory functions such as phagocytosis of immune complexes and modulation of antibody production by B-cells4.029+Up
AK022549CDW92 (CTL1)CDW92 antigenCholine transporter; negative regulation of DC functions; anti-inflammatory role3.908
NM_021221LY6G5BLymphocyte antigen 6 complex, locus G5BGlycosylphosphatidylinositol-anchored molecule, member of the LY6 superfamily2.935
NM_024980GPR157G-protein-coupled receptor 157Member of the GPCR superfamily; G protein activator2.769+
NM_005293GPR20G-protein-coupled receptor 20Member of the GPCR superfamily; G protein activator2.649+  
NM_002346LY6ELymphocyte antigen 6 complex, locus EGlycosylphosphatidylinositol-anchored molecule that belongs to the Ly-6 family, also known as stem cell antigen 2 (SCA2); type I interferon-inducible gene; plays a role in defense response2.650+UpDown
X97671EPORErythropoietin receptorMember of the cytokine receptor family expressed on various hemopoietic cells with immunostimulatory activities3.543+Up
AF212842LIR9 (LILRA5)LIR9Member of the LIR family. Expressed predominantly on myeloid lineage cells; acts as an activating receptor, promoting innate immune responses by triggering secretion of several proinflammatory cytokines2.470+Down
NM_033130SIGLEC10Sialic acid binding Ig-like lectin 10ITIM-containing member of the CD33-related Siglec receptor family; potential role as a pattern-recognition molecule for sialylated pathogens; plays a role in cell adhesion and regulation of inflammation2.446+
AF020314CMRF-35H (CD300a)Leukocyte membrane antigenITIM-containing member of the Ig-like CD300 family of surface receptors; play an inhibitory role on inflammatory mediator production and effector functions of several leukocyte subsets including BM cells, pDC, PMN, T/B ly, and mast cells2.313+Up
AJ130713SIGLEC7Sialic acid binding Ig-like lectin 7ITIM-containing member of the CD33-related Siglec receptor family; acts as an inhibitory receptor; potential role as a pattern-recognition molecule for sialylated pathogens; plays a role in cell adhesion and regulation of inflammation2.209Up
AF165187AGTRAPAngiotensin II receptor-associated proteinTransmembrane protein that interacts with the angiotensin II type I receptor and negatively regulates angiotensin II signaling; regulation of immune response and cell chemotaxis2.163+
AI703188GPR161G-protein-coupled receptor 161Orphan G-protein receptor; activates G protein signaling2.083+
NM_006378SEMA4DSemaphorin 4DIntegral membrane protein with a Ig extracellular domain; controls proliferation, survival, and migration of nervous cells; positive regulator of angiogenesis; plays a role in the activation of both humoral and cellular immunity, including induction of DC maturation, mmunoregulatory functions, and T cells activation and differentiation2.082+UpUp
U90939FCGR2AFc fragment of IgG, low affinity IIa, receptor for (CD32)ITAM-containing subunit of the low affinity IgG receptor; triggers cell activation; involved in myeloid cell effector and regulatory functions such as phagocytosis of immune complexes and modulation of antibody production by B-cells2.004+Up
 Downregulated
AK025833SIGLEC15 (CD33L3)CD33 antigen-like 3Member of the CD33-related Siglec family of sialic acid binding Ig-like lectins; plays a role as modulator of inflammatory and immune responses, cytokine production and DC maturation and Th2 polarization0.186+
NM_000861HRH1Histamine receptor H1Member of the GPCR superfamily; stimulates DC activation and subsequent priming of effector T cells; triggers allergic inflammatory reactions0.219
U41070LTB4RLeukotriene B4 receptorReceptor for leukotrien B4; plays a role in myeloid cell migration; involved in the pathophysiology of many inflammatory diseases, such as bronchial asthma, allergic rhinitis, atherosclerosis, and arthritis0.337
NM_004835AGTR1Angiotensin II receptor, type 1Angiotensin II receptor; mediates regulatory effects of angiotensin II on cell chemotaxis, adhesion to endothelial cells, activation of NF-κβ, enhancement of phagocytosis, production of inflammatory mediators, and DC differentiation; stimulatory role in cellular defense and inflammatory response0.467+
NM_003037SLAMF1Signaling lymphocytic activation molecule family member 1Member of the signaling leukocyte activation Ig subfamily; involved in innate immune-responses; regulates bacterial phagosome functions; costimulatory molecule0.489
Pattern recognition/scavenger receptors
 Upregulated
NM_005582CD180 (LY64/RP105)Lymphocyte antigen 64 homologBelongs to the TLR family; negative regulator of TLR4 signaling and innate immune responses to pathogens (LPS) and danger; expression restricted to APCs; plays a role in autoimmunity; NF-kB activator4.590Up
BC004555GPR132 (G2A)G-protein-coupled receptor 132Subfamily member of the GPCR superfamily; high-affinity receptor for lysophosphatidylcholine (LPC) in oxidized low density lipoprotein; chemotactic for monocytes/macrophages; proatherogenic action4.306
NM_016610TLR8TLR 8Member of the TLR family expressed in cells of the innate immune system; binds single-stranded RNAs from RNA viruses as well as syntetic analog compounds (imidazoquinolines); implicated in the pathophysiology of acute ischemic stroke through activation of inflammatory response2.834
NM_000591CD14CD14 antigenGglycosylphosphatidylinositol-linked, leucine-rich repeat-containing cell surface receptor; involved in bacterial LPS recognition by binding LPS-binding protein (LBP) and delivering LPS-LBP complex to TLR4, activating inflammatory responses2.802+
NM_004271LY86 (MD-1)Lymphocyte antigen 86Member of the MD-2-related lipid-recognition protein family; accessory protein physically associated with CD180 and indispensable for its cell surface expression; involved in LPS recognition and signaling, negative regulator of TLR4/MD-2 in LPS response immune and inflammatory responses2.602
M98399CD36CD36 antigen (thrombospondin receptor)Multifunctional class B scavenger receptor; involved in the uptake and endocytic transport of oxidized LDL, collagen, thrombospondin, anionic phospholipids, and apoptotic cells; adhesion molecule; acts a coreceptor with TLR2-TLR6 to mediate bacterial lipoprotein recognition2.217+UpDown
NM_006018GPR109BPutative chemokine receptorGPCR for the beta-oxidation intermediate 3-OH-octanoic acid2.135+
 Downregulated
NM_004244CD163CD163 antigenMember of the group B scavenger receptors; accounts for the clearance of hemoglobin-haptoglobin complexes; plays a role in anti-inflammatory response; atheroprotective activity0.047+Down
AF051151TLR5TLR 5Member of the TLR family expressed in cells of the innate immune system; recognizes the flagellin protein component of the bacterial flagella; plays a role in the activation of innate immunity mediating production of inflammatory cytokines0.358+Down
AI653117CD59CD59 antigen p18–20 (complement regulatory protein)Cell surface glycoprotein that regulates complement-mediated cell lysis; potent inhibitor of the complement membrane attack complex0.363+Up
NM_015364LY96 (MD-2)Lymphocyte antigen 96Lipid-recognition protein; TLR-4 accessory protein: physically associates with TLR4 on the cell surface and confers responsiveness to LPS indispensable for LPS recognition and signaling; modulates antimicrobial inflammatory responses0.468

To validate the microarray results, the mRNA level of a subset of genes selected among those listed in Table 1 was quantified by qRT-PCR. Relative gene expression levels are shown in Supporting Information Fig. 1. We found full concordance between qRT-PCR and microarray data with regard to the direction of the expression changes. For about half of the genes, expression differences were also of comparable magnitude, whereas they were higher according to microarray for CD180 and CD37 and to qRT-PCR for HLA-DRB6 and FCGRB2, in agreement with previous findings showing that these techniques can often differently estimate the extent of gene modulation [23, 36].

The possible relationship between hypoxia inducibility of genes listed in Table 1 and HRE presence in their promoter was investigated by mapping HRE sequences in the first 2000 bases upstream the transcription initiation site. The frequency of HRE+ genes spotted on the chip was about 60% representing the background of HRE-containing genes in our population. Interestingly, we found that ≈55% of all genes contained at least one member of the HRE family in the promoter, whereas the others were HRE (Table 1), suggesting the involvement of hypoxia-responsive factors other than hypoxia-inducible transcription factors in the transactivation of a substantial number of immune receptor-encoding genes in H-iDCs, similarly to what was previously shown in H-mDCs [23].

TREM-1 is selectively expressed under hypoxic conditions

Among hypoxia-responsive genes, we identified TREM-1 as a common hypoxia molecular target in iDCs, mDCs, and primary monocytes (Table 1), pointing to a critical role of this molecule in the MP response to hypoxia. TREM-1 was previously reported to be constitutively expressed in blood monocytes and completely downregulated during monocyte differentiation into DCs under normoxic conditions [28, 30]. Hence, we were interested in investigating the functional significance of TREM-1 expression in iDCs generated under hypoxia.

To quantify the magnitude of hypoxia effects and address the issue of donor-to-donor variability, we evaluated TREM-1 expression in iDCs generated from seven independent donors under normoxic and hypoxic conditions. As determined by flow cyto-metry (Table 2), H-iDCs expressed the DC marker, CD1a, and displayed an activated phenotype characterized by higher surface levels of CD80 and CD86 costimulatory molecules and the chemokine receptor, CXCR4, compared to iDCs, in agreement with previous data [20]. TREM-1 transcript levels were compared in H-iDCs and iDCs by qRT-PCR. Expression of CAXII was assessed in parallel as an index of response to hypoxia [23]. As depicted in Fig. 1A, TREM-1 mRNA expression was significantly and consistently higher in H-iDCs than in iDCs from all tested samples, paralleling CAXII induction, although with some differences among individual donors ranging from 10- to 21-fold, thus confirming gene inducibility in H-iDCs.

Table 2. Membrane marker expression by iDCs and H-iDCsa
MarkerCulture conditions
 NormoxiaHypoxia
  1. a

    The surface expression of all markers was determined by flow cyto-metry after 4 days’ culture under hypoxic and normoxic conditions.

  2. b

    Data are shown as the mean percentage of positive cells ± SEM from six donors.

  3. c

    p < 0.05, values significantly different from iDCs. Student's t-test.

CD1a95 ± 3b86 ±9
CD4078 ± 277 ± 4
CD8025 ± 448 ± 5c
CD8635 ± 960 ± 12c
CD833 ± 15 ± 2
CXCR44 ± 265 ± 4c
CCR72 ± 14 ± 1
Figure 1.

TREM-1 expression in H-iDCs. iDCs and H-iDCs were generated from different donors. CM was replaced at day 3 of generation with fresh medium supplemented with cytokines, and TREM-1 was analyzed at day four of culture. (A) TREM-1 mRNA expression. Total RNA was reverse-transcribed and tested for TREM-1 expression by quantitative RT-PCR. CAXII mRNA levels were assayed in parallel as positive control. Data are expressed as mean normalized gene expression values, calculated on the basis of triplicate measurements for each experiment/donor, relative to the values obtained for the reference genes. (B) TREM-1 surface expression. iDCs and H-iDCs were stained with anti-CD1a-allophycocyanin and anti-TREM-1-PE Abs and analyzed by flow cytometry. Cells were electronically gated according to their light scatter properties to exclude cell debris. Results from one of seven independent donors are shown (left). Data are expressed as percentage of TREM-1+ cells within CD 1a+ iDCs and H-iDCs generated from seven individual donors (symbols). Horizontal lines represent median values for each group. p-value by the Student‘s t-test is indicated. (C) sTREM-1. Cell-free supernatants were harvested and assayed for sTREM-1 content by ELISA. Data from four of the samples analyzed in (B) are expressed as pg/1 × 106 cells/mL. (D) Effects of reoxygenation on TREM-1 expression. Four-day H-iDCs were cultured for an additional 24 h under 20% O2 (reox) or maintained under 1% (hypo) or 20% (normo) O2 . TREM-1 (black bars) and CAXII (gray bars) mRNA expression was assessed by qRT-PCR (left). Data shown are from one experiment representative of three performed (left). TREM-1 surface expression was evaluated by flow cytometry. Filled histograms represent staining with anti-TREM-1 Ab, whereas open histograms represent staining with isotype-matched control Ab (right). The percentage of TREM-1+ cells is indicated.

TREM-1 surface expression was then measured by flow cytometry in seven individual samples at day 4 of culture. No TREM-1+ iDCs were detectable in any of the donors examined, suggesting that TREM-1 expression is restricted to cells generated under hypoxia (Fig. 1B). A parallel release of the soluble form of TREM-1 (sTREM-1) described in biological fluids during inflammation [37] was demonstrated by ELISA in the supernatants of H-iDCs but not of iDCs, ranging from 80 to 265 pg/106 cells/mL in four different donors (Fig. 1C), consistent with the expression pattern of the membrane-bound form.

H-iDC reoxygenation by exposure to normoxic conditions (reox) for 24 h resulted in a pronounced downregulation of TREM-1 transcript levels (Fig. 1D, left panel). Accordingly, a significant reduction of TREM-1 surface expression was measured upon H-iDC reoxygenation (Fig. 1D, right panel), suggesting the reversibility of hypoxia stimulatory effects on TREM-1 expression.

HIF-1α protein accumulation was reported in hypoxic DCs and paralleled by target gene induction [11, 20-23, 38]. Given the presence of a HRE sequence in TREM-1 gene promoter (Table 1), we investigated HIF-1 role in TREM-1 expression in H-iDCs. To this aim, we added increasing concentrations (0–10 nmol/L) of the HIF-1 DNA-binding inhibitor, echinomycin, at day 3 of H-iDC generation and evaluated TREM-1 expression at day 4 [39]. Expression of the known HIF-1-target gene, VEGF, was assessed in parallel as an index of response to the drug [39]. As shown in Figure 2A, echinomycin strongly decreased vascular endothelial growth factor (VEGF) mRNA, with a 50% inhibition observed with 2 nmol/L and almost complete inhibition with 10 nmol/L, confirming previous data in tumor cells [39]. Treatment with echinomycin also resulted in a dose-dependent downregulation of TREM-1 mRNA levels, although at a lower extent respect to VEGF, with up to 40% of reduction achieved at10 nmol/L. A parallel decrease of TREM-1 surface expression was measured by flow cytometry (Fig. 2B).

Figure 2.

Downmodulation of hypoxia-induced TREM-1 expression by echinomycin. Three-day H-iDCs were treated with the indicated concentrations of echinomycin for 24 h under hypoxic conditions. (A) TREM-1 mRNA expression. Total RNA was isolated and analyzed for TREM-1 mRNA expression (filled diamonds) by qRT-PCR, as described in Fig. 1A. mRNA levels of VEGF (empty diamonds) were tested in parallel as positive control. Results from one experiment representative of three performed are presented as percentage of mRNA remaining after echinomycin treatment, taking as 100% the expression levels of untreated hypoxic cells. (B) TREM-1 surface expression. H-iDCs were stained with anti-TREM-1-PE Abs and analyzed by flow cytometry. Cells were electronically gated according to their light scatter properties to exclude cell debris. Results from one of three independent donors are shown. Filled histograms represent the fluorescent profile of TREM-1-expressing cells, whereas open histograms represent the fluorescent profile of cells stained with the isotype-matched control Ab. The percentage of TREM-1+ cells is indicated.

Overall, these data suggest that TREM-1 expression in H-iDCs is dependent at least in part on HIF-1.

TREM-1 engagement on H-iDCs stimulates their Th1/Th17-polarizing proinflammatory activity

TREM-1 is endowed with proinflammatory and immunoregulatory potential upon cross-linking [29, 30]. To investigate TREM-1 function in H-iDCs, 4-day H-iDCs were plated on a plastic surface coated with a specific anti-TREM-1 agonist mAb or an isotype-matched control anti-HLA-I mAb for 24 h under hypoxia, and the expression of surface antigens was assessed by flow cytometry. As shown in Figure 3A, surface expression of the T-cell costimulatory molecule, CD86, the CD83 maturation marker, and the CCR7 and CXCR4 chemokine receptors was strongly enhanced in response to TREM-1 compared with that from HLA-I triggering, both in terms of mean fluorescence intensity and/or percentage of positive cells, while no modulation of CD40 costimulatory molecule was observed. We analyzed in parallel supernatants for cytokine and chemokine content by ELISA. Enhanced secretion of several proinflammatory, Th1/Th17 cell-priming cytokines and chemokines, such as TNF-α, IL-1β, IL-12, CXCL8, CCL5, CCL17, and osteopontin (OPN), was measured in response to TREM-1 engagement compared with that in cells triggered with anti-HLA-I mAb (Fig. 3B). No substantial differences in phenotype and cytokine secretion were observed in HLA-I-stimulated H-iDCs relative to that of unstimulated cells or cells stimulated with an irrelevant isotype-matched mAb (data not shown), confirming that H-iDC activation by anti-TREM-1 mAb was specific.

Figure 3.

TREM-1 cross-linking on H-iDCs increases the expression of T-cell costimulatory molecules and homing chemokine receptors and the release of proinflammatory cytokines and chemokines. Four-day-H-iDCs were seeded onto plates precoated with agonist anti-TREM-1 mAb or control anti-HLA mAb at 10 μg/mL and cultured for 24 h under hypoxic conditions. (A) Cells were harvested and tested for CD 83, CD 86, HLA-DR, CXCR4, CCR7, and CD 40 surface expression by flow cytometry. Filled histograms represent the fluorescent profile of cells stained with specific conjugated Abs, whereas open histograms represent the fluorescent profile of cells stained with isotype-matched controls. Histograms depict the results obtained from one of five independently tested donors. The mean percentage of positive cells from five different samples and MFI of positive cells ± SEM (between brackets) is indicated in each histogram. p-value by the Student's t-test is indicated. *p≤0.05, **p≤0.01;***p≤0.001 values significantly different from H-iDCs triggered by α-HLA-I mAb. (B) CM was assayed for TNF-α, IL-1β, IL-12, CXCL8, CCL5, CCL17, and OPN content by specific ELISA. Results are expressed as pg or ng/8 × 105 cells/mL and are represented as the mean ± SEM of data from five different experiments. Values significantly different from those of H-iDCs cross-linked with anti-HLA mAb: *p ≤ 0.05; **p ≤ 0.001; ***p ≤ 0.0001, Student's t-test.

To investigate the functional relevance of TREM-1 engagement on H-iDCs, we compared the ability of anti-TREM-1- and anti-HLA-I-stimulated H-iDCs to activate allogeneic T cells in a 5 day MLR assay. As shown in Figure 4A, T-cell proliferation was significantly higher after culture with allogeneic H-iDCs previously cross-linked with anti-TREM-1 mAb than with anti-HLA-I-stimulated H-iDCs. Moreover, T cells alloactivated with TREM-1-triggered H-iDCs showed an increased ability to produce the Th1 and Th17 cytokines, IFN-γ, and IL-17, compared with those cultured with H-iDCs stimulated with anti-HLA-I (Fig. 4B) or unstimulated (data not shown). No significant differences were observed in the secretion of the typical Th2 cytokines, IL-4 and IL-10, by T cells recovered from coculture with TREM-1- and HLA-I-triggered H-iDCs.

Figure 4.

TREM-1 engagement on H-iDCs stimulates T-cell responses and Th1/Th17-priming in MLR. T cells were purified from five different donors. 106/mL T cells were cultured for 5 days with allogeneic anti-TREM-1- or HLA-triggered H-iDCs at 20:1 T:DC ratio. (A) To assess T-cell proliferation, cells were pulsed with 1 μCi of 3H-thymidine during the last 16 h culture, and 3H-thymidine incorporation was measured. Data are expressed as cpm ×10−3. Boxes represent the values falling between the 25th and 75th percentiles; whiskers, the highest and lowest values for each group. Horizontal lines represent median values of three replicates pooled from five different experiments. Values significantly different from those of H-iDCs cross-linked with anti-HLA mAb: *p ≤ 0.05; paired Student's t-test (B) Supernatants were collected at day 4 to determine IFN-γ, IL-17, IL-4, and IL-10 content by ELISA. Results are expressed as pg/1 × 106/mL and are the mean ± SEM of two replicates pooled from three different experiments. Values significantly different from those of H-iDCs cross-linked with anti-HLA mAb: *p ≤ 0.05; **p ≤ 0.001; Student's t-test.

Overall, these data suggest that TREM-1 engagement on H-iDCs induces phenotypic and functional changes typical of maturation, stimulating their Th1/Th17-polarizing proinflammatory activity.

Discussion

DCs immunostimulatory properties are acquired during a complex differentiation and maturation process tightly regulated by a network of inhibitory and activating signals transduced by multiple families of cell surface receptors [3, 8, 9, 25-27]. Understanding how the local pathologic environment modulates DC immunogenic or tolerogenic properties may be critical for the design of more effective DCs-based immunotherapeutic strategies for tumors, chronic inflammatory diseases, and autoimmune disorders [5, 9, 25, 40]. Hypoxia is an important microenvironmental factor to which DCs have to adapt in diseased tissues [10, 11, 16]. Results shown in this study give a strong indication that chronic hypoxic conditions, similar to those present at pathologic sites, can functionally reprogram monocyte-derived iDCs by differentially modulating the expression profile of genes coding for immune-related receptors.

iDCs are specialized for antigen capture and processing and play a critical role in the induction of protective immunity to microbial invasion [3, 5, 12, 27]. Microarray data suggest that iDCs development under chronic hypoxia is associated with the differential expression of various PRR-coding genes. Given the role of these molecules in the recognition of specific pathogen-associated molecular patterns on infectious agents [34], it is conceivable that hypoxia may contribute to the fine tuning of iDC antimicrobial activities through the selective modulation of these receptors. Of relevance is the upregulation of G2A and CD36, which function as endocytic receptors/transporters of lipoproteins and phospholipids and may thus be implicated in lipid-loaded foam cell formation and atherosclerotic plaques development [2, 35]. Moreover, CD163 scavenger receptor, which is endowed with anti-inflammatory and atheroprotective activities, is downregulated [41], consistent with the view that hypoxia exerts a pathogenic role in atherosclerosis [15, 36].

Antigen uptake, in concert with activation stimuli and tissue environmental factors, induces iDCs to mature into mDCs, which have a higher capacity for antigen presentation and T-cell priming [1, 3, 6, 12]. Interestingly, H-iDCs are induced to upregulate genes coding for both classical and nonclassical antigen-presenting receptors as well as molecules that associate with and promote MHC clustering and peptide presentation and T-cell activation [31, 32], suggesting enhanced antigen-presenting ability of iDCs generated at hypoxic sites compared with that of cells in the bloodstream [10, 21, 38].

Hypoxia also affects the expression of a number of genes coding for inhibitory/stimulatory Ig-like immunoregulatory signaling receptors. Of relevance, mRNA for FcγRIIA, FcγRIIB, and FcεRII, which trigger phagocytosis and immune complex clearance, antibody-dependent cell cytotoxicity and respiratory burst [33] is increased. The differential modulation of other Ig-like family members, the most relevant of which are SLAMF9, CD58, TREM-1, LIR9, CMRF-35H, and CD33-related Siglecs, is also noteworthy given the role of these molecules in triggering DCs maturation, proinflammatory cytokine production, and T-cell activating properties [26, 42, 43]. Upregulation of CD69, a member of the lectin superfamily of type II transmembrane receptors and a potent inducer of inflammatory mediator production [36], is another example of the tight regulatory control exerted by hypoxia on iDCs proinflammatory responses. These results point to the role of reduced oxygenation to the pathogenesis of inflammatory disorders and/or autoimmune diseases, which are associated with over-expression of some of these receptors [26, 33, 43].

The influence of low pO2 on the expression profile of immune-related surface receptors has been previously documented in other monocytic lineage cells, such as primary monocytes exposed to short-term hypoxia [36] and monocyte-derived mDCs generated under long-term hypoxic conditions [18, 23], and the results reported here extend to iDCs this trend of response to hypoxia. However, different combinations of receptor-encoding genes are expressed in these cell populations, suggesting that hypoxia may activate a specific transcriptional response in MP depending on their differentiation/maturation stage, which probably represents a mechanism of regulation of the amplitude and duration of inflammatory responses, and the challenge of future studies will be to validate these data in vivo.

TREM-1 is one of the few hypoxia-inducible gene targets in H-iDCs shared with H-mDCs and monocytes. TREM-1 mRNA expression is consistently expressed on H-iDCs generated from different donors but not on the normoxic counterpart, confirming previous evidence of TREM-1 downregulation during monocyte to iDCs differentiation under normoxic conditions [28, 30]. mRNA induction is paralleled by expression of the membrane-bound receptor and its soluble form, detectable in several inflammatory disorders [29, 37, 44]. TREM-1 inducibility by hypoxia is reversible, because cell reoxygenation results in marked decrease of the receptor supporting the role of low pO2 as a TREM-1 inducer in iDCs. In line with these findings, we provide evidence that the HIF/HRE system is implicated, at least in part, in TREM-1 gene inducibility by hypoxia. H-iDCs treatment with echinomycin, a known specific inhibitor of HIF-1 binding to HRE and transcriptional activity [39], downmodulates TREM-1 mRNA and surface protein levels. The potential contribution of other transcription factors, known to mediate hypoxia-dependent gene transactivation in myeloid cells [11, 17, 45], to the regulation of TREM-1 expression in H-iDCs is currently under investigation. These results suggest that TREM-1 expression in iDCs in vivo may vary dynamically with the degree of local tissue oxygenation, which is quite heterogeneous and rapidly fluctuating in diseased tissues [24], giving rise to distinct DC subsets potentially endowed with different functional properties

TREM-1 is functionally active in H-iDCs, as demonstrated by the finding that TREM-1 cross-linking by an agonist mAb on H-iDCs increases surface expression of CXCR4 and CD86 and promotes that of CCR7 and CD83, which play a central role in T-cell migration and activation [46]. These data raise the possibility that TREM-1 engagement on H-iDCs in vivo may deliver a “maturation” signal, thereby increasing their ability to migrate to secondary lymphoid organs and to stimulate adaptive immune responses [6, 8, 13, 14].

TREM-1 engagement also triggers enhanced production of TNF-α, IL-1β, CXCL8, and OPN, suggesting that TREM-1+ H-iDCs infiltrating pathologic tissues are endowed with increased ability to induce angiogenesis and inflammation compared with TREM-1 iDCs present in normoxic tissues [40, 47-51]. These results are in agreement with previous data supporting a role for TREM-1 as an amplifier of inflammation and in the pathogenesis of many infectious and noninfectious inflammatory disorders [23, 29, 30, 37, 44, 52]. Increased OPN secretion is compatible with a Th1 shift of H-iDC responses [47, 48]. The demonstration that TREM-1 engagement triggers production of IL-12, CCL5, and CCL17, which are implicated in the activation of Th1/Th17-polarized immune responses by recruiting inflammatory T cells and restraining expansion of Treg cells [12, 13, 49, 51, 53-57], provides additional evidence that iDCs generated under chronic hypoxia are polarized toward a Th1/Th17 proinflammatory direction. Indeed, we demonstrate that H-iDCs exhibited increased ability to stimulate allogenic T-cell proliferation and Th1/Th17 cell priming upon cross-linking with anti-TREM-1 Ab. These findings highlight TREM-1 potential to contribute to the functional reprogramming of iDCs generated at hypoxic sites toward a more mature, Th1/Th17-polarized inflammatory stage. Given the previously reported evidence that TREM-1 engagement also stimulates the Th1/Th17-polarizing activity of H-mDCs and that both TREM-1+ iDC and mDC subsets are enriched in the inflamed juvenile idiopathic arthritis hypoxic joints [23], it is reasonably to suggest that sustained expression of this molecule in DCs may be of pathologic relevance, representing a potential mechanism of amplification of the local inflammatory process and contributing to chronic inflammation [28, 30, 37].

Although the natural TREM-1 ligand(s) have not been identified, recent studies have suggested a role for this receptor in the recognition of soluble factors released by necrotic cells as a result of inflammation and/or tissue damage, such as the DAMP molecules high-mobility group box 1 and HSP70 [58, 59]. These proteins are present in inflammatory lesions [60] where they can interact with TREM-1 on myeloid cells amplifying inflammatory responses [58, 61], and the challenge of future studies will be to clarify the effective role played in vivo by TREM-1 putative ligand(s) in triggering H-iDC maturation, proinflammatory cytokine/chemokine production, and Th1/Th17-cell polarization via TREM-1 engagement.

In conclusion, our results provide novel mechanistic clues on the contribution of reduced O2 availability to the regulation of immune and inflammatory responses, unraveling the critical role of hypoxia in functionally reprogramming iDCs toward a more mature, Th1/Th17-polarized inflammatory stage.

Materials and methods

DC generation and culture

Monocytes were isolated from venous blood of voluntary healthy donors from the blood bank under an Institutional Review Board-approved protocol, by centrifugation over a Ficoll cushion (Histopaque, Sigma), followed by MACS magnetic bead separation (Miltenyi Biotec) at a purity of >93% CD14+. To generate iDCs, monocytes were plated into six-well culture plates (1.5 × 106 cells/mL) (BD Falcon) in RPMI 1640 (Euroclone) supplemented with 10% heat-inactivated FCS (HyClone) and incubated for 4 days under normoxic (20% O2) or hypoxic (1% O2) conditions, in the presence of GM-CSF and IL-4 (both 100 ng/mL), as detailed [19, 20]. Hypoxic conditions were obtained by culturing cells in an anaerobic workstation incubator (BUGBOX, CARLI Biotec) flushed with a mixture of 1% O2, 5% CO2, and 94% N2. Medium was allowed to equilibrate in a loosely capped flask in the hypoxic incubator for 2 h before use, and pO2 was monitored using a portable oxygen analyzer (Oxi 315i/set, WTW).

Cytokines and antibodies

Human recombinant GM-CSF and IL-4 were from PeproTech; echinomycin was from Alexis Biochemical. mAbs used for flow cytometry: anti-CD83-(PE), anti-CD86-PE (BD Biosciences PharMingen), anti-TREM-1-PE (BioLegend), anti-CXCR4-PE (BioLegend), anti-CCR7-allophycocyanin (BioLegend), anti-CD1a-allophycocyanin (BD), anti-HLA-DR-PE (BD), and anti-CD40-PE (Immunotech). Proper isotype-matched control Abs (BioLegend) were used.

Flow cytometry

Flow cytometry was performed as described [19, 20]. Cells resuspended with FACS buffer (PBS supplemented with 0.2% BSA, 0.01% NaN3) were incubated with fluorochrome-conjugated mAbs for 30 min at 4°C, after blocking nonspecific sites with rabbit IgG (Sigma). Fluorescence was quantitated on a FACSCalibur flow cytometer equipped with CellQuest software (BD-Biosciences). Cells were gated according to their light-scatter properties to exclude cell debris.

RNA isolation, GeneChip hybridization, and array analysis

Gene expression profiling was performed on total RNA from three donor-derived iDCs as described [19]. Briefly, RNA was reverse-transcribed, cDNA was purified and biotin labeled, and labeled cRNA was used for hybridization to Affymetrix HG-U133 plus 2.0 arrays (Genopolis Corporation, Milano) containing 54,000 probe sets coding for 38,500 genes. Data capturing was conducted with Affymetrix analysis software algorithms (Microarray Suite 5.0). Comparative analysis of hypoxic relative to normoxic expression profiles was carried out on GeneSpring Expression Analysis Software Gx9.0 (Silicon Genetics). Gene expression data were normalized using “per chip normalization” and “per gene normalization” algorithms implemented in the GeneSpring program. Gene expression levels were averaged, and fold-change was calculated as the ratio between the average expression level under hypoxia and normoxia. We selected a modulated gene list of greater than or equal to or less than or equal to twofold induction/inhibition. The significance of gene expression differences between the two experimental conditions was calculated using the Mann–Whitney U-test. Only genes that passed the test at a confidence level of 95% (p < 0.05) were considered significant. Complete data set for each microarray experiment was lodged in the Gene Expression Omnibus public repository at NCBI (www.ncbi.nlm.nih.gov/geo/) (accession number GSE6863). Validation of a subset of randomly selected genes was carried out by qRT-PCR.

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 of genes represented in the chip, as detailed [14].

Real-time RT-PCR

Real-time PCR (qRT-PCR) was performed on a 7500 Real Time PCR System (Applied), using SYBR Green PCR Master Mix and sense/antisense oligonucleotide primers designed using Primer-3 software from sequences in the GenBank and obtained from TIBMolbiol (Genova) or from Quiagen (RSP18), as detailed [36]. Expression data were normalized on the values obtained in parallel for three reference genes (ARPC1B, RPS18, RPS19), selected among those not affected by hypoxia in the Affymetrix analysis, using the Bestkeeper software, and relative expression values were calculated using Q-gene software, as detailed [23, 24].

Cross-linking of-TREM-1-positive cells

Twelve-well flat-bottom tissue culture plates (Corning Life Sciences) precoated with 10 μg/mL of agonist anti-TREM-1 mAb (R&D Systems, containing less than 0.1 EU per 1 μg of the antibody by the LAL method), control HLA-I (Serotec), irrelevant isotype-matched Ab, or left uncoated were incubated overnight at 37°C before seeding 8 × 105 H-iDCs/well/mL of fresh RPMI 1640 without cytokines. Plates were briefly spun at 130 g to engage TREM-1. After 24 h stimulation under hypoxic conditions, supernatants were harvested by centrifugation and tested for cytokine/chemokine content by ELISA and H-iDCs were used to stimulate allogeneic T cells.

Mixed leukocyte reaction

T cells were purified by negative selection from peripheral blood mononuclear cells using a PanT kit (Miltenyi Biotec). Total of 1 × 106/mL T cells were cultured with allogeneic H-iDCs previously triggered with anti-TREM-1 mAb or control HLA-I at a 20:1 T:DC ratio. After 4 days, supernatants were collected to measure released cytokines by ELISA. To assess proliferation, T cells were pulsed with 1 μCi of 3H-thymidine (Perkin Elmer) for a further 16 h culture, and 3H-thymidine incorporation was quantified using a TopCount microplates scintillation counter (Canberra Packard). All tests were performed in triplicate. Data are expressed as cpm ×10−3.

ELISA

Conditioned medium (CM) from monocyte-derived iDCs was replaced on day 3 of generation with fresh medium supplemented with cytokines for 24 h, both under normoxic and hypoxic conditions. On day 4, CM were collected, and tested for soluble (s)TREM-1 content by ELISA (R&D Systems). Secreted TNF-α, IL-12, CXCL8, IL-1β, CCL-5, CCL-17, and OPN were measured in the supernatants from iDCs triggered with anti-TREM-1 mAb or control mAbs, whereas IFN-γ, IL-17, IL-4, and IL-10 were quantified in the supernatants of T:DCs cocultures by specific ELISA (R&D Systems). Data were analyzed with the Graph Pad Prism 5 Software.

Statistical analysis

Data are the mean ± SEM of at least three independent experiments, unless differently specified. The Student's t-test was used to determine result significance (p ≤ 0.05).

Acknowledgments

This work was supported by grants from the: Associazione Italiana Ricerca sul Cancro (AIRC, “Code: IG – 10565 Funding source: 5 PER MILLE MIUR 2008 to L.V.; AIRC, Code: IG-9366” to M.G.); the European Network for Cancer Research in Children and Adolescents (ENCCA) to L.V.; Associazione Italiana Glicogenosi (AIG) to L.V.; Progetti di ricerca di Ateneo Università di Torino-Compagnia San Paolo, Special Project Microstructure and Nanostructure to M.G.; Regione Piemonte Progetti strategici Piattaforma innovativa Biotecnologie per le Scienze della Vita: Project IMMONC to F.N. F.R. was supported by a fellowships from AIRC. PBMCs and DCs were derived from the peripheral blood of healthy donors from the blood bank under an Institutional Review Board-approved protocol.

Conflict of interest

The authors declare no financial or commercial conflict of interest.

Abbreviations
CCL

CC chemokine ligand

CM

conditioned medium

CXCR

CXC chemokine receptor

GPCR

G-protein-coupled receptor

H-iDC

hypoxic immature DC

H-mDC

hypoxic mature DC

HIF

hypoxia-inducible factor

HRE

hypoxia-responsive element

LIR

leukocyte Ig-like receptor

OPN

osteopontin

pO2

partial oxygen pressure

PRR

pattern recognition receptor

TREM-1

triggering receptor expressed on myeloid cells

VEGF

vascular endothelial growth factor

Ancillary