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Hypoxia Modifies the Transcriptome of Primary Human Monocytes: Modulation of Novel Immune-Related Genes and Identification Of CC-Chemokine Ligand 20 This information is current as as a New Hypoxia-Inducible Gene of October 1, 2021. Maria Carla Bosco, Maura Puppo, Clara Santangelo, Luca Anfosso, Ulrich Pfeffer, Paolo Fardin, Florinda Battaglia and Luigi Varesio

J Immunol 2006; 177:1941-1955; ; Downloaded from doi: 10.4049/jimmunol.177.3.1941 http://www.jimmunol.org/content/177/3/1941

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Hypoxia Modiﬁes the Transcriptome of Primary Human Monocytes: Modulation of Novel Immune-Related Genes and Identiﬁcation Of CC-Chemokine Ligand 20 as a New Hypoxia-Inducible Gene1

Maria Carla Bosco,2* Maura Puppo,* Clara Santangelo,* Luca Anfosso,‡ Ulrich Pfeffer,† Paolo Fardin,* Florinda Battaglia,* and Luigi Varesio*

Peripheral blood monocytes migrate to and accumulate in hypoxic areas of inflammatory and tumor lesions. To characterize the molecular bases underlying monocyte functions within a hypoxic microenvironment, we investigated the transcriptional profile induced by hypoxia in primary human monocytes using high-density oligonucleotide microarrays. Profound changes in the gene expression pattern were detected following 16 h exposure to 1% O2, with 536 and 677 sequences showing at least a 1.5-fold increase and decrease, respectively. Validation of this analysis was provided by quantitative RT-PCR confirmation of expression differences Downloaded from of selected genes. Among modulated genes, 74 were known hypoxia-responsive genes, whereas the majority were new genes whose responsiveness to hypoxia had not been previously described. The hypoxic transcriptome was characterized by the modulation of a significant cluster of genes with immunological relevance. These included scavenger receptors (CD163, STAB1, C1qR1, MSR1, MARCO, TLR7), immunoregulatory, costimulatory, and adhesion molecules (CD32, CD64, CD69, CD89, CMRF-35H, ITGB5, LAIR1, LIR9), chemokines/cytokines and receptors (CCL23, CCL15, CCL8, CCR1, CCR2, RDC1, IL-23A, IL-6ST). Furthermore, http://www.jimmunol.org/ we provided conclusive evidence of hypoxic induction of CCL20, a chemoattractant for immature dendritic cells, activated/memory T lymphocytes, and naive B cells. CCL20 mRNA up-regulation was paralleled by increased protein expression and secretion. This study represents the first transcriptome analysis of hypoxic primary human monocytes, which provides novel insights into monocyte functional behavior within ischemic/hypoxic tissues. CCL20 up-regulation by hypoxia may constitute an important mechanism to promote recruitment of specific leukocyte subsets at pathological sites and may have implications for the pathogenesis of chronic inflammatory diseases. The Journal of Immunology, 2006, 177: 1941–1955.

eripheral blood monocytes (Mn)3 represent the early coupled receptors differentially expressed and regulated in leuko-

mononuclear phagocyte component of the leukocyte infil- cytes (2). CCL20 (also known as MIP-3␣, liver and activation by guest on October 1, 2021 P trate at sites of inflammation, infection, and tumor growth, regulated chemokine, and Exodus) is a recently described Mn- where they differentiate into inflammatory and tumor-associated derived CC-chemokine which selectively attracts immature den- macrophages (Mf) (1). Mn/Mf are potent regulators of immune dritic cells (iDC), effector/memory T lymphocytes, and naive B and inflammatory reactions. They orchestrate the coordinated re- cells through its specific receptor, CCR6, expressed on these cells cruitment and activation of specific leukocyte subsets at patholog- (for a review, see Ref. 3). ical sites through the local secretion of low m.w. structurally re- Mononuclear phagocyte reactivity in pathological tissues is lated proteins, termed chemokines (1). Chemokines are classified finely tuned by a complex interplay between stimulatory and in- into CXC, CC, C, and CX3C families, which bind to and activate hibitory signals of various nature that include immune-derived members of a superfamily of 7-transmembrane domain, G protein- stimuli (4, 5), viral/bacterial products (5, 6), cell metabolites (4, 7), and tissue-specific signals (8). A common denominator of many pathological processes and an important regulator of gene expres- † *Laboratory of Molecular Biology, G. Gaslini Institute, and Functional Genomics, sion is represented by low partial oxygen pressure (pO ) (reviewed National Cancer Research Institute, Genova, Italy; and ‡University of Insubria, Va- 2 rese, Italy in Ref. 9). Hypoxia occurs in cardiovascular, hematological, and Received for publication November 4, 2005. Accepted for publication May 18, 2006. pulmonary disorders, ischemic wounds, arthritic joints, atheroscle- The costs of publication of this article were defrayed in part by the payment of page rotic plaques, and microbial infections, and experimental and clin- charges. This article must therefore be hereby marked advertisement in accordance ical studies point toward its fundamental role in the pathogenesis with 18 U.S.C. Section 1734 solely to indicate this fact. of these diseases (8–11). Areas of low pO2 are also present in solid 1 This work was supported by grants from the Italian Association for Cancer Re- tumors, where they have been associated with malignant progres- search, Fondazione Italiana per la Lotta al Neuroblastoma, San Paolo Company, Ital- ian Health Ministry, and Ministero Istruzione Universita’ e Ricerca. sion, metastasis formation, resistance to therapy, and poor clinical 2 Address correspondence and reprint requests to Dr. Maria Carla Bosco, Laboratorio outcome (8, 12–14). Transcriptional response to hypoxia is medi- di Biologia Molecolare, Istituto Giannina Gaslini, Padiglione 2, L.go Gerolamo ated primarily by the hypoxia-inducible factor-1 (HIF-1), a het- Gaslini 5, 16147 Genova Quarto, Italy. E-mail address: [email protected] erodimeric basic helix-loop-helix (bHLH) transcription factor 3 Abbreviations used in this paper: Mn, monocyte; Mf, macrophage; iDC, immature composed of HIF-1␤ (also known as the aryl hydrocarbon receptor dendritic cell; pO2, partial oxygen pressure; HIF-1, hypoxia-inducible factor-1; GO, Gene Ontology; EASE, Expression Analysis Systematic Explorer; qRT-PCR, real- nuclear translocator (ARNT)), the constitutive subunit, and HIF- time quantitative PCR; VEGF, vascular endothelial growth factor; HMG, hypoxia- 1␣,2␣,or3␣, the oxygen-sensitive subunits (9, 15). The ␣ sub- modulated gene; IRS, immunoregulatory signaling; ARNT, aryl hydrocarbon receptor nuclear translocator; ECM, extracellular matrix; hMDM, human monocyte-derived units are posttranslationally stabilized under hypoxia and translo- macrophage; MMP, matrix metalloproteinase; bHLH, basic helix-loop-helix. cate to the nucleus where they dimerize with HIF-1␤,

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 1942 TRANSCRIPTIONAL PROFILE OF HYPOXIC MONOCYTES transactivating the hypoxia responsive element present in the pro- conducted by electrophoresis with an Agilent Bioanalyzer 2100 (Agilent Technologies Europe). For each experimental condition, equal amounts of moter of many O2-sensitive genes (9, 15). Regulation of HIF-1 expression and activity by hypoxia is a tightly regulated process Mn RNA from 15 different donors were randomly pooled into three subsets, and the RNA pools were used for probe preparation. Briefly, 20 ␮gof which results from the activity of several oxygen-dependent en- RNA were reverse transcribed into double-stranded cDNA on a GeneAmp zymes and requires interaction and cooperation with various tran- PCR System 2700 thermal cycler (Applied Biosystems), using the Super- scriptional cofactors and other transcription factors (15). Script Double-Stranded cDNA Synthesis kit (Invitrogen Life Technolo- Mononuclear phagocytes accumulate preferentially in hypoxic/ gies) according to the manufacturer’s instructions, except that a T7-(dT)24 primer (high purity salt-free purified) was used in place of the oligo pro- ischemic areas of diseased tissues (1), and hypoxic conditions have vided with the kit. cDNA was purified and used for in vitro transcription been shown to profoundly affect their proinflammatory and immu- with the BioArray High Efficiency RNA Transcript Labeling kit (Enzo Life noregulatory responses by modulating the expression of genes Sciences) in the presence of biotin-11-CTP and biotin-16-UTP. Labeled coding for angiogenic factors, inflammatory cytokines, and extra- cRNA was cleaned up using the Qiagen RNeasy Mini kit, checked for cellular matrix (ECM) components/regulators (reviewed in Ref. 1). quality, and fragmented by incubation in mild alkaline buffer. Recent evidence indicates that hypoxia can also strictly control the GeneChip hybridization and data analysis chemokine network in cells of the monocytic lineage not only by Fragmented cRNA probes were used for hybridization to Affymetrix Hu- regulating the production of specific chemokines but also control- man Genome-U133A 2.0 GeneChips (Genopolis) containing 22,283 probe ling their action through the modulation of their receptors. Up- sets corresponding to 18,400 transcripts. Each RNA pool was hybridized to regulation of CCL3 (16), CXCL1 (1), CXCL8 (1), and CXCR4 an individual chip, and hybridization was performed at 45°C in the pres- (17), and inhibition of CCL2 (16) and CCR5 (18) under hypoxia ence of herring sperm DNA (0.1 mg/ml; Sigma-Aldrich). Chips were then ϫ ϫ were reported. washed with 6 standard saline citrate phosphate/EDTA (1 is 0.15 M

NaCl, 0.01 M sodium phosphate (pH 7.4), and 1 mM EDTA), stained with Downloaded from In the last few years, microarray technology has become an streptavidin-PE, and scanned using a confocal microscope scanner (HP important tool for the characterization of the molecular bases un- GeneArray Scanner 2500) according to Affymetrix’s guidelines. Data cap- derlying cell response to stimulation (19). Recent investigations turing was conducted with standard Affymetrix analysis software algo- have defined the transcriptional profile induced by hypoxia in in rithms (Microarray Suite 5.0), which selects the spots representative of a transcript and subtracts the background from the significant signals (23). vitro-derived human Mf (hMDM) (20, 21). Given their critical role Comparative analysis of hypoxic relative to normoxic expression profiles in the regulation of the initial phases of inflammation (1), it was was conducted on GeneSpring 7.2 software (Silicon Genetics). Gene ex- important to study primary Mn as a model of the early response to pression data for each replicate experiment were normalized using the “per http://www.jimmunol.org/ the hypoxic environment. In this study, we report the first transcrip- chip normalization” and “per gene normalization” algorithms implemented tome analysis of primary human Mn following hypoxia exposure. Our in the GeneSpring program. First, each signal was normalized based upon the median signal in that chip (“per chip normalization”). Each corrected results reveal the regulation by hypoxia of a cluster of novel genes value was then normalized based upon the median of the measurements for relevant to inflammation and immunity coding for surface molecules/ that gene in all samples (“per gene normalization”). This normalization markers, inflammatory cytokines/chemokines, and their receptors, method, which removes the differing intensity scales and binding rates and identify CCL20 as a new hypoxia-inducible gene. from multiple experimental readings, allows the comparison of multiple GeneChip hybridizations (24). Finally, gene expression levels of replicate experiments were averaged, and only genes that were modulated by at least

Materials and Methods 1.5-fold in hypoxic relative to normoxic samples (means of three experi- by guest on October 1, 2021 Cells and culture conditions ments) were considered differentially expressed. The significance of gene expression differences between the two experimental conditions was cal- PBMC were isolated from platelet apheresis of healthy donors, obtained by culated using a one-way ANOVA. Only genes that passed a Student’s the Blood Transfusion Center of the Gaslini Institute (Genova, Italy), by two-sample t test at a confidence level of 95% ( p value Ͻ0.05) were density gradient centrifugation over a Ficoll cushion (Ficoll-Paque PLUS; considered significant. The complete data set for each microarray ex- Amersham Biosciences). Mn were then purified by countercurrent centrif- periment was lodged in the ArrayExpress website ͗www.ebi.ac.uk/ ugal elutriation using a Beckman JE-6 elutriation chamber and Avanti arrayexpress͘. Gene Ontology (GO) data mining (25) for biological process J-20XP rotor system (Beckman Coulter), as described (22), followed by at level 3 and Expression Analysis Systematic Explorer (EASE) biological MACS magnetic bead separation (Human Monocyte Isolation kit-II; Milte- theme analysis (26) were conducted online at ͗http://david.niaid.nih.gov͘ nyi Biotec). The purity of Mn preparations was Ն95%, as assessed by using the Database for Annotation, Visualization, and Integrated Discovery morphology on Giemsa-stained cytocentrifuge slides and flow cytometry (DAVID) 2.0 program (27). with anti-CD14 mAb. Viability, determined by flow cytometry after DNA staining with propidium iodide (5 ␮g/ml in PBS), was Ͼ98%. Real-time RT-PCR Mn were plated in Costar plates (Celbio) in RPMI 1640 (Euroclone; Celbio) supplemented with 10% heat-inactivated FCS (HyClone; Celbio), Real time quantitative PCR (qRT-PCR) of reverse-transcribed cDNA was performed on an I-Cycler (Bio-Rad), using iQ Supermix supplemented 2mML-glutamine, 100 U/ml penicillin, and 100 ␮g/ml streptomycin (Cel- with 10 nM fluorescein (Bio-Rad), 0.1ϫ Sybr-Green I (Sigma-Aldrich), bio) and maintained at 37°C in a humidified incubator containing 21% O2, 5% CO , and 75% N , referred to as normoxic conditions. Hypoxic con- and 300 nM sense and antisense oligonucleotide primers (TIBMolbiol). All 2 2 primer pairs (listed in Table I) were designed using Primer-3 software (28) ditions (i.e., 1% O2) were achieved by incubating and handling the cells at 37°C in a humidified anaerobic work station incubator (Bug Box; ALC from sequences in GenBank with a Tm optimum of 60°C and a product length of 80–150 nt and tested before use to confirm appropriate product International) flushed with a mixture of 94% N2,5%CO2, and 1% O2. Culture medium was allowed to equilibrate for3hinaloosely capped flask size and optimal concentrations. qRT-PCR was conducted in triplicate for in the hypoxic incubator before cell exposure, and pO was monitored each target transcript under the following cycling conditions: initial dena- 2 turation of 3 min during which the well factor was measured, 50 cycles of using a portable trace oxygen analyzer (Oxi 315i/set; WTW). The pO2 in normoxic medium ranged between 149 and 150 mm Hg, values that cor- 15 s at 95°C followed by 30 s at 60°C. Fluorescence was measured during respond to a 21% O concentration in an aqueous solution at 37°C and at the annealing step in each cycle. After amplification, melting curves with 2 80 steps of 15 s and 0.5°C increase were performed to monitor amplicon a barometric pressure of 760 mm Hg, whereas the pO2 attained in the medium under hypoxic conditions was ϳ7.1 mm Hg, which is equivalent identity. Expression data were normalized on the values obtained in parallel for three reference genes (actin-related protein 2/3 complex 1B to an O2 concentration of 1% and is in the range of the hypoxic levels found in inflammatory tissues (8–12). The endotoxin content, determined by the (ARPC1B); lysosomal-associated multispanning membrane protein-5 Limulus amebocyte lysate test (QCL-1000; Bio-Whittaker), was Ͻ0.125 (LAPTM5); and thrombospondin 1 (THBS1)) selected among those not endotoxin units/ml in all reagents. affected by hypoxia in the Affymetrix analysis, using the Bestkeeper software (29). Relative expression values were calculated using Q-gene soft- RNA isolation and cRNA synthesis ware (30). For semiquantitative PCR, reverse-transcribed cDNA was amplified in Total RNA was purified from different donor-derived Mn using the RNeasy triplicate with recombinant TaqDNA polymerase (Invitrogen Life Tech- Mini kit from Qiagen. The physical quality control of RNA integrity was nologies) using the following cycling conditions: denaturation at 94°C, The Journal of Immunology 1943

Table I. Primer pairs used for real-time quantitative RT-PCRa

Geneb Primers Product (bp)

Actin-related protein 2/3 complex, subunit 1B (ARPC1B*) For 5Ј-aacgagaacaagtttgctgtg-3Ј 106 Rev 5Ј-gatgggcttcttgatgtgc-3Ј Activating transcription factor 5 (ATF5) For 5Ј-cccaccttctttcttcagc-3Ј 119 Rev 5Ј-acgaggctctggaggatg-3Ј Adrenomedullin (ADM) For 5Ј-cctgatgtacctgggttc-3Ј 118 Rev 5Ј-ttccctcttcccacgact-3Ј Arginase (ARG1) For 5Ј-attgagaaaggctggtctgc-3Ј 95 Rev 5Ј-cattagggatgtcagcaaagg-3Ј B cell activation gene (RGS1) For 5Ј-tgctgaagtaatgcaatggtct-3Ј 127 Rev 5Ј-caagccagccagaactcaat-3Ј BCL2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) For 5Ј-ttccatctctgctgctctc-3Ј 80 Rev 5Ј-tggtggaggttgtcagac-3Ј Carbonic anhydrase XII (CA12) For 5Ј-cttggcatctgtattgtggtg-3Ј 121 Rev 5Ј-tgggcctcagtctccatc-3Ј CD163 Ag (CD163) For 5Ј-ttcgtcgcattattcttcttgac-3Ј 181 Rev 5Ј-ggcaatagaccctccaag-3Ј CD69 Ag (CD69) For 5Ј-gaacatggtgctactcttgc-3Ј 81 Rev 5Ј-ttcctctctacctgcgtatc-3Ј Chemokine (CC motif) ligand 20 (CCL20) For 5Ј-aatttattgtgggcttcacacg-3Ј 115 Downloaded from Rev 5Ј-acccaagtctgttttggatttg-3Ј Chemokine (CC motif) ligand 23 (CCL23) For 5Ј-ctttgaaacgaacagcgagtg-3Ј 179 Rev 5Ј-cttgtgtcccttcaccttg-3Ј Chemokine (CXC motif) receptor 4 (CXCR4) For 5Ј-gcatctggagaaccagcg-3Ј 111 Rev 5Ј-gaaacagggttccttcatgg-3Ј Fc fragment of IgG, high-afﬁnity Ia, receptor for (FCGR1A) For 5Ј-atcgctacacatcagcagg-3Ј 137 Rev 5Ј-ctgcaagagcaactttgtttc-3Ј

Fc fragment of IgG, low-afﬁnity IIc, receptor for (FCGR2A) For 5Ј-acttctccatcccacaagc-3Ј 112 http://www.jimmunol.org/ Rev 5Ј-gagcttggacagtgatgg-3Ј Fc fragment of IgG, low-afﬁnity IIb, receptor for (FCGR2B) For 5Ј-ttacctgtccttgccactg-3Ј 124 Rev 5Ј-agtttcagcacagcctttgg-3Ј Hypoxia-inducible factor 1, ␣ subunit (HIF1A) For 5Ј-aaatctcatccaagaagcccta-3Ј 118 Rev 5Ј-cgctttctctgagcattctg-3Ј IL-1R antagonist (IL1RN) For 5Ј-tcatgctctgttcttgggaat-3Ј 132 Rev 5Ј-gcttgtcctgctttctgttc-3Ј IL-6 signal transducer (IL-6ST) For 5Ј-ggcctaatgttccagatcc-3Ј 145 Rev 5Ј-tcatttgcttctatttccacaaca-3Ј Leukocyte-associated Ig-like receptor 1 (LAIR1) For 5Ј-cagattccgcattgactcag-3Ј 122 Rev 5Ј-gaggtttctttcaccagcag-3Ј by guest on October 1, 2021 Leukocyte Ig-like receptor, subfamily B, member 7 (LIR9) For 5Ј-gtatggtcagaacccagtg-3Ј 121 Rev 5Ј-tgcgtaatcctgaaggtgtg-3Ј Leukocyte membrane Ag (CMRF-35H) For 5Ј-gcactacgcaaatctggag-3Ј 163 Rev 5Ј-tctgagcagctatcctgttg-3Ј Lysosomal-associated multispanning membrane protein-5 (LAPTM5*) For 5Ј-ggtcacacctctgagtatg-3Ј 131 Rev 5Ј-gtggaggagaagagaaactc-3Ј Macrophage migration inhibitory factor (MIF) For 5Ј-gtccttctgccatcatgc-3Ј 166 Rev 5Ј-gaaggccatgagctggt-3Ј MAX-interacting protein 1 (MXI1) For 5Ј-agatggaacgaatacgaatgg-3Ј 110 Rev 5Ј-gggagaactctgtgctttc-3Ј N-myc downstream-regulated gene 1 (NDRG1) For 5Ј-ccttatcaacgtgaacccttg-3Ј 132 Rev 5Ј-gttactctgcatttcttccttc-3Ј Secreted phosphoprotein 1 (SPP1) For 5Ј-tgacccatctcagaagcag-3Ј 111 Rev 5Ј-atggctttcgttggacttac-3Ј Stabilin 1 (STAB1) For 5Ј-actcttcgtccctgtcaatg-3Ј 157 Rev 5Ј-tcactgatgatgaggctgag-3Ј Thrombospondin 1 (THBS1*) For 5Ј-cagcattctccatcaggaac-3Ј 125 Rev 5Ј-gaggaatggactgttgatagc-3Ј Vascular endothelial growth factor (VEGF) For 5Ј-gcagcttgagttaaacgaacg-3Ј 150 Rev 5Ј-gcagcgtggtttctgtatc-3Ј Vav 3 oncogene (VAV3) For 5Ј-tgttgtgagacgtttggaatg-3Ј 112 Rev 5Ј-tgttcgagaaagtcgtgataatg-3Ј

a The expected PCR product size for each gene is shown. For, forward; Rev, reverse. b The * indicates the reference genes used for data normalization.

annealing at 61°C, and extension at 72°C for 60 s for 30 cycles. Products ized with 0.1% Triton X-100 in PBS for 5 min. Endogenous peroxidases were separated by electrophoresis on a 1.2% agarose gel and visualized by were blocked with 0.3% hydrogen peroxide for 15 min. After rinsing in ethidium bromide staining. PBS, the slides were preincubated for 30 min in blocking buffer (PBS supplemented with 2% human AB serum) and then incubated for 1 h with Immunocytochemistry anti-human CCL20 mAb or an isotype-matched mAb (IgG1; R&D System) ϩ A total of 1 ϫ 105 Mn were applied to polysine glass slides by cytocen- in blocking buffer. mAbs were detected with DakoCytomation Envision trifuging at 900 rpm for 5 min. Cytospin preparations were ﬁxed in 4% System Labeled Polymer-HRP anti-mouse. Peroxidase staining was re- paraformaldehyde in PBS for 30 min at room temperature, and permeabil- vealed by 3-amino-9-ethylcarbazole (DakoCytomation), as a substrate. 1944 TRANSCRIPTIONAL PROFILE OF HYPOXIC MONOCYTES

Slides were counterstained with hematoxylin, coverslipped with 80% up- and down-regulated genes. Moreover, hypoxic Mn showed a glycerol in PBS, and examined with a phase contrast microscope (Olympus prominent expression of genes involved in organogenesis, re- Italia). Photomicrographs were taken with a Zeiss camera. sponse to stress, biosynthesis, and phosphorus metabolism. Inter- ELISA estingly, a significant number of HMGs coded for proteins implicated in cell response to external stimuli, immune response, cell Secreted CCL20 was measured in cell-free supernatants using the Quan- tikine human CCL20 immunoassay kit from R&D Systems (Space Import adhesion and motility, and cell-cell signaling, revealing a trend Export, according to the manufacturer’s instructions. The OD of the plates toward inflammation and immunity (Fig. 2). were determined using a Spectrafluor Plus plate reader from Tecan at 450 nm. All assays were done in duplicate and repeated three times. Comparison of microarray data with known hypoxia-induced changes in gene expression Results Gene expression profile of hypoxic Mn In an initial verification of microarray data, we have cross-refer- enced our results with those of other studies investigating HMGs. Mn purified from 15 independent donors were cultured for 16 h in As summarized in Table II, a large cluster of known HMGs was 1% O , a condition previously shown to effectively modulate gene 2 affected by hypoxia. In addition to the reference gene VEGF, we expression in these cells (17, 31), and the mRNA for the angio- demonstrated up-regulation of 20 genes related to angiogenesis, VEGF genic factor, vascular endothelial growth factor ( ), was as- cell adhesion, transcription, and inflammatory responses, which sessed by RT-PCR as an index of the response to hypoxia. Fig. 1 have been previously identified as hypoxia inducible in cells of the shows VEGF mRNA levels in a representative subset of samples. Mn lineage (Table II). The majority of them, including ad- Mn exposed to normoxic conditions expressed basal levels of renomedullin (ADM), arginase-1 (ARG1), coagulation factor III Downloaded from VEGF mRNA, though showing some degree of donor-to-donor (F3), fibronectin-1 (FN1), IL-1␣ (IL-1A), IL-6, TNF-␣, macro- variation. Incubation under hypoxia caused a strong and consistent phage migration inhibitory factor (MIF), matrix metalloprotein- VEGF up-regulation in all the samples, in agreement with previous ase-1 (MMP-1), osteopontin (SPP1), and VEGF receptor-1 observations (31). (FLT1), were reported to be increased by hypoxia in hMDM and/or The transcriptional profile of hypoxic Mn was then investigated mouse Mf (20, 32), but not in primary Mn. The expression of by microarray analysis. Equal amounts of RNA from the different

another 46 known HMGs characterized in cells types other than http://www.jimmunol.org/ Mn preparations were randomly combined into three pools that mononuclear phagocytes was also triggered in hypoxic Mn (Table were independently hybridized to human Affymetrix HG-U133A II), including classical genes involved in glycolytic metabolism GeneChips, obtaining three biological replicates for each experi- and glucose transport (e.g., glucose transporter 1 and 3 (GLUT1, 3) mental condition. Statistical analysis was performed as described (9, 13, 15, 33), or associated with nonglycolytic metabolism and in Materials and Methods. Pairwise comparison among the data ion transport (e.g., carbonic anhydrase XII (CA12) (13, 33). Genes sets from normoxic and hypoxic samples (average of three exper- coding for two novel HMGs, hypoxia-inducible protein 2 (HIG2) iments) revealed the differential modulation by hypoxia of a large (34) and HIF-1-responsive RTP801 (DDIT4) (15), and other HIF-1 number of transcripts (data not shown). After restricting the profile target genes implicated in HIF-1␣ hydroxylation, such as EGL- Ն to those sequences exhibiting 1.5-fold expression differences in by guest on October 1, 2021 nine homolog-1 (EGLN1) (35) and proline 4-hydroxylases-AI,II hypoxic relative to normoxic samples, we identified 536 up-regu- (P4HA1, A2) (33, 36), were increased in hypoxic Mn (Table II). lated and 677 down-regulated genes. The majority of differentially Finally, several genes related to apoptosis, cell cycle, transcription, expressed genes were identified as unique and named in GenBank, and immune responses, whose responsiveness to hypoxia was pre- whereas the remaining transcripts were either identified as un- viously demonstrated in normal and malignant cells, were also named expressed sequence tags or were hypothetical. A selec- up-regulated in hypoxic Mn. BACH1 transcription repressor (37), tion of hypoxia-modulated genes (HMGs) is presented in Tables bHLH domain-containing B2 transcription factor (BHLHB2/ II and III. DEC1) (15), BCL2/adenovirus E1B-interacting protein 3 (BNIP-3) Functional classification of HMGs (15), hepatocyte growth factor receptor (MET) (15), IL-4 (38), IL-1 receptor antagonist (IL-1RN) (39), MAX-interacting protein-1 Genes displaying at least 1.5-fold differential expression levels (MXI1) (33), and N-myc downstream-regulated gene-1 (NDRG1) were classified into various categories based on the biological (33) represent a few examples (Table II). Interestingly, we also function(s) of the encoded protein to determine the global direction demonstrated inhibition by hypoxia of a cluster of inflammatory of the molecular response to hypoxia. According to GO data min- genes previously found down-regulated in mMf and hMDM, such ing for biological processes (Fig. 2), the transcriptional profile in- as CCL2 (16), CCR5 (18), cathepsin C (CTSC) (21), 2,5-oligoad- duced by hypoxia in Mn was mainly related to cell growth and/or enylate synthetase (OASE) (40), and the ras family member, RAB7 maintenance, signal transduction, nucleic acid, and protein metab- (21). Overall, these data demonstrate that primary Mn share with olism, these functional categories being the most enriched in both Mf and cells of other lineages a cluster of hypoxia-responsive genes.

Identiﬁcation of novel hypoxia-modulated genes in primary Mn To identify genes not previously characterized in terms of responsiveness to hypoxia, the hypoxic transcriptome of primary Mn was further investigated after excluding known HMGs listed in Table FIGURE 1. RT-PCR analysis of VEGF mRNA expression in human II. A list of selected novel hypoxia-modulated genes is presented Mn. Human peripheral blood Mn puriﬁed from different donors were cultured for 16 h under normoxic (Ϫ) or hypoxic conditions (ϩ), and total in Table III. Of interest, several were enzymes or other molecules RNA was reverse transcribed and tested for VEGF mRNA expression by implicated in lipid metabolism with a role in the regulation of fatty semiquantitative RT-PCR, as detailed in Materials and Methods. ␤-actin acid and/or cholesterol biosynthesis or transport (Table III). The mRNA was assayed in parallel as an internal control for input RNA. A most relevant are: apolipoprotein B48 receptor (APOB48R, up- representative experiment of two performed is shown regulated), apolipoprotein E (APOE, down-regulated), fatty acid The Journal of Immunology 1945

Table II. Relative expression of genes previously reported to be modulated by hypoxiaa

Function Gene Symbolb Gene Description Fold Changec Known in Mf/Mnd References

Angiogenesis ADM* Adrenomedullin 2.8 ϩ (hMDM, peritoneal Mf) 9, 13, 15, 20, 33 CALCRL Calcitonin receptor-like 1.5 79 F3* Coagulation factor III (tissue factor) 4.5 ϩ (hMDM) 20 FGF1 Acid ﬁbroblast growth factor 1 1.9 ϩ (hMDM) 43 FLT1 Vascular endothelial growth factor receptor 1 14.8 ϩ (hMDM) 9, 13, 15, 20 MIF* Macrophage migration inhibitory factor 10.1 ϩ (hMDM) 13, 20 SPP1* Secreted phosphoprotein 1 (osteopontin) 28.0 ϩ/Ϫ(hMDM) 13, 20 VEGF Vascular endothelial growth factor 7.5 ϩ (hMDM, hMn) 9, 13, 15, 20, 33 WNT5A Wingless-type 5A 1.7 ϩ (hMDM) 20 Apoptosis BCAT1* Branched chain aminotransferase 1 3.0 33 BNIP3 BCL2/adenovirus E1B 19-kDa-interacting protein 3 16.5 15 BNIP3L BCL2/adenovirus E1B 19-kDa-interacting protein 3-like 4.0 33 MCL1 Myeloid cell leukemia sequence 1 (BCL2-related) 1.8 15 TNF* Tumor necrosis factor 1.6 ϩ (hMDM, peritoneal mMf) 43 Cell adhesion/matrix/ F3* Coagulation factor III (tissue factor) 4.5 ϩ (hMDM) 20 MMP1 Matrix metalloproteinase 1 (interstitial collagenase) 15.6 ϩ (hMDM) 20 FN1 Fibronectin 1 2.0 ϩ (hMDM) 20 SPP1* Secreted phosphoprotein 1 (osteopontin) 28.0 ϩ/Ϫ(hMDM) 13, 20 TIMP-1 Tissue inhibitor of metalloproteinase 1 1.5 ϩ (hMDDC) 44 Cell cycle/ ADM* Adrenomedullin 2.8 ϩ (hMDM, peritoneal Mf) 9, 13, 15, 20, 33 differentiation BCAT1* Branched chain aminotransferase 1 3.0 33 CCNG2 Cyclin G2 1.8 13, 33 INHBA Inhibin, ␤ A (activin A) 4.0 ϩ (hMDM) 20 MET met protooncogene (hepatocyte growth factor receptor) 1.8 15

NDRG1 N-myc downstream regulated gene 1 4.1 33 Downloaded from NPM1* Nucleophosmin 1.5 15 Glucose transport SLC2A1 (GLUT1) Solute carrier family 2 (glucose transporter), member 1 3.3 9, 13, 15 SLC2A3 (GLUT3) Solute carrier family 2 (glucose transporter), member 3 5.8 9, 13, 33 Glycolytic metabolism ALDOA Aldolase A 1.6 9, 13, 15 ALDOC Aldolase C 5.3 9 ENO1* Enolase 1 3.0 9, 13, 15, 33 ENO2 Enolase 2 38.9 33 GAPDH Glyceraldehyde-3-phosphate dehydrogenase 1.6 9, 13, 15 GPI Glucose phosphate isomerase 7.2 15 HK2 Hexokinase 2 1.8 9 http://www.jimmunol.org/ LDHA Lactate dehydrogenase A 2.1 9, 13, 15 PDK1 Pyruvate dehydrogenase kinase, isoenzyme 1 24.5 33 PFKFB4 6-phosphofructo-2-kinasefructose-2,6-biphosphatase 4 2.0 15 PFKP Phosphofructokinase, platelet 7.0 9, 13, 15, 33 PGAM1 Phosphoglycerate mutase 1 2.4 15 PGK1 Phosphoglycerate kinase 1 2.6 9, 13, 15, 33 TPI1 Triose-phosphate isomerase 1 2.3 9 Immune/ responses ADORA2B Adenosine A2b receptor 2.5 80 ARG1* Arginase I 2.8 ϩ (peritoneal mMf) 32 CCL2 Chemokine (CC motif) ligand 2 (MCP-1) 0.6 ϩ (mMf) 16 CCR5 Chemokine (CC motif) receptor 5 0.6 ϩ (mMf) 18 CD55 (DAF) Decay accelerating factor for complement 1.8 81 CTSC Cathepsin C 0.3 ϩ (hMDM) 21 CTSD Cathepsin D 0.5 82 CXCR4 Chemokine (CXC motif) receptor 4 1.9 ϩ (hMn, hMf) 15, 17, 33 by guest on October 1, 2021 IL1A Interleukin 1-␣ 3.2 ϩ (peritoneal, alveolar Mf) 43 IL4 Interleukin 4 1.8 38 IL1RN Interleukin 1 receptor antagonist 3.1 39 IL6 Interleukin 6 3.8 ϩ (peritoneal mMf) 13, 43 MIF* Macrophage migration inhibitory factor 10.1 ϩ (hMDM) 13, 20 OASE1 2,5-Oligoadenylate synthetase 1 0.5 ϩ (mMf) 40 OASE2 2,5-Oligoadenylate synthetase 2 0.5 ϩ (mMf) 40 SPP1* Secreted phosphoprotein 1 (osteopontin) 28.0 ϩ/Ϫ (hMDM) 13, 20 TNF* Tumor necrosis factor 1.6 ϩ (hMDM, peritoneal mMf) 43 Metabolism (nonglycolytic) ADM* Adrenomedullin 2.8 ϩ (hMDM, peritoneal Mf) 13, 15, 20, 33 AK3 Adenylate kinase 3 2.5 9 ARGI* Arginase I 2.8 ϩ (peritoneal mMf) 32 BCAT1* Branched chain aminotransferase 1 3.0 33 CA12 Carbonic anhydrase XII 3.9 13, 33 EGLN1 Egl nine homolog 1 4.6 35 ERO1L ERO1-like 3.3 83 GBE1 Glucan (1,4-␣), branching enzyme 1 3.0 33 P4HA1 Procollagen-proline, 2-oxoglutarate 4-dioxygenasa I 4.5 33, 36 P4HA2 Procollagen-proline, 2-oxoglutarate 4-dioxygenasea II 2.2 33, 36 PAM Peptidylglycine ␣-amidating monooxygenase 4.1 33 PTGS2 Prostaglandin synthase and cyclooxygenase 2 3.6 ϩ (hMDM) 20 VLDLR Very low density lipoprotein receptor 5.3 ϩ (hMn, hMDM) 41 Stress response DD1T4 HIF-1 responsive RTP801 3.1 15 HIG2 Hypoxia-inducible protein 2 9.7 34 WSB1 SOCS box-containing WD protein SWiP-1 2.4 33 Transcription/signaling BACH1 Bach1 transcription repressor 1.8 37 BHLHB2 Basic helix-loop-helix domain containing, class B, 2 2.4 15 EGR1 Early growth response 1 2.2 ϩ (hMn) 43 ENO1* Enolase 1 3.0 9, 13, 15, 33 ID2 Inhibitor of DNA binding 2 1.6 ϩ (hMDM) 15, 21, 33 JMJD1A Zinc finger protein 3.2 33 JUN Jun oncogene 2.2 13 MXI1 MAX-interacting protein 1 4.5 33 RAB7 Member RAS oncogene family 0.6 ϩ (hMDM) 21 NPM1* Nucleophosmin 1.5 15 SNAPC1 Small nuclear RNA activating complex, polypeptide 1 3.6 33 a Gene expression profiling was carried out independently on three RNA pools, each composed by RNA from five different donor-derived Mn cultured under normoxic and hypoxic conditions, and comparative analysis of gene expression differences between the two experimental conditions was conducted as described in Materials and Methods. A function, a common gene symbol, a brief gene description, the fold change value, and published references are specified for each gene. b The* indicates genes with more than one function appearing in multiple functional categories. c The indicated values represent the ratio of hypoxic/normoxic signals (mean of three experiments). A mean ratio Ն1.5 refers to an increase in hypoxic relative to normoxic expression and a mean ratio Յ0.67 refers to a decrease in relative expression. d The ϩ sign indicates genes previously shown to be induced by hypoxia in cells of the monocytic lineage; ϩ/Ϫ indicates genes shown to require HIF-2␣ overexpression. hMn, human monocytes; hMDM, human monocyte-derived macrophages; hMDDC, human monocyte-derived dendritic cells; hMf, human macrophages; mMf, mouse macrophages. 1946 Table III. Relative expression of selected novel genes defining the transcriptome of hypoxic primary monocytesa

Up-regulated Down-regulated

GenBank Symbol Gene Description Fold Upb GenBank Symbol Gene Description Fold Downb

Cytokines/chemokines and receptors NM_020327 ACVR1B Activin A receptor, type IB 1.5 AF031587 CCL15 Chemokine (CC motif) ligand 14 (MIP-1␦) 0.18 NM_004591 CCL20 Chemokine (CC motif) ligand 20 (MIP-3␣) 7.4 AB000221 CCL18 Chemokine (CC motif) ligand 18 (PARC, MIP-4) 0.30 NM_016326 CKLF Chemokine-like factor 3 1.7 U88321 CCL19 Chemokine (CC motif) ligand 19 (Exodus-3) 0.65 NM_000757 CSF1 Colony stimulating factor 1 (macrophage) 1.6 U58913 CCL23 Chemokine (CC motif) ligand 23 (CK␤8, MP1F-1) 0.33 NM_000759 CSF3 Colony stimulating factor 3 (granulocyte) 4.8 A1984980 CCL8 Chemokine (CC motif) ligand 8 (MCP-2) 0.26 M57731 CXCL2 Chemokine (CϫC motif) ligand 2 (GRO-␤) 1.9 NM_001295 CCR1 Chemokine (CC motif) receptor 1 0.53 NM_002090 CXCL3 Chemokine (CϫC motif) ligand 3 (GRO-␥) 2.4 NM_000647 CCR2 Chemokine (CC motif) receptor 2 0.64 NM_002994 CXCL5 Chemokine (CϫC motif) ligand 5 (ENA-78) 3.1 NM_005211 CSF1R Colony stimulating factor 1 receptor (vfms) 0.63 U20350 CX3CR1 Chemokine (Cϫ3C) receptor 1 1.5 NM_000760 CSF3R Colony stimulating factor 3 receptor (granulocyte) 0.66 NM_019618 IL1F9 Interleukin-1 family, member 9 3.5 AF002985 CXCL11 Chemokine (CϫC motif) ligand 11 (1-TAC) 0.49 NM_002182 IL1RAP Interleukin 1 receptor accessory protein 3.2 NM_002993 CXCL6 Chemokine (CϫC motif) ligand 6 (GCP-2) 0.46 NM_016584 IL23A Interleukin 23 p19 subunit 2.3 NM_001953 ECGF1 Platelet-derived endothelial cell growth factor 1 0.54 NM_002184 IL6ST Interleukin 6 signal transducer (gp130) 1.8 NM_001560 IL13RA1 Interleukin 13 receptor, ␣ 1 0.65 NM_007199 IRAK3 Interleukin-1 receptor-associated kinase M 1.5 AI337584 IL18BP Interleukin 18 binding protein 0.63 AI817041 RDC1 G protein-coupled chemokine orphan receptor 4.3 NM_003855 IL18R1 Interleukin 18 receptor 1 0.50 NM_004612 TGFBR1 Transforming growth factor, ␤ receptor 1 2.7 NM_021798 IL21R Interleukin 21 receptor 0.45 BC001281 TNFRSF10B Tumor necrosis factor receptor superfamily 10b 3.5 AI983115 IL27Ra Interleukin 27 receptor a 0.63 BF433902 TNFRSF11B Tumor necrosis factor receptor superfamily 11b 2.2 NM_002253 KDR/FIk1 Kinase domain receptor (VEGFR-2) 0.64 BE568134 TNFRSF21 Tumor necrosis factor receptor superfamily 21 3.7 NM_003242 TGFBR2 Transforming growth factor, ␤ receptor II 0.48 BC006196 TNFRSF9 Tumor necrosis factor receptor superfamily 9 1.5 BC005043 TNFRSF10C Tumor necrosis factor receptor superfamily 10c 0.51 NM_003807 TNFSF14 Tumor necrosis factor (ligand) superfamily 14 4.9 NM_003810 TNFSF10 Tumor necrosis factor superfamily 10 0.51 NM_005118 TNFSF15 Tumor necrosis factor (ligand) superfamily 15 1.7 AF114013 TNFSF13 Tumor necrosis factor superfamily 13 0.47 MONOCYTES HYPOXIC OF PROFILE TRANSCRIPTIONAL NM_021138 TRAF2 TNF receptor-associated factor 2 0.52 Cytoskeleton and ECM components or regulators AI814527 ADAM8 Disintegrin and metalloproteinase 8 (CD156a) 2.3 U48734 ACTN4 Actinin, ␣ 4 0.60 AB011536 CELSR3 Cadherin, EGF LAG seven-pass G-type receptor 3 1.8 NM_021777 ADAM28 Disintegrin and metalloproteinase domain 28 0.49 NM_000094 COL7A1 Collagen, type VII, ␣ 1 3.7 AB002364 ADAMTS3 Disintegrin and metalloprotease with thrombospondin 0.52 AL121981 DLG1 Discs protein 2.0 AI796813 AVIL Advillin 0.33 AI797281 DSC3 Desmocollin 3 1.6 NM_004342 CALD1 Caldesmon 1 0.55 NM_004415 DSP Desmoplakin 1.6 AI341234 CORO1B Coronin, actin-binding protein, 1B 0.21 M62994 FLNB Filamin B, ␤ 3.1 L35594 ENPP2 Autotaxin 0.19 NM_001523 HAS1 Hyaluronan synthase 1 3.2 NM_013281 FLRT3 Fibronectin leucine- rich transmembrane protein 3 0.55 AJ406939 KRTAP4-7 Keratin- associated protein 4.7 1.9 AI633888 FYB FYN-binding protein 0.42 L25541 LAMB3 Laminin S B3 chain 1.5 NM_021991 JUP Junction plakoglobin 0.33 AF342816 LGALS8 Galectin-8, lectin galactoside-binding, soluble, 8 2.8 U76549 KRT8 Cytokeratin 8 0.45 NM_022564 MMP16 Matrix metalloproteinase 16 2.4 J03202 LAMC1 Laminin ␥ 1 0.62 NM_002429 MMP19 Matrix metalloproteinase 19 1.5 NM_006498 LGALS2 Galectin 2, lectin, galactoside-binding, soluble, 2 0.24 NM_015368 PANX1 Pannexin 1 2.4 L16895 LOX Human lysyl oxidase 0.20 AA187563 PARVB Parvin, ␤ 2.1 NM_022718 MMP25 Matrix metalloproteinase 25 0.65 BC006439 PCDHGC3 Protocadherin ␥ subfamily A, 5 2.3 NM_022870 MYH11 Myosin, heavy polypeptide 11 0.12 NM_000297 PKD2 Polycystic kidney disease 2 2.3 NM_004819 SYMPK Gsymplekin 0.54 NM_00292 RGS1 Regulator of G-protein signaling 1 2.4 NM_003247 THBS2 Thrombospondin 2 0.45 NM_006932 SMTN Smoothelin 6.4 NM_005428 VAV1 vav 1 oncogene 0.65 NM_003098 SNTA1 Syntrophin, ␣ 1 2.3 AF118887 VAV3 vav 3 oncogene 0.55 J03225 TFPI1 Tissue factor pathway inhibitor 1 1.6 NM_014000 VCL Vinculin 0.59 L27624 TFPI2 Tissue factor pathway inhibitor 2 5.5 NM_003461 ZYX Zyxin 0.55

NM_022648 TNS1 Tensin 1 16.7 (Table continues)

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Table III. Continued

Up-regulated Down-regulated

GenBank Symbol Gene Description Fold Upb GenBank Symbol Gene Description Fold Downb

Lipid metabolism NM_018361 AGPAT5 Acid acyltransferaseepsilon 2.0 NM_001995 ACSL1 Fatty acid acyl-CoA synthase long-chain family 1 0.56 NM_018690 APOB48R Apolipoprotein B48 receptor 1.7 NM_004457 ACSL3 Fatty acid acyl-CoA synthase long-chain family 3 0.53 NM_001646 APOC4 Apolipoprotein C-IV 1.8 NM_001645 APOC1 Apolipoprotein CI 0.62 NM_000709 BCKDHA Branched chain keto acid dehydrogenase El␣ 1.6 N33009 APOE Apolipoprotein E 0.32 NM_024306 FA2H Fatty acid hydroxylase (FAAH) 1.6 BC004395 APOL2 Apolipoprotein L2 0.40 NM_001442 FABP4 Fatty acid binding protein 4 1.8 NM_000784 CYP27A1 Steroid 27-hydroxylase (cytochrome P450, XXVIIA) 0.30 AI927993 OSBP Oxysterol binding protein 1.7 NM_001444 FABP5 Fatty acid-binding protein 5 0.62 NM_017784 OSBPL10 Oxysterol binding protein-like 10 2.1 AI861942 LDLR Low density lipoprotein receptor 0.65 U78577 PIP5K1A Phosphatidylinositol-4-phosphate 5-kinase ␣ 1.6 BF304759 LRP1 Low density lipoprotein-related protein 1 0.54 BC003393 PIK3CB Phosphoinositide-3-kinase, catalytic, ␤ 3.0 NM_004631 LRP8 Low density lipoprotein receptor-related protein 8 0.61 M61906 PIK3R1 Phosphoinositide-3-kinase polypeptide 1 1.5 W19983 OSBPL1A Oxysterol-binding protein-like 1A 0.41 AK023546 PLCL2 Phospholipase C-like 2 2.1 AW771015 PLCE1 Phospholipase C, ␧ 1 0.58 AF297052 PTGIS Prostacyclin synthase (CYP8A1) 2.0 NM_006226 PLCL1 Phospholipase C-like 1 0.48 AL117515 PLCL2 Phospholipase C-like 2 0.54 Surface molecules/receptors W72082 C1QR1 Complement component 1q, receptor 1 4.2 M81695 CD11C Integrin, ␣ X, leukocyte adhesion p150 0.61 NM_006139 CD28 CD28 Ag (Tp44) 1.7 NM_004334 CD157/BST1 Bone marrow stromal cell Ag 1 0.66 NM_000611 CD59 CD59 Ag p18-20 (protectin) 1.6 Z22970 CD163 CD163 Ag 0.16 L07555 CD69 Early activation Ag CD69 1.8 M98399 CD36 CD36 Ag (thrombospondin receptor) 0.41 NM_001783 CD79A CD79a Ag 1.6 BG230614 CD47 CD47 Ag (integrin-associated protein) 0.55 NM_006889 CD86 CD86 Ag (B7-2 Ag) 1.9 NM_003874 CD84 CD84 Ag (leukocyte Ag) 0.31 NM_006678 CMRF35 Leukocyte Ig-like receptor 2.1 AF200738 CLECSF6 C-type lectin superfamily member 6 (DCIR) 0.56 AJ010102 CMRF-35H Leukocyte membrane Ag 26.0 X14355 FCGR1A Fc fragment of IgG, Ia, receptor for (CD64) 0.33 U73304 CNR1 CB1 cannabinoid receptor 3.4 NM_023914 GPR86 G protein-coupled receptor 86 0.11 NM_030781 COLEC12 Scavenger receptor with G-type lectin .1 1.7 M90686 HLA-G HLA-G histocompatibility Ag class IG 0.61 U56237 FCAR Fc fragment of IgA, receptor for (CD89) 4.2 NM_002162 ICAM3 Intercellular adhesion molecule 3 0.62 NM_021642 FCGR2A Fc fragment of IgG, IIa, receptor for (CD32) 1.9 AI335208 ITGB5 Integrin, ␤ 5 0.17 M31933 FCGR2B Fc fragment of IgG, IIb, receptor for (CD32) 4.3 NM_000889 ITGB7 Integrin, ␤ 7 0.61 NM_000570 FCGR3A Fc fragment of IgG, IIIb, receptor for (CD16) 1.9 AF109683 LAIR1 Leukocyte-associated Ig-like receptor 1 0.49 NM_021624 HRH4 Histamine H4 receptor 1.7 AF009007 LILRB1 Leukocyte Ig-like receptor B1 (CD85j) 0.65 NM_003259 ICAM5 Intercellular adhesion molecule 5 2.0 AF004231 LILRB2 Leukocyte Ig-like receptor B2 (CD85d) 0.52 L76666 KIR3DL2 Killer cell immunoglobulin-like receptor 1.8 AF009635 LILRB3 Leukocyte Ig-like receptor B3 0.56 NM_016523 KLRF1 Killer cell lectin-like receptor F1 1.9 U82979 LILRB4 Leukocyte Ig-like receptor B4 0.57 NM_006770 MARCO Macrophage receptor with collagenous structure 2.0 AF212842 LIR9 Ig-like transcript 11 protein (ILT11) 0.62 NM_002445 MSR1 Macrophage scavenger receptor 1 2.1 NM_002346 LY6E Lymphocyte Ag 6 complex, locus E 0.59 NM_003693 SCARF1 Acetyl LDL receptor, scavenger receptor F1 1.8 NM_006343 MERTK c-mer proto-oncogene tyrosine kinase 0.15 AI949392 SEMA4C Semaphorin 4C 1.7 NM_023068 SN Sialoadhesin (CD169) 0.09 NM_006378 SEMA4D Semaphorin 4D (CD100) 1.8 NM_01513 STAB1 Stabilin 1 0.25 NM_004263 SEMA4F Semaphorin 4F 1.6 AF051151 TLR5 Toll-like receptor 5 0.56 NM_018643 TREM1 Triggering receptor expressed on myeloid cells 1 2.4 NM_016562 TLR7 Toll-like receptor 7 0.24 (Table continues)

1947

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Table III. Continued

Up-regulated Down-regulated

GenBank Symbol Gene Description Fold Upb GenBank Symbol Gene Description Fold Downb

Transcription factors NM_001880 ATF2 Activating transcription factor 2 1.5 NM_001621 AHR Aryl hydrocarbon receptor 0.57 NM_012068 ATF5 Activating transcription factor 5 1.8 NM_014862 ARNT2 Aryl hydrocarbon receptor nuclear translocator 2 0.37 U79751 BLZF1 Basic leucine zipper nuclear factor 1 (JEM-1) 1.6 U85962 CBP CREB-binding protein 0.49 NM_004430 EGR3 Early growth response 3 2.4 NM_004364 CEBPA CCAATenhancer binding protein (CEBP), ␣ 0.51 NM_006732 FOSB Fos homolog B 2.0 AV655640 CEBPD CCAAT/enhancer binding protein (C/EBP), ␦ 0.55 NM_024530 FOSL2 Fos-like Ag 2 1.7 NM_004904 CREB5 cAMP response element-binding protein 5 0.46 BC005329 HSF2 Heat shock transcription factor 2 2.7 NM_000399 EGR2 Early growth response 2 0.55 D13889 ID1 Inhibitor of DNA binding 1 2.2 NM_005890 GAS7 Growth arrest-speciﬁc 7 0.20 NM_002460 IRF4 IFN regulatory factor 4 1.5 NM_001530 HIF1A Hypoxia-inducible factor 1, ␣ subunit 0.50 NM_005655 KLF10 TGFB inducible early growth response 4.5 NM_002383 MAZ MYC-associated zinc ﬁnger protein 0.41 NM_002505 NFYA Nuclear transcription factor Y, ␣ 1.8 NM_000248 MITF Microphthalmia-associated transcription factor 0.55

M21985 NR2C1 TR2 nuclear receptor 2C 1 3.3 NM_005933 MLL Myeloid/lymphoid or mixed-lineage leukemia 0.28 MONOCYTES HYPOXIC OF PROFILE TRANSCRIPTIONAL NM_006186 NR4A2 Nuclear receptor subfamily 4A2 2.2 NM_002432 MNDA Myeloid cell nuclear differentiation Ag 0.20 L34598 RUNX1 Runt-related transcription factor 1 1.9 NM_002467 MYC v-myc avian myelocytomatosis viral oncogene 0.47 L40992 RUNX2 Runt-related transcription factor 2 1.7 U19179 NCOA1 Nuclear receptor coactivator 1 0.49 NM_003112 SP4 Sp4 transcription factor 1.7 NM_016250 NDRG2 N-myc downstream-regulated gene 2 0.53 AF077053 TAF9L Neuronal cell death-related protein 2.9 BF209507 PC4 Activated RNA polymerase II transcription cofactor 4 0.56 AI703074 TCF7L2 Transcription factor 7-like 2 2.8 AV727449 PCAF p300/CBP-associated factor 0.65 BF056105 TCFL4 Transcription factor-like 4 1.6 BC000080 PML Promyelocytic leukemia 0.34 NM_015905 TIF1 Transcriptional intermediary factor 1 1.7 NM_003120 SPI1 Virus proviral integration oncogene spil 0.37 K03199 TP53 p53 cellular tumor Ag 0.40 NM_006991 ZNF197 Zinc ﬁnger protein 197 0.62

a Gene expression proﬁling was carried out independently on three RNA pools, each composed by RNA from ﬁve different donor-derived Mn cultured under normoxic and hypoxic conditions, and comparative analysis of gene expression differences between the two experimental conditions was conducted as described in Materials and Methods. Each gene is given a representative GenBank accession number, a common gene symbol, a brief gene description, and the fold change value. Underlined genes were validated by qRT-PCR. b The indicated values represent the ratio of hypoxic/normoxic signals (means of three experiments). A mean ratio Ն1.5 refers to an increase in hypoxic relative to normoxic expression (fold up) and a mean ratio Յ0.67 refers to a decrease in relative expression

(fold down).

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FIGURE 2. GO data mining. The gene expression profile of hypoxic vs normoxic primary human Mn was analyzed using high-density oligonucleotide arrays, as described in Materials and Methods. Genes showing at least a 1.5-fold change in expression levels between hypoxic and normoxic cells (mean of three experiments) were selected and characterized according to their biological process classification (at level 3) in the GO database. Based on this classification scheme, genes were placed in more than one category if more than one function of the encoded protein had been established. Transcripts without a GO classification were categorized as unclassified. Bars on the right of the y-axis represent up-regulated genes; bars on the left of the y-axis by guest on October 1, 2021 represent down-regulated genes. acyl-CoA synthase long-chain family 1 and 3 (ACSL1,3, down- enhancer-binding protein ␣ (C/EBP␣), CREB-binding protein regulated), fatty acid binding protein 4 (FABP4, up-regulated), low (CBP), HIF-1␣, microphthalmia-associated transcription factor density lipoprotein receptor (LDLR, down-regulated), oxysterol (MITF), MYC oncogene, nuclear receptor coactivator 1 (NCOA1/ binding protein-like 10 (OSBPL10, up-regulated), and steroid 27- SRC-1), p53 tumor Ag (TP53), and zinc finger protein 197 hydroxylase (CYP27A1, down-regulated). Modulation of these (ZNF197/VHLaK) (Table III). These data are indicative of major, genes under low pO2 may have implications for the pathogenesis coordinated changes in transcription and suggest the existence of of atherosclerosis and Alzheimer’s disease, where a role for hyp- both positive and negative O2-driven feedback regulatory mecha- oxia has been suggested (9, 10, 41). nisms of hypoxia transcriptional response Other genes differentially expressed in hypoxic Mn coded for cytoskeleton and ECM components/regulators (Table III). Induc- Differential modulation by hypoxia of immune-related genes ible genes include adhesive proteins of the desmosome type of in Mn cell-cell junction, actin-interacting/regulatory transmembrane mol- As summarized in Table III, a prominent set of novel HMGs have ecules, and secreted proteins. Of note are galectin 8 (LGALS8), immunological relevance. These include genes encoding surface MMP-16 (MT3-MMP), MMP-19, regulator of G-protein signaling immunoregulatory signaling (IRS) receptors, such as early activa- 1(RGS1), tensin 1 (TNS1), and tissue factor pathway inhibitors 1 tion Ag (CD69), leukocyte membrane Ag CMRF-35H, low-affinity and2(TFPI1,2). Among the most significantly down-regulated IgG receptors Fc␥RIIA,B (CD32), IgA receptor Fc␣R(CD89), and genes, we identified advillin (AVIL), autotaxin (ENPP2), galectin triggering receptor expressed on myeloid cells 1 (TREM1), that 2(LGALS2), myosin heavy polypeptide 11 (MYH11), and VAV3. were up-regulated, and high-affinity IgG receptor Fc␥RIA (CD64), Modulation of these genes is likely to regulate Mn adhesion, mo- histocompatibility Ag class IG (HLA-G), leukocyte-associated Ig- tility, and tissue remodeling. like receptor 1 (LAIR1), leukocyte Ig-like receptor 9 (LIR9), and The hypoxic profile also revealed hypoxia inducibility of a set of B1,B2,B3,B4 (LILRB1–4, CD85), that were down-regulated. Sev- genes with transcription regulatory activity, including activating eral scavenging and pattern recognition receptors were also selec- transcription factor (ATF)-2 and ATF-5, Fos homolog B (FOSB), tively induced (complement component 1q receptor 1 (C1qR1); Fos-like Ag 2 (FRA2), and runt-related transcription factors 1 and macrophage receptor with collagenous structure (MARCO); mac- 2(RUNX1,2) (Table III). Moreover, several transcription factor- rophage scavenger receptor 1 (MSR1); scavenger receptor-FI and cofactor-encoding genes were inhibited by hypoxia. Of rele- (SCARF1); scavenger receptor with C-type lectin 1 (COLEC12/ vance are the aryl-hydrocarbon receptor (AHR), ARNT2, CCAAT SRCL)) or repressed (CD163 Ag; stabilin, STAB-1; TLR-5 and -7) 1950 TRANSCRIPTIONAL PROFILE OF HYPOXIC MONOCYTES under hypoxia (Table III). Other differentially expressed genes (CCL23, FCGR1A, LIR9, BNIP-3, Mxl1, and VEGF), in agreement coded for costimulatory and adhesion molecules involved in cell- with previous findings showing that microarray can often under- cell and cell-matrix interaction, such as bone marrow stromal cell estimate the extent of gene regulation compared with qRT-PCR Ag (CD157), CD36, CD84, and CD86 Ags, integrin ␣ X(CD11C), (Varesio et al., unpublished observations). For other genes, such as ␤ ␤ integrin 5 and 7 (ITGB5,7), ICAM3,5, semaphorin 4D (CD100), CMRF-35H, FCGR2B, STAB-1, and MIF, however, higher expres- and sialoadhesin (CD169) (Table III). sion differences were detected by microarray. These results con- The hypoxic transcriptome was also characterized by the mod- firm hypoxia responsiveness of novel genes identified by ulation of a cluster of genes coding for cytokines/chemokines microarray. and/or their receptors. Within the chemokine system, we identified for the first time CCL20, CXCL2, CXCL3, CXCL5, the fractalkine Hypoxia induces CCL20 expression and secretion by human Mn receptor CX3CR1, and the G protein-coupled chemokine orphan We then selected CCL20, one of the novel immune-related genes receptor (RDC1) as hypoxia-inducible genes, whereas CCL15, most strongly up-regulated in hypoxic Mn, for further analysis. CCL18, CCL23, CCL8, CXCL6, CCR1, and CCR2 were the most According to microarray and qRT-PCR data, CCL20 mRNA levels highly hypoxia-repressed genes (Table III). Finally, Mn hypoxic were increased by an average of 7.4- and 5.7-fold, respectively profile included various components of the IL-1 system (IL-1 fam- (Fig. 3). To address the issue of donor-to-donor variability, we ily member 9 (IL1F9); IL-1 receptor accessory protein (IL1RAP); analyzed CCL20 mRNA expression by RT-PCR in a subset of IL-1R associated kinase 3 (IRAK-3); IL-18R1) and members of the samples comprising the pools used for microarrays (Fig. 4A). TNF receptor and ligand superfamilies, as well as IL-23A, IL-6 CCL20 basal expression was detected in all Mn preparations cul- signal transducer (IL-6ST), IL-13RA1, IL-21R, CSF1,3, and tured under normal pO2, although with some variations among Downloaded from CSF1,3Rs (Table III). individual donors. Consistent CCL20 transcript up-regulation was triggered by hypoxia in every donor, independently of the baseline Confirmation of microarray data by qRT-PCR analysis of levels (Fig. 4A), indicating the general inducibility of the gene in selected hypoxia-modulated genes primary Mn. qRT-PCR was also performed to quantify the mag- To validate the microarray results using a different technique, nitude of hypoxia-induced changes. As shown in Fig. 4B, the ex- mRNA levels for a subset of known and novel hypoxia-modulated tent of CCL20 mRNA increase ranged from 3.1- to 11.4-fold genes were quantified by qRT-PCR on a new RNA pool (Fig. 3), among the samples examined. mRNA up-regulation was paralleled http://www.jimmunol.org/ using the primer pairs listed in Table I. For this analysis, we ran- by increased protein expression, as determined by immunocyto- domly selected 27 genes involved in immune regulation, inflam- chemistry (Fig. 5A). Mn isolated from the three donors analyzed matory responses, and transcription. Three reference genes (Table by qRT-PCR were cytocentrifuged and immunostained with a I) were used for data normalization. We found a 100% concor- mAb directed to CCL20. Low levels of CCL20 immunoreactivity dance between qRT-PCR and Affymetrix data with respect to the were detectable in the cytoplasm of normoxic Mn. Hypoxia expo- direction of the expression changes. For the majority of the genes, sure for 16 h resulted in a marked increase in intracellular protein fold-differences were also of comparable magnitude (Fig. 3), al- content in all the samples. No staining was detected when an iso-

though they were higher according to qRT-PCR for six genes type-matched control Ab was used (Fig. 5A). by guest on October 1, 2021

FIGURE 3. qRT-PCR analysis of genes selected from the microarray profile. Equal amounts of total RNA from four donor-derived normoxic or hypoxic Mn were pooled, and RNA pools were subjected to qRT-PCR analysis, as detailed in Materials and Methods. Expression changes of 27 selected genes were evaluated in relation to the values obtained in parallel for three reference genes. Results from one representative experiment of three performed are f expressed as log2 ratio (1% O2 relative to 21% O2) and are the mean of triplicate determinations for each target transcript ( ). Microarray data are shown for comparison (u). The number associated with each bar indicates the linear fold-change of mRNA expression in hypoxic relative to normoxic cells (arbitrarily defined as equal to 1). Positive values indicate that the mRNA level of a particular gene was up-regulated, whereas negative values indicate that .(ء) the transcript was down-regulated. Known hypoxia-inducible genes identified for the first time in cells of the Mn lineage are marked with an asterisk The Journal of Immunology 1951

and statistically valid mean of identifying common changes in a gene expression profile. Previous studies have established the fea- sibility of using this type of approach to overcome interindividual variability and provide reliable results that reflect gene expression in individual donors (42). The suitability of our experimental pro- tocol for identifying bona fide hypoxia-responsive genes in primary cells was inferred by the demonstration that hypoxia modulated 74 genes known from the literature to be responsive to hypoxia. The majority of these genes, involved in glycolysis, apoptosis, cell cycle, and transcription, may constitute a gene cluster commonly induced by hypoxia in cells of different lineages. The general representativeness of the gene expression changes detected by microarray was further established by qRT-PCR, which confirmed the differential expression of 27 genes randomly selected from the microarray profile in a fourth set of pooled RNAs. FIGURE 4. Up-regulation of CCL20 mRNA expression by hypoxia in Two recent reports investigated the hypoxia transcriptome of human Mn. Total RNA was isolated from human peripheral blood Mn hMDM and demonstrated induction of genes encoding angiogen- cultured under normoxic (Ϫ) or hypoxic (ϩ) conditions for 16 h. A, RNA esis and ECM regulators/components (20, 21), some of which from six different donor-derived Mn was reverse transcribed and tested for were also present in our profile. Expression of ADM, F3, FLT1, CCL20 and ␤-actin mRNA expression by semiquantitative RT-PCR. B, Downloaded from FN1, MIF, INHBA, MMP-1, PTGS2, SPP1, and VEGF, among The extent of CCL20 mRNA up-regulation by hypoxia was determined by qRT-PCR analysis on the RNA isolated from the indicated donors, and others, was detectable at significant levels in both primary human relative transcript expression was calculated in relation to the values ob- Mn (this report) and MDM (20), and exposure to hypoxia resulted tained in parallel for three reference genes, as detailed in Materials and in their strong up-regulation in both cell populations. Moreover, Methods. Results from one representative experiment are expressed as fold we demonstrated up-regulation of other genes previously shown to increase of mRNA levels in hypoxic relative to normoxic cells (arbitrarily be inducible by hypoxia in mononuclear phagocytes and coding defined as 1) and are the mean of triplicate determinations. Fold-change for immunoregulatory/proinflammatory proteins and transcription http://www.jimmunol.org/ values are indicated by a number associated with each bar. factors, such as ARG1, CXCR4, EGR1, FGF1, ID2, TIMP-1, and VLDR (17, 21, 32, 41, 43, 44). Interestingly, IL-1␣, IL-6, and TNF-␣, induction in Mn was observed in response to hypoxia Conditioned medium from normoxic and hypoxic Mn cultures alone, whereas up-regulation in Mf and MDM required LPS co- was then analyzed for CCL20 content by ELISA to determine stimulation or a reoxygenation period (43). Furthermore, we con- whether a parallel rise in chemokine secretion was triggered by firmed in primary Mn hypoxic inhibition of a cluster of inflam- hypoxia (Fig. 5B). Normoxic Mn constitutively secreted variable matory genes found down-regulated in mMf and hMDM, such as amounts of CCL20, which accumulated in the culture medium CCL2 (16), CCR5 (18), CTSC (21), OASE (40), and RAB7 (21). by guest on October 1, 2021 ranging from 10 to 410 pg/106 cells/ml in six different donors. These findings indicate that hypoxia is active on different types of Consistent with the mRNA data, chemokine secretion increased by mononuclear phagocytes and across species in regulating the ex-

2.4- to 3.5-fold upon incubation to 1% O2 for 16 h (Fig. 5B) and pression of selected target genes, which may be critical for their was further augmented at longer time points, with almost 7-fold adaptation to the hypoxic environment and ability to function in it higher amounts detectable in the conditioned medium at 72 h of and could be representative of the hypoxic transcriptome of cells culture (Fig. 5C, donor 12). CCL20 is a LPS-inducible chemokine belonging to the monocytic lineage. In contrast, the observation (3) and the response of Mn to LPS was evaluated in parallel ex- that other genes up-regulated in hMDM (20, 21) were not induced periments as a positive control. Interestingly, the extent of CCL20 or were even down-regulated in primary Mn, e.g., the angiogenesis secretion elicited by hypoxia exceeded that triggered by stimula- inducers endothelial cell growth factor-1 (ECGF1) and IL-18 bind- tion with LPS at all the time points analyzed (Fig. 5C), suggesting ing protein (IL-18BP), suggests that hypoxia may also activate in that hypoxia is a more potent stimulus than LPS in inducing mononuclear phagocytes a speciﬁc transcriptional response de- CCL20 production by Mn. pending on their differentiation stage. Taken together, these results indicate that primary human Mn Several HMGs identiﬁed in our analysis were endowed with isolated from different donors produce variable baseline levels of transcription regulatory activity and/or encoded important compo-

CCL20 but respond to low pO2 with consistent CCL20 up-regu- nents of the HIF-1 transcription pathway. Hypoxia increased the lation, confirming CCL20 as a hypoxia-inducible gene. expression of P4HA1, P4HA2, and EGLN1, three members of the prolyl-hydroxylase family which mediate HIF-␣ hydroxylation in Discussion well-oxygenated cells targeting the protein for proteosomal deg- Mn responses that ensue following recruitment at pathological radation by the von Hippel Lindau tumor suppressor protein sites begin in the setting of reduced pO2 (1). Hypoxia regulatory (pVHL) (15), and of BHLHB2, a bHLH family member implicated effects on Mn gene expression have not been characterized in de- in the regulation of pVHL/HIF pathways (15). These data are in tail, and only a limited number of hypoxia-inducible genes have agreement with previous findings in other cell types (15, 35, 36) been identified in these cells (17, 31, 41). In this study, we provide and suggest an O2-driven HIF-1-dependent autoregulatory mech- the first transcriptome analysis of hypoxic primary peripheral anism, required to ensure fast HIF-1␣ degradation upon reoxygen- blood human Mn. ation, also in Mn. Of note is the novel evidence that hypoxia pro- Microarray analysis was conducted on RNA pooled from dif- motes the expression of ATF2 and ATF5, two members of the ATF ferent donor-derived Mn, whose response to hypoxia was demon- family of transcription factors (45) required for efficient activation strated by VEGF up-regulation. Three pools, each composed by of gene transcription by hypoxia (15). Moreover, we demonstrated RNA from five different donors, were investigated per each ex- up-regulation of FosB and FRA-2, two components of the AP-1 perimental condition. RNA pooling is a powerful, cost-effective, complex (45), which is activated by hypoxia and was recently 1952 TRANSCRIPTIONAL PROFILE OF HYPOXIC MONOCYTES Downloaded from http://www.jimmunol.org/

FIGURE 5. Hypoxia induces CCL20 production by primary Mn. Human peripheral blood Mn puriﬁed from the indicated donors were cultured under

normoxic (Ϫ) or hypoxic conditions (ϩ) for 16 h (A and B) or for different time points (C), in the presence or absence of 100 ng/ml LPS. A, CCL20 protein by guest on October 1, 2021 expression was evaluated on cytospin preparations by immunocytochemical analysis using a specific mAb. Staining was conducted with the Envisionϩ System as described in the Materials and Methods. Hematoxylin-counterstained slides were examined under a phase-contrast microscope and photomi- crographed (magnification, ϫ40). CCL20 immunoreactivity is detectable in the cell cytoplasm (brown staining). Staining with an isotype-matched control mAb (IgG1) is shown for donor 7. B and C, Supernatants were harvested and assayed for secreted CCL20 by ELISA. Results from one experiment, representative of three performed, in which each sample was tested in triplicate, are expressed as picogram per 1 ϫ 106 cells/ml. The number associated with each bar indicates the fold induction of CCL20 secretion in hypoxic relative to normoxic cells (arbitrarily defined as equal to 1).

shown to cooperate with HIF-1␣ in the transactivation of hypoxia ceptors for acetylated LDL and may thus be implicated in lipid- inducible genes (46), and of JEM-1, a novel transcriptional cofac- loaded foam cell formation contributing to atherosclerotic plaques tor which enhances AP-1 activity (47). Up-regulation of these development (50). Consistent with the view that hypoxia may exert genes together with down-regulation of ZnF197, a novel VHL- a pathogenetic role in atherosclerosis and Alzheimer’s disease (9, interacting protein that functions as a repressor of HIF-1␣ trans- 10, 41) is also the down-regulation of CD163 and STAB1 scaven- activation (48), may represent a positive regulatory mechanism of ger receptors, which are endowed with atheroprotective activity hypoxia transcriptional response in Mn. Down-regulation of sev- (51, 52), and of various genes involved in the regulation of fatty eral HIF-1 transcriptional cofactors, including CBP, C/EBP␣, acid and/or cholesterol biosynthesis/transport and acting as anti- MITF, and NCOA1/SRC-1, was also observed in hypoxic Mn. All atherogenic factors, e.g., LDLR, ApoE, and CYP27A1 (53, 54). Mn these proteins physically interact with HIF-1␣ enhancing its trans- hypoxic profile also showed up-regulation of a number of other activation function under hypoxia (9, 15, 49), and their down- pattern recognition receptors critical to host defense, the most rel- regulation suggests the existence of a negative feedback mecha- evant of which are the C1qR1, COLEC12, and MARCO. These nism to control Mn hypoxic response. Consistent with this molecules bind specific Ags on bacteria facilitating their recogni- hypothesis, we observed a parallel decrease of the mRNAs encod- tion and phagocytosis (55–57). Interestingly, the TLR family ing HIF-1␣ and one of its dimerization partners, ARNT-2 (9) members TLR5 and TLR7, whose function is also to recognize GO data mining characterized a significant cluster of genes as pathogen or their products and hence initiate innate immune re- being associated with immune regulation and inflammatory responses (58), were down-regulated by hypoxia. Differential mod- sponses, chemotaxis, cell adhesion, and ECM remodeling, the ma- ulation of receptors with similar functions is intriguing and is jority of which has not been previously identified as responsive to likely to contribute to the fine tuning of Mn antimicrobial activities hypoxia. Profound changes were observed in the expression of at sites of infection. scavenger receptors. Of relevance is the up-regulation of MSR1 Myeloid cell immune functions are regulated by a balance of and SCARF1, because these molecules functions as endocytic re- inhibitory and activating signals transduced by multiple families of The Journal of Immunology 1953 cell surface IRS receptors belonging to the Ig superfamily of in- The complexity of the regulation of Mn migratory behavior by hibitory/stimulatory pairs of molecules (59). These receptors are hypoxia is further emphasized by the demonstration of dysregu- either characterized by ITIM/ITAM regulatory components in lated expression of several other migration-related genes. Mn hy- their cytoplasmic domain or pair with ITAM-containing trans- poxic profile was associated with down-regulation of the adhesion membrane adapter proteins (59). Mn exposure to hypoxia resulted molecules CD11C, CD57, and ITGB5,7, which mediate Mn adhe- in the selective modulation of various members of these receptor sion to the endothelium and/or to the ECM (67), and with up- families. Of particular interest is the up-regulation of Fc␥RIIA,B regulation of both the fractalkine receptor, which binds to the and down-regulation of Fc␥RIA. Fc␥RIIA contains a functional CX3C chemokine fractalkine expressed on endothelial cells func- ITAM motif, thus triggering cell activation, whereas Fc␥RIIB en- tioning as a potent adhesion molecule (71), and the RDC1 receptor, codes an ITIM component, leading to repression of cellular re- which share with CXCR4 the chemokine CXCL12/SDF-1 as a sponses (60). Fc␥RIA, which associates for signaling with an natural ligand (72). A critical role in the control of mononuclear ITAM-containing ␥-chain, also belongs to the activatory Fc␥R phagocyte motility is also exerted by MMPs, a group of secreted subclass (60). Fc␥Rs involvement in various autoimmune inflam- enzymes that trigger ECM degradation facilitating leukocyte matory states was reported (60), and our findings suggest the po- movement in tissues (73). Recent reports have shown up-regula- tential role of hypoxia in influencing the pathogenesis of these tion by hypoxia of MMP-1, MMP-7, and MMP12 in hMDM (20, ␥ diseases through the modulation of distinct Fc R genes. 21). Interestingly, only MMP1 was induced also in primary Mn Hypoxic modulation of other ITIM/ITAM Ig family members, that showed specific up-regulation of MMP16 and MMP19 and specifically up-regulation of FCAR, CMRF-35H, and TREM-1 and down-regulation of MMP25. Moreover, the MMP inhibitors, down-regulation of LIR9 and LAIR1, was also observed. These

TIMP1 and TFPI2, which reduce ECM degradation inhibiting cell Downloaded from molecules mediate a variety of effector functions vital to the ad- migratory activity (73, 74), were also modulated in hypoxic Mn. aptative immune response, and their ligation can have both proin- Collectively, these studies indicate that regulation of MMPs and ﬂammatory and immunosuppressive consequences by differen- their inhibitors is a common denominator of mononuclear phago- tially modulating the secretion of pro- or anti-inﬂammatory cyte response to hypoxia, but that different components of these mediators (59–63). The differential expression of inhibitory and families are controlled depending on the cell differentiation stage. activating isoforms of a given receptor family with similar speci-

Altered expression of MMPs has been associated with a variety of http://www.jimmunol.org/ ficities on the same cell is another example of the tight regulatory acute and chronic inflammatory states (73), and hypoxia can prob- role of hypoxia on Mn inflammatory responses. In particular, the ably play a role in the pathogenesis of these diseases by modulat- concomitant down-regulation of the Ig-like inhibitory receptors ing MMP production by infiltrating Mn LILRB1–4 and of their ligand HLA-G, a nonclassical inhibitory Various cytokines/chemokine and/or receptors are modulated by HLA class I Ag, is noteworthy given the role of these molecules in hypoxia in mononuclear phagocytes (1, 43). The Mn hypoxic tran- immune tolerance and immune escape (64). It is conceivable that scriptome confirmed and extended those findings showing differ- inhibition of these molecules may decrease the activation threshold ential expression of other components of the cytokine/chemokine of Mn retained at hypoxic sites. Mn activation under conditions of system. Various members of the IL-1 and the TNFR/ligand super- reduced O availability may also be controlled by the selective 2 families were selectively up- or down-regulated in hypoxic Mn, by guest on October 1, 2021 down- and up-regulation of two C-type lectin receptors, the ITIM- including molecules associated with and mediating signal trans- containing inhibitory molecule CLECSF6 (65) and CD69, a mem- duction from their receptors (e.g., IL1RAP, IRAK3, and the TNFR- ber of the NK receptor family and a potent inducer of Mn inflam- TRAF matory mediator production and cytotoxic activity (66). Although associated factor, ). Because of their pleiotropic effects on a ligand for CD69 has not been identified, previous studies sug- almost every types of cells, coordinated regulation of the TNF and gested a pathogenetic role for CD69 in certain inflammatory states IL-1 systems is likely to represent an important mechanism to characterized by hypoxia, such as rheumatoid arthritis, chronic in- control the amplitude and the duration of inflammatory responses. flammatory liver diseases, and asthma (66). The demonstration that hypoxia induces CSF1 and CSF3, while Mononuclear phagocyte migratory activity is a highly regulated inhibiting their receptors, is noteworthy given the role of these process which depends on a defined repertoire of chemokines/re- factors in the regulation of myeloid cell production, differentiation, ceptors and adhesion molecules, and dysregulated expression of and function (75) and is consistent with previous findings suggest- these proteins may alter their recruitment and activation (67). Var- ing a reciprocal and divergent action of hypoxia on receptor vs ious studies have investigated the mechanisms whereby mononu- ligand expression (16, 18). Accordingly, IL-4 up-regulation and clear phagocytes are retained/concentrated at hypoxic pathological concomitant inhibition of its receptor IL-13RA1 was observed. sites (1). One possibility is that hypoxia inhibits their migration in This interplay is likely to serve as a negative feedback mechanism response to chemokines by decreasing the expression of specific to control the autocrine activation of producing Mn. However, not chemokine receptors, as demonstrated for CCR5 in Mf (18). In all the data presented in this study are consistent with this scenario. agreement with this hypothesis and with previous findings show- Concomitant down-regulation of the Mn chemoattractants CCL2 ing impaired Mn migratory ability to CCL2 under conditions of and CCL8 and of their receptors CCR2 and CCR5 (2) was in fact observed in response to hypoxia. Similarly, we found down-reg- low pO2 (1), we observed CCR5, CCR1, and CCR2 down-regulation. This study also suggests other potential mechanisms for Mn ulation of CCL15, a chemoattractant for neutrophils, monocytes, entrapment in hypoxic areas, such as the up-regulation of RGS1, a and lymphocytes (76), and CCL23, a chemokine mediating resting member of a new class of G protein-signaling deactivators which T cell and Mn chemotaxis (77), and of their common receptor inactivates several chemotactic receptors inhibiting chemoattrac- CCR1. Collectively, these data indicate that a dynamic change in tant-induced Mn migration (68). Furthermore, repression of the chemokine/receptor expression profile occurs in Mn within hy- secreted cell motility-promoting factor, ENPP2 (69), together with poxic tissues. This tight and complex level of control exerted by induction of the antichemotactic cytokine, MIF (13), and the GRO low O2 tension is clearly of pathophysiologic relevance, represent- family chemokines, CXCL2 and CXCL3, which are specialized ing an important mechanism of regulation of leukocyte trafficking Mn-arrest chemokines (70), may also provide a “stop” signal to and function at sites of inflammation. Because some of the mod- Mn within hypoxic tissues. ulated chemokines have angiogenic activity (1, 2, 76), their altered 1954 TRANSCRIPTIONAL PROFILE OF HYPOXIC MONOCYTES

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