This information is current as

of June 10, 2014.

Transcriptional Profiling of the Human

Monocyte-to-Macrophage Differentiation and
Polarization: New Molecules and Patterns of
Gene Expression
Fernando O. Martinez, Siamon Gordon, Massimo Locati and
Alberto Mantovani

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J Immunol 2006; 177:7303-7311; ;

doi: 10.4049/jimmunol.177.10.7303
http://www.jimmunol.org/content/177/10/7303

The Journal of Immunology

Transcriptional Profiling of the Human Monocyte-to-Macrophage

Differentiation and Polarization: New Molecules and Patterns
of Gene Expression1
Fernando O. Martinez,* Siamon Gordon, Massimo Locati,2* and Alberto Mantovani*

onocytes and tissue macrophages provide both immediate defense against foreign agents and assist during
the setting off and development of the adaptive immune response. Monocytes originally derive from CD34 myeloid
progenitor cells in the bone marrow, circulate in the bloodstream,
and enter peripheral tissues where they mature into different types
of resident macrophages, characterized by low oxygen consumption, low protein synthesis rate, and modest cytokine production
(1, 2). Inflammation due to tissue damage or infection results in
resident macrophage activation, which increases the production of
cytokines, chemokines, and other inflammatory mediators, as well
as monocyte recruitment. In the context of specific immune response, the cytokine milieu compels mononuclear phagocytes to
express specialized and polarized functional properties. Mirroring
the Th1/Th2 nomenclature, many refer to polarized macrophages
as M1 and M2 cells (3 6). Classically polarized activated M1
macrophages have long been known to be induced by IFN- alone
or in concert with microbial stimuli as LPS, or cytokines as TNF
and GM-CSF. M1 cells have an IL-12high, IL-23high, IL-10low phenotype, are proficient producers of effector molecules (reactive ox*Istituto Clinico Humanitas, Rozzano, Italy; Institute of General Pathology, University of Milan, Milan, Italy; and Sir William Dunn School of Pathology, University
of Oxford, Oxford, United Kingdom

ygen and nitrogen intermediates) and inflammatory cytokines (IL1, TNF, IL-6), contribute as inducer and effector cells in
polarized Th1 responses, and mediate resistance against intracellular parasites and tumors (711). In contrast, the alternative M2
form of macrophage activation is a generic name used for various
forms of nonclassically activated macrophages resulting from cell
exposure to IL-4 or IL-13, immune complexes, IL-10, glucocorticoid, or secosteroid (vitamin D3) hormones (3, 9, 12). The various
forms of M2 macrophages share an IL-12low and IL-23low phenotype, generally display high levels of scavenger, mannose (13), and
galactose-type receptors (3), and arginine metabolism is shifted to
production of ornithine and polyamines via arginase (14).
Previous studies have addressed the issue of profiling gene expression in M1 or M2 macrophage activation in the mouse, leading
to the identification of new molecules expressed in polarized murine macrophages (e.g., Ym1, Fizz1, MRC1) (13, 15, 16). Data on
human mononuclear phagocytes on the contrary are scanty and
have highlighted important interspecies differences in key molecules, such as arginase and inducible NO synthase, rendering difficult extrapolation (17, 18). In this study, we report for the first
time a whole genome transcriptional profile analysis of the human
monocyte-to-macrophage differentiation and polarized activation
processes, describing distinct molecular signatures which shed
new light on these processes and reveal new candidate markers.

Received for publication March 9, 2006. Accepted for publication August 3, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1

This work was supported by the Italian Association for Cancer Research, Ministero
dellIstruzione dellUniversita` e della Ricerca (Fondo Investimenti Ricerca di Base,
Progetto di Rilevante Interesse Nazionale, and Consiglio Nazionale delle Ricerche
funding), Fondo Interno per la Ricerca Scientifica e Tecnologica (FIRST Project),
Ministero della Salute, Fondazione Cariplo (NOBEL Project), and the European
Commission (Innochem Project, FP6-518167; Mugen Project, LSHG-CT-2005005203). F.O.M. is a recipient of the International PhD program in Cellular and
Molecular Biology fellowship from Vita-Salute San Raffaele University.

Materials and Methods

Reagents
Recombinant human cytokines were obtained from PeproTech. LPS from
Escherichia coli (serotype 055:B5) was obtained from Sigma-Aldrich. Abs
were purchased from Serotec, unless specified. Human cytokines were
measured using commercial ELISA kits purchased from R&D Systems,
according to the manufacturers instructions. All chemicals were obtained
from Sigma-Aldrich, unless specified.

Cell preparation

Address correspondence and reprint requests to Dr. Massimo Locati, Istituto Clinico
Humanitas, Via Manzoni 56, I-20089 Rozzano, Italy. E-mail address: massimo.
locati@humanitas.it
Copyright 2006 by The American Association of Immunologists, Inc.

Human monocytes were obtained from normal blood donor buffy coats by
two-step gradient centrifugation followed by an additional step using the
0022-1767/06/$02.00

Downloaded from http://www.jimmunol.org/ at Otterbein University on June 10, 2014

Comprehensive analysis of the gene expression profiles associated with human monocyte-to-macrophage differentiation and
polarization toward M1 or M2 phenotypes led to the following main results: 1) M-CSF-driven monocyte-to-macrophage
differentiation is associated with activation of cell cycle genes, substantiating the underestimated proliferation potential of
monocytes. 2) M-CSF leads to expression of a substantial part of the M2 transcriptome, suggesting that under homeostatic
conditions a default shift toward M2 occurs. 3) Modulation of genes involved in metabolic activities is a prominent feature
of macrophage differentiation and polarization. 4) Lipid metabolism is a main category of modulated transcripts, with
expected up-regulation of cyclo-oxygenase 2 in M1 cells and unexpected cyclo-oxygenase 1 up-regulation in M2 cells. 5) Each
step is characterized by a different repertoire of G protein-coupled receptors, with five nucleotide receptors as novel M2associated genes. 6) The chemokinome of polarized macrophages is profoundly diverse and new differentially expressed
chemokines are reported. Thus, transcriptome profiling reveals novel molecules and signatures associated with human
monocyte-to-macrophage differentiation and polarized activation which may represent candidate targets in pathophysiology. The
Journal of Immunology, 2006, 177: 73037311.

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TRANSCRIPTIONAL PROFILING OF MACROPHAGE DIFFERENTIATION

Monocyte Isolation kit II (Miltenyi Biotec) as previously described (17).

Macrophages were obtained by culturing monocytes (98% CD14, 13%
CD16) for 7 days in RPMI 1640 (Biochrom) supplemented with 20%
FCS (HyClone) and 100 ng/ml M-CSF in FCS-coated dishes at a density
of 1.5 105/cm2. Macrophage polarization was obtained by removing the
culture medium and culturing cells for an additional 18 h in RPMI 1640
supplemented with 5% FCS and 100 ng/ml LPS plus 20 ng/ml IFN- (for
M1 polarization) or 20 ng/ml IL-4 (for M2 polarization). Five different cell
types were generated: freshly isolated monocytes (Mo), cells at intermediate differentiation (3 days of culture: Md3), resting fully differentiated
macrophages (7 days of culture: M), classical activated macrophages
(M1), alternative activated macrophages (M2).

Transcriptional profile analysis

The transcriptional profile was evaluated in three independent cell preparations, each derived from a different single donor using the Human Genome U133 A and B arrays (HG-U133; Affymetrix) containing a total of
39,000 transcripts. RNA purification and labeling, hybridization, and array scanning were conducted as previously described (19). Scanned images
and raw data were processed using robust multiarray average (20, 21).
Principal component analysis (PCA)3 was conducted on all genes analyzed
to assign the general variability in the data to a reduced set of variables
called principal components (22). Gene expression differences were assessed by means of Students t test, with false discovery rate correction for
multiple testing (23) (R Bioconductor). Genes with an false discovery rate
0.05 and a fold change 2 were considered differentially expressed.
After removal of redundant genes, an expression matrix (3530 15) was
obtained, and figures-of-merit analysis was applied to define the optimal
number of clusters (24). Each cluster was then analyzed by K-means and
hierarchical clustering algorithms with squared Pearson correlation as similarity measurement (25). To identify overrepresented biological categories
within each cluster, the Expression Analysis Systematic Explorer analysis
based on the Gene Ontology (GO) database was applied (26), the percentage of genes within each category per total amount of genes in each cluster
was calculated, and hierarchical clustering was conducted grouping clusters according to their similarities in gene function representation. In each
cluster, the 50 most down-regulated and the 50 most up-regulated genes
were selected for interactome analysis using the ResNet-3.0 literature database (27). The entire data set and technical information requested by
Minimum Information about a Microarray Experiment (MIAME) com3
Abbreviations used in this paper: PCA, principal component analysis; GO, Gene
Ontology; GPCR, G protein-coupled receptor; ALOX5, arachidonate 5-lipoxygenase;
COX, cyclooxygenase; Mo, monocyte; M, macrophage.

FIGURE 2. K-mean clustering of genes differentially expressed during

macrophage differentiation and polarization. Modulated genes were organized
by K-means clustering. The x-axis corresponds to the experimental conditions,
the y-axis to expression levels. Each line represents a gene, with red and green
for high and low expression levels, respectively. Monocyte differentiation correlated with a cluster on early affected genes (A, 478 genes) and a cluster of late
affected genes (B, 390 genes). Two clusters (D, 1108 genes; E, 945 genes) are
associated with M1 polarization, and one cluster (F, 104 genes) with M2 polarization. C (505 genes) includes genes with a complex behavior not directly
linked to a specific cell differentiation state.
pliant are available at the Gene Expression Omnibus (GEO) website
(www.ncbi.nlm.nih.gov/geo), accession number GSE5099.

Gene expression analysis by real-time PCR

Real-time PCR was performed using gene-specific primers designed using
AutoPrime (www.autoprime.de). Primer and probe sequences are
available in the public RTPrimerDB database (http://medgen.UGent.be/
rtprimerdb/) (gene (RTPrimerDB-ID): GPR105 (3478), GPR87 (3479),
P2RY13 (3480), P2RY12 (3481), GPR171 (3482)) (28). Five replicates per
each experimental point were performed, and differences were assessed
with a two-tailed Students t test. Results were normalized using the housekeeping gene GAPDH and the cycle threshold method (19) and are
expressed as relative fold of stimulated over control group, used as
calibrator.

Western blot
After removing the medium, cells were washed in PBS and lysed in icecold lysis buffer (2% Triton X-100, 10 mM Tris-HCl (pH 8), 150 mM
NaCl, 2 mM NaN3, 2 mM EDTA) containing protease inhibitors (Roche
Molecular Biochemicals) for 45 min at 4C. Lysates were harvested and
centrifuged at 13,400 g to eliminate nuclei. Protein concentration was
determined using the bicinchoninic acid assay (Pierce) and 30 g of protein was electrophoresed in a 7.5% SDS-PAGE under nonreducing conditions and transferred to nitrocellulose using standard procedures. PTGS1
and PTGS2 were detected using the specific mAbs CXIII and CX229
(Alexis).

Results
Global transcriptome analysis
The transcriptional events associated with M-CSF-dependent
monocyte-to-macrophage differentiation and subsequent M1 or
M2 cell polarization induced by LPS plus IFN- or IL-4, respectively, were investigated using oligonucleotide microarrays. Results demonstrated the existence of a complex network of gene
regulation and clearly identified specific gene expression patterns
that characterize each phase. PCA analysis was applied to the complete dataset and demonstrated that 98% of the total variance of the

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FIGURE 1. PCA of the transcriptome expressed during macrophage

differentiation and polarization. PCA was carried on all genes under investigation to determine expression trends within the data set. Sample trend
during maturation and polarization is shown in a scatter plot of the principal components 1 and 2, which summarize 98% of the system variance.
Bars represent the SD within the three donors studied. F, monocytes (Mo);
U, monocytes after 3 days of culture with M-CSF (Md3); E, macrophages
after 7 days of culture with M-CSF (M); , M1-polarized macrophages
(M1); , M2-polarized macrophages (M2).

The Journal of Immunology

7305

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FIGURE 3. GO analysis of human macrophage differentiation and polarization. Hierarchical clustering of the percentage of genes associated
with a GO category with respect to the total number of genes in the cluster.
Columns represent clusters of Fig. 2 (the full graph is present in supplemental figure 7SM).

system lies within the first two components. PCA revealed that
monocyte maturation was associated with a significant modification of the global transcriptome (35% of the total variance), with
larger changes taking place in the early phase of the process
(24% variance in the first 3 days of differentiation), followed by
a smaller overhaul (11% variance in the last 4 days of differentiation) in the late phase (Fig. 1). Macrophage polarization was
also associated with significant changes at the transcriptional level,
although the two polarizing conditions were very different, with
M1 polarization profoundly affecting the transcriptional profile
(90% variance in the shift from M to M1), and M2 polarization
resulting in only subtle adjustments (8% variance in the shift
from M to M2) (Fig. 1).
Differentially expressed genes, selected as described in Materials and Methods, were subjected to figures-of-merit analysis,
where K-means clustering performed optimally for 12 clusters,
with no increase in the predictive value of the algorithm for additional behavioral categories (data not shown). Squared Pearson
correlation was used as similarity measurement, cumulating clusters with mirror performances and generating a total of 6 gene
clusters (Fig. 2). To gain insight into the biological processes involved, each cluster was then subjected to hierarchical subclustering (supplemental figures 1 6)4 and GO analysis as included in the
Expression Analysis Systematic Explorer (26) (Fig. 3).
Monocyte-to-macrophage differentiation was associated with
modulation of 868 (2.2%) transcripts in total (Fig. 2, A and B). Of
4

The online version of this article contains supplemental material.

FIGURE 4. Interactome analysis of representative genes involved in

monocyte-to-macrophage differentiation and polarization. A, Prototypical
macrophage was constructed from the best 100 genes representative of
each transitional state. A, Composite of genes changed transiently changed
in maturation. B, Genes stably changed with the maturation and (C) genes
differentially expressed between M1 and M2 cells (C). For A and B, red and
green represent increased and decreased genes, respectively. In C, red represents M1 genes while green stands for M2 genes.

7306

TRANSCRIPTIONAL PROFILING OF MACROPHAGE DIFFERENTIATION

Table I. Genes differentially expressed in M1 vs M2 macrophagesa

Gene Name

M1

M2

M1:M2 Ratio

CCR7
IL2RA
IL15RA
IL7R
GPR86
P2RY5
TGFBR2
HRH1
TLR5
DCL-1
MSR1
CXCR4
DECTIN1
P2RY14
DCSIGN
CLECSF13
MS4A6A
CD36
MS4A4A
MRC1

5,893
4,062
2,867
1,553
342
284
218
182
76
179
7
168
130
16
315
93
247
73
16
173

55
181
178
115
1,852
2,610
2,274
1,918
915
2,265
107
2,678
2,520
416
8,355
2,946
8,064
2,713
688
7,343

107
22
16
13
5
9
10
11
12
13
14
16
19
25
27
32
33
37
43
43

4,084
13,338
11,659
15,121
977
6,468
2,402
198
259
2,354
591
316
2,297
1,636
217
270
290
133

19
103
199
260
47
334
164
20
29
343
83
47
446
370
1,212
3,542
5,457
5,028

212
130
59
58
21
19
15
10
9
7
7
7
5
4
6
13
19
38

BCL2A1
FAS
BIRC3
GADD45G
HSXIAPAF1

4,018
2,243
1,318
682
1,928

117
156
162
144
445

34
14
8
5
4

SLC7A5
SLC21A15
SLC2A6
SLC31A2
SLC21A9
SLC4A7
SLC38A6

2,509
371
2,981
4,022
338
43
99

259
52
513
850
2,476
388
2,370

10
7
6
5
7
9
24

INDO
PLA1A
OASL
CHI3L2
HSD11B1
AK3
SPHK1
PFKFB3
PSME2
PFKP
PSMB9
PSMA2
OAS2
CTSC
HEXB
LIPA

16,605
1,854
3,272
1,078
4,066
702
1,974
2,983
10,918
1,618
3,888
4,957
944
3,161
1,582
2,252

142
55
237
89
476
103
324
509
1,789
310
817
1,199
261
13,800
6,809
9,772

117
33
14
12
9
7
6
6
6
5
5
4
4
4
4
4

CXCL11
CCL19
CXCL10
CXCL9
TNF
CCL5
CCL15
IL12B
IL15
TRAIL
IL6
CCL20
PBEF1
ECGF1
IGF1
CCL23
CCL18
CCL13

(Table continues)

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Membrane receptors
CCR7
Interleukin 2 receptor chain
Interleukin 15 receptor chain
Interleukin 7 receptor
G protein-coupled receptor 86
Purinergic receptor P2Y5
Transforming growth factor receptor II
Histamine receptor H1
Toll-like receptor 5
C-type lectin receptor DCL-1
Macrophage scavenger receptor 1
CXCR4
C-type lectin superfamily member 12
G protein-coupled receptor 105
CD209
C-type lectin superfamily member 13
Membrane-spanning 4-domains, subfamily A, member 6A
CD36
Membrane-spanning 4-domains, subfamily A, member 4
Mannose receptor C type 1
Cytokines and chemokines
CXCL11
CCL19
CXCL10
CXCL9
Tumor necrosis factor ligand superfamily, member 2
CCL5
CCL15
Interleukin 12B
Interleukin 15
Tumor necrosis factor ligand superfamily, member 10
Interleukin 6
CCL20
Visfatin
Endothelial cell growth factor 1
Insulin-like growth factor 1
CCL23
CCL18
CCL13
Apoptosis-related genes
BCL2-related protein A1
Tumor necrosis factor receptor superfamily, member 6
Baculoviral IAP repeat-containing 3
Growth arrest and DNA-damage-inducible,
XIAP associated factor-1
Solute carriers
Solute carrier family 7, member 5
Solute carrier family 21, member 15
Solute carrier family 2, member 6
Solute carrier family 31, member 2
Solute carrier family 2, member 9
Solute carrier family 4, member 7
Solute carrier family 38, member 6
Enzymes
Indoleamine-pyrrole 2,3 dioxygenase
Phospholipase A1 member A
25-oligoadenylate synthetase-like
Chitinase 3-like 2
Hydroxysteroid (11-) dehydrogenase 1
Adenylate kinase 3
Sphingosine kinase 1
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3
Proteasome activator subunit 2
Phosphofructokinase
Proteasome subunit type 9
Proteasome subunit type 2
25-oligoadenylate synthetase 2
Cathepsin C
Hexosaminidase B
Lipase A cholesterol esterase

Gene
Symbol

The Journal of Immunology

7307

Table I. (Continued)
Gene Symbol

Adenosine kinase
Histamine N-methyltransferase
Tyrosylprotein sulfotransferase 2
Ceramide kinase
Heparan sulfate 3-O-sulfotransferase 2
Leukotriene A4 hydrolase
Carbonic anhydrase II
Arachidonate 15-lipoxygenase
Heparan sulfate 3-O-sulfotransferase 1
Extracelullar mediators
Pentraxin 3
Chondroitin sulfate proteoglycan 2
Apolipoprotein L3
Insulin-like growth factor binding protein 4
Apolipoprotein L1
Platelet-derived growth factor
Endothelin 1
Apolipoprotein L2
Inhibin A
Apolipoprotein L6
Transforming growth factor -induced protein
Selenoprotein P1
Chimerin 2
Fibronectin 1
Fibrinogen-like 2
DNA-binding factors
Homeobox expressed in ES cells 1
Interferon regulatory factor 1
Activating transcription factor 3
Interferon regulatory factor 7
Growth arrest-specific 7
Early growth response 2
v-maf musculoaponeurotic fibrosarcoma oncogene homolog

ADK
HNMT
TPST2
CERK
HS3ST2
LTA4H
CA2
ALOX15
HS3ST1

M1

M2

M1-M2 Ratio

385
174
132
255
355
380
118
53
42

1,810
935
824
1,431
2,025
2,556
950
600
544

5
5
6
6
6
7
8
11
13

PTX3
CSPG2
APOL3
IGFBP4
APOL1
PDGFA
EDN1
APOL2
INHBA
APOL6
TGFBI
SEPP1
CHN2
FN1
FGL2

914
1,712
4,453
3,821
3,032
422
539
2,027
688
918
1,120
811
76
330
117

30
107
374
349
465
61
86
315
129
202
8,976
7,641
943
8,382
4,569

30
16
12
11
7
7
6
6
5
5
8
9
12
25
39

HESX1
IRF1
ATF3
IRF7
GAS7
EGR2
MAF

1,238
4,288
4,080
2,694
257
108
26

102
527
509
394
1,350
1,238
1,187

12
8
8
7
5
11
46

a
The table shows a selection of genes strictly associated with macrophage polarization grouped into functional categories. M1 and M2 columns report the mean of the
expression values. In each category, genes are ranked according to their fold difference between M1 and M2.

these, 390 (1.0%) genes were transiently regulated during the first
stage of the differentiation process and returned to basal levels in
fully differentiated macrophages (Fig. 2A; list of genes in supplemental figure 1SM). In this cluster, GO analysis highlighted an
overrepresentation of molecules associated with cell cycle (Fig. 3),
including positive modulators of cell proliferation such as cyclins
(A2, that regulates S phase progression; B1 and B2, that regulate
G2-M phase transition; D1 and D3, which regulate G1 phase progression; E2, participating in the late G1-S phase transition) and
cell division-associated proteins 1, 2, 5, 6, 7, and 20 (29 31) (supplemental figure 1SM). Interactome analysis of transiently modulated genes highlighted three main nodes around the genes CDC2,
BCL2L11, and CCL2, as well as the concerted down-regulation of
HLA members. Also notable was the high number of nuclear proteins and the low number of soluble factors among the main regulated genes (Fig. 4A). A second cluster contained 478 (1.2%)
genes rapidly regulated during the differentiation process, maintained in mature macrophages, and essentially refractory to polarizing stimuli (Fig. 2B; list of genes in supplemental figure 2SM).
These transcripts mostly corresponded to membrane receptors, signal transducers, and extracellular proteins functionally related to
the immune response (Fig. 3). Interactome analysis of stably MCSF-regulated genes showed an inversion in the gene distribution,
with a smaller percentage of nuclear genes and an increase of
membrane receptors and soluble factors. It also highlighted three
main nodes around the down-regulated genes IL-1 and IL-8 and the
up-regulated gene apolipoprotein E. A concerted down-regulation
of chemokine receptors, cystatins, and defensins, and an opposite
increase of complement components was also noticed (Fig. 4B).

As indicated by PCA, M1 polarization had a major effect on cell

transcriptome, affecting 2053 (5.2%) transcripts in total (Fig. 2, C
and D). Of these, 1108 (2.8%) genes were exclusively associated
with classical macrophage activation (Fig. 2C; list of genes in supplemental figure 3SM), while a second cluster of 945 (2.4%) transcripts included a majority of genes associated with classical activation and a minor fraction (20 genes, corresponding to 2% of
the genes) concordantly regulated by IL-4 (Fig. 2D; list of genes in
supplemental figure 4SM). GO analysis revealed an overrepresentation of genes related to DNA transcription and protein metabolism, such as ribosomal proteins and eukaryotic translation initiation factors (Fig. 3).
Although less dramatic than M1, M2 polarization exerted a
significant effect on macrophage transcriptional profile, modulating a total of 104 (0.3%) transcripts (Fig. 2E; list of genes
in supplemental figure 5SM). GO analysis indicated that this set
of genes is particularly rich in immune system-related molecules, including cytokines, chemokines, and G protein-coupled
receptors (GPCR) (Fig. 3).
A peculiar cluster included 505 (1.3%) genes with high expression in Mo and M1 cells and opposite regulation in M and M2
cells (Fig. 2, cluster F; list of genes in supplemental figure 6SM).
Unexpectedly, genes with high expression in Mo and M1 macrophages included prototypic M1 polarization markers, such as the
indoleamine-pyrrole 2,3 dioxygenase (32, 33), the lysosomal-associated membrane protein 3, IL-7R (3, 5), and CCR7, though
despite transcript expression, monocytes did not express membrane CCR7 (data not shown). Similarly, genes with high expression in M and M2 cells included classic M2 polarization markers,

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Gene Name

7308

TRANSCRIPTIONAL PROFILING OF MACROPHAGE DIFFERENTIATION

such as the mannose receptor 1 (34, 35), the scavenger receptors

SR-A and M160 (3, 5, 36). GO analysis revealed that a relevant
percentage of these genes is involved in cellular metabolic activities, such as active transport and oxidoreductase activities (Fig. 3).
To identify genes strictly associated with macrophage polarization, genes included in clusters C to F have been ranked according
to their fold difference between M1 and M2 profiles and further
grouped into functional categories (Table I). The interactome highlights a central role of a restricted panel of molecules, including
CXCR4, TRAIL (TNFSF10/TRAIL), insulin-like growth factor I,
and fibronectin 1, and clearly shows that macrophage polarization
is mainly associated with regulation of membrane receptors and
extracellular proteins with a minor contribution of nuclear factors,
directly opposite to monocyte differentiation (Fig. 4C).
GO analysis of functional categories: lipid metabolism

GO analysis of functional categories: GPCRs

A second category highlighted by GO analysis was represented by
GPCRs (Fig. 3). From a total of 465 entries of GPCRs represented
in the microarray, 53 were detected as differentially expressed during monocyte differentiation and macrophage polarization (Fig.
6A). The hierarchical clustering demonstrates that each stage is
characterized by the expression of a specific group of GPCRs.
Monocytes are characterized by a cluster of 11 highly expressed
genes, 5 of which correspond to chemotactic receptors: CCR2,
CCR5, CCR7, CX3CR1, and FPR1. The combination of IFN- and
LPS has a broad effect with no clear family overrepresentation,
while IL-4 activation is characterized by a cluster of 8 genes with
high expression, 5 of which are nucleotide receptors: GPR86,
GPR105, P2Y8, P2Y11, and P2Y12. Interestingly, GPR86,
GPR105, and P2Y12 are positioned together in chromosome 3.
Real-time PCR confirmed the up-regulation of this M2-associated
gene cluster (P2Y12, GPR105, and GPR86), and revealed minor
overexpression for the closest genomic neighbors GPR87 and
H963 (Fig. 6B).
GO analysis of functional categories: the chemokinome
Macrophage maturation and polarization are characterized by specific patterns of chemokines as suggested by GO ontology analysis
(Fig. 7A). In addition to chemokines already known to be differentially expressed in polarized macrophages (e.g., CXCL10 for
M1; CCL17 for M2), we found new chemokine signatures associated with cell polarization (Fig. 7A). The profiling results were
confirmed by real-time PCR (data not shown) and by measurements of released proteins (Fig. 7, B and C).

Discussion

FIGURE 5. Differential regulation of arachidonate metabolism-related enzymes in human macrophage differentiation and polarization. A, Transcriptional analysis reveals a unique regulatory network of the PG synthases
(PTGS), with M1 polarization resulting in increased expression of PTGS2
(COX2) and inhibition of PTGS1 (COX1), and M2 inducing a mirror regulation. B, Western blot analysis confirmed selective induction of COX2 in M1
macrophages and COX1 in M2 macrophages. Results from one experiment are
shown, representative of three independent experiments.

The present study was designed to characterize the gene expression profile of human monocytes undergoing differentiation into
mature macrophages in the presence of M-CSF and subsequent
polarized activation into M1 or M2 cells. These processes were
associated with major changes in the global transcriptome.
Macrophage polarization to M1 was associated with the most
dramatic change in the transcriptome, whereas stimulation with
IL-4 of M-CSF-differentiated macrophages caused a relatively minor alteration in gene expression. This apparently minor effect of
IL-4 is due to the fact that M-CSF-driven differentiation leads per
se to the acquisition of M2 properties, including expression of
mannose receptor 1 and scavenger receptors SR-A. This finding is
in agreement with previous data showing divergent M1-M2 properties of macrophages differentiated in GM-CSF compared with
M-CSF (38, 39). M-CSF is a homeostatic growth factor circulating
at high levels in normal blood. Thus, drifting toward M2 may be
a default pathway in macrophage differentiation.
In particular, monocyte differentiation in the presence of M-CSF
was associated with early (day 3) dramatic regulation of cell-cycle
genes, including the cyclins A2, B1, B2, D1, D3, E2, and CDCA
1, 2, 5, 6, and 7. Though human mononuclear phagocytes, unlike
mouse macrophages, are generally considered terminally differentiated nonproliferating cells, there are reports of human monocytemacrophage proliferation (40, 41). Evidence for proliferation in

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GO analysis was used to identify functional categories overrepresented in the panel of genes associated with monocyte differentiation and macrophage polarization. Consistent with the well-recognized capability of macrophages to respond and produce a vast
range of lipidic products, one of the most overrepresented categories was lipid metabolism (Fig. 3). In particular, transcriptional
analysis revealed a unique regulation profile for different enzymes
involved in eicosanoid production (Fig. 5A). Monocyte-to-macrophage maturation was associated with a gradual loss of PG-endoperoxide synthases (both PTGS1 and PTGS2), as well as the
arachidonate 5-lipoxygenase (ALOX5), and leukotriene A4 hydrolase. As expected, classical activation was associated with a
marked induction of cyclooxygenase (COX)-2 (37), accompanied
by a significant unexpected further down-regulation of COX-1, leukotriene A4 hydrolase, thromboxane A synthase 1, and ALOX5.
Conversely, alternative activation resulted in the up-regulation of

the M2 marker arachidonate 15-lipoxygenase and unexpectedly

COX-1, here confirmed at the protein level (Fig. 5B).
Sphingolipid mediators, such as sphingosine 1-phosphate, ceramide 1-phosphate, and sphingosine are derived by the enzymatic
breakdown of sphingomyelin, and display potent effects on multiple organ systems. Within this pathway, the most interesting finding is the opposite regulation of the sphingosine and ceramide
kinases, expressed in M1 and M2 macrophages, respectively.

The Journal of Immunology

7309

FIGURE 6. Repertoire of GPCRs expressed during macrophage differentiation and polarization. A, Hierarchical clustering of differentially expressed GPCRs, obtained using average linkage and Pearson correlation as
distance. B, Real-time PCR confirmation of microarray results. f and
represent M1 and M2 cells, respectively. Statistical analysis was performed
with a two-tailed paired t test in five different blood donors. SE was always
below 5% of the individual means (data not shown). All genes were significantly different in M2 vs control macrophages (p 0.05).

the culture was also confirmed in the present study (data not
shown). Thus, the proliferative potential of human monocytes
should not be underestimated and could be exploited and tailored
for cell expansion.
Modulation of genes involved in general cellular metabolic activities is a prominent feature of macrophage differentiation and
polarization. In addition to providing tools for macrophage function in tissues, these changes may have a more subtle significance.
For instance, macrophages are a major component of adipose tissue and play a role in the metabolic syndrome (42).
Macrophages are an active source of pro- and anti-inflammatory
lipid mediators, such as arachidonic acid derivatives and phosphosphingolipids. COX-2 has long been associated with arachi-

donic acid metabolism in M1 cells (37). In contrast, the finding

that M-CSF-differentiated macrophages retain high levels of
COX-1 and that these levels are further augmented by IL-4 is
novel and unexpected. This induction is of functional relevance for
eicosanoid production (data not shown) and may contribute to
pathophysiological reactions, such as toxicity of aspirin and related
drugs in asthma (43, 44). The interconvertible ceramide metabolites sphingosine 1-phosphate and ceramide 1-phosphate have
emerged as potent bioactive agents which regulate critical cellular
functions including cell proliferation, phagocytosis, differentiation,
angiogenesis, chemotaxis, and cell survival (45). Our results suggest that these enzymes can aid in distinguishing between polarized forms of activation, being sphingosine and ceramide kinase
selectively present in M1 and M2 macrophages, respectively
(Table I). M1 polarization is associated with the up-regulation of
ABCA1, the primary gatekeeper for eliminating tissue cholesterol,
and a set of apolipoproteins clustered on chromosome 22q12.3
including APOL1, APOL2, APOL3, and APOL6, which play a
central role in cholesterol transport and atherosclerosis (46).
Different stages of monocyte differentiation and polarization are
characterized by different repertoires of GPCR. In agreement with
previous reports, CCR2 is rapidly down-regulated during monocyte
differentiation (47). CX3CR1 is also down-regulated, but at a slower

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FIGURE 7. Differential expression of chemokines during macrophage

differentiation and polarization. A, Hierarchical clustering of chemokine
expression levels. B and C, ELISA measurement of secreted chemokines in
the supernatant of M1- and M2-polarized macrophages. u and f, M1 and
M2 cells, respectively. All values were statistically significant as assessed
by two-tailed paired t test (p 0.05), and represent data from five different
blood donors. Bars represent the SEM.

7310

TRANSCRIPTIONAL PROFILING OF MACROPHAGE DIFFERENTIATION

not involved in macrophage polarization in the murine system
emerged from this data set as human macrophage alternative activation markers, including fibrinoligase (F13A1) and platelet-derived growth factor C. In contrast, other mouse alternative activation markers such as the GPCR cluster discussed above were
confirmed in our system. Collectively, results indicate that 50%
of macrophage polarization markers selectively apply to one species and not to the other, cautioning against direct mouse-to-human
translation of polarization markers. A direct comparison based on
expression profiling results will be required to fully describe interspecies variability.
Polarization of mononuclear phagocyte function is a useful
simplified conceptual framework, describing a continuum of functional states. Different forms of M2 polarization have been described in vitro and ex vivo (3, 5, 9). The study reported here using
a global profiling approach, describes new molecules and signatures associated with different stages of the human monocyte-tomacrophage differentiation and polarization, which may represent
novel tools and targets in pathophysiology.

Disclosures
The authors have no financial conflict of interest.

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rate, being still expressed on day 3. LPS and IFN- up-regulate CCR7
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polarized chemokines, such as CXCL10 for M1 and CCL17 for
M2 cells, we found high levels of CCL8, CCL15, CCL19, CCL20,
and CXCL13 in M1 cells, and CCL13, CCL14, CCL17, CCL23,
and CCL26 in M2 cells. Association of these molecules with polarized macrophage activation may contribute to pathophysiology.
For instance, we found that M2 cells do not produce detectable
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IL-2R -chain, IL-15R -chain, and IL-7R as previously described
in mice (52, 53). In the other pole, M2 are characterized by the
overexpression of several scavenger receptors able to bind a diverse array of endogenous and foreign molecules (54). Our results
confirm the up-regulation by IL-4 of the mannose receptor 1 (13),
the macrophage scavenger receptor 1(36), the C-type lectin-like
receptor Dectin-1 (55) and DC-SIGN (CD209) (56) and report for
the first time in mature macrophages the up-regulation of DCIR,
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and the less described C-type lectin DCL-1 (58) and CLECSF13.
Alternatively activated macrophages are also characterized by increased expression of fibronectin (59), which is involved in cell
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The solute carrier family of proteins comprises genes whose
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markers in the human system have previously highlighted interspecies discrepancy (17, 61). This report represents the first comprehensive description of the human mononuclear phagocyte system, and provides further evidence of relevant interspecies
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human homolog of the mouse alternative activation markers arginase 1, Fizz1, MMP1 and Ym1. Similarly, a number of molecules

The Journal of Immunology

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