Gene Expression Profiling of Human Decidual
Macrophages: Evidence for Immunosuppressive
Charlotte Gustafsson1*, Jenny Mjo ¨sberg1, Andreas Matussek2, Robert Geffers3, Leif Matthiesen4, Go ¨ran
Berg4, Surendra Sharma5,6, Jan Buer7, Jan Ernerudh1
1Division of Clinical Immunology, Unit for Autoimmunity and Immune Regulation (AIR), Department of Clinical and Experimental Medicine, Faculty of Health Sciences,
University Hospital, Linko ¨ping, Sweden, 2Department of Clinical Microbiology, County Hospital Ryhov, Jo ¨nko ¨ping, Sweden, 3Mucosal Immunity, Helmholtz Centre for
Infection Research (HCI), Braunschweig, Germany, 4Division of Gynecology and Obstetrics, Faculty of Health Sciences, University Hospital, Linko ¨ping, Sweden,
5Department of Pediatrics, Brown University and Women and Infants’ Hospital of Rhode Island, Providence, Rhode Island, United States of America, 6Department of
Pathology, Brown University and Women and Infants’ Hospital of Rhode Island, Providence, Rhode Island, United States of America, 7Institute of Medical Microbiology,
University Hospital Essen, Essen, Germany
Background: Although uterine macrophages are thought to play an important regulatory role at the maternal-fetal
interface, their global gene expression profile is not known.
Methodology/Principal Findings: Using micro-array comprising approximately 14,000 genes, the gene expression pattern
of human first trimester decidual CD14+ monocytes/macrophages was characterized and compared with the expression
profile of the corresponding cells in blood. Some of the key findings were confirmed by real time PCR or by secreted
protein. A unique gene expression pattern intrinsic of first trimester decidual CD14+ cells was demonstrated. A large
number of regulated genes were functionally related to immunomodulation and tissue remodelling, corroborating
polarization patterns of differentiated macrophages mainly of the alternatively activated M2 phenotype. These include
known M2 markers such as CCL-18, CD209, insulin-like growth factor (IGF)-1, mannose receptor c type (MRC)-1 and
fibronectin-1. Further, the selective up-regulation of triggering receptor expressed on myeloid cells (TREM)-2, alpha-2-
macroglobulin (A2M) and prostaglandin D2 synthase (PGDS) provides new insights into the regulatory function of decidual
macrophages in pregnancy that may have implications in pregnancy complications.
Conclusions/Significance: The molecular characterization of decidual macrophages presents a unique transcriptional
profile replete with important components for fetal immunoprotection and provides several clues for further studies of
Citation: Gustafsson C, Mjo ¨sberg J, Matussek A, Geffers R, Matthiesen L, et al. (2008) Gene Expression Profiling of Human Decidual Macrophages: Evidence for
Immunosuppressive Phenotype. PLoS ONE 3(4): e2078. doi:10.1371/journal.pone.0002078
Editor: Derya Unutmaz, New York University School of Medicine, United States of America
Received February 20, 2008; Accepted March 21, 2008; Published April 30, 2008
Copyright: ? 2008 Gustafsson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by grants from the Swedish Research Council, the Medical Research Council of South-east Sweden (FORSS), the County
Council of O¨stergo ¨tland and by a grant from the National Institutes of Health, USA (HD-41701).
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
A distinctive immune tolerance environment is programmed at
the maternal-fetal interface to avoid rejection by the maternal
immune system. The most abundant maternal immune cells in the
decidualized endometrium are NK cells and macrophages.
Although their exact roles are currently being debated, they are
thought to regulate vascular growth and placenta development.
Global gene expression profiling of decidual NK cells provided a
unique pattern compared to their counterparts in blood . In the
case of monocytes/macrophages, accumulating data indicate that
local environment drives them into functionally different subsets.
Classically, macrophages activated by agents like IFN-c, TNF or
bacterial LPS, show profound effector functions like production of
toxic intermediates, essential in the killing of intracellular
microbes. They also secrete high amounts of pro-inflammatory
and Th1 cytokines such as TNF and IL-12. On the other hand,
macrophages activated by Th2 cytokines such as IL-4 and IL-13 as
well as anti-inflammatory cytokine IL-10 induce a suppressive
mode of activation and have been termed alternatively activated
macrophages (, reviewed in [3,4]). In this regard, Mantovani et
al  proposed a nomenclature where M1 and M2 represent
blood derived macrophages activated by inflammatory and anti-
inflammatory cytokines, respectively. However, it is not clear
whether decidual macrophages represent mainly M1 or M2
Due to their role in immune regulation, macrophages in the
uterus are likely to contribute to local immune tolerance in normal
pregnancy. They may do so by phagocytosis of apoptotic cells
harbouring potentially pro-inflammatory and harmful effects on
PLoS ONE | www.plosone.org1 April 2008 | Volume 3 | Issue 4 | e2078
the fetus (reviewed in ). Temporal expression of anti-
inflammatory cytokines such as IL-4 [7–10], IL-13 [11–13] and
IL-10 [7–10,14,15] is evident at the maternal-fetal interface,
suggesting an environment capable of facilitating alternative
activation of macrophages. It is thus likely that decidual
macrophages reveal a suppressive or regulatory phenotype in the
placenta [16,17] as well as in the decidua [9,18–21]. We report
here, for the first time, a global gene expression pattern of decidual
monocytes/macrophages. We compared them with the expression
patterns of blood CD14 positive cells. A clearly distinct gene
expression profile in decidual macrophages is evident, confirming
a selective gene cascade regulation involved in immune modula-
tion and tissue remodelling.
Materials and Methods
Decidual tissue and blood was obtained from eleven women
between 18 and 41 years of age (median age 35 years) with normal
pregnancies, undergoing elective surgical abortions in week 7 to 11
(median week 9) at the department of Gynecology and Obstetrics,
Linko ¨ping University Hospital, Sweden. All pregnancies were
detected viable and dated by crown-rump length measurement
with ultrasound. Misoprostol (CytotecH; Searle, USA) was given to
four of the eleven subjects before the procedure; the data from
these subjects did not differ from the other subjects. Laboratory
analyses performed in the different subjects are shown in table 1.
Written informed consent was obtained from all subjects. The
study was approved by the Local Ethics Committee of Linko ¨ping
Isolation of CD14 positive cells and extraction of total
Mononuclear cells were separated from decidua and blood as
described previously . CD14 positive cells were obtained by two
different methods. Cells from five subjects were sorted by
FACSAria cell sorting, a method resulting in high cell purity but
with the possible disadvantage of causing high mechanical stress to
the cells. Positive immunomagnetic MACS separation, a fast
method with a relatively low physical impact on the cells, was
performed in the six remaining subjects.
For immunomagnetic separation, CD14 positive cells were
selected with anti-CD14 antibody coated beads (Miltenyi Biotec,
Bergish Gladbach, Germany) according to the manufacturer’s
instructions. Flow cytometry in 3 of 6 samples showed purity (%
CD14+cells) of 84, 88 and 92 in blood and 72, 74 and 76 in the
decidua, respectively. For FACSAria (BD Bioscience) cell sorting,
cells were labelled with anti-CD4FITC, CD14APC (Miltenyi
Biotech), CD45PE-Cy5 and CD3 PE-Cy7 antibodies (BD
Bioscience, Stockholm, Sweden). CD14 positive cells were defined
as CD45+CD32CD14+cells and sorted accordingly. Purity of
FACSAria sorted CD14 positive cells in the two subjects used for
micro-array was 98.0 to 99.8% in blood and 96.2–97.2% in
decidua, respectively. In the three subjects used for real-time PCR,
purity was 93–98% in blood and 86–96% in the decidua,
respectively. Following sorting, cells were lysed in RNeasy RLT
lysing buffer and total RNA was extracted according to the
manufacturer’s instructions (Qiagen, West Sussex, UK).
Affymetrix GeneChip Assay
Samples were amplified for GeneChip analysis according to the
recommended protocols by the manufacturer (Affymetrix, Santa
Clara, CA, USA). In all cases, 10 mg of each biotinylated cRNA
preparation was fragmented and placed in a hybridization cocktail
containing four biotinylated hybridization controls (BioB, BioC,
BioD, and Cre), as recommended by the manufacturer. Samples
were hybridized to an identical lot of Affymetrix HG U133 2.0
GeneChips for 16 hours. After hybridization the GeneChips were
washed, stained with SA-PE, and read using an Affymetrix
GeneChip fluidic station and scanner.
Analysis of micro-array data was performed using the
Affymetrix GCOS 1.2 software. For normalization all array
experiments were scaled to a target intensity of 150, otherwise
using the default values of GCOS 1.2. Further downstream
analysis was performed using Array Assist 4.0 (Stratagene, La
Jolla, CA, USA). Data was normalized by the PLIER algorithm
(Affymetrix, Santa Clara, CA, USA) using default parameters.
Genes whose signal maximum intensity did not exceed 100 across
all samples were excluded from further analysis. Student’s T-Test
was applied to identify differences between decidual and blood
monocytes/macrophages. Genes whose p values were below or
equal to 0.05 and mean fold changes more/less than +2/22 fold
in cells from two of the high purity FACSAria separated subjects
Table 1. Methods performed on the different subjects
Subject FACSAria sortingMACS sortingMicro-array Real-time PCR Protein detectionFlow cytometry*
*Flow cytometry for control of purity
Profile of Decidualmacrophages
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were considered differentially regulated in decidual samples
compared with blood cells. These genes were used as a gene
expression signature for the experiment, resulting in 408 regulated
genes. Among these, genes fulfilling the same criteria in cells from
the five MACS separated subjects were selected and resulted in the
final 120 regulated genes. Alongside with these high-stringent
requirements, data analysis with lower stringency was performed.
With the requirement of two-fold increase/decrease and statistical
difference in all seven subjects considered as a common group with
no respect to the separation method used, a much higher number
of genes were revealed regulated (n,1700). We chose to further
analyse the genes obtained by the high-stringency requirements.
These genes were grouped according to plausible functions in the
context of macrophages in early pregnancy, resulting in the groups
immune modulation, tissue remodelling, cell cycle related and cell
To confirm regulated genes in the micro-array, real-time PCR
was performed on flow-cytometrically sorted CD14 positive cells
obtained from three additional subjects. RNA was reversely
transcribed with 500 U SuperScript II reverse transcriptase
(Invitrogen, Carlsbad, California) according to the manufacturers
protocol. Table 2 provides all genes with corresponding primers
(synthesized by TIB Molbiol, Berlin, Germany) used for real-time
PCR. Relative quantification was performed using the 7500 Fast
Real-Time PCR System (Applied Biosystems, Foster City,
California) under the following conditions: 10 min at 95 uC
followed by 40 cycles at 95uC for 15seconds and 60 seconds at
60uC. Amplification of specific PCR products was detected using
the SYBR Green PCR Master Mix (Applied Biosystems) in
duplicates. The housekeeping ribosomal protein S9 (RPS9) gene
was used for normalization. The number of genes analyzed was
restricted by the low amount of lysate available.
Analysis of ex vivo protein secretion from CD14 positive
Blood and decidual immunomagnetically separated CD14
positive cells (50 000 cells) from five subjects were incubated over
night in 200 mL of tissue culture medium (TCM) consisting of
Iscoves modification of Dulbecco’s media (Gibco BRL, Paisley,
Scotland) supplemented with (given as final concentrations in the
medium): L-glutamine (Flow Laboratories, Irvine, Scotland)
292 mg/L; sodium bicarbonate 3.024 g/L; penicillin 50 IE/mL
and streptomycin 50 mg/mL (Flow Laboratories) and 100 x non
essential amino acids 10 mL/L (Flow Laboratories). The cell-free
supernatants were stored at 270uC. The protein level of CCL18
was determined by an in-house double-antibody sandwich ELISA
using a monoclonal anti-human CCL18 (0.5 mg/mL;clone 64507;
R&D Systems, Abingdon, UK) and a polyclonal biotinylated anti-
human CCL18 Antibody (200 ng/ml; R&D Systems). Streptavi-
din-poly-horse radish peroxidase and 3,39,5,59-tetramethylbenzi-
dine liquid substrate (Sigma-Aldrich) were used for detection
(Sandberg et al, unpublished results). The levels of CCL2 and
matrix metalloproteinase (MMP)-9 in cell culture supernatants
were analyzed according to the manufacturer’s instructions. The
kits used were base kits LUH000 and LMP000 together with
analyte kits LUH279 and LMP911 (all purchased fromR&D
Systems, Abingdon, UK). Protein concentrations were analyzed
using the Luminex 100 instrument (Luminex Corporation, Austin,
Texas, USA) and the STarStation software (v 2.3, Applied
cytometry Systems, Sheffield, UK). Genes were selected for
protein analyses based on significance and possibility of detection
due to availability of assay.
Online supplemental material
A complete table of the 120 differentially regulated genes is
provided as supplementary material (Table S1). The entire
microarray dataset is available at GEO database under Acc.
Gene expression data revealed a total of 120 genes differently
regulated (80 up-regulated and 40 down-regulated) in decidual
CD14 positive cells compared to their blood counterparts, when
using the most stringent statistical criteria. A complete list of
differentially regulated genes is provided as supplementary
material and a selection is summarized in Table 3. A major
group of genes encode for proteins with immune regulatory
functions. Further, genes associated with tissue remodelling, cell
proliferation and cell metabolism were commonly regulated.
Table 2. Primers used for real-time RT-PCR
Product59 Sequence 39
IDOATT TGT CTG GCT GGA AAG GCA ACCsense 121 bp59,9 uC
AAG CAC TGA AAG ACG CTG CTT TGGantisense59,9 uC
TREM-2 ACA ACT TGT GGC TGC TGT CCT Tsense 122 bp57,3 uC
TTC CGC AGC GTA ATG GTG AGA GTantisense 59,5 uC
CD 209 AAA TCA GGA AGG CAC GTG GCA ATGsense86 bp59,9 uC
TGT TGG GCT CTC CTC TGT TCC AAT antisense59,9 uC
ICAM-3 CAA TCT CAG CAA CGT GAC TGG CAAsense 90 bp 59,9 uC
ACG GTG ATG TTA GAG GAG CCT GTT antisense59,9 uC
NRP-1 AAG ACC TTC TGC CAC TGG GAA CATsense 103 bp 59,9 uC
AGT TGC CAT CTC CTG TGT GAT CCT antisense 59,9 uC
RPS-9GGC GCA GAC GGT GGA AGCsense 83 bp 59,6 uC
GGT CTC CGC GGG GTC ACA T antisense 60,0 uC
Tm: melting temperature
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Table 3. Selection of genes differentially expressed in decidual compared to blood CD14+cells in early pregnancy.
accession numberFold changea
V-set and immunoglobulin domain containing 4;VSIG4NM_007268 23
Prostaglandin D2 synthase, hematopoietic; PGDSNM_01448513
Chemokine (C-C motif) ligand 18;CCL18, AMAC1 Y137103.8
Membrane bound form
Mannose receptor, C type 1;MRC1NM_00243836
CD209 antigen; DC-SIGNAF29088616
Triggering receptor expressed on myeloid cells 2;TREM2NM_0189659.0
Tumor necrosis factor receptor superfamily, member 21; TNFRSF21NM_016629 7.3
CD9 antigen (p24); CD9NM_0017695.3
Dipeptidase 2;DPEP2 NM_022355
C-type lectin domain family 4, member A;CLEC4A (LLIR) AF200738
Disabled homolog 2, mitogen-responsive phosphoprotein (Drosophila); DAB2AF18829830
V-maf musculoaponeurotic fibrosarcoma oncogene homolog (avian); MAF NM_005360 12
Interleukin-1 receptor-associated kinase 3;IRAK3NM_007199
Secreted phosphoprotein; SPP1 M83248 101
Complement component 3;C3NM_000064 48
Chemokine (C motif) ligand 2; XCL2U237727.6
Chemokine (C-C motif) ligand 2; CCL2, MCP1S697385.8
Chemokine (C motif) ligand 1; XCL1 (lymphotactin)NM_0031755.7
Chemokine (C-C motif) ligand 8;CCL8, MCP2AI9849805.0
Proteolipid protein 2 (colonic epithelium-enriched);PLP2NM_002668
S100 calcium binding protein A8 (calgranulin A);S100A8NM_002964
Properdin P factor, complement;PFCNM_002621
Membrane bound form
SLAM family member 8;SLAMF8 NM_0201254.7
Bone marrow stromal cell antigen 1;BST1 (CD157)NM_004334
Asialoglycoprotein receptor 2;ASGR2, CLEC4H2 NM_001181
Intercellular adhesion molecule 3;ICAM3NM_002162
EGF-like-domain, multiple 5;EGFL5W68084
Selectin L (lymphocyte adhesion molecule 1);SELLNM_000655
Vanin 2;VNN2 NM_004665
Fibronectin 1;FN1 AF130095 68
Complement component 1, q subcomponent, beta polypeptide;C1QB NM_00049158
Collagen, type III, alpha 1;COL3A1AU144167 27
Heat shock 27kDa protein 1; HSPB1NM_00154021
Complement component 1, q subcomponent, alpha polypeptide;C1QANM_015991 16
Endothelial PAS domain protein 1;EPAS1AF05209415
Growth arrest-specific 6;GAS6L13720 11
Collagen, type I, alpha 2;COL1A2 AA78871110
Collagen, type VI, alpha 3;COL6A3NM_004369 9.9
Collagen, type IV, alpha 2;COL4A2X056109.3
Serpin peptidase inhibitor, clade F, member 1;SERPINF1NM_002615 8.2
Nerve growth factor receptor (TNFRSF16) associated protein 1;NGFRAP1NM_0143807.9
Matrix metalloproteinase 9;MMP9, plasminogenNM_0049946.5
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accession numberFold changea
Protein S (alpha);PROS1NM_0003135.3
Insulin-like growth factor 1 (somatomedin C);IGF1 AI9724965.1
Syndecan 2 (heparan sulfate proteoglycan 1);SDC2AL577322 4.4
Integrin, beta 5;ITGB5BE138575 3.7
S100 calcium binding protein A4 (calcium protein, calvasculin, metastasin, murine placental
Chondroitin sulfate proteoglycan 2 (versican);CSPG2BF590263
Peptidyl arginine deiminase, type IV;PADI4NM_012387
Cell cycle related
Glycoprotein (transmembrane) nmb;GPNMBNM_002510 54
Ribonucleotide reductase M2 polypeptide;RRM2BC001886 38
Ankyrin repeat domain 25;ANKRD25NM_0154935.5
Deafness, autosomal dominant 5;DFNA5 NM_0044035.0
CDC20 cell division cycle 20 homolog (S. cerevisiae);CDC20NM_001255 4.6
KIAA0101 NM_014736 3.8
Enolase superfamily member 1;ENOSF1 NM_0175123.7
Pituitary tumor-transforming 1;PTTG1NM_0042193.4
MCM2 minichromosome maintenance deficient 2, mitotin (S. cerevisiae);MCM2 NM_0045263.2
Epithelial membrane protein 1;EMP1NM_001423 3.1
MCM6 minichromosome maintenance deficient 6 (MIS5 homolog,
S. pombe) (S. cerevisiae);MCM6
Hypothetical protein MAC30;MAC30BF038366 2.8
Sprouty homolog 2 (Drosophila);SPRY2NM_005842 2.8
Ribonuclease, RNase A family, 1 (pancreatic);RNASE1NM_002933 2.8
Stathmin 1/oncoprotein 18;STMN1NM_005563 2.7
Ras association (RalGDS/AF-6) domain family 2;RASSF2NM_014737
Chromosome 11 open reading frame 21;C11orf21NM_014144
Selenoprotein P, plasma, 1;SEPP1NM_005410 73
Phospholipid transfer protein;PLTPNM_006227 30
Solute carrier organic anion transporter family, member 2B1;SLCO2B1NM_007256 21
Folate receptor 2 (fetal);FOLR2NM_00080312
Phosphatidic acid phosphatase type 2B;PPAP2BAA628586 11
Ectonucleotide pyrophosphatase/phosphodiesterase 2;ENPP2D45421 10
Phosphatidic acid phosphatase type 2A;PPAP2AAF014403 7.5
Dehydrogenase/reductase (SDR family) member 3;DHRS3NM_004753 6.5
Solute carrier family 2 (facilitated glucose transporter), member 1;SLC2A1NM_0065164.2
ATP-binding cassette, sub-family A (ABC1), member 1;ABCA1NM_0055023.6
Chromosome 9 open reading frame 95/Nicotinamide riboside kinase;C9orf95, NRK1NM_017881 2.8
Hexokinase 3 (white cell);HK3NM_002115
Purinergic receptor P2X, ligand-gated ion channel, 1;P2RX1U45448
RAB11 family interacting protein 1 (class I);RAB11FIP1NM_025151
MOCO sulphurase C-terminal domain containing 1;MOSC1NM_022746
Solute carrier organic anion transporter family, member 3A1;SLCO3A1 NM_013272
RAB27A, member RAS oncogene family;RAB27AU38654
Selenoprotein X, 1;SEPX1NM_016332
Transketolase (Wernicke-Korsakoff syndrome);TKTL12711
aFold change is the factor of regulation of mRNA from CD14+cells in decidua versus CD14+cells in blood. Positive values denote up-regulation and negative values
mean down-regulation. Genes included in this table were up- or down-regulated by a factor of at least 2 in all seven subjects.
Table 3. Cont.
Profile of Decidualmacrophages
PLoS ONE | www.plosone.org5April 2008 | Volume 3 | Issue 4 | e2078
Figure 1 shows a cluster picture of the gene signature of 408
Real-time PCR for confirmation of mRNA expression
Real-time PCR was used to further confirm the expression of
three genes differentially expressed in the array, triggering receptor
expressed on myeloid cells (TREM-2), CD209 and ICAM-3.
Further, two genes, indoleamine 2,3-dioxygenase (IDO) and
neuropilin (NRP-1), with two-fold regulation in all seven subjects
but just below statistical significance were analyzed. PCR results
showed that these five genes were differently regulated in decidual
CD14 positive cells compared to cells from blood (Figure 2).
Luminex and ELISA for confirmation of protein secretion
Altered gene expression observed in the array analysis was also
studied at the protein level by assaying 24-hour cell culture
supernatant of CD14 positive macrophages. Decidual CD14
positive cells secreted significantly higher levels of CCL-18, CCL2
and MMP-9 compared to blood monocytes/macrophages, in
agreement with the array data (Figure 3).
The maternal-fetal interface presents a unique immune site with
the decidua populated with numerous and specialized NK cells
and macrophages. Here we report a distinct and well character-
ized micro-array analysis of human decidual macrophage gene
expression, comprising immunomodulatory and tissue remodelling
genes, as well as genes related to cell proliferation and metabolism.
While only few up-regulated genes were signature of classically
activated M1 macrophage phenotype [22–24], several of the
regulated genes corresponded to markers of alternatively activated
macrophages, including CCL18 , CD209 , mannose
receptor C type (MRC)-1 , fibronectin-1  and insulin-like
growth factor (IGF)-1 . All of these were recently shown to be
up-regulated in blood derived M2 macrophages in a gene
expression comparison with M1 polarized macrophages . In
addition, a number of the differentially regulated genes have
previously been assigned to decidual macrophages and are
confirmed by our data. We also report evidence of other markers
connected to alternative macrophage activation, e.g. TREM-2,
alpha 2 macroglobulin (A2M) and prostaglandin D2 synthase
Among the up-regulated genes a major group could be classified
as immune modulatory with immuno-suppressive or anti-inflam-
matory functions. In agreement with this, a group of activating or
pro-inflammatory genes were down-regulated in decidual macro-
phages compared to their blood counterparts. Several of the up-
regulated genes are surface receptors, among these the often co-
expressed lectins MRC-1 (CD206, macrophage mannose receptor;
MMR) and CD209 as well as the tetraspanin CD9. All have
previously been detected in uterine cells in human pregnancy
[1,27–31]. MRC1 and CD209 (dendritic cell specific intercellular
adhesion molecule-grabbing nonintegrin; DC-SIGN) are generally
associated with alternative activation profile of macrophages.
NRP-1, involved in establishment of the immunological synapse, is
a receptor connected to regulatory T cells . NRP-1 is up-
regulated in decidual macrophages and was recently shown to be
Figure 2. Mean results from three independent real-time PCR
analyses for TREM2, CD209, ICAM3, IDO and NRP-1 showing
fold change of mRNA expression in decidual compared to
blood CD14 positive cells.
Subject 1 2 3 4 5 6 7 Subject 1 2 3 4 5 6 7
Figure 1. Cluster figure of the gene signature of 408 genes
regulated in decidual compared to blood macrophages,
showing all seven subjects (columns). Requirements for regulation
was 2-fold up-/down-regulation and a p-value ,0.05 in the two
subjects with highly pure cellpopulations (subjects 1 and 2).
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up-regulated in M2 cells , indicating a potential role in
TREM-2 expression, to our knowledge, has not been previously
reported in uterine tissue or in relation to pregnancy. In this
regard, we propose a novel mechanism of potential importance of
TREM-2 in maternal-fetal tolerance. In mouse macrophages,
TREM-2 is induced by IL-4 and is down-regulated by IFN-c or
LPS . Information on TREM-2 in humans is very limited,
although it was found to be down-regulated in LPS-stimulated
human monocyte-derived dendritic cells (DCs) . Murine
macrophages express a ligand for TREM-2 on their surface thus
enabling auto-regulation . High expression of TREM-2 in
mouse microglial cells correlate with their ability to phagocytose
apoptotic neurons, hence TREM-2 might be positively regulating
phagocytosis but negatively regulating inflammatory responses
. This proposed dual function of TREM-2 positive cells would
fit well in the context of the pregnant uterus, where the need for
both immune regulation and apoptotic clearance is high.
Chemokines play a central role in polarization of macrophages.
CCL2 and CCL18 were found to be up-regulated, the latter being
involved in recruitment and possibly tolerance indcuction of naı ¨ve
T cells . Interestingly, CCL18 was found to be expressed in
human decidual and amnion tissues during pregnancy whereas
expression was reduced after onset of labour . On the other
hand, CCL2 (monocyte chemoattractant protein-1; MCP-1) is
classically linked with inflammatory responses (reviewed in ).
However, CCL2 was shown to drive Th2 polarization in mice and
stimulation with IL-4/IL-13 in human lung epithelial cells
increased their secretion of CCL2 . Daly et al hypothesized a
dual effect of CCL2 depending on environmental demands .
We therefore suggest a role for CCL2 in pregnancy in recruiting
monocytes/macrophages to the decidua, where other local factors
will be decisive for polarization. PGDS is another up-regulated
soluble immunemodulating molecuble that has been associated
with decidual Th2 cell recruitment and antigen presentation .
IDO, a tryptophan depleting enzyme, was ascribed a role in
murine pregnancy . Although the role of IDO at the maternal-
fetal interface has been questioned , its expression in decidual
macrophages has previously been noted , thus a finding
confirmed in this study.
Considering the plasticity and growth of the feto-placental unit,
functions related to angiogenesis and depletion of apoptotic cells
are important for a successful pregnancy. Accordingly, a large
group of genes up-regulated in decidual CD14 positive cells was
associated with tissue remodelling. Fibronectin increases apoptotic
clearance of cells coated e.g. with C1Qs, both molecules being up-
regulated here. We also found up-regulation of collagen genes as
well as of other molecules promoting clearance or angiogenesis,
e.g. growth arrest-specific (GAS)-6 and protein S alpha (PROS1).
Although MMP9 (plasminogen) is considered a proinflammatory
chemokine, it also cleaves denatured collagens and type IV
collagens in basement membranes, thereby contributing to
remodelling of extracellular matrix and migration of immune
cells. A2M regulates functions of cytokines but in pregnancy it is
suggested to particularly be involved in tissue remodelling during
trophoblast invasion [44,45]. IGF-I is an angiogenic factor that
affects fetal nutrition and size (reviewed in  and its expression
is associated with alternatively activated macrophages [22,47], in
agreement with the angiogenic characteristics of this cell type. We
here confirm the role of uterine macrophages in tissue remodelling
and angiogenesis during placental invasion [6,48], and extend
knowledge by adding a number of regulated genes involved in
Figure 3. Protein concentrations of a) CCL18 as measured with
ELISA, b) CCL2 and c) MMP9 as measured by Luminex multiple
bead technology in 24 hour culture supernatant of CD14
positive monocytes/macrophages isolated from 5 subjects.
Error bars show median values and p,0.01 for differences between
decidual and blood CD14 positive cells for all proteins, using Wilcoxon
signed ranked test.
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A large group of regulated genes was related to cell cycle
functions. Taking into consideration the balance between up- and
down-regulated genes, the net effect was a clear-cut signature of
proliferation, which is in line with the previously demonstrated
proliferative capacity of macrophages [49,50]. Our results also
closely resemble the array data from Martinez et al  where
many genes associated with cell proliferation were up-regulated in
macrophage differentiation and priming of M1 as well of
M2.Genes related to cell metabolism and transport were common
both in the up- and down-regulated groups of genes. This is in line
with Martinez et al where for example lipid metabolites and solute
carriers were regulated in differentiation and priming of
While the present study focused on analysis of the gene
expression profile, previous studies have reported cell surface
expression of CD14+decidual macrophages to include CD209
[29,30], HLA-DR [19,29] and CD68 , whereas the relative
expression of CD80, CD83 and CD86 was lowered [19,29],
indicating a potential to induce T-cell anergy. Previous studies
have also shown a suppression of mixed leukocyte reaction (MLR)
as well as of mitogen-induced proliferation of decidual macro-
phages as compared with their blood counterparts, thus linking
decidual macrophages to a functional suppressive phenotype .
Macrophages show a high degree of plasticity and they adapt to
the tissue environment , often developing properties that do
not precisely fit into the in vitro generated M1 or M2 profiles, e.g.
macrophages in lung tissue  or in tumors (reviewed in ).
Although decidual macrophages mainly fall into the M2 category,
it is important to note that they do exhibit a unique profile. The
precise requirements for this polarization remain to be settled, but
local cytokines and hormones are likely to have important roles.
We also speculate for a role of hepatocyte growth factor (HGF)
since the gene expression profile of monocytes cultured with HGF
 resembled that of decidual CD14 positive cells reported here.
HGF is normally expressed at high levels in the placenta whereas
low levels are associated with pregnancy complications [55–57].
Polarized macrophages do not represent stable lineages, but rather
show reversible adaptations to changes of the environment
[51,52,58]. Thus, while the unique profile of decidual macro-
phages is important in fetal protection, an alteration of this gene
expression could be, in part, involved in complications of
pregnancy. Indeed, a number of genes regulated in this array
were previously found to be associated with pregnancy complica-
tions such as preeclampsia (MR , CCL2 , IGF-1 ,
secreted phosphoprotein (SPP)-1 , MMP-9 ), preterm
labour (CCL18 ) and intrauterine growth restriction (IGF-1
Purity of cell populations is extremely important in gene
expression profiling. Our strategy of using gene expression in the
highly purified samples as a requirement for further consideration
resulted in high-stringent criteria minimizing the risk of reporting
contaminating genes. The distinction between macrophages and
dendritic cells in the decidua is not clear-cut. Dendritic cells may
express the macrophage related CD14  whereas macrophages
may express the DC related CD209. [29,30]. On the other hand,
CD14+ decidual cells were shown to express HLA-DR, CD68 and
CD209, but were negative in CD1a, CD83 and CD86  and, in
addition, CD14+ cells were not able to differentiate into DCs
when cultured in GM-CSF and IL-4 , thus indicating that
CD14+ decidual cells mainly resemble macrophages. In any case,
the expression profile of the major population of CD14 cells is of
relevance irrespective of its relation to dendritic cells.
In conclusion, we present here the global gene expression
pattern of decidual CD14 positive macrophages. The up-
regulation of cell cycle genes indicates a highly proliferative
nature of decidual cells. Although a number of nuclear factors
were regulated, compatible with the differentiation process of
decidua infiltrating macrophages, the main block of the regulated
genes represented membrane receptors or secreted proteins,
pointing to a profound polarization. The expression profile, with
a large number of regulated genes related to immunomodulation
and tissue remodelling, mainly parallels that of M2 polarized
macrophages, including M2 markers such as CCL18, CD209,
IGF-1, MRC-1 and fibronectin-1. Further, the up-regulation of for
example TREM2, A2M and PGDS provide new insights into the
regulating function of macrophages in pregnancy, and several
genes might also be implicated in immune tolerance in general.
Interestingly, some of the regulated genes described here for
normal pregnancies, have been shown to be dysregulated in
complicated pregnancies. Taken together, the mapping of
decidual CD14 positive macrophages showed a unique transcrip-
tional profile that confirms and extends previous knowledge about
these cells as important components of fetal protection.
blood CD14 positive cells in early pregnancy.
Found at: doi:10.1371/journal.pone.0002078.s001 (0.12 MB
Genes differentially expressed in decidual compared to
We thank the Department of Obstetrics and Gynecology for help with
collecting samples for the study, Jan-Ingvar Jo ¨nsson and Florence Sjo ¨gren
for invaluable advice in the FACSAria cell sorting and Karin Backteman
for help with flow cytometry analyses. We also want to thank Tanja To ¨pfer
for performing the array analyses, Kiki Dienus for real-time PCR, Martina
Sandberg and Maria Jenmalm for ELISA analyses, Petra Cassel for
Luminex analyses and Olle Sta ˚hl for helpful input on the gene data.
Conceived and designed the experiments: CG JM AM LM GB JE.
Performed the experiments: RG CG JM AM. Analyzed the data: RG SS
CG JM AM JE. Contributed reagents/materials/analysis tools: RG JB CG
JM AM LM GB JE. Wrote the paper: CG. Other: Revision of manuscript:
LM GB SS JB RG. Drafting of the manuscript: JE AM JM.
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Profile of Decidualmacrophages
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