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PHAGOCYTES, GRANULOCYTES, AND MYELOPOIESIS
Alternatively activated macrophages derived from monocytes and tissue
macrophages are phenotypically and functionally distinct
Uma Mahesh Gundra,1 Natasha M. Girgis,1 Dominik Ruckerl,2 Stephen Jenkins,2 Lauren N. Ward,1 Zachary D. Kurtz,1
Kirsten E. Wiens,1 Mei San Tang,1 Upal Basu-Roy,1 Alka Mansukhani,1 Judith E. Allen,2 and P’ng Loke1
1
Department of Microbiology, New York University School of Medicine, New York, NY; and 2Centre for Immunity, Infection and Evolution, and the Institute
for Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom
Key Points
• Alternatively activated
macrophages derived from
monocytes and tissue
macrophages have distinct
transcriptional profiles and
phenotypes.
• Monocyte-derived AAMs are
more involved with immune
regulation than tissue-derived
AAMs.
Macrophages adopt an alternatively activated phenotype (AAMs) when activated by the
interleukin-4receptor(R)a. AAMs can be derived either from proliferation of tissue resident
macrophages or recruited inflammatory monocytes, but it is not known whether these
different sources generate AAMs that are phenotypically and functionally distinct. By
transcriptional profiling analysis, we show here that, although both monocyte and tissuederived AAMs expressed high levels of Arg1, Chi3l3, and Retnla, only monocyte-derived
AAMs up-regulated Raldh2 and PD-L2. Monocyte-derived AAMs were also CX3CR1-green
fluorescent protein (GFP)high and expressed CD206, whereas tissue-derived AAMs were
CX3CR1-GFP and CD206 negative. Monocyte-derived AAMs had high levels of aldehyde
dehydrogenase activity and promoted the differentiation of FoxP31 cells from naı̈ve CD41
cells via production of retinoic acid. In contrast, tissue-derived AAMs expressed high levels
of uncoupling protein 1. Hence monocyte-derived AAM have properties associated with
immune regulation, and the different physiological properties associated with AAM function
may depend on the distinct lineage of these cells. (Blood. 2014;123(20):e110-e122)
Introduction
There has recently been a major paradigm shift in the field of macrophage biology with the recognition that tissue resident macrophages
can be established independently of definitive hematopoiesis.1-4 These
cells are of embryonic origin and are maintained throughout life by
proliferative self-renewal.5 In contrast, macrophages infiltrating the
tissues during an inflammatory response are derived from blood
monocytes.6,7 This paradigm holds true in many tissues including the
serous cavities1,3 but not for the intestines8,9 and the skin,10 where the
resident cells are of bone marrow origin.
During T helper 2 (TH2)-mediated immune responses, interleukin
(IL)-4 and/or IL-13 can induce macrophage proliferation, leading
to expansion beyond steady-state levels.11,12 Signaling through the
IL-4receptor(R)a also leads to a state of alternative activation.13,14
Alternatively activated macrophages (AAMs) are a critical component
of type 2 immunity during helminth infection15 and allergic
responses.16 However, type 2 immune responses extend beyond just
infection and autoimmunity17 and contribute to the maintenance of
tissue homeostasis,16 damage repair,18 and metabolic homeostasis.19
IL-4 will induce proliferation and alternative activation of macrophages regardless of their embryonic or bone marrow origins.11 This
raises critical questions about the contribution of tissue-resident
macrophages vs blood-derived macrophages to the diverse processes
associated with AAMs. Bone marrow chimeras in which the peritoneal and pleural cavity cells are shielded from irradiation, and thus
Submitted August 8, 2013; accepted March 25, 2014. Prepublished online as
Blood First Edition paper, April 2, 2014; DOI 10.1182/blood-2013-08-520619.
U.M.G. and N.M.G. contributed equally to this work.
This article contains a data supplement.
e110
remain of host origin, allow us to distinguish cells of bone marrow vs
resident origins.11 Using this method, we have demonstrated that
IL-4 induces the proliferation and expansion of resident peritoneal
macrophages, whereas delivery of IL-4 plus thioglycollate caused
recruitment and proliferation of blood monocyte-derived macrophages.11 Here, we demonstrate that AAMs derived from proliferation of local tissue resident macrophages are phenotypically
and functionally distinct from AAMs derived through recruitment of
inflammatory monocytes.
Materials and methods
Mice treatment and infections
C57BL/6, Stat62/2 (Jackson Laboratories) and CX3CR1GFP/1 mice (kindly
provided by Dr Dan Littman) were treated with IL-4/anti-IL-4 monoclonal
antibody (mAb) complexes (IL-4c), prepared as described previously.11 Mice
were injected intraperitoneally (i.p.) with IL-4c on days 0 and 2. Mice were
also treated with 4% thioglycollate alone or in combination with IL-4c for coadministration experiments. Mice were euthanized on day 4. Mice were infected
subcutaneously with 25 Litomososides sigmodontis L3,20 and analysis of
pleural cavity cells was performed 12 days after infection. For Schisotosoma
mansoni infection, mice were infected percutaneously on the abdominal
surface with 25 to 35 cercariae of a Puerto Rican strain (Biomedical Research
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
© 2014 by The American Society of Hematology
BLOOD, 15 MAY 2014 x VOLUME 123, NUMBER 20
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BLOOD, 15 MAY 2014 x VOLUME 123, NUMBER 20
DISTINCT M2 MACROPHAGE PHENOTYPES AND FUNCTIONS
e111
Figure 1. IL-4c and Thio1IL-4c induces alternatively activated macrophages with distinct phenotypes. Mice were untreated (naı̈ve/resident) or injected
i.p. with IL-4c alone (IL-4c) or thioglycollate alone
(Thio) or Thio and IL-4c (Thio1IL-4c) on day 0 and
then with IL-4c again (for IL-4c and Thio1IL-4c) on day
2, and PEC was analyzed on day 4. (A) Representative
FACS plots shows EdU incorporation by peritoneal
macrophages after treatment. EdU was injected
3 hours before analysis. (B) Quantification of EdU
incorporation in macrophages. Results are representative of 3 independent experiments. (C) RNA of peritoneal
macrophages was isolated for RT-PCR analysis for
expression of Chi3l3, Arg1, Retnla, and Raldh2 and
normalized to expression of GAPDH. Graphs depict
mean 6 standard error of the mean of individual
mice pooled from 5 to 6 independent experiments. (D)
EdU incorporation by peritoneal macrophages after
injection of wild-type Stat61/1 mice or Stat62/2 mice
with IL-4c alone, Thio, or Thio1IL-4c. (E) Quantification of EdU incorporation in macrophages. (F) Total
number of F4/801 macrophages recovered. Results
shown are representative of 2 separate experiments.
(G) Representative FACS plots gated on CD11b1
cells for CX3CR1GFP/1 reporter mice treated as
above. (H) The median fluorescent intensity (MFI)
of GFP, gated on CD11b1 cells from the peritoneal
cavity of individual mice. Data are representative of
3 independent experiments. *P , .05 and **P , .01 as
determined by ANOVA.
Institute, Rockville, MD), and analysis of liver leukocytes was performed
8 weeks after infection. This study was carried out in strict accordance with the
recommendations in the Guide for the Care and Use of Laboratory Animals. All
animal procedures were approved by the New York University Institutional
Animal Care and Use Committee under protocol numbers 131004 and 130504,
as well as in accordance with the United Kingdom Home Office requirements.
peritoneal macrophages and 1 mg/mL of soluble anti-CD3 and 5 ng/mL
recombinant human IL-2 (R&D) in complete RPMI. On day 3, cultures
were supplemented with fresh medium containing 5 ng/mL IL-2. Retinoic
acid (RA) receptor inhibitor LE540 (Wako Chemicals USA) was added to
some culture wells. To track cell division, T cells were labeled with Violet
CellTracker (Invitrogen). On day 6, cells were stained for Foxp31 cells
by intracellular nuclear staining.
Flow cytometry
Cells were stained with LIVE/DEAD Aqua or Blue (Invitrogen),
blocked with 4 mg/mL aCD16/32 (2.4G2; Bioxcell), and stained with
CD11b eFluor450 (eBioscience), MHCII APC-Cy7 (Biolegend),
PD-L2 PE (BD Bioscience, Biolegend), F4/80 PE-Cy7 (eBioscience),
CD206 APC (Biolegend), and Siglec-F, DX5, B220, and CD3 PE (BD
Bioscience). Cells were acquired on an LSR II (BD Biosciences) and analyzed
using FlowJo software (Treestar). For 5-ethynyl-29-deoxyuridine (EdU) labeling,
mice were injected i.p. with 0.5 mg EdU (Invitrogen) 3 hours prior to euthanasia
and stained for EdU according to the manufacturer’s instructions. Aldehyde
dehydrogenase (ALDH) activity was measured using the ALDEFLUOR staining
kit (StemCell Technologies) with or without the ALDH inhibitor, diethylaminobenzaldehyde (DEAB) (at a final concentration of 15 mM) as control.
Foxp31 T-regulatory cell differentiation assay
4 3 105 naı̈ve T cells isolated from lymph nodes using the Naive CD41 T Cell
Isolation Kit II (Miltenyi Biotec) were cultured together with 2 3 105
Microarray analysis of sorted cells
CD11b and F4/80 double positive cells (supplemental Figure 3, available on
the Blood Web site) sorted using a BD FACSAria cell were analyzed by using
Sureprint G3 Mouse GE 8x60K array (Agilent) in one color (Cy3)-based
gene expression analysis (Agilent). Processing and downstream analysis of
microarray data (Gene Expression Omnibus accession number GSE54679)
was performed with R/Bioconductor and associated packages and SAM:
Significance Analysis of Microarrays.21 Background correction to logtransformed expression values was performed, and the expression values of
each array was scaled so that the median absolute values are equal for all
arrays. The Clustering of Large Applications function from R was used to find
the best clustering solution that coincided with the global minimum Bayesian
information critera score and a locally maximum silhouette width. Each
cluster was assessed by measuring the Gene Ontology (GO) term overrepresentation by the hypergeometric distribution, as implemented in the
GOstats package using gene annotations from the mouse.db0 package.
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e112
GUNDRA et al
BLOOD, 15 MAY 2014 x VOLUME 123, NUMBER 20
Figure 2. Gene expression profiling of monocytederived and tissue macrophage-derived AAMs. (A)
Unsupervised hierarchical clustering of transcriptional
profiles, displayed as a heat map of log-transformed
expression values, from FACS-purified (CD11b1F4/801)
macrophages of mice untreated (Res) or treated with
IL-4c alone (IL-4c) or thioglycollate alone (Thio) or Thio
and IL-4c (Thio1IL-4c). (B) Comparison of probe
expression for AAMs induced by IL-4c alone or Thio1IL-4c,
indicating that most genes are differentially expressed. (C)
Heat map of log-transformed expression values for a subset
of genes associated with AAMs. (D) Venn diagrams
showing the overlap of genes up-regulated and downregulated by IL-4 in monocyte-derived (Thio1IL-4c vs Thio)
compared with tissue-derived AAMs (IL-4c vs resident).
List of genes are shown in Tables 1 to 8.
RT-PCR and western blot
Quantitative reverse transcription-polymerase chain reaction (RT-PCR)
was performed using the SYBR Green qPCR kit (Applied Biosystems) and
normalized to the housekeeping gene Gapdh by comparative Ct. For western
blots, anti-UCP1 antibody (ab-10983; Abcam) and antiglyceraldehyde-3phosphate dehydrogenase (GAPDH) antibody (sc-32233; SCBT) were used.
Statistical analysis
Significance between groups was determined by analysis of variance
(ANOVA) plus Bonferroni or Dunnett’s correction for multiple comparisons.
Results
Peritoneal AAMs derived from inflammatory monocytes and
tissue macrophages both proliferate and express markers of
alternative activation
Using shielded bone marrow chimeras, we previously showed that
injection of IL-4c into the pleural cavity leads to the expansion of the
resident cell population independently of the bone marrow, whereas
simultaneous injection of IL-4c with thioglycollate (Thio1IL-4c)
generates a large population of AAMs that are derived from
inflammatory blood monocytes.11 We applied this system to the
peritoneal cavity to compare AAMs derived from proliferating tissue
resident macrophages (IL-4c) with AAMs derived from inflammatory monocytes (Thio1IL-4c). Resident macrophages from naı̈ve
untreated animals (Res) and macrophages recruited by thioglycollate
alone (Thio) were used as controls. We confirmed by 3-hour pulse
labeling and EdU staining that both IL-4c treatment and Thio1IL-4c
treatment can induce macrophage proliferation, although Thio1IL-4c
induced macrophages proliferated to a lesser degree with lower
proportion of cells in S phase (Figure 1A-B). Less proliferation may be
due to reduced availability of IL-4 per cell, as the recruited population
will quickly outnumber the resident cells in the Thio1IL-4c-treated
mice.
We next examined expression of the well-defined markers of
alternative activation, arginase I (Arg1), resistin-like molecule a
(Retnla), and Ym1 (or Chi3l3), in both types of AAMs. By real-time
PCR analysis, expression of all 3 genes was highly up-regulated
in both IL-4c- and Thio1IL-4c-induced macrophages (Figure 1C).
This was consistent with our previous studies in the pleural cavity
in which both IL-4c and Thio1IL-4c led to extensive alternative
activation as measured by high percentages of resistin-like molecule
a (RELMa)1 and Ym11 macrophages.11 Stat6 is essential for alternative activation,22,23 but its role in macrophage proliferation has
not been previously demonstrated. Both Thio1IL-4c and IL-4c
activated macrophages from Stat62/2 mice failed to increase proliferation and incorporate EdU in contrast to wild-type littermates
(Figure 1D-E), confirming Stat6 dependence. The failure to proliferate
was reflected in reduced total cell numbers especially among F4/801
macrophages (Figure 1F).
In CX3CR1-green fluorescent protein (GFP)/1 reporter mice,
CX3CR1-GFP is highly expressed by blood monocytes but not by
tissue resident peritoneal macrophages.24 To validate the different
origins of cells derived from IL-4c vs Thio1IL-4c, we injected
CX3CR1-GFP/1 reporter mice with these reagents. Gating on the
CD11b1 cells, the majority of macrophages induced by IL-4c alone
were F480high and CX3CR1-GFPlow, similar to the resident
population in naı̈ve mice. In contrast, the AAMs induced by
Thio1IL-4c were F480int and CX3CR1-GFPhigh (Figure 1G-H).
These results are consistent with a monocyte origin for AAMs
induced by Thio1IL-4c (F480int), whereas those induced by IL-4c
alone are derived from resident macrophages (F480high). Notably,
there is a small CX3CR1-GFPhigh F4/80int macrophage population in
naı̈ve mice and IL-4c alone-treated mice that is likely of monocyte
origin.25
We confirmed that IL-4c induces expansion of the resident cell
population in the peritoneal cavity independently of the bone marrow
by generating bone marrow chimeras in which the serous cavities are
shielded from irradiation and assessing the chimerism in the blood vs
the peritoneal cavity (supplemental Figure 1). The blood chimerism
was ;30% of donor origin, whereas only 1% to 2% of macrophages
in the peritoneal cavity were of donor origin in both phosphatebuffered saline (PBS) and IL-4c-injected mice. Similarly, by
intravenous transfer of purified Ly6C High monocytes from
CX3CR1-GFP/1 mice (CD45.2) into congenic recipient CD45.1
hosts, we confirmed that Thio1IL-4c but not IL-4c alone induces
monocyte recruitment and differentiation (supplemental Figure 1).
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BLOOD, 15 MAY 2014 x VOLUME 123, NUMBER 20
DISTINCT M2 MACROPHAGE PHENOTYPES AND FUNCTIONS
Table 1. List of top 50 genes up-regulated by IL-4 in resident
macrophages
Gene symbol
Description
Expressed sequence AA467197
Chi3l4
Chitinase-like 4 or Ym2
526.5159
Aqp3
Aquaporin 3
295.7499
Sprr2a2
Small proline-rich protein 2A2
149.7646
Cst7
Cystatin F (leukocystatin)
135.3837
Havcr1
Hepatitis A virus cellular receptor 1
116.3776
Mall
Mal, T cell differentiation protein-like
109.5096
Ddx4
DEAD (Asp-Glu-Ala-Asp) box polypeptide 4
107.4824
Tmigd1
Transmembrane and immunoglobulin domain
665.9877
92.0161
containing 1
Timd2
T-cell immunoglobulin and mucin domain containing 2
Chi3l3
Chitinase-like 3 or Ym1
81.1482
Ucp1
Uncoupling protein 1 (mitochondrial, proton carrier)
74.1526
Serpinb7
Serine (or cysteine) peptidase inhibitor, clade B,
90.5118
72.4033
member 7
Tcf23
Transcription factor 23
70.0783
Ear10
Eosinophil-associated, ribonuclease A family,
66.9156
Ear11
Eosinophil-associated, ribonuclease A family,
member 10
63.0005
member 11
Gm4610
Predicted gene 4610
53.6569
Syt10
Synaptotagmin X
52.9130
Gm5150
Predicted gene 5150
49.2676
Klk1b11
Kallikrein 1-related peptidase b11
47.2572
Car12
Carbonic anyhydrase 12
43.5869
Gatm
Glycine amidinotransferase (L-arginine:glycine
42.6830
amidinotransferase)
I830127L07Rik
RIKEN cDNA I830127L07 gene
38.1699
LOC101056540
Unknown
35.2395
Asns
Asparagine synthetase
33.8962
Rrm2
Ribonucleotide reductase M2
32.8288
Chchd10
Coiled-coil-helix-coiled-coil-helix domain
31.9263
Flt1
FMS-like tyrosine kinase 1
31.6849
Slc7a2
Solute carrier family 7 (cationic amino acid
30.7286
Galnt6
UDP-N-acetyl-a-D-galactosamine:polypeptide
containing 10
transporter, y1 system), member 2
30.3134
N-acetylgalactosaminyltransferase 6
Tribbles homolog 3 (Drosophila)
27.4894
A530064D06Rik RIKEN cDNA A530064D06 gene
26.7710
Ccrn4l
25.7457
CCR4 carbon catabolite repression 4-like
AAMs derived from monocytes and tissue macrophages are
transcriptionally distinct
Fold change
AA467197
Trib3
e113
(S cerevisiae)
Grhl3
Grainyhead-like 3 (Drosophila)
25.1532
Entpd3
Ectonucleoside triphosphate diphosphohydrolase 3
24.6177
Kcnn4
Potassium intermediate/small conductance
22.9964
calcium-activated channel, subfamily N,
member 4
Fxyd6
FXYD domain-containing ion transport regulator 6
22.4216
Hist1h1b
Histone cluster 1, H1b
22.4066
Arg1
Arginase, liver
22.1165
Oasl1
2’-59 oligoadenylate synthetase-like 1
21.4259
Gfra2
Glial cell line derived neurotrophic factor family
20.4561
Anpep
Alanyl (membrane) aminopeptidase
20.3326
Shcbp1
Shc SH2-domain binding protein 1
19.8717
Sema3b
Sema domain, immunoglobulin domain (Ig), short
19.7311
receptor a 2
basic domain, secreted, (semaphorin) 3B
Pbk
PDZ binding kinase
Spp1
Secreted phosphoprotein 1
19.4258
19.0967
Ccna2
Cyclin A2
18.2413
Nuf2
NUF2, NDC80 kinetochore complex component,
18.0971
homolog (S cerevisiae)
Ccnf
Cyclin F
17.6265
Plxna2
Plexin A2
17.4434
We next decided to generate a detailed transcriptional profile of
peritoneal AAMs of distinct origins. We fluorescence-activated cell
sorter (FACS) sorted pure populations of CD11b1, F4/80High cells
for resident peritoneal macrophages and IL-4c-induced macrophages and CD11b1, F4/80Int cells for Thio- and Thio1IL-4c-induced
macrophages (supplemental Figure 2). These 4 populations were
subjected to gene expression profiling analysis with whole genome
microarrays (Figure 2). Unsupervised hierarchical clustering
analysis showed coclustering of IL-4c-induced F4/80High AAMs
with resident F4/80High macrophages from untreated mice (Figure 2A),
whereas AAMs induced by Thio1IL-4c coclustered with Thio
induced macrophages (Figure 2A). When AAMs induced by IL-4c
were directly compared with Thio1IL-4c-induced AAMs, expression across all probes was remarkably different between these 2 types
of macrophages (Figure 2B), which is consistent with their different
cellular lineages. A detailed analysis of some genes previously
described in AAMs (Figure 2C) confirmed the up-regulation of
arginase I, RELMa, and Ym1 (or Chi3l3), as well as Tgm226 and
Klf427 in both types of AAMs (Figure 2C). Tgm2 was recently
identified as a universal AAM marker for both humans and mice.26
Aldh1a2 (Raldh2), pdcd1lg2 (PDL2), Socs2, IL-31ra, Ccl17, Ch25h,
Jag2, and Ccl22 were more highly expressed on Thio1IL-4cinduced AAM (Figure 2C).
We then performed supervised comparisons by statistical
analyses of microarrays of IL-4c-induced AAMs vs resident macrophages and Thio1IL-4c-induced AAMs vs Thio-induced macrophages to identify significantly different genes with a false discovery
rate of 0%. With this cutoff, 758 genes were up-regulated in IL-4ctreated macrophages (Table 1) compared with controls, and 368
genes were up-regulated in Thio1IL-4c-treated macrophages
(Table 2) compared with Thio-induced macrophages. One hundred
fifty-three genes were shared between these 2 groups (Table 3;
Figure 2D). Genes up-regulated in the IL-4c-treated macrophages
were enriched by GO analysis for the biological processes involved
in cellular replication (cell cycle, mitosis, DNA replication, etc)
(Figure 3A). Consistent with increased proliferation in both IL-4c
and Thio1IL-4c activated macrophages, genes involved in cellular
replication and metabolism were up-regulated in both types of
AAMs (Figure 3C). Many of the genes up-regulated in Thio1IL-4ctreated macrophages were unclassified in terms of their biological
process (Figure 3B). In contrast to IL-4c-induced macrophages,
up-regulated genes classified as part of the immune system were
enriched in Thio1IL-4c-induced macrophages. Molecular function
GO analysis revealed that cytokine (IL-6, Cish, IL31Ra, Socs6,
Socs2) and chemokine (Ccl17, Ccl2, Ccl7, ccl12, and Ccl24) activity
were the main functions up-regulated in Thio1IL-4c AAMs
(Figure 3D), but these functions were not up-regulated in IL-4ctreated macrophages; instead, chemokine (Ccl3, Cxcl1, Cxcl2,
and Cxcl13) activity was down-regulated (supplemental Figure 3;
Tables 4-6).
To more systematically identify modularity in the expression data
and potentially coregulated genes, we used unsupervised k-means
clustering to identify clusters of genes that have similar expression
profiles (Figure 3E; supplemental Figure 4). We identified 249
clusters based on optimal cluster fitting with the least complexity
(supplemental Figure 4). Twenty-six clusters that had the most
interesting expression patterns were selected for further investigation
(Figure 3E). Figure 3E shows a hierarchical clustering analysis of the
averaged expression for all the genes in each cluster shown as a single
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e114
BLOOD, 15 MAY 2014 x VOLUME 123, NUMBER 20
GUNDRA et al
Table 2. List of top 50 genes up-regulated by IL-4 in
monocyte-derived macrophages
Gene symbol
Description
Table 3. List of 50 genes regulated by IL-4 in both resident and
monocyte-derived macrophages
Fold change
Gene symbol
Description
Fold change*
Ccl24
Chemokine (C-C motif) ligand 24
308.1040
AA467197
Expressed sequence AA467197
367.4197
Ddx4
DEAD (Asp-Glu-Ala-Asp) box polypeptide 4
147.2503
Chi3l4
Chitinase-like 4 or Ym2
310.8889
Ear11
Eosinophil-associated, ribonuclease A family,
129.8195
member 11
Tuba8
Tubulin, a 8
114.0360
Ear10
Eosinophil-associated, ribonuclease A family,
109.5308
Chi3l4
Chitinase-like 4 or Ym2
95.2618
Ear1
Eosinophil-associated, ribonuclease A family,
90.4369
member 10
Eosinophil-associated, ribonuclease A family,
Aquaporin 3
156.1061
DEAD (Asp-Glu-Ala-Asp) box polypeptide 4
127.3663
Ear11
Eosinophil-associated, ribonuclease A family,
88.9709
96.4100
member 11
Ear10
member 1
Ear12
Aqp3
Ddx4
Eosinophil-associated, ribonuclease A family,
88.2232
member 10
Cst7
Cystatin F (leukocystatin)
86.1656
Tuba8
Tubulin, a 8
58.8395
Ear12
Eosinophil-associated, ribonuclease A family,
46.9911
member 12
member 12
Cdh1
Cadherin 1
73.2850
Asns
Asparagine synthetase
46.9515
Il4i1
Interleukin 4 induced 1
71.5692
Chi3l3
Chitinase-like 3 or Ym1
44.8807
44.7643
AA467197
Expressed sequence AA467197
68.8517
Cdh1
Cadherin 1
Asns
Asparagine synthetase
60.0067
Cish
Cytokine inducible SH2-containing protein
33.5730
Ch25h
Cholesterol 25-hydroxylase
59.7103
Gm4610
Predicted gene 4610
28.9111
Cish
Cytokine inducible SH2-containing protein
59.3660
Gm6756
Predicted gene 6756
28.5975
Gm6756
Predicted gene 6756
53.0013
Gfra2
Glial cell line derived neurotrophic factor
28.3951
Ear2
Eosinophil-associated, ribonuclease A family,
47.4012
Phgdh
3-Phosphoglycerate dehydrogenase
28.1069
Slc7a2
Solute carrier family 7 (cationic amino acid
27.6141
member 2
42.3885
family receptor a 2
Vdr
Vitamin D receptor
Timp1
Tissue inhibitor of metalloproteinase 1
40.8995
Pi16
Peptidase inhibitor 16
39.9049
Gm5150
Predicted gene 5150
Fbp1
Fructose bisphosphatase 1
37.6432
A530064D06Rik
RIKEN cDNA A530064D06 gene
26.5447
Cst7
Cystatin F (leukocystatin)
36.9475
Ear2
Eosinophil-associated, ribonuclease A family,
25.9995
Klk8
Kallikrein related-peptidase 8
36.9350
Gfra2
Glial cell line derived neurotrophic factor family
36.3341
Gatm
Glycine amidinotransferase (L-arginine:glycine
transporter, y1 system), member 2
26.6933
member 2
receptor a 2
25.6794
amidinotransferase)
Phgdh
3-phosphoglycerate dehydrogenase
36.1081
Pi16
Peptidase inhibitor 16
23.3797
Tarm1
T cell-interacting, activating receptor on myeloid
36.1055
Flt1
FMS-like tyrosine kinase 1
23.0790
Fbp1
Fructose bisphosphatase 1
22.4783
31.2576
Apoe
Apolipoprotein E
A530064D06Rik RIKEN cDNA A530064D06 gene
26.3184
Clec4d
C-type lectin domain family 4, member d
0.0532
Slc7a2
Solute carrier family 7 (cationic amino acid
24.4996
Apoc2
Apolipoprotein C-II
0.0551
Msx3
msh homeobox 3
cells 1
Ramp3
Receptor (calcitonin) activity modifying protein 3
transporter, y1 system), member 2
0.0499
Atf7
Activating transcription factor 7
0.0663
21.9209
Irf8
Interferon regulatory factor 8
0.0682
Nlrp1a
NLR family, pyrin domain containing 1A
21.6752
Gm11538
Predicted gene 11538
0.0778
Ccl7
Chemokine (C-C motif) ligand 7
21.4119
Fcgr1
Fc receptor, IgG, high affinity I
0.0858
Car6
Carbonic anhydrase 6
20.7420
Rgs1
Regulator of G-protein signaling 1
0.0877
Cebpe
CCAAT/enhancer binding protein (C/EBP), e
20.2090
Ccl3
Chemokine (C-C motif) ligand 3
0.0880
Aldh1a2
Aldehyde dehydrogenase family 1, subfamily A2
19.2205
Prom1
Prominin 1
0.0982
Ccl17
Chemokine (C-C motif) ligand 17
19.2074
Gm1673
Predicted gene 1673
0.1016
Mthfd2
Methylenetetrahydrofolate dehydrogenase
16.9681
Cdkn1c
Cyclin-dependent kinase inhibitor 1C (P57)
0.1045
Olfr871
Olfactory receptor 871
0.1055
(NAD1 dependent), methenyltetrahydrofolate
cyclohydrolase
Neurl3
Neuralized homolog 3 homolog (Drosophila)
0.1063
16.4622
Lyz2
Lysozyme 2
0.1138
Arachidonate 15-lipoxygenase
15.4346
Itgb5
Integrin b 5
0.1160
Resistin like a
15.3394
Lst1
Leukocyte specific transcript 1
0.1253
Ltb4r1
Leukotriene B4 receptor 1
15.2231
Rnf150
Ring finger protein 150
0.1274
Retnlg
Resistin like g
15.1644
Itga6
Integrin a 6
0.1277
Flt1
FMS-like tyrosine kinase 1
14.4730
Cnrip1
Cannabinoid receptor interacting protein 1
0.1286
Olr1
Oxidized low density lipoprotein (lectin-like)
14.4345
Abca1
ATP-binding cassette, sub-family A (ABC1),
0.1294
Cd81
CD81 antigen
14.2763
Cbr2
Carbonyl reductase 2
Slc7a5
Solute carrier family 7 (cationic amino acid
13.8395
1700003F12Rik
RIKEN cDNA 1700003F12 gene
0.1339
Lmo2
LIM domain only 2
0.1416
Ldhb
Lactate dehydrogenase B
0.1431
Aqp3
Aquaporin 3
Alox15
Retnla
member 1
receptor 1
transporter, y1 system), member 5
Fcrls
Fc receptor-like S, scavenger receptor
12.9906
Ap4e1
Adaptor-related protein complex AP-4, e 1
12.5573
Hic1
Hypermethylated in cancer 1
12.2985
Rnase6
Ribonuclease, RNase A family, 6
12.1713
Slc30a4
Solute carrier family 30 (zinc transporter), member 4
12.0286
0.1316
*Calculated as average fold change of genes regulated by IL-4 in resident
macrophage and genes regulated by IL-4 in monocyte-derived macrophages.
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Table 4. Genes up-regulated with IL-4 in monocyte-derived
macrophages and identified by GO analysis of molecular function
to be involved with chemokine activities
Table 6. Genes up-regulated with IL-4 in monocyte-derived
macrophages and identified by GO analysis of molecular function
to be involved with cytokine activities
Gene symbol
Gene symbol
Name
Fold change
Ccl17
Chemokine (C-C motif) ligand 17
19.2074
Gm5150
Predicted gene 5150
Ccl2
Name
Fold change
Gm5150
Predicted gene 5150
4.1190
Ccl12
Chemokine (C-C motif) ligand 12
7.4850
Chemokine (C-C motif) ligand 2
7.4465
Ccl7
Chemokine (C-C motif) ligand 7
21.4119
Ccl7
Chemokine (C-C motif) ligand 7
21.4119
Il6
Interleukin 6
Ccl12
Chemokine (C-C motif) ligand 12
7.4850
Ccl2
Chemokine (C-C motif) ligand 2
Ccl24
Chemokine (C-C motif) ligand 24
308.1040
Cish
Cytokine inducible SH2-containing protein
C3
Complement component 3
Il31ra
Interleukin 31 receptor A
Ccl24
Chemokine (C-C motif) ligand 24
row. The biological significance of each cluster was determined
by measuring the GO term enrichment for biological processes.
Interestingly, genes involved in metabolic processes were highly
enriched in AAMs induced by IL-4c alone. Genes involved in
proliferation and cell cycle were enriched in both types of AAMs,
reflecting the fact that they were both proliferating rapidly with
a high proportion of cells in S phase (Figure 1A). These results
indicate that monocyte-derived AAMs (Thio1IL-4c) and tissue
macrophage-derived AAMs (IL-4c alone) have fundamentally
different molecular signatures and that genes involved in metabolic
processes are especially up-regulated in AAMs induced by IL-4c
alone, and monocyte-derived AAMs up-regulate genes important
in immune responses.
Key markers of alternative activation that are only expressed on
monocyte-derived macrophages
The transcriptional analysis indicated that the message for PD-L2
(also known as B7-DC or Pdcd1lg2), a cell surface marker of alternative activation,22 was up-regulated by IL-4 on monocytederived macrophages but not resident peritoneal cells. This led us to
evaluate cell surface expression of PD-L2, along with the mannose
receptor CD206 (also known as MMR or Mrc1), the first and best
known marker described for AAMs28 (gating strategy; supplemental
Figure 5). F4/80int cells induced by Thio1IL-4c expressed both
PD-L2 and CD206, whereas the majority of IL-4c-induced F4/80high
tissue-derived AAMs did not (Figure 4A-B). CD206 was also
expressed on Thio-elicited macrophages that are not alternatively
activated, whereas PD-L2 was only expressed on Thio-elicited
macrophages in response to IL-4 (Figure 4B). Up-regulation of
PD-L2 was Stat-6 dependent, whereas CD206 was expressed
independently of Stat-6 (Figure 4D-E). We also assessed MHC
class II, which was up-regulated on monocyte-derived AAMs but not
on F4/80high tissue-derived AAMs (Figure 4C). The minor F4/80int
population in naı̈ve and IL-4c alone-treated mice expressed CD206,
and only this minor subset expressed PD-L2 and MHC class II after
exposure to IL-4c (Figure 4A-C). These cells were also CX3CR1GFP1 (Figure 1G) and are phenotypically consistent with a monocyte-derived origin.25 This indicates that the differences in phenotype
we observe are not unique to thioglycollate injection but are a general
feature of monocyte-derived macrophages.
Table 5. Genes down-regulated with IL-4 in resident macrophages
and identified by GO analysis of molecular function to be involved
with chemokine activities
Gene symbol
Name
Fold change
Cxcl13
Chemokine (C-X-C motif) ligand 13
0.0020
Ccl3
Chemokine (C-C motif) ligand 3
0.0439
Cxcl2
Chemokine (C-X-C motif) ligand 2
0.1496
Cxcl1
Chemokine (C-X-C motif) ligand 1
0.1512
Socs6
Suppressor of cytokine signaling 6
Ccl17
Chemokine (C-C motif) ligand 17
Socs2
Suppressor of cytokine signaling 2
4.1190
8.8808
6.9844
59.3660
9.2433
9.6304
308.1040
3.3646
19.2074
7.4007
To determine whether the differences we observed were physiologically relevant to infection, we chose 2 models that induce
high levels of AAMs at the infection site but for which macrophage
origins differed. Infection of mice with S mansoni leads to deposition
of parasite eggs into the liver, with a large accumulation of AAMs in
the granulomas surrounding the eggs. We previously demonstrated
that AAMs induced by S mansoni infection are CX3CR1-GFPhigh,29
suggesting a monocyte origin for these cells. Recent monocyte transfer
and intravital imaging studies have confirmed that these AAM are
monocyte-derived (N.M.G., U.M.G., L.N.W., M. Carbrera,
U. Frevert, and P.L., unpublished data, March 6, 2014). In
contrast, infection with L sigmodontis leads to an expansion of AAMs
in the pleural cavity that are almost entirely derived from the resident
pool, as demonstrated by shielded bone marrow chimeras at 12 days
after infection.11 We examined expression of PD-L2 and CD206 on
AAMs induced by these 2 distinct helminth infections. AAMs induced
by L sigmodontis adult worms residing in the pleural cavity did not
express PD-L2 and CD206 (Figure 5A), the identical expression
pattern to AAMs derived from IL-4c injection. In contrast, injection of
Thio1IL-4c into the pleural cavity induced accumulation of PD-L2and CD206- expressing AAMs (Figure 5A-B). As previously
observed in the peritoneal cavity, macrophages in the pleural cavity
induced by thioglycollate alone expressed CD206 but did not express
PD-L2, which was only induced by Thio1IL-4c injection.
In contrast to L sigmodontis infection, we found that AAMs induced
in the liver by S mansoni infection were similar in phenotype to AAMs
induced by Thio1IL-4c into the peritoneal and pleural cavity. AAMs
that accumulated in the liver granulomas induced by S mansoni eggs
expressed PD-L2 and CD206 (Figure 5C,E). To confirm that PD-L21
and CD2061 macrophages are AAMs, we also stained for intracellular
FIZZ1/RELMa (Figure 5D,F) and gated on the PD-L21CD2061
population compared with the PD-L22CD2062 population. The double
positive cells clearly express more FIZZ1/RELMa than the negative
compartment (Figure 5D,F). Thus, S mansoni and L sigmodontis
infection, which induces AAMs from distinct origins, correspond with
the phenotype of recruited or resident macrophage-derived cells
observed following IL-4c injection into the peritoneal cavity.
AAMs derived from inflammatory monocytes can induce the
differentiation of CD41FoxP31 cells through a retinoic
acid-dependent mechanism
In a further effort to validate the microarray data, we also examined
expression of the enzyme Raldh2 (or Aldh1a2). Raldh2 was chosen
because we previously observed up-regulation in AAMs from the
livers of S mansoni-infected mice,29 and it appeared to be specific to
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GUNDRA et al
Table 7. List of top 50 genes down-regulated by IL-4 in resident
macrophages
Table 8. List of top 50 genes downregulated by IL-4 in
monocyte-derived macrophages
Gene symbol
Gene symbol
Name
Fold change
Name
Fold change
Cxcl13
Chemokine (C-X-C motif) ligand 13
0.0020
Mamdc2
MAM domain containing 2
0.0148
Lyz2
Lysozyme 2
0.0081
Apoe
Apolipoprotein E
0.0148
Atf7
Activating transcription factor 7
0.0131
Dio2
Deiodinase, iodothyronine, type II
0.0169
Lpl
Lipoprotein lipase
0.0177
AF251705
cDNA sequence AF251705
0.0211
0.0226
Irf8
Interferon regulatory factor 8
0.0192
Cdkn1c
Cyclin-dependent kinase inhibitor 1C (P57)
Apoc2
Apolipoprotein C-II
0.0196
Hpgds
Hematopoietic prostaglandin D synthase
0.0232
Dusp6
Dual specificity phosphatase 6
0.0219
Syt10
Synaptotagmin X
0.0287
0.0338
Clec7a
C-type lectin domain family 7, member a
0.0238
C77080
Expressed sequence C77080
Apoc1
Apolipoprotein C-I
0.0300
Mmp27
Matrix metallopeptidase 27
0.0356
Hp
Haptoglobin
0.0339
Mcoln3
Mucolipin 3
0.0364
Gm1673
Predicted gene 1673
0.0344
Tspan7
Tetraspanin 7
0.0368
Gm11538
Predicted gene 11538
0.0367
Htra1
HtrA serine peptidase 1
0.0371
Fcgr1
Fc receptor, IgG, high affinity I
0.0375
Saa3
Serum amyloid A 3
0.0373
Pltp
Phospholipid transfer protein
0.0386
Clec4d
C-type lectin domain family 4, member d
0.0390
Ccl3
Chemokine (C-C motif) ligand 3
0.0439
Cd200
CD200 antigen
0.0412
Abca1
ATP-binding cassette, sub-family A (ABC1),
0.0441
Ms4a7
Membrane-spanning 4-domains, subfamily A,
0.0413
member 1
member 7
Hspa1a
Heat shock protein 1A
0.0494
Sh2d3c
SH2 domain containing 3C
Rgs2
Regulator of G-protein signaling 2
0.0526
Htr2b
5-Hydroxytryptamine (serotonin) receptor 2B
0.0430
0.0452
Lama3
Laminin, a 3
0.0563
Reps2
RALBP1 associated Eps domain containing
0.0489
Nat8l
N-acetyltransferase 8-like
0.0564
Apoc4
Apolipoprotein C-IV
0.0644
Npy
Neuropeptide Y
Clec4d
C-type lectin domain family 4, member d
0.0673
Cnrip1
Cannabinoid receptor interacting protein 1
0.0495
Rab6b
RAB6B, member RAS oncogene family
0.0704
Tmem140
Transmembrane protein 140
0.0502
Emb
Embigin
0.0707
Lhfpl2
Lipoma HMGIC fusion partner-like 2
0.0503
F13a1
Coagulation factor XIII, A1 subunit
0.0713
Arhgap22
r GTPase activating protein 22
0.0507
Fpr1
Formyl peptide receptor 1
0.0714
Prss46
Protease, serine 46
0.0512
Upk1a
Uroplakin 1A
0.0718
Spp1
Secreted phosphoprotein 1
0.0513
Cd63
CD63 antigen
0.0744
Cd93
CD93 antigen
0.0519
Gm4788
Predicted gene 4788
0.0773
Pcp4l1
Purkinje cell protein 4-like 1
0.0525
Ccm2l
Cerebral cavernous malformation 2-like
0.0782
Mfge8
Milk fat globule-EGF factor 8 protein
0.0547
Marco
Macrophage receptor with collagenous structure
0.0804
Gstm1
Glutathione S-transferase, m 1
0.0547
Fpr2
Formyl peptide receptor 2
0.0805
Gpr137b
G protein-coupled receptor 137B
0.0576
Fkbp1a
FK506 binding protein 1a
0.0805
Cd109
CD109 antigen
0.0581
1700003F12Rik RIKEN cDNA 1700003F12 gene
0.0806
Tspan13
Tetraspanin 13
0.0586
Apoe
Apolipoprotein E
0.0850
Gas6
Growth arrest specific 6
Rprm
Reprimo, TP53 dependent G2 arrest mediator
0.0854
4930404N11Rik RIKEN cDNA 4930404N11 gene
0.0631
Mpeg1
Macrophage expressed gene 1
0.0655
Cfhr2
Complement factor H-related 2
0.0855
Tecpr1
Tectonin b-propeller repeat containing 1
0.0680
Abca9
ATP-binding cassette, sub-family A (ABC1),
0.0913
Gprc5b
G protein-coupled receptor, family C, group 5,
0.0703
Cfh
Complement component factor h
0.0924
Pianp
PILR a associated neural protein
Egfl7
EGF-like domain 7
0.0930
Gpr137b-ps
G protein-coupled receptor 137B, pseudogene
0.0734
Olfr871
Olfactory receptor 871
0.0934
Gdpd1
Glycerophosphodiester phosphodiesterase
0.0742
Neurl3
Neuralized homolog 3 homolog (Drosophila)
0.0949
Rgs1
Regulator of G-protein signaling 1
0.0949
Lrrc27
Leucine rich repeat containing 27
0.0748
Rs5-8s1
Unknown
0.0977
Fstl1
Follistatin-like 1
0.0750
Prom1
Prominin 1
0.0986
Pdzk1ip1
PDZK1 interacting protein 1
0.0759
Ldhb
Lactate dehydrogenase B
0.0987
Fhdc1
FH2 domain containing 1
0.0765
Rhbdf1
Rhomboid family 1 (Drosophila)
0.1027
Itgb5
Integrin b 5
0.0792
Fut7
Fucosyltransferase 7
0.1038
Speg
SPEG complex locus
0.0795
Pilra
Paired immunoglobin-like type 2 receptor a
0.1082
Gstm3
Glutathione S-transferase, m 3
0.0799
Lst1
Leukocyte specific transcript 1
0.1086
Rgs1
Regulator of G-protein signaling 1
0.0805
candidate
protein 2
member 9
0.0494
0.0587
member B
the monocyte-derived cells in our microarray analysis. Raldh2 is an
enzyme that regulates production of retinoic acid (RA) with important
implications for immune regulation.30 Raldh2 was up-regulated to a far
greater extent by Thio1IL-4c than IL-4c only, suggesting that AAMs
generated from monocytes preferentially produce RA (Figure 6A). We
used the fluorescent substrate aldefluor (ALD) to confirm enzymatic
activity by flow cytometry and found that Thio1IL-4c induced a
substantial number of ALD1 cells that were F4/80int (Figure 6B;
supplemental Figure 6). As expected from its monocyte origins,
0.0707
domain containing 1
some of the minor F4/80int peritoneal population of naı̈ve untreated
animals and IL-4c-treated animals were also ALD1 (Figure 6C),
although there were many fewer ALD1 cells than in Thio1IL-4ctreated animals.
Because RA can induce Foxp3 expression in CD41 T cells, we
compared the ability of IL-4c- and Thio1IL-4c-induced AAMs to
induce expression of Foxp3 in naı̈ve CD41 T cells. Strikingly, only
Thio1IL-4c induced AAMs were able to induce differentiation
of Foxp31 cells, detected after 7 days of culture (Figure 6D-E).
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Figure 3. GO analysis of transcriptional profiles from monocyte-derived and tissue resident macrophage-derived AAMs. (A) Biological processes (BPs) induced in
IL-4c expanded AAM (IL-4c) relative to resident macrophages (Res). x-axis indicates the amount of statistical significance [as 2log(P)] in enrichment for the indicated
biological process. Arrows depict pathways of interests mentioned in the text. (B) BPs induced in Thio1IL-4c-induced AAMs relative to Thio-induced macrophages. (C) BPs
induced in both monocyte- and tissue-derived AAMs. (D) Molecular function (MF) pathways induced in Thio1IL-4c-induced AAMs relative to Thio-induced macrophages. (E)
Hierarchical clustering analysis of 26 unsupervised k-means clusters of genes that have similar expression profiles. The averaged expression for all the genes in each cluster
is shown as a single row. The biological significance of each cluster was determined by measuring the GO term enrichment for BPs. Results shown are for individual mice.
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Figure 4. AAMs derived from inflammatory monocytes express PD-L2, CD206, and MHC class II, but
not tissue macrophage-derived AAMs. FACS analysis of (A) PD-L2, (B) CD206 and (C) MHC class II
from peritoneal macrophages of mice untreated (naı̈ve/
resident) or injected i.p. with IL-4c alone (IL-4c) or
thioglycollate alone (Thio) or Thio and IL-4c (Thio1IL-4c).
Graphs depict the geometric median fluorescent
intensity (MFI) of PD-L2 and CD206 and percentage
of MHC class II, gated on CD11b1 cells from the
peritoneal cavity of individual mice. Data are representative of 3 independent experiments. (D) Stat6 is
required for regulating PD-L2 but not CD206 expression in monocyte-derived AAMs. (E) Quantitation of
CD11b1 cells that are CD2061 PD-L21 from the
peritoneal cavity of individual mice and (F) the total
number of peritoneal cavity cells recovered from
treated animals. Results shown are representative of 2
independent experiments. *P , .05 and **P , .01 as
determined by ANOVA.
Addition of the synthetic RA receptor antagonist LE540 blocked
the induction of Foxp31 expression by Thio1IL-4c-induced AAMs
(Figure 6D), supporting a direct role for RA in the induction of
Foxp31 T-regulatory (Treg) cells by monocyte-derived AAMs.
Importantly, tissue-derived AAMs did not induce Foxp31 expression on CD41 T cells, despite having the same ability to suppress
T-cell proliferation (Figure 6F). Arginase activity has been shown
to block T-cell proliferation,31,32 and because both types of AAMs
Figure 5. Different helminth infections induce either monocyte-derived or tissue-derived AAMs. (A) Representative FACs plots of pleural cavity macrophages on day 12
after L sigmodontis infection compared with pleural cavity macrophages of mice untreated (Res) or injected i.p. with thioglycollate alone (Thio) or Thio and IL-4c (Thio1IL-4c) or IL-4c
alone (IL-4c). (B) Graphs depict proportion of CD206 and PD-L2 positive pleural cavity macrophages. (C) Representative FACs plots of PD-L2 and CD206 expression gated on CD11b1
cells isolated from the liver of S mansoni-infected mice (8 weeks after infection) compared with CD11b1 cells isolated from the liver of mice untreated (naı̈ve) mice. (D) FIZZ1/RELMa
intracellular staining on PD-L21CD2061 macrophages from S mansoni-infected livers compared with PD-L22CD2062 macrophages. (E) Graph depicts the percentage of PD-L2 and
CD206 double positive cells, gated on CD11b1 cells, from the livers of individual mice. (F) MFI of FIZZ1/RELMa staining on PD-L21CD2061 and PD-L22CD2062 macrophages from
livers of individual infected mice. *P , .05 and **P , .01 as determined by ANOVA. Results shown representative of 2 independent experiments.
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Figure 6. Differential effects of monocyte- and tissue macrophage-derived AAMs on naı̈ve CD41 T cells. (A) RT-PCR analysis of Aldh1a2/Raldh2 expression in
peritoneal macrophages normalized to expression of GAPDH. Graphs depict mean 6 standard error of the mean of individual mice pooled from 5 to 6 independent
experiments. (B) FACS analysis of Aldh activity gated on CD11b1 cells from the peritoneal cavity. Peritoneal cells were stained with aldefluor to detect Aldh activity for 2 hours
prior to staining with cell surface antibodies antibodies. (C) Graph depicts the proportion of CD11b1 cells that are ALD1 from individual mice. Results are pooled from 4
independent experiments. (D) Flow cytometry contour plots showing the percentage of CD251, Foxp31CD41 T cells after 6 days of coculture with peritoneal macrophages
either with or without the RA inhibitor LE540 (1 mM). (E) Quantitation of the percentage of CD251, Foxp31 cells from the CD41 compartment after coculture. Data are shown
from 3 independent experiments. (F) Inhibition of CD41 T-cell proliferation by IL-4c- and Thio1IL-4c-induced AAMs. FACS analysis of activated (anti-CD31IL-2) Cell tracerlabeled naı̈ve CD41 cells cultured with peritoneal macrophages (ratio of 2:1) after 3 days of coculture. Results are representative of 3 independent experiments.
express Arg1 at similarly high levels, this may contribute toward
proliferative inhibition.
Uncoupling protein 1 is expressed only by resident
macrophage-derived AAMs
Having confirmed specific features of monocyte-derived AAMs, we
next validated potential markers for resident macrophage-derived
AAMs. One of the most highly up-regulated genes was Ucp1
(Figure 7A), which is thought be highly selective for brown
adipocytes,33 where it is responsible for thermogenesis.34 We
confirmed by RT-PCR that Ucp1 was highly expressed by IL-4c
treatment in peritoneal macrophages isolated by adherence
(Figure 7B), as well as by FACS sorting on CD11b1F4/801 cells
(Figure 7C), but not expressed by Thio1IL-4c-induced AAMs. To
confirm the expression of Ucp1 by resident-derived AAMs in
a more physiological setting, we used the L sigmodontis infection
model. AAMs induced in the pleural cavity of L sigmodontisinfected mice are resident cell derived11 and up-regulated Ucp1
expression (Figure 7D). Ucp1 expression increased in a linear
fashion over 4 days after injection of IL-4c (Figure 7E) and was
regulated in Stat6-dependent manner (Figure 7F). Finally, we
confirmed that UCP1 protein was abundant in AAMs induced by
IL-4c alone and was not detectable in Thio1IL-4c-induced AAMs
(Figure 7G). Therefore, Ucp1 expression may prove a useful
expression marker for tissue-derived AAMs.
Discussion
Tissue resident macrophages can be of embryonic origin and differ in
cellular lineage from macrophages derived from monocytes that
infiltrate tissues during an inflammatory response.5 In the context of
helminth infection, IL-4/IL-13 can expand tissue resident AAMs11 or
monocyte-derived AAMs,29,35 depending on the type of infection and
the tissue inflicted. A key unanswered question is whether these
2 types of macrophages will respond differently to type 2 cytokines in
ways that reflect their heterogeneous existing transcriptional programs
and/or epigenetic differences.36 In this study, we find that AAMs from
differing origins exhibit functional differences and cellular phenotypes
that may reflect different physiological roles in type 2 responses. These
findings have relevance beyond helminth infection because AAMs are
now implicated in many noninfectious disease processes.37-39
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Figure 7. Ucp1 is a unique marker for tissue resident macrophage-derived AAMs. (A) Microarray data for Ucp1 from AAMs. (B) Real-time PCR measurement of Ucp1
expression (relative to GAPDH) in peritoneal macrophages isolated by adherence. (C) RT-PCR measurement of Ucp1 expression in FACS sorted (CD11b1F4/801)
macrophages. (D) RT-PCR measurement of Ucp1 expression in pleural cavity macrophages on day 12 after L sigmodontis infection. (E) Time course analysis of Ucp1
expression after injection with IL-4c. (F) RT-PCR analysis of Ucp1 expression in peritoneal macrophages of treated Stat6-deficient animals. (G) Western blot analysis of Ucp1
protein from total peritoneal cells of individual mice treated with Thio, Thio1IL-4c, and IL-4c, with GAPDH as a loading control.
This study also identifies potentially useful markers to indicate
whether AAMs in a particular setting are derived from tissue-resident
macrophages or from inflammatory monocytes. PD-L2 has been
validated as a marker of AAM in several in vivo models of type 2
inflammation.23,35,40 Here, we show that IL-4 up-regulation of
PD-L2 is limited to monocyte-derived macrophages. Further, PD-L2
expression is a more specific marker than the mannose receptor
CD206,28 because thioglycollate-elicited macrophages also express
CD206 in the absence of IL-4 and independently of Stat6. Hence, the
absence of PD-L2 expression on macrophages that express Arg1,
Relma, and Chi3l3 could be an indication of tissue-resident origin.
In contrast, Ucp1 was thought to be highly selective to adipocytes,33
but we find here that it is highly expressed in tissue-derived AAMs.
In the context of a type 2 immune response, Ucp1 may prove useful
as a marker to distinguish between tissue-derived and monocytederived macrophages. The physiological relevance of Ucp1 expression by tissue-derived AAMs warrants further study.
The production of RA by dendritic cells (DCs) can drive guttropic immune responses and promote the differentiation of FoxP31
Tregs.30 AAMs also express RALDH2, providing a source of RA
that can induce the differentiation of FoxP31 Tregs.29 The expression of RALDH2 was recently described in human AAMs.26
Here, we show that RALDH2 expression and functional ability
to induce FoxP31 Treg differentiation through RA is restricted
to monocyte-derived AAMs. This restriction may reflect a greater
need for Tregs during inflammatory responses involving monocyte
recruitment. For example, the phagocytosis of apoptotic cells by
inflammatory monocytes, instead of resident macrophages, results
in a more inflammatory response,41 which may require Treg
control. DCs rather than macrophages may be more anatomically
positioned to induce Tregs in draining lymph nodes. Nonetheless,
monocytes have recently been shown to traffic through the tissues
into the lymph nodes.10 Perhaps monocyte-derived AAMs have
more active roles in directing host responses than expanded resident
AAMs. This would be consistent with up-regulated chemokine
activity in monocyte-derived AAMs (Figure 3D) and downregulation in tissue-derived AAMs (supplemental Figure 3). The
preferential expression of MHC class II and thymus and activation
regulated chemokine/Ccl17 by monocyte-derived AAMs (Table 4)42
is also notable because this may attract T cells to specifically interact
with monocyte-derived AAMs.
The differences between global transcriptional profiles of tissuederived AAMs vs monocyte-derived AAMs (Figure 2B) are greater
than differences previously reported in the ImmGen project,43
perhaps because that study focused on tissue-resident macrophages.
Molecules associated with antigen presentation, migration, and
regulation of immune responses were preferentially associated
with monocyte-derived AAMs, as was previously described for
DCs.44 Because infiltrating monocytes during an inflammatory
process can give rise to macrophages and DC-like cells depending
on the environment, a key question is how they differ from
the local resident macrophages and DCs as inflammation resolves. It
is important to note that the interaction of thioglycollate with IL-4
is unclear and may synergistically enhance the observed immune
signature on monocyte-derived AAMs. To exclude the effects of
thioglycollate, future studies will examine the minor population
of CX3CR12GFP1PD-L21 cells (Figure 4) induced by IL-4c
treatment alone.
Egg granulomas from S mansoni infection predominantly recruit
monocyte-derived AAMs, whereas L sigmodontis adult worms in the
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 15 MAY 2014 x VOLUME 123, NUMBER 20
DISTINCT M2 MACROPHAGE PHENOTYPES AND FUNCTIONS
pleural cavity induce tissue-derived AAMs. A proximal explanation
for this difference would be that the egg triggers the release of
inflammatory chemokines (eg, Ccl2/MCP1) that recruit monocytes
to a type 2 environment, whereas L sigmodontis infection does not.
More fundamentally, are monocytes recruited to contain parasites,
whereas resident cells minimize host damage? Although the answer
is likely to be both tissue and parasite specific, it is not yet resolved
whether inflammatory monocytes can eventually become tissueresident F4/80-bright AAMs over time. In this study, we examine
relatively short time periods after stimulation, but it will be important
to know whether conversion occurs over time and whether the
distinct functions we observed in this study are driven by cell origin
or by long-term vs short-term tissue residence.
Acknowledgments
The authors thank Dr Nikollaq Vozhilla for help with breeding
and maintaining mice and Alison Fulton for maintenance of the
L sigmodontis life cycle.
Flow cytometry was performed at the New York University Flow
Cytometry and Cell Sorting Center, which is partially supported by
National Institutes of Health (NIH), National Cancer Institute grant
P30CA16087-31. This work is supported by NIH, National Institute
e121
of Allergy and Infectious Diseases grants AI093811 and AI094166
(P.L.), Ruth L. Kirschstein National Research Service Award fellowship F32AI102502 (N.M.G.), NIH National Cancer Institute training
grant, T32 CA009161; Principle Investigator: Levy (U.B.R.), and
Medical Research Council program grant MR/K01207X/1 (J.E.A.).
Authorship
Contribution: U.M.G. performed research and analyzed data; N.M.G.
performed research and analyzed data; D.R. and S.J. performed
research, analyzed data, and helped draft the manuscript; L.N.W.
performed research; M.S.T. and Z.D.K. analyzed data; K.E.W.
performed research; U.B.R. performed research; A.M. contributed
vital reagents and designed research; and P.L. and J.E.A. designed
research, analyzed data, and wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing
financial interests.
Correspondence: P’ng Loke, Department of Microbiology,
Division of Medical Parasitology, Old Public Health Building,
Room 209, 341 East 25th St, New York, NY 10010; e-mail:
png.loke@nyumc.org; or Judith E. Allen, Institute of Immunology
and Infection Research, University of Edinburgh, Edinburgh EH9
3JT, United Kingdom; e-mail: j.allen@ed.ac.uk.
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2014 123: e110-e122
doi:10.1182/blood-2013-08-520619 originally published
online April 2, 2014
Alternatively activated macrophages derived from monocytes and tissue
macrophages are phenotypically and functionally distinct
Uma Mahesh Gundra, Natasha M. Girgis, Dominik Ruckerl, Stephen Jenkins, Lauren N. Ward,
Zachary D. Kurtz, Kirsten E. Wiens, Mei San Tang, Upal Basu-Roy, Alka Mansukhani, Judith E. Allen
and P'ng Loke
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