Alternatively activated macrophages derived from monocytes and tissue macrophages are phenotypically and functionally distinct

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.

Abstract

Macrophages adopt an alternatively activated phenotype (AAMs) when activated by the interleukin-4receptor(R)α. 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 tissue-derived 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 FoxP3⁺ cells from naïve CD4⁺ 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.

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 cavities^1,3 but not for the intestines^8,9 and the skin,¹⁰ where the resident cells are of bone marrow origin.

During T helper 2 (T_H2)-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)α 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 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, Stat6^−/− (Jackson Laboratories) and CX₃CR1^GFP/+ 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 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.

Flow cytometry

Cells were stained with LIVE/DEAD Aqua or Blue (Invitrogen), blocked with 4 μg/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-2′-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, diethylamino-benzaldehyde (DEAB) (at a final concentration of 15 µM) as control.

Foxp3⁺ T-regulatory cell differentiation assay

4 × 10⁵ naïve T cells isolated from lymph nodes using the Naive CD4⁺ T Cell Isolation Kit II (Miltenyi Biotec) were cultured together with 2 × 10⁵ peritoneal macrophages and 1 µg/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 Foxp3⁺ cells by intracellular nuclear staining.

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 log-transformed 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 over-representation by the hypergeometric distribution, as implemented in the GOstats package using gene annotations from the mouse.db0 package.

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-3-phosphate 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 (Thio+IL-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 (Thio+IL-4c). Resident macrophages from naïve 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 Thio+IL-4c treatment can induce macrophage proliferation, although Thio+IL-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 Thio+IL-4c-treated mice.

Figure 1.

IL-4c and Thio+IL-4c induces alternatively activated macrophages with distinct phenotypes. Mice were untreated (naïve/resident) or injected i.p. with IL-4c alone (IL-4c) or thioglycollate alone (Thio) or Thio and IL-4c (Thio+IL-4c) on day 0 and then with IL-4c again (for IL-4c and Thio+IL-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 ± 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 Stat6^+/+ mice or Stat6^−/− mice with IL-4c alone, Thio, or Thio+IL-4c. (E) Quantification of EdU incorporation in macrophages. (F) Total number of F4/80⁺ macrophages recovered. Results shown are representative of 2 separate experiments. (G) Representative FACS plots gated on CD11b⁺ cells for CX3CR1^GFP/+ reporter mice treated as above. (H) The median fluorescent intensity (MFI) of GFP, gated on CD11b⁺ cells from the peritoneal cavity of individual mice. Data are representative of 3 independent experiments. *P < .05 and **P < .01 as determined by ANOVA.

We next examined expression of the well-defined markers of alternative activation, arginase I (Arg1), resistin-like molecule α (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 Thio+IL-4c-induced macrophages (Figure 1C). This was consistent with our previous studies in the pleural cavity in which both IL-4c and Thio+IL-4c led to extensive alternative activation as measured by high percentages of resistin-like molecule α (RELMα)⁺ and Ym1⁺ macrophages.¹¹ Stat6 is essential for alternative activation,^22,23 but its role in macrophage proliferation has not been previously demonstrated. Both Thio+IL-4c and IL-4c activated macrophages from Stat6^−/− 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 F4/80⁺ macrophages (Figure 1F).

In CX3CR1-green fluorescent protein (GFP)/⁺ 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 Thio+IL-4c, we injected CX3CR1-GFP/⁺ reporter mice with these reagents. Gating on the CD11b⁺ cells, the majority of macrophages induced by IL-4c alone were F480^high and CX3CR1-GFP^low, similar to the resident population in naïve mice. In contrast, the AAMs induced by Thio+IL-4c were F480^int and CX3CR1-GFP^high (Figure 1G-H). These results are consistent with a monocyte origin for AAMs induced by Thio+IL-4c (F480^int), whereas those induced by IL-4c alone are derived from resident macrophages (F480^high). Notably, there is a small CX3CR1-GFP^high F4/80^int macrophage population in naïve 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 phosphate-buffered saline (PBS) and IL-4c-injected mice. Similarly, by intravenous transfer of purified Ly6C^High monocytes from CX3CR1-GFP/⁺ mice (CD45.2) into congenic recipient CD45.1 hosts, we confirmed that Thio+IL-4c but not IL-4c alone induces monocyte recruitment and differentiation (supplemental Figure 1).

AAMs derived from monocytes and tissue macrophages are transcriptionally distinct

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 CD11b⁺, F4/80^High cells for resident peritoneal macrophages and IL-4c-induced macrophages and CD11b⁺, F4/80^Int cells for Thio- and Thio+IL-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 F4/80^High AAMs with resident F4/80^High macrophages from untreated mice (Figure 2A), whereas AAMs induced by Thio+IL-4c coclustered with Thio induced macrophages (Figure 2A). When AAMs induced by IL-4c were directly compared with Thio+IL-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, RELMα, 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 Thio+IL-4c-induced AAM (Figure 2C).

Figure 2.

Gene expression profiling of monocyte-derived 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 (CD11b⁺F4/80⁺) macrophages of mice untreated (Res) or treated with IL-4c alone (IL-4c) or thioglycollate alone (Thio) or Thio and IL-4c (Thio+IL-4c). (B) Comparison of probe expression for AAMs induced by IL-4c alone or Thio+IL-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 down-regulated by IL-4 in monocyte-derived (Thio+IL-4c vs Thio) compared with tissue-derived AAMs (IL-4c vs resident). List of genes are shown in Tables 1 to 8.

We then performed supervised comparisons by statistical analyses of microarrays of IL-4c-induced AAMs vs resident macrophages and Thio+IL-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-4c-treated macrophages (Table 1) compared with controls, and 368 genes were up-regulated in Thio+IL-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 Thio+IL-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 Thio+IL-4c-treated 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 Thio+IL-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 Thio+IL-4c AAMs (Figure 3D), but these functions were not up-regulated in IL-4c-treated macrophages; instead, chemokine (Ccl3, Cxcl1, Cxcl2, and Cxcl13) activity was down-regulated (supplemental Figure 3; Tables 4-6).

Table 1.

List of top 50 genes up-regulated by IL-4 in resident macrophages

Gene symbol	Description	Fold change
AA467197	Expressed sequence AA467197	665.9877
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 containing 1	92.0161
Timd2	T-cell immunoglobulin and mucin domain containing 2	90.5118
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, member 7	72.4033
Tcf23	Transcription factor 23	70.0783
Ear10	Eosinophil-associated, ribonuclease A family, member 10	66.9156
Ear11	Eosinophil-associated, ribonuclease A family, member 11	63.0005
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 amidinotransferase)	42.6830
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 containing 10	31.9263
Flt1	FMS-like tyrosine kinase 1	31.6849
Slc7a2	Solute carrier family 7 (cationic amino acid transporter, y+ system), member 2	30.7286
Galnt6	UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase 6	30.3134
Trib3	Tribbles homolog 3 (Drosophila)	27.4894
A530064D06Rik	RIKEN cDNA A530064D06 gene	26.7710
Ccrn4l	CCR4 carbon catabolite repression 4-like (S cerevisiae)	25.7457
Grhl3	Grainyhead-like 3 (Drosophila)	25.1532
Entpd3	Ectonucleoside triphosphate diphosphohydrolase 3	24.6177
Kcnn4	Potassium intermediate/small conductance calcium-activated channel, subfamily N, member 4	22.9964
Fxyd6	FXYD domain-containing ion transport regulator 6	22.4216
Hist1h1b	Histone cluster 1, H1b	22.4066
Arg1	Arginase, liver	22.1165
Oasl1	2'-5′ oligoadenylate synthetase-like 1	21.4259
Gfra2	Glial cell line derived neurotrophic factor family receptor α 2	20.4561
Anpep	Alanyl (membrane) aminopeptidase	20.3326
Shcbp1	Shc SH2-domain binding protein 1	19.8717
Sema3b	Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3B	19.7311
Pbk	PDZ binding kinase	19.4258
Spp1	Secreted phosphoprotein 1	19.0967
Ccna2	Cyclin A2	18.2413
Nuf2	NUF2, NDC80 kinetochore complex component, homolog (S cerevisiae)	18.0971
Ccnf	Cyclin F	17.6265
Plxna2	Plexin A2	17.4434

Table 2.

List of top 50 genes up-regulated by IL-4 in monocyte-derived macrophages

Gene symbol	Description	Fold change
Ccl24	Chemokine (C-C motif) ligand 24	308.1040
Ddx4	DEAD (Asp-Glu-Ala-Asp) box polypeptide 4	147.2503
Ear11	Eosinophil-associated, ribonuclease A family, member 11	129.8195
Tuba8	Tubulin, α 8	114.0360
Ear10	Eosinophil-associated, ribonuclease A family, member 10	109.5308
Chi3l4	Chitinase-like 4 or Ym2	95.2618
Ear1	Eosinophil-associated, ribonuclease A family, member 1	90.4369
Ear12	Eosinophil-associated, ribonuclease A family, member 12	88.9709
Cdh1	Cadherin 1	73.2850
Il4i1	Interleukin 4 induced 1	71.5692
AA467197	Expressed sequence AA467197	68.8517
Asns	Asparagine synthetase	60.0067
Ch25h	Cholesterol 25-hydroxylase	59.7103
Cish	Cytokine inducible SH2-containing protein	59.3660
Gm6756	Predicted gene 6756	53.0013
Ear2	Eosinophil-associated, ribonuclease A family, member 2	47.4012
Vdr	Vitamin D receptor	42.3885
Timp1	Tissue inhibitor of metalloproteinase 1	40.8995
Pi16	Peptidase inhibitor 16	39.9049
Fbp1	Fructose bisphosphatase 1	37.6432
Cst7	Cystatin F (leukocystatin)	36.9475
Klk8	Kallikrein related-peptidase 8	36.9350
Gfra2	Glial cell line derived neurotrophic factor family receptor α 2	36.3341
Phgdh	3-phosphoglycerate dehydrogenase	36.1081
Tarm1	T cell-interacting, activating receptor on myeloid cells 1	36.1055
Ramp3	Receptor (calcitonin) activity modifying protein 3	31.2576
A530064D06Rik	RIKEN cDNA A530064D06 gene	26.3184
Slc7a2	Solute carrier family 7 (cationic amino acid transporter, y+ system), member 2	24.4996
Msx3	msh homeobox 3	21.9209
Nlrp1a	NLR family, pyrin domain containing 1A	21.6752
Ccl7	Chemokine (C-C motif) ligand 7	21.4119
Car6	Carbonic anhydrase 6	20.7420
Cebpe	CCAAT/enhancer binding protein (C/EBP), ε	20.2090
Aldh1a2	Aldehyde dehydrogenase family 1, subfamily A2	19.2205
Ccl17	Chemokine (C-C motif) ligand 17	19.2074
Mthfd2	Methylenetetrahydrofolate dehydrogenase (NAD+ dependent), methenyltetrahydrofolate cyclohydrolase	16.9681
Aqp3	Aquaporin 3	16.4622
Alox15	Arachidonate 15-lipoxygenase	15.4346
Retnla	Resistin like α	15.3394
Ltb4r1	Leukotriene B4 receptor 1	15.2231
Retnlg	Resistin like γ	15.1644
Flt1	FMS-like tyrosine kinase 1	14.4730
Olr1	Oxidized low density lipoprotein (lectin-like) receptor 1	14.4345
Cd81	CD81 antigen	14.2763
Slc7a5	Solute carrier family 7 (cationic amino acid transporter, y+ system), member 5	13.8395
Fcrls	Fc receptor-like S, scavenger receptor	12.9906
Ap4e1	Adaptor-related protein complex AP-4, ε 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

Table 3.

List of 50 genes regulated by IL-4 in both resident and monocyte-derived macrophages

Gene symbol	Description	Fold change*
AA467197	Expressed sequence AA467197	367.4197
Chi3l4	Chitinase-like 4 or Ym2	310.8889
Aqp3	Aquaporin 3	156.1061
Ddx4	DEAD (Asp-Glu-Ala-Asp) box polypeptide 4	127.3663
Ear11	Eosinophil-associated, ribonuclease A family, member 11	96.4100
Ear10	Eosinophil-associated, ribonuclease A family, member 10	88.2232
Cst7	Cystatin F (leukocystatin)	86.1656
Tuba8	Tubulin, α 8	58.8395
Ear12	Eosinophil-associated, ribonuclease A family, member 12	46.9911
Asns	Asparagine synthetase	46.9515
Chi3l3	Chitinase-like 3 or Ym1	44.8807
Cdh1	Cadherin 1	44.7643
Cish	Cytokine inducible SH2-containing protein	33.5730
Gm4610	Predicted gene 4610	28.9111
Gm6756	Predicted gene 6756	28.5975
Gfra2	Glial cell line derived neurotrophic factor family receptor α 2	28.3951
Phgdh	3-Phosphoglycerate dehydrogenase	28.1069
Slc7a2	Solute carrier family 7 (cationic amino acid transporter, y+ system), member 2	27.6141
Gm5150	Predicted gene 5150	26.6933
A530064D06Rik	RIKEN cDNA A530064D06 gene	26.5447
Ear2	Eosinophil-associated, ribonuclease A family, member 2	25.9995
Gatm	Glycine amidinotransferase (l-arginine:glycine amidinotransferase)	25.6794
Pi16	Peptidase inhibitor 16	23.3797
Flt1	FMS-like tyrosine kinase 1	23.0790
Fbp1	Fructose bisphosphatase 1	22.4783
Apoe	Apolipoprotein E	0.0499
Clec4d	C-type lectin domain family 4, member d	0.0532
Apoc2	Apolipoprotein C-II	0.0551
Atf7	Activating transcription factor 7	0.0663
Irf8	Interferon regulatory factor 8	0.0682
Gm11538	Predicted gene 11538	0.0778
Fcgr1	Fc receptor, IgG, high affinity I	0.0858
Rgs1	Regulator of G-protein signaling 1	0.0877
Ccl3	Chemokine (C-C motif) ligand 3	0.0880
Prom1	Prominin 1	0.0982
Gm1673	Predicted gene 1673	0.1016
Cdkn1c	Cyclin-dependent kinase inhibitor 1C (P57)	0.1045
Olfr871	Olfactory receptor 871	0.1055
Neurl3	Neuralized homolog 3 homolog (Drosophila)	0.1063
Lyz2	Lysozyme 2	0.1138
Itgb5	Integrin β 5	0.1160
Lst1	Leukocyte specific transcript 1	0.1253
Rnf150	Ring finger protein 150	0.1274
Itga6	Integrin α 6	0.1277
Cnrip1	Cannabinoid receptor interacting protein 1	0.1286
Abca1	ATP-binding cassette, sub-family A (ABC1), member 1	0.1294
Cbr2	Carbonyl reductase 2	0.1316
1700003F12Rik	RIKEN cDNA 1700003F12 gene	0.1339
Lmo2	LIM domain only 2	0.1416
Ldhb	Lactate dehydrogenase B	0.1431

^*Calculated as average fold change of genes regulated by IL-4 in resident macrophage and genes regulated by IL-4 in monocyte-derived macrophages.

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 −log(P)] in enrichment for the indicated biological process. Arrows depict pathways of interests mentioned in the text. (B) BPs induced in Thio+IL-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 Thio+IL-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.

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

Gene symbol	Name	Fold change
Ccl17	Chemokine (C-C motif) ligand 17	19.2074
Gm5150	Predicted gene 5150	4.1190
Ccl2	Chemokine (C-C motif) ligand 2	7.4465
Ccl7	Chemokine (C-C motif) ligand 7	21.4119
Ccl12	Chemokine (C-C motif) ligand 12	7.4850
Ccl24	Chemokine (C-C motif) ligand 24	308.1040

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	Name	Fold change
Gm5150	Predicted gene 5150	4.1190
Ccl12	Chemokine (C-C motif) ligand 12	7.4850
Ccl7	Chemokine (C-C motif) ligand 7	21.4119
Il6	Interleukin 6	8.8808
Ccl2	Chemokine (C-C motif) ligand 2	6.9844
Cish	Cytokine inducible SH2-containing protein	59.3660
C3	Complement component 3	9.2433
Il31ra	Interleukin 31 receptor A	9.6304
Ccl24	Chemokine (C-C motif) ligand 24	308.1040
Socs6	Suppressor of cytokine signaling 6	3.3646
Ccl17	Chemokine (C-C motif) ligand 17	19.2074
Socs2	Suppressor of cytokine signaling 2	7.4007

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

Table 7.

List of top 50 genes down-regulated by IL-4 in resident macrophages

Gene symbol	Name	Fold change
Cxcl13	Chemokine (C-X-C motif) ligand 13	0.0020
Lyz2	Lysozyme 2	0.0081
Atf7	Activating transcription factor 7	0.0131
Lpl	Lipoprotein lipase	0.0177
Irf8	Interferon regulatory factor 8	0.0192
Apoc2	Apolipoprotein C-II	0.0196
Dusp6	Dual specificity phosphatase 6	0.0219
Clec7a	C-type lectin domain family 7, member a	0.0238
Apoc1	Apolipoprotein C-I	0.0300
Hp	Haptoglobin	0.0339
Gm1673	Predicted gene 1673	0.0344
Gm11538	Predicted gene 11538	0.0367
Fcgr1	Fc receptor, IgG, high affinity I	0.0375
Pltp	Phospholipid transfer protein	0.0386
Ccl3	Chemokine (C-C motif) ligand 3	0.0439
Abca1	ATP-binding cassette, sub-family A (ABC1), member 1	0.0441
Hspa1a	Heat shock protein 1A	0.0494
Rgs2	Regulator of G-protein signaling 2	0.0526
Lama3	Laminin, α 3	0.0563
Nat8l	N-acetyltransferase 8-like	0.0564
Apoc4	Apolipoprotein C-IV	0.0644
Clec4d	C-type lectin domain family 4, member d	0.0673
Rab6b	RAB6B, member RAS oncogene family	0.0704
Emb	Embigin	0.0707
F13a1	Coagulation factor XIII, A1 subunit	0.0713
Fpr1	Formyl peptide receptor 1	0.0714
Upk1a	Uroplakin 1A	0.0718
Cd63	CD63 antigen	0.0744
Gm4788	Predicted gene 4788	0.0773
Ccm2l	Cerebral cavernous malformation 2-like	0.0782
Marco	Macrophage receptor with collagenous structure	0.0804
Fpr2	Formyl peptide receptor 2	0.0805
Fkbp1a	FK506 binding protein 1a	0.0805
1700003F12Rik	RIKEN cDNA 1700003F12 gene	0.0806
Apoe	Apolipoprotein E	0.0850
Rprm	Reprimo, TP53 dependent G2 arrest mediator candidate	0.0854
Cfhr2	Complement factor H-related 2	0.0855
Abca9	ATP-binding cassette, sub-family A (ABC1), member 9	0.0913
Cfh	Complement component factor h	0.0924
Egfl7	EGF-like domain 7	0.0930
Olfr871	Olfactory receptor 871	0.0934
Neurl3	Neuralized homolog 3 homolog (Drosophila)	0.0949
Rgs1	Regulator of G-protein signaling 1	0.0949
Rs5-8s1	Unknown	0.0977
Prom1	Prominin 1	0.0986
Ldhb	Lactate dehydrogenase B	0.0987
Rhbdf1	Rhomboid family 1 (Drosophila)	0.1027
Fut7	Fucosyltransferase 7	0.1038
Pilra	Paired immunoglobin-like type 2 receptor α	0.1082
Lst1	Leukocyte specific transcript 1	0.1086

Table 8.

List of top 50 genes downregulated by IL-4 in monocyte-derived macrophages

Gene symbol	Name	Fold change
Mamdc2	MAM domain containing 2	0.0148
Apoe	Apolipoprotein E	0.0148
Dio2	Deiodinase, iodothyronine, type II	0.0169
AF251705	cDNA sequence AF251705	0.0211
Cdkn1c	Cyclin-dependent kinase inhibitor 1C (P57)	0.0226
Hpgds	Hematopoietic prostaglandin D synthase	0.0232
Syt10	Synaptotagmin X	0.0287
C77080	Expressed sequence C77080	0.0338
Mmp27	Matrix metallopeptidase 27	0.0356
Mcoln3	Mucolipin 3	0.0364
Tspan7	Tetraspanin 7	0.0368
Htra1	HtrA serine peptidase 1	0.0371
Saa3	Serum amyloid A 3	0.0373
Clec4d	C-type lectin domain family 4, member d	0.0390
Cd200	CD200 antigen	0.0412
Ms4a7	Membrane-spanning 4-domains, subfamily A, member 7	0.0413
Sh2d3c	SH2 domain containing 3C	0.0430
Htr2b	5-Hydroxytryptamine (serotonin) receptor 2B	0.0452
Reps2	RALBP1 associated Eps domain containing protein 2	0.0489
Npy	Neuropeptide Y	0.0494
Cnrip1	Cannabinoid receptor interacting protein 1	0.0495
Tmem140	Transmembrane protein 140	0.0502
Lhfpl2	Lipoma HMGIC fusion partner-like 2	0.0503
Arhgap22	ρ GTPase activating protein 22	0.0507
Prss46	Protease, serine 46	0.0512
Spp1	Secreted phosphoprotein 1	0.0513
Cd93	CD93 antigen	0.0519
Pcp4l1	Purkinje cell protein 4-like 1	0.0525
Mfge8	Milk fat globule-EGF factor 8 protein	0.0547
Gstm1	Glutathione S-transferase, μ 1	0.0547
Gpr137b	G protein-coupled receptor 137B	0.0576
Cd109	CD109 antigen	0.0581
Tspan13	Tetraspanin 13	0.0586
Gas6	Growth arrest specific 6	0.0587
4930404N11Rik	RIKEN cDNA 4930404N11 gene	0.0631
Mpeg1	Macrophage expressed gene 1	0.0655
Tecpr1	Tectonin β-propeller repeat containing 1	0.0680
Gprc5b	G protein-coupled receptor, family C, group 5, member B	0.0703
Pianp	PILR α associated neural protein	0.0707
Gpr137b-ps	G protein-coupled receptor 137B, pseudogene	0.0734
Gdpd1	Glycerophosphodiester phosphodiesterase domain containing 1	0.0742
Lrrc27	Leucine rich repeat containing 27	0.0748
Fstl1	Follistatin-like 1	0.0750
Pdzk1ip1	PDZK1 interacting protein 1	0.0759
Fhdc1	FH2 domain containing 1	0.0765
Itgb5	Integrin β 5	0.0792
Speg	SPEG complex locus	0.0795
Gstm3	Glutathione S-transferase, μ 3	0.0799
Rgs1	Regulator of G-protein signaling 1	0.0805

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 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 (Thio+IL-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 monocyte-derived 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). F4/80^int cells induced by Thio+IL-4c expressed both PD-L2 and CD206, whereas the majority of IL-4c-induced F4/80^high 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 F4/80^high tissue-derived AAMs (Figure 4C). The minor F4/80^int population in naïve 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 CX3CR1-GFP⁺ (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.

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 (naïve/resident) or injected i.p. with IL-4c alone (IL-4c) or thioglycollate alone (Thio) or Thio and IL-4c (Thio+IL-4c). Graphs depict the geometric median fluorescent intensity (MFI) of PD-L2 and CD206 and percentage of MHC class II, gated on CD11b⁺ 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 CD11b⁺ cells that are CD206⁺PD-L2⁺ 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.

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-GFP^high,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 Thio+IL-4c into the pleural cavity induced accumulation of PD-L2- and 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 Thio+IL-4c injection.

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 (Thio+IL-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 CD11b⁺ cells isolated from the liver of S mansoni-infected mice (8 weeks after infection) compared with CD11b⁺ cells isolated from the liver of mice untreated (naïve) mice. (D) FIZZ1/RELMa intracellular staining on PD-L2⁺CD206⁺ macrophages from S mansoni-infected livers compared with PD-L2⁻CD206⁻ macrophages. (E) Graph depicts the percentage of PD-L2 and CD206 double positive cells, gated on CD11b⁺ cells, from the livers of individual mice. (F) MFI of FIZZ1/RELMa staining on PD-L2⁺CD206⁺ and PD-L2⁻CD206⁻ macrophages from livers of individual infected mice. *P < .05 and **P < .01 as determined by ANOVA. Results shown representative of 2 independent experiments.

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 Thio+IL-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-L2⁺ and CD206⁺ macrophages are AAMs, we also stained for intracellular FIZZ1/RELMa (Figure 5D,F) and gated on the PD-L2⁺CD206⁺ population compared with the PD-L2⁻CD206⁻ 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 CD4⁺FoxP3⁺ 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 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 Thio+IL-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 Thio+IL-4c induced a substantial number of ALD⁺ cells that were F4/80^int (Figure 6B; supplemental Figure 6). As expected from its monocyte origins, some of the minor F4/80^int peritoneal population of naïve untreated animals and IL-4c-treated animals were also ALD⁺ (Figure 6C), although there were many fewer ALD⁺ cells than in Thio+IL-4c-treated animals.

Figure 6.

Differential effects of monocyte- and tissue macrophage-derived AAMs on naïve CD4⁺ T cells. (A) RT-PCR analysis of Aldh1a2/Raldh2 expression in peritoneal macrophages normalized to expression of GAPDH. Graphs depict mean ± standard error of the mean of individual mice pooled from 5 to 6 independent experiments. (B) FACS analysis of Aldh activity gated on CD11b⁺ 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 CD11b⁺ cells that are ALD⁺ from individual mice. Results are pooled from 4 independent experiments. (D) Flow cytometry contour plots showing the percentage of CD25⁺, Foxp3⁺CD4⁺ T cells after 6 days of coculture with peritoneal macrophages either with or without the RA inhibitor LE540 (1 µM). (E) Quantitation of the percentage of CD25⁺, Foxp3⁺ cells from the CD4⁺ compartment after coculture. Data are shown from 3 independent experiments. (F) Inhibition of CD4⁺ T-cell proliferation by IL-4c- and Thio+IL-4c-induced AAMs. FACS analysis of activated (anti-CD3⁺IL-2) Cell tracer-labeled naïve CD4⁺ cells cultured with peritoneal macrophages (ratio of 2:1) after 3 days of coculture. Results are representative of 3 independent experiments.

Because RA can induce Foxp3 expression in CD4⁺ T cells, we compared the ability of IL-4c- and Thio+IL-4c-induced AAMs to induce expression of Foxp3 in naïve CD4⁺ T cells. Strikingly, only Thio+IL-4c induced AAMs were able to induce differentiation of Foxp3⁺ cells, detected after 7 days of culture (Figure 6D-E). Addition of the synthetic RA receptor antagonist LE540 blocked the induction of Foxp3⁺ expression by Thio+IL-4c-induced AAMs (Figure 6D), supporting a direct role for RA in the induction of Foxp3⁺ T-regulatory (Treg) cells by monocyte-derived AAMs. Importantly, tissue-derived AAMs did not induce Foxp3⁺ expression on CD4⁺ 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 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 CD11b⁺F4/80⁺ cells (Figure 7C), but not expressed by Thio+IL-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 sigmodontis-infected 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 Thio+IL-4c-induced AAMs (Figure 7G). Therefore, Ucp1 expression may prove a useful expression marker for tissue-derived AAMs.

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 (CD11b⁺F4/80⁺) 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, Thio+IL-4c, and IL-4c, with GAPDH as a loading control.

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

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 monocyte-derived macrophages. The physiological relevance of Ucp1 expression by tissue-derived AAMs warrants further study.

The production of RA by dendritic cells (DCs) can drive gut-tropic immune responses and promote the differentiation of FoxP3⁺ Tregs.³⁰ AAMs also express RALDH2, providing a source of RA that can induce the differentiation of FoxP3⁺ Tregs.²⁹ The expression of RALDH2 was recently described in human AAMs.²⁶ Here, we show that RALDH2 expression and functional ability to induce FoxP3⁺ 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 down-regulation 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 tissue-derived 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 CX3CR1⁻GFP⁺PD-L2⁺ 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 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 tissue-resident 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.

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.