The Prolyl Hydroxylase PHD3 Identifies Proinflammatory Macrophages and Its Expression Is Regulated by Activin A

María M. Escribese, Elena Sierra-Filardi, Concha Nieto, Rafael Samaniego, Carmen Sánchez-Torres, Takami Matsuyama, Elisabeth Calderon-Gómez, Miguel A. Vega, Azucena Salas, Paloma Sánchez-Mateos and Angel L. Corbí
J Immunol August 15, 2012, 189 (4) 1946-1954; DOI: https://doi.org/10.4049/jimmunol.1201064

Abstract

Modulation of macrophage polarization underlies the onset and resolution of inflammatory processes, with polarization-specific molecules being actively sought as potential diagnostic and therapeutic tools. Based on their cytokine profile upon exposure to pathogenic stimuli, human monocyte-derived macrophages generated in the presence of GM-CSF or M-CSF are considered as proinflammatory (M1) or anti-inflammatory (M2) macrophages, respectively. We report in this study that the prolyl hydroxylase PHD3-encoding EGLN3 gene is specifically expressed by in vitro-generated proinflammatory M1(GM-CSF) human macrophages at the mRNA and protein level. Immunohistochemical analysis revealed the expression of PHD3 in CD163+ lung macrophages under basal homeostatic conditions, whereas PHD3+ macrophages were abundantly found in tissues undergoing inflammatory responses (e.g., Crohn’s disease and ulcerative colitis) and in tumors. In the case of melanoma, PHD3 expression marked a subset of tumor-associated macrophages that exhibit a weak (e.g., CD163) or absent (e.g., FOLR2) expression of typical M2-polarization markers. EGLN3 gene expression in proinflammatory M1(GM-CSF) macrophages was found to be activin A dependent and could be prevented in the presence of an anti-activin A-blocking Ab or inhibitors of activin receptor-like kinase receptors. Moreover, EGLN3 gene expression was upregulated in response to hypoxia only in M2(M-CSF) macrophages, and the hypoxia-mediated upregulation of EGLN3 expression was significantly impaired by activin A neutralization. These results indicate that EGLN3 gene expression in macrophages is dependent on activin A both under basal and hypoxic conditions and that the expression of the EGLN3-encoded PHD3 prolyl hydroxylase identifies proinflammatory macrophages in vivo and in vitro.

Introduction

The extreme sensitivity of macrophages to endogenous (e.g., cytokines) and exogenous (e.g., pathogens) stimuli explains their phenotypic and functional heterogeneity under homeostatic and pathological conditions (1). In fact, the existence of a plethora of macrophage polarization states is critical for the adequate onset, regulation, and resolution of immune and inflammatory responses (2). As a representative example, although GM-CSF and M-CSF contribute to macrophage survival and proliferation, they exert distinct actions on macrophage polarization. GM-CSF gives rise to monocyte-derived macrophages that exhibit high Ag-presenting capacity and produce proinflammatory cytokines in response to LPS (3, 4). Conversely, M-CSF generates macrophages with high phagocytic activity and IL-10–producing ability in response to pathogens (3, 4). Based on their respective cytokine profiles, human macrophages generated in the presence of GM-CSF or M-CSF are representative of the classical or alternative macrophage polarization states, respectively, and are considered as proinflammatory or anti-inflammatory macrophages (3, 5).

Variation in the levels of oxygen is a parameter that also determines the state of macrophage polarization (6). In healthy tissues, oxygen levels span from 150 mmHg in the lung to 40–100 mmHg in the circulation and 4–20 mmHg in the tissues (7). Pathologic hypoxia, commonly found in malignant solid tumors, inflammatory lesions, and healing wounds (8–10), also influences macrophage polarization and leads to transcriptional and metabolic changes (6) and the acquisition of effector functions to promote angiogenesis and restore tissue homeostasis (7, 11, 12). Macrophages accumulate in large numbers in damaged hypoxic tissues and respond to hypoxia by altering their gene expression program via upregulation of the hypoxia-inducible factor (HIF) 1 (HIF1α/HIF1β) and HIF2 (HIF2α/HIF1β) transcription factors (13, 14). In the presence of oxygen, both α subunits are prolyl-hydroxylated, recognized by the von Hippel-Lindau tumor suppressor protein (15–17), and degraded by the ubiquitin-proteasome pathway (17). The three HIF prolyl hydroxylases (PHD1–3) (16, 18) require molecular oxygen as a cosubstrate and, therefore, constitute the link between oxygen availability and HIF-dependent transcription (19).

The prolyl hydroxylase PHD3 is encoded by the EGLN3 gene, for which expression is upregulated in platelet-derived growth factor-stimulated rat fibroblasts (20). PHD3 is involved in nerve-growth factor-dependent survival of neurons (21), stimulates pyruvate kinase M2 coactivation for HIF1 (22), and also participates in HIF-independent processes to control cell death, changes in metabolism (23), and IKKβ/NF-κB signaling (24). In the current study, we demonstrate that proinflammatory macrophages constitutively express the EGLN3-encoded PHD3 prolyl hydroxylase in vitro and in vivo and that activin A regulates EGLN3 gene expression both under normoxic and hypoxic conditions.

Materials and Methods

Generation of human monocyte-derived macrophages

Human PBMCs were isolated from buffy coats from normal donors over a Lymphoprep (Nycomed Pharma, Oslo, Norway) gradient according to standard procedures. Monocytes were purified from PBMC by magnetic cell sorting using CD14+ microbeads or the CD16+ Monocyte Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Monocytes (0.5 × 106 cells/ml, >95% CD14+ cells) were cultured in RPMI 1640 supplemented with 10% FCS (completed medium) for 7 d at 37°C in a humidified atmosphere with 5% CO2 and containing 1000 U/ml GM-CSF or M-CSF (10 ng/ml; ImmunoTools, Friesoythe, Germany) to generate M1(GM-CSF) or M2(M-CSF) monocyte-derived macrophages, respectively. Cytokines were added every 2 d. Where indicated, M1(GM-CSF) or M2(M-CSF) macrophages were treated with either IFN-γ (500 U/ml; R&D Systems, Minneapolis, MN) or IL-4 (1000 U/ml; R&D Systems) for 48 h. To generate GM-CSF–free conditioned medium, M1(GM-CSF) macrophages were extensively washed to remove residual GM-CSF and cultured in complete medium for 48 h in the absence of GM-CSF, after which the conditioned medium was collected. Cells were routinely cultured in 21% O2 and 5% CO2 (normoxic conditions). For hypoxic conditions, cells were placed in an in vivo 400 hypoxia Work Station (Ruskinn Technology) that was infused with a mixture of 1% O2, 5% CO2, and 94% N2 (Sociedad Española de Carburos Metálicos) for 24 h and treated or not with the activin receptor-like kinase (ALK)4/5/7 inhibitor SB431542 (Sigma-Aldrich) or an anti-activin A-blocking Ab (R&D Systems).

Quantitative real-time RT-PCR

Oligonucleotides for selected genes were designed according to the Roche software for quantitative real-time PCR (Roche Diagnostics). Total RNA was extracted using the RNeasy kit (Qiagen), retrotranscribed, and individually amplified cDNA were quantified using the Universal Human Probe Roche library (Roche Diagnostics). Assays were made in triplicate and results normalized according to the expression levels of GAPDH RNA. Results were expressed using the ΔΔ threshold cycle method for quantification.

ELISA

Macrophage supernatants from M1(GM-CSF) or M2(M-CSF) macrophages were assayed for the presence of activin A using an ELISA from R&D Systems and according to the protocol supplied by the manufacturer.

Western blot

Cell lysates were obtained in 10 mM Tris-HCl (pH 8), 150 mM NaCl, 1% Nonidet P-40 lysis buffer containing 2 mM Pefabloc, 2 μg/ml aprotinin/antipain/leupeptin/pepstatin, 10 mM NaF, and 1 mM Na3VO4. Ten micrograms cell lysate was subjected to SDS-PAGE and transferred onto an Immobilon polyvinylidene difluoride membrane (Millipore). Protein detection was carried out using an anti-human PHD3 polyclonal Ab (Abcam, Cambridge, U.K.) or an mAb against GAPDH (sc-32233; Santa Cruz Biotechnology, Santa Cruz, CA).

Reporter gene assays

Mv1Lu cells were plated in 24-well plates at 1 × 106 cells/ml in RPMI 1640 with 10% FCS. The luciferase-based plasmid pGL3-EGLN3PEnhA, which contains the firefly luciferase gene under the control of the EGLN3 gene promoter and enhancer, has been previously described (25). Cells were transiently transfected with 2 μg DNA of the reporter construct using Superfect (Qiagen). Twenty to 24 h after transfection, cells were treated with 25 ng/ml human recombinant activin A (PeproTech) for 3 h. To normalize transfection efficiency, cells were cotransfected with an SV40 promoter-based β-galactosidase expression plasmid (RSV-βgal). Measurement of relative luciferase units and β-galactosidase activity was performed using the Luciferase Assay System (Promega) and the Galacto-Ligth kit (Tropix), respectively.

Confocal microscopy and immunohistochemistry

Human tissue samples were obtained from patients undergoing surgical treatment and following the medical ethics committee procedures (Hospital General Universitario Gregorio Marañón, Hospital Clínic de Barcelona). Histopathologic diagnosis was confirmed for each specimen. Acetone-fixed thick sections (4 μm in depth) of cryopreserved tissue were first blocked for 5 min with human Igs and then sequentially incubated with 1–5 μg/ml primary Abs (high m.w. melanoma-associated Ag, BD Pharmingen; FITC-conjugated anti-CD163, Santa Cruz Biotechnology; anti-PHD3, Abcam; anti-CD68, clone PG-M1, DakoCytomation; and anti-FRβ) (26) followed by addition of their corresponding fluorescent secondary Abs. Tissue imaging was performed with a confocal microscope (SP2; Leica Microsystems). For panoramic images, 10× PL-APO NA 0.4 and glycerol immersion 20× PL-APO NA 0.7 objectives were used at 1.5 Airy of pinhole aperture. For more detailed images, the 63× PL-APO NA 1.3 glycerol immersion objective was used. For in vitro cell imaging, cells were plated on coverslips coated with poly-l-lysine (Sigma-Aldrich) for 4 h at 37°C and then fixed with 4% formaldehyde (Sigma-Aldrich). Mean fluorescence intensities were measured in regions of interest manually depicted around CD163+ cells using the LCS 15.37 software from Leica Microsystems. Images were processed with Adobe Photoshop and Illustrator CS (Adobe Systems). Correlation analysis was performed using the SPSS 11.5 software (SPSS).

Statistical analysis

Statistical analysis was performed using Student t test, and a p value <0.05 was considered significant. A two-way ANOVA test was performed for analysis of immunohistochemistry results.

Results

PHD3 is expressed by in vitro-generated proinflammatory M1(GM-CSF) macrophages

After 7 d in culture with GM-CSF, monocytes differentiate into M1 macrophages that, unlike those generated in the presence of M-CSF (M2), exhibit immunogenic, proinflammatory, and antitumor functions (3). Gene expression profiling (GSE27792) (27, 28) revealed that EGLN3 gene expression, which codes for PHD3, is considerably higher in M1(GM-CSF) than in M2(M-CSF) macrophages derived from either CD14+CD16− monocytes (log2 M1/M2 = 6.89; p = 0.0001) or CD14+CD16+ monocytes (log2 M1/M2 = 2.88; p = 0.018) (Fig. 1A), a difference that was further confirmed by quantitative real-time RT-PCR (qRT-PCR) on independent samples (Fig. 1B). Kinetic analysis showed that EGLN3 expression increased along M1(GM-CSF) macrophage differentiation, being detectable after 48 h in culture and reaching maximum levels after 7 d (Fig. 1C). The expression of EGLN3 was considerably increased by either IFN-γ or IL-4 in M1(GM-CSF) macrophages (Fig. 1D). Both cytokines also induced EGLN3 expression in M2(M-CSF) macrophages, although they reached lower levels than in unstimulated M1(GM-CSF) macrophages (Fig. 1D). The differential expression of the EGLN3-encoded PHD3 hydroxylase in both macrophage subtypes was verified by Western blot, as PHD3 was expressed at high levels in lysates from M1(GM-CSF) macrophages, whereas it was barely detectable in M2(M-CSF) macrophages (Fig. 1E). Moreover, immunochemical analysis of in vitro-generated macrophages confirmed that proinflammatory CD68+ M1(GM-CSF) macrophages contained high levels of PHD3, with a predominantly cytoplasmic subcellular distribution (Fig. 1F). By contrast, CD68+ M2(M-CSF) macrophages lacked PHD3 expression (Fig. 1F). The M1(GM-CSF) macrophage-restricted expression of PHD3 was further evidenced upon costaining with an mAb specific for the FRβ, for which expression is only found in M2(M-CSF) macrophages (29). As shown in Fig. 1F, M1(GM-CSF) macrophages exhibited a PHD3+FRβ− phenotype, whereas M2(M-CSF) macrophages were PHD3−FRβ+. Altogether, these results demonstrate the link between expression of the PHD3-coding EGLN3 gene and proinflammatory macrophage polarization, a relationship that was also observed in murine bone marrow-derived M1 and M2 macrophages (data not shown).

FIGURE 1.
FIGURE 1.

Expression of the EGLN3-encoded PHD3 prolyl hydroxylase is restricted to proinflammatory M1(GM-CSF) macrophages. (A) EGLN3 gene expression in M1(GM-CSF) and M2(M-CSF) macrophages derived from either CD14+CD16− or CD14−CD16+ monocytes, as determined by microarray analysis (GSE27792) of three independent samples. (B) EGLN3 gene expression in M1(GM-CSF) and M2(M-CSF) macrophages derived from CD14+CD16− monocytes determined by qRT-PCR on three independent samples. Mean and SD of triplicate determinations are shown. Results are expressed as relative expression (relative to GAPDH RNA levels) and referred to the EGLN3 mRNA levels in M2(M-CSF) cells (***p < 0.001). (C) Kinetics of EGLN3 expression along M1(GM-CSF) macrophage generation. Results are expressed as relative expression (relative to GAPDH RNA levels) and referred to the EGLN3 mRNA levels at 0 h. One representative experiment (out of two) is shown. (D) EGLN3 expression in M1(GM-CSF) and M2(M-CSF) macrophages unstimulated (−) or stimulated for 48 h with IFN-γ or IL-4. Results are expressed as relative expression (relative to GAPDH RNA levels) and referred to the EGLN3 mRNA levels in unstimulated M2(M-CSF) macrophages. (E) Immunoblot analysis of PHD3 in lysates of M1(GM-CSF) and M2(M-CSF) macrophages. GAPDH protein levels were determined as a loading control. (F) Immunofluorescence analysis on M1(GM-CSF) and M2(M-CSF) macrophages, as determined by confocal microscopy using Abs specific for CD68 (white), PHD3 (red), and FRβ (green). No staining was observed upon incubation with isotype-matched irrelevant Abs as negative controls. Nuclei were counterstained with DAPI.

PHD3 expression in macrophages under homeostatic and inflammatory conditions

To determine whether EGLN3 gene expression could be detected in macrophages in vivo, normal human tissues were tested for PHD3-specific reactivity by immunohistochemistry. An extremely low percentage of skin and tonsil CD163+ macrophages were found to express PHD3 (Fig. 2A). Conversely, a moderate percentage of alveolar CD163+ macrophages exhibited PHD3 reactivity (Fig. 2B), indicating that human macrophages can also express PHD3 under homeostatic conditions in vivo. In the case of the gut, a similar picture emerged under homeostatic conditions, as a very low percentage of PHD3+CD163+ macrophages were detected in the colon, regardless of the region analyzed (Q7, Q8, ascending; Q15, sigmoid) (Fig. 3A). However, a higher number of PHD3+ macrophages were detected in CD163+ macrophages from the ileum of patients with Crohn’s disease (Fig. 3B) and from the colon of patients with ulcerative colitis, independently of colon area analyzed (Q5, transverse; Q14, Q16, rectum) (Fig. 3C). To quantitate the level of PHD3 in macrophages in noninflamed and inflamed colon, individual CD163+ macrophages were identified and their PHD3 staining measured. As shown in Fig. 3D, CD163+ macrophages from inflamed tissues exhibit significantly higher intensity of PHD3 staining, thus demonstrating that PHD3+ CD163+ macrophages are found in inflamed gut and that PHD3 levels are higher in macrophages within an inflammatory environment.

FIGURE 2.
FIGURE 2.

Expression of PHD3 in macrophages from noninflamed tissues. (A) Confocal sections of human tonsil (top panels) and skin (bottom panels) after double immunofluorescence analysis for the macrophage-specific marker CD163 (green) and PHD3 (red). In the left panels, nuclei were counterstained with DAPI (blue). In the middle panels, the boundary between dermis and epidermis (skin) and T and B cell areas of the follicle (tonsil) are indicated. (B) Confocal sections of human lung after double immunofluorescence analysis for the macrophage-specific marker CD163 (green) and PHD3 (red) or a control rabbit antiserum (rbIgG, red). In the left and middle panels, nuclei were counterstained with DAPI (blue).

FIGURE 3.
FIGURE 3.

Expression of PHD3 in macrophages from inflamed gut. Confocal sections of samples from normal human intestine (A), ileum from patients with Crohn’s disease (B), and colon from patients with ulcerative colitis (C) after double immunofluorescence analysis for the macrophage-specific marker CD163 (green) and PHD3 (red). Colocalization of both markers yields yellow staining. When indicated, nuclei were counterstained with DAPI (blue). The areas shown at higher resolution are indicated by a square in the left panels. (D) Quantitation of PHD3 staining in CD163+ macrophages from the indicated gut areas of control samples and samples obtained from Crohn’s disease and ulcerative colitis patients. In each case, the background-subtracted PHD3 mean fluorescence intensity (in arbitrary units [a.u.]) from 100 randomly chosen CD163+ cells/sample (from four different ×20 fields) was plotted and statistically processed (two-way ANOVA). (E) Confocal sections of samples from kidney (left and middle panel) and breast (right panel) tumors after double immunofluorescence analysis for the macrophage-specific marker CD163 (green) and PHD3 (red). Colocalization of both markers yields yellow staining. When indicated, nuclei were counterstained with DAPI (blue). (F) Quantitation of PHD3 staining in CD163+ macrophages from the indicated tumor samples. In each case, the background-subtracted PHD3 mean fluorescence intensity (in a.u.) from 150 randomly chosen CD163+ cells/sample (from four different ×20 fields) was plotted.

PHD3 is expressed by tumor-associated macrophages with low levels of M2-specific markers

The pattern of PHD3 expression was also evaluated in tumor-associated macrophages (TAM), for which polarization critically influences tumor progression and metastasis (30). Immunohistochemical analysis revealed PHD3 expression in macrophages from tumors of three different origins (kidney, breast, and colon) (Fig. 3E). In CD163+ TAM, quantitative analysis indicated a heterogeneous expression of PHD3, which ranged from negative to strongly positive (Fig. 3F). A similar result was observed upon analysis of the PHD3 expression in melanoma TAM. A high PHD3 expression was detected in CD68+ macrophages primarily located within melanoma tumor cell clusters and exhibiting a weak expression of CD163 (Fig. 4A). Conversely, high expression of CD163 was detected in macrophages excluded from the tumor cell nests and that also exhibited a high level of expression of the FRβ (Fig. 4B). Like CD163, FRβ expression was primarily restricted to CD68+ macrophages surrounding melanoma cell nests (Fig. 4B). In fact, when coexpression of PHD3 and FRβ was compared, their respective pattern of staining was found to be almost opposite, as PHD3 expression was limited to CD68+FRβ− macrophages that reside within the tumor cell clusters (Fig. 4B). Quantitative analysis of the expression of FRβ and PHD3 in four independent melanoma samples confirmed the negative correlation of their respective expression levels in CD68+ macrophages (Fig. 4C). Therefore, the expression of PHD3 in tumor-associated human macrophages is limited to a macrophage subset that is devoid of anti-inflammatory M2 markers.

FIGURE 4.
FIGURE 4.

EGLN3-encoded PHD3 is expressed by tumor-associated macrophages lacking M2 markers (CD163, FRβ). (A) Confocal sections of a metastatic melanoma sample after triple immunofluorescence analysis of the melanoma marker high m.w. melanoma-associated Ag (HMW-MAA) (white), the macrophage-specific marker CD68 (white), CD163 (green), and PHD3 (red). Magnification of a CD163/CD68 and PHD3 colocalizing area appear enlarged in the bottom panel. In all cases, “T” and “S” indicate the melanoma nests and stroma devoid of tumor cells, respectively. When indicated, nuclei were counterstained with DAPI (blue). (B) Magnifications of an area at the tumor nests/stroma boundary stained with anti-CD163 (white), anti-FRβ (green), and anti-PHD3 (red) Abs and with nuclei counterstained with DAPI (blue). (C) Correlation between FRβ and PHD3 expression levels in CD163+ macrophages from four independent samples of s.c. melanoma metastases. In each case, five fields (750 × 750 μm2) were analyzed, and the background-subtracted mean fluorescence intensity (in arbitrary units [a.u.]) from 360 cells was plotted and statistically processed.

PHD3 expression in macrophages is regulated by activin A

To determine the molecular basis for the restricted expression of PHD3 in proinflammatory macrophages, we first analyzed whether M1(GM-CSF) macrophage-derived soluble factors contribute to EGLN3 expression. As shown in Fig. 5A, M1(GM-CSF)-conditioned medium triggered a dramatic induction of EGLN3 expression in monocytes after 72 h, implying that the differential expression of EGLN3 in M1(GM-CSF) and M2(M-CSF) macrophages might be caused by soluble factors released along M1(GM-CSF) polarization. Because activin A is actively produced by M1(GM-CSF) macrophages, where it constitutes a polarization marker (29), we next evaluated whether activin A had an influence on the M1(GM-CSF)-specific expression of EGLN3. The presence of a blocking Ab against activin A reduced by 50% the acquisition of EGLN3 expression in monocytes exposed to GM-CSF–free M1(GM-CSF)-conditioned medium (Fig. 5B). Furthermore, EGLN3 expression levels were significantly reduced when M1(GM-CSF) macrophages were generated in the presence of inhibitors of TGF-β/activin/nodal type I receptors ALK5/ALK4/ALK7 SB431542 (Fig. 5C) or A-83 (Fig. 5D) or in the presence of a neutralizing anti-activin A Ab (Fig. 5E). Moreover, activin A (25 ng/ml) significantly increased the expression of EGLN3 in M2(M-CSF) macrophages after a 48-h treatment (Fig. 5F) and also enhanced the activity of EGLN3 gene regulatory regions in a heterologous cellular system (Fig. 5G). Taken together, these results indicate that activin A promotes EGLN3 gene expression in M1(GM-CSF) macrophages and contributes to the differential expression of EGLN3 in proinflammatory and anti-inflammatory monocyte-derived human macrophages.

FIGURE 5.
FIGURE 5.

Activin A regulates the expression of the EGLN3-encoded PHD3 prolyl hydroxylase in M1(GM-CSF) macrophages. (A) EGLN3 gene expression in untreated monocytes (Mo) and monocytes exposed for 72 h to the indicated concentrations of M1(GM-CSF)-conditioned medium, as measured by qRT-PCR. Results are expressed as relative expression (relative to GAPDH RNA levels) and referred to the EGLN3 mRNA levels in monocytes cultured for 72 h in the absence of conditioned medium (0%). A representative experiment (out of two) is shown. (B) EGLN3 gene expression in monocytes exposed for 72 h to RPMI 1640 or the indicated concentrations of GM-CSF–free M1(GM-CSF)-conditioned medium and pretreated (1 h) with either an anti-activin A Ab (0.1 μg/ml) or an isotype-matched Ab (Control IgG, 0.1 μg/ml). EGLN3 expression was measured by qRT-PCR, and results are expressed as relative expression (relative to GAPDH RNA levels) and referred to the EGLN3 mRNA levels in monocytes kept in RPMI (0%). A representative experiment (out of two) is shown. EGLN3 expression in M1(GM-CSF) macrophages generated in the presence of DMSO, SB431542 (C), or A-83 (D), as determined by qRT-PCR. Results are expressed as relative expression (relative to GAPDH RNA levels) and referred to the EGLN3 mRNA levels in macrophages generated in the presence of DMSO. In (C), mean and SD of four independent experiments are shown (***p < 0.0001). Two independent experiments are shown in (D). (E) EGLN3 expression in M1(GM-CSF)-macrophages generated in the presence of either a neutralizing anti-activin A Ab (anti-activin A) or an isotype-matched irrelevant Ab (IgG), as determined by qRT-PCR. Results are expressed as relative expression (relative to GAPDH RNA levels) and referred to the EGLN3 mRNA levels in macrophages generated in the presence of control IgG. Two independent experiments were performed, and one of them is shown. (F) EGLN3 expression in M2(M-CSF) either untreated or exposed for 48 h to activin A (25 ng/ml), as determined by qRT-PCR. Results are expressed as relative expression (relative to GAPDH RNA levels) and referred to the EGLN3 mRNA levels in untreated M2(M-CSF) macrophages. Mean and SD of three independent experiments are shown (*p < 0.05). (G) Transcriptional activity of the pGL3-EGLN3PEnhA-Luc reporter construct transfected in Mv1Lu cells and incubated in the absence or in the presence of activin A (25 μg/ml). For normalization, cells were cotransfected with the RSV-βgal expression plasmid, and results are presented as relative promoter activity, which indicates luciferase activity per unit of β-galactosidase activity for each assay condition. Mean ± SD of three independent experiments are shown (***p < 0.001).

Activin A mediates the hypoxia-induced expression of EGLN3 in M2(M-CSF) macrophages

EGLN3 codes for the PHD3 prolyl hydroxylase oxygen sensor (16), and its expression is induced under hypoxic conditions in an HIF-dependent manner (31). The differential expression of EGLN3 in M1(GM-CSF) and M2(M-CSF) macrophages under normoxic conditions prompted us to analyze its expression in both macrophage types under low oxygen tensions. Surprisingly, hypoxia (1% O2, 24 h) did not alter EGLN3 expression in M1(GM-CSF) macrophages (Fig. 6A). However, hypoxia significantly increased the EGLN3 gene expression level in M2(M-CSF) macrophages, whereas it led to a slight downregulation of EGLN1 and EGLN2 gene expression (Fig. 6A). This result implies that the expression of the EGLN genes is distinctly regulated by hypoxia between M1(GM-CSF) and M2(M-CSF) and, more importantly, that both macrophages exhibit a distinct ability to respond to hypoxia.

FIGURE 6.
FIGURE 6.

Activin A mediates the hypoxia-triggered upregulation of EGLN3 gene expression in M2(M-CSF) macrophages. (A) Relative mRNA expression of EGLN3, EGLN2, and EGLN1 in M2(M-CSF) and M1(GM-CSF) macrophages maintained for 24 h under normoxia (Nx; 95% O2) or hypoxia (Hpx; 1% O2), as measured by qRT-PCR. In the case of EGLN3, mean and SD of three independent experiments are shown (***p < 0.001). For EGLN1 and EGLN2, one representative experiment (out of two performed) is shown. Results are expressed as relative expression (relative to GAPDH RNA levels) and referred to the mRNA level of each gene in M2(M-CSF) macrophages maintained in normoxia. (B) Relative mRNA expression of INHBA in M2(M-CSF) and M1(GM-CSF) macrophages maintained for 24 h under normoxia (Nx; 95% O2) or hypoxia (Hpx; 1% O2), as measured by qRT-PCR. Results are expressed as relative expression (relative to GAPDH RNA levels, log scale) and referred to the INHBA mRNA level in M2(M-CSF) macrophages maintained in normoxia. Mean and SD of three independent experiments are shown (*p < 0.05). (C) Activin A protein levels secreted by M2(M-CSF) (left panel) and M1(GM-CSF) (right panel) macrophages exposed to either normoxia or hypoxia for 24 h, as measured by ELISA. Shown are the mean and STD of five independent experiments (*p < 0.05). (D) Relative mRNA expression of EGLN3 in M2(M-CSF) macrophages exposed to either normoxia or hypoxia for 24 h and in the absence or in the presence of DMSO, SB431542 (10 nM), isotype-matched Ab (IgG), or a blocking anti-activin A Ab (0.1 μg/ml). Results are expressed as relative expression (relative to GAPDH RNA levels) and referred to EGLN3 mRNA levels in M2(M-CSF) macrophages exposed to DMSO under normoxia. Mean and SD of three independent experiments are shown (***p < 0.001).

Because activin A directs the expression of EGLN3 in M1(GM-CSF) macrophages and induces EGLN3 gene expression in M2(M-CSF) macrophages (Fig. 5), we hypothesized that activin A might also mediate the hypoxia-dependent induction of EGLN3 expression in M2(M-CSF) macrophages. Determination of activin A levels in both macrophage subtypes after exposure to 1% O2 for 24 h revealed that hypoxia significantly induced the expression of the INHBA gene (Fig. 6B) as well as the secretion of activin A in M2(M-CSF) macrophages (Fig. 6C). By contrast, hypoxia had no relevant effect on INHBA expression or activin A release from M1(GM-CSF) macrophages (Fig. 6B, 6C). To fully test the above hypothesis, we evaluated the influence of a blocking anti-activin A Ab or the SB431542 inhibitor on the hypoxia-mediated upregulation of EGLN3 gene expression in M2(M-CSF) markers. The hypoxia-driven EGLN3 upregulation was significantly reduced in the presence of either anti-activin A or SB431542 (Fig. 6D). Therefore, activin A critically regulates EGLN3 expression in macrophages, mediating both its constitutive expression in proinflammatory M1(GM-CSF) macrophages and its hypoxia-driven upregulation in M2(M-CSF) macrophages.

Discussion

In the present report, we describe the preferential expression of the PHD3 prolyl hydroxylase-encoding EGLN3 gene by human proinflammatory macrophages in vivo and in vitro and that PHD3 expression is restricted to TAM that exhibit weak or absent levels of typical anti-inflammatory/M2 markers. Besides, we demonstrate that activin A regulates the constitutive expression of PHD3 in M1(GM-CSF) macrophages and its hypoxia-inducible expression in M2(M-CSF) macrophages. Considering that activin A production is an exclusive property of proinflammatory M1(GM-CSF) macrophages (29), the activin A–PHD3 axis appears to constitute a specific signature of proinflammatory macrophage polarization. Moreover, because activin A expression is upregulated by hypoxia in M2(M-CSF) macrophages (Fig. 6), the activin A–PHD3 axis might also influence macrophage effector functions under low oxygen pressures, a condition that is common to solid tumors and tissues undergoing active inflammatory responses (32).

The hypoxia-inducible expression of EGLN3 in M2(M-CSF) macrophages is in agreement with results in most cell types (33) and with the presence of a functional hypoxia-responsive element within the first intron of the EGLN3 gene (25). However, the presence of PHD3 in M1(GM-CSF) macrophages in vitro must reflect the existence of hypoxia-independent regulatory mechanisms controlling EGLN3 gene expression, as HIF factors are not detectable in M1(GM-CSF) macrophages under normoxic conditions (data not shown). The presence of PHD3+ macrophages in lung (Fig. 2), where partial pressure of O2 ranges between 150 and 100 mmHg, also supports the hypothesis of such a hypoxia-independent mechanism for regulation of PHD3 expression. Our results indicate that activin A complies with this requirement because it: 1) potentiates the activity of the EGLN3 gene regulatory region; 2) positively regulates EGLN3 expression in M1(GM-CSF) macrophages independently of low oxygen pressure; and 3) mediates the hypoxia-inducible EGLN3 gene expression in M2(M-CSF) macrophages. Therefore, unlike promoter hypermethylation or miR-20a, which inhibit EGLN3 gene expression in tumor cells (34) and cardiomyocytes (35), activin A represents a novel mechanism for positive regulation of EGLN3 expression. Along this line, soluble growth factors may turn out to exert an important control of the hypoxia-independent expression of prolyl hydroxylases in myeloid cells, as EGLN1 gene expression is upregulated by leukemia inhibitory factor in murine osteoclasts (36). In contrast, the contribution of activin A to EGLN3 gene transcription, as well as the hypoxia-inducible expression of activin A in M2(M-CSF) macrophages, is reminiscent of the reciprocal relationships that exist between TGF-β–related factors and hypoxia-regulated signaling and transcription. Besides the synergistic cooperation between hypoxia and TGF-β signaling pathways (37), activin A expression is enhanced in murine cortical neurons in response to ischemia and hypoxia (38), and HIF1α enhances activin A-dependent signaling in murine embryonic stem cells (39).

Huge differences exist between the expression profiles of polarized macrophages of human or mouse origin (40). A recent report has conclusively demonstrated such a difference by comparing the transcriptome of bone marrow-derived murine- and monocyte-derived human macrophages polarized by either GM-CSF or M-CSF (41). Interestingly, the expression of the Egln3 gene has also been found to differentiate between M1(GM-CSF) and M2(M-CSF) bone marrow-derived macrophages, with a significantly higher Egln3 expression in macrophages polarized by GM-CSF (ArrayExpress Archive, http://www.ebi.ac.uk/arrayexpress/, accession number E-MTAB-791). Therefore, the lack of expression of the PHD3-encoding EGLN3 gene appears to correlate with an anti-inflammatory macrophage phenotype. In addition to its identification as a marker for in vitro-generated M1(GM-CSF) macrophages, the relevance of PHD3 expression in macrophages is underscored by the presence of PHD3+ macrophages under homeostatic conditions (e.g., lung), in inflammatory conditions (Crohn’s disease and ulcerative colitis), and within tumor areas. A low percentage of PHD3+ intestinal macrophages was detected in normal noninflamed gut, where macrophages exhibit constitutive IL-10 production and promotion of regulatory T cell responses (42), whereas a high proportion of CD163+PHD3+ macrophages was observed in inflamed gut (Fig. 3). PHD3 expression in macrophages from inflamed tissues can potentially be driven by hypoxia and/or activin A. However, based on the role of activin A in inflammatory responses (43, 44) and its ability to support proinflammatory macrophage polarization and limit the acquisition of anti-inflammatory markers like IL-10 (29), it is tempting to speculate that activin A contributes to PHD3 expression in macrophages within inflamed gut and that macrophage EGLN3 gene expression identifies macrophages exhibiting an M1-skewed proinflammatory polarization. If so, and despite the difficulties in extrapolating polarization marker expression between the human and murine systems (40), our results would be in agreement with the proposed role of PHDs as positive regulators of the LPS-induced inflammatory process (45) and the link between HIF1α induction and M1 polarization (6). The correlation between PHD3 and macrophage M1 polarization is further supported by the restriction of PHD3 expression to a TAM subset that exhibits a low level of M2 polarization-associated markers and for which location differed from that of M2-polarized macrophages (highly positive for CD163 and FRβ). Based on the pro-M2 polarization ability of tumor-conditioned media (46), it seems reasonable to assume that PHD3+ macrophages would give rise to FRβ+ TAM under the influence of tumor-derived factors like M-CSF. Regardless of the ontogenic relationships between both TAM subsets, the appearance of two subsets of CD68+CD163+ TAM macrophages differing in their expression of M2 (FRβ) and M1 (PHD3) polarization markers is compatible with the heterogeneity and the M1/M2 mixed polarization of TAM in murine models of cancer (47, 48), in which M2-like TAMs are enriched within hypoxic areas (48).

As a defining hallmark of the myeloid cell lineage, plasticity allows macrophages to adjust their effector functions to their surrounding environment (49). In response to hypoxic conditions, macrophages modulate their metabolism (50, 51), effector functions in inflammatory responses (52), and ability to handle tumor progression or promote T cell stimulation (53). Because PHD3 regulates HIF protein stability (54) and is itself an HIF target gene (25), it can be hypothesized that the differential EGLN3 gene expression in M1(GM-CSF) and M2(M-CSF) macrophages may underlie a distinct response of both macrophage subtypes to hypoxia. In this regard, it is worth noting that, unlike their M2(M-CSF) counterparts, PHD3-expressing M1(GM-CSF) macrophages exhibited a defective response to hypoxia, at least in terms of EGLN3 and INHBA/activin A upregulation. Therefore, constitutive PHD3 expression appears associated to a certain state of hypoxia resistance that resembles the effects of chronic hypoxia (PHD’s overactivation and desensitization of HIFα) (55). As in the case of exposure to chronic hypoxia, constitutively elevated PHD3 expression might allow macrophages to exert their effector functions in a more efficient manner, protecting them from necrosis (55) or, as in the case of neutrophils (56), enhancing their survival during hypoxia. In this case, the activin A-regulated expression of PHD3 in macrophages would constitute an additional weapon for macrophages to efficiently fight against pathogenic stimuli.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. Luis del Peso for invaluable help with reporter assays and useful suggestions.

Footnotes

  • This work was supported by grants from Ministerio de Economía y Competitividad (BFU2008-01493-BMC and SAF2011-23801), Genoma España (Molecular and Cellular Mechanisms in Chronic Inflammatory and Autoimmune Diseases project), Instituto de Salud Carlos III (Spanish Network for the Research in Infectious Diseases RD06/0008 and Red de Investigación en SIDA RD06/0006/1016), Comunidad Autónoma de Madrid/Fondo Europeo de Desarrollo Regional (Rheumatoid Arthritis: Physiopathology Mechanisms and Identification of Potential Therapeutic Targets Program) (to A.L.C.), Grant SAF2010-1602 from the Ministerio de Ciencia e Innovacion (to P.S.-M.), and Grant Agreement 229673 from the European Community’s Seventh Framework Programme (FP7/2009-2013). M.M.E. is supported by a Sara Borrell postdoctoral contract from Instituto de Salud Carlos III (CD09/00386).

  • Abbreviations used in this article:

    FRβ
    folate receptor β
    HIF
    hypoxia-inducible factor
    PHD3
    prolyl hydroxylase 3
    qRT-PCR
    quantitative real-time RT-PCR
    TAM
    tumor-associated macrophage.

  • Received April 10, 2012.
  • Accepted June 9, 2012.

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