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

This information is current as María M. Escribese, Elena Sierra-Filardi, Concha Nieto, of September 25, 2021. Rafael Samaniego, Carmen Sánchez-Torres, Takami Matsuyama, Elisabeth Calderon-Gómez, Miguel A. Vega, Azucena Salas, Paloma Sánchez-Mateos and Angel L. Corbí J Immunol published online 9 July 2012

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 9, 2012, doi:10.4049/jimmunol.1201064 The Journal of Immunology

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

Marıá M. Escribese,* Elena Sierra-Filardi,* Concha Nieto,* Rafael Samaniego,† Carmen Sańchez-Torres,‡ Takami Matsuyama,x Elisabeth Calderon-Go´mez,{ Miguel A. Vega,* Azucena Salas,{ Paloma Sańchez-Mateos,† and Angel L. Corbı´*

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 Downloaded from 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 http://www.jimmunol.org/ 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. The Journal of Immu- nology, 2012, 189: 000–000.

he extreme sensitivity of macrophages to endogenous existence of a plethora of macrophage polarization states is critical (e.g., cytokines) and exogenous (e.g., pathogens) stimuli for the adequate onset, regulation, and resolution of immune and T explains their phenotypic and functional heterogeneity inflammatory responses (2). As a representative example, although by guest on September 25, 2021 under homeostatic and pathological conditions (1). In fact, the 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 *Laboratorio de Ce´lulas Mieloides, Centro de Investigaciones Biolo´gicas, Consejo Superior de Investigaciones Cientı´ficas, Madrid 28040, Spain; †Hospital General exhibit high Ag-presenting capacity and produce proinflammatory Universitario Gregorio Maranõń, Madrid 28007, Spain; ‡Departamento de Biomedi- cytokines in response to LPS (3, 4). Conversely, M-CSF generates cina Molecular, Centro de Investigacioń y de Estudios Avanzados del Instituto Poli- x macrophages with high phagocytic activity and IL-10–producing tećnico Nacional, Mexico DF 07360, Mexico; Department of Immunology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890- ability in response to pathogens (3, 4). Based on their respective 8544, Japan; and {Institut d’Investigacions Biome`diques August Pi i Sunyer, Centre cytokine profiles, human macrophages generated in the presence Esther Koplowitz, Barcelona 08036, Spain of GM-CSF or M-CSF are representative of the classical or al- Received for publication April 10, 2012. Accepted for publication June 9, 2012. ternative macrophage polarization states, respectively, and are This work was supported by grants from Ministerio de Economıá y Competitividad considered as proinflammatory or anti-inflammatory macrophages (BFU2008-01493-BMC and SAF2011-23801), Genoma Espanã (Molecular and Cel- lular Mechanisms in Chronic Inflammatory and Autoimmune Diseases project), Insti- (3, 5). tuto de Salud Carlos III (Spanish Network for the Research in Infectious Diseases Variation in the levels of oxygen is a parameter that also RD06/0008 and Red de Investigacioń en SIDA RD06/0006/1016), Comunidad determines the state of macrophage polarization (6). In healthy Autońoma de Madrid/Fondo Europeo de Desarrollo Regional (Rheumatoid Arthritis: Physiopathology Mechanisms and Identification of Potential Therapeutic Targets tissues, oxygen levels span from 150 mmHg in the lung to 40–100 Program) (to A.L.C.), Grant SAF2010-1602 from the Ministerio de Ciencia e Inno- mmHg in the circulation and 4–20 mmHg in the tissues (7). vacion (to P.S.-M.), and Grant Agreement 229673 from the European Community’s Pathologic hypoxia, commonly found in malignant solid tumors, 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). inflammatory lesions, and healing wounds (8–10), also influences M.M.E., E.-S.F., C.N., R.S., and C.S.-T. performed research and analyzed data; T.M., macrophage polarization and leads to transcriptional and meta- M.A.V., and P.S.-M. contributed vital reagents; and M.M.E. and A.L.C. designed the bolic changes (6) and the acquisition of effector functions to research, analyzed data, and wrote the paper. promote angiogenesis and restore tissue homeostasis (7, 11, 12). Address correspondence and reprint requests to Dr. Marıá M. Escribese and Dr. Macrophages accumulate in large numbers in damaged hypoxic Angel L. Corbı´, Centro de Investigaciones Biolo´gicas, Consejo Superior de Inves- tigaciones Cientı´ficas, Ramiro de Maeztu 9, Madrid 28040, Spain. E-mail addresses: tissues and respond to hypoxia by altering their gene expression [email protected] and [email protected] program via upregulation of the hypoxia-inducible factor (HIF) 1 Abbreviations used in this article: FRb, folate receptor b; HIF, hypoxia-inducible (HIF1a/HIF1b)andHIF2(HIF2a/HIF1b) transcription factors factor; PHD3, prolyl hydroxylase 3; qRT-PCR, quantitative real-time RT-PCR; TAM, (13, 14). In the presence of oxygen, both a subunits are prolyl- tumor-associated macrophage. hydroxylated, recognized by the von Hippel-Lindau tumor Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 suppressor protein (15–17), and degraded by the ubiquitin-

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201064 2 PHD3 EXPRESSION IN PROINFLAMMATORY MACROPHAGES proteasome pathway (17). The three HIF prolyl hydroxylases medium for 48 h in the absence of GM-CSF, after which the conditioned (PHD1–3) (16, 18) require molecular oxygen as a cosubstrate and, medium was collected. Cells were routinely cultured in 21% O2 and 5% therefore, constitute the link between oxygen availability and HIF- CO2 (normoxic conditions). For hypoxic conditions, cells were placed in an in vivo 400 hypoxia Work Station (Ruskinn Technology) that was independent transcription (19). fused with a mixture of 1% O2,5%CO2, and 94% N2 (Sociedad Espanõla The prolyl hydroxylase PHD3 is encoded by the EGLN3 gene, de Carburos Meta´licos) for 24 h and treated or not with the activin for which expression is upregulated in platelet-derived growth receptor-like kinase (ALK)4/5/7 inhibitor SB431542 (Sigma-Aldrich) or factor-stimulated rat fibroblasts (20). PHD3 is involved in nerve- an anti-activin A-blocking Ab (R&D Systems). growth factor-dependent survival of neurons (21), stimulates py- Quantitative real-time RT-PCR ruvate kinase M2 coactivation for HIF1 (22), and also participates Oligonucleotides for selected genes were designed according to the Roche in HIF-independent processes to control cell death, changes in software for quantitative real-time PCR (Roche Diagnostics). Total RNAwas metabolism (23), and IKKb/NF-kB signaling (24). In the current extracted using the RNeasy kit (Qiagen), retrotranscribed, and individually study, we demonstrate that proinflammatory macrophages con- amplified cDNA were quantified using the Universal Human Probe Roche stitutively express the EGLN3-encoded PHD3 prolyl hydroxylase library (Roche Diagnostics). Assays were made in triplicate and results normalized according to the expression levels of GAPDH RNA. Results in vitro and in vivo and that activin A regulates EGLN3 gene were expressed using the DD threshold cycle method for quantification. expression both under normoxic and hypoxic conditions. ELISA Materials and Methods Macrophage supernatants from M1(GM-CSF) or M2(M-CSF) macrophages Generation of human monocyte-derived 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. Downloaded from Human PBMCs were isolated from buffy coats from normal donors over a Lymphoprep (Nycomed Pharma, Oslo, Norway) gradient according to Western blot standard procedures. Monocytes were purified from PBMC by magnetic cell sorting using CD14+ microbeads or the CD16+ Monocyte Isolation Kit Cell lysates were obtained in 10 mM Tris-HCl (pH 8), 150 mM NaCl, 1% (Miltenyi Biotec, Bergisch Gladbach, Germany). Monocytes (0.5 3 106 Nonidet P-40 lysis buffer containing 2 mM Pefabloc, 2 mg/ml aprotinin/ cells/ml, .95% CD14+ cells) were cultured in RPMI 1640 supplemented antipain/leupeptin/pepstatin, 10 mM NaF, and 1 mM Na3VO4.Ten with 10% FCS (completed medium) for 7 d at 37˚C in a humidified at- micrograms cell lysate was subjected to SDS-PAGE and transferred onto an Immobilon polyvinylidene difluoride membrane (Millipore). Protein mosphere with 5% CO2 and containing 1000 U/ml GM-CSF or M-CSF (10 http://www.jimmunol.org/ ng/ml; ImmunoTools, Friesoythe, Germany) to generate M1(GM-CSF) or detection was carried out using an anti-human PHD3 polyclonal Ab M2(M-CSF) monocyte-derived macrophages, respectively. Cytokines were (Abcam, Cambridge, U.K.) or an mAb against GAPDH (sc-32233; Santa added every 2 d. Where indicated, M1(GM-CSF) or M2(M-CSF) macro- Cruz Biotechnology, Santa Cruz, CA). phages were treated with either IFN-g (500 U/ml; R&D Systems, Min- Reporter gene assays neapolis, 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 Mv1Lu cells were plated in 24-well plates at 1 3 106 cells/ml in RPMI extensively washed to remove residual GM-CSF and cultured in complete 1640 with 10% FCS. The luciferase-based plasmid pGL3-EGLN3PEnhA, by guest on September 25, 2021

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+CD162 or CD142CD16+ 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+ CD162 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 (2) or stimulated for 48 h with IFN-g 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 FRb (green). No staining was observed upon incubation with isotype-matched irrelevant Abs as negative controls. Nuclei were counterstained with DAPI. The Journal of Immunology 3 which contains the firefly luciferase gene under the control of the EGLN3 macrophages lacked PHD3 expression (Fig. 1F). The M1(GM- gene promoter and enhancer, has been previously described (25). Cells CSF) macrophage-restricted expression of PHD3 was further were transiently transfected with 2 mg DNA of the reporter construct using evidenced upon costaining with an mAb specific for the FRb, for Superfect (Qiagen). Twenty to 24 h after transfection, cells were treated with 25 ng/ml human recombinant activin A (PeproTech) for 3 h. To which expression is only found in M2(M-CSF) macrophages (29). normalize transfection efficiency, cells were cotransfected with an SV40 As shown in Fig. 1F, M1(GM-CSF) macrophages exhibited a promoter-based b-galactosidase expression plasmid (RSV-bgal). Mea- PHD3+FRb2 phenotype, whereas M2(M-CSF) macrophages were surement of relative luciferase units and b-galactosidase activity was PHD32FRb+. Altogether, these results demonstrate the link be- performed using the Luciferase Assay System (Promega) and the Galacto- Ligth kit (Tropix), respectively. tween expression of the PHD3-coding EGLN3 gene and proinflammatory macrophage polarization, a relationship that was also Confocal microscopy and immunohistochemistry observed in murine bone marrow-derived M1 and M2 macro- Human tissue samples were obtained from patients undergoing surgical phages (data not shown). treatment and following the medical ethics committee procedures (Hospital General Universitario Gregorio Maranõń, Hospital Clıńic de Barcelona). PHD3 expression in macrophages under homeostatic and Histopathologic diagnosis was confirmed for each specimen. Acetone- inflammatory conditions fixed thick sections (4 mm in depth) of cryopreserved tissue were first blocked for 5 min with human Igs and then sequentially incubated with 1– To determine whether EGLN3 gene expression could be detected 5 mg/ml primary Abs (high m.w. melanoma-associated Ag, BD Pharmin- in macrophages in vivo, normal human tissues were tested for gen; FITC-conjugated anti-CD163, Santa Cruz Biotechnology; anti-PHD3, PHD3-specific reactivity by immunohistochemistry. An extremely Abcam; anti-CD68, clone PG-M1, DakoCytomation; and anti-FRb) (26) low percentage of skin and tonsil CD163+ macrophages were followed by addition of their corresponding fluorescent secondary Abs. found to express PHD3 (Fig. 2A). Conversely, a moderate per- Downloaded from Tissue imaging was performed with a confocal microscope (SP2; Leica + Microsystems). For panoramic images, 103 PL-APO NA 0.4 and glycerol centage of alveolar CD163 macrophages exhibited PHD3 reac- immersion 203 PL-APO NA 0.7 objectives were used at 1.5 Airy of tivity (Fig. 2B), indicating that human macrophages can also pinhole aperture. For more detailed images, the 633 PL-APO NA 1.3 express PHD3 under homeostatic conditions in vivo. In the case of glycerol immersion objective was used. For in vitro cell imaging, cells the gut, a similar picture emerged under homeostatic conditions, 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 as a very low percentage of PHD3 CD163 macrophages were

fluorescence intensities were measured in regions of interest manually detected in the colon, regardless of the region analyzed (Q7, Q8, http://www.jimmunol.org/ depicted around CD163+ cells using the LCS 15.37 software from Leica ascending; Q15, sigmoid) (Fig. 3A). However, a higher number of Microsystems. Images were processed with Adobe Photoshop and Illus- PHD3+ macrophages were detected in CD163+ macrophages from trator CS (Adobe Systems). Correlation analysis was performed using the the ileum of patients with Crohn’s disease (Fig. 3B) and from the SPSS 11.5 software (SPSS). colon of patients with ulcerative colitis, independently of colon Statistical analysis area analyzed (Q5, transverse; Q14, Q16, rectum) (Fig. 3C). To Statistical analysis was performed using Student t test, and a p value ,0.05 quantitate the level of PHD3 in macrophages in noninflamed and was considered significant. A two-way ANOVA test was performed for inflamed colon, individual CD163+ macrophages were identified analysis of immunohistochemistry results. by guest on September 25, 2021 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) mac- + 2 rophages 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-g 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 FIGURE 2. Expression of PHD3 in macrophages from noninflamed PHD3 hydroxylase in both macrophage subtypes was verified by tissues. (A) Confocal sections of human tonsil (top panels) and skin Western blot, as PHD3 was expressed at high levels in lysates (bottom panels) after double immunofluorescence analysis for the macrophage-specific marker CD163 (green) and PHD3 (red). In the left panels, from M1(GM-CSF) macrophages, whereas it was barely detect- nuclei were counterstained with DAPI (blue). In the middle panels, the able in M2(M-CSF) macrophages (Fig. 1E). Moreover, immuno- boundary between dermis and epidermis (skin) and T and B cell areas of chemical analysis of in vitro-generated macrophages confirmed the follicle (tonsil) are indicated. (B) Confocal sections of human lung after + that proinflammatory CD68 M1(GM-CSF) macrophages con- double immunofluorescence analysis for the macrophage-specific marker tained high levels of PHD3, with a predominantly cytoplasmic CD163 (green) and PHD3 (red) or a control rabbit antiserum (rbIgG, red). subcellular distribution (Fig. 1F). By contrast, CD68+ M2(M-CSF) In the left and middle panels, nuclei were counterstained with DAPI (blue). 4 PHD3 EXPRESSION IN PROINFLAMMATORY MACROPHAGES Downloaded from http://www.jimmunol.org/

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 by guest on September 25, 2021 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 320 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 320 fields) was plotted. and their PHD3 staining measured. As shown in Fig. 3D, CD163+ pression of CD163 was detected in macrophages excluded from macrophages from inflamed tissues exhibit significantly higher the tumor cell nests and that also exhibited a high level of ex- intensity of PHD3 staining, thus demonstrating that PHD3+ pression of the FRb (Fig. 4B). Like CD163, FRb expression was CD163+ macrophages are found in inflamed gut and that PHD3 primarily restricted to CD68+ macrophages surrounding mela- levels are higher in macrophages within an inflammatory envi- noma cell nests (Fig. 4B). In fact, when coexpression of PHD3 and ronment. FRb was compared, their respective pattern of staining was found to be almost opposite, as PHD3 expression was limited to CD68+ PHD3 is expressed by tumor-associated macrophages with low FRb2 macrophages that reside within the tumor cell clusters (Fig. levels of M2-specific markers 4B). Quantitative analysis of the expression of FRb and PHD3 in The pattern of PHD3 expression was also evaluated in tumor- four independent melanoma samples confirmed the negative cor- associated macrophages (TAM), for which polarization critically relation of their respective expression levels in CD68+ macro- influences tumor progression and metastasis (30). Immunohisto- phages (Fig. 4C). Therefore, the expression of PHD3 in tumor- chemical analysis revealed PHD3 expression in macrophages from associated human macrophages is limited to a macrophage subset tumors of three different origins (kidney, breast, and colon) (Fig. that is devoid of anti-inflammatory M2 markers. 3E). In CD163+ TAM, quantitative analysis indicated a heteroge- neous expression of PHD3, which ranged from negative to PHD3 expression in macrophages is regulated by activin A strongly positive (Fig. 3F). A similar result was observed upon To determine the molecular basis for the restricted expression of analysis of the PHD3 expression in melanoma TAM. A high PHD3 in proinflammatory macrophages, we first analyzed whether PHD3 expression was detected in CD68+ macrophages primarily M1(GM-CSF) macrophage-derived soluble factors contribute located within melanoma tumor cell clusters and exhibiting to EGLN3 expression. As shown in Fig. 5A, M1(GM-CSF)- a weak expression of CD163 (Fig. 4A). Conversely, high ex- conditioned medium triggered a dramatic induction of EGLN3 The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. EGLN3-encoded PHD3 is expressed by tumor-associated macrophages lacking M2 markers (CD163, FRb). (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-FRb (green), and anti-PHD3 (red) Abs and with nuclei counterstained with DAPI (blue). (C) Correlation between FRb and PHD3 expression levels in CD163+ macrophages from four independent samples of s.c. melanoma metastases. In each case, five fields (750 3 750 mm2) were analyzed, and the background- subtracted mean fluorescence intensity (in arbitrary units [a.u.]) from 360 cells was plotted and statistically processed. expression in monocytes after 72 h, implying that the differential nificantly increased the expression of EGLN3 in M2(M-CSF) expression of EGLN3 in M1(GM-CSF) and M2(M-CSF) macro- macrophages after a 48-h treatment (Fig. 5F) and also enhanced phages might be caused by soluble factors released along M1 the activity of EGLN3 gene regulatory regions in a heterologous (GM-CSF) polarization. Because activin A is actively produced cellular system (Fig. 5G). Taken together, these results indicate by M1(GM-CSF) macrophages, where it constitutes a polarization that activin A promotes EGLN3 gene expression in M1(GM-CSF) marker (29), we next evaluated whether activin A had an influence macrophages and contributes to the differential expression of on the M1(GM-CSF)-specific expression of EGLN3. The presence EGLN3 in proinflammatory and anti-inflammatory monocyte- of a blocking Ab against activin A reduced by 50% the acquisition derived human macrophages. of EGLN3 expression in monocytes exposed to GM-CSF–free M1 (GM-CSF)-conditioned medium (Fig. 5B). Furthermore, EGLN3 Activin A mediates the hypoxia-induced expression of EGLN3 expression levels were significantly reduced when M1(GM-CSF) in M2(M-CSF) macrophages macrophages were generated in the presence of inhibitors of TGF- EGLN3 codes for the PHD3 prolyl hydroxylase oxygen sensor b/activin/nodal type I receptors ALK5/ALK4/ALK7 SB431542 (16), and its expression is induced under hypoxic conditions in an (Fig. 5C) or A-83 (Fig. 5D) or in the presence of a neutralizing HIF-dependent manner (31). The differential expression of EGLN3 anti-activin A Ab (Fig. 5E). Moreover, activin A (25 ng/ml) sig- in M1(GM-CSF) and M2(M-CSF) macrophages under normoxic 6 PHD3 EXPRESSION IN PROINFLAMMATORY MACROPHAGES Downloaded from

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 http://www.jimmunol.org/ either an anti-activin A Ab (0.1 mg/ml) or an isotype-matched Ab (Control IgG, 0.1 mg/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) by guest on September 25, 2021 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 mg/ml). For normalization, cells were cotransfected with the RSV-bgal expression plasmid, and results are presented as relative promoter activity, which indicates luciferase activity per unit of b-galactosidase activity for each assay condition. Mean 6 SD of three independent experiments are shown (***p , 0.001). conditions prompted us to analyze its expression in both macro- the presence of either anti-activin A or SB431542 (Fig. 6D). phage types under low oxygen tensions. Surprisingly, hypoxia (1% Therefore, activin A critically regulates EGLN3 expression O2, 24 h) did not alter EGLN3 expression in M1(GM-CSF) mac- in macrophages, mediating both its constitutive expression in rophages (Fig. 6A). However, hypoxia significantly increased the proinflammatory M1(GM-CSF) macrophages and its hypoxia- EGLN3 gene expression level in M2(M-CSF) macrophages, driven upregulation 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 Discussion of the EGLN genes is distinctly regulated by hypoxia between M1 In the present report, we describe the preferential expression of (GM-CSF) and M2(M-CSF) and, more importantly, that both the PHD3 prolyl hydroxylase-encoding EGLN3 gene by human macrophages exhibit a distinct ability to respond to hypoxia. proinflammatory macrophages in vivo and in vitro and that PHD3 Because activin A directs the expression of EGLN3 in M1(GM- expression is restricted to TAM that exhibit weak or absent levels CSF) macrophages and induces EGLN3 gene expression in M2 of typical anti-inflammatory/M2 markers. Besides, we demon- (M-CSF) macrophages (Fig. 5), we hypothesized that activin A strate that activin A regulates the constitutive expression of PHD3 might also mediate the hypoxia-dependent induction of EGLN3 in M1(GM-CSF) macrophages and its hypoxia-inducible expres- expression in M2(M-CSF) macrophages. Determination of activin sion in M2(M-CSF) macrophages. Considering that activin A A levels in both macrophage subtypes after exposure to 1% O2 for production is an exclusive property of proinflammatory M1(GM- 24 h revealed that hypoxia significantly induced the expression of CSF) macrophages (29), the activin A–PHD3 axis appears to the INHBA gene (Fig. 6B) as well as the secretion of activin A in constitute a specific signature of proinflammatory macrophage M2(M-CSF) macrophages (Fig. 6C). By contrast, hypoxia had no polarization. Moreover, because activin A expression is upregu- relevant effect on INHBA expression or activin A release from M1 lated by hypoxia in M2(M-CSF) macrophages (Fig. 6), the activin (GM-CSF) macrophages (Fig. 6B, 6C). To fully test the above A–PHD3 axis might also influence macrophage effector functions hypothesis, we evaluated the influence of a blocking anti-activin A under low oxygen pressures, a condition that is common to solid Ab or the SB431542 inhibitor on the hypoxia-mediated upregu- tumors and tissues undergoing active inflammatory responses (32). lation of EGLN3 gene expression in M2(M-CSF) markers. The The hypoxia-inducible expression of EGLN3 in M2(M-CSF) hypoxia-driven EGLN3 upregulation was significantly reduced in macrophages is in agreement with results in most cell types (33) The Journal of Immunology 7 Downloaded from

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 http://www.jimmunol.org/ 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 mg/ 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). by guest on September 25, 2021 and with the presence of a functional hypoxia-responsive element ways (37), activin A expression is enhanced in murine cortical within the first intron of the EGLN3 gene (25). However, the neurons in response to ischemia and hypoxia (38), and HIF1a presence of PHD3 in M1(GM-CSF) macrophages in vitro must enhances activin A-dependent signaling in murine embryonic reflect the existence of hypoxia-independent regulatory mecha- stem cells (39). nisms controlling EGLN3 gene expression, as HIF factors are not Huge differences exist between the expression profiles of po- detectable in M1(GM-CSF) macrophages under normoxic con- larized macrophages of human or mouse origin (40). A recent ditions (data not shown). The presence of PHD3+ macrophages in report has conclusively demonstrated such a difference by com- lung (Fig. 2), where partial pressure of O2 ranges between 150 and paring the transcriptome of bone marrow-derived murine- and 100 mmHg, also supports the hypothesis of such a hypoxia- monocyte-derived human macrophages polarized by either GM- independent mechanism for regulation of PHD3 expression. Our CSF or M-CSF (41). Interestingly, the expression of the Egln3 results indicate that activin A complies with this requirement gene has also been found to differentiate between M1(GM-CSF) because it: 1) potentiates the activity of the EGLN3 gene regula- and M2(M-CSF) bone marrow-derived macrophages, with a sig- tory region; 2) positively regulates EGLN3 expression in M1(GM- nificantly higher Egln3 expression in macrophages polarized by CSF) macrophages independently of low oxygen pressure; and GM-CSF (ArrayExpress Archive, http://www.ebi.ac.uk/arrayex- 3) mediates the hypoxia-inducible EGLN3 gene expression in press/, accession number E-MTAB-791). Therefore, the lack of M2(M-CSF) macrophages. Therefore, unlike promoter hyper- expression of the PHD3-encoding EGLN3 gene appears to cor- methylation or miR-20a, which inhibit EGLN3 gene expression in relate with an anti-inflammatory macrophage phenotype. In ad- tumor cells (34) and cardiomyocytes (35), activin A represents a dition to its identification as a marker for in vitro-generated M1 novel mechanism for positive regulation of EGLN3 expression. (GM-CSF) macrophages, the relevance of PHD3 expression in Along this line, soluble growth factors may turn out to exert an macrophages is underscored by the presence of PHD3+ macro- important control of the hypoxia-independent expression of prolyl phages under homeostatic conditions (e.g., lung), in inflammatory hydroxylases in myeloid cells, as EGLN1 gene expression is up- conditions (Crohn’s disease and ulcerative colitis), and within regulated by leukemia inhibitory factor in murine osteoclasts (36). tumor areas. A low percentage of PHD3+ intestinal macrophages In contrast, the contribution of activin A to EGLN3 gene tran- was detected in normal noninflamed gut, where macrophages scription, as well as the hypoxia-inducible expression of activin A exhibit constitutive IL-10 production and promotion of regulatory in M2(M-CSF) macrophages, is reminiscent of the reciprocal T cell responses (42), whereas a high proportion of CD163+ relationships that exist between TGF-b–related factors and PHD3+ macrophages was observed in inflamed gut (Fig. 3). PHD3 hypoxia-regulated signaling and transcription. Besides the syner- expression in macrophages from inflamed tissues can potentially gistic cooperation between hypoxia and TGF-b signaling path- be driven by hypoxia and/or activin A. However, based on the role 8 PHD3 EXPRESSION IN PROINFLAMMATORY MACROPHAGES of activin A in inflammatory responses (43, 44) and its ability to 3. Verreck, F. A., T. de Boer, D. M. Langenberg, M. A. Hoeve, M. Kramer, E. 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