D1 Polarizes Primary Human toward a Proresolution Phenotype through GPR32

This information is current as Mattia Schmid, Claudio Gemperle, Nicole Rimann and of September 26, 2021. Martin Hersberger J Immunol published online 11 March 2016 http://www.jimmunol.org/content/early/2016/03/10/jimmun ol.1501701 Downloaded from

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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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 11, 2016, doi:10.4049/jimmunol.1501701 The Journal of Immunology

Resolvin D1 Polarizes Primary Human Macrophages toward a Proresolution Phenotype through GPR32

Mattia Schmid, Claudio Gemperle, Nicole Rimann, and Martin Hersberger

Resolvin D1 (RvD1) was shown to be a potent anti-inflammatory and proresolution lipid mediator in several animal models of in- flammation, but its mechanism of action in humans is not clear. We show that the RvD1 GPR32 is present on resting, proin- flammatory M(LPS) and alternatively activated primary human M(IL-4) macrophages, whereas TGF-b and IL-6 reduce its membrane expression. Accordingly, stimulation of resting primary human macrophages with 10 nM RvD1 for 48 h maximally reduced the secretion of the proinflammatory cytokines IL-1b and IL-8; abolished to several chemoattractants like chemerin, fMLF, and MCP-1; and doubled the phagocytic activity of these macrophages toward microbial particles. In contrast, these functional changes were not accompanied by surface expression of markers specific for alternatively activated M(IL-4)

macrophages. Similar proresolution effects of RvD1 were observed when proinflammatory M(LPS) macrophages were treated with Downloaded from RvD1. In addition, we show that these RvD1-mediated effects are GPR32 dependent because reduction of GPR32 expression by small interfering RNA, TGF-b, and IL-6 treatment ablated these proresolution effects in primary human macrophages. Taken together, our results indicate that in humans RvD1 triggers GPR32 to polarize and repolarize macrophages toward a proresolution phenotype, supporting the role of this mediator in the resolution of inflammation in humans. The Journal of Immunology, 2016, 196: 000–000. http://www.jimmunol.org/ acrophages are master regulators of inflammation and (7). In addition, it has been shown that macrophages can, to some are key players in the inflammatory response (1). They extent, switch between activation states (8, 9). M are the most plastic cell type of the immune system The lipid mediator 7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z- and have an essential role from the initiation to the resolution of , named resolvin D1 (RvD1), is produced in inflammation and restoration of homeostasis (2). Upon stimula- resolving exudates from v-3 docosahexaenoic acid (10). RvD1 is tion, macrophages undergo a variety of different activation states, known to block proinflammatory migration (11) by from the proinflammatory M1 to the alternatively activated M2, regulating actin polymerization (12), to reduce TNF-a–mediated which play different roles in health and disease (3, 4). The Th1 inflammation in macrophages (10), and to enhance phagocytosis of cytokine IFN-g and TLR-4 ligands induce the proinflammatory apoptotic cells by macrophages (13). These proresolution properties by guest on September 26, 2021 M1 phenotype, which expresses high TNF-a and IL-1b levels and on the cellular level were corroborated in several mouse models of produces reactive oxygen and nitrogen species (5). In contrast, inflammation. RvD1 was shown to reduce inflammatory pain (14, the Th2 cytokines IL-4 and IL-13 lead to the development of 15), to be protective for airway inflammation (16), to reduce second M2 macrophages, characterized by high expression of ALOX15 organ injury induced by ischemia–reperfusion (11), and it enhanced mRNA and high expression of CD206 (mannose receptor) (6). wound healing and suppressed colitis in mouse models of inflam- The M1 macrophages are known to play an important role in the matory diseases (17). RvD1 also lowered adipose tissue inflam- initiation and progression of inflammation, whereas the M2 po- mation and ameliorated insulin sensitivity in mouse models of larization state is associated with the Th2 response against parasite obesity by modifying cytokine and adipokine production in adipose infections, wound healing, tissue repair, and restoration of ho- tissue, which led to a reduction in number and in the meostasis (5). M1 and M2 are only the extremes of a continuum of inflammation state of the fat tissue (18, 19). activation states, and intermediate phenotypes in specific stages of The receptors for RvD1 have recently been identified as the orphan inflammation, such as the resolution phase, have been identified receptor GPR32 and the FPR2/ALX receptor (20). Although RvD1 triggers both receptors, four times lower concentrations are neces- sary for triggering GPR32 than for triggering the FPR2/ALX re- Division of Clinical Chemistry and Biochemistry, University Children’s Hospital Zurich, CH-8032 Zurich, Switzerland; Children’s Research Center, University Child- ceptor (21). The GPR32 is expressed in human ren’s Hospital Zurich, CH-8032 Zurich, Switzerland; and Center for Integrative leukocytes and in adipose tissue, but cell type–specific expression Human Physiology, University of Zurich, CH-8057 Zurich, Switzerland and regulation have not been investigated so far (18). Of interest, Received for publication July 30, 2015. Accepted for publication February 16, 2016. GPR32 has no mouse ortholog, suggesting that the effects seen in This work was supported by the Vontobel Stiftung and the Swiss National Science mice are dependent on FPR2/ALX. In contrast, we have previously Foundation (Grant 320030_125142). shown that the FPR2/ALX receptor is expressed on primary human Address correspondence and reprint requests to Dr. Martin Hersberger, Division of monocytes, but not on macrophages, because of alternative pro- Clinical Chemistry and Biochemistry, University Children’s Hospital Zurich, Stein- wiesstrasse 75, CH-8032 Zurich, Switzerland. E-mail address: martin.hersberger@ moter usage in macrophages and reduced translation of this mRNA kispi.uzh.ch (22), suggesting that GPR32 may be the key player in mediating The online version of this article contains supplemental material. RvD1 effects in human macrophages. Abbreviations used in this article: IL-1ra, IL-1 receptor antagonist; poly I:C, To better understand the mechanism of RvD1-mediated pro- polyinosinic-polycytidylic acid; Q-PCR, quantitative real-time PCR; RvD1, resolvin resolution effects in human inflammation, we analyzed the ex- D1; siRNA, small interfering RNA. pression and function of GPR32 in differently polarized primary Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 human macrophages and the effects of RvD1 on macrophage

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501701 2 RESOLVIN D1 TRIGGERS GPR32 IN HUMAN MACROPHAGES

polarization and repolarization. We show that GPR32 is expressed for 7 d at 37˚C and 5% CO2 in RPMI 1640 (Sigma-Aldrich) supplemented with on the surface of resting M(2), M(LPS), and M(IL-4) macro- 5% FCS (Sigma-Aldrich), 5% human AB serum (Sigma-Aldrich), and 1% phages and that GPR32 is the receptor triggered by RvD1 in penicillin/streptomycin (Sigma-Aldrich). primary human macrophages. We further show that triggering RNA isolation and reverse transcription reaction for GPR32 induces a proresolution macrophage phenotype with re- quantitative real-time PCR duced secretion of proinflammatory cytokines, low chemotaxis, and increased phagocytosis of microbial particles. Total RNA from primary monocytes and macrophages was isolated using the RNeasy Mini Kit from QIAGEN AG (Hilden, Germany). cDNA was prepared by reverse transcription reaction from 1 mg total RNA, using Materials and Methods SuperScript III reverse transcriptase with random primers according to the Materials manufacturer’s instructions (Life Technologies). The TLR4 ligand LPS and the TLR3 ligand polyinosinic-polycytidylic acid Relative quantification of GPR32 expression levels (poly I:C) were purchased from Sigma-Aldrich (St. Louis, MO). The recombinant human cytokines IL-4, IL-6, IL-13, IL-1b, TNF-a, TGF-b, Each quantitative real-time PCR (Q-PCR) was performed in a total volume and IFN-g were purchased from R&D Systems (Minneapolis, MN). The of 20 ml on a LightCycler 480 II System (Roche Diagnostics, Rotkreuz, Switzerland). mRNA quantification included 100 ng cDNA, 4 mM MgCl2, TLR9 ligand CpG was synthesized by Microsynth (Balgach, Switzerland). 3 The TLR7 and TLR8 synthetic ligands 3M001 and 3M002 were purchased 0.5 mM upper primer, 0.5 mM lower primer, and 1 SYBR Green enzyme from 3M Pharmaceuticals (St. Paul, MN). The formylated peptide mix (Roche Diagnostics). Primer sequences are listed in Supplemental fMLF and MCP-1 were purchased from Sigma-Aldrich. Chemerin (149- Table I. The reactions were performed under the following conditions: 157) was purchased from AnaSpec (Fremont, CA). GPR32 rabbit anti- preheating10minat95˚Cfollowedby45cyclesofdenaturation,5sat 95˚C; annealing, 10 s at 60˚C; and extension, 6 s at 72˚C. Relative gene human Ab (GPR32 Ab) and its isotype IgG were purchased from Gene- Downloaded from Tex (Irvine, CA), the secondary Alexa Fluor 488 anti-rabbit Ab from Life expression was normalized to GAPDH. Data were analyzed with the Technologies (Carlsbad, CA). PE-labeled anti-human CD80 Ab, LightCycler 480 software (Roche Diagnostics). allophycocyanin-labeled anti-human CD206 Ab, and the IgG1K isotype FACS analysis control were purchased from R&D Systems. RvD1 (7S,8R,17S-trihydroxy- 4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid) produced by stereospe- Briefly, cells were resuspended in PBS containing 2.5% FCS, and Ab or cific total synthesis (23) was purchased from Cayman Chemicals (Ann isotype control was added and incubated in the dark for 30 min at 4˚C. For Arbor, MI) and was verified by liquid chromatography–mass spectrometry/ GPR32 probes and the respective isotype controls, the cells were washed mass spectrometry analysis in the laboratory (24). once with PBS and incubated with Alexa Fluor 488 anti-rabbit Ab in the http://www.jimmunol.org/ dark for 30 min at 4˚C. All cells were then washed in PBS and resuspended Primary cell purification and cell culture in 2% paraformaldehyde before analysis on a FACSCalibur analyzer (BD WBCs from healthy volunteers were isolated as described previously (25) from Biosciences, Franklin Lakes, NJ). buffy coat (Blutspendezentrum, Zurich, Switzerland), using Histopaque-1077 Small interfering RNA transfection (Sigma-Aldrich) gradient centrifugation. Briefly, monocytes were purified by capture with anti-CD14 Abs coupled to MACS Microbeads (Miltenyi Biotec, Macrophages were grown in six-well plates at a density of 1.5 3 106 Bergisch Gladbach, Germany) and allowed to differentiate into macrophages macrophages per well for 7 d, washed once with PBS, supplied with 1.5 ml by guest on September 26, 2021

FIGURE 1. GPR32 expression on primary human macrophages. (A) Relative mRNA ex- pression of GPR32 measured by Q-PCR after stimulation of primary human macrophages with different stimuli for 24 h (LPS 100 ng/ml, IFN-g 50 ng/ml, IL-1b 10 ng/ml, IL-6 10 ng/ml, TNF-a 25 ng/ml, TGF-b 1 ng/ml, IL-4 10 ng/ml, IL-13 10 ng/ml, CpG 100 ng/ml, poly I:C 1 ng/ml, 3M001 3 mM, 3M002 3 mM). All values are normalized for GAPDH and are presented rela- tive to unstimulated macrophages (Ctrl). Bars indicate the mean and SD of three independent experiments. (B) GPR32 protein expression measured by FACS after stimulation of primary human macrophages with different stimuli for 48 h. Representative graphs of the FACS results and quantification of the difference between the median fluorescence intensity (MFI) of the GPR32 Ab and the isotype control are shown. The isotype control is shown in gray, and cells labeled with GPR32 Ab are in black. Bars indi- cate the mean and SD of three independent ex- periments. *p , 0.05. The Journal of Immunology 3

RPMI 1640 medium with 5% FCS and 5% human AB serum (Sigma- rescence microscope (two photos per membrane and three replicate wells per Aldrich), and then transfected with the ON-TARGETplus GPR32 treatment). Migration indices were calculated over control values. SMARTpool small interfering RNA (siRNA) or nontargeting siRNA at an siRNA end concentration of 200 nM for 3 d (Thermo Scientific, Waltham, Multiplex ELISA MA), as described previously (26). For the formation of lipid–siRNA IL-1b, IL-1 receptor antagonist (IL-1ra), IL-6, IL-8, IL-10, IL-12, GM- complexes, 440 ml nonsupplemented RPMI 1640 medium, 45 ml HiPer- CSF, MCP-1, RANTES, and TNF-a concentrations in supernatants of Fect transfectant (QIAGEN AG), and 15 ml siRNA were mixed for each primary human macrophages were measured using commercially available six-well vial and incubated for 20 min at room temperature. Then 0.5 ml of Bio-Plex Pro human cytokine ELISA (Bio-Rad, Hercules, CA) according the mixture was added drop-wise onto the media of adherent macrophages. to the manufacturer’s instructions. After 6 h, 2 ml RMPI 1640 medium with 5% FCS and 5% human AB serum was added to each well. Phagocytosis assay Ca2+ influx assay After stimulation with RvD1 or vehicle, cells were incubated for 30 min with FITC-labeled zymosan (Life Technologies) (five particles per cell) at 37˚C Macrophages were grown in 24-well plates for 7 d and loaded with the with 5% CO2. After washing the cells with PBS, fluorescence was quenched calcium indicator Fluo-4, using the Fluo-4 Direct Calcium Assay Kit (Life using trypan blue (Sigma-Aldrich) (1:10 diluted), and phagocytosis was 2+ Technologies) according to the manufacturer’s instructions. The Ca mo- assessed using a Tecan plate reader (absorption 485 nm, emission 520 nm). bilization was monitored with a Tecan plate reader (Tecan, Maennedorf, Fluorescence of the phagocytosis assay was adjusted for cell number in each Switzerland) measuring fluorescence (absorption 485 nm, emission 520 nm) well, using DAPI staining (absorption 360 nm, emission 460 nm). every 10 s for 5 min. After 30 s, 10 nM RvD1 was added to the wells. Ca2+ ionophore A-23187 (Life Technologies), 5 mM,wasusedasapositive Statistical analysis control. Fluorescence of the Ca2+ influx assay was adjusted for cell number in each well, using DAPI staining (absorption 360 nm, emission 460 nm). GPR32 expression levels and functional assays were analyzed with ANOVA, using Microsoft Excel 2010 (Microsoft Corporation, Redmond, Downloaded from Chemotaxis assay WA). A p value , 0.05 was considered significant. A total of 5 3 104 macrophages were placed on a 96-well membrane (5.7 mm in diameter, 5-mm pore size; ChemoTX from Neuro Probe, Gai- Results thersburg, MD) in RPMI 1640 containing 0.1% BSA (Sigma-Aldrich). The GRP32 is expressed in resting, M1, and M2 primary human cells were allowed to migrate toward fMLF, MCP-1, and chemerin for 60 macrophages min at the indicated concentrations. Migrated cells were fixed (2% para- formaldehyde) and stained with DAPI (Sigma-Aldrich), and migration was To assess expression of the RvD1 receptor GPR32 in primary http://www.jimmunol.org/ quantified as the total pixel count of DAPI-stained nuclei under the fluo- human macrophages, we measured mRNA and surface protein by guest on September 26, 2021

FIGURE 2. RvD1 induces dose-dependent short-term functional changes in primary human macrophages. RvD1 triggers intracellular Ca2+ release, blocks chemotactic migration, and stimulates phagocytosis of microbial particles with maximal efficiency at 10 nM. (A)Ca2+ monitoring using Fluo-4 as an indicator. At 30 s after the start of the experiment, 1, 10, or 100 nM RvD1 (gray squares), or 5 mMCa2+ ionophore A-23187 (gray line), was given to human mac- rophages. Nonstimulated control cells are shown as black squares. The fluorescence levels are presented relative to the starting value. The symbols indicate the mean, and upper bars represent the SD of three independent experiments. *p , 0.05 for comparison between cells with no stimuli (black squares) and cells stimulated with A-23187 (gray line), #p , 0.05 for comparison between cells with no stimuli (black squares) and cells stimulated with RvD1 (gray squares). In (B)and(C), resting macrophages were stimulated for 15 min in the presence of medium (Med), 1, 10, or 100 nM RvD1, and chemotactic migration or phagocytosis of zymosan particles was measured. (B) Chemotaxis of macrophages toward 10 nM chemerin (Ch), 10 nM formylated peptide (fMLF), and 100 ng/ml MCP-1 was measured. Bars indicate the mean and SD of three independent experiments. *p , 0.05 compared with basal migration of macrophages without stimuli, #p , 0.05 compared with chemotaxis toward Ch, fMLF, or MCP-1 without RvD1 stimulation. (C) Phagocytosis of zymosan particles was measured. Values are presented relative to unstimulated macrophages. Bars indicate the mean and SD of three independent experiments. *p , 0.05. 4 RESOLVIN D1 TRIGGERS GPR32 IN HUMAN MACROPHAGES levels as we have previously done for other G protein–coupled macrophages, measured by a rise in relative fluorescence shortly receptors triggered by lipid mediators (22, 25, 27). GPR32 is after RvD1 stimulation (Fig. 2A). In addition, short-term ex- transcribed and expressed on the surface of resting macrophages posure of macrophages for 15 min with 10 nM RvD1 blocked (Fig. 1), and stimulation with the classical M1 polarization stimuli migration with maximal efficacy toward different well-known IFN-g and LPS, as well as with the M2-polarizing cytokines IL-4 macrophage chemoattractants, namely, the ChemR23 ligand and IL-13, did not alter mRNA transcription (Fig. 1A) or surface chemerin, the formylated peptide fMLF, and MCP-1 (Fig. 2B). protein expression (Fig. 1B). Similarly, stimulation with IL-1b, The same RvD1 concentration increased macrophage phago- TNF-a, and the TLR ligands CpG, poly I:C, 3M001, and 3M002 cytosis of microbial particles (Fig. 2C), indicating that 10 nM also did not affect GPR32 mRNA transcription (Fig. 1A). In RvD1 induces potent functional effects in resting primary contrast, stimulation of macrophages with IL-6 and TGF-b re- human macrophages. duced both GPR32 transcription and surface expression, indicat- RvD1 effects on primary human macrophages are GPR32 ing that resting M(2), M(LPS), and M(IL-4) polarized macrophages dependent show GPR32 expression on their surface, whereas macrophages stimulated with IL-6 or TGF-b do not. It has been shown that RvD1 not only triggers GPR32 but also triggers the FPR2/ALX receptor (20). Because we have previously Short-term treatment of resting primary human macrophages shown that the FPR2/ALX receptor is not expressed on the with 10 nM RvD1 induces intracellular calcium release, membrane of primary human macrophages (22), we investigated reduces chemotaxis, and increases phagocytosis of microbial whether RvD1 effects on primary human macrophages are GPR32 particles dependent. We therefore tested whether a reduction of GPR32 Downloaded from To investigate whether RvD1 induces functional effects on resting membrane expression would reduce RvD1-mediated intracellular primary human macrophages, we treated 7-d-old macrophages with calcium release and RvD1-stimulated phagocytosis of microbial 1, 10, and 100 nM RvD1 and measured intracellular calcium re- particles. Transient transfection of primary human macrophages lease, chemotaxis, and phagocytosis. It was found that 10 nM RvD1 with a pool of four siRNAs specific for GPR32 led to a significant mediated an immediate cytoplasmic calcium release in human decrease of GPR32 mRNA transcripts (Fig. 3A) and ablated http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. RvD1 effects on primary human macrophages are GPR32 dependent. Human primary macrophages transfected with specific siRNA for GPR32 (siGPR32) or nontargeting siRNA (Scramble) were tested for RvD1 responsiveness. (A) Relative mRNA expression of GPR32 measured with Q-PCR after transfection of primary human macrophages with 200 nM siRNA for 72 h. All values are normalized for GAPDH and are presented relative to nontransfected macrophages. Bars indicate the means and SD of three independent experiments. (B) GPR32 protein expression on macrophages by FACS. Representative graphs of the FACS results and quantification of the difference between the median fluorescence intensity (MFI) of the GPR32 Ab and the isotype control are shown. The isotype control is shown in gray and cells labeled with GPR32 Ab are in black. Bars indicate the mean and SD of three independent experiments. (C)Ca2+ monitoring using Fluo-4 as an indicator. At 30 s after the start of the experiment, 10 nM RvD1 (gray squares) or 5 mM Ca2+ ionophore A-23187 (gray line) was given to human macrophages. Nonstimulated control cells are shown as black squares. The fluorescence levels are presented relative to the starting values. The symbols indicate the means, and upper bars represent the SD of three independent experiments. *p , 0.05 for comparison between cells with no stimuli (black squares) and cells stimulated with A-23187 (gray line), #p , 0.05 for comparison between cells with no stimuli (black squares) and cells stimulated with 10 nM RvD1 (gray squares). (D) Phagocytosis of zymosan particles was measured. Values are presented relative to unstimulated macrophages. Bars indicate the mean and SD of three independent experiments. *p , 0.05. The Journal of Immunology 5

GPR32 surface expression after 3 d (Fig. 3B). The observed GPR32 with RvD1 would also induce a long-lasting polarization ablation of GPR32 cell surface expression by a 50% reduction effect on resting macrophages. Stimulation for 48 h with IL-4 in mRNA levels may be explained by the low level of basal clearly induced M(IL-4) macrophages, which secreted lower IL- GPR32 transcripts present in macrophages (threshold cycle 28). 1b,IL-8,andMCP-1andhigherIL-1ra,anddisplayedhigh Macrophages lacking GPR32 did not release Ca2+ intracellu- CD206 on the cell surface (Fig. 4). Similarly, 10 nM RvD1 larly upon 10 nM RvD1 stimulation (Fig. 3C). To corroborate decreased the secretion of IL-1b andIL-8,andshowedaten- these data, we investigated whether the increase in phagocytosis dency to reduce MCP-1 (Fig. 4). It is noteworthy that the lower of microbial particles after 15-min stimulation with RvD1 was secretion of IL-1b was observed without a reduction in IL-1b also GPR32 dependent. Again, the stimulatory effect of 10 nM mRNA levels. Such discrepancies between mRNA expression RvD1 on microparticle phagocytosis by macrophages was abol- and the secretion of cytokines is in line with the posttransla- ished in macrophages treated with GPR32 siRNAs (Fig. 3D). tional regulation of IL-1b secretion (28) and seems to be a Similar results have been obtained for macrophages stimulated with common feature also of other proinflammatory cytokines in TGF-b and IL-6, which also have reduced GPR32 expression (data resolution phase macrophages (29). RANTES, GM-CSF, IL-6, not shown), indicating that the RvD1-mediated effects on primary and IL-12 secretion was not changed either by IL-4 or by RvD1 human macrophages are GPR32 dependent. (data not shown). However, RvD1 did not alter CD206 mem- brane expression characteristic of M(IL-4) macrophage polari- RvD1 treatment of resting primary human macrophages zation. This finding indicates that RvD1 has the potential to induces a new polarization state polarize resting macrophages toward a proresolution phenotype,

Our results indicate that GPR32 is present on resting macrophages which is, however, different from the typical M(IL-4) polari- Downloaded from and that triggering by RvD1 induces intracellular Ca2+ release, zation. No difference was observed for the mRNA expression increases phagocytosis, and blocks chemotaxis toward several of IRF5, ALOX15, ALOX5AP, GATA-3, ADORA3, TGM2, chemoattractants. Next we investigated whether the activation of IL4RA, and HO-1 upon RvD1 treatment (data not shown). http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. RvD1 polarizes resting primary human macrophages to a new proresolution phenotype. Resting primary human macrophages were polarized for 48 h in the presence of medium (Med), LPS, IL-4, or 10 nM RvD1. mRNA and protein expression of cytokines and cell surface markers were measured to characterize polarization of macrophages. In (A)–(D), mRNA levels of TNF-a,IL-1b, IL-10, and TGM2 measured by Q-PCR are presented. All mRNA values are normalized for GAPDH and are presented relative to macrophages polarized with medium alone (Med). In (E)and(F), membrane expression of CD80 and CD206 measured by FACS analysis is shown, with values presented as the difference between the median fluorescence intensity (MFI) of the Abs and their respective isotype controls. In (G)–(L), protein secretion into the supernatants of TNF-a,IL-1b, IL-10, IL-1ra, IL-8, and MCP-1 measured with a multiplex ELISA is shown. Values are presented relative to unstimulated macrophages. Bars indicate the mean and SD of three independent experiments. *p , 0.05. 6 RESOLVIN D1 TRIGGERS GPR32 IN HUMAN MACROPHAGES

FIGURE 5. RvD1-polarized macrophages are less chemotactic and phagocytose more microbial particles. Resting primary human macrophages were polarized for 48 h in the presence of medium (Med), IL-4, or 10 nM RvD1 and afterward tested for chemotactic migration and phagocytosis. (A) Chemotactic migration of macrophages toward 10 nM chemerin (Ch), 10 nM formylated peptide (fMLF), and 100 ng/ml MCP-1. Values are presented relative to the basal migration of macrophages without stimuli. Bars indicate the mean and SD of three independent experiments. *p , 0.05 compared with basal migration of macrophages without stimuli, #p , 0.05 compared with chemotaxis toward Ch, fMLF, or MCP-1 without IL-4 or RvD1 stimulation. (B) Phagocytosis of zymosan particles. Values are presented relative to unstimulated macrophages. Bars indicate the mean and SD of three independent experiments. *p , 0.05. Downloaded from

RvD1-polarized primary human macrophages migrate less microbial particles (Fig. 7), indicating that repolarized human M1 toward different chemoattractant stimuli but phagocytose more macrophages functionally resemble the macrophages observed in microbial particles the RvD1 polarization experiments, displaying a proresolution

To characterize those polarized macrophages on the functional level, phenotype. No difference was observed for the mRNA expression http://www.jimmunol.org/ we performed a chemotaxis assay toward chemerin, the formylated of IRF5, ALOX15, ALOX5AP, GATA-3, ADORA3, IL4RA, and peptide fMLF, and MCP-1. Although control human macrophages HO-1 upon RvD1 treatment (data not shown). migrated toward all three chemoattractants, M(IL-4) macrophages lacked directed migration toward chemerin, the formylated peptide Discussion fMLF, and MCP-1 (Fig. 5A). This lack of migration was accom- In this article, we show that the potent lipid mediator RvD1 po- panied by downregulation of the three chemokine receptors larizes resting primary human macrophages and repolarizes M1 ChemR23, FPR1, and CCR2 (Supplemental Fig. 1) triggered by the macrophages to proresolution-type macrophages by triggering three chemoattractants chemerin, fMLF, and MCP-1, respectively. GPR32. Macrophages are a highly heterogeneous group of cells Similarly, macrophages polarized with 10 nM RvD1 did not migrate that play a fundamental role during the different stages of in- by guest on September 26, 2021 toward any of the tested chemokines (Fig. 5A). However, such flammation and can be characterized by their polarization state RvD1-stimulated macrophages still expressed high mRNA levels of (31). The initiation and sustaining of inflammation are mainly ChemR23, FPR1, and CCR2 (Supplemental Fig. 1), arguing for a associated with the classically activated M1 macrophages, which direct role of GPR32-mediated RvD1 signaling in the regulation of secrete high levels of inflammatory cytokines and produce high actin polymerization. levels of reactive oxygen species (3), whereas polarization of In the second functional read-out, we investigated the effect of macrophages toward an M2-like phenotype seems essential for the RvD1-triggered macrophage polarization on phagocytosis of mi- resolution of inflammation (32). Such alternatively activated M2 crobial particles (Fig. 5B). Polarization of primary human mac- macrophages play an important role in wound healing and tissue rophages with 10 nM RvD1 led to a similar increase in uptake of repair (5). zymosan particles, as seen after IL-4 stimulation, again supporting However, this classification is a simplification of the polarization a role for RvD1 in polarization of macrophages toward a pro- states undergone by macrophages and represents the two extremes resolution phenotype. of a continuum in a universe of activation states (33). In clinical conditions, coexistence of cells in different activation states and RvD1 has similar repolarizing effects on primary human M(LPS) unique or mixed phenotypes have been observed as a reflection of macrophages dynamic changes and complex tissue-derived signals (5). One of Because appear naturally during the resolution phase of these macrophages is the resolution phase macrophage, for which inflammation (30), we investigated whether RvD1 causes a repo- there is in vivo evidence for its presence during the resolution larization of inflammatory M(LPS) macrophages toward a pro- phase of inflammation in mouse models (7, 29, 34, 35). Those resolution macrophage. We therefore sequentially stimulated isolated resolution phase macrophages secrete low levels of primary human macrophages for 3 d with LPS, followed by stim- proinflammatory cytokines and high levels of the anti-inflammatory ulation for 4 d with 10 nM RvD1, and characterized the polarization IL-10, and express high levels of the mannose receptor CD206 on phenotype of these macrophages (Fig. 6). As expected, M(LPS) their surface, all proprieties of M2 polarization (7). In contrast, macrophages exhibited an increased transcription of IL-1b,TNF-a, cyclooxygenase-2 expression was elevated in these resolution phase and cell surface expression of CD80 (Fig. 4), which returned to macrophages in a similar fashion to M1 macrophages, indicating levels comparable to that of untreated macrophages upon removal that resolution phase macrophages have a mixed phenotype (7). of the stimulus for 4 d (data not shown). Sequential stimulation of Transcriptomic analysis of resolution phase macrophages showed these M(LPS) macrophages with 10 nM RvD1 for 4 d further re- high levels of IL-10, COX-2, and CD206 and, despite the low se- duced IL-8, IL-1b, and MCP-1 secretion (Fig. 6H, 6K, 6L) and cretion, also high levels of inflammatory cytokines (29). In the same slightly increased TGM2 expression (Fig. 6D). On the functional mouse model, peritoneal injection of RvD1 induced the formation level, RvD1 reduced chemotaxis and increased phagocytosis of of CD11blow macrophages with high phagocytic activity, reduced The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021 FIGURE 6. RvD1 repolarizes M1 primary human macrophages to a proresolution phenotype. Resting primary human macrophages were polarized toward an M(LPS) phenotype with 100 ng/ml LPS for 3 d. These M1 macrophages were then further incubated for 4 d in the presence of medium (Med), LPS, IL-4, or 10 nM RvD1. To assess the polarization of macrophages, mRNA and protein secretion of cytokines and cell surface markers were measured. In (A)–(D), mRNA levels of TNF-a,IL-1b, IL-10, and TGM2 measured by Q-PCR are presented. All mRNA values are normalized for GAPDH and are presented relative to M(LPS) macrophages repolarized for 4 d in the presence of medium alone (Med). In (E)and(F), membrane expression of CD80 and CD206 measured by FACS analysis is shown, with values presented as the difference between the median fluorescence intensity (MFI) of the Abs and their respective isotype controls. In (G)–(L), protein secretion into the supernatants of TNF-a,IL-1b, IL-10, IL-1ra, IL-8, and MCP-1 measured with a multiplex ELISA is shown. Values are presented relative to M(LPS) macrophages repolarized for 4 d in the presence of medium (Med). Bars indicate the mean and SD of three independent ex- periments. *p , 0.05.

TNF-a, and increased IL-10 secretion, reflecting the proresolution between mice and humans in this context is the lack of a human effect of RvD1 on mouse macrophages (34). Similarly, RvD1 GPR32 ortholog in the mouse. From sequence analyses between treatment of obese mice lowered adipose tissue inflammation, ac- different species, it is thought that only chimpanzees have an companied by a repolarization of adipose tissue M1 macrophages to ortholog of the human GPR32 receptor (20). In contrast, orthologs a proresolution macrophage with lower proinflammatory cytokines, for the second RvD1 receptor, FPR2/ALX, have been identified in higher arginase 1, and higher phagocytic activity (35). mice (36) and rats (37). Hence, all the proresolution effects of Even though some characteristics of the resolution phase mouse RvD1 in mouse models depend on triggering of the FPR2/ALX macrophages described in these studies differ from the charac- receptor (21, 38, 39), whereas in humans both receptors play a teristics of the RvD1-polarized and -repolarized primary human role, depending on the cell type. macrophages in our in vitro experiments, we also observed a de- We have previously shown that the FPR2/ALX receptor is absent crease in proinflammatory cytokine secretion, namely, IL-1b, in primary human macrophages (22), suggesting that LXA4 pro- IL-8, and MCP-1, and an increased phagocytic capacity following resolution actions may be mediated by other receptors in these RvD1 treatment, suggesting that RvD1 does indeed polarize pri- cells (40, 41), and we show in this article that RvD1 signaling in mary human macrophages toward a resolution phase phenotype. human macrophages is mediated by GPR32, involving calcium Differences in receptor expression and distribution as well as the release from intracellular stores. In contrast, RvD1 signaling in experimental set-up may account for the incongruencies between human differs from that of macrophages. Human the in vivo mouse studies and our in vitro human study. Whereas neutrophils have been shown to mobilize FPR2/ALX, but not RvD1 acts through the FPR2/ALX receptor in mice, affecting GPR32, to the cell membrane from secretory granules upon multiple cell types following systemic application, RvD1 acts stimulation with RvD1 (21), and signaling in neutrophils does not through GPR32 on macrophages only in our in vitro study on involve calcium release from intracellular stores (20). This finding isolated primary human macrophages. Indeed, the major difference argues for an elaborate cell type–specific regulation of RvD1 ef- 8 RESOLVIN D1 TRIGGERS GPR32 IN HUMAN MACROPHAGES

FIGURE 7. RvD1-repolarized M1 primary human macrophages are less chemotactic and phagocytose more microbial particles. Resting primary human macrophages were polarized toward an M(LPS) phenotype with 100 ng/ml LPS for 3 d. These M1 macrophages were then repolarized for 4 d in the presence of medium (Med), IL-4, or 10 nM RvD1. Chemotaxis and phagocytosis were measured to characterize the repolarization of macrophages. (A) Chemotactic migration of macrophages toward 10 nM chemerin (Ch), 10 nM formylated peptide (fMLF), and 100 ng/ml MCP-1. Values are presented relative to the basal migration of M1 macrophages repolarized for 4 d in the presence of medium (Med). Bars indicate the mean and SD of three inde- pendent experiments. *p , 0.05 compared with basal migration of M1 macrophages repolarized with medium alone without chemoattractant, #p , 0.05 compared with chemotaxis toward Ch, fMLF, or MCP-1 without IL-4 or RvD1 stimulation. (B) Phagocytosis of zymosan particles. Values are presented Downloaded from relative to M1 macrophages repolarized with medium alone. Bars indicate the mean and SD of three independent experiments. *p , 0.05. fects through the use of two G protein–coupled receptors with on mononuclear cell recruitment also suggest a distinct thera- different sensitivities toward RvD1 (21) and with divergent sig- peutic use for these two mediators, with a preferential triggering

naling pathways for the fine tuning of the proresolution effects of of GPR32 by RvD1 mimetics in diseases for which monocyte and http://www.jimmunol.org/ RvD1 in human inflammation. macrophage immigration may not be beneficial, such as in ath- Despite the different receptor usage between the two species, our erosclerosis. data in humans are in line with the proresolution effects mediated In summary, the findings of this study provide evidence that by RvD1 in mouse models of inflammation. In these mouse models, RvD1 induces a functional switch in isolated primary human RvD1 was shown to reduce neutrophil and macrophage extrava- macrophages toward a proresolution phenotype with blocked sation into the inflamed area and to increase clearance of debris and chemotaxis, reduced secretion of inflammatory cytokines and apoptotic cells by macrophages (10, 20, 34). This finding goes chemokines, and increased phagocytosis of microbial particles. along with the observed effects of RvD1 on primary human We further show that these actions are mediated through GPR32 macrophages, namely, the reduction in IL-8 and MCP-1 secretion; triggering intracellular calcium release, highlighting its key role by guest on September 26, 2021 the reduced chemotaxis of these macrophages toward chemerin, and possible therapeutic use for the resolution of inflammation in fMLF, and MCP-1; and the increased phagocytic activity. diseases for which macrophage immigration may not be bene- In humans, low-nanomolar RvD1 seems to trigger two different ficial. mechanisms leading to reduced extravasation of neutrophils. On the one hand, there is evidence that RvD1 directly reduces human Disclosures neutrophil migration toward IL-8 (42, 43) and (data The authors have no financial conflicts of interest. not shown), and on the other hand, we now show that RvD1-treated macrophages secrete less IL-8. Such a combined effect on the chemotactic activity of neutrophils and on the secretion of the References chemotactic agent by macrophages may well be an efficient 1. Silva, M. T. 2010. When two is better than one: macrophages and neutrophils work in concert in innate immunity as complementary and cooperative partners mechanism to block neutrophil immigration into the inflamed area. of a myeloid phagocyte system. J. Leukoc. Biol. 87: 93–106. In addition, a dual effect of RvD1 seems also to modulate 2. Gordon, S., and P. R. Taylor. 2005. Monocyte and macrophage heterogeneity. human monocyte and macrophage immigration. Although RvD1 Nat. Rev. Immunol. 5: 953–964. 3. Murray, P. J., and T. A. Wynn. 2011. 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