Galectin-2 Induces a Proinflammatory, Anti-Arteriogenic Phenotype in Monocytes and Macrophages

Galectin-2 is a monocyte-expressed carbohydrate-binding lectin, for which increased expression is genetically determined and associated with decreased collateral arteriogenesis in obstructive coronary artery disease patients. The inhibiting effect of galectin-2 on arteriogenesis was confirmed in vivo, but the mechanism is largely unknown. In this study we aimed to explore the effects of galectin-2 on monocyte/macrophage phenotype in vitro and vivo, and to identify the receptor by which galectin-2 exerts these effects. We now show that the binding of galectin-2 to different circulating human monocyte subsets is dependent on monocyte surface expression levels of CD14. The high affinity binding is blocked by an anti-CD14 antibody but not by carbohydrates, indicating a specific protein-protein interaction. Galectin-2 binding to human monocytes modulated their transcriptome by inducing proinflammatory cytokines and inhibiting pro-arteriogenic factors, while attenuating monocyte migration. Using specific knock-out mice, we show that galectin-2 acts through the CD14/toll-like receptor (TLR)-4 pathway. Furthermore, galectin-2 skews human macrophages to a M1-like proinflammatory phenotype, characterized by a reduced motility and expression of an anti-arteriogenic cytokine/growth factor repertoire. This is accompanied by a switch in surface protein expression to CD40-high and CD206-low (M1). In a murine model we show that galectin-2 administration, known to attenuate arteriogenesis, leads to increased numbers of CD40-positive (M1) and reduced numbers of CD206-positive (M2) macrophages surrounding actively remodeling collateral arteries. In conclusion galectin-2 is the first endogenous CD14/TLR4 ligand that induces a proinflammatory, non-arteriogenic phenotype in monocytes/macrophages. Interference with CD14-Galectin-2 interaction may provide a new intervention strategy to stimulate growth of collateral arteries in genetically compromised cardiovascular patients.

Introduction 4 mM β-mercaptoethanol (pH 8.0), and eluted with the same buffer containing higher concentration of imidazole (300 mM). Finally, the protein was desalted using PD-10 columns (GE Healthcare Life Sciences, Uppsala, Sweden) in storage buffer containing 50 mM Tris, 150 mM NaCl, 10% Glycerol, and 4 mM β-mercaptoethanol (pH 8.0). Galectin purity was analyzed by SDS-PAGE on a 15% polyacrylamide gel, and the protein was stained with Coomassie blue stain (Merck, Darmstadt, Germany). After production and purification, both human and mouse galectin-2 appeared as a single homogenous protein band with a molecular weight of 15 kDa. Human galectin-1 appeared as a dimer with a molecular weight of 28 kDa, and as a monomer of 14 kDa (S1 Fig). Galectin concentration was measured using NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Breda, The Netherlands) and stored at -80°C until use.
For macrophage differentiation, the monocytes were seeded at a density of 1 x 10 6 /ml in DMEM complete medium (Invitrogen) supplemented with 5% heat-inactivated human AB serum (Sanquin, Blood bank), 1% penicillin/streptomycin and 2 mM L-glutamine. The macrophages were differentiated by culturing them at 37°C with 5% CO 2 for six days in 145 mm petridishes (Greiner Bio-One).
After six days of culture, the adherent macrophages (M0) were harvested with 4 mg/ml lidocaine hydrochloride monohydrate (Sigma-Aldrich) in PBS at 37°C and 5% CO 2 for 10 minutes.
Macrophages were seeded at a density of 1 x 10 6 /ml in 6 well tissue-culture plates (Greiner Bio-One) in DMEM complete medium and incubated with 10 ng/ml rhIL-4 (Immunotools, Friesoythe, Germany) for differentiation into alternatively activated M2 macrophages for 48 hours. To obtain classically activated M1 macrophages, cells were incubated with 100 U/ml rhIFN-γ (U-Cytech, Utrecht, The Netherlands) and after 7 days of culture, 10 ng/ml LPS (Ultra pure from E. coli 0111:B4; Invivogen) was added and incubated for another 24 hours for full differentiation into M1 macrophages. Recombinant human galectin-2 (10 μg/ml) or storage buffer (vehicle control) was also added at day 7 for the last 24 hours. Control macrophages (M0) were cultured for the same period in DMEM complete medium without additional stimuli. The different macrophage subtypes were used for further experiments as indicated. Phenotypical and functional characterization of macrophages was performed at day 8.

Galectin binding assays
Galectin binding to human monocytes, human monocyte-derived macrophages and-dendritic cells, human T-cells, and mouse macrophage cell line RAW 264.7 was performed. To that end, galectins were labelled with biotin (Sigma-Aldrich) in N-Hydroxysuccinimide (NHS) solution (Sigma-Aldrich). The protein was purified using PD-10 columns (GE Healthcare Life Sciences) in PBS. The galectin concentration was measured using NanoDrop ND-1000 Spectrophotometer. Briefly, cells were suspended in 0.5% bovine serum albumin (BSA; Roche Diagnostics) in PBS containing the indicated amount of biotinylated galectin at 4°C for 30 minutes. For determination of the binding affinity of galectins to monocytes, graded concentrations of biotinylated galectins were used, as indicated. The cells were then washed and incubated with streptavidin-alexa fluor 488 (1:800; Invitrogen) at 4°C for 30 minutes. Background staining was determined by omitting the biotinylated lectin. The binding was also performed in the presence of indicated concentrations of lactose, thiodigalactoside (Carbosynth Compton, UK), PMB (Sigma-Aldrich), or 100 μg/ml anti-human CD14 blocking antibody (Invivogen). For analysis of different subsets of human monocytes, non-specific antibody binding was blocked with 10% normal mouse serum (NMS) in 0.5% BSA/PBS, while cells were labeled with mouse anti-human CD14-PE (Immunotools) and mouse anti-human CD16-APC antibodies (Immunotools). After washing, the cells were analyzed by flow cytometry using Cyan ADP High Performance Research Flow Cytometer (Beckman Coulter, Woerden, Netherlands). The data were analyzed using the Summit V4.3 program (Dako, Fort Collins, CO, USA).

Human monocyte migration assay
Human monocyte migration was studied using 24-well Transwell inserts (6.5 mm) with polycarbonate filters of 5-μm pore size (Corning Life Sciences, Amsterdam, The Netherlands). Briefly, the filters were pre-coated with 10 μg/ml fibronectin (Sigma-Aldrich) in PBS for one hour at RT. Then, 0.5 x 10 6 monocytes diluted in 100 μl of RPMI 1640 medium containing 0.5% BSA were added in the presence or absence of 50 μg/ml recombinant human galectin-2 to the upper chamber of the insert. The lower chamber contained 600 μl of RPMI 1640 medium containing 0.5% BSA without any chemokine. The plates were incubated at 37°C in 5% CO 2 for 24 hours and monocytes that had migrated into the lower chamber were photographed using Leica DM IL microscope at 20 times original magnification (Leica Microsystems B.V., Rijswijk, The Netherlands), counted using flow cytometry by labeling the cells with a nonblocking CD14-PE antibody (Immunotools), and acquiring the cells at a constant acquisition rate over a fixed time-period.
RNA isolation, cDNA synthesis, quantitative real-time PCR Total RNA was isolated from human monocytes or macrophages using RNeasy mini kit (Qiagen) according to the manufacturer's instructions, including a DNase I (Qiagen) digestion step to remove genomic DNA. RNA samples were concentrated by SpeedVac for 30 minutes. RNA purity and concentration was measured using NanoDrop ND-1000 Spectrophotometer. For cDNA synthesis, between 100-500 ng of total RNA per sample was reverse transcribed using RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions. Quantitative real-time polymerase chain reaction (PCR) was carried out in an ABI PRISM 7900HT system (Applied Biosystems, Foster City, CA, USA) with the following primers (Invitrogen) designed by Primer Express version 2.0 (Applied Biosystems): Briefly, in a 10 μl reaction volume, 4 μl of diluted cDNA, 5 μl SYBR Green PCR Master Mix (Applied Biosystems), and 0.5 uM of each gene-specific primers were mixed. Gene expression levels were calculated using an arbitrary standard curve and normalized to the human housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Relative gene expression levels were expressed as a fold change relative to respective untreated samples unless otherwise indicated.

Flow cytometry of human macrophages
The expression of CD40 and mannose receptor (CD206) was analyzed on different human macrophages subtypes by flow cytometry. Briefly, macrophages were detached with 4 mg/ml lidocaine hydrochloride monohydrate in PBS at 37°C, 5% CO 2 for 10 minutes, washed once in PBS, and fixed in 4% paraformaldehyde (PFA; Merck) in PBS at 4°C for 30 minutes. Before labeling with the primary antibodies, cells were washed once with saponin buffer (0.01% saponin, Sigma-Aldrich,0.1% BSA in PBS), and blocked with 10% normal human serum in saponin buffer at RT for 30 minutes. Macrophages were then incubated with mouse anti-human CD40 (AbD Serotec, Oxford, UK) or CD206 (BD Biosciences, San Jose, CA, USA) at RT for one hour:. After washing with saponin buffer, cells were incubated with the secondary goat antimouse alexa fluor 488 antibody (Invitrogen) at 4°C for 30 minutes, and washed with saponin buffer before analysis by flow cytometry. As a negative control, only secondary antibody was used. Results are expressed as a fold change of mean fluorescence intensity (MFI) relative to untreated M0 macrophages.

Actin staining
To analyze the subcellular localization of actin, different human macrophage subtypes were cultured in the presence of storage buffer (vehicle control) or 10 μg/ml recombinant human galectin-2 at day 7 on ibitreat chamber slides (Ibidi, Planegg/Martinsried, Germany) at a density of 1 x 10 6 cells/ml in DMEM complete medium. Macrophages were fixed in 4% PFA in HBSS (Invitrogen) at RT for 10 minutes, and permeabilized with 0.1% Triton X-100 (Merck) in PBS at RT for 5 minutes. Finally, the cells were stained with 0.4 μg/ml phalloidin-tetramethyl rhodamine iso-thiocyanate (TRITC; Sigma-Aldrich) at RT for one hour. Imaging was performed by a confocal laser scanning microscope (Leica TCS SP2 AOBS, Leica Microsystems B. V., Rijswijk, The Netherlands). A total of five randomly selected fields at 63 times original magnification were acquired with Leica confocal software version 2.61 (Leica Microsystems, Wetzlar, Germany).

Motility
Different macrophage subtypes at day 7 were cultured in the presence of storage buffer (vehicle control) or 10 μg/ml recombinant human galectin-2 on ibitreat chamber slides (Ibidi) at a density of 1 x 10 6 cells/ml in DMEM complete medium. The cells were recorded after one hour adhesion using an inverted time-lapse video microscope (Olympus IX81, Zoeterwoude, The Netherlands) housed in a 60% humidified, 5% CO 2 gassed, temperature controlled (37°C) chamber. A total of four randomly selected fields were recorded for 24 hours every 12 minutes at a 20 times original magnification. The movies were converted to avi format with Cell^R software. WCIF Image J software (National Institutes of Health, Bethesda, Maryland, USA) was used to calculate the motility as measured by the travelled distance. Briefly, ten cells per microscopic field were tracked manually. C57BL/6 mice (Charles River, Chatillon-sur-Chalaronne, France), CD14 -/and TLR4 -/mice (C57BL/6 background; The Jackson Laboratory, Bar Harbor, Maine, USA) were bred locally (LUMC). Blood samples were collected from the tail vein from six mice of each strain (male and female, 12-16 weeks old) and diluted 1:50 with RPMI 1640 (Invitrogen), supplemented with penicillin-streptomycin (PAA Laboratories, Cölbe, Germany) and glutamax (Invitrogen). Blood was incubated at 37°C in 5% CO 2 overnight, in the presence of 100 ng/ml LPS (E. coli 0111:B4; Invivogen) or 10 μg/ml recombinant mouse galectin-2, in the absence or presence of 20 μg/ml PMB (Sigma-Aldrich). Cell-free supernatant was collected and TNFα level was measured by ELISA (BD Biosciences) according to the manufacturers protocol.

Mouse models and tissue sampling
Immunofluorescence staining. For immunohistochemistry, we used C57BL/6 mice from a previous study. [11] Briefly, the left deep femoral artery was ligated just distal to the superficial and deep femoral artery bifurcation. The mice were sacrificed at day 7 after ligation and left adductor muscles were dissected, cryopreserved, sectioned (5 μm thick) with a HM-560 cryostat (Thermo Scientific, Runcorn, Cheshire, UK) and stored at -80°C until use.
Before staining, frozen sections were air dried for one hour. Available sections from 7 placebo-treated mice and 5 galectin-2-treated mice were fixed in acetone (10 min). After washing (PBS), sections were incubated with 10% NMS in 0.1% BSA/PBS for 30 minutes to block nonspecific antibody binding, and stained with rat anti-murine F4/80 (1:100; AbD Serotec) for one hour. After washing, sections were incubated with goat anti-rat Alexa fluor 647 secondary antibody (Invitrogen) for one hour to identify macrophages. Next, sections were washed, blocked with 10% normal rat serum (NRS) in 1% BSA/PBS for 30 minutes, and incubated with both rabbit anti-murine alpha-smooth muscle actin (1:100; Abcam, Cambridge, UK) and rat antimurine CD40 biotin (M1; 1:400; eBioscience, Vienna, Austria) or rat anti-murine CD206 biotin (M2; 1:50; Biolegend, London, UK) at 4°C overnight. After washing, sections were incubated for one hour with goat anti-rabbit Alexa fluor 488 secondary antibody (Invitrogen) to visualize smooth muscle cells or streptavidin Alexa fluor 555 (Invitrogen) to identify the two macrophage subsets. After washing, nuclei were counterstained with Hoechst (1:50.000; Invitrogen) for 3 minutes. Sections were photographed using Leica DM6000 microscope with LAS AF software, at 20 times original magnification. The ratio of CD40 to F4/80 or CD206 to F4/ 80-positive cells was quantified of defined collateral arteries in the adductor muscles in a blinded fashion.

Statistical analysis
Intergroup comparisons were performed using Student's t-test (two-sided). Mann-Whitney U tests were used for group comparisons requiring nonparametric analytic approaches. P-values less than 0.05 were considered significant. All experiments were performed with at least three independent donors, and data are presented as mean ± standard error of the mean (SEM), unless otherwise indicated. All statistics were performed using GraphPad Prism version 6.0 (Graphpad Software, San Diego, CA, USA).

Galectin-2 binds monocytes and macrophages through CD14 and activates TLR4 signaling
We evaluated the binding of human and mouse biotinylatedgalectin-2 to the surface of human monocytes by flow cytometry. Human monocytes bound both human and mouse galectin-2 with a high affinity (apparent Kd of 1.1 μg/ml (77 nM) and 2.5 ug/ml, respectively, S2 Fig). Both galectins bound in a concentration dependent manner with near maximal binding at10 μg/ml and an apparent Bmax of 93.6 and80.7 for human and mouse galectin-2, respectively. By contrast, a much lower binding was observed for human galectin-1 at equal fluorescent labeling (apparent Kd 0.2 μg/ml, Bmax of 28.9). Next, the carbohydrate-binding specificity of human galectin-2 was determined by adding either lactose or thiodigalactoside together with human galectin-2 to human monocytes. These disaccharides did not inhibit binding of human galectin-2 (Fig 1A), suggesting that the binding is carbohydrate-independent. Interestingly, different human monocyte subsets (classical CD14 ++ CD16 -; intermediate CD14 ++ CD16 + ; and non-classical CD14 + CD16 + monocytes) showed differential binding of human galectin-2 ( Fig 1B). CD14 low monocytes (non-classical) showed a lower binding of human galectin-2 than CD14 high monocytes (classical and intermediate), reaching significance for the comparison with intermediate monocytes (Fig 1B). These results suggest that binding is associated with expression levels of CD14. Additionally, binding of human galectin-2 to different types of Because of the association of galectin-2-monocyte binding with CD14 expression levels, we examined whether galectin-2 is a ligand for CD14.CD14 antibodies that neutralize activation of the CD14-associated TLR2 and TLR4 receptors [23,24] caused a 50% reduction in binding of human galectin-2 to human monocytes, whereas binding of human galectin-1 was not affected by these antibodies (Fig 2A).Next, we examined whether binding of galectin-2 to CD14 leads to TLR activation and the possible role of the two CD14-associated TLRs in activation. We analyzed whether galectin-2 is able to induce IFN-β gene expression. IFN-β activity is induced by the TLR4 ligand LPS, but not by the TLR2 ligand PGN (Fig 2B). [25]The observation that galectin-2 was able to induce IFN-β expression, comparable to LPS and irrespective of TLR2 stimulation, suggests that galectin-2 activates TLR4.To further explore this possibility, we stimulated mouse whole blood cells deficient for TLR4 or CD14 with m-galectin-2. Induction of TNF-α protein production was determined by ELISA, showing that both LPS and mgalectin-2 required CD14 and TLR4 for signaling (Fig 2C). The LPS response, but not the galectin-2-induced response was inhibited by polymyxin B. These results indicate that galectin-2 acts independent of LPS, and requires both CD14 and TLR4 to mediate its effects. Also in human monocytes, galectin-2 required CD14 for IFN-β induction. Neutralizing CD14 antibodies indeed inhibited the induction of IFN-β expression by approximately 50% (Fig 2D).

Galectin-2 induces monocytes to express proinflammatory genes, inhibits the expression of pro-arteriogenic factors and inhibits monocyte migration
To study the biological consequences of monocyte/galectin-2 interaction, we determined the effect of galectin-2 on pro-and anti-inflammatory gene expression. We observed a significant increase in the expression of proinflammatory genes such as TNF-α, IL-6, IL12-p40 and IFN-β upon incubation of monocytes with human galectin-2. In contrast to LPS-induced expression of these cytokines, their galectin-2 induced expression was not affected by polymyxin B (Fig 3A). Consistently, involvement of LPS in binding of galectin-2 to monocytes was excluded by FACS analysis, as binding was not affected by Polymyxin B (data not shown). These results
Arteriogenesis depends on the migration of circulating monocytes into the vessel wall and local proliferation of tissue-resident macrophages [39,40]. Given the vital role of monocytes in arteriogenesis, we assessed the effect of human galectin-2 on human monocyte migration in a Galectin-2 Is Proinflammatory and Anti-Arteriogenic transwell system followed by flow cytometry. Galectin-2 treatment significantly inhibited monocyte migration (Fig 4A).

Galectin-2 inhibits macrophage motility, induces macrophages to express M1 proinflammatory surface proteins and cytokines, and suppresses an M2 phenotype
Recent studies have shown a role for M2 macrophages in arteriogenesis. [14][15][16] Given the proinflammatory effect of galectin-2 on monocytes, we next hypothesized that galectin-2 might impair arteriogenesis by preventing pro-arteriogenic M2 macrophage differentiation Because M1 and M2 macrophages display distinct morphologies, associated with their differential migratory properties [41], we first studied the effect of human galectin-2 on the actin distribution of different human macrophage subtypes (M0, M1 [IFN-γ + LPS], and M2 ). Galectin-2 treatment of M0 and M2 macrophages, which are typically round, caused an elongated cell shape, characteristic of M1 macrophages (Fig 4B). The LPS-induced elongated cell shape of  Galectin-2 Is Proinflammatory and Anti-Arteriogenic M1 macrophages was not further affected by galectin-2. We have previously shown that M1 macrophages are less motile than M2 macrophages [41]. Therefore we assessed the effects of galectin-2 on macrophage motility by video time-lapse microscopy (S1 File, Videos 1-6). Galectin-2 treatment reduced the motility of M0 and M2 macrophages to a level comparable to M1 macrophages (Fig 4C).
To test whether galectin-2 could reduce M2 markers, we measured the effects of galectin-2 on a set of distinctive M2-expressed marker genes [17][18][19][20][21]i.e. mannose receptor (CD206), PDGF-C, CCL26, and CCL18. RT-PCR data showed that galectin-2 significantly reduced mRNA expression of the mannose receptor by M0-and M2 macrophages, and reduced PDGF-C in M0macrophages (Fig 6A). Galectin-2 did not significantly inhibit the expression of the other M2 markers, CCL26 and CCL18. The reduced gene expression of the mannose receptor was confirmed at the protein level by flow cytometry (Fig 6B). Collectively, these data indicate that galectin-2 can partially modify differentiated M2 macrophages to M1 macrophages.

Galectin-2 increases the number of M1 macrophages and reduces M2 macrophages around collateral arteries in vivo
We previously showed that systemic administration of galectin-2 inhibits arteriogenesis after arterial ligation in the murine hind limb model [11]. Diminished arteriogenesis, as detected by Laser Doppler Perfusion Analysis and histology, was accompanied by a reduced number of macrophages surrounding the collateral vessels in the adductor muscle. We now used tissue sections from this study to assess the effect of human galectin-2 treatment on the presence of M1 and M2 macrophages around the collateral arteries in vivo by immunohistochemistry. M1-like macrophages were defined here as CD40 + F4/80 + -positive cells while M2-like macrophages were detected as CD206 + F4/80 + -cells, in close proximity to actively remodeling collateral arteries in the adductor muscle. This revealed that the number of M1 macrophages was significantly increased (24%) in mice treated with galectin-2 compared to placebo-treated mice. Furthermore, the number of M2 macrophages showed a significant decrease by 40% following galectin-2 treatment compared to placebo-treated mice (Fig 7). These results indicate that galectin-2 treatment in vivo promotes M1 and inhibits M2 macrophage accumulation during expansive arterial remodeling.

Discussion
Galectin-2 expression in monocytes is associated with a low natural arteriogenic response in patients suffering from ischemic heart disease. in vivo data from a murine model of arteriogenesis showed that galectin-2 administration results in reduced arteriogenesis [11].
In the present study we describe that arteriogenesis can be affected by galectin-2 through modulation of monocyte/macrophage phenotype and physiology. Overall, we show that galectin-2 acts as a endogenous ligand that induces a proinflammatory status, in monocytes and macrophages, highly comparable to the effects of the well known exogenous modulator LPS.
We confirm and extend the previous finding that galectin-2 binds to monocytes [42], by showing its dose-dependency and, more importantly, that galectin-2 binding to human monocytes is carbohydrate-independent. Indeed, galectins may interact through protein-protein binding [1], as has been described earlier for the structurally related galectin-1 with H-ras [43]. The described affinity of galectin-2 and galectin-1 for oligosaccharides is in the micromolar range [44,45], whereas the affinity of galectin-2 for the surface of monocytes appeared to be in the nanomolar range. Together with the observed absence of an inhibiting effect of lactose on binding, this strongly indicates a highly specific protein-protein interaction. Consistent with this, we also did not observe any reduction in galectin-2-induced proinflammatory gene expression in human monocytes by lactose. This implies that also the effector functions of galectin-2 are independent of carbohydrate interaction.
Here, we provide several lines of evidence that galectin-2 requires CD14 for its binding to monocytes. CD14 high monocytes (i.e. classical and intermediate) show a higher binding to human galectin-2 than CD14 low monocytes (non-classical). Furthermore, immature DCs expressing low levels of CD14 [46] and T cells (CD14-negative) showed consistent lack of galectin-2 binding in our experiments. Finally, CD14 blocking antibodies reduced the binding of human galectin-2 to human monocytes.
CD14 is a known co-receptor for TLR4 and in rare instances also for TLR2 [23,24]. CD14 itself does not induce signaling, because it lacks a transmembrane part or cytoplasmic tail [47]. Signaling through CD14/TLR4 is MyD88-dependent, while the induction of IFN-β is MyD88-independent, and requires TRIF and IRF3 [23]. TLR2 signaling is also MyD88-dependent, but does not activate the TRIF-dependent pathway and therefore does not induce IFN-β expression. Galectin-2 strongly induced IFN-β gene expression in human monocytes to similar levels as LPS, indicating signaling through TLR4, although signaling through TLR2 is not entirely excluded. Our experiments with CD14-and TLR4-deficient murine cells confirmed that both CD14 and TLR4 are required for the NFκB-dependent induction of TNFα protein secretion by galectin-2.
Possible effects of LPS contamination were ruled out by including the LPS-neutralizing antibiotic polymyxin B as a control, at levels that completely blocked LPS responses. The possibility that galectin-2 would bind to TLR4 on monocytes via LPS was ruled out as well, by showing that polymyxin B, while preventing binding of LPS to macrophages [48], did not inhibit binding of galectin-2. Cytokine induction by galectin-2 was dependent on CD14, as established in two different models, being human cells in the presence of neutralizing CD14 antibodies and murine CD14-or TLR4-deficient cells.
Incubation of human monocytes with human galectin-2 led to increased expression of proinflammatory genes (IL-6, TNF-α, IL-12p40 and IFN-β), decreased expression of the antiinflammatory and proarteriogenic cytokine TGFβ1 [49] and decreased expression of proarteriogenic factors MMP2, MMP9, VEGF-A, PDGF-B and HGF. These effects establish a key role for galectin-2 in monocyte proinflammatory activation status. Galectin-2 also bound human M0 macrophages and stimulated their polarization to an M1-like phenotype, as evidenced by a characteristic elongated cell shape, reduced motility and induction of proinflammatory expression of surface proteins and cytokines. These findings were corroborated by a highly increased surface protein expression of the proinflammatory M1 marker CD40. In contrast to M0 and M2 macrophages, M1 macrophages do not form filopodia upon stimulation with a chemoattractant, which may explain their reduced motility and migration relative to M0 and M2 macrophages [41]. An inhibiting effect of galectin-2 on monocyte migration has been described before [42]. Here, we confirm and extend these observations, showing that migration and motility of monocytes as well as M0 -, and M2 macrophages were almost completely blocked by galectin-2. Galectin-2 stimulation of M2 macrophages also resulted in a change from a roundto an elongated cell shape accompanied by a markedly reduced motility and a reduced expression of the M2-specific marker CD206, at both mRNA and protein level.
Together, these data show that galectin-2 polarizes monocytes and macrophages to a proinflammatory M1 state, while preventing pro-arteriogenic M2 differentiation.
These in vitro results were further substantiated by our observation that galectin-2 treatment increased the number of M1-like, CD40-positive macrophages and reduced the number of M2-like CD206-positive macrophages in close proximity of actively expanding collateral arteries in a murine model for arteriogenesis.
In summary, galectin-2 is shown to be an endogenous ligand for CD14 on human monocytes, responsible for inducing a proinflammatory phenotype in monocytes and macrophages by provoking TLR4-dependent signaling. Targeting of galectin-2-mediated responses in monocytes and macrophages may provide new therapeutic strategies in coronary artery disease patients with a low arteriogenic response and a high expression of galectin-2. (TIF) S1 File. Video files: recombinant human galectin-2 induces the M1 phenotype in macrophages. Human monocyte-derived macrophage subtypes were stimulated at day 7 with vehicle (control) or 10 μg/ml recombinant human galectin-2, and morphological changes and movement were recorded by an inverted time-lapse video microscope (Olympus IX81) for 24 hours. Experiment is performed at 60% humidity, 37°C in 5% CO 2 and recorded with a 20x objective lens. The movies were converted to avi format with Cell^R software. Bar represents 200 μm.