Chemical Modulation of in Vivo Macrophage Function with Subpopulation-Specific Fluorescent Prodrug Conjugates

Immunomodulatory agents represent one of the most promising strategies for enhancing tissue regeneration without the side effects of traditional drug-based therapies. Tissue repair depends largely on macrophages, making them ideal targets for proregenerative therapies. However, given the multiple roles of macrophages in tissue homeostasis, small molecule drugs must be only active in very specific subpopulations. In this work, we have developed the first prodrug–fluorophore conjugates able to discriminate closely related subpopulations of macrophages (i.e., proinflammatory M1 vs anti-inflammatory M2 macrophages), and employed them to deplete M1 macrophages in vivo without affecting other cell populations. Selective intracellular activation and drug release enabled simultaneous fluorescence cell tracking and ablation of M1 macrophages in vivo, with the concomitant rescue of a proregenerative phenotype. Ex vivo assays in human monocyte-derived macrophages validate the translational potential of this novel platform to develop chemical immunomodulatory agents as targeted therapies for immune-related diseases.


■ INTRODUCTION
The regenerative capacity of organisms is largely influenced by the local immune response to tissue damage, with macrophages being a central component. 1 Macrophages are multifunctional phagocytic cells, which play a pivotal role in the repair of most tissues 2 and exhibit different phenotypes depending on their microenvironment. 3 Conventionally, macrophages are categorized into proinflammatory M1 and tissue-repairing M2 phenotypes. 4 Tissue regeneration depends largely on macrophages, making them promising targets for proregenerative immunomodulatory therapies. 5 However, given the plasticity and multiple roles of macrophages in tissue homeostasis, it is essential that macrophage-targeting therapies deplete only specific subpopulations. Whereas this can be partially achieved by genetic methods in transgenic models, 6 there are no chemical agents that can be clinically translated which can effectively target subpopulations of macrophages in vivo and modulate tissue regeneration.
Our group and others have developed chemical tools to examine macrophage activity in vivo. 7−10 The groups of Schultz and Bogyo described Forster resonance energy transfer (FRET) probes to monitor the enzymatic activity of macrophages in pulmonary inflammation (e.g., matrix metalloprotease 12, MMP12) 11 and cancer (e.g., cathepsins in tumor-associated macrophages), respectively. 12−14 Chang and co-workers recently reported near-infrared fluorophores with preferential uptake in macrophages and enhanced spectral properties for in vivo imaging. 15,16 These imaging probes provide generic readouts of macrophage activity but cannot modulate their function in vivo.
Prodrug−fluorophore conjugates have been described as effective tools to deliver fluorescent and therapeutic loads into target cells with enhanced selectivity and reduced side effects. 17 Most conjugates have been reported in the context of cancer therapy, either for photodynamic cell ablation 18 or for fluorescence-guided removal of tumor cells, 19,20 as healthy and cancer cells can be readily discriminated by several biomarkers. In the context of immune modulation, very few prodrugs have been reported to activate in subsets of immune cells. Blum et al. described cathepsin-regulated theranostic photosensitizers to ablate macrophages in aggressive breast cancer mouse models and in atherosclerotic plaques. 21,22 To the best of our knowledge, there are no prodrug−fluorophore conjugates that can monitor and modulate the function of M1 macrophages in vivo.
The 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) scaffold has been widely used in the development of fluorescent probes due to its excellent photophysical and cell permeability properties. 23−27 Our group has demonstrated the suitability of BODIPY fluorophores for imaging phagosomal acidification in macrophages in real time. 28 Given the phagosomal pH and hydrolytic activity of M1 murine macrophages, 29 we envisaged that prodrug−fluorophore conjugates targeting acidic phagosomes would allow us to reprogram the immune function of M1 macrophages with high precision and marginal side effects while tracking the fate of drug-treated macrophages in vivo.
Herein we have developed BODIPY−prodrug conjugates targeting M1 macrophages with negligible effects in other macrophage subpopulations. The specific release of BODIPY activatable fluorophores and cytotoxic drugs into M1 macrophages enabled blocking of the proinflammatory macrophage phenotype in vivo in a model of tissue regeneration. Besides, the translational potential of these novel conjugates in human macrophages opens multiple avenues in precision medicine for the chemical modulation of immune cell function.

■ RESULTS AND DISCUSSION
Synthesis and Characterization of Fluorescent Prodrug Activatable Conjugates for M1 Macrophages. Activatable prodrugs have been widely described in the literature as enhanced delivery molecules to improve therapeutic efficacy in target cells while reducing potential side effects in off-target cells. 30,31 For instance, glutathioneactivatable prodrugs have been successfully developed as theranostic agents with enhanced cytotoxic activity in cancer cells. 32,33 Similar chemical strategies have been also used in the construction of smart nanomaterials with increased responseto-noise ratios in specific tissues. 34,35 Among the different subpopulations of macrophages, M1 macrophages contain intracellular acidic phagosomes. Acidic phagosomes present pH values between 4.5 (late phagosomes) and 6.5 (early phagosomes), depending on their maturation state. 36,37 We envisioned that the conjugation of cytotoxic drugs to pHactivatable BODIPY fluorophores through phagosome-cleavable spacers would allow us to specifically deplete the proinflammatory activity of M1 macrophages. In order to construct BODIPY−prodrug conjugates that would be specifically cleaved in M1 macrophages but not in other macrophages, we synthesized the core scaffold of a pH-activatable fluorophore including two ester spacers at different positions of the BODIPY structure (3 and 4, Figure 1). Compounds 3 and 4 were obtained in good yields by derivatization of their corresponding isonitriles 1 and 2 via Ugi multicomponent reaction at the meso position of the BODIPY scaffold with diethylamine and methyl N-ethylglycinate, respectively ( Figure 1).
The key intermediates in the synthesis of 1 and 2 were their corresponding BODIPY nitro derivatives [see the Supporting Information for full synthetic and characterization details], which were hydrogenated, formylated with ethyl formate, and dehydrated with POCl 3 to render the corresponding pHinsensitive BODIPY isonitriles (1 and 2). First, we evaluated the fluorescence properties of the esters 3 and 4 at different pH values in order to assess their suitability to monitor phagosomal acidification in M1 macrophages. The two BODIPY esters (3 and 4) exhibited pH-dependent fluorescence emission, however very divergent pK a values ( Figure 2A and Figure S5). Compound 3 showed a pK a of 6.0 as well as strong fluorescence staining in lipopolysaccharide (LPS)-stimulated M1 macrophages but not in nonactivated macrophages ( Figures 2C and  2D). On the other hand, compound 4 (pK a : 3.2) and the pHinsensitive isonitrile 2 were unable to label M1 macrophages. These results point at the diethylamine group in compound 3 as the key structural feature to achieve pH sensitivity in the appropriate range for M1 macrophage sensing (i.e., between 4.5 and 6.5). The dissimilar behavior of compound 4 suggests that any modifications in the diethylamine group might alter not only the pH-dependent fluorescence response but also the acidotropic accumulation of BODIPY fluorophores in macrophages.
In view of these results, we derivatized compound 3 with an acid-cleavable N-acylhydrazone group for controlled release of both fluorescent and therapeutic molecules in the acidic phagosomes of M1 macrophages. 38 In order to target and block the proinflammatory function of M1 macrophages, we conjugated the profluorophore 3 to doxorubicin as a cytotoxic molecule for M1 macrophage depletion. 39 We envisaged that

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Research Article the phagosomal pH would accelerate the cleavage of the Nacylhydrazone group to activate both fluorescent and functional responses in M1 macrophages. We prepared the BODIPY− doxorubicin hydrazone 5 by hydrolysis of the ethyl ester 3 in acidic conditions, followed by one-pot hydrazide formation via activation as succinidimyl ester and reaction with hydrazine in THF. The conjugation of the hydrazide to the carbonyl group of doxorubicin was performed in MeOH using catalytic amounts of trifluoroacetic acid to render compound 5 as an activatable BODIPY−prodrug with a pH-labile linker ( Figure  1). The nonlabile BODIPY−doxorubicin amide conjugate 6 was also prepared as a negative control. In this case, activation of compound 3 with (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)-dimethylamino-morpholino-carbenium hexafluorophosphate (COMU) and DIPEA in DMF, followed by conjugation to the amine group of doxorubicin, yielded the amide derivative 6. Furthermore, hydrazone and amide BODIPY−doxorubicin conjugates of the ester 4 (compounds 7 and 8, respectively) were also prepared as additional negative controls of BODIPY− prodrug conjugates including labile groups not matching the phagosomal pH in M1 macrophages ( Figure 1). Notably, the spectral characterization of all derivatives showed that excitation and emission wavelengths of the conjugates remained unaltered after conjugation to doxorubicin (Table S1 and Figure S7), with the BODIPY core being the main contributor to the fluorescence emission ( Figure S8).
Functional Assays in M1 and M2 Macrophage Subpopulations. First, we evaluated the suitability of the BODIPY−doxorubicin conjugates as efficient prodrugs for macrophages. We compared the cell viability of nonactivated RAW264.7 murine macrophages after treatment with doxor-ubicin alone and with the conjugates 5−8 ( Figure S6). Whereas doxorubicin induced significant dose-dependent cell toxicity, none of the BODIPY conjugates showed any effects in the cell viability of macrophages, indicating that the cytotoxic action of doxorubicin was effectively blocked via derivatization with Nacylhydrazone (5, 7) or amide (6,8) groups. Furthermore, we also confirmed that the BODIPY fluorophores on their own (compounds 3 and 4) did not induce any cytotoxicity in macrophages ( Figure S6).
Next, we assessed the functional effects of compound 5 in separate M1 and M2 macrophage subpopulations. We employed reported protocols to activate macrophage cells toward classical M1 (treatment with LPS) or alternative M2 phenotypes (treatment with IL-4) and incubated them with different concentrations of compound 5. As shown in Figure  3A, the phagosomal acidification in M1 macrophages enabled the release of the cytotoxic doxorubicin in a dose-dependent manner, whereas no functional effects were observed in either quiescent or M2 macrophages. Polarization of macrophages toward M1 or M2 phenotypes was confirmed by expression of cell-surface markers in flow cytometry analysis (i.e., CD86 for M1 macrophages, 40 CD206 in M2 macrophages 41 ) ( Figure 3B) and nitric oxide (NO) production 42 ( Figure 3C). Notably,  represented as means ± SD (n = 4). n.s. for p > 0.05, * for p < 0.05, ** for p < 0.01, *** for p < 0.001.

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Research Article incubation of M1 and M2 macrophage subpopulations with the noncleavable amide derivative 6 did not cause any cytotoxicity, indicating the phagosomal pH-induced cleavage as the primary mechanism for the specific release of doxorubicin in M1 macrophages ( Figure S9). HPLC−MS analysis of the acidcleavable hydrazone 5 confirmed the cleavage of doxorubicin only under acidic environments, with no drug release observed at neutral pH or for the noncleavable amide conjugate 6 ( Figure S10). We further characterized the functional effects of the hydrazone 5 in M1 macrophages by immunochemical analysis ( Figure S11). In these assays, we measured the levels of several cytokines in LPS-stimulated M1 macrophages that had been treated or not with hydrazone 5 and observed a reduction in the production of key cytokines and chemokines (e.g., TNFα) associated with the M1 macrophage phenotype. In addition, we corroborated that IL-4-stimulated macrophages presented increased arginase activity as a result of their M2 polarization 43 ( Figure S12).
Fluorescence Analysis of M1 Macrophages. Prodrug− fluorophore conjugates are advantageous in that they enable monitoring cell fate upon drug release using fluorescence readouts. 44 As such, we compared the fluorescence staining of nonactivated macrophages and M1 macrophages after incubation with compound 5 by flow cytometry. Nonactivated macrophages displayed low BODIPY fluorescence staining ( Figure 4A), while strong fluorescence emission was observed in LPS-stimulated M1 macrophages (Figures 4B), indicating the efficient cell uptake of compound 5 and its intracellular activation upon phagosomal acidification. Furthermore, we compared the apoptotic index of both macrophage subpopulations by coincubation with Annexin V as a direct functional readout of cellular apoptosis due to doxorubicin release inside macrophages. The low apoptotic index in nonactivated macrophages increased 3-fold in LPS-and 5-treated M1 macrophages ( Figure 4C). Moreover, apoptotic cells (i.e., Annexin V stained cells) displayed also strong BODIPY fluorescence ( Figure 4B), confirming the simultaneous release of doxorubicin and the BODIPY fluorophore in M1 macrophages.
Next, in order to assess the subcellular localization of the released cargo in live macrophages, we imaged RAW264.7 macrophages after incubation with compound 5 under fluorescence confocal microscopy. Macrophages were stimulated with zymosan, a glycan isolated from yeast cells that induces classical M1 activation. 45 Zymosan beads were conjugated to the pH-insensitive fluorophore Texas red so that two-color fluorescence images could be acquired. As shown in Figure 4E, strong colocalization was observed between the green BODIPY fluorophore and red-fluorescent zymosan in the intracellular phagosomes of live macrophages. This observation confirms that M1 macrophages uptaking zymosan beads turn on the pH-activatable BODIPY fluorophore. To confirm this hypothesis, we preincubated live macrophages with bafilomycin Aan inhibitor of the vacuolar-type H + -ATPase required for phagosomal acidification 46 and then treated them with red zymosan beads and compound 5. The images shown in Figure  4F confirmed that phagosomal acidification was the main intracellular mechanism for the activation of the pH-sensitive BODIPY fluorophore, as bafilomycin-treated M1 macrophages were not green fluorescently stained despite having taken up red zymosan beads. Together with the functional assays, these results assert the potential of compound 5 as an effective prodrug and activatable BODIPY imaging agent to visualize and modulate the function of M1 macrophages.
Subpopulation Discrimination of Macrophages and ex Vivo Assays in Human Macrophages. To evaluate the selective activation of compound 5 in mixtures of cells with different phenotypes, we cultured M1 and M2 macrophages in a Transwell assay to coincubate both subpopulations with the same concentration of compound 5. RAW264.7 macrophages were plated and polarized toward M1 and M2 populations and then treated for 20 h with compound 5 under physiological conditions. The two subpopulations were exposed to the same experimental conditions, being only separated by a membrane permeable to small molecules ( Figure 5A). After incubation, both subpopulations were isolated, treated with Annexin V, and analyzed by flow cytometry. As shown in Figure 5B, M1 macrophages showed around 4-fold higher apoptotic index than M2 macrophages (indicated by Annexin V staining, 72% vs 19%). Annexin V positive cells were also stained with the BODIPY fluorophore, confirming the enhanced hydrolytic activity of M1 over M2 macrophages after 20 h. These results confirm the preferential activation of compound 5 (i.e., doxorubicin intracellular release and fluorescence emission) in M1 macrophages, even in the presence of other macrophage subpopulations.
Whereas in vivo cell ablation has been achieved by incorporating genetically encoded receptors (e.g., diphtheria toxin receptor, DTR) into specific cell lineages, 47 including macrophages, such approaches are poorly translatable to clinical environments. In order to examine the translational potential of compound 5 to deplete subpopulations of human M1 macrophages, we isolated and cultured monocyte-derived macrophages from human peripheral blood. Macrophages were stimulated with LPS and polarized toward the M1 phenotype, which was confirmed by increased expression of the cell-surface marker CD86 (Figures 5C and 5D) and the tumor necrosis factor alpha (TNF-α) 48 ( Figure S13). Under these conditions, human macrophages were treated with both activatable and nonactivatable BODIPY−prodrug conjugates (i.e., compounds 5 and 6, respectively). The incubation of LPSstimulated human M1 macrophages with compound 5 led to significant enhancement of the fluorescence emission ( Figures  5C and 5D) and effective doxorubicin release that resulted in reduced cell viability (Figures 5E and 5F). Flow cytometric analysis confirmed the double-positive staining of human M1 macrophages with a fluorescently labeled anti-CD86 antibody and compound 5 ( Figure S14), indicating preferential activation of compound 5 in human M1 macrophages. Furthermore, we also corroborated that the treatment of human M1 macrophages with the noncleavable BODIPY− prodrug 6 did not cause any cytotoxic effect ( Figure S15). These results suggest that phagosomal pH is a speciesindependent activation mechanism to release prodrugs in defined subpopulations of macrophages and also assert the potential of compound 5 for translational studies in human macrophages.
In Vivo Functional and Imaging Assays. In view of the excellent properties of 5 as a BODIPY−prodrug conjugate to deplete subpopulations of macrophages, we investigated its application to modulate immune cell function in a zebrafish model of tissue regeneration. When their tail fin is amputated at 2 days postfertilization (dpf), zebrafish larvae regenerate their

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Research Article fin within 72 h postwounding (hpw). Under these conditions, macrophages migrate toward the amputation site within 1−2 hpw and their number is typically maintained to enable tissue regeneration. 49 We explored the applicability of compound 5 to visualize recruited macrophages by fluorescence imaging as well as to alter tissue regeneration in situ by depleting M1 macrophages in vivo. Upon tail fin amputation, we treated zebrafish larvae with compound 5 and performed time-lapse imaging by live fluorescence confocal microscopy. As shown in Figure 6A and Movie S1, mCherry-expressing macrophages were recruited to the injury site but only a few macrophages showed green fluorescence corresponding to the intracellular activation of compound 5. This is in agreement with reported observations that correlate the initial recruitment of macrophages to injury sites to the M2 wound-healing phenotype and not the M1 proinflammatory phenotype. 50 Images at longer time points [i.e., 24 h post-treatment (hpt), Figure 6B and Movie S2] showed enhanced green fluorescence in the intracellular phagosomes and reduced cell mobility, likely due to an increased polarization toward the M1 phenotype. However, we did not see significant changes in macrophage numbers at 48 hpw after the treatment with compound 5 ( Figure 6E), which suggests that M1 macrophages may represent a relatively small subpopulation in the tail fin under these experimental conditions. Therefore, we treated wounded larvae with LPS, which leads to a marked increase in TNF-α M1 macrophages in the regenerating fin in vivo ( Figure S16). Indeed, whereas most macrophages in 5-treated larvae showed weak green fluorescence and unaffected morphology ( Figure  6C), brighter fluorescence emission was detected in the wound edges of zebrafish treated with LPS and compound 5, with most macrophages showing green fluorescent phagosomes and rounded cellular morphology, characteristic of apoptotic and necrotic cells due to the release of doxorubicin ( Figure 6D).
We further confirmed this observation using TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining, which is used to detect apoptotic cells that undergo extensive DNA degradation, and observed increased apoptosis in zebrafish larvae that had been cotreated with LPS and compound 5 ( Figure S17). Under these conditions, a significant reduction in the macrophage numbers within the regenerating fin was observed when compared to the DMSO control ( Figure 6E and Figure S18).
Whereas there is experimental evidence that macrophages are required for tail fin regeneration, the exact contribution of specific macrophage subpopulations in the regenerative process is not fully understood. Thus, we measured the regeneration of the tail fin area under different experimental conditions ( Figure  6F and Figure S19). First, we observed a significant reduction in the regenerated area for 5-treated larvae, suggesting that the depletion of M1 macrophages can impair tissue regeneration. Second, in vivo stimulation with LPS, which enhances M1 macrophage polarization, and cotreatment with compound 5 were sufficient to rescue tail fin regeneration to a similar level as the DMSO control. These results are in agreement with the recent observations described by Nguyen-Chi et al. in which TNF-α positive M1 macrophages but not TNF-α negative macrophage subpopulations were required for tissue regeneration in zebrafish. 51

■ CONCLUSIONS
We have developed novel fluorogenic BODIPY−prodrug conjugates targeting proinflammatory subpopulations of macrophages. To the best of our knowledge, these are the first chemical probes able to modulate the function of M1 macrophages in vivo without affecting other macrophage subpopulations. Specific cleavage of the prodrug conjugates within the acidic phagosomes of M1 macrophages led to the

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Research Article intracellular release of a pH-activatable fluorophore as well as the cytotoxic drug doxorubicin for in situ cell tracking and subpopulation-specific macrophage depletion. We have demonstrated the applicability of these conjugates in vivo using regeneration models to image and deplete phagocytic M1 macrophages. Notably, we have observed a proregenerative role for TNF-α expressing M1 macrophages in vivo, which opens new avenues to noninvasively interrogate the contribution of macrophage subpopulations in multiple pathologies without the need for transgenic modifications. This platform will accelerate the development of chemical immunomodulatory agents as subpopulation-specific targeted therapies for immune-related disorders.

■ EXPERIMENTAL SECTION
General Materials. Commercially available reagents were used without further purification. Thin-layer chromatography was conducted on Merck silica gel 60 F254 sheets and visualized by UV (254 and 365 nm). Silica gel (particle size 35−70 μm) was used for column chromatography. 1 H and 13 C spectra were recorded in a Bruker Avance 500 spectrometer (at 500 and 125 MHz, respectively). Data for 1 H NMR spectra are reported as chemical shift δ (ppm), multiplicity, coupling constant (Hz), and integration. Data for 13 C NMR spectra are reported as chemical shifts relative to the solvent peak. HPLC− MS analysis was performed on a Waters Alliance 2695 separation module connected to a Waters PDA2996 photodiode array detector and a ZQ Micromass mass spectrometer (ESI-MS) with a Phenomenex column (C 18 , 5 μm, 4.6 × 150 mm). Conjugates were purified using a Waters semipreparative HPLC system using a Phenomenex column (C 18 Axial, 10 μm, 21.2 × 150 mm) and UV detection.

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Research Article BODIPY Hydrazone 7. N 2 was bubbled through a suspension of doxorubicin (12 mg, 0.020 mmol) and the corresponding hydrazide BODIPY 20 ( Figure S3) (15 mg, 0.030 mmol) in anhydrous MeOH (6 mL) for 30 min. Then TFA (0.5 mL) was added, and the reaction mixture was stirred for 48 h at rt in the dark. Finally, volatiles were removed under reduced pressure, and the resulting residue was purified by semipreparative HPLC to obtain 7 as a red solid, synthetic yield 17%. HPLC (H 2 O−ACN with 0.1% HCOOH): t R 5.58 min. 1 Figure S3) (7 mg, 0.015 mmol) in DMF (0.5 mL) was added DIPEA (8 μL, 0.045 mmol). The solution was stirred for 15 min, and COMU (9 mg, 0.022 mmol) was added predissolved in DMF (0.2 mL). After 6 h stirring at rt, the crude mixture was extracted with CH 2 Cl 2 , and the organic extracts were dried over MgSO 4 , filtered, and evaporated under reduced pressure. The mixture was purified by semipreparative HPLC to obtain 8 as a red solid, synthetic yield 26%. HPLC (H 2 O−ACN with 0.1% HCOOH): t R 6.6 min. 1  Spectral Characterization of BODIPY−Prodrug Conjugates. Spectroscopic and quantum yield data were recorded on a Synergy HT spectrophotometer (Biotek). Compounds were dissolved at the indicated concentrations, and spectra were recorded at rt. Spectra are represented as means from at least two independent experiments with n = 3. Quantum yields were calculated by measuring the integrated emission area of the fluorescence spectra and comparing it to the area measured for fluorescein in basic EtOH as reference (QY: 0.97). 52 Cell Culture and Polarization to M1 and M2 Macrophages. RAW264.7 macrophages were grown in DMEM cell culture media supplemented with 10% FBS, antibiotics (100 U mL −1 penicillin, 100 mg mL −1 streptomycin), and 2 mM Lglutamine in a humidified atmosphere at 37°C with 5% CO 2 . For M1 polarization, macrophages were treated with LPS (100 ng mL −1 ) for 18 h as reported. 53 M2 macrophages were isolated upon treatment with IL-4 (100 ng mL −1 ) for 18 h as reported. 54 Flow cytometry analysis of M1 and M2 mouse macrophage populations was performed as detailed below using fluorescent anti-CD86-APC (2 μg mL −1 , Biolegend) and anti-CD206-APC antibodies (2 μg mL −1 , Biolegend) as M1 and M2 markers, respectively.
For NO production assays, cell supernatants from RAW264.7 macrophages (∼90−100% confluence in 24-well plates) that had been polarized or not toward M1 or M2 as described above were collected. NO production was determined using the Griess reagent (Sigma) in which equal volumes (100 μL) of Griess reagent and cell supernatants were mixed and incubated in the dark for 15 min before determining the absorbance at 540 nm in a spectrophotometer.
For arginase activity assays, mouse macrophages were polarized or not toward M1 or M2 as detailed above and the production of urea generated by the arginase-dependent hydrolysis of L-arginine was measured as reported. 55 Cell Viability. Cell viability was determined using a TACSR MTT Cell Proliferation assay (Trevigen) following the manufacturer's instructions. Briefly, RAW264.7 macrophages were plated on 96-well plates and stimulated as described above when appropriate, reaching 90−95% confluence on the day of the experiment. Compounds were added to the cells at indicated concentrations and incubated at 37°C overnight. Then cells were washed and treated according to the manufacturer's instructions, and their absorbance values (570 nm) were measured in a Synergy HT spectrophotometer (Biotek). Data analysis was performed using GraphPrism 5.0. Cell viability data was normalized to the proliferation of the cells in cell culture medium.
Cytokine Immunochemical Profiling. Cytokine levels were determined using a Mouse Cytokine Array Panel A (Proteome Profiler, R&D System) following the manufacturer's instructions. Briefly, RAW264.7 macrophages were plated on 12-well plates on the day before the experiment, reaching 70− 80% confluence on the day of the experiment. Macrophages were polarized toward the M1 phenotype with LPS (100 ng mL −1 ) and incubated in the presence or absence of compound 5 (10 μM) at 37°C for 24 h. Cell supernatants were collected, centrifuged, and treated following the manufacturer's instructions. Membranes were developed in an X-ray Ecomax Processor (Photon Imaging System), scanned, and analyzed using ImageJ.
Flow Cytometry. RAW264.7 macrophages were plated on 24-well plates 4 h before the experiment and incubated at 37°C . Macrophages were stimulated (with LPS or IL-4), and compounds were added to the cells at the indicated concentrations and incubated at 37°C for 20 h. Cells were detached, resuspended in CaCl 2 buffer (20 mM HEPES Buffer, 140 mM NaCl, 2 mM CaCl 2 , 0.1% BSA), and analyzed by flow cytometry (BD FACSCalibur cytometer, Becton Dickinson) using Annexin V-AF647 (Invitrogen) as the marker for apoptotic cells and/or compound 5 as indicated. Data analysis was performed with the software Flowjo.
Live-Cell Fluorescence Confocal Microscopy. Cells were plated on glass chamber slides Lab-Tek II (Nunc), stimulated with Texas Red conjugated Zymosan A S. cerevisiae BioParticles (0.05 mg mL −1 ), washed with PBS, and incubated with compound 5 (150 nM) at 37°C. Cells were imaged under a Zeiss LSM510 META fluorescence confocal microscope equipped with a live cell imaging stage. Fluorescence and brightfield images were acquired using a 40× oil objective. Fluorophores were excited with 488 nm (BODIPY) or 543 nm (Texas Red) lasers. All images were analyzed and processed with ImageJ.

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