In Vitro Multiparameter Assay Development Strategy toward

Article pubs.acs.org/molecularpharmaceutics

In Vitro Multiparameter Assay Development Strategy toward Differentiating Macrophage Responses to Inhaled Medicines Ewelina Hoffman,†,#,○ Abhinav Kumar,†,○ Varsha Kanabar,†,‡ Matthew Arno,§ Lucas Preux,† Val Millar,∇ Clive Page,†,‡ Helen Collins,∥ Ian Mudway,⊥ Lea Ann Dailey,*,† and Ben Forbes† †

Institute of Pharmaceutical Science, ‡Sackler Institute of Pulmonary Pharmacology, §Genomics Facility, ⊥MRC-PHE Centre for Environment and Health, Division of Analytical and Environmental Sciences, Franklin-Wilkins Building, King’s College London, 150 Stamford Street, London SE1 9NH, United Kingdom ∥ Division of Immunology, Infection and Inflammatory Diseases, Guy’s Campus, King’s College London, 15-16 Newcomen Street, London SE1 1UL, United Kingdom # Medical University of Łódź, Muszyńskiego 1, 90-151 Łódź, Poland ∇ GE Healthcare Life Sciences, The Maynard Centre, Forest Farm Road, Whitchurch, Cardiff CF14 7YT, United Kingdom ABSTRACT: Although foamy macrophages (FMΦ) are commonly observed during nonclinical development of medicines for inhalation, there are no accepted criteria to differentiate adaptive from adverse FMΦ responses in drug safety studies. The purpose of this study was to develop a multiparameter in vitro assay strategy to differentiate and characterize different mechanisms of drug-induced FMΦ. Amiodarone, staurosporine, and poly(vinyl acetate) nanoparticles were used to induce distinct FMΦ phenotypes in J774A.1 cells, which were then compared with negative controls. Treated macrophages were evaluated for morphometry, lipid accumulation, gene expression, apoptosis, cell activation, and phagocytosis. Analysis of vacuolization (number/area vacuoles per cell) and phospholipid content revealed inducer-dependent distinctive patterns, which were confirmed by electron microscopy. In contrast to the other inducers, amiodarone increased vacuole size rather than number and resulted in phospholipid accumulation. No pronounced dysregulation of transcriptional activity or apoptosis was observed in response to sublethal concentrations of all inducers. Functionally, FMΦ induction did not affect macrophage activation by lipopolysaccharide, but it reduced phagocytic capacity, with different patterns of induction, severity, and resolution observed with the different inducers. An in vitro multiparameter assay strategy is reported that successfully differentiates and characterizes mechanisms leading to FMΦ induction by different types of agents. KEYWORDS: alveolar macrophage, foamy macrophage, toxicology, vacuolation, morphometry

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INTRODUCTION Foamy macrophages (FMΦ) are a common observation during nonclinical development of medicines for inhalation.1 FMΦ is a term used to describe an alveolar macrophage with a vacuolated cytoplasmic appearance when viewed by light microscopy,1,2 and safety concerns related to their induction contribute significantly to compound attrition rates in the development of new inhaled medicines.1,3 Inhaled drugs may induce the foamy appearance in alveolar macrophages via different pharmacological and nonpharmacological mechanisms. In some cases, the development of FMΦ may simply be a physiological adaptive and reversible response to inhaled particulates,1,4 whereas similar-looking FMΦ can be indicative of an adverse reaction, especially if other signs of inflammation are present. It is important to be able to screen for macrophage responses that produce detrimental effects in the lungs and distinguish them from molecules that induce an adaptive FMΦ response, i.e., false positives in safety terms that may lead to potentially useful drugs being withdrawn from development prematurely. Currently, there are no universally accepted criteria to differentiate adaptive from adverse responses when FMΦ are © 2015 American Chemical Society

observed in inhaled drug safety studies. High doses are often required nonclinically in toxicology studies to establish safety margins for a component to move into clinical development, enhancing the possibility of observing abnormal macrophage responses.5,6 Deficiencies in our knowledge of different macrophage phenotypes and their significance to lung biology remain a significant challenge in the development of inhaled medicines. This is particularly so in terms of our current poor understanding of FMΦ biology. Ideally, compounds that induce FMΦ should be identified in early development using in vitro studies and characterized according to induction mechanisms and FMΦ phenotypes that are well-understood in terms of their safety implications. This would screen out unsuitable drug candidates at an early stage, provide more efficient medicines development, and reduce Special Issue: Advances in Respiratory and Nasal Drug Delivery Received: Revised: Accepted: Published: 2675

January 16, 2015 April 27, 2015 May 5, 2015 May 5, 2015 DOI: 10.1021/acs.molpharmaceut.5b00048 Mol. Pharmaceutics 2015, 12, 2675−2687

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Molecular Pharmaceutics

Figure 1. Multiparameter in vitro assay strategy for differentiating alveolar macrophage responses to inhaled pharmaceuticals.

Table 1. Panel of FMΦ Inducers/Controls Chosen To Explore Different Mechanisms of FMΦ Development in the Hierarchical in Vitro Screening Strategy inducers/controls

H2O solubility (25 °C)

FMΦ inducers

controls

amiodarone

∼1 mM

staurosporine poly(vinyl acetate-co-alcohol) nanoparticles (PVAc NP)

∼10−20 μM insoluble

salbutamol (base) poly(vinyl alcohol) solution (PVA)

∼60 mM ∼630 mg/mL

rationale for study inclusion cationic amphiphilic drug exposure Mechanism: drug complexation with phospholipids prevents metabolism, direct inhibition of phospholipases16,17 apoptosis Mechanism: protein kinase C inhibitor and potent inducer of apoptosis, some stages of apoptotic macrophages take on a foamy appearance18,19 poorly soluble nanomaterial Postulated mechanism: intracellular polymer hydrolysis or other degradation process leading to lysosomal destabilization and induction of autophagy21 established inhaled pharmaceutical vehicle control for PVAc NP20

alcohol) nanoparticles (PVAc NP);20 we used poly(vinyl alcohol) (PVA; a low molecular weight polymer used as an amphiphilic coating material for PVAc NP)20 and salbutamol base (a widely used inhaled pharmaceutical not previously reported to induce FMΦ) as controls. It was hypothesized that FMΦ induction could be quantified in a dose-dependent manner and a unique FMΦ profile could be established for each inducer, which would include information not only on the chemistry of vacuolar bodies but also on the transcriptional, metabolic, and functional status of the cells following exposure to different inducers. The compilation of such a profile for a variety of pharmaceutical inducers is the first step toward a decision-tree-based approach, which could be used to screen out compounds early in the development process if they show potential for causing adverse macrophage responses in vivo.

unnecessary animal use in nonclinical studies. Knowledge of the different mechanisms by which the FMΦ phenotype can arise and resolve is key to understanding the safety implications of this phenomenon. FMΦ are induced by phagocytosis of poorly soluble drug particles, stimulation of excess lung surfactant (taken up by alveolar macrophages), impaired metabolism of intracellular phospholipids, phagocytosis of surfactant phospholipid−drug complexes, apoptosis, and autophagy.1−3 FMΦ are also observed during progression of different lung disorders, such as pulmonary alveolar proteinosis7 and tuberculosis infection.8 The aim of this study was to design and evaluate a multiparameter in vitro assay strategy (Figure 1) that will identify agents that induce the FMΦ, distinguish between different mechanisms resulting in FMΦ induction, and provide information on the cellular and functional status of FMΦ. The strategy combines a variety of techniques, which have been used previously, often separately, to characterize FMΦ. These include cellular imaging to quantify morphometry and phospholipid accumulation,9,10 toxicogenomic approaches,10−12 and functional assays.13−15 A panel of three model pharmaceutical compounds, one nanoparticle formulation, and one polymer solution (Table 1) was chosen for a pilot study of the FMΦ assay strategy. Three of the panel agents are known inducers of the FMΦ phenotype: amiodarone,16,17 staurosporine,18,19 and poly(vinyl-acetate-co-

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MATERIALS AND METHODS Materials. Amiodarone hydrochloride, staurosporine, salbutamol base, low molecular weight poly(vinyl acetate) (12.8 kDa), and poly(vinyl alcohol) (PVA; 8−12 kDa) were purchased from Sigma-Aldrich (Dorset, UK). All other materials were of analytical grade. Cell Culture and Reagents. J774A.1 cells (derived from Balb/c mice) were chosen as a model macrophage-like cell line due to their accessibility, constant and rapid growth rate, and suitability as a comparator cell line for in vivo alveolar 2676

DOI: 10.1021/acs.molpharmaceut.5b00048 Mol. Pharmaceutics 2015, 12, 2675−2687

Article

Molecular Pharmaceutics macrophage responses observed in a Balb/c murine model.20 Cells were cultured in a humidified atmosphere of 5% CO2 at 37 °C. Culture media consisted of high glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Life Technologies, UK) supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies, UK), 1 μg/mL streptomycin and penicillin (Gibco, Life Technologies, UK), and 1 mM sodium pyruvate (Gibco, Life Technologies, UK). Cells were routinely subcultured when 70−80% confluent and seeded in multiwell plates. Manufacture and Characterization of PVAc NP. Nanoparticles were manufactured from a 12.8 kDa poly(vinyl acetate-co-alcohol) polymer with approximately 34 mol % hydroxyl groups and 66 mol % residual acetate groups, as confirmed by NMR analysis. Nanoparticles were prepared and characterized as described by Jones et al.20 PVAc NP size was characterized by photon correlation spectroscopy (Zetasizer NanoZS, Malvern, UK), and the mean hydrodynamic diameter of all batches produced was 170 ± 33 nm. Cytotoxicity. J774A.1 cells were seeded at a density of 25 000 cells/well in a 96-well plate and cultured overnight. The cells were then exposed to various concentrations of inducers for 6, 24, and 72 h. At the end of each treatment period, cells were washed with culture medium before incubating with 0.8 mg/mL 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) in culture medium for 4 h to allow MTT conversion by mitochondrial enzymes within the cells to form insoluble blue formazan crystals. The crystals were solubilized with 10% (w/v) SDS in 0.01 M HCl, and the color change was quantified at 570 nm. The metabolic activity was expressed as a percentage of the mean value for untreated cells. Cell Proliferation. J774A.1 cells were seeded at a density of 25 000 cells/well in a 96-well plate and cultured overnight. The cells were then exposed to various concentrations of inducers/ controls diluted in cell culture medium for 6, 24, and 72 h. At the end of each treatment period, all treatments were aspirated and cells were fixed with 4% (v/v) paraformaldehyde for 30 min at room temperature. The fixative was then aspirated, and 1% (w/v) methylene blue in 0.01 M sodium borate buffer, pH 8.5, was added for 30 min at room temperature. Cells were washed thoroughly with 0.01 M sodium borate buffer, and the dye was finally eluted with ethanol and 0.1 M HCl mixture and quantified at 660 nm. The cell proliferation rate was expressed as a percentage of the mean value for untreated cells. Cell Morphology and Morphometric Analysis. J774A.1 cells were seeded at a density of 2500 cells/well in a 96-well plate and cultured for 48 h prior to treatment with all compounds over a range of eight concentrations. Cell staining was performed following the protocol of the manufacturer (Invitrogen, UK). Briefly, macrophages were incubated with HCS LipidTOX Red phospholipidosis detection kit to detect macrophage phospholipid accumulation in the presence of eight concentrations of FMΦ inducers/controls for 24 or 72 h. Following LipidTOX Red staining, cells were fixed with 4% paraformaldehyde in PBS containing Hoechst 33342 dye for nuclei staining. Each plate was incubated for 30 min at room temperature, and after washing with PBS, the cell cytoplasm was stained using HCS Cell Mask for 1 h at room temperature. Afterward, cells were washed with PBS and imaged using an IN Cell Analyzer 2200 (GE Healthcare, UK). Data analysis was performed using the Developer Toolbox within the IN Cell Investigator image analysis software (GE Healthcare, UK). Two quantitative outputs, mean vacuole number and mean vacuole

area as a percentage of the total cell area, were analyzed to characterize FMΦ development. Data are reported as the mean ± SD from three individual experiments using three different cell passage numbers. Gene Expression Studies. J774A.1 cells were seeded at a density of 106 cells/well in a 6-well plate and cultured overnight. The cells were then exposed to amiodarone (5 μM), staurosporine (1 nM), PVAc NP (1 mg/mL), or PVA solution (0.4 mg/mL) diluted in cell culture medium for 6 and 24 h. Total RNA was extracted from approximately 106 cells using lysis buffer provided with RNeasy plus mini kit (Qiagen) following the manufacturer’s instructions and quantified with a Nanodrop 1000 (Thermo Scientific). The high capacity RNAto-cDNA reverse transcription kit (Applied Biosystems) was employed for reverse transcription of RNA into cDNA. q-RTPCR was carried out using the Applied Biosystems 7900HT Fast PCR system with TaqMan probes for specific genes of interest. β-actin, GAPDH, and POLDIP3 were used as the reference gene for normalization of cycle thresholds. A hot start at 95 °C for 10 min was followed by 40 cycles at 95 °C for 15 s and 65 °C for 1 min. Amplification data were analyzed using SDS 2.4 and DataAssist (V3) software packages. The comparative cycle threshold method (ΔCT) was employed for data analysis.22 The CT values for all genes of interest (GOI) were normalized against the CT value of the housekeeping gene (HKG), i.e., ΔCT (GOI) = CT (GOI) − CT (HKG). For treatment groups, ΔΔCT value of treated cells was calculated relative to their respective untreated control, i.e., ΔΔCT (GOI) = (ΔCT (GOI compound treatment) − ΔCT (GOI untreated control)). This was used to calculate the foldchange (FC) of treated cells relative to control cells for each compound (FC = 2−ΔΔCT). Statistical significance of FC GOI from treatment groups relative to FC in GOI of untreated control cells was considered for P < 0.05 using a paired, twotailed Student’s t test. The volcano score, which takes into account both the magnitude of transcript regulation (FC) and the statistical significance of the change (p-value), was calculated for each GOI using the FC and p-values calculated above. The volcano score is defined as v = log (FC) × log (p-value), and transcripts were considered potentially dysregulated after treatment when v ≤ −0.103, when FC > 1.2 and p < 0.05. The volcano plot was prepared according to the method of Glaves et al.23 Apoptosis. J774A.1 cells were seeded at 300 000 cells/well in a 24-well plate and incubated for 48 h prior to treatment. The cells were then exposed to various concentrations of inducers/controls diluted in cell culture medium for 6 and 24 h. At the end of each treatment period, cells were washed with icecold PBS and lysed, and the lysate was collected for colorimetric assessment of caspase-3 activity (Sigma-Aldrich, UK). Briefly, 5 μL of lysate or caspase-3 positive control (5 μg/ mL) was diluted with 90 μL of assay buffer in a 96-well plate and mixed gently with 5 μL of the caspase-3 substrate, AcDEVD-pNA (20 mM). The mixture was incubated overnight at 37 °C, and the enzyme-catalyzed release of pNA (pnitroaniline) was monitored at 405 nm using a microtiter plate reader. The caspase-3 activity was calculated according to the manufacturer’s instructions. Classical (M1) Activation and Response to LPS Challenge. J774A.1 cells were seeded at a density of 25 000 cells/well in a 96-well plate and cultured overnight. Two dosing schemes were explored; the first evaluated whether the normal macrophage response to LPS challenge was altered by the 2677


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Figure 2. Cell proliferation (left) and metabolic activity (right) of J774A.1 cells treated with different foamy macrophage inducer and control compounds: (A) amiodarone, (B) staurosporine, (C) PVAc NP/PVA solution, and (D) salbutamol base over a range of concentrations. Cell proliferation and metabolic activity were measured after 6, 24, and 72 h incubation periods. Data represent the mean ± SD from three individual experiments. Difference from untreated cells: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

The second dosing scheme evaluated whether activated and nonactivated macrophages respond differently to inducer compounds. In this case, cells were first primed with fresh medium ± IFN-γ (500 IU) for 12 h prior to addition of inducers/controls diluted in cell culture medium at the concentrations listed above ± LPS (100 ng/mL). Following a further 24 h incubation period, the amount of nitrite present in the supernatant was measured as described above. Phagocytosis. J774A.1 cells were seeded at a density of 300 000 cells/well in a 24-well plate and cultured overnight. The cells were then exposed to two concentrations of inducers/ controls diluted in cell culture medium for 6 and 24 h. At the end of each treatment period, the cell culture medium was removed and replenished with 200 μL of fresh cell culture medium containing 1 μm polysterene beads (ThermoFischer Scientific, USA) at a concentration of 2 × 108 particles/mL and

FMΦ state. To this purpose, FMΦ were exposed 24 h to amiodarone (5 μM), staurosporine (1 nM), PVAc NP (1 mg/ mL), and the controls (PVA solution (0.4 mg/mL) and salbutamol base (4 mM)) diluted in cell culture medium. The treatment medium was then removed and replaced with fresh medium ± IFN-γ (500 IU) followed by a 12 h incubation before replacement with fresh medium ± LPS (100 ng/mL). Following a further 24 h incubation period, the supernatant was collected and nitric oxide production was determined in the form of total nitrites using the Greiss reaction. Briefly, 50 μL of supernatant was mixed with 50 μL of Greiss reagent (1:1 mixture of 1% sulfanilamide in 5% phosphoric acid and 0.1% αnaphthylamine in distilled water). The mixture was incubated for 5 min at room temperature, the absorbance was measured at 540 nm, and the amount of nitrite was calculated from a standard curve of sodium nitrite. 2678


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Molecular Pharmaceutics incubated for 2 h. The cells were then washed three times with 1 mL of PBS and lysed in N-methyl-pyrrolidone/25 mM Tris buffered saline, pH 8.1 (1:3). The fluorescence of the cell lysate was measured with exitation at 468 nm and emission at 504 nm, and the concentration of internalized beads was calculated using a calibration curve. The mean ± SD is reported from n = 3 individual experiments with different passage numbers. Statistics. All experiments were repeated independently at least three times. When applicable, statistical comparisons were made either using ANOVA or Student’s t test. Values of p < 0.05 were considered to be significant and denoted *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

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RESULTS Determination of the Sublethal Dose Range. The J774A.1 macrophage-like cell line, derived from a Balb/c mouse, was chosen for this study to compare with previous in vivo observations of foamy alveolar macrophage development in this strain of mouse.20 Exploratory studies were carried out to determine the highest concentration of inducer and control compounds that did not cause significant cytotoxicity over the 72 h study period. Two measures of cytotoxicity were evaluated, i.e., cell proliferation rate and metabolic activity (Figure 2). Each compound was tested over a concentration range that was dependent upon the pharmacological and toxicological activity of the compound. To facilitate comparisons with the literature, molar concentrations were provided for amiodarone, staurosporine, and salbutamol base, whereas mass concentrations were used for the PVAc NP and the PVA vehicle control. On the basis of the data presented in Figure 2, an upper concentration limit was determined for each compound, above which no further experiments were performed (except when used as a positive control in selected experiments): amiodarone (5 μM), staurosporine (10 nM), PVAc NP (5 mg/mL), PVA solution (0.4 mg/mL), and salbutamol base (4 mM). Assessment of Vacuolation and Phospholipid Accumulation. When FMΦ develop as a response to inhaled pharmaceuticals, they are typically identified visually by a histopathologist during light microscopic assessment of lung tissue slices.2 The mechanism of FMΦ induction cannot be easily determined by visual assessment, as the FMΦ phenotype appears similar regardless of mechanism. Visual and quantitative image analysis of transmission electron micrographs can provide greater insight into FMΦ characteristics, such as cell status and vacuole content, e.g., presence of phospholipid lamellar bodies or cell debris, but it is too expensive and timeconsuming for routine implementation. Illustrative transmission electron micrographs (Figure 3) allow comparison of untreated J774A.1 cells with cells exposed 24 h to amiodarone (5 μM), staurosporine (1 nM), or PVAc NP (1 mg/mL). Superficially, the vacuolated cells in the treatment groups are quite similar in terms of number and size of the vacuoles, although, upon closer inspection, the contents of the amiodarone-treated cells show evidence of the characteristic electron-dense lamellar bodies of phospholipid inclusion16,17 compared to that of staurosporineand PVAc NP-treated cells. To address the shortcomings of microscopic evaluation of FMΦ, a cell imaging method was utilized for concurrent analysis of cell morphometrics, vacuolation, and lipid accumulation. A significant correlation (p < 0.0001) was observed between the number of vacuoles per cell and percentage area of vacuoles per cells for all treatment groups

Figure 3. Representative transmission electron micrographs of (A) untreated J774A.1 cells and cells treated for 24 h with (B) amiodarone (5 μM), (C) staurosporine (1 nM), and (D) PVAc NP (1 mg/mL).

(Figure 4, left panel), although different inducers showed different relationships between these two parameters. For example, amiodarone induced a small increase in the percentage area of vacuoles but virtually no increase in vacuole 2679


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Figure 4. (Left) Linear relationship between the percentage vacuolar area per cell and the mean number of vacuoles per cell; (Right) percentage vacuolar area per cell plotted against the average LipidTOX Red fluorescence intensity (au) per cell. Data points represent the mean value of n = 3 replicate experiments and reflect combined data over a range of concentrations incubated at both t = 24 and 72 h: (A) amiodarone, 0.03−40 μM; (B) staurosporine, 0.06−8 nM; (C) PVAc NP, 0.06−8 mg/mL; (D) PVA solution, 0.03−3 mg/mL; (E) salbutamol base, 0.002−0.3 mM; and (F) untreated cells (n = 15).

number. Staurosporine and PVAc NP treatment, in contrast, produced a substantial increase in vacuole number compared to vacuole size. This pattern was also observed in TEM micrographs of J774A.1 cells exposed to different treatments (Figure 3). Interestingly, the mean fluorescence intensity per

cell (in arbitrary units; au) of the selective phospholipid dye, LipidTOX Red, did not correlate significantly with either percentage vacuole area per cell (Figure 4, right panel) or number of vacuoles. Cells treated with increasing concentrations of amiodarone were the only samples from the 2680


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apoptosis (Figure 6). Arrows highlight the FMΦ inducer/ control concentrations that were evaluated in the gene expression assay. Significantly elevated levels of caspase-3 activity were observed only for concentrations of FMΦ inducers/controls that were found to be cytotoxic (Figure 2), and cells exposed to all concentrations of amiodarone showed no significant caspase-3 activity at all. Taken together, these data indicate that the FMΦ that developed as a response to sublethal inducer concentrations were not significantly dysregulated in their transcriptional activity and were not undergoing active apoptosis under the conditions tested. Assessment of FMΦ Functional Activity. The phagocytic capability of FMΦ was compared with untreated macrophages by treating J774A.1 cells with sublethal concentrations of inducer and control compounds for 6, 24, and 72 h followed by incubation of the treated cells with 1 μm diameter polystyrene particles (Figure 7). Higher concentrations of amiodarone and PVAc NP impaired the phagocytosis of 1 μm particles to ∼50% of the untreated control at early time points, but a recovery of phagocytic activity occurred by 72 h. In contrast, staurosporine showed a trend toward a reduction in phagocytic activity over time, which was not significant, and salbutamol base treatment resulted in a significantly reduced phagocytic activity primarily at the higher concentration tested, most likely due to an increased prevalence of apoptotic cells. In addition to phagocytic activity, the ability of FMΦ to undergo classical (M1) activation and respond to LPS challenge was assessed. The first treatment scheme was designed to evaluate whether the normal macrophage response to LPS challenge was altered by the FMΦ state. Cells were pretreated with sublethal concentrations of compound for 24 h to generate FMΦ and were then differentiated into resting or M1-primed cells through addition of either IFN-γ-free or IFN-γsupplemented medium, followed by exposure to LPS-free or LPS-supplemented medium. Only salbutamol base treatment (4 mM) inhibited the response of M1-activated cells to LPS (Figure 8A,B), albeit at a relatively high concentration compared to the concentrations used clinically, which was attributed to a pharmacological effect (i.e., disruption of CD14/ toll-like receptor 4 complexation, which inhibits M1 activation24) rather than a toxicological effect. In a second experiment, a treatment scheme was designed to evaluate whether the cellular response to LPS challenge was altered by concomitant dosing of the inducer and control compounds (Figure 8C,D). In this case, cells were first differentiated into two groups, M1-primed or resting macrophages, through addition of medium with or without IFN-γ, respectively, followed by exposure of both groups to compound in LPS-free or LPS-supplemented medium. Interestingly, resting macrophages responded to PVAc NP exposure with a slight, but significant, stimulation of nitric oxide regardless of the presence of LPS. IFN-γ-primed cells also produced significant amounts of nitric oxide when exposed to sublethal concentrations of salbutamol base, amiodarone, staurosporine, or PVAc NP in the absence of LPS, although responses of primed cells to compounds and LPS together did not differ from those of untreated control cells exposed to LPS challenge.

inducer/control panel to show significant phospholipid accumulation. Assessment of FMΦ Cellular Status. As the transmission electron micrographs and morphometric data highlight, FMΦ development occurred at sublethal treatment concentrations across the range of inducers tested. To determine whether FMΦ generated by different inducers displayed unique gene expression profiles indicative of the mechanism of FMΦ induction or their functional status, targeted gene expression profiling was conducted at 6 and 24 h for cells treated with amiodarone (5 μM), staurosporine (1 nM), PVAc NP (1 mg/ mL), or PVA solution (0.4 mg/mL). Due to time and cost constraints, salbutamol base treatment was not included in this analysis. A panel of 27 transcripts representing key regulators of oxidative stress, inflammation, cell death, autophagy, phospholipidosis, and macrophage functionality were selected for evaluation (Table 2). Table 2. Classification of the Selected Transcripts Chosen to Explore Gene Expression Changes in J774A.1 Cells Exposed to Inducers of Foamy Macrophage Morphology process/function oxidative stress inflammation cell death autophagy phospholipidosis macrophage functionality housekeeping genes

transcripts KEAP1, GCLC, SOD1, HMOX1 CHUK, TNF, IL-6, IL-10, NOS2, CXCL1 CASP3, BCL2, GADD45A, Parp1/Parp2, FAS MAP1LC3A, Atg7, Becn1 ASAH1, LSS, PLA2G15, LYPLA2, SMPD1, ADFP or Plin2, LAMP-2 TGF-β, ARG2 β-actin, GAPDH, POLDIP3

The volcano plots in Figure 5 depict those genes from the panel which express a fold-change ≥ 1.2 after treatment with inducer and control compounds for 6 and 24 h. All transcripts for which gene expression was significantly upregulated are located in the bottom right quandrant of the plot, whereas genes that were significantly downregulated are depicted in the bottom left quandrant. For amiodarone, we observed no significant increase in genes related to the induction of apoptosis, autophagy, or inflammation, with only an increase in HMOX1 evident at the 6 h exposure time point, consistent with an adaptive response to mild oxidative stress. Notably, despite the evidence of phospholipid accumulation in the vacuoles of amiodarone treated J774A.1 cells, we saw no change in the expression of the panel of genes related to phospholipidosis at either the 6 or 24 h time point. For staurosporine, a known apoptosis inducer, increased expression of TNFα was noted at the 6 h time point, with decreased expression of BCL2 evident at 24 h. A significant downregulation of IL-10 was also observed at the later time point, although other markers of autophagy and apoptosis were unaltered at either time point. The most pronounced transcriptional response was observed in the PVAc NP challenge cells, with an upregulation of TNFα and FAS and a downregulation of Atg7 and Bcl2 at the 6 h time point, consistent with the induction of apoptosis. TNFα expression remained elevated at the 24 h time point, where downregulation of both PLA2G15 and IL-10 was also evident. To augment the information provided by evaluation of gene expression at 6 and 24 h treatment exposure, caspase-3 activity was also measured at the same time points as an indicator of

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DISCUSSION An effective in vitro screening strategy for the FMΦ phenotype should be able to (1) identify pharmaceutical compounds at risk of inducing FMΦ in vivo, (2) differentiate FMΦ based on 2681


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Figure 5. Volcano plots of gene expression following (A) 6 h and (B) 24 h treatment with amiodarone (5 μM), staurosporine (1 nM), PVAc NP (1 mg/mL), or PVA solution (0.4 mg/mL). Each data point represents the mean value of n = 3 replicate experiments with different cell passage numbers.

antiarrhythmic agent is associated with serious pulmonary toxicity, including acute or subacute pneumonitis, alveolitis, pulmonary infiltrates, pleural disease, and fibrosis.25,29 Similar to other CAD’s, the postulated mechanism of FMΦ development is based on the ability of the amphiphilic CAD molecule to bind to phospholipids and prevent enzyme-mediated metabolism, as well as direct inhibition of phospholipases themselves.16,30 The strategy developed here was able to successfully distinguish amiodarone-treated J774A.1 cells from other treatment groups. Amiodarone-treated cells displayed a characteristic morphology, which has been described previously as consisting of a single or a few very large, distended vacuoles per cell observed at concentrations > 5 μM.27 This qualitative description corresponded well with the CellMask staining

morphometric and mechanistic information, and (3) underpin a deeper understanding of FMΦ biology.1−3 The multiparameter in vitro screening strategy presented here has been developed as a first step toward achieving these aims. Despite the limited number of compounds tested in this pilot study, the assay strategy was able to successfully distinguish FMΦ inducers from noninducers, reveal compound-dependent differences phospholipid accumulation, vacuole number, and vacuole area, and provide data on the impact of inducers on FMΦ cellular health and functional status. Amiodarone, a prototypic cationic amphiphilic drug (CAD), was chosen for its well-known pulmonary toxicity profile and propensity to induce phospholipidosis in the alveolar macrophage population.13,16,17,25−29 Oral administration of this 2682


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Figure 6. Caspase-3 activity in J774A.1 cells treated with different inducers and controls over a range of concentrations for 6 and 24 h: (A) amiodarone, (B) staurosporine, (C) PVAc NP, and (D) salbutamol base. Data represent the mean ± SD of n = 3 replicate experiments with different cell passage numbers. Arrows above selected bars indicate concentrations that were tested in gene expression studies. Difference from untreated cells: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 7. Phagocytic activity of J774A.1 cells treated with sublethal concentrations of inducers and controls for 6, 24, and 72 h: (A) amiodarone (B) staurosporine (C) PVAc NP/PVA solution, and (D) salbutamol base. Data are presented as a percentage of the phagocytic activity of untreated J774A.1 cells. Data represent the mean ± SD of n = 3 replicate experiments with different cell passage numbers. Difference from untreated cells: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

profile, which revealed an increase in mean percentage vacuole area at amiodarone concentrations ≥ 20 μM but virtually no increase in vacuole number. Importantly, amiodarone was the only compound from the current panel to induce a significant

increase in LipidTOX Red staining intensity, which is indicative of phospholipidosis.9,10 J774A.1 cells exposed to a moderate dose (5 μM) of amiodarone for 24 h did not show significant increases in gene expression of the phospholipid biomarkers 2683


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Figure 8. (Top) Ability of (A) resting FMΦ and (B) M1-primed FMΦ to respond to LPS challenge. (Bottom) Response of (C) resting and (D) M1-primed J774A.1 cells to concomitant administration of inducer or control compounds and LPS. Data represent the mean ± SD of n = 3 replicate experiments with different cell passage numbers. Difference from untreated cells: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

percentage area of vacuoles (CellMask staining), with no increase in LipidTOX Red fluorescence intensity. Cells treated with a moderate dose of staurosporine (1 nM) showed an early (6 h) increase in the expression of TNF-α but no increase in caspase-3 activity or altered response to LPS challenge under these exposure conditions. However, phagocytic activity, while similar to untreated cells over 24 h, seemed to decrease slightly over time. These data suggest that staurosporine-induced vacuolation may occur as a defensive response prior to onset of apoptosis at later time points or upon exposure to higher concentrations. PVAc NP were included in the study as an inducer of unknown mechanism following previous observations that intratracheal administration of PVAc NP led to significant dosedependent development of coarsely vacuolated alveolar macrophages in Balb/c mice.20 Interestingly, in vivo exposure to a relatively hydrophilic grade of PVAc NP (40% hydroxyl group content) generated a higher percentage of vacuolated alveolar macrophages with no accompanying neutrophilic inflammatory response, whereas a more hydrophobic grade of PVAc NP (20% hydroxyl content) provoked a substantial neutrophilic inflammatory response but a lower incidence of vacuolated macrophages. Similar to this study, a PVA solution of 0.4 mg/ mL was used as a vehicle control for previous in vivo

selected for study, although this was not entirely unexpected since amiodarone exposure to human hepatoma HepG2 cells (8 μM for 6 and 24 h) showed fewer genes that were significantly up- or downregulated after treatment and smaller magnitudes of fold-change (0.35−2.15) compared to those with other CAD molecules tested.11 No caspase-3 activity was observed in amiodarone-treated J774A.1 cells, which is in line with a previous report that rats treated with amiodarone exhibited a reduced caspase-3 activity in lung tissue compared to control groups.31 Finally, amiodarone exposure to J774A.1 cells did not alter the macrophage response to LPS challenge,32 although a small transient reduction in phagocytic activity was observed, which contrasts with reports that amiodarone can stimulate phagocytic activity both in vitro33 and in vivo.32 Staurosporine, a protein kinase C inhibitor known to induce apoptosis via caspase-dependent and -independent pathways,18,19 was chosen to elucidate quantitative differences between cells with phospholipidosis and cell vacuolation caused by alternative mechanisms, such as apoptosis. Apoptosis is often accompanied by morphological changes to the cell, such as cytoplasm division into apoptotic bodies that can appear as large vacuoles in light and transmission electron micrographs.34,35 Staurosporine exposure to J774A.1 cells induced a time- and dose-dependent increase in both vacuole number and 2684


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vacuolation with the use of this well-known β-agonist but also due to the moderately poor solubility profile of this drug. It was initially envisioned that salbutamol base might also act as a model for poorly soluble pharmaceuticals at concentrations above its solubility limit, a topic of considerable interest in the field.1 Although salbutamol cytotoxicity limited dosing to concentrations below the solubility limit, it was nonetheless useful as a negative control as it established that cell morphometrics, especially vacuole number and total area, were very similar in profile to untreated cells. While the data generated from this multiparameter in vitro assay strategy are information-rich, the approach is not yet sufficiently validated as a tool to support early decision making on compound progression or termination; this requires further research with a much larger library of inducer and control compounds. However, the current study provides a useful starting point for further development. First, several of the assays performed in this study (e.g., assessment of cell viability, morphometrics, phospholipid accumulation, apoptosis, and phagocytosis) are compatible with cell image analysis. Integration of multiple parameters using multidye cocktails and assessment with one measurement technique would make important efficiency savings while maintaining a high degree of data richness. Second, to develop a decision tree tool for early compound selection studies, it would be important to define and validate critical threshold values for specified parameters to use as a basis for progression/termination decisions. Thresholds for mean vacuole number, total vacuole area, and phospholipid content could be generated for specific cell lines or primary cells by testing an appropriate number of noninducer compounds across a sublethal dose range (similar to the salbutamol example in this study). Using the data from this study, example threshold values for J774A.1 cells might be defined as >16 vacuoles per cell, > 14% vacuolar area per total cell area, or >2000 au LipidTOX Red fluorescence intensity. It will also be important to investigate in vitro−in vivo correlations (IVIVC) in macrophage responses. Clements and Thomas (2014)39 recently described a methodology that uses multiparameter cluster analysis to compare a series of cell-based assay results with drug-induced cardiotoxicity in preclinical models. Using this strategy, they were not only able to identify in vitro parameters predictive of in vivo cardiotoxicity but also could rank compounds according to their potential for causing in vivo toxicity. In the case of IVIVC studies assessing alveolar macrophage responses to inhaled pharmaceuticals, substantial development work is still required to determine the appropriate in vivo metrics. For example, tissue histopathology reports from drug safety studies rarely include quantitative descriptors for FMΦ, which makes it difficult to compare with in vitro data. One alternative may be to evaluate whether comparison with measurements on bronchoalveolar lavage samples is suitable for IVIVC purposes. Lavage samples provide an easily accessible cell population in sufficient cell numbers for evaluation using the same integrated panel of assays used for in vitro screening, although cell sorting may be required prior to analysis. A disadvantage of this approach is that it would require the generation of a large and expensive in vivo data set created specifically to explore this issue.

experiments and showed no incidence of vacuolated alveolar macrophages or acute inflammatory response following a single administration.20 Exposure of PVAc NP (40% hydroxyl group content) to J774A.1 cells also induced a vacuolated state that was visually very similar to the murine alveolar macrophages obtained by bronchoalveolar lavage.20 CellMask staining profiles of PVAc NP-treated cells revealed dramatic increases in both vacuole number and percentage vacuole area per cell, supporting visual assessments using light microscopy and transmission electron microscopy. We established that PVAc NP did not cause intracellular phospholipid accumulation and therefore did not induce phospholipidosis. Interestingly, the profound vacuolation did not seem to reduce the capacity for cell proliferation, although the metabolic activity of cells treated with ≥1 mg/mL showed a very unique dose-dependent, time-independent reduction in metabolic activity. PVAc NP treatment (1 mg/ mL for 6 and 24 h) resulted in the most robust transcriptional response relative to that of the other tested inducers, with TNF-α and FAS significantly upregulated and Atg7 significantly downregulated (P < 0.05) at 6 h and with gene expression profiles returning nearly to normal by 24 h (except for TNF-α expression). Exposure of cells to many types of nanomaterials have been associated with two forms of autophagic dysfunction, either excessive autophagy induction or blockade of autophagy flux.21 Interestingly, the blockade of autophagy flux is often observed to be accompanied by an accumulation of dysfunctional mitochondria and elevated oxidative stress levels. The combined reduction in metabolic activity, the morphology and appearance of the vacuoles, and transcriptional changes observed in the PVAc NP-treated cells may be indicators of PVAc NP-induced blockage to autophagic flux mechanisms, a mechanism that may be investigated in further detailed studies. It is thought that nanoparticle-induced lysosomal blockade may result from a variety of causes, including lysosomal overload with indigestible material, disruption of lysosomal fusion with other compartments, and disruption of the cytoskeleton.21 Interestingly, some nanoparticulate therapeutic systems, such as alumina vaccines, are designed to exploit the mechanism of autophagic dysfunction to achieve an enhanced therapeutic effect.36 However, as autophagy is typically seen as a prosurvival response, it is currently uncertain what the longer term impacts of autophagic dysfunction may be. Finally, it is interesting to note that alveolar macrophages from smokers with a known autophagic defect (i.e., blockade of autophagy flux) observed a marked reduction in the ability of the macrophages to phagocytose and respond to bacterial pathogens.21 In the current study, PVAc NP also showed a transient reduction in phagocytosis at high exposure concentrations, but it otherwise did not impair the response of macrophages to LPS challenge. The two control compounds utilized in the study to distinguish between FMΦ inducers and noninducers, PVA solution and salbutamol base, were, for the most part, successful as negative controls. However, at concentrations > 1 mg/mL, PVA solution did increase vacuole number and percentage area per cell, which is not completely unexpected, as cell treatment with other types of polymeric solutions (e.g., therapeutic pegylated proteins37 and poly(vinylpyrrolidone) plasma expanders38) has also been associated with enhanced cellular vacuolation and possibly autophagic dysfunction.21 Salbutamol base was chosen as an interesting negative control compound, not only due to the lack of reports associating cellular

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CONCLUSIONS A pilot study evaluating the ability of a multiparameter in vitro screening strategy to differentiate macrophage responses to inhaled pharmaceuticals was described here. The strategy was 2685


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(10) Nioi, P.; Perry, B. K.; Wang, E. J.; Gu, Y. Z.; Snyder, R. D. In vitro detection of drug-induced phospholipidosis using gene expression and fluorescent phospholipid-based methodologies. Toxicol. Sci. 2007, 99, 162−173. (11) Sawada, H.; Takami, K.; Asahi, S. A toxicogenomic approach to drug-induced phospholipidosis: analysis of its induction mechanism and establishment of a novel in vitro screening system. Toxicol. Sci. 2005, 83, 282−92. (12) Sawada, H.; Taniguchi, K.; Takami, K. Improved toxicogenomic screening for drug-induced phospholipidosis using a multiplexed quantitative gene expression ArrayPlate assay. Toxicol. In Vitro 2006, 20, 1506−13. (13) Reasor, M. J.; Kacew, S. Drug-induced phospholipidosis: are there functional consequences? Exp. Biol. Med. 2001, 226, 825−30. (14) Brasey, A.; Igue, R.; Djemame, L.; Seguin, S.; Renzi, P.; Ferrari, N.; Seguin, R. The effect of in vitro exposure to antisense oligonucleotides on macrophage morphology and function. J. Nucleic Acids Invest. 2011, 2, e12. (15) Brasey, A.; Igue, R.; Ferrari, N.; Seguin, R. Phenotypic but not functional changes in macrophages following exposure to antisense oligonucleotides in vitro. Am. J. Respir. Crit. Care Med. 2010, 181, A1282 DOI: 10.1164/ajrccm-conference.2010.181.1_MeetingAbstracts.A1282. (16) Reasor, M. J.; Kacew, S. An evaluation of possible mechanisms underlying amiodarone-induced pulmonary toxicity. Proc. Soc. Exp. Biol. Med. 1996, 212, 297−304. (17) Reasor, M. J.; Kacew, S. Amiodarone pulmonary toxicity: morphologic and biochemical features. Proc. Soc. Exp. Biol. Med. 1991, 196, 1−7. (18) Yamaki, K.; Hong, J.; Hiraizumi, K.; Ahn, J. W.; Zee, O.; Ohuchi, K. Participation of various kinases in staurosporine induced apoptosis of RAW 264.7 cells. J. Pharm. Pharmacol. 2002, 54, 1535− 1544. (19) Belmokhtar, C. A.; Hillion, J.; Segal-Bendirdjian, E. Staurosporine induces apoptosis through both caspase-dependent and caspase-independent mechanisms. Oncogene 2001, 20, 3354− 3362. (20) Jones, M. C.; Jones, S. A.; Riffo-Vasquez, Y.; Spina, D.; Hoffman, E.; Morgan, A.; Patel, A.; Page, C.; Forbes, B.; Dailey, L. A. Quantitative assessment of nanoparticle surface hydrophobicity and its influence on pulmonary biocompatibility. J. Controlled Release 2014, 183, 94−104. (21) Stern, S. T.; Adiseshaiah, P. P.; Crist, R. M. Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Part. Fibre Toxicol. 2012, 9, 20. (22) Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 2001, 25, 402−408. (23) Glaves, P. D.; Tugwood, J. D. Generation and analysis of transcriptomics data. Methods Mol. Biol. 2011, 691, 167−85. (24) Wang, W.; Xu, M.; Zhang, Y. Y.; He, B. Fenoterol, a beta(2)adrenoceptor agonist, inhibits LPS-induced membrane-bound CD14, TLR4/CD14 complex, and inflammatory cytokines production through beta-arrestin-2 in THP-1 cell line. Acta Pharmacol. Sin. 2009, 30, 1522−1528. (25) Papiris, S. A.; Triantafillidou, C.; Kolilekas, L.; Markoulaki, D.; Manali, E. D. Amiodarone: review of pulmonary effects and toxicity. Drug Saf. 2010, 33, 539−558. (26) Pauluhn, J. Inhaled cationic amphiphilic drug-induced pulmonary phospholipidosis in rats and dogs: time-course and doseresponse of biomarkers of exposure and effect. Toxicology 2005, 207, 59−72. (27) Reasor, M. J.; Ogle, C. L.; Kacew, S. Amiodarone-induced pulmonary toxicity in rats: biochemical and pharmacological characteristics. Toxicol. Appl. Pharmacol. 1989, 97, 124−133. (28) Reasor, M. J.; Ogle, C. L.; Walker, E. R.; Kacew, S. Amiodaroneinduced phospholipidosis in rat alveolar macrophages. Am. Rev. Respir. Dis. 1988, 137, 510−518.

able to differentiate successfully in vitro responses to amiodarone, which induces a well-characterized phospholipidosis, from other treatments based on the inducer-specific patterns of quantitative morphometric parameters, such as the correlation of vacuole number with percentage vacuole area per cell and LipidTOX Red staining. With further development and validation, the strategy may be employed usefully in a pharmaceutical research and development environment to reduce inhaled compound attrition rates during drug development as a result of FMΦ-related safety concerns as well as to deepen our understanding of FMΦ biology.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions ○

E.H. and A.K. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was funded by the National Centre for Replacement, Refinement and Reduction of Animals in Research. The authors would like to thank Drs. Dave Hassall, Joanne Rhodes, and Michael Clements for valuable contributions to the study design and manuscript review.

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REFERENCES

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