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Automobile diesel exhaust particles induce lipid droplet formation in macrophages in vitro
Environmental Toxicology and Pharmacology 40 (2015) 164–171
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Automobile diesel exhaust particles induce lipid droplet formation in macrophages in vitro Yi Cao ∗ , Kim Jantzen, Ana Cecilia Damiao Gouveia, Astrid Skovmand, Martin Roursgaard, Steffen Loft, Peter Møller Department of Environmental Health, Section of Environmental Health, University of Copenhagen, Øster Farimagsgade 5A, DK-1014 Copenhagen K, Denmark
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Article history: Received 4 March 2015 Received in revised form 4 June 2015 Accepted 6 June 2015 Available online 9 June 2015 Keywords: Automobile diesel exhaust particles Oxidative stress Lysosomal dysfunction Lipid droplet
a b s t r a c t Exposure to diesel exhaust particles (DEP) has been associated with adverse cardiopulmonary health effects, which may be related to dysregulation of lipid metabolism and formation of macrophage foam cells. In this study, THP-1 derived macrophages were exposed to an automobile generated DEP (A-DEP) for 24 h to study lipid droplet formation and possible mechanisms. The results show that A-DEP did not induce cytotoxicity. The production of reactive oxygen species was only significantly increased after exposure for 3 h, but not 24 h. Intracellular level of reduced glutathione was increased after 24 h exposure. These results combined indicate an adaptive response to oxidative stress. Exposure to A-DEP was associated with significantly increased formation of lipid droplets, as well as changes in lysosomal function, assessed as reduced LysoTracker staining. In conclusion, these results indicated that exposure to A-DEP may induce formation of lipid droplets in macrophages in vitro possibly via lysosomal dysfunction. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Exposure to particulate matter (PM), especially PM with an aerodynamic diameter below 2.5 m (PM2.5) and 0.1 m (ultrafine particles, UFP) has been associated cardiopulmonary health effects (Hiraiwa and van Eeden, 2013), which are considered to be related to oxidative stress and inflammation (Miller et al., 2012; Moller et al., 2014). Elevated levels of lipid peroxidation products have been observed in humans living in urban areas and in controlled trials with well-defined exposure to PM (Moller and Loft, 2010). Inhalation or instillation of ambient air PM in animals has likewise been associated with elevated levels of lipid peroxidation products in lung tissue (Moller et al., 2014). Alveolar macrophages are central in the defense against deposited PM as well as in the inflammatory pathways related to lung and cardiovascular diseases attributed to exposure to air pollution (Hiraiwa and van Eeden, 2013; Ling and van Eeden, 2009). Interestingly, a recent study showed that pharyngeal aspiration of diesel exhaust particles (DEP), the major source of UFP in urban environments, was associated with accumulation of lipid droplets in alveolar
∗ Corresponding author at: Section of Environmental Health, Department of Public Health, University of Copenhagen, Øster Farimagsgade 5A, Building 5B, 2nd Floor, DK-1014 Copenhagen K, Denmark. Tel.: +45 3533 7210. E-mail address: [email protected] (Y. Cao). http://dx.doi.org/10.1016/j.etap.2015.06.012 1382-6689/© 2015 Elsevier B.V. All rights reserved.
macrophages and lipid peroxidation in lung tissue (Yanamala et al., 2013). Moreover, hyperlipidemic ApoE−/− mice developed pulmonary emphysema apparently related to lipid accumulation in macrophages with subsequent cholesterol efflux and activation of the Toll-like receptor 4 signaling pathway (Goldklang et al., 2012). Another study showed that inhalation of DEP in mice was associated with elevated levels of lipid peroxidation products in the lungs, plasma and liver, which was considered to be due to dysfunction of high-density lipoproteins (Yin et al., 2013a). Such alteration in lipid homeostasis and subsequent lipid accumulation in the vascular system is implicated in the development of atherosclerosis (Moore et al., 2013). Interestingly, recent animal studies showed that exposure to DEP changed the characteristics of atherosclerotic plaques with increased lipids and formation of macrophage foam cells (Miller et al., 2013; Bai et al., 2011). Intracellular lipids stored as triglycerides in lipid droplets are taken up by autophagosomes and subsequently delivered to lysosomes for degradation, a process known as lipophagy (Liu and Czaja, 2013). Lipophagy was initially discovered in hepatocytes (Singh et al., 2009) and was later reported to occur in macrophages (Ouimet et al., 2011). Lipophagy dysfunction in macrophages plays a crucial role in lipid accumulation, foam cell formation and development of atherosclerosis (Moore et al., 2013; Ouimet and Marcel, 2012). In addition, recent studies showed that exposure to a variety of nanomaterials induced lipid accumulation in hepatocytes (Vesterdal et al., 2014) and macrophages (Cao et al., 2014a,b).
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In this study, we used human THP-1 monocyte differentiated macrophages as an in vitro model to alveolar macrophages to study lipid droplet formation and possible mechanisms after exposure to an automobile generated DEP (A-DEP). Cytotoxicity was measured using both 2-(4-iodophenyl)-3-(4-nitrophenyl)5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) and calcein acetoxymethyl (calcein AM). Intracellular reduced glutathione (GSH) levels and production of reactive oxygen species (ROS) were measured to indicate oxidative stress using fluorescent probes. The formation of lipid droplets in macrophages was determined using Bodipy 493/503 and lysosomal function was investigated using LysoTrackerTM green.
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Size-distribution of A-DEP was analyzed using a NanoSight LM20 (NanoSight, Amesbury, UK); 5 g A-DEP/ml RPMI-1640 medium with 10% FCS were analyzed in triplicates on three independent days. The Nanoparticle Tracking Analysis (NTA) version 2.3 was used to track and analyze the particles and means of ‘Mean’ (the mean size of the particles) and ‘Mode’ (the size of the most frequently occurring particles) were calculated from three independent experiments (n = 3). Comparative studies indicated that NTA is a good alternative to transmission electron microscopy for the measurement of particle sizes (Roursgaard et al., 2014; De Temmerman et al., 2014). 2.3. Cytotoxicity assay
2. Materials and methods 2.1. Cell line The monocytic cell line THP-1, obtained from the American Type Culture Collection (Manassas, VA, USA), were cultivated in supplemented RPMI-1640 medium with 10% fetal bovine serum (FBS) and differentiated into macrophage-like cells by treatment with 10 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma, St. Louis, MO, USA) as previously described (Jantzen et al., 2012). It has been shown that PMA differentiated THP-1 macrophages resemble primary human monocyte-derived macrophages, and thus serves as a good in vitro model for human macrophages (Daigneault et al., 2010). We have previously used THP-1 derived macrophages to study the formation of macrophage foam cells (Cao et al., 2014a,b; Del Bo et al., 2015). Prior to particle exposure, cells were seeded at either a density of 5 × 104 cells/well in 0.1% gelatin pre-coated 96-well plates or 8-well microscopy chamber slides (Ibidi, Munich, Germany), or in a density of 5 × 105 cells/well in pre-coated 12-well plates overnight. 2.2. Particles A-DEP was provided as a gift from Dr. Hiromi Izawa (Gifu University, Gifu, Japan). This type of A-DEP was collected from an automobile engine as previously described (Izawa et al., 2007), using the same combustion conditions and collection method as described (Sagai et al., 1993). The A-DEP material has been thoroughly characterized and compared to a standard reference material (SRM 2975) DEP from the National Institute of standards and Technology (Singh et al., 2004; DeMarini et al., 2004). Chemical analysis showed that A-DEP contains carbon (elemental carbon and organic carbon), extractable organic material and polycyclic aromatic hydrocarbons, and scanning electron micrographs of A-DEP showed aggregated >50 m particles, whereas SRM 2975 contained particles with sizes <10 to > 50 m (Singh et al., 2004; DeMarini et al., 2004). Exposure of mice to A-DEPs induced influx and activation of macrophages and inflammatory response in the lungs (Singh et al., 2004). For the exposure, A-DEP suspensions were prepared by sonication of 1 mg/ml particles in RPMI-1640 medium with 10% FCS for 8 min (with alternating 10 s pulses and 10 s pauses and continuously cooling on ice) using a Branson Sonifier S-450D (Branson Ultrasonics Corp., Danbury, CT, USA) equipped with a disrupter horn (Model number: 101-147-037), and then diluted in RPMI1640 medium with 10% FCS to the concentrations needed for exposure. Cells were exposed to 200 l/well (in 96-well plate) or 2 ml/well (in 12-well plate) 2.5 g/ml, 5 g/ml and 10 g/ml A-DEP, which equate to 1.6 g/cm2 , 3.1 g/cm2 and 6.3 g/cm2 , respectively. For fluorescence microscopy, cells were exposed to 200 l/well 10 g/ml A-DEP. The suspensions of A-DEP were applied freshly after each sonication.
Cytotoxicity was measured after exposure to particles using WST-1 reagent assay (Roche Diagnostics GmbH, Mannheim, Germany) and calcein AM staining (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions. WST-1 is a water soluble tetrazolium salt which is cleaved to a formazan dye by succinate-tetrazolium reductase systems in metabolically active cells. Cells were seeded on pre-coated 96-well plates and exposed to various concentrations of A-DEP for 24 h. Then, cells were washed using Hanks solution, followed by incubation with 100 l RPMI-1640 medium with 10% FCS and 10% WST-1 reagent at 37 ◦ C for 2 h. The absorbance of the formazan product was measured at 450 nm with 630 nm as background reference using an ELISA reader (Labsystems, Multiskan Ascent). Calcein AM can be converted to green-fluorescence by intracellular esterases in living cells. Following A-DEP exposure, cells on pre-coated black 96-well plates were washed twice using Hanks solution, followed by incubation with 0.5 g/ml calcein AM in RPMI-1640 medium at 37 ◦ C for 30 min. The green fluorescence was subsequently measured (ex: 485 nm; em: 538 nm) by a fluorescence spectrophotometer (Fluoroskan Ascent FL; Labsystems). To confirm the results, microscopy was used to visualize the morphology of the cells. A-DEP exposed THP-1 macrophages seeded on pre-coated 8-well microscopy chamber slides (Ibidi) were visualized by differential Interference Contrast (DIC) in a Leica AF6000 inverted widefield microscope with 40× magnifications (Leica Microsystems GmbH, Wetzlar, Germany). 2.4. Reduced GSH measurement The effect of A-DEP exposure on intracellular reduced GSH levels was estimated using a fluorescent probe ThioGlo® -1 (Covalent Inc., Woburn, MA) as previously described (Cao et al., 2011). ThioGlo® -1 is a membrane permeable probe which becomes highly fluorescent upon reacting with thiol groups (Cao et al., 2011). Because most of the intracellular thiols are reduced GSH, it can be used to estimate levels of intracellular reduced GSH. For the experiment, THP-1 macrophages on pre-coated 96-well plates were exposed to various concentrations of A-DEP for 24 h. After exposure cells were washed once in Hanks solution and incubated with 10 M probe in PBS at 37 ◦ C for approximately 5 min. The fluorescence was measured at excitation 355 nm and emission 460 nm by a fluorescence spectrophotometer (Fluoroskan Ascent FL; Labsystems). 2.5. ROS measurement The ROS production was estimated using 2 ,7 -dichlorofluorescein diacetate (DCFH-DA; Invitrogen A/S Taastrup, Denmark) and CellROX deep red (Invitrogen, Eugene, OR, USA). DCFH-DA is a cell permeable probe which is deacetylated inside the cells and further oxidized to a fluorescent probe 2 ,7 -dichlorofluorescein (DCF) upon reacting with a variety of ROS, making it a general indicator of intracellular ROS production
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(Dikalov et al., 2007). The fluorescence of DCF was quantified using a fluorescence spectrophotometer as previously described with slight modifications (Dikalov et al., 2007). Briefly, THP-1 macrophages seeded on pre-coated black 96-well plates were exposed to various concentrations of A-DEP for 24 h. Following exposure, cells were washed twice in Hanks solution and incubated with 10 M DCFH-DA in RPMI-1640 medium at 37 ◦ C for 30 min (Dikalov et al., 2007). After incubation the cells were washed twice and fluorescence was measured (ex: 355 nm; em: 460 nm) using a fluorescence spectrophotometer (Fluoroskan Ascent FL; Labsystems). For benchmark purposes, A-DEP exposure was also compared to exposure to the general benchmark DEP SRM 2975 (NIST, Gaithersburg, MD, USA). In this setup, cells were pre-loaded with DCFH-DA probe, washed and exposed to particles for 3 h, followed by the measurement of the fluorescence as described above. Fluorescence microscopy was used to supplement the results obtained with DCF by CellROX deep red, a specific probe for cytoplasmic ROS. The exposed THP-1 macrophages seeded on precoated 8-well microscopy chamber slides were washed twice using warm RPMI-1640 medium, and then stained with 5 M probe in RPMI-1640 medium at 37 ◦ C for 30 min. Hereafter, cells were washed twice in warm RPMI-1640 medium, and then visualized by combined DIC and red fluorescence in a Leica microscope with 63× magnification, with each image captured as z-stack in 5 randomly selected areas (n = 5), followed by 3D deconvolution and 3D projection using Leica LAS AF 2.6.0.7266 software. The integrated density of fluorescence was measured by ImageJ (NIH) and normalized by the number of cells in each randomly selected area. 2.6. Measurements of lipid droplet formation The formation of lipid droplets was measured using Bodipy 493/503 (Molecular Probes, Eugene, OR). For the quantitative measurement of lipid droplets, THP-1 macrophages were seeded on pre-coated 12-well plates and exposed to various concentrations of particles for 24 h. After exposure, the cells were washed once in Hanks solution and incubated with 1 g/ml Bodipy 493/503 in RPMI-1640 medium at 37 ◦ C for 30 min. Hereafter, the cells were washed in Hanks solution, harvested with trypsin, centrifuged, and re-suspended in cold PBS prior to flow cytometric analysis using a flow cytometer (BD AccuriTM C6, Cambridge, UK) equipped with CFlow plusTM software for data analysis. A total of 1 × 105 events were analyzed for each sample with an unexposed and unstained sample as negative control, and fluorescence was collected from FL1 channel. The formation of lipid droplets was also measured by fluorescence microscopy as previously described (Cao et al., 2014b). Briefly, A-DEP exposed THP-1 macrophages seeded on pre-coated 8-well microscopy chamber slides were washed twice with PBS and fixed by 4% paraformaldehyde for 30 min at room temperature. It has previously been shown that fixation of cells by paraformaldehyde did not affect the structure of lipid droplets or lipid contents (DiDonato and Brasaemle, 2003). Following fixation the cells were washed again with PBS and stained with 1 g/ml Bodipy 493/503 for 15 min, followed by washing and addition of mounting media (Ibidi). The green fluorescence was quantified using a Leica microscope with 40× magnification in 5 randomly selected areas (n = 5), with each sample captured as a z-stack. After 3D deconvolution and 3D projection, the fluorescent areas in each independent area were analyzed by ImageJ and normalized by the number of cells (Cao et al., 2014b).
LysoTracker signal could be due to either increase of lysosomal pH leading to poor retention of the dye or the increase of lysosomal membrane permeabilization leading to the rupture of lysosomes, both of which indicate lysosomal dysfunction (Emanuel et al., 2014). For the measurement, THP-1 macrophages were seeded on pre-coated 12-well plates and exposed to various concentrations of particles for 24 h. After exposure, the cells were washed once in Hanks solution and incubated with 50 nM LysoTracker green in RPMI-1640 medium at 37 ◦ C for 30 min. Hereafter, the cells were washed in Hanks solution, harvested with trypsin, centrifuged, and re-suspended in cold PBS prior to flow cytometric analysis using a flow cytometer (BD AccuriTM C6, Cambridge, UK) equipped with CFlow plusTM software for data analysis. A total of 1 × 105 events were analyzed for each sample with an unexposed and unstained sample as negative control, and fluorescence was collected from FL1 channel. 2.8. Statistics One-way ANOVA followed by Tukey HSD test using R-3.0.2; P values <0.05 were considered statistically significant. 3. Results 3.1. Size of A-DEP The representative size distribution of particles suspended in RPMI-1640 medium with 10% FCS analyzed using NTA is as shown in Fig. 1. The ‘Mean’ of A-DEPs (the mean size of the particles) was calculated as 192 ± 4 nm, and ‘Mode’ (the size of the most frequently occurring particles) was calculated as 154 ± 7 nm (mean ± SE of means of three independent experiments; n = 3). 3.2. Cytotoxicity The results from WST-1 (Fig. 2A) and calcein AM (Fig. 2B) assays showed no statistically significant changes in WST-1 product formation or calcein AM fluorescence (p > 0.05). In addition, the microscopic images (Fig. 2C) showed no obvious changes in the morphology of the A-DEP exposed cells. These results combined indicate that A-DEP exposure did not induce cytotoxicity in THP-1 macrophages.
2.7. Lysosomal activity Lysosomal activity was indicated using LysoTracker® green (Invitrogen, Carlsbad, CA) that stains lysosomes. A decrease of the
Fig. 1. Representative nanosight (NTA) measurements of particle size distribution in RPMI-1640 medium with 10% FCS. The black line indicates particle size and the gray line indicates SE.
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Fig. 2. Effect of 24 h exposure to A-DEP on cytotoxicity. (A) WST-1 assay. (B) Calcein AM staining assay. Data is expressed as mean ± SE of means of three independent experiments (n = 4 for each experiment). (C) Representative images from two independent experiments showing the morphology of THP-1 macrophages after 24 h exposure to A-DEP.
Fig. 3. Effect of 24 h exposure to A-DEP on oxidative stress. (A) ROS production. Data is expressed as mean ± SE of means of three independent experiments (n = 4 for each experiment). (B) Representative images from two independent experiments showing CellROX deep red fluorescence. (C) Quantitative analysis of integrated density of CellROX fluorescence/cell, respectively. Data is expressed as mean ± SE in 5 randomly selected independent areas (n = 5). The unexposed cells are normalized to 100%. (D) Reduced GSH levels. Data is expressed as mean ± SE of means of three independent experiments (n = 4 for each experiment). *p < 0.05 compared with unexposed cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 4. ROS production in THP-1 macrophages after 3 h exposure to A-DEP using SRM 2975 as benchmark. Data is expressed as mean ± SE of means of four independent experiments (n = 3 for each experiment). *p < 0.05 compared with unexposed cells.
not statistically significant (p > 0.05). Representative fluorescence microscope images are shown in Fig. 3B. No obvious increase in CellROX deep red signal was observed after particle exposure using fluorescence microscopy. Quantitative analysis of the integrated density of CellROX deep red (Fig. 3C) indicates that ROS production was at equal levels between A-DEP exposed cells and controls. The results from reduced GSH measurements (Fig. 3D) showed that 24 h exposure to A-DEP increased intracellular levels of reduced GSH, and a statistically significant increase of reduced GSH was observed after exposure to 5 g/ml A-DEP (p < 0.05; p = 0.087 at concentration of 10 g/ml). A benchmark comparison of the effect of A-DEP and the often used benchmark DEP SRM2975 showed that A-DEP can induce ROS production in THP-1 macrophages after 3 h exposure at high concentrations (p < 0.05 at 25 g/ml and p < 0.01 at 100 g/ml, compared with control), whereas the SRM 2975 induced significant increase in ROS production at 100 g/ml (p < 0.01, compared with control; Fig. 4). 3.4. Lipid droplet
3.3. Oxidative stress The results from ROS measurements are shown in Fig. 3A. Exposure to A-DEP was associated with a modest increase in ROS production as measured by DCFH-DA, but the increase was
The formation of lipid droplets was measured by Bodipy 493/503 and results are shown in Fig. 5. Exposure to A-DEP increased the formation of lipid droplets in a concentrationdependent manner, and a statistically significant increase of lipid
Fig. 5. Lipid droplet formation as measured by Bodipy 493/503 staining in THP-1 macrophages after 24 h exposure to A-DEP. (A) Quantitative measurement of lipid droplet formation by flow cytometry. Data is expressed as means ± SE of means of three independent experiments, with 1 × 105 events analyzed in each sample. The unexposed cells are normalized as 100%. *p < 0.01 compared with unexposed cells. (B) Representative images from two independent experiments showing Bodipy 493/503 green fluorescence. (C) Quantitative analysis of integrated Bodipy 493/503 fluorescence area/cell. Data is expressed as mean ± SE in 5 randomly selected areas (n = 5). The unexposed cells are normalized to 100%. *p < 0.05 compared with unexposed cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 6. Lysosomal activity as measured by LysoTracker green staining after 24 h exposure to A-DEP. Data is expressed as means ± SE of means of three independent experiments, with 1 × 105 events analyzed in each sample. The unexposed cells are normalized as 100%. *p < 0.05 compared with unexposed cells.
droplet formation was observed after 24 h exposure to 10 g/ml A-DEP (p < 0.01, compared with control; Fig. 5A). Representative fluorescence microscope images are shown in Fig. 5B. Lipid droplets were sparse but detectable inside the unexposed cells (Fig. 5B, left panel), whereas cells exposed to A-DEP (Fig. 5B, right panel) contained large lipid droplets. The quantitative analysis of fluorescence microscope images (Fig. 5C) indicates that the average area of lipid droplets/cell was significantly increased after exposure to A-DEP (p < 0.05). 3.5. Lysosomal activity Fig. 6 shows lysosomal activity as indicated by LysoTracker green staining. Exposure to A-DEP resulted in a statistically significant decrease in LysoTracker signal at 2.5 g/ml (p < 0.05), 5 g/ml (p < 0.01) and 10 g/ml (p < 0.05) as compared with the control. 4. Discussion In the present study, THP-1 monocyte derived macrophage was used as an in vitro model to study A-DEP exposure induced lipid droplet formation and possible mechanisms. Using different assays, no obvious increase in cytotoxicity (Fig. 2) or ROS production (Fig. 3) was observed after 24 h exposure of up to 10 g/ml A-DEP, but a transient increase in ROS production was seen after 3 h exposure to A-DEP as well as the benchmark DEP SRM2975 (Fig. 4). Other studies have shown that exposure to PM induced oxidative stress, which generally is considered to play an important role in PM induced toxicity (Moller et al., 2014; Madl et al., 2014) and adverse vascular effects (Araujo and Nel, 2009; Donaldson et al., 2013; Miller et al., 2012; Shrey et al., 2011). Previous work also showed that exposure to 100 g/ml, but not lower concentrations of DEP SRM 2975, significantly induced ROS in THP-1 macrophages (Jantzen et al., 2012). Similarly, our study shows that exposure to A-DEP only significantly induced ROS production in THP-1 macrophages at high concentration of 25 or 100 g/ml (Fig. 4). Thus, high exposure concentrations of DEP may induce oxidative stress, whereas low exposure concentrations as used in this study may be associated with adaptation to oxidative stress. This effect could be explained by particle-induced cellular survival responses through the activation of adaptation factors such as nuclear factor erythroid 2-related factor 2 and consequently upregulated expression of antioxidant defense genes (Kensler et al., 2007). Previously, it has been shown that 20 h exposure of human lung epithelial cells to fly ash
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particles increased levels of the antioxidant enzyme heme oxygenase-1 and increased GSH levels (Diabate et al., 2011). Six hours exposure of human lung epithelial cells to high concentrations of fullerene C60 nanoparticles increased GSH levels (Wang et al., 2014). Our previous work showed that 3 h exposure to DEP or carbon-based particles induced ROS in a number of different types of human cells (Cao et al., 2014a,b; Forchhammer et al., 2012; Frikke-Schmidt et al., 2011; Jantzen et al., 2012; Vesterdal et al., 2012, 2014). However, exposure of primary human endothelial cells to carbon black nanoparticles (Printex90) only significantly increased reduced GSH levels at 24 h, but did not significantly affect it or expression of antioxidant enzymes (glutamate-cysteine ligase modifier subunit and heme oxygenase 1) after 3 h (Cao et al., 2014b). This indicated that particle exposure could induce a transient production of ROS after 3 h, but an adaptive response to oxidative stress could be induced after a longer exposure period. Our result is also in keeping with a current hypothesis describing a hierarchical tiered oxidative stress response that minor oxidative stress induces defense mechanisms; whereas higher levels initiate specific signaling pathways that lead to injury and cytotoxicity (Madl et al., 2014). It has previously been demonstrated that exposure to a variety of particles could induce lipid accumulation in different types of cells in vitro (Cao et al., 2014a,b; Khatchadourian and Maysinger, 2009; Vesterdal et al., 2014; Przybytkowski et al., 2009). Some studies indicated that oxidative stress may be involved in the regulation of lipid accumulation, as treatment with antioxidants prevented particle-induced lipid accumulation (Cao et al., 2014a; Khatchadourian and Maysinger, 2009). Another study showed that antioxidant vitamin E attenuated oxidized low-density lipoprotein induced lipid accumulation in macrophages by modulating nuclear factor B related pathway (Huang et al., 2012). However, our recent study indicated that carbon black nanoparticles induced lipid accumulation in macrophages independently of oxidative stress, as addition of antioxidants did not affect particle-induced lipid accumulation and particles promoted lipid accumulation at concentrations not associated with increased ROS production (Cao et al., 2014b). Increased oxidative stress may not completely explain particle-induced lipid accumulation. In the present study, we found that A-DEP significantly induced formation of lipid droplets in macrophages (Fig. 5) at concentrations that was not associated with increased ROS production as measured by different methods (Fig. 3). These results combined indicated that A-DEP induced lipid droplet formation in macrophages is not directly related with ROS production. We did not attempt to measure lipid droplet formation after 3 h, although we observed an increase in ROS production after 3 h (Fig. 4), because our previous study showed that exposure of THP-1 macrophages to high concentrations of free fatty acids was only associated with significantly increased lipid accumulation after 24 h, but not after 3 h (Cao et al., 2014a). Therefore, it probably takes longer time than 3 h for the newly synthesized lipid droplets to be observed. The results presented in this study indicate that A-DEP alters lysosomal activity in macrophages as we observed a significantly reduced LysoTracker green signal (Fig. 6). Our previously published study showed that exposure to ZnO nanoparticles resulted in reduced LysoTracker signal especially in the presence of free fatty acids (Cao et al., 2015). The results are also in agreement with a previous report, showing that exposure of macrophages to DEP SRM 2975 induced lysosomal dysfunction (Chaudhuri et al., 2012). Recently, it was shown that oxidized low-density lipoprotein treatment resulted in lysosomal dysfunction evidenced by observations of enlargement of lysosomes, loss of LysoTracker signal and increase of lysosomal pH in cultured macrophages. Lysosomal biogenesis induced by transcription factor overexpression was observed to revert lysosomal dysfunction and enhance
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cholesterol efflux, which suggests a central role of lysosomes in the protection of macrophages against atherogenic lipid loading (Emanuel et al., 2014). Lysosomes also play an important role in the degradation of engulfed particles, and nanoparticle exposure may impair the fusion of autophagosomes with lysosomes (Stern et al., 2012). A recent study showed that carbon nanotube exposure induced autophagosome accumulation due to a blockade of the autophagic flux, whereas pharmacological stimulation of the autophagic flux promoted extracellular efflux of nanoparticles and attenuated nanoparticle-induced toxicity (Orecna et al., 2014). Thus, it is possible that the altered lysosomal activity may impair digestion and efflux of particles and lipids, which may be the mechanisms of A-DEP induced lipid droplet formation in macrophages. It may also be possible that DEP synergize with lipids to induce vascular effects because particles have been suggested as the cause for oxidative stress (Miller et al., 2012). For example, it has been shown before that DEP and oxidized phospholipids synergistically enhanced inflammatory gene expression both in human endothelial cells in vitro and mice livers in vivo (Gong et al., 2007). DEP exposure in vitro caused high-density lipoprotein dysfunction, and dysfunctional high-density lipoproteins further promoted DEP oxidation (Yin et al., 2013b). Under in vivo conditions, dysfunction of high-density lipoproteins mediated systemic effects in plasma and liver induced by inhalational exposure to DEP (Yin et al., 2013a). Animal studies have shown that exposure to DEP can induce influx of macrophages (Bai et al., 2011; Miller et al., 2013; Singh et al., 2004). In the present study we observed increased formation of lipid droplets in macrophages after exposure to 10 g A-DEP/ml (6.3 g/cm2 ). This concentration is within the concentrations used in previous particle toxicology studies (Moller et al., 2014). The concentration of DEP in urban is typically about 3 g/m3 , which corresponds to a deposited daily dose of 7.2 pg/cm2 , assuming inhalation of 12 m3 /day, 20% deposition and a surface area of 100 m2 . A theoretical program predicted that life-time exposure to 1 mg/m3 spherical nanoparticles may lead to 20–48.9 g/cm2 particles in lung surface, depending on the size of particles (Gangwal et al., 2011). The concentration of A-DEP shown to significantly induce lipid droplet formation may be achievable to alveolar macrophages to induce similar effects in vivo after high concentrations of A-DEP exposure. This in theory supports the finding that DEP exposure induced accumulation of lipid droplets in alveolar macrophages in mice (Yanamala et al., 2013). Monocytes in blood can also be exposed to particles penetrated through the alveolarcapillary barrier. However, it is unclear if enough particles are able to translocate to induce the effects (Donaldson et al., 2013). Collectively, the results of the present study showed that A-DEP exposure promoted formation of lipid droplets in macrophages, which is possible through mechanisms related with lysosomes but not directly associated with ROS production. Dysregulation of lipid metabolism and subsequent macrophage foam cell formation may be involved in mediating adverse cardiopulmonary health effects induced by DEP exposure. Conflict of interest The authors declare no conflict of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgement This work was supported by the Danish Research Council for Health and Disease (grant no. 12-126262).
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Automobile diesel exhaust particles induce lipid droplet formation in macrophages in vitro Yi Cao ∗ , Kim Jantzen, Ana Cecilia Damiao Gouveia, Astrid Skovmand, Martin Roursgaard, Steffen Loft, Peter Møller Department of Environmental Health, Section of Environmental Health, University of Copenhagen, Øster Farimagsgade 5A, DK-1014 Copenhagen K, Denmark
a r t i c l e
i n f o
Article history: Received 4 March 2015 Received in revised form 4 June 2015 Accepted 6 June 2015 Available online 9 June 2015 Keywords: Automobile diesel exhaust particles Oxidative stress Lysosomal dysfunction Lipid droplet
a b s t r a c t Exposure to diesel exhaust particles (DEP) has been associated with adverse cardiopulmonary health effects, which may be related to dysregulation of lipid metabolism and formation of macrophage foam cells. In this study, THP-1 derived macrophages were exposed to an automobile generated DEP (A-DEP) for 24 h to study lipid droplet formation and possible mechanisms. The results show that A-DEP did not induce cytotoxicity. The production of reactive oxygen species was only significantly increased after exposure for 3 h, but not 24 h. Intracellular level of reduced glutathione was increased after 24 h exposure. These results combined indicate an adaptive response to oxidative stress. Exposure to A-DEP was associated with significantly increased formation of lipid droplets, as well as changes in lysosomal function, assessed as reduced LysoTracker staining. In conclusion, these results indicated that exposure to A-DEP may induce formation of lipid droplets in macrophages in vitro possibly via lysosomal dysfunction. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Exposure to particulate matter (PM), especially PM with an aerodynamic diameter below 2.5 m (PM2.5) and 0.1 m (ultrafine particles, UFP) has been associated cardiopulmonary health effects (Hiraiwa and van Eeden, 2013), which are considered to be related to oxidative stress and inflammation (Miller et al., 2012; Moller et al., 2014). Elevated levels of lipid peroxidation products have been observed in humans living in urban areas and in controlled trials with well-defined exposure to PM (Moller and Loft, 2010). Inhalation or instillation of ambient air PM in animals has likewise been associated with elevated levels of lipid peroxidation products in lung tissue (Moller et al., 2014). Alveolar macrophages are central in the defense against deposited PM as well as in the inflammatory pathways related to lung and cardiovascular diseases attributed to exposure to air pollution (Hiraiwa and van Eeden, 2013; Ling and van Eeden, 2009). Interestingly, a recent study showed that pharyngeal aspiration of diesel exhaust particles (DEP), the major source of UFP in urban environments, was associated with accumulation of lipid droplets in alveolar
∗ Corresponding author at: Section of Environmental Health, Department of Public Health, University of Copenhagen, Øster Farimagsgade 5A, Building 5B, 2nd Floor, DK-1014 Copenhagen K, Denmark. Tel.: +45 3533 7210. E-mail address: [email protected] (Y. Cao). http://dx.doi.org/10.1016/j.etap.2015.06.012 1382-6689/© 2015 Elsevier B.V. All rights reserved.
macrophages and lipid peroxidation in lung tissue (Yanamala et al., 2013). Moreover, hyperlipidemic ApoE−/− mice developed pulmonary emphysema apparently related to lipid accumulation in macrophages with subsequent cholesterol efflux and activation of the Toll-like receptor 4 signaling pathway (Goldklang et al., 2012). Another study showed that inhalation of DEP in mice was associated with elevated levels of lipid peroxidation products in the lungs, plasma and liver, which was considered to be due to dysfunction of high-density lipoproteins (Yin et al., 2013a). Such alteration in lipid homeostasis and subsequent lipid accumulation in the vascular system is implicated in the development of atherosclerosis (Moore et al., 2013). Interestingly, recent animal studies showed that exposure to DEP changed the characteristics of atherosclerotic plaques with increased lipids and formation of macrophage foam cells (Miller et al., 2013; Bai et al., 2011). Intracellular lipids stored as triglycerides in lipid droplets are taken up by autophagosomes and subsequently delivered to lysosomes for degradation, a process known as lipophagy (Liu and Czaja, 2013). Lipophagy was initially discovered in hepatocytes (Singh et al., 2009) and was later reported to occur in macrophages (Ouimet et al., 2011). Lipophagy dysfunction in macrophages plays a crucial role in lipid accumulation, foam cell formation and development of atherosclerosis (Moore et al., 2013; Ouimet and Marcel, 2012). In addition, recent studies showed that exposure to a variety of nanomaterials induced lipid accumulation in hepatocytes (Vesterdal et al., 2014) and macrophages (Cao et al., 2014a,b).
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In this study, we used human THP-1 monocyte differentiated macrophages as an in vitro model to alveolar macrophages to study lipid droplet formation and possible mechanisms after exposure to an automobile generated DEP (A-DEP). Cytotoxicity was measured using both 2-(4-iodophenyl)-3-(4-nitrophenyl)5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) and calcein acetoxymethyl (calcein AM). Intracellular reduced glutathione (GSH) levels and production of reactive oxygen species (ROS) were measured to indicate oxidative stress using fluorescent probes. The formation of lipid droplets in macrophages was determined using Bodipy 493/503 and lysosomal function was investigated using LysoTrackerTM green.
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Size-distribution of A-DEP was analyzed using a NanoSight LM20 (NanoSight, Amesbury, UK); 5 g A-DEP/ml RPMI-1640 medium with 10% FCS were analyzed in triplicates on three independent days. The Nanoparticle Tracking Analysis (NTA) version 2.3 was used to track and analyze the particles and means of ‘Mean’ (the mean size of the particles) and ‘Mode’ (the size of the most frequently occurring particles) were calculated from three independent experiments (n = 3). Comparative studies indicated that NTA is a good alternative to transmission electron microscopy for the measurement of particle sizes (Roursgaard et al., 2014; De Temmerman et al., 2014). 2.3. Cytotoxicity assay
2. Materials and methods 2.1. Cell line The monocytic cell line THP-1, obtained from the American Type Culture Collection (Manassas, VA, USA), were cultivated in supplemented RPMI-1640 medium with 10% fetal bovine serum (FBS) and differentiated into macrophage-like cells by treatment with 10 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma, St. Louis, MO, USA) as previously described (Jantzen et al., 2012). It has been shown that PMA differentiated THP-1 macrophages resemble primary human monocyte-derived macrophages, and thus serves as a good in vitro model for human macrophages (Daigneault et al., 2010). We have previously used THP-1 derived macrophages to study the formation of macrophage foam cells (Cao et al., 2014a,b; Del Bo et al., 2015). Prior to particle exposure, cells were seeded at either a density of 5 × 104 cells/well in 0.1% gelatin pre-coated 96-well plates or 8-well microscopy chamber slides (Ibidi, Munich, Germany), or in a density of 5 × 105 cells/well in pre-coated 12-well plates overnight. 2.2. Particles A-DEP was provided as a gift from Dr. Hiromi Izawa (Gifu University, Gifu, Japan). This type of A-DEP was collected from an automobile engine as previously described (Izawa et al., 2007), using the same combustion conditions and collection method as described (Sagai et al., 1993). The A-DEP material has been thoroughly characterized and compared to a standard reference material (SRM 2975) DEP from the National Institute of standards and Technology (Singh et al., 2004; DeMarini et al., 2004). Chemical analysis showed that A-DEP contains carbon (elemental carbon and organic carbon), extractable organic material and polycyclic aromatic hydrocarbons, and scanning electron micrographs of A-DEP showed aggregated >50 m particles, whereas SRM 2975 contained particles with sizes <10 to > 50 m (Singh et al., 2004; DeMarini et al., 2004). Exposure of mice to A-DEPs induced influx and activation of macrophages and inflammatory response in the lungs (Singh et al., 2004). For the exposure, A-DEP suspensions were prepared by sonication of 1 mg/ml particles in RPMI-1640 medium with 10% FCS for 8 min (with alternating 10 s pulses and 10 s pauses and continuously cooling on ice) using a Branson Sonifier S-450D (Branson Ultrasonics Corp., Danbury, CT, USA) equipped with a disrupter horn (Model number: 101-147-037), and then diluted in RPMI1640 medium with 10% FCS to the concentrations needed for exposure. Cells were exposed to 200 l/well (in 96-well plate) or 2 ml/well (in 12-well plate) 2.5 g/ml, 5 g/ml and 10 g/ml A-DEP, which equate to 1.6 g/cm2 , 3.1 g/cm2 and 6.3 g/cm2 , respectively. For fluorescence microscopy, cells were exposed to 200 l/well 10 g/ml A-DEP. The suspensions of A-DEP were applied freshly after each sonication.
Cytotoxicity was measured after exposure to particles using WST-1 reagent assay (Roche Diagnostics GmbH, Mannheim, Germany) and calcein AM staining (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions. WST-1 is a water soluble tetrazolium salt which is cleaved to a formazan dye by succinate-tetrazolium reductase systems in metabolically active cells. Cells were seeded on pre-coated 96-well plates and exposed to various concentrations of A-DEP for 24 h. Then, cells were washed using Hanks solution, followed by incubation with 100 l RPMI-1640 medium with 10% FCS and 10% WST-1 reagent at 37 ◦ C for 2 h. The absorbance of the formazan product was measured at 450 nm with 630 nm as background reference using an ELISA reader (Labsystems, Multiskan Ascent). Calcein AM can be converted to green-fluorescence by intracellular esterases in living cells. Following A-DEP exposure, cells on pre-coated black 96-well plates were washed twice using Hanks solution, followed by incubation with 0.5 g/ml calcein AM in RPMI-1640 medium at 37 ◦ C for 30 min. The green fluorescence was subsequently measured (ex: 485 nm; em: 538 nm) by a fluorescence spectrophotometer (Fluoroskan Ascent FL; Labsystems). To confirm the results, microscopy was used to visualize the morphology of the cells. A-DEP exposed THP-1 macrophages seeded on pre-coated 8-well microscopy chamber slides (Ibidi) were visualized by differential Interference Contrast (DIC) in a Leica AF6000 inverted widefield microscope with 40× magnifications (Leica Microsystems GmbH, Wetzlar, Germany). 2.4. Reduced GSH measurement The effect of A-DEP exposure on intracellular reduced GSH levels was estimated using a fluorescent probe ThioGlo® -1 (Covalent Inc., Woburn, MA) as previously described (Cao et al., 2011). ThioGlo® -1 is a membrane permeable probe which becomes highly fluorescent upon reacting with thiol groups (Cao et al., 2011). Because most of the intracellular thiols are reduced GSH, it can be used to estimate levels of intracellular reduced GSH. For the experiment, THP-1 macrophages on pre-coated 96-well plates were exposed to various concentrations of A-DEP for 24 h. After exposure cells were washed once in Hanks solution and incubated with 10 M probe in PBS at 37 ◦ C for approximately 5 min. The fluorescence was measured at excitation 355 nm and emission 460 nm by a fluorescence spectrophotometer (Fluoroskan Ascent FL; Labsystems). 2.5. ROS measurement The ROS production was estimated using 2 ,7 -dichlorofluorescein diacetate (DCFH-DA; Invitrogen A/S Taastrup, Denmark) and CellROX deep red (Invitrogen, Eugene, OR, USA). DCFH-DA is a cell permeable probe which is deacetylated inside the cells and further oxidized to a fluorescent probe 2 ,7 -dichlorofluorescein (DCF) upon reacting with a variety of ROS, making it a general indicator of intracellular ROS production
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(Dikalov et al., 2007). The fluorescence of DCF was quantified using a fluorescence spectrophotometer as previously described with slight modifications (Dikalov et al., 2007). Briefly, THP-1 macrophages seeded on pre-coated black 96-well plates were exposed to various concentrations of A-DEP for 24 h. Following exposure, cells were washed twice in Hanks solution and incubated with 10 M DCFH-DA in RPMI-1640 medium at 37 ◦ C for 30 min (Dikalov et al., 2007). After incubation the cells were washed twice and fluorescence was measured (ex: 355 nm; em: 460 nm) using a fluorescence spectrophotometer (Fluoroskan Ascent FL; Labsystems). For benchmark purposes, A-DEP exposure was also compared to exposure to the general benchmark DEP SRM 2975 (NIST, Gaithersburg, MD, USA). In this setup, cells were pre-loaded with DCFH-DA probe, washed and exposed to particles for 3 h, followed by the measurement of the fluorescence as described above. Fluorescence microscopy was used to supplement the results obtained with DCF by CellROX deep red, a specific probe for cytoplasmic ROS. The exposed THP-1 macrophages seeded on precoated 8-well microscopy chamber slides were washed twice using warm RPMI-1640 medium, and then stained with 5 M probe in RPMI-1640 medium at 37 ◦ C for 30 min. Hereafter, cells were washed twice in warm RPMI-1640 medium, and then visualized by combined DIC and red fluorescence in a Leica microscope with 63× magnification, with each image captured as z-stack in 5 randomly selected areas (n = 5), followed by 3D deconvolution and 3D projection using Leica LAS AF 2.6.0.7266 software. The integrated density of fluorescence was measured by ImageJ (NIH) and normalized by the number of cells in each randomly selected area. 2.6. Measurements of lipid droplet formation The formation of lipid droplets was measured using Bodipy 493/503 (Molecular Probes, Eugene, OR). For the quantitative measurement of lipid droplets, THP-1 macrophages were seeded on pre-coated 12-well plates and exposed to various concentrations of particles for 24 h. After exposure, the cells were washed once in Hanks solution and incubated with 1 g/ml Bodipy 493/503 in RPMI-1640 medium at 37 ◦ C for 30 min. Hereafter, the cells were washed in Hanks solution, harvested with trypsin, centrifuged, and re-suspended in cold PBS prior to flow cytometric analysis using a flow cytometer (BD AccuriTM C6, Cambridge, UK) equipped with CFlow plusTM software for data analysis. A total of 1 × 105 events were analyzed for each sample with an unexposed and unstained sample as negative control, and fluorescence was collected from FL1 channel. The formation of lipid droplets was also measured by fluorescence microscopy as previously described (Cao et al., 2014b). Briefly, A-DEP exposed THP-1 macrophages seeded on pre-coated 8-well microscopy chamber slides were washed twice with PBS and fixed by 4% paraformaldehyde for 30 min at room temperature. It has previously been shown that fixation of cells by paraformaldehyde did not affect the structure of lipid droplets or lipid contents (DiDonato and Brasaemle, 2003). Following fixation the cells were washed again with PBS and stained with 1 g/ml Bodipy 493/503 for 15 min, followed by washing and addition of mounting media (Ibidi). The green fluorescence was quantified using a Leica microscope with 40× magnification in 5 randomly selected areas (n = 5), with each sample captured as a z-stack. After 3D deconvolution and 3D projection, the fluorescent areas in each independent area were analyzed by ImageJ and normalized by the number of cells (Cao et al., 2014b).
LysoTracker signal could be due to either increase of lysosomal pH leading to poor retention of the dye or the increase of lysosomal membrane permeabilization leading to the rupture of lysosomes, both of which indicate lysosomal dysfunction (Emanuel et al., 2014). For the measurement, THP-1 macrophages were seeded on pre-coated 12-well plates and exposed to various concentrations of particles for 24 h. After exposure, the cells were washed once in Hanks solution and incubated with 50 nM LysoTracker green in RPMI-1640 medium at 37 ◦ C for 30 min. Hereafter, the cells were washed in Hanks solution, harvested with trypsin, centrifuged, and re-suspended in cold PBS prior to flow cytometric analysis using a flow cytometer (BD AccuriTM C6, Cambridge, UK) equipped with CFlow plusTM software for data analysis. A total of 1 × 105 events were analyzed for each sample with an unexposed and unstained sample as negative control, and fluorescence was collected from FL1 channel. 2.8. Statistics One-way ANOVA followed by Tukey HSD test using R-3.0.2; P values <0.05 were considered statistically significant. 3. Results 3.1. Size of A-DEP The representative size distribution of particles suspended in RPMI-1640 medium with 10% FCS analyzed using NTA is as shown in Fig. 1. The ‘Mean’ of A-DEPs (the mean size of the particles) was calculated as 192 ± 4 nm, and ‘Mode’ (the size of the most frequently occurring particles) was calculated as 154 ± 7 nm (mean ± SE of means of three independent experiments; n = 3). 3.2. Cytotoxicity The results from WST-1 (Fig. 2A) and calcein AM (Fig. 2B) assays showed no statistically significant changes in WST-1 product formation or calcein AM fluorescence (p > 0.05). In addition, the microscopic images (Fig. 2C) showed no obvious changes in the morphology of the A-DEP exposed cells. These results combined indicate that A-DEP exposure did not induce cytotoxicity in THP-1 macrophages.
2.7. Lysosomal activity Lysosomal activity was indicated using LysoTracker® green (Invitrogen, Carlsbad, CA) that stains lysosomes. A decrease of the
Fig. 1. Representative nanosight (NTA) measurements of particle size distribution in RPMI-1640 medium with 10% FCS. The black line indicates particle size and the gray line indicates SE.
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Fig. 2. Effect of 24 h exposure to A-DEP on cytotoxicity. (A) WST-1 assay. (B) Calcein AM staining assay. Data is expressed as mean ± SE of means of three independent experiments (n = 4 for each experiment). (C) Representative images from two independent experiments showing the morphology of THP-1 macrophages after 24 h exposure to A-DEP.
Fig. 3. Effect of 24 h exposure to A-DEP on oxidative stress. (A) ROS production. Data is expressed as mean ± SE of means of three independent experiments (n = 4 for each experiment). (B) Representative images from two independent experiments showing CellROX deep red fluorescence. (C) Quantitative analysis of integrated density of CellROX fluorescence/cell, respectively. Data is expressed as mean ± SE in 5 randomly selected independent areas (n = 5). The unexposed cells are normalized to 100%. (D) Reduced GSH levels. Data is expressed as mean ± SE of means of three independent experiments (n = 4 for each experiment). *p < 0.05 compared with unexposed cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 4. ROS production in THP-1 macrophages after 3 h exposure to A-DEP using SRM 2975 as benchmark. Data is expressed as mean ± SE of means of four independent experiments (n = 3 for each experiment). *p < 0.05 compared with unexposed cells.
not statistically significant (p > 0.05). Representative fluorescence microscope images are shown in Fig. 3B. No obvious increase in CellROX deep red signal was observed after particle exposure using fluorescence microscopy. Quantitative analysis of the integrated density of CellROX deep red (Fig. 3C) indicates that ROS production was at equal levels between A-DEP exposed cells and controls. The results from reduced GSH measurements (Fig. 3D) showed that 24 h exposure to A-DEP increased intracellular levels of reduced GSH, and a statistically significant increase of reduced GSH was observed after exposure to 5 g/ml A-DEP (p < 0.05; p = 0.087 at concentration of 10 g/ml). A benchmark comparison of the effect of A-DEP and the often used benchmark DEP SRM2975 showed that A-DEP can induce ROS production in THP-1 macrophages after 3 h exposure at high concentrations (p < 0.05 at 25 g/ml and p < 0.01 at 100 g/ml, compared with control), whereas the SRM 2975 induced significant increase in ROS production at 100 g/ml (p < 0.01, compared with control; Fig. 4). 3.4. Lipid droplet
3.3. Oxidative stress The results from ROS measurements are shown in Fig. 3A. Exposure to A-DEP was associated with a modest increase in ROS production as measured by DCFH-DA, but the increase was
The formation of lipid droplets was measured by Bodipy 493/503 and results are shown in Fig. 5. Exposure to A-DEP increased the formation of lipid droplets in a concentrationdependent manner, and a statistically significant increase of lipid
Fig. 5. Lipid droplet formation as measured by Bodipy 493/503 staining in THP-1 macrophages after 24 h exposure to A-DEP. (A) Quantitative measurement of lipid droplet formation by flow cytometry. Data is expressed as means ± SE of means of three independent experiments, with 1 × 105 events analyzed in each sample. The unexposed cells are normalized as 100%. *p < 0.01 compared with unexposed cells. (B) Representative images from two independent experiments showing Bodipy 493/503 green fluorescence. (C) Quantitative analysis of integrated Bodipy 493/503 fluorescence area/cell. Data is expressed as mean ± SE in 5 randomly selected areas (n = 5). The unexposed cells are normalized to 100%. *p < 0.05 compared with unexposed cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 6. Lysosomal activity as measured by LysoTracker green staining after 24 h exposure to A-DEP. Data is expressed as means ± SE of means of three independent experiments, with 1 × 105 events analyzed in each sample. The unexposed cells are normalized as 100%. *p < 0.05 compared with unexposed cells.
droplet formation was observed after 24 h exposure to 10 g/ml A-DEP (p < 0.01, compared with control; Fig. 5A). Representative fluorescence microscope images are shown in Fig. 5B. Lipid droplets were sparse but detectable inside the unexposed cells (Fig. 5B, left panel), whereas cells exposed to A-DEP (Fig. 5B, right panel) contained large lipid droplets. The quantitative analysis of fluorescence microscope images (Fig. 5C) indicates that the average area of lipid droplets/cell was significantly increased after exposure to A-DEP (p < 0.05). 3.5. Lysosomal activity Fig. 6 shows lysosomal activity as indicated by LysoTracker green staining. Exposure to A-DEP resulted in a statistically significant decrease in LysoTracker signal at 2.5 g/ml (p < 0.05), 5 g/ml (p < 0.01) and 10 g/ml (p < 0.05) as compared with the control. 4. Discussion In the present study, THP-1 monocyte derived macrophage was used as an in vitro model to study A-DEP exposure induced lipid droplet formation and possible mechanisms. Using different assays, no obvious increase in cytotoxicity (Fig. 2) or ROS production (Fig. 3) was observed after 24 h exposure of up to 10 g/ml A-DEP, but a transient increase in ROS production was seen after 3 h exposure to A-DEP as well as the benchmark DEP SRM2975 (Fig. 4). Other studies have shown that exposure to PM induced oxidative stress, which generally is considered to play an important role in PM induced toxicity (Moller et al., 2014; Madl et al., 2014) and adverse vascular effects (Araujo and Nel, 2009; Donaldson et al., 2013; Miller et al., 2012; Shrey et al., 2011). Previous work also showed that exposure to 100 g/ml, but not lower concentrations of DEP SRM 2975, significantly induced ROS in THP-1 macrophages (Jantzen et al., 2012). Similarly, our study shows that exposure to A-DEP only significantly induced ROS production in THP-1 macrophages at high concentration of 25 or 100 g/ml (Fig. 4). Thus, high exposure concentrations of DEP may induce oxidative stress, whereas low exposure concentrations as used in this study may be associated with adaptation to oxidative stress. This effect could be explained by particle-induced cellular survival responses through the activation of adaptation factors such as nuclear factor erythroid 2-related factor 2 and consequently upregulated expression of antioxidant defense genes (Kensler et al., 2007). Previously, it has been shown that 20 h exposure of human lung epithelial cells to fly ash
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particles increased levels of the antioxidant enzyme heme oxygenase-1 and increased GSH levels (Diabate et al., 2011). Six hours exposure of human lung epithelial cells to high concentrations of fullerene C60 nanoparticles increased GSH levels (Wang et al., 2014). Our previous work showed that 3 h exposure to DEP or carbon-based particles induced ROS in a number of different types of human cells (Cao et al., 2014a,b; Forchhammer et al., 2012; Frikke-Schmidt et al., 2011; Jantzen et al., 2012; Vesterdal et al., 2012, 2014). However, exposure of primary human endothelial cells to carbon black nanoparticles (Printex90) only significantly increased reduced GSH levels at 24 h, but did not significantly affect it or expression of antioxidant enzymes (glutamate-cysteine ligase modifier subunit and heme oxygenase 1) after 3 h (Cao et al., 2014b). This indicated that particle exposure could induce a transient production of ROS after 3 h, but an adaptive response to oxidative stress could be induced after a longer exposure period. Our result is also in keeping with a current hypothesis describing a hierarchical tiered oxidative stress response that minor oxidative stress induces defense mechanisms; whereas higher levels initiate specific signaling pathways that lead to injury and cytotoxicity (Madl et al., 2014). It has previously been demonstrated that exposure to a variety of particles could induce lipid accumulation in different types of cells in vitro (Cao et al., 2014a,b; Khatchadourian and Maysinger, 2009; Vesterdal et al., 2014; Przybytkowski et al., 2009). Some studies indicated that oxidative stress may be involved in the regulation of lipid accumulation, as treatment with antioxidants prevented particle-induced lipid accumulation (Cao et al., 2014a; Khatchadourian and Maysinger, 2009). Another study showed that antioxidant vitamin E attenuated oxidized low-density lipoprotein induced lipid accumulation in macrophages by modulating nuclear factor B related pathway (Huang et al., 2012). However, our recent study indicated that carbon black nanoparticles induced lipid accumulation in macrophages independently of oxidative stress, as addition of antioxidants did not affect particle-induced lipid accumulation and particles promoted lipid accumulation at concentrations not associated with increased ROS production (Cao et al., 2014b). Increased oxidative stress may not completely explain particle-induced lipid accumulation. In the present study, we found that A-DEP significantly induced formation of lipid droplets in macrophages (Fig. 5) at concentrations that was not associated with increased ROS production as measured by different methods (Fig. 3). These results combined indicated that A-DEP induced lipid droplet formation in macrophages is not directly related with ROS production. We did not attempt to measure lipid droplet formation after 3 h, although we observed an increase in ROS production after 3 h (Fig. 4), because our previous study showed that exposure of THP-1 macrophages to high concentrations of free fatty acids was only associated with significantly increased lipid accumulation after 24 h, but not after 3 h (Cao et al., 2014a). Therefore, it probably takes longer time than 3 h for the newly synthesized lipid droplets to be observed. The results presented in this study indicate that A-DEP alters lysosomal activity in macrophages as we observed a significantly reduced LysoTracker green signal (Fig. 6). Our previously published study showed that exposure to ZnO nanoparticles resulted in reduced LysoTracker signal especially in the presence of free fatty acids (Cao et al., 2015). The results are also in agreement with a previous report, showing that exposure of macrophages to DEP SRM 2975 induced lysosomal dysfunction (Chaudhuri et al., 2012). Recently, it was shown that oxidized low-density lipoprotein treatment resulted in lysosomal dysfunction evidenced by observations of enlargement of lysosomes, loss of LysoTracker signal and increase of lysosomal pH in cultured macrophages. Lysosomal biogenesis induced by transcription factor overexpression was observed to revert lysosomal dysfunction and enhance
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cholesterol efflux, which suggests a central role of lysosomes in the protection of macrophages against atherogenic lipid loading (Emanuel et al., 2014). Lysosomes also play an important role in the degradation of engulfed particles, and nanoparticle exposure may impair the fusion of autophagosomes with lysosomes (Stern et al., 2012). A recent study showed that carbon nanotube exposure induced autophagosome accumulation due to a blockade of the autophagic flux, whereas pharmacological stimulation of the autophagic flux promoted extracellular efflux of nanoparticles and attenuated nanoparticle-induced toxicity (Orecna et al., 2014). Thus, it is possible that the altered lysosomal activity may impair digestion and efflux of particles and lipids, which may be the mechanisms of A-DEP induced lipid droplet formation in macrophages. It may also be possible that DEP synergize with lipids to induce vascular effects because particles have been suggested as the cause for oxidative stress (Miller et al., 2012). For example, it has been shown before that DEP and oxidized phospholipids synergistically enhanced inflammatory gene expression both in human endothelial cells in vitro and mice livers in vivo (Gong et al., 2007). DEP exposure in vitro caused high-density lipoprotein dysfunction, and dysfunctional high-density lipoproteins further promoted DEP oxidation (Yin et al., 2013b). Under in vivo conditions, dysfunction of high-density lipoproteins mediated systemic effects in plasma and liver induced by inhalational exposure to DEP (Yin et al., 2013a). Animal studies have shown that exposure to DEP can induce influx of macrophages (Bai et al., 2011; Miller et al., 2013; Singh et al., 2004). In the present study we observed increased formation of lipid droplets in macrophages after exposure to 10 g A-DEP/ml (6.3 g/cm2 ). This concentration is within the concentrations used in previous particle toxicology studies (Moller et al., 2014). The concentration of DEP in urban is typically about 3 g/m3 , which corresponds to a deposited daily dose of 7.2 pg/cm2 , assuming inhalation of 12 m3 /day, 20% deposition and a surface area of 100 m2 . A theoretical program predicted that life-time exposure to 1 mg/m3 spherical nanoparticles may lead to 20–48.9 g/cm2 particles in lung surface, depending on the size of particles (Gangwal et al., 2011). The concentration of A-DEP shown to significantly induce lipid droplet formation may be achievable to alveolar macrophages to induce similar effects in vivo after high concentrations of A-DEP exposure. This in theory supports the finding that DEP exposure induced accumulation of lipid droplets in alveolar macrophages in mice (Yanamala et al., 2013). Monocytes in blood can also be exposed to particles penetrated through the alveolarcapillary barrier. However, it is unclear if enough particles are able to translocate to induce the effects (Donaldson et al., 2013). Collectively, the results of the present study showed that A-DEP exposure promoted formation of lipid droplets in macrophages, which is possible through mechanisms related with lysosomes but not directly associated with ROS production. Dysregulation of lipid metabolism and subsequent macrophage foam cell formation may be involved in mediating adverse cardiopulmonary health effects induced by DEP exposure. Conflict of interest The authors declare no conflict of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgement This work was supported by the Danish Research Council for Health and Disease (grant no. 12-126262).
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