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PMID: 28286432 was deleted because it is a duplicate of PMID: 29368911
.2016 Dec;12(12):2139-50.
doi: 10.1166/jbn.2016.2320.

Decreased Uptake and Enhanced Mitochondrial Protection Underlie Reduced Toxicity of Nanoceria in Human Monocyte-Derived Macrophages

Decreased Uptake and Enhanced Mitochondrial Protection Underlie Reduced Toxicity of Nanoceria in Human Monocyte-Derived Macrophages

Salik Hussain et al. J Biomed Nanotechnol.2016 Dec.

Abstract

Cerium dioxide nanoparticles (nanoceria), currently used as catalysts including additives to diesel fuel, also present potential as a novel therapeutic agent for disorders involving oxidative stress. However, little is known about the effects of nanoceria on primary human cells involved in the innate immune response. Here, we evaluate nanoceria effects on monocyte derived macrophages (MDMs) from healthy human subjects. Peripheral blood monocytes were isolated from healthy human volunteers. MDMs were obtained by maturing monocytes over a five-day period. MDMs were exposed to well-characterized nanoceria suspensions (0, 5, 10, 20 μg/mL) for 24 or 48 hours. We evaluated particle uptake, ultrastructural changes, cytotoxicity, and mitochondrial damage in MDMs through transmission electron microscopy (TEM), confocal imaging, flow cytometry, spectrometry, western blots, and immunofluorescence techniques. The role that intracellular concentration of nanoceria plays in the toxicity of MDMs was evaluated by 3D image analysis and compared to monocytes as a nanoceria sensitive cell model. Nanoceria failed to induce cytotoxicity in MDMs at the tested doses. Nanoceria-exposed MDMs showed no mitochondrial damage and displayed significant accumulation of anti-apoptotic proteins (Mcl-1 and Bcl-2) during the maturation process. TEM and confocal analyses revealed efficient uptake of nanoceria by MDMs, however 3D image analyses revealed lower nanoceria accumulation per unit cell volume in MDMs compared to monocytes. Taken together, our results suggest that mitochondrial protection and reduced volume-corrected intracellular nanoparticle concentration account for the lower sensitivity of human MDMs to nanoceria.

Keywords: Nanoceria; Nanotoxicity; Monocyte-Derived Macrophages; Mitochondrial Damage.

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Conflict of interest statement

Conflict of Interest: The authors declare no competing financial interest.

Figures

Figure 1
Figure 1. Experimental Layout and Maturation of Human Peripheral Blood Monocytes into Monocyte-Derived Macrophages
A) Experimental time points and TEM images of monocytes (Day 0) and MDMs (Day 5). B) Cell size estimation (Day 0 - Day 7) by flow cytometry. C) Time course analysis of cell surface expression of mature macrophage markers (25F9) and intracellular expression of CD68 expression by flow cytometry. Data were analyzed by analysis of variance (ANOVA) followed by Tukey’s post hoc test. Graphs show average ± SEM of three independent experiments with triplicates of each condition. ****p< 0.0001 (compared to Day 0)
Figure 2
Figure 2. Characterization of nanoceria
A) The physicochemical characteristics of nanoceria. B) TEM image of nanoceria. Image courtesy of Dr. A R Badireddy. C) Intensity-weighted size distributions of freshly prepared nanoceria suspensions in exposure medium (Ex-vivo medium + 1% serum), PBS, and water.
Figure 3
Figure 3. Characterization of fluorescein-modified nanoceria
Fluorescein-modified nanoceria nanoparticles were characterized by A) FT-IR and B) UV-VIS and fluorescence spectroscopies. DLS analyses of particle suspension stability with fluorescein-modified and unmodified nanoceria in C) exposure medium (Ex vivo medium + 1%serum) and D) water and PBS. Particle suspensions were characterized at 0, 2, 16, 24 and 48 hours. Error bars and values in parentheses correspond to standard deviation of the measurements.
Figure 4
Figure 4. Ultrastructural (TEM) analyses of human MDMs after nanoceria exposure for 24 hours
A) Control and B) nanoceria treated MDMs both at day 6. Arrows indicate ceria accumulation in the vesicles. Arrow head indicates free ceria in the cytoplasm
Figure 5
Figure 5. Nanoceria does not induce cytotoxicity and mitochondrial damage in human MDMs
A) MDMs were exposed to 0–20 µg/mL nanoceria for 24 or 48 hours and cell death was quantified by flow cytometry (propidium iodide staining). B) MDMs were exposed to 20 µg/mL nanoceria for 24 or 48 hours, labeled with JC-1 probe, and analyzed by fluorescent microscopy. C) MDMs were immunostained with an antibody against activated Bax (6A7) and analyzed by fluorescent microscopy. D) MDMs were labeled for AIF protein and analyzed by flow cytometry. H2O2 and Staurosporine (STS) were used as positive controls. Data are represented as mean ± SEM of at least 3–6 independent experiments with triplicates of each condition. Data were analyzed by analysis of variance (ANOVA) followed by Tukey’s post hoc test or students t-test. **p < 0.01, ****p < 0.0001 compared to untreated control
Figure 6
Figure 6. Differentiation dependent increase in expression of anti-apoptotic Mcl-1 and Bcl-2 proteins in human MDMs
A) Representative images of Mcl-1 expression in cells during different time points of differentiation. B) Corrected total cell fluorescence (CTCF) for Mcl-1 during the maturation period. C) Representative western blot analysis of BcL-2 expression during the maturation of monocytes to MDMs with or without nanoceria exposure. D) Densitometry data of the Bcl-2 expression experiments (n=3). Data are presented as mean ± SEM and analyzed by ANOVA followed by Tukey’s posthoc test. *p< 0.05,**p< 0.01,****p< 0.0001 (compared to Day 1).
Figure 7
Figure 7. Quantification of nanoceria uptake in monocytes (CD14+ cells) and MDMs
A) Validation of nanoceria toxicity in CD14+ cells and its comparison with MDMs by flow cytometry using PI as a marker of toxicity. B) LSCM images of nanoceria treated monocytes (CD14+) and MDMs. Cell nuclei are marked with DAPI (blue), particles are green, and cell membranes are marked with Cellmask deep red (red). C) Quantification of cell volume after nanoceria exposure (20µg/mL) for 24 or 48 hours. D) Quantification of nanoparticle volume internalized by cell using 3D image analysis. E) Cell volume corrected uptake of nanoceria by monocytes (CD14+) and MDM after 24 and 48 hours exposure to 20 µg/mL nanoparticles. Data are presented as mean ± standard error of mean and were analyzed by Mann-Whitney U test. *p< 0.05, **p< 0.01, ***p< 0.001
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