Edited by: Diana Boraschi, Consiglio Nazionale Delle Ricerche (CNR), Italy
Reviewed by: Paolo Decuzzi, The Methodist Hospital Research Institute, USA; Albert Duschl, University of Salzburg, Austria
†These authors have contributed equally to this work.
‡Present address: Toshiro Hirai, Departments of Dermatology and Immunology, University of Pittsburgh, Pittsburgh, PA, USA
Specialty section: This article was submitted to Inflammation, a section of the journal Frontiers in Immunology
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The application of nanotechnology in the health care setting has many potential benefits; however, our understanding of the interactions between nanoparticles and our immune system remains incomplete. Although many of the biological effects of nanoparticles are negatively correlated with particle size, some are clearly size specific and the mechanisms underlying these size-specific biological effects remain unknown. Here, we examined the pro-inflammatory effects of silica particles in THP-1 cells with respect to particle size; a large overall size range with narrow intervals between particle diameters (particle diameter: 10, 30, 50, 70, 100, 300, and 1,000 nm) was used. Secretion of the pro-inflammatory cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α induced by exposure to the silica particles had a bell-shaped distribution, where the maximal secretion was induced by silica nanoparticles with a diameter of 50 nm and particles with smaller or larger diameters had progressively less effect. We found that blockade of IL-1β secretion markedly inhibited TNF-α secretion, suggesting that IL-1β is upstream of TNF-α in the inflammatory cascade induced by exposure to silica particles, and that the induction of IL-1β secretion was dependent on both the NLRP3 inflammasome and on uptake of the silica particles into the cells
The application of nanotechnology is a promising means of developing novel diagnostic and imaging technologies, photothermal therapies, vaccines, and drug delivery systems (
There are two main factors that make nanoparticles not only more effective but also more hazardous than the bulk material. The first is their ability to cross biological barriers [e.g., blood–brain barrier (
The pro-inflammatory effects of nanoparticles are well described in the literature and are a major issue for the development of safe nanomedicines (
In the present study, we examined the effects of particle size on the pro-inflammatory response of THP-1 cells to exposure to silica particles within a large overall size range (10–1,000 nm) that included narrow intervals between the particle diameters. We also explored the mechanisms underlying this size-specific inflammatory response in our model, although it should be noted that the experimental conditions were not chosen to represent human exposure scenarios.
Amorphous silica particles (silica particles) with diameters of 10, 30, 50, 70, 100, 300, or 1,000 nm (nSP10, nSP30, nSP50, nSP70, nSP100, mSP300, and mSP1000, respectively) were purchased from Micromod Partikeltechnologie (Rostock/Warnemünde, Germany). Crystalline silica particles (Min-U-Sil-5; crystalline silica in diameter of not bigger than 5 μm) were purchased from Pennsylvania Sand Glass Corporation (Pittsburgh, PA, USA). The endotoxin level of each size of silica particle (50 μg/mL in cell culture media) was 0.25, 0.15, 0.11, 14.88, 1.23, 0.01, and <0.01 endotoxin units/mL for nSP10, nSP30, nSP50, nSP70, nSP100, mSP300, and mSP1000, respectively, as determined by a Pyros Kinetix turbidity assay instrument with a limit of detection of 0.001 endotoxin units/mL. Endotoxin testing was performed on our behalf by nanoComposix (San Diego, CA, USA). Immediately prior to use, the dispersions of the particles were sonicated at 400 W for 5 min at 25°C and then vortexed for 1 min.
Phorbol 12-myristate 13-acetate (PMA), polyinosinic acid potassium salt (poly I), cytochalasin D, bafilomycin A1, BMS345541, and adenosine 5′-triphosphate disodium salt hydrate (ATP) were purchased from Sigma Aldrich (St. Louis, MO, USA). zYVAD-fmk and UNC569 were purchased from Merck (Darmstadt, Germany).
THP-1 cells (human acute monocytic leukemia cell line) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured at 37°C (95% room air, 5% CO2) in RPMI1640 (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% fetal bovine serum, 1% antibiotic cocktail (10,000 U/mL penicillin, 10,000 μg/mL streptomycin, and 25 μg/mL amphotericin B; Gibco, BRL, Bethesda, MD, USA), and 2-mercaptoethanol (50 μM; Gibco).
THP-1 cells (3.0 × 104 cells/well) were seeded in flat-bottom 96-well plates (Nunc, Rochester, NY, USA) and then differentiated into macrophages by incubation with 0.5 μM PMA at 37°C for 24 h. After incubation, the cells were washed with the cell culture media and treated with the silica particles, crystalline silica, or ATP. After incubation for 6, 12, or 24 h, the supernatants were collected. To determine cell viability after exposure to the test materials, the concentration of lactate dehydrogenase in the supernatants was measured by using a Cytotoxicity LDH Assay Kit (Wako, Osaka, Japan) in accordance with the manufacturer’s instructions. To evaluate the pro-inflammatory response to exposure to the test materials, the concentrations of the pro-inflammatory cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α, and of the receptor antagonist (RA) IL-1RA, in the supernatants were assessed by ELISA kits (IL-1β, BD Pharmingen, San Diego, CA, USA; TNF-α, eBioscience, San Diego, CA, USA; IL-1RA, R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturers’ instructions. In inhibitory and neutralizing antibody assays, cytochalasin D, zYVAD-fmk, BMS345541, bafilomycin A1, anti-human scavenger receptor (SR) A1 monoclonal antibody (351620) (R&D Systems) or its mouse IgG1 isotype control (BioLegend, San Diego, CA, USA), anti-human macrophage receptor with collagenous structure (MARCO) antibody (PLK1) (Hycult Biotech, Uden, The Netherlands) (
THP-1 cells (9.0 × 105 cells/well) were seeded in 6-well plates (Nunc) and then differentiated into macrophages by incubation with 0.5 μM PMA at 37°C for 24 h. After incubation, the cells were washed with the cell culture media and treated with the silica particles (50 μg/mL), crystalline silica (500 μg/mL), or ATP (3 mM). After incubation for 6, 12, or 24 h, the cells were washed twice with phosphate-buffered saline and lysed with Mammalian Protein Extraction Reagent (M-PER; Thermo Fisher Scientific, Rockford, IL, USA). Protein samples (1 μg) were loaded on a 20% sodium dodecyl sulfate–polyacrylamide gel. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (GE Healthcare, Buckinghamshire, UK). The blots were blocked with 1% BSA in phosphate-buffered saline with 0.02% Tween 20 for 2 h at room temperature. The blots were incubated with monoclonal antibody to human IL-1β/IL-1F2 (8516) (R&D systems) at 1 h. HRP-conjugated goat anti-mouse antibody (SouthernBiotech, Birmingham, AL, USA) was added to the membranes, which were then incubated for 1 h at room temperature. The protein bands on the membrane were visualized with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific), and the images were captured by LAS4000 mini (GE Healthcare). The densities of the bands in the captured image were analyzed by using the ImageJ software (version 1.46r, National Institutes of Health).
THP-1 cells (1.4 × 107 cells/dish) were seeded in 150-mm dishes and differentiated into macrophages by incubation with 0.5 μM PMA at 37°C for 24 h. After incubation, the cells were washed with phosphate-buffered saline and incubated with 50 μg/mL of each test material for 6, 12, or 24 h. In a neutralization assay, PMA-differentiated THP-1 cells were pre-incubated for 30 min with anti-human SR-A1 or its isotype control at a concentration of 0.4 μg/mL. After incubation with the test materials, the supernatant was removed and the cells were washed twice with phosphate-buffered saline. The cells were then detached from the dish surface using trypsin, washed with the cell culture media, and collected. After the cells were collected, samples from three dishes were pooled for analysis. The pooled cells were counted, suspended in 1 mL of MilliQ water, and sent to Japan Food Research Laboratories (Osaka, Japan), where the samples were prepared for ICP-AES analysis as follows: the cells were heated to 500°C and ash melted with sodium carbonate. Water was added to the residue and the mixture was heated for 30 min before being passed through filter paper. The filtrates were then brought to a volume of 50 mL with ultrapure water. The mass of silicon in each sample was then measured with a Vista-MPX ICP-AES instrument (Varian, Palo Alto, CA, USA) on our behalf by Kiyokawa Plating Industry Co., Ltd. (Fukui, Japan). Silicon uptake by the cells was calculated as the amount of silicon in silica particle-treated cells minus the silicon level in non-silica-treated cells.
Statistical analyses were performed by using the Ekuseru-Toukei 2012 software (Social Survey Research Information Co., Ltd., Tokyo, Japan). Data are presented as mean ± SD. Significant differences between the control group and experimental group were assessed by using Student’s
Methods used in the Supplementary Figures are in the supplementary figures file.
The hydrodynamic diameters of the silica particles dispersed in the cell culture medium (5 mg/mL), as measured by means of dynamic light scattering, were 10.0, 24.3, 48.3, 64.7, 86.0, 285.7, and 1,164.3 nm for nSP10, nSP30, nSP50, nSP70, nSP100, mSP300, and mSP1000, respectively (Table S1 in Supplementary Material). These hydrodynamic diameters suggest that the silica particles were well dispersed in the cell culture medium. Transmission electron microscopy images of the silica particles used in the present study are provided in our previous reports (
We first evaluated the cytotoxicity of the silica particles in THP-1 cells by means of a lactate dehydrogenase cytotoxicity assay (Figure
To examine the effect of particle size on the pro-inflammatory effects of the silica particles in THP-1 cells, we measured the concentration of the pro-inflammatory cytokines IL-1β and TNF-α in the culture supernatant after incubation of the cells with the silica particles for 6, 12, or 24 h (Figure
It has been reported that the induction of TNF-α production by crystalline silica is mediated by IL-1β (
Two processes are involved in the secretion of mature IL-1β: NF-κB-dependent pro-IL-1β synthesis and NLRP3 inflammasome (caspase-1)-dependent cleavage of pro-IL-1β (
Particulate matter such as crystalline silica and alum is known to activate the NLRP3 inflammasome
We next examined the effects of silica particle size on the induction of pro-IL-1β. Since we detected pro-IL-1β in untreated cells, PMA-differentiation has induced a certain amount of pro-IL-1β, which is consistent with a previous report (Figures
It is well known that endocytosis of particulate matter triggers the pro-inflammatory responses. We, therefore, evaluated whether the size-specific pro-inflammatory effect of silica particles was endocytosis dependent. Blocking actin-dependent endocytosis with cytochalasin D, a potent inhibitor of actin polymerization, completely suppressed the induction of IL-1β in the silica particle- or crystalline silica-treated cells (Figure
Class A scavenging receptors (SR-A) are a group of receptors reported to be involved in the uptake into cells of environmental particles, including artificial nanoparticles such as amorphous silica nanoparticles (
A remaining question is why exposure to nSP50 had a greater effect on the induction of IL-1β than exposure to nSP100 even though both appeared to be taken up
The present results suggest that SR-A1-mediated endocytosis underlies silica particle-induced IL-1β secretion, and that the size-specific pro-inflammatory effects of silica particles are a result of the ligand size specificity of this SR-A1-mediated endocytosis. The present results also suggest that silica particles with a diameter of 50 nm induced the strongest pro-inflammatory response. SR-A1 is known to mediate both pro- and anti-inflammatory responses due to its broad ligand specificity (
The results of the present study also suggest that the uptake of nSP10 is independent of SR-A1-mediated endocytosis (Figure
The present results suggest that nSP50-mediated MerTK signaling increased the induction of IL-1β, although MerTK signaling itself is often discussed in an anti-inflammatory, immunosuppressive context mainly due to its relationship to the uptake of apoptotic cells by macrophages (
In previous studies, we observed greater induction of IL-1β in THP-1 cells treated with mSP1000 than in those treated with smaller particles (i.e., nSP30, nSP50, nSP70, mSP300, and mSP1000), although higher concentrations of silica particles were used than in the present study (i.e., 100 μg/mL; 6 h incubation) (
Scavenger receptors are a potentially useful target for vaccines for vaccine development (
The results of the present study suggest that SR-A1-mediated uptake of nanoparticles led to a size-specific inflammatory response in THP-1 cells. Since nanoparticles also have size-specific effects in non-phagocytic cells (
NN, TH, and YY designed the experiments and interpreted the results. NN, TH, KM, and MA performed the experiments and analyzed the data. NN, TH, and YY wrote the manuscript; EK, KI, and KH provided technical support and conceptual advice. YT supervised the project. All authors have read, discussed, and approved the final manuscript.
YY is employed by the Research Foundation for Microbial Diseases of Osaka University. All other authors declare no competing financial interests.
The authors thank Ms. Kaori Murayama, Ms. Risako Nagahashi, and Ms. Nobuyo Hashino for supporting them in the lab.
This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. JP16K01437 to KH, no. JP25136712 to YY, no. JP12J00488 to TH, no. JP26242055 to YT, and no. JP15K12540 to YT); by a Health Labour Sciences Research Grant from the Ministry of Health, Labour, and Welfare of Japan (no. H25-kagaku-ippan-005 to YT); and by the Uehara Memorial Foundation (to YY).
The Supplementary Material for this article can be found online at
nSP10, nSP30, nSP50, nSP70, nSP100, mSP300, and mSP1000, amorphous silica particles with diameters of 10, 30, 50, 70, 100, 300, and 1,000 nm, respectively; silica particles, amorphous silica particles; crystalline silica, crystalline silica particles; PMA, phorbol 12-myristate 13-acetate; poly I, polyinosinic acid potassium salt; ATP, adenosine 5′-triphosphate disodium salt hydrate; ICP-AES, inductively coupled plasma atomic emission spectrometry; FLICA, fluorescent-coupled YVAD inhibitor to the activated form of caspase-1; SR, scavenger receptor; MARCO, macrophage receptor with collagenous structure.