Materials
Amorphous silica nanoparticles (50 nm diameter without surface modification; 50nm-plain with amine surface modification; 50nm-NH2) and microparticles (3 μm diameter; 3μm-plain) used in this study were purchased from Micromod Partikeltechnologie GmbH (Rostock, Germany). In some experiments, nanoparticles were labeled with FITC for their localization. Prior to in vitro and in vivo studies, the particles were vortexed for 60 s.
Monoclonal rat anti-CD68 antibody (Clone FA-11) was purchased from Bio-rad (Hercules, CA, USA). Anti-F4/80 antibody (CI: A3‐1) was from (Caltag Laboratories, Burlingame, CA, USA). Anti-Ly-6G-antibody (Clone REA526) was from Miltenyi Biotec (Bergisch Gladbach, Germany). Hoechst33342 was from Dojindo (Kumamoto, Japan). L-leucyl-L-leucine methyl ester (hydrochloride) was from Cayman Chemical (Ann Arbor, MI, USA). N-acetyl-L-cysteine (NAC) was from Wako Pure Chemicals (Osaka, Japan). Diphenyleneiodonium chloride (DPI) was from Cayman Chemical. gp91ds-tat was from Anaspec (Fremont, CA, USA).
Murine silica-induced lung injury model
C57BL/6J female mice (8–10 wks old) were purchased from Jackson Laboratory (Charles River Laboratories Japan, Yokohama, Japan). They were kept in isolated and ventilated cages and maintained under specific pathogen-free conditions. All mouse care and handling protocols were approved by the University Committee on Use and Care of Animals at Nagoya University Graduate School of Medicine. Vehicle (sterile water) alone or each silica particle in the vehicle (400 µg/body) were administered intratracheally to mice under anesthesia with inhaled isoflurane. Their body weight was measured at baseline and 24 and 72 h after the administration. Mice were euthanized 72 h after intratracheal administration for analysis of bronchoalveolar lavage and harvesting lung tissues.
Bronchoalveolar lavage fluid (BALF) collection and cell counts
To collect bronchoalveolar lavage fluid (BALF), the trachea was cannulated, the lungs were lavaged three times with PBS (0.7 ml each time), and ~1.5ml of the instilled fluid was consistently recovered. Total cell numbers were counted with a standard hemocytometer.
BALF was centrifuged at 3000 rpm for 5 min at 4°C. After centrifugation, supernatants were collected and stored in -80°C until the subsequent analysis, while cell pellets were used to prepare cytospins. Smears of BALF cells were prepared with cytocentrifugation using Cytofuge2 (StatSpin, Norwood, MA, USA) at 1000 rpm for 5 min and then stained with May-Gruenwald and Giemsa stain. Cell differentiation was examined by counting at least 100 cells using standard hemocytologic criteria to classify the cells as monocytes/macrophages, neutrophils, or lymphocytes.
BALF protein concentration
Total protein concentration in BALF were determined using a Bicinchoninic Acid Protein Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer's instruction.
Mouse lung histological analysis
For histological analysis, lungs were fixed in formalin and embedded in paraffin. Four micrometer section were stained with hematoxylin and eosin (H&E). Representative images of >3 lung lobes from each mouse are obtained using a BZ-8000 microscope (Keyence, Osaka, Japan) with low and high magnification views. For the quantification of injured lung areas, image processing and digital stitching were performed using BZ-X analyzer (Keyence). The ratio of injured area to whole lobe were assessed in three lobes per animal, and the average ratio of three lobes was calculated.
Cell culture and RNA extraction
The mouse macrophage cell line RAW 264.7 was purchased from American Type Culture Collection (Manassas, VA, USA). RAW 264.7 cells were cultured in Dulbecco’s Modified Eagle Medium (Sigma-Aldrich) supplemented with 10% fatal calf serum and 1% antibiotics. All cultures were incubated at 37°C in a humidified atmosphere with 5% CO2.
RAW 264.7 macrophages (5.0×105 cells in 1 ml per well) were seeded on 12-well cell culture plates. Twenty-four hours later cells were stimulated with each silica particle (100 µg/ml). In the experiment to assess ROS involvement, an ROS inhibitor (NAC; final concentration 10 mM), or gp91ds-tat (final concentration 20 µM) was added simultaneously with silica particles. Six hours later, the cell-culture dishes were washed with PBS and 1 ml TRIzol (Life Technologies Corp., Carlsbad, CA, USA) was added to isolate total RNA. Isolation of RNA was conducted according to the manufacturer’s protocol.
Quantitative RT-PCR
Realtime PCR assays were carried out using the GoTaq 1-Step RTqPCR System (Promega, Madison, WI, USA) The sequences of oligonucleotide primers used in this study are summarized in Supplemental Table 1. Relative expression levels of each target were normalized to the 18s rRNA expression signals.
Flow cytometry
For the analysis of alveolar macrophages in BALF, Fc receptors (FcRs) were blocked with FcR Blocking Reagent (#130-092-575, Myltenyi Biotic, Bergisch Gladbach, Germany) before staining. Alveolar macrophages then were stained with CD68-biotin antibody (1:100) and streptavidin-Alexa Fluor 594 conjugate. For analysis of the Raw 264.7 cell experiment in vitro, after incubation with silica NPs, Raw 264.7cells were trypsinized and harvested for FACS analysis. Cells then were analyzed using an FACS Canto II flow cytometer (Becton-Dickinson Japan, Tokyo, Japan). A total of 10,000 events were acquired for each analysis. Dead cells and silica particles were gated out depending on FSC and SSC. The percentage of the fluorescent cells relative to the control was taken into account.
Assessment of intracellular ROS level
Levels of intracellular ROS in RAW 264.7 cells were determined using a DCFDA/H2DCFDA-Cellular ROS Assay Kit (ab113851, Abcam, Cambridge, UK) according to the manufacturer’s protocol. Briefly, Raw 264.7 macrophages (5.0×105 cells in 1 ml per well) were seeded on 12-well plates and stimulated with silica particles (100 µg/ml) 24 h later. After 3 h stimulation, cells were incubated with 20 µM DCFDA for 30 min. Cells were then trypsinized and washed once with ice-cold PBS. An FACS Canto II flow cytometer were used to determine intracellular ROS levels by DCF fluorescence.
Immunofluorescent studies for cell surface markers
Lung and cell immunostaining were conducted as previously described [47]. For lung tissue, frozen sections were incubated with primary antibodies against CD68 (1:300 dilution) or Ly-6G (1:100 dilution) at 4°C overnight. For BAL cells, slides with cytospins were incubated with primary antibodies against CD68 (1:300 dilution) after fixation and permeabilized with 0.2% Triton X-100. For RAW 264.7 cells immunostaining, cells were grown in chamber slides and stimulated with silica particles (100 µg/ml). Six hours later, cells were briefly washed with PBS and fixed in 4% paraformaldehyde for 15 min. The slides were then incubated with primary antibody against F4/80 (1:500 dilution) at 4°C overnight. In each experiment the slides were then washed with PBS, and reacted with an Alexa Fluor 594-conjugated secondary antibody at room temperature. After nuclear staining with Hoechst 33342, the slides were mounted and scanned by confocal laser scanning microscopy (TiEA1R; Nikon Instech Co., Tokyo, Japan). For localization of FITC signals from silica nanoparticles, an imaging software (NIS-Elements AR; Nikon Instech Co.,) was utilized for analyzing the fluorescence intensities of silica nanoparticles, F4/80, CD68, and the nucleus.
Immunofluorescent studies of intracellular distribution of silica nanoparticles
Assessment of intracellular localization of silica nanoparticles in RAW264.7 cells was conducted as follows. Briefly, the RAW264.7 cells (4.7 x 104 cells) were cultured on 8-well chamber plates (µ-Plate, ibidi GmbH, Grafelfing, Germany) for 24 h, and the cells were treated with silicas nanoparticles (5 μg/ml) for 24 hours in the presence with Texas Red-dextran (Life Technologies) (70 kDa, final concentration 0.5 mg/ml) as an endosomal marker. In addition, the cells were treated with LysoTracker-Red (Life Technologies) (final concentration 500 nM) as a lysosomal marker and Hoechst 33342 (80 nM) (Life Technologies) for 15 min prior to the microscopic observation. Images of the culture slides were captured using an FV1200 confocal laser scanning microscope (Olympus, Tokyo, Japan) for visualization of colocalized signals of FITC-labeled silica NPs, organelle markers, and nuclei counterstained with Hoechst33342.
Raw264.7 cells with LMP reporter
The pmCherry-Gal3 construct was a gift from Prof. Hemmo Meyer (Addgene plasmid # 85662; http://n2t.net/addgene:85662; RRID: Addgene_85662). Raw264.7 cells (2.0×106) were transfected with 2 µg of pmCherry-Gal3 plasmid DNA using Nucleofector2b (Kit V, protocol D-032; Lonza) and seeded on 6-well plates. Twenty-four hour after transfection, cells expressing Gal3 with an N-terminal mCherry-tag were selectively grown in complete medium supplemented with 1000 μg/mL of G418 (Life Technologies) for 5 d. Monoclonal cell lines were further obtained by limited dilution in 96-well plates. Only clones with positive Gal3 puncta signals with LLoMe stimulation observed by fluorescent microscopy were used for the subsequent study.
Evaluation of endosomal ROS signal
Intra-endosomal ROS production was detected using OxyBURST H2HFF Green BSA (Thermo Fisher Scientific, Austin, TX, USA) as described previously [48] [49]. Briefly, Raw264.7 cells were incubated in the presence of 100 µg/ml OxyBURST H2HFF Green BSA for 1 h at 37°C and then stimulated by the addition of phorbol myristate acetate (PMA) (300 nM) or each silica particle (100 µg/ml). For the assessment of NOX2 inhibition, gp91ds-tat (5 µM) was added simultaneously with PMA or silica particles. Cells were briefly washed with PBS and then fixed in 4% paraformaldehyde for 15 min and evaluated by confocal laser scanning microscopy (TiEA1R). For the quantification of endosomal ROS signal, the green puncta observed by confocal laser scanning microscopy in each cell were counted, and the average number of puncta per cell was calculated. Experiment were performed three times, and data were expressed as mean ±SEM.
Statistical analysis
For comparison of data from more than two groups, one-way ANOVA was employed, and the significance of the difference among the groups were tested by Tukey’s multiple comparison test. In case of time-dependent weight change analysis, two-way ANOVA was employed. SPSS statistics software (Ver.25) (IBM, Chicago, IL, USA) and GraphPad Prism Ver.8 (GraphPad Software, San Diego, CA, USA) were used to conduct statistical analyses and draw graphs. P-values <0.05 were considered statistically significant.