Reagents
The following particles were purchased from Sigma; aluminum oxide nanoparticles (Al2O3, 642991), cerium (IV) oxide nanopowder (CeO2, 700290), cobalt iron oxide nanopowder (CoFe2O4, 7733352), copper (II) oxide nanopowder (CuO, 544868), diamond nanopowder (C: 636428), iron oxide (II, III) nanoparticles (Fe3O4, 900062), nickel (II) oxide nanopowder (NiO, 637130), silicon dioxide (SiO2, S5631), silver nanopowder (Ag, 576832), titanium (IV) oxide anatase nanopowder (TiO2, 637254), uric acid sodium salt (C5H3N4O3Na, U2875), zinc oxide nanoparticles (ZnO, 721077). The following chemicals were purchased from Sigma; LOPAC 1280 library (LO4200), CA-074 methyl ester (ME) (C5857), cyclosporin A (30024), ferrostatin-1 (SML0583), necrostatin-1 (N9037), nigericin (N7143), PD173952 (PZ0113), PF431396 (PZ0185), PF562271 (PZ0387), rucaparib (PZ0036), wortmannin (W1628). The following chemicals were purchased from Cayman Chemical; AC-YVAD-CMK (10014), Bafilomycin A1 (11038), PF573228 (14924), Z-DEVD-FMK (14414). MCC950 (AG-CR1-3615) and Z-VAD-FMK (AG-CP3-0002) were purchased from AdipoGen. Other chemicals were purchased from following vendors; BAY61-3606 (Chemscene, CS-0235), Cytochalasin D (Enzo, BML-T109-0001), GSK143 (Tocris, 6362), Z-LEHD-FMK (R&D Systems, FMK008). poly(dA:dT)/LyoVec (tlrl-patc) was purchased from InvivoGen. The following antibodies were purchased from Biolegend; IgG2a isotype control antibody (400516), anti-mouse Cd18 antibody (101410), IgG2b isotype control antibody (400644), anti-mouse/human Cd11b antibody (101248). Anti-Cd36 antibody (ab80080) was purchased from Abcam and anti-mouse Cd16/Cd32 antibody was purchased from BD Biosciences (553140). Chemicals were prepared in DMSO between 10 to 40 mM stock concentrations except for nigericin (5 mM in ethanol) and poly(dA:dT)/LyoVec (50 µg/mL in water).
Bone marrow derived macrophage (BMDM)
Bone marrow cell suspensions from femurs and tibias of C57BL/6 mice (6- to 10-week old) were seeded at 5 × 106 cells per plate in non-treated sterile 10 cm plates and grown for three days in 10 mL of “BMDM media” consisting of DMEM/F-12 (Thermo, 10565–018), supplemented with 10% heat-inactivated defined FBS (HyClone, SH30070.03), 1 × penicillin/streptomycin, 1 × non-essential amino acids (Thermo, 11140–050), and 20 ng/mL mouse M-CSF (BioLegend, 576406). On day three, 5 mL of BMDM media were added on top of 10 mL media already present in the plate and grown until day seven. A step-by-step protocol for bone marrow isolation and culturing [64] were followed. Prior to nanoparticle or compound treatment, BMDM were primed with 200 ng/mL of ultrapure E. coli K12 lipopolysaccharide (LPS) (InvivoGen, LPS-EK Ultrapure) for 3 h in BMDM media. All cells were incubated in a 5% CO2 incubator at 37 °C. Data for each figure panel were obtained from the same batch of cells.
Particle preparation and addition
Particles were prepared as 20 mg/mL stock solution by adding particles to sterile distilled deionized water except for Fe3O4 which came as 5 mg/mL stock. Stock solutions were sonicated for 10 min (200W, 15 s on/5 s off cycles) in ice-cold sonicator bath (Bioruptor UCD-200TM, Cosmo Bio) and added to OPTI-MEM media (Thermo, 31,985–070) at 400 µg/mL concentration (2×). This 2× particle solution was mixed with equal volume of OPTI-MEM (either containing compound or antibody) to achieve a final concentration of 200 µg/mL.
Particle characterization
Stock solutions of particles were sonicated as above and added to OPTI-MEM media at 200 µg/mL concentration. The hydrodynamic diameter (dynamic light scattering (DLS) method) and zeta potential were measured using Zeta-potential and particle size analyzer (Otsuka Electronics, ELSZ-2000).
Chemical screen for particle toxicity (LDH assay) in BMDM
Bone marrow derived macrophage (BMDM) were seeded at 50,000 cells per well in 96-well plates (Corning, 3596), one day before the screen. On the day of the screen, BMDM were primed for 3 h with 200 ng/mL of LPS in BMDM media. Cells were then washed once with phosphate buffer saline (PBS) (Thermo, 10010023) and treated with 50 µL of OPTI-MEM containing 20 µM of the chemicals from LOPAC library and 200 µg/mL of particles. After 3 h incubation, levels of lactate dehydrogenase (LDH) released into supernatants were measured using a Cytotoxicity Detection Kit (LDH) Plus (Roche, 04 744 934 001) according to the manufacturer’s protocol. In brief, 100 µL of LDH assay reagent were added to each well, incubated for 5 min at room temperature, and reaction was stopped by adding 50 µL of stop solution. Absorbance was measured at 490 nm and 600 nm on Synergy HTX multi-mode reader. Background signal (600 nm) was subtracted from assay signal (490 nm) to obtain LDH assay value. LDH assay values of all wells were normalized to the in-plate basal (untreated; n = 4) and maximum (lysis buffer treated; n = 4) wells as; (target LDH value–basal LDH value)/(maximum LDH value–basal LDH value) × 100%. The normalized LDH value of compound treated wells were normalized to in-plate particle-treated wells (n = 8) and expressed as; (Compound and nanoparticle treated LDH value–Nanoparticle only LDH value)/(Nanoparticle only LDH value) × 100%. Details of screening including workflow are described in Additional file 1: Table S1, Fig. S9.
Particle treatment in additional cell types
C2C12 mouse myoblasts (CRL-1772) and Hepa1-6 mouse hepatocarcinoma cells (CRL-1830) were purchased from ATCC. Derivative of HEK293T human embryonic kidney cells (632180) were purchased from Clontech. All cells were grown in DMEM (Sigma, D6429) supplemented with 10% heat-inactivated defined FBS and 1 × penicillin/streptomycin. HEK293T and Hepa1-6 were seeded at 50,000 cells per well and C2C12 were seeded at 20,000 cells per well at 100 μL volume per well in 96-well plate one day before the screen. Cells were incubated with particles (200 μg/mL) in OPTI-MEM media for 3 h.
Immunoblotting
BMDM were seeded at 5 × 105 cells per well in 1 mL of BMDM media in 12-well plates (Corning, 353043) the day before treatment. Cells were primed with 200 ng/mL LPS for 3 h followed by particle and compound treatment in OPTI-MEM at 500 µL volume.
OPTI-MEM were collected as supernatant and centrifuged at 14,000 ×g for 1 min to eliminate cell debris. The remaining cells were collected in 100 µL of RIPA buffer (Thermo, 89901) containing 1 × concentration of protease/phosphatase inhibitors (Thermo, 78440). Proteins in the cell lysates were quantified using BCA assay kit (Thermo, 23227) according to manufacturer’s protocol. Amount of total protein were adjusted with RIPA buffer for equal loading and 1 volume of 4 × Laemmli buffer (Bio-Rad, 1610747) containing 1.43 M β-mercaptoethanol (Sigma, M-3148) were added to 3 volume of adjusted protein lysate. To extract proteins from supernatant, 500 µL of methanol and 125 µL of chloroform were added to 500 µL of supernatant (OPTI-MEM) and vortexed for 10 s. The mixture were centrifuged at 14,000 ×g for 5 min and upper phase was removed. The middle protein layer and bottom layer were washed by gently adding 500 µL of methanol and centrifuged again at 14,000 ×g for 5 min. The supernatant was removed and remaining protein pellet was dried at 50 °C for 5 min. Protein pellet was resuspended in 60 µL of 1 × Laemmli buffer containing 355 mM β-mercaptoethanol. All protein samples were heated for 5 min at 95 °C for denaturation and were separated by SDS-PAGE, transferred to PVDF membrane, and were immunoblotted. The primary antibodies were mouse IL-1β (R&D, AF-401-NA; 1:1,000 (v/v) dilution), mouse caspase-1 (Santa Cruz, sc-514; 1:1,000 (v/v) dilution), human and mouse beta actin (Cell Signaling, 4970; 1:2,000 (v/v) dilution). Secondary antibodies were HRP-conjugated anti-rabbit IgG (Cell Signaling, 7074) and anti-goat IgG (R&D, HAF019) at a 1:2,000 (v/v) dilution. Blocking and primary antibody incubation were performed in 5% bovine serum albumin (BSA) (Sigma, A7906) in T-TBS (Tris-buffered saline (TBS) containing 0.1% Tween-20 (Sigma, P1379)). Secondary antibody incubation were performed in T-TBS without BSA. Blocking was performed for 1 h at room temperature, primary antibody incubation for overnight at 4 °C, and secondary antibody incubation for 1 h at room temperature.
Lysosomal membrane permeabilization
Lysosomal membrane permeabilization was detected by measuring cathepsin B release into the cytosol following a published protocol [65]. Briefly, 2.5 × 105 BMDM cells were seeded in 24-well plates the day before the assay and primed for 3 h with 200 ng/mL LPS on the day of the assay. BMDM were then treated with particles at 200 µg/mL concentration for 30 min in 250 µL of OPTI-MEM per well. BMDM were washed once with PBS. Plasma membrane permeabilization was performed by adding 200 µL of digitonin extraction buffer (250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 20 mM HEPES, 0.5 mM Pefabloc (Roche, 11873601001), pH 7.5) containing 15 µg/mL of digitonin (Sigma, D141) and incubated on ice for 10 min with gentle rocking. Permeabilization of both the plasma membrane and the lysosomal membrane were performed by adding 200 µL of digitonin extraction buffer containing 200 µg/mL of digitonin and incubated on ice for 10 min with gentle rocking. Cathepsin B activity in digitonin extraction buffer were measured by adding 50 µL of samples extracted with digitonin extraction buffer with 50 µL of reaction buffer containing 50 mM sodium acetate, 4 mM EDTA, 8 mM DTT, 0.5 mM Pefabloc, 50 μM zFR-AFC (Enzo, ALX260-129-M005) in black-walled clear-bottom 96-well plates (BD Falcon, 353219). The reaction was monitored by measuring fluorescence (Ex 360 nm/Em 528 nm) once every minute for 20 min at 30 °C temperature using Synergy HTX multi-mode reader. The change in fluorescence over time was used to calculate enzyme activity. To normalize for variation in plasma membrane permeabilization, LDH activity in the digitonin extraction buffer were assessed using Cytotoxicity Detection Kit (LDH) Plus. Cathepsin B activity were then divided by LDH release value to calculate normalized cathepsin B activity. Normalized cathepsin B activity for 15 μg/mL digitonin wells were divided by normalized cathepsin B activity from 200 μg/mL digitonin wells to calculate lysosomal membrane permeabilization, represented as percent of maximum cathepsin B activity.
MTT assay
BMDM were seeded at 50,000 cells per well in clear 96-well plates at 100 µL volume per well (Corning, 3596), one day before the assay. Following 3 h LPS priming and 3 h particle treatment in OPTI-MEM, cells were incubated with phenol-free DMEM (Thermo, 31053028) containing 0.5 mg/mL of MTT (M6494) for 1 h in 37 °C CO2 incubator. Media containing MTT were removed and 50 µL of DMSO were added to each well to dissolve formazan crystal. Absorbance was recorded at 540 nm on Synergy HTX multi-mode reader. The data were normalized to the average value of untreated negative control wells and represented as percent of no particle control.
Intracellular ATP
BMDM were seeded at 50,000 cells per well at 100 µL volume per well in white-walled clear-bottom 96-well plates (BD Falcon, 353377). Intracellular ATP was measured using CellTiter Glo (Promega, G7571) according to the manufacturer’s protocol. In brief, 50 µL of reconstituted CellTiter Glo reagent was added to each well, incubated for 10 min at room temperature in dark, and luminescence was measured using Synergy HTX multi-mode reader. The data were normalized to the average value of untreated negative control wells and represented as percent of no particle control.
Intracellular potassium
BMDM were seeded at 5 × 105 cells per well in 1 mL of BMDM media in 12-well plates (Corning, 353043). Following 3 h LPS priming and 3 h particle treatment, cells were washed with PBS twice, and cell lysates were collected in 2 mL 10% nitric acid (Wako, 149–06845). Intracellular potassium was measured using a BWB-XP flame photometer (BWB Technologies) and KCl (Sigma, 24–5550) dilution series was used to generate standard curve. The data were normalized to the average value of untreated negative control wells and represented as percent of no particle control.
E. coli uptake assay
BMDM were seeded at 50,000 cells per well in black-walled clear-bottom 96-well plates (BD Falcon, 353,219). Following 3 h LPS priming, cells were treated with 5 µM of compounds and 0.5 mg/mL of pHrodo Red E. coli (Thermo, P35361) in OPTI-MEM at 100 µL volume. After 1 h incubation in 37 °C CO2 incubator, cells were washed twice with PBS and replaced with 100 µL of PBS. Fluorescence signal (Ex 560 nm/Em 585 nm) was measured using a Synergy HTX multi-mode reader. Fluorescent signals were normalized to the in-plate basal (without pHrodo Red E. coli; n = 4) and maximum (with pHrodo E. coli; n = 4) wells as; (target signal – basal signal)/(maximum signal – basal signal) × 100%.
Flow cytometric analysis
BMDM were seeded at 5 × 105 cells per well in 1 mL media in 12-well tissue culture treated plates. For reactive oxygen species (ROS) measurement, cells were incubated with 500 µL OPTI-MEM containing 10 µM CM-H2DCFDA (Thermo, C6827) and incubated for 20 min at 37 °C in a CO2 incubator. Cells were detached using TrypLE (Thermo, 12604–021) and analyzed using a FACS Calibur (BD Bioscience) with Ex 488 nm/Em 530 nm. Gating strategies are described in Additional file 1: Fig. S7. All FACS data were analyzed with FlowJo (v10.8).
Transmission electron microscopy (TEM)
BMDM were fixed in 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min at 4 °C. Cells were then fixed in 2% glutaraldehyde in 0.1 M phosphate buffer overnight at 4 °C. Cells were washed three times in 0.1 M phosphate buffer and post-fixed with 2% osmium tetroxide in 0.1 M phosphate buffer for 1 h at 4 °C. Cells were then dehydrated as follows; 50% ethanol (5 min, 4 °C), 70% ethanol (5 min, 4 °C), 90% ethanol (5 min, room temperature), 100% ethanol (5 min, room temperature, total 3 times). The cells were then transferred to an EM embedding resin Quetol-812 (Nissin EM, 340-H) and polymerized for 48 h at 60 °C. The polymerized resins were sectioned at 70 nm with a diamond knife using Ultracut-UCT ultramicrotome (Leica), mounted on copper grids, and stained with 2% uranyl acetate at room temperature for 15 min. Samples were then washed with distilled water followed by secondary stain with Lead stain solution (Sigma, 18–0875-2-25ML-J) at room temperature for 3 min. The grids were observed on a transmission electron microscope (JEOL, JEM-1400Plus) at an accelerated voltage of 100 kV. Digital images (3296 × 2472 pixels) were taken with a CCD camera (JEOL, EM-14830RUBY2).
Data visualization
Heatmap view of assay data were generated using matrix visualization and analysis software Morpheus (https://software.broadinstitute.org/morpheus). Average linkage hierarchical clustering was performed using Euclidean distance for visualizing patterns in the inhibitory profiles.
Statistical analysis
Statistical significance was assessed using ordinary one-way and two-way ANOVA followed by Bonferroni’s multiple comparison with Prism 8 (8.4.3). Spearman rank correlation values and four-parameter dose–response curves were generated with Prism 8 (8.4.3). For chemical screens, Z values corresponding to the 50% decrease, a threshold used to identify primary hits, are indicated in Additional file 1: Fig. S5.
