Phorbol-12-myristate

Human peripheral blood mononuclear cells (PBMC) were isolated by Ficoll Paque (GE healthcare) density centrifugation from whole blood donated by healthy volunteers. The South Sheffield Research Ethics Committee approved the studies, and subjects gave written, informed consent. Monocytes were enriched from freshly isolated PBMC using MACS Monocyte Isolation Kit II and MACS LS Columns (Miltenyi Biotec), yielding an average 98% purity. To differentiatiate PBMC into monocyte-derived macrophages (MDM) 2×10⁶ PBMC/mL were plated in RPMI 1640 media (Lonza) with 2 mmol/L L-glutamine (Gibco BRL) containing 10% human AB serum (First Link (UK) LTD) in 24-well plates (Costar). After 24 h, non-adherent cells were removed, and adherent cells were cultured in RPMI with 10% heat-treated fetal bovine serum (FBS; Bioclear) in 5% CO₂ at 37°C to give a final concentration of approximately 2×10⁵ MDM/ml at 14d [17] (link). The THP1 cell line was obtained from ATCC, and maintained at 2×10⁵ cells/ml in RPMI 1640 medium supplemented with 10% FCS and 2 mmol/L L-glutamine. THP1 cells (2×10⁵/ml) were differentiated using 100 nM Vitamin D₃ (VD₃, Sigma-Aldrich) or 200 nM phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) for 3d. Differentiation of PMA treated cells was enhanced after the initial 3d stimulus by removing the PMA-containing media then incubating the cells in fresh RPMI 1640 (10% FCS, 1% L-glutamine) for a further 5d (PMAr).

Human monocytic THP-1 cells were maintained in culture in Roswell Park Memorial Institute medium (RPMI 1640, Invitrogen) culture medium containing 10 % of heat inactivated fetal bovine serum (Invitrogen) and supplemented with 10 mM Hepes (Gibco, #15630-056), 1 mM pyruvate (Gibco, #11360-039), 2.5 g/l D-glucose (Merck) and 50 pM ß-mercaptoethanol (Gibco; 31350–010). THP-1 monocytes are differentiated into macrophages by 24 h incubation with 150 nM phorbol 12-myristate 13-acetate (PMA, Sigma, P8139) followed by 24 h incubation in RPMI medium. Macrophages were polarized in M1 macrophages by incubation with 20 ng/ml of IFN-γ (R&D system, #285-IF) and 10 pg/ml of LPS (Sigma, #8630). Macrophage M2 polarization was obtained by incubation with 20 ng/ml of interleukin 4 (R&D Systems, #204-IL) and 20 ng/ml of interleukin 13 (R&D Systems, #213-ILB). HepG2 and A549 cells were respectively cultivated in Dulbecco’s modified Eagle's minimal essential medium (DMEM medium 1 g glucose/l) (Gibco) and Minimum Essential Medium Eagle medium (MEM) (Gibco), both containing 10 % fetal bovine serum. In the co-culture experiments, THP-1 monocytes were differentiated in 6 Transwell inserts (membrane pore size of 0.4 μm, Corning, #3450). Macrophages and HepG2 cells were co-cultured in CO₂ independent medium supplemented with 0.5 mM L-glutamine (Sigma, # G3126) and 3.75 g/l of D-glucose (Sigma, #50-99-7) for 16 h before being incubated with or without 50 μM etoposide (Sigma, #E1383) for 24 h. Macrophages and A549 cells were co-cultured in CO₂ independent medium supplemented with 0.5 mM L-glutamine and 2.5 g/l of D-glucose for 24 h before being incubated with or without 50 μM etoposide for 16 h. In the monoculture experiments, 0.8 x 10⁶ THP-1 monocytes were differentiated and polarized in 6 well plates. Next, they were incubated in CO₂ independent medium supplemented with 0.5 mM L-glutamine (Sigma, # G3126) and 3.75 g/l of D-glucose (Sigma, #50-99-7) for 16 h before being incubated with or without 50 μM etoposide (Sigma, #E1383) for 24 h.

ENMs and reagents. The CPPs obtained ZnO from Meliorum Technologies Inc. (Rochester, NY). TiO₂-P25 (81% anatase and 19% rutile) was purchased from Evonik (Parsippany, NJ); TiO₂-A was provided by P. Biswas (Washington University, St. Louis, MO); and the CPPs prepared the TiO₂-NBs as previously described (Hamilton et al. 2009 (link)). The CPPs obtained the O-MWCNT stock in powder form from Cheap Tubes Inc. (Brattleboro, VT); obtained the P-MWCNT by treating O-MWCNT with dilute acids, chelating agents, and mild conditions to minimize oxidized or damaged tubes; and created F-MWCNT through further acid treatment of P-MWCNT, which introduced carboxyl groups on 5.27% of the carbon backbone (on a per weight basis) (Chen and Mitra 2008 ; Wang et al. 2011 (link)).
The CPPs purchased low-endotoxin bovine serum albumin (BSA) from Gemini Bio-Products (West Sacramento, CA); dipalmitoylphosphatidylcholine, phorbol 12-myristate, 13-acetate (PMA), and lipopolysaccharide (LPS from Escherichia coli 0127:B8) from Sigma-Aldrich (St. Louis, MO); and 1,25-dihydroxy-vitamin D₃ from EMD Millipore (Billerica, MA). The CPPs purchased the cytotoxicity assays CellTiter 96 (MTS assay) and CytoTox 96 [LDH (lactate dehydrogenase) assay] from Promega (Madison, WI).
Preparation of ENMs in cell culture media. The CPPs prepared ENM stock solutions (5 mg/mL) from dry powder using endotoxin-free sterile water and then prepared all ENM suspensions in cell culture media using the stock solutions as needed. Briefly, the CPPs vortexed and then sonicated ENM stock solutions (with the exception of TiO₂-NB, which was stirred to prevent mechanical shear) using a water bath sonicator or cup horn sonicator (depending on laboratory availability) immediately before diluting the solutions into complete cell culture media.
Cell culture and co-incubation with EMN. The CPPs grew all cells at 37°C in a 5% CO₂ atmosphere. RLE-6TN cells, a rat alveolar type II epithelial cell line, from American Type Culture Collection (ATCC; Manassas, VA) were cultured in Ham’s F12 medium (ATCC) supplemented with l-glutamine, bovine pituitary extract (BPE), insulin, insulin growth factor (IGF)-1, transferrin, and epithelial growth factor (EGF), supplemented with 10% fetal bovine serum (FBS). THP-1 cells, a human acute monocytic leukemia cell line (ATCC) were cultured in HEPES-buffered RPMI 1640 supplemented with l-glutamine (Mediatech, Corning, NY), 0.05 mM β-mercaptoethanol, and 10% FBS (PAA Laboratories, Dartmouth, MA). BEAS-2B cells (ATCC) were cultured in bronchial epithelial growth medium (BEGM) obtained from Lonza Inc. (Walkersville, MD) supplemented with BPE, insulin, hydrocortisone, human EGF, epinephrine, triiodothyronine, transferrin, gentamicin/amphotericin-B, and retinoic acid. For the THP-1 differentiation performed in the first series of experiments (phase I), the CPPs pretreated cells with 1.62 µM (1 µg/mL) PMA for 18 hr. However, the CPPs identified excessive cell clumping and cell death during the phase I studies. Therefore, the CPPs alternatively pretreated THP-1 cells with vitamin D₃ at 150 nM overnight and then 5 nM PMA in order to obtain the differentiated macrophage-like cells used during the second series of experiments (phase II). For the IL-1β release, co-culturing THP-1 cells with 10 ng/mL LPS was necessary to initiate transcription of pro-IL-1β. The CPPs initiated aggressive phagocytic activity by adding PMA just before particle exposure.
Before ENM exposure, the CPPs cultured aliquots of 1.5 × 10⁴ cells (for THP-1 cells, 10⁵ cells were seeded into each well of a 96-well plate) in 0.2 mL of the cell culture media in 96-well plates (Costar, Corning, NY) at 37°C for 24 hr. The CPPs freshly prepared all of the ENM suspensions at final concentrations of 10, 25, 50, and 100 µg/mL in the cell culture media. After exposure of the cells to the ENMs for 24 hr at 37°C, the CPPs collected supernatants to measure LDH and IL-1β production then used the remaining cells to test cellular viability by MTS assay.
Physicochemical characterization of ENMs. The CPPs identified the primary particle size and morphology of the ENMs by using a transmission electron microscope (TEM; model 100CX) and a scanning electron microscope (SEM; model JSM-7600F) (both from JEOL Ltd., Tokyo, Japan). In addition, the CPPs characterized the particle hydrodynamic size in H₂O and cell culture media using dynamic light scattering (DLS) (Ji et al. 2010 (link)). The CPPs characterized particle crystallinity and structure using X-ray diffraction measurements and measured particle surface area by Brunauer–Emmett–Teller (BET) surface area analysis. The CPPs performed zeta-potential measurements of the ENM suspensions using a ZetaSizer Nano-ZS instrument (Malvern Instruments, Worcestershire WR, UK). Finally, the CPPs determined the elemental composition of the particles as well as ZnO dissolution rate using inductively coupled plasma mass spectrometry (ICP-MS) (model SCIEX Elan DRCII; PerkinElmer, Norwalk, CT).
Endotoxin analysis of ENMs. CPPs measured the endotoxin content of ENM stock suspensions, as well as dispersions in PBS and tissue culture media, using the colorimetric Limulus amebocyte lysate assay (Lonza Inc.). The LPS content of all ENM suspensions was < 0.3 EU/mL.
Determination of cell viability. The CPPs determined cellular viability using MTS (CellTiter 96) and LDH (CytoTox 96; both from Promega) according to the manufacturer’s protocols. To avoid the interference created by ENMs while measuring formazan absorbance at 490 nm, the CPPs introduced a centrifugation (2000 × g for 10 min) procedure in phase II experiments to collect particles in the wells after incubation with the MTS reagents. CPPs then followed this centrifugation step with a brief mixing and transfer of the supernatant to a new 96-well plate before measuring the formazan absorbance at 490 nm. The CPPs eliminated interference of any residual LDH in FBS by heat-inactivation (70°C water bath for 5 min).
ELISA for IL-1β quantification. The CPPs determined IL-1β production in the THP-1 culture supernatant using a human IL-1β ELISA kit (R&D Systems Human IL-1β DuoSet™; R&D Systems, Minneapolis, MN) following the manufacturer’s instructions.
Statistical analysis. The CPPs used the two-way analysis of variance followed by Tukey or Bonferroni correction for multiple comparisons of means for statistical analysis of responses across ENMs and cell lines. In order to define interlaboratory comparisons across two harmonization rounds, the CPPs conducted a meta-analysis of LDH, MTS, and IL-1β assays across eight different laboratories for three cell lines (BEAS-2B, RLE-6TN, and THP-1) exposed to several ENMs (TiO₂-P25, TiO₂-A, TiO₂-NBs, ZnO, O-MWCNT, P-MWCNT, and F-MWCNT). The CPPs combined information within assays and cell lines using a robust two-stage hierarchical model of toxicity. For all quantities of interest, the CPPs obtained Monte Carlo inference by implementing a custom Gibbs sampler in the R computing environment (R Foundation for Statistical Computing, Vienna, Austria). To normalize data, the CPPs subtracted background negative control values (MTS, LDH, and IL-1β) and provided adjustments for positive control values in the case of LDH assays. Details about the statistical model used for analysis are provided in Supplemental Material, p. 8 (http://dx.doi.org/10.1289/ehp.1306561).

To generate THP-1 derived macrophages (TDM), THP-1 cells (in 0.4 μm transwell insert for 6-well plate) were treated with 10 ng/ml phorbol 12-myristate 13-acetate (PMA) for 24 hours. Then, the cells were cultured with 25 ng/ml IL-4 or LPS for another 48 hours to generate THP-1-derived macrophages. Meanwhile, THP-1 cells were treated with 10 ng/ml phorbol 12-myristate 13-acetate (PMA) for 24 hours before further treatment (B. thetaiotaomicron, heat killed B. thetaiotaomicron, B. thetaiotaomicron culture medium or acetic acid).

Reactivation of Kasumi-3 cells and CD14+ monocytes with phorbol 12-myristate 13-acetate (PMA, Merck) was achieved at a concentration of 20 ng/mL (O’Connor and Murphy, 2012 (link); Poole et al, 2021 (link)).