McDaniel et al., 2019 - Pulmonary Exposure to Magnéli Phase Titanium Suboxides Results in Significant Macrophage Abnormalities and Decreased Lung Function. Frontiers in immunology   10:2714 Full text @ Front Immunol

Figure 1

Magnéli phase phagocytosis results in increased cell death in bone marrow-derived macrophages. (A–C) Characterization of Magnéli phases used in this study. (A) Schematic illustrating Magnéli phase generation. (B,C) TEM images of Magnéli phases formed by annealing P25 TiO2 nanoparticles with coal in a pure N2 atmosphere for 2 h at 900°C. Electron diffraction patterns were characteristic of Magnéli phases and confirmed these as predominately Ti6O11 particles. Particles were between 10 and 200nm in size. (D) Un-treated bone marrow-derived macrophages (1 × 106 cells/well) and (E) macrophages treated with Ti6O11 (1, 10, 100, or 1,000 ppm) were visualized using TEM (Scale bar: 5 μm). Magnéli phases appear as punctate dark dots in the macrophages and are concentrated in phagolysosomes. (F) Macrophages containing Magnéli phases demonstrate morphological features consistent with apoptosis, including cell shrinking and membrane blebbing (red arrows) (Scale bar: 1μm). (G,H) Cytotoxicity was evaluated using trypan blue exclusion (G) across the Ti6O11 dose range and (H) at 100 ppm over a 24 h time course. (I,J) Inflammation was evaluated by assessing the production of pro-inflammatory cytokines, such as (I) IL-1β, (J) IL-6, and (K) TNF in the cell free supernatant following exposure to different doses of Magnéli phases. (L) Macrophage function was evaluated by assessing the ability of macrophages to phagocytose fluorescent Escherichia coli 24 h post-exposure to 100 ppm Magnéli phases. Data are expressed as mean ± SEM (n = 3 independent experiments). **p < 0.01.

Figure 2

Macrophage exposure to Magnéli phases activates gene expression profiles associated with apoptosis and mitochondria dysfunction. (A) Gene expression was evaluated using real time PCR based Superarrays (Qiagen). For each gene target on the array, fold change was calculated based on ΔΔCt values. Genes found to be ±2-fold change in expression from untreated specimens were defined as significant. The resultant data was evaluated using Ingenuity Pathway Analysis (IPA) to define pathways and global correlations between gene expression profiles and biological functions. Cell death signaling, specifically apoptosis, was the top pathway up-regulated in the macrophages following Magnéli phase exposure. IPA further identified gene expression profiles consistent with mitochondria dysfunction as a potential factor associated with the increased apoptosis signaling. (B) Pathways associated with inflammation and specifically inflammation associated cell death (i.e., pyroptosis and necroptosis) were significantly down-regulated at the level of gene transcription. Either fold change in gene expression or z-score values are displayed for each node as appropriate.

Figure 3

Alterations in cellular energetics and mitochondrial membrane potential in macrophages treated with Ti6O11. (A) Oxygen consumption rate (OCR) and (B) extracellular acidification rate (ECAR) were measured using Agilent Seahorse XF96 Analyzer in mock and Ti6O11 treated BMDMs at either 100 ppm or 1,000 ppm. (C) Mitochondrial membrane potential was evaluated using tetramethylrhodamine (TMRM) in macrophages treated with vehicle (blue) or 100 ppm Magnéli phases (green), with H2O2 used to induce maximum loss of membrane potential in each group. (D) Cellular oxidative stress was evaluated using dihydrodichlorofluorescein (DCF) fluorescence. All data are expressed as mean ± SEM (n = 3/group). AU, Arbitrary Units. *p < 0.05, #p < 0.05, **p < 0.01.

Figure 4

Magnéli phases concentrate in pulmonary macrophages following a single exposure and are retained in the lung. (A) Following a single airway exposure to 100 ppm, titanium associated with the Magnéli phases were retained in the lungs and detected using ICP-MS for over 7 days post-expsure. Control tissues were collected from animals exposed to 100 ppm Ti6O11 and immediately euthanized. (B–D) Representative H&E stained tissue sections used for histopathology evaluation. (B) Vehicle control showing no nanoparticles or pathology. (C) Significant airway inflammation was observed in LPS treated animals, but no evidence of inflammation was found in the mice treated with Magnéli phases at any timepoint evaluated. (D) Larges areas of macrophages containing Magnéli phases were found in all treated animals (yellow box). (E–G) The majority of pulmonary macrophages contain Magnéli phases, with no other cell types appearing to contain or associate with the nanoparticles determined using dark field microscopy. Darkfield images were taken of (E) Ti6O11 nanoparticles alone or the lungs of mice 7 days post-exposure to either (F) PBS or (G) 100 ppm Magnéli phases. (G) Particles in the tissues were identified by bright punctate dots, almost exclusively localized in macrophages. Representative H&E stained lung sections (Scale bar = 50 μm). All data are expressed as mean ± SEM (n = 7/group). **p < 0.01.

Figure 5

Magnéli phases concentrate in pulmonary macrophages, resulting in significant dysfunction. (A) Representative images of BALF cytology from PBS and Ti6O11 (100 ppm) treated mice. (B) Differential cell counts in the BALF after treatment with PBS, Ti6O11, or LPS. (C) Gene expression profiling and analysis using Ingenuity Pathway Analysis software identified gene transcription patterns associated with increases in apoptosis, formation of reactive oxygen species, and wound healing/fibrosis. Data are expressed as mean ± SEM (n = 3 mice per PBS treated group, n = 7 mice per Ti6O11 treated group, and n = 4 per LPS treated group). *p < 0.05.

Figure 6

Repeated exposures concentrates Magnéli phases in pulmonary macrophages. (A) Using ICP-MS, we quantified the amount of titanium in the lungs following multiple airway exposures to 100 ppm Ti6O11 over a 30 day period. (B) Histopathology using H&E stained tissue sections reveled significant airway inflammation LPS treated animals, but no evidence of inflammation in the mice treated with Magnéli phases. Larges areas of macrophages containing Magnéli phases were found in all treated animals (Scale bar = 20 μm). (C) Darkfield images were taken of mouse lungs post-repeated exposures, on day 30. Particles in the tissues were identified by bright punctate dots. Almost all particles were concentrated in macrophages (green arrows). However, small numbers of Ti6O11 nanoparticles were found associated with alveolar epithelial cells (green arrows). Representative H&E stained lung sections (Scale bar = 50 μm). (D) Phagocytic Index (number of BALF macrophages containing Magnéli phases per 100 cells). (E–G) APAF1 immunohistochemistry staining from (E) saline, (F) LPS, and (G) Magnéli Phase exposed lungs. (G) Following Magnéli Phase exposure, only endothelial cells and macrophages containing particles were broadly positive for APAF1. (E) In saline exposed mice, only endothelial cells were positive for APAF1; (F) whereas, LPS exposure resulted in a range of cell types staining positive for APAF1. Macrophages in each image are identified by red arrows. All data are expressed as mean ± SEM (n = 7/group). **p < 0.01.

Figure 7

Chronic exposure to Magnéli phases significantly attenuates lung function. (A–D) Baseline pulmonary function parameters including (A) total respiratory system resistance (R), (B) Newtonian resistance (Rn), (C) elastance (E), and (D) dynamic compliance (C) were measured by FlexiVent in control and Ti6O11 exposed mice. (E–H) Assessment of AHR to methacholine (MCh) in control and Ti6O11 exposed mice. (E) Total respiratory system resistance (R), (F) Newtonian resistance (Rn), (G) elastance (E), and (H) dynamic compliance (C) dose response curves following challenge with increasing concentrations of MCh in aerosolized saline (0, 1.5, 3, 6, 12, and 24 mg/ml) for control and Ti6O11 exposed mice were examined on FlexiVent. All data are expressed as mean ± SEM (n = 7/group). *p < 0.05, ****p < 0.0001, compared to the PBS treated control group.

Acknowledgments:
ZFIN wishes to thank the journal Frontiers in immunology for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Front Immunol