Nickel nanoparticles cause exaggerated lung and airway remodeling in mice lacking the T-box transcription factor, TBX21 (T-bet)

Abstract

Background: Nickel nanoparticles (NiNPs) are increasingly used in a variety of industrial applications, including the manufacturing of multi-walled carbon nanotubes (MWCNTs). While occupational nickel exposure is a known cause of pulmonary alveolitis, fibrosis, and cancer, the health risks of NiNPs are not well understood, especially in susceptible individuals such as asthmatics. The T-box transcription factor Tbx21 (T-bet) maintains Th1 cell development and loss of T-bet is associated with a shift towards Th2 type allergic airway inflammation that characterizes asthma. The purpose of this study was to determine the role of T-bet in susceptibility to lung remodeling by NiNPs or MWCNTs.

Methods: Wild-type (WT) and T-bet-/- mice were exposed to NiNPs or MWCNTs (4 mg/kg) by oropharyngeal aspiration (OPA). Necropsy was performed at 1 and 21 days. Bronchoalveolar lavage fluid (BALF) was collected for differential counting of inflammatory cells and for measurement of cytokines by ELISA. The left lung was collected for histopathology. The right lung was analyzed for cytokine or mucin (MUC5AC and MUC5B) mRNAs.

Results: Morphometry of alcian-blue/periodic acid Schiff (AB/PAS)-stained lung tissue showed that NiNPs significantly increased mucous cell metaplasia in T-bet-/- mice at 21 days (p < 0.001) compared to WT mice, and increased MUC5AC and MUC5B mRNAs (p < 0.05). MWCNTs also increased mucous cell metaplasia in T-bet-/- mice, but to a lesser extent than NiNPs. Chronic alveolitis was also increased by NiNPs, but not MWCNTs, in T-bet-/- mice compared to WT mice at 21 days (P < 0.001). NiNPs also increased IL-13 and eosinophils (p < 0.001) in BALF from T-bet-/- mice after 1 day. Interestingly, the chemokine CCL2 in the BALF of T-bet-/- mice was increased at 1 and 21 days (p < 0.001 and p < 0.05, respectively) by NiNPs, and to a lesser extent by MWCNTs at 1 day. Treatment of T-bet-/- mice with a monoclonal anti-CCL2 antibody enhanced NiNP-induced mucous cell metaplasia and MUC5AC mRNA levels (p < 0.05), yet marginally reduced NiNP-induced alveolitis.

Conclusion: These findings identify T-bet as a potentially important susceptibility factor for NiNP exposure and to a lesser extent for MWCNT exposure, and suggests that individuals with asthma are at greater risk.

Figure 1

Mucous cell metaplasia in response to NiNP exposure in WT and T-bet^-/- mice. A) Quantification of mucus producing cells at 1 or 21 days post-exposure determined using ImageJ analysis software (NIH). Data presented as the percentage of AB/PAS-positive stained area per total area. ***p < 0.001 compared to the control group of the same genotype or as indicated. All data represent mean values ± SEM of at least three measurements per lung of 5–7 mice per exposure group. B) Low magnification (10×) of AB/PAS stained lung tissue sections (open arrows) at 21 days after WT and T-bet^-/- mice were exposed to a 0.1% pluronic solution or NiNP. Asterisk indicates area of fibrosis. C) High magnification (40× and 100×) of insert from 10× in (B). Black arrows indicate alveolar macrophages containing NiNP agglomerates.

Figure 2

MUC5AC and MUC5B mRNA levels at 1 and 21 days after exposure. Taqman quantitative real-time RT-PCR was used to measure changes in whole lung mRNA levels of A) MUC5AC and B) MUC5B. Values are means ± SEM (n = 5–7 animals/group). *p < 0.05, **p < 0.01, ***p < 0.001 as compared to the control group of the same genotype or as indicated.

Figure 3

Differential cell counts in the BALF 1 and 21 day after NiNP exposure. Immune cells numbers for A) eosinophils, B) lymphocytes, C) neutrophils, and D) macrophages are presented as the mean values ± SEM out of a total of 500 cells counted per animal for 5–7 animals per dose group at 20× magnification. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the time matched control group of the same genotype or as indicated. E) Photomicrographs representing each cell type. Macrophages and neutrophils demonstrated phagocytosis of NiNP in both genotypes at both time points in response to exposure (100×).

Figure 4

Histopathological analysis of inflammation in the lungs of WT and T-bet^-/- mice in response to NiNPs. A) Lung pathology was scored in mice for inflammation 1 and 21 days after initial exposure. All data represent mean values ± SEM. *p < 0.05, ***p < 0.001 compared to the control group of the same genotype (n = 5–7 animals/group). B) Representative photomicrographs at low magnification (10×) of H&E stained lung sections at 21 days after mice were exposed. TB: Terminal bronchiole, ADB: alveolar duct bifurcation, AD: alveolar duct. C) Higher magnification (40× and 100×) of images from 10× in (B).

Figure 5

The effects of NiNP on airway fibrosis and parenchymal alveolitis in WT and T-bet^-/- mice. A) Cross-sections of airways stained with trichrome were measured for the area to perimeter ratio of collagen deposition. B) Representative images of results from (A) of airways stained with trichrome (open arrows) at low magnification (10×) at 21 days post exposure. C) Lung pathology was then scored for parenchymal lesions in WT and T-bet^-/- mice. D) Low magnification (10×) of trichrome stained lung parenchyma sections (open arrows) at 21 days after WT and T-bet^-/- mice were initially exposed. Images are a representation of the data shown in (C). Black arrows indicate NiNP aggregates. Data are the mean values ± SEM (n = 5–7 animals/group). *p < 0.05, ***p < 0.001 compared to the control group of the same genotype or as indicated.

Figure 6

CCL2 mRNA and protein levels in the lungs of mice after NiNP exposure. A) CCL2 mRNA expression was measured by qRT-PCR in whole lung tissue at 1 and 21 days post exposure while B) CCL2 protein in BALF was analyzed by ELISA after 1 or 21 days of initial exposure (nd, not detectable). *p < 0.05, **p < 0.01, ***p < 0.001 as compared to the control group of the same genotype or as indicated. Data are the mean values ± SEM (n = 5–7 animals/group).

Figure 7

Mucous cell metaplasia and alveolitis in response to anti-CCL2 mAb treatment in T-bet^-/- mice 21 days post-exposure. A) Cells stained with AB/PAS were quantitated for mucin protein expression using ImageJ (NIH) analysis for percentage of positive stained area per total area in mice treated with IgG2B Isotype Control or anti-CCL2 mAb and exposed to either a 0.1% pluronic solution or NiNPs. B) MUC5AC and C) MUC5B whole lung mRNA expression were quantitated using qRT-PCR analysis. D) Cross sections of lungs stained with trichrome were scored for parenchymal lesions in WT and T-bet^-/- mice 21 days after initial NiNP exposure. Lungs were scored in a blinded manner by three independent reviewers. E) Soluble collagen content was measured using the Sircol Assay kit in whole lung homogenates and expressed as μg/mg of protein. F) Whole lung col1a2 mRNA expression measured by qRT-PCR. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the time matched control group of the same genotype or as indicated. Data are the mean values ± SEM (n = 3–5 animals/group).