Surface area- and mass-based comparison of fine and ultrafine nickel oxide lung toxicity and augmentation of allergic response in an ovalbumin asthma model

Abstract

Background: The correlation of physico-chemical properties with mechanisms of toxicity has been proposed as an approach to predict the toxic potential of the vast number of emerging nanomaterials. Although relationships have been established between properties and the acute pulmonary inflammation induced by nanomaterials, properties' effects on other responses, such as exacerbation of respiratory allergy, have been less frequently explored.Methods: In this study, the role of nickel oxide (NiO) physico-chemical properties in the modulation of ovalbumin (OVA) allergy was examined in a murine model. Results: 181 nm fine (NiO-F) and 42 nm ultrafine (NiO-UF) particles were characterized and incorporated into a time course study where measured markers of pulmonary injury and inflammation were associated with NiO particle surface area. In the OVA model, exposure to NiO, irrespective of any metric was associated with elevated circulating total IgE levels. Serum and lung cytokine levels were similar with respect to NiO surface area. The lower surface area was associated with an enhanced Th2 profile, whereas the higher surface area was associated with a Th1-dominant profile. Surface area-normalized groups also exhibited similar alterations in OVA-specific IgE levels and lung neutrophil number. However, lung eosinophil number and allergen challenge-induced alterations in lung function related more to particle size, wherein NiO-F was associated with an increased enhanced pause response and NiO-UF was associated with increased lung eosinophil burden.Conclusions: Collectively, these findings suggest that although NiO surface area correlates best with acute pulmonary injury and inflammation following respiratory exposure, other physico-chemical properties may contribute to the modulation of immune responses in the lung.

Keywords: Nanotoxicology; hypersensitivity; immune response; nickel oxide nanoparticles; ovalbumin allergy model.

Figure 1.

Treatment groups, corresponding exposures, and timeline of treatments in the OVA asthma model. Two groups of mice were exposed to dispersion medium (DM) or NiO particles at doses identical to those of the time course study on day 0 via oropharygenal aspiration (asp). One group of each treatment was not sensitized to OVA to serve as non-sensitized particle control groups and another group of each treatment was sensitized to OVA. Accordingly, on days 1 and 10, mice were intraperitoneally injected (i.p.) with PBS (non-sensitized) or OVA + alum in PBS (sensitized). On day 14, 150 μL blood was collected via the tail vein to evaluate circulating OVA-specific IgE levels, indicative of successful sensitization to OVA (green circle). On days 19 and 28, mice were aspirated with OVA or PBS and placed in whole body plethysmography (WBP) chambers to assess airway response to allergen challenge (blue circle). On day 29, all mice were euthanized.

Figure 2.

Electron micrographs of NiO-F (A) and NiO-UF (B) particles in DM illustrating size and morphology differences.

Figure 3.

Rate of dissolution for NiO-F (solid lines) and NiO-UF (dotted lines) in different simulated biological fluids shown as percent of the original Ni concentration measured as soluble fraction. Purple lines represent particles suspended in dispersion media (DM, pH 7.2), pink lines indicate particles suspended in Gamble’s solution (GS, pH 7.4), and orange lines indicate particles suspended in artificial lysosomal fluid (ALF, pH 4.5). n = 3, p < 0.05, * indicates statistically different from the same particle in GS and DM suspensions.

Figure 4.

Markers of pulmonary injury and inflammation measured in mice from the NiO time course study at 1, 10, 19, and 29 d post NiO exposure following bronchoalveolar lavage (BAL). (A) Lung lactate dehydrogenase (LDH) levels in BAL fluid; (B) total cell number in BAL; and (C) total neutrophil number in BAL are shown for DM control (gray), NiO-1 (green), NiO-2 (blue), and NiO-3 (red) groups. n = 8, p < 0.05, * indicates statistically significant from all other groups, # indicates statistically significant from DM control.

Figure 5.

(A) Total cell number in the mediastinal lymph node recovered from each group and (B) cell phenotypes by percent at 1, 10, 19, and 29 d post-exposure in the NiO time course study. n = 8, p < 0.05, * indicates statistically significant from all other groups, # indicates statistically significant from DM, ^ indicates statistically significant from DM and NiO-1.

Figure 6.

Levels of several notable Th1/pro-inflammatory cytokines that were elevated in the BALF of animals in the NiO time course study. BALF IL-6 (A), IFN-γ (B), and TNF-α (C) responses were generally conserved with respect to NiO surface area, wherein the higher dose (NiO-3) caused more pronounced and persistent increases. n = 8, p < 0.05, * indicates statistical significance over all other groups at the corresponding time point, # indicates statistical significance over DM only.

Figure 7.

Whole lung nickel levels following aspiration of 40 μg the NiO-1 fine particle (green) or the NiO-3 ultrafine particle (red) in mice were measured by ICP-MS on dried lung tissue at 1, 10, 19, and 29 d post-exposure. Data is expressed as a percentage of the originally-administered dose. n = 6, p < 0.05, * indicates statistically significant difference between particles at the same time point.

Figure 8.

Dark field and light micrographs of alveolar macrophages recovered by BAL from each group at 10 d post-exposure. Images of macrophages isolated from the DM (A-C), NiO-1 (D-F), NiO-2 (G-I), and NiO-3 (J-L) groups showed variations in the degree of particle loading at 10 d, and size/morphology of internalized particles.

Figure 9.

Levels of circulating total (n = 3–5) and OVA-specific (n = 8) IgE from serum of mice collected on day 14 of the OVA allergy model, following NiO aspiration and two sensitization procedures. Sensitized groups are shown in purple and non-sensitized groups are shown in orange. Total IgE levels are represented by the entire bar, OVA-specific IgE levels are represented by the hatched portion of the bar, and OVA-specific: total IgE ratio is expressed as a percentage over the corresponding bars. p < 0.05, “a” indicates statistically significant from all non-sensitized groups.

Figure 10.

Whole body plethysmography results at day 28 of the OVA model. P_enh was recorded every 30 s for 6 h immediately following OVA aspiration challenge for each mouse. (A) A time course view of the P_enh response is shown for a single, representative animal from the DM, OVA, NiO-1A, NiO-2A, and NiO-3A groups. (B) Area under the curve was calculated, and averaged for animals from each group and expressed as fold-change over OVA control average. n = 8, p < 0.05, “A” indicates fold-change with statistical significance over all other groups.

Figure 11.

(A) Total cell number and fraction of eosinophils (pink), polymorphonuclear cells (orange), lymphocytes (red), and alveolar macrophages (yellow) recovered from BAL at day 29 in the OVA asthma model. n = 8, p < 0.05, “a” indicates total BAL cell number significant over all sensitized groups, “b” indicates significance over DM, NiO-1, and NiO-2. Percent eosinophils (B) and neutrophils (C) of total BAL cells are expressed as fold change over OVA control levels (dotted line). n = 8, p < 0.05. “A” indicates fold change significantly different from all other groups, “B” indicates fold change significantly different from NiO-1A.

Figure 12.

(A) Total number of cells (whole bar) and phenotypes [CD4+ T-cells (orange), CD8+ T-cells (red), and B-cells (yellow)] collected from the mediastinal lymph nodes at day 29 in the OVA asthma model. (B) The table shows the total cell number and percent of cell phenotypes in the OVA-exposed groups at day 29 depicted in the bar graph. n = 8, p < 0.05. “a” indicates statistical significance over all non-sensitized groups; “b” indicates statistical significance over DM, NiO-1, NiO-2; “c” indicates statistical significance over DM; “d” indicates statistical significance over all non-sensitized groups, OVA; “e” indicates statistical significance over all other groups; “f” indicates statistical significance over all non-sensitized groups, NiO-3A.

Figure 13.

Levels of circulating total and OVA-specific IgE from serum of mice collected on day 29 of the OVA allergy model following NiO aspiration, two sensitization procedures, and two OVA challenges. Sensitized groups are shown in purple and non-sensitized groups are shown in orange. Total IgE levels are represented by the entire bar, OVA-specific IgE levels are represented by the hatched portion of the bar, and OVA-specific: total IgE ratio is expressed as a percentage over the corresponding bars. n = 8, p < 0.05. “a” indicates statistical significance over all non-sensitized groups; “d” indicates statistical significance over all non-sensitized groups, OVA; “f” indicates statistical significance over all non-sensitized groups, NiO-3A.

Figure 14.

Th1/17 and Th2 cytokine levels in BAL fluid collected at day 29 from NiO-exposed/OVA-sensitized mice expressed as fold change over OVA control values. n = 8, p < 0.05. “A” indicates fold-change with statistical significance over all other groups; “C” indicates fold-change with statistical significance over OVA, NiO-3A; “D” indicates fold-change with statistical significance over OVA, NiO-2A.

Figure 15.

Th1/17 and Th2 cytokine levels in serum from NiO-exposed/OVA-sensitized mice expressed as fold change over OVA control values. n = 8, p < 0.05. “A” indicates fold-change with statistical significance over all other groups; “C” indicates fold-change with statistical significance over OVA, NiO-3A; “E” indicates fold-change with statistical significance over OVA.