Exposure to Nickel Oxide Nanoparticles Induces Acute and Chronic Inflammatory Responses in Rat Lungs and Perturbs the Lung Microbiome

Abstract

Nickel oxide nanoparticles (NiO NPs) are highly redox active nanoparticles. They can cause acute and chronic inflammation in rat lungs. Unlike the gut microbiome, the association between the lung microbiome's role and pulmonary inflammatory response to inhaled nanoparticles remains largely unexplored. We aimed to explore the interaction between the lung microbiome and inflammatory responses in rats exposed to NiO NPs. Thirty female Wistar rats were randomly categorized into control and low- (50 cm²/rat), and high- (150 cm²/rat) dose NiO NPs exposure groups. NiO NPs were intratracheally instilled, and cytological, biochemical, proinflammatory cytokine, and lung microbiome analyses of bronchoalveolar lavage fluid were performed at 1 day and 4 weeks after instillation. NiO NPs caused a neutrophilic and lymphocytic inflammatory response in rat lung. We demonstrated that exposure to NiO NPs can alter the lung microbial composition in rats. In particular, we found that more Burkholderiales are present in the NiO NPs exposure groups than in the control group at 1 day after instillation. Dysbiosis in the lung microbiome is thought to be associated with acute lung inflammation. We also suggested that Burkholderiales may be a key biomarker associated with lung neutrophilic inflammation after NiO NPs exposure.

Keywords: inflammatory response; lung microbiome; nickel oxide nanoparticles.

Figure 1

Histological changes in the lungs of rats at 1 day and 4 weeks after instillation of nickel oxide nanoparticles (NiO NPs). Tissues were stained with hematoxylin and eosin. Compared with the control group, the instillation of NiO NPs in rats caused the narrowing of alveolar ducts (AD) and alveoli (A) and blocking of bronchioles (B). In the high-dose group, NiO NPs produced foamy macrophages and exhibited deposition of proteinaceous materials in the alveoli on day 28 after instillation. (Indicator; macrophage, bar = 50 μm). C: control group; L: low-dose group (50 cm²/rat); H: high-dose group (150 cm²/rat).

Figure 2

Cytological analysis of bronchoalveolar lavage fluid (BALF) after intratracheal instillation of nickel oxide nanoparticles (NiO NPs). The number of total cells, percentage of macrophages, neutrophils, eosinophils, granulocytes, and lymphocytes is indicated (A). Diff–Quik staining images of immune cells in BALF after intratracheal instillation of NiO NPs. Macrophages (arrowhead) were observed after 1 day in C, L, and H. However, foamy macrophages (arrow) were observed only at 4 weeks in the L and H (B). Although neutrophils (dotted line) were recruited in the BALF of L and H, lymphocytes (solid line) were recruited at 4 weeks after instillation in BALF of L and H. Eosinophils (circle) were then observed 1 day after instillation in H from the cell image. Data are presented as mean ± standard deviation (SD) of each group. One-way analysis of variance (ANOVA) was performed for the comparison between the NiO NPs-treated and control groups with statistical significance indicated by ** p < 0.01 and *** p < 0.001. C: control group; L: low-dose group (50 cm²/rat); H: high-dose group (150 cm²/rat).

Figure 3

Proinflammatory cytokine analysis of bronchoalveolar lavage fluid (BALF) after intratracheal instillation of nickel oxide nanoparticles (NiO NPs). Levels of cytokine-induced neutrophil chemoattractant-3 (CINC-3), eotaxin, monocyte chemoattractant protein (MCP)-1, interferon (IFN)-γ, and interleukin (IL)-1β are indicated. Data are presented as mean ± standard deviation (SD) of each group. One-way analysis of variance (ANOVA) was performed for the comparison between the NiO NPs-treated and control groups with statistical significance indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001. C: control group; L: low-dose group (50 cm²/rat); H: high-dose group (150 cm²/rat).

Figure 4

Biochemical analysis of bronchoalveolar lavage fluid (BALF) after intratracheal instillation of nickel oxide nanoparticles (NiO NPs). Lactate dehydrogenase (LDH) levels, total protein levels, turbidity, and phospholipid levels are indicated. Turbidity was measured as optical density at 600 nm. LDH, total protein, phospholipids, and turbidity level in the BALF significantly increased at 4 weeks after instillation. Data are presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was performed for the comparison between the NiO NPs-treated and control groups with statistical significance indicated by * p < 0.05 and *** p < 0.001. C: control group; L: low-dose group (50 cm²/rat); H: high-dose group (150 cm²/rat).

Figure 5

Alpha diversity, which estimates bacterial diversity and species richness. Bacterial diversity (Shannon; right side) and richness estimates (chao1; left) are divided into two groups, reflecting 1 day (light gray) and 4 weeks (dark gray) after intratracheal instillation of nickel oxide nanoparticles (NiO NPs), by using a boxplot. Community diversity and richness were evaluated using the alpha diversity and were not significantly different between exposure groups of NiO NPs and control group. Median values are shown as a line within the box and error bars indicate the standard error of the samples C: control group; L: low-dose group (50 cm²/rat); H: high-dose group (150 cm²/rat).

Figure 6

Beta diversity of principal coordinates analysis (PCoA) of weighted unifrac distances (A). Unweighted pair group method with arithmetic mean (UPGMA) cluster analysis based on community composition of each sample in at phylum level (B). The similarities of lung microbial composition between instillation groups of NiO NPs and control group were not significant at the phylum level. C: control group; L: low-dose group (50 cm²/rat); H: high-dose group (150 cm²/rat).

Figure 7

Bacterial taxonomic composition analysis after exposure to nickel oxide nanoparticles (NiO NPs). Bacterial taxonomic assignment was at the phylum level and samples were compared according to NiO NPs concentrations at 1 day and 4 weeks after instillation. NiO NPs induced the change in lung microbial composition in rats at 1 day after instillation. C: control group; L: low-dose group (50 cm²/rat); H: high-dose group (150 cm²/rat).