Lung exposure of titanium dioxide nanoparticles induces innate immune activation and long-lasting lymphocyte response in the Dark Agouti rat

Figures & data

Figure 1.  Dose-dependent differences in the number of cells in bronchoalveolar lavage fluid from rats 24 h after intratracheal instillation with nanosized TiO₂. One-way ANOVA with Dunnett’s post-test; value is significantly (*P < 0.05 and **P < 0.01) different vs. phosphate-buffered saline (PBS) control. Data are presented as mean ± SEM (n = 6).

Table 1.  Proportion of natural killer (NK) cells expressing NKR-P1A^bright.

Days	0 (n = 10)	1 (n = 14)	2 (n = 15)	8 (n = 11)	16 (n = 5)	30 (n = 6)
% Bright	36 ± 3	30 ± 2	72 ± 2**	70 ± 3**	62 ± 2**	43 ± 4

Expressed as mean percentage of total NK numbers ± SD.

One-way ANOVA with Dunnett’s post-test; value significantly (**P < 0.01,) different as compared with control.

Figure 2.  The number of cells in bronchoalveolar lavage fluid from nano-TiO₂ (5 mg/kg)-exposed rats 0 (n = 10), 1 (n = 20), 2 (n = 15), 8 (n = 11), 16 (n = 5), 30 (n = 6), and 90 (n = 6) days post-intratracheal instillation. One-way ANOVA with Dunnett’s post hoc test was employed, and values significantly changed vs. nonexposed control animals (timepoint 0) are indicated (*P < 0.05, **P < 0.01, ***P < 0.001). A Student’s t-test was performed to compare TiO₂-exposed rats with that of rats exposed for phosphate-buffered saline (PBS) only at Days 1, 16, 30, and 90 (^$$P < 0.01, ^$$$P < 0.001). Data are presented as mean ± SEM.

Figure 3.  Numbers of NK T-cells (CD3⁺ NKR-P1A⁺), NK cells (CD3⁻ NKR-P1A⁺), B-cells (CD3⁻ CD45⁺), and dendritic cells (CD3⁻ CD45RA⁻ OX-62⁺ OX-6⁺) in bronchoalveolar lavage fluid from nanosized TiO₂ (5 mg/kg) exposed rats 0 (n = 5), 1 (n = 7), 2 (n = 7), 8 (n = 10), 16 (n = 5), 30 (n = 5), and 90 (n = 4) days after a single intratracheal instillation. Kruskal–Wallis test with Dunn’s post-test; value is significantly (*P < 0.05, **P < 0.01, ***P < 0.001) different vs. control.

Figure 4.  Numbers of cytotoxic T-cells (CD3⁺ CD8⁺) and CD4⁺ T-cells, and median intensity of CD25 receptor expression on CD4⁺ T-cells in bronchoalveolar lavage fluid from nanosized TiO₂ (5 mg/kg) exposed rats 0 (n = 5), 1 (n = 7), 2 (n = 7), 8 (n = 10), 16 (n = 5), 30 (n = 5), and 90 (n = 4) days after a single intratracheal instillation. Kruskal–Wallis test with Dunn’s post-test; value is significantly (*P < 0.05, **P < 0.01, ***P < 0.001) different vs. control.

Figure 5.  Concentration of cytokines in bronchoalveolar lavage fluid from nanosized TiO₂ (5 mg/kg)-exposed rats 0 (n = 10), 1 (n = 14), 2 (n = 15), 8 (n = 11), 16 (n = 5), 30 (n = 6), and 90 (n = 6) days after a single intratracheal instillation. Kruskal–Wallis test with Dunn’s post-test; value is significantly (*P < 0.05, **P < 0.01, ***P < 0.001) different vs. control.

Figure 6.  Concentration of cytokines in serum from nano-TiO₂ (5 mg/kg) exposed rats 0 (n = 10), 1 (n = 13), 2 (n = 15), 8 (n = 11), 16 (n = 5), 30 (n = 6), and 90 (n = 5) days after a single intratracheal instillation. Kruskal–Wallis test with Dunn’s post-test; value is significantly (*P < 0.05, **P < 0.01) different vs. control.

Figure 7.  Lung sections from rat tissue showing particle uptake at Days 2 (A and B), 30 (C and D), and 90 (E and F) post-nano-TiO₂ exposure. Particle aggregates (arrows) can be found in alveolar macrophages at Days 2 and 30, and mainly in the interstitium at Day 90. The tissues were stained with hematoxylin–eosin.