Effects of titanium dioxide nanoparticle exposure on neuroimmune responses in rat airways

Abstract

Exposure to ambient nanoparticles (defined as particulate matter [PM] having one dimension <100 nm) is associated with increased risk of childhood and adult asthma. Nanomaterials feature a smaller aerodynamic diameter and a higher surface area per unit mass ratio compared to fine or coarse-sized particles, resulting in greater lung deposition efficiency and an increased potential for biological interaction. The neurotrophins nerve growth factor and brain-derived neurotrophic factor are key regulatory elements of neuronal development and responsiveness of airway sensory neurons. Changes in their expression are associated with bronchoconstriction, airway hyperresponsiveness, and airway inflammation. The neurogenic-mediated control of airway responses is a key pathophysiological mechanism of childhood asthma. However, the effects of nanoparticle exposure on neurotrophin-driven airway responses and their potential role as a predisposing factor for developing asthma have not been clearly elucidated. In this study, in vivo inhalation exposure to titanium dioxide nanoparticles (12 mg/m(3); 5.6 h/d for 3 d) produced upregulation of lung neurotrophins in weanling (2-wk-old) and newborn (2-d-old) rats but not in adult (12-wk-old) animals compared to controls. This effect was associated with increased airway responsiveness and upregulation of growth-related oncogene/keratine-derived chemokine (GRO/KC; CXCL1, rat equivalent of human interleukin [IL]-8) in bronchoalveolar lavage fluid. These data show for the first time that exposure to nanoparticulate upregulates the expression of lung neurotrophins in an age-dependent fashion and that this effect is associated with airway hyperresponsiveness and inflammation. These results suggest the presence of a critical window of vulnerability in earlier stages of lung development, which may lead to a higher risk of developing asthma.

FIGURE 1

The left panel shows an actual picture of the exposure system used for this study. (A) Fluidized-bed aerosol generator containing TiO₂ powder. (B) Air drier unit, which introduces dry clean air into the generator to fluidize the air and disperse the particles into the exposure chamber. (C) Exposure chamber containing the unrestrained animals. The right panel illustrates the schematic diagram of the inhalation exposure system.

FIGURE 2

A typical number-based particle size distribution of TiO₂ aerosol where C_n is the number concentration of the aerosol and D_g is the geometric (mobility) diameter.

FIGURE 3

(A) In adult rats, NGF and BDNF expression is unchanged following exposure to TiO₂ nanoparticulate. (B) Consistent with their agonists, the expression of neurotrophin receptors was unaffected except for a decreased expression of the BDNF receptor, TrKb, which may be due to a physiologically diminished activity of neurotrophins associated with age. Data were generated by densitometric analysis of the intensity of the target gene bands normalized for the standard reference gene, β-actin, and are expressed as mean ± SE (n = 6 for both groups; asterisk indicates signficant at p < .05).

FIGURE 4

(A) In weanling rats, exposure to TiO₂ nanoparticles upregulates NGF and BDNF expression in lung homogenates. This effect is associated with a significant increase of the high-affinity receptor for BDNF, TrKb. The high-affinity receptor for NGF, TrKa, is also upregulated, although the magnitude of the change is not enough to reach statistical significance, whereas the expression of the NGF low-affinity receptor, p75, is unaffected (B). The intensity of the bands for the target genes was analyzed by computer densitometry and normalized for the standard reference gene, β-actin, as previously described. A representative gel image of the NGF and BDNF bands is also shown. Data are expressed as mean ± SE (n = 6 for both groups; asterisk indicates signficant at p < .05).

FIGURE 5

(A) Newborn rats show increased lung NGF expression following exposure to TiO₂ nanoparticles compared to their age-matched controls. This effect appears to be of a higher magnitude compared to weanlings exposed to the same level of inhaled particulate. BDNF expression was unaffected. (B) Consistent with its agonist, the NGF low-affinity receptor, p75, is upregulated in the lungs of newborn animals exposed to TiO₂ nanoparticles compared to control, whereas for the NGF high-affinity receptor, TrKa, levels are undetectable. TrKb expression is slightly yet significantly increased despite the lack of response of its agonist, BDNF, indicating a possible increase of TrKb-mediated BDNF activity. Data shown in figure are obtained by densitometric analysis of the target genes band as previously described and are expressed as mean ± SE (n = 6; asterisk indicates signficant at p < .05).

FIGURE 6

Photomicrographs of hematoxylin and eosin-stained sections from the lung of weanling rats (A) sacrificed after the last day of exposure to air or TiO₂ nanoparticles show the absence of any significant pathological changes compared to age-matched controls, despite the fact that tissue neurotrophins were significantly upregulated. In adult rats (B), the absence of exposure-related histopathological changes is consistent with the absence of any significant changes in neurotrophin expression (magnification 20×; micrographs are representative of 3 rats per group).

FIGURE 7

(A) In weanling rats, baseline specific airway resistance (sRaw; cm H₂O-s) is increased in animals exposed to TiO₂ compared to their age-matched controls 1 h after the end of the last exposure. In the same animals, the airway response to increasing concentration of inhaled methacholine is increased in exposed rats compared to controls (B). Data are expressed as mean ± SE (n = 5–6; asterisk indicates significant at p < 0.05 and double asterisk indicates signficant at p < 0.01).

FIGURE 8

The analysis of BAL fluid by multiplexed quantification using Luminex 200 System and xMAP technology revealed the presence of increased concentrations of the cytokine GRO/KC (rat equivalent of human IL-8) in the airways of exposed weanling animals compared to their age-matched controls. Data are expressed as picograms of analyte per microgram of total protein and are mean ± SE (n = 6 for both groups; asterisk indicates signficant at p < .05).