Allergic airway inflammation by nasal inoculation of particulate matter (PM2.5) in NC/Nga mice

Abstract

To evaluate the effect of airborne particulate matter 2.5 (PM2.5) in winter on airway inflammation, water-soluble supernatant (Sup) and water-insoluble precipitate (Pre) in PM2.5 were inoculated in NC/Nga mice with high sensitivity to mite allergens. Sup with aluminum oxide was injected intraperitoneally for sensitization. Five days later, Sup, Pre or both Sup and Pre were inoculated via the nasal route five times for more sensitization and a challenge inoculation on the 11th day in NC/Nga mice. On the 12th day, mice were examined for airway hyperresponsiveness (AHR), BALF cell count and IL-1β concentration, mRNA expression of Th1 and Th2 cytokines, chemokines such as eotaxin 1 and eotaxin 2, inflammasomal complex molecules such as IL-1β, caspase 1 and the nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) in lung tissue as well as histopathology. The synergistic effect of Sup and Pre was observed in terms of increases in AHR, BALF cells, the mRNA expression of IL-13, eotaxin1 and IL-1β, and the IL-1β concentration in BALF. Intracellular deposits of insoluble particulates were observed in macrophages around inflammatory granulation of the mouse group treated with Sup and Pre. These results suggest that PM2.5 can induce airway hyperresponsiveness in mice with genetically high sensitivity to mite allergens by an inflammasome-associated mechanism and synergistic action of insoluble particulates and soluble components.

Figure 1. AHR to acetylcholine after exposure to PM.

Effect of control (sterilized physiological saline), Sup (supernatant fraction containing 50 μg of protein), Pre (200 μg of precipitate in 25 μl of PB) and Sup plus Pre administered intranasally in NC/Nga mice. The bronchoconstriction (%) is expressed as mean ± SEM of 5–8 animals (n = 8, 7, 5 and 5 for saline control, Sup, Pre and Sup+Pre groups, respectively). Two-way ANOVA showed P<0.0001 concerning responses to acetylcholine and mouse groups. *P<0.05, **P<0.01 and ***P<0.001 represent multiple-comparison test results of the dose responses to acetylcholine in the mouse groups of Sup, Pre and control vs. the mouse group of Sup plus Pre.

Figure 2. BALF cell numbers of total cell fraction, eosinophils, lymphocytes and macrophages.

The counts of these cells in the BALF are expressed as mean ± SEM of 4–8 animals (n = 8, 6, 4 and 4 for Control, Sup, Pre and Sup+Pre groups, respectively). P for ANOVA is expressed in the upper portion of each figure. Tukey’s multiple-comparison test results are shown in the mouse groups of control, Sup and Pre compared with the mouse group of Sup plus Pre as ***P<0.001 and the Pre group compared with the control as # P<0.05.

Figure 3. IL-1β concentration of BALF was determined using ELISA.

Data are expressed as the mean ± SEM from 5–8 mice (n = 8, 7, 8 and 5 for Control, Sup, Pre and Sup+Pre groups, respectively). P for ANOVA is expressed in the upper portion of each figure. *P<0.05 and **P<0.01 represent Tukey’s multiple-comparison test results of each group compared with Sup plus Pre.

Figure 4. Changes in mRNA expression for cytokines and chemokines in the lung tissue after exposure to PM.

Mice were administered saline control, supernatant (Sup) containing 50 μg of protein, 200 μg of precipitate (Pre) and Sup plus Pre. Real-time PCR for the cytokines IL-4, IL-5, IL-13, eotaxin-1, eotaxin-2, IFN-γ and TNF-α was performed under optimized conditions. Relative expression was calculated with GAPDH as an internal standard. Data are expressed as the means ± SEM from 5–8 mice (n = 8, 7, 8 and 5 for Control, Sup, Pre and Sup+Pre groups, respectively). P for ANOVA is expressed in the upper portion of each figure. *P<0.05 and ***P<0.001 represent Tukey’s multiple-comparison test of control, Sup and Pre compared with Sup plus Pre.

Figure 5. Changes in mRNA expression for IL-1β, caspase 1 and NLRP3 in the lung tissue after exposure to PM.

Data are expressed as the mean ± SEM from 5–8 mice (n = 8, 7, 8 and 5 for Control, Sup, Pre and Sup+Pre groups, respectively). P for ANOVA is expressed in the upper portion of each figure. *P<0.05 and ***P<0.001 represent Tukey’s multiple-comparison test of each group compared with Sup plus Pre.

Figure 6. Histopathological findings of the lungs after exposure to PM.

Paraffin-embedded sections were stained with hematoxylin-eosin for the saline control (A) and Sup plus Pre (B). The periodic acid-Schiff (PAS) staining of Sup plus Pre mice is shown in C. High magnification of H&E staining of the site of inflammatory granulation of Sup plus Pre mice (D).