Early-life exposure to three size-fractionated ultrafine and fine atmospheric particulates in Beijing exacerbates asthma development in mature mice

Abstract

Background: Epidemiological studies have suggested that elevated levels of air pollution contribute to an increased incidence or severity of asthma. Although late-onset adult asthma seems to be more attributable to environmental risk factors, limited data is available on the impact of early-life exposure to size-fractionated ambient particulate matter (PM) on asthma in adults. We aimed to determine the effect on the development and exacerbation of asthma in the adult after the mice were exposed as juveniles to three size-fractionated ambient particulates collected from Beijing.

Methods: The three size-fractionated ambient particulates were collected from urban Beijing in winter, heavily affected by traffic and coal-fired emissions. The typical morphological and major chemical components of the PM were characterized first. Oxidative stress and expression of DNA methyltransferases (DNMTs) were then examined in vitro and in the lungs of mouse pups 48 h after exposure to PM by oropharyngeal aspiration. When the exposed and control juvenile mice matured to adulthood, an antigen-induced asthma model was established and relevant bio-indices were assessed.

Results: PM with different granularities can induce oxidative stress; in particular, F1, with the smallest size (< 0.49 μm), decreased the mRNA expression of DNMTs in vitro and in vivo the most significantly. In an asthma model of adult mice, previous exposure as juveniles to size-fractionated PM caused increased peribronchiolar inflammation, increased airway mucus secretion, and increased production of Th2 cytokines and chemokines. In general, F1 and F2 (aerodynamic diameter < 0.95 μm) particulates affected murine adult asthma development more seriously than F3 (0.95-1.5 μm). Moreover, F1 led to airway inflammation in the form of both increased neutrophils and eosinophils in BALF. The activation of the TGF-β1/Smad2 and Smad3/Stat3 signaling pathways leading to airway fibrosis was more profoundly induced by F1.

Conclusion: This study demonstrated that exposure to ambient PM in juvenile mice enhanced adult asthma development, as shown by increased Th2 responses, which might be associated with the persistent effects resulting from the oxidative stress and decreased gene expression of DNMTs induced by PM exposure. The observed differences between the effects of three size-fractionated particulates were attributed to particle sizes and chemical constituents, including heavy metals and also PAHs, since the amounts of PAH associated with more severe toxicity were enriched equivalently in the F1 and F2 fractions. Relative to the often mentioned PM2.5, PM with an aerodynamic diameter smaller than 0.95 μm had a more aggravating effect on asthma development.

Keywords: Adulthood; Allergic asthma; Early-life exposure; Particulate matter.

Fig. 1

Characteristics of PM. Scanning electron micrographs (a) and transmission electron microscopy (b) were used to characterize the external morphology and internal features of the fractions of collected particulate matter. c Typical TEM images and relevant EDX spectra of elemental mapping results of F1

Fig. 2

The proportion of PAH with different numbers of benzene rings in F1 (a) and F2 (b). The proportion of PAHs with two to six benzene rings was determined; the proportions of BAP, IND and DBA with five benzene rings, which may be associated with more severe toxicity, are also listed separately at the side

Fig. 3

PM induced oxidative stress in vitro and in vivo. a and b The increased expression of heme oxygenase (HO-1) in THP-1 cells induced by PM at different exposure times and concentrations was determined. c and d The effects of PM on HO-1 expression in RAW 264.7 cells at different exposure times and concentrations. HO-1 expression was increased significantly following a 5 h exposure to PM (F1, F2, and F3, 25 μg/ml). PM induced HO-1 expression in a dose dependent manner, and the effect was inhibited by the antioxidant NAC. Cells were treated with PM (10, 25, or 50 μg/ml) for 5 h. RAW 264.7 cells were incubated with 20 mM NAC for 1 h before addition of 25 μg/ml PM for 5 h. e and f Schematic of PM exposure protocol. PM (F1, F2 and F3: 50 μg per dose; F1(s): 15 μg per dose) or PBS was administered by oropharyngeal aspiration to juvenile BALB/c mice on 17, 19 and 21 days after birth. HO-1 expression in lung tissues was measured by western blotting 48 h after the last exposure to PM or PBS. n = 3–5 mice/group

Fig. 4

Effects of PM exposure on DNMTs in vitro and in vivo. a and b The effect of PM treatment on mRNA expression of DNMTs in vitro. RAW 264.7 cells were exposed to 25 μg/ml PM for 5 h. c and d mRNA expression of DNMTs in lung tissues of mice was assessed 48 h following exposure to PM three times on the 17th, 19th and 21st days of postnatal age. n = 5 mice/group. Data are expressed as mean ± SEM. *p < 0.05 vs. Con group, ^&p < 0.05 vs. OVA/PM (F1, F2 or F3)

Fig. 5

PM exposure in infant mice enhances pulmonary inflammation in adulthood after induction of allergic asthma in vivo. a The exposure protocol to PM and induction of asthma model. PM (F1, F2 and F3: 50 μg per time; F1(s): 15 μg per time) or PBS was administered to juvenile BALB/c mice at 17, 19 and 21 days after birth by oropharyngeal aspiration (OA). An OVA-model of asthma was established at six weeks of age. b The test groups are shown in the table. c Total WBC and the differential counts for Monocyte, Neutrophil, Lymphocyte, Basophil and Eosinophil in BALF were evaluated by differential counting 24 h following the final protocol day. d Serum antibody concentration measured by ELISA. The data are expressed as the mean ± SEM of 5 mice per group. *p < 0.05 vs. Con group; #p < 0.05 vs. OVA group, ^&p < 0.05 vs. OVA/PM (F1, F2 or F3)

Fig. 6

Exposure of infant mice to PM enhances pulmonary inflammation and airway mucus secretion in an asthma model. a Morphologic features of mouse lung inflammation stained with H&E. Arrows depict peribronchiolar inflammation. Scale bar = 50 μm. b Representative micrographs of PAS-stained mucus in airway epithelium. Scale bar = 100 μm. c The mRNA expression of Muc5ac and Muc5b in mouse lung tissues. d The degree of PAS staining in the airway epithelium was quantified using ImagePro-Plus 7.0 (Media Cybernetics, USA). Data are plotted as means ± SEM (n = 5 mice/group). *p < 0.05 vs. Con group; #p < 0.05 vs. OVA group, ^&p < 0.05 vs. OVA/PM (F1, F2 or F3)

Fig. 7

The mRNA expression levels of Th2 cytokines and chemokines in mouse lungs were measured by real-time PCR. a The regulation of Th2 type cytokines and IL-33 was observed in an asthma model with mice previously exposed to PM as infants. b mRNA expression of eosinophil-attracting chemokine Ccl11, and Th2 chemokines was more strongly triggered in mouse lungs following PM exposure as infants. Data are expressed as means ± SEM (n = 5 mice/group). *p < 0.05 vs. Con group; #p < 0.05 vs. OVA group, ^&p < 0.05 vs. OVA/PM (F1, F2 or F3)

Fig. 8

F1 exposure enhanced the OVA-induced activation of the TGF-β1/Smad2 (a) and Smad3/Stat3 (b) pathways involved in airway fibrosis. The expression of the relevant proteins was detected 24 h after the last OVA challenge according to the schedule shown in Fig. 5. n = 5 mice/group

Fig. 9

Early-life exposure to PM in Beijing exacerbates asthma development in adult mice