Fine particulate matter contributes to COPD-like pathophysiology: experimental evidence from rats exposed to diesel exhaust particles

Abstract

Background: Ambient fine particulate matter (PM_2.5) is considered a plausible contributor to the onset of chronic obstructive pulmonary disease (COPD). Mechanistic studies are needed to augment the causality of epidemiologic findings. In this study, we aimed to test the hypothesis that repeated exposure to diesel exhaust particles (DEP), a model PM_2.5, causes COPD-like pathophysiologic alterations, consequently leading to the development of specific disease phenotypes. Sprague Dawley rats, representing healthy lungs, were randomly assigned to inhale filtered clean air or DEP at a steady-state concentration of 1.03 mg/m³ (mass concentration), 4 h per day, consecutively for 2, 4, and 8 weeks, respectively. Pulmonary inflammation, morphologies and function were examined.

Results: Black carbon (a component of DEP) loading in bronchoalveolar lavage macrophages demonstrated a dose-dependent increase in rats following DEP exposures of different durations, indicating that DEP deposited and accumulated in the peripheral lung. Total wall areas (WAt) of small airways, but not of large airways, were significantly increased following DEP exposures, compared to those following filtered air exposures. Consistently, the expression of α-smooth muscle actin (α-SMA) in peripheral lung was elevated following DEP exposures. Fibrosis areas surrounding the small airways and content of hydroxyproline in lung tissue increased significantly following 4-week and 8-week DEP exposure as compared to the filtered air controls. In addition, goblet cell hyperplasia and mucus hypersecretions were evident in small airways following 4-week and 8-week DEP exposures. Lung resistance and total lung capacity were significantly increased following DEP exposures. Serum levels of two oxidative stress biomarkers (MDA and 8-OHdG) were significantly increased. A dramatical recruitment of eosinophils (14.0-fold increase over the control) and macrophages (3.2-fold increase) to the submucosa area of small airways was observed following DEP exposures.

Conclusions: DEP exposures over the courses of 2 to 8 weeks induced COPD-like pathophysiology in rats, with characteristic small airway remodeling, mucus hypersecretion, and eosinophilic inflammation. The results provide insights on the pathophysiologic mechanisms by which PM_2.5 exposures cause COPD especially the eosinophilic phenotype.

Keywords: COPD; Diesel engine exhaust; Emphysema; Eosinophils; PM2.5; Small airway remodeling.

Fig. 1

Study protocol and structural changes in different compartments of rat lung. The DEP generator and exposure chamber were shown in (A), while the schema of study protocol was demonstrated in (B). Total wall area (WAt) of large airways showing no significant difference following 2-wk, 4-wk and 8-wk DEP exposures, respectively, as compared to the filtered air controls (C-D). DEP exposures for three durations induced significant increases in WAt of small airways (E-F). Compared to the filtered air control, 4-wk and 8-wk DEP exposures, respectively, resulted in significant increases in MLI of alveoli (G-H). Data were expressed as mean (95% CI). ns = not significant, *** p < 0.001. n = 6 rats/group. Bars in all the HE staining images = 100 μm

Fig. 2

Changing trend of DEP concentrations during exposure period. The changing trends of particle mass concentration (PMC, A) and particle number concentration (PNC, B) during 4 h DEP exposures were shown. Correlation analysis showed that the changing trend of PMC was significantly correlated with that of PNC during the exposure period (C, r² = 0.96, p < 0.0001)

Fig. 3

Carbon loading in alveolar macrophages and lung function parameters. Carbon uptake areas in BALF macrophages (A, red arrows) following DEP exposures for 2-wk, 4-wk and 8-wk significantly increased, in a dose-dependent manner, compared to carbon uptake areas following filtered air exposure (*** p < 0.001, ## p < 0.01, ### p < 0.001, B). Bar = 8 μm. Total lung capacity (TLC) increased significantly following 2-wk, 4-wk and 8-wk DEP exposures as compared to filtered air controls, respectively (** p < 0.01, C). Also, lung resistance increased significantly following 4-wk and 8-wk DEP exposures, respectively (** p < 0.01, D). Data were expressed as mean (95% CI). ns = not significant

Fig. 4

Remodeling and fibrosis of the peripheral lung. Of the small airways, the relative expression of α-SMA (stained in green) of smooth muscle to total wall area increased significantly in DEP exposure for 4-wk and 8-wk, respectively (*** p < 0.001, A-B). In the alveolar region, the number of α-SMA positive cells (white arrow head, C) increased significantly post-DEP exposures (*** p < 0.001, D). The fibrosis areas (stained in blue, E) around the small airways also significantly increased following 4-wk and 8-wk DEP exposures, respectively (* p < 0.05, ** p < 0.01, F). The contents of hydroxyproline in lung tissue significantly increased in rats exposed to DEP for 4 weeks and 8 weeks, respectively (** p < 0.01, G). Data were expressed as mean (95% CI). Bars in the immunofluorescent staining images = 50 μm, while bar in the Masson Trichrome staining image = 100 μm. SA: small airways, ns = not significant

Fig. 5

Mucus production and epithelial cell morphology of the small airways. The MUC5AC-immunopositive cells (red arrows, A) and PAS-positive goblet cells (blue arrows, C) were indicated. Quantification analysis showed that the number of MUC5AC positive cells of the superficial epithelia was increased significantly following DEP exposures over 4-wk and 8-wk (** p < 0.01, B). The number of PAS positive cells was also increased significantly following DEP exposures for 4-wk and 8-wk (** p < 0.01, D). Data were expressed as mean (95% CI). Magnification of the lower panel images in A and C, 1000 x, ns = not significant

Fig. 6

Recruitment of inflammatory cells to the small airways and inflammatory mediators in BAL fluid. Eosinophil (black arrow head, A) infiltrated around small airways increased significantly in rats exposed DEP for 4 weeks and 8 weeks (** p < 0.01, B). For the mast cells (red arrows head, C), exposure of rats to DEP for 8 weeks (* p < 0.05, D), but not for shorter durations, demonstrated a significant increase around the small airways. The infiltrations of Iba-1 immuno-positive macrophages (blue arrow head, E) were significantly increased in small airways following 4-wk (*** p < 0.001) or 8-wk DEP exposure (** p < 0.01), as compared to the filtered air controls, respectively (F). Bar in the images (Panel A, C, E) = 100 μm. ns = not significant. SA = small airways. Cell differentials in BALF showed significant increases of total leukocyte, macrophage as well as neutrophil, but not eosinophils, following 8-week DEP exposure, as compared to filtered air control, respectively (** p < 0.01, ns, not significant, G-J). DEP exposure for 4-wk or 8-wk significantly increased BALF concentrations of TNF-α, IL-6, IL-13 and IL-17 A, respectively (** p < 0.01, ns, not significant, K-N). Data were expressed as mean (95% CI)

Fig. 7

Oxidative stressor concentrations. As compared to filtered air control, the serum levels of 8-OHdG increased significantly in rats exposed to DEP for 4-wk or 8-wk (** p < 0.01, *** p < 0.001, A). Exposure of rats to DEP for 3 durations resulted in significant increases of serum MDA levels, as compared to the control, respectively (* p < 0.05, *** p < 0.001, B). Data were expressed as mean (95% CI)