Exposure to Ambient Particulate Matter Induced COPD in a Rat Model and a Description of the Underlying Mechanism

While the health effects of air pollution have been an international public health concern since at least the 1950s, recent research has focused on two broad sources of air pollution, namely, biomass fuel (BMF) and motor vehicle exhaust (MVE). Many studies have shown associations between air pollution PM and exacerbations of pre-existing COPD, but the role of air pollution PM in the development and progression of COPD is still uncertain. The current study indicates that rats can develop pronounced COPD following chronic exposure to air pollution PM (BMF and MVE), as characterized by lung function reduction, mucus metaplasia, lung and systemic inflammation, emphysema, and small airway remodeling. Comparative analyses demonstrate that both BMF and MVE activate similar pathogenesis that are linked to the development of COPD. These findings also show that some differences are found in the lungs of rats exposed to BMF or MVE, which might result in different phenotypes of COPD.


Determination of Gas Concentrations in the Exposure Rooms During Air Pollution PM
Exposure. We measured gas concentrations inside the chambers because gaseous co-pollutants are generated by combustion. The O 2 , CO, NO X and SO 2 levels in the exposure rooms during BMF and MVE exposure are shown in Fig. S1. The CO and NO X levels were higher in the BMF group than in the MVE group, whereas the O 2 levels were not significantly different between the two groups. The SO 2 levels were low in both groups, and the SO 2 levels in the MVE group were slightly higher than those in the BMF group. Figure S1. The O 2 , CO, NO X and SO 2 levels in the exposure rooms during air pollution PM exposure. A, B, C, D: The CO and NO X levels were higher in the BMF group than those in the MVE group, whereas the O 2 levels were not significantly different between the two groups.
The SO 2 levels were low in both groups, the SO 2 levels in the MVE group were slightly higher than those in the BMF group.
Effects of Air Pollution PM on Survival and Body Weight. Consistent with human patients 3 with COPD, we found that rats with COPD showed restless behavior, wheezing, and shortness of breath within the first 2 months of developing COPD. As COPD progressed to later stages, the rats showed progressive and gradual reductions in body weight. As shown in Fig. S2, there were no significant differences among the rats in all three groups in terms of initial body weight. From 4 months of exposure onward, the mean weight gain was lower in the BMF group and the MVE group than in the control group. Furthermore, there was a significant difference (p<0.05) between the two exposure groups and the control group regarding weight gain after 5 months of exposure. All of the rats were fed a standard diet throughout the experimental period. At the end of the experiment, after 7 months of exposure, the mean weight gain was 155.25±35.26 g in the control group, 102.15±32.23 g in the BMF group, and 100.21±22.64 g in the MVE group. These results show that the BMF-and MVE-exposed rats had attenuated rates of weight gain compared to the control group. Figure S2. Effects of air pollution PM on body weight. The body weights (g) of the rats from the different study groups were measured from 1 week to 29 weeks (7 months) of the exposure period. Values are expressed as the mean weight gain. After 5 months of exposure, 4 the mean weight gain was lower in the BMF group and the MVE group than in the control group. There was a significant difference (p< 0.05) between the exposure group and the control group regarding weight gain (n=14-32). Data are expressed as the mean±SEM (A-D). *P < 0.05, **P < 0.01.

Air pollution PM Exposure Induces Apoptosis of Alveolar Septal Cells, Leading to
Airspace Enlargement. Walter A and colleague found diesel (DIE)-exposed animals showed higher levels of apoptosis in lung sections evaluated using TUNEL staining S1 . In addition, Francesca found cigarette smoke increased alveolar septal cell apoptosis in NHPs S2 . We evaluated airway apoptosis by detecting DNA fragmentation using TUNEL staining. The present study revealed that significant increases in DNA fragmentation counts were detected after 3 and 5 months of BMF or MVE exposure. The results indicated that the BMF exposure-induced alveolar septal cell apoptosis was more severe (Fig. S3). In addition to inflammation, apoptosis might be another important mechanism leading to alveolar enlargement in rats after air pollution PM exposure. Figure S3. Air pollution PM exposure induces apoptosis of alveolar septal cells. TUNEL staining of lung tissues harvested from rats after 3 and 5 months of BMF or MVE exposure (n=8). Compared with controls, significant increases in TUNEL-positive cells were detected after 3 and 5 months of BMF or MVE exposure (p< 0.01). The TUNEL-positive cells in the BMF-exposed rats were higher than those in the MVE-exposed rats. The white arrow 5 indicates a TUNEL-positive cell. Data are shown as the mean±SEM. *P < 0.05, **P < 0.01.

Supplemental Materials and Methods
Exposure System. PM mass concentrations and particle size distribution were measured using DustTraks (TSI 8533, USA) with a time resolution of 1 s S3,S4 . A DustTrak has a flow rate of 1.7 LPM and measures mass concentrations of total suspended particles (PM total) as well as PM 10 , PM 2.5 , and PM in the range of 0.001-150 mg/m 3 . NO, SO 2 , CO and O 2 concentrations were measured using an electrochemical gas analyzer (Testo 340, Germany) S5 . This instrument was capable of measuring NO concentrations within a range of 0-3000 ppm, SO 2 concentrations within a range of 0-5000 ppm, CO concentrations within a range of 0-10,000 ppm, and O 2 concentrations within a range of 0-25%. The measurement accuracies for these four components were ±5%, ±5%, ±5% and ±1%. NOx and CO concentrations were corrected to a 0% O 2 level in the product stream, and the NOx concentration was measured as the sum of the amounts of NO and NO 2 .

Measurement of the Apoptosis of Alveolar Septal Cells. Apoptotic cells in lung tissues
were evaluated using Terminal-deoxynucleoitidyl transferase mediated nick end labeling was performed using cold, sterile, pyrogen-free, Ca 2+ and Mg 2+ -free phosphate-buffered saline (PBS) at a volume of 2.5 ml for the first lavage and 3 ml for subsequent lavages.
Approximately 8 ml of BALF per rat was collected in sterile centrifuge tubes. Pooled BALF cells for each rat were washed in PBS followed by centrifugation (800 × g for 10 min at 4 °C).
Cell-free first fractions of BALF aliquots were frozen at −80 °C for protein and cytokine evaluations S6 .
BALF Cell Counting and Differentials. Cells were collected from BALF through centrifugation, and the cell pellets were resuspended in 1 ml of PBS. A 10-μl aliquot of each sample was stained with trypan blue and analyzed using a hemocytometer to determine cell number and viability. PBS (100 μl) was then added to the cells, which were resuspended onto slides. After being fixed with 10% paraformaldehyde for 24 h, the slides were stained with hematoxylin and eosin (H&E) (Solarbio Tech; Beijing, China) and analyzed at x400 by light microscopy. A total of 400 cells per sample were counted to determine the percentages of alveolar macrophages, neutrophils and lymphocytes.
Sampling Lung Tissues and Blood. Rats were sacrificed by intraperitoneal injection of sodium pentobarbital (100 mg/kg). The left or right lungs were inflated and fixed using 4% paraformaldehyde (pH 7.40) at 25 cm H 2 O pressure for 24 h. The lungs were then embedded in paraffin and cut into 4-µm-thick sections. The right lung tissues were snap frozen in liquid nitrogen and stored at -80°C for Western blot analysis. The body cavity was opened and blood samples were collected from the inferior vena cava; serum was stored at -80°C for ELISA.

Morphometric Measurements.
To avoid observer bias, all microscope slides were coded 7 before analysis by one observer and were read blindly. Photographs were taken on a Zeiss Axio Imager 2 Microscope (Carl Zeiss, Germany), and morphometric analysis was performed using Image-Pro Plus (IPP) 7.0 software (Media Cybernetics, Silver Spring, USA). Airspace size was quantified in lung tissues stained with H&E using the mean linear intercept S7,S8 , and an algorithm was applied to perform quantitative characterization of airspace enlargement S9 .
The thickness of the small airway wall was analyzed according to previously described methods S9,S10 . Small airways cut transversely and with a basement membrane perimeter (Pbm) of less than 2000 μm were examined. The results were standardized for airway size based on the Pbm (μm 2 /μm). At least five small airways were counted per slide S11 . Collagen deposition around small airways and vessels was assessed in paraffin-embedded and formalin-fixed lung sections stained with Masson's trichrome using a commercial kit (Sigma-Aldrich). The areas of collagen deposited around airways and vessels were quantified based on areas that were stained blue by Masson's trichrome using image analysis. Periodic acid-Schiff (PAS) staining is mainly used to visualize structures containing a high proportion of carbohydrate macromolecules, such as connective tissues, mucus, the glycocalyx, and basal laminae. Lung sections were stained with AB-PAS using commercial kits (Sigma-Aldrich, St. Louis, MO) following the manufacturer's instructions.
Immunohistochemistry and Immunofluorescence. Immunohistochemical (IHC) evaluation was performed as described elsewhere S12 . The sections were incubated with primary antibodies against a-SMA (Sigma-Aldrich) or MUC5AC (Abcam; Cambridge, UK). A horseradish peroxidase (HRP)-conjugated secondary antibody was also used and visualized with diaminobenzidine using an Immunohistochemistry Detection Kit (Gene Tech;Shanghai,8 China) according to the manufacturer's protocols. Photographs were taken using identical conditions for light setting and contrast. Color segmentation protocols in the IPP7.0 system were utilized to obtain data. The area of a-SMA-immunostaining in the small airway wall was quantitatively analyzed S13 . Immunofluorescence evaluation was performed as described elsewhere S14 . The tissue sections were then incubated with primary antibodies (against E-cadherin, vimentin, or FSP1) and detected using an appropriate fluorochrome-linked secondary antibody. DAPI was used as a nuclear counterstain. Images were acquired using a Zeiss Axio Imager 2 microscope (Carl Zeiss, Germany). Double immunofluorescence staining of E-cadherin (BD Biosciences; California, USA) and vimentin or FSP1 (Abcam; Cambridge, UK) was performed using a MultiVision polymer detection system (Thermo Scientific; Utah, USA) according to the manufacturer's protocols.
Western Blot Analysis. Levels of MMP9 and MMP2 proteins in the lung were evaluated by Western blotting as previously described S15 . Lung tissues were ground into powder using liquid nitrogen in a mortar. Tissue lysates were collected into EP tubes and centrifuged at 10,000 rpm for 10 min at 4°C. The protein content in the supernatant was measured using a BCA Protein Assay Kit (Keygenbio, Nanjing). A total of 80 μg protein from each group of samples was assayed by SDS-PAGE. Following electrophoretic transfer, membranes were treated at RT for 1 hour with 5% skim milk. Then, the membranes were incubated overnight at 4°C with primary antibodies against MMP9, MMP2 (Abcam; Cambridge, UK), and GAPDH (Santa Cruz Biotechnology; California, USA). The membranes were then incubated with secondary antibodies conjugated to HRP (Santa Cruz Biotechnology; California, USA).
Immunodetection was performed by chemiluminescence (ECL, Millipore). Relative protein 9 levels were quantified and normalized to GAPDH protein levels.
Measurement of Cytokines Using Bio-Plex. Cytokines in BALF and blood sera from rats exposed to BMF and MVE were analyzed using a Bio-Plex system (Bio-Rad, CA, USA).