SI Materials and Methods

Materials and chemicals

The DEP and PM used in this work are the Standard Reference Material 2975 and

Standard Reference Material 1648a, respectively, from the National Institute of

Standards and Technology (NIST, USA). 1-nitropyrene, 1-nitrofluoranthene (BCR305),

naphthalene (91489), fluoranthene (F807) and phenanthrene (P11409) were purchased

from Sigma-Aldrich Co. LLC (USA). 6-hydroxynitropyrene (6-OHNP) and 8-

hydroxynitropyrene (8-OHNP) were synthesized as previously described (1).

Subject characterization

We recruited 600 healthy volunteers from 3 cities with discrepant outdoor air pollution

levels, including 100 male (mean age 65 years, range: 57-72 years) and 100 female

(mean age 66 years, range: 56-72 years) in Zhangjiakou; 100 male (mean age 64 years,

range: 57-71 years) and 100 female (mean age 64 years, range: 55-72 years) in Nanjing;

as well as 100 male (mean age 66 years, range: from 60-73 years) and 100 female (mean

age 64, range: 58-73) in Shijiazhuang. All volunteers were healthy non-smokers, free

of any diagnosis of respiratory diseases, and lived in urban communities. Blood and

mid-stream urine samples were collected between Dec.10th to Dec. 20th, 2014.

For the panel study, we recruited 30 male (mean age 64 years, range: 58-71 years) and

30 female (mean age 65 years, range: 57-73 years) retired COPD patients in Nanjing;

30 male (mean age 65 years, range: 59-72 years) and 30 female (mean age 64 years,

range: 57-71 years) retired COPD patients in Shijiazhuang. All subjects were living in

urban communities and had been diagnosed with mild-to-moderate COPD in Nanjing

Chest Hospital or the Second Hospital of Hebei Medical University according to the

classification of Global Initiative for Chronic Obstructive Lung Disease (GOLD). We

included the stable COPD patients and excluded those who were current active smokers,

www.pnas.org/cgi/doi/10.1073/pnas.2019025117 passive smokers (living with a current smoker), or had severe comorbidities or

inflammatory diseases. In addition, 60 healthy volunteers were recruited in each city

among the first batch of healthy volunteers. Blood and mid-stream urine samples of

COPD patients and healthy volunteers were collected in Jan. 20th and Jan. 28th, 2015 in

Nanjing or in Jan. 8th and Jan. 16th, 2015 in Shijiazhuang. The Institutional Review

Board in the School of Public Health at Southeast University approved the study protocol. Written consent forms were obtained from all subjects.

Spirometry Post-bronchodilator (BD) spirometry testing was performed immediately after blood

sampling with the EasyOne Spirometer Model 2001-2S (NDD Medical Technologies,

Switzerland). Data of forced expiratory volume in 1 second (FEV1) and Forced vital capacity (FVC) were collected.

Collection of particulate matter

The 120 COPD patients and 120 healthy individuals wore sampling equipment for 10 h /per day (from 9:00 am to 7:00 pm), from Jan. 21th to 27th, 2015 in Nanjing and from

Jan. 9th to 15th in Shijiazhuang. The sampling equipment consisted of AirChek 2000

pumps (SKC Inc, USA) with Triplex PM2.5 cyclones, which sampled at 1.5L/min through 37 mm Teflon filers. Filters were frozen until analysis.

Measurement of 1-NP and 1-NP metabolites

Half of PM filter were recovered in 10 ml acetone/hexane mixture (1:1) by microwave- assisted extraction. Then the extracts were analyzed using an Agilent 00A GC/MS

Triple Quadrupole System (7890A- 7000 series MS, Agilent Technologies Inc, USA) as described(2). The quantitative determination of 1-NP was performed in Multiple

Reaction Monitoring (MRM) mode. Metabolites of 1-NP, 6-OHNP, and 8-OHNP in urine samples were measured by HPLC tandem quadrupole MS/MS as described (1). Lentivirus transduction

C/EBPα, NDUFA1, NDUFA2, NDUFC2, NDUFS4, and ATP5H overexpression

lentiviruses, as well as C/EBPα shRNA, were generated by co-transfection with

packaging plasmids, pSPAX2 and pMD2G. The shRNA lentivirus harbored a short-

hairpin RNA sequence to target . The overexpression lentivirus harbored a target

coding sequence, which was tagged with c-Myc. Lentivirus (MOI = 30) was added

to the HBE cells, and subsequently treated with Blasticidin S for two weeks to obtain stable transduction HBE cells. For experiments, HBE cells were thawed and allowed to grow for three passages before use. The wild-type (WT) HBE cells and lentivirus stable transduction (LST) HBE cells were then treated with 50 μg/ml DEP for 24 h RNA and were collected for further analysis.

RNA microarray and analysis

HBE cells were seeded in 10 cm culture dishes and exposed to 50 μg/ml DEPs with

three biological replicates. Complete medium was removed after 24 h treatment.

Adherent cells were then collected. The total RNA was extracted using the TRIzol

reagent (Invitrogen, USA) according to the manufacturer’s instructions.

An Agilent Array platform (Agilent Technologies, Santa Clara, CA, USA) was

employed for microarray analysis of total RNA extracted from HBE cells as described

(3). An absolute fold change of 2 or more and 0.05 adjusted P-value were set as cut-off

to evaluate the significance of gene expression differences of raw data. Database for

Annotation, Visualization, and Integrated Discovery (DAVID 6.7) functional

annotation tool was used to analyze differentially expressed genes. The P-value was set

to 0.05 to denote the significance of GO enrichment in the differentially expressed

mRNA list. The pathway analysis for differentially expressed mRNAs was performed on the KEGG database (P-value was set as 0.05).

Cell growth assays

Cellular growth was evaluated using the Cell Counting Kit-8 (Nanjing Jiancheng

Bioengineering Institute, China). HBE cells were plated at a density of 1 × 104 per well

in a 96-well plate and treated with 0, 0.1, 1, 10 μg/ml 1-NP coupled with or without

C/EBPα lentivirus infection with 8 biological replicates for each concentration.

Accordingly, 10 μl of CCK-8 was added to each well. The cells were incubated for 4 h

at 37°C. Absorbance was determined at 450 nm. Cell viability affected by 1-NP was

monitored every 24 h up to 3 days.

Transmission electron microscopic observation

HBE cells were treated with 0 or 10 μg/1-N ml P for 24 h, then collected and fixed.

Ultra-thin sections were stained with uranyl acetate and lead citrate, and then observed

in a transmission electron microscope (JEOL-1010, Japan).

Metabolomics analysis

GC/TOFMS analysis was performed using an Agilent 7890 gas chromatography system

coupled with a Pegasus 4D time-of-flight mass spectrometer. HBE cells were exposed

to DEPs (0, 10, 20, and 50 μg/ml) for 24 h with 9 biological replicates. The cell lysate sample and post-exposure blood samples from 120 COPD patients and 120 healthy volunteers were then prepared and analyzed as previously described(4).

Animal experiments

Male C57BL/6 mice (23–25 g) were purchased from Vital River Laboratory Animal

Technology (China). Homozygous Atg7fl/fl mice on a C57BL/6 background were

generated by insertion of a loxP sequences within introns 13 and 14 of Atg7 gene. The conditional lung Atg7-/- were modeled as previously described(5). Briefly, 6-week-old mice were anesthetized and 50 μL Lenti-Cre virus (105 infectious particles) was administered dropwise into one nostril until the virus was completely inhaled. The intranasal delivery method was completed in 3-5 min per mouse.

The first batch of animal experiments included three groups (with ten C57BL/6 mice in each group): control with a sham treatment of 10 μL PBS; mice treated with a single exposure of 5 μg DEPs (10 μL DEPs at a concentration of 0.5 μg/μL); or mice exposed to 50 μg DEPs (10 μL DEPs with a concentration of 5 μg/μL) through intratracheal instilation. Mice were sacrificed on the 29th day of DEP administration.

A second batch of mice were divided into three groups (with ten C57BL/6 mice in each group): control with a sham treatment of 10 μL PBS; mice treated with a single exposure of 1 μg 1-NP (10 μL 1-NP with a concentration of 0.1 μg/μL); or mice exposed to 5 μg

1-NPs (10 μL 1-NPs with a concentration of 0.5 μg/μL) through intratracheal instilation.

Mice were sacrificed on the 29th day of 1-NP administration.

A third batch of mice was divided into four groups (with ten C57BL/6 mice in each group): control mice treated with control shRNA lentivirus in PBS; mice treated with single C/EBPα shRNA lentivirus in PBS; mice treated with single 5 μg/mouse 1-NP treatment; and mice treated with 5 μg/mouse 1-NP coupled with C/EBPα shRNA lentivirus. Mice received a single intranasal instilation with 1 × 108 TU/mouse one week before DEP exposure. A single dose of 5 μg 1-NPs was administered through intratracheal instillation. Mice were sacrificed on the 29th day of 1-NP administration.

A fourth batch of mice was divided into four groups (with ten C57BL/6 mice in each group): control mice treated with control vector lentivirus in PBS; mice treated with single C/EBPα vector lentivirus in PBS; mice treated with single 5 μg/mouse 1-NP treatment; and mice treated with 5 μg/mouse 1-NP coupled with C/EBPα vector lentivirus. Mice received a single intranasal instillation with 1 × 108 TU/mouse one week before 1-NP exposure. A single dose of 5 μg 1-NP was administered through intratracheal instillation. Mice were sacrificed on the 29th day of 1-NP administration.

A fifth batch of mice included four groups (with ten mice in each group): control wild type (WT) C57BL/6 mice (sham); 5 μg/mouse 1-NP treatment of WT mice; control

ATG7-knock out (KO) mice (sham); 5 μg/mouse 1-NP treatment of ATG7-KO mouse.

The mice were intratracheally instilled with a single dose of 5 μg 1-NP. Mice were sacrificed on the 29th day of 1-NP administration.

A sixth batch of mice was divided into eight groups (with ten mice in each group): control WT mice (sham); control ATG7-KO mice (sham); 5 μg/mouse 1-NP treated WT mice; 5 μg/mouse 1-NP treated ATG7-KO mice; 3-MA (15 mg/kg) + 1-NP (5 μg/mouse) treated WT mice; 3-MA (15 mg/kg) + 1-NP (5 μg/mouse) treated ATG7-KO mice; 1-

NP (5 μg/mouse) + 3-MA (15 mg/kg) + taurine (150 mg/Kg) treated WT mice; and 1-

NP (5 μg/mouse) + 3-MA (15 mg/kg) + taurine (150 mg/Kg) treated ATG7-KO mice.

The mice were administered a single dose of 5 μg 1-NP through intratracheal instillation.

3-MA and (or) taurine were injected intraperitoneally (I.P.) 30 min before the 1-NP administration. Mice were sacrificed on the 29th day of 1-NP administration.

A seventh batch of mice was divided into six groups (with ten mice in each group): control WT mice (sham); control ATG7-KO mice (sham); 50 μg/mouse DEP treated

WT mice; 50 μg/mouse DEP treated ATG7-KO mice; DEP (50 μg/mouse) + 3-MA (15 mg/kg) + taurine (150 mg/Kg) treated WT mice; and DEP (50 μg/mouse) + 3-MA (15 mg/kg) + taurine (150 mg/Kg) treated ATG7-KO mice. The mice were administered a single dose of 50 μg DEP through intratracheal instillation. 3-MA and taurine were injected intraperitoneally (I.P.) 30 min before the DEP administration. Mice were sacrificed on the 29th day of DEP administration.

An eighth batch of mice was divided into six groups (with ten mice in each group):

control WT mice (sham); control ATG7-KO mice (sham); 200 μg/mouse PM treated

WT mice; 200 μg/mouse PM treated ATG7-KO mice; PM (200 μg/mouse) + 3-MA (15

mg/kg) + taurine (150 mg/Kg) treated WT mice; and PM (200 μg/mouse) + 3-MA (15

mg/kg) + taurine (150 mg/Kg) treated ATG7-KO mice. The mice were administered a

single exposure of 200 μg PM through intratracheal instillation. 3-MA and (or)

taurine was injected intraperitoneally (I.P.) 30 min before the PM administration. Mice

were sacrificed on the 29th day of PM administration.

Mouse airway resistance measurement

The specific airway resistance (sRAW) was evaluated in conscious mice, and was measured using the FinePointe non-invasive airway mechanics (DIS Buxco, USA) on

the 28th day after the single dose of 1-NP, DEP or PM challenge. Each animal was

restrained in a special chamber which allowed for the independent measurement of nasal and thoracic flows. Each mouse was monitored for five consecutive minutes.

Bronchoalveolar lavage and histopathological analysis of mice lung tissue

Bronchoaleolar lavage of 4 mice in each group was harvested via the tracheal canula with 0.4 ml PBS × 3 times before sacrifice. Leukocytes were quantified with a

hemocytometer, and cell differentiation was performed by counting 200 total cells after

Wright-Giemsa stain. One piece of lung tissue from six mice within each group was collected and stored in liquid nitrogen for mRNA, ChIP and immunoblot assays.

Another piece of lung tissue was immediately prepared for ROS, MDA, and ATP level

analyses. Another piece of lung tissues were stored in PFA for 24 h at 4°C, embedded

in paraffin, serially sectioned (5 μm) and mounted on silane-covered slides. The

sections selected from each mouse were stained after dewaxing with hematoxylin and eosin (H&E) and evaluated under a light microscope (400×) to examine tissue histology.

The severity of alveolitis was scored as previously described (3). The mean linear

intercept (Lm) was quantified to characterize the enlargement of airspaces in

emphysema. Three random fields from eath section at ×10 magnification under microscopy were qualitifed by the indirect stereological methods(6). Sections were

stained with Masson’s trichrome following standard protocols.

After dewaxing, IHC staining was performed as previously described(3), and samples

were incubated overnight at 4°C with mouse monoclonal antibodies against ATG7

(1:100) (ab53255, abcam, USA), LC3B (1:100) (ab48394, abcam, USA),

NDUFA1(ab131423, abcam, USA), and NDUFA2 (1:100) (ab198196, abcam, USA).

Antibody binding to tissue sections was visualized with a biotinylated rabbit anti-mouse

IgG antibody (1:400; DAKO) and developed using diaminobenzidine (DAB) as a

substrate. For the negative controls, the primary antibodies were omitted. Each section

was examined under microscopy by two histologists. The score of IHC for each protein

were estimated by the percentage of positive cells (1: ≥0% and ≤25%; 2: >26% and

≤50%; 3: >50% and ≤75%; 4: >76% and ≤100%) × intensity of staining (0: negative;

1: weak; 2: moderate; 3: strong).

RNA isolation and quantitative real-time PCR assay

HBE cells were seeded in 6-well plates at a density of about 1 × 106 cells per well. The

cells were then exposed to 10, 20, or 50 μg/ml DEPs or control medium for 24 h, or

treated with 1 or 10 μg/ml 1-NP, 3-nitrofluoranthene, naphthalene, fuoranthene, phenanthrene, respectively, for 24 h. Subsequently, the cells were trypsinized and collected. Lung tissues stored in liquid nitrogen were homogenized in ice-cold 50 mM Tris–HCl buffer (pH 7.55). Then HBE cells were treated with control medium, 3-MA

(5 μM), 3-MA (5 μM) + taurine (25 mM), 1-NP (10 μg/ml), 1-NP (10 μg/ml) + 3-MA

(5 μM) or 1-NP (10 μg/ml) + 3-MA (5 μM) + taurine (25 mM). Accordingly, HBE cells

were pre-treated with 3-MA or taurine 1 h before 1-NP treatment. Complete medium

was removed after 24 h treatment. Adherent cells were then collected. The total RNA

was extracted using the TRIzol reagent (Invitrogen, USA) according to the

manufacturer’s instructions.

Total RNA of HBE cells and lung tissues was extracted using a GenElute™ Mammalian

Total RNA Miniprep Kit (Sigma, USA) according to the manufacturer’s protocol. The

mRNA levels for modulated genes were determined by reverse transcription of total

RNA followed by qRT-PCR on a Quant Studio 6 Flex System (Applied Biosystems,

Life Technologies, USA) using SYBR PCR Master Mix reagent kits (Takara, Japan) following the manufacturer’s protocol. Primers were designed for the modulated genes and are provided in Supplemental Tables S9 and S10. All experiments were performed in triplicate. The mRNA levels provided were normalized to cyclophilin A.

ChIP assay

ChIP was performed using the ChIP-IT™ Express Magnetic assay kit (Active Motif,

USA). Briefly, HBE cells were briefly fixed with 4% formaldehyde for 10 min. Mice

lung tissues were ground into powder in liquid nitrogen and fixed for 10 min at 37 °C

with 4% formaldehyde. Glycine was added after incubation to terminate reaction with

a final concentration of 125 mM. The samples were then centrifuged at 2,000 g for

2 min, and washed once with cold PBS plus protease inhibitors. Pellet was subsequently

collected. The ChIP reaction antibody was a normal rabbit IgG (NI01, EMD Chemicals,

Inc., Gibbstown, NJ) and an anti-C/EBP Alpha (1:500 dilution; ab40764, Abcam, USA). Precipitated genomic DNA was analyzed by quantitative PCR in triplicate

measurements for each sample using appropriate primers (Tables S11 and S12).

Autophagic flux

The Cyto-ID autophagy detection kit (Enzo, USA) was used to determine the presence

of autophagic vacuoles and monitor autophagic flux in HBE cells according to the

manufacturer’s instructions. After 24 h treatment, cells were collected for

centrifugation at 400×g for 5 min and washed once by PBS. The pellet was resuspended

in 500 μl of the Cyto-ID green detection reagent, incubated for 30 min in the dark at

37°C, and subsequently analyzed by a fluorescent microscope and flow cytometry.

ELISA

The levels of inflammatory cytokine in serum were measured using Human IL-6 ELISA

Kit (Multi Sciences, China). The levels of IL-6 in BALF were measured by a Mouse

IL-6 ELISA kit (Multi Sciences, China) according to the manufacturer’s instructions.

Each sample was assayed in triplicate.

Immunoblotting assay

Proteins were analyzed by immunoblotting with anti-C/EBP Alpha (1:500) (Abcam,

USA), anti--tubulin (1:10,000) (Sigma, USA), ATG7 (1:500) (abcam, USA), LC3B

(1:1000) (2775S, CST, USA) or anti-c-Myc (1:5,000 dilution; Santa Cruz

Biotechnology, USA) antibodies as indicated. References

1. Miller‐Schulze JP, et al. (2013) Evaluation of urinary metabolites of 1‐nitropyrene as biomarkers for exposure to diesel exhaust in taxi drivers of Shenyang, China. Journal of exposure science & environmental epidemiology 23(2):170‐175. 2. Tutino M, Di Gilio A, Laricchiuta A, Assennato G, & de Gennaro G (2016) An improved method to determine PM‐bound nitro‐PAHs in ambient air. Chemosphere 161:463‐469. 3. Li X, et al. (2016) An acetyl‐L‐carnitine switch on mitochondrial dysfunction and rescue in the metabolomics study on aluminum oxide nanoparticles. Part Fibre Toxicol 13:4. 4. Xu B, et al. (2014) Metabolomic profiles delineate the potential role of glycine in gold nanorod‐induced disruption of mitochondria and blood‐testis barrier factors in TM‐4 cells. Nanoscale 6(14):8265‐8273. 5. DuPage M, Dooley AL, & Jacks T (2009) Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase. Nature protocols 4(7):1064‐1072. 6. Knudsen L, Weibel ER, Gundersen HJ, Weinstein FV, & Ochs M (2010) Assessment of air space size characteristics by intercept (chord) measurement: an accurate and efficient stereological approach. J Appl Physiol (1985) 108(2):412‐421.

Figure legends

Fig. S1 DEP exposure triggers major mitochondrial damage.

(A) Volcano diagram depicting the differentially expressed genes in HBE cells exposed to DEPs. Each red point corresponds to one differentially expressed gene. (B) Cellular components (CC), (C) molecular functions (MF) and (D) Sub-ontology biological processes (BP) of down-regulated gene oncology enrichment, which showed that mitochondria-related functions are the mostly involved enrichment categories. The mitochondrial-related enrichments were labeled with red. (E) Down-regulated genes are mainly involved in oxidative phosphorylation KEGG pathways.

Fig. S2 DEP exposure inhibited mitochondrial-related gene expression levels.

(A) and (B) Gene expression levels in HBE exposed to DEPs (n=6). (C) and (D) Gene expression levels in lung tissues of mice exposed to DEPs (n=6). (E) and (F) Fold change of modulated genes in cells treated with 50 μg/ml DEPs compared to array data.

** P<0.01, *** P<0.001, compared with control.

Fig. S3 Pathologic alterations observed in DEP- treated mouse lung tissues.

(A) Representative image of mouse lung tissue in control group by H&E staining (100×,

scale bar equals to 200 μm). (B) Enlarged airspace in distal alveolar is observed in DEP-

treated mouse lung tissue (100×, scale bar equals to 200 μm). (C) Alveolitis is observed

in DEP-treated mouse lung tissue (100×, scale bar equals to 200 μm). (D) Score of Lm

and alveolitis of mouse lung tissue (airspace enlargement: n=18; alveolitis: n=6). (E)

Representative image of mouse lung tissue in control group by Masson’s staining (100×, scale bar equals to 200 μm). (F) Augment of collagen fiber is observed surround small airway in DEP-treated mouse lung tissue (100×, scale bar equals to 200 μm).

Fig. S4 1-NP is a predominant toxic component of DEPs.

(A) mRNA expression levels in HBE cells treated with PAHs or nitro-PAHs chemicals.

(n=6) (B) mRNA expression of five mitochondria-related genes in 1-NP-treated mouse lung tissues. (n=6) (C) Representative images of 1-NP-treated HBE cells by TEM. The normal morphology of mitochondria was observed in control group. Swollen mitochondria and autophagosome was observed in 1-NP-treated HBE cells and are shown by white or black arrows, respectively. M-Mitochondria, N-Nucleus, CY-

Cytoplasm, AP-autophagosome The right image of each group is magnified of the left one highlighted with black box).

Fig. S5 1-NP exposure increases inflammatory factor levels in COPD patients.

Associations between 1-NP metabolites and IL-6 levels of COPD patients exposed to

(A) light air pollution or (B) moderate air pollution levels were analyzed by Pearson correlation (n=60). No significant correlations of 6-OHNP and 8-OHNP levels in urine to IL-6 levels in serum were found in healthy subjects from (C) Nanjing city and (D)

Shijiazhuang city (n=60).

Fig. S6 Overexpression of C/EBPα rescue expression of target genes.

(A) The binding sites of C/EBPα in gene promoter region. ChIP assays suggested that the binding of C/EBPα to (B) NDUFA2, (C) NDUFC2, (D) NDUFS4 and (E) ATP5H was affected by 1-NP exposure.

Fig. S7 C/EBPα overexpression could partially rescue 1-NP-induced alveolitis in mouse lung tissues. (A) Representative images of H&E staining in mouse lung tissues (100×, scale bar

equals to 200 μm). (B) Histological score of alveolitis. *P < 0.05, ***P < 0.001, compared with control vector-treated group; #P < 0.05, compared with 1-NP and control

vector-treated group (n=6).

Fig. S8 Histological lesion of mouse lung tissue and cell viabilities following 1-NP

exposure.

(A) Representative images of H&E staining showed emphysema in mouse lung tissues

(100×, scale bar equals to 200 μm). (B) Lm of alveoli. *P < 0.05, ***P < 0.001, compared

with the control LTV-treated group (n=18). (C) Representative images of H&E staining

showed alveolitis in mouse lung tissues (100×, scale bar equals to 200 μm). (D)

Histological score of alveolitis. *P < 0.05, ***P < 0.001, compared with the control LTV-

treated group (n=6). (E) Cell growth of HBE cells was inhibited by 1-NP exposure and

rescued by C/EBPα overexpression. *P < 0.05, **P < 0.01, ***P < 0.001, compared with

control group of each day (n=8).

Fig. S9 Suppression of autophagy attenuates alveolitis in mice.

(A) Representative images of H&E staining in mouse lung tissue (100×, scale bar equals

to 200 μm). (B) Score of alveolitis. *P < 0.05, **P < 0.01, compared with WT control

group; #P < 0.05, compared with 1-NP-treated WT group (n=6)

Figure S10 Representative images of IHC staining in mouse lung tissues (400×, scale bar equals to 50 μm).

Fig. S11 Gene expression levels in HBE.

(A) 3-MA and taurine restore the collapse of MMP in 1-NP-treated HBE cells (200×).

(B) Taurine but not 3-MA rescued the mRNA expression of NDUFC2, NDUFS4 and ATP5H in HBE treated with 1-NP. ***P<0.001, compared with control. ###P<0.001,

compared with control within each group (n=6).

Fig. S12 Taurine and 3-MA ameliorate 1-NP-induced alveolitis.

(A) Representative images of H&E staining in mouse lung tissues (100×, scale bar

equals to 200 μm). (B) Score of alveolitis. *P < 0.05, **P < 0.01, compared with WT

control group (n=6).

Fig. S13 Gene expression levels in mouse lung tissues.

Taurine rescued the mRNA expression of (A) Ndufc2, (B) Ndufs4 and (C) Atp5h in lung

tissue of mouse treated with 1-NP **P<0.01, ***P<0.001, compared with WT control

(n=6).

Fig. S14 Taurine and 3-MA partially recuse PM-induced COPD-like lesions in

mice

(A) Representative images of H&E staining in DEP-treated mouse lung tissue (100×,

scale bar equals to 200 μm). (B) Histological scores of mouse lung tissues (n=18). (C)

Airway responsiveness of mice (n=6). (D) Number of inflammatory cells in BALF

(n=6). (E) Representative images of H&E staining in PM-treated mouse lung tissues

(100×, scale bar equals to 200 μm). (F) Histological scores of mouse lung tissues (n=18).

(G) Airway responsiveness of mice (n=6). (H) Number of inflammatory cells in BALF

(n=6). *P < 0.05, **P < 0.01, ***P < 0.001 compared with WT control. #P < 0.05, ##P <

0.01, compared with the WT within each group

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Table S1 KEGG terms of down regulated genes in HBE cells KEGG terms Genes NDUFA2, NDUFS4, COX7B, NDUFC2, NDUFA1, hsa00190:Oxidative phosphorylation COX17, ATP5H, UQCRHL HNRNPA3, U2AF2, SNRPD3, SYF2, MAGOHB, hsa03040:Spliceosome HSPA1A, HNRNPC, BUD31 NDUFA2, NDUFS4, COX7B, NDUFC2, NDUFA1, hsa05012:Parkinson's disease ATP5H, UQCRHL NDUFA2, NDUFS4, COX7B, NDUFC2, NDUFA1, hsa05010:Alzheimer's disease ATP5H, UQCRHL NDUFA2, NDUFS4, COX7B, NDUFC2, NDUFA1, hsa05016:Huntington's disease ATP5H, UQCRHL hsa04130:SNARE interactions in SNAP29, TSNARE1, VTI1A vesicular transport hsa03050:Proteasome PSMB6, SHFM1, PSMD7

Table S2 Modulated genes involved in mitochondrial functions in HBE cells Gene Main functions Adjusted P value Fold change Symbol NDUFA1 NADH dehydrogenase 1 alpha subcomplex subunit 1 3.93E-08 0.109347 NDUFA2 NADH dehydrogenase 1 alpha subcomplex subunit 2 3.08E-07 0.152849 isoform 1 NDUFS4 NADH dehydrogenase iron-sulfur protein 4, 3.38E-07 0.162564 mitochondrial precursor NDUFC2 NADH dehydrogenase [ubiquinone] 1 subunit C2 3.93E-08 0.06755 isoform 1 ATP5H ATP synthase subunit d, mitochondrial isoform b 3.70E-07 0.181061 NOXO1 NADPH oxidase organizer 1 isoform c 0.006617087 0.22359 COX18 mitochondrial inner membrane protein COX18 0.005612627 6.901499 PRDX2 peroxiredoxin-2 isoform a 0.011049891 0.216709 SH3GLB1 endophilin-B1 isoform 1 0.030137285 6.328394 UQCRHL ubiquinol-cytochrome c reductase hinge protein-like 1.91E-06 0.230638 RAC1 ras-related C3 botulinum toxin substrate 1 isoform 0.003592806 0.496794 Rac1b

Table S3 Mass fraction values for selected PAHs and Nitro-PAHs in SRM2975 Mass Fraction (mg/kg) Fluoranthene 30.9±0.5 Phenanthrene 20.7±0.3 Naphthalene 4.0±0.13 1-Nitropyrene 35.2±2.2 3-Nitrofluoranthene 3.8±0.24

Table S4 Urinary 1-NP metabolite concentrations in subjects exposed to different levels of air pollution (mean ± SD) Air pollution level Good Slightly polluted Lightly polluted Creatinine-adjusted 6-OHNP (pg/mg creatinine) Detectable rate 67% 73.5% 83% Geomean (95% CI) 1.676 2.017* 2.229 *** (1.538 to (1.851 to 2.197) (2.041 to 2.434) 1.827) Median 1.650 1.818 2.068 Range 0.738 to 5.613 0.822 to 5.800 0.799 to 10.44 Creatinine-adjusted 8-OHNP (pg/mg creatinine) Detectable rate 68% 69.5% 80.5% Geomean (95% CI) 1.413 1.829 ** 1.982 *** (1.284 to (1.641 to 2.038) (1.783 to 2.202) 1.554) Median 1.624 1.788 1.846 Range 0.446 to 5.242 0.467 to 7.750 0.464 to 9.333 *P<0.05, **P<0.01, ***P<0.001 compared with individual from city with good air quality (Kruskal- Wallis H test, n=200).

Table S5 API and air pollutants levels in cities during observation

Level of PM2.5 PM10 SO2 NO2 CO Date AQI pollution μg/m3 μg/m3 μg/m3 μg/m3 mg/m3 Nanjing Jan. 21th, 2015 159 Light 121 209 33 70 1.51 Jan. 22th, 2015 157 Light 118 166 35 58 1.64 Jan. 23th, 2015 165 Light 125 183 33 83 1.53 Jan. 24th, 2015 240 Moderate 189 275 46 118 2.06 Jan. 25th, 2015 234 Moderate 184 239 21 71 1.8 Jan. 26th, 2015 281 Heavy 230 282 18 60 2.04 Jan. 27th, 2015 111 Slight 79 101 12 32 0.85 Shijiazhuang Jan. 09th, 2015 253 Heavy 203 306 178 119 4.61 Jan. 10th, 2015 221 Moderate 178 265 167 110 4.45 Jan. 11th, 2015 125 Slight 94 150 105 75 2.37 Jan. 12th, 2015 133 Slight 100 148 104 67 2.19 Jan. 13th, 2015 243 Moderate 195 284 173 93 3.41 Jan. 14th, 2015 263 Heavy 212 303 148 101 3.43 Jan. 15th, 2015 358 Severe 310 418 144 112 5.48

Table S6 1-NP level and urinary 1-NP metabolite concentrations in subjects included in panel study (mean ± SD) Healthy subjects COPD patients Lightly polluted duration (Nanjing) Pre- Pre-exposure Post-exposure Post-exposure exposure IL-6 (pg/mL) 7.50±2.01 7.82±2.07 8.29±2.28 9.48±2.26*** Creatinine-adjusted 6-OHNP 1.60±0.74 1.97±0.70*** 1.52±0.53 2.06±0.61*** (pg/mg creatinine) Creatinine-adjusted 8-OHNP 1.39±0.76 1.58±0.57** 1.34±0.44 1.55±0.48** (pg/mg creatinine) 1-NP (ng/m3) 0.112±0.035 0.114±0.036 Moderately polluted duration (Shijiazhuang) Pre- Pre-exposure Post-exposure Post-exposure exposure IL-6 (pg/mL) 7.54±1.81 7.91±1.75 8.42±1.69 8.86±1.62** Creatinine-adjusted 6-OHNP 2.10±0.71 2.47±0.63* 2.23±0.82 2.69±1.03** (pg/mg creatinine) Creatinine-adjusted 8-OHNP 1.72±0.61 1.95±0.52** 1.89±0.84 2.08±0.87** (pg/mg creatinine) 1-NP (ng/m3) 0.135±0.040 0.135±0.038

*P<0.05, **P<0.01, ***P<0.001 compared with pre-exposure levels (Kruskal-Wallis H test, n=60)

Table S7 Significantly modulated metabolite in serum of COPD patients compared with that of healthy subjects (Wilcoxon signed-rank test, n=120)

Metabolite HMDB ID Regulation FC P value Dimethylbenzimidazole HMDB03701 Up 2.972 0.011 L-Malic acid HMDB00156 Down 0.668 0.022 Cyclic AMP HMDB00058 Down 0.663 0.008 L-3-Phenyllactic acid HMDB00748 Down 0.655 0.004 Pyridoxal 5'-phosphate HMDB01491 Down 0.639 0.001 (-)-Matairesinol HMDB35698 Down 0.604 0.009 L-Cysteine HMDB00574 Down 0.59 <0.001 3-Methyladenine HMDB11600 Down 0.586 0.003 Taurine HMDB00251 Down 0.585 0.02 Hypotaurine HMDB00965 Down 0.547 0.001 Inosinic acid HMDB00175 Down 0.539 <0.001

Table S8 Significantly modulated metabolites in HBE lysates after DEP treatment* DEP (μg/ml) 10 20 50 metabolite HMDB ID FC P value FC P value FC P value Taurine HMDB00251 0.6228 0.049 0.4244 0.013 0.3404 0.003 3-Methyladenine HMDB11600 0.6015 0.043 0.4949 0.017 0.4071 0.002 L-Aspartyl-L- HMDB00706 0.8497 0.046 2.0242 0.049 2.1975 0.0019 Phenylalanine *Kruskal-Wallis H test, n=9

Table S9 Primer sequences of human genes for RT-PCR assay

Forward Reverse NDUFA1 5’-GCGTACATCCACAGGTTCACT-3’ 5’-GCGCCTATCTCTTTCCATCAGA-3’ NDUFA2 5’-GCAGCAAGTCGAGGAGTCG-3’ 5’-CGTTTCTCAATGAAGTCCCTGA-3’ NDUFS4 5’-TGCTCGCAATAACATGCAGTC-3’ 5’-GATCAGCCGTTGATGCCCAA-3’ NDUFC2 5’-CGGCCTGATTGATAACCTAATCC-3’ 5’-AAGCTGGCGATGCAAACCA-3’ ATP5H 5’-GCTGGGCGAAAACTTGCTCTA-3’ 5’-CCAGTCGATAGCTGGTGGATT-3’ NOXO1 5’-CTGTTGGAAACCTATTCTCGGAG-3’ 5’-GGTGCGAAGAAGCCAGTGAT-3’ COX18 5’-GGGCAGCATTCTGCTCTCC-3’ 5’-CCCAACTGATTTGCACGAACT-3’ PRDX2 5’-GAAGCTGTCGGACTACAAAGG-3’ 5’-TCGGTGGGGCACACAAAAG-3’ RAC1 5’-ATGTCCGTGCAAAGTGGTATC-3’ 5’-CTCGGATCGCTTCGTCAAACA-3’ SH3GLB1 5’-CAGGAACAGCTTATGGTAATGCC-3’ 5’-GGCTGACGTTTGAATCAGTTCTC-3’ ATG7 5’-ATGATCCCTGTAACTTAGCCCA-3’ 5’-CACGGAAGCAAACAACTTCAAC-3’ ATG5 5’-AAAGATGTGCTTCGAGATGTGT-3’ 5’-CACTTTGTCAGTTACCAACGTCA-3’ ATG12 5’-CTGCTGGCGACACCAAGAAA-3’ 5’-CGTGTTCGCTCTACTGCCC-3’ LC3B 5’-AAGGCGCTTACAGCTCAATG-3’ 5’-CTGGGAGGCATAGACCATGT-3’ P62 5’-GCACCCCAATGTGATCTGC-3’ 5’-CGCTACACAAGTCGTAGTCTGG-3’ LAMP1 5’-TCTCAGTGAACTACGACACCA-3’ 5’-AGTGTATGTCCTCTTCCAAAAGC-3’ C/EBPα 5’- AACTCACCGCTCCAATGCC-3’ 5’- CCCTATGTTTCCACCCCTTTC-3’ CYPA 5’CCCACCGTGTTCTTCGACATT3’ 5’GGACCCGTATGCTTTAGGATGA3’

Table S10 Primer sequences of mouse genes for RT-PCR assay

Forward Reverse Ndufa2 5’-TTGCGTGAGATTCGCGTTCA-3’ 5’-ATTCGCGGATCAGAATGGGC-3’ Ndufs4 5’-CTGCCGTTTCCGTCTGTAGAG-3’ 5’-TGTTATTGCGAGCAGGAACAAA-3’ Ndufc2 5’-GGCCATGAGCCCTTAAAATTCT-3’ 5’-CCGTGCAGTAGCCCAACAA-3’ Ndufa1 5’-ATGTGGTTCGAGATTCTCCCT-3’ 5’-TGGTACTGAACACGAGCAACT-3’ Atp5h 5’-GCTGGGCGTAAACTTGCTCTA-3’ 5’-CAGACAGACTAGCCAACCTGG-3’ Noxo1 5’-CTTGGTGCAAATGGACCGACT-3’ 5’-CCAGCTCCTCCGCACAAAT-3’ Cox18 5’-CGAGTGGCTAGACTCACCTAT-3’ 5’-AAATGCCGTCTGAATGTGTGG-3’ Prdx2 5’-GGTAACGCGCAAATCGGAAAG-3’ 5’-TCCAGTGGGTAGAAAAAGAGGT-3’ Rac1 5’-GAGACGGAGCTGTTGGTAAAA-3’ 5’-ATAGGCCCAGATTCACTGGTT-3’ Sh3glb1 5’-AACCTCCTTAGCAAAGCTGAATG-3’ 5’-GGGTTGTTTATACGACTTGGTGC-3’ Atg7 5’-TCTGGGAAGCCATAAAGTCAGG-3’ 5’-GCGAAGGTCAGGAGCAGAA-3’ Atg5 5’-TGTGCTTCGAGATGTGTGGTT-3’ 5’-GTCAAATAGCTGACTCTTGGCAA-3’ Atg12 5’-TCCCCGGAACGAGGAACTC-3’ 5’-TTCGCTCCACAGCCCATTTC-3’ Lc3b 5’-TTATAGAGCGATACAAGGGGGAG-3’ 5’-CGCCGTCTGATTATCTTGATGAG-3’ P62 5’-AGGATGGGGACTTGGTTGC-3’ 5’-TCACAGATCACATTGGGGTGC-3’ Lamp1 5’-CAGCACTCTTTGAGGTGAAAAAC-3’ 5’-ACGATCTGAGAACCATTCGCA-3’ Cypa 5’GAGCTGTTTGCAGACAAAGTTC3’ 5’CCCTGGCACATGAATCCTGG3’

Table S11 Primer sequences of human genes for ChIP assay

Forward Reverse NDUFA1 5’-CTGATCCACCGCCTTTCCT-3’ 5’-CAGTCCTCTTCCGTCAGTGTCT-3’ NDUFA2 5’-GGGCTCAAGCGTCACACATA-3’ 5’-GACAGCGAAAGAACGATAACA-3’ NDUFC2 5’-CCAAGCTGGTCTTGAACTCCT-3’ 5’-ACACTCCCTCCAAAGCCTAAA-3’ NDUFS4 5’-CACCAGTCCTAACAAGCAGTAA-3’ 5’-TCCTTCGTTCTCAAGCCAGT-3’ 5’-GGAAGATCTTTCCTCAGCCAACC- ATP5H 5’-AGCTGCAGTTCCGCCATCTT-3’ 3’

Table S12 Primer sequences of mouse genes for ChIP assay

Forward Reverse Ndufa1 5’-CCCCGATAGATCTTGTCATCTTCC-3’ 5’-GGTTCATTCTCGTCCTCGCT-3’ Ndufc2 5’-GTTTCCTAGCCTTTGCCCAC-3’ 5’-CTTCTAAGTGGCCCACAACG-3’ Ndufa2 5’-TCGCGAAATGTCCCCCTAAC-3’ 5’-CGATTGGATGACGCAGGGAA-3’ Ndufs4 5’-GGGCTTCTCAGCCTCAATGT-3’ 5’-AGGGACTCACTTCCTCCGTT-3’ Atp5h 5’-GACTCCATGCTCATTGGCTG-3’ 5’-GTTGCCGGAAGTTCTCTCCT-3’