Methods in the present study have been reported previously [21, 22] but are restated herein for reader accessibility.
2.1. Ambient PM2.5 Collection
Ambient winter PM2.5 collection occurred in Sacramento, California; Jinan, Shandong; and Taiyuan, Shanxi. The California sampling site was located in downtown Sacramento, at the northeast corner of T and 13th streets, on the rooftop of a two-story building (N38°34’, W121°29’). Downtown Jinan’s collection site was located on the rooftop of a three-story primary school, Wang She Ren (N36°40’, E117°09’). The sampling site in downtown Taiyuan was located on the rooftop of the five-story College of Environmental Science and Resources building at Shanxi University (N37°47’, E112°34’). All three sampling locations are proximal to major freeways and surrounded by a combination of residential, commercial, and industrial sites.
In Sacramento, a high-volume sampler system (Tisch Environmental Inc., TE‑6070V2.5-HVS) was used to collect PM2.5 for seven days. The system had a PM2.5 size-selective head (Tisch Environmental Inc., TE-6001) that ran at a flow rate of 40 cubic feet per minute (cfm), and was fitted with Teflon-coated borosilicate glass microfiber filters (Pall Corporation, TX40H120WW). The filters were pre-cleaned by repeated sonication in a mixture of Milli-Q water, dichloromethane, and hexane prior to sampling 
In Jinan and Taiyuan, PM2.5 was collected for one day using a high-volume particle collector (Thermo Anderson, HVAIR100) operated at a flow rate of 40 cfm. The shorter collection times in China were due to higher density and heavier loading of PM relative to the sampling site in California. The sampler was fitted with a PM2.5 size-selective opening and 90 mm diameter quartz microfiber filters (Whatman, WHA1851090). Twenty-four hours before collection, the quartz filters were pre-heated at 450°C for the removal of any potential endotoxin from the filters.
2.2. PM Sample Extraction
Following sample collection in Sacramento and Jinan, the filters were post-weighed to calculate the collected PM mass (post weight – pre weight = PM mass), placed in Milli-Q water, and sonicated for 1 hour to obtain PM extracts. The sonicated PM extracts were passed through 0.2 µm pore size syringe filters, and the collected solution (approximately 100 mL) was lyophilized and stored at ‑80°C. A more detailed description of PM extraction methods is provided by Bein and Wexler .
Filters from Taiyuan were post-weighed to calculate the collected PM mass, cut into segments for placement in a 250 mL conical flask with 30 mL Milli-Q water, and sonicated for 30 minutes (3 cycles of 10 minutes each) to produce a PM extract. The extract was filtered through six layers of sterile gauze, lyophilized to powder, and stored at -80°C.
Later, the frozen, lyophilized PM extracts from Sacramento, Jinan, and Taiyuan were weighed, resuspended in Milli-Q water, and sonicated for 20 minutes to derive PM extract samples (PMCA, PMSD, and PMSX, respectively) of the same concentration (1 μg/ μL; Figure 1). All PM samples were frozen at -20°C until needed.
2.3. Chemical Characterization of Stock Extract Samples via High-Resolution Aerosol Mass Spectrometry (HR-AMS)
Chemical characterization was performed at the University of California, Davis. Frozen stock PM extracts were thawed to 4°C, sonicated for 20 minutes for thorough PM dispersal, and diluted by Milli-Q water to 0.1 μg/μL. Subsequently, 1 mL of each diluted stock sample was individually passed through a constant output aerosol generator with an inert argon carrier gas, and a silica gel diffuser where the sample became atomized. HR-AMS was used to determine the chemical composition of each extract sample. Chemical species such as nitrate, sulfate, chloride, ammonium, and organic compounds were quantified . The organic compounds were fragmented and further analyzed to determine their elemental composition (as regards hydrogen, carbon, oxygen, and nitrogen) and average degree of oxidation as detailed by Aiken et al. , Ghio et al. , Sun et al. , and Zhang et al. .
2.4. Animals and Exposure
The UC Davis Institutional Animal Care and Use Committee approved of all animal procedures and housing practices. Sixty-three male, 6-week old BALB/C mice were purchased (Envigo, Hayward, CA) and randomly assigned to one of four groups exposed to 1) vehicle control (Milli-Q water; n=9), 2) PMCA (n=18), 3) PMSD (n=18); or 4) PMSX (n=18). All mice were acclimated for 2 weeks prior to the start of the exposure period, maintained on a 12-hour light/dark cycle, and housed three per cage with sterile laboratory bedding and ad libitum access to food and water.
Immediately before exposure, the stock extracts of PMCA, PMSD, and PMSX were defrost, sonicated for 20 minutes and diluted by Milli-Q water to a concentration of 20 µg/50 µL. Single oropharyngeal aspiration (OPA) exposures occurred five times, once per day on 1, 4, 7, 10, and 14 days (Figure 2). During exposure, the mice were sedated by inhaling of isoflurane with oxygen at a 3:1 ratio . Each mouse was given 50 µL of Milli-Q water, PMCA, PMSD, or PMSX via OPA such that the exposure volume was consistent across all groups as was each single PM mass (20 µg) for all PM-exposed groups. A single dose of this nature has been used previously  to examine PM effects. With high ambient PM levels noted in each of the sampled cities (in particular China), a total PM dose of 100 μg administered over a two-week period could approach levels experienced by inhalation of ambient air over this same period of time.
2.5. Bronchoalveolar Lavage Fluid (BALF)
Necropsies and biological sample collections were completed on Days 1, 2, and 4 after the final OPA exposure (15, 16 and 18 days, respectively). On each of these days, mice were randomly selected from each group (n=3 controls, and 6/PM group), weighed, and euthanized for necropsy with a 0.2 mL intraperitoneal injection of Beuthanasia-D pentobarbital solution (65 mg/kg body weight; Nembutal, Cardinal Health, Sacramento, CA). Each mouse was cannulated intratracheally, and the left mainstem airway was clamped while the right lung was lavaged with two aliquots (0.6 mL/aliquot) of Dulbecco’s Phosphate Buffered Saline (PBS; Sigma-Aldrich, St Louis, MO). The collected BALF was centrifuged at 2000 ´ g at 4°C for 15 minutes to pellet the cells. BALF supernatant was decanted, frozen in liquid nitrogen, and stored at -80°C. Cell pellets were resuspended in 500 mL PBS for determination of total cell numbers and viability using a hemocytometer and 0.4% Trypan Blue solution for viability detection (Sigma-Aldrich).
BALF cytospin slides were prepared using a Shandon Cytospin (Thermo Shandon, Inc., Pittsburg, PA). The slides were then stained with DippKwik Differential Stain (American MasterTech, Lodi, CA) to determine the proportions of BAL macrophages, neutrophils, eosinophils, and lymphocytes in cell counts (500 cells/animal) performed using Brightfield microscopy. The experimental protocol is shown in Figure 3.
2.6. Lung Collection
The left lungs were inflated-fixed at 30 cm of pressure for one hour, stored in 4% paraformaldehyde for 48 hours, and subsequently transferred into 70% ethanol for later tissue processing for histopathology. The right lung lobes of each mouse were placed in two cryovials—one vial for the cranial and middle lobes, and the other for the caudal and accessory lobes. All vials were stored at -80°C until further use.
2.7. Semi-Quantitative Lung Histopathology
Following placement of the left lung in 70% ethanol, four transverse slices (levels) were prepared as a method to uniformly sample histological changes throughout the entire lobe. These tissue slices were dehydrated, embedded in paraffin, sectioned at a thickness of 5 µm, placed on glass slides, stained with Harris hematoxylin and eosin (H&E; American MasterTech, Lodi, CA), and cover slipped.
Four transverse slices were stained for each mouse (n=9/control or 18/PM group). All slides were examined independently by two blinded observers (WY and SCV) for the presence of inflammation, cellular infiltrates, and cellular/tissue remodeling (i.e. desquamation and squamous metaplasia of airway epithelial cells, septal wall thickening) in alveolar ducts and airways using a semi-quantitative scoring scale. Previously published rubrics  were used to rank the severity (absent to marked; 0-3) and extent (0: no changes, 1: less than one-third of the slide, 2: one-half of the slide, 3: two-thirds of the slide) of the observed remodeling and inflammation. For each mouse, the final score for a given parameter (e.g. alveolitis) was the averaged product of the severity and extent scores for the four transverse slices. Use of the products resulted in scores ranging from 0-9 thus increasing the probability of finding significant (p < 0.05) differences between groups . Detailed semi-quantitative scoring guidelines are shown in supplemental Table S1.
2.8. Gene Expression Analysis
Ribonucleic acid (RNA) was isolated from the right caudal and accessory lung lobes using TRI Reagent (Sigma-Aldrich) and Quick-RNA Miniprep kit (Zymo Research, Irvine, CA). RNA was converted to copy deoxyribonucleic acid (cDNA) using a high-capacity cDNA Reverse Transcription Kit (Applied Biosystem’s, Indianapolis, IN). Gene-specific mouse primers (0.2 μM; IDT, Coralville, IA), cDNA (2 μL/reaction), and SYBR Green (Applied Biosystem) DNA-binding stain were used for quantitative polymerase chain reaction (qPCR) measurements. Expression of genes for inflammatory cytokines, interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α), and neutrophil chemokines, chemokine (C-X-C Motif) ligands-3 and -5 (CXCL-3 and CXCL-5) were examined. Expression was assessed using the ΔΔ-Ct method and standardized to the expression of elongation factor 1-alpha 1 (EEF1a1) housekeeping genes . Mouse gene primers were designed using Primer3 primer design software . Primers used in this study are detailed in Supplemental Table S2.
2.9. Enzyme-Linked Immunosorbent Assay (ELISA)
ELISAs (Biolegend, San Diego, CA) were performed on the homogenized cranial and middle lobes of the right lung to analyze concentrations of specific proteins including TNF-a; monocyte chemoattractant protein-1 (MCP-1), a chemoattractant responsible for monocyte and macrophage recruitment; CXCL-1, seen in inflammation or wound healing; and IL-1b and IL-6, both mediators of inflammatory responses. The cranial and middle lobes from control- and PM-exposed animals and standards from the R&D Systems ELISA kits (1000 μg/mL to 7.8 μg/mL) were prepared and examined in duplicate in 96-well plates using a SpectroMax plate reader (Molecular Devices, Sunnyvale, CA). Duplicate readings were averaged. All concentrations were normalized to total lung protein and reported in pg of specific protein per mg of lung tissue.
2.10. Statistical analysis
No data points were excluded prior to statistical analysis. All statistical tests were performed using GraphPad PRISM 8.0 software. A value of p < 0.05 was considered statistically significant. For each measured endpoint, Shapiro-Wilk tests were first used to detect normality, a one-way analysis of variance (ANOVA) and post hoc Tukey’s test were performed to determine differences due to treatment. All data in the present document are expressed as the mean ± standard error of the mean (SEM).