Pulmonary Health Effects Following Repeated Exposure and Cessation to Wintertime Particulate Matter from California and China

Background: Epidemiological studies show a strong association between fine particulate matter (PM 2.5 ) air pollution and adverse pulmonary effects. While PM concentration can vary by time and location, PM toxicity has been most recently linked to both physicochemical composition and exposure scenario. To study the relevance of particle characteristics to toxicity, winter PM 2.5 samples were obtained from three geographically similar regions (Sacramento, California, USA; Jinan, Shandong, China; and Taiyuan, Shanxi, China), with typically high atmospheric PM 2.5 emissions. PM extract samples (PM CA , PM SD , and PM SX , respectively) were administered by oropharyngeal aspiration (OPA) to different groups of BALB/C mice, at equal mass concentrations [0 (water vehicle control only) or 20 µg/50 µL], on five different occasions over a two-week period, for a cumulative PM dose of 0 or 100 µg/mouse. Mice were necropsied on Days 1, 2 and 4 after the final exposure, and pulmonary effects were evaluated by bronchoalveolar lavage (BAL), histopathology, quantitative polymerase chain reaction tests, and enzyme-linked immunosorbent assays. Results: Unique differences were noted in the chemical composition for each geographic region with PM SX containing the highest concentration of sulfates (organic and inorganic). A systematic examination of the time lag effects of repeated PM exposure demonstrated unique differences. In mice administered PM SX versus the control, BAL neutrophilia, alveolitis, and bronchiolitis were observed on Days 1 and 4. By Day 4, PM SX -exposed mice also exhibited increased gene expression for multiple inflammatory cytokines/chemokines (interleukin 1 beta, tumor necrosis factor alpha, chemokine C-X-C motif ligands-3 and -5), and increased levels of monocyte chemoattractant protein-1 relative to control-, PM CA -, or PM SD -exposed mice . Conclusions: Direct comparison of the toxic effects of three geographically different PM

eosinophils, and lymphocytes in cell counts (500 cells/animal) performed using Brightfield microscopy. The experimental protocol is shown in Figure 3.

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.

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 [27] 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 [28]. Detailed semi-quantitative scoring guidelines are shown in supplemental Table S1.
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 [29]. Mouse gene primers were designed using Primer3 primer design software [30]. 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.

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).

Chemical Composition of PM Extracts
Organic compounds comprised the largest fraction of the total mass in each of the extract samples accounting for 54%, 57%, and 45% in PM CA , PM SD , and PM SX , respectively ( Fig. 4A-C). Within the organic fraction, carbon and oxygen accounted for at least 87% of the mass (Fig. 4D-F). Of the other compounds measured by HRAMS, nitrate and sulfate fractions were highly variable while those of chloride and ammonium were relatively similar among the California and China PM extracts. Nitrate was more abundant in California PM compared to China PM, while sulfate was most prevalent in PM SX versus the other two PM extracts ( Fig. 4A-C). The sulfate and nitrate measured in all three PM samples were principally inorganic, identified by the presence of anions in both the ammonium sulfate and ammonium nitrate. However, based on ion balance analysis, a significant fraction of the sulfate in PM SX was found to be organic.

Cell Differentials in BALF
Recovery time did not appear to play a role in the responses observed in BALF as there were no statistically significant differences between necropsy days (Fig. 5). No significant (p < 0.05) exposure-related differences were noted for total cell counts of mice exposed to vehicle control versus PM CA , PM SD , or PM SX irrespective of the recovery time post-OPA ( Fig. 5A). In contrast, significantly increased neutrophil numbers were observed on post-OPA Days 1 and 4 in all PM-exposed groups relative to controls (p < 0.05 for all comparisons), and on Day 4 in PM SX -exposed mice relative to their PM CA -and PM SDexposed counterparts (p = 0.0326 and p = 0.0196, respectively; Fig. 5B). Interestingly, no statistically significant treatment-related effects were observed on Day 2 (Fig. 5B), as neutrophil numbers in all PM-exposed groups dropped temporarily to near-control levels. Figure 5. Oropharyngeal aspiration of regional particulate matter (PM) extracts produced acute and subacute neutrophilia in mice up to four days post exposure. Animals were exposed on five separate days over a two-week period to Milli-Q water (control; n = 9), or a PM extract (20 µg/50µL) from Sacramento, CA; Jinan, Shandong; or Taiyuan, Shanxi (PM CA , PM SD , or PM SX , respectively). Each mouse received a 50 µL aspirate on the exposure days (total PM dose = 0 or 100 µg/mouse). Mice from each group were then necropsied on Day 1, 2 or 4 after the last exposure. Graphs show counts of total bronchoalveolar lavage fluid (BALF) cells (A) and neutrophils (B) collected at different post-exposure time-points. Total cell and neutrophil data were analyzed separately via one-way ANOVA tests and are presented as the mean ± standard error of the mean. * indicates a significant (p < 0.05) difference from control. Brackets indicate significant (p < 0.05) differences between PM-exposed groups.

Histological analysis
There were no significant (p < 0.05) differences in bronchiolar inflammation scores on post-OPA Day 1 (Fig. 6A). However, on Day 2, PM SX− exposed mice exhibited significantly (p = 0.0179) more bronchiolar inflammation than controls (Fig. 6A). By Day 4, both groups given Chinese PM extracts exhibited more severe bronchiolitis than controls (p = 0.0399 for PM SD ; p = 0.0027 for PM SX ; Fig. 6A), with neutrophilic influxes into the peribronchiolar and alveolar regions of the lungs that were not observed in other groups (Figs. 7A-D). Mice exposed to PM SX were found to have visible black particles (likely due to coal combustion) in macrophages throughout the alveolar regions of the lungs.
In contrast to the bronchioles, perivascular and subpleural regions, the alveoli appeared to be most affected by PM. On post-OPA Days 1 and 2, significantly greater alveolitis was observed in groups administered PM SD ( p = 0.0206 and 0.0361, respectively) or PM SX ( p = 0.0057 and 0.0172, respectively) versus vehicle control (Fig. 6B). Over these two days, no significant intra-group differences in alveolitis were observed within the PM SD or PM SX groups. By Day 4, all three PM-exposed groups exhibited higher scores for alveolitis than controls ( Fig. 6B; p = 0.0012, p = 0.0043, and p = 0.0001 for PM CA , PM SD , and PM SX , respectively). PM SD -and PM SX -exposed mice had average scores for alveolitis of 1.8 and 2.4, respectively, with notable changes in alveolar wall thickening, and numerous free macrophages distributed throughout the parenchyma (Fig. 8A-D), while control mice had an average score of 0.7, with thin-walled alveolar septa and relatively few lumenal macrophages (Figs. 6B and 8). Although inter-group differences were not statistically significant among the PM-exposed mice, those administered PM SX appeared to demonstrate the greatest effects (Figs. 8A-D) with cellular debris and numerous foamy, particle-laden macrophages aggregated in the alveolar airspaces (Figs. 8D).    Day 4, IL-1β in PM CA -exposed mice relative to controls (p = 0.0049) and in PM SX -exposed mice relative to their PM SD -exposed counterparts (p = 0.0152; Figs. 9A-B). Gene expression of CXCL-3 and CXCL-5 were also significantly elevated in PM-versus control-  (n = 18/group). OPA occurred on five separate days over a two-week period yielding a total PM dose of 0 or 100 µg/mouse. Gene levels were analyzed for each animal and averaged for each treatment group. Gene expression is shown relative to the housekeeping gene, EEf1a1. Data are shown as the mean ± SEM. One-way ANOVA and Tukey tests were performed at a significance level of p < 0.05. * indicates a significant (p < 0.05) difference from vehicle control; brackets indicate significant (p < 0.05) differences between noncontrol groups. Expression of CXCL-3 and CXCL5 genes (A and B, respectively) was measured for each animal, averaged for each treatment group, and assessed relative to the housekeeping gene, EEf1a1. One-way ANOVA and Tukey tests were performed at a significance level of p < 0.05. Resulting data are shown as the mean ± SEM. * indicates a significant (p < 0.05) difference from sham control; brackets indicate significant (p < 0.05) differences between non-control groups.

Quantification of Cytokines by ELISA
Although six cytokines were examined, concentrations of three proteins (TNF-α, CXCL-1, and IL-6) were too low to quantify reliably with limits of detection at 5.16, 66.521, 8.301 ng/mL, respectively. Of the two remaining cytokines, IL-1β (data not shown) and MCP-1 (Fig. 11), only protein levels of the latter varied significantly among the PM-and control-exposed groups, with PM SX producing increases relative to control on Days 2 and 4 (p = 0.0306 and p < 0.0001, respectively); and PM CA and PM SD producing similar increases on Day 18 alone (p = 0.0037 and p = 0.0437, respectively).

DISCUSSION
Global air pollution in the form of PM emissions is of major concern. The associated effects can be seasonal with sustained levels of elevated ambient particles over time, especially during the winter season. PM exposures can arise from a multitude of sources and range from short-term to prolonged and/or intermittent. Various sizes of PM can affect children, the elderly, and those with pre-existing health issues. However, long-term exposure to PM 2.5 is most associated with morbidity and premature mortality [32], and has been described as one of the leading risk factors for premature mortality contributing to 800,000 premature deaths each year [1].
Several reviews have concluded that chemical PM components can play an important role in post-exposure health effects [33,34]. Therefore, to enact the proper exposure controls We report herein that repeated PM SX exposure produced higher BALF neutrophil numbers than PM CA and PM SD on post-OPA Day 4 (Fig. 5B), and higher (p < 0.05) bronchiolar and alveolar inflammation scores relative to controls on more days than PM CA and PM SD ( Fig. 6A-B). Repeated exposure to PM can lead to an increase in neutrophils found in BAL because neutrophil influx is causally associated to lung injury [31,36], with the relative number of neutrophils entering the lungs typically reflective of the severity of the biological response. Neutrophils are the first non-resident cells to be recruited to the site of inflammation. For this study, neutrophil influx occurs by egress of these cells from the capillaries of the airways, and transport through the epithelium into the airway lumen and alveoli [37]. This was confirmed in our study by the histological evidence of neutrophils in the pulmonary interstitium and among epithelial cells (Fig. 7).
Compared to control-, PM CA -and PM SD -exposed mouse groups, mice exposed to PM SX also exhibited significantly increased mRNA for TNF-α and IL-1β, cytokines involved in pulmonary inflammatory reactions [38], at Days 2 and 4 (Figs. 9A-B); and CXCL-3 and CXCL-5 neutrophil chemokines [39,40] at Day 4 (Figs. 10A-B). MCP-1 protein was increased in all PM-exposed groups relative to controls at Day 4, but PM SX produced the longest-lasting response that was also stronger than that for PM SD (Fig. 11). MCP-1, is a chemoattractant responsible for monocyte and macrophage recruitment. However, growing evidence shows MCP-1 may also be involved in attracting neutrophils [41]. Therefore, increased MCP-1 protein levels could have contributed to the BALF neutrophilia observed in PM-exposed mice (Figs. 5 and 11).
Although statistically significant differences were only occasionally observed between PM SX -and other PM-exposed groups, PM SX exposure most frequently produced differences relative to control. Cumulatively, BALF neutrophil and lung histopathology results suggested the chemical composition of PM SX may be contributing to its seemingly greater toxicity relative to PM CA and PM SD on an equal PM mass basis. Although we were unable to definitively identify the chemical constituent(s) that produced the observed biological effects, we believe organosulfates and polyaromatic hydrocarbons (PAHs) likely contributed to the differential induction effects observed.
The three PM extracts tested in the present study had similar chemical compositions, with ≤ 10% difference in the fractions of organic compounds, chloride, and ammonium.
However, a major difference was observed when comparing sulfate levels, which measured at 2%, 14%, and 26% in PM CA , PM SD , and PM SX , respectively (Figs. 4A-C). Several studies indicated sulfate-associated particles (i.e., fossil fuel combustion products) are among the most toxic in terms of effects on annual cardiopulmonary and cardiovascular diseases as well as lung cancer mortality [33,42]; and sulfate has been associated with increased percentages of BALF neutrophils [33].
Particulate air pollution-characterized by high secondary aerosol concentrations including organosulfates-has been a serious environmental problem during recent winters in China [43,44]. Therefore, higher fractions of organosulfates are plausible in PM SX and PM SD relative to PM CA . While in the present study sulfates were not speciated, and inorganic sulfate is likely quite innocuous, future exposure studies should quantify organic and inorganic sulfates to better understand the health risks they pose.
In addition to organosulfates, based on the source, PM may also be composed of polyaromatic hydrocarbons (PAHs). PAHs have received considerable attention due to their potential toxic, carcinogenic, and mutagenic effects, and coal combustion is known to produce PAHs [45]. Coal consumption in China in 2012 reached 2.75 billion tons of standard coal, approximately one-half of the global coal consumption, and Shanxi province is one of the largest coal production centers in China [46]. A study by Zhang et al. Although animals in the present study were not exposed by inhalation, OPA is the most effective method to simulate inhalation of equal mass dose to compare PM extracts from diverse geographic locations. Findings from the present study provide further evidence for

Conclusions
In summary, the results of this study suggest when evaluating PM CA , PM SD and PM SX samples on an equal mass basis, elevated sulfate-associated chemical composition may be an important factor in PM toxicity. Following repeated exposure over a two-week period, mice exposed to PM SX demonstrated the greatest inflammatory response found in the increases of pulmonary neutrophil numbers, pro-inflammatory cytokines and chemokines levels that is thought to be due to its highest sulfate than other PMs. Furthermore, exposure to all three PM samples produced greater toxicity at post-OPA day 4, compared exposure to air pollution and stroke: systematic review and meta-analysis. BMJ.      Oropharyngeal aspiration of regional particulate matter (PM) extracts produced acute and subacute neutrophilia in mice up to four days post exposure. Animals were exposed on five separate days over a two-week period to Milli-Q water Total cell and neutrophil data were analyzed separately via one-way ANOVA tests and are presented as the mean ± standard error of the mean. * indicates a significant (p < 0.05) difference from control. Brackets indicate significant (p < 0.05) differences between PM-exposed groups.     genes (A and B, respectively) was measured for each animal, averaged for each treatment group, and assessed relative to the housekeeping gene, EEf1a1. Oneway ANOVA and Tukey tests were performed at a significance level of p < 0.05.
Resulting data are shown as the mean ± SEM. * indicates a significant (p <0.05) difference from sham control; brackets indicate significant (p < 0.05) differences between non-control groups.

Figure 11
Mice exhibited subacute increases in Monocyte Chemoattractant Protein (MCP)-1 levels following exposure to particulate matter (PM) extracts from California or China. The bar graph shows the result from enzyme-linked immunosorbent assays performed on lung tissues of mice culled on 1, 2 or 4 day(s) after the last 50 µL oropharyngeal aspiration (OPA) exposure to Milli Q water (vehicle control; n=9), or a PM extract (20 μg/50μL) from Sacramento, CA; Jinan, Shandong; or Taiyuan, Shanxi (n=18/group). OPA occurred on five separate days over a two-week period yielding a total PM dose = 0 or 100 µg/mouse. Separate ANOVAs were performed for each time-point to determine inter-group differences due to exposure. * indicates a significant (p < 0.05) difference from control; brackets indicate significant (p < 0.05) differences between non-control groups.

Supplementary Files
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