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Fine Particulate Air Pollution and the Expression of microRNAs and Circulating Cytokines Relevant to Inflammation, Coagulation, and Vasoconstriction.

Introduction

Cardiovascular disease (CVD) hospitalization and mortality attributable to fine particulate matter (P[M.sub.2.5]) air pollution continues to be one of the greatest public health concerns worldwide because of ubiquitous exposure, particularly in developing countries (GBD 2015 Risk Factors Collaborators 2016; Yang et al. 2013). However, biological mechanisms that link P[M.sub.2.5] exposures with CVD events remain to be fully elucidated. It is widely accepted that PM air pollution plays a causal role in CVDs, including stroke, hypertension, heart failure, acute myocardial infarction, coronary heart disease, and atherosclerosis (Brook et al. 2010), and it has been proposed that inflammatory responses to P[M.sub.2.5] contribute to these effects (Franklin et al. 2015). Consistent with this mechanism, a recent panel study of healthy adult nonsmokers reported that serum concentrations of pro-inflammatory cytokines that may contribute to systemic inflammation, platelet activation, and endothelial dysfunction were elevated in association with ambient P[M.sub.2.5] concentrations during the previous 24 h (Pope et al. 2016).

In recent decades, the field of epigenetics has attracted increasing interest because it can serve as a bridge between environmental exposures and health-relevant events (Vrijens et al. 2015). As a main epigenetic regulatory mechanism, microRNAs (miRNAs) have been proposed to play a crucial role in regulating cellular events (Marques-Rocha et al. 2015). miRNAs comprise a large family of small noncoding RNA molecules, which function in messenger RNA (mRNA) silencing and gene expression control at the posttranscriptional level. In most cases, miRNAs lead to the inhibition of protein synthesis either by degrading mRNA transcripts or by repressing the translational process (Sevignani et al. 2006). It has been suggested that miRNA dysregulation may be involved in the development of a wide range of CVDs (Maegdefessel 2014).

Exposure to air pollutants has the potential to change the profiles of miRNAs (Jardim 2011; Rider et al. 2016). However, there is far less available evidence on miRNAs than on clinical or subclinical outcomes. In a recent systematic review, Vrijens et al. (2015) noted that although miRNA expression has been associated with air pollution exposures, findings for specific miRNAs have been inconsistent with regard to the direction of associations and among in vitro, in vivo, and human studies. Furthermore, few studies have explored the role of miRNAs as potential mediators of biological pathways linking air pollutants and CVDs (Motta et al. 2016).

We hypothesized that an acute change in P[M.sub.2.5] exposure would alter the expression of candidate miRNAs in peripheral blood. We further speculated that these miRNA alterations might mediate the expression (mRNA and protein) of cytokines relevant to systemic inflammation, coagulation, and vasoconstriction. To facilitate this investigation, we designed a randomized crossover trial with a panel of healthy young adults in which ambient P[M.sub.2.5] was naturally inhaled.

Materials and Methods

Study Design and Setting

We designed a double-blind, randomized crossover trial with two distinctly different scenarios of P[M.sub.2.5] exposure, which were established by the alternative use of true and sham air purifiers in Shanghai, China, where particulate air pollution is severe compared with developed countries. We recruited 60 healthy students from 17 dormitory rooms at the Jiangwan campus of Fudan University. Smokers and those living with smokers were excluded from enrollment. These rooms were randomized into two groups (8 and 9 rooms per group, respectively). From November 2015 to December 2015, one group used a true air purifier placed in the center of the room for 9 consecutive days (two weekends and five weekdays), followed by a washout period, and then used a sham air purifier under the same conditions for another 9 consecutive days (two weekends and five weekdays). The other group received the same intervention at the same time but in reverse order. A 12-d washout period was selected because we believed that it would provide sufficient time to eliminate carry-over effects from the first intervention because previous epidemiological studies have suggested that the duration of acute air-pollutant effects is <1 wk (Chen et al. 2015b; Wang et al. 2015). This period was also short enough to reduce the likelihood of confounding by time-varying factors that might influence cytokine or miRNA expression. All participants and research staff were blinded to the group assignment. To ensure blinding, the study participants were not informed that this was a crossover study; in addition, a note reading "Do not open or move" was affixed to all purifiers.

All air purifiers were identical (model KJEA200E; 3M, Shanghai, China) except for removal of the filter gauze (i.e., the high efficiency particulate air filter) in the sham group. The purifier has an airflow volume of up to <301 [m.sup.3]/h and an effective area of 20 [m.sup.2]. All rooms were distributed among three adjacent dormitory buildings. The room areas were <20 [m.sup.2]; the rooms did not have carpets and were equipped with simple wooden furniture (beds, desks, and chairs) that had been used for [greater than or equal to] 5 y. Inhabitants were asked to keep doors and windows closed during the study period so that air exchange rates would be similar across all rooms. To reduce indoor air pollution sources, all dormitory rooms were cleaned by sweeping/mopping, without the use of cleaning agents, before the onset of each intervention stage. Inhabitants were not allowed to clean or cook in the rooms during the entire study period. Therefore, similar indoor PM levels and compositions were expected for the same group at the same stage. Participants were allowed to leave their dormitories to attend classes and to go to the cafeteria but were required to stay in their rooms [greater than or equal to] 12 hours per day on weekdays and [greater than or equal to] 19 hours per day on weekends during each intervention period.

Our study protocol was approved by the Institutional Review Board of the School of Public Health, Fudan University, and was registered at ClinicalTrials.gov (NCT02712333). All participants provided written informed consent at enrollment.

Exposure Measurements

We calculated time-weighted personal P[M.sub.2.5] exposures for each subject over each intervention period. Specifically, we requested each subject to record their time-location information every half-hour. We used P[M.sub.2.5] measurements (per half-hour) monitored at the rooftop of the campus main building as surrogates of the exposure levels for all time spent away from the dormitory room within the campus, regardless of whether participants were inside a building. We also obtained hourly P[M.sub.2.5] data from 13 fixed-site monitoring stations across Shanghai for approximating an occasional exposure outside the campus (for example, for an internship or field work) in terms of proximity. Finally, we calculated individual exposure to P[M.sub.2.5] during each intervention period by mapping the time and location data to the corresponding P[M.sub.2.5] monitoring data. Indoor P[M.sub.2.5] was measured using MicroPEM Personal Exposure Monitors (RTI International), which can record data-logged real-time P[M.sub.2.5] concentrations in 10-s cycles. Outdoor P[M.sub.2.5] on the campus was monitored using a GRIMM EDM180 environmental dust monitor (GRIMM Aerosol Technik Ainring GmBH & Co.). To ensure the comparability of P[M.sub.2.5] data measured by the MicroPEMs and the GRIMM EDM180, parallel calibrations were performed before the start of our project.

We calculated time-weighted personal daily levels of temperature and relative humidity during each intervention period using indoor measurements obtained from a HOBO data logger (Onset Computer Corporation) in each dormitory room and ambient measurements obtained from a monitor at a fixed site of the Shanghai Meteorological Bureau.

Cytokines Test

Fasting venous peripheral blood samples (two samples from each participant) were drawn at the same time of day by a certified nurse immediately after completion of each intervention stage. The blood samples were collected at the same time of day to reduce variation due to circadian rhythms. Serum was extracted within 1 h (after centrifugation) from one of the two samples, transported directly to our laboratory, and stored at -80[degrees]C. The second blood sample was prepared for RNA analysis as described below. We measured blood levels of 10 cytokines including 6 pro-inflammatory cytokines [interleukin-1 (IL-1; gene name: IL1), interleukin-6 (IL-6; gene name: IL6), tumor necrosis factor-[alpha] (TNF-[alpha]; gene name: TNF), toll-like receptor-2 (TLR-2; gene name: TLR2), CD40 ligand (CD40L; gene name: CD40LG), and intercellular adhesion molecule-1 (ICAM-1; gene name: ICAM1)], two procoagulant cytokines [tissue factor (F3; gene name: F3) and plasminogen activator inhibitor-1 (PAI-1; gene name: SERPINE1)], and two vasoconstrictors [endothelin-1 (ET-1; gene name: EDN1) and angiotensin converting enzyme-1 (ACE-1; gene name: ACE)]. These cytokines were selected because they were associated with particulate air pollution in at least two previous studies of Shanghai residents or university students performed by our group (Chen et al. 2015a, 2015b; Wang et al. 2015, 2016). We measured these biomarkers using enzyme-linked immunosorbent assays (ELISAs). All tests were performed according to the manufacturer's instructions (see Table S1 for the manufacturers and the limits of detection (LODs) of each assay kit).

RNA Analyses

The second blood sample collected after each intervention was used for RNA analyses. Total RNA was extracted using a QIAamp RNA Blood Mini kit (Qiagen) immediately (within 2 h) after the blood samples were drawn. All extractions were performed according to the manufacturer' s protocols. Isolated RNA samples were stored at -80[degrees] C. We measured mRNA expression for each of the 10 cytokines listed above. In addition, we selected up to two miRNAs for each cytokine based on information supporting the cytokine as a "primary target" of the miRNA(s), including "strong evidence" (reporter assay, Western blot, or qPCR) in the miRTarBase database (http:// mirtarbase.mbc.nctu.edu.tw/php/search.php#target), validation of the miRNA-mRNA relationship in the EXIQON database (https://www.exiqon.com/miRSearch), or a high score ([greater than or equal to] 95) for the miRNA-mRNA relationship in the miRDB database (http:// www.mirdb.org/miRDB/). miRNAs related to a cytokine in more than one database were preferentially selected over other miRNAs for that cytokine. Each cytokine gene (mRNA) was related to 1-2 miRNAs, and each of the selected miRNAs was related to 1-3 cytokines (see Table S2).

Complementary DNA was synthesized using total RNA. Briefly, reverse transcription (RT) reactions were performed in a volume of 20 [micro]L containing total RNA (250 ng), dNTPs (2.5mM each, 2 [micro]L), 10xRT buffer (2 [micro]L), random primers (1[micro]M, 1 [micro]L), MMLV reverse transcriptase (Promega; 10 U/[micro]L, 2 [micro]L), OligodT primer (0.5 [micro]L), RNAase inhibitor (40 U/[micro]L, 0.3 [micro]L) and RNase-free water ([less than or equal to] 20 [micro]L). The RT reaction was conducted at 16[degrees]C for 30 min, followed by 40 min at 42[degrees]C and 5 min at 85[degrees]C. Then, the products were stored at -20[degrees]C for later use. The RT mixtures were then subjected to real-time PCR reactions, which were performed using the Applied Biosystems[R] ViiA[TM] 7 Real Time PCR System (Thermo Fisher Scientific). Primers were designed with AlleleID 7 software (PREMIER Biosoft). For each sample, triplicate measurements were performed on 384-well reaction plates. The data were analyzed using ViiA7[TM] Software v1.2 (Thermo Fisher Scientific) with the automatic Cq setting. For both mRNA and miRNA analyses, real-time PCR was performed using TaqMan miRNA assay hydrolysis probes and Gene Expression Real-Time PCR Master Mix (both from Applied Biosystems, Thermo Fisher Scientific). A [DELTA]Cq value in a random sample was used as a calibrator, and the [DELTA][DELTA]Cq was calculated using the [DELTA]Cq of each sample minus the calibrator. The relative quantity (RQ) of each sample was calculated using the standard relative quantification formula (RQ = [2.sup.-[DELTA][DELTA]Cq]).

Statistical Analysis

Measured values for protein expression (cytokines), mRNAs and miRNAs were natural-log transformed for statistical analyses (Fossati et al. 2014). We also calculated the time-weighted daily levels of P[M.sub.2.5], temperature, and relative humidity during each intervention period for all subjects according to their self-administered time-location questionnaires. We used linear mixed-effect models with random-effect intercepts for each subject to estimate effects of the air purification intervention (dichotomized as "true" or "sham") and continuous P[M.sub.2.5] exposures (time-weighted averages based on indoor and outdoor exposures for each participant) on each outcome, accounting for the repeated measures for each participant (Chen et al. 2015a, 2016). All models were adjusted for age (in years), gender, body mass index (continuous), and daily average temperature and relative humidity (continuous) during each intervention period.

Furthermore, we conducted a mediation analysis to estimate the proportion of the association between P[M.sub.2.5] and cytokine expression (mRNAs or cytokines) mediated by the association between P[M.sub.2.5] and miRNA expression (Chen et al. 2016; Imai et al. 2010; Wang et al. 2016). This approach decomposes the total estimated effect of P[M.sub.2.5] on cytokine expression into an estimate of the direct effect and an estimate of the indirect effect of P[M.sub.2.5] on cytokine expression mediated by miRNA. To allow for a comparable analysis, we first standardized the exposure (P[M.sub.2.5]), the mediator (miRNA), and the outcome (cytokine or mRNA) by subtracting the mean and dividing by the standard deviation (Bind et al. 2016). Estimates of the proportion of the effect of P[M.sub.2.5] on cytokine expression mediated by miRNA were generated using the "mediation" (version 4.4.6) package in R (version 3.1.2; R Foundation for Statistical Computing), with uncertainty estimated using the quasi-Bayesian Monte Carlo method with 1,000 simulations based on normal approximation (Imai et al. 2010). Because the assumptions required for causal interpretation were not specifically evaluated and P[M.sub.2.5] exposure was not randomized by the crossover design, we performed mediation analyses using the repeated-measures data. The mediation analyses were conducted in conjunction with the aforementioned linear mixed-effect models. We did not evaluate the possible exposure-mediator interactions because of the small sample size and the lack of sufficient variation in P[M.sub.2.5] concentrations in each interventional scenario. We report the results of mediation analyses only when there was a consistent negative direction in the pathway of P[M.sub.2.5]--miRNA--mRNA or of P[M.sub.2.5]--miRNA--cytokine because the negative regulation of miRNAs has been widely hypothesized.

In addition, we examined whether there were carryover effects by comparing the median responses of the two treatment orders (true-to-sham air purification vs. sham-to-true air purification) by incorporating a dummy variable of order as a fixed-effect term (i.e., "1" for the true-to-sham order, and "0" for the sham-to-true order) in the mixed-effect models with P[M.sub.2.5]. The period effects were similarly assessed by adding a fixed-effect term of period (i.e., "1" for the first stage, and "0" for the second stage) in the models. Finally, an intervention x period interaction term was evaluated in the models.

All statistical analyses were performed in R (version 3.1.2; R Foundation for Statistical Computing). Effect estimates are reported as percentage differences [with 95% confidence intervals (CIs)] in outcomes associated with the air purification intervention or as interquartile range (IQR) increases in time-weighted daily average P[M.sub.2.5] concentrations. In all analyses, the Benjamini-Hochberg false discovery rate (FDR) method was used to account for multiple testing with FDRs <0.05 and <0.10 considered as "significant" and "borderline significant," respectively.

Results

Descriptive Statistics

Five participants dropped out of the study before the second intervention was completed, leaving 55 in the final analysis (28 males and 27 females), with a mean age of 20 y (range 18-22 y) and an average body mass index of 21kg/[m.sup.2] (range 17-28). According to the self-administered questionnaires, participants spent approximately 75% of the time on average inside their dormitories during the two intervention scenarios (see Table 1), and no participants left the urban areas of Shanghai during the whole study period. Table 1 also shows 82% (on average for all rooms combined) lower P[M.sub.2.5] concentrations during true purification than during sham purification; accordingly, the time-weighted personal P[M.sub.2.5] exposure was 54% lower during true purification. P[M.sub.2.5] exposures only varied slightly among participants in the same interventional scenarios. Temperature and relative humidity were quite similar between the two interventional scenarios and did not differ much among subjects in the same interventional scenarios.

Table 2 summarizes the descriptive statistics for miRNAs, mRNAs and cytokines. There were no missing data and no data below the LODs. In general, we observed higher values for miRNAs and lower values for mRNAs and cytokines (in geometric means) in the true-purification scenario than in the shampurification scenario.

Regression Results

As shown in Table 3, compared with the sham intervention, the true purification intervention was associated with significantly lower expression of IL1 mRNA and protein and significantly higher expression of the single miRNA predicted for IL-1 (miR-21-5p); with significantly lower TNF mRNA (but not TNF-[alpha] protein) and significantly higher expression of the miRNA predicted for this cytokine (miR-187-3p); with significantly lower TLR2 mRNA (but not TLR-2 protein), and significantly higher expression of one of the two miRNAs predicted for this cytokine (miR-146a-5p); and with significantly lower EDN1 mRNA and ET-1 protein and significantly higher expression of both miRNAs predicted for this cytokine (miR-1-3p and miR-199a-5p). Finally, the intervention was associated with significantly lower IL6 mRNA (but not IL-6 protein) but was not significantly associated with expression of the miRNA predicted for this cytokine (miR-26a-5p); similarly, the intervention was associated with significantly lower expression of F3 protein but was not associated with F3 mRNA or with the expression of either miRNA predicted for this gene (miR-19b-3p and miR-93-5p).

Patterns of associations with an IQR increase in time-weighted P[M.sub.2.5] exposure (28[micro]g/[m.sup.3]) were very consistent with those for the filter intervention, with inverse associations with predicted miRNAs (Table 4) and positive associations with mRNA and protein (Table 5) as exposure increased. IQR increases in personal continuous P[M.sub.2.5] exposures (time-weighted) were associated with lower estimated mean expression of 8 of the 10 miRNAs and with higher estimated mean values for all of the mRNAs and cytokines. Specifically, an IQR increase in P[M.sub.2.5] was associated with significantly higher expression of mRNA and protein for IL1, IL6 (FDR 0.10 for protein), F3, and EDN1; and with significantly lower expression of the same miRNAs associated with the intervention (miR-21-5p for IL1, miR-1-3p for EDN1). Similarly, an IQR increase in P[M.sub.2.5] was associated with significantly higher TNF mRNA (but not TNF-[alpha] protein) and significantly lower expression of the predicted miRNA (miR-187-3p) for this cytokine; and with higher TLR2 mRNA (FDR 0.053) (but not TLR-2 protein) and significantly lower expression of one of the two miRNAs for this cytokine (miR-146a-5p). miR 146a-5p was also predicted as a miRNA for CD40LG, which had a borderline increase in protein (FDR 0.07) but not mRNA expression (FDR 0.17) in association with P[M.sub.2.5]. Unlike the finding for the filter intervention, F3 mRNA expression was significantly increased in association with P[M.sub.2.5]. However, consistent with the findings for the filter intervention, F3 protein was significantly higher in association with P[M.sub.2.5], and neither of the predicted miRNAs for F3 was associated with exposure.

Estimates of the proportion of associations between P[M.sub.2.5] and mRNA or protein expression mediated by predicted miRNAs for each cytokine varied greatly, and none was significant at FDR <0.05 (Table 6). We estimated that miR-187-3p mediated 25% (95% CI: 6%, 43%), and that miR-146a-5p mediated 20% (95% CI: 2%, 38%), of the association between P[M.sub.2.5] and TNF mRNA (FDR 0.08 and 0.15, respectively); that miR-1-3p mediated 22% (95% CI: 5%, 39%) of the association with EDN mRNA (FDR = 0.08); that miR-199a-5p mediated 21% (95% CI: 5%, 38%) of the association with ET-1 protein (FDR = 0.13); and that miR-21-5p mediated 41% (95% CI: 6%, 75%) of the association with IL-1 protein (FDR = 0.13). Otherwise, estimated mediation proportions were close to the null, imprecise, or both.

In the mixed-effect models with P[M.sub.2.5] and various biomarkers, the dummy terms for order (i.e., "1" for the true-to-sham order and "0" for the sham-to-true order), period (i.e., "1" for the first stage and "0" for the second stage), and the intervention X period interaction were not significant at the 0.05 FDR level (see Table S3).

Discussion

We performed a randomized crossover study comparing miRNA and cytokine expression following an air-filtration intervention to reduce indoor P[M.sub.2.5] and a sham intervention; we also estimated associations between the outcomes and time-weighted P[M.sub.2.5] exposure estimates that accounted for both ambient and indoor P[M.sub.2.5] concentrations during the two intervention periods. The results of both analyses suggested that P[M.sub.2.5] exposure increased the expression of cytokines involved in inflammation, coagulation, and endothelial dysfunction while decreasing the expression of miRNAs that may regulate expression of these cytokines. Collectively, these results provide preliminary evidence supporting our hypothesis that short-term exposure to P[M.sub.2.5] may contribute to the pathogenesis of CVD, at least in part, by affecting the expression of circulating miRNAs.

Our findings from a randomized crossover study were consistent with those of previous epidemiological studies showing that circulating cytokines were sensitive to a short-term exposure to PM (Chen et al. 2015a, 2015b; Wang et al. 2015; Wu et al. 2012). Our results suggest that P[M.sub.2.5] might contribute to the pathophysiology of cardiovascular diseases by increasing the expression of cytokines that induce systemic inflammation (IL-1), coagulation (F3), or vasoconstriction or endothelial dysfunction (ET-1) (Franklin et al. 2015). In addition to finding evidence consistent with effects on protein transcription, we also found associations between P[M.sub.2.5] and mRNA expression for IL-1, F3, ET-1, IL-6, TNF, and TLR-2.

The mechanism by which P[M.sub.2.5] may induce cytokine expression has not been fully characterized (Marques-Rocha et al. 2015). miRNAs have emerged as important regulators in gene expression, and miRNA expression is crucial to the regulation of cellular processes. However, findings from studies of miRNA expression and air pollution exposure have been inconsistent (Jardim 2011). For example, in a recent systematic review, Vrijens et al. (2015) noted that findings for specific miRNAs from in vitro, in vivo, and human studies varied with regard to both the presence and direction of associations with air pollutants.

We utilized three online databases to identify miRNAs likely to target the cytokines of interest. However, different tools did not always predict the same miRNAs for each cytokine, reinforcing the fact that the linkages between the selected miRNAs and cytokines are not specific and are not known with certainty. Expression of eight of the 10 miRNAs measured in our study population was higher following the filter versus the sham intervention and lower in association with an IQR increase in time-weighted P[M.sub.2.5] exposure. Our findings of inverse associations between P[M.sub.2.5] and miR-21-5p, miR-1-3p, and miR-146a-5p were consistent with findings from the Boston Area Normative Aging Study (Fossati et al. 2014). In contrast, PM total mass was not associated with miR-21 or miR-146 in foundry workers, although some metal PM components were inversely associated with miR-146 measured by real-time PCR (Bollati et al. 2010). miR-146a expression was significantly up-regulated in post-exposure versus baseline samples from the same population based on a microarray analysis of 847 human miRNAs (Motta et al. 2013). In addition, miR-21 expression was higher following exposure to diesel exhaust (vs. filtered air) in a controlled-exposure crossover study of 14 asthma patients (Yamamoto et al. 2013). Inconsistent findings among studies of PM and miRNA expression may reflect differences in PM characteristics, study participants, or study designs, although random error and noncausal explanations must also be considered. Nevertheless, although this field is still in its preliminary stages, our findings and those of previous investigators merit further investigation and confirmation.

Altered miRNA expression may trigger a cascade of cytokines involved in the complex biological processes that contribute to cardiovascular diseases (Motta et al. 2013), but our understanding of the role of miRNAs in complex molecular networks is limited. Consistent with our a priori hypothesis, we found preliminary evidence supporting positive mediation of the effects of P[M.sub.2.5] at both the transcriptional (mRNA) and translational (protein expression) levels by miRNAs. However, the validity of the estimates from the mediation analysis depends on several unverifiable assumptions, including the absence of uncontrolled confounding of associations between the exposure and the outcomes, between the exposure and the mediator, and between the mediator and the outcomes. In particular, we note that although the filter intervention was randomized, the time-weighted P[M.sub.2.5] exposures used as the basis of the mediation analysis were not. In addition, the analysis was further complicated by the fact that each miRNA can target multiple mRNAs/proteins, and a single mRNA/ protein is often the target of multiple miRNAs (Motta et al. 2013).

Our study had several strengths. First, we enrolled healthy students living in university dormitories to reduce potential confounding from indoor air pollution, smoking, alcohol drinking, medication use, dietary structure, time-activity patterns, sociodemographic characteristics, and socioeconomic status (Chen et al. 2015a, 2016). Second, this interventional crossover design with true and sham air purifiers created large self-contrasts in P[M.sub.2.5] exposures that are common in developing countries, making it easier to capture potential molecular changes in response to P[M.sub.2.5] (Chen et al. 2016). Third, circulating cytokines, mRNAs, and miRNAs were evaluated simultaneously in our analysis, allowing for a preliminary assessment of the potential mediation or modulation of miRNAs in the cardiovascular effects of P[M.sub.2.5].

Some limitations should also be noted. First, power and precision were somewhat limited because of the small sample size. Second, studying young, healthy subjects and special exposure scenarios reduced the potential for uncontrolled confounding but may limit generalizability to other populations and contexts. Third, we cannot assume that estimates from our mediation analyses represent causal mediation of an effect of P[M.sub.2.5] by miRNAs because it is not possible to confirm underlying assumptions required for this to be true, including the absence of some uncontrolled confounding of associations between P[M.sub.2.5] and miRNAs, between miRNAs and the outcomes, and between P[M.sub.2.5] and the outcomes (Valente et al. 2017). Fourth, experimental control was limited because students left their dormitory rooms to attend classes and to go to the cafeteria, and the intervention was therefore not the sole influence on P[M.sub.2.5] exposures during the sham or the filter intervention periods. We also estimated the effects of time-weighted P[M.sub.2.5] exposures that accounted for ambient P[M.sub.2.5] concentrations and time-activity patterns, but these exposures were not randomized, and some degree of exposure misclassification and confounding was likely. Fifth, we did not have data on the proportions of different leukocyte subpopulations in the participants' blood samples, which may influence both miRNA and cytokine expression (Fossati et al. 2014) and may vary over short periods of time. Therefore, we cannot rule out the possibility of uncontrolled bias from this source of variation. Finally, we measured P[M.sub.2.5] only and cannot rule out the possibility that larger or smaller PM might have been responsible for the estimated associations with P[M.sub.2.5].

Conclusion

In summary, we performed a randomized crossover trial of a sham versus true air filtration intervention and estimated associations with time-weighted P[M.sub.2.5] exposures (indoor and outdoor exposures) in healthy university students in Shanghai, China. We identified several miRNAs that were inversely associated with P[M.sub.2.5] exposure and found that the expression of several cytokines (mRNA and/or protein) predicted to be targets of the same miRNAs was positively associated with P[M.sub.2.5] exposure. These findings need to be confirmed in other populations and experimental models, but they provide preliminary evidence that acute responses and pathophysiologic mechanisms that may link P[M.sub.2.5] exposure with cardiovascular disease, including systemic inflammation and endothelial dysfunction, may be partly mediated by effects of P[M.sub.2.5] on miRNA expression.

Acknowledgments

This work was funded by the National Natural Science Foundation of China (91643205), the Public Welfare Research Program of the National Health and Family Planning Commission of China (201502003), the Shanghai 3-Year Public Health Action Plan (GWTD2015S04 and 15GWZK0202), and the Research Program of the Shanghai Environmental Protection Bureau (2016-11).

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Renjie Chen, (1,2,3) *, Huichu Li, (1,2) *, Jing Cai, (1,2,3) *, Cuicui Wang, (1,2) Zhijing Lin, (1,2) Cong Liu, (1,2) Yue Niu, (1,2) Zhuohui Zhao, (1,2) Weihua Li, (4) and Haidong Kan (1,2,4)

(1) School of Public Health, Key Lab of Public Health Safety of the Ministry of Education, Fudan University, Shanghai, China

(2) School of Public Health, Key Lab of Health Technology Assessment of the Ministry of Health, Fudan University, Shanghai, China

(3) Shanghai Key Laboratory of Meteorology and Health, Shanghai, China

(4) Key Laboratory of Reproduction Regulation of National Population and Family Planning Commission, Shanghai Institute of Planned Parenthood Research, Institute of Reproduction and Development, Fudan University, Shanghai, China

* These authors contributed equally to this work.

Address correspondence to H. Kan, Department of Environmental Health, School of Public Health, Fudan University, P.O. Box 249, 130 Dong-An Road, Shanghai 200032, China. Email: kanh@fudan.edu.cn

Supplemental Material is available online (https://doi.org/10.1289/EHP1447).

The authors declare they have no actual or potential competing financial interests.

Received 1 December 2016; Revised 28 November 2017; Accepted 30 November 2017; Published 17 January 2018.

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Table 1. Descriptive statistics of daily average environmental data in
sham-purified air and true-purified air scenarios for all subjects
during the two intervention scenarios.

                                            Sham-purified air

Variables                       Minimum   Mean   Median   SD    Maximum

P[M.sub.2.5] in dormitories      33.5     46.8    46.5    9.1    69.5
  ([micro]g/[m.sup.3])
Time in dormitories (%)          54.4     74.9    76.4    8.3    90.5
PM25 ([mirco]g/[m.sup.3]) (a)    36.3     53.1    53.2    9.4    79.5
Temperature ([degrees]C) (a)     15.9     19.9    19.8    1.7    23.4
Relative humidity (%) (a)        47.3     64.3    65.1    7.4    77.9

                                            True-purified air

Variables                       Minimum   Mean   Median   SD    Maximum

P[M.sub.2.5] in dormitories       6.6     8.6     9.9     4.0    17.1
  ([micro]g/[m.sup.3])
Time in dormitories (%)          56.1     75.5    75.7    7.0    90.0
PM25 ([mirco]g/[m.sup.3]) (a)    12.8     24.3    24.4    5.3    37.3
Temperature ([degrees]C) (a)     16.1     20.2    21.0    2.1    23.4
Relative humidity (%) (a)        47.3     64.6    66.0    7.8    74.1

Note: P[M.sub.2.5], particulate matter with an aerodynamic diameter
<2.5 [micro]m; SD, standard deviation.

(a) Data were calculated as time-weighted personal exposures by
mapping the time and location data to the corresponding monitoring
data.

Table 2. Descriptive statistics of miRNAs, mRNAs, and cytokines in
sham-purified air and true-purified air scenarios for all subjects
during the two intervention stages.

                                      Sham-purified air

                                      Mean
Variables           Minimum    P25     (a)     P50     P75    Maximum

miRNAs (b)
  miR-21-5p          0.02     0.10    0.32    0.34    0.95     4.11
  miR-26a-5p         0.06     0.13    0.24    0.21    0.34     2.28
  miR-187-3p         0.01     0.02    0.05    0.03    0.09     0.49
  miR-146a-5p        0.01     0.04    0.08    0.08    0.16     1.45
  miR-19b-3p         0.04     0.08    1.61    5.59    19.64    51.85
  miR-93-5p          0.06     0.14    0.33    0.38    0.72     2.89
  miR-145-5p         0.01     0.02    0.08    0.07    0.25     1.14
  miR-1-3p           0.01     0.02    0.07    0.06    0.23     1.88
  miR-199a-5p        0.01     0.03    0.04    0.03    0.06     0.12
  miR-4b492          0.01     0.03    0.10    0.15    0.37     12.37
mRNAs (b)
  IL1                0.06     0.19    0.43    0.41    0.64     22.26
  IL6                0.22     1.88    2.92    2.72    4.45     67.97
  TNF                0.17     0.39    0.62    0.50    0.86     11.61
  TLR2               0.09     0.15    0.28    0.23    0.39     6.39
  CD40LG             0.07     0.52    0.83    0.75    1.17     19.05
  ICAM1              0.03     0.12    0.21    0.19    0.33     5.40
  F3                 0.08     0.25    0.51    0.39    0.62     30.10
  SERPINE1           0.28     0.54    1.12    1.12    1.67     24.62
  EDN1               0.16     0.92    1.30    1.52    2.03     9.20
  ACE                0.09     0.51    0.88    0.86    1.10     32.24
Cytokines
  IL-1 (ng/mL)         7       13      23      19      48       94
  IL-6 (ng/mL)         1       49      88      116     203      623
  TNF-[alpha]         15       43      57      55      76       149
    (ng/mL)
  TLR-2 (ng/mL)       0.2      0.5     0.7     0.7     1.0      2.7
  CD40L (pg/mL)       84       130     184     202     235      935
  ICAM-1 (ng/mL)       4        5       7       7       8       27
  F3 (pg/mL)          37       53      82      68      113      394
  PAI-1 (ng/mL)       60       105     120     124     146      209
  ET-1 (pg/mL)         2        5       8       7      12       186
  ACE-1 (ng/mL)       93       151     182     187     223      428

                                      True-purified air

                                              Mean
Variables           Minimum    P25     P50     (a)     P75    Maximum

miRNAs (b)
  miR-21-5p          0.05     0.30    0.66    0.79    1.63     7.86
  miR-26a-5p         0.02     0.12    0.28    0.37    0.55     8.02
  miR-187-3p         0.01     0.03    0.13    0.14    0.28     7.27
  miR-146a-5p        0.01     0.06    0.20    0.18    0.54     10.42
  miR-19b-3p         0.02     0.09    1.21    1.74    15.76    46.72
  miR-93-5p          0.04     0.11    0.27    0.33    0.59     2.13
  miR-145-5p         0.01     0.03    0.14    0.10    0.36     17.4
  miR-1-3p           0.01     0.06    0.22    0.19    0.49     11.70
  miR-199a-5p        0.01     0.03    0.07    0.06    0.10     1.28
  miR-4b492          0.01     0.03    0.14    0.18    0.38     14.05
mRNAs (b)
  IL1                0.05     0.15    0.27    0.21    0.40     6.75
  IL6                0.18     1.08    1.59    1.35    2.92     35.56
  TNF                0.17     0.25    0.44    0.44    0.58     6.86
  TLR2               0.03     0.12    0.20    0.19    0.29     2.56
  CD40LG             0.12     0.41    0.64    0.70    0.87     8.98
  ICAM1              0.04     0.10    0.17    0.14    0.22     3.15
  F3                 0.09     0.16    0.35    0.25    0.66     2.89
  SERPINE1           0.15     0.61    0.96    0.91    1.41     12.56
  EDN1               0.09     0.30    0.66    0.66    1.24     10.16
  ACE                0.11     0.34    0.67    0.71    1.25     31.44
Cytokines
  IL-1 (ng/mL)         3        8      14      15      21       54
  IL-6 (ng/mL)         1       38      59      81      173      443
  TNF-[alpha]         10       37      44      50      58       143
    (ng/mL)
  TLR-2 (ng/mL)       0.1      0.4     0.5     0.6     0.8      2.8
  CD40L (pg/mL)       21       131     167     182     234      418
  ICAM-1 (ng/mL)       4        5       6       6       7       13
  F3 (pg/mL)          28       49      76      66      107      419
  PAI-1 (ng/mL)       24       92      113     116     140      215
  ET-1 (pg/mL)         2        5       7       6      10       88
  ACE-1 (ng/mL)       88       134     176     180     220      341

Note: ACE-1, angiotensin converting enzyme-1; CD40L, CD40 ligand; ET-
1, endothelin-1; F3, tissue factor 3; ICAM-1, intercellular adhesion
molecule-1; IL-1, interleukin-1; IL-6, interleukin-6; miRNA, microRNA;
mRNA, messenger RNA; P, percentile; PAI-1, plasminogen activator
inhibitor-1; TLR-2, toll-like receptor-2; TNF-[alpha], tumor necrosis
factor-[alpha].

(a) Geometric means.

(b) Calculated using the relative quantifications formula
([2.sup.-[DELTA][DELTA]Cq]).

Table 3. Percent changes (mean and 95% confidence intervals) in
miRNAs, mRNAs, and circulating cytokines comparing the true-purified
air scenario with the sham-purified air scenario.

Genes            Mean    95% CI        p-Value    FDR

miRNAs
  miR-21-5p      133     76, 191       0.002      0.006
  miR-26a-5p     15      -34, 64       0.542      0.773
  miR-187-3p     143     81,205        0.002      0.006
  miR-146a-5p    111     53, 169       0.006      0.013
  miR-19b-3p     6       -87, 99       0.895      0.895
  miR-93-5p      7       -37, 52       0.725      0.805
  miR-145-5p     58      -21, 137      0.180      0.300
  miR-1-3p       148     -48, 78       0.006      0.013
  miR-199a-5p    76      41, 112       0.001      0.006
  miR-4492       15                    0.618      0.773
mRNAs
  IL1            -63     - 102, -24    0.010      0.025
  IL6            -74     - 103, -46    <0.001     0.002
  TNF            -38     -58, -18      0.002      0.009
  TLR2           -44     -74, -14      0.013      0.027
  CD40LG         -25     -51, 0        0.073      0.102
  ICAM1          -26     -61, 9        0.164      0.164
  F3             -61     - 110, -12    0.039      0.065
  SERPINE1       -22     -46, 1        0.081      0.102
  EDN1           -87     - 132, -42    0.004      0.013
  ACE            -29     -68, 9        0.161      0.164
Cytokines
  IL-1           -56     -88, -23      0.006      0.021
  IL-6           -55     - 105, -4     0.064      0.128
  TNF-[alpha]    -5      -26, 17       0.658      0.731
  TLR-2          -2      -26, 22       0.853      0.853
  CD40L          -19     -35, -2       0.039      0.097
  ICAM-1         -8      -28, 13       0.466      0.583
  F3             -22     -31, -13      <0.001     0.001
  PAI-1          -6      - 17,6        0.339      0.485
  ET-1           -21     -35, -8       0.006      0.032
  ACE-1          -4      - 12,4        0.306      0.484

Note: ACE-1, angiotensin converting enzyme-1; CD40L, CD40 ligand; CI,
confidence interval; ET-1, endothelin-1; F3, tissue factor 3; FDR,
false discovery rate; ICAM-1, intercellular adhesion molecule-1; IL-
1, interleukin-1; IL-6, interleukin-6; miRNA, microRNA; mRNA,
messenger RNA; PAI-1, plasminogen activator inhibitor-1; TLR-2, toll-
like receptor-2; TNF-[alpha], tumor necrosis factor-[alpha].

Table 4. Percent changes (means and 95% CIs) in miRNAs in association
with an interquartile range change (28 [mirco]g/[m.sup.3]) of
P[M.sup.2.5] concentrations.

miRNAs        Targeted cytokines    Percent changes    p-Value    FDR

miR-21-5p     IL-1, ICAM-1          -119 (-169, -70)   <0.001    0.008
miR-26a-5p    IL-6                   -14 (-57, 29)      0.511    0.639
miR-187-3p    TNF-[alpha]            -51 (-91, -12)     0.028    0.051
miR-146a-5p   TLR2, CD40L, ICAM-1    -76 (126, -27)     0.012    0.030
miR-19b-3p    TLR2, F3              -83 (-178, 12.0)    0.130    0.217
miR-93-5p     ICAM-1, F3               0(-39, 38)       0.983    0.983
miR-145-5p    PAI-1                   11 (-80, 58)      0.723    0.803
miR-1-3p      ET-1                  -74 (-129, -19)     0.005    0.017
miR-199a-5p   ET-1                   -64(110, -18)      0.002    0.001
miR-4492      ACE                     38 (-16, 92)      0.192    0.274

Note: ACE-1, angiotensin converting enzyme-1; CD40L, CD40 ligand; CI,
confidence interval; ET-1, endothelin-1; F3, tissue factor 3; FDR,
false discovery rate; ICAM-1, intercellular adhesion molecule-1; IL-
1, interleukin-1; IL-6, interleukin-6; miRNA, microRNA; mRNA,
messenger RNA; PAI-1, plasminogen activator inhibitor-1;
P[M.sup.2.5], particulate matter with an aerodynamic diameter less
than 2.5 [micro]m; TLR-2, toll-like re-ceptor-2; TNF-[alpha], tumor
necrosis factor-[alpha].

Table 5. Percent changes (means and 95% CIs) in mRNAs and cytokines in
association with an interquartile range change (28 [micro]g/
[m.sup.3]) of P[M.sub.2.5] concentrations.

                          mRNAs

Name          Percent change    p-Value     FDR

IL1             49 (16, 82)      0.014     0.028
IL6             54 (27, 85)      0.002     0.010
TNF             28 (9, 47)       0.010     0.025
TLR2            34 (6, 62)       0.032     0.053
CD40LG          19 (-5, 42)      0.138     0.167
ICAM1           24 (-7, 56)      0.150     0.167
F3             77 (37, 117)      0.004     0.020
SERPINE1        19 (-2, 41)      0.100     0.143
EDN1            57 (23, 91)      0.008     0.025
ACE             6 (-29, 42)      0.713     0.713

                        Cytokines

Name          Percent change    p-Value     FDR

IL-1            41 (13,69)       0.013     0.043
IL-6            51 (6, 96)       0.050     0.100
TNF-[alpha]     10 (-9, 29)      0.301     0.431
TLR-2           9 (-12, 31)      0.388     0.431
CD40L           17 (3, 32)       0.028     0.070
ICAM-1          3 (-15,21)       0.741     0.741
F3               18(9,27)        <0.001    0.006
PAI-1           5 (-5, 15)       0.350     0.431
ET-1            21 (9, 34)       0.003     0.015
ACE-1           3 (-4, 11)       0.359     0.431

Note: ACE-1, angiotensin converting enzyme-1; CD40L, CD40 ligand; CI,
confidence interval; ET-1, endothelin-1; F3, tissue factor 3; FDR,
false discovery rate; ICAM-1, intercellu-lar adhesion molecule-1; IL-
1, interleukin-1; IL-6, interleukin-6; mRNA, messenger RNA; PAI-1,
plasminogen activator inhibitor-1; P[M.sub.2.5], particulate matter
with an aerodynamic di-ameter less than 2.5 [micro]m; TLR-2, toll-
like receptor-2; TNF-[alpha], tumor necrosis factor-[alpha].

Table 6. The mediation proportion (%, means and 95% confidence
intervals) by microRNAs in associations of P[M.sub.2.5] with mRNAs and
circulating cytokines.

                                           mRNAs

miRNAs           Name             Percent           p-Value     FDR

miR-21-5p      IL1           8.41 (17.35,34.17)       0.51     0.85
miR-26a-5p     IL6          4.27 (-7.52, 16.06)       0.47     0.85
miR-187-3p     TNF          24.65 (5.92, 43.38)       0.01     0.08
miR-146a-5p    TNF          20.01 (2.26, 37.76)       0.03     0.15
miR-146a-5p    TLR2        12.04 (-53.51, 77.59)      0.71     0.99
miR-19b-3p     TLR2                  --                --       --
miR-146a-5p    CD40LG       28.47 (-13.17,70.11)      0.19     0.71
miR-21-5p      ICAM1       13.74 (-73.13, 100.61)     0.76     0.99
miR-93-5p      ICAM1         0.50 (-4:94, 5.94)       0.86     0.99
miR-146a-5p    ICAM1        5.32 (-10.24,20.88)       0.49     0.85
miR-19b-3p     F3           5.47 (-4.46, 15.40)       0.28     0.84
miR-93-5p      F3            0.21 (-5:28, 5.70)       0.94     0.99
miR-145-5p     SERPINE1     1.76 (-17.40, 20.92)      0.85     0.99
miR-1-3p       EDN1         22.39 (5.38, 39.40)       0.01     0.08
miR-199a-5p    EDN1         4.52 (-7.96, 17.00)       0.47     0.85
miR-4492       ACE           0.13 (-8.36, 8.62)       0.99     0.99

                                        Cytokines

miRNAs         Name               Percent           p-Value     FDR

miR-21-5p      IL-1         40.50 (6.43, 74.57)       0.02     0.13
miR-26a-5p     IL-6         2.19 (-7.57, 11.95)       0.65     0.99
miR-187-3p     TNF-a        2.57 (-8.88. 14.02)       0.66     0.99
miR-146a-5p    TNF-a       10.04 (-19.33, 39.41)      0.50     0.99
miR-146a-5p    TLR-2        8.44 (-16.25, 33.13)      0.49     0.99
miR-19b-3p     TLR-2        5.60 (-6.33, 17.53)       0.36     0.99
miR-146a-5p    CD40L                 --                --       --
miR-21-5p      ICAM-1        0.10 (-6.43, 6.63)       0.99     0.99
miR-93-5p      ICAM-1        0.30 (-7.05, 7.65)       0.95     0.99
miR-146a-5p    ICAM-1       4.39 (-4.96, 13.74)       0.36     0.99
miR-19b-3p     F3                    --                --       --
miR-93-5p      F3            0.75 (-6.60, 8.10)       0.84     0.99
miR-145-5p     PAI-1         0.29 (-4:08, 4.66)       0.91     0.99
miR-1-3p       ET-1         2.92 (-19.97, 25.81)      0.79     0.99
miR-199a-5p    ET-1         21.40 (5.14, 37.66)       0.01     0.13
miR-4492       ACE-1                 --                --       --

Note: --, invalid results of mediation analyses when there was a
positive direction in the pathway of P[M.sub.2.5]-miRNA--mRNA or of
P[M.sub.2.5]-miRNA-cytokine according to our hypothe-sis; ACE-1,
angiotensin converting enzyme-1; CD40L, CD40 ligand; CI, confidence
interval; ET-1, endothelin-1; F3, tissue factor 3; FDR, false
discovery rate; ICAM-1, intercellular adhesion molecule-1; IL-1,
interleukin-1; IL-6, interleukin-6; miRNA, microRNA; mRNA, messenger
RNA; PAI-1, plasminogen activator inhibitor-1; P[M.sub.2.5],
particulate matter with an aerodynamic diameter less than 2.5 im; TLR-
2, toll-like receptor-2; TNF-[alpha], tumor necrosis factor-[alpha].
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Title Annotation:Research
Author:Chen, Renjie; Li, Huichu; Cai, Jing; Wang, Cuicui; Lin, Zhijing; Liu, Cong; Niu, Yue; Zhao, Zhuohui;
Publication:Environmental Health Perspectives
Article Type:Report
Date:Jan 1, 2018
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