The human circulating miRNome reflects multiple organ disease risks in association with short-term exposure to traffic-related air pollution
Introduction
Numerous epidemiological studies have associated exposure to traffic-related air pollution (TRAP) with increased risk of cardiovascular disease, (Brook et al., 2010; Lee et al., 2014) respiratory disease, (Xing et al., 2016) several types of cancer, including lung and breast cancer, (Hamra et al., 2014; Tagliabue et al., 2016) and more recently also of neurodegenerative diseases (Cacciottolo et al., 2017; Chen et al., 2017) and kidney disease (Bowe et al., 2017). A number of gaseous pollutants such as nitrogen dioxide (NO2) as well as particulate matter components (PM) are routinely monitored to characterize TRAP exposure. PM is a complex mixture of fine particles with a diameter of 10 μm or less (PM10), a diameter of 2.5 μm or less (PM2.5), black carbon (BC), ultrafine particles with a diameter of 0.1 μm or less (UFP) and soot (Falcon-Rodriguez et al., 2016). Upon inhalation PM penetrates deeply into the lungs from where, depending on their size, particles are capable of entering the circulation and being distributed to distal organs such as the heart, spleen or liver (Falcon-Rodriguez et al., 2016; Li et al., 2015; Yaghjyan et al., 2017). It has even been reported that ultrafine PM crosses the blood-brain barrier and translocates from the circulation to the brain (Oberdorster et al., 2004). Further, it has been demonstrated that PM triggers oxidative stress in the respiratory tract and that this might induce a systemic inflammatory cascade, thus increasing the risk for respiratory and cardiovascular diseases (Bollati et al., 2015). Presumably, pollutants, once distributed over the whole body, may cause a similar cascade of oxidative stress and inflammation in target organs, thereby increasing risks for cancer and neurodegenerative disease (Block & Calderon-Garciduenas, 2009). However, the precise molecular mechanisms that link TRAP exposure to increased disease risks are still poorly understood which hampers the development of dedicated biomarkers capable of informing on relevant molecular mechanisms of action.
Several studies have thus highlighted the impact of environmental exposure on gene expression profiles, (van Leeuwen et al., 2006) DNA-methylation patterns, (Georgiadis et al., 2016) and p53 status (Intarasunanont et al., 2012). More recently, environmental exposure-induced alterations in microRNA (miRNA) levels have been described (Krauskopf et al., 2017a). These small non-coding RNAs are involved in the posttranscriptional regulation of gene expression, and consequently are involved in virtually all cellular processes (Bartel, 2009). Furthermore, while these fine-tuners of gene expression are capable of adjusting to internal and external conditions, they also exhibit tissue/organ specific expression patterns (Landgraf et al., 2007). As a consequence of organ injury, cells may leak their content including the highly stable protein-bound miRNAs, into the peripheral circulation (Turchinovich et al., 2012). Given the fact that certain miRNAs are more abundantly expressed in specific organs, circulating miRNA (cmiRNA) signatures may thus also reflect organ-specific responses to exposure (Krauskopf et al., 2015). Furthermore, through active secretion, extracellular vesicle-bound cmiRNAs may act as mediators in intercellular and interorgan communication (Hunter et al., 2008). Therefore, cmiRNAs leaked or released from organs into the circulation, have become a new promising class of biomarkers capable of non-invasively interrogating organ pathogenesis and organ-toxic mechanisms from so called ‘liquid biopsies’ (Krauskopf et al., 2015).
To date, most reported air pollution-induced changes in miRNA expression have been identified in solid tissues in animal models (Vrijens et al., 2015). The first evidence on PM exposure-related modifications in cmiRNA levels in humans was provided through investigating healthy steel plant workers. This study identified 2 vesicle-associated miRNAs that were elevated after occupational exposure to metal-rich PM (Bollati et al., 2015). Additionally, a study on long term exposure to ambient air pollution (6 month or 1 year) identified the elevation of 5 vesicle-associated cmiRNAs in the serum of healthy subjects (Rodosthenous et al., 2016). Another study among children identified 2 cmiRNAs in the extracellular fraction of saliva to be significantly altered with long-term ultrafine PM exposure (Vriens et al., 2016).
These studies provided evidence that the extracellular miRNA genome (miRNome) is affected by TRAP exposure through utilizing targeted approaches, and were consequently restricted to analyzing a priori known air pollution-associated miRNAs. In the current study, we present for the first time a global analysis of the circulating miRNome by applying next generation sequencing technology and real-time exposure measurements in an experimental cross-over study of human volunteers (n = 24) following short-term traffic-related air pollution exposure. This study demonstrates the potential of circulating miRNAs as novel biomarkers for health risk assessment in relation to environmental exposure-induced target tissue pathogenesis.
Section snippets
Selection of the population
Plasma samples were collected during a randomized experimental crossover study in which non-smoking participants, either healthy or suffering from ischemic heart disease (IHD) or chronic obstructive pulmonary disease (COPD), walked for 2 h along Oxford Street in London (where only diesel-powered buses and taxicabs are permitted). In a separate session the same subjects also walked for 2 h through traffic-free Hyde Park. In order to balance between sufficient exposure and what is acceptable for
Exposure range
The subjects analyzed for cmiRNAs were exposed to a mean ambient air NO2 level of 7.9 (CI 5.9–9.8) μg/m3 in Hyde Park and 18.1 (CI 15.1–21.1) μg/m3 in Oxford Street. For PM2.5 the mean exposure level in Hyde Park was 5.6 (CI 4.5–6.8) μg/m3 and 25.6 (CI 21–30.2) μg/m3 in Oxford Street. For BC the exposure level was 1.0 (CI 0.8–1.3) μg/m3 in Hyde Park and 11.4 (CI 9.9–12.8) μg/m3 in Oxford Street and for PM10 16.0 (CI 12.5–19.5) μg/m3 in Hyde Park and 37.0 (CI 32.2–41.7) μg/m3 in Oxford Street.
Discussion
In this study, we evaluated the global circulating miRNome in plasma from human subjects exposed to ambient TRAP for only 2 h by using NGS. We identified 54 cmiRNAs that appear to be involved in the molecular response to NO2, UFP, PM2.5, BC and PM10 exposure. Next, we gathered information on tissue-specific miRNAs from those organs known to be targeted by ambient air pollutants. We found that the most abundant cmiRNAs present in plasma are equally expressed in all organs known to be targeted by
Author contributions
J.K., T.M.K. and J.C.K. designed the research. K.F.C., P. Cu., P. Co., B.B., F.J.K. and P.V. organized the epidemiologic part of the work. J.K. and R.S. performed the experiments. J.K., F.C., K.V., M.C. and R.V. analyzed the data. T.M.K. and J.C.K. supervised the project. J.K., T.M.K. and J.C.K. co-wrote and all authors commented on the paper.
Additional information
The authors declare no competing financial interests.
Acknowledgments
This work has been supported by the European Union within the frame of the Exposomics (226756) project and the British Heart Foundation (PGF/10/82/28608).
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