Final Report: Cardiovascular Consequences of Immune Modification by Traffic-Related Emissions

EPA Grant Number: R834796C003
Subproject: this is subproject number 003 , established and managed by the Center Director under grant R834796
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: University of Washington Center for Clean Air Research
Center Director: Vedal, Sverre
Title: Cardiovascular Consequences of Immune Modification by Traffic-Related Emissions
Investigators: Campen, Matthew J. , Lund, Amie K. , McDonald, Jacob D. , Rosenfeld, Michael
Institution: Lovelace Respiratory Research Institute , University of New Mexico , University of Washington
EPA Project Officer: Callan, Richard
Project Period: December 1, 2010 through November 30, 2015 (Extended to November 30, 2017)
RFA: Clean Air Research Centers (2009) RFA Text |  Recipients Lists
Research Category: Health Effects , Air

Objective:

Traffic-related emissions are associated with the incidence and progression of acute and chronic cardiovascular sequelae in human population studies. Such phenomena of near-roadway health effects have yet to be characterized toxicologically. Because of overlapping issues related to noise, socioeconomic status, ethnicity, etc., there is a need to better understand the biological plausibility that fresh mixtures of vehicular emissions have a more potent than expected impact on human health.

We hypothesized that the complex mixtures produced by traffic are inherently more toxic due to the combined presence of both particulates and volatile organic emissions. 

Furthermore, we hypothesized that emissions-induced oxidation of certain endogenous phospholipids, presumably from the pulmonary surfactant, can stimulate the activity of immune cells through such receptors and in turn promote the invasion of existing vascular lesions. This latter hypothesis evolved to developing a battery of assays geared to assess the endothelial inflammatory potential of serum, which had immense value for both studies of the mechanisms of air pollution systemic health effects and for clinical research efforts.

Approach: This project uses complex roadway mixtures as generated and characterized in the laboratory. In Aim 1, we will ascertain (1) the potentiating effects of physical and photochemical aging on fresh emissions, and (2) interactions of vehicular emissions with pertinent copollutants (ozone, road dust), both in terms of driving systemic vascular oxidative stress. In Aim 2, we will examine effects of the emissions-induced oxidative modifications to endogenous phospholipids in terms of activating immune-modulating receptors such as LOX-1, CD-36, TLR-2 and TLR-4. In this Aim, we will utilize transgenic models to examine the roles of these receptors and characterize the lipidomic alterations in various tissues. In Aim 3, we will further explore the role of specific immune cell populations as participants in the innate and adaptive responses to emissions-induced phospholipid modifications. In this Aim, we will utilize mouse models of immunodeficiency, including SCID and B-cell deficient models. Additionally, we will pursue bone-marrow transplants from mice lacking those receptors described in Aim 2 to mechanistically establish the involvement of the oxidatively modified phospholipids.

Owing to suggestions from the advisory committee, we have focused on the nature and bioactivity of circulating factors induced by pollutant exposures, as these appear to be ligands that interact with the scavenger receptors of interest in Aims 2 and 3. This has been an area of significant progress for the past year.

Summary/Accomplishments (Outputs/Outcomes):

Two major hypotheses were tested in this project. The results related to each of the hypotheses are presented in the two sections following:

A. Testing whether the complex mixtures produced by traffic are inherently more toxic due to the combined presence of both particulates and volatile organic emissions

Using a variety of approaches enabled by the technical advancements in Project 2, we were able to manipulate the complex emissions of gasoline and diesel exhausts to test the extent to which combined particles and gases led to more toxic effects. In a first major study, we utilized a common hypercholesterolemic mouse model (Apolipoprotein E-null, or ApoE-/-) exposed to mixed vehicle emission (MVE-—combined gasoline and diesel exhausts) for 6 hours/day × 50 days, with additional permutations of removing PM by filtration and also removing gaseous species from particulate matter (PM) by denudation. Several vascular bioassays, including matrix metalloproteinase-9 (MMP9) protein, 3-nitrotyrosine and plasma-induced vasodilatory impairments, highlighted that the whole emissions, containing both particulate and gaseous components, was collectively more potent than MVE-derived PM or gas mixtures, alone. Thus, we concluded that inhalation of fresh whole emissions induce greater systemic vascular toxicity than either the particulate or gas phase alone. These findings support the hypothesis that the near-roadway environment may have a more focused toxic impact due to gas-particle interactions.

To further confirm the gas-PM interactions as a major driver of near-roadway health effects, we conducted a highly innovative project involving the denudation of freshly generated PM with a smaller size distribution that we compared to an exposure where vehicular particulates were collected and resuspended at a larger size fraction (via a Wright Dust Feeder), to specifically examine the role of surface area as a driver of gas-PM enhancements in toxicity (Tyler et al., 2016). To determine the validity of the hypothesis that a greater surface area would allow for greater gaseous interaction with PM (and therefore greater toxicity), we examined pulmonary and neurological inflammatory outcomes in C57BL/6 and ApoE-/- male mice after acute and chronic exposure to vehicle engine-derived particulate matter, generated as ultrafine (UFP) and fine (FP) sizes, with additional exposures using UFP or FP combined with gaseous copollutants derived from fresh gasoline and diesel emissions, labeled as UFP + G and FP + G.

The UFP and UFP + G exposure groups resulted in the most profound pulmonary and neuroinflammatory effects. Phagocytosis of UFP + G particles via resident alveolar macrophages was substantial in both mouse strains, particularly after chronic exposure, with concurrent increased proinflammatory cytokine expression of CXCL1 and TNFα in the bronchial lavage fluid. In the acute exposure paradigm, only UFP and UFP + G induced significant changes in pulmonary inflammation and only in the ApoE-/- animals. Similarly, acute exposure to UFP and UFP + G increased the expression of several cytokines in the hippocampus of ApoE-/- mice, including Il-1β, IL-6, Tgf-β and Tnf-α, and in the hippocampus of C57BL/6 mice, including Ccl5, Cxcl1, Il-1β and Tnf-α. Interestingly, Il-6 and Tgf-β expression were decreased in the C57BL/6 hippocampus after acute exposure. Chronic exposure to UFP + G increased expression of Ccl5, Cxcl1, Il-6, and Tgf-β in the ApoE-/- hippocampus, but this effect was minimal in the C57BL/6 mice, suggesting compensatory mechanisms to manage neuroinflammation in this strain.

Inflammatory responses in the lung and brain were most substantial in ApoE-/- animals exposed to UFP + G, suggesting that the surface area-dependent interaction of gases and particles is an important determinant of toxic responses. As such, freshly generated UFP in the presence of combustion-derived gas phase pollutants may be a greater health hazard than would be predicted from PM concentration alone, lending support for epidemiological findings of adverse neurological outcomes associated with roadway proximity.

B. Testing the inflammatory potential of circulating factors arising from pollution exposures.

Inhaled pollutants induce the release of vasoactive factors into the systemic circulation, but little information is available regarding the nature of these factors or their receptors. The pattern recognition receptor CD36 interacts with many damage-related circulating molecules, leading to activation of endothelial cells and promoting vascular inflammation; therefore, we hypothesized that CD36 plays a pivotal role in mediating cross-talk between inhaled ozone (O3)-induced circulating factors and systemic vascular dysfunction (Robertson et al., 2013). O3 exposure (1 ppm × 4 hours)-induced lung inflammation in wild-type (WT) mice, which was absent in the CD36 deficient (CD36-/-) mice. Acetylcholine (ACh)-evoked vasorelaxation was impaired in isolated aortas from O3-exposed WT mice but not in vessels from CD36-/- mice. To delineate whether vascular impairments were caused by lung inflammation or CD36-mediated generation of circulating factors, naïve aortas were treated with diluted serum from control or O3-exposed WT mice, which recapitulated the impairments of vasorelaxation observed after inhalation exposures. Aortas from CD36-/- mice were insensitive to the effects of O3-induced circulating factors, with robust vasorelaxation responses in the presence of serum from O3-exposed WT mice. Lung inflammation was not a requirement for production of circulating vasoactive factors, as serum from O3-exposed CD36-/- mice could inhibit vasorelaxation in naïve WT aortas. These results suggest that O3 inhalation induces the release of circulating bioactive factors capable of impairing vasorelaxation to ACh via a CD36-dependent signaling mechanism. Although lung inflammatory and systemic vascular effects were both dependent on CD36, the presence of circulating factors appears to be independent of CD36 and inflammatory responses.

 In a second major study testing the role of circulating factors in causing endothelial cell dysfunction and inflammation, we tested comparisons between single pollutants and common combustion mixtures, in terms of driving such serum inflammatory and vasoactive effects (Aragon et al., 2016). Healthy C57BL/6 mice were exposed to a single 6-hour period of contrasting pollutant atmospheres: road dust, MVE particulate matter, MVE gases, road dust plus ozone, road dust plus MVE, and hardwood smoke. Serum obtained from mice 24 hours after these exposures was used as a stimulus to assess inflammatory potential in two assays: incubated with primary murine cerebrovascular endothelial cells for 4 hours to measure inflammatory gene expression or applied to naïve aortic rings in an ex vivo myographic preparation. Road dust and wood smoke exposures were most potent at inducing inflammatory gene expression, while MVE atmospheres and wood smoke were most potent at impairing vasorelaxation to ACh. Responses are consistent with recent reports on MVE toxicity but reveal novel serum bioactivity related to wood smoke and road dust. These studies suggest that the compositional changes in serum and resultant bioactivity following inhalation exposure to pollutants may be highly dependent on the composition of mixtures.

Conclusions:

First, our major findings support both hypotheses. This is essential to better understanding the toxic effects of traffic-related particulate matter health effects. While all particulates may be assumed to be toxic, the freshly generated particles near a roadway may have enhanced toxicity due to the coexistence of adhered volatile and semivolatile compounds. Such particles will therefore be in greater concentration near a roadway but will also exhibit greater toxicity on a per-particle basis as a result of this gas-particle association. Second, our studies confirm that circulating factors arising from inhalation exposures to various pollutants have the potential to inhibit normal vasoactivity (via the CD36 receptor) and induce inflammatory pathways in endothelial cells. These effects appeared more potent with complex combustion mixtures, especially vehicular emissions. Such findings may help us explain the pathways that lead from inhalation of toxicants to systemic health outcomes like atherosclerosis and neurological disorders.

References:

  1. Aragon MJ, Chrobak I, Brower J, Roldan L, Fredenburgh LE, McDonald JD, Campen MJ. Inflammatory and vasoactive effects of serum following inhalation of varied complex mixtures. Cardiovascular Toxicology 2016;16(2):163-171.
  2. Campen M, Robertson S, Lund A, Lucero J, McDonald J. Engine exhaust particulate and gas phase contributions to vascular toxicity. Inhalation Toxicology 2014;26(6):353-360.


Journal Articles on this Report : 9 Displayed | Download in RIS Format

Other subproject views: All 26 publications 16 publications in selected types All 16 journal articles
Other center views: All 187 publications 87 publications in selected types All 86 journal articles
Type Citation Sub Project Document Sources
Journal Article Aragon MJ, Chrobak I, Brower J, Roldan L, Fredenburgh LE, McDonald JD, Campen MJ. Inflammatory and vasoactive effects of serum following inhalation of varied complex mixtures. Cardiovascular Toxicology 2016;16(2):163-171. R834796 (2015)
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  • Journal Article Brower JB, Doyle-Eisele M, Moeller B, Stirdivant S, McDonald JD, Campen MJ. Metabolomic changes in murine serum following inhalation exposure to gasoline and diesel engine emissions. Inhalation Toxicology 2016;28(5):241-250. R834796 (2016)
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  • Journal Article Campen MJ, Lund A, Rosenfeld M. Mechanisms linking traffic-related air pollution and atherosclerosis. Current Opinion in Pulmonary Medicine 2012;18(2):155-160. R834796 (2012)
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  • Journal Article Campen M, Robertson S, Lund A, Lucero J, McDonald J. Engine exhaust particulate and gas phase contributions to vascular toxicity. Inhalation Toxicology 2014;26(6):353-360. R834796 (2014)
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  • Journal Article Paffett ML, Zychowski KE, Sheppard L, Robertson S, Weaver JM, Lucas SN, Campen MJ. Ozone inhalation impairs coronary artery dilation via intracellular oxidative stress: evidence for serum-borne factors as drivers of systemic toxicity. Toxicological Sciences 2015;146(2):244-253. R834796 (2014)
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  • Journal Article Robertson S, Colombo ES, Lucas SN, Hall PR, Febbraio M, Paffett ML, Campen MJ. CD36 mediates endothelial dysfunction downstream of circulating factors induced by O3 exposure. Toxicological Sciences 2013;134(2):304-311. R834796 (2013)
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  • Journal Article Schisler JC, Ronnebaum SM, Madden M, Channell M, Campen M, Willis MS. Endothelial inflammatory transcriptional responses to an altered plasma exposome following inhalation of diesel emissions. Inhalation Toxicology 2015;27(5):272-280. R834796 (2015)
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  • Journal Article Tyler CR, Zychowski KE, Sanchez BN, Rivero V, Lucas S, Herbert G, Liu J, Irshad H, McDonald JD, Bleske BE, Campen MJ. Surface area-dependence of gas-particle interactions influences pulmonary and neuroinflammatory outcomes. Particle and Fibre Toxicology 2016;13(1):64 (18 pp.). R834796 (2016)
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  • Journal Article Yin F, Lawal A, Ricks J, Fox JR, Larson T, Navab M, Fogelman AM, Rosenfeld ME, Araujo JA. Diesel exhaust induces systemic lipid peroxidation and development of dysfunctional pro-oxidant and pro-inflammatory high-density lipoprotein. Arteriosclerosis, Thrombosis, and Vascular Biology 2013;33(6):1153-1161. R834796 (2013)
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  • Supplemental Keywords:

    Coronary artery disease, oxidized phospholipids, atherosclerosis, particulate matter, volatile organic compounds, carbon monoxide, ozone, Health, Scientific Discipline, Air, ENVIRONMENTAL MANAGEMENT, Air Quality, air toxics, Health Risk Assessment, Risk Assessments, mobile sources, Biochemistry, Environmental Monitoring, Risk Assessment, ambient air quality, atmospheric particulate matter, particulate matter, aerosol particles, air pollutants, motor vehicle emissions, vehicle emissions, air quality models, motor vehicle exhaust, airway disease, bioavailability, air pollution, particle exposure, atmospheric aerosols, ambient particle health effects, vascular dysfunction, cardiotoxicity, atmospheric chemistry, exposure assessment

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    Progress and Final Reports:

    Original Abstract
  • 2011 Progress Report
  • 2012 Progress Report
  • 2013 Progress Report
  • 2014
  • 2015 Progress Report
  • 2016 Progress Report

  • Main Center Abstract and Reports:

    R834796    University of Washington Center for Clean Air Research

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R834796C001 Exposure Mapping – Characterization of Gases and Particles for ExposureAssessment in Health Effects and Laboratory Studies
    R834796C002 Simulated Roadway Exposure Atmospheres for Laboratory Animal and Human Studies
    R834796C003 Cardiovascular Consequences of Immune Modification by Traffic-Related Emissions
    R834796C004 Vascular Response to Traffic-Derived Inhalation in Humans
    R834796C005 Effects of Long-Term Exposure to Traffic-Derived Particles and Gases on Subclinical Measures of Cardiovascular Disease in a Multi-Ethnic Cohort