Exposure to air pollution is an established risk factor for asthma, reduced lung function as well as inflammatory and oxidative processes which are in turn linked with obesity and diabetes. Risk for these adverse outcomes begins early in childhood and well-documented racial/ethnic differences and social disparities render minority children especially vulnerable, yet these populations are underrepresented in the literature. Analysis for truly representative effect estimates should consider a wide array of genetic, social and environmental factors, biologic pathways from exposure to disease, with consideration for causal mediators and interactions. Mediation and interaction analysis can also aid in determining the transportability of estimated effects from one population to another. However, estimation and interpretability of target parameters in mediation analysis is complicated by issues such as mediator-outcome interactions, non-linearities, and exposure-induced confounding, which cannot be addressed using traditional regression approaches. My long-term career goals are to assess optimal interventions on environmental health risk factors that best reduce overall risk in populations of interest. I will utilize advanced epidemiologic methods maximinzing internal validity and efficiency of estimation of target. I will expand on existing methods for estimation of effects in the presence of time varying exposures and covariates as well as exposure-induced mediator outcome confounding (estimation of controlled direct effects and the randomized intervention analogues for the natural direct and indirect effects) which cannot be addressed with traditional regression approaches. The proposed methodology will be suitable for assessment of potential interventions on continuous exposures, which will be especially beneficial in the area of environmental epidemiology. In Aims 1 & 2 of this proposal I will use these proposed methods to assess direct, indirect and total effects of air pollution exposures on risk of asthma, overall lung function and metabolic syndrome, within a counterfactual framework. I am well suited to perform the proposed research based on 1) my past experience in environmental health and advanced methods and counterfactual approaches 2) the exceprional interdisciplinary mentoring team I have assembled and 3) the unique research opportunity offered by the datasets in the proposal, comprised largely of minority children. This proposed study will enable me to quantify mediated effects of air pollution exposures in especially vulnerable populations. I will be advised by a world-class team of mentors to expand my expertise in integrating advanced epidemiologic methods with causal inference applications (e.g., machine learning and efficient estimators of causal inference parameters) in environmental epidemiological studies; epigenetic and exposomic factors as potential modifiers or mediators of effect; and health disparities and social factors associated with environmental exposures. The proposed research and training will enable me to establish an independent career as a leader in intervention assessment and causal inference in environmental epidemiology

Particulate matter (PM) air pollution is considered a top-10 contributor to (and the leading environmental risk factor for) the global burden of disease. To date, evidence on PM health effects has been gathered primarily from medium-to-large scale epidemiology studies, which have traditionally relied upon relatively crude measures of human exposure (i.e., fixed site sampling for PM mass with little to no PM composition analyses). As a result, these studies tend to emphasize the effects of PM on more sedentary populations (such as the elderly) and/or that live close to air monitoring sites. The field now recognizes that air pollution exposure is highly heterogeneous and that exposure measurement error substantially limits the linkage of exposures to specific pathologies. Recently, epidemiologic interest in mobile populations (e.g., school-aged children or working adults) has increased and the exposure assessment field has shifted towards measures and models of personal exposure to specific PM chemical constituents (and PM properties) suspected to drive human morbidity and mortality. Unfortunately, existing technologies for both sampling and analysis are limited by cost, and usability. Thus, a need exists for personal PM sensors that are inexpensive, wearable (with low-burden), yet still highly sensitive and capable of measuring specific PM properties. Our team has developed technology that meets these needs: a small, portable, inexpensive micro environmental sampler and a low cost sensing chemistry that can quantify PM chemical composition both quickly and cost-effectively. During this project, we propose to 1) Evaluate and validate the sampling and analysis methods using laboratory, field, and limited personal exposure studies (R21 phase) and 2) Demonstrate performance and added scientific value through application in the Children's Health and Air Pollution in the San Joaquin Valley (CHAPS-SJV) study (R33 phase). In the first phase, we will demonstrate the usability of our technology by engaging multiple test populations (college students, 9-11 year olds in Fort Collins, CO and high school students in Fresno, CA). In the second phase, we will use the system to provide first of its kinds information on micro environmental exposures and PM composition as it relates to inflammation biomarkers for acute exposures. The resulting data will be used to improve models of air pollution exposure for children in the San Joaquin Valley with the long-term goal of improving the health of these children.

Our overall objective is to determine the molecular mechanisms by which immune dysregulation leads to human disease, specifically the atopic diseases of food allergy, allergic rhinitis, allergic conjunctivitis and allergic asthma in the children exposed to high levels of PAHs (polycyclic aromatic hydrocarbons). We previously published work on an important link between PAH exposure in ambient air pollution (AAP) and changes at the DNA level in immune cells that led to their impaired function. This decrease in cell function was directly associated with increases in Th2 cytokines, IL-4 and IL-13, and subsequent clinical outcomes of allergy and asthma, including decreased lung function, in the same pediatric subjects. However, the effect of these cellular changes on allergic disorders was not evaluated at the time; therefore, we will focus on the effects of PAH and will determine whether PAH exposure is associated with systemic immune dysregulation, leading to atopic diseases (food allergy, allergic rhinitis, allergic conjunctivitis, and allergic asthma). T cells are key mediators of the adaptive immune system and play critical roles in modulating inflammation (specifically regulatory T cells or Treg) and inducing allergy (i.e., Th2 effector cells which can lead to isotype switching of B cells to synthesize IgE, a molecule associated with allergies). To date, we have focused our studies on isolated T cell subsets; and, in this proposal, we will conduct studies on Th subsets to be able to elucidate the full mechanisms of how PAHs modulate the immune system. To do this, our approach is to perform a cross-sectional analysis in a well-defined "piece-wise" continuum of all pediatric ages in immune development for whom detailed information will be collected on human disease outcomes and for whom blood, saliva, and urine samples will be collected at repeated time points. We plan to use novel and innovative immunological studies including epigenetic pyrosequencing studies on single cells and mass spectrometry based flow cytometry for detection of 38 simultaneous immune cell parameters. In this way, we will be able to determine key time points of sensitivity of the immune system to PAH exposure by defining the molecular mechanisms that play a role in immune system impairment during immune development.

Metabolic syndrome is increasingly prevalent, is usually associated with obesity and glucose dysregulation, and leads to increased risk of cardiovascular disease. Risks of obesity and diabetes type 2 begin in eariy childhood, including in utero programming, and environmental factors likely play a role in these risks. The effect of exposure to ambient air pollution (AAP) on risk of obesity and diabetes among children has been well studied. Our primary hypothesis is that oxidative stress induced by exposure to AAP leads to systemic inflammation which in turn leads to increased risk of obesity and diabetes. A secondary mechanistic hypothesis is that AAP-induced Treg dysfunction increases risks of obesity and diabetes. To test these hypotheses, analyses of children in the different stages of development represented in a piecewise, natural history design will be conducted (ages 0-2, 7-9, 16-19, and 19-22). Detailed historical information, anthropometric data, and blood samples will be collected for all subjects. Exposures to AAP will be estimated from in utero onward. The proposed study has the following aims: a) to determine whether chronic exposure to AAP, especially polycyclic aromatic hydrocarbons (PAHs), is associated with increased HbAlc, BMI, and 8-isoprostane (biomarker of oxidative stress), CRP (biomarker of systemic inflammation), leptin, adiponectin, and high-density lipoprotein (biomarkers of abnormal fat and glucose metabolism); b) to determine whether AAP-induced dysfunction of T regulatory (Treg) and T effector cells is associated with increased HbAlc and BMI; and c) to determine whether epigenetic modification of Foxp3 underiies the associations between Treg dysfunction and HbAlc or BMI. Using an experienced field center staff, 220 children in each ofthe two younger age groups and 100 subjects in each of the older groups will be recruited from Fresno and followed for variable durations depending on age. Working with the Exposure Core to generate lifetime pollutant exposure histories and the Biostatistics-Epidemiology Core to build marginal structural models, the multidisciplinary research team will conduct analyses of the associations between chronic exposures to air pollutants (or Treg functional parameters) and HbAlc, BMI, or the biomarkers of interest.

The basic biological mechanisms by which ambient air pollution affects the human body have been studied mainly in the areas of oxidative stress, DNA damage, and airway epithelial changes [Galli, 2008]. Some studies have focused on the downstream, unregulated inflammatory responses that result from Th2 polarization associated with ambient air pollution exposure[Bernstein, 2004;Devouassoux, 2002;Diaz-Sanchez, 1999;Diaz-Sanchez, 2000;Diaz-Sanchez, 2000;Diaz-Sanchez, 2005;Diaz-Sanchez, 1996;Diaz-Sanchez, 1997;Finkelman, 2004;Fujieda, 1998;Riedl, 2005;Sawant, 2008 ;Saxon, 2005]. However, I propose to study the effects of ambient air exposure on Treg, and test the hypothesis that specific decreases in Treg function consequent to this exposure are a major component of the immunopathology of asthma. In fact, the lack of normal Treg function in the lung is associated with asthma in children [HartI, 2007]. Treg represent the basic counterregulatory arm of the immune system in human development; however, little is known on how ambient air pollution affects Treg differentiation and function. My laboratory has performed studies on Treg isolated from blood samples of children living in the Central Valley in collaboration with the Fresno Asthma Children's Environmental Study (FACES) [Margolis, 2008; Tager, 2006; Tager, 2005; Tager, 1998] in which chronic exposure to ambient air pollution has been measured. I have found that Treg function is attenuated by up to 10 fold compared to controls; in addition, this impairment is associated with direct decreases in Foxp3, a gene associated with Treg development and function in humans [Hori, 2003; Ono, 2007; Sakaguchi, 2003]. Combined with rigorous epidemiological studies, the innovative functional and molecular tools proposed in Project 3 would allow for observations about environmental exposure in children to become more fundamental at the basic science level. Specifically, 1 propose: Specific Aim 1 to define the mechanisms of Treg impairment in non asthmatic and asthmatic children and Specific Aim 2: to evaluate if Treg dysfunction correlates with estimate individual exposure. The study will generate a unique body of scientific knowledge of detailed exposure and individual follow-up data linked to immune system changes that are otherwise not currently available to advance the field of environmental effects on health. Our approach will address the biologic plausibility of the association between the increase in asthma and ambient air pollution exposure.