Ilona Jaspers - US grants

DESCRIPTION (provided by applicant): Exposure to air pollutants, such as diesel exhaust (DE), is associated with airway inflammation, increased susceptibility to viral infection, and exacerbation of underlying respiratory disorders including allergic asthma. Based on studies in our laboratory and others, the mechanisms by which air pollutants cause these adverse effects likely involve alteration of inflammatory and antiviral signaling pathways linked to cellular oxidant/antioxidant imbalance. Since the previous grant we have expanded our experimental models to investigate 1.) how pre-existing allergic airway disease modifies the ability of DE to increase susceptibility to influenza, 2.) the role of NK cells and T cells in influenza-induced responses, and 3.) the effects of environmental pollutants on susceptibility to influenza virus in humans in vivo using inoculation with the live-attenuated influenza virus (LAIV) vaccine. We are therefore well positioned to test the hypotheses that exposure to DE increases allergic inflammation and susceptibility to influenza in humans, that oxidative stress-induced suppression of NK cell function mediates these effects, and that supplementation with SFN can prevent DE-induced alterations of antiviral immune responses. Aim 1 will determine how DE exposure modifies inflammatory and antiviral responses to LAIV in normal volunteers and subjects with allergic rhinitis (AR). This will be a randomized, prospective comparison study comparing cohorts of normal or AR subjects randomized to receive either DE (100-300 (g/m3 x 2hr at rest) or placebo (clean air), followed by a standard dose of LAIV. Nasal lavage fluids and biopsies will be sampled at intervals during the resulting self-limited infection. Endpoints will include inflammatory mediators, antiviral factors, virus clearance, and effects of antioxidant genotype on exposure outcomes. Aim 2 will determine how exposure to DE modifies NK and T cell activation in the context of influenza infection by assessing changes in activation, cytotoxic potential, and cytokine production (a) in nasal NK and T cells from subjects exposed to DE prior to infection with LAIV and (b) in in vitro models to further define potential mechanisms of DE-induced changes in NK cell activity. Aim 3 will determine whether antioxidant supplementation with Sulforaphane (SFN) prevents the effects of DE exposure on virus-induced inflammation, antiviral defense response, and immune cell competence. These studies are expected to increase our understanding of whether and how DE enhances susceptibility to influenza virus, especially in the setting of allergic inflammation and the role of NK and T cells in these responses. We further anticipate that these studies will provide a model template useful for assessment of the impact of other environmental agents on respiratory mucosal defense in the context of viral infections, and for determination of the efficacy of intervention strategies. PUBLIC HEALTH RELEVANCE: This project will utilize a novel, safe, and currently approved protocol for controlled assessment of the combined effects of diesel exhaust and virus exposures on nasal inflammation in humans with allergic rhinitis in a "real-life" setting. Using samples obtained from volunteers this project will examine mechanisms by which exposure to air pollutants modify immune responses in humans. It will also test the impact of dietary antioxidants (sulforaphane-rich broccoli sprouts) on the nasal response to diesel exhaust and virus. Given the high frequency of exposures to diesel exhaust, the tendency of influenza to occur in large scale epidemics, and the high prevalence of allergic airway diseases, the potential scientific and public health impact of the project is large.

Project Title: Fate, transport, and toxicity of engineered nanoparticles in the atmosphere
PI: Richard M, Kamens, kamens@unc.edu; Ilona Jaspers (co-PI) ilona_jaspers@med.unc.edu Michael P. Tolocka (co-PI) fizzchem@hotmail.com,
Institution: University of North Carolina at Chapel Hill Chapel Hill, NC, 27599

Submitted to: Interfacial Transport and Thermodynamics Program, # 1414 NSF (EPA# NI-108)
NSF project Officer: Dr. Robert M. Wellek, and Deputy Director, CBET, Division of Engineering, NSF

Project Objective: Determine the atmospheric fate of engineered nanoparticles (ENP) and the effect of aging on the toxicity of this class of materials.

Intellectual merit: The first focus is on the chemical kinetics of ENP atmospheric transformations. ENP will react faster than their micron-sized counterparts found in dusts. The PIs aim to understand how they behave as seed for secondary organic aerosol (SOA) reactions. Because ENP act as photocatalysts, the yield of SOA will be affected. The second focus is to understand the toxicity of this class of materials. They will rapidly screen the ENP for the production of reactive oxygen species (ROS) as measured by a chemical method. Easily reducible species, such as Ni3+ in NixOy will enhance ROS production. The last step is to measure the actual toxicological response of human bronchial epithelial cells. As the materials age, the metal center becomes more oxidized and therefore easily reduced, resulting in increased inflammatory and other biological response to ENP.

For the kinetics experiments, both flow tube and smog chamber studies will be performed. The flow tube studies will be used to determine kinetic parameters of uptake onto their surfaces as a function of relative humidity and particle size. In this way, we can use these parameters for the models that will be developed for their chemical transformations in real atmospheres. To simulate those atmospheres, the smog chamber experiments will be used to determine the fate of ENP as well as their toxicity measured using chemical and in vitro biological methodologies. The chemical method involves the reaction of the suspended particulate matter with dithiothreitol. For the toxicity analysis, they will use differentiated human bronchial epithelial cells or A549 cells grown on membrane support. Cells will be exposed to the fresh or aged ENP. Basolateral supernatants and RNA will be analyzed for the levels of inflammatory cytokines and chemokines and for the levels of intracellular inflammatory mediators as well as markers of oxidative stress.

Broader Impacts: The unique aspect of this research is that the PIs aim to understand the fate of ENP in the atmosphere and their toxicological effect. Accurate measurements of the oxidation and transport of ENP in the atmosphere are essential. The present uncertainties in understanding ENP fate and transport can strongly affect conclusions from studies that may underestimate the potential toxicity of this class of materials, their derivatives, and their influence on atmospheric processes. Detailed kinetic data will benefit modeling efforts by providing a basis to estimate the fate and transport of ENP. Adding these data to existing databases for air-shed models will benefit officials of governmental organizations who use these models to set policies regarding risk assessment and control strategies for generators of ENP. Health effects researchers will benefit from the toxicological data acquired from our experiments as a guide for exposure assessment. In addition, epidemiological studies that aim to locate a causal mechanism for the deleterious effects of ambient particulate matter will benefit from a more precise understanding of what compounds are associated with the aerosol and their toxicity. The proposed research program not only enables the identification of ENP reactions and their kinetics in real time, but also the rapid reaction and data analysis provides greater throughput allowing a greater range of nanoparticle studies.

Supplemental Keywords: air, ambient air, atmosphere, tropospheric, chemical transport, exposure, effects, health effects, human health, dose-response, stressor, chemicals, toxics, particulates, oxidants, intermediates.

Affect; airway epithelium; Antiviral Agents; Antiviral Response; Attenuated; base; Biological Markers; Biopsy; Cells; Chronic; Cigar; Cigarette; cigarette smoke; cigarette smoke-induced; Cigarette Smoker; cigarette smoking; Complex; Data; defense response; design; DNA Methylation; Environmental Tobacco Smoke; environmental tobacco smoke exposure; epigenomics; Epithelial; Epithelial Cells; Epithelium; Experimental Models; experimental study; Exposure to; fighting; Gene Expression; genome-wide; Genomics; Habits; hookah; Host Defense; Human; Human Volunteers; Immune; Immune response; Immunologic Markers; Impairment; In Vitro; in vitro Model; in vivo; Incidence; Individual; Inflammation Mediators; Inflammatory Response; Influenza; influenzavirus; insight; interest; Life; Link; Lung; Measurable; Measures; methylation pattern; Modeling; Mucosal Immune Responses; Mucositis; Nasal Epithelium; Non-smoker; non-smoking; Nose; novel; Outcome; Play; Population; primary outcome; Publishing; respiratory; respiratory infection virus; respiratory virus; response; response biomarker; Role; Sampling; Severities; Smoke; Smoker; Smoking; Structure of respiratory epithelium; System; Testing; Tobacco; tobacco exposure; tool; translational study; Viral; Virus Diseases; Virus Replication; volunteer;

DESCRIPTION (provided by applicant): Ozone is one of the most commonly encountered environmental pollutants and human exposures to increased levels of this reactive molecule are clearly linked to proinflammatory responses and exacerbation of respiratory illness. Unsaturated lipids are particularly vulnerable to ozone and a number of electrophilic aldehydes and epoxides are known as primary products of lipid ozone exposure. Cholesterol, for example, reacts readily with ozone and gives oxysterol products that result from conversion of the ring- B double bond of the sterol to reactive carbonyl and epoxide functionality. Endogenously formed oxysterols are well-known ligands for the liver-X-receptor (LXR), which regulates the expression of genes involved in cholesterol homeostasis, fatty acid synthesis, and reverse cholesterol transport. More recent studies also implicate LXR in having potent anti-inflammatory and immune regulatory function. In contrast to endogenously formed oxysterols, some of the ozone-derived oxysterols inhibit LXR function, yet the role of this mechanism in ozone pathogenesis is known. Ozone is known to modify cellular function and activate epithelial cells but the biochemical mechanisms of these processes have yet to be defined. Ozone-derived oxysterols are electrophiles that react with common nucleophilic residues present in proteins, forming stable adducts. We contend that the known chemical reactivity of ozone, its presumed exposure to lipids in the airway, the oxidative stress that results from these exposures, and the association of human diseases with environmental insults calls for coordinated studies aimed at discovering the fundamental chemical and biological events linking lipids, environmental exposures and the pathophysiological response. Ozone-derived oxysterols are a common theme in the proposed research and we outline strategies here that are designed to: 1. Provide chemically pure ozone-derived oxysterols to assess the effect of these compounds and their metabolites on the function of epithelial cells, with specific focus on the LXR signaling pathway. 2. Develop methods based on click chemistry and synthetic alkynyl sterol analogs that permit the isolation and identification of oxysterol-protein adducts and define the oxysterol adduction proteome in epithelial cells and macrophages. 3. Utilize synthetic sterol and oxysterol analogs to track and identify lipids in various epithelial cellular compartments. 4. Develop assays for biomarkers of lipid-protein adduction based on our cellular studies. The laboratories at Vanderbilt University and University of North Carolina at Chapel Hill provide a unique combination of expertise for the study of an important environmental problem. This expertise includes skills in chemical synthesis, isolation and characterization coupled with experience studying adverse health effects induced upon exposure to air pollutants and the cellular mechanisms mediating these responses.

PROJECT SUMMARY/ABSTRACT Ozone (O3) continues to be of great public health concern with more than 1/3 of the U.S. population, 122 million people, currently living in areas exceeding the National Ambient Air Quality Standard (NAAQS), which are exposure levels known to cause inflammatory responses in humans. O3 is highly reactive and known to oxidize biomolecules, including unsaturated lipids, such as cholesterol. Yet, how O3-induced chemical reactions translate into intracellular effects presents a knowledge gap. O3-derived products of cholesterol include electrophiles, such as oxysterol, which have the ability to form adducts with nucleophilic centers of proteins, thus affecting cellular signaling. The overall objective of this application is to determine how formation of oxysterols and oxysterol-protein adducts link O3-induced chemical reactions with biological effects. We developed experimental protocols in which airway epithelial cells (ECs) are treated with alkyne-modified O3- derived oxysterols followed by reacting the cell lysates with an azido biotin reagent under ?click? cycloaddition conditions, resulting in the biotinylation of any protein that forms a covalent bond with alkyne-modified oxysterol. This biotinylated protein mixture can be ?pulled down? for proteomic analysis of the ?adductome? or individual oxysterol-protein adduct formation. Using a proteomic screen of oxysterol-protein adducts formed in ECs, we identified NLRP2 as a potential target. Specific Aim 1 will expand these initial studies, characterize the overall protein ?adductome? generated by O3-derived oxysterol in ECs, and focus on the role of oxysterol- adducted NLRP2 in O3-induced pro-inflammatory responses. Specific Aim 2 will focus on how O3-derived oxysterols affect macrophage function and whether similar to ECs, oxysterols form protein adducts in macrophages, thus affecting cellular function. Using a co-culture system composed of ECs and macrophages, this aim will also determine whether oxysterols formed at or near EC membranes communicate with macrophages. Specific Aim 3 will focus on the relationship between 7-dehydrocholesterol (7-DHC), the last step during cholesterol biosynthesis, and O3-induced inflammation. 7-DHC is more susceptible to O3-induced oxysterol formation and we have evidence that increased lung 7-DHC levels correlate with O3-induced inflammation in humans in vivo. Furthermore, we show that modifying 7-DHC levels by commonly prescribed small molecule antidepressants enhance O3-induced inflammation. Using linked in vitro, mouse in vivo, and human in vivo experiments this aim is designed to determine how pharmacologically modulating pulmonary 7- DHC levels could increase the susceptibility to O3-induced inflammation. The findings developed in this study will uncover novel interactions between oxidized lipids and modification of cellular function in the context of O3 exposure and foster a better understanding of how commonly prescribed drugs could sensitize to O3-induced inflammation.

PROJECT SUMMARY/ABSTRACT The need for a diverse environmental health workforce has growing urgency as the population of the planet nears eight billion people and the environmental health impacts of natural disasters and drought become evident on every continent. In addition to understanding how environmental stressors impact human health, scientists of the next generation will need to work across disciplines to develop viable solutions to environmental and social challenges. Yet the disciplines that underpin environmental health sciences?biomedical, clinical and behavioral sciences?lack the diversity that bolsters effective problem solving (Ferrini-Mundy, 2013). More broadly, women, persons with disabilities, and three racial and ethnic groups?people of African American, Hispanic, and/or American Indian and Alaska Native descent?remain underrepresented in science and engineering education programs and employment (NAS, 2011; NSF, 2019). Pursuit of STEM careers requires additional support for all students, especially underrepresented minority and women students who contend with systemic racial and gender bias. For these students, the likelihood of persisting in STEM majors is influenced by opportunities to meaningfully participate in the academic community and develop supportive relationships with faculty and peers (Figueroa et al., 2015; PCAST, 2012; Tsui, 2007). Within this context, the Curriculum in Toxicology & Environmental Medicine (CiTEM) in the UNC- Chapel Hill School of Medicine, together with the UNC Institute for the Environment (UNC-IE) and the Department of Biological and Biomedical Sciences at NC Central University (NCCU), propose an environmental health-focused undergraduate research program to expand and diversify the pool of students entering the environmental health workforce. The 21st Century Environmental Health Scholars program will provide mentored research experiences and professional development for students from diverse backgrounds, with a goal of preparing these students for further studies or research careers in environmental health sciences. We will accomplish this goal through the following aims. Aim 1. Facilitate summer undergraduate research opportunities in environmental health sciences. Aim 2. Facilitate academic-year undergraduate research experiences and broaden professional development to include environmental health sciences careers, science communication, and community engagement. Aim 3. Implement robust mentorship training that incorporates evidence-based practices in mentoring diverse students and cohort building opportunities. Over five years, this program will prepare 25 diverse undergraduates for careers in environmental health sciences, introducing them to cutting-edge research topics and facilitating essential skills development. At the same time, participating mentors will enhance their ability to create and sustain inclusive learning environments. Together, these activities will encourage the participation of individuals from diverse backgrounds to pursue environmental health sciences research and careers.

PROJECT SUMMARY/ABSTRACT Chronic obstructive pulmonary disease (COPD) is a progressive pulmonary disease characterized by frequent episodes of acute deterioration termed acute exacerbations of COPD (AECOPD). The underlying biological mechanisms and risk profiles contributing to AECOPD are poorly understood. The nasal mucosal environment, including the inflammatory state and nasal microbiome, play a role in host defenses against viral infections. Susceptibility to viral infections, a trigger of AECOPD, is a potential mechanism for the COPD frequent exacerbator phenotype. Our research group has established approaches to rigorously characterize and quantify the nasal mucosal immune environment and microbiome, as well as determine the functional status of the nasal immune environment with a controlled Live Attenuated Influenza Vaccine (LAIV) exposure. Our overall objective is to determine the immune alterations and functional implications of changes in the nasal immune environment and microbiome present in individuals with frequent AECOPD. We hypothesize that dysfunctional nasal immunity underlies the frequent AECOPD phenotype and therefore presents a novel mechanism of increased AECOPD risk. We hypothesize that these immune alterations will impact responses to controlled viral infection and could predict the course of naturally occurring AECOPD. We will define the nasal immune environment (Aim 1) and microbiome (Aim 2) of frequent exacerbators, infrequent exacerbators, and healthy non-smokers, using cluster analyses to define the nasal ?biological fingerprint? of the frequent AECOPD phenotype. Through longitudinal assessments embedded in these Aims we will define the variations of these measures during stable state and during AECOPD. We will then use a Live Attenuated Influenza Vaccine (LAIV) model to determine the functional implications of differences in the respiratory immune environment of frequent and infrequent exacerbators during a controlled viral challenge (Aim 3). Successful completion of these aims will lead to the first rigorous characterization of the nasal mucosal and microbiome changes associated with frequent exacerbators of COPD, validate these findings in a controlled experimental setting, and advance a novel hypothesis that the frequent exacerbator phenotype arises from altered nasal immune responses and microbiome constitution.