Publications by authors named "Ilona Jaspers"

113 Publications

Distinguishing Human Peripheral Blood NK Cells from CD56CD16CD69CD103 Resident Nasal Mucosal Lavage Fluid Cells.

Sci Rep 2018 02 21;8(1):3394. Epub 2018 Feb 21.

Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Natural killer (NK) cells are members of the innate lymphoid cells group 1 (ILC1s), which play a critical role in innate host defense against viruses and malignancies. While many studies have examined the role of circulating peripheral blood (PB) CD56 NK cells, little is known about the resident CD56 cell population. Therefore, matched CD56 cells from nasal lavage fluid (NLF) and PB of smokers and non-smokers were compared phenotypically, via flow cytometry, and functionally, via NK-cell specific gene expression. NLF and PB CD56 cells had similar expression of CD56, but differentially expressed tissue residency (CD69 and CD103) and cytotoxicity (CD16) markers. In addition, NLF CD56 cells expressed lower levels of cytotoxicity-associated genes, perforin (PRF1) and granzyme B (GZMB), and increased levels of cytokines and cell signaling molecules, TRAIL, IFNGR2, and IL8, as compared to PB CD56 cells. In smokers, ITGA2 was downregulated in NLF CD56 cells, while markers of cytotoxic function were primarily downregulated in PB CD56 NK cells. Overall, NLF CD56 cells are a unique cell population that likely play a role in orchestrating innate immune responses in the nasal cavity, which is distinct from their role as a non-antigen-restricted cytotoxic CD56 lymphocytes in the PB.
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http://dx.doi.org/10.1038/s41598-018-21443-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821812PMC
February 2018

Interleukin-13 stimulates production of nitric oxide in cultured human nasal epithelium.

In Vitro Cell Dev Biol Anim 2018 Mar 29;54(3):200-204. Epub 2018 Jan 29.

The Center for Environmental Medicine, Asthma, and Lung Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

The diversity and extent of signaling functions of nitric oxide (NO) in cell physiology as well as its presence and influence as a common component of ambient air pollution and tobacco smoke are gaining increasing research attention relative to both health and disease. While cellular NO production is typically associated with inflammatory cells and processes, the airway epithelium particularly of the paranasal sinuses, has been documented to be a rich source of excreted NO. Inasmuch as excreted NO derives from both mucosal and inflammatory cell sources, distinguishing the individual contribution of these compartments to total excreted cellular NO is potentially problematic. We simulated an inflammatory mucosal environment by stimulating human nasal epithelial cultures with interleukin-13 (IL-13), a mediator produced by eosinophils in asthma, allergic rhinitis, and sinusitis. While a consistent baseline of NO excretion in control cultures was documented, widely variable individual responses to IL-13 exposure were observed in companion cultures maintained under identical conditions and tested at the same time. These studies suggest that cellular NO excretion by the healthy epithelial mucosa is subject to considerable individual variability and may be significantly elevated among some individuals in the presence of IL-13 stimulation.
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http://dx.doi.org/10.1007/s11626-018-0233-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6337725PMC
March 2018

Effect of secondary organic aerosol from isoprene-derived hydroxyhydroperoxides on the expression of oxidative stress response genes in human bronchial epithelial cells.

Environ Sci Process Impacts 2018 Feb;20(2):332-339

Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Isoprene-derived secondary organic aerosol (SOA), which comprise a large portion of atmospheric fine particulate matter (PM), can be formed through various gaseous precursors, including isoprene epoxydiols (IEPOX), methacrylic acid epoxide (MAE), and isoprene hydroxyhydroperoxides (ISOPOOH). The composition of the isoprene-derived SOA affects its reactive oxygen species (ROS) generation potential and its ability to alter oxidative stress-related gene expression. In this study we assess effects of isoprene SOA derived solely from ISOPOOH oxidation on human bronchial epithelial cells by measuring the gene expression changes in 84 oxidative stress-related genes. In addition, the thiol reactivity of ISOPOOH-derived SOA was measured through the dithiothreitol (DTT) assay. Our findings show that ISOPOOH-derived SOA alter more oxidative-stress related genes than IEPOX-derived SOA but not as many as MAE-derived SOA on a mass basis exposure. More importantly, we found that the different types of SOA derived from the various gaseous precursors (MAE, IEPOX, and ISOPOOH) have unique contributions to changes in oxidative stress-related genes that do not total all gene expression changes seen in exposures to atmospherically relevant compositions of total isoprene-derived SOA mixtures. This study suggests that amongst the different types of known isoprene-derived SOA, MAE-derived SOA are the most potent inducer of oxidative stress-related gene changes but highlights the importance of considering isoprene-derived SOA as a total mixture for pollution controls and exposure studies.
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http://dx.doi.org/10.1039/c7em00439gDOI Listing
February 2018

Nasospheroids permit measurements of CFTR-dependent fluid transport.

JCI Insight 2017 11 16;2(22). Epub 2017 Nov 16.

Marsico Lung Institute/Cystic Fibrosis Research Center.

Expansion of novel therapeutics to all patients with cystic fibrosis (CF) requires personalized CFTR modulator therapy. We have developed nasospheroids, a primary cell culture-based model derived from individual CF patients and healthy subjects by a minimally invasive nasal biopsy. Confocal microscopy was utilized to measure CFTR activity by analyzing changes in cross-sectional area over time that resulted from CFTR-mediated ion and fluid movement. Both the rate of change over time and AUC were calculated. Non-CF nasospheroids with active CFTR-mediated ion and fluid movement showed a reduction in cross-sectional area, whereas no changes were observed in CF spheroids. Non-CF spheroids treated with CFTR inhibitor lost responsiveness for CFTR activation. However, nasospheroids from F508del CF homozygotes that were treated with lumacaftor and ivacaftor showed a significant reduction in cross-sectional area, indicating pharmacologic rescue of CFTR function. This model employs a simple measurement of size corresponding to changes in CFTR activity and is applicable for detection of small changes in CFTR activity from individual patients in vitro. Advancements of this technique will provide a robust model for individualized prediction of CFTR modulator efficacy.
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http://dx.doi.org/10.1172/jci.insight.95734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752372PMC
November 2017

E-Cigarette Use Causes a Unique Innate Immune Response in the Lung, Involving Increased Neutrophilic Activation and Altered Mucin Secretion.

Am J Respir Crit Care Med 2018 02;197(4):492-501

1 Marsico Lung Institute.

Rationale: E-cigarettes have become increasingly popular and little is known about their potential adverse health effects.

Objectives: To determine the effects of e-cigarette use on the airways.

Methods: Induced sputum samples from cigarette smokers, e-cigarette users, and nonsmokers were analyzed by quantitative proteomics, and the total and individual concentrations of mucins MUC5AC and MUC5B were determined by light scattering/refractometry and labeled mass spectrometry, respectively. Neutrophil extracellular trap (NET) formation rates were also determined for the same groups.

Measurements And Main Results: E-cigarette users exhibited significant increases in aldehyde-detoxification and oxidative stress-related proteins associated with cigarette smoke compared with nonsmokers. The levels of innate defense proteins associated with chronic obstructive pulmonary disease, such as elastase and matrix metalloproteinase-9, were significantly elevated in e-cigarette users as well. E-cigarette users' sputum also uniquely exhibited significant increases in neutrophil granulocyte-related and NET-related proteins, such as myeloperoxidase, azurocidin, and protein-arginine deiminase 4, despite no significant elevation in neutrophil cell counts. Peripheral neutrophils from e-cigarette users showed increased susceptibility to phorbol 12-myristate 13-acetate-induced NETosis. Finally, a compositional change in the gel-forming building blocks of airway mucus (i.e., an elevated concentration of mucin MUC5AC) was observed in both cigarette smokers and e-cigarette users.

Conclusions: Together, our results indicate that e-cigarette use alters the profile of innate defense proteins in airway secretions, inducing similar and unique changes relative to cigarette smoking. These data challenge the concept that e-cigarettes are a healthier alternative to cigarettes.
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http://dx.doi.org/10.1164/rccm.201708-1590OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821909PMC
February 2018

Electronic Cigarettes: Their Constituents and Potential Links to Asthma.

Curr Allergy Asthma Rep 2017 Oct 5;17(11):79. Epub 2017 Oct 5.

Curriculum in Toxicology, School of Medicine, University of North Carolina, Chapel Hill, NC, USA.

Purpose Of Review: Vaping is gaining popularity in the USA, particularly among teens and young adults. While e-cigs are commonly represented as safer alternatives to tobacco cigarettes, little is known regarding the health effects of their short- or long-term use, especially in individuals with pre-existing respiratory diseases such as asthma. Flavored e-cig liquids (e-liquids) and e-cig aerosols contain airway irritants and toxicants that have been implicated in the pathogenesis and worsening of lung diseases. In this review, we will summarize existing data on potential health effects of components present in e-cig aerosols, such as propylene glycol, vegetable glycerin, nicotine, and flavorings, and discuss their relevance in the context of asthma.

Recent Findings: Recent survey data indicate that adolescents with asthma had a higher prevalence of current e-cig use (12.4%) compared to their non-asthmatics peers (10.2%) and conveyed positive beliefs about tobacco products, especially e-cigs. Similarly, a study conducted among high school students from Ontario, Canada, indicated a greater likelihood of e-cig use in asthmatics as compared to their non-asthmatic peers. Availability of different flavorings is often cited as the main reason among youth/adolescents for trying e-cigs or switching from cigarettes to e-cigs. Occupational inhalation of some common food-safe flavoring agents is reported to cause occupational asthma and worsen asthmatic symptoms. Moreover, workplace inhalation exposures to the flavoring agent diacetyl have caused irreversible obstructive airway disease in healthy workers. Additionally, recent studies report that thermal decomposition of propylene glycol (PG) and vegetable glycerin (VG), the base constituents of e-liquids, produces reactive carbonyls, including acrolein, formaldehyde, and acetaldehyde, which have known respiratory toxicities. Furthermore, recent nicotine studies in rodents reveal that prenatal nicotine exposures lead to epigenetic reprogramming in the offspring, abnormal lung development, and multigenerational transmission of asthmatic-like symptoms. Comparisons of the toxicity and health effects of e-cigs and conventional cigarettes often focus on toxicants known to be present in cigarette smoke (CS) (i.e., formaldehyde, nitrosamines, etc.), as well as smoking-associated clinical endpoints, such as cancer, bronchitis, and chronic obstructive pulmonary disease (COPD). However, this approach disregards potential toxicity of components unique to flavored e-cigs, such as PG, VG, and the many different flavoring chemicals, which likely induce respiratory effects not usually observed in cigarette smokers.
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http://dx.doi.org/10.1007/s11882-017-0747-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5995565PMC
October 2017

Gene Expression Profiling in Human Lung Cells Exposed to Isoprene-Derived Secondary Organic Aerosol.

Environ Sci Technol 2017 Jul 5;51(14):8166-8175. Epub 2017 Jul 5.

Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, ‡Center for Environmental Medicine, Asthma and Lung Biology, School of Medicine, and §Department of Pediatrics, School of Medicine, The University of North Carolina , Chapel Hill, North Carolina 27599, United States.

Secondary organic aerosol (SOA) derived from the photochemical oxidation of isoprene contributes a substantial mass fraction to atmospheric fine particulate matter (PM). The formation of isoprene SOA is influenced largely by anthropogenic emissions through multiphase chemistry of its multigenerational oxidation products. Considering the abundance of isoprene SOA in the troposphere, understanding mechanisms of adverse health effects through inhalation exposure is critical to mitigating its potential impact on public health. In this study, we assessed the effects of isoprene SOA on gene expression in human airway epithelial cells (BEAS-2B) through an air-liquid interface exposure. Gene expression profiling of 84 oxidative stress and 249 inflammation-associated human genes was performed. Our results show that the expression levels of 29 genes were significantly altered upon isoprene SOA exposure under noncytotoxic conditions (p < 0.05), with the majority (22/29) of genes passing a false discovery rate threshold of 0.3. The most significantly affected genes belong to the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor network. The Nrf2 function is confirmed through a reporter cell line. Together with detailed characterization of SOA constituents, this study reveals the impact of isoprene SOA exposure on lung responses and highlights the importance of further understanding its potential health outcomes.
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http://dx.doi.org/10.1021/acs.est.7b01967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5610912PMC
July 2017

Flavored e-cigarette liquids and cinnamaldehyde impair respiratory innate immune cell function.

Am J Physiol Lung Cell Mol Physiol 2017 08 11;313(2):L278-L292. Epub 2017 May 11.

Curriculum in Toxicology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina;

Innate immune cells of the respiratory tract are the first line of defense against pathogenic and environmental insults. Failure of these cells to perform their immune functions leaves the host susceptible to infection and may contribute to impaired resolution of inflammation. While combustible tobacco cigarettes have been shown to suppress respiratory immune cell function, the effects of flavored electronic cigarette liquids (e-liquids) and individual flavoring agents on respiratory immune cell responses are unknown. We investigated the effects of seven flavored nicotine-free e-liquids on primary human alveolar macrophages, neutrophils, and natural killer (NK) cells. Cells were challenged with a range of e-liquid dilutions and assayed for their functional responses to pathogenic stimuli. End points included phagocytic capacity (neutrophils and macrophages), neutrophil extracellular trap formation, proinflammatory cytokine production, and cell-mediated cytotoxic response (NK cells). E-liquids were then analyzed via mass spectrometry to identify individual flavoring components. Three cinnamaldehyde-containing e-liquids exhibited dose-dependent broadly immunosuppressive effects. Quantitative mass spectrometry was used to determine concentrations of cinnamaldehyde in each of the three e-liquids, and cells were subsequently challenged with a range of cinnamaldehyde concentrations. Cinnamaldehyde alone recapitulated the impaired function observed with e-liquid exposures, and cinnamaldehyde-induced suppression of macrophage phagocytosis was reversed by addition of the small-molecule reducing agent 1,4-dithiothreitol. We conclude that cinnamaldehyde has the potential to impair respiratory immune cell function, illustrating an immediate need for further toxicological evaluation of chemical flavoring agents to inform regulation governing their use in e-liquid formulations.
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http://dx.doi.org/10.1152/ajplung.00452.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5582929PMC
August 2017

Temporal structure/function variation in cultured differentiated human nasal epithelium associated with acute single exposure to tobacco smoke or E-cigarette vapor.

Inhal Toxicol 2017 02;29(3):137-144

d The Department of Medicine , The University of North Carolina at Chapel Hill , Chapel Hill , NC , USA.

Objective: Mucociliary clearance sustains a baseline functionality and an "on demand" capability to upregulate clearance upon irritant exposure involving mucus hypersecretion and accelerated ciliary beat frequency (CBF) modulated by nitric oxide (NO). This study characterized these elements as well as cellular and exogenous NO concentrations subsequent to a single exposure to tobacco smoke (TS) or e-cigarette vapor (EV) on cultured human airway epithelium.

Materials And Methods: Air-liquid interface (ALI) airway epithelial cultures per nonsmoking human subjects were subjected to single TS or EV exposures. Measures of ciliary function and secretion were performed and cellular and exogenous NO concentrations under control and experimental conditions were assessed.

Results: Both TS and EV exposures resulted similar patterns of decline in CBF within 1 min of the completion of exposure followed by a gradual return often exceeding baseline within 1 h. Post-exposure examination of exposed cultures suggested morphologic differences in secretory function relative to controls. The relative NO concentrations of TS and EV chamber air were sharply different with EV NO being only slightly elevated relative to cellular NO production.

Discussion And Conclusions: Epithelial remodeling and mucociliary dysfunction have been clearly associated with TS exposure. However, information contrasting epithelial structure/function following a single acute TS or EV exposure is limited. This study demonstrates a similar pattern of epithelial response to acute TS or EV exposure. Inasmuch as NO may contribute to an inflammatory milieu and generation of toxic metabolites, it is plausible that recurrent exposures over time may be contributory to chronic pathologies.
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http://dx.doi.org/10.1080/08958378.2017.1318985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5545111PMC
February 2017

Alterations in airway microbiota in patients with PaO2/FiO2 ratio ≤ 300 after burn and inhalation injury.

PLoS One 2017 30;12(3):e0173848. Epub 2017 Mar 30.

National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Chapel Hill, North Carolina, United States of America.

Background: Injury to the airways after smoke inhalation is a major mortality risk factor in victims of burn injuries, resulting in a 15-45% increase in patient deaths. Damage to the airways by smoke may induce acute respiratory distress syndrome (ARDS), which is partly characterized by hypoxemia in the airways. While ARDS has been associated with bacterial infection, the impact of hypoxemia on airway microbiota is unknown. Our objective was to identify differences in microbiota within the airways of burn patients who develop hypoxemia early after inhalation injury and those that do not using next-generation sequencing of bacterial 16S rRNA genes.

Results: DNA was extracted from therapeutic bronchial washings of 48 patients performed within 72 hours of hospitalization for burn and inhalation injury at the North Carolina Jaycee Burn Center. DNA was prepared for sequencing using a novel molecule tagging method and sequenced on the Illumina MiSeq platform. Bacterial species were identified using the MTToolbox pipeline. Patients with hypoxemia, as indicated by a PaO2/FiO2 ratio ≤ 300, had a 30% increase in abundance of Streptococcaceae and Enterobacteriaceae and 84% increase in Staphylococcaceae as compared to patients with a PaO2/FiO2 ratio > 300. Wilcoxon rank-sum test identified significant enrichment in abundance of OTUs identified as Prevotella melaninogenica (p = 0.042), Corynebacterium (p = 0.037) and Mogibacterium (p = 0.048). Linear discriminant effect size analysis (LefSe) confirmed significant enrichment of Prevotella melaninognica among patients with a PaO2/FiO2 ratio ≤ 300 (p<0.05). These results could not be explained by differences in antibiotic treatment.

Conclusions: The airway microbiota following burn and inhalation injury is altered in patients with a PaO2/FiO2 ratio ≤ 300 early after injury. Enrichment of specific taxa in patients with a PaO2/FiO2 ratio ≤ 300 may indicate airway environment and patient changes that favor these microbes. Longitudinal studies are necessary to identify stably colonizing taxa that play roles in hypoxemia and ARDS pathogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0173848PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373524PMC
August 2017

Novel applications for a noninvasive sampling method of the nasal mucosa.

Am J Physiol Lung Cell Mol Physiol 2017 Feb 23;312(2):L288-L296. Epub 2016 Dec 23.

Curriculum in Toxicology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina;

Reliable methods for sampling the nasal mucosa provide clinical researchers with key information regarding respiratory biomarkers of exposure and disease. For quick and noninvasive sampling of the nasal mucosa, nasal lavage (NL) collection has been widely used as a clinical tool; however, limitations including volume variability, sample dilution, and storage prevent NL collection from being used in nonlaboratory settings and analysis of low abundance biomarkers. In this study, we optimize and validate a novel methodology using absorbent Leukosorb paper cut to fit the nasal passage to extract epithelial lining fluid (ELF) from the nasal mucosa. The ELF sampling method limits the dilution of soluble mediators, allowing quantification of both high- and low-abundance soluble biomarkers such as IL-1β, IL-8, IL-6, interferon gamma-induced protein 10 (IP-10), and neutrophil elastase. Additionally, we demonstrate that this method can successfully detect the presence of respiratory pathogens such as influenza virus and markers of antibiotic-resistant bacteria in the nasal mucosa. Efficacy of ELF collection by this method is not diminished in consecutive-day sampling, and percent recovery of both recombinant IL-8 and soluble mediators are not changed despite freezing or room temperature storage for 24 h. Our results indicate that ELF collection using Leukosorb paper sampling of ELF provides a sensitive, easy-to-use, and reproducible methodology to collect concentrated amounts of soluble biomarkers from the nasal mucosa. Moreover, the methodology described herein improves upon the standard NL collection method and provides researchers with a novel tool to assess changes in nasal mucosal host defense status.
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http://dx.doi.org/10.1152/ajplung.00476.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5336583PMC
February 2017

Ozone-derived Oxysterols Affect Liver X Receptor (LXR) Signaling: A POTENTIAL ROLE FOR LIPID-PROTEIN ADDUCTS.

J Biol Chem 2016 Nov 4;291(48):25192-25206. Epub 2016 Oct 4.

From the Curriculum in Toxicology, Departments of Pediatrics and Microbiology and Immunology, Center for Environmental Medicine, Asthma, and Lung Biology, University of North Carolina, Chapel Hill, North Carolina 27599,

When inhaled, ozone (O) interacts with cholesterols of airway epithelial cell membranes or the lung-lining fluid, generating chemically reactive oxysterols. The mechanism by which O-derived oxysterols affect molecular function is unknown. Our data show that in vitro exposure of human bronchial epithelial cells to O results in the formation of oxysterols, epoxycholesterol-α and -β and secosterol A and B (Seco A and Seco B), in cell lysates and apical washes. Similarly, bronchoalveolar lavage fluid obtained from human volunteers exposed to O contained elevated levels of these oxysterol species. As expected, O-derived oxysterols have a pro-inflammatory effect and increase NF-κB activity. Interestingly, expression of the cholesterol efflux pump ATP-binding cassette transporter 1 (ABCA1), which is regulated by activation of the liver X receptor (LXR), was suppressed in epithelial cells exposed to O Additionally, exposure of LXR knock-out mice to O enhanced pro-inflammatory cytokine production in the lung, suggesting LXR inhibits O-induced inflammation. Using alkynyl surrogates of O-derived oxysterols, our data demonstrate adduction of LXR with Seco A. Similarly, supplementation of epithelial cells with alkynyl-tagged cholesterol followed by O exposure causes observable lipid-LXR adduct formation. Experiments using Seco A and the LXR agonist T0901317 (T09) showed reduced expression of ABCA1 as compared with stimulation with T0901317 alone, indicating that Seco A-LXR protein adduct formation inhibits LXR activation by traditional agonists. Overall, these data demonstrate that O-derived oxysterols have pro-inflammatory functions and form lipid-protein adducts with LXR, thus leading to suppressed cholesterol regulatory gene expression and providing a biochemical mechanism mediating O-derived formation of oxidized lipids in the airways and subsequent adverse health effects.
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http://dx.doi.org/10.1074/jbc.M116.732362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5122785PMC
November 2016

Response to comments by Emma et al.

Authors:
Ilona Jaspers

Am J Physiol Lung Cell Mol Physiol 2016 08;311(2):L526

Department of Pediatrics, School of Medicine, University of North Carolina, Chapel Hill, North Carolina; Curriculum in Toxicology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina; and Center for Environmental Medicine, Asthma, and Lung Biology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina

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http://dx.doi.org/10.1152/ajplung.00313.2016DOI Listing
August 2016

E-cigarette use results in suppression of immune and inflammatory-response genes in nasal epithelial cells similar to cigarette smoke.

Am J Physiol Lung Cell Mol Physiol 2016 07 10;311(1):L135-44. Epub 2016 Jun 10.

Curriculum in Toxicology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina; Center for Environmental Medicine, Asthma, and Lung Biology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina; and Division of Clinical Pharmacology, Departments of Medicine and Bioengineering & Therapeutic Sciences, University of California San Francisco, San Francisco, California

Exposure to cigarette smoke is known to result in impaired host defense responses and immune suppressive effects. However, the effects of new and emerging tobacco products, such as e-cigarettes, on the immune status of the respiratory epithelium are largely unknown. We conducted a clinical study collecting superficial nasal scrape biopsies, nasal lavage, urine, and serum from nonsmokers, cigarette smokers, and e-cigarette users and assessed them for changes in immune gene expression profiles. Smoking status was determined based on a smoking history and a 3- to 4-wk smoking diary and confirmed using serum cotinine and urine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) levels. Total RNA from nasal scrape biopsies was analyzed using the nCounter Human Immunology v2 Expression panel. Smoking cigarettes or vaping e-cigarettes resulted in decreased expression of immune-related genes. All genes with decreased expression in cigarette smokers (n = 53) were also decreased in e-cigarette smokers. Additionally, vaping e-cigarettes was associated with suppression of a large number of unique genes (n = 305). Furthermore, the e-cigarette users showed a greater suppression of genes common with those changed in cigarette smokers. This was particularly apparent for suppressed expression of transcription factors, such as EGR1, which was functionally associated with decreased expression of 5 target genes in cigarette smokers and 18 target genes in e-cigarette users. Taken together, these data indicate that vaping e-cigarettes is associated with decreased expression of a large number of immune-related genes, which are consistent with immune suppression at the level of the nasal mucosa.
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http://dx.doi.org/10.1152/ajplung.00170.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967187PMC
July 2016

Diesel exposure suppresses natural killer cell function and resolution of eosinophil inflammation: a randomized controlled trial of exposure in allergic rhinitics.

Part Fibre Toxicol 2016 05 6;13(1):24. Epub 2016 May 6.

Center for Environmental Medicine, Asthma and Lung Biology, University of North Carolina at Chapel Hill, 104 Mason Farm Rd, Campus Box 7310, Chapel Hill, NC, 27599-7310, USA.

Exposure to diesel exhaust (DE) is known to exacerbate allergic inflammation, including virus-induced eosinophil activation in laboratory animals. We have previously shown that in human volunteers with allergic rhinitis a short-term exposure to DE prior to infection with the live attenuated influenza virus (LAIV) increases markers of allergic inflammation in the nasal mucosa. Specifically, levels of eosinophilic cationic protein (ECP) were significantly enhanced in individuals exposed to DE prior to inoculation with LAIV and this effect was maintained for at least seven days. However, this previous study was limited in its scope of nasal immune endpoints and did not explore potential mechanisms mediating the prolonged exacerbation of allergic inflammation caused by exposure to DE prior to inoculation with LAIV. In this follow-up study, the methods were modified to expand experimental endpoints and explore the potential role of NK cells. The data presented here suggest DE prolongs viral-induced eosinophil activation, which was accompanied by decreased markers of NK cell recruitment and activation. Separate in vitro studies showed that exposure to DE particles decreases the ability of NK cells to kill eosinophils. Taken together, these follow-up studies suggest that DE-induced exacerbation of allergic inflammation in the context of viral infections may be mediated by decreased activity of NK cells and their ability to clear eosinophils.
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http://dx.doi.org/10.1186/s12989-016-0135-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4859992PMC
May 2016

Assessment of biological responses of EpiAirway 3-D cell constructs versus A549 cells for determining toxicity of ambient air pollution.

Inhal Toxicol 2016 ;28(6):251-9

a Department of Environmental Sciences & Engineering , University of North Carolina at Chapel Hill , Chapel Hill , NC, USA .

Context: EpiAirway™ 3-D constructs are human-derived cell cultures of differentiated airway epithelial cells that may represent a more biologically relevant model of the human lung. However, limited information is available on their utility for exposures to air pollutants at the air-liquid interface (ALI).

Objective: To assess the biological responses of EpiAirway™ cells in comparison to the responses of A549 human alveolar epithelial cells after exposure to air pollutants at ALI.

Methods: Cells were exposed to filtered air, 400 ppb of ozone (O3) or a photochemically aged Synthetic Urban Mixture (SynUrb54) consisting of hydrocarbons, nitrogen oxides, O3 and other secondary oxidation products for 4 h. Basolateral supernatants and apical washes were collected at 9 and 24 h post-exposure. We assessed cytotoxicity by measuring lactate dehydrogenase (LDH) release into the culture medium and apical surface. Interleukin 6 (IL-6) and interleukin 8 (IL-8) proteins were measured in the culture medium and in the apical washes to determine the inflammatory response after exposure.

Results: Both O3 and SynUrb54 significantly increased basolateral levels of LDH and IL-8 in A549 cells. No significant changes in LDH and IL-8 levels were observed in the EpiAirway™ cells, however, IL-6 in the apical surface was significantly elevated at 24 h after O3 exposure.

Conclusion: LDH and IL-8 are robust endpoints for assessing toxicity in A549 cells. The EpiAirway™ cells show minimal adverse effects after exposure suggesting that they are more toxicologically resistant compared to A549 cells. Higher concentrations or longer exposure times are needed to induce effects on EpiAirway™ cells.
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http://dx.doi.org/10.3109/08958378.2016.1157227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4913276PMC
January 2017

Effect of Broccoli Sprouts and Live Attenuated Influenza Virus on Peripheral Blood Natural Killer Cells: A Randomized, Double-Blind Study.

PLoS One 2016 28;11(1):e0147742. Epub 2016 Jan 28.

Center for Environmental Medicine, Asthma and Lung Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

Unlabelled: Enhancing antiviral host defense responses through nutritional supplementation would be an attractive strategy in the fight against influenza. Using inoculation with live attenuated influenza virus (LAIV) as an infection model, we have recently shown that ingestion of sulforaphane-containing broccoli sprout homogenates (BSH) reduces markers of viral load in the nose. To investigate the systemic effects of short-term BSH supplementation in the context of LAIV-inoculation, we examined peripheral blood immune cell populations in non-smoking subjects from this study, with a particular focus on NK cells. We carried out a randomized, double-blinded, placebo-controlled study measuring the effects of BSH (N = 13) or placebo (alfalfa sprout homogenate, ASH; N = 16) on peripheral blood mononuclear cell responses to a standard nasal vaccine dose of LAIV in healthy volunteers. Blood was drawn prior to (day-1) and post (day2, day21) LAIV inoculation and analyzed for neutrophils, monocytes, macrophages, T cells, NKT cells, and NK cells. In addition, NK cells were enriched, stimulated, and assessed for surface markers, intracellular markers, and cytotoxic potential by flow cytometry. Overall, LAIV significantly reduced NKT (day2 and day21) and T cell (day2) populations. LAIV decreased NK cell CD56 and CD158b expression, while significantly increasing CD16 expression and cytotoxic potential (on day2). BSH supplementation further increased LAIV-induced granzyme B production (day2) in NK cells compared to ASH and in the BSH group granzyme B levels appeared to be negatively associated with influenza RNA levels in nasal lavage fluid cells. We conclude that nasal influenza infection may induce complex changes in peripheral blood NK cell activation, and that BSH increases virus-induced peripheral blood NK cell granzyme B production, an effect that may be important for enhanced antiviral defense responses.

Trial Registration: ClinicalTrials.gov NCT01269723.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0147742PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4731143PMC
July 2016

An Allergic Lung Microenvironment Suppresses Carbon Nanotube-Induced Inflammasome Activation via STAT6-Dependent Inhibition of Caspase-1.

PLoS One 2015 19;10(6):e0128888. Epub 2015 Jun 19.

Department of Biological Sciences, Environmental and Molecular Toxicology Program, North Carolina State University, Raleigh, North Carolina, United States of America.

Background: Multi-walled carbon nanotubes (MWCNTs) represent a human health risk as mice exposed by inhalation display pulmonary fibrosis. Production of IL-1β via inflammasome activation is a mechanism of MWCNT-induced acute inflammation and has been implicated in chronic fibrogenesis. Mice sensitized to allergens have elevated T-helper 2 (Th2) cytokines, IL-4 and IL-13, and are susceptible to MWCNT-induced airway fibrosis. We postulated that Th2 cytokines would modulate MWCNT-induced inflammasome activation and IL-1β release in vitro and in vivo during allergic inflammation.

Methods: THP-1 macrophages were primed with LPS, exposed to MWCNTs and/or IL-4 or IL-13 for 24 hours, and analyzed for indicators of inflammasome activation. C57BL6 mice were sensitized to house dust mite (HDM) allergen and MWCNTs were delivered to the lungs by oropharyngeal aspiration. Mice were euthanized 1 or 21 days post-MWCNT exposure and evaluated for lung inflammasome components and allergic inflammatory responses.

Results: Priming of THP-1 macrophages with LPS increased pro-IL-1β and subsequent exposure to MWCNTs induced IL-1β secretion. IL-4 or IL-13 decreased MWCNT-induced IL-1β secretion by THP-1 cells and reduced pro-caspase-1 but not pro-IL-1β. Treatment of THP-1 cells with STAT6 inhibitors, either Leflunomide or JAK I inhibitor, blocked suppression of caspase activity by IL-4 and IL-13. In vivo, MWCNTs alone caused neutrophilic infiltration into the lungs of mice 1 day post-exposure and increased IL-1β in bronchoalveolar lavage fluid (BALF) and pro-caspase-1 immuno-staining in macrophages and airway epithelium. HDM sensitization alone caused eosinophilic inflammation with increased IL-13. MWCNT exposure after HDM sensitization increased total cell numbers in BALF, but decreased numbers of neutrophils and IL-1β in BALF as well as reduced pro-caspase-1 in lung tissue. Despite reduced IL-1β mice exposed to MWCNTs after HDM developed more severe airway fibrosis by 21 days and had increased pro-fibrogenic cytokine mRNAs.

Conclusions: These data indicate that Th2 cytokines suppress MWCNT-induced inflammasome activation via STAT6-dependent down-regulation of pro-caspase-1 and suggest that suppression of inflammasome activation and IL-1β by an allergic lung microenvironment is a mechanism through which MWCNTs exacerbate allergen-induced airway fibrosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0128888PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4474696PMC
April 2016

A comparison of three dispersion media on the physicochemical and toxicological behavior of TiO2 and NiO nanoparticles.

Chem Biol Interact 2015 Jul 9;236:74-81. Epub 2015 May 9.

Center for Environmental Medicine, Asthma, and Lung Biology, School of Medicine, University of North Carolina-Chapel Hill, Chapel Hill, NC, USA. Electronic address:

Unlabelled: Nanomaterials represent a burgeoning field of technological innovation. With the onset of environmental release and commercial product exposure associated with nanomaterial manufacture and proliferation, the concomitant effects on human health remain unknown and demand further investigation. Agglomeration of nanomaterials in biologically relevant media used in in vitro methods further complicates dosing in toxicological study.

Objective: to compare the effects of in vitro dispersion techniques on the physicochemical and toxicological dosimetry of TiO2 (<50 nm) and NiO (<20 nm) nanoparticles and some resulting toxicological endpoints to test for potential effects.

Methods: three media were prepared for A549 and 16hbe14o cells with varying concentrations of TiO2 and NiO nanoparticles. Physicochemical effects were analyzed with dynamic light scattering, ICP-MS, SEM, and TEM. Toxicological effects were determined after stimulation of cells with nanoparticles for 4 and 24h followed by analysis of inflammatory and oxidative stress markers with ELISA and RT-PCR. Our data show that dispersion media differentially affect physicochemical properties and toxicological endpoints. Therefore, we conclude that in vitro nanotoxicology models that use re-suspension methods of exposure yield inconsistent and misleading biological results due to physicochemical variation of particle characteristics and transport processes.
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http://dx.doi.org/10.1016/j.cbi.2015.05.001DOI Listing
July 2015

Respiratory protease/antiprotease balance determines susceptibility to viral infection and can be modified by nutritional antioxidants.

Am J Physiol Lung Cell Mol Physiol 2015 Jun 17;308(12):L1189-201. Epub 2015 Apr 17.

Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; Center for Environmental Medicine, Asthma and Lung Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; and Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

The respiratory epithelium functions as a central orchestrator to initiate and organize responses to inhaled stimuli. Proteases and antiproteases are secreted from the respiratory epithelium and are involved in respiratory homeostasis. Modifications to the protease/antiprotease balance can lead to the development of lung diseases such as emphysema or chronic obstructive pulmonary disease. Furthermore, altered protease/antiprotease balance, in favor for increased protease activity, is associated with increased susceptibility to respiratory viral infections such as influenza virus. However, nutritional antioxidants induce antiprotease expression/secretion and decrease protease expression/activity, to protect against viral infection. As such, this review will elucidate the impact of this balance in the context of respiratory viral infection and lung disease, to further highlight the role epithelial cell-derived proteases and antiproteases contribute to respiratory immune function. Furthermore, this review will offer the use of nutritional antioxidants as possible therapeutics to boost respiratory mucosal responses and/or protect against infection.
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http://dx.doi.org/10.1152/ajplung.00028.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4587599PMC
June 2015

Analysis of sphingosine kinase activity in single natural killer cells from peripheral blood.

Integr Biol (Camb) 2015 Apr;7(4):392-401

Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599, USA.

Sphingosine-1-phosphate (S1P), a lipid second messenger formed upon phosphorylation of sphingosine by sphingosine kinase (SK), plays a crucial role in natural killer (NK) cell proliferation, migration, and cytotoxicity. Dysregulation of the S1P pathway has been linked to a number of immune system disorders and therapeutic manipulation of the pathway has been proposed as a method of disease intervention. However, peripheral blood NK cells, as identified by surface markers (CD56(+)CD45(+)CD3(-)CD16) consist of a highly diverse population with distinct phenotypes and functions and it is unknown whether the S1P pathway is similarly diverse across peripheral blood NK cells. In this work, we measured the phosphorylation of sphingosine-fluorescein (SF) and subsequent metabolism of S1P fluorescein (S1PF) to form hexadecanoic acid fluorescein (HAF) in 111 single NK cells obtained from the peripheral blood of four healthy human subjects. The percentage of SF converted to S1PF or HAF was highly variable amongst the cells ranging from 0% to 100% (S1PF) and 0% to 97% (HAF). Subpopulations of cells with varying levels of S1PF formation and metabolism were readily identified. Across all subjects, the average percentage of SF converted to S1PF or HAF was 37 ± 36% and 12 ± 19%, respectively. NK cell metabolism of SF by the different subjects was also distinct with hierarchical clustering suggesting two possible phenotypes: low (<20%) or high (>50%) producers of S1PF. The heterogeneity of SK and downstream enzyme activity in NK cells may enable NK cells to respond effectively to a diverse array of pathogens as well as incipient tumor cells. NK cells from two subjects were also loaded with S1PF to assess the activity of S1P phosphatase (S1PP), which converts S1P to sphingosine. No NK cells (n = 41) formed sphingosine, suggesting that S1PP was minimally active in peripheral blood NK cells. In contrast to the SK activity, S1PP activity was homogeneous across the peripheral blood NK cells, suggesting a bias in the SK pathway towards proliferation and migration, activities supported by S1P.
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http://dx.doi.org/10.1039/c5ib00007fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4566154PMC
April 2015

Association between early airway damage-associated molecular patterns and subsequent bacterial infection in patients with inhalational and burn injury.

Am J Physiol Lung Cell Mol Physiol 2015 May 13;308(9):L855-60. Epub 2015 Mar 13.

Department of Pediatrics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; Center for Environmental Medicine, Asthma and Lung Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; and.

Bacterial infection is a major cause of morbidity affecting outcome following burn and inhalation injury. While experimental burn and inhalation injury animal models have suggested that mediators of cell damage and inflammation increase the risk of infection, few studies have been done on humans. This is a prospective, observational study of patients admitted to the North Carolina Jaycee Burn Center at the University of North Carolina who were intubated and on mechanical ventilation for treatment of burn and inhalational injury. Subjects were enrolled over a 2-yr period and followed till discharge or death. Serial bronchial washings from clinically indicated bronchoscopies were collected and analyzed for markers of tissue injury and inflammation. These include damage-associated molecular patterns (DAMPs) such as hyaluronic acid (HA), double-stranded DNA (dsDNA), heat-shock protein 70 (HSP-70), and high-mobility group protein B-1 (HMGB-1). The study population was comprised of 72 patients who had bacterial cultures obtained for clinical indications. Elevated HA, dsDNA, and IL-10 levels in bronchial washings obtained early (the first 72 h after injury) were significantly associated with positive bacterial respiratory cultures obtained during the first 14 days postinjury. Independent of initial inhalation injury severity and extent of surface burn, elevated levels of HA dsDNA and IL-10 in the central airways obtained early after injury are associated with subsequent positive bacterial respiratory cultures in patients intubated after acute burn/inhalation injury.
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http://dx.doi.org/10.1152/ajplung.00321.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4421787PMC
May 2015

Application of chemical vapor generation systems to deliver constant gas concentrations for in vitro exposure to volatile organic compounds.

Environ Sci Process Impacts 2014 Dec;16(12):2703-10

Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Exposure to volatile organic compounds from outdoor air pollution is a major public health concern; however, there is scant information about the health effects induced by inhalation exposure to photochemical transformed products of primary emissions. In this study, we present a stable and reproducible exposure method to deliver ppm-ppb levels of gaseous standards in a humidified air stream for in vitro cell exposure through a direct air-liquid interface. Gaseous species were generated from a diffusion vial, and coupled to a gas-phase in vitro exposure system. Acrolein and methacrolein, which are major first-generation photochemical transformation products of 1,3-butadiene and isoprene, respectively, were selected as model compounds. A series of vapor concentrations (0.23-2.37 ppmv for acrolein and 0.68-10.7 ppmv for methacrolein) were investigated to characterize the exposure dose-response relationships. Temperature and the inner diameter of the diffusion vials are key parameters to control the evaporation rates and diffusion rates for the delivery of target vapor concentrations. Our findings suggest that this exposure method can be used for testing a wide range of atmospheric volatile organic compounds, and permits both single compound and multiple compound sources to generate mixtures in air. The relative standard deviations (%RSD) of output concentrations were within 10% during the 4-hour exposure time. The comparative exposure-response data allow us to prioritize numerous hazardous gas phase air pollutants. These identified pollutants can be further incorporated into air quality simulation models to better characterize the environmental health risks arising from inhalation of the photochemical transformed products.
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http://dx.doi.org/10.1039/c4em00465eDOI Listing
December 2014

Phenotypic modification of human airway epithelial cells in air-liquid interface culture induced by exposure to the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

Ultrastruct Pathol 2015 Apr 2;39(2):104-9. Epub 2014 Oct 2.

Department of Pediatrics, The University of North Carolina at Chapel Hill , Chapel Hill, NC , USA and.

The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogen. We used an air-liquid interface epithelial cell culture system to model changes associated with NNK exposure relative to pathologies documented in human tobacco-related illnesses. Although in vitro systems exhibit certain limitations, they often offer accentuation of subtle pathologies. While the distribution of cell types in control cultures typically favors the ciliated cell phenotype, NNK-exposed cultures transitioned to non-ciliated cell phenotypes as well as reflecting features consistent with squamous metaplasia. We conclude that NNK impacts normal growth and differentiation of human airway epithelium in a short interval of time in vitro.
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http://dx.doi.org/10.3109/01913123.2014.960546DOI Listing
April 2015

Interaction with epithelial cells modifies airway macrophage response to ozone.

Am J Respir Cell Mol Biol 2015 Mar;52(3):285-94

1 Curriculum in Toxicology.

The initial innate immune response to ozone (O3) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived signals affect Mac response to O3. Macs from the bronchoalveolar lavage (BAL) of healthy volunteers were cocultured with the human bronchial epithelial (16HBE) or alveolar (A549) epithelial cell lines. Cocultures, Mac monocultures, and epithelial cell monocultures were exposed to O3 or air, and Mac immunophenotype, phagocytosis, and cytotoxicity were assessed. Quantities of hyaluronic acid (HA) and IL-8 were compared across cultures and in BAL fluid from healthy volunteers exposed to O3 or air for in vivo confirmation. We show that Macs in coculture had increased markers of alternative activation, enhanced cytotoxicity, and reduced phagocytosis compared with Macs in monoculture that differed based on coculture with A549 or 16HBE. Production of HA by epithelial cell monocultures was not affected by O3, but quantities of HA in the in vitro coculture and BAL fluid from volunteers exposed in vivo were increased with O3 exposure, indicating that O3 exposure impairs Mac regulation of HA. Together, we show epithelial cell-Mac coculture models that have many similarities to the in vivo responses to O3, and demonstrate that epithelial cell-derived signals are important determinants of Mac immunophenotype and response to O3.
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http://dx.doi.org/10.1165/rcmb.2014-0035OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4370258PMC
March 2015

The Gillings Sampler--an electrostatic air sampler as an alternative method for aerosol in vitro exposure studies.

Chem Biol Interact 2014 Sep 7;220:158-68. Epub 2014 Jul 7.

Department of Environmental Sciences & Engineering, University of North Carolina at Chapel Hill, United States. Electronic address:

There is growing interest in studying the toxicity and health risk of exposure to multi-pollutant mixtures found in ambient air, and the U.S. Environmental Protection Agency (EPA) is moving towards setting standards for these types of mixtures. Additionally, the Health Effects Institute's strategic plan aims to develop and apply next-generation multi-pollutant approaches to understanding the health effects of air pollutants. There's increasing concern that conventional in vitro exposure methods are not adequate to meet EPA's strategic plan to demonstrate a direct link between air pollution and health effects. To meet the demand for new in vitro technology that better represents direct air-to-cell inhalation exposures, a new system that exposes cells at the air-liquid interface was developed. This new system, named the Gillings Sampler, is a modified two-stage electrostatic precipitator that provides a viable environment for cultured cells. Polystyrene latex spheres were used to determine deposition efficiencies (38-45%), while microscopy and imaging techniques were used to confirm uniform particle deposition. Negative control A549 cell exposures indicated the sampler can be operated for up to 4h without inducing any significant toxic effects on cells, as measured by lactate dehydrogenase (LDH) and interleukin-8 (IL-8). A novel positive aerosol control exposure method, consisting of a p-tolualdehyde (TOLALD) impregnated mineral oil aerosol (MOA), was developed to test this system. Exposures to the toxic MOA at a 1 ng/cm(2) dose of TOLALD yielded a reproducible 1.4 and 2-fold increase in LDH and IL-8 mRNA levels over controls. This new system is intended to be used as an alternative research tool for aerosol in vitro exposure studies. While further testing and optimization is still required to produce a "commercially ready" system, it serves as a stepping-stone in the development of cost-effective in vitro technology that can be made accessible to researchers in the near future.
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http://dx.doi.org/10.1016/j.cbi.2014.06.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4252865PMC
September 2014

Effect of broccoli sprouts on nasal response to live attenuated influenza virus in smokers: a randomized, double-blind study.

PLoS One 2014 9;9(6):e98671. Epub 2014 Jun 9.

Center for Environmental Medicine, Asthma and Lung Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America; Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

Background: Smokers have increased susceptibility and altered innate host defense responses to influenza virus infection. Broccoli sprouts are a source of the Nrf2 activating agentsulforaphane, and short term ingestion of broccoli sprout homogenates (BSH) has been shown to reduce nasal inflammatory responses to oxidant pollutants.

Objectives: Assess the effects of BSH on nasal cytokines, virus replication, and Nrf2-dependent enzyme expression in smokers and nonsmokers.

Methods: We conducted a randomized, double-blind, placebo-controlled trial comparing the effects of BSH on serially sampled nasal lavage fluid (NLF) cytokines, viral sequence quantity, and Nrf2-dependent enzyme expression in NLF cells and biopsied epithelium. Healthy young adult smokers and nonsmokers ingested BSH or placebo (alfalfa sprout homogenate) for 4 days, designated Days -1, 0, 1, 2. On Day 0 they received standard vaccine dose of live attenuated influenza virus (LAIV) intranasally. Nasal lavage fluids and nasal biopsies were collected serially to assess response to LAIV.

Results: In area under curve analyses, post-LAIV IL-6 responses (P = 0.03) and influenza sequences (P = 0.01) were significantly reduced in NLF from BSH-treated smokers, while

Nad(p)h: quinoneoxidoreductasein NLF cells was significantly increased. In nonsmokers, a similar trend for reduction in virus quantity with BSH did not reach statistical significance.

Conclusions: In smokers, short term ingestion of broccoli sprout homogenates appears to significantly reduce some virus-induced markers of inflammation, as well as reducing virus quantity. Nutritional antioxidant interventions have promise as a safe, low-cost strategy for reducing influenza risk among smokers and other at risk populations.

Trial Registration: ClinicalTrials.gov NCT01269723.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0098671PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4049587PMC
June 2015

Inflammatory response of monocytes to ambient particles varies by highway proximity.

Am J Respir Cell Mol Biol 2014 Dec;51(6):802-9

1 School of Public Health, Xinxiang Medical University, Henan Province, China.

Epidemiological studies have demonstrated associations of chronic respiratory disease with near-roadway pollutant exposure, effects that were independent of those of regional air pollutants. However, there has been limited study of the potential mechanisms for near-roadway effects. Therefore, we examined the in vitro effect of respirable particulate matter (PM) collected adjacent to a major Los Angeles freeway and at an urban background location. PM was collected on filters during two consecutive 15-day periods. Oxidative stress and inflammatory response (intracellular reactive oxygen species [ROS], IL-1β, IL-6, IL-8, and TNF-α) to PM aqueous extract was assessed in THP-1 cells, a model for evaluating monocyte/macrophage lineage cell responses. The near-roadway PM induced statistically significantly higher levels of IL-6, IL-8, and TNF-α (P < 0.01) and a near significant increase in IL-1β (P = 0.06) but did not induce ROS activity (P = 0.17). The contrast between urban background and near-roadway PM-induced inflammatory cytokines was similar in magnitude to that corresponding to temporal differences between the two collection periods. PM-induced proinflammatory protein expression was attenuated by antioxidant pretreatment, and PM stimulation enhanced the activity of protein kinases, including extracellular signal-regulated kinase and c-Jun N-terminal kinase. Pretreatment of THP-1 cells with kinase inhibitors reduced PM-induced proinflammatory mediator expression. The proinflammatory response was also reduced by pretreatment with polymyxin B, suggesting a role for endotoxin. However, the patterns of PM-induced protein kinase response and the attenuation of inflammatory responses by antioxidant or polymyxin B pretreatment did not vary between near-roadway and urban background locations. We conclude that near-roadway PM produced greater inflammatory response than urban background PM, a finding consistent with emerging epidemiologic findings, but these differences were not explained by PM endotoxin content or by MAPK pathways. Nevertheless, THP-1 cells may be a model for the development of biologically relevant metrics of long-term spatial variation in exposure for study of chronic disease.
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http://dx.doi.org/10.1165/rcmb.2013-0265OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4291543PMC
December 2014

Air toxics and epigenetic effects: ozone altered microRNAs in the sputum of human subjects.

Am J Physiol Lung Cell Mol Physiol 2014 Jun 25;306(12):L1129-37. Epub 2014 Apr 25.

Center for Environmental Medicine, Asthma, and Lung Biology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina; and Department of Pediatrics, University of North Carolina, School of Medicine University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

Ozone (O3) is a criteria air pollutant that is associated with numerous adverse health effects, including altered respiratory immune responses. Despite its deleterious health effects, possible epigenetic mechanisms underlying O3-induced health effects remain understudied. MicroRNAs (miRNAs) are epigenetic regulators of genomic response to environmental insults and unstudied in relationship to O3 inhalation exposure. Our objective was to test whether O3 inhalation exposure significantly alters miRNA expression profiles within the human bronchial airways. Twenty healthy adult human volunteers were exposed to 0.4 ppm O3 for 2 h. Induced sputum samples were collected from each subject 48 h preexposure and 6 h postexposure for evaluation of miRNA expression and markers of inflammation in the airways. Genomewide miRNA expression profiles were evaluated by microarray analysis, and in silico predicted mRNA targets of the O3-responsive miRNAs were identified and validated against previously measured O3-induced changes in mRNA targets. Biological network analysis was performed on the O3-associated miRNAs and mRNA targets to reveal potential associated response signaling and functional enrichment. Expression analysis of the sputum samples revealed that O3 exposure significantly increased the expression levels of 10 miRNAs, namely miR-132, miR-143, miR-145, miR-199a*, miR-199b-5p, miR-222, miR-223, miR-25, miR-424, and miR-582-5p. The miRNAs and their predicted targets were associated with a diverse range of biological functions and disease signatures, noted among them inflammation and immune-related disease. The present study shows that O3 inhalation exposure disrupts select miRNA expression profiles that are associated with inflammatory and immune response signaling. These findings provide novel insight into epigenetic regulation of responses to O3 exposure.
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http://dx.doi.org/10.1152/ajplung.00348.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4060009PMC
June 2014

Live attenuated influenza vaccine strains elicit a greater innate immune response than antigenically-matched seasonal influenza viruses during infection of human nasal epithelial cell cultures.

Vaccine 2014 Mar 30;32(15):1761-7. Epub 2014 Jan 30.

The Center for Environmental Medicine, Asthma and Lung Biology, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA; Department of Pediatrics, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA.

Influenza viruses are global pathogens that infect approximately 10-20% of the world's population each year. Vaccines, including the live attenuated influenza vaccine (LAIV), are the best defense against influenza infections. The LAIV is a novel vaccine that actively replicates in the human nasal epithelium and elicits both mucosal and systemic protective immune responses. The differences in replication and innate immune responses following infection of human nasal epithelium with influenza seasonal wild type (WT) and LAIV viruses remain unknown. Using a model of primary differentiated human nasal epithelial cell (hNECs) cultures, we compared influenza WT and antigenically-matched cold adapted (CA) LAIV virus replication and the subsequent innate immune response including host cellular pattern recognition protein expression, host innate immune gene expression, secreted pro-inflammatory cytokine production, and intracellular viral RNA levels. Growth curves comparing virus replication between WT and LAIV strains revealed significantly less infectious virus production during LAIV compared with WT infection. Despite this disparity in infectious virus production the LAIV strains elicited a more robust innate immune response with increased expression of RIG-I, TLR-3, IFNβ, STAT-1, IRF-7, MxA, and IP-10. There were no differences in cytotoxicity between hNEC cultures infected with WT and LAIV strains as measured by basolateral levels of LDH. Elevated levels of intracellular viral RNA during LAIV as compared with WT virus infection of hNEC cultures at 33°C may explain the augmented innate immune response via the up-regulation of pattern recognition receptors and down-stream type I IFN expression. Taken together our results suggest that the decreased replication of LAIV strains in human nasal epithelial cells is associated with a robust innate immune response that differs from infection with seasonal influenza viruses, limits LAIV shedding and plays a role in the silent clinical phenotype seen in human LAIV inoculation.
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http://dx.doi.org/10.1016/j.vaccine.2013.12.069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979967PMC
March 2014
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