This study identifies transcriptional phenotypes of sputum samples from normal volunteers and atopic asthmatics exposed to ozone. Network analyses suggest that asthmatics elevate immune signaling following oxidative stress, while nonasthmatics attempt to mitigate the ozone-induced response

Alexis, N.E.

Brickey, W.J.

Fry, R.C.

Hernandez, M.

Peden, D.B.

Rager, J.E.

Ting, J.P.Y.

Zhou, B.

Zhou, H.

Carolina Digital Repository

Airway cells from atopic asthmatics exposed to ozone displayan enhanced innate immune gene profileMichelle Hernandez, MDa,b, Willie June Brickey, PhDc, Neil E. Alexis N, PhDa,b, Rebecca C.Fry, PhDd, Julia E. Rager, MSd, Baiming Zhou, MSe, Jenny PY Ting, PhDe, Haibo Zhou,PhDa,e,*, and David B. Peden, MD,MSa,b,c,*aCenter for Environmental Medicine, Asthma and Lung Biology The University of North Carolinaat Chapel Hill School of Medicine Chapel Hill, North CarolinabDepartment of Pediatrics The University of North Carolina at Chapel Hill School of MedicineChapel Hill, North CarolinacDepartment of Microbiology and Immunology The University of North Carolina at Chapel HillSchool of Medicine Chapel Hill, North CarolinadEnvironmental Sciences & Engineering, The Gillings School of Global Public HealtheBiostatistics, The Gillings School of Global Public HealthCapsule SummaryThis study identifies transcriptional phenotypes of sputum samples from normal volunteers andatopic asthmatics exposed to ozone. Network analyses suggest that asthmatics elevate immunesignaling following oxidative stress, while nonasthmatics attempt to mitigate the ozone-inducedresponse.KeywordsAsthma; Ozone; Induced Sputum; Profiling; Oxidative StressRespiratory complications caused by ozone (O3) represent a significant public healthburden1–4. Ozone and a range of other pollutants are thought to exert their effects viaoxidative stress responses in the lower airway. Despite studies suggesting that pulmonaryoxidative insults by air pollutants lead to deleterious health outcomes, treatment options forenvironmentally-induced oxidative stress are limited, partially due to the lack ofunderstanding of exactly how these pollutants affect susceptible populations.© 2011 American Academy of Allergy, Asthma and Immunology. Published by Mosby, Inc. All rights reservedCorresponding Author: Michelle L. Hernandez, MD Center for Environmental Medicine, Asthma and Lung Biology 104 Mason FarmRoad UNC School of Medicine Chapel Hill, NC 27516 Michelle_Hernandez@med.unc.edu phone 919-843-5383 fax 919-966-9863.*These authors contributed equally to this effort as senior authorsPublisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to ourcustomers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review ofthe resulting proof before it is published in its final citable form. Please note that during the production process errors may bediscovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Disclosures: N. E. Alexis has contracts with Centocor Pharmacueticals, GlaxoSmithKline and MedImmune. D. B. Peden has been aconsultant to GlaxoSmithKline and Aquinox Pharmaceuticals and a contract with MedImmune. J. PY. Ting had a one-year contractwith GlaxoSmithKline (Philadelphia). The rest of the authors have declared that they have no conflicts of interest.NIH Public AccessAuthor ManuscriptJ Allergy Clin Immunol. Author manuscript; available in PMC 2013 January 1.Published in final edited form as:J Allergy Clin Immunol. 2012 January ; 129(1): 259–261.e2. doi:10.1016/j.jaci.2011.11.007.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptOur group recently reported that atopic asthmatics (AA) had enhanced sputum inflammatoryresponses compared to normal volunteers (NV) after a 2 hour, 0.4 ppm O3 exposure,characterized by increases in macrophage TLR4 expression and concentrations of IL-1β,IL-6, and IL-8 in sputum samples5. This report expands on those results by examining geneexpression profiles of sputum inflammatory cells from NV and AA volunteers as a means ofidentifying additional mechanisms that may explain the differences in inflammatoryresponse between these two cohorts, and to confirm the airway inflammatory phenotype in alarger cohort of AA and NV.Subject recruitment, sample collection and analysis techniques are identical to those we haverecently reported5. Demographic data from a total of 51 subjects (34 NV, 17 AA) arepresented in Table I. Statistical tests include paired t-tests to assess a specific cohortresponse to O3 for cell differentials; non-parametric paired Wilcoxon signed rank test toassess O3 responses for induced sputum cytokines; and non-parametric t tests (Mann-Whitney Test) to examine baseline differences in cytokines or cell differentials between AAand NV.Data for induced sputum cell differentials and cytokines from 9 NV and 6 AA were added toour previously published cohort5. The composition of sputum cellularity was similarbetween the two cohorts, with the exception of increased sputum eosinophils in the AAcohort at baseline compared to NV (Table I). AA continued to show evidence of increasedlevels of IL-1β, IL-6, and IL-8 after O3 exposure (Table I), with reduced levels of IL-10 andincreased levels of IL-1β and IL-8 at baseline. The larger sample size supports the notionthat despite similarities in sputum cellularity, AA display increased levels of airway pro-inflammatory cytokines after O3 exposure compared to NV.Of the 51 volunteers described above, adequate mRNA was recovered from 18 NV and 13AA for microarray analysis using a custom-designed microarray representing more than2000 immune response gene targets6. Raw expression data were first normalized using thedown-weighting loess fit7 to remove systematic ratio-intensity dependence. The normalizedred/green expression ratio was log2 transformed and uploaded to Significant Analysis ofMicroarrays software package8 in R (version 2.10) to identify differentially expressed genes.To control for multiple comparisons, we used false discovery rate (FDR), estimated by 5000permutations for each target.At FDR = 0.05, we found a total of 102 genes that showed differential expression after O3exposure compared to baseline conditions (Figure 1A). Fifty-five genes had significantexpression changes with O3 exposure in NV; 47 genes had significant changes in geneexpression with O3 exposure in AA. Only one gene overlapped between the two cohorts,aurora kinase B.In order to identify potential biological pathways affected by O3 exposure, the differentiallyexpressed gene profiles for each cohort were overlaid onto protein-protein maps enabledthrough the Ingenuity Pathways' Knowledge Base6. Statistical significance of each networkwas calculated using a Fischer's exact test. This test generated a p-value signifying theprobability that each network was associated with the mRNA targets by chance alone.For each of the two gene lists, significant networks were identified. The set of 55 genes thatshowed O3-induced changes in NV could be integrated into four networks ranging insignificance from 10−16 to 10−44. The most significant NV network is enriched for proteinsinvolved in the ERBB2 (also known as HER-2) pathway, with most of the signalingpredicted to be downregulated in this pathway (Table E1). The 47 differentially expressedgenes associated with the O3 challenge in AA were integrated into four networks ranging inHernandez et al. Page 2J Allergy Clin Immunol. Author manuscript; available in PMC 2013 January 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptsignificance from 10−9 to 10−45. The most significant AA network is enriched for genesinvolved in immune response signaling such as NFκB (Table E2).Of the genes that showed the most statistically significant changes in gene expression,confirmatory quantitative PCR was performed on induced sputum mRNA from 6 NV and 8AA using previously published methods9. A comparative CT approach was used to discernchanges in relative expression due to O3 with β-Actin as the housekeeping gene and pre-O3as untreated control (Figure 1B).The expression levels of HER-2 pathway genes such as ERBB2, cyclin D1 (CCND1), andmatrix metalloprotease II (MMP2) were significantly increased in AA compared to NV(Figure 1B). Furthermore, AA had significantly increased expression of genes involved incellular detoxification after oxidative stress (GPX3, GSTM4, NQO1) and those involved inneutrophil oxidative burst (NCF2) compared to NV (Figure 1B). This is consistent with theidea that asthmatics have a differential oxidative stress response following O3 exposurecompared to NV. The expression levels of several innate immune genes, such as the MHC-IImolecule HLA-DPA1, the integrin ICAM-1, and the pro-inflammatory cytokines IL-6, IL-8,IL-18, and TNFα were significantly increased in AA compared to NV upon exposure to O3(Figure 1B).In summary, gene expression profiles for sputum cells are distinctly different in NV and AAdespite similar neutrophil and macrophage proportions after acute O3 exposure. Comparedto NV, AA showed increased immune signaling, elevated pro-inflammatory cytokines, andup-regulated expression of the HER-2 gene network, indicative of enhanced epithelial cellproliferation. These data suggest that unlike NV, AA cannot limit epithelial cell proliferativeresponses following O3 -induced oxidative stress. HER-2 blockade has been found to reducelung fibrosis and remodeling in a model of bleomycin-induced lung injury10. Limitations ofthis study include small sample size and mixed sputum cellularity that prevented cellspecific determination of oxidative stress responses in these populations. Future studies willfocus on dissecting the contributions of distinct cell types on determining responses to O3 ina larger cohort of individuals, as well as including baseline measures of inflammation. Thisinformation is important for the judicious development of therapeutics that will prevent orreduce the harmful effects of oxidant lung injury.AcknowledgmentsFunding Sources: MLH is supported by NIH KL2RR025746; WJB, NEA, JPYT, and DBP are supported by NIAIDU19AI077437; RCF and JR are supported by P30ES010126. This work was also funded by CR 83346301 from theUS Environmental Protection Agency.AbbreviationsO3 OzoneAA atopic asthmaticNV normal volunteerppm part per millionIL-1β interleukin -1 betaIL-8 interleukin-8IL-6 interleukin-6TLR4 toll-like receptor 4Hernandez et al. Page 3J Allergy Clin Immunol. Author manuscript; available in PMC 2013 January 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptTLR2 toll-like receptor 2TNFα tumor necrosis factor alphaICAM1 Inter-Cellular Adhesion Molecule 1HLA-DP1 major histocompatibility complex, class II, DPGSTM4 glutathione S-transferase mu 4GPX3 glutathione peroxidase 3NCF2 neutrophil cytosolic factor 2ERBB2 v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian)CCND1 cyclin D-1MMP2 matrix metalloprotease IINQO1 NAD(P)H dehydrogenase, quinone 1References1. Bell ML, McDermott A, Zeger SL, Samet JM, Dominici F. Ozone and short-term mortality in 95US urban communities, 1987-2000. JAMA. 2004; 292:2372–8. [PubMed: 15547165]2. Chuang GC, Yang Z, Westbrook DG, Pompilius M, Ballinger CA, White CR, et al. Pulmonaryozone exposure induces vascular dysfunction, mitochondrial damage, and atherogenesis. Am JPhysiol Lung Cell Mol Physiol. 2009; 297:L209–16. [PubMed: 19395667]3. Jerrett M, Burnett RT, Pope CA 3rd, Ito K, Thurston G, Krewski D, et al. Long-term ozoneexposure and mortality. N Engl J Med. 2009; 360:1085–95. [PubMed: 19279340]4. Stafoggia M, Forastiere F, Faustini A, Biggeri A, Bisanti L, Cadum E, et al. Susceptibility factors toozone-related mortality: a population-based case-crossover analysis. Am J Respir Crit Care Med.2010; 182:376–84. [PubMed: 20339147]5. Hernandez ML, Lay JC, Harris B, Esther CR Jr. Brickey WJ, Bromberg PA, et al. Atopic asthmaticsubjects but not atopic subjects without asthma have enhanced inflammatory response to ozone. JAllergy Clin Immunol. 2010; 126:537–44. e1. [PubMed: 20816188]6. Alexis NE, Brickey WJ, Lay JC, Wang Y, Roubey RA, Ting JP, et al. Development of an inhaledendotoxin challenge protocol for characterizing evoked cell surface phenotype and genomicresponses of airway cells in allergic individuals. Ann Allergy Asthma Immunol. 2008; 100:206–15.[PubMed: 18426139]7. Dudoit S, Yang YH, Callow MJ, Speed TP. Statistical methods for identifying differentiallyexpressed genes in replicated cDNA microarray experiments. Statistica Sinica. 2002; 12:111–39.8. Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizingradiation response. Proc Natl Acad Sci U S A. 2001; 98:5116–21. [PubMed: 11309499]9. Brickey WJ, Alexis NE, Hernandez ML, Reed W, Ting JP, Peden DB. Sputum inflammatory cellsfrom patients with allergic rhinitis and asthma have decreased inflammasome gene expression. JAllergy Clin Immunol. 2011; 128:900–3. [PubMed: 21868073]10. Faress JA, Nethery DE, Kern EF, Eisenberg R, Jacono FJ, Allen CL, et al. Bleomycin-inducedpulmonary fibrosis is attenuated by a monoclonal antibody targeting HER2. J Appl Physiol. 2007;103:2077–83. [PubMed: 17916677]Hernandez et al. Page 4J Allergy Clin Immunol. Author manuscript; available in PMC 2013 January 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1. O3-induced transcript profiles in NV and AAA) Heat map of O3-modulated genes in NV and AA with low (blue) and high (red)expression. B) Changes with O3 in PCR-amplified ERBB2 pathway, oxidant response andinnate immune transcripts relative to ACTB (n=6 NV, 8 AA). Mean changes in geneexpression with O3 (± SEM). Nonparametric t testing (Mann Whitney testing) was used tocompare differences in NV and AA.Hernandez et al. Page 5J Allergy Clin Immunol. Author manuscript; available in PMC 2013 January 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptHernandez et al. Page 6Table ICharacteristics of ParticipantsClinical Characteristic Normal Volunteer Atopic Asthmaticn 34 17Age (y), mean (SD) 24.2 (3.9) 24.4 (5.5)Gender 20 Female / 14 Male 10 Female / 7 MaleRace 27 White; 4 African American; 3 Asian 13 White; 3 African American 1 AsianPre O3 Post O3 Pre O3 Post O3Induced Sputum Cellularity% Neutrophils, Mean (SD) 40.9 (19.3) 64.3 (15.7)* 35.8 (19.4) 58.4 (23.4)*% Eosinophils, Mean (SD) 0.12 (0.2) 0.5 (0.2) 1.95 (2)** 2.9 (4.7)% Macrophages, Mean (SD) 55.6 (18.7) 32.1 (14.8) 59.6 (19.7) 34.2 (20.7)Induced Sputum Cytokines (pg/mlsputum)IL-1β, Median (25%–75%) 208 (162–691) 221 (77–475) 648+ (244–1247) 1121++ (295–2018)IL-8, Median (25%–75%) 4694 (1892–11373) 3622 (837–14071) 12496^ (8347–25669) 23278++ (7558–46020)IL-6, Median (25%–75%) 110 (46–172) 123 (39–233) 32 (18–138) 139# (31–666)IL-10, Median (25%–75%) 36 (0–307) 7 (0–240) 0** (0–6) 0 (0–53)*p<0.001 comparing pre O3 to post O3 values**p<0.01 comparing baseline values of AA to NV+p=0.03 comparing baseline values of AA to NV++p=0.03 comparing pre O3 to post O3 values^p=0.02 comparing baseline values of AA to NV#p=0.01 comparing pre O3 to post O3 valuesJ Allergy Clin Immunol. 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Airway cells from atopic asthmatic patients exposed to ozone display an enhanced innate immune gene profile

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Airway cells from atopic asthmatic patients exposed to ozone display an enhanced innate immune gene profile

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