To determine if the GSTM1 null genotype is a risk factor for increased inflammatory response to inhaled endotoxin

Alexis, Neil E.

Bromberg, Philip A.

Devlin, Robert B.

Diaz-Sanchez, David

Dillon, Madeline A.

Harris, Bradford

Hernandez, Michelle L.

Kleeberger, Steven

Lay, John C.

Peden, David B.

Reed, William

Zhou, Haibo

Zou, Baiming

PubMed

Carolina Digital Repository

Enhancement of systemic and sputum granulocyte response toinhaled endotoxin in people with the GSTM1 null genotypeMadeline A Dillon1, Bradford Harris1, Michelle L Hernandez1, Baiming Zou1, William Reed1,Philip A Bromberg1, Robert B Devlin2, David Diaz-Sanchez2, Steven Kleeberger3, HaiboZhou1, John C Lay1, Neil E Alexis1, and David B Peden11Center for Environmental Medicine, Asthma and Lung Biology, School of Medicine, University ofNorth Carolina at Chapel Hill, Chapel Hill, North Carolina, USA2Environmental Public Health Division, National Health and Environmental Effects ResearchLaboratory, United States Environmental Protection Agency, Research Triangle Park, NorthCarolina, USA3Laboratory of Respiratory Biology, National Institute of Environmental Health Sciences,Research Triangle Park, North Carolina, USAAbstractObjective—To determine if the GSTM1 null genotype is a risk factor for increased inflammatoryresponse to inhaled endotoxin.Methods—35 volunteers who had undergone inhalation challenge with a 20 000 endotoxin unitdose of Clinical Center Reference Endotoxin (CCRE) were genotyped for the GSTM1 nullpolymorphism. Parameters of airway and systemic inflammation observed before and afterchallenge were compared in GSTM1 null (n=17) and GSTM1 (n=18) sufficient volunteers.Results—GSTM1 null volunteers had significantly increased circulating white blood cells(WBCs), polymorphonuclear neutrophils (PMNs), platelets and sputum PMNs (% sputum PMNsand PMNs/mg sputum) after CCRE challenge. GSTM1 sufficient volunteers had significant, butlower increases in circulating WBCs, PMNs and % sputum PMNs, and no increase in platelets orPMNs/mg sputum. Linear regression analysis adjusted for baseline values of the entire cohortrevealed that the GSTM1 null genotype significantly increased circulating WBCs, platelets and %sputum PMNs after challengeConclusion—These data support the hypothesis that the GSTM1 null genotype is a risk factorfor increased acute respiratory and systemic inflammatory response to inhaled CCRE. These dataare consistent with other observations that the GSTM1 null genotype is associated with increasedrespiratory, systemic and cardiovascular effects linked to ambient air particulate matter exposureCorrespondence to David B Peden, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7310,USA; peden@med.unc.edu.Competing interests BH has received research support from the National Institute of Environmental Health Sciences, USEnvironmental Protection Agency and Purdue Pharmaceuticals-Quintiles. PAB has received support from the US EnvironmentalProtection Agency and National Institute of Health. DBP has consulted for GlaxoSmithKline and Funxional Therapeutics and hasreceived research support from the National Institute of Environmental Health Sciences, the National Center for Complementary andAlternative Medicine, the National Heart, Lung, and Blood Institute, the National Institute for Allergy and Infectious Diseases, the USEnvironmental Protection Agency and the National Center for Research Resources. The rest of the authors have declared that theyhave no competing interests.Ethics approval This study was conducted with the approval of the IRB at the University of North Carolina.Provenance and peer review Not commissioned; externally peer reviewed.NIH Public AccessAuthor ManuscriptOccup Environ Med. Author manuscript; available in PMC 2013 October 28.Published in final edited form as:Occup Environ Med. 2011 October ; 68(10): . doi:10.1136/oem.2010.061747.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptand indicate that the GSTM1 null genotype should be considered a risk factor for adverse healtheffects associated with exposure to environmental endotoxin.INTRODUCTIONExposure to environmental endotoxin is linked to a number of clinical diseases, includingbyssinosis, occupational lung disease in animal workers, and asthma exacerbation.1Endotoxin is a component of bioaerosols associated with exposure to animals in agriculturaland domestic settings, particulates produced in tobacco and biomass fuel smoke, and hasbeen found in coarse and fine mode ambient and indoor air particulate matter (PM).1Ambient air PM and passive smoke exposure have been associated with increasedrespiratory tract2 and cardiovascular morbidity.3 Additionally, ambient air PM exposures areassociated with increased markers of systemic inflammation.4 Taken together, these reportssuggest that inhaled PM can exert local effects on the airway and impact other organsystems via increased systemic inflammation. As endotoxin is an important constituent ofmany bioaerosol and PM species,1 and is the primary ligand for the TLR4 receptor onmacrophages and neutrophils,1 it is very likely that environmental endotoxin contributes tosystemic morbidity in PM exposed populations.Endotoxin promotes oxidative burst activity and production of reactive oxygen species byphagocytic and innate immune cells, which is thought to be an important mechanism forinducing exacerbation of lung disease.5 Inhalation of endotoxin also increases markers ofsystemic oxidant stress. We hypothesise that the effect of inhaled endotoxin on airway andsystemic inflammation will be enhanced in persons with genetic deficiencies in antioxidantdefences. A number of polymorphisms of antioxidant genes have been associated withincreased response to environmental agents. Among these is a gene deletion (null)polymorphism of the glutathione-S-transferase Mu1 (GSTM1) gene. The homozygousGSTM1 null genotype is prevalent in approximately 40–50% of the population6 and hasbeen associated with increased risk for cardiovascular disease events associated withexposure to ambient air PM,7 increased risk of wheezing in children exposed to tobaccosmoke during the perinatal period, and increased risk of acute exacerbation of asthma relatedto ambient air O3 exposure6.We have developed a low dose endotoxin challenge protocol which models occupationaland environmental exposure to airborne endotoxin using 20 000 endotoxin units (EU) ofClinical Center Reference Endotoxin (CCRE), which approximates the amount of endotoxinthat a worker would be exposed to during an 8 h work shift in a concentrated animal farmingoperation. We have used this level of inhaled CCRE in studies of normal volunteers andsubjects with asthma and find that it causes increased sputum neutrophilia without any fever,systemic symptoms or change in lung function. In this report, we identified 35 healthyvolunteers who had undergone inhalation challenge with CCRE and stratified them on thebasis of GSTM1 genotype. We compared systemic (circulating) white blood cells (WBCs)and polymorphonuclear neutrophil (PMN) counts and sputum PMNs, expressed as % oftotal cells that are PMNs (% sputum PMNs) and PMNs/mg sputum, 4 h after challenge ofGSTM1 null and sufficient volunteers to test the hypothesis that absence of GSTM1enhances inflammatory response to inhaled CCRE.METHODSThirty-five healthy non-allergic, adults without asthma who had undergone endotoxininhalation challenge with a 20 000 EU dose of CCRE between 2003 and 2009 in identicalprotocols approved by the University of North Carolina IRB were included in this study. Allvolunteers had normal lung function, normal methacholine challenge and negative skin testsDillon et al. Page 2Occup Environ Med. Author manuscript; available in PMC 2013 October 28.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptand were genotyped only after challenge and assessment of samples was completed, thusavoiding selection bias. Venipuncture, induced sputum, spirometry, cytokine measurementsin sputum and genotyping for GSTM1 were performed as previously described.89Circulating total WBCs, PMNs and platelet counts, sputum PMNs and mediators inrecovered sputum samples, spirometry measures and symptom scores were assessed atbaseline (pre-challenge) and 4 h after CCRE challenge was complete in GSTM1 null andsufficient cohorts. Baseline sputum and blood samples were obtained 24–48 h prior toCCRE challenge. CCRE inhalation had no effect on symptoms or spirometry.To determine if CCRE had an overall effect on circulating and sputum inflammatoryresponses, data were log-transformed to ensure a normal distribution and paired t tests ofpre- and post-endotoxin values were performed. To examine the effect of the GSTM1 nullgenotype on WBCs, PMNs, platelets and sputum PMNs (% sputum PMNs and PMN/mgsputum) following CCRE challenge across the entire cohort (n=35), we used the log-transformed values of these analytes in a linear regression model after adjusting for baselineanalyte values, age, gender, ethnicity and body mass index (BMI).RESULTSFigure 1 depicts the levels of baseline and post-CCRE challenge circulating WBCs andPMNs (figure 1A,B), % sputum PMNs (figure 1C) and sputum PMNs expressed as PMN/mg sputum (figure 1D) in the GSTM1 sufficient and null groups. In GSTM1 sufficientvolunteers, CCRE significantly increased circulating WBCs (p=0.006) and PMNs(p=0.001), as well as the % sputum PMNs (p=0.01), but not PMN/mg sputum (p=0.11). InGSTM1 null volunteers, CCRE increased circulating WBCs (p=0.001) and PMNs(p=0.003), as well as both sputum measures of PMNs, % sputum PMNs (p=0.002) andPMNs/mg sputum (p=0.005).We also found a signiffcant CCRE-induced increase in blood platelets in GSTM1 nullvolunteers (27.3 ± 1.8 ×106/ml baseline vs 28.4±1.8 ×106/ml post-CCRE, p=0.01) but not inGSTM1 suffcient volunteers (25.3 ± 1.5 ×106/ml baseline vs 25.8±1.6 ×106/ml post-CCRE,p=0.41). CCRE induced significant changes in both GSTM1 cohorts in sputum TNFα(43±22 pg/ml baseline vs 63±33 pg/ml post-CCRE in GSTM1 sufficient volunteers, p=0.05and 26±7 pg/ml baseline vs 118±63 pg/ml post-CCRE in GSTM1 null volunteers, p=0.002)and IL-1β levels (369±297 pg/ml baseline vs 517±377 pg/ml post-CCRE in GSTM1sufficient volunteers, p=0.01 and 234±66 pg/ml baseline vs 598±255 pg/ml post-CCRE inGSTM1 null volunteers, p=0.007). Linear regression analysis adjusted for baseline valuesrevealed that the GSTM1 null genotype significantly increased post-challenge circulatingWBCs (β=0.120, SE=0.059, p=0.04), platelets (β=0.054, SE=0.021, p=0.02) and % sputumPMN (β=0.263, SE=0.126, p=0.05).DISCUSSIONAs endotoxin induces host innate immune cells to generate reactive oxygen species, wehypothesised that the GSTM1 null genotype would be associated with increasedinflammatory response to inhaled endotoxin. When considered as a single cohort of 35volunteers, these subjects had increased sputum and systemic PMNs 4 h followingendotoxin (CCRE) challenge, without exhibiting any change in lung function, malaise orfever. These results are similar to those reported by our group and others10 using this levelof endotoxin.With stratification of the group on the basis of GSTM1 genotype, we found that 17 GSTM1null volunteers had a significant increase in PMNs/mg sputum following inhalation ofDillon et al. Page 3Occup Environ Med. Author manuscript; available in PMC 2013 October 28.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptCCRE, whereas 18 GSTM1 sufficient volunteers showed no significant change in PMNs/mgsputum following CCRE challenge. However, when differential counts of sputum cells areconsidered, both groups do have a significant increase in % sputum PMNs. Sputum IL-1βand TNFα are also increased in both groups after challenge, suggesting that airwayinflammatory responses, although muted in the GSTM1 sufficient group, are induced byinhaled CCRE in both GSTM1 genotypes. We also found that while both the GSTM1 nulland sufficient groups had systemic PMN and WBC responses to CCRE, the GSTM1 nullvolunteers had a larger increase in circulating PMNs and WBCs, as well as an increase inplatelets not observed in GSTM1 sufficient volunteers.We employed linear regression analysis to further assess the role of the GSTM1 genotype inmodifying the response to CCRE. Because we had previously reported that BMI correlatedwith airway neutrophilic response to CCRE,11 and others have found that obesity is also arisk factor for adverse effects associated with ambient air PM exposure,7 we adjusted forBMI as well as ethnicity, gender, age, baseline levels of analytes, and GSTM1 genotype.Our linear regression analysis revealed that the GSTM1 null genotype was a significantmodifier of the 4 h response of circulating WBCs, platelets and sputum PMNs (% sputumPMNs) to CCRE challenge. Overall, our results indicate that while those with the GSTM1sufficient or null genotype have systemic and respiratory inflammatory response to inhaledendotoxin, this response is exaggerated in GSTM1 null volunteers compared to those whoare GSTM1 sufficient.Our observations are consistent with other studies in which GSTM1 null subjects haveincreased response to other pro-oxidant pollutants. In addition to the epidemiological studiesoutlined in the introduction, GSTM1 null volunteers with asthma have increased nasalresponse to allergen after experimental diesel exhaust and tobacco smoke exposure,6 and wehave shown that those with the GSTM1 null genotype have increased PMN influx to theairway 24 h following exposure to 0.4 ppm O3.9 In a cohort of volunteers who were exposedto 0.4 ppm O3 and 20 000 EU CCRE on separate occasions, we found a strong within-individual correlation between O3 and CCRE-evoked PMN response.12 Thus, susceptibilityto O3 also correlated with susceptibility to endotoxin (CCRE). While this initial study islimited by a relatively small sample size, these results suggest that compared to the GSTM1sufficient genotype, the GSTM1 null genotype confers greater risk for adverse health effectsassociated with exposure to a wide variety of environmental pollutants, includingenvironmental endotoxin found in bioaerosols, airborne PM and second-hand tobaccosmoke.AcknowledgmentsFunding This research was supported in part by grants R01ES012706, RC1ES018417 and P30010126 from theNational Institute of Environmental Health Sciences, U19AI077437 from the National Institute for Allergy andInfectious Diseases, P01AT002620 from the National Center for Complementary and Alternative Medicine, andKL2RR025746, M01RR00046 and UL1RR025747 from the National Center of Research Resources of the NationalInstitutes of Health, as well as CR 83346301 from the US Environmental Protection Agency. Although the researchdescribed herein has been funded in part by the United States Environmental Protection Agency, it has not beensubjected to the EPA’s required peer and policy review. The findings contained in this report do not necessarilyreflect the views of the Environmental Protection Agency or the National Institutes of Health, and no officialendorsement should be inferred.References1. Radon K. The two sides of the “endotoxin coin”. Occup Environ Med. 2006; 63:73–8. [PubMed:16361410]Dillon et al. Page 4Occup Environ Med. Author manuscript; available in PMC 2013 October 28.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript2. Medina-Ramon M, Zanobetti A, Schwartz J. The effect of ozone and PM10 on hospital admissionsfor pneumonia and chronic obstructive pulmonary disease: a national multicity study. Am JEpidemiol. 2006; 163:579–88. [PubMed: 16443803]3. Chuang KJ, Coull BA, Zanobetti A, et al. Particulate air pollution as a risk factor for ST-segmentdepression in patients with coronary artery disease. Circulation. 2008; 118:1314–20. [PubMed:18779445]4. Urch B, Speck M, Corey P, et al. Concentrated ambient fine particles and not ozone induce asystemic interleukin-6 response in humans. Inhal Toxicol. 2010; 22:210–18. [PubMed: 20088738]5. Romieu I, Castro-Giner F, Kunzli N, et al. Air pollution, oxidative stress and dietarysupplementation: a review. Eur Respir J. 2008; 31:179–97. [PubMed: 18166596]6. London SJ, Romieu I. Gene by environment interaction in asthma. Annu Rev Public Health. 2009;30:55–80. [PubMed: 18980546]7. Schwartz J, Park SK, O’Neill MS, et al. Glutathione-S-transferase m1, obesity, statins, andautonomic effects of particles: gene-by-drug-by-environment interaction. Am J Respir Crit CareMed. 2005; 172:1529–33. [PubMed: 16020798]8. Alexis NE, Lay JC, Almond M, et al. Acute LPS inhalation in healthy volunteers induces dendriticcell maturation in vivo. J Allergy Clin Immunol. 2005; 115:345–50. [PubMed: 15696093]9. Alexis NE, Zhou H, Lay JC, et al. The glutathione-S-transferase Mu 1 null genotype modulatesozone-induced airway inflammation in human subjects. J Allergy Clin Immunol. 2009; 124:1222–8.[PubMed: 19796798]10. Michel O, Nagy AM, Schroeven M, et al. Dose-response relationship to inhaled endotoxin innormal subjects. Am J Respir Crit Care Med. 1997; 156:1157–64. [PubMed: 9351616]11. Alexis NE, Peden DB. Inflammatory response of the airway to inhaled endotoxin correlates withbody mass index in atopic patients with asthma but not in normal volunteers. J Allergy ClinImmunol. 2006; 117:1185–6. [PubMed: 16675350]12. Hernandez ML, Harris B, Lay JC, et al. Comparative airway inflammatory response of normalvolunteers to ozone and lipopolysaccharide challenge. Inhal Toxicol. 2010; 22:648–56. [PubMed:20540623]Dillon et al. Page 5Occup Environ Med. Author manuscript; available in PMC 2013 October 28.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptWhat this paper adds► Persons with the GSTM1 null genotype have increased respiratory andsystemic inflammatory responses to experimental inhalational challenge tolow dose endotoxin.► As GSTM1 is an antioxidant enzyme, activation of oxidative responses ininnate immune cells by endotoxin may play a role in mediating subsequentinflammatory responses to this agent.► These results suggest that the GSTM1 null genotype should be considered arisk factor for adverse health effects associated with exposure to a widevariety of environmental pollutants, including environmental endotoxinfound in bioaerosols, airborne particulate matter and second-hand tobaccosmoke.Dillon et al. Page 6Occup Environ Med. Author manuscript; available in PMC 2013 October 28.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1.Baseline versus post-CCRE (endotoxin) responses (mean±SEM) of (A) circulating whiteblood cells (WBCs), (B) polymorphonuclear neutrophils (PMNs), (C) % PMNs in sputumand (D) sputum PMNs/mg sputum in GSTM1 sufficient and GSTM1 null volunteers.Dillon et al. Page 7Occup Environ Med. Author manuscript; available in PMC 2013 October 28.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

Enhancement of systemic and sputum granulocyte response to inhaled endotoxin in people with the GSTM1 null genotype

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Enhancement of systemic and sputum granulocyte response to inhaled endotoxin in people with the GSTM1 null genotype

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