Hypoxia potentiates allergen induction of HIF-1α...

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Hypoxia potentiates allergen induction of HIF-1α, chemokines, airway inflammation, TGF-β1, and airway remodeling in a mouse model Kwang Je Baek a , 1 , 2 , Jae Youn Cho a , 1 , Peter Rosenthal a , Laura E. Crotty Alexander b , a , Victor Nizet c , d , David H. Broide a , a Department of Medicine, University of California, San Diego, La Jolla, CA, USA b Pulmonary Critical Care Section, Veterans Affairs San Diego Healthcare System, San Diego, CA, USA c Department of Pediatrics, University of California San Diego, San Diego, CA, USA d Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, San Diego, CA, USA Received 4 January 2013; accepted with revision 12 February 2013 KEYWORDS Hypoxia; Neutrophil; Eosinophil; KC; Eotaxin-1 Abstract Whether hypoxia contributes to airway inflammation and remodeling in asthma is unknown. In this study we used mice exposed to a hypoxic environment during allergen challenge (simulating hypoxia during an asthma exacerbation) to investigate the contribution of hypoxia to airway inflammation and remodeling. Although neither hypoxia alone, nor OVA allergen alone, induced significant neutrophil influx into the lung, the combination of OVA and hypoxia induced a synergistic 27 fold increase in peribronchial neutrophils, enhanced expression of HIF-1α and one of its target genes, the CXC-family neutrophil chemokine KC. The combination of hypoxia and OVA allergen increased eotaxin-1, peribronchial eosinophils, lung TGB-β1 expression, and indices of airway remodeling (fibrosis and smooth muscle) compared to either stimulus alone. As hypoxia is present in N 90% of severe asthma exacerbations, these findings underscore the potential of hypoxia to potentiate the airway inflammatory response, remodeling, and accelerate the decline of lung function in asthma exacerbations. © 2013 Elsevier Inc. All rights reserved. Institute where work was done: University of California San Diego. Corresponding author at: Department of Medicine, University of California San Diego, Biomedical Sciences Building, Room 5080, 9500 Gilman Drive, La Jolla, CA 92093-0635, USA. Fax: +1 858 534 2110. E-mail addresses: [email protected] (K.J. Baek), [email protected] (J.Y. Cho), [email protected] (P. Rosenthal), [email protected] (L.E. Crotty Alexander), [email protected] (V. Nizet), [email protected] (D.H. Broide). 1 Co-first authors as contributed equally to the performance of the research. 2 Present address of KJB: Department of Emergency Medicine, Konkuk University School of Medicine, Konkuk University Medical Center, Seoul, Republic of Korea. 1521-6616/$ - see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clim.2013.02.004 available at www.sciencedirect.com Clinical Immunology www.elsevier.com/locate/yclim Clinical Immunology (2013) 147, 2737

Transcript of Hypoxia potentiates allergen induction of HIF-1α...

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ava i l ab l e a t www.sc i enced i r ec t . com

C l i n i ca l Immuno logy

www.e l sev i e r . com/ l o ca t e / y c l im

Clinical Immunology (2013) 147, 27–37

Hypoxia potentiates allergen induction of HIF-1α,chemokines, airway inflammation, TGF-β1, and airwayremodeling in a mouse model☆

Kwang Je Baek a, 1, 2, Jae Youn Cho a, 1, Peter Rosenthal a,Laura E. Crotty Alexander b, a, Victor Nizet c, d, David H. Broide a,⁎

a Department of Medicine, University of California, San Diego, La Jolla, CA, USAb Pulmonary Critical Care Section, Veterans Affairs San Diego Healthcare System, San Diego, CA, USAc Department of Pediatrics, University of California San Diego, San Diego, CA, USAd Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, San Diego, CA, USA

Received 4 January 2013; accepted with revision 12 February 2013

☆ Institute where work was done: Un⁎ Corresponding author at: Departm

Gilman Drive, La Jolla, CA 92093-0635E-mail addresses: [email protected]

[email protected] (L.E. Crotty Alexand1 Co-first authors as contributed equ2 Present address of KJB: Departmen

Seoul, Republic of Korea.

1521-6616/$ - see front matter © 2013http://dx.doi.org/10.1016/j.clim.201

KEYWORDSHypoxia;Neutrophil;Eosinophil;KC;Eotaxin-1

Abstract Whether hypoxia contributes to airway inflammation and remodeling in asthma isunknown. In this study we used mice exposed to a hypoxic environment during allergen challenge(simulating hypoxia during an asthma exacerbation) to investigate the contribution of hypoxia toairway inflammation and remodeling. Although neither hypoxia alone, nor OVA allergen alone,induced significant neutrophil influx into the lung, the combination of OVA and hypoxia induced asynergistic 27 fold increase in peribronchial neutrophils, enhanced expression of HIF-1α and one ofits target genes, the CXC-family neutrophil chemokine KC. The combination of hypoxia and OVA

allergen increased eotaxin-1, peribronchial eosinophils, lung TGB-β1 expression, and indices ofairway remodeling (fibrosis and smooth muscle) compared to either stimulus alone. As hypoxia ispresent in N90% of severe asthma exacerbations, these findings underscore the potential of hypoxiato potentiate the airway inflammatory response, remodeling, and accelerate the decline of lungfunction in asthma exacerbations.© 2013 Elsevier Inc. All rights reserved.

iversity of California San Diegoent of Medicine, University of, USA. Fax: +1 858 534 2110.c.kr (K.J. Baek), angelajyc858er), [email protected] (V. Nizetally to the performance of thet of Emergency Medicine, Kon

Elsevier Inc. All rights reserve3.02.004

.California San Diego, Biomedical Sciences Building, Room 5080, 9500

@gmail.com (J.Y. Cho), [email protected] (P. Rosenthal),), [email protected] (D.H. Broide).research.kuk University School of Medicine, Konkuk University Medical Center,

d.

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1. Introduction

Exacerbations of severe asthma are associated with hypox-emia that can persist for several days in approximately 90% ofsubjects as assessed by arterial blood gas analysis [1]. Thecause of the hypoxemia in the majority of asthma exacerba-tions is due to altered ventilation perfusion ratios [1]. Inaddition, laboratory studies in asthmatics have demonstratedthat hypoxia impairs the perception of symptoms includingdifficulty breathing, chest tightness, and breathlessness, allof which may contribute to treatment delay during asthmaexacerbations [2]. Asthma exacerbations are also associatedwith neutrophilic airway inflammation in adults [3–5],eosinophilic and neutrophilic inflammation in children [6],and a greater decline in lung function [7,8]. At present there islimited information regarding whether hypoxia during exac-erbations of asthma contributes to neutrophilic and/oreosinophilic airway inflammation and subsequent remodelingor decline in lung function. In this study we have used a mousemodel to investigate whether mice exposed to a hypoxicenvironment during allergen challenge (to simulate hypoxiaduring an asthma exacerbation) have evidence of increasedneutrophilic and/or eosinophilic airway inflammation andenhanced airway remodeling.

Hypoxia induces the transcription factor hypoxia-induciblefactor (HIF) which regulates expression of over 100 genes,many of which are potentially relevant to inflammation andremodeling in asthma [9–11]. For example, hypoxia inducesexpression of pro-inflammatory cytokines (IL-1β, TNFα, IL-8,VEGF) [9–11], which have been detected at increased levels inthe airway of asthmatics [12–14]. IL-8 in particular is achemokine regulating neutrophil recruitment that may con-tribute to the neutrophilic airway inflammation noted duringexacerbations of asthma [3–5]. Hypoxia in asthma exacerba-tions may also contribute to airway remodeling as neonatalcalves exposed to chronic hypoxia develop increased airwayfibrous tissue and smooth muscle [15], mice exposed tochronic hypoxia develop increase lung type III fibrillar and typeIV basement membrane collagen after ten days of hypoxia[16], and hypoxia can increase the proliferation of rat airwaysmooth muscle cells in vitro [17].

The ubiquitously expressed and best-studied form of HIFis HIF-1, a heterodimer consisting of the oxygen-regulatedalpha subunit (HIF-1α) and a constitutively expressed betasubunit HIF-1β (also known as aryl hydrocarbon receptornuclear translocator protein or ARNT) [9–11]. Less wellstudied isoforms HIF-2 and HIF-3 exhibit more restricted tissueexpression [9]. In previous studies we have demonstrated,using conditional myeloid HIF-1α knockout mice and pharma-cologic HIF-1α inhibitors, that myeloid cell expression ofHIF plays an important role in the development of airwayhyperresponsiveness under normoxic conditions [18]. Inter-estingly, HIF may also be induced by local tissue hypoxia asopposed to systemic hypoxia in inflamed tissues that are oftenhypoxic as a result of decreased perfusion, edema, vascularinsult and/or influx of oxygen-consuming immune cells orpathogens [19]. These localized areas of lung tissue hypoxiamay be pertinent not only to severe asthma, but may alsooccur in mild and moderate asthmatics. Thus, activation ofHIF-1α in the context of inflammation can occur in bothnormoxic as well as hypoxic external environments. Additionalstudies using mouse models of asthma have demonstrated

under normoxic conditions that HIF-1 pharmacologic inhibitors[20,21], HIF siRNA knockdown [21], and conditional HIF-1βdeficient mice [22] influence levels of airway inflammationand/or airway remodeling. Human studies have also demon-strated under normoxic conditions increased levels of HIF-1αin lung tissue and bronchial fluid of patients with asthma, andin the nasal fluid of patients with rhinitis after allergenchallenge [22].

In this study we have used a mouse model of allergeninduced asthma studied under hypoxic conditions (to simulatesevere asthmatics having hypoxic asthma exacerbation) todetermine the influence of hypoxia on levels of airwayinflammation and remodeling. These studies under hypoxicconditions differ critically from prior studies investigating therole of HIF in allergen-induced inflammation and remodelingunder normoxic conditions [18,20–22]. Overall our studiesdemonstrate that in a mouse model simulating hypoxia duringan asthma exacerbation, the combined hypoxia and allergenstimulus significantly enhanced HIF-1α expression, airwayinflammation (in particular neutrophilic but also eosinophilic),as well as lung levels of KC (the murine equivalent of IL-8),eotaxin-1, and TGF-β1, with resultant increased airwayremodeling. As hypoxia is present in N90% of severe asthmaexacerbations, these findings underscore the potential ofhypoxia to potentiate the airway inflammatory response,increase levels of remodeling, and contribute to the decline inlung function in severe asthmatic exacerbations.

2. Material and methods

2.1. Mouse model of acute OVA challenge and/orhypoxia exposure

The following four groups of BALB/C mice aged 6–8 weeks(n=8 mice/group) (Jackson Labs) were studied. 1) Nohypoxia+no OVA; 2) No hypoxia+OVA; 3) Hypoxia+no OVA;and 4) Hypoxia+OVA. The hypoxia and allergen exposures aswell as all procedures involving experimental animals wereapproved by the Animal Care and Use Committee of theUniversity of California San Diego.

2.2. Hypoxia exposure

Mice in the hypoxia groups (groups 3 and 4) were placed in aplexiglass chamber maintained at 10% O2, while normoxicgroups (groups 1 and 2) were placed in a control chamberopen to room air (21% O2). The duration of the hypoxia ornormoxia exposure was 7 days based on pilot time coursestudies. CO2, water vapor, and ammonia were removed fromthe chambers by Drierite (anhydrous calcium sulfate) (FisherScientific, Atlanta, GA).

2.3. Acute OVA protocol

BALB/C mice were immunized s.c. on days 0, 7, 14, and 21with 25 μg of OVA (OVA, grade V; Sigma) adsorbed to 1 mgof alum (Aldrich) in 200 μl normal saline as previouslydescribed [23]. OVA-challenged mice received intranasalOVA (20 μg OVA in 50 μl PBS) on days 27, 29 and 31 underisoflurane (Vedco, Inc. St Joseph, MO) anesthesia. The

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non-OVA control mice (age and sex matched) were sensi-tized but not challenged with OVA.

2.4. Combined hypoxia and acute OVA protocol

Mice exposed to either hypoxia or normoxia started theirchamber exposures on day 33, two days after the mice hadreceived their last intranasal OVA challenge. The protocol wasdesigned to address whether addition of hypoxia to an ongoingepisode of allergic inflammation (to simulate the developmentof hypoxia after the initiation of the exacerbation) wouldinfluence levels of inflammation and remodeling 7 days later(a sufficient time period to allow remodeling to develop).

2.5. Processing of lungs for immunohistology

Mice were sacrificed 24 h after the final OVA and/or hypoxiachallenge and broncho-alveolar lavage (BAL) fluid and lungswere analyzed as previously described [23–25]. In brief, BALwas obtained by flushing 0.8 ml PBS through an intratrachealtube. Lungs in the different groups of mice were equiva-lently inflated with an intratracheal injection of a similarvolume of 4% paraformaldehyde solution (Sigma Chemicals,St Louis, MO) to preserve the pulmonary architecture. Lungsfrom the different experimental groups were processed as abatch for either histologic staining or immunostaining underidentical conditions. Stained and immunostained slides were allquantified under identical light microscope conditions, includ-ing magnification (20×), gain, camera position, and backgroundillumination. The quantitative histologic and image analysis ofall coded slides was performed by blinded research associates.

2.6. Quantitation of peribronchial eosinophils andneutrophils

The number of peribronchial major basic protein (MBP)positive eosinophils and neutrophil elastase positive neutro-phils were quantitated as previously described [23–26]. Inbrief, lung sections were processed for MBP immunohistochem-istry using an anti-mouse MBP Ab (kindly provided by JamesLee PhD, Mayo Clinic, Scottsdale, Arizona) or anti-neutrophilelastase antibody (Santa Cruz Biotechnology, Santa Cruz, CA).The number of individual cells staining positive for either MBPor neutrophil elastase in the peribronchial space were countedusing light microscopy. Results are expressed as the number ofperibronchial cells staining positive for either MBP or neutro-phil elastase per bronchiole with 150–200 μm of internaldiameter. At least ten bronchioles were counted in each slide.

2.7. Lung levels of chemokines/cytokines thatregulate recruitment of neutrophils and eosinophils.

Levels of neutrophil chemoattractant KC themurine equivalentof human IL-8 [26], eosinophil chemoattractant eotaxin-1, Th2cytokines (IL-5, IL-13), and innate cytokines (IL1β) weremeasured in BAL by ELISA (R&D System Inc, Minneapolis,Minn). The IL-1β, IL-5, IL-13, KC, and eotaxin-1 assays had asensitivity of 15 pg/ml.

2.8. Detection of peribronchial cells expressing KC

Lung sections were immunostained with both a KC primaryAb (R&D systems, Minneapolis, MN) and an anti-MBP Ab todetermine whether MBP+peribronchial cells express KC. Thetwo different primary antibodies were detected using twodifferent horseradish peroxidase (HRP) enzyme-labeled sec-ondary antibodies with tyramide signal amplification (MolecularProbes) according to the manufacturer's instructions as previ-ously described [25]. The anti-MBP Ab was detected with Alexa546 (red), while the anti-KC Ab was detected with Alexa 488(green). Cells co-expressing MBP and KC had a merged yellowcolor.

2.9. Peribronchial fibrosis

The area of peribronchial trichrome staining (as an index ofcollagen deposition) in paraffin embedded lung was outlinedand quantified using a light microscope (Leica DMLS, LeicaMicrosystems Inc., NY) attached to an image analysis system(Image-Pro plus, Media Cybernetics, MI) as previously de-scribed [25]. Results are expressed as the area of trichromestaining per μm length of basement membrane of bronchioles150–200 μm of internal diameter.

2.10. Thickness of the peribronchial smooth musclelayer

Lung sections were immunostained with an anti-α-smoothmuscle actin primary antibody (Sigma-Aldrich). The area ofα-smooth muscle actin staining was outlined and quantifiedusing a light microscope attached to an image analysissystem as previously described [25]. Results are expressed asthe area of α-smooth muscle actin staining per μm length ofbasement membrane of bronchioles 150–200 μm of internaldiameter.

2.11. Mucus

The number of PAS positive and PAS negative, airway epithelialcells in individual bronchioles were counted as previouslydescribed in this laboratory [25]. At least ten bronchioles werecounted in each slide. Results are expressed as the percentageof PAS-positive cells per bronchiole which is calculatedfrom the number of PAS-positive epithelial cells per bronchusdivided by the total number of epithelial cells of eachbronchiole.

2.12. Peribronchial TGF-β1+ cells

The number of peribronchial cells expressing TGF-β1 wereassessed in lung sections processed for immunohistochemistryusing an anti-TGF-β1 primary Ab (Santa Cruz Biotechnology,Inc, Santa Cruz, CA), the immunoperoxidase method, andimage analysis quantitation as previously described [25].Results are expressed as the number of TGF-β1 positivecells/bronchus [25].

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2.13. HIF-1α expression

Levels of lung HIF-1α mRNA were quantitated by qPCR. TotalRNA obtained from lung tissue was prepared with RNeasy®MiniKit (Qiagen) according to the manufacturer's protocol. Synthe-sis of cDNA was performed from 1ug of total RNA withSuperscript®II (Invitrogen) according to the manufacturer'srecommendations. qPCR was performed on a Max 3000 s(Stratagene) using RT2SYBR®Green Rox™qPCR master mix(Qiagen). Mouse HIF1α primers for qPCR were purchased fromOrigene Technologies. Levels of lung GAPDH in each samplewere used as controls.

To identify which lung cells express HIF-1α, lung sectionswere immunostainedwith an anti-HIF-1α Ab (Novus Biologicals)using the immunoperoxidasemethod and a TSA detection kit aspreviously described in this laboratory [25].

2.14. Statistical analysis

Results in the different groups of mice were compared bya Student t test. All results are presented as mean±SEM. Astatistical software package (Graph Pad Prism, San Diego,CA) was used for the analysis. P values of b0.05 wereconsidered statistically significant.

3. Results

3.1. Effect of hypoxia on HIF-1α expression in thelung

Exposure ofmice to hypoxia alone induced a 3.2 fold inductionof lung HIF-1α mRNA (hypoxia vs normoxia) (Fig. 1A). Incontrast, exposure of mice to allergen alone in the absence ofhypoxia induced a slight 1.3 fold induction of lung HIF-1αmRNA (Fig. 1A). However, the combination of hypoxia andOVA allergen induced a significant 5.3 fold increase in levels oflung HIF-1α mRNA compared to OVA and normoxia (p=0.03)(Fig. 1A), and a 2.2 fold increase compared to hypoxia and noOVA (p=0.05) (Fig. 1A). Immunohistochemistry studies dem-onstrated that epithelial cells were the predominant cellexpressing increased levels of HIF-1α following exposure tohypoxia (Fig. 1C). In contrast, following either OVA allergen orOVA+hypoxia exposure, peribronchial inflammatory cells aswell as airway epithelium expressed increased levels of HIF-1α(Figs. 1D, E).

3.2. Effect of hypoxia on OVA induced eosinophiland neutrophil airway inflammation

In the absence of allergen, peribronchial eosinophils did notdiffer significantly betweenmice exposed to hypoxia (2.2±0.1MBP+cells/bronchus) and their normoxic controls (0.7±0.2)(p=NS, Fig. 2A). Likewise, peribronchial neutrophils weresimilar in the hypoxia (3.9±1.2) and normoxia (0.3±0.04)groups without OVA challenge (p=NS, Fig. 2B). In normoxiaOVA-challenged mice had greatly increased peribronchialeosinophils (86.9±5.4 vs. 0.7±0.2 MBP+cells/bronchus, OVAvs No-OVA, pb0.0001, Fig. 2A), and a slight but statisticallyinsignificant change in peribronchial neutrophils (p=NS,Fig. 2B).

Synergy was observed in combination of hypoxia withallergen challenge. During OVA-challenge, mice exposed tohypoxia had 1.8 fold more peribronchial eosinophils (155.9±7.5) compared to normoxia controls (86.9±5.4) (pb0.001,Fig. 2A). An even more dramatic effect was observed with OVAallergen induced recruitment of peribronchial neutrophils,which increased approximately 27.3-fold in hypoxia (62.7±4.1)compared to normoxia (2.3±0.9) (pb0.0001, Fig. 2B).

3.3. Effect of hypoxia on levels of lung neutrophilchemokine KC

As the combination of hypoxia and OVA allergen synergisticallyincreased levels of peribronchial neutrophils, we examinedwhether hypoxia influenced levels of lung KC a majorneutrophil chemoattractant [26]. Exposure of mice to hypoxiaalone in the absence of OVA allergen challenge did not induce asignificant increase in BAL KC (hypoxia vs normoxia in theabsence of OVA) (p=NS) (Fig. 3A), which corresponded to theobservation that hypoxia alone did not induce significantperibronchial neutrophil recruitment (Fig. 2B). Similarly, micechallenged with OVA allergen alone in normoxic conditions hada slight but statistically insignificant increase in levels of BALKC (OVA vs no OVA in normoxia) (p=NS) (Fig. 3A) andperibronchial neutrophils (p=NS) (Fig. 2B). However, hypoxiain combination with OVA allergen challenge induced a largesynergistic increase in levels of BAL KC (hypoxia+OVA vsnormoxia+OVA) (p=0.03) (Fig. 3A) which corresponded to thesynergistic increase in peribronchial neutrophils in miceexposed to hypoxia+OVA (Fig. 2B).

To determine the cellular source of lung KC in hypoxia andOVA allergen challengedmice, we immunostained lung sectionsand demonstrated that peribronchial cells rather than epithe-lial cells were the predominant source of KC (Fig. 3B). Doublelabel immunofluorescence microscopy studies demonstratedthat MBP+eosinophils were one of the peribronchial inflamma-tory cells expressing KC (Figs. 3B–D).

3.4. Effect of hypoxia on levels of chemokines(eotaxin-1), Th2 cytokines (IL-5, IL-13), and innatecytokines (IL-1β) that regulate eosinophilicinflammation

As hypoxia also increased OVA induced peribronchial eosino-phil recruitment (Fig. 2A) we examined whether hypoxiainfluenced levels of mediators known to influence levels oflung eosinophils, including chemokines (eotaxin-1) [27], Th2cytokines, (IL-5, IL-13) [25,28], or innate cytokines (IL-1β)[29]. Exposure of mice to hypoxia in the absence of OVAallergen induced a significant increase in levels of BAL IL1β(78.7±18.3 vs 23.8±10.7 pg/ml) (hypoxia vs normoxia inthe absence of OVA) (p=0.04), but not other chemokines/cytokines studied including eotaxin-1 (p=NS), IL-13 (p=NS), orIL5 (p=NS). OVA allergen challenge alone induced significantincreases in BAL IL-1β (109.5±15.8 vs 23.8±10.7 pg/ml)(OVA vs no OVA in normoxia) (p=0.003), eotaxin-1 (35.6±11.7 vs 15.8±8.4 pg/ml) (p=0.04), IL13 (151.2±28.1 vs 16.3±4.1 pg/ml) (p=0.001) and IL-5 (113.7±24.8 vs 4.0±1.4 pg/ml)(p=0.003). The combination of hypoxia and OVA allergenchallenge induced a significant increase in eotaxin-1 comparedto OVA alone (52.8±16.2 vs 35.6±11.7 pg/ml) (hypoxia+OVA

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Figure 1 Effect of hypoxia and OVA allergen on lung HIF-1α expression. Different groups of BALB/c mice (n=8 mice/group) weresensitized and challenged with OVA allergen. Mice were then exposed to normoxia or hypoxia in exposure chambers. Mice weresacrificed 24 h after the final OVA and/or hypoxia challenge. Lungs were processed for qPCR (to detect HIF-1α and GAPDH) (panel A)and immunofluoresence microscopy (panels B–E) to detect HIF-1α expression. The combination of hypoxia and OVA allergen induceda significant 5.3 fold increase in levels of lung HIF-1α mRNA compared to OVA and normoxia (p=0.03) (panel A), and a 2.2 foldincrease compared to hypoxia and no OVA (p=0.05) (panel A). Immunofluorescence studies demonstrated that epithelial cells werethe predominant cell expressing increased levels of HIF-1α following exposure to hypoxia (panel B). In contrast, following either OVAallergen or OVA+hypoxia exposure, peribronchial inflammatory cells as well as epithelium expressed increased levels of HIF-1α(panels D–E).

31Hypoxia, Inflammation and Remodeling

vs normoxia+OVA) (p=0.02) but did not induce significantincreases in BAL IL-1β (p=NS), IL-13 (p=NS), or IL-5 (p=NS).

3.5. Effect of hypoxia on OVA induced peribronchialfibrosis

In the absence of OVA allergen, exposure of mice to hypoxiaalone challenge induced a significant increase in peribronchialfibrosis (0.25±0.06 vs 1.07±0.03 μm2/μm trichrome stainedperibronchial area) (normoxia vs hypoxia) (pb0.001) (Figs. 4A,C). Likewise, OVA allergen challenge alone under normoxicconditions induced a significant increase in peribronchial

fibrosis (0.25±0.01 vs 1.86±0.03) (no OVA vs OVA in normoxia)(pb0.001) (Figs. 4A, B, C). Hypoxia in combination with OVAallergen challenge significantly increased levels of peribronchialfibrosis compared to either hypoxia alone (pb0.001) (Figs. 4A,E), or compared to OVA allergen alone (pb0.001) (Fig. 4A).

3.6. Effect of hypoxia on OVA induced changes inperibronchial smooth muscle layer

Exposure of mice to hypoxia alone in the absence of OVAallergen induced significant increases in peribronchial smoothmuscle as assessed by the α-smooth muscle actin (α-SMA)

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Figure 2 Effect of hypoxia and OVA allergen on peribronchialeosinophils and neutrophils. Different groups of BALB/cmice (n=8mice/group) were sensitized and challenged with OVA allergen.Mice were then exposed to normoxia or hypoxia in exposurechambers. Mice were sacrificed 24 h after the final OVA and/orhypoxia challenge. Lungs were processed for immunohistochem-istry using either an anti-MBP Ab to detect eosinophils (panel A)or an anti-neutrophil elasatase Ab to detect neutrophils (panel B).OVA allergen challenge in normoxic conditions induced asignificant increase in peribronchial eosinophils (no OVA vs OVAin normoxic conditions) (pb0.0001) (panel A), and a very slightbut statistically insignificant increase in peribronchial neutrophils(p=ns) (panel B). Hypoxia in combination with OVA allergenchallenge increased peribronchial eosinophils (OVA in normoxia vsOVA in hypoxia) (pb0.001) (panel A) and peribronchial neutrophils(OVA in normoxia vs OVA in hypoxia) (pb0.0001) (panel B).

32 K.J. Baek et al.

immunostained area (1.0±0.1 vs 1.8±0.2 μm2/μm α-SMAimmunostained peribronchial area) (normoxia vs hypoxia inthe absence of OVA) (p=0.004) (Figs. 5A–C). OVA allergenchallenge alone also induced a similar increase in theperibronchial α-SMA immunostained area (OVA vs no OVA innormoxia) (pb0.001) (Figs. 5A, B, D). The combination ofhypoxia and OVA allergen significantly increased the area ofperibronchial α-SMA staining compared to either hypoxiaalone (pb0.01) (Figs. 5A, C, E) or OVA alone (pb0.05)(Figs. 5A, D, E).

3.7. Effect of hypoxia on the number of cellsexpressing the remodeling mediator TGF-β1

OVA allergen challenge alone induced an increase in thenumber of peribronchial TGF-β1 positive cells (2.4±0.4 vs31.8±3.5 TGF-β1 positive cells/bronchus) (OVA vs No OVAin normoxia) (p=0.003) (Fig. 6). In contrast, exposure ofmice to hypoxia alone did not increase the number ofperibronchial TGF-β1 positive cells (hypoxia vs normoxia inthe absence of OVA allergen (p=NS) (Fig. 6). However, thecombination of hypoxia and OVA allergen significantlyincreased the number of peribronchial TGF-β1 positive cellsin a synergistic fashion as compared to OVA allergen alone(31.8±3.5 vs 101.7±14.9 TGF-β1 positive cells/bronchus)(OVA+normoxia vs OVA+hypoxia) (pb0.0001) (Fig. 6).

3.8. Effect of hypoxia on OVA induced changes inmucus production

OVA allergen challenge alone induced a significant increase inmucus production compared to the non-OVA challenged groupunder conditions of normoxia (63.4±1.2 vs 2.0±0.1 PAS+cells/bronchus) (OVA vs no OVA in normoxia) (pb0.0001). Exposureof mice to hypoxia alone in the absence of OVA allergeninduced a very slight but statistically significant increase inmucus production (5.4±1.9 vs 2.0±0.2 PAS+cells/bronchus)(hypoxia vs normoxia in the absence of OVA) (p=0.03).However, the level of mucus production induced by hypoxiaalone (5.4±1.9 PAS+cells/bronchus) was significantly less thanthat induced by OVA alone (63.4±1.2 PAS+cells/bronchus)(OVA vs hypoxia) (pb0.0001). The combination of hypoxia andOVA allergen (75.3±1.8 PAS+cells/bronchus) induced a signif-icant increase in levels of mucus production compared tohypoxia alone (5.4±1.9 PAS+cells/bronchus) (p=0.03) but notcompared to OVA (p=NS).

4. Discussion

Using a mouse model simulating hypoxia in an allergeninduced asthma exacerbation, we have demonstrated thatthe combination of hypoxia and allergen is a strongerstimulus than either stimulus alone in inducing increasedlung HIF-1α expression, peribronchial inflammation (neutro-philic and eosinophilic), expression of lung chemokines (KC,eotaxin-1), and lung remodeling cytokines (TGF-β1) withresultant increased airway remodeling (peribronchial fibro-sis, peribronchial smooth muscle). As hypoxia is present inover 90% of severe asthma exacerbations [1], these findingsunderscore how hypoxia can not only potentiate the airwayinflammatory response, but also increase remodeling insevere asthma exacerbations associated with hypoxia. Asexacerbations are common in severe asthmatics [30,31], andare associated with an accelerated decline in lung function[7,8], further studies are needed to determine whetherearly institution of oxygen therapy in an asthma exacerba-tion associated with hypoxia could reduce levels of airwayinflammation and subsequent remodeling which lead todecline in lung function.

We found that the combination of hypoxia and allergenis stronger than either stimulus alone in enhancing HIF-1α

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A

KC KC+MBP MBP

B C D

0

250

500

750

No OVA OVAHypoxiaNormoxia

No OVA OVA

KC

(p

g/m

l BA

L) p=0.03

Figure 3 Effect of hypoxia and OVA allergen on lung KC expression. Different groups of BALB/c mice (n=8 mice/group) weresensitized and challengedwith OVA allergen. Mice were then exposed to normoxia or hypoxia in exposure chambers. Mice were sacrificed24 h after the final OVA and/or hypoxia challenge. BAL was used to measure levels of KC by Elisa (panel A), and lungs were processed forimmunoflurescence microscopy using anti-KC and anti-MBP Abs (panels B–D). Hypoxia in combination with OVA allergen induced asynergistic increase in levels of BAL KC (hypoxia+OVA vs normoxia+OVA) (p=0.03) (panel A).Double label immunofluorescencemicroscopy studies demonstrated that MBP+eosinophils were one of the peribronchial inflammatory cells expressing KC (merged yellow)(panel B).

33Hypoxia, Inflammation and Remodeling

expression in the lung. Previous studies in mouse modelsundergoing allergen challenge have demonstrated underconditions of normoxia that inhibiting HIF reduces levels ofinflammation and remodeling [19–22]. As HIF is inducedunder normoxic conditions by allergen in these [19–22] andour current study, it suggests that allergen induced inflamedtissues may be hypoxic as a result of decreased perfusion,edema, vascular insult and/or influx of oxygen-consumingimmune cells [19]. Human studies have also demonstratedunder normoxic conditions increased levels of HIF-1α in lungtissue and bronchial fluid of patients with asthma, and in thenasal fluid of patients with rhinitis after allergen challenge[22]. Our study extends these observations to demonstratethat under hypoxic conditions allergen challenge induces amuch greater increase in HIF-1α expression compared toeither stimulus alone. In addition, we demonstrate thatwhile hypoxia predominantly induces HIF-1α expression inairway epithelium, the combination of allergen and hypoxiainduces HIF-1α expression in peribronchial inflammatorycells and airway epithelium.

The importance of our demonstration of increased HIF-1αexpression in response to allergen and hypoxia is underscoredby the detection of increased expression of the HIF-1αregulated chemokine KC and the associated highly signifi-cant 27 fold increase in peribronchial neutrophils. Thishypoxia induced potentiation of allergen induced neutrophilicinflammation may be particularly important to adults withasthma [3–5] as well as approximately 50% of children [6] inwhom exacerbations are frequently associated with neutro-philic airway inflammation. In life threatening severe exacer-bations of asthma (i.e. intubation for management of statusasthmaticus), high levels of neutrophils and IL-8 have beendetected in BAL fluid [4]. The importance of the large numberof neutrophils and neutrophil derived pro-inflammatory medi-ators to the pathogeneses of either asthma exacerbations orremodeling awaits studies with neutrophil specific antagonists.Studies of neutrophilic inflammation in human asthma exacer-bations are also frequently confounded by variables includingcorticosteroid administration (which can increase neutrophilicinflammation by reducing neutrophil apoptosis) [32], or

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0

1

2

3

4

Tri

chro

me

Sta

ined

Are

a/B

ron

chu

s(μm

2/μm

)

No OVA OVAHypoxiaNormoxia

No OVA OVA

p<0.001

p<0.001

A

Figure 4 Effect of hypoxia and OVA allergen on peribronchial fibrosis. Different groups of BALB/c mice (n=8 mice/group) weresensitized and challenged with OVA allergen. Mice were then exposed to normoxia or hypoxia in exposure chambers. Mice weresacrificed 24 h after the final OVA and/or hypoxia challenge. Lungs were stained with trichrome to detect the area of peribronchialtrichrome staining as an index of collagen deposition. Exposure of mice to hypoxia alone in the absence of OVA allergen challengeinduced a significant increase in peribronchial fibrosis (normoxia vs hypoxia) (pb0.001) (panels A, C). OVA allergen challenge alone innormoxic conditions induced a significant increase in peribronchial fibrosis (no OVA vs OVA in normoxia) (pb0.001) (panels A, B, C).Hypoxia in combination with OVA allergen challenge significantly increased levels of peribronchial fibrosis compared to eitherhypoxia alone (pb0.001) (panels A, E), or compared to OVA allergen alone (pb0.001) (panel A).

34 K.J. Baek et al.

infection (which can independently provoke neutrophilicinflammation). In this mouse model, we were able todemonstrate that the combination of hypoxia and allergen (inthe absence of confounding variables such as corticosteroidtherapy or infection) significantly induces neutrophilic inflam-mation. Further studies in human asthma exacerbations areneeded to determine the relative contribution of hypoxia toneutrophilic airway inflammation during an exacerbation.

Exacerbations of asthma are also known to be associatedwith eosinophilic inflammation in children [6] and adults[33–36]. For example asthmatics with refractory eosinophilic

inflammation have significantly higher exacerbation rates[33], while eosinophil targeted therapies such as anti-IL-5significantly reduce asthma exacerbation rates [33–36]. Inthis study, the absolute number of peribronchial eosinophils(155.9±7.5) was greater than the absolute number ofperibronchial neutrophils (62.7±4.1), even though the com-bined hypoxia and allergen stimulus had its most dramaticeffect on increasing peribronchial neutrophils. Studies by theSevere Asthma Network have identified that each of the fivesevere asthma phenotypes based on cluster analysis have highrates of asthma exacerbations (3.4–4.6 exacerbations/year)

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0

1

2

3

4

No OVA OVA No OVA OVA

HypoxiaNormoxia

P<0.001

P<0.01

p=0.004

α−S

MA

Sta

ined

Are

a( μ

m2/

μm)

A

B C

D E

Figure 5 Effect of hypoxia and OVA allergen on peribronchial smooth muscle. Different groups of BALB/c mice (n=8 mice/group)were sensitized and challenged with OVA allergen. Mice were then exposed to normoxia or hypoxia in exposure chambers. Mice weresacrificed 24 h after the final OVA and/or hypoxia challenge. Lungs were processed for immunohistochemistry with an anti-α-smoothmuscle actin Ab to detect the area of peribronchial α-smooth muscle actin (α-SMA) staining. Exposure of mice to hypoxia alone in theabsence of OVA allergen induced significant increases in the peribronchial α smooth muscle actin immunostained area (normoxia vshypoxia in the absence of OVA) (p=0.004) (panels A–C). OVA allergen challenge alone also induced a similar increase in the peribronchialα-SMA immunostained area (OVA vs no OVA in normoxia) (pb0.001) (panels A, B, D). The combination of hypoxia and OVA allergensignificantly increased the area of peribronchial α-SMA staining compared to either hypoxia alone (pb0.01) (panels A, C, E) or comparedto OVA alone (p=0.05) (panels A, D, E).

35Hypoxia, Inflammation and Remodeling

[37], compared to meta-analysis studies of mild asthmatics(0.2 exacerbations/year in mild asthmatics) [38]. Interestingly,early onset atopic severe asthma with sputum eosinophilia hadthe highest exacerbation rate (4.6/year) [37].

We demonstrated that hypoxia also increases levels ofperibronchial eosinophilic inflammation and that this isassociated with increased levels of eotaxin-1. Since eotaxin-1is not one of the N100 genes known to be directly regulatedby HIF-1α [10,11], the increased levels of eotaxin-1 may bea result of activation of either HIF-1α target genes whichthrough downstream cascades subsequently regulate eotaxin-1expression, and/or a HIF-1α independent effect on eotaxin-1expression. HIF-1α is expressed in eosinophils and regulates

eosinophil chemotaxis and survival [19,39]. Deletion of HIF-1αin eosinophils decreased their chemotaxis under normoxia[19]. In addition, in vitro studies of human peripheral bloodeosinophils have demonstrated that hypoxic eosinophilsupregulated HIF-1α expression as well as anti-apoptoticBcl-XL protein levels more than pro-apoptotic Bax levels, andwere more viable than normoxic eosinophils [39]. Thus, theincreased eosinophils we have detected in lungs of miceexposed to hypoxia and allergen may be mediated by theincreased eotaxin-1 we have detected, as well as by potentialalternate pathways including HIF effects on eosinophilchemotaxis and survival. Increased eosinophilic inflammationmay itself contribute to increased neutrophilic inflammation

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No OVA OVA No OVA OVAHypoxiaNormoxia

0

50

100

TG

F-β

+ C

ells

/ Bro

nch

us

p=0.003

p<0.0001

p<0.0001 150

Figure 6 Effect of hypoxia and OVA allergen on peribronchialTGF-β1+ cells. Different groups of BALB/cmice (n=8mice/group)were sensitized and challengedwith OVA allergen. Micewere thenexposed to normoxia or hypoxia in exposure chambers. Mice weresacrificed 24 h after the final OVA and/or hypoxia challenge.Lungs were processed for immunohistochemistry using ananti-TGF-β1 Ab to quantitate the number of peribronchialTGF-β1+ cells. OVA allergen challenge alone induced an increasein the number of peribronchial TGF-β1 positive cells (OVA vs NoOVA in normoxia) (p=0.003). Exposure of mice to hypoxia alonedid not increase the number of peribronchial TGF-β1 positive cells(hypoxia vs normoxia in the absence of OVA allergen (p=NS). Thecombination of hypoxia and OVA allergen significantly increasedthe number of peribronchial TGF-β1 positive cells in a synergisticfashion as compared to OVA allergen alone (OVA+normoxia vsOVA+hypoxia) (pb0.0001).

36 K.J. Baek et al.

(through eosinophil expression of KC) as well as to airwayremodeling (through increased expression of TGF-β1) [25]. Wehave demonstrated that the combination of hypoxia andallergen significantly increased levels of the pro-remodelingcytokine TGF-β1 as well as indices of airway remodeling,including increased peribronchial fibrosis and increasedsmooth muscle. We have also previously demonstrated theimportance of eosinophil expressed TGF-β1 to airway remod-eling in mouse models of asthma [25,40]. Similar results havealso been observed in humans with asthma in which airwayeosinophil expression of TGF-β1 and indices of airwayremodeling are significantly reduced by eosinophil targetedanti-IL-5 therapy [41].

In summary, this study demonstrates that hypoxia maysignificantly potentiate allergen induced lung HIF-1α expres-sion, chemokine expression, airway inflammation (neutrophilic,eosinophilic), expression of the pro-remodeling cytokineTGF-β1, and indices of airway remodeling (fibrosis, andsmooth muscle). Studies have shown that synergism betweenasthma triggers (viral infection, allergen exposure, and atopicsensitization) conferred the greatest risk of triggering severeasthma exacerbations [42]. As over 90% of severe asthmaexacerbations are associated with hypoxia [1], hypoxia maybe another asthma trigger contributing to synergism withallergen or viral infection. Although exposure of mice to 10%hypoxia is a standard model of investigating hypoxia in mousemodels of lung disease [43], how this level of hypoxia in micetranslates to in vivo studies in humans with severe asthma andhypoxia is unknown. Further study is thus needed to determine

whether hypoxia contributes to increased neutrophilic and/oreosinophilic airway inflammation in human subjects withasthma. Moreover, as exacerbations of asthma are associatedwith an accelerated decline in lung function, further study isneeded to determine whether the hypoxia induced increasedremodeling in our mouse model is evident in asthmaticswith exacerbations associated with hypoxia and contributeto previously observed enhanced decline in lung functionassociated with severe asthma exacerbations [7,8].

Conflict of interest statement

The author(s) declare that there are no conflicts of interest.

Acknowledgment

This research was supported by NIH AI 38425, AI 70535, and AI 72115(D Broide), a Veterans Affairs Career Development Award-2 (LE CrottyAlexander) and a Stephen Silberstein Senior Fellowship Award fromthe American Asthma Foundation (V Nizet).

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