OMICS A Journal of Integrative Biology

Volume 13, Number 5, 2009

Original Article
ª Mary Ann Liebert, Inc.
DOI: 10.1089=omi.2009.0042

The Nutrigenomics of Asthma:

Molecular Mechanisms of Airway Neutrophilia
following Dietary Antioxidant Withdrawal

Katherine J. Baines,1,2 Lisa G. Wood,1,2 and Peter G. Gibson1,2

Abstract

Depletion of antioxidants through consumption of a low antioxidant diet has been reported to increase neu-
trophilic airway inflammation and worsen symptoms of asthma. Using a nutrigenomics approach, this study
explores the mechanisms of airway neutrophilic inflammation due to depletion of dietary antioxidants. Induced
sputum samples were collected at baseline and after participants consumed a low antioxidant diet for 14 days.
Genome-wide gene expression profiles were generated from sputum RNA samples from participants with a
>10% change in sputum neutrophils using Illumina Humanref-8 expression microarrays. There were 104 genes
differentially expressed following the dietary change. Upregulated genes were involved in the innate immune
response and included the innate immune receptors TLR2, IL1R2, CD93, the signaling molecules IRAK2, IRAK3,
and neutrophil proteases MMP25 and CPD. Downregulated genes included those involved in endogenous
antioxidant defenses (GSTA1, GSTA2) and protease inhibition (SLPI, SERPINB3). Altered expression of five
genes (TLR2, IRAK2, IL1R2, C20orf114, and SERPINB3) was confirmed using real-time polymerase chain reaction
(PCR). These observations suggest that depletion of dietary antioxidants in asthma may result in upregulation of
genes involved in the innate immune response. A diet low in antioxidants may be contributing to the devel-
opment of neutrophilic asthma through activation of the innate immune response.

Introduction inflammatory cells such as eosinophils and neutrophils and

the production of reactive oxygen species (ROS). This leads to

A sthma is a chronic inflammatory disease of the

airways, which is associated with variable airflow
obstruction, airways hyperresponsiveness, and respiratory
symptoms. Airway inflammation in asthma is reliably as-
sessed using hypertonic saline induced sputum. This is a
noninvasive technique that samples cellular and soluble ma-
terial from the lower airways. Sputum samples have previ-
ously been used for measurement of inflammatory cells,
cytokines, enzymes, and gene expression (Grissell et al., 2005).
Through the use of induced sputum it has been identified that
airway inflammation in asthma is heterogeneous; both eo-
sinophilic and noneosinophilic subtypes of asthma have been
recognized (Simpson et al., 2006), whereas each subtype is
associated with different inflammatory pathways. Eosino-
philic asthma typically occurs with an allergen driven Th2
type response, where noneosinophlic asthma has been asso-
ciated with the innate immune pathway (Simpson et al., 2006).
Regardless of the mechanistic pathway leading to airway in-
flammation, the end result involves accumulation of activated
oxidative stress, as ROS overwhelms the host antioxidant
defenses (Nadeem et al., 2003).
Host defense against ROS is provided by a range of anti-
oxidants that are present in the respiratory tract lining
fluid including antioxidant enzymes (superoxide dismutase,
glutathione peroxidase, catalase), metal-binding proteins
(lactoferrin, transferring, ceruloplasmin), and low molecular
weight antioxidants (ascorbate, urate, glutathione) (van der
Vliet et al., 1999). These compounds are either synthesized by
the body (endogenous) or obtained in the diet (exogenous).
Dietary antioxidants have been shown to be important to re-
spiratory health. Various dietary antioxidant deficiencies
have been reported in asthma, including vitamin C (Kelly
et al., 1999), vitamin E (Kelly et al., 1999), and carotenoids,
including lycopene (Wood et al., 2005). Further evidence re-
lating antioxidants to asthma comes from large epidemio-
logical studies, which collectively report that a variety of
antioxidants are protective against asthma (Ford et al., 2004,
Rubin et al., 2004). To this end, we have previously observed a

1
Priority Research Centre for Asthma and Respiratory Diseases, University of Newcastle, Callaghan NSW Australia.
2
Department of Respiratory and Sleep Medicine, Hunter Medical Research Institute, John Hunter Hospital, New Lambton NSW Australia.

355
356
direct effect of antioxidants on airway inflammation in asth-
ma. We demonstrated that consumption of a low antioxidant
diet for 10 days can decrease lung function, worsen asthma
symptoms, and modulate airway inflammation by increasing
sputum neutrophils and airway neutrophil activation (Wood
et al., 2008). We also found that this increase in airway in-
flammation can be reversed using antioxidant supplementa-
tion in the form of lycopene-rich treatments (Wood et al.,
2008). Taken together, these studies indicate a significant in-
fluence of dietary antioxidant status on asthma, and none-
osinophilic asthma in particular. On the other hand, it is
noteworthy that the molecular mechanisms of these effects
have not been identified to date.
Numerous cellular processes, including gene expression
and cell fate, can be modulated by subtle changes in the
oxidant–antioxidant balance. At the molecular level, in-
creased levels of ROS have been implicated in instigating
inflammatory responses in the lungs through the activation of
transcription factors such as nuclear factor-kB (NF-kB) and
activator protein (AP)-1, signal transduction, chromatin re-
modeling, and gene expression of pro-inflammatory media-
tors (Rahman. 2003). Understanding the mechanisms by
which dietary antioxidants affect airway inflammation in
asthma is crucial in developing therapeutic and nutritional
supplementation strategies to improve respiratory outcomes
in asthma and airway diseases more generally.
Nutrigenomics refers to an exciting new application of
omics technologies to nutritional science, and assesses how
diet integratively influences gene expression, protein expres-
sion and metabolism (Kussmann et al., 2006). The study of
gene expression using microarray technology (transcrip-
tomics) in response to changing environmental exposures
such as nutritional interventions is a powerful tool for the
comprehensive measurement of genome–environment inter-
actions. Although it appears that intake of foods rich in anti-
oxidants are beneficial for respiratory health, the molecular
mechanisms of such clinical observations remain elusive and
have not been studied in airway samples in asthma.
The present study investigates the molecular mechanisms
of airway neutrophilia induced by withdrawal of antioxidants
using a nutrigenomics approach. Based on prior observations
of innate immune dysfunction in neutrophilic asthma and the
oxidant sensitive status of the key innate immune transcrip-
tion factors (e.g., NF-kB), we hypothesized that dietary anti-
oxidant withdrawal would alter expression of genes involved
in the innate immune response.
Methods
Participants
Adults with stable asthma were recruited from the John
Hunter Hospital Asthma Clinic, NSW, Australia. Asthma was
diagnosed according to American Thoracic Society guidelines
based upon current (past 12 months) episodic respiratory
symptoms, doctor’s diagnosis (ever) and demonstrated evi-
dence of airway hyperresponsiveness to hypertonic saline.
Exclusion criteria included: recent (past month) respiratory
tract infection, recent asthma exacerbation, recent unstable
asthma or change in maintenance therapy, current smoking
and use of vitamin or mineral supplements. All participants
gave written informed consent prior to their inclusion in the
study, and the Hunter New England Area Health Service and
BAINES ET AL.
The University of Newcastle Research Ethics Committee ap-
proved the study.
Dietary protocol
Following collection of baseline data, participants were in-
structed to consume a low antioxidant diet for 14 days. This
diet included no more than one piece of fruit and two servings
of vegetables per day and the avoidance of tea, coffee, red
wine, fruit juices, nuts, seeds, vitamin or mineral supplements,
and aspirin (Record et al., 2001). Adherence was monitored by
using 24-h recall dietary records and by measurement of
plasma levels of antioxidants (tocopherols and carotenoids) at
baseline and following the 2-week intervention.
Sputum induction and analysis
Spirometry (KoKo PD Instrumentation, Louisville, CO)
and sputum induction with hypertonic saline (4.5%) were
performed as previously described (Gibson et al., 1998). Fol-
lowing spirometry, 4.5% saline was inhaled from an ultra-
sonic nebuliser for doubling time periods. A fixed sputum
induction time of 15 min was used for all participants. For
inflammatory cell counts, selected sputum was dispersed
using dithiothreitol (DTT). The suspension was filtered, and a
total cell count of leucocytes and cell viability was performed.
Cytospins were prepared, stained (May-Grunwald Giemsa)
and a differential cell count obtained from 400 nonsquamous
cells. For gene expression microarray analysis, 100 mL of se-
lected sputum was added to Buffer RLT (Qiagen, Hilden,
Germany) and stored at
808C.
Whole genome gene expression microarrays
Participants (n ¼ 10) were selected for gene expression
analysis if they had a >10% increase in sputum neutrophils
after the dietary change. RNA was extracted from induced
sputum samples using the RNeasy Mini Kit (Qiagen) and
quantitated using the Quant-iT RiboGreen RNA Quantitation
Assay Kit (Molecular Probes Inc., Invitrogen, Eugene, OR).
Fluorescence was measured at wavelengths 485 nm (excita-
tion) and 520 nm (emission) (FLUOstar Optima, BMG Lab-
tech, VIC, Australia). RNA (500 ng) was reverse transcribed
into cRNA and biotin-UTP labeled using the Illumina Total-
Prep RNA Amplification Kit (Ambion, Austin, TX). A total of
750 ng of cRNA was hybridized to the Illumina Sentrix
HumanRef-8 v2 Expression BeadChips (Illumina, San Diego,
CA) using standard protocols (see http:==www.illumina.com=
pages.ilmn?ID¼51 for further details on chip design). Each
BeadChip measured the expression of 20,590 genes, was
scanned using the Illumina Bead Station and captured using
BeadScan 3.5.11 (Illumina).
Quantitative real-time polymerase chain reaction
(PCR) validation
RNA (200 ng) was reverse-transcribed to cDNA using the
high-capacity cDNA reverse transcription kit as per manu-
facturers instructions (Applied Biosystems, Foster City, CA).
Taqman qPCR primer and probes for the selected targets that
were differentially expressed in the microarrays including
TLR2, IRAK2, IL1R2, C20orf114, and SERPINB3 were pur-
chased in kit form (Applied Biosystems). PCR primers and
probes were combined with the reference gene eukaryotic 18S
MOLECULAR MECHANISMS OF ANTIOXIDANT WITHDRAWAL IN ASTHMA 357

ribosomal RNA in duplex real-time PCRs (ABI GeneAmp
7700 cycler, Perkin-Elmer, Norwalk, CT). Results were cal-
culated using 2
DDCt relative to both the housekeeping gene
(18S) and the baseline result.
Plasma antioxidant measurement
High-pressure liquid chromatography (HPLC) meth-
odology was used to measure plasma antioxidants, includ-
ing carotenoids (b-carotene, lycopene, a-carotene, lutein=
zeaxanthin and b-cryptoxanthin) and tocopherols (a-, b-, d-,
and g-tocopherol) (Wood et al., 2005). Ethanol: ethyl acetate
(1:1) containing internal standards (canthaxanthin and to-
copherols acetate) and BHA was added to the sample. Anti-
oxidants were extracted using a series of ethyl acetate and
hexane washes. The solvents were evaporated with nitrogen
and the sample reconstituted in HPLC grade hexane. Chro-
matography was performed on a Hypersil ODS column
(100 mm2.1 mm5 mm) with a flow rate of 0.3 mL=min.
Analysis used a mobile phase of acetonitrile: dichloro-
methane: methanol 0.05% ammonium acetate (85:10:5 v=v)
and a diode array detector set at 470 nm and 290 nm for ca-
rotenoids and tocopherols, respectively.
Statistical analysis
Data were analyzed using Stata 9 (Stata Corporation, Col-
lege Station, TX) and reported as mean (SD) for normally
distributed data and median (Q1–Q3) for data not normally
distributed. Statistical comparisons were performed using the
paired Student’s t-test for normally distributed data (% pre-
dicted FEV1, % predicted FVC, % FEV1=FVC, asthma control
score, exhaled nitric oxide), and the Wilcoxon test was ap-
plied to data not normally distributed (total cell count, %
neutrophils, % eosinophils, % macrophages, % lymphocytes);
p < 0.05 was considered significant.
For the whole genome gene expression, data were nor-
malized using cubic spline in Illumina’s BeadStudio 3.0 soft-
ware (Illumina) and exported to GeneSpring GX 10 software
(Agilent Technologies, Santa Clara, CA). Data were further
normalized to the median of all samples and a paired t-test
was applied to identify genes that were differentially ex-
pressed before and after 14 days of the low antioxidant diet.
Genes were judged to be differentially regulated only when
(1) the gene was detected as present or marginal in 10 out of 20
samples studied (<0.05 detection p value), (2) the extent of
difference in expression was statistically significant ( p < 0.05
paired t-test adjusted for multiple comparisons using the
Benjamini Hochberg method), and (3) the difference in ex-
pression was >1.5-fold. Hierarchical clustering analysis was
performed by creating a horizontal dendrogram for the con-
ditions (revealing significant relationships between the ex-
pression profiles of the samples) and a vertical dendrogram
for the entities (revealing significant relationships between
the expression levels of genes across all samples). These
dendrograms were generated using the Pearson uncentered

Table 1. Clinical Characteristics and Induced

Sputum Inflammatory Cell Counts of Participants
with Asthma at Baseline and Following 14 Days
of the Low Antioxidant Diet

Following
14 days on a low
Baseline antioxidant diet p

N 10 10
Plasma antioxidants 13.1 (1.4) 12.4 (1.7) 0.036
(carotenoids and
tocopherols) mg=L,
mean (SD)
% predicted FEV1, 78.7 (17.6) 78.3 (16.4) 0.959
mean (SD)
% predicted FVC, 96.2 (11.2) 95.4 (9.4) 0.865
mean (SD)
% FEV1=FVC, 65.4 (11.4) 65.6 (10.9) 0.968
mean (SD)
Asthma 0.71 (0.53) 0.78 (0.56) 0.777
control score,
mean (SD)
Exhaled nitric oxide, 36.2 (23.9–49.7) 30.0 (17.8–30.9) 0.248
median (Q1–Q3)
Total cell 2.9 (2.0–5.5) 3.0 (1.0–6.0) 0.870
count106=mL,
median (Q1–Q3)
Neutrophils %, 35.8 (14.3–43.3) 61.9 (45.3–73.5) 0.007
median (Q1–Q3)
Eosinophils %, 11.9 (0.3–46.3) 3.9 (0.0–9.5) 0.341
median (Q1–Q3)
Macrophages %, 39.1 (32.0–73.0) 30.5 (14.0–43.8) 0.364
median (Q1–Q3) FIG. 1. Volcano plot displaying differentially expressed
Lymphocytes %, 0.3 (0.3–0.8) 0.1 (0.0–0.8) 0.391 genes (dark gray squares) that were both significant (paired
median (Q1–Q3) t-test p < 0.05, horizontal line) and altered greater than 1.5-
fold (vertical line).
358 BAINES ET AL.

Table 2. Selected Genes of Interest Differentially Expressed in Airway Samples from Asthmatic
Subjects following 14 Days of a Low Antioxidant Diet: Fold Change Compared to Baseline

Gene name Gene symbol GenBank ID p Value Fold change

Interleukin-1 receptor associated kinase 2 IRAK2 NM_001570.3 0.036 1.85

CASP8 and FADD like regulator CFLAR NM_003879.3 0.019 1.73
Interleukin-1 receptor, type II IL1R2 NM_173343.1 0.047 1.72
Carboxypeptidase D CPD NM_001304.3 0.026 1.63
E2F transcription factor 3 E2F3 NM_001949.2 0.004 1.63
Calcium=calmodulin-dependent protein kinase kinase 2 CAMKK2 NM_153500.1 0.023 1.61
C-type lectin domain family 4, member E CLEC4E NM_014358.2 0.024 1.60
Matrix metalloproteinase 25 MMP25 NM_022468.4 0.020 1.60
Leptin receptor overlapping transcript LEPROT NM_017526.2 0.027 1.60
Interleukin-1 receptor associated kinase 3 IRAK3 NM_007199.1 0.011 1.59
Bromodomain adjacent to zinc finger domain, 1A BAZ1A NM_013448.2 0.008 1.56
Mitogen activated kinase kinase kinase 8 MAP3K8 NM_005204.2 0.034 1.53
Anthrax toxin receptor 2 ANTXR2 NM_058172.3 0.042 1.51
CD93 molecule CD93 NM_012072.3 0.036 1.51
Interferon-related developmental regulator 1 IFRD1 NM_001550.2 0.006 1.51
Wilms tumor 1 associated protein WTAP NM_004906.3 0.045 1.51
RING1 and YY1 binding protein RYBP NM_012234.4 0.041 1.50
Toll-like receptor 2 TLR2 NM_003264.3 0.012 1.50
Fibroblast growth factor binding protein 1 FGFBP1 NM_005130.3 0.034
1.50
S100 calcium binding protein A16 S100A16 NM_080388.1 0.042
1.51
Insulin-like growth factor binding protein 2 IGFBP2 NM_000597.2 0.006
1.55
Secretory leukocyte peptidase inhibitor SLPI NM_003064.2 0.048
1.56
Calpain 9 CAPN9 NM_016452.1 0.011
1.60
Mucin 20, cell surface associated MUC20 NM_001098516.1 0.025
1.60
Nedd4 family interacting protein NDFIP2 NM_019080.1 0.049
1.63
Discoidin domain receptor tyrosine kinase 1 DDR1 NM_013993.2 0.025
1.64
Forkhead box A1 FOXA1 NM_004496.2 0.037
1.64
Charcot-Leyden crystal protein CLC NM_001828.4 0.046
1.83
Polymeric immunoglobulin receptor PIGR NM_002644.2 0.044
1.85
CD24 molecule CD24 NM_013230.2 0.010
1.93
Forkhead box J1 FOXJ1 NM_001454.2 0.018
1.96
Arachidonate 15-lipoxygenase ALOX15 XM_001131480.1 0.025
2.00
Trefoil factor 3 (intestinal) TFF3 NM_003226.2 0.017
2.09
Tryptase alpha=beta 1 TPSAB1 NM_003294.3 0.030
2.10
Prominin 1 PROM1 NM_006017.1 0.014
2.14
Glutathione S-transferase alpha 1 GSTA1 NM_145740.2 0.011
2.36
Glutathione S-transferase alpha 2 GSTA2 NM_000846.3 0.009
2.42
Chromosome 20 open reading frame 114 C20orf114 NM_033197.2 0.011
2.51
Serpin peptidase inhibitor, clade B (ovalbumin), member 3 SERPINB3 NM_006919.1 0.021
2.92

algorithm that clusters the samples based on their correlation
coefficients, with average linkage.
Results
Clinical parameters
Ten participants who had sufficient paired sputum RNA
samples (100 mL in buffer RLT) were selected for the micro-
array analysis, based on the criterion that they had a >10%
increase in sputum neutrophils after 14 days of the low anti-
oxidant diet. They had a mean age (SD) of 63 (11) years. This
included six males and four females, with 7=10 (70%) of the
participants being atopic. Asthma pattern was classified as
intermittent (n ¼ 5, 50%), moderate (n ¼ 3, 30%), or severe
persistent (n ¼ 2, 20%). Median (Q1–Q3) daily dose of inhaled
corticosteroids was 800 (500–1,000) mg beclomethasone
equivalents=day. No participants were current smokers, and
60% (n ¼ 6) were never smokers with 40% (n ¼ 4) being ex-
smokers, with a median (Q1–Q3) pack years of 7.2 (2.2–36).
Plasma antioxidant (carotenoid and tocopherol) levels de-
creased significantly following 10 days on the low antioxidant
diet (Table 1). There was no change in clinical parameters
(Table 1).
Whole genome gene expression microarrays
A gene was considered to be expressed if it was flagged as
present or marginal in 10 out of 20 samples tested, and this
resulted in 17,659 genes out of 22,184 (79.6%) being expressed.
Following dietary antioxidant withdrawal, the expression of
1,654 genes out of 17,659 genes were significantly altered
( p < 0.05 paired t-test) at visit 2 compared to baseline. A
volcano plot analysis was used to further refine this list and
select those genes that were both significantly different
( p < 0.05 paired t-test adjusted for multiple comparisons using
the Benjamini Hochberg method) and a fold change of 1.5-fold
or greater (Fig. 1). This resulted in 104 genes that were dif-
ferentially expressed following the dietary change. Each of the
MOLECULAR MECHANISMS OF ANTIOXIDANT WITHDRAWAL IN ASTHMA 359

Table 3. Differentially Expressed Genes in Airway Samples from Asthmatic Subjects

following 10 Days of a Low Antioxidant Diet: Fold Change Compared to Baseline

Gene name Gene symbol GenBank ID Fold change

Interleukin-1 receptor associated kinase 2 IRAK2 NM_001570.3 1.85

CASP8 and FADD like regulator CFLAR NM_003879.3 1.73
Interleukin-1 receptor, type II IL1R2 NM_173343.1 1.72
Carboxypeptidase D CPD NM_001304.3 1.63
E2F transcription factor 3 E2F3 NM_001949.2 1.63
Norrie disease protein NDP NM_000266.1 1.62
Calcium=calmodulin-dependent protein kinase kinase 2 CAMKK2 NM_153500.1 1.61
C-type lectin domain family 4, member E CLEC4E NM_014358.2 1.60
Matrix metalloproteinase 25 MMP25 NM_022468.4 1.60
Chromosome 5 open reading frame 41 C5orf41 NM_153607.1 1.60
Leptin receptor overlapping transcript LEPROT NM_017526.2 1.60
Interleukin-1 receptor associated kinase 3 IRAK3 NM_007199.1 1.59
Transmembrane protein 154 TMEM154 NM_152680.1 1.57
Bromodomain adjacent to zinc finger domain, 1A BAZ1A NM_013448.2 1.56
Mitogen activated kinase kinase kinase 8 MAP3K8 NM_005204.2 1.53
Anthrax toxin receptor 2 ANTXR2 NM_058172.3 1.51
CD93 moelcule CD93 NM_012072.3 1.51
Chromosome 1 open reading frame 24 C1orf24 NM_052966.1 1.51
Interferon-related developmental regulator 1 IFRD1 NM_001550.2 1.51
Wilms tumor 1 associated protein WTAP NM_004906.3 1.51
RING1 and YY1 binding protein RYBP NM_012234.4 1.50
Toll-like receptor 2 TLR2 NM_003264.3 1.50
Fibroblast growth factor binding protein 1 FGFBP1 NM_005130.3
1.50
Histone cluster 1, H2bd HISTH2BD NM_138720.1
1.50
Dehydrogenase=reductase (SDR family) member 9 DHRS9 NM_199204.1
1.51
Tctex 1 domain containing 2 TCTEX1D2 NM_152773.2
1.51
S100 calcium binding protein A16 S100A16 NM_080388.1
1.51
Aldehyde dehydrogenase 1 family, member L1 ALDH1L1 NM_012190.2
1.51
Leucine rich repeat containing 48 LRRC48 NM_031294.2
1.54
Myoglobin MB NM_005368.2
1.54
Insulin-like growth factor binding protein 2 IGFBP2 NM_000597.2
1.55
Midkine MDK NM_002391.3
1.56
Secretory leukocyte peptidase inhibitor SLPI NM_003064.2
1.56
NDRG family member 2 NDRG2 NM_201539.1
1.57
ATPase, Hþ=Kþ transporting, nongastric, alpha polypeptide ATP12A NM_001676.4
1.57
Cingulin CGN NM_020770.1
1.58
Chromosome 1 open reading frame 225 LOC388610 NM_001013642.2
1.58
Junction plakoglobin JUP NM_002230.1
1.60
Calpain 9 CAPN9 NM_016452.1
1.60
Mucin 20, cell surface associated MUC20 NM_001098516.1
1.60
Solute carrier family 27 (fatty acid transporter) member 2 SLC27A2 NM_003645.2
1.61
Creatinine kinase, brain CKB NM_001823.3
1.62
Spermatogenesis associated 18 homologue SPATA18 NM_145263.2
1.62
WD repeat domain 54 WDR54 NM_032118.2
1.62
Alcohol dehydrogenase 1C (class 1), gamma polypeptide ADH1C NM_000669.3
1.62
Transforming, acidic coiled-coil containing protein 2 TACC2 NM_206860.1
1.63
Nedd4 family interacting protein NDFIP2 NM_019080.1
1.63
Discoidin domain receptor tyrosine kinase 1 DDR1 NM_013993.2
1.64
Forkhead box A1 FOXA1 NM_004496.2
1.64
EPS8 like 1 EPS8L1 NM_133180.2
1.64
Rhophilin, Rho GTPase binding protein 2 RHPN2 NM_033103.3
1.64
Aquaporin 5 AQP5 NM_001651.1
1.65
tubulin polymerization-promoting protein family member 3 TPPP3 NM_015964.2
1.66
Tetraspanin 13 TSPAN13 NM_014399.3
1.68
Phospholipase A2, group X PLA2G10 NM_003561.1
1.69
Transmembrane protein 98 TMEM98 NM_001033504.1
1.70
Claudin 7 CLDN7 NM_001307.4
1.70
EF-hand calcium binding domain 1 EFCAB1 NM_024593.2
1.70
Iroquois homeobox 3 IRX3 NM_024336.1
1.71
Chromosome 9 open reading frame 135 C9orf135 NM_001010940.1
1.72
Family with sequence similarity 3, member D FAM3D NM_138805.2
1.74
Cortexin 1 CTXN1 NM_206833.2
1.74
(Continued)
360 BAINES ET AL.

Table 3. (Continued)

Gene name Gene symbol GenBank ID Fold change

Hypothetical protein LOC646723 LOC646723 XR_017241.1
1.75

calcium channel, voltage-dependent, gamma subunit 6 CACNG6 NM_031897.2
1.75
Tetraspanin 8 TSPAN8 NM_004616.2
1.76
Tight junction protein 3 (zona occludens 3) TJP3 NM_014428.1
1.78
Tetraspanin 1 TSPAN1 NM_005727.2
1.78
solute carrier family 44, member 4 SLC44A4 NM_025257.2
1.78
Hypothetical protein FLJ23834 FLJ23834 NM_152750.3
1.80
Membrane-spanning 4-domains, subfamily A, member 8B MS4A8B NM_031457.1
1.81
Chromosome 13 open reading frame 30 C13orf30 NM_182508.1
1.81
Leucine rich repeat containing 23 LRRC23 NM_201650.1
1.82
CDC42 effector protein (Rho GTPase binding) 5 CDC42EP5 NM_145057.2
1.82
Dynein, light chain, roadblock-type 2 DYNLRB2 NM_130897.1
1.82
Charcot-Leyden crystal protein CLC NM_001828.4
1.83
Chloride intracellular channel 6 CLIC6 NM_053277.1
1.83
Calcyphosine CAPS NM_080590.1
1.84
Zinc finger, MYND-type containing 10 ZMYND10 NM_015896.2
1.84
Chromosome 19 open reading frame 33 C19orf33 NM_033520.1
1.84
Chromosome 9 open reading frame 116 C9orf116 NM_144654.2
1.85
Polymeric immunoglobulin receptor PIGR NM_002644.2
1.85
V-set and immunoglobulin domain containing 2 VSIG2 NM_014312.3
1.89
Chromosome 2 open reading frame 40 C2orf40 NM_032411.1
1.92
CD24 molecule CD24 NM_013230.2
1.93
Cytochrome P450, family 4, subfamily B, polypeptide 1 CYP4B1 NM_000779.3
1.94
Chromosome 20 open reading frame 85 C20orf85 NM_178456.2
1.95
Tektin 1 TEKT1 NM_053285.1
1.95
Forkhead box J1 FOXJ1 NM_001454.2
1.96
Anterior gradient homolog 3 (Xenopus laevis) AGR3 NM_176813.3
1.97
Arachidonate 15-lipoxygenase ALOX15 XM_001131480.1
2.00
G protein-coupled receptor 56 GPR56 NM_201524.1
2.01
Keratin 19 KRT19 NM_002276.4
2.05
Radial spoke head 1 homolog (Chlamydomonas) RSPH1 NM_080860.2
2.05
Keratin 8 KRT8 NM_002273.2
2.08
Trefoil factor 3 (intestinal) TFF3 NM_003226.2
2.09
Tryptase alpha=beta 1 TPSAB1 NM_003294.3
2.10
Chromosome 9 open reading frame 24 C9orf24 NM_147169.1
2.11
Prominin 1 PROM1 NM_006017.1
2.14
Placenta-specific 8 PLAC8 NM_016619.1
2.16
Calcyphosine CAPS NM_004058.2
2.23
Glutathione S-transferase alpha 1 GSTA1 NM_145740.2
2.36
Glutathione S-transferase alpha 2 GSTA2 NM_000846.3
2.42
Chromosome 20 open reading frame 114 C20orf114 NM_033197.2
2.51
Serpin peptidase inhibitor, clade B (ovalbumin), member 3 SERPINB3 NM_006919.1
2.92

104 genes was investigated and are reported in Table 2 if they
had a known and biologically relevant immune function. The
complete list of these genes is provided in Table 3. There were
22 genes that were upregulated and 82 genes that were
downregulated following the low antioxidant diet. A hierar-
chical cluster containing these 104 differentially expressed
genes was constructed, and significant differences in the gene
expression profiles between the airway samples obtained from
participants at baseline and post low antioxidant diet were
observed (Fig. 2).
Interestingly, antioxidant withdrawal resulted in the spe-
cific upregulation of genes involved in the inflammatory and
immune responses including the innate immune receptors
TLR2, IL1R2, CD93, ANTXR2, the innate immune signaling
molecules IRAK2, IRAK3, MAP3K8, and neutrophil proteases
MMP25 and CPD. A molecule important in the regulation of
apoptosis, CFLAR, was also upregulated after the dietary
change. The downregulated genes included those involved in
antioxidant and protease defense, cellular structure, metabo-
lism, and processes. Many of the downregulated genes were
also of unknown function (n ¼ 35, 43%). Importantly, genes
involved in endogenous antioxidant defenses such as gluta-
thione metabolism (GSTA1, GSTA2) as well as genes involved
in protease inhibition (SLPI, SERPINB3) were downregulated.
Interestingly, the expression of genes associated with eosin-
ophil (CLC) and mast cell (TPSAB1) responses was also re-
duced. Table 2 displays selected genes relating to immune
responses that were differentially expressed.
Selected upregulated genes involved in inflammatory and
immune responses (TLR2, IRAK3, IL1R2, IRAK2, and CD93)
and proteolysis (CFLAR, MMP25, and CPD) were evaluated
and found to be significantly correlated with sputum neu-
trophil % and each other, indicating that expression of these
genes plays an important role in the development of neutro-
MOLECULAR MECHANISMS OF ANTIOXIDANT WITHDRAWAL IN ASTHMA 361

philic airway inflammation resulting from antioxidant with-

drawal (Table 4). At baseline, several genes involved in the
innate immune response were significantly correlated with
the level of plasma antioxidants, including IRAK3 (r ¼
0.83;
p ¼ 0.003; Fig. 3), IL1R2 (r ¼
0.65; p ¼ 0.043), and CD93
(r ¼
0.66; p ¼ 0.038).

Real-time PCR validation

Quantitative real-time PCR (qPCR) was used to confirm
the altered expression of five genes after antioxidant with-
drawal, in the nine pairs of samples with sufficient remaining
RNA. These included three upregulated genes TLR2, IRAK2,
and IL1R2, and two downregulated genes C20orf114 and
SERPINB3. qPCR consistently confirmed the results of the
microarray (Fig. 4, Table 5).

Discussion
This is the first study to investigate the molecular mecha-
nisms of airway neutrophilia induced by withdrawal of die-
tary antioxidants in asthma. Participants consumed a diet low
in antioxidants for 14 days, and the whole genome gene ex-
pression of induced sputum samples was studied before and
after the dietary change. It was found that airway gene ex-
pression was significantly altered by the low antioxidant diet,
with increased expression of many genes involved in the in-
nate immune response, including receptors, signaling pro-
teins, and proteases. This was accompanied by decreased
expression of genes associated with the endogenous antioxi-
dant glutathione, as well as protease inhibition. Expression of
selected inflammatory and immune related genes were highly
correlated with sputum neutrophil %, indicating that they
play an important role in neutrophilic airway inflammation
induced by antioxidant depletion. Altered expression of five
genes (TLR2, IL1R2, IRAK2, C20orf114, and SERPINB3) were
further confirmed using qPCR. These genes were chosen for
their biologically relevant roles in the innate immune and
inflammatory responses. Additionally, these genes were dif-
ferentially expressed at a range of fold changes providing
further validation of the microarray results.
There is a large body of evidence suggesting that dietary
antioxidants are important in protecting against activation of
the innate immune response. Vitamin C has a direct effect on
innate immune activation, and has been shown to suppress
NF-kB activity (Carcamo et al., 2002), and enhance neutrophil
function (Sharma et al., 2004). Lycopene (Kim et al., 2004), and
b-carotene (Bai et al., 2005) are also inhibitors of NF-kB acti-
vation. As dietary antioxidants are usually consumed to-
gether; combinations of antioxidants are most likely to affect
innate immune responses. For example, a combination of vi-
tamin C and E were shown to inhibit intracellular ROS pro-
duction and inhibit the NF-kB, PKR, eIF-2a, protein kinase C,
and p38 MAPK pathways (Tan et al., 2005). We have also
recently demonstrated that antioxidant levels modify innate

FIG. 2. Hierarchical cluster showing gene expression pro-

files of participants with asthma (n ¼ 10). The dendrogram at
the top of the figure shows the relationships between par-
ticipants with asthma at baseline (red branches) and after
nutritional intervention (blue branches). The dendrogram on
the left represents the relationships between the expression
levels of each gene across all samples.
362 BAINES ET AL.

Table 4. Spearman Rank Correlation Analysis between the Expression

of Evaluated Upregulated Genes and Sputum Neutrophil %

Genes involved in the inflammatory and immune response Genes involved in proteolysis

TLR2 IRAK3 IL1R2 IRAK2 CD93 CFLAR MMP25 CPD

b a b b b c c
Sputum N% 0.60 0.49 0.57 0.64 0.60 0.77 0.68 0.66b
TLR2 0.69c 0.88c 0.69c 0.82c 0.65b 0.75c 0.86c
IRAK3 0.82c 0.80c 0.74c 0.59b 0.79c 0.83c
IL1R2 0.79c 0.92c 0.58b 0.82c 0.90c
IRAK2 0.81c 0.77c 0.78c 0.91c
CD93 0.58b 0.69c 0.88c
CFLAR 0.63b 0.68c
MMP25 0.83c
CPD

Data shows the Spearman rank correlation coefficient (r).

a
p < 0.05, bp < 0.01; cp < 0.001.

immune activity in the airways. We reported that the anti-
oxidant withdrawal diet led to an increase in airway inflam-
mation, involving an increase in the proportion of sputum
neutrophils in the airways (Wood et al., 2008). The present
nutrigenomics study extends these findings by examining the
effect of antioxidant withdrawal on airway inflammation,
investigating these mechanisms at a molecular level using
whole genome gene expression.
The toll-like receptor (TLR) family is the main signaling
receptors of innate immunity. TLR2 is a receptor for a variety
of microbial ligands, including lipoteichoic acids (LTAs) and
peptidoglycans associated with gram-positive bacteria (Kurt-
Jones et al., 2002). Activation of TLR2 regulates several im-
portant proinflammatory neutrophil functions through the
activation of the NF-kB (Sabroe et al., 2005). TLR2 has also
recently been shown to be essential to sensing of oxidant in-
duced inflammation in vivo (Paul-Clark et al., 2009) and our
data show significant upregulation of TLR2 gene expression
in airway cells during a pro-oxidant state. TLR signaling is
known to involve interleukin-1 receptor associated kinases
(IRAKs) of which, IRAK2 plays a central role (Keating et al.,
2007). Furthermore, the IRAK3 (IRAKM) gene has been re-
ported to be associated with early onset persistent asthma
(Balaci et al., 2007). We found evidence for increased gene
expression for each of these molecules following antioxidant
withdrawal. Consistent with these effects, our data confirm
involvement of many genes of the innate immune pathway in
the response of asthmatic airways to antioxidant withdrawal.
The factors driving neutrophil responses in asthma are not
well established. Although transient neutrophilia is associ-
ated with infections, the cause of chronic neutrophilia is not
known. Potential candidates are air pollutants, personal and
environmental tobacco smoke exposure, and dietary influ-
ences. Each of these factors can be associated with activation
of innate immune mechanisms. This study suggests that die-
tary deficiencies in antioxidants may contribute to the neu-
trophil influx in noneosinophilic asthma via similar innate
immune mechanisms. Given the global changes in diet, the
rise in asthma prevalence, and the association of new onset
asthma following migration with noneosinophilic mecha-
nisms, the observations in this study suggest a mechanism
whereby changes in nutritional intake can modulate innate
immunity and asthma.
Glutathione is an important endogenous antioxidant in
the airways. The glutathione S-tranferases (GSTs) are a large

FIG. 3. Baseline levels of plasma antioxidants were signif- FIG. 4. The expression of three upregulated genes (TLR2,
icantly correlated with airway gene expression of IRAK3 IL1R2, and IRAK2) and two downregulated genes (C20orf114
(Spearman r ¼
0.83, p ¼ 0.003). and SERPINB3) were further validated using real-time PCR.
MOLECULAR MECHANISMS OF ANTIOXIDANT WITHDRAWAL IN ASTHMA
Table 5. Quantitative Real-Time PCR Validation
of Microarray Results
Microarray QPCR
Pairs Pairs Paired
Mean fold changed Mean fold changed t-test
change (>1.5-fold) change (>1.5-fold) p value
TLR2 1.50 7=10 2.78 8=9 0.002
IL1R2 1.72 6=10 3.07 6=9 0.028
IRAK2 1.85 7=10 3.85 6=9 0.090
C20orf114
2.51 8=10
7.29 7=9 0.035
SERPINB3
2.92 7=10
7.82 7=9 0.007
family of phase II enzymes that function in the detoxification
of various carcinogens, environmental toxins, therapeutic
drugs, products of oxidative stress, and also triggers of airway
inflammation such as cigarette smoke (Strange et al., 2001).
Genes in the GST family are crucial for protecting cells from
the damaging effects of ROS and are highly polymorphic. GST
genotypes have been associated with decline in lung function
(Imboden et al., 2007) and development of asthma (Imboden
et al., 2008). Interestingly, the anti-inflammatory and oxidant
scavenging activities of GSTs can be modulated by the innate
immune response; for example, GSTA1 and GSTA2 gene ex-
pression is repressed by IL-1b (Ng et al., 2007). Here we have
shown a downregulation of two GST genes of the alpha class
GSTA1 and GSTA2 after the low antioxidant diet, suggesting
that endogenous antioxidant defenses may be impaired. This
is in agreement with previous studies that have shown that
the activity of endogenous antioxidants is dependent on the
presence of dietary antioxidants. For example, antioxidant
treatment combining vitamin C, vitamin E, and b-carotene in
athletes resulted in increased activities of superoxide dis-
mutase, catalase, and glutathione reductase (Tauler et al.,
2002). The underlying molecular mechanism for these effects
may reside in the ability of carotenoids to activate the anti-
oxidant response element Nrf2, and thereby modulate gene
transcription (Ben-Dor et al., 2005).
Proteolytic enzymes play an important role in tissue re-
modelling and repair in the airways. Levels of proteolytic
enzymes are increased in asthma (Vignola et al., 1998), and
this increase is thought to indicate an imbalance in the
protease=antiprotease system. The current study links dietary
intake of antioxidants with altered gene expression of pro-
teases and antiproteases, including the upregulation of mol-
ecules with proteolytic activity including MMP25, CPD, and
CFLAR and downregulation of molecules with antiproteolytic
activity, such as SLPI and SERPINB3. Increased presence of
ROS are also known the influence the activity of neutrophil
granule proteins (Reeves et al., 2002). CFLAR is also an im-
portant antiapoptotic gene that is dependent on NF-kB
(Kanetaka et al., 2008). CFLAR can also modulate cell motility
by promoting the expression of matrix metalloproteinase
(MMP)-9 (Park et al., 2008), and its gene expression is also
modulated by antioxidant status (Payton et al., 2007).
Nutrigenomics is a new field being explored that will
promote an increased understanding about how nutrients act
at the molecular level and ultimately allow effective dietary
intervention strategies to be developed (Muller and Kersten,
2003). In this context, the latest powerful technology involv-
363
ing DNA microarrays makes it possible to assess the effects of
particular dietary interventions on the expression of the entire
genome. Few nutrigenomics studies have been applied in
humans, where an important barrier to identifying molecular
markers and mechanisms is the inaccessibility to relevant
tissue samples. Here, importantly, we have shown that in-
duced sputum samples are a suitable and noninvasive source
of RNA to study the molecular mechanisms of airway in-
flammation using gene expression microarrays.
Although the present study is the first to demonstrate
changes to the gene expression profile of airway samples due to
dietary change, there are a number of limitations that need to be
addressed in future studies. Future studies should include a
control group receiving a placebo (i.e., without a change to their
diet), to rule out putative temporal changes in gene expression
that might have occurred without a dietary intervention.
Confounding factors such as age, gender, inhaled steroid use,
asthma severity, smoking, and atopic status should also be
investigated for their relationship to airway gene expression.
However, this study looked at intraindividual changes over a
2-week period during which these potential confounders did
not change. Furthermore, intraindividual differences in base-
line antioxidant status may have an influence on airway gene
expression, and the use of a dietary stabilization period be-
fore the intervention would be useful. The intraindividual
differences in antioxidant levels at baseline are largely reflec-
tive of differences in recent dietary intake of participants.
Interestingly, baseline antioxidant levels correlated negatively
with baseline gene expression of several genes that were
subsequently upregulated with antioxidant withdrawal. This
suggests that irrespective of the starting point, antioxidant
withdrawal further modifies expression of these genes.
In conclusion, the present nutrigenomics study suggests
that the depletion of dietary antioxidants in asthma may re-
sult in upregulation of genes involved in the innate immune
response. These observations indicate that a low antioxidant
diet may lead to neutrophilic airway inflammation in asthma
through potentiation of innate immune pathways. These
findings may have significance in providing a mechanism to
link changes in diet and clinical asthma, as well as to highlight
potential new therapeutic targets, because innate immune
activation could be modified by blocking receptors, signaling
cascades, or the subsequent cytokine responses. A deeper
understanding of the molecular mechanisms and pathways
underpinning the antioxidant actions in asthma also holds
substantial promise for the development of future nutri-
genomics interventions aimed at preventing asthma and=or
improving clinical management of asthma.
Funding
The authors acknowledge funding received from the Na-
tional Health and Medical Research Council (NHMRC). Dr.
Katherine Baines holds an HMRI Xstrata Coal Asthma Re-
search Fellowship, Dr. Lisa Wood holds a University of
Newcastle Brawn Fellowship, and Professor Peter Gibson
holds an NHMRC Practitioner Fellowship.
Acknowledgments
The authors acknowledge Lakshitha Gunawardhana,
Hayley Scott, Joanne Smart, and Emma Hall for their technical
assistance.
364
Author Disclosure Statement
The authors declare that no conflicting financial interests
exist.
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Address correspondence to:
Dr. Katherine J. Baines
Level 3, HMRI, John Hunter Hospital
Locked Bag 1, Hunter Region Mail Centre
Newcastle, NSW 2310, Australia
E-mail: katherine.baines@newcastle.edu.au