Disruption of the transcription factor Nrf2 promotes pro-oxidative dendritic cells that stimulate Th2-like immunoresponsiveness upon activation by ambient particulate matter.

Abstract

Oxidative stress is important in dendritic cell (DC) activation. Environmental particulate matter (PM) directs pro-oxidant activities that may alter DC function. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates expression of antioxidant and detoxification genes. Oxidative stress and defective antioxidant responses may contribute to the exacerbations of asthma. We hypothesized that PM would impart differential responses by Nrf2 wild-type DCs as compared with Nrf2(-/-) DCs. We found that the deletion of Nrf2 affected important constitutive functions of both bone marrow-derived and highly purified myeloid lung DCs such as the secretion of inflammatory cytokines and their ability to take up exogenous Ag. Stimulation of Nrf2(-/-) DCs with PM augmented oxidative stress and cytokine production as compared with resting or Nrf2(+/+) DCs. This was associated with the enhanced induction of Nrf2-regulated antioxidant genes. In contrast to Nrf2(+/+) DCs, coincubation of Nrf2(-/-) DCs with PM and the antioxidant N-acetyl cysteine attenuated PM-induced up-regulation of CD80 and CD86. Our studies indicate a previously underappreciated role of Nrf2 in innate immunity and suggest that deficiency in Nrf2-dependent pathways may be involved in susceptibility to the adverse health effects of air pollution in part by promoting Th2 cytokine responses in the absence of functional Nrf2. Moreover, our studies have uncovered a hierarchal response to oxidative stress in terms of costimulatory molecule expression and cytokine secretion in DCs and suggest an important role of heightened oxidative stress in proallergic Th2-mediated immune responses orchestrated by DCs.

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Disruption of the Transcription Factor Nrf2 Promotes
Pro-Oxidative Dendritic Cells That Stimulate Th2-Like
Immunoresponsiveness upon Activation by Ambient
Particulate Matter1
Marc A. Williams,2,3*†Tirumalai Rangasamy,2* Stephen M. Bauer,* Smruti Killedar,*
Matthew Karp,* Thomas W. Kensler,‡Masayuki Yamamoto,¶Patrick Breysse,‡
Shyam Biswal,‡§and Steve N. Georas3*†
Oxidative stress is important in dendritic cell (DC) activation. Environmental particulate matter (PM) directs pro-oxidant activ-
ities that may alter DC function. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive transcription factor that
regulates expression of antioxidant and detoxification genes. Oxidative stress and defective antioxidant responses may contribute
to the exacerbations of asthma. We hypothesized that PM would impart differential responses by Nrf2 wild-type DCs as compared
with Nrf2?/?DCs. We found that the deletion of Nrf2 affected important constitutive functions of both bone marrow-derived and
highly purified myeloid lung DCs such as the secretion of inflammatory cytokines and their ability to take up exogenous Ag.
Stimulation of Nrf2?/?DCs with PM augmented oxidative stress and cytokine production as compared with resting or Nrf2?/?
DCs. This was associated with the enhanced induction of Nrf2-regulated antioxidant genes. In contrast to Nrf2?/?DCs, coincu-
bation of Nrf2?/?DCs with PM and the antioxidant N-acetyl cysteine attenuated PM-induced up-regulation of CD80 and CD86.
Our studies indicate a previously underappreciated role of Nrf2 in innate immunity and suggest that deficiency in Nrf2-dependent
pathways may be involved in susceptibility to the adverse health effects of air pollution in part by promoting Th2 cytokine
responses in the absence of functional Nrf2. Moreover, our studies have uncovered a hierarchal response to oxidative stress in
terms of costimulatory molecule expression and cytokine secretion in DCs and suggest an important role of heightened oxidative
stress in proallergic Th2-mediated immune responses orchestrated by DCs. The Journal of Immunology, 2008, 181: 4545–4559.
A
major public health concern is the global increase in
urban and roadside traffic pollution. Despite its impor-
tance, there is poor appreciation and understanding of
how exposure to particulates contained in environmental airborne
pollution affect the immune system. Although there is currently a
lack of data indicating the mechanisms involved, some studies
have suggested that inhaled particulate matter (PM)4derived from
industry, power stations, or diesel exhaust particles contribute to
the increased incidence of asthma, allergic conditions, pulmo-
nary infections, cardiovascular disease, and mortality in the in-
fant and adult populations (1–5). In addition, different types of
air pollution can have profoundly different qualitative effects on
human health (3).
Dendritic cells (DCs) are the key components of innate immu-
nity that rapidly responds to diverse environmental cues. Because
DCs are highly efficient in activating naive T cells, they link innate
and adaptive immunity during episodes of infection or cellular
damage and tissue necrosis. In this way, DCs efficiently activate
naive T cells, resulting in their clonal expansion and differentiation
into different effector lineages (6–9). Although DCs are poised to
*Division of Pulmonary and Critical Care Medicine and†Department of Environmental
Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY
14642;
School of Public Health, and§Division of Pulmonary and Critical Care Medicine, Johns
Hopkins University School of Medicine, Baltimore, MD 21205; and¶Center for Tsukuba
Advanced Research Alliance, University of Tsukuba, Tsukuba, Japan
‡Department of Environmental Health Sciences, Johns Hopkins Bloomberg
Received for publication June 29, 2007. Accepted for publication July 23, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This research was supported in part by a New Investigator Project from the Center for
Childhood Asthma in the Urban Environment (funded by National Institute of Environ-
mental Health Sciences (NIEHS) and U.S. Environmental Protection Agency Grant
P01ES09606 to S.N.G. and M.A.W.) as well as by the Department of Medicine, Univer-
sity of Rochester School of Medicine and Dentistry (Rochester, NY). The research was
also supported by National Institutes of Health (NIH) Research Grants R01 HL073952
and HL071933 (to S.N.G.), COPD SCCOR P50 HL084945 and CA 94076 (to T.W.K.)
and by NIH NIEHS Pilot Project Grants P30 ES03819 and P30 ES001247 (to M.A.W.).
M.A.W. wrote the paper, performed the research, analyzed the data, and was responsible
for the design and implementation of the experiments. T.R. cowrote the paper, performed
the research with M.A.W., and assisted with the analysis of the data as well as with the
design and implementation of the experiments. S.K. assisted with the technical aspects of
the experiments and also contributed important elements for the design and implemen-
tation of some of the experiments. M.K. provided important technical assistance and data
analysis in the execution of some of the experimental assays. S.M.B. assisted with the
design and implementation of the data derived from lung DCs and naive CD4?T cell
coculture studies as well as with the measurement of cytokine production. T.W.K. pro-
vided vital new reagents and analytical tools and assisted with analysis of some of the
data. M.Y. contributed vital materials to this study. P.B. contributed vital materials to this
studyaswellasmethodologicalapproachesinusingparticulatematter.S.B.assistedinthe
experimental design and data analysis. S.N.G. assisted in the experimental design, critical
discussion, and data analysis.
2M.A.W. and T.R. contributed equally to this work.
3Address correspondence and reprint requests to Dr. Marc. A. Williams or Dr. Steve N.
Georas, Division of Pulmonary and Critical Care Medicine, University of Rochester
SchoolofMedicine,601ElmwoodAvenue,Box692,Rochester,NY14642-8692.E-mail
addresses: marc_williams@urmc.rochester.edu or steve_georas@urmc.rochester.edu
4Abbreviations used in this paper: PM, particulate matter; APM, ambient particulate
matter; CD40L, CD40 ligand; DC, dendritic cell; DCF, dichlorofluorescein; DCFH-
DA, 2?,7?-dichlorofluorescein diacetate; DX, dextran; GCLc, glutathione cysteine li-
gase catalytic subunit; GCLm, GCLc modifier subunit; HO-1, hemeoxygenase-1; KC,
keratinocyte; MFI, mean fluorescence intensity; NAC, N-acetyl cysteine; Nrf2, nu-
clear erythroid 2 p45-related factor 2; PDCA-1, plasmacytoid DC Ag-1; ROS, reac-
tive oxygen species; VEGF, vascular endothelial growth factor; wt, wild type.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
www.jimmunol.org

Page 2

respond to inhaled pollutants such as ambient particulate matter
(APM), very little is known about how APM affects the activation
of DCs. Interestingly, other groups have recently reported that hu-
man airway epithelial cells exposed to diesel exhaust particles se-
crete chemokines and other mediators that recruit DCs and induce
their maturation (10, 11). Thus, DCs will be among the first cells
to sense and respond to inhaled particulate pollution.
Airway inflammation in allergic asthma reflects aberrant im-
mune responses against otherwise harmless inhaled allergens (9).
Although DCs reside in the airway and are richly interdigitated
throughout the bronchial epithelium (12), little is known about
how inhaled environmental exposures affect DCs. We have previ-
ously shown that ambient urban PM instructs a novel pathway of
DC maturation and directs them to stimulate a complex pattern of
Th1- and Th2-associated T cell responses (2). Oxidative stress is
important in DC maturation and could influence the ultimate pat-
tern of immune responses (13, 14). DCs also require a balanced
intracellular redox state for proper functional development (15–
19). Depletion of glutathione in murine APCs in vivo resulted in
suppressed Th1 and elevated Th2 activity (19). Oxidative stress
occurs when oxidants overwhelm antioxidant defenses that may
also involve signaling via the transcription factor NF?B under con-
ditions of accumulated hydrogen peroxide (20). To counteract the
deleterious effects of oxidative stress, all cells have evolved an
elaborate defense mechanism to maintain redox homeostasis. This
system includes a series of antioxidant detoxification enzymes
(21–26).
The nuclear erythroid 2 p45-related factor 2 (Nrf2) has been
shown by our group and others to be a key regulatory transcription
factor that induces antioxidant and detoxification genes that protect
against the deleterious effects of reactive oxygen species (ROS)
(22–27). Nrf2 is a redox-sensitive, basic leucine zipper transcrip-
tion factor. During oxidative stress, Nrf2 is activated following its
detachment from a cytosolic inhibitor called Keap1 and then trans-
locates to the nucleus where it binds to the antioxidant response
element in the promoter region of target genes, leading to their
transcriptional induction (22–28).
We have shown that genetic deletion of Nrf2 renders mice more
susceptible to Th2-driven allergic airway inflammation (25). This
suggested that Nrf2 normally functions to maintain allergen-driven
immune responses in check. However, the mechanisms of how
Nrf2 regulates immune responses and responds to diverse envi-
ronmental danger signals remains poorly understood. Clues to the
potential importance of Nrf2 in host immunity come from studies
in Nrf2-deficient mice (29). These mice were more susceptible to
sepsis in part due to the augmented transcription of several innate
immune response genes (29). This study suggested that Nrf2 may be
important in regulating innate immunity. Interestingly, Li and col-
leagues showed that Nrf2 is activated by diesel exhaust particles in
epithelial cells and macrophages (22, 23), but how Nrf2 function in
dendritic cells remains poorly understood.
In the present study, we exposed bone marrow-derived DCs
from Nrf2?/?and Nrf2?/?mice to urban airborne PM to assess
whether a defective antioxidant defense in DCs would alter their
responses to an important environmental airborne pollutant. Dis-
ruption of Nrf2 in DCs leads to increased oxidative stress and a
dysregulated pattern of immunological responsiveness in PM-ex-
posed DCs that was also characterized by an enhanced promotion
of a Th2-type immune response upon the coculture of APM-stim-
ulated Nrf2?/?lung DCs with naive CD4?OT-II T cells. These
studies point to a crucial role for Nrf2 in innate immunity and
oxidative defense mechanisms in response to airborne particulate
pollution.
Materials and Methods
Use of wild-type (wt) and Nrf2 gene-disrupted mice
Nrf2-deficient CD1:ICR mice were generated as previously described (30).
Mice were genotyped for Nrf2 expression by PCR amplification of
genomic DNA extracted from the tail using three different primers (19) as
follows: 5?-TGGACGGGACTATTGAAGGCTG-3? (sense for both geno-
types); 5?-CGCCTTTTCAGTAGATGGAGG-3? (antisense for wt Nrf2
mice); and 5?-GCGGATTGACCGTAATGGGATAGG-3? (antisense for
LacZ). All investigations done with mice met the approval of the Johns
Hopkins University Animal Care and Use Committee and were conducted
in strict accordance with guidelines set by the U.S. Animal Welfare Acts
and National Institutes of Health guidelines. Male OT-II transgenic mice
expressing the TCR specific for OVA323–339 were also used in this study
(see below) and maintained in the laboratory animal research facility of the
University of Rochester (Rochester, NY) concordant with the approval of
the University of Rochester Animal Care and Use Committee. Both strains
of mice were propagated in specific pathogen-free conditions, fed an AIN-
76A diet and water ad libitum, and housed in polycarbonate cages with
hard wood chip bedding in a conventional animal facility maintained under
controlled conditions (temperature at 23 ? 2°C, humidity of 55 ? 5%, and
continuous light/dark cycles of 12 h).
Generation of murine DCs and stimulation
Myeloid DCs were generated from bone marrow-derived precursors of
naive Nrf2?/?and Nrf2?/?mice as described using a highly reproducible
protocol that generates conventional myeloid DC (online supplemental ma-
terial for Ref. 2) in static culture at 37°C in a fully humidified 5% CO2/95%
air incubator. Bone marrow precursors were harvested from the pooled
femurs and tibiae of female mice (8 wk old; five mice per genotype per
independent experiment) by flushing with ice-cold complete RPMI 1640
culture medium (Dutch modification) supplemented with 20 mM HEPES
buffer, 2 mM L-glutamine, 2.5 ?g/ml gentamicin sulfate, and 8% (v/v)
FBS; aggregates were gently disbursed by repeated pipetting in ice-cold
culture medium. Cells were centrifuged at 400 ? g for 10 min at 8°C and,
following two washes in ice-cold divalent cation-free PBS (pH 7.4), the
cells were resuspended and the erythrocytes were removed by lysis in ACK
buffer (150 mM NH4Cl, 1.0 mM KHCO3, and 0.1 mM Na2EDTA (pH 7.4))
for 3 min at room temperature. The lysis reaction was quenched by the
addition of ice-cold culture medium and centrifugation at 400 ? g for 10
min at 8°C. Cells were resuspended in PBS containing 10 mM EDTA,
0.1% BSA, and 10 mM HEPES, and centrifuged twice at 200 ? g for 10
min at 8°C to deplete platelets (we have found that platelets can adversely
block the development of conventional myeloid DC and reduce the yield;
thus, we prefer to remove them). Cell pellets were next resuspended in
culture medium and seeded into 6-well tissue culture clusters at a density
of 2.5 ? 105cells per well in a total volume of 4 ml. Cells were cultured
at 37°C in a sterile filtered atmosphere of 5% CO2/95% air and a fully
humidified incubator. Cultures were pulsed at day 0 and every 48 h with a
combination of IL-4 (10 ng/ml) and GM-CSF (25 ng/ml) to propagate
immature myeloid DC as we have previously described (2). After 8 days of
culture, immature DCs were harvested, washed, and seeded at a density of
8 ? 105cells/ml in 12-well culture dishes in a total volume of 2.0 ml.
Immature DCs propagated by this method were conventional myeloid DCs
with an end of culture viability of at least 95.6 ? 2.9% (by 0.2% (v/v)
trypan blue exclusion and light microscopic evaluation). Immature myeloid
DCs were characterized as moderate expressing CD11c?, high expressing
CD11b?, and moderate expressing MHC class II (Ia/Ie haplotype) cells, as
described in Fig. 1. Immature DCs were next stimulated with culture me-
dium alone (resting immature DC), 100 ng/ml LPS (Escherichia coli-de-
rived endotoxin, serotype 055:B5, in endotoxin-free water), or 10 ?g/ml
Baltimore city ambient particulate matter (PM10) in endotoxin-free PBS
with 20 mM HEPES buffer (pH 7.4). In some experiments, carbon black
particles were used as a negative control to test for any PM-mediated,
contact-dependent activation of DCs at 10 ?g/ml (Sigma-Aldrich). Fol-
lowing 48 h of culture, we harvested DCs and culture supernatant to assess
cell function and secretion of cytokines.
Isolation and purification of conventional myeloid lung DCs
In some experiments, we confirmed our observations made with bone mar-
row-derived DC from Nrf2?/?as compared with Nrf2?/?by studying the
functional and activation-dependent responses of CD11c-selected conven-
tional myeloid lung DC using a protocol developed in our laboratory. In
three independent experiments, we enriched for pulmonary myeloid
CD11c?PDCA-1?DC (Fig. 1; where PDCA is plasmacytoid DC Ag-1).
Although we have reproducibly applied this protocol in our laboratory, it
4546Nrf2-DISRUPTED DCs ARE PROINFLAMMATORY

Page 3

has not been previously published by us; for that reason, it is described in
full in this article.
Groups of four to five mice per genotype were euthanized one at a time
by i.p. injection of 200 mg/kg sodium pentobarbital euthanasia solution,
and then cervical dislocation, consistent with University of Rochester In-
stitutional Animal Care and Use Committee protocols and the most recent
guidelines on euthanasia from the American Veterinary Medical Associa-
tion. Upon confirmation of euthanasia, the abdominal aorta was severed
and immediately blotted with sterile surgical gauze, and the diaphragm as
well as the rib cage was then excised. Next, the right and left ventricles of
the heart were perfused with two successive 5.0-ml volumes of ice-cold
PBS (divalent cation-free) supplemented with 20 IU/ml sodium heparin.
Next, the heart was clamped and excised after the removal of pericardiac
membrane. Using forceps, the trachea was grasped and cut close to the
head of animal so that the lungs and trachea could be removed from the
mouse. The trachea was then resected, leaving ?2–4 mm above the lungs
for manipulation of the organs. Up to five whole lungs were deposited into
20 ml of digest buffer (divalent cation-free PBS, 10 mM HEPES-NaOH
(pH 7.4) supplemented with 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, and
1.8 mM CaCl2) on ice and, using a 10-cm2petri culture-dish, the lungs
were sliced into fragments of ?2-mm2using two sterile scalpel blades. The
culture dish was washed with an additional 5 ml of digest buffer to collect
residual lung fragments for a total volume of 25 ml. Just before incubation
at 37°C in a standard cell culture incubator, the digest buffer was supple-
mented with tissue-digesting enzymes, including collagenase II (Worth-
ington Laboratories) at 2.0 mg/ml, dispase II (Sigma-Aldrich) at 0.25 mg/
ml, and DNase I (Sigma-Aldrich) at 2.0 mg/ml. The crude tissue
homogenate was incubated for 30–45 min with periodic agitation by in-
verting the tube 8–10 times every 5 min. The suspension was then diluted
with an equal volume of filter-sterilized GKN buffer (11 mM D-glucose, 5.5
mM KCl, 137 mM NaCl, 25 mM Na2HPO4, and 5.5 mM NaH2PO4) sup-
plemented with 2 mM EDTA and 5% (v/v) FBS that was held at 37°C until
use. Next, the digest was passed over a 70-?m nylon cell strainer to liberate
a single cell suspension, which was washed twice with 10 ml of GKN plus
5% FBS; the washings were collected into the same tube, centrifuged at
450 ? g for 10 min at 25°C, and the supernatant was discarded carefully.
The cell pellet was resuspended in 10 ml of culture medium, and the con-
taminating CD11c?macrophages in this cell suspension were removed by
adherence to tissue culture grade 10-cm2plastic dishes for 1 h at 37°C. The
macrophage-depleted, cell-rich supernatant was harvested and centrifuged
at 400 ? g for 8 min at 20°C. The cell pellet was then carefully resus-
pended in ice-cold MACS cell separation buffer (divalent cation-free PBS
supplemented with 2 mM EDTA and 2.5% (v/v) FBS) to 10 ml and cen-
trifuged at 250 ? g for 10 min at 8°C. Next, plasmacytoid DCs were
removed by following the direct selection procedure for immunomagnetic
isolation of PDCA-1-expressing plasmacytoid DCs (Miltenyi Biotech).
The PDCA-1 depleted lung DCs were then subjected to the conventional
myeloid CD11c DC direct enrichment procedure following the manufac-
turer’s protocol (Miltenyi Biotec). Using the above protocol, from groups
of four mice we routinely harvested 4.3 ? 105to 1.25 ? 106CD11c?DCs
(90.8–97.6% pure by flow cytometric quantitation of CD11c expression) of
purified DC. The low yield of highly purified lung DCs that one obtains
using the above procedure relative to the vast numbers that one liberates
from bone marrow precursors favors much of our work to be modeled
using bone marrow-derived DCs and not lung DCs. Nonetheless, sufficient
numbers are obtained that permit basic flow cytometric and cytokine se-
cretion-type assays upon the activation of CD11c lung DCs as well as
carefully designed naive allogeneic CD4?T cell/DC cocultures.
Baltimore city ambient particulate matter
Ambient PM was collected in the spring of 2001 (April-June) using a
high-volume cyclone collector with a theoretical cut point of 0.85-?m
aerodynamic diameter as we have described in detail (2). Collected APM
was pooled, stored under nitrogen gas, and then refrigerated until use.
Before use, 10 mg/ml APM was suspended in 20 mM HEPES-buffered,
divalent cation-free PBS (pH 7.4), vortexed at a high speed for 5 min, and
used immediately. The toxicity of APM was tested against murine bone
marrow-derived DCs by monitoring trypan blue exclusion. After 48 h of
culture, the toxic dose of APM that induced 50% killing (TD50) was ?540
?g/ml. All experiments were done using APM at 10 ?g/ml. We excluded
the possibility that endotoxin contamination of APM may provoke DC
activation by the Limulus amebocyte lysate QCL-1000 assay (Cambrex).
We found contaminating levels to be ?50 pg of endotoxin per 100 ?g/ml
APM. We have previously shown in detailed titration analyses against
immature murine DC that the very low concentrations of endotoxin we
FIGURE 1.
iments described herein and the highly purified rare myeloid conventional DCs subpopulation enriched from the pooled lungs of Nrf2?/?(B) and Nrf2?/?
(C) mice. Cell surface expressions, shown here as flow cytofluorographic histograms, were typical of several enrichments conducted in our laboratory
and were determined by real-time flow cytometry (FACScalibur and CellQuest software). For bone-marrow-derived DCs, the myeloid DC phenotype
(A) was confirmed by high expression of an CD11c-allophycocyanin conjugate and coexpression of CD11b-PE, with low-moderate expression of
MHC class II-FITC typical of resting state immature DCs. For purified lung DCs, the myeloid DC phenotype was confirmed by expression of
CD11c-PE, absence of the plasmacytoid DC marker PDCA-1-FITC, and low to very low expression of B220-PE in both Nrf2?/?DCs and Nrf2?/?
DCs. Data are described as MFI.
Determination of the cell surface markers that characterize bone marrow-derived DCs (BM-DC) in liquid static culture (A) for the exper-
4547The Journal of Immunology

Page 4

found to be present in atmospheric APM (?50 pg of endotoxin per 100
?g/ml APM) did not affect either the cell surface expression of activation
markers or the secretion of inflammatory cytokines, nor did it alter signif-
icantly the interaction of DCs with naive CD?T cells (online supplemental
material for Ref. 2). In these experiments, we investigated the dose-depen-
dent effects of APM on DCs by reciprocal 10-fold dilutions and contrasted
the observations against the equivalent levels of endotoxin found to be
present in APM by reciprocal 10-fold dilutions (online supplemental ma-
terial for Ref. 2).
Treatment with N-acetyl cysteine (NAC)
To determine the effect of PM (Sigma-Aldrich) instructing DC activation
by an oxidative stress-mediated mechanism, we cultured DCs as described
and, on day 8, stimulated the DCs in the absence/presence of APM (10
?g/ml) with/without NAC (5 mM) for 48 h. DCs were analyzed by flow
cytometry for cell surface markers or by ELISA for cytokine secretion. To
maximize antioxidant activity, DCs were pretreated with NAC for 1 h
before adding PM for the remaining 48-h incubation period.
Characterization of cell membrane-expressed maturation
markers of DC
We used multiparameter flow cytometry to measure the expression of func-
tion-associated molecules by bone marrow-derived as well as highly pu-
rified, freshly isolated myeloid lung DC as previously described (2). DCs
were harvested 48 h after stimulation (as described above) and resuspended
in divalent cation-free PBS supplemented with 2% (v/v) FCS and 0.2%
(w/v) sodium azide (FACS buffer). DC preparations were blocked in 5%
(v/v) normal mouse serum for 15 min at 4°C, washed twice in FACS buffer,
and then secondarily blocked for 15 min at 4°C in anti-mouse CD16/
CDw32 (mouse BD Fc block, clone 2.4G2; BD Pharmingen) to prevent
nonspecific Fc-? receptor-mediated binding of specific detection Abs. DC
preparations were next stained immediately with the following FITC or PE
fluorochrome-conjugated mAbs (BD Pharmingen): anti-MHC class II-PE
or FITC (polymorphic Ia/Ie determinants), anti-CD11b-PE, anti-CD11c-
PE, anti-CD80-PE, anti-CD86-PE, and anti-CD40-PE. In assays where the
enrichment and purity of bone marrow-derived DCs and lung DCs required
confirmation, we also used anti-CD45R/B220-PE (BD Pharmingen), anti-
PDCA-1-FITC (clone JF05-1C2.4.1; Miltenyi Biotec), and anti-CD11c-
allophycocyanin (clone N418; Miltenyi Biotec). Samples were washed
twice in FACS buffer by centrifugation at 400 ? g for 6 min at 4°C and
fixed in 2% (v/v) paraformaldehyde in FACS buffer before analysis. We
analyzed samples on a FACSCalibur flow cytometer using CellQuest 3.1
software (BD Biosciences). The instrument had a standard optical filter
configuration with band pass filters of 530/30 nm and 585/44 nm for FL1
(FITC-conjugated antibodies) and FL2 (PE-conjugated antibodies) data ac-
quisition, respectively. For the analysis of forward angle light scatter, side
angle light scatter, and cell surface receptor expression, data were acquired
in real time. Cell surface expression data were acquired in real time as
geometric mean fluorescence intensity (MFI). The instrument was stan-
dardized before phenotypic analysis with calibration beads (FluoroSpheres
6-Peak; DakoCytomation) and cleaned with sequential washes of distilled
water, 10% (v/v) hypochlorite, and distilled water before data acquisition.
Cytokine measurements
Cell-free culture supernatants were enumerated for cytokine concentrations
by commercial ELISA. We measured the secretion of IL-6 and IL-10 (both
from E-Bioscience with a sensitivity of 2.5 pg/ml cytokine), IL-18 (from
MBL International with a sensitivity of 12.5 pg/ml cytokine), and IL-12p40
and TNF-? (both from Invitrogen-BioSource with limits of sensitivity of
7.5 pg/ml cytokine). We also measured levels of vascular endothelial
growth factor (VEGF)-A (R&D Systems). The VEGF ELISA possessed
limits of sensitivity of 7.8 pg/ml. Cytokine secretion was measured in both
bone marrow-derived DCs as well as the myeloid lung DCs purified from
Nrf2?/?and Nrf2?/?mice. In CD11c?lung DCs, we measured the time-
dependent secretion of TNF-?, IL-12p40, and keratinocyte-derived che-
mokine (KC/CXCL1) at 24, 48, and 72 h poststimulation with PM such
that the duration and peak production of these inflammatory cytokines of
PM-stimulated DC could be determined. We reported data as picograms of
secreted product per 106DC. All measurements were done according to the
manufacturer’s guidelines.
Enrichment of highly pure naive CD4?T cells from OT-II mice
Male OT-II transgenic mice expressing the TCR specific for OVA323–339
and MHC I-Abon the C57BL/6 genetic background were provided by Dr.
D. Topham (University of Rochester School of Medicine, Rochester, NY).
These mice were housed in dedicated pathogen-free facilities at the Uni-
versity of Rochester. For purposes of isolating and purifying naive CD4?
T cells, pools of four mice were euthanized by CO2asphyxiation followed
by cervical dislocation and removal of the superficial inguinal, lumbar,
sacral, renal, axillary, brachial, and cervical nodes. A single mononuclear
cell suspension in complete RPMI 1640 culture medium was prepared from
the lymph nodes, which were next washed twice at 300 ? g for 10 min at
4°C. After the final wash, cells were resuspended in 8 ml of ice-cold,
serum-free culture medium and layered over a cushion of 5 ml of Lym-
pholyte M at a density of 1.0875 g/cm3(pH 6.9) (Cedarlane). This is a
density cell separation medium specifically optimized for the isolation
of viable lymphocytes from murine lymphoid cell suspensions. This
procedure depleted cell debris, fatty tissue, erythrocytes, platelets, and
nonviable cells from the lymphocyte preparation. The interface lym-
phocytes were harvested and washed twice in ice-cold complete culture
medium and once in MACS buffer (divalent cation-free PBS supple-
mented with 2.5% FBS and 2 mM EDTA) at 300 ? g for 10 min at 4°C.
Cells were then subjected to a procedure that enriched and purified the
naive CD4?CD62L?helper T cells by an indirect immunomagnetic bead
purification protocol according to the manufacturer’s instructions (Miltenyi
Biotec, product no.130-093-227).
Assay of cytokine responses of lung DC and naive CD4?OT-II
T cells
To determine the stimulatory function of highly enriched pulmonary my-
eloid CD11c?Nrf2?/?DCs as compared with Nrf2?/?DC counterparts,
DCs were exposed to PM (at 10 ?g/ml) for 48 h, washed in complete
culture medium, and then seeded into 24-well plates in duplicate at a den-
sity of 5 ? 104DCs per well at 500 ?l/well. DC were cocultured with
2.5 ? 105naive CD4?CD62L?T cells per well also at 500 ?l/well for a
total volume of 1.0 ml at a stimulator to responder cell ratio of 1:5, re-
spectively. Cell culture supernatants were harvested after 6 days and quan-
tified by conventional ELISA for the elaboration of either a Th1-type re-
sponse (IL-12p70, with a sensitivity limit of 7.2 pg/ml obtained from
Invitrogen-BioSource, and IFN-?, with a sensitivity limit of 5.6 pg/ml ob-
tained from BD Biosciences-BD Pharmingen) or a Th2-type immune re-
sponse (IL-5 and IL-13, both with a sensitivity limit of 4 pg/ml and ob-
tained from eBioscience). Data were collected from duplicate measures in
the ELISA platforms described above and defined as mean picograms of
secreted cytokine per milliliter.
Analysis of extracellular Ag uptake by DCs
The uptake of FITC-conjugated dextran (DX) (40 kDa; Molecular Probes)
by resting or activated DCs was measured by our previously published
procedure (2, 31). Resting or activated DCs (as described above) were
washed and then incubated in complete culture medium plus 1 mg/ml
FITC-DX for 0, 10, 20, 40, and 80 min at 37°C (to measure energy-de-
pendent uptake) or at 4°C to monitor the background fluorescence of the
receptor and cell membrane-immobilized FITC-DX that could not be taken
up into the cell at this temperature. Active uptake of FITC-DX by cells at
37°C was determined by subtracting the background geometric MFI of DC
labeled with FITC-DX at 4°C from the MFI of FITC-DX that was specif-
ically taken up by DC at 37°C (2, 31).
Determination of free radical generation in activated DCs
We quantified free radical production in DC as previously described (32,
33). This assay quantifies the oxidation of nonfluorescent 2?,7?-dichlo-
rofluorescein diacetate (DCFH-DA; Eastman-Kodak) to fluorescent dichlo-
rofluorescein (DCF) in the presence of intracellularly accumulated hydro-
gen peroxide. On day 8 of culture, immature DCs were assayed for free
radical production. This was done by first loading 200 ?l of a DC suspen-
sion (2.0 ? 105cells total) with 100 ?l of 5 mM (final concentration)
DCFH-DA diluted in PBSg buffer (pH 7.4) (10 mM HEPES, 0.1% (w/v)
gelatin, and 10 mM D-glucose) at 37°C for 15 min with agitation. DC were
then stimulated with 200 ?l of the following agents in control diluent:
control diluent alone (PBSg (pH 7.4)), APM (10 ?g/ml), carbon black
particles (10 ?g/ml, as negative control), or the positive controls LPS (100
ng/ml) or CD40 ligand (CD40L) trimer (50 ng/ml). Samples were stimu-
lated for 80 min and then washed, suspended in 1.0% (v/v) paraformalde-
hyde in FACS buffer, and read on a FACSCalibur flow cytometer using
CellQuest 3.1 software (BD Biosciences). We acquired MFI data in the
FL1 channel of intracellular DCF fluorescence in real time and transformed
it into the percentage increase in respiratory burst activity relative to non-
DCFH-DA-loaded resting/nonstimulated DCs for each time point.
4548 Nrf2-DISRUPTED DCs ARE PROINFLAMMATORY

Page 5

Determination of oxidative stress in activated DCs
We measured mitochondria-derived H2O2by chemiluminescence from the
luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) reagent using a
Berthold Biolumat LB9505 luminometer (PerkinElmer) as described (34).
To detect extracellular H2O2, 10 ?M luminol and 10 ?g/ml HRP were
added to 1 ml of PBS (supplemented with 2.5 mM MgCl2and 5 mg of
glucose) containing 1 ? 106immature DCs and 10 ?g/ml APM. Resting,
nonstimulated DCs were used as control. Immediately following the addi-
tion of luminol and HRP, we measured the resultant chemiluminescence
continuously at 37°C for 60 min. We expressed the data from these ex-
periments as an integrated area under the curve and as mean ? SD from the
product of three independent experiments.
Determination of Nrf2-regulated antioxidant genes in DCs
We used quantitative real-time PCR to measure the mRNA levels of Nrf2-
regulated genes using a previous published procedure (25). We measured
the expression levels of the glutathione cysteine ligase catalytic subunit
(GCLc), the GCLc modifier subunit (GCLm), and heme oxygenase-1
(HO-1) in DCs using commercially available assay kits (Applied Biosys-
tems). Quantitative RT-PCR was performed using the fluorescent dye
SYBR Green master mix following standard protocols on an ABI PRISM
7900 system (Applied Biosystems). Total RNA was extracted from DCs
and used for first-strand cDNA synthesis. The reverse transcription reaction
was performed with 1 ?g of DNase-treated total RNA, 0.5 ?g of anchored
oligo(dT)15primer, and 500 ?M dNTPs (Invitrogen). The levels of cDNA
for GAPDH or each antioxidant gene generated from total cellular RNA
were calculated by using standard curves generated with cDNAs for
GAPDH or each antioxidant gene in which there was a linear relationship
between the number of cycles required to exceed the threshold and the
number of copies of cDNA added. The data were analyzed using the se-
quence detector software SDS 2.0 (Applied Biosystems). All PCRs were
performed in triplicate.
Data analysis
We expressed data as mean ? SD, and these were the product of two
(endocytosis assays) to six independent experiments as indicated (at least
three mice per genotype were used for each independent experiment).
Comparisons between paired and unpaired data were tested for significant
differences using one- and two-way ANOVA, Student’s t test, and post hoc
correction according to the Bonferroni method. Statistical significance was
set at an alpha value of at least p ? 0.05 as indicated. Statistical measure-
ments were done using SigmaStat version 2.03 software and Microsoft
Excel statistical analysis software.
Results
Bone marrow and pulmonary DC expression of
function-associated molecules
To test the hypothesis that PM differentially activates Nrf2?/?as
compared with Nrf2?/?DC, we exposed DC to PM and analyzed
the expression of CD11c, CD40, CD80, CD86, and MHC class II
by flow cytometry (Fig. 2). In the first series of experiments, we
enumerated cell surface expression of these markers in DCs de-
rived from bone marrow progenitors (Fig. 2, A–C). In addition, in
the second series of experiments we measured the cell surface
expression of activation markers on freshly isolated and highly
purified PDCA-1?CD11c?myeloid lung DCs (Fig. 2D).
Although the resting expression of CD40 was similar between
genotypes (Fig. 2, A and C), the resting expression of MHC class
II was somewhat greater on Nrf2?/?DCs (p ? 0.079; Fig. 2, A
and C). Stimulation of DCs augmented the expression of both
CD40 and MHC class II (Fig. 2A) on Nrf2?/?as well as Nrf2?/?
FIGURE 2.
DCs. A and B, Bone marrow-derived DCs from Nrf2?/?and Nrf2?/?mice were stimulated in vitro with particulate matter at 10 ?g/ml for 48 h and surface
expressions of CD40 and MHC class II (A) CD80 and CD86 (B) were determined by multiparameter flow cytometry. C, In addition, flow histograms of
those cell surface markers present on bone marrow-derived DCs of Nrf2?/?and Nrf2?/?mice from one of the experiments is shown. D, Expressions of
CD11c, CD40, CD80, CD86, and MHC class II were found on highly purified lung DCs of Nrf2?/?and Nrf2?/?mice. Data are described as geometric
mean ? SD of data collected as MFI from n ? 6 age- and sex-matched mice per group. Data inserted in the representative flow histograms is also defined
as geometric MFI units. Absolute levels of significance (p values) are shown in the figures.
Flow cytometric determination of cell surface expression of function-associated molecules by bone marrow-derived DCs and purified lung
4549The Journal of Immunology

Page 6

DCs with the highest levels of MHC class II still evident on
Nrf2?/?DCs (Fig. 2A). The resting expression of CD80 (p ?
0.086) and CD86 (p ? 0.068; Fig. 2, B and C) was lower on
Nrf2?/?as compared with Nrf2?/?DC. This was concordant with
our observations made for MHC class II. Stimulation of DC with
PM augmented the expression of both CD80 and CD86 by
Nrf2?/?as compared with Nrf2?/?DC (Fig. 2, B and C), and
although there were no statistically significant differences between
genotypes in the expression of CD80, after the exposure of DCs to
PM the expression of CD86 was markedly greater on Nrf2?/?DCs
as compared with their wt counterparts (Fig. 2, B and C; p ?
0.0023).
We repeated the above studies in lung DCs. We found that there
were marked differences in the constitutive (resting) expression of
cell surface function-associated molecules between genotypes
(Fig. 2D). Although the expression of CD40 was not statistically
different between Nrf2?/?lung DCs and their wt counterparts, the
expression of CD11c and CD80 (to a lesser extent) and the ex-
pression of CD86 and MHC class II in particular were markedly
greater on lung DCs from Nrf2?/?DCs as compared with their wt
counterparts (Fig. 2D). Thus, Nrf2?/?lung DCs were at a consti-
tutively greater level of activation in the resting state than Nrf2?/?
lung DCs.
Upn the activation of lung DCs by APM we observed a striking
disparity in the overall hyperresponsiveness of Nrf2?/?DC as
compared with the expected modest augmentation in cell surface
markers by Nrf2?/?DCs (Fig. 2D). In both Nrf2?/?and Nrf2?/?
lung DCs, the expression of CD11c was augmented upon activa-
tion by APM. There was only modest augmentation in CD40 ex-
pression upon the stimulation of Nrf2?/?DCs with APM, whereas
the expression of this receptor doubled upon the activation of
Nrf2?/?DCs by APM (Fig. 2D). In addition, although there was
a modest up-regulation in the expression of CD80 and a more
significant augmentation in both CD86 and MHC class II expres-
sion upon the stimulation of Nrf2?/?DC with APM, we observed
remarkable responses by Nrf2?/?DCs upon activation by APM
(Fig. 2D). Expression levels of CD80, CD86, and MHC class II
were all markedly enhanced as compared with those for resting
DCs as well as when contrasted with their wt counterparts. These
observations were concordant, at least in part, with those obser-
vations made for the functional response of bone marrow-derived
DCs to particulate matter exposure (Fig. 2, A–C).
Effects of NAC on receptor expression by DCs
NAC is a widely used antioxidant molecule that possesses immu-
nomodulatory effects, including an ability to dampen the expres-
sion of cell surface-expressed molecules upon the activation of
DC. We were interested in determining the ability of NAC to sup-
press PM-mediated DC activation. Thus, in a separate series of
experiments (Fig. 3) we pretreated DCs with 5 mM NAC for 1 h
and then exposed DCs to PM for 48 h before phenotypic analysis.
First, we confirmed that Nrf2?/?as well as Nrf2?/?DCs re-
sponded appropriately to stimulation with PM, as we had shown
previously in our initial observations discussed above (Fig. 2) for
all surface markers studied (Fig. 3). Next, we stimulated DCs with
or without PM in the presence or absence of NAC as shown
(Fig. 3).
In resting Nrf2?/?wt DC, NAC inhibited the expression of
CD40 (Fig. 3A), CD80 (Fig. 3B), and CD86 (Fig. 3C) while at the
same time promoting the expression of MHC class II (Fig. 3D).
FIGURE 3.
cell surface expression of CD40 (A), CD80 (B), CD86 (C), and MHC class II (D) on bone marrow-derived DCs from Nrf2?/?and Nrf2?/?mice. Data
shown are mean ? SD of geometric MFI from n ? 6 age- and sex-matched mice per group. Levels of tests of significance between treatments are shown
and are as described in the text.
Flow cytometric quantitation of function-associated molecule expression by DCs showing the effects of NAC on PM-induced alterations in
4550Nrf2-DISRUPTED DCs ARE PROINFLAMMATORY

Page 7

Although most of these effects of NAC were evident in Nrf2?/?
DCs, we did not see a statistically significant decrease in CD40
expression in NAC treated Nrf2?/?DCs and a modest diminution
in expression of CD40 by their wt counterparts (p ? 0.066; Fig.
3A). However, a consistent observation was that in PM-stimulated
Nrf2?/?and Nrf2?/?DCs NAC attenuated the PM-driven en-
hancement of MHC class II, CD80, and CD86 expression. How-
ever, only in Nrf2?/?DCs did NAC significantly attenuate CD40
expression (p ? 0.05; Fig. 3A). Thus, CD40 expression in resting
and PM-stimulated Nrf2?/?DCs appeared to be refractory to NAC
(Fig. 3A).
Particulate matter directs an Nrf-2-dependent pattern of
cytokine production by DCs
We examined a panel of cytokines known to be important in al-
lergic diseases and the differentiation of T cells. Thus, we assessed
the constitutive ability of Nrf2?/?and Nrf2?/?knockout DCs to
release inflammatory cytokines in the resting state and following
activation by PM (Fig. 4). We found that Nrf2?/?DCs released
minimal levels of IL-12p40 and IL-6 (Fig. 4A) and IL-10 and
TNF-? (Fig. 4B), yet proportionately elevated levels of IL-18 (Fig.
4C) and VEGF (Fig. 4D) in the resting state. By contrast, resting
Nrf2?/?DCs constitutively released greater levels of IL-12p40
(p ? 0.0012), IL-6 (p ? 0.0006), IL-10 (p ? 0.0038), and TNF-?
(p ? 0.0001), as well as VEGF (p ? 0.001), than their wt coun-
terparts, although the levels constitutively released by both
Nrf2?/?and Nrf2?/?DCs were modest (Fig. 4). By contrast,
Nrf2?/?DCs released constitutively lower levels of IL-18 than
their Nrf2?/?DC counterparts (Fig. 4C; p ? 0.0024) by mecha-
nisms that may be dependent in part on Nrf2 activity.
Activation of both Nrf2?/?and Nrf2?/?DC with PM enhanced
the secretion of all cytokines measured with the exception of IL-18
(Fig. 4, A–C). In response to PM stimulation, Nrf2?/?DCs se-
creted markedly less IL-18 as compared with resting DCs (Fig. 4C;
p ? 0.0003). This contrasted with the up-regulation in IL-18 pro-
duction by Nrf2?/?DCs as compared with resting DCs (Fig. 4C;
p ? 0.00038), further supporting the notion that the production of
IL-18 by DCs is dependent on the expression of Nrf2. Under con-
ditions of disrupted Nrf2 we saw suppressed constitutive expres-
sion of IL-18 and enhanced production of this cytokine following
stimulation with PM, whereas in wt DCs the converse was true.
This is a novel and previously unreported effect.
Moreover, we repeated the above studies in freshly isolated,
highly purified CD11c?myeloid lung DCs by investigating the
time-dependent production of the secretion of TNF-? and IL-
12p40, representing two cytokines thought to be important in pul-
monary inflammation and the inflammatory chemokine KC/
CXCL1 (Fig. 5, A and B, respectively). The secretion of TNF-?
and IL-12p40 are important in mouse models of pulmonary and
allergic inflammation (35–38). In addition, we studied the tempo-
ral secretion of KC (Fig. 5B) in response to PM (10 ?g/ml), be-
cause this chemokine is an important mediator in lung inflamma-
tion and we have also found that secretion of KC is a very sensitive
marker of murine myeloid DC activation (39, 40).
In our experiments, we found that peak secretion of TNF and
IL-12p40 by Nrf2?/?and Nrf2?/?DC occurred 48 h after stim-
ulation by PM (Fig. 5). However, as was concordant for Nrf2?/?
bone marrow-derived DCs, their pulmonary myeloid DC counter-
parts also exhibited markedly greater levels of TNF and IL-12p40
secretion than was observed for wt pulmonary DC at all of the time
points studied. In addition, the constitutive secretion of both cy-
tokines by Nrf2?/?DCs in the resting state (time 0 h) was also
markedly greater than the levels observed for Nrf2?/?DCs (Fig.
5), consistent with the observations made for bone marrow-derived
DCs. By contrast, peak secretion of KC followed a more rapid
pattern of secretion that peaked at 24 h poststimulation for both
genotypes (Fig. 5B). Concordant with the absolute levels of TNF
and IL-12p40 secretion, we found that Nrf2?/?DCs secreted sig-
nificantly greater levels of KC both constitutively in the resting
state and upon activation by PM. Thus, both bone marrow-derived
FIGURE 4.
IL-12p70 and IL-6 (A), IL-10 and TNF (B), as well as IL-18 (C) and VEGF (D) is shown. Bone marrow-derived DCs from the indicated strains were
incubated with or without (Resting) particulate matter for 48 h, followed by analysis of cell-free supernatants by commercially available ELISA. Data are
expressed as picograms per million cells and are the product of the mean ? SD of n ? 6 sex- and age-matched mice per group.
Quantitation of inflammatory cytokine production by PM-exposed DCs from Nrf2?/?and Nrf2?/?mice. The secretion of immunoreactive
4551 The Journal of Immunology

Page 8

and pulmonary myeloid DCs from Nrf2?/?mice possess a height-
ened state of inflammatory activation.
Effects of NAC on inflammatory cytokine production by DCs
In a separate series of experiments, we next looked at the effects of
NAC on cytokine production by both Nrf2?/?and Nrf2?/?DCs in
the resting state as well as following PM activation (Table I). First,
we confirmed the cytokine responses of both Nrf2?/?and Nrf2?/?
DCs as defined in Fig. 4. Second, we pretreated DCs with 5 mM
NAC for 1 h and then exposed them to PM for 48 h before as-
sessing the secretion of cytokines by commercial ELISA (Table I).
NAC provoked a complex pattern of cytokine production. In Nrf2
wt DCs, NAC enhanced the secretion of IL-6, IL-10, IL-18, and
VEGF and suppressed the secretion of IL-12p40 and TNF. By
contrast, in Nrf2?/?DCs NAC enhanced the secretion of TNF-?
and VEGF and dampened the production of IL-12p40, IL-6, IL-10,
and IL-18 (Table I).
Although cytokine production by both Nrf2?/?and Nrf2?/?DC
populations responded appropriately following exposure to PM
(Table I), NAC significantly attenuated the production of most of
the cytokines by PM-exposed DCs. However, the production of
TNF-? by PM-exposed Nrf2?/?DCs remained unaltered after
NAC treatment, whereas the production of VEGF remained unaf-
fected by NAC in Nrf2?/?DCs. It is currently unknown why the
secretion of TNF-? and VEGF should show such differences be-
tween Nrf2?/?and Nrf2?/?DCs following PM exposure in the
presence of NAC (Table I). This pattern of cytokines secreted by
PM-exposed DCs is unusual and different from that associated
with classical activators of DCs such as LPS or CD40L, which
typically induce IL-6, TNF-?, and IL-12 in a coordinated fashion.
Pulmonary myeloid Nrf2?/?DCs promote Th2-like cytokine
responses by naive CD4 T cells
In 6-day differentiated cocultures of highly purified naive
CD4?CD62L?T cells stimulated by either Nrf2?/?pulmonary
myeloid DCs or their wt counterparts, we were interested in de-
termining how PM pre-exposure affected the ability of DCs to
influence T cell activation, especially the production of Th1 vs Th2
cytokines (Fig. 6). To discriminate Th2 cytokine responses we
measured the secretion of IL-13 and IL-5 (Fig. 6A) and, by con-
trast, to identify Th1 cytokine responses we measured the secretion
of IL-12p70 and IFN-? (Fig. 6B). To better appreciate the bias of
the Th2-type cytokine responsiveness of PM-exposed Nrf2?/?
DCs contrasted with that of Nrf2?/?DCs, we compared the levels
of IL-13 secretion relative to the levels of either IFN-? or IL-
12p70 by ratiometric analysis (Fig. 6C).
We observed that the secretion of IL-13 was enhanced in PM-
stimulated pulmonary DC/T cell cocultures. However, the amounts
seen in coculture with Nrf2?/?DCs were at least 2.8-fold greater
than those seen using wt DC in cocultures (Fig. 6A). Similarly,
FIGURE 5.
time-dependent secretion of inflam-
matory cytokines of resting (unstimu-
lated) lung DCs from Nrf2?/?and
Nrf2?/?mice as compared with PM-
exposed DCs. The
TNF-? (A) and KC (B) was assessed
by commercial ELISA protocols at
24, 48, and 72 h following the expo-
sure of DCs to PM. Data are de-
scribed as mean picograms per mil-
lionDCs
?
1
significance as indicated (???) are
p ? 0.01.
An analysis of the
secretionof
SD.Levelsof
Table I. Effects of NAC on cytokine production by both Nrf2?/?and Nrf2?/?DCs in the resting state as well as following PM activationa
Treatment IL-12p40IL-6 IL-10TNFIL-18 VEGF
Resting Nrf2?/?
Resting Nrf2?/?NAC
481.9 ? 128.4
352.4 ? 193.1
(p ? 0.017)
122.3 ? 22.4
171.3 ? 35.6
(p ? 0.048)
15.1 ? 35.6
79.5 ? 12.3
(p ? 0.021)
110.6 ? 9.1
93.5 ? 9.8
(p ? 0.021)
430.8 ? 37.3
603.9 ? 104.2
(p ? 0.047)
597.4 ? 186.2
652.5 ? 181.3
(p ? NS)
Resting Nrf2?/?
Resting Nrf2?/?NAC
1004 ? 128.9
423.1 ? 112.7
(p ? 0.004)
356.6 ? 39.3
97.7 ? 12.5
(p ? 0.0039)
45.5 ? 25.4
30.1 ? 21.1
(p ? 0.042)
288.6 ? 30.6
409.4 ? 85.5
(p ? 0.063)
200.5 ? 37.7
74.9 ? 18.8
(p ? 0.021)
1143 ? 131.2
1417 ? 144.1
(p ? 0.01)
APM Resting Nrf2?/?
APM Resting Nrf2?/?NAC
9895 ? 1966
947.4 ? 161.7
(p ? 0.001)
11467 ? 3590
5638 ? 1791
(p ? 0.065)
971.8 ? 236.1
52.9 ? 7.2
(p ? 0.0026)
4178 ? 452.6
1547 ? 436.6
(p ? 0.002)
206.9 ? 96.6
23.2 ? 9.5
(p ? 0.031)
1615 ? 286.6
1460 ? 122.1
(p ? NS)
APM Resting Nrf2?/?
APM Resting Nrf2?/?NAC
29572 ? 3764
24835 ? 2966
(p ? 0.017)
28170 ? 2355
18570 ? 2030
(p ? 0.024)
2535 ? 732.9
1889 ? 455.1
(p ? 0.059)
9825 ? 1873
9272 ? 1174
(p ? ns)
603.3 ? 98.2
161.2 ? 50.7
(p ? 0.004)
2066 ? 473.6
1294 ? 250.2
(p ? 0.034)
aImmature DCs were stimulated in the absence or presence of APM (10 ?g/ml) with or without NAC (5 mM) for 48 h. After this stimulation, we analyzed DC cytokine
secretion by ELISA. To maximize antioxidant activity, cells were pretreated with NAC for 1 h prior to addition of APM for the remaining 48-h incubation period. Levels of
cytokine secretion are shown as mean picograms per million cells ? SD (n ? 3 independent experiments). The p values for levels of significance between pairs of data are shown
in parenthesis for each cytokine. This was done for resting unstimulated DCs in the absence or presence of NAC for both Nrf2?/?and Nrf2?/?DCs and again for DCs stimulated
without or with APM in the absence or presence of NAC.
4552 Nrf2-DISRUPTED DCs ARE PROINFLAMMATORY

Page 9

although PM-stimulated pulmonary DC promoted enhanced pro-
duction of IL-5 in coculture with OT-II T cells, the relative
amounts seen in Nrf2?/?DC cocultures were at least 3.5-fold
greater than those seen in wt DC cocultures (Fig. 6A). These data
suggest an enhanced and default pro-Th2 bias of Nrf2?/?DCs
seen upon contact with naive CD4?T cells. Although PM-stim-
ulated Nrf2?/?DC also enhanced the production of IL-13 and IL-5
by naive CD4?T cells, the levels secreted were markedly lower.
In addition, resting Nrf2?/?DCs stimulated greater levels of both
IL-13 and IL-5 than their wt counterparts on coculture with naive
CD4?T cells, supporting our suggestion of a pro-Th2 bias in
Nrf2?/?DCs (Fig. 6A).
PM-exposed DCs also stimulated enhanced production of both
Th1-type cytokines, IL-12p70 and IFN-? (Fig. 6B). However, in
contrast to the markedly greater levels of Th2-type cytokines that
we found in Nrf2?/?DC/T cell cocultures as compared with their
wt counterparts, this was not true for the observed levels of IL-
12p70 and IFN-? between genotypes (Fig. 6B). In this case, we
found that there was only a 1.5-fold greater level of IL-12p70
secretion and IFN-? in cocultures stimulated by Nrf2?/?DCs as
compared with cocultures stimulated by Nrf2?/?DCs (Fig. 6B).
When expressing the data described as a ratio of IL-13 secretion
relative to either IL-12p70 or IFN-? (Fig. 6C), we found that those
cocultures stimulated by PM-exposed Nrf2?/?DCs indeed pro-
moted a more dramatic pro-Th2 bias of cytokine responsiveness by
naive CD4?T cells than did their wt counterparts.
APM and differential Ag uptake by DCs
During the functional maturation of DCs, there is an initial aug-
mentation of Ag uptake followed by a diminished internalization
of uptake in favor of Ag processing and presentation. To determine
the endocytic activity of DCs, we measured the time-dependent
uptake of dextran as a model exogenous Ag (see Materials and
Methods).
In the resting state (Fig. 7A) we found that both Nrf2?/?and
Nrf2?/?efficiently took up Ag, although DCs from Nrf2?/?mice
showed a lowered ability to take up FITC-DX as compared with
their Nrf2?/?counterparts that was significantly different at 30
min. We also measured the ability of DCs to take up dextran fol-
lowing activation with PM (Fig. 7B). Under these circumstances,
Nrf2?/?DCs retained an efficient time-dependent ability to take up
exogenous Ag as well as an improved ability to do so at the con-
clusion of the assay as compared with their resting counterparts
(p ? 0.084; Fig. 7). By contrast, PM-exposed Nrf2?/?DCs ex-
hibited a greatly diminished ability to take up exogenous FITC-DX
as compared with PM-exposed Nrf2?/?DCs (Fig. 7B) as well as
their resting counterparts (Fig. 7A; p ? 0.013). This highlights a
functional difference between murine DCs that express and those
that lack Nrf2 gene expression and suggests that the disruption of
FIGURE 6.
(IL-13 and IL-5) cytokines by OVA-pulsed (50 ?g/ml) lung DCs from
Nrf2?/?and Nrf2?/?mice and coculture with naive CD4?CD45RA?allo-
geneic OT-II T cells at a stimulator (DC) to responder T cell ratio of 1:5 (see
Materials and Methods). The secretion of IL-13 and IL-5 (A) and the secretion
IL-12p70 and IFN-? (B) are shown. In addition, a ratiometric analysis of the
secretion of IL-13 produced by PM-stimulated Nrf2?/?and Nrf2?/?DCs
relative to either IFN-? or IL-12p70 secretion is shown (C). Data are described
as picograms of cytokine per milliliter produced in the coculture.
Determination of Th1-type (IL-12p70 and IFN-?) vs Th2-type
FIGURE 7.
titation of the endocytic uptake of
FITC-DX (40 kDa) by resting (A) or
PM-exposed (B) DCs from Nrf2?/?
(WT, wild type) and Nrf2?/?(Nrf2
ko (knockout)) mice. The time-de-
pendent endocytosis of FITC-dextran
is shown and levels of tests of signif-
icance between Nrf2?/?as compared
with the Nrf2?/?DCs are also de-
scribed for comparison. Data are de-
scribed as geometric MFI.
Flow cytometric quan-
4553 The Journal of Immunology

Page 10

Nrf2-mediated signaling mechanisms may impair endocytosis by
activated DCs (See Discussion).
PM-induced oxidative stress in Nrf2-deficient DCs
We assessed oxidative stress activity in resting and APM-exposed
DCs by quantifying intracellular H2O2accumulation. We did this
by using the reporter molecule DCFH-DA, which is nonfluorescent
in the unexcited state but becomes rapidly oxidized during normal
basal metabolism and even more so during cellular activation.
DCFH-DA is highly specific for H2O2accumulation and is oxi-
dized by products of NO reacting with oxygen-free radicals. Thus,
for this reason DCFH-DA is an important reporter of alterations of
the intracellular redox state of cells.
Both resting Nrf2?/?and Nrf2?/?DCs exhibited a basal level
of H2O2production that was somewhat elevated in Nrf2?/?DCs
(Fig. 8; p ? 0.024). Activation of DCs with PM (Fig. 8A) pro-
voked rapid increases in H2O2production in both Nrf2?/?and
Nrf2?/?DCs as compared with resting DCs (p ? 0.001). The
greatest levels of activity were seen in Nrf2?/?DCs as compared
with their wt counterparts at all time points (Fig. 8A; p ? 0.01).
Importantly, we did not observe significant increases in H2O2ac-
cumulation in DCs exposed to carbon black particles (Fig. 8B),
indicating that H2O2production by DCs was a specific effect of
components contained in PM. Also, LPS promoted enhanced H2O2
accumulation equally well in both Nrf2?/?and Nrf2?/?DCs as
compared with resting DCs (Fig. 8C). This was in stark contrast
with the responses of Nrf2?/?and Nrf2?/?DCs to CD40L stim-
ulation (Fig. 8D). Under these conditions, we found that Nrf2?/?
DCs were particularly sensitive to the effects of CD40L as com-
pared with Nrf2?/?DCs showing a rapid (within 10 min) and
marked increase in H2O2production as compared with resting and
wt DCs at 10 and 20 min poststimulation (p ? 0.001, Fig. 8D).
We confirmed the effects of PM on DCs independently by mea-
suring a luminol-based oxidative stress assay (Fig. 9). In the cur-
rent study, we have used this assay to measure H2O2formation by
DCs in the presence/absence of PM. Concordant with our obser-
vations above, we found that while resting DCs did not produce
significant amounts of H2O2by this assay, PM directed a marked
FIGURE 8.
titation of H2O2production and accu-
mulation of resting DCs as compared
with PM-exposed DCs. Data are geo-
metric MFI ? SD as a function of
DCF fluorescence (oxidized DCFH-
DA). The respiratory burst of DCs de-
rived from Nrf2?/?and Nrf2?/?mice
is shown following stimulation with
PM (A) and as compared with the
negative control carbon black (CB)
particulates (B), bacterial LPS/endo-
toxin (C), and trimeric CD40L (D).
ko, Knockout.
Flow cytometric quan-
FIGURE 9.
stress in Nrf2?/?DCs as compared with Nrf2?/?DCs. In this assay, real-
time H2O2formation was measured by a peroxidase luminol chemilumi-
nescence (CL) method. The CL response was initiated by adding 5 ?M
luminol and 10 ?g/ml HRP and continuously monitored at 37°C for 1 h.
Data are mean ? SD of three independent experiments. Levels of signif-
icance between resting and PM-treated DCs are described as absolute val-
ues on the figure; ?? represents enhanced production of H2O2by Nrf2?/?
DCs as compared with their wt counterparts at p ? 0.001. ko, Knockout.
The effect of PM and the induction of enhanced oxidative
4554Nrf2-DISRUPTED DCs ARE PROINFLAMMATORY

Page 11

augmentation in H2O2production as compared with resting DCs
(p ? 0.001, Fig. 9). Moreover, luminol-derived chemilumines-
cence was significantly higher in PM-stimulated Nrf2?/?DCs than
the Nrf2?/?counterparts. Thus, using two independent assays we
found that PM induced excess oxidative stress in Nrf2-disrupted
DCs as compared with Nrf2?/?DCs.
Attenuation of antioxidant gene expression in Nrf2-defienct DCs
Oxidative stress may be important in the maturation of DCs. An-
tioxidants inhibit some aspects of DC maturation. However, very
little is known about the expression of antioxidant genes by DCs as
a function of their activation and/or maturation state. We therefore
assessed the induction of three classic Nrf2-regulated genes in
Nrf2?/?and Nrf2?/?DCs before or after exposure to PM, namely
GCLc (Fig. 10A), the GCLc modifier subunit GCLm (Fig. 10B), as
well as HO-1 (Fig. 10C). We found that while LPS and CD40L
enhanced the induction of expression of GCLc (3.1- and 2.3-
fold, respectively; Fig. 10A), PM dramatically augmented the
expression of this gene as compared with unstimulated Nrf2?/?
DCs (6.1-fold induction; Fig. 10A). In Nrf2?/?DCs we ob-
served a lower level of induction of GCLc in response to stim-
ulation by LPS (1.4-fold) or CD40L (1.3-fold), whereas the
stimulation of Nrf2?/?DCs with PM was completely without
effect (Fig. 10A). Under all conditions, the induction of GCLc
by Nrf2?/?DCs following exposure to LPS, CD40L, or PM
was significantly greater than the levels of induction seen in
Nrf2?/?DCs (p ? 0.001; Fig. 10A).
We observed similar responses of DCs at the level of GCLm
induction (Fig. 10B). In Nrf2?/?DCs, LPS (5.1-fold), CD40L
(4.8-fold), and PM (9.92-fold) augmented the induction of this
gene to levels that were markedly greater than those in Nrf2?/?
DCs (p ? 0.001, Fig. 10B). In addition, the stimulation of Nrf2?/?
DCs with PM was completely without effect, and stimulation with
LPS or CD40L directed only partial induction (1.11- and 1.14-
fold, respectively). Finally, whereas CD40L only modestly in-
duced expression of HO-1 (4.4-fold; Fig. 10C) in Nrf2?/?DCs,
it failed to induce any expression by Nrf2?/?DCs. By contrast,
LPS markedly induced the expression of HO-1 by wt DCs
(12.5-fold) and only marginally did so in Nrf2?/?DCs (2.2-fold
induction). In Nrf2?/?and Nrf2?/?DCs, PM directed a massive
induction of HO-1, particularly in Nrf2?/?DCs (28.6- and 8.7-
fold respectively).
In summary, Nrf2?/?DCs expressed greater constitutive levels
of expression of GCLc, GCLm, and HO-1 as compared with their
Nrf2?/?counterparts (Fig. 10). In addition, these data show that
PM is a highly potent inducer of three important antioxidant genes
in an Nrf2-dependent manner.
Discussion
The link between innate immunity and subsequent functional re-
sponses to environmental particulate exposures, such as ambient
urban PM, remains poorly defined. Similarly, the link between PM
exposure, antioxidant defense mechanisms, and allergic immunity
warrants further investigation. DCs are the key component of the
innate immune system that evolved to rapidly sense and respond to
diverse environmental stimuli. In this work, we used a well-char-
acterized source of ambient urban PM (1, 2) to probe the role of
oxidative stress in DC activation. Oxidative stress plays an impor-
tant role in promoting DC activation and its functional maturation
(19, 41–43).
In the current study, we have provided a comprehensive analysis
of the effects of endotoxin-free APM (1, 2) on the functional re-
sponses of murine bone marrow-derived DCs as well as pure pop-
ulations of pulmonary CD11c?myeloid DCs generated from
Nrf2?/?and Nrf2?/?mice. We show that Nrf2 regulates a phys-
iologically relevant and intrinsic antioxidant defense system that
protects DCs from ambient urban particles. Our studies indicate
that Nrf2 plays a previously underappreciated role in innate im-
munity and suggests that a deficiency of Nrf2-dependent pathways
may be involved in susceptibility to the adverse health effects of air
pollution.
We showed that PM drives many aspects of DC activation that
are crucial in innate immunity and host defense. Specifically, when
contrasted with Nrf2?/?DCs, we showed that cell surface expres-
sion of costimulatory molecules and MHC class II was higher on
Nrf2?/?DCs. This indicated that DCs from Nrf2?/?mice were
already in a state of relative heightened activation as compared
with their wt counterparts, possibly due to oxidant signals gener-
ated during their in vitro differentiation. In addition, the cell sur-
face expression of CD80, CD86, and MHC class II could be aug-
mented by PM-exposed Nrf2?/?DCs to levels that were seen on
Nrf2?/?DCs. The developmental pathway of DC maturation in
Nrf2?/?DCs warrants further investigation, but it does exemplify
FIGURE 10.
Nrf2?/?DCs as compared with Nrf2?/?DC. Quantitative real-time RT-
PCR analysis showed increased levels of mRNA for genes such as ?GCLc
(A), ? GCLm (B), and HO-1 (C) in PM-exposed wt Nrf2?/?DCs as com-
pared with Nrf2?/?DCs. Results are mean ? SD of three independent
experiments. Levels of statistical significance (??, p ? 0.05) are for
Nrf2?/?DCs as compared with Nrf2?/?DCs. For comparative purposes,
the inductions of ?GCLc (A), ? GCLm (B), and HO-1 (C) in response to
both LPS and trimeric CD40L are shown. In each case, PM was more
effective at inducing antioxidant gene expression than either LPS or
CD40L in this model. Ct, Cycle threshold.
Increased transcriptional induction of antioxidant genes in
4555The Journal of Immunology

Page 12

the hypothesis that ROS play a crucial role in the maturation of
DCs. In addition, our data support the hypothesis that Nrf2 may
guard against inappropriate maturation of DCs until the resting
DCs sense and respond to danger signals.
We confirmed the importance of reactive oxidants contributing
to the maturation of DCs by using the antioxidant molecule NAC.
Treatment of DCs with the antioxidant NAC inhibited the matu-
ration of DCs (43), and this was concordant with our observations.
In resting Nrf2?/?DCs we showed that NAC dampened the ex-
pression of costimulatory molecules. We also observed enhanced
cell surface expression of MHC class II molecules by Nrf2?/?
DCs following exposure to NAC. However, in Nrf2?/?DCs the
expression of CD40 was unaffected by NAC. This suggests that
NAC targets the expression of CD40 in an Nrf2-dependent man-
ner. Under these circumstances, it is likely that the DC is held in
a state of immaturity and is poised to sample and associate endo-
genously processed Ag by MHC class II. Others have shown that
ROS serve a critical role in the activation of DCs as well as the
inhibition of DC maturation by antioxidants (16, 43–45). In human
monocyte-derived DCs, ROS generated by xanthine oxidase in-
duced early phenotypic maturation of augmented cell surface ex-
pression of the costimulatory molecules CD80 and CD86 as well
as the DC maturation marker CD83. NAC also attenuated the PM-
driven augmentation of cell surface expression of MHC class II,
CD80, and CD86 in both Nrf2?/?and Nrf2?/?DCs while the
expression of CD40 by APM-stimulated Nrf2?/?DCs was less
sensitive to the effects of NAC, although some minimal inhibition
was observed.
Consistent with the observations made above, we found that
inflammatory and immunomodulatory cytokines were affected by
stimulating DCs with PM. We found that Nrf2?/?DCs secreted
constitutively greater levels of IL-12p40, IL-6, IL-10, TNF-?, and
VEGF than their wt counterparts, and yet DCs lacking Nrf2 se-
creted constitutively lower levels of IL-18 as compared with wt
DCs. This pattern of cytokine production is consistent with a con-
stitutive and heightened level of DC activation in the absence of
functional Nrf2. Further, it suggests heightened and relatively un-
checked production of ROS as a potential mechanism responsible
for enhanced cytokine production.
Activation of DCs from wt mice and those lacking functional
Nrf2 with PM enhanced the production of all cytokines measured,
with one notable exception. The secretion of IL-18 was strikingly
dampened in Nrf2?/?DCs and enhanced in DCs lacking Nrf2.
This novel finding suggests that the regulated production of IL-18
is dependent, at least in part, on Nrf2 activity and free radical
production. IL-18 is an important cytokine with roles in septic
shock and inflammatory diseases (46). In macrophages, at least,
two signals are necessary for the production and secretion of IL-18
(47–49). For IL-18 to be released from the producing cell, a prim-
ing and activating signal is required. The priming signal may in-
clude a pathogen-associated molecular pattern (such as the classic
bacterial danger signal LPS that occupies and transduces a signal
via TLR4), but secretion requires cleavage by caspase I. If caspase
I is switched off or remains as inactive procaspase I, then IL-18
secretion is dampened (47, 48). It is possible that, in murine DCs
expressing Nrf2, stimulation with PM is not seen as a “classic
danger signal.” Alternatively, Nrf2 (and ROS) may play a role in
regulating caspase I activity. Future experiments will be needed to
distinguish between these and other possibilities.
In addition, we studied the functional and phenotypic status of
highly purified CD11c?lung myeloid Nrf2?/?and Nrf2?/?DCs
upon activation by particulate matter, where we observed remark-
able concordance with their bone marrow-derived DCs counter-
parts. For example, the expression of the costimulatory molecules
followed a similar pattern between bone marrow-derived and lung
DCs. We noted a consistently dampened expression of CD40 in
Nrf2?/?pulmonary DCs relative to their wt counterparts, whereas
the expression of both CD80 and particularly CD86 were present
at greater levels on Nrf2?/?DCs (Fig. 2D). The consistently low-
ered constitutive expression of CD40 on Nrf2?/?bone marrow as
well as lung DCs is of interest because CD40 serves crucial roles
in cell-mediated as well as humoral-mediated immunity, particu-
larly in the context of the class switching of Ig to IgE (50, 51). Our
data imply an important and as yet unrecognized role for Nrf2 in
regulating the cell surface expression of CD40. However, we know
that CD40-CD40L interactions between DCs and T cells, respec-
tively, are required for optimal IgE responses and atopy (52). In
human subjects with asthma, CD40 expression is markedly up-
regulated on a number of different cell types, including macro-
phages (53), eosinophils (54), and epithelial cells in the conducting
airways (55).
In addition, upon activation by particulate matter Nrf2?/?lung
(or bone marrow-derived) DCs and their wt counterparts gave aug-
mented levels of expression of CD40, although this was not sta-
tistically significant in bone marrow-derived Nrf2?/?DCs (Fig.
2A). The synergistic increase in both CD40 expression by Nrf2?/?
lung DCs and their ability to promote IL-13 secretion in coculture
with naive CD4?T cells would suggest that Nrf2 normally func-
tions to inhibit proallergic DC phenotypes in vivo. We previously
reported that Nrf2-deficient mice develop higher IgE levels in as-
sociation with more severe allergic airway inflammation after sen-
sitization and challenge with OVA (25). The data contained in this
report suggest that this was due at least in part to a greater differ-
entiation of proallergic DCs in the absence of Nrf2. It will be
important in future studies to define the contribution of Nrf2 in
specific cell types to protection from allergen-driven Th2 immune
responses in vivo.
Both wt and Nrf2?/?lung DCs promoted an enhanced pro-Th2
cytokine response upon activation by PM, but the magnitude of
this response was markedly greater using Nrf2?/?lung DCs. Ra-
tiometric analyses (Fig. 6C) revealed that Nrf2?/?lung DCs pro-
moted ?5-fold more IL-13 than IFN-? than their wt counterparts
and ?4-fold greater amounts of IL-13 than IL-12p70 in the DC/
CD4?T cell coculture system. By contrast, Nrf2?/?lung DCs
promoted only a 2.8-fold increase in IL-13:IFN-? and a 2.2-fold
increase in IL-13:IL-12p70 (Fig. 6C), conditions that favor a Th2
bias, but markedly lower than the ratios observed for Nrf2?/?
lung DCs.
The observation that Nrf2?/?DCs exhibit an inherent ability to
promote pro-Th2 cytokine secretion by responding CD4?T cells
is of considerable interest. The importance of DCs in directing a
Th1-type or Th2-type Ag-specific activation of naive T cells in
regional draining lymph nodes is now fairly well established and is
thought to be largely dependent on the local cytokines secreted by
in the immunological synapse with CD4?T cells as well as other
signals (56–58). In the context of allergic Th2-mediated inflam-
mation, effector CD4?Th2 cells rapidly exit the lymph nodes and
migrate to sites of established inflammation whereupon they in-
teract with IgE-bearing tissue DCs to further augment the Th2
cytokine pool, including enhanced production of proallergic cyto-
kines such as IL-5, IL-9, and IL-13 (59–61).
Our data add to a growing body of work indicating that oxida-
tive stress in DCs is an important determinant of the Th1/Th2
balance. For example, it was recently shown that Nrf2 inhibits
NF-?B-mediated signal transduction, which is critical for the elab-
oration of IL-12 and TNF-? secretion as well as costimulatory
molecules such as CD80, CD86, and CD54 by APCs, including
DCs (62). In this elegant study it was further shown that a change
4556Nrf2-DISRUPTED DCs ARE PROINFLAMMATORY

Page 13

in the intracellular redox status of DCs upon activation by partic-
ulates such as diesel exhaust particulates disrupt the normal ability
of TLR agonists to mature DCs. This perturbation of DC function
was also associated with dampened IFN-? and augmented IL-10
secretion in Ag-specific T cells (62), which is in keeping with our
findings using Nrf2?/?DCs, lung myeloid DCs, and ambient PM
(Figs. 4 and 6). Our data support the idea that restoring the oxidant/
antioxidant balance in DCs may have a therapeutic benefit in Th2-
dominant allergic diseases (11, 63).
The effects of NAC on inflammatory cytokine production by wt
as well as by Nrf2?/?DCs was complex. In resting Nrf2?/?wt
DCs, NAC suppressed only the secretion of IL-12p40 and TNF
and actually enhanced the production of all others, including IL-18
in this model. Thus, in resting DCs with active Nrf2, exposing cells
to NAC can actually enhance somewhat the release of IL-6, IL-10,
IL-18, and VEGF. By contrast, in resting DCs lacking active Nrf2,
exposing cells to NAC enhanced secretion of only TNF-? and
VEGF. The regulation in VEGF secretion between Nrf2-express-
ing DCs and those lacking Nrf2 was of considerable interest, be-
cause we found that Nrf2?/?DCs produced markedly more VEGF
at baseline than their wt counterparts (Table I).
VEGF is an important mitogen and chemotactic agent that plays
diverse roles in tumor growth and survival, wound repair, angio-
genesis, microvascular permeability, and asthma (64–66). For ex-
ample, VEGF dampens IL-12 synthesis and down-modulates the
differentiation of CD4?Th1 cells following their interaction with
LPS-matured DCs (67). In addition, VEGF may attenuate the dif-
ferentiation and maturation of DCs from hematopoietic progeni-
tors (68, 69). Others have shown that VEGF is sensitive to alter-
ations in oxygen tension and to increases in intracellular levels of
ROS (70, 71). Indeed, it has been shown that VEGF signaling is
associated with the redox state of the cell (68, 69).
In our experiments, VEGF was elevated in Nrf2?/?DCs. This
would be consistent with the notion that VEGF is linked to the
redox state of the cell. We also observed increased expression of
HO-1 and other Nrf2-regulated antioxidant genes (Fig. 10C), in
particulate matter-exposed DCs from both Nrf2-expressing and
Nrf2-disrupted mice. Because HO-1 enzymatic activity is an im-
portant stimulus for VEGF production (70), one interesting possi-
bility is that Nrf2 induces VEGF in a HO-1-dependent manner.
VEGF may also positively feed back and enhance HO-1 expres-
sion in vivo and in vitro (71, 72). This would imply a possible
interaction between Nrf2-mediated antioxidant signaling and
VEGF during DC maturation.
Direct evidence for a pro-oxidative effect of PM on the func-
tional activation of DCs came from studies where we assessed the
accumulation of H2O2by DCFH-DA assay and quantified ROS
synthesis by a luminol-based assay. In these experiments, we
found that PM enhanced the accumulation of H2O2in Nrf2?/?and
Nrf2??DCs. DCs lacking expression of Nrf2 also accumulated
more H2O2than Nrf2?/?DCs, presumably by a compromised
ability to detoxify H2O2. The ambient PM used in our studies is a
complex mixture of heavy and transition metals and other particles
coalesced around a carbon core (1, 2). The mechanisms by which
ambient PM induces oxidative stress include the effects of metals
and possibly hydrocarbon and aryl hydrocarbon-containing com-
ponents. Unlike commonly used diesel exhaust particles that are
generated from test engines under experimental conditions, the
ambient PM used in our studies reflects “real-world” exposures
and is likely derived from multiple sources. Although this adds to
the complexity of PM, we feel that this is also a more clinically
relevant compound to test in exposure models.
We also looked at the expression of other antioxidant genes to
determine the ROS defense systems present as well as the total
antioxidative capacity in Nrf2?/?as compared with Nrf2?/?wt
DCs following their exposure to PM. In addition to HO-1, we
found that there was a heightened constitutive level of expression
of GCLc and HO-1 in resting Nrf2?/?DCs as compared with
Nrf2?/?DCs. It will be interesting in future studies to determine
how compromised expression of antioxidant genes in Nrf2-defi-
cient DCs leads to enhanced expression of cell surface markers and
increased secretion of inflammatory cytokines in response to PM.
Alternative and perhaps complementary explanations for the en-
hanced constitutive activation of Nrf2?/?DCs, particularly fol-
lowing stimulation by PM, may include decreased expression of
HO-1 in Nrf2?/?DCs relative to their wt counterparts. The mech-
anisms responsible are complex but may involve the ability of
HO-1 to otherwise protect cells against oxidative stress, cellular
injury, and inflammation, an activity lacking in Nrf2?/?DCs (73).
When present in cells expressing Nrf2, the antioxidant enzyme
HO-1 metabolizes heme to biliverdin, free divalent iron, and car-
bon monoxide (74). The relevance of this is that biliverdin is fur-
ther metabolized to bilirubin and both are powerful antioxidant and
immunosuppressive proteins (75).
In summary, Nrf2-disrupted DCs exhibit a heightened and con-
stitutively proinflammatory state. These observations indicate an
important role for Nrf2 in gauging an appropriate pattern of in-
flammatory activation of DCs in response to danger signals such as
environmental particulate matter or other allergens. Disruption of
the Nrf2 gene may potentially enhance host susceptibility to var-
ious allergic or infectious diseases, although this awaits formal
demonstration.
Acknowledgments
We thank Dr. Peyton Eggleston and his team at the Johns Hopkins National
Institute of Environmental Health Sciences Center for Childhood Asthma
in the Urban Environment, Baltimore, MD for their enthusiastic support
and Dr. John McDyer (Johns Hopkins University, Baltimore, MD) for pro-
viding trimeric CD40L. We also thank Dr. David Topham, Department of
Immunology and Microbiology, University of Rochester, Rochester, NY
for kindly providing the male OT-II mice used in these studies.
Disclosures
The authors have no financial conflict of interest.
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