IMMUNOLOGY ORIGINAL ARTICLE

Endogenous ligands of the aryl hydrocarbon receptor regulate lung

dendritic cell function

Thomas H. Thatcher,1 Marc A. Summary

Williams,1,2,† Stephen J. Pollock,2,3
Claire E. McCarthy,2 Shannon H.
Lacy,2 Richard P. Phipps2,3 and
Patricia J. Sime1,2,3
1
Division of Pulmonary and Critical Care
Medicine, University of Rochester, Rochester,
NY, 2Department of Environmental Medicine,
University of Rochester, Rochester, NY, 3Lung
Biology and Disease Program, University of
Rochester, Rochester, NY, USA
doi:10.1111/imm.12540
Received 4 August 2015; revised 28
September 2015; accepted 1 October 2015.
†
Present address: Army Public Health
Center [Provisional], Toxicology Portfolio –
Health Effects Research Program, Aberdeen
Proving Ground, Edgewood, MD 21010,
USA
Correspondence: Dr P. J. Sime, University
of Rochester, Division of Pulmonary and
Critical Care Medicine, 601 Elmwood Ave-
nue, Box 692, Rochester, NY 14642, USA.
Email: Patricia_Sime@urmc.rochester.edu
Senior author: Patricia J. Sime
The aryl hydrocarbon receptor (AhR) is a transcription factor that has
been extensively studied as a regulator of toxicant metabolism. However,
recent evidence indicates that the AhR also plays an important role in
immunity. We hypothesized that the AhR is a novel, immune regulator of
T helper type 2 (Th2) -mediated allergic airway disease. Here, we report
that AhR-deficient mice develop increased allergic responses to the model
allergen ovalbumin (OVA), which are driven in part by increased den-
dritic cell (DC) functional activation. AhR knockout (AhR / ) mice sensi-
tized and challenged with OVA develop an increased inflammatory
response in the lung compared with wild-type controls, with greater
numbers of inflammatory eosinophils and neutrophils, greater T-cell
proliferation, greater production of Th2 cytokines, and higher levels of
OVA-specific IgE and IgG1. Lung DCs from AhR / mice stimulated
antigen-specific proliferation and Th2 cytokine production by naive T
cells in vitro. Additionally, AhR / DCs produced higher levels of tumour
necrosis factor-a and interleukin-6, which promote Th2 differentiation,
and expressed higher cell surface levels of stimulatory MHC Class II
and CD86 molecules. Overall, loss of the AhR was associated with
enhanced T-cell activation by pulmonary DCs and heightened pro-
inflammatory allergic responses. This suggests that endogenous AhR
ligands are involved in the normal regulation of Th2-mediated immunity
in the lung via a DC-dependent mechanism. Therefore, the AhR may rep-
resent an important target for therapeutic intervention in allergic airways
inflammation.
Keywords: allergy; antigen presentation/processing; dendritic cells; lung; T
helper type 1/type 2 cells.

In addition to its regulation of xenobiotic responses, it

Introduction
The aryl hydrocarbon receptor (AhR) is a ligand-activated
transcription factor that has been extensively studied as a
mediator of toxicant metabolism. Dioxin (2,3,7,8-tetra-
chlorodibenzo-p-dioxin, TCDD), a persistent organic pol-
lutant and by-product of industrial processes, was the
first AhR ligand identified, and the AhR is an important
mediator of toxicant metabolism and excretion.1
is increasingly evident that the AhR plays an important
role in immune processes. The AhR is widely expressed
in tissues of the immune system.2 Further, AhR signalling
plays a key role in immunological functions at mucosal
surfaces, including the lung.3 Data from our laboratory
and others suggests that the AhR regulates pulmonary
inflammation. For example, AhR-deficient mice develop
heightened inflammatory responses to cigarette smoke

Abbreviations: AhR, aryl hydrocarbon receptor; BrDU, bromodeoxyuridine; Con A, concanavalin A; DCs, dendritic cells; DRE,
dioxin response element; FICZ, 6-formylindolo[3,2-b]carbazole; IDO, indoleamine 2,3-dioxygenase; IFN-c, interferon-c; IL-6, in-
terleukin-6; LPS, lipopolysaccharide; OVA, ovalbumin; PE, phycoerythrin; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; Th2, T
helper type 2; TNF-a, tumour necrosis factor a

ª 2015 John Wiley & Sons Ltd, Immunology, 147, 41–54 41

T. H. Thatcher et al.
and bacterial lipopolysaccharide (LPS), which is associ-
ated with increased nuclear factor-jB activity.4 Similarly,
AhR-deficient mouse lung fibroblasts and AhR-deficient
human pulmonary endothelial cells produce elevated
levels of inflammatory cytokines and chemokines in
response to inflammatory stimuli.5,6
Several studies have reported that AhR ligands sup-
press allergic lung inflammation. In rodent models of
asthma-like disease using ovalbumin (OVA) or house
dust mite, TCDD reduces eosinophilia, cytokine produc-
tion and IgE levels.7–10 Evidence suggests that the AhR
regulates the balance of immunosuppressive regulatory T
lymphocytes and effector T helper type 2 (Th2) and
Th17 cells. For example, TCDD promotes the expansion
of regulatory T cells and inhibits activation of helper
and cytotoxic T cells.11,12 Th17 cell differentiation can
be enhanced or inhibited by AhR signalling, depending
on the AhR ligand and timing of AhR activation.13,14 In
the context of allergic hypersensitivity, AhR agonists
inhibit Th2-mediated cytokine production and allergic
responses in mouse models of atopic dermatitis, food
allergy and asthma-like disease.10,15,16 However, when
using whole animal models, it is difficult to determine
whether the effects of AhR ligands are due to direct
effects on T cells or to effects on T-cell programming by
antigen-presenting cells. Interestingly, wild-type recipients
of adoptively transferred AhR-deficient T cells displayed
TCDD-induced inhibition of T-cell proliferation in
response to influenza virus,11,17 suggesting that antigen-
presenting cells play a key a role in AhR modulation of
T-cell function.
Dendritic cells (DCs) are potent antigen-presenting
cells. In the lung, DCs reside in the epithelium but extend
cellular processes into the airways to sample the environ-
ment.18 During the pathogenesis of allergic asthma, DCs
take up inhaled allergen and traffic to the local lymph
nodes where they stimulate the induction of Th2-
mediated allergic sensitization. The AhR is constitutively
expressed by DCs, and data suggest that the AhR plays a
role in mediating the functions of these antigen-present-
ing cells. For example, TCDD increased DC numbers and
altered DC phenotypes in a mouse model of peanut
allergy.19 TCDD also inhibited DC activation of CD8+ T
cells during the Th1-mediated response to influenza
infection.20
However, these studies may be limited by the use of
TCDD as a model AhR ligand.21 TCDD has a higher
affinity for the AhR and a longer half-life than naturally
occurring ligands,21 and the signals it delivers are qualita-
tively as well as quantitatively different from naturally
occurring and endogenous ligands.22–25 Interestingly,
three different non-dioxin AhR ligands all failed to sup-
press the allergic response in a mouse model of peanut
allergy.26 As a result, the role of endogenous AhR ligands
on immune cell function remains unclear.
42
Using AhR knockout (AhR / ) mice we have investi-
gated the role of endogenous AhR ligands in allergic
airways inflammation. AhR / mice developed increased
inflammation and Th2 responses when sensitized and
challenged with OVA. Importantly, we co-cultured T cells
with wild-type and AhR / DCs and demonstrated that
AhR / DCs promoted increased Th2 responses in autol-
ogous T cells. Overall, our results suggest that endoge-
nous AhR ligands act via the AhR to regulate immune
responses.
Materials and methods
Mice
Aryl hydrocarbon receptor knockout mice (AhR / ,
strain B6
129-Ahrtm1Bra) were purchased from The Jack-
son Laboratory (Bar Harbor, ME) and bred at the
University of Rochester. This strain carries a targeted
deletion of exon 2 of the Ahr gene and was backcrossed
for 12 generations onto C57BL/6.27 Controls were age-
and gender-matched C57BL/6J mice (The Jackson Labo-
ratory), or age- and gender-matched AhR+/ heterozy-
gous litter mates. No functional differences were observed
between AhR+/+ wild-type and AhR+/ heterozygous
mice. OT-II mice, which carry a transgenic T-cell receptor
gene specific for OVA,28 were also purchased from The
Jackson Laboratory. For some experiments, AhR / mice
were cross-bred with the OT-II mice; offspring that were
OT-II+ and AhR / were identified by PCR of tail snip
DNA. All animal procedures were performed under the
supervision of the University Committee on Animal
Research.
Ovalbumin allergy models
Mice were sensitized to OVA (Fraction V; Sigma, St Louis,
MO) with a single intraperitoneal injection of 8 lg OVA
with 4 mg aluminium hydroxide (Sigma) in 0
5 ml nor-
mal saline on day 0, followed by a single 1-hr exposure to
OVA or saline aerosol on day 5. Ovalbumin aerosol expo-
sures were performed by placing the mice in individual
compartments of a wire cage, which was placed inside a
closed plastic box connected to a nebulizer (Salter, Arvin,
CA) containing OVA (5 mg/ml in normal saline) and
attached to medical grade compressed air at 6 psi. Control
animals were injected with saline plus aluminium hydrox-
ide alone and exposed to saline aerosol in an identical
apparatus. On day 19, all mice were challenged with two
1-hr exposures to OVA aerosol. Mice were killed on days
20, 22 and 26 (i.e. at 1, 3 and 7 days post-challenge) and
tissues and cells were harvested.
To evaluate mucosal immunity, mice were sensitized
with OVA and LPS as described elsewhere.29,30 Briefly,
AHR / and heterozygous (AHR+/ ) litter mate controls
ª 2015 John Wiley & Sons Ltd, Immunology, 147, 41–54

were sensitized to OVA by inhalation of 100 lg OVA
plus 100 ng LPS (Pseudomonas, 600 000 EU/mg; Sigma)
in 40 ll of saline on days 0 and 7. The mice were chal-
lenged with OVA aerosol on day 19 and harvested on
days 20, 22 and 26 (i.e. at 1, 3 and 7 days post-challenge)
as described above.
Tissue harvest and bronchoalveolar lavage
Mice were anaesthetized with 2,2,2-tribromoethanol
(Avertin, 250 mg/kg intraperitoneally) and killed by
exsanguination. Blood was collected by cardiac puncture
and the serum fraction was stored for later analysis. Total
and OVA-specific immunoglobulin in serum was mea-
sured as previously described.31 The heart and lungs were
removed en bloc and the lungs were lavaged twice with
0
5 ml PBS. The lavage fluid was centrifuged and the
cell-free supernatants were frozen for later analysis. The
bronchoalveolar lavage (BAL) cell pellet was resuspended
in PBS and the total cell number was determined by
counting on a haemocytometer. Differential cell counts
(minimum of 300 cells per slide) were performed on
Cytospin-prepared slides (Thermo Shandon, Pittsburgh,
PA) stained with 3-Step Stain (Richard-Allan Scientific,
Kalamazoo, MI). The lungs were frozen in liquid nitrogen
for later analysis. Some lungs were inflated and fixed in
10% neutral-buffered formalin without undergoing
lavage. Tissues were embedded in paraffin, sectioned
(5-lm), and stained with haematoxylin & eosin or with
periodic acid–Schiff’s reagent (PAS) (Richard-Allan). To
quantify PAS-stained goblet cells, the left bronchus was
photographed using an Olympus BX-51 microscope
equipped with an InSight camera (Spot Diagnostic Instru-
ments, Sterling Heights, MI). The length of the imaged
segment was determined using the software calibration
tool provided and the number of PAS-positive cells per
100 µm was determined. Two sections per mouse and
three mice per group were counted by a technician who
was blinded to the treatment groups.
Histological scoring
Slides were reviewed by a veterinary pathologist who was
blinded to the treatment groups. The abundance of
perivascular aggregates was scored 0–3 as follows: 0, none
present; 1, perivascular inflammatory cell aggregates (at
least two cells thick) surround ≤ 20% of blood vessels; 2,
perivascular inflammatory cell aggregates surround 21–
40% of blood vessels; 3, perivascular inflammatory cell
aggregates surround > 40% of blood vessels. Eosinophilic
inflammation was scored 0–3 as follows: 0, none present;
1, inflammatory cell aggregates are composed of ≤ 25%
eosinophils; 2, inflammatory cell aggregates are composed
of 26–50% eosinophils; 3, inflammatory cell aggregates
are composed of > 50% eosinophils.
ª 2015 John Wiley & Sons Ltd, Immunology, 147, 41–54
AhR ligands regulate dendritic cells
Lymph node cell proliferation assays
At harvest, the peribronchial and mediastinal lymph
nodes were removed and lymph node mononuclear cells
were harvested by compressing the nodes between sterile
frosted glass slides. Pooled lymph node cells (four mice
per group) were plated in a 96-well round bottom plate
at a density of 1 9 105 cells per well and cultured in
200 ll of HL-1 serum-free medium (Biowhittaker, Walk-
ersville, MD) as described previously31,32 with the indi-
cated concentration of OVA, or with concanavalin A
(Con A, 0
75 lg/ml, Sigma) as a positive control. After
3 days of culture, 75 ll of medium was removed and
saved for cytokine analysis, and replaced with 75 ll of
fresh medium containing bromodeoxyuridine (BrDU).
Proliferation was measured on the fourth day using a
BrDU incorporation assay according to the manufac-
turer’s protocol (Roche, Indianapolis, IN).
DC/T-cell co-culture
Lung DCs were isolated as previously described.33–35
Naive mice were killed and the lungs were perfused with
saline, subsequently removed, and placed in ice-cold
medium. Lungs from four to six mice were pooled per
isolation. After all the lungs were harvested, the lung tis-
sue was minced with a sterile scalpel and then digested
with 2
5 mg/ml Collagenase Type 2 (Worthington Bio-
chemical Corporation, Lakewood, NJ) and 5 units/ml
Dispase (BD Biosciences, Bedford, MA) for 45 min at
37°. The lung tissue was disrupted by passing five times
through a 20-ml syringe, and then passed through a 100-
µm cell strainer. The resulting single-cell suspension was
washed with medium, and plated on tissue-culture-trea-
ted dishes in RPMI-1640 medium (Invitrogen, Carlsbad,
CA) containing 8% fetal bovine serum (Sigma). The cells
were incubated overnight to remove macrophages and
other adherent cells. The following day, the non-adherent
cells were gently washed off, collected by centrifugation,
and CD11c+ cells were isolated by positive selection with
antibody-coupled magnetic beads according to the manu-
facturer’s directions (Miltenyi Biotech, Auburn, CA). DCs
were counted and cultured overnight (100 000 DCs per
well of a 24-well plate) in RPMI-1640 medium and stim-
ulated with 100 ng/ml Pseudomonas aeruginosa LPS
(Sigma) with or without 20 lg/ml OVA. After 18–20 hr,
the DC supernatants were removed and saved for analy-
sis, and the DCs were washed with HL-1 medium,
counted and co-cultured with T cells as described below.
T cells were harvested by collecting peripheral lymph
nodes from naive mice. The nodes were compressed
between sterile frosted glass slides to release the T cells,
from which CD4+ T cells were isolated by negative selec-
tion with antibody-coupled magnetic beads (Miltenyi Bio-
tech) according to the manufacturer’s protocol. The T
43
T. H. Thatcher et al.
cells were then washed and resuspended in HL-1 medium
and co-cultured with DCs (quadruplicate cultures for
each condition) for 4 days in round-bottom 96-well cul-
ture plates (4000 DCs and 40 000 T cells per well for a
DC : T-cell ratio of 1 : 10). Ovalbumin (20 lg/ml) was
added to wells containing OVA-pulsed DCs but addi-
tional LPS was not added. After 3 days of culture, 75 ll
of medium was removed and saved for cytokine analysis,
and replaced with 75 ll of fresh medium containing
1 lCi/well [methyl-3H]thymidine (Dupont NEN Prod-
ucts, Boston, MA). After 18 hr, the cells were harvested
onto glass fibre filters using a Packard Micromate 96 cell
harvester (Packard Co., Meriden, CT) and 3H incorpora-
tion was counted with a Packard TopCount Liquid Scin-
tillation Counter. Background proliferation was measured
in wells without added antigen. Con A (Sigma) was
added to some wells at a final concentration of 1
5 lg/ml
as a positive control.
Cytokine analysis
Cytokines were measured in BAL fluid and cell culture
supernatants by cytokine multiplex analysis (Milliplex
MAP, Millipore, Temecula, CA) and were read on a
Luminex 100 System (Luminex, Austin, TX), except for
interleukin-6 (IL-6) and tumour necrosis factor-a (TNF-
a), which were measured by ELISA (R&D Systems, Min-
neapolis, MN) according to the manufacturer’s protocols.
Flow cytometry
Lung DCs were harvested as described and cultured for
48 hr with 100 ng/ml LPS. The DCs were harvested and
washed with PBS, then stained with Live/Dead Fixable
Violet cell stain (Invitrogen) as per the manufacturer’s
instructions. Non-specific antibody binding was blocked
with PBS containing 1% BSA and 10% Mouse Fc Block
(BD Biosciences) for 15 min. Three independent experi-
ments were performed using different antibodies and
instruments. For the first experiment, the DCs were
stained with anti-CD11c (clone N418, Miltenyi Biotech)
and either anti-class II MHC-phycoerythrin (PE) or
anti-CD86-PE (BD Pharmingen, San Jose, CA) and
analysed on a FACSCalibur flow cytometer using CELL-
QUEST 3
1 software (BD Biosciences). For the second
and third experiments, DCs were stained with anti-
CD11c-allophycocyanin (BD Biosciences), anti-MHC
class II-PE (eBioscience, San Diego, CA), anti-CD205-
PE-Cy7 (eBioscience), and anti-CD86-biotin (eBio-
science) antibodies for 30 min. Cells were then washed
with PBS containing 1% BSA followed by staining with
streptavidin-allophycocyanin-eFL780 (eBioscience) for
20 min to label the anti-CD86-biotin antibodies. Cells
were analysed by flow cytometry using a FACS Canto II
flow cytometer (BD Biosciences) using the Violet B
44
(405 nm laser; 450/50 nm), Red C (633 nm laser; 660/
20 nm), Blue D (488 nm laser; 585/42 nm), Blue A
(780/60 nm) and Red A (780/60 nm) channels for
detection of Live/Dead stain, CD11c, MHC class II,
CD205 and CD86, respectively. Subsequent analysis was
performed using FLOWJO software (Ashland, OR). Only
live cells, excluding cells positive for Live/Dead cell stain,
were included for analysis of surface marker expressions.
Because the three experiments used different instruments
and antibodies, geometric mean fluorescence intensity
(GMFI) for each condition was normalized to resting
control cells.
Statistical analysis
Statistical significance was assessed by one-way or two-
way analysis of variance as described in the figure legends,
using PRISM version 5 (GraphPad Software, La Jolla, CA).
A P-value < 0
05 was considered significant.
Results
AhR / mice develop an exaggerated allergic airway
response
To investigate the role of the AhR in the allergic immune
response, AhR / mice and C57BL/6 control mice were
sensitized to the model allergen OVA as previously
described.31 The mice were harvested 1, 3 and 7 days fol-
lowing a challenge with inhaled OVA aerosol. Mice sensi-
tized and challenged with OVA exhibited an increase in
total BAL cells, with increased numbers of macrophages,
eosinophils, neutrophils and lymphocytes (Fig. 1).
Although the peak response at day 3 was not significantly
different between AhR / and wild-type mice, the AhR /
mice exhibited significantly increased numbers of macro-
phages, eosinophils and neutrophils at day 1, and signifi-
cantly increased macrophages and eosinophils at day 7.
This suggested that the inflammatory response in AhR /
mice was more rapid and more prolonged.
AhR / mice develop increased lung inflammation
and goblet cell hyperplasia
The OVA sensitization protocol used in this study results
in perivascular accumulation of infiltrating lymphocytes
and eosinophils (Fig. 2). AhR / mice exhibited signifi-
cantly increased perivascular aggregates compared with
wild-type controls, and these aggregates contained
increased numbers of eosinophils. AhR / mice also
exhibited increased parenchymal involvement, with
increased presence of alveolar macrophages, neutrophils
and eosinophils (Fig. 2c,d and Table 1). Another impor-
tant hallmark of allergic airways inflammation is
increased mucus production, which was detected by PAS
ª 2015 John Wiley & Sons Ltd, Immunology, 147, 41–54
 AhR ligands regulate dendritic cells

(a) 16 Total cells (b) 7 Macrophages

Cell number x 105

Cell number x 105

12 *
** 5
*
8
3

4
1
0 0
Saline Day 1 Day 3 Day 7 Saline Day 1 Day 3 Day 7

(c) 8 Eosinophils (d) Neutrophils

4
*
C57BL/6

Cell number x 105

Cell number x 105

6 AhR–/– 3
**

4 2
Figure 1. Aryl hydrocarbon receptor-deficient
(AhR / ) mice develop increased allergic air- *
2 1
way inflammation. C57BL/6 mice (grey bars)
or AhR / mice (black bars) were sensitized
and challenged with ovalbumin (OVA) as 0 0
Saline Day 1 Day 3 Day 7 Saline Day 1 Day 3 Day 7
described, and harvested 1, 3 and 7 days after
challenge, or were sensitized to saline alone (e)
1·4 Lymphocytes
and harvested 3 days after challenge. Differen-
tial counts were performed on bronchoalveolar
Cell number x 105

lavage (BAL) cells and the total cell number 1·0

(a), number of macrophages (b), eosinophils
(c), neutrophils (d) and lymphocytes (e) are
0·6
reported. Results shown are the means  stan-
dard error for n = 6 mice per group and are
representative of two independent experiments. 0·2
*P ≤ 0
05, **P ≤ 0
01 by two-way analysis of 0
variance with Bonferroni post-test. Saline Day 1 Day 3 Day 7

staining for goblet cells. The allergic response to OVA AhR / mice develop increased OVA-specific
provokes goblet cell hyperplasia and mucus production in antibodies
the large airways, as indicated by increased PAS-positive
The allergic response to inhaled OVA includes high titre
staining of airway epithelial cells. Comparison of airways
OVA-specific IgG1 and IgE antibodies. Interestingly,
of similar calibre revealed that AhR / mice have a dra-
OVA-sensitized and -challenged AhR / mice developed
matic increase in the number and density of PAS-positive
a dramatic IgE response, with significant increases in both
cells (Figs 2e and f, Table 1).
total serum IgE and OVA-specific IgE that were not seen
in C57BL/6 controls (Fig. 4). Both strains exhibited
/
AhR mice have an increased Th2 cytokine profile increased total serum IgG1 following immunization but
only the AhR / mice developed high titre OVA-specific
The allergic response to OVA includes transient expres-
IgG1 antibodies.
sion of Th2 cytokines in the lungs immediately after chal-
lenge.31 AhR / mice exhibited increased Th2 cytokines
AHR deficiency also promotes mucosal immune
in BAL fluid compared with C57BL/6 controls, including
responses
IL-5, IL-10, IL-13 and monocyte chemoattractant protein
1 (Fig. 3). However, IL-4 levels were similar in both Although the OVA/aluminium hydroxide/intraperitoneal
strains. Levels of the Th1 cytokine IL-12 were also similar, injection model is widely used, most airway allergens are
but interferon-c (IFN-c) was significantly reduced in the typically encountered by inhalation. To determine
AhR / mice. TNF-a, IL-6 and IL-17 were elevated on whether AhR deficiency also promoted direct airway sen-
day 1 following aerosol challenge but were not signifi- sitization, we tested a model of mucosal immunity in
cantly different between AhR / and C57BL/6 control which OVA is given by inhalation in combination with
mice. LPS.29 As previously reported in this model, sensitization

ª 2015 John Wiley & Sons Ltd, Immunology, 147, 41–54 45

T. H. Thatcher et al.
C57BL/6
(a) (b)
Saline
(c) (d)
V aerosol challenge. Lungs were inflated and
V
OVA
V
(e) (f)
OVA
with OVA plus LPS via inhalation leads to an inflamma-
tory response involving both neutrophils and eosino-
phils.29,30 Here, AhR / mice developed significantly
higher eosinophilia than controls, with significantly
higher levels of total and OVA-specific IgE (Fig. 5).
/
AhR lymph node cells have stronger antigen recall
responses
To more closely examine the role of the AhR in the aller-
gic response to OVA, lymph node cells were harvested
from the peribronchial lymph nodes of OVA-sensitized
and challenged C57BL/6 and AhR / mice at 3 and
7 days after challenge. After stimulation with OVA
in vitro, proliferation was measured by BrDU incorpora-
tion assay, and cytokine production was measured by
multiplex assay as described. Lymph node cells from
AhR / mice harvested 3 days (data not shown) and
7 days after challenge exhibited significantly greater pro-
liferation in vitro, in response both to OVA and to the
non-specific stimulus Con A (Fig. 6a). Similar to the
cytokine results from BAL fluid, increased proliferation
was accompanied by increased production of the Th2
cytokines IL-4, IL-5, IL-10 and IL-13 and decreased
production of the Th1-associated cytokine IFN-c
46
AhR–/–
Figure 2. Aryl hydrocarbon receptor-deficient
(AhR / ) mice develop increased lung inflam-
mation and goblet cell hyperplasia. Mice were
sensitized with ovalbumin (OVA) or saline as
described and were killed 7 days after OVA
fixed with formalin, and sections were stained
with haematoxylin & eosin. (a, b) Saline-
exposed mice have normal alveoli and airways.
(c) OVA-challenged wild-type mice develop
extensive lymphocytic inflammation around
blood vessels after OVA challenge. (d) OVA-
challenged AhR-deficient mice develop larger
perivascular infiltrates, as well as inflammation
of the parenchymal space, with increased num-
bers of alveolar macrophages, neutrophils and
eosinophils (arrows). V, vascular lumen. (e, f)
Periodic acid–Schiff’s (PAS) stain reveals a dra-
matic increase in PAS+ cells in the airways of
AhR-deficient mice challenged with OVA (ar-
rows). Bar = 100 lm. The results shown are
representative of two independent experiments
with four to six mice per group in each experi-
ment.
(Fig. 6b). The difference in recall proliferation between
wild-type and AhR / lymph node cells was more pro-
nounced at lower OVA concentrations, but the effect on
cytokine production was seen at all concentrations tested.
AhR / T cells respond better to priming by wild-
type DCs
The increased T-cell proliferation and cytokine produc-
tion in AhR / mice could be the result of a defect in
T-cell function or to an alteration in T-cell programming
by antigen-presenting cells. To determine whether AhR
deficiency has a direct effect on antigen responses by T
cells, we cross-bred the AhR / mice to the OT-II strain
that carries a transgenic T-cell receptor specific for a class
II epitope of OVA.28 Co-cultures were established using
AhR+/+:OT-II and AhR / :OT-II T cells as responders
and lung DCs from C57BL/6 (AhR+/+) mice as stimula-
tors. AhR / T cells had a stronger proliferative response
to OVA with resting or LPS-treated DCs (Fig. 7a). Addi-
tionally, AhR / T cells had a significantly stronger pro-
liferative response to the non-specific stimulus Con A
than control T cells (Fig. 7a). The OVA-stimulated T cells
did not produce detectable levels of cytokines, but
Con A-stimulated T cells produced detectable levels of
ª 2015 John Wiley & Sons Ltd, Immunology, 147, 41–54
 AhR ligands regulate dendritic cells

Table 1. Lung histology scoring

C57BL/6 AhR–/–

300 IL-4 8000 IL-5

C57BL/6 AhR knockout
***
6000
Abundance of 1
25 (n = 4) 2
533 (n = 4) 200

pg/ml

pg/ml
perivascular aggregates 4000
(0–3)1 100
2000
Eosinophilic 1
0 (n = 4) 2
53 (n = 4)
inflammation (0–3)2 0 0
Saline Day 1 Day 3 Saline Day 1 Day 3
PAS-positive cells (per 7
8  1
1 (n = 3) 11
2  1
64 (n = 3)
100 lM of bronchial IL-6 IL-10
800 120
wall, mean  SEM) *
600
80

pg/ml
pg/ml
1
Perivascular inflammatory cell aggregates (at least two cells thick) 400
were scored as; 0, none present; 1, surrounding ≤ 20% of blood ves- 40
200
sels; 2, 21–40% of blood vessels; 3, > 40% of blood vessels.
2
Eosinic inflammation was scored as; 0, none present; 1, Inflamma- 0 0
Saline Day 1 Day 3 Saline Day 1 Day 3
tory cell aggregates are composed of ≤ 25% eosinophils; 2, 26–50%
eosinophils; 3, > 50% eosinophils. IL-12p70 IL-13
25 300
3
P < 0
05 by Mann–Whitney non-parametric test. *
20
4
P < 0
05 by Student’s t-test. 200
15

pg/ml

pg/ml
10
100
the Th2-associated cytokines IL-4 and IL-13 and the Th1- 5
associated cytokine IFN-c, with AhR / T cells producing 0 0
significantly more IL-4 and less IFN-c (Fig. 7b). Saline Day 1 Day 3 Saline Day 1 Day 3

25 IL-17 250 MCP-1

/ *
Antigen priming by AhR DCs alters T-cell 20 200
behaviour 15 150
pg/ml

pg/ml
10 100
To investigate whether AhR deficiency alters T-cell prim- 5 50
ing by lung DCs, we harvested conventional CD11c+ DCs 0 0
from lung tissue of C57BL/6 and AhR / mice by enzy- Saline Day 1 Day 3 Saline Day 1 Day 3
matic tissue digestion and magnetic bead purification.
IFN-γ 12 TNF-α
The purified DCs were stimulated overnight with LPS 40 ** **
and subsequently pulsed with OVA. These DCs were used 30 8
pg/ml
pg/ml

as stimulators in co-cultures with wild-type (AhR+/+) 20

OT-II T cells. Proliferation was measured by thymidine
incorporation and cytokines were measured by multiplex
assay as described. DCs from AhR / mice promoted sig-
nificantly more OVA-specific proliferation in OT-II T
cells than C57BL/6 DCs, whether resting or stimulated
with LPS (Fig. 8a). AhR / DCs also promoted the
release of higher levels of cytokines IL-4, IL-5, IL-13 and
IL-17 than did wild-type DCs. (Fig. 8b).
AhR /
lung DCs are more highly activated
One route by which DCs can promote a Th2 response is
through production of the cytokines TNF-a and IL-6.34
After stimulating DCs overnight with LPS, the culture
supernatants were saved for analysis while the DCs were
washed with fresh media and co-cultured with T cells.
Resting DCs produce very little TNF-a or IL-6, but LPS
strongly promotes the production of TNF-a and IL-6 by
lung DCs, and cytokine production was significantly
higher in AhR / DCs (Fig. 9a,b). We also examined
expression of activation markers on lung DCs by flow
4
10
0 0
Saline Day 1 Day 3 Saline Day 1 Day 3
Figure 3. Aryl hydrocarbon receptor-deficient (AhR / ) mice exhi-
bit increased T helper type 2 (Th2) cytokines following ovalbumin
(OVA) challenge. C57BL/6 mice (grey bars) or AhR / mice (black
bars) were sensitized and challenged with OVA as described, and
cytokines in bronchoalveolar lavage (BAL) fluid were measured by
multiplex assay or ELISA as described in the Materials and methods.
Results shown are mean  standard error for four (saline) or six
(OVA) mice per group, and are representative of two independent
experiments. *P ≤ 0
05, **P ≤ 0
01 by two-way analysis of variance
with Bonferroni post-test.
cytometry. There were no differences between wild-type
and AhR / CD11c+ DCs in the per cent expressing
MHC class II or CD86; however, AhR / DCs exhibited
significantly higher fluorescence intensity after LPS
stimulation (Fig. 9c and d). Interestingly, fewer AhR /
DCs express CD205, a surface marker associated with

ª 2015 John Wiley & Sons Ltd, Immunology, 147, 41–54 47

T. H. Thatcher et al.

(a)
20
Total IgE (b)
20 000
OVA-specific IgE phase I and phase II detoxification pathways. However, it
*
C57BL/6 has become increasingly evident that the AhR is also an
AhR–/–

Titre units/ml
15 15 000 important mediator of immunological functions. The
µg/ml

10 *** 10 000
*** * AhR plays an important role in the immune defence and
*
allergic sensitization of mucosal tissues, especially the
5 5000 lung. We have previously reported that AhR / mice dis-
0 0 play exaggerated lung inflammation when challenged by
Day 1 Day 3 Day 7 Day 1 Day 3 Day 7 cigarette smoke or LPS.4 Similarly, AhR / lung cells pro-
duce greater levels of pro-inflammatory mediators when
(c) Total IgG1 (d) OVA-specific IgG1
2000 70 * stimulated with injurious stimuli.5,6 This suggests that the
Tiiter units/ml x 105 60 AhR plays an important role in regulating pulmonary
1500 50
inflammatory responses. Therefore, we hypothesized that
40
µg/ml

1000
30
20
500
10
0 0
Day 1 Day 3 Day 7
Figure 4. Aryl hydrocarbon receptor-deficient (AhR / ) mice exhi-
bit higher antibody levels following ovalbumin (OVA) challenge.
C57BL/6 (grey bars) or AhR / (black bars) mice were sensitized
and challenged with OVA as described, and total and OVA-specific
IgE and IgG1 was measured in serum as described.31 (a) Total IgE,
(b) OVA-specific IgE. (c) Total IgG1, (d) OVA-specific IgG1. Results
shown are mean  standard error for n = 4 to n = 10 mice per
group. The data were non-normally distributed and were analysed
by one-way analysis of variance using the Kruskal–Wallis non-para-
metric test with Dunn’s multiple comparison test. *P ≤ 0
05,
***P ≤ 0
001.
regulatory DCs, after LPS stimulation, compared with
wild-type DCs (Fig. 9e).
The AhR ligand FICZ inhibits DC priming of T cells
Since AhR / DCs promote greater T-cell activation, we
hypothesized that one function of the AhR is to down-
regulate antigen priming by DCs in concert with endoge-
nous AhR ligands. To test this hypothesis, we isolated
wild-type DCs and co-stimulated them with LPS and
OVA plus the AhR ligand 6-formylindolo[3,2-b]carbazole
(FICZ). The DCs were washed extensively and then used
to stimulate AhR / OT-II T cells. As it has been previ-
ously reported that AhR ligands can suppress activation
of T cells,11,14,17,36 we used AhR / T cells that would be
incapable of responding independently to any trace
amounts of AhR ligands remaining after washing the
DCs. As shown in Fig. 10, 100 nM and 200 nM FICZ
markedly inhibited T-cell priming by treated DCs. Prim-
ing by AhR / DCs was not inhibited by FICZ (not
shown).
Discussion
The AhR is well known for its ability to respond to envi-
ronmental pollutants like dioxins by up-regulating the
endogenous activation of the AhR protects the lung dur-
ing immune responses against inhaled allergens, and that
AhR / mice would develop exaggerated allergic airway
inflammation.
Day 1 Day 3 Day 7
To test this hypothesis, we sensitized and challenged
wild-type and AhR / mice to inhaled OVA in a model
of asthma-like disease. AhR / mice challenged with
OVA developed a more pronounced inflammatory
response with higher levels of BAL neutrophils and eosi-
nophils, as well as greater tissue inflammation. Interest-
ingly, there were significantly more BAL cells in the
AhR / mice at day 1 and day 7 (Fig. 1), suggesting that
the allergic response to aerosol challenge occurs faster
and does not resolve as quickly in AhR / mice. The
results also suggest a hyper-reactive Th2 response in
AhR / mice. We found increased BAL levels of Th2
cytokines, such as IL-5, IL-13, monocyte chemoattractant
protein 1 and IL-10, and a decrease in the Th1 cytokine
IFN-c in AhR / mice (Fig. 3). As mucus production by
airway epithelial cells is dependent on Th2 cytokines,37,38
the increase in goblet cell metaplasia in the AhR / mice
is another indication of an enhanced Th2 response
(Fig. 2e,f). Further, AhR / mice developed high titres of
OVA-specific IgE and lymph node cells exhibited antigen-
dependent production of IL-4, IL-5 and IL-10. This phe-
notype is similar to the stereotypical Th2 response of
BALB/c mice to OVA, and contrasts with the low levels
of IgE and lymph node cell production of Th2 cytokines
detected in wild-type C57BL/6 mice. AhR / mice also
developed exaggerated inflammation and IgE antibodies
when challenged with LPS and OVA in a pure inhalation
model (Fig. 5), suggesting that the AhR and AhR ligands
regulate the immune response to inhaled antigens under
conditions that may be more representative of natural
allergic responses than the OVA/alum model.29,30 Overall,
these results are consistent with our hypothesis that the
AhR is an important negative regulator that acts as a
brake on pulmonary allergic responses.
Previous studies found that the treatment of rodents
with potential endogenous AhR ligands, such as FICZ,
indole-3-carbinole and quercetin, attenuated OVA-
induced allergic inflammation.10,39,40 In these models,
AhR activation led to reduced eosinophilia, inhibited Th2

48 ª 2015 John Wiley & Sons Ltd, Immunology, 147, 41–54

 AhR ligands regulate dendritic cells

(a) Total cells (b) Macrophages

10 8
* C57BL/6 *

Cell number x 105

Cell number x 105
8
6 AhR–/–
6 *
4
4
2
2

0 0
Day 1 Day 3 Day 7 Day 1 Day 3 Day 7

(c) Eosinophils (d) Eosinophil percent

2.0 50
*

Cell number x 105

40 ***
1·5

Cell percent
30
1·0
20
0·5
10

0 0
Day 1 Day 3 Day 7 Day 1 Day 3 Day 7

Figure 5. Aryl hydrocarbon receptor-deficient (e) Neutrophils (f) Neutrophil percent

1·5 25
(AhR / ) mice exhibit increased allergic air- * *
*
Cell number x 105

way inflammation after inhalation challenge. 20

AhR / mice or AhR+/ litter mate controls 1·0

Cell (%)
15
were sensitized to ovalbumin (OVA) by inhala-
tion of OVA plus lipopolysaccharide (LPS) as 10
0·5
described in the Materials and methods, and 5
harvested 1, 3 or 7 days after challenge. Differ-
0 0
ential counts were performed on bronchoalve-
Day 1 Day 3 Day 7 Day 1 Day 3 Day 7
olar lavage (BAL) cells and the total cell
number (a), number of macrophages (b), neu-
(g) Total IgE (h) OVA-specific IgE
trophils (c), percentage of neutrophils (d), 8 100 000
number of eosinophils (e) and percentage of **
***
eosinophils (f) are reported. Serum levels of 6 10 000
Titre units/ml

(g) total IgE and (h) OVA-specific IgE were

µg/ml

determined as described. Results shown are 4 * 1000

mean  standard error of the mean for n = 4
2 100
mice per group. *P ≤ 0
05, **P ≤ 0
01,
***P ≤ 0
001 by two-way analysis of variance 0 10
with Bonferroni post-test. Day 1 Day 3 Day 7 Day 1 Day 3 Day 7

cytokine and IgE production, increased Th1 cytokine
levels, and decreased expression of the Th2 transcription
factor GATA3.10,39,40 However, these studies did not
investigate whether the AhR ligands were acting directly
on T cells or on accessory cells that may programme the
T-cell responses. Our lymph node antigen recall experi-
ments showed that T-cell activation in AhR / lymph
node cells was significantly stronger than was seen in con-
trols, with greater antigen-specific proliferation and cyto-
kine production (Fig. 6), but likewise did not allow us to
determine the relative contributions of T cells and DCs to
the response. Therefore, we co-cultured AhR / T cells
with wild-type lung DCs, and wild-type T cells with
AhR / lung DCs. Our results indicated that AhR / T
cells were more prone to activation by lung DCs, and that
AhR / lung DCs had an increased potential to activate
wild-type T cells.
We confirmed the role of antigen-presenting cells in
AhR modulation of CD4+ T-cell allergic responsiveness
by co-culturing conventional CD11c+ DCs that had been
isolated from C57BL/6 and AhR / lung tissue with wild-
type OT-II T cells and OVA. We found that AhR / lung
DCs promoted greater non-specific and OVA-specific
T-cell proliferation, as well as increased production of
IL-4, IL-5 and IL-13 (Fig. 8). Interestingly, AhR / DCs
also produced higher levels of TNF-a and IL-6 (Fig. 9a,b)
and expressed increased surface levels of MHC Class II
and the co-stimulatory molecule CD86 (B7.2) (Fig. 9c
and d). Finally, the non-dioxin AhR ligand FICZ partially
attenuated the ability of wild-type lung DCs to prime

ª 2015 John Wiley & Sons Ltd, Immunology, 147, 41–54 49

T. H. Thatcher et al.

(a) 1·200 Proliferation

1·000 * *
*
0·800
*
OD450 0·600 C57BL/6
AhR–/–
0·400

0·200

0·000
50 5 1 ConA
OVA (µg/ml)
(b)
40 IL-4 600 IL-5
***
30 ***
* * 400 ***
pg/ml

pg/ml

20 **
200
10

0 0
50 5 1 ConA 50 5 1 ConA
OVA (µg/ml) OVA (µg/ml)
Figure 6. Lymph node cells from aryl hydro-
600 IL-13 200 IFN-γ carbon receptor-deficient (AhR / ) mice have
*** higher antigen recall responses. C57BL/6 mice
*** 150
400 (grey bars) or AhR / mice (black bars) mice
were sensitized and challenged with ovalbumin
pg/ml
pg/ml

** 100
(OVA) as described. Seven days after challenge,
200 the mice were killed, the peribronchial lymph
50
nodes were collected, pooled by group, and
0 0 single cell suspensions were prepared and cul-
50 5 1 ConA 50 5 1 ConA tured in vitro for 4 days with the indicated
OVA (µg/ml) OVA (µg/ml) concentration of OVA, or with 0
75 lg/ml
concanavalin A (Con A). (a) Proliferation was
150 IL-12p70 measured by bromodeoxyuridine (BrDU)
incorporation and detected by absorbance at
100 450 nm. (b) Cytokines were measured in cul-
ture supernatants by multiplex assay as
pg/ml

described. The lower limit of detection was

50
1
5 pg/ml. Results shown are the mean  stan-
dard deviation of quadruplicate wells, and are
0 representative of two independent experiments.
50 5 1 ConA *P ≤ 0
05, **P ≤ 0
01, ***P ≤ 0
001 by two-
OVA (µg/ml) way analysis of variance.

T cells (Fig. 10). Hence, endogenous AhR ligands appear allergy therapeutics with AhR binding activity suppressed
to play a role in DC activation of Th2 immune responses. T-cell activation by DCs in mouse models of allergic air-
Although previous reports are generally in agreement ways inflammation,36,44,45 but several non-dioxin AhR
that exogenous AhR ligands can attenuate the ability of ligands failed to inhibit peanut-specific responses in a
DCs to prime and activate T cells, the results are some- food allergy model.26 The relative contribution of AhR
what ligand and model specific. Schulz et al. found that ligands to immune regulation via T cells or DCs is also
splenic DCs from mice treated with TCDD suppressed difficult to assess in whole animal models. Herein, we
IL-5 and IL-13 production by T cells in response to pea- have shown that endogenous AhR ligands contributed to
nut extract, a food allergen in susceptible individuals,19 the regulation of lung immune responses via lung DCs,
whereas the AhR ligand 4-n-nonylphenol reduced ear such that AhR / DCs, unable to respond to these
swelling in a mouse model of contact hypersensitivity.41 ligands, exhibited a pro-inflammatory and pro-allergic
However, TCDD was also reported to increase activation phenotype.
of CD8+ T cells by splenic DCs42 and to induce DC The AhR has also been reported to regulate the devel-
maturation and apoptosis.43 Similarly, two novel anti- opment of Th17 responses.13,14 A previous report showed

50 ª 2015 John Wiley & Sons Ltd, Immunology, 147, 41–54

 AhR ligands regulate dendritic cells

150 * (a) Proliferation

8 **
(a) Proliferation
20
* C57BL/6 DCs
*

Stimulation index
6 AhR–/– DCs
100
Stimulation index

15
AhR+/+ T cells
AhR–/– T cells 4
10
*
50 2
5
0
0 Resting LPS
0
Resting LPS ConA OVA – + – +
OVA – + – +
(b) IL-4 IL-5
(b) IL-4 IL-13 10 20
100 25
* *
* 20 15
80

pg/ml
pg/ml
60 15
pg/ml
pg/ml

5 10
40 10
5
20 5
0 0 0 0
Resting LPS ConA Resting LPS ConA Resting LPS Resting LPS
OVA – + – + OVA – + – + OVA – + – + OVA – + – +
80 IFN- γ
IL-13 IL-17
6 150 *
60 *
pg/ml

* 4
40 100
pg/ml

20
0
Resting LPS ConA
OVA – + – + Resting
Figure 7. Increased proliferation and altered cytokine production in
aryl hydrocarbon receptor-deficient (AhR / ) T cells co-cultured
with AhR wild-type dendritic cells (DCs). Lung DCs were harvested
from C57BL/6 (AhR+/+) mice and cultured with lipopolysaccharide
(LPS) and ovalbumin (OVA) as described. T cells were harvested
from the peripheral lymph nodes of OT-II/AhR+/+ and OT-II/AhR /
mice and used as responders in a co-culture experiment at a ratio
of 4000 DCs to 40 000 T cells (i.e. a ratio of 1 : 10). (a) Prolifera-
tion was measured after 4 days by thymidine incorporation assay.
The results are reported as stimulation index normalized to OT-II/
AhR+/+ T cells cultured with resting DCs. (b) The indicated cytoki-
nes were determined in culture supernatants by multiplex assay as
described. Results shown are the mean  standard deviation of qua-
druplicate wells, and are representative of two independent experi-
ments. *P ≤ 0
05 by two-way analysis of variance.
pg/ml
2 50
0 0
LPS Resting LPS
OVA – + – + OVA – + – +
Figure 8. Aryl hydrocarbon receptor-deficient (AhR / ) dendritic
cells (DCs) promote T helper type 2 (Th2) -like responses from
naive AhR wild-type T cells. Lung DCs were harvested from
C57BL/6 (AhR+/+) and AhR / mice and stimulated overnight
in vitro with lipopolysaccharide (LPS) plus or minus ovalbumin
(OVA). T cells were harvested from the peripheral lymph nodes
of OT-II (AhR+/+) mice and co-cultured with the DCs at a ratio
of 4000 DCs to 40 000 T cells. (a) Proliferation was measured
after 4 days by thymidine incorporation assay. The results are
reported as stimulation index normalized to resting C57BL/6 DCs.
(b) Cytokines were determined in culture supernatants by multi-
plex assay as described. Results shown are the mean  standard
deviation of quadruplicate wells, and are representative of three
independent experiments. *P ≤ 0
05, **P ≤ 0
01 by two-way anal-
ysis of variance.

that bone-marrow-derived DCs from AhR / mice

increased T-cell production of IL-17 after stimulation with
Th1-promoting bacterial ligands.46 Although IL-17 is not a
significant factor in our model of allergic lung inflamma-
tion in vivo (Fig. 3), we also observed increased IL-17 pro-
duction by T cells stimulated with AhR / lung DCs
(Fig. 8). Therefore, our study agrees with previous observa-
tions that the anti-Th17 effect of AhR ligands is due at least
in part to programming by antigen-presenting cells.13
Whereas the endogenous AhR ligands responsible for
attenuating DC activation of T cells have not been
definitively identified, a strong candidate is a family of
tryptophan metabolites called kyneurins. Kyneurins are
synthesized from tryptophan by the enzyme indoleamine
2,3-dioxygenase (IDO), previously hypothesized to be an
important immune suppressor.47 Kyneurins activate the
AhR, and the AhR is required to up-regulate expression
of IDO. Kyneurins also act on bone-marrow-derived
DCs to suppress Th17 responses and induce regulatory
T cells.46,48 In the absence of IDO, lung DCs promote
allergic responses.49 Our results support the concept that

ª 2015 John Wiley & Sons Ltd, Immunology, 147, 41–54 51

T. H. Thatcher et al.

(a) TNF- α (b) IL-6

5000 * 4000 *
C57BL/6
4000 AhR–/–
3000
3000

pg/ml

pg/ml
2000
2000

1000
1000

0 0
Resting LPS Resting LPS

(c) MHC class II (d) CD86 (e) CD205

Control resting
Control +LPS
AhR–/– resting
Cell number

Cell number

Cell number
AhR–/– +LPS
Unstained

Fluorescence intensity Fluorescence intensity Fluorescence intensity

Per cent positive Per cent positive Per cent positive

80 100 15 *

80

Per cent CD205+

Per cent MHC II+

Per cent CD86+

60
10
60
40
40
5
20
20

0 0 0
Resting LPS Resting LPS Resting LPS

MHC class II CD86 CD205

†† ††
3 3 1·5
Normalized GMFI

Normalized GMFI

Normalized GMFI

2 2 1·0

1 1 0·5

0 0 0
Resting LPS Resting LPS Resting LPS

Figure 9. Aryl hydrocarbon receptor-deficient (AhR / ) dendritic cells (DCs) express higher levels of cytokines and altered cell surface markers.
Lung DCs were harvested from C57BL/6 (AhR+/+) and AhR / mice and stimulated overnight in vitro with lipopolysaccharide (LPS). (a) Inter-
leukin-6 (IL-6) and (b) tumour necrosis factor-a (TNF-a) were determined in the culture supernatants by ELISA. Results shown are the
mean  standard deviation of quadruplicate wells, and are representative of two independent experiments. *P < 0
05 by two-way analysis of
variance. (c–e) CD11c+ DCs were analysed by flow cytometry for MHC class II (c), CD86 (d) and CD205 (e). A representative histogram is
shown (top). Graphs show the percentage of CD11c+ DCs also staining positive for the indicated marker (middle), and normalized geometric
mean fluorescence intensity (GMFI), with resting control cells set to 1
0 (bottom) and represent mean  standard error for three independent
experiments (for MHC and CD86) and mean  standard deviation for two independent experiments for CD205. ††P ≤ 0
01 by paired t-test.
*P < 0
05 by repeated measures two-way analysis of variance.

IDO-derived products act on the AhR to dampen activa- Because the role of AhR in regulating T-cell function is
tion of lung DCs and subsequent allergic responses to relatively better studied, we have focused largely on the
inhaled antigens. role of the AhR in regulating lung DC function. However,

52 ª 2015 John Wiley & Sons Ltd, Immunology, 147, 41–54


Proliferation
8
6 funded in part by the U.S. Army Medical Department. The
Stimulation index
***
***
4
2
0
LPS + + + + + +
OVA – + + + + +
FICZ (nM) 0 0 10 50 100 200
Figure 10. The aryl hydrocarbon receptor (AhR) ligand 6-formylin-
dolo[3,2-b]carbazole (FICZ) inhibits T-cell priming by DCs. Lung
DCs were harvested from wild-type (AhR+/+) mice as described, and
incubated overnight with lipopolysaccharide (LPS), ovalbumin
(OVA) and the indicated concentration of FICZ. After washing, the
DCs were co-cultured with AhR / :OT-II T cells at a ratio of 1 : 10,
and proliferation was determined by thymidine incorporation assay.
Results are mean  standard deviation for six replicate wells per
condition normalized to resting DCs, and are representative of three
independent experiments. ***P < 0
001 by one-way analysis of vari-
ance with Bonferroni post-correction.
we also showed that AhR / T cells are more easily acti-
vated than wild-type T cells, regardless of the DC geno-
type used (Fig. 7 and data not shown), and more readily
adopt a Th2/Th17 phenotype than wild-type T cells.
These results are consistent with previous studies showing
that activation of the AhR with TCDD decreased cyto-
toxic and helper T-cell expansion in a mouse model of
influenza,11,50,51 and suggest that AhR ligands act as a
brake on inflammatory responses in T cells similarly to in
DCs. In the absence of a functional AhR, T cells seem to
be intrinsically prone to increased Th2 activation.
Overall, using both the conventional and mucosal
immune models, we have demonstrated that the absence
of functional AhR expression by lung DCs leads to
increased allergic responses to inhaled allergens. We
report the novel finding that AhR / pulmonary DCs
promote CD4+ T-cell activation, and enhance both Th2
and Th17 pro-inflammatory cytokine production in
response to allergen. Consequently, endogenous AhR sig-
nalling in DCs appears to play a protective role in allergic
airway disease and could be further investigated as a ther-
apeutic target for Th2-mediated lung diseases, including
asthma.
Acknowledgements
This research was funded in part by UL1RR024160,
UL1TR000042, T32ES01247, T32HL66988, DE011390,
ª 2015 John Wiley & Sons Ltd, Immunology, 147, 41–54
AhR ligands regulate dendritic cells
AI071064, HL088325 and HL120908. These funders had no
role in study design, data collection and analysis, decision
to publish, or in the preparation of the manuscript. SHL is
views expressed herein are those of the author and do not
reflect the official policy or position of the Department of
the Army, Department of Defense, or the U.S. Government.
Author contributions
THT, MAW, RPP and PJS conceived the study and
designed the experiments. THT, MAW and SJP per-
formed experiments and collected data. SHL reviewed
and scored the histology. THT, MAW, SJP, CMM, SHL,
RPP and PJS analysed the data and wrote the manuscript.
Disclosures
The authors have no conflicts of interest to declare.
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