Cyclooxygenase-2 (COX-2) Regulates Th17 Cell Differentiation During
Allergic Lung Inflammation
Hong Li1, J. Alyce Bradbury1, Ryan T. Dackor1, Matthew L. Edin1, Joan P. Graves1, Laura M.
DeGraff1, Ping Ming Wang1, Carl D. Bortner2, Shuichiro Maruoka1, Fred B. Lih3, Donald N.
Cook1, Kenneth B. Tomer3, Anton M. Jetten1 and Darryl C. Zeldin1*
Laboratories of Respiratory Biology1, Signal Transduction2, and Structural Biology3, National
Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina 27709
*Correspondence and Reprint Requests to:
Darryl C. Zeldin, M.D.
Division of Intramural Research, NIH/NIEHS
111 T.W. Alexander Drive, Building 101, Room A222,
Research Triangle Park, NC 27709
Phone: 919-541-1169 Fax: 919-541-4133
Funding: Division of Intramural Research, NIH/NIEHS.
Running Title: COX-2 regulates Th17 cell differentiation
Classification: 5.2, Eicosanoids, Immune Effects
Word count total: 5765
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AJRCCM Articles in Press. Published on April 7, 2011 as doi:10.1164/rccm.201010-1637OC
Copyright (C) 2011 by the American Thoracic Society.
Online Supplement: This article has an online data supplement, which is accessible from this
issue's table of content online at www.atsjournals.org
Contributions of Authors:
The work presented here was carried out in collaboration with all authors.
Hong Li: Conceived, designed, performed and interpreted experiments. Wrote the manuscript.
J. Alyce Bradbury: Acquisition and analysis of in vivo, western blot, and ELISA experiments.
Ryan T. Dackor: Acquisition and analysis of prostanoid mini-pump data. Revision of manuscript.
Matthew L. Edin: Acquisition and analysis of LC-MS data. Revision of manuscript.
Joan P. Graves: Acquisition and analysis of RT-PCR data.
Laura M. DeGraff: Acquisition and analysis of prostanoid mini-pump data.
Ping Ming Wang: Acquisition and analysis of lung immunostaining data.
Carl D. Bortner: Acquisition and analysis of FACS data.
Shuichiro Maruoka: Acquisition and analysis of in vivo experimental data.
Fred B. Lih: Acquisition and analysis of LC-MS data.
Kenneth B. Tomer: Design, acquisition and analysis of LC-MS data.
Donald N. Cook: Hypothesis, conception, and design of the study
Anton M. Jetten: Hypothesis, conception, and design of the study.
Darryl C. Zeldin: Hypothesis, conception, and design of the study. Interpretation of data.
Revision of the manuscript.
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At a Glance Commentary:
Scientific Knowledge on the Subject
Cyclooxygenase (COX) enzymes are known to be important regulators of Th1-Th2 balance in
allergic lung disease; however, it is not known whether COX-1 or COX-2-derived eicosanoids
regulate Th17 cell differentiation or function, or the mechanisms involved.
What This Study Adds to the Field
This study identifies COX-2 as a key regulator of Th17 cell differentiation and function in
allergic lung inflammation via an autocrine loop that involves PGI2, PGF2α and their respective
cell surface receptors.
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Rationale: Th17 cells comprise a distinct lineage of pro-inflammatory T helper cells that are
major contributors to allergic responses. It is unknown whether cyclooxygenase (COX)-derived
eicosanoids regulate Th17 cells during allergic lung inflammation.
Objective: To determine the role of COX metabolites in regulating Th17 cell differentiation and
function during allergic lung inflammation.
Methods: COX-1-/-, COX-2-/- and wild type mice were studied in an in vivo model of
ovalbumin-induced allergic inflammation and an in vitro model of Th17 differentiation using
flow cytometry, cytokine assays, confocal microscopy, RT-PCR and immunoblotting. In addition
the role of specific eicosanoids and their receptors was examined using synthetic prostaglandins,
selective inhibitors and siRNA knockdown.
Measurements and Main Results: Th17 cell differentiation in lung, lymph nodes and BALF was
significantly lower in COX-2-/- mice following ovalbumin sensitization and exposure in vivo. In
vitro studies revealed significantly impaired Th17 cell differentiation of COX-2-/- naïve CD4+ T
cells with decreased Stat3 phosphorylation and RORt expression. Synthetic PGF2α and PGI2
enhanced Th17 cell differentiation of COX-2-/- CD4+ T cells in vitro. The selective COX-2
inhibitor, NS-398, and FP and IP receptor siRNA knockdown significantly decreased Th17 cell
differentiation in vitro. Administration of synthetic prostaglandins increased Th17 cell
differentiation in vitro and restored accumulation of Th17 cells in lungs of allergic COX-2-/- mice
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Conclusions: COX-2 is a critical regulator of Th17 cell differentiation during allergic lung
inflammation via autocrine signaling of PGI2 and PGF2α through their respective cell surface
Total word count: 241
Key Words: Th17 cell • COX-2 • asthma • prostaglandins • IL-17 • allergic lung inflammation
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CD4+ T cells play an important role in the initiation of the immune response by providing
help to other cells and by taking on a variety of effector functions during the immune response.
Upon antigenic stimulation, naïve CD4+ T cells activate, expand and differentiate into various
effector subsets that are characterized by distinct functions and cytokine profiles (1). Th17 cells
are a recently discovered CD4+ helper T cell subset characterized by the production of IL-17A,
IL-17F and IL-22 (2). While Th1 and Th2 cell differentiation depend on single effector cytokines
(IL-12 and IL-4, respectively), Th17 differentiation is induced by the combined activity of
transforming growth factor- (TGF-) and IL-6 in mice, or TGF- and IL-21 (naive T-cells) or
IL-1 (memory T-cells) in humans (3). In both species, these cytokines affect IL-17 production
by activating and inducing the expression of key lineage-specific transcription factors such as
Stat3 and the orphan nuclear receptor RORt (4). IL-17A plays important roles in immune
responses such as delayed-type hypersensitivity, contact hypersensitivity and allergic airway
inflammation (5). IL-17A promotes inflammation by inducing various pro-inflammatory
cytokines and chemokines, recruiting neutrophils, enhancing antibody production and activating
T cells (6). It was previously reported that IL-17A is upregulated in asthma and nasal polyposis,
and in the latter condition, its expression is resistant to topical steroids (7, 8).
Cyclooxygenases (COXs) are responsible for the formation of prostaglandins (PGs)
which are involved in regulating inflammatory responses (9, 10). The two COX isoforms,
COX-1 and COX-2, are expressed at varying levels in different tissues (11). COX-1 is
constitutively expressed in most mammalian tissues and cells, whereas COX-2 is inducible in
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macrophages and other cell types at sites of inflammation (12, 13). Prostaglandins exert their
actions by binding to a family of G-protein-coupled receptors. These include the DP1 and DP2
subtypes of the PGD2 receptor, the EP1, EP2, EP3 and EP4 subtypes of the PGE2 receptor, the
PGF2α receptor (FP), the PGI2 receptor (IP) and the TxA2 receptor (TP) (14). It is known that
some of these receptors are expressed during T cell differentiation, a process which is important
in regulating inflammation and immune responses (15).
Recent studies suggest that COX-1 and COX-2 may play important roles in regulating the
Th1-Th2 balance in allergic and non-allergic lung diseases (16). These findings demonstrate
COX-2-dependent regulation of TGF- and IL-6, key cytokines that are produced by
macrophages and involved in the differentiation of naïve CD4+ T cells to Th17 cells. The
pro-inflammatory effects of PGE2 in experimental inflammatory bowel disease (17) and
collagen-induced arthritis in mice (18) are mediated through IL-17. In addition, PGE2 favors
Th17 expansion and IL-17 production through activation of dendritic cells via a paracrine
mechanism that also involves upregulation of the IL-23 and IL-1 receptors on T cells (19, 20).
However, it remains unknown whether 1) COX-1 and/or COX-2 are critically involved in the
Th17 cell differentiation process, 2) COX-derived prostaglandins other than PGE2 are involved
in Th17 cell differentiation, and 3) prostaglandins directly influence Th17 cells during
differentiation, independent of effects on dendritic cells. Therefore, in the present study we used
COX-1-/- and COX-2-/- mice to examine whether prostaglandins regulate Th17 cell differentiation
in a model of allergic lung inflammation. Our results show that the number of Th17 cells in lung
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and lymph nodes was dramatically decreased in COX-2-/- mice, but not COX-1-/- mice, compared
to wild type (WT) following ovalbumin sensitization/exposure in vivo. There was also
significantly impaired Th17 cell differentiation and IL-17A production in COX-2-/- T cells in
vitro. COX-2 was expressed in Th17 cells and regulated dose-dependently by IL-6. Prostaglandin
production was increased during Th17 differentiation, a phenomenon that was largely COX-2
dependent. Importantly, synthetic PGF2α and PGI2 partially restored the Th17 cell differentiation
defect in COX-2-/- cells in vitro, and systemic administration of prostaglandins restored
accumulation of Th17 cells in lungs of allergic COX-2-/- mice in vivo. Finally, Th17 cells
expressed prostanoid receptors, and Th17 cell differentiation was significantly reduced by FP and
IP receptor antagonists and siRNA knockdown. Together, these results indicate that Th17 cell
differentiation during allergic lung inflammation is critically regulated by COX-2-derived PGF2α
and PGI2 via an autocrine pathway that involves binding to FP and IP receptors on T cells.
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Materials and Methods
Additional details are provided in an online methods supplement, which is accessible from this
issue's table of contents online at www.atsjournals.org.
Six to ten week old male COX-1-/- mice, COX-2-/- mice and WT controls on a hybrid
C57BL6J x 129/Ola genetic background were purchased from Taconic (16). All animal
experiments were performed according to the NIH Guide for the Care and Use of Laboratory
Animals and approved by the NIEHS Animal Care and Use Committee.
Ovalbumin-induced allergic airway inflammation model
Mice were immunized with ovalbumin (20 g emulsified in 0.2 ml of aluminum
hydroxide adjuvant) or vehicle (adjuvant only) by i.p. injection on days 0 and 1. Starting on day
fourteen, mice were exposed to 1% ovalbumin (or saline) via nebulizer for 30 minutes per day
for four consecutive days. Forty-eight hours after the last exposure, mice were euthanized for
Bronchoalveolar lavage fluid (BALF) and isolation of lung and spleen CD4+ T cells
BALF was collected in 2 ml of PBS. Naïve or allergic CD4+ T cells were isolated using
kits from Miltenyi Biotec. Additional experiments used cell sorting to obtain naïve
CD4+ CD25− CD44+low CD62L+ T cells at >99% purity. Cell cultures were maintained in RPMI
1640 with 10% FBS. Th17 differentiation was induced as described (4).
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Flow cytometric analysis and intracellular cytokine staining
Naïve CD4+ T cells were differentiated with TGF- and IL-6 for 5 days and re-stimulated
for 4 hours with 12-0-tetradecanoyl-phorbol-13-acetate, ionomycin and brefeldin A. Intracellular
cytokine staining of fixed cells was analyzed using an LSRII flow cytometer (Becton Dickinson).
siRNA knockdown of IP and FP receptors
Freshly isolated naïve CD4+ T cells from spleens of WT mice were transfected with 20
nmol of siRNAs to the IP receptor, the FP receptor, or negative control using mouse T cell
Nucleofector solution (Amaxa). Transfected cells were induced to differentiate into Th17 cells
and analyzed by FACS on day 5.
Analysis of cytokine levels
Blood, BAL fluid or supernatants from T cell cultures were analyzed for cytokines using
Histopathology and Immunostaining of lung tissue
Lungs were perfusion-fixed and stained with hematoxylin/eosin. For immunstaining of
lung tissue, the frozen-lung sections were fixed in methanol with 0.3% H2O2 at 4oC,
permeabilized with Triton X-100 (0.8%), and stained with fluorescent antibodies. Adjacent
sections were stained with hematoxylin/eosin.
Implantation of osmotic minipumps for delivery of synthetic prostaglandins
PGE2, PGF2, and the PGI2 analog iloprost (Cayman, Ann Arbor MI) or vehicle (15%
ethanol/sterile saline) were delivered by osmotic minipumps (Alzet) one week prior to
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Eicosanoid analysis in blood and BAL fluid
Eicosanoid levels in BAL fluid were analyzed by liquid chromatography-tandem mass
spectrometry as previously described (21).
Data are presented as means ± standard error of the mean (SEM). Statistical comparisons
were performed by randomized-design two-way analysis of variance (ANOVA) followed by the
Newman-Keuls post-hoc test for more than two groups, or by unpaired Student’s t-test for two
groups. Statistical significance was defined as p<0.05.
Total word count: 491
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COX-2-/- mice have reduced Th17 cells during allergic lung inflammation in vivo
Allergic lung diseases such as asthma are hypothesized to result from dysregulated
immune responses. Previous studies have demonstrated that COX products play an important
role in regulating Th1-Th2 balance in both allergic and non-allergic lung diseases (16). Recent
progress in characterizing the pro-inflammatory IL-17 cytokine family has added an additional
layer of complexity to our understanding of the regulation of allergic lung inflammation (22).
Therefore, to investigate the role of COX-1 and COX-2 in regulating Th17 cells in vivo, we used
an established model of ovalbumin sensitization/exposure to induce allergic lung inflammation
in COX-1-/-, COX-1+/+, COX-2-/- and COX-2+/+ mice. Following ovalbumin
sensitization/exposure, the percentages of IL-17+ CD4+ (Th17) cells in spleen, lymph nodes,
blood, lung and BALF of COX-1-/- and COX-1+/+ mice were comparable (Figure 1A, 1C, 1D and
1E). In contrast, COX-2-/- mice showed significantly reduced percentages of Th17 cells in lung
(5.3% vs. 10.9%), BALF (12.7% vs. 20.3%) and lymph nodes (3.1% vs. 6.5%), but not in spleen
and blood, compared to COX-2+/+ mice (Figure 1B, 1F, 1G and 1H). The absolute number of
IL-17+ CD4+ T cells in lung and BALF was also significantly decreased in COX-2-/- mice (Figure
E1, available in online data supplement 2). Consistent with this finding, we found that IL-17A
levels in blood and BALF were significantly decreased in COX-2-/- mice compared to COX-2+/+
mice after ovalbumin exposure (Figure 1I and 1J). Interestingly, levels of IL-6 were also
significantly decreased in blood and BALF from COX-2-/- mice compared to COX-2+/+ mice
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after ovalbumin sensitization/exposure (Figure 1I and 1J). Together, these data suggest that Th17
cell numbers, and IL-17A and IL-6 cytokine levels, are reduced in COX-2-/- mice relative to WT
during allergic lung inflammation in vivo.
To further implicate a role for COX-2 in regulating Th17 cells in vivo, and to exclude the
contribution of compensatory pathways such as altered levels of TGF-, IL-6 or leukocytes in
the observed phenotype, we performed an acute COX-2 inhibitor study in WT mice exposed to
ovalbumin. Th17 cell percentages in lung (5.18±1.13 vs. 7.25±1.47; n=6, p<0.01) and blood
(4.05±0.59 vs. 6.07±0.85, n=6, p<0.01) were significantly decreased in the COX-2 inhibitor
group compared to control after ovalbumin exposure (Figure E2, available in online data
supplement 2). Th17 cell percentages in BALF (19.12 ± 3.66 vs. 22.42 ± 2.14; n=6, p>0.05),
lymph nodes (12.03 ± 1.47 vs. 14.21 ± 0.94; n=6, p>0.05) and spleen (6.50 ± 1.17 vs. 8.40 ±
0.68; n=6, p>0.05) also tended to be lower in the COX-2 inhibitor group, but these differences
did not reach statistical significance.
Localization of Th17 cells to sites of allergic lung inflammation
Analysis of lung tissue sections stained with hematoxylin/eosin revealed that allergic
COX-2+/+ and COX-2-/- had increased inflammation compared to non-allergic mice that were not
sensitized/exposed to ovalbumin (Figure 2A, 2E and 2I). Consistent with our previous work
demonstrating an inhibitory effect of prostaglandins on Th2 immune responses (16, 23), lungs
from allergic COX-2-/- mice showed increased airway inflammation compared to lungs from
allergic COX-2+/+ mice (Figure 2A and 2E). To examine the localization of Th17 cells in this
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model, we stained adjacent lung tissue sections with anti-CD4 and anti-IL-17A. In COX-2+/+
mice, there were abundant Th17 cells localized to sites of allergic inflammation (Figure 2B, 2C
and 2D). In contrast, allergic COX-2-/- mice showed markedly reduced numbers of Th17 cells at
sites of allergic inflammation (Figure 2F, 2G and 2H). Th17 cells were not observed in
non-allergic mice that were not exposed to ovalbumin (Figure 2J, 2K and 2L). To quantify the
number of Th17 cells in vivo, multiple randomly selected regions (n=22-29) from each lung
section were counted and quantified using MetaMorph software. The results demonstrate the
presence of significantly more lung IL-17+/CD4+ T cells in COX-2+/+ mice than in COX-2-/- mice
after ovalbumin exposure (Figure 2M, p<0.01). Lung IL-17+/CD4+ T cells of both COX-2+/+ and
COX-2-/- mice were significantly increased after ovalbumin exposure compared to control (no
OVA exposure). Together, these data indicate that Th17 cells localize to sites of allergic lung
inflammation and confirm reduced numbers of Th17 cells in inflammatory loci of COX-2-/- mice
exposed to ovalbumin in vivo.
Regulation of COX-1 and COX-2 expression during Th17 cell differentiation in vitro and in
COX-2 is primarily expressed in activated monocytes and macrophages (24); however, T
cells are reported to express COX-2 and produce PGE2 after retroviral infection (25). COX-2
expression and PGE2 production are also known to be involved in the inhibitory mechanism of
Treg cells (26). To further elucidate the role of COX enzymes in Th17 cell differentiation, the
expression of COX-1 and COX-2 in CD4+ T cells during Th17 cell differentiation in vitro was
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investigated. Naïve CD4+ T cells purified from WT mouse spleens were stimulated for 4-5 days
to induce Th17 cell differentiation, and COX-1 and COX-2 mRNA and protein levels were
examined by RT-PCR and immunoblotting, respectively. Untreated, naïve CD4+ T cells
expressed low levels of COX-2 mRNA and protein (Figure 3A-D). Treatment of naïve CD4+ T
cells with anti-CD3, anti-CD28, anti-IFNγ, TGF-β and IL-6 increased COX-2 mRNA and protein
levels in an IL-6 dose-dependent fashion. In contrast, COX-1 was constitutively expressed in
naïve CD4+ T cells and mRNA/protein levels were unchanged during Th17 cell differentiation
(Figure 3A-D). We also used flow cytometry to measure intracellular expression of COX-1 and
COX-2 during Th17 differentiation. IL-6 significantly and dose-dependently increased the
expression of COX-2 protein, but not COX-1 protein, in Th17 cells differentiated from naïve
CD4+ T cells isolated from spleen (Figure 3E and 3F) and lung (Figure 3G and H). To provide
further evidence of COX-2 expression in CD4+ T cells that is linked to regulation of Th17 cells
in vivo, we isolated CD4+ T cells from lung after ovalbumin exposure and examined COX-2
expression by real time PCR. The results showed that COX-2 mRNA levels were significantly
increased in lung CD4+ cells after OVA exposure (Figure 3I). Together, these results demonstrate
that COX-2 is upregulated during Th17 cell differentiation in vitro and in vivo.
Impaired Th17 cell differentiation of COX-2-/- naïve CD4+ T cells in vitro
To examine the role of COX-2 in Th17 cell differentiation in vitro, we treated equal
numbers of naïve CD4+ T cells (>95% pure) isolated from WT and COX-2-/- mouse spleens with
anti-CD3, anti-CD28, anti-IFN-γ, TGF-β and IL-6, and quantified IL-17–producing CD4+ T cells
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