Regulation of type 17 helper T-cell function by nitric oxide during inflammation.
ABSTRACT Type 17 helper T (Th17) cells are implicated in the pathogenesis many of human autoimmune diseases. Development of Th17 can be enhanced by the activation of aryl hydrocarbon receptor (AHR) whose ligands include the environmental pollutant dioxin, potentially linking environmental factors to the increased prevalence of autoimmune disease. We report here that nitric oxide (NO) can suppress the proliferation and function of polarized murine and human Th17 cells. NO also inhibits AHR expression in Th17 cells and the downstream events of AHR activation, including IL-22, IL-23 receptor, and Cyp1a1. Conversely, NO did not affect the polarization of Th17 cells from mice deficient in AHR. Furthermore, mice lacking inducible nitric oxide synthase (Nos2(-/-)) developed more severe experimental autoimmune encephalomyelitis than WT mice, with elevated AHR expression, increased IL-17A, and IL-22 synthesis. NO may therefore represent an important endogenous regulator to prevent overexpansion of Th17 cells and control of autoimmune diseases caused by environmental pollutants.
- SourceAvailable from: Rodolfo Thomé[Show abstract] [Hide abstract]
ABSTRACT: Chloroquine (CQ), an antimalarial drug, has been shown to modulate the immune system and reduce the severity of experimental autoimmune encephalomyelitis (EAE). The mechanisms of disease suppression are dependent on regulatory T cell induction, although Tregs-independent mechanisms exist. We aimed to evaluate whether CQ is capable to modulate bone marrow-derived dendritic cells (DCs) both phenotypically and functionally as well as whether transfer of CQ-modulated DCs reduces EAE course. Our results show that CQ-treated DCs presented altered ultrastructure morphology and lower expression of molecules involved in antigen presentation. Consequently, T cell proliferation was diminished in coculture experiments. When transferred into EAE mice, DC-CQ was able to reduce the clinical manifestation of the disease through the modulation of the immune response against neuroantigens. The data presented herein indicate that chloroquine-mediated modulation of the immune system is achieved by a direct effect on DCs and that DC-CQ adoptive transfer may be a promising approach for avoiding drug toxicity.Immunology and Cell Biology advance online publication, 12 November 2013; doi:10.1038/icb.2013.73.Immunology and Cell Biology 11/2013; · 4.21 Impact Factor
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ABSTRACT: Th9 cells protect hosts against helminthic infection but also mediate allergic disease. Here we show that nitric oxide (NO) promotes Th9 cell polarization of murine and human CD4(+) T cells. NO de-represses the tumour suppressor gene p53 via nitrosylation of Mdm2. NO also increases p53-mediated IL-2 production, STAT5 phosphorylation and IRF4 expression, all essential for Th9 polarization. NO also increases the expression of TGFβR and IL-4R, pivotal to Th9 polarization. OVA-sensitized mice treated with an NO donor developed more severe airway inflammation. Transferred Th9 cells induced airway inflammation, which was exacerbated by NO and blocked by anti-IL-9 antibody. Nos2(-/-) mice had less Th9 cells and developed attenuated eosinophilia during OVA-induced airway inflammation compared with wild-type mice. Our data demonstrate that NO is an important endogenous inducer of Th9 cells and provide a hitherto unrecognized mechanism for NO-mediated airway inflammation via the expansion of Th9 cells.Nature Communications 08/2014; 5:4575. · 10.74 Impact Factor
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ABSTRACT: The immune system is regulated by distinct signaling pathways that control the development and function of the immune cells. Accumulating evidence suggest that ligation of aryl hydrocarbon receptor (Ahr), an environmentally responsive transcription factor, results in multiple cross talks that are capable of modulating these pathways and their downstream responsive genes. Most of the immune cells respond to such modulation, and many inflammatory response-related genes contain multiple xenobiotic-responsive elements (XREs) boxes upstream. Active research efforts have investigated the physiological role of Ahr in inflammation and autoimmunity using different animal models. Recently formed paradigm has shown that activation of Ahr by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3,3'-diindolylmethane (DIM) prompts the differentiation of CD4(+)Foxp3(+) regulatory T cells (Tregs) and inhibits T helper (Th)-17 suggesting that Ahr is an innovative therapeutic strategy for autoimmune inflammation. These promising findings generate a basis for future clinical practices in humans. This review addresses the current knowledge on the role of Ahr in different immune cell compartments, with a particular focus on inflammation and autoimmunity.BioMed research international. 01/2014; 2014:520763.
Regulation of type 17 helper T-cell function by
nitric oxide during inflammation
Wanda Niedbalaa,1, Jose C. Alves-Filhoa,b,1, Sandra Y. Fukadaa,c,1, Silvio Manfredo Vieirab, Akio Mitania,d,
Fabiane Sonegoa, Ananda Mirchandania, Daniele C. Nascimentoa, Fernando Q. Cunhab, and Foo Y. Liewa,2
aInstitute of Infection, Immunity, and Inflammation, University of Glasgow, Glasgow G12 8TA, Scotland;bDepartment of Pharmacology, School of Medicine
of Ribeirão Preto, University of São Paulo,14049-900, Ribeirão Preto, Brazil;cDepartment of Physics and Chemistry, Faculty of Pharmaceutical Sciences,
University of São Paulo, 14040-903, Ribeirão Preto, Brazil; anddDepartment of Periodontology, School of Dentistry, Aichi Gakuin University, Nagoya,
Edited by Yoichiro Iwakura, Institute of Medical Science, University of Tokyo, Tokyo, Japan, and accepted by the Editorial Board April 19, 2011 (received for
review January 13, 2011)
Type 17 helper T (Th17) cells are implicated in the pathogenesis
many of human autoimmune diseases. Development of Th17 can
be enhanced by the activation of aryl hydrocarbon receptor (AHR)
whose ligands include the environmental pollutant dioxin, poten-
tially linking environmental factors to the increased prevalence of
autoimmune disease. We report here that nitric oxide (NO) can
suppress the proliferation and function of polarized murine and
human Th17 cells. NO also inhibits AHR expression in Th17 cells
and the downstream events of AHR activation, including IL-22,
IL-23 receptor, and Cyp1a1. Conversely, NO did not affect the
polarization of Th17 cells from mice deficient in AHR. Furthermore,
mice lacking inducible nitric oxide synthase (Nos2−/−) developed
more severe experimental autoimmune encephalomyelitis than
WT mice, with elevated AHR expression, increased IL-17A, and
IL-22 synthesis. NO may therefore represent an important endog-
enous regulator to prevent overexpansion of Th17 cells and con-
trol of autoimmune diseases caused by environmental pollutants.
are associated with pathogenesis of human autoimmune diseases,
including multiple sclerosis, rheumatoid arthritis, inflammatory
bowel disease, and psoriasis (1, 2). Thus, there likely exist rigorous
endogenous control mechanisms to limit Th17 development. We
now show that nitric oxide (NO) can potently inhibit Th17 pro-
liferation and function.
NO is a key mediator of a variety of biological functions, in-
cluding vascular relaxation, platelet aggregation, neurotrans-
mission, tumoricidal and microbicidal activities, and immuno-
suppression (3–5). It is also associated with some of the most
important immunopathologies, including rheumatoid arthritis,
diabetes, systemic lupus erythematosus, and septic shock. NO is
derived from the guanidino nitrogen atom(s) and molecular
oxygen in a reaction catalyzed by the enzyme nitric oxide syn-
thase (NOS). There are three forms of NOS. The endothelial
form (eNOS or NOS3) and neuronal form (nNOS or NOS1)
produce the amount of NO required for physiological functions.
The cytokine-inducible form (iNOS or NOS2) is activated by
a number of immunological stimuli, such as IFN-γ, TNF-α, and
LPS, and catalyses the high output of NO, which can be cytotoxic
and kill intracellular pathogens.
NO plays a key role in the immune response (3, 5). NO down-
regulates the expression of selectins (P and E), vascular cell
adhesion molecule-1, and intracellular adhesion molecule-1 in-
duced by cytokine activation on the vessel wall, resulting in
inhibition of rolling of leukocytes along the endothelium and
migration of cells from vessels to the tissues (6). We have pre-
viously reported that low doses of NO selectively enhanced Th1-
cell differentiation and expansion under Th1 polarizing con-
ditions. This was mediated by enhanced IL-12 receptor (IL-12R)
β2 expression through a cGMP-dependent pathway (7). We have
also shown that NO can induce a subset of CD4+CD25+Foxp3−
regulatory T (NO-Treg) cells from CD4+CD25−T cells via p53,
IL-2, and OX40 in a cGMP-independent manner (8). NO-Tregs
ype 17 helper T (Th17) cells have recently been defined as
a new lineage of CD4+T cells that produce IL-17. Th17 cells
suppressed the proliferation of freshly purified CD4+CD25−
effector cells in vitro and ameliorated the effector T cell-mediated
colitis and collagen-induced arthritis in the mouse in an IL-10–
dependent manner. Both Th1 and Treg cells are pivotal to auto-
immune diseases, indicating that NO may be a key player in mod-
ulating inflammatory disease.
Th17 cell differentiation is dependent on IL-6, IL-1, and TGF-
β (with potential species-specific differences) and is enhanced
by activation of the aryl hydrocarbon receptor (AHR) (9–11).
AHR ligands include the environmental pollutant dioxin and the
ultraviolet-B light-induced tryptophan metabolite 6-formylindolo
[3, 2-b] carbazole (FICZ), potentially linking environmental fac-
tors to the increased prevalence of autoimmune diseases.
We now show that NO potently inhibits the proliferation and
function of mouse and human Th17 cells. Furthermore, we show
that NO suppresses the expression of AHR in Th17 cells in-
dependent of IL-2, IL-10, retinoid-related orphan receptor α
(RORα), and RORγt. NO had no effect on the polarization of
Th17 cells from CD4+T cells of Ahr−/−mice. In vivo, Nos2-
deficient mice developed exacerbated experimental autoimmune
encephalomyelitis (EAE) accompanied by elevated numbers of
Th17 cells and AHR expression in the spinal cord. Thus, NO is
an endogenous negative regulator of Th17-cell development and
may prevent autoimmune diseases, including those induced by
NO Suppresses Th17-Cell Polarization. CD4+T cells were purified
(>98% pure) from the lymph nodes and spleens of BALB/c mice
or B6 mice by immunomagnetic beads and polarized under Th17
differentiation conditions (TGF-β + IL-6 + IL-1β or these cyto-
kines + anti–IL-2 + anti–IL-4 and anti–IFN-γ) in the presence of
graded concentrations of an NO-donor (NOC-18) for up to 5 d.
NO inhibited the production of IL-17A protein and expression of
mRNA in a dose- and time-dependent manner. The maximum
inhibition was achieved at 200 μM NOC-18, and the inhibition was
evident from 72 h after Th17 polarization (Fig. 1A). NO inhibited
the percentage of IL-17–producing T cells but had no apparent
effect on the low number of IL-10–producing cells as determined
by intracellular flow cytometry (FACS). NO also blocked the
Th17 polarization-driven cell cycle as estimated by FACS on [5-
(-6) carboxyfluorescein diacetate succinimidyl ester] (CFSE) di-
lution (Fig. 1B). The NO-mediated inhibition of Th17 was not
Author contributions: W.N., J.C.A.-F., S.Y.F., F.Q.C., and F.Y.L. designed research; W.N.,
J.C.A.-F., S.Y.F., S.M.V., A. Mitani, F.S., A. Mirchandani, and D.C.N. performed research;
F.Q.C. contributed new reagents/analytic tools; W.N., J.C.A.-F., S.Y.F., and F.Y.L. ana-
lyzed data; and W.N., J.C.A.-F., and F.Y.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. Y.I. is a guest editor invited by the Editorial Board.
1W.N., J.C.A.-F., and S.Y.F. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| May 31, 2011
| vol. 108
| no. 22www.pnas.org/cgi/doi/10.1073/pnas.1100667108
attributable to increased apoptosis or necrosis of the treated cells
as evident by the similar percentage of viability (propidium iodide-
negative and Annexin V-negative cells) of the NO-treated and
control untreated cells (Fig. 1C). Despite the inhibitory effect of
NO on cell cycle during the first 3 d of culture, NO did not reduce
but did increase the total number of T cells at the end of the
culture (before culture: 1.5 × 105cells/mL; at the end of the 5-d
culture: without NO, 3.5 ± 0.3 × 105/mL; with 100 μM NOC-18,
6.3 ± 1.5 × 105/mL; with 200 μM NOC-18, 10.7 ± 1.1 × 105/mL).
NO also inhibits the protein synthesis and mRNA expression of
IL-17F in a dose- and time-dependent manner (Fig. 1D). The
inhibition of IL-17F by NO was evident at 48 h and earlier than
that for IL-17A. NO also blocked the expression of IL-21, IL-22,
and IL-23R (Fig. 1E). In contrast, NO (at 200 μM NOC-18)
markedly increased the synthesis of IL-2 during the Th17 polari-
zation as determined by flow cytometry using a cytokine secretion
assay and in the culture supernatant harvested at day 5 of the
culture (polarizing condition without anti–IL-2) (Fig. 1F). IFN-γ
and IL-4 production was low and barely detectable by ELISA or
by intracellular staining in culture with or without anti–IFN-γ and
anti–IL-4 antibodies. NOC-18 has a half-life of 20 h in solution.
To demonstrate that the effect on Th17 is mediated by NO pro-
duced by NOC-18 and not by other stable residual products of
NOC-18, we incubated the medium containing NOC-18 (200 μM)
at 37° C and 5% (vol/vol) CO2for 5 d. We then compared the
conditioned medium (CM) with freshly prepared NOC-18 solu-
tion for its effect on Th17 development. Whereas the freshly
prepared NOC-18 medium suppressed the polarization of Th17
from CD4+T cells, the spent CM did not (Fig. S1A). We have
also further titrated the concentration of NOC-18 used. At con-
centrations below 60 μM, NOC-18 modestly but not significantly
affected the polarization of Th17; NOC-18 is cytotoxic beyond 300
μM (Fig. S2). We then determined whether the cells could re-
spond to IL-23 during the suppression. In the primary Th17 po-
larization culture in the presence of NO, the cells were not
responsive to exogenously added IL-23 (Fig. S3A). We also in-
vestigated whether the continuous presence of NO is required to
maintain the suppression of Th17-cell development. Th17 cells
were harvested after 5 d of polarization in the presence of 200 μM
NOC-18 and restimulated with IL-23 and anti-CD3 with or
without freshly added NOC-18. The NO-treated Th17 cells re-
covered from suppression in the absence of additional NO but
remained suppressed in the presence of NO (Fig. S3 B and C).
We next compared the effect of NO on Th1-, Th2-, and Th17-cell
polarization in parallel cultures. In the condition in which NO
markedly suppressed Th17 polarization, NO had little effect on
Th1 differentiation but enhanced the low levels of Th2 de-
velopment (Fig. S4).
Together, these results demonstrate that at the concentrations
used, NO selectively suppressed Th17-cell proliferation and the
cytokines and cytokine receptor associated with IL-17. Further-
more, the NO-mediated suppression was not attributable to cy-
totoxicity or NO-induced metabolites.
NO Also Suppresses the Proliferation of Established Th17 Cells. We
next investigated whether NO can also affect the functions of
established Th17 cells. CD4+T cells from BALB/c mice were
polarized as above for 5 d. The cells were washed and then
recultured for 3 d with anti-CD3, IL-23, and IL-1β as indicated in
the presence of graded concentrations of NOC-18. The cells were
then harvested for intracellular IL-17A and RORγt (a major
transcription factor of Th17) staining and the concentrations of
cytokines in the culture supernatant determined by ELISA.
Around 75% of Th17 cells were RORγt+. NO inhibited the per-
centage of IL-17+RORγt+cells in a dose-dependent manner and
had only a modest effect on the IL-17−RORγt+cells (Fig. 2 A
and B). NO also inhibited the proliferation of the polarized Th17
cells as shown by CFSE staining analysis in a dose-dependent
manner (Fig. 2C). The concentrations of IL-17A, IL-17F, and IL-
22 in the culture supernatant were also significantly reduced by
NO in a dose-dependent manner (Fig. 2D). In contrast, the con-
from BALB/c mice were cultured in round-bottom 96-well plates with mi-
tomycin C-treated antigen-presenting cells as well as anti-CD3, anti-CD28,
IL-6, TGF-β, and IL-1 with graded concentration of an NO donor (NOC-18)
for up to 120 h. In some cultures, anti–IL-2, anti–IL-4, and anti–IFN-γ anti-
bodies were also added with similar results, except with a higher percentage
of IL-17+cells. (A) IL-17A in the culture supernatant was measured by ELISA,
and Il17a expression in the cells was determined by qPCR. Ctrl, control. (B)
IL-17– and IL-10–producing cells were determined by intracellular staining
(day 4), and cell cycle was estimated by CFSE dilution (day 3) using flow
cytometry (FACS). (C) Viability of the cells cultured above was determined
by propidium iodide and Annexin V staining and measured by FACS on day 4
of culture. (D) Production of IL-17F was determined by ELISA, and the ex-
pression of Il17f mRNA was measured by qPCR. (E) mRNA levels of Il21, Il22,
and Il23r in cells were determined by qPCR. (F) Concentrations of IL-2 in the
culture supernatant (day 4, 200 μM NOC-18) were determined by ELISA, and
the percentage of IL-2+and IL-10+cells was assayed by FACS using a cytokine
secretion assay. Data are expressed as mean ± SEM (n = 5), representative of
three independent experiments. *P < 0.05 compared with control without
NO suppresses the polarization of Th17 cells. Purified CD4+T cells
Niedbala et al.PNAS
| May 31, 2011
| vol. 108
| no. 22
centration of IL-10 in the culture supernatant was not affected by
NO (Fig. 2D). These results demonstrate that NO selectively
inhibits the proliferation and functions of established Th17 cells.
NO Suppresses Th17 Differentiation Independent of IL-2, IL-10, cGMP,
or ETS1.Various cytokines regulate the polarization of Th17 cells:
TGF-β, IL-6, IL-1β, and IL-21 are positive inducers, whereas IL-
2, IL-4, IL-27, IL-35, and IFN-γ are negative regulators (12–20).
Because NO treatment led to a marked increase in IL-2 synthesis
(Fig. 1F), we investigated the potential role of IL-2 in the NO-
mediated inhibition of Th17 development. As expected (19), the
number of Th17 cells polarized was dramatically enhanced in
CD4+T cells from Il2−/−mice compared with WT cells (Fig.
3A). However, NO inhibited the polarization of Th17 cells to
a similar extent whether the cells were from the WT mice or the
Il2−/−mice (Fig. 3 A and B). NO also inhibited the polarization
of Th17 cells from CD4+cells of IL-10–deficient mice to the
same degree as that of the WT mice (Fig. 3C). These results
demonstrate that IL-2 and IL-10 are unlikely the mediators of
NO-induced inhibition of Th17 development. IFN-γ, IL-4, and
IL-27 were not detected in the culture supernatants. Further-
more, NO was equally effective in suppressing Th17 polarization
in the presence of anti–IFN-γ and anti–IL-4 antibodies (Fig. 1A),
thus excluding the potential role of these cytokines in NO-
mediated suppression of Th17. We then examined the possibility
that NO may exert its influence via the activation of soluble
guanylyl cyclase (sGC), thus elevating cGMP, a well-established
NO-effector pathway (21). CD4+T cells from BALB/c mice
were cultured for 3 d under the same Th17 polarizing condition
as above with 0.5–1 mM 8-Bromo–cGMP (8-Br–cGMP, an an-
alog of cGMP). 8-Br–cGMP had no effect on IL-17 synthesis
(Fig. S5). Conversely, we polarized Th17 cells in the presence of
NO and 10 μM [1H-oxodiazolo-(1,2,4)-(4,3-a) guinoxaline-1-one]
(ODQ), a competitive inhibitor of the activation of sGC. ODQ
was not able to reverse the inhibitory effect of NO on IL-17
production (Fig. S5). Similar negative results were obtained with
established Th17 cells. Therefore, NO is unlikely to affect Th17
polarization and function via cGMP. We also examined the ef-
fect of NO on Ets1, a recently discovered transcriptional acti-
vator that is a negative regulator of Th17 development (22).
CD4+T cells from BALB/c mice were polarized to Th17 with or
without NOC-18. Cells were harvested 12 or 24 h later, and Ets1
mRNA expression was determined by quantitative PCR (qPCR).
NO did not affect the expression of Ets1 (Fig. S6). Together,
these results show that NO suppresses Th17 differentiation in-
dependent of IL-2, IL-10, cGMP, or ETS-1.
NO Suppresses the Expression of AHR on Th17 Cells. Recently, it has
been reported that AHR expression and activation enhanced the
development of Th17 cells (9–11). We therefore investigated
whether NO would have an effect on AHR expression during
Th17 differentiation. CD4+T cells were polarized under the
Th17 condition as described above in the presence of 200 μM
NOC-18. The cells were harvested at regular intervals for up to
48 h, and the synthesis of AHR was determined by Western blot
analysis. As expected, naive cells did not produce a detectable
level of AHR, whereas differentiating Th17 cells expressed AHR
in a time-dependent manner (Fig. 4A). NO markedly inhibited
the production of AHR in a time-dependent (Fig. 4A) and dose-
dependent (Fig. 4B) fashion. Protein synthesis was significantly
suppressed by 100 μM NOC-18 and reduced by at least fivefold
by 200 μM NOC-18. NO also potently inhibited the Ahr mRNA
in a time-dependent manner (Fig. 4C). The suppression of AHR
by NO was evident 24 h after Th17 polarization, and this was
sustained for at least 72 h.
The orphan nuclear receptors, RORα and RORγt (encoded
by Rora and Rorc, respectively, in mice) have been identified as
the key transcription factors that determine the differentiation of
Th17 (23, 24). We therefore investigated the effect of NO on the
expression of Rora and Rorc. NO had no effect on the expression
of Rora or Rorc at any of the time points tested during the dif-
ferentiation of Th17 cells (Fig. 4D).
AHR, also known as dioxin receptor, is a transcription factor
member of the basic helix-loop-helix periodicity/AHR nuclear
translocator/single–minded (Per/ARNT/Sim) family (25). AHR
binds to its high-affinity ligands, such as FICZ; translocates to
the nucleus and dimerizes with ARNT; and could cause a variety
of toxicological effects (26). The best-studied downstream target
of AHR is the Cyp1a1 gene family that encodes cytochrome p450
family drug-metabolizing enzymes. We therefore investigated
polarized for 5 d as in Fig. 1. The cells were then recultured for 3 d with IL-23
(20 ng/mL), anti-CD3 (4 μg/mL), and anti-CD28 (1.5 μg/mL) antibodies in the
presence of the NO donor NOC-18 (200 μM) (A) or as indicated. (A) Percen-
tages of IL-17A+cells and RORγt+CD4+T cells in the control and NO-treated
cells, together with the isotype control and unactivated cells (Th0), were
determined by intracellular staining using FACS, and representative data are
shown. Ctrl, control. (B) Quantitation of the FACS data in A. (C) Cell cycle of
the established Th17 cells treated with graded concentrations of NOC-18
was determined by CFSE staining. (D) IL-17A, IL-17F, IL-22, and IL-10 con-
centrations in the culture supernatant were measured by ELISA. Data are
mean ± SEM (n = 5), representative of four independent experiments. *P <
0.05 (compared with control without NOC-18).
NO suppresses the function of established Th17. CD4+T cells were
WT B6 mice or B6 Il2−/−or Il10−/−mice were purified and polarized to Th17 in
the presence of 200 μM NOC-18 for 4 d as in Fig. 1. IL-17A+and CD4+T cells
were determined by FACS (A). IL-17A concentrations in the culture super-
natants of Il2−/−(B) or Il10−/−cells (C) were determined by ELISA. Data are
mean ± SEM (n = 5), representative of two independent experiments. *P <
0.05 (compared with control without NOC-18). Ctrl, control.
NO suppresses Th17 independent of IL-2 or IL-10. CD4+T cells from
| www.pnas.org/cgi/doi/10.1073/pnas.1100667108 Niedbala et al.
whether NO would also affect the expression of the downstream
events of AHR activation. As expected, FICZ enhanced the
synthesis of IL-17A and increased the expression of Il17f, Il22,
Il23r, and cyp1a1 mRNA during Th17-cell polarization (Fig. 4 E
and F). NO markedly suppressed the enhanced IL-17A synthesis
induced by FICZ (Fig. 4E). NO also significantly suppressed the
FICZ-induced enhanced mRNA expression of Il17f, Il22, Il23r,
and Cyp1a1 (Fig. 4F). We also found that NO has no effect on
Rorc expression whether CD4+T cells were exposed to FICZ or
not during Th17 polarization (Fig. S7). These results are con-
sistent with an earlier report that the FICZ-AHR activation
pathway did not affect the expression of Rorc during Th17 po-
larization (10). Together, these results indicate that NO sup-
presses Th17 development, at least in part, via the inhibition of
AHR expression. To test this possibility directly, CD4+T cells
from WT or Ahr−/−mice were polarized to Th17 in the presence
or absence of NOC-18. As expected, Ahr−/−cells produced 50%
less Th17 than the WT cells (10). NO markedly suppressed the
Th17 polarization of WT cells but failed to suppress the Th17
polarization of Ahr−/−cells (Fig. 4G). IL-22 was not produced by
Ahr−/−cells (Fig. 4G).
We further examined the effect of NO on the expression of IL-
1R, IL-6R, and TGF-βRII. NO reduced the expression of IL-
1R1, had no effect on IL-6R, and modestly enhanced the ex-
pression of TGF-βRII (Fig. S8A). These results indicate that NO
may also inhibit Th17-cell development via the down-regulation
of IL-1R1. We then investigated the effect of NO on Th17 po-
larization in T cells from Il1r1−/−mice (27). IL-1R–deficient cells
produced about 50% less Th17 than the WT cells. NO com-
pletely and similarly suppressed Th17 polarization in WT and
Il1r1−/−cells (Fig. S8 B and C), indicating that IL-1R1 is unlikely
to be the major target in the NO-mediated suppression of Th17
polarization. We then investigated the effect of NO on the ex-
pression of Foxp3, IRF4, and Stat3 phosphorylation, all of which
have been implicated to play a significant role in Th17 differ-
entiation (17, 24, 28, 29). Furthermore, it has been suggested
that iNOS inhibits IL-6–mediated STAT3 activation (30). Under
the Th17 polarizing condition, NO had no effect on Foxp3 (Fig.
S9A) or IRF4 (Fig. S9C) expression but significantly inhibited
Stat3 phosphorylation (Fig. S9B).
Together, these results suggest that NO is unlikely to affect
Th17 differentiation via modulating Foxp3 or IRF4 expression;
rather, NO suppresses Th17 differentiation and function, at least
in part, via the inhibition of AHR expression and Stat3 phos-
phorylation. The molecular mechanism by which NO inhibits
AHR expression is unknown at present. It is also unclear whether
NO suppresses the functions of other AHR-bearing cells. These
questions are now amenable to investigation.
NO Suppresses Human Th17-Cell Differentiation. Next, we inves-
tigated whether NO also has an effect on the polarization of
human Th17 cells. CD4+T cells were purified from the pe-
ripheral blood of healthy donors by immunomagnetic beads and
cultured for 4 d with anti-human (h) CD3, anti-hCD28, hIL-6,
hTGF-β, hIL-1β, hIL-21, hIL-23, anti–hIFN-γ, and anti–hIL-4
in the presence of graded concentrations of NOC-18. NO sup-
pressed the polarization of IL-17–producing cells in a dose-
dependent manner (Fig. 5A). When the culture supernatants
were analyzed by ELISA, NO again potently suppressed IL-17
and IL-22 synthesis but had no effect on IL-10 production (Fig.
5B). The AHR agonist FICZ strongly enhanced the expression
of IL-23R. This increase was abrogated by NO (Fig. 5C). There
was also no difference in the percentage of viable cells between
the NO-treated culture and the control untreated cells, with a
low percentage of propidium iodide-positive and Annexin V-
positive cells in both cultures (Fig. 5D). These results therefore
recapitulate the observation seen with the murine cells, again
indicating the selective suppressive effect of NO on human
Th17-cell differentiation and that this effect was not attributable
to potential cytotoxic effects of NO.
Nos2−/−Mice Developed Exacerbated EAE, Elevated Numbers of Th17
Cells, and Enhanced AHR Expression. Th17 plays a prominent role in
the pathogenesis of autoimmune diseases, such as EAE, and NO
has been reported to suppress EAE (31–35). We therefore in-
vestigated the effect of NO on Th17 development and functions
in the murine model of EAE in vivo. WT and Nos2−/−mice of
the C57BL/6 background were immunized with the myelin oli-
godendrocyte glycoprotein (MOG35–55) peptide in complete
Freund’s adjuvant. Nos2−/−mice developed earlier and more
severe EAE compared with WT mice (Fig. S10A). Whereas WT
BALB/c mice were cultured (for 3 d, unless indicated otherwise) as in Fig. 1 in
the presence of NOC-18 (200 μM, unless indicated otherwise). AHR protein
synthesis was analyzed by Western blot in a time- and dose-dependent
manner and was also expressed as the ratio to the housekeeping protein
β-actin. Ctrl, control; ND, not detectable. Data are mean ± SEM (n = 3). *P <
0.05. The expression of Ahr (C) and Rora and Rorc (D) mRNA was also ana-
lyzed by qPCR. Data are mean ± SEM (n = 3), representative of three in-
dependent experiments. *P < 0.05 (compared with control without NOC-18).
(E and F) CD4+T cells from BALB/c mice were polarized to Th17 for 3 d as
above in the presence of NOC-18 (200 μM) ± FICZ (200 nM). The concen-
tration of IL-17A in the culture supernatant was determined by ELISA (E),
and the expression of mRNA of Il17f, Il22, Il23r, and Cyp1a1 was determined
by qPCR (F). Data are mean ± SEM (n = 3), representative of three in-
dependent experiments.#P < 0.05 compared with control; *P < 0.05 com-
pared with the group with FICZ but without NOC-18. (G) CD4+T cells from
WT B6 mice or B6 Ahr−/−mice were polarized to Th17 for 4 d ± NOC-18 (100
μM) in flat-bottom 96-well plates. IL-17A+cells were analyzed by intracellular
staining, and IL-22 concentration was determined by ELISA. Data are mean ±
SEM (n = 3). *P < 0.05; **P < 0.01 compared with WT mice not treated with
NOC-18. The difference between NOC-18–treated WT cells and NOC-18–
treated Ahr−/−cells was not significant.
NO suppresses the expression of AHR. (A and B) CD4+T cells from
Niedbala et al. PNAS
| May 31, 2011
| vol. 108
| no. 22
mice developed EAE with a mean day of onset of 14 d, Nos2−/−
mice developed EAE with earlier kinetics (onset at 11.5 d). Se-
rum collected on day 17 from the Nos2−/−mice contained
a higher concentration of IL-17A than that of the WT mice (Fig.
S10B). The draining lymph node cells of the Nos2−/−mice also
produced larger amounts of IL-17A than produced by the cells
from the WT mice in the recall response to MOG35–55antigen in
vitro (Fig. S10C). Cells from both groups of mice produced
similarly high levels of IL-17A in response to the polyclonal T-cell
mitogen, Con A, demonstrating antigen specificity. There was,
however, no difference in the amount of IFN-γ produced by both
strains of mice, showing that the effect of NO is cell type-specific
and in agreement with an earlier finding that AHR is principally
expressed on Th17 cells (10). Our results are in agreement with
a recent report (which used a reverse protocol) that mice given an
NO donor (GSNO) developed less severe EAE accompanied by
reduced IL-17 but not IFN-γ production (36).
In addition, we found that the spinal cord tissue from Nos2−/−
mice expressed a significantly higher level of Ahr, Il17a, and Il22
compared with that of the WT mice (Fig. S10D). There was no
difference in the Il21 mRNA between the two groups. Histo-
logical examination shows that the spinal cord of the Nos2−/−
mice contained markedly more CD3+IL-17A+T cells compared
with the WT mice (Fig. S10E). A higher magnification version of
the pictures is provided in Fig. S11. Sections were also stained
with H&E and examined under light microscopy. Nos2−/−mice
exhibited significantly more perivascular and subpial infiltrates of
different size compared with the WT mice.
To obtain direct evidence that the exacerbated disease in EAE
Nos2−/−mice was mediated by enhanced Th17 response, we
treated EAE Nos2−/−mice with a neutralizing anti–IL-17 anti-
body. As expected, Nos2−/−mice developed significantly more
severe EAE compared with the WT mice. The disease in the
Nos2−/−mice was completely abolished by the treatment with the
anti–IL-17 antibody (Fig. S10F). Together, these results indicate
that NO produced during an inflammatory disease may attenuate
the disease development by inhibiting Th17 differentiation as-
sociated with the suppression of AHR in vivo.
Data reported here demonstrate that NO can suppress the pro-
of the expression of AHR. This finding provides a hitherto un-
recognized function of NO, a molecule of pervasive and pivotal
pathophysiological roles. Our results also demonstrate a mecha-
nism for preventing excessive activation of Th17 cells, and hence
inflammatory diseases. This observation also suggests that en-
dogenously produced NO could attenuate environmental-linked
NOS2, which catalyses the production of high levels of NO, is
induced by cytokines like IFN-γ and TNF-α following an adaptive
immune response and is expressed in a variety of cells. Although
NOS2 is not readily detected in human peripheral blood mono-
cytes, its overall in vivo role in humans is not in doubt. NOC-18
at a rate of 100–200 μM constantly releases 200–400 nM NO with
a half-life of 20 h (37). This dose of NO occurs in vivo in sites of
acute infection and inflammation (38) and has been used rou-
tinely in experiments in vitro (8, 39). The expression of iNOS
at the site of inflammation during EAE has been extensively
reported (33, 40–42). We show here by qPCR and immunofluo-
rescent staining that Th17 is expressed at the site of inflammation.
Whether Th17 cells differentiated at the site of inflammation
is unknown, and investigation is limited by currently available
may be sufficient to suppress the differentiation of Th17 in the
lymph nodes. Furthermore, NO may also suppress fully differ-
entiated Th17 cells at the site of inflammation. We have not used
Nos1- and Nos3-deficient mice in our study because the low levels
to affect a highly inflammatory disease model like EAE.
It is important to note that NO is not cytotoxic at the dose
used, because there was no evidence of any increase in apoptosis
or necrosis under the culture conditions. Furthermore, the NO-
treated cells produced elevated levels of IL-2 synthesis and no
reduction of IL-10 production. These findings also indicate
a high degree of selectivity of the action of NO in the polariza-
tion of Th17 cells. The effect of NO on Th17 development
appears to be independent of the classic sGC-cGMP pathway,
because at the concentrations known to be physiologically ef-
fective, 8-Br–cGMP had no effect on Th17 differentiation and
ODQ could not reverse the inhibitory effect of NO. It is also
unlikely that NO mediates the suppression via IL-2, IL-10, or
IFN-γ, because NO could effectively suppress Th17 differentia-
tion in the absence of these cytokines.
It should be noted that NO down-regulates Th17 proliferation
but does not reprogram Th17 cells. Following a resting period in
the absence of NO, Th17 cells can resume proliferation in the
presence anti-CD3 and IL-23. This finding suggests that NO,
generated during inflammation, could limit collateral damage by
shutting down Th17 cells for as long as it takes without perma-
nently eliminating the cells, which may be required for defense
against future infections.
Our results provide direct evidence that the suppressive effect
of NO on Th17 cell proliferation and function is associated with
the inhibition of Ahr mRNA transcription and AHR protein
synthesis. NO also inhibits the expression of the known down-
stream events of AHR-ligand binding, including IL-22 and
CYP1a1, reinforcing the notion that NO suppresses the expres-
sion of AHR. Furthermore, NO has no apparent effect on
RORα or RORγt expression, the canonical pathway of Th17
differentiation. This, however, is in agreement with a previous
report that AHR enhances Th17 polarization independent of
Rorc (10) and further supports the notion that the NO-mediated
purified from peripheral blood of healthy donors and cultured for 4 d with
bead-coated anti-CD3 + anti-CD28, IL-6, TGF-β, IL-1β, IL-21, IL-23, anti–IFN-γ,
and anti–IL-4 antibodies in the presence of graded concentrations of NOC-18.
(A) Intracellular FACS analysis of IL-17A. Ctrl, control. (B) Concentrations of IL-
17A, IL-22, and IL-10 were determined by ELISA. (C) Expression of IL-23R was
also determined by FACS. The histogram shows isotype control (gray), Th17
polarized without NOC-18 or FICZ (green), Th17 polarized with 2 μM FICZ
(blue), and Th17 polarized with 100 μM FICZ + NOC-18 (red). (D) Percentage of
viable cells at the end of culture was analyzed by propidium iodide and
Annexin V staining. Data are mean ± SEM (n = 5), representative of three
independent experiments. *P < 0.05 (compared with control without NOC-18).
NO suppresses human Th17 cell differentiation. CD4+T cells were
| www.pnas.org/cgi/doi/10.1073/pnas.1100667108Niedbala et al.
suppression of Th17 cells is closely associated with the inhibition
of AHR expression. Nevertheless, given the pleiotropic nature of
NO, it is likely that NO may affect other molecules in Th17
polarization. NO also suppressed the expression of IL-1R1 and
IL-23R (but not IL-6R or TGF-βRII). IL-1 is a key driver of
Th17 polarization (14, 43, 44). However, because NO similarly
suppresses Th17 polarization of WT as well as Il1r1−/−cells, IL-
1R1 is unlikely a major target of NO. In contrast, NO markedly
suppressed Th17 proliferation in WT cells but not in Ahr−/−cells.
It is therefore likely that AHR plays a major role in the NO-
mediated inhibition of Th17 differentiation.
In our hands, the expression of IL-23R is, at least in part, linked
to the activation of AHR. FICZ markedly promoted IL-23R ex-
pression in murine and human Th17 cell differentiation, and this
enhancement was abrogated by NO. NO also significantly sup-
pressed Stat3 phosphorylation. It is not clear whether Stat3 mod-
ulatesRORγtgene transcription,but activatedStat3bindsdirectly
to the Stat-binding sites in the IL-17 gene promoter and increases
IL-17 gene transcription (28). Thus, the inhibition of Stat3 phos-
phorylation may represent an additional mechanism by which NO
suppresses Th17 differentiation independent of RORγt.
Our results demonstrate that NO could effectively inhibit Th17
proliferation and function, and hence attenuate inflammation. This
finding opens the intriguing possibility that NO could be a consti-
tutive negative regulator of a range of inflammatory diseases ini-
tiated by, among others, environmental factors. The demonstration
that human Th17 cell function is similarly affected by NO further
suggests the therapeutical potential of NO in inflammation.
Materials and Methods
Mice. Full details of mouse strains and induction of EAE are presented in SI
Materials and Methods.
Cell Culture. Cell culture was carried out in vitro with purified CD4+T cells
according to generally used protocols as cited in the text. NOC-18 was added
at the beginning of the cultures (SI Materials and Methods).
Induction and Measurement of EAE. EAE was induced with MOG35–55peptide
in complete Freund’s adjuvant containing Myobacterium tuberculosis
H37Ra (Difco Laboratories). Clinical signs of EAE were assessed daily
according to scores based on a 10-point scale or a 5-point scale (SI Mate-
rials and Methods).
ACKNOWLEDGMENTS. We thank Dr. Brigitta Stockinger for providing the
Il2−/−and Ahr−/−cells and Dr. Jean Langhorne for providing the Il10−/−cells
(both from the National Institute of Medical Research); and Dr. Bernard
Ryffel (Centre National de la Recherche Scientifique) for providing the
il1r1−/−cells. This work was supported by The Wellcome Trust, the Medical
Research Council of the United Kingdom, the European Union (F.Y.L.), and
by the State of São Paulo Research Foundation, Brazil (F.Q.C.).
1. Murphy CA, et al. (2003) Divergent pro- and antiinflammatory roles for IL-23 and IL-12
in joint autoimmune inflammation. J Exp Med 198:1951–1957.
2. Langrish CL, et al. (2005) IL-23 drives a pathogenic T cell population that induces
autoimmune inflammation. J Exp Med 201:233–240.
3. Bogdan C, Röllinghoff M, Diefenbach A (2000) The role of nitric oxide in innate
immunity. Immunol Rev 173:17–26.
4. Moncada S, Higgs A (1993) The L-arginine-nitric oxide pathway. N Engl J Med 329:
5. Niedbala W, Cai B, Liew FY (2006) Role of nitric oxide in the regulation of T cell
functions. Ann Rheum Dis 65(Suppl 3):iii37–iii40.
6. De Caterina R, et al. (1995) Nitric oxide decreases cytokine-induced endothelial
activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules
and proinflammatory cytokines. J Clin Invest 96:60–68.
7. Niedbala W, et al. (2002) Nitric oxide preferentially induces type 1 T cell
differentiation by selectively up-regulating IL-12 receptor beta 2 expression via cGMP.
Proc Natl Acad Sci USA 99:16186–16191.
8. Niedbala W, et al. (2007) Nitric oxide induces CD4+CD25+ Foxp3 regulatory T cells from
CD4+CD25 T cells via p53, IL-2, and OX40. Proc Natl Acad Sci USA 104:15478–15483.
9. Quintana FJ, et al. (2008) Control of T(reg) and T(H)17 cell differentiation by the aryl
hydrocarbon receptor. Nature 453:65–71.
10. Veldhoen M, et al. (2008) The aryl hydrocarbon receptor links TH17-cell-mediated
autoimmunity to environmental toxins. Nature 453:106–109.
11. Kimura A, Naka T, Nohara K, Fujii-Kuriyama Y, Kishimoto T (2008) Aryl hydrocarbon
receptor regulates Stat1 activation and participates in the development of Th17 cells.
Proc Natl Acad Sci USA 105:9721–9726.
12. Park H, et al. (2005) A distinct lineage of CD4 T cells regulates tissue inflammation by
producing interleukin 17. Nat Immunol 6:1133–1141.
13. Harrington LE, et al. (2005) Interleukin 17-producing CD4+ effector T cells develop via
a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol 6:1123–1132.
14. Sutton C, Brereton C, Keogh B, Mills KHG, Lavelle EC (2006) A crucial role for
interleukin (IL)-1 in the induction of IL-17-producing T cells that mediate autoimmune
encephalomyelitis. J Exp Med 203:1685–1691.
15. Batten M, et al. (2006) Interleukin 27 limits autoimmune encephalomyelitis by suppressing
the development of interleukin 17-producing T cells. Nat Immunol 7:929–936.
16. Stumhofer JS, et al. (2006) Interleukin 27 negatively regulates the development of
interleukin 17-producing T helper cells during chronic inflammation of the central
nervous system. Nat Immunol 7:937–945.
17. Zhou L, et al. (2007) IL-6 programs T(H)-17 cell differentiation by promoting
sequential engagement of the IL-21 and IL-23 pathways. Nat Immunol 8:967–974.
18. Wei L, Laurence A, Elias KM, O’Shea JJ (2007) IL-21 is produced by Th17 cells and
drives IL-17 production in a STAT3-dependent manner. J Biol Chem 282:34605–34610.
19. Laurence A, et al. (2007) Interleukin-2 signaling via STAT5 constrains T helper 17 cell
generation. Immunity 26:371–381.
20. Niedbala W, et al. (2007) IL-35 is a novel cytokine with therapeutic effects against
collagen-induced arthritis through the expansion of regulatory T cells and sup-
pression of Th17 cells. Eur J Immunol 37:3021–3029.
21. Arnold WP, Mittal CK, Katsuki S, Murad F (1977) Nitric oxide activates guanylate
cyclase and increases guanosine 3′:5′-cyclic monophosphate levels in various tissue
preparations. Proc Natl Acad Sci USA 74:3203–3207.
22. Moisan J, Grenningloh R, Bettelli E, Oukka M, Ho IC (2007) Ets-1 is a negative
regulator of Th17 differentiation. J Exp Med 204:2825–2835.
23. Ivanov II, et al. (2006) The orphan nuclear receptor RORgammat directs the
differentiation program of proinflammatory IL-17+ T helper cells. Cell 126:1121–1133.
24. Yang XO, et al. (2008) T helper 17 lineage differentiation is programmed by orphan
nuclear receptors ROR alpha and ROR gamma. Immunity 28:29–39.
25. Burbach KM, Poland A, Bradfield CA (1992) Cloning of the Ah-receptor cDNA reveals
a distinctive ligand-activated transcription factor. Proc Natl Acad Sci USA 89:8185–8189.
26. Ohtake F, et al. (2003) Modulation of oestrogen receptor signalling by association
with the activated dioxin receptor. Nature 423:545–550.
27. Labow M, et al. (1997) Absence of IL-1 signaling and reduced inflammatory response
in IL-1 type I receptor-deficient mice. J Immunol 159:2452–2461.
28. Chen Z, et al. (2006) Selective regulatory function of Socs3 in the formation of IL-17-
secreting T cells. Proc Natl Acad Sci USA 103:8137–8142.
29. Brüstle A, et al. (2007) The development of inflammatory T(H)-17 cells requires
interferon-regulatory factor 4. Nat Immunol 8:958–966.
30. Villavicencio RT, et al. (2000) Induced nitric oxide inhibits IL-6-induced stat3 activation
and type II acute phase mRNA expression. Shock 13:441–445.
31. Kahn DA, Archer DC, Gold DP, Kelly CJ (2001) Adjuvant immunotherapy is dependent
on inducible nitric oxide synthase. J Exp Med 193:1261–1268.
32. Zielasek J, et al. (1995) Administration of nitric oxide synthase inhibitors in experimental
autoimmune neuritis and experimental autoimmune encephalomyelitis. J Neuroimmunol
33. Okuda Y, Sakoda S, Fujimura H, Yanagihara T (1997) Nitric oxide via an inducible
isoform of nitric oxide synthase is a possible factor to eliminate inflammatory cells from
the central nervous system of mice with experimental allergic encephalomyelitis.
J Neuroimmunol 73:107–116.
34. Dalton DK, Wittmer S (2005) Nitric-oxide-dependent and independent mechanisms of
protection from CNS inflammation during Th1-mediated autoimmunity: Evidence
from EAE in iNOS KO mice. J Neuroimmunol 160:110–121.
35. Fenyk-Melody JE, et al. (1998) Experimental autoimmune encephalomyelitis is
exacerbated in mice lacking the NOS2 gene. J Immunol 160:2940–2946.
36. Nath N, Morinaga O, Singh I (2010) S-nitrosoglutathione a physiologic nitric oxide
carrier attenuates experimental autoimmune encephalomyelitis. J Neuroimmune
37. Keefer LK, Nims RW, Davies KM, Wink DA (1996) “NONOates” (1-substituted diazen-
1-ium-1,2-diolates) as nitric oxide donors: Convenient nitric oxide dosage forms.
Methods Enzymol 268:281–293.
38. Kim YM, Chung HT, Simmons RL, Billiar TR (2000) Cellular non-heme iron content is
a determinant of nitric oxide-mediated apoptosis, necrosis, and caspase inhibition.
J Biol Chem 275:10954–10961.
39. Macphail SE, et al. (2003) Nitric oxide regulation of human peripheral blood
mononuclear cells: Critical time dependence and selectivity for cytokine versus
chemokine expression. J Immunol 171:4809–4815.
gene expression in experimental allergic encephalomyelitis. J Immunol 160:5955–5962.
41. Staykova MA, Paridaen JT, Cowden WB, Willenborg DO (2005) Nitric oxide
contributes to resistance of the Brown Norway rat to experimental autoimmune
encephalomyelitis. Am J Pathol 166:147–157.
42. Dasgupta S, Roy A, Jana M, Hartley DM, Pahan K (2007) Gemfibrozil ameliorates
relapsing-remitting experimental autoimmune encephalomyelitis independent of
peroxisome proliferator-activated receptor-alpha. Mol Pharmacol 72:934–946.
43. Chung Y, et al. (2009) Critical regulation of early Th17 cell differentiation by
interleukin-1 signaling. Immunity 30:576–587.
44. Gulen MF, et al. (2010) The receptor SIGIRR suppresses Th17 cell proliferation via
inhibition of the interleukin-1 receptor pathway and mTOR kinase activation. Immunity
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| vol. 108
| no. 22