An endogenous aryl hydrocarbon receptor ligand acts on dendritic cells and T cells to suppress experimental autoimmune encephalomyelitis.
ABSTRACT The ligand-activated transcription factor aryl hydrocarbon receptor (AHR) participates in the differentiation of FoxP3(+) T(reg), Tr1 cells, and IL-17-producing T cells (Th17). Most of our understanding on the role of AHR on the FoxP3(+) T(reg) compartment results from studies using the toxic synthetic chemical 2,3,7,8-tetrachlorodibenzo-p-dioxin. Thus, the physiological relevance of AHR signaling on FoxP3(+) T(reg) in vivo is unclear. We studied mice that carry a GFP reporter in the endogenous foxp3 locus and a mutated AHR protein with reduced affinity for its ligands, and found that AHR signaling participates in the differentiation of FoxP3(+) T(reg) in vivo. Moreover, we found that treatment with the endogenous AHR ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) given parenterally or orally induces FoxP3(+) T(reg) that suppress experimental autoimmune encephalomyelitis. ITE acts not only on T cells, but also directly on dendritic cells to induce tolerogenic dendritic cells that support FoxP3(+) T(reg) differentiation in a retinoic acid-dependent manner. Thus, our work demonstrates that the endogenous AHR ligand ITE promotes the induction of active immunologic tolerance by direct effects on dendritic and T cells, and identifies nontoxic endogenous AHR ligands as potential unique compounds for the treatment of autoimmune disorders.
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ABSTRACT: Recent studies show that the aryl hydrocarbon receptor (AhR) is involved in immune responses. AhR is activated following interaction with its ligands, such as 6-formylindolo[3,2-b]carbazole (FICZ) and 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). In this study, we investigated the role of AhR activation by its endogenous ligands in the pathogenesis of ocular Behcet's disease (BD). The expression of AhR was significantly decreased in active BD patients as compared to inactive BD patients and normal controls. Both FICZ and ITE inhibited Th1 and Th17 polarization and induced the expression of IL-22 by PBMCs and by CD4(+)T cells in active BD patients and normal controls. Stimulation of purified CD4(+)T cells with FICZ or ITE caused a decreased expression of RORC, IL-17, IL-23R, and CCR6 and an increased phosphorylation of STAT3 and STAT5. The present study suggests that a decreased AhR expression is associated with disease activity in BD patients. The activation of AhR by either FICZ or ITE was able to inhibit Th1 and Th17 cell polarization. Further studies are needed to investigate whether modulation of AhR might be used in the treatment of BD.Mediators of Inflammation 01/2014; 2014:195094. · 3.88 Impact Factor
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ABSTRACT: Trung Nguyen and Taisuke Nakahama have contributed equally to this work. Aryl hydrocarbon receptor (AHR) is thought to be a crucial factor in the regulation of immune responses. Many AHR-mediated immunoregulatory mechanisms have been discovered, and this knowledge may enhance our understanding of the molecular pathogenesis of autoimmune inflammatory syndromes such as collagen-induced arthritis, experimental autoimmune encephalomyelitis, and experimental colitis. Recent findings have elucidated the critical link between AHR and indoleamine 2,3-dioxygenase (IDO) in the development of regulatory T cells and Th17 cells, which are key factors in a variety of human autoim-mune diseases. Induction of IDO and IDO-mediated tryptophan catabolism, together with its downstream products such as kynurenine, is an important immunoregulatory mecha-nism underlying immunosuppression, tolerance, and immunity. Recent studies revealed that induction of IDO depends on AHR expression. This review summarizes the most cur-rent findings regarding the functions of AHR and IDO in immune cells as they relate to the pathogenesis of autoimmune diseases in response to various stimuli. We also discuss the potential link between AHR and IDO/tryptophan metabolites, and the involvement of several novel related factors (such as microRNA) in the development of autoimmune dis-eases. These novel factors represent potential therapeutic targets for the treatment of autoimmune disorders. Aryl hydrocarbon receptor (AHR) is a ligand-activated member of the Per–Arnt–Sim (PAS) family of basic helix–loop–helix (HLH) transcription factors. AHR mediates cellular responses to toxins or its ligands, including TCDD, 6-formylindolo[3,2-b]carbazole (FICZ), kynurenine, and 2-(1 H-indole-3 -carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) (1–4). AHR forms an active complex in the cytoplasm with chaperone proteins such as heat shock protein 90 (HSP90), AHR-interacting protein (AIP), and p23 (5–7). Once bound to its ligands, the AHR complex translo-cates to the nucleus and binds AHR nuclear translocator (Arnt). The resultant AHR–Arnt heterodimers bind specific motifs, called dioxin-responsive elements (DREs), in the promoter region of tar-get genes. These targets, the so-called AHR battery genes, include CYP1A1, CYP1A2, CYP1B1, and other members of cytochrome P450 family (8–11). Several pathways are involved in the regula-tion of AHR, including proteasomal degradation of AHR, ligand metabolism by CYP1A1, and formation of the AHR–Arnt complex (12, 13). One of these pathways involves an inhibitory peptide. Mimura et al. isolated a cDNA clone that encode a polypeptide with high similarity to the sequence of the bHLH/PAS of AHR (14). This polypeptide can repress the transcriptional activity of AHR by competing with AHR for binding to Arnt and by binding to the enhancer sequence XRE, upstream of the AHRR gene; therefore, this peptide is designated AHR repressor or AHRR. Expression of AHRR is induced by the AHR/Arnt heterodimer through binding to XRE, resulting in feedback inhibition of AHR. In addition, sev-eral transcription factors can interact and regulate AHR signaling; these include STAT-1, STAT3, STAT5, Pai-2, Sp1, c-maf, and Bach2 in certain cell types (15–24). AHR is activated in many immune cell types, including T cells, B cells, NK cells, macrophages, and dendritic cells (DCs), as well as in epithelial cells, Langerhans cells, innate lymphoid cells, intraepithelial lymphocytes, and microglia (15, 16, 20, 21, 25–40). Depending on the presence of specific lig-ands, AHR activation may suppress or exacerbate experimental autoimmune diseases. For examples, TCDD and ITE can suppress experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS), whereas FICZ exacerbates disease devel-opment (17, 41–43). Differences in the stability and structure of these ligands, as well as their affinity for AHR, should be taken into account when considering their mode of action in the activa-tion of AHR. In addition, AHR seems to be regulated by unraveled factors such as transcription factors, tryptophan metabolites, feed-back regulation of the cytokine network, and microRNA (miR). Below, we will discuss in detail the factors that may interact with AHR to modulate immune responses.Frontiers in immunology. 11/2014; 5.
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ABSTRACT: IL-22 as a cytokine is described with opposing pro-inflammatory and anti-inflammatory functions. Cell regeneration, tissue remodelling and balance between commensal bacteria in the gut and host immune system are considered as anti-inflammatory features of IL-22, whereas production of IL-22 from Th17 cells links this cytokine to pro-inflammatory pathways. Th17 cells and group 3 innate lymphoid cells (ILC3) are two major producers of IL-22 and both cell types express ROR-ct and Aryl hydrocarbon receptor (AhR) transcription factors. Typically, the immune system cells are the main producers of IL-22. However, targets of this cytokine are mostly non-hematopoietic cells such as hepatocytes, keratinocytes, and epithelial cells of lung and intestine. Association of IL-22 with other cytokines or transcription factors in different cell types might explain its contrasting role in health and disease. In this review we discuss the regulation of IL-22 production by AhR- and IL-23-driven pathways. A clear understanding of the biology of IL-22 will provide new opportunities for its application to improve human health involving many debilitating conditions.Cytokine 01/2015; · 2.52 Impact Factor
An endogenous aryl hydrocarbon receptor ligand acts
on dendritic cells and T cells to suppress experimental
Francisco J. Quintana1, Gopal Murugaiyan, Mauricio F. Farez, Meike Mitsdoerffer, Ann-Marcia Tukpah, Evan J. Burns,
and Howard L. Weiner1
Center for Neurologic Diseases, The Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115
Edited by Hartmut Wekerle, The Max Planck Institute for Psychiatry, Martinsried, Germany, and accepted by the Editorial Board October 4, 2010 (received for
review June 26, 2010)
The ligand-activated transcription factor aryl hydrocarbon receptor
(AHR)participatesin the differentiation of FoxP3+Treg, Tr1cells, and
IL-17–producing T cells (Th17). Most of our understanding on the
role of AHR on the FoxP3+Tregcompartment results from studies
using the toxic synthetic chemical 2,3,7,8-tetrachlorodibenzo-p-di-
oxin. Thus, the physiological relevance of AHR signaling on FoxP3+
Tregin vivo is unclear. We studied mice that carry a GFP reporter in
the endogenous foxp3 locus and a mutated AHR protein with re-
duced affinity for its ligands, and found that AHR signaling partic-
ipates in the differentiation of FoxP3+Tregin vivo. Moreover, we
found that treatment with the endogenous AHR ligand 2-(1′H-in-
dole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) given
parenterally or orally induces FoxP3+Tregthat suppress experimen-
tal autoimmune encephalomyelitis. ITE acts not only on T cells, but
also directly on dendritic cells to induce tolerogenic dendritic cells
that support FoxP3+Tregdifferentiation in a retinoic acid-dependent
manner. Thus, our work demonstrates that the endogenous AHR
ligand ITE promotes the induction of active immunologic tolerance
by direct effects on dendritic and T cells, and identifies nontoxic
endogenous AHR ligands as potential unique compounds for the
treatment of autoimmune disorders.
FoxP3 control immune autoreactivity in healthy individuals
(1). FoxP3+Tregare generated in the thymus (natural Treg, nTreg)
and also in the periphery (induced Treg, iTreg). The importance of
FoxP3+Tregfor immunoregulation is highlighted by the immune
mice and humans (1). Conversely, the induction of FoxP3+Tregis
viewed as a promising approach for the treatment of immune-
mediated disorders (2).
We (3) and others (4–8) have found that the ligand-activated
transcription factor aryl hydrocarbon receptor (AHR) controls
the differentiation of Treg, Tr1 cells (9), and IL-17–producing T
cells (Th17) in vitro and in vivo. AHR activation by its high-af-
finity ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vivo
results in the expansion of the CD4+CD25+Foxp3+Tregcom-
partment (3). These CD4+CD25+Foxp3+Tregare functional and
suppress the development of experimental autoimmune enceph-
alomyelitis (EAE) (3), experimental autoimmune uveoretinitis
(7), and spontaneous autoimmune diabetes (10).
TCDD is a valuable tool to study the immunological effects of
AHR activation, but TCDD is not a natural AHR ligand and its
toxicity rules out its therapeutic use. Thus, it is not yet known
whether there is a physiological role for AHR in FoxP3+Treg,
and whether nontoxic AHR ligands exist which can expand
FoxP3+Tregin vivo to treat autoimmunity.
To address these questions, we used mice carrying a GFP re-
porter in foxp3 and a mutant AHR protein with reduced affinity
for its ligands. In addition, we investigated the effect and mech-
anisms of action of the nontoxic mucosal AHR ligand 2-(1′H-
indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE)
on FoxP3+Tregboth in vitro and in vivo in the model of EAE.
egulatory T cells (Treg) that express the transcription factor
AHR Activation by Endogenous Ligands Promotes the Differentiation
of FoxP3+iTreg. FoxP3+Tregare classified as FoxP3+nTreg(gener-
ated in the thymus) and FoxP3+iTreg(generated in the periphery)
(1). The gut-associated lymphoid tissue is a major physiological site
the thymus and mesenteric lymph nodes (MLN). AHR-d Foxp3gpf
mice carry a GFP reporter in the foxp3 gene (12), and harbor the
d allele of ahr (AHR-d), which codes for an AHR protein with re-
the frequency of FoxP3+Tregcells in the MLN of AHR-d Foxp3gfp
mice (Fig. S1A); no difference was detected in the frequency
of thymic FoxP3+Treg(Fig. S1B). WT and AHR-d Foxp3+Treg
showed comparable suppressive activity in vitro (Fig. S2).
To determine whether the reduced frequency of FoxP3+Tregin
the MLN of AHR-d Foxp3gfpmice resulted from an impaired
generation of FoxP3+iTregin vivo, we used a transfer model (14).
CD4+FoxP3:GFP−T cells from naive AHR-d Foxp3gfpor
Foxp3gfpmice were transferred to RAG-1-deficient mice, and
the reconstituted mice were immunized with MOG35–55(14). We
found a significant reduction in the frequency of CD4+FoxP3:
GFP+Tregin mice that received AHR-d FoxP3:GFP−T cells
(Fig. S1C). These results suggest a physiological role for endog-
enous AHR ligands in the generation of FoxP3+iTregin vivo.
Given our results on the role of AHR on FoxP3+iTregin vivo,
we analyzed the role of AHR on the differentiation of FoxP3+
iTregtriggered in vitro with TGF-β1 and IL-2 (3, 15). Naive CD4+
T cells from AHR-d Foxp3gfpmice showed a significant impair-
ment in their differentiation into FoxP3+Treg(Fig. S1D), with or
endogenous ligands in Tcells affects FoxP3+iTregdifferentiation.
We investigated the mechanism by which AHR signaling in T
cells might influence the development of FoxP3+iTreg. Stat1 acti-
vation interferes with the differentiation of Th17 (16) and FoxP3+
Treg(17). AHR interacts with Stat1 and limits its activation during
the differentiation of Th17 cells (5). Thus, we analyzed Stat1 phos-
mice activated with TGF-β1 and IL-2 for 48 h. We found increased
Stat1phosphorylation in naiveCD4+TcellsfromAHR-dFoxp3gfp
Author contributions: F.J.Q., G.M., M.F.F., M.M., A.-M.T., E.J.B., and H.L.W. designed re-
search; F.J.Q., G.M., M.F.F., M.M., A.-M.T., and E.J.B. performed research; F.J.Q. and M.M.
contributed new reagents/analytic tools; F.J.Q., G.M., M.F.F., M.M., A.-M.T., E.J.B., and
H.L.W. analyzed data; and F.J.Q., G.M., and H.L.W. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. H.W. is a guest editor invited by the Editorial
See Commentary on page 20597.
1To whom correspondence may be addressed. E-mail: email@example.com or
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| November 30, 2010
| vol. 107
| no. 48www.pnas.org/cgi/doi/10.1073/pnas.1009201107
mice (Fig. S1E); no changes were detected in the phosphorylation
of Stat5 (Fig. S3). Although an interaction between AHR and
Stat3 was not found by Kimura et al. (5), we also investigated Stat3
phosphorylation in Tregand Th17 cells differentiated from naive
in the phosphorylation of Stat3 in Treg(1.9% in WT vs. 1.1% in
the differentiation of FoxP3+iTreg.
AHR Activation by the Nontoxic Endogenous Ligand ITE Suppresses
EAE. The results above suggested that endogenous AHR ligands
function in vivo to induce FoxP3+iTreg. Several endogenous
AHRligandshave beendescribed. ITE,for example,isa nontoxic
mucosal AHR ligand (18). Based on the role of AHR on the
generation of FoxP3+iTreg, and the potential of FoxP3+Treg
expansion as a therapeutic approach for the management of au-
toimmune disorders (2), we investigated the ability of AHR ac-
tivation by ITE to induce functional FoxP3+Tregand treat EAE.
day of EAE induction suppressed EAE in WT B6 mice (Fig. 1A).
ITE did not suppress EAE in AHR-d mice, indicating that the
effects of ITE on EAE were mediated by AHR (Fig. 1A). Oral
EAE. Although the liver is highly sensitive to the toxic effects of
TCDD (19), ITE administration did not result in liver toxicity
(Table S1). Thus, ITE suppresses EAE by an AHR-dependent
mechanism that does not involve toxicity and is active orally.
study the suppression of EAE by AHR activation with ITE, we
MOG35–55. ITE treatment suppressed the recall proliferative re-
sponse to MOG35–55(Fig. 1C); no differences were seen upon
activation with mitogenic antibodies to CD3 (Fig. S4), indicating
that ITE treatment did not result in global immunosuppression.
Moreover, draining lymph node cells from ITE-treated mice se-
cretedhigher amounts ofTGF-β1 and IL-10and loweramounts of
IFN-γ and IL-17 upon activation with MOG35–55(Fig. 1D).
To investigate the therapeutic potential of ITE, we used the
relapsing-remitting model of EAE induced in SJL mice by im-
munization with PLP139–151. We initiated treatment with ITE on
day 17 (daily, 200 μg/mice, intraperitoneally), during the re-
mission that followed the first attack. Treatment with ITE re-
duced the disability in the relapse and the encephalitogenic
response to PLP139–151(Fig. 1 E and F). Thus, AHR activation by
ITE suppresses the encephalitogenic T-cell response and EAE in
both preventive and therapeutic experimental paradigms.
AHR Activation by the Nontoxic Endogenous Ligand ITE Expands the
FoxP3+Treg Compartment. We (3) and others (4, 5, 7, 10) have
shown that AHR activation in vivo can expand the FoxP3+Treg
compartment; thus, we investigated the effect of ITE on FoxP3+
Treg. We found that ITE administration (200 μg/mice, intra-
peritoneally) to Foxp3gfpmice starting on the day of EAE in-
duction increased the frequency of CD4+FoxP3:GFP+Treg,
mainly CD4+CD25+FoxP3:GFP+Treg(Fig. 2A). Similar results
were observed in SJL mice treated with ITE from day 17 after
EAE induction (Fig. S5). Hence, ITE expands the FoxP3+Treg
To further investigate the roleof AHRactivation by ITE on the
generation of Foxp3+iTregin vivo, we transferred CD4+Foxp3:
GFP−T cells from FoxP3gfpmice into RAG-1-deficient mice. The
ITE suppressesEAE. (A) EAEwasinducedinnaivewild-type B6
or AHR-d mice,andITE orvehicle as control was administered
i.p. daily from the day of immunization until the termination
of the experiment. The course of EAE is shown as the mean
EAE score + SEM. Shown are representative data of one of
three experiments that produced similar results. **P < 0.001
compared to control-treated WT mice or ITE treated AHR-
vehicle as control was administered orally daily from the day
course of EAE is shown as the mean EAE score + SEM. **P <
0.001 compared to control-treated WT mice. (C) Proliferative
response to MOG35-55of lymph node cells from ITE or control
treated animals 10 d after immunization with MOG35-55in
CFA. Cell proliferation is indicated as cpm + SD in triplicate
wells. *P < 0.05 and **P < 0.001 compared to cells from
control-treated mice. (D) Cytokine secretion triggered by
MOG35-55in lymph node cells from ITE or control treated
animals 10 d after immunization with MOG35-55in CFA. **P <
0.001 compared to cells taken from control-treated mice. (E)
was administered i.p. daily from day 17 after the immuniza-
tion till the termination of the experiment. The course of EAE
in these mice is shown as the mean EAE score + SEM. **P <
0.001 when compared to control-treated WT mice. (F) Pro-
liferative response to PLP139-151of splenocytes taken from ITE
or control treated SJL mice 30 days after immunization with
PLP139-151/CFA. Cell proliferation is indicated as cpm + SD in
triplicate wells. **P < 0.001 when compared to cells taken
from control-treated mice. Representative data of 1 of at
least 2 experiments that produced similar results.
AHR activation by the nontoxic endogenous ligand
Quintana et al.PNAS
| November 30, 2010
| vol. 107
| no. 48
reconstituted mice were immunized with MOG35–55in IFA (14)
and treated daily with 200 μg per mouse ITE for 1 wk. Three
weeks later the frequency of FoxP3:GFP+Tregwas analyzed by
FACS. ITE treatment led to a significant increase in the conver-
sion of CD4+Foxp3:GFP−donor T cells into CD4+Foxp3:GFP+
iTreg(Fig. 2B). Thus, the expansion of the FoxP3+Tregcom-
partment triggered by AHR activation with ITE is caused, at least
in part, by the induction of CD4+Foxp3+iTreg.
We then analyzed the effect of ITE on the induction of
MOG35–55-specific FoxP3+Treg. CD4+FoxP3:GFP+Tregfrom
ITE or control treated mice were studied for their ability to sup-
press the proliferative response of 2D2+CD4+CD62L+Foxp3:
GFP−T cells triggered by MOG35–55 and irradiated APCs.
Transgenic 2D2+T cells express a T-cell receptor reactive with
MOG35–55(20). We found that FoxP3:GFP+Tregfrom ITE-
treated mice show a significant increase in their ability to suppress
the proliferation of 2D2+CD4+CD62L+Foxp3:GFP−T cells
(Fig. 2C), suggesting that ITE expanded the MOG35–55-specific
FoxP3+Tregcompartment. Moreover, to further investigate the
CD4+Foxp3+Treg, we analyzed the recall response of Foxp3gfp
mice treated with ITE or vehicle. CD4+T cells from ITE-treated
mice showed a reduction in their proliferative recall response to
MOG35–55(Fig. 2D), which was abrogated by the removal of
FoxP3:GFP+T cells (Fig. 2D), suggesting that AHR activation by
ITE induces FoxP3+Treg that suppress the encephalitogenic
We performed adoptive transfer experiments to study the role
of Tregon the suppression of EAE by ITE administration. The
transfer of 5 × 106CD4+T cells from ITE-treated mice before
immunization with MOG35–55resulted in a significant suppres-
sion of EAE; CD4+T cells from control-treated mice had no
effect (Fig. 2E). The suppressive activity of CD4+T cells from
ITE-treated mice was lost when CD4+CD25+T cells were de-
pleted (Fig. 2E). Thus, AHR activation by ITE results in the
generation of CD4+Foxp3+Tregcells that suppress the enceph-
AHR Activation by the Nontoxic Endogenous Ligand ITE Induces
Tolerogenic Dendritic Cells. Dendritic cells (DCs) control the acti-
vation and polarization of T cells (21) and the FoxP3+Treg
compartment in vivo (22). Based on the reported expression of
functional AHR by DCs (4, 23, 24), we studied the effects of ITE
on DCs. To determine if DCs are affected by treatment with ITE
in vivo, we immunized WT mice with MOG35–55in complete
Freund’s adjuvant (CFA), treated them daily with ITE or vehicle
(administered intraperitoneally), and analyzed splenic DCs by
FACS 10 d after immunization. Splenic DCs from ITE-treated
mice (DCITE) showed slightly decreased CD86 expression and an
increased CD103 expression; no changes in the expression of
MHC-I, MHC-II, CD40, and CD80 or the size of the splenic DC
compartment were observed (Fig. 3A). We did find, however, that
IL-1β, IL-6, IL-12, IL-23, and osteopontin, and more TGF-β1 and
IL-10 (Fig. 3B). DCITEalso showed a decreased ability to trig-
ger the proliferation of naive 2D2+T cells with MOG35–55(Fig.
3C). In addition, naive 2D2+T cells activated with DCITEand
MOG35–55secreted lower amounts of IL-17 and IFN-γ, and more
IL-10 and TGF-β1 (Fig. 3D). Hence, treatment with the AHR
ligand ITE induces tolerogenic DCs in vivo.
FoxP3+Tregcan induce a tolerogenic phenotype in DCs (25).
Thus, it is possible that AHR activation by ITE in vivo modifies
the activity of DCs indirectly, as a result of the expansion of the
FoxP3+Treg compartment. To investigate if the tolerogenic
phenotype of DCITEwas a result of the direct effects of AHR
activation by ITE on DCs, we generated bone marrow-derived
DC (BM-DC) (26) and treated them with ITE 100 nM or vehicle
during the last 24 h of their differentiation. Treatment with ITE
did not affect the number or the phenotype of BM-DC at the
end of the culture (Fig. S6A). However, purified ITE-treated
CD11c+BM-DC (BM-DCITE) showed a decreased expression of
the proinflammatory cytokines IL-1β, IL-6, IL-12, IL-23, and
osteopontin, and a concomitant increase in the expression of
TGF-β1 and IL-10 (Fig. S6B). Moreover, BM-DCITEshowed
a reduced ability to activate 2D2 T cells with MOG35–55
and trigger the production of IL-17 or IFN-γ (Fig. S6 C and
D). Conversely, 2D2 T cells activated with BM-DCITE and
MOG35–55produced increased levels of IL-10 and TGF-β1 (Fig.
S6D). Taken together, these results demonstrate that AHR ac-
tivation by ITE in DC induces tolerogenic DC.
DCITEPromote the Differentiation of FoxP3+Tregby a Retinoic Acid-
Dependent Mechanism. DCs drive the differentiation of effector
(21) and regulatory cells (22, 27); thus, we investigated the role
of AHR on the differentiation of Th17 cells and FoxP3+iTregby
dogenous ligand ITE expands the FoxP3+Treg
compartment. (A) Frequency of CD4+Foxp3:
GFP+Tregin splenocytes from ITE- or control-
treated mice, 21 d after EAE induction. (B)
CD4+Foxp3:GFP–T cells from naive Foxp3gfp
were transferred into RAG-1 deficient hosts,
the recipients were immunized with MOG35–55
in IFA, treated daily with ITE for 1 wk, and the
frequency of CD4+FoxP3:GFP+Tregwas ana-
lyzed in the spleen 3 wk after immunization.
*P < 0.05 compared with mice transferred
with WT cells. (C) Suppressive activity of CD4+
Foxp3:GFP−Tregfrom ITE- or control-treated
Foxp3gfpmice coincubated with naive 2D2
Foxp3:GFP−T cells activated with MOG35–55
and irradiated APC. **P < 0.001 compared
with cells taken from control-treated mice. (D)
Recall response to MOG35–55of CD4+T cells
and CD4+Foxp3:GFP−T cells lymph node cells
from ITE- or control-treated Foxp3gfpmice
10 d after immunization with MOG35–55 in
CFA. Cell proliferation is indicated as cpm ±
SD in triplicate wells. *P < 0.05 and **P <
0.001 compared with cells taken from con-
trol-treated mice. (E) CD4+or CD4+CD25−T cells (5 × 106) were purified from ITE- or control-treated mice 10 d after immunization with MOG35–55in CFA and
transferred into naive mice. After 1 d, EAE was induced in the recipients with MOG35–55in CFA. The course of EAE in these miceis shown as the mean EAE score ±
SEM. **P < 0.001 compared with mice transferred with CD4+T cells from control-treated mice or CD4+CD25−T cells from ITE-treated cells. Representative data
of one of at least three experiments that produced similar results.
AHR activation by the nontoxic en-
| www.pnas.org/cgi/doi/10.1073/pnas.1009201107Quintana et al.
DCs. For these experiments, naive 2D2+CD4+CD62L+FoxP3:
GFP−T cells were activated with DCITEor control DCs in the
presence of MOG35–55or antibodies to CD3. We found that
DCITEshowed an increased ability to promote the differentia-
tion of FoxP3+iTregupon activation with MOG35–55or anti-
bodies to CD3 and TGF-β1 and IL-2 (Fig. 4A). Similarly, BM-
DCITE were more efficient at inducing the differentiation of
FoxP3+Tregand showed a significant decrease in their ability to
promote Th17 differentiation (Fig. 4B). These effects were me-
diated by the activation of AHR by ITE, as they were not ob-
served using BM-DCITEdifferentiated from AHR-d mice. Thus,
AHR activation by ITE induces tolerogenic DCs that promote
the differentiation of FoxP3+Treg.
It has been recently reported that DCs interfere with the de-
velopment of Th17 cells and promote the differentiation of
FoxP3+iTregby means of the production of retinoic acid (RA)
(11, 28, 29). The synthesis of RA is controlled by retinal dehy-
drogenases, which are encoded by members of the Aldh1a gene
family (30). To investigate the role of RA biosynthesis in the
tolerogenic phenotype of DCITE, we measured the expression of
aldh1a1 and aldh1a2 in DCITEby qPCR. DCITEwere found to
express significantly higher levels of aldh1a1, but no changes
toxic endogenous ligand ITE induces
tolerogenic DC. (A) FACS analysis
of splenic DC from ITE- (DCITE) or
control- (DC) treated mice. Numbers
indicate the percent of positive cells;
the staining obtained with isotype
control antibodies is shown in gray.
(B) Quantitative PCR analysis of cyto-
kine expression by DC or DCITE. *P <
0.05; **P < 0.01; and ***P < 0.001
compared with DC. (C and D) Naive
2D2+CD4+FoxP3:GFP−T cells were
stimulated with MOG35–55and DC or
DCITEand, and proliferation (C) and
cytokine secretion (D) was analyzed.
compared with T cells incubated with
control DC. Representative data of
one of at least three experiments that
produced similar results.
AHR activation by the non-
Tregdifferentiation by an RA-de-
pendent mechanism. (A) Naive
MOG35–55, and TGF-β1 + IL-2, and
the frequency of FoxP3:GFP+T
CD4+FoxP3:GFP−T cells were
stimulated with MOG35–55 and
control (BM-DC) or ITE-treated
BM-DC (BM-DCITE) derived from
WT or AHR-d mice, in the pres-
ence of TGF-β1 + IL-6 or TGF-β1 +
IL-2 and the frequency of IL-17+
T cells and FoxP3:GFP+T cells was
analyzed, respectively. (C) Quan-
titative PCR analysis of aldh1a2
expression by DC or DCITEfrom
WT or AHR-d mice; results are
presented relative to GAPDH
mRNA. **P < 0.001 compared
with DC from WT mice or DCITE
from AHR-d mice. (D) Naive 2D2+
CD4+FoxP3:GFP−T cells were
stimulated with DC or DCITE,
MOG35–55and TGF-β1 +IL-2, with
or without the specific inhibitor
of RA signaling LE135 (iRA). Rep-
DCITE promote FoxP3+
Quintana et al.PNAS
| November 30, 2010
| vol. 107
| no. 48
were detected on the expression of aldh1a2 (Fig. 5C). Similar
results were found when the expression of aldh1a1 and aldh1a2
was analyzed on BM-DCITEcells (Fig. S7). To investigate the
significance of RA in the ability of DCITEto promote the dif-
ferentiation of FoxP3+Treg, we used the specific inhibitor of RA
signaling LE135. We found that the ability of DCITEto induce
the differentiation of FoxP3+Tregwas significantly reduced in
the presence of LE135 (Fig. 4D); no effects were observed on the
ability of control DCs to promote the differentiation of FoxP3+
Treg. Taken together, these data demonstrate that AHR activa-
tion by ITE induces anti-inflammatory DCs that promote iTreg
differentiation via the secretion of RA.
Passive Transfer of BM-DCITE Suppresses EAE. To investigate the
relevance of the tolerogenic DCs induced by ITE treatment on
the suppression of EAE, we performed adoptive transfer
experiments. Control BM-DC and BM-DCITEwere incubated
for 1 h with MOG35–55and transferred ip to naive mice (2 × 106
per mouse); this procedure was repeated three times, once every
4 d. Four days after the last transfer of BM-DC, EAE was in-
duced. We found that the adoptive transfer of BM-DCITEfrom
WT mice resulted in a significant suppression of EAE (Fig. 5A);
no protective effects were observed when control BM-DC or
AHR-d BM-DCITEwere transferred (Fig. 5A). The suppression
of EAE by WT BM-DCITEcorrelated with the suppression of the
encephalitogenic response to MOG35–55 (Fig. 5B). BM-DC
and AHR-d BM-DCITEhad no effect on the recall response
to MOG35–55(Fig. 5B). Moreover, mice transferred with WT
BM-DCITEhad increased numbers of FoxP3+Tregthan recipi-
ents of wild type BM-DC or AHR-d BM-DCITE, and a concom-
itant reduction in the frequency of Th1 and Th17 cells (Fig. 5C).
Thus, the DCITEinduced by treatment with ITE contribute to
the expansion of FoxP3+Tregand the suppression of EAE.
The induction of FoxP3+Tregis viewed as a promising approach
for the treatment of human autoimmune disorders (2). Several
methods have been described to differentiate and expand human
FoxP3+Treg in vitro, but their ability to produce significant
numbers of functional FoxP3+Tregin a consistent manner is
limited (31). Thus, strategies aimed at the induction of functional
FoxP3+Tregin vivo are more likely to be translated into clinical
practice. We and others have shown that AHR activation indu-
ces functional FoxP3+Tregthat suppress the development of
experimental autoimmunity and transplant rejection (3, 4, 7, 10);
thus, AHR is an attractive target for the induction of functional
FoxP3+Treg. However, to date, studies on the effect of AHR
ligands in models of autoimmunity have mostly focused on
TCDD, a synthetic toxin. Because of its lack of toxicity (32), the
endogenous AHR ligand ITE is a potential compound to induce
functional FoxP3+Tregin vivo and treat autoimmune diseases.
We found an impaired ability of AHR-d CD4+FoxP3−T cells
to differentiate into FoxP3+iTregboth in vitro and in vivo. The
mutant AHR protein in AHR-d Foxp3gfpmice displays a signif-
icant reduction in its affinity for AHR ligands (13). Thus, the
reduced differentiation of AHR-d CD4+FoxP3−T cells into
FoxP3+iTregsuggests that AHR activation in T cells by endog-
enous ligands plays a physiological role in the differentiation of
FoxP3+iTreg. This interpretation is in agreement with the im-
paired differentiation of AHR-deficient CD4+T cells into
FoxP3+iTregdescribed by Kimura et al. (5). The specific AHR
ligands that modulate T-cell differentiation in vitro and in vivo
are, however, still largely unknown.
AHR limits the activation of Stat1 during Th17 differentiation
Stat1, AHR interferes with the antagonistic activity of Stat1 sig-
naling on FoxP3+Tregdifferentiation. Note that Stat1 activation
interferes with the differentiation of iTreg(17, 33) but not nTreg
(33); indeed, several cytokines, signaling pathways, and genomic
elements have differential contributions to the generation of iTreg
and nTreg(34, 35). The control of Stat1 activation by AHR might
preferentially favor the differentiation of FoxP3+iTregduring an
active immune response by interfering with IFN-γ signaling. To-
Tregsurvival (4), its effects on Stat1 activation might participate in
the control of immunopathology during the immune response.
AHR activation has been shown to modulate the function and
maturation of DCs (4, 23, 24) and macrophages (36, 37). Indeed,
AHR in macrophages limits LPS-induced inflammation (36, 37),
and AHR in DCs mediates the anti-inflammatory activities of
lipoxin A4 (23). We found that the AHR ligand ITE induces DCs
that promote the differentiation of FoxP3+Treg in a RA-
dependent manner. Mucosal CD103+DC promote the differen-
tiation of FoxP3+Tregvia RA (11, 28, 29), and RA (38) and IL-10
(39) also have autocrine anti-inflammatory effects on DCs. Thus,
our results support a model in which AHR activation induces
tolerogenic DCs that promote the generation of FoxP3+Tregvia
the production of RA and the concomitant down-regulation of
proinflammatory cytokines that interfere with FoxP3+Tregdif-
ferentiation (12, 14). Moreover, it is possible that under physio-
logical conditions, endogenous AHR ligands participate in the
DCITE suppresses EAE. (A) Naive
mice received BM-DC and BM-DCITE
(2 × 106per mouse, three times
every 4 d), derived from WT or
AHR-d mice and EAE was induced.
The course of EAE is shown as the
mean EAE score ± SEM. **P <
0.001 compared with mice trans-
ferred with BM-DC from WT mice
or BM-DCITEfrom AHR-d mice. (B)
Recall response to MOG35–55 in
splenocytes 21 d after EAE in-
duction. Cell proliferation is in-
dicated as cpm ± SD in triplicate
wells. **P < 0.001 compared with
cells from mice transferred with
BM-DC from WT mice or BM-DCITE
from AHR-d mice. (C) Frequency
of CD4+Foxp3+Tregin splenocytes
21 d after EAE induction, and fre-
and CD4+IFN-γ+T cells in splenocytes 21 d after EAE, following activation with MOG35–55for 5 d. Representative data of one of at least two
experiments that produced similar results.
Passive transfer of BM-
| www.pnas.org/cgi/doi/10.1073/pnas.1009201107 Quintana et al.
development of the mucosal CD103+DC that promote the dif-
ferentiation of FoxP3+iTreg.
In conclusion, our work demonstrates that the endogenous
AHR ligand ITE, given either orally or parenterally, acts on DCs
and T cells to promote the induction of functional FoxP3+Treg
that suppress EAE. We recently reported that AHR activation
promotes the differentiation of suppressive human regulatory T
cells in vitro (40). Thus, nontoxic endogenous AHR ligands like
ITE are potential new compounds for the treatment of auto-
Materials and Methods
Mice and Reagents. Foxp3gfpknock-in mice have been described (12). C57BL/
6-, AHR-d–, SJL-, and RAG-1–deficient mice were purchased from The Jack-
son Laboratories. All experiments were carried out in accordance with the
guidelines of the standing committee of animals at Harvard Medical School.
ITE was purchased from Sigma-Aldrich and from Tocris Bioscience.
EAE Induction. EAE was induced by subcutaneous immunization with 100 μg
of MOG35–55peptide or 50 μg of PLP139–151peptide (HSLGKWLGHPDKF) as
T-Cell Differentiation in Vitro. Naive CD4+CD62LhighCD44lowFoxp3:GFP−
T cells were stimulated for 5 to 6 d (3) using plate-bound antibody to CD3
(145-2C11, 1 μg/mL) plus soluble antibody to CD28 (PV-1, 2 μg/mL) and hu-
man TGF-β1 (3 ng/mL, unless otherwise indicated) and mouse IL-2 (50 units/
mL) or mouse IL-6 (30 ng/mL). Alternatively, naive CD4 T cells were activated
with BM-DC or purified DC at a 5:1 T cell-to-DC ratio, and activated with
soluble antibody to CD3 (0.5 μg/mL) or MOG35–55(20 μg/mL).
Adoptive Transfer Experiments. RAG-1 deficient mice received 1 × 106FACS-
sorted CD4+Foxp3−T cells. One month after transfer, host mice were
checked for reconstitution of CD4+T cells, immunized with MOG35–55in IFA,
and 3 wk later, Foxp3:GFP expression was tested in splenocytes by FACS.
Real-Time PCR. Real-time PCR was performed as described (3). All values were
expressed relative to the expression of GAPDH.
Cell Proliferation and Cytokine Production. Cells were cultured in serum-free
X-VIVO 20 media (BioWhittaker) and cell proliferation and cytokine pro-
duction were analyzed as described (3).
FACS. For intracellular cytokine staining, cells were stimulated with PMA (50
ng/mL) (Sigma-Aldrich), ionomycin (1 μg/mL) (Calbiochem), and GolgiStop
(BD Biosciences) for 4 h and stained, as described (3). For the analysis of Stat
phosphorylation, T cells were activated with plate bound antibodies to CD3
and CD28, with or without the addition of cytokines, and 48 h after the
initiation of the cultures the cells were stained with antibodies to Stat1 or
Stat5, following the manufacturer’s instructions (BD Biosciences).
IL-4 (10 ng/mL) and GM-CSF (10 ng/mL). On day 6, the cells were treated with
ITE or vehicle, and 24 h later, they were analyzed by FACS or purified with
ACKNOWLEDGMENTS. We thank Deneen Kozoriz for the FACS sorting. This
work was supported by Grants AI435801 and NS38037 from the National
Institutes of Health (to H.L.W.), Grants 1K99AI075285 from the National
Institutes of Health and RG4111A1 from the National Multiple Sclerosis
Society (to F.J.Q.), and Grant MI 1221/1-1 from the Deutsche Forschungsge-
meinschaft (to M.M.).
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Quintana et al. PNAS
| November 30, 2010
| vol. 107
| no. 48