Differential Regulation of Foxp3 and IL-17 Expression in CD4
T Helper Cells by IRAK-11
Urmila Maitra,* Sarah Davis,* Christopher M. Reilly,†and Liwu Li2*
Host immune responses are finely regulated by the opposing effects of Th17 and T regulatory (Treg) cells. Treg cells help to
dampen inflammatory processes and Th17 cells facilitate various aspects of immune activation. The differentiation of Th cells
depends on a unique combination of stimulants and subsequent activation of diverse transcription factors. In particular, coop-
erative activation of NFAT and Smad3 leads to the induction of Treg cells, and cooperation among STAT3 and Smad3 switches
to the induction of Th17 cells. We have previously shown that the IL-1 receptor associated kinase 1 (IRAK-1) selectively activates
STAT3 and inactivates NFAT. Physiological studies have shown that IRAK-1?/?mice are protected from developing various
inflammatory diseases, including experimental autoimmune encephalomyelitis and atherosclerosis with unknown mechanism. In
this study, we demonstrate that IRAK-1 plays a critical modulatory role in the differentiation of Th17 and Treg cells. Following
stimulation with TCR agonists and TGF?, IRAK-1?/?CD4 Th cells display elevated nuclear NFATc2 levels and increased
interaction of NFATc2 and Smad3, resulting in increased expression of Foxp3, a key marker for Treg cells. IRAK-1?/?mice have
constitutively higher populations of Treg cells. In contrast, when stimulated with TCR agonists together with IL-6 and TGF-?,
IRAK-1?/?CD4 Th cells exhibit attenuated STAT3 Ser727 phosphorylation and reduced expression of IL-17 and ROR?t com-
pared with wild-type cells. Correspondingly, IRAK-1 deletion results in decreased IL-17 expression and dampened inflammatory
responses in acute and chronic inflammatory mice models. Our data provides mechanistic explanation for the anti-inflammatory
phenotypes of IRAK-1?/?mice. The Journal of Immunology, 2009, 182: 5763–5769.
expressing inhibitory T regulatory (Treg)3cells (1–4). Th1 cells
are responsible for the cell-mediated elimination of intracellular
pathogens, whereas promotion of humoral and allergic response is
largely dependent on Th2 responses. The significance of Th17
cells is increasingly recognized due to their involvement in organ-
specific autoimmunity and numerous inflammatory complications.
Elevated levels of IL-17 are associated with the pathogenesis of
multiple sclerosis, atherosclerosis, and diabetes (5). In contrast,
Treg cells play a central role in dampening various inflammatory
effects of effector Th cells and are beneficial for the resolution of
inflammatory processes (6, 7).
The counteractions of Th17 and Treg cells are not only reflected
in their opposing immune modulatory functions, but also during
their differentiation processes. Recent studies have identified reti-
D4?Th cells play a crucial role in mediating host in-
flammatory responses. Naive CD4?T cells can differ-
entiate into Th1, Th2, Th17 effector cells, or the Foxp3
noic acid-related orphan receptor ?t (ROR?t), a member of the
nuclear hormone receptor super family, as a key transcription fac-
tor driving the Th17 differentiation program (8). In contrast, the
Treg cells are defined by the expression of the forkhead family
transcription factor, Foxp3. TGF? is involved in the differentiation
of both Th17 and Treg cells (9, 10). However, in the case of Treg
differentiation, TGF?-induced transcription factor Smad3 cooper-
ates with NFAT to induce the expression of Foxp3 (11). In con-
trast, TGF? and IL-6 trigger the coordinated activation of Smad3
and STAT3 to induce the transcription factor ROR?t necessary for
Th17 differentiation (4).
The activation statuses of NFATc2 and STAT3 are controlled
by signal-dependent phosphorylations mediated by upstream ki-
nases and phosphatases (12, 13). Maximal activation of STAT3
requires phosphorylation at both Tyr705 and Ser727 sites (14).
Although JAK2 kinase is shown to be responsible for phosphor-
ylating STAT3 at Tyr705, we have shown that IL-1 receptor
associated kinase 1 (IRAK-1) is capable of selectively phosphor-
ylating STAT3 at Ser727 (15). Regarding NFAT, calcium-depen-
dent phosphatase calcineurin dephosphorylates and activates
NFAT. We have recently demonstrated that IRAK-1 is responsible
for phosphorylating NFAT and maintaining NFAT in an inactive
state (16). We therefore hypothesize that IRAK-1 may facilitate
the differentiation of Th17 and dampen the induction of Treg cells
by affecting the activation status of STAT3 and NFAT. Our hy-
pothesis is further supported by previous functional studies using
IRAK-1?/?mice, which are protected from diverse inflammatory
diseases such as experimental autoimmune encephalomyelitis
(EAE), atherosclerosis, and septic shock (16–19).
In this study, we examined the expression of Foxp3, IL-17A,
and ROR?t in wild-type and IRAK-1?/?CD4 T cells. Mechanis-
tically, the activation status of STAT3 and NFATc2 following
TGF-? or IL-6 plus TGF-? were evaluated. We found that IRAK-
1?/?CD4 T cells have decreased STAT3 phosphorylation at
Ser727 and decreased expression of ROR?t and IL-17A following
*Laboratory of Innate Immunity and Inflammation, Department of Biology, Virginia
Polytechnic Institute and State University and†Virginia College of Osteopathic Med-
icine, Blacksburg, VA 24061
Received for publication January 14, 2009. Accepted for publication February
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by a research grant from the National Institutes of Health
AI064414 (to L.L.).
2Address correspondence to: Liwu Li, Life Science 1 Building, Washington Street,
Department of Biology, Virginia Polytechnic Institute and State University, Blacks-
burg, VA 24061. E-mail address: email@example.com
3Abbreviations used in this paper: Treg cells, T regulatory cells; ChIP, chromatin
immunoprecipitation; EAE, experimental autoimmune encephalomyelitis; IRAK-1,
interleukin-1 receptor associated kinase 1; PKC, protein kinase C; ROR?t, retinoic
acid-related orphan receptor ?t.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
TGF?1 and IL-6 treatment. In contrast, we observed that IRAK-
1?/?CD4 T cells have elevated levels of nuclear NFATc2 and
increased expression of Foxp3 upon TGF-? treatment. IRAK-1?/?
mice also exhibit constitutively elevated levels of CD4?CD25?
Foxp3?Treg cell population. In contrast, IRAK-1?/?mice chal-
lenged with various inflammatory agents exhibit reduced IL-17
production, leading to alleviated inflammatory symptoms. Our
study reveals a novel contribution of IRAK-1 to the differentiation
of Th17 and Treg cells, with significant implications in the patho-
genesis of diverse inflammatory diseases.
Materials and Methods
The Ab against Smads and STAT3 (total and phosphorylated) was obtained
from Cell Signaling Technology. Anti-laminB, ?-actin, and NFAT-c2 Abs
were purchased from Santa Cruz Biotechnology. Recombinant TGF?-1
was purchased from R&D Systems and IL-6 was obtained from BD Bio-
sciences. The TCR agonists, anti-mouse CD3 and CD28, were purchased
from BD Biosciences. LPS (E. coli O11:B4) was obtained from
Wild-type C57BL/6 mice were obtained from Charles River Laboratories.
IRAK-1?/?mice on C57BL/6 background were provided by Dr. James
Thomas (University of Texas Southwestern Medical School, Dallas, TX).
ApoE?/?/IRAK-1?/?mice were obtained by breeding ApoE?/?mice
(The Jackson Laboratory) with IRAK-1?/?mice. All mice were bred and
housed at Derring Hall Animal Facility at Virginia Polytechnic Institute
and State University, in compliance with approved Animal Care and Use
Western blot analysis and immunoprecipitation assays
Isolation of whole cell lysates and cytoplasmic and nuclear extracts were
performed as described previously (20). Briefly, cells were rinsed in PBS
and subsequently lysed on ice in the lysis buffer (10 mM HEPES, pH 7.9,
1.5 mM MgCl2, 10 mM KCl, 0.5 mM EDTA, 0.5 mM DTT, 0.5 mM
PMSF, 1 ?g/ml leupeptin, 1 ?g/ml pepstatin) for 30 min, followed by
addition of 10% Triton X-100. The samples were centrifuged for 10 min at
5000 rpm and the supernatant fractions were transferred and saved as cy-
toplasmic extracts. Pellets containing the intact nuclei were lysed and sol-
ubilized with the high salt buffer (20 mM HEPES, pH 7.9, 1.5 mM MgCl2,
0.4 M NaCl, 0.2 mM EDTA, 0.5 mM DTT, 1 mM PMSF) for 30 min on
ice followed by centrifugation at maximum speed for 20 min. The super-
natant was saved as the nuclear extract. Western blotting analysis of the
protein samples were performed as described previously (21). Immuno-
blots were developed by using the Amersham ECL Plus Western Blotting
Chemiluminescent Detection System (GE Healthcare).
For immunoprecipitations, the cell lysates were treated with 2 ?g pri-
mary Ab or normal IgG in the presence of 35 ?l of protein AG plus agarose
(Santa Cruz Biotechnology) and incubated on a rotating wheel at 4°C over-
night. The samples were centrifuged briefly and the pellets were washed
three times in immunoprecipitation buffer containing protease inhibitors.
The bound proteins were eluted from the agarose beads by boiling with 2?
Laemmli sample buffer for 5 min. The eluted proteins were loaded on a
7.5% SDS-polyacrylamide gel for electrophoresis followed by Western
blot analysis and immunoblotting with the indicated Abs.
Splenocytes were harvested from wild-type and IRAK-1?/?mice as pre-
viously described (20). Harvested splenocytes were stained with PE-rat
anti-mouse CD4 and allophycocyanin-rat anti-mouse CD25. Cells were
subsequently fixed and permeabilized with Cytofix/Cytosper solution from
BD Biosciences, and stained with PE-conjugated anti-mouse Foxp3 Ab
according to the manufacturer’s protocol. Flow cytometry analyses were
performed on a FACSCanto cytometer and data were analyzed using the
FACSDiva software (both from BD Biosciences).
In vitro CD4 T cell differentiation
Naive CD4 T cells were isolated from the spleen of wild-type and IRAK-
1?/?mice using the MACS CD4 Microbeads (Miltenyi Biotec), according
to the manufacturer’s protocol, and differentiated in vitro. For Th17 dif-
ferentiation, the cells were incubated with plate-bound anti-CD3 (5 ?g/ml)
mAbs and soluble anti-CD28 (5 ?g/ml) in the presence of TGF?1 (5 ng/
ml) and IL-6 (20 ng/ml) at 37°C for 3 days. For Treg differentiation, the
naive CD4 T cells were incubated with TGF?1 (5 ng/ml) in the presence
of plate-bound anti-CD3 mAbs and anti-CD28 at 37°C for 3 days. Fol-
lowing differentiation the cells were washed with PBS and used for sub-
sequent experiments as indicated.
Real time RT-PCR
Total RNAs were prepared from either untreated or treated CD4 Th17 or
Treg cells using Trizol (Invitrogen) according to the manufacturer’s pro-
tocol. Reverse transcription was conducted using the High-Capacity cDNA
reverse transcription kit (Applied Biosystems) and subsequent real-time
PCR analyses were performed using the SYBR green supermix on an IQ5
thermocycler (Bio-Rad). The relative levels of Foxp3, ROR?t, and IL17A
transcripts were calculated using the ??Ct method after normalizing with
GAPDH as the internal control. The relative levels of mRNAs in untreated
wild-type cells were adjusted to 1 and served as the basal reference value.
Chromatin immunoprecipitation (ChIP) assays
The isolated splenocytes from wild-type and IRAK-1?/?mice were either
untreated or treated with TGF? (5 ng/ml) alone or in combination with
IL-6 (20 ng/ml), followed by cross-linking with 1% formaldehyde in RPMI
1640 complete medium for 15 min with gentle rocking at room tempera-
ture. Cells were then washed twice with ice-cold PBS and treated with
glycine solution for 5 min to stop the cross-linking reaction. Cells were
then lysed in buffer containing SDS and protease inhibitor mixture. Sam-
ples were sonicated six times with 30-s pulses at 4°C followed by centrif-
ugation to collect the sheared chromatin. The sheared chromatin was used
to set up immunoprecipitation reactions with the indicated Abs using the
CHIP-IT Express kit (Active Motif) as per manufacturer’s recommenda-
tions. The immunoprecipitated DNA fragments were analyzed by PCR
using primers spanning the IL-17A or Foxp3 promoter regions.
Acute and chronic inflammatory treatments
Acute treatment. Wild-type and IRAK-1?/?mice of matched gender
and age were injected with LPS (E. coli O11:B4; Sigma-Aldrich) (25
mg/kg) or PBS i.p. Total blood was drawn 6 hours after the injection.
Plasma was collected and diluted 1/5 in sample diluent (Bio-Plex Di-
luent kit no. 171-305-008; Bio-Rad). Cytokine levels were assayed us-
ing a multiplex bead-based immunoassay as described by the manufac-
turer’s protocol (Bio-Rad).
High-fat diet feeding and analyses. ApoE?/?and ApoE?/?/IRAK-1?/?
mice of matched gender and age were fed with a Western Diet (TD.94059;
Harlan Laboratories) for 3 mo. Plasma levels of IL-17 were measured as
Statistical significance was determined using the unpaired two-tailed Stu-
dent’s t test. Values of p less than 0.05 were considered statistically
Elevated induction of Foxp3 in IRAK-1?/?CD4?T cells
following stimulation with TGF?
To test whether IRAK-1 is involved in the induction of Foxp3 Treg
cells in vitro, we treated CD4?T cells harvested from wild-type
and IRAK-1?/?splenocytes with plate-bound anti-CD3 Ab and
soluble anti-CD28 Ab in the presence or absence of TGF?. Three
days after stimulation, total RNAs were harvested and the relative
levels of Foxp3 expression were determined by real-time RT-PCR.
As shown in Fig. 1A, the levels of expressed Foxp3 message were
three times higher in IRAK-1?/?CD4?T cells compared with
wild-type CD4?T cells.
We further studied the CD4?CD25?Foxp3?T regulatory cell
populations in vivo. Splenocytes were harvested from age and gen-
der equivalent wild-type and IRAK-1?/?mice and used to per-
form flow cytometry analyses. As shown in Fig. 1B, IRAK-1?/?
mice have significantly higher levels of CD4?CD25?Foxp3?T
cells when compared with wild-type mice (p ? 0.05). There
was no significant difference regarding the frequencies of
CD4?CD25?T cells between wild-type and IRAK-1?/?mice
(data not shown). This is consistent with the established notion
5764DIFFERENTIAL INDUCTION OF Foxp3 AND IL-17 BY IRAK-1
that CD25 expression is not the definitive marker for Treg cells
in both mice and humans. Instead, Foxp3 is the most pertinent
indicator for Treg cells. Our data explain previous findings
demonstrating that IRAK-1?/?mice have attenuated inflamma-
tory symptoms, including EAE and atherosclerosis (17–19, 22).
Therefore, the higher percentage of Foxp3-expressing Treg
cellsmight explainthe protective
Elevated NFATc2 levels contribute to increased Foxp3
induction in IRAK-1?/?CD4?T cells
The transcription of Foxp3 is induced by the cooperative action of
Smad3 and NFAT transcription factors in response to TGF? (11).
We have previously shown that IRAK-1 attenuates the transcrip-
tional activity of NFAT in both macrophages and fibroblasts (16).
IRAK-1 phosphorylates NFAT at its Ser-Pro-rich motifs and helps
to maintain NFAT in an inactive form (16). To further examine the
molecular mechanism underlying the elevated Foxp3 gene expres-
sion in IRAK-1?/?CD4?T cells, we examined the nuclear levels
of NFATc2 in wild-type and IRAK-1?/?CD4?T cells. As shown
in Fig. 2A, TCR activation plus TGF? treatment induced signifi-
cantly higher nuclear levels of NFATc2 in IRAK-1?/?CD4?T
cells compared with the wild-type cells. The elevated nuclear
NFATc2 levels in IRAK-1?/?cells can also be observed with
TCR activation alone (Fig. 2B).
We further examined the nuclear levels of Smad2 and Smad3
in wild-type and IRAK-1?/?CD4?T cells. TGF? treatment
equally induced the nuclear accumulation of Smad2 and Smad3
in wild-type and IRAK-1?/?cells (Fig. 2C). Our data demon-
strate that IRAK-1 is preferentially involved in modulating
TCR-induced NFATc2 levels, instead of TGF?-induced Smad3.
Because NFAT cooperates with Smad3 in promoting the ex-
pression of Foxp3 (11), we subsequently examined the interac-
tion between NFATc2 and Smad3 in wild-type and IRAK-1?/?
cells. As shown in Fig. 2D, TCR ligation and TGF? treatment
induced significantly elevated interaction between Smad3 and
NFATc2 in IRAK-1?/?cells as compared with the wild-type
We also examined the association of Smad3 with the endog-
enous Foxp3 promoter region using the ChIP assay. As shown
in Fig. 2E, TCR ligation and TGF? treatment induced signifi-
cantly higher levels of Smad3 association with the Foxp3 pro-
moter region in IRAK-1?/?CD4?T cells. Taken together, al-
though IRAK-1 is not affecting the total nuclear levels of
Smad2/3 proteins, IRAK-1 deletion indirectly contributes to in-
creased Smad3 binding with Foxp3 promoter through increas-
ing the nuclear levels of NFATc2.
sion. A, Naive CD4 T cells isolated from wild-type and IRAK-1-deficient
mice were stimulated with anti-CD3 and anti-CD28 for 3 days in the pres-
ence or absence of TGF?. Total RNA was isolated and expression of
Foxp3 was determined by real-time RT-PCR. Data are representative of
three independent experiments and expressed as mean ? SEs. ??, p ?
0.01. B, Splenocytes were isolated from wild-type and IRAK-1-deficient
mice and stained with PE-anti-mouse CD4, allophycocyanin-anti-mouse
CD25, and anti-mouse Foxp3 Abs, followed by flow cytometry analyses.
The results shown are representative of three separate experiments and
expressed as mean ? SD. n ? 4. ?, p ? 0.05. WT, Wild type.
Loss of IRAK-1 enhanced TGF?-induced Foxp3 expres-
T cells contributes to higher induction of Foxp3. A, Naive CD4 T cells
isolated from wild-type and IRAK-1-deficient mice were activated with
anti-CD3 and anti-CD28 for 24 h in the presence or absence of TGF?.
Nuclear lysates from indicated conditions were separated by SDS-PAGE
gel and NFATc2 levels were detected by immunoblotting. The same blots
were probed with LaminB-specific Abs as a marker for loading control. B,
The expression of NFATc2 was analyzed by immunoblotting following
stimulation with TCR agonists for the indicated time points. The same
samples were probed for LaminB as a loading control, C, Naive CD4 T
cells were stimulated with TCR agonists with or without TGF? as specified
and the nuclear distribution of Smad2 and Smad3 were analyzed by im-
munoblotting. Data are representative of at least three separate experi-
ments, D, CD4 T cells were stimulated with anti-CD3 and anti-CD28 in the
presence of TGF? as indicated. The lysates were immunoprecipitated (IP)
with anti-Smad3 followed by immunoblotting (IB) with NFATc2-specific
Abs. The arrowhead points to the specific bands. The specific location of
Ig heavy (IgGH) is indicated. E, Chromatin immunoprecipitation assays
were performed in unstimulated cells or cells stimulated with TGF? plus
anti-CD3 and anti-CD28 using Smad3-specific Abs. NTC, No template
control. The input DNA was used as the loading control. Data are repre-
sentative of three independent experiments.
Increased nuclear NFATc2 expression in IRAK-1-deficient
5765 The Journal of Immunology
Decreased expression of IL-17 and ROR?t in IRAK-1?/?
CD4?T cells following stimulation with TGF? and IL-6
To determine the role of IRAK-1 in the induction of Th17 cells, we
stimulated CD4?T cells harvested from wild-type and IRAK-
1?/?splenocytes with plate-bound anti-CD3 Ab and soluble anti-
CD28 in the presence or absence of TGF? plus IL-6. Three days
after stimulation, total RNAs were harvested and the relative levels
of IL-17 and ROR?t were determined by real-time RT-PCR. As
shown in Fig. 3, the levels of induced IL-17A and ROR?t were
significantly higher in wild-type cells compared with IRAK-1?/?
cells (p ? 0.05).
IRAK-1?/?T cells have decreased IL-6-stimulated STAT3
Th17 cells are generated by the cooperative action of the cytokines
TGF? and IL-6 (4, 23, 24). IL-6 acts through the activation of
STAT3 to induce the expression of IL-17A as well as the tran-
scription factor ROR?t (8). ROR?t further contributes to the dif-
ferentiation and stable maintenance of Th17 cells. We have pre-
viously shown that IRAK-1 phosphorylates STAT3 at its Ser727
site and contributes to the full activation of STAT3 (15). To elu-
cidate further the role of STAT3, we determined the levels of
activated STAT3 in CD4 T cells isolated from wild-type and
IRAK-1?/?mice in response to IL-6 and TGF?. As shown in Fig.
4A, STAT3 Ser727 phosphorylation induced by IL6 and TGF?
was significantly attenuated in IRAK-1?/?cells. Furthermore,
consistent with our previous finding, IRAK-1 deletion did not af-
fect STAT3 Tyr705 phosphorylation induced by IL-6 (Fig. 4A).
To define the functional implication for decreased STAT3
Ser727 phosphorylation in IRAK-1?/?cells, we examined the
binding of STAT3 with the endogenous IL-17 promoter region
through ChIP assay. As shown in Fig. 4B, compared with wild-
type cells, IRAK-1?/?cells have significantly decreased levels of
STAT3 bound with the promoter region of IL-17.
Decreased IL-17 expression and attenuated inflammatory
responses in IRAK-1?/?mice
IL-17 plays a key role in exacerbating various forms of inflam-
matory responses (5). Although previous studies have demon-
strated that IRAK-1 is associated with the pathogenesis of various
inflammatory diseases, including septic shock, EAE, and athero-
sclerosis, there has been no report regarding the induced levels of
IL-17 in IRAK-1?/?mice under various inflammatory conditions
(18, 19, 22). Based on our mechanistic studies and other pheno-
typic findings, we hypothesize that IRAK-1 deletion would atten-
uate IL-17 expression in vivo. To test this, we set up several acute
and chronic inflammatory animal studies. As shown in Fig. 5A,
acute LPS injection capable of inducing septic shock significantly
induced IL-17 plasma levels in wild-type mice. In sharp contrast,
the levels of IL-17 in IRAK-1?/?mice following the same dose of
LPS injection were significantly lower (p ? 0.05). This correlated
with higher survival rate in IRAK-1?/?mice following LPS in-
oculation (data not shown).
Furthermore, we evaluated the plasma levels of IL-17 in
ApoE?/?and ApoE?/?/IRAK-1?/?mice fed with high-fat diet
for 3 months. As we have previously reported, ApoE?/?mice fed
with high-fat diet developed severe atherosclerosis whereas
ApoE?/?/IRAK-1?/?mice had significantly lower atherosclerotic
plaques (16). In our current study, we found that the levels of
plasma IL-17 in ApoE?/?mice were significantly elevated fol-
lowing high-fat diet feeding (Fig. 5B). In contrast, the levels of
IL-17 in ApoE?/?/IRAK-1?/?mice were significantly lower (p ?
0.01). These results suggest that IRAK-1 plays a major role in
T cells in response to IL6 and TGF?1. A, Naive CD4 T cells from wild-
type (WT) and IRAK-1-deficient mice were stimulated with anti-CD3 and
anti-CD28 for 3 days with or without TGF?1 together with IL-6 as indi-
cated. Expression of IL-17A mRNA was analyzed by real-time RT-PCR.
Results represent the mean ? SD of three independent samples. B,
CD4?T cells were activated with anti-CD3 and anti-CD28 mAbs for 3
days in the presence or absence of TGF?1 together with IL-6. Relative
mRNA levels of ROR?t were examined by real-time RT-PCR. Results
are expressed as mean ? SD from three independent experiments. ?,
p ? 0.05; ??, p ? 0.01.
Decreased induction of IL-17A and ROR?t in IRAK-1?/?
CD4 T cells isolated from wild-type (WT) and IRAK-1-deficient mice. A,
Naive CD4 T cells were stimulated with TCR agonists in the presence or
absence of TGF? and IL-6. Cell lysates were harvested for the determi-
nation of STAT3 phosphorylation status (Ser727 and Tyr705) by immu-
noblotting. The same blots were probed with STAT3-specific Abs to com-
pare the total levels of STAT3 in wild-type and IRAK-1-deficient T cells.
ns, Nonspecific bands. B, Chromatin immunoprecipitation (IP) assays were
performed in unstimulated cells or cells stimulated with TGF? and IL-6
plus anti-CD3 and anti-CD28 using STAT3-specific Abs. The input DNA
was used as the loading control. Data are representative of three indepen-
Differential effect of IL-6 on STAT3 phosphorylation in
5766 DIFFERENTIAL INDUCTION OF Foxp3 AND IL-17 BY IRAK-1
promoting inflammation in diverse disease models through the ex-
pression of IL-17.
We have herein demonstrated that IRAK-1 plays an important role
in modulating the balance between the differentiation of Treg and
Th17 cells. IRAK-1 fulfills this function by attenuating TCR-me-
diated activation of NFATc2, while facilitating the IL-6-mediated
activation of STAT3 (Fig. 6).
Our data provides a mechanistic explanation for several previ-
ous reports demonstrating that IRAK-1 deletion can provide pro-
tection from various inflammatory diseases. Deng et al. initially
reported that IRAK-1?/?mice are protected from developing EAE
(19). At the time of their discovery, neither Treg cells nor Th17
cells were fully defined. Instead, elevated Th1 response was
thought to be responsible for the development of various inflam-
matory diseases. Deng et al. examined in detail the respective Th1
response and did not observe significant difference among wild-
type and IRAK-1?/?mice. Besides autoimmune diseases, studies
done by others and us indicate that IRAK-1 is closely associated
with the pathogenesis of diverse chronic inflammatory diseases,
including atherosclerosis (17–19, 25). A study using human patient
samples has shown elevated IRAK-1 levels in human atheroscle-
rotic plaques (26). Our study using ApoE?/?/IRAK-1?/?mice
demonstrated that deletion of IRAK-1 renders protection form
high fat diet-induced atherosclerosis (16). In addition, IRAK-1 has
also been associated with the risk of lupus and kidney inflamma-
tion in humans (26, 27). Our unpublished observation indicated
that IRAK-1?/?mice are protected from experimental kidney in-
flammation using the mouse model of anti-glomerular basement
membrane and LPS injection. Mechanistically, this is the first
study to define that IRAK-1 deletion contributes to attenuated
IL-17 expression and Th17 differentiation, while facilitating Foxp3
expression and Treg differentiation. Recently, Treg cells have been
increasingly recognized to be essential in attenuating various in-
flammatory processes and lessening the progression of inflamma-
tory diseases (28). In contrast, IL-17-producing Th17 cells play a
critical role in propagating inflammatory processes that were pre-
viously thought to be mediated by the Th1 cells (3). Our data
presented in this report provides mechanistic explanation of the
protective phenotype of IRAK-1?/?mice and defines a significant
connection between IRAK-1 and the balance of Treg cells and
Excessive expression of IL-17 cytokine is not only associated
with chronic inflammatory diseases, but also with acute injury and
inflammation. Several recent reports indicate that IL-17 is highly
induced following acute endotoxemia in laboratory animals (29–
31), and may contribute to septic shock and multiorgan failure.
IRAK-1?/?mice are known to have alleviated symptoms from
LPS-induced septic shock (32, 33). Furthermore, humans carrying
variant IRAK-1 genes are at a higher risk of getting septic shock
(22). Intriguingly, we found that compared with wild-type mice,
IRAK-1?/?mice have significantly lower plasma levels of IL-17
following acute lethal dose of LPS injection. The mechanism lead-
ing to the rapid expression of IL-17 with septic shock is not
known. Besides CD4 Th cells, many other cell types, such as NK
cells, ??T cells, lymphoid tissue inducer-like cells, neutrophils,
paneth cells, and epithelial cells are also capable of producing
IL-17 (34–37). LPS is a potent inducer of acute phase proteins,
including IL-6 during septic shock (38). IL-6 produced may
further induce the activation of STAT3 and subsequent tran-
scription of IL-17 in these cells. We cannot rule out the possi-
bility that LPS alone or in combination with other stimulatory
factors may directly contribute to STAT3 activation and IL-17
expression. Previous studies done by others and us have shown that
LPS can induce STAT3 phosphorylation at both tyrosine and serine
residue (15, 39), and LPS-mediated STAT3 serine phosphoryla-
tion is dependent on IRAK-1. Future studies are needed to clar-
ify the source of IL-17 expression as well as the molecular
IRAK-1-mediated STAT3 and NFATc2 phosphorylation may
differentially modulate the activities of STAT3 and NFATc2. As
demonstrated previously, the kinase domain of IRAK-1 may adopt
a tertiary structure that resembles the cyclin-dependent-kinase (40,
41). Consequently, IRAK-1 preferentially phosphorylates sub-
strates with serine/threonine-proline rich motifs. To date, all of the
well-defined IRAK-1 substrates, including IRAK-1 itself, STAT3,
NFATc2/c4, and IRF5/7, contain such a motif (15, 42, 43). It has
been reported that STAT3 serine phosphorylation is required for
sponses in IRAK-1-deficient mice. A, Wild-type (WT) and IRAK-1?/?
mice (n ? 4 per group) were injected with 500 ?g LPS or PBS (Control)
i.p. followed by isolation of plasma 6 h postinjection. IL-17 levels were
assayed using a Bio-Rad multiplex bead-based immunoassay kit. ?, p ?
0.05. B, ApoE?/?and ApoE?/?/IRAK-1?/?mice were fed with high-fat
diet for 3 mo and the plasma levels of IL-17 were measured using the
Bio-Rad multiplex bead-based assay kit. At least four mice were analyzed
in each group. Results are expressed as mean ? SD. ??, p ? 0.01.
Decreased IL-17 expression and reduced inflammatory re-
Treg and Th17 cells. Treg and Th17 cells have opposing phenotypes, and
can be induced by distinct sets of stimulants. IRAK-1 promotes the dif-
ferentiation of Th17 and attenuates the differentiation of Treg cells. Mech-
anistically, our data reveal that IRAK-1-mediated STAT3-S727phosphor-
ylation facilitates IL-6-induced transcription of ROR?t and IL-17, thereby
contributing to the differentiation of Th17 cells. In contrast, IRAK-1 at-
tenuates the nuclear translocation of NFAT-c2, dampening the expression
of Foxp3 and Treg cell differentiation. Consequently, deletion of IRAK-1
tilts the balance toward Foxp3-expressing Treg cells and provides protec-
tion against various inflammatory diseases.
A model for IRAK-1-mediated differential regulation of
5767 The Journal of Immunology
the maximum activation of STAT3 transcriptional activity (14). In
contrast, NFAT phosphorylation has been shown to facilitate nu-
clear export of NFAT and attenuate NFAT transcriptional activity
(13). Consistent with previous findings, our current study clearly
indicates that IL6-mediated STAT3 Ser727 phosphorylation is
greatly reduced in IRAK-1?/?cells. This translates into decreased
binding of STAT3 to the endogenous promoter of IL-17 and re-
duced IL-17 expression in IRAK-1?/?CD4 T cells. On the con-
trary, we found that NFATc2 nuclear levels are significantly higher
in TCR-ligated IRAK-1?/?CD4 T cells as compared with the
wild-type cells. This phenomenon closely correlates with elevated
interaction between NFATc2 and Smad3 in the nucleus of IRAK-
1?/?CD4 T cells following TCR ligation and TGF? stimulation.
The induced Foxp3 expression levels are significantly higher in
IRAK-1?/?cells compared with wild-type cells. Consequently,
IRAK-1 contributes to the exacerbation of inflammation by induc-
ing the expression of IL-17 and decreasing the expression of
Our study expands the repertoire of diverse signaling networks
involving IRAK-1. Initially defined as a downstream component of
the IL-1 receptor complex (44), IRAK-1 was later recognized as a
key molecule in other cellular signaling networks downstream of
multiple receptors and coreceptors such as TLRs, CD26, neuro-
trophin nerve growth factor, and insulin (19, 25, 45–48). Its close
homologue IRAK-4 was also implicated in the TCR-mediated sig-
naling process (48). Our present data demonstrate that IRAK-1 is
involved in the signaling process downstream of both IL-6 and
TCR agonists. However, it is not clear how IRAK-1 can be re-
cruited in response to IL-6 and TCR agonists. One of the potential
connectors could be protein kinase C (PKC). In fact, we and others
have reported that several isoforms of PKC can interact, phosphor-
ylate, and activate IRAK-1 (49–51). Both IL-6 and TCR agonists
are well-known activators of PKC, which may subsequently acti-
vate IRAK-1. Future studies are needed to further determine the
molecular basis for this connection.
Taken together, we have revealed a novel role of IRAK-1 during
the induction of ROR?t, IL-17, and Foxp3. IRAK-1 serves as a
critical link modulating the delicate balance between the differen-
tiation of Th17 and Treg cells (Fig. 6). In combination with func-
tional studies, we posit that IRAK-1 may serve as a viable target
for future development of therapeutic interventions of diverse in-
flammatory diseases ranging from acute septic shock to chronic
inflammatory diseases including atherosclerosis and autoimmune
We thank Lin Zhang from our laboratory for assistance with flow cytom-
etry analyses and Samantha Baglin for critical reading of the manuscript.
The authors have no financial conflict of interest.
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