hirano t 2008 il-17 autoimmunity feedback loop il-6

Immunity

Article

Interleukin-17 Promotes Autoimmunityby Triggering a Positive-Feedback Loopvia Interleukin-6 InductionHideki Ogura,1,4 Masaaki Murakami,1,4 Yuko Okuyama,1 Mineko Tsuruoka,1 Chika Kitabayashi,1 Minoru Kanamoto,1

Mika Nishihara,1 Yoichiro Iwakura,2 and Toshio Hirano1,3,*1Laboratory of Developmental Immunology and the CREST Program of the Japan Science and Technology Agency,

Graduate School of Frontier Biosciences, Graduate School of Medicine, and WPI Immunology Frontier Research Center,Osaka University, Osaka 565-0871, Japan2Center for Experimental Medicine and the CREST Program of the Japan Science and Technology Agency, Institute of Medical Science,

University of Tokyo, Tokyo 108-8639, Japan3Laboratory of Cytokine Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama 230-0045, Japan4These authors contributed equally to this work

*Correspondence: [email protected]

DOI 10.1016/j.immuni.2008.07.018

SUMMARY

Dysregulated cytokine expression and signaling aremajor contributors to a number of autoimmune dis-eases. Interleukin-17A (IL-17A) and IL-6 are importantin many disorders characterized by immune self-rec-ognition, and IL-6 is known to induce the differentia-tion of T helper 17 (Th17) cells. Here we describedan IL-17A-triggered positive-feedback loop of IL-6signaling, which involved the activation of the tran-scription factors nuclear factor (NF)-kB and signaltransducer and activator of transcription 3 (STAT3)in fibroblasts. Importantly, enhancement of this loopcaused by disruption of suppressor of cytokinesignaling 3 (SOCS3)-dependent negative regulationof the IL-6 signal transducer gp130 contributed tothe development of arthritis. Because this mecha-nism also enhanced experimental autoimmune en-cephalomyelitis (EAE) in wild-type mice, it may bea general etiologic process underlying other Th17cell-mediated autoimmune diseases.

INTRODUCTION

Autoimmune diseases—a heterogeneous group of disorders

that are controlled by many genetic and environmental fac-

tors—are most simply defined by the presence of mediators of

autoimmunity, such as autoantibodies or autoreactive lympho-

cytes. Various complex molecular processes differentially inter-

act in each of these disease states, which often manifest with

similar symptoms, making it difficult to identify general etiologic

mechanisms (Andersson and Holmdahl, 2005; Davidson and

Diamond, 2001; Falcone and Sarvetnick, 1999; Marrack et al.,

2001; Mathis and Benoist, 2004; Wandstrat and Wakeland,

2001). A common feature among them, however, is that disease

expression depends on the activation status of the immune sys-

tem. Thus, current research is focused on identifying the mech-

anisms that result in the causative dysregulation of the immune

628 Immunity 29, 628–636, October 17, 2008 ª2008 Elsevier Inc.

system. For example, it is believed that inflammation is critical

for the induction of autoimmune diseases, because some auto-

immune and autoimmune-related diseases, including rheuma-

toid arthritis, are characterized by robust immune responses

that result in unresolved inflammation (Baccala et al., 2007;

Hirano, 2002; O’Shea et al., 2002).

In the immune system, CD4+ T cells serve as an important

source of proinflammatory cytokines. Interestingly, cytokines,

and in particular such proinflammatory cytokines as tumor

necrosis factor-a (TNF-a) and interleukin-6 (IL-6), are critically in-

volved in autoimmune diseases (Hirano, 2002; Ioannou and Isen-

berg, 2000; O’Shea et al., 2002). In fact, anti-TNF-a and anti-IL-6

receptor therapies have been successfully employed for a num-

ber of chronic autoimmune diseases, including rheumatoid

arthritis and Crohn’s disease (Andreakos et al., 2002; Nishimoto

and Kishimoto, 2004). Although classification of CD4+ T cells

into T helper 1 (Th1) and Th2 cells has provided a framework for

understanding the roles of various CD4+ T cell subtypes in the de-

velopment of autoimmune diseases (Glimcher and Murphy, 2000;

Mosmann and Coffman, 1989; Zhu et al., 2006), recent studies

have identified Th17 cells as a previously unknown arm of the

CD4+ T cell effector response. These cells secrete several proin-

flammatory cytokines, including IL-17A (Bettelli et al., 2007; Cua

et al., 2003; Harrington et al., 2005; Park et al., 2005; Veldhoen

et al., 2006), deficiency of which in mice results in resistance to

such autoimmune diseases as collagen-induced arthritis, exper-

imental autoimmune encephalomyelitis (EAE), and the arthritis

disorders that develop in SKG and IL-1 receptor antagonist-defi-

cient mice (Afzali et al., 2007; Bettelli et al., 2007; Hirota et al.,

2007; Iwakura and Ishigame, 2006). The development of Th17

cells requires a combination of T cell receptor (TCR) stimulation,

TGF-b, and IL-6 followed by retinoic acid-related orphan recep-

tor-gt expression via signal transducer and activator of transcrip-

tion 3 (STAT3) activation (Bettelli et al., 2007; Ivanov et al., 2006;

Nishihara et al., 2007; Veldhoen et al., 2006). Moreover, it was re-

ported that T cells serve as a source of TGF-b during Th17 cell dif-

ferentiation (Li et al., 2007). Among these mediators, the multi-

functional cytokine IL-6 has been suggested to play a central

role in a number of diseases (Hirano, 1998; Ishihara and Hirano,

2002). Indeed, IL-6 deficiency suppresses the development of

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Immunity

IL-17 and IL-6 Signaling in Autoimmune Diseases

many of the aforementioned autoimmune diseases as well as the

arthritis that develops in mice carrying a Y759F mutation in the IL-

6 receptor subunit gp130 (F759 mice), which enhances the re-

ceptor signaling (Boe et al., 1999; Campbell et al., 1991; Hirota

et al., 2007; Okuda et al., 1998; Sasai et al., 1999; Sawa et al.,

2006). Considering the requirement of IL-6 for Th17 cell develop-

ment, these results suggest that IL-6 acts upstream of IL-17A to

induce the onset of various autoimmune diseases. How IL-17A

contributes to autoimmune diseases and/or inflammation, how-

ever, has yet to be elucidated. Thus, identification of the down-

stream targets of IL-17A that directly contribute the development

of autoimmune diseases should advance our understanding of

the mechanisms underlying IL-17A-mediated diseases.

Our research has focused on IL-6 and the associated signaling

pathways. We have established several knockin mouse lines

expressing mutated variants of gp130, a transducer of IL-6

signaling (Ohtani et al., 2000), and showed that F759 mice spon-

taneously develop an rheumatoid arthritis-like disease that

depends on mature lymphocytes (Atsumi et al., 2002). Because

Y759 of gp130 is essential for the suppressor of cytokine signal-

ing 3 (SOCS3)-dependent negative regulation of gp130 signaling

(Ilangumaran et al., 2004; Yoshimura et al., 2007), IL-6- and

gp130-mediated STAT3 activation is enhanced in F759 mice.

In other words, SOCS3-mediated negative feedback is specifi-

cally defective in the gp130 signaling pathway of F759 mice.

The development of disease in the F759 mice is at least partially

dependent on IL-6 (Sawa et al., 2006). The disease severity in the

F759 mice is also accelerated in a manner dependent on IL-6 by

crossing the mice with human T cell leukemia virus 1 (HTLV-1)

p40-Tax transgenic mice in which nuclear factor (NF)-kB signal-

ing was enhanced (Ishihara et al., 2004). These results suggest

that IL-6 is one of critical factors for the disease in F759 mice.

Moreover, F759 mice develop arthritis in a manner dependent

on the homeostatic proliferation of CD4+ T cells, although the

F759 mutation is not required in T cells for the disease. Rather,

the F759 mutation in nonhematopoietic cells is essential for

IL-7-mediated CD4+ T cell homeostatic proliferation as well as

disease development (Sawa et al., 2006).

Here, we showed that IL-6 not only functioned upstream of

IL-17A but also acted as a critical downstream target of IL-17A

and, unexpectedly, that IL-17A together with IL-6 triggered

a positive-feedback loop of IL-6 expression through the activa-

tion of NF-kB and STAT3 in fibroblast cells. We showed that

blockade of the IL-6 loop significantly suppresses the develop-

ment of arthritis in F759 mice and EAE. Thus, we concluded

that IL-17A promotes autoimmune diseases by triggering a

positive-feedback loop via IL-6 induction.

RESULTS

An IL-17A-Triggered Positive-Feedback Loopof IL-6 Expression in FibroblastsBecause many autoimmune diseases are dependent on both

IL-17A and IL-6, and because IL-17A induces the expression

of IL-6 in fibroblasts (Yao et al., 1995), we hypothesized that

IL-6 functions not only as an inducer of Th17 cell differentiation

but also as a downstream effector of IL-17A. Moreover, both

IL-17A and IL-6 may function together during the effector phases

of autoimmune diseases. We first investigated the roles of IL-17A

and IL-6 signaling in fibroblasts because we previously used chi-

meric mice with transplanted bone-marrow cells to show that the

F759 mutation in nonhematopoietic cells but not in hematopoi-

etic cells results in spontaneous autoimmune arthritis (Sawa

et al., 2006). We prepared mouse embryonic fibroblast (MEF)

cells from WT mice. After the MEF cells were stimulated with

IL-17A in the presence or absence of IL-6, we analyzed the

expression of various target molecules of the IL-17A–NF-kB

signaling pathway, including IL-6. The expression of each of

the IL-17A–NF-kB targets tested (IL-6, keratinocyte-derived cy-

tokine [KC], and MIP2) was synergistically enhanced by IL-17A

and IL-6 (Figures 1A and 1B and Figure S1 available online).

Depleting the MEF cells of molecules involved in either IL-17A

or IL-6 signaling, such as NEMO or STAT3, respectively, showed

that IL-6 expression is completely dependent on NF-kB signaling

and that the synergistic effect of IL-17A and IL-6 on IL-6 expres-

sion was mediated by STAT3 signaling (Figures 1C and 1D).

IL-1b- and TLR ligand-triggered IL-6 expression has been re-

ported to depend on IkB-z, another target of NF-kB (Yamamoto

et al., 2004). Similarly, the expression of IkB-z was synergistically

enhanced by IL-17A plus IL-6 in MEF cells, and this effect was

ameliorated by knocking down STAT3 expression (Figure 1E).

Furthermore, deleting IkB-z completely suppressed IL-6 expres-

sion even after treatment with IL-17A and IL-6 (Figure 1F). Con-

sistent with these results, IL-17A overexpression increased

the serum IL-6 concentration in WT animals (Figure 1G). Taken

together, the data showed that fibroblasts contain an IL-17A-

triggered positive-feedback loop of IL-6 expression, which is

mediated through NF-kB and STAT3.

Enhancement of the IL-17A-TriggeredPositive-Feedback Loop of IL-6 Expression in F759 MiceWe next hypothesized that excess IL-6 signaling resulting from

an enhancement of this positive-feedback loop in fibroblasts is

involved in autoimmune diseases. To test this hypothesis, we

used F759 mice as an animal model; the IL-6–gp130-STAT3 sig-

naling pathway is specifically enhanced in these mice because of

the lack of SOCS3-dependent negative feedback. As expected,

compared to the amounts observed in WT cells, F759 MEF cells

expressed higher IL-6 and the NF-kB target KC in response to

treatment with IL-17A plus IL-6 (Figures 2A and 2B), indicating

that the IL-17A-triggered positive-feedback loop of IL-6 expres-

sion is enhanced in F759 MEF cells. Importantly, overexpression

of IL-17A resulted in a larger increase in the serum IL-6 concen-

tration in young F759 mice (2 months old and arthritis free) com-

pared with that observed in age-matched WT animals (Figures

2C). These results demonstrated that the IL-17A-triggered pos-

itive-feedback loop of IL-6 expression is enhanced in F759

mice. This conclusion was further supported by the significantly

higher serum IL-17A concentrations in older F759 mice com-

pared to young F759 or age-matched WT mice (Figure 2D), the

increased numbers of Th17 cells in older F759 mice compared

with control mice (Figure 2E), and the elevated serum IL-6

concentration observed in older F759 mice (Figure 2F). Thus,

F759 mice especially in age had an enhanced IL-17A-triggered

positive-feedback loop of IL-6 expression.

Soluble IL-6 receptor (IL-6R) has been detected in the serum

of such autoimmune-prone mice as MRL/lpr, BWF1, and BXSB

mice (Suzuki et al., 1993). We examined whether the amount of

Immunity 29, 628–636, October 17, 2008 ª2008 Elsevier Inc. 629

Immunity


soluble IL-6R is increased in F759 mice and found that the serum

concentration of soluble IL-6R age-dependently increased in

F759 mice (Figure S2A), although young F759 and age-matched

Figure 1. IL-17A-Triggered Positive-Feedback Loop of IL-6 Expres-

sion in Fibroblasts of WT Mice

(A and B) MEF cells were prepared from WT C57BL/6 mice and then stimulated

with human IL-6 + soluble IL-6R and/or IL-17A for 24 hr. The culture superna-

tant was collected and assessed with ELISAs specific for (A) IL-6 or (B)

keratinocyte-derived cytokine (KC).

(C) MEF cells derived from NEMO-deficient mice were stimulated with human

IL-6 + soluble IL-6R and/or IL-17A for 24 hr. The culture supernatant was

collected and assessed with ELISAs specific for IL-6.

(D and E) MEF cells from WT mice were transfected with siRNA specific for

STAT3 or a scrambled control. The cells were then stimulated with human

IL-6 + soluble IL-6R and/or IL-17A. The culture supernatant was collected

24 hr after stimulation and assessed with ELISAs specific for (D) IL-6 or (E)

the expression of IkB-z mRNA was calculated after 1 hr with real-time PCRs.

(F) MEF cells derived from IkB-z-deficient mice or WT controls were stimulated

with human IL-6 + soluble IL-6R and/or IL-17A for 24 hr. The culture superna-

tant was collected and assessed with ELISAs specific for IL-6.

(G) WT C57BL/6 mice (2 months old) were intravenously injected with mock or

IL-17A expression vector. Serum concentrations of IL-6 were determined with

ELISAs.

Graphs in (A)–(F) show mean ± SD of more than three independent ex-

periments. p values were calculated with Student’s t tests. **p < 0.01;

***p < 0.005; n.s., not significant.


WT mice show similar amounts (data not shown). We also

showed that soluble IL-6R enhanced IL-6 production in MEF

cells in the presence of IL-6 and IL-17A (Figure S2B). Thus, sol-

uble IL-6R may play a role to IL-17A-triggered positive-feedback

loop of IL-6 expression in F759 mice with age.

IL-6 Downstream of IL-17A Is Involvedin the Development of ArthritisWe then looked for a link between the enhanced IL-17A-trig-

gered positive-feedback loop of IL-6 expression and the devel-

opment of autoimmune arthritis in the F759 mice. We first estab-

lished double mutant animals by crossing mice carrying the

Y759F mutation in gp130 with IL-17A-deficient (Il17a�/�) mice.

Depleting F759 mice of IL-17A significantly suppressed the

development of arthritis (Figure 3A), indicating that the arthritis

Figure 2. The IL-17A-Triggered Positive-Feedback Loop of IL-6Expression Is Enhanced in F759 Mice

(A and B) MEF cells were prepared from F759 mice and then stimulated with

human IL-6 + soluble IL-6R and/or IL-17A for 24 hr. The culture supernatant

was collected and assessed with ELISAs specific for (A) IL-6 or (B) keratino-

cyte-derived cytokine (KC).

(C) F759 mice (2 months old) were intravenously injected with mock or IL-17A

expression vector. Serum concentrations of IL-6 were determined with ELISAs.

(D–F) Young F759 (2 months old), older F759 (10–12 months old), or control

mice were sacrificed and T cells isolated from their lymph nodes and spleens

were subjected to intracellular labeling of IL-17A. (E) The actual number of

CD4+CD44hiIL-17A+ T cells was calculated. Serum concentrations of IL-17A

(D) and IL-6 (F) were determined with ELISAs.

All graphs show mean ± SD of more than three independent experiments.

p values were calculated with Student’s t tests. *p < 0.05; **p < 0.01; ***p < 0.005.

Immunity


in F759 mice is dependent in IL-17A. To confirm this result, we

overexpressed IL-17A by using the hydrodynamics-based trans-

fection method (Zhang et al., 1999) in young F759 mice (2 months

old and arthritis free) and used the mice by the time they reached

4–5 months of age, because we have found that the clinical

scores of F759 mice normally show a significant difference

when the mice reach 6 months of age and that the clinical scores

of Il6�/� F759 or Il17a�/� F759 mice do not significantly differ

from those of control F759 mice when the mice are younger

than 4–5 months old (Figure 3A; Atsumi et al., 2002; Sawa

et al., 2006). As previously demonstrated, this treatment signifi-

cantly increased the serum concentrations of IL-6 (Figure 2C).

In fact, IL-17A overexpression induced arthritis in young F759

mice but not in control WT mice (Figure 3B and Figure S3A).

These results demonstrated that the arthritic condition that de-

Figure 3. IL-17A Is Critically Involved in the Development of Arthritis

in F759 Mice

(A) The clinical arthritis scores of Il17a�/� F759 mice (filled circles, n = 17),

mean ± SD were determined by clinical assessment method of arthritis as

described in the Experimental Procedures and compared to Il17a+/� F759 or

F759 mice (open circles, n = 17).

(B) Two-month-old and arthritis-free F759 mice (mock, n = 12; IL-17A, n = 16)

or WT control mice (mock, n = 5; IL-17A, n = 5) were intravenously injected with

mock or IL-17A expression vector every 8–10 days, and the clinical score was

determined as described in the Experimental Procedures.

(C) Two-month-old and arthritis-free F759 mice (mock, n = 8; IL-17A, n = 8) or

WT control mice (mock, n = 4; IL-17A, n = 4) were intravenously injected with

mock or IL-6 expression vector every 8–10 days. They were then assessed for

signs of arthritis.

Data represent the mean ± SD (A) or SEM (B, C) of the scores. p values were

calculated with Student’s t tests. *p < 0.05; **p < 0.01; ***p < 0.005.

velops in F759 mice is an IL-17A-mediated disease and strongly

supported the idea that enhanced IL-6 signaling driven by an

IL-17A-triggered positive-feedback loop is critical for the devel-

opment of arthritis in these mice.

To examine whether or not IL-6 is a critical downstream target

of IL-17A for the development of arthritis in F759 mice, we over-

expressed IL-6 via the hydrodynamics-based transfection

method in young F759 mice (2 months old and arthritis free)

and used the mice by the time they reached 4–5 months of

age. IL-6 overexpression resulted in the development of arthritis

in the young F759 mice but not in control C57BL/6 mice

(Figure 3C and Figure S3B) as was observed for IL-17A overex-

pression (Figure 3B and Figure S3A), although IL-6 overexpres-

sion did not markedly increase the serum concentrations of

IL-17A in the F759 mice (data not shown). These results indi-

cated that the enhanced IL-6 signaling contributes to arthritis

in F759 mice and suggested that IL-6 is a critical downstream

target of IL-17A during this process.

To examine the relative positions of IL-17A and IL-6 in this sig-

naling hierarchy directly, we used the two double mutant mouse

lines: Il6�/� F759 mice and Il17a�/�F759 mice. We then overex-

pressed IL-17A and IL-6 in the Il6�/� F759 and Il17a�/� F759

mice, respectively; IL-6 expression induced arthritis in the

Il17a�/� F759 mice, whereas IL-17A did not result in the develop-

ment of arthritis in the Il6�/� F759 mice (Figures 4A and 4B and

Figure S3A and S3B). These data showed that IL-6 is a down-

stream target of IL-17A in the IL-17A-dependent arthritis that de-

velops in F759 mice. Taken together, the results show that excess

IL-6expression, which is induced by an IL-17A-triggered positive-

feedback loop, leads to an autoimmune arthritis in F759 mice.

IL-6–STAT3 Signaling in Fibroblasts Plays a Rolefor the Development of Arthritis in F759 MiceTo investigate whether the F759 mutation in nonhematopoietic

cells is required for the development of the spontaneous arthritis,

Figure 4. IL-6 Is a Downstream Target of IL-17A in the Development

of Arthritis in F759 Mice

(A) Il17a�/� F759, F759, or Il17a+/� F759 mice (2 months old and arthritis free)

were intravenously injected with mock (F759 mice, open circles, n = 12;

Il17a�/� F759 mice, open triangles, n = 14) or IL-6 expression vector (F759

mice, closed circles, n = 14; Il17a�/� F759 mice, filled triangles, n = 19) every

8–10 days, and assessed for signs of arthritis.

(B) Il6�/� F759 and F759 mice (2 months old and arthritis free) were intrave-

nously injected with mock (F759 mice, open circles, n = 10; Il6�/� F759 mice,

open triangles, n = 5) or IL-17A expression vector (F759 mice, closed circles,

n = 10; Il6�/� F759 mice, filled triangles, n = 6) every 8–10 days, and assessed

for signs of arthritis.

Data represent the mean ± SEM of the scores. p values were calculated with

Student’s t tests. *p < 0.05; **p < 0.01; ***p < 0.005.


Immunity


we deleted STAT3 from the type I collagen+ fibroblasts of F759

mice by using the Cre-loxP system. The development of

arthritis was significantly suppressed in the type I collagen-

Cre-STAT3flox/flox F759 mice compared with controls (Figure 5).

These results showed that IL-6–STAT3 signaling, a component

of the IL-17A-triggered positive-feedback loop of IL-6 expres-

sion, in type I collagen+ fibroblasts is important for the develop-

ment of arthritis in F759 mice.

The IL-17A-Mediated Positive-Feedback Loop of IL-6Expression Contributes to Enhanced Autoimmunityin EAETo determine whether the IL-17A-mediated positive-feedback

loop of IL-6 expression promotes and/or enhances autoimmu-

nity in WT mice, we performed several in vivo transfer experi-

ments in WT control mice by using pathogenic Th17 cells from

mice that had challenged EAE. We cultured CD4+ T cells from

EAE mice with bone marrow-derived dendritic cells in the pres-

ence of MOG peptide plus IL-23, resulting in IL-17A-producing

MOG-specific CD4+ T cells (Figure S4E). The transfer of Th17

cells induced EAE and increased the serum IL-6 concentration

in WT mice (Figure 6A). This result was consistent with the

IL-17A-mediated IL-6 expression contributing to EAE in WT mice.

To further examine this possibility, we transferred the Th17

cells into Il6�/�, type I collagen-Cre-STAT3flox/flox, or control WT

mice to examine the development of EAE. We showed that con-

trol animals developed EAE disease. Importantly, both Il6�/� and

type I collagen-Cre-STAT3flox/flox mice showed at least partial

suppression of EAE after the Th17 cells were transferred (Figures

6B and 6C). These results showed that IL-6–STAT3 signaling in

nonhematopoietic cells (type I collagen+ cells) contributes to

Th17 cell-mediated EAE in WT mice. Additionally, we induced

Th17 cell development from Il17a�/�and control mice immunized

with MOG peptide by the same method. The percentages and

numbers of CD4+ T cells in the inguinal lymph nodes were similar

in Il17a�/� and control mice immunized with MOG peptide on

day 11 (data not shown). The percentages and numbers of

CD4+ T cells expressing high amounts of CD44 as well as those

Figure 5. IL-6–STAT3 Signaling in Type I Collagen+ Fibroblasts

Contributes to the Development of Arthritis in F759 Mice

Clinical scores for arthritis in type I collagen-Cre-STAT3flox/flox F759 mice (filled

circles; n = 15) were determined as described in the Experimental Procedures

and compared to those in control type I collagen-Cre-STAT3flox/+ F759

and STAT3flox/flox F759 mice (open circles; n = 20). Data represent the

mean ± SEM of the scores. p values were calculated with Student’s t tests.

*p < 0.05; ***p < 0.005.


of CD4+CD25+ cells among the CD4+CD44hi T cells were also

similar in these mice (Figures S4A–S4D). Importantly, the expres-

sion of IL-17F and IFN-g were also comparable between Il17a�/�

and WT T cells cultured in vitro (Figures S4F and S4G), whereas

IL-17A was not detected in cultured CD4+ T cells from Il17a�/�

mice (Figure S4E). These results suggested that IL-17A defi-

ciency does not markedly affect Th17 cell development. Then,

we transferred the CD4+ T cells into WT mice and investigated

the development of EAE. We showed that a lack of IL-17A expres-

sion in Th17 cells attenuated the development of EAE (Figure 6D).

Together, the results shown in Figures 1 and 6 support the path-

ologic roles of the positive-feedback loop between IL-17A and

IL-6 signaling in EAE.

DISCUSSION

We found here that IL-17A together with IL-6 synergistically

increased IL-6 expression in MEF cells through NF-kB and

STAT3 signaling. Indeed, in vivo expression of IL-17A increased

Figure 6. The Positive-Feedback Loop between IL-17A and IL-6

Enhances EAE in WT Mice

(A) WT mice (n =10; 2-month-old C57BL/6 mice) were intravenously injected

with Th17 cells from WT mice with EAE. Serum IL-17A concentrations were

determined with ELISAs.

(B) Il6�/� mice and WT mice (2 months old) were intravenously injected with

Th17 cells from WT mice with EAE. The clinical EAE scores of Il6�/�mice (filled

circles, n = 5) were determined as described in the Experimental Procedures

and compared to those of WT mice (open circles, n = 12).

(C) Type I collagen-Cre-STAT3flox/flox and WT mice (2 months old) were intra-

venously injected with Th17 cells from WT mice with EAE. The clinical EAE

scores of type I collagen-Cre-STAT3flox/flox mice (filled circles, n = 3) were de-

termined as described in the Experimental Procedures and compared to those

of control mice such as STAT3flox/flox mice (open circles, n = 5).

(D) WT mice (2-month-old C57BL/6 mice) were intravenously injected with

Th17 cells from WT mice with EAE and Il17a�/�mice. The clinical EAE scores

of WT mice (filled circles, n = 7) injected with Th17 cells from Il17a�/�mice with

EAE were determined as described in the Experimental Procedures and com-

pared to those of WT mice with EAE (open circles, n = 6).

Data represent the mean ± SEM of the scores. p values were calculated with

Student’s t tests. *p < 0.05; **p < 0.01.

Immunity


the serum IL-6 concentration. These results demonstrate that an

IL-17A-triggered positive-feedback loop of IL-6 expression is

present in WT fibroblasts. IL-17A and IL-6 also synergistically

induced the expression of various NF-kB target genes, including

KC, MIP2, and IkB-z. IL-17A mediated the expression of these

genes in a manner that was IL-6 dose dependent. Thus, these

results suggested that STAT3 may directly or indirectly interact

with NF-kB, as reported previously (Battle and Frank, 2002;

Hagihara et al., 2005; Yang et al., 2007).

We hypothesized that dysregulation of this feedback loop may

contribute to the development of various IL-17A- and IL-6-medi-

ated autoimmune diseases. We first selected F759 mice as an

animal model to examine the dysregulation of the loop and dem-

onstrated that the spontaneously developing arthritis in F759

mice is dependent on IL-17A: F759 mice lacking IL-17A showed

significantly reduced clinical arthritis scores, and overexpression

of IL-17A in young arthritis-free F759 mice induced the develop-

ment of arthritis. Importantly, overexpression of IL-17A did not

trigger arthritis in Il6�/� F759 mice, whereas overexpression of

IL-6 induced the disorder in Il17a�/� F759 mice, indicating that

IL-6 is a critical downstream target of IL-17A. Consistent with

this result, IL-17A expression greatly increased the expression

of IL-6 in F759 mice.

To detail the relationship between the enhancement of the

IL-17A-triggered positive-feedback loop of IL-6 signaling and

arthritis in F759 mice, we have performed several in vivo and

in vitro experiments, which led to the following findings: (1) the

serum IL-6 and IL-17A concentrations markedly increased in

an age-dependent manner in F759 mice compared to the

concentrations observed in WT mice; (2) IL-17A overexpression

produced a larger increase in the serum IL-6 concentration in

F759 mice than that in WT mice; and (3) IL-6 expression via the

IL-17A-triggered positive-feedback loop was enhanced in F759

MEF compared to WT MEF cells. Collectively, the data under-

score the central role of the enhanced signaling mediated

through the IL-17A-triggered positive-feedback loop of IL-6

expression in fibroblasts in the development of arthritis in F759

mice. In fact, specific deletion of the essential loop component

STAT3 in type I collagen+ fibroblasts suppressed the develop-

ment of arthritis in F759 mice. Because F759 mice lack SOCS3-

mediated negative feedback only in the gp130 signaling axis, it

is reasonable to speculate that specifically deleting SOCS3 in

nonhematopoietic cells would also increase the risk of autoim-

mune and/or inflammatory diseases. Consistent with this notion,

SOCS3 deficiency in liver cells increased the degree of liver fibro-

sis (Ogata et al., 2006), which likely mirrors NF-kB activation in fi-

broblasts. In other words, dysregulated NF-kB activation in type

I collagen+ fibroblasts may trigger the feedback loop to increase

IL-6 expression in F759 mice, in which IL-6-mediated STAT3

activation is already dysregulated. Consistent with this scenario,

we previously showed that transgenic expression of HTLV-1–

Tax, which activates NF-kB, enhanced the IL-6-dependent

development of arthritis in F759 mice (Ishihara et al., 2004). More-

over, viral IL-17A from herpesvirus saimiri was shown to activate

NF-kB and induce IL-6 production in fibroblasts (Yao et al., 1995).

These results suggest that viral infections, which stimulate the

NF-kB signaling pathway in fibroblasts, may result in an in-

creased risk for the development of autoimmune diseases by

triggering the positive-feedback loop controlling IL-6 expression.

Considering the requirement of IL-6 for Th17 cell development

(Bettelli et al., 2007; Nishihara et al., 2007), upregulation of IL-6

expression may expand the Th17 cell population on one hand,

while also amplifying the IL-17A-triggered positive-feedback

loop. This hypothesis is supported by results showing that

both the number of Th17 cells and the serum IL-17A concentra-

tion were elevated in older F759 mice compared with WT control

mice. Therefore, we propose that two cell populations, type I col-

lagen+ fibroblasts and Th17 cells, are components of an IL-6 sig-

naling amplifier in vivo. Under normal conditions, the IL-6 ampli-

fier is controlled by several negative-feedback mechanisms,

which maintain homeostasis of IL-6 and IL-17A signaling. Under

pathological conditions, in which Th17 cells trigger an autoim-

mune disease, the IL-6 amplifier abnormally enhances signaling;

the dysregulated enhancement mediated by the amplifier may

be induced by dysregulated activation of NF-kB and/or STAT3

in fibroblasts via a variety of stochastic environmental and/or

genetic factors.

IL-17A is a member of cytokine subfamily and is highly homol-

ogous to IL-17F, a cytokine known to stimulate the NF-kB path-

way. Because IL-17A deficiency resulted in a lower but recogniz-

able arthritis in F759 mice, we hypothesized that IL-17F in

addition to IL-17A may contribute to disease development.

We found that IL-17F was barely detectable in F759 mice and

Il6�/� F759 mice regardless of age, whereas it was present in

Il17a�/�/F759 mice (data not shown). These results suggested

that, under specific conditions, IL-17F may be involved in the

development of autoimmune arthritis.

Numerous studies support strategies that target TNF-a for the

treatment of rheumatoid arthritis as well as other chronic autoim-

mune diseases (Feldmann and Maini, 2001). Moreover, TNF-a

acts in concert with IL-17A to induce IL-6 production (Awane

et al., 1999; Chen et al., 2003; Ruddy et al., 2004). Therefore,

TNF-a may also enhance the IL-6 amplifier proposed here. Con-

sistent with this idea, we observed that depletion of TNF-a in F759

mice partially but significantly inhibited the development of arthri-

tis (data not shown). In addition, TLR-mediated signaling is

known to induce IL-6 expression (Akira et al., 2001; Medzhitov

et al., 1997); for example, spontaneous arthritis develops in

SKG mice in response to pathogens, whereas the mice remain

healthy under specific pathogen-free conditions (Sakaguchi

et al., 2003). This pathogen response can be mimicked via zymo-

san, which triggers IL-6 release (Hata et al., 2004). It is possible

that IL-6 expression induced by TLR-mediated signaling triggers

the IL-6 amplifier especially in the presence of activation of Th17

cells via the pathogens. Thus, a number of factors, including such

cytokines as TNF-a and other IL-6 family cytokines, TLR-medi-

ated signaling, and soluble IL-6R, may act upstream of the IL-6

amplifier, which may explain why targeting IL-6 signaling shows

therapeutic efficacy in humans with rheumatoid arthritis (Nishi-

moto and Kishimoto, 2004).

It should be noted, however, that other mechanisms can in-

duce autoimmune diseases, because not all rheumatoid arthritis

patients respond to anti-IL-6R therapy and depletion of IL-6

does not universally suppress the development of autoimmune

diseases in experimental models. Indeed, recent data has

highlighted a critical role for IL-23 in generating pathogenic

Th17 cells, whereas IL-6 alone generates T cells that produce

both IL-10 and IL-17A (McGeachy et al., 2007), suggesting that


Immunity


IL-6-independent mechanisms can induce Th17-mediated auto-

immune diseases. Thus, the mechanisms underlying the develop-

ment of autoimmune diseases are complex. Here we have shown

that dysregulation of an IL-17A-triggered positive-feedback loop

of IL-6 expression in fibroblasts, at least in part, drives the IL-17A-

mediated autoimmune arthritis observed in F759 mice.

It is important to show that this IL-17A-triggered positive-feed-

back loop of IL-6 expression in type I collagen+ fibroblasts medi-

ates other autoimmune diseases. Because it was reported that

a neuroinflammation induced activation of fibroblasts followed

by type I collagen expression in spinal cord (Okada et al.,

2007), we used EAE as another experimental model. In addition,

to exclude a possible involvement of IL-6 in Th17 cell develop-

ment in vivo, we employed a Th17 cell-transfer system to show

that the feedback loop may play a role in EAE in WT mice. Injec-

tion of established MOG-specific Th17 cells into WT mice

increased IL-6 expression, which was followed by encephalomy-

elitis, and IL-6 deficiency in the recipient mice attenuated the de-

velopment of EAE after Th17 cells were injected. Moreover, spe-

cific depletion of STAT3 in type I collagen+ fibroblasts attenuated

EAE development. Finally, depletion of IL-17A from the injected

MOG-specific Th17 cells at least partially suppressed the devel-

opment of EAE. Together, these results strongly support a role

for an IL-17A-triggered positive-feedback loop of IL-6 expres-

sion in nonhematopoietic cells in the development of EAE in

WT mice. Therefore, we hypothesize that the IL-17A-triggered

positive-feedback loop of IL-6 expression in nonhematopoietic

cells may provide a general etiologic mechanism for various

autoimmune diseases.

EXPERIMENTAL PROCEDURES

Mouse Strains

C57BL/6 mice were purchased from Japan SLC. The F759 mouse line carrying

a human version of gp130 (S710L) was established previously (Atsumi et al.,

2002; Ishihara et al., 2004; Sawa et al., 2006). IL-6-deficient mice were pro-

vided by M. Kopf (Max-Planck-Institute of Immunobiology, Germany), back-

crossed with C57BL/6 mice more than 10 times, and crossed with F759

mice. Type I collagen-Cre mice were provided by G. Karsenty (Baylor College

of Medicine, Houston, TX) and crossed with STAT3flox/flox mice that were pro-

vided by S. Akira (Osaka University, Japan) (Takeda et al., 1998). IL-17A-defi-

cient mice in a C57BL/6 background (Iwakura and Ishigame, 2006) were

crossed with F759 mice. All mice were maintained under specific pathogen-

free conditions according to the protocols of the Osaka University Medical

School. All animal experiments were performed according to the guidelines

of the Institutional Animal Care and Use Committees of the Graduate School

of Frontier Bioscience and Graduate School of Medicine, Osaka University.

Clinical Assessment of Arthritis and Histological Analysis

Mice were inspected and assessed for signs of arthritis as described previously

(Atsumi et al., 2002; Ishihara et al., 2004; Sawa et al., 2006). In some experi-

ments, knee joints were fixed in Bouin’s liquid and embedded in paraffin. His-

tological analysis was performed with sections stained with hematoxylin-eosin.

Cytokine Expression In Vivo

Mouse IL-6 and IL-17A genes were cloned into the pCAGGS vector. The

resulting vectors or mock vectors were intravenously injected (IL-6 vector,

1 mg in 2 ml; IL-17A vector, 50 mg in 2 ml) into mice every 8–10 days as

described previously (Zhang et al., 1999). Twenty-four hours after injection,

the IL-6 and IL-17A concentrations in serum after IL-6 and IL-17A vector injec-

tions were 40 ng/ml and 20 ng/ml, respectively, whereas the levels of these

cytokines did not significantly increase with mock vectors comparing to the

levels observed in untreated mice.


Intracellular Cytokine Staining

The in vivo number of Th17 cells was determined as previously described

(Nishihara et al., 2007). In brief, T cells from spleen and lymph nodes were

stimulated with CD3 and CD28 antibodies in the presence of Golgi-plug (BD

Biosciences) for 6 hr. Intracellular IL-17A was labeled with IL-17A antibodies

after surface staining, fixation, and permeabilization.

Antibodies and Reagents

APC-conjugated anti-CD4 (BioLegend); FITC-conjugated anti-CD44 (eBio-

sciences); PE-conjugated anti-CD25 (eBiosciences), anti-IL-17A (eBiosciences)

or control-IgG2a (eBiosciences); PE-Cy5-conjugated anti-CD4 (BioLegend);

and biotin-conjugated anti-CD8 (eBiosciences), anti-CD19 (eBiosciences),

anti-I-A/I-E (BioLegend), or anti-NK1.1 (eBiosciences) antibodies as well as

streptavidin-conjugated Pacific Blue (Invitrogen) were used for cell staining.

ELISA

IL-6, IFNg, KC, MIP-2, IL-17A, IL-17F, and soluble IL-6R levels in serum or in

cell-culture supernatant were determined with ELISA kits (BD Biosciences,

eBiosciences, or R&D).

Flow Cytometry

For cell-surface labeling, 106 cells were incubated with fluorescence-conju-

gated antibodies for 30 min on ice. The cells then were analyzed with CyAn

flow cytometers (BeckmanCoulter) and the collected data were analyzed

with Summit software (BeckmanCoulter) and/or Flowjo software (Tree Star).

Cell Preparation and Stimulation

MEF cells from WT and F759 fetuses (14–15 days postcoitus) were prepared

as described previously (Sawa et al., 2006). NEMO-deficient MEF cells were

kindly provided by M. Pasparakis (University of Cologne), M. Saito, and S.

Akira (Osaka University, Japan). IkBz-deficient MEF cells were kindly provided

by M. Yamamoto and S. Akira (Osaka University, Japan). MEF cells were

plated in 6-well plates (2 3 105 cells/well) and stimulated with IL-6 plus soluble

IL-6R (20 ng/ml each) and/or IL-17A (50 ng/ml) for 24 hr after 2 hr of serum star-

vation. Human IL-6 (Toray), human soluble IL-6R (R&D Systems), and mouse

IL-17A (R&D Systems) were used for the experiments. For knockdown of

STAT3, MEF cells were transfected with specific siRNA (Sigma-Aldrich) with

GenePORTER transfection reagent (Genlantis) 48 hr before the cells were

stimulated with the cytokines. Cells were harvested, total RNA was prepared

for real-time PCRs, and cell-culture supernatant was collected for ELISA

assays.

Induction of Th17 Cell Differentiation and EAE Development

WT or IL-17A-deficient mice were injected with MOG(33-55) peptide (synthe-

sized by Sigma) in complete Freund’s adjuvant (Sigma-Aldrich) at the base of

the tail on day 0 followed by intravenous injection of pertussis toxin (List

Biological Laboratories) on days 0, 2, and 7. On day 11, CD4+ T cells were

then sorted with anti-CD4 microbeads (BD Biosciences). The resulting

CD4+ T cell-enriched population (2 3 106 cells) was cocultured with rIL-23

(20 ng/ml; R&D) in the presence of MOG peptide-pulsed bone marrow-derived

dendritic cells (5 3 105 cells) for 3 days. Cells (5 3 105 cells) were then injected

intravenously into sublethally irradiated (5 gray) recipients on day 0 followed by

intravenous injection of pertussis toxin on days 0, 2, and 7. The clinical score

used is described previously (Huseby et al., 2001). Surface markers for CD4+

T cells, such as TCR, CD4, CD44, and CD25, were examined with the Th17

cells. For cell-surface labeling, 106 T cells were incubated with fluorescence-

conjugated antibodies for 30 min on ice. We routinely detected more than

20% IL-17A+ cells and less than 2% Foxp3+ cells among the WT CD4+

T cells after culture (data not shown). Cytokine concentrations in supernatants

of cultured Th17 cells from IL-17A-deficient and WT control mice were deter-

mined with ELISAs.

Real-Time PCRs

A GeneAmp 5700 sequence detection system (ABI, Warrington, USA) and

SYBER green PCR Master Mix (Sigma-Aldrich) were used to quantify the levels

of IkBz mRNA, IL-6 receptor mRNA, and HPRT mRNA. Total RNA was

prepared from MEF cells with a GenElute Mammalian Total RNA kit (Sigma-Al-

drich) and DNase I (Sigma-Aldrich). The PCR primer pairs used for real-time

Immunity


PCRs were as follows: mouse HPRT primers, 50-GATTAGCGATGATGAACC

AGGTT-30 and 50-CCTCCCATCTCCTTCATGACA-30; mouse IkB-z primers,

50-GTGGCAGGTAGAGCAGGAAG-30 and 50-CCACCTGAAAGGCACTCTGT-30;

and mouse IL-6 receptor primers, 50-ACAGTGTGGGAAGCAAGTCC-30 and

50-CCGTGAACTCCTTTGACCAT-30. The conditions for the real-time PCRs

were 40 cycles of 94�C for 15 s followed by 60�C for 60 s. The relative mRNA

expression levels were normalized to the levels of HPRT mRNA.

Statistical Analysis

Student’s t tests (two-tailed) were used for statistical testing between two

groups.

SUPPLEMENTAL DATA

Supplemental Data include four figures and can be found with this article online

at http://www.immunity.com/cgi/content/full/29/4/628/DC1/.

ACKNOWLEDGMENTS

We thank M. Kopf (Max-Planck-Institute of Immunobiology, Germany) for IL-6-

deficient mice, G. Karsenty (Baylor College of Medicine, Houston, TX) for type I

collagen-Cre mice, S. Akira (Osaka University, Japan) for STAT3flox/flox mice,

and A. Kumanogo (Osaka University, Japan) for kind advice about the induc-

tion of EAE. We appreciate the excellent technical assistance provided by J.

Jiang, R. Sato, E. Iketani, and T. Hayashi and thank R. Masuda for her excellent

secretarial assistance. We thank D. Kamimura for careful reading of this man-

uscript. This work was supported by KAKENHI (H.O., M.M., and T.H.), Uehara

Foundation (M.M.), and the Osaka Foundation for the Promotion of Clinical Im-

munology (M.M. and T.H.). The authors declare they have no conflicting finan-

cial interests.

Received: March 4, 2008

Revised: June 11, 2008

Accepted: July 11, 2008

Published online: October 9, 2008

REFERENCES

Afzali, B., Lombardi, G., Lechler, R.I., and Lord, G.M. (2007). The role of T

helper 17 (Th17) and regulatory T cells (Treg) in human organ transplantation

and autoimmune disease. Clin. Exp. Immunol. 148, 32–46.

Akira, A., Takeda, K., and Kaisho, T. (2001). Toll-like receptors: Critical proteins

linking innate and acquired immunity. Nat. Immunol. 2, 675–680.

Andersson, A., and Holmdahl, R. (2005). A genetic basis for shared autoimmu-

nity in mouse models. Autoimmunity 38, 209–217.

Andreakos, E., Foxwell, B., Brennan, F., Maini, R., and Feldmann, M. (2002).

Cytokines and anti-cytokine biologicals in autoimmunity: Present and future.

Cytokine Growth Factor Rev. 13, 299–313.

Atsumi, T., Ishihara, K., Kamimura, D., Ikushima, H., Ohtani, T., Hirota, S.,

Kobayashi, H., Park, S., Saeki, Y., Kitamura, Y., and Hirano, T. (2002). A point

mutation of Tyr-759 in interleukin 6 family cytokine receptor subunit gp130

causes autoimmune arthritis. J. Exp. Med. 196, 979–990.

Awane, M., Andres, P.G., Li, D.J., and Reinecker, H.C. (1999). NF-kB-inducing

kinase is a common mediator of IL-17-, TNFa-, and IL-1b-induced chemokine

promoter activation in intestinal epithelial cells. J. Immunol. 162, 5337–5344.

Baccala, R., Hoebe, K., Kono, D.H., Beutler, B., and Theofilopoulos, A.N.

(2007). TLR-dependent and TLR-independent pathways of type I interferon

induction in systemic autoimmunity. Nat. Med. 13, 543–551.

Battle, T.E., and Frank, D.A. (2002). The role of STATs in apoptosis. Curr. Mol.

Med. 2, 381–392.

Bettelli, E., Oukka, M., and Kuchroo, V.K. (2007). T(H)-17 cells in the circle of

immunity and autoimmunity. Nat. Immunol. 8, 345–350.

Boe, A., Baiocchi, M., Carbonatto, M., Papoian, R., and Serlupi-Crescenzi, O.

(1999). Interleukin 6 knock-out mice are resistant to antigen-induced experi-

mental arthritis. Cytokine 11, 1057–1064.

Campbell, I., Kay, T., Oxbrow, L., and Harrison, L. (1991). Essential role for

interferon-g and interleukin-6 in autoimmune insulin-dependent diabetes in

NOD/Wehi mice. J. Clin. Invest. 87, 739–742.

Chen, Y., Thai, P., Zhao, Y.H., Ho, Y.S., DeSouza, M.M., and Wu, R. (2003).

Stimulation of airway mucin gene expression by interleukin (IL)-17 through

IL-6 paracrine/autocrine loop. J. Biol. Chem. 278, 17036–17043.

Cua, D.J., Sherlock, J., Chen, Y., Murphy, C.A., Joyce, B., Seymour, B.,

Lucian, L., To, W., Kwan, S., Churakova, T., et al. (2003). Interleukin-23 rather

than interleukin-12 is the critical cytokine for autoimmune inflammation of the

brain. Nature 421, 744–748.

Davidson, A., and Diamond, B. (2001). Autoimmune diseases. N. Engl. J. Med.

345, 340–350.

Falcone, M., and Sarvetnick, N. (1999). Cytokines that regulate autoimmune

responses. Curr. Opin. Immunol. 11, 670–676.

Feldmann, M., and Maini, R.N. (2001). Anti-TNF alpha therapy of rheumatoid

arthritis: What have we learned? Annu. Rev. Immunol. 19, 163–196.

Glimcher, L.H., and Murphy, K.M. (2000). Lineage commitment in the immune

system: The T helper lymphocyte grows up. Genes Dev. 14, 1693–1711.

Hagihara, K., Nishikawa, T., Sugamata, Y., Song, J., Isobe, T., Taga, T., and

Yoshizaki, K. (2005). Essential role of STAT3 in cytokine-driven NF-B-mediated

serum amyloid A gene expression. Genes Cells 10, 1051–1063.

Harrington, L.E., Hatton, R.D., Mangan, P.R., Turner, H., Murphy, T.L., Murphy,

K.M., and Weaver, C.T. (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.

Hata, H., Sakaguchi, N., Yoshitomi, H., Iwakura, Y., Sekikawa, K., Azuma, Y.,

Kanai, C., Moriizumi, E., Nomura, T., Nakamura, T., and Sakaguchi, S. (2004).

Distinct contribution of IL-6, TNF-alpha, IL-1, and IL-10 to T cell-mediated

spontaneous autoimmune arthritis in mice. J. Clin. Invest. 114, 582–588.

Hirano, T. (1998). Interleukin 6 and its receptor: Ten years later. Int. Rev. Immu-

nol. 16, 249–284.

Hirano, T. (2002). Cytokines in autoimmune disease and chronic inflammatory

proliferative disease. Cytokine Growth Factor Rev. 13, 297–298.

Hirota, K., Hashimoto, M., Yoshitomi, H., Tanaka, S., Nomura, T., Yamaguchi,

T., Iwakura, Y., Sakaguchi, N., and Sakaguchi, S. (2007). T cell self-reactivity

forms a cytokine milieu for spontaneous development of IL-17+ Th cells that

cause autoimmune arthritis. J. Exp. Med. 204, 41–47.

Huseby, E.S., Sather, B., Huseby, P.G., and Goverman, J. (2001). Age-depen-

dent T cell tolerance and autoimmunity to myelin basic protein. Immunity 14,

471–481.

Ilangumaran, S., Ramanathan, S., and Rottapel, R. (2004). Regulation of the

immune system by SOCS family adaptor proteins. Semin. Immunol. 16,

351–356.

Ioannou, Y., and Isenberg, D.A. (2000). Current evidence for the induction of

autoimmune rheumatic manifestations by cytokine therapy. Arthritis Rheum.

43, 1431–1442.

Ishihara, K., and Hirano, T. (2002). IL-6 in autoimmune disease and chronic

inflammatory proliferative disease. Cytokine Growth Factor Rev. 13, 357–368.

Ishihara, K., Sawa, S., Ikushima, H., Hirota, S., Atsumi, T., Kamimura, D., Park,

S., Murakami, M., Kitamura, Y., Iwakura, Y., and Hirano, T. (2004). The point

mutation of tyrosine 759 of the IL-6 family cytokine receptor gp130 synergizes

with HTLV-1 pX in promoting rheumatoid arthritis-like arthritis. Int. Immunol.

16, 455–465.

Ivanov, I.I., McKenzie, B.S., Zhou, L., Tadokoro, C.E., Lepelley, A., Lafaille,

J.J., Cua, D.J., and Littman, D.R. (2006). The orphan nuclear receptor ROR-

gammat directs the differentiation program of proinflammatory IL-17+ T helper

cells. Cell 126, 1121–1133.

Iwakura, Y., and Ishigame, H. (2006). The IL-23/IL-17 axis in inflammation.

J. Clin. Invest. 116, 1218–1222.

Li, M.O., Wan, Y.Y., and Flavell, R.A. (2007). T cell-produced transforming

growth factor-beta1 controls T cell tolerance and regulates Th1- and Th17-

cell differentiation. Immunity 26, 579–591.


http://www.immunity.com/cgi/content/full/29/4/628/DC1/

Immunity


Marrack, P., Kappler, J., and Kotzin, B. (2001). Autoimmune disease: why and

where it occurs. Nat. Med. 7, 899–905.

Mathis, D., and Benoist, C. (2004). Back to central tolerance. Immunity 20,

509–516.

McGeachy, M.J., Bak-Jensen, K.S., Chen, Y., Tato, C.M., Blumenschein, W.,

McClanahan, T., and Cua, D.J. (2007). TGF-beta and IL-6 drive the production

of IL-17 and IL-10 by T cells and restrain T(H)-17 cell-mediated pathology. Nat.

Immunol. 8, 1390–1397.

Medzhitov, R., Preston-Hurlburt, P., and Janeway, C.A.J. (1997). A human

homologue of the Drosophila Toll protein signals activation of adaptive immu-

nity. Nature 24, 394–397.

Mosmann, T.R., and Coffman, R.L. (1989). TH1 and TH2 cells: Different

patterns of lymphokine secretion lead to different functional properties.

Annu. Rev. Immunol. 7, 145–173.

Nishihara, M., Ogura, H., Ueda, N., Tsuruoka, M., Kitabayashi, C., Tsuji, F.,

Aono, H., Ishihara, K., Huseby, E., Betz, U.A., et al. (2007). IL-6-gp130-

STAT3 in T cells directs the development of IL-17+ Th with a minimum effect

on that of Treg in the steady state. Int. Immunol. 19, 695–702.

Nishimoto, N., and Kishimoto, T. (2004). Inhibition of IL-6 for the treatment of

inflammatory diseases. Curr. Opin. Pharmacol. 4, 386–391.

O’Shea, J.J., Ma, A., and Lipsky, P. (2002). Cytokines and autoimmunity. Nat.

Rev. Immunol. 2, 37–45.

Ogata, H., Chinen, T., Yoshida, T., Kinjyo, I., Takaesu, G., Shiraishi, H., Iida, M.,

Kobayashi, T., and Yoshimura, A. (2006). Loss of SOCS3 in the liver promotes

fibrosis by enhancing STAT3-mediated TGF-beta1 production. Oncogene 25,

2520–2530.

Ohtani, T., Ishihara, K., Atsumi, T., Nishida, K., Kaneko, Y., Miyata, T., Itoh, S.,

Narimatsu, M., Maeda, H., Fukada, T., et al. (2000). Dissection of signaling cas-

cades through gp130 in vivo: Reciprocal roles for STAT3- and SHP2-mediated

signals in immune responses. Immunity 12, 95–105.

Okada, M., Miyamoto, O., Shibuya, S., Zhang, X., Yamamoto, T., and Itano, T.

(2007). Expression and role of type I collagen in a rat spinal cord contusion in-

jury model. Neurosci. Res. 58, 371–377.

Okuda, Y., Sakoda, S., Bernard, C., Fujimura, H., Saeki, Y., Kishimoto, T., and

Yanagihara, T. (1998). IL-6-deficient mice are resistant to the induction of

experimental autoimmune encephalomyelitis provoked by myelin oligoden-

drocyte glycoprotein. Int. Immunol. 10, 703–708.

Park, H., Li, Z., Yang, X.O., Chang, S.H., Nurieva, R., Wang, Y.H., Wang, Y.,

Hood, L., Zhu, Z., Tian, Q., and Dong, C. (2005). A distinct lineage of CD4

T cells regulates tissue inflammation by producing interleukin 17. Nat. Immu-

nol. 6, 1133–1141.

Ruddy, M.J., Wong, G.C., Liu, X.K., Yamamoto, H., Kasayama, S., Kirkwood,

K.L., and Gaffen, S.L. (2004). Functional cooperation between interleukin-17

and tumor necrosis factor-alpha is mediated by CCAAT/enhancer-binding

protein family members. J. Biol. Chem. 279, 2559–2567.


Sakaguchi, N., Takahashi, T., Hata, H., Nomura, T., Tagami, T., Yamazaki, S.,

Sakihama, T., Matsutani, T., Negishi, I., Nakatsuru, S., and Sakaguchi, S.

(2003). Altered thymic T-cell selection due to a mutation of the ZAP-70 gene

causes autoimmune arthritis in mice. Nature 426, 454–460.

Sasai, M., Saeki, Y., Ohshima, S., Nishioka, K., Mima, T., Tanaka, T., Katada,

Y., Yoshizaki, K., Suemura, M., and Kishimoto, T. (1999). Delayed onset and

reduced severity of collagen-induced arthritis in interleukin-6-deficient mice.

Arthritis Rheum. 42, 1635–1643.

Sawa, S., Kamimura, D., Jin, G.H., Morikawa, H., Kamon, H., Nishihara, M.,

Ishihara, K., Murakami, M., and Hirano, T. (2006). Autoimmune arthritis asso-

ciated with mutated interleukin (IL)-6 receptor gp130 is driven by STAT3/

IL-7-dependent homeostatic proliferation of CD4+ T cells. J. Exp. Med. 12,

1459–1470.

Suzuki, H., Yasukawa, K., Saito, T., Narazaki, M., Hasegawa, A., Taga, T., and

Kishimoto, T. (1993). Serum soluble interleukin-6 receptor in MRL/lpr mice is

elevated with age and mediates the interleukin-6 signal. Eur. J. Immunol. 23,

1078–1082.

Takeda, K., Kaisho, T., Yoshida, N., Takeda, J., Kishimoto, T., and Akira, S.

(1998). Stat3 activation is responsible for IL-6-dependent T cell proliferation

through preventing apoptosis: Generation and characterization of T cell-

specific Stat3-deficient mice. J. Immunol. 161, 4652–4660.

Veldhoen, M., Hocking, R., Atkins, C., Locksley, R., and Stockinger, B. (2006).

TGF in the context of an inflammatory cytokine milieu supports de novo differ-

entiation of IL-17-producing T cells. Immunity 24, 179–189.

Wandstrat, A., and Wakeland, E. (2001). The genetics of complex autoimmune

diseases: Non-MHC susceptibility genes. Nat. Immunol. 2, 802–809.

Yamamoto, M., Yamazaki, S., Uematsu, S., Sato, S., Hemmi, H., Hoshino, K.,

Kaisho, T., Kuwata, H., Takeuchi, O., Takeshige, K., et al. (2004). Regulation of

Toll/IL-1-receptor-mediated gene expression by the inducible nuclear protein

IkappaBzeta. Nature 430, 218–222.

Yang, J., Liao, X., Agarwal, M.K., Barnes, L., Auron, P.E., and Stark, G.R.

(2007). Unphosphorylated STAT3 accumulates in response to IL-6 and acti-

vates transcription by binding to NFkappaB. Genes Dev. 21, 1396–1408.

Yao, Z., Fanslow, W.C., Seldin, M.F., Rousseau, A.M., Painter, S.L., Comeau,

M.R., Cohen, J.I., and Spriggs, M.K. (1995). Herpesvirus Saimiri encodes

a new cytokine, IL-17, which binds to a novel cytokine receptor. Immunity 3,

811–821.

Yoshimura, A., Naka, T., and Kubo, M. (2007). SOCS proteins, cytokine signal-

ling and immune regulation. Nat. Rev. Immunol. 7, 454–465.

Zhang, G., Budker, V., and Wolff, J.A. (1999). High levels of foreign gene

expression in hepatocytes after tail vein injections of naked plasmid DNA.

Hum. Gene Ther. 10, 1735–1737.

Zhu, J., Yamane, H., Cote-Sierra, J., Guo, L., and Paul, W.E. (2006). GATA-3

promotes Th2 responses through three different mechanisms: induction of

Th2 cytokine production, selective growth of Th2 cells and inhibition of Th1

cell-specific factors. Cell Res. 16, 3–10.

hirano t 2008 il-17 autoimmunity feedback loop il-6

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