Induction of interleukin-10 in the T helper type 17 effector popula-tion by the G protein coupled estrogen receptor (GPER) agonist G-1

Introduction

CD4+ helper T lymphocytes orchestrate adaptive immune

responses to invading pathogens, and are critical to the

pathogenesis of numerous disease processes, including

autoimmunity and cancer. They are an attractive drug

target because of their central role in immunity, and their

implication in a wide variety of diseases. There are several

distinct lineages of CD4+ helper T cells, each specialized

in enhancing specific branches of the immune system.

The original paradigm described by Mossman and Coff-

mann1 divided CD4+ helper T lymphocytes into the T

helper type 1 (Th1) and Th2 populations, with Th1 cells

producing interferon-c (IFN-c) and coordinating cellular

immunity responses and Th2 cells secreting humoral

immunity mediators such as interleukin-4 (IL-4), IL-5

and IL-13. In 2005, the Th1–Th2 paradigm was expanded

as the Th17 population emerged as a third class of

helper/effector T cell. Th17 cells are characterized by

expression of the transcription factor RORct,2,3 and

secrete pro-inflammatory cytokines including IL-214 and

IL-17A/F. These cells are important to controlling infec-

tions by extracellular pathogens, but also appear to play a

deleterious role in human health by contributing to the

pathogenesis of numerous autoimmune diseases.5 In mice,

Th17 differentiation depends on transforming growth

Ryan L. Brunsing1 and Eric R.

Prossnitz1,2

1Department of Cell Biology and Physiology,

University of New Mexico Health Science Cen-

ter, Albuquerque, NM, and 2UNM Cancer

Center, University of New Mexico Health

Science Center, Albuquerque, NM, USA

doi:10.1111/j.1365-2567.2011.03471.x

Received 28 February 2011; revised 16 May

2011; accepted 3 June 2011.

Correspondence: E. R. Prossnitz, Department

of Cell Biology & Physiology, University of

New Mexico, Albuquerque, NM 87131, USA.

Email: eprossnitz@salud.unm.edu

Senior author: Eric R. Prossnitz

Summary

Interleukin-10 (IL-10) is a potent suppressor of the immune system, com-

monly produced by CD4+ T cells to limit ongoing inflammatory responses

minimizing host damage. Many autoimmune diseases are marked by large

populations of activated CD4+ T cells within the setting of chronic

inflammation; therefore, drugs capable of inducing IL-10 production in

CD4+ T cells would be of great therapeutic value. Previous reports have

shown that the small molecule G-1, an agonist of the membrane-bound

G-protein-coupled estrogen receptor GPER, attenuates disease in an ani-

mal model of autoimmune encephalomyelitis. However, the direct effects

of G-1 on CD4+ T-cell populations remain unknown. Using ex vivo cul-

tures of purified CD4+ T cells, we show that G-1 elicits IL-10 expression

in T helper type 17 (Th17) -polarized cells, increasing the number of IL-

10+ and IL-10+ IL-17A+ cells via de novo induction of IL-10. T-cell cul-

tures differentiated in the presence of G-1 secreted threefold more IL-10,

with no change in IL-17A, tumour necrosis factor-a, or interferon-c.

Moreover, inhibition of extracellular signal-regulated kinase (but not p38

or Jun N-terminal kinase) signalling blocked the response, while analysis

of Foxp3 and RORct expression demonstrated increased numbers of IL-

10+ cells in both the Th17 (RORct+) and Foxp3+ RORct+ hybrid T-cell

compartments. Our findings translated in vivo as systemic treatment of

male mice with G-1 led to increased IL-10 secretion from splenocytes fol-

lowing T-cell receptor cross-linking. These results demonstrate that G-1

acts directly on CD4+ T cells, and to our knowledge provide the first

example of a synthetic small molecule capable of eliciting IL-10 expression

in Th17 or hybrid T-cell populations.

Keywords: estrogen receptor; G-protein-coupled estrogen receptor 1;

GPR30; hybrid T cell; interleukin-10; T helper type 17; regulatory T cells

� 2011 The Authors. Immunology � 2011 Blackwell Publishing Ltd, Immunology, 134, 93–106 93

I M M U N O L O G Y O R I G I N A L A R T I C L E

factor-b (TGF-b) and IL-6- or IL-21-mediated signal

transducer and activator of transcription 3 (STAT3) acti-

vation,5 while IL-23 signalling plays a critical role in sta-

bilizing the Th17 phenotype.6 Although Th1, Th2 and

Th17 effector T cells coordinate a robust and diverse arse-

nal of adaptive immune responses necessary for the main-

tenance of human health, mechanisms of restraint must

limit effector responses to protect the host from immune-

mediated damage.

A major breakthrough in elucidating the mechanisms

of adaptive immune regulation emerged with the identifi-

cation of an array of regulatory T (Treg) -cell popula-

tions. The best defined class of Treg cells expresses the

forkhead transcription factor Foxp3 and suppresses

numerous animal models of autoimmune disease,7

whereas loss of Foxp3 function in humans and mice pre-

cipitates a fatal multi-organ autoimmune condition

marked by the inability to control T-cell responses.8,9 The

T reg cells function to dampen immune responses

through a variety of approaches, including contact-medi-

ated inhibition, secretion of perforin and granzyme A/B,

sequestration of key growth factors such as IL-2, and

secretion of suppressive cytokines including TGF-b, IL-10

and IL-35.7 Interleukin-10 in particular plays an impor-

tant role in immune homeostasis, both in mice10 and

humans,11 suggesting that it has several non-redundant

roles in regulating inflammatory responses. Many cell

types in addition to Foxp3+ cells12 can produce IL-10,

most notably several lineages of CD4+ T cells,13 including

Th1,14–16 Th214,17 and Th1718–20 cells, as well as various

types of Treg cells.21 In a feed-forward mechanism, IL-10

can drive its own expression through the induction of an

IL-10-producing Treg-cell population termed Tr1

cells.22,23 Conversely, IL-10 can also be induced indepen-

dently of IL-10 signalling in both Foxp3+ and Foxp3)

Treg-cell populations.24 Given its potent anti-inflamma-

tory effects, various strategies are being explored to target

IL-10 for therapeutic intervention.25

The intimate interplay between the critical factors in

development of Treg and Th17 cells, along with the dual

reliance on TGF-b signalling for their differentiation,26

has led to conceptualization of a Treg–Th17 axis. From a

therapeutics perspective, the identification of drugs

that promote pro-inflammatory or anti-inflammatory

responses by influencing differentiation along this axis

has gained momentum as examples of T-cell plasticity

continue to be characterized,27 in particular within the

Treg-cell and Th17-cell populations.28 Moreover, several

reports have characterized ‘hybrid’ T-cell populations

where Foxp3 is expressed in various effector T-cell popu-

lations,29 and IL-10 can be produced by Th1, Th2 and

Th17 cells.12 These results suggest that it may be possible

to treat disease by shifting the balance along the Treg–

Th17 axis in situ during ongoing immune responses. For

example, one mechanism to dampen inflammation would

be to induce IL-10 expression within Th17 cells partici-

pating in pathological inflammation. To that end, target-

ing non-cytokine signalling pathways may be a viable

option. For example, ATP,30 sphinogosine-1-phosphate31

and vitamin D32 can modulate Th17 development,

whereas antigen-presenting cell (APC)-derived indolamine

2,3-dioxygenase33 and retinoic acid34 can promote Treg-

cell populations, highlighting the importance of non-cyto-

kine signalling pathways to this paradigm.

Estrogen is a well-documented modulator of immune

function in humans and mice, capable of increasing the

expression of Foxp335 and IL-10.36 These effects translate

to human disease wherein patients with multiple sclerosis

experience a decrease in symptoms during pregnancy,37

and to murine models of autoimmune disease where

estrogen inhibits development of and reverses experimen-

tal autoimmune encephalomyelitis (EAE),38 an animal

model of multiple sclerosis. Although the effects of estro-

gen are presumed to be mediated by the classical estrogen

receptors, ERa and ERb, recent studies have pointed to

the newly described G protein-coupled estrogen receptor

GPR30/GPER as contributing to many of these responses.

We and others have recently shown that, like estradiol

(E2), the GPER-selective agonist G-1 can attenuate

EAE.38,39 In the current work we show that G-1 can evoke

IL-10 expression and secretion from CD4+ T cells differ-

entiated under Th17-polarizing conditions. G-1-mediated

IL-10 expression was blocked by the GPER-directed

antagonist G15,40 and was dependent on extracellular sig-

nal-regulated kinase (ERK) signalling, consistent with

known mechanisms of IL-10 production within effector

T-cell populations.12 Analysis of IL-17A, Foxp3 and

RORct expression demonstrated that these responses

occurred in cells expressing both IL-17A and RORct, as

well as in a population of Foxp3+ RORct+ hybrid T cells.

Taken together, our results demonstrate a novel immuno-

modulatory property for G-1. In addition, these data sug-

gest that the family of GPER-directed small molecules

may serve as model compounds for a new class of T-cell-

targeted pharmaceuticals in the treatment of autoimmune

disease and cancer.

Materials and methods

Mice

Male (7–11 weeks old) C57BL/6 and Foxp3egfp mice were

used for this study. Mice were purchased from Jackson

Laboratory (Bar Harbor, ME), and subsequently housed,

bred and cared for according to the institutional guide-

lines in the Animal Resource Facility at the University of

New Mexico. Foxp3-IRES-GFP (Foxp3egfp) transgenic

mice, which contain egfp under the control of an internal

ribosomal entry site (IRES) inserted downstream of the

foxp3 coding region, have been previously described.41

94 � 2011 The Authors. Immunology � 2011 Blackwell Publishing Ltd, Immunology, 134, 93–106

R. L. Brunsing and E. R. Prossnitz

Purification of T-cell populations

T cells were obtained from single cell suspensions follow-

ing homogenization of spleens and lymph nodes by

mechanical disruption and passage through a 70-lm

nylon filter. Suspensions were stained with anti-CD4,

anti-CD62 ligand (CD62L) and anti-CD44 antibodies

(Biolegend, San Diego, CA). Enriched populations of

CD4+ CD62Lhi and CD4+ CD44lo CD62Lhi naive T cells

were collected by flow cytometric cell sorting on a MoFlo

cell sorter (Cytomation, Carpinteria, CA). Purity was reg-

ularly > 96%. In most cases, experiments were repeated

with both types of sorted naive T cells, and no differences

were noted. Alternatively, CD4+ cells were collected from

the single cell suspensions by magnetic bead sorting,

using CD4 microbeads (Miltenyi, Bergisch Gladbach,

Germany) and positive selection on an AutoMACS (Milt-

enyi). This yielded populations with a purity > 90%.

Culture conditions

All experiments and cell purification were carried out in

RPMI-1640 medium supplemented with fetal bovine serum

(FBS), penicillin/streptomycin, L-glutamine, HEPES,

sodium pyruvate and 2-mercaptoethanol. Phenol red-free

buffers and charcoal-stripped FBS were used to minimize

exposure to estrogens or phyto/xenoestrogens that could

have confounded our results. Cells were stimulated in cul-

ture with soluble anti-CD3e (1�0 lg/ml) and anti-CD28

(2�5 lg/ml) antibodies (Biolegend), and supplemented

with various combinations of TGF-b (0�5–10 ng/ml), IL-6

(20 ng/ml) and IL-23 (20 ng/ml) as described (Biolegend

and eBiosciences, San Diego, CA). G-1 and DMSO were

added concurrently with the stimulatory antibodies and

cytokines. Non-polarizing conditions (Th0) contained no

exogenous cytokines. Th17 conditions contained TGF-

b + IL-6 ± IL-23. Experiments were carried out using 96-

well plates with 2 · 105 cells per well (106 cells/ml). For

experiments using GPER and mitogen-activated protein

(MAP) kinase inhibitors, cells were pre-incubated for 60–

90 min with 25 lM PD98059 [MAP kinase kinase (MEK)

inhibitor], 250 nM Jun N-terminal kinase (JNK) II inhibi-

tor, 100 nM SB203580 (p38 inhibitor), or 500 nM G15

(GPER antagonist,40 provided by Dr Jeffrey Arterburn at

New Mexico State University) where indicated, before the

addition of stimulatory antibodies or cytokines. All com-

pounds used in the study were dissolved in DMSO. All cul-

tures were incubated at 37� (+ 5% CO2).

Intracellular cytokine staining and analysis

Following 4 days in culture, cells were washed with med-

ium and ‘rested’ for 60–90 min at 37� (+ 5% CO2). Cul-

tures were then treated with PMA (50 ng/ml) and

ionomycin (500 ng/ml) for 4–5 hr in the presence of Bre-

feldin A (Biolegend) followed by fixation in Fixation Buf-

fer (Biolegend). Samples were then washed and stained

for intracellular proteins in Permeabilization Wash buffer

(Biolegend) for 2 hr at room temperature, and washed

with excess Permeabilization Wash buffer for 15 min at

room temperature before centrifugation and analysis.

Immediately after staining, data were collected on a FAC-

Scalibur (Becton Dickinson, Franklin Lakes, NJ). Data

analysis was performed using FLOWJO software (TreeStar,

Ashland, OR). Antibodies for staining included anti-IL-

10-allophycocyanin, anti-IL-10-phycoerythrin, anti-IL-

17A-phycoerythrin, and IL-17A-peridinin chlorophyll

protein and anti-IFN-c-allophycocyanin all from Bioleg-

end, as well as anti-RORct-phycoerythrin from eBio-

sciences.

Proliferation studies

For analysis of proliferation, freshly sorted T cells were

stained with 2�5 lM eFluor670 according to the manufac-

turer’s protocols (eBiosciences). Cells were then cultured,

stained and analysed as indicated above. Geometric mean

fluorescence intensity (GMFI) of eFluor670 was deter-

mined using FLOWJO software (TreeStar), and unstimu-

lated controls were used to differentiate between

proliferating and non-proliferating cells.

Luminex multiplex assays

Following 4 days in culture, T cells were washed with cold

medium to remove any cytokines in solution, resus-

pended in fresh medium, and counted. For cultures of

splenocytes, single cells suspensions were obtained follow-

ing homogenization of spleens by mechanical disruption

and passage through a 70-lm nylon filter. Cells were then

plated in a 96-well plate with 2 · 105 cells per well

(106 cells/ml), allowed to incubate for 60–90 min at 37�(+5% CO2), and re-stimulated with soluble anti-CD3e(2�5 lg/ml) antibody. Following the indicated stimulation

time, culture medium was collected and spun down to

remove any residual cells. The concentration of IL-4, IL-

6, IL-10, IL-17A, IFN-c and tumour necrosis factor-a(TNF-a) in the cell-free culture medium was analysed

using custom bead arrays from Millipore, and quantified

on a Luminex 100 system (Austin, TX) with the Luminex

XY plate handling platform. Assays were performed

according to the manufacturer’s protocols. Duplicate wells

were assayed for each sample, and data are representative

of the average median value for each sample. Analysis was

performed using IS 2.3 software (Luminex).

In vivo treatment with G-1

A vehicle consisting of 90% emulsion solution

(PBS + 0�9% Tween-20 + 0�9% BSA) and 10% ethanol

� 2011 The Authors. Immunology � 2011 Blackwell Publishing Ltd, Immunology, 134, 93–106 95

IL-10 induction by GPER agonist G-1

was used. For delivery of compounds, E2 or G-1 was dis-

solved in ethanol and added at appropriate concentrations

such that 100 ll per animal per injection was used. The

compound was added to each injection as part of the

10% ethanol found in the vehicle, so it was diluted such

that < 10 ll per animal per injection was required. Injec-

tions were administered in the afternoon, and to limit

stress from the long series of injections inherent in this

study, animals were sedated using isofluorane before

injection. Compound was delivered subcutaneously on

the dorsum adjacent to the hind limb, and the side of the

injection was alternated every 2 days.

Results

G-1 elicits IL-10 production in CD4+ T cells underTh17 polarizing conditions

To investigate the direct effects of G-1 on CD4+ T cells,

we chose to use purified cultures of naive T cells activated

by polyclonal stimulation with anti-CD3e and anti-CD28

antibody. This eliminated secondary effects caused by the

activity of G-1 on APCs within the culture. Furthermore,

primary cells from male mice were used throughout the

study to avoid potential confounding effects of either; (i)

varying estrogen levels in female mice, or (ii) the inflam-

matory effects of ovariectomy. We have also determined

that CD4+ CD44loCD62Lhi naive T-cell and CD4+ Foxp3+

T-reg cell populations express the G-1 target GPER (R. L.

Brunsing and E. R. Prossnitz, manuscript in preparation).

Given that G-1 can protect mice from EAE38,39 and

the importance of the the Th17 lineage to this model,3

we began by determining the effects of G-1 on naive

T-cell differentiation under Th17-polarizing conditions

(TGF-b/IL-6 ± IL-23). Hence, naive T cells from 7- to

11-week-old male C57BL/6 mice were collected by FACS

and stimulated for 4 days ex vivo, supplemented with

combinations of TGF-b, IL-6 and IL-23. Following

4 days of stimulation, cells were analysed for expression

of IFN-c, IL-17A and IL-10 by intracellular cytokine

staining. Expression of IL-10 was present exclusively in

cultures treated with IL-6 (Fig. 1a), consistent with pre-

vious findings using ex vivo culture systems where treat-

ment with TGF-b alone blocks IL-10 expression in

differentiating CD4+ T cells,13 and efficient induction of

IL-10 secretion from effector T-cell populations requires

one of the STAT activating cytokines (for example,

IL-6).19 As expected, IL-17A expression was also largely

dependent on Th17-polarizing conditions (i.e. treatment

with both TGF-b and IL-6; Fig. 1a), although a small

number of IL-17A+ cells was observed in the TGF-b-

treated cultures (data not shown), and was enhanced by

the addition of IL-23. G-1 treatment resulted in an

increase in the percentage of IL-10+ cells within Th 17

cell-polarized cultures (Fig. 1b), including within cultures

supplemented with IL-23 (Fig. 1c), which is known to be

important in stabilizing the phenotype of Th17 popula-

tions.6 This G-1-mediated IL-10 expression was specific

as no increase in the prevalence IL-17A+ cells was

observed in either of the Th17-polarizing conditions

(Fig. 1b,c). In addition, G-1 treatment had no effect on

IFN-c expression in cultures stimulated with CD3/28

alone (Fig. 1d); however, few IFN-c+ cells were detected

in the other culture conditions tested (see Supplementary

material, Fig. S1).

To determine whether the induction of IL-10+ cells

translated into a specific increase in the secretion of IL-10

from G-1-treated cultures, naive T cells were collected

and stimulated as above, in the presence of TGF-b and

IL-6. After 4 days of differentiation, DMSO-treated and

G-1-treated cells were collected, washed with medium to

remove any cytokines released over the course of differen-

tiation, and re-plated at 106 cells/ml. Cells were then

re-stimulated with anti-CD3e antibody for 24 hr, after

which culture medium was analysed for the presence of

newly secreted IL-6, IL-10, IL-17A, TNF-a and IFN-c by

Luminex multiplex assay. Cells differentiated in the pres-

ence of G-1 produced approximately threefold more IL-10

than control cultures (Fig. 2a), consistent with our obser-

vation that G-1 induced an IL-10-producing population.

No difference in the secretion of IL-6, IL-17A, TNF-a or

IFN-c was detected (Fig. 2b–e), again suggesting that G-1

was specifically driving the production of the anti-inflam-

matory cytokine IL-10, and not pro-inflammatory media-

tors such as TNF-a and IFN-c. Taken together, these data

show that G-1 can specifically drive IL-10 expression

within, and secretion from, CD4+ T-cell populations.

Induction of an IL-10+ IL-17A+ and IL-10+ IL-17A)

population by G-1

As G-1-induced IL-10 expression was dependent on

Th17-polarizing conditions, we sought to determine the

relationship between G-1-induced IL-10+ cells and those

expressing the characteristic Th17 cytokine IL-17A.

Hence, naive T cells were again collected by FACS and

polyclonally stimulated in the presence of TGF-b and

IL-6. Cells were cultured with increasing doses of G-1

(1–500 nM) and analysed for IL-17A and IL-10 by intra-

cellular cytokine staining (Fig. 3a). Our data reveal a

dose-dependent increase in IL-10+ IL-17A) (Fig. 3a,b)

and IL-10+ IL-17A+ cells (Fig. 3a,c) within G-1-treated

cultures. A similar trend was observed under IL-23 polar-

izing conditions (Fig. 1a and data not shown). In addi-

tion, G-1-mediated IL-10 expression was blocked by the

recently described GPER antagonist G15.40 The induction

of a population of IL-10+ IL-17A+ cells suggests that G-1

can elicit IL-10 expression within cells that have differen-

tiated to the Th17 lineage. Taken together, these data

show that G-1 can elicit IL-10 production within the

96 � 2011 The Authors. Immunology � 2011 Blackwell Publishing Ltd, Immunology, 134, 93–106

R. L. Brunsing and E. R. Prossnitz

Th17 compartment, a response that is blocked by the

GPER-selective antagonist G15.

ERK signalling is critical for G-1-mediated IL-10expression

Interleukin-10 production within Th populations has

been shown to be dependent on signalling through extra-

cellular signal-regulated kinases ERK1/2,12,13 one of three

MAP kinase cascades, the others comprising JNK1/2 and

p38. GPER has been shown to activate the ERK pathway,

although predominantly in cancer cells.42 To test whether

G-1-mediated induction of IL-10 was dependent on MAP

kinase signalling, naive T cells were treated with either

PD98059, an inhibitor of the ERK pathway, SB203580,

an inhibitor of the p38 pathway, or the JNK II inhibitor,

and stimulated under Th17-polarizing conditions as

before. Consistent with other published reports,13 we

found that inhibition of p38 had no effect on IL-10

expression in Th17-polarized cells. Similarly, JNK signal-

ling appeared not to be required for G-1-mediated

induction of IL-10 (Fig. 4a). In contrast, there was no

difference in the percentage of IL-10+ cells observed

between control and G-1-treated cultures when cells were

cultured with the ERK inhibitor PD98059 (Fig. 4a,b),

consistent with a role for ERK signalling specifically in

DM

SO

IL-1

0

IL-1

0

IL-1

0

IL-1

0

IL-1

0IL

-10

IL-1

0

IL-1

0

IL-1

0

IL-1

0

IL-1

0

G-1

IL-17A IL-17A IL-17A IL-17A IL-17A

IL-17A IL-17AIL-17AIL-17AIL-17A

IL-17A

0·057

0·095

1·25 0·096

0·36

1·31 0·11

0·12

1·08 0·37 4·82 0·067

0·61

21·2 9·12

5·12

19·8 9·27

9·470·56

0·11

0·57

16·4 6·08

7·11

CD3/28

TGF-β1

IL-6

IL-23

1·27 3·82 14·5 6·4

11·5

+ +

+

+ +

+

+

+

+

+

+

+

–

–

–

–

– –

–

–

(a)

TGF-β /IL-6/IL-23

IFNγ +

TGF-β /IL-6

2·5

2·0

1·5

1·0

0·5

0·0IL-10+ IL-17A+

2·5

2·0

1·5

1·0

0·5

0·0IL-10+ IL-17A+

DMSODMSOG-1G-1

NS

NS

2·5

2·0

1·5

1·0

0·5

0·0

CD3/28 alone

NS

DMSO

G-1

Rel

ativ

e %

Rel

ativ

e %

Rel

ativ

e %

*

***

(b) (c) (d)

Figure 1. The G-protein-coupled estrogen receptor (GPER) -directed agonist G-1 induces interleukin-10 (IL-10) production from CD4+ T cells.

CD4+ CD44lo CD62Lhi naive CD4+ T cells were collected by FACS and cultured for 4 days ex vivo with various combinations of transforming

growth factor-b (TGF-b), IL-6 and IL-23, and supplemented with 100 nm G-1 or vehicle (DMSO, control). Cells were subsequently stained for

intracellular IFN-c, IL-17A and IL-10, then analysed by flow cytometry. (a) Representative plots from the various conditions showing intracellular

IL-17A and IL-10. (b) Quantification of data from five to seven independent experiments showing relative number of total IL-10+ cells and total

IL-17A+ cells in cultures treated with TGF-b + IL-6. (c) Quantification of data from four to seven independent experiments showing relative

prevalence of total IL-10+ cells and total IL-17A+ cells in cultures treated with TGF-b + IL-6 + IL-23. (d) Quantification of IFN-c+ cells in cul-

tures stimulated with anti-CD3/28 in non-polarizing conditions (i.e. without the addition of any cytokines). P-values determined by Student’s t-

test; *P < 0�05; ***P < 0�0005. Error bars = SEM; NS, not significant.

� 2011 The Authors. Immunology � 2011 Blackwell Publishing Ltd, Immunology, 134, 93–106 97

IL-10 induction by GPER agonist G-1

G-1-mediated IL-10 induction. These data suggest that

G-1 mediates IL-10 expression by activating ERK signal-

ling in CD4+ T cells.

The ERK pathway is known to be a potent activator of

cell proliferation. To determine if G-1-mediated increases

in IL-10 were the result of increased proliferation of cells

expressing IL-10 rather than induction of IL-10 de novo,

naive T cells were stained with the proliferation dye eFlu-

or670 before stimulation in culture. We were unable to

detect any significant difference in the proportion of

dividing cells following G-1 treatment. The observation

that G-1-treated cultures demonstrate attenuated dilution

of the eFluor dye compared with the DMSO-treated cul-

tures (Fig. 5) indicates that the increase in IL-10+ cells

following G-1 treatment is not the result of an increase in

cell proliferation, and in fact shows that proliferating cells

are going through fewer divisions when treated with G-1,

perhaps because of the action of IL-10. In addition, the

dramatic increase in the number of non-dividing cells

expressing IL-10 in G-1-treated cultures (as indicated in

the upper right quadrant in Fig. 5b) suggests that G-1

can specifically drive expression of IL-10 independent of

cell division. Taken together, these data show that G-1

stimulates de novo IL-10 expression within differentiating

Th17 cells through direct action on T cells via an ERK-

dependent mechanism.

IL-10 induction occurs within a hybrid T-cellpopulation

An emerging paradigm in T-cell biology is the induction

of ‘hybrid’ T-cell populations that express one of the

canonical effector T-cell transcription factors (for example

T-bet from the Th1 lineage) as well as Foxp3.29 These

cells appear to play a role in the regulation of specific

types of inflammatory responses, where the expression of

Foxp3 imparts a suppressive phenotype, and the expres-

sion of the lineage-specific factor such as T-bet leads to a

repertoire of gene products (e.g. chemokine receptors)

that allow for targeting to sites of inflammation. Presum-

ably, this provides a mechanism for the recruitment of

regulatory T cells to sites on ongoing inflammatory

responses. To investigate the expression of Foxp3 together

with RORct, naive T cells were collected from Foxp3egfp

transgenic mice.41 Cells were stimulated for 4 days in the

presence of TGF-b and IL-6 with or without G-1 added

to the culture. Following differentiation, IL-10, IL-17A,

RORct and Foxp3 were analysed by intracellular cytokine

staining or detection of endogenous GFP expression

by flow cytometry. G-1 was equally effective at inducing

IL-10 production within Foxp3) RORct+ Th17 cells as in

Foxp3+ RORct+ hybrid T cells (Fig. 6). The Th17 (i.e.

RORct+) subset yielded an increase in both IL-10+ IL-

17A+ and IL-10+ IL-17A) cells, while only IL-10+ IL-17A)

cells were detected in the hybrid T-cell population. In fact

no IL-17A+ cells were present in the Foxp3+ population

(data not shown). These data demonstrate the ability of

G-1 to induce IL-10 within the recently described hybrid

Th17 population in addition to conventional (Fox-

p3) RORct+) Th17 cells.

In vivo treatment with G-1 leads to increased IL-10secretion from splenocytes

Our results show that treatment of naive T cells with G-1

in culture can lead to increased IL-10 expression and

secretion. To determine if these findings translated

in vivo, wild-type mice were injected subcutaneously with

G-1 for 7 consecutive days, after which isolated spleno-

cytes were stimulated in culture with anti-CD3e and anti-

CD28 antibodies. Samples of supernatant were collected

24, 48 and 72 hr after stimulation and analysed for

10

5

0DMSO G-1

G-1

G-1

G-1

NS

6

4

2

0DMSO

DMSODMSO

DMSO

G-1

NS NS

NS

200

200

200

150

100

50

00

0

600

400

400

300

100

**

IL-1

0 (n

g/m

l)IL

-6 (

pg/m

l)

IL-1

7A (

ng/m

l)

IFN

- γ (

pg/m

l)

TN

F- α

(pg

/ml)

(a) (b)

(d)(c)

(e)

Figure 2. Cytokine secretion following ex vivo treatment with G-1.

CD4+ CD62Lhi naive CD4+ T cells were collected by FACS and cul-

tured for 4 days ex vivo with anti-CD3/28 + interleukin-6 (IL-

6) + transforming growth factor-b (TGF-b) in the presence of

100 nm G-1 (black bars) or DMSO (white bars). Cells were washed

on day 4 and re-stimulated with anti-CD3e alone. Culture medium

was collected after 24 hr and analysed for the presence of secreted

(a) IL-10, (b) IL-17A, (c) interferon-c (IFN-c), (d) tumour necrosis

factor-a (TNF-a), and (e) IL-6 by Luminex multiplex assay. Data are

the means from three independent experiments done in triplicate.

P-values determined by Student’s t-test; *P < 0�02. Error bars =

SEM; NS, not significant.

98 � 2011 The Authors. Immunology � 2011 Blackwell Publishing Ltd, Immunology, 134, 93–106

R. L. Brunsing and E. R. Prossnitz

secreted IL-6, IL-10, IL-17A, IFN-c and TNF-a by Lumin-

ex multiplex assay. No trends were observed for any of

the analytes following 24 hr of stimulation (Fig. 7). As

postulated, following 72 hr of stimulation cells from the

G-1 treated mice produced significantly more IL-10

(Fig. 7a), in agreement with our results with cultured

DM

SO

G-1

IL-1

0IL

-10

IL-1

0IL

-10

IL-1

0IL

-10

IL-1

0IL

-10

15·2 3·29

4·95

15·7 3·74

5·01

14·5 3·9

5·39 6·18

5·0117·4 32·6 10·6

3·9

39·9 12·6

2·87

5·73

4·9417·94·42

5·77

17·2

IL-17A IL-17A IL-17A IL-17A

IL-17AIL-17AIL-17AIL-17A

G-1

(a)

50

40

30

20

10

01 10 100 500

15

10

5

01 10 100 500

G-1 (nM)

DMSO

G-12·0

1·5

1·0

0·5

0·0+ G15

% o

f tot

al p

op.

% o

f tot

al p

op.

***

***

IL-10+IL-17A– IL-10+IL-17A+

Rel

ativ

e %

IL-1

0+

G-1 (nM)

*

*

TGF-β/IL-6

G-1

DMSODMSO

G-1

**

*

(b)(c)

(d)

Figure 3. The G-protein-coupled estrogen receptor (GPER) -directed agonist G-1 induces interleukin-10 (IL-10) production in IL-17A+ cells.

CD4+ CD44lo CD62Lhi or CD4+ CD62Lhi naive CD4+ T cells were collected by FACS and cultured for 4 days ex vivo with anti-CD3/28 + IL-

6 + transforming growth factor-b (TGF-b). Increasing doses of G-1 (1–500 nm, black bars) or equivalent amounts of vehicle (DMSO, white bars)

were added, or cultures were pre-treated with the GPER antagonist G15. Cells were subsequently stained for intracellular IL-17A and IL-10, and anal-

ysed by flow cytometry. (a) Representative plots from the various conditions showing intracellular IL-17A and IL-10. (b–c) Quantification of data

from one of two independent experiments showing the percentage of cells that are (b) IL-10+ IL-17A) and (c) IL-10+ IL-17A+ for the given condi-

tions. (d) Summary of data from two independent experiments showing that the GPER-directed antagonist G15 (500 nm) can block G-1 (100 nm)-

mediated IL-10 induction. P-values determined by Student’s t-test; *P < 0�01; **P < 0�001; ***P < 0�0001. Error bars = SD (b,c) or SEM (d).

� 2011 The Authors. Immunology � 2011 Blackwell Publishing Ltd, Immunology, 134, 93–106 99

IL-10 induction by GPER agonist G-1

naive T cells. Moreover, there was a statistically significant

difference between the time–course of IL-10 secretion for

the cells from G-1-treated mice compared with those

from vehicle-treated animals, as determined by analysis of

variance (Fig. 7a).

Some unexpected results where obtained as well. We

observed that G-1-treated splenocytes demonstrated a sta-

tistically significant increase in the secretion of IL-17A at

48 hr (Fig. 7b). This differed from our findings in Fig. 1,

where no change in the prevalence IL-17A+ cells was

observed, and Fig. 2 where naive T cells cultured with

G-1 produced similar levels of IL-17A compared with

control cells. Additionally, splenocytes from G-1-treated

mice produced decreased levels of IFN-c relative to those

that were treated with vehicle alone (Fig. 7c), suggesting

that in addition to driving production of IL-10 and IL-

17A, G-1 may act systemically to reduce the levels of

IFN-c. This result differed from those shown in Figs 1

and 2 as well, where no changes in IFN-c expression were

noted. These observations probably reflect the complex

nature of the in vivo environment, with secondary effects

resulting from activity on other immune populations. No

changes in the secretion of TNF-a (Fig. 7d) or IL-6

(Fig. 7e) were detected, in agreement with our findings

from Fig. 2. Collectively, these data suggest that pharma-

cological stimulation of GPER in vivo leads to an increase

in the production of the cytokines IL-10 and IL-17A, and

decreased production of the pro-inflammatory cytokine

IFN-c following T-cell activation, yielding an overall anti-

inflammatory environment.

Discussion

It is known that CD4+ T cells play a critical role in the

pathogenesis of many of the most prominent diseases of

the Western world, including cancer, autoimmunity and

infectious diseases. The cytokine IL-10 is a potent sup-

pressor of immune responses, capable of acting on a mul-

titude of cell types to dampen inflammatory responses to

and limit host damage by infection and autoimmune dis-

ease. In this study, we demonstrated that the GPER-direc-

ted agonist G-1 can drive IL-10 production from Th17-

(a) DMSOG-1

NS3

2

1

0

DM

SO

(b)

IL-1

0IL

-10

IL-17A

IL-10+IL-17A+

2·0

1·5

1·0

0·5

0·0Control PD98059

DMSOG-1

DMSOG-1

DMSOG-1

Control PD98059Control PD98059

2·0

1·5

1·0

0·5

0·0

2·0

1·5

1·0

0·5

0·0

Rel

ativ

e %

(c) IL-10+IL-17A–

NS

IL-10–IL-17A+

Rel

ativ

e %

Rel

ativ

e %

G-1

25·5 5·22

6·29

38 10·4 27·6 15

IL-1

0IL

-10

4·44 11

12·5

14·326·3PD98059No inhibitor

Rel

ativ

e %

IL-1

0+

TGF-β /IL6 + PD + JNK inh + SB

IL-17A

IL-17A IL-17A

*****

**

**

***

***

Figure 4. G-1-induced interleukin-10 (IL-10) production is extracellular signal regulated kinase (ERK) dependent. CD4+ CD44lo CD62Lhi naive

CD4+ T cells were collected by FACS and cultured for 4 days ex vivo with anti-CD3/28 + IL-6 + transforming growth factor-b (TGF-b) in the

presence of 100 nm G-1 (black bars) or DMSO (white bars). Cultures were supplemented with inhibitors of the ERK (PD98059), Jun N-terminal

kinase (JNK II inhibitor), or p38 (SB203580) signalling cascades. Following culture, cells were collected and stained for intracellular IL-10 and

IL-17A, and analysed by flow cytometry. (a) Graphs represent summary of data from three independent experiments. (b) Representative plots

from one of four independent experiments with PD98059. (c) Summary of data from four PD98059 experiments analysing induction within pop-

ulations expressing combinations of IL-10 and IL-17A. P-values determined by Student’s t-test; *P < 0�05; **P < 0�005; ***P < 0�0005. Error

bars = SEM; NS, not significant.

100 � 2011 The Authors. Immunology � 2011 Blackwell Publishing Ltd, Immunology, 134, 93–106

R. L. Brunsing and E. R. Prossnitz

polarized CD4+ T-cell populations. We observed an

increase in the number of cells expressing IL-10 within,

and increased IL-10 secretion from, the G-1-treated

cultures. This response was not the result of global

changes in cytokine production as G-1 had no effect on

the expression of IL-17A under Th17-polarizing condi-

tions, or in the induction of IFN-c in non-polarizing

(Th0) conditions. We also observed no significant change

in the secretion of IL-6, IL-17A, TNF-a or IFN-c from G-

1-treated cultures, demonstrating high selectivity for the

mechanism of G-1-mediated IL-10 induction.

We did occasionally detect fewer cells in G-1-treated

cultures relative to those treated with DMSO (RLB and

ERP, unpublished observation), but this was not a con-

sistent finding. This observation may reflect variability in

the temporal dynamics of IL-10 induction between

different experiments or G-1-mediated induction of reg-

ulatory T-cell populations. As noted above, we observed

a slight but significant decrease in proliferation of G-1-

treated cultures (Fig. 5). Additionally, we noted a small

but significant increase in expression of the apoptotic/

cell death marker Annexin V in G-1-treated cultures

(see Supplementary material, Fig. S2). Either of these

effects may be contributing to a decrease in cell number

in G-1-treated cultures. Further studies will be required

to determine whether these findings reflect the direct

effects of G-1 on cellular proliferation and viability, or

are secondary to the induction of regulatory T-cell

populations.

Systemic delivery of G-1 drove IL-10 production from

splenocytes following T-cell activation in culture. It is

notable that this effect does not require overt in vivo anti-

gen recognition. This result may reflect that G-1-mediated

signalling in naive T cells leads to an alteration in their

resting state, perhaps through transcriptional mechanisms.

Another possibility is that there is carryover of G-1 dur-

ing purification of splenocytes before culture, where anti-

gen presentation is mimicked using stimulatory

antibodies, or that the effects are the result of the low lev-

els of T-cell activation inherent in naive mice. Along

those lines, we have consistently found a small population

of memory cells within the spleen of untreated mice, sug-

gesting low levels of immune activation in ‘naive’ animals

(data not shown). It is also possible that pre-existing

memory T cells are responsible for G-1’s effect in this set-

ting, as G-1 can drive IL-10 secretion from this popula-

tion (unpublished observation). In agreement with our

observations from cultured T cells (Fig. 2), systemic

administration of G-1 had no effect on IL-6 or TNF-asecretion. Conversely, we did detect increased secretion of

IL-17A following in vivo treatment with G-1, while also

observing a decrease in the production of IFN-c. These

differences from results with purified T-cell cultures may

reflect the effects of G-1 on other immune populations

following in vivo treatment. Such populations may also be

contributing to the observed IL-10 secretion, directly or

indirectly. Another possibility includes G-1-mediated

IL-10 production during the week-long injections of G-1,

leading to inactivation of splenic APCs and a decrease in

the secretion of Th1-polarizing cytokines like IL-12, and

hence to lower IFN-c production.

Th17 cells are localized in high numbers to sites of

autoimmune inflammation. Our data suggest that it may

be possible to induce IL-10 in situ where large numbers

of Th17 cells persist, through systemic treatment with

G-1. The feasibility of this therapeutic approach is suggested

by experiments in which IL-10+ Th17 cells differentiated

with TGF-b and IL-6 alone inhibited the development of

EAE following adoptive transfer of neuropeptide-reactive

Th17 cells.19 This effect was dependent on IL-10 produc-

tion19 and suggests that such cells can inhibit fully differ-

entiated pathogenic T-cell populations through the

secretion of IL-10 in situ, as would likely be required in

the case of a viable therapeutic intervention based on the

results of our study. While our finding that systemic G-1

could increase IL-17A secretion from murine splenocytes

warrants further attention, it must be noted that IL-17A

has been shown to exhibit immunosuppressive properties

in several settings, including in the development of ath-

erosclerosis43–45 and the induction of T-cell-mediated

colitis.46 Moreover, the IL-10+ Th17 cells discussed above

that were shown to exhibit bystander suppressive effects

in EAE also produced high levels of IL-17A.19 Conse-

quently, the induction of IL-17A is reconcilable with its

(a) DMSO G-1

NS1·0

0·8

0·6

0·4

0·2

0·0% Proliferating ∝ 1/(MFI)

IL-1

0

15·8

64·9 18·7

0·61

DMSO

G-1

20·2 3·37

52·7 23·7

eFluor670

eFluor670

(b) 104

104

103

103

102

102

101

101

100

IL-1

0

104

103

102

101

100

100

104

103

10210

110

0

A.U

.

**

Figure 5. G-1 effects are not dependent on proliferation.

CD4+ CD62Lhi naive CD4+ T cells were collected by FACS and

stained with the proliferation dye eFluor670 before culture. Follow-

ing differentiation for 4 days ex vivo in culture with anti-CD3/

28 + interleukin-6 (IL-6) + transforming growth factor-b (TGF-b)

in the presence of 100 nm G-1 (black bars) or DMSO (white bars),

cells were stained for intracellular IL-10. (a) Percentage of cells

proliferating and the inverse of geometric mean fluorescence inten-

sity (GMFI), a measure of total proliferation. (b) Sample plots show-

ing IL-10 expression and eFluor670 staining. The upper right

quadrant shows cells expressing IL-10 without evidence of prolifera-

tion. Data show one of two independent experiments. P-values

determined by Student’s t-test; **P < 0�02. Error bars = SD; NS, not

significant, A.U., arbitrary units.

� 2011 The Authors. Immunology � 2011 Blackwell Publishing Ltd, Immunology, 134, 93–106 101

IL-10 induction by GPER agonist G-1

ability to attenuate EAE, despite the established impor-

tance of Th17 cells to EAE induction,3,47,48 and the fact

that systemic neutralization of IL-17A/F attenuates clinical

symptoms in this model.49 However, there is also clear

evidence that IL-17A can contribute to pathogenic

inflammation.5 Future studies aimed at determining the

context in which G-1 or any related compounds elicit

critical Th17 factors like IL-17A/F, IL-21, IL-22, IL-23

and the aryl hydrocarbon receptor will be critical to

determining the setting(s) in which G-1 has therapeutic

potential.

The observation of G-1-induced IL-17A secretion may

offer some insight into autoimmune pathophysiology.

There is a longstanding debate about how the apparent

immunosuppressive activities of E2 can be reconciled with

the higher prevalence of autoimmune disease in women.

It is possible that E2-mediated activation of GPER may

drive increased IL-17A production under specific circum-

stances, and that this contributes to augmented autoim-

mune pathogenesis in women. Future studies aimed at

investigating this possibility should be directed at delin-

eating the specific conditions in which GPER activation

leads to IL-17A, and perhaps IL-17F, production. It

would be interesting to correlate these findings with stud-

ies investigating the expression of ERa, ERb and GPER,

which may vary over time. An explanation for the sexual

dimorphism in the prevalence of autoimmune disease

may reside in identifying a setting where GPER plays a

predominant role in estrogen signalling, perhaps as the

result of down-regulation of ERa and ERb within specific

cell populations, under conditions where GPER activation

leads to production of IL-17A or even IL-17F.

If these properties can be definitively described, there is

also the possibility that G-1 may serve a role in T-cell-

Isotype control “Hybrid” T cells

RORgtRORgt

IL-10+IL-17A–

Th17 cells

DMSO

G-1

Rel

ativ

e %

IL-1

0+IL

-17A

–

Rel

ativ

e %

IL-1

0+IL

-17A

–

Rel

ativ

e %

IL-1

0+IL

-17A

–

Rel

ativ

e %

IL-1

0–IL

-17A

+

Rel

ativ

e %

IL-1

0–IL

-17A

+

Rel

ativ

e %

IL-1

0+IL

-17A

+

Rel

ativ

e %

IL-1

0+IL

-17A

+

Total pop Th17 cells Hybrid T cells

NSNS

Th17 cellsTotal popTh17 cellsTotal pop

DMSO G-1 DMSO G-1

0 0 0 0

1 1 1 1

2 2 2 2

3 *** ***

0

1

2

3

0

1

2

3

0

1

2

3

3 33

IL-10+IL-17A+IL-10–IL-17A+

0·027

0·65

Fox

p3

Fox

p3

****** *

(a)

(b)

(c) (d)

Figure 6. G-1 induces interleukin-10 (IL-10) production within the hybrid T-cell population. CD4+ CD62Lhi naive CD4+ T cells were collected

by FACS from Foxp3egfp mice and cultured for 4 days ex vivo with anti-CD3/28 + IL-6 + transforming growth factor-b (TGF-b) in the presence

of 100 nm G-1 (black bars) or DMSO (white bars). Cells were collected and stained for intracelluar IL-10, IL-17A, and RORct, and analysed by

flow cytometry. Cells that were Foxp3+ RORct+ were designated as hybrid T cells, whereas those that were Foxp3) RORct+ were designated as T

helper type 17 (Th17) cells. (a) Gating logic to determine hybrid T-cell and Th17 populations. (b–d) Graphs represent summary of data from

three independent experiments showing the relative percentage of (b) IL-10+ IL-17A), (c) IL-10+ IL-17A+, and (d) IL-10) IL-17A+ populations.

P-values determined by Student’s t-test; *P < 0�05; **P < 0�005; ***P < 0�0005. Error bars = SEM; NS, not significant.

102 � 2011 The Authors. Immunology � 2011 Blackwell Publishing Ltd, Immunology, 134, 93–106

R. L. Brunsing and E. R. Prossnitz

based tumour vaccine strategies. Evidence suggests that

polarization of tumour-specific T-cells towards a Th17

phenotype before adoptive transfer can enhance tumour

eradication.50 G-1 or a related compound may serve as a

cost-effective and safe alternative to recombinant cyto-

kines during T-cell culture, or even as a systemic adjuvant

treatment to help stabilize the cells following adoptive

transfer, especially given the fact that we observed

increased IL-17A production following in vivo G-1 treat-

ments. Moreover, further delineating the role of GPER in

polarization along the Treg–Th17 axis, may uncover other

pharmacological approaches, such as the use of G15, that

can elicit anti-tumour responses by driving conversion of

Treg cells into Th17 populations. This strategy was vali-

dated in principle through the use of indoleamine 2,3-

dioxygenase inhibitors in the B16 melanoma model.33

Our findings also suggest that GPER-mediated induc-

tion of IL-10 may play a role in estrogen’s ability to

suppress autoimmune diseases. Two previous reports

demonstrated that G-1 can suppress EAE.38,39 In one

study, the authors found that G-1’s protective effects cor-

related with increased programmed death-1 (PD-1)

expression on Foxp3+ Treg cells, and were dependent on

PD-1 expression as PD-1 knockout mice were not pro-

tected from disease by G-1.38 Notably, the authors also

observed increased IL-10 production from G-1-treated

splenocytes collected from diseased animals compared

with placebo controls, an effect lost in the PD-1 knockout

mice.38 This correlates well with our results in Fig. 7, as

we observed increased IL-10 production from splenocytes

of G-1-treated mice. Notably, IL-10 production in CD4+ T

cells can inhibit the development of EAE,18 a disease

whose pathogenesis is dependent on RORct expression.3

The fact that we demonstrated G-1 leads to an increase in

IL-10 within RORct+ cells, and that IL-10 induction

occurs even in the presence of IL-23, leads to the hypothe-

sis that G-1 suppressed EAE through the induction of

IL-10 production from RORct+ cells specifically within the

600

4

IL-1

7A (

ng/m

l)

IL-1

0 (p

g/m

l)IL

-6 (

pg/m

l)IF

N-γ

(ng

/ml)

TN

F-α

(pg

/ml)

3

2

1

0

400

400

300

200

100

500

200

0

0

0

0

20

40

60

80 Vehicle (n = 3)

G-1 (n = 5)Vehicle (n = 3)G-1 (n = 5)

Vehicle (n = 3)G-1 (n = 5)

Vehicle (n = 3)G-1 (n = 5)

Vehicle (n = 3)

***

*

*

G-1 (n = 5)

10

2-way ANOVA: P = 0·05

2-way ANOVA: P = 0·05

2-way ANOVA: P = 0·09

20

30

24

24

48

48

Time in culture (hr) Time in culture (hr)

Time in culture (hr)Time in culture (hr)

Time in culture (hr)

72 24 48 72

24 48 7272

24 48 72

(a)(b)

(d)(c)

(e)

Figure 7. Cytokine secretion following in vivo treatment with G-1. Seven- to eleven-week-old male wild-type C57BL/6 mice were injected with G-1

(5 lg/day) or vehicle for 7 consecutive days. One day following the last injection, splenocytes were collected and cultured in the presence of anti-

CD3e (1�0 lg/ml) and anti-CD28 (2�5 lg/ml) antibody. Culture medium was collected after 24, 48, and 72 hr and analysed for the presence of

secreted (a) IL-10, (b) IL-17A, (c) interferon-c (IFN-c), (d) tumour necrosis factor-a (TNF-a) and (e) interleukin-6 (IL-6) by Luminex multiplex

assay. Graphs are mean data with three to five mice per group. P-values comparing G-1 and vehicle at a given time-point were determined by Stu-

dent’s t-test; *P � 0�05, **P � 0�01. P-values comparing G-1 and vehicle curves were determined by two-way anova as indicated. Error bars = SEM.

� 2011 The Authors. Immunology � 2011 Blackwell Publishing Ltd, Immunology, 134, 93–106 103

IL-10 induction by GPER agonist G-1

central nervous system via a PD-1-dependent mechanism.

It has also been recently shown that estrogen can protect

mice from EAE in a Foxp3-indpendent manner.51 Again

an increase in IL-10 was noted, though it is not known

what cells were responsible for this effect. Additionally,

other studies have shown that: (i) E2 can increase IL-10

production in vivo in a GPER-dependent manner,36 and

(ii) the in vitro suppressive activity of Treg cells from PD-

1 knockout mice was enhanced following in vivo treatment

with E2, without changing the expression levels of Foxp3.52

One hypothesis to explain these results may be that E2 sig-

nalling through classical estrogen receptors substitutes for

PD-1-mediated signalling in the induction of IL-10 from

effector populations when E2 is used in lieu of G-1. Fur-

ther studies using conditional knockouts of IL-10 within

the CD4+ compartment, and analysis of GPER, ERa, and

ERb signalling in Foxp3+ and Foxp3) populations, includ-

ing the specific requirement of PD-1 expression, will be

needed to definitively address these questions.

G-1 has been characterized as a selective agonist for the

G protein-coupled estrogen receptor GPER,53 a recently

identified non-classical member of the estrogen receptor

family.54 Consistent with this mechanism of action, G-1-

mediated IL-10 expression was inhibited by the addition

of the GPER-directed antagonist G15.40 Our results are

also supported by observations that G-1-mediated inhibi-

tion of EAE is dependent on GPER expression.38

Although small molecules can be subject to off-target

activity, it is unlikely that both G-1 and G15 would exhi-

bit off-target profiles that mimic their established activi-

ties towards GPER. Nevertheless, further investigation

into the G-1 target(s) in T cells is warranted. Although

GPER)/) mice exist, they exhibit higher levels of apopto-

sis in double-negative thymocytes and lack the E2-medi-

ated increase in this population following systemic

estrogen administration.55 Therefore studies aimed at ver-

ifying GPER as the target of G-1 within the T-cell popula-

tion will need to employ inducible knockout strategies or

retroviral RNAi targeting of GPER to avoid the con-

founding effects of aberrant thymic T-cell development

observed in GPER)/) mice.

Our results have begun to elucidate the mechanisms by

which G-1 induces IL-10 expression and production. Addi-

tion of the MEK1 inhibitor PD98059 blocked G-1-medi-

ated IL-10 induction, whereas addition of inhibitors of the

p38 and JNK pathways was without effect. These findings

are consistent with reports that ERK signalling is necessary

for the induction of IL-10 in Th1 and Th2 cells, and con-

tributes to IL-10 expression in Th17 populations, with no

detectable difference when p38 signalling is blocked.13 Why

addition of PD98059 led to a mild increase in the number

of IL-10+ cells within control (DMSO) cultures is unclear

(Fig. 4b). This stands in contrast to the previous reports

discussed above,12,13 yet we consistently observed this

effect. Interestingly, in the work by Saraiva et al.13 blockade

of ERK signalling only led to a partial inhibition of IL-10

induction from Th17 cultures. This suggests there are two

pathways of IL-10 induction in Th17 cells, the ‘ERK-

dependent pathway’ described above, and an alternative

pathway. One hypothesis to explain the discrepancy

between our findings and previous reports would be that

this alternative pathway: (i) is inhibited by ERK signalling

(an ‘ERK-sensitive pathway’), and (ii) is the predominant

pathway for IL-10 induction in culture conditions using

charcoal-stripped FBS in lieu of normal FBS, as we have

done here. Given that ERK signalling is implicated in IL-10

expression within Th1 and Th2 cells, it will be interesting

to determine whether G-1 can drive IL-10 production

under Th1- or Th2-polarizing conditions. The lack of IL-

10 expression in unpolarized (Th0) cells is not unexpected.

Interleukin-10 production in Th populations requires

STAT activation via IL-4, IL-6, IL-12, IL-21 and IL-27.18,20

However, these cytokines are produced by APCs and dif-

ferentiated T-cell populations and are likely to be in lim-

ited supply in the pure cultures of naive T cells that we

employed. We observed that G-1 was unable to induce IL-

10 production in differentiating naive T cells without the

addition of both TGF-b and IL-6 to the culture medium,

suggesting that G-1 cannot replace any of the critical sig-

nals necessary to induce IL-10 in Th17 cells. It appears that

the function of TGF-b in Th17 development is to block the

differentiation of Th1 and Th2 cells.56 Hence our observa-

tion that G-1 treatment with IL-6 alone does not consis-

tently elicit IL-10 production despite detectable levels of

IL-10+ cells perhaps reflects a dependence on Th17 differ-

entiation. Future studies will need to address this question.

Finally, the IL-10+ IL-17A+ cells we identified appear to

be part of the autoregulatory pathway,21 as they express

RORct but not Foxp3. In fact we detected virtually no IL-

17A+ cells within the Foxp3+ population. While not com-

pletely unexpected, because Foxp3 can inhibit some of

the transcriptional activity of RORct,57 Foxp3+ IL-17A+

cells have been previously reported.58 Our observation

that G-1 induces IL-10 expression in Foxp3+ RORct+

hybrid T cells suggests that, in addition to generating IL-

10 production in populations already localized at the site

of inflammation, G-1 may also enhance the suppressive

function of Treg populations drawn in from the circula-

tion. Such a response would not be unprecedented as

T-bet-induced CXCR3 expression in Foxp3+ cells has

been shown to play a role in targeting Treg cells to sites

of Th1-type inflammation.59 If IL-10 can be stably

induced in hybrid T-cell populations following in vivo G-

1 treatment, their suppressive activity may be enhanced as

they are recruited to sites of ongoing inflammation.

Numerous attempts have been made to harness the

immunosuppressive properties of IL-10 for therapeutic

benefit, many of which have been based on the use of

biologics.25 To our knowledge, this is the first evidence that

a synthetic small molecule can shift the balance along the

104 � 2011 The Authors. Immunology � 2011 Blackwell Publishing Ltd, Immunology, 134, 93–106

R. L. Brunsing and E. R. Prossnitz

Treg–Th17 axis in favour of IL-10 production, in this case

by acting directly on T-cell populations. These data build on

previous results demonstrating that dexamethasone and

retinoic acid can elicit IL-10 from polyclonally stimulated

naive T cells when IL-4, IL-12 and IFN are neutralized.60

Also worth noting is the fact that it is becoming increas-

ingly clear that GPER probably plays a smaller role in the

majority of classical estrogen responses, such as uterine

imbibition, as compared with its better known counter-

part ERa.40 Hence G-1 may be associated with a more

tolerable adverse effect profile.

Our findings suggest that the membrane-permeable

small molecule G-1 may serve as a novel T-cell-targeted

immunosuppressive agent in settings where large popula-

tions of Th17 cells exist, for example in rheumatoid

arthritis, inflammatory bowel disease, or psoriasis. G-1

may also prove useful for in vitro generation of IL-10-

producing cells for adoptive immunotherapy. Future

studies delineating the specific signalling mechanisms and

targets of G-1 and other related compounds will be semi-

nal to the continued development of this new class of

immunoregulatory estrogenic small molecules. The selec-

tivity of G-139,53 and its attractive pharmacological prop-

erties38 make this compound a strong candidate for

pharmaceutical development, paving the way for the

development of novel T-cell targeted immunotherapeu-

tics.

Acknowledgements

This work was supported by National Institutes of Health

grants R01 CA116662, CA118743 and CA127731 (E.R.P.).

Data were generated in the Flow Cytometry Shared

Resource Center supported by the University of New

Mexico Health Sciences Center and the University of New

Mexico Cancer Center. The authors would like to thank

Drs Rick Lyons and Mary Lipscomb for insightful discus-

sions, Kristin Owens and Lori Deihl for their technical

assistance, and Dr Helen Hathaway for her expertise in

the care and use of mice.

Authorship contributions and disclosure ofconflict of interest

R.L.B. and E.R.P. designed and interpreted experiments

and wrote the manuscript. R.L.B. carried out experiments

and E.R.P. holds a U.S. patent on G-1 and G15.

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Figure S1. G-1 does not alter IFNc expression.

Figure S2. G-1 increases Annexin V expression.

Please note: Wiley-Blackwell are not responsible for the

content or functionality of any supporting materials sup-

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ing material) should be directed to the corresponding

author for the article.

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R. L. Brunsing and E. R. Prossnitz