LETTER
1038/nature11984
Induction of pathogenic TH17 cells by inducible
salt-sensing kinase SGK1
Chuan Wu1*, Nir Yosef1,2*, Theresa Thalhamer1{*, Chen Zhu1, Sheng Xiao1, Yasuhiro Kishi1, Aviv Regev2,3 & Vijay K. Kuchroo1,2
TH17 cells (interleukin-17 (IL-17)-producing helper T cells) are
highly proinflammatory cells that are critical for clearing extracel-
lular pathogens and for inducing multiple autoimmune diseases1.
IL-23 has a critical role in stabilizing and reinforcing the TH17
phenotype by increasing expression of IL-23 receptor (IL-23R)
and endowing TH17 cells with pathogenic effector functions2,3.
However, the precise molecular mechanism by which IL-23 sus-
tains the TH17 response and induces pathogenic effector functions
has not been elucidated. Here we used transcriptional profiling of
developing TH17 cells to construct a model of their signalling net-
work and nominate major nodes that regulate TH17 development.
We identified serum glucocorticoid kinase 1 (SGK1), a serine/
threonine kinase4, as an essential node downstream of IL-23 sig-
nalling. SGK1 is critical for regulating IL-23R expression and sta-
bilizing the TH17 cell phenotype by deactivation of mouse Foxo1, a
direct repressor of IL-23R expression. SGK1 has been shown to
govern Na1 transport and salt (NaCl) homeostasis in other cells5–8.
We show here that a modest increase in salt concentration induces
SGK1 expression, promotes IL-23R expression and enhances TH17
cell differentiation in vitro and in vivo, accelerating the develop-
ment of autoimmunity. Loss of SGK1 abrogated Na1-mediated
TH17 differentiation in an IL-23-dependent manner. These data
demonstrate that SGK1 has a critical role in the induction of patho-
genic TH17 cells and provide a molecular insight into a mechanism
by which an environmental factor such as a high salt diet triggers
TH17 development and promotes tissue inflammation.
To determine the molecular mechanisms by which naive T cells
develop into effector TH17 cells, we measured genome-wide messenger
RNA expression profiles using microarrays along 18 time points over
72 h, following the in vitro exposure of naive T cells to TH17 polarizing
conditions (transforming growth factor b1 (TGF-b1) with IL-6). To
examine the role of IL-23 in TH17 development, we added IL-23 at the
late time points (48–72 h) and monitored the transcriptional response
in both wild-type and Il23r–/– cells. We ranked the genes according to
their extent of induction in cells treated with TGF-b1 and IL-6 (relative
to non-polarized activated T cells) and repression in Il23r–/– cells
(relative to wild-type cells) (Methods, Fig. 1a and Supplementary
Table 1). Murine Sgk1 was one of the top ranking genes, whose tran-
scriptional regulation was strongly associated with both IL-23R sig-
nalling and TH17 cell differentiation (Fig. 1a). Quantitative polymerase
chain reaction (qPCR) analysis showed that Sgk1 is induced at low
levels by TGF-b1 (induced regulatory T (iTreg) cells), and not induced
in other T cell subsets (TH0, TH1, TH2). As expected, it is most highly
expressed under TH17 differentiation conditions (Fig. 1b). Sgk1
expression is strongly induced during the first 2 h after stimulation
of naive T cells under TH17-polarizing conditions. This is followed by a
sharp decline by 10 h to a steady expression level that is still substan-
tially higher than in the control population (Fig. 1c and Supplementary
Fig. 1a). Furthermore, Sgk1 expression is specifically induced and
1
Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. 2Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge,
Massachusetts 02142, USA. 3Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02140, USA. {Present address: Department of
Molecular Biology, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria.
*These authors contributed equally to this study.
©2013 Macmillan Publishers Limited. All rights reserved
doi:10.
maintained by exposure to IL-23 (Fig. 1c and Supplementary Fig.
1b). Although Il23r–/– T cells initially produce Sgk1 mRNA, they can-
not sustain this expression (Supplementary Fig. 1b, c). Finally, the
kinase activity of SGK1 is also significantly higher in TH17 cells than
in other T cell subsets (Supplementary Fig. 1d), and restimulation of
differentiated TH17 cells with IL-23 elevates further SGK1 kinase activ-
ity (Supplementary Fig. 1e). Thus, IL-23 signalling is critical for main-
taining Sgk1 expression during TH17 cell differentiation.
Network analysis of the transcriptional changes in Il23r–/– T cells
using the ANAT software9 singled out SGK1 as a potential nodal point
downstream of IL-23R signalling. Based on a curated database of
protein–protein interactions (PPIs), we constructed a network model
that connects known proteins of the IL-23R signalling pathway
(Methods) to the transcription factors whose function is dysregulated
in Il23r–/– cells (Methods, Fig. 1d and Supplementary Fig. 1f). We
ranked the network’s nodes based on a centrality measure, defined
as the fraction of IL-23R-affected transcription factors downstream
of that node in the network (Methods and Supplementary Table 1).
SGK1 was the highest-ranking node (Supplementary Fig. 1g), suggest-
ing that it acts both as a transcriptional target of IL-23R signalling and
as a kinase that may mediate the transcriptional effects of the pathway.
Using Sgk1–/– mice, we studied the impact of loss of SGK1 on TH17
differentiation in vitro. We observed no abnormality of SGK1-
deficient T cells during primary differentiation into TH17 cells
(Fig. 1e). However, Sgk1–/– TH17 cells restimulated with IL-23 showed
impaired IL-17 production (Fig. 1e and Supplementary Fig. 2b).
Memory Sgk1–/– T cells also showed a defect in IL-17 production upon
IL-23 stimulation, but not under stimulation with TGF-b1 and IL-6
(Supplementary Fig. 2a). To study the function of SGK1 specifically in
IL-17-producing T cells that carry the CD4 antigen (CD41 T cells),
we generated Il17f CreSgk1fl/fl mice in which SGK1 was deleted in cells
producing IL-17F, enabling us to analyse the function of SGK1 in the
maintenance of TH17 phenotype. Il17f CreSgk1fl/fl T cells also showed
no defect in primary TH17 differentiation, but displayed reduced IL-17
production when restimulated with IL-23 (Fig. 1f and Supplementary
Fig. 2c). IL-23R expression was also significantly reduced in Sgk1–/– T
cells (Fig. 1g). Thus, loss of SGK1 does not affect primary TH17 dif-
ferentiation, but profoundly affects their stability and IL-23R expres-
sion. One possible explanation for the dispensability of SGK1 during
primary TH17 differentiation is redundancy with other kinases, such as
its homologue AKT10. However, SGK1 seems to be indispensable for
IL-23R-dependent stability and maintenance of TH17 cells.
Microarray analysis of Sgk1–/– versus wild-type TH17 cells restimu-
lated with IL-23 showed a significant overlap in differentially expres-
sed genes with the Il23r–/– versus wild-type IL-23-restimulated TH17
cell profiles, supporting further the functional relatedness of the SGK1
and IL-23R pathways (Fisher exact test, P , 0.001) (Fig. 1h and
Supplementary Fig. 2d). Consistent with this, genes downregulated
in Sgk1–/– cells are significantly enriched (Fisher exact test, P , 0.001)
2 5 A P R I L 2 0 1 3 | VO L 4 9 6 | N AT U R E | 5 1 3
 RESEARCH LETTER

TH17 (50–72 h)

Network score
Il23r–/– (<60 h)
Il23r–/– (>60 h)
TH17 (0–48 h)
a b
TH17 (0–2 h)
c d
300

(relative expression)
Gene Pdk1
600 150

(relative expression)
Sgk1 mRNA
Il17a

Sgk1 mRNA
200
Timp1 400 100
Aqp3 Akt

15
1

SGK1
Igfbp4
Sgk1 100 200 50
Socs3
Centrality score
Fold change (log2)

Smox
Glipr2 0 0
0 0.5 1 2 4 8 12 24 36 48 60 72 Mapk1
Frmd4b 0 Foxo3 Creb1
Nedd4l
Ier3 Time after stimulation (h)

β
0

Node size
+ β+ 6
1 21
re

-1
-2
iT

-2
-
Transcription factor

H
T

IL
Anxa2

-2 IL-

IL
IL

IL
+

+
+

+
Cpd IL-23R signalling

3
TGF-β + IL-6

6
F-

-2
-
-
Prnp

IL
TG

F- GF
TGF-β + IL-6 + IL-23 25% 0

IL
SGK1

IL

+
Cd24a Centrality score

TG T

+
TH0

β
-6
β

F-
Gem

IL
TG TG

+
Mgll
–1

β
0

Fold change (log2)

F-
Cdkn2d
Ets1 h
Rorc TH17 –1 1
Cd28 Cell-surface molecules Cytokines
Il23r–/–
e f g Sgk1–/–

Sgk1+/+Il23rgfp
1º TGF-β + IL-6 2º IL-23 1º TGF-β + IL-6 2º IL-23 1º TGF-β + IL-6 2º IL-23

Slc44a1
Cd86
Cd93
Nt5e
Sco1
Procr
Plxnc1
Klrd1
Cd68
Cd80
Klrc1
Ifngr1
Cd48
Cd83
Btla
Cd38
Ifitm3
Slamf7
Pgp

Lta
Il9
Il4
Il2
Ifng
Il13
Il1r1
Il7r
Cxcr3
Il17a
Il1r2
Ccr6
Ccr1
Il13ra1
Csf2
Il21
Ccl3
Ccl4
Tnfsf8
Il2ra
Il2rb
Il23a
Tnfsf10
Il4i1
Ccl22
Il3
Il5
Spp1
Gene
Abcb1a
Il17fCreSgk1+/+ Il17fCreSgk1fl/fl

104 10 4 104 104

104 104
38.4 0.01 33.8 0.73 10
37.6 0.13 10 18.5 3.36
3 3 103 103
103 103
WT

102 102 102 102

102 102
101
3.81 101
28.3
101 101
101 101
0.21 4.94
IL-17A

IL-17A

CD4

100
0.15 100 0
1.93 10 0
10 0 100
100 101 102 103 104
100 0
10 101 102 103 104 Transcription factors
10 101 102 103 104 100 101 102 103 104 100 101 102 103 104
100 101 102 103 104
104
104
Il23r–/–

Sgk1–/–Il23rgfp
104 104 104
104
41.2 0.01 14.3 0.63 35.3 0.09 9.61 1.06 103
103 103 103
Sgk1–/–
Sgk1–/–

103 103
102 102

Tfdp1
Tradbp
Myc
Phb
Rpl7l1
Hivep3
Pa2g4
Repin1
Mllt3
Ruvbl1
Phf17
Mybbp1a
Jund
Trip13
Prmt7
Sox4
Msc
Tbx21
Fus
Gata3
Egr3
Egr2
Satb1
Rora
Pou6f1
Creb3l2
Nfe2l2
Ddit3
Fos
Bcl6
Bach2
Mxd1
Bcl3
Runx3
Pycr1
Klf4
Ncoa1
Runx2
Mbnl3
Smad4
Ebf1
Jun
Foxp1
Etv3
Pja2
Nab1
Zeb1
Id3
Atf3

Ahr

Atf4

Gene
2 2
102 10 10
102
4.25 101
15.2
101 101 101
101 101
0.25 3.48 0.16 2.31 100 100
100 100 0 100 0 100 101 102 103 104 100 101 102 103 104
100 0 10 101 102 103 104 10 101 102 103 104
10 101 102 103 104 100 101 102 103 104

IFN-γ IFN-γ IL-23R GFP

Figure 1 | SGK1 is specifically induced in TH17 cells and is important for are sized proportionally to their centrality score. e–g, Naive CD41 T cells from
their maintenance. a, Top candidate genes ranked by their average of fold- Sgk1–/– (e), Il17f CreSgk1 fl/fl (f) or Sgk1–/–Il23r gfp (g) and control mice were
increase in TH17 conditions (TGF-b1 with IL-6 compared to TH0) and fold differentiated into TH17 cells with TGF-b1 and IL-6 (left) or restimulated with
decrease in Il23r–/– (knockout versus wild-type cells, TGF-b1, IL-6 and IL-23 IL-23 (right). IL-17 and IFN-c or IL-23R (GFP) expression were assessed.
condition). The centrality score of a given protein is the percentage of IL-23R- Numbers in the graphs indicate the percentage of cells in that quadrant. h, Heat
affected transcription factors downstream of that protein in the network map displaying microarray data, fold change of selected gene subsets in the two
(Methods). b, Sgk1 mRNA expression in different T cell subsets. c, Kinetic analysis experimental settings: Sgk1–/– versus wild-type, and Il23r–/– versus wild-type
of Sgk1 gene expression in activated naive wild-type CD41 T cells differentiated TH17 cells (TGF-b1 and IL-6, restimulated with IL-23). Only genes with a
with TGF-b, IL-6 and IL-23. d, IL-23R PPI network model (this is an enlargement significant fold change in the Sgk1–/– TH17 cells are presented. Data are
of the SGK1 sub-network from the full network of Supplementary Fig. 1f). Nodes representative of at least two independent experiments. Error bars, s.d.

for genes that are upregulated in wild-type TH17 cells compared to To determine the reason for fewer TH17 cells being found in SGK1-
other T cell subsets11 (Methods and Supplementary Fig. 2e). Selected deficient mice, we transferred purified GFP1 cells from differentiated
genes were confirmed by qPCR analysis (Supplementary Fig. 2f). Genes Cd4CreSgk1fl/flIl17agfp or control TH17 cells to congenic Ly5.1 mice and
from several other pathways are also enriched (over- or underex- traced the IL-17 GFP1 cells in different organs after immunization
pressed) (Supplementary Table 2), including cell cycle and prolifera- with MOG35-55 (Fig. 2c). Starting with the same number of CD41IL-
tion, which may be related to the known role of SGK1 as a regulator of 171 T cells, we found that 7 and 12 days after transfer, SGK1-deficient
proliferation and apoptosis7,8,10. Although our analysis strongly associ- TH17 cells failed to maintain IL-17 production, particularly in the
ates SGK1 with the TH17 program, genes important for development central nervous system (CNS) (Fig. 2d and Supplementary Fig. 4c).
and function of other T cell subsets, such as Ifng, Tbx21 or Gata3 were Next, we crossed Il17f CreRosa26ReYFP mice onto the SGK1-deficient
also dysregulated in Sgk1–/– cells, suggesting possible additional effects background, and analysed the expression of IL-17 in T cells that had
of this kinase in other T cell subsets. turned on the Il17f gene as determined by enhanced yellow fluorescent
To determine the role of SGK1 in vivo, we immunized Cd4CreSgk1fl/fl protein (eYFP) expression. We induced EAE in these mice and analysed
mice with myelin oligodendrocyte glycoprotein (MOG) peptide 35–55 the frequency of eYFP1 cells producing IL-17 in infiltrating CD41 T
(MOG35-55) to induce experimental autoimmune encephalomyelitis cells in the lymph nodes and CNS. The Sgk1–/– reporter mice showed a
(EAE). SGK1-deficient mice exhibited significantly reduced EAE smaller proportion of CD41eYFP1 T cells in both organs. Furthermore,
incidence and severity. IL-17 production from infiltrated CD41 T cells there was a dramatic loss of IL-17 expression by eYFP1 T cells in the
in different organs of SGK1-deficient mice was also reduced, whereas SGK1-deficient mice, indicating that TH17 cells could not stably retain
interferon-c (IFN-c) levels were unaffected (Fig. 2a and Sup- IL-17 production during EAE (Fig. 2e and Supplementary Fig. 4d).
plementary Fig. 3a). When we restimulated the isolated T cells from To understand better the molecular role of SGK1 in TH17 cells, we
immunized mice with IL-23 in the presence of MOG35-55, the SGK1- conducted another network analysis, using PPI data to connect SGK1
deficient T cells also showed impaired IL-17 but normal IFN-c pro- to the transcription factors whose activity is dysregulated in Sgk1–/–
duction (Supplementary Fig. 3b, c). Next, using Il23r gfp reporter mice, TH17 cells (Methods). The analysis suggested Foxo1 as one of the
we observed reduced IL-23R–GFP (green fluorescent protein) expres- highest-ranking nodes downstream of SGK1 (Fig. 3a, Supplementary
sion on infiltrating CD41 T cells in different organs of SGK1-deficient Table 1 and Supplementary Fig. 5a). Foxo1 phosphorylation by SGK1
mice undergoing EAE (Supplementary Fig. 4a). Similar to the res- in adipocytes has been shown previously to lead to its deactivation and
ponse of Cd4CreSgk1fl/fl mice, reduced TH17 differentiation and disease translocation from the nucleus to the cytoplasm12. Consistent with this
severity were also observed in Il17f CreSgk1fl/fl mice during EAE observation, we found that SGK1 phosphorylates Foxo1 (Supplemen-
(Fig. 2b). In addition, to exclude any effects of SGK1-deficient tary Fig. 5b). Immunoblot analysis of Sgk1–/– TH17 cells restimulated
bystander cells, we transferred purified Il17f CreSgk1fl/fl CD41 T cells with IL-23 confirmed that there is not only reduced phosphorylation
into Rag2–/– mice and induced EAE. Mice that received SGK1- of Foxo1 in the nucleus but increased mRNA and protein expression of
deficient T cells developed less severe disease compared to mice that Foxo1 (Fig. 3b, c), suggesting that compromised phosphorylation of
received wild-type T cells (Supplementary Fig. 4b). Foxo1 can result in its own transcriptional upregulation. It has been
5 1 4 | N AT U R E | VO L 4 9 6 | 2 5 A P R I L 2 0 1 3
©2013 Macmillan Publishers Limited. All rights reserved
 LETTER RESEARCH

shown previously that Foxo1 can regulate its own expression13 and we promoter transcriptional activity (Fig. 3g). Additionally, the inhibition
have found that Foxo1 binds to a site located about 1 kilobase (kb) of Il23r transcription by a phosphorylation-insensitive triple alanine
upstream of the first exon in the Foxo1 locus (Supplementary Fig. 6a). mutant of Foxo1, Foxo1 AAA, was not reduced in the presence of
Transfection of a Foxo1 luciferase reporter in the presence of Foxo1 SGK1 (Supplementary Fig. 6h). Furthermore, we observed an endogen-
led to increased luciferase activity (Supplementary Fig. 6b), whereas ous Foxo1–RORct interaction in primary TH17 cells (Fig. 3h). These
increasing expression of SGK1 in the presence of Foxo1 resulted in a data support a model in which some of the effects of SGK1 are due to
dose-dependent decrease in reporter activity, suggesting that SGK1 phosphorylation of Foxo1, which may be a key step in relieving RORct
inhibited Foxo1-mediated transactivation of its own promoter (Fig. 3d). from Foxo1-mediated inhibition, enhancing the expression of IL-23R.
To decipher the consequences of Foxo1 expression on TH17 cell SGK1 has been reported to act as a mediator for sodium homeostasis.
development, we used Foxo1–/– CD41 memory T cells and observed It can be induced by exogenous sodium chloride and is one of the major
higher expression of Il23r and Il17a in these cells than in wild-type cells, kinases that regulates Na1 intake by phosphorylation of epithelial
indicating that Foxo1 may act as a repressor of TH17 cell development sodium channels (ENaCs)4,5. Considering the defects in TH17 develop-
and of IL-23 signalling (Fig. 3e and Supplementary Fig. 6c). We also ment in Sgk1–/– mice, this raised the hypothesis that increasing sodium
found potential binding sites of Foxo1 located about 1 kb upstream of concentration may affect the TH17 cell phenotype through SGK1. To test
the first exon of the Il23r locus by chromatin immunoprecipitation this, we first activated naive T cells in the presence of additional NaCl,
(ChIP)-PCR (Supplementary Fig. 6d). Moreover, there is significantly but in the absence of any polarizing cytokines. Microarray analysis of
enriched binding of Foxo1 on the Il23r promoter region in Sgk1–/– cells these NaCl-treated cells showed a significant upregulation of Sgk1 and of
compared to that in wild-type T cells, indicating enhanced suppression multiple other genes associated with TH17 development (Fisher exact
of Il23r transcription in the absence of SGK1 (Fig. 3f). Retinoic-acid- test; P , 0.001; Supplementary Table 2 and Supplementary Fig. 7a),
receptor-related orphan receptor ct (RORct) has been suggested to be which we confirmed by qPCR analysis of selected genes (Sup-
the master transcription factor of TH17 development and ChIP-seq14 plementary Fig. 7b). We also found increased mRNA and protein levels
(ChIP coupled with high-throughput DNA sequencing) and our of IL-17 and IL-23R with additional NaCl under various TH17 polarizing
ChIP-PCR analysis confirmed that IL-23R is one of the targets of conditions (Fig. 4a, b and Supplementary Fig. 7c). Furthermore, a
RORct (Supplementary Fig. 6e). Indeed, we observed that the Il23r sodium-induced increase in TH17 development and IL-23R expression
promoter is transactivated by RORct in IL-23-restimulated TH17 was not observed in SGK1-deficient T cells, specifically in the context of
cells and it can be inhibited by Foxo1 in a dose-dependent manner IL-23–IL-23R signalling (Fig. 4c, d). Importantly, culturing cells with
(Supplementary Fig. 6f, g). While Foxo1 inhibited RORct-mediated mannitol did not alter TH17 cell differentiation, excluding the possibility
Il23r expression, co-expression of SGK1 together with RORct and that the TH17 program is initiated simply by the alteration of osmotic
Foxo1 abrogated the suppressive effects of Foxo1 and rescued Il23r pressure (Supplementary Fig. 7d).

a b
LN Spleen Blood CNS 104

Il17fCreSgk1+/+ Il17fCreSgk1fl/fl
104
Cd4CreSgk1+/+ Cd4CreSgk1fl/fl

3 Cd4CreSgk1+/+ 104
10.3 1.35
104
12.5 1.93
104
14.2 5.26
104
20.4 10.7 Il17fCreSgk1+/+ 103 103 24.7
15.7
Cd4CreSgk1fl/fl 103 103 103 103
Il17fCreSgk1fl/fl
Mean clinical score

Mean clinical score

3 102 102
102 102 102 102
101 101
2 101 101 101 101

IFN-γ
100 100

IL-17
2.45 5.86 100 11.2 100 17.2
2
IL-17

100 100 100 101 102 103 104 100 101 102 103 104
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
104 104
104 104 104 104
1 6.38 0.51 4.31 0.46 8.64 3.11 11.5 3.27 103 103 20.3
103 103 103 103 7.68
1 102 102
102 102 102 102
101 101
101 101 101 101
0 0.98 4.12 9.63 15.7 0 100 100
0 10 15 20 25 100 100 100 100 100 101 102 103 104 100 101 102 103 104
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 0 10 15 20 25
Time after immunization (d) Time after immunization (d) CD4
IFN-γ

c CD45.2 TGF-β + IL-6 e

In vitro culture Sort out IL-17A GFP+ LN
CD4+ T cells 104
7.64 32.7
103

Cd4CreSgk1+/+Il17agfp 102 Il17fCreRosa26ReYFPSgk1+/+

or 101
Counts
eYFP

Cd4CreSgk1fl/flIl17agfp Transfer 100

100 101 102 103 104 100 101 102 103 104
104
103
3.18 3.55

CD45.1 MOG + CFA 102 Il17fCreRosa26ReYFPSgk1–/–

immunization 7–10 days Spleen, LN, blood, 101
100
CNS analysis on 100 101 102 103 104 100 101 102 103 104
EAE onset CD45.2+ cells
CD4 IL-17
Day 5
Cd4CreSgk1+/+Il17agfp
d Cd4CreSgk1fl/flIl17agfp CNS
Cd4CreSgk1+/+Il17agfp Cd4CreSgk1fl/flIl17agfp

dLN Spleen Blood CNS

CD45.2 + CD4 + IL-17+ cells (%)

25 104
104 104 104 104 * *** 103 22.8
26.1
103 10.4 103 20.5 103 18.5 103 21.4
20
** 102 Il17fCreRosa26ReYFPSgk1+/+
102 102 102 102
101
IL-17A GFP

101 101 101 101

Counts
eYFP

100
100 100 100 100 15 * 100 101 102 103 104 100 101 102 103 104
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 104
104 104 104 104 7.54
10 103 12.7
103 5.22 103 7.31 103 6.33 103 2.76
102 Il17fCreRosa26ReYFPSgk1–/–
102 102 102 102
5 101
101 101 101 101
100
100 100 100 100 100 101 102 103 104 100 101 102 103 104
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 0
CD4 IL-17
N

CD45.2
e

oo

N
dL

le

C
Bl
Sp

Figure 2 | SGK1-deficient mice are resistant to EAE, owing to a defect in from the donor CD41 cells collected from the indicated organs 12 days after
maintaining the TH17 phenotype. a, EAE development in Cd4CreSgk11/1 and transfer; representative histograms (left) and quantification of the flow
Cd4CreSgk1fl/fl mice (left), and IL-17 and IFN-c secretion by CD41 T cells cytometry data (right; means and s.d. are shown in red, n 5 10). dLN, draining
isolated from indicated organs at the peak of disease (right) (n 5 12). b, EAE lymph nodes. e, IL-17A production by CD41eYFP1 T cells isolated from lymph
development in Il17f CreSgk11/1 and Il17f CreSgk1fl/fl mice (left), and IL-17 and nodes or CNS of wild-type and SGK1-deficient Il17f CreRosa26ReYFP fate-
IFN-c secretion by CD41 T cells within the CNS (right) (n 5 10). c, Schematic reporter mice 17 days after MOG35-55–CFA immunization (n 5 10). *P , 0.05,
illustration of adoptive transfer experiments shown in d. d, IL-17 production **P , 0.01 and ***P , 0.001 (Student’s t-test). Error bars, s.d.

2 5 A P R I L 2 0 1 3 | VO L 4 9 6 | N AT U R E | 5 1 5
©2013 Macmillan Publishers Limited. All rights reserved
 RESEARCH LETTER

a Transcription
factor b WT Sgk1–/– c d
SGK1 SGK1 α-CD3/28 α-CD3/28
α-CD3/28 + IL-23 α-CD3/28 + IL-23
Foxo1 WT Sgk1–/– Foxo1 luciferase
0 10 30 60 10 30 60 0 10 30 60 10 30 60 min

–
2

1 –/
(relative expression)
pFoxo1

k
Foxo1

activity (RU)
Sg
Foxo1

Luciferase
150 1.5
**

mRNA
Smad4 Dyrk1a Foxo1 1
Foxo1 100
Cry2
0.5
Jun
E2f4 50
Tubulin 0
Histone H3 0 Foxo1 – – + + + + +

Node size
SGK1 – + –

25% 0
Centrality score

e f Foxo1 enrichment g Il23r luciferase h IP IP

te
0.5 1

at

sa
WT

s
Il17a Il23r IL-17A

t
ly

W o1

ly
(relative expression)

Rγ
4 0.4 Sgk1–/– 0.8

le

le
x
2.5 2

activity (RU)

RO

Fo
Luciferase

ho

ho
***
Input (%)

G
**

α-

α-
W
Ig

Ig
2 3 1.5 *** 0.3 0.6
ng ml–1
mRNA

1.5 2 IB: α-Foxo1 IB: α-RORγt

1 0.2 0.4
1
0.5 1 0.5 0.1 0.2
IB: α-RORγt IB: α-Foxo1
0 0 0 0 0
Cre +/+ 1 2 3 4 5 6
Cd4 Foxo1 RORγt – + – – + – + + + + +
Cd4CreFoxo1fl/fl 0.5 kb Il23r Foxo1 – – + – – + + + + + +
SGK1 – – – + + + –
1 2 3 4 5 6

Figure 3 | SGK1 signalling promotes IL-23R expression through from Foxo1–/– mice were stimulated for 24 h with PMA (phorbol myristate
phosphorylation of Foxo1. a, SGK1 PPI network model. Left, network acetate) and ionomycin, and levels of IL-17A and IL-23R expression were
composed of all the protein nodes with a P value of under 0.0001 (Methods); right, determined by qPCR or ELISA (enzyme-linked immunosorbent assay) (IL-17A).
enlargement of the Foxo1 sub-network. Nodes are sized relative to their centrality f, The binding of Foxo1 to the Il23r promoter in wild-type and Sgk1–/– IL-23-
score. In the subnetwork, directed edges (arrows) from one protein to another restimulated TH17 cells was assayed by ChIP-PCR. Six horizontal bars represent
correspond to post-translational modifications of the second protein by the first. the locations of the Foxo1-binding sites on the Il23r locus detected by real-time
Non-directed edges (lines without arrowheads) correspond to PPIs between one PCR. Two thick vertical bars represent exons 1 and 2 of the Il23r locus. g, Il23r
protein and another with no known directionality. b, Phosphorylated Foxo1 promoter activity was measured in HEK293T cells transfected with an Il23r
(p-Foxo1; phosphorylation site Ser 256) and total Foxo1 levels were assessed in promoter-driven luciferase reporter along with the indicated plasmids.
nuclear extracts after restimulation of wild-type (WT) and Sgk1–/– TH17 cells. h, Immunoprecipitation (IP) (control IgG (immunoglobulin-c), anti-RORct or
c, Levels of mRNA (left) and protein (right) of Foxo1 were analysed 3 days anti-Foxo1) of lysates of wild-type IL-23-restimulated TH17 cells, followed by
after IL-23 restimulation of differentiated TH17 cells. d, HEK293T cells were immunoblot (IB) analysis with indicated antibodies. **P , 0.01 and
transfected with a Foxo1 promoter-driven luciferase reporter along with the ***P , 0.001 (Student’s t-test). Data are representative of three independent
indicated plasmids, and promoter activity was assessed. e, Memory CD41 T cells experiments. RU, relative units. Error bars, s.d.

Recent studies have shown that different components in the daily in vivo, we fed a high salt diet (HSD) to wild-type or Cd4CreSgk1fl/fl
diet and gut microbiota can strongly affect the frequency of effector T mice. After 3 weeks on HSD, we observed that un-immunized wild-
cells in the gut15,16. Furthmore, previous data indicate that molecules type mice showed a marked increase in the frequency of TH17 cells in
related to sodium homeostasis can influence TH17 cell responses17,18. the lamina propria, whereas no notable changes were observed in the
To understand further the effect of NaCl on TH17 cell generation mesenteric lymph nodes or spleen. Conversely, SGK1-deficient mice

a None TGF-β + IL-6 IL-23 IL-6 + IL-23 b None TGF-β + IL-6 IL-23 IL-6 + IL-23 Figure 4 | NaCl potentiates TH17 cell
104 104 104 104
104
3
0.01 0.01
104
3
39.2 0.01
104
3
0.76 0.02
104
3
0.39 0.01 103 103 103 103
differentiation in vitro and in vivo, enhancing
10 10 10 10
102 102 102 102 Control
102
0.23
102
0.17
102
1.75
102
5.42
Control EAE induction. a, IL-17 and IFN-c production by
101 101
0.01
101
3.65
101
4.74
101 101 101 101
naive wild-type CD41 T cells stimulated with
IL-17

2.48 10 0
10 0
10 100 0
CD4

100 0 100 100 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
10 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
104 104 104 104 104 104 104 104 indicated cytokines for 3 days with or without
2.12 0.01 59.4 0.01 12.9 0.01 22.8 0.01
103 103 103 103 103 103 103 103
40 mM NaCl. Ctrl, control. b, IL-23R (GFP)
NaCl 102 102 102 102 NaCl
expression in Il23rgfp CD41 T cells stimulated
102 102 102 102
0.88 0.43 12.6 56.7
10 1
101
10 1
10 1 101 101 101 101
0.25 0.01 0.46 0.21
100 100 100 100
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
100 100 100 100
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 under conditions described in a. c, IL-17 and IFN-c
IFN-γ IL-23R GFP production by wild-type (WT) and Sgk1–/– CD41 T
NaCl cells stimulated with the indicated cytokines in the
c NaCl d
None None TGF-β + IL-6 IL-23 IL-6 + IL-23 presence of 40 mM NaCl. d, IL-23R (GFP)
Sgk1+/+Il23rgfp Sgk1–/–Il23rgfp

None TGF-β + IL-6 IL-23 IL-6 + IL-23 104 104 104 104 104
104
1.33 0.12
104
44.2 0.01
104
4.55 0.74
104
25.3 1.52 103 103 103 103 103 expression in Sgk1–/–Il23rgfp and control CD41 T
103 103 103 103
102 102 102 102
WT
102

101
0.33
102

101
1.54
102

101
2.19
102

101
55.3
102

101
77.6 cells stimulated under conditions described in
101 101 101 101 c. e, Clinical scores of EAE in Cd4CreSgk11/1 and
CD4

0 0 0 0
10 10 10 10 100 0
12.5 0.12 7.64 1.26 100 101 102 103 104
1 2 3
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 10 10 10 10 10
4
IL-17

Cd4CreSgk1fl/fl mice fed with high salt diet (HSD) or

0 0 0 0
10 0 10 10 10
10 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 104 104 104 104 104
104 104 104 104
0.67 0.23 47.1 0.01 2.23 0.59 13.2 1.21 103 103 103 103 103
103 103 103 103
Sgk1–/–
102
0.46
102
0.42
102 102
24.4
102
40.8
control diet (n 5 15). f, Quantification of CD41IL-
102 102 102 102 1.78
101 101 101 101
1.13
101
100
10
101
0
10
101
0
10 100
101
0
101
171 or CD41IFN-c1 T cells from the indicated
organs of Cd4CreSgk11/1 and Cd4CreSgk1fl/fl mice
10 0
10
11.8 0
10 0
0.0110 0 0
8.45 0
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
1 2 3 4 1 2 3 4
100 101 102 103 104 100 101 102 103 104 10 10 10 10 10 10 10 10 10 10
IL-23R GFP
IFN-γ fed HSD or control diet on day 17 after
e 3.5 f Cd4CreSgk1+/+ immunization with MOG35-55–CFA. *P , 0.05
Cd4CreSgk1fl/fl
3 (Student’s t-test). Data are representative of three
Mean clinical score

60 80
CD4+IL-17+ cells (%)

CD4+IFN-γ+ cells (%)

2.5 * * * independent experiments. mLN, mesenteric lymph

2 40
60 node. Error bars, s.d.
*
1.5 40
1 20
* *
20
0.5
0 0 0
7 10 13 16 19 22 25
trl

SD

trl

SD

trl

SD

trl

SD

trl

SD

trl

SD
C

C
H

Time after immunization (d)

Cd4CreSgk1+/+ Cd4CreSgk1+/+ HSD Spleen mLN CNS Spleen mLN CNS
Cd4CreSgk1fl/fl Cd4CreSgk1fl/fl HSD

5 1 6 | N AT U R E | VO L 4 9 6 | 2 5 A P R I L 2 0 1 3
©2013 Macmillan Publishers Limited. All rights reserved

showed a much milder enhancement of TH17 cell frequency in the gut,
whereas there was no increase in IFN-c production in any of the mice
fed with HSD (Supplementary Fig. 8a, b).
Finally, we studied whether HSD would affect the development of
TH17 and EAE in vivo. Wild-type mice fed HSD had more severe EAE
than mice fed a normal diet, and this increased severity was dramat-
ically reduced in SGK1-deficient mice (Fig. 4e and Supplementary Fig.
8c,d). We also observed a significantly higher frequency of TH17 cells
in mesenteric lymph nodes and CNS of wild-type mice fed with HSD
than in those of SGK1-deficient mice fed with HSD. The percentage of
IFN-c producing T cells in the CNS, but not in the peripheral immune
compartments, of wild-type mice was increased in mice fed HSD,
suggesting that HSD may increase infiltration but not expansion of
IFN-c1 effector T cells in the target organs (Fig. 4f and Supplementary
Fig. 8e). Consistent with our in vitro data, we observed elevated IL-17
but not IFN-c production from CD41 T cells isolated from EAE-
immunized wild-type mice fed with HSD and restimulated in vitro
with MOG35-55, compared to production from cells from EAE mice
fed a normal diet (Supplementary Fig. 8f). The data presented here
indicate that high sodium intake potentiates TH17 cell generation in
vivo in an SGK1-dependent manner and therefore has the potential to
increase the risk of promoting autoimmunity.
In conclusion, we used a combination of microarray data analysis,
large-scale PPI network analysis and experimental data from several
different knockout mice to establish IL-23R-SGK1-Foxo1 as a critical
axis in TH17 stabilization. We show that Foxo1 acts as a repressor of IL-
23R expression by binding directly to the Il23r promoter and inhibi-
ting RORct-mediated Il23r transactivation. Phosphorylation of Foxo1,
mediated by SGK1, leads to its deactivation and promotes unopposed
RORct-mediated Il23r transcription. SGK1 has been studied exten-
sively in the context of NaCl transport19,20. Modest increase of the
NaCl concentration induces SGK1 expression in T cells with increased
IL-23R expression and TH17 cell generation in vitro. Interestingly, even
in un-immunized mice fed with HSD, enhancement of TH17 differ-
entiation was observed in vivo in the gut and gut-associated lymphoid
tissue, and this increase in TH17 cells can be recalled at other peripheral
sites after immunization. Although our data suggest an essential role
for SGK1 in this process, it is likely that other immune cells and path-
ways are also influenced by increased salt intake. Furthermore, our
results do not exclude additional alternative mechanisms by which
an increase in NaCl affects TH17 cells. Nevertheless, the elevated in
vivo TH17 differentiation resulting from HSD raises the important
issue of whether increased salt in westernized diets and in processed
foods contributes to an increased generation of pathogenic TH17 cells
and for an unprecedented increase in autoimmune diseases.
METHODS SUMMARY
Microarrays and network analysis. For gene-expression analysis Affymetrix
microarray chips were used. Data were processed using the GenePattern suite21. Dif-
ferentially expressed genes were detected using fold-change and t-test analysis (for
Sgk1–/– and NaCl-treated T cells) or a consensus of fold-change, the EDGE software22
and a novel sigmoid-based method23 (for the Il23r–/– TH17 cell time-course data). A
command-line version of the ANAT software9 was used for network analysis.
In vitro T cell differentiation. Naive T cells were FACS-sorted, stimulated with
plate-bound anti-CD3 and anti-CD28 antibodies and the indicated cytokines or
NaCl, and cells were analysed by qPCR or flow cytometry at different time points.
Experimental autoimmune encephalomyelitis model. Mice were immunized
subcutaneously with MOG35-55 in complete Freud’s adjuvant (CFA), and heat-
inactivated Mycobacterium tuberculosis and with intraperitoneal injection of
Bordatella pertussis toxin.
In vivo cell transfer. Naive T cells were differentiated towards TH17 cells, then
transferred into MOG35-55–CFA-immunized hosts and T cells isolated from vari-
ous organs were analysed by flow cytometry at 7–12 days after onset of EAE.
Western blot and immunoprecipitation. Differentiated T cells or transfected
HEK293T cells were lysed in whole cell extract (WCE) buffer (containing 50 mM
Tris buffer, pH 7.5, 100 mM NaCl, 0.1% Triton X-100, 10% v/v glycerol, 1 mM
DTT, 1 mM PMSF and protease inhibitors (Sigma)), and lysates were subjected to
western blot or immunoprecipitation analysis.
©2013 Macmillan Publishers Limited. All rights reserved
LETTER RESEARCH
Promoter-activity reporter assay. HEK293T cells were transfected with lucifer-
ase reporter constructs and expression vectors, and luciferase expression was
determined after 48 h.
Received 30 May 2012; accepted 6 February 2013.
Published online 6 March 2013.
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Supplementary Information is available in the online version of the paper.
Acknowledgements We thank D. Kozoriz for cell sorting and A. Waisman for providing
Il17f Cre mice. L. Zhou, D. Accili, J. Demoulin and K. Sato provided reagents. This work was
supported by the US National Institutes of Health (NS030843, NS045937, AI073748 and
AI045757 to V.K.K.; 1P01HG005062-01, 1P50HG006193-01 and DP1-OD003958-01
to A.R.; and K01DK090105 to S.X.); the National MS Society (RG2571 to V.K.K.); the
Howard Hughes Medical Institute (A.R.); the Klarman Cell Observatory; Guthy Jackson
Foundation; and the Austrian Science Fund (FWF, J 3091-B12 to T.T.).
Author Contributions C.W, N.Y. and T.T. carried out experiments and wrote the
manuscript. C.Z., S.X. and Y.K. carried out experiments. N.Y. analysed the data. A.R. and
V.K.K. supervised the study and edited the manuscript.
Author Information The microarray data sets have been deposited in the Gene
Expression Omnibus database under accession numbers GSE43956, GSE43957 and
GSE43969. Reprints and permissions information is available at www.nature.com/
reprints. The authors declare no competing financial interests. Readers are welcome to
comment on the online version of the paper. Correspondence and requests for
materials should be addressed to A.R. (aregev@broad.mit.edu) or V.K.K.
(vkuchroo@rics.bwh.harvard.edu).
2 5 A P R I L 2 0 1 3 | VO L 4 9 6 | N AT U R E | 5 1 7
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