Download Development, cytokine profile and function of human interleukin 17

© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
ARTICLES
Development, cytokine profile and function of human
interleukin 17–producing helper T cells
Nicholas J Wilson1, Katia Boniface1, Jason R Chan1,4, Brent S McKenzie1, Wendy M Blumenschein2,
Jeanine D Mattson2, Beth Basham2, Kathleen Smith2, Taiying Chen2, Franck Morel3, Jean-Claude Lecron3,
Robert A Kastelein1, Daniel J Cua1, Terrill K McClanahan2, Edward P Bowman1 & Rene de Waal Malefyt1
TH-17 cells are a distinct lineage of proinflammatory T helper cells that are essential for autoimmune disease. In mice,
commitment to the TH-17 lineage is dependent on transforming growth factor-b and interleukin 6 (IL-6). Here we demonstrate
that IL-23 and IL-1b induced the development of human TH-17 cells expressing IL-17A, IL-17F, IL-22, IL-26, interferon-c, the
chemokine CCL20 and transcription factor RORct. In situ, TH-17 cells were identified by expression of the IL-23 receptor and
the memory T cell marker CD45RO. Psoriatic skin lesions contained IL-23-producing dendritic cells and were enriched in the
cytokines produced by human TH-17 cells that promote the production of antimicrobial peptides in human keratinocytes. Our
data collectively indicate that human and mouse TH-17 cells require distinct factors during differentiation and that human
TH-17 cells may regulate innate immunity in epithelial cells.
CD4+ helper T cells produce large quantities of cytokines in response
to antigen-specific activation. Classically, T helper cells are defined as
T helper type 1 (TH1) or TH2 on the basis of their cytokine-expression
profiles1. TH1 cells develop in response to interleukin 12 (IL-12) and
produce interferon-g (IFN-g), which enhances cellular immunity and
is important for the clearance of intracellular pathogens and bacterial
infection. TH2 cells develop in response to IL-4 and produce IL-4, IL-5
and IL-13, which enhance humoral immunity and are important for
the elimination of parasitic infection2.
The TH-17 subset of T helper cells was identified on the basis of its
ability to produce IL-17A, IL-17F and IL-22 (refs. 3–8). TH-17 cells
provide protection in certain infections but, more importantly, have
also been linked to the development of autoimmune disease, a
function previously assigned to TH1 cells and IFN-g. TH-17 cells
mediate pathology in experimental autoimmune encephalomyelitis,
inflammatory bowel disease and collagen-induced arthritis9–13 and
were first recognized during assessment of the involvement of IL-23 in
autoimmune disease.
IL-23 is a member of the IL-6 family of cytokines; it consists
of a unique p19 subunit coupled to the p40 subunit of IL-12 (ref. 14).
IL-23 may be more important for the survival and population
expansion of TH-17 cells than for TH-17 lineage commitment.
Transforming growth factor-b (TGF-b), in the presence of IL-6, elicits
the differentiation of naive CD4+ T cells into TH-17 cells15–17. The
nuclear receptor RORgt acts as a key transcription factor in this
lineage-commitment process18. In addition, IL-1 and tumor necrosis
factor enhance TH-17 development, whereas IL-4, IFN-g, IL-27 and
IL-2 suppress TH-17 differentiation4,19–22. Although the precise influences of IL-23, TGF-b, IL-6 and other cytokines on the differentiation
and function of TH-17 cells are yet to be fully determined, the
resistance of mice lacking IL-23 to experimental autoimmune
encephalomyelitis, inflammatory bowel disease and collagen-induced
arthritis emphasizes the importance of IL-23 in mediating autoimmune disease.
In addition to its involvement in mediating autoimmune pathology
in the brain, gut and joints, IL-23 can initiate inflammation in the
skin. Repeated intradermal injection of IL-23 results in epidermal
hyperplasia and inflammation that resembles psoriasis23. Persistent
psoriatic lesions in humans contain immune infiltrates, including
dendritic cells (DCs) and T cells, as well as larger amounts of
various cytokines, including IFN-g24. Psoriatic lesions also contain
higher concentrations of small antimicrobial molecules, including
b-defensins and the calcium-binding proteins S100A8 and S100A9,
among others25. These antimicrobial molecules are produced by
epithelial and immune cells and protect the skin against bacterial,
fungal and viral infection26.
Although IL-23 and TH-17 cells contribute to the pathogenesis of
autoimmune disease in mice, their influence on immune function in
humans remains unclear. Here we demonstrate that human naive
CD4+ T cells develop into TH-17 cells in response to IL-23 or IL-1b. In
contrast to what might have been predicted based on experiments in
mice, TGF-b and IL-6 did not induce TH-17 differentiation and even
1Department of Discovery Research and 2Department of Experimental Pathology and Pharmacology, Schering-Plough Biopharma (formerly DNAX Research), Palo Alto,
California 94304-1104, USA. 3Laboratoire Cytokines et Inflammation EA 3806, Universite de Poitiers, Centre Hospitalier Universitaire de Poitiers, Pole Biologie Sante,
Pineau, 86022 Poitiers, France. 4Present address: Entelos, Foster City, California 94404, USA. Correspondence should be addressed to R.d.W.M.
([email protected]).
Received 16 April 2007; accepted 26 June 2007; published online 5 August 2007; doi:10.1038/ni1497
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stimulated the cells for 5–6 d with T cell–activation beads in the
presence of medium, IL-12, IL-23, TGF-b, IL-6, IL-1b, or TGF-b plus
IL-6 and then maintained the cells thereafter with various cytokines
and IL-2 for an additional 5–6 d before reactivating them. Enzymelinked immunosorbent assay (ELISA) showed that IL-23 induced a
TH-17 phenotype, as indicated by the production of IL-17A, IL-17F
and IL-22 (Fig. 1a and Supplementary Fig. 1a online). Moreover,
IL-23-treated CD4+ T cells also produced IFN-g, albeit less than that
produced by TH1 cells. We confirmed these findings by quantitative
RT-PCR analysis (Fig. 1b) and intracellular staining (Supplementary
Fig. 1b online). IL-23 increased the percentage of cells producing
IL-17A or IFN-g. A large proportion of IL-23-derived IL-17Aproducing T cells also produced IFN-g. This observation suggests
that IFN-g production is a characteristic of a subset of human TH-17
cells. IL-2, TGF-b or IL-6 alone did not induce TH-17 differentiation,
although CD4+ T cells cultured with IL-2 alone had high expression of
RESULTS
IL-23 and IL-1b drive the differentiation of human TH-17 cells
As TGF-b, IL-6 and IL-23 are central to the development and function
of mouse TH-17 cells, we first analyzed the involvement of these
cytokines in the development of human TH-17 cells. For comparison,
we examined the cytokine profiles of T cells cultured with IL-2 or
IL-12 (TH1-polarizing conditions). We isolated naive CD4+CD45RA+
T cells from the peripheral blood of normal human donors and
3,000
120
10,000
2,500
100
8,000
350
300
250
200
150
100
50
0
70
2,000
250
40
20
60
50
2
1
2,000
1,500
1,000
500
4,000
200
100
50
IL-6
+ TGF-β
IL
IL 2
-1
2
IL
-2
TG 3
FTG T
β
F- GF IL
-β -6
β
+
+
IL
-6 IL-6
+
IL
-2
3
200
100
α-IL-2
+ α-IL-2R
2
-4
IL
-2
-1
IL
IL
2
3
2
-2
-1
IL
IL
3
-2
-2
IL
2
-1
IL
IL
-2
1
10
10
0
IL-6
1
10
10
0
1
1
10
TGF-β
+ IL-6
300
0
0
0
400
-4
200
150
500
-1
400
200
600
IL-17A (pg/ml)
600
e
250
IL-17A (pg/ml)
800
0.
100
IL
d
1,000
1
1
10
1,500
0
0
1,200
0.
2,000
-2
0
-2
IL
IL 2
-1
2
IL
-2
TG 3
FTG T
β
F- GF I
-β Lβ
6
+
IL + IL
-6
-6
+
IL
-2
3
400
IL
0
IL
50
IL
2,000
TGF-β
100
500
100
IL
IL 2
-1
2
IL
TG 23
FTG T
β
F- GF IL
-β -6
β
+
+
IL
-6 IL-6
+
IL
-2
3
200
6,000
IL
IL 2
-1
2
IL
TG 23
FTG T
β
F- GF IL
-β -6
β
+
+
IL
-6 IL-6
+
IL
-2
3
300
150
2,500
600
IL22 (RU)
8,000
IL
IL 2
-1
2
IL
-2
TG 3
FTG T
β
F- GF I
L
β
+ β + -6
IL
-6 IL-6
+
IL
-2
3
IL
-2
IL
-1
2
IL
-2
TG 3
FTG T
β
F- GF
I
β
L
+ β + -6
IL
-6 IL+ 6
IL
-2
3
10,000
400
IFNG (RU)
500
200
0
0
IL
c
4,000
0
IL
-2
IL
-1
2
IL
-2
TG 3
FTG T
β
F- GF I
β
- L
+ β + -6
IL
-6 IL+ 6
IL
-2
3
Donor 1
IL17A (RU)
b
6,000
IL-17F (pg/ml)
IFN-γ (ng/ml)
500
60
IL17F (RU)
1,000
80
IL-22 (pg/ml)
1,500
IL-17F (pg/ml)
0
2,000
IL-22 (pg/ml)
0
IFN-γ (ng/ml)
0
7,000
6,000
5,000
4,000
3,000
2,000
1,000
0
IL
IL 2
-1
2
IL
TG 23
FTG T
β
F- GF IL
β
+ β + -6
IL
-6 IL-6
+
IL
-2
3
Donor 2
IL-17A (pg/ml)
Donor 1
IL-17A (pg/ml)
a
IL-17A (pg/ml)
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
blocked IL-23-induced development of TH-17 cells. Human TH-17
cells derived in vitro expressed a unique profile of cytokines and
chemokines. We identified TH-17 cells in situ as a subpopulation of
CD4+ memory T cells expressing the IL-23 receptor (IL-23R).
Furthermore, involvement of the IL-23–TH-17 pathway in human
psoriasis indicates an important function for TH-17 cytokines in
regulating the innate immunity of epithelial cells.
IL-23
Figure 1 IL-23, but not a combination of TGF-b and IL-6, promotes the development of TH-17 cells from human naive CD4+ T cells. Naive CD4+ T cells
were activated with beads coated with anti-CD2, anti-CD3 and anti-CD28 and then were cultured for 10–12 d in the presence of IL-2, IL-12, IL-23, TGF-b
and/or IL-6. (a) ELISA of IL-17A, IFN-g and IL-22 and electrochemiluminescence assay of IL-17F in cell-free supernatants of cultured T cells restimulated
for 48 h with beads in the presence of IL-2. (b) Real-time quantitative RT-PCR of transcript expression at 24 h after activation, presented relative to
expression of transcripts encoding ubiquitin (RU, relative units). (c) ELISA of IL-17A production induced by TGF-b alone (0.1–10 ng/ml) or in combination
with IL-6 (30 ng/ml), or IL-6 alone (1–100 ng/ml) or in combination with TGF-b (10 ng/ml). (d) ELISA of IL-17A production induced by various cytokines
(horizontal axis) in the presence or absence of neutralizing anti-IL-2 (a-IL-2; 10 mg/ml) and anti-IL-2R (a-IL-2R; 10 mg/ml). (e) ELISA of IL-17A production
induced by various combinations of cytokines (horizontal axis). Data are representative of five (a), three (b,e) or two (c,d) experiments; error bars,
mean + s.e.m. of duplicates.
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2,000
6,000
4,000
2,000
1β
1β
+
IL
-
23
23
IL
-
IL
IL
-
3
β
-1
-2
β
+
IL
IL
3
2
-2
IL
-1
+ β
IL
-2
3
-1
IL
IL
IL
-1
β
IL
2
3
-2
IL
-1
-2
IL
IL
3
β
-1
-2
-1
β
+
IL
IL
3
-2
IL
-1
2
0
-1
200
-2
400
IL
600
7,000
6,000
5,000
4,000
3,000
2,000
1,000
0
IL
4,000
IL17F (RU)
8,000
-2
2
IL
+
IL
-
IL22 (RU)
800
IL
500
1β
1β
1,000
12,000
**
1,000
IL
-
23
23
1β
IL
-
IL
-
2
12
IL
-
+
IL
-
1β
23
23
16,000
IL
**
1,500
0
IL
-
IFNG (RU)
500
2,000
8,000
IL
-
IL
-
IL
-
2
12
IL
-
23
+
3
-2
IL
1,000
0
0
+
β
1,500
12
10,000
IL-22 (pg/ml)
70
60
50
40
30
20
10
0
1β
-1
β
-1
IL
4,000
0
IL
3
-2
IL
IL
2
-1
IL
6,000
0
0
-2
Donor 2
300
250
200
150
100
50
0
IL
b
2,000
IL
-
10
*
IL-17F (pg/ml)
20
*
IL-17F (pg/ml)
8,000
IL-22 (pg/ml)
10,000
30
IL
-
23
1β
IL
-
IL
-
2
IL
-
12
IFN-γ (ng/ml)
1,400
1,200
1,000
800
600
400
200
0
40
IL
-
IFN-γ (ng/ml)
1,200
1,000
800
600
400
200
0
IL
-
IL-17A (pg/ml)
Donor 2
IL17A (RU)
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
Donor 1
IL-17A (pg/ml)
a
Figure 2 IL-23 and IL-1b drive the differentiation of human TH-17 cells. Naive CD4+ T cells were activated with beads coated with anti-CD2, anti-CD3
and anti-CD28 and were then cultured for 10–12 d in the presence of IL-2, IL-12, IL-23 and/or IL-1b. (a) ELISA of IL-17A, IFN-g and IL-22 and
electrochemiluminescence assay of IL-17F in cell-free supernatants of T cells restimulated for 48 h with coated beads in the presence of IL-2. *, over
10,000 pg/ml; **, over 2,000 pg/ml. (b) Real-time quantitative RT-PCR of transcripts encoding IL-17A, IFN-g, IL-22 and IL-17F at 24 h after activation,
presented relative to expression of transcripts encoding ubiquitin. Data are representative of seven (a) or three (b) experiments.
IL-22. These data suggest a positive effect of IL-2 on IL-22 production.
In agreement with published data7, IL-6 enhanced and TGF-b
inhibited IL-22 production in these conditions (Fig. 1). Notably,
over a wide range of concentrations, TGF-b, IL-6 or a combination
of these cytokines did not induce the differentiation of human TH-17
cells (Fig. 1c). In fact, these cytokines actually inhibited the
IL-23-induced production of IL-17A, IL-17F, IL-22 and IFN-g
(Fig. 1a). These data collectively indicate the existence of a speciesspecific divergence in cytokine action.
Analysis of the function of IL-2 in the differentiation of human
TH-17 cells demonstrated additional differences in the development of
TH-17 cells in humans versus mice22 (Fig. 1d). Neutralization of IL-2
completely inhibited the development of human TH-17 cells. This
effect was not specific to the TH-17 lineage, as the development of TH0
and TH1 cells was also impaired by the removal of IL-2, indicating that
IL-2 may be required for T cell survival. In contrast, other cytokines
exerted similar effects on the development of TH-17 cells in humans
and mice; both IL-12 and IL-4 prevented IL-23-induced TH-17
development (Fig. 1e).
As expected, TH1 cells developing in the presence of IL-12 produced
large quantities of IFN-g and small amounts of IL-17A, IL-17F and
IL-22. The production of IL-17A, IL-17F and IL-22 in response to
IL-12 was generally lower than that of cultures with IL-2 alone,
suggesting that IL-12 suppresses the differentiation of human TH-17
cells (Fig. 1a and Supplementary Fig. 1a–c). When cultured with IL-2
alone, a small percentage of T cells produced either IL-17A or IFN-g;
only a few cells produced both cytokines. IL-12 strongly enhanced the
percentage of cells producing IFN-g as well as IFN-g production
on a per-cell basis; IL-12 also suppressed the percentage of IL-17Aproducing cells (Supplementary Fig. 1b).
Like IL-23, IL-1b induced the expression of IL-17A, IL-17F, IL-22
and IFN-g by naive CD4+ T cells (Fig. 2a). The IL-1b-induced
952
increase in IFN-g production was modest compared with that induced
by IL-12, but IL-1b induced more IL-17F production than that
induced by IL-23. A combination of IL-23 and IL-1b did not result
in additive or synergistic effects. We confirmed these results by real
time RT-PCR analysis (Figs. 1b and 2b). These findings suggest that
IL-23 and IL-1b, but not TGF-b and/or IL-6, promote the in vitro
differentiation and/or population expansion of human TH-17 cells
that produce IL-17A, IL-17F, IL-22 and IFN-g.
Cytokine profile of human TH-17 cells
As IL-23 promoted the population expansion of CD4+ T cells
producing IL-17A, IL-17F and IL-22, we searched for additional
cytokines and chemokines produced by these cells that could further
define this cell type. We used Affymetrix microarrays to analyze the
transcription of genes encoding cytokines and chemokines in CD4+
T cells cultured in various conditions. IL17A was the gene most
induced by IL-23; however, IL22 and IL26 and CCL20 (encoding the
chemokine CCL20) had expression profiles similar to IL17A (data not
shown). We confirmed the microarray data by quantitative RT-PCR
(Supplementary Fig. 1c,d). IL-23 strongly upregulated the expression
of IL17A, IL17F, IL22, IL26 and CCL20, whereas expression of these
genes was downregulated or unaffected by IL-12. In addition, IL-23
upregulated whereas IL-12 downregulated expression of the gene
encoding human RORgt. Again, IL-23 and IL-12 promoted the
expression of IFN-g mRNA and protein (Supplementary Fig. 1e).
An extended time-course experiment tracking the appearance of
this cytokine profile showed that both IL-12 and IL-23 induced IFNG
mRNA expression on days 6, 12 and 16 (Supplementary Fig. 1f).
Transcripts of IL17A, IL17F, IL22 and IL26 mRNA were not modulated
by IL-23 at day 6, but all were higher after stimulation by days 12 and
16. IL-12 suppressed the expression of IL17A and IL22 at all time
points tested and did not induce the expression of IL17F or IL26 at any
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T cells had higher expression of IL17A, IL17F, IL22, IL26 and CCL20
mRNA than their IL-23R– counterparts (Fig. 3f). These data show
that a population of IL-23R+CD4+ memory T cells present in the
peripheral blood of healthy humans is able to produce large quantities
of IL-17A and IFN-g and to have high expression of IL17A, IL17F,
IL22, IL26 and CCL20 after T cell receptor stimulation with beads
coated with anti-CD3, anti-CD28 and anti-CD2. The coordinated
expression of this set of molecules in a naturally occurring T cell
population defines a ‘signature’ cytokine and chemokine profile of
human TH-17 cells.
IL-23R defines human TH-17 cells in situ
Given that IL-23 induced TH-17 populations in vitro and that IL-23
stimulates the proliferation of CD4+ memory T cells rather than naive
CD4+ T cells14, we hypothesized that circulating human TH-17 cells
should include a population of memory CD4+ T cells expressing
IL-23R. To determine whether IL-23R expression defines a population
of circulating TH-17 cells, we analyzed the expression of IL-23R as well
as CD45RA (expressed on naive human T cells) and CD45RO
(expressed on memory human T cells) on freshly isolated CD4+ T
cells from healthy human donors. A subset of human CD4+CD45RA–
CD45RO+ memory T cells but not CD4+CD45RA+CD45RO– naive
T cells expressed IL-23R (Fig. 3a). The stimulation of freshly isolated
human CD4+CD45RO+ memory T cells with IL-23 increased IL-17A
and IFN-g production in a dose-dependent way (Fig. 3b,c), indicating
that IL-23R expressed on human CD4+ memory T cells is functional.
In addition, IL-23 induced slight cellular proliferation (Fig. 3d). Next
we sorted human CD3+CD4+CD45RO+ memory T cells into IL-23R+
and IL-23R– fractions and activated the cells with beads coated with
antibody to CD3 (anti-CD3), anti-CD28 and anti-CD2 in the presence
of IL-2. ELISA showed that IL-23R+ memory T cells from healthy
donors produced more IL-17A than did IL-23R– memory T cells,
whereas both IL-23R+ and IL-23R– T cells produced large quantities of
IFN-g (Fig. 3e). Furthermore, real-time RT-PCR showed that IL-23R+
200
3.27
120
80
40
c
0
100 101 102 103 104
CD45RA
80
40
0
100 101 102 103 104
CD45RO
[H] (c.p.m. × 103)
120
3
2
1
7
6
5
4
3
2
1
0
100
250
60
150
20
0
50
0
40
Donor 4
1,600
1,200
800
20
1 10 100
IL-23 (ng/ml)
4
400
–
+
–
+
–
0
+
0.45
6
10
9
0.35
5
4
8
7
6
3
2
5
4
3
1
2
0.25
0.15
0.05
0
0
d
13.43
160
0
60
0
0
3
200
20
0
IFN-γ (ng/ml)
IL-23R
160
40
Donor 3
350
–
0
+
–
0
+
–
1
0
+
–
+
–
+
1 10 100
IL-23 (ng/ml)
f
500
4,000
450
160
400
3,000
350
120
300
200
100
0
1 10 100
IL-23 (ng/ml)
0
–
+
2,000
1,000
0
–
+
250
150
50
0
–
+
90
CCL20 (RU)
0
100 101 102 103 104
CD4
60
Donor 2
140
IL26 (RU)
40
Donor 1
80
IL22 (RU)
80
80
The TH-17 cytokine profile of human psoriasis
Like TH1 cells, TH2 cells and regulatory T cells, each of which
has a specific cytokine profile that dictates cellular function, human
TH-17 cells may influence particular immune functions and pathological processes through the coordinated expression of IL-17A,
IL-17F, IL-22, IL-26 and CCL20. Published data indicate that transcripts encoding IL-23p19 and IL-12p40, the subunits that compose
human IL-23, are expressed during human psoriasis23,27, a T cell–
mediated inflammatory disease of the skin. Analysis of lesional skin
sections from patients with psoriasis (with psoriasis activity skin index
scores (indicating disease severity) of 8.5–26) by immunohistochemistry with anti-IL-23p19 demonstrated IL-23p19 protein in cells with
DC morphology distributed throughout the dermis (Fig. 4a).
Costaining with an antibody to DC-LAMP, a protein expressed
specifically in lysosomes of DCs, confirmed that these cells were
DCs (Fig. 4b). These IL-23p19-expressing DCs were not present in
IL17F (RU)
IL-17A (pg/ml)
IL-23R
120
e
100
IL-17A (pg/ml)
b
15.75
160
IFN-γ (ng/ml)
200
IL17A (RU)
a
IL-23R
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
time point. These data indicate that IL-23 regulates the expression of a
specific set of cytokines and chemokines that includes IL-17A, IL-17F,
IL-22, IL-26, IFN-g and CCL20.
80
40
0
–
+
70
50
30
10
0
–
+
Figure 3 Human memory CD4+ T cells express IL-23R, produce substantial amounts of IL-17A and express the same set of cytokines as in vitro–polarized
TH-17 cells. (a) Flow cytometry of CD4+ T cells isolated from peripheral blood. Numbers in plots indicate percent IL-23R+CD4+ cells (top), IL-23R+CD45RA+
cells (middle) or IL-23R+CD45RO+ cells (bottom). Data are from one experiment per donor and are representative of four experiments with four different
donors. (b,c) ELISA of IL-17A (b) and IFN-g (c) in cell-free supernatants of CD4+CD45RO+ memory T cells cultured for 3 d with IL-2 alone or in the
presence of increasing concentrations of IL-23. Data represent three experiments. (d) Proliferation of CD4+CD45RO+ memory T cells treated for 3 d with
IL-23, assessed by incorporation of [3H]thymidine. Data represent three experiments. (e,f) ELISA of IL-17A and IFN-g in supernatants of CD3+CD4+
CD45RO+IL-23R+ T cells (+) and CD3+CD4+CD45RO+IL-23R– T cells (–) sorted from peripheral blood and activated for 5 d with beads coated with antiCD2, anti-CD3 and anti-CD28 in the presence of IL-2. Data are from four experiments with four different healthy donors. (f) Real-time quantitative RT-PCR
of the expression of IL-23-regulated genes among mRNA collected after 5 d, presented relative to the expression of transcripts encoding ubiquitin. Data are
from one experiment per donor and are representative of two experiments with two different donors. Error bars, mean + s.d. of triplicates.
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a
Donor 1
Donor 3
Donor 3
IL-23p19
b
TH-17 cytokines induce antibacterial peptides
IL-17A, IL-22 and IL-26 regulate immunity in various epithelial cell
types by enhancing the expression of genes encoding proinflammatory
cytokines and antimicrobial molecules28–31. Psoriatic lesions contain
larger amounts of antimicrobial peptides, including b-defensin 2,
b-defensin 3, and the calcium-binding proteins S100A7 (psoriasin),
S100A8 and S100A9, than does healthy skin25. We confirmed that both
lesional and nonlesional skin of patients with psoriasis had higher
expression of the genes encoding b-defensin 2 (Fig. 4c) and
b-defensin 3 (data not shown) than did healthy skin. To assess whether
there was a relationship between the production of TH-17 cytokines
and the higher expression of genes encoding antimicrobial products,
we examined the regulation of the expression of antimicrobial peptides in normal human epithelial keratinocytes (NHEKs). Genes
encoding the receptors for IL-17A, IL-17F and IL-22 were expressed
in NHEKs, whereas we did not detect the gene encoding a component
of the IL-26 receptor (Fig. 5a). These data indicate that NHEKs may
be responsive to most cytokines produced by human TH-17 cells.
Indeed, IL-17A, IL-17F and IL-22 induced in NHEKs, in a dosedependent way, expression of the genes encoding b-defensin 2 and
b-defensin 3, as well as S100A8 and S100A9 (Supplementary Fig. 2
online), in accordance with published reports8,30. IL-17A most
NL
L
***
1.45
1.20
0.95
0.70
0.45
0.20
–0.05
N
NL
L
2.25
1.75
1.25
0.75
0.25
–0.25
–0.75
–1.25
*
N
***
NL
L
***
3.0
2.5
2
1.5
1
0.5
N
NL
L
IL22 expression
(log transformed)
N
IL-23p19 + DC-LAMP
1.25
0.75
0.25
–0.25
–0.75
–1.25
–1.75
–2.25
DEFB4 expression
(log transformed)
***
**
IL17F expression
(log transformed)
0
1.5
1.0
0.5
0.0
–0.5
–1.0
–1.5
–2.0
–2.5
DC-LAMP
IL1B expression
(log transformed)
***
1
RORC expression
(log transformed)
2
IL17A expression
(log transformed)
IL-23p19
c
–1
skin from healthy donors (data not shown).
–2
These data confirmed that IL-23 is expressed
N
NL L
in human psoriatic skin and suggest that DCs
***
*
1.5
are a source of IL-23. Next we measured the
1.0
0.5
0.0
expression of IL17A, IL17F, IL22, IL26 and
–0.5
–1.0
IFNG in ‘punch biopsies’ of nonlesional and
–1.5
–2.0
–2.5
lesional skin from a large group of patients
N
NL
L
with psoriasis and of skin of healthy donors.
IL17A, IL17F, IL22, IL26 and IFNG had significantly higher expression in lesional skin than in nonlesional or
healthy skin (Fig. 4c). Notably, transcripts encoding IL-1b were
also upregulated in psoriatic skin lesions, and, consistent with the
existence of an IL-23–TH-17 pathway, the gene encoding RORgt
was also upregulated in psoriatic skin. These data suggest that
IL-23, which is expressed by DCs in the skin of patients with
psoriasis, may promote the development and activation of TH-17
cells, which might account for the increase in TH-17 cytokines in
psoriatic lesions.
954
Donor 2
Isotype
IFNG expression
(log transformed)
Figure 4 IL-23p19 and TH-17 effector
cytokines are associated with human psoriasis.
(a) Immunohistochemistry of IL-23p19 in
psoriatic skin samples from three donors. Scale
bars, 50 mm. Far right, enlargement of adjacent
image (original magnification, 400).
(b) Costaining of psoriatic skin samples with
Texas Red–conjugated anti-IL-23p19 and
fluorescein isothiocyanate–conjugated anti-DCLAMP. Arrows indicate positive staining for
IL-23p19 and DC-LAMP. Original magnification,
800. (c) Real timeRT-PCR of the expression
of transcripts encoding IL-17A, IL-17F, IL-22,
IL-26, IFN-g, RORgt (RORC), IL-1b and bdefensin 2 (DEFB4) in normal human skin (NS)
and in nonlesional (NL) and lesional (L) human
psoriatic skin. Each dot represents one specimen;
horizontal bars indicate the median. *, P o
0.05; **, P o 0.01; ***, P o 0.001. Data are
representative of three experiments (a,b) or at
least one experiment per gene (c).
IL26 expression
(log transformed)
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
ARTICLES
5
4
3
2
1
0
–1
***
N
NL
L
***
***
N
NL
L
potently induced the expression of this set of genes, whereas IFN-g
did not substantially modulate their expression (Supplementary
Fig. 2 and data not shown).
To address the physiological importance of those findings, we
obtained supernatants from activated IL-23-differentiated TH-17
cells or T cells derived from lesional skin of patients with psoriasis
and compared their effects on the expression of antimicrobial proteins
and their genes. TH-17 supernatants induced higher expression of
these genes in NHEKs than did TH1 or TH0 supernatants (Fig. 5b–d).
More notably, neutralization of IL-17A in TH-17 cell supernatants
suppressed the induction of b-defensin 2 to the degree induced by TH0
supernatants, but neutralization of IFN-g did not (Fig. 5e), suggesting
a central function for IL-17A in the expression of this antimicrobial
peptide. Similarly, neutralization of IL-17A completely blocked the
expression of genes encoding antimicrobial proteins in NHEKs
induced by supernatants of psoriatic skin T cells (Fig. 5f). These
results show that IL-17 from in vitro–derived and disease-associated
TH-17 cells is crucial for the induction of antimicrobial peptides in
NHEKs. Our results collectively indicate that the IL-23–TH-17 pathway is important for innate epithelial immunity and is involved in
human psoriasis.
DISCUSSION
Here we have examined the development, cytokine and chemokine
profile, and function of human TH-17 cells. Our data have shown
that IL-23 and IL-1b are able to drive human naive CD4+ T cells
toward a TH-17 phenotype. Studies of the differentiation of mouse
TH-17 cells have established requirements for TGF-b in combination
with IL-6 (refs. 15–17) and have demonstrated that IL-23 may be
required for the population expansion and/or survival of TH-17 cells
rather than for polarization toward the TH-17 lineage. In contrast
to those data generated in mice, here we found that when used in
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NATURE IMMUNOLOGY
0
TH0 TH1 TH-17
20
10
30
20
10
0
0
TH0 TH1 TH-17
β-defensin 2
(pg/ml)
500
30
e
40
900
500
300
100
0
TH0 TH1 TH-17
TH0 TH1 TH-17
f
500
400
TH0
TH-17
700
IgG
S100A7
('fold increase')
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
IL
17
IL RA
17
IL RC
10
IL RB
2
IL 0RA
22
R
A1
0
1,000
d
40
DEFB4
('fold increase')
100
c
1,500
6
5
4
3
2
1
0
S100A9
(RU × 10 3 )
200
b
S100A8
(RU × 10 3 )
300
β-defensin 2
(pg/ml)
400
DEFB4 (RU × 10 3 )
a
Expression (RU)
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α-IL-17 α-IFN-γ
12
10
8
6
4
2
0
300
Figure 5 TH-17 effector cytokines elicit the expression of antimicrobial molecules from human
200
keratinocytes. (a) Quantitative PCR of the expression of transcripts encoding the receptors for IL-17A
100
(IL17RA and IL17RC), IL-17F (IL17RC) and IL-22 (IL10RB and IL22RA), as well as a component of
0
the IL-26 receptor (IL20RA), in NHEKs. (b–d) Real-time RT-PCR of transcripts encoding b-defensin
IgG
α-IL-17
IgG
α-IL-17
2 (b, left), S100A8 (c) and S100A9 (d) and ELISA of b-defensin 2 protein (b, right) in human
Psoriatic T cells
Psoriatic T cells
keratinocytes stimulated for 48 h with conditioned media from CD4+ T cells cultured with IL-2 (TH0),
IL-12 (TH1) or IL-23 (TH-17). RT-PCR results are relative to the expression of transcripts encoding ubiquitin. (e) ELISA of b-defensin 2 in supernatants of
NHEKs stimulated for 48 h with conditioned media from CD4+ T cells cultured with IL-2 (TH0) or IL-23 (TH-17) in the presence of control IgG (20 mg/ml),
anti-IL-17A or anti-IFN-g (20 mg/ml). (f) Real time RT-PCR of b-defensin 2 and S100A7 transcripts in NHEKs stimulated for 48 h with supernatants of
T cells isolated from lesional psoriatic skin in the presence of control IgG (40 mg/ml) or anti-IL-17A (40 mg/ml). Results are relative to those of the control
culture. Data are representative of four (a, mean + s.d.) or two (e,f) experiments or four experiments with different donors (b–d).
similar, physiologically relevant concentrations, TGF-b and IL-6
did not induce the development of human TH-17 cells. Instead,
our data showed that a combination of TGF-b and IL-6 reduced the
IL-23-driven production of IL-17A, IL-17F and IL-22. We confirmed
that IL-6 alone enhanced the IL-2-induced production of IL-22 by
human CD4+ T cells7; this IL-2-induced IL-22 production was
also inhibited by TGF-b. These results indicate that a combination
of TGF-b and IL-6 is not sufficient to drive TH-17 differentiation in
the human system and suggest instead that TGF-b may actually
suppress the differentiation of human TH-17 cells. We also found
that neutralization of IL-2, which enhances the differentiation of
mouse TH-17 cells22, did not enhance the development of human
TH-17 cells. However, IL-12 and IL-4 exerted similar effects on the
differentiation of human and mouse TH-17 cells. These data emphasize some critical differences and similarities in human and mouse
TH-17 cell biology.
Our in vitro studies showed that IL-23 expands a population of
human T helper cells (TH-17 cells) with a cytokine and chemokine
expression profile distinct from that of TH1 or TH2 cells. These human
TH-17 cells produced IL-17A, IL-17F, IL-22, IL-26 and CCL20.
It is well established that IL-23 regulates the expression of IL-17A,
IL-17F and IL-22 in mouse CD4+ T cells3,7,8; however, we found
that IL-23 also induced the production of IL-26 and the chemokine
CCL20 in human CD4+ T cells. Our data confirm published findings
describing the production of IL-22 and CCL20 by mouse TH-17
cells8,9. In addition, we have identified a population of
CD4+CD45RO+ memory T helper cells present in human peripheral
blood that expressed IL-23R and produced more IL-17A than their
IL-23R– counterparts. These IL-23R+ memory helper T cells had the
same distinct ‘signature’ cytokine and chemokine profile as in vitro–
polarized TH-17 cells, suggesting that human helper T cells derived
by IL-23 in vitro constitute an accurate representation of a subset
of helper T cells normally present in the peripheral blood of
healthy humans.
Pathologies associated with TH-17 cells probably occur after dysregulation of the appropriate ‘checks’ that control TH-17 cell proliferation or cytokine production. That idea is supported by the
observation that ubiquitous overexpression of the p19 subunit of
IL-23 in mice results in multiorgan inflammation, including infiltration of lymphocytes and macrophages into the liver, lungs, digestive
tract and skin32. In addition, intradermal injection of IL-23 into mice
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results in a psoriasis-like phenotype characterized by epidermal
thickening, cellular infiltrates and greater production of proinflammatory cytokines, including the TH-17 cytokines IL-17A, IL-17F and
IL-22. The human autoimmune disease psoriasis presents many of the
features associated with overexpression or intradermal injection of
IL-23 in mice, including epidermal hyperplasia and infiltration of
macrophages, DCs and lymphocytes into lesional skin23,24,32. Moreover, a genome-wide screen has identified the gene encoding IL-23R as
a ‘psoriasis-susceptibility gene’33. Here we found that lesional skin
samples from patients with psoriasis, but not skin samples from
healthy donors, contained DCs that expressed IL-23p19. These data
confirm published results showing that IL23 mRNA is upregulated in
psoriasis23,24,27 and suggest that IL-23 may participate in the pathology of psoriasis. Supporting that conclusion, a phase 2 clinical trial
has shown that an antibody to IL-12p40 that neutralizes both IL-12
and IL-23 demonstrates efficacy in patients with psoriasis34. We found
that the TH-17-derived cytokines IL-17A, IL-17F, IL-22 and IL-26 were
more abundant in lesional and nonlesional skin of patients with
psoriasis than in normal skin. As IL-23p19 and IL-23-derived
TH-17-associated molecules are more abundant in human psoriatic
skin than in normal skin and IL-23 induces psoriasis-like disease in
mice23, psoriasis may be considered a TH-17-associated disease rather
than a TH1-associated disease.
TH-17 cells probably have a specific role in normal immune function
through the coordinated action of their effector cytokines and chemokines, similar to the well established functions of TH1 and TH2 cells in
regulating cellular immunity and antibody production. The ‘signature’
cytokine and chemokine profile of TH-17 cells suggests that these cells
regulate the immune function of epithelial cells rather than cells of the
classical immune system. The IL-23-regulated expression of IL-17A,
IL-17F, IL-22 and IL-26 by a subset of CD4+ T cells is particularly
notable, as receptors for IL-17A, IL-17F, IL-22 and IL-26 are expressed
on the epithelial and stromal cells of tissues that include skin, lung,
colon and brain12,28,29,31,35. The nonhematopoietic expression of the
receptors for these cytokines suggests that they function on nonimmune cells to modulate host defense. Therefore, IL-23-derived TH-17
cells provide a link in communication between the adaptive immune
system and nonspecific immunity in epithelial cells.
It is becoming increasingly apparent that epithelial cells are key to
host defense through the production of inflammatory cytokines and
antimicrobial molecules26,36. The antimicrobial peptides produced by
955
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
ARTICLES
epithelial cells include the b-defensins and S100A7, S100A8 and
S100A9. These molecules protect surfaces exposed to the external
environment, such as those lining the skin, lungs and gut, against
bacterial, fungal and viral infection. The IL-23-regulated cytokines
IL-17A, IL-22 and IL-26 regulate the expression of antimicrobial
peptides and proinflammatory cytokines in various epithelial cell
types30,31,37,38. Our data have shown that b-defensin 2 and b-defensin
3 were upregulated in skin of patients with psoriasis and that the
TH-17 cytokines IL-17A, IL-17F and IL-22 induced the expression of
b-defensin 2, b-defensin 3, S100A8 and S100A9 in NHEKs; in contrast
the TH1 cytokine IFN-g had minimal effects. Moreover, supernatants
from activated IL-23-derived TH-17 cells enhanced the expression of
these antimicrobial peptides in NHEKs. In addition, anti-IL-17A, but
not anti-IFN-g, suppressed the production of b-defensin 2 induced by
TH-17 culture supernatants. These data suggest that although IFN-g is
present in TH-17 supernatants, IL-17A rather than IFN-g is critical for
the production of b-defensin 2 by NHEKs. In support of that
conclusion, of the cytokines present TH-17 supernatants, IL-17A
most potently elicited b-defensin 2 from NHEKs; IL-17F and IL-22
also functioned, albeit less efficiently, in this capacity. The function of
IL-26 awaits clarification; however, the expression pattern of the IL-26
receptor indicates that IL-26 may be more prominent in colon or
brain than in the skin37,39.
In addition to the small antibacterial molecules induced by TH-17derived cytokines, the TH-17-selective chemokine CCL20 also has
direct antimicrobial activity40,41. CCL20 and b-defensin 2 share direct
antimicrobial activity against certain bacteria and, despite having
minimal sequence similarity, CCL20 and b-defensin 2 bind to the
G protein–coupled receptor CCR6 (ref. 40). CCL20 enhances the
migration of Langerhans cells and immature CD11b+ DCs from
Peyer’s patches, as well as memory and effector T cells that home to
skin and mucosal surfaces42–44; b-defensin 2 also induces the chemotaxis of DCs and T cells by means of the chemokine receptor CCR6
(ref. 45). Thus, it is possible that CCL20 produced by TH-17 cells not
only directly enhances epithelial immunity via its antimicrobial
activity but also contributes to the infiltration of inflammatory cells
in IL-23-mediated autoimmune diseases.
It seems that TH-17 cells are uniquely suited to initiate host defense
at epithelial surfaces through the coordinated action of cytokines
(IL-17A, IL-17F, IL-22 and IL-26) that induce different classes of
antimicrobial molecules such as S100A7 and b-defensin 2, which
along with CCL20 have dual functions in direct microbial killing
as well as attracting immature DCs and effector and memory
T cells. Specific functions for the IL-23 and IL-17A pathway in the
homeostatic regulation of granulopoiesis46 and in the protection
against certain bacterial infections such as Klebsiella pneumoniae47,48
and Cryptococcus neoformans49 have been described. Our results
further extend the involvement of TH-17 cells in innate immunity
to neutrophil-independent mechanisms of antimicrobial protection.
METHODS
Isolation and culture of human naive CD4+ T cells. Peripheral blood
mononuclear cells were prepared from buffy coats obtained from healthy
donors (Stanford Blood Center) by centrifugation through Ficoll (Histopaque
1077; Sigma). CD4+ T cells were isolated by two rounds of magnetic bead
depletion of CD19+, CD14+, CD56+, CD16+, CD36+, CD123+, CD8+, T cell
receptor-g and T cell receptor-d–positive and glycophorin A–positive cells
(CD4+ T Cell Isolation Kit II) on an AutoMACS instrument (Miltenyi Biotec).
Subsequently, naive CD45RA+ T cells were obtained by two rounds of depletion
with anti-CD45RO magnetic beads (Miltenyi Biotec). T cells (CD4+CD45RO–)
were cultured for 5–6 d in 96-well flat-bottomed plates (Falcon) at a density of
1 105 cells per well in Yssel’s media containing human AB serum (Gemini
956
Bio-Products) along with beads coated with anti-CD2, anti-CD3 and anti-CD28
(1 bead per 10 cells; T Cell Activation/Expansion Kit, Miltenyi Biotec) in
nonpolarizing conditions (no cytokines), TH1-polarizing conditions (human
IL-12 (5 ng/ml); R&D Systems) or TH-17-polarizing conditions (human IL-23
(50 ng/ml; DNAX) and human IL-1b (50 ng/ml; R&D Systems)), or with or
without IL-6 (30 ng/ml; R&D Systems) and TGF-b (10 ng/ml; R&D Systems).
Cells were split and were cultured for an additional period of 5–6 d in the
presence of various cytokines and IL-2 (100 U/ml; R&D Systems). Where
indicated, after 10–12 d cells were cultured for an additional 5–6 d with IL-2
and the indicated cytokines and were analyzed (on days 15–18) or additional
reagents, including human IL-4 (10 ng/ml; DNAX), anti–human IL-2 (17H2;
10 mg/ml; DNAX) and anti–human IL-2R (B-B10; 10 mg/ml; Diaclone), were
added. For analysis of cytokine production, 5 105 cells per ml were
stimulated with T cell–activation beads in the presence of IL-2 and cells or
culture supernatants were collected at 24 h (for RNA) or 48 h. The analysis of
T cell proliferation is described in the Supplementary Methods online.
Isolation and culture of T cells from psoriatic skin. T cells that had infiltrated
lesional psoriatic skin were isolated with Expander beads (Dynal) and were
cultured for 10–14 d in the presence of IL-2 (ref. 50). T cells (2 106 cells per
ml) were activated for 24 h with anti-CD3 (SPV-T3b; Beckman Coulter) and
anti-CD28 (L293; BD Biosciences) and cell-free supernatants were collected.
NHEK culture. NHEKs (Cambrex) were cultured for 48 h with or without
20% T cell culture supernatants in the presence or absence of anti-IL-17A
(20 mg/ml (16C10; DNAX) or 40 mg/ml (41809; R&D Systems)) or anti-IFN-g
(B27; 20 mg/ml; DNAX).
ELISA and electrochemiluminescence. ELISA of IFN-g was done with antibodies from BD Biosciences (BD551221 and BD554550). IL-22 and CCL20
ELISA kits were from R&D Systems, and the b-defensin 2 ELISA kit was from
Phoenix Pharmaceuticals. The IL-17A ELISA and IL-17F electrochemiluminescence assay were developed ‘in-house’ with anti-IL-17A (430D10 and 12B12;
DNAX) and with biotinylated anti-IL-17F (JL20-21A11.B5; DNAX) and polyclonal anti-IL-17F (AF1335; R&D Systems), respectively, and are described in
the Supplementary Methods online.
Immunohistochemistry of skin tissue. The acquisition of human skin specimens is described in the Supplementary Methods online. Paraffin sections from
normal and lesional psoriatic skin tissue were fixed in 4% (wt/vol) neutralbuffered formalin, then were incubated at 25 1C with 2% (vol/vol) human
serum, 4% (vol/vol) normal horse serum and avidin-biotin (Vector Labs) and
were immunostained for 2 h with anti-IL-23p19 (12F12; DNAX) or isotype
control antibody (5 mg/ml; BD550878; BD Biosciences). After being washed in
1% (wt/vol) BSA in PBS, slides were incubated for 1 h with biotinylated goat
anti-rat (10 mg/ml; BA4000; Vector Labs), followed sequentially by Vector ABC
elite peroxidase complex (45 min) and Vector Nova Red substrate. Sections
were then counterstained with hematoxylin before being mounted. For
costaining experiments, frozen sections of lesional psoriatic skin were blocked
with 4% (vol/vol) horse serum and were incubated with anti–human IL-23p19
(12F12; DNAX) or mouse immunoglobulin G1 (IgG1; 20 mg/ml; BD550878;
BD Biosciences), followed by Texas Red–conjugated goat anti–mouse IgG
(Jackson ImmunoResearch). Slides were blocked with 5% (vol/vol) mouse
serum (Sigma) and were incubated with fluorescein isothiocyanate–conjugated
anti-DC-LAMP (104.G4; Immunotech), were mounted in DAPI (4,6-diamidino-2-phenylindole) mounting medium and were examined with a Leica TCS SP
confocal microscope.
Flow cytometry and cell sorting. For sorting of IL-23R+ CD4+ T cells, CD4+
T cells were partially purified (over 95% purity) from peripheral blood
mononuclear cell samples with the RosetteSep human CD4+ T cell enrichment
kit (Stem Cell Technologies) according to the manufacturer’s instructions.
The CD4+ T cell population (1 108 cells) was stained for 1 h on ice with
anti-CD3 (MHCD0306; CalTag), anti-CD45RO (BD555492; BD Biosciences)
and anti-IL-23R (BAF1400; R&D Systems) and was washed with PBS.
CD3+CD45RO+IL-23R+ and CD3+CD45RO+IL-23R– populations were sorted
to over 99% purity with a FACSvantage cell sorter (Becton Dickinson).
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RNA isolation, real-time quantitative PCR, and intracellular staining. These
procedures are described in the Supplementary Methods online.
Statistical analysis. Data from human skin samples were analyzed with the
Kruskal-Wallis test with Dunn’s multiple-comparison post-test; t-tests were
done on data from in vitro–polarized T cells. P values of less than 0.05 were
considered statistically significant.
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
Note: Supplementary information is available on the Nature Immunology website.
ACKNOWLEDGMENTS
Supported by the National Health and Medical Research Council of Australia
(N.J.W.).
COMPETING INTERESTS STATEMENT
The authors declare competing financial interests: details accompany the full-text
HTML version of the paper at http://www.nature.com/natureimmunology/.
Published online at http://www.nature.com/natureimmunology
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions
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