The phosphatase DUSP2 controls the activity of the ... - Nature

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The phosphatase DUSP2 controls the activity of the transcription activator STAT3 and regulates TH17 differentiation © 2015 Nature America, Inc. All rights reserved.

Dan Lu1,5,6, Liang Liu1,2,6, Xin Ji1, Yanan Gao3, Xi Chen1, Yu Liu1, Yang Liu1, Xuyang Zhao1, Yan Li3, Yunqiao Li1, Yan Jin1, Yu Zhang3, Michael A McNutt1 & Yuxin Yin1,2,4 Deregulation of the TH17 subset of helper T cells is closely linked with immunological disorders and inflammatory diseases. However, the mechanism by which TH17 cells are regulated remains elusive. Here we found that the phosphatase DUSP2 (PAC1) negatively regulated the development of TH17 cells. DUSP2 was directly associated with the signal transducer and transcription activator STAT3 and attenuated its activity through dephosphorylation of STAT3 at Tyr705 and Ser727. DUSP2deficient mice exhibited severe susceptibility to experimental colitis, with enhanced differentiation of T H17 cells and secretion of proinflammatory cytokines. In clinical patients with ulcerative colitis, DUSP2 was downregulated by DNA methylation and was not induced during T cell activation. Our data demonstrate that DUSP2 is a true STAT3 phosphatase that modulates the development of TH17 cells in the autoimmune response and inflammation. The TH17 subset of helper T cells, defined by the secretion of interleukin 17A (IL-17A), IL-17F, IL-21 and IL-22, is well documented as being involved in the pathogenesis of autoimmune disorders1. The differentiation of TH17 cells typically requires the cytokines TGF-β and IL-6, as well as signaling via the STAT3 transcription activator1. Other transcription factors, such as RORγt and RORα, are necessary for TH17 differentiation, but most of these are targets or partners of STAT3 (ref. 2). However, T cell–specific deletion of STAT3 impairs the development of TH17 cells3. In addition, the inhibitory effect of TGF-β and IL-2 on IL-17A expression is dependent on STAT3, and Stat3−/− regulatory T cells (Treg cells) show reduced inhibitory effects on TH17 differentiation4,5. STAT3 is thus a transcription factor that is indispensable for IL-17 expression but is also crucial for the differentiation and proinflammatory function of TH17 cells. Human inflammatory bowel disease (IBD) includes Crohn’s disease and ulcerative colitis (UC), characterized by idiopathic chronic recurrent intestinal inflammation in a genetically susceptible host6. Accumulating evidence suggests that STAT3 in T cells has an important role in the pathogenesis of IBD. STAT3 in intestinal T cells is constitutively activated both in patients with Crohn’s disease and in those with UC7,8. Moreover, genome-wide association studies have shown that polymorphisms in genes encoding components of STAT3 signaling (for example, IL23R, JAK2 and STAT3) are correlated with increased susceptibility to IBD9,10. In agreement with clinical studies, a T cell–transfer

model of colitis has demonstrated that STAT3 serves a critical function in driving colitis by promoting the population expansion of T cells and modulating the balance of the differentiation of TH17 cells and that of Treg cells, which highlights the importance of STAT3 in the pathogenesis of IBD11. Although STAT3-associated kinases such as JAK, Abl and PKC have been characterized12,13, relatively little is known about the nuclear phosphatases that modulate the transcriptional activity of STAT3. In mammalian cells, the dual-specificity phosphatase (DUSP) family includes at least 30 typical and atypical DUSPs, which dephosphor ylate both threonine residues and tyrosine residues of the substrates they target14. On the basis of sequence alignment, subcellular localization and substrate specificity, the typical DUSPs can be subcategorized into three subfamilies, which include DUSPs specific to the mitogen-activated protein kinases (MAPKs) Jnk and p38 (DUSP8, DUSP10 and DUSP16), which localize in the nucleus and cytoplasm; cytoplasmic Erk-specific DUSPs (DUSP6, DUSP7 and DUSP9); and mitogen- and stress-inducible nuclear DUSPs (DUSP1, DUSP2, DUSP4 and DUSP5)15. Gene-ablation studies in mice have helped define the regulatory roles of DUSPs in cells of the immune system. For example, DUSP1 inhibits the secretion of proinflammatory cytokines from dendritic cells though blockade of p38 signaling 16; DUSP5 negatively modulates the IL-33-dependent survival and function of eosinophils17; and DUSP4, DUSP14 and DUSP22 affect T cell response through inhibiting IL-2 signaling18–20.

1Institute

of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China. 2Department of Pathology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China. 3Department of Immunology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China. 4Peking-Tsinghua Center for Life Sciences, Peking University Health Science Center, Beijing, China. 5Present address: Department of Immunology, Tianjin Key Laboratory of Cellular and Molecular Immunology, Tianjin Medical University, Tianjin, 300070, China. 6These authors contributed equally to this work. Correspondence should be addressed to Y.Y. ([email protected]). Received 1 July; accepted 24 August; published online 19 October 2015; corrected online 10 November 2015 (details online); doi:10.1038/ni.3278

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© 2015 Nature America, Inc. All rights reserved.

Articles DUSP2 (PAC1) localizes in the nucleus and is expressed predominantly in the immune system21. Published work has demonstrated that DUSP2 is a downstream target of the tumor suppressor p53 that is required for the cellular response to oxidative stress22. DUSP2 is also involved in transcription factor E2F1–mediated apoptotic signaling23. Like other members of the DUSP family, DUSP2 interacts with MAPKs; however, the target protein for its phosphatase activity is controversial21,24,25. Other non-MAPK proteins, such as histone H3, FAK and Lck, have also been described as targets of the DUSP family20,26,27; however, the underlying mechanism that governs the affinity of DUSPs for different targets is not well understood. In this study, we characterized the function of nuclear DUSP2 in a T cell–mediated adaptive immune response and inflammation. Through the use of experimental models of colitis, we found DUSP2 deficiency induced TH17 differentiation by directly enhancing the transcriptional activity of STAT3. Mechanistic studies showed that dephosphorylation of STAT3 by DUSP2 was crucial for the inhibition of STAT3-mediated transactivation of the gene encoding IL-17 and other downstream genes. Our study therefore identifies DUSP2 as a negative regulator of STAT3 signaling that participates in development of the TH17 lineage of T cells. RESULTS Dimerization of DUSP2 with DUSPs antagonizes IL-2 signaling Comprehensive quantification of mRNA encoding members of the DUSP family in various mouse tissues identified DUSP2 as the unique member of this family expressed exclusively in tissues showing enrichment for T cells, including thymus, spleen and lymph nodes (Fig. 1a). To investigate the potential function of DUSP2 in lymphocyte development, we measured Dusp2 mRNA expression in various lymphoid populations. Dusp2 expression was higher in all mature lymphocytes, including CD4+ or CD8+ single-positive thymic T cells and peripheral B cells and T cells than in immature (CD4−CD8− or CD4+CD8+) T cells in the thymus (Supplementary Fig. 1a). Of particular interest, we also found that Dusp2 expression varied among helper T cell subsets (Supplementary Fig. 1a). As the initiation of T cell differentiation requires active signals from the T cell antigen receptor and co-stimulatory molecules, we analyzed Dusp2 expression during T cell activation. Dusp2 mRNA expression was transiently increased in the early stage of T cell activation in a manner similar to that of mRNA encoding CD69, which has been described as a marker for the activation of T cells or B cells (Supplementary Fig. 1b). In accordance with that finding, treatment with the phorbol ester PMA plus ionomycin, which induces calcium influx and mimics the activation of T cells and B cells, also increased the expression of DUSP2 protein (Supplementary Fig. 1c). We found that in addition to Dusp2, genes encoding other members of the DUSP family, including Dusp1, Dusp4, Dusp5, Dusp10, Dusp11 and Dusp14, were induced during T cell activation (Supplementary Fig. 1d). However, these DUSPs differed not only in their expression patterns in T cells stimulated by antibody to the T cell antigen receptor and to the co-receptor CD28 but also in their expression profiles in various helper T cells (Supplementary Fig. 1e,f), which indicated that the expression of DUSPs exhibited considerable spatial and temporal variation during the activation and differentiation of T cells and that these characteristics participated in the dynamic process of T cell development. Since several DUSPs have been reported to inhibit T cell proliferation through IL-2 signaling18–20, we used a human IL2 promoter–driven luciferase reporter assay to determine whether all inducible DUSPs affected IL-2 signaling. Overexpression of DUSP1, DUSP2 or DUSP4 substantially suppressed IL2 promoter luciferase activity relative to the 1264

effect on its activity of other DUSPs, which had little or no repressive effect (Fig. 1b). We also observed similar effects after stimulation of MOLT4 cells (a human acute T cell lymphoblastic leukemia line) with PMA and ionomycin (Fig. 1c). Moreover, co-transfection of vector encoding DUSP2 or/and DUSP1 robustly augmented the suppressive effect of DUSP4 on IL2 activity (Fig. 1d), which suggested that DUSP2 functioned collaboratively with DUSP1 and DUSP4. To evaluate the relationship of DUSP2 with other DUSPs during T cell activation, we screened a panel of DUSPs, in which we fused the open reading frames of a total of 22 DUSP-encoding genes with sequence encoding a FLAG tag, then transfected those into 293T human embryonic kidney cells together with vector encoding green fluorescent protein (GFP)-tagged DUSP2 (DUSP2-GFP). Co-immunoprecipitation and subsequent immunoblot analysis showed that there was substantial interaction of DUSP2 with itself and with DUSP1 or DUSP4, as well as moderate association of DUSP2 with DUSP12 or DUSP22 (Fig. 1e). However, there was only minimal precipitation of DUSP2 together with DUSP5 or other members of the DUSP family (Fig. 1f). Moreover, in vitro binding assays further confirmed those findings and demonstrated DUSP2 was directly associated with DUSP1 and DUSP4 (Fig. 1g). In conclusion, these data indicated that DUSP2 formed homodimers with itself or formed heterodimers with DUSP1 or DUSP4 to produce a complex that modulated T cell activation; this underscored the importance of DUSP2 in the development and function of T cells. DUSP2 deficiency promotes IL-17 in vitro and in vivo To further investigate the role of DUSP2 in the activation and differentiation of T cells, we generated mice with conventional knockout of DUSP2 (Supplementary Fig. 2a). Both quantitative RT-PCR and immunoblot analysis showed undetectable expression of Dusp2 and DUSP2 in lymphoid homogenates from Dusp2−/− mice (Supplementary Fig. 2b,c). Similar to mice deficient in other DUSPs, Dusp2−/− mice developed apparently normally, and there were no gross differences between wild-type mice and Dusp2−/− mice in their T cell subsets (from thymus, lymph nodes and spleen) (Supplementary Fig. 2d–h). Consistent with our IL2 luciferase data, Dusp2−/− T cells had higher expression of Il2 mRNA and underwent slightly more cell divisions in response to stimulation with antibody to the invariant signaling protein CD3 (anti-CD3) and anti-CD28 than did wild-type T cells (Supplementary Fig. 3a,b), which suggested that DUSP2 deficiency enhanced the activation and proliferation of T cells via stronger IL-2 signaling. We next sought to determine whether DUSP2 deficiency had a role in T cell differentiation. When activated with anti-CD3 and anti-CD28 under various helper T cell–polarizing conditions, the production of interferon-γ (IFN-γ), IL-4 and IL-10 by Dusp2−/− naive T cells seemed to be normal (Fig. 2a,b). However, there was greater production of IL-17A in Dusp2−/− T cells than in wild-type T cells and a lower proportion of induced Treg cells (iTreg cells) among Dusp2−/− T cells than among wild-type T cells (Fig. 2a,b). To more precisely delineate the role of DUSP2 in T cell polarization, we compared the efficiency of wild-type and Dusp2−/− naive T cells in their differentiation into TH17 cells or iTreg cells under various skewing conditions (Supplementary Fig. 3c–e). We found DUSP2 deficiency promoted TH17 differentiation with increasing doses of IL-6 (Supplementary Fig. 3c). Moreover, deficiency in DUSP2 resulted in a much smaller proportion of iTreg cells when we used low or intermediate concentrations of TGF-β, whereas increasing or decreasing the dose of IL-2 did not affect iTreg cell development in mice of either genotype (Supplementary Fig. 3d,e). These results suggested that the regulation of T cell development by VOLUME 16 NUMBER 12 DECEMBER 2015 nature immunology

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+ – + – – – + ++ – – – +

Ve D ctor U D SP U 2 D SP -FL U 8 A D SP -FL G U 1 A SP 0- G 16 FL -F AG LA G

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Ve c D tor U D SP U 2 D SP -FL U 1 D SP 7- AG U 1 F D SP 8- LA U 2 F G D SP 1-FLAG U 2 L D SP 2-F AG U 2 L SP 3- A 28 FL G -F AG LA G

Expression (log2 fold)

80

Ve D cto U r D SP U 1 D SP -FL U 2 A D SP -FL G U 4 A SP -F G 5- LA FL G AG

Dusp1 Dusp2 Dusp3 Dusp4 Dusp5 Dusp6 Dusp7 Dusp8 Dusp9 Dusp10 Dusp11 Dusp12 Dusp13 Dusp14 Dusp15 Dusp16 Dusp18 Dusp19 Dusp21 Dusp22 Dusp23 Dusp26 Dusp27 Dusp28

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b IL2-Luc repression (%)

H ea Sk rt Br ele a ta Lu in l m n us St g cl o e Smma ch a C ll o in Li lon tes v tin Ki er e d Te ney st U is te Bo rus Th ne m Ly ymu arr o m Sp ph s w Emleen nod e br yo

a

Figure 1 DUSP2 dimerizes with DUSPs and antagonizes IL-2 signaling. (a) Quantitative RT-PCR (qRT-PCR) analysis of the DUSP2-GFP + + + + + + + + (kDa) DUSP2-GFP + + + + + + + + (kDa) DUSP2-GFP DUSP2-GFP expression of genes encoding members of the DUSP family –55 –55 lgH lgH –35 –40 IP: FLAG IP: FLAG (right margin) in mouse tissues (outlined area indicates tissues –25 DUSPs DUSPs related to the immune system); results were normalized to –25 * –35 –40 those of the gene encoding β-actin, and relative expression was –25 DUSPs DUSPs Input Input calculated by the change-in-threshold method. (b) IL2 luciferase –25 –55 DUSP2-GFP (IL2-Luc) reporter activity in HEK293T cells 24 h after DUSP2-GFP –55 transfection of an IL2 luciferase reporter plasmid and a control DUSP1-GFP – – + + – – + + – – – – DUSP1-GFP renilla luciferase reporter, together with vectors encoding the DUSP2-GFP – – + + – – – – – – + + DUSP2-GFP – – – – + + DUSP5-GFP DUSP4-GFP + + + + + + transcription factor NFATc2 (for the transactivation of IL2) and Vector Vector + – + – + – + – + – + – various DUSPs (horizontal axis; bracketing indicates grouping – + – + – + (kDa) DUSP2-FLAG DUSP2-FLAG – + – + – + (kDa) –70 –70 DUSPs DUSPs of DUSP subcategories). (c) IL2 luciferase reporter activity in lgH –55 lgH –55 IP: FLAG IP: FLAG MOLT4 cells transfected with an IL2 luciferase reporter plasmid DUSP2-FLAG DUSP2-FLAG –35 –35 and a control renilla luciferase reporter, together with vectors –70 DUSPs –70 DUSPs Input Input encoding NFATc2 and DUSP1, DUSP2, DUSP4 or DUSP5, and DUSP2-FLAG –35 DUSP2-FLAG –35 then, 24 h after transfection, stimulated for 4 h with PMA and DUSP1-FLAG – + – + – + – + DUSP4-FLAG ionomycin. (d) IL2 luciferase reporter activity in HEK293T – – + + (kDa) DUSP2-His DUSP2-His – – + + (kDa) cells after mock transfection (Mock) or transfection of an IL2 lgH –55 –55 lgH DUSP2-His DUSP2-His –40 –40 luciferase reporter plasmid and a control renilla luciferase IP: FLAG IP: FLAG DUSP1-FLAG DUSP4-FLAG –40 –35 reporter, together with vector expressing NFATc2 and various DUSP1-FLAG –40 DUSP4-FLAG –35 combinations (below graph) of vectors encoding DUSP1, DUSP2 Input Input DUSP2-His DUSP2-His –40 –40 and DUSP4 (++ indicates an amount double that indicted by +). (e) Immunoassay of HEK293T cells transfected to express DUSP2-GFP and vector only or FLAG-tagged members of the DUSP family (above lanes), assessed by immunoprecipitation of lysates with anti-FLAG and immunoblot analysis with anti-GFP; below (Input), immunoblot of samples above without immunoprecipitation (throughout). IgH, immunoglobulin heavy chain; *, nonspecific bands. Right margin, molecular sizes, in kilodaltons (kDa). (f) Immunoassay (as in e) of HEK293T cells transfected to express various combinations (above lanes) of DUSP2-FLAG and vector or GFP-tagged DUSP1, DUSP2, DUSP5 or DUSP4. (g) Immunoassay of a mixture of purified histidine-tagged DUSP2 (DUSP2-His) plus FLAG-tagged DUSP1 or DUSP4, assessed by immunoprecipitation with anti-FLAG and immunoblot analysis with anti-DUSP2. Data are representative of two independent experiments (a–g) with three biological replicates per condition (b–d) (error bars (b–d), s.e.m.).

f

g

DUSP2 was associated with the concentration of IL-6 and TGF-β, but not with the concentration of IL-2, consistent with the finding that DUSP2 deficiency enhanced the IL-6-mediated phosphorylation of STAT3 but did not affect the IL-2-driven phosphorylation of STAT5 (Supplementary Fig. 3f,g). In agreement with the results reported above, the expression of mRNA from TH17 cell signature genes, including Rorc, Rora, Il17a and Il17f, was markedly upregulated in Dusp2−/− T cells under TH17-polarizing conditions, relative to such expression in wild-type T cells under TH17-polarizing conditions (Fig. 2c). To further confirm the role of DUSP2 in T cell differentiation in vivo, we immunized wild-type and Dusp2−/− mice with keyhole limpet hemocyanin (KLH) in complete Freund’s adjuvant. One week later, we analyzed the expression of IL-17A, IFN-γ or the transcription factor Foxp3 in lymphocytes from these immunized mice. We consistently observed nature immunology VOLUME 16 NUMBER 12 DECEMBER 2015

a greater frequency of IL-17A-producing cells and a lower proportion of Foxp3+ cells in the CD4+ T cell population in Dusp2−/− mice than in their wild-type counterparts, whereas the number of IFN-γ+ cells was identical in these two groups (Supplementary Fig. 4a). In addition to the T cell response, the production of antigen-specific antibodies in the serum after immunization with KLH was much greater in Dusp2−/− mice than in wild-type mice (Supplementary Fig. 4b), which suggested that DUSP2 also participated in humoral immunity. Together these results provided evidence that loss of DUSP2 facilitated the induction of TH17 cell development and inhibited iTreg cell differentiation in vitro. Deletion of DUSP2 exacerbates DSS-induced colitis in mice To further support the findings reported above, we used a dextran sulfate sodium (DSS)-induced mouse colitis model that has been 1265

Articles widely used to analyze the contribution of distinct T cell subsets in mucosal damage response28. We induced acute colitis in the mice through oral administration of 3% DSS in drinking water over a 10day period. DSS-induced acute colitis was exacerbated in Dusp2−/− mice relative to that in wild-type mice, as determined by weight loss, clinical scores, survival curves and blood glucose (Fig. 2d and

a

Supplementary Fig. 5a). In addition, both gross tissue evaluation and histological analysis revealed distinct mucosal hyperemia, massive colon ulceration and increased infiltration by inflammatory cells in the colonic tissues of DSS-treated Dusp2−/− mice (Fig. 2e). We also observed similar effects after treatment of mice with 5% DSS in the acute and recovery phases (Supplementary Fig. 5b). Consistent

55.4

12.7

5.96

15.4

51.2

14.9

12.2

14.0

51.7

WT

102

3

101

2

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Foxp3

0102 103 104 105

1,500 1,000 500 0

TH2

600

WT Dusp2 –/–

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16 32 48 64 80 96 Time after stimulation (h)

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60 0

e

2 4 6 8 10 Time after 3% DSS (d)

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WT

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120

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30

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1

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iTreg

WT Dusp2 –/–

150 100 50 0

0


***

WT Dusp2 –/–

90 60 30 0

0

2

4 6 8 10 12 14 16 Time after 3% DSS (d)

–/–

Dusp2

*

8

Day 4

30

120

0 1 2 3 4 5 6 7 8 9 10 11 12 13 Time after 3% DSS (d)

Day 4

***

45

200


2

Day 0

WT –/– Dusp2

+

+

60

0

60

+

Tr1

90

* * *

Dusp2 –/– WT

UT

WT Dusp2 –/–

3

WT

Dusp2 –/–

0

WT Dusp2 –/–

4

0

12

Dusp2

TH17

5 Clinical score

***** ****** *** *** **

80

UT

400

WT Dusp2 –/–

100

0

5

Rora expression (fold)

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Survival (%)

WT Dusp2 –/–

2,500

0

+

0

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Histological score

TH1

9

Rorc expression (fold)

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20

+

+

+

6

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15

CD4 IL-10 cells (%)

CD4 IL-17A cells (%)

9

ll17f expression (fold)

ll17a expression (fold)

0

d 120 Body weight (%)

12

+

15

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15 CD4 IL-4 cells (%)

+

45

+

CD4 IFN-γ cells (%)


100 101 102 103

CD25

NS

60

30

c

100

CD4

b

24.2

CD4 CD25 Foxp3 cells (%)

10

4

IL-4

–/–

IFN-γ

Dusp2

103

IL-10

10

5

IL-17A

10

WT Dusp2 –/–

6 4

NS

2 0

Day 4

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Figure 2 DUSP2 deficiency promotes TH17 differentiation in vitro and enhanced susceptibility of Dusp2−/− mice to DSS-induced colitis. (a) Flow cytometry analyzing the staining of IFN-γ, IL-4, IL-17A, IL-10, Foxp3 and CD25 (assessing differentiation efficiency) in wild-type (WT) and Dusp2−/− naive (CD4+CD25−CD62LhiCD44lo) T cells activated with plate-bound anti-CD3 plus anti-CD28 and cultured under various polarizing conditions (as in b), then re-stimulated for 5 h with PMA and ionomycin. Numbers in top right corners (left) indicate percent marker-positive CD4 + cells; numbers adjacent to outlined areas (far right) indicate percent Foxp3 +CD25+ cells. (b) Frequency of IFN-γ+, IL-4+, IL-17A+, IL-10+, CD25+ or Foxp3+ cells among wild-type and Dusp2−/− peripheral lymphocytes left untreated (UT) or naive T cells cultured under various polarizing conditions (horizontal axes), then re-stimulated for 5 h with PMA and ionomycin. (c) qRT-PCR analysis of mRNA in wild-type and Dusp2−/− naive T cells cultured under TH17polarizing conditions (presented as in Fig. 1a). (d) Body weight (left; relative to initial weight, set as 100%), clinical score (middle) and survival (right; log-rank (Mantel-Cox) test) of wild-type and Dusp2−/− mice treated with 3% DSS. (e) Morphological analysis (left) of colons from wild-type and Dusp2−/− mice on day 4 of treatment with 3% DSS, and hematoxylin-and-eosin staining (middle) and histological score (right) of colonic sections from wild-type and Dusp2−/− mice on days 4 and 9 of administration of 3% DSS. Outlined areas (left) indicate regions enlarged 10× at far left. Scale bars (middle), 50 µm. Each symbol (b,e) represents an individual mouse. NS, not significant (P > 0.05); *P < 0.05, **P < 0.01 and ***P < 0.001 (unpaired Student’s t-test). Data are representative of three independent experiments with three to five mice per group (a,b,d,e; error bars, s.e.m.) or two independent experiments with two biological replicates from six mice per group (c; error bars, s.e.m.).

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0

0

1.28

b

LP

PP 1.51

0

3.33

10

105

0

98.7

0

98.5

0

96.7

0

2.74

0

4.94

0

8.61

4

10

IL-17A

Dusp2–/–

3

10

IL-17A+ cells (%)

WT

2

10 0

0

97.3 2

3

4

0

95.1

0

91.4

0

2.43

0

27.6

*

8 6 4

* MLN

PP

LP

98.1

0

97.6

0

72.4

0

3.79

0

5.66

0

31.6

104

IFN-γ

103 102 0

20 10 5

0

96.2

0

94.3

0

26.3

15.4

0

0

17.7

0

73.7

0

84.6

0

82.3

0

21.7

0

12.3

0

16.8

104

Dusp2

–/–

103 102 0

Foxp3+ cells (%)

30

WT

105

78.3

0

87.7

0

83.2

0

6.93

0

10.3

NS NS

MLN

PP

0

93.1

0

89.7

LP

105 0

4.66

0

5.05

0

11.1

4

10

Dusp2–/–

NS NS

IL-10

0

95.3 2

3

4

0

94.9

0

*

45 30 15 0

MLN

PP

WT –/– Dusp2

9

120

LP

NS

PP

60 30 NS

60

NS

MLN

NS

90

0

NS

3

2

10 0

40

6

0

3

10

80

60

18

6

*

120

IL-4 (pg/ml)

95.9

IL-10+ cells (%)

0

80

0

WT –/– Dusp2 NS

12

12 WT

160

160

0102 103 104 105 4.10

240

0

24

0 0

NS

68.4

0102 103 104 105

*

320

40

0 0

70

IL-1β (pg/ml)

Dusp2–/–

140

IFN-γ (pg/ml)

105

0

WT –/– Dusp2

TNF (pg/ml)

WT

IFN-γ+ cells (%)

60

210

400

5

1.88

*

280

0

2 0

WT –/– Dusp2 350

**

010 10 10 10 0

0

LP

88.9

45 30 15 0

5

010 10 10 10


4 2 0 UT

DSS

4 2 0 UT

DSS

6 4 2 0 UT

DSS

10 8 6 4 2 0

Foxp3 expression (fold)

6

6

HIx expression (fold)

DUSP2 modulates the accumulation of 0 TH17 cells in colon UT DSS −/− To assess the association of Dusp2 with T cell differentiation in the exacerbation of colitis, we isolated lymphocytes from mesenteric lymph nodes, Peyer’s patches and lamina propria during the most severe phase of acute colitis and chronic colitis (day 8 and day 28, respectively) and stained intracellular cytokines in those cells. Dusp2−/− CD4+ T cells showed an enhanced inflammatory response, with an increased proportion of IL-17A+ cells, relative to that of their wild-type counterparts (Fig. 3a and Supplementary Fig. 6). There was no significant difference between wild-type mice and Dusp2−/− mice in the frequency of Foxp3+ cells or IL-10+ cells in the CD4+ T cell population (Fig. 3a and Supplementary Fig. 6). In addition, although the amount of IL-4 and IFN-γ that was produced remained similar, we detected larger amounts of proinflammatory cytokines (IL-6, IL-17, TNF and IL-1β) in Dusp2−/− mice than in wildtype mice (Fig. 3b). Consistent with the finding that the Dusp2−/− colon had a greater frequency of TH17 cells, real-time PCR analysis revealed higher expression of TH17 cell signature genes (Il17a and Il17f), with no change in the level of mRNAs encoding TH1 cell transcription factors (Tbx21 and Hlx) or the Treg cell transcription factor Foxp3, in the colon of Dusp2−/− mice after treatment DSS, relative to that of their wild-type counterparts (Fig. 3c). Therefore, TH17 cells seemed to have a dominant role in Dusp2−/− mice after DSS administration. Together with the observation that Dusp2 expression was increased in DSS-induced colon inflammation (Fig. 3c), these data suggested that DUSP2 limited the development of TH17 cells during

8

Tbx21 expression (fold)

180 150 120 40 20

ll17f expression (fold)

c

CD4

ll17a expression (fold)

with those findings, in a chronic colitis model induced by three cycles of DSS treatment for 6 d, followed by 14 d of DSS-free water, Dusp2−/− mice developed colitis that was more severe than that of their wild-type littermates (Supplementary Fig. 5c–f). In conclusion, Dusp2−/− mice showed enhanced susceptibility to DSS-induced colitis.

WT –/– Dusp2

IL-6 (pg/ml)

MLN

IL-17 (pg/ml)

a

Foxp3

Figure 3 DUSP2 deficiency facilitates the TH17 response during DSS-induced colitis. (a) Flow cytometry analyzing the intracellular staining of IL-17A, IFN-γ, Foxp3 or IL-10 and surface staining of CD4 of lymphocytes isolated from the mesenteric lymph nodes (MLN), Peyer’s patches (PP) and lamina propria (LP) of wild-type and Dusp2−/− mice treated for 6 d with 2.5% DSS in drinking water, followed by 14 d of untreated (DSS-free) drinking water, with this cycle repeated once, assessed day 8 after the beginning of the second DSS cycle. Right, frequency of cells positive for markers assessed at left. Numbers in quadrants (left) indicate percent cells in each. (b) Enzymelinked immunosorbent assay of IL-6, IL-17, TNF, IL-1β, IFN-γ and IL-4 in serum from wildtype and Dusp2−/− mice on day 8 of treatment with 3% DSS. (c) qRT-PCR analysis of genes in wild-type and Dusp2−/− colonic cells left untreated or stimulated for 8 d with 3% DSS (presented as in Fig. 1a). Each symbol (a,b) represents an individual mouse. *P < 0.05 and **P < 0.01 (unpaired Student’s t-test). Data are representative of two independent experiments with four or five (a), three or five (b) or three (c) mice per group (error bars, s.e.m.).

Dusp2 expression (fold)


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UT

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colitis and supported our hypothesis that DUSP2 participates in the adaptive immune response during inflammation. DUSP2 deficiency drives T cell–mediated colitis To delineate the involvement of DUSP2 in chronic intestinal inflammation, we used a T cell–transfer model of colitis in which we reconstituted immunodeficient CB-17 mice of the severe combined immunodeficiency (SCID) strain with CD4+CD45RBhi naive T cells from wild-type or Dusp2−/− mice and monitored the recipients for the development of wasting disease. Mice reconstituted with Dusp2−/− T cells began to lose weight 2 weeks earlier than those that received wild-type T cells (Fig. 4a). We confirmed the development of colitis clinically by gross tissue evaluation and histological analysis at 7 weeks, when mice reconstituted with Dusp2−/− T cells developed marked inflammation in the colon (Fig. 4b–e). In addition to the pathological changes in the colon, we also found that mice reconstituted with Dusp2−/− T cells developed psoriaform lesions (data not shown), which has been reported to be a T H17 cell–driven inflammatory skin disease29. The T cell–transfer model of colitis is characterized by the accumulation of TH1 and TH17 cells in the colon30. Mice that received Dusp2−/− T cells exhibited a significantly greater number of CD4+ T cells in the mesenteric lymph nodes, spleen and colon than did recipients of wild-type T cells (Fig. 4f), which suggested that deletion 1267

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2 0

T

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MLN 0

Spl 1.76

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98.9

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95.6

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9.69

IL-17A

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WT Dusp2–/–

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95.5 2

010

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4

0

99.2

0

90.3

0

75.3

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5.72

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36.5

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24.7

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94.3

0

63.5

0

38.7

0

56.8

IFN-γ

10

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15.8

010

3

4

0

61.3

0

43.2

NS

80

LP

***

**

60 40 20 0

2

2

h of DUSP2 drove T cell accumulation in the intestine. + + 25 Consistent with those findings, the frequency of IL-17A CD4 T cells was much higher in mesenteric lymph nodes and 20 colon of mice reconstituted with Dusp2−/− T cells than in 15 that of mice reconstituted with wild-type T cells (Fig. 4g). 10 To some extent, we also observed a greater proportion of IFN-γ+CD4+ T cells in some lymphoid tissues (spleen and 5 lamina propria but not mesenteric lymph nodes) of mice 0 reconstituted with Dusp2−/− T cells than in those of mice reconstituted with wild-type T cells (Fig. 4g). In agreement with those results, Dusp2−/− T cells from lamina propria had higher expression of mRNA from TH17 cell signature genes, including Il17a, Il17f, Il22 and Il23r (Fig. 4h), which further substantiated the concept that DUSP2-deficient T cells exhibited a pronounced TH17 phenotype. Moreover, Dusp2−/− T cells upregulated genes encoding signature pathogenic TH17 cell molecules31–33, including proinflammatory cytokines (Il33, Tnf and Csf2), chemokines and receptors (Ccl3, Ccl4, Ccl9 and Ccr6), transcription factors (Tbx21 and Hlx) and the effector molecule granzyme B (Gzmb) (Fig. 4h). Conversely, there was no noticeable change in DUSP2-deficient T cells in the expression of Ahr, Maf, Il9, Ccl20, Ikzf3 or Cxcr6 (Fig. 4h), which are recognized as non-pathogenic TH17 cell signature genes31,32. Ablation of DUSP2 thus induced a pathogenic type of TH17 cells in colitis. Given the finding DUSP2 deficiency resulted in a decreased frequency of Treg cells in vitro (Fig. 2a,b), we sought to determine whether deletion of DUSP2 functionally affected the capacity of Treg cells to control colitis. We transferred CD45.1+CD4+CD45RBhi cells into CB-17 SCID mice alone or together with Treg cells from wild-type or Dusp2−/− mice. As expected, the transfer of CD4+CD45RBhi cells alone resulted in severe intestinal inflammation accompanied by progressive weight loss and colon thickening (Supplementary Fig. 7a,b). However, Treg cells of either genotype effectively prevented the wasting disease (Supplementary Fig. 7a,b), which indicated that Dusp2−/− Treg cells had suppressive

+

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1268

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r6

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ll1 7f R or a R or c ll2 2 ll2 3r ll3 3 C sf 2 Tn f C cr 6 Tb x2 1 H Ix C cl 3 C cl 4 C cl 9 G zm b Ah r M af

ll1 7a

Gene expression (fold)


Figure 4 DUSP2 controls T cell–dependent intestinal inflammation. (a) Body weight of CB-17 SCID recipient mice given wild-type or Dusp2−/− naive CD4+CD45RBhi T cells. (b) Morphological analysis of colons from recipient mice at 7 weeks after transfer as in a. (c) Ratio of colon weight to colon length in recipient mice as in b. (d) Hematoxylin-and-eosin staining of colonic sections from recipient mice as in b. Scale bars, 50 µm. (e) Histological scores of recipient mice as in b. (f) Total CD4+ T cells in mesenteric lymph nodes, spleen (Spl) and lamina propria of recipient mice as in b. (g) Expression of IL-17A and IFN-γ in CD4+ T cells isolated from the mesenteric lymph nodes, spleen and lamina propria of recipient mice as in b. (h) qRT-PCR analysis of mRNA levels of various genes (horizontal axis) in mononuclear cells from the lamina propria of recipient mice as in b (presented as in Fig. 1a). Each symbol (c,e–g) represents an individual mouse. *P < 0.05, **P < 0.01 and ***P < 0.001 (unpaired Student’s t-test). Data are representative of three (a–f) or two (g,h) independent experiments with nine (a), six (d,e), five (b,c,f,g) or four (h) mice per group (error bars, s.e.m.).

CD4 T cells (×105)

6

WT Dusp2–/– 100

–/ –

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8

D

0 1 2 3 4 5 6 7 8 Time after transfer (weeks)

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***

D

–/–

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T

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120

**

0.15

Histological score

c

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WT –/– Dusp2 130

Colon weight/colon length (g/cm)

a

functions similar to those of wild-type Treg cells in this model of transfer colitis. Accordingly, mice that received Dusp2−/− or wild-type Treg cells exhibited an identical increase in the fraction of CD4+Foxp3+cells present in the mesenteric lymph nodes and spleen, relative to that of recipients of PBS (Supplementary Fig. 7c). DUSP2 was thus not required for the response or function of Treg cells in intestinal inflammation. Collectively, these data demonstrated that DUSP2 deficiency promoted the population expansion of CD4+ T cells and pathogenic TH17 polarization but had no substantial effect on Treg cell responses in colitis. DUSP2 is an essential modulator of STAT3 signaling The DUSPs are a group of phosphatases that modulate phosphorylationdependent signaling pathways via the dephosphorylation of serine, threonine and tyrosine residues. To investigate the molecular mechanism by which deficiency in DUSP2 phosphatase activity aggravated colitis with an enhanced TH17 response, we performed quantitative analysis of phosphorylated proteins (phosphoproteomic analysis) of colon homogenates from Dusp2−/− mice treated with DSS and their wild-type counterparts (Supplementary Table 1). On the basis of evaluation of expression profiles and subcellular localization, we identified various phosphorylated peptides of candidate proteins, including STAT3, which is essential for IL-6-mediated VOLUME 16 NUMBER 12 DECEMBER 2015 nature immunology

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b

c

p2

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Events (% of max)

D us

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Figure 5 DUSP2 negatively modulates the UT DSS WT STAT3 signaling pathway. (a) Immunoblot WT Dusp2–/– –/– Dusp2 100 (kDa) analysis of total and phosphorylated (p-) STAT3, p-STAT3 - 70 80 Erk1/2, p38 and Akt in colon homogenates H&E 60 STAT3 - 70 40 from wild-type and Dusp2−/− mice left untreated - 40 p-Erk1/2 20 or stimulated for 8 d with 3% DSS (below blots). Erk1/2 - 40 0 Anti-p(b) Microscopy of colonic sections from wild0102 103 104 105 p-p38 - 40 STAT3 type and Dusp2−/− mice after treatment p-STAT3 p38 - 40 with DSS, stained with hematoxylin and eosin p-Akt - 55 WT 15 Dusp2–/– ** Akt - 55 (H&E), antibody to phosphorylated STAT3 12 Control lgG GAPDH - 35 (Anti-p-STAT3) or control antibody IgG. 9 α-tubulin - 55 Scale bars, 50 µm. (c) Flow cytometry (top) 6 NS β-actin - 40 analyzing the intracellular staining of STAT3 3 + UT DSS phosphorylated at Tyr705 (p-STAT3) in CD4 0 DSS UT T cells isolated from Peyer’s patches of wildWT type and Dusp2−/− mice left untreated or Dusp2–/– treated for 3 d with 3% DSS (above plots). 15 80 ** 10 * 5 * 12 Gray shaded curves, isotype-matched control 8 60 4 9 6 antibody. Below, frequency of cells as above 40 6 4 with phosphorylated STAT3 (p-STAT3+); 3 NS NS 20 3 2 each symbol represents an individual mouse. NS 2 0 0 0 (d) qRT-PCR analysis of mRNA from STAT3 UT DSS UT DSS UT DSS 1 target genes in colonic cells from wild-type NS and Dusp2−/− mice left untreated or 0 25 NS ** 200 300 Mock + + – – – – + – – – – + – – – – stimulated for 8 d with 3% DSS (presented 20 240 150 RORγt – + + + + + – – – – – + + + + + as in Fig. 1a). (e) Luciferase activity of 15 180 STAT3 – – – – – – + + + + + + + + + + 100 HEK293T cells transfected with an Il17a 10 120 DUSP2WT – – – – – – – NS 50 5 luciferase reporter plasmid together with 60 NS NS DUSP2C/S – – – – – + – – – – + – – – – + 0 0 various combinations (below graphs) of 0 UT DSS UT DSS UT DSS mock transfection or transfection of vector encoding RORγt, STAT3, increasing amounts (wedges) of wild-type DUSP2 (DUSP2 WT), or the DUSP2 C257S mutant (DUSP2C/S), and then, 24 h after transfection, stimulated for 4 h with PMA, ionomycin and IL-6; results are presented in relative units (RU) relative to the activity of renilla luciferase. *P < 0.05 and **P < 0.01 (unpaired Student’s t-test). Data are representative of three (a–c,e) or two (d) independent experiments with three (a,b,d) or four (c) mice per group or three biological replicates per condition (e) (error bars, s.e.m.).


Luciferase activity (RU)

Cox2 expression (fold)

Cox1 expression (fold)

Ccl7 expression (fold)

TH17 differentiation. However, in this analysis we did not find members of the MAPK family (Erk1, Erk2 and p38) that have been reported as canonical substrates of DUSP2. To confirm the phosphoproteomic data, we assessed distinct phosphorylation-dependent pathways by immunoblot analysis. Although the amount of phosphorylated STAT3 was identical in wild-type and Dusp2−/− mice under quiescent conditions, the amount was greater in the colon of Dusp2−/− mice following DSS treatment than in that of their wild-type counterparts (Fig. 5a). Phosphorylation of Erk (Erk1/2), p38 and the kinase Akt was substantially greater in the colon of Dusp2−/− mice than in that of wild-type mice, without DSS treatment, and administration of DSS led to downregulation of these pathways (Fig. 5a), which suggested that MAPK and phosphatidylinositol-3-OH kinase–Akt signaling pathways were not involved in the process of acute colitis. Notably, immunohistological analysis also indicated greater amounts of phosphorylated STAT3 in Dusp2−/− colon than in wild-type colon, following DSS treatment (Fig. 5b). In addition, we consistently observed that CD4+ T cells in Peyer’s patches from DSS-treated Dusp2−/− mice had greater amounts of phosphorylated STAT3 than did those from wild-type mice (Fig. 5c). Genes that have been reported as targets of STAT3, including Rora, Cox1, Cox2, Ccl5, Ccl7 and Cxcl1, were also upregulated concomitant with STAT3 activation in Dusp2−/− mice after DSS treatment (Fig. 5d). Moreover, unlike its catalytically inactive mutant C257S 34, wild-type DUSP2 inhibited STAT3-mediated transactivation of the Il17a promoter in a dose-dependent manner, as shown by luciferase reporter assays (Fig. 5e). Together these results demonstrated that STAT3 was an important target of the phosphatase DUSP2, without which the STAT3 pathway was maintained at a high level of activity under inflammatory conditions.

Cxcl1 expression (fold)

Rora expression (fold)

e

Ccl5 expression (fold)


d

DUSP2 directly catalyzes STAT3 dephosphorylation To investigate the molecular mechanism by which DUSP2 deficiency selectively promoted STAT3 signaling, we used immunoprecipitation followed by mass spectrometry (MS) to identify DUSP2-associated proteins. We identified multiple DUSP2-associated proteins, including members of the MAPK family (MAPK2, MAPK4, MAPK6 and MAPK14), as well as other proteins (RPA1 and IGF2BP3) (Fig. 6a). However, a notable finding was the identification of three STAT3 peptides (Fig. 6a). Subsequent co-immunoprecipitation assays demonstrated that FLAG-tagged DUSP2 (DUSP2-FLAG) was precipitated with endogenous STAT3 in HEK293T cells (Fig. 6b), which further confirmed a physical association between DUSP2 and STAT3. We next sought to determine whether other members of the STAT family interacted with DUSP2. We transfected vector encoding DUSP2 into HEK293T cells together with vector encoding STAT1, STAT3, STAT3β, STAT4 or STAT6. Co-immunoprecipitation assays showed that with the exception of STAT4, all STAT proteins interacted, to varying extents, with DUSP2, but STAT3 had the greatest interaction in vivo (Fig. 6c). We co-expressed DUSP2-GFP with a series of truncated forms of STAT3 to map the DUSP2-binding region of STAT3; the results demonstrated that the amino-terminal domain of STAT3 was essential for its interaction with DUSP2 (Fig. 6d). In addition, we found that the DUSP2 residue Cys257 was indispensable not only for DUSP2’s phosphatase activity but also for its physical association with STAT3, as shown by reciprocal precipitation experiments with these two proteins (Fig. 6e). To definitively determine whether DUSP2 served as a phosphatase for STAT3, we used an in vitro phosphatase assay. Through the use of S-protein beads, we precipitated phosphorylated STAT3 from IL-6-stimulated HEK293T cells transfected to express a fusion of 1269

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Coiled coil

DNA binding

Coiled coil

DNA binding

Linker

Ve ct or ST AT 1 ST -FL AT AG ST 3-F AT LA 3 G ST β-F AT LA G 4F ST AT LAG 6FL AG +

+

+

IP: FLAG

55

STATs IgH

100 55

STATs

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(kDa)

DUSP2

35

STAT3

100

SH2

TAD

SH2

TAD

SH2

TAD

SH2

TAD

SH2

TAD

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55

DUSP2-GFP N terminus

∆CCD

STAT3

DNA binding

N terminus

STAT3∆DBD

Coiled coil

Linker

Linker

∆C C D

FL

or ST AT ST 3 AT ST 3 AT 3 ST AT 3 ST AT ST 3 AT 3

Linker

(kDa)

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STAT3∆NT

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DUSP2-GFP IgH

IP: FLAG

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STAT3 truncations

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DUSP2-GFP DNA binding

Y705

∆RRR

RHO

DUSP2

C/S

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RRR

STAT3

D

S TPP

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Input

Variant DUSP2 STAT3

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R ∆R

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TPP

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DUSP2

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RHO RRR

STAT3-FLAG TPP

D DUSP2

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TPP

KK

C

FL

D

RHO KKK

2 SP D 2 U SP D 2 U SP D 2 U SP 2

C

SP

DUSP2KKK

TPP D

U

RRR

70

S727

U

RHO

DUSP2FL

STAT3 truncations

D

e

55

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D

STAT3

N terminus

ct

∆LD

Ve


+

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N terminus

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DUSP2-GFP

∆N T

FL

STAT3

35

∆2 –

d

DUSP2

D

MAPK14 DUSP2

35

100

∆D BD

MQQLEQMLTALDQMR IVELFRNLMK VCIDKDSGDVAALRGSR

MAPK2

STAT3

∆L

IGF2BP3 STAT3

55

In pu t IP :I gG IP :F LA G

(kDa)

170 100

c

In pu t IP :I gG IP :S TA T3

(kDa)

b

D U SP 2FL AG

Pr ot ei n la Ve dd ct er or

a

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(kDa) 55 100 70 55 100

C

Figure 6 DUSP2 physically interacts with STAT3. (a) MS analysis of DUSP2-associated proteins after immunoprecipitation (via anti-FLAG beads) from lysates of HEK293T cells transiently transfected to express DUSP2-FLAG. Right (outlined text), three matched peptide sequences that correspond to STAT3. (b) Immunoassay of HEK293T cells transfected to express DUSP2-FLAG; lysates immunoprecipitated with anti-FLAG or anti-STAT3 were analyzed by immunoblot with anti-STAT3 or anti-FLAG. (c) Immunoassay of HEK293T cells transfected to express DUSP2-GFP and vector only or FLAGtagged STAT1, STAT3, STAT3β, STAT4 or STAT6 (above lanes); lysates immunoprecipitated with anti-FLAG were analyzed by immunoblot with anti-GFP. (d) Immunoassay of HEK293T cells transfected to express DUSP2-GFP and vector only or plasmids encoding FLAG-tagged STAT3 truncation mutants (above lanes; as at left); lysates immunoprecipitated with anti-FLAG were analyzed by immunoblot with anti-GFP. STAT3 FL, full length STAT3; STAT3∆2-30, STAT3 lacking amino acids 2–30; STAT3∆NT, STAT3 lacking the amino (N) terminus; STAT3∆CCD, STAT3 lacking the coiled-coil domain; STAT3∆DBD, STAT3 lacking the DNA-binding domain; STAT3∆LD, STAT3 lacking the linker domain; SH2, Src-homology 2 domain; TAD, transactivation domain. (e) Immunoassay of HEK293T cells transfected to express STAT3-FLAG and vector only or plasmids encoding GFP-tagged DUSP2 mutant (above lanes; as at left); lysates immunoprecipitated with anti-FLAG were analyzed by immunoblot with anti-GFP. DUSP2 FL, full-length DUSP, with three arginine resides (RRR) (positions 56–58) in the rhodanese (RHO) domain, an aspartic acid residue (D) at position 226, and a cysteine residue (C) at position 257 in the tyrosine-protein phosphatase (TPP) domain; DUSP2 KKK, DUSP2 with replacement of those arginine residues with lysine (K); DUSP2 ∆RRR, DUSP2 with deletion of those arginine residues; DUSP2 C/S, DUSP2 with replacement of the cysteine residue with serine (S); DUSP2 D/A, DUSP2 with replacement of the aspartic acid with alanine (A). Data are representative of two independent experiments.

STAT3 and the peptide S-tag (Fig. 7a). We purified DUSP2 or control protein from HEK293T cells transfected with vector encoding DUSP2FLAG or empty vector by extraction with anti-FLAG beads and elution with FLAG peptides, and incubated DUSP2 or the control protein with a fusion of phosphorylated STAT3 and S-protein beads in phosphatase buffer. We then eluted bound proteins and subjected them to MS. Incubation of STAT3 with DUSP2 led to dephosphorylation of two STAT3 residues that have been reported to promote its activity, Tyr705 and Ser727, compared with incubation of STAT3 with the control protein, which had less of an effect on the phosphorylation of STAT3 (Fig. 7a). To further support that finding, we evaluated the binding of DUSP2 to two STAT3 mutants with substitution of those two residues (Y705F and S727A). While the effect of DUSP2 on STAT3 appeared

to be reduced by use of the Y705F mutant (Fig. 7b), we observed no effect on protein association by use of the S727A mutant (data not shown). However, the interaction of DUSP2 and STAT3 was strengthened in the presence of IL-6, which induces STAT3 phosphorylation (Fig. 7c). These findings indicated that these phosphorylated residues were involved in the direct interaction of STAT3 and DUSP2. To further investigate DUSP2’s phosphatase function in vivo, we evaluated the ability of DUSP2 to dephosphorylate ectopically expressed or endogenous phosphorylated STAT3. PMA and ionomycin had an enhancing effect on Dusp2 expression in T cells; we compared the amount of phosphorylated STAT3 in wild-type and Dusp2−/− lymphocytes following stimulation with PMA and ionomycin. After that stimulation, we found enhanced phosphorylated

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2+ b18 -H2O,

3+

2+ b18 -NH3,

+

05 Y7

+ y10 -NH3–P

y9 , 983.34

3+

y21 -H2O, y21 -NH3 800.93

IP: FLAG

DUSP2-GFP DUSP2-GFP IgH Variant STAT3

+ + + (kDa) 55 100

Intensity

SP 2 U D

M oc k

Pr ot ei

n

la

dd

er

4,000

F

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YcCR P E S Q E H P E A D P G S A A P pY L K

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Y705

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STAT3 70

1,000

BSA

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0

+

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500

c

2+

y 11,b 9 -H2O,b21 –P 1169.45 +

b8 1050.36 3+ [M+3H] -NH3-H2O + 2+ 3+ y 6 ,b12 ,b20 –P 849.50 742.48

b3 480.15

40

+

b9 1187.36

+

+ y4 y9 -H2O 600.23 965.35 2+ y12 –P 2+ b17 -H2O 601.24+ 947.47 y5 + 2+ 671.19 b7 ,b 16 2+ 2+ y13 ,b11 -H2O 921.18 2+ b9 698.46 + 2+ ++ y 8 ,y16 -H2O b5 594.33 886.41 706.24

2,000

130

+

2+

b21 1218.63

+

b 11 -H2O,y13 1395.48

1,000

+

IP: FLAG

b13 1599.64

1,500

2,000

Input

m/z

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STAT3 Input Variant DUSP2-GFP

2+

b18 2+ 992.04 b17 956.51

IL-6 STAT3-FLAG DUSP2-GFP DUSP2-GFP IgH STAT3-FLAG

– – + – + + + + +

(kDa) 55 70 100 70

STAT3-FLAG DUSP2-GFP

S727 STAT3–S-tag

+

F I cC V T P T T cC S N T I D L P oxM pS P R

5,000

+

2+

+

2+

d

WT Dusp2–/– (kDa) p-STAT3

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b12 -H2O, y5 , b12 -NH3, y18 –P 683.12

100 STAT3 Figure 7 DUSP2 catalyzes the PMA + 4,000 iono DUSP2 35 dephosphorylation of STAT3. (a) MS GAPDH 35 analysis of the phosphorylation of STAT3 3,000 p-STAT3 Tyr705 (top right) and STAT3 Ser727 70 100 STAT3 (bottom right) in a mixture of proteins UT DUSP2 35 from HEK293T cells mock transfected 2,000 GAPDH 35 or transfected to ectopically express DUSP2-FLAG (purified with an anti-FLAG 1,000 M2 affinity gel) (above lanes, left), plus phosphorylated S-tagged STAT3 (STAT3– 0 S-tag) obtained from IL-6-stimulated 500 1,000 1,500 2,000 HEK293T cells (below lanes, left), incubated m/z together in phosphatase buffer; bound proteins 5 Mock Mock DUSP2WT DUSP2C/S were eluted from bands excised after SDS-PAGE DUSP2WT 4 IL-6 (h) 0 0.5 1 2 4 6 0 0.5 1 2 4 6 0 0.5 1 2 4 6 (kDa) (left). In peptide sequences (above plots at right), DUSP2C/S p-STAT3 70 3 angled lines on the peptide backbone correspond STAT3 70 2 to b- and y-type ion series observed in the spectra. 35 GAPDH 1 (b) Immunoassay of HEK293T cells transfected to Variant DUSP2 35 express a combination of DUSP2-GFP and vector 0 IL-6 (h) 0 1 2 3 4 5 6 only or FLAG-tagged wild-type STAT3 (STAT3WT) or the Y705F mutant of STAT3 (STAT3Y705F); lysates immunoprecipitated with anti-FLAG were analyzed by immunoblot analysis anti-GFP. (c) Immunoassay (as in b) of HEK293T cells transfected for 24 h to express DUSP2-GFP alone or with STAT3-FLAG (above lanes), then left unstimulated (−) or stimulated (+) for 2 h with IL-6. (d) Immunoblot analysis of total and phosphorylated STAT3 in mononuclear cells obtained from the peripheral lymph nodes of wild-type or Dusp2−/− mice and then left untreated (UT) or stimulated for 5 h with PMA and ionomycin (PMA + iono). (e) Immunoblot analysis of total and phosphorylated STAT3 in HEK293T cells transfected to express STAT3-FLAG and mock-transfected for 24 h (left) or transfected for 24 h to express GFP-tagged wild-type DUSP (DUSP2 WT) (middle) or DUSP with substitution of serine for cysteine (as in Fig. 6e; DUSP2C/S), followed by stimulation for various times (above lanes) with IL-6. Right, quantification of phosphorylated STAT3 relative to total STAT3 in HEK293T cells treated as in left. Data are representative of two independent experiments.

Intensity

y +7 -H2O,y 2+ 15 893.45 2+

b15 862.57

+

b5 621.25

2+

b12 691.86

3+

2+ b15 -NH3 854.07

[M+3H] -NH3-H2O 790.89

y 3+ -H2O, b3+ 421.11 2+

3+

y5 ,y8

342.14

+ b4

+ b5-H2O

520.21 603.24 y 2+ y 4+ 8 512.54 586.21

3+ y 18 -H2O 710.18

2+ y 2+ 16 -H2O,y 17 -H2O–P 935.07

y182+-H2O,y182+-NH3 1064.68 2+

2+

y16 ,y17 –P 943.96

2+ y 13 -H2O 785.50

2+

y18

1073.54

2+ y19 –P,b9+

1080.52

+

b12 1382.61

b12+-H2O 1364.41

e

p-STAT3


b162+, y7+ 911.27

STAT3 in lymphocytes from Dusp2−/− mice but not in those from wild-type mice (Fig. 7d). In addition, ectopic expression of wild-type DUSP2 inhibited the phosphorylation of overexpressed STAT3 upon stimulation with IL-6, but its catalytically inactive mutant C257S, which affected its association with STAT3, did not show this effect, and the total amount of STAT3 remained unaffected in both cases (Figs. 6e and 7e). These data indicated that DUSP2 directly bound to and dephosphorylated STAT3 Tyr705 and Ser727. Inactivation of DUSP2 by CpG methylation in patients with UC The results reported above for the mouse models and our molecular findings led us to further assess the status of DUSP2 in clinical inflammatory disease. Notably, DUSP2 mRNA expression was much lower in peripheral blood mononuclear cells (PBMCs) from patients with UC than in those from healthy control subjects (Fig. 8a). DUSP2 mRNA expression in PBMCs from patients with UC increased only slightly following stimulation with PMA and ionomycin, and a similar increase in DUSP2 protein was undetectable by immunoblot analysis (Fig. 8b,c). To determine the mechanism by which DUSP2 expression was repressed, we carried out bioinformatics analysis; this revealed nature immunology VOLUME 16 NUMBER 12 DECEMBER 2015

that DUSP2 has a typical DNA sequence in the promoter region that fulfils the criteria for a CpG island (Fig. 8d). Subsequent methylation-specific PCR analysis showed that the DUSP2 promoter covering the specific sites bound by p53 and the transcription factor HIF-1α22,35 was methylated in 12 of 20 PBMCs from patients with UC, and methylation of DUSP2 was not induced after stimulation with PMA and ionomycin (Fig. 8e,f). In contrast, we found no methylation of the DUSP2 promoter in PBMCs from 12 healthy control subjects (Fig. 8e). Collectively, these data supported the proposal that DUSP2 is transiently induced during T cell activation under normal conditions, and an increased amount of DUSP2 inhibits STAT3 activity and thereby blocks polarization of naive T cells into TH17 cells, which maintains immunological homeostasis. However, in pathological conditions, the gene encoding DUSP2 is silenced by DNA methylation, which leads to deregulation of TH17 cells and subsequently contributes to inflammatory changes (Supplementary Fig. 8a). DISCUSSION The differentiation and function of TH17 cells is mediated mainly through IL-6–STAT3 signaling1, but little is known about the role of 1271

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Figure 8 DUSP2 expression is reduced by frequent 4 methylation in patients with UC. (a) qRT-PCR analysis UC-U 2 of DUSP2 mRNA in PBMCs from healthy control subjects (n = 44) or patients with UC (n = 24) (presented as in Fig. 1a). UC-M 0 Each symbol represents an individual subject; small horizontal Healthy UC UC Methylation – + – lines indicate the mean (± s.e.m.). (b) qRT-PCR analysis of DUSP2 mRNA in PBMCs obtained from healthy control subjects (n = 6) or patients with UC (n = 20) and left untreated (−) or stimulated for 2 h with PMA and ionomycin (+) (presented as in Fig. 1a). (c) Immunoblot analysis of DUSP2 in PMBCs as in b. Each lane represents an individual subject. (d) Human DUSP2 promoter, showing primers for methylation-specific PCR (MSP); angled arrow indicates the transcription start site. HRE, hypoxia-response element. (e) Methylation-specific PCR analysis of the DUSP2 promoter in PBMCs from patients with UC or healthy control subjects. M, methylated; U, unmethylated. Each lane represents an individual subject. (f) DUSP2 expression in PBMCs obtained from patients with UC and healthy control subjects and treated with PMA and ionomycin, relative to that in PBMCs left untreated. Each symbol represents an individual subject; small horizontal lines indicate the mean (± s.e.m.). *P < 0.01 and **P < 0.001 (unpaired Student’s t-test). Data are representative of three independent experiments (error bars, s.e.m.).

phosphatases in the regulation of this pathway. Our study showed that the phosphatase DUSP2, which was upregulated in T cell activation, dimerized with other DUSPs to antagonize IL-2 signaling, and that this molecule was a negative regulator of STAT3 during commitment of cells to the TH17 lineage. We observed that DUSP2-deficient naive T cells in vitro showed a ‘preference’ for differentiation into TH17 cells, whereas differentiation into iTreg cells was inhibited. Therefore, Dusp2−/− mice had more TH17 cells in the colon and lymph nodes than did their wild-type counterparts in colitis models. Although both in vitro studies and the KLH assay showed that the differentiation of Dusp2−/− T cells into iTreg cells was inhibited, DUSP2 had little effect on the response or suppressive function of Treg cells in the setting of colitis. Further in vitro investigation suggested that DUSP2 regulated iTreg cell differentiation in a TGF-β signaling dependent manner. Given the concept that DUSPs, including DUSP2, ‘preferentially’ regulate different signaling pathways in different milieus15, it is thus conceivable that DUSP2 does not affect TGF-β-mediated iTreg cell differentiation in the context of intestinal inflammation. Additional work will be required for full understanding of the role of DUSP2 in regulation of Treg cells in different diseases. In our study, the expression of several DUSPs was upregulated in T cells upon stimulation with anti-CD3 and anti-CD28, which indicated that these inducible DUSPs might participate in T cell activation. IL2 luciferase assays showed that unlike other inducible DUSPs, DUSP2, DUSP1 and DUSP4 substantially suppressed IL-2 signaling. Combined with the finding that DUSP2 facilitated DUSP4 to inhibit IL2 expression, we conclude that DUSP2 is not only related to other DUSPs but is also distinct from these molecules. For DUSPs, in vitro assays do not always reflect in vivo physiological roles15. In our study, we found that DUSP2 formed a homodimer or formed a complex with other DUSPs and thus gave rise to different substrate ‘preferences’. For example, our data showed that the DUSP2DUSP4 heterodimer had greater association with STAT3 than did the DUSP2-DUSP2 homodimer (data not shown). Moreover, despite the finding of higher levels of phosphorylated Erk1/2 or p38 in the colon of Dusp2−/− mice than in that of wild-type mice under quiescent conditions, we found higher levels of phosphorylated STAT3, but not of phosphorylated Erk1/2 or p38, in the colon of Dusp2−/− mice after DSS treatment than in that of their wild-type littermates. This indicated that different stimuli affect DUSP2’s substrate specificity.

Our observations thus support the concept that DUSP2’s modulation of STAT3’s activity is specific to cell type and stimulus. Phosphatases such as PTPN6, PTPN22 and DUSP1 have been shown to serve a crucial role in regulation of the immune response36,37. Therefore, we speculate DUSP2 expressed exclusively in immunological tissues might also participate in this process. The results of our model were proinflammatory, as the severity of colitis in Dusp2−/− mice was exacerbated due to enhanced development of TH17 cells. That finding differs from the results of another study, in which Dusp2−/− mice given injection of arthritogenic K/BxN serum showed diminished susceptibility to rheumatoid arthritis, and DUSP2 deficiency negatively regulated proinflammatory signaling in mast cells and macrophages25. However, this unexpected difference in these two model systems might result from the use of different effector immunological cells in different inflammatory milieus. In support of that interpretation, another published study has reported conflicting results for DUSP10-deficient macrophages that produced greater levels of proinflammatory cytokines in the innate immune response and DUSP10-deficient mice that showed resistance to autoimmune disease due to impaired T cell function38. It is therefore entirely plausible that expression of DUSP2 in different cells of the immune system could result in varied effects on the immune response. Phosphorylation of the ‘TH17 program’ initiator STAT3 in T cells leads to activation of the transcription factors RORγt and IκBζ and to the production of IL-17 and IL-21 after stimulation with signaling via IL-6 and its receptor gp130, which results in the commitment of cells to the TH17 lineage2,39,40. It is thus conceivable that nuclear phosphatases participate in the commitment of cells to the TH17 lineage through regulation of STAT3 activity during T cell activation. Here we found that transiently inducible DUSP2 dephosphorylated the STAT3 residues Tyr705 and Ser727 and thus inhibited its transcriptional activity. Although STAT3 is also necessary for the differentiation of induced type 1 regulatory T cells (Tr1 cells), here we found DUSP2 deficiency had no substantial effect on the differentiation of Tr1 cells in in vitro or in vivo experimental colitis models. Moreover, we found similar expression of the transcription factors AhR and c-Maf in wild-type and Dusp2−/− T cells; these factors are essential for the induction of IL-10-producing Tr1 cells. Thus, our data demonstrated that transiently inducible DUSP2 was not involved in Tr1 differentiation. Therefore, under normal conditions, DUSP2 repressed

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Articles development of the TH17 lineage during colitis via dephosphorylation and consequent deactivation of STAT3. Among the factors that contribute to the pathogenesis of IBD, the role of genetic predisposition has been highlighted by genetic studies and mouse models, and numerous candidate genes associated with IBD susceptibility have been identified, including STAT3 and other genes encoding proteins of the TH17 program41. In the mouse models of IBD we studied here, Dusp2−/− mice developed more severe inflammatory disease, with increased STAT3 activity and augmented production of TH17 cells. This has fueled interest in the genetic status of DUSP2 in human IBD. Notably, DUSP2 was downregulated by methylation and failed to be induced during T cell activation in PBMCs from patients with UC, which would indicate the potential of targeting DUSP2 for therapeutic intervention in human IBD. In summary, our data have identified a previously unknown function of DUSP2 in TH17 differentiation. DUSP2, encoded by a gene that was induced during T cell activation and was inactivated by CpG methylation in patients with UC, modulated the T H17 program– initiator STAT3. Thus, our identification of the role of DUSP2 in T cell development provides a potential therapeutic target for treatment of inflammatory diseases. Methods Methods and any associated references are available in the online version of the paper. Accession codes. ProteomeXchange (PRIDE partner repository): MS proteomics data, PXD002743. Note: Any Supplementary Information and Source Data files are available in the online version of the paper. Acknowledgments We thank C. Huang for help with the generation of Dusp2−/− mice; Y. Li for technical support; Y. Zhou for analysis of mouse phenotypes; J. Zhang for critical reading of the manuscript and comments; and C.Y. Wang for long-term support. Supported by the National Natural Science Foundation of China (81430056, 31420103905 and 81372491 to Y.Y.), the China National Major Scientific Program (2010CB912202 to Y.Y.), the Shu Fan Education Foundation and the Lam Chung Nin Foundation for Systems Biomedicine. AUTHOR CONTRIBUTIONS D.L. and L.L. designed the study, performed most of the experiments, and analyzed the data; X.J. helped with some experiments; Y.G. and Yu Liu contributed to the flow cytometry and enzyme-linked immunosorbent assays; X.C. provided animal care and technical assistance; X.Z. did the mass spectrum analysis; Yang Liu provided clinical material; Yunqiao Li prepared the polyclonal antibody to DUSP2; Yan Li and Y.J. provided technical support; Y.Z. participated in designing the study and provided expertise; M.A.M. discussed and revised the manuscript; Y.Y. designed the project and supervised the experiments; and D.L., L.L. and Y. Y. wrote the manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. 1. Korn, T., Bettelli, E., Oukka, M. & Kuchroo, V.K. IL-17 and Th17 cells. Annu. Rev. Immunol. 27, 485–517 (2009). 2. Yang, X.O. et al. T helper 17 lineage differentiation is programmed by orphan nuclear receptors RORα and RORγ. Immunity 28, 29–39 (2008). 3. Yang, X.O. et al. STAT3 regulates cytokine-mediated generation of inflammatory helper T cells. J. Biol. Chem. 282, 9358–9363 (2007). 4. Yang, X.P. et al. Opposing regulation of the locus encoding IL-17 through direct, reciprocal actions of STAT3 and STAT5. Nat. Immunol. 12, 247–254 (2011). 5. Chaudhry, A. et al. CD4+ regulatory T cells control TH17 responses in a Stat3dependent manner. Science 326, 986–991 (2009). 6. Abraham, C. & Cho, J.H. Inflammatory bowel disease. N. Engl. J. Med. 361, 2066–2078 (2009).


7. Lovato, P. et al. Constitutive STAT3 activation in intestinal T cells from patients with Crohn′s disease. J. Biol. Chem. 278, 16777–16781 (2003). 8. Musso, A. et al. Signal transducers and activators of transcription 3 signaling pathway: an essential mediator of inflammatory bowel disease and other forms of intestinal inflammation. Inflamm. Bowel Dis. 11, 91–98 (2005). 9. Barrett, J.C. et al. Genome-wide association defines more than 30 distinct susceptibility loci for Crohn′s disease. Nat. Genet. 40, 955–962 (2008). 10. McGovern, D.P. et al. Genome-wide association identifies multiple ulcerative colitis susceptibility loci. Nat. Genet. 42, 332–337 (2010). 11. Durant, L. et al. Diverse targets of the transcription factor STAT3 contribute to T cell pathogenicity and homeostasis. Immunity 32, 605–615 (2010). 12. Danial, N.N., Pernis, A. & Rothman, P.B. Jak-STAT signaling induced by the v-abl oncogene. Science 269, 1875–1877 (1995). 13. Jain, N., Zhang, T., Kee, W.H., Li, W. & Cao, X. Protein kinase C δ associates with and phosphorylates Stat3 in an interleukin-6-dependent manner. J. Biol. Chem. 274, 24392–24400 (1999). 14. Wei, W. et al. Dual-specificity phosphatases 2: surprising positive effect at the molecular level and a potential biomarker of diseases. Genes Immun. 14, 1–6 (2013). 15. Jeffrey, K.L., Camps, M., Rommel, C. & Mackay, C.R. Targeting dual-specificity phosphatases: manipulating MAP kinase signalling and immune responses. Nat. Rev. Drug Discov. 6, 391–403 (2007). 16. Huang, G., Wang, Y., Shi, L.Z., Kanneganti, T.D. & Chi, H. Signaling by the phosphatase MKP-1 in dendritic cells imprints distinct effector and regulatory T cell fates. Immunity 35, 45–58 (2011). 17. Holmes, D.A., Yeh, J.H., Yan, D., Xu, M. & Chan, A.C. Dusp5 negatively regulates IL-33-mediated eosinophil survival and function. EMBO J. 34, 218–235 (2015). 18. Huang, C.Y. et al. DUSP4 deficiency enhances CD25 expression and CD4+ T-cell proliferation without impeding T-cell development. Eur. J. Immunol. 42, 476–488 (2012). 19. Yang, C.Y. et al. Dual-specificity phosphatase 14 (DUSP14/MKP6) negatively regulates TCR signaling by inhibiting TAB1 activation. J. Immunol. 192, 1547–1557 (2014). 20. Li, J.P. et al. The phosphatase JKAP/DUSP22 inhibits T-cell receptor signalling and autoimmunity by inactivating Lck. Nat. Commun. 5, 3618 (2014). 21. Rohan, P.J. et al. PAC-1: a mitogen-induced nuclear protein tyrosine phosphatase. Science 259, 1763–1766 (1993). 22. Yin, Y., Liu, Y.X., Jin, Y.J., Hall, E.J. & Barrett, J.C. PAC1 phosphatase is a transcription target of p53 in signalling apoptosis and growth suppression. Nature 422, 527–531 (2003). 23. Wu, J., Jin, Y.J., Calaf, G.M., Huang, W.L. & Yin, Y. PAC1 is a direct transcription target of E2F–1 in apoptotic signaling. Oncogene 26, 6526–6535 (2007). 24. Chu, Y., Solski, P.A., Khosravi-Far, R., Der, C.J. & Kelly, K. The mitogen-activated protein kinase phosphatases PAC1, MKP-1, and MKP-2 have unique substrate specificities and reduced activity in vivo toward the ERK2 sevenmaker mutation. J. Biol. Chem. 271, 6497–6501 (1996). 25. Jeffrey, K.L. et al. Positive regulation of immune cell function and inflammatory responses by phosphatase PAC-1. Nat. Immunol. 7, 274–283 (2006). 26. Kinney, C.M. et al. Histone H3 as a novel substrate for MAP kinase phosphatase-1. Am. J. Physiol. Cell Physiol. 296, C242–C249 (2009). 27. Li, J.P., Fu, Y.N., Chen, Y.R. & Tan, T.H. JNK pathway-associated phosphatase dephosphorylates focal adhesion kinase and suppresses cell migration. J. Biol. Chem. 285, 5472–5478 (2010). 28. Wirtz, S., Neufert, C., Weigmann, B. & Neurath, M.F. Chemically induced mouse models of intestinal inflammation. Nat. Protoc. 2, 541–546 (2007). 29. Ma, H.L. et al. IL-22 is required for Th17 cell-mediated pathology in a mouse model of psoriasis-like skin inflammation. J. Clin. Invest. 118, 597–607 (2008). 30. Ostanin, D.V. et al. T cell transfer model of chronic colitis: concepts, considerations, and tricks of the trade. Am. J. Physiol. Gastrointest. Liver Physiol. 296, G135–G146 (2009). 31. Lee, Y. et al. Induction and molecular signature of pathogenic TH17 cells. Nat. Immunol. 13, 991–999 (2012). 32. Ghoreschi, K. et al. Generation of pathogenic TH17 cells in the absence of TGF-β signalling. Nature 467, 967–971 (2010). 33. El-Behi, M. et al. The encephalitogenicity of TH17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF. Nat. Immunol. 12, 568–575 (2011). 34. Farooq, A. et al. Solution structure of the MAPK phosphatase PAC-1 catalytic domain. Insights into substrate-induced enzymatic activation of MKP. Structure 11, 155–164 (2003). 35. Lin, S.C. et al. Suppression of dual-specificity phosphatase-2 by hypoxia increases chemoresistance and malignancy in human cancer cells. J. Clin. Invest. 121, 1905–1916 (2011). 36. Hammer, M. et al. Increased inflammation and lethality of Dusp1−/− mice in polymicrobial peritonitis models. Immunology 131, 395–404 (2010). 37. Vang, T. et al. Protein tyrosine phosphatases in autoimmunity. Annu. Rev. Immunol. 26, 29–55 (2008). 38. Zhang, Y. et al. Regulation of innate and adaptive immune responses by MAP kinase phosphatase 5. Nature 430, 793–797 (2004). 39. Okamoto, K. et al. IkappaBzeta regulates TH17 development by cooperating with ROR nuclear receptors. Nature 464, 1381–1385 (2010). 40. Wei, L., Laurence, A., Elias, K.M. & O’Shea, J.J. IL-21 is produced by Th17 cells and drives IL-17 production in a STAT3-dependent manner. J. Biol. Chem. 282, 34605–34610 (2007). 41. Neurath, M.F. Cytokines in inflammatory bowel disease. Nat. Rev. Immunol. 14, 329–342 (2014).

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Mice. To generate Dusp2−/− mice, a targeting vector with a neomycin-resistant cassette (positive-selection marker) flanked by two loxP sites, as well as a fragment encoding DTA (negative-selection marker) was constructed. The region of Dusp2 spanning exon 1 to exon 4 was targeted by homologous recombination, resulting in the deletion of the Dusp2 coding region. Targeted embryonic stem cell clones were selected and injected into C57BL/6 blastocysts. Chimeras were used for the generation of homozygous mice. Dusp2−/− mice were backcrossed with C57BL/6 mice for ten generations, and littermates were used as a control. CB-17 SCID mice were purchased from Vital River Laboratory Animal Technology. All animals were maintained in a special pathogen–free facility, and the animal study protocols used were approved by the ethics committee of Peking University Health Science Center (approval number bjmu20110301). Patients and specimens. Human blood specimens were obtained from 66 subjects with ulcerative colitis (UC) at Peking University Third Hospital and from 64 healthy subjects at the China-Japan Friendship Hospital. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by FicollHypaque centrifugation (Amersham Biosciences). Informed consent was provided by each subject before sample collection (according to the Helsinki Declaration), and all procedures were approved by the ethics committee of Peking University Health Science Center. Preparation of lymphocytes and flow cytometry. Cell suspensions from thymus, spleen, mesenteric lymph nodes, Peyer’s patches and lamina propria were prepared as described42. For in vitro T cell differentiation, naive T cells (CD4+CD25−CD62LhiCD44lo) sorted by flow cytometry from peripheral lymph nodes were activated with 2 µg/ml of plate-bound anti-CD3 (145-2C11; Quantobio) and 1 µg/ml of antiCD28 (37.51; Quantobio). For TH1 polarization, 10 µg/ml anti-IL-4 (11B11; Quantobio) and 10 ng/ml IL-12 (R&D) were used. For TH2 polarization, 10 µg/ml anti-IFN-γ (XMG1.2; Quantobio) and 20 ng/ml IL-4 (R&D) were used. For TH17 polarization, 20 ng/ml IL-6 (R&D), and 5 ng/ml TGF-β (R&D) with anti-IFN-γ and anti-IL-4 were used. For iTreg cell differentiation, 1 ng/ml TGF-β and 4 ng/ml IL-2 (R&D) with anti-IFN-γ and anti-IL-4 were used. For Tr1 cell differentiation, 50 ng/ml IL-27 (eBioscience) with anti-IFN-γ and anti-IL-4 (identified above) were used. After 48 or 72 h of culture, cells were collected for RNA extraction. For intracellular cytokine analysis, cells were re-stimulated with 100 ng/ml PMA and 500 ng/ml ionomycin in the presence of GolgiPlug and GolgiStop (BD) for 5 h. Cells were then fixed (eBioscience), permeabilized (Biolegend) and analyzed for expression of IFN-γ, IL-4, IL-17 or IL-10 (antibodies identified below), and for expression of Foxp3 with the Foxp3 Staining Buffer Set (eBioscience). For flow cytometry analysis of phosphorylated STAT3, cells were fixed with 2% paraformaldehyde at 37 °C for 10 min and permeabilized in 90% ice-cold methanol for 30 min, followed by two washes with flow buffer (PBS with 1% FBS) before incubation for 1 h at room temperature with antibody to STAT3 phosphorylated at Tyr705 (612569; BD Biosciences) or antibody to STAT5 phosphorylated at Tyr694 (612599; BD Biosciences). KLH immunization. 6- to 8-week-old mice were immunized at the base of the tail with 0.5 mg/ml KLH emulsified in complete Freund’s adjuvant (100 µl per mouse). After 7 d, the draining lymph nodes were removed and cells were stimulated with PMA and ionomycin for 5 h before intracellular staining of IL-17A and IFN-γ or Foxp3 (antibodies identified below). KLHspecific antibodies in serum collected from immunized mice were measured by enzyme-linked immunosorbent assay. Serum samples were added in threefold serial dilutions onto plates pre-coated with 10 µg/ml KLH, followed by goat antibody to mouse IgM,IgG, IgG1, IgG2a, IgG2bor IgG3 (SBA Clonotyping System-HRP Kit; 5300-05; Southern Biotech). Co-immunoprecipitation and immunoblot analysis. HEK293T cells were lysed in co-immunoprecipitation lysis buffer containing 150 mMNaCl, 0.1 mM EDTA, 10% glycerol, 0.5% NP40 and a ‘cocktail’ of protease inhibitors. Cell lysates were subjected to SDS-PAGE and immunoblot analysis was performed with the appropriate antibodies (identified below). The lysates were immunoprecipitated with IgG or the appropriate antibodies (identified below), and

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the precipitants were washed six times with PBS-T (PBS with 0.1% Tween-20), followed by immunoblot analysis. In vitro binding assays. Histidine- or histidine-FLAG-tagged proteins were expressed in Escherichia coli and were purified with nickel–nitrilotriacetic acid agarose. The DUSP2-His preparation was mixed with His-FLAG-tagged DUSP1 or DUSP4 at 4 °C. This mixture was immunoprecipitated with anti-FLAG, followed by immunoblot analysis. Antibodies. The following commercial antibodies were used in this study: anti-STAT3 (124H6), antibody to phosphorylated STAT3 (D3A7), anti-Erk (137F5), antibody to phosphorylated Erk (D13.14.4E), anti-p38 (9212), antibody to phosphorylated p38 (9211), anti-Akt (9272) and antibody to phosphorylated Akt (9275) (all from Cell Signaling Technology); anti-IFN-γ (XMG1.2), anti-IL-17A (eBio17B7), anti-IL-4 (11B11), anti-Foxp3 (FJK-16s), anti-IL-10 (JES5-16E3), anti-CD62L (MEL-14), and anti-CD4 (4SM95) (all from eBioscience); antibody to phosphorylated STAT3 (4/P-STAT3; 612569) and antibody to phosphorylated STAT5 (47/Stat5(pY694); 612599) (both from BD Biosciences); anti-CD4 (GK1.5), anti-CD45RB (C363-16A), anti-CD25 (3C7) and anti-CD44 (IM7) (all from Biolegend); anti-FLAG (M2; SigmaAldrich); anti-GAPDH (IC4) and anti-GFP (9F6) (both from Sungenebiotech); anti-β-actin (PM053) and ant-α-tubulin (2F9) (both from MBL). To prepare DUSP2 antibody, DNA corresponding to amino acids 91–290 of DUSP2 of mouse origin was subcloned into the vector pET28a, and the resultant protein was purified by histidine affinity chromatography for antibody production in mouse. The specificity of the polyclonal antibody against DUSP2 was assessed by immunoblot and immunoprecipitation assays (Supplementary Fig. 8b). DSS-induced colitis. Wild-type and Dusp2−/− mice (8–10 weeks of age) received 3% (wt/vol) DSS (molecular weight, 36,000-50,000; MP Biochemicals) in their drinking water to model acute colitis, and 5% (wt/vol) DSS to model recovery. In addition, to model chronic colitis, wild-type and Dusp2−/− mice were subjected to 2.5% DSS for 6 d, followed by 14 d of DSS-free water, and this cycle was repeated twice. Body weight and stool were monitored daily starting from day 1 of treatment. On day 9, or when mice had lost more than 20% of initial body mass, the colon was removed and measured, photographed and prepared for histological examination. Colon specimens were fixed in 10% neutral buffered formalin and paraffin-embedded sections were stained with hematoxylin and eosin (H&E). Intestinal inflammation was assessed histologically by methods described43. Immunostaining of phosphorylated STAT3 on colonic sections was performed according to the manufacturer’s instructions (Cell Signaling Technology). Images of H&E staining and immunohistological staining were obtained and measured on an Olympus Microscope IX51 and a DP72 camera together with ‘cellSens’ software (Olympus). T cell–transfer model of colitis. Naive CD4+CD45RBhi cells sorted from wildtype or Dusp2−/− mice were injected intraperitoneally into 6- to 8-week-old SCID recipients (5 × 105 cells per mouse). For the co-transfer model, 7.5 × 105 wild-type CD45.1+CD4+CD45RBhi cells alone or together with 2.5 × 105 CD4+CD25+ Treg cells from wild-type or Dusp2−/− mice were transferred into SCID recipients. Body weight change of each mouse was monitored weekly after transfer. Mice were sacrificed when significant weight loss occurred in the experimental groups. Transfection and luciferase reporter assay. IL2 promoter luciferase assays were performed as described44. HEK293T cells were co-transfected with pGL3-IL2 luciferase reporter plasmid and a control pRL-TK renilla luciferase reporter, together with vector encoding NFATc2 plus vector encoding various DUSPs. 24 h after transfection, luciferase activity was measured with a Dual-Luciferase Reporter Assay System (Promega). For analysis of the effect of DUSP2 on Il17a promoter activity, HEK293T cells were transfected with the pGL3-Il17a luciferase reporter plasmid and a control pRL-TK renilla luciferase reporter together with vector encoding RORγt or/and STAT3 plus vector encoding wild-type DUSP2 or the DUSP2C/S mutant, at different concentrations. Cells were stimulated for 4 h with PMA, ionomycin and IL-6. Luciferase activity was measured.

doi:10.1038/ni.3278


Quantitative real-time PCR and enzyme-linked immunosorbent assay. Following isolation with TRIzol reagent (Invitrogen), mRNA was specifically purified with a StarRNA mRNA Purification Kit (GenStarBiosolutions). First-strand cDNA was then obtained with the Transcript Reverse Transcription System (Promega), and TransStart Top Green qPCRSuperMix (TransGen Biotech) was used for real-time PCR (all primers, Supplementary Table 2); all procedures were done according to the manufacturer’s specifications. The value obtained for each gene was normalized to that of the gene encoding β-actin, and relative expression was calculated by change in threshold. Enzyme-linked immunosorbent assay s for IL-6, IL-17, TNF, IL-1β, IL-4 and IFN-γ in mouse serum were performed according to the manufacturer’s instructions (eBioscience). Protein purification and DUSP2 phosphatase assay. Protein purification was performed as described45. HEK293T cells were transfected with control vector or vector encoding DUSP2 together with vector encoding FLAG tag or STAT3 with an S-tag. 24 h later, cells transfected to express STAT3 were stimulated for 1 h with IL-6. All cells were then harvested with co-immunoprecipitation lysis buffer (described above). Precipitation assays were performed to purify DUSP2 protein with anti-FLAG M2 affinity gel (Sigma-Aldrich) and phosphorylated STAT3 protein with S-protein agarose beads (Novagen). DUSP2 protein was eluted with FLAG peptide for 1 h at room temperature. To analyze DUSP2 phosphatase activity, phosphorylated STAT3 protein with S-protein agarose was added to the phosphatase reaction buffer containing DUSP2 protein or control, followed by incubation at room temperature for 1 h. Beads were then washed with PBS, and the bound proteins were eluted by boiling and subjected to SDS-PAGE. Proteins on the gel were identified with a Pierce Silver Stain Kit per the manufacture’s instruction (Thermo), and the associated bands were excised for analysis by MS. Mass spectrometry. After silver staining of gels, excised gel segments were subjected to in-gel trypsin digestion and dried. Peptides were dissolved in 10 µl 0.1% formic acid and auto-sampled directly onto a 100-µm × 10-cm

doi:10.1038/ni.3278

fused silica emitter made in-house packed with reversed-phase ReproSil-Pur C18-AQ resin (3 µm and 120 Å; Ammerbuch). Samples were then eluted for 50 min with linear gradients of 5–32% acetonitrile in 0.1% formic acid at a flow rate of 300 nl/min. Mass spectra data were acquired with an LTQ Orbitrap Elite mass spectrometer (ThermoFisher) equipped with a nanoelectrospray ion source (ProxeonBiosystems). Fragmentation in the LTQ was performed by collision-induced dissociation (normalized collision energy, 35%; activation Q, 0.250; activation time, 10 ms) with a target value of 3,000 ions. The raw files were searched with the SEQUEST engine against a database from the Uniprot protein sequence database. Parameters were set as follows: protein modifications were carbamidomethylation (C) (fixed), oxidation (M) (variable) and phosphorylation (S, T, Y) (variable); the enzyme specificity was set to trypsin; amaximum missed cleavages were set to 2; the precursor ion mass tolerance was set to 10 ppm, and MS/MS tolerance was 0.5 Da. Methylation-specific PCR. Methylation-specific PCR analysis was performed as described46 (primer sequences, Supplementary Table 2). Statistical analysis. Prism GraphPad software v6.01 was used for analysis. The statistical significance of differences between wild-type and Dusp2−/− groups was calculated with an unpaired Student’s t-test or log-rank (Mantel-Cox) test. P values of 0.05 or less were considered significant.

42. Ivanov, I.I. et al. The orphan nuclear receptor RORγt directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126, 1121–1133 (2006). 43. Alex, P. et al. Distinct cytokine patterns identified from multiplex profiles of murine DSS and TNBS-induced colitis. Inflamm. Bowel Dis. 15, 341–352 (2009). 44. van Loosdregt, J. et al. Stabilization of the transcription factor Foxp3 by the deubiquitinase USP7 increases Treg-cell-suppressive capacity. Immunity 39, 259–271 (2013). 45. Gu, T. et al. CREB is a novel nuclear target of PTEN phosphatase. Cancer Res. 71, 2821–2825 (2011). 46. Lu, D. et al. The tumor-suppressive function of UNC5D and its repressed expression in renal cell carcinoma. Clin. Cancer Res. 19, 2883–2892 (2013).

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Erratum: The phosphatase DUSP2 controls the activity of the transcription activator STAT3 and regulates TH17 differentiation Dan Lu, Liang Liu, Xin Ji, Yanan Gao, Xi Chen, Yu Liu, Yang Liu, Xuyang Zhao, Yan Li, Yunqiao Li, Yan Jin, Yu Zhang, Michael A McNutt & Yuxin Yin Nat. Immunol. 16, 1263–1273 (2015); published online 19 October 2015; corrected after print 10 November 2015

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In the version of this article initially published, in Figure 2a, the middle plot in the bottom row was a duplicate of another plot, and in Figure 5c, the top right plot was a duplicate of another plot. These errors have been corrected for the PDF and HTML versions of this article.

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