Midkine Inhibits Inducible Regulatory T Cell Differentiation by Suppressing the Development of Tolerogenic Dendritic Cells

This information is current as Yoshifumi Sonobe, Hua Li, Shijie Jin, Satoshi Kishida, of September 23, 2021. Kenji Kadomatsu, Hideyuki Takeuchi, Tetsuya Mizuno and Akio Suzumura J Immunol 2012; 188:2602-2611; Prepublished online 8 February 2012;

doi: 10.4049/jimmunol.1102346 Downloaded from http://www.jimmunol.org/content/188/6/2602

Supplementary http://www.jimmunol.org/content/suppl/2012/02/08/jimmunol.110234 Material 6.DC1 http://www.jimmunol.org/ References This article cites 48 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/188/6/2602.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Midkine Inhibits Inducible Regulatory T Cell Differentiation by Suppressing the Development of Tolerogenic Dendritic Cells

Yoshifumi Sonobe,* Hua Li,* Shijie Jin,* Satoshi Kishida,† Kenji Kadomatsu,† Hideyuki Takeuchi,* Tetsuya Mizuno,* and Akio Suzumura*

Midkine (MK), a -binding , reportedly contributes to inflammatory diseases, including Crohn’s disease and rheumatoid arthritis. We previously showed that MK aggravates experimental autoimmune encephalomyelitis (EAE) by decreas- ing regulatory CD4+CD25+Foxp3+ T cells (Tregs), a population that regulates the development of autoimmune responses, although the precise mechanism remains uncertain. In this article, we show that MK produced in inflammatory conditions suppresses the

development of tolerogenic dendritic cells (DCregs), which drive the development of inducible Treg. MK suppressed DCreg- Downloaded from mediated expansion of the CD4+CD25+Foxp3+ Treg population. DCregs expressed significantly higher levels of CD45RB and produced significantly less IL-12 compared with conventional dendritic cells. However, MK downregulated CD45RB expression and induced IL-12 production by reducing phosphorylated STAT3 levels via src homology region 2 domain-containing phosphatase-2 in DCreg. Inhibiting MK activity with anti-MK RNA aptamers, which bind to the targeted to suppress the function of the protein, increased the numbers of CD11clowCD45RB+ dendritic cells and Tregs in the draining lymph nodes and suppressed the severity of EAE, an animal model of multiple sclerosis. Our results also demonstrated that MK was produced http://www.jimmunol.org/ by inflammatory cells, in particular, CD4+ T cells under inflammatory conditions. Taken together, these results suggest that MK aggravates EAE by suppressing DCreg development, thereby impairing the Treg population. Thus, MK is a promising therapeutic target for various autoimmune diseases. The Journal of Immunology, 2012, 188: 2602–2611.

egulatory T cells (Tregs), characterized by the expression the combination of activated T cells and Smad3 (5, 6). Although of Foxp3, are potent immunomodulators that suppress these in vitro cytokine-mediated iTreg differentiation models help R proliferation and the release of proinflammatory cyto- to elucidate mechanisms of immune regulation, they do not nec- kines from effector T cells (1). Modulating Treg differentiation is essarily reflect the precise in vivo processes. critical for controlling various autoimmune diseases, including Th cell differentiation requires Ag presentation by APCs, such by guest on September 23, 2021 multiple sclerosis (MS), type 1 diabetes, and inflammatory bowel as dendritic cells (DCs). Although these cells can promote adap- disease (2–4). Stimulation of naive CD4+ T cells with TGF-b tive immune responses, stimulating DCs with such suppressive causes inducible Treg (iTreg) differentiation, which is character- factors as TGF-b, IL-10, galectin-1, vasoactive intestinal peptide, ized by Foxp3 expression via the RUNX transcription factors or and 1,25-dihydroxyvitamin D3 induces self-tolerance in vitro and in vivo (7–11). Previous reports showed that DCs stimulated with TGF-b and IL-10 elicit CD4+CD25+Foxp3+ Treg differentiation *Department of Neuroimmunology, Research Institute of Environmental Medicine, + Nagoya University, Nagoya 464-8601, Japan; and †Department of Biochemistry, from naive CD4 T cells (8, 12, 13). These results suggest that Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan regulation of these Treg-inducing tolerogenic DCs (DCregs) may Received for publication August 16, 2011. Accepted for publication January 6, 2012. provide therapeutic approaches for autoimmune diseases. This work was supported in part by the YOKOYAMA Foundation for Clinical The heparin-binding growth factor midkine (MK) was originally Pharmacology; a Grant-in-Aid for Scientific Research (Young Scientists B) and the identified as a expressed in response to (14). MK Global Centers of Excellence Program “Integrated Functional Molecular Medicine for Neuronal and Neoplastic Disorders” from the Ministry of Education, Culture, is thought to play a role in a number of inflammatory diseases, Sports, Science and Technology of Japan; Health and Labor Science Research Grants including cisplatin-induced renal damage, Crohn’s disease (CD), for Research on Intractable Diseases from the Ministry of Health, Labor and Welfare; and rheumatoid arthritis (RA) (15–18). Our previous study showed the Program for Promotion of Fundamental Studies in Health Sciences of the Na- tional Institute of Biomedical Innovation; and a grant from the New Energy and that inhibiting MK activity alleviated experimental autoimmune Industrial Technology Development Organization of Japan. encephalomyelitis (EAE), an animal model of MS, by expanding Address correspondence and reprint requests to Dr. Yoshifumi Sonobe, Department the Treg population (19). However, the precise mechanisms that of Neuroimmunology, Research Institute of Environmental Medicine, Nagoya Univer- regulate Treg proliferation remain largely unknown. sity, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. E-mail address: sonobe@riem. nagoya-u.ac.jp In this article, we show that inhibiting MK activity expands the The online version of this article contains supplemental material. DCreg population, which causes iTreg differentiation from naive CD4+ T cells. MK suppressed the development of DCregs in vitro Abbreviations used in this article: CD, Crohn’s disease; DC, dendritic cell; DCcon, conventional dendritic cell; DCreg, regulatory T cell-inducing tolerogenic dendritic and in vivo, and it induced IL-12 production by these cells by cell; dLN, draining lymph node; EAE, experimental autoimmune encephalomyelitis; downregulating levels of phosphorylated STAT3 (pSTAT3) via src iTreg, inducible regulatory T cell; KO, knockout; MK, midkine; MOG, myelin oli- godendrocyte glycoprotein; MS, multiple sclerosis; PD-L1, programmed cell death homology region 2 domain-containing phosphatase (SHP)-2. The ligand 1; pSTAT, phosphorylated STAT; RA, rheumatoid arthritis; rm, recombinant overall result was reduced DCreg-mediated iTreg differentiation. murine; SHP, src homology region 2 domain-containing phosphatase; Treg, regula- Inhibiting MK using RNA aptamers significantly suppressed EAE. tory T cell; WT, wild-type. MK was produced by inflammatory cells, in particular CD4+ Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 T cells under inflammatory conditions. Taken together, these www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102346 The Journal of Immunology 2603 results suggest that MK produced by CD4+ T cells aggravates 500 ng/ml ionomycin. After staining with PerCP–anti-CD4 Abs, cells were EAE by suppressing DCreg development and subsequent iTreg fixed and permeabilized with Cytofix/Cytoperm reagent (BD Biosciences) differentiation. Thus, expanding the iTreg population by inhibiting and stained with FITC–anti-IFN-g (XMG1.2; BD Biosciences) and PE– anti-IL-17 (TC11-18H10; BD Biosciences) Abs. The samples were ex- MK activity and DCreg proliferation may provide a useful strat- amined with a Cytomics FC500 system (Beckman Coulter) and analyzed egy for treating autoimmune diseases, including MS. with CXP Software Ver. 2.0 (Beckman Coulter).

Materials and Methods MLR Animals CD4+CD252 T cells were isolated from the spleens of BALB/c mice using a MACS system (Miltenyi Biotec). CD4+CD252 T cells (H2d) were b d Female C57BL/6 (H2 ) and BALB/c (H2 ) mice were obtained from Japan cocultured for 4 d with DCcons, MK-treated DCcons, DCregs, or MK- SLC. MK-knockout (KO) mice (C57BL/6) were generated as described treated DCregs from C57BL/6 mice (H2b) at 1:1 ratios in the presence previously (19). Mice were bred in the animal facility of the Research or absence of rIL-12p70, anti–IL-12p40 Abs (C17.8; BD Biosciences), Institute of Environmental Medicine at Nagoya University. The protocols or SHP-2 inhibitor (NSC87877; Calbiochem). The percentage of CD4+ for animal experiments were approved by the Animal Experiment Com- CD25+Foxp3+ Tregs was determined by flow cytometry. The levels of mittee of Nagoya University. IFN-g in the culture supernatant was measured by ELISA. Generation of DCs Suppression assay DCs were generated as previously described (20). In brief, immature CD4+CD252 T cells were isolated from spleens of BALB/c mice by conventional DCs (DCcons) were generated by incubating bone marrow MACS system. CD4+CD252 T cells and MACS-sorted CD4+ T cells ex- cells in complete DMEM containing 10% FBS, 2 mM L-glutamine, and panded by DCcons, MK-treated DCcons, DCregs, or MK-treated DCregs 20 ng/ml GM-CSF (R&D Systems) for 6 d, with or without 100 ng/ml in MLR were cultured in the presence of 5 mg/ml plate-bound anti-CD3 Downloaded from recombinant MK. Immature DCregs were generated with 20 ng/ml GM- (145-2C11; BD Biosciences) and 2 mg/ml soluble anti-CD28 (37.51; BD CSF, 20 ng/ml recombinant murine (rm)IL-10, and 20 ng/ml recombinant Biosciences) Abs for 3 d at the indicated ratio. BrdU was added for the human TGF-b1, with or without 100 ng/ml recombinant MK. Immature final 16 h, and proliferation was evaluated using a BrdU cell-proliferation DCcons or DCregs were then stimulated to become mature DCs with 100 assay (Calbiochem). ng/ml LPS for 24 h. Adoptive transfer of DCs Flow cytometry

Immature DCcons or DCregs treated or not with MK were pulsed for 24 h http://www.jimmunol.org/ Cells were resuspended in FACS buffer (PBS supplemented with 2% FBS with 20 mg/ml myelin oligodendrocyte glycoprotein (MOG)35–55 and 100 and 0.01% sodium azide). Nonspecific staining was blocked using rat anti- 5 ng/ml LPS and were transferred i.v. into wild-type (WT) mice (5 3 10 FcR Abs (BD Biosciences) for 30 min at 4˚C, after which the cells were cells/mouse). One day later, EAE was induced as described below. stained with FITC–anti-CD11c (HL-3), PE–anti-CD45RB (16A), PE–anti- MHC class I (AF6-88.5), PE–anti-MHC class II (M5/114.15.2), PE–anti- EAE CD80 (16-10A1), and PE–anti-CD86 (GL-1) Abs from BD Biosciences, as well as PE–anti-programmed cell death ligand 1 (PD-L1; MIH5) Abs from MOG-induced EAE was induced as described previously (19). In brief, eBioscience. To detect Tregs, cells were incubated with PerCP–anti-CD4 mice were injected s.c. with 0.2 ml emulsion containing 200 mg MOG35–55 (RM4-5; BD Biosciences) and FITC–anti-CD25 (7D4; BD Biosciences) in PBS combined with an equal volume of CFA containing 300 mg heat- Abs for 20 min at 4˚C, treated with cell permeabilization solution (eBio- killed Mycobacterium tuberculosis H37Ra. Mice were injected i.p. with science), and stained with PE–anti-Foxp3 Abs (FJK-16s; eBioscience). For pertussis toxin on the day of immunization and 2 d after immuniza- by guest on September 23, 2021 cytokine staining, cells were stimulated for 4 h with 50 ng/ml PMA and tion (200 ng/mouse). Mice were scored daily according to the following

FIGURE 1. MK suppresses DCreg development. (A and B) Allogeneic CD4+CD252 T cells from BALB/c mice were cocultured with each subset of DCs from C57BL/6 mice for 4 d. Percentages of CD4+CD25+Foxp3+ Tregs were assessed by flow cytometry (n = 6). *p , 0.05, ***p , 0.001. (C) Treg function was evaluated using proliferation assays in which CD4+CD252 T cells and MACS-sorted CD4+ T cells expanded by DCcons, MK-treated DCcons, DCregs, or MK-treated DCregs in MLR were cultured in the presence of 5 mg/ml plate-bound anti-CD3 and 2 mg/ml soluble anti-CD28 Abs for 3 d at the indicated ratio. Proliferation was evaluated using a BrdU cell-proliferation assay kit (Calbiochem). Results obtained with CD4+CD252 T cell were set to 1 (n = 3). 2604 MIDKINE SUPPRESSES THE DEVELOPMENT OF TOLEROGENIC DCs scale: 0, normal; 1, limp tail or mild hind limb weakness; 2, moderate hind Histological analysis limb weakness or mild ataxia; 3, moderate to severe hind limb weakness; 4, severe hind limb weakness, mild forelimb weakness, or moderate ataxia; EAE mice were intracardially perfused with PBS at 28 d after immuni- 5, paraplegia with moderate forelimb weakness; and 6, paraplegia with zation; spinal cords were fixed with 4% paraformaldehyde and then em- severe forelimb weakness, severe ataxia, or moribundity. bedded in paraffin. Ten-micrometer-thick sections were stained with H&E, as described previously (19). Stained sections were observed under a mi- RNA aptamers croscope. Inflammatory foci were identified as perivascular clusters con- taining $20 mononuclear cells. Anti-MK RNA aptamers were produced by Ribomic, essentially as de- scribed previously (21). In brief, RNA aptamers were raised against MK Isolation of CNS mononuclear cells using systematic evolution of ligands by exponential enrichment (SELEX). The functionally optimized 49-mer RNA molecule was modified with CNS mononuclear cells were prepared from the brains and spinal cords of fluorine and O-methyl at the 29 position of each ribose and with a cho- EAE mice intracardially perfused with PBS from the left ventricle 28 d after lesterol moiety and inverted dT tags at the 59 and 39 ends, respectively. immunization (22). The tissues were then passed through a nylon mesh Anti-MK RNA aptamers were administered to EAE mice at a dose of 15 (NB 60; NBC). The CNS-infiltrating mononuclear cells were enriched mg/kg every other day from the day of immunization. using a 30% Percoll gradient. Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 2. MK induces IL-12 production in DCregs. Each subset of DCs was expanded, as described in Materials and Methods. After treatment with LPS (100 ng/ml) for 24 h, levels of surface molecules and cytokines were assessed. (A and B) Expression of surface molecules in each DC subset was assessed using flow cytometry. Representative data are shown in (A), whereas average results are shown in (B)(n = 3). (C) The levels of IL-12, IL-6, TGF-b, IL-10, and IL-27 in culture supernatant containing each DC subset were analyzed using ELISAs. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 2605

Cytokine ELISA for 4 d with plate-bound anti-CD3 Abs and soluble anti-CD28 Abs in RPMI 1640 medium supplemented with 2 mM sodium pyruvate, 2 mM ELISA kits specific for mouse IL-12p70, IL-6, IL-10, and IFN-g were L-glutamine, and 10% FBS in the presence of recombinant cytokines. obtained from BD Biosciences. An ELISA kit specific for mouse TGF-b T cells were polarized with rmIL-12 (10 ng/ml; R&D Systems), rmIFN-g was purchased from R&D Systems, and kits specific for IL-23 and IL-27 (5 ng/ml; R&D Systems), and anti–IL-4 Abs (11B.11; 10 mg/ml; BD were obtained from eBioscience. Biosciences) for Th1 cells; mouse IL-4 (2 ng/ml; R&D Systems) and anti– Western blotting IFN-g Abs (XMG1.2; 10 mg/ml, BD Biosciences) for Th2 cells; human TGF-b1 (5 ng/ml), IL-6 (30 ng/ml), and anti–IFN-g Abs for Th17 cells; Cells were lysed in TNES buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, human TGF-b1 (5 ng/ml) for iTregs; and IL-4 (2 ng/ml) and human TGF- 1% Nonidet P-40, 2 mM EDTA, and 0.1% SDS) with a protease inhibitor b1 (5 ng/ml) for Th9 cells. mixture (Roche) and a phosphatase inhibitor mixture (Sigma). Thirty micrograms of protein from total cell lysates was examined on Western Statistical analysis blots, as previously described (23), using anti–SHP-2 (polyclonal; Cell Differences between the mean values were analyzed using the two-tailed Signaling), anti-pSTAT3 (D3A7; Cell Signaling), anti-STAT3 (84; BD Student t test, with Welch’s correction. The p values , 0.05 were con- Biosciences), anti-MK (H-65; Santa Cruz Biotechnology), and anti–a-tu- sidered significant. bulin (DM1A; Sigma) Abs. Quantitative RT-PCR Results RNAwas extracted with an RNeasy Mini kit (Qiagen) and reverse transcribed MK suppresses Treg differentiation by inhibiting DCreg with oligo(dT)12–18 (Invitrogen), according to the manufacturer’s protocol. development The cDNA served as a template to amplify in real-time PCRs with We analyzed the effects of MK on DCreg development in the

TaqMan assays (Applied Biosystems), Universal PCR Downloaded from Master Mix (Applied Biosystems), and Rotor-Gene Q (Qiagen). Expression presence of TGF-b and IL-10. Allogeneic responses of BALB/ + 2 levels of target genes were calculated using a comparative method and nor- c-derived CD4 CD25 T cells with C57BL/6-derived DCregs malization to GAPDH expression levels. The following primers and probes induced CD4+CD25+Foxp3+ Treg differentiation (Fig. 1A, 1B). were obtained from Applied Biosystems: IL-12p35, Mm00434165_m1; However, MK suppressed DCreg-mediated differentiation of IL-12p40, Mm00434174_m1; IL-23p19, Mm00518984_m1; GAPDH, + + + Mm99999915_g1; and MK, Mm00440279_m1. CD4 CD25 Foxp3 Tregs (Fig. 1A, 1B). The levels of MK were negligible in the culture supernatant (Supplemental Fig. 1). To

Th cell differentiation examine the suppressor activity of these in vitro-induced CD4+ http://www.jimmunol.org/ + 2 CD4+CD62L+ T cells were isolated using MACS beads, according to the T cells, CD4 CD25 effector T cells were stimulated with anti- manufacturer’s protocol (Miltenyi Biotec). CD4+ T cells were stimulated CD3 and anti-CD28 Abs in the presence of CD4+ T cells ex- panded by each subset of DCs. CD4+ T cells that developed in response to DCregs suppressed anti-CD3 Ab- and anti-CD28 Ab- induced proliferation of the CD4+CD252 effector T cells (Fig. 1C). However, MK-stimulated DCregs did not suppress prolifer- ation of effector T cells (Fig. 1C). Taken together, these results suggest that MK suppresses expansion of the DCreg population. by guest on September 23, 2021 MK suppresses DCreg-mediated iTreg differentiation by inducing IL-12 production We next analyzed molecules related to Ag presentation, includ- ing MHCs, B7 family molecules, and cytokines, in each DC subset using flow cytometry and ELISA. B7-1 (CD80) and B7-2 (CD86) expression levels in DCregs were significantly lower than

FIGURE 4. MK-treated DCregs do not induce IFN-g production. Al- logeneic responses were induced using CD4+CD252 cells (BALB/c) and FIGURE 3. MK suppresses iTreg differentiation by inducing IL-12 pro- DCs (C57BL/6). The levels of IFN-g in culture supernatant were analyzed duction in DCregs. (A and B) Allogeneic CD4+CD252 T cells from BALB/c using ELISA. MK-treated DCregs did not induce IFN-g production by micewerecoculturedwitheachsubsetofDCsfromC57BL/6micefor4din T cells. In contrast, addition of soluble anti-CD28 Abs (2 mg/ml) to MK- the presence or absence of rIL-12p70, anti-IL-12p40 Abs. Percentages of treated DCregs increased IFN-g production. Data are representative of CD4-gated CD25+Foxp3+ cells were assessed by flow cytometry. *p , 0.05. three independent experiments. ***p , 0.001. 2606 MIDKINE SUPPRESSES THE DEVELOPMENT OF TOLEROGENIC DCs in DCcons (Fig. 2A, 2B). Expression levels of MHC class I and II, suppressed DCreg-induced iTreg differentiation by inducing IL-12 B7-1 (CD80), B7-2 (CD86), and B7-H1 (PD-L1) were similar production. with and without MK treatment (Fig. 2A, 2B), showing that MK did not affect the expression of molecules related to Ag presen- MK suppresses iTreg differentiation by upregulating SHP-2 tation. However, DCregs expressed significantly higher levels of expression and inducing IL-12 production CD45RB, and addition of MK decreased the expression (Fig. 2A, Because we previously showed that MK induced SHP-2 expression 2B). In addition, levels of the inhibitory cytokines IL-10 and TGF- in CD4+ T cells (19), we next assessed SHP-2 expression in bone b, as well as the type 1 regulatory T cell-inducing cytokine IL-27, marrow cells by Western blotting. MK tripled the expression of in the culture supernatant were similar for the various DC subsets SHP-2 in bone marrow cells (Fig. 5A). Moreover, IL-10–induced (Fig. 2C). In contrast, DCregs produced significantly less IL-6 and STAT3a phosphorylation was inhibited by MK, an effect that was IL-12 compared with DCcons. IL-23 concentrations were less than reversed by the SHP-2 inhibitor NSC87877 (Fig. 5B). This SHP-2 the detection threshold in the culture supernatant (data not shown). inhibitor also inhibited IL-12 production by MK-stimulated Notably, MK significantly increased IL-12 production in DCregs, DCregs (Fig. 5C), as well as CD4+CD25+Foxp3+ Treg differen- whereas IL-6 and IL-23 levels were not affected (Fig. 2C). These tiation from CD4+CD252 T cells induced by MK-treated DCregs results suggest that MK-stimulated DCregs do not cause naive in allogeneic responses (Fig. 5D, 5E). Taken together, these results CD4+ T cells to differentiate into iTregs because of IL-12 pro- suggest that MK suppresses expansion of the DCreg population by duction in the DCregs. To address this hypothesis, we examined downregulating pSTAT3 levels via SHP-2. the effects of IL-12 produced by MK-treated DCregs on iTreg differentiation using rIL-12– and anti–IL-12–neutralizing Abs. MK-treated DCregs do not suppress autoimmune inflammation Downloaded from rIL-12 downregulated DCreg-mediated iTreg differentiation dur- in vivo ing the allogeneic responses (Fig. 3). Furthermore, MK-mediated To determine whether MK-induced suppression of DCreg devel- suppression of iTreg differentiation by DCregs was ameliorated by opment impairs CD4+CD25+Foxp3+ Treg differentiation in vivo, anti–IL-12–neutralizing Abs (Fig. 3). Although the addition of C57BL/6 mice were injected with one of four DC subsets anti–IL-12–neutralizing Abs upregulated the percentage of CD4+ (DCcons, DCregs, MK-stimulated DCcons, and MK-stimulated + + CD25 Foxp3 Tregs in MLR using DCcons, it was not signifi- DCregs), and EAE was induced. Injecting DCregs significantly http://www.jimmunol.org/ cantly different compared with isotype control. However, MK- suppressed the development of EAE, whereas the disease was treated DCregs did not induce the differentiation of IFN-g–pro- significantly more severe in mice injected with MK-stimulated ducing Th1 cells (Fig. 4). Together, these results suggest that MK DCregs compared with mice injected with untreated DCregs by guest on September 23, 2021

FIGURE 5. MK induces IL-12 production in DCregs by upregulating SHP-2 levels. (A) Bone marrow cells were cultured with MK for 6 d. Levels of SHP-2 in whole-cell lysates were analyzed by Western blotting. (B) Levels of pSTAT3 in cells cultured for 6 d with the indicated cytokines and SHP-2 inhibitor (SHP2I). (C) Levels of IL-12 in supernatant containing DCcons, DCregs, or MK-treated DCregs in the presence or absence of SHP2I were analyzed by ELISA. (D and E) Percentages of CD4-gated CD25+Foxp3+ cells in allogeneic responses induced by DCcons, DCregs, or MK-treated DCregs, in the presence or absence of SHP2I, were assessed by flow cytometry. *p , 0.05. The Journal of Immunology 2607

(Fig. 6A). Injection of DCregs reduced the infiltration of cells in also noted in dLN DCs (Fig. 7E). In contrast, significantly fewer the spinal cord at 28 d after immunization, whereas inflammatory IFN-g– and IL-17–producing CD4+ T cells had infiltrated into the foci were increased in meninges of mice that received MK- CNS in mice treated with anti-MK RNA aptamers compared with stimulated DCregs compared with mice injected with untreated PBS-treated mice (Fig. 7F). DCregs (Fig. 6B). The percentage and number of CD4+CD25+ MK-KO mice showed significantly milder EAE symptoms Foxp3+ Tregs among the cells obtained from draining lymph compared with WT mice (Supplemental Fig. 4A). The percentages nodes (dLNs) of EAE mice injected with DCregs were signifi- of CD11clowCD45RB+ DCregs and CD4+CD25+ Foxp3+ Tregs in cantly higher than in any of the other mice with EAE (Fig. 6C). dLNs of MK-KO mice were higher than in WT mice (Sup- Similar changes were not observed in the CNS, however. (Fig. 6C, plemental Fig. 4B, 4C). In contrast, fewer IFN-g– or IL-17–- Supplemental Fig. 2). Accordingly, the percentage and number of producing CD4+ T cells infiltrated into the CNS of MK-KO mice IL-17– and IFN-g–producing CD4+ T cells obtained from the compared with WT mice (Supplemental Fig. 4D). Taken together, CNS of EAE mice injected with DCregs were significantly lower these results support MK as a suppressor of DCreg expansion in than data obtained from mice with EAE injected with MK-treated mice with EAE. DCregs, whereas the groups showed similar IL-17– and IFN-g– MK is produced by CD4+ cells in vitro and in vivo producing CD4+ T cell populations from the dLNs (Supplemental Fig. 3A, 3B). These results suggest that MK inhibits the ability of We next attempted to identify the cellular source of MK. Ex- DCregs to induce Treg differentiation in vivo. pression levels of MK mRNA and MK protein were significantly elevated in the dLNs of EAE mice compared with naive mice (Fig. MK suppresses expansion of the DCreg population in vivo + + 8A, 8B). MK mRNA expression was induced in CD11b , CD4 , Downloaded from We then examined the number of CD11clowCD45RB+ DCregs in and CD8+ cells from the dLNs of EAE mice (Fig. 8A). MK MK-KO mice and EAE mice treated with anti-MK RNA aptam- protein expression was also induced in CD4+ T cells from the ers, which bind to MK to suppress the function of the protein (19). dLNs of EAE mice (Fig. 8C). Because CD4+ T cells play a critical Anti-MK RNA aptamers reduced the clinical EAE scores (Fig. role in the development of EAE, we focused on MK expression in 7A) and infiltration of cells in the spinal cord 28 d after immu- CD4+ T cells in vitro. MK mRNA and MK protein expression low +

nization (Fig. 7B). The percentages and numbers of CD11c were significantly upregulated in CD4 T cells stimulated with http://www.jimmunol.org/ CD45RB+ DCregs and CD4+CD25+Foxp3+ Tregs in dLNs of mice anti-CD3 and anti-CD28 Abs or Con A (Fig. 8D, 8E). In addition, treated with anti-MK RNA aptamers were higher than in PBS- CD4+CD62L+ naive T cells showed markedly increased MK treated mice (Fig. 7C, 7D). Reduced levels of IL-12p40 mRNA mRNA levels in response to anti-CD3 and anti-CD28 Abs (Fig. expression, but not IL-12p35 or IL-23p19 mRNA expression, were 8F). Interestingly, Th1 cells, which were induced in the presence by guest on September 23, 2021

FIGURE 6. MK inhibits the suppressive functions of DCregs in EAE. (A) Clinical EAE scores in mice that underwent adoptive transfer of each DC subset (n = 6). (B) H&E staining of lumbar spinal cords from EAE mice that underwent adoptive transfer of each DC subset. Arrows show inflammatory foci. Boxed area: original magnification 320. Scale bars, 200 mm. (C) Number of CD4+CD25+Foxp3+ cells in the CNS and dLN harvested 28 d after immunization. Cells were counted by multiplying the percentage of CD4+CD25+Foxp3+ cells by the absolute number of cells obtained from the CNS or dLN. *p , 0.05, ***p , 0.001. 2608 MIDKINE SUPPRESSES THE DEVELOPMENT OF TOLEROGENIC DCs Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 7. MK suppresses DCreg development in vivo. (A) Clinical EAE scores from mice injected with PBS or anti-MK RNA aptamers (n = 5). (B) H&E staining of lumbar spinal cords from PBS- or anti-MK RNA aptamer-treated EAE mice. Arrows show inflammatory foci. Scale bars, 200 mm. Numbers of CD11clowCD45RB+ DCregs (C) and CD4+CD25+Foxp3+ Tregs (D) in dLNs of EAE mice. (E) Levels of IL-12p35, IL-12p40, and IL-23p19 mRNA in CD11c+ cells obtained from the dLNs of EAE mice using MACS beads. (F) Numbers of IFN-g– and IL-17–producing cells in the CNS. *p , 0.05, **p , 0.01. of IL-12 and IFN-g, expressed significantly higher levels of MK which induced CD4+CD25+Foxp3+ Treg differentiation. However, mRNA compared with Th0 cells (Fig. 8G). These results suggest it is still unclear how DCregs induce iTreg differentiation. We also that MK produced by CD4+ T cells plays an important role in the showed that DCregs expressed higher levels of PD-L1 and lower symptoms of EAE by suppressing DCreg expansion. levels of IL-12. PD-L1 reportedly induces iTreg development and maintains this cellular population (24). Thus, PD-L1 may con- Discussion tribute to iTreg differentiation from naive T cells. Furthermore, Our previous report showed that inhibiting MK induced Treg DCcons induce Th1 cell differentiation owing to higher levels expansion and suppressed autoimmune responses. In this study, we of CD80 and CD86 expression and IL-12 production. CD28- showed that MK suppressed Treg development by inhibiting ex- mediated signaling is known to induce T cell activation to pro- pansion of the DCreg population. Stimulating bone marrow cells mote Th1 cell differentiation in the presence of IL-12. Although with TGF-b and IL-10 resulted in the development of DCregs, our data indicated that CD80, CD86, and PD-L1 were highly The Journal of Immunology 2609 Downloaded from http://www.jimmunol.org/

FIGURE 8. MK expression is induced in CD4+ T cells during EAE. (A) CD11b+, CD11c+, CD4+, and CD8+ cells were sorted from the dLNs of mice with EAE 14 d after immunization with MOG35–55, and MK mRNA levels were assessed using quantitative RT-PCRs. *p , 0.05, naive versus EAE. Western blotting was used to analyze MK protein levels in all dLN cells (B) and CD4+ cells sorted from the dLNs (C) of EAE mice. Levels of MK mRNA (D) and protein (E) in CD4+ cells stimulated with anti-CD3 and anti-CD28 Abs or Con A. MK mRNA levels in naive or memory CD4+ T cells (F) and in various Th subsets (G). *p , 0.05, **p , 0.01, ***p , 0.001. expressed in DCcons, high doses of soluble CD28 were reported neutralizing Abs (37) and KO mice (38–40) showed that the Th1 + to overcome PD-L1–induced negative regulation of CD4 T cells cytokine IFN-g plays an important role in suppressing EAE. In by guest on September 23, 2021 (25). Overall, higher expression levels of PD-L1 and lower ex- contrast, MOG-reactive Th1 cells are reported to induce EAE (41, pression levels of CD80 and CD86 may be important for DCreg- 42). These findings suggest that factors other than IFN-g mediate mediated iTreg-induction processes. the effects of Th1 cells in EAE. In the current study, we showed STAT3 activation is important for the development of tolero- that MK is produced by CD4+ cells during EAE development. In genic DCs or suppression of DCs that lead to aggressive immune addition, in vitro studies revealed that MK expression was strongly responses (26–28). Injecting small interfering RNA specific for induced in Th1 cells compared with naive Th cells. Thus, MK may STAT3 in myeloid cells led to immune activation and antitumor be one of the factors that contributes to Th1 cell-mediated exac- activity (29). In particular, IL-10 reduces IL-12 production in DCs erbation of EAE. Of note, IFN-g KO mice show accelerated by inhibiting NF-kB recruitment to the IL-12p40 promoter in collagen-induced arthritis, a model of RA (43), whereas anti–IL- a STAT3-dependent manner (30). Consistent with this, we showed 12 Abs prevent this disease in IFN-g KO mice (44), suggesting that MK downregulated the levels of pSTAT3 and induced IL-12 that Th1 cells also have a prominent role in RA. In addition, Th1 production in DCregs. Thus, decreasing pSTAT3 levels in DCs cells contribute to CD (45). Because MK has been detected in the exacerbated inflammatory processes by inducing IL-12 produc- synovial fluid of patients with RA (18) and is more strongly tion. We also showed that MK suppressed STAT3 activation in expressed in CD (16), MK may play a role in these Th1 cell- DCs by inducing SHP-2 expression. In fact, mice lacking SHP-2 induced autoimmune diseases by suppressing DCreg develop- signaling show prolonged STAT3 phosphorylation (31). In addi- ment and subsequently expanding the Treg population. tion, SHP-2 is required for discoidin domain receptor-induced In this study and in a previous article, we demonstrated the suppression of STAT3 phosphorylation (32). Interestingly, low- efficacy of anti-MK RNA aptamers in mice with EAE, suggesting density lipoprotein receptor-related protein, which is thought to that a similar approach may be useful for MS. Aptamers are single- be the receptor for MK, was shown to associate with SHP-2 in stranded oligonucleotides that bind to with high affinity a human follicular thyroid carcinoma cell line (33). These results and affect function of the target (46). Toxicologic studies have suggest that SHP-2 signaling plays an important role in MK- shown that aptamers do not produce the same adverse effects induced suppression of STAT3 phosphorylation in DCs. MK- observed with antisense oligonucleotides (47–49), such as im- induced SHP-2 signaling also inhibits IL-10–induced suppres- mune stimulation, complement activation, and anticoagulation. sion of IL-12 production, which is a STAT3-mediated response. In this article, we showed that administration of anti-MK RNA Therefore, inhibiting MK may be a useful strategy to treat auto- aptamers enhanced DCreg development in dLNs during EAE. immune diseases, because such an approach would upregulate Cytokine production and Ag presentation by DCs are critical for p-STAT3 levels and regulate immune responses in myeloid cells. differentiation of Th cells to initiate autoimmune processes. Thus, Recently, Th17 cells, a CD4+ Th cell subset, have been im- expansion of the DCreg population in response to anti-MK RNA plicated in EAE development (34–36). Moreover, studies using aptamers regulates Treg development at early stages. 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