Neuropeptide Signaling Activates Dendritic Cell-Mediated Type 1 Immune Responses through Neurokinin-2 Receptor

This information is current as Hidemitsu Kitamura, Minoru Kobayashi, Daiko Wakita and of October 3, 2021. Takashi Nishimura J Immunol 2012; 188:4200-4208; Prepublished online 2 April 2012; doi: 10.4049/jimmunol.1102521 http://www.jimmunol.org/content/188/9/4200 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2012/04/02/jimmunol.110252 Material 1.DC1 http://www.jimmunol.org/ References This article cites 46 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/188/9/4200.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

Neuropeptide Signaling Activates Dendritic Cell-Mediated Type 1 Immune Responses through Neurokinin-2 Receptor

Hidemitsu Kitamura,* Minoru Kobayashi,* Daiko Wakita,* and Takashi Nishimura*,†

Neurokinin A (NKA), a neurotransmitter distributed in the central and peripheral nervous system, strictly controls vital responses, such as airway contraction, by intracellular signaling through neurokinin-2 receptor (NK2R). However, the function of NKA–NK2R signaling on involvement in immune responses is less-well defined. We demonstrate that NK2R-mediated neuro- peptide signaling activates dendritic cell (DC)-mediated type 1 immune responses. IFN-g stimulation significantly induced NK2R mRNA and remarkably enhanced surface expression levels of bone marrow-derived DCs. In addition, the DC-mediated NKA production level was significantly elevated after IFN-g stimulation in vivo and in vitro. We found that NKA treatment induced type 1 IFN mRNA expressions in DCs. Transduction of NK2R into DCs augmented the expression level of surface MHC class II and promoted Ag-specific IL-2 production by CD4+ T cells after NKA stimulation. Furthermore, blockade of NK2R by an antagonist significantly suppressed IFN-g production by both CD4+ T and CD8+ T cells stimulated with the Ag-loaded DCs. Downloaded from Finally, we confirmed that stimulation with IFN-g or TLR3 ligand (polyinosinic-polycytidylic acid) significantly induced both NK2R mRNA and surface protein expression of human PBMC-derived DCs, as well as enhanced human TAC1 mRNA, which encodes NKA and Substance P. Thus, these findings indicate that NK2R-dependent neuropeptide signaling regulates Ag-specific T cell responses via activation of DC function, suggesting that the NKA–NK2R cascade would be a promising target in chronic inflammation caused by excessive type 1-dominant immunity. The Journal of Immunology, 2012, 188: 4200–4208. http://www.jimmunol.org/ sthma, affecting .300 million people worldwide, is els were steroid resistant, suggesting that both Th1 and Th17 a major public health problem; symptoms include re- cause steroid-resistant severe airway inflammation. Furthermore, A current wheezing, coughing, and shortness of breath. we performed intranasal injection with IFN-g and confirmed the Generally, bronchial asthma, characterized as a Th2 cell-mediated direct effect of IFN-g on the increased AHR (11). Then, we airway inflammation, causes elevation of serum IgE level, eosin- demonstrated that the expression level of neurokinin-2 receptor ophilia in the lung, and airway hyperresponsiveness (AHR) (1–3). (NK2R) was remarkably enhanced in the Th1-dependent asthma However, some patients with asthma occasionally develop severe models and in the lung after IFN-g injection. In addition, we found neutrophilia-like chronic obstructive pulmonary disease (COPD), that administration of NK2R antagonist significantly blocked the by guest on October 3, 2021 and the pathology shows steroid drug resistance (4–8). The precise IFN-g–dependent increase in AHR. Therefore, these findings mechanisms of severe asthma, compared with classic allergic strongly suggest that NK2R-mediated neuropeptide signaling asthma, have not been elucidated. might be closely related to IFN-g–induced airway inflammation. We established Th1-, Th2-, and Th17-dependent asthma models Substance P (SP) and neurokinin A (NKA), encoded by a single by adoptive transfer of Ag-specific Th1, Th2, or Th17 cells into TAC1 (Tac1) , are well-known neuropeptides, composed of wild-type mice (9, 10). Then, we found that the Th2-mediated structurally and evolutionarily conserved peptides characterized asthma model was steroid sensitive, but the Th1 and Th17 mod- by the common C-terminal sequence FXGLM-NH2. The precur- sor of tachykinins, called preprotachykinins, are digested into smaller peptides by posttranslational proteolytic processing. It *Division of Immunoregulation, Section of Disease Control, Institute for Genetic was demonstrated that they are located in the excitatory non- Medicine, Hokkaido University, Sapporo 060-0815, Japan; and †Division of ROYCE’ Health Bioscience, Section of Disease Control, Institute for Genetic Medicine, Hok- adrenergic and noncholinergic nerves of the mammalian respira- kaido University, Sapporo 060-0815, Japan tory tract (12, 13). Therefore, release of tachykinins by the Received for publication September 2, 2011. Accepted for publication February 26, excitation of these nerve systems is thought to be involved in the 2012. pathogenesis of airway allergy in humans (14, 15). Because those This work was supported in part by a Grant-in-Aid for a National Project “Knowl- factors are produced in the airway tissues during inflammatory edge Cluster Initiative” (second stage, “Sapporo Biocluster Bio-S”), a Grant-in Aid for Scientific Research (B) (22300331 to T.N.), Grants-in Aid for Young Scientists responses, developing therapy that blocks the tachykinin-depen- (B) (22700894 to D.W. and 22790370 to H.K.) from the Ministry of Education, dent signaling cascade might be beneficial for the treatment of Culture, Sports, Science and Technology, Japan, and by the Joint Research Program allergic inflammation (16–18). of the Institute for Genetic Medicine, Hokkaido University. Generally, SP secreted by neurons in response to local tissue Address correspondence and reprint requests to Dr. Takashi Nishimura, Division of Immunoregulation, Institute for Genetic Medicine, Hokkaido University, Kita-15, damage is capable of inducing and augmenting many inflammatory Nishi-7, Kita-ku, Sapporo 060-0815, Japan. E-mail address: [email protected]. responses. Recent articles indicated that SP regulates the function ac.jp of various leukocytes, such as vascular endothelial growth factor Abbreviations used in this article: 7AAD, 7-aminoactinomycin D; AHR, airway secretion from mast cells (19), cytotoxicity of NK cells (20), cy- hyperresponsiveness; BALF, bronchoalveolar lavage fluid; COPD, chronic obstruc- tive pulmonary disease; DC, dendritic cell; DT, diphtheria toxin; DTR, diphtheria tokine production by monocytes and T cells (21, 22), induction of toxin receptor; GR, GR 159897; hNK2R, human neurokinin-2 receptor; hTAC1, chemokine or chemokine receptor of macrophages (23, 24), and human TAC1; NKA, neurokinin A; NK2R, neurokinin-2 receptor; poly I:C, Ag presentation of dendritic cells (DCs) (25). In contrast, NKA is polyinosinic-polycytidylic acid; SP, substance P; Tg, transgenic. known to control various vital responses, such as airway con- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 traction, vasodilatation, and vascular permeability, in humans (15, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102521 The Journal of Immunology 4201

16, 26). However, the function of NKA in the immune system is medium containing 10% FCS in the presence of murine GM-CSF (20 ng/ less-well defined compared with the role of SP. ml) or the culture supernatant of GM-CSF–producing CHO cells, as de- The neurokinin receptors NK1R, NK2R, and NK3R encode scribed previously (35). Loosely adherent clustering cells were harvested on days 6–8, and CD11c+ DCs were isolated by the IMag Cell Separation TACR1 (Tacr1), TACR2 (Tacr2), and TACR3 (Tacr3) in System with anti-CD11c mAb-bound beads or FACSAria (BD Bio- mammals, respectively. All of them belong to the rhodopsin-like sciences). The sorted CD11c+ DCs were immature (CD11chigh MHC class family 1 of -coupled receptors, which plays a key role in IIlow), and the purity was .95% (data not shown). These DCs were used cell signaling (27, 28). It is well known that SP exhibits prefer- for the present experiments. The generated DCs were treated with murine IFN-g (10 ng/ml), NKA (1 mM), or LPS (1 mg/ml) for 6 h and used for the ential binding to NK1R, which is expressed on immune cells, such evaluation of mRNA expression levels. For analysis of NK2R protein as monocyte and macrophages, in addition to cells of the nervous expression levels, DCs were treated with IFN-g (10 ng/ml) for 24 h. For system. NKA has more affinity to NK2R, although naturally oc- NKA assays, DCs were treated with IFN-g (2, 4, or 20 ng/ml) for 24 h. curring tachykinins can stimulate any of the neurokinin recep- Depletion of CD11c+ population with DT in lung of tors. Recent articles reported that NK2R expression was observed CD11c-DTR Tg mice in neurons (29), murine macrophages (30), alveolar macrophages from patients with COPD (31), tissue from chronic pancreatitis PBS or DT (1 mg) was injected intranasally into CD11c-DTR Tg mice. (32), and airway smooth muscle (33). After 24 h, recombinant murine IFN-g (3 mg) was administered intrana- sally into the mice. Total cells were collected from collagenase-treated In the present study, we first found that IFN-g stimulation lung tissues or bronchoalveolar lavage fluid (BALF) 24 h after the ad- significantly enhanced surface NK2R expression levels on both ministration of IFN-g into the PBS- or DT-treated CD11c-DTR Tg mice, murine and human DCs and then confirmed that DCs increase the and the number of cells was counted. CD11c- and CD11b-expressing populations were analyzed by flow cytometry, and the number of NKA, a neuropeptide, level after IFN-g stimulation in vivo, as + CD11c cells was calculated. NKA levels in the collected BALF were Downloaded from well as in vitro. In contrast, NKA stimulation significantly induced measured by enzyme immunoassay 24 h after IFN-g administration. the mRNA expression of type 1 IFN, IFN-a, and IFN-b in DCs. Finally, we demonstrated that the NKA–NK2R signaling cascade Transduction of NK2R gene in bone marrow-derived DCs + + activates DC-mediated CD4 T and CD8 T cell responses. From Mouse NK2R with FLAG cDNAwas constructed by reverse transcription of these findings, we propose a novel paradigm of NK2R-dependent total RNA from bone marrow-derived DCs with specific primers. The cross-talk between the neural and immune systems in Th1/IFN-g– resulting cDNA was inserted into pMX-IRES-GFP retroviral vector ob- dependent chronic inflammation, including severe asthma. tained from Dr. T. Kitamura (University of Tokyo). The transduction of http://www.jimmunol.org/ pMX-IRES-GFP (Mock) or pMX-NK2R-IRES-GFP into DCs was per- formed as described (36). For the following analyses, we sorted GFP+ cells Materials and Methods from the retrovirus-infected DCs using FACSAria. Briefly, the DCs were harvested by pipetting and stained with anti-CD11c and 7AAD. The DCs Mice 2 gated by 7AAD CD11c+GFP+ populations were sorted by FACSAria. Wild-type BALB/c and C57BL/6 mice were purchased from Charles River Flow cytometry showed that the purity of the Mock- or NK2R-transduced Japan (Yokohama, Japan). Ifnar2/2 mice were kindly provided by Dr. Y. DCs was .95% (data not shown). The NK2R-transduced DCs totally Iwakura (University of Tokyo, Tokyo, Japan). Stat12/2 mice were kindly expressed NK2R-FLAG protein on the cell surface (data not shown). provided by Dr. S. Akira (Osaka University, Osaka, Japan). CD11c- diphtheria toxin (DT) receptor (DTR) transgenic (Tg) mice were pur- Generation of human DCs from PBMCs by guest on October 3, 2021 chased from The Jackson Laboratory (Bar Harbor, ME). H-2b– and I-Ab– Research protocols for human subjects were approved by the Institutional restricted OVA peptide-specific TCR Tg (OT-I and OT-II) mice were Review Boards of Hokkaido University Graduate School of Medicine and provided by F.R. Carbone (University of Melbourne, Victoria, Australia). the Institute for Genetic Medicine. Written informed consent was obtained All mice were maintained in specific pathogen-free conditions according to from each subject. PBMCs were obtained from healthy individuals by the guidelines of our institute’s animal department and were used at 6–8 Ficoll-Hypaque (Amersham Bioscience, Uppsala, Sweden) gradient cen- wk of age. trifugation after obtaining their informed consent. PBMCs (2 3 105 cells) Abs and reagents were cultured with AIM-V (Invitrogen, Carlsbad, CA) in the presence of recombinant human IL-4 or IL-3 plus GM-CSF for 7 d, as described Allophycocyanin-conjugated anti-CD11c mAb (HL3), PE-conjugated anti- previously (37). We evaluated phenotypes of the generated human DCs. CD11b mAb (M1/70), PE-conjugated anti–IFN-g mAb (XMG1.2), and We confirmed that .60% of whole-cell populations expressed CD11c and PE-conjugated anti–I-Ab (AF6-120.1) were purchased from BD Biosci- HLA class II, although .95% of adherent cells were CD11c+ and HLA ence (San Diego, CA). 7-aminoactinomycin D (7AAD) was purchased class II+ populations (data not shown). The generated human DCs were left from Beckman Coulter (Miami, FL). A rabbit polyclonal Ab against NK2R untreated (control) or were treated with IFN-g (20 ng/ml) or poly I:C (20 (M-48) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), ng/ml) for 24 h, and the expression levels of human NK2R (hNK2R) or which reacted with NK2R of mouse, rat, and human origin. Control rat human TAC1 (hTAC1) were evaluated. IgG was purchased from MP Biomedicals (Eschwege, Germany). Allo- phycocyanin-conjugated anti-human CD11c, FITC-conjugated HLA-DP/ PCR DQ/DR, and FITC-conjugated anti-human HLA-A/B/C were purchased Total RNA was extracted from murine and human DCs with an Isogen RNA g from BD Biosciences. Recombinant mouse IFN- , murine GM-CSF, hu- extraction kit (Nippon Gene, Toyama, Japan). First-strand cDNA was man IFN-g, human GM-CSF, and human IL-4 were purchased from synthesized using 1 mg total RNA with the oligo deoxythymidine primer PeproTech (Rocky Hill, NJ). LPS and DT were purchased from Sigma- (Invitrogen), at a 1:20 ratio, with Superscript III reverse transcriptase Aldrich (St. Louis, MO). Polyinosinic-polycytidylic acid (poly I:C) was (Invitrogen). Genes for murine NK2R (Tarc2), IL-12p40 (Il12b), IFN-a purchased from InvivoGen (San Diego, CA). NKA agonist (NKA 140- (Ifna), IFN-b (Ifnb), b-actin (Actb), hNK2R (TACR2), hTAC1 (TAC1), and 07171) was purchased from Wako Chemical Industries (Osaka, Japan). human GAPDH (GAPDH) were amplified using a thermal cycler (Light- NK2R selective antagonist GR 159897 (GR) was purchased from Tocris Cycler; Roche, Indianapolis, IN). The primer sequences used in this study Bioscience (Bristol, U.K.). GR was a potent selective antagonist of NK2R were as follows: NK2R (forward: 59-AATGACAACGGAGGCAAGAT-39, and had negligible activity against NK1R or NK3R (34). The OT-I reverse: 59-AAGCTGCAGGAATCACCACT-39, Universal probe: #4), IL- (chicken OVA257–264, SIINFEKL) and the OT-II (chicken OVA323– 12p40 (forward: 59-GATTCAGACTCCAGGGGACA-39, reverse: 59-TGG- 339, ISQAVHAAHAEINEAGR) peptides were kindly supplied by Dr. H. TTAGCTTCTGAGGACACATC-39, Universal probe: #27), IFN-a (for- Tashiro (Fujiya, Hadano, Japan). OVA protein was purchased from Sigma- ward: 59-CCTGTGTGATGCAGGAACC-39, reverse: 59-TCACCTCCCA- Aldrich Japan (Tokyo, Japan). pMX-IRES-GFP retrovirus vectors were GGCACTGA-39, Probe: 59-AGACTCCCTGCTGGCTGTGAGGACA-39), kindly provided by Dr. T. Kitamura (University of Tokyo). IFN-b (forward: 59-ATGAGTGGTGGTTGCAGGC-39, reverse: 59-TGAC- Generation of bone marrow-derived DCs CTTTCAAATGCAGTAGATTCA-39, Probe: 59-AAGCATCAGAGGCGG- ACTCTGGGA-39), TNF-a (forward: 59-GTTCTCTTCAAGGGACAAG- Bone marrow cells were prepared from wild-type, Ifna2/2, and Stat12/2 GCTG-39, reverse: 59-TCCTGGTATGAGATAGCAAATCGG-39, Probe: mice. DCs were generated from bone marrow cells with RPMI 1640 59-TACGTGCTCCTCACCCACACCGTCA-39), IL-6 (forward: 59A--GCT 4202 NEUROKININ A SIGNALING CONTROLS DC-MEDIATED T CELL RESPONSES

CCAAACTGGATATAATCAGGA-39, reverse: 59-CCAGGTAGCTATGG- Confocal microscopy TACTCCAGAA-39, Probe: #6), b-actin (forward: 59-AGCCATGTACGT- AGCCATCCA-39, reverse: 59-TCTCCGGAGTCCATCACAATG-39,Probe: DCs generated on glass coverslips in 35-mm culture dishes (Iwaki) were 59-TGTCCCTGTATGCCTCTGGTCGTACCA-39), hNK2R (forward: 59- stimulated with IFN-g for 24 h and fixed with 4% paraformaldehyde in TCTACTCCATGACCGCCATT-39, reverse: 59-GGTGCTGGGAGCTGAA- PBS. Immunostaining of DCs was performed with permeabilization by BD AG-39, Universal probe: #11), hTAC1 (forward: 59-AGAATGTCACGTG- Perm/Wash Buffer containing Abs and 1% goat serum. The cells were GGTCTGG-39, reverse: 59-CTCAGTGCCTTGCGGTATTT-39, Universal washed with PBS and mounted with Mounting Medium (DakoCytoma- probe: #75), and human GAPDH (forward: 59-CTCTGCTCCTCCTGT- tion). We used the anti-NK2R mAb at 1:50 dilution. Hoechst 33352 was TCGAC-39, reverse: 59-ACGACCAAATCCGTTGACTC-39, Universal used for nuclear staining. Fluorescence signals were detected with the probe: #60). Sample signals were normalized to the spectral Confocal Scanning system (FV300; Olympus, Tokyo, Japan) through a 633/1.4 Oil DIC I lens. The signals were visualized with the b-actin, according to the DDCt method: DCt = DCtsample 2 DCtreference. Percentages against the control sample were then calculated for each standard software (Olympus). sample. In some experiments, murine NK2R, IL-12p40, and b-actin were amplified using a thermal cycler (PerkinElmer) with specific primers on T cell-activation assay the basis of intron insertion. After amplification, PCR products with the Mock- or NK2R-overexpressing DCs were used as APCs. OVA peptide- appropriate size were separated by electrophoresis in 1% agarose gels specific CD4+ and CD8+ T cells were prepared from OT-II and OT-I containing ethidium bromide and visualized by UV light illumination. TCR-Tg mice using a nylon wool column and an IMag Cell Separation 9 Primer sequences were as follows: NK2R (forward: 5 -TGCTGGTGGC- System with anti-CD4 or anti-CD8 mAb-bound beads, respectively. The TGTAACAGGCAACG-39, reverse: 59-TAGAAACATTGTGGGGAGGC- purities of CD4+ or CD8+ T cells were .95% (data not shown). Mock- or GAGAGC-39), IL-12p40 (forward: 59-TCCTGAAGTGTGAAGCACCA- NK2R-DCs (2 3 104 cells) were cultured with OT-II CD4+ T cells (1 3 AA-39, reverse: 59-GAGTGCTCCAGGAGTCAGGGTA-39), and b-actin 105 cells) in the presence of vehicle or NKA (1 mM) with OT-II peptide (0, (forward: 59-GTGATGGTGGGAATGGGTCAG-39, reverse: 59-TTTGAT- 0.25, 1 mg/ml) for 48 h. [3H]thymidine was added to the culture media GTCACGCACGATTTCC-39). 3

during the last 4 h. [ H]thymidine incorporation by the proliferated cells Downloaded from Flow cytometry was counted using MicroBeta2 (PerkinElmer). IL-2 production was assayed with Immunoassay kits (BD Bioscience), as described (35). In For analysis of cell surface molecules, the DCs were stained with 7AAD another experiment, normal DCs from bone marrow of wild-type mice and fluorescence-labeled anti-CD11b, anti-CD11c, and anti-NK2R mAbs. were sorted and used as APCs. OT-II or OT-I TCR-Tg CD4+ or CD8+ Expression levels were evaluated by FACSCalibur or FACSCanto (BD T cells were prepared by the same methods described above. Normal DCs Biosciences). were cultured together with OT-II CD4+ T cells or OT-I CD8+ T cells in the http://www.jimmunol.org/

FIGURE 1. IFN-g significantly upregulates NK2R by guest on October 3, 2021 mRNA level of DCs in a STAT1-dependent manner. (A) Bone marrow-derived DCs were generated from wild-type mice. Quantitative PCR analysis was used to evaluate NK2R mRNA expression level in the DCs treated with IFN-g (10 ng/ml) for 6 h. Mean and SD were calculated from data of three independent experiments (n = 6/group). (B) NK2R mRNA expres- sion levels in wild-type and Stat12/2 DCs treated with IFN-g (10 ng/ml) for 6 h were evaluated by PCR. One representative of at least three independent experi- ments is shown. Similar results were obtained in the experiments. (C) NK2R protein expression of untreated DCs (left panel; control) or DCs treated with 10 ng/ml IFN-g for 24 h (right panel) was evaluated by confocal microscopy. One representative of at least three inde- pendent experiments is shown. Scale bars, 5 mm. (D) Surface NK2R expression levels of the control and IFN-g–treated DCs were analyzed by flow cytometry. One representative of at least three independent experiments is shown. (E) Mean fluorescence intensity (MFI; left panel) and percentage (right panel)of NK2R-expressing cells in the control and IFN-g– treated DCs. Mean and SD were calculated from data of three independent experiments (n = 6/group). *p , 0.05, Student t test. The Journal of Immunology 4203 presence of NK2R antagonist (GR: 10 mM) and OT-II peptide (0, 0.25, 1 NKA level in the culture supernatant of bone marrow-derived DCs mg/ml), OT-I peptide (0, 0.25, 1 mg/ml) for 24–72 h, or OVA protein (0, 25, treated with IFN-g. As a result, the NKA level increased signifi- 100 mg/ml) for 72 h, respectively. IL-2, IFN-g, IL-4, and IL-17 production cantly at 24 h after the IFN-g stimulation (Fig. 2C). Taken to- was evaluated with the Immunoassay kits. gether, these findings indicate that DCs are at least one of the ELISA responsible populations for the increase in NKA levels in response IL-2, IFN-g, IL-4, IL-17, TNF-a, and IL-6 levels in the culture super- to IFN-g stimulation. natants were measured using OptEIA mouse IL-2, IFN-g, IL-4, IL-17, TNF-a, and IL-6 ELISA kits, respectively (BD Biosciences). IFN-a and NKA stimulation significantly induces IFN-a/b mRNA IFN-b levels in the culture supernatants were measured with VeriKine expression levels on DCs Mouse IFN-a and IFN-b ELISA kits (PBL InterferonSource, Piscataway, NJ). ELISA kits were used according to the manufacturer’s instructions. We further investigated the effect of NKA on the production of The NKA level was determined using a Neurokinin A Enzyme Immuno- cytokines by DCs. NKA stimulation significantly enhanced mRNA assay kit (Peninsula Laboratories, San Carlos, CA). expression levels of both IFN-a (Fig. 3A) and IFN-b (Fig. 3B), Statistical analysis whereas IL-12p40 increased only slightly (Fig. 3C), and TNF-a (Fig. 3D) and IL-6 (Fig. 3E) were unchanged. In this study, we All experiments were independently repeated at least three times. Mean and confirmed that LPS, a TLR4 ligand, stimulation significantly en- SD were calculated for in vitro murine data, and mean and SE were cal- culated for in vivo murine and in vitro human data. Significant differences in hanced IFN-b, IL-12p40, TNF-a, and IL-6 but not IFN-a. These the results were determined by the two-tailed Student t test; p , 0.05 was findings suggest that NKA stimulation induces type 1 IFN mRNAs considered statistically significant. by DCs. Furthermore, we evaluated IFN-a,IFN-b, IL-12p70, TNF-a, and IL-6 protein production by NKA-stimulated DCs. Results However, we could not detect these protein production levels after Downloaded from IFN-g stimulation enhances NK2R expression level of DCs the NKA stimulation in the present experiments (data not shown). To address the effect of neuropeptide signaling on DC-mediated Transduction of NK2R gene into DCs enhances Ag-specific immune responses, we first investigated the expression level of IL-2 production by CD4+ T cells a receptor for NKA, NK2R, on DCs in a type 1 immune condition. To evaluate the effects of NK2R-mediated NKA stimulation on DC DCs generated from bone marrow cells of wild-type mice were

functions, we constructed the pMX-FLAG-NK2R-IRES-GFP ret- http://www.jimmunol.org/ stimulated with recombinant murine IFN-g in vitro. As a result, IFN-g treatment significantly induced NK2R mRNA expression on DCs (Fig. 1A, 1B). The NK2R mRNA induction by IFN-g stimulation was remarkably reduced in STAT-1–deficient DCs (Fig. 1B). These data indicate that IFN-g stimulation induces NK2R mRNA expression of DCs in a STAT-1–dependent manner. In addition, we found that LPS stimulation induced NK2R mRNA expression on DCs (Supplemental Fig. 1A). The LPS-dependent

augmentation of NK2R mRNA level was not observed in by guest on October 3, 2021 IFNAR1- or STAT-1–deficient DCs (Supplemental Fig. 1A, 1B). These data suggest that type 1 IFN produces after LPS stimula- tion induced NK2R mRNA expression of DCs by an autocrine mechanism in a STAT-1–dependent manner. We further evaluated whether IFN-g enhanced NK2R protein expression levels of DCs. Confocal microscopy showed that NK2R protein level was enhanced in the IFN-g–stimulated DCs (Fig. 1C). We confirmed that both the surface expression levels of NK2R on DCs (Fig. 1D) and the percentage of NK2R-expressing cells were significantly enhanced by IFN-g stimulation (Fig. 1E). These data suggest that IFN-g, type 1 cytokine stimulation acti- vates the NK2R-mediated signaling cascade of DCs in a STAT-1– dependent manner. DCs elevate the NKA, a neuropeptide, level in response to IFN-g stimulation in vivo and in vitro In the current study, we found that intranasal injection of IFN-g into wild-type mice significantly enhanced the NK2R mRNA expression level of lung tissue and the NKA level in BALF, resulting in increased airway inflammation (11). In the current FIGURE 2. DCs are required for IFN-g–dependent NKA increase. IFN-g study, we investigated whether DCs were required for the increase (3 mg) was intranasally administered into CD11c-DTR Tg mice preinjected in NKA. CD11c-DTR Tg mice were intranasally injected with with PBS (n = 7/group) or DT (n = 6/group). (A) The percentage and total + PBS as a control or DT and then IFN-g was injected into the mice. number of CD11c cells in lung tissue (left panels) and BALF (right We confirmed that CD11c+ populations in the lung tissue and panels) were analyzed by flow cytometry at 24 h after IFN-g adminis- tration. Mean and SE were calculated from data of three independent BALF were remarkably reduced by DT treatment (Fig. 2A). At 24 experiments (n = 6–7/group). (B) NKA levels in the collected BALF at 24 h h after the IFN-g injection, NKA level in BALF was significantly + after IFN-g treatment in vivo. Mean and SE were calculated from data of reduced by the depletion of the CD11c population (Fig. 2B). three independent experiments (n = 6–7/group). (C) NKA levels in the + These findings suggest that CD11c populations, such as pulmo- culture supernatants of bone marrow-derived DCs treated with IFN-g (10 nary DCs and alveolar macrophages, contribute to IFN-g–depen- ng/ml) for 24 h. Mean and SD were calculated from data of three inde- dent NKA elevation in the lung. Furthermore, we determined that pendent experiments (n = 6–7/group). *p , 0.05, Student t test. 4204 NEUROKININ A SIGNALING CONTROLS DC-MEDIATED T CELL RESPONSES

FIGURE 3. NKA stimulation enhances both IFN- a/b and NK2R mRNA expression levels of DCs. DCs generated from wild-type mice were treated with NKA (1 mM) or LPS (1 mg/ml) for 6 h. The mRNA ex- pression levels of IFN-a (A), IFN-b (B), IL-12p40 (C), TNF-a (D), and IL-6 (E) were determined by quanti- tative PCR. Mean and SD were calculated from data of three independent experiments (n = 6/group). *p , Downloaded from 0.05, Student t test. http://www.jimmunol.org/ by guest on October 3, 2021 roviral vector. We then confirmed the surface expression level transduction into DCs, even in the absence of extraneous NKA of FLAG-tagged NK2R protein in Plat E cells by flow cytometry stimulation. We found that the culture medium with 10% FCS (data not shown) and alteration of the morphology of NK2R- contained NKA, as shown in the normal culture condition (Fig. overexpressed RAW264.7 cells compared with Mock-RAW264.7 2C), suggesting that the NKA in the culture medium also affected cells after NKA stimulation (Supplemental Fig. 2A). MHC class II expression of NK2R-transduced DCs without the We transduced NK2R gene into DCs with a retrovirus-infection further addition of NKA. system, and the Mock- or NK2R-transduced DCs were isolated Blockade of NK2R-mediated signaling pathway significantly using a cell sorter. We found that the NK2R-DCs enhanced surface + + MHC class II expression level after NKA stimulation compared inhibits Ag-specific CD4 and CD8 T cell responses with Mock-DCs (Fig. 4A), although IL-12 production by Mock- or To confirm the effect of NKA–NK2R signaling on Ag-specific NK2R-DCs was not observed by flow cytometry (Supplemental T cell responses, we cultured spleen cells obtained from OT-II Fig. 2C). Then, we cocultured OT-II CD4+ T cells with Mock- or or OT-I mice in the presence of NK2R antagonist GR for 48 h. NK2R-transduced DCs in the presence or absence of OT-II pep- Immune responses to each OVA Ag peptide were evaluated by tides plus NKA or vehicle. The transduction of NK2R gene into [3H]thymidine-incorporation assay. The Ag-specific proliferation DCs appeared to promote the Ag-specific responses to CD4+ of spleen cells from OT-II and OT-I mice was significantly sup- T cells (Fig. 4B). Then, we evaluated the proliferation by [3H]thy- pressed by the addition of GR (10 mM; Supplemental Fig. 3A). midine-incorporation assay and confirmed that NK2R-DCs sig- We confirmed that there was no difference in the ratio of 7AAD+ nificantly enhanced cell proliferation compared with Mock-DCs. Annexin V+ apoptotic cells between GR and DMSO control In addition, NKA stimulation increased the enhancement of cell groups at 8 h (Supplemental Fig. 3B). proliferation (Fig. 4C). Furthermore, we examined IL-2 produc- Then, we cocultured OT-II CD4+ T cells with DCs in the tion by CD4+ T cells cocultured with Mock- or NK2R-transduced presence of GR (10 mM). The blockade of NK2R-mediated sig- DCs in the presence of NKA with or without OT-II peptide. naling by antagonist remarkably reduces IFN-g and IL-2 pro- NK2R-DCs significantly enhanced Ag-specific IL-2 production by duction by CD4+ T cells in the presence of OT-II peptide or OVA CD4+ T cells compared with Mock-DCs. NKA stimulation aug- protein (Fig. 5A). Under the same conditions, neither IL-17 nor mented IL-2 production by CD4+ T cells cocultured with NK2R- IL-4 production was observed in the present coculture system DCs (Fig. 4D). These data suggest that NK2R-dependent NKA (data not shown). Furthermore, we examined Ag-specific re- stimulation increases Ag-presentation of DCs to activate CD4+ sponses of OT-I CD8+ T cells cocultured with DCs in the presence T cells. of GR. The blockade of NK2R-mediated signaling also suppresses In the present experiments, the proliferation and IL-2 produc- IFN-g production by CD8+ T cells after stimulation with OT-I tion by CD4+ T cells were significantly increased by the NK2R peptide or OVA protein (Fig. 5B). The Journal of Immunology 4205 Downloaded from http://www.jimmunol.org/

FIGURE 4. NK2R-mediated NKA signaling in DCs promotes the acti- vation of Ag-specific CD4+ T cells. DCs generated from wild-type mice were transduced with Mock or NK2R genes with the retroviral infection system using pMX-IRES-GFP or pMX-NK2R-IRES-GFP, respectively. FIGURE 5. Blockade of NK2R-mediated signaling cascade suppresses (A) Surface MHC class II (I-Ab) expression levels of Mock- and NK2R- IFN-g and IL-2 production by Ag-specific T cells. DCs were generated transduced DCs treated with vehicle or NKA for 24 h were evaluated by from bone marrow cells of wild-type mice in the presence of GM-CSF for flow cytometry. Data are from one representative experiment of at least 6d.(A) The generated DCs (2 3 104 cells) were cocultured with OT-II three independent experiments. Similar results were obtained in the CD4+ T cells (1 3 105 cells) in the absence or presence of NK2R an- by guest on October 3, 2021 experiments. (B) OT-II CD4+ T cells (1 3 105 cells) were cocultured with tagonist (GR, 10 mM) with OT-II peptide (right panels) or OVA protein Mock- or NK2R-transduced DCs (2 3 104 cells) in the presence of vehicle (left panels) for 24–72 h. IFN-g production by the OT-II CD4+ T cells at 72 h or NKA (1 mM) with OT-II peptide (1 mg/ml). Photomicrographs of the (top panels) and IL-2 production at 24 h (bottom panels) were evaluated by culture cells at 48 h after stimulation. Data are from one representative ELISA. Mean and SD were calculated from data of three independent experiment of at least three independent experiments. Scale bars, 250 mm. experiments (n = 6/group). (B) The generated DCs (2 3 104 cells) were (C) Proliferation of OT-II CD4+ T cells cocultured with NK2R- or Mock- cocultured with OT-I CD8+ T cells (1 3 105 cells) in the absence or transduced DCs in the presence of vehicle or NKA (1 mM) with OT-II presence of NK2R antagonist (GR, 10 mM) with OT-I peptide (right panel) peptide (0, 0.25, 1 mg/ml) were evaluated by [3H]thymidine-incorporation or OVA protein (left panel) for 72 h. IFN-g production by the OT-I CD8+ assay. Mean and SD were calculated from the representative data of three T cells were evaluated by ELISA. Mean and SD were calculated from data independent experiments (n = 3/group). (D) IL-2 production levels of of three independent experiments (n = 6/group). *p , 0.05, Student t test. OT-II CD4+ T cells cocultured with Mock- or NK2R-transduced DCs in the presence of vehicle or NKA (1 mM) with OT-II peptide were evaluated by ELISA. Mean and SD were calculated from data of three independent levels were significantly enhanced by IFN-g stimulation (Fig. 6D, n p , t experiments ( = 3/group). * 0.05, Student test. 6E). Furthermore, human DCs were stimulated with poly I:C, dsRNA, and IFN-g for 24 h. We found that poly I:C, a TLR3 li- Thus, these findings strongly suggest that the NKA–NK2R- gand, enhanced both hNK2R and hTAC1 mRNA expression levels signaling cascade is involved in DC-mediated type 1 immunity in human DCs (Fig. 6D, 6E). In this study, we confirmed that including Ag-specific CD4+ and CD8+ T cell responses. human DCs enhanced HLA class II expression levels after NKA plus IFN-g stimulation compared with IFN-g alone, whereas NKA Human DCs induce NK2R and TAC1 genes after IFN-g and stimulation did not induce cytokine production (data not shown). poly I:C stimulation These findings suggest that NKA–NK2R signaling could regulate Finally, we examined whether human DCs induce expression DC function in human immunity. of NK2R (hNK2R) and TAC1 (hTAC1) after IFN-g stimulation. CD11c-, HLA class II-, and HLA class I-expressing DCs were Discussion generated from adherent cells of human PBMCs and stimulated Excitatory transmitters, such as tachykinins including SP and with recombinant human IFN-g for 24 h. As a result, we con- NKA, are widely distributed within both the central and the pe- firmed that both hNK2R protein level (Fig. 6A) and surface ex- ripheral nervous systems. Although the tachykinins were initially pression levels (Fig. 6B, 6C) were remarkably enhanced by IFN-g considered neurotransmitters, many articles reported that expres- stimulation, similar to the case observed in mouse DCs. In this sion of their receptors, NK1R and NK2R, was observed in non- study, we found that both hNK2R and hTAC1 mRNA expression neural tissues, including endothelial cells, fibroblasts, smooth 4206 NEUROKININ A SIGNALING CONTROLS DC-MEDIATED T CELL RESPONSES

FIGURE 6. Human DCs induce both NK2R and TAC1 gene after IFN-g or poly I:C stimulation. Hu- man DCs generated from the adherent cells of PBMCs were left untreated (control) or were treated with IFN-g (20 ng/ml) or poly I:C (20 ng/ml) for 24 h. (A) hNK2R protein expression levels of the control (left panel) and IFN-g–treated (right panel) human DCs were evaluated by confocal microscopy. One repre- sentative experiment of at least three independent ex- periments is shown. Similar results were obtained in the experiments. Scale bars, 5 mm. (B) Surface hNK2R expression levels of the control and IFN-g–treated human DCs were determined by flow cytometry. One representative experiment of at least three independent experiments is shown. (C) Mean fluorescence intensity Downloaded from (MFI; left panel) and percentage (right panel)of hNK2R-expressing cells in the control and IFN-g– treated human DCs were evaluated by flow cytometry. Mean and SE were calculated from data of three in- dependent experiments (n = 6/group). (D) Relative expression levels of hNK2R and TAC1 mRNAs in the human DCs treated with IFN-g, poly I:C, or NKA for http://www.jimmunol.org/ 24 h were determined by quantitative PCR. Mean and SE were calculated from data of three independent experiments (n = 6/group). (E) hTAC1 mRNAs in the human DCs treated with IFN-g or poly I:C for 24 h were determined by quantitative PCR. Mean and SE were calculated from data of three independent ex- periments (n = 6/group). *p , 0.05, Student t test. by guest on October 3, 2021

muscle cells, and inflammatory cells, suggesting that tachykinins (38, 39). Mice lacking NK1R reduced schistosomiasis granuloma function between the nervous system and other organs (27–33). formation (40). In a posttreatment-reactive encephalopathy model In this study, we found that both murine and human DCs, one of of Trypanosoma brucei, NK1R-deficient mice have a significantly the most powerful APCs, significantly enhanced NKA production reduced clinical impairment, but a more severe neuroinflammatory levels and expressed NK2R after IFN-g stimulation (Figs. 1, 6). In response, compared with wild-type mice (41). The critical roles of addition, stimulation of DCs with TLR ligands, such as LPS and SP were reported in T cell-mediated experimental autoimmune poly I:C, which mimic bacterial and virus infection, respectively, encephalomyelitis models (42, 43). The findings strongly suggest enhanced NK2R expression levels (Fig. 6, Supplemental Fig. 1). that the SP–NK1R–signaling cascade contributes to type 1 im- In addition, we revealed in this study that murine DCs stimulated munity, as well as inflammatory responses. Although NK1R is with NKA significantly induced mRNA expression of IFN-a and ubiquitously expressed in addition to the CNS, it was demon- IFN-b, type 1 IFNs (Fig. 3). Although the protein expressions of strated that NK2R is mostly expressed in peripheral tissues (29– these cytokines were not detected in the present experiments, we 33). Although the precise function of the NKA–NK2R–signaling confirmed the blocking effects of anti–IFN-b and anti–IL-12 pathway, especially in immune responses, is less-well known mAbs on NKA-dependent augmentation of IFN-g production af- compared with that of NK1R, we demonstrated in this study that ter OVA Ag stimulation (data not shown). Therefore, we specu- NK2R-mediated NKA stimulation enhances DC function, such lated that NKA-mediated cytokine production might function in as IFN-a/b production (Fig. 3A, 3B), MHC class II expression the microenvironment, including cell-to-cell interaction. Taken (Fig. 4A), and Ag presentation to CD4+ and CD8+ T cells (Figs. together, these findings strongly suggest that NK2R-mediated 4B, 4C, 5). neuropeptide signaling is involved in host immune responses, NK2R, as well as NK1R, belongs to the rhodopsin-like family 1 including cytokine production and Ag presentation by DCs. of G protein-coupled receptors, which plays a crucial role in in- Previous works indicated that blockade of NK1R influences host tracellular signaling (27, 28). Previously, we indicated that the responses against various infections. In salmonellosis, mice treated TRIF–GEFH1–RhoB pathway regulated MHC class II expression with an SP antagonist have increased bacterial burden and reduced on DCs, which was required for the subsequent CD4+ T cell ac- IFN-g and IL-12 production compared with appropriate controls tivation (44). In this study, we indicated that the surface MHC The Journal of Immunology 4207 class II expression level of NK2R-transduced DCs was increased tients with steroid-resistance disorders, including severe inflam- by NKA stimulation (Fig. 4A). Moreover, the morphology of matory diseases. NK2R-transduced DCs was altered by the addition of NKA (Sup- In the present work, we found that human DCs also induced plemental Fig. 2B) in addition to NK2R-transduced RAW264.7 expression of hNKA and hTAC1 mRNAs in response to IFN-g cells (Supplemental Fig. 2A). These findings suggest that NK2R- (Fig. 6). Furthermore, stimulation of the TLR3 ligand poly I:C mediated signaling in DCs might be related to membrane traf- enhanced both genes. Although human CD11c+ and HLA class II+ ficking of MHC class II vesicles and alteration of cytoskeleton cells were possible contaminants of both monocyte and macro- mediated by a posttranscriptional mechanism. phages in the present experiments, these findings indicate that NKA stimulation significantly enhanced mRNA expression NK2R-dependent cross-talk between the neural and immune levels of type 1 IFNs of DCs rather than IL-12p40, although it did systems would function in human subjects. Therefore, we are now not induce mRNA expression levels of TNF-a and IL-6 (Fig. 3, evaluating the effect of NK2R antagonist on the activation of Supplemental Fig. 2C). In general, plasmacytoid DCs induce both human immune cells and confirming the several suppression IFN-a and IFN-b production in response to foreign nucleic acids effects of cytokine production by human DCs and T cells. through TLR7 and TLR9 (45). In contrast, conventional DCs and We conclude that NK2R-dependent neuropeptide signaling macrophages produce IFN-b and various proinflammatory cyto- activates DC-mediated immune responses, suggesting that the kines by TLR3, TLR7, and TLR9 ligands after stimulation (46). NKA/NK2R-mediated signaling pathway would be a promising Although DCs generated from bone marrow with GM-CSF in this target in therapy for patients with chronic inflammation caused by study showed phenotypes of conventional DCs, IFN-a and IFN-b excessive activation of type 1 immunity, including steroid-resistant mRNA expression was induced by NKA stimulation (Fig. 3A, severe asthma. 3B). The present data suggest that NK2R-dependent neuropeptide Downloaded from signaling uses a different mechanism than the TLR-dependent Acknowledgments pathway for cytokine production by conventional DCs. We thank Dr. T. Kitamura (University of Tokyo) for providing pMX vector Recently, we demonstrated that intranasal administration of plasmid and Dr. T. Sudo (Toray, Tokyo, Japan) for providing GM-CSF– IFN-g remarkably induced NKA and NK2R expression in the lung producing CHO cells. tissues and blockade of NKA–NK2R signaling significantly sup- http://www.jimmunol.org/ pressed IFN-g–induced AHR and the symptom of Th1 cell- Disclosures transferred asthma model (11). These findings suggested that the The authors have no financial conflicts of interest. NKA–NK2R cascade must be related to neuro-immune cross-talk in type 1 immunity including airway inflammation. 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