Monophosphate/Protein Kinase ′ Adenosine 5 Calcitonin Gene-R

Calcitonin Gene-Related Peptide and Cyclic Adenosine 5 ′-Monophosphate/Protein Kinase A Pathway Promote IL-9 Production in Th9 Differentiation Process This information is current as of September 28, 2021. Norihisa Mikami, Yayoi Miyagi, Kaori Sueda, Miku Takatsuji, So-ichiro Fukada, Hiroshi Yamamoto and Kazutake Tsujikawa J Immunol 2013; 190:4046-4055; Prepublished online 15 March 2013; Downloaded from doi: 10.4049/jimmunol.1203102 http://www.jimmunol.org/content/190/8/4046 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2013/03/15/jimmunol.120310 Material 2.DC1 References This article cites 56 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/190/8/4046.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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Calcitonin Gene-Related Peptide and Cyclic Adenosine 59-Monophosphate/Protein Kinase A Pathway Promote IL-9 Production in Th9 Differentiation Process

Norihisa Mikami, Yayoi Miyagi, Kaori Sueda, Miku Takatsuji, So-ichiro Fukada, Hiroshi Yamamoto, and Kazutake Tsujikawa

Th9 cells are a novel Th cell subset that produces IL-9 and is involved in type I hypersensitivity such as airway inﬂammation. Although its critical roles in asthma have attracted interest, the physiological regulatory mechanisms of Th9 cell differentiation and function are largely unknown. Asthma is easily affected by psychological factors. Therefore, we investigated one of the physiological mediators derived from the nervous system, calcitonin gene-related peptide (CGRP), in asthma and Th9 cells because

CGRP and activation of the cAMP/protein kinase A (PKA) pathway by CGRP are known to be important regulators in several Downloaded from immune responses and allergic diseases. In this study, we demonstrated that the CGRP/cAMP/PKA pathway promotes IL-9 production via NFATc2 activation by PKA-dependent glycogen synthase kinase-3b inactivation. Moreover, CGRP also induces the expression of PU.1, a critical transcriptional factor in Th9 cells, which depends on PKA, but not NFATc2. Additionally, we demonstrated the physiological importance of CGRP in IL-9 production and Th9 differentiation using an OVA-induced airway inflammation model and T cell–specific CGRP receptor-deficient mice. The present study revealed a novel regulatory mechanism comprising G protein–coupled receptor ligands and nervous system-derived substances in Th9 cell differentiation and type I http://www.jimmunol.org/ hypersensitivity. The Journal of Immunology, 2013, 190: 4046–4055.

D4+ Th cells play central roles in acquired immunity. Th fluorobenzene. In contrast, Th2 cells produce IL-4, IL-5, and IL- cells can be divided into several subsets, including Th1, 13, which drive many allergic diseases, such as atopic dermatitis. C Th2, Th17, and regulatory T cells. These subsets dif- Recently,anewThcellsubsetthatsecretesmainlyIL-9was ferentiate themselves from naive T cells under specific cytokine identified and named the Th9 cell (5, 6). Th9 cells differentiate conditions during the stimulation by APC. Each Th cell differ- from naive Th cells in the presence of both TGF-b and IL-4, or entiation is regulated by a specific transcriptional factor. It is from Th2 cells in the presence of TGF-b (6). IL-9 influences known that T-bet, GATA3, and RORgt are master transcriptional many cell types, including T cells, B cells, mast cells, eosino- by guest on September 28, 2021 factors for Th1, Th2, and Th17 cell differentiation, respectively phils, lung epithelial cells, and hematopoietic progenitors (7–10). (1–3). Regulatory T cells also require stable expression of the The essential roles of IL-9 in the pathogenic progression of type I transcriptional factor Foxp3 for their development and suppressor hypersensitivity have been demonstrated using IL-9–transgenic phenotype (4). In addition to the obligatory role in acquired im- mice and IL-9–deficient mice. IL-9–transgenic mice exhibit ac- munity, Th cells contribute to induction of a number of immune celerated hypersensitivity, and the condition was suppressed in diseases. Th1 cells mediate type IV–delayed allergies such as IL-9–deficient mice (11, 12). Although type I hypersensitivity contact dermatitis elicited by trinitrochrolobenzene or dinitro- had been considered a Th2 cytokine (IL-4, IL-5, and IL-13)- mediated reaction, recent findings showed that the blockade of IL-4 signaling had little effect on human asthma, and currently Laboratory of Molecular and Cellular Physiology, Graduate School of Pharmaceuti- cal Sciences, Osaka University, Osaka 565-0871, Japan IL-9 is attracting attention as a new therapeutic target of type I Received for publication November 9, 2012. Accepted for publication February 19, hypersensitivity (13). 2013. PU.1 is reported to be an important transcriptional regulator of This work was supported by Grant-in-Aid for Scientific Research 22590062 from the Th9 cell differentiation (14). In addition, a few studies indicated Ministry of Education, Culture, Sports, Science, and Technology of Japan; a grant that other factors, such as NF-kB, NFATc2, and IFN regulatory from the Japan Chemical Industry Association Long-Range Research Initiative; a grant from the Osaka Foundation for Promotion of Clinical Immunology; and factor 4 (IRF-4), regulate IL-9 production in Th9 cells (15–17). a grant-in-aid for scientific research from the Japan Society for the Promotion of However, the molecular mechanisms and physiological regulators Science fellows. of Th9 differentiation are largely unknown; thus, it is extremely Address correspondence and reprint requests to Dr. So-ichiro Fukada, Laboratory of important to investigate how Th9 cell differentiation is regulated Molecular and Cellular Physiology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail address: in the clinical condition of type I hypersensitivity. [email protected] There are many lines of evidence that an elevated cAMP level The online version of this article contains supplemental material. in immune cells affects several immune responses, such as cyto- Abbreviations used in this article: 6-bnz-cAMP, N6-benzoyl-cAMP; CGRP, calcito- kine production, cell proliferation, apoptosis, chemotaxis, and phago- nin gene-related peptide; ChIP, chromatin immunoprecipitation; 8-CPT-cAMP, 8-(4- cytosis (18–22). Generally, the intracellular cAMP level increases chlorophenylthio)-29-O-methyl-cAMP; EPAC, exchange factor directly activated by cAMP; GPCR, G protein–coupled receptor; GSK, glycogen synthase kinase; IRF-4, when the ligand binds to its G protein–coupled receptor (GPCR) IFN regulatory factor 4; PACAP, pituitary adenylate cyclase–activating polypeptide; and adenylate cyclase is activated. PKA, protein kinase A; RAMP1, receptor activity modifying protein 1; VIP, vaso- There are many GPCR ligands in neurotransmitters, and it is active intestinal peptide. well known that the immune system and allergic responses are Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 affected by the nervous system. In fact, the impact of psychological www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203102 The Journal of Immunology 4047 factors on the pathogenesis of asthma has been well studied (23). (Osaka, Japan), and bCGRP was purchased from Phoenix Pharmaceuticals Calcitonin gene-related peptide (CGRP) is a 37-aa neuropeptide (Burlingame, CA). Human rTGF-b1 and mouse rIL-4 were purchased that is released from sensory neurons by several types of stimula- from PeproTech (Rocky Hill, NJ). Dibutyryl cAMP and lithium chloride were bought from Wako Pure Chemical Industries (Osaka, Japan). N6- tion, such as protons, heat, chemical, and psychological events, and benzoyl-cAMP (6-bnz-cAMP), 8-(4-chlorophenylthio)-29-O-methyl- increases the intracellular cAMP level through its specific recep- cAMP (8-CPT-cAMP), and GSK-3b inhibitor VII came from Calbio- tors. We hypothesized that CGRP is a potent Th9 regulator because chem (San Diego, CA). PGE2 and the PKA inhibitors (H89 and KT5720) its expression is increased in asthma (24, 25), and CGRP also has were purchased from Sigma-Aldrich (St. Louis, MO). PGD2 was purchased from Cayman Chemical (Ann Arbor, MI). LPS-free OVA was potent regulatory effects on Th cell differentiation (26–28). The bought from Seikagaku (Tokyo, Japan). CGRP receptor is a compound of receptor activity modifying protein 1 (RAMP1), which has the binding site of CGRP, and calci- Isolation and differentiation of naive T cells tonin receptor-like receptor, which is coupled with Ga proteins. CD4+CD252CD44lowCD62L+ naive Th cells, which purified from spleen CGRP receptors are expressed not only in nerve cells, but also in cells by using a FACS Aria II (BD Biosciences, San Diego, CA), had various types of immune cells, and many effects of CGRP on im- purity .99% (data not shown). The purified naive Th cells were stimulated mune functions are reported in addition to Th cell differentiation. with plate-bound anti-CD3 mAb (2C11) and soluble anti-CD28 mAb (PV- 1; Beckman Coulter) for 48 h. The culture medium was supplemented with For instance, CGRP inhibits T cell proliferation, Ag presentation, rIL-4 (10 ng/ml) or rIL-4 (10 ng/ml) plus TGF-b (5 ng/ml) for Th2 or Th9 and Ab secretion (29–31). We previously generated RAMP1- cell differentiation, respectively. CGRP, analogs, and all inhibitors were deficient mice and investigated the physiological effects of CGRP supplied at the start of stimulation. We confirmed that these molecules had (32); we found that CGRP suppresses the functions of dendritic and no effect on the cell viability and activation at the concentrations used.

Th1 cells, but promotes Th2 and Th17 cell differentiation in contact For restimulation assays, Th2 and Th9 cells were differentiated as above Downloaded from for 4 d. Th2 cells were further differentiated into Th9 cells by culturing with hypersensitivity and experimental autoimmune encephalomyelitis, TGF-b (5 ng/ml) for 2 d. These differentiated cells were restimulated with respectively (27, 28, 33). Our investigations also showed that anti-CD3 mAb (2C11) for 24 h in the presence or absence of CGRP. RAMP1-deficient mice were resistant to OVA-induced food allergy, although the regulatory mechanism has not been revealed (34). Intracellular staining and ELISA Moreover, it is reported that aCGRP-deficient mice exhibit less For IL-9, GATA3, and NFATc2 intracellular staining, cells were fixed and

airway inflammation (35). Therefore, CGRP may be a physiological stained using a BD Cytofix/Cytoperm kit, according to the manufacturer’s http://www.jimmunol.org/ molecule that promotes type I hypersensitivity, and it is possible instructions. IL-9 concentrations in cell supernatants were measured using a Mouse IL-9 Tissue Culture ELISA Ready-SET-Go! set (eBiosscience, that CGRP promotes these inflammations by promoting Th9 cell San Diego, CA). IL-4, IFN-g, and IL-10 were measured, as previously functions. described (28). In this study, we show that the CGRP/cAMP/protein kinase A (PKA) pathway is important in the regulation of Th9 differen- Immunocytochemistry tiation. Moreover, our study found that PKA inhibits glycogen Stimulated Th cells were collected on glass slides using cytospin and fixed synthase kinase (GSK)–3b function and enhances NFATc2 binding with cooled acetone or 4% paraformaldehyde for 15 min. After blocking to the IL-9 promoter region as a new regulatory mechanism in Th with 5% BSA, the cells were stained with anti-NFATc1, anti-NFATc2,

anti–pGSK-3b, or anti-PU.1 Ab at 4˚C overnight. After the first stain- by guest on September 28, 2021 cells. CGRP/cAMP/PKA pathway also controls the expression ing, the cells were reacted with secondary Abs conjugated with Alexa of PU.1 via NFATc2-independent manner. Our findings have Fluor 488 or Alexa Fluor 568 (Molecular Probes, Eugene, OR). Speci- implications for the roles of CGRP and facilitate investigation mens were mounted using Vectashield with DAPI (Vector Laboratories, of the relationship between the neurotransmitters and airway Burlingame, CA), and the signals were recorded photographically using inflammation. a fluorescence microscope BX51 (Olympus, Tokyo, Japan) equipped with a DP70 CCD camera (Olympus). RNA extraction and RT-PCR Materials and Methods Mice Total RNA was extracted using TRIzol reagent (Invitrogen). Total RNA was reverse transcribed into cDNA using a PrimeScript RT reagent kit C57BL/6 mice and BALB/c mice (6–10 wk old) were purchased from (Takara, Kyoto, Japan), according to the manufacturer’s instructions. Semi- Charles River Japan (Kanagawa, Japan). Lck-Cre transgenic mice were quantitative RT-PCR was performed using a Thermal Cycler (Bio-Rad, purchased from The Jackson Laboratory (Bar Harbor, ME). RAMP1- Hercules, CA), and quantitative real-time RT-PCR was performed using a deficient mice were generated (32), and heterozygous knockout mice were Lightcycler System (Roche, Basel, Switzerland). The primers used for PCR backcrossed for 12 generations to C57BL/6 or BALB/c mice and used to were as follows: IL-9 forward, 59-CTGATGATTGTACCACACCGTGC- generate the offspring used in the current study. All procedures, including 39; IL-9 reverse, 59-GCCTTTGCATCTCTGTCTTCTGG-39;T-betfor- experimental animals, were approved by the Experimental Animal Care ward, 59-CTAACACACACGTCTTTACT-39; T-bet reverse, 59-AACA- and Use Committee of the Graduate School of Pharmaceutical Sciences, GATGCGTACATGGA-39; GATA3 forward, 59-CGGAAACTCCGTCA- Osaka University. GG-39; GATA3 reverse, 59-ACGTCTTGGAGAAGGG-39; NFATc1 forward, 59-GGAGCGGAGAAACTTTGCG-39; NFATc1 reverse, 59-GTGA- Abs and reagents CACTAGGGGACACATAACT-39; NFATc2 forward, 59-GCACATAAGG- CCATCAGCTCA-39; NFATc2 reverse, 59-TCGCCAGAGAGACTGGCA- PE-conjugated anti-mouse CD25 mAb (PC61) was purchased from BD A-39; c-Maf forward, 59-ACTTCGACGACCGCTTCTC-39; c-maf reverse, Pharmingen (San Diego, CA). Allophycocyanin-Cy7–conjugated anti- 59-TTCTCGGAAGCCGTTGCTCAC-39; PU.1 forward, 59-AGAGCTA- mouse CD4 mAb (GK1.5) and PE-conjugated anti-mouse IL-9 mAb TACCAACGTCCAATGC-39; PU.1 reverse, 59-TTCTCAAACTCGTTG- (RM9A4) were purchased from BioLegend (San Diego, CA). FITC- TTGTGGAC-39; Foxp3 forward, 59-CCTCTTCTTCCTTGAACCC-39; conjugated anti-mouse c-kit mAb (2B8) and PE-conjugated anti-mouse ε Foxp3 reverse, 59-GTGGACGCACTTGGAG-39; GAPDH forward, 59-C- Fc RIa mAb (MAR-1) were obtained from eBioscience (San Diego, ATGCCATCACTGCCACCC-39; and GAPDH reverse, 59-GGTAGGAA- CA). Anti–phosphor-GSK-3b (ser 9) polyclonal Ab was purchased from CACGGAAGGCC-39. Bioss (Woburn, MA). Anti-PU.1 polyclonal Ab was purchased from Bio- world Technology (Saint Louis Park, MN). We bought allophycocyanin- Chromatin immunoprecipitation assay conjugated anti-mouse CD44 mAb (IM7.8.1) from Miltenyi Biotec (Auburn, CA). FITC-conjugated anti-mouse CD62L mAb (REG56) was acquired A chromatin immunoprecipitation (ChIP) assay was performed using from Immunotech (Marseille, France). Anti-GATA3 polyclonal Ab, anti- the EpiQuick ChIP kit (Epigentek, Brooklyn, NY), according to the NFATc1 polyclonal Ab, anti-NFATc2 polyclonal Ab, and NFAT activation manufacturer’s instructions. The PCR primers used were as follows: inhibitor III (INCA-6) were purchased from Santa Cruz Biotechnology IL-9 promoter forward, 59-GATTCTCACAACCAGAATTCCTGC-39; (Santa Cruz, CA). aCGRP was purchased from the Peptide Institute IL-9 promoter reverse, 59-CTACCAGCATCTTCCAGTCTAGC-39. 4048 CGRP PROMOTES Th9 CELL DIFFERENTIATION AND IL-9 PRODUCTION

FIGURE 1. CGRP promotes IL-9 production by Th9 cells. (A) The percentage of IL-9+ cells was analyzed by FACS after 48-h CD3/CD28 stimulation in the presence of 1 nM CGRP. Data are representative of four independent experiments. (B) IL-9 mRNA expression was measured by real-time PCR. Data are Downloaded from represented as mean 6 SD from three mice. (C) IL-9 production was measured by ELISA. Data are represented as mean 6 SD from three mice. (D) Th9 cells were differentiated from naive Th cells for 4 d or from differentiated Th2 cells with TGF-b for 2 d. These cells were stimulated with anti-CD3 mAb for 24 h, and IL-9 production was measured by ELISA. Data are represented as mean 6 SD from three mice. *p , 0.05, **p , 0.01 versus control.

Lung lymph nodes were collected at 24 h after the last challenge, and Airway inflammation and ex vivo IL-9 production http://www.jimmunol.org/ mRNAwas extracted from sorted Th cells. Spleen Th cells of challenged To induce the airway inflammation, mice were immunized with 250 mg mice were also harvested at 24 h after the last challenge, and cell OVA suspended in 4 mg aluminum hydroxide hydrate [Al(OH)3.xH2O] gel suspensions were prepared. The sorted Th cells were stimulated with on days 0 and 7 (i.p.). To elicit inflammation, mice were challenged daily anti-CD3 and anti-CD28 mAb for 72 h and used for IL-4 and IL-9 with 1% OVA in PBS using a nebulizer (Omron, Kyoto, Japan) for 30 min ELISA. from day 14 to 16. Twenty-four hours after the last challenge, lung tissues and lung lymph nodes were collected. For histology, tissues were fixed Statistics with 4% paraformaldehyde and frozen in liquid nitrogen. Transverse cryosections (10 mm) were stained with H&E. The histological scoring A significant difference was further examined by unpaired Student t test was performed according to Table I. or Student–Newman–Keuls test after nonrepeated measures ANOVA. A , , For flow cytometry, lung tissue was homogenized with a frosted glass probability of 5% (p 0.05) was considered statistically significant. by guest on September 28, 2021 slide, and the mononuclear cells were then stained with mAbs specific for We use the means of results from individual mice for the all statistical CD4, FcεR, and c-kit. IL-4 and IL-9 in lung homogenate supernatants were analysis, and we confirmed the reproducibility of the experiments at least also measured by ELISA. two times.

FIGURE 2. cAMP/PKA pathway promotes IL-9 production in Th9 cells. (A) Dibutyryl cAMP (dbcAMP) promotes Th9 differentiation. IL-9 production was measured by ELISA. (B) IL-9 production was measured by ELISA in the presence of a PKA-speciﬁc (6-bnz) or EPAC-speciﬁc (8-CPT) analog. (C) PKA inhibitor completely blocked the promoting effect of CGRP. IL-9 production was measured by ELISA in the presence of 1 nM CGRP and 5 mM H89

(PKA inhibitor). (D) IL-9 production was measured by ELISA in the presence of 1 nM CGRP and 1 mM KT5720 (PKA inhibitor). (E) PGE2 also promotes IL-9 production via cAMP/PKA pathway. IL-9 production was measured by ELISA in the presence of 1 mM PGE2 and 5 mM H89. (F) PGD2 also promotes IL-9 production via cAMP/PKA pathway. IL-9 production was measured by ELISA in the presence of 0.5 mM PGD2 and 5 mM H89. These data are represented as mean 6 SD from three mice. *p , 0.05, **p , 0.01 versus IL-4 1 TGF-b. The Journal of Immunology 4049

Results no effect (Fig. 2B). To further examine whether IL-9 production CGRP upregulates IL-9 production from naive T cells via its by CGRP is mediated by PKA activation, the PKA inhibitor H89 receptor or KT5720 was added to the Th9-skewed medium with CGRP. As shown in Fig. 2C and 2D, the promoting effect of CGRP dis- First, we evaluated whether CGRP affects IL-9 production by T appeared when PKA inhibitor was present. These results demon- cells. Naive splenic Th cells from C57BL/6 mice were sorted and strated that CGRP promotes Th9 cell differentiation through the stimulated under the Th9 condition in the presence or absence of cAMP/PKA pathway. CGRP. Because two isoforms of CGRP exist, the effects of both PGE and PGD are well known as one of the GPCR ligands. aCGRP and bCGRP were examined in the following experiments. 2 2 They also upregulate intracellular cAMP level through specific As shown in Fig. 1A, the percentage of IL-9+ cells increased when GPCRs and promote IL-4 or IL-17 production through PKA aCGRP or bCGRP was added to the Th9 conditioning medium. activation (38). Therefore, the effects of PGE2 and PGD2 on IL- Expression of IL-9 mRNA and IL-9 in the supernatant was also 9 production were also investigated. As expected, PGE2 and upregulated in the presence of each CGRP isoform through its PGD2 promoted IL-9 production in Th9 cells, and these effects specific receptor because such an increment was not detected in were blocked by H89 (Fig. 2E, 2F). Thus, we concluded that the RAMP1-deficient T cells (Fig. 1B, 1C). Naive splenic Th cells cAMP/PKA pathway is an important factor for IL-9 production from BALB/c mice produced similar results (data not shown). The in Th9 cells. dose dependence of CGRP was also confirmed (Supplemental Fig. 1A). CGRP induced the elevation of IL-9 production in a CGRP and PKA induce PU.1 and GATA3 expression and NFATc2 activation

bell-shaped manner, and a high concentration of CGRP rather Downloaded from suppressed IL-9 production due to its inhibitory effects on T cell To examine the molecular mechanism by which CGRP induces proliferation or activation (Supplemental Fig. 1B). Incidentally, IL-9 production in Th9 cells, we investigated the relationship we estimate that the physiological concentration of CGRP is be- between CGRP and Th cell–related transcriptional factors in Th9 tween 0.1 and 10 nM based on measurements of the CGRP con- cells using RT-PCR. As a result, we found that CGRP upregulated centration in serum (32). Therefore, the concentration (1 nM) of mRNA expression of PU.1 and GATA3 in Th9 cells (Fig. 3A). CGRP for promoting IL-9 seems to be adequate. We also observed

PU.1 is reported to be the major transcriptional factor that induces http://www.jimmunol.org/ the promoting effect of CGRP on IL-9 production even when a Th9 cell-skewed condition was arranged using different concentrations of IL-4 and TGF-b (Supplemental Fig. 1C). These results suggest that CGRP upregulates IL-9 production in Th9 cells through its speciﬁc receptor, the RAMP1/calcitonin receptor- like receptor heterodimer on Th cells. It has been reported that Th9 cells can be generated from Th2 cells in the presence of TGF-b, and that CGRP promotes Th2

differentiation (Supplemental Fig. 1D). Therefore, there is a pos- by guest on September 28, 2021 sibility that CGRP indirectly upregulates IL-9 production by promoting IL-4 production or Th2 differentiation. To clarify this point, we measured IL-4 production in a Th9 cell-skewed condition. CGRP slightly increased IL-4 production in Th9 cells, but the amount of IL-4 in Th9 cells was smaller than that in Th2 cells (Supplemental Fig. 1D). We conﬁrmed that such IL-4 levels (,1 ng/ml) in the Th9-skewed culture did not affect IL-9 production (data not shown). In addition, CGRP showed no effect on IFN-g and IL-10 productions (Supplemental Fig. 1E, 1F). Furthermore, we investigated whether CGRP affects IL-9 production in differentiated Th9 cells from naive Th cells or Th2 cells. As shown in Fig. 1D, CGRP signiﬁcantly promotes IL-9 production from differentiated Th9 cells. These results indicate that CGRP directly promotes IL-9 production in Th9 cells independent of other cytokine productions.

CGRP induces IL-9 production through cAMP/PKA pathway We have reported that CGRP elevates the intracellular cAMP concentration through CGRP receptors in Th cells (28). Therefore, we examined whether CGRP promotes IL-9 production via cAMP pathways in Th9 cells. First, we used a membrane-permeable FIGURE 3. CGRP promotes expression of PU.1 and GATA3. (A)Th cAMP analog, dibutyryl cAMP, and observed that the cAMP an- cell–related gene expressions were analyzed by RT-PCR. Data are repre- alog increased IL-9 production in Th9 cells in a dose-dependent sentative of three independent experiments. (B) GATA3 and PU.1 mRNA manner as well as CGRP did (Fig. 2A). It is well known that the expressions after 48-h stimulation were measured by real-time PCR. Data 6 C effects of cAMP in immune systems are mediated by activating are represented as mean SD from three mice. ( ) GATA3 expressions were analyzed by FACS after 48-h stimulation. Data are representative of PKA or exchange factor directly activated by cAMP (EPAC) two independent experiments. (D) Immunofluorescence staining of PU.1 (36, 37). Therefore, we performed the experiment using PKA- or protein was performed after 48-h stimulation. Scale bar, 20 mm. Data are EPAC-specific cAMP analogs. In the Th9-skewed condition, 6- representative of three independent experiments. *p , 0.05, **p , 0.01 bnz-cAMP, which is a PKA-specific activator, promoted IL-9 versus IL-4 1 TGF-b. a,1nMaCGRP; b,1nMbCGRP; 6b, 50 mM6- production, but an EPAC-specific activator, 8-CPT-cAMP, had bnz-cAMP; 8C, 50 mM 8-CPT-cAMP. 4050 CGRP PROMOTES Th9 CELL DIFFERENTIATION AND IL-9 PRODUCTION

Th9 cells and IL-9 expression (14). GATA3 is a putative tran- IL-9 enhancer regions (Fig. 4B, 4C). We also observed that the scriptional factor because GATA3-null mice exhibit a loss of IL-9 binding of GATA3 to the IL-9 region was increased in the pres- (6). Therefore, we focused on these two factors and confirmed the ence of CGRP (Fig. 4B, 4C). Next, we performed further experi- increased mRNA expression due to a CGRP- or PKA-specific ments using a NFAT inhibitor. In the presence of the NFAT cAMP analog using a real-time PCR system (Fig. 3B). Increases inhibitor, CGRP showed no promoting effect on the production of in GATA3 and PU.1 protein levels were also observed by FACS IL-9 (Fig. 4D). These results suggest that NFATc2 activation is and immunofluorescence staining, respectively (Fig. 3C, 3D). also essential in the effect of CGRP on IL-9 production. These results suggest that these factors may be central regulators b of CGRP signaling in IL-9 production. CGRP and PKA induce GSK-3 phosphorylation Recently, it was also reported that NFATc2 induces IL-9 trans- Next, we attempted to elucidate the mechanism by which the activation in Th cells (16). Moreover, one report demonstrated CGRP/cAMP/PKA pathway induces the translocation of NFATc2. that IRF-4, which synergizes with NFATc2 in the IL-4 expression GSK-3b is well known as a regulator of NFATc2 localization. process, is also crucial for the development and function of IL-9 GSK-3b phosphorylates NFATc2 and inhibits its nuclear localiza- production in Th9 cells (17). In contrast, we previously reported tion (39). In contrast, PKA inactivates GSK-3b by phosphorylating that CGRP enhances the transcriptional activity of NFATc2 in it at Ser9 in HEK293 cells (40). Therefore, it is possible that Th2 and Th17 cell differentiation (27, 28). Under a Th9-skewed PKA promotes the nuclear localization of NFATc2 by inhibiting condition, although NFATc2 expression was not changed at either GSK-3b activity in Th cells. To investigate this possibility, phos- mRNA or protein levels by CGRP (Fig. 3A and data not shown), phorylated GSK-3b was detected using its specific Ab. In the immunofluorescent staining revealed that CGRP induces the Th9-skewed condition, both CGRPs induced GSK-3b (Ser9) Downloaded from translocation of NFATc2 into nuclei (Fig. 4A). We also investi- phosphorylation compared with nontreated cells (Fig. 4E). In gated the expression of NFATc1, and found that CGRP did not addition, a PKA activator, but not an EPAC activator, induced the affect the expression and localization of NFATc1 (Fig. 3A, 4A). phosphorylation of GSK-3b (Fig. 4E). These results suggest that Therefore, we hypothesized that the translocation of NFATc2 is CGRP induced IL-9 production through PKA-dependent GSK-3b one of the key mechanisms by which CGRP affects Th9 differ- phosphorylation.

entiation. To examine this hypothesis, a ChIP assay was per- We next examined IL-9 production in the presence of a GSK-3b http://www.jimmunol.org/ formed. CGRP upregulated the amount of NFATc2 binding to the inhibitor. As shown in Fig. 4F, nuclear localization of NFATc2 by guest on September 28, 2021

FIGURE 4. CGRP promotes NFATc2 transcriptional activity and GSK-3b phosphorylation. (A) Immunofluorescence staining of NFATc1 and NFATc2 protein was performed after 24-h stimulation. Data are representative of three independent experiments. (B and C) ChIP was performed using anti-NFATc2 or anti-GATA3 Abs. PCR was performed using specific primers for IL-9 promoter. Data are represented as mean 6 SD from three mice. (D) IL-9 production under the Th9-skewed condition was measured by ELISA. Data are represented as mean 6 SD from three mice. (E) Phosphorylated GSK-3b (Ser9) was determined by immunofluorescence staining. Data are representative of three independent experiments. (F) The effect of GSK-3b inhibitor and NFAT inhibitor on NFATc2 localization was confirmed by immunocytochemistry. Data are representative of two independent experiments. Scale bars, 20 mm. (G– I) IL-9 production was measured by ELISA in the presence of GSK-3b inhibitor, NFAT inhibitor, and CGRP under the Th9-skewed condition. Lithium chloride (LiCl) was used as GSK-3 inhibitor. Data are represented as mean 6 SD from three mice. *p , 0.05, **p , 0.01 versus IL-4 1 TGF-b. a,1nM aCGRP; b,1nMbCGRP; 6-bnz, 50 mM 6-bnz-cAMP; 8-CPT, 50 mM 8-CPT-cAMP; dbc, 20 mM dbcAMP. The Journal of Immunology 4051 was increased by treatment with the GSK-3b inhibitor, and this effect both 24 and 48 h (Fig. 5B). It suggests that CGRP has other effect was diminished by the NFAT inhibitor. Indeed, the inhibi- mechanisms in IL-9 production at the late stage of Th9 differ- tion of GSK-3b induced IL-9 production as well as CGPP or entiation independence of GSK-3b/NFATc2 pathway. Thus, we dbcAMP (Fig. 4G), and the NFAT inhibitor suppressed the effect confirmed the time-dependent effect of CGRP and inhibitors on of the GSK-3b inhibitor (Fig. 4H). Furthermore, both the specific IL-9 production using ELISA. As results, CGRP promotes IL-9 GSK-3b inhibitor and lithium chloride (a traditional GSK-3 in- production after 72- and 96-h stimulation (Fig. 5C). Noteworthily, hibitor) diminished the effects of CGRP in IL-9 production (Fig. although CGRP could not promote IL-9 in the presence of GSK- 4I). We confirmed that GSK-3b expression was not affected by 3b inhibitor after 48 h (as similar to Fig. 4I), GSK-3b inhibition CGRP or GSK-3b inhibitor (data not shown). These results sug- did not diminish CGRP effects after 72 and 96 h (Fig. 5C). Ad- gested that the effect of GSK-3b inhibitor overlapped with the ditionally, PKA inhibitor completely diminished CGRP effect at CGRP signaling pathway, and thus the CGRP effect was diminished every time point. Therefore, the presence of the PKA-dependent in GSK-3b inhibitor-treated cells. Taken together, these results in- but GSK-3b independent pathway was suggested. dicate that cAMP/PKA activation with CGRP induces the NFATc2 We also investigated the mRNA expression of GATA3 and nuclear transition by phosphorylation of GSK-3b (Ser9), which PU.1. As shown in Fig. 5D, the NFAT inhibitor also blocked leads to IL-9 production. CGRP effects on the mRNA expression of GATA3. In contrast, PU.1 mRNA expression is increased by CGRP even in the presence of CGRP and PKA induce PU.1 expression via the inhibitor of NFAT or GSK-3b, although the PKA inhibitor b GSK-3 –independent pathway H89 completely blocked the promoting effect of CGRP on PU.1

Next, we investigated whether mRNA expression of IL-9 depends expression (Fig. 5E). These results suggest that NFATc2 activated Downloaded from on NFATc2 or GSK-3b. Both the NFAT inhibitor and the GSK-3b the expression of GATA3 and IL-9 in the CGRP signaling. In inhibitor blocked CGRP effects on the mRNA expression of IL-9 contrast, CGRP induced PU.1 expression independent of inacti- at 24 h after stimulation (Fig. 5A). However, these inhibitors did vation of GSK-3b or activation of NFATc2. Therefore, it was not inhibit the CGRP effect on the mRNA expression of IL-9 at suggested that PKA activation occurs through at least two path- 48 h after stimulation, although PKA inhibitor H89 inhibited the ways, as follows: the GSK-3b–dependent activation of NFATc2 at http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. The effects of CGRP on mRNA expressions in the presence of inhibitors. (A and B) The effect of NFAT, GSK-3b, and PKA inhibitors on the mRNA expression of IL-9 was analyzed by real-time PCR after 24-h (A) and 48-h (B) stimulation under the Th9-skewed condition. (C) The time-dependent effect of CGRP and inhibitors on IL-9 production. IL-9 was measured by ELISA after 48-, 72-, and 96-h stimulation under the Th9-skewed condition. (D) The effect of NFAT, GSK-3b, and PKA inhibitors on the mRNA expression of GATA3 was analyzed by real-time PCR after 24-h stimulation. (E) The effect of NFAT, GSK-3b, and PKA inhibitors on the mRNA expression of PU.1 was analyzed by real-time PCR after 24-h stimulation. These data are represented as mean 6 SD from three mice. *p , 0.05, **p , 0.01 versus IL-4 1 TGF-b, #p , 0.05 versus IL-4 1 TGF-b 1 inhibitor. a,1nMaCGRP; b,1nM bCGRP. 4052 CGRP PROMOTES Th9 CELL DIFFERENTIATION AND IL-9 PRODUCTION

Table I. Histological score of airway inﬂammation

Score 0 Score 1 Score 2 Score 3 Around airway 0 cell layers thick 1–4 cell layers thick 5–10 cell layers thick .10 cell layers thick Around vessel 0 cell layers thick .30% vessels have ,70% vessels have .70% vessels have .0 cell layers thick .5 cell layers thick .5 cell layers thick Total score is determined as airway score plus vessel score. an early phase and the GSK-3b–independent expression of PU.1 (Fig. 6A, 6B). We also observed a decrease in the number of at a late phase. Th cells and mast cells infiltrated into lung tissue in RAMP1- deficient mice (Fig. 6C). These results suggest that CGRP recep- CGRP enhances IL-9 production in vivo tor signaling is necessary for promoting airway inflammation. It Finally, we investigated the physiological role of CGRP in Th9 cell has been reported that aCGRP-deficient mice also show resistance development using RAMP1-deficient mice, which indicates a to airway inflammation, but that the IgE levels and number of specific deletion of CGRP signaling (32). OVA-induced airway eosinophils in BALF do not change (35). Consistent with this inflammation is a type I hypersensitivity, and this model is char- observation, serum IgE levels made no difference in wild-type acterized by the infiltration of inflammation cells and airway re- and RAMP1-deficient mice in our experiments (Fig. 6D).

sistance. As results of H&E staining and its scoring (Table I), after We next investigated the IL-9 production in these mice. First, Downloaded from the OVA challenge, RAMP1-deﬁcient mice showed less inﬁltrat- we evaluated IL-4 and IL-9 concentrations in spleen Th cells after ing cells and lower clinical scores compared with wild-type mice an OVA challenge. As shown in Fig. 6E, in spleen, only IL-4 http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. RAMP1-deficient mice show resistance to airway inflammation. (A and B) Inflammation of lung tissue was evaluated by H&E staining. Scale bar, 200 mm. (C) The number of cells infiltrated into lung tissue was measured by FACS. (D) The serum OVA-specific IgE concentrations were measured by ELISA. (E) IL-4 and IL-9 productions in CD4+ spleen cells from challenged mice were measured by ELISA. (F) IL-4 and IL-9 concentrations in lung tissue were measured by ELISA. (G) IL-4 and IL-9 mRNA expressions in CD4+ lymphocytes from challenged mice were measured by real-time PCR. These data are represented as mean 6 SD from six mice per group. *p , 0.05, **p , 0.01. The Journal of Immunology 4053 production was increased after the challenge. In contrast, the IL-9 other calcitonin family peptides (41). However, both Th0 and Th9 concentration in lung tissue was strongly increased, and we found cells do not express calcitonin receptor, and recombinant calcito- a decreased level of IL-9 in RAMP1-deficient mice compared nin did not affect the production of IL-9 in vitro (data not shown). with wild-type mice (Fig. 6F). We also demonstrated that freshly Moreover, in airway inflammation, the phenotype of aCGRP- isolated Th cells derived from RAMP1-deficient lymph nodes deficient mice is similar with that of RAMP1-deficient mice (35). exhibited low IL-9 mRNA expression in comparison with wild- Therefore, these results strongly suggest that CGRP/CGRP re- type mice (Fig. 6G). However, the IL-4 level in lung tissue was not ceptor signaling promotes Th9 cell differentiation through up- increased after the challenge, and its expression was not signifi- regulation of IL-9 production, which enhances IL-9–mediated cantly different in control and RAMP1-deficient mice (Fig. 6F). inflammation. Moreover, the IFN-g concentration in lung tissue was not de- tectable even after the challenge (data not shown). These obser- Discussion vations suggest that CGRP promotes IL-9 expression in Th9 cells It is well known that IL-9 is mainly secreted by T cells (Th9 cells) that are activated in the lung-draining lymph nodes and have and that it plays critical roles in type I hypersensitivities such as infiltrated into the lung tissue. asthma. As a cause of airway inflammation and asthma, IL-4 has To further clarify the roles of CGRP in Th9 cells, we used been considered to be the best therapeutic target in asthma. T cell–specific RAMP1-deficient mice by crossing Lck-Cre trans- However, several clinical and fundamental studies have demon- genic and RAMP1 flox/flox (f/f) mice. The T cell–specific RAMP1- strated that IL-9 and IL-13 are more important cytokines in the deficient mice also showed both lower clinical scores and a de- onset of allergic asthma than IL-4 (13, 42–44). Moreover, it was crease in infiltrations of Th and mast cells into the lung tissue also demonstrated that IL-9 induces IL-13 expression in airway Downloaded from compared with control mice (Cre-negative RAMP1-f/f mice) after epithelial cells and promotes airway inflammation (45). Therefore, an OVA challenge (Fig. 7A–C), although serum OVA-specific IgE the molecular regulation of IL-9 production is one of the great levels in T cell–specific RAMP1-deficient and control mice were challenges in this research field because IL-9 seems to be directly similar (Fig. 7D). Moreover, the IL-9 concentration in lung tissue related to the treatment of asthma. was low in T cell–specific RAMP1-deficient mice (Fig. 7E). The It is also reported that IL-1 promotes IL-9 production from

level of IL-4 was not changed in those mice. RAMP1 can also murine bone marrow–derived mast cells and human eosinophils http://www.jimmunol.org/ interact with calcitonin receptor,andworkasthereceptorfor (46, 47). In mast cells, the LPS/p38 MAPK pathway also promotes by guest on September 28, 2021

FIGURE 7. T cell–specific RAMP1-deficient mice also show resistance to airway inflammation. (A and B) Inflammation of lung tissue was evaluated by H&E staining. Scale bar, 200 mm. (C) The number of cells infiltrated into lung tissue was measured by FACS. (D) The serum OVA-specific IgE concentrations were measured by ELISA. (E) IL-4 and IL-9 concentrations in lung tissue were measured by ELISA. These data are represented as mean 6 SD from four mice per group. *p , 0.05, **p , 0.01. 4054 CGRP PROMOTES Th9 CELL DIFFERENTIATION AND IL-9 PRODUCTION

IL-9 production via NF-kB and GATA1 activation (48). As a novel specific expression pattern of these molecules in lymph nodes regulation of IL-9 in Th cells, some cytokines such as IL-25, IFN- because only aCGRP mRNA expression could be detected in g, and IL-21 can upregulate IL-9 production in Th9 cells (15, 49). lymph nodes (data not shown). We have also confirmed that These studies show that IL-9 production is regulated by many CGRP-positive nerve endings are distributed in lung, skin, and physiological substances and molecular pathways. around lymph nodes, but not in spleen (data not shown). Actu- The IL-9 promoter has sites to which several transcription ally, the cytokine expression in spleen Th cells was affected in factors, such as c-Jun, IRF-4, NF-kB, NFAT, and GATA3, are able RAMP1-deficient mice neither in an airway inflammation model, to bind (50). Actually, NF-kB has been shown to bind and activate contact hypersensitivity, nor experimental autoimmune enceph- the IL-9 promoter in mast cells and Th9 cells (15, 48). In addition, alomyelitis experiments, although the production in lymph nodes it has been also reported that PU.1 and IRF-4 bind to the IL-9 was dramatically regulated (28, 33). To reveal the different promoter and play essential roles in Th9 cell differentiation (14, effects of CGRP, VIP, and PACAP, it is necessary to investigate 17). Our study also demonstrated that GATA3 and NFATc2 bind to whether VIP and PACAP physiologically affect Th cell differ- the IL-9 promoter region and contribute to the transcription of IL- entiation in an airway inflammation model like CGRP. 9. This observation supports the decreased expression of IL-9 in In conclusion, we revealed that CGRP promotes Th9 cell dif- GATA3-deficient mice or NFATc2-deficient Th cells (6, 16). ferentiation via the cAMP/PKA pathway and airway inflam- In this study, we showed that the neuropeptide CGRP, which is mation. IL-9 affects many cells, including B cells, mast cells, a GPCR ligand, promotes IL-9 production from Th9 cells via the eosinophils, T cells, and goblet cells; therefore, the regulation of cAMP/PKA pathway. In addition, we demonstrated that CGRP IL-9 production will be directly targeted in the treatment of airway promotes the expression of both GATA3 and PU.1 and the binding inflammation. Because Th9 cells produce most of the IL-9, tar- Downloaded from of NFATc2 to the IL-9 promoter via nuclear translocation. Our geting the CGRP pathway might open new prospects for designing study using a NFAT inhibitor also indicated that CGRP increased therapeutic strategies for the treatment of airway inflammation. GATA3 mRNA expression via NFATc2. The correctness of this result is substantiated by previous reports that showed that the Disclosures GATA3 promoter also has NFATc2 binding sites and that NFATc2 The authors have no financial conflicts of interest. bindingtotheGATA3promoter enhances the expression of http://www.jimmunol.org/ GATA3 (51). It is known that GSK-3b inhibits NFATc2 activity (39, 52) and References that GSK-3b activity is inhibited by phosphorylation of its Ser9 in 1. Szabo, S. J., S. T. Kim, G. L. Costa, X. Zhang, C. G. Fathman, and L. H. Glimcher. 2000. A novel transcription factor, T-bet, directs Th1 lineage GSK-3b (53). It was also reported that GSK-3b is phosphorylated commitment. Cell 100: 655–669. by PKA (40). In this study, we showed that CGRP inactivates 2. 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