Identification of Functional Roles for Both IL-17RB and IL-17RA in Mediating IL-25-Induced Activities

This information is current as Erika A. Rickel, Lori A. Siegel, Bo-Rin Park Yoon, James B. of September 29, 2021. Rottman, David G. Kugler, David A. Swart, Penny M. Anders, Joel E. Tocker, Michael R. Comeau and Alison L. Budelsky J Immunol 2008; 181:4299-4310; ;

doi: 10.4049/jimmunol.181.6.4299 Downloaded from http://www.jimmunol.org/content/181/6/4299

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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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Identification of Functional Roles for Both IL-17RB and IL-17RA in Mediating IL-25-Induced Activities

Erika A. Rickel,* Lori A. Siegel,* Bo-Rin Park Yoon,* James B. Rottman,† David G. Kugler,* David A. Swart,* Penny M. Anders,* Joel E. Tocker,* Michael R. Comeau,* and Alison L. Budelsky1*

IL-25 (IL-17E) is a unique IL-17 family ligand that promotes Th2-skewed inflammatory responses. Intranasal administration of IL-25 into naive mice induces pulmonary inflammation similar to that seen in patients with allergic asthma, including increases in bronchoalveolar lavage fluid , bronchoalveolar lavage fluid IL-5 and IL-13 concentrations, goblet cell hyperplasia, and increased airway hyperresponsiveness. IL-25 has been reported to bind and signal through IL-17RB (IL-17BR, IL-17Rh1). It has been demonstrated recently that IL-17A signals through a heteromeric receptor composed of IL-17RA and IL-17RC. We sought to determine whether other IL-17 family ligands also utilize heteromeric receptor complexes. The required receptor Downloaded from subunits for IL-25 biological activities were investigated in vitro and in vivo using a combination of knockout (KO) mice and antagonistic Abs. Unlike wild-type mice, cultured splenocytes from either IL-17RB KO or IL-17RA KO mice did not produce IL-5 or IL-13 in response to IL-25 stimulation, and both IL-17RB KO and IL-17RA KO mice did not respond to intranasal admin- istration of IL-25. Furthermore, treatment with antagonistic mAbs to either IL-17RB or IL-17RA completely blocked IL-25- induced pulmonary inflammation and airway hyperresponsiveness in naive BALB/c mice, similar to the effects of an antagonistic http://www.jimmunol.org/ Ab to IL-25. Finally, a blocking Ab to human IL-17RA prevented IL-25 activity in a primary human cell-based assay. These data demonstrate for the first time that IL-25-mediated activities require both IL-17RB and IL-17RA and provide another example of an IL-17 family ligand that utilizes a heteromeric receptor complex. The Journal of Immunology, 2008, 181: 4299–4310.

nterleukin-25 (also known as IL-17E) is a member of the a soluble IL-17RB.Fc or an antagonistic Ab to IL-25 in- structurally related IL-17 family of that were iden- hibits many of the components of the Ag-induced Th2-type pul- I tified based on to IL-17A (1, 2). Unlike monary inflammation (9, 10). the proinflammatory effects associated with IL-17A, IL-25 pro- The biological effects of IL-25 are reported to be mediated motes Th2-type immune responses. For example, transgenic ex- through IL-17RB (also known as IL-17BR or IL-17Rh1), a mem- by guest on September 29, 2021 pression of IL-25 in mice results in increased blood eosinophilia, ber of the IL-17R family of receptors (1, 2). IL-25 binds serum IgE, IgG1, IL-13, and IL-5 concentrations and expres- with high affinity to and activates signaling pathways through IL- sion of IL-4, IL-5, IL-10, and IL-13 in multiple tissues (3, 4). In 17RB (11–14). It has, however, recently been reported that IL-17A contrast, IL-25 knockout (KO)2 mice exhibit an impaired Th2 re- and IL-17F signal through a heteromeric receptor complex con- sponse to infection by the parasitic helminthes Nippostrongylus taining IL-17RA and IL-17RC (15). In of this information, brasiliensis and Trichuris muris, resulting in increased suscepti- we investigated the receptor subunits required for IL-25 activities bility to infection and chronic inflammation (5, 6). using a combination of KO mice and antagonistic Abs. The roles The effects of IL-25 specifically in the lung have also been in- for both IL-17RB and IL-17RA in mediating IL-25-induced events vestigated. Intranasal administration of either IL-25-expressing ad- were first investigated using IL-17RB KO and IL-17RA KO mice. enovirus or rIL-25 protein increases bronchoalveolar lavage fluid KO mouse experiments were validated by studies using antago- (BALF) and lung tissue eosinophilia, airway hyperresponsiveness, nistic Abs to these receptors. Finally, the mouse data were fol- mucus secretion, and lung mRNA levels of IL-4, IL-5, IL-13, and lowed up by examining the role for IL-17RA in IL-25-mediated CCL11/eotaxin (7, 8). In an OVA-induced pulmonary inflamma- activity in a human primary cell-based assay. Taken together, these tion model, IL-25 mRNA expression increases in the lung after data provide compelling evidence that IL-25 biological activities aerosolized OVA challenge, and antagonism of IL-25 using either require both IL-17RB and IL-17RA, identifying IL-25 as another IL-17 family cytokine that utilizes IL-17RA in a heteromeric re-

*Department of Inflammation Research, Amgen, Seattle, WA 98119; and †Depart- ceptor complex. ment of Pathology, Amgen, Cambridge, MA 02139 Received for publication March 17, 2008. Accepted for publication July 20, 2008. The costs of publication of this article were defrayed in part by the payment of page Materials and Methods charges. This article must therefore be hereby marked advertisement in accordance Mice with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Address correspondence and reprint requests to Dr. Alison Budelsky, Amgen, 1201 C57BL/6 and BALB/c wild-type (WT) mice were obtained from Charles Ϫ/Ϫ Amgen Court West, Seattle, WA 98119. E-mail address: [email protected] River Laboratories and Taconic Farms. The generation of IL-17RA 2 (IL-17RA KO) mice has been described previously (16), and the IL-17RA Abbreviations used in this paper: KO, knockout; WT, wild type; BALF, bronchoal- Ϫ/Ϫ veolar lavage fluid; BAL, bronchoalveolar lavage; AHR, airway hyperresponsiveness; KO mouse breeding colony is maintained at Taconic Farms. IL-17RB MCh, methacholine; PAS, periodic acid-Schiff; TSLP, thymic stromal lymphopoietin; (IL-17RB KO) mice were generated as follows. A gene targeting vector i.n. ϭ intranasal; PENH, enhanced pause; TRAF6, TNFR-associated factor 6. was constructed with a 2107-bp 5Ј arm of homology and a 5889-bp 3Ј arm of homology flanking a neomycin resistance cassette. A thymidine kinase Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 cassette was inserted into the 5Ј end of the vector. The targeting vector was www.jimmunol.org 4300 IL-17RB AND IL-17RA ARE REQUIRED FOR IL-25 ACTIVITIES

designed to remove a region of exon 3 of the murine IL-17RB gene en- Intranasal cytokine challenge protocol coding aa H38-R73. 129-derived embryonic stem cells were electroporated Mice were intranasally dosed with 0.5 ␮g of murine IL-25 (Amgen), 0.5 with the targeting vector and selected in the presence of G418 and ganci- ␮ ␮ clovir as previously described (17). Embryonic stem cell clones carrying g of murine IL-13 (Biosystems), 0.5 g of murine IL-17F (R&D Sys- tems), or an equal volume (50 ␮l) of vehicle. Cytokines were diluted in the targeted insertion in IL-17RB were identified by a combination of PCR ␮ and genomic Southern blot analyses and were injected into Swiss Black PBS (Life Technologies) containing 10 g/ml mouse serum albumin blastocysts. PCR genotyping was performed using primers 3291-82 (5Ј- (Sigma-Aldrich) or in PBS alone. IL-25, IL-13, and IL-17F intranasal chal- CCAGGATCCAGGAGTTTAGCTT-3Ј) and 3291-83 (5Ј-CCCTATGTC lenge was performed once daily for 4 consecutive days. All mice were analyzed 24 h after the final intranasal challenge. CTTCCCGTTTCTA-3Ј) that produce a 336-bp product for the WT allele and a 280-bp product for the KO allele. Male chimeras were crossed to Ab treatment protocol Swiss Black females to generate mice heterozygous for the IL-17RB mu- tation (IL-17RBϩ/Ϫ). IL-17RBϩ/Ϫ mice were subsequently intercrossed to BALB/c mice were injected i.p. once daily for 4 consecutive days with 250 generate IL-17RBϪ/Ϫ mice (IL-17RB KO). IL-17RB KO mice were ␮g of anti-IL-25 mAb (Amgen), 250 ␮g of anti-IL-17RB mAb (Amgen), moved to a C57BL/6 background using Marker-Assisted Accelerated 200 ␮g of anti-IL-17RA-mAb (Amgen), 200 ␮g of anti-IL-17A mAb (Am- Backcrossing (MAX-BAXSM) technology at Charles River Laboratories. gen), or appropriate protein control 3 h before receiving the intranasal Mice that were identified to be 99.5% C57BL/6 were used to establish a challenge. breeding colony at Charles River Laboratories to produce mice for exper- imental use. Mice for experimental use were housed at Amgen in accor- Bronchoalveolar lavage fluid cellularity and cytokine dance with Amgen’s Internal Animal Care and Use Committee under spe- concentrations cific pathogen-free conditions and according to federal guidelines. Mice were used at 8–12 wk of age and were age and sex matched within Bronchoalveolar lavage (BAL) was performed by intubating mice anes- ␮ experiments. thetized with a 300- l i.p. injection of 2.5% Avertin (2,2,2-tribromoetha- nol; Sigma-Aldrich) and flushing the lungs with 2 ϫ 600-␮l volumes of Downloaded from ice-cold Dulbecco’s PBS (Life Technologies). BAL fluid cells were pel- leted by centrifugation at 0.1 ϫ g in an Eppendorf centrifuge model 5415D Generation of mAbs to mouse IL-17RB, mouse IL-25, mouse for 10 min, resuspended in PBS plus 5% FBS (HyClone), and analyzed IL-17RA, and mouse IL-17A using an Advia 120 hematology machine (Bayer). A correlation between the BALF cellularity counts provided by the AutoBAL software and by A mAb to mouse IL-17RB was generated in IL-17RB KO mice using manual differentials was confirmed using a linear regression analysis. The standard hybridoma techniques. Briefly, IL-17RB KO mice were immu- cleared BAL fluid was analyzed by ELISA for IL-5, IL-13, IL-17A, or nized with recombinant mouse IL-17RB.huFc (R&D Systems). Spleens http://www.jimmunol.org/ IL-17F levels according to the manufacturer’s directions (R&D Systems). and inguinal lymph nodes from mice with positive serum Ab titers to The limit of detection for each ELISA used in this study is as follows: IL-5, mouse IL-17RB were fused with equal numbers of NS-1 mouse myeloma 31 pg/ml; IL-13, 61 pg/ml; IL-17A, 15 pg/ml; and IL-17F, 23 pg/ml. Sam- cells. Ag-positive hybridomas were identified by ELISA and the resulting ples with cytokine concentrations below the limit of detection were re- hybridomas were cloned by limiting dilution. IgG was purified from cell ported as the limit of detection for the ELISA. culture supernatants from subcloned hybridoma lines and tested for their ability to block IL-25-induced IL-5 production in a mouse splenocyte as- Airway hyperresponsiveness measurements say. An Ab to mouse IL-17RB was chosen for in vivo use that completely ␤ blocked IL-25-induced IL-5 production in a mouse splenocyte assay (IC50 Airway hyperresponsiveness (AHR) to acetyl- -methylcholine (MCh; ϳ8 nM). Sigma-Aldrich) challenge was measured noninvasively in conscious, un- restrained mice using a whole-body plethysmograph (Buxco Electronics)

A mAb to mouse IL-25 was generated using standard hybridoma tech- by guest on September 29, 2021 niques in Lewis rats. Briefly, Lewis rats were immunized with mouse IL-25 as described previously (19). Briefly, mice were placed into individual (R&D Systems). Spleen and inguinal lymph node cells from rats with a plethysmograph chambers and exposed to aerosolized vehicle (distilled positive serum Ab titer were fused with NS-1 mouse myeloma cells, and water) followed by increasing concentrations of aerosolized MCh (3–50 the resulting Ag-positive hybridomas were cloned by limiting dilution. IgG mg/ml). Enhanced pause (PENH) was recorded and expressed as the per- was purified from cell culture supernatants from subcloned hybridoma cent change relative to baseline in response to MCh challenge. lines and tested for their ability to block IL-25- induced IL-5 production in AHR to MCh challenge was also measured in anesthetized and mechan- a mouse splenocyte assay. An Ab to mouse IL-25 was chosen for in vivo ically ventilated mice. Mice were sedated with xylazine hydrochloride (20 use that completely blocked IL-25-induced IL-5 production in a mouse mg/kg i.p.) and anesthetized with sodium pentobarbital (100 mg/kg i.p.). ϳ The trachea was cannulated with a metal needle and the mouse was con- splenocyte assay (IC50 37 nM). A mAb to mouse IL-17RA was generated using standard hybridoma nected to a small animal ventilator (flexiVent, SCIREQ; Scientific Respi- techniques in Lewis rats. Briefly, rats were immunized with recombinant ratory Equipment). Each mouse was ventilated with sinusoidal inspiration mouse IL-17RA.Fc (R&D Systems), spleens and inguinal lymph node cells and passive expiration with a rate of 150 breaths/minute and amplitude of were fused with NS-1 mouse myeloma cells, and the resulting Ag-positive 10 ml/kg mouse weight. A positive end expiratory pressure of 3.0 cm of hybridomas were cloned by limiting dilution. IgG was purified from cell H2O was established by the connection of the mouse to a water column. culture supernatants from subcloned hybridoma lines and tested for their After the mouse was ventilated for 1 min, the lungs were expanded twice ability to block IL-17A-induced IL-6 production from cultured NIH-3T3 to total lung capacity (amplitude pressure of 30 cm of H2O). An aerosol of cells (18). An Ab to mouse IL-17RA was chosen for in vivo use that either saline or increasing concentrations of MCh was delivered to the lung completely blocked IL-17A-induced IL-6 production in the 3T3 cell assay for 15 s followed by 15 s of ventilation. Following aerosol and ventilation, (IC ϳ500 nM). This Ab does not bind mouse IL-17RB.HIS or mouse a 2.5-Hz volume-driven oscillation was applied to the airway opening. 50 Each of the 10- to 2.5-Hz volume-driven oscillations had 0.20-ml ampli- IL-17RC.HIS by ELISA. tude and lasted 1.25 s. Before the next dose of MCh, lungs were expanded A mAb to mouse IL-17A was developed as described previously (18). twice to total lung capacity. Pressure and volume measurements over time in the respiratory system were recorded by the small animal ventilator, and respiratory system resistance (R) was calculated by fitting the data to the Splenocyte cultures ϭ ϩ single compartment model of the respiratory system where Ptr RV ϩ ϭ ϭ ϭ ϭ EV PO (Ptr tracheal pressure, V volume/time, E elastance Spleens were removed aseptically from C57BL/6 or BALB/c mice and ϭ ϭ treated with 0.4 mg/ml collagenase D (Roche) and 0.1% DNase I (Roche) pressure/volume, V volume, PO baseline pressure). in RPMI 1640 (Life Technologies) to generate single-cell suspensions. Lung histology Splenocytes were cultured for 72 h at 37°C in a 5% CO2 humidified in- cubator at 2.0 ϫ 107 cells/ml in complete DMEM (Life Technologies) Mouse lungs were perfused with 1 ml of 10% neutral-buffered formalin alone or with the addition of 1 ␮g/ml Con A (Sigma-Aldrich), mouse IL-25 solution (Sigma-Aldrich) instilled directly into the trachea and then im- (Amgen), mouse IL-17A (R&D Systems), or mouse IL-17F (R&D Sys- mersed in neutral-buffered formalin solution for 18 h. Lungs were then tems) at the indicated final concentrations. Inhibition experiments were processed, embedded in paraffin, sectioned at a thickness of 6 ␮m, and done in the presence of 100 ␮g/ml anti-mouse IL-17RB mAb, anti-mouse stained with H&E or periodic acid-Schiff (PAS). Lung inflammation was IL-17RA mAb, or a control mAb. Abs were preincubated with splenocytes assessed by a pathologist blinded to the treatment groups. The following for 45 min before addition of mouse IL-25. The cell supernatants were scoring system was used for assessing goblet cell hyperplasia in PAS- examined for IL-5 and IL-13 concentrations by ELISA (R&D Systems). stained sections: 0 ϭ normal; 1 ϭ minimal, goblet cell hyperplasia in large The Journal of Immunology 4301

bronchioles; 2 ϭ mild, goblet cell hyperplasia in large and medium bron- compared with stimulation with medium alone; however, IL-17RB chioles; 3 ϭ moderate, goblet cell hyperplasia in large, medium, and some KO splenocyte responses were lower than those of WT spleno- ϭ small bronchioles; and 4 marked, goblet cell hyperplasia in all airways. cytes (Fig. 1A). These data suggest a requirement for IL-17RB in Quantitative real-time PCR analysis of whole lung tissue mediating IL-25-induced activities. A potential role for IL-17RA in mediating IL-25-induced re- Total RNA was isolated from individual frozen lungs using a Qiagen RNeasy Maxi following the manufacturer’s instructions. cDNA was produced from sponses was next analyzed in splenocytes from IL-17RA KO mice. lung total RNA using a High Capacity cDNA Archive Kit (Applied Biosys- Cultured splenocytes from IL-17RA KO mice also did not produce tems) following the manufacturer’s instructions. The following Assay on De- IL-5 or IL-13 in response to IL-25 stimulation (Fig. 1B). Similar to mand TaqMan primers were purchased (PE Applied Biosystems): mouse IL-17RB KO splenocytes, IL-17RA KO splenocytes produced sig- CCL2/MCP-1 (Mm00441242_m1), CCL11/eotaxin (Mm00441238_m1), IL-5 (Mm00439646_m1), IL-9 (Mm00434305_m1), IL-10 (Mm004396 nificantly increased concentrations of both IL-5 and IL-13 in re- 16_m1), IL-13 (Mm00434204_m1), IL-17A (Mm00439619_m1), IL- sponse to Con A stimulation compared with stimulation with me- 17RA (Mm00434214_m1), IL-17RB (Mm00444706_m1), and IL-17RC dium alone (Fig. 1B). These data suggest for the first time that (Mm01184649_m1). TaqMan analysis was performed on the Applied Bio- IL-17RA is required for mediating IL-25-induced activities. systems Prism 7900HT Fast RT-PCR System (PE Applied Biosciences). Interestingly, IL-17RA KO splenocytes produced significantly Each data point represents expression results from individual mouse lungs. The relative expression of each gene to GAPDH (PE Applied Biosystems) more IL-5 and IL-13 compared with WT splenocytes in response in each treatment group was determined by SDS 2.2.3 (PE to Con A stimulation (Fig. 1B). It is unclear at this time why Con Applied Biosciences). A stimulation elicited a lower response from IL-17RB KO spleno- Human PBMC IL-25 bioassay cytes compared with WT splenocytes, but elicited a higher re- sponse from IL-17RA KO splenocytes compared with WT spleno- Downloaded from PBMC were isolated from heparinized human whole blood obtained from cytes; however, one possibility is that a cell type important for normal donors and set up in culture at 5 ϫ 106 cells/ml for 24 h with 100 ng/ml recombinant human thymic stromal lymphopoietin (TSLP; Amgen) normal immune responses is absent in these KO mice spleens. To in X-VIVO 15 (Lonza) plus 5% human AB serum (Lonza). PBMC were further investigate the roles of IL-17RB and IL-17RA in WT then collected and set up in restimulation cultures as single-cell suspen- splenocytes, antagonistic Abs to IL-17RB or IL-17RA were tested sions at 4 ϫ 106 cells/well in 48-well plates with 0.5-ml final volume/well in splenocyte experiments using cells from WT BALB/c mice. in the presence of 10 ng/ml recombinant human IL-2 (R&D Systems), 10 IL-25 induced more IL-5 production from BALB/c splenocytes http://www.jimmunol.org/ ng/ml recombinant human IL-25 (R&D Systems), or various concentra- tions of recombinant human IL-17A (R&D Systems). Inhibition experi- compared with C57BL/6 splenocytes (Fig. 1C), as might be ex- ments were performed in the presence or absence of 10 ␮g/ml soluble pected because Th2 responses are easier to induce in BALB/c mice human IL-17RB.Fc (R&D Systems), 10 ␮g/ml goat anti-human IL-17RA compared with C57BL/6 mice (21). Anti-mouse IL-17RB mAb ␮ Ab (R&D Systems; catalog no. AF177), 10 g/ml mouse anti-human IL- and anti-mouse IL-17RA mAb treatment significantly blocked IL- 17RA mAb clone 133621 (R&D Systems; catalog no. MAB1771), 10 ␮g/ml soluble human IL-17RA.Fc (Amgen) (20), 10 ␮g/ml mouse anti- 25-induced IL-5 production from WT BALB/c splenocytes (Fig. human IL-17A mAb clone 41809 (R&D Systems; catalog no. MAB317), 1C). The combination of splenocyte data from IL-17RB KO mice, or 10 ␮g/ml of an appropriate protein control. Soluble human IL-17RB.Fc IL-17RA KO mice, and the antagonistic Ab experiments provide and soluble human IL-17RA.Fc were preincubated with IL-2 plus IL-25 for the first in vitro evidence that IL-25 activities may require both 30 min before addition to the PBMC cultures. Abs to human IL-17RA and IL-17RB and IL-17RA. by guest on September 29, 2021 IL-17RB were preincubated with PBMC for 30 min at room temperature before restimulation with IL-2 plus IL-25. The Ab to human IL-17A was IL-17RA has been reported previously to be required for activity added to the PBMC culture at the time of IL-2 plus IL-25 restimulation. of both IL-17A and IL-17F (15). We therefore next examined the After 3 days of culture, supernatants were harvested and assessed for IL-5 potential role of IL-17A and IL-17F in mediating IL-25-induced production by ELISA (R&D Systems). The goat anti-human IL-17RA Ab activities in this BALB/c splenocyte assay. IL-17A and IL-17F (R&D Systems; catalog no. AF177) and mouse anti-human IL-17A mAb clone 41809 (R&D Systems; catalog no. MAB317) are reported by the concentrations were not detectable by ELISA in the cell culture manufacturer to inhibit IL-17A-induced IL-6 production by normal human supernatants from IL-25-stimulated splenocytes (data not shown), dermal fibroblasts. The ability of the mouse anti-human IL-17RA mAb suggesting that IL-25 does not induce IL-17A or IL-17F produc- clone 133621 (R&D Systems; catalog no. MAB1771) to inhibit IL-17A tion. In addition, IL-17A and IL-17F stimulation of mouse spleno- activities is not reported by the manufacturer. cytes did not induce IL-5 or IL-13 production even at concentra- Statistics tions up to 100 ng/ml (data not shown). Together these data suggest that IL-25-induced IL-5 and IL-13 production from Statistical analyses were performed using GraphPad Prism software ver- sion 5.00 or SAS. Statistical significance was reported if p Ͻ 0.05. The splenocytes is not dependent on IL-17A or IL-17F, providing sup- statistical test used to analyze each set of data is indicated appropriately in port for a direct role for IL-17RA in mediating IL-25 responses. the figure legends. IL-17RB KO and IL-17RA KO mice do not show increased Results BALF cellularity, BALF IL-5 and IL-13 concentrations, or lung Splenocytes from IL-17RB KO and IL-17RA KO mice do not proinflammatory gene mRNA levels in response to intranasal produce IL-5 or IL-13 in response to IL-25 stimulation administration of IL-25 To begin investigating the relationship between IL-25 and IL- The in vivo roles for IL-17RB and IL-17RA in mediating IL-25- 17RB, we generated IL-17RB KO mice on a C57BL/6 back- induced activities were next examined using an intranasal cytokine ground. IL-17RB KO mice develop normally and produce litters challenge protocol in IL-17RB KO mice and IL-17RA KO mice. of normal size with equivalent numbers of males and females. As previously reported (22), intranasal IL-25 increased total BALF There was no obvious phenotype when IL-17RB KO mice were leukocyte numbers in WT mice, including increases in BALF eo- examined using routine anatomic and clinical pathology tests. A sinophils, neutrophils, and, to a lesser extent, lymphocytes and role for IL-17RB in mediating IL-25-induced responses was first macrophages (Fig. 2A). Intranasal IL-25, however, did not increase analyzed in splenocytes from IL-17RB KO mice. Cultured spleno- BALF leukocyte numbers in IL-17RB KO mice (Fig. 2A). Intra- cytes from IL-17RB KO mice did not produce IL-5 or IL-13 in nasal IL-13 administration increased total BALF leukocytes, in- response to IL-25 stimulation, unlike WT splenocytes (Fig. 1A). cluding BALF eosinophils, neutrophils, and lymphocytes to a sim- IL-17RB KO splenocytes did produce significantly increased con- ilar degree in both IL-17RB KO mice and WT mice (Fig. 2B), centrations of IL-5 and IL-13 in response to Con A stimulation providing evidence that these mice can recruit leukocytes to the 4302 IL-17RB AND IL-17RA ARE REQUIRED FOR IL-25 ACTIVITIES

FIGURE 1. IL-17RB and IL-17RA are required for IL-25-induced IL-5 and IL-13 production from splenocyte cultures. A, Splenocytes from WT C57BL/6 mice and IL-17RB KO mice were cultured in medium alone, various concentrations of IL-25, or 1 ␮g/ml Con A for 72 h. IL-5 and IL-13 con- centrations were measured in cell cul- ture supernatants by ELISA. The splenocyte cultures are pools of five in- dividual spleens. Bars represent the mean Ϯ SE from triplicate wells. Data are representative of four independent experiments. B, Splenocytes from WT C57BL/6 mice and IL-17RA KO mice were cultured in medium alone, various concentrations of IL-25, or 1 ␮g/ml Con A for 72 h. IL-5 and IL-13 con- centrations were measured in cell cul- Downloaded from ture supernatants by ELISA. The splenocyte cultures are pools of five in- dividual spleens. Bars represent the mean Ϯ SE from triplicate wells. Data are representative of four independent experiments. Statistical analyses of

IL-25 stimulation compared with me- http://www.jimmunol.org/ dium stimulation were performed using a nonparametric one-way ANOVA: p Ͻ ,ءءء p Ͻ 0.01; and ,ءء ;p Ͻ 0.05 ,ء 0.001. Statistical analyses of Con A-stimulated WT splenocytes and KO splenocytes were performed using a t test. A value of p Ͻ 0.05 was consid- ered significant. C, Splenocytes from BALB/c mice were stimulated with IL-25 in the presence and absence of by guest on September 29, 2021 control Ab, anti-mouse IL-17RB mAb, or anti-mouse IL-17RA mAb for 72 h. IL-5 concentrations were measured in cell culture supernatants by ELISA. The splenocyte cultures are pools of five individual spleens. Bars represent the mean Ϯ SE from triplicate wells. Data are representative of three inde- pendent experiments. Statistical analy- ses were performed using a nonpara- metric one-way ANOVA.

BALF in response to a stimulus other than IL-25. Intranasal IL-25 lungs (Fig. 2G), providing evidence that disruption of expression also increased BALF IL-5 and IL-13 concentrations in WT mice, of IL-17RB by homologous recombination did not affect expres- but not in IL-17RB KO mice (Fig. 2C). sion of these other IL-17R family members. Together with the The induction of proinflammatory in the lungs in response BALF data, the lung mRNA analyses show that IL-17RB KO mice to intranasal IL-25 challenge was next examined in WT and IL- do not respond to IL-25 stimulation, providing evidence that IL-25 17RB KO mice. Transcript levels of proinflammatory cytokines activities require IL-17RB. and previously identified as being up-regulated by The effects of intranasal IL-25 administration in WT and IL- IL-25 or other IL-17 receptor and ligand family members were 17RA KO mice were next examined. Intranasal IL-25 administra- chosen for evaluation (4, 7, 23). Intranasal IL-25 administration tion did not increase total BALF leukocyte numbers in IL-17RA significantly increased lung expression of CCL2 and CCL11 KO mice, but did in WT mice (Fig. 3A). Intranasal IL-13 admin- mRNAs in WT mice, but did not increase expression of either of istration, however, increased total BALF leukocytes, including those genes in IL-17RB KO mice (Fig. 2, D and E). Intranasal BALF eosinophils, neutrophils, and lymphocytes to an equal ex- IL-25 also induced IL-5, IL-13, IL-9, and IL-10 mRNA expression tent in both IL-17RA KO and WT mice (Fig. 3B), demonstrating in WT mice, and none of these genes was up-regulated in IL-17RB that these mice could recruit BALF leukocytes in response to a KO mice (data not shown). There was a trend toward an increase different cytokine stimulus. Intranasal IL-25 administration also in IL-17A mRNA in IL-25-treated WT mice, but not in IL-25- did not increase BALF IL-5 and IL-13 concentrations in IL-17RA treated IL-17RB KO mice (Fig. 2F). Expression of IL-17RA and KO mice, but did in WT mice (Fig. 3C). Finally, intranasal IL-25 IL-17RC mRNAs was equivalent in WT and IL-17RB KO mice administration did not significantly increase the lung mRNA levels The Journal of Immunology 4303

AB 150,000 250,000 p = 0.0253 IN Vehicle, WT IN Vehicle, WT IN Vehicle, IL-17RB KO IN Vehicle, IL-17RB KO 200,000 IN IL-25, WT IN IL-13, WT IN IL-25, IL17RB KO 100,000 IN IL-13, IL-17RB KO p = 0.0161

150,000 p = 0.0007 p = 0.0077

100,000 Cells BALF

BALF Cells 50,000

50,000

0 0

cytes pho Leukocytes Eosinophils Neutrophils Leukocytes Eosinophils Neutrophils Lym Macrophages LymphocytesMacrophages

C D p=0.0062 p=0.0048 400 p = 0.0015 0.020 IL-5 Downloaded from

IL-13 p = 0.0012 300 0.015

200 0.010 pg/mL

100 0.005 http://www.jimmunol.org/ Relative to GAPDH Relative CCL2 Gene Expression

0 0.000 WT IL-17RB KO WT IL-17RB KO WT IL-17RB KO WT IL-17RB KO

IN Vehicle IN IL-25 IN Vehicle IN IL-25

EFp=0.001 p=0.0005 0.8 0.005 by guest on September 29, 2021

0.004 0.6

0.003 0.4 0.002 0.2 Relative to GAPDH Relative Relative to GAPDH Relative 0.001 IL-17A Gene Expression CCL11 Gene Expression 0.0 0.000 WT IL-17RB KO WT IL-17RB KO WT IL-17RB KO WT IL-17RB KO

IN Vehicle IN IL-25 IN Vehicle IN IL-25

G WT 0.015 IL-17RB KO

0.010

0.005 Gene Expression Relative to GAPDH to Relative

0.000 IL-17RA IL-17RC FIGURE 2. Intranasal IL-25 administration did not increase BALF cellularity, BALF IL-5 and IL-13 concentrations or expression of proinflammatory genes in IL-17RB KO mice. IL-17RB KO or WT mice (n ϭ 5 per group) were i.n. dosed with vehicle, 0.5 ␮g of IL-25, or 0.5 ␮g of IL-13 for 4 days, and BALF and lungs were analyzed 24 h after the final dose. A and B, Differential counts were performed on the BALF. The mean total number of BALF cells Ϯ SE is shown for each group. C, BALF IL-5 and IL-13 concentrations were measured by ELISA from individual mice. The mean concentrations are plotted Ϯ SE. RNA was isolated from individual lungs (n ϭ 4 per group), and expression levels of CCL2 (D), CCL11 (E), IL-17A (F), IL-17RA (G), and IL-17RC (G) were evaluated by TaqMan. Gene expression levels were normalized to GAPDH expression. The data are representative of two independent experiments. Statistical analyses of WT and KO mice were performed using a t test. 4304 IL-17RB AND IL-17RA ARE REQUIRED FOR IL-25 ACTIVITIES

A B 800,000 250,000 IN Vehicle, WT IN Vehicle, WT IN Vehicle, IL17RA KO IN Vehicle, IL-17RA KO

p = 0.0015 200,000 600,000 IN IL-25, WT IN IL-13, WT IN IL-25, IL17RA KO IN IL-13, IL17RA KO 150,000 400,000 p = 0.0116

p = 0.0099 100,000 p = 0.034 BALF Cells p = 0.0002 BALF Cells 200,000 50,000

0 0

Leukocytes Eosinophils Neutrophils Leukocytes Eosinophils Neutrophils LymphocytesMacrophages LymphocytesMacrophages

C 4000 D 0.010 p=.0001 p=.0002 3500 IL-5 p = 0.0126 IL-13 0.008 Downloaded from 3000 2500 p = 0.0239 0.006 2000 400

pg/mL 0.004 300 200 0.002 http://www.jimmunol.org/ 100 Relative to GAPDH to Relative CCL2 Gene Expression 0 0.000 WT IL-17RA KO WT IL-17RA KO WT IL-17RA KO WT IL-17RA KO IN Vehicle IN IL-25 IN Vehicle IN IL-25 E F p=.0001 p=0.0002 0.0015 0.20 by guest on September 29, 2021

0.15 0.0010 p=0.0002

0.10 0.0005 Relative to GAPDH Relative Relative to GAPDH CCL11 Gene Expression

0.05 IL-17A Gene Expression

0.0000 0.00 WT IL-17RA KO WT IL-17RA KO WT IL-17RA KO WT IL-17RA KO IN Vehicle IN IL-25 IN Vehicle IN IL-25

G 0.025 0.020 WT 0.015 IL-17RA KO 0.010 0.005

0.0005 0.0004 0.0003 Relative to GAPDH Gene Expression 0.0002 0.0001 0.0000 IL-17RB IL-17RC FIGURE 3. Intranasal IL-25 administration did not increase BALF cellularity, BALF IL-5 and IL-13 concentrations, or expression of proinflammatory genes in IL-17RA KO mice. IL-17RA KO or WT mice (n ϭ 5 per group) were i.n. dosed with vehicle, 0.5 ␮g of IL-25, or 0.5 ␮g of IL-13 for 4 days, and BALF and lungs were analyzed 24 h after the final dose. A and B, Differential counts were performed on the BALF. The mean total number of BALF cells Ϯ SE is shown for each group. C, BALF IL-5 and IL-13 concentrations were measured by ELISA from individual mice. The mean concentrations are plotted Ϯ SE. RNA was isolated from individual lungs (n ϭ 4 per group), and expression levels of CCL2 (D), CCL11 (E), IL-17A (F), IL-17RB (G), and IL-17RC (G) were evaluated by TaqMan. Gene expression levels were normalized to GAPDH expression. Data are representative of two independent experiments. Statistical analyses of WT and KO mice were performed using a t test. The Journal of Immunology 4305 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. Intranasal IL-25 administration did not induce histological signs of lung inflammation in IL-17RB KO or IL-17RA KO mice. WT mice (A and B), IL-17RB KO mice (C and D), and IL-17RA KO mice (E and F)(n ϭ 5 per group) were i.n. dosed with vehicle for 4 days, and lungs were harvested for histological analysis 24 h after the final dose. WT mice (G and H), IL-17RB KO mice (I and J), and IL-17RA KO mice (K and L)(n ϭ 5 per group) were i.n. dosed with 0.5 ␮g of IL-25 for 4 days, and lungs were harvested for histological analysis 24 h after the final dose. Formalin-fixed lung tissue sections were stained with H&E (A–L) or PAS (M and N) for analysis. Goblet cell hyperplasia was assessed in PAS-stained sections as described in Materials and Methods. Original magnification, ϫ20 in A, C, E, G, I, and K. Original magnification, ϫ400 in B, D, F, H, J, and L. Statistical analyses of WT and KO mice were performed using a nonparametric one-way ANOVA. of CCL2, CCL11, or IL-17A in IL-17RA KO mice, but did in WT IL-17RB KO or IL-17RA KO mice were unable to extravasate into mice (Fig. 3, D–F). In addition, expression of IL-5, IL-13, IL-9, the airways, the lungs of intranasally dosed WT, IL-17RB KO, and and IL-10 mRNAs were not up-regulated in IL-17RA KO mice IL-17RA KO mice were examined histologically for signs of in- treated with intranasal IL-25, but were in WT mice (data not flammation. Intranasal administration of vehicle alone had no in- shown). Lung expression levels of IL-17RB and IL-17RC mRNAs flammatory effect on the lung pathology of any of the mice exam- in IL-17RA KO mice were similar to those seen in WT mice, ined (Fig. 4, A–F). Intranasal IL-25 administration in WT mice providing evidence that disruption of IL-17RA did not affect ex- induced a prominent inflammatory response in the region around pression of these other IL-17R family members (Fig. 3G). These the vessel and in the vessel wall of most of the pulmonary blood data show that IL-17RA KO mice do not respond to IL-25 stim- vessels, including pulmonary arteries, arterioles, veins, and ulation, providing the first in vivo evidence that IL-25 activities venules, but not capillaries (Fig. 4, G and H). There was a robust require IL-17RA. accumulation of eosinophils beneath the endothelium lining these vessels and prominent endothelial hyperplasia (Fig. 4H). In addi- IL-17RB KO and IL-17RA KO mouse lungs do not display tion to eosinophils, the inflammatory cell infiltrate also consisted IL-25-induced histological signs of inflammation of neutrophils, lymphocytes, and monocytes/macrophages (Fig. 4, Intranasal administration of IL-25 caused a significant increase in G and H). The inflammation extended beyond the blood vessels to BALF cellularity in WT mice but not in IL-17RB KO or IL-17RA adjacent bronchioles, and the inflammatory cells traversed the KO mice. To address the possibility that leukocytes in the lungs of walls to reach the lumen of the airways (Fig. 4G). In addition to the 4306 IL-17RB AND IL-17RA ARE REQUIRED FOR IL-25 ACTIVITIES

A B

500,000 p < 0.01 3000 IN IL-25, Control

p < 0.001 α 400,000 IN IL-25, -IL-17RB IN IL-25, α-IL-25 2000 300,000 IN Vehicle

200,000 1000

BALF Leukocytes 100,000 PENH [% Above Baseline] PENH [% Above 0 0 Control Control α-IL-17RB α-IL-25 1 10 100 [Methacholine] (mg/mL) IN Vehicle IN IL-25 C D p < 0.01 400,000 p < 0.01 50,000 p < 0.001 40,000 Downloaded from 300,000 30,000 200,000 20,000

100,000 BALF Leukocytes BALF Eosinophils 10,000 http://www.jimmunol.org/

0 0

Control Control Control Control α-IL-17A α-IL-17A α-IL-17A α-IL-17A α-IL-17RA α-IL-17RA α-IL-17RA α-IL-17RA IN Vehicle IN IL-25 IN Vehicle IN IL-25 E F p < 0.05 by guest on September 29, 2021 250,000 p < 0.01 500

IL-5 p < 0.01

p < 0.01 IL-13 200,000 400

150,000 300

100,000 pg/mL 200 BALF Neutrophils 50,000 100

0 0

Control Control Control Control -IL-17A α-IL-17A α-IL-17A α-IL-17A α α-IL-17RA α-IL-17RA α-IL-17RA α-IL-17RA IN Vehicle IN IL-25 IN Vehicle IN IL-25 G H

2500 IN IL-25, Control IN vehicle, Control 10 IN IL-25, α-IL-17RA IN vehicle, α-IL-17RA IN IL25, Control IN IL-25, α-IL-17A IN vehicle, α-IL-17A IN IL25, α-IL17RA 2000 8 p = 0.0101 IN Vehicle

1500 0s/mL) 6 2

1000 4 R (cmH 500 2 [PENH, % Above Baseline] [PENH, % Above 0 0 1 10 100 0 10 20 30 [Methacholine] (mg/mL) [Methacholine] (mg/mL) FIGURE 5. Intranasal IL-25-induced lung inflammation was blocked by anti-IL-25 mAb, anti-IL-17RB mAb, and anti-IL-17RA mAb treatment. BALB/c mice (n ϭ 5 per group) were treated with either control Ab or blocking Abs to IL-25, IL-17RB, IL-17RA, or IL-17A 3 h before each i.n. dose of IL-25. Mice were i.n. dosed with 0.5 ␮g of IL-25 for 4 days. BALF and lungs were analyzed 24 h after the final IL-25 dose. A, Total number of BALF The Journal of Immunology 4307 inflammatory cell infiltrate observed in H&E-stained sections, PAS staining revealed robust goblet cell hyperplasia in the lungs of WT mice intranasally dosed with IL-25 (Fig. 4, M and N). In contrast, intranasal IL-25 administration did not induce any of these histological changes in either IL-17RB KO mice (Fig. 4, I, J, and M) or IL-17RA KO mice (Fig. 4, K, L, and N). The combined BALF, lung mRNA, and lung histology analyses from IL-17RB KO and IL-17RA KO mice administered intranasal IL-25 provide the first in vivo evidence that both IL-17RB and IL-17RA are required for IL-25 responsiveness.

Anti-mouse IL-17RB mAb treatment or anti-mouse IL-17RA mAb treatment blocks IL-25-induced lung inflammation in BALB/c mice To further investigate the essential role of IL-17RB in mediating IL-25 activities in vivo, we compared the effects of an antagonistic Ab to mouse IL-17RB with the effects of an antagonistic Ab to mouse IL-25 on IL-25-induced inflammation in the lung. Naive BALB/c mice were treated with either anti-IL-17RB mAb or anti- Downloaded from IL-25 mAb before intranasal challenge with IL-25 in the 4-day challenge model. Both anti-IL-17RB mAb and anti-IL-25 mAb treatment inhibited IL-25-induced activities in the lung, including increased BALF leukocytes (Fig. 5A), BALF IL-5 and IL-13 con- centrations (data not shown), and AHR (Fig. 5B).

The role of IL-17RA in mediating IL-25 activities in vivo was http://www.jimmunol.org/ next examined using an antagonistic Ab to mouse IL-17RA. Naive BALB/c mice were treated with anti-IL-17RA mAb before intra- nasal challenge with IL-25 in the 4-day challenge model. Anti-IL- 17RA mAb treatment blocked all measured IL-25-induced re- sponses in the lung, including BALF cellularity (Fig. 5, C–E), BALF IL-5 and IL-13 concentrations (Fig. 5F), and AHR (Fig. 5, G and H). Although we were unable to detect IL-17A protein in the BALF of IL-25-treated WT BALB/c mice (data not shown), the up-reg- by guest on September 29, 2021 ulation of IL-17A lung transcript by IL-25 (Figs. 2F and 3F) sug- gested the possibility that IL-17A might be playing a role in IL-25-mediated activities. To more closely examine the role of IL-17A in IL-25-induced biological activities, the effects of an antagonistic Ab to mouse IL-17A were also examined in mice FIGURE 6. Abs to IL-17RB, IL-17RA, or IL-25 block IL-25-induced intranasally dosed with IL-25. Anti-IL-17A mAb treatment inhib- histological signs of pulmonary inflammation. BALB/c mice were i.n. ited IL-25-induced BALF neutrophilia (Fig. 5E), but had no effect dosed with vehicle (A)or0.5␮g of IL-25 (B–F) for 4 days, and lungs were on IL-25-induced BALF eosinophils (Fig. 5D), lymphocytes (data harvested for histological analysis 24 h after the final dose. Mice (n ϭ 5 per not shown), and macrophages (data not shown), BALF IL-5 and group) were treated with control Ab (B) or blocking Abs to IL-17RB (C), IL-13 concentrations (Fig. 5F), or AHR (Fig. 5G). These data pro- IL-25 (D), IL-17RA (E), or IL-17A (F) 3 h before each i.n. dose. Formalin- fixed lung tissue sections were stained with H&E (A–F) or PAS (G) for vide evidence that IL-17A may be playing a role in IL-25-induced analysis. A–F, Original magnification, ϫ200. G, Goblet cell hyperplasia BALF neutrophilia in this mouse model, but that all other IL-25- was assessed in PAS-stained lung sections as described in Materials and induced activities are independent of IL-17A activity. Methods. Statistical analyses were performed using a nonparametric one- The abilities of the anti-IL-17RB mAb and of the anti-IL-17RA way ANOVA. mAb to block IL-25-induced activities were further investigated through histological analysis of the lungs from these mice. Intra- ilar to that described in WT C57BL/6 mice (Fig. 4, G, H, M, and nasal IL-25 administration into BALB/c mice induced pulmonary N). Pulmonary inflammation induced by intranasal IL-25 admin- inflammation (Fig. 6B) and goblet cell hyperplasia (Fig. 6G) sim- istration was at the level of background in mice treated with either

leukocytes in mice i.n. dosed with IL-25 and treated with control Ab, anti-IL-25 mAb, or anti-IL-17RB mAb. Each dot represents BALF cellularity from one mouse. B, AHR in mice treated with control Ab, anti-IL-17RB mAb, or anti-IL-25 mAb. The mean percent change in PENH relative to baseline is reported for each treatment group Ϯ SE. Total BALF leukocytes (C), eosinophils (D), and neutrophils (E) from mice treated with control Ab, anti-IL-17RA mAb, or anti-IL-17A mAb. Each dot represents BALF cellularity from one mouse. F, BALF IL-5 and IL-13 concentrations from mice treated with control Ab, anti-IL-17RA mAb, or anti-IL-17A mAb. The bars represent the mean concentration Ϯ SE. G, AHR in mice treated with control Ab, anti-IL-17RA mAb, or anti-IL-17A mAb. The mean percent change in PENH relative to baseline is reported for each treatment group Ϯ SE. H, AHR was also measured in mechanically ventilated mice treated with vehicle and control Ab (n ϭ 5), IL-25 and control Ab (n ϭ 7), or IL-25 and anti-IL-17RA mAb (n ϭ 8). Mean airway resistance (R) is shown for each treatment group Ϯ SE. Data shown are representative of two independent experiments for A and B, six independent experiments for C–F, and two independent experiments for G and H. Statistical analyses of control and Ab-treated mice were performed using a one-way ANOVA (A and C–F) or a repeated measure ANOVA (H). 4308 IL-17RB AND IL-17RA ARE REQUIRED FOR IL-25 ACTIVITIES

AB 800 300

600 200

400

100 [huIL-5] (pg/mL) 200 [huIL-5] (pg/ml)

0 0

IL-2 IgG 2b IL-2 IL-25 G IL-25 Medium Medium Goat -hIL17RA -hIL17RA Control.Fc IL-2 + IL-25Control IL-17RB.FcFc α α IL-2 + IL-25 IL-17RB.FcIL-17RA.Fc IL-17A 1ng/mL MouseMu IgG2b α-huIL-17A Mouse MuIg IL-17A 10ng/mL Goat IL-17A 100ng/mL IL-2 + IL-25 IL-2 IL-2 + IL-25 Downloaded from FIGURE 7. Goat anti-human IL-17RA Ab treatment but not mouse anti-human IL-17A mAb treatment can block IL-25-induced IL-5 production in an in vitro human PBMC assay. A, Human PBMC were cultured in TSLP for 24 h, harvested, and restimulated with IL-2 and IL-25 for an additional 72 h in the presence or absence of soluble IL-17RB.Fc protein, IL-17RA Abs, or appropriate control . The production of IL-5 was determined in the culture supernatants by ELISA and is reported as the mean Ϯ SD. Data shown are from one PBMC donor and are representative of three experiments performed using three different donors. B, Human PBMC were cultured in TSLP for 24 h, harvested, and restimulated with IL-2, IL-2 and IL-25, or IL-2 and IL-17A for an additional 72 h. Inhibition experiments were done in the presence or absence of soluble IL-17RB.Fc, soluble IL-17RA.Fc protein, a

blocking IL-17A mAb, or an appropriate control protein. The production of IL-5 was determined in the culture supernatants by ELISA and is reported as http://www.jimmunol.org/ the mean Ϯ SD. Data shown are from one PBMC donor and are representative of two experiments performed using two different donors. the anti-IL-17RB mAb (Fig. 6C), anti-IL-25 mAb (Fig. 6D), or human IL-17RA Ab can block IL-17A activities, unlike the poly- anti-IL-17RA mAb (Fig. 6E), similar to that seen in mice intrana- clonal Ab to IL-17RA which is known to block IL-17A activities. sally dosed with vehicle (Fig. 6A). Moreover, IL-25-induced gob- Given the polyclonal anti-IL-17RA Ab we tested is reported to let cell hyperplasia was dramatically reduced in mice treated with be capable of neutralizing IL-17A bioactivity and completely in- anti-IL-17RB mAb, anti-IL-25 mAb, or anti-IL-17RA mAb (Fig. hibited the response to IL-25, the role of IL-17A in this PBMC 6G). Anti-IL-17A mAb treatment had no effect on IL-25-induced assay was next examined. TSLP-stimulated PBMC did not pro- by guest on September 29, 2021 pulmonary lesions (Fig. 6F) or goblet cell hyperplasia (Fig. 6G), duce IL-5 in response to IL-17A stimulation alone (data not providing further evidence that although IL-17A may be involved shown) or in response to IL-17A plus IL-2 (Fig. 7B). In addition, in IL-25-induced BALF neutrophilia, all other IL-25- induced ac- IL-17A plus IL-25 plus IL-2 produced similar amounts of IL-5 as tivities measured in this study are independent of IL-17A activity. IL-25 plus IL-2 (data not shown). These data provide evidence that the TSLP-stimulated PBMC do not respond to IL-17A. An Ab to IL-17RA can block IL-5 production from We have not been able to detect endogenous IL-17A protein in IL-25-stimulated human PBMC cultures the PBMC culture medium (data not shown); however, to more The data generated in mice provide substantial evidence of a role closely examine the role of endogenous IL-17A in this PBMC for IL-17RA in IL-25-induced activities, leading us to next inves- assay, the effects of two IL-17A antagonists on IL-25-induced IL-5 tigate this relationship in human cells. We first developed a human production were tested. Blockade of endogenous IL-17A using ei- IL-25 cell-based bioassay and then analyzed the effects of anti- ther soluble IL-17RA.Fc or a neutralizing mAb to IL-17A did not human IL-17RA Abs in that assay. TSLP-stimulated dendritic cells inhibit IL-5 production in response to stimulation by IL-25 plus have been shown to induce expression of IL-17RB mRNA in IL-2 (Fig. 7B). The lack of inhibition seen with IL-17A antagonists memory Th2 T cells and to expand these IL-25 responsive cells provide further evidence that IL-17A does not play a role in the (24, 25). We tested whether or not TSLP stimulation could create IL-25 plus IL-2- induced IL-5 production in this PBMC assay. an IL-25 responsive environment in a culture of PBMC. In fact, Taken together, these data demonstrate that IL-17RA plays a func- TSLP-stimulated PBMC produced IL-5 in response to IL-25 stim- tional role in the human IL-25R complex in vitro that is indepen- ulation in the presence of IL-2, and this IL-5 production could be dent of IL-17A, providing an encouraging complement to our data blocked to the level of IL-2 stimulation alone by treatment with generated in mice. soluble IL-17RB.Fc but not by treatment with an irrelevant Fc protein (Fig. 7A). PBMC cultures not prestimulated with TSLP Discussion showed variable responses to IL-25 plus IL-2 stimulation at a In the present study, we have provided evidence that IL-25-in- lower level (data not shown). The effects of two Abs to human duced activities require both IL-17RB and IL-17RA. IL-17RB has IL-17RA were next tested in this assay. Complete blockade of been shown previously to bind IL-25 and to induce activation of IL-25-induced IL-5 production in these cell cultures was observed multiple signaling molecules in response to IL-25 stimulation (11, with a polyclonal goat Ab to human IL-17RA but not with an 12, 14). Our experiments using IL-17RB KO mice and an antag- irrelevant control polyclonal goat Ab (Fig. 7A). Treatment with a onistic Ab to IL-17RB provide additional functional data support- mouse mAb to human IL-17RA, however, was not able to block ing a role for IL-17RB in mediating IL-25-induced activities. A IL-25-induced IL-5 production in this PBMC assay (Fig. 7A). It is surprising finding was that IL-17RA KO mice also did not respond currently unknown whether or not this mouse monoclonal anti- to IL-25 stimulation, and our subsequent experiments showed that The Journal of Immunology 4309 antagonistic Abs to IL-17RA could completely inhibit IL-25- in- BALB/c splenocytes (data not shown) and intranasal IL-17F did duced activities in the mouse and in a human primary cell assay. not increase BALF leukocytes or BALF IL-5 and IL-13 concen- The observation that IL-17RA was involved in IL-25 activities trations (data not shown). These data suggest that IL-17F and was unexpected because it has been reported that this ligand and IL-25 do not have similar activities in vitro or in vivo. Further- receptor do not associate. Human 293 kidney cells transfected with more, IL-25 did not induce detectable concentrations of IL-17F in an expression vector for IL-17RA bound IL-17A.Fc but not IL- stimulated splenocyte cultures or in BALF from intranasally dosed 25.Fc (11). In addition, we have not detected an interaction be- mice (data not shown). Together, these data provide initial evi- tween IL-25 and IL-17RA.Fc using BIAcore technology (our un- dence that IL-17F is not involved in IL-25-mediated activities. published data). However, there are other examples where a However, future experiments using antagonistic Abs to IL-17F subunit does not bind ligand but is required for will provide further clarity as to the potential role IL-17F may have activating signaling pathways in response to that ligand. For ex- in mediating IL-25-induced activities. ample, IL-1RAcP does not bind either IL-1␣ or IL-1␤, but it is The signaling pathways activated by IL-17A and IL-25 include required along with IL-1RI to form a fully functional IL-1R com- many of the same proteins. For example, stimulation of different plex (reviewed in Ref. 26). cell types with either IL-17A or IL-25 activates TNFR-associated In addition to investigating the receptor subunits involved in factor 6 (TRAF6), ERK, JNK, and p38, but the mechanisms by IL-25-mediated activities, we also investigated the potential in- which these signaling pathways are activated have been reported to volvement of IL-17A. IL-25 consistently induced IL-17A lung be different (12–14). For example, IL-17RA does not contain a mRNA expression in our mouse experiments, suggesting the pos- TRAF6-binding motif but it has been reported to recruit TRAF6 by sibility that many of the effects of IL-25 that we were measuring directly associating with the adaptor protein Act1 which does con- Downloaded from could in fact be due to IL-17A acting through IL-17RA, rather than tain a TRAF6-binding motif (29). IL-17RA and Act1 interact IL-25 acting directly through IL-17RA. We have been unable to through homotypic interaction of their SEFIR (similar expression detect IL-17A protein in BALF or lung homogenates of mice in- to fibroblast genes and IL-17R) domains, which tranasally dosed with IL-25 or in cell culture supernatants of either share similar residues with two of the three conserved motifs mouse splenocytes stimulated with IL-25 or human PBMC stim- present in TIR (Toll/IL-1R) domains (30). Signaling through IL-

ulated with IL-25 plus IL-2 (our unpublished data), but to directly 17RB, in contrast, has been reported to be through direct interac- http://www.jimmunol.org/ test the role of IL-17A we examined the ability of IL-17A to in- tion of the receptor with TRAF6 (12). Unlike IL-17RA, the cyto- duce IL-5 and IL-13 in a mouse splenocyte assay and the effects of solic region of IL-17RB contains a TRAF6-binding motif but does antagonistic Abs to IL-17A on IL-25-mediated activities in both not contain a SEFIR domain (12, 30). Interestingly, IL-17RC, our mouse and human experimental systems. IL-17A did not in- which has also been reported to interact with IL-17RA, also does duce IL-5 or IL-13 production in our mouse splenocyte assay, not contain a SEFIR domain (15, 30). It is possible that the SEFIR demonstrating that it does not have the same activity as IL-25 in domain of IL-17RA may be important for initiating intracellular this in vitro assay. Our experiments using a neutralizing anti-IL- signaling pathways, either in response to IL-17A when complexed 17A mAb in mice revealed a possible role for IL-17A in mediating with IL-17RC or in response to IL-25 when complexed with IL- IL-25-induced BALF neutrophil accumulation, but also showed 17RB. Elucidation of the precise functional role of each of these by guest on September 29, 2021 that IL-17A did not play a role in any other IL-25-induced activity receptors in activating signaling pathways in response to ligand measured (Figs. 5 and 6). In our human PBMC assay, a mouse stimulation will require continued investigation. anti-human IL-17A mAb did not inhibit IL-25-induced IL-5 pro- Collectively, these data demonstrate for the first time that IL- duction (Fig. 7B). IL-17A mediates recruitment of neutrophils 25-mediated activities require the presence of both IL-17RB and (22); therefore, it is not surprising that this component of our IL-17RA, identifying IL-25 as another IL-17 ligand family cyto- mouse experiment could be attributed to IL-17A. The fact that all kine that utilizes a receptor comprised of at least two distinct sub- of the other IL-25-induced activities were independent of IL-17A units. Additional studies are required to determine how IL-17RA activity, both in our mouse and human experiments, supports our interacts with IL-17RB and in what stoichiometry for mediating hypothesis that IL-25 acts directly through a receptor complex IL-25-induced activities. IL-17RA has now been identified to be comprised of at least IL-17RB and IL-17RA. We did not expect involved in the receptor complex for IL-17A, IL-17F, and IL-25, IL-17A to mediate all of the downstream effects of IL-25 that were and it will be interesting to compare how IL-17RA interacts with measured because it has been shown that IL-17A and IL-25 me- other IL-17R family members in these different receptor diate different activities in vivo and in vitro. For example, adeno- complexes. viral expression of IL-17A in mice induced neutrophil influx into the lung while adenoviral expression of IL-25 induced Acknowledgments influx (22). In addition to increasing BALF neutrophils, we have We thank Richard Armitage, Guang Chen, Carlos Escobar, Kathy Shaffer, shown that intranasal administration of IL-17A increased BALF Kim Shigenaka, and Sam Tran for their technical assistance and/or helpful KC concentrations 1 h after dosing, while IL-25 did not (our un- discussions. published data). Finally, in our human PBMC assay, we have Disclosures shown that IL-25 but not IL-17A induced IL-5 production from E. Rickel, L. Steffen, B. Yoon, J. Rottman, D. Kugler, D. Swart, P. Anders, TSLP-stimulated PBMC (Fig. 7B and data not shown). J. Tocker, M. Comeau, and A. Budelsky are all stockholding employees of Another IL-17 family ligand reported to require IL-17RA for Amgen. activity is IL-17F (15, 27). Overexpression of IL-17F in the lung has been reported to increase numbers of neutrophils in BALF (24) References and lung tissue (22). In contrast, intranasal administration of IL- 1. Kolls, J. K., and A. Linden. 2004. -17 family members and inflam- 17F to naive BALB/c mice has been reported to not induce BALF mation. Immunity 21: 467–476. 2. Moseley, T. A., D. R. Haudenschild, L. Rose, and A. H. Reddi. 2003. Interleu- neutrophils (28). To investigate the potential role for IL-17F in kin-17 family and IL-17 receptors. Cytokine Growth Factor Rev. 14: 155–174. mediating IL-25-induced activities, especially BALF neutrophilia, 3. Kim, M. R., R. Manoukian, R. Yeh, S. M. Silbiger, D. M. Danilenko, S. Scully, J. Sun, M. L. DeRose, M. Stolina, D. Chang, et al. 2002. Transgenic overexpres- we compared the effects of IL-17F and IL-25 in vitro and in vivo. sion of human IL-17E results in eosinophilia, B-lymphocyte hyperplasia, and IL-17F did not induce IL-5 and IL-13 production from cultured altered antibody production. Blood 100: 2330–2340. 4310 IL-17RB AND IL-17RA ARE REQUIRED FOR IL-25 ACTIVITIES

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